DESC:
A SYNERGISTIC HERBAL COMPOSITION FOR IMPROVING FOR SEXUAL WELLNESS IN FEMALES
FIELD OF THE INVENTION
The present invention describes an oral synergistic herbal sexual wellness formulation for females. More specifically the present invention relates to a synergistic herbal formulation having the combination of herbs Tribulus terrestris and Trigonella foenum-graecum.
BACKGROUND OF THE INVENTION
The sexual response cycle in both men and women is a complex interplay between the autonomic nervous system, endocrine system, and circulatory system, that involves a plethora of biochemical factors like neurotransmitters, vasoactive agents and endocrine factors, etc. It is well described in the scientific and medical literature that subjects having low levels of sex hormones are more likely to be depressed than those with normal hormonal levels. They also suffer from complex outcomes like loss of libido, sexual arousal disorder, orgasmic disorder, sexual pain disorder, etc. A multitude of pathways operates in a well-orchestrated manner in every stage of the sexual responsive cycle namely desire (libido), excitation (arousal), orgasm and resolution. To address the various problems of the sexual response cycle, the availability of drugs is scarce. Phosphodiesterase (PDE5) inhibitors for erectile dysfunction (ED), ospemifene (estrogen receptor modulator) for dyspareunia, flibanserin (serotonin receptor modulator) for hypoactive sexual desire disorder (HSDD) are a few drugs available in the market to date. Further, there are several products in the marketplace to address virility and female sexual dysfunction (ED).
There are supplements available in the market, purported to alleviate human sexual problems. But they are marketed with little or no scientific evidence and rely on traditional belief and personal testimonials. Companies are focusing on investing in the discovery of new ingredients that fall primarily into 3 main functional categories: estrogen therapy (Ospemifene), testosterone boosters, and stimulators of nitric oxide (NO) levels. Several research has been done and documented as patents, out of which few are listed here.
US10149878B2 discloses a method of enhancing female libido by oral administration of an effective amount of fenugreek hydro alcoholic seed extract. The change in female libido has been measured by behavioural and/or biological factors.
EP3560488AI describes an invention comprising extracts of Trigonella foenum graecum, Tumera diffusa, and Gingko biloba at a weight ratio from 6:1:1 to 24:6:1. The invention provides a pharmaceutical composition comprising of the above herbal combinations together with pharmaceutically accepted excipients for the treatment of female sexual dysfunction caused by hormonal imbalance conditions such as pregnancy, postpartum, breastfeeding, hormonal contraception, perimenopause and post menopause. The lack of mainstream medication and various other reasons including side effects and ineffectiveness in certain populations has led to the growing popularity of Dietary supplements (DS) or Nutraceuticals for better management of sexual problems. Thus, the need for the present invention arises that can overcome the problems associated with the existing prior art.
OBJECTIVE OF THE INVENTION
The main objective of the present invention is to develop a synergistic herbal formulation that up-regulates sex hormones thereby improves sexual health and cellular function in females.
Yet another objective of the present invention is to provide a palatable hence ‘easy to consume’ beverage despite being an herbal composition.
Yet another objective of the present invention is to provide a scientifically validated and functional nutraceutical formulation.
Yet another objective of the present invention is to provide a synergistic herbal formulation that has an extended shelf life.
Yet another objective of the present invention is to provide a synergistic herbal formulation that improves libido and helps maintain sexual balance.
Further objectives, advantages, and features of the present invention will become apparent from the detailed description provided herein below, in which various embodiments of the disclosed invention are illustrated by way of example.

