NUTRACEUTICAL COMPOSITIONS AND METHODS OF USE OF SAFED

MUSLI EXTRACTS

TECHNICAL FIELD OF THE INVENTION:

The invention relates to the nutraceutical compositions of safed musli as extract and juice and method of use of the same in improving or enhancing the aphrodisiac activity. The compositions are also used to alleviate stress and tiredness in individuals, thus improving sexual desire and behaviour.

BACKGROUND AND PRIOR ART OF THE INVENTION:

Search for an effective aphrodisiac dates back to centuries. Aphrodisiacs are classified as agents that increase libido, potency, and/or sexual pleasure. Various substances of animal and plant origin have been used as aphrodisiacs in different cultures; pharmacological basis of some has been worked out that has resulted in the understanding of their mechanisms of action. For increasing libido, ambrein, a major constituent of Ambra grisea, is used in Arab countries. This tricyclic triterpene alcohol increases the concentration of several anterior pituitary hormones and serum testosterone. Bufo toad skin and glands containing bufotenine is the active ingredient in 'love stone' popular in West Indies and in the Chinese medication chan su. For increasing potency, panax ginseng is often used in traditional Chinese medicine, works as an antioxidant by enhancing nitric oxide synthesis in the endothelium of many organs. Furthermore, Cantharidin ("Spanish fly"), a chemical compound with vesicant properties derived from blister beetles, has been used as a sexual stimulant for increasing sexual pleasure. Its mode of action is believed to be by inhibition of phosphodiesterase and protein phosphatase activity and stimulation of beta-receptors, inducing vascular congestion and inflammation. Sildenafil (Viagra ^® ) is a synthetic compound that has been effective for erectile dysfunction. Safed musli is a traditional medicinal plant in India, which belongs to Liliaceae family and grows as a wild plant in Bastar Forest (MadhyaPradesh), Dangs Forest (Gujarat), Mount Abu, Mahi, Aravalli Hills (Rajasthan) of India.

In India about eight species of safed musli are reported - Chlorophytum borivillianum, Chlorophytum arundinaceum, Chlorophytum tuberocum, Chlorophytum malabericum,

Chlorophytum attenuatum, Chlorophytum breviscapum, Asparagus fllicinus, A. gonoclados. Of these, Chlorophytum borivillianum, Chlorophytum arundinaceam, and Chlorophytum tuberosum was collected by our tribes from the forest.

Chlorophytum borivillianum is the only species that is commercially cultivated and is widely cultivated in different parts of India like Andhra Pradesh, Rajasthan, Madhya Pradesh, Maharashtra and Gujarat. It is a potent herb whose root tubers have been used for its aphrodisiac and health promoting properties since thousands of years in India. It contains alkaloids, steroid, saponin, polysaccharides, carbohydrates, proteins, minerals, vitamins etc and is often used in Ayurvedic medicine.

Safed musli supports physiological balance or homeostasis. It improves access to energy stores, helps to improve stamina and resistance to stress and tiredness, helps in mobilization of the body's nutritional resources, helps to normalize various indices such as blood pressure or blood sugar and improves function and well being. Although its action is primarily supportive and restorative, it treats male sexual inadequacies like oligospermia, lack of libido, impotency, general debility and so on.

International PCT application WO 03017784 describes herbal health protective, promotive and neutraceutical formulation for women and expectant mothers, wherein the formulation comprises extract obtained from herbs of medicinal plants of genera Centella, Chlorophytum, Boerhaavia, Sida and optionally Withania somnifera, Pueraria tuberose, Asparagus recemosus and Saraca indica. The formulations referred in the aforesaid application are selected from the genera Chlorophytum, particularly Chlorophytum tuberosum.

Though safed musli is a potent herb and there are plenty of references in many Ayurveda classics, there is no prior publication on Chlorophytum borivillianum species regarding its extraction, formulation, processes and method of use in enhancing or improving aphrodisiac activity and other parameters that causes sexual dysfunction such as stamina, stress and tiredness.

