FIELD OF THE INVENTION:

The present invention relates to extraction of bio-active compounds from a Fungus. More particularly the present invention relates to the extraction of bio-active compounds exhibiting anticancer property from Lentinus tuberregium.

PRIOR ART:

US 7,514,085 disclose immune modulating ompounds obtained from fungi (Lentinus species). The compositions comprises of polypeptides and polysaccharides. The invention also discloses methods of producing these compositions using filamentous fungi cultivated in liquid medium

US Patent Application Number 20110008384 discloses unique antiviral property exhibiting compounds, prepared from medicinal mushroom mycelium, extracts and derivatives. The compositions are derived from Fomitopsis and blends of medicinal mushroom species and are useful in preventing and treating viruses including Poxviridae and Orthopox viruses.

US Patent Application Number 20090130138 discloses compounds having unique antiviral and antibacterial properties which are prepared from medicinal mushroom mycelium, extracts and derivatives. The compositions are derived from Fomitopsis, Piptoporus, Ganoderma, Inonotus, Trametes, Pleurotus, and blends of medicinal mushroom species and are useful in preventing and treating viruses including Poxyiridae and Orthopox viruses, flu viruses including bird flu (H5N1), SARS and Hepatitis C(HCV), as well as infections from Mycobacterium tuberculosis, Staphylococcus aureus and Escherichia coli.

US Patent Application 20060171958 discloses compounds having unique antiviral properties which are prepared from medicinal mushroom mycelium, extracts and derivatives. The compositions are derived from Fomitopsis, Piptoporus, Ganoderma and blends of medicinal mushroom species and are useful in preventing and treating viruses including Orthopox viruses, influenza, avian influenza, Venezuelan Equine Encephalitis, yellow fever, West Nile, Dengue, New World and Old World arenaviruses, hantavirus, Rift Valley fever, sandfly fever, hantavirus, SARS, Rhinovirus and other viruses.
WO2001027305 discloses process for producing, methods and compositions of cholesterol lowering agents extracted from higher basidiomycetes mushrooms.
Gunatilaka et al., 1981 discloses anti cancer compounds extracted from marine sponges like Axinella cannabina, Tethya auratia and Raphidostila incise.

The prior art discussed above discloses various bio-active compounds that are being extracted from various varieties of fungi (mushroom). From this it is evident that mushrooms are rich in bioactive compounds. Further the prior art reveals that anti-cancer compounds can be extracted from marine sponges. However the commercial cultivation of marine sponge is so difficult. So there exists a need to extract anti cancer compounds from easily cultivated mushrooms Lentinus tuberregium, an Indian non-toxic edible mushroom.

OBJECT OF THE INVENTION:

The main object of the present invention is to extract bio-active compounds exhibiting anticancer property from Lentinus tuberregium.
Another object of the present invention is to cultivate Lentinus tuberregium economically.

Yet another object of the present invention is to utilize fruit bodies and mycelium of the Lentinus tuberregium to extract the bio-active compounds exhibiting anticancer property.
Yet another object of the present invention is to extract anticancer property exhibiting bio-active compound 5a,8a-epidioxy-24£-methylcholesta-6,22-dien-33-ol from Lentinus tuberregium.

Yet another object of the present invention is to extract anticancer property exhibiting bio-active compound Ergosta-5, 7, 22-trien-3(3-ol from Lentinus tuberregium.

Further object of the present invention is to utilize the extracted anticancer property exhibiting bio-active compounds 5a,8a-epidioxy-24i;-methylcholesta-6,22-dien-3 β -ol and Ergosta-5, 7, 22-trien-3B-ol to formulate anticancer drug and as nutraceutical food additive.

SUMMARY OF THE INVENTION:

The present invention discloses a process for extraction of anticancer property exhibiting bio-active compounds 5a,8α -epidioxy-24^-methylcholesta-6,22-dien-3p-ol (LT-I) and Ergosta-5, 7, 22-trien-3 β -ol (LT-II) from Lentinus tuberregium. The process involves drying and pulverizing the fruit bodies and mycelium of Lentinus tuberregium and extracting LT-I and LT-II by shaking the pulverized dried fruit bodies and mycelia with more than one appropriate organic solvent to obtain a suspension. Further the suspension was separated by centrifugation followed by filtration.

