In vitro Antimicrobial Activity and Phytochemical ...

P. Saravana Kumar et al / Int. J. Pharm. Phytopharmacol. Res. 2014; 3 (4): 323-326

ISSN (Online) 2249-6084 (Print) 2250-1029

International Journal of Pharmaceutical and Phytopharmacological Research (eIJPPR) [Impact Factor – 0.7826] Journal Homepage: www.eijppr.com

Research Article In vitro Antimicrobial Activity and Phytochemical Analysis of Anisomeles malabarica (Linn.) (Lamiaceae) Leaf and Inflorescences 1

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P. Saravana Kumar , Naif Abdullah Al-Dhabi , V. Duraipandiyan , S. Ignachimuthu * 1 2

Division of Microbiology, Entomology Research Institute, Loyola College, Chennai – 600 034, India. Department of Botany and Microbiology, College of Science, King Saud University, P.O.Box.2455, Riyadh 11451, Saudi Arabia.

Article info Article History: Received 27 November 2013 Accepted 27 January 2014

Keywords: Anisomeles malabarica, Antibacterial, Antifungal, MIC, Phytochemical

Abstract The crude extracts of Anisomeles malabarica were tested for antimicrobial activity using the methods such as zone of inhibition and MIC against selected Gram positive, Gram negative bacteria and fungal pathogens. Hexane and ethyl acetate extracts of leaf and inflorescence showed maximum zones of inhibition against Gram positive and Gram negative bacteria. However, the extracts from both leaf and inflorescence did not show good antifungal activity. The minimum inhibitory concentration of hexane and ethyl acetate extracts of leaf ranged from 1000-125g/ml and that of inflorescence extract ranged from 1000-31.25g/ml against Gram positive and Gram negative bacteria. The present study showed that the hexane and ethyl acetate inflorescence extracts of Anisomeles malabarica exhibited maximum antibacterial activity when compared with leaf extracts.

1. INTRODUCTION Traditional and folklore medicines play important role in health services around the globe. About three quarter of the world’s population relies on plants and plant extracts for healthcare. India 1 has an extensive forest cover enriched with plant diversity . Over the past 20 years, there has been an increased interest in the investigation of natural materials as source of new antifungal and antibacterial agents. Different extracts and essential oils from traditional medicinal plants have been tested to identify the source of therapeutic effects. Natural products of higher plants may be new sources of antimicrobial agents with possibly novel mechanism 2 of action . A wide range of medicinal plant parts is used as raw drugs with varied medicinal properties. The different parts used include root, stem, flower, fruit, twigs exudates and modified plant organs. While some of these raw drugs are collected in smaller quantities by the local communities and folk healers for local use, many other raw drugs are collected in larger quantities and traded 3 in the market as the raw material for many herbal industries . Although hundreds of plant species have been tested for antimicrobial properties, vast majority has not been adequately 4 evaluated . Anisomeles malabarica (L.) (Malabar catmint) Cogn.Syn. Nepeta malabarica L., (Family: Lamiaceae) is a medicinal plant used as a folkloric m edicine to treat amentia, 5 anorexia, fevers, swellings and rheumatism . It is distributed throughout India. The herb is reported to possess anticancer, allergenic, anthelmintic, antiallergic, antianaphylactic, antibacterial, anticarcinomic, antiedemic, antihistaminic, antiinflammatory, antileukemic, antinociceptive, antiplasmodial, antiseptic and 6,7 antiperotic properties . Not many studies are there for antimicrobial activity of this plant. The aim of this study was to evaluate the antimicrobial activity of Anisomeles malabarica leaves and inflorescences.

