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

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.

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.

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 nourishing Yin, and clearing phlegm are used to treat and prevent the formation of lung carbuncles².

Marsdenia tenacissima (MT) is a medicinal plant belonging to the family Asclepiadaceae (Marsdenia ssp.), also known as Wuguteng. It has the effects of clearing heat and detoxification, relieving cough and asthma, and has antitumor properties³. Xiaoaiping injection, a preparation made from a single herb of MT, has been widely used in clinics and shows good therapeutic anticancer effect⁴. However, with population increase, the demand for MT has increased sharply. Accordingly, the supply of wild MT resources is insufficient, and the quality of cultivated medicinal materials is uneven. There are problems such as poor quality of herb materials, unstable content of ingredients, and low bioavailability, which seriously threaten the development of MT. Among these bioactive compounds, saponins from MT have been highly investigated. More than 100 saponins from MT have been identified; they can be divided into two major types: (1) C₂₁ steroidal glycosides in MT, including tenacissoside A-P, marsdenoside A-M, tenacigenoside A-L, and tenacigenin A-D. (2) Pentacyclic triterpenes, such as oleanolic acid and ursolic acid. Many studies have indicated that the anticancer activity of petroleum ether extracts is higher than the highly polar molecules from MT. Hence, it is advantageous to convert the major polysaccharide of saponins from MT into another minor aglycone of MT with higher bioavailability and bioactivities⁵.

Biotransformation, also known as biocatalysis, refers to the physiological and biochemical reactions of the transformation or structural modification of exogenous substrates by using related enzymes in biological systems⁶. It is more economical, safer, regionally selective, and stereoselective than traditional chemical synthesis and can produce some bioactive compounds that are difficult to prepare by conventional synthetic chemistry^7,8. Therefore, the development of traditional Chinese medicine and natural drugs is essential in promoting the modernization and internationalization of traditional Chinese medicine.

The co-fermentation system of MT was established using biotransformation. The original medicinal materials were transformed by microbial enzyme systems. On the one hand, the extract of MT was used as a substrate to produce the fermentation culture of fungi in this mode. At the same time, fungal fermentation shortened the breeding cycle of bacteria, reduced production costs, and improved production efficiency⁹. On the other hand, the increased precipitation of active components by fungal fermentation is beneficial, producing chemical components and an improved efficacy¹⁰. The use of liquid two-way fermentation technology is helpful to protect the active ingredients of medicinal materials, improve efficacy, save drug sources, and may produce better effectiveness by providing techniques for the structural modification of active ingredients of herb materials. It is of great significance to improve the modernization level of traditional Chinese medicine.

In this study, G. lucidum, O. sinensis, and Inonotus obliquus (I. obliquus) were selected as the fermentation microorganisms in the early stage after the literature review, and the strains were preliminarily screened. Then, the final fermentation strains were determined by exploring the efficacy of the screened strains through the tumor mouse model. Finally, the primary metabolites and secondary metabolites of the fermentation products were systematically analyzed and evaluated by LC-MS, and we obtained a monomeric compound, tenacigenin A, from fermentation products.

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 lung cancer cell line in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified atmosphere at 37 °C under 5% CO₂.
3. Keep the C57BL/6J mice, aged 6-8 weeks and weighing 19 ± 2 g, in an environmentally controlled room with a 12 h/12 h light/dark cycle and with unlimited access to food and water. Disinfect them regularly.

2. Preliminary screening of strains

1. Inoculate three strains of fungi into potato dextrose agar (PDA) solid medium using sterile techniques and culture them in the dark at 28 °C. After the dishes are fully covered with fungi, store them at 4 °C.
2. Inoculate the strains preserved in the PDA solid medium in the seed solution of the first-stage shake flask and culture them at 28 °C, with shaking at 120 rpm for 4-5 days. Inoculate the first-stage fungi liquid into the shake flask containing the secondary fungi liquid at a volume ratio of 1:20 in a constant temperature shaker for 4-5 days.
3. Dilute the MT with water to 100 mg/L (MT/water) and add the solution to agar to form a solid medium. Inoculate 1 mL of the secondary fungal liquid of each of the three strains into the solid culture containing only MT, and then divide into G. lucidum-MT, I. obliquus-MT, and O. sinensis-MT culture systems. Place these culture systems at 28 °C without shaking in the dark.
4. After some time, observe the growth of the strains in the above three culture systems, and select the strains that can only grow with MT for further screening.

