TECHNICAL FIELD OF THE INVENTION:

The present invention relates to novel processes for the isolation and identification of pharmacologically active polysaccharide from Safed Musli roots and its enrichment is achieved by in vivo techniques.

BACKGROUND AND PRIOR ART:

The Genus Chlorophyium belongs to the botanical family of Liliaceae and more than 175 species of Chlorophyium have been reported in the world. In India. 13 species of Chlorophyium have been reported. All these species are differed in appearance and the native species are sold as Safed Musli in the Indian drug market. Chlorophyium horivilianum Santapau & Fernandes produces the highest yield and robust tubers and it is considered as true Safed Musli. Other Indian Chlorophyium species include C. arundinaceum, C. tuberosum, C. laxum, C. malabaricum and C. breviscapum.
The wild variety of Safed Musli (C. horivilianum) grows in Bastar forest in Madhya Pradesh, Dangs forest in Gujarat, Mount Abu, Mahi, Aravalli hills of Rajasthan and forest areas of Western Ghat of southern India.

The origin of Safed Musli can be traced back in the oldest mountain ranges on the Indian sub-continent, presently known in the states of Gujarat, Rajasthan, Madhya Pradesh and the central Deccan Plateau (Andhra Pradesh). It grows well in tropical and sub-tropical climates with altitudes up to 1500 meters. Because of the significant medicinal properties, C. horivilianum popularly known as Safed Musli has got maximum demand and commercial value and it is widely cultivated in the states of Madhya Pradesh, Uttar Pradesh, Chattisgarh, Maharashtra, Gujarat, Andhra Pradesh, Karnataka and Tamil Nadu.
For increasing sexual potency, Panax ginseng is often used in traditional Chinese medicine and it works as an antioxidant by enhancing nitric oxide synthesis in the endothelium of the organs. Some of the clinical studies on Safed Musli also prove

similar pharmacological property. Sildenafil citrate (Viagra®) is a synthetic compound that has been effective for erectile dysfunction. Since Safed Musli is also proved to be useful in erectile dysfunction, it is called as "Herbal Viagra".

Safed Musli has been described in the ancient Ayurvedic literature such as Bhavaprakash Nighantu, Rasendra Sarsangrah. and Raja Ballabh Nighantu as "Vajeekarana", means an aphrodisiac drug which is recommend for "ksheena sukra" (oligospermia and impotency). Safed Musli is regarded as a well known aphrodisiac drug prescribed by the practitioners of traditional Indian system of medicine since long time (since 12th century). By virtue of its aphrodisiac properties, Safed Musli has been an ingredient of many aphrodisiac formulations.

Recent pharmacological research indicates that the steroidal saponins present in the Safed Musli roots (tubers) are mainly responsible for the aphrodisiac activity.

However, other than the above mentioned medicinal property as an aphrodisiac drug, Safed Musli has one more valuable medicinal property as adaptogen. The lead for this medicinal property is available in some Traditional Indian Medical literatures like Sharangadhar Samhita. Bhava Prakash, Bhaishajya Rathnavali and Vangaseua. It is described that Safed Musli has the significant property of rejuvenating systems of the human body (a rejuvenator). Consequently, it is well obvious that Safed Musli has an immunomodulatory activity (adaptogenic property).

Some other medicinal herbs have also been reported to possess adaptogenic properties and they are included in the category of "Adaptogens". Charaka Samhita, an exhaustive compendium of Ayurveda (published in 1000 A.D.) describes the use of some medicinal herbs as adaptogens. An exclusive chapter in the book conceptualizes a category of drug activity as "Rasayana". The word 'Rasayana' is the combination of two words 'Rasa' meaning ELIXIR and 'Ayana" meaning HOUSE. Therefore, the word signifies adaptogenic activity of a drug, which simply means that Rasayana therapy prevents the diseases and counteracts with the aging process by means of optimization or homeostasis.

In Ayurveda many herbal drugs, including Safed Musli, are categorized as rasayana for the management of neurodegenerative diseases and useful as immunomodulators and aphrodisiac supplements,

US Application No.US20060l5963 titled "Commercially Viable Process for In-Vitro Mass Culture of Chlorophytum Borivilianum" relates to the commercially viable process for in-vitro mass culture of Chlorophytum borivilianum using media with low concentration of nutrients and phytohormones, for large-scale multiplication of the true to type clones of elite variety with disease free root tubers of uniform quality which can survive in the field at the rate of as much as about 100%.

SUMMARY OF THE INVENTION:

The present invention discloses a novel method for the isolation and identification of pharmacologically active polysaccharide from Safed Musli roots and its enrichment by in vivo techniques thereof. The said polysaccharides are useful as immunomodulators and aphrodisiac supplements.

