2.1 Instruments, chemicals and reagents

Melting points were determined using digital melting point apparatus (Sonar) India, whereas Rudolf autopol model polarimeter measured the optical rotations. The solvents, hexane, ethyl acetate, methanol, ethanol, glacial acetic acid, sulphuric acid, hydrochloric acid, were purchased from E-Merck Ltd, India. Pre-coated TLC plates (layer thickness 0.25 mm), silica gel for column chromatography (70–230 mesh ASTM) and LiChroprep RP-18 (40–63 μm) were from Merck (Germany). Authentic standards of chemicals were purchased from Sigma-Aldrich (USA). Ultraviolet–visible spectroscopy was measured with TU-1800_PC UV–vis spectrophotometer. Both ¹H and ¹³C NMR spectra were recorded in CDCl₃ and CD₃OD on a Bruker DRX-300 & 500 model spectrometers operating at 300 and 75 and 500 and 125 MHz, respectively. NMR spectra were obtained in deuterated chloroform, methanol using tetramethylsilane (TMS) as an internal standard, FAB-MS data were recorded on a JEOL SX-102 spectrometer, and Electrospray ionization (ESI) mass spectra were recorded in positive mode on an API-3000, LC/MS/MS (Applied Biosystem/MDS SCIEX, Toronto, Canada) mass spectrometer using a standard ESI source coupled with LC separation system and HR ESI MS in the positive mode was recorded on an agilent 6520 QTOF (ESI-HRMS). Infrared spectroscopy was recorded on an FT-IR spectrophotometer Shimadzu 8201 PC (4000–400 cm⁻¹). Thin layer chromatography was performed on precoated silica gel 60 F₂₅₄, plates (Merck, layer thickness 0.25 mm). Visualization of the TLC spots was performed using 5% H₂SO₄ in ethanol spray reagent.

2.2 Plant material

The fruits of Z. armatum were purchased from the local market of Lucknow, State of Uttar Pradesh, India in the month of March 2018 and identified by the Department of Botany and Pharmacognosy, CIMAP. A voucher specimen (ZA/F/14) was deposited in the herbarium of the CSIR-CIMAP, India.

2.3 Extraction of fruits

Dried fruits of Z. armatum (14.5 Kg) in powdered form were extracted with methanol (70 L) by refluxing 8 h for three days and concentrated in vacuo to obtain a semisolid brown mass to yield (2.9 kg) of an extract, which was suspended in water and extracted with hexane, ethyl acetate and, n-butanol, successively, to produce 1.5 kg, 300 g, 258 g and, 603 g extracts, respectively. Isolated compounds (1–7), ethyl acetate fraction (8), and n-butanol fraction (9) was evaluated for cytotoxicity and anti-oxidant potential.

2.4 Isolation of the compounds from ethyl acetate extract of Z. armatum

The ethyl acetate extract (206 g) was subjected to silica gel column (1.5 kg; 60–120 mesh size) and eluted with solvent of n-hexane, n-hexane-EtOAc (9:1–1:9, v/v), EtOAc, and MeOH to give 37 fractions (frs.; each of 10 L). Fractions were checked by TLC and showing complex mixtures, fraction 7 (11 g, n-hexane-EtOAc 7:3) were chromatographed over silica gel column (90 g; 60–120 mesh size, each fraction of 50 ml) yields. The elution was sequentially performed with CHCl₃, CHCl_3-MeOH (9.5:0.5, 9:1, 8:2 v/v) Compound 1 (10 mg), fraction 26 (3.4 g, obtained in EtOAc) were re-chromatographed over silica gel column (50 g; 200–400 mesh size; each fraction of 20 ml). The elution was sequentially performed with CHCl₃, CHCl_3-MeOH (1%, 2%, 3%, 4%, 5% v/v) to yield 20 fractions yield Compound 2 (25 mg).

