1. A polyherbal nanosuspension for the treatment of polycystic ovarian syndrome associated conditions and related infertility, said nanosuspension comprising of bioactive fractions prepared from Cedrus deodara extract, Rhododendron arboreum extract, Prunus persica extract, surfactant and a polymer.

2. The polyherbal nanosuspension as claimed in claim 1, wherein the fraction is obtained from, bark, flower and seed extracts.

3. The polyherbal nanosuspension as claimed in claim 1, wherein the Cedrus deodara fraction is obtained from bark extract, Rhododendron arboreum fraction is obtained from flower extract and Prunus persica fraction is obtained from seed extract.

4. The polyherbal nanosuspension as claimed in claim 1, wherein ratio between Cedrus deodara bark extract, Rhododendron arboreum flower extract, Prunus persica seed extract is in the range from 1 to 10: 1 to10 : 1 to 10, respectively.

5. The polyherbal nanosuspension as claimed in claim 1, wherein the ratio between fractions of Cedrus deodara bark extract, Rhododendron arboreum flower extract and Prunus persica seed extract is 1:1:1.

6. The polyherbal nanosuspension claimed in claim 1, wherein the extract is a solvent extract.

7. The polyherbal nanosuspension as claimed in claim 6, wherein the solvent is selected from butanol, water, methanol, hydromethanol, acetone, dichloromethane, hexane, ether, chloroform.

8. The polyherbal nanosuspension as claimed in claim 1, wherein the surfactant is selected from surfactants sorbitan fatty acid esters, (Spans), polysorbates (Tweens), and poloxamer (Pluronics).

9. The polyherbal nanosuspension as claimed in claim 1, wherein the poloxamer is Poloxamer 407.

10. The polyherbal nanosuspension as claimed in claim 1, wherein the polymer is selected from surfactants sorbitan fatty acid esters (Spans), polysorbates (Tweens), and poloxamer (Pluronics).

11. The polyherbal nanosuspension as claimed in claim 1, wherein the surfactant is selected from hydroxypropylmethyl cellulose, hydroxypropyl cellulose, methyl cellulose, soy lecithin and hydrogenated soy lecithin.

12. The polyherbal nanosuspension as claimed in claim 1, wherein the said nanosuspension is further lyophilized to obtain lyophilized powder form.

13. The polyherbal formulation as claimed in claim 1 wherein the particle size of the nanosuspension is between 50 to 4000 nm.

14. A process for preparing the polyherbal nanosuspension as claimed in claim 1, said process comprising, a) preparing solvent extracts of extracts of Cedrus deodara bark, Rhododendron arboreum flowers and Prunus persica seeds separately by extraction, b) investigating uterine smooth muscles relaxant activity of solvent extracts of each of the extracts obtained in (a) against carbachol induced contraction in isolated uterine smooth muscles of rats, c) selecting the best extract which showed uterine smooth muscles relaxant activity and carrying out fractionation of the best extracts through a series of liquid-liquid partitions, d) investigating the prepared fractions in (c) for uterine smooth muscles relaxant activity against carbachol induced contraction in isolated uterine smooth muscles of rats, e) selecting the fractions of Cedrus deodara bark, Rhododendron arboreum flowers and Prunus persica seeds from (d) with higher uterine smooth muscle relaxant activity to prepare polyherbal nanosuspension, f) mixing the fractions of (e) of Cedrus deodara bark, Rhododendron arboreum flowers and Prunus persica seeds in a particular ratio in an inert solvent to obtain a mixture, g) preparing an aqueous solution of the surfactant, h) preparing an aqueous solution of the polymer, i) mixing (g) and (h) in a beaker and stirring using a magnetic stirrer, j) homogenizing the mixture obtained in (f), k) mixing (i) and (j), l) conducting probe sonication for 1 min to obtain the nanosuspension.

15. The process as claimed in claim 14, wherein the fractionation is done through a series of liquid-liquid partitions using solvents such as chloroform, butanol and water.

16. The process as claimed in claim 14, wherein the inert solvent is selected from N-methyl-2-pyrrolidone (NMP), N,N dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and N,N-dimethylacetamide (DMAc).

17. The process as claimed in claim 14, wherein the nanosuspension thus obtained is lyophilized with a cryoprotectant to obtain polyherbal formulation in powder form.

18. The process as claimed in claim 17, wherein the cryoprotectant is selected from sucrose, lactose, mannitol, and polyethylene glycol.

Field of the Invention
The present invention relates to a formulation, particularly in form of polyherbal nanosuspension for the treatment of polycystic ovarian syndrome related infertility. More particularly, the present invention relates to a polyherbal nanosuspension for the treatment of polycystic ovarian syndrome related infertility. The present invention is also related to the preparation of polyherbal nanosuspension for the treatment of polycystic ovarian syndrome related infertility.

