In this article we will discuss about:- 1. Introduction to Phytosterols 2. Materials and Methods used for Phytosterols 3. Results and Discussion.
Introduction to Phytosterols:
Phytosterols are considered potential raw material for partial synthesis of pharmaceutically active steroids. The increasing demand for steroidal drugs has resulted in the depletion of many natural resources. Hence, an alternative source for a starting material is imperative. In this context presence of phytosterols in bamboo had been reported.
Microbial transformation have been most widely exploited in the field of steroids and several processes have’ been developed for the selective bacterial degradation of the side chain of naturally occurring sterols. Conversion of sterols to C19 steroids provide valuable intermediates for industrial steroids drug production. Succulent bamboo shoots is proposed as an excellent source of phytosterols and its biotransformation into Androsta-I, 4-diene-3, 17-diene (ADD) using Arthrobacter globiformis in the presence of metabolic inhibitors are described which appear promising for exploitation at commercial scale.
Phytosterols content were estimated in the succulent bamboo shoots of different species of bamboo found in Manipur was conducted which ranges from 0.12 to 0.19% dry weight basis. Other portions of the plant also contain considerable amount of phytosterols (0.001 to 0.34% dry wt.). Fresh succulent bamboo shoot samples from five of these bamboo species were subjected to fermentation which resulted in an enrichment of phytosterols from 0.3 to 0.6% dry weight as compared to that of fresh one.
Further, extraction and purification of the crude phytosterols were done to isolate different phytosterols by TLC, UV, Mass and IR spectral analysis. The isolated phytosterols i.e. stigmasterol, β-sitosterols and campesterol were then subjected to microbial bioconversion which yielded a considerable amount of Androsta-1, 4-diene-3, 17-diene (ADD) in the incubation mixture in presence of metabolic inhibitors (α, α1-dipyridyl and sodium arsenate). Gradual increase in the production of ADD was seen after inhibitors were added and high rate of production of ADD was observed with 24 h incubation.
Materials and Methods used for Phytosterols:
In order to screen out whether all parts of the plant material contain phytosterols, each of the plant material (rhizome, stem sheath, bristles on the stem sheath, mature stem scraped, leaves, succulent bamboo shoots, inflorescence) were collected from different localities of Manipur and were oven dried as above. The dried samples were powdered and the concentration of total phytosterols was determined calorimetrically using Liebermann-Burchard reagent.
For enrichment of sterols during anaerobic digestion, fermentation of the fresh succulent bamboo shoots were done by inoculating thin slices with the exudates obtained from already fermented slices of bamboo shoots (traditionally fermented) sold in the local market in the name of ‘Soibum’. After inoculation, the samples were kept in an incubator at 30°C ± 2°C for a period of 35 days, during which period fermentation took place. Weekly interval analyses on the changes in the level of total phytosterols were carried out during fermentation using Liebermann Burchard reaction.
To purify phytosterols, the first step was extraction of phytosterols from the fermented samples. Extraction was done in a 1 litre Soxhlet extraction flask using a mixture of Benzene, petroleum ether and 2N ethanolic KOH (10: 5: 1) as the refluxing solvent. After selective solubilization of the crude phytosterols with Acetonitrile, three type of creamy coloured precipitates were obtained; one at the surface layer of the solution, another cream crystalline form sticking to the side wall of the contained and the third is a brownish coloured precipitate residue at the bottom of the contained.
These precipitates were further partially purified as a result of differential solubilization with acetonitrile. The partially purified different phytosterols were then subjected to TLC. The TLC analysis was performed on Silica- Gel-G (0.25 mm thick) plates (20 x 20 cm) using Benzene- Diethyl ether (7: 3).
Melting point of the crystals was determined and UV spectral analyses were conducted. For UV spectral studies, the absorbance of the compounds and the authentic sample (Sigma Chemical, U.S.A.) were measured from 225 nm to 400 nm on a Beckman DU-64- Spectrophotometer. Further, analysis of IR, NMR and Mass spectra were done at CDRI, Lucknow for confirmation of the compound as compared to that of the authentic sample (stigmasterol, β-sitosterols and campesterol).
For the study of biotransformation of the three different phytosterols extracted from succulent bamboo shoots, it was considered necessary to obtain suitable microorganisms capable of utilising sterols. For this Arthrobacter globiformis (IMTECH, Chandigarh) was used as the organism for conversion of sterols into C19 steroids.
The medium on which the bacterium was grown contained (g/1) NaHSO4 ,2 H2O, 3.0; KH2 PO4 , 3.0; NaCl, 0.2; (NH4)2 SO4, 1.0; MgS04, 7.H2 O, 0.3; CaCl2, 2H2 O, 0.1; FeSO4,7H2O, 0.001; Sodium molybdate, 0.0001. The pH was adjusted to 7.2 and fine suspension of, β- sitosterols (authentic sample) as well as the three different phytosterols (stigmasterol, β-sitosterols and campesterol) isolated from the succulent bamboo shoot was made separately in the above medium.
