Preparation of a Traditional Poly Herbal Gel for Abnormal Vaginal Discharge

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*Corresponding author: hajimehd@sbmu.ac.ir
© 2018. Open access. This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (https://creativecommons.org/licenses/by-nc/4.0/)
Research Journal of Pharmacognosy (RJP) 6(4), 2019: 27-35
Received: 13 July 2019
Accepted: 18 Sep 2019
Published online: 22 Sep 2019
DOI: 10.22127/rjp.2019.93506 Original article
Preparation of a Traditional Poly Herbal Gel for Abnormal Vaginal
Discharge
Maedeh Rezghi1,2 , Homa Hajimehdipoor2* , Sara Zakerin1,2, Shirin Fahimi2
1Student Research Committee, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
2Traditional Medicine and Materia Medica Research Center and Department of Traditional Pharmacy,
School of Traditional Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Abstract
Background and objectives: Plants are usually used for treatment of different diseases according to
their traditional application. The use of the plant based medications is increasingly becoming popular
all over the world. Traditional herbal medicines should be transformed into new forms to make better
acceptance and more comfortable use by patients. The aim of the present study was to formulate a
poly herbal gel based on Iranian traditional medicine (ITM), and determine its physicochemical
characteristics. Methods: The gel formulation consisted of Boswellia spp., Carum carvi L., Punica
granatum L. and Myrtus communis L. extracts. The product was evaluated for the physicochemical
parameters. The laboratory stability tests, microbiological tests and rheological behavior were
performed on the gel. Additionally, total tannin content of the gel was measured as pyrogallol using
Folin-Ciocalteu reagent and hide powder. Results: Stability and physicochemical evaluations as well
as microbiological tests showed that the prepared formulation was stable towards physical changes
with no growth of pathogenic microorganisms. Moreover, it represented plastic behavior which is in
favor of a topical product and also total tannins contents were determined to be 0.27 % in the gel.
Conclusion: The product was acceptable due to its physical characterisitcs and so appropriate for
topical use. Regarding the antibacterial, antifungal and anti-inflammatory properties of the medicinal
herbs in the gel and also, the role of tannins as astringent agents, this formulation could be a suitable
candidate for abnormal vaginal discharge with regards to its traditional application in ITM.
Keywords: Boswellia; Carum carvi; Iranian traditional medicine; Myrtus communis; Punica
granatum
Citation: Rezghi M, Hajimehdipoor H, Zakerin S, Fahimi Sh. Preparation of a traditional poly herbal gel for
abnormal vaginal discharge. Res J Pharmacogn. 2019; 6(4): 27-35.
Introduction
The history of herbal products mainly began with
our ancestors who learned that using certain
herbs resulted in healing of some diseases [1].
Traditional medicine is known as a useful source
of potentially valuable compounds for prevention
and treatment of wide range of diseases [2].
About 60-80% of the world trust in herbal drugs
[3]. World Health Organization (WHO) remarks
that 74% of the plant based medicines are used in
modern medicine that their modern usage
correlates with their traditional application as
herbal drugs [4]. In the United States, usage of
complementary and alternative medicine (CAM)
has developed as well [5].
Abnormal vaginal discharge is a common
gynecological complaint among women in
reproductive ages. Persistent vaginal discharge
can cause considerable distress to many women
and it can be one of the major reasons for women
to visit gynecologist. Physiological reasons for
increased vaginal discharge are high estrogen
levels during mid-cycle, pregnancy and sexual

Rezghi M. et al.
28 Res J Pharmacogn 6(4): 27-35
arousal [6,7]. The pathological causes can be
divided into those that are infective or non-
infective. The most common pathological causes
of vaginal discharge are trichomoniasis, bacterial
and fungal infections and inflammation, which
vaginitis is one of its subsets [6-8].