Background of the invention
Polycystic ovarian syndrome (PCOS) or Stein-Leventhal syndrome often called as hyperandrogenic anovulation is one of the widely prevalent endocrine disorders of females at their reproductive age It is mainly characterized by menstrual irregularities, hyperandrogenism and different size of ovarian cysts in females. Around 25-30% women with oligo anovulations have PCOS, 33% women with PCOS are suffering from metabolic syndrome and 50 % of women with PCOS facing difficulties like infertility and recurrent miscarriages. It has been widely prevailing in the society, however very few therapies are available which are associated with severe side effects.
US11382945 discloses a polyherbal composition for preventing and alleviating Polycystic ovary syndrome. The composition comprises Cinnamomum zeylyanicum/ Trigonella foenum, Garcinea cambogia/ Emblica officinalis, Linum usittatissimum/Tribulus terrestris, Vitex agnus-castus /Trachyspermum ammi, Zingiber officinale/ Putranjiva roxburghii, Ocimum sanctum / Glycyrrhiza glabra /Mentha spicate, Withania somnifera /Sesamum indicum Oryza sativa / Cicer arietinum, Ferula foetida /Prunus amygdalus. The document discloses Zingiber officinale and Putranjiva roxburghii as a cyst shrinking agent.
IN202111011591 discloses a polyherbal menstrual cramp relief tea formulation comprising Mimosa Pudica, Symplocos racemose, Zingiber officinale, Trigonella foenumgraecum, Camellia sinensis and Matricaria chamomilla.

US 11,364,274 discloses a composition for the treatment and management of polycystic ovarian syndrome comprising Saraca indica, Symplocos racemosa, Boerhavia diffusa, Tinospora cordifolia, Terminalia arjuna, Saccharum officinarum and Commiphora mukul, or extracts thereof; Shilajit; and bhasmas.
Sreedharan et al., 2018 reports the use of C. deodara bark to remove PCOS associated multiple uterine fibroids, ovarian cysts and to treat infertility in females
Midlekoop and Labadie, 1983 discloses an Ayurvedic preparation containing R. arboreum i.e., Ashoka Arishta has been reported to possess prostaglandin synthetase inhibitory activity and estrogenic activity .
Swaminathan and Kochhar, 2019 discloses the use of the flowers of R. arboretum in treating menstrual disorders. Adding to the ethnomedicinal relevance of medicinal plants, P. persica has been utilized as a major source of traditional medicine for the treatment of various disorders among females in many countries. Also, it is reported to regulate aromatase and estrogen synthesis on choriocarcinomal cells of human placenta (Dhingra et al., 2018).
Despite having tremendous potential, the plants have not yet been scientifically explored for the management of PCOS and other female disorders. Therefore, for the very first time, an effort has been made to blend these plants into a single formulation in the form of nanosuspension to evaluate the therapeutic potential of these plants in the effective management of PCOS and associated conditions.

Object of the invention
The primary object of the present invention is to provide a polyherbal nanosuspension for the treatment of polycystic ovarian syndrome associated conditions and related infertility.
It is another object of the present invention to provide a polyherbal nanosuspension comprising bioactive fractions prepared from Cedrus deodara , Rhododendron arboreum, and Prunus persica extracts for the treatment of polycystic ovarian syndrome associated conditions and related infertility.
It is further object of the present invention to provide a polyherbal nanosuspension comprising bioactive fractions prepared from Cedrus deodara, Rhododendron arboreum, and Prunus persica extracts showing reversed letrozole effects by restoring the irregular estrous cycle, disturbed hormone levels, abnormal levels of oxidative stress markers and disturbed ovarian dynamics in female rats.
It is yet another object of the present invention to provide a polyherbal formulation comprising bioactive fractions prepared from Cedrus deodara , Rhododendron arboreum, and Prunus persica extracts for the treatment of polycystic ovarian syndrome associated conditions and related infertility.
It is yet another object of the present invention to provide a polyherbal nanosuspension and formulation thereof that improves gestation rate and fertility index in female rats.
It is yet another object of the present invention to provide a safe, non-toxic, herbal formulation for the treatment of polycystic ovarian syndrome associated conditions and related infertility.
It is yet another object of the present invention to provide process of preparation of a polyherbal nanosuspension for the treatment of polycystic ovarian syndrome associated conditions and related infertility.

Summary of the invention
Accordingly, the present invention provides a polyherbal nanosuspension for the treatment of polycystic ovarian syndrome associated conditions and related infertility, said formulation comprising bioactive fractions prepared from Cedrus deodara, Rhododendron arboreum, and Prunus persica extracts, along with surfactant and a polymer.
In one embodiment the present invention provides a polyherbal nanosuspension for the treatment of polycystic ovarian syndrome related infertility, said formulation comprising bioactive fractions prepared from Cedrus deodara, Rhododendron arboreum and Prunus persica extracts wherein the extract is prepared from leaf, seed, bark, flower and seed of Cedrus deodara, Rhododendron arboretum and Prunus persica.
In another embodiment, the present invention displayed uterine smooth muscle relaxant activity of bioactive fractions prepared from Cedrus deodara, Rhododendron arboreum and Prunus persica extracts.
In another embodiment the present invention opted nanoprecipitation process for the preparation of polyherbal nanoformulation comprising bioactive fractions of Cedrus deodara, Rhododendron arboreum and Prunus persica extracts.

