Inhibitory Activity of Chemical Constituents from Vitellaria paradoxa Gaertn. (Sapotaceae) Against Pathogens Associated with Respiratory Tract Infections

doi:10.21203/rs.3.rs-3967817/v1

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Inhibitory Activity of Chemical Constituents from Vitellaria paradoxa Gaertn. (Sapotaceae) Against Pathogens Associated with Respiratory Tract Infections

https://doi.org/10.21203/rs.3.rs-3967817/v1

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Vitellaria paradoxa Gaertn. is a commonly used medicinal plant for the treatment of tuberculosis (TB) and other respiratory infections in Africa. Therefore, its phytochemicals can be explored in rational drug discovery, development, and design against respiratory-tract infections. The aim of this study is to identify chemical composition of V. paradoxa hexane stem-bark extract, and further isolate possible antimicrobial agents from its crude extract. 48 phytochemicals, including1-heptacosanol, 2-nonenal, (E)-, and hexadecanoic acid, methyl ester were identified using GC-MS. Column chromatography led to the isolation of three compounds, including 11-hydroxy β-amyrin cinnamate (1), α-amyrin cinnamate (2), and sitosterol cinnamate (3). All the compounds showed activity against all the tested pathogens, with compound (3) showed better activity against all bacterial strains, with minimum inhibitory concentrations (MICs) varying from 0.0625 to 0.25 mg/ml. Compounds (1) and (2) showed no cytotoxicity against the cervical cancer cell line (HeLa) (IC₅₀; 82.53 ± 4.07 and 77.82 ± 10.5 respectively). To our knowledge, compounds 2 and 3 are reported here from the stem-bark part of the plant for the first time. Similarly, inhibitory activities of compounds 1–3 against the tested strains are studied here for the first time. These results provide in part scientific justification for the traditional uses of V. paradoxa for treating respiratory tract infections, including tuberculosis in Africa.

Medicinal Chemistry

Vitellaria paradoxa

Sapotaceae

Respiratory tract infections

Tuberculosis

Cytotoxicity

Sitosterol cinnamate

Respiratory tract infections (RTIs), such as pneumonia impose immense health burden in the world, accounting for over four millions fatalities yearly (Forum of International Respiratory Societies and European Respiratory Society, 2017). It is particularly a leading cause of death among children within the age of 5 years than older children or adults in developing countries. It is estimated that over 2 million children within this age group die due to pneumonia in developing nations, and over 43% of global death rates from acute RTIs are linked to developing countries, including Nigeria, Ethiopia, Democratic Republic Congo (Demissie et al., 2021). These figures do not include the over 1.2 million deaths recorded as due to tuberculosis (TB), which is another fatal respiratory-cum-lung infection (WHO, 2020). Together, respiratory tract infections and tuberculosis are two significant contributors to the incessant rising in the global burden of communicable diseases (Michaud, 2009). Despite significant breakthroughs in the rollout of antibiotics to curb the menace of these infections over the past decades, there are still the problem of antimicrobial resistance in addition to the adverse effects of these antibiotics on the host-like hypersensitivity. Drug-resistant bacterial infections are on the increase worldwide, and this has called for continued surveillance and desperate search for new drug pipelines against menace of tuberculosis and other bacterial pathogens associated with respiratory infections, including methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Enterococcus faecalis, Klebsiella oxytoca, Escherichia coli, Klebsiella aerogenes, and Enterobacter cloacae (Mirzayev et al., 2021; Prat and Lacoma, 2016; Yoo et al., 2020; Husain et al., 2021; Passarelli-Araujo et al., 2019; Lee et al., 2019; Zhong et al., 2018).

