Abstract
Antimicrobial activities of crude extracts and extracted phenols from sporophytic parts of epiphytic fern Arthromeris himalayensis (Hook.) Ching belonging to the family Polypodiaceae were studied in summer and winter seasons against Bacillus subtilis AR-2 (Gr +ve) and Escherichia coli XL1-Blue (Gr -ve). Both the crude extracts and extracted phenols from sporophytic plant parts showed antimicrobial activities. In both the seasons of summer and winter, maximum phenol contents were more or less same. Each and every sporophytic plant parts showed antimicrobial activities by crude extract as well as by extracted phenols. Detailed observations revealed that crude extract shows better antimicrobial activity than extracted phenol.
Introduction
Arthromeris himalayensis (Hook.) Ching belongs to the family Polypodiaceae and is a warm temperate fern, exclusively epiphytic in nature and is distributed in India throughout the Himalayan region from Eastern Himalayas to Western Himalayas. This epiphytic fern is also distributed in the mountains of China, Nepal and Burma. This fern is found in between 2700-3600 m altitude. Currently microbial resistance to antibiotics has been a global concern, as all known classes of natural compounds for antimicrobial therapy are becoming resistant3. Many fern species are important medicinal plants161314 however their antimicrobial properties is not known79. So far, there is no record of antimicrobial property of Arthromeris himalayensis. So, there is a urgent need of new compounds for antimicrobial therapy and as such this study was conducted to test potentiality of this plant as an antimicrobial agent. Present investigation reports the antimicrobial effects of extracted phenols and crude extracts of soprophytic plant body of Arthromeris himalayensis.
Materials and Methods
Crude Extract: Different parts of the sporophytic plant body (Leaves, rachis, root and rhizome) of Arthromeris himalayensis were collected from the different parts of Himalayas at an altitudes of 2700-3600 m. All the experiments were done by fresh plant materials. In one set, 100 mg of each of root, rhizome, rachis, sterile and fertile leaves were collected in summer and winter. Each sample of this 100 mg plant parts was crushed with mortar and pestle and the compounds were extracted in 80% boiled of ethanol. This ethanolic mixture was centrifuged at 4000 RPM for 10 min. Then the supernatant was taken out and its total volume was made to 5 ml with 80% boiled ethanol. To that 4 ml distilled water was added and was kept on a hot plate at 40oC to evaporate the alcohol. Thus the crude extract comes in the water solution with a concentration of 2.5% v/v. For each extraction of plant parts, 10 replicates were made for summer and winter.
Total Phenol: In second set of experiment, total phenols from each 100 mg fresh wt of different plant parts collected in summer and winter were extracted and established according to the method of Bray & Thorp4. The biochemical analysis of the crude extracts was done512. Plant materials were extracted in 80% boiled ethanol, the extract contains plants total protein, total phenols and total soluble and insoluble carbohydrates. As extracted phenols and crude extracts were made from 100 mg plant tissue, the phenol contents were same in both. Total protein content was determined by a method of Moore and Stein11. Total carbohydrate content was determined following the methods of Mc Cready et al.10. For all the biochemical analysis, 10 replicates were made for each season. The treatment consisted of three factors- 1) Sporophyte 2) Plant parts (Rhizome, rachis, sterile and fertile leaves 3) Bacteria (Bacillus subtilis AR-2 and Escherichia coli XL-1 Blue).
Antibacterial Activity: Antibacterial activity was measured using ‘Agar cup’ method15. In the agar cup assay, nutrient agar plates of 2 cm thickness were prepared. One set was inoculated with Bacillus subtilis AR-2 and the other with Escherichia coli XL-1 Blue. Cups of 9 mm diameter were made in the plates in a systematic manner with cork borer. The 0.1 ml water extract of the different parts of sporophytic plant body and their extracted phenols were applied in separate cups and incubated at 37oC. After 24 hrs, diameter of the hallow zones formed due to bacterial lyses were measured. Distilled water was used as a control. In each of the experiments 6 replicates were made.
Phytochemistry: Total protein was estimated following Moore and Stein211 method. Soluble and insoluble carbohydrates were estimated by the methods of Mc Cready et al.10.
