INTRODUCTION:

Many efforts have been made to discover new antimicrobial compounds from various kinds of sources such as micro-organisms, animals, and plants. One of such resources is folk medicines. Systematic screening of them may result in the discovery of novel effective compounds (Tomoko et al. 2002)¹. The increasing prevalence of multidrug resistant strains of bacteria and the recent appearance of strains with reduced susceptibility to antibiotics raises the specter of untreatable bacterial infections and adds urgency to the search for new infection-fighting strategies (Sieradski et al. 1999)². Many microorganisms can cause several diseases and in spite of the tremendous advancement of medical science and technology, diseases are the leading health problem particularly in the under privileged population in the remote rural areas in the developing countries.

Nature has been a source of medicinal agents for thousands of years and an impressive number of modern drugs have been isolated from natural sources, many based on their use in traditional medicine. Medicinal plants are used locally in the treatment of infections caused by fungi, bacteria, viruses and parasites and over 60% of people in Nigeria rural areas depend on the traditional medicine for the treatment of their ailments (Ghani et al., 1989)³. Different plants have been used as a source of inspiration in the development of novel drugs (Robbers et al., 1996)⁴. Plant derived medicines are widely used because they are relatively safer than the synthetic alternatives, they are easily available and cheaper (Iwu et al., 1999)⁵. Many plant species have been evaluated for their antimicrobial activity in the past 20 years (Castello et al., 2000)⁶. Since then efficacy of many medicinal plants in the treatment of many diseases have been put to test in many laboratories (Shajahan and Ramesh, 2004)⁷.

Memecylon umbellatum Burm. (Family: Melastomataceae) is small evergreen shrub or tree having young terete branches and bears numerous umbellate cymes. The plant is known as “Anjani” in Sanskrit and “Ironwood tree” in English. Plants are distributed mostly in coastal regions of the Deccan peninsula, the eastern and southern part of India all along Western Ghats and in the Andaman islands.^8,
9. The leaves have been reported to possess astringent properties and are given to treat leucorrhoea and gonorrhoea. Lotion prepared from leaves is used to treat eye troubles. The decoction of the root is used in the treatment of excessive menstrual discharge.¹⁰ Leaves are also reported to possess antiviral activity.¹¹ Bark is used in the treatment of bruises externally as lepa along with coconut kernels (Dymock).The literature survey reveal that leaves and roots of Memecylon umbellatum have been investigated for its hypoglycemic activity using alloxan induced hyperglycemic wistar albino rats.^{12, 13} Wound healing activity of ethanolic extract of leaves has also been reported.¹⁴ Root of the plant has been reported for antipyretic, analgesic and anti-inflammatory, hepatoprotective, anthelmintic and anti-insect activities.^15-18 Plant contains wide variety of phytoconstituents such as umbellactone, amyrine, oleanolic acid, ursolic acid, sitosterol and organic acids. ^{19, 20}

Taking into account that the mostly leaves and root of the plant have been studied but no literature on medicinal property of inflorescence is reported. Hence the present study was planned to explore any possible potential of Memecylon umbellatum (MU) inflorescence extracts along with phytochemical investigation.

MATERIALS AND METHODS:

Materials:

The inflorescences of Memecylon umbellatum were collected in the month of March-April from Gaganbavada region (Kolhapur Dist.), Maharashtra, India. The plant material was taxonomically indentified by Dr. S. R. Yadav, Department of Botany, Shivaji University, Kolhapur, (M.S.). The voucher specimen (SGK-1) is deposited in department of Pharmacognosy, Bharati Vidyapeeth College of Pharmacy, Kolhapur. Microbial cultures were procured from NCIM Pune, Culture media from Hi-Media and all the reagents and chemicals used were of AR grade (Loba, S.D. Fine, and Merck)

Methods:

Collection drying and powdering of plant material:

Inflorescences were collected in the month of March-April and dried under shade for one week. Dried sample was powdered using electric blender (Bajaj) and fine powder was obtained by passing through # 80. Powder sample (1kg) was used for extraction.

