Abubidentin A, New Oleanane-type Triterpene Ester from Abutilon bidentatum and its antioxidant, cholinesterase and antimicrobial activities

Chemicals and reagents

All the chemicals and reagents used for this study including; ethanol (96%), methanol (99.8%), petroleum ether (40–60 °C), dichloromethane (≥99.8%), chloroform (99.9%), n-butanol (99.8%), dimethyl sulfoxide (DMSO), hydrochloric acid (HCl), potassium hydroxide (KOH, ≥85%), 1,1-diphenyl2-picrylhydrazyl (DPPH, 95%), 2, 20-azino-bis[3-ethyl benzo thiazoline-6-sulphonic acid] (ABTS), potassium persulfate (K₂S₂O₈), acetylthiocholine iodide (≥99.0%), S-butyrylthiochoilne iodide (≥98.0%), sodium phosphate (Na₃PO₄, 96%), ascorbic acid, resazurin, 2-nitrobenzoic acid and were obtained from Sigma Aldrich (Hamburg, Germany). Muller Hinton agar and Muller Hinton broth were procured from HiMedia (Himedia Laboratories Pvt Ltd., Mumbai, India). Donepezil, galantamine and chloramphenicol were purchased from the local drug store.

Instrumentation

Optical rotation: Perkin-Elmer Model 341 LC polarimeter (Perkin-Elmer, Waltham, MA, USA). UV spectra: in MeOH using a Perkin-Elmer Lambda 25 UV/VIS spectrophotometer (Perkin-Elmer, Waltham, MA, USA). IR spectra: Shimadzu Infrared-400 spectrophotometer (Shimadzu, Kyoto, Japan). ESIMS spectra: Agilent 6320 Ion trap mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) equipped with an electrospray ionization interface. NMR spectra: Bruker DRX 700 spectrometer (Bruker, Rheinstetten, Germany). Column chromatographic separations: silica gel 60 (0.04–0.063 mm, Merck, Darmstadt, Germany). TLC analyses: pre-coated silica gel F254 aluminum sheets (Merck, Darmstadt, Germany).

Enzymes and pathogenic strains

The acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were obtained from the mouse brain and human blood at the Pharmacology Department of King Saud University, SaudiArabia. Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Staphylococcus aureus were provided by Microbiology Department, King Khaled University Hospital (KKUH), Saudi Arabia.

Plant material

The fresh aerial parts of Abutilon bidentatum Hochst. were collected from Jazan city, Aseer region, Saudi Arabia in March 2009. The plant was kindly identified by Dr. Mohamed Yusuf at the Pharmacognosy Department, College of Pharmacy, King Saud University. A voucher specimen (#16022) was deposited in the herbarium of the Pharmacognosy Department.

Extraction and isolation

Air dried-powdered aerial parts of A. bidentatum (900 g) were exhaustively extracted by maceration with 96% EtOH (3L × 7) with shaking. The combined EtOH extract was dried under reduced pressure at 45 °C to give a dry total extract weighing 89 g (EE). A part of the dried residue (87 g) was suspended in 450 mL distilled water and fractionated by shaking with petroleum ether (500 mL × 6), dichloromethane (500 mL × 4), ethyl acetate (500 mL × 5), and n-BuOH (500 mL × 6), respectively. Each combined extract was individually evaporated under reduced pressure to give the petroleum ether (PEF, 13 g), dichloromethane (DCF, 4 g), ethyl acetate (EAF, 6 g), n-BuOH (BF, 9 g), and aqueous soluble (AF, 55 g) fractions. Therefore, part of PEF (11.5 g) was chromatographed on SiO₂ column (330 g, 5 × 90 cm). Elution was started with petroleum ether and the polarity was gradually increased with CH2Cl2, followed by CH₂Cl₂: MeOH mixtures up to 100% MeOH to afford seven subfractions: AB-1 to AB-7. TLC pattern of subfractions AB-2 and AB-5 showed the presence of prominent compounds and were selected for further column chromatography. Subfraction AB-2 (33 mg) was chromatographed over silica gel using petroleum ether:CH₂Cl₂ gradient to give 1 (6.7 mg, white powder). Subfraction AB-5 (311 mg) was applied on SiO₂ column (15 g, 70 × 1 cm), using petroleum ether:CH₂Cl₂ (95:5 to 80:20) to give two subfractions AB-5.1 and AB-5.2 containing two major spots on TLC. Subfraction AB-5.1 (43 mg) was purified on SiO₂ column, eluted with petroleum ether:CH₂Cl₂ gradient to yield 2 (10 mg, white amorphous powder). Sub fraction AB-5.2 was similarly treated as subfraction AB-5.1 to give 3 (12 mg, white amorphous powder).