There is an increasing trend in the use of chemically developed products to treat male sexual inadequacies. Prescription drugs such as Viagra (Sildenafil, a cGMP phosphodiesterase inhibitor) are used to treat erectile dysfunction. However such compounds are reported to be fraught with several side effects and carry an additional risk to persons with cardiovascular history. With the available remedies, there seems to be a need to develop a formulation with lower risk factors, decreased side effects and higher benefits thus making it safe for use.

SUMMARY OF THE INVENTION:

The invention provided herein describes the compositions of safed musli extract and their use in improving and enhancing aphrodisiac activity in human beings.

The composition, particularly herbal ready-to-drink compositions, comprises extracts of safed musli [Chlorophytum borivillianum), which is used for its comprehensive adaptogenic and invigorating effects, ex: as a health tonic. A process for preparation of safed musli composition is also disclosed herein. More particularly, the present stable herbal ready to drink compositions comprises safed musli with or without Aloe vera and process for preparation thereof. Use of stevia in the ready-to-drink juices is also discussed.

One aspect of the invention is to provide effective amount of safed musli extract alone or in combination with Aloe vera for treatment of sexual dysfunctions, erectile dysfunction and sperm autogenesis.

Another aspect of the invention is to provide effective amount of safed musli extract in combination with Aloe vera and other herbs including Morinda citrifolia, Stevia reboudiana, Asparagus racemosus, Glycyrrhiza glabra and Hemidesmus indicus or combination of safed musli with any one or more number of herbs.

In another aspect, the invention provides use of safed musli extract for reducing stress and in turn balancing the mood in stressful conditions in subjects comprising administering effective amount of safed musli compositions. The composition described herein can be

combined with one or more of herbal compounds such as Aloe vera and one or more number of herbs to produce the desirable effect.

Yet another aspect of the invention is to increase the antioxidant activity and nitric oxide levels in subjects to treat sexual dysfunctions, erectile dysfunction and sperm autogenesis by administering the safed musli compositions. Safed musli extract also contains steroidal saponins, and the saponins are in general a good antioxidant agent, since they are the free radical scavengers. Moreover safed musli root contains considerable quantity of zinc and iron, wherein the nutritive value analysis of safed musli in the present invention depicts the content of zinc as 3.75mg/100g and iron as 4.65mg/100g. Zinc is also responsible for the antioxidant property of safed musli.

The invention also provides safed musli composition in a ready-to-drink form comprising the safed musli extract at a concentration of 0.7-3% v/v, 7-15% w/v sweetening agents, 0.4% v/v flavours, 120 ppm preservatives, pH adjusting agents, viscosity adjusting agents and distilled water, adjusted to a pH of 3.0-3.5. The preservatives in the said composition are 0.1%w/v sorbate and 0.08%w/v benzoate, the viscosity-adjusting agent used is 0.5%w/v xanthan and the pH adjusting-agent is citric acid.

The present invention also discloses different method of preparation of safed musli extract as powder form by using either water or any organic solvents or aqueous organic solvents in different ratios as extracting medium. Further it is also disclosed here the method of preparation of safed musli juice from roots.

Another aspect of the invention provides a process for the preparation of stable composition of safed musli, the said process comprises of:

(a) Dissolving safed musli juice, sucrose, flavour and sorbate in distilled water and making up volume to 200 ml;

(b) Adjusting pH of the solution with citric acid, adding xanthan, stirring and filtering the solution;

(c) Heating at 8O ⁰ C, cooling, filling and sealing in a container.

DETAILED DESCRIPTION OF THE INVENTION:

Herbal compounds are considered as better treatment options for a variety of physiological conditions due to their fewer side effects. According to World Health Organization, 80% of the world population use herbal medicines. Absence of synchronization among physical, mental and emotional states may lead to lack of sexual drive thus resulting in sexual dysfunctions. Sexual dysfunction refers to a problem during any phase of the sexual response cycle that prevents the individual from experiencing satisfaction from the sexual activity.

Sexual dysfunction can be a result of physical or psychological causes both of which can be treated. The physical causes include heart problems, diabetes, neurological disorders, chronic diseases and so on. Hormonal imbalance is also an important aspect that may dampen the sexual response in an individual. The psychological causes include stress, anxiety, relationship problems and depression.

