Carbohydrate Modifications of Neoandrographolide for Improved Reactive Oxygen Species-Mediated Apoptosis through Mitochondrial Pathway in Colon Cancer.

Modifications at the carbohydrate moiety of neoandrographolide, isolated from the medicinal plant Andrographis paniculata, result in more potent and less toxic derivatives, namely, 4',6'-benzylidene neoandrographolide (2b) and 4'6'-p-methoxybenzylidene neoandrographolide (2c). These showed improved cytotoxicity against SW-620, PC-3, and A549 cancer cell lines. Nuclear morphology studies were conducted on compound 2b by 4',6-diamidino-2-phenylidole staining and detection of intracellular reactive oxygen species (ROS) accumulation. It showed an increase in the generation of cellular and mitochondrial ROS level. The probable relation of B-cell lymphoma-2 (Bcl-2, an apoptosis inhibitor) to B-cell lymphoma-2-associated X protein (Bax, an apoptosis promoter) ratio with caspase-3 (apoptosis coordination enzyme) in the colon cancer cell line SW-620 was investigated, and it was discovered that upon 2b treatment, the expression of caspase-3 Bax increased remarkably. However, in 2b-treated cells, the expression of Bcl-2 was downregulated as compared to untreated cells.

Introduction

Biological mechanisms in normal as well as pathogenic cells are being governed to a large extent by reactive oxygen species (ROS). These species which include superoxide (O2•), nitroxyl radical (NO•), hydroxyl (•OH), alkoxyl (•OR), peroxyl (ROO•), and so forth act as chemical messengers and are fatal factors for DNA and proteins.[1] In normal conditions, ROS are generated through different endogenous or exogenous ways and then abolished by antioxidant systems to regulate cellular homeostasis. Strangely, mitochondria functions as a production place (mitochondrial electron transport chain) as well as a target of ROS. The sources of ROS also include xanthine oxidase, cytochrome P450, lipoxygenases, and NADPH oxidases (NOX) which are endogenous and are considered essential. In malignant cells, oxidative stress-mediated signaling phenomenon influences the cell behavior. The progression of cell cycle, cell survival and apoptosis, proliferation, cell morphology, cell motility, energy metabolism, angiogenesis, and tumor maintenance includes the involvement of ROS in neoplasm.[2] Natural products have been used as significant and potent antineoplastic entities for a long time, and carbohydrate units adjoining a natural compound change the biocompatible properties of that molecule, such as the cardiac glycoside, etoposide (glycoside of podophyllotoxin with a d-glucose derivative). It has been proved that carbohydrate modification can make a remarkable change in the activity of the bioactive molecule. Similar outcomes were observed with the glucoside modifications in the labdane diterpenoid neoandrographolide.

An ethnopharmacologically important plant, Andrographis paniculata (commonly known as king of bitters, Kalmegh,and Chirait), is a rich source of labdane diterpenoids,[3,4] viz., andrographolide followed by neoandrographolide and 14-deoxy-11,12-didehydroandrographolide. Although medicinal chemistry of andrographolide is well explored,[5−10] limited information is available on the semisynthetic studies of the second-principle phytoconstituent, neoandrographolide.[11] Therefore, during the present investigation, neoandrographolide was isolated from A. paniculata followed by the synthesis of its semisynthetic derivatives, and their cytotoxic potential was explored.

Neoandrographolide is a labdaneglucoside with molecular formula C26H40O8, molecular mass of 480.597, and has a melting point of 165–166 °C. Its structure is a C19-O-β-d-glucopyranoside diterpene with an α,β-unsaturated lactone.[12] The biosynthesis of neoandrographolide is reported via glucosyltransferase (ApUGT)-catalyzed 19-O-glucosylation of andrograpanin.[13] It is soluble in methanol, ethanol, propanol, acetone, pyridine, and acetic acid. It is reported to be more a potent anti-inflammatory metabolite than andrographolide.[14] The current investigation was carried out to isolate neoandrographolide from the aerial parts of A. paniculata and to carry out carbohydrate modifications in it. The effect of the modified neoandrographolide was studied against different cancer cell lines. Its benzylidene derivatives 2b and 2c were shown to have more drug-like properties, with more potency and less toxicity than the parent molecule against the cell lines SW-620 and PC-3, respectively. Also, these compounds were found to be more biofriendly than the promising natural product andrographolide reported from the genus Andrographis. The molecules 2b and 2c were found to induce apoptosis on SW-620 cells via the mitochondria-dependent pathway mediated by ROS. In the present study, the molecules 2b and 2c were found to have subsided mitochondrial membrane potential and increased ROS generation in colon cancer cell lines, leading to apoptosis via the Bax-/Bcl-2-dependent apoptosis pathway. The third most common cause of neoplastic morbidity and mortality is the colon cancer. Therefore, we emphasize the execution of naturally derived biocompatible molecules on the lethal cell line SW-620.

