Evaluation of Antimicrobial Activity of Casuarina equisetifolia Frost (Casuarinaceae)

 

Anil Kumar Aher1, Subodh Pal1, Sadahev Yadav2, Umesh Patil2 and Snehendu Bhattacharya3

1Natural Product Lab.,Department of Pharmacognosy, N.D.M.V.P.S. College of Pharmacy, Gangapur road, Nashik-422002

2V.N.S. Institute of Pharmacy, Bhopal-462044

3Dr. Bhanuben Nanavati College of Pharmacy, Mithibai College Campus, V.M. Road ,Vile Parle (West),Mumbai- 400 056.

 

ABSTRACT

The crude methanolic extracts of bark, wood, leaf and fruits of Casuarina equisetifolia and chromatographically isolated compounds were studied for antibacterial and antifungal activity by Cup and Plate method. Various parts of plant were collected from near by Nashik region identified as Casuarina equisetifolia Linn Family Casuarinaceae by P. S. N. Rao, Joint Director, Botanical Survey of India, Pune (M.S.).The plant materials were dried in oven at 40 0C. The structures of isolated compounds were confirmed by spectroscopic techniques. The compounds ANA 01, ANA 02 and ANA 04 were confirmed as Catechin, Ellagic acid and Gallic acid from bark; Quercetin (ANA 03) and (ANA 05) Lupeol were characterized from leaf and fruit respectively. The screenings of antibacterial and antifungal activities of isolated compounds were compared with Ampicillin (10units/disc) and Ketokonazole (10units/disc).

 

The isolated compounds ANA01-05 have shown activity against Gram negative bacteria and less activity against Gram positive bacteria. Among these, ANA04 (Gallic acid) and ANA05 (Lupeol) have shown good activity against Gram-negative (E. coli and Pseudomonas aeuroginosa) bacteria.

 

Methanolic extracts of wood, bark and fruit has shown good activity (10.0, 12.0 and 10.0 mm respectively) against Gram positive microorganisms (Staph. aureous) while the extracts were without any effect against Gram negative microorganism. Fruit extract has resulted in good antifungal activity against Candida albicans. Lupeol was isolated from fruit which has shown similar (8.0 mm) antifungal activity.

 

Key words: - Casuarina equisetifolia, Antibacterial, Antifungal, Cup and Plate method.

 

INTRODUCTION

Casuarina equisetifolia (Casuarinaceae) is handsome tree with drooping branches, 10-50 m high1. It is found in dry hill sides of open forests in India, Sri lanka and Australia2. The following phytoconstituents have been isolated from the plant so far; kaempferol, quercetin3, alicyclic acids   (Shikimic and Quinic acid),amino acids4,taraxwrol, lupenone, lupeol, gallic acid,b-sitosterol5, catechin and gallocatechin6,7. The plant is used as astringent1, in diarrhoea8, cough, ulcers, toothache, lotion for swelling9 and diabetes10.  

 

The biological activities, viz. anticancer, antibacterial9, hypoglycemic, antifungal2 of the leaf has been reported. Antibacterial activity was shown by isolated compounds 24-ethyl cholest-5, 22-dien-3β-ol, 24-ethyl cholest-5-en-3β-ol, 24-methyl cholest-5-en-3β-ol and cholesterol from leaves against Staph. aureus11.

 

Literature review suggests that the antimicrobial activity of the plant has not been comparatively studied and hence in the present study the same was investigated for isolated compounds.

 

 

 

MATERIALS AND METHODS: -

    Preparation of Plant extracts: -

Coarsely powdered materials of leaf, bark, wood and fruit (100 g each) were subjected to reflux with 250 ml of methanol for 4 hrs coded as MEL, MEB, MEW and MEF followed by subsequent filtration and evaporation to yield extract. The extracts were autoclaved at 121oC and 15 lbs pressure and stored at 4oC. The extracts were dissolved in DMSO (5%w/v) for screening of activity.

 

Separation and isolation of Phytoconstituents by chromatographic methods: -

Gradient fractionation of acetone soluble part from methanolic bark extract by column chromatography was performed by the using Toluene: Ethyl acetate: Methanol (4:3:3) followed by Methanol resulting in the isolation of compounds designated as ANA 01(Catechin), ANA 04 (Gallic acid) and ANA 02(Ellagic acid).

 

The suitable solvent systems were developed for the identification of active phytoconstituents by Thin layer chromatography. Lupeol was identified from Petroleum-ether extract of bark and fruits of the Casuarina equisetifolia by Benzene: Ethyl acetate (9:1) followed by spraying with Vanillin-sulphuric acid reagent. The Petroleum-ether extract of fruit was processed by PTLC to isolate compound designated as ANA 05 (Lupeol). Methanolic extract of leaf was chromatographed with Quercetin by Toluene: Ethyl acetate: Formic acid (5:4:1), followed by spraying with Ferric chloride. The presence of quercetin was evidenced in U.V. light. The methanolic extract of leaf was processed by PTLC to isolate compound designated as ANA 03 (Quercetin).