The present invention discloses safed musli compositions and its use in enhancing or improving aphrodisiac activity and improving or treating stressful conditions. The instant invention shows increase in antioxidant activity, nitric oxide concentrations and sex hormones has also been described in the instant invention that result in the improving the sexual response cycle.

The said composition is in the form of ready-to-drink juice comprising extract of safed musli {Chlorophytum borivillianum) more particularly, the present invention discloses a process for the preparation of aqueous extract of safed musli for medicinal use.

Safed musli belongs to family of Liliaceae. It is a rich source of over 25 alkaloids, vitamins, minerals, proteins, carbohydrates, steroid, saponins, polysaccharides etc. Roots of safed musli {Chlorophytum borivillianum) are used for the preparation of nutritive tonic. Dried roots of Chlorophytum borivilianum, safed musli, belonging to family Liliaceae are used for the preparation of the extract.

Stable ready-to-drink juices of safed musli alone or in combination with Aloe vera juice and herbs (Morinda citrifolia, Stevia reboudiana, Asparagus racemosus, Glycyrrhiza glabra and Hemidesmus indicus) and with any one or more number of herb are developed by the process described herein. While examples of juices alone or in combination, using sugar is described, such combinations using Morinda citrifolia, Stevia reboudiana, Asparagus racemosus, Glycyrrhiza glabra and Hemidesmus indicus in conventional dosage forms are also discussed.

Safed musli has many additional beneficial effects including properties of adaptogen, immunomodulation and anti-aging and is also a rich source of zinc and iron.

Zinc increases the activity of natural killer cells and boosts the production of antibodies in response to infections. As Zinc is required for the production of the antioxidant enzyme superoxide dismutes, which neutralizes potentially damaging free radicals, it may also help to fight cancer. The mineral also aids in the production of testosterone and boosts sperm production. Physiological supplementation of zinc in ageing and in age - related degenerative diseases corrects immune defects, reduces infection relapse and prevents ageing. Zinc is also an antioxidant mineral that is important in protecting sperm against free radical attack.

Iron helps in the formation of hemoglobin, which is present in the red blood cells, and myoglobin, a molecule that transports oxygen in muscles. It also prevents iron deficiency anemia, as well as stimulates the immune system. It is also used to improve muscular and athletic performance, and prevents fatigue.

Composition and Process of Preparation of safed musli extracts:

Process of preparation of safed musli extract is disclosed in patent Application No. 1262/MUM/2003 titled 'Process for Extraction of safed musli and characterization of the extraction thereof and compositions of safed musli has been described in patent Application No. 603/MUM/2004 titled 'Safed Musli compositions and process of preparation thereof both of which are incorporated herein by reference. Evaluation of aphrodisiac property of safed musli has been disclosed in Application No.

984/MUM/2006 titled 'Process for Standardisation and evaluation of aphrodisiac property of Safed Musli' dated 22/06/2006 is also incorporated herein by reference.

This safed musli extract is further used to prepare the said composition. 10% of safed musli extract (0.7 -3.0% w/v) is used for preparation of ready-to-drink juice.

Physical evaluation of safed musli extract carried out is tabulated as follows:

Table 1: Physical Evaluation

Chemical evaluation of the said safed musli extract is carried out and tabulated as follows:

Table 2.1: Chemical evaluation (Qualitative test)

Table 2.2: TLC (Thin Layer Chromatography) Profile of the Safed Musli extract

Two spots were obtained with the extract.

Table 2.3: HPLC of the isolated compound is supposed to be the marker

Single peak at 246 nm

Stable herbal compositions are prepared in the form of ready-to-drink juice. The said stable herbal compositions comprise safed musli extract (juice) along with other excipients like sucrose, flavor, sorbate etc. The process for preparation of the stable herbal composition in ready-to-drink juice form of safed musli comprises a) Mixing safed musli juice, sucrose, flavor and sorbate; b) Dissolving the said mixture in distilled water to make 200 ml solution; c) Adjusting the pH of the said solution to 3.0 - 3.5 with citric acid followed by the addition of xanthan gum while stirring; and d) Filtering and heating the solution to 80 ° C. e) The aforesaid preparation of safed musli juice is filled and sealed in storage containers at 4 ⁰ C and is stable for a period of 3 months. The said stable herbal compositions comprise safed musli extract (juice) in combination with Aloe vera juice along with other excipients like sucrose, flavor, sorbate etc. The extract is also provided in combination with the herbs (Morinda citrifolia, Stevia reboudiana, Asparagus racemosus, Glycyrrhiza glabra and Hemidesmus indicus).

