DESC:
FORM 2
The Patents Act 1970
(39 of 1970)
&
The Patent Rules 2003
NON-PROVISIONAL SPECIFICATION
(See Section 10 and rule 13)

TITLE OF THE INVENTION:
A POLY-HERBAL COMPOSITION FOR FEMALE GONODAL AND GENERAL WELLNESS AND METHOD OF PREPARATION THEREOF

APPLICANT
SARVOTHAM CARE LIMITED
#1-20-248, 1st Floor, Umajay Complex,
Rasoolpura, Secunderabad,
Telengana, India – 500003

PREAMBLE OF THE DESCRIPTION:
THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED
CROSS- REFERENCE TO RELATED APPLICATION
This application claims the priority of the provisional application with serial number 202141037013 filed on 16th of Aug, 2021 with the title, “A POLY-HERBAL COMPOSITION FOR FEMALE GONODAL AND GENERAL WELLNESS AND METHOD OF PREPARATION THEREOF (SurvoNutraDivaTM)” and the contents of which is incorporated in entirety.

A) TECHNICAL FIELD OF INVENTION
[001] The present invention generally relates to a poly-herbal composition and more particularly relates to the poly-herbal composition for ameliorating female infertility and improve general wellbeing in females. The present invention further relates to a method of synthesizing the same.

B) BACKGROUND OF INVENTION
[002] The complex and fragile nature of the human body and its associated physiology is widely known. Throughout a normal human lifespan, human body suffers from various psychological, pathological, and physiological stress / abnormalities. These health problems are most destructive and even cause death. One such condition that specifically affects female is known as ‘Female Sexual Disorders’ or ‘Female Infertility.’ These disorders are defined in The Diagnostic and Statistical Manual of Mental Disorders (“DSM”) and include the following three categories: (1) Genito pelvic pain/penetration disorder; (2) Sexual interest/arousal disorder; and (3) Female orgasmic disorder. All these problems are common and known to significantly reduce the quality of life for millions of females.
[003] A wide range of ‘nutrients’ are essential for women, as they undergo periodical menstrual cycle, in addition, nourishing the embryo and new-born during pregnancy and lactation, respectively are the nutrient driven biological processes. The wellness of women, the quality of life in terms of health aspects such as reproduction, hormonal cycles, gastrointestinal, etc. are important. Infertility in female depends on several factors such as age, ovulation cycle, day-to-day stress, and health. The female infertility may be due to ovulation disorders (polycystic ovary syndrome (PCOS), hypothalamic dysfunction, premature ovarian failure, excess prolactin), damage to fallopian tube (tubal infertility), endometriosis, uterine or cervical causes.
[004] Various remedies include treatment with chemically synthesized preparations, or other therapies, or use of prescription medications such as steroids and non-steroids drugs for female infertility. These drugs and therapies address these medical problems with varying degrees of effectiveness by increasing the production of reproductive hormones and/or by regulating the ovulation cycle, but often have unwanted serious side-effects. Also, these therapies overlook one of the most important factors in treatment: psychological well-being. Moreover, allopathic medications also have increased risk of Ovarian Hyper-stimulation Syndrome (OHSS) and ovarian tumours in females.
[005] Hence there is a need to develop an improved formulation for treating female infertility or female sexual disorders, without the adverse effects associated with conventional modes of treatment along with improving female wellbeing.
[006] The present disclosure overcome the problems in the art and provides a poly-herbal composition for treatment of infertility in females which shows synergistic effect by ameliorating fertility rate in female, without any side-effects.
[007] The value additions and above-mentioned shortcomings, disadvantages and problems are addressed herein, as detailed below.

C) OBJECTIVES OF INVENTION
[008] The primary objective of the present invention is to provide a poly-herbal composition for use in the treatment of female gonadal disorders and general wellness.
[009] Another objective of the present invention is to provide a poly-herbal composition comprising of a powdered admixture of pericarp extract of Myristica fragrans, seeds of Citrullus lanatus and aerial part extract of Centella asisatica; one or more herbal base additives extracts of Shatavari, Ashoka, Lodhra, Dhataki, Punarnava, Khadira, Kamala, Narikela, and Fructus Lyceum; a pharmaceutically acceptable excipient; a preservative and one or more lubricating agents for treating Female Sexual Disorder (FSD).
[0010] Yet another objective of the present invention is to provide a poly-herbal formulation which is absolutely free from synthetic chemicals.
[0011] Yet another objective of the present invention is to provide simple and less-time consuming method of synthesizing a poly-herbal infertility composition for use in the treatment of female gonadal disorders and general wellness.
[0012] These and other objectives and advantages of the embodiments herein will become readily apparent from the following detailed description taken in conjunction with the accompanying drawings.

