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In vitro antioxidant and antibacterial activity of Rhaphidophora pertusa stem
Fitoterapia 77 (2006) 605 – 607 www.elsevier.com/locate/fitote
Short report
In vitro antioxidant and antibacterial activity of Rhaphidophora pertusa stem J.M. Sasikumar ⁎, P.A. Doss Department of Biotechnology, Karpagam Arts and Science College, Coimbatore, 641 021, India Received 11 March 2005; accepted 20 June 2006 Available online 6 July 2006
Abstract Methanol extract of Rhaphidophora pertusa stem was analyzed for its antioxidant (DPPH, reducing power and Fe3+ metal chelation methods) and antibacterial activities. The extract was found effective against the three antioxidant test models and exhibited strong and moderate antibacterial activity against the tested pathogenic bacteria. © 2006 Elsevier B.V. All rights reserved. Keywords: Rhaphidophora pertusa; Antioxidant activity; Antibacterial activity
1. Plant Rhaphidophora pertusa Schott. (Araceae) stems, collected from the Southern slopes of the Nilgiris, Tamil Nadu, India, were authenticated by scientists from the Botanical Survey of India, Southern Circle, Coimbatore. A voucher specimen was deposited in the Department of Biotechnology, Karpagam Arts and Science College, Coimbatore, India. 2. Uses in traditional medicine and reported activities Fresh juice of the stem is administrated orally for treating ascites, and enlarged spleen and liver [1]. The stem is bitter, acrid, astringent, digestive and carminative, and good for ulcers, odontolgia and bronchitis [2]. Stem juice is administrated to treat pain in the colon and abdominal tumours [3]. Tribal people of the Nilgiris use the stem to treat stomach complaints and tumours (personal communication of J. M. Sasikumar). Stem extracts were found antiulcerogenic [4]. 3. Previously isolated compounds No reports.
⁎ Corresponding author. Fax: +91 422 2611043. E-mail address: [email protected] (J.M. Sasikumar). 0367-326X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2006.06.004
606
J.M. Sasikumar, P.A. Doss / Fitoterapia 77 (2006) 605–607
Table 1 Phytochemical screening of R. pertusa stem extracts Compounds
PE
BE
CE
EE
ME
Alkaloids Saponins Flavonoids Steroids/sterols Terpenoids Anthracene glycosides Cardiac glycosides Phenolic compounds
− − − + + − − −
− − − + + − − −
− − − − + − − −
− − − − − − − +
− + − + − + + +
PE — petroleum ether (60 °C–80 °C); BE — benzene; CE — chloroform; EE — ethyl acetate; ME methanol; “+” — detected; “−” — not detected.
Table 2 Antioxidant activity of the R. pertusa stem methanol extract MeOH extract (μg/ml)
DPPH reduction a Inhibition (%)
Control 1000 500 250 125 50 25 12.5 BHA c
0 91.86 ± 2.026 70.96 ± 2.117 63.8 ± 3.055 57.8 ± 1.265 45.96 ± 7.44 37.36 ± 3.08 28.76 ± 3.799
a b c d
Ic50 (mg/ ml) b
54.56 ± 0.205 (55.44–53.67)
53.27 ± 0.727
MeOH extract (μg/ml)
Fe3+ chelation a Absorbency (460 nm)
% chelation
Concentration (μg/ml)
Reducing power a Absorbency (700 nm)
Control 1000 500 250 125
0.229 ± 0.003 0.055 ± 0.035 0.075 ± 0.002 0.155 ± 0.003 0.210 ± 0.005
0 90.68 ± 2.353 75.97 ± 1.553 67.1 ± 1.147 50.25 ± 1.261
Control 1000 500 250 125
0.076 ± 0.005 0.665 ± 0.021 0.445 ± 0.021 0.312 ± 0.129 0.222 ± 0.008
EDTA d
0.009 ± 0.02
96.08 ± 1.645
BHA c 100(μg/ml)
0.683 ± 0.008
Values are Mean ± S.D. (N = 3 for test groups and standards). Confidential limits 95% in parentheses. BHA, butylated hydroxy anisole, reference standard. EDTA, ethylene diamine tetra acetic acid, reference standard.