Fraction I (105 gm) was subjected to silica gel column (500 gm; 60–120 mesh size) and eluted with solvent of n-hexane, n-hexane-EtOAc (1:1, 3:7, 2:8, 1:9 v/v), EtOAc, and MeOH to give 17 fractions (each of 15 L). Fraction 2 (25gm, n-hexane-EtOAc 1:1) was chromatographed over silica gel column (50 g; 60–120 mesh size, each fraction of 20 ml) elution was sequentially performed with CHCl₃, CHCl_3-MeOH (0.5%, v/v) yields 10 fractions yield Compound 3 (10 mg). Fraction 7&8 (41 g, n-hexane-EtOAc 7:3) were chromatographed over silica gel column (120 g; 60–120 mesh size, each fraction of 100 ml). The elution was sequentially performed with CHCl₃, CHCl_3-MeOH (1%, 2%, 5% v/v) to yields 25 fractions yields Compound 5 (15.2 mg), Compound 6 (20 mg), Fraction 13&14 (20 g, n-hexane-EtOAc 9:1) were chromatographed over silica gel column (100 g; 60–120 mesh size, each fraction of 50 ml) and elution was sequentially performed with CHCl₃, CHCl_3-MeOH (0.5%, 1%, 2%, 3%, v/v) to yield 12 fractions yields Compound 4 (22 mg), and the isolation of Compound 7 from the method described by Nooreen et al. (2017a).

2.5 Cell viability study on HepG2 cells

The HepG2 (Liver) and lung (A549) cancer cell lines were obtained from the German Collection of Moicroorganisms and Cell Cultures (DSMZ), Braunschweig, Germany. The cell viability of isolated compounds (1–7), ethyl acetate extract and, n-butanol extract was tested using a protocol of Fraga et al. (2008) with slight modifications, and this assay was used to detect the influence of isolated compounds on cell viability using the following protocol (Fraga et al., 2008). The effect of different compounds on the viability of normal and transformed cells was determined by MTT (3- [4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay. Briefly, cells were seeded in a 96-well plate at a density of 1 × 10⁴ cells/well. Cells were treated with different concentrations of samples (10 µg, 20 µg, 30 µg, 40 µg, 50 µg, 100 µg, 150 µg, and 200 µg) at 24 h, 48 h, and 72 h to determine the toxic and sub-toxic doses. At the end of treatment, 20 µL MTT (5 mg/mL) was added to each well. After incubation for 3 h, media along with MTT was removed. 200 µL DMSO was added to dissolve the formazan crystal, and absorbance was recorded at 540 nm using an ELISA plate reader. The plot of percent cell viability versus different compounds concentrations was used to calculate the concentration lethal to 50% of the cells (IC₅₀). The cellular morphological changes were observed under inverted phase-contrast microscopy (Nikon Eclipse Ti-S, Tokyo, Japan). A similar test of different compounds was also performed on WRL-68 to examine if the treatment had a distinguishable effect between normal and cancer cells.

2.6 Quantification of intracellular reactive oxygen species (ROS)

Intracellular reactive oxygen species (ROS) generation was analyzed by using fluorescence microscopic imaging technique as per previous protocol (Siddiqui, 2015). Cells (1 × 10⁴ per well) were exposed with compound 1with sub IC₅₀ value and IC₅₀ value, i.e., 25.375 µg/ml and 50.75 µg/ml for 24 h and 48 h. Subsequently, cells were incubated with Dichloro-dihydro-fluorescein diacetate (DCFH-DA, 10 mM) at 37 °C for 30 min and washed with PBS. The intracellular fluorescence intensity of cells was visualized by an inverted fluorescence microscope (Nikon ECLIPSE Ti-S, Tokyo, Japan). For quantitative fluorometric analysis, cells (1 × 10⁴per well) were seeded and treated with different compounds in 96-well black bottom culture plate. Fluorescence intensity was measured with a multiwall microplate reader (Synergy H1 Hybrid Multi-Mode Microplate Reader, BioTek, Winooski, VT) at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. ROS production was quantified using Image J software (Image J, National Institutes of Health, and Bethesda, MD). Data were expressed as a percentage of fluorescence intensity relative to the control wells.

2.7 DPPH radical inhibition activity

The free radical inhibition activity of different compounds (1–7), ethyl acetate extract (8), and butanol extract (9) were determined by 1, 1-diphenyl-2-picrylhydrazyl (DPPH Assay) (Blois, 1958). 3 ml reaction mixture was prepared which contained different concentrations of the crude extract and 0.1 mM methanolic DPPH solution and was incubated for 30 min. The absorbance of each reaction mixture using a spectrophotometer was taken at 517 nm where lower absorbance recorded for the reaction mixture indicated elevated free radical inhibition activity. All extracts were analyzed in triplicates. Ascorbic acid was taken as a standard. $$\mathit{DPPH}inhibitionactivity\left(\%\right)=\left(A_0-A_1/A_0\right)\times 100$$ Where, A₁- Sample absorbance; A₀ - Absorbance of the control.