Abubidentin A (1α,3β,30-Trihydroxy-29-carboxy-olean-9(11), 12-diene-3-dotriacontanoate; 3)

White amorphous powder. [α]_D +45 (c = 0.1, MeOH). UV (MeOH) λ_max (log ɛ): 212 (3.10), 275 (2.21) nm. IR (KBr): 3,420, 2,947, 1,732, 1,712, 1,636, 1,245 cm⁻¹. NMR data (CDCl₃, 700 and 176 MHz) see Table 1. (+) ESI-MS m/z: 971.4 [M+Na]⁺; (-) ESI-MS m/: z 947.8 [M-H]^-.

Alkaline hydrolysis of compound 3

Compound 3 (4 mg) was dissolved in five mL of 3% KOH/MeOH solution and kept undisturbed for 15 min at ambient temperature. After 15 min, 1 N HCl/MeOH was added to the solution for the neutralization. The solution was then partitioned with CHCl3, CHCl3 layer was separated, evaporated and the obtained residue was taken up for column chromatography on SiO2 (mesh 60–120) using as eluent hexane: EtOAc gradient to provide methyl ester of dotriacontanoic acid, which was verified by GC/MS (Ibrahim, Mohamed & Ross, 2016; El-Shanawany et al., 2015; Al-Musayeib et al., 2013). A 500-Clarus Perkin-Elmer GC/MS (Waltham, MA, USA) was applied for GC/MS analysis. The integrator combined with software (4.5.0.007 version) controller was turbo mass. A 5 MS/GC elite capillary (30 × 0.25 mm × 0.5 µm) column and helium (He) a carrier gas at 2 ml/min flow rate (55.8 cm/s flow initial with 32 p.s.i., split; 1:40) were applied. Temperature conditions including; source temperature, inlet line temperature, emission trap and electron energy were adjusted at 150 °C, 200 °C, 100 °C and 70 eV, respectively. The injector temperature at 220 °C was maintained. Whereas, the temperature of the column was set at 50 °C for 5 min, raised to 220 °C at the 20 C/min rate. MS was scanned from 50 to 650 m/z.

Antioxidant activity

DPPH radical scavenging activity

The ability of the samples to scavenge DPPH radical was determined by Wang, Chen & Hou (2019) method with slight modification (Wang, Chen & Hou, 2019). A total of 20 µL of different sample concentrations (5.25–50 mg mL⁻¹) solutions was reacted with 180 µL of DPPH^• (6–5 M) dissolved in methanol (80%) in a 96-well plate. Samples were incubated at ambient temperature under dark conditions for 30 min and the absorbance wavelength was measured at 517 nm against a blank sample. DPPH^• solution and ascorbic acid were used as blank samples and positive control, respectively. The inhibitory concentration (IC₅₀) was expressed as the concentration that inhibits the 50% of DPPH and calculated using the following equation ${\displaystyle \text{Inhibition percentage}\left(\text{\%}\right)=\frac{\left({\mathrm{A}}_{\mathrm{b}}-{\mathrm{A}}_{\mathrm{s}}\right)}{{\mathrm{A}}_{\mathrm{b}}}\times 100}$

where A_b and A_s are the absorbance values of the blank and test samples, respectively.

ABTS radical scavenging activity

ABTS radical cation decolorization assay was performed to measure the total antioxidant activity of the samples by obeying the previously described method (Re et al., 1999). In brief, ABTS^•+ radicals were generated by treating 7 mM ABTS⁺ aqueous solution with 2.4 mM potassium persulfate for 12-16 h at room temperature in the dark. Prior to use, this solution was diluted with ethanol (approx. 1:89 v/v) and equilibrated to 0. 7,000 ± 0.02 at 300 °C absorbance at 734 nm to obtain the working solution. Afterward, a 1.0 mL of working solution was reacted with 20 µL (1 mg mL⁻¹) samples and incubated for 30 min. After incubation, samples were scanned at 734 nm absorbance wavelength and the percentage of inhibition was determined. Ascorbic acid (AA) was applied reference standard.