Table 3 Antibacterial activity of the R. pertusa stem methanol extract Microorganisms Gram (+) Staphylococcus aureus MTCC 737 Streptococcus pyogenes Gram (−) Escherichia coli MTCC 443 Bacillus subtilis Klebsiela pneumoniae Pseudomonas aeruginosa Salmonella typhi MTCC 734 MIC — minimum inhibitory concentration. a Ampicilin, reference standard.
MIC (mg/ml)
Amp a (mg/ml)
2.0 2.0
0.015 0.015
4.0 2.0 1.0 2.0 2.0
0.031 0.015 0.031 0.031 0.015
J.M. Sasikumar, P.A. Doss / Fitoterapia 77 (2006) 605–607
607
4. Tested material Methanol extracts of shade-dried and milled stem. Petroleum ether 60–80 °C (7.12%), benzene (10.72%), CHCl3 (2.16%), EtOAc (7.21%) and MeOH (19.7%) extracts for phytochemical screening [5,6] (Table 1). 5. Studied activity Antioxidative effect (Table 2) by scavenging DPPH (1,1-Diphenyl-2-picryl hydrozyl) free radicals by modified Blois method [7], Fe3+ metal chelation by thiocyanate method [8] and reducing power according to the method of Oyaizu [9]. Antibacterial activity (Table 3) by determination of minimum inhibitory concentration (MIC) using broth dilution method [10]. Bacterial seeding concentration was done with a cell suspension containing 5 × 104 cfu/ml. 6. Used microorganisms Listed in Table 3. Escherichia coli MTCC 443, Salmonella typhi MTCC 734 and Staphylococcus aureus MTCC 737 were procured from the Institute of Microbial Technology (IMTECH), Chandigarh, India. Other microorganisms were clinical isolates obtained from the Department of Microbiology, Karpagam Arts and Science College, Coimbatore, India. 7. Results Reported in Tables 1, 2 and 3. 8. Conclusions The methanol extract from the stem of R. pertusa showed potent antioxidant activity based on DPPH, reducing power and Fe3+ chelation tests in a dose dependent manner. The extract strongly scavenged DPPH radicals with the IC50 being 54.56 ± 0.205 μg/ml and the activity is comparable to that of BHA. The extract strongly chelated Fe3+ ions at 1000 μg/ml concentration (90.68 ± 2.353). It also caused significant elevation of reducing power. The higher absorbancy at high concentration indicates the strong reducing power potential. The presence of phenolic compounds might be responsible for the antioxidant activity. Methanol extract of the stem exhibited moderate antibacterial activity against all the bacterial pathogens with MIC values of 1.0–4.0 mg/ml. Thus, the medicinal claims of the plant being used in the treatment of liver disorders, abdominal tumours and stomach complaints may be in part due to the antioxidant activity. Further isolation of antioxidant constituents and in vivo antioxidant studies are warranted. Acknowledgement The authors are grateful to the Management, Karpagam Educational Institutions for the generous support and encouragement. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]
Pushpangadan P, Asha V. Anc Sci Life 1998;17:235. Warrier PK, Nambiar VPK, Ramankutty C. Indian medicinal plants: a compendium of 500 species. Madras: Orient Longman Ltd; 1995. Prajapati ND, Purohit SS, Sharma AK, Kumar T. A handbook of medicinal plants. Jodhpur, India: Agrobios; 2003. p. 438. Karpagakumar Sundari SK, Valarmathi R, Akilandeswari RSS, Koshy RP, Hemamalini K. Indian Drugs 2002;38:588. Harborne JB. Phytochemical methods. London: Chapman and Hall Ltd; 1987. Trease GE, Evans MC. Textbook of pharmacognosy. 12th ed. London: Balliere-Tindal; 1979. p. 343. Chindambara Murthy KN, Vanitha A, Mahadeva Swamy M, Ravishankar GA. J Med Food 2003;6:99. Jeffery GH, Basset J, Mandem J, Dennry RC. Vogels text book of quantitative analysis ELBS. UK: Bathpress; 1989. Oyaizu M. Jpn J Nutr 1986;44:314. Rasoanaivo P, Ratsimamanga Urverg S. Monograph for the IFS-NAPRECA bioassays. Madagascar: Antanonarive; 1993. p. 72.
