A Scientific Evaluation of Traditional Chinese Medicine (TCM): Houttuynia cordata Thunb Saururaceae - A Case Study

Abstract

Traditional Chinese Medicine (TCM) is one of the oldest medical systems in the world, still used for prevention or treatment of diseases in the modern China. It is a unique and holistic system, guided by ancient Chinese philosophy. The fish mint herb Houttuynia cordata Thunb (Yu xing cao 鱼腥草) is widely used as a TCM herb in China for the treatment of ailments including cancer, coughs, fever, dysentery, enteritis, and cognitive deficits. The H. cordata medicine is used as a decoction prepared by traditional aqueous extraction of the dried plant material, which is the most used formulation in TCM. Previous studies exploring organic solvent extracts of H. cordata have been shown to contain secondary metabolites including flavonoids, alkaloids, phenolics and volatile components. These extracts demonstrate anti- inflammatory, anti-obesity, and anti-cancer effects as well as activity in inflammatory bowel disease (IBD). However, there is a big gap in studies, with few investigations into the chemical composition and biological activity of the H. cordata traditional aqueous decoction. Therefore, research on the chemical fingerprinting and biological studies for H. cordata traditional aqueous extract of is demanding. In this thesis, sixteen H. cordata samples purchased from three different geographical regions (Sichuan, Hubei, Anhui) in China were investigated. Plant identity for each material was confirmed by microscopic means in comparison with monographs. Three extraction methods (traditional aqueous decoction extraction, phytochemical extraction, and steam distillation) of H. cordata were carried out. The chemically fingerprints for each extract were generated using analytical techniques including Nuclear Magnetic Resonance (NMR) spectroscopy, High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) and Gas Chromatography-Mass Spectrometry (GC-MS) as appropriate. The main focus of the study was on the aqueous extract, which corresponds to the traditional medicinal decoction of H. cordata. Principal component analysis (PCA) based on NMR spectroscopy metabolomics was used to analyse the metabolomic data for the aqueous and phytochemical extracts, and this allowed us compare small differences that become clearer in the metadata. This project is centred on the traditional aqueous decoction, and biological activities of the corresponding aqueous extracts were assessed and correlated with the chemistry. We initially investigate the ability of the extracts to inhibit the cholinesterase enzymes including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The extracts only demonstrated significant inhibition of AChE activity. The anti-inflammatory activity of aqueous extracts was then evaluated in vitro (TNF-α activated Caco-2 monolayers) by assessing intestinal inflammation associated mediators (ZO-1, Occludin, EGFR, IL-1β, IL-6, IL-10, MAPK ERK 1/2) through RT-qPCR, ELISA and Western-blot. The anti-inflammatory role of H. cordata was then further evaluated in a DSS-induced murine colitis model in vivo. In this model both prevention and recovery effects of the H. cordata aqueous extract were evaluated by disease scores (weight loss, bloody stool, diarrhoea), hematoxylin and eosin staining of colonic tissue, and mediators associated with inflammation (MPO, Occludin, TNF- α,EGFR, IL-1β, IL-6, IL-10). Principal component analysis (PCA) of the 1H NMR spectroscopy metadata for the 16 samples analysed distinguished three aqueous outliers. Three outliers were also distinguished between the samples, from their phytochemical (MeOH) extracts. The outliers identified in the two types of extract differed from each other, showing that the plants biosynthesis different chemical metabolites depending on their area of cultivation and methods of preparation and storing. Fifteen compounds were identified and quantified by HPLC-MS. Using this technique qualitative and quantitative differences between the aqueous outliers and quality control (QC) sample can be distinguished. Twenty-eight volatile compounds were identified by GC-MS in the essential oil recovered by steam distillation of H. cordata plant material. The main component identified in this oil is 2-undecanone known as the ‘fish mint’ marker. H. cordata aqueous extracts showed protective effects on the intestinal inflammation in vitro by enhancing ZO-1, Occludin, IL-10 and suppressing EGFR, IL-1β and IL-6. A mechanism explaining this activity via an EGFR dependent MAPK (ERK) 1/2 signalling pathway is proposed. The three chemically different aqueous outliers, are shown to be pharmacologically different, in these biological studies. An international Good Manufacture Practice (GMP) standardised product of H. cordata plant material from Hubei showed more potency. A beneficial intestinal improvement observed in vivo, was seen at a molecular level within colonic tissues for both the prevention and recovery trial, which agrees with in vitro findings, although no apparent enhancement in recovery symptoms was observed. In conclusion, aqueous extracts from H. cordata sourced from different geographical regions have been shown to be chemically different, which may be a result of biotic and abiotic factors. these observations underpin the need for scientific evaluation of TCM herbal material to ensure its consistent quality, safety, and efficacy. Extracts were prepared using traditional aqueous and phytochemical extraction methods. These extracts have different chemical fingerprints. The resulting H. cordata aqueous decoctions also demonstrated varying intensity of pharmacological activity. Inhibition of the enzyme AChE supports the use of H. cordata in improving cognitive health and is worthy of further study. Confirmation of the role of the traditional decoction of H. cordata in the support of intestinal barrier integrity is very significant and validates the traditional use of H. cordata decoction in the treatment of inflammatory gut conditions such as inflammatory bowel disease (IBD).