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Antibacterial activity and cytotoxicity of extractives from Ravenia spectabilis
Fitoterapia 75 (2004) 510–513
Short report
Antibacterial activity and cytotoxicity of extractives from Ravenia spectabilis M.H. Sohrab, R. Chowdhury, K.M. Rahman, C.M. Hasan, M.A. Rashid* Phytochemical Research Laboratory, Faculty of Pharmacy, University of Dhaka, Dhaka 1000, Bangladesh Received 8 August 2003; accepted 30 March 2004
Abstract A methanolic extract of Ravenia spectabilis, an isolated alkaloid, arborinine plus a fraction comprising arborinine and g-fagarine (VLC), showed mild to significant in vitro antibacterial activity. In a brine shrimp lethality bioassay, the extract and the fraction were found to exhibit moderate cytotoxicity having LC50 of 76.26 mgyml and 14.98 mgyml, respectively. 䊚 2004 Elsevier B.V. All rights reserved. Keywords: Ravenia spectabilis; Antibacterial; Cytotoxicity
Plant. Ravenia spectabilis Engl. (Rutaceae), stems collected from the Balda Garden of Dhaka district, Bangladesh in October 1999 and identified at the Bangladesh National Herbarium (voucher specimen number: DACB-28090). Uses in traditional medicine. No ethnomedical use has been reported for this species. Previously isolated classes of constituents. Alkaloids w1–5x. *Corresponding author. Tel.: q880-2-9661900-59x6062; fax: q880-2-8615583. E-mail address: [email protected] (M.A. Rashid). 0367-326X/04/$ - see front matter 䊚 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2004.03.008
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Table 1 Antibacterial activity of test samples of R. spectabilis Microorganism
Aeromonus hydrophilia Bacillus cereus Bacillus megaterium Bacillus subtilis Escherichia coli Klebsiella sp. Pseudomonas aeruginosa Salmonella paratyphi A Salmonella paratyphi B Salmonella typhi Sarcina lutea Shigella boydii Shigella dysenteriae Shigella flexneriae Shigella sonnei Staphylococcus aureus Vibrio cholerae Vibrio mimicus Vibrio parahemolyticus
Strains
AM 10481 QL 29 QL 38 QL 40 ATCC 25922 – ATCC 27853 AM 16590 AM 15961 AM 16406 QL 166 ATCC 13147 ATCC 26131 Y 976 C 182 ATCC 25923 569 B N 1967 AM 16362
Diameter of zone of inhibition (mm) MeOH extract (500 mgydisc)
VLC fraction-23 (400 mgydisc)
Kanamycin (30 mgydisc)
15 28 25 10 08 – 08 10 08 – 15 10 17 – 08 13 – 25 09
17 17 15 18 23 – 13 24 19 – 15 13 22 – 10 – – 21 08
19 24 31 22 36 20 25 34 22 20 22 20 31 22 24 31 23 21 21
‘-’ Indicates no sensitivity.
New-isolated constituents. Arborinine w6x (yield: 0.021%), g-fagarine w7x (yield: 0.018%), stigmasterol and stigmasta-4,22-dien-3-one.
Tested material. Methanol extract (yield: 2.61% of air-dried plant material), VLC fraction 23 (petroleum ether-EtOAc 1:3) of the crude extract and an isolated alkaloid, arborinine from the same fraction.
Studied activity. Antibacterial activity by the standardized disc diffusion method w8x and cytotoxicity by brine shrimp lethality bioassay w9x. DMSO was used as a solvent and control in the cytotoxicity study. Tests were done in triplicate.
Used organisms. The bacterial strains listed in Table 1 were collected as pure cultures from the Institute of Nutrition and Food Science (INFS), University of Dhaka, Bangladesh. Brine shrimp (Artemia salina) nauplii (Ocean 90, USA) were used for cytotoxicity study. For hatching, shrimp eggs were kept in 3.8% brine solution with a constant oxygen supply for 48 h; the mature nauplii were then used in the experiment.
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512
Table 2 Cytotoxicity of extractives from R. spectabilis bark on brine shrimps Concentration (mgyml) 6.25 12.50 25.00 50.00 100.00 200.00
Methanol extract Mortality %) 10.00"1.00 23.33"0.58 40.00"0.00 43.33"0.58 48.67"0.58 66.67"0.58
a
LC50 (mgyml)
b
76.26 (69.83–83.80)
Concentration (mgyml)
VLC fraction Mortality (%)a
LC50 (mgyml)b
6.25 12.50 25.00 50.00 100.00 200.00
40.00"0.00 48.33"0.58 53.33"0.58 63.33"0.58 80.00"1.00 93.33"0.58
14.98 (13.65–16.33)
0.0390 0.078125 0.15625 0.3125 0.625 1.25
Vincristine sulfate 21.67"2.88 29.17"1.44 31.00"1.73 0.583 43.33"2.88 (0.512–0.676) 48.33"2.88 61.66"2.88
a
Values are mean"S.D. (ns3 for test and ns2 for positive control). 95% Confidential limits in parentheses. Vincristine sulfate: positive control. b
Results. Reported in Table 1 (antibacterial) and Table 2 (cytotoxicity).
