A mechanistic review on medicinal plants used for rheumatoid arthritis in

traditional Persian medicine.

Farzaei MH1,2,3, Farzaei F2, Abdollahi M4, Abbasabadi Z1, Abdolghaffari
AH5,6,7, Mehraban B8.
Author information
Abstract
OBJECTIVES:
Rheumatoid arthritis (RA) is a chronic, inflammatory, autoimmune disease,
which affects synovial tissue in multiple joints. Although conventional
treatments of RA commonly alleviate the symptoms, high incidence of
adverse reactions leads to research tendency towards complementary and
alternative medicine. As various medicinal plants are traditionally used for
the management of symptomatologies associated with RA in Persian
medicine, we reviewed medicinal literature to confirm their efficacy in the
management of RA.
KEY FINDINGS:
Scientific evidence revealed that traditional medicaments exert beneficial
effects on RA through several cellular mechanisms including downregulation
of pro-inflammatory cytokines such as TNF-, IL-6 and NF-B, suppression of
oxidative stress, inhibition of cartilage degradation with destructive
metalloproteinases and enhancement of antioxidant performance. Various
active constituents from different chemical categories including flavonols,
lignans, coumarins, terpenes, glycosylflavons, dihydroflavonols,
phytoestrogens, sesquiterpene lactones, anthraquinones, alkaloids and
thymoquinones have been isolated from the medicinal plants.
SUMMARY:
The pharmacological mechanisms of the medicinal plants traditionally used
for RA in Persian medicine are discussed in the current review. Further
investigations are mandatory to focus on bioefficacy of these phytochemicals
for finding novel natural drugs.
2016 Royal Pharmaceutical Society.
KEYWORDS:
biological mechanisms; complementary and alternative medicine;
inflammatory cytokines; metalloproteinases; mitogen-activated protein
kinases; natural drugs; nuclear factors; phytochemicals; phytotherapy;
transduction signalling pathways

The role of ultrasound in pharmaceutical production:

sonocrystallization
http://onlinelibrary.wiley.com/doi/10.1111/jphp.12614/full
Authors
Laura de los Santos Castillo-Peinado,
Mara Dolores Luque de Castro
First published: 27 July 2016Full publication history
DOI: 10.1111/jphp.12614View/save citation
Cited by: 0 articlesCitation tools
Article has an altmetric score of 1
Funding Information
Abstract
Objectives
The main aim of this review was to develop a critical discussion of the key
role ultrasound (US) can play on the production of active pharmaceutical
ingredients (APIs) by discussing the versatile effect this type of energy
produces.
Methods
The different crystallization techniques that can be assisted and improved by
US are discussed in the light of the available US devices and the effect
pursued by application of US energy. Simple and complex analytical methods
to monitor API changes are also discussed.
Key findings
The countless achievements of API US-assisted production are summarized in
a table, and outstanding effects such as narrower particle size distribution;
decreased particle size, induction time, metastable zone and supersaturation
levels; or a solubility increase are critically discussed.
Conclusions
The indisputable advantages of sonocrystallization over other ways of API
production have been supported on multiple examples, and pending goals in
this field (clarify the effect of US frequency on crystallization, know the
mechanism of sonocrystallization, determine potential degradation owing to
US energy, avoid calculation of the process yield by determining the
concentration of the target drug remaining in the solution, etc.) should be
achieved.
Introduction

Pharmaceutical materials science has as aim product development. The solid

form, crystallization and particle engineering are core elements linking the
final steps of the synthesis pathway of active pharmaceutical ingredient (API)
manufacture to the drug product attributes. Crystallization is the final step of
API manufacture that, from a regulatory perspective, must be controlled and
reproducible, providing API of suitable quality in terms of both purity and
appropriate physical properties for dosage form design and robust product
processing.[1] In addition, advances in drug delivery systems require
specially engineered drug particles to meet biopharmaceutical and
processing needs.[2] Accordingly, development of engineered drug particles
has become a major research area due to limitations of conventional particle
formation and pretreatment processes in fine-tuning. Techniques such as
micronization,[3] spray-drying,[4] spray-freezing,[5] supercritical fluid
processing,[6] spherical crystallization,[7] solution atomization and
crystallization by sonication (SAXS),[8] sonocrystallization[9] and
sonofragmentation,[10] among others, are used to provide particles with
novel physicochemical properties. Once dissolved, orally ingested drugs
must have sufficient lipophilicity to move across cell membranes, but also
sufficient hydrophilicity to be transported within the body. However, the rates
of dissolution after intake of moderately hydrophobic drugs can be
problematic, unless their crystal size is small enough. Aerosol drugs also
require control of the particle size and size distribution to administer dosage
successfully: particles too large will not get into the deep lung and particles
too small will be less easily trapped but more easily absorbed. Parenteral
(injected) drugs must also control particle size because potentially fatal
embolisms can result with particles larger than ca. 5 m.[11]
The crystallization phenomenon
Crystallization from the melt or from solution is a major separation and
purification unit operation applied in the chemical and pharmaceutical
industries.[12] The main driving force for crystallization is supersaturation,
the conditions for which can be reached by different ways, each with typical
limitations. Most times, lowering the temperature of the preheated saturated
solution decreases the solubility of the solute in the solvent leading to
supersaturation and crystal formation as a result this approach is not
applicable to compounds having weak or no dependency of solubility on the
temperature. Supersaturation can also be achieved by solvent evaporation
it is not suitable for crystallization of heat-sensitive materials,
pharmaceutical products and explosives. Antisolvent supersaturation[13, 14]
is an alternative approach to overcome the difficulties in evaporative and
cooling supersaturation by addition of an antisolvent in which the solute
solubility is very low or negligible, thus leading to crystallization by an
energy-saving process as compared to the cooling and evaporative
crystallization. Major disadvantages of antisolvent crystallization are the

chances of improper mixing of the antisolvent and the original solvent, in

addition to the problems of solvent recovery process. The improper mixing of
antisolvent can result in the undesired spontaneous nucleation and highly
localized supersaturation leading to agglomerated crystals with a large
crystal size.[15] Combination of cooling and antisolvent crystallization for
specific compounds is another approach to achieve the desired
supersaturation and crystal size distribution.
Crystal and particle engineering involves control of the crystallization
kinetics (nucleation, growth, agglomeration). It can also involve particle
engineering technologies such as dry impact, high shear wet milling (HSWM)
[12] or sonofragmentation[16] to reduce particle size via particle breakage.
Sonocrystallization[17] is the technique based on the use of ultrasound (US)
to direct the generation of the desired morphology and particle size
distribution (PSD), as this energy has shown to increase the nucleation rate
in the target medium as compared to experimentation carried out without
US[9, 18-20] and, therefore, to produce smaller particle size with narrower
size distribution. Controversial information on the effects ascribed to USassisted crystallization (increased or retarded crystal growth rate,[20-23]
increased[24] or decreased induction periods[25-29]) but also conclusive
information on decreased metastable zone width (MZW),[9, 22, 30]
enhanced solubility,[31] decreased mean particle size[19, 24, 25, 32] and
homogenous particle size distribution and crystal morphology [21-23, 33]
appear in the literature.
A matter closely related to crystallization is supramolecular self-assembly, a
powerful tool to develop functional advanced materials. Applications of
supramolecular materials require precise control of morphology of
hierarchical structures and the macroscale structural properties, because
properties of supramolecular materials not only are decided by the
properties of the precursor molecule, but are also sensitively dependent on
both shape and size. However, control of supramolecular self-assembly from
given molecules with a desirable morphology, and therefore function,
remains a great challenge.[34] Recently, US has been found to play an
unexpected role in affecting supramolecular self-assembly, thus providing a
new way to modulate supramolecular self-assembly.[34]
Ultrasound: devices and features
The characteristics of US are highly influenced by the type of device that
provides this type of energy,[35] but only aspects that seem to influence
crystallization are considered in this section. General properties of US,
frequency zone and mechanisms of US energy transmission can be found
elsewhere.[35]
US equipment

There are three main types of US devices at the laboratory scale: cleaning
baths, probes (also referred to as US horns) and reactors or plate
transducers. Despite the well-known deficiencies of US cleaning baths when
used for tasks different from cleaning or degassing, for which they have been
designed,[35] a number of researchers use this device, omnipresent in most
laboratories. The irreproducible performance of US cleaning baths and the
decline of power with the working time are not taken into account by the
authors who use them for crystallization purposes without considering the
influence of these aspects on the results. US probes are the most used
devices to favour drug crystallization, usually commercial probes that apply
their characteristic effect on either, commercial[36-38] or laboratorydesigned cells,[2, 39] working in a batch[40] or flow regime.[40, 41] US
laboratory reactors (also known as US processors), either commercial or
especially designed, are usually more versatile, as they can provide different
US frequencies in narrow[42] or wide ranges,[40] and variable US power that
can reach more than 1000 W.[19]
US probes or tips are scaled down from the conventional laboratory size to
work with g-to-mg samples close to the discovery phase of pharmaceutical
development where only very small amounts of material are available.[43]
These devices are usually made on titanium, except when they work in the
presence of microwaves that require probes or tips of glass.
Scale-up US devices for industrial applications are of two main types: (1)
high-intensity probes operating in a flow cell[21, 44] or immersed into a large
volume,[45] always in direct contact with the processed system and applying
their energy in a no-focused manner; (2) opposing parallel transducers or
arranged around a duct through which the processed system flows are
devices that usually provide higher US energy than probes, but also with the
characteristic no homogeneous distribution. The number of transducers can
be one or several.[46]
Features of US
The chemical and physical effects of US do not come from any direct
interaction with molecular species. Instead, sonochemistry (more properly, it
should be ultrasonochemistry) arises from ultrasonic cavitation: the
formation, growth and implosive collapse of bubbles in a liquid.[47-50] In
homogeneous liquids, collapse of bubble clouds produces intense local
heating with a temperature of ca. 5000 K, pressures of ca. 105 kPa and
enormous heating and cooling rates above 1010 K/s within isolated
submicron reactors.[50-53] The physical effects of US that arise in
heterogeneous, solidliquid systems are also derived from cavitation. When a
cavitating bubble (i.e. ca. 50 m at maximum size under 20 kHz) collapses in
a significantly larger surface, the bubble undergoes a markedly asymmetric
collapse, which generates a high-speed jet of liquid with a velocity higher

than 100 m/s that smashes into the surface.[54] The impingement of this jet
can cause localized erosion, surface pitting, ultrasonic cleaning and
enhancement of surface by generating shear forces.[54] Cavitational
collapse also releases shockwaves that have velocities as high as ca. 4000
m/s and high pressure amplitudes of 106 kPa, which can easily produce
plastic deformation of malleable metals and induce high-velocity collisions
between micron-sized solid particles.[55-57]
Ultrasound nomenclature
A traditional misuse of the US nomenclature is not distinction between the
audible zone and the US zone, including both in the former (e.g.
sonochemistry, sonocrystallization). Some authors justify this combination by
a higher simplicity, which is to the detriment of clarity and proper use of
terms, in the authors opinion. As a consequence of this misuse, the
adjective acoustic is also used in dealing with frequencies above 16 kHz
and within all range encompassed by US (viz., 16 kHz to GHz).[35] Because
of the general use of sonocrystallization, this term is used in this review,
despite ultrasonocrystallization is the right form.
No many authors seem to know that US is not of radiant nature; therefore,
US irradiation is frequently found in the literature on this matter.[10, 17, 42,
58-61] Less frequent but also found in US-related publications is the use of
wavelength instead of frequency.[10]
Sonocrystallization history
Despite Richards and Loomis reported in 1927 the first study on the effects
of US on crystallization,[62] the research in this field was delayed owing to
inconsistent results and the lack of proper US devices. Revitalization of the
technique occurred in the 1950s and 1960s when many of the benefits of
sonocrystallization discussed in current literature were first observed. A
review on this subject by Kapustin emphasized the reduction in grain size in
melt crystallizations via US,[63] Turner et al.[64] reported that a short burst
of high-intensity US induced crystallization of sugar syrups that were
resistant to crystallization, and other authors reported that micronized,
uniform crystals of pharmaceuticals were achieved via US-based approaches.
[65-67] Over fifty years have elapsed since early reviews on
sonocrystallization discussed the proposed mechanisms of action[67, 68]
attempting to understand the fundamentals and control of
sonocrystallization. In a recent review, the effects of US on crystallization
have been reviewed and the mechanisms of the driving force of US to
achieve a desired product critically discussed.[16]
The role of US in drug crystallization
The properties of drug substances and dosage forms can be highly affected
by the particle size, a critical process parameter in pharmaceutical
production. The fundamental issue with particle size analysis is the variety of

equivalent particle diameters generated by different techniques, which is

largely attributable to the particle shape and particle dispersion mechanism
involved. Thus, to enable selection of the most appropriate or optimal sizing
technique, cross-correlation among different techniques may be required. For
these reasons, particle design techniques (e.g. spherical crystallization,
extrusion spheronization, pastillation, fragmentation and fluid-jet and air-jet
milling) have been developed to modify the physicochemical, micromeritic
and biopharmaceutical properties of the drugs.[69] Pharmaceutical
sonocrystallization (i.e. the use of US to favour API crystallization) is under
very active investigation for its ability to influence particle size and size
distribution, and also to reduce the MZW, induction time and supersaturation
levels required for nucleation, to improve reproducibility of crystallization, to
control polymorphism and to reduce or avoid the need for seed crystals or
other foreign materials.[42, 58] Also US can favour postcrystallization
processes such as particle rounding or fragmentation, among others.[69]
Objectives of pharmaceutical crystallization under US application
The key challenge in pharmaceutical active crystallization is manufacturing
the optimum solid form with the desired chemical and physical properties,
which can involve to produce either a metastable or stable crystalline form,
to study the possibilities of obtaining an amorphous drug product, to
manufacture the right size particle in principal manufacture and to optimize
crystallization yield. The main objectives of sonocrystallization were as
follows: (1) to reduce the MZW as nucleation occurs at a lower degree of
supersaturation, (2) to enhance crystal productivity by a rapid induction of
primary nucleation, (3) to reduce the crystal size, (4) to inhibit
agglomeration, (5) to allow manipulation of crystal size distribution, (6) to
modify the morphology by polymorph control, (7) to reduce impurities and
(8) to improve solidliquid separation. Moreover, it can help to increase
secondary nucleation by disrupting crystals or agglomerates. How US
assistance allows obtaining a high density of crystal distribution and a lower
particle size as compared to conventional crystallization, milling hammer (to
decrease particle size) and optimized and non-optimized HSWM is shown in
Figure 1. In this way, particle size reduction and the potential change in
crystal habit may solve solubility/permeability problems and also facilitate
generic formulation.[70] Therefore, sonocrystallization is a viable
manufacturing option whether by continuous flow mode, batch mode or insitu generation of seed crystals.
Figure 1.
Figure 1. Open in figure viewerDownload Powerpoint slide
Measurements of particle size and size distribution obtained. Reproduced
from [41] with permission of the American Association of Pharmaceutical
Scientists.
Sonocrystallization and sonopost-crystallization techniques

While sonocrystallization is under very active investigation for its ability to

favourably influence crystallization-related parameters, reports on the effects
of US on postcrystallization particles are surprisingly limited.
The most frequently used US-assisted crystallization techniques are
discussed below:
Evaporation-based and cold-based sonocrystallization
The two classical ways to reach saturation evaporation (when the
thermostability of the system was appropriate)[61, 70-72] or cooling (when
the decrease in solubility of the target drug was significant enough)[40-42,
60, 73] have been assisted by US, and the typical crystallization
parameters have been improved in all instances.[42, 72]
Salting-out sonocrystallization
This technique involves fast addition to the solute solution of enough
precipitant (salting-out agent) to yield the complete precipitation and
immediate or simultaneous application of US, which travels throughout the
crystallization vessel to mix, cavitate and blend the precipitant with the
solution uniformly. The solubility of the solute in the solvent is sharply
reduced, and the solution immediately reaches its maximum supersaturation
so that primary nucleation and crystal growth occur rapidly. The exact
amount of precipitant is determined by the required yield of the product and
its solubility. The maximum supersaturation must be established at the
beginning of the sonocrystallization process to provide enough driving force
for continuous nucleation and growth.[17]
Melt sonocrystallization
This particle engineering technique involves application of US energy to the
soft or viscous molten mass dispersed in an immiscible liquid[74]; thus,
solidification/crystallization from emulsified melt is carried out under the
influence of US energy. The technique, initially used to produce sintered
crystals and porous glassy beads, allows extending the US energy received
by the melt in the emulsified state and determines the properties of the
resultant particles, which are dependent on US energy input and frequency,
and solidification rate of the melt. In turn, this last variable depends on the
temperatures of glass transition of the material and that of the medium.
Application of US at temperatures above the transition temperature favours
crystallization, whereas processing below the transition temperature results
in an amorphous state. The mechanical stress due to ultrasonication results
in sintered crystals or porous beads. The porous nature and potential for
producing crystalline particles as well as amorphous particles offer flexibility
to the technology and are looked upon for improving the solubility of poorly
soluble pharmaceuticals.

Antisolvent sonocrystallization
This technique involves selection of an antisolvent that can successfully
precipitate the dissolved compounds from their solutions favoured by the
application of US during the crystallization step. The role of the antisolvent is
to reduce the solubility of a solute in the solution and induce prompt
crystallization, thus making unnecessary the use of thermal energy that can
degrade the activity of materials sensitive to temperature, and avoiding
expensive energy-intensive equipment required for evaporation-based
crystallization. Therefore, the core of antisolvent crystallization is the
selection of the antisolvent, while application of US during this step
facilitates the process by circumventing the problems associated with
antisolvent crystallization using polar antisolvents as water for hydrophobic
pharmaceutical compounds[75] or no polar antisolvents as carbon dioxide for
hydrophilic compounds. The main advantages of a polar antisolvent as water
for crystallization of hydrophobic drugs are the nearly zero solubility of such
compounds and complete miscibility with many polar organic solvents, in
addition to be an environmentally safe option easily separated from final
products. A disadvantage of water as antisolvent successfully circumvented
by US application is a delayed mixing rate between the solution and
antisolvent owing to a low diffusivity of water in organic solutions.[33] The
low mixing rate could hinder the fast local supersaturation, thus decreasing
the nucleation rate and subsequent particle formation, which lead to the
formation of large crystals with a broad size distribution. The use of carbon
dioxide as antisolvent is promoted by its relatively high miscibility with
organic solvents and low solubility towards polymers and pharmaceuticals.
However, the requirements of high-pressure equipment along with high
operating costs increase the expenses of the crystallization process.[76-78]
Spray sonocrystallization
This recent technique is a sophisticated mode of antisolvent crystallization in
which the drug solution is sprayed into the flow of the antisolvent by a
channel drilled down in the centre of a specially designed flow-through US
probe. In this way, the drug solution is atomized upon its exit from the horn
into the flowing stream of antisolvent. The momentum transfer and
micromixing created by acoustic cavitation form a fine dispersion of the drug
solvent into the flowing antisolvent within 100 ms, which substantially
increases the rate of solventantisolvent mixing and leads to a rapid
formation of nanocrystals at the 100-nm scale.[72]
Solution atomization and crystallization by sonication
This technique, with principles similar to the previous two, was developed to
produce APIs with a narrow particle size distribution, centred in the optimum
particle size for reproducible and maximum therapeutic efficacy.[8] An SAXS
process consists of three interdependent steps: (1) production of aerosol
droplets of the solute into a carrier solvent using a suitable aerosol

generator, (2) collection of the highly supersaturated droplets in a

crystallization vessel containing a non-solvent of the drug and (3) application
of US energy to a crystallization vessel to controllably induce homogeneous
nucleation and crystal growth. By combining these steps and controlling
relevant parameters, high-purity micron-sized sphere-like crystalline particles
are produced. The rapid evaporation of solvent from the micron-sized aerosol
enhances the kinetically limited diffusion within the droplets, while
application of US energy minimizes agglomeration, thus improving the
powder-handling properties of crystallized particles. Only on application of
US, does the droplets in the antisolvent undergo nucleation and
crystallization? Without US, the particles solidify into semicrystalline and
poorly defined particles. The major advantage of this technique relates to the
use of any suitable aerosol generator and development of the whole process
under atmospheric pressure and ambient conditions. SAXS results in
spherical crystalline particles within well-defined particle size range; it has
the potential for batch and continuous processing at an industrial scale, but
the limitation of requiring a suitable organic solvent to dissolve the drug that
undergoes evaporation at the required rate.
Studies about the effects of US on postcrystallization are surprisingly limited.
[79] Two of the main techniques used for reduction and homogenization of
crystal particles after crystallization are discussed below:
Particle rounding/sonofragmentation technology
Particle rounding, a technique invented for shaping of explosives,[10]
consists of suspending the starting material in a partial solvent liquid, and
applying US energy to the suspension, whereby the starting material is
shaped and ground to produce a final suspension of ground and rounded
particles. In heterogeneous solidliquid systems, the interface produces a
perturbation in the US field that induces an asymmetric collapse of the
cavitational bubbles. At extended interfaces several times larger than the
resonance cavitation size, the result is a microjet of liquid passing through
the cavitation that impinges on the solid surface at ca. 100 m/s. The
phenomenon is the origin of the erosion effect, used for ultrasonic shaping
and surface modification; therefore, this technology is applied for shaping of
drugs and excipients with the main benefits of (1) improving packing density
of powders, (2) enhancing flow of powders, (3) reducing electrostatic
charges, (4) manufacturing by direct compression of material without
granulation and (5) achieving higher filler loading in composite pastes.
Recently, Zeiger and Suslick[73] have reviewed the potential mechanisms for
the breakage of molecular crystals under high-intensity US
(sonofragmentation) and related their experimental and modelling studies of
sonocrystallization and fragmentation of acetylsalicylic acid (aspirin) crystals
as a model API. The authors suggested as primary mechanism of
sonofragmentation of molecular crystals the direct particleshockwave and

related particleturbulent shear interactions. They evaluated the effect of

particle concentration on rates of sonofragmentation (e.g. final particle size
after sonication for fixed time and intensity) and established that if the rate
of particle fragmentation is strictly first order in particle concentration, then
the average particle size after a given ultrasonic exposure time would be
zero order in concentration of particles. On the contrary, if the rate of
fragmentation is dominated by interparticle collisions, the rate of
fragmentation would be second order in particle concentration, and the
particle size would be linearly related to the initial particle concentration (i.e.
mass loading in a slurry). No dependence of average particle size after
fragmentation on the mass loading (i.e. initial particle concentration) was
demonstrated; therefore, interparticle collisions do not dominate the
mechanism of sonofragmentation. Particlehorn collisions and particlewall
collisions were considered minor contributors to sonofragmentation
mechanisms.
Mechanisms and variables affecting sonocrystallization
The variables that affect sonocrystallization are a consequence of the
involved mechanisms, which are discussed first.
Mechanisms involved in sonocrystallization
The well-known beneficial effects of US on crystallization processes have
been attributed to different mechanisms which partially or totally explain
these effects and the final results. A widely used explanation to these
phenomena is the so-called hot spot theory, which attributes nucleation to
local hot spots, created by the concentration of kinetic energy in the
collapsing cavity or due to rapid cooling afterwards. Other popular
mechanism is based on the pressure shockwave caused by cavity collapse
that creates localized high pressures. There are substances for which the
solubility decreases by increasing the pressure, thus increasing local
supersaturation and inducing nucleation. A hypothesis related to the
shockwave effect states that nucleation starts by segregation of the solute
and solvent near the bubble wall.[58] This is caused by high pressures
occurring in the ultimate phase of bubble collapse. Yet, another hypothesis
suggests that nucleation occurs during bubble expansion: solvent
evaporating into the bubble or cooling of the liquid interface layer increases
local supersaturation, which could lead to nucleation around the cavity. It is
assumed that nucleation is induced due to the bubble surface acting itself as
nucleation centre so that the mechanism seems to be of heterogeneous
nature. To prove this assumption, gassing has been investigated as
nucleation inductor during batch cooling crystallization: the gas bubbles are
just expanding, not collapsing.[59] Also of interest is the electrical theory,
which proposes the consequences of cavitation as caused by electrical
charges on the cavity interface layer.