Brief description of the drawings
Fig. 1 depicts entrapment efficacy (%) obtained from 15 batches of formulation.
Fig. 2 depicts vaginal cytology inspection in rats to determine the effects of polyherbal nanosuspension on estrous cycle (a) control group (b) positive control group (c) standard group (d) nanosuspension (0.5mL) (e) nanosuspension (1mL) group rats. Data represented most prominent phase in each group, n=5, where D= Diestrus phase, P= Proestrus phase, E= Estrus phase, M= Metestrus phase
Fig. 3 depicts effect of nanosuspension on endocrine variables (a) FSH, (b) LH, (c) Estradiol, (d) Aromatase.
Fig. 4 depicts effect of nanosuspension on ovarian levels of (a) MDA, (b) catalase and (c) SOD.
Fig. 5 depicts histological section of ovaries from different experimental groups (a) control, (b) LTZ (1mg/kg), (c) CC (1mg/kg), (d) nanosuspension (0.5mL), (e) nanosuspension (1mL). PF= Primary follicles, TF= Tertiary follicles, O= Mature oocytes, GF= Graafian follicles, CL= Corpora lutea, CF= Cystic follicles.
Fig. 6 depicts in vitro release profiles of pure fractions from nanosuspension and the fractions solution.
Detailed description of the invention
Polycystic ovarian syndrome (PCOS) is mainly characterized by menstrual irregularities, hyperandrogenism and different size of ovarian cysts in females. It leads to infertility and other complications.
The inventors of the present invention surprisingly found that a formulation comprising bioactive fractions of Cedrus deodara, Rhododendron arboreum and Prunus persica is effective in the treatment of PCOS related infertility. Accordingly, the present invention relates to a herbal nanosuspension for the treatment of polycystic ovarian syndrome (PCOS) related infertility. The present invention also provides a polyherbal nanoformulation for the treatment of polycystic ovarian syndrome.
Cedrus deodara, commonly known as Deodar Cedar is a large evergreen tree belonging to the Pinaceae, or pine family. Its various parts / extract can be used in cough, asthma, fever, joint problems, diabetes and skin problems.
Rhododendron arboreum, commonly known as Tree Rhododendron is an evergreen shrub or small tree belonging to the family, Ericaceae. Its various parts / extract can be used in the prevention and treatment of heart diseases, dysentery, diarrhea, detoxification, inflammation, fever, constipation, bronchitis and asthma.
Prunus persica, commonly known as Peach is a fruit tree of Rosaceae family. Its various parts / extract can be used antiasthmatic, antitussive, emollient, haemolytic, laxative and sedative.
In one embodiment the present invention provides a polyherbal nanosuspension for the treatment of polycystic ovarian syndrome associated conditions and related infertility, said formulation comprising bioactive fractions of Cedrus deodara, Rhododendron arboreum, and Prunus persica extracts, surfactant and a polymer.
The extract can be obtained from any part of the plant such as shoot, root, stem, axillary buds, seeds, stipules, leaves, petals, flowers, ovules, bracts, branches, petioles, node, internodes, bark, pubescence, tillers, rhizomes, fronds, blades, pollen, stamen, microtubers, and the like. Preferable, seed, bark or flower is used.
The plant part is dried and pulverized. Drying can be done by sun drying, shade drying or drying in hot air oven under controlled condition.
The dried and pulverized plant part is further subjected to extraction. Extraction can be done by various methods known in the art such as cold maceration, infusion, percolation, digestion, decoction, hot continuous extraction (Soxhlet), aqueous-alcoholic extraction by fermentation, counter-current extraction, microwave-assisted extraction, ultrasound extraction (sonication), supercritical fluid extraction, and phytonic extraction, hydrodistillation techniques (water distillation, steam distillation, water and steam distillation), hydrolytic maceration followed by distillation, expression and enfleurage (cold fat extraction) headspace trapping, solid phase micro-extraction, protoplast extraction, microdistillation and their combinations. In a preferred embodiment, the extraction is done by successive extraction employing cold maceration and hot extraction.
