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Details OF Research Programs / Projects
| Sr. No. |
Title & scope of the R&D project |
Name of the project leader |
Duration of the project |
Remarks (Indicate specific reasons,If any, for proposing the R&D project) |
| 1 |
Bioremedial measures for environmental contaminants.
a) Isolation of scavenger microbial systems from petroleum sludge to clean petroleum waste dumps and oil spills.
b) Isolation, purification of enzyme HRPO for the treat-ment of industrial waste water.
|
Dr. S. S. Save
Dr. M. Sundaresan
Dr. A. M. Bhagwat
|
3 years |
This is a project of great environmental significance. The bioremedial measures are likely to prove more environmental friendly a than the tra-tidional/Con-ventional methods. Further the bioremedial methods are likely to be less expen-sive. |
| 2 |
Isolation and identification of active antidiabetic principles in the fruits of Ficus species. |
Dr. P. T. Muthe
Dr. A. M. Bhagwat
Mrs. K. Ozarkar
|
3 years |
Fruits of Ficus species have been reported to have hypo-glycemic factors. In this project a systematic effort will be made to isolate & identify such factors and examine their effect on induced diabetic ani- mals as well as human diabetic volunteers. |
| 3 |
Studies on pigment production from fungi |
Dr. S. S. Save |
3 years |
To develop food grade colour for food industry. |
| 4 |
Irradiation studies of some Ayurvedic raw materials : Microbiological and chemical aspects |
Dr. S. S. Save
Ms. Chhaya Sawant
|
3 years |
The project aims to evaluate the effect of gamma rediation used to improve shelf life of ayurvedic powders on chemical stability of the drug. |
| 5 |
Characterization of some Ayurvedic Bhasmas with respect to Metallofullere-nes, their analogs or simply inorganic Buckyballs. |
Dr. I. C. Bhoir
Dr. M. Sundaresan
Dr.(Mrs) S.I. Bhoir
Dr. A. M. Bhagwat
|
3 years |
To chara-terise Ayur-vedic Bhasma & to evaluate the pusence of metallofullerenes or simply inorganic buckey balls. |
| 6 |
Feasibility studies for replacement of RP-HPLC/UV by a PC-SFC/UV for pharmaceutical analysis. |
Dr. M. Sundaresan |
1998 |
In-house project. The RP-HPLC uses solvents that are toxic and environmentally unfriedly. PC SFC on the other hand is environmentally friendly, cheaper and faster compared as to RP-HPLC. |
| 7 |
Comparative Biochemical survey of plants poisonous to fish and other animals. |
Dr. P. T. Muthe |
1997 |
Some of the substances have been identified as excellent piscicedes to be used in prawn culture because of their highly specific action. |
| 8 |
Micropropagation, Chemical and pharmaceutical studies of certain medicinal plants |
Dr. S. M. Karmarkar |
1997 |
Major thrust is for the identification of marker compounds / active compunds as medicinal plants. Micropropagation as well as embryogenesis for medi-cinally important rare / endangered species is being undertaken. |
| 9 |
Effect of environmental contamination on raptor population. |
Dr. A. M. Bhagwat
(joint project with BNHS, Mumbai.)
|
1997 |
Environmental con-tamination due to heavy application of pesticides is affe-cting Birds of prey populations world over. This project addresses the problem in India. |
| 10 |
An indigenous method for the synthesis of essential Amino acid
L-phenylalanine
|
Dr. S. S. Save |
1998 |
Project concluded in August 2001. |
| 11 |
Irradiation studies of some ayurvedic raw materials for export |
Dr. S. S. Save
Dr.M. Sundaresan
|
1998 |
Project concluded in June 2001. |
| 12 |
In vitro propagation of the ornamental plant species, diephenbachia, staghorn fern, and syngonium and development of protocol for multiple shoot formation |
Dr. S. M. Karmarkar |
1999 |
Project concluded in December 2000. |
| 13 |
Studies on the effect of environmental contaminants on raptors with special reference to the sheheen, Falco peleginoide. |
Dr. A. M. Bhagwat |
1998 |
Project concluded in May 2001. |
| 14 |
DAE project on solid fluid Extraction of Metals using Supercritical Fluid Carbon dioxide. |
Dr. I.C.Bhoir |
2000 |
Novel method for fine purification of metals in the form of metal complexed. The method is extremely ecofriendly as it does not use highly consive inorganic acids |
Reasearch Activity
Summary :
The study was carried out in an attempt to evaluate the usefulness of gamma radiation for sterilization of Ayurvedic raw materials such as : Amrita, Karela
Methodology :
1. irradiation of raw materials
2. microbiological analysis of non-irradiated and irradiated samples with included
a) Determination of moisture content
b) Determination of microbial load before and after irradiation
c) Sterility testing.