D) SUMMARY OF INVENTION
[0013] The various embodiments of the present invention provide a poly-herbal composition for use in the treatment of female gonadal disorders and general wellness is provided. The formulation comprises a powdered admixture of pericarp extract of Myristica fragrans, seeds of Citrullus lanatus and aerial part extract of Centella asisatica; one or more herbal base additives extracts of Shatavari, Ashoka, Lodhra, Dhataki, Punarnava, Khadira, Kamala, Narikela, and Fructus Lyceum; a pharmaceutically acceptable excipient; a preservative and one or more lubricating agents.
[0014] According to an embodiment of the present invention, the herbal base additives are extracts of Shatavari, Ashoka, Lodhra, Dhataki, Punarnava, Khadira, Kamala, Narikela, and Fructus Lyceum.
[0015] According to an embodiment of the present invention, the pericarp extract of Myristica fragrans, seeds of Citrullus lanatus and aerial part extract of Centella asisatica is present in a ratio of 60:20:20.
[0016] According to an embodiment of the present invention, the powdered admixture of pericarp extract of Myristica fragrans, seeds of Citrullus lanatus and aerial part extract of Centella asisatica is present in an amount of 11% (w/w).
[0017] According to an embodiment of the present invention, the extract of Shatavari is present in an amount ranging from 100 to 120 mg.
[0018] According to an embodiment of the present invention, the extract of Ashoka is present in an amount ranging from 100 to 120 mg.
[0019] According to an embodiment of the present invention, the extract of Lodhra is present in an amount ranging from 50 to 70 mg.
[0020] According to an embodiment of the present invention, the extract of Dhataki is present in an amount ranging from 40 to 50 mg.
[0021] According to an embodiment of the present invention, the extract of Punarnava is present in an amount ranging from 40 to 50 mg.
[0022] According to an embodiment of the present invention, the extract of Khadira is present in an amount ranging from 20 to 30 mg.
[0023] According to an embodiment of the present invention, the extract of Kamala is present in an amount ranging from 20 to 30 mg.
[0024] According to an embodiment of the present invention, the extract of Narikela is present in an amount ranging from 20 to 30 mg.
[0025] According to an embodiment of the present invention, the extract of Fructus Lyceum is present in an amount ranging from 8 to 10 mg.
[0026] According to an embodiment of the present invention, the said pharmaceutically acceptable excipient is microcrystalline cellulose.
[0027] According to an embodiment of the present invention, the said pharmaceutically acceptable excipient is present in an amount ranging from 400 to 420 mg.
[0028] According to an embodiment of the present invention, the said preservative is sodium benzoate.
[0029] According to an embodiment of the present invention, the said preservative is present in an amount ranging from 1 to 3 mg.
[0030] According to an embodiment of the present invention, one or more lubricating agents are colloidal silicon dioxide and magnesium stearate.
[0031] According to an embodiment of the present invention, the colloidal silicon dioxide is present in an amount ranging from 5 to 9 mg.
[0032] According to an embodiment of the present invention, the magnesium stearate is present in an amount ranging from 5 to 7 mg.
[0033] According to an embodiment of the present invention, the said preservative is sodium benzoate.
[0034] According to an embodiment of the present invention, the said preservative is present in an amount ranging from 1 to 3 mg.
[0035] According to another embodiment of the present invention, a method of synthesizing a poly-herbal composition for use in the treatment of female gonadal disorders and general wellness is provided. The method comprises: a) preparing a dried powdered admixture of pericarp extract of Myristica fragrans, seeds of Citrullus lanatus and aerial part extract of Centella asisatica; b) preparing a hydro-alcoholic extract by soaking the dried powdered admixture of step (a) in an aqueous alcohol; c) rota-evaporation and drying the hydro-alcoholic extract of step (b) to obtain a dried mass; d) adding one or more powdered herbal base additives to the dried mass, wherein the herbal base additives are extracts of Shatavari, Ashoka, Lodhra, Dhataki, Punarnava, Khadira, Kamala, Narikela, Fructus Lyceum; e) blending the dried mass with the said powdered herbal base additives to obtain a powdered blend; f) granulating the powdered blend with the said pharmaceutically acceptable excipient at 50 – 60? in drying chamber; g) cooling the granulated blend of step (f) at room temperature; h) milling and sieving the granulated blend of step (g) using 40-60 mesh size; i) lubricating the granulated blend of step (h) with one or more lubricating agents; j) adding the said preservative to the granulated blend; and k) encapsulating the lubricated granules of step (j) in capsule shells.
[0036] According to an embodiment of the present invention, the dried powdered admixture is prepared by mixing pulverized coarse powdered extract of pericarp of Myristica fragrans, seeds of Citrullus lanatus and aerial part of Centella asisatica / Gotu Kola in a ratio of 60:20:20.
[0037] According to an embodiment of the present invention, the ratio of water and alcohol is 50:50.
[0038] According to an embodiment of the present invention, the powdered admixture is added in an amount of 11% (w/w).
[0039] According to an embodiment of the present invention, the extract of Shatavari is added in an amount ranging from 100 to 120 mg.
[0040] According to an embodiment of the present invention, the extract of Ashoka is added in an amount ranging from 100 to 120 mg.
[0041] According to an embodiment of the present invention, the extract of Lodhra is added in an amount ranging from 50 to 70 mg.
[0042] According to an embodiment of the present invention, the extract of Dhataki is added in an amount ranging from 40 to 50 mg.
[0043] According to an embodiment of the present invention, the extract of Punarnava is added in an amount ranging from 40 to 50 mg.
[0044] According to an embodiment of the present invention, the extract of Khadira is added in an amount ranging from 20 to 30 mg.
[0045] According to an embodiment of the present invention, the extract of Kamala is added in an amount ranging from 20 to 30 mg.
[0046] According to an embodiment of the present invention, the extract of Narikela is added in an amount ranging from 20 to 30 mg.
[0047] According to an embodiment of the present invention, the extract of Fructus Lyceum is added in an amount ranging from 8 to 10 mg.
[0048] According to an embodiment of the present invention, the said pharmaceutically acceptable excipient is microcrystalline cellulose.
[0049] According to an embodiment of the present invention, the said pharmaceutically acceptable excipient is added in an amount ranging from 400 to 420 mg.
[0050] According to an embodiment of the present invention, the said lubricants are colloidal silicon dioxide and magnesium stearate.
[0051] According to an embodiment of the present invention, the colloidal silicon dioxide is added in an amount ranging from 5 to 9 mg.
[0052] According to an embodiment of the present invention, the magnesium stearate is added in an amount ranging from 5 to 7 mg.
[0053] According to an embodiment of the present invention, the said preservative is sodium benzoate.
[0054] According to an embodiment of the present invention, the sodium benzoate is added in an amount ranging from 1 to 3 mg.
[0055] According to an embodiment of the present invention, the rota-evaporation is done at a bath temperature 60°C with condenser of chilled water circulation and the vacuum of 74.5 torr / 4 – 5 h.
[0056] According to an embodiment of the present invention, in the freeze drying step the concentrated liquid is allowed to distribute as a thin layer on inner-walls of flask by rotating slowly in the Chilled Methanol bath. The freezed contents are subjected to vacuum for removing of moisture through sublimation / 7 – 8 h.
[0057] These and other aspects of the embodiments herein will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following descriptions, while indicating preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments herein without departing from the spirit thereof, and the embodiments herein include all such modifications.

E) BRIEF DESCRIPTION OF DRAWINGS
[0058] The other objectives, features and advantages will occur to those skilled in the art from the following description of the preferred embodiment and the accompanying drawings in which:
[0059] FIG. 1 is a schematic representation showing the steps involved in the preparation of a poly-herbal composition for use in the treatment of female gonadal disorders and general wellness, according to an embodiment of the present invention.
[0060] FIG. 2 demonstrates study design for in vivo acute oral toxicity testing, according to an embodiment of the present invention.

