FORMULATION AND EVALUATION OF POLY HERBAL OINTMENT

CONTAINING EXTRACTS OF OCIMUM SANCTUM AND

ADHATHODA VASICA

MASTER OF PHARMACY DISSERTATION PROTOCOL

SUBMITTED TO THE

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA,

BANGALORE.

BY
Mr. Sandesh Bhat B.Pharm
Under The Guidance of

Mr. Shripathy D

M. Pharm

ASST.PROFESSOR

DEPARTMENT OF PHARMACEUTICS.
SRINIVAS COLLEGE OF PHARMACY, VALACHIL, MANGALORE 574143
2013-2015

RAJIV GANDHI UNIVERSITY OF HEALTH

SCIENCESBANGALORE, KARNATAKA
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1.

Name of the Candidate and

Address:

MR. SANDESH BHAT

1stYEARM.PHARM,
DEPT. OF PHARMACEUTICS,
SRINIVAS COLLEGE OF PHARMACY,
VALACHIL, MANGALORE-574143.

2.

Name of the Institution:

3.

Course of Study and Subject:

MASTER OF PHARMACY
(PHARMACEUTICS)

4.

Date of Admission:

25/07/2013

5.

Title of the Project:

SRINIVAS COLLEGE OF PHARMACY,

VALACHIL, FARANGIPETE POST,
MANGALORE-574143.

FORMULATION AND EVALUATION OF POLY HERBAL OINTMENT

CONTAINING EXTRACTS OF OCIMUM SANCTUM AND ADHATHODA
VASICA

Brief Resume of the intended work:

6.1

Need of the study:

Medicinal herbs as a potential source of therapeutic aids have attained a significant role in health
system all over the world for both humans and animals, not only in the diseased condition but also as
potential material for maintaining good health. Since time immemorial, medicinal plants, natures
hidden and to a large extent unexplored treasure, have been used virtually in all human cultures
around the world (over 75 % of the population) as a source of safe and effective medicines1.
Herbal drugs have found wide spread use in many countries not only because they are easily
available and are cheaper but an important reason has been the notion that they are safer than
synthetic drugs which may not always be true. According to WHO guidelines, an herbal product
needs to be standardized with respect to safety before releasing it into the market2.
Microorganisms are the causative agents of almost all kinds of acute and chronic diseases. Many
kinds of diseases have been treated with herbal medications throughout the history of mankind. The
therapeutic value of medicinal plants depends upon the presence of one or more constituents
possessing certain physiological and pharmacological activity. Plants based antimicrobials have
enormous therapeutic potential. They are effective in the treatment of infectious diseases while
simultaneously mitigating many of the side effects that are often associated with synthetic
antimicrobials. Many commercially proven drugs used in modern medicine were initially used in
crude form in traditional or folk healing practices or for other purposes that suggested potentially
useful biological activity2.
Wounds are inescapable events in life. Wounds may arise due to physical, chemical or microbial
agents. Healing is survival mechanism and represents an attempt to maintain normal anatomical
structure and function. Wound healing is a process by which tissue regeneration occurs. It is
complex, dynamic process of restoring integrity and tissue layer, which involves an array of interrelated and concomitant events. The management of wound healing is a complicated and expensive
programme. Many herbs have proved to possess significant pro-healing properties in different types
of wounds3.
Several medicinal plants have been used since time immemorial for treatment of cuts, wounds and
burns and showed promising effects. Some very common plants like Aloe vera , Azadirachta
indica , Carica papaya , Celosia argentea , Centella asiatica , Cinnamomum zeylanicum , Curcuma
longa , Nelumbo nucifera , Ocimum sanctum , Phyllanthus emblica , Plumbago zeylanica ,

Adhatoda vasica Pterocarpus santalinus , Terminalia arjuna and Terminalia chebula have been
extensively reported in Ayurveda, Siddha and Unani systems of medicines for their wound healing
potential4.

The plant Ocimum sanctum and Ocimu gratissimum has a versatile role to play in traditional
medicines. These are popularly known as Thulasi also tulsi, or Holy Basil is an aromatic plant in
family Lamiaceae. Ocimum sanctum

is cultivated for its medicinal purpose as herbaltea in

Ayurveda and religious purpose as performing worship with leaves across South Asia. It was well
documented already on the bioactive compounds of Ocimum sanctum

for medicinal aspects i.e.