Different solvents used for extraction are selected from water, methanol, ethanol hexane, dichloromethane, acetic acid, ethyl acetate, benzene, 2-butanol, cyclohexane, dichloromethane, chloroform carbon tetrachloride, hexane, ethanol, ethyl ether, petrol ether, propanetriole. methanol, propanol, toluene, hydromethanol (70% methanol) and mixtures thereof. Mixture of two solvents such water–dichloromethane, water–ether, water–hexane, water-methanol or water-hydromethanol can be used.
The extracts are then formulated to a nanosuspension using surfactant and a polymer.
Example of polymer that can be used are selected from hydroxypropylmethyl cellulose, hydroxypropyl cellulose, methyl cellulose, soy lecithin and hydrogenated soy lecithin.
Example of surfactant that can be used are selected from sorbitan fatty acid esters, (Spans), polysorbates (Tweens), and poloxamer (Pluronics). In a preferred embodiment, the surfactant is poloxamer 407.
In another embodiment the present invention provides a polyherbal nanosuspension for the treatment of polycystic ovarian syndrome associated conditions and related infertility, said nanosuspension comprising bioactive fractions prepared from Cedrus deodara bark, Rhododendron arboreum flower,Prunus persica seed extracts, Polaxomer 407 and soy lecithin.
The ratio between Cedrus deodara bark extract, Rhododendron arboreum flower extract, Prunus persica seed extract in the polyherbal nanosuspension is in the range from 1 to 10: 1 to10 : 1 to 10, respectively.
In a preferred embodiment, the ratio between fractions of Cedrus deodara bark, Rhododendron arboreum flower, and Prunus persica seed extracts is 1:1:1.
In yet another embodiment, the present invention provides a polyherbal nanosuspension for the treatment of polycystic ovarian syndrome associated conditions and related infertility, said formulation comprising bioactive fractions prepared from Cedrus deodara bark, Rhododendron arboreum flower, Prunus persica seed extracts, Polaxomer 407 and soy lecithin wherein the ratio of bioactive fractions of Cedrus deodara bark, Rhododendron arboreum flower, and Prunus persica seed extracts is 1:1:1.
In another embodiment, the present invention provides a process for preparing the polyherbal nanosuspension comprising bioactive fractions of Cedrus deodara, Rhododendron arboreum, Prunus persica, surfactant and a polymer said process comprising:
a) preparing solvent extracts of extracts of Cedrus deodara bark, Rhododendron arboreum flowers and Prunus persica seeds separately by extraction,
b) investigating uterine smooth muscles relaxant activity of solvent extracts of each of the extracts obtained in (a) against carbachol induced contraction in isolated uterine smooth muscles of rats,
c) selecting the best extract which showed uterine smooth muscles relaxant activity and carrying outfractionation of the best extracts through a series of liquid-liquid partitions,
d) investigating the prepared fractions in (c) for uterine smooth muscles relaxant activity against carbachol induced contraction in isolated uterine smooth muscles of rats,
e) selecting the fractions of Cedrus deodara bark, Rhododendron arboreum flowers and Prunus persica seeds from (d) with higher uterine smooth muscle relaxant activity to prepare polyherbal nanosuspension,
f) mixing the fractions of (e) of Cedrus deodara bark, Rhododendron arboreum flowers and Prunus persica seeds in a particular ratio in an inert solvent to obtain a mixture,
g) preparing an aqueous solution of the surfactant,
h) preparing an aqueous solution of the polymer,
i) mixing (g) and (h) in a beaker and stirring using a magnetic stirrer,
j) homogenizing the mixture obtained in (f),
k) mixing (i) and (j),
l) conducting probe sonication for 1 min to obtain the nanosuspension.
The inert solvent for use in step (f) is selected from N-methyl-2-pyrrolidone (NMP), N,N dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and N,N-dimethylacetamide (DMAc). Preferably, dimethyl sulfoxide is used.
The nanosuspension thus obtained is lyophilized with a cryoprotectant to obtain polyherbal formulation in powder form. The cryoprotectant is selected from sucrose, lactose, mannitol, and polyethylene glycol.
Examples