Results and Discussion

In the current study, 128 g (5.1%) of methanolic extract was obtained from shed, air-dried shoot plant material (2.5 kg) of A. paniculata. The extract was subjected to liquid–liquid partition, subsequently with hexane–methanol and dichloromethane–methanol solvents. Neoandrographolide (0.1%) was isolated as white needle-like crystals with mp 174–75 °C. It is chemically identified as ent-19-hydroxy-8(17),13-labdadien-16,15-olide 19-O-β-d-glucopyranoside.

The sugar residue of the isolated labdane glucoside diterpene was subjected to acetonide protection, following the method reported previously,[15] and the ester protection was done in the presence of a base (Scheme ). 4′,6′-Diol-protected neoandrographolides were confirmed by their spectral analysis. In the 1H nuclear magnetic resonance (NMR) spectrum of benzylidene derivative, a peak near δ 5.1 ppm was assigned to acetal proton, and the peak at δ 101.88 ppm in 13C NMR belongs to acetal carbon. The tetraacetate derivative was characterized by four signals between δ 170.7 and 169 ppm (four acetate carbonyl carbons) in its 13C NMR spectrum. In the 1H NMR spectrum of the tetrabenzoate derivative, twenty aromatic protons appeared at δ 8.03–7.29 ppm as multiplets and four benzoate carbonyl carbons were displayed between 166.12 and 165.00 ppm in its 13C NMR spectrum.

Scheme 1

Semisynthetic Acetal and the Ester Derivatives of Neoandrographolide

The anticancer activity of the parent compound and its derivatives was investigated against three human cancer cell lines PC-3 (prostate), SW-620 (colon), and A549 (lung) as well as in normal cell lines. Among seven molecules, two compounds, 4′,6′-benzylidene neoandrographolide (2b) and 4′6′-p-methoxybenzylidene neoandrographolide (2c), were found to show effective cytotoxic activity against the three human cancer cell lines. The IC50 value of compound 2b against PC-3, A549, and SW-620 was 6.2, 2.65, and 1.75, respectively, and IC50 of compound 2c against these three cancer cell lines was 4.65, 9.91, and 7.52, respectively (Table ).

Table 1

Effect of Neoandrographolide and Its Derivatives on the Panels of Human Cancer Cell Linesa

tissue	prostate adenocarcinoma	lung adenocarcinoma	colon adenocarcinoma	normal breast epithelial
cell line	PC-3	A549	SW-620	FR-2
code	IC50 (μM)
A	14 ± 0.11	15 ± 1.23	15 ± 2.11	6 ± 0.45
1	12.7 ± 0.34	5.53 ± 0.34	9.5 ± 0.89	4.2 ± 0.13
2b	6.2 ± 0.21	2.65 ± 0.78	1.75 ± 0.33	11.2 ± 0.67
2c	4.65 ± 0.45	9.91 ± 0.22	7.52 ± 0.56	10 ± 1.21
3	30.24 ± 0.02	>100	>50	32 ± 0.027
4	>100	>50	>100	23 ± 0.05
paclitaxel	0.065 ± 0.02	<0.01 ± 0.001	0.110 ± 0.003

(A): Andrographolide; (1): neoandrographolide; (2b): 4′,6′-benzylidene neoandrographolide; (2c): 4′,6′-p-methoxybenzylidene neoandrographolide; (3): 2′,3′,4′,6′-tetra-O-acetyl neoandrographolide; (4): 2′,3′,4′,6′-tetra-O-benzoyl neoandrographolide.