Soxhlet extraction and Evaluation of Extracts^21-23

Air dried powder sample (1Kg) was used for extraction. Sample powder was packed gently in previously washed and dried cloth bag and solvent was placed from top with the help of funnel to moisten the drug sample. 3.5lit of solvent was placed in distillation flask and assembly was made air tight with sealing wax. Solvents were selected on the basis of extractive values and with their increasing order of polarity. Extraction was carried out at or slightly above the boiling point of each solvent using thermostatic heating mantle for 18h or on the basis of clarity of dropping solvent (saturation). The solvent was collected every time after completion of process and powder was dried in hot air oven for 24h at 45⁰C. The process was repeated for all the next solvents and finally the dried powder was macerated for 48h with 3.5 lit. of chloroform water IP (0.25% v/v) at room temperature in macerating bottle with frequent shaking. All the liquid extracts were subjected for physical analysis and are concentrated in rotary film evaporator and finally dried under reduce pressure. The residue was weighed and % yield was calculated (Table no.1). All the extracts were further dried over anhydrous calcium chloride and preserved in vacuum desiccators for further studies.

Table No. 1: Physical analysis of liquid inflorescences extracts and % yield.

Name of extract	Colour of extract	pH	Fluorescence			Sp.gr.	Density	Viscosity	yield of solids (g)	Nature of solid extract
Name of extract	Colour of extract	pH	D	S	L	Sp.gr.	Density	Viscosity	yield of solids (g)	Nature of solid extract
PEEI	G	5.6	-	G	R	0.6326	0.6457	0.6493	0.210	Waxy
ChEI	G	6.0	B	O	DB	1.0896	1.1245	0.6976	0.510	Lumpy
EAEI	YG	4.3	-	R	-	0.9016	0.9827	0.5804	0.418	Powder
AEI	B	5.4	-	RB	-	0.7815	0.8463	0.5649	0.321	Powder
MEI	YB	5.0	-	G	PG	0.8215	0.8769	0.9684	7.17	Waxy
AqEI	FY	5.2	-	G	G	1.0242	1.0565	1.0438	6.62	Powder

PEEI-Petroleum Ether extract Inflorescence, ChEI-Chloroform Extract Inflorescence, EAEI- Ethyl Acetate Extract Inflorescence, AEI-Acetone Extract Inflorescence, MEI-Methanol Extract Inflorescence, AqEI-Aqueous Extract Inflorescence, Y-Yellow, G-Green, O-Orange, YB-Yellowish Brown, YG-Yellowish Green, B-Brown, RB-Reddish Brown, R-Red, PG-Pale Green, FY- Faint Yellow.

Table No. 2: Phytochemical screening of inflorescence extracts

Extract

Sugars

Alk.

Tannins

Glycosides

steroids

proteins

Org. acids

AqEI

MEI

EAEI

ChEI

PEEI

AqEI- Aqueous Extract Inflorescence, MEI-Methanol Extract Inflorescence, EAEI- Ethyl Acetate Extract Inflorescence, ChEI- Chloroform Extract Inflorescence, PEEI- Petroleum Ether Extract Inflorescence, Alk-Alkaloids, Gly-Glycosides, Org. acids-Organic acids, R-Reducing sugars, NR-Non Reducing sugars, HT-Hydrolysable Tannins, CT-Condensed Tannins, a-anthracene glycosides, c-Cardiac glycosides, s-saponin glycosides, f-flavanoidal glycosides, co-coumarin glycosides, + Positive, - Negative.

Phytochemical screening: All the dried extracts were dissolved in small quantity of respective solvents and tested for phytoconsituents using standard reagents and tests²⁴. The results are given in Table no.2.

Screening of Antimicrobial Activity:

Bacterial and fungal strains: The test organisms were purchased from NCIM, NCL Pune. The organisms were sub-cultured in the media specified. The organisms, their ATCC code, Media in which they are sub-cultured are given in Table No.3. Bacteria were incubated at 37⁰C in incubator for 24 h and fungus at 27⁰C for 48 h. They were further stored at 4⁰C in the refrigerator to maintain stock culture.

Table No. 3: Microorganisms with their ATCC Codes and media used for subculture

Sr. No.	Name of microorganism	ATCC Code	Media
1.	Escherichia coli	8739	Nutrient Agar
2.	Bacillus subtilus	6633	Nutrient Agar
3.	Micrococcus luteus	9341	Nutrient Agar
4.	Pseudomonas aruginosa	27853	Nutrient Agar
5.	Staphylococcus aureus	25923	Nutrient Agar
6.	Aspergillus niger	16404	Saboraud dextrose agar
7.	Candida albicans	10231	Saboraud dextrose agar
8.	Penicillium notatum	28682	Saboraud dextrose agar

a) Antibacterial Activity ^25-28

Preparation of Media:

Nutrient Agar: Accurately weighed 28 gm of Nutrient agar (Hi-Media) was dissolved in the 1000 ml of distilled water by heating with frequent agitation. The media was finally sterilized in autoclave at 121⁰C for 15 min.

