Research Article ISSN 2250-0480 VOL 6/ ISSUE 3/JULY 2016

IN VITRO ANTIMICROBIAL AND ANTIDIABETIC ACTIVITY OF LEAF EXTRACTS OF SWIETENIOIDES AND HOMALIUM ZEYLANICUM

V.ANURADHA DEVI* AND K.MALLIKARJUNA1

1Department of Botany and Microbiology, Acharya Nagarjuna University *Department of Botany, Government junior college, Guntur.

ABSTRACT

This study was performed to evaluated the antimicrobial and antidiabetic activities of Indian medicinal Schrebera swietenioides and Homalium zeylanicum. The leaves of the plants were extracted with ethyl acetate, methanolic and aqueous solvents to give respective extracts. The ethyl acetate, methanolic and aqueous solvent extracts were evaluated for the medicinal activities. Antimicrobial activity of the extracts were evaluated by determining the minimum inhibitory concentration values. Different pathogenic organisms including four Bactria (B.subtilis, S. aureus, P.aeruginosa, E.coli) and one fungus (A.niger) were evaluated for antimicrobial activity. Antidiabetic activity was studied by inhibition of α- amylase enzyme using Spectrophotometer. The Methanolic extracts of both plants shows high inhibition activity on E.coli where as water extract inhibits the other pathogenic strains in the study. Both the plants in the study were found to having α- amylase inhibiting activity. H.zeylanicum extract concentration of 200µg/ml was found to be sufficient for 50% inhibition of α- amylase. In S swietenioides, methanolic extract at 250µg/ml and aqueous extract of 200µg/ml concentration of was found to be sufficient for 50% inhibition of α- amylase. This study indicates that the plant leaves contain a variety of compounds contributing to antimicrobial and antidiabetic activity, which could be used for food additives and the development of useful natural compounds.

KEYWORDS: Schrebera swietenioides, Homalium zeylanicum, antidiabetic activity, antimicrobial Activity

INTRODUCTION tall, with thick grey bark. Various studies and reviews proved that Schrebera swietenioides contains different medicinal properties [3-7]. The plant parts Schrebera swietenioides have been used for different treatments like leaves of S. swietenioides generally called as bullakaya, S.swietenioides used in treatment of enlargement of magalinga, tondamukkudi in telugu and Weaver's spleen and in urinary discharges, roots used in Beam tree in english, Banpalas in hindi is belongs to leprosy, bark in boils and bums and the fruits used in the family and found in Peru, Tropical and hydrocel treatment. Southern Africa, and Southeast Asia [1, 2]. It is a moderate sized deciduous tree, growing up to 20 m

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Research Article ISSN 2250-0480 VOL 6/ ISSUE 3/JULY 2016

Figure 1 Schrebera swietenioides plant

Homalium zeylanicum including rheumatism, antiinflammatory, H. zeylanicum Benth plant generally called as Ceylon hepatoprotective and antidiabetic agent in homalium in english, kaluvaluka, Manthalamukhi in Rayalaseema region of Andhra Pradesh. In Nigeria, it telugu belongs to Flacourtiaceae family and is is used as traditional medicine for the treatment of distributed in evergreen and semi-evergreen forests, malaria, ulcer, and inflammatory diseases and as an native to South India and Srilanka [9]. It is also found aphrodisiac. Various studies have been conducted to in , , , Nepal, and evaluate the medicinal properties of plant Vietnam [6]. The various parts of plant including, anthelimintic, anti-diabetic, antidyslipidemic and bark and leaf having many traditional uses, mainly in hepatoprotective activities are reported by various diabetes, rheumatism and wound healing. It has been extracts of H.zeylanicum [9-13]. traditionally used for treating several ailments

Figure 2 Homalium zeylanicum plant

The present study aimed to investigate the anti MATERIALS AND METHODS diabetic [15, 16] and antibiotic [17] properties of leaf extracts of Homalium zeylanicum and Schrebera Instrumentation swietenioides plants. The phytochemcial composition Teccomp UV-2301 double beam UV-Visible of both plants have been recently reported by us [14]. spectrophotometer was used to carry out spectral analysis and the data was recorded by Hitachi P - 2 Pharmaceutical Science Plant science

