FORM 2 THE PATENT ACT 1970 (39 of 1970) &
The Patents Rules, 2003 PROVISIONAL SPECIFICATION(See section 10 and rule 13)
1. TITLE OF THE INVENTION:
"Process for Standardisation and evaluation of aphrodisiac property of Safed Musli"
2. APPLICANT (S)
(a) NAME: NANDAN BIOMATRIX LIMITED
(b) NATIONALITY: Indian Company incorporated under the Indian Companies ACT, 1956
(c) ADDRESS: C-002/C-003, Ground Floor, Gokul Plaza, Thakur Complex, Kandivli (East), Mumbai - 400 101, Maharashtra, India.
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention.


The contents of the specification of the following related patent applications may be
treated as incorporated as herein by reference.
1262/MUM/2003 titled "Processes for extraction of Safed musli and characterization of
the extracts thereof.”
603/MUM/2004 titled "Safed Musli compositions and process for preparation thereof.”
Technical Field of the invention:
The present invention relates to providing a validated process for evaluation of aphrodisiac and antioxidant properties of Safed musli (Chlorophytum borivillianum) and for quantifying the therapeutic benefits of the herbal formulation by in-vivo animal studies.
Background and Prior Art:
Safed Musli belongs to Liliaceae family and grows as a wild plant in Bastar Forest (Madhya Pradesh), 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 fdicinus, A. gonoclados. Of these Chlorophytum borivillianum, Chlorophytum arundinaceam and Chlorophytum tuberosum are collected by our tribes from the forest.
Chlorophytum borivillianum (Safed Musli) is the only species which is under commercial cultivation and is widely cultivated in different parts of India like Andhra Pradesh, Rajasthan, Madhya Pradesh, Maharashtra, Gujarat.
Safed Musli contains alkaloids, steroid, saponin, polysaccharides, carbohydrates, proteins, minerals, vitamins etc.
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Chlorophytum borivillianum (Safed Musli) is a divine grace herb which offers all the effects required for the achievements of health par excellence or for attaining the ultimate positive health, i.e. Balya - provides energy to each cell, Rasayana - rejuvenates the entire body-mind-soul complex, Vrishya - effectuates both qualitative and quantitative increase in Shukra -the terminal of seven physiological systems (dhatus) resulting in experiencing bliss in sexual intercourse and producing better pregnancy.
Safed Musli restores and maintains the physical harmony - the equipoise of Dosha-Dhatu- Mala so as to render these microcosms to interplay in symphony with the macrocosm. This state of harmony is the ultimate response of human.
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, etc.
United States Patent Application 20060015963 discloses commercially viable process for in-vitro mass culture of Chlorophytum borivilianum.
United States Patent Application 20060062863 discloses an obesity control agent with health-restorative and health-promotional benefits to humans comprising the extract of Chlorophytum species.
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. Herb for the formulations is selected from genera Chlorophytum, particularly Chlorophytum tuberosum.
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International PCT Application WO 03006036 describes pharmaceutical or medicinal preparation comprising mixture of eleven herbs namely Withania somnifera, Chlorphyton borivilianum, Boerhavia diffusa, Elephantopus scaber, Moringa oleifera, Tecoma undulata, Bauhinia purpurea, Ficus racemosa, Cyperus rotundus, Sphaeranthus acmella and Tinospora cordifolia. The herbal formulation is effective for the treatment of cancer, in particular squamous cell carcinomas, tumours and other metastatic states, including lung cancer.
The Prescription drugs such as Viagra (Sildenafil, a cGMP phosphodiesterase inhibitor), is used to treat erectile dysfunction. However such compounds are fraught with several side effects and carry an additional risk to persons with cardiovascular history.
Herbal drugs are safe, effective and have fewer side effects. According to World health Organisation, 80% of the world population uses the herbal medicines. There is a lack of synchronization among physical, mental and emotional states and their existing problems such as male sexual dysfunction. There arises a need for an effective drug for treating the male sexual dysfunction. Chlorophytum borivillianum (Safed Musli) herb is used for aphrodisiac activity. The present invention provides an evaluation study for the aphrodisiac property of Safed Musli.
Objects of the Invention:
The main object of the present invention is to provide a process for evaluation of the aphrodisiac property of Safed Musli (Chlorophytum borivillianum).
It is another object of the present invention to provide scientific validation and the therapeutic profile of Safed Musli with respect to aphrodisiac activity, antioxidant activity, nitric oxide concentration, testosterone concentration and also aphrodisiac activity of Safed Musli on stressed conditions.
