Apparatus and reagents
2,2-Diphenyl-1-picrylhydrazyl (DPPH) was purchased from Sigma-Aldrich Co., USA. Silica gel and silica gel F254 plates were purchased from Merck, Germany. Nutrient agar media, standard disc of kanamycin and ketoconazole were purchased from Hi media, India. The NMR spectra were documented by 400 MHz NMR spectrometer (Bruker, Switzerland) using deuterated chloroform and deuterated methanol purchased from Sigma-Aldrich Co., USA. All the chemicals and solvents used were of analytical grades.
Plant collection and identification
The aerial part of T. maxima was collected from Rangamati, Chittagong Hill Tracts, Bangladesh on August 20, 2015. The taxonomical identification of the tested plant was authenticated from Bangladesh National Herbarium (BNH), Mirpur, Dhaka and voucher specimen was deposited in the BNH with the accession number DACB 42267.
Preparation of the crude extract
A total of 1 kg powdered coarse plant materials were subjected to maceration in dichloromethane:methanol (1:1) mixture using 2.5 l of each solvent in an air-tight container for 5 days with intermittent shaking. The solvent mixture with most of the extractable compounds was collected by filtration with cotton plug followed by filter paper. The solvent was removed using rotary evaporator (Heidolph, Germany) at temperature around 40 °C - 50 °C under reduced pressure. The above process was repeated two times with fresh solvents to gather more concentrated extract. Finally, all the filtrates were mixed together to get the crude extract (19 g).
Chromatographic procedures
The crude extract was fractionated by vacuum liquid chromatography (VLC) on silica gel (60G) eluted with gradient solvent system as petroleum ether-CH2Cl2-MeOH to obtain 27 fractions. These fractions were screened by thin layer chromatography (TLC) with aluminum plates coated with silica gel (F254) and the plates were envisaged under UV light at 254 nm, 365 nm and by spraying with vanillin/1% H2SO4 solution followed by heating. The fractions having similar TLC patterns were combined to give 8 fractions (F-1 to F-8). Column chromatography (silica gel 70-230 and 230-400, mesh) and preparative TLC (PTLC) were performed for separation and purification of the compounds from the VLC fractions.
Bioassay screening of the fractions
DPPH free radical scavenging activity
The free radical scavenging ability of T. maxima VLC fractions were screened by measuring the reduced absorbance of methanolic DPPH solution [9]. Methanolic DPPH stock solution (20 μg/mL) was added (200 μL) to methanolic sample solution to obtain final 4 ml solution of different concentration (200 μg/mL to 12.5 μg/mL). The absorbance was measured at 517 nm by using UV-VIS spectrophotometer (Analytic Jena AG, Germany) after the solutions were mixed properly and kept in dark for 20 min. The result was expressed using the following formula as the percentage inhibition:
$$ \left[\left({\mathrm{A}}_0-{\mathrm{A}}_1\right)/{\mathrm{A}}_0\right]\ \mathrm{x}100, $$
where A0 is the absorbance of the control and A1 is the absorbance of the fractions/standard.
Antimicrobial assay
Disc diffusion method was used for preliminary antimicrobial assay [10] against four bacterial strains (Bacillus megaterium, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli) and two fungal strains (Aspergillus flavus and Aspergillus niger). A total of 100 μL of suspension of each microorganism containing approximately 100-150 CFU/mL was spread over the nutrient agar for bacteria and potato dextrose agar media for fungi. Filter paper discs around 6 mm diameter were sterilized, steeped with 200 μg of different fraction solutions and placed gently in the spreaded agar plates. Kanamycin (30 μg/disc) and ketoconazole (30 μg/disc) were used as positive control and solvent discs were used as negative control in this study. The zone of inhibition was measured as diameter in mm after 24-h incubation at 37 °C for bacteria and 48-h incubation at 28 °C for fungi.