Research Article ISSN 2250-0480 VOL 6/ ISSUE 3/JULY 2016 software. Standard cuvettes of 10mm path length are leaves of both selected plants S. swietenioides and H. used for analysis. Ultrasonicator (1.3L) was used for zeylanicum by ethyl ecetate, methanol and water sonicating the standard and formulation sample. solvents using soxhlet extractor. Standard and sample drugs were weighed by using Denver electronic analytical balance (SI-234). Procedure The extracts showing positive results for Flavonoids Chemicals and reagents were used for the determination of α- amylase Alpha amylase enzyme was purchased from Sigma inhibition activity Aldrich chemicals, all the other chemicals used were purchased from Fisher Scientific, Mumbai. Nutrient In vitro α- amylase inhibition activity by agar medium for bacterial strains and czapek-dox Spectrophotometric method medium for fungi are used for studying the 1ml of alpha amylase and 1 ml of plant extract were antimicrobial property. Fluconazole antibiotic was taken in a test tube and incubated at 37◦C for 10 min. used as standard for antifungal activity and After pre-incubation, 1ml of 1% (v/v) starch solution amoxicillin drug was used as a standard for was added to each tube and incubated at 37◦C for antibacterial activity. 15min. The reaction was terminated with 2 m DNSA reagent, placing in boiling water bath for 5min, Cultures used for antimicrobial activity cooled down to room temperature, diluted, and the The microorganisms used were as follows, absorbance was measured at 546 nm. The control Pseudomonas aeruginosa NCIM 2036, reaction representing 100% enzyme activity did not Staphylococcus aureus ATCC BAA 1026, Bacillus contain any plant extract. To eliminate the subtilis ATCC 11774, Escherichia coli ATCC 10536 absorbance produced by plant extract, appropriate and Aspergillus niger- NCIM 616. extract controls were also used. % inhibition of alpha

amylase by each plant extract can be calculated using Preparation of plant extract Series of extraction methods were conducted with the the following formula.

Anti fungal and Antibacterial Activity by Agar well dishes. The respective clinical strain was spread diffusion method separately on the agar medium. The wells were Each petri dish containing nutrient agar medium was created using a stainless steel sterilized cork borer inoculated with one bacterial culture by spreading the under aseptic conditions. The ethyl acetate, suspension of the organism with a sterile glass rod methanolic and aqueous solvent extracts of two with a bended tip. In each plate cups of 6mm plants were tested at different concentration. diameter were made at equal distances using sterile Flucanazole was used as standard. The plates were cork borer. The extracts of two plants were tested. incubated at room temperature for 48 h and zones of All plates were kept in the refrigerator for 30 minutes inhibition were measured. Diameter of the zone of to allow the diffusion of sample to the surrounding inhibition was measured and the average diameter for agar medium. The petri dishes were incubated at each sample was calculated. The diameter obtained 370C for 24 hrs. For antifungal activity sterilised for the test samples were compared with standard Czapek-Dox medium was poured into sterile petri

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Research Article ISSN 2250-0480 VOL 6/ ISSUE 3/JULY 2016

Table 1 α- amylase inhibition of Schrebera swietenioides

S. Concentration of % of alpha Amylase % of alpha Amylase inhibition % of alpha Amylase No Sample inhibition of of Methanolic Extract inhibition of Aqueous Ethyl Acetate Extract extract 1 50µg/ml 7.234 13.302 16.919 2 100µg/ml 16.686 18.903 32.789 3 150µg/ml 19.953 40.723 42.940 4 200µg/ml 27.071 47.841 56.943 5 250µg/ml 28.705 53.792 63.594 6 300µg/ml 30.222 63.360 65.578 7 350µg/ml 34.306 66.744 73.629 8 400µg/ml 35.589 70.47841 75.146