F) DETAILED DESCRIPTION OF DRAWINGS
[0061] In the following detailed description, a reference is made to the accompanying drawings that form a part hereof, and in which the specific embodiments that may be practiced is shown by way of illustration. The embodiments are described in sufficient detail to enable those skilled in the art to practice the embodiments and it is to be understood that the logical, mechanical, electronic and other changes may be made without departing from the scope of the embodiments. The following detailed description is therefore not to be taken in a limiting sense.
[0062] According to an embodiment of the present invention, a poly-herbal composition for use in the treatment of female gonadal disorders and general wellness is provided. The formulation comprises a powdered admixture of pericarp extract of Myristica fragrans, seeds of Citrullus lanatus and aerial part extract of Centella asisatica; one or more herbal base additives; a pharmaceutically acceptable excipient; a preservative and one or more lubricating agents.
[0063] According to an embodiment of the present invention, the herbal base additives are extracts of Shatavari, Ashoka, Lodhra, Dhataki, Punarnava, Khadira, Kamala, Narikela, and Fructus Lyceum.
[0064] According to an embodiment of the present invention, the admixture comprises the dried pulverized coarse powder (20 to 30 mesh) of pericarp of Myristica fragrans (nutmeg), seeds of Citrullus lanatus (watermelon) and aerial part of Centella asisatica/Gotu Kola (spade leaf) in the designated ratio.
[0065] According to an embodiment of the present invention, the admixture more particularly comprises the dried pulverized coarse powder (20 to 30 mesh) of pericarp of Myristica fragrans (nutmeg), dried pulverized coarse powder (20 to 30 mesh) of seeds of Citrullus lanatus (watermelon) and dried pulverized coarse powder (20 to 30 mesh) of aerial part of Centella asisatica/Gotu Kola (spade leaf) in the ratio of 60:20:20.
[0066] According to an embodiment of the present invention, the powdered admixture of pericarp extract of Myristica fragrans, seeds of Citrullus lanatus and aerial part extract of Centella asisatica is present in an amount of 11% (w/w).
[0067] According to an embodiment of the present invention, the extract of Shatavari is present in an amount ranging from 100 to 120 mg.
[0068] According to an embodiment of the present invention, the extract of Ashoka is present in an amount ranging from 100 to 120 mg.
[0069] According to an embodiment of the present invention, the extract of Lodhra is present in an amount ranging from 50 to 70 mg.
[0070] According to an embodiment of the present invention, the extract of Dhataki is present in an amount ranging from 40 to 50 mg.
[0071] According to an embodiment of the present invention, the extract of Punarnava is present in an amount ranging from 40 to 50 mg.
[0072] According to an embodiment of the present invention, the extract of Khadira is present in an amount ranging from 20 to 30 mg.
[0073] According to an embodiment of the present invention, the extract of Kamala is present in an amount ranging from 20 to 30 mg.
[0074] According to an embodiment of the present invention, the extract of Narikela is present in an amount ranging from 20 to 30 mg.
[0075] According to an embodiment of the present invention, the extract of Fructus Lyceum is present in an amount ranging from 8 to 10 mg.
[0076] According to an embodiment of the present invention, the said pharmaceutically acceptable excipient is microcrystalline cellulose.
[0077] According to an embodiment of the present invention, the said pharmaceutically acceptable excipient is present in an amount ranging from 400 to 420 mg.
[0078] According to an embodiment of the present invention, the said preservative is sodium benzoate.
[0079] According to an embodiment of the present invention, the said preservative is present in an amount ranging from 1 to 3 mg.
[0080] According to an embodiment of the present invention, one or more lubricating agents are colloidal silicon dioxide and magnesium stearate.
[0081] According to an embodiment of the present invention, the colloidal silicon dioxide is present in an amount ranging from 5 to 9 mg.
[0082] According to an embodiment of the present invention, the magnesium stearate is present in an amount ranging from 5 to 7 mg.
[0083] According to an embodiment of the present invention, the said preservative is sodium benzoate.
[0084] According to an embodiment of the present invention, the said preservative is present in an amount ranging from 1 to 3 mg.
[0085] According to another embodiment of the present invention, a method of synthesizing a poly-herbal composition for use in the treatment of female gonadal disorders and general wellness is provided.
[0086] FIG. 1 is a schematic representation showing the steps involved in the preparation of a poly-herbal composition for use in the treatment of female gonadal disorders and general wellness, according to an embodiment of the present invention. With respect to FIG. 1, the method comprises: preparing a dried powdered admixture of pericarp extract of Myristica fragrans, seeds of Citrullus lanatus and aerial part extract of Centella asisatica (101); preparing a hydro-alcoholic extract by soaking the dried powdered admixture in an aqueous alcohol (102); rota-evaporation and freeze drying the hydro-alcoholic extract to obtain a dried mass (103); adding one or more powdered herbal base additives to the dried mass, wherein the herbal base additives are extracts of Shatavari, Ashoka, Lodhra, Dhataki, Punarnava, Khadira, Kamala, Narikela, Fructus Lyceum (104); blending the dried mass with the said powdered herbal base additives to obtain a powdered blend (105); granulating and drying the powdered blend with the said pharmaceutically acceptable excipient at 50 to 60? for 24 – 48 hours in dryer (106); cooling the granulated blend at room temperature (107); milling and sieving the granulated blend using 40-60 mesh size (108); lubricating the granulated blend with one or more lubricating agents for 30 minutes (109); adding the said preservative to the granulated blend (110); and encapsulating the lubricated granules in capsule shells (111).
[0087] According to an embodiment of the present invention, the pericarp of Myristica fragrans is taken, cleaned and dried in sunlight. The dried pericarp of Myristica fragrans are crushed and grounded to obtain a pulverized coarse powder (20 to 30 mesh).
[0088] According to an embodiment of the present invention, the rota-evaporation is done at a bath temperature 60°C with condenser of chilled water circulation and the vacuum of 74.5 torr / 4 – 5 h.
[0089] According to an embodiment of the present invention, in the freeze drying step the concentrated liquid is allowed to distribute as a thin layer on inner-walls of flask by rotating slowly in the Chilled Methanol bath. The freezed contents are subjected to vacuum for removing of moisture through sublimation / 7 – 8 h.
[0090] According to an embodiment of the present invention, the seeds of Citrullus lanatus is taken, cleaned and dried in sunlight. The dried seeds of Citrullus lanatus are crushed and grounded to obtain a pulverized coarse powder (20 to 30 mesh).
[0091] According to an embodiment of the present invention, the aerial part of Centella asisatica/Gotu Kola is taken, cleaned and dried in sunlight. The dried aerial part of Centella asisatica/Gotu Kola are crushed and grounded to obtain a pulverized coarse powder (20 to 30 mesh).
[0092] According to an embodiment of the present invention, the dried powdered admixture is prepared by mixing pulverized coarse powdered extract of pericarp of Myristica fragrans, seeds of Citrullus lanatus and aerial part of Centella asisatica / Gotu Kola in a ratio of 60:20:20.
[0093] According to an embodiment of the present invention, the ratio of water and alcohol is 50:50.
[0094] According to an embodiment of the present invention, the powdered admixture is added in an amount of 11% (w/w).
[0095] According to an embodiment of the present invention, the extract of Shatavari is added in an amount ranging from 100 to 120 mg.
[0096] According to an embodiment of the present invention, the extract of Ashoka is added in an amount ranging from 100 to 120 mg.
[0097] According to an embodiment of the present invention, the extract of Lodhra is added in an amount ranging from 50 to 70 mg.
[0098] According to an embodiment of the present invention, the extract of Dhataki is added in an amount ranging from 40 to 50 mg.
[0099] According to an embodiment of the present invention, the extract of Punarnava is added in an amount ranging from 40 to 50 mg.
[00100] According to an embodiment of the present invention, the extract of Khadira is added in an amount ranging from 20 to 30 mg.
[00101] According to an embodiment of the present invention, the extract of Kamala is added in an amount ranging from 20 to 30 mg.
[00102] According to an embodiment of the present invention, the extract of Narikela is added in an amount ranging from 20 to 30 mg.
[00103] According to an embodiment of the present invention, the extract of Fructus Lyceum is added in an amount ranging from 8 to 10 mg.
[00104] According to an embodiment of the present invention, the said pharmaceutically acceptable excipient is microcrystalline cellulose.
[00105] According to an embodiment of the present invention, the said pharmaceutically acceptable excipient is added in an amount ranging from 400 to 420 mg.
[00106] According to an embodiment of the present invention, the said lubricants are colloidal silicon dioxide and magnesium stearate.
[00107] According to an embodiment of the present invention, the colloidal silicon dioxide is added in an amount ranging from 5 to 9 mg.
[00108] According to an embodiment of the present invention, the magnesium stearate is added in an amount ranging from 5 to 7 mg.
[00109] According to an embodiment of the present invention, the said preservative is sodium benzoate.
[00110] According to an embodiment of the present invention, the sodium benzoate is added in an amount ranging from 1 to 3 mg.
[00111] According to an embodiment of the present invention, an efficacy of poly herbal compound (PHC) SurvoNutraDivaTM for wellness, sexuality and to ameliorate infertility in females is evaluated.