3. Strain selection rescreening

NOTE: After preliminary screening, only G. lucidum and O. sinensis could grow normally on solid medium containing MT. To further select the system with good antitumor effect, the antitumor efficacy was tested in a transplanted tumor mouse model.

1. Inoculate G. lucidum and O. sinensis into liquid medium containing only MT-MT with G. lucidum co-fermentation (MGF) and MT with O. sinensis co-fermentation (MOF), and ferment for 10 days at 28 °C with shaking at 180 rpm.
2. After fermentation, filter the mycelium through gauze and dry it. Extract with 80% ethanol 3 x 12 h.
3. Check the antitumor activity of the fermented and unfermented MT in LLC tumor cell-bearing C57BL/6J mice.
   1. Subcutaneously inject LLC cells (1.0 × 10⁶ cells in 0.2 mL of phosphate-buffered saline (PBS) per mouse) into the right flank of male C57BL/6J mice (except in the control group).
   2. To confirm successful establishment of the model, touch the tumor nodules and verify they are ~5 mm in size. Randomly group the tumor-bearing mice (n = 10 per group) into the model group, MGF group, MOF group, MT group, and cisplatin group (positive control). Administer saline to the control group (no tumor cells) and the model group. Sacrifice the mice after 14 days of treatment and collect the tumor tissue.

4. Chemical composition analysis of fermentation products

NOTE: After verifying the efficacy of fermentation, G. lucidum was selected as the fermentation strain in this study. We performed metabolomics analysis of MGF by liquid chromatography-mass spectrometry (LC-MS)¹¹.

1. Extract 10 mg of MT, G. lucidum, and MT with G. lucidum co-fermentation (MGF) with 500 µL of methanol:H₂O (7:3), shake at 4 °C for 1 min, sonicate for 30 min, and centrifuge for 20 min at 1,200 × g. Collect the supernatants, filter them through a 0.22 µm polyvinylidene fluoride membrane, and inject into the liquid chromatography-tandem mass spectrometry setup (LC-MS/MS).
   ​NOTE: Ultra performance liquid chromatography (UPLC)-separated metabolites and a quality control (QC) sample library was constructed by high-resolution mass spectrometry (see Table of Materials).
2. Use the following UPLC conditions: a C18 column (100 mm x 2.1 mm; 8 µm), mobile phase A-0.1% (v/v) formic acid in ultrapure water, mobile phase B-acetonitrile. Set the gradient elution profile to be 0 min 5% B; 2 min 5% B; 14 min 98% B; 17 min 98% B; 17.1 min 5% B; 20 min 5% at a flow rate of 0.3 mL/min.
3. Detect and identify the metabolites by high-resolution mass spectrometry. Set the column temperature to 40 °C, automatic injector temperature to 4 °C, and injection volume to 2 µL. Maintain the following MS conditions: the voltage of the electrospray ion source: +5,500/-4,500 V; the pressure of the curtain gas, ion source 1, and ion source 2: 35, 60, and 60 psi, respectively; the temperature of the ion source: 600 °C; the depolymerization potential: ± 100 V.
4. Prepare the quality control samples (QCs) by mixing equal volumes of MT, GL, and MGF extracts, and name the three replicates QC 1-3, respectively. Process and detect quality control and analysis samples by following the same steps. Insert a QC sample for every three samples to investigate the repeatability of detection and analysis.

5. Extraction and isolation of fermentation products

1. Dissolve the fermentation extract obtained from the fermentation broth in distilled water and stir thoroughly.
   NOTE: Here, 2 kg of fermentation extract was obtained from 40 L of fermentation broth.
2. From section 4, elute 10%, 30%, 50%, 70%, and 95% ethanol fractions using a macroporous adsorption resin column and collect the fractions.
   NOTE: Here, 10.72 g of the 95% ethanol fraction, 34.72 g of the 70% ethanol fraction, 61.57 g of the 50% ethanol fraction, and 79.7 g of the 30% ethanol fraction were obtained.
3. Based on the polarity of the fractions, elute the 70% ethanol fraction using silica gel column chromatography with ethyl acetate:methanol elution and collect the fractions.
   NOTE: Here, Fr. 1-Fr. 7 were collected.
4. Perform Sephadex LH-20 column chromatography to further purify the fractions (here, Fr. 3 and Fr. 4) followed by semi-preparative liquid phase separation after purification using 1.0 g of each fraction.
   NOTE: By using semi-preparative liquid chromatography and silica gel column chromatography, we isolated and purified the 95% ethanol fraction.
5. Perform microspectroscopy and nuclear magnetic resonance spectroscopy and compare the structure of the compound(s) with structures from databases for 600M and 700M and determine the planar structure of the compound(s).
   NOTE: Here, a hybrid mass spectrometer was used to determine the molecular weight of the mixture. We identified the structure of one compound (see Supplemental Figure S1).