SUMMARY OF THE INVENTION
The present invention describes the synergistic effect produced by 2 herbal extracts in the context of female sexual health. The effectiveness of this unique combination is evaluated both in cell based mechanistic assays in vitro and in 40 healthy female volunteers. The results obtained from cell lines and Human volunteers have strongly proven the fact that the unique herbal formula comprising of this combination of herbs very effectively helps in improvement of overall sexual health of women when consumed on a daily basis. The herbal sexual wellness formulation comprising of an extract of Tribulus terrestris in the range of 0.01µg/ml to 500µg/ml and an extract of ingredient B in the range of 0.01µg/ml to 500µg/ml. Herein, aqueous extract of T.terrestris (henceforth mentioned as ingredient A) is an extract from the fruit of the plant but not limited to stem, leaf, inflorescence, or root extract and aqueous extract of T.f.graecum (henceforth mentioned as ingredient B) is an extract from the seed of the plant but not limited to fruit, leaf, stem, or inflorescence extract. Herein, the herbal extracts used in the study were aqueous extract but not limited to ethanol, methanol, acetone, ethyl acetate extracts and contains the bio-actives protodioscin, alkaloids, steroidal saponins, furostanol saponins, flavonoid glycosides, coumarins, alkaloids, and polyphenol compounds, and other bioactives. In an embodiment, the ingredient A in the herbal sexual wellness formulation is obtained from Tribulus terrestris but not limited to T.terrestris orientalis, T.terrestris var robustus, T.terrestris var brachyceras, T.terrestris var inermis, T. terrestris var sericeus, T. terrestris var rajasthanensis, T.terrestris var macrocarpus, T.terrestris var desertorum, T.terrestris var biocornutus and the ingredient B is obtained from Trigonella feonum-graecum but not limited to Trigonella caerulea. In the embodiment of the present invention, the herbal sexual wellness formulation has been evaluated in vitro using immortal cell lines, wherein the cell lines are selected from but not limited to mammalian, avian, Piscean, or arthropod origin. In another aspect of the invention, the cell type for the desired cell lines is HeLa cells but not limited to other cervical carcinoma cell lines. In an embodiment of the present invention, at least one pharmaceutically accepted excipient is selected from, but not limited to, coloring agents such as allura red and flavoring agents such as strawberry powder, acidity regulator such as sodium citrate and citric acid but not limited to acetic acid, malic acid, lactic acid, adipic acid, and other salts, the preservative is sodium benzoate but not limited to other similar preservatives, emulsifier/stabilizer/gelling agent/ thickener is xanthan gum but not limited to other similar agents, added in the range 0.05% to 0.5%. In an embodiment of the invention, the herbal sexual wellness formulation is an oral administration formulation selected from, but not limited to, a pill, tablet, capsule, concentrated syrup, food additive, suspension, emulsion, powdered drink mix, carbonated/non-carbonated beverage and a chewable solid. In an embodiment of the present invention, the in vitro evaluation of the herbal sexual wellness formulation using HeLa cell lines, wherein the HeLa cell lines in a specific combination of ingredient A and ingredient B in a concentration range of 0.1µg – 50 µg/ 8x103cells, show synergistic effects on NO-cGMP pathway and regulate the levels of estradiol, indicate involvement in relaxation of smooth muscles and influence on libido and sexual health in females. In another aspect of the invention, the specific combination of ingredient A and ingredient B provide synergistic effects by modulating nitric oxide levels, which in turn influence intracellular calcium levels and leads to phosphorylation/dephosphorylation of myosin light chain thereby influencing smooth muscle relaxation. In yet another aspect of the invention, the specific combination of ingredient A and ingredient B, wherein the ingredients when taken in a ratios falling into the range of 1:1 to 1:500, acts upon the smooth muscles found in corpus cavernosum but is not limited to the myometrium, vaginal muscularis, and other smooth muscles of the female genital tract. In yet another aspect of the invention, the specific combination of Ingredient A and Ingredient B provide synergistic effects by modulating the levels of hormone such as estradiol which in turn controls various genomic/nongenomic pathways related to female libido. It also regulates the levels of estrogen which in turn activates several genes responsible for sexual maturation and gestation, maturation and stimulation of ovary and vaginal lubrication
The main advantage of the present invention is to develop a synergistic herbal formulation that up-regulates sex hormones thereby improves sexual health and cellular function in females.
Yet another advantage of the present invention is that the present invention provides a palatable hence easy to consume beverage despite being an herbal composition.
Yet another advantage of the present invention is that the present invention provides a scientifically validated and functional nutraceutical composition.
Yet another advantage of the present invention is that the present invention provides synergistic herbal composition that has an extended shelf life.
Yet another advantage of the present invention is that the present invention provides a synergistic herbal composition that improves libido and helps maintain sexual balance.
Further objectives, advantages, and features of the present invention will become apparent from the detailed description provided herein below, in which various embodiments of the disclosed invention are illustrated by way of example.

DETAILED DESCRIPTION OF THE INVENTION
Definition
The term “a” or “an”, as used herein, is defined as one. The term “plurality”, as used herein, is defined as two as or more than one. The term “another”, as used herein, is defined as at least a second or more. The terms “including” and/or “having”, as used herein, are defined as comprising (i.e., open language).