EXPERIMENTAL DETAILS
[00112] A method of synthesizing a poly-herbal composition for use in the treatment of female gonadal disorders and general wellness, comprises
[00113] a) preparing a dried powdered admixture of an extract of Myristica fragrans, Citrullus lanatus, Centella asiatica,
[00114] b) The pulverized coarse powders of pericarp of Myristica fragrans, seeds of Citrullus lanatus and aerial part of Centella asisatica /Gotu Kola are are mixed at a ratio of 60:20:20, respectively to obtain an admixture.
[00115] c) The admixture is soaked in hydroalcohol (50:50) solvent system for 24 hours. Then the supernatant decanted and the residual slurry was once again soaked with hydro-alcohol solvent and the processed repeated thrice.
[00116] d) The pooled hydro-alcoholic solvent possessing the extract subjected to concentration through roto-evaporation and freeze-drying processes.
[00117] e) Wherein the herbal base additives are selected and extracts were obtained from Shatavari, Ashoka, Lodhra, Dhataki, Punarnava, Khadira, Kamala, Narikela, Fructus Lyceum,
[00118] f) Such herbal base powdered additive was mixed with Hydro-alcoholic extract of Myristica fragrans, Citrullus lanatus and Centella asisatica.
[00119] g) blending the said admixture of step (d) with one or more herbal base additives of step (e) to obtain blend;
[00120] h) preparing a hydro-alcoholic extract (Kindly provide details of how the hydro alcoholic extract is prepared and the ratio), wherein the hydro-alcoholic extract is obtained by soaking the blend of step (c) in an aqueous alcohol;
[00121] i) The ‘Microcrystalline Cellulose PH102 (FLOCEL 102)’ (MCCP) is added to blend of hydro-alcoholic extract and herbal extract additives and subjected for granulation.
[00122] j) Granulation of MCCP + Hydro-alcohol extract + Herbal additives is done at 50 – 60 o c in drying chambers, followed by cooling granulated material to room temperature.
[00123] k) The granulated lumps were subjected to 40 – 60 mesh granules through milling and sieving through 40 – 60 mesh.
[00124] l) Granules of 40 – 60 mesh were lubricated with addition of Colloidal Silicon Dioxide and Mg Stearate using double cone blender.
[00125] Such lubricated granules were filled into HPMC zero size capsules using G40 Automatic Capsule Filler.
[00126] According to an embodiment of the present invention, the rota-evaporation is done at a bath temperature 60°C with condenser of chilled water circulation and the vacuum of 74.5 torr / 4 – 5 h.
[00127] According to an embodiment of the present invention, in the freeze drying step the concentrated liquid is allowed to distribute as a thin layer on inner-walls of flask by rotating slowly in the Chilled Methanol bath. The freezed contents are subjected to vacuum for removing of moisture through sublimation / 7 – 8 h.

IN VITRO CYTOTOXICITY STUDY
[00128] Cytotoxicity studies
[00129] The monolayer cell culture was trypsinized and the cell count was adjusted to 100,000 cells/ml using RPMI 1640-containing 10% FBS. To each well of the 96 well microtiter plate, 0.1 ml of the diluted cell suspension was added. After 24 hours upon formation of partial monolayer, the supernatant was flicked off, the monolayer was washed once with medium. 100µL of different test concentrations of test preparation was added on to the partial monolayer in microtiter plates. The plates were then incubated at 37°C for 1 day in 5% CO2 atmosphere. After 24 hours, microscopic examination was carried out and observations were noted. The drug solutions in the wells were discarded and 50µL of MTT in DPBS was added to each well. The plates were gently shaken and incubated for 3 hours at 37º C in 5% CO2 atmosphere. The supernatant was removed and 100µL of DMSO was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader at a wavelength of 570 nm.
[00130] The percentage growth inhibition was calculated and the concentration of test preparation that inhibited the cell growth by 50% (CTC50) values were generated from the dose-response values for each cell line.