Natural products have historically been acting as an invaluable source of therapeutical agents in many disease areas, very much in the area of infections due to pathogenic bacteria (Atanasov et al., 2021). In fact, they have been at the origin of many clinically used drugs, such as streptomycin and rifamycin against tuberculosis and other related infections (Dong et al., 2017). Natural plant-based drugs could serve as anti-infective agents with novel modes of actions (Porras et al., 2021). Many plant species in the family Sapotaceae have been reported to possess several pharmacological activities, including antimycobacterial (da Costa et al., 2013; Kumatia and Appiah-Opong, 2021; McGaw et al., 2015). The species Vitellaria paradoxa Gaertn. (Fig. 1) is a widely distributed medicinal plant in sub-Sahara Africa that has been traditionally used in the treatment of respiratory tract infections and related symptoms, including tuberculosis (Ogbole and Ajaiyeoba, 2009), bronchitis, coughs and whooping cough (Ariyo et al., 2020; Lawal et al., 2020), and lung disorders (Nadembega et al., 2011). It has been reported to possess antioxidant, anti-inflammatory, anticancer (Ojo et al., 2021), antidiarrhoeal, and insecticidal activities (Buxton et al., 2020). Prior phytochemical investigation led to the identification of Androseptoside A, Lupeol acetate (Akihisa et al. 2010), gallic acid, ferulic acid, and rutin (Ojo et al., 2021). However, little is known about its anti-infective potential against causative agents of respiratory infections. The present study, thus, described GC-MS metabolites profiling of V. paradoxa, and the chromatographic isolation of cinnamoyl compounds from the hexane extract. The study also described the pharmacological activities of the isolated compounds against Mycobacterium smegmatis which is used as “surrogate” model for the virulent Mycobacterium tuberculosis, together with six respiratory-tract-associated bacterial pathogens, including Escherichia coli, Klebsiella aerogenes, Staphylococcus aureus, Enterobacter cloacae, Klebsiella oxytoca, and Enterococcus faecalis.

2.1 General instrumentations

Brucker Avance III NMR spectrometer was used to record the ¹H (frequency; 500 MHz), ¹³C (frequency; 125 MHz) and 2D-NMR spectra of the isolated compounds using tetramethylsilane (TMS) as internal standard. Also, deuterated chloroform (d-CHCl₃) was employed as solvent for running the NMR analysis. Mass spectra were done and recorded on a Brucker Compact mass spectrometer (University of the Witwatersrand, South Africa). IR was performed using Perkin Elmer FTIR 600 series. Column chromatography was performed using column (3 cm × 55 cm) with silica gel (mesh size; 60–120). Thin-layer chromatography (TLC) analysis was carried out on pre-coated silica gel 60 F₂₅₄ plates (Macherey-Nagel GmbH & Co. KG) with layer thickness of 0.20 mm. The plates were treated with p-anisaldehyde-sulphuric acid reagent, and visualized under UV light (254 and 365 nm). Chemicals used in the experiment were purchased from Sigma-Aldrich, South Africa.

2.2 Plant material collection

The V. paradoxa (Fig. 1) stem-bark sample was collected from the University of Ibadan, Oyo State, Western Nigeria (Geographical location: 7^o 23̛ 28N 3^o 54̛ 60E) during the month of September, 2019. The plant sample was identified and authenticated taxonomically by Mr. D. P. O. Esimekhuai, a Chief Plant Technologist at the Botany Department, University of Ibadan, Nigeria. The voucher specimen, UIH-22897, has been deposited in the same department. The stem-bark sample was thoroughly washed and air-dried for two weeks in an open shaded place at room temperature. The air-dried samples were milled into fine powder with the aid of an industrial grinder and kept in a sealed polythene bag until further use at room temperature.

2.3 Extraction

500 g of V. paradoxa stem-bark was extracted (5 times) with n-hexane (1 L) at room temperature for 24 hrs. The extract was concentrated after filtration using a rotary evaporator under reduced pressure at 40 ^oC to give a yellowish waxy substance.