DETAILED DESCRIPTION OF THE INVENTION:

The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

It is scientifically proved that the active chemical constituents present in the medicinal herbs are responsible for most of their pharmacological activities. The modern research on medicinal herbs and their chemical constituents suggests that fructo-oligosaccharides have more potential benefits for a variety of health conditions ranging from bone tumor to colorectal cancer, from immunity to satiety and weight management. Thus, the biological polymers particularly, sugar polymers (polysaccharides) from natural source are well incorporated in the medicinal perspective.

The fructo-oligosaccharides present in the Safed Musli root is implicated for its adaptogenic activity. The fructo-oligosaccharides are used as prebiotic supplements and which are more important in immunostimulant activity. Another report suggests that Safed Musli extract influences the alterations in the microbial population of the large intestine, thereby improving digestive health, which is vital to maintain overall health and well-being throughout the life.

In one embodiment, a novel method is developed for the isolation of mucilaginous polysaccharides from the roots of Safed Musli and identified as fructo-oligosaccharides. Further, its identity is compared and confirmed with known fructo-oligosaccharides.

Another embodiment describes about the in vivo methods for improving the synthesis of enhanced quantity of polysaccharides in the Safed Musli roots.

Accordingly, authentic seeding material (tubers) of Safed Musli was obtained from Nandan Agro Farms, Hyderabad, India. The tubers were sowed and multiplied in the R&D experimental field for further studies. The fully matured tubers were collected and the epidermis was peeled off. The tubers were dried under the sunlight and hygienically stored. For the isolation of polysaccharides, Safed Musli root powder was extracted in 20 volume of water for 5 hr at 45°C under stirring condition. These are the optimum conditions for the maximum release of polysaccharides into water. The extract was filtered through a 5 micron filter cloth to remove the marc from the filtrate. The brix of the extract was 1,5-1.7°, which was checked by using the refractometer.

Thereafter, in the next step, double the volume of any one of the water miscible organic solvents like methanol, ethanol, isopropyl alcohol or acetone etc. was added to the filtrate and the reaction mixture was stirred for 1 hr. The polysaccharides were separated out as mucilage and the mucilage was filtered through muslin cloth. The mucilage was purified by re-dissolving in distilled water and re-precipitated in the same manner. The procedure was repeated thrice to get good quality mucilage. Finally, mucilage solution was treated with trichloroacetic acid in order to remove any adherent proteins. The residual protein and nucleic acid materials in the mucilage were removed by using enzymes like protease,

DNAse and RNAse. The purified mucilage was separated and then subjected to freeze drying and stored in air-tight container until further use.

The mucilage is a white, odorless, amorphous powder. When dissolved in water, it gives a neutral (pH=7.2), slightly turbid, colloidal and viscous solution; the mucilage solution does not show optical rotation. Total yield of mucilage by ethyl alcohol precipitation was found to be 28% w/w. Moisture content of mucilage powder was found to be 8.63%. Mucilage decomposes above 200°C, which is a characteristic of most of the polysaccharide.

The other physicochemical values for the mucilage were as follows: ash value: 0.93%; acid insoluble ash value: 0.29%; water insoluble ash: 0.49%; sulphated ash: 1.09%; swelling index: 8.45; calcium content: 0.26%; arsenic content: less than lppm; heavy metals: less than 10 ppm and foreign matter: not more than 0.5% w/w. It does not show the presence of chloride, sulphate and uronic acids. Rheological behaviour of 1.0-4.0% mucilage solution was studied by using Brookfield viscometer and observed the pseudo plastic flow.

The mucilage was hydrolyzed and the resulting solution was neutralized using barium carbonate and filtered. The filtrate was concentrated and subjected to thin layer chromatography on silica gel G plates (Merck) sprayed with 0.3 % w/v boric acid, using glacial acetic acid: Chloroform: Water (8:5:1) as mobile phase. The spots were visualized with aniline - phthalate reagent. The hydrolyzed mixture was also subjected to osazone formation.

The mucilage did not reduce Fehling's solution; this indicates that mucilage is non -reducing in nature. However, hydrolyzed product gave positive Fehling's test due to the production of reducing sugars. The shape and time of formation of osazone crystals from the hydrolysate mixture indicates the presence of fructose and glucose (yellow needles, 5 min) and traces of galactose. Further, acid hydrolysate of mucilage when chromatographed on silica ge) G plates (Merck) sprayed with 0.3% w/v boric acid, using

glacial acetic acid: chloroform: water (8:5:1) as mobile phase gave 1 spot corresponding to that of standard fructose with aniline-phthalate reagent.