DETERMINATION OF IN VITRO ANTI-OXIDANT ACTIVITY
[00131] Method:
[00132] Preparation of test solution:
[00133] For the study, each weighed test substance was separately dissolved in DMSO and volume was made up with RPMI-1640 cell culture media supplemented with 2% inactivated FBS to obtain a stock solution of 10 mg/ml concentration and sterilized by filtration. Serial two-fold dilutions were prepared from the stock solution of 10 mg/ml concentration for carrying out cytotoxic studies.
[00134] Cell Line and Culture medium
[00135] Human Ovarian Cancer cells (SKOV3) was obtained from National Centre for Cell Sciences (NCCS, Pune, India) and were cultured in RPMI-1640 media supplemented with 10% inactivated Fetal Bovine Serum (FBS), penicillin (100 IU/mL), streptomycin (100 µg/mL) and amphotericin B (5 µg/ml) in a humidified atmosphere of 5% CO2 at 37°C until confluent. The cells were dissociated with TPVG solution (0.2% trypsin, 0.02% EDTA, 0.05% glucose in PBS). The stock cultures were grown in 25 cm2 culture flasks and all experiments were carried out in 96 well micro-titre plates.
[00136] Hydrogen Peroxide (H2O2) induced cytotoxicity assay
[00137] The monolayer of cells was trypsinized and the cell count was adjusted to 1.0 x 105 cells/ml using respective media viz., RPMI-1640 containing 10% FBS. To each well of the 96 well microtiter plate, 0.1ml of the diluted cell suspension was added. After 24 hours upon formation of partial monolayer, the supernatant was flicked off, the monolayer was washed once with medium and cells were treated with 500µM of H2O2 (prepared in medium with 2% FBS) 3h prior to the addition of test preparation. After 3 hours, the non-toxic concentrations (250 or 500µg/mL) of the test substance were added to the cells. The plates were then incubated at 37°C for 24 hours in 5% CO2 atmosphere, and microscopic examination was carried out and observations were noted after 24 hours.
[00138] The drug solutions in the wells were discarded and 50µL of MTT in DPBS was added to each well. The plates were gently shaken and incubated for 3 hours at 37º C in 5% CO2 atmosphere. The supernatant was removed and 100µL of DMSO was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader at a wavelength of 570 nm.
[00139] The percentage growth inhibition was calculated and the concentration of test drug needs to inhibit the cell growth by 50% (CTC50) values were generated from the dose-response values for each cell line.

DETERMINATION OF IN VITRO ANTI-INFLAMMATORY ACTIVITY
[00140] Preparation of test solution
[00141] For the study, 10mg of test substance was separately dissolved and volume was made up with RPMI-1640 supplemented with 2% inactivated FBS to obtain a stock solution of 1 mg/ml concentration and sterilized by filtration. Serial two-fold dilutions were prepared from the stock solution for carrying out cytotoxic studies.
[00142] Cell line and Culture medium:
[00143] SKOV3 cell line was cultured in RPMI-1640 media supplemented with 10% inactivated Fetal Bovine Serum (FBS), penicillin (100 IU/ml), streptomycin (100 µg/ml) and amphotericin B (5 µg/ml) in a humidified atmosphere of 5% CO2 at 37? until confluent. The cells were dissociated with TPVG solution (0.2% trypsin, 0.02% EDTA, 0.05% glucose in PBS).
[00144] In vitro LPS-induced toxicity
[00145] Step I: Induction of Toxicity on SKOV3 cells
[00146] SKOV3 cells were seeded into 96 well culture dishes at a cell population 100, 00 cells/mL in RPMI-1640 with 10% FBS. After 24 hours, the cells were treated with a known non-toxic concentration of test preparations along with 100 µg/mL of lipopolysaccharide (LPS) and incubated at 37°C with 5% CO2 for 24 h.
[00147] Step II: Estimation of cyto-protection by MTT assay
[00148] After 24 hours, microscopic examinations were carried out and observations were noted. The drug solutions in the wells were discarded and 50µL of MTT in PBS was added to each well. The plates were gently shaken and incubated for 3 hours at 37º C in 5% CO2 atmosphere. The supernatant was removed and 100µL of DMSO was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader at a wavelength of 570 nm.

IN VIVO ACUTE ORAL TOXICITY TESTING
[00149] FIG. 2 demonstrates study design for in vivo acute oral toxicity testing, according to an embodiment of the present invention.
[00150] Table 1 illustrates test system used for in vivo acute oral toxicity testing, according to an embodiment of the present invention.

Table 1: Test system used for in vivo acute oral toxicity testing
Species Rat (Rattus norvegicus)
Strain Sprague Dawley
Sex Female (nulliparous and non-pregnant)
Number of animals Step I: 3 animals;
Step II: 3 animals;
Step III: 3 animals;
Step IV: 3 animals
Total 12 animals were used in the study
Age at the start of dosing Step I : 8 - 9 weeks,
Step II : 8 - 9 weeks,
Step III : 9 - 10 weeks,
Step IV: 9 - 10 weeks
Body weight at the start of dosing

Step I : 179.05 to 188.49 g,
Step II : 187.29 to 191.06 g,
Step III: 197.94 to 201.86 g,
Step IV: 196.91 to 210.95 g,
The body weight variation did not exceed ± 20% of the mean weight of any previously dosed animal.
Source Animal Facility (AFT), Vanta Bioscience Limited.
Health status Prior to inclusion into the study, animals were examined by the Study Veterinarian for the health status and their suitability for
use in the experiment.

[00151] Preparation of test solution
[00152] The test substance was weighed and triturated in mortar pestle. 2 mL of Milli Q water was added to it and transferred to a measuring cylinder with a syringe. The mortar and pestle were rinsed twice with 2 mL of the Milli Q water and transferred to measuring cylinder. The required volume was made up using Milli Q water.
[00153] Animal Receipt and Acclimatization
[00154] A total number of 12 female Sprague Dawley rats were received from the Animal Facility (AFT) of Vanta Bioscience Limited. Animals were acclimatized to the experimental conditions for a period of 5, 7, 12 and 15 days for step I, II, III and IV, respectively; prior to start of the administration.
[00155] Initial dose of Sarvonutra-Diva™: 300 mg/kg body weight
[00156] Dose Volume: A dose volume of 10 m.L/kg body weight was maintained to administer the required dose.
[00157] Route of Administration: Oral route
[00158] Administration of the Test preparation
[00159] Prior to dosing, animals were fasted (feed was withheld) for 12 hours 48 minutes, 13 hours, 13 hours 9 minutes and 12 hours 56 minutes for Step I, Step II, Step III and Step IV, respectively; with free access to drinking water ad libitum. The test preparation was administered orally to animals based on the body weight recorded on dosing day (fasting bodyweight) using a stainless-steel ball-tipped intubation cannula attached to a calibrated syringe. Further, feed was withheld for a period of 03 hours 30 minutes, 03 hours 8 minutes, 03 hours 21 minutes and 03 hours 22 minutes for Step I, Step II, Step III and Step IV, respectively; after administration of the test item.
[00160] Parameters evaluated:
[00161] Mortality and General Clinical Signs: Mortality and morbidity was observed twice daily throughout the experiment. The animals were observed for toxic signs at various time points on the day of administration: at 30 minutes and at 1, 2, 3 and 4 hours after administration and once daily till day 14 of observation period.
[00162] Body Weight: Individual animal body weight were recorded on dosing day (prior to test item administration) and on days 7 and 14 for all the animals.
[00163] Necropsy and Gross Pathology: All dead animals were subjected to gross necropsy as soon as possible after death. All the surviving animals were necropsied at the end of the observation period. Animals were euthanized via CO2 asphyxiation. All necropsy observations were recorded.

