Development And Preliminary Clinical Application Of Lithocarpus Polystachyus Oral Liquid

Necrotic Enteritis（NE）caused by Clostridium perfringens（C.perfringen）infection in livestock and poultry is one of the most important diseases with a wide prevalence,causing huge economic losses every year and seriously threatening the development of the farming industry.Currently,antimicrobial drugs or feed additives are still the first choice for the control of NE,but the increasing problem of bacterial resistance due to the long-term massive and irregular use of antimicrobial drugs,coupled with the limitations of antibiotic use during the rest period of chicken feeding,poses a great challenge for the control of NE.In response to the widespread bacterial resistance problem worldwide,different countries and regions are taking corresponding countermeasures.In the context of “antibacterial ban”,“antibacterial restriction”and“antibacterial reduction” policies advocated by the Ministry of Agriculture and Rural Affairs of China,it is urgent to develop alternative antibiotic strategies or novel drugs for the control of C.perfringen infected chicken NE in order to further meet the needs of green,healthy and ecological farming.C.perfringen types A and C are the main serotypic pathogenic bacteria causing NE,and the organism plays a key role in the pathogenic process by adhering directly to host intestinal cells with the help of the Type IV Pili（TFP）function.It was shown that the pathogenicity of C.perfringen was significantly reduced or even lost after TFP knockout or deletion,but the normal growth of the bacterium was not affected.Targeting C.perfringen TFP for alternative antibiotic drug screening and new veterinary drug development by targeting bacterial non-life essential components becomes an ideal solution strategy.Chinese medicine is a treasure of the Chinese nation,and its effectiveness has been tested by clinical use for a long time and has played a significant positive role in the world health safety events in recent years.Compared with traditional antibiotics,active ingredients from natural plants have the advantages of more pharmacological activity,lower price,clear sources and low induced resistance,which have very broad research value and application prospects.In our laboratory,the TFP inhibitor screening platform was established,and after extensive screening,we found that the TFP-mediated adhesion function was significantly inhibited by the active ingredient from traditional Chinese medicine,and it has good antibacterial activity in vitro,which has good therapeutic effect on chicken NE caused by C.perfringen infection.Based on the previous study,we backtracked the Chinese herbal medicines according to their active ingredients and found that phloretin is held in small amount in most natural plants,which is difficult to extract and utilize effectively.After reviewing the literature and preliminary screening,it was found that the main component of Lithocarpus Polystachyus is phlorizin,which is relatively abundant and easy to extract.Phlorizin will be completely converted to phloretin after enzymatic digestion in the intestine.This research intends to select Lithocarpus Polystachyus with good druggability property,and prepare the oral liquid of Chinese medicine by prescription processes for the prevention of NE and suitable for group administration mode in chicken farming industry.Firstly,the herbal origin of Lithocarpus Polystachyus was screened for its content,and the best extraction process was determined by orthogonal test;the prescription of oral solution was determined by screening the excipients according to the physicochemical properties of phlorizin and the characteristics of oral solution dosage form;after the laboratory trial and pilot production test,the prescription components and preparation process of Lithocarpus Polystachyus oral liquid（LPOL）were determined as follows: Lithocarpus Polystachyus was added to 25 times the volume of purified water in advance.The extracts were filtered to remove the residue,combined with the filtrate,decompressed and concentrated to a relative density of >1.04,added anhydrous sodium sulfite according to the prescribed amount,adjusted the p H value to7.0,added water to the full amount and then filled and sterilized.Next,the content of phlorizin in LPOL was detected by high performance liquid chromatography（HPLC）and validated methodologically,the stability of LPOL was examined by accelerated test and long-term test investigation,the expiration date of LPOL was formulated,and the quality standard of LPOL was initially established.Next,the in vitro phenotypic identification tests such as biofilm,sliding movement and cell adhesion were conducted to verify the inhibitory effect of LOPL on TFP function.Finally,we investigated the preventive effect of the LPOL on NE in artificially infected chickens by establishing a model of NE,and examined the indexes of material to meat ratio,immune organ index,ileal histopathological sections and ileal colony number,and found that the LPOL had good preventive effect on C.perfringen-infected chickens with NE.In conclusion,this study successfully prepared a stable and effective LPOL,which can significantly inhibit the sliding movement,biofilm formation and cell adhesion of C.perfringen,and then inhibit the function of TFP;it has a good effect on the prevention of NE in chickens caused by C.perfringen infection.This study will provide a new drug development strategy for the infection and prevention and control of drug-resistant bacteria in veterinary clinics,and also provide an effective herbal preparation for NE caused by C.perfringen infection.