IC50 value μg/ml 561.97 232.38 142.35

Table 2 α- amylase inhibition of Homalium zeylanicum

S. No Concentration of % of alpha Amylase % of alpha Amylase % of alpha Amylase Sample inhibition of Ethyl inhibition of inhibition of Acetate Methanolic Extract Aqueous Extract 1 50µg/ml 11.435 31.739 18.086 2 100µg/ml 22.637 45.274 25.087 3 150µg/ml 27.071 62.894 32.905 4 200µg/ml 29.755 73.2789 50.058 5 250µg/ml 31.855 74.912 53.909 6 300µg/ml 36.406 77.246 63.010 7 350µg/ml 37.456 78.063 66.744 8 400µg/ml 40.607 78.880 69.311

IC50 value μg/ml 492.53 119.26 199.77

Table 3 Antimicrobial activity of Schrebera swietenioides

Name of the Type of Size of zones (in mm) observed for different concentrations (in microorganism extract µg/ml) 1000 500 250 100 50 10 (µg/ml) (µg/ml) (µg/ml) (µg/ml) (µg/ml) (µg/ml) E.coli Methanol 25 19 10.2 9.5 8.6 - Ethyl acetate 21.3 19.5 10.1 9.6 6.5 - Aqueous 20 17.2 12.8 9.2 - - Staph. aureus Methanol 25.3 15.2 9.8 8.6 5.9 - Ethyl acetate 24.8 19.8 11.3 4.9 - - Aqueous 19.3 14.2 10.6 8.7 7.6 - P. aeruginosa Methanol 20 15 10.1 7.5 5.1 - Ethyl acetate 21.4 18.6 13.2 9.3 - - Aqueous 20.2 15.4 9.7 10.1 7.6 - B.subtilis Methanol 25.6 15.4 9.3 7.9 4.9 - Ethyl acetate 24 17 11.3 4.1 - - Aqueous 19.2 9.8 7.7 6.9 4.2 - A. niger Methanol 20.2 16.4 6.2 3.8 - - Ethyl acetate 20.7 14.2 5.9 4.5 - - Aqueous 10.3 - - - - -

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Research Article ISSN 2250-0480 VOL 6/ ISSUE 3/JULY 2016

Table 4 Antimicrobial activity of Homalium zeylanicum

Name of the Type of Size of zones (in mm) observed for different concentrations (in microorganism extract µg/ml) 1000 500 250 100 50 10 (µg/ml) (µg/ml) (µg/ml) (µg/ml) (µg/ml) (µg/ml) E.coli Methanol 25.4 23.2 17.6 7.3 4.7 - Ethyl acetate 21.9 19.8 15.7 5.8 4.2 3.9 Aqueous 22.8 18.4 11.6 6.8 - - Staph. aureus Methanol 22.6 20.3 18.1 6.2 - - Ethyl acetate 23.9 21.8 16.7 6.4 - - Aqueous 23.7 21.4 18.1 8.1 4.3 - P. aeruginosa Methanol 25.1 22.8 16.7 5.3 - - Ethyl acetate 26.1 24.9 18.4 7.9 4.8 - Aqueous 24.7 22.3 19.4 8.7 5.6 - B.subtilis Methanol 25.4 21.2 16.2 4.7 - - Ethyl acetate 23.9 21.7 19.7 10.2 6.7 - Aqueous 22.7 19.8 16.3 7.6 5.7 - A.niger Methanol 9.8 6.7 4.9 4.1 - - Ethyl acetate 10.1 7.8 6.5 4.6 3.9 - Aqueous 12.3 10.1 5.7 4.1 - -

(a) (b) (c) (d)

Figure A Antimicrobial activity study results (zone of inhibition) of Schrebera swietenioides extracts

(e) (f) (g) (h)

Figure B Antimicrobial activity study results (zone of inhibition) of Homalium zeylanicum extracts

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Research Article ISSN 2250-0480 VOL 6/ ISSUE 3/JULY 2016 a. Schrebera swietenioids methanolic extract (Escherichia coli) b. Schrebera swietenioids methanolic extract (Bacillus subtilis) c. Schrebera swietenioids ethylacetate extract (Bacillus subtilis) d. Schrebera swietenioids methanolic extract (Staphylococcus aureus) e. Homalium zeylanicum ethylacetate extract (Escherichia coli) f. Homalium zeylanicum aqueous extract (Escherichia coli) g. Homalium zeylanicum ethylacetate extract (Staphylococcus aureus) h. Homalium zeylanicum ethylacetate extract (Bacillus subtilis)