2.8 Ferric reducing antioxidant power (FRAP) assay

This assay is based upon the measurement of the change in absorbance at 593 nm occurring due to the development of blue color. A blue-colored ferrous; Fe²⁺ − 1,2,5 tripyridyltriazine compound was formed from the colorless oxidized ferric Fe³⁺ form due to the electron-donating antioxidants (Benzie and Strain, 1996). The FRAP assay solutions consisted of 300 mM acetate buffer pH 3.6, 10 mM, TPTZ (2,4,6- tripyridyltriazine) added in 40 mM HCl and 20 mM FeCl₃·6H2O FeSO₄ was taken as a standard. Results of percentage scavenging were compared with the standard i.e. Ascorbic acid. $$\mathit{FRAP}scavengingactivity\left(\%\right)=\left(A_0-A_1/A_0\right)\times 100$$ Where, A₁- sample absorbance A₀ – absorbance of the control.

2.9 Assay of antioxidant enzyme (SOD and Catalase) activities

The samples containing the antioxidant enzymes to be tested were prepared by the following steps described by Mukherjee and Choudhuri (1983) with some modifications, Samples were finely ground by pestle in an ice-cold motor in 10 ml of phosphate buffer (KH₂PO₄/K₂HPO₄) (100 mM) with pH 7.0, having Na₂EDTA (0.1 mM) and also 0.1 g of polyvinylpyrrolidone (PVP) was added to the samples. Filtering of the homogenate using filter paper was done, centrifuged at 15000Xg for 10 min at 4 °C, the supernatant was re-centrifuged at 18000Xg for 10 min, the supernatant was stored at 4 °C for enzyme assay.

2.10 Statistical analysis

All data are expressed as mean ± standard deviation (SD). Statistical analyses were performed using One-way ANOVA. Differences as p < 0.05 were considered statistically significant

3.1 Characterization of compounds

Compound 1 (Fig. 1), was obtained as yellow semi-solid mass from ethyl acetate extract and its IR spectrum displayed absorption bands for hydroxyl group (3403 cm⁻¹), ester function (1721 cm⁻¹), unsaturation (1633 cm⁻¹), and aliphatic chain (931 cm⁻¹). Its molecular ion peak was determined at m/z 291 [M+H]⁺ on the basis of ESIMS and ¹³C NMR spectra corresponding to the molecular formula of an aromatic ester with aliphatic chain C₁₈H₂₇O₃.

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Fig. 1

Chemical structures of compounds (1 – 7) isolated from Z. aramtum fruits.

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The ¹H NMR spectrum of compound 1, showed deshielded signals as a triple-doubles at δ 5.94 (J = 1.5, 3.0, 8.5), 5.92 (J = 1.5, 1.5, 8.6 Hz) and multiplets at δ 6.71, 5.99, 5.96, 5.59 and 5.56 were assigned to vinylic H-5, H-6 and aromatic protons H-2′, H-3′, H-4′, H-5′ and H-6′. Twelve three-proton signals as a broad singlet at δ 1.09, as doublets at δ 1.67 (J = 7.5 Hz), and multiplets at δ 2.20, 3.23 were associated with tertiary C-1, 9, 10, 11 methyls, and C-7, 3, 4 methylene protons. The ¹³C NMR spectrum of 1, exhibited a signal for ester carbon at δ 169.20 (C-7′), and vinylic carbons appeared at δ 125.18 (C-5), 129.93 (C-6) were assigned. The six aromatic carbons were appeared at δ 145.29 (C-1′), 132.98 (C-2′), 133.25 (C-3′), 131.60 (C-4′), 133.25 (C-5), and 132.82 (C-1′). The methyl and vinylic carbons appeared at δ 18.48 (C-1, 10), 27.36 (C-9, 11) and at δ 125.18 (C-5), 129.93 (C-6), respectively. The methylene and methane carbons were appeared at δ 33.10 (C-3), 32.70 (C-4), 51.22 (C-7), and 71.78 (C-2), 71.78 (C-8), respectively. The presence of C-7′ signal in the deshielded region at δ 169.20, and C-1′ signal also deshielded in at δ 145.29 suggested ketone linkage of the benzene ring.

