The effect of Phela, a traditional medicine on the immune system of a rat model
| dc.contributor.advisor | Walubo, A. | |
| dc.contributor.author | Lekhooa, 'Makhotso Rose | |
| dc.contributor.other | Du Plessis, J. B. | |
| dc.contributor.other | Matsabisa, M. G. | |
| dc.date.accessioned | 2015-09-16T10:32:22Z | |
| dc.date.available | 2015-09-16T10:32:22Z | |
| dc.date.copyright | 2010-08 | |
| dc.date.issued | 2010-08 | |
| dc.date.submitted | 2010-08 | |
| dc.description.abstract | English: Phela is a herbal traditional medicine product prepared using well defined parts of four African medicinal plants. The aim of the study was to determine the mechanism of action by which phela boosted the immune system of a rat model. Unfortunately, subsequent literature research revealed that very little is known about phela. As such chromatographic methods were developed by which to identify phela and for quality control purposes. Of note, the developed methods complemented each other; they were compiled into a comprehensive method for phela fingerprinting. It involved sample extraction of the phela by either acidic extraction or a simple “saltingout” method, followed by Thin Layer Chromatography (TLC), and/or preparative Column Chromatography (CC) that were supported by High Performance Liquid Chromatography with UV-detector (HPLC_UV), HPLC with fluorescence detector (HPLC_FL), HPLC with photo diode array detector (HPLC_PDA) and Gas Chromatography-Mass selective detector (GC_MSD) spectrometry. The method was successfully used to differentiate phela from another herbal product made from Hypericum perforatum (St John’s wort) illustrating its high potential for application to other herbal medicines in development. Thereafter a HPLC_UV method for monitoring phela in plasma was developed and validated. However, due to its pitfalls, a HPLC_FL method was developed as well. The HPLC_UV method had a linear regression equation of y = 0.02x – 0.59, correlation coefficient of r2 = 0.9983 and accuracy within 15 %. For HPLC_FL three marker peaks were selected and standardized by the retention time. The developed HPLC_UV and HPLC_FL methods were tested by analysing blood collected from rats treated with phela after 1, 2, 4, 6 and 8 hours respectively. Unfortunately, analysis by HPLC_UV method had no peaks whereas, during HPLC_FL method analysis, a new peak was observed at 9.2 minutes. The peak was depicted as a metabolite for phela and was then utilized as the plasma marker. Since the metabolite is unknown and therefore no standard exists by which to express its plasma concentration in conventional units, the changes in peak area per unit volume (L) of plasma (peak-area/L) were used to derive the appropriate pharmacokinetic parameters hence peak-kinetics. The predictive parameters show that concentration of the metabolite at steady state would be 48 peak-area/ml, hence no accumulation of the drug. The final part of the study was undertaken to understand the mechanism of action of phela using a rat model by observing for changes in the levels of TH1 and TH2 cytokines. Rats were divided into six groups. The first group was not treated with anything. The four groups were treated daily with either normal-saline, orally (control); cyclosporine in olive oil, subcutaneously (negative control); Phela, orally (test-group 1) and phela+cyclosporine (test-group 2). The last group was inoculated once with influenza vaccine. The treatment period was 14 days and in each group rats were sacrificed after 7 and 14 days. Haematology tests, biochemistry tests, and cyclosporine level analysis were done. Serum cytokine analysis for TH1 (IL-2, IFN-γ and TNF-α) and TH2 (IL-4 and IL-10) cytokines was measured by ELISA. On days 7 and 14, the concentrations of TH1 cytokines in the Phela-only treated group were similar to the control. However, the TH1 cytokines were higher in the Phela+cyclosporine-A treated group than in the cyclosporine-A group, and cyclosporine- A concentrations were similar in both groups. These results show that Phela did not affect TH1 cytokines of a normal immune system but stimulated them when the immune system was suppressed by cyclosporine-A. This implies that Phela is a cyclosporine-A antagonist that may stimulate a compromised immune system. In conclusion, Phela is an immune stimulant. | en_ZA |