2.4 GC-MS analysis

The hexane crude extract was subjected to GC-MS analysis after dissolving in HPLC-grade methanol and filtering through a 0.2 µm PTFE syringe-driven filter into GC-MS vials. The GC-MS instrument Agilent Technologies (GC-7890B: MS-5977AMSB) was employed in the analysis of the extract. The injector temperature was set at 290 ⁰C, while column oven temperature was set at 50°C to 150°C at 3°C/min, then held isothermal for 10 min and finally raised to 300°C at 10°C/min. The sample injection was 1 𝜇L. Carrier gas was pure Helium gas (99.9%) 1 ml/min in a splitless mode. The total GC run time was 19 mins with a mass detector. The ion source temperature was set at 350°C with ionization voltage of 70 eV. The inlet temperature was 250°C, with solvent delay of 10 mins. The mass scan (m/z) was run from 40 to 1000 amu. The identification of the phytochemicals present in the crude extract was achieved by comparing their spectra with the spectra fingerprint of the known compounds in the curated database of National Institute Standard and Technology, NIST version—2011. The name, molecular formula, molecular weight, and chemical structure of the phytochemicals in the analyzed extract were identified (Table S1) provided in Supplementary List. Relative quantity of the phytochemicals presents in the stem-bark extract of V. paradoxa were individually expressed as percentage (%) based on peak areas produced in the chromatogram.

2.5 Column chromatographic isolation

10 g of the crude extract was fractionated using gradient elution [starting from hexane (100%), with 10% increase in chloroform to 100% chloroform] on silica gel column (3 cm × 55 cm) to yield twelve fractions (VPH 1- VPH 10) on the basis of their thin-layer chromatography (TLC) profiles. All fractions were screened against Mycobacterium smegmatis to evaluate their in vitro antimycobacterial activity using micro-dilution technique. The most bioactive fractions VPH 6 (109.5 mg), VPH 7 (2.20 g), and VPH 10 (220.9 mg) were subjected to further fractionation. Fraction VPH 6 was re-chromatographed on silica gel column, eluting with hexane-ethyl acetate (1:9 to 0:10) as mobile phase to yield four sub-fractions VPH 6A-D. The sub-fraction VPH 6A appeared as a white solid substance was recrystallized from dichloromethane/methanol to give compound 1 (11-hydroxy β-amyrin cinnamate, 15.4 mg). Fraction VPH 7 was further purified on silica gel column (mobile phase: hexanes-ethyl acetate 97:3 to 10:90) to give three sub-fractions VPH 7A-C. The sub-fraction VPH 7A gave compound 2 (α-amyrin cinnamate, 26.2 mg) after recrystallization from dichloromethane/methanol. Sub-fraction VPH 7B was subjected to further purification to give additional quantity of amyrin cinnamate (9.7 mg). Fraction VPH 10 was chromatographed on silica gel column (mobile phase: hexanes-ethyl acetate 85:15 to 10:90) to give three sub-fractions VPH 10 A-C. Sub-fraction VPH 10C (22.10 mg) after recrystallization from dichloromethane/methanol gave compound 3 (sitosterol cinnamate, 9. 6 mg). The structures of the compounds 1–3 were unambiguously elucidated on the basis of their obtained physical and spectroscopic data as well as comparison with those published in the literature.