Short report
In vitro antioxidant and antibacterial activity of Rhaphidophora pertusa stem J.M. Sasikumar ⁎, P.A. Doss Department of Biotechnology, Karpagam Arts and Science College, Coimbatore, 641 021, India Received 11 March 2005; accepted 20 June 2006 Available online 6 July 2006
Abstract Methanol extract of Rhaphidophora pertusa stem was analyzed for its antioxidant (DPPH, reducing power and Fe3+ metal chelation methods) and antibacterial activities. The extract was found effective against the three antioxidant test models and exhibited strong and moderate antibacterial activity against the tested pathogenic bacteria. © 2006 Elsevier B.V. All rights reserved. Keywords: Rhaphidophora pertusa; Antioxidant activity; Antibacterial activity
1. Plant Rhaphidophora pertusa Schott. (Araceae) stems, collected from the Southern slopes of the Nilgiris, Tamil Nadu, India, were authenticated by scientists from the Botanical Survey of India, Southern Circle, Coimbatore. A voucher specimen was deposited in the Department of Biotechnology, Karpagam Arts and Science College, Coimbatore, India. 2. Uses in traditional medicine and reported activities Fresh juice of the stem is administrated orally for treating ascites, and enlarged spleen and liver [1]. The stem is bitter, acrid, astringent, digestive and carminative, and good for ulcers, odontolgia and bronchitis [2]. Stem juice is administrated to treat pain in the colon and abdominal tumours [3]. Tribal people of the Nilgiris use the stem to treat stomach complaints and tumours (personal communication of J. M. Sasikumar). Stem extracts were found antiulcerogenic [4]. 3. Previously isolated compounds No reports.
⁎ Corresponding author. Fax: +91 422 2611043. E-mail address: [email protected] (J.M. Sasikumar). 0367-326X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2006.06.004
606
J.M. Sasikumar, P.A. Doss / Fitoterapia 77 (2006) 605–607
Table 1 Phytochemical screening of R. pertusa stem extracts Compounds
PE
BE
CE
EE
ME
Alkaloids Saponins Flavonoids Steroids/sterols Terpenoids Anthracene glycosides Cardiac glycosides Phenolic compounds
− − − + + − − −
− − − + + − − −
− − − − + − − −
− − − − − − − +
− + − + − + + +
PE — petroleum ether (60 °C–80 °C); BE — benzene; CE — chloroform; EE — ethyl acetate; ME methanol; “+” — detected; “−” — not detected.
Table 2 Antioxidant activity of the R. pertusa stem methanol extract MeOH extract (μg/ml)
DPPH reduction a Inhibition (%)
Control 1000 500 250 125 50 25 12.5 BHA c
0 91.86 ± 2.026 70.96 ± 2.117 63.8 ± 3.055 57.8 ± 1.265 45.96 ± 7.44 37.36 ± 3.08 28.76 ± 3.799
a b c d
Ic50 (mg/ ml) b
54.56 ± 0.205 (55.44–53.67)
53.27 ± 0.727
MeOH extract (μg/ml)
Fe3+ chelation a Absorbency (460 nm)
% chelation
Concentration (μg/ml)
Reducing power a Absorbency (700 nm)
Control 1000 500 250 125
0.229 ± 0.003 0.055 ± 0.035 0.075 ± 0.002 0.155 ± 0.003 0.210 ± 0.005
0 90.68 ± 2.353 75.97 ± 1.553 67.1 ± 1.147 50.25 ± 1.261
Control 1000 500 250 125
0.076 ± 0.005 0.665 ± 0.021 0.445 ± 0.021 0.312 ± 0.129 0.222 ± 0.008
EDTA d
0.009 ± 0.02
96.08 ± 1.645
BHA c 100(μg/ml)
0.683 ± 0.008
Values are Mean ± S.D. (N = 3 for test groups and standards). Confidential limits 95% in parentheses. BHA, butylated hydroxy anisole, reference standard. EDTA, ethylene diamine tetra acetic acid, reference standard.