3. Physiochemical analysis
Moisture content of each powder was determined by drying the powder at 110oC in hot air oven. At different time intervals, percentage loss of moisture content was monitored by taking the weight of the powder. From the percentages loss of moisture, actual moisture content was calculated.
The raw material for sterilization by gamma radiation was packed in low-density polyethylene bags. Samples were exposed to a range of irradiation doses i.e. 2, 5, 6, 9, 10, 15 & 20 kGy to determine effective dose of irradiation required for complete sterilization.
Microbial analysis of irradiated and non-irradiated material included,
1) Enrichment
2) Determination of total aerobic, anaerobic and fungal viable count.
3) Sterility testing for checking shelf life after irradiation
4) Biological activity of Guggul.
Enrichment makes use of selective culture media and incubation conditions to isolate microorganisms directly from natural samples.
For total viable count and sterility testing methodology used was pour plate technique. It was performed using selective media for aerobic bacteria, anaerobic bacteria and fungi.
Biological activity of guggul was tested before and after irradiation using Ditch Plate Technique. This method is an agar diffusion technique used for measuring activity of an anti-microbial agent. It is mainly used when the compound is insoluble.
All four commercially obtained powders had a very heavy microbial load prior to irradiation, however it was significantly reduced after radiation treatment.
There was no change in the physiochemical characteristic of all four powders.
Conclusion :
It was observed that at 2 kGy dose, there was substantial decrease in the number of viable microorganisms of all four powders.
At 10 kGy complete sterilization of powders was achieved. This sterility was maintained for 7 months. In addition it was also noted that irradiation does not change the anti-microbial activity of guggul.
Summary
Methodology
Sample preparation and packing of material. Samples were prepared for extraction in the following manner.
A portion of the materials, both irradiated and nonirradiated, equivalent to 5 gm was weighed out and transferred to the extraction used which was previously packed on one side with glass wool to prevent any physical carry over. This was in addition to the filter which was already provided. The layer of glass wool was ~2 mm thick. Glass rod bits of size ~2-3 nm. Several of them, had already been placed over the glass wood and sample in order to avoid a large void volume. After the transfer of the material another layer of glass wool was placed over the material and the glass bits and the cap was screwed in. the flanges were tightened and the cylinder was fitted to the equipment.
Carbon dioxide was let into the equipment at the required rate. Initially for 30 sec. The gas, after attaining supercritically, passed through the cylinder and was let off into the atmosphere. Thereafter the outlet of the extraction vessel was led into a glass tube with a conical end containing a suitable solvent. The pressure and the temperature of the gas had already been adjusted to the desired values.
Each extraction was carried out for 30 min., except in case where methanol was used as the co-solvent where the time was restricted to 10 min. The flow rates were constant at 5 ml/min. The co-solvent extractant was 3.85% (v/v) of methanol dissolved in SCF CO2 at the specified pressure and temperature.
Chromatography
In all cases the technique of PC SFC (packed column supercritical fluid chromatography) was utilised for the characterisation.
In this case extraction vessel was disconnected and a column used in HPLC was connected in its place. The oven used for making supercritical fluid CO2 was used for the chromatographic experiments also. After a series of experiments involving the change of variable parameters like modifier concentration, pressure, temperature etc. the following chromatographic conditions were established.