The said safed musli composition comprises of 0.7 to 3.0 % v/v of safed musli extract; 7 to 15% w/v of sucrose, flavor, sorbate, xanthan and citric acid. The pH of the composition is adjusted to 3.0 to 3.5. The preferred concentration of safed musli extracts in the said composition is in the range of 1.25 to 2.5 % w/v of the total composition. The preferred concentration of sucrose in the said composition is 5.0% of the total composition. Flavor is used at a concentration of 0.4% v/v of the total composition. Sorbate is used at a concentration of 120ppm. Xanthan is used as viscosity modifier at a concentration of 0.5 w/v of the total composition.

A process for the preparation of the safed musli compositions in the form of ready-to- drink juice comprises dissolving safed musli extracts, sucrose, flavor, sorbate in distilled water to making volume to 200ml; adjusting the pH of the said solution to 3.0 - 3.5 with citric and; adding xanthan to the said solution while stirring; filtering the said solution; heating the said solution to 80 ⁰ C; filing and sealing the said composition in storage containers at 4°C.

Evaluation of Aphrodisiac property of safed musli {Chlorophytum borivilianum) in Wistar rats:

The scientific validation and the therapeutic profile of safed musli with respect to aphrodisiac, antioxidant activities are investigated in the present study and compared with the known herbal and synthetic preparation from the market. The evaluation is carried out in the animals and the data is interpreted as behavioural changes supported by biochemical estimation of male sexual hormones and other parameters such as nitric oxide and antioxidant properties as given in examples.

The Aphrodisiac study was performed in Wistar rats of either sex weighing between 150- 200gms. Tentex forte is administered in the form of 1% gum acacia suspension. All the drugs are prepared fresh every day and administered orally using soft gavages. Adult rats are used for the aphrodisiac study, and maintained under standard conditions. To quantify the total sexual behaviour, mounting female rats are paired with males treated with single dose. Animals are observed for three hours with gap of 15 minutes and their behaviours recorded.

In stress and sexual behavioural studies Wϊstar rats of either sex are used. The animals are divided into eight groups and each group contained 6 males and 6 females, and the doses that are administered to animals are based on the human dose. Immobilization and starvation for 24 hrs are selected as a method for stress. Sexual response such as number of sniffing, genital grooming, mounts and intromission are visually monitored for 1 hour. The experiment procedure is repeated after a gap of 8 days for 3 times.

In antioxidant study DPPH (2, 2 -diphenyl-2-picryl-hydrazyl) method is used to monitor and compare the antioxidant activity of foods and drugs. Trolox is used as a standard reference.

In the estimation of nitric oxide concentration, the nitric oxide levels are observed after 45 days aphrodisiac study and aphrodisiac study after stress. Production of Nitric oxide is estimated by measuring the amount of nitrite in the serum sample collected on 0, 15 ^th , 30 ^th , and 45 ^th using a spectrophotometric assay based on Griess reaction (PROMEGA, US).

The estimation of sex hormone comprises, determining the concentration of serum testosterone and progesterone in duplicate using competitive ELISA kits supplied by IBL (HAMBURG). Testosterone levels are observed after 45 days aphrodisiac study and aphrodisiac study after stress.

Definitions:

The term 'aphrodisiac' in the present invention refers to an agent that, increases the semen volume and total sperm count, improves or enhances sexual response, overall sex drive, sexual potency and a general tingling sensation.

By 'combination', it is meant that safed musli extract or preparation is combined with one or more herbs (ex: Morinda citrifolia, Stevia reboudiana, Asparagus racemosus, Glycyrrhiza glabra, Hemidesmus indicus and Aloe vera).

The second agent, e.g. Aloe vera and herbs (Morinda citrifolia, Stevia reboudiana, Asparagus racemosus, Glycyrrhiza glabra and Hemidesmus indicus) are added to safed

musli extract in an amount that is considered effective and non-toxic. The second agent is preferably mixed with the safed musli preparation. Alternatively, it is also provided subsequently.