It is further an object of the present invention, wherein the evaluation has been carried out in the animals and the data has been interpreted with behavioural changes supported by
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biochemical estimation of male sexual hormones and other parameters such as nitric oxide and antioxidant properties.
It is yet another object of the present invention to provide an evaluation study making use of Wistar rats for evaluation of the aphrodisiac property of Safed Musli by monitoring and recording the parameters such as sniffing, genital grooming, mounting and intromission.
In yet another object of the invention the aphrodisiac activities of Safed Musli are compared with the known herbal and synthetic preparations available like Ashwagandha dry root powder, Sildenafil citrate (Viagra), Korean Ginseng methanolic extract and Tentex Forte.
Summary of the Invention:
The present invention relates to process for standardization and evaluation of aphrodisiac property of Safed Musli (Chlorophytum borivillianum). The scientific validation and the therapeutic profile of Safed Musli with respect to aphrodisiac, antioxidant activities are compared with the known herbal and synthetic preparations available in the market like Ashwagandha dry root powder, Sildenafil citrate (Viagra), Korean Ginseng methanolic extract and Tentex Forte.
The effect of the Safed Musli on aphrodisiac property, stress conditions, antioxidant activity and on levels of nitric oxide release and testosterone are studied in the present invention. The evaluation study as per the present invention makes use of Wistar rats for the evaluation of the aphrodisiac property of Safed Musli by monitoring and recording the parameters such as sniffing, genital grooming, mounting and intromission.
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Detailed Description:
The present invention describes the process for standardization and evaluation of aphrodisiac property of Safed Musli. Safed Musli (Chlorophytum borivillianum) is a potent herb and its root tubers are used for human consumption for aphrodisiac property (increased sexual response and overall sex drive, improved sexual potency). Safed Musli contains alkaloids, steroid, saponin, polysaccharides, carbohydrates, proteins, minerals, vitamins etc.
The evaluation as per the present invention has been carried out in the animals and the data has been interpreted with behavioural changes supported by biochemical estimation of male sexual hormones and other parameters such as nitric oxide and antioxidant properties.
The effect of the Safed Musli on aphrodisiac property, stress conditions, antioxidant activity and on levels of nitric oxide release and testosterone are studied in the present invention. The evaluation study as per the present invention makes use of Wistar rats for evaluation of the aphrodisiac property of Safed Musli by monitoring the parameters such as sniffing, genital grooming, mounting and intromission.
The scientific validation and the therapeutic profile of Chlorophytum borivillianum with respect to aphrodisiac, antioxidant activities are compared with the known herbal and synthetic preparation available in the market.
The present invention further describes the evaluation of aphrodisiac property of Safed Musli wherein the aphrodisiac property of Safed Musli is compared with that of Ashwagandha dry root powder, Sildenafil citrate (Viagra), Korean Ginseng methanolic extract and Tentex Forte which are the known herbal and synthetic preparation from the market.
For the evaluation study of Safed Musli, the Safed Musli dry powder, Safed Musli methanolic extract and Safed Musli aqueous extract are made use of.
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Process for preparation of aqueous extract of Safed Musli:
The process for preparation of aqueous extract of Safed Musli comprises powdering the raw Safed Musli root to facilitate extraction, extracting the powder with water (1:10 ratio) by heating till aliquot gives constant weight on drying which indicates a time sufficient to extract all water soluble constituents, separating the marc from extract by centrifugation, drying the extract at temperature not more than 60° C, powdering the extract after complete drying and weighing the dry extract. The yield was approximately 50 % of the dry weight of raw powder.
The temperature range for the water extraction is 30 to 100° C, particularly 50 to 70° C more particularly 60° C.
The time required for complete water extraction of Safed Musli depends on the temperature used for the extraction. At optimum temperature of 60° C time required for extraction was upto 2 hours.
Process for preparation of non-aqueous /methanolic 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 or 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 more particularly at 50 to 55° C.
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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.
