The Study Of Stilbenes From Cajanus Cajan Leaves By LC-MS And Its Anti-MRSA Activity

ObjectiveAs the largest organ of the human body,the skin,resides in the outermost layer of the human body which is the first line of defense of the human immune system.Once damaged,such as common clinical burns,surgical trauma,accidental trauma,the risk of skin infection with bacteria do increase.The most common skin infection is Staphylococcus aureus which can cause life-threatening infections.It is going to be the leading cause of death in hospitalized patients for the resistance of the genes carried by this strain to many antibiotics,including the most effective and widely used Penicillin and its derivatives.Since the advent of penicillin,the use rate of penicillin has continued to increase,and natural selection has gradually made some Staphylococcus aureus show resistance.Methicillin has effectively controlled the infection of resistant bacteria,derived from which MRSA and MSSA were coming into being.Not only to methicillin,but also aminoglycosides,tetracyclines,and macrolides is MRSA resistant to.Since the discovery of MRSA in the United Kingdom in 1961,the MRSA infecting rate is increasing globally and becoming a common clinically bacteria.However,the abuse of antibiotics brings about bacterial resistance,without control which may enter the "post-antibiotic era" with no drug available.The broad-spectrum resistance problem of MRSA infection has become a difficult problem globally.The extraction of monomer compounds from Chinese herbal medicines for the treatment of clinical diseases is playing an increasingly important role in modern medical systems.The merits,not easy to resist,low price,obvious effect with small side effects and so on,make it suitable for patients to take.In the 21st century,strengthening the research and development of Chinese herbal medicines can not only promote the knowledge and experience of the Chinese nation for thousands of years,but also provide practical and effective experiences and treatment measures for the prevention and control of worldwide diseases.In this study,LC-MS-oriented separation technology was used to obtain a series of stilbene compounds 1-5 in pigeon pea leaves.A clinically isolated standard strain MRSA JCSC4744 was selected to conduct the first series of studies on the antibacterial activity and mechanism of stilbene compounds in vitro and in vivo looking for leading compounds against MRSA infections.Methods1.Enrichment and separation of stilbene compounds.Pigeon pea leaves were dried and weighed(20 kg),and immersed in 95%industrial alcohol at room temperature for 7 days,3 times.The extracts were combined and concentrated under reduced pressure to a non-ethanol flavor to obtain a total extract(3.53 kg).The extract was suspended in water,extracted with ethyl acetate,and the extract was concentrated under reduced pressure to obtain an ethyl acetate(EtOAc)portion(1.34 kg).According to the literature,the molecular weight of stilbene compounds in pigeon pea is basically in the range of 250-350.LC-MS analysis of the EtOAc fractions obtained is combined with the characteristics of strong ultraviolet absorption of the structure of stilbene compounds to screen out stilbene Based on the retention time of stilbene-like compounds in HPLC,appropriate column chromatography methods were used to further enrich and isolate and purify the stilbene-like compounds to obtain monomer compounds.For the isolated compounds,the structure of the compounds was determined by1D NMR data such as 1H,13C,and DEPT,combined with physicochemical constants such as mass spectrometry.2.Resazurin colorimetric method was used for MIC and the MBC of compounds 1-5.The microdilution method was used in a 96-well plate to make the drug concentration from wells 1 to 10 to 100μg/mL,50μg/mL,25 μg/mL,12.