Embodiments of a present disclosure relates to a herbal composition.
More particularly, the present disclosure relates to a synergistic herbal
formulation for treating vaginal infections and method thereof.
BACKGROUND OF THE INVENTION
[2] Female vaginal mucosa forms an ecosystem for microorganisms such as
bacteria, fungi, etc. The gram-positive rods usually called “vaginal normal flora”
is prevalently found in vaginal mucosa. Majority of these large gram-positive rods
belongs to Lactobacilli. They are capable of producing acids by metabolizing
substances such as glycogen in vaginal mucous epithelial cells so as to maintain
the vaginal acidity within a pH value ranging from 3.5 to 4.5, inhibit pathogens,
and resist infection. This is the most common symbiotic arrangement known in
humankind, as they play a very important role in the health of female genital tract.
The abnormal changes of the vaginal flora and acidity usually causes a series of
disorder including genital tract infections.
[3] Many factors may affect the vaginal pH apart from the bacterial flora,
this includes the physiological changes, environment, stress and hormonal
changes. These changes impact the vaginal secretion, consequently effecting the
intimate hygiene. The pH value of vaginal secretion would be elevated to 4.8,
even 5.4 or higher. Complaints such as vulvodynia, pruritus vulvae, algopareunia,
abnormal leucorrhea with fishy smell, etc., would be common in individuals. The
vaginal resistance of the patient against infections usually decreases and thereby
increases the risk of STD and HIV infections. The abnormal changes of vaginal
pH may also induce infections in urinary systems, and is especially harmful to the
health of pregnant women and fetus, including serious consequences such as
abortion, premature delivery, intrauterine growth retardation of fetus, etc.
[4] Among various vaginal infections, Candidal vaginitis, Bacterial
vaginosis (BV), Lactobacilliosis (LB) and Cytolytic vaginosis (CV) are four
3
common diseases. However, their pathogenesis are all related to the abnormality
of vaginal flora and vaginal acidity.
[5] Candidal vaginitis is commonly deemed as an endogenous infection and
usually relates to the overgrowth of monilia and the toxin produced thereby in
acidic microenvironment, which is formed by acids produced by lactobacilli in
vagina. The monilial hypha or spore could be found in the patient’s vaginal
secretion. The pH value of vaginal secretion usually is lower than 4.5. The clinical
symptoms are usually most serious before menstruation and alleviated during and
after menstruation.
[6] Various therapeutic methods include the administration of various antifungal agents or antibiotics such as ketoconazole, nystatin, etc. Due to widespread
infection and the general problem of recurrence, several treatments have been
used over the years. While the exploit of substances such as vaginal douches,
betadine, etc., is beneficial, their action, conversely is short-lived. Systemic
administration of azole and other drugs although effective, have adverse effects
and are indicated in pregnant women.
[7] Antifungal therapy is highly effective for individual symptomatic
attacks but does not prevent recurrences. In fact, maintenance therapy with an
efficacious anti-Candida drug lengthens the time to recurrence but does not
provide a long-term cure. These limitations necessitate a safer and more efficient
remedy in this field. Also, the predominant factors that has stimulated the search
for safer and more effective intimate hygiene products has been increasing.
Incidence of certain strains of Candida albicans becoming resistant to azole
antifungals are on rise. Recurrences may be caused by the other species of
Candida that are not equally susceptible to the usual first-line treatments. In vitro
studies have shown that imidazole antifungal agents such as miconazole and
clotrimazole, are not effective against the non-Candida albicans fungi. Candida
tropicalis and Candida glabrate are 10 times less sensitive to miconazole than is
Candida albicans. Topical boric acid has also been used in the treatment of vulvo-
4
vaginal candidiasis, although effective it can be systematically absorbed through
the vaginal mucosa.
[8] Therefore, there is a need for developing a wider variety of effective
formulations useful in maintaining natural hygiene and pH of vagina.
SUMMARY OF THE INVENTION
[9] In accordance with an embodiment of the present invention, a
synergistic herbal formulation for treating vaginal infections comprises of an
effective amount of plant extracts selected from the group consisting of
Azadirachta indica in the range of 0.25% to 2% (w/v), Ficus benghalensis in the
range of 0.10 - 1% (w/v), Ficus glomerata in the range of 0.10%-1% (w/v), Ficus
religiosa in the range of 0.10 - 1%(w/v), Thespesia populnea in the range of 0.10 -
1% (w/v), Ficus infectoria in the range of 0.10 - 1% (w/v) and Melaleuca
alternifolia in the range 0.02 - 1% (w/v).
[10] In accordance with an embodiment of the present invention, wherein
the synergistic herbal formulation comprises of a lactic acid from alpha hydroxy
acids in the range 0.2 - 1.00% (w/v).
[11] In accordance with an embodiment of the present invention, wherein
the synergistic herbal formulation comprises of a natural fatty acids of coconut oil
in the range of 16 – 33.00% (w/v).
[12] In accordance with an embodiment of the present invention, wherein
the synergistic herbal formulation comprises of a soap nut extract as a surfactant
in the range of 2.0 – 4.00% (w/v).
[13] In accordance with an embodiment of the present invention, wherein
the synergistic herbal formulation comprises of a glycerine in the range of 1.00%
(w/v).
5
[14] In accordance with an embodiment of the present invention, wherein
the synergistic herbal formulation comprises of a benzalkonium chloride in the
range of 0.10 – 0.20% (w/v).
[15] In accordance with an embodiment of the present invention, wherein
the synergistic herbal comprises of a soya protein in the range of 0.10 to 1.00%
(w/v) and a wheat protein in the range of 0.05 – 2.00% (w/v).
[16] In accordance with an embodiment of the present invention, wherein
the synergistic herbal formulation comprises of a sodium chloride in the range of
0.5 – 1.40% (w/v).
[17] In accordance with an embodiment of the present invention, wherein
the synergistic herbal formulation comprises of a potassium sorbate in the range
of 0.10 – 1% (w/v).
[18] In accordance with an embodiment of the present invention, wherein
the synergistic herbal formulation comprises of a fragrance in the range of 0.10 –
0.20%.
[19] In accordance with an embodiment of the present invention, wherein
the synergistic herbal formulation can be formulated as water soluble gels,
foaming solution, soft gel, solutions, aerosols, creams, ointments, capsules,
microcapsules, suppositories or tablets, preferably water soluble gels, capsules or
tablets.
[20] In accordance with an embodiment of the present invention, wherein
the synergistic herbal formulation is effective in treatment of Vulvo-vaginal
candidiasis.
[21] In accordance with an embodiment of the present invention, wherein
the synergistic herbal formulation maintains the vaginal secretion pH in the range
of 3.5 to 4.5.
6
[22] In accordance to an embodiment of the present invention, a method for
preparing a synergistic herbal formulation in a form of water-soluble gel, the
method comprises of: processing of a leaf water soluble extract of Azadirachta
indica in the range of 0.25% - 2% (w/v) with a dried extracts from a decoction of
the bark from Ficus benghalensis in the range of 0.10% - 1% (w/v), Ficus
glomerata in the range of 0.10%-1% (w/v), Ficus religiosa in the range of 0.10 -
1%(w/v), Thespesia populnea in the range of 0.10 - 1% (w/v), Ficus infectoria in
the range of 0.10 - 1% (w/v) and adding the mixture obtained from step a) to a
mixture of lactic acid from AHA complex in the range of 0.2% - 1.00% (w/v)
along with natural fatty acids of coconut oil in the range of 16% - 33.00% (w/v),
soapnut extract in the range of 2.0% - 4.00% (w/v), glycerine in the range of
1.00% (w/v), Melaleuca alternifolia in the range of 0.02 - 1% (w/v),
benzalkonium chloride in the range of 0.10 - 0.20 % (w/v), soya protein in the
range of 0.10 - 1 % (w/v), wheat protein in the range of 0.05 - 2% (w/v), sodium
chloride in the range of 0.5-1.40% (w/v), potassium sorbate in the range of 0.10 -
0.20% (w/v), fragrance in the range of 0.10 - 0.20%.