Preparation of Test and Standard Drug Solutions:

Preparation of Test Extracts: All the five test extracts were prepared freshly by dissolving 1 gm of previously dried extract in the 10 ml of respective solvent in which they were extracted. This gives the 100 mg ml^-1 of stock solution. From which 0.1 ml was used for test.

Preparation of Normal Saline Solution: Accurately weighed 0.9g sodium chloride was dissolved in 100 ml of distilled water. Normal saline was sterilized before preparation of microbial suspension at 121⁰C for 15 min. in autoclave.

Preparation of Standard Drug Solutions:

Doxycyclin: Weighed accurately 100 mg doxycyclin, and dissolved in 100 ml of 0.1M hydrochloric acid to get 1000μg/ml stock solution. This was then diluted further with distilled water to get solution of 10 µg/ml and 0.1ml was used in well.

Ciprofloxacin: Weighed accurately 100 mg ciprofloxacin, and dissolved in 100 ml 0.1 M hydrochloric acid to get 1000 μg/ml stock solution. This was then diluted further with distilled water to get solution of 10 μg/ml and 0.1 ml was used in well.

Fluconazole: Weighed accurately 100 mg fluconazole, and dissolved in 100 ml of dimethyl formamide (DMF) to give 1 mg/ml stock solution. This solution was further diluted with buffer (which was prepared by dissolving 2g di-potassium hydrogen phosphate and 8 gm potassium dihydrogen phosphate in distilled water to produce 100 ml) to get solution of 10 µg/ml.Working solution 0.1 ml of each was used in the well as positive control which will have test concentration of 1µg each of Doxycyclin, Ciprofloxacin and Fluconazole in the well.

Sterilization of Equipments and Media:

Dry Heat Sterilization: All the glass wares previously washed were sterilized in hot air oven. Petri-dishes, pipettes, test tubes were wrapped separately in the paper and kept in the hot air oven for sterilization at 180⁰C for 1h.

Moist Heat Sterilization: Normal saline solution and nutrient Medias were sterilized in autoclave at 121⁰C for 15 min.

Table No. 4: Antibacterial Activity of Memecylon umbellatum Inflorescences Extracts

Part of plant	Sample/ extract	Conc. used	Zone of inhibition in mm.*
Part of plant	Sample/ extract	Conc. used	*E.coli*	*P. aeruginosa*	*M. luteus*	*B. subtilus*	*S. aureus*
Control-1	2% acacia suspension	0.2ml	--	--	--	--	--
Control-2	DMSO	0.2ml	--	--	--	--	--
Standard-1	Doxycycline	01µg	22	18	23	25	23
Standard-2	Ciprofloxacin	01µg	25	21	27	25	26
Inflorescence	Pet. ether	3mg 6mg	-- --	-- --	09 10	12 14	10 11
	Chloroform	3mg 6mg	-- --	-- --	09 10	12 14	10 11
	Ethyl acetate	0.5mg 1.0mg	25 27	-- --	16 18	20 23	22 25
	Acetone	0.5mg 1.0mg	09 10	-- --	10 10	-- --	10 11
	Methanol	0.5mg 1.0mg	21 22	-- --	20 21	18 19	18 20
	Aqueous	3mg 6mg	15 16	-- --	-- --	11 12	16 17

* Average of triplicates

Table No. 5: Antifungal Activity of Memecylon umbellatum Inflorescences Extracts

Part of plant	Sample/ extract	Conc. used	Zone of inhibition in mm.*
Part of plant	Sample/ extract	Conc. used	*A. niger*	*C. albicans*	*P. notatum*
Control-1	2% acacia suspension	0.2ml	--	--	--
Control-2	DMSO	0.2ml	--	--	--
Standard	Fluconazole	5μg	16	17	17
Inflorescence	Pet. ether	3mg 6mg	11 12	-- --	11 12
	Chloroform	3mg 6mg	11 12	11 12	11 12
	Ethyl acetate	0.5mg 1.0mg	11 12	11 12	14 16
	Acetone	3mg 6mg	10 11	11 12	10 11
	Methanol	0.5mg 1.0mg	11 12	10 11	11 12
	Aqueous	3mg 6mg	-- --	12 13	12 13