Compound 1, 11-hydroxy β-amyrin cinnamate: White powder; ESI-MS: [M + H]⁺ m/z = 573.3100; MF: C₃₉H₅₆O₃; soluble in CHCl₃; ¹H-NMR (500 MHz, δ ppm, CHCl₃-d = 7.24 ): 7.64 (d, 1H, J = 15.9 Hz, H_− 3'), 7.51 (m, 2H, J = 4.2 Hz, H_− 6'/8') and 7.50 (1H, H_− 7'), 7.35 (d, 2H, J = 4.9 Hz, H_− 5'/9'), 6.44 (d, 1H, J = 10.0 Hz, H_− 2'), 5.56 (dd, 1H, H_− 12), 4.62 (dd, 1H, J = 9.80 and 5.3 Hz, H_− 3), 4.10 (t, 1H, J = 3.5 Hz, H_− 11), 0.77–1.03 (s, 8×3H); ¹³C-NMR (125 MHz, δ ppm, CHCl₃-d = 77.23 ): 38.75 (CH₂, C_− 1), 23.96 (CH₂, C_− 2), 80.85 (CH, C_− 3), 37.09 (C, C_− 4), 55.58 (CH, C_− 5), 18.51 (CH₂, C_− 6), 32.17 (CH₂, C_− 7), 42.32 (C, C_− 8), 47.92 (CH, C_− 9), 39.89 (C, C_− 10), 81.27 (CH, C_− 11), 124.59 (CH, C_− 12), 139.88 (C, C_− 13), 42.35 (C, C_− 14), 26.87 (CH₂, C_− 15), 28.37 (CH₂, C_− 16), 33.15 (C, C_− 17), 59.34 (CH, C_− 18), 41.79 ( CH₂, C_− 19), 31.50 (C, C_− 20), 34.00 (CH₂, C_− 21), 40.31 (CH₂, C_− 22), 28.98 (CH₃, C_− 23), 15.99 (CH₃, C_− 24), 14.33 (CH₃, C_− 25), 17.14 (CH₃, C_− 26), 25.40 (CH₃, C_− 27), 28.98 (CH₃, C_− 28), 17.74 (CH₃, C_− 29), 22.93 (CH₃, C_− 30), 167.02 (C, C_− 1'), 144.48 (CH, C_− 2'), 119.13 (CH, C_− 3'), 134.84 (C, C_− 4'), 128.26 (CH, C_− 5'/9'), 129.07 (CH, C_− 6'/8'), 130.33 (CH, C_− 7'). These data supports those reported for 11α-hydroxy-β-amyrin by Ikuta and Morikawa (1992) and its cinnamate by Sirignano et al. (2021) in the literature.

Compound 2, α-amyrin cinnamate: White solid powder; ESI-MS: [M + H]⁺ m/z = 557.4117; MF: C₃₉H₅₆O₂; m.p = 231–235 (uncorrected); soluble in CHCl₃; ¹H-NMR (500 MHz, δ ppm, CHCl₃-d = 7.24 ): 7.64 (d, 1H, J = 16.0 Hz, H_− 3'), 7.52 (m, 2H, J = 3.95 Hz, H_− 6'/8') and 7.50 (1H, H_− 7'), 7.35 (d, 2H, J = 4.15 Hz, H_− 5'/9'), 6.42 (d, 1H, J = 16.0 Hz, H_− 2'), 5.76 (dd, 1H, H_− 12), 4.61 (dd, 1H, J = 9.80 and 6.30 Hz, H_− 3), 0.76–1.20 (s, 8×3H); ¹³C-NMR (125 MHz, δ ppm, CHCl₃-d = 77.23 ): 38.72 (CH₂, C_− 1), 23.61 (CH₂, C_− 2), 81.22 (CH, C_− 3), 37.05 (C, C_− 4), 55.54 (CH, C_− 5), 18.49 (CH₂, C_− 6), 33.11 (CH₂, C_− 7), 40.27 (C, C_− 8), 47.88 (CH, C_− 9), 38.17 (C, C_− 10), 23.93 (CH₂, C_− 11), 124.55 (CH, C_− 12), 139.84 (C, C_− 13), 42.31 (C, C_− 14), 26.84 (CH₂, C_− 15), 28.33 (CH₂, C_− 16), 33.96 (C, C_− 17), 59.30 (CH, C_− 18), 39.83 (CH, C_− 19), 39.88 (CH, C_− 20), 31.48 (CH₂, C_− 21), 41.76 (CH₂, C_− 22), 28.36 (CH₃, C_− 23), 17.10 (CH₃, C_− 24), 15.97 (CH₃, C_− 25), 17.13 (CH₃, C_− 26), 23.47 (CH₃, C_− 27), 28.97 (CH₃, C_− 28), 17.72 (CH₃, C_− 29), 21.61 (CH₃, C_− 30), 166.97 (C, C_− 1'), 144.46 (CH, C_− 2'), 119.08 (CH, C_− 3'), 134.79 (C, C_− 4'), 128.23 (CH, C_− 5'/9'), 129.03 (CH, C_− 6'/8'), 130.30 (CH, C_− 7'); FTIR (KBr) υ_max: 2854.2, 2933.3 (C-H, aliphatic), 1706.9 (C = O), 1634.1 (C = C) cm^− 1. These data agree with the those reported by Akihisa et al. (2010) and Miranda et al. (2000) in the literature for α-amyrin cinnamate.