Chromatographic conditions
| Pressure | : | 100 kg/cm2 |
| Temperature | : | 45oC |
| Column | : | (250 x 4.6 nm) 10 um. |
| Mobile phase | : | SFC CO2 with 7.2% (v/v) meth. |
| Injection volumen | : | 20 ul and |
| Flow rate of CO2 | : | 2 ml/min. |
| Flow rate of methanol | : | 0.15 ml/min. |
| Wavelength of detection | : | 254 nm |
| Run time | : | 10-15 nm. |
After each extraction the residue was dissolved in 5 ml methanol with slight heating and 20 ul of this solution was injected into the column.
Result and discussion
A persual of the data obtained on the SFC profiles of the Ayurvedic natural products shows that the profiles of the irradiated and nonirradiated materials significantly differ in both total peak areas, peak heights and the retention times of the components of the extracts. Even the number of components varies in most of the cases. It was thought that as irradiation was carried out at 25 kGy. The changes may be due to the high doses received. However patterns obtained at even lower doses revealed a similar picture even though lesser discrepancies were noticed in these profiles.
In the case of Parijat these differences were least in that most of the cases the number of components obtained were the same for the irradiarted and nonirradiated materials were nearly the same in at least 4 of the 6 extracts obta3ined at different pressures and temperatures.
With karela in only 3 out of the 8 extracts the number of components was the same. Retention times and peak areas and heights obtained were different. The total area evaluation could also not yield any tangible results. In guggul the same picture was revealed. In Amrita, however, the components of the irradiated product were found to be higher giving rise to a connection that degradation due to irradiation gave rise to more components i.e., degradation acompanied decomposition.
Normal phase chromatography also revealed disturbed chromatograms with non steady base lines.
Profiles provide the advantages of assessing degradation by producing an array of compound or compound groups. This method is definitely more useful than estimating a “marker” or active compound. This method lacks the sensitivity for assessing the therapeutic utility of a natural substances as the therapeutic activity of the compound is associated with synergistic activity of the accompanying compounds.
It is tuhs concluded that a more exhaustive examination of these Ayurvedic products should be conducted, preferably with a photodiode array detector before coming to definite conclusions.
Production of L-phenylalanine : A biotechnological approach
Objectives :
1) Development of methodology for the invitro production of L-Phenylalanine using Rhodotorula yeast cells.
2) Product recovery, identification and quantitation.
3) Detailed investigation with respect to stabilisation of this biotechnological process involving the use of conventional immobilisation technique, for the purpose of commercial viability.
Various methods, both chemical and biological, exist for the synthesis of L-Phenylalanine. However, in a chemical raction, equimolar mixtures of L and D isomers would be obtained. Resolution of the stereoisomers and provision of toxicological data for each of the isomers as well as for the racemate is time consuming and involves sophisticated and expensive methods. Enzymatic and fermentative methods score widely for the above reasons. Among the bioconversions, the use of L-Phenylalanine Ammonia Lyase (PAL, E.C.4.3.1.5) has been receiving increasing attention because of the low price of the raw feed stock, t-Cinnamic acid, as well as the stereospecific production of L-Phe.
A strain of the red yeast, Rhodotorula glutinis was chosen for this study. The cells are known to contain high levels of the enzyme PAL that converts t-CA to L-Phe.
Physiological manipulations asing media components, pH, temperature required for growth etc., were studied for obtaining maximum growth and optimum PAL activity. A standardised procedure was developed for the estimation of PAL activity.
The next step will involve the optimisation of the reaction of interest i.e. the production of L-Phe. From t-CA. A variety of parameters were manipulated and the effects studied to arrive at standardised protocol for the reversal of the normal physiological reaction catalysed by PAL.
To make the process viable cells need to be immobilised to allow recycling, easy product recovery and a stable enzyme. Various methods to immobilise cells were studied.
The data generated have been successfully used in the conversion of trans cinnanamic acid to L-Phenylalanine on a bench scale.
Isolation and Application of Zymomonas CBP 99
A. The present work deals with the isolation, identification, characterisation and application of bacterium from sugarcane molasses.
The organisms were isolated on standard media, viz. MYGP and MacConkeys media. After studying its colony characters, the isolates were subjected to various biochemical tests. After through biochemical investigations one of the isolates showing interesting characteristics was selected and taken up for a systematic study of its growth parameters involving.