antimicrobial, adaptogenic, anti-diabetic, hepato-protective, anti-inflammatory, anti-carcinogenic,

radio-protective, immunomodulatory, chemopreventive, cardioprotective, and safe guarding against
possible deficiences. Ocimum sanctum extracts are effective against Staphylococcus aureus, E.
coli, P.aeruginosa, S. typhimurium and used as better alternative in food preservation5.
Adhatoda vasica Linn. (Acanthaceae) known as chue Mue, grows as weed in almost all parts of the
India. Leaves and stems of the plant have been reported to contain an alkaloid mimosine, leaves also
contain mucilage and root contains tannins. Adhatoda vasica is used for its anti- hyperglycemic,
anti-diarrhoeal, anti-convulsant and cytotoxic properties. The plant also contains turgorins, leaves
and roots are used in treatment of piles and fistula. Paste of leaves is applied to hydrocele. The
methanolic, chloroform and Diethyl ether extract ointment (10%w/w) of Adhatoda vasica has
significant wound healing activity6.
These two important herbs have reported significant anti-bacterial and wound healing activities
which are complementary to wound healing process. The growing popularity of natural and herbal
medications easy availability of raw materials, cost effectiveness and paucity of reported
adverse reaction, prompted us formulate a poly herbal topical preparation of both anti-microbial
and wound healing activity. Thus here is an attempt for the formulation of poly herbal ointment
having both the effect.
6.2 Review

of literature:

Exhaustive literature survey was carried out on the proposed topic using Internet, RGUHS (Helinet
Consortium) and referring journals. The survey reveals that no work has been carried out on the
proposed topic and some related work is quoted below;

 Rajashree PH Vidya V, Merin C, Jincy E, Ranjith S.,formulated and evaluated an

antiseptic poly herbal ointment. Ointments were formulated using methanolic extracts (by
continuous hot percolation-soxhletation) of Azadirachta indica , Chromolaena odorata
,Mimosa pudica ,Samadera indica and were evaluated for its physicochemical property,
antibacterial and antioxidant activity. Ointments were prepared using different concentrations
of the extracts such as 2%, 4%, 6% w/w by fusion method using emulsifying ointment as
base.. The results showed that the scavenging activity of the formulations increased with
increase in concentration and this is due to the presence of flavanoids and tannins. The
presence of both antibacterial and antioxidant activity reveals that the prepared ointment can
also be used for wound healing7.

Pavitra S, Saranya D, Sankar P, Murugesan S., had worked on the effect of Tulasi (Ocimum
sanctum) as a disinfection for water treatment. The present study was focused on evaluation
of antimicrobial activity of Ocimum sanctum leaf extract in normal tap and river water. The
antimicrobial effect was studied with different concentration (100 to 600 mg l-1) of Tulsi leaf
extract in tap and river water. In this, 600 mg l-1 concentration of plant extract treated water
shown effective antimicrobial activity at 15 to 16 hrs than the other concentration of extract.
The 500 mg l-1 of extract treated water showed 95 to 98% antibacterial activity in 14 to 16
hrs. The minimum bacterial concentration (MBC) was observed in 500 and 600 mg l-1
extract concentration. The concentration of the bacterial cells inhibited gradually for an hour
was studied by spread plate method8.

Anupama S.A, Khatal P.P, Priyanka G.G, Shaikh H.M., formulated and evaluated an
ointment containing Aloe vera and Ocimum sanctum for wound healing activity.Aloe vera
and Ocimum sanctum was extracted using methanol and the extract was formulated as an
ointment. The evaluation of wound healing effect of herbal ointment formulated with Aloe
vera and Ocimum sanctum embedded in the ointment bases having strength of 10%, and 15%
was carried out using the in vivo excision wound healing model and in vivo incision wound
healing model. The evaluation of wound healing was compared with that of Nitrofurazone
ointment (0.2% w/w). There was a progressive decrease in wound area with time, indicating
the efficacy of the formulations in healing the induced wounds. By the 21st day, the ointment
containing 15% w/w of Aloe vera and the Ocimum sanctumshowed 100% healing. The
ointment containing 15% w/w of Aloe vera and the Ocimum sanctum methanol extract
exhibited synergistic approach and also minimized the time of wound healing9.

 Mohini P, Prag K, Shailesg K, Meghana L, Santosh B, Pravin C., evaluated an antiinflammatory activity of herbal gel formulation. This study evaluated a new herbal gel
preparation containing extract from leaves of Ocimum Sanctum for its topical antiinflammatory activity against carrageenan induced oedema and anti-nociceptive effect.
Gelling agent used in this study was 1% w/w concentration of carbopol- 940. The studies
were conducted on wistar rats of either sex (160-180 g). Change in oedema volume of the rat
hind paw was measured. From the study we concluded that combination of Ocimum Sanctum
potentiated the anti-inflammatory and anti-nociceptive effect topically10.

