(v) Kaposi's sarcoma and a number of other human tissue proliferating diseases. In addition, this compound demonstrated moderate in vivo activity against the P - 388, P -1534, L - 1210 marine leukaemia, the walker 256 carcinosarcoma, sarcoma 180 and Lewis lung tumour test systems.
Unlike other antimitotic drugs, Taxol promotes the formation of highly stably microtubules that resist the depolymerization by specifically binding to the N - terminal region of beta - tubulin. Taxol binding alters the confirmation of the tubulin sub unit, thus greatly retarding tubulin heterodimer dissociation. Cell division is then blocked at the mitotic stage and the cell dies. Besides this central mechanism, Taxol exerts numerous other cellular efforts. Observations made the Taxol resistant murine and human tumors cells make it increasingly clear that cellular transport of Taxol and its microtubule binding activity are important factors in the development of resistance to this drug.
The mechanism of action of the drug is tubulin polymerisation in the cancer cells and it works very well in combination with other anticancer drugs. The drug is available as injections. Paclitaxel anticancer - activity was discovered in the 1960 s. during large scale plant - screening program sponsored by the National Cancer Institute (NCI, U.S.A). In 1985 NCI began conducting clinical trials of Taxol's safety and its effectiveness against various types of cancers. Total biosynthesis of Taxol was achieved in 1994, which is not suitable for commercial production.
The primary source of Taxol is the dried inner bark of Taxus brevifolia (Pacific Yew). Three species of Taxus, namely, T. baccata, T. brevifolia and T. yannanensis produce very small quantities of Taxol, Pacific Yew is a slow growing evergreen coniferous tree native to the tropical forests of northwestern United States.
To obtain 1kg of Taxol requires about 10,000 kg of bark (Vidensek, N., P. Lim, A. Campbell and C. Carlson. 1990. Taxol content in bark, wood, root, leaf, twig and peeling from several Taxus species. Journal of Natural Products 53: 1609 - 1610) and several thousand trees must be cut to produce this quantity of bark. Production of Taxol is about 0,01% to

0.03% of dry weight, yet as much as 2g of purified Taxol is required for a full regimen of anti-tumour treatment.
The scarcity of Taxol and the ecological impact of harvesting it have prompted extensive searches for the alternative sources including semi synthesis, cellular culture production and chemical synthesis. The alternative sources, while having been accomplished are not economically feasible. A few years ago pharmaceutical nature of product's search has tended to favour compounds derived from micro organisms because of their diversity and ease to culture and possibility of manipulating them to enhance the production of active compounds.
Many investigations on endophytic fungi have been carried out using hosts from temperate conditions, mainly from the Northern Hemisphere and New Zealand. The information available from tropical regions are scarce. However, the available data show that tropical plant hosts contain a great diversity of endophytic fungi. Potentially, they are of biotechnological importance as new pharmaceutical compounds, secondary metabolites, agents of biological control, and other useful characteristics could be found by further exploration of tropical endophytes. Also, a better understanding of plant-endophyte relationships in tropical conditions can be achieved from these studies. Some endophytic microorganisms are able to produce compounds of biotechnological value as antibiotics and antitumor drugs.
After several years of effort, a Taxol producing endophytic fungus, Taxomyces andreane was discovered (Strobel, G.A., A. Stierle and W.M. Hess. 1993. Taxol formation in Yew-Taxus. Plant Science 84: 65 - 74; Stierle, A., G.A. Strobel and D. Stierle. 1993. Taxol and taxanes production by Taxomyces anclreanae, an endophytic fungus of Pacific yew. Science, 260: 214-216; Stierle, A., G.A. Strobel and D. Stierle. 1995. The search for a Taxol producing microorganism among the endophytic fungi of the Pacific Yew, Taxus brevifolia. Journal of Natural Products. 58:1315 -1324). .
Recent work carried out reported that plants other than yew have endophytic fungi associated with them that also make this drug. Some endophytic fungi belonging to different genera such as Taxomyces andreanae, Pestalotiopsis microspora, Alternaria alternata, Periconia sp., Pithomyces sp., Monochaetia sp,.Seimatoantlerium nepalense (Dahiya, J.S. 1996.