The doses of eight groups are listed below in Table 1:

Sr. No. COMPOUND HUMAN DOSE(Perday/70 Kgman) RAT DOSE(Conversion factor from humandose to rat = 0.018) (mg/kg)
1 Ashwagandha Dry root powder 6000 mg 540.00
2 Sildenafil citrate (Viagra) 100 mg 9.00
3 Safed Musli Dry root powder 6000 mg 540.00
4 Safed Musli methanolic Extract 260 mg 23.40
5 Safed Musli Aqueous Extract 500 mg 45.00
6 Ginseng methanolic Extract 900 mg 81.00
7 Tentex forte 2000 mg 180.00
8 Control Administered distil water as per the body wt i.e. 10ml/kg
Example:
Safed Musli dry root powder - 6 gm/day
6000 x 0.018 = 108 mg/rat (200g)
200g-108mg
1000-540 mg/kg
540mg-1000g
x-120g
64.8mg/120g rat = 390mg/6 rats

Study of aphrodisiac property.
The evaluation study makes use of Wistar rats for evaluation of the aphrodisiac property of Safed Musli by monitoring the parameters such as sniffing, genital grooming, mounting and intromission.
Process for evaluation of aphrodisiac property:
Wistar rats of either sex weighing between 150-200 gms were made use for the evaluation study. The adult Wistar rats were used for the aphrodisiac study. Tentex forte were administered in the form of 1% gum acacia suspension. All the drugs were prepared fresh every day and administered orally using soft gavages. The rats were maintained under standard conditions. To quantify the total sexual behaviour of mounting; female rats were paired with males treated with single dose. Animals were observed for three hours with gap of 15 minutes and their behaviours were monitored and recorded.
Blood samples were collected from male rats for Biochemical, Hormonal and Nitric oxide estimation on 0, 15th, 30th and 45th day. On the 46th day all the male rats were sacrificed for histopathological evaluation by cervical dislocation.
Stress is an important factor, which shows influence on male sexual behaviour. In the evaluation study as per the present invention; the aphrodisiac activity has been observed after subjecting the Wistar rats to stress conditions by monitoring the same parameters such as sniffing, genital grooming, mounting and intromission. The monitored parameters were graded according to their activity.
Process for evaluation of stress and sexual behavioural:
Wistar rats of either sex weighing between 150-200 gms and 10 weeks old 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 selected as a method for causing stress. Sexual response such as number of sniffing, genital grooming, mounts and intromission were visually monitored for 1 hour. The whole experiment procedure was repeated after a gap of 8 days for 3 times.
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An antioxidant is a chemical that prevents the oxidation of other chemicals. Antioxidants intercept free radicals and protect cells from the oxidative damage. Antioxidants prevent injury to blood vessel membranes. Antioxidants are believed to play a role in preventing the development of such chronic diseases as, Cancer, Heart diseases, Stroke, Cataract, Atherosclerosis and Rheumatoid arthritis.
Process for evaluation of antioxidant activity:
DPPH (2, 2 -diphenyl-2-picryl-hydrazyl) method has been used to monitor and compare
the antioxidant activity of foods and drugs. Trolox, an antioxidant was used as a reference
standard.
Nitric Oxide is an important signaling molecule that acts in many tissues to regulate a diverse range of physiological processes. Nitric oxide has now been demonstrated to play a role in a variety of biological processes, including neurotransmission, immune defense, and apoptosis etc. Reports suggest that nitric oxide has a specific role in penile erection. The physiological mechanism of erection involves release of nitric oxide (NO) in the corpus cavernosum during sexual stimulation. Nitric oxide then activates the enzyme guanylate cyclase which results in increased levels of cyclic guanosine monophosphate (cGMP), producing smooth muscle relaxation in the corpus cavernosum and allowing inflow of blood. Decrease in nitric oxide concentration would cause erectile dysfunction. Erectile dysfunction can be a total inability to achieve erection, an inconsistent ability to do so, or a tendency to sustain only brief erections.
Process for evaluation of nitric oxide levels:
Nitric oxide levels were observed after 45 days of aphrodisiac study and aphrodisiac study after stress. Production of Nitric oxide was estimated by measuring the amount of nitrite in the serum sample collected on 0, 15th, 30th, and 45th using a spectrophotometric assay based on Griess reaction (PROMEGA, US).
Testosterone and progesterone hormones play an important role in the male sexual behaviour. The evaluation study provides the effect on testosterone level, which is a key factor in male sexual behaviour.
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Process for estimation of testosterone level:
The present study was designed to estimate the levels of male sex hormones. The groups were studied for the aphrodisiac activity. Serum testosterone and progesterone concentration were determined in duplicate using competitive ELISA kits supplied by IBL (HAMBURG). Testosterone levels were observed after 45 days of aphrodisiac study and aphrodisiac study after stress.
