The effects of 19 traditional Chinese medicine extracts on the growth and biofilm of marine pathogenic Vibrio alginolyticus

Vibrio is a type of bacteria widely present worldwide, and the outbreaks and epidemics of vibriosis caused by it not only cause huge economic losses to the marine aquaculture industry, but also pose a serious threat to human health. Vibrio alginolyticus is a halophilic Gram negative short rod-shaped bacterium belonging to the Vibrio genus of the Vibrionaceae family. It is the most abundant marine Vibrio and is widely distributed in seawater and estuaries worldwide. It is a common marine pathogen that can cause massive mortality of marine fish, shrimp, and shellfish. In natural habitats, bacteria of the Vibrio genus can almost always form self protective biofilms, which not only make it easier for bacteria to resist external environmental pressures, but also provide an ideal platform for them to more easily invade and infect other organisms.
Bacterial biofilm is a structural bacterial community composed of extracellular matrix (including extracellular polysaccharides, proteins, and eDNA) secreted by bacterial communities to encapsulate themselves. Its formation enables bacteria to resist the host immune system and the killing effect of antibiotics, resulting in high resistance to antibiotics and making infections difficult to clear. At present, the prevention and treatment of bacterial biofilms has become one of the research hotspots. However, due to the characteristics of bacterial biofilms, they have high drug resistance. To fundamentally eliminate bacteria, drugs not only need to have bactericidal effects on bacteria, but also need to be able to pass through the biofilm without being blocked or neutralized by it. However, there are currently few drugs that can achieve such an effect. In response to this challenge, traditional treatment methods and antibiotic use are increasingly restricted. Therefore, it is of great significance to study and find new and sustainable methods to control the growth and infection of Vibrio alginolyticus. Among them, the potential of traditional Chinese medicine has aroused widespread interest. China is a country with abundant resources of traditional Chinese medicine, which has a long history of use, clear sources and effects. It has unique resource advantages in the screening and research of antibacterial drugs. Compared with synthetic antibiotics, the active ingredients extracted from traditional Chinese medicine contain a large number of small molecule chemical components with unique and diverse structural types, which have great therapeutic potential and minimal side effects. Therefore, it provides a broad space for finding new mechanisms of action for anti biofilm drugs. This study selected 19 traditional Chinese medicines as research objects and extracted them with different solvents to investigate the effects of their extracts on the growth and biofilm formation of marine Vibrio alginolyticus, providing guidance for the development of traditional Chinese medicine drugs against biofilm formation.

Bacteria in the biofilm state not only have significantly improved resistance to antibiotics, but also their pathogenic ability has become stronger. Therefore, traditional antibiotic treatment for bacterial biofilm infections is greatly limited. At present, the prevention and control of bacterial biofilm has become a research hotspot. Numerous studies have shown that plant extracts and their active ingredients have the ability to resist bacterial biofilm infections, and have the advantages of low cost, easy processing, good antibacterial properties, and less susceptibility to bacterial resistance. Therefore, exploring suitable plant extracts and their active ingredients will become an effective strategy for preventing and treating bacterial biofilm infections.
This study selected 19 traditional Chinese medicines and extracted them using different solvents to investigate the effects of their extracts on the growth and biofilm formation of marine Vibrio alginolyticus. The results showed that different solvent extracts of 19 traditional Chinese medicines exhibited different antibacterial activities against Vibrio alginolyticus, among which 12 water extracts of traditional Chinese medicines had antibacterial activity, 17 ethanol extracts of traditional Chinese medicines had antibacterial activity, and 15 ethyl acetate extracts of traditional Chinese medicines had antibacterial activity. Among the different solvent extracts of 19 kinds of traditional Chinese medicines, most of the extracts promoted the formation of the biofilm of Vibrio alginolyticus, while Gongding perfume extract, Paris polyphylla and Anemarrhena asphodeloides ethanol extract, Myrtle and Forsythia suspensa ethyl acetate extract significantly inhibited the formation of the biofilm of Vibrio alginolyticus.