Differentiating the normal and abnormal vaginal
discharge is essential in early detection of
pathological discharge in order to prevent the
possible complications of delayed treatment for
reproductive tract infections such as infertility,
ectopic pregnancy, increased risk of HIV
transmission and infant death [7,9]. In Iranian
traditional medicine (ITM), also known as
Persian medicine, herbal medicines have been
recommended for prevention and treatment of
different diseases. Various prescriptions were
used for abnormal uterine discharge presented in
different dosage forms including topical
formulations [10-12]. Among them, a mixture of
frankincense (Boswellia spp.), caraway (Carum
carvi L.), pomegranate (Punica granatum L.) and
myrtle (Myrtus communis L.) (equal amounts)
has been used as a topical preparation
traditionally named “Zemad” [10]. Since
traditional forms should be converted to modern
dosage forms for better acceptance by patients
and due to faster release and better percutaneous
absorption of the drug in the gel-based
formulation compared to other topical forms
[13,14], in the present investigation, a topical
poly herbal gel, containing the above mentioned
herbs which was retrieved from ITM, has been
formulated and its physicochemical
characteristics was determined.
Material and methods
Ethical considerations
Shahid Beheshti University of Medical Sciences
approved this research with the code of
IR.SBMU.RETECH.REC.1395.330
Chemicals
Propylene glycol (Merck), triethanolamine
(Fluka), ethanol (Merck), Folin-Ciocalteu (Merck)
and Hide powder (Sigma-Aldrich) were used in
the experiment. Other solvents and chemicals
were of analytical grade.
Plant material
Oleo gum resin of Boswellia spp., fruit peels of
Punica granatum L., fruits of Carum carvi L. and
leaves of Myrtus communis L. were purchased
from Tehran herbal market. The samples were
identified in the Herbarium of Traditional
Medicine and Materia Medica Research Center
(TMRC), Shahid Beheshti University of Medical
Sciences, Tehran, Iran. Herbal Market Samples
(HMS) of Boswellia spp (No. 454), Punica
granatum L. (No. 452), Carum carvi L. (No. 453)
and Myrtus communis L. (No. 455) have been
stored at the Herbarium of TMRC. The
specimens were kept separately in well-closed
containers.
Plant material analysis
Quality control tests were performed on the
herbal samples according to their monographs in
pharmacopoies [15,16].
Extraction
All dried samples were powdered. Then, aqueous
extracts of P. granatum fruit peels and M.
communis leaves, ethanol extract of Boswellia
spp. oleo gum resin and hydro-alcoholic (ethanol
70%) extract of C. carvi fruits were prepared
using decoction and maceration methods for
aqueous and alcoholic extracts, respectively
(plant: solvent, 1:10 w/v). The extracts were
concentrated under vacuum pressure up to ten
times of their primary volume
Formulation of the topical herbal gel
Poly herbal gel was made by using 5% of the
each mentioned plants. Gel base was prepared by
applying carbopol in water (0.5, 1 and 2%) and
the most suitable formulation was selected.
Punica granatum, M. communis and C. carvi
extracts were added to the gel base, respectively
and were mixed well. Then various percentages
of propylene glycol were added to frankincense
extract (to increase solubility) and mixed with the
gel. In the final step, triethanolamine was added
to the mixture to form the gel.
Evaluation of the topical gel
Physical examination
Physical properties of the product such as
appearance, homogeneity, odor and color were
examined.
Mechanical stability
In this experiment, samples of the product were
centrifuged for 15 min at 3750 rpm. Then
physical changes, including phase separation
were evaluated in the gel [17].

Poly herbal gel for abnormal vaginal discharge
29
Determination of pH
pH amount of the final product, diluted 1:10 in
distilled water, was determined using a pH meter
(Mettler-Toledo AG, Seven easy model,
Switzerland). The tests were done three times at
room temperature and their average was recorded.
Viscosity determination and assessment of
rheological features of the gel
Viscosity of the final formulation was measured
using Brookfield Rheometer (Brookfield, DV2
RV model, USA) with spindle SC4-29 at room
temperature. Various shear stresses and shear
rates were used on the sample and the resulting
rheogram was plotted to highlight the viscosity
and rheological behavior of the herbal gel.
Temperature stability
In temperature stability tests, six samples of the
gel were selected. Three samples were placed in
refrigerator at 4° C and the rest were put in
incubator at 40° C. After two weeks, the samples
were replaced. Subsequent to 28-days cycle, the
samples were evaluated for apparent changes
such as homogeneity, uniformity, odor and color
[17].
Total tannins content of the gel
Total tannins content was determined in the gel
as a marker using Folin-Ciocalteu reagent and
hide powder in accordance with British
pharmacopoeia with some modifications [15].