[23] To further clarify the advantages and features of the present invention, a
more particular description of the invention will follow by reference to specific
embodiments thereof, which are illustrated in the appended figures. It is to be
appreciated that these figures depict only typical embodiments of the invention
and are therefore not to be considered limiting in scope. The invention will be
described and explained with additional specificity and detail with the appended
figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[24] The disclosure will be described and explained with additional specificity
and detail with the accompanying figures in which:
[25] FIG. 1a is a bar graph illustrating the percentage ratio of symptoms of
pruritis among the patients of the clinical study in accordance with an
embodiment of the present disclosure.
7
[26] FIG. 1b is a bar graph illustrating mean score of symptoms of pruritis
among the patients of the clinical study in accordance with an embodiment of the
present disclosure.
[27] FIG. 2a is a bar graph illustrating percentage ratio of symptoms of
irritation among the patients of the clinical study in accordance with an
embodiment of the present disclosure.
[28] FIG. 2b is a bar graph illustrating mean score of symptoms of irritation
among the patients of the clinical study in accordance with an embodiment of the
present disclosure.
[29] FIG. 3a is a bar graph illustrating percentage ratio of symptoms of burning
sensation among the patients of the clinical study in accordance with an
embodiment of the present disclosure.
[30] FIG. 3b is a bar graph illustrating mean score of symptoms of burning
sensation among the patients of the clinical study in accordance with an
embodiment of the present disclosure.
[31] FIG. 4a is a bar graph illustrating percentage ratio of symptoms of
erythema among the patients of the clinical study in accordance with an
embodiment of the present disclosure.
[32] FIG. 4b is a bar graph illustrating mean score ratio of symptoms of
erythema among the patients of the clinical study in accordance with an
embodiment of the present disclosure.
[33] FIG. 5a is a bar graph illustrating percentage ratio of symptoms of oedema
among the patients of the clinical study in accordance with an embodiment of the
present disclosure.
[34] FIG. 5b is a bar graph illustrating percentage ratio of symptoms of oedema
among the patients of the clinical study in accordance with an embodiment of the
present disclosure.
8
[35] FIG. 6 is a bar graph illustrating percentage ratio of symptoms of
excoriation of vulva/vagina among the patients of the clinical study in accordance
with an embodiment of the present disclosure.
[36] FIG. 7 is a bar graph illustrating percentage ratio of vaginal discharge
observed among the patients of the clinical study in accordance with an
embodiment of the present disclosure.
[37] FIG. 8 is a bar graph illustrating mean vaginal pH observed among the
patients of the clinical study in accordance with an embodiment of the present
disclosure.
[38] FIG. 9 is a bar graph illustrating the percentage ratio of patients and
physician’s global assessment of the clinical study in accordance with an
embodiment of the present disclosure.
[39] Further, those skilled in the art will appreciate that elements in the figures
are illustrated for simplicity and may not have necessarily been drawn to scale.
Furthermore, in terms of the construction of the device, one or more components
of the device may have been represented in the figures by conventional symbols,
and the figures may show only those specific details that are pertinent to
understanding the embodiments of the present disclosure so as not to obscure the
figures with details that will be readily apparent to those skilled in the art having
the benefit of the description herein.
DETAILED DESCRIPTION
[40] For the purpose of promoting an understanding of the principles of the
disclosure, reference will now be made to the embodiment illustrated in the
figures and specific language will be used to describe them. It will nevertheless be
understood that no limitation of the scope of the disclosure is thereby intended.
Such alterations and further modifications in the illustrated system, and such
further applications of the principles of the disclosure as would normally occur to
9
those skilled in the art are to be construed as being within the scope of the present
disclosure.
[41] The terms "comprises", "comprising", or any other variations thereof,
are intended to cover a non-exclusive inclusion, such that a process or method that
comprises a list of steps does not include only those steps but may include other
steps not expressly listed or inherent to such a process or method. Similarly, one
or more components, compounds, and ingredients preceded by "comprises... a"
does not, without more constraints, preclude the existence of other components or
compounds or ingredients or additional components. Appearances of the phrase
"in an embodiment", "in another embodiment" and similar language throughout
this specification may, but not necessarily do, all refer to the same embodiment.
[42] Unless otherwise defined, all technical and scientific terms used herein
have the same meaning as commonly understood by those skilled in the art to
which this disclosure belongs. The system, methods, and examples provided
herein are only illustrative and not intended to be limiting.
[43] In the following specification and the claims, reference will be made to
a number of terms, which shall be defined to have the following meanings. The
singular forms “a”, “an”, and “the” include plural references unless the context
clearly dictates otherwise.
[44] According to an embodiment of the present invention, a synergistic
herbal formulation for treating vaginal infections comprises of an effective
amount of plant extracts selected from the group consisting of Azadirachta indica
in the range of 0.25% to 2% (w/v), Ficus benghalensis in the range of 0.10 - 1%
(w/v), Ficus glomerata in the range of 0.10%-1% (w/v), Ficus religiosa in the
range of 0.10 - 1%(w/v), Thespesia populnea in the range of 0.10 - 1% (w/v),
Ficus infectoria in the range of 0.10 - 1% (w/v) and Melaleuca alternifolia in the
range 0.02 - 1% (w/v).
10
[45] According to an embodiment of the present invention, wherein the
synergistic herbal formulation comprises of a lactic acid from alpha hydroxy acids
in the range 0.2 - 1.00% (w/v).
[46] According to an embodiment of the present invention, wherein the
synergistic herbal formulation comprises of a natural fatty acids of coconut oil in
the range of 16 – 33.00% (w/v).
[47] According to an embodiment of the present invention, wherein the
synergistic herbal formulation comprises of a soap nut extract as a surfactant in
the range of 2.0 – 4.00% (w/v).
[48] According to an embodiment of the present invention, wherein the
synergistic herbal formulation comprises of a glycerine in the range of 1.00%
(w/v).
[49] According to an embodiment of the present invention, wherein the
synergistic herbal formulation comprises of a benzalkonium chloride in the range
of 0.10 – 0.20% (w/v).
[50] According to an embodiment of the present invention, wherein the
synergistic herbal formulation comprises of a soya protein in the range of 0.10 to
1.00% (w/v) and a wheat protein in the range of 0.05 – 2.00% (w/v).
[51] According to an embodiment of the present invention, wherein the
synergistic herbal formulation comprises of a sodium chloride in the range of 0.5
– 1.40% (w/v).
[52] According to an embodiment of the present invention, wherein the
synergistic herbal formulation comprises of a potassium sorbate in the range of
0.10 – 1% (w/v).
11
[53] According to an embodiment of the present invention, wherein the
synergistic herbal formulation comprises of a fragrance in the range of 0.10 –
0.20%.
[54] According to an embodiment of the present invention, wherein the
synergistic herbal formulation can be formulated as water soluble gels, foaming
solution, soft gel, solutions, aerosols, creams, ointments, capsules, microcapsules,
suppositories or tablets, preferably water soluble gels, capsules or tablets.
[55] According to an embodiment of the present invention, wherein the
synergistic herbal formulation is effective in treatment of Vulvo-vaginal
candidiasis.
[56] According to an embodiment of the present invention, wherein the
synergistic herbal formulation maintains the vaginal secretion pH in the range of
3.5 to 4.5.
[57] According to an embodiment of the present invention, a method for
preparing a synergistic herbal formulation in a form of water-soluble gel, the
method comprises of: processing of a leaf water soluble extract of Azadirachta
indica in the range of 0.25% - 2% (w/v) with a dried extracts from a decoction of
the bark from Ficus benghalensis in the range of 0.10% - 1% (w/v), Ficus
glomerata in the range of 0.10%-1% (w/v), Ficus religiosa in the range of 0.10 -
1%(w/v), Thespesia populnea in the range of 0.10 - 1% (w/v), Ficus infectoria in
the range of 0.10 - 1% (w/v) and adding the mixture obtained from step a) to a
mixture of lactic acid from AHA complex in the range of 0.2% - 1.00% (w/v)
along with natural fatty acids of coconut oil in the range of 16% - 33.00% (w/v),
soapnut extract in the range of 2.0% - 4.00% (w/v), glycerine in the range of
1.00% (w/v), Melaleuca alternifolia in the range of 0.02 - 1% (w/v),
benzalkonium chloride in the range of 0.10 - 0.20 % (w/v), soya protein in the
range of 0.10 - 1 % (w/v), wheat protein in the range of 0.05 - 2% (w/v), sodium
chloride in the range of 0.5-1.40% (w/v), potassium sorbate in the range of 0.10 -
0.20% (w/v), fragrance in the range of 0.10 - 0.20%.