| dc.description.abstract | Afrikaans: Phela is ‘n kruie tradisionele medisinale produk voorberei uit goed-gedefineerde dele van vier Afrika medisinale plante. Die doel van die studie was om die meganisme van werking te bepaal waardeur phela die immuunsisteem versterk in ‘n rotmodel. Ongelukkig toon ‘n literatuur soektog dat daar baie min bekend is omtrent phela. As sulks is chromatografiese metodes ontwikkel vir die identifikasie van phela en vir kwaliteitsbeheer-doeleindes. Van belang is die feit dat die ontwikkelde metodes mekaar komplementeer; hulle is saamgevoeg in ‘n omvattende metode vir die bepaling van ‘n phela vingerafdruk. Dit behels monster ekstraksie van phela deur middel van ‘n suurekstraksie of ‘n eenvoudige “uit-soutings” metode, gevolg deur Dunlaag Chromatografie (TLC), en/of voorbereidende kolom chromatografie (CC) ondersteun deur Hoë verrigting Vloeistof Chromatografie met UV-detektor (HPLC_UV); HPLC met fluoresensie detektor (HPLC_FL); HPLC met foto-diode-rangskikking detektor (HPLC_PDA), en Gas Chromatografie-Massa selektiewe detektor (GC_MSD) spektrometrie. Die metode is suksesvol gebruik om phela te onderskei van ‘n ander kruieproduk gemaak van Hypericum perforatum (St John’s wort). Dit illustreer die hoë potensiaal vir applikasie in ander kruiemedisynes in ontwikkeling. Daarna is ‘n HPLC_UV metode ontwikkel en gevalideer vir die monitering van phela in plasma. Weens die inherente probleme van die metode is ‘n HPLC_FL metode ook ontwikkel. Die HPLC_UV metode het ‘n liniêre regressievergelyking van y = 0.02x – 0.59, korrelasiekoëffisiënt van r2 = 0.9983 en akkuraatheid binne 15 %. Vir HPLC_FL is drie merker pieke geselekteer en gestandardiseer volgens die retensietyd. Die ontwikkelde HPLC_UV en HPLC_FL metodes is getoets deur die analise van bloed versamel vanaf rotte behandel met phela na onderskeidelik 1, 2, 4, 6 en 8 ure. Ongelukkig was daar geen pieke tydens analise met die HPLC_UV metode nie, terwyl ‘n nuwe piek waargeneem is teen 9.2 minute met die HPLC_FL metode. Die piek is geïnterpreteer as ‘n metaboliet vir phela en is toe gebruik as die plasmamerker. Aangesien die metaboliet onbekend is en daar dus geen standaard is waarvolgens die plasma konsentrasie in konvensionele eenhede uitgedruk kan word nie, is die veranderinge in piekarea per eenheid volume (L) van plasma (piek-area/L) gebruik om die toepaslike farmakokinetiese parameters te bepaal, dus piek-kinetika. Die voorspellende parameters wys dat die konsentrasie van die metaboliet by gelykvlak 48 piek-area/ml behoort te wees en dat daar, dus geen ophoping van die middle is nie. Die finale deel van die studie is onderneem om die meganisme van werking van phela te verstaan. ‘n Rotmodel is gebruik om die verandering in die vlakke van TH1 en TH2 sitokiene waar te neem. Die rotte is in ses groepe verdeel. Die eerste groep is nie behandel nie. Vier groepe is daagliks behandel met òf normale soutoplossing, oraal (kontrole); siklosporien in olyfolie subkutaan (negatiewe kontrole), Phela, oraal (toetsgroep 1) en phela+siklosporien-A (toetsgroep 2). Die laaste groep is een maal ingeënt met influenza vaksien. Die behandelingsperiode was 14 dae en in elke groep is rotte geslag na 7 en na 14 dae. Hematologie-toetse, biochemie-toetse, en siklosporienvlak-bepalings is gedoen. Serum sitokien analise vir TH1 (IL-2, IFN-γ en TNF-α) en TH2 (IL-4 en IL-10) sitokiene is gemeet met ELISA. Op dag 7 en dag 14 was die konsentrasies