Antimicrobial activity and GC-MS analysis of Ocimum tenuiflorum and Acalypha hispida extract against Streptococcus pyogenes

RESEARCH ARTICLE

Open Access Full Text Article

Antimicrobial activity and GC-MS analysis of

Ocimum tenuiflorum

and

Acalypha hispida

extract against

Streptococcus pyogenes

D Elamparithi

_,

_{P Mani}

_{, V Moorthy}

1_{Department of Biotechnology, Annai College of Arts and Science, Kumbakonam, Tamil Nadu, India}

* For correspondence e-mail: [email protected]

Article Info: Received 26 Nov 2014; Revised: 19 Dec 2014; Accepted 26 Dec 2014

ABSTRACT

The antimicrobial activities of single and combined fraction of leaves from Ocimum tenuiflorum and

Acalypha hispida were analyzed against human pathogenic bacteria Streptococcus pyogenes. Antimicrobial properties of both species fractions were tested using disc diffusion method. Analysis of the data revealed that, the ethanol commix methanol active fractions of the O. tenuiflorum exhibited the highest antibacterial

activity. It showed antibacterial activity against S. pyogenes: 9 mm zone at 2:18 concentration. The ethanol

commix methanol fraction of A. hispida showed the least antibacterial activity against S. pyogenes (5 mm).

Results were compared concurrently to standard drugs. In the GC-MS analysis 18 and 47 bioactive

phytochemical compounds were identified in the ethanolic fraction of O. tenuiflorum and A. hispida

respectively. Based on the current findings, it can be concluded that these plant fractions have antimicrobial activity, which is as potent as standard antimicrobial drugs against certain microorganisms.

Keywords: Ocimum tenuiflorum, Acalypha hispida, Antibacterial activity, Zone of inhibition.

1. INTRODUCTION

Streptococcus pyogenes is a common human

commensal organism and also a major cause of nosocomial infections worldwide associated with high death rates, prolonged hospitalization and increased medical costs [1]. It is responsible for a wide range of diseases, including endocarditis, osteomyelitis, toxic-shock syndrome, pneumonia, food poisoning and carbuncles [2], and this bacteria contain a variety of virulence products or systems that make it difficult to treat associated infections. Streptococcus pyogenes also causes a number of di-

-verse infections the growing antibiotic resistance, including Methicillin-resistant. MRSA strains are resistance against almost all clinically available antibiotics [3]. Hence, search for effective and safer antimicrobial agents has become an area of current research. According to World Health Organization, medicinal plants are the best sources to obtain a variety of novel drugs [4].

Ocimum tenuiflorum L., (Lamiaceae) is an Aromatic

plant; it is use folk medicine that has been used for the management of various diseases. Different part of

Malaya

Journal of

Biosciences

this plant has been used for management of anti-diabetic [5] anti-diarrhoeal [6] anti-inflammatory [7] antispasmodic [8] anti-cough [9] anticancer [10] and hepatoprotective [11]. Another traditional medicinal species A. hispida (Euphorbiaceae) is a flowering shrub and this plant is also known as the Philippines Medusa, red hot cat's tail andfox tail. It has been used for thousands of years to treat against several chronic diseases. The antifertility [12], antifungal [13], antiviral [14], antitumor [15] properties were some of the studies of the earlier workers. Phytochemical investigations of these two species have been reported on the Lipids, Alkaloids, Steroids, Tannins, Polyphenols, Terpenoids, Flavonoids and Saponins [16]. Hence, the present study was carried out to test the antimicrobial efficacy of the Ocimum tenuiflorum

and Acalypha hispida leaves fractions against

Streptococcus pyogenes strain.

2.

MATERIALS AND METHODS

2.1. Plant material

The leaves of O. tenuiflorum and A. hispida were

collected from various areas of kolimalai, Namkkal district, Tamil Nadu, India. A voucher specimen of the plant has been deposited at the Department herbarium at Vels University, Chennai, Tamil Nadu, India. The dried leaves were cut into small pieces and powdered using an electric mill.

2.2. Preparation of the fractions of extract

500 g of O. tenuiflorum and A. hispida powdered was extracting with absolute ethanol using soxhlet apparatus over 48h and then the extract was reduced to dryness in a rotary evaporator. Each plant crude ethanol extract was sequentially partitioned with water, ethanol, methanol, acetone, hexane and butanol fractions, respectively.

2.3. Test Organisms

Clinical pathogenic organisms Streptococcus

pyogenes was used for this studyand it wasisolated from skin infected patients at skin clinics in and around Thanjavur and Chennai Tamil Nadu, India. The isolated species were kept in vials and stored in Refrigerator.