12
[58] Example Formulation
[59] The synergistic herbal formulation of the present disclosure is
preferably a vaginal water-soluble gel composition, characterized in that:
Azadirachta indica in the range of 0.25 - 2% (w/v),
Ficus benghalensis in the range of 0.10 - 1% (w/v),
Ficus glomerata in the range of 0.10%-1% (w/v),
Ficus religiosa in the range of 0.10 - 1%(w/v),
Thespesia populnea in the range of 0.10 - 1% (w/v),
Ficus infectoria in the range of 0.10 - 1% (w/v),
Melaleuca alternifolia in the range of 0.02 - 1% (w/v),
Lactic Acid from AHA complex in the range of 0.2 - 1.00% (w/v)
[60] Further the total amount of natural fatty acids of coconut oil in the
range of 16-33.00% (w/v), soapnut extract based surfactant in the range of 2.0-
4.00% (w/v), glycerine in the range of 1.00% (w/v), benzalkonium chloride in the
range of 0.10 - 0.20 % (w/v), soya protein in the range of 0.10 - 1 % (w/v), wheat
protein in the range of 0.05 - 2% (w/v), sodium chloride in the range of 0.5-1.40%
(w/v), potassium sorbate in the range of 0.10 -0.20% (w/v), fragrance in the range
of 0.10 - 0.20%, Aqua QS;
[61] The inactive auxiliary components are non-flowable, viscous, watersoluble gel matrix, wherein the gel matrix is preferably natural fatty acids of
coconut oil. The formulation optionally comprises one or more antibacterial
agents and/or bactericides, wherein when the said antibacterial agents and/or
bactericides are Azadirachta indica in the range of 0.25 - 2% (w/v), Melaleuca
alternifolia in the range of 0.02 - 1% (w/v), Benzalkonium Chloride in the range
of 0.10 - 0.20 % (w/v).
13
[62] Also, the synergistic herbal formulation further comprises one or more
phyto actives from Ficus benghalensis, Ficus glomerata, Ficus religiosa,
Thespesia populnea, and Ficus infectoria. The synergistic herbal formulation is
packaged in a sterilizing and sealing manner, preferably a single dose packaged in
a sterilizing and sealing manner, and does not contain any live bacterium, fungus
or other microorganism. The synergistic herbal formulation further comprises of
color of fruit in the form of anthocyanin and alpha hydroxy acids and the
formulation further comprises of fragrance of fruit and essential oil. The
synergistic herbal formulation may further comprises of natural soapnut extract
based surfactant in the range of 2.0- 4.00% and natural fatty acids of coconut oil
in the range of 16-33.00% (w/v).
[63] Example Method of Preparation of the Synergistic Herbal Formulation
[64] The method of preparation process of synergistic herbal formulation of
the present invention is preferably a vaginal water-soluble gel composition,
comprises of processing leaf water soluble extract of Azadirachta indica in the
range of 0.25 - 2% (w/v) with a dried extracts from the decoction of the bark from
Ficus benghalensis in the range of 0.10 - 1% (w/v), Ficus glomerata in the range
of 0.10%-1% (w/v), Ficus religiosa in the range of 0.10 - 1%(w/v), Thespesia
populnea in the range of 0.10 - 1% (w/v), Ficus infectoria in the range of 0.10 -
1% (w/v).
[65] Further the above obtained mixture is added to the mixture of lactic
acid from AHA complex in the range of 0.2 - 1.00% (w/v), along with the total
amount of natural fatty acids of coconut oil in the range of 16-33.00% (w/v),
soapnut extract based surfactant in the range of 2.0- 4.00% (w/v), glycerine in the
range of 1.00% (w/v), Melaleuca alternifolia in the range of 0.02 - 1% (w/v),
benzalkonium chloride in the range of 0.10 - 0.20 % (w/v), soya protein in the
range of 0.10 - 1 % (w/v), wheat protein in the range of 0.05 - 2% (w/v), sodium
chloride in the range of 0.5-1.40% (w/v), potassium sorbate in the range of 0.10 -
0.20% (w/v), fragrance in the range of 0.10 - 0.20%, Aqua QS.
14
[66] The above obtained synergistic herbal formulation can be transformed
into water soluble gels, foaming solution, softgel, solutions, aerosols, creams,
ointments, capsules, microcapsules, suppositories, or tablets, preferably water
soluble gels, capsules or tablets.
[67] Also, synergistic herbal formulation of the present disclosure in the
form of foaming solution creates neutral buffer when comes in contact of the
vaginal flora and does not require rinsing when used in 1-2 ml/output as foaming
liquid.
EXPERIMENTAL RESULTS
In-vitro Clinical Evaluation of the synergistic herbal formulation of the present
invention in Vulvovaginal candidiasis.
[68] Vulvovaginal candidiasis is a common disease affecting about one-third of
all women at least once their lifetime. The present line of treatment of
vulvovaginal candidiasis is expensive and associated with adverse effects. In view
of this an herbal-based formulation is developed as described in the present
disclosure that is safe and provides anti-Candida activity.
[69] Screening of several herbs of the synergistic herbal formulation of the
present invention revealed that the liquid formulation of synergistic the herbal
formulation of the present invention possess activity against Candida species
including the antibiotic resistant strains. The fruits of the plants used in the herbal
formulation were obtained and extracted with organic solvent to get liquid, which
was authenticated by physicochemical tests. This liquid was then tested against a
panel of organisms by in vitro assays to get the Minimum Inhibitory
Concentration (MIC) value.
[70] In vitro study, a comparison of the synergistic herbal formulation of the
present invention was made with the widely used tea tree oil, wherein the herbal
formulation of the present disclosure was found to be better in terms of anti-
15
Candida activity (as assessed by MIC values). The toxicity studies conducted in
laboratory animals also proved safety of the liquid.
[71] According to an embodiment of the present invention, a local formulation
in the form of foam was prepared using the synergistic herbal formulation of the
present disclosure and tested against Candida species. The synergistic herbal
formulation retained the activity of the herbs and appears to be useful as an antiinfective agent. The product is herbal based, safe and helps to reduce the risk of
drug resistance.
[72] The herbals forming part of the synergistic herbal formulation of the
present disclosure is used in diseases of genitourinary organs such as gonorrhoea
and other vaginal discharges of women. Vulvovaginal candidiasis is a mucosal
infection of genital and gastrointestinal tract caused by Candida species. An
estimated 75% of all women experience an episode of Candida vaginitis in their
lifetime and the symptomatic vaginal candidiasis is present with symptoms that
include itching, burning, soreness, an abnormal vaginal discharge, and
dyspareunia and signs that include vaginal and vulvar erythema and edema.
[73] Vaginal candidiasis is often associated with conditions such as diabetes
mellitus, antibiotic therapy, corticosteroid therapy and pregnancy, although in
many cases, there are no clear predisposing factors. Due to widespread infection
and the general problem of recurrence, several treatments have been used over the
years. While the exploit of substances such as vaginal douches, Betadine, etc., is
beneficial, their action, conversely, is short-lived. Systemic administration of
azole and other drugs although effective, have adverse effects and are not
indicated in pregnant women. These limitations necessitate a safer and more
efficient remedy in this field. Also, the predominant factor that has stimulated the
search for safer and more effective antifungal agents has been the increasing
incidence of systemic mycoses in immunologically compromised patients and the
unfortunate incidence of certain strains of Candida albicans becoming resistant to
azole antifungals. Therefore, there is a need for developing wider variety of
antifungal agents useful in the treatment of fungal diseases.