Total carbohydrate content was estimated by using anthrone reagent. Total carbohydrate in the mucilage was found to bd 84.25%. The fractional precipitation curve showed only one inflection point, which indicates that the mucilage is a homopolysaccahride. Further, the mucilage was tested for its application as a binding suspension and emulsifying agent.
To determine the nature of the polysaccharide, it was hydrolyzed by (V) the conventional method of using dilute acids (such as hydrochloric acid or sulphuric acid) (ii) in other method, a fungus isolated from' the Safed Musli rhizosphere was used as hydrolyzing agent. The confirmation of the identity of the polysaccharide was subjected to chromatographic techniques such as two dimensional paper chromatography, TI.C, HPTLC and HPLC (reverse pha$e anion exchange chromatography).

The isolated polysaccharide is identified as oligo-fructose polymer or fructo-oligosaccharides. This is further confirmed by the TLC and HPTLC analysis of the hydrolyzed material in comparison with reference standards of fructan, fructose and glucose. The chromatographic studies confirmed that the polysaccharide isolated from the Safed Musli root is inulin type of fructan.

The morphological character and shape determination of the final product is done by observing under optical microscope and the polysaccharide is having crystalline structure with sphaeroraphidal characters.

The quantitative determination of polysaccharide was done by colorimetric method using fructose as standard. The quantity of fructan in the root is invariably found as an average value of 11% on dry weight of the root.

In another embodiment, the invention provides a method for the improvement of the quantity of extractable polysaccharide by employing a starch solubilizing bacteria.

Consequently, the present invention also discloses a method to obtain more quantity of fructo-oligosaccharide from Safed Musli root, by employing a starch solubilizing bacterium. The starch solubilizing bacterium was isolated from the rhizosphere of Safed Musli root. The bacterium was sub-cultured to obtain pure culture. Safed Musli root was coarsely powdered and suspended in 20 volumes of water and stirred for 1 hour at room temperature. The inoculum of starch solubilizing bacterium (5000cfu/ml) was inoculated in the root powder-water mixture and stirred for 1 hour at room temperature. This mixture was incubated in a biological incubator for 3 days. The solid root particles were filtered through 5 micron filter cloth again the same solution was filtered through 45ii filter apparatus to remove bacterial cells. The resultant filtrate was precipitated for polysaccharides by using water miscible organic solvents as described elsewhere in the embodiment.

The content of purified polysaccharide is enriched up to 19.5% on the dry weight of the root by the method of employing starch solubilizing bacteria.

In a further embodiment, the invention provides a method for the enhancement of biomass production and improvement in the quality traits such as content of carbohydrate, content of protein, content of saponin and content of polysaccharide in the root by using growth hormones (Gibberllic acid and cytokinin) as foliar spray.
The content of polysaccharide in the Safed Musli root is found enhanced when the plant is treated with the growth hormone (1) gibberllic acid and (2) cytokinin. Accordingly, the Safed Musli tubers were planted during June- 2007 season. The experiment was designed in 3 plots of 0.25 acre each. Plot-1: Control plants sprayed with only demineralized water; plot-2: plants sprayed with gibberllic acid (100 ppm) and plot-3: plants sprayed with cytokinin (50 ppm). Replicates of the experimental plots were maintained for each treatment. The plants were raised in the beds uniformly and usual cultivation practices were followed for all the plots. At the age of 2 months, when the plants were at full

foliage stage, they were sprayed (Is' foliar spray) with commercial grade Gibberllic acid (100 ppm) and cytokinin (50 ppm). The 2nd foliar spray was done exactly after 30 days.
The tubers were uniformly harvested during the month of April 2008. The dried tubers were analyzed for the following growth parameters: (1) biomass production (tubers+ crowns) (2) total carbohydrate content, (3) total protein content (4) total saponin content and (5) total polysaccharide content.

Both the growth hormones significantly increased the contents of all the 4 growth parameters when compared to control plants. Among the 2 growth hormones investigated, cytokinin pronounced greater yield and registered more amount of saponin and polysaccharide production (2 fold increase) in the roots (on dry weight basis).

Further, in another embodiment, a method was developed for the enrichment of biomass production and improvement in the quality traits like content of carbohydrate, content of protein, content of saponin and content of polysaccharide in the roots by the application of Azosprillum spp., Pseudomonas fluorescence and Vesicular Arbuscular Mycorrihizal (VAM) fungus as biofertilizers.

Accordingly, the biofertilizer mixture consisting of Azosprillum spp., Pseudomonas fluorescence and Vesicular Arbuscular Mycorrhizal (VAM) fungal culture was prepared and amended in the soil before the tubers were sowed. One month after sowing, the Azosprillum population in the rhizosphere soil and the VAM fungal infection in the fibrous roots were checked. The application of biofertilizers enhanced the quantity of biomass yield and 3 fold increments in the content of carbohydrate, proteins, saponins and polysaccharide was recorded. But it is noticed that the chemical nature of polysaccharide was unaltered.

