# Oleanane and Stigmasterol-Type Triterpenoid Derivatives from the Stem Bark ofAlbizia gummifera and Their Antibacterial Activities

Authors: Tamrat Tesfaye Ayele; Getahun Tadesse Gurmessa; Zelalam Abdissa; Girmaye Kenasa; Negera Abdissa
Journal: Journal of Chemistry
 (2022)

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## Abstract

Chromatographic separation of methanol extract of stem bark ofAlbizia gummifera led to the isolation of two oleanane-type triterpenoids (1 and 2), a stigmasterol derivative (3), and stigmasterol (4). The structures of the compounds were elucidated based on 1D and 2D NMR spectroscopic data and comparing with reported literature values. The crude extract and the isolated compounds were evaluated for their antibacterial activities against five bacterial strains. Compounds 1, 2, and 4 showed marginal antibacterial activity with the growth inhibition zone ranging from 6.8 to 13.2 mm against the tested bacterial strains, with compounds 2 and 4 showing significant inhibition (13.0 and 13.2 mm) against S. flexneri and S. typhimurium, respectively. While, the standard drug, gentamycin showed inhibition zone 20.0 and 24.0 mm against S. flexneri and S. typhimurium, respectively.


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## Body

## 1. Introduction

The genusAlbizia (family, Fabaceae) comprising of more than 160 species is widely distributed in subtropical and tropical regions with the major diversity in Africa and South America [1, 2]. Albizia gummifera C.A. Smith is a species of native to sub-Saharan Africa and Madagascar [3] and commonly known in the indigenous medical system to treat various human ailments [1, 4, 5]. The extracts of different solvents have reported to possess antimicrobial [6], antigonorrheal [7], antiplasmodial, and antileishmanial [8] activities. The plant elaborates triterpenoids [5, 9] as a common metabolite with proapoptotic and antiplasmodial activities. The stem bark of A. gummifera has been commonly visited by traditional healers for the treatment of bacterial infection in the eastern part of Ethiopia. However, the phytochemical and biological profile of this plant is not exhaustively studied. Thus, hereby, we report the isolation of four compounds (1–4) (Figure 1) from the stem bark of A. gummifera along with their antibacterial activities.Figure 1
Structures of the isolated compounds from stem bark ofA. gummifera.


## 2. Materials and Methods

### 2.1. General Experimental Procedures

Solvents and reagents used for extraction and purification of the compounds were of analytical and HPLC grade. Analytical TLC precoated sheets ALUGRAM® Xtra SIL G/UV254 (layer: 0.20 mm silica gel 60 with the fluorescent indicator UVF254/365) were used for purity analysis. Silica gel 100–200 mesh was used for column chromatography. Visualization was made on TLC by spraying with 10% H2SO4 acid solution and heating on a hot plate. NMR spectra were recorded on an Av600 MHz spectrometer (Bruker, Billerica, MA, USA). Chemical shifts were expressed in parts per million (ppm) downfield of trimethylsilane (TMS) as an internal reference. Whatman filter paper No. 3, 1% dimethylsulfoxide (DMSO), Petri dishes, and gentamycin were used for antibacterial analysis.


### 2.2. Plant Materials

The stem bark ofA. gummifera was collected from Wollega University, Nekemte campus, in September 2019. The plant was authenticated by botanist, Dr. Fikadu Gurmessa, and a voucher specimen (TTA004Ag) has been deposited in Wollega University Herbarium.