1) Study of various enzymes produced by the isolate
2) Determination of optimum pH and temperature for the growth of the isolate,
3) Study of growth response to different growth conditions, which included
a) Sucrose, Glucose, Salt and Ethanol tolerance
b) The effect of dyes on the growth
c) Growth in presence of antibiotics
d) Effect of KCN on growth
e) Effect of a preservative-sodium benzoate
f) Amino acid utilization
g) Determination of TDP and TDT
h) Study of the Growth-cycle and the generation time.
B. The applications of Zymononas
1) Dough fermentation : the dough fermentation capability of the newly isolated organism was compared to the commercially available dry active yeast.
2) Alcohol production : Various media were used for assessing maximum ethanol production by Zymomonas (CBP 99). Optimization of parameters involved, effect of varying concentrations of sucrose and glucose on alcohol production, effect of aeration on the same etc. alcohol formed was assayed by the Potassium dichromate method.
3) Phenylalanine production by freshly isolated cells of Zymomonas CBP 99: According to the literature survey carried out we are reporting for the first time the presence of Phenylalanine ammonia lyase (PAL) in a wild type strain of Zymomonas. The enzyme is responsible for conversion of trans-cinnamic acid to L-Phenylalanine. Te conditions for maximum production of Phenylalanine were checked. This included optimization of media components, reaction parameters etc. paper chromatography was performed to detect the amino acid formed.
4) Production of sorbitol and glycerol : The ability of the organisms to produce glycerol and sorbitol in the fermentation media will also be evaluated by HPLC.
Title : Studies on in-vitro growth and phytochemical constituents in Catharanthus roseus L.(G) Don.
The main objectives of the present project were
1) Standardisation of protocols for micropropagation of Catharanthus roseus
2) Standardisation of protocol for development of callus and its growth for procuring a maximum yield of the biomolecules ajmalicine, reserpine, serpentine.
3) Development of suspension cultures for obtaining maximum yield of biomolecules ajmalicine, reserpine, serpentine.
4) Testing of in-vitro cultures for their bioactivity against bacterial and fungal strains.
5) To compare C.roseus root extract, leaf extract, extracts of in-vitro cultures of C.roseus with the extracts of Rauvolfia serpentina roots in terms of their phytochemicals constitutents.
Some cultures have been transferred and maintained through subcultures on a medium which supports the growth of callus so as to bring about optinal growth of biomass. Growth kinetics of callus have been studied. The fully growtn callus has been subcultured for rhizogenesis. The kinetics of growth as well as rhizogenesis have been studied with a view to optimising the yield of the root products.
Roots developed in-vitro as well as callus developed from leaves have been analysed for their phytoconstituents (alkaloids) and compared with those of roots of naturally growtn C.roseus. in addtion the in-vitro cultures of C.roseus have been compared with the natural roots of Rauvolfia serpentina since R.serpentina is a major source of the same hypotensive indole alkaloids (ajmalicine, reserpine and serpentine). For this purpose in-vitro cultures of R.serpentina have been raised.
The alcoholic extracts of C.roseus cultures, natural leaves, natural roots were tested against some bacterial and fungal strains. The bioactivity of these extracts against the bacterial and fungal pathogens is found to be retained in the in-vitro cultures.
C.roseus is a rich source of alkaloids and is cultivated through seeds. The root alkaloid ajmalicine which is hypotensive, is very highly priced and hence efforts are being made to increase the propagation rate of C.roseus and thereby increase the production of this highly priced alkaloid, ajmalicine.
The multiple shoot cultures of C.roseus were initiated from nodal explant on Murashige and Skoog medium (1962) containing BAP and NAA.
The callus cultures were raised on MS medium with NAA and kinetin for increasing the production of alkaloids. Callus, when subcultured on a growth medium of lower strength supplemented with growth hormones, tends towards rhizogenesis, resulting in a good yield of the root biomass.
Suspension cultures were developed in MS medium with lower concentrations for the same growth hormones and these were also found to give a higher yield of biomass.
