Potential Anti-Tuberculosis Activity of the Extracts and Their Active Components of Anogeissus Leiocarpa (DC.) Guill. and Perr. with Special Emphasis on Polyphenols

Abstract

In Sudanese traditional medicine, decoctions of the stem bark of Anogeissus leiocarpa are used for the treatment of tuberculosis (TB). However, this plant has not been investigated before for its antimycobacterial effects. Our screening results show, for the first time, that many extracts of various parts of A. leiocarpa exhibit growth inhibitory activity against Mycobacterium smegmatis. Minimum inhibitory concentration (MIC) values ranged between 625 and 5000 µg/mL, with an ethyl acetate extract of the root showing the lowest MIC value. The good antimycobacterial effects of the root part could be due to its high concentration of ellagic acid derivatives, ellagitannins, and flavonoids. Thin layer chromatography (TLC) fractionation resulted in some fractions with better activity than the starting point crude methanol extract (MIC 2500 µg/mL). Those fractions with the lowest MIC values contained a high number of antioxidant compounds. Fractions 3 and 4 (MIC 1500 and 1000 µg/mL, respectively) contained high concentrations of di-methyl ellagic acid ([M-H] ^- 329.0318). Fraction 6 (MIC 2000 µg/mL) contained a lower concentration of di-methyl ellagic acid and was not as growth inhibitory as fractions 3 and 4. Moreover, in fraction 3, an acetylated ellagic acid derivative ([M-H] ^- 343.0477) and di-methyl-ellagic acid xyloside ([M-H]^- 461.0739) were tentatively characterized. Di-methyl ellagic acid xyloside was also present in fraction 4 and could strongly contribute to the antimycobacterial effect of this fraction. Additionally, protocatechuic acid ([M-H]-at m/z 153.0196) was present in fraction 4. Our antimycobacterial results obtained from this research justify the use of A. leiocarpa in Sudanese folk medicine against cough related to TB. Roots, stem bark, and leaves of A. leiocarpa are sources for new potent anti-TB drug lead compounds.

Keywords: Africa; Anogeissus leiocarpa; Mycobacterium smegmatis; ellagic acid derivatives; ellagitannins; flavonoids; stilbenes; tuberculosis.

Figure 1

(A) Occurrence of Anogeissus leiocarpus in Africa according to various vegetation mapping projects. (TAFP), red dots; Naturalis Herbarium specimen, yellow spots; Africa Nord project, orange spots; FLOTRO project, violet dots (Source: African Plant Database); (B) Anogeissus leiocarpa growing in semi-arid and woodland savannah zone in Sudan; (C) leaves and flowers; (D) scaly bark. Photos: E. Y. A. Salih and H. H. Gibreel 2006.

Figure 2

Trend in the number of research papers on Anogeissus leiocarpa from 1998 to 2017. The figure illustrates that most of the research papers have dealt with other research aspects than antimicrobial or phytochemical research. Thus, there is a need for more research in the mentioned research areas. Source: Scopus research data base.

Figure 3

Chemical structure of ellagic acid derivatives and other chemical constituents detected earlier from A. leiocarpa: (A) dimethyl ellagic acid xylopyranoside, (B) dimethyl ellagic acid, (C) ellagic acid xylopyranoside, (D) epsilon-Viniferin, (E) avicularin, (F) taxifolin (dihydroquercetin), (G) methyl-taxifolin (dihydroisorhamnetin), (H) aromadendrin (dihydrokaempferol), (I) ampelopsin (dihydromyricetin), (J) pinocembrin, (K) guaijaverin, (L) pterostilbene, (M) pinosylvin (trans-dihydroxystilbene), (N) methyl pinosylvin, and (O) kynurenic acid [18,26,27]. Source for molecular structures: (CAS), 2020.

Figure 4

(I) RP₁₈-TLC fractions (Fr1–Fr12) of a Soxhlet extract obtained with methanol from the root of A. leiocarpa. (A) Fractions at 366 nm, (B) fractions at 254 nm, and (C) fractions sprayed with the 2,2-diphenoyl-1picrylhydrazy (DPPH) reagent, with the yellow color indicating antioxidant activity. (II) Thin layer chromatography fingerprints of the standard compounds corilagin (R_f 0.549), ellagic acid (R_f 0.173), and gallic acid (R_f 0.721) (A) at 254 nm, (B) at 366 nm, and (C) after being sprayed with the DPPH reagent. Fractions marked with bold font (Fr 3, Fr 4, Fr 6, and Fr 8) were obtained in sufficient amount for antimycobacterial testing.

Figure 5

(A) Methanolic Soxhlet extract of the root of A. leiocarpa. (1) Gallic acid, (2) protocatechuic acid, (3a) flavonoid, (3b) alkaloid, (4a) flavonoid, (4b) ellagitannin, (5) aromadendrin, (6) di-galloyl-β-d-glucose, (7) ampelopsin, (8) methyltaxifolin, (9a*) flavonoid, (9b) isomer of methyltaxifolin, (9c) flavonoid, (10) ellagic acid derivatives, (11) taxifolin, (12) ellagic acid derivatives, (13) ellagic acid derivatives, (14) ellagic acid derivative, (15) di-methyl ellagic acid glucoside, (16) pentagalloylglucose, (17) ellagic acid derivatives, (18) di-methyl ellagic acid xylopyranoside, (19) ellagitannin, (20) ellagitannins, (21) di-methyl ellagic acid, (22a) ellagitannin, (22b) ellagitannin, (23) pinosylvin, (24) acetylated ellagic acid derivative, (25) ellagic acid derivatives, and (26) methylpinosylvin. (B) TLC chromatograms of RP₁₈ TLC fractions (Fr3–Fr8) obtained from the methanol Soxhlet extract (A); ** Unknown compounds; (EA der), ellagic acid derivatives; (Di-Me EA xyl), dimethyl ellagic acid xyloside; (ET), ellagitannin; (Ace-EA der), acetylated ellagic acid derivatives; and (PCA), protocatechuic acid. Main compounds with bold text.

Figure 6

Percentage of extraction yield. (A) ethyl acetate (EtOAc), aqueous (aqu), and acetone (acet) in sequential extraction; (B) hot (HH₂O) and cold water (H₂O*) extraction; and (C) methanol extraction, Soxhlet extraction (Me sox), and cold methanol extraction (Me*); (A.L.), Anogeissus leiocarpa; (Rb), root bark; (R), root; (W), stem wood; (B), stem bark; and (L), leaf. Extracts obtained using the same solvent are marked with the same color or color–texture.

Figure 7

Chemical structure of ellagitannins identified from Anogeissus spp. and A. leiocarpa: (A) flavano-ellagitannin anogeissinin A, (B) anogeissinin B, (C) cornusiin B, (D) anogeissinin, (E) casuarinin, (F) castalagin (R1 = H; R2 = OH) and vescalagin (R1 = OH; R2 = H), (G) corilagin, and (H) isomer of corilagin sanguiin H4. DHHDP: dehydro-hexahydroxydiphenoyl; HHDP: hexahydroxydiphenoyl. Source for molecular structures: CAS, 2020.