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The Effect of Cultivation and Plant Age on the Pharmacological Activity of Merwilla Natalensis Bulbs

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The Effect of Cultivation and Plant Age on the Pharmacological Activity of Merwilla Natalensis Bulbs South African Journal of Botany 2005, 71(2): 191–196 Copyright © NISC Pty Ltd Printed in South Africa — All rights reserved SOUTH AFRICAN JOURNAL OF BOTANY ISSN 1727–9321 The effect of cultivation and plant age on the pharmacological activity of Merwilla natalensis bulbs SG Sparg, AK Jäger and J van Staden* Research Centre for Plant Growth and Development, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa * Corresponding author, e-mail: [email protected] Received 5 March 2004, accepted in revised form 27 August 2004 Merwilla natalensis bulbs were cultivated at two thesis by COX-1, with activity decreasing as the bulbs different sites under different treatments. Bulbs were matured. The cultivation treatments had a significant harvested every six months for a period of two years effect on the antihelmintic activity of bulbs cultivated at and were tested for antibacterial, anti-inflammatory and the Fort Hare site. Results suggest that irrigation might anthelmintic activity. The cultivation treatments had no increase the antihelmintic activity of the bulbs if culti- significant effect (P ≤ 0.05) on neither the antibacterial vated in areas of low rainfall. The age of the bulbs at activity, nor the anti-inflammatory activity. However, the both sites had a significant effect on the antihelmintic age of the bulbs had a significant effect against the test activity, with activity increasing with plant maturity. bacteria and on the inhibition of prostaglandin syn- Introduction Merwilla natalensis (Planchon) Speta (synonym Scilla varied when the plants were grown in different geographical natalensis) is ranked as one of the more commonly-sold regions. The concentrations of compounds also varied medicinal plants at many of the informal medicinal markets seasonally. in South Africa (Cunningham 1988, Mander 1997). The Very little research has been done on seasonal variation bulbs are used for a wide range of ailments such as gastro- in pharmacological activity or the effect that plant growth intestinal disorders, sprains and fractures, boils and sores, (age) has on the pharmacological activity. In a study by Light internal tumours and even gynaecological disorders (Watt et al. (2002) the effect of senescence in Siphonochilus and Breyer-Brandwijk 1962, Hutchings 1989, Van Wyk et al. aethiopicus on its pharmacological activity was investigated. 1997). In a previous study (Sparg et al. 2002), bulb extracts Taylor and Van Staden (2001) investigated the effect of age, of M. natalensis were screened for pharmacological activity season and growth conditions on the anti-inflammatory in vitro. The results showed M. natalensis to have anti- activity of Eucomis autumnalis plant extracts. Significant inflammatory, anthelmintic, antischistosomal and antibac- differences in activity were found between plants harvested terial activity, although the latter was not very significant. before and after the growing season. The effect of cold According to Bernath (1986), ecological factors have storage on the anti-inflammatory activity of E. autumnalis decisive impacts on the chemical composition of plants. autumnalis also showed differences in activity (Taylor and Therefore, the variability in natural product content and Van Staden 2002). composition are functions of both growth and development, In this study, the effects of different cultivation treatments and play a role in determining the optimal harvest time of and age of the bulbs on the pharmacological activity of M. certain species. Prance (1994) suggested that the chemistry natalensis was investigated. within individual plant species varies according to the ecology, the soil and the climate. Yaniv and Palevitch (1982) Materials and Methods stated that, depending on the species, the quantity of secondary metabolites increases, decreases, or remains Cultivation and harvesting constant when under water stress. Seasonal variation of alkaloids in Crinum moorei was studied by Elgorashi et al. Two sites in South Africa were selected to cultivate tissue- (2002). No clear trend in seasonal variation was noted, with cultured M. natalensis bulbs as a crop on a small-scale alkaloid yields varying from organ to organ as well as from basis. The two sites selected were the smallholding H2A season to season. Dong et al. (2003) showed that the active Botanicals (Hillcrest, KwaZulu-Natal) and premises at the constituents of Panax notoginseng, including saponins, University of Fort Hare (Alice, Eastern Cape). 