Syringa is a dried flower bud of the genus Syringa in the family Myrtle, widely used as a spice and medicinal herb. Chen et al. used boiling water decoction method to study the antibacterial effect of 100 Chinese herbal medicines on Vibrio alginolyticus. The results showed that after adjusting the pH value of the medicine to neutral, male cloves could effectively inhibit and kill Vibrio alginolyticus, with a MIC value of 125mg/mL. This study used reflux extraction method and different solvents for drug extraction. The results showed that all three solvent extracts of Eucommia ulmoides had significant antibacterial effects. The MIC of the water extract reached 50mg/mL, and the extraction effect of the active ingredients of the drug was significantly improved. Moreover, the organic solvent extracted Eucommia ulmoides had stronger antibacterial activity. The MIC of the ethanol extract and ethyl acetate extract reached 1.6mg/mL and 3.1mg/mL, respectively. It is speculated that this is closely related to the chemical properties of the antibacterial ingredients it contains. Syringa is rich in eugenol, acetyl eugenol, eugenol, methyl salicylate, methyl n-pentyl ketone, and benzaldehyde, among which eugenol is the most important antibacterial component. Therefore, compared with the water extract of Syringa, organic solvent extract has higher antibacterial activity. In addition, the extract of Gongding perfume also showed a significant inhibitory effect on the biofilm formation of Vibrio alginolyticus, and the extract of Gongding Perfume and ethyl acetate still had a significant inhibitory effect on the biofilm formation of Vibrio alginolyticus at a low working concentration (0.8mg/mL). Therefore, both as antibacterial agents and anti biofilm preparations, Syringa has high utilization value.
Chonglou is a general term for plants in the Liliaceae family, belonging to the genus Chonglou. Currently, 241 compounds have been isolated and identified, such as steroidal saponins, pentacyclic triterpenoids, sterols, flavonoids, polysaccharides, alkaloids, etc., which have analgesic, hemostatic, anti-tumor, antibacterial and other activities. Among them, there are 136 steroidal saponins, which are the main active ingredients of Chonglou. This study found that both ethanol extract and ethyl acetate extract of Chonglou have strong antibacterial activity, with MIC of 12.5mg/mL. Ethanol extract has a significant inhibitory effect on the formation of biofilm of Vibrio alginolyticus, while water extract has neither antibacterial nor anti biofilm activity. This is because the main active ingredient of Chonglou, steroidal saponins, is insoluble in water and easily soluble in organic solvents such as methanol, ethanol, chloroform, and ether. Therefore, ethanol extract has better antibacterial and anti biofilm effects.
The active ingredients, efficacy, and toxicity of traditional Chinese medicine will change after processing. Zhimu is the dried rhizome of the lily family plant Zhimu, which mainly contains active ingredients such as steroidal saponins, flavonoids, polysaccharides, lignans, organic acids, and benzophenones. Among them, steroidal saponins are the most biologically active ingredients in Zhimu. Bai et al. conducted a comparative study on the changes in the content of saponins, flavonoids, and polysaccharides in Ganoderma lucidum before and after processing. The results showed that after salt roasting, the content of various monomeric saponins, mangiferin, and polysaccharides in Ganoderma lucidum increased; After being roasted with alcohol, the content of saponins I, AIII, mangiferin, neomangiferin, isomangiferin, and polysaccharide components in Anemarrhena chinensis increased; After stir frying, the content of sarsapogenin, saponins I from Anemarrhena chinensis, mangiferin, and polysaccharide components all increased. This study compared and analyzed the effects of different solvent extracts of Ganoderma lucidum and its three processed products, salt Ganoderma lucidum, wine Ganoderma lucidum, and stir fried Ganoderma lucidum, on the growth and biofilm formation of Vibrio alginolyticus. The results showed that the antibacterial activity varied with different processing methods, but the effects on biofilm formation of Vibrio alginolyticus showed certain similarities. It is speculated that the changes in its activity are closely related to the changes in the chemical composition of Ganoderma lucidum after processing, but further research is needed on the corresponding relationship between its activity and chemical composition.
The research on plant extracts and their active ingredients has shown broad application prospects in the field of antibacterial biofilm. The development of antibacterial biofilm preparations has important reference significance for the prevention and treatment of aquatic animal diseases caused by pathogenic Vibrio. In the following research, we will identify the active ingredients of traditional Chinese medicine extracts and analyze their inhibitory mechanisms on the formation of pathogenic Vibrio biofilms, in order to develop safe and effective antibacterial biofilm preparations.