Study On Isolation, Identification And Mechanism Of Active Components From Arisaema Sinii Krause Against Mycobacterium Tuberculosis

Tuberculosis（TB）is a highly infectious and highly lethal chronic infectious disease caused by Mycobacterium tuberculosis（MTB）.In recent years,due to the emergence of multidrug-resistant tuberculosis（MDR-TB）and extensively drug resistant tuberculosis（XDR-TB）,and the cross-infection of tuberculosis and AIDS,the death rate of tuberculosis has surpassed that of AIDS,ranking ninth.Health poses a serious threat.Although there are drugs for treating tuberculosis,the treatment time is too long（6-9 months）,causing some patients to not take the medicine,causing the emergence of drug-resistant bacteria;in addition,due to the obvious hepatotoxicity of some anti-tuberculosis drugs,long-term medication can lead to the patient’s liver damage.Therefore,the discovery of new drugs and shortening the treatment time of tuberculosis are hot topics in recent years.A large number of studies have shown that Mycobacterium tuberculosis forms biofilm（BF）,which can produce phenotypic resistance to Mycobacterium tuberculosis,which is one of the important mechanisms of Mycobacterium tuberculosis resistance,if it inhibits the formation of Mycobacterium tuberculosis biofilm.Or destroying the formed biofilm will help the bacteria to recover the sensitivity against sputum drugs,thereby improving the treatment effect of tuberculosis and shortening the treatment cycle.Chinese herbal medicine is one of the important sources of new drug discovery.China’s Chinese herbal medicine resources are rich and diverse.The project team used the diversity of Guizhou Chinese herbal medicine resources and rich ethnic medicine resources in the early stage to conduct screening tests and found that the Yi people’s folk treatment of tuberculosis is single.Arisaema sinii Krause has the activity of inhibiting the biofilm of Mycobacterium tuberculosis.In this study,the active constituents of Mycobacterium tuberculosis biofilm were inhibited and the preliminary mechanism was discussed.First,using the hot reflux method,the ethanol extract of Arisaema sinii Krause was obtained.Then,the extraction section is carried out to obtain the extraction part of the petroleum ether layer,the extraction part of the ethyl acetate layer,and the remaining layer.The biofilm formation scoring method was used to evaluate the biofilm resistance.The experimental results showed that the petroleum ether layer extraction site had a good activity to inhibit the biofilm formation of Mycobacterium tuberculosis,showing a significant dose-effect relationship.When the drug concentration was 1 mg/mL,The biofilm meter is divided into 2.0,and the biofilm score is 1.3 when the drug concentration is 2 mg/mL.The in vitro anti-tuberculosis activity of different parts was determined by the modified Roche medium absolute concentration method.It was found that the extraction of petroleum ether layer did not inhibit the activity of Mycobacterium tuberculosis,while the ethyl acetate fraction had antibacterial activity（MIC=1mg/mL）.The experimental results suggest that the ethanol extract of Arisaema sinii Krause has both compounds that inhibit the biofilm of Mycobacterium tuberculosis,and compounds against Mycobacterium tuberculosis.In order to separate the Arisaema sinii Krause biofilm inhibitor,the petroleum ether layer was further segmented by silica gel column chromatography,and the activity of different components was tested.After repeated column purification,the compound monomer inhibiting the biofilm of Mycobacterium tuberculosis was isolated.YS-01（80mg）.YS-MS m/z:601.2[M+H]⁺.¹H NMR（500 MHz,CDCl₃-d6）:7.70（s,-NH-,1H）,7.53（s,-NH-,1H）,6.23-5.49（t,3H）,6.03（d,1H）,5.50（s,J=8.2Hz,Dp-CH-,2H）,5.45（dt,J=18.1 Hz,3H）,5.21-4.67（m,7H）,2.48（dt,4H）,2.21（dt,4H）,1.87（dt,J=14.9 Hz,4H）,1.63（t,2H）,1.64-0.97（d,15H）,1.34-0.86（d,9H）,0.95-0.77（t,3H）.¹³C NMR（100 MHz,CDCl₃-d6）δ199.76,171.82,167.77,132.44,130.88,128.80,123.72,68.16,55.99,55.87,53.80,45.81,42.38,39.61,38.72,,36.11,35.61,33.97,32.96,32.04,30.35,29.69,28.92,28.19,26.04,24.18,23.74,22.98,21.02,19.81,19.01,18.69,17.38,14.05,11.94,10.95.It is identified as:3,6,8-trimethylindol-1-en-1-yl 3,6-dioxo-5-（4,8,10,13-tetramethyltetradecane-2,6-Methyl-2-diyl）-piperazine-2-carboxylate.Using the biofilm scoring method,it was found that YS-01 inhibited the formation of Mycobacterium tuberculosis biofilm at a concentration of 4μg/mL,and the biofilm could not form at 8μg/mL.Scanning electron microscopy revealed that the Mycobacterium tuberculosis biofilm that had formed could be destroyed when the active monomer drug concentration was 32μg/mL.YS-01 was combined with first-line anti-caries drugs,and in vitro anti-BCG activity experiments showed that YS-01 and first-line anti-tuberculosis drugs isoniazid（1/2 MIC）,rifampicin（1/2 MIC）and pyrazinamide（1/2 MIC）,they all showed good synergy.The Pks1 gene is involved in the formation of Mycobacterium tuberculosis biofilm.The expression product of this gene is polyketide synthase,which is the main component of Mycobacterium tuberculosis biofilm.The effect of active monomer compound YS-01 on the regulation of Pks1 gene expression in Mycobacterium tuberculosis H37Rv strain was preliminarily explored by real-time PCR.The results were analyzed by 2^-??Ct method.In comparison,the expression of Pks1 was significantly up-regulated in Mycobacterium tuberculosis after intervention with compound monomer YS-01,and its 2^-??value was 93 times that of the blank control.To this end,we speculate that YS-01 is a polyketide synthase inhibitor of Mycobacterium tuberculosis.In summary,this paper evaluated the biofilm activity of Mycobacterium tuberculosis in Arisaema sinii Krause,traced and isolated the effective monomer compounds,identified the structure of the compounds,and preliminarily discussed the molecular mechanism of inhibiting the biofilm formation of Mycobacterium tuberculosis.The development of anti-tuberculosis drugs provides new ideas.