The most accepted explanation of the small size particle achieved by

sonocrystallization is related to the characteristic effects of US (streaming
and cavitation as the most important): the stirring effect causes a reduction
in thickness of the diffusion layers in the vicinity of the crystal surfaces by
the high-energy shockwaves impinging on the particle surface. This can
create high-velocity interparticle collisions that can alter the particle
morphology and size dramatically. It was reported that these interparticle
collisions occur with such a great force that even metal particles tend to melt
together.[55] Microjet formation in the vicinity of the particle surface is
another well-established mechanism for accounting the effect of cavitation in
solidliquid systems that seems to occur only if the surface is several times
larger than the resonant bubble size.[80] The production of fines and the
erosion of the parent seed crystals by the action of US caused a rapid
increase in the total surface area available for growth. It is likely that the
rapid healing of the fractured parent crystals and the outgrowth of fines after
US application in the growth experiments either camouflage any differences
in crystal habit at the end or produce morphological changes, depending on
both the target system and the involved variables.
Control experiments without supersaturation showed that during US
application, seed crystals were disrupted and many fines appeared. Thus,
this approach can be applied to systems that are difficult to nucleate.
Scanning electron microscopy (SEM) and particle size analyses showed that
alteration in the habit and size of the target crystals by US is possible. The
increased surface area available for crystal growth resulted in the observed
crystallization rate enhancement.[59]
As the exact mechanisms behind US-assisted crystallization are not known
yet, the following hypotheses seem to be the most accepted: (1) the effect of
US is not directly caused by vibrations of the US waves but by the cavitation
bubbles formed by the US field[42, 69, 81]; (2) both the amount and the size
of the cavitation bubbles affect the nucleation rate; (3) higher US intensities
produce more cavitation bubbles and nucleation increases; (4) larger US
frequencies produce smaller cavitation bubbles which have a smaller impact
on the nucleation rate; and (5) the segregation and cavitation bubble
theories link the nucleation rate to the size of the cavitation bubbles.[25]
In addition, the following US effects have been experimentally supported: (1)
improved microscale mixing occurs from cavitation and associated
turbulence, accelerating diffusion rates of reactants and reducing induction
times for crystallization as a result.[17, 26, 30, 82] Reduced induction time
increases the rate of nucleation by increasing the growth rates of embryonic
crystallites, which prevents their redissolution.[83] (2) By similar
phenomena, US also reduces the MZW (i.e. the range of metastability of a
supersaturated solution in either temperature or antisolvent concentration),
which diminishes the rate of crystal growth and decreases crystal size.[9, 23,

84] (3) The increase in gasliquid interfaces produced by bubble formation,

collapse and fragmentation enhances nucleation rates.[83] (4) Rates of
secondary nucleation are also increased by US by breakage of primary
crystals due to interparticle collisions or, more importantly, shockwave
fragmentation during ultrasonication (i.e. sonofragmentation[16, 79])
increases the number of secondary nucleation sites, which results in
increased number of smaller crystals. (5) Turbulent flow from cavitation also
diminishes crystal aggregation, which produces smaller solid particulates
with narrower size distribution. (6) Antisolvent sonocrystallization, which
generates a high level of supersaturation quickly and induces higher
nucleation rates,[83, 85-88] benefited by enhanced mixing between the
antisolvent and solution.
Variables affecting sonocrystallization
The sonocrystallization process is influenced by US variables such as
frequency, intensity, power, duty cycle, but also by physical variables such
as temperature, pressure, time, volume of the cell, size of the probe, and
chemical variables such as API concentration, pH. Modelization of the process
helps to better understanding the influence of the variables involved in
sonocrystallization.[70]
The effect of the US frequency on sonocrystallization has been scarcely
studied as most of the research on this process has not taken into account
this variable (sometimes even the value of US frequency does not appear in
the article). The most common situation is the use of a single-frequency
probe plunged into the target system. When a probe working at several
frequencies in a narrow range (1530 kHz at an intensity of 26 kW/l) was
used for sonocrystallization of sepctinomycin hydrochloride, no significant
differences in crystal size or shape were detected when varying the
frequency.[19] This was explained by the fact that the wavelengths are much
larger than the size of the nuclei at these frequencies. A wider frequency
range (203610 kHz) at smaller intensities (167 W/l) was tested in the
crystallization of adipic acid.[42] These different frequencies caused no
significant difference in MZW, although smaller particles and less
agglomeration were observed at the lowest frequencies. The effect of
frequencies of 355.5 and 1046 kHz on the crystallization of dodecanedioic
acid,21 for the same power supplied to the transducers, proved that the
power inside the reaction medium was not constant for the different
frequencies. Therefore, no conclusions could be made about the single effect
of the frequency on the MZW, but only a reduction in it was detected at high
US intensities. These observations, together with the results in other papers
about sonochemical reactions, show the importance of keeping constant the
power inside the reaction medium when comparing different frequencies.[22,
31, 89] Also the effect of US frequencies of 20, 44, 139, 500 and 647 kHz on
the antisolvent crystallization of sodium chloride showed that similar size

distributions were observed at the different frequencies, while the effect of

these frequencies on the MZW was, however, not studied.[90]
The unfeasibility of general conclusions from the above research because of
the use of limited frequency ranges, the lack of calibration of the dissipated
power and the use of different intensities, crystallization products and
reactor geometries led to recent studies on the effects of the US frequency
and intensity on nucleation. Taking into account for the first time, the effect
of US frequency on the MZW and crystal size distribution (CSD) was
investigated over a broad frequency range of 161140 kHz in one single
reactor geometry on one single product.[40] In contrast to previous studies,
the power inside the reaction medium was kept constant for all frequencies.
Furthermore, the effect of the US intensity was investigated in a batch
reactor and a US flow cell. The MZW and CSD of both reactor set-ups were
compared at different US intensities. Finally, the optimal US frequency and
intensity for enhancement of the nucleation were defined. The effect of US
frequencies ranging from 16 to 1140 kHz on the MZW, CSD and crystal shape
was tested at a constant intensity of 53 W/l. Figure 2 shows the average
reduction in MZW as a function of the applied frequency. A reduction in the
MZW, as compared to no US conditions, was observed at all frequencies, but
at 850 kHz, it was attributed to degradation products formed by
ultrasonochemical degradation of the target drug (paracetamol). Either
significant inhibition or promotion of the crystal growth and the appearance
of multiple nucleation bursts in the presence of even trace amounts of
impurities was already reported in the literature,[91, 92] but a degradation
level of more than 10% higher at 850 kHz as compared to 580 or 1140 kHz
was observed by other authors.[40] Therefore, higher level of degradation
could significantly affect the nucleation process and the observed MZWs.
Figure 2.
Figure 2. Open in figure viewerDownload Powerpoint slide
Effect of the studied range of US frequency, from 16 to 1140 kHz, on the
metastable zone width (MZW). Reproduced from [40] with permission of
Elsevier.
The effect of US intensity on the sonocrystallization process seems to be
strongly dependent on the target system, the crystallization method and
time at which US was applied during the process. Figure 3 shows the MZW
and volume mean diameter as a function of the ultrasonic intensity (Ical) in
the batch and flow cell US reactors.[40] In the former, no significant
differences in MZW or mean diameter were observed due to the limited
intensity range. In the flow cell, however, significant higher US intensities
were reached due to the smaller reactor volume. In this mode, the MZW
could be significantly reduced, according to an almost linear trend, from 13.5
C at 570 W/l to 6.4 C at 1784 W/l. The batch set-up seems to be more
efficient to reduce the MZW compared with the flow cell as lower intensities
are needed to achieve an MZW of 8 C compared with the flow cell, which

can be attributed to continuous exposition to US in the former vs pulsed US

by recirculation in the flow cell. The smallest MZW can be, however,
achieved in the flow cell due to the higher intensities in this set-up. It is
worth emphasizing that SEM analysis showed no significant difference
among the US intensities or between ultrasonicated and no ultrasonicated
crystals.[40]
Figure 3.
Figure 3. Open in figure viewerDownload Powerpoint slide
Comparison of the metastable zone width (MZW) and volume mean diameter
obtained by different US intensities using a US batch or US flow cell.
Reproduced from [40] with permission of Elsevier.
Increased values of both US intensity and diameter of the horn tip also
increase the crystallization rate. An increase in US intensity seems to result
in heavier flow, while one in horn tip diameter leads to more uniform flow
patterns, which allows concluding that the effect of cavitation known as
microstreaming contributes no significantly to crystallization, which it is more
markedly affected by macrostreaming.[93]
The influence of US power output (5, 10 and 15 W) on the crystal size and
morphology as compared to the no ultrasonicated process for roxithromycin
showed that US significantly reduced the particle size of drug roxithromycin
without modifying the overall crystal habit (rhombic tabular habit) of the
particles, with a higher reduction by increasing the power output. The
average sizes of particles produced with the power outputs 5, 10 and 15 W
were 27.7, 19.3 and 14.6 m, respectively, while the average size of
particles obtained without US was 58.4 m.[72]
Increasing the US application time gives rise to the following sequence: at
short times, US fails to blend the solution and precipitant uniformly, so little
precipitate is obtained after ultrasonication; longer times produce apparent
crystals the size of which decreases for longer US application.[39] Figure 4
shows the drastic reduction in the crystal size by increasing the time of US
application. These results demonstrated that it is possible to tailor a crystal
size distribution between the extreme cases of a short burst of US to
nucleate at lower levels of supersaturation and allow growth to large
crystals, and the production of small crystals via continuous US application
(or a longer single burst) throughout the duration of the process, which can
facilitate prolific nucleation at higher levels of supersaturation at the
expenses of some crystal growth. Intermittent application of US can give
intermediate effects. In any event, the optimum value needs to be
determined experimentally. Particle size control has been demonstrated for a
number of molecules including sorbitol hexaacetate, where more regularly
shaped crystals can be formed.[94]
Figure 4.

Figure 4. Open in figure viewerDownload Powerpoint slide

Crystal sizes as a function of the time of US application. Reproduced from
[39] with permission of Wiley-VCH.
The temperature negatively influences sonocrystallization, the significance of
the effect largely depending on the influence of this variable on the solubility
of the target system.[61] It was also found that US induces the reduction in
the particle size only when this energy was applied to the solution at the
initial stage of crystallization.[61] According to the crystallization theory, the
rate of nucleation is inversely proportional to the temperature.[93] Moreover,
the mass transfer coefficient that governs the crystal growth rate is strongly
influenced by the temperature of the target system. Therefore, the
temperature is a key governing factor that can control the final particle size
and distribution.
Analytical tools to study the effect of US on drug crystallization
Development of a sonocrystallization method at the laboratory scale requires
application of a number of analytical techniques based on very different
principles, which encompasses the analysis of the target active principle,
through the evolution of the crystallization steps to the in-depth study of the
characteristics of the crystallized product. The formed crystals are studied
from the analytical and biopharmacological points of view, always in
comparison with the target characteristics of the control product obtained in
the absence of US energy. The analytical tools used along the overall process
(and in some cases the suggestion of those that could substitute the present
ones) are discussed below.
One of the simplest instruments in an analytical laboratory, a molecular
absorption spectrophotometer or photometer, is frequently used from the
beginning of the process, to monitor the absorptiometric characteristics of
the API, through the process to know how the liquid phase is being
impoverished in the target active principle as crystallization progresses, and
at the end of the process to calculate the yield by determining the amount of
API remaining in the liquid phase. The main shortcoming of this simple
instrument is the poor analytical information (regarding both sensitivity and
selectivity) it provides. Thus, impurities of the raw materials, reactions
between the active component and impurities or potential degradation of the
active component under US application can be hardly detected by this
instrument. When the presence of some of these undesirable compounds is
suspected, more complex instrumentation as a mass spectrometer (MS),
preferably of the time-of-flight (TOF) type, is required for identification, with a
view on their potential toxicity.[36]
The almost omnipresence of a high-performance liquid chromatograph
(HPLC) with molecular absorptiometric detector in any laboratory is
considered by most of the researchers as an analyser by itself as they
express its application to a method as HPLC analysis (or assay) with not

mention to the detector, indispensable unit as the role of the chromatograph

is only separation. It is the most used tool for determining the purity of the
target API, monitoring crystallization evolution and even to detect the
presence of degradation products if enough molar absorptivity and
concentration of the products allow obtaining a peak in the chromatogram.
The known low sensitivity of molecular absorptiometry could make
mandatory the coupling of an MS detector to the chromatograph if low
concentrations of undesirable compounds are suspected in the system
(preferable a triple-quad MS when the concentrations are very low).
Micromeritics makes use of instrumentation based on very different
principles that allows analysis of small crystal particles from different
aspects. Thus, PSD is mainly studied by laser diffractometry.[2] Nevertheless,
also particle uniformity after blending has been determined in a more coarse
way by ultraviolet (UV) photometry at 276 nm in the case of salbutamol
sulfate.[2] For the analysis of particle morphology, techniques such as
differential scanning calorimetry (DSC), Fourier transform infrared (FT-IR)
spectroscopy, or X-ray powder diffraction (XRPD) spectroscopy can be used,
with special emphasis on the wide type of information (e.g. texture, chemical
composition, crystalline structure, orientation of materials) provided by
sophisticated instruments based on SEM and related techniques.[95]
Flowability of a target system is evaluated by determining the angle of
repose, bulk density, packing rate and compressibility of the powder through
conventional procedures using well-established equations, which are also
used to show the differences between ultrasonicated and no ultrasonicated
crystallization processes.[70] The study of friability, or condition of being
easily crumbled or pulverized, is carried out by milling the target system and
using given equations as a function of time.[36]
Table 1 lists the differences in both micromeritic and flow properties of
flurbiprofen crystallized in the presence and absence of US.[71] The
unquestionable improvement in these properties in the former product is
clearly shown in the table. A recent, representative example of the use of
present analytical instrumentation in this field is the research on overcoming
the poor tabletability of paracetamol (pca) by sonocrystallization that
provided a mixture of nano-sized and micrometre-sized crystals of the
monoclinic form of this drug.[95] Figure 5 shows that XRPD analysis of the
sonocrystallized bulk material, in addition to selected-area electron
diffraction (SAED) measurements of individual nanocrystals, revealed that
pca crystallizes as form I (Figure 5ac). The presence of the orthorhombic
form II could not be detected by XRPD or SAED. A thorough inspection of the
SEM and TEM (transmission electron microscopy) images indicated that the
sonocrystallized sample exhibits a narrow particle size distribution and also
an improved tabletability attributed to a greater compatibility. Specifically,
the micrographs revealed the presence of aggregated nano-sized and

micrometre-sized crystals with plate and prism morphologies and prismatic

crystals in the bulk (Figure 5dh).
Table 1. Properties of untreated and treated flurbiprofen. Reproduced from
[71] with permission of Wiley-Liss Inc
Parameters Untreated flurbiprofen Treated flurbiprofen
Particle size (m) 62.4 47.68.5 5.1
Elongation (E)
1.95 1.61
Angle of repose ()
44.1 1.5 38.1 0.5
Bulk density (g/ml)
0.32 0.0 0.33 0.0
Compressibility (a)
2.25 0.81
Packing rate or cohesiveness (b)
0.004 0.023
Carr index (%)
38 0.6
26 0.4
Hausner ratio
1.6 1.36
Figure 5.
Figure 5. Open in figure viewerDownload Powerpoint slide
Characterization of the sonocrystallized bulk of material: (a) X-ray powder
diffraction (XRPD) patterns (1 calculated form I; 2 sonocrystallized form I;
3 sonocrystallized form I after compaction; 4 calculated form II). (b)
selected-area electron diffraction (SAED) patterns of individual nanocrystals
(form I) in 010 zone axis. (c) SAED patterns of individual nanocrystals
(form I) in 211 zone axis, (dh) SEM and TEM images which depict the
particle size distribution of sonocrystallized form I. Reproduced from [95]
with permission of Wiley-VCH.
Conventional parameters usually determined in drug development with
conventional analytical equipment, but deserving proper updating, are as
follows: (1) Moisture content. This is a common determination in
pharmaceutical development that has been traditionally performed by
thermogravimetry.[70] At the industrial scale, once the method has been
exhaustively assessed, the faster and cheaper method is based on near
infrared spectrometry (NIRS), which has no sense to be implemented at the
laboratory scale the small number of analyses required during optimization
of the method does not justify the development of the empirical calibration
curve required when applying NIRS.[96, 97] (2) Stability studies. The stability
of the obtained form of a given drug can be measured at room temperature
for a number of hours, then days if the stability persists, and a final study of
accelerated stability, usually adjusted to strict guidelines of temperature and
relative humidity for a given time of at least 3 months. All these stability
studies can be monitored by the typical instrumentation that provides
information on the crystal characteristics (and changes, if produced) as is the
case with the stability of celecoxib porous particles, investigated by SEM,
DSC and XRPD.[38] Changes in the crystal structure can be caused by
degradation, the pathway and final products of which cannot be studied by
the above instrumentation. Crystal dissolution and proper separation of the
components followed by identification by MS is the best way to know the
target products and even the degradation pathway. (3) Intrinsic dissolution

rate or dissolution rate studies make use of the USP paddle apparatus; then
determining the target active principle by photometry provides poor
information about potential changes in the principle that do not affect
dramatically to its absorptivity. Once again, MS instruments provide a more
accurate information.
No material equipment, but crucial in the development of a
sonocrystallization method, is an appropriate optimization design. An
example of the usefulness of a well-selected optimization approach (together
with the appropriate selection of the variables to be studied) was provided by
Patel and Murthy who used the Taguchi method for sonocrystallization of
lactose from whey, and selected as target variables ultrasonication time,
solvent volume, initial concentration of lactose in the whey and initial pH of
the system.[98, 99] This systematic and efficient optimization method for
conducting experimentation allows determining near optimum design
parameters for the performance and cost by constructing a special set of
general design guidelines for factorial experiments using special sets of
orthogonal arrays.[100] How selection of the appropriate optimization design
influences the final product is shown in Figure 6.[101] The drastic reduction
in the crystal size after optimization is clear from the photographs in the
figure.
Figure 6.
Figure 6. Open in figure viewerDownload Powerpoint slide
Lactose crystals: (a) recovered at optimized conditions, (b) pure. Reproduced
from [99] with permission of Wiley-VCH.
Achievements in drug sonocrystallization
Table 2 summarizes examples that allow discussing the outstanding
achievements in the improvement of drugs crystallization by US assistance.
Table 2. Outstanding applications of sonocrystallization in pharmacy
Drug Features
Aim of the research
Improvement by
sonocrystallizationa
Sonocrystallization technique US equipment
Optimized parameters Analytical tools Ref.
a The typical achievements of sonocrystallization are not included.
Benzoic acid
Selected model To process modelling
Better
tabletability by greater compatibility
Antisolvent US processor, 20 kHz,
750 W
Benzoic acid conc., antisolvent addition rate, US power, T =
constant. HPLCUV (228 nm)
[70]
Sodium acetate Model for cooling sonocrystallization acetate
To
avoid/minimize seeding Avoidance of seeding, crystals with specific shapes
Cooling
US probe, 20 kHz, 120 W
Sodium acetate conc.,
seeding T, solvent, time of US application, US power Coulter counter [60]
Paracetamol
Poor tabletability To improve tabletability by favouring a
given crystalline form Better compaction properties of monoclinic crystals

Antisolvent US processor, 25 kHz, 250 W No optimization study

XRPD, SAED, SEM, TEM [95]
To study the influence of US frequency and intensity Process intensification
Cooling
US reactor, 161140 kHz (seven different frequencies)
Flow and batch modes Laser diffraction (particle size analyser) [40]
Gemfibrozil Selected model in water
To compare two sonocrystallization
methods
Variety of crystallization forms and selection of the best for
generic dosage Melt emulsification and solvent diffusion US processor, 24
kHz, 200 W Ultrasonic bath US power, pH, T, time, solvent
XRPD, SEM
[58]
Flurbiprofen
Poor flowability, solubility and dissolution rate To improve
bioavailability
Increased solubility and bioavailability
Melt US probe,
20 kHz, 500 W, 40/50 s duty cycle No optimization study HPLCUV (248
nm), SEM, DSC, FT-IR, XRPD [71]
Celecoxib Crystalline, poor solubility
To obtain an amorphous soluble
form Production of celecoxib porous particles with given biopharmaceutical
properties Melt US probe, 24 kHz, 200 W, 0.8/1.0 s duty cycle No
optimization study
HPLCUVvs (252 nm), SEM, DSC, XRPD [38]
Progesterone
Polymorphism
To maximize production of the required
form Obtainment of the most stable form
Melt with and without US
assistance, in solution with US
US probe, 24 kHz, 200 W, 0.8/1.0 s duty
cycle, T = 40 C Slight changes of US amplitude and duty cycle XRPD, DSC,
FT-IR, FT-Raman [102]
l-glutamic acid
Polymorphism
To maximize the required form
Polymorphism control by controlling supersaturation Saturation by
lowering the pH US microtip, 20 kHz, 600 W US power, US application
time, continuous, discontinuous application
Coulter counter, Raman
spectroscopy, SEM, ATR-FT-IR [39]
Ibuprofen Poor flowability, compaction, hydrophobicity
To improve
crystal characteristics Agglomerated formation, increased solubility, specific
surface area, intrinsic dissolution rate and compressional properties
Melt
US probe, 24 kHz, 200 W, 0.8/1.0 s duty cycle US application time
SEM, DSC, XRPD, FT-IR, surface area analysis (BET) [36]
Salbutamol sulfate
Coarse needles To obtain fine needles Crystal
shape appropriate for inhalation
Melt US tip, 20 kHz, 750 W, T control
T, US application time and power Laser diffraction (particle size
distribution range of 0.022000 m), SEM, thermal analysis DRIFTS, DSC,
XRPD [2]
Lactose
High biochemical oxygen demand (BOD) of whey with lactose
To optimize typical crystallization conditions (Taguchi method)
Acceleration of lactose recovery
Antisolvent US bath, 22 kHz, 120 W
T, US application time, initialfinal pH, stirring, seeding
Conventional titrator, UVvis spectrophotometer
[98]
See Figure 6
US application time, antisolvent and lactose conc., initial
pH, constant T
UVvis spectrophotometer, microscope [99]