'Effective amount' as described herein means amounts that can trigger the said aphrodisiac effect without causing physical or physiological toxicity to human being.

'Sexual dysfunction' refers to a problem during any phase of the sexual response cycle that may prevent the individual from experiencing satisfaction from the sexual activity. The sexual response cycle has four phases: excitement, plateau, orgasm and resolution. Sexual dysfunction as described in the invention also means lack of sexual drive to initiate sexual activity.

'Antioxidant agent' as described herein, is the scavenging free radicals from the body that increases antioxidant activity and also leads to anti-aging, adaptogenic and immunomodulatory activities.

The term 'adaptogen' refers to an agent that increases stamina, endurance, concentration, mental clarity, strength and also reduces tiredness.

The term 'anti-ageing' in the present invention refers to prevention or treatment of immune defects in age-related degenerative diseases, prevention of infection relapse related to ageing.

The following examples are for the purpose of illustration of the invention only and are not intended in any way to limit the scope of the invention.

A] Process of Preparation of safed musli composition and attributes of safed musli composition:

Example 1: Preparation of non-aqueous extract of safed musli:

The crude powder of safed musli root was extracted with solvents like ether and primary alcohol more particularly methanol and petroleum ether. The crude powder of safed musli

was first extracted with petroleum ether to remove fatty materials from the powder. Subsequently the material was subjected to methanol extraction at 50-55° C for a time sufficient to extract all methanol soluble constituents, which was approximately upto 2 hours. The mixture was then cooled and filtered. The filtrate so obtained was concentrated by removing the solvent by distillation under reduced pressure. The yield obtained was 5 % on the dry weight of raw safed musli powder.

The temperature range for the non-aqueous extraction is 30 to 70° C, particularly 50 to 55° C. The time required for complete non-aqueous extraction of safed musli depends on the temperature used for the extraction. At optimum temperature of 50-55° C time required for extraction was upto 2 hours.

Characterization of the non-aqueous extracts:

The above non-aqueous extract was subjected to analysis of saponins, steroids and other phyto-constituents of secondary metabolite etc. Various constituents were separated from the respective class by solvent fractionation or chromatographic methods. The isolated compounds were characterized by chemical methods, physical parameters, chromatographic techniques (TLC, CC, PC, HPLC etc) and spectroscopic examination (UV, IR, MS, NMR etc).

Example 2: Preparation of ready to drink juice (1.25%):

Safed musli juice, sucrose, flavor and sorbate are all dissolved in distilled water to make 200 ml. The pH of the solution is adjusted to 3.0 - 3.5 with citric acid followed by the addition of xanthan while stirring. The solution is finally filtered, heated to 80° C, filled and sealed into bottles and refrigerated.

Table 3: Ingredients used for the preparation of ready to drink juice 1.25%

Example 3: Preparation of ready to drink juice (2.5%)

Safed musli juice, sucrose, flavor and sorbate are all dissolved in distilled water to make 200 ml. The pH of the solution is adjusted to 3.0 - 3.5 with citric acid followed by the addition of xanthan while stirring. The solution is finally filtered, heated to 80° C, filled and sealed into bottled and refrigerated.

Table 4: Ingredients used for the preparation of ready to drink juice 2.5%

Example 4: Preparation of ready to drink juice comprising safed musli extract with Aloe vera (2.5%):

Safed musli juice, Aloe vera juice, sucrose, flavor, benzoate and sorbate are mixed. The pH of the solution is adjusted to 3.0 - 3.5 with citric acid followed by the addition of xanthan gum while stirring. The solution is finally filtered, filled and sealed into bottles and refrigerated.

Table 5: Ingredients used for the preparation of ready to drink juice comprising safed musli extract with Aloe vera (2.5%)

The said ready-to-drink juice form of the said compositions is optionally, sweetened and flavored suitably with added stabilizers, as mentioned in the detailed description. The said composition can be formulated optionally with stevia in a conventional dosage form.

Stability at 3 months showed no changes in the juice for 3 months from the date of preparation except for the slight browning of the colour of the juice.