6. Statistical analyses

1. Repeat all the fermentation experiments six times. Assess statistical significance using Student's t-test. Express all data as the mean ± SD, repeat all experiments in triplicate, and set p < 0.05 for statistical significance (*0.01 ≤ p ≤ 0.05, **0.001 ≤ p ≤ 0.01, ***p ≤ 0.001).
2. Use SCIEX OS software to identify metabolites mainly based on mass error, retention time, isotope match, MS/MS library purity score, and computational molecular formula. Use the following library setting parameters: mass error < 5 ppm, isotope ratio difference < 20, and library score > 50.

Preliminary screening results of strains
To explore fungi capable of co-fermenting with MT, we selected three fungi: G. lucidum, I. obliquus, and O. sinensis. First, the strains were activated: G. lucidum, I. obliquus, and O. sinensis were inoculated in the PDA medium as shown in Figure 1A-C; Figure 1D-F shows the results after inoculation into the liquid PDA medium. The second step was to inoculate the fungi in the secondary seed solution into the solid medium containing only MT; only G. lucidum and O. sinensis could grow normally (Figure 1G,H). The fungi were inoculated in MT liquid medium, and the results after 10 days of culture are shown in Figure 1I,J. G. lucidum and O. sinensis were preliminarily screened based on their growth in the presence of MT.

Figure 1: Fungi on various media. PDA solid medium:(A) G. lucidum, (B) I. obliquus, (C) O. sinensis. PDA liquid medium: (D) G. lucidum, (E) I. obliquus, (F) O. sinensis. MT solid medium: (G) O. sinensis, (H) G. lucidum. MT liquid medium: (I) O. sinensis, (J) G. lucidum. Please click here to view a larger version of this figure.

Rescreening strains
To further explore the antitumor effect of fungal fermentation of MT compared with that of just MT, we selected a mouse tumor model. The results showed that the tumor volume decreased in all mice except the control group, and the MGF group had the smallest tumors (Figure 2A). The tumor weight of each group was lower than that of the model group, those of the MGF and MOF groups were significantly lower than the MT group, and the tumor weight of the MGF group was lower than the MOF group, indicating that the antitumor effect of MGF was significantly stronger than that of MT and MOF (Figure 2B).

Figure 2: Antitumor effects in vivo. (A) Images of tumor tissue. (B) Mouse tumor weight. *p≤ 0.05; **p≤ 0.01; ***p≤ 0.001; ****p≤ 0.0001. Please click here to view a larger version of this figure.

Characteristic features of C₂₁ steroidal saponins in Marsdenia tenacissima with Ganoderma lucidum co-fermentation
C₂₁ steroidal glycosides are the most studied active chemical constituents in MT in recent years, and they are also the main components that have anticancer activity. Their characteristics are that the 11 and 12 positions of aglycone are often esterified with common organic acids such as benzoic acid (Bz), tiglic acid (Tig), cinnamic acid (Cin), and acetic acid (Ac), and the three positions of aglycone are linked to a variety of β-deoxyglucose molecules^12,13,14. A total of 13 kinds of C₂₁ steroidal glycosides were identified in MGF, and five kinds of tenacigenin, including 11α-O-2-Methylbutyryl-12β-O-2-benzoyltenacigeninB, 11α, 12β-Di-O-tigloyltenacigeninB, 11α-O-Tigloyl-12β-O-benzoyl-marsdenin, tenacigenin A, and tenacigenin B, which were upregulated in MT with GL co-fermentation. Nine kinds of C₂₁ steroidal glycosides were identified in MT, including tenacissimoside H, 11α-O-2-Methylbutyryl-12β-O-2-benzoyltenacigeninB, tenacissoside I, 11α-O-Tigloyl-12β-O-acetyltenacigenin B, 11α-O-Togloyl-17β-tenacigenin B, 11α-O-Tigloyl-12β-O-benzoyl-marsdenin, tenacissoside I, 11α-O-2-Methylbutyryl-12β-O-2-tigloyltenacigeninB, and tenacigenin C, which were downregulated in MT with GL co-fermentation (Figure 3).