The mineral medium containing the phytosterols were homogenised in warming blender for 10-15 mins and it was then sterilised in the Autoclave (15 lbs pressure) for 15 min. The bacterium was then allowed to grow in a 250 ml flask containing 50 ml of the medium on a reciprocal shaker (80 strokes/min) at 30 ± 2°C for 24 h.
This was followed by further addition of inhibitors (0.1%, α, α1-dipyridyl and 0.1% sodium arsenate). The incubation was then carried out for 72 hrs after addition of inhibitor. At regular interval, flasks were withdrawn from the shaker and a representative sample (5 ml) was withdrawn from each flask and was extracted with dried and re-distilled ethylacetate (1:1 v/v). Suitable aliquots of the elthyl extract were used for the estimation of residual sterol by Liebermann- Burchard reaction and estimation of ADD by Zimmermann reaction.
Results and Discussion Related to Phytosterols:
The result showed that the level of total phytosterols in fresh samples ranges from 0.12 to 0.19% on dry wt. basis in different species of bamboos. The highest concentration was found in Dendrocalamus halmiltonii and Bambusa pallida (0.19%) followed by B. balcooa, Cephalostachyum pergracile, D. strictus and Arundinaria callose. The lowest concentration was found in B. khasiana with 0.12% dry wt. basis (Table 1).
The level of phytosterol varies in different parts of the plant (Table 2). Since the rhizome, mature stem scraps, stem sheath and bristles on stem sheath possess less quantity of phytosterols, these sources cannot be preferred for phytosterol extraction.
The inflorescence, on the other hand, contains very high content of phytosterols but it is rarely available. The result showed that succulent shoots of bamboo possessed a considerable amount of phytosterols. Therefore, it seems essential to proceed for a detailed study on extraction and enrichment of phytosterols through biochemical means.
In the fermentation experiments, there was an increasing trend in the concentration of total phytosterols from the initial stage of fermentation (0-day) till day 35 (Table 2). The increase in the level of phytosterols in the fermented samples was due to anaerobic digestion by microorganisms that cause degradation of the organic matter and resulted in the enrichment of phytosterols.
The TLC plates of the three different phytosterols extracted from bamboo shoots developed with Liebermann-Burchard reagent showed three spots each. The Co-chromatography with standard samples revealed that the middle spots of the surface floating fractions tallied with that the β-sitosterols, stigmasterol (Sigma), similarly the middle spot of the side wall sticking fraction tallied with (Sigma) and the middle spot of the bottom residue fraction tallied with the campesterol (Sigma). The data showing Rf values are given in Table 3-5.
Melting point of each fraction phytosterols (Table 4) was determined and were found to be 170°C for the upper surface floating crystals, 140°C for the side wall sticking crystals and 95°C for the bottom residue fractions.
The UV spectrall analysis of the authentic sample and the different fractions showed similar peaks. The IR spectral data and the Mass spectra of the compound showed similarity with those obtained of the authentic samples of stigmasterol, β-sitosterols and campesterol (Sigma Chemicals, St. Luis U.S.A.).
These phytosterols (stigmasterol, β-sitosterols and campesterol) isolated from fermented bamboo shoots are used for microbial conversion into Androsta-1, 4-diene-3, 17-diene (ADD) using Arthrobacter globiformis. Phytosterols Consumption (residual phytosterol) and ADD accumulation in the culture broth in the presence and absences of metabolic inhibitors (α, α1-dipyridyl and sodium arsenate) were estimated colorimetrically in Beckman DU-64 Spectrophotometer. The level of residual phytosterols which remains unutilised during microbial transformation in the broth were maximum in the beginning and were found even upto 48h incubation and decline later in presence of each inhibitor (Table 6).
But in the case of accumulation of ADD, gradual increase in the production of ADD was seen after inhibitors α, α1-dipyridyl and sodium arsenate) were added and high rate of production of ADD was observed with 24h incubation (Table 7).
The maximum accumulation of ADD was more or less maintained for a prolong period of incubation (36h) but decline later. This showed that the inhibitor protected the sterol nucleus from being used-up by the bacterium but the consumption of the side chain at C17 position continued. As a result, 17 ketosteroids (ADD) got accumulated in the culture broth, which was determined by Zimermann reactions.
There observation also showed that Arthrobacter globiformis is not only efficient in the bioconversion of β-sitosterols into ADD as reported earlier but it is equally effective in the bioconversion of partially purified other phytosterols. For the first time β-sitosterols, stigmasterols and campesterol had been isolated from bamboo species and has been microbiologically converted into ADD using Arthrobacter globiformis. The present work attempts at utilizing bamboo shoot which are easily available in huge quantity as the raw material for the isolation of phytosterols and its microbial bio-conversion into ADD.