RESULTS AND DISCUSSION Antimicrobial activity Plants are an important source of potentially useful Antidiabetic activity structures for the development of new Many natural sources have been investigated with chemotherapeutic agents. The first step towards this respect to suppression of glucose production from the goal is the in vitro antimicrobial activity assay (Tona, carbohydrates in the gut or glucose absorption from 1998). These observations will help in identifying the the intestine. Alpha-amylase catalyses the hydrolysis active principle responsible for such activities and in of alpha-1,4-glycosidic linkages of starch, glycogen the developing drugs for the therapeutic use in and various oligosaccharides. Alpha-glucosidase Human beings. The antibacterial activity of the ethyl further breaks down the disaccharides to simple acetate, methanolic and aqueous solvents leaf sugars, readily available for intestinal absorption. The extracts of two plants was depends on their phyto inhibition of their activity in the digestive tract of chemical composition. The diameters of the humans is considered to be effective tool to control inhibition zones were measured in millimetre. The diabetes. In addition, these effects may leads to preliminary phytochemical components of the plants diminished absorption of monosaccharides. were studied various bioactive compounds were Therefore, effective and nontoxic inhibitors of alpha- reported (14). The demonstration of antimicrobial amylase and alpha-glucosidase have long been bacterial activity against both gram positive and gram sought. The results of antidiabetic activity using - negative bacteria and fungi may be indicative of the amylase inhibitory assay of the ethyle acetate, presence of broad spectrum antibiotic compounds. methanol and aqueous extracts of S. swietenioides Both leaf extract has shown excellent antibacterial and H. zeylanicum leaves are shown in Table 1 and 2. activity against gram negative and gram positive All the tested extracts exhibited the dose dependent organism. When compared to both plants, Schrebera inhibition of enzyme. The extracts was exhibited swietenioides showed the high antibacterial activity IC50 less than 100µg/mL will be considered active in on gram positive than gram negative and Homalium comparison with other tested extracts. MeOH leaf zeylanicum shown the high antibacterial activity on extract of S. swietenioides exhibited better gram negative than gram positive. Results of zone of antidiabetic activity with 75.14% enzyme inhibition inhibition were presented in tables 3 and 4. This will at 0.40 mg/ml concentration and IC50 value was be of immense advantage in fighting the menace of found to be 0.25 mg/ml for S. swietenioides. Similar antibiotic refractive pathogens that are so prevalent in results were found with the methanolic extract also. recent times. However, actual antibacterial This reflected that methanolic and aqueous extract of ingredients need to be extracted and identified also its S. swietenioides leaves contains certain compound(s) tolerable levels in the human body as well as any with alpha glucosidase inhibitory activity. On the toxic effects on human and animal tissues are other side H. zeylanicum leaf methanoilc extract investigated accordingly. exhibited the high antidiabetic activity by reducing CONCLUSION 78.88% enzyme inhibition at 0.40 mg/ml concentration and IC50 value was found to be 0.15 The study clearly shows that the extracts have either mg/ml. This reflected that methanolic extract of H. protected the cells from the toxic effect of alloxan or zeylanicum leaves contains certain compound(s) with the cells recovered after the initial injury. The alpha glucosidase inhibitory activity. preliminary phytochemical analysis shows the presence of many constituents which would have played a role in the pharmacological activities P - 6 Pharmaceutical Science Plant science

Research Article ISSN 2250-0480 VOL 6/ ISSUE 3/JULY 2016 studied. The present study provided results to justify extracts of Schrebera swietenioides and Homalium the traditional claim of herbs for anti diabetic zeylanicum, proved that both plants were having activity. The reported activities of the extracts make antimicrobial compounds. The current work will the leaves of plant more valuable in treating not only provide new reference data for the drug development diabetes but also the associated secondary disorders. and possesses the ability to inhibit pathogenic The plant can also be further explored for its activity bacteria. Further studies should be done on against wide spectrum of microbes and can be fractionation and identification of bioactive developed into a powerful antibiotic. The screening constituents which are responsible for antibacterial of antimicrobial activity performed with different activity.

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