Activité antimycobacterienne et cibles cellulaires des huiles essentielles de quatre plantes utilisées dans le traitement de la tuberculose à Yabassi et Nkondjock (littoral-Cameroun)

Abstract

Tuberculosis (TB) is an airborne infection caused by complex bacteria Mycobacterium tuberculosis. Despite treatment and preventive (vaccine) measures that have been put in place, one-third of the world’s population is infected by these bacteria. Recently, population became ill with multi-drug-resistant tuberculosis (resistant to Isoniazid and Rifampicin) and extensively drug-resistant TB with additional resistance to fluoroquinolones and injectable drugs (Amikacin, Kanamycin, Capreomycin). This shows an existing need to search for new molecules to prevent the spread of this infection. This study aimed at carrying out an ethnobotanical survey of medicinal plants used to treat tuberculosis in two localities of Nkam sub-divisions and showed the effect of four essential oils (EOs) on some cellular targets of Mycobacterium tuberculosis. The essential oils were obtained by hydrodistillation using Clevenger apparatus and analyzed by gas chromatography coupled by Flamme Ionisator Detector (GC-FID) and gas chromatography associated coupled to mass spectrophotometry (GC-SM). Base on the chemical composition of the EOs, three triplicates of EOs’s combinations were realized in proportion of (1/1/2), (1/2/1) and (2/1/1) using the manual book of aromatherapy and these combinations have been analysed by GC FID and GC-MS. The inhibition parameters such as Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) of EOs, their combinations, Ofloxacin and Rifampicin were determined against two clinical isolates IS53 and IS310 and the reference strain H37Rv by using microdilution methods. The impact of EOs on the cells membrane of Mycobacterium tuberculosis was evaluated by quantification of extracellular biomolecules (DNA, RNA and proteins) by spectrophotometry using NanoDrop 1000 and the results were statistically analysed by ANOVA one way. The effect on DNA and MIRU 04 gene of M. tuberculosis was tested by quantification of total DNA and amplicons of MIRU gene at the NanoDrop and separation on agarose gel electrophoresis of cells exposed at MIC and MBC of EOs and Ofloxacin. The results of ethnobotanical survey included 37 traditional healers and 14 amongst had identified 12 plants used to treat tuberculosis in Yabassi and Nkondjock sub-divisions. These 12 plants belonging to 7 botanical families among which Allium sativum, Drypetes gosseweileri, Echinops giganteus and Pentadiplandra brazzeana were selected because they are used alone in a medical recipe to treat tuberculosis contrary to the others. The chemical analysis of EOs revealed that benzyl isothiocyanate was present in both Drypetes gossweileri and Pentadiplandra brazzeana EOs at 91,3 % and 96,00 % respectively, while 2- methyl propyl trisulfide, methyl allyl trisulfide and diallyl sulfide were in A. sativum EO at 51,0 %; 12,8 % and 11,1 % respectively.The EO of E.giganteus showed the presence of tricyclic sesquiterpens silphiperfol-6-ene at 27,4 %; silphiperfolan-6--ol at 11,3 %, persilphiperfol-7-ene at 7,4 % and caryophyllene at 7,0 %. Benzyl isothiocyanate was the main compound in all the combinations but at lower percentage than in EO with the decrease in proportion of 2,2 times in E1, 3,8 times in E2 and 2,5 times in E3. The most active EO showed the MIC equal to 4.88 µg/mL and 9.76 µg/mL against H37Rv, IS53 and IS310 strains for Drypetes gossweileri and Pentadiplandra brazzeana. The least activity was observed for the OE of E. giganteus showed the MIC equal to 156,25µg/mL on the H37Rv cells or IS53 isolate and 312,5µg/mL on IS310 isolate. The interaction between EOs were all antagonists with MIC ranging from 39,06 to 156,25 µg/mL against M. tuberculocis. The release of biomolecules was significant in treated cells at MIC of EOs compared to cells treated by Ofloxacin and untreated cells of M. tuberculosis showing lost of cell permeability. The inhibition effect of EOs against M. tuberculosis released significant quantities of DNA ranging from 70 to 506,1 ng/µL, RNA from 42 to 340 ng/µL and proteins ranging from 37 to 1650 104ng/µL proteins at MIC and MBC. The inhibition has been confirmed by observing no band of DNA in agarose profile of treated cells by EOs or Ofloxacin while a single band of DNA appearing for untreated cells. DNA and MIRU 04 gene inhibition were observed at MBC and confirmed by the decrease of DNA from 0,2 to 7,40 ng/µL for total DNA and 4,10 to 282 ng/µL for amplicon of MIRU 04 gene present at MIC but absent at MBC in treated cells.The results obtained justified the traditional use of these plants for the treatment of respiratory diseases and tuberculosis in the two localities of Nkam sub-divisions.