Development of a model to characterize the effect of Phela on selected immune markers in immune-suppressed rats
Lekhooa, 'Makhotso Rose
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The therapeutic potential of several plant species and necessity for scientific validation of the use of plant derived medicines has prompted interest in field of traditional medicines. According to the WHO, in Africa alone, up to 80% of the population use herbal medicines to meet their primary health care needs and most of them have not been scientifically tested. Understanding the mechanism of action of herbal medicines is necessary for their proper use with regard to indications and limitations. One of South African traditional herbal medicines, Phela is currently being developed for use in immune compromised patients; hence there is a need to establish its mechanism of immunomodulation. Unfortunately, there is no appropriate animal model for the testing of immune-boosters. The current models involve either in vitro or ex vivo models. Furthermore, an ideal model would be a disease specific model, but this would not tell much about the mechanism of action, and would call for testing of every product in each disease model. As such, based on the understanding of the model of immune response in particular diseases, an in vivo model in which the cell mediated, humoral or non-specific immune response can be studied is more appropriate. Hence an animal model by which to evaluate purported immune boosters and traditional medicine to understand their mechanism of action on the immune system is essential. Here, it was proposed to undertake a study to develop a rat model by which to characterize the effect of Phela on selected immune markers in immune-suppressed rats. The above mentioned aim was achieved through six objectives outlined below. Firstly, an HPLC method with two detectors was applied to ensure consistency of all batches of Phela that were used throughout the study before undertaking an in vivo study. Two mark peaks were observed after analysis of Phela by HPLC-DAD. Phela fingerprint was confirmed by comparing the current results from both methods with those obtained previously. Secondly, an HPLC-UV assay was developed, validated and applied for the simultaneous determination of cyclophosphamide and dexamethasone concentration in rat plasma. The retention time was at 4.2, 5.7 and 8.1 minutes for cyclophosphamide, dexamethasone and internal standard, respectively. The method was linear with regression and correlation coefficients of y = 0.04x+0.11 and 0.999 for cyclophosphamide, and y = 0.32x–1.52 and 0.998 for dexamethasone, and their respective recoveries of 102 – 108 % and 99 – 107 %. The drugs were stable at -20 °C up to a month. Thereafter, the slide-a-lyzer technique was used to rule out potential interactions of Phela with the immunesuppresants [cyclosporine, cyclophosphamide and dexamethasone] before co-administration in rats experiment. Despite wide variations, the results indicated that there was no significant difference between the free fractions of drug-only group when compared with drug+Phela group. As thus, the above mentioned drugs could be co-administered with Phela without interference. In order to develop an animal model, three rat experiments were undertaken. For the first experiment, rats were treated with three escalading doses of Phela for three weeks, along with levamisole a known immune stimulant and a control group. Five rats were sacrificed once weekly per group. Physiological function tests and immune markers (CD4, CD8, IgG, IgM, IL-2 and IL-10) concentration was determined. Phela caused increase in white cell count, which correlates with elevated lymphocyte sub-sets (i.e. CD4 and CD8 count) after treatment with all three doses and this observation peaked by day 14 of treatment. Moreover, Phela led to ample stimulation of the immune system as indicated by increased CD4 cell count and IL-2 at doses of 5 and 15.4 mg/kg. This selective effect implies that Phela can be indicated in diseases that interfere with CD4 and IL-2 count, but this needed to be confirmed in a diseased model. The 15.4 mg/kg dose was selected to be used in subsequent studies. For the second experiment, the aim was to determine the optimum dose and time it takes to achieve optimum immune suppression by known immune suppressants; cyclosporine, cyclophosphamide and dexamethasone. Different groups of twelve rats each were treated with cyclosporine, cyclophosphamide and dexamethasone only, along with a control group in each case. Physiological and immune tests described in the first experiment were also done. As expected, the animals exhibited abnormal physiological function tests in association with progressive immunesuppression. Cyclosporine inhibited the cell mediated immunity, while cyclophosphamide suppressed the humoral immunity and the suppressive effect of dexamethasone was multi-systemic. In all cases, the immunesuppression continued up to the end of the study period. The optimum dose and time of each drug was established. This implies that a rat model of drug induced immune suppression was successfully developed. This rat model was to be validated when the immune suppressed rat model was co-administered with a test drug, in this case Phela, to understand its mechanism of immune modulation which is described in the experiment that follows. The aim of the third experiment was to apply the rat model to establish the mechanism of action of a purported immune booster Phela on the immune system. Different groups of fifteen rats each were pre-treated with cyclosporine, cyclophosphamide or dexamethasone only to induce immunesuppression. Thereafter, the control-groups continued on the immunesuppresant only and the test groups we co-treated with an immunesuppresants (CsA/CP/Dex) and Phela for 21 days. Tests described in the first experiment were similarly done. Phela stopped the progression of immunesuppression in rats treated with cyclosporine as indicated by the reversal and/or resistance to CsA induced changes in the WCC, neutrophils, lymphocytes, CD4, CD8 cells and IL-2 count. Furthermore, Phela prevented progression of CP-induced body and thymus weight loss, suppression of IgG and IgM, and minimal effect on CD4 and CD8 cell count. Observations from the results indicated that the mechanism of immunomodulation of Phela in rats is cell mediated. Therefore, Phela would be candidate for testing against diseases or disorders associated with suppressed CMI, such as HIV/AIDS and Tuberculosis. In conclusion, a rat immunesuppression model has been successfully developed and applied to establish the mechanism of immunomodulation of Phela in rats. Characterizing the mechanism of Phela in rats has indicated the scope of its application for use during diseases with a loss of cell mediated immunity. This model is a necessity in South Africa and across the world at large where many traditional herbal medicines and their products are purported as immune stimulants but lack proof of indication and a scope of application. Furthermore, this model is a tool and/approach that can be used to scientifically validate any immune stimulant and/or traditional medicine to establish its mechanism of action on the immune system, describing its limitations and contra-indications thereof. Lastly, the rat model was applied using Phela a known South African immune stimulant to establish its mechanism of.