Compound 3, sitosterol cinnamate: White powder; ESI-MS: [M + H]⁺ m/z = 545.8512; Molecular formula: C₃₈H₅₆O₂; soluble in CHCl₃; ¹H-NMR (500 MHz, δ ppm, CHCl₃-d = 7.24 ): 7.64 (d, 1H, J = 15.8 Hz, H_− 3'), 7.51 (m, 2H, J = 4.2 Hz, H_− 6'/8') and 7.50 (1H, H_− 7'), 7.35 (d, 2H, J = 4.9 Hz, H_− 5'/9'), 6.42 (d, 1H, J = 16.0 Hz, H_− 2'), 5.12 (dd, 1H, J = 5.8 Hz, H_− 6), 3.57 (dd, 1H, H_− 3), 0.77-1.00 (s, 6×3H); ¹³C-NMR (125 MHz, δ ppm, CHCl₃-d = 77.23 ): 38.23 (CH₂, C_− 1), 29.91 (CH₂, C_− 2), 71.28 (CH, C_− 3), 39.72 (CH₂, C_− 4), 139.78 (C, C_− 5), 119.12 (CH, C_− 6), 29.57 (CH₂, C_− 7), 32.10 (CH, C_− 8), 51.48 (CH, C_− 9), 34.47 (C, C_− 10), 21.79 (CH₂, C_− 11), 37.40 (CH₂, C_− 12), 43.53 (C, C_− 13), 55.37 (CH, C_− 14), 23.25 (CH₂, C_− 15), 28.71 (CH₂, C_− 16), 56.17 (CH, C_− 17), 12.28 (CH₃, C_− 18), 19.23 (CH₃, C_− 19), 40.52 (CH, C_− 20), 21.29 (CH₃, C_− 21), 31.71 (CH₂, C_− 22), 25.61 (CH₂, C_− 23), 49.71 (CH, C_− 24), 41.01 (CH, C_− 25), 13.25 (CH₃, C_− 26), 21.59 (CH₃, C_− 27), 22.90 (CH₂, C_− 28), 12.45 (CH₃, C_− 29), 167.0 (C, C_− 1'), 144.5 (CH, C_− 2'), 117.6 (CH, C_− 3'), 138.36 (C, C_− 4'), 128.25 (CH, C_− 5'/9'), 129.06 (CH, C_− 6'/8'), 129.06 (CH, C_− 7'); FTIR (KBr) υ_max: 2854.2, 2920.1 (aliphatic, C-H), 1706.9 (C = O), 1634.1 (C = C), 1165.8 (C-O) cm^− 1. On the basis of ¹H and ¹³C-NMR, MS and FTIR data above and comparison with those reported in the literature (Buxton et al., 2020), compound 3 is identified as sitosterol cinnamate.