Isolation of the compounds
Phytochemical investigation for compound isolation on fractions F-2, F-3 and F-4 were performed based on the preliminary chemical profiling using TLC, antimicrobial activity of F-2 and F-3 and antiradical activity of F-4. Polar fractions F-5 to F-8 eluted with CH2Cl2/0.1-8% MeOH showed very poor resolution of the compounds on TLC due to matrix effect. Therefore, phytochemical investigation for isolation of compounds from fraction F-5 to F-8 could not carry out in spite of their strong antiradical property.
An aliquot (80 mg) of VLC fraction F-3 (583.4 mg) eluted with petroleum ether/35-45% CH2Cl2was subjected to preparative thin layer chromatography (PTLC) (stationary phase: silica gel F254, mobile phase: toluene/10% EtOAc, two developments; thickness of plates: 0.25 mm) to afford compound 1 (3.0 mg, 3.75%). Compound 2 (2.5 mg, 3.12%) was obtained by subjecting 80 mg of the VLC fraction F-4 (1.38 g) eluted with petroleum ether/50-85% CH2Cl2 to PTLC (mobile phase: toluene/30% EtOAc, 2 developments). Fraction F-3 was subjected to silica gel column chromatography and eluted with n-hexane-CH2Cl2-MeOH solvent system with increasing polarity. Column fraction eluted with n-hexane/35-40% CH2Cl2 gave brown amorphous mass which was purified by solvent treatment to afford compound 3 (2.2 mg, 2.75%). Fraction F-4 was rechromatographed by silica gel column chromatography eluted with petroleum ether-CH2Cl2-MeOH in gradients of increasing polarities. Fraction obtained by petroleum ether/85-95% CH2Cl2 was further purified by PTLC (mobile phase: toluene/5% EtOAc, 2 developments) to yield compound 4 (3.0 mg). One slightly impure white crystal obtained from the VLC fraction of petroleum ether/20-30% CH2Cl2 (F-2, 287.5 mg) was purified by treatment with different solvents to get compound 5 (3.2 mg, 4%).
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4-Hydroxybenzaldehyde (1): (3.0 mg, brown amorphous powder); 1H NMR (400 MHz, CDCl3): δ 9.78 (1H, s, −CHO), 7.74 (2H, d, J = 8.6 Hz, H-2, H-6), 6.91 (2H, d, J = 8.6 Hz, H-3, H-5). 13C NMR (100 MHz, CDCl3): δ 191.4 (−CHO), 163.2 (C-4), 132.4 (C-2, C-6), 128.8 (C-1), 115.8 (C-3, C-5).
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4-Hydroxycinnamic acid (2): (2.5 mg, white amorphous powder); 1H NMR (400 MHz, CDCl3/1% CD3OD): δ 7.37 (2H, d, J = 8.2 Hz, H-2, H-6), 6.79 (2H, d, J = 8.2 Hz, H-3, H-5), 7.60 (1H, d, J = 16.0 Hz, H-7), 6.23 (1H, d, J = 16.0 Hz, H-8). 13C NMR (100 MHz, CDCl3): δ 169.3 (C-9), 158.8 (C-4), 145.3 (C-7),132.0 (C-2, C-6), 125.9 (C-1), 115.5 (C-3, C-5), 114.6 (C-8).
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4-Hydroxybenzoic acid (3): (2.2 mg, brown amorphous powder); 1H NMR (400 MHz, CD3OD): δ 7.88 (2H, d, J = 8.4 Hz, H-2, H-6), 6.78 (2H, d, J = 8.4 Hz, H-3, H-5). 13C NMR (CD3OD, 100 MHz): δ 171.4 (−COOH), 163.2 (C-4), 132.4 (C-2, C-6), 128.9 (C-1), 115.8 (C-3, C-5).