2. MATERIALS AND METHODS 2.1. Collection of Plant Material Fresh leaves and inflorescences of Anisomeles malabarica (Linn.) (Free from diseases) were collected from Vengodu, Thiruvannamalai district, Tamil Nadu, India (Latitude: 12º54´2383´´, North; Longitude: 79º 69´9216´´, East Elevation ft/m 227.5/65.4). The plant was identified by Dr. V. Duraipandiyan, Division of Botany, Entomology Research Institute, Loyola College. A voucher specimen (No.ERI/ETHPH/TA235) was deposited at Entomology Research Institute, Loyola College, Chennai, India. 2.2. Preparation of Crude Extract Leaves and inflorescences of A.malabarica were shade dried, powdered and extracted successively with hexane, ethyl acetate, and methanol respectively. The extracts were then filtered through a blotting paper. The filtrates were evaporated using a rotary º evaporator at 45 C. The concentrated extracts were transferred to º 8 glass screw cap tubes and stored at 4 C for further use . 2.3. Test Microbes Gram negative bacteria such as Enterobacter aerogens MTCC 111, Salmonella typhimurium (SPT), Klebsiella pneumoniae MTCC 109, Shigella flexneri MTCC 1457, Proteus vulgaris MTCC 1771, Gram positive bacteria such as Micrococcus luteus MTCC 106, Bacillus subtilis MTCC 441, Methicillin resistant Staphylococcus aureus (MRSA) and fungi such as Candida albicans MTCC 227, Candida krusei CK, Candida parapsilosis CP and Malassesia pachydermatis MP were used as test pathogens. The reference bacterial cultures were obtained from the Institute of Microbial Technology (IMTECH), Chandigrah, 160 036, India and all the fungal cultures were obtained from the Department of Microbiology, Christian Medical College, Vellore, Tamil Nadu, India. 2.4. Preparation of Inoculums Bacterial inoculums were prepared by growing cells in Muller Hinton broth (MHB) Himedia for 24h at 37º C. The filamentous fungi were grown on sabouraud dextrose agar (SDA) slants at 28º C for 10 days and the spores were collected using sterile double

*Corresponding Author: Dr. S. Ignacimuthu, Director, Entomology Research Institute, Loyola College, Nungambakkam, Chennai – 600 034, Tamil Nadu, India. Email: [email protected] , Tel: 044 2817 8348, Fax: 044 2817 5566.

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distilled water and homogenized. Yeast was grown on Sabouraud dextrose broth (SDA) at 28º C for 48 h.

known in Indian traditional medicine. This plant is known to contain various active principles of therapeutic value and to possess biological activity against a number of diseases. Previously the methanolic extract of A. marabalica showed 19 mm inhibition against Pseudomonas aeruginosa, Salmonella typhimurium at 14 100mg/ml . In the minimal inhibitory concentration (MIC) study the hexane and ethyl acetate extracts of leaf showed moderate activity (Figure 5). Hexane and ethyl acetate extracts of Inflorescence showed significant activity against Shigella flexneri and Enterbacter aerogenes at 125g/ml, 3l.25g/ml respectively (Figure 6). Methanolic extracts of leaf and inflorescence did not show any activity against tested microbes. The antimicrobial activity against Gram positive bacteria was more pronounced than Gram negative 15,16 bacteria . Medicinal plants are rich in secondary metabolites which include alkaloids, glycosides, terpenoids, flavonoids, insecticides, steroids and other active metabolites. As shown in Table 1 leaf and inflorescence extracts of A.malabarica showed the presence of steroid; terpenoid was present in all the extracts of leaf and inflorescence. Flavonoid was present only in methanol extract of the leaf and inflorescence. Phenol/tannin, alkaloid and glycosides were present in ethyl acetate and methanol extracts of leaf and inflorescence. Saponin was present only in methanol extract of leaves and inflorescences. Quinonoids were totally absent in all the extracts. On the basis of different in RF values, TLC showed abundant occurrence of few of these compounds such as alkaloid, flavonoid, phenol and saponin (Table 2). Flavonoids, the major group of phenolic compounds, are reported for their antimicrobial and antiviral activity. They are important for prevention of diseases associated with oxidative damage of membrane, proteins and DNA. Flavanoids have been reported to exert multiple biological effects such as anti-inflammatory, anti-allergic, anti-viral 17 and anti-cancer activities . The ethyl acetate inflorescence extract showed high activity when compared to other extracts; this may be due to the presences of phenols which were in high amount. Steroids and terpenoids were present in high am ount in the hexane extract of inflorescence of A.malabarica; this may be responsible 18 for its antimicrobial activity . There are reports that plants rich in tannins have antibacterial potential due to their basic character that allows them to react with proteins to form stable water soluble compounds thereby killing the bacteria by directly damaging its cell 19 membrane . Tannins also decrease the bacterial proliferation by 20 blocking key enzymes at the microbial metabolism . They have been found to have anti-viral, anti-bacterial, fanti-parasitic, anti21, 22, 23 inflammatory, anti-ulcer and anti-oxidant properties . The ethyl acetate extract of inflorescence showed high amount of tannins. Alkaloids possess anti-inflammatory, anti-asthmatic, and anti24,25 anaphylactic properties . In the present study we also confirmed the presence of alkaloids in ethyl acetate extract of inflorescence of A.malabarica; this may have contributed for its antibacterial activity. Saponins possess antimicrobial, anti-inflammatory, anti-feedent, 26,27 and hemolytic properties . The presence of saponins in Anisomeles malabarica may have contributed for its antibacterial activity.