Table 3 Antibacterial activity of the R. pertusa stem methanol extract Microorganisms Gram (+) Staphylococcus aureus MTCC 737 Streptococcus pyogenes Gram (−) Escherichia coli MTCC 443 Bacillus subtilis Klebsiela pneumoniae Pseudomonas aeruginosa Salmonella typhi MTCC 734 MIC — minimum inhibitory concentration. a Ampicilin, reference standard.
MIC (mg/ml)
Amp a (mg/ml)
2.0 2.0
0.015 0.015
4.0 2.0 1.0 2.0 2.0
0.031 0.015 0.031 0.031 0.015
J.M. Sasikumar, P.A. Doss / Fitoterapia 77 (2006) 605–607
607
4. Tested material Methanol extracts of shade-dried and milled stem. Petroleum ether 60–80 °C (7.12%), benzene (10.72%), CHCl3 (2.16%), EtOAc (7.21%) and MeOH (19.7%) extracts for phytochemical screening [5,6] (Table 1). 5. Studied activity Antioxidative effect (Table 2) by scavenging DPPH (1,1-Diphenyl-2-picryl hydrozyl) free radicals by modified Blois method [7], Fe3+ metal chelation by thiocyanate method [8] and reducing power according to the method of Oyaizu [9]. Antibacterial activity (Table 3) by determination of minimum inhibitory concentration (MIC) using broth dilution method [10]. Bacterial seeding concentration was done with a cell suspension containing 5 × 104 cfu/ml. 6. Used microorganisms Listed in Table 3. Escherichia coli MTCC 443, Salmonella typhi MTCC 734 and Staphylococcus aureus MTCC 737 were procured from the Institute of Microbial Technology (IMTECH), Chandigarh, India. Other microorganisms were clinical isolates obtained from the Department of Microbiology, Karpagam Arts and Science College, Coimbatore, India. 7. Results Reported in Tables 1, 2 and 3. 8. Conclusions The methanol extract from the stem of R. pertusa showed potent antioxidant activity based on DPPH, reducing power and Fe3+ chelation tests in a dose dependent manner. The extract strongly scavenged DPPH radicals with the IC50 being 54.56 ± 0.205 μg/ml and the activity is comparable to that of BHA. The extract strongly chelated Fe3+ ions at 1000 μg/ml concentration (90.68 ± 2.353). It also caused significant elevation of reducing power. The higher absorbancy at high concentration indicates the strong reducing power potential. The presence of phenolic compounds might be responsible for the antioxidant activity. Methanol extract of the stem exhibited moderate antibacterial activity against all the bacterial pathogens with MIC values of 1.0–4.0 mg/ml. Thus, the medicinal claims of the plant being used in the treatment of liver disorders, abdominal tumours and stomach complaints may be in part due to the antioxidant activity. Further isolation of antioxidant constituents and in vivo antioxidant studies are warranted. Acknowledgement The authors are grateful to the Management, Karpagam Educational Institutions for the generous support and encouragement. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]
Pushpangadan P, Asha V. Anc Sci Life 1998;17:235. Warrier PK, Nambiar VPK, Ramankutty C. Indian medicinal plants: a compendium of 500 species. Madras: Orient Longman Ltd; 1995. Prajapati ND, Purohit SS, Sharma AK, Kumar T. A handbook of medicinal plants. Jodhpur, India: Agrobios; 2003. p. 438. Karpagakumar Sundari SK, Valarmathi R, Akilandeswari RSS, Koshy RP, Hemamalini K. Indian Drugs 2002;38:588. Harborne JB. Phytochemical methods. London: Chapman and Hall Ltd; 1987. Trease GE, Evans MC. Textbook of pharmacognosy. 12th ed. London: Balliere-Tindal; 1979. p. 343. Chindambara Murthy KN, Vanitha A, Mahadeva Swamy M, Ravishankar GA. J Med Food 2003;6:99. Jeffery GH, Basset J, Mandem J, Dennry RC. Vogels text book of quantitative analysis ELBS. UK: Bathpress; 1989. Oyaizu M. Jpn J Nutr 1986;44:314. Rasoanaivo P, Ratsimamanga Urverg S. Monograph for the IFS-NAPRECA bioassays. Madagascar: Antanonarive; 1993. p. 72.