Variation in chemical composition of Harpagophytum species as function of age and locality

Abstract

English: Dried tubers, growing on secondary roots of Harpagophytum procumbens subsp. procumbens are widely used as an analgesic and anti-inflammatory medicine against arthritis and other chronic pain conditions. Tubers are harvested in large quantities from South Africa, Botswana and Namibia. The dried material is exported to European countries. This industry makes a big contribution to poverty alleviation in rural areas. The large quantities exported (about 600 tons per annum), has raised concerns about the sustainability of wild harvesting. Apart from threatening the plant with extinction, overexploitation of the resource will also threaten the economic survival of rural communities. This thesis aims to study the variation in chemical composition between samples from different regions, species, harvesting regimes and age of tubers. Only tubers from secondary roots are removed and a large percentage of harvested plants survive. This thesis is part of a bigger study of which the aim is to determine the optimum wild harvesting regime resulting in the best yield of the active ingredient in a sustainable harvesting industry. Plant material (tubers) was collected from December 2007 to February 2008 in the North West Province, Limpopo Province, Namibia, Caprivi in Zimbabwe. In the North West Province, plant materials were collected in 6 different areas and/or farms: Cassel, Ganyesa, Moswana, Molopo Nature Reserve, Terra firma and Lafras. Samples from Cassel represents tubers from the same plants with different inter harvest periods (one to five years). The freshly collected tubers were sliced, sun dried and analysed with HPLC-UV for six different analytes (harpagide, harpagoside, 8-p-coumaroylharpagide, verbascoside, isoverbascoside and 6-acetylacteoside). These analytes can be divided into two structurally related groups. An analytical method was developed to quantify the six analytes routinely. The method is based on water and methanol as eluent and a reverse phase analytical column. A stepwise isocratic procedure ( 3% MeOH for 1 min, 50% MeOH for 20 min, column cleanup with 95% MeOH for 5 min and regeneration with 3% MeOH for 5 min) was found to be the best for our purposes. The method was validated for specificity, accuracy, precision, robustness and linearity. Internal standards were used for calibration. The data from more than one thousand analyzed samples were statistically processed using StatSoft, inc. (2008), STATISTICA, version 8.0. to answer the following questions: 1. Are there meaningful differences in chemical composition between populations from the five different inter harvest periods (1 to 5 years) at Cassel in the North West Province (Are there meaningful differences in chemical composition of tubers that are one, two, three, four or five years old?). 2. Are there meaningful differences in chemical composition between the six populations from the North West Province (Cassel, Ganayesa, Moswana, Terra Firma, Lafras and Molopo Nature Reserve)? 3 Are there meaningful differences in the chemical composition of populations from North West Province and Namibia? 4. Are there meaningful differences in the chemical composition of populations from North West Province/ Namibia (H. procumbens subsp. procumbens, from the Northern Province (H. zeyheri subsp. zeyheri) and from Zimbabwe (Victoria Falls) (H. zeyheri subsp. sublobatum)? Factor analysis of the six variables (harpagide, harpagoside, 8-p-coumaroylharpagide, verbascoside, isoverbascoside and 6-acetylacteoside) yielded two, three or four factors depending on the level of degree of variance that we required. Multivariate and univariate analysis (MANOVA and ANOVA) of normal and transformed data indicated highly significant differences. Levine’s test was used to test homogeneity of variances. In correlation analysis all six variables were used because the factors did match clusters based on the chemical structure of the six variables. In all four experiments significant variations were observed and described. Cluster analysis, using scatter plots of three factors (factor 1: verbascoside, isoverbascoside and 6-acetylacteoside, factor 2: harpagide and factor 3: 8-p-coumaroylharpagide) identified three distinct populations. These three populations are from three different geographical regions and correspond with the corresponding taxonomic classification of the different populations. (H. zeyheri subsp. zeyheri from the Limpopo Province, H. zeyheri subsp. sublobatum from Zimbabwe (Victoria Falls) and Captivi and H. procumbens subsp. procumbens from the North West province and Namibia.

Afrikaans: Gedroogte sekondêre wortels van Harpagophytum procumbens subsp. procumbens (Burch.) de Candolle ex Meissner word algemeen vir medisinale gebruik geneem vir pynverligtende en anti-inflamatoriese werking teen artitis en ander chroniese pyn toestande. Die plant groei wild in veral Suid Afrika, Botswana en Namibïe waar die oes en verkope hiervan ’n groot bydrae maak tot armoede verligting in arm afgeleë gebiede. Na raming word tot 600 ton gedroogte wortels per jaar na Europa uitgevoer en ontstaan die vrese dat hierdie wild groeiende plant se volhoubare ontginning bedreig word. Nie alleen kan oorontginning die plant uitwis nie, maar dit kan die ekonomiese oorlewing van arm gemeenskappe bedreig wat hierop berus as bron van inkomste. Die doel van hierdie projek was om die chemiese samestelling te ontleed van a) wortels uit verskillende streke, b) wortels van verskillende Harpagophytum spesies, c) die gereeldheid van oesting en d) plant ouderdom. Hierdie tesis vorm deel van ’n groter projek om die oes kondisies te bepaal wat sal lei tot die produksie van wortels met verhoogde vlakke van aktiewe komponente met medisinale waarde, met ’n oog op volhoubare verbouing van die plant. Plant materiaal is tussen Desember 2007 en Februarie 2008 versamel in die Noord Wes Provinsie, Limpopo Provinsie, Namibia, Caprivi en Zimbabwe. Die plante is oopgegrawe en slegs die sywortels is versigtig geoes om nie die voortbestaan van die plant te benadeel nie. Die vesamelde wortels is verwerk en met HPLC geanaliseer en die vlakke van ses verskillende analiete (harpagied, harpagosied, 8-p-rumaroiehylharpagied, verbaskosied, isoverbaskosied en 6-asetielakteosied) is gekwantifiseer. Meer as ’n duisend datapunte is statisties ontleed om te bepaal of daar betekenisvolle verskille is in die chemiese samestelling van sywortels is wat: van een tot vyf jaar oud is vanaf dieselfde lokaliteit? vanuit ses verskillends populasies in die Noord Wes Provinsie? vaniut die Noord Wes Provinsie en Namibia? vanuit die Noord Wes Provinsie/Namibie, die Limpopo Provinsie en Zimbabwe? Die rekenaar program, Statistica, is gebruik vir die analisering van die data en het ingesluit die faktor analise van die ses veranderlikes (die ses geanaliseerde analiete) wat twee, drie en vier faktore onderskeidelik opgelewer het. Multivariate en univariate analises van ongetransformeerde en getransformeerde data het hoogs betekenisvolle verskille uitgewys, terwyl korrelasie analises betekenisvolle variasies uitgewys het. Kluster analises het drie diskrete populasies uitgewys wat korreleer met die diskrete geografiese areas waar materiaal versamel is asook die verskillende spesies wat in hierdie areas aangetref word.