Example 1: Preparation of plant extracts: The pulverized plant materials i.e., C. deodara bark, R. arboreum flowers and P. persica seeds were subjected for successive extraction employing cold maceration and hot extraction techniques with different solvents such as methanol, hydromethanol (70% methanol) and water. The content in the flask was stirred occasionally and this procedure was performed repeatedly for three consecutive cycles. (The voucher specimen numbers for C. deodara bark, R. arboreum flowers and P. Persica seeds (BURI-1447/2022; BURI-1448/2022; BURI-1446/2022) were deposited in the herbarium of Banasthali Vidyapith, Rajasthan, India for future reference).
Example 2: Preparation of fractions from plant extracts
The hydromethanolic extracts of Cedrus deodara bark, Rhododendron arboreum flower and Prunus persica seed showed strong uterine smooth muscle relaxant activity against carbachol induced uterine contractions in isolated uterine smooth muscles of rats. In view of these findings, fractions were prepared from hydromethanolic extracts of Cedrus deodara bark, Rhododendron arboreum flowers and Prunus persica seeds through a series of liquid-liquid partitions using chloroform, butanol and water as solvents. The prepared fractions were then evaluated for uterine smooth muscle relaxant activity against carbachol induced uterine contractions in isolated uterine smooth muscles of rats. The chloroform fraction of Cedrus deodara bark extract, butanol fraction of Rhododendron arboreum flower extract and butanol fraction of Prunus persica seed extract showed higher uterine smooth muscles relaxant activity. Therefore, chloroform fraction of Cedrus deodara bark, butanol fraction of Rhododendron arboreum and butanol fraction of Prunus persica seed was selected to prepare polyherbal nanoformulation.
Example 3: Preparation of polyherbal nanosuspension (PHF): PHF was prepared by nanoprecipitation method. The three plant extracts were taken in a beaker and dissolved in 1mL (5% DMSO) solution. Simultaneously measured amount of soya lecithin was taken in another beaker and was dissolved in water. The accurately weighed poloxamer 407 (1%) (stabilizer) was dissolved in water and kept under stirring in Remi Mortar using magnetic bead and stirring was maintained at 500 rpm at room temperature. The non- aqueous solution was homogenised at 6000 rpm for 30 min and aqueous solution was added to it with the help of 14-gauge size needle. The prepared nanosuspension was then probe sonicated for 1 min. The volume of nanosuspension prepared in each trial was 10mL. The optimized formulation was lyophilized using mannitol as a cryoprotectant (1:1) and was utilized for the further studies (Joshi, 2022; Shinkar et al., 2022).
Example 4: Stability studies of the nanosuspension: Stability studies of nanosuspension were carried out to evaluate the change in its Particle size (PS), Zeta potential (ZP) and polydispersity index (PDI) over a period of 6 months (1, 3 and 6 months) of storage at three different temperature 4, 25 and 40°C. No visual sedimentation of particles was observed in PHF at 25°C and 40°C after subsequent period. This study examined the stability of PHF on a long-term basis (Papdiwal et al., 2014). It was observed that the particle size remained almost same at 25°C and 40°C but increased from 1444nm to 3605nm when stored at 4°C upto 6 months. The PDI was also found same at 25°C and 40°C. Findings of the stability studies are enlisted in Table 1.
Table 1: Stability studies of nanosuspension for 6 months
Time intervals Parameters Storage conditions
4°C 25°C 40°C
1 month PS (nm) 1444 187.4 142.5
ZP (mV) -44.6 -30.5 -19.3
PDI 0.584 0.280 0.219
3 months PS (nm) 2350 180.8 133.9
ZP (mV) -57.3 -27.2 -17.8
PDI 0.146 0.235 0.233
6 months PS (nm) 3605 178.5 130.1
ZP (mV) -42.5 -17.8 -6.84
PDI 1 0.230 0.291
Example 5 : Determination of Entrapment efficiency, EE (%): About 10 ml of nanosuspension was centrifuged at 11000 rpm for 20 min at a controlled temperature of 4° C using ultracentrifuge (ThermoFisher Scientific, Germany) to determine the entrapped drug present in the nanosuspension. The supernatant was separated carefully using the micropipette without disturbing the sediment and measured spectrophotometrically at 220 nm in triplicates (Dekate et al., 2018). The amount of drug entrapped in a nanosuspension was determined using the formula,