Asha BA, Nagabhushan, GH. Shashikala., studied the wound healing activity of topical
Ocimum sanctum Linnin albino rats. The animals were divided into two groups of of topical
test, control with 6 animals in each group. The time taken for 50% wound contraction and
complete epithelisation by topical Ocimum sanctum was significantly (p<0.001) less
compared to topical control. Histopathological studies showed early inflammatory changes,
dense collagen and neovascularisation in wounds treated with topical Ocimum sanctum,
compared to control. Mean tensile strength of topical Ocimum sanctum treated wound was
significantly great (p<0.001) compared to control. Topical Ocimum sanctum promoted better
granulation tissue, early and complete epithelisation and better tensile strength compared to
control11.

Smita J, UK. Patil., formulated and characterised a poly herbal cream for wound healing
activity. The herbal cream formulations were designed by using ethanolic extracts of
Argemone mexicana, Cassia tora, Evolvulus alsinoides , Ocimum centum , Curcumis
sativus . Excision wound measuring about 177 mm2 was created on the albino rats placed in
groups (n=5) and the ointment applied topically on the wounded area which was measured at
intervals of 3 days until epithelialization and complete wound closure.. Application of the
herbal cream formulation containing the Argemone mexicana L. extract (1.0 g/100g
ointment) produced the highest rate of wound healing, reducing the epithelialization period
to 14.9 days compared to the control formulation treatment with epithelialization period of
18.3 days. Therefore, it can be concluded that the formulating A. maxicana L. extracts
(0.1g/100g of cream base) into an herbal cream is most effective and safer usage in wound
repairs process12.

 Satish N, Atul D, Damodar G., developed and evaluated an antimicrobial formulation

containing extracts of Anthocephalus cadamba (ROXB) MIQ. Leaves. The purpose of this
study was to develop a stable preparation of well known Indian medicinal plants
Anthocephalus Cadamba(Roxb) Miq in the treatment of infectious skin diseases, by in vitro
determination of their antimicrobial potential against selected microorganism. The result
showed that the formulation containing 0.5%W/W showed good antimicrobial activity
compared to other formulations. All formulation showed good stability during storage and no
major changes observed after carrying out other physic chemical evaluations and during
entire storage period13.

Vidya V, NA Alekutty, B. Jayakar, Subin MZ, Litha T., developed and formulated an
antimicrobial herbal formulation containing the methanolic extract of Samadera indica for
skin diseases. The methanolic extract prepared from the dried leaves of Samadera indica by
continuous hot percolation, were used to examine the toxicity, according to the OECD 423
guidelines, in Swiss Albino mice. Topical formulations were prepared by incorporating
Samadera indica (5% w / w) in an emulsifying ointment and a carbopol gel base and
evaluated for physical parameters and in-vitro antimicrobial activity (S. aureus, P.
aeruginosa and C. albicans). Thus, the present study concludes that the formulated ointment
and gel are safe and efficient antimicrobial formulations for the topical delivery of the
methanolic extract of Samadera indica14.

Objectives of the study :

6.3

The objective of the study is to prepare poly herbal ointment containing the methanolic extract of
Ocimum sanctum and Adhatoda vasica.
Specific objectives of the present investigation are as follows:

To carry out preformulation study of drug.

To carry out drug polymer interaction study.

To formulate the ointment containing extracts of Ocimum sanctum and Adhatoda vasicausing

various polymers by suitable method.

To evaluate physic-chemical properties of the herbal ointment.

To carry out in-vitro drug release study of prepared formulation.

To find out antimicrobial activity.

To evaluate wound healing property.

To carry out acute skin irritation study.

To carry out stability study by ICH guidelines.

Materials and Methods:

Materials:
Drug

: Methanolic extract of Ocimum sanctum and Adhatoda vasica.

Excipients

: Emulsifying wax, White soft paraffin, Liquid paraffin

Methyl paraben, propyl paraben etc.(preservatives)
Distilled water, etc .

Method:
Extraction of active principles:
The shade dried, powdered whole herb of Ocimum sanctum and Adhatoda vasica were defatted by
extracting with petroleum ether (60-800C), followed by extraction with methanol using Soxhlets
extractor. The methanolic extract was then concentrated using rotary flash evaporator to a syrupy
consistency. The residual solvent was removed by drying the extract in vacuum oven.
Preparation of herbal ointment:
Required quantity of the ingredients are weighed and melted. To this, adequate quantities of
methanolic extract of the plants were added and stirred well until a homogeneous mass were
obtained.

7.1

Source of data:

1.Review of literature from:

a) Journals such as : Journal of pharmaceutical and Scientific Innovation.
International Journal of Pharma and Bio Sciences.
Asian Pacific Journal of Tropical Disease.
International Journal of Pharmacy and Life Sciences.
Contemporary Investigations and Observations In Pharmacy.
Scholars Research Library.
Journal of Chemical And Pharmaceutical Research.
Internet browsing.