Valdecoxib Poor solubility of crystals

Crystal habit changes
Agglomerate formation easy to dissolve Melt US probe, 24 kHz, 200
W, 0.8/1.0 s duty cycle No details SEM, DSC, XRPD, FT-IR [37]
Spectinomycin hydrochloride, roxithromycin, creatine, sucrose, sodium
hyposulphite
Organic and inorganic modelsTo establish the influence of
the sonosalting-out technique
Typical improvements, no differences in
the effect of US on organic and inorganic compounds Salting out US probe, 9
s on/13 s off, from 0 to 1200 W
Constant T, US application time, US
frequency SEM, XRPD, Laser transmission, Coulter counter
[19]
Adipic acid Model of agglomerate formation
To clarify US effect and
gassing effect on nucleation Typical parameters comparable in both modes
Cooling
US bath, variable frequency US frequency, US application
time, initial supersaturation, T
Balance, SEM, XRPD
[42]
Roxithromycin
Granular form precipitate
To micronize the drug by US
Typical parameters
Antisolvent US probe, 22.5 kHz
US power,
US application time, solution injection rate, solution conc, T FE-SEM, XRPD,
particle size analyser [72]
2-Carboxyphenyl salicylate Model drug To homogenize small particle size
Particle size 75175 nm Spray antisolvent US probe, 20 kHz, 130 W
US power, antisolvent and solute flow rate, drug conc.
SEM, XRPD
[61]
Aspirina
Sonocrystallized To study the fragmentation rate
Information
on fragmentation mechanism Cooling
US probe, 20 kHz, 600 W
US probe, 20 kHz, 750 W
US power, T
Fluorescence image analyser [73]
Mannitol
Model
To integrate drug substance and drug product design
Integration drug substance and drug product design
(sonocrystallizationsonofragmentation) Cooling
US flow cell, 20 kHz,
150 W/l
Previously studied
Optical light microscope
[41]
Most of the drugs to which the studies have been devoted were selected as
models of some characteristics that deserved to be improved: poor
tabletability or solubility, coarse particles, etc.
The aim of a given study has most times been to improve typical parameters
of the crystallization process; therefore, a narrower particle size distribution;
a decrease in particle size, induction time, metastable zone and
supersaturation levels; or a solubility increase are general achievements of
sonocrystallization as compared to conventional crystallization. The known
phenomena caused by US (e.g. induction of acoustic streaming,
microstreaming and highly localized temperature and pressure within a fluid)
seem to be the reasons for the rapid induction of primary nucleation, the
reduction in crystal size, inhibition or promotion of agglomeration and the
manipulation of crystal size distribution, among others.[22, 36, 96]

A typical example of increased solubility of a given API is flurbiprofen. By

comparing the features of this anti-inflammatory drug obtained in the
presence or absence of US assistance, its solubility increased about 35% in
the former product, thus involving a twofold increase in its intrinsic
dissolution rate as compared with the no ultrasonicated form, with a similar
increase in bioavailability. Also the dissolution rate studies revealed that 90%
of the drug was released within 20 min for the product obtained by
sonocrystallization as compared with 60% released for the conventionally
crystallized drug.[71] This behaviour is most times due to the formation of
highly porous particles, as is the case with sonocrystallized celecoxib, the
particles of which were in the range of 100 m to 1.1 mm, with yields in the
range 89.290.0% and loss of the drug in the aqueous phase less than 0.1%
w/w.[38] As commented in Section 'Analytical tools to study the effect of US
on drug crystallization', potential degradation of a target drug under US
application has been scarcely studied and only by low-sensitive and selective
detectors after chromatographic separation (molecular absorptiometric
detectors in all instances).[38]
Effects of US on polymorphic forms
Polymorphism is a characteristic that affects a number of pharmaceuticals
(e.g. 23% steroids, 60% sulfonamides and 70% of barbiturates), with a
dissimilar effect on molecule-like solubility, dissolution rate, stability,
hygroscopicity and bioavailability. Therefore, the study of drug
polymorphism has become vital, as it can directly affect the in-vivo
performance of the given API. An example of this behaviour is progesterone,
which exhibits two polymorphs, form I (-form) with a melting point at 128
C and form II (-form) with melting point at 122 C, the former being
thermodynamically more stable than the latter. The comparative study of
melt sonocrystallization (MSC) with melt, and sonocrystallization (SC) for
induction of polymorphism in progesterone was developed, and the
polymorphs were characterized by DSC, XRD, FT-IR and FT-Raman
spectroscopy, showing that melt sonocrystallized progesterone contained
both polymorphs, more soluble form II along with less soluble form I, whereas
melt progesterone and sonocrystallized progesterone contained only form I.
Therefore, the last method was found to provide the best final product, as it
also provided the less particle size and stability.[102]
As the different polymorphs exhibit different solubilities, their prevalence in
the solution can be controlled by the supersaturation level. This was the case
of optimizing l-glutamic acid production, as this drug presents two
polyphorms: the -polymorph is stable (needle-shaped or flake-shaped
crystals that difficult postprocessing), whereas the -polymorph is
metastable (prismatic or granular crystals that facilitated postprocessing).
US-initiated nucleation produced pure -polymorph (over 99.5 wt%) in the

end product crystals at 1 mol/L supersaturation. Seeded crystallization with

the pure polymorph also produced almost pure -polymorph crystals (over
93.0 wt%), whereas spontaneous nucleation produced a polymorph mixture
(from 30 to 70 wt%) under the same operating conditions.[39] This indicates
that supersaturation-controlled sonocrystallization can be a way to control
polymorphism in crystals, being another way seeding with target
polymorphs, if the seed crystals are available.
Versatility of US-assisted crystallization
One of the most outstanding aspects of sonocrystallization is versatility,
which allows manipulation of the product as a function of the required final
characteristics. By increasing the applied US power and duration of the
application, the crystallization technique and other variables as required,
target characteristics can be achieved. Although small particle size is the
most frequent required characteristic, agglomeration can also be the
pursued characteristic. This is the case with ibuprofen as the commonly used
needle-shaped crystals of this analgesic show poor flowability, compaction
behaviour and sticking to the punches due to its high cohesivity and
adhesivity. Besides these properties, ibuprofen shows poor dissolution
behaviour because of its hydrophobic nature, an undesirable property as a
rapid drug release is preferable for analgesic drugs. For this reason, a melt
sonocrystallization process was developed for ibuprofen in which ibuprofen
melt was poured in deionized water maintained at 25 C and simultaneously
subjected to US energy. The agglomerates obtained were evaluated using
SEM, DSC, XRPD, FT-IR, intrinsic dissolution rate, solubility, image analysis,
Heckel plot analysis and friability studies. The obtained irregular
agglomerates with porous surface comprised crystals having different habits
such as needles, plates and some hollow tubes. Solubility, specific surface
area and intrinsic dissolution rate increased with US treatment. SEM and
XRPD confirmed crystal habit changes, which resulted in improvement in
compressional properties and reduction in sticking. Loss of drug in the
aqueous phase was found to be less than 2% (w/w) for different sonication
time batches, the loss increasing by increasing US duration as a
consequence of breakage of agglomerates by US cavitation; therefore, the
time of US application must be strictly controlled to obtain the optimum
production.[36] A very different final product is required for drug
aerosolization, as is the case with pulmonary delivery of salbutamol sulfate
(SS). Application of US during antisolvent crystallization resulted in fine
elongated crystals, which compared favourably with aggregates of large
irregular crystals obtained in the absence of US. Figure 7 shows the key
influence of US variables and chemical and physical variables on the
obtainment of the required characteristics in this case. Thus, the increase in
the per cent of US power (nominal power 750 W) and decrease in
temperature (Figure 7a and 7b, respectively), the scant influence of US
duration after a given time (Figure 7c) and the decisive influence of the API

concentration illustrate how optimization of these variables is the key to

obtain the pursued characteristics for a given product.[2]
Figure 7.
Figure 7. Open in figure viewerDownload Powerpoint slide
Particle size distribution obtained by sonocrystallization at different: (a)
percentages of US nominal power, (b) temperatures, (c) time of US
application. Reproduced from [2] with permission of Elsevier.
Nevertheless, the versatility of sonocrystallization offers different possibilities
depending of the nature of the target API. The applicability of the results
found in a study need to be modified when applied to other drug as a
function of the similarity of its physical and chemical characteristics to the
model. Therefore, no general guidelines established in a previous research
should be applied to other API with different physical and chemical
characteristics. Sonocrystallization studies on organic and inorganic
compounds (viz., spectinomycin hydrochloride, roxithromycin, creatine,
sucrose and sodium hyposulphite) were developed by Li et al.[19] in 2003,
based on the salting-out technique, a narrow range of US frequencies (1530
kHz), power between 100 and 1000 W and 145 s US application, but the
results were not properly compared and discussion about different
behaviours was not in the publication. An in-depth study on this subject
would be of great interest to establish guidelines as a function of the
chemical nature of the API, the sonocrystallization technique used and the
pursued results.
It is worth emphasizing that studies on comparison of different
sonocrystallization techniques on a given API should involve comparison of
each of the steps to obtain the final product. Thus, comparison of the results
obtained by different sonocrystallization techniques and a given API
(gemfibrozil) showed that the melt emulsification method did not lead to a
desirable result (small and no agglomerated crystals) because the fast
cooling and spray-drying applied in this procedure were not enough for
micronization. On the contrary, when the solvent diffusion technique was
used, the critical role of the drying procedure was evidenced: lyophilization
drying provided larger particles than spray-drying because the temperature
decrease in the former was not sufficient to inhibit crystal growth.
Sonocrystallization from solution with spray-drying resulted in the smallest
particles (20 times smaller relative to those of pure gemfibrozil) and the
narrowest monodisperse distribution (15 m) with a large specific surface
area (2.34 m2/g).[58]
Trends
From the literature in the field, it can be concluded that the advantages
involved in sonocrystallization are indisputable; therefore, its role in the
pharmaceutical industry will grow as the parameters involved in the overall

process of drug production are better controlled. This requires more in-depth
studies at the laboratory scale with the following aims:
To clarify the influence of US frequency on crystallization. As it does not seem
to influence crystallization dramatically, in both a short[19] and large
frequency range,[40] a pending study is that devoted to know the influence
of US frequency on the process as a function of either crystal structure or
crystal composition.
To know the mechanism(s) behind US-assisted crystallization. Despite the
number of theories, the exact mechanism is not known yet.[40] Knowledge
of this mechanism would facilitate to foresee the behaviour of a given API
and the values of the variables to be used as a function of its characteristics.
To establish guidelines to start a given optimization process based on a
narrow interval of the target variables to be studied. This will be the result of
an in-depth study of US, physical and chemical variables within a wide range.
To determine potential degradation owing to US energy of the target drug by
sensitive and selective analysis of the solution and crystal composition. Once
detected the presence of degradation products, determination of their
toxicity will be the pending goal.
To avoid calculation of the process yield by determining the concentration of
the target drug remaining in the solution, which can be the result of a
number of side undesirable processes.
The continuous flow manufacturing processes, each time more demanded,
can find an excellent allied in US when applied in the pharmaceutical field.
[103, 104]
Declaration
Acknowledgement
Funding by the Andalusian Regional Government (Junta de Andaluca) and
FEDER Program through project FQM-1602 is gratefully acknowledged.

Curcumin prevents paracetamol-induced liver mitochondrial

alterations
http://onlinelibrary.wiley.com/doi/10.1111/jphp.12501/full
Authors
Luis Fernando Granados-Castro,
Daniela Sarai Rodrguez-Rangel,
Berenice Fernndez-Rojas,
Juan Carlos Len-Contreras,
Rogelio Hernndez-Pando,
Omar Noel Medina-Campos,
Dianelena Eugenio-Prez,
Enrique Pinzn,
Jos Pedraza-Chaverri
First published: 15 January 2016Full publication history
DOI: 10.1111/jphp.12501View/save citation
Cited by: 1 articleCitation tools
Article has an altmetric score of 34
Funding Information
Abstract
Objective
In the present study was evaluated if curcumin is able to attenuate
paracetamol (PCM)-induced mitochondrial alterations in liver of mice.
Methods
Mice (n = 56/group) received curcumin (35, 50 or 100 mg/kg bw) 90 min
before PCM injection (350 mg/kg bw). Plasma activity of alanine
aminotransferase (ALT) and aspartate aminotransferase (AST) was
measured; histological analyses were done; and measurement of
mitochondrial oxygen consumption, mitochondrial membrane potential, ATP
synthesis, aconitase activity and activity of respiratory complexes was
carried out.
Key findings
Curcumin prevented in a dose-dependent manner PCM-induced liver
damage. Curcumin (100 mg/kg) attenuated PCM-induced liver histological
damage (damaged hepatocytes from 28.3 7.7 to 8.3 0.7%) and
increment in plasma ALT (from 2300 150 to 690 28 U/l) and AST (from
1603 43 to 379 22 U/l) activity. Moreover, curcumin attenuated the
decrease in oxygen consumption using either succinate or malate/glutamate
as substrates (evaluated by state 3, respiratory control ratio, uncoupled
respiration and adenosine diphosphate/oxygen ratio), in membrane potential,
in ATP synthesis, in aconitase activity and in the activity of respiratory
complexes I, III and IV.

Conclusions
These results indicate that the protective effect of curcumin in PCM-induced
hepatotoxicity is associated with attenuation of mitochondrial dysfunction.
Introduction
Paracetamol (PCM), also known as N-acetyl p-aminophenol, has been one of
the widespread most frequently used drugs for analgesic and antipyretic
purposes for the last 30 years.[1] PCM is a safe and effective drug at
recommended doses: for adults 325650 mg orally every 46 h with a
maximum of 4 g per day and for children 1015 mg/kg every 45 h with a
maximum of 5075 mg/kg per day.[2] PCM overdose is able to induce
hepatotoxicity and acute liver failure. As a matter of fact, in 2006 alone, the
American Association of Poison Control Centers implicated PCM in nearly 140
000 poisoning cases, in which more than 100 patients died.[3] It is
responsible for more emergency room visits than any other drug on the
market.[2]
In this context, several compounds have been used to attenuate PCMinduced hepatotoxicity, including N-acetylcysteine,[4, 5] sulforaphane,[6] Sadenosylmethionine,[7] green tea polyphenols,[7] (RS)-n-propylthiazolidine4(R)-carboxylic acid,[7] -lipoic acid,[8] tymoquinone,[9] diphenyl diselenide,
[10] methylene blue[11] and curcumin[12-15] among others.
Curcumin prevents PCM-induced increase in plasma alanine
aminotransferase (ALT),[13, 14] liver necrosis[12-15] and apoptosis,[13, 14]
malondialdehyde (MDA) content,[12, 13, 15] inflammatory cytokines[12] and
liver deoxyribonucleic acid (DNA) fragmentation[14] among others.
The reactive toxic metabolite of PCM is N-acetyl-p-benzoquinoneimine
(NAPQI).[11] The PCM toxicity was found to consist of two phases: the initial
glutathione (GSH) depletion and covalent binding of NAPQI to target proteins
and the subsequent increase in the mitochondrial permeability transition and
nitration of proteins.[16] The impairment of GSH antioxidant system has
been noticed to enhance the susceptibility to mitochondrial dysfunction from
oxidative stress, resulting in the collapse of mitochondrial membrane
potential and adenosine triphosphate (ATP) depletion.[16]
The mitochondrial alterations associated to PCM-induced liver toxicity include
decrease in mitochondrial oxygen consumption,[5, 16-19] ATP synthesis,[16,
17, 19] mitochondrial membrane potential,[16] and activity of mitochondrial
complexes I (NADH dehydrogenase), II (succinate dehydrogenase) and IV
(cytochrome c oxidase).[20] PCM inhibits complex II.[11] It has been shown
that N-acetylcysteine,[5] diphenyl diselenide[16] and methylene blue[11]
protect against mitochondrial dysfunction in PCM-induced liver toxicity.
Interestingly, it has not been established whether curcumin may attenuate

the following PCM-induced liver mitochondrial alterations: oxygen

consumption, membrane potential, ATP synthesis, and activity of aconitase
and respiratory complexes I, II, III and IV. In this context, it has been found
that curcumin is able to attenuate mitochondrial alterations in the following
experimental models: potassium dichromate-induced hepatotoxicity[21, 22]
and nephrotoxicity,[23] cardiac damage-induced by chronic renal failure[24]
and maleate-induced nephrotoxicity.[25] By the above mentioned reasons it
was hypothesized that curcumin may ameliorate PCM-induced mitochondrial
alterations. The present study aimed to evaluate if the protective effect of
curcumin in PCM-induced hepatotoxicity is associated with attenuation of
mitochondrial dysfunction. This is the first work that aimed to address these
points.
Materials and Methods
Reagents
The following reagents were from Sigma-Aldrich (St. Louis, Missouri, USA):
Curcumin (catalogue number C7727, purity 80%), D-mannitol, sodium
succinate dibasic, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES), safranin-O, glucose, glucose-6-phosphate dehydrogenase (G6PDH),
bovine serum albumin fat free (BSA), -nicotinamide adenine dinucleotide
phosphate hydrate, carboxymethylcellulose sodium salt (CMC), hexokinase,
adenosine 5-diphosphate sodium salt (ADP), carbonyl cyanide 4(trifluoromethoxy)phenylhydrazone (FCCP), ethylenediaminetetraacetic acid
(EDTA), percoll, rotenone, sucrose, 2,6-dichlorophenolindophenol sodium salt
hydrate (DCPIP), antimycin A, -nicotinamide adenine dinucleotide hydrogen
(NADH), decylubiquinone (DuB), malonic acid, cytochrome c and sodium
borohydride. Sodium phosphate dibasic, sodium phosphate monobasic,
potassium cyanide (KCN), Tween 20 detergent, magnesium chloride (MgCl2)
and sodium dithionite were from Merck (HES, Darmstadt, Germany).
Potassium phosphate dibasic (K2HPO4) and potassium chloride (KCl) were
from Mallinckrodt (Paris, Kentucky, USA). ALSL-0430 and ASSL-0430 kits to
measure the plasma activity of ALT and aspartate aminotransferase (AST)
were obtained from ELITechGroup (Princeton, New Jersey, USA). BIO-RAD
Protein Assay (BIO-RAD Laboratories, Hercules, California, USA) was used for
protein quantification. As sedative, Sedalphorte MR Reg. SAGARPA Q-7503003 (Sodium pentobarbital, Mexico city) was used. Other compounds and
reagents used were of high purity and were obtained commercially.
Animals
Male CD1 mice with an initial body weight of 3540 g were used. Animals
were housed on a 12-h light/dark cycle with ad libitum access to purified
water and food (Teklad Global 2018S; Harlan Laboratories, Indianapolis,
Indiana, USA). The Local Committee for the Care and use of Laboratory
Animals (CICUAL) approved the project (FQ/CICUAL/044/12) and the

experiments were conducted in accordance with the Guide for the Care and
Use of Laboratory Animals.
Model of PCM hepatotoxicity
All animals were withdrawn from food 4 h before any manipulation. Six
groups of animals were studied (n = 56): Control group (CT): Animals
received 0.05% CMC (curcumin's vehicle) 90 min before an ip saline solution
(PCM's vehicle) injection. Paracetamol group (PCM): animals received 0.05%
CMC 90 min before an ip PCM injection (350 mg/kg bw). Curcumin +
paracetamol (PCM + CUR): mice received 35, 50 or 100 mg/kg bw of
curcumin 90 min before an ip PCM injection. The doses of curcumin were
chosen based in previous pilot experiments. Curcumin (CUR): mice received
curcumin (100 mg/kg bw) 90 min before an ip saline solution injection. Mice
were sacrificed 14 h after the last administration. Animals were
anaesthetized with sodium pentobarbital (80 mg/kg). Blood was obtained
from the axillary vein in heparinized Eppendorf tubes and plasma was
separated by centrifugation. Liver was immediately removed and prepared
for histological and ultrastructural study.
Markers of liver injury
Hepatic injury was determined by measuring in plasma the activity of ALT
and AST with commercial kits.
Histological analysis
Transverse slices of approximately 1 mm were obtained from freshly
extracted liver. The slices were immersed in 10% formaldehyde diluted in
phosphate buffered saline (PBS) for a week to fix the tissue. Then, the fixed
tissue was immersed in different concentrations of ethylic alcohol and
subsequently in xylol for later inclusion in paraffin wax; sections of 4 m
thickness were made and mounted on glass slide. After dewaxing, sections
were stained with haematoxylin-eosin staining. The histological analysis was
circumscribed to the hepatocytes located around the portal areas (150200
m width) because hepatocytes in this zone are the first to metabolize PCM.
Ultrastructural studies
To study the mitochondrial ultrastructural morphology, immediately after
animal sacrifice, small liver tissue fragments were obtained and fixed by
immersion into 10% glutaraldehyde dissolved in cacodylates buffer pH 7.2
during 24 h at 4C. Then, tissue fragments were post-fixed with osmium
tetraoxide, dehydrated in graded ethylic alcohol solutions and embedded in
Epon resin (London Resin Company, London, UK). Thin sections from 70 to 90
nm were placed on cooper grids, contrasted with lead and uranium salts and
examined with a FEI Technei electron microscope. The histological and
ultrastructural studies were performed only with the curcumin dose of 100
mg/kg.
Isolation of liver mitochondria

Liver mitochondria were isolated from the whole liver using differential
centrifugation with two different Percoll gradients according to Ratner et al.
[26] The mitochondrial protein content was measured according to Bradford,
[27] adapted to a 96 well plate, to assure the use of equal protein quantity
among all subsequent determinations.
Determination of oxygen consumption
Measurement of mitochondrial oxygen consumption was carried out using a
Clark-type electrode attached to a micro-chamber with a constant
temperature of 37C (Strathkelvin instruments, ML, Scotland, UK) according
to previous studies of our group.[23, 25]
Mitochondrial membrane potential (MMP)
A method based on a previous work[28] was used to determine the MMP. In
brief, 2 m safranin-O was used to measure fluorometrically the MMP at
excitation and emission wavelengths of 530 and 590 nm, respectively, in a
black 96 well plate.
ATP synthesis assay
Thirty g of freshly isolated mitochondria were loaded in a well containing
cold ATP-buffer (9 U/ml hexokinase, 220 mm glucose, 140 mm sodium
succinate, 1.9 U/ml G6PDH and 2.6 mg NADP+; pH 7.2). A basal ATP
production was measured indirectly for 3 min at 340 nm, the ATP production
was initiated with the addition of 1.2 mm ADP and it was monitored each 30
s for 5 min at 340 nm.
Activity of mitochondrial complexes
The enzymatic activities of all complexes were determined
spectrophotometrically at 37C with 5 g of isolated liver mitochondria. For
the activity assays of complexes I and II, mitochondria were broken with four
freezing and thawing cycles. For complexes III and IV, Tween 20 was added to
the solution as the freezing process has shown a decrease in these
complexes enzymatic activities.[29] The activity of mitochondrial complexes
was carried out independently in a 96 well plate in presence of the
respective complex inhibitor. The specific activity of all complexes was
determined by the subtraction of the activity in the presence of the specific
inhibitor from the total activity. The final volume of each well was 300 l.
Complex I activity
In each well, 5 g of broken mitochondria were added to a mix solution
containing 41 mm potassium phosphate buffer, 3.5 mg/ml BSA, 67 m
DCPIP, 1 m antimycin A, 0.2 mm NADH and 0.2 mm KCN, pH 7.4. To inhibit
complex I and to determine the non-enzymatic activity, 13 m rotenone
were added in an identic parallel well. The mix was incubated for 5 min at
37C and the baseline was monitored by a kinetic reading for 2 min at 600

nm. The reaction was started by the addition of 3.12 mm DuB and the
subsequent decrease in absorbance was followed for 3 min.
Complex II activity
In each well, 5 g of broken mitochondria were added to a solution
containing 30 mm potassium phosphate buffer, 0.4 mg/ml BSA, 67 m
DCPIP, 1 m antimycin A, sodium 15 mm succinate and 0.2 mm KCN, pH 7.4.
To inhibit complex II and to determine the non-enzymatic activity, 10 mm
malonic acid was added in an identic parallel well. The plate was incubated
inside the spectrophotometer at 37C for 10 min and the baseline activity
was read at 600 nm for 2 min. The reaction was started by the addition of
3.12 mm DuB and the subsequent decrease in absorbance was followed for 3
min.
Complex III activity
In each well, 5 g of hepatic mitochondria were added to a mix solution
containing 30 mm potassium phosphate buffer, 0.4 mg/ml BSA, 220 m
Tween 20, 1 m rotenone, 0.4 mm KCN, 0.6 mm MgCl2, 0.1 mm EDTA and
17 m oxidized cytochrome c, pH 7.4. To inhibit complex III and to determine
the non-enzymatic activity, 1.8 mm antimycin A was added in an identic
parallel well. The mix was incubated for 10 min at 37C and the baseline was
monitored by a kinetic reading at 550 nm for 2 min. The reaction was started
by the addition of 3.12 mm decylubiquinol and the increase in absorbance at
550 nm was followed for 3 min.
Complex IV activity
In each well, a freshly prepared solution containing 37 mm potassium
phosphate buffer, 0.4 mg/ml BSA, 220 m Tween 20 and 17 m reduced
cytochrome c, pH 7.0, was placed. To inhibit the complex IV and to
determine the non-enzymatic activity, 0.25 mm KCN was added to a similar
parallel well. The plate was incubated inside the spectrophotometer at 37C
for 10 min and the baseline activity was read at 500 nm for 2 min. The
reaction was started by the addition of 5 g of hepatic mitochondria and the
decrease in the absorbance at 550 nm was followed for 3 min.
Aconitase activity
Mitochondria were broken by sonication for 2 min in aconitase buffer (20 mm
isocitrate, 0.6 mm MnCl2, 50 mm Tris-HCl; pH 7.4). Fifteen g of
mitochondrial protein were used for each well, the mixture was incubated for
2 min at 25C and then the production cis-aconitate was followed for 3 min
at 240 nm.[25]
Statistical analysis
Results were expressed as means standard error of the mean (SEM). Data
were analysed by one-way analysis of variance followed by multiplecomparisons according to Turkey test using Prism 6.0 software (GraphPad,