 

Characterization of Isolated Compounds: -

The isolated compounds were characterized with help of the instrumental techniques UV spectroscopy, IR spectroscopy, Mass spectrometry, NMR spectroscopy.

 

Characterization of Compound ANA01

UV (MeOH) max: 219,279 nm; 

FT-IR: (KBr): 3820.10, 3391.85 (O-H), 2933.20, 2892.27, 1627.56(C=O), 1522.37, 1471.52, 1289.96(C-O-C), 1245.99, 1198.02(C-O) and 868.70 cm-1.

LC-ESI-MS: m/e: 290(5%, M+ ,C15H 16O+6 ),  289(12%, M+-1),245(32%, M+-CH3O2), 205(100%, M+-C2H6O4) ,203 (40%) and 179(5%).

13C-NMR (DMSO, 300 MHz): ): d 156.59(C-7), 156.33(C-5), 155.51(C-3’), 144.99(C-4’), 130.75(C-8), 118.64(C-6), 115.26(C-6’), 114.64(C-9),99.23(C-10),95.27(C-5’),94.02(C-2’),81.12(C-3),66.45(C-2) and 27.98(C-4).

1HNMR (DMSO, 300 MHz): d 9.01(s), 6.72(1H,H-6,8,s), 6.68(1H,H-2’,5’,s), 6.61(1H,H-6’,s), 5.88(s), 5.70(1H,H-5,7,s), 4.92(1H,H-3’,4’,s), 4.48(1H,H-3,s), 3.56(1H,H-3,s),2.39(s),2.37(s) and 2.64(2H,H-4,d).

 

Characterization of compound ANA02

UV (MeOH) max: 365.0, 254.0 nm; 

FT-IR: (KBr): 3072.72, 3596.20(O-H), 1699.60(C=O),1622.21(C=C),1581.82,1509.10,1447.52,1398.43(C-O),1195.11 and ,882.06 cm-1.

LC-ESI-MS: m/e: 301.8(10%, M+- C14H 6O+8)  300.7(10%),283.8(40%), 271.9(4%) ,256.8 (50%, M+-H2O+O2), 228.9(100%, M+-CH3O4), 212.9(8%),200.9(30%) and 184.9(60%).

13C-NMR (C6D5N, 300MHz): ): d 164.23(C-7,7’),142.09(C-2,3,2’,3’), 124.48(s), 123.11(C-4,5,6,4’,5’,6’) and 111.92(C-1,1’).

1HNMR (C6D5N, 300 MHz): d 8.14(1H,H-1’,s) and 5.28(1H,H-2,3,2’,3’,s).

 

Characterization of compound ANA03

UV (MeOH) max: 255, 372nm; 

FT-IR: (KBr): 3405.97(O-H), 3100.53, 1667.12(C=O), 1627.56(C=C), 1522.37, 1471.52, 1319.97(C-O), 1262.97(C-O-C), 1198.94

and 868.70 cm-1.

LC-ESI-MS: m/e: 301.7 (0.5%, M+,C15H 10O+6 ),  272.9 (6%),256.7 (15%), 229.2(8%) ,192.8 (10%),178.8(100%, M+-C6H6O2) and 150.9(65%, M+-C7H6O3).

13C-NMR (d6- acetone, 300 MHz): ): d 177.70 (C-4), 165.95 (C-7), 162.87 (C-5), 158.58 (C-3), 149.13 (C-3’), 148.35 (C-4’), 146.59 (C-2), 137.61 (C-1’), 124.51 (C-9), 122.04 (C-10), 116.98 (C-8), 116.36 (C-6), 104.88 (C-6’), 99.60 (C-2’) and 94.78 (C-5’).

1HNMR (d6- acetone, 300 MHz): d 7.74(1H,H-6,s), 7.73(1H,H-8,s), 7.65(1H,H-2’,s), 7.62(1H,H-5’,s), 6.89(1H,H-6’,s), 6.86(1H,H-3,), 6.38(1H,H-7,s), 6.18(H-5,s) and 4.88(1H,H-3’,4’,s).

 

Characterization of compound ANA04

UV (MeOH) lmax: 272.8 nm; 

FT-IR: (KBr): 3285.28(O-H), 3072.72, 1703.34(C=O), 1615.63(C=C), 1501.90, 1446.96, 1339.41, 1245.99 and 867.96 cm-1.