2.4. Determination of antimicrobial activity

Culture supernatants and fractions of extract of the both plants were used in the disc-diffusion method separately. Streptococcus pyogenes swabbed on the surface of the sabouraud agar plates and discs (Whatman No.1 filter paper with 9 mm diameter) impregnated with the 50 µl of each plant sample was place on the surface individually. To compare the anti-bacterial activities, Nystatin (20 µg/disc) used as standard antibiotic and negative control, a blank disc impregnated with solvent followed by drying was used. The plates (triplicates) were incubated 28°C for 72 h. The antimicrobial potency of the test samples was measured by determining the diameter of the zones of inhibition in millimeter.

2.5. GC-MS analysis

30 g powdered sample of O. tenuiflorum and A. hispida were soaked and dissolved in 75 ml of methanol for 24 h. Then the filtrates were collected by evaporated under liquid nitrogen. The GC-MS analysis was carried out using a Clarus 500 Perkin- Elmer (Auto System XL) Gas Chromatograph equipped and coupled to a mass detector Turbo mass gold – Perking Elmer Turbomas 5.2 spectrometer with an Elite-1 (100% Dimethyl ply siloxane), 300 m x 0.25 mm x 1 µm df capillary column. The instrument was set to an initial temperature of 110°C, and maintained at this temperature for 2 min. At the end of this period, the oven temperature was raised upto 280°C, at the rate of an increase of 5°C/min, and maintained for 9 min. Injection port temperature was ensured as 250°C and Helium flow rate as 1 ml/min. The ionization voltage was 70 eV. The samples were injected in split mode as 10:1. Mass Spectral scan range was set at 45-450 (mhz). The chemical constituents were identified by GC-MS. The fragmentation patterns of mass spectra were compared with those stored in the spectrometer database using National Institute of Standards and Technology Mass Spectral database (NIST-MS). The percentage of each component was calculated from relative peak area of each component in the chromatogram.

3. RESULTS AND DISCUSSION

Table 1. Antimicrobial activity of O. tenuiflorum and A. hispida individual fraction tested against Streptococcus pyogenes

by disk diffusion method.

Table 2. Antimicrobial activity of ethanol and methanol combined fractions of the O. tenuiflorum and A. hispida tested against

Streptococcus pyogenes by disk diffusion method

Plant Zone of inhibition (mm)

Water Ethanol Methanol Acetone Hexane Butanol

O. tenuiflorum 2 7 3 2 2 0.5

A. hispida 3 6 4 0.5 1 2

Plant sample/ Fraction

concentration

Zone of inhibition (mm)

18:2

(E/M)

16:4

(E/M)

14:6

(E/M)

12:8

(E/M)

10:10

(E/M)

8:12

(E/M) 6:14

(E/M) 4:16

(E/M) 2:18

(E/M)

