Co-Fermentation of Marsdenia tenacissima with Ganoderma lucidum and Anti-Lung Cancer Effect of the Fermentation Products

Summary

Here, we describe a protocol to establish a systematic and comprehensive co-fermentation system to produce biotransformation products rich in pungent saponins by using Marsdenia tenacissima (Roxb.) Wight et Arn. (MT) as a fermentation medium for Ganoderma lucidum. This will serve as a methodological reference for the development of other ethnic drugs.

Abstract

Marsdenia tenacissima (Roxb.) Wight et Arn. (MT), as a traditional Chinese and Dai herbal medicine, has anti-inflammatory, antibacterial, and antitumor properties. However, most of its main active substances are aglycones, such as tenacigenin A and tenacigenin B. As the bioavailability of MT is low and its medicinal active components are challenging to synthesize, it is primarily studied by biotransformation. This study aims to produce biotransformation products rich in pungent saponins by using MT as a fermentation medium for Ganoderma lucidum (G. lucidum).

Through the preliminary screening of three medicinal fungi, it was found that G. lucidum and Ophiocordyceps sinensis (O. sinensis) can generally grow in the medium for MT; hence, the efficacy of the fermentation of the two types of fungi was screened using a mouse model of lung cancer. Finally, the co-fermentation of G. lucidum and MT was selected for further investigation. Non-target metabolomics analysis was performed on the products of MT with G. lucidum co-fermentation. We identified 12 specific saponins of MT from the fermentation products, and obtained a monomeric compound, tenacigenin A, from fermentation products.

Most of the tenacigenin showed a significant upward trend, through tenacigenin A and tenacigenin B levels. The results showed that the efficacy of MT improved after fermentation by G. lucidum. Furthermore, the biotransformation of C₂₁ steroidal glycosides in MT was the central reaction in this fermentation process. In summary, this study established a systematic and comprehensive co-fermentation system and pharmacodynamic evaluation method for MT, which not only enhanced the full utilization of effective active substances in MT but also provided a methodological reference for the development of other ethnic drugs.

Introduction

Lung cancer belongs to the category of "lung carbuncle" in traditional Chinese medicine. The pathogenesis of lung carbuncle is a weakness of the healthy "Qi" of patients, imbalance of Yin and Yang, toxins, and stagnation in the lung, leading to lung dysfunction, blood blockage, and fluid loss in the lung. Thus, long-term blood stasis and sputum toxins in the lungs form a lung mass¹. Therefore, strengthening the Qi and eliminating pathogenic factors is the basic principle of treating lung cancer. The methods of nourishing Yin to balance Yin and Yang, clearing heat, detoxifying and dispersing stagnation, supplementing Qi and nouri....

Protocol

This study was conducted following the recommendations of the Experimental Animal Center of Minzu University (No. ECMUC2019008AA). The protocol was approved by the Experimental Animal Ethical Committee of Minzu University. MT was collected from Kunming, Yunnan province.

1. Preparation for the study

1. Extract MT with hot water (100 °C; MT/water = 1/10, w/v) for 30 min and collect the MT extraction residue as the test material.
2. Maintain the LLC mouse lun.......

Discussion

After strain screening experiments, we found that not all medicinal fungi can survive normally on herb materials. Without any additional medium, the survival of medicinal fungi depends on the degradation of the components in medicinal materials through their own enzymes to synthesize the required carbon and nitrogen sources. It can be inferred that I. obliquus may not contain enzymes capable of degrading saponins of MT. For G. lucidum and O. sinensis that can be co-fermented with MT, both medic.......

Materials

Name	Company	Catalog Number	Comments
Acetonitrile	Tonguang Fine Chemicals Company, Beijing, China	20200923
Agar	Sinopharm Chemical ReagentCo., Ltd., USA	NO.20080107
Autoclave	BinJiang Medical Co., Ltd., Jiangyin, China	LS-50LD
constant shaking incubator	Zhicheng Inc. All rights reserved., Shanghai, China	ZWY-100D
Ganoderma lucidum	BeNa Culture Collection, Beijing, China	31732
Inonotus obliquus	BeNa Culture Collection, Beijing, China	117822
LLC Mouse lung cancer cell	National Infrastructure of Cell Line Resource, Beijing, China	PUMC000673
Male C57BL/6J mice	Vital River Laboratory Animal Technology Co., Ltd., Beiijng, China	No.110011210107024684
Methanol	Tonguang Fine Chemicals Company, Beijing, China	20210723
Ophiocordyceps sinensis	BeNa Culture Collection, Beijing, China	118371
Poly tetra fluoroethylene	Jinteng Experiment Equipment Co., Ltd., Tianjing,China	No.997
QTRAP 5500 LC/MS	AB Sciex Pte. Ltd., USA	CV20231711
Rotary Evaporator	BUCHI Co., Ltd., Shanghai, China	R-300
Syringe	Zhiyu Medical Instrument Co., Ltd., Jinagsu, China	V500111
Ultra-clean bench	BOXUN Medica Bioological Instrument Co., Ltd., Shanghai, China	SW-CJ-LFD