RESULT
[00164] IN VITRO CYTOTOXICITY STUDY
[00165] Table 2 shows cytotoxic properties of test substance (Sarvonutra-DivaTM) against SKOV3 cell line, according to an embodiment of the present invention. With respect to Table 2, the results suggest no-cytotoxicity at tested concentrations up to 500 µg/ml of Sarvonutra-DivaTM and a minimal cytotoxicity (12.95±0.88%) was noted even at 1000 µg/ml to of Sarvonutra-DivaTM.
[00166] In view of this, the CTC50 of Sarvonutra-DivaTM is considered > 1000µg/ml, hence further in vitro efficacy testing for anti-oxidant and anti-inflammatory activities were evaluated at a concentration of 250 and 500 µg/ml to of Sarvonutra-DivaTM.
Table 2: Cytotoxic properties of test substance (Sarvonutra-DivaTM) against SKOV3 cell line
Sr. No. Name of Test substance Test Conc. (µg/ml) % Cytotoxicity CTC50 (µg/ml)
1 RR210565
(SarvonutraDivaTM) 1000 12.95±0.88 >1000
500 6.94±1.19
250 4.51±0.61
125 3.00±0.61
62.5 2.72±0.13
31.25 1.82±0.26
15.625 1.32±0.18
7.5125 1.29±0.23

[00167] DETERMINATION OF IN VITRO ANTI-OXIDANT ACTIVITY
[00168] Based on the CTC50 value of cytotoxicity study, 250 and 500 µg/ml concentration of test substance (SarvonutraDivaTM) are considered as non-toxic concentrations. Hence, the test substance (SarvonutraDivaTM) was evaluated for in vitro anti-oxidant activity at non-toxic concentrations 250 and 500 µg/ml of test substance (SarvonutraDivaTM) against Hydrogen Peroxide induced toxicity.
[00169] Table 3 shows anti-oxidant activity of test substance in SKOV3 cells against Hydrogen peroxide induced toxicity, according to an embodiment of the present invention. With respect to Table 3, the test substance showed dose dependent protection against Hydrogen Peroxide induced toxicity. Also, the test product showed dose-dependent anti-oxidant activity.

Table 3: Anti-oxidant activity of test substance in SKOV3 cells against Hydrogen peroxide induced toxicity
Sr. No. Samples Concentration tested
(µg/ml) % Protection
1 RR210565
(Sarvonutra-DivaTM ) 500 53.31±6.33
250 28.41±5.52

[00170] DETERMINATION OF ANTI-INFLAMMATORY ACTIVITY
[00171] Based on the CTC50 value of cytotoxicity study, 250 and 500 µg/ml concentration of test substance (Sarvonutra-DivaTM) are considered as non-toxic concentrations. Hence, the test substance (Sarvonutra-DivaTM) was evaluated for in vitro anti-inflammatory activity at non-toxic concentrations 250 and 500 µg/ml of test substance (Sarvonutra-DivaTM) against LPS-induced toxicity.
[00172] Table 4 shows percentage protection of Test Substance (SarvonutraDivaTM) in SKOV3 cell line against LPS-induced toxicity, according to an embodiment of the present invention. With respect to Table 4, the test substance showed dose dependent protection against LPS-induced toxicity. Also, the test product showed dose-dependent anti-inflammatory activity.
Table 4: Percentage protection of Test Substance (Sarvonutra-DivaTM) in SKOV3 cell line against LPS-induced toxicity

Sr. No. Samples Concentration tested
(µg/ml) % Protection over
LPS Control
1 RR210565
(Sarvonutra-DivaTM ) 500 46.76± 5.43
250 33.95 ± 4.52

[00173] IN VIVO ACUTE ORAL TOXICITY TESTING
[00174] Morbidity/Mortality and Clinical Signs
[00175] Table 5 shows animal mortality and morbidity rate, according to an embodiment of the present invention. With respect to Table 5, there was no mortality or clinical signs of toxicity observed throughout the observation period in all animals of step I and step II at 300 mg/kg body weight and in all animals of step III and step IV at 2000 mg/kg body weight.

Table 5: Animal Mortality and Morbidity
Step Dose (mg/kg body weight) Animal Number Sex Mortality/Morbidity
I 300 1 Female 0/3
2 Female
3 Female
II 300 4 Female 0/3
5 Female
6 Female
III 2000 7 Female 0/3
8 Female
9 Female
IV 2000 10 Female 0/3
11 Female
12 Female

[00176] Table 6 shows animal cage side clinical sign observation, according to an embodiment of the present invention. With respect to Table 6, no abnormal clinical sign observed in test animals up to 14 days.

Table 6: Animal Cage Side Clinical Sign Observation

Animal Number Observation days
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
30 mins. 1 hr 2 hr 3 hr 4 hr
1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
11 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
12 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
*Key: 0 = Normal

[00177] Body weight
[00178] Body weight gain was observed in all animals treated with test item at 300 mg/kg body weight (step I and II) and for animals treated with test item at 2000 mg/kg body weight (Step III and Step IV) on day 7 and day 14. Table 7 shows result of animal body weights and body weight gain (grams), according to an embodiment of the present invention.