San Diego, California, USA). The comparisons with a P < 0.05 were
considered significant.
Results
Curcumin was able to attenuate, in a dose-dependent way, the PCM-induced
increase in the plasma activity of ALT and AST (Figure 1). The increase in ALT
activity was prevented with 50 and 100 mg/kg of curcumin and the increase
in AST activity was prevented with the three doses of curcumin. Curcumin
alone was unable to induce changes in the activity of these enzymes (Figure
1).
Figure 1.
Figure 1. Open in figure viewerDownload Powerpoint slide
Curcumin (CUR) prevents, in a dose-response way, the paracetamol (PCM)induced increase in the activity of alanine aminotransferase (ALT) and
aspartate aminotransferase (AST). Data are presented as mean SEM, n =
56. aP < 0.05 vs Control, bP < 0.05 vs PCM, cP < 0.05 vs PCM + CUR 35, dP
< 0.05 vs PCM + CUR 50.
Moreover, curcumin (100 mg/kg) attenuated the PCM-induced liver
histological damage (Figure 2). Four parameters were determined as tissue
damage markers: (1) the percentage of the area occupied by inflammatory
cells into and around portal zones based in a total area of 150 000 m2; (2)
percentage of periportal normal hepatocytes in an area of 50 000 m2; (3)
percentage of necrotic or apoptotic hepatocytes in the same areas; and (4)
percentage of regenerative liver cells (binucleated hepatocytes or with very
large nucleus, mitotic figures). Figure 2 shows that PCM-induced increase in
the percentage of damaged hepatocytes and inflammation area was
prevented by curcumin pretreatment. Animals treated with curcumin alone
showed normal liver histology. These changes showed good correlation with
the ultrastructural analysis, liver cells from PCM-treated animals showed
numerous cytoplasmic vacuoles and swollen mitochondria with fragmented
or effacement cristae and numerous electrondense amorphous deposits in
the matrix, while these changes were seen occasionally in mice treated with
PCM and curcumin (Figure 3). There were not ultrastructural abnormalities in
animals treated only with curcumin.
Figure 2.
Figure 2. Open in figure viewerDownload Powerpoint slide
Curcumin (CUR, 100 mg/kg) prevents the paracetamol (PCM)-induced
histological liver injury. Samples were stained with hematoxylin and eosin
(H&E). The analysis was carried out only with the 100 mg/kg curcumin dose,
as it was the most effective one. *Shows inflammation area, +shows
damaged hepatocytes and arrows show regenerating hepatocytes.
Quantification of all parameters measured; inflammation area was obtained
from a total of 150 000 m2 and all cellular counts were made from a total of

50 000 m2. Data are presented as mean SEM, n = 56. aP < 0.05 vs
Control (CT), bP < 0.05 vs PCM, eP < 0.05 vs PCM + CUR 100.
Figure 3.
Figure 3. Open in figure viewerDownload Powerpoint slide
Representative electromicroscopy micrographs of mitochondria from the
different experimental groups. (a) Normal mitochondria structure in
hepatocyte from control mouse that received only the vehicle solution. (b)
Swollen mitochondria with rupture of external membrane (arrows),
effacement of cristae and electrondense deposits in the matrix induced by
paracetamol (PCM) administration. (c) Curcumin (100 mg/kg) prevents the
mitochondrial damage induced by PCM, only some mitochondria show
electrondense material deposited in the matrix. (d) Administration of
curcumin alone did not induce mitochondrial damage.
Paracetamol induced decrease in oxygen consumption (Figures 4 and 5).
Curcumin was able to attenuate the decrease in state 3, respiratory control
ratio (State 3/State 4), uncoupled respiration, ADP/O (mol of ADP/mol of
oxygen consumed during State 3) and increase in state 4 using either
succinate (Figure 4) or malate/glutamate (Figure 5) as substrates of the
electron chain transport. Protective effect was observed with 50 and 100
mg/kg of curcumin.
Figure 4.
Figure 4. Open in figure viewerDownload Powerpoint slide
Curcumin (CUR) prevents paracetamol (PCM)-induced alterations in
mitochondrial respiration using succinate as substrate of the electron
transport chain. (a) state 3, initiated with adenosine diphosphate (ADP)
addition in the chamber of respiration, (b) state 4, reached when the ADP is
depleted, (c) Respiratory control ratio (RCR), (d) ADP/oxygen (O) ratio, (e)
uncoupled respiration, initiated with carbonyl cyanide 4(trifluoromethoxy)phenylhydrazone (FCCP) addition. Data are presented as
mean SEM, n = 56. aP < 0.05 vs Control, bP < 0.05 vs PCM, cP < 0.05 vs
PCM + CUR 35, dP < 0.05 vs PCM + CUR 50, eP < 0.005 vs PCM + CUR 100.
Figure 5.
Figure 5. Open in figure viewerDownload Powerpoint slide
Curcumin prevents paracetamol (PCM)-induced alterations in mitochondrial
respiration with glutamate/malate as substrate of the electron transport
chain. (a) state 3, initiated with adenosine diphosphate (ADP) addition in the
chamber of respiration, (b) state 4, reached when the ADP is depleted, (c)
Respiratory control ratio (RCR), (d) ADP/oxygen (O) ratio, (e) uncoupled
respiration, initiated with carbonyl cyanide 4(trifluoromethoxy)phenylhydrazone (FCCP) addition. Data are presented as
mean SEM, n = 56. aP < 0.05 vs Control, bP < 0.05 vs PCM, cP < 0.05 vs
PCM + CUR 35, dP < 0.05 vs PCM + CUR 50.
Curcumin (100 mg/kg) prevented the PCM-induced decrease in membrane
potential (Figure 6), ATP synthesis (Figure 7) and aconitase activity (Figure 7).

Figure 6.
Figure 6. Open in figure viewerDownload Powerpoint slide
Curcumin (CUR, 100 mg/kg) prevents paracetamol (PCM)-induced decrease
in mitochondrial membrane potential (MMP) that was obtained with the
fluorescence of safranin-O. FU = Change in fluorescent units. Data are
presented as mean SEM, n = 56. aP < 0.05 vs Control, bP < 0.05 vs PCM.
Figure 7.
Figure 7. Open in figure viewerDownload Powerpoint slide
Effect of curcumin on paracetamol (PCM)-induced alterations in enzymatic
activity of mitochondrial complexes (a) I, (b) II, (c) III and (d) IV, (e) ATP
synthesis and (f) aconitase activity. ATP synthesis by the mitochondria was
measured using glutamate/malate as substrate of the electron transport
chain. Aconitase activity was determined as it is a mitochondrial marker of
oxidative stress. The PCM + CUR 35 group was no longer determined as it
showed no significant difference vs the Control group in various previous
determinations. Data are presented as mean SEM, n = 56. aP < 0.05 vs
Control, bP < 0.05 vs PCM, dP < 0.05 vs PCM + CUR 50, eP < 0.05 vs PCM +
CUR 100.
Paracetamol induced decrease in the activity of the respiratory complexes I,
II, III and IV. Curcumin was able to prevent the decrease in the activity of
respiratory complexes I, III and IV (Figure 7).
Curcumin alone increased the activity of respiratory complex III (Figure 7)
and membrane potential. ATP synthesis, aconitase activity and activity of
complex I, II and IV remained unchanged in the curcumin treated group.
Discussion
Paracetamol-induced hepatotoxicity has been a significant issue for several
years because PCM is a widespread used drug for analgesic and antipyretic
purposes[1] and it is responsible for more emergency room visits than any
other drug on the market.[2] During overdose, PCM is mainly metabolized by
cytochrome P450 into NAPQI, which reacts directly with GSH causing its
depletion[16] (Figure 8). Consequently, NAPQI reacts with mitochondrial
membrane proteins which causes reactive oxygen species (ROS) production,
[30] mitochondrial DNA damage[31] mitochondrial transition pore (MTP)
opening and decrease of ATP production[16, 17, 19] (Figure 8).
Figure 8.
Figure 8. Open in figure viewerDownload Powerpoint slide
Graphical representation of the paracetamol (PCM), N-acetyl-pbenzoquinoneimine (NAPQ1) and curcumin interaction in mice hepatocytes.
Curcumin is able to induce diverse cellular responses to decrease the PCM
overdose alteration. ADP, Adenosine diphosphate; PCM or N-acetyl-paminophenol; ARE, Antioxidant response elements; ATP, Adenosine
triphosphate; Ca2+, Calcium cation; CAT, Catalase; CI, NADH:ubiquinone
oxidoreductase; CII, Succinate dehydrogenase; CIII, Coenzyme Q:

cytochrome c-oxidoreductase; CIV, cytochrome c oxidase; CUR, Curcumin

(Diferuloylmethane); CYP2E1, Cytochrome P450 2E1; Cyt c, Cytochrome c;
Glu/mal, Glutamate and malate; GPx, Glutathione peroxidase; GSH,
Glutathione; GST, Glutathione-S-transferase; HO-1, Heme oxygenase 1; IMS,
Intermembrane space; Keap 1, Kelch-like ECH-associated protein 1; Maf,
Small maf proteins; MTP, Mitochondrial transition pore; NADH, Nicotinamide
adenine dinucleotide hydrogen; NAPQI, N-acetyl-p-benzoquinone imine;
NQO1, NAD(P)H dehydrogenase [quinone] 1; Nrf2, nuclear factor (erythroidderived 2)-like 2; RNS, Reactive nitrogen species; ROS, Reactive oxygen
species; SOD, Superoxide dismutase; m, Mitochondrial membrane
potential.
Moreover, it has been found translocation of the membrane protein B-cell
lymphoma-2 (Bcl-2)-associated-X protein (BAX), which combines with Bcl-2
antagonist killer 1 (Bak) in the outer mitochondrial membrane to form pores
and allow the release of intermembrane proteins such as cytochrome c[32]
(Figure 8). Consistently, previous studies have shown that PCM and NAPQI
inhibit mitochondrial respiration.[5, 19, 33, 34] Furthermore, in an earlier
study, the interaction between NAPQI and the respiratory chain was
investigated using submitochondrial particles. Succinate dehydrogenase
(associated with respiratory complex II) was found to be very sensitive to
NAPQI, while NADH dehydrogenase (respiratory complex I) was inhibited to a
lesser extent.[19] The well-documented PCM-induced alterations in
mitochondrial function may also be related to the fact that PCM affects the
expression of mitochondrial protein-encoding genes related to the subunits
of electron transport chain (ETC.) complexes.[31] These abnormalities may
affect the assembly, stability and structure of ETC complexes.[31]
From the above described alterations it is clear that mitochondrial damage
plays a critical role in the PCM-induced hepatotoxicity. Therefore, compounds
with the potential protective effects on mitochondrial dysfunction may be
useful to prevent PCM-induced hepatotoxicity. The antioxidant curcumin has
been found to protect against mitochondrial dysfunction in several
experimental models.[[21-25] reviewed in [35]] In potassium dichromateinduced nephrotoxicity model, curcumin was able to prevent the following
mitochondrial alterations[23]: decrease in oxygen consumption, in oxidative
stress, in ATP content, in calcium retention, and in mitochondrial membrane
potential as well as in the activity of complexes I, II, II-III and V. In potassium
dichromate-induced hepatotoxicity model, curcumin was able to prevent the
decrease in the aconitase activity, in ATP content, in oxygen consumption,
and in the activity of respiratory complex I as well as the increase in
oxidative stress and in permeability transition pore opening.[21, 22] In
maleate-induced nephrotoxicity model, it was able to prevent reduced
oxygen consumption and activities of complex I and aconitase.[25] In
addition, curcumin was also able to prevent the decrease in aconitase
activity and in oxygen consumption in hearts of rats with chronic renal
failure.[24] In addition, Subudhi et al.[36] found effects of curcumin on

oxygen consumption. They reported that curcumin administration

ameliorates hyperthyroidism-induced increase in state 3 and state 4 as well
as in respiration in complex I in mitochondria from rat liver.
In the present study was found that curcumin indeed exerts a
hepatoprotective effect against PCM-induced damage. Curcumin attenuated
PCM-induced increment in ALT and AST plasma activity, which are sensitive
indicators for the hepatic function; their excessive leakage into the blood
shows the disruption of integrity of hepatic cell membranes. The
hepatoprotective effect of curcumin was also confirmed by histological
observations. The protective effect observed was consistent with the findings
of Somanawat et al.[12] Li et al.,[13] Bulku et al.,[14] and Yousef et al.[15]
Remarkably, the hepatoprotection observed in our study was associated with
prevention of PCM-induced mitochondrial alterations. In fact, curcumin
prevented the PCM-induced decrease in oxygen consumption (State 3, RCR,
uncoupled respiration and ADP/O), in membrane potential, in ATP synthesis,
in the aconitase activity and in respiratory complexes I, II and IV (Figures 48). These mitochondrial functional data obtained in isolated mitochondria
were supported by ultrastructural observations seen in the liver of these
animals. Figure 3 illustrates swollen mitochondria and disruption of external
membrane, effacement of cristae and electrondense deposits in the matrix
induced by PCM treatment (Figure 3b). Curcumin prevents these PCMinduced mitochondrial alterations since only some mitochondria show
electrondense material accumulated in the matrix (Figure 3c).
To our knowledge, this is the first demonstration that curcumin prevents
mitochondrial alteration in the PCM-induced hepatotoxicity. These data are
consistent with the findings in other experimental models.[21-25]
Interestingly, in all cases, the improvement of mitochondrial function was
associated with the prevention of the decrease in the activity of aconitase, a
marker of oxidative stress.[37] This suggests that the antioxidant effect of
curcumin is key in the protective effect of mitochondrial function. In fact, it
has been well-established that curcumin is a direct antioxidant[38] that may
be responsible of the protection against mitochondrial dysfunction in this
experimental model (Figure 8). In addition, curcumin may also exert its
antioxidant effect through an indirect mechanism via the transcription factor
nuclear factor (erythroid-derived 2)-like 2 (Nrf2) (Figure 8). We have no
enough data in this work to support the indirect antioxidant effect of
curcumin in this experimental model. It is unknown if curcumin may be able
to interfere with the direct inhibition of mitochondrial proteins by NAPQI. This
deserves to be further analysed.
Our data suggest that curcumin may be useful to attenuate the PCM-induced
liver damage in humans. Studies should be conducted in the future to
evaluate this potential effect of curcumin in human beings.

Together, these mechanisms might explain, at least in part, some of the

cytoprotective effects of curcumin on the PCM-induced hepatic damage.
Conclusion
These results indicate that the protective effect of curcumin in PCM-induced
hepatotoxicity is associated with attenuation of mitochondrial dysfunction.
Declarations
Acknowledgements
This work was partially supported by PAPIIT IN210713 and CONACYT (220046
and 252008).

A mechanistic review on medicinal plants used for rheumatoid

arthritis in traditional Persian
medicine
Authors
Mohammad Hosein Farzaei,
Fatemeh Farzaei,
Mohammad Abdollahi,
Zahra Abbasabadi,
Amir Hossein Abdolghaffari,
Bahman Mehraban
First published: 15 July 2016Full publication history
DOI: 10.1111/jphp.12606View/save citation
Cited by: 0 articlesCitation tools
Article has an altmetric score of 1
Abstract
Objectives
Rheumatoid arthritis (RA) is a chronic, inflammatory, autoimmune disease,
which affects synovial tissue in multiple joints. Although conventional
treatments of RA commonly alleviate the symptoms, high incidence of
adverse reactions leads to research tendency towards complementary and
alternative medicine. As various medicinal plants are traditionally used for
the management of symptomatologies associated with RA in Persian
medicine, we reviewed medicinal literature to confirm their efficacy in the
management of RA.
Key findings
Scientific evidence revealed that traditional medicaments exert beneficial
effects on RA through several cellular mechanisms including downregulation
of pro-inflammatory cytokines such as TNF-, IL-6 and NF-B, suppression of
oxidative stress, inhibition of cartilage degradation with destructive
metalloproteinases and enhancement of antioxidant performance. Various
active constituents from different chemical categories including flavonols,
lignans, coumarins, terpenes, glycosylflavons, dihydroflavonols,
phytoestrogens, sesquiterpene lactones, anthraquinones, alkaloids and
thymoquinones have been isolated from the medicinal plants.
Summary
The pharmacological mechanisms of the medicinal plants traditionally used
for RA in Persian medicine are discussed in the current review. Further
investigations are mandatory to focus on bioefficacy of these phytochemicals
for finding novel natural drugs.
Introduction

Inflammatory processes play a pivotal role in the development of various

diseases. Chronic inflammation arises in pathologic conditions including
exposure to toxic agents, resistant infections caused by certain
microorganisms as well as autoimmunity. Rheumatoid arthritis (RA) is a
chronic, inflammatory, autoimmune disease, which affects synovial tissue in
multiple joints and is characterized by joint swelling, cartilage damage,
synovial inflammation and bony erosion, which can finally result in joint
destruction.[1-3] The total world prevalence of RA is about 0.51% and is
three to four times more common in women.[2] The initiation of RA is
unknown, although interactions between genetic factors, sex hormones and
an immune triggering agent (endogen or environmental substances which
triggers an immune response) are considered as contributing factors.[2, 4]
In RA, synoviocytes and macrophages produce joint-damaging cytokines.
These pro-inflammatory cytokines inhibit the synthesis of proteoglycans and
collagen and increase their degradation. They also activate proteolytic
enzymes (i.e. MMPs and collagenases) promoting destruction of cartilage.[35] Pro-inflammatory cytokines, such as interleukin-1 (IL-1), IL-6 and
tumour necrosis factor- (TNF-), stimulate articular manifestations of RA
through increasing inflammatory cell infiltration, particularly T cells, B cells,
and macrophages, and bone erosion. T-cell activation results in autoantibody
production and release of TNF- and IL-6; in consequence, the articular and
extra-articular manifestation of RA occurs. Chemotactic cytokines, termed
chemokines which attract lymphocytes, monocytes and neutrophils, possess
chemotactic activity and play a significant role in tissue destruction and
synovitis. The pro-inflammatory enzyme, cyclooxygenase (COX)-2, converts
arachidonic acid into prostaglandin-E2 (PGE2) and prompts hyperplasia and
pannus formation in synovial joints resulting in suppression of apoptosis in
synovial fibroblasts and induction of proliferation. Regulation of arachidonic
acid metabolism and modulation of other mediators secreted by
macrophages and other immune cells can be potential targets for the
treatment of chronic inflammatory conditions by inhibiting enzymes like COX
and lipooxygenase (LOX).[3, 5-7]
The goal of conventional treatment is to slow or reverse cartilage
degradation and decrease the pain without toxic pharmacological
consequences. Disease-modifying antirheumatic drugs (DMARDs) and
biological agents including anti-TNF- are used for the prevention of swelling,
pain and joint destruction in RA. Nevertheless, current treatment with
DMARDs is not effective in all patients. Immunosuppressants, nonsteroidal
anti-inflammatory drugs (NSAIDs) and steroidal anti-inflammatory drugs are
commonly used for alleviation of RA complication, but potential side effects
limit their use. For example, NSAIDs and steroids can cause adverse
haematologic, gastrointestinal and renal complications. Long-term
administration of these drugs may lead to adverse effects such as increased
risk of gastrointestinal ulceration and bleeding due to suppression of COX-l.

Therefore, many patients look for complementary and alternative medicine

(CAM) approaches for the management of debilitating diseases with chronic
pain, such as RA.[3, 8, 9]
Traditional medicines all over the world suggest a wide range of remedies for
the management of symptomatologies associated with chronic disorders as
in RA. Scientists are exploring within nations traditional medicine to find
alternative anti-arthritic drugs. Medicinal plants and plant-derived natural
agents are among the most important resources of traditional medicines.[1013] Traditional Persian medicine dates back to thousands of years. History of
medicine in Iran was initiated almost in the fourth century BC.[14, 15] It is
suggested that traditional Persian medicine has played a key role in historical
progress of pharmaceutical sciences.[16-19]
In traditional Persian literature, the term rheumatoid arthritis is expressed as
a disturbance in humoral balance of the body, which commonly occurs in
subjects with cold and moist temperaments. Patients with this humoral
disorder suffer from chronic pain in the joints waja-e-mafasel, mainly due
to accumulation of phlegm in the joints.[20, 21]
To our knowledge, there is no mechanistic and evidence-based review on
traditional medicinal plants used for RA in Persian medicine. As various
medicinal plants have traditionally been used for RA managements in Persian
medicine, we reviewed medicinal literature to confirm their efficacy and
safety in the management of RA. In addition, molecular and biological
mechanisms of action along with active phytochemical agents were
highlighted, in favour of plating future drugs for the treatment of RA.
Method
In the current study, a list of medicinal plants traditionally used topically or
orally for the treatment of RA was collected from traditional Persian
literature, including Al-Qanoon fi al-Tibb (The Canon of Medicine, written by
Avicenna in 1025 CE),[20] Makhzan-ol-advieh (written by Aghili Alavi
Khorasani in 1771 CE),[22] Qarabadin-e-kabir (the largest pharmaceutical
manuscript of Persian medicine, written by Aghili Alavi Khorasani in 1772 CE)
[22] and Tohfat-ol-Moemenin (written by Mohammad Tonkaboni in 1670 CE).
[23] Subsequently, electronic databases including Scirus, PubMed, Scopus,
Web of Science, Google Scholar and Cochrane library were explored for each
of these plants, and all retrieved articles were evaluated to ascertain any in
vitro, in vivo or clinical evidence for their efficacy and pharmacological
mechanisms. The retrieved studies demonstrated either apparent efficiency
of these remedies or their indirect effectiveness on the mechanisms involved
in the management of RA. Data were collected from 1970 to 2014
(December). Only published articles were included in this review. Language
restriction was considered, and English language articles were included. The
search terms were rheumatoid arthritis or arthritis or inflammation and

the name of each mentioned plant in the whole text. Results from primary
search were screened by two independent investigators. References of finally
included articles were reviewed for relevant studies. Included articles were
reviewed to extract scientific names of plants, part and extract of the plants,
active components (if mentioned), type of inflammation or arthritis, animal
model for in vivo and type of cell line for in vitro studies. Results were
considered to determine differences between test groups and control groups
in terms of pro-inflammatory cytokines, joint swelling, cartilage degradation,
tissue oedema, granuloma formation, inflammatory reactions, leucocyte
infiltration, number of inflammatory cells, antioxidant enzymes and factors,
disease activity index and proteolytic enzymes. Results are summarized in
Tables 1, 2, 3 and 4. Table 1 demonstrates the selected medicinal plants used
for the treatment of RA in traditional Persian medicine. Tables 2, 3 and 4
show in vitro, in vivo and clinical evidence for the efficacy of the medicinal
plants in rheumatoid disorders. In human studies, factors such as study
design, number of patients, interventions, duration of treatment and efficacy
and tolerability of the herbal treatment were also collected.
Table 1. Medicinal plants traditionally used for the treatment of RA in
traditional Persian medicine
Scientific names Family
Vernacular name(s)
Traditional uses [20,
22, 23]
Alhagi camelorum Fisch.
Fabaceae Khar-e-shotor (resin)
Diuretic,
antinociceptive, joint pain and RA
Althaea officinalis L.
Malvaceae Khatmi (flower) Respiratory disorders,
peptic ulcer, colitis, inflammation, antinociceptive, joint pain and RA
Arctium lappa L. Asteraceae Arghitun
Fissure, burn wound, joint pain and
RA
Artemisia absinthium L. Asteraceae Afsantin (aerial part)
Gastric tonic,
neuralgia, diuretic, inflammation, joint paint and RA
Astragalus arbusculinus Bornm. & Gauba Fabaceae Anzarut (gum)
Antinociceptive, joint pain and arthritis
Cassia angustifolia M. Vahl
Fabaceae Sanaa (leaf)
Laxative,
migraine, purgative of phlegm, RA
Citrus medica L. Rutaceae Otroj, Toranj (fruit)
Inflammation, goat,
antidepressant, headache, joint pain and RA
Clematis ochroleuca Aiton
Ranunculaceae Zeyan, Yasamin-e-zard
Respiratory disorders, headache, joint pain and RA
Colchicum autumnale L.
Colchicaceae
Surenjan, Golhasrat (corm)
Inflammation, goat, haemorrhoid, hepatitis, joint pain and RA
Convolvulus arvensis L. Convolvulaceae Lablab-e-saghir Inflammation,
respiratory disorders, joint pain and RA
Cuscuta epithymum L. Convolvulaceae Aftimun, Sos-e-shabdari (fruit)
Headache, joint pain and RA
Dolichos lablab L. Fabaceae Lablab-e-kabir
Headache, inflammation,
diuretic, wound, joint pain and RA

Dorema ammoniacum D. Don.