Evaluation of cholinesterase inhibitory activities

The cholinesterase inhibitory effect of test sample was investigated on two enzymes (acetylcholinesterase and butyrylcholinesterase) by spectrophotometric method by following Ellman et al. procedure with little modification (Ellman et al., 1961). Crude enzymes, AChE (acetylcholinesterase) and BChE (butyrylcholinesterase) were collected from brain of mice and blood of humans, respectively, by obeying earlier described procedure (Asaduzzaman et al., 2014; Uddin et al., 2015). The AChE and BChE assays were tested by using two chemical substrates acetylthiocholine iodide and S-butyrylthiochoilne iodide, respectively. In brief, 10 µL of each enzyme was reacted individually with equal volume (10 µL) of different concentration (25–400 µgmL⁻¹) test sample and reference standard followed by incubation at 37 °C for 15 min for the complete interaction. Afterwards, 2-nitrobenzoic acid (1 mM, 62 µL), sodium phosphate buffer (50 mM, 25 µL, pH 8) provided with bovine albumin serum (0.1%) and acetylcholine iodide (0.5 mm, 13 µL) were added separately into each reaction mixture. Each reaction mixture was individually incubated further for 15 mis at 37 °C and absorbance at 405 nm was immediately noted against the blank. Donepezil and galantamine were used as reference compounds for AChE and BChE activity, respectively. All the experiments were performed in triplicates to avoid error and the results were estimated through the two-tailed Student’s t-test at a p < 0.05 significance. The inhibition percentage of cholinesterase activity was calculated using the following equation ${\displaystyle \mathrm{I}\text{\%}=\frac{{\mathrm{A}}_{\mathrm{C}}-{\mathrm{A}}_{\mathrm{s}}}{{\mathrm{A}}_{\mathrm{C}}}\times 100}$

where A_c and A_s is the absorbance of control and sample or reference compound. IC50 values could be determined from the dose response curve obtained by plotting the percent inhibition values against test concentrations of each compound.

Antimicrobial activity

Minimum inhibitory concentration (MIC) assay was performed to evaluate the in vitro antimicrobial activities using a broth microdilution method (Nascente, 2009). Three bacterial strains Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Staphylococcus aureus were applied to examine the antimicrobial activity of samples. Briefly, the microbial strains were transferred to Muller Hinton agar (MHB, HiMedia, India) and 24-h colonies were individually suspended in 10 mL Muller Hinton broth (MHB, HiMedia, India). The suspension of each microbial strain was standardized at 575 nm wavelength using a spectrophotometer (CRAIC Technologies, CA, USA), to match the McFarland scale (1.5 × 108 CFU mL⁻¹). The standardized suspension was further diluted to obtain a final concentration of 5 × 105 CFU mL⁻¹. The samples prepared in DMSO at 1 mgmL⁻¹ and different concentrations (30–500 µgmL⁻¹) were attained after dilution in Mueller Hinton broth. Three inoculated wells containing different concentrations of DMSO (4% to 1% range), one non-inoculated well without an antimicrobial agent and negative controls were included. The inoculated well was used to monitor whether the broth was sufficient for the microbial strain to grow. Chloramphenicol (500 to 30 mgmL⁻¹) was applied as a positive control. The sample treated 96-well microplates were sealed and incubated for 24 h at 37 °C After a 1-day incubation, 30 ml of 0.02% resazurin solution was added into each well to examine the viability of the microbial strain (Palomino et al., 2002). The minimum used concentration of test sample that inhibited the microorganism growth (MIC value) was calculated as the minimum concentration of the test samples required to prevent the color change of the resazurin solution. All the assays were performed in triplicates.

Statistical analysis

The experiments were conducted in triplicates and results were expressed as mean ± standard deviation. Statistical and graphical analysis were performed on Graph Pad Prism (version 8.0.1) and Microsoft Excel 2010. T-test was carried out to determine the statistical significance between the average values and (p < 0.05) was considered significant.