Then the filtered suspension was dried in vacuum to obtain a crude organic extract. The crude extract along with silica gel is loaded into a column packed with silica gel and LT-I and LT-II were eluted separately from the column by elution using more than one appropriate organic solvents. The eluted LT-I and LT-II were purified to obtain pure LT-I and LT-II.

DETAILED DESCRIPTION OF THE INVENTION:

The present invention discloses a process for extraction of anticancer property exhibiting bio-active compounds 5a,8a-epidioxy-24-methylcholesta-6,22-dien-3 β -ol (LT-I) and Ergosta-5, 7, 22-trien-3B-ol (LT-II) from Lentinus tuberregium. The fungus Lentinus tuberregium is a non toxic and highly nutritious edible mushroom. It has an added advantage where no toxicity has reported hence these compounds can be easily purified and can be used to formulate anticancer drug. Further the whole mushroom can be processed and be utilized as nutraceutical food additives.

The mass Cultivation of Lentinus tuberregium is highly economical. The substrate for the cultivation of L tuberregium is an important criterion and should be a cost-effective one besides being available round the year. In the present study, paddy straw, sugarcane bagasse, sawdust and rice husk have been used as substrates for the cultivation of L tuberregium. Mushroom cultivation involves in two major steps namely preparation of spawn and preparation of substrate.

For spawn preparation one kg of healthy sorghum grains (Sorghum vulgare) was washed thoroughly under tap water, after which the grains were half-cooked in 1.5 L of water. Excess water was drained, air dried and the grains were mixed with 20 g of calcium carbonate to make the grains free and homogeneous. Two hundred grams each of such boiled grains were taken in 500 mL bottles or polypropylene bags and aseptically inoculated with 8 mm discs of pure culture of L tuberregium.

The inoculated polypropylene bags or bottles were incubated at 25±1°C for 14-18 days.
For preparation of substrates for cultivation, all the four different substrates such as paddy straw, sugar cane bagasse, sawdust and rice husk, were soaked in water overnight and drained, resulting in a moisture content of approximately 70 %. All the substrates were chopped before use and thoroughly mixed by hand. Each substrate was filled in polypropylene bag and steamed at 80°C for 90 min. Steamed substrate bags were inoculated with grain spawn at a rate of 6 % (w/w), and transferred to the incubation room for spawn run. Spawn was mixed with the substrate by layer method. The pinholes were also made in the bags manually to ensure ventilation. The bags were incubated at 20°C under dark condition.

Spawn running room was mainly used to keep the beds for running spawn. The temperature of the spawn running room was maintained between 25 to 30°C. Light was not required in the spawn running room.

Thatched shed was preferred for mushroom growing. Sheds were built in east west direction to avoid direct effect of sun and to reduce the temperature inside the mushroom shed. Entry of rats, squirrels, snakes etc was prevented by a mesh. The sides of the shed were covered with coconut plant leaves. The floor of the shed was filled with sand to a uniform height up to 15 cm. Racks were built to accommodate the mushroom beds and inner side of the shed was covered with jute gunny bags. Water was sprinkled twice a day on the floor and gunny bags to maintain the required temperature (28-30°C) and relative humidity (75 - 85 %).

After incubation for 20 days, bags were transported to the fruiting room. Colonization of mycelia was witnessed with the naked eyes in these bags. Polypropylene bags were cut-off and hanged the whole mycelia colonized compact substrate using thread to initiate primordial formation. All the bags remained at the fruiting site for 8-10 weeks exposed to light at 20°C. Necessary ventilation was facilitated with the temperature of the shed maintained at 26°C to 35°C. In addition, relative humidity was maintained at over 85 %. Pin heads of growth appeared after 8-10 d and mature fruit bodies were harvested at right stage.

The bags were retained under same conditions for 40-45 days for the second and third flushes. All the treatments with the substrates were performed in triplicates.
Mushroom cultivation has two important phases viz, spawn running and fructification, temperature and humidity are two vital factors involved in both phases. The temperature and humidity of the bags were maintained by spraying of water twice a day. Slits were also cut in the bags with the help of paper pins for ventilation. The bags were watered thrice a day during cropping. The experiment was laid out in a completely randomized design (CRD) with three replications and six treatments. The data were analyzed statistically.