192 Sparg, Jäger and Van Staden Cultivation at each site was carried out on three plots. The eight replicates for each treatment for each harvest. first plot was the control treatment, which received no Indomethacin (20µM) was used as a positive control. fertiliser, additives or supplementary irrigation. The plants were grown in tilled topsoil and relied on rain for their water Anthelmintic bioassay source. The second plot was only fertilised and not irrigated (referred to as the fertiliser treatment). In the case of the H2A The nematode Caenorhabditis elegans var. Bristol (N2) was Botanicals site, the fertiliser used for the second treatment cultured on nematode growth agar seeded with E. coli was a mixture of composted horse and chicken manure at a according to the method of Brenner (1974). The anthelmintic ratio of 2:1, whereas inorganic fertiliser, NPK 232(22) assay used was that of Rasoanaivo and Ratsimamanga- (Nitrochem), was used for the second treatment at the Fort Urverg (1993) as described by McGaw et al. (2000). Hare site. The third plot was irrigated and fertilised (referred Methanol extracts were in ethanol and tested at a final to as the irrigation treatment). The plants were planted concentration of 1mg m–1 for anthelmintic testing. Four approximately 30cm apart in rows with 50cm spacing extracts per treatment per harvest were tested. One between rows. replicate of each of the extracts was performed. The assay Bulbs were harvested every six months from each of the was repeated a second time giving a total of eight replicates three treatments (20 bulbs per treatment per harvest period) for each treatment for each harvest. Levamisole (5µg ml–1 ) over a period of two years. The harvested bulbs were dried and a solvent blank were used as positive and negative at 50°C for 72h. Bulbs were divided into groups of fives to controls respectively. Using a dissecting microscope, the form four sample groups. The five bulbs were ground percentage worm mortality was estimated and compared together and stored in airtight containers in the dark at with the controls. ±25°C (room temperature). This was done for each of the treatments for each harvest from both the H2A Botanicals Statistical analysis and Fort Hare sites. The results from each assay were analysed statistically Extract preparations using analysis of variance (ANOVA) to determine interactions and the effect the treatments and bulb age had Methanol and dichloromethane extracts were prepared for on the pharmacological activity. Comparisons were made each of the four samples for each treatment of each harvest. using Fisher’s pairwise comparisons at a 5% level of Two grams of dried material were extracted with 20ml significance. extraction solvent. The material was allowed to extract for 24h with constant agitation. The extracts were filtered under Results and Discussion vacuum through Whatman No. 1 filter paper discs using a Büchner funnel. The filtered extracts were taken to dryness Minimum inhibitory concentrations (MIC) were calculated for and stored in sealed pill vials in the dark at 5°C. an average of eight replicates for each treatment of each harvest. The lower the MIC value, the higher the Antibacterial bioassay antibacterial activity. The results from the antibacterial screening of bulbs cultivated at the H2A Botanicals site are The microdilution antibacterial assay as described by Eloff summarised in Table 1. No significant differences (P ≤ 0.05) (1998) was used to screen the extracts for antibacterial in activity between treatments against both the Gram- activity. Methanol extracts were dissolved in ethanol to positive bacteria were observed. Twelve month-old bulbs concentrations of 50mg ml–1 for use in the assay. Four harvested from the fertiliser treatment gave the highest extracts per treatment per harvest were tested. The assay activity (3.9mg ml–1) against B. subtilis. Bulbs harvested at was repeated a second time, giving a total of eight replicates six months from the fertiliser treatment had the highest for each treatment for each harvest. The extracts were activity (2.34mg ml–1) against S. aureus. Analysis of the tested against two Gram-positive bacteria, Bacillus subtilis results showed no significant interactions between the (ATCC 6051) and Staphylococcus aureus (ATCC 12600), treatments and bulb age on the antibacterial activity against and two Gram-negative bacteria, Escherichia coli (ATCC both B. subtilis and S. aureus. Both the treatments and the 11775) and Klebsiella pneumoniae (ATCC 13883). bulb age had no significant effect (P ≤ 0.05) on the Neomycin and solvent blanks were used as positive and antibacterial activity when tested against B. subtilis. negative controls respectively. However, the bulb age had a significant effect on the antibacterial activity (P = 0.002) against S. aureus, with the Anti-inflammatory bioassay antibacterial activity decreasing as the plants matured. The six month-old bulbs had significantly higher antibacterial Extracts were screened using the COX-1
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