O. tenuiflorum 1 2 2 1 8 2 0.5 - 9

Table 3. Compounds identified by GC-MS in the O. tenuiflorum methanolic extract

S.No. Compound (Tentative ID) Retention

time

Peak Area %

1. Name: Butane, 1,1-diethoxy Formula: C8H1802

MW: 146

2.89 1.99

2. Name: -Pentane, 1,1idiethoxy Formula: C9H20O2

MW: 160

3.99 0.30

3. Name: 4H-Pyran-4-one,2,3-dihydro-3-5- dihydroxy-6-methyl

Formula: C6H8O4

MW: 144

7.56 0.85

4. Name: Copaene Formula: C15H24

MW ;204

13.54 8.55

5. Name: Caryophyllene Formula: C15H24

MW: 204

14.66 38.34

6. Name: α- Caryophyllene Formula: C15H24

MW: 204

15.46 4.36

7. Name ; 1H-Benzocycloheptene, 2,4 a,5,6,7,,8,9,9a-octahydro-3,5,5-trimethyl-9-methyllene- (4aS-cis)-

Formula: C15H24

MW;204

16.40 2.23

8. Name: Naphthalene, 1,2,3,4,4 a,7-hexahydro-1,6-dimethyl-4-(1-methylethyl)- Formula: C15H24

MW: 204

17.14 3.62

9. Name: 1,6,10-Dodecatrien-3-ol, 3,7,11-trimethyl-,[S-(Z)]-Nerolidol Formula: C15H26O

MW: 222

17.47 4.85

10. Name: Caryophyllene oxide Formula: C15H24O

MW: 220

18.42 7.89

11. Name: ledol Formula: C15H26O

MW: 222

18.95 2.38

12. Name: tetracyclo [6.3.2.0 (2.5). 0 (1,8)] tridecan-9-ol, 4,4-dimethyl Formula: C15H24O

MW: 220

19.56 10.89

13. Name: Caryophyllene oxide Formula: C15H24O

MW: 220

20.25 2.80

14. Name: cis-Z-α- Bisabolene epoxide Formula: C15H24O

MW: 220

23.21 1.64

15. Name: 4,4,8- Trimethyltricyclo [6.3.1.0 (1,5)]dodecane-2,99diol Formula: C15H26O2

MW: 238

24.61 1.21

16. Name: n- Hexadecanoic acid Formula: C16H3202

MW: 256

25.90 7.55

17. Name: - Hexadecanoic acid, ethyl ester Formula: C18H36O2

MW: 284

Table 4. Compounds identified by GC-MS in A. hispida methanolic extract

S.No. Compound (Tentative ID) Retention

time

Peak Area %Peak Area

1. Name: dl-Homoserine Formula: C4H9NO3 MW: 119

3.36 425931 0.1589

2. Name: 2-Furanmethanol Formula: C5H6O2 MW: 98

3.73 798689 0.2979

3. Name: 2-Cyclopentene-1,4-dione Formula: C5H4O2

MW: 96

4.14 1701291 0.6345

4. Name: Propanoic acid, 2-hydroxy-2-methyl- Formula: C4H8O3

MW: 104

4.43 7186447 2.6803

5. Name: Butanoic acid, 4-hydroxy- Formula: C4H8O3

MW: 104

4.56 4324497 1.6129

6. Name: (+-)-4-Amino-4,5-dihydro-2(3H)-furanone Formula: C4H7NO2

MW: 101

4.85 545852 0.2036

7. Name: 1-Pyrrolidineethanamine Formula: C6H14N2

MW: 114

5.00 1586724 0.5918

8. Name: 2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-one Formula: C6H8O4

MW: 144

5.52 1225662 0.4571

9. Name: Glycerin Formula: C3H8O3 MW: 92

5.60 3930203 1.4658

10. Name: 4(H)-Pyridine, N-acetyl- Formula: C7H9NO

MW: 123

6.22 406378 0.1516

11. Name: Aziridine, 2-isopropyl-1,3-dimethyl-, trans- Formula: C7H15N

MW: 113

6.34 1265760 0.4721

12. Name: Butane, 1-(ethenyloxy)-3-methyl- Formula: C7H14O

MW: 114

6.50 411661 0.1535

13. Name: 2,3-Pentanedione, 4-methyl- Formula: C6H10O2

MW: 114

6.72 2228251 0.8311

14. Name: Butanamide Formula: C4H9NO MW: 87

7.13 168775 0.0629

15. Name: 1,3,5-Triazine-2,4,6-triamine Formula: C3H6N6

MW: 126

7.36 5533534 2.0638

16. Name: 4-(4-Methyl-piperazin-1-yl)-1,5,-dihydro-imidazol-2-one Formula: C8H14N4O

MW: 182

7.93 3955657 1.4753

17. Name: Acetone, 1-[4-(dimethylaminoethoxy)phenyl]- Formula: C13H19NO2

MW: 221

18. Name: 3-Amino-2-oxazolidinone Formula: C3H6N2O2

MW: 102

8.47 1216423 0.4537

19. Name: 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- Formula: C6H8O4

MW: 144

8.59 13576916 5.0637

20. Name: N-Methylpyrrole-2-carboxylic acid Formula: C6H7NO2

MW: 125

9.13 1284539 0.4791

21. Name: 4H-Pyran-4-one, 3,5-dihydroxy-2-methyl- Formula: C6H6O4

MW: 142

9.30 663332 0.2474

22. Name: Proline, N-methyl-, butyl ester Formula: C10H19NO2

MW: 185

9.50 945063 0.3525

23. Name: L-Proline, 1-methyl-5-oxo-, methyl ester Formula: C7H11NO3

MW: 157

10.90 253479 0.0945

24. Name: 2-Methoxy-4-vinylphenol Formula: C9H10O2

MW: 150

11.50 4220484 1.5741

25. Name: Phenol, 2,6-dimethoxy- Formula: C8H10O3

MW: 154

12.09 1145897 0.4274

26. Name: Phenol, 2-methoxy-3-(2-propenyl)- Formula: C10H12O2