Fermentation for Taxol production. WO 96/32490; Li, J.Y., R.S. Sidhu, A. Bollon and G.A. Strobel. 1998a. Stimulation of Taxol production in liquid cultures of Pestalotiopsis microspora. Mycological Research 102: 461 - 464; Li, J.Y., R.S. Sidhu, EJ. Ford, W.M. Hess and G.A. Strobel. 1998b. the induction of Taxol production in the endophytic fungus Periconia sp.from Torreya grandifolia. Journal of Industrial Microbiology and Biotechnology 20: 259 - 264) are reported to produce Taxol.
Thus, if a microbial source of the drug would be available, it would eliminate the need to harvest and extract the slow growing and relatively rare yew trees for this drug. The price for the drug would then be reduced.
Therefore the main objective of the present invention is to provide novel microorganisms isolated from endophytic fungi from medicinal plants namely Pestalotiopsis menezesiana and Botryodiplodia theobromae
Another objective of the present invention is to provide novel microorganisms designated as MTCC 7380 & MTCC 7381 both dated 16-09-2005 from Pestalotiopsis menezesiana and Botryodiplodia theobromaeresptctively deposited at Institute of Microbial Technology, (IMTECH), a CSIR Institution, Chandigarh
Yet another objective of the present invention is to provide processes for the isolation of novel microorganisms designated MTCC 7380 & MTCC 7381 both dated 16-09-2005 from Pestalotiopsis menezesiana and Botryodiplodia theobromae respectively deposited at Institute of Microbial Technology, (IMTECH), a CSIR Institution, Chandigarh.
Still another objective of the present invention is to provide an improved process for the preparation of taxol having the formula I given above from the novel microorganisms designated as MTCC 7380 & MTCC 7381 both dated 16-09-2005 from Pestalotiopsis menezesiana and Botryodiplodia theobroma respectively deposited at Institute of Microbial Technology, (IMTECH) a CSIR Institution, Chandigarh.
Yet another objective of the present invention is to provide an improved process for the preparation of Taxol .useful for the treatment of cancer from the endophytic fungi from

medicinal plants namely Pestalotiopsis menezesiana and Botryodiplodia theobromae deposited at Institute of Microbial Technology, (IMTECH) , a CSIR Institution, Chandigarh and having the accession no MTCC 7380 and MTCC 7381 respectively both dated 16-09-2005 wherein the concentration of Taxol produced has a purity of 99.9 % identical to that obtained from hitherto known sources / methods .
Still another objective of the present invention is to provide an improved process for the preparation of Taxol useful for the treatment of cancer from the endophytic fungi from medicinal plants namely Solarium nigrum and Terminalia arjuna deposited at Institute of Microbial Technology, (IMTECH), a CSIR Institution, Chandigarh and having the accession nos MTCC 7380 and MTCC 7381 respectively both dated 16-09-2005 wherein Taxol is released into the culture medium, thereby making it easily to isolate making the process less expenditure.
The isolation of the above said microorganisms can be effected by employing the known extraction procedure in the literature (Strobel, G.A., X. Yang, J. Sears, R. Kramer, R.S. Sidhu and W.M. Hess. 1996. Taxol from Pestalotiopsis microspora, an endophytic fungus of Taxus wallachiana. Microbiology 142:435 - 440).
Accordingly the present invention provides novel microorganism designated as MTCC 7380 dated 16-09-2005 deposited at IMTECH, Chandigarh useful for the preparation of Taxol of the formula I given above which is useful for the treatment of Cancer
According to another aspect of the present invention there is provided novel microorganisms designated as MTCC 7381 dated 16-09-2005 deposited at IMTECH, Chandigarh useful for the preparation of Taxol of the formula I given above which is useful for the treatment of Cancer
According to another aspect of the present invention there is provided a process for the isolation of the microorganisms designated as MTCC 7380 from Solanum nigrum which comprises
(i) Cutting the parts of the plant Solanum nigrum into small pieces (ii) Cleaning the pieces to remove unwanted materials adhering on them (iii) Further sterilizing with sodium hypochlorite solution , and ethanol

