Ethnobotanical, pharmacological, and chemical studies of Paris polyphylla var. yunnanensis (Chong- Lou herb) from Yunnan province, China

Abstract

Cancer is one of the two leading causes of death around the world. The main treatments for cancer are currently surgery, radiation therapy, chemotherapy, targeted therapy, and immunotherapy. However, immunotherapy always followed with limitations, while other therapies always accompanied many disadvantages for muti-drug resistance, and side effects. Traditional Chinese medicine (TCM) is often used as an adjunct therapy to mitigate adverse reactions to radiotherapy and chemotherapy. Many preparations are recorded in Chinese Pharmacopoeia (2020 version) for the adjuvant treatment of cancer, consisting of various of TCM herbs. Among them, several commercially available preparations include Paris polyphylla var. yunnanensis (Franch.) Hand.- Mazz. (PPvY), which have been used in both the treatment and adjuvant treatment of cancer. This thesis was divided into three parts according to the objectives of this study. Firstly, ethnobotanical methods were used to record the traditional medicinal knowledge (TMK) of PPvY and folk prescriptions contained PPvY. Secondly, anti-cancer activity for pro-apoptotic and cytotoxic effects of cultivated PPvY was verified through phytochemical approach and in vitro cell experiments. Lastly, we tried to reduce the toxicity of cultivated PPvY to normal human cells and increase its inhibition rate against human cancer cells through TCM processing methods for fermentation. The TMK of PPvY and folk prescriptions related to PPvY was carried out by literatures and ethnobotanical investigation. The results revealed that PPvY and prescriptions contained PPvY primarily addressed cancer, inflammation, and the functions of clearing heat and detoxifying. The TCM herbs most frequently used alongside PPvY were Engleromyces sinensis and Glycyrrhiza yunnanensis. The commonly employed processing methods primarily utilize PPvY in both its dried and fresh forms. Based on the results of traditional medicine knowledge for PPvY, a phytochemical approach was used to study the substance basis of cultivated PPvY. Five crude extracts were obtained from the rhizomes in cultivated PPvY. The methods of High-performance liquid chromatography (HPLC) and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium (MTS) were used to determine the total saponins contents and cell inhibition rate respectively. According to fraction A3 had the highest of total saponin content (59.38%) and cytotoxicity activity, six pure compounds (Paris saponin Pb, Paris saponin H, Paris saponin III, Paris saponin I, Paris saponin VII, Paris saponin II) were isolated and identified by Nuclear Magnetic Resonance (NMR) and Mass spectrometry (MS). All six compounds were all lower than a commonly used chemotherapy drug-cisplatin (positive control), with IC50 (Half maximal inhibitory concentration) values of 17.83 μM (SMMC-7721), 5.67 μM (HepG2), 21.02 μM (SK-HEP-1), and 26.08 μM (A549). The results indicated that these six Paris saponins induced significant cell cycle arrest and apoptosis in three cancer cells (A549 & SMMC-7721 & HepG2), which was correlated with a decrease in mitochondrial membrane potential. Total concentration (6.96%) of three Paris saponins (I + II + VII) in cultivated PPvY were with levels ten times higher than wild and those standards (0.6%) in 2020 version of Chinese Pharmacopoeia. These above results supported the potent anti-cancer efficacy of widely cultivated PPvY, which was consistent with the record in literatures and ancient books. Two fermentation trials including solid and liquid fermentation were conducted on the effects for anti-cancer and toxicity with PPvY. Firstly, three kinds of Jiuqu pies from different origins were used as starter cultures. Eight single strains were isolated and identified from three Jiuqu pies, including six bacterium and two fungi through plate method. The HPLC method was used to determine the total saponins contents before and after fermentation. According to total saponin content was increased after solid fermentation, the PPvY was fermented to further comparing the anti-cancer activity and toxicity before and after solid fermentation. Two human cancer cells (NCI-H460 and A549) and one normal human lung cell line BEAS-2B were tested. The results from comparison of antitumor efficacy of PPvY before and after fermentation showed that the anti-cancer effect on cell viability rate for these two human cancer cells in the comparison of BF (before fermentation) and NC (negative control without Jiuqu pies) was not increased by three Jiuqu pies, but increased in three single microbial strains (JQJD6, JQJD7 and JQJD8) for NCI-H460 in solid fermentation. In the comparison of BF and NC, the results from comparison of toxicity of PPvY before and after fermentation showed that the toxicity on the cell viability rate of one normal human lung cell line was reduced in JQP1 in liquid fermentation and in two single microbial strains (JQJD1 and JQJD3). Furthermore, new sources of microorganisms should be found in the fermentation of PPvY to better increase anti-cancer activity and reduce toxicity. The possibility of using solid fermentation should be used to other TCM fermentation in the future. In summary, the above results indicated that cultivated PPvY can serve as a substitute for wild resources, providing more case studies for biodiversity conservation.