16
[74] An effective synergistic herbal formulation for Vulvovaginal candidiasis is
prepared as disclosed in the present invention and screened for anti-Candida
activity, wherein the herbal formulation exhibited synergistic activity against a
panel of species including the azole resistant strains of Candida. The activity
against resistant species was found to be significant and better than the reference
standard of Tea tree oil.
[75] The antifungal property of the synergistic herbal formulation of the present
disclosure has been identified and local formulation i.e., foam/liquid is being
developed to treat Vulvovaginal candidiasis. Also, the synergistic herbal
formulation is active against azole resistant strains of Candida and has advantage
over marketed synthetic drugs in present situation, where the resistance to
synthetic drug is increasing. Importantly, its safety is proven in the laboratory
animals and no adverse events known.
[76] Also, it may be appreciated that a synergistic effect exists wherever the
action of a combination of active ingredients are higher than the action of each of
the ingredients taken alone. As a result, the synergistic effective amount or an
effective amount of a synergistic composition or formulation is an amount that
exhibits greater effectiveness against treatment of Vulvo-vaginal candidiasis than
the sum of anti-fungal properties of the individual ingredients.
[77] Experiment 1
The synergistic action of the inventive herbal formulation of the present
disclosure is demonstrated by the following experiments. Preclinical studies of
Anti-Candida activity of the synergistic herbal formulation of the present
disclosure.
[78] The aim of these experimental studies was to evaluate the anti-Candida
potential of the synergistic herbal formulation of the present disclosure.
Bioactivity by agar-well diffusion method
17
[79] A stock solution of 20 mg/mL was prepared by dissolving of the
synergistic herbal formulation in methanol and water. Various dilutions were
further prepared to get a series of concentration. These samples were tested
against a panel of microorganisms, including Candida albicans in vivo (Ca),
Candida albicans 10231, Candida krusei (GO3 and GO6), Candida glabrate
(HO4 and HO5), Aspergillus fumigatus (Asp.), Microsporum gypseum (Mg),
Trichophyton mentagrophytes (Tm), Staphylococcus aureus 209P (Sa), and E.coli
by agar well diffusion assay.
[80] Table 5 below shows the qualitative bioassay of the synergistic herbal
formulation of the present disclosure. Key: - No zone of inhibition, numbers
indicate diameter of zone of inhibition in mm.
[81] The results reveals broad-spectrum activity of the synergistic herbal
formulation of the present disclosure and further experiments were conducted
against Candida species to determine its anti-Candida activity.
[82] Experiment 2
In vitro susceptibility testing by Macro broth dilution method
[83] MIC of the oil was calculated using the standard method of Clinical
Laboratory Standards Institute (CLSI) with some modifications so as to suit
18
herbal oil. In this method, series of dilutions of the liquid was subjected to test
cultures and incubated at specific conditions. The amount of growth in the tubes
containing the extract was compared visually with the amount of growth in the
growth control tubes. The MIC was defined as the lowest concentration showing
100% growth inhibition. Candida albicans in vivo- fluconazole sensitive,
Candida albicans 10231 fluconazole resistant, Candida krusei, (GO3-fluconazole
resistant and GO6 fluconazole sensitive) Candida glabrata (HO5 fluconazole
resistant and HO4 fluconazole sensitive), Aspergillus fumigatus, Microsporum
gypseum, Trichophyton mentagrophytes, Staphylococcus aureus and Escherichia
coli were used as test organisms. The liquid was serially diluted to obtain a
dilution range of 0.5 to 0.01% (vol/vol) of the liquid in the final tubes using
DMSO. In another set, liquid dilutions were prepared in saline with Tween 80
(0.001%) to determine the solubility of the Liquid. Both the solvent controls were
included in the assay. The standard anti-fungal agent Ketoconazole was used as a
positive control.
[84] Table 2 below illustrates MIC results of the synergistic herbal formulation
of the present disclosure.
Conc
(%)
Candida
albicans
Candida
Fr
albicans
10231
Candida
kruseiFr
G03
Candida
krusei Fs
G06
Conc
(%)
Candida
albicans
Candida
Fr
albicans
10231
Candida
Fr
krusei
G03
Candida
kruseiFs
G06
0.5 - - - - 0.5 - - - -
0.25 - - - - 0.25 - - - -
0.12 - - - - 0.12 vsg
vsg
+
+
+
- - -
0.06 vvsg - - - 0.06 - vsg
vsg
vsg
+
-
0.03 s
g - vsg - 0.03 - -
0.01 + - vsg vsg 0.01 - vsg
1%
DMSO + + + +
T80
+ +
MIC 0.12 <0.01 0.06 0.03 MIC 0.25 <0.01 0.12 0.03
19
[85] Key: Fr- fluconazole resistant, Fs- fluconazole sensitive, vvsg- very very
slight growth, vsg-very slight growth, sg- slight growth, + growth, - no growth.
[86] Initial experiments using DMSO and Tween 80 as solvents revealed better
results with DMSO as compared to Tween 80. Therefore, further experiments
were carried with MSO.
[87] Table 3 illustrates MIC results of the synergistic herbal formulation of the
present disclosure against Candida species.
Candida
Fr
Candida
Fr
Conc (%)
Candida albicans Candida glabrata Aspergillus
glabrata Fs Candida Candida
albicans 10231 H04 H05 kruseiFrG03 krusei FsG06 fumigatus
0.5 - - - - - - -
0.25 - - - - - - -
0.12 - - - - - - -
0.06 sg - - - - - -
0.03 sg - + - vsg - -
0.01 + - + - vsg vsg -
0.005 + + + vsg vsg vsg -
MIC 0.12 0.01 0.06 0.01 0.06 0.03 <0.005
20
[88] Key: Fs- fluconazole sensitive, Fr- fluconazole resistant, vvsg- very very
sight growth, vsg- very slight growth, sg- slight growth, + growth, - no growth.
[89] Table 4 below illustrates the MIC results of the synergistic herbal
formulation of the present disclosure against Bacterial and Dermatophyte species.
[90] Key: vvsg- very very slight growth, vsg- very slight growth, sg- slight
growth, + growth, - no growth.
[91] Table 5 below illustrates the MIC results of standard ketoconazole.
21
[92] Key: Fs- fluconazole sensitive, Fr- fluconazole resistant, sg- slight growth,
+ growth, - no Growth.
[93] The results show that the liquid is active against all the strains of Candida
species tested. The liquid exhibits good activity against the azole resistant strains
of Candida as well.
[94] Table 6 below illustrates the compiled MIC results of all the batches of the
synergistic herbal formulation of the present disclosure.
[95] Key: Fr-Fluconazole resistant strain, Fs-Fluconazole sensitive strain, NDNot done.
[96] The above table shows the compiled values of MICs of 4 batches of herbal
formulation of the present disclosure against various Candida species. The MIC
value against Candida albicans is 0.12 or 0.25% (which is one log difference)
therefore the oil shows consistent activity.
22
[97] All the batches of the synergistic herbal formulation was tested in
triplicates (n=3) and showed consistent activity. The liquid synergistic herbal
formulation is active against azole resistant strains of Candida with good MIC
values. Therefore, the synergistic herbal formulation has potential to be developed
as anti-Candida formulation.
[98] Comparison of activity with marketed sample
[99] The liquid was compared with Tea tree oil (which is a known herbal oil
with anti-infective property) for its MIC value. The assay was done against
sensitive and resistant strains of Candida, Aspergillus and dermatophytes.
[100] Table 7a below illustrates MIC Values of the synergistic herbal
formulation of the present invention against Candida species.
[101] Key: vsg-very sight growth, sg-slight growth, + growth, - no growth, FrFluconazole resistant strain, Fs-Fluconazole sensitive strain.
[102] Table 7b below illustrates MIC values of Tea tree oil against Candida
species
23
[103] Key: vsg-very slight growth, sg-slight growth, + growth, - no growth, FrFluconazole resistant strain, Fs-Fluconazole sensitive strain.
[104] Table 7c below illustrates activity of the synergistic herbal formulation of
the present invention and Tea tree oil against Dermatophytes MIC results.
