By Dr. Megha Agrawal, Dr. Shyamasri Biswas, Dr.

Kim Van Vliet, Contributing Editors

Vacuum Based Processes for

Polyphenol Biofilms to Combat
Complex Diseases

he application of vacuum technology in the processing

and synthesis of clinically important bio-compounds and
films of polyphenols is expanding. Novel vacuum-based
processes have been employed to develop hybrid polyphenols
for a range of biomedical applications to combat complex diseases that include prevention of cerebral stroke, cancer, Alzheimers
and renal failures. In addition, sophisticated and state-of-the-art
high-vacuum and ultra-high-vacuum technology based surface
characterization techniques have been implemented to gain insights of these pharmaceutically important and relevant biofilms.
It is believed that further advances in vacuum based processes
in synthesizing delicate biofilms of polyphenols would make
it possible to optimize polyphenols in combination with other
clinically important biomolecules to develop new biomedical
therapeutics and design of drugs. In this column, we describe
advances in vacuum-based processing of polyphenols for various biomedical applications. We first describe polyphenols, their
important compounds and their health benefits.
Polyphenols are a structural class of naturally occurring or
synthetic or semisynthetic organic compounds. They can be developed as thin biofilms for extremely efficient neuroprotective
drug agents for prevention in ischemic cerebral stroke related injuries and other biomedical applications. Resveratrol (3,5,4'-trihydroxy-trans-stilbene) and flavanols, notably ()-epicatechin,
are important natural polyphenolic materials. Neuroprotective
functions of resveratrol have been researched extensively and
documented. The application of resveratrol can potentially be
beneficial to prevent ischemic cerebral stroke. Ischemic stroke is
a serious medical condition that occurs when there is insufficient
blood flow to the brain to meet metabolic demand. For effective
cellular metabolism and activity, oxygen availability in tissues is

critically important. Also, normal brain functions require regular

supply of glucose. Thus, the role of resveratrol in reducing the
toxic effects of inadequate supply of oxygen (hypoxia) and glucose is of immense interest to investigate.
Oxygen-glucose deprivation OGD, can trigger a multifaceted cellular response that leads to cerebral injuries and cellular
death. Reactive oxygen species ROS induced oxidative stress
and apoptosis are reported as key events in such serious health
conditions. Resveratrol has been shown to be effective in modulating ROS.
Therefore, the design and development of these polyphenols
in their thin film forms with or without the combination of other
biomolecules is of immense interest for biotechnologists and thin
film experts as these polyphenols are known for their potential
health benefits for their antioxidant activity and neuroprotective
role in the vascular and nervous systems [1, 2]. However, formidable technological and medical challenges associated with
resveratrol for its clinical success are its low water solubility
and stability. Hence, new dry processes such as novel vacuum
deposition or evaporation based processes need to be developed
for its effective preparation for the realization of the full clinical
potential of resveratrol.
Thus, many collaborative research and developmental efforts
between vacuum technologists, biotechnologists, thin film scientists and engineers, physicists, biochemists and biologists are
currently taking place across the globe to investigate these clinically important polyphenolic thin biofilms specifically for their
role in reducing the toxic effects of inadequate supply of oxygen
(known as hypoxia) and glucose in human body [1-4]
Resveratrol can be extracted from red grapes in typical vacuum evaporation amber. The chamber is then filled with dry nitro-

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July 2014 Vacuum Technology & Coating

Figure 1. Fabrication of resveratrol loaded HNTs via a vacuum deposition process that eventually generates dried resveratrol loaded drug carriers
for cancer treatment [Credit: Viviana Vergaro, Yuri M. Lvov, Stefano Leporatti, Macromol. Biosci. 2012, 12, 12651271].

gen until the pressure reaches 1.1 bars. Recently, resveratrol in

combination with halloysite nanotubes (HNTs) has been synthesized via a vacuum deposition based process for advanced drug
delivery system to destroy cancer cells (Figure 1). Halloysite
clay is considered green and natural carrier material for hydrophobic drugs encapsulation applications [5]. The researchers
demonstrated synthesis of resveratrol- loaded clay nanotubes using a vacuum based slow drying deposition process that slowed
48-hour release with a constant release rate with time. Loading
of the HNTs with resveratrol was carried out based on vacuum
deposition cycling of halloysite suspension in saturated solution
containing resveratrol. The air located inside the tubes was replaced by the drug solution during this process (Figure 1) [5].
They also employed an additional functionalization for controlling the release rate that was made using layer-by-layer polyelectrolyte multilayer coating onto the tube external surface and
they demonstrated further decrease of release rate. The authors
concluded that resveratrol combined with halloysite clay nanotubes can be used as novel natural pharmaceutical carriers [5].
Researchers showed that low soluble drugs using their saturated
solutions in organic solvents can be loaded with halloysite material, which is natural alumino-silicate clay with hollow tubular
structure. Resveratrol was loaded inside these clay nanotubes
lumens. Resveratrol-loaded clay nanotubes were then added to
breast cell cultures for toxicity tests [5].
In another work that employed vacuum evaporation of polyphenols, liposomes were prepared in a round bottom vial by mixing the appropriate amounts of stock solutions, which were 2102
M in chloroform for lipids and 0.1 M in ethanol for resveratrol.
The process was followed by a specific vacuum evaporation
performed overnight to obtain a dry lipid film (with or without
Resveratrol) [6]. Further, the application of vacuum technology
was extended for the characterization that was based on a special
ultra-violet quantification of trans-resveratrol on liposomes that
employed high-vacuum pumps for a low-pressure environment
to get rid of the scattering background (scaling as 4) due to the
large aggregates in solution, which can affect precise intensity
evaluation [7].
Vacuum-evaporation based processes have been employed to
recover and stabilize polyphenolics from muscadine grape pomace following juice manufacture and that have been evaluated in
laboratory-scale and pilot-scale trials. In a related study, extracts
were concentrated using spray-drying and vacuum evaporation