(iv) Treating the cleaned pieces with a Carbon source for a period in the range of 2-7 days at a temperature in the range of 28-32° C to form fungal masses
(v) Separating the fungal masses of the microorganism designated as MTCC 7380 by conventional methods and
(vi) Purifying the resultant microorganism designated as MTCC 7380 by/conventional methods
According to another aspect of the present invention there is provided a process for the isolation of the microorganisms designated as MTCC 7381 from Terminalia arjuna which comprises
(i) Cutting the parts of the plant Terminalia arjuna into small pieces
(ii) Cleaning the pieces to remove unwanted materials adhering on them
(iii) Further sterilizing with sodium hypochlorite solution and ethanol
(iv) Treating the cleaned pieces with a Carbon source for a period in the range of 2-7
days at a temperature in the range of 28-32° C to form fungal masses
(vi) Separating the fungal masses of the microorganism designated as MTCC 7381 by
conventional methods and
(vi) Purifying the resultant microorganism designated as MTCC 7381 by conventional
methods


useful for the treatment of cancer which comprises
(i) growing the novel microorganism designated as MTCC 7380 or MTCC 7381 in a medium containing Carbon, Nitrogen and Carbon sources , at a temperature in the range of 28-32° C, at a pH in the range of 6.5 -7.0 for a period in the range of 15-25 days.
(ii) separating the fungal mass formed from the culture medium by filtration
(iii) adding to the culture medium Sodium bi carbonate to avoid fatty acid contamination.
(iv) extracting the resulting medium with methylene chloride
(v) separating the organic phase from the liquid culture medium phase by known methods
(vi) Drying the Organic phase at reduced pressure and at a temperature in the range of 28-35°C.
(vii) Dissolving the resulting dried powder with methylene chloride
(viii) Eluting the solution through silica gel column employing different solvent systems starting with 70 ml of 100% methylene chloride and mixtures of methylene chloride / ethyl acetate at 20:1,10:1,6:1,3:1 and 1:1 (v/v).
(ix) Separating the fractions coming out at 6:1 & 1:1 containing Taxol and mixing them followed by evaporation.
(x) Drying the fractions to get Taxol.
The microorganisms designated as MTCC 7380 & MTCC 7381 are not hitherto known . Hence the process for the preparation of Taxol from the said new organisms are also not hitherto known. This is the first time Taxol has been prepared from the above said fungi, and therefore the process is novel.


FeCl3 - mg, MnS04 - 2.5mg, H3BO3 - 1.4mg, KI- 0.7mg, Distilled Water - 1000ml.) and incubated for21 days at21°C
Stimulation of Taxol production in liquid cultures of Pestalotiopsis microspore Mycological Research 102: 461 - 464) reported that the subtitution of ammonium tartrate by peptone, or of casein enzyme hydrolysate, leucine, glycine or serine had negative, little, or no effect on Taxol production of fungal growth. The sulphate salts of Mn, Mg and Zn had optimum effect on Taxol production at 0.1 and 0.001 respectively. Although carbon source may influence secondary metabolism, an increase of sucrose up to 100 g / L had no significant effect on Taxol production. Therefore, the above ingredients of the M-1D medium are essential for the growth as well as the Taxol production of the test fungi.
In the process of the present invention the fungi were grown preferably in M-1D (Modified liquid medium), Potato Dextrose Broth (PDB), Oat Meal Broth (OMB) and Corn Meal Broth (CMB) medium for the production of Taxol. But the above said, M-1D medium is found to be the best medium for the production of Taxol and the fungal extracts of the test fungi namely, Pestalotiopsis menezesiana and Botryodiplodia theobromae was found to the maximum amount of Taxol producers.
The cultures were found to grow very well at 25 - 33 °C at room temperature. The culture filtrate was passed through layered cheesecloth preferably of four layers. The resulting culture fluid was extracted with methylene chloride, preferably two equal volumes and the organic phase was evaporated to dryness under reduced pressure at 3 a temperature in then range of 28-35°C, preferably at 35° C. The residue was dissolved in 1ml methylene chloride and placed on a column of silica gel and eluted in a step-wise manner with different solvent systems starting with 70 ml of 100% methylene chloride and mixtures of methylene chloride / ethyl acetate at 20:1,10:1, 6:1, 3:1 and 1:1 (v/v). A compound having the same mobility as authentic Taxol was found from the 6:1 and 1:1 fractions. These fractions were combined and evaporated to dryness.
The formation of Taxol was confirmed by analytical methods using Thin Layer Chromatography (TLC), High Performance Thin Layer Chromatography (HPTLC), High Performance Liquid Chromatography (HPLC), Ultraviolet (CV), Infra Red (IR), Mass Spectrometry (MS). In the present invention twenty one day old culture filtrates of Pestalotiopsis