Time was recorded in days for the completion of growth of mycelia on substrates, appearance of pin heads and maturation of fruiting bodies under different treatments. The data were also recorded for the yield of fruiting bodies and biological efficiency of substrates. The total biological efficiency was worked out against the dry weight of each substrate.


Isolation and Characterization of compounds of subject matter was carried out as follows. The pulverized dried fruit bodies of L tuberregium were extracted with different organic solvents viz., hexane, dicholoromethane, choloroform, ethyl acetate and ethanol with an increasing order of polarity. The extraction flasks were kept in a shaker. The obtained suspension containing bioactive substances were separated by centrifugation. The fine particles in the suspension were removed by filtration through Whatman No.1 filter paper. The organic portion was allowed to dry in vacuum to get dried materials, and then the dried materials were named as crude organic extracts.

To detect the number of compounds present in the crude ethyl acetate extract, TLC was performed on precoated silica gel plate. The extract (5uL ) was spotted on the TLC plate approximately 1 cm above the bottom of the plate and it was developed in the presaturated TLC tank with different solvent systems at different ratios (hexane: chloroform (1:1); dicholoromethane:choloroform (1:1) and dicholoromethane : methanol and chloroform: methanol (increasing concentration of methanol) . The presence of bands was visualized under UV at 254 and 365 nm (Raaman, 2006).

A glass column was packed with silica gel (100-200 mesh) with chloroform as the packing solvent. The column was packed up to a predetermined length using the formula V=Trr2h (V-packing volume of the column; TT-3.14; r2- multiply the radius of the column; h-total length of the column).

The concentrated ethyl acetate extract was ground well with silica gel and loaded on to the column and eluted with 100% chloroform. Later, the column was eluted with an increasing percentage of methanol. Each fraction with 5 mL/min was collected in 96 test tubes and the presence of compound was checked by TLC with different solvent systems. The fractions that showed a similar pattern were pooled together and allowed to evaporate. Eight partially purified fractions were obtained and named as 1-VIII. Based on the quality and purity, the compounds II (fractions 11-26-27-42) were taken up for further purification.

To identify the group of the active principle, TLC was performed. The TLC plates that had been spotted with pure compound were allowed to develop (Ishikawa et al., 2001). The plates were subjected to spray with spray reagent such as Vanillin sulphuric acid,

AICI3, Dragendorff reagent, Folin-ciocalteu reagent and freshly prepared KOH reagent (Wagner et a/., 1984; Krebs etal., 1969).

The extraction of the bio- active compounds from L. tuberregium is as follows. One kg of dried powder of L. tuberregium was used for the extraction of compounds with different organic solvents such as hexane, dichloromethane, ethyl acetate and methanol by successive shake flask method.

The resulting extract from each solvent was evaporated in a rotary evaporator at 40°C and the appearance and consistency were recorded. The crude hexane extract (220 mg/100 g) appeared yellow in colour and its consistency was yellow oily. The yields of dichloromethane and ethyl acetate extracts were found as lesser than hexane and ethanol extracts. Dicholoromethane extract (170 mg/100 g) and ethyl acetate extract (155 mg / 100 g) were observed with similar appearance and consistency (yellow/brown solid). The ethanolic extract (270 mg/100 g) appeared as brown and solid in nature.

The isolation, detection and purification of bioactive compounds from extracts of Lentinus tuberregium fruit body and mycelium was carried out. The compounds were isolated from the test organism, L tuberregium by employing various chromatographic techniques (vide Material and Methods). The TLC plate was spotted with crude ethyl acetate extract and run with the solvent system chloroform: methanol (95:5).

The developed TLC plate was observed under UV at 365 nm. It was noticed that the second band emitted bright greenish fluorescence and the third band emitted bluish colour, while the remaining spots were pale greenish. The Rf values of the spots 1-3 were 0.31, 0.55 and 0.84, respectively. However, five bands were observed at 254 nm with the same Rf of 0.06, 0.13, 0.27, 0.51 and 0.84, respectively. Interestingly, a band at the Rf value 0.51, and 0.84 showed the major compounds in the ethyl acetate extract and it also showed significant antimicrobial activity in the screening tests (well diffusion method). Hence, it was targeted in the column chromatographic separation.

