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The anti-inflammatory and antibacterial activities of Amaryllidaceae alkaloids
South African Journal of Botany 2003, 69(3): 448–449 Printed in South Africa — All rights reserved
Copyright © NISC Pty Ltd
SOUTH AFRICAN JOURNAL OF BOTANY ISSN 0254–6299
Research Note
The anti-inflammatory and antibacterial activities of Amaryllidaceae alkaloids EE Elgorashi, S Zschocke and J van Staden* Research Centre for Plant Growth and Development, School of Botany and Zoology, University of Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa * Corresponding author, e-mail: [email protected] Received 7 March 2003, accepted in revised form 8 April 2003
In the course of a continued investigation of the alkaloid constituents of the family Amaryllidaceae, 15 Amaryllidaceae alkaloids isolated from Crinum bulbispermum and C. moorei were tested for anti-inflammatory activity using cyclooxygenase assay (COX-1 and COX-2) and antibacterial activity against Bacillus sub-
tilis, Staphylococus aureus, Escherichia coli and Klebsiella pneumoniae. The alkaloids showed only low inhibitory activity on COX-1 and COX-2. No bacterial growth inhibition was observed at the final concentration of 250μg ml-1 used in this study.
Crinum species (Amaryllidaceae) are used traditionally in South African folk medicine for rheumatism, backache, septic sores, abscesses and cold, kidney and bladder infections (Roberts 1990, Hutchings et al. 1996). The genus Crinum is a true representative of the family Amaryllidaceae which is the exclusive source of the Amaryllidaceae alkaloids. The biological activities associated with these alkaloids covers analgesic, central nervous system, antiviral, antitumor and antimalarial effects (Ghosal et al. 1985, Lin et al. 1995, Campbell et al. 1998). Experiments on the analgesic effects of these alkaloids, which are attributed to their resemblance to morphine and codeine skeletons, were carried out exclusively on laboratory animals (Ghosal et al. 1985). Despite the wide range of biological activities involved, there have been no reports concerning the activity of these alkaloids in terms of cyclooxygenese-1 (COX-1) and cyclooxygenese-2 (COX-2) anti-inflammatory inhibition. In terms of antibacterial activity there is only a single report (Viladomat et al. 1996). In the course of our investigation of the alkaloid constituents of the family Amaryllidaceae, 15 alkaloids were isolated from Crinum bulbispermum [(Brum f.) Milne-Redh. & Schweick] and C. moorei Hook f. (Elgorashi et al. 1999, 2001). These were tested for antiinflammatory activity using COX-1 and COX-2 and antibacterial activity using four bacterial strains. COX-1 and COX-2 assays were performed as described by Jäger et al. (1996) and Noreen et al. (1998). Inhibition refers to reduction of prostaglandin formation in comparison to untreated samples. Enzyme solution (60μl) and sample solution (in 12.5% ethanol) were incubated at room temperature for 5min. The reaction was started by adding 20μl[l-14C] arachidonic acid (30μM). Samples were incubated for 10min
at 37°C and the reaction terminated by adding 10μl 2N HCl. Results given are the mean of three experiments. IC50 values were determined by regression analysis of the results at three different concentrations of the sample. Positive control measurements were carried out with indomethacin. Antibacterial activity was determined using the microtitre plate method for determining the minimum inhibitory concentration of plant extracts (Eloff 1998) with a final concentration of 250μg ml–1. Neomycin was used as a reference compound. Bacterial strains used were Bacillus subtilis, Staphylococus aureus, Escherichia coli and Klebsiella pneumoniae. The isolated alkaloids exhibited only very low inhibitory activity on COX-1 and COX-2 (Table 1). Furthermore, no bacterial growth inhibition was observed for any of the alkaloids under investigation at the final concentration of 250μg ml–1. Alkaloids are a group of structurally very complex, biologically, often highly active, and biogenetically diverse natural products. They have yielded a large number of medically and/or toxicologically important natural products like morphine, vinca alkaloids and galanthamine (Heinrich 2002). The antibacterial and antifungal activities of Amaryllidaceae plants including Crinum species are well known (Ghosal et al. 1985). Despite this, none of the alkaloids tested showed antibacterial and anti-inflammatory activity. In many studies the biological activity of Amaryllidaceae extracts was attributed to their non-basic constituents. For instance, the non-basic extract (hot water extract) of bulbs of Crinum delagoense yielded an active compound against BL6 mouse melanoma cells (Nair et al. 1998). The activity shown by water extracts of Clivia species, Narcissus species and Hymenocalis littoralis was