LC-ESI-MS: m/e: 169.9 (M+, C6H 6O+5,) 168.9 (100%, M+-1) and 125 (40%, M+-COOH).

13C-NMR (d6- acetone, 300 MHz):: d 170.936 (C-7), 146.755 (C-3,C-5), 140.023 (C-4), 122.792 (C-1)and 110.79 (C-2,C-6).

1HNMR (d6- acetone, 300 MHz): d 9.136 (1H,H-7,s), 7.08 (1H,H-2,H-6,s) and 5.011 (1H,H-3,H-4,H-5,s).

 

Characterization of Compound ANA05

UV (MeOH) max: 211.4nm; 

FT-IR: (KBr): 3325.02, 3083.22, 2937.88, 1642.30(C=C), 1451.71, 1378.36(O-H, bend) and 1106.83 cm-1;

LC-ESI-MS: m/e: 426.7 (0.5%, M+, C30H 50O+),  410.4 (12%, M+-H2O),366.0 (5%), 353.2 (6%) ,339.4 (20%), 325.5(2%), 311.4(4%),299.4(27%),285.3(52%),271.3(80%, M+-C9H19),257.3(100%,M+-C10H21), 243.2(80%, M+-C11H23),229.4(75%),215.3(60%),201.3(40%),189.3(25%),175.4(52%),159.3(25%)  and 137.2(7%);

13C-NMR (d6- acetone, 300 MHz): d 151.48 (C-20), 124.55 , 109.97 (C-25), 78.49 (C-3), 56.233(C-5),51.271(C-9),48.989(C-18),48.778(C-19),43.68(C-17),43.518(C-14),41.592(C-8),40.572(C-22),39.504(C-4),38.938(C-1),37.886(C-13),36.219(C-10),35.069 (C-16),28.531(C-21),28.272(C-23),28.175(C-15),25.957(C-12),21.604(C-11),19.451(C-30),19.046(C-6),18.270(C-2),16.586(C-24),16.392(C-28),16.068(C-25),14.903(C-27).

1HNMR (d6- acetone, 300 MHz): d 4.70(1H,H-29,d), 4.56(1H,H-27,dd), 3.32(1H,H-3,s), 3.13(1H,-3,s), 2.83(2H,H-1,2,6,7,11,12,15,16,m), 2.44(H-20,H-21,sex), 1.69(3H,H-22,23,24,25,26,28), 1.07(s), 0.99(s),0.86(multiplet) and 0.76(s).

 

Preparation of isolated compounds: -

The isolated compounds ANA01-05 (0.001 % w/v solutions in DMSO) obtained from column chromatography were used for antimicrobial screening of activity.

Table 1: - Antimicrobial activity of Casuarina equisetifolia

 

 

 

Sr.No.

 

 

Test compound

Zone of inhibition ( in mm) [Mean±S.D.]

Microorganism

E. coli

NCIM 2109

Staph. aureus NCIM 2079

Pseudomonas aeuroginosa NCIM 2036

Candida albicans

MTCC 227

1         

MEL

7.0

--

8.0

--

2         

MEB

--

10.0

--

--

3         

MEW

7.0

12.0

--

--

4         

MEF

--

10.0

--

8

5         

ANA01

7.0

8.0

7.0

--

6         

ANA02

11.0

--

7.0

--

7         

ANA03

10.0

--

8.0

--

8         

ANA04

11.0

--

14.0

--

9         

ANA05

7.0

8.0

12.0

8

10      

Gentamycin

24

24

24

--

11      

Kitokonazole

--

--

--

23

12      

 

 

 

 

 

 

 

Antimicrobial activity of Methanolic extracts of Casuarina equisetifolia with Pseudomonas  aeruginosa

Photo 01: -Antimicrobial activity of Methanolic extracts of Casuarina equisetifolia with E.coli and Pseudomonas aeruginosa

 

Test organisms: -

Pathogenic bacterial strains, Staphylococcus aureus NCIM 2079, (Gram positive), Escherichia coli NCIM 2109, Pseudomonas aeruginosa NCIM 2036 (Gram Negative) and fungi like Candida albicans MTCC 227 were used for screening of antimicrobial activity. The microbial strains were obtained from National Chemical Laboratory, Pune, India. The stock cultures were maintained on nutrient agar medium at 4oC. The microorganism was activated by inoculating a loopful of the strain in the nutrient broth (25 ml).