* Average of triplicates

Table No. 5: MIC for Different Memecylon umbellatum Inflorescences Extracts

Test organisms	MIC in mg for different extracts
Test organisms	Pet. ether	Chloroform	Ethyl acetate	Acetone	Methanol	Aqueous
E.coli	15	15	0.5	0.5	0.5	3
P. aeruginosa	15	15	1.0	15	1.0	15
M. luteus	3	3	0.5	0.5	0.5	7
B. subtilus	3	3	0.5	7	0.5	3
S. aureus	3	3	0.5	0.5	0.5	3
A. niger	7	3	0.5	3	0.5	15
C. albicans	15	3	0.5	3	0.5	3
P. notatum	7	3	0.5	3	0.5	3

Preparation of Microbial Suspension: Microbial suspensions were prepared by transferring one loop full of stock culture to the 10 ml of normal saline solution. All the procedure was conducted in the laminar air flow in aseptic area.

a) Antibacterial Activity:

Cylinder-Plate or Cup-Plate Method: All the sterilized materials were kept in the aseptic area in the Ultra-Violet laminar air flow. Bacterial suspensions (3 ml) were then poured in the petriplates. As soon as nutrient agar attained 50⁰C temperature, 20 ml was poured in to the petriplates containing bacterial suspension and plates were rotated to mix the suspension with media. When the agar got solidified bores were made in the plate with sterile borer of 8 mm diameter. In each plate six bores were made. Out of which one is meant for addition of standard, two for negative control of blank solvents of standard and sample and remaining three bores for addition of same concentrations of sample. 0.1 ml of sample was added in each cylinder. The plates were kept to allow diffusion at room temperature for three hours and then incubated in the upright position in incubator at 37⁰C for about 21 h for bacterial growth. The diameter of zone of inhibition was accurately measured for bacterial growth in each treated plate as shown in Table No.4. The zone of inhibition of bacterial growth by the test solution was compared with the zone of inhibition by the standard at tested concentrations.

b) Antifungal Activity:

For fungus, SDA (Saboraud Dextrose Agar) was poured in the petriplates, allowed to solidify. The fungal suspensions were then sprayed uniformly over the surface of agar. All the procedure was same as that of for antibacterial activity. Fungi were incubated at room temperature for 48h. Zone of inhibitions of fungal growth were recorded as shown in Table No. 5.

Determination of MIC:

Promising extracts were tested for MIC using serial broth dilution technique²⁹. 10 ml of sterile nutrient broth without agar was taken in a test tube and concentrations of 0.5, 1, 3, 7, 15 mg of test samples were added aseptically. 1 ml of inoculum of each test organism in normal saline (10⁵CFU) was added in each sample. The tubes were incubated at 37°C for 24 h (or 48 h for the fungi) and then observed for the Minimum Inhibitory Concentration (MIC). The growth of organisms was observed as turbidity determined by a double beam spectrophotometer (Jasco V-530) at 620 nm. Control tubes without the tested extracts were assayed simultaneously. All samples were tested in triplicate and results are given in table no. 6.

RESULT AND DISCUSSION:

The yield was found maximum for methanol and minimum for light petroleum. Polar extracts showed presence of tannins glycosides sugars and organic acids while non polar extracts showed steroids and triterpens. Ethyl acetate and methanol extracts have shown better antimicrobial activity compare to other extracts. All the extracts failed to show activity against P. aeruginosa. Petroleum ether and chloroform extracts exhibited activity at higher concentration (3mg) compare to ethyl acetate and methanol (0.5mg). The activity shown by ethyl acetate was comparable with standards at tested concentrations. MIC for ethyl acetate and methanol was found to be 0.5mg for the entire organism except P. Aeruginosa (1mg) compare to 3-15mg for other extracts. Acetone and aqueous extracts also showed antimicrobial activity but at higher concentrations (3 -15mg). Different extracts showed presence of tannins, glycosides, triterpens and steroids as important constituents. These constituents may be responsible for antimicrobial activity as per the reported activities such as Camellia sinensis,²⁰ Rhizophora apiculata bark,²¹ blue and white flowering Silybum marianum,²² roots of Tecoma stans,²³ punicalagin from the peel of Punica granatum, tannic acid from galls and prodelphinidin oligomers from the bark of Elaeocarpus sylvestris var. ellipticus etc.²⁴. Further study is necessary to isolate the constituent responsible for activity from ethyl acetate and methanol extracts.

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Received on 15.10.2011 Accepted on 23.11.2011

Asian J. Pharm. Res. 1(4): Oct. - Dec. 2011; Page 114-118