The term “comprising” is not intended to limit the present invention with such terminology rather is used in a wider sense. Any invention using the term comprising could be separated into one or more claims using “consisting” or “consisting of”. The term “comprising” may be used interchangeably with the terms “having” or “containing”.
Reference in this document to “one embodiment”, “certain embodiments”, “an embodiment”, “another embodiment”, and “yet another embodiment” or similar terms, throughout the document means that a specific feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of such phrases in various places, this specification throughout are not necessarily all referring to the same embodiment. Furthermore, the specific features, structures, or characteristics are combined in any suitable manner in one or more embodiments without limitation.
The term “or” as used herein is to be interpreted as inclusive or meaning any one or more combinations. Therefore, “A, B or C” means any of the following: “A; B; C; A and B; A and C; B and C; A, B and C”. An exception to this definition will occur only when a combination of elements, functions, steps, or acts are in mutually exclusive, inherently.
As used herein, the term "one or more" generally refers to, but is not limited to, singular as well as the plural form of the term.
The drawings featured in the figures are to illustrate certain convenient embodiments of the present invention and are not to be considered as a limitation to that. Term "means" preceding a present participle of operation indicates the desired function for which there is one or more embodiments, i.e., one or more methods, for achieving the desired function and that one skilled in the art could select from these or their equivalent in view of the disclosure herein and use of the
Fig. 1 illustrates MTT assay. 10ng to 500µg/ml of both T.terrestris and T.f.graecum were incubated with HeLa cells for 24 hrs and MTT performed at the end of 24hrs incubation
Figure 2 illustrates dose dependent increase in estradiol with herbal combination. A. Standard curve obtained with the Estradiol standard provided with Cayman kit. X axis indicates the concentrations of standards used. Y-axis indicates the Logit (B/B0) values obtained with the excel worksheet provided by Cayman for calculation of Estradiol. B. Dose dependent increase in concentration of estradiol estimated from culture supernatant after incubation of herbs with Hela cells for 24hrs. Herb A-T.terrestris. Herb B-T.f.graecum. T.terrestris at 500µg/ml produces 56.1pg/ml estradiol, which increases to 57.7, 62.4 and 83.4pg/ml with additive effect of 10, 100 and 500µg of T.f.graecum respectively
Figure 3 illustrates the dose dependent increase in Nitrate with herbal combination. Nitrate/nitrite are byproducts of nitric oxide produced in cells during the upregulation of NO/cGMP signalling. A. Standard curve obtained with the Nitrate standard. X axis indicates the concentrations of standards used. Y-axis indicates the OD at 540nm. B. There was a dose dependant increase in the Nitric oxide levels at 24 hrs as evident from the increase in Nitrate/Nitrite levels (by products of Nitric oxide). L-NAME and Sodium nitroprusside were used as negative controls in the assay. Endogenous Nitrate/Nitrite levels from 8 X 103 HeLa cells were 3.2 µM, which was improved to 4.2 µM with 500µg/ml of herb B. This increase further increases in a dose dependant manner up to 7.4µg/ml with both herbs A and B at equal ratios.

Figure 4 illustrates the dose dependent increase in Testosterone with T.terrestris and T.f.graecum. A. Standard curve obtained with the Testosterone standard provided with Cayman kit. X axis indicates the concentrations of standards used. Y-axis indicates the Logit (B/B0) values obtained with the excel worksheet provided by Cayman for calculation of Testosterone. B. Dose dependent increase in concentration of testosterone estimated from culture supernatant after incubation of herbs with Hela cells for 24hrs. Herb A-T.terrestris. Herb B-T.f.graecum. T.terrestris at 100 and 500µg/ml produces 22 and 39pg/ml testosterone respectively. T.f.graecum at 100 and 500µg/ml produces 27 and 45pg/ml testosterone respectively
Figure 5 illustrates the box & Whisker Plot for the Total Score of SIPFBQ by Two Treatment Arms. Total score of Subject Investigational product Feedback Questionnaire at the end of the 2 month study compared between 2 treatment arms: (1) Test arm (2) placebo- control arms
Figure 6 illustrates the improvement in FSFI score comprising of 5 questions over 2 month study period. Q1-Level of Sexual Desire (Level of Desire) Q2. Satisfaction with Arousal (Arousal) Q3. Maintain Lubrication (Maintain Lubrication) Q4. Ability to reach orgasm (Orgasm) Q5. Overall Satisfaction with sexual desire (Satisfaction with Desire)
Figure 7 illustrates Schematic representation of Clinical trial- Study design
Figure 8 illustrates Composition of drinks used for Clinical trial. ‘Amore-For her’ is the female sexual wellness drink whose clinical efficacy was evaluated against placebo control drink that had all other ingredients except the herbs that act synergistically.