The elution from the crude ethyl acetate extract (4.2 g) on silica gel column (3 x 90 cm) with increasing solvent polarity (100 % chloroform initially, later increasing percentage of methanol) yielded 96 fractions. The fractions which showed a similar pattern were
pooled together. Based on the quality and purity, the partially purified compound l(fractions 11-26) and II (27-42) were further purified, resulting purified compound LT-1 130 mg, LT-ll-110 mg/500g of dried powder respectively. TLC of the purified compound LT-1 was carried out with the solvent system chloroform: methanol in the ratio of 95:5. It, showed the greenish band at the Rf value of 0.432 whereas, the purified compound of LT-II was carried out with the solvent system of hexane : ethyl acetate in 90:10. It showed a bluish band with the Rf value of 0.532.

To ascertain the phytochemical nature of the isolated compound, the developed TLC plate was sprayed with different spray reagents such as methanol, sulphuric acid, aluminium chloride, Dragendroff reagent, Folin - Ciocalteu reagent and potassium hydroxide. Among them, methanol sulphuric acid exhibited a distinct purple violet spot, which indicated the steroid nature of the purified compound LT-1 and II.

The compound LT-1 was analysed by an 1H NMR spectrum in order to enumerate the hydrogen atoms present in the compound. 1H NMR spectrum showed resonances for six methyl groups at 5 0.81 (3H, s, Me-18), 0.88 (3H, s, Me-19), 0.98 (3H, d, J=6.31 Hz, Me-21), 0.81 (3H, d, J=6.3 Hz, Me-26), 0.83 (3H, d, J=6.93 Hz, Me-27), 0.91 (3H, d, J=6.63 Hz, Me-28). The resonances at 6 3.96 (1H, m, H-3), 6.25 (1H, d, J=8.83 Hz, H-6) and 6.49 (1H, d, J=8.83 Hz, H-7) suggested a A6, mono hydroxylated 5a, 8a-epidioxysteroidal compound.

The compound LT-1 was analysed by an 13C NMR spectrum in order to enumerate the
number of carbon atoms present in the compound. This is supported by the 13C NMR
signals at 82.14 and 79.41 of C-5 and C-8, respectively. The presence of two olefenic
protons at 5 5.08 (1H, ddd, J=15.2, 8.83, 2.5 Hz) and 5.12 (1H, ddd, J=15.6, 8.81,
2.3Hz) was indicative of A22 unsaturation and the same assigned to H-23 and H-22
respectively, which were also confirmed by their 13C NMR resonances at 135.39 and
132.39 ppm for C-22 and C-23, respectively.

The B-configuration of hydroxy group at position 3, 6H 3.96 (1H, m, H-3) and 5C 66.44 ppm (d, C-3) suggesting the molecular formula- C28H4603.

In 1H - NMR Analysis of Bioactive Compound LT-2, Six methyl groups have shown up at 50.63 (3H,s.Me-18), 0.94(3H,s.Me-19), 1.03(3H, d, J=6.4 Hz, Me-21), 0.84 (3H, d,J=6.4 Hz, Me-26),0.80 (3H, d,J=6.4 Hz, Me-27), 0.91 (3H, d, J=6A Hz, Me-28). A broad singlet at 53.62 suggested the presence of 30- hydroxyl group.C-3 appeared at 71.7ppm.

In the 13C NMR spectrum of LT-2, the presence of two olefinic protons at 55.38 (1H, ddd, J=, 8.83,5.6, 2.5 Hz) and 5.56(1 H, dd, J=8.81, 2.3Hz) was indicative of A22 unsaturation and assigned to H-23 and H-22 respectively, which were also confirmed by their 13C NMR resonances at 135.5 and 131.9 ppm for C-22 and C-23, respectively. The other two olefinic protons at 65.18 (2H,m) were assigned to the protons on C-6 and C-7. 13C NMR resonances appeared at 6 139.8, 119.6, 116.2 and 141.3 ppm for the carbons at C-5, C-6, C-7 and C-8 respectively suggesting the molecular formula-C28H4403.

