Evaluation Of The Antimycobacterial Activity Of Tibetan Herbs In Vitro

Tuberculosis remains a serious health threat,and the rapid spread of a new coronavirus has set back global tuberculosis control efforts since early 2020,with data indicating that the incidence and mortality of tuberculosis are expected to increase significantly by the end of 2022.Currently available anti-tuberculosis drugs and their efficacy are very limited,especially for drug-resistant tuberculosis.In recent years,more and more studies have shown that many herbal medicines have good antibacterial activity,relatively low adverse effects and are less likely to develop drug resistance.Tibetan medicine has a long history,and most traditional Tibetan medicines are taken from the Qinghai-Tibet Plateau.Due to the special ecological environment,Tibetan herbs have unique characteristics and often have better biological activity.In view of this,the present study was conducted to investigate the in vitro anti-tuberculosis activity of some commonly used Tibetan herbs with reference to Tibetan medicine literature,with a view to developing new drugs for the treatment of tuberculosis.In this study,ethanolic crude extracts of 14 commonly used Tibetan herbs and their organic solvent extracts were prepared by traditional methods,and the in vitro anti-Mycobacterium tuberculosis activity of each extract was detected by MABA method;five Tibetan herbs with good anti-Mycobacterium tuberculosis effect were selected from the 14 Tibetan herbs,and the potential mechanisms of their drug active ingredients against Mycobacterium tuberculosis were further explored by using molecular docking technique,KEGG pathway analysis and GO functional analysis.The in vitro anti-tuberculosis activity of the ethanolic extracts of 14 Tibetan herbs showed that the MIC values of the crude extracts of 14 Tibetan herbs against Mycobacterium tuberculosis strain H37 Ra ranged from 15.08～937.5 μg/ml,with the best antibacterial effect of the ethanolic extracts of Saxifraga with MIC values of 15.08 μg/ml;the ethanolic extracts of Rhodiola and Erysimum cheiranthoides L.also showed good activity with MIC values of 34.58 μg/ml and 38.57μg/ml,respectively.The MIC values of the petroleum ether extracts of 14 Tibetan herbs against Mycobacterium tuberculosis strain H37 Ra ranged from 9.52～218.75 μg/ml,among which the petroleum ether extracts of Delphinium brunonianum Royle,Saxifraga,Lonicera semenovii Regel,and Primula calderiana had the best effect against Mycobacterium tuberculosis,with MIC values ranging from 9.52～28.9 μg/ml.The MIC values of the petroleum ether extracts of 14 Tibetan herbs against Mycobacterium tuberculosis strain H37 Ra ranged from 17.82～687.5 μg/ml,with the ethyl acetate extracts of Saxifraga having the best effect,with MIC values of 17.82 μg/ml,and the ethyl acetate extracts of Delphinium brunonianum Royle,Erysimum cheiranthoides L.and Primula calderiana also showed good effects,with MIC values of 18.5 μg/ml.The MIC values were18.7μg/ml,20μg/ml and 29.3μg/ml,respectively.The MIC of the butylalcohol extract of Saxifrage was 20.7 μ g/ml against Mycobacterium tuberculosis in vitro;The MIC values of the butylalcohol extracts from Delphinium brunonianum Royle 、Leiospora exscapa 、 Erysimum cheiranthoides L.、 Gentianopsis paludosa(Nepal)、Aster tataricus L.f.、Lonicera semenovii Regel and Primula calderiana were 134.28～500 μg/ ml in vitro;The MIC values of the remaining herbs were >500 μg/ml.In vitro,the MIC of the extracts from Delphinium brunonianum Royle 、 Rhodiola 、Saxifraga、Lonicera semenovii Regel were 242.96～293.8 μg/ml;The MIC values of the remaining herbs were >1.0 mg/ml.According to the results of molecular docking,hesperidin and luteolin had the highest binding activity,followed by Loniceroside,Neochlorogenic acid,8-Epiloganic acid,Dibutyl phthalate and Tetrahydroberberine.gua B2、mdh、pkg、tuf、tsf、tpi、pps C、gro ES were the core target genes of M.Tuberculosis with the highest binding activity to the selected drug active components.The results of GO function show that,the active components of the selected drugs may play the role of anti-mycobacterium tuberculosis through cytosolic,cell membrane,plasma membrane,Polyketide synthase complex,RNA polymerase and secondary metabolite biosynthesis signaling pathway.The best in vitro anti-Mycobacterium tuberculosis activity was found in the ethanolic crude extracts of three Tibetan herbs,namely Rhodiola,Saxifraga and Erysimum cheiranthoides L.;the petroleum ether,ethyl acetate and butylalcohol extracts of Saxifraga showed better in vitro anti-Mycobacterium tuberculosis activity;In vitro antibacterial effects of petroleum ether and ethyl acetate extracts of Delphinium brunonianum Royle,Lonicera semenovii Regel,and Primula calderiana also showed good antibacterial effects against Mycobacterium tuberculosis.The 21 active ingredients of the five Tibetan herbs may exert their anti-Mycobacterium tuberculosis effects mainly through multi-functional,multi-pathway and multipathway effects on the production of some essential functional proteins in Mycobacterium tuberculosis,nitrogen metabolism in the cells of Mycobacterium tuberculosis,affecting cell wall production and physiological and biochemical functions such as glycoisomerization,glycolysis and production of toxic products in Mycobacterium tuberculosis.