### 2.3. Extraction and Isolation

The maceration extraction was employed [10] in this experiment to extract secondary metabolites from the stem bark of A. gummifera. About 940 g of sample was extracted with methanol (2 L) three times each. The filtrate was evaporated using a rotary evaporator to yield 10 g of semisolid extract. The crude extract (9 g) was suspended in water and extracted with petroleum ether and chloroform to yield 3 g (yellow semisolid) and 4 g (brown semisolid), respectively. Then, 2.50 g of crude extract of petroleum ether was adsorbed on 3 g of silica gel and subjected to column chromatography. The column was eluted with n-hexane (100%) and continued with n-hexane/ethyl acetate (95/5, 90/10, 85/15, 80/20, …, 25/75, v/v) to obtain 77 fractions, each 200 mL. The fractions were grouped into six subfractions (1–6) based on their TLC profile. Subfraction 2 showed a precipitate and washed repeatedly with petroleum ether to obtain a white powder compound (4) (20 mg). Subfraction 4 was subjected to small column chromatography over silica gel and eluted with n-hexane in increasing gradient of ethyl acetate to afford a light yellow powder compound (1) (20 mg).The chloroform crude (3.50 g) was also subjected to column chromatography over silica gel and eluted with dichloromethane/ethyl acetate (80/20, 70/30, 60/40, and 50/50), each 200 mL. Sixty-four fractions were collected and combined into five (1–5) subfractions. Subfraction 5 was again subjected to small column chromatography over silica gel and eluted withn-hexane/ethyl acetate (80/20, 70/30, and 60/40) to afford a white powder compound (3) (21 mg) and light yellow compound (2) (13 mg).


### 2.4. Pathogenic Bacterial Strains

Five pathogenic bacterial strains, one Gram-positive (Staphylococcus aureus (ATCC25923)) and four Gram-negative (Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC27853), Salmonella typhimurium (ATCC13311), and Shigella flexneri (ATCC29903)) were obtained from the Department of Biology, Wollega University, and used for evaluation of antibacterial activities.


### 2.5. Antibacterial Assay of the Isolated Compounds

The paper disc diffusion method was employed to determine the antibacterial activities of the isolated compounds as described by Kumara et al. [11] with slight modification. The pathogens were seeded on Muller–Hinton agar media (model), and simultaneously, sterile circular discs of 6 mm in diameter were prepared from Whatman filter paper No. 3. The solutions of crude extract 20 μg/mL, each and compounds (1, 2 and 4) (10 μg/mL, each), were prepared in 1% dimethylsulfoxide (DMSO). The discs were impregnated with these solutions and applied aseptically to the Petri dishes of microbial culture [12]. The applied substances let to diffuse to the agar for half an hour and then incubated at 35°C for 24 h. Both DMSO (1%) and gentamycin (impregnated disc, 10 μg/mL) were used as negative and positive controls, respectively. All tests were performed in triplicate, and zone of inhibition was measured from the edge of each disc with a ruler.


### 2.6. Statistical Analysis

Data were measured as mean ± SD. Statistical analysis was performed using SPSS version 20 Software. The global comparison was done using two-way ANOVA followed by the least significant difference (LSD) multiple comparison test. Pairwise testing was done with the help of the unpaired post hoc test.P<0.05 implies that it is statistically significant.


## 2.1. General Experimental Procedures

Solvents and reagents used for extraction and purification of the compounds were of analytical and HPLC grade. Analytical TLC precoated sheets ALUGRAM® Xtra SIL G/UV254 (layer: 0.20 mm silica gel 60 with the fluorescent indicator UVF254/365) were used for purity analysis. Silica gel 100–200 mesh was used for column chromatography. Visualization was made on TLC by spraying with 10% H2SO4 acid solution and heating on a hot plate. NMR spectra were recorded on an Av600 MHz spectrometer (Bruker, Billerica, MA, USA). Chemical shifts were expressed in parts per million (ppm) downfield of trimethylsilane (TMS) as an internal reference. Whatman filter paper No. 3, 1% dimethylsulfoxide (DMSO), Petri dishes, and gentamycin were used for antibacterial analysis.


## 2.2. Plant Materials

The stem bark ofA. gummifera was collected from Wollega University, Nekemte campus, in September 2019. The plant was authenticated by botanist, Dr. Fikadu Gurmessa, and a voucher specimen (TTA004Ag) has been deposited in Wollega University Herbarium.