The ester protection of the hydroxyl groups of carbohydrate moiety does not show significant activity, whereas the acetonides were found to have good bioactivity. Compounds 2b and 2c (4′,6′-benzylidene neoandrographolide and 4′,6′-p-methoxybenzylidene neoandrographolide) were found to be more potent against the SW-620, PC-3, and A549 cell lines than the other derivatives.

In nuclear morphology study, it was observed that 2b induced nuclear fragmentation as the concentration advanced (Figure ). To assess the molecular mechanism of the molecule imposing apoptosis in SW-620 colon cancer cells, the mitochondrial effect of 2b on SW-620 cells was assessed by examining the accumulation of cellular ROS. In SW-620 cells, ROS production was induced by 2b which appeared to play a critical role in the induction of apoptotic cell death in a dose-dependent manner agreeable with the increase in concentration. ROS production was also induced by cells exposed to H2O2 which were used as a positive control (Figure ).

Figure 1

Nuclear fragmentation was observed with membrane blebbing (E,F) at various concentrations of 2b (C–F) compared to untreated control (A) cells that remain intact, with no blebbing and fragmentation. Paclitaxel was used as a positive control (B).

Figure 2

Generation of increased level of ROS by 2b (E,F) in a concentration-dependent manner (C–F) leading to apoptosis in SW-620 cells as compared with H2O2 (B), a major contributor to oxidative damage, whereas no significant change of fluorescence was observed in control cells (A), indicating the selective targeting of 2b in liberating ROS in SW-620 colon cancer cells and not in the control cells. Mean ± SD, n = 3,***P < 0.001.

MitoSOX Red fluorescence was used to confirm that mitochondria were crucial sites of ROS production in SW-620 cells. It recognizes superoxide synthesis to quantify mitochondrial ROS (Figure ). It was found that an increase in the concentration of 2b increased the production of mitochondrial superoxide. In addition, 2b significantly inhibited mitochondrial ROS generation in SW-620 cells as compared to the positive control. These observations confirmed that 2b plays a pivotal role in mitochondria-derived ROS-induced apoptosis. Also, with the increase in the concentration of 2b, the antiapoptotic Bcl-2 and proapoptotic Bax protein expressions in SW-620 cells were found to be decreased and increased, respectively. Furthermore, preincubation with 2b significantly increased the activation of caspase-3/7 compared to the control cells, which concluded an enhancement in the rate of apoptotic proteins (Figure ).

Figure 3

2b increased mitochondrial superoxide production (A,B): SW-620 cells were incubated at the indicated concentration of 2b and incubated with MitoSOX Red for 20 min and then analyzed by a fluorescence microscope. Significant decrease in fluorescence intensity (C–F), indicative of superoxide production, was detected in SW-620 cells compared with the control cells. Mean ± SD, n = 3, **P < 0.01 ***P < 0.001.

Figure 4

(A): Expression of caspase-3 protein in SW-620 cells treated with 2b at indicated concentrations for 48 h. The treated samples showed significantly increased caspase 3/7 activity compared to the untreated one (control). Mean ± SD, n = 3, *P < 0.05 ***P < 0.001. The data were analyzed by one-way analysis of variance. (B) Effect of 2b on the expression of Bax and Bcl-2 proteins in SW-620 cells for 48 h. Mean ± SD, n = 3, *P < 0.05.

Experimental Section

Instrumentation and Chemicals

The commercially available reagents were purchased from Sigma-Aldrich and solvents from SD Fine Chemicals. Melting point was measured on a Perfit melting point apparatus. The progression of reaction and purity of final products were monitored on silica gel-precoated aluminum sheets (60F254, Merck).The spots on thin-layer chromatography (TLC) plates were visualized by exposure to ultraviolet (UV) light at 254 nm, iodine vapors, and 1% cerric ammonium sulfate in water containing 30% H2SO4 (by volume). Column chromatography was executed on silica gel (60–120 mesh). IR spectra were recorded on a PerkinElmer spectrophotometer. Tetramethylsilane was used as an internal standard to record 1H NMR and 13C NMR spectra on a Bruker AC-400 spectrometer. Liquid chromatography/mass spectrometry (LC/MS) analysis was performed on an Agilent 6410 LC/MS–MS (Agilent Technologies, USA).