The present invention describes a herbal sexual wellness formulation for females that provide synergistic effects in vitro. The herbal sexual wellness formulation comprising of an extract of ingredient A and ingredient B in the range of 1µg/ml to 500 µg/ml. Herein, the ingredient A is an extract from the fruit of the plant but not limited to stem, leaf, inflorescence, or root extract and the ingredient B is an extract from the seed of the plant but not limited to fruit, leaf, stem, or inflorescence extract. Herein, the herbal extract of ingredients A and B is an aqueous extract but not limited to ethanol, methanol, acetone, ethyl acetate extracts and contains the bio-actives protodioscin, alkaloids, steroidal saponins, furostanol saponins, flavonoid glycosides, coumarins, alkaloids, and polyphenol compounds, and other bioactives.
In an embodiment, the ingredient A in the herbal sexual wellness formulation is obtained from Tribulus terrestris but not limited to T.terrestris orientalis, T.terrestris var robustus, T.terrestris var brachyceras, T.terrestris var inermis, T. terrestris var sericeus, T. terrestris var rajasthanensis, T.terrestris var macrocarpus, T.terrestris var desertorum, T.terrestris var biocornutus and the ingredient B is obtained from Trigonella feonum-graecum but not limited to Trigonella caerulea
In the embodiment of the present invention, the herbal sexual wellness formulation has been evaluated in vitro using a cell line, wherein the cell lines are selected from but not limited to mammalian, avian, Piscean, or arthropod origin. In another aspect of the invention, the cell type for the desired cell lines is HeLa cells but not limited to other cervical carcinoma cell lines.

In an embodiment of the present invention, an at least one pharmaceutically accepted excipient is selected from, but not limited to, coloring agents such as allura red and flavoring agents such as strawberry powder, acidity regulator such as sodium citrate and citric acid but not limited to acetic acid, malic acid, lactic acid, adipic acid, and other salts, preservative such as sodium benzoate but not limited to other similar preservatives, emulsifier/stabilizer/gelling agent/ thickener such as xanthan gum but not limited to other similar agents, added in the range 0.05% to 0.5% w/v%.

In an embodiment of the invention, the herbal sexual wellness formulation is an oral administration formulation selected from, but not limited to, a pill, tablet, capsule, concentrated syrup, food additive, suspension, emulsion, powdered drink mix, carbonated/non-carbonated beverage and a chewable solid.

In an embodiment of the present invention, the in vitro evaluation of the herbal sexual wellness formulation using HeLa cell lines, wherein the HeLa cell lines in a specific combination of ingredient A and ingredient B in a concentration range of 0.1ng to 50µg/ 8x103cells each, show synergistic effects on NO-cGMP pathway by modulating nitric oxide levels, which in turn influence intracellular calcium levels and leads to phosphorylation/dephosphorylation of myosin light chain thereby influencing smooth muscle tone.

In yet another aspect of the invention, the specific combination series of ingredient A and ingredient B, wherein the ingredients when taken in a ratio in the range between 1:1 to 1:500 regulates female sexual responsive cycle by upregulating the levels of female sex hormone Estradiol.

In yet another aspect of the invention, the specific combination of Ingredient A and Ingredient B provide synergistic effects by modulating the levels of hormone such as estradiol which in turn controls various genomic/nongenomic pathways related to female libido. It also regulates the levels of estrogen which in turn activates several genes responsible for sexual maturation and gestation, maturation and stimulation of ovary and vaginal lubrication. The present invention claims an increase in both NOS activity and estrogen upregulation using the synergistic combination of extracts from Ingredient A and Ingredient B. The effect of these extracts on female sexual wellness has been validated using HeLa cell lines.
Materials and methods:

Cell culture
HeLa cell lines obtained from NCCS, Pune, India was cultured in MEM supplemented with 10% fetal bovine serum (FBS). Cell lines were cultivated in the presence of benzylpenicillin and streptomycin and are maintained at 37o C in a humidified atmosphere of 5 % CO2.