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Stigmast-4-en-3-one (4): (3 mg, white crystal); 1H NMR (400 MHz, CDCl3): δ 5.75 (1H, s, H-4), 1.20 (3H, s, H-19), 0.94 (3H, d, J = 5.2 Hz, H-21), 0.91 (3H, d, J = 6.4 Hz, H-29), 0.87 (3H, d, J = 5.6 Hz, H-26), 0.84 (3H, d, J = 5.6 Hz, H-27), 0.73 (3H, s, H-18).13C NMR (100 MHz, CDCl3): δ 198.9 (C-3), 171.4 (C-5), 123.6 (C-4), 55.9 (C-17), 55.8 (C-14), 53.7 (C-9), 45.7 (C-24), 42.5 (C-13), 39.7 (C-12), 38.6 (C-10), 36.0 (C-20), 35.7 (C-8), 35.6 (C-1), 34.0 (C-22), 33.9 (C-2), 32.9 (C-6), 32.1 (C-7), 29.1 (C-25), 28.1 (C-16), 26.1 (C-23), 24.1 (C-15), 23.1 (C-28), 21.1 (C-11), 19.7 (C-26), 19.1 (C-27), 18.7 (C-21), 17.5 (C-19), 11.9 (C-18), 11.9 (C-29).
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β-stigmasterol (5): (3.2 mg, white needle); 1H NMR (400 MHz, CDCl3): δ 5.37 (1H, d, J = 4.8 Hz, H-6), 5.18 (1H, dd, J = 15.0, 8.6 Hz, H-22), 5.04 (1H, dd, J = 15.2, 8.4 Hz, H-23), 3.55 (1H, m, H-3), 1.03 (3H, s, H-19), 0.94 (3H, d, J = 6.4 Hz, H-21), 0.86 (3H, d, J = 2.8 Hz, H-29), 0.85 (3H, d, J = 1.2 Hz, H-26), 0.82 (3H, d, J = 7.2 Hz, H-27), 0.71 (3H, s, H-18). 13C NMR (100 MHz, CDCl3): δ 140.7 (C-5), 138.4 (C-22), 129.2 (C-23), 121.6 (C-6), 71.8 (C-3), 56.9 (C-14), 56.8 (C-17), 51.2 (C-24), 50.1 (C-9), 45.8 (C-25), 42.3 (C-13), 42.2 (C-4), 40.4 (C-20), 39.8 (C-12), 37.2 (C-1), 36.4 (C-10), 33.9 (C-8), 31.7 (C-7), 29.2 (C-16), 28.1 (C-2), 24.4 (C-28), 24.3 (C-15), 21.1 (C-11), 21.0 (C-21), 19.7 (C-27), 19.3 (C-26), 19.0 (C-19), 12.1 (C-29), 12.0 (C-18).
Evaluation of cytotoxicity of pure compounds
Cytotoxic activity was studied against Vero cell line (Vero cell line, CLS 605372, Germany) using slight modification of the Trypan Blue Exclusion Method [11, 12]. Cells were cultivated in 75cm2 flasks in 5% (v/v) CO2 at 37 °C with media described at Khan et al, 2018 [10]. According to study design, cells were grouped into 3 with three replica each. Treatment groups were evaluated with vehicle group. Vero cells were split the day before experiments. The freshly prepared doses (0.1, 0.5, 1.0, 5.0, 10 and 20 μg/mL) were administered into 1 day before cultured T- flasks with approximately 2.5 × 106 cells. Negative control corresponds to the cells cultured with medium with 0.6% DMSO. After 24 h of incubation period cells were harvested using 0.5% trypsin. The number of dead cells was calculated by automated cell counter (LUNA-II™, South Korea) [13] using trypan blue (0.4% w/v). Percentage of dead cells was calculated following the mathematical formula:
$$ \mathrm{Percentage}\ \mathrm{of}\ \mathrm{dead}\ \mathrm{cells}=\frac{\mathrm{No}\ \mathrm{of}\ \mathrm{stained}\ \left(\mathrm{dead}\right)\ \mathrm{cells}}{\mathrm{Total}\ \mathrm{number}\ \mathrm{of}\ \mathrm{cells}}\times 100 $$
Statistical analysis
All values for antioxidant and antimicrobial evaluation were determined as mean ± standard deviation (SD) where n = 3. Continuous variables between groups were compared with one-way analysis of variance (ANOVA) with post hoc tukey’s test for the analysis of the cytotoxicity results of pure compounds. Mean values between groups were compared using independent student t-test for equality of variances. Statistical significance was accepted when P < 0.001.