Plant Material

A. paniculata was procured from the experimental plots of School of Biotechnology, University of Jammu. The plant material was taxonomically identified, accessioned, and deposited in the herbarium of Department of Botany, University of Jammu, for future reference (accession number: 15796).

Preparation of Methanolic (MeOH) Extract

Fresh shoot parts (about 8 kg) of A. paniculata were collected, cleaned, air-dried, and crushed to powdered form (2.5 kg, 31.2%). Extraction of the powdered plant material was done in double-distilled methanol (5 L) at room temperature (RT) for 24 h, filtered, and evaporated under a reduced pressure (Buchi Rotary Evaporator, R-210). The filtrate was evaporated and combined.

Isolation of Neoandrographolide

The methanolic extract was defatted by liquid–liquid partition (three times) with hexane and methanol. After concentration, the methanol extract was extracted with dichloromethane and finally subjected to column chromatography on a 60–120 mesh silica gel using dichloromethane–methanol as the solvent system.[16] A 2.5 g (0.1%) neoandrographolide was isolated from 5% methanol in dichloromethane fraction. Colorless crystals (mp 165–166 °C) were obtained after subjecting it to crystallization in ethanol.

General Procedure for the Semisynthesis of Neoandrographolide Analogues

4′,6′-Isopropylidene Neoandrographolide (2a)

Neoandrographolide (48.0 mg, 0.1 mmol), 2,2-dimethoxypropane (14.70 μL, 0.12 mmol), and camphor sulfonic acid catalyst (1.16 mg, 0,005 mmol) were dissolved in a 1:5 mixture of dry dimethylformamide and dry toluene. The reaction was carried out under a nitrogen atmosphere at RT. The progress of the reaction was monitored over TLC. After the completion of the reaction (4 h), toluene was evaporated on a rotary evaporator and the content was diluted with ethyl acetate (15 mL). The reaction mixture was treated with a saturated sodium bicarbonate solution (5 mL) and water (5 mL) to quench the remaining catalyst and was then extracted with ethyl acetate (10 mL × 3). The organic layers were collected followed by drying over anhydrous sodium sulfate and were concentrated on the rotary evaporator. The concentrated mass obtained was subjected to column chromatography over silica (60–120 mesh). Hexane/ethyl acetate (1:1) was used as an eluant to obtain 4′,6′-isopropylidene neoandrographolide with 85% yield (44 mg).

4′,6′-Benzylidene Neoandrographolide (2b) and 4′,6′-p-Methoxybenzylidene Neoandrographolide (2c)

4′,6′-Benzylidene neoandrographolide (50.1 mg, 88% yield) and 4′,6′-p-methoxybenzylidene neoandrographolide (51.5 mg, 86% yield) were obtained with benzaldehyde dimethyl acetal(18 μL, 0.12 mmol) and p-methoxybenzaldehyde dimethyl acetal(20 μL, 0.12 mmol), respectively, using the same procedure as mentioned above (Scheme ).[15]

Scheme 2

1,3-Diol Protection of Neoandrographolide: (a) 2,2-Dimethoxypropane (0.12 mmol)/(b) Benzaldehyde Dimethyl Acetal (0.12 mmol)/(c) p-Methoxybenzaldehyde Dimethyl Acetal (0.12 mmol), RT

Tetraacetate Neoandrographolide (3)

Acetic anhydride (19 μL, 0.2 mmol) and a catalytic amount of dimethylaminopyridine  (1.2 mg, 0.01 mmol) were added at RT[1] to neoandrographolide (48.0 mg, 0.1 mmol) in pyridine (9.66 μL, 0.12 mmol).[17] After the completion (3–4 h) of the reaction, which was checked using TLC, it was extracted with ethyl acetate. Pyridine was quenched with CuSO4 solution. The resultant, after column chromatography over silica (60–120 mesh), yielded tetraacetate neoandrographolide with a yield of 76% using the solvent system hexane:ethyl acetate (7:3) as the eluent (Scheme ).

Scheme 3

Acetylation of Neoandrographolide

Tetrabenzoate Neoandrographolide (4)

For benzoylation reaction,[18] benzoyl chloride (55.8 μL, 0.48 mmol) was added dropwise at 0 °C to a stirred solution of neoandrographolide (48.0 mg, 0.1 mmol) in 1 mL dichloromethane and triethylamine (67 μL, 0.48 mmol). After the completion (3–4 h) of the reaction, it was concentrated under reduced pressure. Tetrabenzoate neoandrographolide was yielded (yield 68%) from the resultant, after column chromatography over silica (60–120 mesh), using the solvent system hexane:ethylacetate (6:4) as the eluant (Scheme ).