## 2.3. Extraction and Isolation

The maceration extraction was employed [10] in this experiment to extract secondary metabolites from the stem bark of A. gummifera. About 940 g of sample was extracted with methanol (2 L) three times each. The filtrate was evaporated using a rotary evaporator to yield 10 g of semisolid extract. The crude extract (9 g) was suspended in water and extracted with petroleum ether and chloroform to yield 3 g (yellow semisolid) and 4 g (brown semisolid), respectively. Then, 2.50 g of crude extract of petroleum ether was adsorbed on 3 g of silica gel and subjected to column chromatography. The column was eluted with n-hexane (100%) and continued with n-hexane/ethyl acetate (95/5, 90/10, 85/15, 80/20, …, 25/75, v/v) to obtain 77 fractions, each 200 mL. The fractions were grouped into six subfractions (1–6) based on their TLC profile. Subfraction 2 showed a precipitate and washed repeatedly with petroleum ether to obtain a white powder compound (4) (20 mg). Subfraction 4 was subjected to small column chromatography over silica gel and eluted with n-hexane in increasing gradient of ethyl acetate to afford a light yellow powder compound (1) (20 mg).The chloroform crude (3.50 g) was also subjected to column chromatography over silica gel and eluted with dichloromethane/ethyl acetate (80/20, 70/30, 60/40, and 50/50), each 200 mL. Sixty-four fractions were collected and combined into five (1–5) subfractions. Subfraction 5 was again subjected to small column chromatography over silica gel and eluted withn-hexane/ethyl acetate (80/20, 70/30, and 60/40) to afford a white powder compound (3) (21 mg) and light yellow compound (2) (13 mg).


## 2.4. Pathogenic Bacterial Strains

Five pathogenic bacterial strains, one Gram-positive (Staphylococcus aureus (ATCC25923)) and four Gram-negative (Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC27853), Salmonella typhimurium (ATCC13311), and Shigella flexneri (ATCC29903)) were obtained from the Department of Biology, Wollega University, and used for evaluation of antibacterial activities.


## 2.5. Antibacterial Assay of the Isolated Compounds

The paper disc diffusion method was employed to determine the antibacterial activities of the isolated compounds as described by Kumara et al. [11] with slight modification. The pathogens were seeded on Muller–Hinton agar media (model), and simultaneously, sterile circular discs of 6 mm in diameter were prepared from Whatman filter paper No. 3. The solutions of crude extract 20 μg/mL, each and compounds (1, 2 and 4) (10 μg/mL, each), were prepared in 1% dimethylsulfoxide (DMSO). The discs were impregnated with these solutions and applied aseptically to the Petri dishes of microbial culture [12]. The applied substances let to diffuse to the agar for half an hour and then incubated at 35°C for 24 h. Both DMSO (1%) and gentamycin (impregnated disc, 10 μg/mL) were used as negative and positive controls, respectively. All tests were performed in triplicate, and zone of inhibition was measured from the edge of each disc with a ruler.


## 2.6. Statistical Analysis

Data were measured as mean ± SD. Statistical analysis was performed using SPSS version 20 Software. The global comparison was done using two-way ANOVA followed by the least significant difference (LSD) multiple comparison test. Pairwise testing was done with the help of the unpaired post hoc test.P<0.05 implies that it is statistically significant.