Antioxidant activity assessments

DPPH and ABTS⁺ assays are widely applied to measure the compound’s ability to determine its antioxidant potential. Both the spectrophotometric methods used for evaluating antioxidant activity are based on electron transfer reactions and visually rely on the reduction of a colored oxidant. The obtained results from these two assays expressed good correlation. Fig. 3A and Table 2 represents the free radical inhibition of isolated compounds (1, 2, and 3) and ascorbic acid (AA, standard) at different concentrations. The results showed that the DPPH scavenging potential of compounds 1, 2, and 3 incrementally increased with the increase in concentration of compounds. The IC₅₀ values obtained were 10.82 ± 0.24, 7.60 ± 0.42, and 4.67 ± 0.28 µgmL⁻¹ for compounds 1, 2, and 3 were, respectively, indicating that compound 3 possesses the highest radical scavenging potential and was 1.55 fold lower than ascorbic acid (IC₅₀ 3.12 ± 0.24 µgmL⁻¹), followed by compounds 2 and 1 (p < 0.05).

The ABTS⁺ assay is an additional important procedure for the quantification of radical scavenging potential that can provide parallel results to those obtained in the DPPH assay. The results showed that all the three isolated compounds exerted significant ABTS free radical scavenging activity and had an antioxidant potential proportional to that of ascorbic acid (Fig. 3B and Table 2). Compound 3 was found to be the most active radical scavenger with an IC₅₀ value of 6.42 ± 0.25 µgmL⁻¹ and 3.12 fold lower than that of ascorbic acid, followed by compound 2 and 1 with IC₅₀ values of 8.18 ± 0.13 and 11.45 ± 0.37 µgmL⁻¹, respectively ( p < 0.05). Compound 3 displayed both the highest DPPH and ABTS radical scavenging activities. A correlation between DPPH and ABTS methods applied to determine the antioxidant potential of the tested compounds was examined. The DPPH radical activity showed a strong correlation with ABTS radical activity (R₂ = 96.67%).

Cholinesterase inhibitory activity

The soluble fraction of the extract and compounds (1–3) were screened for AChE and BChE inhibition at different concentrations. The percent inhibition of AChE by the test compounds were presented in Fig. 4A. Donepezil used as a reference AChE inhibitor showed an IC₅₀ value of 9.32 ± 0.38 µgmL⁻¹. The result revealed that all the investigated compounds (1–3) showed inhibition of the AChE enzyme in a dose-dependent manner. Among the compounds, high activity was displayed by compound 3 with IC₅₀ values of 38.13 ± 0.07 µgmL⁻¹, followed by compound 2 (IC₅₀ value = 68.65 ± 0.56 µgmL⁻¹). However, compound 1 showed low activity with IC₅₀ of 121.97 ± 1.61 µgmL⁻¹ (Table 2). Similarly, in BChE inhibitory assay, compounds 3 and 2 exerted high inhibitory potentials with IC₅₀ of 32.68 ± 0.37 and 49.52 ± 0.35 µgmL⁻¹, respectively (Table 2, Fig. 4B). The IC₅₀ value of compound 1 was 137.76 ± 0.67 µgmL⁻¹ exhibited very negligible effects. Thus, compound 3 had appreciable activity towards both AChE and BChE enzymes.

Antimicrobial activity

The antimicrobial potential of isolated compounds (1–3) was assessed by calculating MIC values and their ability to inhibit the growth of the tested microbial strain. The assay was performed in 96-well microplates by applying resazurin as a developer. The well plates that exhibited the blue color after the addition of resazurin were considered as the MIC value for each microorganism (Ostrosky et al., 2008). The pure isolated compounds 1–3 were examined against E. coli, P. aeruginosa, and S. aureus bacterial strains for their antimicrobial potential. All the tested compounds exerted antimicrobial effect in the range of 125–1,000 µgmL⁻¹ towards the pathogenic strains (Table 3). However, the prominent activity was shown by compound 2 with MIC ≤125 gmL⁻¹, ≤250 µgmL⁻¹, ≤150 µgmL⁻¹ and compound 3 with ≤150 µgmL⁻¹, ≤125 µgmL⁻¹, ≤125 µgmL⁻¹, against E. coli, P. aeruginosa, and S.aureus, respectively. There is no consistent classification with respect to MIC values (Aligiannis et al., 2001), but the values obtained ≤1,000 µgmL⁻¹ were considered as satisfactory and sensitive (Webster et al., 2008). Thus, the MIC value of compounds 3 and 2 can be considered as promising.