2.6 Antimicrobial activity

2.6.1 Microbial strains and culture

The reference strains of Mycobacterium smegmatis (MC 2155), Escherichia coli (ATCC 25922), Klebsiella aerogenes (ATCC 13882), Staphylococcus aureus (ATCC 25923), Enterobacter cloacae (ATCC 13047), Klebsiella oxytoca (ATCC 8724) and Enterococcus faecalis (ATCC 13047) were received as gifts from the Department of Food and Biotechnology, University of Johannesburg, Doornfontein, South Africa. The strains were subsequently maintained at the microbiology laboratory of the department at -80°C until use. M. smegmatis strain was cultured on Middlebrook 7H11 agar under hygienic conditions, supplemented with 2% (v/v) glycerol and 10% (v/v) oleic acid, albumin, dextrose and catalase, and allowed to grow for 24 h. Middlebrook 7H9 broth was employed for the determination of minimum inhibitory concentrations (MICs) of the compounds on M. smegmatis. Nutrient agar was used for the activation of other bacterial strains. The Mueller-Hinton broth was used for assessing the MIC values of these strains (Kuete et al., 2012).

2.6.2 Minimum inhibitory concentrations (MICs) determination

MIC was expressed as the smallest concentration of the tested compounds that irreversibly converted the blue dye of resazurin solution into pink and exhibited total inhibition of the growth of bacteria (Eloff, 1999) (Table 1). The antimicrobial activity of the test compounds was assessed using resazurin micro-titer assay in a 96-well plate against the studied strains as previously described (Kuete et al., 2012), with minimal adjustment. Test samples were dissolved in pure dimethyl suphoxide (DMSO) to afford a final concentration of 1.0 mg/ml, and serially diluted (two folds) to afford working concentrations from 1.0 to 0.0313 mg/ml in a 96-well plate to 7H9 broth for M. smegmatis, and Mueller-Hinton broth for other strains. 100 µl of each concentration was seeded with 100 µl of standardized suspension of inoculums (1.5×10⁶ CFU/ml) in duplicate under aseptic conditions. The plates were sealed with sterile seals and then incubated at 37°C for 24 h. Viable bacterial cells were confirmed calorimetrically by the addition of resazurin dye (40 µL of 200 µg/mL) after 2 h incubation at 37°C as they enzymatically reduced resazurin dye (blue colour) to the resorufin (pink colour), and remained blue in death cells. Muller-Hilton broth (50% v/v in DMSO) was used as negative control. The concentration of DMSO in the well had no experimental influence on the bacterial growth. Streptomycin and nalidixic acid, two broad-spectrum antibiotics, were used as positive controls.

2.7 in vitro cytotoxicity

The compounds were evaluated for their in vitro cytotoxicity against human cervical cancer cell line (HeLa cells) employing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) assay as described by Parthasarathy and his colleagues (2020) with minimal adjustment. A fixed concentration (50 µg/ml) of tested samples was prepared in 100% dimethyl sulphoxide (DMSO) and incubated with HeLa cells in 96-well plate (1 × 10⁴ cells/mL) cultured and maintained in DMEM with 10% (v/v) fetal bovine serum (FBS) at 37 ^oC for 1 day in an environment of 5% carbon dioxide. Control cells were supplemented with 100 µl with pure DMSO. The number of HeLa cells that survived the tested samples exposure were determined by employing the resazurin-based reagent, and subsequently reading resorufin fluorescence in a multiwell micro-plate reader (VERSAmax, USA) at 595 nm. The experiments were performed in duplicate, and a mean standard deviation (SD) was calculated for each. The results were expressed as percentage (%) cell viability based on the fluorescence reading in treated wells against untreated control wells (Table 1).

