Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Culture – maintenance – or preservation techniques – per se
Reexamination Certificate
1998-03-31
2001-04-17
Witz, Jean C. (Department: 1651)
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
Culture, maintenance, or preservation techniques, per se
C424S537000
Reexamination Certificate
active
06218183
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a novel screening method for the identification and selection of plants possessing antimicrobial activity and tolerance to abiotic stresses.
BACKGROUND OF THE INVENTION
Many human pathogens have developed resistance against commonly used antibiotics. This has necessitated a search for new anti-microbial substances. Besides other organisms, plants are also considered as a good source of such compounds. So far, known screening methods for testing antimicrobial activities of plants have resulted into identification of very few potential plants. To increase the chances of discovering new antimicrobials from plants, there is a need to develop alternate methods. Plants identified by the method of the present invention have shown high antimicrobial activity and tolerance to various abiotic stresses like high salt concentration, paucity of water etc. These plants can be grown in normally uncultivable lands and may be utilized as the potential source of antibiotics against selected human pathogens.
Antimicrobials have a large market share globally. Extensive screening programs are being carried out throughout the world to identify the potential plant source with new antimicrobial compounds. More than 2800 Indian plant species have so far been screened at random for different biological activities (Dhar et al. 1968, Indian J. Exp. Biol. 6:232; ibid, 1973, Indian J. Exp. Biol. 11:43; ibid, 1974, Indian J. Exp. Biol., 12:512; Bhakuni et al. 1969, Indian J. Exp. Biol. 7:250; ibid, 1971, Indian J. Exp. Biol. 9:91, ibid, 1988, Indian J. Exp. Biol., 26:883; ibid, 1990, Indian J Exp. Biol., 28:619; Dhawan et al., 1977, Indian J. Exp. Biol., 15:208; ibid, 1980, Indian J. Exp. Biol., 18:594, Aswal et al., 1984, Indian J. Exp. Biol., 22:312). However, antimicrobial activity was observed only in 45 plant species (1.6%); among these, 21 species (0.75%) exhibited antibacterial and antifungal activites. Antimicrobial activites were also observed in seeds of 35 species of Angiospems and it was found that only the seeds of 5 species (14%) had this activity. This shows that, the methods of random selection of plants for screening purposes are time consuming and costly. Besides, these offer little chance of discovering a new plant as a potential source of antibiotics (Evans, 1989, Pharmacognosy, ELBS, Bailliere Tindale, London pp. 670).
Another method used commonly for identifying plants with antimicrobial activity is through interpretation of ancient literatures and ethno-botanical data. These are merely confirmation of the activities already mentioned in these literatures. This way of identifying active plants has been moderately successful (Brantner & Grain, 1994, J. Ethnopharmacology, 44:35; Grosverhor et al., 1995, J. Ethnopharmacology, 45:97; Ieven et al., 1979, Planta Med., 36: 311; Taylor et al., 1995, J. Ethnopharmacology, 46:153; Muanza et al., 1994, Int. J. Pharmacology, 32:337; Vlietinck et al., 1995, J. Ethnopharmacology, 46:31; Weber et al., 1992, Planta Med., 58:417).
The common method currently used for testing antimicrobial activity of plant materials is as follows.
Extraction: Powdered plant materials of known quantity are exhaustively extracted with known suitable quantity of methanol either at room temperature for 2-3 weeks or using a Soxhlet extractor for 24 h. The solvent is removed at a low temperature under reduced pressure to yield a thick syrup which is suspended in methanolic water mixture (1:9) and then successively extracted with hexane, chloroform and ethyl acetate. Afforded organic solutions are dried over anhydrous sodium sulfate, filtered and concentrated to give organic extracts. Samples from methanol, hexane, chloroform and ethyl acetate extracts, as well as lyophilized aqueous fractions are further used to test for the antibacterial and antifungal activities (Ieven et al., 1979, Planta Med., 36:311).
Antimicrobial tests: Testing of the antimicrobial activity is usually performed according to the general agar plate diffusion method (Bauer et al., 1966, Am J. Clin Path. 45:493). About 10 mg of plant extract is dissolved in methanol (0.5-1 ml). Sterile blank filter paper discs of 6 mm diameter are impregnated with the resulting solutions and then asceptically deposited on the surface of innoclated plates each containing a specific test microorganism. After 48 hr of incubation at 36° C. for bacteria and 72 hr of incubation at 25° C. for fungi, positive results are established by the presence of clear zones of inhibition around active extracts.
Quantitative estimation of antimicrobial activity: The degree of activity is recorded in four grades according to the internal diameter (in mm) of the zones of the inhibition, incorporating the diameter of the disc (6 m): +3 (strongly active, more than 15 mm of the internal diameter), +2 (moderately active, 14-10 mm of the internal diameter), +1 (less active, internal diameter less than 9 mm) and 0 (inactive). Discs of different concentrations of common antibiotics are used as a positive control (Muanza et al., 1994, Int. J. Pharmacog., 32:337).
DISADVANTAGES OF THE PRIOR ART
The method described above for screening plants for antimicrobial activity is lengthy, time consuming and costly on account of the use of different solvents for extraction of plant materials. On the other hand, the present screening procedure for identifying potent antimicrobial plant/plant parts is quick as extraction process/procedure is completely deleted, involves less chemicals and is almost certain with reliability. The plants identified so far by this method have exhibited 100% activity and many of them are new records. These findings may lead to the discovery of new chemical agents with microbial activity which are not yet discovered. These plants are also tolerant to abiotic stresses and can be grown/cultivated in any drought prone and/or saline soils.
OBJECTS THE INVENTION
The main object of the present invention is to develop a novel screening process for the identification and selection of plants possessing antimicrobial activity and tolerance to abiotic stresses.
Another object of the present invention is to provide an improved, reliable and accurate quantitative estimation method to determine the antimicrobial potential of seeds/plant parts using them directly in measured quantities, instead of their extracts.
SUMMARY OF THE INVENTION
The above-objects are achieved by providing a novel screening process for the identification and selection of plants possessing high antimicrobial activity and tolerance to abiotic stresses; which comprises identification of plant species that grow naturally and survive on the fresh/decomposing (10-15 days old) cattle dung, a medium rich in different microbes and high salt concentration; testing of their seeds against selected microbes directly in measured quantities, eliminating the extraction process completely; quantitative estimation of antimicrobial potency using the radius of inhibition zone as well as the ratio of inhibition zone and weight of the tested seed; calibration of antimicrobial potency of seeds with known antibiotics and assessment of their salt tolerant capability. The same applies to the other organs of the plant eg. root, stem, leaf, flower and fruit.
DESCRIPTION OF THE INVENTION
In India, decomposed cattle dung is commonly used as organic manure for garden and farm plants. However, it is found that several medicinal, aromatic and horticultural plants when grown in pots containing mixture of fresh cattle dung and soil, do not survive. It is further observed that upon deposition of cattle dung, small herbaceous plants decay rapidly and often die. The area outside the dung patch gets rapidly covered by grass, while the dung patch itself remains unpopulated or is sparsely populated by grasses for many months. This shows that fresh or decomposing cattle dung is a harsh and unsuitable medium for plant growth. A majority of the seeds are not able to produce seedlings on it. The dung excreted by cattle is a complex substance, rich in
Bagchi Gurudas
Darokar Mahendra Pandurang
Kumar Sushil
Council of Scientific & Industrial Research
Fish & Richardson P.C.
Witz Jean C.
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