2.5. Antimicrobial Activity The antimicrobial susceptibility was tested by disc diffusion 9 method . 20 ml of agar medium was poured into the plates to obtain uniform depth and was allowed to solidify. The standard inoculum suspension was streaked over the surface of the media using sterile cotton swab to ensure the confluent growth of the organism and the plates were allowed to dry for 5 min. After drying, the discs with concentrations of 2.5, 1.25 and 0.625mg/disc were placed on the surface of the plate with sterile forceps and gently pressed to ensure contact with the inoculated agar surface. Ampicillin for bacteria and Ketocanozole for fungi (10μg/disc) were used as positive controls; 5 % DMSO was used as a blind control in these assays. Finally the inoculated plates were incubated for 24h at 37º C for bacteria and 48h at 30ºC for fungi. The inhibition zones and the diameter of the discs were measured in millimeters. All the experiments were done in triplicates and the results were presented as mean ± SEM. Statistical analysis of all the data obtained was evaluated using one-way ANOVA. 2.6. In Vitro Antimicrobial Assay The crude extracts of leaf and inflorescence of Anisomeles malabarica (8mg) were dissolved in 0.2 ml of dimethylsulfoxide (DMSO and used for antimicrobial study using standard broth 10 microdilution method . The MIC was calculated. Mueller Hinton broth (Himedia, Mumbai) was prepared and sterilized by 0 autoclaving at121 C, 15lbs for 15minutes. The required concentration of the extract (1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ ml, 0.0625 mg/ml, and 0.03125 mg/ml) was added to 96 well micro titer plate containing 0.1 ml broth. The 3l of log phase culture was introduced into respective wells and the final inoculum 5 o size was 1x10 cfu/ml. The plates were incubated at 37 C for 18 h. Negative and solvent control (DMSO) was also included. Streptomycin for bacteria was included in the assay as positive control. 5l of the test broth was introduced on plain Mueller Hinton agar plates to observe the viability of the organism. MIC was determined as the lowest concentration which inhibited complete growth. 2.7. Primary and Secondary Qualitative Phytochemical Analysis Primary qualitative phytochemical analysis of leaves and inflorescences was done according to standard procedure. Analysis of some phytochemicals such as steroid, terpenoid, phenol, flavonoid, quinonoid, alkaloid, glycoside, and saponin were 11 also done . Secondary qualitative chemical analysis of phenol, 12,13 flavonoid, alkaloid and saponin were also done . 3. RESULTS AND DISCUSSION Plant derived medicines have made significant contribution towards human health. The increasing prevalence of resistant microorganisms and the emergence of multi-drug resistant strains have led to the search of new compounds. The present study was conducted to analyze the antimicrobial activity of A.marabalica. The results of the antibacterial and antifungal screening of six extracts from leaves and inflorescence are given in figures 1-4. The inhibitory effects of the extracts were directly proportional to the increasing concentration. The mean zone of inhibition for the extract against Gram positive and Gram negative bacteria ranged between 10 and 19 mm. The ethyl acetate extract of inflorescence showed highest mean zone of inhibition (14 mm) against Gram positive bacterium Shigella flexneri at 0.625mg/disc. The hexane extract of A.marabalica (inflorescence) showed significant activity against Shigella flexneri and Enterbacter aerogens with 14mm and 13mm at 0.625mg/disc respectively. A moderate level of activity was shown by ethyl acetate leaf extract with 13, 11, 10mm against Shigella flexneri, Klebsiella pneumonia, MRSA and fungus Malassesia pachydermatis at 0.625mg/disc, respectively. Methanol extract showed least activity with 10mm against MRSA and Klebsiella pneumonia and fungus Malassesia pachydermatis at 1.25mg/disc. However, extracts from both leaf and inflorescence did not show much antifungal activity against the fungal isolates tested in the present study. The presence of antimicrobial substances in higher plants is well established. The medicinal properties and pharmacological actions of A.malabarica are well

Hexane 2.5mg/disc Hexane 1.25mg/disc Hexane 0.625mg/disc

Methanol 2.5mg/disc Methanol 1.25mg/disc Methanol 0.625mg/disc

Ethyl acetate 2.5mg/disc Ethyl acetate 1.25mg/disc Ethyl acetate 0.625mg/disc

40

Ampicillin 10ug/disc

Zone of inhibition

30

20

10

us ur e S. a

bt ili s B

.s u

te us M .lu

ur iu m

ris

hi m S. ty p

er i ex n

P. vu lg a

e S. fl

on ia um

.p ne K

E. ae r

og en s

0

Fig.1: Effects of Anisomeles marabalica leaf extracts on bacteria using disc diffusion assay