The present study reports GC-MS analysis and the isolation of bioactive principles of V. paradoxa, a multipurpose medicinal plant used in African traditional medicine for managing respiratory infections, including tuberculosis and cough. GC-MS profiling of the extract revealed the occurrence of a total of 60 peaks of which 48 chemical compounds were identified. The identified compounds with their peak number, retention time (RT), and peak area (%) are presented in Table S1 (Supplementary Information). Out of the 48 detectable chemical constituents in the chromatogram (Supplementary Information), the major abundant bioactive compounds include hexadecane (11.23%), tetradecane (7.61%), 1-nonadecene (4.81%), 9-octadecenoic acid, methyl ester, (E)- (4.69%), n-pentadecanol (4.43%), 1-heptacosanol (4.15%), hexadecanoic acid, methyl ester (3.58%), glycidyl oleate (3.55%), and 1-hexadecene (3.34%). In addition, the phytochemicals identified in this present study belong to different classes of compounds, and most of them could be employed as antimicrobial agents (Table S1 and Fig. 2). 1-heptacosanol, a long-chain fatty alcohol, is reported as antimicrobial agent against E. coli and S. aureus (Abdi et al., 2020). 2-nonenal, (E)- is a phytoconstituent reported from Brownea grandiceps (Jacq.) leaves oils with promising antimycobacterial activity against M. tuberculosis (Korany et al., 2021). Hexadecanoic acid, methyl ester was reported to show promising antimicrobial effect against clinical pathogenic bacteria, including Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and K. pneumoniae (Shaaban et al., 2021). The presence of these compounds could be one of the reasons for the ethnopharmacological uses of the species in the management of respiratory-related infections. In addition, it could also be linked to these identified lipophilic compounds - hexadecane, tetradecane, and 1-nonadecene among others (Table S1). These lipophilic compounds possess the ability to weaken the binding mechanism of bacterial cell membranes as well as disrupt the membrane balance, which subsequently lead to cytoplasmic leakage and bacterial cell death (Savoia, 2012).

Similarly, the hexane stem-bark extract was subjected to column chromatography on silica to afford three compounds, including 11-hydroxy β-amyrin cinnamate (1) (Sirignano et al., 2021), α-amyrin cinnamate (2) (Miranda et al., 2000), and sitosterol cinnamate (3) (Buxton et al., 2020) (Fig. 3). The structures of the three compounds were established by analysis of their ¹H, ¹³C and 2D-NMR, as well as their mass spectroscopy (Provided in Supplementary List), and by comparison with previously reported data in the literature. Notably, to our knowledge, compounds 2 and 3 are reported here from the stem-bark of the plant for the first time. In order to explore the contribution of compounds 1–3 to the ethnobotanical uses of the plant in the treatment of tuberculosis and cough, they were screened against M. smegmatis (used as model for the pathogenic strain M. tuberculosis) together with six other bacterial pathogens associated with respiratory tract infections (Table 1) employing 96-well plate microbroth dilution method, and also investigated the cytotoxicity of compounds with the exception of isolate 3 which was not assessed to due low amount available.

Table 1

Antimicrobial and cytotoxicity of tested compounds 1–3 from *Vitellaria paradoxa*
	Antimicrobial (MIC, mg/mL)							Cytotoxicity (IC₅₀ µg/mL)
Strains:	Ms	Ec	Ka	Sa	Ecl	Ko	Ef	HeLa
Compound 1	0.50	0.50	0.50	0.50	0.50	0.50	0.50	82.53 ± 4.06
Compound 2	1.00	0.50	0.50	0.50	1.00	0.50	0.50	77.82 ± 10.5
Compound 3	0.125	0.0625	0.250	0.0625	0.0625	0.0625	0.0625	Not determined
Streptomycin	0.004	0.064	0.016	0.256	0.512	0.016	0.128
Nalidixic acid	0.512	0.512	0.256	0.064	0.016	0.008	> 0.512
Ms - Mycobacterium smegmatis; Ec - Escherischia coli; Ka - Klebsiella aerogenes; Sa - Staphylococcus aureus; Ecl - Enterobacter cloacae; Ko - Klebsiella oxytoca; Ef - Enterococcus faecalis; HeLa – Human cancer cell line