Based on the 1H NMR and 13C NMR, DEPT, APT, FT-IR the data revealed that the compound LT-1 was 5a, 8a-epidioxy-24£-methylcholesta-6,22-dien-3p-ol C28H48O3
Mdting point: 354.81 °C Mol.Wt 428.65 Structure of LT-1 (5a,8a-epidioxy-24^-methylcholesta-6,22-dien-3p-ol) LT-1 was isolated as crystalline white needles, with [a]D of - 5° (c 0.35CHCI3). EI-MS showed molecular ion peak at m/z 428 [M]+ and fragment ions at m/z 396 [M-(02)]+, 378 [M-(02 + H20)]+, 363 [M-(02 + H20 + CH3)]+, 271 [M-(02 + side chain)]+, and 253 [M-(02 + side chain + H20)]+ suggesting the molecular formula-C28H4603.

Based on the 1H NMR and 13C NMR, DEPT, APT, FT-IR, the data revealed that the compound LT-2 was Ergosta-5, 7, 22-trien-3B-ol C2RH44O Exact Mass: 396.34 Mo!, Wt: 396,65 m/e: 396.34 (100.0%), 397,34 (30.3%), 398,35 (4.6%) C 84.79; HJl.18; 0,4.03 Structure of LT-2 (Ergosta-5,7,22-trien-3p-ol)

LT-2 was a colourless, amorphous powder: 164-165°C;[a]D22=-103°(c 0.1.CHCI3) suggesting the molecular formula C28H44O. The compound LT 1 and LT 2 was also detected in the mycelium of L tuberregium.

In one of the preferred embodiment the present invention shall disclose a process for extraction of anticancer property exhibiting bio-active compounds 5a,8o>epidioxy-24^-methylcholesta-6,22-dien-3p-ol (LT-I) and Ergosta-5, 7, 22-trien-3p-ol (LT-II) from Lentinus tuberregium comprising of fruit bodies and mycelium.

The process involves drying and pulverizing the fruit bodies and mycelium of Lentinus tuberregium followed by extracting the bio-active compounds LT-I and LT-II by shaking the pulverized dried fruit bodies and mycelia with more than one appropriate organic solvent for a predetermined time at predetermined rpm to obtain a suspension.

Then, the suspension is separated by centrifugation at predetermined rpm for predetermined time and filtered. Subsequently, the filtered suspension is dried in vacuum to obtain a crude organic extract. The obtained crude organic extract is mixed with silica gel and loaded in to a column packed with silica gel to absorb LT-I and LT-II on to the column. Then more than one appropriate organic solvent is charged into the column to selectively remove LT-I by elution and the eluate containing LT-I was collected from the column. Again more than one appropriate organic solvent is charged into the column to selectively remove LT-II by elution and the eluate containing LT-II was collected from the column. Further the eluted LT-I and LT-II was purified to obtain purified LT-I and LT-II.

As per the invention organic solvents are selected from the group comprising of hexane, dichloromethane, chloroform, ethylacetate, ethanol or combinations thereof.
According the invention crude organic extract containing LT-I has thin layer chromatogram with a spot around Rf value 0.432 with the solvent system of chloroform : methanol in the ratio of 95 : 5, and spraying reagent of methanol : sulphuric acid 90 :10, arising from LT-I.

In accordance with the invention the crude organic extract containing LT-II has a thin layer chromatogram with a spot around Rf value 0.532 with the solvent system of
hexane : ethyl acetate in the ratio of 90 : 10, and spraying reagent of methanol : sulphuric acid 90 : 10, arising from LT-II.

In another preferred embodiment the present invention shall disclose an anticancer property exhibiting bio-active compound 5ct,8a-epidioxy-24i;-methylcholesta-6,22-dien-3p-ol (LT-I) prepared from the process as discussed above.

In another preferred embodiment the present invention shall disclose an anticancer property exhibiting bio-active compound Ergosta-5, 7, 22-trien-3p-ol (LT-II) prepared from the process as discussed above.

EXAMPLES

Extraction of Compounds from L tuberregium One kg of both fruitbody and mycelium dried powder of L tuberregium were used for the extraction of compounds with different organic solvents such as hexane, dichloromethane and ethyl acetate by successive shake flask method. The resulting extract from each solvent was evaporated in a rotary evaporator at 40°C and the appearance and consistency were recorded.

The crude hexane extract (220 mg/100 g) appeared yellow in colour and its consistency was yellow oily. The yields of dichloromethane and ethyl acetate extracts were found as lesser than the hexane and ethanol extracts. Dicholoromethane extract (170 mg/100 g) and ethyl acetate extract (155 mg/100 g) were observed with similar appearance and consistency (yellow/brown solid).