Herbal extracts
T.terrestris aqueous dry extracts of whole fruit purchased from Herbo Nutra, India.
T.f.graecum aqueous dry extract of whole seed purchased from Amruta herbals Pvt Ltd, India.
Cell viability assessment – MTT assay
MTT (Dimethylthiazol-diphenyltetrazolium bromide) is a colorimetric assay that assess cell viability by determining these mitochondrial functional state. Mitochondria in a living cell secretes NAD(P)H-oxidoreductases which reduces yellow coloured MTT taken up by cells due to its net positive charge to form violet coloured formazan crystals. For this study, HeLa cells were seeded in 96 well plate at a concentration of 6000 cells/well (100ul cell suspension) and incubated for 24 hours. Herbal extracts ranging from 0.5mg/ml to 10ng/ml concentration were added after the incubation. MTT assays were performed for 2hr, 4hr, 8hr and 24 hrs (only 24hrs read out depicted in this patent), by adding 10µl of 5mg/ml MTT reagent and was incubated at 37o C in dark for 2hrs. After the incubation time, 100µl of crystal dissolving solution (solution containing 20% SDS and 50% DMF) was added and then incubated in dark until the crystals were dissolved. OD was measured at 560nm, cell viability was calculated as follows:

Nitric oxide estimation using Griess reagent:
Cells are seeded at a density of 8 X 103 cells /well. At the end of 24-hour incubation, the cells are treated with 1mg/ml concentration of herbs. The total nitrate/nitrite in the cell supernatant is estimated using colorimetric assay kit (cat no: 760871) Cayman chemicals (USA). Average OD values from 3 separate assays are subtracted from the blank values. Standard curve is plotted using Nitrate/Nitrite standards (1-100 µM). Concentration of nitrate/nitrite in the samples was determined using the standard curve.
Competitive ELISA for estimation of Estradiol:
Cells are seeded at a density of 8 X 103 cells /well. At the end of 24-hour incubation, the cells are treated with various concentrations of herbs. Estradiol in the culture supernatant of the cervical cell is estimated with competitive ELISA kits obtained from cayman chemicals (USA) (cat no 501890). B/B0 values are calculated for all the samples (B indicates ‘% bound’ i.e., percentage of analyte that competes with the given tracer in the sample. B0 indicates ‘maximum bound’ i.e., the value obtained with the maximum binding of tracer in the absence of analyte). With the B/B0 values obtained for standards, the 4-parameter logistic fit standard curve is plotted as mentioned in the kit. Samples concentrations were estimated using the equation obtained from the standard curve.
Competitive ELISA for estimation of Testosterone:
Cells were seeded at a density of 8 X 103 cells /well. At the end of 24-hour incubation, the cells are treated with 100 and 500µg/ml of T.terrestris and T.f.graecum. After 24hrs of incubation with herbs, Testosterone in the culture supernatant of HeLa was estimated with competitive ELISA kits obtained from cayman chemicals (USA) (cat no 582701). B/B0 values were calculated for all the samples (B indicates ‘% bound’ i.e., percentage of analyte that competes with given tracer in the sample. B0 indicates ‘maximum bound’ i.e., value obtained with maximum binding of tracer in the absence of analyte). With the B/B0 values obtained for standards, 4-parameter logistic fit standard curve is plotted as mentioned in the kit. Samples concentrations were estimated using the equation obtained from the standard curve.

Statistical analysis:
All data are represented as mean ±standard deviation (SD). Differences between the groups were determined with Analysis of variance (ANOVA) followed by the LSD multiple comparison tests, using SPSS version 25. The data is considered statistically significant if P<0.5
Double blinded, prospective, randomized, comparative, placebo controlled clinical trial:
Design: This clinical study was designed a double blinded, prospective, randomized, comparative, placebo controlled clinical study to evaluate the effectiveness, safety and tolerability of “Amore – Female Sexual Wellness Drink” of Sipwise Beverages Pvt. Ltd. in improving Sexual Wellness in Healthy Adult Female Subjects. Healthy adult females between 21 and 65 years of age who were in a heterosexual, monogamous relationship with an active sexual life were screened for the clinical study. Apart from the inclusion exclusion criteria, the subjects were also screened using the Female Sexual Function Index Questionnaire.