The isolated compounds 1–3 pharmacologically showed the antimycobacterial activity, with sitosterol cinnamate (3) exhibited the best activity against the inhibition of M. smegmatis (MIC; 0.125 mg/ml). It also showed the best activity against other tested bacterial strains, with MICs varying from 0.0625 to 0.25 mg/ml (Table 1). When compared with one of the positive controls, compound 3 exhibited better or equal activity to the nalidixic acid against M. smegmatis, E. coli, K. aerogenes, S. aureus, and E. faecalis. It also displayed better or equal activity against E. coli, S. aureus, E. cloacae, and E. faecalis in comparison with streptomycin, an antibiotic for treating tuberculosis. Unarguably, structure dictates functions. Structurally, all the isolates 1–3 (Fig. 3) contain cinnamoyl groups at the C-3. It is well-known that cinnamoyl functionality is a component of many drug-like molecules with enhanced biological activities, including antimycobacterial and antimalarial (De et al., 2012; Tanachatchairatana et al., 2008). Presumably, the better activity of compound 3 might be connected with structural difference to those of 1 and 2, which mainly consist of triterpenoid skeleton. Meanwhile, in setting clinical benchmark, Gibbons (2004) defined that a plant extract or its natural product has little clinical relevance if its MIC values greater than 1.0 mg/ml. Thus, compounds 1–3 fall under Gibbons' benchmark, and their inhibitory activities against the tested strains are also reported here for the first time. Also, compounds 1 and 2 showed a non-toxicity at the single concentration (50.0 µg/ml) tested against human cancer cell line (HeLa), with compound 1 had better toxicity profile with IC₅₀ of 82.53 ± 4.06 µg/ml (Table 1). Plant natural products are considered toxic if the IC₅₀ value ≤ 4 µg/ml (Pezzuto, 2002) according to the benchmark by the American National Cancer Institute (NCI). However, it might be erroneous to draw conclusion on the cytotoxicity of the tested compounds. Thus, further studies using normal human cell lines and/or more than one cancer cell lines in in vivo studies would be valuable to unravel the toxicity of these compounds in rational drug design and development against respiratory infection pathogens, including TB.

The present study reported identification of 48 phytochemicals using GC-MS, and the chromatographic isolation of three compounds, including 11-hydroxy β-amyrin cinnamate (1), α-amyrin cinnamate (2), and sitosterol cinnamate (3) from Vitellaria paradoxa. The compounds 1–3 showed inhibition of M. smegmatis, E. coli, K. aerogenes, S. aureus, E. cloacae, K. oxytoca, and E. faecalis, and their antimicrobial activities are reported here for the first time to our knowledge. Likewise, compounds 2 and 3 are isolated from the stem-bark of the plant for the first time in this study. Antibacterial activities of these compounds could provide some scientific justifications for the ethnomedicinal uses of V. paradoxa in the treatment of respiratory disorders, including tuberculosis.

Conflicts of Interest

The authors declare that they have no conflict of interest.

Funding

This work was supported financially by the South African TWAS-NRF Fellowships (Ref: Grant Numbers 116110 & 139057).

Acknowledgement

The authors are grateful to the National Research Foundation, South Africa, as well as The World Academy of Sciences, Italy for providing research fund for this work.

CRediT authorship contribution statement

Olusesan Ojo: Conceptualization, Methodology, Data curation, Data analysis, Writing - original draft, Funding acquisition. Edwin M. Mmutlane: Writing - review & editing, Supervision. Derek T. Ndinteh: Conceptualization, Supervision, Writing - review & editing, Funding acquisition

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The authors declare no competing interests.

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Inhibitory Activity of Chemical Constituents from Vitellaria paradoxa Gaertn. (Sapotaceae) Against Pathogens Associated with Respiratory Tract Infections

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Abstract

Figures

1 Introduction

2 Materials and methods

2.1 General instrumentations

2.2 Plant material collection

2.3 Extraction

2.4 GC-MS analysis

2.5 Column chromatographic isolation

2.6 Antimicrobial activity

2.6.1 Microbial strains and culture

2.6.2 Minimum inhibitory concentrations (MICs) determination

2.7 in vitro cytotoxicity

3 Results and discussion

4 Conclusion

Declarations

References

Additional Declarations

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