Briefly, the proper dilution of the gel sample was
oxidized with Folin-Ciocalteu reagent and the
reaction was neutralized with aqueous solution of
sodium carbonate (29%, w/v). The sample was
placed in dark for 30 min. The absorbance of the
resulting blue color was determined at 760 nm
using water as compensation liquid to get the
amounts of total phenolics. Measurement of
tannins was conducted in continuation of the
above mentioned method by mixing the same
dilution of sample with hide powder to separate
tannins from other polyphenols. Subsequent to
shaking vigorously for 60 min, the compound
was filtrated and the above colorimetric
procedure was conducted on the filtrates to
measure the amounts of polyphenols which were
not adsorbed by hide powder. Tannins content of
the solutions was determined according to the
following equation:
Content of tannins = [Total phenolics content–non-
adsorbed polyphenols content)]
Quantification was done on the basis of the
standard curve of pyrogallol. Results were
mentioned as gram of pyrogallol equivalent per
100 gram of the gel. All measurements were
performed at room temperature in triplicate.
Microbiological tests
Microbial tests were performed in accordance
with WHO instructions [18].
Results and Discussion
In this research, a poly herbal gel suggested (for
abnormal vaginal discharge according to ITM
prescriptions) was prepared. The results of the
analysis of the plants contained in the product
have been shown in table 1. The results were
within the acceptable range of pharmacopoeias
[15,16].
One of the main ingredients of the formulation is
the gelling agent. The concentration of gel
forming agent is very important because lower
concentrations will result in a mixture with very
low consistency, while high concentrations can
lead to the formation of a gel with high viscosity
causing a non-uniform distribution of drug in the
formulation and the lack of proper dispersion on
the skin [19]. In this study, various percentages
of gelling agent were tested to choose the most
suitable concentration for the formulation. The
gel base including 0.5 % of carbomer 940 was a
diluted gel whose liquefaction was observed 24 h
later and it was inappropriate for gel formulations.
With increasing the carbomer concentration to
1.0 %, the uniform and smooth gel base was
observed. The gel base containing 2.0 % of
carbomer 940 was thick and sticky with lack of
proper distribution; therefore, 1.0% of carbomer
940 was determined as the most appropriate
concentration for gel forming. The ingredients of
the final gel formulation along with their quantity
have been presented in table 2. The prepared gel
showed acceptable physicochemical properties
including appearance, homogeneity, uniformity
as well as smooth and soft feeling after using on
the skin, without being sticky or feeling of
dryness. The pH of the polyherbal gel was
measured to be sure that the formulated could be
used without the risk of irritancy and sensitivity
to the skin. The pH was within the appropriate
range for the skin products (pH= 4-6) [20]. No
signs of phase separation and physical changes
were observed during centrifugation and
temperature stability tests.

Rezghi M. et al.
30 Res J Pharmacogn 6(4): 27-35
Table 1. Analysis of Boswellia spp, Carum carvi, Punica granatum, Myrtus communis
Plant materials
Total ash%
Acid
insoluble
ash%
Water
soluble ash%
Sulfated
ash%
Loss on
drying%
Essential
oil%
Total
polyphenolics
%
Boswellia spp.
Carum carvi
Punica granatum
Myrtus communis
5.55± 0.1
4.66± 0.2
5.04± 0.2
-
-
-
-
2.83± 0.1
-
-
-
2.04± 0.1
-
-
-
4.06± 0.2
6.4± 0.5
8.45± 0.7
-
-
5.0± 0.5
5.0± 0.4
-
1.5± 0.1
-
-
16.5± 0.6
-
Table 2. Composition of the ingredients in the herbal gel
Table 3. Physicochemical characteristics of the poly herbal
gel
Moreover, the results of microbiological tests
were in agreement with requirements [18] (table
3). The product was satisfactory with regards to
its physical parameters and so suitable for topical
application with no need for any preservatives.
The flow properties of semi solid products
influence each step of the pharmaceutical
development process, such as filling, mixing,
packing and removal from the container before
the application on the action site. The time-
dependent change in viscosity provides
pharmaceutical formulations with the flexible
rheological manifestation, which subsequently
affects the release profiles of loaded drugs.
Therefore, it is essential for researchers to outline
the flow properties and influencing factors, and
their effects on the pharmacological efficacy of
thixotropic formulations, particularly gels,
ointments, emulsions and colloids [21].