Treatment arms:
Subjects who were eligible were enrolled into the study and randomized into two treatment arms:
Treatment Arm I : 33 Subjects : Amore – Female Sexual Wellness Drink
Treatment Arm II : 11 Subjects : Placebo
Subjects consumed 2 drinks/day (1-ounce liquid shots) for a period of 2 months and FSFI analysis done (a) at the time of induction (b) end of 1 month and (c) end of 2 months study period
Outcome measure:
Efficacy was analysed using Female Sexual Function Index (FSFI) which is a standardized questionnaire to evaluate the improvement in sexual wellness in adult females. There are five questions considered in this outcome measure FSFI and those questions are related to the following five attributes: 1. Level of Sexual Desire (Level of Desire) 2. Satisfaction with Arousal (Arousal) 3. Maintain Lubrication (Maintain Lubrication) 4. Ability to reach orgasm (Orgasm) 5. Overall Satisfaction with sexual desire (Satisfaction with Desire).
An improvement of = 20% from the baseline score in comparison with placebo is set as the endpoint with the expectation of higher score indicating better efficacy levels. Since it is a positive trend questionnaire a total score of = 20 from baseline in comparison is set as the endpoint with the expectation of higher score indicating better efficacy levels.
Statistical analysis:
All statistical analysis is performed in accordance with the ICH E9 guideline for Statistical Principles for Clinical Trials, using SAS® (Version 9.4 or higher). The efficacy of each individual treatment arm was determined by Single Proportion Test and comparative efficacy for proportion of subjects was determined by Two Proportion Test. For absolute scores, the efficacy of each treatment arm was determined by Single Mean Test and the comparative efficacy was determined using Two Mean Test. All point estimates, such as mean and standard deviation and interval estimates such as 95% confidence intervals will be provided for the estimated parameters.
Detailed discussion of results and observations:
MTT assay was performed to test the cytotoxicity levels of the herbs used in the assay. A wide range of concentration series from 10ng to 500µg/ml was found to be non toxic to the cells. The viability was more than 85% even in the highest concentration tested. Fig 1 depicts the cell viability at the concentrations tested at 24 hrs post incubation with herbs.
Estradiol, the female sex hormone is endogenously expressed by the HeLa cells and were secreted into the cell supernatant at the concentration of 1pg/ml. i.e., 0.1pg estradiol was secreted by 8 X 103 HeLa cells as shown in Fig 2. The 1pg/ml shoots up to 56.1pg/ml with 500ug/ml of aqueous T.f.graecum. This increase in estradiol synthesis in the presence of T.f.graecum is further synergistically increased to 57.7, 62.4 and 83.4pg/ml with additive effect of 10ug, 100mg and 500ug of T.terrestris respectively. This synergistic increase in estradiol production in cervical cancer cell line like HeLa very obviously gives strong evidence for the positive outcome of this herbal combination in improving female sex and general health.
NO-cGMP signaling is believed to play a major role in vaginal blood flow and smooth muscle relaxation during sexual arousal, which is mostly attributed to the regulation of endothelial Nitric oxide production. Studies have indicated the post-translational regulation of nitric oxide synthase by estrogen. As we already are aware of the increase in estrogen levels in HeLa cell line (shown in Fig 2), we further wanted to check if there is a relevant increase in Nitric oxide levels in HeLa incubated with the herbs T.terrestris (A) and T.f.graecum (B) aqueous extracts at ratios 1:1 to 1:500 (A:B) as depicted in Fig 3. There was a dose dependent increase in the Nitric oxide levels as evident from the increase in Nitrate/Nitrite levels (by products of Nitric oxide). L-NAME and Sodium nitroprusside were used as negative controls in the assay. Endogenous Nitrate/Nitrite levels from 8 X 103 HeLa cells were 3.2 µM, which was improved to 4.2 µM with 500µg/ml of herb B. This increase further increases in a dose dependent manner up to 7.4µg/ml with both herbs A and B at equal ratios. This Synergistic effect of the herbal combination in in increasing the Nitric oxide levels in female cervical cancer cell line is a strong evidence of the potential roles of this herbal combination in improvement of female sexual functions.