The alcoholic extracts of leaves of C.roseus roots and leaves of plants developed in-vitro, roots of plants growing in a natural environment as well as callus were tested against several bacterial and fungal pathogens. The root extracts were found to be active against the fungus Tricoderma sp. And Aspergillus nigir. This activity was found to be retained in the roots of invitro cultures also.
The roots and in-vitro cultures of C.roseus were extracted upto a single spot detection of the alkaloids ajmalicine, reserpine and serpentine on TLC employing cerric amtnonium sulphate (CAS).
Work to be done :
A quantitative analysis of the extracts of plant parts of in-vitro cultures as well as those of Crosses growing naturally needs to be undertaken so as to determine whether Crosses could be used as a substitute for R.serpentina in terms of its phytochemical constituents of medicinal use.
Synopsis on Hypoglycemic effects of phytochemicals from various medicinal plants using diabetic mouse model.
Introduction
Diabetes mellitus is a group of physiological disorders that all lead to an elevation of glucose in the blood (hyperglycemia). As hyperglycemia increases, there is loss of glucose in the urine. The two major types of diabetes are Type-1 and Type II. The first one is called as insulin dependent diabetes mellitus (IDDM). It is also known as juvenile onset diabetes since it occurs in people younger than the age of 20 and persists throughout life. Type-II diabetes is very common in people who are above 40 and overweight. It is called as non-insulin dependent diabetes mellitus (NIDDM) or mature onset diabetes. (Tortora & Grabowski, 1992).
Sulphonylureas and biguanides are the major groups of oral hypoglycemic agents (OHA) which freely available in the market and are extensively used in the management of diabetes. Insulin pumps are also used to treat diabetes. Diabetes is a diasease which needs to be monitored regularly. Most of the modern medicines are long term but also expensive. They also show some adverse effects like hypoglycemia allergic reactions, headache, blurred vision etc., it taken for a long period of time (Goodman & Gillman).
The Indian region with a vast heritage of diverse ethnic culture and rich biodiversity is said to be a great emporium of ethnobotanical wealth. The numerous ethnic population in different parts of the country still practice herbal remedies for all aliments including diabetes (T.S.Rana, K.K.Singh & R.R.Rao 1999).
A transition from modern medicinal system to traditional or natural system demands standardization as per scientific methods. This helps not only in better acceptance of traditional system but will also aid in uplifting this system at par with modern medicines.
Various indiganous plants are reported to have anti diabetic properties and Lewis 1949 and Mukherjee 1957 screened many of them from time to time for their hypoglycemic activities as reviewed.
Based on the literature survey, which was carried out, some plants having antidiabetic properties were selected for further scrutiny. These include Cinnamomum tamala, Ficus species, Asparagus racemosa, Catharanthus roseus.
Work plan :
Objective : To evaluate anti diabetic effect of varorious medicinal plants.
Methodology : The first step will include selection of a plant and collecting the plant material from the particular source. Then extenhsive pharmacognosy will be carried out to confirm the plant material. Various parts will be extracted with solvents i.e. methanol, ethanol, iso-propyl alcohol. Etc. to isolate and identify potential hypoglycemic phytochemicals and these will be evaluated against the hole plant extract.
Pilot study : The extracts will first be tested on animals models to evaluate their toxic levels and accordingly doses will be fixed. The extracts will then be screened to confirm their individual anti diabetic effect using alloxan treated diabetic mice as model. The lab is CPCSEA approved.
Experinental : The extracts, which are found to give positive results, will no be used for separation of various active components. To obtain isolate active components, the plant extract will be first qualitatively analysed by TLC and screened to determine the biological activity. For purification and isolation, the active plant extracts will be sequentially fractionated and each fraction will be subjected to bioassay and toxicity evaluation in animals.
The miximum effective component will be further studied to confirm its molecular structure, mode of action and maximum effective dose in mice model.
The following tests will be carried out which show the hypoglycemic effect of extracts as compared to standards.
1) Blood glucose
2) Glucose-6 phosphate dehydrogenase (G-6PD)
3) Blood cholesterol
4) Triglycerides
5) Hepatic glycogen.