Scheme 4

Synthesis of Tetrabenzoate Neoandrographolide from Neoandrographolide

Spectral Analysis

Neoandrographolide (1)

C26H40O8 mol. wt 480.59 amu, mp 174–75 °C, elemental analysis: observed: C, 64.08; H, 8.11; calcd: C, 64.98; H, 8.39; IR (KBr) νmax: 3385, 2920, 2850, 1745, 1650, 1453, 1072 cm–1; 1H NMR (400 MHz, CD3OD): δ 7.36 (m, 1H), 4.84 (m, 2H),4.86–4.64 (m, 2H), 4.19–4.12 (m, 3H), 3.87–3.69 (m, 2H), 3.33–3.28 (m, 2H), 3.23–3.18 (m, 2H), 1.27 (m, 2H), 1.08 (m, 2H), 2.17–2.07 (m, 1H), 1.97–1.89 (m, 3H), 1.85–1.75 (m, 3H), 1.65 (m, 3H), 1.47 (m, 2H), 1.43–1.38 (m, 1H), 1.36 (d, J = 3.9 Hz, 1H), 1.33–1.24 (m, 1H), 1.15–1.09 (m, 1H), 1.05 (s, 3H), 0.73 (s, 3H). 13C NMR (100 MHz, CDCl3): δ ppm 175.41, 147.8, 146.1, 133.4, 105.85, 103.6, 76.8, 76.3, 73.8, 72.2, 70.6, 70.2, 61.3, 56.3, 48.2, 47.8, 47.4, 47.7, 46.9, 39.2, 38.8, 38.2, 26.9, 24.1, 21.5, 14.4.

C29H44O8 mol. wt 520.65 amu, mp 193–194 °C, elemental analysis: observed: C, 65.82; H, 8.17; calcd: C, 66.90; H, 8.52. Colorless crystals (85% yield). IR (KBr) νmax: 3373, 2922, 2852, 1742, 1457, 1377, 1260, 1021 cm–1; 1H NMR (400 MHz, CDCl3): δ 7.11 (m, 1H), 4.86 (m, 1H), 4.78–4.59 (m, 2H), 4.32–4.17 (m, 2H), 4.01 (m, 2H), 3.95–3.89 (m, 2H), 3.79 (m, 2H), 3.64–3.58 (m, 2H), 3.49–3.35 (m, 2H), 3.28–3.22 (m, 2H), 3.19 (m, 3H), 2.44–2.08 (m, 4H), 2.08 (s, 1H), 1.76–1.58 (m, 3H), 1.51 (s, 3H), 1.43 (s, 3H), 1.24 (s, 3H), 1.17–1.06 (m, 2H), 1.00 (s, 3H), 0.66–0.57 (m, 1H). 13C NMR (100 MHz, CDCl3): δ ppm: 171.2, 147.3, 143.8, 134.8, 106.9, 103.7, 99.7, 74.9, 72.5, 70.8, 67.3, 66.8, 64.3, 62.0, 56.0, 52.1, 49.5, 48.3, 47.8, 47.1, 46.3, 41.3, 38.2, 37.8, 32.2, 28.9, 26.5, 23.5, 14.6.

4′,6′-Benzylidene Neoandrographolide (2b)