Testosterone, being the precursor for estradiol in steroid biosynthesis pathway, it is important to look into the levels of endogenous and induction levels of testosterone with the chosen herbs in HeLa cell line. Testosterone was estimated from the HeLa cell supernatant obtained after a 24 incubation with herbs A and B. As shown in Fig 4, HeLa cells secrete endogenously a concentration of 11pg/ml of testosterone, which increased up to 22 and 39pg/ml with 100 and 500µg/ml with Herb A respectively. Herb B also increase the amount of testosterone to 27 and 45 pg/ml with 100 and 500µg/ml of Herb B respectively. These results strongly indicate the synergistic increase in Estradiol concentration (shown in Fig 2) with the herbal combination used in the study is directly related to upregulation of steroid biosynthesis, as evident from the increase in its precursor Testosterone (shown in Fig 4).
Double blinded clinical trial were conducted on healthy women volunteers for 2 months and have produced promising results on the efficacy of the drink in improvement of overall sexual functions. Two mean test was done to test whether Treatment Arm-I (Female SWD) is better than Treatment Arm-II (Placebo drink) in terms of whether the mean total Score of SIPFBQ is greater than 20. As shown in Fig5, to compare the two treatment arms in terms of mean total Score of SIPFBQ, the hypothesis can be stated as follows: Null hypothesis, H0: Mean total score of SIPFBQ by Treatment Arm-I is not significantly different from that of Treatment Arm-II (i.e., µ1 = µ2) Alternate hypothesis, H1: Mean total score of SIPFBQ by Treatment Arm-I is significantly greater than that of Treatment Arm-II (i.e., µ1 > µ2) Statistical Test to be applied: Two Mean Test (or Independent Samples t-test) Result: According to the result of Two-Mean Test, the null hypothesis is rejected at 5% level of significance (t = 15.91, p = 0.000 < 0.05). In addition, the mean total score of SIPFBQ by Female SWD (22.06) is greater than that of Placebo (13.33). Hence, the evidence is sufficient to conclude that the mean total score of SIPFBQ by Treatment Arm-I is significantly greater than that of Treatment Arm-II.
As shown in Fig 6, there a very significant improvement of in FSFI index in the subjects who consumed the test drink for 2 months. The table in Figure 6 clearly shows the improvement in the score obtained for all the 5 questions of the FSFI questionnaire. The mean percentage score for test Vs placebo arm for all the questions were strikingly significant numbers.

In an embodiment of the present invention, the formulation of Female sexual drink formulation used in clinical trial is disclosed. The formulation comprising of Step 1:Disperse weighed quantity of xanthan gum in about 30 mL of warm, purified water to make a uniform dispersion of gum and allow to rest for about 15 minutes. Step 2: Dissolved the herbal extracts in about 20 mL of purified water under stirring. Step 3: To the above herbal extract solution add weighed quantities of sodium citrate, sucralose and sodium benzoate one after the other and mix for about 5 minutes. Add the solution of herbs slowly to xanthan gum under stirring. Step 4: To 30 mL of purified water add spray dried fruit powder and mix well without formation of lumps. Add fruit powder solution to the xanthan gum mixture and mix well. Step 5: To this mixture add colour and flavour one after the other and mix well. Step 6: To about 10 mL of water add citric acid, dissolve and add to the above mixture. Step 7: Transfer the mixture to a 100 mL measuring cylinder and make up the volume with purified water. Step 8: Transfer again to the mixing vessel, mix for about 5 minutes. Step 9: Transfer the drink to the primary packing container, label and store in room temperature.

Although this invention has been described by examples and with reference to possible embodiments thereof, it is to be understood that modifications or improvements may be made thereto without deviating from the scope of the invention. Further objectives, advantages, and features of the present invention will become apparent from the detailed description provided herein, in which various embodiments of the disclosed present invention are illustrated by way of example and appropriate reference to accompanying drawings. Those skilled in the art to which the present invention pertains may make modifications resulting in other embodiments employing principles of the present invention without deviating from its spirit or characteristics, particularly upon considering the foregoing teachings. Accordingly, the described embodiments are to be considered in all respects only as illustrative, and not restrictive, and the scope of the present invention is, therefore, indicated by the appended claims rather than by the foregoing description or drawings. Consequently, while the present invention has been described with reference to particular embodiments, modifications of structure, sequence, materials and the like apparent to those skilled in the art still fall within the scope of the invention as claimed by the applican ,CLAIMS:I/ WE CLAIM
1.A herbal sexual wellness formulation for females providing synergistic effects in vitro comprising of.
a.an extract of ingredient A used in the range of 1 ng/ml to 500 µg/ml.
b.an extract of ingredient B used in the range of 1ng/ml to 500 µg/ml.