In yet another embodiment, the invention provides a method for the preparation of a health drink (functional beverage) from Safed Musli root extract which is enriched with functional active molecules like total saponins and fructo-oligosaccharides. Accordingly,

the polysaccharide was extracted from 100 g of Safed Musli with 20 volume of water and total solid (TSS) was adjusted to 1°X. Acceptable amount of food grade preservatives such as sodium benzoate and potassium sorbate, parabens and other similar chemical preservatives were added to this juice. The pH of the juice was adjusted to 4.3 to 4.5 by using any one of the organic acids like citric acid, malic acid, tartaric acid etc. The total saponin and fructo-oligosaccharides in the juice were estimated and they were found invariably 38-42% and 27-30% respectively.

We Claim,
1. Processes for isolation of mucilaginous polysaccharides, and for in vivo improvement and enhancement of the quantity of polysaccharides from Safed Musli roots to obtain an enriched extract and a process for preparation of health drink (functional beverage) from the said enriched extracts.

2. A process for isolation of mucilaginous polysaccharides from Safed Musli roots as in Claim 1, wherein the said process comprises,

a. extracting pre-sundried Safed Musli roots with 1:20 volumes of water for 5 hours
with stirring at 45°C;

b. filtering the aqueous extract through a 5u, filter to remove the marc from the
filtrate to obtain extract with brix of 1.5-1.7° as checked using refractometer;

c. doubling the volume of aqueous extract by mixing water miscible solvent selected
from methanol, ethanol, isopropyl alcohol or acetone and mixing for 1 hour
before separation of the mucilage by filtration;

d. purifying the mucilage by re-dissolving in purified water and re-precipitating
repeatedly as in earlier step;

e. treating the purified mucilage with trichloroacetic acid to remove the adherent
proteins;

f. removing residual proteins and nucleic acid materials in the mucilage by treating
with enzyme such as protease, DNAse and RNAse;

g. separating the purified mucilage in 28% w/w yield, wherein the mucilage is
obtained as white, odorless, amorphous powder decomposing at around 200°C
and having ash value: 0.93%; acid insoluble ash value; 0.29%; water insoluble ash
0.49%; sulphated ash: 1.09%; swelling index 8.45; calcium content 0.26%;
arsenic content less then 1ppm; heavy metals less then 10ppm and foreign matter
not more then 0.5% w/w; Rheological behavior of 1.0-4.0%;

h. hydrolyzing the mucilage solution by neutralizing with barium carbonate;

i. concentrating the filtered solution and partially subjecting to osazone formation and hydrolysis to characterize the isolated polysaccharide as fructo-oligosaccharide of the inulin type fructan in an yield of 11% w/w from the Safed Musli root.

3. A process for improvement and enrichment of the quantity of extractable polysaccharide an in Claim 1, by employing a starch solubalizing bacterium isolated from the Rhizosphere of Safed Musli root.

4. A process for improvement and enrichment as in Claim 1, wherein the biomass production and quality traits such as content of carbohydrates, protein saponin and polysaccharide are enhanced by using growth hormones such as Gibberllic acid and cytokinine as foliar spray.

5. A process for improvement and enrichment as in Claim I, wherein the biomass production is enhanced and the quality traits such as contents of carbohydrates, proteins, saponin and polysaccharides in the Safed Musli roots are enhanced by the application of Azosprillum spp., Pseudomona florescence and Vasicular Arbusclur Mycorrihizal (VAM) fungus as biofertilizers,.

6. A process for preparation of a health drink (functional beverage) from enriched Safed Musli root extract as in Claim 1, wherein the said process comprises;

a. prepartion of extract from lOOgm of powdered Safed Musli root with 20 volumes
of water to have a total solid (TSS) content of 1°X, 38-42% of total saponins and
27-30% of fructo-oligosaccharides;

b. formulating the health drink (functional beverage) by mixing pharmaceutically
acceptable quantities of preservatives selected from Na-benzoate, K-sorbate and
parabens;

c. adjusting the pH of the juice to 4.3-4.5 by using organic acid selected from citric
acid, malic acid and/or tartaric acid.

7. A process for isolating the starch solubalizing bacterium from the rhizosphere of Safed Musli root as in Claim 3, wherein the said process comprises;

a. coarsely powdering Safed Musli root, suspending in 20 volumes of water and
stirring for 1 hour at room temperature;

b. inoculating the inoculum of starch solubalizing bacterium (5000 cfu/ml) in the
root powder-water mixture and stirring for 1 hour at room temperature
c. incubating the mixture in a biological incubator for 3 days;

d. filtering the solid root particles through 5 micron filter and further through 45
micron filter to remove bacterial cells for use in enrichment process.