ABSTRACT

         Coelomycetous fungi Pesalotiopsis breviseta (Sacc.) Steyaert was screened for the production of taxol an anticancer drug. Taxol production was confirmed by Ultra Violet (UV) spectroscopy, Infra-Red (IR) spectroscopy, High Performance Liquid Chromatography (HPLC) and Liquid Chromatography Mass Spectrum (LC - MS) analysis. The compound produced by the fungus with and without elicitors was compared with authentic taxol, identified and confirmed as taxol. Minimum of 150 µg/L taxol was produced with biotic fungal elicitor, while 160 µg/L was produced without any elicitors. Maximum of 250 µg/L taxol was produced with abiotic elicitor, copper sulphate (CuSO₄). The tested fungus P. breviseta, has a great potential for commercial exploitation in the future, for better taxol production.

Introduction

         Taxol is the commercial trade name used by Bristal-Myers Squibb for the compound, paclitaxel. It has poor water solubility and a complex chemical structure. Taxol along with a chemically similar analogue, Taxotere (Docetaxel) (Guenard et al., 1993) has unique anti-tumour mechanism of action. The drugs are believed to block cell cycle progression during mitosis by binding to and stabilizing microtubules (Schiff et al., 1979, Nicolaou et al., 1994). During cell division, taxol interferes with the development of the microtubules needed for cell duplication thus inhibiting the faster growing tumor cells. This is different from other anti-cancer agents that work by interfering with the DNA of tumor cells.

         Clinical development of taxol progressed slowly because of the extremely small amount of drug obtained from the crude bark extract of the tree Pacific yew. Taxol was approved for the treatment of ovarian cancer by the Food and Drug Administration (FDA) in 1992 and subsequently in the same year was used for treatment of breast cancer. Since then clinical use of taxol had increased and its use has been extended for treatment of lung cancer, squamous cancers of the head and neck and various other cancers (Eisenhaver and Vermorken, 1998). Naturally Pacific yew grows slow but under intensive nursery culture, can grow quite quickly. The tree lives 200-300 years with some species 400 years or more and Taxol is found in small quantities (0.001 to 0.01% of the dry bark weight) and the content can vary in them. It is generally considered to take 3 to 10 trees per patient. Based on the year 1992 data from the US Forest Service, 36000 trees are required to provide 327200 kg of bark (about 9 kg/tree) from which only 24 kg of taxol can be extracted (about 0.66 g/tree). Hence there is an urgent need to search for the alternative sources for this promising anticancer drug.

         The plant cell culture of Taxus species appeared to be promising to obtain taxol and related taxane compounds. Ketchum and Gibson (1996) investigated this using free and immobilized cells of Taxus cuspidata using perfusion culture reactors and achieved continuous taxol production at a rate of 0.3 mg/g DCW (dry cell weight) per day for 40 days.

         The presence of various microorganisms from the bark of the yew tree was found to be capable of producing taxol. The fungi Taxomyces andreanae (Stierle et al., 1993) isolated from the inner bark of a yew tree growing in North-western Montana was able to produce taxol and other taxanes in denovo fashion when grown in semi-synthetic liquid media but the yield was very low (24-50 ng/L). Pestalotiopsis microspora, a coelomycetous endophyte in Taxus brevefolia was observed to produce taxol (Stroebel et al., 1996) in considerable amount more than the Taxus sp. There are atleast 12 distinct enzymatic reactions involved in taxol biosynthesis (Croteau et al., 1995; Floss and Mocek, 1995). The genetic manipulation of fungi could be achieved more easily than that of plants and it may be possible to improve the production significantly with the help of genetic engineering. Keeping this in mind there is an urgent need to search for the alternative sources for this promising anticancer drug. The present study deals with the stimulation of selected endophytic fungi Pesalotiopsis breviseta for the production of taxol.

Materials and methods

Experimental designing

         Biotic and abiotic elicitors were used for enhancement of taxol production from the fungus. The taxol produced without any elicitors by the fungus served as control. One week after the inoculation of fungi in M1D broth media, the abiotic elicitor copper sulphate (CuSO₄) 50 mg/L was added. Later after two weeks another dose of the abiotic elicitor of same concentration was elicitated. For biotic, fungal elicitors was used. Initially the fungi were inoculated in M1D broth media without sucrose for a week after which it was elicitated using 50 mg/L of fungal carbohydrate.