Summary and conclusion :
On basis of data generated effectiveness of herbal medicines on diabetes mellitus will be predicted. The animal model will indicate the effective doses to be used in the treatment of human patients.
Studies on costus speciosus (Koen.ex.Retz.) sims.
The rhizomes of Costus speciosus required for the above mentioned study ware collected from their natural habitats. Initially, the method of morris et al., (1958) for extraction of diosgenin from the rhizomes was standardised. Since this method was not found to be completely satisfactory, slight modifications in the methodology were attempted. With these modifications, it was possible to collect sizeable samples of diosgenin crude extract. Crude diosgenin content in the nine samples of Costus speciosus obtained from different habitats was quantified by colorimetric method using antimony pentachloride.
From the above data, it was apparent that the sample from Ernakulam, Cochin, Kerala showed the highest diosgenin content while amongst samples from Maharashtra, the highest values for diosgenin have been recorded in the plants obtained form Kolshet, Thane.
A few other phytochemical studies were also undertaken.
1) An attempt was made to correlate rhizome diameter with diosgenin content. It can be concluded that as the rhizome diameter increases, the diosgenin content decreases.
2) Diosgenin conetnt of Costus speciosus was also recorded at different growth stages. It is clear that the diosgenin content increases, when the rhizome sprouts in June-July and continues to increase till flowering starts in August.
Diosgenin content was maximum in the month of August, but later it started to decline till aerial parts of the plant became dry by November-December. Apparently, diosgenin synthesis commences soon after germination and continues to increase till it reaches maximum at the time of flowering. Subsequently, synthesis of diosgenin in the rhizome does not occur probably because the sugar moiety is utilised for seed setting.
As another aspect of study, the rhizomes of the plant were utilised for separation of the sapogenins in the crude extract, following the technique of thin layer chromatography (TLC).
Five components were identified
Sapogenin
Diosgenin
Lanosterol
B-sitosterol
Stigmasterol
Tigogenin
Combinations of chloroform : benzene as solvent system in the series 9:1, 8:2, 7:3, 6:4, 5:4, 4:6, 3:7, 2:8 and 1:9 were also tried. Of these, chloroform : benzene (9:1) gave the best results. This system was then used as the final standard system and all the plant samples collected from various natural habitats were subjected to TLC analysis. Diosgenin was detected in all the samples as was evident from comparison of its Rf with standard diosgenin. (Hi Media Lab. Ltd., Mumbai).
In another set of experiments on chromatography, a column chromatographic procedure is also being standardised. Two different columns were run and the evaluates from the column were analysed for separation of diosgenin. Although, gradient elutions of with hexane and ethyl acetate gave a separation of diosgenin from the other components, the results were not satisfactory. Hence another column with was eluated with a gradient chloroform and benzene combination. This column gave better separation and diosgenin could be separated. Further UV and IR analysis work is in progress.
The sample from Nagaon, Alibaug was employed for the experiments on in-vitro growth of C.speciosus. The initial experiments involved sterilisation and germination of seeds of C. speciosus. It was observed that the seeds had a low viablility. While the germination percentage in vivo (soil + peat moss 1:1) was only 20%, the percentage of in vitro germination was much lower at 5%.
Simultaneously, another part of the study was taken up involving experiments on tissue culture for developing a protocol for the mass propagation of these plants. Buds developed on the rhizome and aerial part of the rhizome of plants from the natural environment were used for tissue culture.
In the initial experiments, Murashige and Skoog (1962) medium was used but was not found to be satisfactory. In further experiments, Murashige and Skoog’ RT medium as described by Khanna and Staba (1968) was employed with addition of 30 g sucrose and 0.7% agar per litre.
In the presence of BAP alone callus formation occurred after 45 days in all tubes accompanied by shoot formation from axillary buds in about 50% of the tubes. With 2,4-D alone, only callusing occurred and the callus showed browning. This is periodically being subcultured at 3-4 weeks intervals on 1 ppm 2, 4-D in RT MS medium.