C33H44O8 mol. wt 568.69 amu, mp 142–143 °C, elemental analysis: observed: C, 69.70; H, 7.83; calcd: C, 69.69; H, 7.80; colorless solid (88% yield). IR (KBr) νmax: 3384, 2923, 2856, 1740, 1707,1610, 1460, 1370, 1255, 1170, 1041, 795, 700, 667 cm–1; 1H NMR (400 MHz, CDCl3): δ 8.01 (m, 1H), 7.50–7.11 (m, 5 Ar H), 5.53 (s, 1H), 4.88 (m, 1H), 4.78 (m, 2H), 4.60 (m, 1H), 4.37–4.29 (m, 2H), 4.05 (d, J = 9.4 Hz, 1H), 3.80 (dd, J = 17.3, 9.5 Hz, 2H), 3.56 (t, J = 9.3 Hz, 1H), 3.48 (d, J = 8.7 Hz, 1H), 3.43 (m, 1H), 3.23 (d, J = 9.4 Hz, 1H), 2.45 (m, 1H), 2.18–2.03 (m, 2H), 1.97–1.87 (m, 2H), 1.79 (m, 2H), 1.61 (m, 2H), 1.56 (m, 2H), 1.51–1.33 (m, 2H), 1.27–1.07 (m, 3H), 1.03 (s, 3H), 0.99–0.93 (m, 2H), 0.68 (s, 3H). 13C NMR (100 MHz, CDCl3): δ ppm: 174.3, 162.6, 147.3, 143.9, 137.0, 134.8, 129.2, 128.3, 126.3, 106.9, 103.7, 101.8, 80.6, 76.7, 74.8, 73.1, 72.8, 70.1, 68.7, 66.3, 56.4, 56.2, 39.5, 38.9, 38.4, 38.2, 36.5, 35.9, 27.7, 24.5, 21.7, 18.9, 15.4.

4′,6′-p-Methoxybenzylidene Neoandrographolide (2c)

C34H46O9 mol. wt 598.72 amu, mp 147–148 °C, elemental analysis: observed: C, 68.18; H, 7.72; calcd: C, 68.21; H, 7.74; colorless solid (86% yield). IR (KBr) νmax: 3385, 2924, 2853, 1742, 1707,1606, 1457, 1376, 1258, 1168, 1101, 1041, 794, 699, 667 cm–1; 1H NMR analysis: δ 7.43–6.90 (m, 4Ar H), 5.50 (s, 1H), 4.88 (m, 3H), 4.79 (m, 3H), 4.60 (m, 2H), 4.31 (d, J = 7.6 Hz, 1H), 4.19–3.98 (m, 4H), 3.91 (s, 3H), 3.85–3.71 (m, 4H), 3.70–3.47 (m, 3H), 3.22 (m, 4H), 2.96–2.90 (m, 4H), 2.08 (d, J = 7.0 Hz, 1 H), 1.27 (m, 3H), 1.02 (s, 3H), 0.68 (s, 3H). 13C NMR (100 MHz, CDCl3): δ ppm: 171.2, 162.8, 160.2, 147.3, 143.9, 134.8, 129.5, 127.6, 114.3, 113.6, 107.0, 103.7, 101.8, 80.5, 74.7, 73.1, 70.1, 66.4, 60.4, 56.4, 56.2, 55.3, 39.5, 38.4, 38.2, 27.7, 24.5, 21.7, 21.0, 20.6, 18.9, 15.4, 14.1, 1.0.

C34H48O12 mol. wt 648.73 amu, mp 155–158 °C, elemental analysis: observed: C, 62.93; H, 7.44; calcd: C, 62.95; H, 7.46; colorless solid (76% yield); IR (KBr) νmax: 3079, 2933, 2851, 1747, 1643, 1370, 1230, 1037, 896, 829, 755, 700, 667, 602 cm–1; 1H NMR (400 MHz, CDCl3): δ 7.05 (m,, 1H), 5.20 (m, 1H), 5.08 (m, 1H), 5.00 (m, 1H), 4.85 (m, 1H), 4.79 (m, 1H), 4.60 (m, 2H), 4.39 (m, 1H), 4.28 (m,, 1H), 4.14 (m, 2H), 3.97 (d, J = 9.2 Hz, 1H), 3.68 (m, 3H), 3.18 (d, J = 9.3 Hz, 1H), 2.40 (m, 4H), 1.99 (m, 4H), 1.71 (m, 4H), 1.62 (m, 3H), 1.50 (m, 3H), 1.27 (m, 4H), 1.04 (m, 3H), 0.94 (s, 3H), 0.67 (s, 3H). 13C NMR (100 MHz, CDCl3): δ ppm: 174.4, 170.9, 170.7, 169.2, 143.8, 143.2, 135.6, 134.8, 121.3, 109.2, 106.8, 101.2, 80.1, 77.3, 77.0, 76.7, 71.6, 70.1, 69.6, 64.8, 61.7, 54.7, 41.3, 38.6, 38.2, 36.7, 27.6, 24.7, 23.8, 22.7, 22.7, 21.1, 20.7, 15.2.

