The far-reaching significance and prospect of microbial fermentation of compound Chinese medicine tonics I. History of Chinese medicine fermentation 1. History The ancient people of China were very wise and started to apply fermentation method in Chinese medicine concoction more than three thousand years ago . Traditional Chinese medicines, such as: Liu Shen Qu, Tempeh, Sempervivum Qu, Red Qu, Shen Xiang Qu, Dou Huang, etc., are all drugs formed by using the solid fermentation of microorganisms in nature. Such as the manufacture of the half summer tune, “the compendium of materia medica” recorded: “half summer research and powder, ginger juice, alum soup and as a cake, kozo leaves wrapped in a basket, to be born yellow coat, dried in the sun.” Its properties are bitter, pungent, and flat, and it can dispel phlegm and cough, eliminate food stagnation, and treat diarrhea [1], and the fermentation of Semen is itself toxic to some extent, but its original efficacy is enhanced after fermentation, while its toxicity is greatly reduced. Tempeh fermentation is “two or three buckets of black soybeans, soaked in water overnight, drained yellow cooked, spread on the mat … … Artemisia cover, Hou yellow coat on all over …… an urn built solid, mulberry leaves cover three inches thick, sealed clay … … …so seven times” is made, bitter and cold in nature, has the function of relieving symptoms and eliminating irritation, promoting depression and detoxification. Douhuang is also made of fermented black soybeans, “with a bucket of black soybeans. Yellow cooked, spread on a mat covered with artemisia, such as blanching sauce method, to be taken out of the upper yellow sun-dried. Its nature is sweet and warm, can dispel dampness and itch, strengthen the spleen and benefit the qi. Unfermented black soybeans, on the other hand, are bitter and flat in nature and have the effect of activating the blood, diuresis and detoxification. From the above, it can be seen that this traditional fermentation method involves mixing herbs with excipients and fermenting them under natural conditions at room temperature and pressure, using naturally occurring microorganisms, which results in improved efficacy, altered medicinal properties, reduced toxic side effects, and even new efficacy. This traditional fermentation method has been used for a long time. In modern times, there has been a lot of progress in the preparation of single herbs by fermentation, and many studies have been conducted not only on solid fermentation but also on liquid fermentation, and it has been found that both the composition and the original function of the herbs have changed greatly before and after fermentation, which is consistent with the results of the ancient traditional fermentation method. Even more valuable effects, such as anti-cancer, were found. In solid fermentation such as the fermentation of pentospermum, rhubarb, astragalus and so on. It was found that the astringent and antidiarrheal function of pentaphyllum was greatly enhanced after fermentation, while gallic acid was produced, resulting in new pharmacological effects such as antibacterial, antiviral, antifungal, antitumor, anti-allergic and choleretic and bronchodilator. Unfermented pentospermum has none of these effects[2] . Dai Wansheng et al. used fermentation method to concoct rhubarb and changed the content of anthraquinones in the active ingredients of rhubarb. After yeast fermentation, it was found that more rhubarb insoluble quinones were preserved than the traditional method of fermentation, and the content of bound anthraquinones with laxative effect was reduced, which moderated the strong laxative effect of rhubarb and its adverse effects on the gastrointestinal tract in clinical application; meanwhile, the content of free insoluble quinones, which are the main active ingredients of antibacterial and antitumor, was increased by about six times, extending its application[3] . Dr. Wu Zhiyong, a Chinese medicine doctor in Hong Kong, collaborated with Lin Lushan, a professor from the mainland. The fermentation of Astragalus membranaceus using biotechnology has been successful. The medicinal effect was greatly improved, and the fermented Astragalus contained up to five times more Astragalus polygoni than the traditional process, and animal experiments proved that the dosage of fermented Astragalus was 28 times less than that made by the traditional method to produce the same medicinal effect[4] . The strong antitumor activity of camptothecin, along with serious gastrointestinal toxicity, such as inhibiting bone marrow function and causing hemorrhagic cystitis, restricts its further clinical application. 