A modified Schenk’s and Hildebrandt’s (1972) medium supplemented with various concentrations of of kinetin and 6-benzyl aminopurine were used for producing multiple shoots. Axillary buds and rhizome buds were used as explants. Axillary buds gave the best results. The modified SH medium, wherein the concentration of vitamins and nitrates was increased gave better results than normal SH medium. Kinetin at higher concentrat5ions with 1 mg/1 Indole 3-acetic acid and adenine sulphate gave good results. Two to five shoots were seen within a span of 6 to 8 weeks.
Title : Studies on Cassia tora, Linn.
It is well known that ‘Ayurveda’, the indigenous system of medicine is a well organized traditional healthcare system which, because of its comparatively lower cost and the general absence of side effects, finds favour among a large section of rural as well as urban population of India. There is ample proof of various recipes of Indian herbs in curing many maladies.
One of the plants reported in Ayurvedic scripts is Cassia tora Linn. (Sans. : Chakramardha; Mar.: Takala, Tanleli, tarvat). A literature survey indicated its use against various skin diseases such as ringworm, eczema, scabies. Because of the high incidence of skin diseases, especially among the weaker strata of the Indian population, it was felt worthwhile undertaking research on this plant.
The main objectives of the project were :
1] Morphological identification and, pharmacognostical and phytochemical characterization of the plant.
2] Evaluation of the plant for its antimicrobial properties.
3] Separation of different fractions from the crude drug for the study of the antimicrobial activity of these fractions and their identification.
4] Micropropagation of the plant through multiple shoot formation.
Plant material of Cassia tora, Linn. Was collected locally and identified on the basis of available literature. Pharmacognostic studies have been undertaken so as to bring about the standardization of plant material.
A phytochemical screening of the plant extracts employing TLC indicated that these extracts as well as callus extracts contain-glycosides, flavonoids, bitter principles, and anthrone and anthrancene derivatives.
To evaluate antimicrobial activity, plant parts were cleaned and extracted in n-Hexane, Benzene, Chloroform, and Methanol. Extractive values of these extracts were best in methanol, and they ranged from 1.85% (Root) to 7.4% (Seed).
As per the previously published reports, light petroleum ether was used as a solvent for extracting oil from the seeds. The yield was higher (5.8%) than the previous reports (5.0%).
Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans were tested in-vitro for their susceptibility or resistance to all the above extracts and oil. P. aeruginosa was found to be resistant to the oil. Among the various extracts, the test organisms exhibited maximum susceptibility to the methanolic seed extract.
The extracts and the oil were also tested in-vitro for their shelf life with respect to their activity. The active extracts and the oil have been observed to retain activity for a period of six months, after which the activity gradually declines.
Before separation of fractions, the oil was analysed for its physico-chemical properties such as saponification value, acid value, ester value, specific gravity.
Initially thin layer chromatographic experiments were undertaken to determine the most suitable solvent system for the resolution of the oil sample into its components. Among the following solvent systems – Benzene + Ethyl acetate (90 : 10), Chloroform + Ethanol + Glacial acetic acid (94 : 5 : 1), n-Hexane, n-Hexane + Benzene (70: 30, 60 : 40, 50 : 50, 40 : 60, 30 : 70); Benzene, benzene + Chloroform (1 : 1), chloroform, chloroform + Ethanol (1 : 1), Ethanol, Ethanol + Methanol (1 : 1), Methanol – the system n-Hexane + Benzene (1 : 1) proved the best. Using this solvent system oil sample was eluted on a silica gel column employing a gradient system so as to obtain, as far as possible, single band fractions. These fractions are yet to be identified.
Tissue culture studies were undertaken wherein the axillary bud and the hypocotyl of the seedling were used as explants. Multiple shoot formation was achieved using Murashige and Skoog medium (1962) supplemented with 1 ppm Benzyl amino purine.
The methanolic extracts of the callus which developed in in-vitro cultures exhibited antimicrobial activity against the same test organisms metntioned above. The activity was comparable with that of the in-vivo samples. The extract of the callus which developed on MS medium (1962) supplemented with higher concentrations of BAP exhibited a significant inhibition of growth of the test organisms.
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