Rheological behaviors consist of plastic, viscous
and elastic features and a mixture of these,
viscoelasticity, is one of the most important
parameters in the evaluation of topical
formulations [17,22,23] that ultimately leads to a
suitable combination with favorable viscosity,
stability and durability on the surface of the skin
[24,25]. The rheogram of the formulated gel was
measured using stainless steel Brookfield
rheometer (figure 1). According to the figure, the
rheogram was non-linear indicating non-
Newtonian behavior. Since the curve did not start
from the origin of the coordinates, it can be
deduced that the formulation exhibited plastic
(Bingham) rheological behavior and also the
curve was transformed to a line that represented a
typical feature of plastic behavior that was
predictable for semisolid compounds. In order to
make non-linear region as linear as possible and
determine the y-intercept more accurately, the log
values of shear stress were drawn against the log
values of shear rate (figure 2). By calculating the
antilog of y-intercept of the equation (y =
0.253x+ 1.631) corresponding to the linear plot,
the yield value was defined (42.75 Pa). The
calculated plastic viscosity and Bingham yield
stress of the gel were 2.19 Pa.s and 94.35 Pa,
respectively, obtained from the slope and y-
intercept of the equation (y = 2.190x+ 94.35)
corresponding to the linear part of the rheogram
(figure 3).
Abnormal vaginal discharge is a common
gynaecological complaint among women in
reproductive age. Due to the role of bacterial and
fungal infections as well as inflammation as
pathological causes [6-8], it seems that
antimicrobial, antifungal and anti-inflammatory
agents could improve the problem.
Figure 1. Rheogram of the poly herbal gel, representing the
presence of a plastic behavior
Natural remedies are more acceptable regarding
that they are safer with less side effects compared
to synthetic ones.
Myrtus communis L. is an evergreen perennial
shrub belonging to the Myrtaceae family [26].
Ingredients
Quantity (%)
Plant extracts
Carbopol 940
Propylene glycol
Triethanolamine
Distilled water
5% from each one (based on dried plant)
1%
7%
0.5%
Up to 100%
Parameters
Results
Color
Odor
Homogeneity
pH
Tannins %
yellow-brown
Characteristic with dominant odor of frankincense
Good
5.35 ± 0.04
0.27±0.01

Poly herbal gel for abnormal vaginal discharge
31
Figure 2. The linear plot of log shear stress-log shear rate
for the poly herbal gel
Figure 3. The linear plot of the poly herbal gel rheogram
The extracts of the plant have been investigated
for their polyphenolics and tannins previously
[27]. The antibacterial activity of methanol,
ethanol, and ethyl acetate extracts of M.
communis leaves has been revealed [28]. Myrtus
communis has shown antibacterial activity
against Bacillus subtilis and E. coli as well as
Staphylococcus aureus, Proteus mirabilis and
Kleibsiella pneumonia [27,29,30]. Eslami et al.
proved that the plant methanol extract showed a
strong antibacterial activity on regular and IMP-
producing P. aeruginosa strains [31].
Furthermore, M. communis L. essential oil (EO)
has shown to be effective against Candida
species [32,33]. Mehrabani et al. showed that
ethyl acetate and total methanol extracts of M.
communis leaves demonstrated the most
considerable antifungal effects among other
extracts of the plant [26]. In another study, the
aqueous leaf extract of the plant showed toxic
activity against of Trichomoniasis vaginalis at
pH 4.65 [34]. Anti-inflammatory activity of n-
hexane and acetone extracts, as well as the
essential oils obtained from the berries and leaves
of M. communis were determined based on the
inhibition of the increase in vascular permeability
[35]. Also, the anti-inflammatory effects of
Myrtucommulone and semimyrtucommulone
found in the leaves of myrtle, were demonstrated
through in vivo model [36-38].
About 50% of the total fruit weight of Punica
granatum Linne (pomegranate) corresponds to
the peel, which is an important source of
bioactive compounds such as phenolics,
flavonoids, ellagitannins (ETs), and
proanthocyanidin compounds [39]. Pomegranate
peel is rich in hydrolyzable tannins, mainly
punicalin, pedunculagin, and punicalagin [40].