Table 7: Animal Body Weights and Body Weight Gain (grams)
Step Dose (mg/kg body weight) Animal Number Sex Dose Volume (mL) Dosing Day (prior to dosing) (g) Day 7 (g) Day 14 (g) Weight change (0-7) (g) Weight change (7-14) (g)
I 300 1 Female 1.8 179.05 212.29 240.26 33.24 27.97
2 Female 1.8 180.54 203.31 222.35 22.77 19.04
3 Female 1.8 188.49 217.38 243.12 28.89 25.74
Mean 182.69 210.99 235.24 28.3 24.25
Standard Deviation 5.08 7.12 11.26 5.26 4.65
II 300 4 Female 1.9 189.31 205.38 220.97 16.07 15.59
5 Female 1.9 191.06 209.64 225.48 18.58 15.84
6 Female 1.9 187.29 201.95 218.21 14.66 16.26
Mean 189.22 205.66 221.55 16.44 15.9
Standard Deviation 1.89 3.85 3.67 1.99 0.34
III 2000 7 Female 2.0 198.71 231.19 247.62 32.48 16.43
8 Female 2.0 201.86 235.4 249.64 33.54 14.24
9 Female 2.0 197.94 216.07 232.64 18.13 16.57
Mean 199.5 227.55 243.3 28.05 15.75
Standard Deviation 2.08 10.17 9.29 8.61 1.31
IV 2000 10 Female 2.1 210.95 227.08 242.6 16.31 15.52
11 Female 2.0 200.87 216.43 231.88 15.56 15.45
12 Female 2.0 196.91 213.87 229.11 16.96 15.24
Mean 202.91 219.13 234.53 16.22 15.4
Standard Deviation 7.24 7.01 7.12 0.70 0.15

[00179] Necropsy and Gross Pathology
[00180] Table 8 shows animal gross pathology findings, according to an embodiment of the present invention. With respect to Table 8, necropsy observations revealed no abnormalities externally and internally in all the animals of step I, step II, step III and step IV.
Table 8: Animal Gross Pathology Findings
Step Dose (mg/kg body weight) Animal Number Sex Mode of death Gross pathology observations
External Internal
I 300 1 Female TS NAD NAD
2 Female TS NAD NAD
3 Female TS NAD NAD
II 300 4 Female TS NAD NAD
5 Female TS NAD NAD
6 Female TS NAD NAD
III 2000 7 Female TS NAD NAD
8 Female TS NAD NAD
9 Female TS NAD NAD
IV 2000 10 Female TS NAD NAD
11 Female TS NAD NAD
12 Female TS NAD NAD
*NAD = No abnormality detected; TS = Terminal sacrifice

DISCUSSION & CONCLUSION
[00181] Test preparation at varied concentrations was evaluated for in vitro cytotoxicity study by MTT assay. The results suggest no-cytotoxicity at tested concentrations up to 500 µg/ml of Sarvonutra-DivaTM and a minimal cytotoxicity (12.95±0.88%) was noted even at 1000 µg/ml to of Sarvonutra-DivaTM. Thus, it is concluded that test preparation (Sarvonutra-DivaTM) shows no or minimal toxicity.
[00182] The in vitro anti-oxidant activity of test preparation (Sarvonutra-DivaTM) was evaluated in human ovarian cancer cell line (SKOV3) at non-toxic concentrations 250 µg/ml & 500 µg/ml. When the cells were treated with the test preparation (Sarvonutra-DivaTM) post-exposure of hydrogen peroxide, the percentage protection exhibited by the test preparation was found to be 53.31±6.33 percent at 500µg/mL concentration and 28.41±5.52 percent at 250 µg/ml concentration, respectively. In this study, it was determined that Sarvonutra-DivaTM plays potential role in preventing the oxidative damage induced by hydrogen peroxide. On basis of the study, it is concluded that the Sarvonutra-DivaTM is able to reduce the oxidative stress and ameliorate the toxic effects induced by hydrogen peroxide (H2O2) in SKOV3 cells.
[00183] The in vitro anti-inflammatory activity of test preparation (Sarvonutra-DivaTM) was evaluated in human ovarian cancer cell line (SKOV3) at non-toxic concentrations 250 µg/ml & 500 µg/ml. The test substance, Sarvonutra-DivaTM exhibited significant protection against LPS-induced cytotoxicity in a dose dependent manner. The highest concentration tested (500µg/mL) exhibited 46.76± 5.43% protection over LPS control. These results show that Sarvonutra-DivaTM was effective in protecting SKOV3 cells from LPS-induced cytotoxicity, suggesting that the test preparation (Sarvonutra-DivaTM) has potential anti-inflammatory properties.
[00184] Based on in vivo acute oral toxicity study, it is concluded that the LD50 cut-off value of the test preparation (Sarvonutra-DivaTM) after single oral (gavage) administration in female Sprague Dawley rats is "5000 mg/kg body weight" and classified under "Category 5 or unclassified" according to Globally Harmonized System of Classification and Labelling of Chemicals (GHS).
[00185] Centella asiatica has nutritional value and rich source of protein, vitamins and minerals. In addition, it’s use has been widely accepted in improving the mental abilities, to enhance vascular support, as an anti-stress agent, to enhance immunity, as a lipid-lowering agent, and for many other valuable properties. Citrullus lanatus seed has essential oils helps in improving the anti-oxidant and anti-inflammatory functions upon consumption. Citrulline, major phytoconstituent of C. lanatus has smooth muscle relaxation activity thus attributed for prevention of preterm deliveries through action myometrium.
[00186] The in vitro anti-oxidant and anti-inflammatory activities of Sarvonutra-DivaTM, evaluated in SKOV3 cells exposed to H2O2 and LPS, respectively demonstrated substantial protective potential. Such activities of compound is due to the presence of various phytoconstituents of the selected herbs. In addition, the in vitro cytotoxicity assessed in SKOV3 cells, suggest minimal cytotoxicity at 1000 µg/mL of Sarvonutra-DivaTM, hence the CTC50 is also above 1000 µg/mL.
[00187] These observations were validated with ‘acute oral administration’ testing at a concentrations of 300 mg/kg and 2000 mg/kg body weight in rats. No morbidity and mortality was noted among the rats up to 14 days. Measurement of body weight and food consumption are being considered as two important and sensitive parameters for in vivo nonclinical toxicity/ safety evaluation studies, to assess the ‘illness’, if any, related to test compound administration.
[00188] The results of the current study have shown a proportionate increase in body weight gain and food intake by the animals during the course of experiment suggesting no evidence of toxicity.

G) ADVANTAGES OF INVENTION
[00189] The present invention relates to a poly-herbal composition for ameliorating female infertility. The “additive effect” of admixture of extracts of Myristica fragrans, Citrullus lanatus, Centella asiatica with one or more herbal base additives consisting of extracts of Shatavari, Ashoka, Lodhra, Dhataki, Punarnava, Khadira, Kamala, Narikela, Fructus Lyceum promotes general health and wellbeing of the females and ameliorates female infertility in treatment of Female Sexual Disorders (FSD).
[00190] The poly-herbal infertility composition of the present invention is eco-friendly since the ingredients utilized are natural and free from synthetic chemicals.
[00191] The method of preparing a poly-herbal infertility composition of the present invention is simple as it involves single extraction step in aqueous alcohol and less time consuming.
[00192] It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the claims presented in the complete specification or non-provisional application.