Extraction of taxol

          The extraction procedure was followed by the method of Strobel et al. (1996). The fungus was grown in 2 litre Erlenmeyer flasks containing 500 ml of M1D medium as described in experimental setup and supplemented with 1g soytone L^-1 (Pinkerton and Strobel, 1976). After incubation for 21 days at 26 ± 1°C the culture filtrate was obtained by passing through four-layered cheesecloth. To avoid fatty acid contamination 0.25 g of sodium carbonate (NaCO₃) was added to the filtrate and extracted with equal volumes of solvent dichloromethane twice. The organic phase was collected and evaporated to dryness under reduced pressure at 35ºC. The dry solid residue was re-dissolved in methanol and loaded on a 1.5 X 30 cm column of silica gel (Baker 40 micron). Elution of the column was performed in a stepwise manner starting with 70 mL of 100% methylene chloride followed by mixtures of organic solvents methylene chloride:ethyl acetate at different proportions 20:1, 10:1, 6:1, 3:1 and 1:1 v/v. Fractions were subjected to TLC and those having same mobility as the authentic taxol were combined and evaporated to dryness. The compound obtained was analysed further for the presence of taxol in the fungal samples.

Thin layer chromatography (TLC):

          Chromatography was done on 0.25 mm Merck precoated silica gel plates. The plates were developed successively in each of the following solvent systems as follows; A - Chloroform/ Methanol 7:1 v/v, B - Chloroform/ Acetonitrile 7:3 v/v, C - Ethyl acetate/2-Propanol 95:5 v/v, D- Methylene chloride/ Tetra hydro furan 6:2 v/v and E- Methylene chloride/ Methanol/ Dimethylformamide 90:9:1 v/v/v. Taxol was detected with 1% w/v vanillin/sulphuric acid (H₂SO₄) reagent after gentle heating (Cardellina, 1991). It appeared as a bluish spot fading to dark grey after 24 hours.

Ultra Violet (UV) spectroscopy

          The sample containing taxol was analysed spectroscopically. After chromatography, the area of plate containing putative taxol was carefully removed by scrapping off the silica at the appropriate Rf and exhaustively eluted with methanol. The UV spectral analysis of fungal sample was superimposed on authentic taxol at 273 nm.

High Pressure Liquid Chromatography (HPLC)

          Isolated samples were analysed by HPLC (Shimadzu 9A model) using a reverse phase C₁₈ column with a UV detector. Sample (20 µl) was injected each time and detected at 232 nm. The mobile phase was methanol/acetonitrile/water (25:35:40 v/v) at flow rate of 1.0 ml min-1. Taxol was quantified by comparing the peak area of the samples with that of the standard using the formula,

Taxol Content = Standard concentration × Total area of the sample

Total area of the standard

High Pressure Liquid Chromatography (HPLC)

          The IR spectra of the compound were recorded on Shimadzu FT IR 8000 series instrument. The purified taxol was ground with spectra grade potassium bromide (KBr) (1:10) pressed in to pellets under vacuum using spectra lab Pelletiser and compared with authentic Taxol. The IR spectrum was recorded in the region 4000 – 500 cm^-1nm.

Liquid Chromatography - Mass Spectrometry

          The sample from Pesalotiopsis breviseta was subjected to electron spray mass spectroscopic analysis for the confirmation of presence of taxol. The sample was dissolved in methanol:water:acetic acid (50:50:1 v/v). It was injected with a spray of 2 µL and spray voltage of 50 V by the loop injection method.

Results and Discussion

          In the present study the test fungus Pestalotiopsis breviseta a Coelomycete, was tested for production and enhancement of taxol by biotic and abiotic elicitors in a semi-synthetic medium M1D, for its growth. The taxol was extracted using dichloromethane and the solvent was removed by evaporation under vacuum, the resulting residue was re-dissolved in methanol and treated as the sample containing taxol to further studies for its confirmation.