Gullon et al. demonstrated antibacterial activity
of peel of P. granatum and determined its total
phenolics and tannins contents [41]. Colak
proved the antifungal and antibacterial activities
of pomegranate skin extract and it was concluded
that pomegranate skin extract could be used as a
preservative and coloring material [42]. The anti-
Trichomoniasis vaginalis effect of P. granatum
extract (in-vitro and in-vivo) showed
considerable results [43,44]. The results of
another study revealed that hydrolysable tannins
of pomegranate inhibited NO production and
iNOS expression in RAW 264.7 cells and could
be used as a standard marker for the anti-
inflammatory activity of P. granatum [45]. In a
study performed in 2017, Houston et al.
demonstrated that pomegranate rind extract down
regulated expression of COX-2 more than total
pomegranate tannins verifying that the rind
extract penetrated the skin and modulated COX-2
regulation in the viable epidermis [46]. Ben Saad
et al. showed that ellagic acid, gallic acid and
punicalagin A&B obtained from P. granatum
potentially inhibited LPS-induced NO, PGE-2
and IL-6 production and could be considered as
the compounds responsible for anti-inflammatory
potential of the plant [47].
Marichali et al. demonstrated that the leaves
extracts of Carum carvi L. (Umbelliferae) were
rich in total flavonoids and phenols [48]. Also,
Thies showed that the caraway possess tannins
[49]. Antibacterial activity of the essential oil
extracted from fruits of C. carvi was determined
against Gram-positive and Gram-negative
bacterial species and also it has been revealed
that aqueous extract of the plant can be used as a
potent antibacterial agent for human pathogens
[50-53]. In a study, it was found that caraway had
a high inhibitory effect against Aspergillus
species [54]. It has been revealed that C. carvi
aqueous extract inhibited the production of
inflammatory mediators such as iNOS, COX-2,
IL-6 and TNF-α. Moreover, C. carvi exhibited
anti-neuroinflammatory effects via regulation of

Rezghi M. et al.
32 Res J Pharmacogn 6(4): 27-35
NF-kappa B signaling [55].
The chemical analysis of oleo-gum resin water
extract of Boswellia dalzielii (Burseraceae)
demonstrated the presence of total phenols,
flavonoids, anthocyanins and tannins in the herb
[56]. Afsar et al. showed that the methanol
fraction of B. serrata leaves contained high
amounts of total phenolics, flavonoids and
tannins and exhibited strong in vitro anti-
inflammatory activity [57]. Methanol extract of
the resin of B. carterii exhibited marked anti-
inflammatory activity for their inhibitory effect
against 12-O-tetradecanoylphorbol-13-acetate
(TPA)-induced inflammation in mice [58]. In
addition, anti-inflammatory effect of B. elongate
Balf. F has been established as well [59].
Moreover, B. carterii oil has demonstrated
antimicrobial activity [60]. Adelakun et al.
investigated that methanol and aqueous extracts
of B. dalziellii stem bark exhibited antibacterial
and antifungal activity [61].
Quantitative assay in our research demonstrated
the presence of tannins in the prepared gel due to
the presence of plants with high content of
tannins in the gel. Tannins have astringent
properties, resulting in faster healing of wounds
and inflamed mucous membranes [62]. Also,
tannin-rich fruits have been proven to have anti-
inflammatory properties [63]. Moreover, the
antimicrobial activities of tannins are well
documented. Furthermore, it has been revealed
that the growth of many fungi, yeasts, bacteria,
and viruses was inhibited by tannins [64]. Due to
antimicrobial, antifungal and anti-inflammatory
effects of phytochemical components of the gel
ingredient, the formulated herbal gel might be
effective in abnormal vaginal discharge with
respect to its traditional use.
Acknowledgements
The project was granted by Traditional Medicine
and Materia Medica Research Center, Shahid
Beheshti University of Medical Sciences, Tehran,
Iran (grant no.: 173).
Author contributions
Homa Hajimehdipoor and Shirin Fahimi
supervised the project; Maedeh Rezghi and Sara
Zakerin performed the experimental parts;
Maedeh Rezghi prepared the manuscript.
Declaration of interest
The authors declare that there is no conflict of
interest. The authors alone are responsible for the
accuracy and integrity of the paper content.
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Abbreviations
ITM: Iranian traditional medicine; HMS: Herbal
Market Samples