The ¹H–¹H COSY spectrum of 1 showed correlations of H-2′ with H-3′ and H-4′; H-7 with H-9, 11 methyls and H-6 methine; H-3 with H-1, and 10 methyls. The HSQC experiment of 1 showed key-correlations between the protons H-2′ at δ 5.99 and C-2′ at δ 132.98; H-6′ at δ 5.96 and C-6′ at δ 132.82; H-5 at δ 5.94 and C-5 at δ 128.18; H-6 at δ 5.92 and C-6 at δ 129.93; H-3 at δ 2.20 and C-5 at δ 33.10; H-4 at δ 3.23 and C-4 at δ 32.70; H-7 at δ 1.67 and C-7 at δ 51.22. The HMBC spectrum of 1 exhibited interaction of H-2′, H-3′ and H-6′ with C-1′; H-3′ and H-4′ with C-5′; H-1 and h-10 with C-2; H-4 with C-5 and C-6; H-7 with C-5, C-6, and C-8; H-9, 11 with C-8. On the basis of the evidence of spectroscopic studies, the structure of 1 was established as (cis)-2, 8-dimethyl-non-5-en-8-ol-2-olyl benzoate (1). This is a new compound and reported the first time in nature.

Compound 2 (Fig. 1), was obtained as yellow semi-solid mass from ethyl acetate extract and its IR spectrum displayed absorption bands for hydroxyl group (3415 cm⁻¹), ester function (1725 cm⁻¹), unsaturation (1630 cm⁻¹), and aliphatic chain (906 cm⁻¹). Its molecular ion peak was determined at m/z 319 [M+H] ⁺ (C₂₀H₃₁O₃) on the basis of ESIMS and ¹³C NMR spectra corresponding to the molecular formula of an aromatic ester with an aliphatic chain (C₂₀H₃₁O₃).

The ¹H NMR spectrum of compound 1, showed deshielded signals as a triple doublet at δ 5.67 (J = 2.5, 7.0, 7.5), 5.63 (J = 8.0, 5.5, 7.6 Hz) and multiplets at δ 6.78, 6.05, 6.01, 5.83 and 5.80 were assigned to vinylic H-8, H-7 and aromatic protons H-2′, H-3′, H-4′, H-5′ and H-6′. Twelve three-proton signals as broad singlet at δ 1.16, as doublet at δ 1.72 (J = 7.0 Hz), and multiplets at δ 3.24, 2.58, 2.29 and 2.21 were associated with tertiary C-1, 11, 12, 13 methyls, C-9, 3–6 methylene protons. The ¹³C NMR spectrum of 1, exhibited signals for ester carbon at δ 169.20 (C-7′), vinylic carbons at δ 125.18 (C-5), 129.93 (C-6) and aromatic carbons δ 145.29 (C-1′), 132.98 (C-2′), 133.25 (C-3′), 131.60 (C-4′), 133.25 (C-5) and 132.82 (C-1′).

The ¹H–¹H COSY spectrum of 2 showed correlations of H-2′ with H-3′ and H-4′; H-7 with H-8 and H-6; H-9 with H-11 methyls and H-9 methylene; H-3 with H-1. The HSQC experiment of 1 showed key-correlations between the protons H-2′ at δ 6.05 and C-2′ at δ 132.01; H-6′ at δ 6.01 and C-6′ at δ 131.61; H-7 at δ 5.63 and C-7 at δ 123.60; H-8 at δ 5.67 and C-8 at δ 129.36; H-3 at δ 2.58 and C-3 at δ 32.06; H-4 at δ 2.29 and C-4 at δ 31.87; H-9 at δ 1.72 and C-9 at δ 50.19. The HMBC spectrum of 1 exhibited interaction of H-2′, H-3′ and H-6′ with C-1′; H-3′ and H-4′ with C-5′; H-1 and H-10 with C-2; H-6 with C-7 and C-8; H-9 with C-7, C-8 and C-9; H-11, 13 with C-10. On the basis of these evidences of spectroscopic studies, the structure of 2 was established as (cis)-2, 10-dimethyl-undec-7-en-10-ol-2-olyl benzoate (2). This is a new compound and reported the first time in nature.

