Clinical applications and mechanisms of action of Gui Zhi Fu Ling Wan for the management of menopausal hot flushes: literature reviews and computational analyses
Creators: name: LI, Mingdi
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 420803
subject: Traditional Chinese medicine and treatments

description: Background
Menopausal hot flushes (MHF) are a sudden and transient onset of erythema with warmth or burning sensation on the face or neck which appears during the menopausal transition (pre-menopausal and peri-menopausal, menopausal and post-menopausal). It significantly impacts on the quality of life (QoL) of both the sufferers and their families. Menopausal hormone therapies are the most popular management of MHF. However, adverse effects (e.g. nausea, dizziness, dry mouth) and contraindications with hormone-dependent diseases (e.g. breast cancer) exist. In recent years, an increasing number of menopausal women have applied Chinese herbal medicine (CHM) for the management of MHF symptoms. <br><br>

Aim
This research project aimed to evaluate clinical applications and investigate the mechanisms of action of a classic CHM formula in the management of MHF. <br><br>
Phase I: Identification of a Chinese herbal formula for menopausal hot flushes
A systematic review was conducted following the Cochrane Handbook and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist to identify a classic CHM formula popularly investigated for MHF by analysing modern randomised controlled trials (RCTs) from seven databases. Following a set of prescribed search strategies, data selection and extraction, and statistical analyses using Review Manager 5.3, Gui Zhi Fu Ling Wan (GZFLW) was identified from this study as it was found to be the most frequently investigated classic formula for managing MHF and was selected to be the main formula investigated in this research project (Chapter 4, Publication 1, Appendix 1).<br><br>
Phase II: A classic literature review of Gui Zhi Fu Ling Wan
A comprehensive review of classic texts was performed by searching the Zhong Hua Yi Dian CD-ROM to obtain classic literature evidence of GZFLW for MHF, explore its development and variations and identify its standard ingredients used in clinical practice. Through different dynasties, 64 articles from 49 classic books containing information on GZFLW were identified. Classic texts showed that a flushed complexion is one symptom of GZFLW's indications. GZFLW was developed approximately 1,800 years ago in the Han dynasty (206 BC - 220 AD) and has been continuously used since then. The standard version of GZFLW consists of five ingredients: Gui zhi/Rou gui, Shao yao/Bai shao/Chi shao, Mu dan pi, Tao ren and Fu ling. Even though the same ingredients were recorded under different names, GZFLW was the only formula name used throughout all dynasties (Chapter 5).<br><br>

Phases III and IV: Modern clinical and experimental study reviews
Comprehensive reviews of modern clinical and experimental studies were carried out to evaluate the therapeutic effects, safety, phytochemistry, pharmacodynamics and pharmacokinetics of GZFLW for MHF. Articles that investigated standard versions of GZFLW from different evidence levels were obtained from 20 English and Chinese databases. Data were synthesised and analysed using Review Manager 5.3 where applicable. The quality of the included studies was assessed based on different study types.
Two RCTs, one parallel controlled study, four case series, 34 phytochemical studies, five pharmacokinetic studies, four pharmacodynamic studies, one phytochemical study and one pharmacokinetic study were included. 
Modern clinical studies have shown that GZFLW is a safe formula with minor gastrointestinal adverse events. By means of comparison of the before and after data, dose-dependent skin temperature changes were detected. GZFLW can inhibit the blood flow to the upper body while activating the blood flow in the lower body, and it can reduce the calcitonin gene-related peptide (CGRP) level in plasma. Case series studies pointed out that GZFLW may target a specific human oestrogen receptor ß (ESR2) genotype. Thus, ESR2 and CGRP receptors were proposed to be potentially related to the mechanisms of action of GZFLW on MHF.
The included studies on phytochemicals and pharmacokinetics reported 169 known compounds and four unknown compounds from GZFLW and 13 metabolites. Dose-dependent skin temperature changes were also detected in pharmacodynamic studies. The effective dose suggested in the in vivo study was 1,000 mg/kg, which was four times the dose in the RCT (12.5 g/day) when the participant weighed 50 kg. GZFLW had similar effects to oestradiol (E2) on normalising CGRP level. This supports the proposal of the CGRP receptor being associated with the mechanisms of action of GZFLW on MHF (Chapters 6 and 7, Publication 2, Appendix 2).<br><br>

Phase V: Computational analyses
Computational molecular modelling and automated docking approaches were used to investigate the mechanisms of action of GZFLW on possible newly identified biological pathways and synergistic effects of GZFLW. The overall computational strategy adopted involved the following steps: 1) identification of potential target proteins; 2) molecular docking predictions; and 3) proposal of targeted signalling pathways and ligand-target interactions. Target proteins were identified by searches of four electronic databases and their amino acid sequences were obtained from Uniprot and three-dimensional structures modelled by the Swiss-model (Schwede et al., 2003) or I-TASSER (Zhang, 2008). Compounds and target protein models were docked using AutoDock Vina. Networks of chemical compounds and targets identified through the determination of highly predicted affinity compound-target complexes produced from the docking process were established to illustrate the correlation between the herbs/formula and MHF. Promising active chemical compounds with substantial therapeutic potential were identified.
Among the identified 82 proteins with 171 protein structural models (some of which describe the same protein in different conformational states), the protein-protein interaction network contained a small number of highly connected hub proteins, and based on examination of network statistical descriptors, these proteins were proposed to be at least partially biologically connected as a group. Top 10 high-binding affinity chemical compounds were mainly from two herbal ingredients: Shao yao and Mu dan pi. Based on histograms describing the binding affinity values of all compounds bound to each protein, the top 20 proteins favoured by the formula were predicted to behave differently in their interactions with all compounds from the formula. A total of 17 ligand-target interactions were selected as key interactions. Chemical compounds from GZFLW may manage MHF through affecting reproductive hormone changes, thermoregulation and peripheral nerve system/periphery. The formula was predicted to act on 11 proteins related to MHF. Eight chemical compounds were predicted as bioactive chemical compounds as they were both the high binding affinity multi-targeting compounds for all MHF-related proteins and had the highest affinity to the main proteins identified by molecular modeling, namely 4'-O-methylgalloylpaeoniflorin, benzoylpaeoniflorin sulfate, ß-amyrin acetate, galloyloxypaeoniflorin, galloylpaeoniflorin sulfonate, hexagalloyl glucose, mudanpioside H isomer and pentagalloylglucose (Chapters 8 and 9). <br><br>

Conclusions
This project investigated the mechanisms of action of GZFLW for the management of MHF. GZFLW was predicted to have multi-targeting effects through pathways related to reproductive hormone changes, thermoregulation and the peripheral nervous system and periphery. Shao yao and Mu dan pi were found to be the herbal ingredients of GZFLW which provide the most bioactive chemical compounds predicted to affect MHF. This study supports the further investigation of these specific bioactive compounds in GZFLW for the management of MHF.