Conclusions. The mean zones of inhibition produced by the crude methanolic extract was found to be 8–28 mm at 500 mgydisc against most of the test organisms. The activity was particularly significant against Bacillus cereus, B. megaterium and Vibrio mimicus (ZI 28, 25 mm and 25 mm, respectively). The antibacterial spectrum of VLC fraction-23 was in line with expectations. The mean zones of inhibition were found to be 8–24 mm at 400 mgydisc dose level against 14 out of the 19 organisms. The fraction exhibited good activity against Bacillus cereus, B. subtilis, Aeromonus hydrophilia, Escherichia coli, Salmonella paratyphi A, S. paratyphi B, Shigella dysenteriae and Vibrio mimicus (Table 1). The purified compound, arborinine, demonstrated only mild in vitro antibacterial activity against Escherichia coli and Shigella dysenteriae with zones of inhibition of 7 mm and 9 mm, respectively, at a dose of 200 mgydisc. The crude extract and the VLC fraction-23 demonstrated LC50 of 76.26 (Gs0.004824) mgyml and 14.98 (Gs0.004464) mgy ml, respectively, against brine shrimp nauplii (Table 2). No mortality was observed in the negative control (DMSO). Acknowledgments The authors wish to thank the Institute of Nutrition and Food Science (INFS), University of Dhaka, Dhaka-1000, Bangladesh for supplying the test microorganisms.
M.H. Sohrab et al. / Fitoterapia 75 (2004) 510–513
513
References w1 x w2 x w3 x w4 x w5 x w6 x w7 x
Khan MA, Waterman PG. Fitoterapia 1990;61:282. Bhattacharyya P, Chakraborty A, Chowdury BK. Phytochemsitry 1984;23:2409. Talapatra SK, Maitim BC, Talaparta B, Das BC. Tetrahedron Lett 1969;54:4789. Paul BD, Bose PK. J Indian Chem Soc 1968;45:552. Paul BD, Bose PK. J Indian Chem Soc 1969;7:678. Sultana N, Hartley TG, Waterman PG. Phytochemistry 1999;50:1249. Mai HD, Van-Dufat HT, Michel S, Tillequin F, Bastien D, Sevenet T. Z Naturforsch wCx 2001;56:492. w8x Bauer AW, Kirby WMM, Sherris JC, Turck M. Am J Clin Pathol 1966;45:493. w9x Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DE, McLaughlin JL. Planta Med 1982;45:31.
Short report
Antibacterial activity and cytotoxicity of extractives from Ravenia spectabilis M.H. Sohrab, R. Chowdhury, K.M. Rahman, C.M. Hasan, M.A. Rashid* Phytochemical Research Laboratory, Faculty of Pharmacy, University of Dhaka, Dhaka 1000, Bangladesh Received 8 August 2003; accepted 30 March 2004
Abstract A methanolic extract of Ravenia spectabilis, an isolated alkaloid, arborinine plus a fraction comprising arborinine and g-fagarine (VLC), showed mild to significant in vitro antibacterial activity. In a brine shrimp lethality bioassay, the extract and the fraction were found to exhibit moderate cytotoxicity having LC50 of 76.26 mgyml and 14.98 mgyml, respectively. 䊚 2004 Elsevier B.V. All rights reserved. Keywords: Ravenia spectabilis; Antibacterial; Cytotoxicity
Plant. Ravenia spectabilis Engl. (Rutaceae), stems collected from the Balda Garden of Dhaka district, Bangladesh in October 1999 and identified at the Bangladesh National Herbarium (voucher specimen number: DACB-28090). Uses in traditional medicine. No ethnomedical use has been reported for this species. Previously isolated classes of constituents. Alkaloids w1–5x. *Corresponding author. Tel.: q880-2-9661900-59x6062; fax: q880-2-8615583. E-mail address: [email protected] (M.A. Rashid). 0367-326X/04/$ - see front matter 䊚 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2004.03.008
M.H. Sohrab et al. / Fitoterapia 75 (2004) 510–513
511
Table 1 Antibacterial activity of test samples of R. spectabilis Microorganism
Aeromonus hydrophilia Bacillus cereus Bacillus megaterium Bacillus subtilis Escherichia coli Klebsiella sp. Pseudomonas aeruginosa Salmonella paratyphi A Salmonella paratyphi B Salmonella typhi Sarcina lutea Shigella boydii Shigella dysenteriae Shigella flexneriae Shigella sonnei Staphylococcus aureus Vibrio cholerae Vibrio mimicus Vibrio parahemolyticus
Strains
AM 10481 QL 29 QL 38 QL 40 ATCC 25922 – ATCC 27853 AM 16590 AM 15961 AM 16406 QL 166 ATCC 13147 ATCC 26131 Y 976 C 182 ATCC 25923 569 B N 1967 AM 16362
Diameter of zone of inhibition (mm) MeOH extract (500 mgydisc)
VLC fraction-23 (400 mgydisc)
Kanamycin (30 mgydisc)
15 28 25 10 08 – 08 10 08 – 15 10 17 – 08 13 – 25 09
17 17 15 18 23 – 13 24 19 – 15 13 22 – 10 – – 21 08
19 24 31 22 36 20 25 34 22 20 22 20 31 22 24 31 23 21 21
‘-’ Indicates no sensitivity.