[105] Key: vvsg-very very slight growth vsg-very slight growth, sg-slight
growth, + growti growth, Fr-Fluconazole resistant strain, Fs-Fluconazole sensitive
strain no.
[106] Table 8 illustrates below the compiled values of MIC of the synergistic
herbal formulation of the present disclosure, Tea tree oil and Ketoconazole.
MIC % (v/v) MIC (mcg/ml) MIC (mcg/ml)
Organisms Herbal Formulation
0.12
Tea Tree oil Herbal
Formulation
Tea Tree oil
2260
Ketoconazole
C.albicans 0.25 1130 0.5
C.albicans 10231 0.01 0.25
0.06
94
282
2260 >8
C.krusei G06 0.03 564 8
C.krusei G03 0.06 0.06 564 564 >8
C.glabrata H04 0.12 0.03 1130 282 4
C.glabrata H05 0.01 0.25 94 2260 >8
A.fumigatus <0.005 0.06 <47 564 1
Key: Fr-Fluconazole resistant strain, Fs-Fluconazole sensitive strain
Herbal Formulation
24
200
50
control
MIC (mg/ml) 50
10
25 9 vsc
-
100 -
-
conc (mg/ml) Candida
albicans
[107] Remarks: From the compiled values it is evident that the synergistic herbal
formulation of the present disclosure is better than Tea tree oil in terms of activity
against Candida species, especially against the azole resistant strains.
[108] Experiment 3
[109] Anti-Candida activity of the synergistic herbal formulation of the
present invention.
[110] The aim of this study was to evaluate the anti-Candida activity of V-tablet
prepared using the herbal formulation of the present disclosure extract by Agar
dilution method (in vitro assay) (7, 28). The tablet was dissolved in saline and
calculated amount was added to Sabourauds medium with 1.2% agar. A growth
control plate without any extract or formulation was included in the study. The
assay was conducted thrice. The MIC was defined as the lowest concentration of
extract or formulation giving no visible growth or causing almost complete
inhibition of growth in the plates.
[111] Table 9 illustrates below the anti-Candida activity of V-tablet
[112] Key: - no growth, vsc very small colonies, numbers indicate, number of
colonies at respective concentration.
25
[113] Remarks: The V-tablet containing the synergistic herbal formulation of
the present disclosure is active against Candida albicans as seen from the results
and hence the formulation has retained the activity of the herbs.
[114] Experiment 4
[115] Acute toxicity of the synergistic herbal formulation of the present
disclosure in female Wistar rats.
[116] The synergistic herbal formulation of the present disclosure was
administered orally to female Wistar rats to study acute toxicity. Fasted rats were
weighed and administered 2000 mg/kg body weight of the herbal formulation of
the present disclosure. The rats were observed for any gross changes or mortality
on the day of administration as well as for 14 days post administration. On 14th
day their stomach were dissected and the mucosa was examined for presence of
inflammatory lesions. On post-mortem the stomach mucosa was found to be
normal and had no lesions whatsoever. Administration of the herbal formulation
of the present disclosure (single dose of 2000 mg/kg) led to only mild and
transient inflammatory symptoms on the day of administration in fasted female
Wistar rats. These symptoms subsided the next day and no other symptoms were
observed during the 14-day observation period. Based on the results it was
concluded that is safe.
[117] Experiment 5 In vivo studies
[118] Efficacy testing of plant extracts of the synergistic herbal formulation
of the present disclosure against Candida albicans in murine oral candidiasis
model.
[119] The objective of this study was to test the efficacy of the synergistic herbal
formulation and compare the same to that of Tea tree oil a standard herbal
antifungal product against Candida albicans in a murine oral candidiasis model. In
this model, mice were immunosuppressed with prednisolone7 and were given
tetracycline hydrochloride. They were orally infected with 10 viable cells of
Candida albicans by means of a cotton swab and enough chlorpromazine chloride
26
had been injected to keep them in a sedative state for about 3 hr after inoculation.
Three hours post infection the herbal formulation of the present disclosure and
Tea tree oil were applied orally to the respective group of 5-6 animals. From day 3
to day 7 post inoculation, 10-10 cfu of Candida were recovered from the oral
cavity and whitish, curd-like patches were observed on most parts of tongue of
infected mice that received no treatment. On day 4, herbal Liquid of the herbal
formulation of the present disclosure showed 0.5 log and 1 log reduction in fungal
cfu at 1000 and 2000mg/kg, respectively. However, Tea tree oil showed 1 log, 1.5
log and 1.5 log reduction in cfu at 500, 1000 and 2000mg/kg dose, respectively.
[120] Conclusion: Herbal oil of the synergistic herbal formulation showed
comparable efficacy against candidiasis compared to Tea tree oil.
[121] Based on the above, the in vitro studies conducted in house showed the
MIC range of liquid to be 1.13 to 2.26 mg/ml. Thus, the effective concentration of
liquid according to 100 times the MIC value will be between 113 to 226 mg.
Therefore, a dose of 100 mg, twice daily should ideally be enough to show the
efficacy.
[122] Clinical Trial Results of the Synergistic Herbal Formulation of the
present disclosure
[123] Experiment 6
Efficacy and safety of the synergistic herbal formulation of the present disclosure
in the treatment of Vulvo-Vaginal Candidiasis.
Method and Materials
[124] The present study was an open label study to the clinical (elimination of
signs and symptoms) and mycological effectiveness (negative mycological
studies) of the herbal formulation of the present disclosure in the management of
vulvo-vaginal candidiasis (VVC).
27
[125] Females of age 18 & above diagnosed with vulvo vaginal candidiasis on
mycological culture were included. Patients who have taken systemic or vaginal
antibiotics, probiotics, systemic or vaginal anti-fungal agents or systemic
corticosteroids in previous 30 days, patients with history of HIV, diabetes
mellitus, immunosuppressive disorders & pregnant ladies were excluded.
[126] Primary end point was a negative mycological swab after treatment.
Cotton tipped swabs were used to collect specimens from lateral wall of vagina of
each woman. This was used to determine the presence of yeast by direct wet
mount microscopy using a drop of 10% potassium hydroxide solution.
Mycological swab was done on before enrolment & after the end of the study (day
10) to assess the effectiveness of the herbal formulation of the present disclosure.
[127] Secondary end points were changes in vaginal pH & VVC questionnaire.
VVC questionnaire was assessed at every follow-up visit after enrolment. The
questionnaire evaluated signs & symptoms including itching, burning, irritation,
edema, erythema and/or excoriation of the vagina/vulva. Each evaluated sign
and/or symptom was given a numerical rating based on severity (absent = 0; mild
= 1; moderate = 2; severe = 3; very severe=4). The patient had a minimum
composite signs/symptoms score equal to 2. The evaluable patients had clinical
evidence of disease of at least moderate severity at entry, defined as having a
minimal composite score of 7. Severe disease was defined as a minimal composite
sign/symptom score of 13.
[128] A total of 22 women were screened, of whom 20 were found eligible for
enrolment. The total study period was 10 days. Patients were administered with
the herbal formulation of the present disclosure for 10 consecutive nights. The
herbal actives of the present disclosure would release slowly and was directly
available to act against the organisms. Follow up was done on days 3, 7, 10 after
enrolment.
28
[129] Table No: 10
No. of
Screenings
No. of
enrolments
Drop-outs Adverse
Events
Complete
cases
22 20 None 2 20
[130] Patients were not allowed to take any other medicines for the disease
under consideration during the course of the trial. Patients who were required to
take medicines for any other complaints during the course of the consumed
medicines only after consultation with the Investigator and record of it was
maintained in the Case Report Form. One patient suffered from itching all over
body, investigator administered injection Pheniramine maleate of 2 ml I.V.
followed by tablet Pheniramine maleate 45.3 mg three times a day for 2 Days.
CBC, SGPT, Sr. Creatnine were carried out in order to assess the safety of the
patient at the time of enrolment & after treatment.