South African Journal of Botany 2003, 69(3): 448–449
Table 1: Anti-inflammatory activity of the Amaryllidaceae alkaloids (500μM) and Indomethacin on COX-1 and COX-2 Compound 8α-ethoxyprecriwelline N-desmethyl-8α-ethoxypretazettine N-desmethyl-8β-ethoxypretazettine Bulbispermine 1-O-Acetyllycorine Epivittatine Crinamine 3-O-Acetylhamayne 6-Hydroxycrinamine Epibuphanisine Powelline Crinine Cherylline Crinamidine 1-Epideacetylbowdensine Indomethacin IC50*
Inhibitory activity (%) COX-1 COX-2 11 14 32 0 24 0 23 0 4 0 17 0 9 0 17 0 21 0 4 0 4 0 16 0 0 0 10 0 3 0 62
* At a concentration of 5μM and 200μM on COX-1 and COX-2 respectively
explained by the wide occurrence of polyphenolic compounds that are soluble in water (Ieven et al. 1979). In a recent study in our laboratory on a number of Cyrtanthus and Gethyllis species, antibacterial and anti-inflammatory activities were detected in the dichloromethane extracts (non-basic extracts) rather than the methanolic extracts where alkaloids were only detected using Dragendorff’s reagent (unpublished). In conclusion, the rationale behind uses of Crinum species in traditional medicine attributed largely to their alkaloid content needs to be carefully revised and revisited since the basic constituents showed very low or lack of activity in antiinflammatory and antibacterial activity. Acknowledgements — The financial support of the University of Natal Research Fund and the National Research Foundation, Pretoria is gratefully acknowledged.
Edited by JN Eloff
449
References Campbell WE, Nair J, Gammon DW, Bastida J, Codina C, Viladomat F, Smith PJ, Albercht CF (1998) Cytotoxic and antimalarial alkaloids from Brunsvigia littoralis. Planta Medica 64: 91–93 Elgorashi EE, Drewes SE, Van Staden J (1999) Alkaloids from Crinum bulbispermum. Phytochemistry 52: 533–536 Elgorashi EE, Drewes SE, Van Staden J (2001) Alkaloids from Crinum moorei. Phytochemistry 56: 637–640 Eloff JN (1998) A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Medica 64: 711–713 Ghosal S, Saini KS, Razdan S (1985) Crinum alkaloids: their chemistry and biology. Phytochemistry 24: 2141–2156 Heinrich M (2002) Book review: Alkaloids — nature’s curse or blessing. Journal of Ethnopharmacology 83: 269–270 Hutchings A, Scott AH, Lewis G, Cunnigham AB (1996) Zulu Medicinal Plants. University of Natal Press, Pietermaritzburg. ISBN 0–86980–893–1 Ieven M, Van den Berge DA, Mertens F, Vlietinck A, Lammens E (1979) Screening of higher plants for biological activities 1. Antimicrobial activity. Planta Medica 36: 311–321 Jäger AK, Hutchings A, Van Staden J (1996) Screening Zulu medicinal plants for prostaglandin-synthesis inhibitors. Journal of Ethnopharmacology 52: 95–111 Lin LZ, Hu SJ, Chai HB, Pensuparp J, Pezutto JM, Cordell GA, Ruangrungsi N (1995) Lycorine alkaloids from Hymenocallis littoralis. Phytochemistry 40: 1295–1298 Nair JJ, Campbell WE, Gammon DW, Albrecht CF, Viladomat F, Codina C, Bastida J (1998) Alkaloids from Crinum delagoense. Phytochemistry 49: 2539–2543 Noreen Y, Ringbom T, Perera P, Danielson H, Bohlin L (1998) Development of a radiochemical cyclooxygenase-1 and -2 in vitro assay for identification of natural products as inhibitors of prostaglandin biosynthesis. Journal of Natural Products 61: 2–7 Roberts M (1990) Indigenous Healing Plants. Southern Book Publishers, Halfway House. ISBN 1–86812–317–0 Viladomat F, Almanza GR, Codina C, Bastida J, Campbell WE, Mathee S (1996) Alkaloids from Brunsvigia orientalis. Phytochemistry 43: 1379–1384
Copyright © NISC Pty Ltd
SOUTH AFRICAN JOURNAL OF BOTANY ISSN 0254–6299
Research Note
The anti-inflammatory and antibacterial activities of Amaryllidaceae alkaloids EE Elgorashi, S Zschocke and J van Staden* Research Centre for Plant Growth and Development, School of Botany and Zoology, University of Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa * Corresponding author, e-mail: [email protected] Received 7 March 2003, accepted in revised form 8 April 2003
In the course of a continued investigation of the alkaloid constituents of the family Amaryllidaceae, 15 Amaryllidaceae alkaloids isolated from Crinum bulbispermum and C. moorei were tested for anti-inflammatory activity using cyclooxygenase assay (COX-1 and COX-2) and antibacterial activity against Bacillus sub-
tilis, Staphylococus aureus, Escherichia coli and Klebsiella pneumoniae. The alkaloids showed only low inhibitory activity on COX-1 and COX-2. No bacterial growth inhibition was observed at the final concentration of 250μg ml-1 used in this study.