Apiaceae Oshagh, Vasha (rhizome)
Diuretic, antinociceptive, respiratory disorders, hepatitis, joint pain and
RA
Ferula assa-foetida L. Apiaceae Anjedan, Anghuze (oleo-gum resin)
Gastric tonic, appetizer, hepatitis, diuretic, joint pain and RA
Ferula persica L. Apiaceae Sakbinaj (oleo-gum resin)
Goat, headache,
haemorrhoid, joint pain and RA
Inula helenium L. Asteraceae Rasan, Zanjabil-e-shami
Antidepressant,
inflammation, liver disorder, diuretic, joint pain and RA
Narcissus tazzeta L.
Amaryllidaceae Narges
Wound healing, goat,
antinociceptive, joint pain and RA
Nepeta menthoides Boiss. & Buhse Lamiaceae Ostokhodus (aerial part)
Gastric tonic, inflammation, antidepressant, haemorrhoid, joint pain
and RA
Nigella sativa L. Ranunculaceae Shuniz, Siah dane (seed)
Diuretic,
hepatitis, headache, haemorrhoid, joint pain and RA
Opopanax chironium W.D.J.Koch
Apiaceae Javshir
Antinociceptive,
diuretic, goat, headache, joint pain and RA
Peganum harmala L.
Nitrariaceae
Harmal, esfand Diuretic,
headache, liver disorder, antinociceptive, joint pain and RA
Rheum palmatum L.
Polygonaceae
Rivand-e-chini
Diuretic,
haemorrhoid, Respiratory disorders, joint pain and RA
Smilax china L. and S. glabra Roxb. Smilacaceae
Ashabe-ye- maghrebi
Inflammation, gastric tonic, goat, haemorrhoid, diuretic, joint pain and
RA
Strychnos nux-vomica L.
Loganiaceae
Azaraghi, Marg-e-moosh
Joint pain and RA
Table 2. In vitro studies on medicinal plants traditionally used for the
treatment of RA
Plant Part/extraction
Result
Active constituent
References
NO, nitric oxide; IL, interleukin; TNF-, tumour necrosis factor-alpha; COX-2,
cyclooxygenase-2; iNOS, inducible NO synthase; LPS, lipopolysaccharide; NFB, nuclear factor B; 5-LOX, 5-lipoxygenase; IFN-, interferon- ; IgG,
Immunoglobulin G; MAPKs, Mitogen-activated protein kinase; Con A,
concanavalin A; CYP P450, cytochrome P450; PHC, Primary human
chondrocytes; JNK, c-Jun N-terminal kinase; ERK, extracellular signalregulated kinase; LT-B4, leukotriene-B4; PGE2, prostaglandin-E2; Caco-2,
colon adenocarcinoma-2; VEGF, vascular endothelial growth factor. and
show increase and decrease in mentioned variables, respectively.
Arctium lappa
Seeds/Ethanolic extract Anti-inflammatory activity via NO,
iNOS, TNF- and IL-6 on LPS-induced RAW 264.7 macrophage
Arctigenin [33]
Root/Butanolic extract Anti-inflammatory activity via IL-4, IL-5, NF-B
and MAPKs phosphorylation on Con A-induced primary splenocytes

[34]

Seeds/Arctigenin showed anti-inflammatory activity by IL-1, IL-6

and TNF- on PGN- and LPS-induced peritoneal macrophages Arctigenin
and glycoside arctiin
[35]
Artemisia capillaris
Shoot/Hot water extract
Anti-inflammatory
activity via NO, PGE2, iNOS, COX-2, TNF-, IL-1 and IL-6 on IFN- and LPS-stimulated RAW 264.7 macrophagesScoparone [37]
Artemisia sylvatica
Aerial parts/Methanol extract Anti-inflammatory
activity by NF-B, NO, TNF-, COX-2 and iNOS on LPS-induced
RAW264.7 macrophages
Artemisolide, 3-methoxytanapartholide,
deacetyllaurenobiolide, moxartenolide as well as arteminolides B and D[40]
Citrus medica
Peel/Ethanol extract
Anti-inflammatory activity by NO,
TNF-, PGE2, iNOS, COX-2, NF-B, IL-1,IL-6, JNK and ERK on
LPS-stimulated mouse RAW 264.7 macrophages
Limonene and terpinene [45]
Clematis chinensis
Root/Acetone extract Anti-inflammatory activity via
PGE2, MMP-3 and COX-2 on IL-1-and LPS-stimulated PHC Saponins
[48]
Clematis mandshurica Root/Ethanolic extract Anti-inflammatory activity
byPGE2 and NO on LPS- and IFN -stimulated mouse peritoneal
macrophages as well as IL-2 and IFN- on Con A-activated splenocytes

[47]
Cuscuta campestris
Seeds/Ethanolic extract Anti-inflammatory activity by
NO on LPS-induced murine RAW264.7 macrophages Quercetin [55]
Cuscuta reflexa Seeds/Ethanolic extract Anti-inflammatory activity by
TNF-, COX-2 and NF-B on LPS-induced murine RAW264 macrophages

[56]
Inula falconeri
Aerial part/Ethanolic extract Anti-inflammatory activity via
NO on LPS-induced RAW264.7 macrophages Guaiane, pseudoguaiane,
xanthane, eudesmane, germacrane, rare secocaryophyllane, chromolaevane
and carabrane
[64]
Inula viscosa
Aerial part/Dichloromethanic extract
Antiinflammatory activity via 5-LOX and LT-B4 in peritoneal rat neutrophils
7-O-methylaromadendrin, 3-acetyl-7-O-methylaromadendrin and
sakuranetin [63]
Rheum palmatum L.
Root/-Anti-inflammatory activity via TNF-, IL-6
,IL-8, PGE2, MMP-1, COX-2 and VEGF in IL-1 and LPS-stimulated
synoviocytes
Emodin
[70]
Smilax china L. Rhizome/Ethanolic extracts Anti-inflammatory activity via
NO, TNF- and NF-B in LPS-induced murine macrophages
[72]
Smilax glabra
Rhizome/Aqueous extract
Anti-inflammatory activity via
IL-1 and NO on LPS-induced peritoneal macrophages, as well as T
lymphocyte proliferation and IL-2 production on Con A-induced splenocytes

[71]
Table 3. In vivo studies on medicinal plants traditionally used for the
treatment of RA

Plant Part/extraction
Method/route of administration
Animal
Result
Active constituents
References
TPA,12-O-tetradecanoylphorbol-13-acetate; MPO, Myeloperoxidase; CFA,
complete Freund's adjuvant; IL-6, interleukin-6; PGE2, prostaglandin-E2; PGF,
prostaglandin-F; IFN-, interferon- ; TNF-, tumour necrosis factor-alpha; NFB, nuclear factor B; NO, nitric oxide; MDA, malondialdehyde; COX1,
cyclooxygenase-1; iNOS, inducible NO synthase; ND, not determined; LT-B4,
leukotriene-B4; EPP, ethyl phenylpropiolate; 5-HT, 5-hydroxytryptamine; LOX,
lipoxygenase.
Althaea officinalis Flower/Aqueous extract
Carrageenan- and formalininduced oedema/Oral administration
Rat Inflammation in both model

[25]
Althaea rosea
Flower/Ethanolic extract
Acetic acid-induced increase
permeability of abdominal capillaries and carrageenan- and dextran-induced
paw oedema/Oral administration
Rat Permeability of abdominal
capillaries, paw oedema in all models by release of PGE from
inflammatory tissue

[24]
Arctium minus
Leaves/Ethanolic extract
Carrageenan-induced paw
oedema/Oral administration Mouse
Ethanolic extract showed paw
oedema

[36]
Artemisia capillaris
Aerial part/Ethanolic extract Arachidonic acidinduced ear oedema/Oral administration Mouse
Ear oedema
Scopoletin, scopolin, scoparone, esculetin, quercetin, capillarisin,
isorhamnetin, 3-O-robinobioside, isorhamnetin 3-O-galactoside and
chlorogenic acid [41]
Artemisia douglasiana Aerial parts/Ethanol extract CFA-induced oedema
and cotton pellet-induced granuloma/Oral administration Rat
Inflammation via NF-B and granuloma formation
Dehydroleucodine
[39]
Cassia alata
Leaf/Hexane extract
CFA-induced arthritis/Oral
administration
Rat RA with swelling and cartilage degradation in
the knee joint and leucocyte of synovial fluid
[42]
Clematis chinensis
Root/-Collagen-induced arthritis/Oral administration
Rat Anti-arthritic activity via TNF-, IL-1 levels in peripheral
blood and COX-2 in synovial membrane Triterpene saponin (AR-6)
[51]
Clematis vitalba L.
Aerial parts/Hydroalcoholic extract Carrageenan-,
serotonin- and PGE2-induced paw oedema and CFA-induced arthritis/Oral
administration
Mouse
Paw oedema in all models and arthritis and
oedema of knee joint C-glycosylflavon, 4-O-coumaroyl-isovitexine or
vitalboside [50]
Colchicum luteum
Corm/Hydroalcoholic extract Formaldehyde and
CFA-induced arthritis/Oral administration Rat Joint swelling and arthritis
in both model via IL-6, IL-1 and TNF-

[53]
Corm/Hydroalcoholic extract Cotton pellet-induced granuloma formation and
carrageenan-induced paw oedema/Oral administration
Rat

Granuloma formation and paw oedema through IL-6, IL-1 and

TNF-

[54]
Ferula hermonis Root/Dichloromethanic extract
Carrageenan-induced
paw oedema/Oral administration
Rat Paw oedema
Ferutinin, teferin
and teferidin
[57]
Inula viscosa
Aerial part/Dichloromethanic extract
TPA-induced ear
oedema and PLA2-induced paw oedema/Topical administration
Mouse
Ear and paw oedema 3-acetyl-7-O-methylaromadendrin, sakuranetin,
7-O-methylaromadendrin and sakuranetin
[63]
Smilax china
Rhizome/Methanol extract
Carrageenan-induced paw
oedema/Oral administration Mouse
Paw oedema by LOX
Sieboldogenin
[73]
Smilax glabra
Rhizome/Ethanolic extracts Carrageenan-induced paw
oedema/Oral administration Rat Paw oedema by leucocyte migration

[72]
Rhizome/Aqueous extract
Adjuvant-induced arthritis/Oral administration
Rat Arthritis and swelling with activated macrophages

[71]
Strychnos nux-vomica Seed/Alkaloid fractions Carrageenan-induced paw
oedema, acetic acid-induced vascular permeability and CFA-induced
arthritis/Intraperitoneal administration
Rat Paw oedema byPGE2,
vascular permeability, arthritis by 6-keto-PGF1a and 5-HT in rat's
blood plasma
Brucine and brucine N-oxide [75]
Table 4. Clinical studies on medicinal plants traditionally used for the
treatment of RA
Plant Preparations/Route of administration
Study design
Disease
No. of patients
Treatment duration
Result
References
Treatment group Control group
TID, tree times per day; BID, two times per day.
Clematis mandshurica Capsule 200 mg (TID) containing Clematis
mandshurica, Trichosanthes kirilowii, and Prunella vulgaris/Oral
administration
Celecoxib 200 mg (BID) Multicentre, randomized, doubleblind, double-dummy, Phase III, noninferiority controlled clinical trial
Patients with rheumatoid arthritis 183 6-week
No significant
difference between response rate of plant and control group indicating the
herbal capsule was as efficacious as celecoxib. No serious adverse effects
were observed. [52]
Nigella sativa
Nigella sativa oil capsules/Oral administration Placebo
A placebo-controlled study
Patients with rheumatoid arthritis 40
1-month
Disease activity score significantly, number of swollen
joints and improvement in the duration of morning stiffness (P = 0.017)[81]
Finding and Results
Scientific and vernacular names of medicinal plants used for the treatment of
RA in traditional Persian literature are shown in Table 1. Moreover, details of
in vitro and in vivo findings that support their efficacy in RA are

demonstrated in Tables 2 and 3. Table 4 shows clinical trials on mentioned

medicinal plants. Below, these medicinal plants with their possible
mechanisms of action along with active phytochemical agents in the
management of RA are described.
Althaea officinalis L. (Malvaceae)
The flowers of A. officinalis, commonly known as marshmallow, have
traditionally been used for the treatment of inflammatory diseases and
chronic pain from the ancient time. In traditional Persian medicine, this
remedy possesses a wide range of therapeutic indications such as
respiratory disorders, colitis and joint pains. The flowers of A. officinalis
possess anti-inflammatory activity and decrease capillaries permeability by
attenuating release of PGE from inflammatory tissue.[24, 25]
Scopoletin, one of the main constituent of the leaves of A. officinalis, can
improve RA through inhibiting the release of the pro-inflammatory cytokines
PGE2, TNF-, IL-1 and IL-6 and suppressing the expression of COX-2.[26,
27] Scientific literature suggests activation of a transcription factor, namely
nuclear factor (NF), which binds to significant consensus DNA factors that
exist on the promoter of specific genes, to trigger the expression of proinflammatory cytokines. Scopoletin significantly downregulates Rel A (p65)
that is a subfamily of NF-B in nucleus of human mast cell line. It is reported
that activation of NF-B is mediated by phosphorylation and degradation of
inhibitor of NF-B (IB), resulting in the nuclear migration of NF-B binding to
DNA, and stimulates cytokine genes expression. Suppression of IB
phosphorylation and degradation in cytoplasm of human mast cell by
scopoletin has a pivotal role in anti-inflammatory potential of this natural
remedy.[28]
Likewise, scopoletin improves histological architecture of arthritic joints,
limits synovial hyperplasia, reduces the formation of new blood vessel within
the synovial tissues and inhibits erosive changes in the bone and cartilage.
Synovial macrophages are stimulated to secrete vascular endothelial growth
factor (VEGF), which binds to specific receptors on local endothelium and
initiates angiogenesis and migration into the joint cavity, with a resultant
enhancement in vascular permeability.[29] Deregulated expression of VEGF
in RA suggests a potential role for VEGF in the disease pathogenesis. There is
considerable interest in targeting angiogenesis and its related growth factors
to discover novel therapeutic approaches for successful protection and
treatment of RA. A high dose of scopoletin reduces the overexpression of
VEGF, basic fibroblast growth factor (bFGF)-2 and IL-6 in the synovial tissues
of animals with adjuvant-induced arthritis. Thus, this natural compound
possesses therapeutic benefits in RA through anti-angiogenic alterations and
a decrease in neovascularization mediated by suppressing IL-6, VEGF and
FGF-2 overexpression.[30, 31]

Arctium lappa L. (Asteraceae)

Different species of Arctium have been used in traditional medicine for
managing topical and systemic inflammatory conditions like rheumatoid
disorders and chronic inflammatory bowel disease. Arctigenin is a lignan
compound considered as one of the main constituents of Arctium lappa
seeds. Upon inflammatory condition of RA pathogenesis, macrophages
release pro-inflammatory cytokines and also nitric oxide (NO). Experimental
investigations showed that arctigenin and its glycoside, arctiin, exhibit antiinflammatory activity by suppressing a wide range of interleukins like IL-1,
IL-6, IL-4 and IL-5, as well as TNF-. This natural compound also alleviates
the level of NO, which is mediated by suppressing the activity and
expression of inducible NO synthase (iNOS). The cellular mechanism of antiarthritic and anti-inflammatory activity of arctigenin is attributed to inhibiting
nuclear signalling pathway (NF-B) and mitogen-activated protein kinases
(MAPKs) phosphorylation. MAPK is a major molecular target component that
increases the expression of mediators of inflammation, which are central to
the pathophysiology of RA. The -isoform is important to the intracellular
signalling pathway for the generation of TNF- or IL-1. It also regulates the
expression of COX-2, the enzyme that regulates PGE2 in inflammation.[32]
Inhibitors of MAPK such as arctigenin block the production of TNF- and IL-1
in monocytes and in synovial tissue of arthritic animals.[33-35] Likewise, the
leaf of A. minus (Hill) Bernh. exhibits anti-inflammatory potential in animal
model of carrageenan-induced paw oedema. [36]
Artemisia absinthium L. (Asteraceae)
In traditional Persian medicine, the aerial part of A. absinthium is one of the
ancient drugs that possess medicinal effects on neuralgia, rheumatoid
disorder, as well as inflammatory diseases. Scoparone, one of the main
active constituents of A. capillaris Thunb., suppresses inflammatory cascade
produced by macrophages significantly in IFN-- and LPS-stimulated RAW
264.7 cell mediated by reducing the release of NO and PGE2.[37] Any
decrease in the level of NO is mediated by inhibition of iNOS expression.
Likewise, inhibition of COX-2 expression by scoparone has a pivotal role in
reduction in inflammatory reaction mediators.[37] Expression of COX-2 and
synthesis of cytokines, such as TNF-, IL-1, IL-6 and IL-8, in RA condition is
mediated by nuclear signalling pathway.[38]
Aerial parts of A. sylvatica Maxim. and A. douglasiana Besser suppress
nuclear signalling pathway (NF-B), so they play an important role in the
reduction in RA symptoms.[37, 39] Phytochemical investigations have shown
that numerous chemical constituents are considered as responsible agents
for anti-arthritis and anti-inflammatory potentials of Artemisia spp including,
artemisolide, 3-methoxytanapartholide, deacetyllaurenobiolide,
moxartenolide, arteminolides, dehydroleucodine, scopoletin, scopolin and
esculetin.[37, 40, 41]

Cassia angustifolia M. Vahl (Fabaceae)

Cassia angustifolia is one of the important traditional remedies used for
clinical symptoms of RA. There is no scientific evidence on the efficacy of this
species in managing rheumatoid disorders. However, the leaf of C. alata L.
improves RA symptoms, including swelling, and cartilage degradation, and
inhibits leucocyte infiltration into synovial fluid of rat knee joint.[42]
Citrus medica L. (Rutaceae)
Citrus medica commonly known as citron is cultivated worldwide, and the
peel, leaves and root have been used in folk medicine of Asian nations
particularly India and Iran. In traditional medicine, this natural drug is
suggested to be useful for the treatment of rheumatism, hepatitis and
arthritis. It has been confirmed that the fruits possess antioxidant and antiinflammatory activity. The peels of C. medica and fruits of C. unshiu
(Swingle) Marcow. suppress inflammatory response in rheumatoid condition.
These natural remedies execute anti-inflammatory activity in terms of
suppressing inflammatory cytokines such as TNF-, PGE2, IL-1, as well as
IL-6, which regulate different vascular and intercellular cell adhesion agents,
leading to the recruitment of leucocytes to sites of inflammation. Citrus fruits
also inhibit the release of NO via suppressing the expression of iNOS
enzyme. Limonene as one of the active agents of C. medica is effective in
inhibiting the production of NO and decreases the expression of iNOS and
COX-2 proteins. It also decreases the expression of TNF-, IL-1 and IL-6.[4345] The inhibitory effect of this medicinal plant on pro-inflammatory
cytokines and mediators is mediated by suppressing the nuclear signalling
pathway.[45]
MAPK pathway is considered as one of the most broadly investigated cellular
signal transduction pathways regulating inflammatory process in arthritic
condition. In vitro investigations have shown that this transduction pathway
possesses a crucial role in modulating iNOS and COX-2 enzymes expression,
as well as stimulating the production of RA-associated cytokines in
macrophages and synovial cells. In addition, TNF-, IL-1 and IL-6 are the
major inducers of extracellular signal-regulated kinases (ERK), c-Jun Nterminal kinase (JNK) and p38 MAPK activation in cultured human synovial
cells. Citrus constituents possess therapeutic effects on RA-associated
inflammation via reducing the phosphorylation of MAPK subsets, JNK and
ERK.[45, 46]
Clematis ochroleuca Aiton (Ranunculaceae)
Clematis ochroleuca is traditionally used for several rheumatoid disorders as
RA. Saponin-enriched extracts from the root of C. chinensis Osbeck possess a
significant therapeutic potential on LPS-stimulated acute inflammatory
arthritis in rabbit. This natural plant can significantly elevate matrix collagen
II levels in the immunohistochemical assay in animal models. Likewise, the

