Development of HPTLC fingerprints and assessment of Neutropoiesis activity of cucurbita maxima leaf extracts on chemotherapy induced neutropenic wistar rats

Abstract

Introduction: Chemotherapy-induced neutropenia (CIN) is a major cause of mortality among cancer patients worldwide. Although granulocyte colony-stimulating factors like filgrastim and antibiotic prophylaxis are commonly used to manage CIN, their high cost and limited availability pose significant challenges, particularly in low-resource settings. At the Uganda Cancer Institute, patients have turned to pumpkin leaves (Cucurbita maxima) for CIN management, though there is limited scientific evidence on their efficacy and phytochemical composition. This study aimed to develop High-Performance Thin Layer Chromatography (HPTLC) fingerprints and assess the neutropoiesis activity of Cucurbita maxima leaf extracts in chemotherapy-induced neutropenic Wistar rats. Methods: HPTLC fingerprints for the aqueous and methanolic extracts of Cucurbita maxima leaves were developed using a CAMAG HPTLC automatic machine. The HPTLC plates were scanned at wavelengths of 190 nm, 260 nm, and 366 nm, and visualized under T white light, RT white light, R white light, UV 254 nm, and UV 366 nm. The results were presented in images, graphs, and tables, with Rf values expressed as mean Rf ± standard deviation. Fifty Wistar rats were randomly divided into five groups, each with ten rats. Neutrophil counts were measured intermittently in both the test and control groups. Cyclophosphamide was used to induce neutropenia, while filgrastim served as the positive control. Results were analyzed using STATA Version 16/MP and presented in graphs and tables. Results: The HPTLC analysis showed significant variability in the phytochemical components depending on the extraction solvent and the geographical source of the plants. At 190 nm, aqueous extracts exhibited fewer bands (3 and 4 for Lira and Kampala sources, respectively), while methanolic extracts showed a higher number of bands (12 for Lira and 9 for Kampala). At 260 nm, aqueous extracts from both regions showed more bands (5 and 6) compared to 190 nm, whereas methanolic extracts had the lowest absorbance with only 3 bands each. At 366 nm, methanolic extracts from Lira and Kampala showed 12 and 11 bands, respectively, indicating a higher number of phytochemical components compared to aqueous extracts. Geographical variations were noted, with aqueous extracts from Kampala showing a slightly higher compound composition compared to those from Lira, while methanolic extracts from Lira displayed a more complex profile than the Kampala sample. The Kruskal-Wallis test showed statistically significant differences in neutrophil counts between the groups (p = 0.0099). Group 4 (methanolic extract) had the highest rank sum (292.50), outperforming Group 3 (aqueous extract) (p = 0.0380). No significant difference was observed between Group 4 (methanolic extract) and Group 5 (filgrastim) (p = 0.1711), indicating comparable efficacy in reducing chemotherapy-induced neutropenia. Conclusions: HPTLC fingerprints of Cucurbita maxima extracts demonstrated moderate geographical variability, underscoring the influence of growing conditions on chemical composition. The methanolic extract exhibited superior phytochemical diversity and was more effective in mitigating neutropenia in Wistar rats, showing comparable efficacy to filgrastim. These findings suggest the potential of Cucurbita maxima as a natural alternative for managing CIN. The study emphasizes the need for stringent quality control and standardization of herbal medicines to address geographical and solvent-related variations and calls for further research to isolate and characterize bioactive compounds to ensure safe and effective treatments.