Result: The EE (%) obtained from 15 batches of nanosuspension was in the range of 55.39 to 91.45 % (Figure 1). The EE (%) of the optimized nanosuspension was found 91.70 %.
Example 6: Determination of Drug loading : The drug loading is calculated by directly measuring the concentration of drug in nanosuspension. About 1 ml (30 mg) of nanosuspension was taken and diluted with pH 7.4 phosphate buffer. The solution was measured in UV-VIS spectrophotometer at 220 nm in triplicates (Mahlawat et al., 2022). The amount of drug loaded in a nanosuspension was determined employing the formula,

The total drug content obtained from 15 batches of nanosuspension was in the range of 15.67% to 38.82 %, in which the total content of the optimized nanosuspension (F11) was found 38.82%.
Example 7: In vivo studies
Example7 (a) Induction of PCOS in rats by Letrozole (LTZ) : 50 female rats were used to investigate the therapeutic potential of polyherbal nanosuspension on PCOS associated symptoms and related infertility. Briefly, PCOS was induced by the oral administration of LTZ according to the protocol described (Ndeingang et al., 2019) with slight modifications. PCOS in rats was induced by oral administration of 1mg/kg LTZ (dissolved in distilled water) daily for 21 consecutive days. From the day of LTZ administration, vaginal smears from each rat were collected every morning to determine their estrous cyclicity. At day 22, the rats were allocated into 5 groups containing 10 rats per each group.
Group No of rate Treatment
Group 1
(Control) 10 0.9% saline without LTZ administration
Group 2
(Positive Control) 10 1mg/kg LTZ only
Group 3
(Standard) 10 1mg/kg clomiphene citrate (CC)
Group 4
(treatment group) 10 0.5 ml polyherbal nanosuspension
Group 5
(treatment group) 10 1 ml polyherbal nanosuspension
Example 7 (b) Determination of the estrous phases: The estrous cyclicity in rats was determined by the presence of predominant cell types observed in the vaginal smears collected from the rats throughout the entire studies. Figure 2 represents estrous cyclicity of rats in different experimental groups. The rats in control group exhibited regular estrous cycle which lasted for five days with successive estrous phases i.e., proestrus, estrus, metestrus and two phases of diestrus (Figure 2a). In positive control (LTZ, 1mg/kg) group, estrus cycle in rats was remain consistent in the diestrus phase (Figure 2b). The rats administered orally with 1mg/kg CC exhibited normal estrous cycle starting with the proestrus phase after 12 days of administration (Figure 2c). Treatment with nanosuspension resulted in restoration of estrous cycle in rats from the proestrus phase after 15 days of administration at 0.5mL (Figure 2d) and 11 days of administration at 1mL (Figure 2e) respectively.
Example 7 (c) Determination of the effect of nanosuspension on body weight, abdominal fat weight, relative uterine weight and uterine epithelial height: At the end of 21 days treatment, 5 rats from each group were sacrificed with high dose of anesthesia after fasting overnight. Blood samples were then collected from the rats in ethylenediamine tetraacetic acid (EDTA) vials and centrifuged for 15 min at 3000 rpm using ultracentrifuge. The serum was then separated and stored at -20 for further use. From the beginning till end of the study, body weight of rats was recorded weekly. The abdominal fat and uterine horns from each rat were collected for estimation of relative abdominal fat weight, relative uterine weight and uterine epithelial height. Simultaneously, ovaries from each rat were dissected out, cleaned and fixed in PBS and 10% formalin for biochemical and histopathological analysis respectively (Movondo et al., 2020). The result is as in Table 2 below:
Table 2: Effect of nanosuspension on body weight, abdominal fat weight, relative uterine weight and uterine epithelial height
Groups Body weight
(g) Abdominal fat weight (g/100g of body weight) Relative uterine weight (mg/100g of body weight) Uterine epithelial height (?m)
Control 180±0.31 1.76±1.25 53.8±0.14 106.4±3.21
LTZ (1mg/kg) 225±0.78 3.27±1.93 20.2±0.78 92.6±2.64
CC (1mg/kg) 200±0.29*** 1.70±1.32 51.6±0.36*** 115.9±3.82***
nanosuspension (0.5mL) 215±0.63*** 2.23±0.83 32.4±0.29*** 98.1±3.49*
nanosuspension (1mL) 200±0.45*** 1.68±0.57** 46.7±0.91*** 108.5±3.69***
Values are represented as Mean±SD, n=5. *p<0.05, **p<0.01, ***p<0.001
Example 7 (d) Effect of nanosuspension on endocrine variables in rats: Serum levels of hormones viz. follicle stimulating hormone (FSH) and luteinizing hormone (LH) were estimated by commercially available ELISA kits (USCN, Wuhan) and serum levels of estradiol and enzyme aromatase were estimated by rat estradiol and rat aromatase ELISA kits (MyBioSource, US) respectively.
Example 7 (e) Effect of nanosuspension on endocrine variables in rats: Serum levels of FSH, LH, estradiol and aromatase were determined to evaluate the effects of nanosuspension on endocrine variables in rats. Findings of the study are enlisted in Table 3 and illustrated in Figure 3. Administration of LTZ resulted in lower serum levels of FSH, estradiol and aromatase in rats, the effect of LTZ was significantly reversed by the oral administration of PHF at 0.5mL and 1mL in a dose dependent manner. Nanosuspension treated rats exhibited marked elevation in serum levels of FSH, estradiol and aromatase as compared to the positive control rats. Moreover, nanosuspension also attenuated enhanced serum level of LH in LTZ treated rats in a dose dependent manner. The ameliorative effect of nanosuspension was found similar as that of standard CC (1mg/kg).
Table 3: Effect of nanosuspension on endocrine variables in rats
Groups FSH (mIU/mL) LH (mIU/mL) Estradiol (pg/mL) Aromatase (pg/mL)
Control 6.23±0.02 4.25±0.36 64.3±0.12 1.23±0.40
LTZ (1mg/kg) 2.21±0.06 5.96±0.21 12.4±0.28 0.46±0.16
CC (1mg/kg) 6.12±0.03*** 4.42±0.28*** 56.9±0.49*** 1.14±0.26***
nanosuspension (0.5mL) 5.36±0.04*** 4.98±0.42*** 38.3±0.65*** 1.01±0.32***
nanosuspension (1mL) 6.01±0.05*** 4.62±0.19*** 51.6±0.72*** 1.11±0.29***
Values are represented as Mean±SD, n=5. ***p<0.001
Example 7 (f) Effect of nanosuspension on fertility index and gestation rate: To evaluate the effect of nanosuspension on PCOS related infertility, the remaining female rats (5 per group) from each group were crossbred with male rats of proven fertility (1 male for 2 females) for 5 days. During this, the vaginal smears from female rats were collected every morning to determine the presence of spermatozoa, which confirmed the gestation in female rats (Awounfack et al., 2018). When appearance of spermatozoa was confirmed, the female rats were separated from male rats in other cages and this day was considered as the first day of gestation (Awounfack et al., 2018). The pregnant female rats were observed till parturition and at the completion of the study, fertility index (FI) and gestational rate (GR) were calculated (Awounfack et al., 2018) by the following formula,