saponin fraction encompasses preventive effects on monosodium

iodoacetate-induced animal arthritis. Macrophages of synovial tissue trigger
matrix metalloproteinase (MMP)-3-associated cartilage damage. The saponin
compounds exhibited inhibitory effects against LPS-stimulated
overexpression of MMP-3 and MMP-13, indicating its benefits in
inflammatory-associated joint degradation. Wen et al. showed that liposome
cream from this medicinal plant alleviated arthritic complication as well as
the levels of IL-1 and TNF- in the synovial fluid of arthritic rabbits
stimulated by intra-articular injections of papain. Positron emission
tomography (PET) imaging has confirmed the therapeutic potential of this
remedy in animal model of RA. It significantly reduces 2-18F-fluoro-2-deoxyd-glucose (18F-FDG) uptake in terms of standard uptake value being
assessed by PET uptake in the animal arthritic joints. Reduction in the PGE2
level in primary human chondrocytes mediated by suppressing COX-2
expression is among the main contributors in its anti-arthritic potential.[4749]
The root of C. mandshurica Rupr. exhibits a remarkable anti-inflammatory
activity. The roots significantly lower LPS- and IFN -stimulated PGE2 and NO
production in mouse peritoneal macrophages and lessen IL-2 and IFN- in
Con A-activated splenocytes.[47, 48] In addition, triterpene saponin, Cglycosylflavon and 4-O-coumaroyl-isovitexine are among the responsible
agents for anti-arthritic potential of Clematis spp.[50, 51] In a randomized
double-blind clinical trial performed by Song et al., intake of C. mandshuricacontaining capsules improved therapeutic response in patients with RA
similar to celecoxib. During the clinical trial, the plant was safe and no major
adverse effect was observed.[52]
Colchicum autumnale L. (Colchicaceae)
The corm of C. autumnale is an important natural drug with long history of
efficacious use for managing various inflammatory disorders like goat,
haemorrhoid, hepatitis and rheumatism. Hydroalcoholic extract from the
corm of C. luteum Baker exhibited remarkable anti-arthritic potential in
animal model of formaldehyde-induced arthritis and was superior to
indomethacin in alleviating the joint swelling during the observation period.
Likewise, corm extract showed a strong therapeutic effect on complete
Freund's adjuvant (CFA)-induced arthritis, which has a wide range of
pathological and immunological features with human RA. The anti-arthritic
potential of this remedy is mediated by inhibiting the production of proinflammatory cytokines TNF-, IL-6 and IL-1 as well as the expression of
TNF-R1 in the synovium. Scientific studies have confirmed that the receptor
subtype, TNF-R1, is involved in pathophysiological effects of TNF- leading to
arthritic conditions.[53, 54]
It has been reported that colchicine, the active phytochemical agent of C.
luteum, suppresses pro-inflammatory cells like macrophages through its

interaction with cellular tubulin protein, demonstrating that this medicinal

plant obviously improves rat paw oedema symptoms including granuloma
formation mediated by suppressing the inflammatory cytokines TNF-, IL-6
and IL-1 in inflamed tissue.[53, 54]
Cuscuta epithymum L. (Convolvulaceae)
Cuscuta epithymum, commonly known as dodder, is a traditional medicinal
plant, which has been administered by Persian physicians for a wide range of
diseases. In vitro assessment showed that methanolic extract from the seeds
of C. campestris Yuncker. significantly lowers the production of nitrite in
activated macrophages. Quercetin as one of the main active constituent in
the seeds of C. campestris plays a critical role in anti-inflammatory potential
of this plant. Lee et al.[55] reported that processed seeds have higher level
of quercetin leading to enhancement of inhibiting inflammatory reaction in
RAW264.7 cells. Likewise, C. reflexa Roxb. inhibits NF-B binding to its
relevant motif and consequent initiating transcription activity, which leads to
regulating various inflammatory signalling pathways. It is confirmed that
downregulation of the cytokines involved in inflammatory arthritis, COX-2
and TNF-, by treatment of this natural agent is mediated via suppressing
NF-B expression.[55, 56]
Ferula asafoetida L. and F. persica L. (Apiaceae)
The oleo-gum resin of both F. assa-foetida and F. persica is among the
important remedies of traditional Persian medicine, which has been used for
various disorders particularly inflammatory illnesses. Experimental study
showed that the active phytoconstituents, ferutinin and teferin play a central
role in improvement in inflammatory response by Ferula species.[57]
Ferutinin, a phytoestrogen compound which is abundant in Ferula genus, has
a strong osteoprotective activity. Daily intake of ferutinin for 2 months
significantly prevents osteoporosis due to estrogen deficiency in
ovariectomized rats. Histomorphometrical assessment of trabecular and
cortical bone from femur and lumbar vertebrae has demonstrated that this
natural molecule has higher anti-osteoporotic effect than estradiol benzoate
on bone mass.[58] Methyl 3,5-O-dicaffeoylquinate and 3,5-O-dicaffeoylquinic
acid from the flower of F. lutea Poir. significantly inhibit 5-LOX enzyme, which
catalyses the dioxygenation of polyunsaturated fatty acids to produce
Hydroperoxyeicosatetraenoic acids and Hydroxyeicosatetraenoic Acids.[59,
60] Crude extract from F. persica and isolated active ingredients,
persicasulphide and umbelliprenin, significantly inhibit MMP-2 and 9, a family
of endopeptidase which regulates destruction of extra cellular matrix and is
involved in inflammatory arthritis.[61]
Inula helenium L. (Asteraceae)
Inula helenium commonly known as horseheal is a perennial plant with
various medicinal indications in folk and traditional medicine.
Dihydroflavonols elicited from aerial part of I. viscosa L. including

sakuranetin, 7-O-methylaromadendrin, 3-O-acetylpadmatin and 3-acetyl-7O-methylaromadendrin have demonstrated both in vitro and in vivo antiinflammatory activity. Dihydroflavonol compounds of I. viscosa significantly
suppressed the metabolism of arachidonate in rat peritoneal leucocytes,
which is stimulated by cation ionophore.[62, 63] Given the fact that
arachidonic acid through COX pathway is metabolized into thromboxane A2
and PGs, and also via the LOX pathway metabolized into leukotrienes,
Hydroperoxyeicosatetraenoic acids and Hydroxyeicosatetraenoic Acids,
biosynthetic cascade of arachidonic acid possesses a pivotal role in several
pathological conditions as inflammatory reaction or arthritis.[3, 5] Likewise,
the flavonoid components of I. viscosa remarkably reduce the production of
leukotriene (LT)-B4, in vitro in rat peritoneal leucocytes. Among the
dihydroflavonols, sakuranetin can inhibit the activity of 5-LOX in vitro.[62-64]
Elastin is a key extracellular matrix protein, which has a mechanical effect on
different tissues like ligaments, arteries and skin. Neutrophil elastase is a
proteolytic enzyme, with the potential to degrade fibrous, fibronectin and
cartilage proteoglycans. Therefore, elastase is a key contributor in the
pathology of RA. Hernndez et al.[63] reported that sakuranetin and 7-Omethylaromadendrin can significantly decrease the elastase release and
suppress the activity of elastase enzyme.
The sesquiterpenoid compounds, ilicic acid and inuviscolide, from I. viscosa
demonstrated inhibitory effect on phorbol ester- or ethyl phenylpropiolateinduced ear oedema as well as the phospholipase (PL)A2- or serotonininduced paw oedema.[62] In addition, sesquiterpene lactones like ergolide
and granilin isolated from I. falconeri Hook. f. aerial part exhibit inhibitory
effect on NO production in RAW264.7 macrophages.[63, 64]
Nigella sativa L. (Ranunculaceae)
The seeds of N. sativa, commonly known as kalonji or black caraway, grow in
south and south-west Asia. The seeds have traditionally been administered
by the physicians of Middle and Far East as a medicinal remedy for managing
various diseases. In a placebo-controlled clinical trial reported by Gheita and
Kenawy, intake of N. sativa oil reduced clinical signs of RA including the
number of swollen joints and disease activity score in patients with RA.[81]
The seeds of N. sativa exhibited ear and paw oedema alleviation in animal
models.[66] Thymoquinone, the major active compound derived from N.
sativa, is a bioflavonoid with strong anti-inflammatory, antioxidant,
neuroprotective, as well as anticarcinogenic effects. In vivo studies exhibited
the ability of thymoquinone for managing inflammatory-associated diseases;
21-day administration of thymoquinone in Wistar rat with collagen-induced
arthritis demonstrated anti-arthritic effects and significantly improved clinical
signs of joints. This substance reduces articular elastase and
myeloperoxidase (MPO) activity. MPO is released from stimulated

granulocytes within the inflammatory condition and is directly associated

with the activity and accumulation of leucocytes in the arthritic joints.
Thymoquinone also suppresses the expression of pro-inflammatory cytokines
including IL-1, TNF-, IL-10, IFN-, PGE2 and IL-6, which are highly
expressed in the rheumatoid joint and are a key contributor in the
pathogenesis of RA. In addition, antioxidative damage is another mechanism
of N. sativa constituents in improving rheumatoid disorders through elevating
the activity of antioxidant enzymes as well as inhibiting the products of lipid
peroxidation and NO.[65-68]
Rheum palmatum L. (Polygonaceae)
Preclinical studies have shown that the root of R. palmatum demonstrates
strong anti-inflammatory activity. Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is an anthraquinone component derived from rhizomes and
roots of R. palmatum with a strong potential to inhibit the overexpression of
inflammatory agents including TNF, iNOS and IL-10 as well as NF-B p65.
This molecule can significantly suppress the proliferation of RA synoviocytes,
which are induced by IL-1 as well as LPS under hypoxic condition. Hypoxia
is defined as a pathological situation with deficient oxygen supply, resulting
in the stimulating transcription factor hypoxia-inducible factor 1 (HIF-1) and
also VEGF. Literature evidence has shown that VEGF is obviously elevated in
RA synovial fluid with a specific role in angiogenesis of rheumatoid joints.
Emodin remarkably suppresses hypoxia-associated RA through reducing the
overexpression of HIF-1 and VEGF. Likewise, this natural molecule
suppresses the expression of pro-inflammatory cytokines like TNF-, IL-6, IL8 and PGE2, which is mediated by inhibiting the expression and activity of
COX-2 in the rheumatoid condition, indicating its therapeutic action on RA
progress.[69, 70]
Accumulated evidence suggests that MMPs released from synoviocytes are
associated with joint destruction in rheumatoid pathogenesis. Intervention
with both IL-1 and LPS has obviously stimulated upregulation of MMP-1 and
MMP-13 expressions in synoviocytes suppressed by emodin. It has been
confirmed that the intracellular signalling pathways, p38 MAPK and NF-B,
are involved in the transcriptional activation of MMP expression. However,
the potential of emodin in suppressing MMP-1 and MMP-13 is not related to
MAPK and NF-B pathways.[69, 70]
Histone deacetylases (HDACs) encompass a class of enzymes with
modulatory effect on inflammatory cascade and MMP function in
synoviocytes. It has been found that attenuation of HDAC activity
particularly HDAC1 is among the main mechanisms of emodin in managing
IL-1- and LPS-induced RA in hypoxic condition.[69, 70]
Smilax china L. and S. glabra Roxb. (Smilacaceae)

The rhizome of S. china and S. glabra has a long history of use for
inflammation, gastric tonic, goat, haemorrhoid, as well as joint disorders in
Persian medicine. Several pharmacological studies have shown antiinflammatory, anticancer and antinociceptive activity of this plant. In
traditional Chinese medicine, this remedy has various therapeutic effects,
particularly chronic pelvic inflammation. Experimental studies on animal
models of inflammation have confirmed the anti-inflammatory potential of
this natural drug, which is mediated by attenuating the overexpression of
pro-inflammatory mediators, TNF-, NO and IL-2. In addition, regulation of
nuclear signalling NF-B is the possible mechanism of its anti-arthritic effect.
T lymphocyte has a significant role in immunological events of pathological
processes in RA. In vitro studies have revealed that this remedy can
significantly inhibit the proliferation of T lymphocyte.[71, 72] In addition, S.
glabra has exhibited improvement in RA symptoms through inhibiting
leucocyte migration and suppressing activated macrophages in vivo.[71-73]
Seiboldogenin is a steroidal saponin, which is derived from ethyl acetate
fraction of the S. china and S. glabra crude extract. It has been reported that
seiboldogenin has modulatory effect on biphasic inflammatory reactions
including early phase of the inflammation through suppressing the release of
histamine and serotonin as well as later phase of inflammatory response
mediated by regulating the activity of kinin-like agents, proteases and PGs,
in animal models.[74] Inhibitory potential of this molecule on LOX indicates
its ability to manage inflammatory conditions as RA.[71, 73]
Strychnos nux-vomica L. (Loganiaceae)
One of natural drugs, which has traditionally been used for inflammatory
disorders, especially rheumatoid condition, is S. nux-vomica. In traditional
medicine, this plant is assumed to have palliative effect on rheumatic pain.
Experimental investigations have shown that the seeds of S. nux-vomica
possess anti-inflammatory activity in terms of suppressing PGE2 and
decreasing vascular permeability. Brucine and brucine N-oxide are two
natural alkaloids, which are isolated from the seeds of S. nux-vomica.[75, 76]
In hot-plate and writhing test, the alkaloids of S. nux-vomica has shown
protective effect on thermic and chemical stimuli. Their analgesic activity has
been long lasting in comparison with pethidine. Likewise, the alkaloids have
demonstrated inhibitory effect on carrageenan-induced rat paw oedema.[77]
Brucine and brucine N-oxide remarkably alleviate clinical signs of CFAinduced RA mediated by reducing the levels of 6-keto-PGF1a. 5Hydroxytryptamine (5-HT) is expressed in excited sensory neurons of
inflammatory sites and is a key contributor in the sensation of pain from
arthritic joints. Brucine and brucine N-oxide significantly decrease the levels
of 5-HT in CFA-induced arthritis rat's blood plasma and elevate 5hydroxytryindole-3-acetic acid, the main metabolite of degradation of 5-HT

by MAO, indicating role of MAO activity in regulating 5-HT pathway by these

natural agents.[75, 77]
Brucine and brucine N-oxide obviously suppress the release of PGE2 in
inflamed tissue and reduce levels of 6-keto-PGF1a in blood plasma, with no
significant effect on the level of thromboxane B2. Therefore, the mechanism
of action of the alkaloids is not entirely similar to NSAIDs.[75]
For some of the medicinal plants traditionally been used for the management
of RA including Astragalus arbusculinus Bornm. & Gauba (Fabaceae),
Convolvulus arvensis L. (Convolvulaceae), Dolichos lablab L. (Fabaceae),
Dorema ammoniacum D. Don. (Apiaceae), Narcissus tazzeta L.
(Amaryllidaceae), Nepeta menthoides Boiss. & Buhse (Lamiaceae),
Opopanax chironium W.D.J.Koch (Apiaceae) and Peganum harmala L.
(Nitrariaceae), no scientific evidence was observed. Cellular and preclinical
studies for scientific evaluation of the efficacy of these remedies are
required.
Discussion and Conclusion
This review assessed the evidence and molecular mechanisms of medicinal
plants used for the treatment of RA in traditional Persian medicine. In
addition, active phytochemical agents of these plants for plating future
natural drugs were discussed.
Investigations have indicated that people suffering from chronic debilitating
diseases and also patients dissatisfied with conventional treatments often
seek alternative treatments. Traditional and folklore approaches for the
management of diseases are among the main alternative sources of
medicine. However, in spite of the growing level of tendency towards
traditional medicine, evidence for safety and effectiveness of these
alternative medicines is limited.[78-80] Although conventional treatments of
RA commonly alleviate the symptoms, high incidence of adverse reactions of
these drugs has resulted in exploration of alternative methods, particularly
traditional remedies, for symptomatic relief of RA.
Numerous medicinal plants have traditionally been used for the management
of RA in Persian medicine. Various experimental studies on these medicinal
plants were gathered, and efficacious and pharmacological aspects of this
natural therapy were presented. These studies comprise cell, animal and
human studies, which are summarized in details in Tables 2, 3 and 4,
respectively.
Scientific evidence has revealed that traditional medicaments exert
beneficial effects on RA through several cellular and molecular mechanisms
including downregulation of pro-inflammatory cytokines such as IL-12, IL-2,
IL-8, TNF-, IFN-, IL-1, IL-6 and IL-8 as well as inhibiting initiation of

inflammatory response. It is suggested that some traditional natural agents

targeting these cytokines act as anti-IL-1 receptor antagonist and anti-TNF
inhibitors. Accumulated evidence has shown the association between
oxidative stress in pathological processes of inflammatory arthritis and
rheumatoid disorders and excessive generation of free radicals and oxidants
in arthritic joints.[81-83] Therefore, suppression of oxidative-associated
damage of arthritic tissue mediated by attenuating free radicals and NO as
well as downregulation of iNOS expression is among the anti-arthritic
mechanisms of natural remedies in traditional Persian medicine. Likewise,
enhancement of antioxidative performance through stimulating the
expression and activity of antioxidant enzymes including catalase, SOD and
GPx is another mechanism of the natural remedies (Figure 1).
Figure 1.
Figure 1. Open in figure viewerDownload Powerpoint slide
Possible biochemical pathways and mechanisms in the pathogenesis of
rheumatoid arthritis where medicinal plants can have an effect. MAPK,
Mitogen-activated protein kinase; NF-B, nuclear factor B; Nrf2, nuclear
factor E2-related factor 2; IL-1, Interleukin-1; TNF-, tumor necrosis factoralpha; COX1, cyclooxygenase-2; ERK, extracellular signal-regulated kinase;
JNK, c-Jun N-terminal kinase; PGE2, prostaglandin-E2; MMP, matrix
metalloproteinases; CAT, catalase; SOD, superoxide dismutase; ROS, reactive
oxidative species.
Matrix metalloproteinases (MMPs) consist of more than 20 proteinases, which
are expressed abundantly in chondrocytes and synovial cells within arthritic
joints and have a principal role in the degradation of the matrix in RA.[84] It
has been found that traditional natural agents inhibit cartilage degradation
through downregulation of destructive metalloproteinases (e.g. MMP-9, MMP3). Results of the current review article show that modulation of
transcriptional and transduction signalling pathways including NF-B and
MAPK, with upstream regulatory activity on inflammatory as well as oxidative
stress cascade, plays a central role in therapeutic potential of natural
remedies on pathological condition of RA.
The results of in vitro and preclinical studies are preliminary; nevertheless,
they suggest the promising potential of mentioned natural drugs in the
improvement in the associated symptoms of RA. In addition, good tolerance
of most of the herbal remedies along with the long history of consumption in
traditional medicine was demonstrated. However, nonclinical studies are
mandatory to determine the toxicity profiles of almost all medicinal plants in
common use for the management of RA.
Based on reviewed cellular and animal studies, various active phytochemical
agents derived from mentioned medicinal plants are potentially efficacious
on RA. These phytoconstituents are from different chemical categories
including flavonols (quercetin), lignans (arctigenin), coumarins (scopoletin

and scoparone), oxyanthraquinones, terpenes (limonene), triterpene

saponin, steroidal saponin (seiboldogenin), glycosylflavons, phytoestrogens
(ferutinin), sesquiterpenes (umbelliprenin), sesquiterpenoid (ilicic acid and
inuviscolide), sesquiterpene lactones (ergolide and granilin), dihydroflavonols
(sakuranetin and 7-O-methylaromadendrin), anthraquinones (emodin),
alkaloids (brucine and brucine N-oxide), as well as thymoquinone. Figure 2
illustrates the chemical structures of the principle components of medicinal
plants traditionally been used for the management of RA in Persian medicine.
Further research is mandatory to focus on bioefficacy and safety aspects of
these phytochemical agents for finding novel natural drugs.
Figure 2.
Figure 2. Open in figure viewerDownload Powerpoint slide
Chemical structures of principle components of medicinal plant traditionally
used for management of RA in Persian medicine. a: Arctigenin, b: Scoparone,
c: Artemisolide, d: Limonene, e: -terpinene, f: Quercetin, g: Guaiane, h:
germacrane, i: 3-acetyl-7-O-methylaromadendrin, j: Emodin, k: Scopoletin, l:
isorhamnetin, m: Dehydroleucodine, n: vitalboside, o: Ferutinin, p:
sakuranetin, q: Brucine.
To conclude, numerous in vitro, preclinical and clinical studies have
confirmed the beneficial effects of traditionally used medicinal plants for the
management of RA pathogenesis and its complications in traditional Persian
medicine. Limited human studies suggest that traditional medicinal plants
used for RA have less adverse effects than conventional drugs. Results
obtained from pharmacological studies indicate a necessity to establish
bioefficacy, optimum dosage and duration of treatment. Further welldesigned human clinical trials are required to evaluate the effects of
traditional natural remedies in terms of symptomatic, functional and
biological outcomes. Current natural agents may also be tested as adjunctive
therapies in combination with conventional drugs for RA.
Declarations
Conflicts of interest
Authors have no conflict of interest.
Acknowledgements
The authors thank the National Elite Foundation for the support of MH.
Farzaei postdoctoral program under supervision of M. Abdollahi.

A mechanistic review on medicinal plants used for rheumatoid arthritis in

traditional Persian medicine
Authors
Mohammad Hosein Farzaei,
Fatemeh Farzaei,
Mohammad Abdollahi,
Zahra Abbasabadi,
Amir Hossein Abdolghaffari,
Bahman Mehraban
First published: 15 July 2016Full publication history
DOI: 10.1111/jphp.12606View/save citation
Cited by: 0 articlesCitation tools
Article has an altmetric score of 1
Abstract
Objectives
Rheumatoid arthritis (RA) is a chronic, inflammatory, autoimmune disease,
which affects synovial tissue in multiple joints. Although conventional
treatments of RA commonly alleviate the symptoms, high incidence of
adverse reactions leads to research tendency towards complementary and
alternative medicine. As various medicinal plants are traditionally used for
the management of symptomatologies associated with RA in Persian
medicine, we reviewed medicinal literature to confirm their efficacy in the
management of RA.
Key findings
Scientific evidence revealed that traditional medicaments exert beneficial
effects on RA through several cellular mechanisms including downregulation
of pro-inflammatory cytokines such as TNF-, IL-6 and NF-B, suppression of
oxidative stress, inhibition of cartilage degradation with destructive
metalloproteinases and enhancement of antioxidant performance. Various
active constituents from different chemical categories including flavonols,
lignans, coumarins, terpenes, glycosylflavons, dihydroflavonols,
phytoestrogens, sesquiterpene lactones, anthraquinones, alkaloids and
thymoquinones have been isolated from the medicinal plants.
Summary
The pharmacological mechanisms of the medicinal plants traditionally used
for RA in Persian medicine are discussed in the current review. Further
investigations are mandatory to focus on bioefficacy of these phytochemicals
for finding novel natural drugs.
Introduction
Inflammatory processes play a pivotal role in the development of various
diseases. Chronic inflammation arises in pathologic conditions including

exposure to toxic agents, resistant infections caused by certain

microorganisms as well as autoimmunity. Rheumatoid arthritis (RA) is a
chronic, inflammatory, autoimmune disease, which affects synovial tissue in
multiple joints and is characterized by joint swelling, cartilage damage,
synovial inflammation and bony erosion, which can finally result in joint
destruction.[1-3] The total world prevalence of RA is about 0.51% and is
three to four times more common in women.[2] The initiation of RA is
unknown, although interactions between genetic factors, sex hormones and
an immune triggering agent (endogen or environmental substances which
triggers an immune response) are considered as contributing factors.[2, 4]
In RA, synoviocytes and macrophages produce joint-damaging cytokines.
These pro-inflammatory cytokines inhibit the synthesis of proteoglycans and
collagen and increase their degradation. They also activate proteolytic
enzymes (i.e. MMPs and collagenases) promoting destruction of cartilage.[35] Pro-inflammatory cytokines, such as interleukin-1 (IL-1), IL-6 and
tumour necrosis factor- (TNF-), stimulate articular manifestations of RA
through increasing inflammatory cell infiltration, particularly T cells, B cells,
and macrophages, and bone erosion. T-cell activation results in autoantibody
production and release of TNF- and IL-6; in consequence, the articular and
extra-articular manifestation of RA occurs. Chemotactic cytokines, termed
chemokines which attract lymphocytes, monocytes and neutrophils, possess
chemotactic activity and play a significant role in tissue destruction and
synovitis. The pro-inflammatory enzyme, cyclooxygenase (COX)-2, converts
arachidonic acid into prostaglandin-E2 (PGE2) and prompts hyperplasia and
pannus formation in synovial joints resulting in suppression of apoptosis in
synovial fibroblasts and induction of proliferation. Regulation of arachidonic
acid metabolism and modulation of other mediators secreted by
macrophages and other immune cells can be potential targets for the
treatment of chronic inflammatory conditions by inhibiting enzymes like COX
and lipooxygenase (LOX).[3, 5-7]
The goal of conventional treatment is to slow or reverse cartilage
degradation and decrease the pain without toxic pharmacological
consequences. Disease-modifying antirheumatic drugs (DMARDs) and
biological agents including anti-TNF- are used for the prevention of swelling,
pain and joint destruction in RA. Nevertheless, current treatment with
DMARDs is not effective in all patients. Immunosuppressants, nonsteroidal
anti-inflammatory drugs (NSAIDs) and steroidal anti-inflammatory drugs are
commonly used for alleviation of RA complication, but potential side effects
limit their use. For example, NSAIDs and steroids can cause adverse
haematologic, gastrointestinal and renal complications. Long-term
administration of these drugs may lead to adverse effects such as increased
risk of gastrointestinal ulceration and bleeding due to suppression of COX-l.
Therefore, many patients look for complementary and alternative medicine