μg/mL,6.25 μg/mL,3.12μg/mL.,1.56 μg/mL,0.78μg/mL;0.39μg/mL,0 μg/mL.Add 100 μL of MH medium containing 106 CFU/mL of test bacteria and resazurin developer(0.06 mg/mL)respectively.Vancomycin severed as the positive group.Three replicates for each antibacterial agent.After 12 h incubation,the MIC was defined as the minimum concentration to maintain a blue color.MBC is the lowest drug concentration capable of killing>99.9%of the initial bacterial content in the CLSI.3.Dynamic bactericidal curve of compounds 1-5 against MRSA.Take longistyline A(LLA)as an example,1.8 mL of MRS A bacteria solution diluted to 1 × 106 CFU/mL into a 24-well plate,and add 0.2 mL DMSO,7.8μg/mL vancomycin,62.5μg/mL vancomycin,15.6 μg/mL LLA and 125μg/mL LLA.Then DMSO(negative control),0.78 μg/mL(1 ×MIC)vancomycin,6.25μg/mL(8 × MIC)vancomycin,1.56μg/mL(1 × MIC)LLA,12.5μg/mL(8 × MIC)LLA treatment solution;incubate 24-well plates in a 37℃ incubator,and incubate at 0 h,0.5 h,2 h,4 h,8 h,and 24 h were used to determine the number of bacteria in each group;cultivating time was taken as the abscissa,and the logarithm of the number of bacteria was taken as the ordinate to draw the bactericidal curve of LLA and analyzed dynamic bactericidal effect.The bactericidal curve of compound 2-5 was determined by the same method.4.Anti-MRSA activity test of LLA in vivo.48 female Balb/c mices(aged 6-8 weeks)divided into control group,MRSA group,MRSA+Van and MRSA+LLA group.After shaving hair from the back of the mice,a full thickness wound(1 cm in diameter)was created.Each wound was inoculated with 50 μL of MRS A JCSC4744 cells containing 106 CFU,except for the control group.Then,5 min later the same concentration of drug(10 μg of LLA or Van)dissolved in 50 μL of PBS was applied to the wound for the LLA and Van treatment groups,respectively.The weight and wound size of each mouse were recorded every two days;on the third day of MRSA infection,4 mice were randomly selected to measure the number of wound bacteria,serum tumor necrosis factor-alpha(TNF-α),Interleukin-6(IL-6)content;on the 3rd day after MRSA infection,4 mice were randomly selected and the wound skin tissues Harvested tissue was fixed in 4%paraformaldehyde.Solutions of Gram stain(Solarbio)and Giemsa stain(Sigma)were used for histological staining.Antibodies against CD8a were utilised for immunohistochemistry(ICH)examination was performed by three independent investigators.Images were taken using a BX-53 microscope.5.Cytoplasmic membrane depolarisation assay.MRSA JCSC4744 was suspended and washed at least three times with 5 mM HEPES buffer solution to obtain a suspension with an optical density at 600 nm(OD600)of 0.1.After culturing the suspension with 0.4μM of 3,3-dipropylthiacarbocyanine(DiSC3-5)at 37℃ in the dark for 20 min,100 mM KCl was added to the mixture.Serial concentrations of LLA or an equal volume of DMSO(negative control)were added and the fluorescence signal was detected at an excitation wavelength of 660 nm and an emission wavelength of 675 nm.SYTOX Green assay.Briefly,MRSA JCSC4744 cells were adjusted in 0.85%NaCl buffer to an OD600 of 0.2 and were cultured with indicated concentrations of LLA,melittin or DMSO(negative control),respectively,for 4 h.SYTOX Green was added to the mixture to achieve a final concentration of 3 μM and the mixture was incubated for 5 min.Fluorescence was monitored with 504 nm as excitation wavelength and 523 nm as emission wavelength.Scanning electron microscopy(SEM).MRSA JCSC4744 cells treated with LLA at 8 × MIC or the same volume of DMSO(negative control)were incubated for 4 h at 37℃.After washing at least three times with PBS,cells were pre-fixed in PBS containing 2.5%paraformaldehyde and 2%glutaraldehyde at 4℃ overnight.Samples were then post-fixed for 2.5 h in 1%osmium tetroxide.After washing another three times with PBS,cells were dewatered by graded ethanol series and were freeze-dried.Subsequently,samples were coated with gold and were examined and photographed.Results1.Under the guidance of LC-MS,5 compounds were isolated and purified from the ethyl acetate fractions of pigeon pea leaves,and their structures were identified by comparing NMR data with the literature:longistyline A(1),cajanine(2),longistyline C(3),2-hydroxy-4-methoxy-3-isopentenyl-6-phenethylbenzoic