Crinum species (Amaryllidaceae) are used traditionally in South African folk medicine for rheumatism, backache, septic sores, abscesses and cold, kidney and bladder infections (Roberts 1990, Hutchings et al. 1996). The genus Crinum is a true representative of the family Amaryllidaceae which is the exclusive source of the Amaryllidaceae alkaloids. The biological activities associated with these alkaloids covers analgesic, central nervous system, antiviral, antitumor and antimalarial effects (Ghosal et al. 1985, Lin et al. 1995, Campbell et al. 1998). Experiments on the analgesic effects of these alkaloids, which are attributed to their resemblance to morphine and codeine skeletons, were carried out exclusively on laboratory animals (Ghosal et al. 1985). Despite the wide range of biological activities involved, there have been no reports concerning the activity of these alkaloids in terms of cyclooxygenese-1 (COX-1) and cyclooxygenese-2 (COX-2) anti-inflammatory inhibition. In terms of antibacterial activity there is only a single report (Viladomat et al. 1996). In the course of our investigation of the alkaloid constituents of the family Amaryllidaceae, 15 alkaloids were isolated from Crinum bulbispermum [(Brum f.) Milne-Redh. & Schweick] and C. moorei Hook f. (Elgorashi et al. 1999, 2001). These were tested for antiinflammatory activity using COX-1 and COX-2 and antibacterial activity using four bacterial strains. COX-1 and COX-2 assays were performed as described by Jäger et al. (1996) and Noreen et al. (1998). Inhibition refers to reduction of prostaglandin formation in comparison to untreated samples. Enzyme solution (60μl) and sample solution (in 12.5% ethanol) were incubated at room temperature for 5min. The reaction was started by adding 20μl[l-14C] arachidonic acid (30μM). Samples were incubated for 10min
at 37°C and the reaction terminated by adding 10μl 2N HCl. Results given are the mean of three experiments. IC50 values were determined by regression analysis of the results at three different concentrations of the sample. Positive control measurements were carried out with indomethacin. Antibacterial activity was determined using the microtitre plate method for determining the minimum inhibitory concentration of plant extracts (Eloff 1998) with a final concentration of 250μg ml–1. Neomycin was used as a reference compound. Bacterial strains used were Bacillus subtilis, Staphylococus aureus, Escherichia coli and Klebsiella pneumoniae. The isolated alkaloids exhibited only very low inhibitory activity on COX-1 and COX-2 (Table 1). Furthermore, no bacterial growth inhibition was observed for any of the alkaloids under investigation at the final concentration of 250μg ml–1. Alkaloids are a group of structurally very complex, biologically, often highly active, and biogenetically diverse natural products. They have yielded a large number of medically and/or toxicologically important natural products like morphine, vinca alkaloids and galanthamine (Heinrich 2002). The antibacterial and antifungal activities of Amaryllidaceae plants including Crinum species are well known (Ghosal et al. 1985). Despite this, none of the alkaloids tested showed antibacterial and anti-inflammatory activity. In many studies the biological activity of Amaryllidaceae extracts was attributed to their non-basic constituents. For instance, the non-basic extract (hot water extract) of bulbs of Crinum delagoense yielded an active compound against BL6 mouse melanoma cells (Nair et al. 1998). The activity shown by water extracts of Clivia species, Narcissus species and Hymenocalis littoralis was