Example 7 (g) Effect of nanosuspension on fertility index and gestation rate in rats
The gestation phase in female rats was confirmed by the presence of spermatozoa detected through vaginal cytology. The fertility index in LTZ treated rats was found 0% whereas in contrast, the rats treated with nanosuspension at 0.5mL and 1mL enhanced the fertility rate by 60% and 70% respectively as similar as that of control group rats (80%) and standard CC treated rats (80%) (Table 4).
Likewise, nanosuspension treated rats (0.5mL, 1mL) also enhanced gestation rate by 100% as compared to the LTZ treated rats which showed 0% gestation rate. The findings attained by PHF treatment was found consistent with the findings obtained from the control group rats as well as CC treated rats (Table 4).
Table 4: Effect of nanosuspension on fertility index and gestation rate
Groups Fertility index (%) Gestation rate (%)
Control (0.9% saline) 80 100
LTZ (1mg/kg) 0 0
CC (1mg/kg) 80 100
nanosuspension (0.5mL) 60 100
nanosuspension (1mL) 75 100
Example 8: Biochemical studies
Determination of the effect of nanosuspension on ovarian levels of MDA, catalase and SOD : The ovarian tissue homogenate was prepared by taking known amount of ovarian tissue in a mortal pestle, and homogenized in PBS (0.1M, pH 7.4) for 3-4 min. The mixture was then centrifuged and the supernatant was collected and utilized for the estimation of malondialdehyde (MDA, catalase and superoxide dismutase (SOD) levels. Ovarian levels of MDA, catalase and SOD were determined to evaluate the effect of nanosuspension on biochemical alterations. Findings revealed that administration of LTZ exhibited marked reduction in the ovarian levels of protective enzymes viz. catalase and SOD whereas CC treated rats showed enhanced levels of these enzymes. Similarly, administration of nanosuspension at both doses i.e., 0.5mL and 1mL also resulted in marked elevation in ovarian levels of these enzymes in a dose dependent manner (Table 5, Figure 4) when compared with the positive control rats. Moreover, ovarian level of MDA was found higher in LTZ treated rats. The oxidative effect of LTZ was markedly reversed by the administration of PHF (0.5mL, 1mL) in a dose dependent manner which exhibited significant decline in ovarian levels of MDA similarly as that of CC treated rats (Table 5, Figure 4).
Table 5: Effect of nanosuspension on ovarian levels of MDA, catalase and SOD
Groups MDA
(nM MDA/g) Catalase
(?M/min/mg protein) SOD
(mg/100g wet tissue)
Control (0.9% saline) 2.3±0.48 5.3±0.05 2.98±0.93
LTZ (1mg/kg) 8.6±0.36 0.89±0.06 0.19±0.76
CC (1mg/kg) 2.4±0.52*** 5.12±0.08*** 4.42±0.62***
nanosuspension (0.5mL) 5.9±0.63*** 3.8±0.19*** 3.26±0.54***
nanosuspension (1mL) 2.8±0.29*** 4.9±0.03*** 4.28±0.38***
Values are represented as Mean±SD, n=5. ***p<0.001
Example 9: Histopathological studies: The histological images of the ovaries from different experimental groups are illustrated in Figure 5a-5e, where control group exhibited predominance of mature follicles viz. graafian and tertiary follicles and presence of corpora lutea (indicator of ovulation) and mature oocytes, thus indicating normal ovarian physiology. In contrast, ovarian sections of LTZ treated rats demonstrated predominance of cystic follicles and few tertiary follicles. The ovary images of CC treated rats exhibited normal ovarian physiology nearly as that of control group images with the presence of corpora lutea. Following treatment with nanosuspension (0.5mL) showed presence of graafian follicles, primary follicles, tertiary follicles and corpora lutea with fewer cystic follicles in ovarian sections. The ovarian sections of rats treated with nanosuspension (1mL) demonstrated similar ovarian physiology as that of control group with the presence of corpora lutea and mature follicles predominantly. The predominance of corpora lutea in PHF treated ovarian sections of rats indicated ameliorating potential of PHF in anovulation and cystic conditions.
Example 10: In vitro drug release study: In vitro studies were performed with optimized nanosuspension in two different stimulated body fluids mainly PBS (pH, 7.4) and buffer (pH, 1.2) using dialysis membrane method. It was observed that the release in PBS 7.4 (86.27 %) was more than buffer 1.2 (29.72 %) within 48h. Therefore, PBS 7.4 was chosen as the release media for further studies. The release study of nanosuspension and fractions solution was performed in PBS 7.4 upto 48h to observe the maximum release. The nanosuspension exhibited initial burst release upto 20% in 2 h followed by slower exponential phase, thereby showing a pattern of rapid release within the first few hours and then a steady constant release during the remaining time observed. The cumulative drug release (% CDR) from nanosuspension was found 86.27 % in 48h which was slower than the % CDR from the fractions solutions which was found 80% at 4h. The release behavior of nanosuspension for 48h confirmed its optimum and sustained release as compared to the release profile of fractions solution. Figure 6 shows the in vitro release profiles of pure fractions from nanosuspension and the fractions solution.
Advantages of the invention:
1. The polyherbal nanosuspension of the present invention was found to be stable for six months at two different storage conditions, i.e., 30° C / 65 ± 5% relative humidity and 40° C / 75 ± 5% relative humidity.
2. The in-vitro drug release studies polyherbal nanosuspension of the present inventio indicated sustained release behaviour at intestinal buffer (pH 7.4) for 48h as compared to the control formulation (fraction’s mixture).
3. The acute toxicity study reported that polyherbal nanosuspension of the present inventio was found safe up to 2000mg/kg dose.
4. The polyherbal nanosuspension of the present invention significantly reversed the letrozole effects by restoring the irregular estrous after 15 (0.5mL) and 11 days (1mL) of treatment.
5. The polyherbal nanosuspension of the present inventio markedly increased the serum levels of follicle stimulating hormone in rats as compared to the diseased rats.
6. The polyherbal nanosuspension of the present invention decreased the serum levels of luteinizing hormone in rats as compared to the diseased rats. 13. The serum levels of estradiol were also significantly enhanced in rats treated with the polyherbal nanosuspension of the present invention as compared to the diseased rats.
7. The polyherbal nanosuspension of the present invention significantly reversed the effect of Letrozole (aromatase inhibitor) by enhancing the serum level of aromatase in rats.
8. The administration of polyherbal nanosuspension of the present invention markedly restored the abnormal ovarian levels of oxidative stress markers viz. malondialdehyde, catalase and superoxide dismutase in the rats.
9. In histopathological studies, polyherbal nanosuspension of the present invention has shown tremendous potential in ameliorating anovulation and cystic conditions in rats as evidenced by the presence of mature follicles and corpora lutea (indicator of ovulation) in the histological ovarian sections.
10. The polyherbal nanosuspension of the present invention is found to be useful in ameliorating fertility rate and gestational index in female rats.