 

Photo 02: - Antimicrobial activity of Methanolic extracts of Casuarina equisetifolia with Staph. aureous and Candida albicans

 

Antimicrobial activity: -

Antimicrobial activity of the crude extracts and isolated compounds were    investigated against  pathogenic bacterial  and fungal strains by the Cup- plate method12. Muller Hinton agar and Sabouraud Dextrose agar media were used for antibacterial and antifungal activities respectively.  Disc of Gentamycin and Ketoconazole  (Hi-media Lab., Mumbai) were served as reference standards. In each of well (6 mm in diameter), 0.1 ml of the tests were transferred to cups aseptically. The plates containing bacterial strains and fungal strains were incubated at 37 ±0.5oC and 28±0.5oC for 48 hrs respectively. The zone of inhibition (mm) was calculated by measuring the diameter of the zone of bacterial and fungal growth around the cup (Photo 1 and 2). The average of three independent determinations was recorded (Table 1).

 

RESULTS AND DISCUSSIONS: -

Spectroscopic data showed the chemical nature of compounds which were confirmed by comparative TLC with respective standards. Methanolic extract of bark resulted in separation of ANA01 (Catechin), ANA02 (Ellagic acid) and ANA04 (Gallic acid). Methanolic extract of leaf and petroleum extract of fruit showed the presence of ANA03 (Quercetin) and ANA05 (Lupeol) respectively.

 

On antibacterial activity screening, the isolated compound ANA 04 and 05 showed activity against Gram negative bacteria, E. coli and Pseudomonas aeuroginosa. ANA 04 identified as Gallic acid showed comparable activity with standard Gentamycin (14 mm).

 Methanolic extracts of wood, bark and fruit has shown good activity against Gram positive microorganisms (Staph. aureus) while the extracts were without any effect against Gram negative microorganism. The Gram positive bacteria were more susceptible than Gram negative bacteria which may be the result of differences in cell wall structure  between Gram positive and Gram negative bacteria where Gram negative bacteria has outer membrane acting as barrier to many environmental substances including antibiotics13.

 

Fruit extract and its isolate ANA 05 (Lupeol)  have shown similar antifungal activity (8 mm)against Candida albicans.

 

The results of the present study support the folkloric usage by Cook Islanders in the form of an infusion of the grated bark to treat mouth and urinary tract infections2  The bark extract possess Gallic acid with antibacterial properties that can be used as antimicrobial agents in new drugs for the therapy of infectious diseases caused by pathogens.

 

ACKNOWLEDGEMENT

Authors are thankful to the Dr. S.B.Wagh, Principal, NDMVPS College of Pharmacy, Nashik for providing necessary facilities.  

                                                                                                                  

REFERENCES: -

1.       Mhaskar KS, Blatter E and Caius JF(Ed.). Kirtikar and Basu’s Illustrated Indian Medicinal Plants, Sri Satguru  Publications, Delhi (India). 2000: 3248-50.

2.       Han ST. Medicinal Plants in South Pacific, Western Pacific Series No.19, WHO Regional publications, Geneva.  1998:41.

3.       Ansary EL et al. Flavonol glycosides of Casuarina equisetifolia. Biosci. 1977:32C:444 -445.

4.       Madhusudanamma W et al. Isolation and Characterization of Alicyclic acids, Polyols &Amino acids from Casuarina equisetifolia. Leather Sci. (Madras). 1978: 25(8): 369-371.

5.       Rastogi RP and Mehrotra BN. Compendium of Indian  Medicinal Plants, Central drug Research Institute, Lucknow and National Institute of Science Communications, New Delhi.1998: 183.

6.       Roux, DG. d-Gallocatechin from the bark of Casuarina equisetifolia Linn. Nature. 1957: 179:158-159.

7.        Madhulata W et al. Phenolic constituents present in Casuarina fruits and wood. Leather Sci. (Madras).1985: 32(2): 38-39.

8.       Chopra  RN, Nayar SL and Chopra IC. Glossary of Indian Medicinal Plants, National Institute of Science Communications (C.S.I.R.), New Delhi. 1956: 55.

9.       Anonymous. Wealth of India, Raw Materials, Vol.3. NISCIR, New Delhi,India. 1992: 380-85.

10.     Prajapati ND et al. Hand book of Medicinal Plants, Agrobios (India), Jodhpur, India. 2003: 121-22.

11.     Rastogi RP and Mehrotra BN. Compendium of Indian  Medicinal Plants, Vol.4,CDRI Lucknow and NISC New Delhi, India. 2002: 165.

12.     Cimanga RK et al. Antibacterial and antifungal activities of some extracts and fractions of Mitracarpus scaber Zucc. (Rubiaceae). J. of Natural Remedies. 2004: 4:17-25.

13.     Chandrasekaran M et al. Antibacterial activity of faty acid metrhyl esters of Ipomoea pes-caprae L. Indian Drugs. 2005:42(5):  275-281.

 

Received on 17.04.2009

Accepted on 12.05.2009     

© A&V Publication all right reserved

Research Journal of Pharmacognosy  and Phytochemistry. 1(1): July.-Aug. 2009, 64-68