(CAM) approaches for the management of debilitating diseases with chronic

pain, such as RA.[3, 8, 9]
Traditional medicines all over the world suggest a wide range of remedies for
the management of symptomatologies associated with chronic disorders as
in RA. Scientists are exploring within nations traditional medicine to find
alternative anti-arthritic drugs. Medicinal plants and plant-derived natural
agents are among the most important resources of traditional medicines.[1013] Traditional Persian medicine dates back to thousands of years. History of
medicine in Iran was initiated almost in the fourth century BC.[14, 15] It is
suggested that traditional Persian medicine has played a key role in historical
progress of pharmaceutical sciences.[16-19]
In traditional Persian literature, the term rheumatoid arthritis is expressed as
a disturbance in humoral balance of the body, which commonly occurs in
subjects with cold and moist temperaments. Patients with this humoral
disorder suffer from chronic pain in the joints waja-e-mafasel, mainly due
to accumulation of phlegm in the joints.[20, 21]
To our knowledge, there is no mechanistic and evidence-based review on
traditional medicinal plants used for RA in Persian medicine. As various
medicinal plants have traditionally been used for RA managements in Persian
medicine, we reviewed medicinal literature to confirm their efficacy and
safety in the management of RA. In addition, molecular and biological
mechanisms of action along with active phytochemical agents were
highlighted, in favour of plating future drugs for the treatment of RA.
Method
In the current study, a list of medicinal plants traditionally used topically or
orally for the treatment of RA was collected from traditional Persian
literature, including Al-Qanoon fi al-Tibb (The Canon of Medicine, written by
Avicenna in 1025 CE),[20] Makhzan-ol-advieh (written by Aghili Alavi
Khorasani in 1771 CE),[22] Qarabadin-e-kabir (the largest pharmaceutical
manuscript of Persian medicine, written by Aghili Alavi Khorasani in 1772 CE)
[22] and Tohfat-ol-Moemenin (written by Mohammad Tonkaboni in 1670 CE).
[23] Subsequently, electronic databases including Scirus, PubMed, Scopus,
Web of Science, Google Scholar and Cochrane library were explored for each
of these plants, and all retrieved articles were evaluated to ascertain any in
vitro, in vivo or clinical evidence for their efficacy and pharmacological
mechanisms. The retrieved studies demonstrated either apparent efficiency
of these remedies or their indirect effectiveness on the mechanisms involved
in the management of RA. Data were collected from 1970 to 2014
(December). Only published articles were included in this review. Language
restriction was considered, and English language articles were included. The
search terms were rheumatoid arthritis or arthritis or inflammation and
the name of each mentioned plant in the whole text. Results from primary

search were screened by two independent investigators. References of finally

included articles were reviewed for relevant studies. Included articles were
reviewed to extract scientific names of plants, part and extract of the plants,
active components (if mentioned), type of inflammation or arthritis, animal
model for in vivo and type of cell line for in vitro studies. Results were
considered to determine differences between test groups and control groups
in terms of pro-inflammatory cytokines, joint swelling, cartilage degradation,
tissue oedema, granuloma formation, inflammatory reactions, leucocyte
infiltration, number of inflammatory cells, antioxidant enzymes and factors,
disease activity index and proteolytic enzymes. Results are summarized in
Tables 1, 2, 3 and 4. Table 1 demonstrates the selected medicinal plants used
for the treatment of RA in traditional Persian medicine. Tables 2, 3 and 4
show in vitro, in vivo and clinical evidence for the efficacy of the medicinal
plants in rheumatoid disorders. In human studies, factors such as study
design, number of patients, interventions, duration of treatment and efficacy
and tolerability of the herbal treatment were also collected.
Table 1. Medicinal plants traditionally used for the treatment of RA in
traditional Persian medicine
Scientific names Family
Vernacular name(s)
Traditional uses [20,
22, 23]
Alhagi camelorum Fisch.
Fabaceae Khar-e-shotor (resin)
Diuretic,
antinociceptive, joint pain and RA
Althaea officinalis L.
Malvaceae Khatmi (flower) Respiratory disorders,
peptic ulcer, colitis, inflammation, antinociceptive, joint pain and RA
Arctium lappa L. Asteraceae Arghitun
Fissure, burn wound, joint pain and
RA
Artemisia absinthium L. Asteraceae Afsantin (aerial part)
Gastric tonic,
neuralgia, diuretic, inflammation, joint paint and RA
Astragalus arbusculinus Bornm. & Gauba Fabaceae Anzarut (gum)
Antinociceptive, joint pain and arthritis
Cassia angustifolia M. Vahl
Fabaceae Sanaa (leaf)
Laxative,
migraine, purgative of phlegm, RA
Citrus medica L. Rutaceae Otroj, Toranj (fruit)
Inflammation, goat,
antidepressant, headache, joint pain and RA
Clematis ochroleuca Aiton
Ranunculaceae Zeyan, Yasamin-e-zard
Respiratory disorders, headache, joint pain and RA
Colchicum autumnale L.
Colchicaceae
Surenjan, Golhasrat (corm)
Inflammation, goat, haemorrhoid, hepatitis, joint pain and RA
Convolvulus arvensis L. Convolvulaceae Lablab-e-saghir Inflammation,
respiratory disorders, joint pain and RA
Cuscuta epithymum L. Convolvulaceae Aftimun, Sos-e-shabdari (fruit)
Headache, joint pain and RA
Dolichos lablab L. Fabaceae Lablab-e-kabir
Headache, inflammation,
diuretic, wound, joint pain and RA

Dorema ammoniacum D. Don.

Apiaceae Oshagh, Vasha (rhizome)
Diuretic, antinociceptive, respiratory disorders, hepatitis, joint pain and
RA
Ferula assa-foetida L. Apiaceae Anjedan, Anghuze (oleo-gum resin)
Gastric tonic, appetizer, hepatitis, diuretic, joint pain and RA
Ferula persica L. Apiaceae Sakbinaj (oleo-gum resin)
Goat, headache,
haemorrhoid, joint pain and RA
Inula helenium L. Asteraceae Rasan, Zanjabil-e-shami
Antidepressant,
inflammation, liver disorder, diuretic, joint pain and RA
Narcissus tazzeta L.
Amaryllidaceae Narges
Wound healing, goat,
antinociceptive, joint pain and RA
Nepeta menthoides Boiss. & Buhse Lamiaceae Ostokhodus (aerial part)
Gastric tonic, inflammation, antidepressant, haemorrhoid, joint pain
and RA
Nigella sativa L. Ranunculaceae Shuniz, Siah dane (seed)
Diuretic,
hepatitis, headache, haemorrhoid, joint pain and RA
Opopanax chironium W.D.J.Koch
Apiaceae Javshir
Antinociceptive,
diuretic, goat, headache, joint pain and RA
Peganum harmala L.
Nitrariaceae
Harmal, esfand Diuretic,
headache, liver disorder, antinociceptive, joint pain and RA
Rheum palmatum L.
Polygonaceae
Rivand-e-chini
Diuretic,
haemorrhoid, Respiratory disorders, joint pain and RA
Smilax china L. and S. glabra Roxb. Smilacaceae
Ashabe-ye- maghrebi
Inflammation, gastric tonic, goat, haemorrhoid, diuretic, joint pain and
RA
Strychnos nux-vomica L.
Loganiaceae
Azaraghi, Marg-e-moosh
Joint pain and RA
Table 2. In vitro studies on medicinal plants traditionally used for the
treatment of RA
Plant Part/extraction
Result
Active constituent
References
NO, nitric oxide; IL, interleukin; TNF-, tumour necrosis factor-alpha; COX-2,
cyclooxygenase-2; iNOS, inducible NO synthase; LPS, lipopolysaccharide; NFB, nuclear factor B; 5-LOX, 5-lipoxygenase; IFN-, interferon- ; IgG,
Immunoglobulin G; MAPKs, Mitogen-activated protein kinase; Con A,
concanavalin A; CYP P450, cytochrome P450; PHC, Primary human
chondrocytes; JNK, c-Jun N-terminal kinase; ERK, extracellular signalregulated kinase; LT-B4, leukotriene-B4; PGE2, prostaglandin-E2; Caco-2,
colon adenocarcinoma-2; VEGF, vascular endothelial growth factor. and
show increase and decrease in mentioned variables, respectively.
Arctium lappa
Seeds/Ethanolic extract Anti-inflammatory activity via NO,
iNOS, TNF- and IL-6 on LPS-induced RAW 264.7 macrophage
Arctigenin [33]
Root/Butanolic extract Anti-inflammatory activity via IL-4, IL-5, NF-B
and MAPKs phosphorylation on Con A-induced primary splenocytes

[34]

Seeds/Arctigenin showed anti-inflammatory activity by IL-1, IL-6

and TNF- on PGN- and LPS-induced peritoneal macrophages Arctigenin
and glycoside arctiin
[35]
Artemisia capillaris
Shoot/Hot water extract
Anti-inflammatory
activity via NO, PGE2, iNOS, COX-2, TNF-, IL-1 and IL-6 on IFN- and LPS-stimulated RAW 264.7 macrophagesScoparone [37]
Artemisia sylvatica
Aerial parts/Methanol extract Anti-inflammatory
activity by NF-B, NO, TNF-, COX-2 and iNOS on LPS-induced
RAW264.7 macrophages
Artemisolide, 3-methoxytanapartholide,
deacetyllaurenobiolide, moxartenolide as well as arteminolides B and D[40]
Citrus medica
Peel/Ethanol extract
Anti-inflammatory activity by NO,
TNF-, PGE2, iNOS, COX-2, NF-B, IL-1,IL-6, JNK and ERK on
LPS-stimulated mouse RAW 264.7 macrophages
Limonene and terpinene [45]
Clematis chinensis
Root/Acetone extract Anti-inflammatory activity via
PGE2, MMP-3 and COX-2 on IL-1-and LPS-stimulated PHC Saponins
[48]
Clematis mandshurica Root/Ethanolic extract Anti-inflammatory activity
byPGE2 and NO on LPS- and IFN -stimulated mouse peritoneal
macrophages as well as IL-2 and IFN- on Con A-activated splenocytes

[47]
Cuscuta campestris
Seeds/Ethanolic extract Anti-inflammatory activity by
NO on LPS-induced murine RAW264.7 macrophages Quercetin [55]
Cuscuta reflexa Seeds/Ethanolic extract Anti-inflammatory activity by
TNF-, COX-2 and NF-B on LPS-induced murine RAW264 macrophages

[56]
Inula falconeri
Aerial part/Ethanolic extract Anti-inflammatory activity via
NO on LPS-induced RAW264.7 macrophages Guaiane, pseudoguaiane,
xanthane, eudesmane, germacrane, rare secocaryophyllane, chromolaevane
and carabrane
[64]
Inula viscosa
Aerial part/Dichloromethanic extract
Antiinflammatory activity via 5-LOX and LT-B4 in peritoneal rat neutrophils
7-O-methylaromadendrin, 3-acetyl-7-O-methylaromadendrin and
sakuranetin [63]
Rheum palmatum L.
Root/-Anti-inflammatory activity via TNF-, IL-6
,IL-8, PGE2, MMP-1, COX-2 and VEGF in IL-1 and LPS-stimulated
synoviocytes
Emodin
[70]
Smilax china L. Rhizome/Ethanolic extracts Anti-inflammatory activity via
NO, TNF- and NF-B in LPS-induced murine macrophages
[72]
Smilax glabra
Rhizome/Aqueous extract
Anti-inflammatory activity via
IL-1 and NO on LPS-induced peritoneal macrophages, as well as T
lymphocyte proliferation and IL-2 production on Con A-induced splenocytes

[71]
Table 3. In vivo studies on medicinal plants traditionally used for the
treatment of RA

Plant Part/extraction
Method/route of administration
Animal
Result
Active constituents
References
TPA,12-O-tetradecanoylphorbol-13-acetate; MPO, Myeloperoxidase; CFA,
complete Freund's adjuvant; IL-6, interleukin-6; PGE2, prostaglandin-E2; PGF,
prostaglandin-F; IFN-, interferon- ; TNF-, tumour necrosis factor-alpha; NFB, nuclear factor B; NO, nitric oxide; MDA, malondialdehyde; COX1,
cyclooxygenase-1; iNOS, inducible NO synthase; ND, not determined; LT-B4,
leukotriene-B4; EPP, ethyl phenylpropiolate; 5-HT, 5-hydroxytryptamine; LOX,
lipoxygenase.
Althaea officinalis Flower/Aqueous extract
Carrageenan- and formalininduced oedema/Oral administration
Rat Inflammation in both model

[25]
Althaea rosea
Flower/Ethanolic extract
Acetic acid-induced increase
permeability of abdominal capillaries and carrageenan- and dextran-induced
paw oedema/Oral administration
Rat Permeability of abdominal
capillaries, paw oedema in all models by release of PGE from
inflammatory tissue

[24]
Arctium minus
Leaves/Ethanolic extract
Carrageenan-induced paw
oedema/Oral administration Mouse
Ethanolic extract showed paw
oedema

[36]
Artemisia capillaris
Aerial part/Ethanolic extract Arachidonic acidinduced ear oedema/Oral administration Mouse
Ear oedema
Scopoletin, scopolin, scoparone, esculetin, quercetin, capillarisin,
isorhamnetin, 3-O-robinobioside, isorhamnetin 3-O-galactoside and
chlorogenic acid [41]
Artemisia douglasiana Aerial parts/Ethanol extract CFA-induced oedema
and cotton pellet-induced granuloma/Oral administration Rat
Inflammation via NF-B and granuloma formation
Dehydroleucodine
[39]
Cassia alata
Leaf/Hexane extract
CFA-induced arthritis/Oral
administration
Rat RA with swelling and cartilage degradation in
the knee joint and leucocyte of synovial fluid
[42]
Clematis chinensis
Root/-Collagen-induced arthritis/Oral administration
Rat Anti-arthritic activity via TNF-, IL-1 levels in peripheral
blood and COX-2 in synovial membrane Triterpene saponin (AR-6)
[51]
Clematis vitalba L.
Aerial parts/Hydroalcoholic extract Carrageenan-,
serotonin- and PGE2-induced paw oedema and CFA-induced arthritis/Oral
administration
Mouse
Paw oedema in all models and arthritis and
oedema of knee joint C-glycosylflavon, 4-O-coumaroyl-isovitexine or
vitalboside [50]
Colchicum luteum
Corm/Hydroalcoholic extract Formaldehyde and
CFA-induced arthritis/Oral administration Rat Joint swelling and arthritis
in both model via IL-6, IL-1 and TNF-

[53]
Corm/Hydroalcoholic extract Cotton pellet-induced granuloma formation and
carrageenan-induced paw oedema/Oral administration
Rat

Granuloma formation and paw oedema through IL-6, IL-1 and

TNF-

[54]
Ferula hermonis Root/Dichloromethanic extract
Carrageenan-induced
paw oedema/Oral administration
Rat Paw oedema
Ferutinin, teferin
and teferidin
[57]
Inula viscosa
Aerial part/Dichloromethanic extract
TPA-induced ear
oedema and PLA2-induced paw oedema/Topical administration
Mouse
Ear and paw oedema 3-acetyl-7-O-methylaromadendrin, sakuranetin,
7-O-methylaromadendrin and sakuranetin
[63]
Smilax china
Rhizome/Methanol extract
Carrageenan-induced paw
oedema/Oral administration Mouse
Paw oedema by LOX
Sieboldogenin
[73]
Smilax glabra
Rhizome/Ethanolic extracts Carrageenan-induced paw
oedema/Oral administration Rat Paw oedema by leucocyte migration

[72]
Rhizome/Aqueous extract
Adjuvant-induced arthritis/Oral administration
Rat Arthritis and swelling with activated macrophages

[71]
Strychnos nux-vomica Seed/Alkaloid fractions Carrageenan-induced paw
oedema, acetic acid-induced vascular permeability and CFA-induced
arthritis/Intraperitoneal administration
Rat Paw oedema byPGE2,
vascular permeability, arthritis by 6-keto-PGF1a and 5-HT in rat's
blood plasma
Brucine and brucine N-oxide [75]
Table 4. Clinical studies on medicinal plants traditionally used for the
treatment of RA
Plant Preparations/Route of administration
Study design
Disease
No. of patients
Treatment duration
Result
References
Treatment group Control group
TID, tree times per day; BID, two times per day.
Clematis mandshurica Capsule 200 mg (TID) containing Clematis
mandshurica, Trichosanthes kirilowii, and Prunella vulgaris/Oral
administration
Celecoxib 200 mg (BID) Multicentre, randomized, doubleblind, double-dummy, Phase III, noninferiority controlled clinical trial
Patients with rheumatoid arthritis 183 6-week
No significant
difference between response rate of plant and control group indicating the
herbal capsule was as efficacious as celecoxib. No serious adverse effects
were observed. [52]
Nigella sativa
Nigella sativa oil capsules/Oral administration Placebo
A placebo-controlled study
Patients with rheumatoid arthritis 40
1-month
Disease activity score significantly, number of swollen
joints and improvement in the duration of morning stiffness (P = 0.017)[81]
Finding and Results
Scientific and vernacular names of medicinal plants used for the treatment of
RA in traditional Persian literature are shown in Table 1. Moreover, details of
in vitro and in vivo findings that support their efficacy in RA are

demonstrated in Tables 2 and 3. Table 4 shows clinical trials on mentioned

medicinal plants. Below, these medicinal plants with their possible
mechanisms of action along with active phytochemical agents in the
management of RA are described.
Althaea officinalis L. (Malvaceae)
The flowers of A. officinalis, commonly known as marshmallow, have
traditionally been used for the treatment of inflammatory diseases and
chronic pain from the ancient time. In traditional Persian medicine, this
remedy possesses a wide range of therapeutic indications such as
respiratory disorders, colitis and joint pains. The flowers of A. officinalis
possess anti-inflammatory activity and decrease capillaries permeability by
attenuating release of PGE from inflammatory tissue.[24, 25]
Scopoletin, one of the main constituent of the leaves of A. officinalis, can
improve RA through inhibiting the release of the pro-inflammatory cytokines
PGE2, TNF-, IL-1 and IL-6 and suppressing the expression of COX-2.[26,
27] Scientific literature suggests activation of a transcription factor, namely
nuclear factor (NF), which binds to significant consensus DNA factors that
exist on the promoter of specific genes, to trigger the expression of proinflammatory cytokines. Scopoletin significantly downregulates Rel A (p65)
that is a subfamily of NF-B in nucleus of human mast cell line. It is reported
that activation of NF-B is mediated by phosphorylation and degradation of
inhibitor of NF-B (IB), resulting in the nuclear migration of NF-B binding to
DNA, and stimulates cytokine genes expression. Suppression of IB
phosphorylation and degradation in cytoplasm of human mast cell by
scopoletin has a pivotal role in anti-inflammatory potential of this natural
remedy.[28]
Likewise, scopoletin improves histological architecture of arthritic joints,
limits synovial hyperplasia, reduces the formation of new blood vessel within
the synovial tissues and inhibits erosive changes in the bone and cartilage.
Synovial macrophages are stimulated to secrete vascular endothelial growth
factor (VEGF), which binds to specific receptors on local endothelium and
initiates angiogenesis and migration into the joint cavity, with a resultant
enhancement in vascular permeability.[29] Deregulated expression of VEGF
in RA suggests a potential role for VEGF in the disease pathogenesis. There is
considerable interest in targeting angiogenesis and its related growth factors
to discover novel therapeutic approaches for successful protection and
treatment of RA. A high dose of scopoletin reduces the overexpression of
VEGF, basic fibroblast growth factor (bFGF)-2 and IL-6 in the synovial tissues
of animals with adjuvant-induced arthritis. Thus, this natural compound
possesses therapeutic benefits in RA through anti-angiogenic alterations and
a decrease in neovascularization mediated by suppressing IL-6, VEGF and
FGF-2 overexpression.[30, 31]

Arctium lappa L. (Asteraceae)

Different species of Arctium have been used in traditional medicine for
managing topical and systemic inflammatory conditions like rheumatoid
disorders and chronic inflammatory bowel disease. Arctigenin is a lignan
compound considered as one of the main constituents of Arctium lappa
seeds. Upon inflammatory condition of RA pathogenesis, macrophages
release pro-inflammatory cytokines and also nitric oxide (NO). Experimental
investigations showed that arctigenin and its glycoside, arctiin, exhibit antiinflammatory activity by suppressing a wide range of interleukins like IL-1,
IL-6, IL-4 and IL-5, as well as TNF-. This natural compound also alleviates
the level of NO, which is mediated by suppressing the activity and
expression of inducible NO synthase (iNOS). The cellular mechanism of antiarthritic and anti-inflammatory activity of arctigenin is attributed to inhibiting
nuclear signalling pathway (NF-B) and mitogen-activated protein kinases
(MAPKs) phosphorylation. MAPK is a major molecular target component that
increases the expression of mediators of inflammation, which are central to
the pathophysiology of RA. The -isoform is important to the intracellular
signalling pathway for the generation of TNF- or IL-1. It also regulates the
expression of COX-2, the enzyme that regulates PGE2 in inflammation.[32]
Inhibitors of MAPK such as arctigenin block the production of TNF- and IL-1
in monocytes and in synovial tissue of arthritic animals.[33-35] Likewise, the
leaf of A. minus (Hill) Bernh. exhibits anti-inflammatory potential in animal
model of carrageenan-induced paw oedema. [36]
Artemisia absinthium L. (Asteraceae)
In traditional Persian medicine, the aerial part of A. absinthium is one of the
ancient drugs that possess medicinal effects on neuralgia, rheumatoid
disorder, as well as inflammatory diseases. Scoparone, one of the main
active constituents of A. capillaris Thunb., suppresses inflammatory cascade
produced by macrophages significantly in IFN-- and LPS-stimulated RAW
264.7 cell mediated by reducing the release of NO and PGE2.[37] Any
decrease in the level of NO is mediated by inhibition of iNOS expression.
Likewise, inhibition of COX-2 expression by scoparone has a pivotal role in
reduction in inflammatory reaction mediators.[37] Expression of COX-2 and
synthesis of cytokines, such as TNF-, IL-1, IL-6 and IL-8, in RA condition is
mediated by nuclear signalling pathway.[38]
Aerial parts of A. sylvatica Maxim. and A. douglasiana Besser suppress
nuclear signalling pathway (NF-B), so they play an important role in the
reduction in RA symptoms.[37, 39] Phytochemical investigations have shown
that numerous chemical constituents are considered as responsible agents
for anti-arthritis and anti-inflammatory potentials of Artemisia spp including,
artemisolide, 3-methoxytanapartholide, deacetyllaurenobiolide,
moxartenolide, arteminolides, dehydroleucodine, scopoletin, scopolin and
esculetin.[37, 40, 41]

Cassia angustifolia M. Vahl (Fabaceae)