We claim:

1. A polyherbal nanosuspension for the treatment of polycystic ovarian syndrome associated conditions and related infertility, said nanosuspension comprising of bioactive fractions prepared from Cedrus deodara extract, Rhododendron arboreum extract, Prunus persica extract, surfactant and a polymer.
2. The polyherbal nanosuspension as claimed in claim 1, wherein the fraction is obtained from, bark, flower and seed extracts.
3. The polyherbal nanosuspension as claimed in claim 1, wherein the Cedrus deodara fraction is obtained from bark extract, Rhododendron arboreum fraction is obtained from flower extract and Prunus persica fraction is obtained from seed extract.
4. The polyherbal nanosuspension as claimed in claim 1, wherein ratio between Cedrus deodara bark extract, Rhododendron arboreum flower extract, Prunus persica seed extract is in the range from 1 to 10: 1 to10 : 1 to 10, respectively.
5. The polyherbal nanosuspension as claimed in claim 1, wherein the ratio between fractions of Cedrus deodara bark extract, Rhododendron arboreum flower extract and Prunus persica seed extract is 1:1:1.
6. The polyherbal nanosuspension claimed in claim 1, wherein the extract is a solvent extract.
7. The polyherbal nanosuspension as claimed in claim 6, wherein the solvent is selected from butanol, water, methanol, hydromethanol, acetone, dichloromethane, hexane, ether, chloroform.
8. The polyherbal nanosuspension as claimed in claim 1, wherein the surfactant is selected from surfactants sorbitan fatty acid esters, (Spans), polysorbates (Tweens), and poloxamer (Pluronics).
9. The polyherbal nanosuspension as claimed in claim 1, wherein the poloxamer is Poloxamer 407.
10. The polyherbal nanosuspension as claimed in claim 1, wherein the polymer is selected from surfactants sorbitan fatty acid esters (Spans), polysorbates (Tweens), and poloxamer (Pluronics).
11. The polyherbal nanosuspension as claimed in claim 1, wherein the surfactant is selected from hydroxypropylmethyl cellulose, hydroxypropyl cellulose, methyl cellulose, soy lecithin and hydrogenated soy lecithin.
12. The polyherbal nanosuspension as claimed in claim 1, wherein the said nanosuspension is further lyophilized to obtain lyophilized powder form.
13. The polyherbal formulation as claimed in claim 1 wherein the particle size of the nanosuspension is between 50 to 4000 nm.
14. A process for preparing the polyherbal nanosuspension as claimed in claim 1, said process comprising,
a) preparing solvent extracts of extracts of Cedrus deodara bark, Rhododendron arboreum flowers and Prunus persica seeds separately by extraction,
b) investigating uterine smooth muscles relaxant activity of solvent extracts of each of the extracts obtained in (a) against carbachol induced contraction in isolated uterine smooth muscles of rats,
c) selecting the best extract which showed uterine smooth muscles relaxant activity and carrying out fractionation of the best extracts through a series of liquid-liquid partitions,
d) investigating the prepared fractions in (c) for uterine smooth muscles relaxant activity against carbachol induced contraction in isolated uterine smooth muscles of rats,
e) selecting the fractions of Cedrus deodara bark, Rhododendron arboreum flowers and Prunus persica seeds from (d) with higher uterine smooth muscle relaxant activity to prepare polyherbal nanosuspension,
f) mixing the fractions of (e) of Cedrus deodara bark, Rhododendron arboreum flowers and Prunus persica seeds in a particular ratio in an inert solvent to obtain a mixture,
g) preparing an aqueous solution of the surfactant,
h) preparing an aqueous solution of the polymer,
i) mixing (g) and (h) in a beaker and stirring using a magnetic stirrer,
j) homogenizing the mixture obtained in (f),
k) mixing (i) and (j),
l) conducting probe sonication for 1 min to obtain the nanosuspension.
15. The process as claimed in claim 14, wherein the fractionation is done through a series of liquid-liquid partitions using solvents such as chloroform, butanol and water.
16. The process as claimed in claim 14, wherein the inert solvent is selected from N-methyl-2-pyrrolidone (NMP), N,N dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and N,N-dimethylacetamide (DMAc).
17. The process as claimed in claim 14, wherein the nanosuspension thus obtained is lyophilized with a cryoprotectant to obtain polyherbal formulation in powder form.
18. The process as claimed in claim 17, wherein the cryoprotectant is selected from sucrose, lactose, mannitol, and polyethylene glycol.