acid(4),3-methoxy-5-hydroxy-2-(3-methyl-2-butenylbibenzyl)(5).2.The results of MIC and MBC experiments show that for MRSA(JCSC4744,JCSC4469),the MIC and MBC of 1 are both 1.56 μg/mL;the MIC and MBC of 2 are both 6.25 μg/mL;MIC and MBC of 3 both are 12.5 μg/mL;MIC and MBC of 4 are 3.12-6.25;MIC of 5 is 50μg/mL;Van MIC is 0.78 μg/mL,MBC is 1.56 μg/mL.Compounds 1-5 also show good bactericidal activity against S.aureus CMCC26003;For B.cereus CMCC63302 strain,compounds 1-4 have smaller MIC and MBC values than Van,however they have no antibacterial activity against E.coil ATCC8739.3.MRSA JCSC4744 was selected to perform a time-kill dynamic study.A remarkable antimicrobial efficacy of 1-5 was observed,with a rapid reduction in CFU within the first 0.5 h of exposure at as concentration of 8 × MIC.At 1 × MIC,a 3-log decrease(99.9%reduction)in MRSA survival was rapidly achieved after 8 h of exposure,whereas Van required 24 h to attain a 3-log reduction with the same fold MIC value4.Day 3,infected mice treated with LLA or Van showed initial healing of lesions.In contrast,apparent suppuration collected in the excision region could be easily observed in infected mice without treatment,and they died within 5 days.By Day 7,scabs could be seen in all of the remaining groups,with the control group(no MRSA)showing the best wound healing efficacy,followed by the LLA treated group.Moreover,the LLA and Van treatment groups shared almost identical full closure times with the control group,as well as significantly alleviated weight loss induced by MRSA infection.Gram staining of sections from wounded tissue was performed.Gram-positive micro-organisms were almost absent in infected mice treated with LLA,similar to treatment with Van.To compare the antibacterial efficacy of local treatment with LLA and Van,lesion sections were subsequently cultured to calculate the CFU of MRSA after 3 days.Consistent with the Gram staining results,bacterial outgrowth was significantly decreased due to treatment with LLA compared with the MRSA group(P<0.01).Moreover,LLA treatment was significantly more efficient at reducing MRSA infection than Van treatment(P<0.05).Staining against CD8 was dense in the vicinity of the wound for untreated mice,whilst it was remarkably reduced in the presence of LLA.In addition,the serum concentrations of inflammatory ytokines in the infected group were 606.57 ± 68.99 pg/mL for IL-6 and 280.58±42.27 pg/mL for TNF-α,whilst LLA treatment significantly reduced the induction of both cytokines to 180.74±10.78 pg/mL and 87.25± 10.19 pg/mL(P<0.05,P<0.01),respectively.5.The fluorescent probe DiSC3-5 was utilised in a cytoplasmic membrane depolarisation assay and the results demonstrated that membrane depolarisation by 1-5 was associated with its bactericidal ability,the fluorescence signal was positively correlated with time and dose;SYTOX green experimental results showed that compared with the blank group,the concentration was 0.5 × MIC,1 × MIC,2 × MIC,4 × MIC,8 × MIC stilbene compounds After 4 h of 1-5 treatment,the fluorescence intensity increased in a concentration-dependent manner;compared with the normal group.SEM was further consistent with the aforementioned studies,showing that 1-5 could induce bacterial cell lysis.Conclusion1.Pigeon pea are rich in stilbene compounds.2.The compounds 1-5 show different levels of antibacterial activity against MRSA.Among them,the MIC of LLA against MRSA JCSC4744 is 1.56 μg/mL,showing strong antibacterial activity.3.The time-killing curve showed that the compounds 1-5 showed high bactericidal activity at the concentrations of 1 x MIC and 8 x MIC.4.In vivo,antibacterial experiments show that LLA is less toxic and alleviate weight loss caused by MRSA infection,accelerate wound healing,reduce mortality,reduce the number of wound bacteria and TNF-α and IL-6 inflammatory factors.5.The antibacterial mechanism of compounds 1-5 are related to the dissipation of bacterial cell membrane potential,destruction of membrane permeability,and induction of bacterial lysis.