[131] Trial was carried out according to the protocol design & ICH-GCP
Guidelines. Investigators followed the procedure as mentioned in the protocol at
each stage of the study i.e., taking proper patient consent, screening assessment,
recruitment of only those patients who fulfil all the criteria, follow-up of patients
on their scheduled visit, dispensing of medication, assessment of all the
parameters at all visit, maintaining the investigational reports of all the patients,
maintaining drug accountability record, proper filling-up of case report forms
(CRF) with black ball point pen, giving proper instructions to the patients about
use of medication & their scheduled visits.
[132] Monitoring Subject Compliance
[133] The patients were dispensed with 10 day’s drug supply on enrolment. The
compliance was checked by counting the number of unused pessaries and was
recorded in the CRF.
29
[134] Data recorded on CRF
[135] All data were recorded at the time of examining the patient and were
maintained as source data. The source data includes the patient’s name, address,
Tel No, vital sign examination values, safety parameters laboratory values,
clinical examination, medical history, medication consumption. For all laboratory
examination, values were recorded in the CRF and the Investigators signed the lab
reports as soon as they were received from the lab. These lab reports were
maintained as the source data for verification by the monitors. Although, the
detailed study conduct is described above, the study flow as per visits in brief is
mentioned hereinafter.
[136] Screening Visit
[137] During the first/screening visit to the site, the physician evaluated if the
patient fulfilled the criteria to participate in the study. After patient signed
informed consent form, investigator performed detailed clinical, physical &
systemic examination Investigator recorded the disease history, present signs and
symptoms and treatment taken in past. Blood sample was collected for CBC,
SGPT, Sr.Creatnine. Vaginal swab was taken for mycological examination & pH.
Based on these examination, physician informed the patients about their eligibility
for the study. In case patient was not eligible to enter into study physician
informed the patient and provide the patient with suggestive recommendations.
[138] Baseline Visit (day 0)
[139] The day patient was included in the study; physician performed detailed
clinical, physical & systemic examination and the assessed laboratory
investigations. Signs & symptoms of VVC were assessed from the VVC
questionnaire. The patient was dispensed with the study medication for 10 days.
Patient was recommended to insert 1 pessery at night daily. Patient was also
informed about the date and time of the next visits.
[140] 1
st Follow-up (day 3)
30
[141] Physician performed detailed clinical, physical & systemic examination
along with assessment of VVC questionnaire. The history of disease in past 3 days
was noted. Patient was also informed about the date and time of the next visit.
[142] 2
nd Follow-up (day 7)
[143] Physician performed detailed clinical, physical and systemic examination
along with assessment of VVC questionnaire. The history of Disease in past 4
days was noted. Test request form for safety lab investigations of CBC, SGPT,
Sr.Creatnine was given to the patient for 3rd follow-up visit.
[144] 3
rd Follow-up (day 10)
[145] Physician performed detailed physical and systemic examinations along
with assessment of VVC questionnaire. Assessment for lab investigations was
done. The history of disease in past 3 days was noted. Vaginal swab for
mycological examination & pH was done. The past medication was measured to
check the compliance and the empty container of the study medication was taken
back. Physician’s & patients global assessment of therapy was done during this
visit.
[146] Recording Adverse Drug Reactions
[147] Adverse Events (AE), Serious Adverse Events (SAE) and Adverse Drug
Reactions (ADR) if any were recorded at every visit. Adverse events were
recorded in two patients. One patient with itching all over the body. Another
patient had giddiness, vomiting & tremors. Both adverse events were unrelated to
the study drug.
[148] Statistical analysis
[149] Data were analyzed using SPSS/PC+10.0 statistical package. Descriptive
statistics were given as Mean ± SD with n as the number of observations.
Student’s paired t tests were applied t compare basal & final values. Friedman
(non-parametric 2 ways ANOVA) tests were applied to detect overall change
31
during treatment considering all time points. Descriptive Statistics were calculated
on to Per Protocol Population (completed patients). All the tests were two tailed.
Level of significance was taken as P=0.05.
[150] Abbreviations: SD=Standard Deviation, S=Significant, NS= Not
Significant, DF= Degree of Freedom, P=Actual Probability value, Sign.
=Significance, NA=Not Applicable, n = number of observations, MISS=missing.
[151] Results
[152] All 20 patients who were recruited in the study completed the full course
of treatment. The average age was 34.10 + 5.47 (minimum 23, maximum 45). All
vital signs and symptoms were normal before treatment and there were no
significant changes in them after treatment.
[153] Table 11: Vital signs & physical examination (Mean ± SD)
Variable Baseline Day 3 Day 7 Day 10
Pulse Rate
(Per minute)
77.95 ± 5.54 77.55 ± 3.85 77.80 ± 4.06 77..20 ± 5.16
Systolic BP
(mm/Hg)
123.60 ± 10.77 124.40 ± 8.17 125.20 ± 6.14 123.90 ± 9.66
Diastolic BP
(mm/Hg)
82.90 ±7.44 83.40 ± 6.90 83.80 ± 6.99 83.30 ± 7.43
Temperature (ºC) 36.93 ± 0.41 37.04 ± 0.16 36.87 ± 0.37 36.77 ± 0.35
Resp. Rate 20.60 ± 2.06 20.65 ± 1.79 20.80 ± 1.77 20.50 ± 1.36
Weight (kg) 61.10 ± 6.39 61.20 ± 6.34 61.20 ± 6.34 61.20 ± 6.33
[154] In the efficacy assessment, mycological culture test was positive in all the
patients before treatment and mycological cure was found in 17 patients (85%)
after 10 days treatment whereas only 3 patients (15%) were remained positive for
mycological culture. Clinically significant improvement in signs and symptoms of
VVC were found in majority of the patients after 10 days treatment. All the
domains of VVC questionnaire on signs and symptoms were alleviated
32
significantly (p < 0.001) (Table 12 & 13, Fig 2 - 9). The individual symptom
results were as follows:
[155] Pruritus: 13(65%) patients had complete relief of symptom, 6(30%)
patients had mild symptom and 1(05%) patient had moderate symptom.
[156] Burning: 16(80%) patients had complete relief of symptom, 3(15%)
patients had mild symptom and 01(05%) patient had moderate symptom.
[157] Irritation: 16(80%) patients had complete relief of symptom and 4(20%)
patients had mild symptom.
[158] Oedema: 19(95%) patients had complete relief of symptom and 01(05%)
patient had mild symptom.
[159] Erythema: 18(90%) patients had complete relief of symptom and 2(10%)
patients had mild symptom.
[160] FIG. 4a is a bar graph illustrating percentage ratio of symptoms of
erythema among the patients of the clinical study in accordance with an
embodiment of the present disclosure. FIG. 4b is a bar graph illustrating mean
score ratio of symptoms of erythema among the patients of the clinical study in
accordance with an embodiment of the present disclosure. FIG. 5a is a bar graph
illustrating percentage ratio of symptoms of oedema among the patients of the
clinical study in accordance with an embodiment of the present disclosure. FIG.
5b is a bar graph illustrating percentage ratio of symptoms of oedema among the
patients of the clinical study in accordance with an embodiment of the present
disclosure. FIG. 6 is a bar graph illustrating percentage ratio of symptoms of
excoriation of vulva/vagina among the patients of the clinical study in accordance
with an embodiment of the present disclosure. FIG. 7 is a bar graph illustrating
percentage ratio of vaginal discharge observed among the patients of the clinical
study in accordance with an embodiment of the present disclosure.
33
[161] In laboratory investigations there were significant changes in
haemoglobin, pH, eosinophils, basophils, neutrophils, monocytes and in serum
Creatinine but the levels were within the normal range (Table 14).
[162] One patient had itching all over the body and was given appropriate
treatment for 3 days. Another patient had giddiness, tremors of thigh, vomiting,
dysuria for 2 days, but no medication given. The patient recovered within 2 days.
According to the investigator the relationship of these adverse events with study
drug were unlikely.
[163] In Global assessment about the efficacy of the test drug about 35% felt
it was excellent, 40% said it was good and only 25% said it was fair. None of the
patients said it was poor. Similarly, the physician opinion was same as those of
patients’. (Table 15, Fig 13). About tolerability both patients and physician said it
was good.