3.2 Cytotoxicity evaluation

All the isolated compounds (1–7), ethyl acetate extract (8), and butanol extract (9) were evaluated for cytotoxic activity against the growth of HepG2 and A549 cancer cells in a dose-dependent and time-dependent manner using MTT assay to assess cell inhibition. The results are shown in Table 1. Nine treatments (1 to 9) were screened by using MTT cell viability assay. Compound 1 was the most potent inhibitor of HepG2 cancer cells compared to compounds 2 and 7. It significantly affected the viability of cells till 48 h. After which there was no effect. The IC₅₀ value of 1 was approximately 50 ± 0.05 µg/mL against HepG2 at 24 h. and 25 ± 0.02 µg/mL at 48 hrs against standard drug doxorubicin IC₅₀ of 0.95 ± 0.08 µg/mL at 24 hrs and 1.7 ± 0.01 µg/mL at 48hrs against HepG2 cancer cells. A similar IC₅₀ value for doxorubicin has been reported by other researchers (Khazaei et al., 2017). Compound 2 and 7 also exhibited a certain degree of cytotoxicity with an IC₅₀ value of 52.37 ± 0.04/78 ± 0.02 in 24 hrs/48 hrs and 52.40 ± 0.04/78 ± 0.02 in 24hrs/48 hrs, respectively.

3.3 Quantification of intracellular reactive oxygen species (ROS) assay

The HepG2 cells treated with compound 1 showed a significant increase in ROS intensity in a dose- and time-dependent manner as compared to untreated cells as shown in Fig. 2. (a) Control cells for 24 h. (b) Cells treated for 24 h with 25.375 µg/mL of compound 1. (c) Cells treated for 24 h with 50.75 µg/mL of 1. (d) Control cells for 48 h. (e) Cells treated for 48 h with 25.37 µg/mL of compound 1. (f) Cells treated for 48 h with 50.02 µg/mL of compound 1. The results of the quantitative measurement of ROS showed that 25.37 µg/mL of compound 1 induced a 122.43% increment in ROS production as compared to untreated cells. Moreover, ROS production was increased by 165.53% (p < 0.001) at 50.75 µg/mL of compound 1 compared to untreated cells. Those findings are properly supported via previous reports, in which the induction inside the ROS generation was documented because of the effect of herbal bioactive compounds (Karimi et al. 2010; Ghali et al., 2014).

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Fig. 2

Photomicrographs of HepG2 cell line stained with DCFH-DA dye. Detection of reactive oxygen species in HepG2 cell line where; (A) & (D) are control cells; (B), (C), (E) & (F) are treated cells.

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3.4 Antioxidant activity

DPPH free radical inhibition activity of various compounds in various concentrations is depicted in Table 2A, Fig. 3A. The inhibition activity of the different compounds revealed that the highest inhibition was caused by compound 1 followed by 2 and 7 compounds. Inhibition shown by these compounds was far higher than the other compounds and to some extent was comparable to the standard i.e. ascorbic acid over the IC₅₀ value 14.90, 15.44, and 15.53 (Table 3, Fig. 4). Compound 5 recorded the least inhibition having an IC₅₀ value of 20.61 and the rest tested compounds demonstrated moderate inhibition. Results on Fe (III) reduction demonstrated that all the treated extracts and compounds had lower reducing ability than the radical inhibiting activity (Table 2B). 1, 2, and 7 compounds showed the highest reducing ability at an EC₅₀ value of 19.44, 20.44, and 22.46 respectively (Table 3). Compounds showed reducing ability which was nearly comparable to the standard used i.e. FeSO4 (Table 3, Fig. 4). The reducing effect of all the compounds increased with an increase in their concentrations. The highest activity of SOD (427.14 ± 0.52) was reported for compounds 1(Table 4, Fig. 5A). In contrary to this a remarkable decrease in SOD enzyme activity was recorded in compound 6 which was 121.11 ± 0.50 (Fig. 3B).

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Fig. 3a

DDPH free radical inhibition caused by different compounds.

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Fig. 3b

Ferric ion scavenging activity of different compounds.

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Fig. 4

Antioxidant enzyme activity of the different compounds in terms of IC₅₀ and EC₅₀ (µg/mL).

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3.5 Catalase activity

Compounds 1, 2, and compound 7 recorded the highest enhancement in enzymatic activities 5212.50 ± 0.61, 5045.83 ± 0.48, and 5030.83 ± 0.70 respectively (Table 5, Fig. 5B). Compound 3 reported the least activity 3995.83 ± 0.60. Considerably higher catalase enzyme activity was recorded in the compounds, particularly which might have aroused due to a large amount of H₂O₂ generated as the finishing product of SOD catalyzed reaction which has to be neutralized by the optimized treated compounds when compared to control.

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Fig. 5a

Effect of different compounds and extracts on enzymatic activity (SOD).

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Fig. 5b

Effect of different compounds and extracts on enzymatic activity (CAT).

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