          Thin layer chromatography (TLC) was developed on a 0.25 mm (10 x 20 cm) silica gel plate in different solvent system with authentic taxol (Sigma, Cat. No. T-7402) as standard. The sample showed identical Rf and UV characteristics with the standard and reacted positively with Vanillin/H₂SO₄ spray reagent, yielding a blue spot which turned grey after 12 - 24 h. After chromatography the taxol was eluted with methanol. The UV spectral analysis of samples were superimposed on that of authentic taxol with two maxima at 273 nm and 235 nm which showed that the purified sample might to be a taxol (Fig.1a-d).

Figure 1: Ultra Violet (UV) Absorption of standard taxol against taxol isolated from the fungus under different conditions (control, biotic and abiotic elicitors)

             The sample containing taxol was analysed by HPLC using a reverse phase C₁₈ column with a UV detector for the quantification using the authentic taxol as standard. The amount of taxol produced by the fungus was quantified by comparing the peak area of the samples (Fig. 2a-d) with that of the taxol standard using the methods and formula described earlier. The effect of biotic and abiotic elicitors on the fungus Pestalotiopsis breviseta for taxol production was quantified. The maximum taxol production was observed in the medium containing chemical elicitor CuSO₄ (250 μg/l) and minimal of 150 μg/L of taxol in medium containing fungal elicitor. The cultures grown without any elicitors i.e. control produced 160 µg/L. Thus further convincing evidence for the identity of taxol was obtained by High Pressure Liquid Chromatography.

Figure 2: High Pressure Liquid Chromatography (HPLC) analysis of standard taxol against taxol isolated from the fungus under different conditions (control, biotic and abiotic elicitors)

          The presence of taxol was further confirmed by using Infra-Red analysis of the compound isolated from the fungus. The appearances of bands convincingly illustrated the identical feature of the taxol isolated from the fungus without any elicitor (Control) (Fig. 3b) and taxol isolated from the fungus treated with abiotic elicitor (Fig. 3c) with the authentic taxol used as standard (Fig. 3a). Infra-Red analysis showed that the presence of alcoholic O - group in the sample is evident by its OH stretching vibration at 3448 cm^-1. The aliphatic CH - stretch at 2931 cm^-1, the C=O stretch positioned 1724 cm^-1 and the amide C=O stretch is shifted to lower value at 1652 cm^-1. The intense peak at 1247.16 cm^-1 is due to COO stretch. The alkyl C-O stretch of ester is observed at 1072 cm^-1. The peak at 707 cm^-1 is due to aromatic C, H bond. In extracted sample though the intensity of the bands are very much diminished in the fingerprinting region, appearance of overtone 2362 cm^-1 convincingly illustrates the identical nature of the extracted sample with authentic taxol. The taxol isolated from fungus treated with biotic elicitor was not subjected to further analysis as its yield was very low comparatively.

Fig. 3a&b: (a) IR spectrum of Authentic Taxol and (b) taxol produced by Pestalotiopsis breviseta without elicitors (Control).

Fig. 3c: IR spectrum of Taxol extracted from Pestalotiopsis breviseta using abiotic elicitor.

          The fungal compound produced an identical LC mass spectrum when compared with the standard. Characteristically, authentic taxol yielded both an (M+H)+ peak at 855 and an (M+Na)+ peak at 856 (Fig. 4a) and by comparison, fungal taxol produced in the presence of abiotic elicitor also had these characteristics peak (Fig. 4b). Based on these results it was confirmed that the compound produced by the fungus Pestalotiopsis breviseta was taxol.

Fig. 4a: Liquid Chromatography-Mass Spectrometry (LC-MS) analysis of standard taxol.

Fig. 4b: Liquid Chromatography-Mass Spectrometry (LC-MS) analysis of taxol isolated from Pestalotiopsis breviseta treated with abiotic elicitor.

          The amount of taxol produced by the fungus could be increased by improving culture techniques, addition of various elicitors, application of genetic engineering and gene expression studies. This would lead to improved production of taxol and overrule its production from plant source.

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