Table 12: Signs and Symptoms (Percentage of patients)
[164] Scoring: 0=Absent, 1= Present
Variable Baseline Day 3 Day 7 Day 10
Pruritus 0=0(0%),
1=20(100%)
0=0(0%),
1=20(100%)
0=6(30%),
1=14(70%)
0=16(80%),
1=4(20%)
Irritation 0=0(0%),
1=20(100%)
0=1(5%),
1=19(95%)
0=7(35%),
1=13(65%)
0=17(85%),
1=3(15%)
Burning 0=0(0%),
1=20(100%)
0=1(5%),
1=19(95%)
0=9(45%),
1=11(55%)
0=18(90%),
1=2(10%)
Erythema 0=2(10%),
1=18(90%)
0=5(25%),
1=15(75%)
0=14(70%),
1=6(30%)
0=18(90%),
1=2(10%)
Excoriati
on of
vulva/va
gina
0=10(50%),
1=10(50%)
0=15(75%),
1=5(25%)
0=17(85%),
1=3(15%)
0=19(95%),
1=1(5%)
Oedema 0=12(60%),
1=8(40%)
0=17(85%),
1=3(15%)
0=17(85%),
1=3(15%)
0=19(95%),
1=1(5%)
Vaginal discharge 0=0(0%),
1=20(100%)
0=1(5%),
1=19(95%)
0=2(10%),
1=18(90%)
0=9(45%),
1=11(55%)
Table 13: Signs and Symptoms (Actual Values) (Mean + S.D.)
34
[165] Scoring: None=0, Mild=1, Moderate=2, Severe=3, Very Severe=4
Variable Baseline Day 3 Day 7 Day 10
Friedman’s
Chi sq. value,
DF,Sign., P
value
Pruritus 1.95 ± 0.76 1.95 ± 0.76 0.85 ± 0.99 0..40 ± 0.60 Chi sq. = 52.3, DF=3,
S, P<0.001
Burning 1.75 ± 0.72 1.35 ± 0.93 0.75 ± 0.72 0..25 ± 0.55 Chi sq. = 47.1, DF=3,
S, P<0.001
Irritation 1.70 ± 0.80 1.20 ± 0.77 0.70 ± 0.57 0..20 ± 0.41 Chi sq. = 42.4, DF=3,
S, P<0.001
Oedema 0.45 ± 0.51 0.21 ± 0.41 0.10 ± 0.31 0..05 ± 0.22 Chi sq. = 17.5, DF=3,
S, P<0.001
Erythema 1.15 ± 0.59 0.55 ± 0.51 0.30 ± 0.47 0..10 ± 0.31 Chi sq. = 36.5, DF=3,
S, P<0.001
DF – Degrees of Freedom, S- Significant
[166] Table 14: Laboratory Investigations. Mean ± SD or Number
(Percentage)
Variable Before After t test, Sign., P value
Hb g/dl 9.40 ± 1.02 9.55 ± 1.01 t = 2.6, S, P=0.02
PH 3.55 ± 0.51 5.55 ± 1.05 t =9.2, S, P<0.001
Total RBC 6550000 ± 1036441 6495000 ± 960523 t = 0.9, NS, P=0.4
Total WBC 6605.0 ± 518.6 6575.0 ± 600.0 t = 0.5, NS, P=0.6
Eosinophil 6.35 ± 2.74 4.70 ± 2.20 t = 5.1, S, P<0.001
Basophil 0.00 ± 0.00 0.70 ± 0.73 t = 4.3, S, P<0.001
Neutrophil 47.85 ± 4.60 49.10 ± 5.06 t =2.3, S, P=0.04
Monocytes 0.00 ± 0.00 0.55 ± 0.60 t = 4.1, S, P<0.001
Lymphocytes 45.80 ± 3.62 44.90 ± 4.79 t = 1.6, NS, P=0.1
Serum
creatinine
0.75 ± 0.10 0.67 ± 0.12 t = 3.8, S, P=0.001
35
SGPT 28.70 ± 7.29 29.50 ± 6.60 t = 1.6, NS, P=0.1
pH 3.55 ± 0.51 05.55 ± 1.05 t = 9.2, S, P<0.001
S-Significant, NS – Not Significant
[167] FIG. 8 is a bar graph illustrating mean vaginal pH observed among the
patients of the clinical study in accordance with an embodiment of the present
disclosure.
Table 15: Patient’s and Physician’s Global Assessment [Number (Percentage)]
[168] Recovery Score 1=Excellent, 2= Good, 3= Fair, 4=Poor, 5=Very Poor
Recovery Score Patient’s Global
Assessment
Physician’s Global
Assessment
Excellent =1 7 (35.0 %) 7 (35.0 %)
Good = 2 8 (40.0 %) 8 (40.0 %)
Fair =3 5 (25.0 %) 5 (25.0 %)
Poor = 4 0 (0.0 %) 0 (0.0 %)
Very Poor = 5 0 (0.0 %) 0 (0.0 %)
Total 25 (100 %) 25 (100 %)
[169] FIG. 9 is a bar graph illustrating the percentage ratio of patients and
physician’s global assessment of the clinical study in accordance with an
embodiment of the present disclosure.
[170] Discussion
[171] Yeast was isolated from the vaginal swab of all the 20 females
screened. Vaginal swab of 17 (85%) out of 20 enrolled was found to be negative
after treatment with the herbal formulation of the present invention. This finding
is consistent with of miconazole or any imidazole therapy which has a success
rate of 85.4%.
36
[172] There was a complete remission of the infection in 85% of the patients
in 10 days. The mycological culture test was negative after treatment with the
herbal formulation of the present invention. However, the Positive culture may
not be considered as a parameter for confirming VVC because, Candida is
omnipresent. Clinical signs and symptoms are more reliable indications to assess
the efficacy of the drug. Signs and symptoms are assessed as per the VVC
questionnaire. All the symptoms which were present before had completely
disappeared after treatment (p < 0.001).
[173] The herbal extracts were found to exhibit in vitro antioxidant and
antimicrobial activities which could be the possible mechanisms for reduction in
local vaginal symptoms of burning, irritation, and excoriation. There was a
significant improvement in the local vaginal symptoms such as erythema, oedema,
burning. This may be due to the anti-inflammatory properties of piper cubeba as
observed with other studies.
[174] It was found that the synergistic herbal formulation of the present
disclosure was as effective as anti-fungal medicine in improving the symptoms
associated with vulvo-vaginal candidiasis, though it was slow in action.
[175] This clinical study describes 20 cases of persistent vaginal candidiasis,
unresponsive to azole drug treatment. From the enrolled patients, 8 patients had a
history of chronic recurrent vaginal candidiasis which was untreated with
antibiotics have resolved with the herbal formulation of the present invention,
showing the efficacy of the herbal formulation in cases of resistant infections.
[176] Adverse events were reported in 10% patients. One patient had itching
all over the body for 3 days which resolved with symptomatic therapy. Patient
gave history of fish consumption prior to the itching which may be the cause of
allergic reaction. One patient had a history of giddiness, tremors, vomiting after
37
consumption of the study drug which resolved without any symptomatic therapy
but patient reported of improvement in vaginal discharge.
[177] One patient with H/O controlled Diabetes Mellitus also reported an
improvement in the symptoms of vaginal discharge with the study drug. This
shows the efficacy of the herbal formulation of the present disclosure in diabetic
patients, although further studies in this regard may be required to confirm its
effect.
[178] The present clinical study has shown that the treatment with the
synergistic herbal formulation of the present invention has effectively controlled
the signs and symptoms in patients with Vulvo-vaginal candidiasis. Mild adverse
event were noted which unlikely have relationship with study medication. So, it
may be concluded that the synergistic herbal vaginal formulation of the present
disclosure is effective and safe in the treatment of Vulvo-vaginal Candidiasis.
[179] Overall, it may be appreciated that the synergistic herbal formulation of
the present disclosure aides in maintaining natural hygiene and pH of vagina.