324


Zone of inhibition

40 Hexane 2.5mg/disc Hexane 1.25mg/disc Hexane 0.625mg/disc

30 20


10

Methanol 2.5mg/disc Methanol 1.25mg/disc Methanol 0.625mg/disc Ampicillin 10ug/disc

er m

at is

si s ac hy d

M .p

C. p

ar ap si lo

C. kr us ei

C

.a

lb i

ca ns

0

Fig. 2: Effects of Anisomeles marabalica leaf extracts on fungi using disc diffusion assay Hexane 2.5mg/disc Hexane 1.25mg/disc Hexane 0.625mg/disc

40


Figure 6: Minimum inhibitory concentration of Anisomeles marabalica inflorescences extract


Table 1: Qualitative analysis of secondary metabolites


Class of compound Zone of inhibition

++ ++

Leaf Ethyl acetate ++ ++

-

++

++

-

+++

++

-

++

+++ +

-

+++

+++ ++

-

+

++

-

+++

+++

-

-

++

-

-

+++

Hexane

30

Steroid Terpenoid Phenol/ Tannin Flavonoid Quinonoid Alkaloid Glycosides/ Sugars Saponin

20

10

ur eu s

s ub til i

te us

im ur i

+ +

S. a

B

ph

P

S. fl

.p K

.s

Table 2: Qualitative analysis of secondary metabolites by thin layer chromatography

M

Fig.3: Effects of Anisomeles marabalica inflorescences extracts on bacteria using disc diffusion assay

.v u

+++; Strongly positive, ++; moderately positive, +; weakly positive, ; Negative

E. a

S. ty

.lu

um

s lg ar i

ex ne ri

ne um on ia

e

er og en s

0

Inflorescence Ethyl Methanol acetate +++ ++ + +++ ++ +

Methanol Hexane

Class of compound

Zone of inhibition

40 Hexane 2.5mg/disc Hexane 1.25mg/disc Hexane 0.625mg/disc

30 20


10


Alkaloids

Flavonoids


er m

at is

si s ac hy d

Phenolics

M .p

ar ap si lo

C. kr us ei

C. p

C

.a

lb i

ca ns

0

Fig.4: Effects of Anisomeles marabalica inflorescences extracts on fungi using disc diffusion assay

Saponins

Solvent system Methanol: Conc. NH4OH (200:3)

Leaf extract Hexane Ethyl acetate Methanol Hexane Ethyl acetate

Chloroform: Methanol Methanol (19:1) Hexane Chloroform: Ethyl acetate Methanol (27:0.3) Methanol Chloroform: Hexane Glacial Ethyl acetic acid acetate :Methanol: Water Methanol (64:34:12:8)

Total RF bands value 0

-

0

-

1 0

0.14

1

0.59

1

0.59

0

0

1

0.66

1 1

0.22 0.88

1

0.94

2

0.55, 1.0

Infloresc Total RF ences bands value extract Hexane 0 Ethyl 0 acetate Methanol 1 0.90 Hexane 1 0.94 Ethyl 0.25, 2 acetate 0.94 0.21, Methanol 3 0.53, 0.96 Hexane 0 0 Ethyl 1 0.70 acetate Methanol 1 073 Hexane 1 0.90 Ethyl 1 0.92 acetate Methanol

2

0.55, 0.92

Spray reagent

Dragendroffs reagent

No Reagent UV light

Ferric chloride Reagent

Iodine Vapour

4. CONCLUSION Plants have been reported to have multiple biological effects including antimicrobial activity. The present study confirmed that the extracts of inflorescence of A.malabarica contained high amounts of antibacterial compounds. Promising antibacterial activity was shown by hexane and ethyl acetate inflorescence extracts; moderate antibacterial activity was shown by leaf hexane and ethyl acetate extracts. However poor activity was shown by methanolic extract. Further investigation on the isolation and identification of antimicrobial component(s) in the active extracts may lead to chemical entities for clinical use. 5. ACKNOWLEDGEMENT The authors are grateful to Entomology Research Institute, Loyola College, Chennai. The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through the research group project no. RGPVPP-213'

Figure 5: Minimum inhibitory concentration of Anisomeles marabalica leaf extract

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6. CONFLICT OF INTEREST We declare that we have no conflict of interest. 15. REFERENCES 1.

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