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Seasonal Variation in Uterotonic Activity Of SEASONAL VARIATION IN depending on where the plant was harvested.' Temporal variation is another possible source of variability in chemical UTEROTONIC ACTIVITY OF constituents and biological activity of plants. This is a well­ RHOICISSUS TRIDENTATA documented phenomenon.'·' The expression of genes coding for chemical constituents of plants is under the control of EXTRACTS various factors, such as the developmental stage of the organism, duration and intensity of light, nutrient supply, L C Katsoulis, D J H Veale, I Havlik triggering of internal signals and sequential expression of genes coding for secondary metabolites.' All the above factors vary temporally, which in turn causes seasonal fluctuations in Background. Rhoicissus tridentata lignotubers are widely used the levels of both primary and secondary metabolites within in southern African traditional pregnancy-related remedies. plant tissues. Objectives. To determine the seasonal variation in contractile The aim of this study was to determine whether the activity of extracts from different parts of the plant. contractile activity of R. tridentata varies seasonally. Methods. Isolated rat uterus tissue was used to compare the contractile activity of crude aqueous extracts of R. tridentata METHODS made from plant material harvested every 3 months over a R. tridentata subsp. cuneifolia plant material was collected from period of 2 years. Suikerbosrand Nature Reserve, 60 km south of Johannesburg. Results and conclusions. The activity of the plant extracts Three plants were harvested every 3 months from May 1996 to from plants harvested in summer and autumn were 4 - 5- April 1998. The identity of the plants was validated by fold higher than extracts from plants harvested in winter or L Katsoulis, and one voucher specimen for each individual spring. The tubers stimulated the greatest degree of contrac­ plant harvested was lodged in the C E Moss Herbarium tions, followed by the stems, roots and leaves. These results (voucher specimen numbers of the three plants: 086928, 082929 highlight the need to standardise the timing of harvesting and 082930). R. tridentata. Milled plant material was boiled for an hour, settled, and S Afr Med J 2002; 92: 375-377. the supernatant lyophilised. Sections of uterine tissue obtained from the central portion of the uterine horns of oestrogenised virgin Sprague-Dawley rats were mounted in 50 ml organ Decoctions of the roots and lignotubers of Rhoicissus tridentata baths containing aerated (5% C02 in 0 2) Tyrode solution. The (subsp. cuneifolia) are used by southern African women in uterine tissue was maintained at 26°C to decrease spontaneous ethnic herbal remedies for delayed childbirth and to facilitate contractility. The organs were rinsed repeatedly while being childbirth.'" R. tridentata decoctions are prepared by boiling allowed to return to baseline, before any investigations. dried milled roots or lignotubers for approximately an hour. Control challenges with acetylcholine were done between each This is done by traditional healers who sell the bottled test challenge. The activity of the extracts was tested by decoctions. The plant is widely distributed throughout the incubating the tissues with 1.3 mg/ml of the various plant eastern portion of southern Africa,' and is found in most extracts for 5 minutes before adding acetylcholine traditional healer vending stores around Johannesburg. cumulatively. This was done to determine whether the plant Pharmacological investigations using isolated rat uterus and extracts alter the organ's response to acetylcholine. Isotonic ileum suggest that the contractile response to hot water plant contractions were measured against 1 g resistance and extracts may be mediated by muscarinic receptors and the expressed relative to the maximal response of the same tissues stimulation of prostaglandin synthesis.' While doing to acetylcholine. More details of the methods are given in the preliminary work on the pharmacology of the plant extracts it study by Veale et al.' became evident that there was a high degree of variability in the activity of extracts from different plant samples. The pharmacological response to R. tridentata has been shown RESULTS AND DISCUSSION to vary geographically. The plant extracts acted either Fig. 1 illustrates how the different parts of the plants harvested agonistically or antagonistically on muscarinic receptors in winter and spring all yielded aqueous extracts with low levels of contractile activity, whereas the extracts from different Department of Experimental and Clinical Pharmacology, University of the Witwatersrand, Johannesburg parts of the plants harvested during summer and autumn were L C Katsoulis, PhD considerably more active. During summer the tubers yielded D J H Veale, PhD the most active aqueous extracts, followed by the stems, roots I Havlik, PhD and leaves. The autumn plants exhibited a similar pattern of ORIGINAL ARTICLES Spring Summer ...0 100 100 Ill ~ Ill 80 80 0 c: c.