C54H56O12 mol. wt 897.01 amu, mp 145–147 °C, elemental analysis: observed: C, 72.28; H, 6.20; calcd: C, 72.30; H, 6.29; colorless solid (68% yield), IR (KBr) νmax: 3065, 2931, 2853, 1731, 1601, 1451, 1267, 1106, 1095, 975, 853, 802, 686, 700, 667, 502 cm–1; 1H NMR (400 MHz, CDCl3): δ ppm 8.03–7.29 (m, 20 Ar–H), 7.09 (m, 1H), 5.91 (t, J = 9.6 Hz, 1H), 5.66 (t, J = 9.6 Hz, 1H), 5.55 (t, J = 8.6 Hz, 1H), 4.76 (m, 2H), 4.64 (m, 1H), 4.58–4.45 (m, 1H), 4.14 (m, 1H), 4.00 (d, J = 9.2 Hz, 1H), 3.65–3.45 (m, 1H), 3.27 (d, J = 9.0 Hz, 1H), 2.51–2.33 (m, 1H), 2.17–2.03 (m, 2H), 1.84–1.65 (m, 3H), 1.53 (d, J = 14.8 Hz, 1H), 1.44 (m, 3H), 1.25 (m, 4H), 1.12 (m, 2H), 1.02–0.85 (m, 2H), 0.81 (s, 3H), 0.57 (s, 3H). 13C NMR (100 MHz, CDCl3): δ ppm: 174.4, 166.1, 165.8, 165.0, 147.3, 143.8, 134.8, 133.4, 133.2, 129.7, 129.6, 129.4, 129.1, 128.8, 128.7, 128.3, 126.3, 121.4, 109.3, 106.8, 106.7, 101.5, 101.2, 77.4, 77.0, 76.7, 76.7, 73.2, 72.8, 71.9, 71.7, 71.6, 70.1, 69.6, 64.8, 63.2, 61.7, 60.4, 56.4, 55.9, 47.8, 37.5, 38.9, 38.6, 39.5, 38.9, 38.3, 37.8, 36.4, 27.5, 24.6, 24.5, 21.6, 18.9, 15.1, 11.0.

Biological Screening Methodology

Sulforhodamine B Assay

The assay was performed by seeding a cell suspension of favorable cell density in 96-well flat-bottom plates (NUNC). Cell densities for each well used in the screen for SW-620-10, 000, A549-7000 and PC-3-7000, 100 μL of cell/well was plated. The cells were then incubated for 48 h and exposed to various concentrations of test materials containing a complete growth medium. Paclitaxel was used as a positive control. The plates were further incubated under the same conditions for another 48 h at 37 °C. The cells were then fixed with cold trichloroacetic acid for 1 h at 4 °C. After 1 h, the plates were rinsed three times with water and allowed to air dry. After drying, 100 μL of 0.4% sulforhodamine B (SRB) dye was added and kept for half an hour at RT. The plates were subsequently washed three times with 1% v/v acetic acid to remove the unbound SRB. After drying at RT, the bound dye was solubilized by adding 100 μL of 10 mM Tris (tris(hydroxymethyl)aminomethane) buffer (pH 10.4) to each well. To solubilize the protein-bound dye, the plates were kept on the shaker for 5 min. Finally, OD was taken at 540 nm in a microplate reader. IC50 values were determined by plotting OD against concentration.[19]

Nuclear Morphology Assessment

Nuclear morphology study was done using the dye 4′,6-diamidino-2-phenylidole (DAPI). DAPI binds to the minor groove of double-stranded DNA, with a preference for the adenine–thymine clusters in the interphase of the mitotic cell. The formation of apoptotic bodies was confirmed by performing DAPI staining to visualize whether the cell nucleus in the fragmented form remains intact after treatment with the molecule. Human cancer cells SW-620 (colon cancer) were seeded at 1.5 × 105 cells/mL/well in six-well tissue culture plates in RPMI medium supplemented with 10% fetal bovine serum. This was followed by incubation for 24 h at 37 °C in a CO2 incubator. After 24 h, the cells were treated with this different concentration of stock solution and with paclitaxel (positive control) and incubated for 48 h. After 48 h, the incubation medium was removed, and the cells were fixed with methanol followed by incubation for 20 min at 37 °C. After fixation, the cells were stained with DAPI (1 μg/mL) and incubated for 20 min in the dark at RT. The dye was removed and washed with 1% phosphate-buffered saline and examined under an inverted fluorescence microscope (Olympus, 1 × 81).[20]

