Study on the effect of traditional Chinese medicine extracts on the biofilm of Staphylococcus aureus
Bacterial biofilm (BF) refers to a special functional structure formed by bacteria to adapt to the natural environment and gradually attach to the surface of living or non living organisms during the growth process. It is a bacterial community composed of bacterial cells and the water containing polymeric substrates secreted by the bacteria themselves. The formation of BF is equivalent to an effective barrier against external invasion, which can enable bacteria to resist the host immune system and the killing effect of antibiotics, thereby developing resistance to antibiotics (10-1000 times higher than in a planktonic state), making it difficult to clear infections and becoming a major potential source of difficult to treat infections today. Therefore, using only traditional antibiotics to treat bacterial biofilm infections will greatly reduce the effectiveness.
Staphylococcus aureus is a common foodborne pathogen that is widely present in natural environments. An important characteristic of Staphylococcus aureus is the ability to form biofilms at the site of infection, making it more difficult to treat infections caused by these strains. To treat Staphylococcus aureus in the state of biofilm, higher concentrations of antibiotics must be used, but excessive use of antibiotics can lead to the development of drug resistance and may cause the occurrence of synergistic bacterial infections. In recent years, some new strategies have been reported to address biofilm infections, including density sensing system quenchers and antibiotic combination density sensing system quenchers, but these methods have not been approved for use. Therefore, finding natural antibacterial agents from nature that can inhibit biofilm formation has become an important choice.
The World Health Organization predicts that 25% of modern medicines worldwide are directly or indirectly related to medicinal plants, of which 94 out of 162 approved antimicrobial drugs from 1981 to 2019 are derived from natural products. Medicinal plants are one of the main sources of bioactive molecules, but the screening of antimicrobial agents against biofilms from medicinal plants is still in its early stages. At present, it has been reported internationally that extracts from medicinal plants such as Scutellaria baicalensis, Scutellaria baicalensis, and Curcuma longa exhibit activity in inhibiting the biofilm of Staphylococcus aureus. Traditional Chinese medicine has the characteristics of multiple components, multiple targets, and multiple effects, and is not prone to developing drug resistance, therefore it has good application prospects in antibacterial therapy. At present, there are also some reports on the inhibition of Staphylococcus aureus biofilm by Chinese herbal medicine: Wang et al. found that Ginkgo biloba outer seed coat extract inhibited the formation of Staphylococcus aureus biofilm in a concentration dependent manner; Li et al. found that traditional Chinese medicine Hezi can significantly inhibit the formation of biofilms in Staphylococcus aureus, and also has good antibacterial effects; Liao et al. found that the aqueous extract of Houttuynia cordata can effectively inhibit the formation of biofilm in Staphylococcus aureus. Therefore, medicinal plants are an important source for exploring pathogen biofilm inhibitors. However, most of the research on these medicinal plants is based on analyzing their biofilm function under the premise of their antibacterial activity. This may interfere with the judgment of test results due to the antibacterial effect of extracts, or may miss the screening of plant extracts that do not have antibacterial activity but have biofilm inhibitory activity. Therefore, this study selected 16 local characteristic Chinese medicinal herbs such as rhubarb as research objects, analyzed the effects of different solvent extracts on the growth of Staphylococcus aureus, and based on this, selected sub inhibitory concentration extracts to analyze their effects on biofilm formation, aiming to provide guidance for the research and drug development of traditional Chinese medicine anti biofilm formation.

Antibiotics are currently the main strategy for treating cases related to Staphylococcus aureus infection. However, the formation of Staphylococcus aureus biofilm enhances its resistance to antibiotics, thereby reducing the effectiveness of antibiotics. Therefore, inhibiting or eliminating biofilm formation before using antibiotics will greatly improve the effectiveness of antibiotics. This is also an effective way to solve bacterial resistance and chronic infections caused by biofilm formation in clinical practice. This experiment selected 16 traditional Chinese medicinal herbs to study their antibacterial activity. The results showed that most of the extracts had antibacterial activity against Staphylococcus aureus, while Longgu Feng had no antibacterial activity against Staphylococcus aureus, which is consistent with the experimental results of other researchers. Longgu Feng is the stem of the Cyatheaceae plant Cyathea, which mainly contains chemical components such as terpenes, flavonoids, phenolic acids, and steroids; At present, it has been found to have various pharmacological activities such as dispelling wind and dampness, promoting blood circulation and dispersing blood stasis, clearing heat and detoxifying, as well as anti-tumor effects. Although the antibacterial effect of Longgu Feng on Staphylococcus aureus is not significant, there is still potential for exploring other bioactive functions. Many plant derived extracts have antibacterial activity against Staphylococcus aureus, but the composition of the extracts is complex and the target of action is unclear. Therefore, it is still a long way to completely replace antibiotics with traditional Chinese medicine.