## 3. Results and Discussion

Compound1 was obtained as a light yellow solid. The 1H NMR spectrum (Table 1) displayed seven singlets at δH 0.99 (3H, s, H-23), 0.78 (3H, s, H-24), 0.93 (3H, s, H-25), 0.77 (3H, s, H-26), 1.13 (3H, s, H-27), 0.92 (3H, s, H-29), and 1.43 (3H, s, H-30), integrated for three protons each for seven methyl groups; a triplet olefinic protons resonating at δH 5.27(1H, t, J = 3.7 Hz, H-12), a doublet of doublets methine proton at δH 3.21 (1H, dd, J = 11.3, 4.3 Hz, H-3), and two methine protons signals at δH 1.56 (1H, m, H-9) and 2.82 (1H, dd, J = 13.8, 4.7 Hz, H-18), which are the typical characteristics of an oleanane-type of triterpene [13–15]. Furthermore, di-ortho/meta coupling protons at δH 7.53 (2H, td, J = 5.8, 3.5 Hz, H-4′ and H-5′) and the deshielded proton signals at δH 7.71(2H, dd, J = 12.5, 5.7, 3.3 H z, H-3′ and H-6′) are a characteristic of ortho-disubstituted benzene moiety [15]. The oxygenated methylene protons at δH 4.08 (2H, d, J = 6.7 Hz, H-1″), multiple proton signals for methine protons at δH 2.04 (1H, m, H-2″), 1.32 (1H, m, H-6″), two doublet for methyl protons at δH 0.99 (3H, d, J = 6.4 Hz, H-9″), 0.90 (3H, d, J = 6.0 Hz, H-10″), and one triplet for methyl protons at δH 0.92 (3H, t, J = 3.7 Hz, H-8″) indicate the presence of geranyl unit (monoterpene) linked through an ester bond.Table 1
1H NMR and 13C NMR (600 MHz) spectroscopic data for compounds 1–4 in CDCl3.
C/H1234δH (m, J in Hz)δC (ppm)δH (m, J in Hz)δC (ppm)δH (m, J in Hz)δC (ppm)δH (m, J in Hz)δC (ppm)11.62 (m)38.41.62 (m)38.41.14 (m)37.31.14 (m)37.321.64 (m)27.21.64 (m)27.21.55 (m)31.61.55 (m)31.733.21 (dd, 11.3, 4.3)79.03.22 (dd,11.3,4.3)79.03.52 (td, 11.2, 5.7)71.83.52 (td, 11.2, 5.7)71.84—38.8—39.32.30 (d, 5.0)42.22.30 (d, 5.0)42.250.74 (m)55.20.75 (m)55.2—140.7—140.861.57 (m)18.31.56 (m)18.35.35 (dd, 5.4, 1.9)121.75.35 (dd, 5.4, 1.9)121.771.45 (m)32.61.36 (m)32.61.83 (m)31.91.83 (m)31.98—39.3—37.11.45 (m)31.91.45 (m)31.991.56 (m)47.61.56 (m)47.60.93 (m)50.20.93 (m)50.210—37.1—38.8—36.5—36.5111.90 (m)23.41.90 (m)23.41.30 (m)22.71.30 (m)21.1125.27 (t, 3.7)122.65.28 (t, 3.7)122.61.83 (m)39.71.83 (m)39.713—143.6—143.6—42.3—42.314—41.6—41.61.00 (m)56.91.00 (m)56.9151.32 (m)28.91.08 (m)27.71.55 (m)24.41.55 (m)24.4160.94 (m)23.61.42 (m)23.61.33 (m)28.91.33 (m)28.917—46.5—46.51.15 (m)56.01.15 (m)56.0182.82 (dd, 13.8, 4.7)40.92.82 (dd, 13.8, 4.7)41.00.68 (s)12.10.68 (s)12.0191.62 (m)45.91.15; 1.64 (m)45.91.03 (s)19.81.03 (s)19.820—30.7—30.72.04 (m)40.52.04 (m)40.5211.70 (m)38.71.70 (m)38.71.04 (m)21.11.04 (m)21.2224.22 (dd, 11.5, 5.9)68.24.22 (dd, 11.4, 5.9)68.25.15 (m)138.35.15 (m)138.3230.99 (s)28.11.00 (s)28.15.03 (dd, 15.1, 8.8)129.35.03 (dd, 15.1, 8.8)129.3240.78 (s)15.50.76 (s)15.51.55 (m)51.31.55 (m)51.2250.93 (s)15.30.93 (s)15,31.45 (m)31.945.8260.77 (s)17.10.75 (s)17.10.94 (m)11.00.82 (d, 6.6)21.1271.13 (s)25.91.16 (s)25.91.42 (m)23.70.80 (d, 6.6)19.028—183.7—183.51.70 (m)38.70.71 (s)25.4290.92 (s)33.10.94 (s)33.14.22 (dd, 11.4, 5.9)68.21.03 (s)12.3301.43 (s)23.71.43 (s)23.8——1′—132.4—132.5-132.52′—132.57.70 (dd, 5.7, 3.3)128.87.70 (dd, 5.7, 3.3)128.83′7.71 (dd, 12.5, 5.7, 3.3)128.87.53 (dd, 5.7, 3.3)130.97.53 (dd, 5.7, 3.3)130.94′7.53 (dt, 5.8, 3.5)130.97.53 (dd, 5.7, 3.3)130.97.53 (dd, 5.7, 3.3)130.95′7.53 (dt, 5.8, 3.5)130.97.53 (dd, 5.7, 3.3)130.97.53 (dd, 5.7, 3.3)130.96′7.71 (dd, 12.5, 5.7, 3.3)128.97.70 (dd, 5.7, 3.3)128.87.70 (dd, 5.7, 3.3)128.87′—167.8—167.8—167.88′—167.7—178.59′4.08 (d, 6.7)71.82.35 (t, 7.5)33.810′2.04 (m)27.71.65 (dt, 34.2, 7.1)24.711′1.01 (m)27.71.86 (m)31.912′1.62 (m)22.91.55 (m)31.913′1.78 (m)32.41.30–1.45 (m)29.714′1.32 (m)33.81.30–1.45 (m)29.515′1.50 (m)30.41.30–1.45 (m)29.316′0.94 (t, 3.7)11.01.30–1.45 (m)29.517′0.99 (d, 6.4)19.21.30–1.45 (m)29.618′0.90 (d, 6.0)14.11.30–1.45 (m)29.419′1.30–1.45 (m)29.720′1.30–1.45 (m)29.721′1.30–1.45 (m)29.722′1.30–1.45 (m)29.723′1.27 (m)30.424′1.33 (m)23.025′0.91 (t, 7.5)14.1The13C NMR spectrum (Table 1) showed carbon signals for 48 carbon atoms, of which 30 carbon atoms are for aglycone core, 8 carbon atoms for phthalate moiety, and 10 carbon atoms for the two isoprene chains. The downfield shifted carbon signals at δC 167.8 (C-7′), 167.7 (C-8′), 132.5 (C-2′), 132.4 (C-1′), 130.9 (C-4′and C-5′), and 128.9 (C-3′ and C-6′) are carbons for phthalate moiety, while the signals resonate at δC 71.8 (C-1″) and 27.7 (C-2″ and C-3″), three methyl carbons resonate at 11.0 (C-8″), 19.2 (C-9″), and 14.1 (C-10″) for the geranyl unit (monoterpene), whereas the signals of seven methyls of aglycone core (C-23, C-24, C-25, C-26, C-27, C-29, and C-30) are resonated at δC 28.1, 15.5, 15.3, 17.1, 25.9, 33.1, and 23.7, respectively. The signals resonating at δC 38.4 (C-1), 79.0 (C-3), 122.6 (C-12), 143.6 (C-13), 40.9 (C-18), and 183.7 (C-28) are in accordance with the reported literature data for oleanoloate [13–16] except signal at δC 68.2 (C-22) which could be due to the oxygen attachment with the phthalate moiety. The gross structure of the aglycone and position of the substituent was determined by analysis of 1H-1H COSY and HMBC experiments (Figure 2). The 1H-1H COSY cross-peaks showed the coupling of hydroxymethine proton H-3 (δH 3.21) with H-2 (δH 1.64), H-12 (δH 5.27) with H-11(δH 1.90), H-15 (δH 1.32) with H-16 (δH 0.94), H-18 (δH 2.82) with H-19 (δH 1.61), and H-21 (δH1.71) with H-22 (δH 4.22) enabled to construct the structure of aglycone. The correlation of proton H-3′ (δH 7.71) with H-4′ and H-5′ (δH 7.53) through ortho/meta coupling further confirm the phthalate moiety. Similarly, the cross-peaks from oxymethylene proton H-1″ (δH 4.08) with H-2″ (δH 2.04) and H-5″ (δH 1.78) with H-6″ (δH 1.32) also revealed the geranyl unit (monoterpene).Figure 2
Key HMBC (curved arrow) and1H-1H COSY (bold line) correlations of compounds 1 and 2.The long-range HMBC coupling (Figure2) from oxymethine proton at δH 4.22 (H-22) to carbon signal at δC 23.6 (C-16), δC 38.7 (C-21), δC 30.7 (C-20), and δC 167.8 (C-7′) enabled to establish the phthalate moiety at C-22 through an ester bond. The oxymethylene protons peaks at δH 4.08 (H-1″ showed an HMBC correlation to carbon signals at δC 167.7 (C-8′) and δC 27.7 (C-2″)) confirmed the attachment of geranyl unit at C-8′ of phthalate. Thus, based on the above spectroscopic analysis, the structure of compound 1 was deduced to be a 3-hydroxy-22-(2-((2, 6-dimethyloctyloxy) carbonyl) benzoyloxy)olean-12-en-28-oic acid.Compound2 was obtained as a yellow powder. The NMR spectral data showed