Investigating the antidiabetic potential of the combination of Catharanthus roseus and Portulacaria afra leaf extracts

MSc (Pharmaceutical Sciences), North-West University, Potchefstroom Campus Background: Diabetes mellitus is a chronic disease that causes high blood sugar. The cost and side effects of modern antidiabetic drugs have become a major burden to patients suffering from diabetes, especially those from developing countries. As a result, medicinal plants are considered a favourable alternative for antidiabetic treatment in developing countries. In South Africa there is anecdotal evidence that the 1:1 combination of Catharanthus roseus (C. roseus) and Portulacaria afra (P. afra) are being used to treat symptoms of diabetes mellitus. There are multiple scientific reports on the in vitro and in vivo antidiabetic potential of C. roseus, whilst there is insufficient data on the antidiabetic effects of P. afra and its combination with C. roseus. Aim: This present study investigated the antidiabetic potential of the aqueous leaf extract of Catharanthus roseus and Portulacaria afra, independently and in combination (1:1), in both in vitro and in vivo models. Methods: In the in vitro study, the safety of the plants was determined by measuring the cytotoxicity against HepG2 cell line. In addition, the antidiabetic potentials of the plant extracts were evaluated by measuring the enzyme activity (α-amylase, α-glucosidase and hexokinase) — common markers of diabetes therapy —in the HepG2 cells. An in vivo study was conducted using a chemically induced diabetic rat model. Diabetes was induced in male Sprague Dawley rats (50) by a single intravenous injection of streptozotocin at 55 mg/kg body weight. Animals varying between seven to nine weeks of age were randomly assigned to six groups; non-diabetic (n = 10), diabetic non-treated (n = 8), metformin (500 mg/kg body weight, n = 8), C. roseus (CR, 100 mg/kg body weight, n = 8), P. afra (PA, 100 mg/kg body weight, n = 8) and 1:1 combination of C. roseus and P. afra (CR:PA, 100 mg/kg body weight, n = 8). The rats were treated by receiving an oral administration of the aqueous leaf extracts of CR, PA and CR:PA for 28 consecutive days. Results: The 1:1 extract combination of C. roseus and P. afra, with a IC50 value of 4.10 μg/mL (i.e. NR assay), had the highest cytotoxicity on the HepG2 cell line, with statistical significance (p < 0.05), compared to P. afra (IC50 = 27.93 μg/mL). Extract of C. roseus (IC50 = 6.02 μg/mL i.e. NR assay) was also cytotoxic towards liver cells, whilst P. afra had the least cytotoxic effect on the liver cells (IC50 = 27.93 μg/mL i.e. NR assay). Further cytotoxic investigation (i.e. MTT assay) showed that the interference of copper compounds with MTT formazan, rendered the MTT assay to be less suitable for cell viability measurements when compared to the neutral red assay. The results of the in vitro assays showed that the plant extracts had no effects on the α-glucosidase activity in HepG2 cells. However, the inhibition activity of P. afra and CR:PA extracts on α-amylase and the activation of the liver hexokinase enzyme by the plant extracts, suggested potential antidiabetic effects that are commonly involved in the reduction of blood glucose levels. The in vivo study demonstrated that daily oral gavage with C. roseus effectively lowered plasma glucose in diabetic rats. In addition, administration of C. roseus showed improved reversal effects in the relative weights of the heart, liver and kidneys, whilst also maintaining the body weights in diabetic rats. In contrast, oral treatment with P. afra resulted in elevated plasma glucose levels and caused a supplemental increase in the relative liver and kidney weights. The treatment with P. afra to diabetic rats resulted in a significant decline in body weights (p < 0.05). The administration of CR:PA to diabetic rats exhibited similar results in the liver and kidneys, thus displaying an antagonistic effect between C. roseus and P. afra. While C. roseus lowered the blood glucose levels in diabetic rats, P. afra raised it. Further investigation showed that the relative weights of the pancreas were unaffected for all treatment groups, except for CR:PA-treated rats, which displayed a significant decrease in comparison to non-diabetic rats (p < 0.05). The reason for this incident is still not well understood and would require further evaluation through organ histology. Understanding the major physiological function of these two plant extracts would be crucial for future studies. Conclusion: The present in vitro data displayed potential antidiabetic effects of aqueous leaf extracts of P. afra and its 1:1 combination with C. roseus, whilst this was not the case for the in vivo study. The leaf extract of P. afra caused hyperglycaemic effects in diabetic rats, meaning that the alleged antidiabetic effects of P. afra, as presented by the inhibition activity of α-amylase and activation of hexokinase (as seen by the in vitro data), had no lowering effects on the blood glucose levels in animal models. The in vivo data demonstrated that the combination of CR:PA displayed antagonistic effects in glycaemic control, as an effect that was produced by the contrasting actions of C. roseus and P. afra. The current study evidently revealed the shortcomings associated with in vitro studies, one of which was the misleading antidiabetic results that was presented by the in vitro data. Our study has conclusively shown that the efficacy of drugs is better evaluated when using complex in vivo models such as animal. However, one of the disadvantages of STZ is that it specifically targets β-cells in the pancreas. Therefore, the STZ model could contain potential limitations to the proposed testing of the antidiabetic effects of the plants. In addition, one would need to perform a carbohydrate-loading test to see whether the increased postprandial blood glucose levels are decreased by the plant extracts. In conclusion, P. afra and its 1:1 combination with C. roseus (CR:PA) leaf extracts have shown no antidiabetic effects in Sprague Dawley rats, whereas leaf extracts of C. roseus did show antidiabetic activities. Masters