Screening Of Active Ingredients Of Pomegranate Flower And Evaluation Of Therapeutic Effect In Mouse Mastitis Model

Purpose and significance:Nowadays,antibiotics are still the first choice for the treatment of mastitis in dairy cows.With the introduction of policies such as"reducing and limiting antibodies"by the Ministry of Agriculture and Rural Affairs of China,veterinary clinics need a safe,efficient,and less toxic drug to replace them for the treatment of related diseases.In our laboratory,we found that the pomegranate flower has certain antibacterial and anti-inflammatory effects,not only can inhibit the growth of E.coli,S.aureus,and S.agalactiae,but also can inhibit the ear swelling caused by xylene in mice.Therefore,this study firstly extracted polysaccharides,polyphenols,alkaloids,and flavonoids active substances from pomegranate flowers and evaluated their effects through in vitro antibacterial tests,safety tests,in vivo anti-inflammatory tests,and mastitis treatment in mice.The aim is to explore the medicinal components of pomegranate flowers and to explore the potential biological efficacy.The significance is to provide data support for the establishment of the standard of active ingredients of pomegranate flower and to provide a scientific basis for the development and utilization of antibacterial and anti-inflammatory drugs,mastitis treatment drugs for dairy cows,and local medicinal herbs in Xinjiang.Methods:1.The active substances of polysaccharides,polyphenols,alkaloids,and flavonoids in pomegranate flowers were extracted and determined by solvent extraction and ultrasound-assisted extraction.The antibacterial effects of crude extracts were detected by punching method and micro broth method.2.The safety of crude extracts of active substances was determined by acute toxicity pre-test and maximum tolerance test,and the crude extracts of pomegranate polysaccharides,polyphenols,alkaloids,and flavonoids of 1 800 mg/kg,900 mg/kg,and 450 mg/kg were intragastrically administered to each group of mice induced by xylene to calculate the degree of auricle swelling and observe pathological changes to evaluate the anti-inflammatory effect of crude extracts of pomegranate flowers in vivo.3.A mouse mastitis model was established by injecting Staphylococcus aureus into the fourth breast area,and each group was treated with 20 m L/kg saline,0.13 g/kg ciprofloxacin,1.8 g/kg pomegranate polysaccharide,1.8 g/kg pomegranate polyphenols,and 1.8 g/kg pomegranate flavonoids by gavage after successful treatment.The therapeutic effects of each crude extract of Garnet on mastitis in mice were evaluated by examining the content of IL-6,TNF-α,the number of colonies,and pathological changes in the mammary tissues of each group at 3,5,and 7 days of treatment.Results:1.The contents of corresponding active substances in the crude extracts of pomegranate flower polysaccharides,polyphenols,alkaloids,and flavonoids were 35.59±0.27%,43.15±0.90%,34.87±0.42%,and 28.18±0.19%,respectively.The crude extracts of polysaccharides,polyphenols,and flavonoids had an inhibitory effect on S.aureus.The bacteriostatic effect at the same concentration was as follows:crude extract of polyphenols>crude flavonoids>crude extract of polysaccharides.2.The LD₅₀values of pomegranate flower polysaccharides,polyphenols,alkaloids,and flavonoids crude extracts were all greater than 1 800 mg/kg,and the maximum tolerated dose was 4.5 g/kg,which was 45 times of the adult dose and belonged to non-serious acute toxic substances.Among them,the medium dose of pomegranate polysaccharide,high dose of polysaccharide,high dose of polyphenol,and high dose of flavonoid could significantly inhibit the swelling of mouse auricle,with the inhibition rate of 31.14%,40.12%,31.14%,and 32.93%,respectively,and also reduce the degree of inflammatory cell infiltration,and the effect was not significantly different from that of dexamethasone（P>0.05）3.The crude extracts of pomegranate flower polyphenols and flavonoids could reduce the degree of inflammatory cell infiltration and tissue damage in mouse mammary tissue,and significantly decrease the number of colonies in mouse mammary tissue（P<0.05）,and the effect of inhibiting IL-6 and TNF-αsecretion was not significantly different from that of the positive drug ciprofloxacin（P>0.05）.Conclusion:Pomegranate flowers contain active ingredients such as polysaccharides,polyphenols,alkaloids,and flavonoids.Among them,pomegranate flower polysaccharides,polyphenols,and flavonoids crude extracts have certain antibacterial and anti-inflammatory effects,not only can inhibit the growth of S.aureus,but also have certain safety and can reduce the ear swelling caused by xylene in mice under the condition of ensuring the safety of mice.In addition,the crude extract of pomegranate flower polyphenols and flavonoids also had good therapeutic effects on mastitis caused by Staphylococcus aureus infection in mice,which could significantly reduce the number of colonies and cytokine content in the mammary tissue of mice and reduce the degree of inflammatory cell infiltration and tissue damage.