Further the synergistic herbal formulation along with alpha hydroxy acids and the
natural surfactants to of the present disclosure can be formulated in various forms
such as creams, foam, liquid, a deodorizing agent, a cosmetic, a disinfectant
composition, or herbal/natural composition, etc., to treat and prevent unwanted
microbial growth against resistant organisms and to maintain the pH of vagina.
The synergistic herbal formulation of the present disclosure along with the herbal
oils would be released slowly and directly available to act against the organisms.
Also, the synergistic herbal formulation is active against azole resistant strains of
Candida and has an advantage over other synthetic drugs used since many
patients exhibit resistance to synthetic drugs.
[180] Also, it may be appreciated that the surfactant and the herbal actives are
used in the proportions which forms the active ingredients of the synergistic
38
herbal formulation in the form of foam does not require rinsing. Since it is in the
balanced state that when it comes with contact with acidic to normal vaginal
mucosa, it gets neutralized and after maintaining the pH of the vaginal flora with
the range from 3.5 to 4.5. Further, the herbal formulation comprises of natural
color from fruit extracts in the form of alpha hydroxy acids, which resembles
anthocyanin pigment and also fragrance is generated with natural action of fruit
extracts and essential oils.
[181] More importantly, the synergistic herbal formulation of the present
invention provides relief from vaginal discharge and odor seen during vaginitis,
which is effective over the conventional method involving application of the
composition and cleansing the genital area. The synergistic herbal formulation of
the present disclosure provides a synergistic effect by blending the plant extracts,
essential oil and the auxiliary material in a specific range. The synergistic herbal
formulation can be stored in a dispensing bottle or bottle with foaming pump with
the herbal formulation of the present disclosure.
[182] It may be appreciated that the herbal formulation of the present
disclosure can also used for treatment of abnormal increase of vaginal acidity,
cytolytic vaginosis, lactobacillosis and candida vaginitis. The use of synergistic
herbal formulation of the present invention also promotes the angiogenesis of
vaginal mucosa, epithelial cornification of vaginal mucosa, and epithelial damage
healing in vagina, thereby further enhancing therapeutic effects of the composition
manufactured according to the present invention.
[183] Overall, the synergistic herbal formulation of the present disclosure
which can be formulated as a cleaning-hygiene agent comprises of plant extracts
of Azadirachta indica, Ficus benghalensis, Ficus glomerata, Ficus religiosa,
Thespesia populnea, Ficus infectoria, and Melaleuca alternifolia along with lactic
acid from AHA complex as active components and further comprising one or
more inactive auxiliary components suitable for human vagina, wherein the
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natural fatty acids of coconut oil, soapnut extract based surfactant, glycerine,
benzalkonium chloride, soya protein, wheat protein, sodium chloride, potassium
sorbate or mixtures thereof. This synergistic admixture of the present disclosure
exhibits excellent synergistic properties in treating Vulvo-vaginal Candidiasis
compared to their individual actions.
[184] Also, the synergistic herbal formulation according to the present
disclosure can be a cleaning-hygiene product, a deodorizing agent, a cosmetic, a
disinfectant or a herbal (non-prescription product or prescription products), the
dosage form of which includes but is not limited to water soluble gels, foaming
solution, softgel, solutions, aerosols, creams, ointments, capsules, microcapsules,
suppositories or tablets, preferably water soluble gels, capsules or tablets.
[185] While specific language has been used to describe the invention, any
limitations arising on account of the same are not intended. As would be apparent
to a person skilled in the art, various working modifications may be made to the
method in order to implement the inventive concept as taught herein.
[186] The figures and the foregoing description give examples of
embodiments. Those skilled in the art will appreciate that one or more of the
described elements may well be combined into a single functional element.
Alternatively, certain elements may be split into multiple functional elements.
Elements from one embodiment may be added to another embodiment. For
example, order of processes described herein may be changed and are not limited
to the manner described herein. Moreover, the actions of any flow diagram need
not be implemented in the order shown; nor do all of the acts need to be
necessarily performed. Also, those acts that are not dependent on other acts may
be performed in parallel with the other acts. The scope of embodiments is by no
means limited by these specific examples.

WE CLAIM:
1. A synergistic herbal formulation for treating vaginal infections, comprising
an effective amount of plant extracts selected from the group consisting of
Azadirachta indica in the range of 0.25% to 2% (w/v), Ficus benghalensis
in the range of 0.10 - 1% (w/v), Ficus glomerata in the range of 0.10%-1%
(w/v), Ficus religiosa in the range of 0.10 - 1%(w/v), Thespesia populnea in
the range of 0.10 - 1% (w/v), Ficus infectoria in the range of 0.10 - 1%
(w/v) and Melaleuca alternifolia in the range 0.02 - 1% (w/v).
2. The synergistic herbal formulation as claimed in claim 1, wherein the herbal
formulation comprises of a lactic acid from alpha hydroxy acids in the range
0.2 - 1.00% (w/v).
3. The synergistic herbal formulation as claimed in claim 1, wherein the herbal
formulation comprises of a natural fatty acids of coconut oil in the range of
16 – 33.00% (w/v).
4. The synergistic herbal formulation as claimed in claim 1, wherein the herbal
formulation comprises of a soap nut extract as a surfactant in the range of
2.0 – 4.00% (w/v).
5. The synergistic herbal formulation as claimed in claim 1, wherein the herbal
formulation comprises of a glycerine in the range of 1.00% (w/v).
6. The synergistic herbal formulation as claimed in claim 1, wherein the herbal
formulation comprises of a benzalkonium chloride in the range of 0.10 –
0.20% (w/v).
7. The synergistic herbal formulation as claimed in claim 1, wherein the herbal
comprises of a soya protein in the range of 0.10 to 1.00% (w/v) and a wheat
protein in the range of 0.05 – 2.00% (w/v).
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8. The synergistic herbal formulation as claimed in claim 1, wherein the herbal
formulation comprises of a sodium chloride in the range of 0.5 – 1.40%
(w/v).
9. The synergistic herbal formulation as claimed in claim 1, wherein the herbal
formulation comprises of a potassium sorbate in the range of 0.10 – 1%
(w/v).
10. The synergistic herbal formulation s claimed in claim 1, wherein the herbal
formulation comprises of a fragrance in the range of 0.10 – 0.20%.
11. The synergistic herbal formulation as claimed in claim 1, wherein the herbal
formulation can be formulated as water soluble gels, foaming solution, soft
gel, solutions, aerosols, creams, ointments, capsules, microcapsules,
suppositories or tablets, preferably water soluble gels, capsules or tablets.
12. The synergistic herbal formulation as claimed in claim 1, wherein the herbal
formulation is effective in treatment of Vulvo-vaginal candidiasis.
13. The synergistic herbal formulation as claimed in claim 1, wherein the herbal
formulation maintains the vaginal secretion pH in the range of 3.5 to 4.5.
14. A method for preparing a synergistic herbal formulation in a form of watersoluble gel, the method comprises of:
processing of a leaf water soluble extract of Azadirachta indica in
the range of 0.25% - 2% (w/v) with a dried extracts from a decoction of
bark obtained from Ficus benghalensis in the range of 0.10% - 1% (w/v),
Ficus glomerata in the range of 0.10%-1% (w/v), Ficus religiosa in the
range of 0.10 - 1%(w/v), Thespesia populnea in the range of 0.10 - 1%
(w/v), Ficus infectoria in the range of 0.10 - 1% (w/v); and
adding the mixture obtained from step a) to a mixture of lactic acid
from alpha-hydroxy complex in the range of 0.2% - 1.00% (w/v) along with
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natural fatty acids of coconut oil in the range of 16% - 33.00% (w/v),
soapnut extract in the range of 2.0% - 4.00% (w/v), glycerine in the range of
1.00% (w/v), Melaleuca alternifolia in the range of 0.02 - 1% (w/v),
benzalkonium chloride in the range of 0.10 - 0.20 % (w/v), soya protein in
the range of 0.10 - 1 % (w/v), wheat protein in the range of 0.05 - 2% (w/v),
sodium chloride in the range of 0.5-1.40% (w/v), potassium sorbate in the
range of 0.10 -0.20% (w/v), fragrance in the range of 0.10 - 0.20%.