that demonstrated the potential to extract and concentrate polyphenolic-rich extracts for use in value-added applications [8].
Type II diabetes mellitus (T2DM) is a complex disease characterized by progressive deposition of amyloid in the extracellular matrix of -cells. Researchers investigated the interaction of
the islet amyloid polypeptide (IAPP) with lipid model raft mixtures and INS-1E cells using high-vacuum based fluorescence
microscopy techniques. Based on the state-of-the-art vacuum
fluorescence microscopy, researchers demonstrated the inhibitory effect of the red wine compound resveratrol on IAPP fibril
formation that revealed its potential use in developing therapeutic strategies against T2DM [9].
Bacterial infection and the mechanism of antimicrobial resistance can be understood by the biofilm formation. One viable
alternative strategy to antibiotics is the antibiofilm approach. In
a recent study, the antibiofilm activities of resveratrol and five of
its oligomers, namely, -viniferin, suffruticosol A, suffruticosol
B, vitisin A, and vitisin B, were investigated against enterohemorrhagic Escherichia coli O157:H7 and Pseudomonas aeruginosa PA14. Vitisin B, a stilbenoid tetramer, was found to inhibit
biofilm formation by the two bacteria the most effectively and at
5 g/mL inhibited E. coli O157:H7 biofilm formation by more
than 90% [10] (Figure 2). The authors employed a slow vacuum
evaporation process to the biofilm formation. These results suggest significance of resveratrol in applications where a vacuum
deposited thin-film strategy is required to design and develop
therapeutics for advanced biomedical applications.
In another application of vacuum evaporation of polyphenols,
vacuum based processing has been employed for the growth of
fruit based polyphenols. Juices, jams and confectionery products
use berry fruits (phenolic compounds) that are very popular raw
materials due to their pleasant flavor, odor and color. They are
usually with high nutritional value being rich in antioxidants.
Development of appropriate processing and preservation technologies are required for effective growth of these molecules.
Vacuum drying and vacuum evaporation are two most popular
effective technologies in fruit processing industry providing exclusion of high temperature and presence of oxygen [11].
Flavonol is an important polyphenolic compound and known
for its applications in neurodegenerative diseases. Their efficient
synthesis in the laboratory is key to develop this clinically and
pharmaceutically important compound for new therapeutic designs for prevention of ischemic cerebral stroke. High vacuum

Vacuum Technology & Coating July 2014 www.vactechmag.com or www.vtcmag.com 

Figure 2. Biofilm formation by resveratrol oligomers [Credit: Jin-Hyung Lee, Yong-Guy Kim, Shi Yong Ryu, Moo Hwan Cho, and Jintae Lee, J.
Nat. Prod., 2014, 77 (1), pp 168172].

evaporation has been shown to be an effective method to prepare

solvent free high-quality flavonol. In this regard, flavonol containing single compounds were subjected to vacuum evaporation
and further tested for advanced biomedical applications [12].
Further, Researchers have encountered problems in the isolation
of cranberry monomeric flavonols, anthocyanins and PAC isolates using chromatography that were reported especially problematic because of their labor intensive nature, low yield, low

Figure 3. Vacuum evaporation method for encapsulation of polyphenols [Credit: Aude Munin and Florence Edwards-Lvy, Pharmaceutics.
Dec 2011; 3(4): 793829].