Table 1 shows that, compared with MT, only tenacigenin was detected after fermentation by G. lucidum, whereas C₂₁ steroidal glycosides in MT connected with glycosidic ligands were almost not detected. Previous research has showed that tenacissoside I and tenacissimoside H are the main components of saponins, with multiple glycosidic ligands from MT. We found that these two compounds were downregulated metabolites after fermentation.

Figure 3: A total of 13 kinds of C₂₁ steroidal glycoside structures. Please click here to view a larger version of this figure.

Table 1: A total of 13 types of C₂₁ steroidal glycosides were identified in MGF. Abbreviation: RT-EM = retention time-exact mass.

Identifying compounds of fermentation products
From the fermentation product, we isolated a monomeric compound. Tenacigenin A has the chemical formula C21H32O5 according to its 1H NMR (600 MHz, CD3OD-d4) δ 1.13 (s, Me(19)); 1.19 (s, Me(21)); 1.19 (s, Me(18)); 1.86 (br. s, H-C(17)); 2.31 (br. s, H-C(9)); 3.56-4.19 (m, H-C(3)); 3.32 (d, J = 2.4, H-C(12)); 3.90 (br. d, J = 2.6, H-C(11)). 13C NMR (150 MHz, CD3OD-d4) δ 39.5 (C-1), 31.3 (C-2), 73.1 (C-3), 38.0 (C-4), 47.6 (C-5), 28.6 (C-6), 33.9 (C-7), 79.9 (C-8), 59.8 (C-9), 36.8 (C-10), 71.9 (C-11), 71.8 (C-12), 45.3 (C-13), 82.1 (C-14), 35.3 (C-15), 24.1 (C-16), 55.3 (C-17), 17.8 (C-18), 16.3 (C-19), 100.9 (C-20), and 24.6 (C-21). In general, the above data are consistent with those reported in the literature, and the compound is identified as tenacigenin A¹⁵.

Figure 4: Total ion chromatograms of MGF. Please click here to view a larger version of this figure.

Supplemental Figure S1: NMR of tenacigenin A. Please click here to download this File.

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 medicinal fungi can grow normally on the medium with MT as substrate. The antitumor effect of the fermented product is the best test for its drug use.

Through the establishment of a mouse tumor model, we found that the antitumor effect of MT was significantly enhanced after fungal fermentation. From this, it can be concluded that after the fermentation of medicinal fungi, the active ingredients linked to difficult absorption and high polarity in MT may be decomposed into substances with high bioavailability and low polarity. Many studies have indicated that the anticancer activity of petroleum ether extracts is higher than the micropolar molecules from MT¹⁶. The antitumor effect of MGF was stronger than that of MOF, indicating that the effect of G. lucidum on MT was stronger than that of O. sinensis through biological transformation. Therefore, it is necessary to analyze the components of MGF in detail.

For the preparation stage of the seed liquid of G. lucidum, I. obliquus, and O. sinensis, an aseptic operation is needed to avoid mutual contamination of the fungi. It is unnecessary to add any carbon source or nitrogen source when configuring the culture medium of MT. After high-temperature sterilization, it is made into a sterile culture medium containing only MT. When the fungi were inoculated into the medium containing only MT by aseptic operation, with time, it was important to observe whether there was mycelium on the surface of the medium. Fungal growth temperature is generally controlled at 28 °C; the culture time should not exceed 10 days; too high temperature and too long incubation affects the growth of fungi and thus influences the fermentation process.

This study proposed a method of co-fermentation of G. lucidum and MT for the first time, and the efficacy of fermentation products was preliminarily explored. This method is convenient, efficient, and environmentally friendly for expanded production. Because the fermentation culture operation of this study needs to be completed in a sterile environment, it is necessary to ensure the standardization of the aseptic procedure and a clean experimental environment.

At present, the fermentation of traditional Chinese medicine is still in its infancy. The basic research mainly focuses on the fermentation process and process control and is limited to the fermentation of a single strain. Compound strains ferment traditional Chinese medicine to produce more biological enzymes and metabolites, with high biological conversion efficiency and more development potential and value. In the future, it may become an important direction for the research of traditional Chinese medicine fermentation.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

This work was supported by grants from the National Natural Science Foundation of China (81973977). The MT samples were identified by Professor Tongxiang-Liu and kept at the School of Pharmacy, Minzu University of China.