10-hydroxycamptothecin, a structural analogue of camptothecin, has significant efficacy against a variety of cancers with minimal toxic side effects, and the non-toxic Aspergillus flavus strain was used to convert camptothecin into 10-hydroxycamptothecin with a conversion rate of 50 The conversion rate is more than 50%. The conversion rate was more than 50%, which greatly reduced the gastrointestinal toxicity of camptothecin [5]. After fermentation of Panax ginseng roots by Bacillus subtilis in conjunction with Yunnan University and Kunming Institute of Botany, Li Guohong et al. In a study of the saponin composition therein, it was found that ginsenoside RH4 was isolated from the fermented Panax ginseng . This compound was not detected before fermentation. It indicates that this compound was produced by fermentation, probably during the fermentation process. Some saponin components of Panax ginseng fibrous roots were converted into ginsenosides by microorganisms [6]. In liquid fermentation, for example, Jin Jianming et al [7] isolated a new compound, agave glycoside C, from the fermentation broth after fermentation using agave …… Wang Lin et al. fermented Ganoderma lucidum with four herbs, Ephedra, Lycopodium, Jinyinhua, and forsythia, and found that these four herbs had a significant promotion and increase in the biomass of Ganoderma lucidum and made The cough-suppressing and expectorant effects of the fermentation solution of Ganoderma lucidum were higher than those before fermentation with Chinese herbs [8]. Wang Feng et al. studied the fermentation medium of Ganoderma lucidum with the addition of low toxicity, medicinal and dietary herbs, and the results showed that the fermentation broth with the addition of bitter melon had significant hypoglycemic effect [9]. In conclusion, there are many changes in the composition, efficacy, etc. of single herbs, whether solid or liquid, after fermentation. This may be due to the fact that microorganisms have a very powerful ability to decompose and transform substances, and can produce abundant secondary metabolites. In their metabolism and growth and reproduction, they break down various organic substances of Chinese herbs. This results in new medicinal effects. Thus, this fermentation method can enhance and adjust the medicinal properties, improve the efficacy, reduce the toxic side effects and expand the indications more significantly than the general physical or chemical preparation methods. If single herbs have such powerful changes after fermentation, what will happen if compound herbal tonics themselves, which produce very complex changes during decoction and infusion, are fermented again? Such a study has already been done. (1) Current situation in China The research on fermentation of compound Chinese medicine tonics has been started in China since the 80s, but the development is still slow and the reports are not common, and the ones applied to the clinic and the society are even less, but the economic and social benefits brought by the existing research results are very great. For example, Pientzehuang has become a traditional Chinese medicine protected by the state. Sanju Oral Liquid is popular among consumers as a health product beneficial to digestive tract health. Kangfu Ling, as an anti-tumor medicine, is widely used in clinical application. Pientzehuang is the microbial ferment of famous traditional Chinese medicine such as musk, cow’s yellow, snake bile and panax pseudoginseng, which is a good medicine to reduce yellow and swelling in clinic; Sanju Oral Liquid is a nutritional health food, which is made of beef broth, soybean sprout infusion, yeast cream, cane sugar and glucose with the joint fermentation of Bifidobacterium, Lactobacillus acidophilus and DL bacteria, and the product is beneficial to keep the health of gastrointestinal tract. “The main ingredients include Ganoderma lucidum fermenting bacteria, Radix et Rhizoma ginseng, Radix et Rhizoma maitake, Poria lacryma, Coix lacrymae and Radix et Rhizoma Huaiyang, etc. Most of the samples were transformed by biological fermentation and had the effect of inhibiting the growth of sarcoma in mice. Other clinical reports on the fermentation of Liu Wei Di Huang decoction, Yu Ping Feng San decoction were only seen. For example, Dong Mei and Guo Fang [11 ] reported that the fermentation of the traditional formula Liu Wei Di Huang Tang and its fermentation solution had a significant inhibitory effect on liver cancer in mice. 0.3 g/kg of Liu Wei Di Huang fermentation solution administered continuously for two weeks significantly inhibited the growth of liver cancer H22 in mice with a tumor inhibition rate of 3O% , while the same dose of Liu Wei Di Huang decoction had no significant tumor inhibition effect. Moreover, both Liu Wei Di Huang fermentation solution and Liu Wei Di Huang decoction had the effect of elevating peripheral blood leukocytes on bone marrow inhibition caused by cyclophosphamide in mice, and the number of leukocytes in Liu Wei Di Huang decoction