Thin Layer Chromatography (TLC) The compounds were isolated from the test organism, L tuberregium by employing various chromatographic techniques (vide Material and Methods). The TLC plate was spotted with crude ethyl acetate extract and run with the solvent system choloroform: methanol (95:5).

The developed TLC plate was observed under UV at 365 nm. It was noticed that the second band emitted bright greenish fluorescence and the third band emitted bluish colour, while the remaining spots were pale greenish. The Rf values of the spots 1-3 were 0.31, 0.55 and 0.84, respectively. However, five bands were observed at 254 nm with the same Rf of 0.06, 0.13, 0.27, 0.51 and 0.84, respectively. Interestingly, a band at the Rf value 0.51, and 0.84 showed the major compounds in the ethyl acetate extract and it also showed significant antimicrobial activity in the screening tests (well diffusion method). Hence, it was targeted in the column chromatographic separation.

Column Chromatography The elution from the crude ethyl acetate extract (4.2 g) on silica gel column (3 x 90 cm) with increasing solvent polarity (100 % chloroform initially, later increasing percentage of methanol) yielded 96 fractions. The fractions which showed a similar pattern were pooled together. Based on the quality and purity, the partially purified compound I (fractions 11-26) and II (27-42) were further purified, resulting purified compound LT-I (130mg) LT-I I -(110 mg/500g) of dried powder respectively. TLC of the purified compound LT-1 was carried out with the solvent system chloroform: methanol in the ratio of 95:5. It, showed the greenish band at the Rf value of 0.432, whereas, the purified compound of LT-II was carried out with the solvent system of hexane : ethyl acetate in 90:10. It showed a bluish band with the Rf value of 0.532.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

WE CLAIM,

1. A process for extraction of anticancer property exhibiting bio-active compounds 5a,8a-epidioxy-24£-methylcholesta-6,22-dien-3p-ol (LT-I) and Ergosta-5, 7, 22-trien-3P-ol (LT-II) from Lentinus tuberregium comprising of fruitbodies and mycelium, the said process comprises of following steps.

a. drying and pulverizing the fruit bodies and mycelium of Lentinus tuberregium.,

b. extracting the said bio-active compounds LT-I and LT-II by shaking the pulverized dried fruit bodies and mycelia of step(a) with atleast one appropriate organic solvent for a predetermined time at predetermined rpm to obtain a suspension,

c. separating the suspension obtained in step(b) by centrifugation at predetermined rpm for predetermined time,

d. filtering the separated suspension of step(c),

e. drying the filtered suspension of step(d) in vacuum to obtain a crude organic extract,

f. loading a mixture comprising of the crude organic extract of step (e) and silica gel in to a column packed with silica gel to absorb LT-I and LT-II on to the column,

g. charging atleast one appropriate organic solvent into the column to selectively remove LT-I by elution

h. collecting the eluate containing LT-I from the column, i. charging atleast one appropriate organic solvent into the column to selectively remove LT-II by elution j. collecting the eluate containing LT-II from the column, k. purifying the eluted LT-I of step (h) and LT-II of step (j) to obtain purified LT-I and LT-II.

2. The process as claimed in claim 1 wherein the said organic solvents is selected from the group comprising of hexane, dichloromethane, chloroform, ethylacetate, ethanol or combinations thereof.

3. The process as claimed in claim 1 , wherein the said crude organic extract of step1(e) has thin layer chromatogram with a spot around Rf value 0.432 with the solvent system of chloroform : methanol in the ratio of 95 : 5, and spraying reagent of methanol: sulphuric acid 90 :10, arising from LT-I.

4. The process as claimed in claim 1, wherein the said crude organic extract of step1(e) has a thin layer chromatogram with a spot around Rf value 0.532 with the solvent system of hexane : ethyl acetate in the ratio of 90 : 10, and spraying reagent of methanol: sulphuric acid 90 :10, arising from LT-II.

5. An anticancer property exhibiting bio-active compound 5a,8a-epidioxy-24^- methylcholesta-6,22-dien-3B-ol (LT-I) prepared from the process as claimed in claim 1.

6. An anticancer property exhibiting bio-active compound Ergosta-5, 7, 22-trien-3p-ol (LT-II) prepared from the process as claimed in claim 1.