7.2 Method of Collection of Data:

1. The data related to physiochemical properties of the drug will be collected from drug
information centre, various standard books, journals & other sources like research literature
data bases such as science direct etc. and laboratory experiments.
2. Identification of the chemical constituents.:

Test for alkaloids.

Test for tannins.
Test for glycosides.
Test for saponins.

 Test for resins.

Test for volatile oils.

3. Evaluation of formulated herbal ointment, as follows:

pH15.
Viscosity15.
Spreadability15.
Extrudability15.
In-vitro diffusion study15.

Anti-microbial study15.
Acute skin irritant study15.
Wound healing activity15.
Stability studies15.

7.3 Does the study require any investigations or interventions to be Conducted on patients or
other humans or animals? If so, please
describe briefly.
-applicable7.4 Has ethical clearance been obtained from your institution in case of 7.3?
-obtainedRef: SCP/CPCSEA/P13/F209/2013

List of references:
1. Sandya D, Akbar A, Mahesh G, Bhaskar G, Yogesh M. Comparative evaluation of poly
herbal formulation for anti-inflammatory and analgesic activity in rats and mice.
Pharmacia, 2010;2(1)285-290.

Der

2. Arnabaditya M, Chandan D, Sujit D, Durga CS. Physico-Chemical and antimicrobial

study of polyherbal formulation Int J Compre Pharm2010;1(4):
3. Shikha S and

Nidhi M Evaluation of poly herbal formulation for wound healing activity

Der Pharmacia Lettre 2009;1(1)157-161.

4. Dash GK and Murty PN. The wound healing effects of a new poly herbal formulation Der
Pharmacia Lettre 2011;3(1):342-349.
5

N. Prasannabalaji, G.Muralidharan, RN. Sivanandan, S. Kumaran, S.R. Pugazhvendan.

Asian Pac J Tropical Disease 2012;S291-5.

Gukzar A, Manjul PS, Anita S. Wound Healing Potential Of Some Medicinal Plants
2011;9(1):136-145.

Rajashree PH, Vidya V, Merin C, Jincy E, Ranjith S Formulation And Evaluation Of

Antiseptic Polyherbal Ointment. Int J Pharm & Life Sci 2012;3(10)

Pavitra S, Saranya D, Sankar P, Murugesan S Effect Of Tulsi As Disinfectant For Water

Treatment. Hitek J Bio Sci & Bioengg 2012;1(1):1-7.

Anupama S.A, Khatal P.P, Priyanka G.G, Shaikh H.M. Evaluation Of Aloe vera And
Ocimum sanctum For Wound Healing Activity CIOP 2013;2(2):52-8.

10 Mohini P, Prag K, Shailesg K, Meghana L, Santosh B, Pravin C. Evaluation Of Antiinflammatory Activity Of Herbal Gel Formolation. J Nat Plant Resour 2011;1(2):105-9.
11 Asha BA, Nagabhushan, GH. Shashikala Study Of Wound Healing Activity Of Ocimum
sanctum In Albino Rats . J Chem Pharm Res 2011;3(6):122-6.
12 Smita J, UK. Patil. Formulation And Characterization Of Poly Herbal Cream With
Wound Healing Activity 2011;3(1):1470-7.
13 Satish N,
Formulation

Atul D,

Damodar G, Development

Containing

IJPRD 2011;3(10):8-12.

Extract

And

Evaluation Of

Antimicrobial

Of Anthocephalus cadamba(ROXB) MIQ

Leaves.

14 Vidya V, NA Alekutty, B. Jayakar, Subin MZ, Litha t. Developed And Evaluated An

Antimicrobial Herbal Formulation Containing The Methanolic Extracts Of Samodera
indica For Skin Disease. J Adv Pharm Technol Res 2012;3(2):106-111.
15 Prabhudutta P, Arpita G, Formulation And Evaluation Of Topical Doseage Form Of
Eupatropium odoratum And Their Wound Healing Activity. IJPBS 2010;1(2);1-13.

Signature of the candidate

9.
(MR. SANDESH BHAT)
The work, which is assigned to
10.

Remarks of the Guide

MR. SANDESH BHAT is under my guidance.

Mr.SHRIPATHY.DM.Pharm.

11.

11.1 Name and Designation of the

Guide

Srinivas College of Pharmacy

Valachil, Mangalore- 574143

11.2 Signature

11.3Head of the Department

Dr. A. R. SHABARAYA M.Pharm.,Ph.D.

HOD and Principal,
SrinivasCollege of Pharmacy,
Valachil, Mangalore- 574143
Recommended and forwarded for

12.

12.1 Remarks of the Principal

12.2 Signature

consideration.

favourable