similar spectral feature with compound 1 except for some substituted positions. The 1H NMR spectrum (Table 1) displayed five aromatic protons at δH7.70 (1H, dd, J = 5.7, 3.3 Hz, H-2′ and H-6′) and δH 7.53 (1H, dd, J = 5.7, 3.3 Hz, H-3′, H-4′, and H-5′), containing HSQC correlations with carbons at δC 128.8 (C-2′ and C-6′) and 130.9 (C-3′, C-4′, and C-5′), respectively. However, the 13C NMR spectrum (Table 1) showed an esteric carbonyl carbon at δC 167.8 (C-7′) and one quaternary carbon at δC 132.5 (C-1′), indicating the benzoate linked to oleanolic moiety. The long-range HMBC correlations (Figure 2) between oxymethine protons at δH 4.22 (H-22) and carbon signals at δC 38.7 (C-21), δC 30.7 (C-20), and δC 23.6 (C-16) and carbonyl carbon at δC 167.8 (C-7′) established the linkage of benzoate at C-22. Therefore, based on the above findings, the structure of compound 2 was defined to be 22-(benzoyloxy)-3-hydroxyolean-12-en-28-oic acid.Compound3 was obtained as a white solid. The 1H and 13C NMR spectra data are similar to that of stigmasterol (4) (coisolated from the same fraction) except for some signals for C-28, C-29, and C-3 of the stigmasterol (could be due to the substitution effect). The 1H NMR spectrum displayed doublet of doublets oxymethylene protons at δH 4.22 (2H, dd, J = 11.4, 5.9 Hz, H-29). These protons showed a long-range HMBC cross-coupling (Figure 3) with carbonyl easter of benzoate moiety, whose presence is evidenced from strongly deshielded doublet of doublets at δH7.70 (2H, dd, J = 5.7, 3.3 Hz) for H-2″ and H-6″ and multiplet at δH 7.53 (3H, for H-3″, H-4″, and H-5″), indicating that the position of the benzoate substituent at C-29.Figure 3
Key HMBC (curved arrow) and1H-1H COSY (bold line) correlations of compound 3.The1H NMR spectrum also showed signals for aliphatic carboxylate (Table 1). In this regard, 13C NMR spectrum showed that a downfield shifted carbon signal at δC 178.5 is assignable to carbonyl carbon (C-1′) and the signal at δC 33.8 for C-2′. The HSQC spectrum confirmed the connectivity between protons at δH 2.35 (2H, t, J = 7.5 Hz), 1.65 (2H, dt, J = 34.2, 7.1 Hz), 1.33 (2H, m), and 0.91(3H, t, J = 6.8 Hz) with carbon atoms at δC 33.8 (C-2′), 24.7 (C-3′), 23.0 (C-17′), and 14.1 (C-18′), respectively.The triplet methylene protons atδH 2.35 (2H, t, J = 7.6 Hz) showed strong HMBC correlations with carbon at δC 178.5 (carbonyl carbon, C-1′) and δC 24.7 (C-3′), respectively. Based on the above spectral data, the structure of compound 3 was defined as 29-benzoyl-3-octadecanoyl stigmasterol.Compound4 was isolated as a white powder. The 1H NMR spectrum (Table 1) displayed three doublet of doublets peaks at δH 5.35 (1H, dd, J = 5.2, 2.2 Hz), 5.15(1H, dd, J = 15.1, 8.7 Hz), and 5.01(1H, dd, J = 15.2, 8.7 Hz), which are typical signals for olefinic proton (H-6) of the steroidal skeleton and olefinic protons (H-22 and H-23), respectively. The proton signal at δH 3.52(1H, dd, J = 11.2, 6.5, 4.7 Hz) integrating for 1H was indicative of hydroxymethine proton of H-3. Two singlet peaks at δH 0.86 (3H, s) and 1.05(3H, s) were assigned to two tertiary methyl groups attached to C-13 (H-18) and C-10 (H-19), respectively. The spectrum further revealed three doublet peaks at δH 1.01 (3H), 