There are multiple mechanisms of drug resistance in Staphylococcus aureus, among which the problem of drug resistance caused by biofilm formation has received increasing attention. At present, research has been conducted to control the phenomenon of pathogenic bacteria resistance caused by biofilm formation by removing or inhibiting biofilm formation through chemical or biological methods. In particular, the development of traditional Chinese medicine drugs against Staphylococcus aureus biofilm has been used to effectively reduce its resistance, especially different types of flavonoids have the activity of inhibiting the growth and biofilm formation of Staphylococcus aureus. This experiment found that the water and alcohol extracts of rhubarb, Polygonum cuspidatum, Qianghuo, Ligustrum lucidum, Scutellaria baicalensis, Scutellaria baicalensis, Scutellaria baicalensis, Scutellaria baicalensis, and peppermint all have significant inhibitory effects on the biofilm of Staphylococcus aureus. Literature review of the compound components of the above herbs revealed that rhubarb, Polygonum cuspidatum, Ligustrum lucidum, Scutellaria baicalensis, Scutellaria baicalensis, Scutellaria baicalensis, Scutellaria baicalensis, Scutellaria baicalensis, and peppermint all contain flavonoids. However, whether the flavonoids contained in them are related to their inhibition of the formation of Staphylococcus aureus biofilm still needs further verification in subsequent experiments. In addition, research has reported that rhubarb and Polygonum cuspidatum contain a large amount of anthraquinone substance emodin, and Xiang et al. have also confirmed that aloe vera contains anthraquinone substance emodin and has the activity of inhibiting the formation of Staphylococcus aureus biofilm. Therefore, it is speculated that the inhibition of Staphylococcus aureus biofilm by rhubarb and Polygonum cuspidatum extracts in this experiment may be related to their presence of anthraquinone substance emodin.

At present, plant active ingredients have great potential in intervening in bacterial biofilms, as traditional antibiotics are difficult to penetrate into bacterial biofilms and are prone to residue and resistance issues. Therefore, using plant active ingredients alone is difficult to control bacterial biofilm infections. However, using low residue and low toxicity plant active ingredients together with antibiotics can effectively reduce the amount of drugs used, reduce their toxic side effects, and provide the ability for drugs to penetrate bacterial biofilms. Dey et al. found that combining naringin with tetracycline and ciprofloxacin can enhance the ability of these two antibiotics to inhibit biofilm formation in Pseudomonas aeruginosa. Yan et al. found that the combination of thymol and benzylpenicillin can significantly enhance the inhibitory and clearing effects on the biofilm of Staphylococcus aureus. In addition, traditional Chinese medicine extracts can also be made into nano formulations, as traditional antibiotics are difficult to penetrate into highly structured mature biofilms. However, plant active ingredients can be made into nano inhibitors, which are beneficial for drugs to enter bacterial biofilms and exert their therapeutic effects. For example, nano berberine can inhibit the formation of biofilms of Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus, clear their mature biofilms, and reduce the expression of virulence factor genes related to their biofilms. So combining plant active ingredients with other drugs for the treatment of biofilm infections may achieve good results and have important application prospects.

In addition, the extraction method of traditional Chinese medicine can also affect the results of drug activity experiments. Chu et al. used five different extraction methods to extract the active substances of traditional Chinese medicine Luo Xianzi, and found that the product obtained by water extraction had the strongest antibacterial activity. Zhang et al. used water extraction, alcohol extraction, and supercritical CO2 to extract the active ingredients of Schisandra chinensis. They found that the antibacterial activity of the drug active substance obtained by alcohol extraction was significantly higher than that of the other two solvent extracts. Iqbal et al. found that the methanol, n-hexane, ethyl acetate, n-butanol, and water extracts of Araceae have different antibacterial effects on the tested pathogens. Compound composition analysis revealed significant differences in the types and abundance of components in these different solvent extracts. Zhang et al. used 7 different solvents to extract Angelica root and found significant differences in the biological activity and compound composition of the extracts from different solvents. These results suggest that the types and abundances of components in plant extracts extracted with different solvents may vary. In this experiment, water and ethanol were used as solvents to extract active substances from traditional Chinese medicine. The results showed that the water extracts of four drugs, namely Tie Lingxian, Pixian Grass, Feng Wei Grass, and Jing Jie, had a promoting effect on the formation of Staphylococcus aureus biofilm, while their alcohol extracts had an inhibitory effect on the formation of Staphylococcus aureus biofilm. It is speculated that the reason for this may be the difference in the composition and abundance of the substances extracted by the two different extraction methods, which leads to different effects on the formation of Staphylococcus aureus biofilm. This discovery provides us with some hints that substances that inhibit biofilms and substances that promote biofilms can be extracted using different solvents. If future development of inhibitors for Tielingxian, Pixian Grass, Phoenix Tail Grass, and Schizonepeta tenuifolia is to consider extracting them with water first to remove impurities, and then extracting their extracts again with ethanol. Therefore, the results of this experiment may provide a reference for the development of natural drugs with inhibitory effects on Staphylococcus aureus biofilm, thereby reducing the use of antibiotics.
The results of this study showed that most of the extracts from these 16 Chinese medicinal herbs have inhibitory effects on the biofilm of Staphylococcus aureus, but some extracts have high concentrations of action, such as the water extracts of Qianghuo and Ligustrum lucidum with concentrations exceeding 60mg/mL. The actual value of these high concentration extracts is not high, so the development of these medicinal herbs needs to comprehensively consider factors such as the content or potency of the effective active ingredients in their extracts. At present, the research on traditional Chinese medicine for anti biofilm treatment in China is still in the exploratory stage, and the study of its active ingredients and targets is not deep enough. In the future, based on the screening of active medicinal plants for anti biofilm treatment, we also need to identify the components of their active compounds and study their targets and mechanisms of action at the molecular biology level.