Results and Discussion
It has been found that different parts of Arthromeris himalayensis accumulate different amounts of secondary metabolites in different climatic conditions. Fertile leaf accumulates highest amount of phenols (770-720 µg/mg fresh wt) followed by root (684-619.20 µg/mg fresh wt) in winter and summer season. Sterile leaf stood third position in respect to accumulation of phenol (666-616.17 µg/mg fresh wt.). In rhizome a large variation is found in accumulation of phenols, which is 640 µg/mg fresh wt of in winter and 519 µg/mg fresh wt in summer. The total concentration of all the biochemical compounds except phenols, were maximum in favourable condition i.e. in summer (as summer is pleasant in mountains). The total phenols content in summer is less than winter (Table: 1).
Table: 1- Total phenols, total protein, soluble and insoluble carbohydrate contents in plant parts of Arthromeris himalayensis in two conditions.
The trend of accumulation of biochemical compounds reverse to phenols was observed in cases of total proteins, soluble carbohydrates and insoluble carbohydrates. Increase in protein was very little in summer in respect to winter season. Here also the fertile leaf accumulates highest concentration of protein 137 µg/mg fresh wt in summer and 130 µg/mg fresh wt in winter, followed by sterile leaf (131.27-124.84 µg/mg fresh wt) > Root (62.52-58.58 µg/mg fresh wt) > Rachis (57.20-53.27 µg/mg fresh wt) > rhizome (53.12-50.52 µg/mg fresh wt). Soluble and insoluble carbohydrates also follow the same trend regarding seasonal changes. Rhizome which is the main accumulator of soluble carbohydrate contains 93.21-91.02 µg/mg fresh wt, followed by sterile leaf (87.20-83.12 µg/mg fresh wt) > root (83.13-80.21 µg/mg fresh wt) > fertile leaf (79.12-73.10 µg/mg fresh wt)> rachis (78.30-71.00 µg/mg fresh wt). Accumulation of amount of insoluble carbohydrates was greater than the soluble carbohydrates. Rhizome accumulates maximum amount of insoluble carbohydrates (99.31-97.13 µg/mg fresh wt) during summer and winter respectively among the plant parts studied, followed by sterile leaf (92.90-89.93 µg/mg fresh wt) > fertile leaf (83.30-82.13 µg/mg fresh wt) > rachis (76.40-73.30 µg/mg fresh wt) > root (75.00-72.21 µg/mg fresh wt) (Table: 1).
Regarding antimicrobial activity, both crude extracts and extracted phenols showed bacterial lyses against gram positive (gr +ve) and gram negative (gr –ve) bacteria. Here crude extracts showed higher inhibitory property than extracted phenols. It has been found that gr +ve bacteria were more prone to extracted phenol than gr –ve bacteria(Table: 2). Inhibition zone (hallow area) created by fertile leaf extract was larger than any other parts of the plant in respect to crude extracts as well as extracted phenols (Fig. 1).
Table: 2. Effect of crude extracts and extracted phenols of Arthromeris himalayensis on hallow zone size in the plates of Bacillus subtilis AR-2 and Escherichia coli XL-1 Blue.
Legend to the hallow zones in Fig. 1: 1. Root; 2. Rhizome; 3. Rachis; 4. Sterile leaf; 5. Fertile leaf.Fig. 1: Effect of the extracts from Arthromeris himalayensis (Hook.) Ching on bacterial culture plates: 1. Effect on extracted phenol on E.coli 2. Effect of crude extracts on Bacillus subtilis 3. Effect of crude extracts on E. coli 4. Effect on extracted phenol on Bacillus subtilis.
From the above results it was found that, the plant extracts of Arthromeris himalayensis showed significant inhibitory activity against bacterial strains (grm +ve and gr –ve). Crude extracts of the plant parts were more potential than the extracted phenols regarding antimicrobial property. It is probably due to the presence of some unknown compounds and phenols which cumulatively inhibit bacterial growth. Antimicrobial property of this plant might be attributed to the presence of high levels of phenolic compounds. This is supported by the presence of high phenol concentration (588-770µg/mg wt) in each parts of the plant.