The nutritional value of safed musli — Aloe vera combination juice is tabulated hereunder:

Table 6: Nutritional value of safed musli - Aloe vera combination juice

B] Efficacy and Behavioral Studies:

Example 5: Efficacy Studies on Safed Musli:

5.1 Study Design:

A detailed pre clinical study was conducted to show the potent aphrodisiac properties of safed musli. For this purpose (1) aqueous extract of safed musli (2) methanolic extracts of safed musli, (3) dry root powder of safed musli were used; and efficacy was experimentally compared with dry root powder of (1) Ashwagandha (Withania somniferά), (2) Methanolic extract of Korean Ginseng (Panax ginseng), (3) Sildenafil citrate (VIAGRA ^® ) - a known synthetic aphrodisiac agent and (4) Tentex Forte (a polyherbal aphrodisiac formulation manufactured and marketed by Himalaya HealthCare Company, India). The study was done on the healthy, adult Wistar rats of both sexes. The dose of the drug was determined based on the approved human dosages and the same has been described in the Table No. 7.

Table 7: Details of the drugs their dosage administered to animals.

Methodology:

Aphrodisiac activity of safed musli extracts and other drugs were studied in Wistar rats. To evaluate the aphrodisiac activity, four sexual behavioural parameters were selected i.e., (i) sniffing, (ii) genital grooming, (iii) mounting and (iv) intromission.

Healthy and adult Wistar rats of either sex weighing between 150-200 gm were used. Tentex Forte was administered in the form of 1% gum acacia suspension. All other drugs were prepared fresh every day and administered orally to male rats using soft gavages. The rats were maintained under standard animal house conditions. To monitor the total sexual behaviours, the female rats were paired with males treated with single dose of the drug. Animals were observed for 3 hours with an interval of 15 minutes and their behaviours were recorded.

Biochemical studies:

To understand the physiological changes in the treated rates, the following biochemical aspects were analyzed. Further these physiological factors are supportive to the aphrodisiac activity.

(1) Estimation of testosterone and progesterone

(2) Estimation of nitric oxide production

(3) Antioxidant activity

Blood samples were collected from male rats on 0, 15 ^th , 30 ^th and 45 ^th day. On 46 ^th day the male rats were sacrificed for histopathological evaluation by cervical dislocation and the analysis was done.

5.2 Estimation of testosterone and progesterone:

Testosterone and progesterone concentrations were determined in duplicate using competitive ELISA method. Levels of hormones were estimated on 0, 15 ^th , 30 ^th , 45 ^th day after the administration of the drugs in both aphrodisiac study and aphrodisiac study after stress induced condition (Table 8 and Table 9).

Table 8: Testosterone estimation after 45 days in male rats:

Table 9: Testosterone estimation after stress:

Results and observation:

The level of sexual hormones was significantly increased in safed musli aqueous and methanolic extract, and refers to table 8 and table 9.

5.3 Estimation of nitric oxide production:

The production of nitric acid is an important physiological phenomenon for the successful sexual act, since nitric oxide is one of the key agents for blood flow in to the penis and

thus it is helpful for the sustained penile erection. Nitric oxide levels were estimated at 0, 15 ^th , 30 ^th and 45th day after the administration of the drugs in both aphrodisiac study and aphrodisiac study after stress induced condition (Table 10 and Table 11). Production of nitric oxide was estimated by measuring the amount of nitrite present in the serum samples using a spectrophotometry assay method based on Griess reaction.

Table 10: Nitric oxide estimation after 45 days in male rats

Table 11: Nitric oxide estimation after stress:

Results and observation:

Nitric oxide concentrations significantly increased only in animal groups treated with aqueous and methanolic extract of safed musli, methanolic extract of ginseng and sildenafil citrate (Viagra). The extent of intromission was found maximum in safed musli aqueous extract, which suggests that it helps in erectile dysfunction in which the nitric oxide concentration may play a vital role (Table 10 and Table 11).