South African Journal of Botany 2003, 69(3): 448–449
Table 1: Anti-inflammatory activity of the Amaryllidaceae alkaloids (500μM) and Indomethacin on COX-1 and COX-2 Compound 8α-ethoxyprecriwelline N-desmethyl-8α-ethoxypretazettine N-desmethyl-8β-ethoxypretazettine Bulbispermine 1-O-Acetyllycorine Epivittatine Crinamine 3-O-Acetylhamayne 6-Hydroxycrinamine Epibuphanisine Powelline Crinine Cherylline Crinamidine 1-Epideacetylbowdensine Indomethacin IC50*
Inhibitory activity (%) COX-1 COX-2 11 14 32 0 24 0 23 0 4 0 17 0 9 0 17 0 21 0 4 0 4 0 16 0 0 0 10 0 3 0 62
* At a concentration of 5μM and 200μM on COX-1 and COX-2 respectively
explained by the wide occurrence of polyphenolic compounds that are soluble in water (Ieven et al. 1979). In a recent study in our laboratory on a number of Cyrtanthus and Gethyllis species, antibacterial and anti-inflammatory activities were detected in the dichloromethane extracts (non-basic extracts) rather than the methanolic extracts where alkaloids were only detected using Dragendorff’s reagent (unpublished). In conclusion, the rationale behind uses of Crinum species in traditional medicine attributed largely to their alkaloid content needs to be carefully revised and revisited since the basic constituents showed very low or lack of activity in antiinflammatory and antibacterial activity. Acknowledgements — The financial support of the University of Natal Research Fund and the National Research Foundation, Pretoria is gratefully acknowledged.
Edited by JN Eloff
449
References Campbell WE, Nair J, Gammon DW, Bastida J, Codina C, Viladomat F, Smith PJ, Albercht CF (1998) Cytotoxic and antimalarial alkaloids from Brunsvigia littoralis. Planta Medica 64: 91–93 Elgorashi EE, Drewes SE, Van Staden J (1999) Alkaloids from Crinum bulbispermum. Phytochemistry 52: 533–536 Elgorashi EE, Drewes SE, Van Staden J (2001) Alkaloids from Crinum moorei. Phytochemistry 56: 637–640 Eloff JN (1998) A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Medica 64: 711–713 Ghosal S, Saini KS, Razdan S (1985) Crinum alkaloids: their chemistry and biology. Phytochemistry 24: 2141–2156 Heinrich M (2002) Book review: Alkaloids — nature’s curse or blessing. Journal of Ethnopharmacology 83: 269–270 Hutchings A, Scott AH, Lewis G, Cunnigham AB (1996) Zulu Medicinal Plants. University of Natal Press, Pietermaritzburg. ISBN 0–86980–893–1 Ieven M, Van den Berge DA, Mertens F, Vlietinck A, Lammens E (1979) Screening of higher plants for biological activities 1. Antimicrobial activity. Planta Medica 36: 311–321 Jäger AK, Hutchings A, Van Staden J (1996) Screening Zulu medicinal plants for prostaglandin-synthesis inhibitors. Journal of Ethnopharmacology 52: 95–111 Lin LZ, Hu SJ, Chai HB, Pensuparp J, Pezutto JM, Cordell GA, Ruangrungsi N (1995) Lycorine alkaloids from Hymenocallis littoralis. Phytochemistry 40: 1295–1298 Nair JJ, Campbell WE, Gammon DW, Albrecht CF, Viladomat F, Codina C, Bastida J (1998) Alkaloids from Crinum delagoense. Phytochemistry 49: 2539–2543 Noreen Y, Ringbom T, Perera P, Danielson H, Bohlin L (1998) Development of a radiochemical cyclooxygenase-1 and -2 in vitro assay for identification of natural products as inhibitors of prostaglandin biosynthesis. Journal of Natural Products 61: 2–7 Roberts M (1990) Indigenous Healing Plants. Southern Book Publishers, Halfway House. ISBN 1–86812–317–0 Viladomat F, Almanza GR, Codina C, Bastida J, Campbell WE, Mathee S (1996) Alkaloids from Brunsvigia orientalis. Phytochemistry 43: 1379–1384