group and fermentation solution group was significantly higher than that in the model control group (P< 0.01), and the number of leukocytes in Liu Wei Di Huang fermentation solution group was more than that in the decoction group (P< 0.01). In terms of immunomodulation, the efficacy of Liu Wei Di Huang fermentation solution was significantly better than that of Liu Wei Di Huang decoction [12]. Specifically, the fermentation solution of Liu Wei Di Huang was administered to mice at 300 mg/kg by gavage once daily, and after two weeks of continuous administration, the fermentation solution of Liu Wei Di Huang significantly enhanced the delayed metabolic response (P< 0.05) and the hemolytic value of half was significantly higher (P< 0.05) than that of Liu Wei Di Huang decoction. This indicates that the fermented solution was significantly better than Liu Wei Di Huang decoction in enhancing cellular and humoral immune functions in mice (p< 0.01).8 Xu Qihua [13] et al. fermented the Chinese herbal compound Yu Ping Feng San decoction, and the results showed that the fermented solution of Yu Ping Feng San was more effective than Yu Ping Feng San decoction in enhancing immune functions in mice. Yang Hailong et al [14] took 15 g of each of 10 Chinese herbs including Astragalus and made aqueous extract to study its effect on the biomass and extracellular polysaccharides of Ganoderma lucidum liquid fermentation, and the results showed that at a dose of 187.5 g of raw drug amount per liter of medium, the aqueous extracts of Coix seed, Lycium barbarum, Angelica sinensis, Radix et Rhizoma ginseng, Chinese yam, Radix et Rhizoma codonopsis, Dendrobium and Aloe vera all promoted the growth of Ganoderma lucidum (biomass increase) except for Astragalus , with the significant effect of hyssop and yam, with 2.35 and 2.12 times the biomass of the control, respectively. It can be seen that compound Chinese medicine tonics also have many good effects after fermentation, so can it have toxic side effects on human body? A study has been made specifically on this. Han Chun Yang et al. used a fermented solution of a compound herbal preparation consisting of Astragalus, Radix Codonopsis, Poria, Glycyrrhiza, Semen, Malt and Hawthorn with immune enhancing and growth promoting effects, and studied the toxicological effects of the preparation to evaluate its safety. The mice were found to have normal activity, mental status and diet on the day of administration, and no death or other abnormalities were observed within 1 week, and no abnormal changes were found in the heart, liver, spleen, lung and kidney of each mouse after post-mortem examination. The maximum tolerance of the mice was also measured. The results showed that the mice showed no abnormal changes in mental, appetite and activity compared with the control group, and no abnormalities were found in the internal organs. Therefore, it can be concluded that the preparation is safe for short-term use and the toxicity level should be "practically non-toxic", which is a relatively safe preparation [15]. (2) Foreign research on the fermentation of Chinese herbal medicines is less reported abroad. In the early 1990s, Japanese Kyoichi Kobashi discovered that herbal ingredients such as senna glycosides could be transformed into laxative active ingredients with the help of intestinal bacteria and play a therapeutic role. There are also reports related to the biotransformation of active components of herbal medicines such as glycosides, flavonoids, and coumarins through chemical modification by intestinal bacteria. [16] Japanese, fermented soybeans with Bacillus bacteria, because Bacillus bacteria enzyme system is rich, including amylase, cellulase and protease, and can increase vitamin K containing lus subtilis bacteria ring can eliminate pathogenic bacteria in the small intestine, and its extract has obvious anti-cancer activity and hypotensive effect.6 Korea in recent years is very keen on the research of fermented herbal medicine III. Compound herbal tonics The advantages of fermentation (1) The original active ingredients of traditional Chinese medicine can be preserved Microbial fermentation is a biological transformation under normal temperature and pressure, so it can protect the active ingredients of traditional Chinese medicine from destruction to the maximum extent. (2) It is more conducive to the absorption and utilization of the active ingredients and reduces the burden on the stomach and intestines. In the process of microbial fermentation, large molecules of active ingredients that cannot be directly utilized after the drug enters the body are degraded by microorganisms into small molecules of active substances and are directly utilized, which greatly improves the efficacy of Chinese medicine. Small molecule active substances have higher activity than large