menezesiana ( Accession no MTCC 7380) and Botryodiplodia theobromae. (Accession No MTCC 7381) were found to be maximum amount of Taxol producer.
Thin Layer Chromatography (TLC) analysis of Taxol
The fungal extracts .of Pestalotiopsis menezesiana (JMCAS 1023) and Botryodiplodia theobromae. were subjected to Thin Layer Chromatography (TLC) on a 0.25 mm (10 x 20 cm) silica gel plate developed in different solvent systems with authentic Paclitaxel (Sigma). Both these fungi showed positive reaction with 1% w/v Vanillin / Sulphuric acid reagent after gentle heating. It appeared as a bluish spot that faded to dark gray after 24 hours. They had Rf values identical to that of authentic Taxol (Sigma).
High performance Thin Layer Chromatography (HPTLC) analysis
The presence of Taxol was further confirmed by subjecting the two fungal samples to HPTLC (CAMAG - Planar HPTLC, Anchrom, Central Instrumentation facility meant for Herbal Science program). With Automatic TLC Sampler, 5 μl of the sample was injected and developed in a CAMAG flat bottom chamber with Chloroform ; Methanol, 9 : 1 for 20 min. Following chromatography the plate was dried for 10 min. and scanned using TLC Scanner 3 with winCATS software, fluorescence measurement at 254 nm and 366 nm.
High Performance Liquid Chromatography (HPLC) analysis
To confirm further the presence of Taxol in the fungal extracts, the samples were subjected to HPLC (Shimadzu 9A model) using a reverse phase Cig column with a UV detector. A 20 μl of sample was injected each time and detected at 232 nm. The mobile phase was methanol / acetonitrile / water (25:35:40, by vol.) at 1.0 ml min"1. Taxol was quantified by comparing the peak area of the samples with that of the Taxol standard using the formula:

Standard concentration x Total area of the sample
Taxol content =
Total area of the Standard By using the above formula, the amounts of Taxol estimated from the culture filtrates were estimated to be 170 μg / L and 150 |ig / L from Pestalotiopsis menezesiana (Accession No MTCC 7380) and Botryodiplodia theobromae (Accessions no MTCC 7381) respectively as shown in Figs la & lb respectively.
Ultra Violet (UV) absorption spectral analysis
After chromatography, the two potential coelomycetous endophytic fungi were analyzed spectroscopically for further confirmation of Taxol. The area of TLC plate containing putative Taxol was carefully removed by scrapping off the silica at the appropriate Rf and exhaustively eluting it with methanol. The UV spectral analysis of two fungal samples were analysed using a Beckman DU - 40 Spectrophotometer at 273 nm. The samples showed absorbance at 273 nm similar to that of authentic Taxol.
Infra Red (IR) spectroscopic analysis
The IR spectra of the compound were recorded on Shimadzu FT IR 8000 series instrument. The sample was ground with IR grade Potassium BroM-lDe (KBr) (1:10) pressed into discs under vacuum using spectra lab Pelletiser and the spectrum was recorded from 4000 -500 cm-1 nm and compared with authentic Taxol.
In the present invention , IR spectrum of the two fungal extracts showed a broad peak in the range of 3336 - 3436 cm"1, which is assigned for the presence of O - group in the parent compound, is evident by its OH (Hydroxyl group) stretch. The aliphatic CH stretch is observed in the range of 2920 - 2939 cm"1. The registration peak observed in the range of 2356 - 2364 cm"1 in all the samples is due to NH (amine group) stretching frequency. The C=0 (carboxyl / keto group) stretch was positioned in the range of 1593 - 1735 cm"1. The COO (carboxyl group) stretching frequency was observed in the range of 1261 - 1276 cm-1. The peak in the range of 1045 - 1068 cm"1 is due to the presence of aromatic C, H bends