For, SARVOTHAM CARE LIMITED

BY THEIR AGENT
(DR. BABITHA THARAPPAN)
IN/PA-1614
ATV-LEGAL

,CLAIMS:Claims:
We claim:
1. A poly-herbal composition for use in the treatment of female gonadal disorders and general wellness, comprises:
a) a powdered admixture of pericarp extract of Myristica fragrans, seeds of Citrullus lanatus and aerial part extract of Centella asisatica;
b) one or more herbal base additives, wherein the herbal base additives are extracts of Shatavari, Ashoka, Lodhra, Dhataki, Punarnava, Khadira, Kamala, Narikela, Fructus Lyceum; and
c) a pharmaceutically acceptable excipient;
d) a preservative; and
e) one or more lubricating agents.
2. The poly-herbal composition as claimed in claim 1, wherein the pericarp extract of Myristica fragrans, seeds of Citrullus lanatus and aerial part extract of Centella asisatica is present in a ratio of 60:20:20.
3. The poly-herbal composition as claimed in claim 1, wherein the powdered admixture of pericarp extract of Myristica fragrans, seeds of Citrullus lanatus and aerial part extract of Centella asisatica is present in an amount of 11% (w/w).
4. The poly-herbal composition as claimed in claim 1, wherein the extract of Shatavari and the extract of Ashoka is present in an amount ranging from 100 to 120 mg.
5. The poly-herbal composition as claimed in claim 1, wherein the extract of Lodhra is present in an amount ranging from 50 to 70 mg.
6. The poly-herbal composition as claimed in claim 1, wherein the extract of Dhataki and the extract of Punarnava is present in an amount ranging from 40 to 50 mg.
7. The poly-herbal composition as claimed in claim 1, wherein the extract of Khadira, the extract of Kamala and the extract of Narikela is present in an amount ranging from 20 to 30 mg.
8. The poly-herbal composition as claimed in claim 1, wherein the extract of Fructus Lyceum is present in an amount ranging from 8 to 10 mg.
9. The poly-herbal composition as claimed in claim 1, wherein the said pharmaceutically acceptable excipient is microcrystalline cellulose, and wherein the said pharmaceutically acceptable excipient is present in an amount ranging from 400 to 420 mg.
10. The poly-herbal composition as claimed in claim 1, wherein the said preservative is sodium benzoate, and wherein the said preservative is present in an amount ranging from 1 to 3 mg.
11. The poly-herbal composition as claimed in claim 1, wherein one or more lubricating agents are colloidal silicon dioxide and magnesium stearate, and wherein the colloidal silicon dioxide is present in an amount ranging from 5 to 9 mg, and wherein the magnesium stearate is present in an amount ranging from 5 to 7 mg.
12. A method of synthesizing a poly-herbal composition for use in the treatment of female gonadal disorders and general wellness, comprises:
a) preparing a dried powdered admixture of pericarp extract of Myristica fragrans, seeds of Citrullus lanatus and aerial part extract of Centella asisatica;
b) preparing a hydro-alcoholic extract by soaking the dried powdered admixture of step (a) in an aqueous alcohol;
c) rota-evaporating and freeze drying the hydro-alcoholic extract of step (b) to obtain a dried mass;
d) adding one or more powdered herbal base additives to the dried mass, wherein the herbal base additives are extracts of Shatavari, Ashoka, Lodhra, Dhataki, Punarnava, Khadira, Kamala, Narikela, Fructus Lyceum;
e) blending the dried mass with the said powdered herbal base additives to obtain a powdered blend;
f) granulating and drying the powdered blend with the said pharmaceutically acceptable excipient at 50 to 60? for 24 – 48 hours in dryer;
g) cooling the granulated blend of step (f) at room temperature;
h) milling and sieving the granulated blend of step (g) using 40-60 mesh size;
i) lubricating the granulated blend of step (h) with one or more lubricating agents for 30 minutes;
j) adding the said preservative to the granulated blend; and
k) encapsulating the lubricated granules of step (j) in capsule shells.
13. The method as claimed in claim 12, wherein the dried powdered admixture is prepared by mixing pulverized coarse powdered extract of pericarp of Myristica fragrans, seeds of Citrullus lanatus and aerial part of Centella asisatica / Gotu Kola in a ratio of 60:20:20.
14. The method as claimed in claim 12, wherein the ratio of water and alcohol is 50:50.
15. The method as claimed in claim 12, wherein the powdered admixture is added in an amount of 11% (w/w).
16. The method as claimed in claim 12, wherein the rota-evaporation is done at a bath temperature of 60°C with a condenser of chilled water circulation and the vacuum of 74.5 torr / 4 – 5 h.
17. The method as claimed in claim 12, wherein the freeze drying is done by distributing the hydro-alcoholic extract as a thin layer on inner-walls of flask by rotating slowly in the chilled Methanol bath.
18. The method as claimed in claim 12, wherein the extract of Shatavari and the extract of Ashoka is added in an amount ranging from 100 to 120 mg, each.
19. The method as claimed in claim 12, wherein the extract of Lodhra is added in an amount ranging from 50 to 70 mg.
20. The method as claimed in claim 12, wherein the extract of Dhataki and the extract of Punarnava is added in an amount ranging from 40 to 50 mg, each.
21. The method as claimed in claim 12, wherein the extract of Khadira and the extract of Kamala and the extract of Narikela is added in an amount ranging from 20 to 30 mg, each.
22. The method as claimed in claim 12, wherein the extract of Fructus Lyceum is added in an amount ranging from 8 to 10 mg.
23. The method as claimed in claim 12, wherein the said pharmaceutically acceptable excipient is microcrystalline cellulose and wherein the said pharmaceutically acceptable excipient is added in an amount ranging from 400 to 420 mg.
24. The method as claimed in claim 12, wherein the said lubricants are colloidal silicon dioxide and magnesium stearate, wherein the colloidal silicon dioxide is added in an amount ranging from 5 to 9 mg, and wherein the magnesium stearate is added in an amount ranging from 5 to 7 mg.
25. The method as claimed in claim 12, wherein the said preservative is sodium benzoate, and wherein the sodium benzoate is added in an amount ranging from 1 to 3 mg.

For, SARVOTHAM CARE LIMITED

BY THEIR AGENT
(DR. BABITHA THARAPPAN)
IN/PA-1614
ATV-LEGAL