molecule substances because they can easily pass the blood-brain barrier and bind to human cell proteins. The molecular weight of fermented Chinese medicine is smaller, thus it has the characteristics of faster and more complete absorption and better therapeutic effect in human body. For example, soy isoflavones are mainly composed of nine isoflavone glycosides and three corresponding ligands. After the action of intestinal microorganisms, some of the glycosides detach to release free dihydroxyisoflavones (soy glycosides) and trihydroxyisoflavones (genistein), which can be effectively absorbed by the body. (3) Generate new effects and expand the scope of indications. Since microorganisms produce various enzymes during the growth process, such as cellulase, ligninase, amylase, protease, lipase, etc. These rich and powerful enzymes can decompose and transform the components of traditional Chinese medicine to form new active ingredients and produce new efficacy, and the microorganisms themselves can also produce rich secondary metabolites during the growth process in the special environment of traditional Chinese medicine, and some of the secondary metabolites themselves are drugs with good efficacy; the secondary metabolites of the microorganisms and certain substances in traditional Chinese medicine have chemical reactions to produce new compounds, thus forming new active ingredients or changing the active ingredients. The secondary metabolites of microorganisms and certain substances in Chinese medicine undergo chemical reactions to produce new compounds, thus forming new active ingredients or changing the content of each active ingredient, and thus having new therapeutic, preventive and health care functions. (4) Reducing the toxic side effects of the drugs themselves and reducing heavy metals, pesticides, antibiotics and other pollutants The decomposition effect of microorganisms can also decompose the toxic substances in Chinese medicine, thus reducing the toxic side effects of the drugs. The powerful decomposition effect of microorganisms may also decompose the heavy metals, pesticides and antibiotics in Chinese medicine into other harmless substances. The above examples of successful fermentation of compound herbal tonics, although few, fully demonstrate the great significance and prospect of fermentation. From them, we can also get new ideas and inspiration. Chinese traditional Chinese medicine has been developed for thousands of years, and there are countless classical and experimental prescriptions available today. From the SARS rampage around the world a few years ago to the seriousness of the bird flu epidemic around the world today, we can also go from there to find new antiviral drugs. Many Chinese medicines have been fermented to produce anti-tumor effects, so there are new avenues for future research into new anti-tumor drugs without toxic side effects. Also, there are many kinds of Chinese medicine preparations, such as pills, creams, powders, etc. Among them, decoction is the most easily absorbed, but it is well known that no one likes the taste of Chinese medicine soup, which is a major reason to hinder the development of Chinese medicine to the world. If we ferment the Chinese medicine soup, not only has a lot of good effects, but also a huge change in taste, not the original bitter and difficult to drink, but become sour and sweet men and women love to drink, then the resulting economic and social benefits will be immeasurable. However, there is no research report on whether the taste of Chinese medicine has changed after fermentation. The ancient people fermented Chinese medicine through natural fermentation, while modern research methods are using a single strain of bacteria to artificially cause fermentation, imagine if we also use natural fermentation of Chinese medicine tonics as the ancients did, similar to kimchi fermentation, what will be the results of its efficacy and taste? The author will try to see. In conclusion, due to the unique advantages of compound Chinese medicine itself in the research and development of new Chinese medicine, the application of microbial fermentation method can not only expand the scope of Chinese medicine treatment, but also provide new ideas and technical means in the research of dosage form improvement, taste change and creation of new drugs, which is expected to inject new vitality and bring explosive changes to the research and development of new Chinese medicine, with broad prospects and huge development space. It is promising and has great development potential. It is also conducive to promoting the modernization and internationalization of Chinese medicine, improving the international competitiveness of Chinese medicine, and making new contributions to the development of Chinese medicine to the world for the benefit of all mankind.