Wherein, ingredient A is an extract from the fruit of the plant but not limited to stem, leaf, inflorescence, or root extract.
Wherein, ingredient B is an extract from the seed of the plant but not limited to fruit, leaf, stem, or inflorescence extract.
Wherein, the herbal extract of ingredient A and B is an aqueous extract but not limited to ethanol, methanol, acetone, ethyl acetate extracts and contains the bio-actives protodioscin, alkaloids, steroidal saponins, furostanol saponins, flavonoid glycosides, coumarins, alkaloids, and polyphenol compounds, and other bioactives.
2.The herbal hangover formulation as claimed in claim 1, wherein the ingredient A is obtained from Tribulus terrestris but not limited to T.terrestris orientalis, T.terrestris var robustus, T.terrestris var brachyceras, T.terrestris var inermis, T. terrestris var sericeus, T. terrestris var rajasthanensis, T.terrestris var macrocarpus, T.terrestris var desertorum, T.terrestris var biocornutus.
3.The herbal hangover formulation as claimed in claim 1, wherein the ingredient B is obtained from Trigonella feonum-graecum but not limited to Trigonella caerulea
4.The herbal formulation as claimed in claim 1, has been evaluated in vitro using cell lines, wherein the cell lines is selected from of mammalian, avian, piscean, and arthropod origin, wherein the cell type is HeLa cells but not limited to other cervical carcinoma cell lines.
5.The herbal hangover formulation as claimed in claim 1, wherein the herbal hangover formulation comprises an at least one pharmaceutically accepted excipient selected from, but not limited to, coloring agents , flavoring agents, acid stabilizers, preservatives, emulsifier/stabilizer/gelling agent, and thickener, wherein, the coloring agents is allura red, flavoring agents is strawberry, acidity regulator is sodium citrate and citric acid is selected from but not limited to acetic acid, malic acid, lactic acid,and adipic acid, the preservative is sodium benzoate but not limited to other similar preservatives, emulsifier/stabilizer/gelling agent/ thickener is xanthan gum.
6.The herbal hangover formulation as claimed in claim 1, wherein the formulation is an oral administration selected from, but not limited to, a pill, tablet, capsule, concentrated syrup, food additive, suspension, emulsion, powdered drink mix, carbonated/non-carbonated beverage and a chewable solid.
7.The in vitro evaluation using HeLa cell lines as claimed in claim 4, wherein the HeLa cell lines in a specific combination of ingredient A and ingredient B in a concentration of 0.1ng – 50 µg/ 8x103cells and 0.1 – 50 µg/ 8x103cells respectively are used and have synergistic effects on NO-cGMP pathway and regulate the levels of estradiol, indicate involvement in relaxation of smooth muscles and influence on libido and sexual health in females.
8.A specific combination of ingredient A and ingredient B as claimed in claim 7, provide synergistic effects by modulating nitric oxide levels, which in turn influence intracellular calcium levels and leads to phosphorylation/dephosphorylation of myosin light chain thereby influencing smooth muscle relaxation.
9.A specific combination of ingredient A and ingredient B as claimed in claim 7, wherein the ingredients when taken in a ratio of 1:5, 1:50 and 1:500 acts upon the smooth muscles found in corpus cavernosum but is not limited to the myometrium, vaginal muscularis, and other smooth muscles of the female genital tract.
10.A specific combination of Ingredient A and Ingredient B as claimed in claim 7, provide synergistic effects by modulating the levels of hormone such as estradiol which in turn controls various genomic/nongenomic pathways related to female sexual libido and also regulates the levels of estrogen which in turn activates several genes responsible for sexual maturation and gestation, influences maturation and stimulation of ovary and increases vaginal lubrication.
11.The synergistic herbal formulation as claim in claim1, wherein the formulation comprising of T.terrestris and T.f.graecum along with other food grade ingredients improved the overall sexual function in healthy female volunteers.
12.The clinical efficiency of the sexual wellness drink as claimed in claim 1, wherein result of significant positive outcome was measured by Female sexual Function Index (FSFI), but not limited to other methods to measure the outcome of improvement of female sexual function
13.The clinical efficiency of the sexual wellness drink as claimed in claim 1, resulted in an average improvement from baseline to end of study was 36.63% (t = 6.32, p = 0.000 < 0.05), compared to 3.73% in Placebo (t = –49.22, p = 1.000 > 0.05).