5.4 Antioxidant activity:

There are several in-vitro methods available to study the antioxidant property of a drug or any chemical compound. DPPH (2, 2 -diphenyl-2-picryl-hydrazyl) method was used in this study to monitor and compare the antioxidant activity of safed musli and other drugs. This is a simple, rapid and inexpensive method to measure antioxidant capacity that involves the use of free radical. DPPH is widely used to test the ability of compounds to act as free radical scavenger by donating its hydrogen atoms that can be estimated on the basis of colour development by using spectrophotometry analysis.

Trolox (6-hydroxy 2,5,7,8- tetramethylchroman^-carboxylic acid) was used as a reference standard, which is a water-soluble derivative of vitamin E with potent antioxidant property.

Table 12: HPLC Analysis (Anti-Oxidant Property)

Table 13: Colorimetric Analysis (Anti-Oxidant Property)

Results and observation:

Aqueous and methanolic extracts of safed musli and methanolic extract of Ginseng had shown good antioxidant activity. The radical scavenging activity of safed musli aqueous extract was found to be significantly higher than methanolic extract of safed musli (Table 12 and Table 13).

Further, histopathological studies conducted in the liver, lung, kidney, spleen brain and testis of the animals identify side effects and toxicity of the drugs. The histopathological study reveals that the organs studied did not show any specific toxicity in safed musli

treated group when compared to control groups. The animals treated with Viagra and

Ashwagandha have shown mild degree of toxicity to liver, lung, kidney, spleen, brain and testis.

No significant changes were observed in levels of glucose, albumin, and total protein.

Example 6: Studies on the aphrodisiac activity after inducing Stress.

In this investigation four sexual behavioral parameters had been employed to ascertain the efficacy of safed musli after inducing the stress. Stress is an important factor, which shows influence on male sexual behaviour. In this study aphrodisiac activity after stress conditions was estimated with the parameters of sniffing, genital grooming, mounting and intromission.

Method:

Healthy Wistar rats of both the sexes weighing between 150-200 gm and 10 weeks older animals were used. The animals were divided into eight groups and each group contained 6 males and 6 females. The doses that are administered to animals are based on the human dose. Immobilization and starvation for 24 hrs were the 2 parameters selected as stressors (stress inducing agents). Sexual response such as number of sniffing, genital grooming, mounting and intromission were visually monitored for 1 hour. The whole experiment was repeated 3 times after an interval of 8 hour between each experiment.

Results and observation:

It was clearly observed that administration of aqueous extract of safed musli alleviate the mental stress of the rats, which were induced stress. Safed musli aqueous extract significantly increased the number of sniffs and genital grooming. The extent of mounting was also observed maximum with safed musli aqueous extract treatment. Intromission did not occur in stressful condition with all the groups.

While the present invention is described above in connection with preferred or illustrative embodiments, these embodiments are not intended to be exhaustive or limiting of the invention. Rather, the invention is intended to cover all alternatives, modifications and equivalents included within scope, as defined by appended claims.

BRIEF DESCRIPTION OF THE FIGURES:

FIGURE 1 illustrates UV spectrum of marker compound. The compound shows maximum absorption at 275nm.

FIGURE 2 illustrates the IR spectrum of marker compound.

The IR spectra of the compound shows peaks at 3434.9 cm ^" ', 3111.7 cm ^" ', 2853.8 cm ^λ ,

1669.3 cm ⁴ , 151.9 cm ^"1 , 1358 cm ^"1 , 1280.2 cm ^"1 , 1046.7 cm - ¹ , 1036.3 cm - ¹ , 979.2 cnT ¹

953.3 cm ⁴ , 828 cm ^'1 , 771.7 cm ^"1 .

Groups like C=O, O-H found to be present, the other bands are due to CH ₃ and CH ₂ bending and stretching.

FIGURE 3 illustrates the Proton NMR spectrum of the marker compound.

The proton NMR spectrum shows peaks at 1.642, 4.589, 4.639, 4.689, 6.569, 6.578,

7.215, 7.225 and 9.634 ppm.

FIGURE 4 illustrates the C ¹³ NMR spectrum of the marker compound.

The C ¹³ NMR spectrum shows peak at 64.731, 111.944, 121.911, 152.942, 157.357 and

177.803 ppm. The peak at 76.872, 77.192 and 77.513 ppm is those of the solvent spectra enclosed.

FIGURE 5 shows Mass spectrum of the marker compound.