Toxicology Data:
To observe the toxic effect of the Safed Musli and other groups, the histopathological parameters were investigated. After sacrificing the animals the vital organs like liver, kidney, spleen, lungs, testis, and brain were collected and checked for the toxicity.
Effect of the evaluation study:
1) Aphrodisiac study: Safed Musli aqueous extract and solvent extract significantly increased the number of sniffs. Safed Musli aqueous extract showed significant genital grooming, increased number of mounts and maximum intromission.
2) Stress study: After stress conditions Safed Musli aqueous extract significantly increased the number of sniffs. Genital grooming was only significant in Safed Musli aqueous extract. Maximum extent of mounting was found with Safed Musli aqueous extract. Intromission did not occur in stressful condition with all the groups.
3) Antioxidant study: In the present study it has been found that Safed Musli aqueous extract, Safed Musli methanolic extract and Ginseng methanolic extract showed good antioxidant activity. The radical scavenging activity of Safed Musli aqueous extract was found to be significantly higher than Safed Musli methanolic extract.
4) Nitric oxide estimation: Nitric oxide concentrations are significantly decreased in all the groups except Safed Musli aqueous extract, Safed Musli methanolic extract, ginseng methanolic extract and Sildenafil citrate (Viagra). The extent of intromission was found maximum in Safed Musli aqueous extract; which suggests that it helps in conditions of erectile dysfunction.
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5) Testosterone estimation: The level of testosterone hormone was significantly increased
in Safed Musli aqueous and solvent extract. The histopathological study revealed that the
organs which had been studied did not show any specific toxicity in Safed Musli treated
group; when compared to control groups. The animals treated with Viagra and
Ashwagandha showed mild degree of toxicity to liver, lung, kidney, spleen, brain and
testis.
No significant changes were observed in Glucose, Albumin, and total protein.
Benefits of the evaluation study:
1. Safed Musli aqueous extract and methanolic extract showed good response in the aphrodisiacs parameters such as sniffing, genital grooming, mounting, and intromission suggestive of potent herb for aphrodisiac use.
2. Aphrodisiac activity studied with the parameters such as sniffing, genital grooming, mounting, and intromission under stress conditions also proved the potency of Safed Musli aqueous extract and methanolic extract. Hence it reduces the stress and act as a mood balancer in stressful conditions.
3. Antioxidant intercept free radicals and protect the cells from oxidative damage which leads to aging and disease. It is clear that antioxidants act as rejuvenator and thus helps to be an aphrodisiac. In the present study it has been found that Safed Musli aqueous extract has shown good antioxidant activity. The radical scavenging activity of Safed Musli aqueous extract was found to be significantly higher than Safed Musli methanolic extract and ginseng methanolic extract proving additional point of the usefulness of Safed Musli as an aphrodisiac.
4. Nitric oxide is an important signaling molecule that has been found to be involved in regulating male sexual behaviour. Reports suggest that nitric oxide has a specific role in penile erection. The erection of the penis during sexual excitation is mediated by nitric oxide released from nerve endings close to the blood vessels of the penis. Relaxation of these vessels causes increase in the blood flow and produces penile
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erection. The extent of intromission was found maximum in Safed Musli aqueous extract which suggest that it helps in erectile dysfunction by maintaining the nitric oxide concentration.
5. It has been proved beyond doubt that the hormones such as testosterone and progesterone play an important role in the male sexual behaviour. Much of the information is on testosterone, which is a key factor. Hence, the present study was designed to estimate the male sex hormones and found that the level of testosterone hormone was significantly increased in Safed Musli aqueous and methanolic extract suggestive of potent herb in preventing the depletion of testosterone hormone which is very important for sexual function and libido.
6. Zinc is an antioxidant mineral that is also important in protecting sperm against free radical attack. Semen is rich in zinc. This would imply that it plays an important role in sperm autogenesis. Nutritive value of Safed Musli suggests that it is a rich source of Zinc(3.75mg/100gm).
Safed Musli is rich source of zinc and therefore has the following advantages.
(i) Safed Musli helps in protecting the sperm against free radical attack.
(ii) Safed Musli helps in successful fertilization.
(iii) Safed Musli increases the sperm activity, speeding them up and thereby acts like

DR. GOPAKUMAR G. NAIR
Agent for the Applicant GOPAKUMAR NAIR ASSOCIATE
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a mineral turbo change,
(iv) Safed Musli also helps in preventing the early discharge of the acrosome reaction.