Bioactive Polyphenolic and Terpenoid Compounds from Stem Bark and Flowers of Terminalia brownii

2.1. Plant Materials, Extraction and Isolation

Stem bark and flowers of T. brownii were collected in April, 2021 in Mwingi; Kitui County, Kenya (1°02'18.7”S 37°55'01.9”E) and identified at the Herbarium of South Eastern Kenya University (voucher number SKU.JULY/2023/01). The plant name was checked with the plant list database (http:/www.theplantlist.org) to confirm plant taxonomy. The air – dried plant materials were powdered and sequentially extracted using n-hexane, dichloromethane, 50% dichloromethane/methanol and methanol through maceration [15].

50% dichloromethane/methanol and methanol extracts of T. brownii’s stem bark and flowers which exhibited strong antimicrobial activity [16], were separately subjected to column chromatography on a silica gel and eluted with gradient of ethyl acetate/n-hexane (40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100.0, v/v) and methanol/ethyl acetate (5:95, 10:90, 15:85, 20:80, v/v). The fractions were purified by preparative thin layer chromatography and pure isolates characterized using ¹H NMR, ¹³C NMR, IR, and HRESI-MS.

3.1. Isolated Compounds

Bioassay-guided study of 50% dichloromethane/methanol and methanol fractions led to separation and detection of seven known metabolites and three new compounds. The isolates were assessed using ¹H NMR, ¹³NMR,IR, HRESI-MS and results compared with previously reported data for the seven known compounds, termiglaucescin, 69 mg (1) [17], arjunglucoside – I, 130 mg (2) [18], sericoside, 23 mg (3) [19], 23-galloyl arjungenin, 95 mg (4) [17], 28-O-β-D-glucopyranosyl-2,3,6-trihydroxy-23-galloylolean-12-dien-28-oate, 48 mg (5) [20], 3,3',4',5 tetrahydroxy-7-methoxy flavone, 61 mg (9) [21] and 3,3’,4’,5,7-penta hydroxy flavone, 98 mg (10) [22]. The three newly isolated compounds were identified as 1,4, 7-tri-O-galloyl-6-deoxyheptose, 11mg (6) [23] 1,2,4-tri-O-galloyl-8,9 dideoxynonose, 10 mg (7) [24] and rhamnetin-3-O-2,3,6-tri-galloyl-β-D-glucopyranoside, 40 mg (8) [25]. Fig. (1) illustrates the molecular structures of the isolates.

Compounds 1 – 3 and 4 -5 were isolated from methanol and 50% dichloromethane/methanol extract of T.brownii stem bark respectively while compound 6 – 10 were obtained from 50% dichloromethane:methanol extract of T.brownii flower. Compound 1, 4,5,6,7 and 8 were eluted by 15% methanol/ethyl acetate while compound 2, 3, 9 and 10 were eluted by 20% methanol/ethyl acetate solvent system.

Compound 6 identified as 1,4, 7-tri-O-galloyl-6-deoxyheptose was isolated as light brown amorphous substance (11 mg) with melting point range of 176 – 178 °C and molecular formula C₂₈H₂₆O₁₈ as determined by HRESIMS (m/z 650.1106). The infrared Absorption at 3295, 1708 and 1611 cm^-1 revealed existence of hydroxyl, ester carbonyl and C=C functional group respectively in the molecule.

Compound 6 was generated based on spectral data evaluation [23, 26]. C – 6 substitution evidenced by lack of hydroxyl proton at C-6, ¹H NMR spectrum (Fig. 2) which is typically diagnostic of hydroxyl group on C – 6 in heptose [27, 28]. The ¹H NMR signal at C – 1, C – 4 and C – 7 of heptose moiety were significantly downfield compared to those of 6-deoxyheptose [23]. ¹³C NMR spectrum (Table 1) exhibited seven aliphatic carbon indicating presence of 6-deoxyheptose and 15 sp² carbon signals corresponding to three galloyl groups. COSY and HMBC correlations (Fig. 3) revealed the atomic connectivity within the 6-deoxyheptose framework and confirmed the substitution pattern, supporting the identification of Compound 6.

Compound 7 for 1,2,4-tri-O-galloyl-8,9 dideoxynonose was retrieved as brown powder (10 mg) with melting point range of 186 – 188 ^oC and molecular formula C₃₀H₃₀O₁₈ as determined by HRESIMS analysis (m/z 678.1536). The identification of compound 7 was based on spectral data analysis [24, 29]. The ¹H NMR spectrum (Fig. 4) three aromatic proton signifying existence of three galloyl units. In the sugar regions, three protons were observed significantly downfield compared to those of nonose moiety implying the attachment of galloyl entities to their respective carbons [30]. ¹³C NMR spectrum (Table 1) revealed eight aliphatic carbons associated with 8,9 dideoxynonose moiety and 15 sp² carbon representing the three galloyl groups. HMBC correlations (Fig. 5) showed the inter – atomic interaction of 8,9 dideoxynonose and galloyl skeletons and the substitution pattern of the 8,9 dideoxynonose system, thus confirming compound 7.

Compound 8 for rhamnetin-3-O-(2,3,6-tri-galloyl)-β-D-glucopyranoside was acquired as pale green powder (40 mg) with melting point range of 156 – 157 °C and molecular formula C₄₃H₃₄O₂₄ as confirmed by HRESIMS analysis (m/z 934.1524). Infrared absorption at 3201, 1705 and 1607 cm^-1 confirmed the existence of hydroxyl, carbonyl and C-C stretching at aromatic system respectively. The compound was established based on spectroscopic data analysis [25, 31]. The ¹H NMR signal (Fig. 6) at C – 2 and C – 3 of the glucosyl moiety were significantly downfield compared to those of rhamnetin-3-O-6-galloyl)-β-D-glucopyranoside [31, 32]. The ¹³C NMR data (Table 2) indicated three galloyl groups. HMBC correlations interactions (Fig. 7) revealed inter – atomic interactions in rhamnetin-3-O-6-galloyl)-β-D-glucopyranoside framework and substitution on glucosyl moiety, confirming the structure of compound 8.

3.2. Antimicrobial Activity

All isolated compounds showed activity against the tested pathogens (Table 3). E. coli, was more susceptible to compound 6 (16.5±0.7). Compound 1 exhibited excellent antifungal activity, with zone of inhibition of 16.0±2.7 mm. All the identified compounds had reduced antimicrobial efficacy compared to the positive control. According to [33], plant extracts containing flavonoids and phenolic exhibit antibacterial activity against a wide spectrum of harmful bacteria due to their chemical structures and biological properties. Flavonoids and phenolics contain hydroxyl group that interact with bacterial cell wall and membranes, creating disruption of cell integrity, inhibition of enzyme function and interference with bacterial DNA and protein synthesis [34]. Additionally, these compounds may act as antioxidants reducing oxidative stress in cells inhibiting bacterial growth hence making them effective against various bacterial strains [35, 36]. Terpenes and their derivatives exhibit potent antimicrobial activity against drug – resistant pathogens bacteria and fungi owing to their ability to erode microbial cell membranes and obstruct integral cellular process [13, 37]. These results align with previous studies that documented the antimicrobial properties of termiglaucescin (1), arjunglucoside – I (2), Sericoside (3), 23-galloyl arjungenin (4) [17, 38]. This corroboration reinforces the potential of these compounds as viable option for developing new antimicrobial agent particularly in the face of rising drug resistance.

The study proposes the isolation and characterization of n-hexane, chloroform and ethylacetate extract of T.brownii flowers and carry out toxicity and antioxidant tests on T.brownii flowers extracts.