0.86 (3H), and 0.93 (3H) which are assigned to methyl groups at C-20 (H-21) and C-25 (H-26 and H-27), respectively.The13C NMR spectrum (Table 1) also showed the presence of 29 carbon atoms, of which the signal at δC 71.8 is assignable to C-3. The signals at δC 140.8, 121.7, 138.3, and 129.3 are corresponding to olefinic carbons at C-5, C-6, C-22, and C-23, respectively. Based on these data and comparison to the reported literature [17–19], compound 4 was found to be stigmasterol. This is the first report of compound 4 from A. gummifera.Compounds1, 2, and 4 were evaluated for their antibacterial activities against five pathogenic bacteria strains: Staphylococcus aureus (ATCC25923), Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC27853), Salmonella typhimurium (ATCC13311), and Shigella flexneri (ATCC29903) (Table 2) using the disk diffusion method.Table 2
Bacterial growth inhibition zone in diameter (mm) of crude extracts (20μg/mL each) and isolated compounds (1, 2, and 4) (10 μg/mL each).
Compounds/extractsTested micro-organismS. aureusE. coliP. aeruginosaS. typhimuriumS. flexneriME9871010PE897117CHE—711714∗1777.2——2—7.3106.81347.477.313.26.9Gentamycin1621142024—, not active; ME, methanol extract; PE, petroleum ether extract; CHE, chloroform extract. All values are mean values ± standard deviation of three replicates.The crude extracts showed weak to moderate activity on both Gram-positive and Gram-negative bacterial strains with zone of inhibition ranging 7–14 mm with chloroform extract demonstrating the highest zone of growth inhibition (14 mm)P<0.05 against S. flexneri. This is in agreement with the report that the hydroalcoholic extract of A. gummifera exhibited growth inhibition against bacterial pathogens [6, 20]. The isolated compounds also showed moderate antibacterial activity against the test strains. These activities vary from strain to strain. Compound 4 showed significant inhibition (13.2 mm) against S. typhimurium (ATCC13311). Gram-negative bacteria showed greater susceptibility to 2 and 4 than Gram-positive. It might be due to the thickness of the peptidoglycan layer. It is worth to highlight that stigmasterol has been reported to inhibit several proinflammatory and matrix degradation mediators typically involved in human osteoarthritis- (OA-) induced cartilage degradation, at least in part through the inhibition of the NF-κB pathway [21]. It also showed good membrane stabilizing activity in HRBC with minimum stabilization 62.74% at 10 μg/mL and maximum stabilization 65.19% at 40 μg/mL [22].The antibacterial activity results confirm the traditional value of the plant (A. gummifera), provided that these results further supported with in vivo and cytotoxicity tests.


## 4. Conclusion

Four triterpenoids1, 2, 3, and 4 were isolated from stem bark of A. gummifera. This is the first report of compounds 1, 2, and 3. The crude extract and isolated compounds showed moderate antibacterial activity with compound 4 exhibiting the highest. The observed antibacterial activities of the extracts and pure compounds could give insight about potential of the traditionally used medicinal plants for development of antibacterial drugs.



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