Cassia angustifolia is one of the important traditional remedies used for
clinical symptoms of RA. There is no scientific evidence on the efficacy of this
species in managing rheumatoid disorders. However, the leaf of C. alata L.
improves RA symptoms, including swelling, and cartilage degradation, and
inhibits leucocyte infiltration into synovial fluid of rat knee joint.[42]
Citrus medica L. (Rutaceae)
Citrus medica commonly known as citron is cultivated worldwide, and the
peel, leaves and root have been used in folk medicine of Asian nations
particularly India and Iran. In traditional medicine, this natural drug is
suggested to be useful for the treatment of rheumatism, hepatitis and
arthritis. It has been confirmed that the fruits possess antioxidant and antiinflammatory activity. The peels of C. medica and fruits of C. unshiu
(Swingle) Marcow. suppress inflammatory response in rheumatoid condition.
These natural remedies execute anti-inflammatory activity in terms of
suppressing inflammatory cytokines such as TNF-, PGE2, IL-1, as well as
IL-6, which regulate different vascular and intercellular cell adhesion agents,
leading to the recruitment of leucocytes to sites of inflammation. Citrus fruits
also inhibit the release of NO via suppressing the expression of iNOS
enzyme. Limonene as one of the active agents of C. medica is effective in
inhibiting the production of NO and decreases the expression of iNOS and
COX-2 proteins. It also decreases the expression of TNF-, IL-1 and IL-6.[4345] The inhibitory effect of this medicinal plant on pro-inflammatory
cytokines and mediators is mediated by suppressing the nuclear signalling
pathway.[45]
MAPK pathway is considered as one of the most broadly investigated cellular
signal transduction pathways regulating inflammatory process in arthritic
condition. In vitro investigations have shown that this transduction pathway
possesses a crucial role in modulating iNOS and COX-2 enzymes expression,
as well as stimulating the production of RA-associated cytokines in
macrophages and synovial cells. In addition, TNF-, IL-1 and IL-6 are the
major inducers of extracellular signal-regulated kinases (ERK), c-Jun Nterminal kinase (JNK) and p38 MAPK activation in cultured human synovial
cells. Citrus constituents possess therapeutic effects on RA-associated
inflammation via reducing the phosphorylation of MAPK subsets, JNK and
ERK.[45, 46]
Clematis ochroleuca Aiton (Ranunculaceae)
Clematis ochroleuca is traditionally used for several rheumatoid disorders as
RA. Saponin-enriched extracts from the root of C. chinensis Osbeck possess a
significant therapeutic potential on LPS-stimulated acute inflammatory
arthritis in rabbit. This natural plant can significantly elevate matrix collagen
II levels in the immunohistochemical assay in animal models. Likewise, the

saponin fraction encompasses preventive effects on monosodium

iodoacetate-induced animal arthritis. Macrophages of synovial tissue trigger
matrix metalloproteinase (MMP)-3-associated cartilage damage. The saponin
compounds exhibited inhibitory effects against LPS-stimulated
overexpression of MMP-3 and MMP-13, indicating its benefits in
inflammatory-associated joint degradation. Wen et al. showed that liposome
cream from this medicinal plant alleviated arthritic complication as well as
the levels of IL-1 and TNF- in the synovial fluid of arthritic rabbits
stimulated by intra-articular injections of papain. Positron emission
tomography (PET) imaging has confirmed the therapeutic potential of this
remedy in animal model of RA. It significantly reduces 2-18F-fluoro-2-deoxyd-glucose (18F-FDG) uptake in terms of standard uptake value being
assessed by PET uptake in the animal arthritic joints. Reduction in the PGE2
level in primary human chondrocytes mediated by suppressing COX-2
expression is among the main contributors in its anti-arthritic potential.[4749]
The root of C. mandshurica Rupr. exhibits a remarkable anti-inflammatory
activity. The roots significantly lower LPS- and IFN -stimulated PGE2 and NO
production in mouse peritoneal macrophages and lessen IL-2 and IFN- in
Con A-activated splenocytes.[47, 48] In addition, triterpene saponin, Cglycosylflavon and 4-O-coumaroyl-isovitexine are among the responsible
agents for anti-arthritic potential of Clematis spp.[50, 51] In a randomized
double-blind clinical trial performed by Song et al., intake of C. mandshuricacontaining capsules improved therapeutic response in patients with RA
similar to celecoxib. During the clinical trial, the plant was safe and no major
adverse effect was observed.[52]
Colchicum autumnale L. (Colchicaceae)
The corm of C. autumnale is an important natural drug with long history of
efficacious use for managing various inflammatory disorders like goat,
haemorrhoid, hepatitis and rheumatism. Hydroalcoholic extract from the
corm of C. luteum Baker exhibited remarkable anti-arthritic potential in
animal model of formaldehyde-induced arthritis and was superior to
indomethacin in alleviating the joint swelling during the observation period.
Likewise, corm extract showed a strong therapeutic effect on complete
Freund's adjuvant (CFA)-induced arthritis, which has a wide range of
pathological and immunological features with human RA. The anti-arthritic
potential of this remedy is mediated by inhibiting the production of proinflammatory cytokines TNF-, IL-6 and IL-1 as well as the expression of
TNF-R1 in the synovium. Scientific studies have confirmed that the receptor
subtype, TNF-R1, is involved in pathophysiological effects of TNF- leading to
arthritic conditions.[53, 54]
It has been reported that colchicine, the active phytochemical agent of C.
luteum, suppresses pro-inflammatory cells like macrophages through its

interaction with cellular tubulin protein, demonstrating that this medicinal

plant obviously improves rat paw oedema symptoms including granuloma
formation mediated by suppressing the inflammatory cytokines TNF-, IL-6
and IL-1 in inflamed tissue.[53, 54]
Cuscuta epithymum L. (Convolvulaceae)
Cuscuta epithymum, commonly known as dodder, is a traditional medicinal
plant, which has been administered by Persian physicians for a wide range of
diseases. In vitro assessment showed that methanolic extract from the seeds
of C. campestris Yuncker. significantly lowers the production of nitrite in
activated macrophages. Quercetin as one of the main active constituent in
the seeds of C. campestris plays a critical role in anti-inflammatory potential
of this plant. Lee et al.[55] reported that processed seeds have higher level
of quercetin leading to enhancement of inhibiting inflammatory reaction in
RAW264.7 cells. Likewise, C. reflexa Roxb. inhibits NF-B binding to its
relevant motif and consequent initiating transcription activity, which leads to
regulating various inflammatory signalling pathways. It is confirmed that
downregulation of the cytokines involved in inflammatory arthritis, COX-2
and TNF-, by treatment of this natural agent is mediated via suppressing
NF-B expression.[55, 56]
Ferula asafoetida L. and F. persica L. (Apiaceae)
The oleo-gum resin of both F. assa-foetida and F. persica is among the
important remedies of traditional Persian medicine, which has been used for
various disorders particularly inflammatory illnesses. Experimental study
showed that the active phytoconstituents, ferutinin and teferin play a central
role in improvement in inflammatory response by Ferula species.[57]
Ferutinin, a phytoestrogen compound which is abundant in Ferula genus, has
a strong osteoprotective activity. Daily intake of ferutinin for 2 months
significantly prevents osteoporosis due to estrogen deficiency in
ovariectomized rats. Histomorphometrical assessment of trabecular and
cortical bone from femur and lumbar vertebrae has demonstrated that this
natural molecule has higher anti-osteoporotic effect than estradiol benzoate
on bone mass.[58] Methyl 3,5-O-dicaffeoylquinate and 3,5-O-dicaffeoylquinic
acid from the flower of F. lutea Poir. significantly inhibit 5-LOX enzyme, which
catalyses the dioxygenation of polyunsaturated fatty acids to produce
Hydroperoxyeicosatetraenoic acids and Hydroxyeicosatetraenoic Acids.[59,
60] Crude extract from F. persica and isolated active ingredients,
persicasulphide and umbelliprenin, significantly inhibit MMP-2 and 9, a family
of endopeptidase which regulates destruction of extra cellular matrix and is
involved in inflammatory arthritis.[61]
Inula helenium L. (Asteraceae)
Inula helenium commonly known as horseheal is a perennial plant with
various medicinal indications in folk and traditional medicine.
Dihydroflavonols elicited from aerial part of I. viscosa L. including

sakuranetin, 7-O-methylaromadendrin, 3-O-acetylpadmatin and 3-acetyl-7O-methylaromadendrin have demonstrated both in vitro and in vivo antiinflammatory activity. Dihydroflavonol compounds of I. viscosa significantly
suppressed the metabolism of arachidonate in rat peritoneal leucocytes,
which is stimulated by cation ionophore.[62, 63] Given the fact that
arachidonic acid through COX pathway is metabolized into thromboxane A2
and PGs, and also via the LOX pathway metabolized into leukotrienes,
Hydroperoxyeicosatetraenoic acids and Hydroxyeicosatetraenoic Acids,
biosynthetic cascade of arachidonic acid possesses a pivotal role in several
pathological conditions as inflammatory reaction or arthritis.[3, 5] Likewise,
the flavonoid components of I. viscosa remarkably reduce the production of
leukotriene (LT)-B4, in vitro in rat peritoneal leucocytes. Among the
dihydroflavonols, sakuranetin can inhibit the activity of 5-LOX in vitro.[62-64]
Elastin is a key extracellular matrix protein, which has a mechanical effect on
different tissues like ligaments, arteries and skin. Neutrophil elastase is a
proteolytic enzyme, with the potential to degrade fibrous, fibronectin and
cartilage proteoglycans. Therefore, elastase is a key contributor in the
pathology of RA. Hernndez et al.[63] reported that sakuranetin and 7-Omethylaromadendrin can significantly decrease the elastase release and
suppress the activity of elastase enzyme.
The sesquiterpenoid compounds, ilicic acid and inuviscolide, from I. viscosa
demonstrated inhibitory effect on phorbol ester- or ethyl phenylpropiolateinduced ear oedema as well as the phospholipase (PL)A2- or serotonininduced paw oedema.[62] In addition, sesquiterpene lactones like ergolide
and granilin isolated from I. falconeri Hook. f. aerial part exhibit inhibitory
effect on NO production in RAW264.7 macrophages.[63, 64]
Nigella sativa L. (Ranunculaceae)
The seeds of N. sativa, commonly known as kalonji or black caraway, grow in
south and south-west Asia. The seeds have traditionally been administered
by the physicians of Middle and Far East as a medicinal remedy for managing
various diseases. In a placebo-controlled clinical trial reported by Gheita and
Kenawy, intake of N. sativa oil reduced clinical signs of RA including the
number of swollen joints and disease activity score in patients with RA.[81]
The seeds of N. sativa exhibited ear and paw oedema alleviation in animal
models.[66] Thymoquinone, the major active compound derived from N.
sativa, is a bioflavonoid with strong anti-inflammatory, antioxidant,
neuroprotective, as well as anticarcinogenic effects. In vivo studies exhibited
the ability of thymoquinone for managing inflammatory-associated diseases;
21-day administration of thymoquinone in Wistar rat with collagen-induced
arthritis demonstrated anti-arthritic effects and significantly improved clinical
signs of joints. This substance reduces articular elastase and
myeloperoxidase (MPO) activity. MPO is released from stimulated

granulocytes within the inflammatory condition and is directly associated

with the activity and accumulation of leucocytes in the arthritic joints.
Thymoquinone also suppresses the expression of pro-inflammatory cytokines
including IL-1, TNF-, IL-10, IFN-, PGE2 and IL-6, which are highly
expressed in the rheumatoid joint and are a key contributor in the
pathogenesis of RA. In addition, antioxidative damage is another mechanism
of N. sativa constituents in improving rheumatoid disorders through elevating
the activity of antioxidant enzymes as well as inhibiting the products of lipid
peroxidation and NO.[65-68]
Rheum palmatum L. (Polygonaceae)
Preclinical studies have shown that the root of R. palmatum demonstrates
strong anti-inflammatory activity. Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is an anthraquinone component derived from rhizomes and
roots of R. palmatum with a strong potential to inhibit the overexpression of
inflammatory agents including TNF, iNOS and IL-10 as well as NF-B p65.
This molecule can significantly suppress the proliferation of RA synoviocytes,
which are induced by IL-1 as well as LPS under hypoxic condition. Hypoxia
is defined as a pathological situation with deficient oxygen supply, resulting
in the stimulating transcription factor hypoxia-inducible factor 1 (HIF-1) and
also VEGF. Literature evidence has shown that VEGF is obviously elevated in
RA synovial fluid with a specific role in angiogenesis of rheumatoid joints.
Emodin remarkably suppresses hypoxia-associated RA through reducing the
overexpression of HIF-1 and VEGF. Likewise, this natural molecule
suppresses the expression of pro-inflammatory cytokines like TNF-, IL-6, IL8 and PGE2, which is mediated by inhibiting the expression and activity of
COX-2 in the rheumatoid condition, indicating its therapeutic action on RA
progress.[69, 70]
Accumulated evidence suggests that MMPs released from synoviocytes are
associated with joint destruction in rheumatoid pathogenesis. Intervention
with both IL-1 and LPS has obviously stimulated upregulation of MMP-1 and
MMP-13 expressions in synoviocytes suppressed by emodin. It has been
confirmed that the intracellular signalling pathways, p38 MAPK and NF-B,
are involved in the transcriptional activation of MMP expression. However,
the potential of emodin in suppressing MMP-1 and MMP-13 is not related to
MAPK and NF-B pathways.[69, 70]
Histone deacetylases (HDACs) encompass a class of enzymes with
modulatory effect on inflammatory cascade and MMP function in
synoviocytes. It has been found that attenuation of HDAC activity
particularly HDAC1 is among the main mechanisms of emodin in managing
IL-1- and LPS-induced RA in hypoxic condition.[69, 70]
Smilax china L. and S. glabra Roxb. (Smilacaceae)

The rhizome of S. china and S. glabra has a long history of use for
inflammation, gastric tonic, goat, haemorrhoid, as well as joint disorders in
Persian medicine. Several pharmacological studies have shown antiinflammatory, anticancer and antinociceptive activity of this plant. In
traditional Chinese medicine, this remedy has various therapeutic effects,
particularly chronic pelvic inflammation. Experimental studies on animal
models of inflammation have confirmed the anti-inflammatory potential of
this natural drug, which is mediated by attenuating the overexpression of
pro-inflammatory mediators, TNF-, NO and IL-2. In addition, regulation of
nuclear signalling NF-B is the possible mechanism of its anti-arthritic effect.
T lymphocyte has a significant role in immunological events of pathological
processes in RA. In vitro studies have revealed that this remedy can
significantly inhibit the proliferation of T lymphocyte.[71, 72] In addition, S.
glabra has exhibited improvement in RA symptoms through inhibiting
leucocyte migration and suppressing activated macrophages in vivo.[71-73]
Seiboldogenin is a steroidal saponin, which is derived from ethyl acetate
fraction of the S. china and S. glabra crude extract. It has been reported that
seiboldogenin has modulatory effect on biphasic inflammatory reactions
including early phase of the inflammation through suppressing the release of
histamine and serotonin as well as later phase of inflammatory response
mediated by regulating the activity of kinin-like agents, proteases and PGs,
in animal models.[74] Inhibitory potential of this molecule on LOX indicates
its ability to manage inflammatory conditions as RA.[71, 73]
Strychnos nux-vomica L. (Loganiaceae)
One of natural drugs, which has traditionally been used for inflammatory
disorders, especially rheumatoid condition, is S. nux-vomica. In traditional
medicine, this plant is assumed to have palliative effect on rheumatic pain.
Experimental investigations have shown that the seeds of S. nux-vomica
possess anti-inflammatory activity in terms of suppressing PGE2 and
decreasing vascular permeability. Brucine and brucine N-oxide are two
natural alkaloids, which are isolated from the seeds of S. nux-vomica.[75, 76]
In hot-plate and writhing test, the alkaloids of S. nux-vomica has shown
protective effect on thermic and chemical stimuli. Their analgesic activity has
been long lasting in comparison with pethidine. Likewise, the alkaloids have
demonstrated inhibitory effect on carrageenan-induced rat paw oedema.[77]
Brucine and brucine N-oxide remarkably alleviate clinical signs of CFAinduced RA mediated by reducing the levels of 6-keto-PGF1a. 5Hydroxytryptamine (5-HT) is expressed in excited sensory neurons of
inflammatory sites and is a key contributor in the sensation of pain from
arthritic joints. Brucine and brucine N-oxide significantly decrease the levels
of 5-HT in CFA-induced arthritis rat's blood plasma and elevate 5hydroxytryindole-3-acetic acid, the main metabolite of degradation of 5-HT

by MAO, indicating role of MAO activity in regulating 5-HT pathway by these

natural agents.[75, 77]
Brucine and brucine N-oxide obviously suppress the release of PGE2 in
inflamed tissue and reduce levels of 6-keto-PGF1a in blood plasma, with no
significant effect on the level of thromboxane B2. Therefore, the mechanism
of action of the alkaloids is not entirely similar to NSAIDs.[75]
For some of the medicinal plants traditionally been used for the management
of RA including Astragalus arbusculinus Bornm. & Gauba (Fabaceae),
Convolvulus arvensis L. (Convolvulaceae), Dolichos lablab L. (Fabaceae),
Dorema ammoniacum D. Don. (Apiaceae), Narcissus tazzeta L.
(Amaryllidaceae), Nepeta menthoides Boiss. & Buhse (Lamiaceae),
Opopanax chironium W.D.J.Koch (Apiaceae) and Peganum harmala L.
(Nitrariaceae), no scientific evidence was observed. Cellular and preclinical
studies for scientific evaluation of the efficacy of these remedies are
required.
Discussion and Conclusion
This review assessed the evidence and molecular mechanisms of medicinal
plants used for the treatment of RA in traditional Persian medicine. In
addition, active phytochemical agents of these plants for plating future
natural drugs were discussed.
Investigations have indicated that people suffering from chronic debilitating
diseases and also patients dissatisfied with conventional treatments often
seek alternative treatments. Traditional and folklore approaches for the
management of diseases are among the main alternative sources of
medicine. However, in spite of the growing level of tendency towards
traditional medicine, evidence for safety and effectiveness of these
alternative medicines is limited.[78-80] Although conventional treatments of
RA commonly alleviate the symptoms, high incidence of adverse reactions of
these drugs has resulted in exploration of alternative methods, particularly
traditional remedies, for symptomatic relief of RA.
Numerous medicinal plants have traditionally been used for the management
of RA in Persian medicine. Various experimental studies on these medicinal
plants were gathered, and efficacious and pharmacological aspects of this
natural therapy were presented. These studies comprise cell, animal and
human studies, which are summarized in details in Tables 2, 3 and 4,
respectively.
Scientific evidence has revealed that traditional medicaments exert
beneficial effects on RA through several cellular and molecular mechanisms
including downregulation of pro-inflammatory cytokines such as IL-12, IL-2,
IL-8, TNF-, IFN-, IL-1, IL-6 and IL-8 as well as inhibiting initiation of

inflammatory response. It is suggested that some traditional natural agents

targeting these cytokines act as anti-IL-1 receptor antagonist and anti-TNF
inhibitors. Accumulated evidence has shown the association between
oxidative stress in pathological processes of inflammatory arthritis and
rheumatoid disorders and excessive generation of free radicals and oxidants
in arthritic joints.[81-83] Therefore, suppression of oxidative-associated
damage of arthritic tissue mediated by attenuating free radicals and NO as
well as downregulation of iNOS expression is among the anti-arthritic
mechanisms of natural remedies in traditional Persian medicine. Likewise,
enhancement of antioxidative performance through stimulating the
expression and activity of antioxidant enzymes including catalase, SOD and
GPx is another mechanism of the natural remedies (Figure 1).
Figure 1.
Figure 1. Open in figure viewerDownload Powerpoint slide
Possible biochemical pathways and mechanisms in the pathogenesis of
rheumatoid arthritis where medicinal plants can have an effect. MAPK,
Mitogen-activated protein kinase; NF-B, nuclear factor B; Nrf2, nuclear
factor E2-related factor 2; IL-1, Interleukin-1; TNF-, tumor necrosis factoralpha; COX1, cyclooxygenase-2; ERK, extracellular signal-regulated kinase;
JNK, c-Jun N-terminal kinase; PGE2, prostaglandin-E2; MMP, matrix
metalloproteinases; CAT, catalase; SOD, superoxide dismutase; ROS, reactive
oxidative species.
Matrix metalloproteinases (MMPs) consist of more than 20 proteinases, which
are expressed abundantly in chondrocytes and synovial cells within arthritic
joints and have a principal role in the degradation of the matrix in RA.[84] It
has been found that traditional natural agents inhibit cartilage degradation
through downregulation of destructive metalloproteinases (e.g. MMP-9, MMP3). Results of the current review article show that modulation of
transcriptional and transduction signalling pathways including NF-B and
MAPK, with upstream regulatory activity on inflammatory as well as oxidative
stress cascade, plays a central role in therapeutic potential of natural
remedies on pathological condition of RA.
The results of in vitro and preclinical studies are preliminary; nevertheless,
they suggest the promising potential of mentioned natural drugs in the
improvement in the associated symptoms of RA. In addition, good tolerance
of most of the herbal remedies along with the long history of consumption in
traditional medicine was demonstrated. However, nonclinical studies are
mandatory to determine the toxicity profiles of almost all medicinal plants in
common use for the management of RA.
Based on reviewed cellular and animal studies, various active phytochemical
agents derived from mentioned medicinal plants are potentially efficacious
on RA. These phytoconstituents are from different chemical categories
including flavonols (quercetin), lignans (arctigenin), coumarins (scopoletin

and scoparone), oxyanthraquinones, terpenes (limonene), triterpene

saponin, steroidal saponin (seiboldogenin), glycosylflavons, phytoestrogens
(ferutinin), sesquiterpenes (umbelliprenin), sesquiterpenoid (ilicic acid and
inuviscolide), sesquiterpene lactones (ergolide and granilin), dihydroflavonols
(sakuranetin and 7-O-methylaromadendrin), anthraquinones (emodin),
alkaloids (brucine and brucine N-oxide), as well as thymoquinone. Figure 2
illustrates the chemical structures of the principle components of medicinal
plants traditionally been used for the management of RA in Persian medicine.
Further research is mandatory to focus on bioefficacy and safety aspects of
these phytochemical agents for finding novel natural drugs.
Figure 2.
Figure 2. Open in figure viewerDownload Powerpoint slide
Chemical structures of principle components of medicinal plant traditionally
used for management of RA in Persian medicine. a: Arctigenin, b: Scoparone,
c: Artemisolide, d: Limonene, e: -terpinene, f: Quercetin, g: Guaiane, h:
germacrane, i: 3-acetyl-7-O-methylaromadendrin, j: Emodin, k: Scopoletin, l:
isorhamnetin, m: Dehydroleucodine, n: vitalboside, o: Ferutinin, p:
sakuranetin, q: Brucine.
To conclude, numerous in vitro, preclinical and clinical studies have
confirmed the beneficial effects of traditionally used medicinal plants for the
management of RA pathogenesis and its complications in traditional Persian
medicine. Limited human studies suggest that traditional medicinal plants
used for RA have less adverse effects than conventional drugs. Results
obtained from pharmacological studies indicate a necessity to establish
bioefficacy, optimum dosage and duration of treatment. Further welldesigned human clinical trials are required to evaluate the effects of
traditional natural remedies in terms of symptomatic, functional and
biological outcomes. Current natural agents may also be tested as adjunctive
therapies in combination with conventional drugs for RA.
Declarations
Conflicts of interest
Authors have no conflict of interest.
Acknowledgements
The authors thank the National Elite Foundation for the support of MH.
Farzaei postdoctoral program under supervision of M. Abdollahi.