Mass Spectroscopic (MS) analysis
The molecular weight of Taxol produced by the fungi was confirmed by Mass Spectroscopic analysis. The FAB Mass spectra of the fungal Taxol were recorded on a JEOL X 102 / DA-600, m-nitro benzyl alcohol (NBA) was used as the matrix.
According to the process of the present invention, the amounts of Taxol estimated from the culture filtrates were 170 pg / L and 150 pg / L from Pestalotiopsis menezesiana ( Accession No MTCC 7380) and Botryodiplodia theobromae. (Accession No MTCC 7381) respectively. The amounts of Taxol produced according to the present invention are higher than those already reported in the hitherto known processes.
Cytotoxicity tests for fungal taxol
In vitro solid tumor assay (Dimethyl sulfoxide i.e. DMSO - method)
Crude samples of the two fungi viz., Botryodiplodia theobromae and Pestalotiopsis menezesiana were tested for their cytotoxicity against cancer cell lines by disk diffusion assay. The disk diffusion assay defines the differential killing among the 7 cell types examined:
1. L1210-Murine Leukemia
2. CCRF-CEM-Human Leukemia
3. Murine Colon 38-Solid tumor
4. Human Colon HI 16-Solid tumor
5. Human Lung H125-Solid tumor
6. Haematopoietic Progenitor Cell-Murine
7. CFU-GM-Human Normal Cell
Both antiproliferative response and differential activity of fungal taxol were observed. 1 The samples were dissolved in DMSO. A volume of 15 μl of the sample was dropped onto a 6.5

mm disks (Baxter filter disks). The disks are allowed to dry overnight and then placed on the edge of the Petri dish. The plates are incubated for 7-10 days and examined by an inverted stereomicroscope (10X) for the measurement of inhibition measured from the edge of the filter disk to the beginning of the normal-sized colony formation.
Cytotoxicity test of the crude sample of B. theobromae by disk diffusion method showed distinct antiproliferative and differential activity on L1210, 250 units and after 1/4 dilution, 200 units and on Colon 38 > 1000 units which means all the cells were wiped out and after lA dilution 600 units (Table -1).
Cytotoxicity test of the crude sample of P. menezesiana by disk diffusion method showed distinct antiproliferative and differential activity on L1210, 200 units and after lA dilution, 250 units and on Colon 38, 700 units and after lA dilution also 700 units, which means that there is potential anti cancerous activity (Table -1).


Advantages of the invention:
Two new microorganisms designated as MTCC 7380 & MTCC 7381 are produced which are useful for the preparation of TAXOL
The Process of preparing Taxol from the new microorganisms designated as MTCC 7380 & MTCC 7381 is simple, economical and is industrially applicable resulting the possibility of making taxol and making the important drug available to needy persons at affordable prices.
The concentration of Taxol produced from the process of the invention is identical with that produced by the hitherto known processes (99.9 % purity).
The process of preparation of Taxol can be effected at room temperature (between 25°C and 33°C) thereby making the process simple & Economical.
Taxol prepared according to the process of the present invention, is formed in the culture medium and therefore can be isolated without employing any expensive steps thereby again making the process simple and economical.
Cytotoxicity test were conducted for the two selected fungi viz., Botryodiplodia theobromae and Pestalotiopsis menezesiana by disk diffusion assay on various cancer cell lines. It was observed that both the fungi showed high toxicity on cancer cell lines.