van TH1 sitokiene in die groep war slegs met Phela behandel is soortgelyk aan die kontrole. Die TH1 sitokiene was egter hoër in die Phela+siklosporien-A behandelde groep as in met die siklosporien-A groep, en siklosporien-A konsentrasies was soortgelyk in beide groepe. Hierdie resultate wys dat phela nie TH1 sitokiene in ‘n normale immuunsisteem affekteer nie, maar die immuunsisteem stimuleer wanneer dit onderdruk word deur siklosporien-A. Dit impliseer dat phela ‘n siklosporien-A antagonis is wat ‘n ingekorte immuunsisteem stimuleer. Ter opsomming kan dus aanvaar word dat phela ‘n immuunstimulant is. Ongelukkig was daar geen pieke tydens analise met die HPLC_UV metode nie, terwyl ‘n nuwe piek waargeneem is teen 9.2 minute met die HPLC_FL metode. Die piek is geïnterpreteer as ‘n metaboliet vir phela en is toe gebruik as die plasmamerker. Aangesien die metaboliet onbekend is en daar dus geen standaard is waarvolgens die plasma konsentrasie in konvensionele eenhede uitgedruk kan word nie, is die veranderinge in piekarea per eenheid volume (L) van plasma (piek-area/L) gebruik om die toepaslike farmakokinetiese parameters te bepaal, dus piek-kinetika. Die voorspellende parameters wys dat die konsentrasie van die metaboliet by gelykvlak 48 piek-area/ml behoort te wees en dat daar, dus geen ophoping van die middle is nie. Die finale deel van die studie is onderneem om die meganisme van werking van phela te verstaan. ‘n Rotmodel is gebruik om die verandering in die vlakke van TH1 en TH2 sitokiene waar te neem. Die rotte is in ses groepe verdeel. Die eerste groep is nie behandel nie. Vier groepe is daagliks behandel met òf normale soutoplossing, oraal (kontrole); siklosporien in olyfolie subkutaan (negatiewe kontrole), Phela, oraal (toetsgroep 1) en phela+siklosporien-A (toetsgroep 2). Die laaste groep is een maal ingeënt met influenza vaksien. Die behandelingsperiode was 14 dae en in elke groep is rotte geslag na 7 en na 14 dae. Hematologie-toetse, biochemie-toetse, en siklosporienvlak-bepalings is gedoen. Serum sitokien analise vir TH1 (IL-2, IFN-γ en TNF-α) en TH2 (IL-4 en IL-10) sitokiene is gemeet met ELISA. Op dag 7 en dag 14 was die konsentrasies van TH1 sitokiene in die groep war slegs met Phela behandel is soortgelyk aan die kontrole. Die TH1 sitokiene was egter hoër in die Phela+siklosporien-A behandelde groep as in met die siklosporien-A groep, en siklosporien-A konsentrasies was soortgelyk in beide groepe. Hierdie resultate wys dat phela nie TH1 sitokiene in ‘n normale immuunsisteem affekteer nie, maar die immuunsisteem stimuleer wanneer dit onderdruk word deur siklosporien-A. Dit impliseer dat phela ‘n siklosporien-A antagonis is wat ‘n ingekorte immuunsisteem stimuleer. Ter opsomming kan dus aanvaar word dat phela ‘n immuunstimulant is. | af |
| dc.identifier.uri | http://hdl.handle.net/11660/1233 | |
| dc.language.iso | en | en_ZA |
| dc.publisher | University of the Free State | en_ZA |
| dc.rights.holder | University of the Free State | en_ZA |
| dc.subject | Drugs -- Testing | en_ZA |
| dc.subject | Pharmacology, Experimental | en_ZA |
| dc.subject | Traditional medicine | en_ZA |
| dc.subject | HIV infections -- South Africa | en_ZA |
| dc.subject | AIDS (Disease) -- South Africa | en_ZA |
| dc.subject | Rats as laboratory animals | en_ZA |
| dc.subject | Traditional-herbal-medicine-products | en_ZA |
| dc.subject | Pharmacokinetics | en_ZA |
| dc.subject | Phela | en_ZA |
| dc.subject | Dissertation (M.Med.Sc. (Pharmacology))--University of the Free State, 2010 | en_US |
| dc.subject | Cyclosporine-Aantagonist | en_ZA |
| dc.subject | Cytokines | en_ZA |
| dc.subject | Peak-kinetics | en_ZA |
| dc.subject | Immune-stimulant | en_ZA |
| dc.subject | Chromatographic-fingerprinting | en_ZA |
| dc.title | The effect of Phela, a traditional medicine on the immune system of a rat model | en_ZA |
| dc.type | Dissertation | en_ZA |