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isolate purity and poor stability [13]. Vacuum evaporation based

extraction of the compound has been shown to be effective to
overcome processing problems [13].
There have been reports on the combination of polyphenols
and other compounds for unique functional properties. For example, in a work on composites of resveratrol, a resveratrol-phospholipid complex was prepared assisted by vacuum evaporation
and the complex was found to be highly bioavailable antioxidant
for the treatment of ageing and cellular degeneration [14].
To overcome the problem of instability of polyphenols under
heat and light, encapsulation of polyphenols has been proposed.
In the vacuum evaporation method, dichloromethane a volatile
solvent that present in a very low miscibility with water is chosen. It is followed by the active compound that gets dissolved
or dispersed in the polymer solution. The mixture is then finely
emulsified in a large amount of water containing surfactants that
results in an oil-in-water (O/W) emulsion. Vacuum evaporation
of the solvent is subsequently realized under vacuum. This method can be applied to the preparation of micro- or nanoparticles
[15].
In conclusion, vacuum process based technologies are promising for the synthesis of clinically important polyphenols for new
therapeutics and drug design to combat complex diseases. The
field of vacuum technology based research and development of
polyphenols is still growing. It is anticipated that this field will
continue to grow with the advent of new vacuum processes and
deposition tools. A number of collaborative opportunities between vacuum technologists and biotechnologists exist where
vacuum technologists can design the processing tools for biotechnologists to optimize the polyphenolic comounds and their
antioxidant derivatives to develop new medical pathways to treat
and prevent a number of serious health conditions.
References for Further Reading
1.  Agrawal M, Kumar V, Kashyap MP, Khanna VK, Randhawa GS,
Pant AB. Ischemic insult induced apoptotic changes in PC12 cells:

July 2014 Vacuum Technology & Coating

protection by trans resveratrol. Eur J Pharmacol, 2011; 666: 5-11.

2. Agrawal M, Kumar V, Singh AK, Kashyap MP, Khanna VK, Siddiqui MA, Pant AB. trans-Resveratrol protects ischemic PC12
Cells by inhibiting the hypoxia associated transcription factors and
increasing the levels of antioxidant defense enzymes. ACS Chem
Neurosci, 2013; 4: 285-94.
3. Leonardo CC*, Agrawal M*, Singh N, Moore JR, Biswal S, Dore
S. Oral administration of the flavanol (-)-epicatechin bolsters endogenous protection against focal ischemia through the Nrf2 cytoprotective pathway. Eur J Neurosci, 2013; 38: 3659-68.
4. Singh N*, Agrawal M*, Dore S. Neuroprotective properties and
mechanisms of resveratrol in in vitro and in vivo experimental cerebral stroke models. ACS Chem Neurosci, 2013; 4: 1151-62.
5. Viviana Vergaro, Yuri M. Lvov, Stefano Leporatti, Halloysite Clay
Nanotubes for Resveratrol Delivery to Cancer Cells, Macromol.
Biosci. 2012, 12, 12651271].
6. Clark D1, Tuor UI, Thompson R, Institoris A, Kulynych A, Zhang
X, Kinniburgh DW, Bari F, Busija DW, Barber PA, Protection
against recurrent stroke with resveratrol: endothelial protection,
PLoS One. 2012;7(10):e47792.
7. Claudia Bonechi, Silvia Martini, Laura Ciani, Stefania Lamponi,
Herbert Rebmann, Claudio Rossi, Sandra Ristor, Using Liposomes as Carriers for Polyphenolic Compounds: The Case of
Trans-Resveratrol, PLoS ONE 7(8): e41438. doi:10.1371/journal.
pone.0041438.
8. Jorge A. Cardona, Joon-Hee Lee and Stephen T. Talcott, Color and
polyphenolic stability in extracts produced from muscadine grape
(Vitis rotundifolia) pomace, J. Agric. Food Chem., 2009, 57 (18),
pp 84218425.
9. Diana Radovan, Norbert Opitz, Roland Winter, Fluorescence microscopy studies on islet amyloid polypeptide fibrillation at heterogeneous and cellular membrane interfaces and its inhibition by
resveratrol, FEBS Letters, 583, 1439 (2009).
10. Jin-Hyung Lee, Yong-Guy Kim, Shi Yong Ryu, Moo Hwan Cho,
and Jintae Lee, Resveratrol Oligomers Inhibit Biofilm Formation
of Escherichia coli O157:H7 and Pseudomonas aeruginosa, J. Nat.
Prod., 2014, 77 (1), pp 168172.
11. Brquez, R.M., Canales, E.R., Redon, J.P. (2010) Osmotic dehydration of raspberries with vacuum pretreatment followed by microwave-vacuum drying. Journal of Food Engineering 99: 121127.
12. Abida Taskeen, Ismat Naeem and Hifsa Mubeen, Isolation of flavonols from Euphorbia wallichii by preparative High Performance
Liquid Chromatography, Nature and Science, 2009;7(8)
13. Ajay P. Singh, Ted Wilson, Amanda J Kalk, James Cheong,and
Nicholi Vorsa, Isolation of specific cranberry flavonoids for biological activity assessment, Food Chem. Oct 15, 2009; 116(4): 963
968.
14. 
Giorgio Pifferi, Pirgiorgio Anzaghi, Rosanna Stefli, Resveratrol-phospholipids complexes, their preparation, and pharmaceutical and cosmetic composition containing same, US Patent
US20040116386 A1, 2004.
15. Aude Munin and Florence Edwards-Lvy, Encapsulation of Natural Polyphenolic Compounds; a ReviewPharmaceutics, Dec 2011;
3(4): 793829.

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