= en o 60 60 Ql ..c: ... u - :;. 40 40 • Tubers (N=6) ~CJ • Tubers (N = 6) • Stems (N= 13) ')( ~ 20 • Stems (N= 1s) 20 v Leaves (N = 14) ~ o Roots (N= 14) o Roots (N = 14) 0 0 + + + Acetylcholine (N= 16) + Acetylcholine (N= 16) Rh -8.5 -7.5 -6.5 -5.5 -4.5 -3.5 -2.5 Rh -8.5 -7.5 -6.5 -5.5 -4.5 -3.5 -2.5 Log acetylcholine concentration (M) Log acetylcholine concentration (M) 100 Autumn 100 Winter ....0 Cl) Cl) 80 80 ;I/ c: QI 0 c: c..:.: 60 60 Ul 0 Cl) .s::. ... I..) c; :;. 40 • Tubers (N= 12) 40 E Gi • Sfems (N= 19) I..) "' Stems (N= 17) .iE ")( 20 I 20 cu cu v Leaves (N = 22) 3'!/ o Roots (N= 1s) .--r-··· E o Roots (N= 13) • Tubers (N= 18) + --+----t-··-1'·-· 0 + + --+---+-·--!'- ~ 0 + + Acetylcholine (N= 16) " + Acetylcholine (N= 16) Rh -8.5 -7.5 -6.5 -5.5 -4.5 -3.5 -2.5 Rh -8.5 -7.5 -6.5 -5.5 -4.5 -3.5 -2.5 Log acetylcholine concentration (M) Log acetylcholine concentration (M) Fig. 1. Dose response curves of isolated rat uterus when pre-treated with 1.3 mg/ml aqueous extracts of all parts of Rhoicissus tridentata subsp. cuneifolia harvested during spring before the cumulative addition of acetylcholine. Each point represents the mean with the vertical bars representing the standard error of the mean (SEM). variation except that the roots were the least active part. The The root and stem extracts showed a similar pattern in autumn and summer tubers were four and five times more seasonal variation in uterotonic activity to that of the tuber active than the winter and spring extracts. The stems displayed extracts. That is, the activity of the extracts started increasing similar seasonal variability. when plants were harvested after the first rains. The activity Statistical differences between the direct contractile activity increased throughout the growing season (or rainy season), of the plants, shown by 'Rh' on the dose response curves, are reaching a peak in autumn. The activity of the extracts then fell given in Table I. · as the plants lost their leaves and became dormant during winter. The rise in activity as the rains started could have been All parts of the plant harvested in summer and autumn caused by the improved uptake of minerals from the soil were able to stimulate contractions of the isolated uterus following the mineralisation flush after the first rains, which directly. Traditional healers usually use the lignotubers, which usually leads to a sharp rise in the nitrogen content within the yield the most uterotonically active extracts. The activity of the plant.10 tuber extracts is highest during the growing season and decreases during the seasons when the plant is dormant. Variability in the contractile activity of the extracts could have been caused by: (i) altered concentrations of the active principle(s); (ii) a change in the quality or nature of the active Table I. Statistical differences .between the contractile activity of principle(s); or (iii) changes in the production of compounds extracts from different parts of Rhoicissus plants harvested during different seasons such as saponins which may have altered extraction of the Stems pharmacologically active component(s). Tubers ,. Roots Spring v.. summ.er Spring ~. sµmmer * Spring v. summert Spring v. autumn t Summer v. autumn* RELEVANCE TO CONSERVATION Summer v. win~r t Autumn v. wintert Aµ\ufiU1 v. winter* The stem extracts yielded a similar response to the most active part, namely the lignotubers. This is significant from an environmental perspective, as harvesting stems for the trade in traditional remedies would have a much lower impact on the May 2002, Vol. 92, No. 5 SAM] ORIGINAL ARTICLES plant populations of R. tridentata than harvesting the PATHOGENIC MECHANISM OF lignotubers and roots. With current use of the lignotubers, the plant has to be uprooted for lignotubers to be harvested. TYPE 2 DIABETES IN GHANAIANS - THE IMPORTANCE OF BETA CELL CLINICAL RELEVANCE SECRETION, INSULIN SENSITIVITY Variability in the uterine response to the decoctions from AND GLUCOSE EFFECTIVENESS different seasons highlights a severe flaw in the use of traditional herbal remedies. Collectors and vendors have no Albert G B Amoah, Samuel K Owusu, Dara P Schuster, way of determining the chemical composition of the plant Kwame Osei material harvested. Eight traditional healers in and around Johannesburg were questioned on whether they were aware of any seasonal variation in the efficacy of R. tridentata. None of Objective. To assess insulin sensitivity and beta cell secretion them seemed to be aware of the concept. in indigenous Ghanaian subjects with a spectrum of glucose Traditional healers were also unaware of any toxic effects of intolerance. the plant (personal communication) despite reports implicating 11 12 Research and methods. We evaluated beta cell secretion, insulin the plant in fatal poisonings. • This suggests that only certain sensitivity (Si) and glucose effectiveness (Sg) in three groups: plant samples may be toxic.
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