We Claim
1. Novel microorganism designated as MTCC 7380 dated 16-09-2005 deposited at
IMTECH, Chandigarh useful for the preparation of Taxol of the formula I given above which is useful for the treatment of Cancer
2. Novel microorganism designated as MTCC 7381 dated 16-09-2005 deposited at IMTECH,
Chandigarh useful for the preparation of Taxol of the formula I given above which is useful for the treatment of Cancer
3. A process for the isolation of the microorganisms designated as MTCC 7380 from Solarium
nigrum which comprises
(i) Cutting the parts of the plant Solatium nigrum into small pieces
(ii) Cleaning the pieces to remove unwanted materials adhering on them
(iii) Further sterilizing with sodium hypochlorite solution, and ethanol
(iv) Treating the cleaned pieces with a Carbon source for a period in the range of 2-7 days
at a temperature in the range of 28-32° C to form fungal masses
(v) Separating the fungal masses of the microorganism designated as MTCC 7380 by
conventional methods and
(vi) Purifying the resultant microorganism designated as MTCC 7380 by conventional
methods
4. A process for the isolation of the microorganisms designated as MTCC 7381 from
Terminalia arjuna which .comprises
(i) Cutting the parts of the plant Terminalia arjuna into small pieces
(ii) Cleaning the pieces to remove unwanted materials adhering on them
(iii) Further sterilizing with sodium hypochlorite solution and ethanol
(iv) Treating the cleaned pieces with a Carbon source for a period in the range of 2-7
days at a temperature in the range of 28-32° C to form fungal masses
(vii) Separating the fungal masses of the microorganism designated as MTCC 7381 by
conventional methods and

(vi) Purifying the resultant microorganism designated as MTCC 7381 by conventional methods

(i) growing the novel microorganism designated as MTCC 7380 in a medium containing Carbon, Nitrogen and Carbon sources , at a temperature in the range of 28-32° C, at a'pH in the range of 6.5 -7.0 for a period in the range of 15-25 days.
(ii) separating the fungal mass formed from the culture medium by Alteration
(iii) adding to the culture medium Sodium bi carbonate to avoid fatty acid contamination.
(iv) extracting the resulting medium with methylene chloride
(v) separating the organic phase from the liquid culture medium phase by known methods
(vi) Drying the Organic phase at reduced pressure and at a temperature in the range of 28-35°C.
(vii) Dissolving the resulting dried powder with methylene chloride
(viii) Eluting the solution through silica gel column employing different solvent systems starting with 70 ml of 100% methylene chloride and mixtures of methylene chloride / ethyl acetate at 20:1,10:1, 6:1,3:1 and 1:1 (y/v).
(ix) Separating the fractions coming out at 6:1 & 1:1 containing Taxol and mixing them followed by evaporation.
(x) Drying the fractions to get Taxol.


(i) growing the novel microorganism designated as MTCC 7381 in a medium containing Carbon, Nitrogen and Carbon sources , at a temperature in the range of 28-32° C, at a pH in the range of 6.5 -7.0 for a period in the range of 15-25 days.
(ii) separating the fungal mass formed from the culture medium by Alteration
(iii) adding to the culture medium Sodium bi carbonate to avoid fatty acid contamination.
(iv) extracting the resulting medium with methylene chloride
(v) separating the organic phase from the liquid culture medium phase by known methods
(vi) Drying the Organic phase at reduced pressure and at a temperature in the range of 28-35°C.
(vii) Dissolving the resulting dried powder with methylene chloride
(viii) Eluting the solution through silica gel column employing different solvent systems starting with 70 ml of 100% methylene chloride and mixtures of methylene chloride / ethyl acetate at 20:1,10:1,6:1,3:1 and 1:1 (v/v).
(ix) Separating the fractions coming out at 6:1 & 1:1 containing Taxol and mixing them followed by evaporation.
(x) Drying the fractions to get Taxol.
7. Novel microorganisms designated as MTCC 7380 and MTCC 7381 both dated 16-09-2005
deposited at IMTECH, Chandigarh useful for the preparation of Taxol of the formula I given above which is useful for the treatment of Cancer substantially as herein described

8. Processes for the preparation of novel microorganisms designated as MTCC 7380 and
MTCC 7381 both dated 16-09-2005 deposited at IMTECH, Chandigarh useful for the preparation of Taxol of the formula I given above which is useful for the treatment of Cancer substantially as herein described
9. An improved process for the preparation of Taxol having the formula I given above
useful for the treatment of cancer from the new microorganisms designated as MTCC 7380
and MTCC 7381 both dated 16-09-2005 deposited at IMTECH, Chandigarh , substantially as herein described