MW: 164

12.16 1733884 0.6467

27. Name: 1,5-Diazabicyclo[4.4.0]dec-5-en-2-one Formula: C8H12N2O

MW: 152

13.10 212085 0.0791

28. Name: 4-Isopropenyl-4,7-dimethyl-1-oxaspiro[2.5]octane Formula: C12H20O

MW: 180

15.78 671903 0.2506

29. Name: 2(4H)-Benzofuranone, 5,6,7,7a-tetrahydro-4,4,7a-trimethyl- Formula: C11H16O2

MW: 180

15.86 726446 0.2709

30. Name: Ethanone, 1-(3,4-dimethoxyphenyl)- Formula: C10H12O3

MW: 180

16.22 643116 0.2399

31. Name: (7,7-Dimethyl-2-oxobicyclo[2.2.1]hept-1-yl)methanesulfonic acid, methyl ester

Formula: C11H18O4S MW: 246

16.73 1183133 0.4413

32. Name: Ethyl N-(2-methylphenyl)carbamate Formula: C10H13NO2

MW: 179

16.96 377459 0.1408

33. Name: Quinoline, 2-ethyl- Formula: C11H11N MW: 157

17.12 231405 0.0863

34. Name: 2-Butynoic acid, 4-cyclohexyl-4-oxo-, ethyl ester Formula: C12H16O3

MW: 208

17.34 694432 0.2590

35. Name: 5-Isopropenyl-1,2-dimethylcyclohex-2-enol Formula: C11H18O

17.46 374403 0.1396

37. Name: 3-Buten-2-one, 4-(4-hydroxy-2,2,6-trimethyl-7-oxabicyclo[4.1.0]hept-1-yl)-

Formula: C13H20O3 MW: 224

18.34 53236 0.0199

38. Name: 1,4-Benzenediol, 2,6-dimethyl- Formula: C8H10O2

MW: 138

18.49 281005 0.1048

39. Name: 5-Isopropyl-3,3-dimethyl-2-methylene-2,3-dihydrofuran Formula: C10H16O

MW: 152

19.00 383211 0.1429

40. Name: 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol Formula: C10H12O3

MW: 180

19.23 4266483 1.5913

41. Name: 5-Bromopentanoic acid, 2-isopropoxyphenyl ester Formula: C14H19BrO3

MW: 314

19.39 2941348 1.0970

42. Name: 3,7,11,15-Tetramethyl-2-hexadecen-1-ol Formula: C20H40O

MW: 296

20.08 7262203 2.7085

43. Name: 4-Decenoic acid, 3-methyl-, (E)- Formula: C11H20O2

MW: 184

20.39 2769435 1.0329

44. Name: à-D-Glucopyranoside, à-D-glucopyranosyl Formula: C12H22O11

MW: 342

22.87 162387312 60.5648

45. Name: 3-Heptanol, 3,5-dimethyl- Formula: C9H20O

MW: 144

23.61 12472845 4.6519

46. Name: Bicyclo[4.4.0]dec-2-ene-4-ol, 2-methyl-9-(prop-1-en-3-ol-2-yl)-

Formula: C15H24O2 MW: 236

34.16 2846962 1.0618

47. Name: Cholesta-4,6-dien-3-ol, (3á)- Formula: C27H44O

MW: 384

35.56 4740284 1.7680

Inhibition results of all the individual fractions of O. tenuiflorum (2, 7, 3, 2, 2, 0.5 mm zone) and A. hispida (3, 6, 4, 0.5, 1, 2 mm zone) are summarized. In Table 2, dynamic solvent ethanol and methanol fractions of both species were selected and treating various combinations against bacterial strains. The O.

tenuiflorum ethanol combined methanol solvent

active fraction (2:18) showed significant

antimicrobial activity such as 9 mm zone of inhibition in diameter, against novel S. Pyogenes

strain. However, leaves sample of A. hispida showed

mild to moderate antimicrobial activity and its ethanol combined methanol fraction (2:18) at ranged from 5 mm zone of inhibition in diameter against novel S. Pyogenes. This fraction show lower antimicrobial activity as comparable to Nystatin and

O. tenuiflorum fraction.

The result of the GC-MS analysis of O. tenuiflorum

sample were presented in Table 3. Total, 18 compounds were identified in the methanol fractions

of O. tenuiflorum. However, totally 47 compounds identified from the methanol fractions of the A.

hispida were presented in Table 4. All these

compounds are of pharmacological importance as they possess the properties such as analgesic, anti-diabetic, antibacterial, and antifungal.

4. CONCLUSION

In conclusion, the study suggests that O. tenuiflorum

and A. hispida ethanol with methanol compounding fractions may serve as good source for new anti-microbial agent with therapeutic potentials.

Moreover, further investigations, including

toxicological evaluation, are required to harness the possible potential use of both samples either as drug.

Conflict of Interest

Acknowledgement

The authors wish to thank Department of Biotechnology, Annai College of Arts and Science, Kumbakonam, Tamilnadu.

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