New-isolated constituents. Arborinine w6x (yield: 0.021%), g-fagarine w7x (yield: 0.018%), stigmasterol and stigmasta-4,22-dien-3-one.
Tested material. Methanol extract (yield: 2.61% of air-dried plant material), VLC fraction 23 (petroleum ether-EtOAc 1:3) of the crude extract and an isolated alkaloid, arborinine from the same fraction.
Studied activity. Antibacterial activity by the standardized disc diffusion method w8x and cytotoxicity by brine shrimp lethality bioassay w9x. DMSO was used as a solvent and control in the cytotoxicity study. Tests were done in triplicate.
Used organisms. The bacterial strains listed in Table 1 were collected as pure cultures from the Institute of Nutrition and Food Science (INFS), University of Dhaka, Bangladesh. Brine shrimp (Artemia salina) nauplii (Ocean 90, USA) were used for cytotoxicity study. For hatching, shrimp eggs were kept in 3.8% brine solution with a constant oxygen supply for 48 h; the mature nauplii were then used in the experiment.
M.H. Sohrab et al. / Fitoterapia 75 (2004) 510–513
512
Table 2 Cytotoxicity of extractives from R. spectabilis bark on brine shrimps Concentration (mgyml) 6.25 12.50 25.00 50.00 100.00 200.00
Methanol extract Mortality %) 10.00"1.00 23.33"0.58 40.00"0.00 43.33"0.58 48.67"0.58 66.67"0.58
a
LC50 (mgyml)
b
76.26 (69.83–83.80)
Concentration (mgyml)
VLC fraction Mortality (%)a
LC50 (mgyml)b
6.25 12.50 25.00 50.00 100.00 200.00
40.00"0.00 48.33"0.58 53.33"0.58 63.33"0.58 80.00"1.00 93.33"0.58
14.98 (13.65–16.33)
0.0390 0.078125 0.15625 0.3125 0.625 1.25
Vincristine sulfate 21.67"2.88 29.17"1.44 31.00"1.73 0.583 43.33"2.88 (0.512–0.676) 48.33"2.88 61.66"2.88
a
Values are mean"S.D. (ns3 for test and ns2 for positive control). 95% Confidential limits in parentheses. Vincristine sulfate: positive control. b
Results. Reported in Table 1 (antibacterial) and Table 2 (cytotoxicity).
Conclusions. The mean zones of inhibition produced by the crude methanolic extract was found to be 8–28 mm at 500 mgydisc against most of the test organisms. The activity was particularly significant against Bacillus cereus, B. megaterium and Vibrio mimicus (ZI 28, 25 mm and 25 mm, respectively). The antibacterial spectrum of VLC fraction-23 was in line with expectations. The mean zones of inhibition were found to be 8–24 mm at 400 mgydisc dose level against 14 out of the 19 organisms. The fraction exhibited good activity against Bacillus cereus, B. subtilis, Aeromonus hydrophilia, Escherichia coli, Salmonella paratyphi A, S. paratyphi B, Shigella dysenteriae and Vibrio mimicus (Table 1). The purified compound, arborinine, demonstrated only mild in vitro antibacterial activity against Escherichia coli and Shigella dysenteriae with zones of inhibition of 7 mm and 9 mm, respectively, at a dose of 200 mgydisc. The crude extract and the VLC fraction-23 demonstrated LC50 of 76.26 (Gs0.004824) mgyml and 14.98 (Gs0.004464) mgy ml, respectively, against brine shrimp nauplii (Table 2). No mortality was observed in the negative control (DMSO). Acknowledgments The authors wish to thank the Institute of Nutrition and Food Science (INFS), University of Dhaka, Dhaka-1000, Bangladesh for supplying the test microorganisms.
M.H. Sohrab et al. / Fitoterapia 75 (2004) 510–513
513
References w1 x w2 x w3 x w4 x w5 x w6 x w7 x
Khan MA, Waterman PG. Fitoterapia 1990;61:282. Bhattacharyya P, Chakraborty A, Chowdury BK. Phytochemsitry 1984;23:2409. Talapatra SK, Maitim BC, Talaparta B, Das BC. Tetrahedron Lett 1969;54:4789. Paul BD, Bose PK. J Indian Chem Soc 1968;45:552. Paul BD, Bose PK. J Indian Chem Soc 1969;7:678. Sultana N, Hartley TG, Waterman PG. Phytochemistry 1999;50:1249. Mai HD, Van-Dufat HT, Michel S, Tillequin F, Bastien D, Sevenet T. Z Naturforsch wCx 2001;56:492. w8x Bauer AW, Kirby WMM, Sherris JC, Turck M. Am J Clin Pathol 1966;45:493. w9x Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DE, McLaughlin JL. Planta Med 1982;45:31.