Current microbial resistance to antibiotics has been a global concern, as it spans nearly all known classes of natural compounds. So, there is a urgent need of new compounds for antimicrobial therapy. It has been found that phenolic compounds in plants have strong antimicrobial properties against gr +ve and gr –ve bacteria. Pharmacological, pharmaceutical, phytopathological and food processing industries are some of the fields where phenolic compounds can be applied as bio- preservatives. Arthromeris himalayensis showed potential evidence for its ethno-pharmacological use and promising broad spectrum antimicrobial drug. Further work is needed to isolate those active principles responsible for antimicrobial properties.
Conclusion
From the above result and discussion we can conclude that Arthromeris himalayensis (Hook.) Ching has the potentiality to establish itself as a broad spectrum ethno-pharmacological antimicrobial drug. Current microbial resistance to antibiotics has been a global concern, as it spans nearly all known classes of natural compounds. So, this type of research work for searching new sources of antimicrobial agents and new antimicrobial compounds is required to enrich the medical science. Further work is on the progress to isolate the compounds responsible for antimicrobial properties.
Acknowledgements
The authors are thankfully acknowledging the financial assistance of UGC (MRP), and CAS Department of Botany, and Department of Microbiology, Burdwan University for providing necessary facilities.References
- Asolkar LV, Kakkar KKN and Chakkre OJ. 1992. Glossary of Indian Medicinal Plants with Active Principles. Part-I (A-K) second supplement, CSIR, New Delhi.
- Berenbaum MR and Zangri AR. 1996. Phytochemical diversity: Adaptation or random variation. Recent Advances in Phytochemistry. 30:1-24.
- Boller T. 1995. Chemoperception of microbial signals in plant cells. Annual Review of Plant Physiology and Molecular Biology. 46:189-214.
- Bray HG and Thorp WV. 1954. Analysis of phenolic compounds of interest in metabolism, Methods of Biochemical Analysis (Eds., D.Blick). New York, Interscience Publication.Vol-1:27-52.
- Britto AJD. Manickam VS and Gopalakrishnan S. 1994. Chemotaxonomical study on Thelypteroid ferns of the western Ghats of south India. Journal of Economic and Taxonomic Botany. 18:639-644.
- Cready MC, Gruggolz RM, Silveria J and Owens HS. 1950. Determination of starch and amylase in vegetables. Analytical Chemistry. 22:1156-1158.
- Guha (Ghosh) P, Mukhopadhyay R, Pal PK and Gupta K. 2004. Antimicrobial activity of crude extracts and extracted phenols from gametophyte and sporophytic plant parts of Adiantum capillus-veneris L. Allelopathy Journal. 13(1):57-66.
- Hain R, Reif HJ, Krause E, Langebartels R, Kindl H, Vorman B, Wiese W, Schmelzer E, Schreier PH, Stoecker RH and Stenzel K. 1993. Disease resistance nature from foreign phytoalexin expression in a novel plant. Nature. 361:153-156.
- Khandelwal S, Gupta MC and Kaushik JP. 1985. Antimicrobial activity of oil of Ophioglossum L. Indian Perfumes. 27:50-53.
- Moore S and Stein WW. 1948. Photometric ninhydrin method for use in the chromatography of amino acids. Journal of Biological Chemistry. 176:367-368.
- Patric RD, Manickam VS, Britto AJD, and Gopalakrishnan S, Ushioda TM, Tanimura A, Fuchino H and Tanaka N. 1995. Chemical and Chemotaxonomical Studies od Dicranopteroid species. Chemical and Pharmaceutical Bulletin.43:1800-1803.
- Puri GS and Arora RK. 1961. Some medicinal ferns from Western India, Indian Forester. 87:179-183.
- Sharma BD. 1989. Ferns and fern allies of Rajasthan: Experimental and phytochemical studies. Indian Fern Journal. 6:195-203.
- Tortura GJ, Funke BR and Case CL. 2001. The Control of Microbial Growth, Microbiology- An Introduction. 7th Edn. San Fransisco, USA: The Benjamin/Cummings Publishing Company, Inc. 390 Bridge, Parkway. 167-188.
- Vyas MS, Rathore D and Sharma BD. 1989. Phytochemistry of Rajasthan Pteridophytes: Study of Phenols in relation to stress. Indian Fern Journal. 6:244-246.