Detection of Intracellular and Mitochondrial ROS Levels

Cellular and mitochondrial ROS levels were determined using DCFDA and MitoSOX Red, respectively, according to the manufacturer’s instructions. Briefly, SW-620 cells (2 × 105 mL/well) were seeded in six-well tissue culture plates and incubated at 37 °C for 24 h. After 24 h, the cells were treated with 2b at different concentrations (0.8, 1.7, 3.5, and 7 μM) and incubated for 48 h. After this, the media were removed, and DC-FDA and MitoSOX Red dye were added and further incubated for 20 min at 37 °C. Before the addition of the dye, the cells were incubated with H2O2 (0.05%) as a positive control for 15 min. The total cellular and mitochondrial ROS levels were monitored by a fluorescence microscope (Olympus, 1 × 81).[21]

Caspase-3/7 Activity Assay

The activity of caspase-3/7 in SW-620 cell lysates was performed by using the fluorescent assay (caspase-3/7 activity assay kit #5723). It contains a fluorogenic substrate (N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin or Ac-DEVD–AMC) for caspase-3/7 and cleaves this substrate between DEVD and AMC, generating a highly fluorescent AMC proportional to the number of apoptotic cells in the sample. This is detected using a fluorescence reader with excitation at 380 nm and emission between 420 and 460 nm. The SW-620 cells were seeded at 1 × 105 cells/well in a 96-well plate and kept overnight. After 24 h, the cells were treated with different concentrations of 2b for 48 h and then lysed in 30 μL of lysis buffer (1×) (supplied with the kit). The cell lysate was mixed with the substrate solution, incubated at 37 °C in the dark for 2 h, and visualized using a relative fluorescent unit.[22]

Western Blotting Analysis

The SW-620 cells were centrifuged at 1200g for 10 min, and the cell pellet was collected and lysed with a lysis buffer (50 mM Tris-HCl pH 8.0, 120 mM NaCl, 0.5% NP-40, 1 mM phenylmethylsulfonyl fluoride). A 50 μg of protein was loaded onto 10% sodium dodecyl sulfate–polyacrylamide gel and electrophoresed. After electrophoresis, the protein was transferred to a polyvinylidene difluoride membrane (Millipore, IPVH00010, USA), and the membrane was blocked with 5% nonfat dry milk in tris-buffered saline-Tween buffer 7 (0.12 M Tris base, 1.5 M NaCl, 0.1% Tween 20) at RT for 2 h. The membrane was then incubated with primary mouse antibody against β-actin (1:2000), Bax (1:2000 #5023), and Bcl-2 (1:2000# 15701) (Cell Signaling Technologies) overnight at 4 °C followed by incubation with horseradish peroxidase-conjugated secondary antibody (antimouse #7076 and antirabbit #7074 1:1000) for 1 h at RT. Protein–antibody complexes were detected using chemiluminescence (Immobilon Western chemiluminescent HRP substrate, MERCK Millipore, USA).[23]

Conclusions

Various studies have reported that increased intracellular ROS can cause cell membrane potential damage and thus block the cell cycle, get into the cell nucleus to cause DNA damage, further activate the endogenous pathway, and ultimately lead to cell death. In the current study, neoandrographolide was isolated from A. paniculata to generate its hydroxyl-protected 19-O-β-glucoside analogues. An increase in the anticancer activity was observed in its acetonides. These derivatives were assessed for enhancement in their anticancer potential as compared to the parent molecule. 4′,6′-Benzylidene neoandrographolide inhibited the proliferation of SW-620 colon cancer cells by increasing the intracellular ROS levels, which resulted in an increased oxidative stress, and, subsequently, induced apoptosis. Taken together, 4′,6′-benzylidene neoandrographolide induced apoptosis on SW-620 cells via ROS-mediated mitochondria-dependent pathway. To find out the molecular mechanism of the active molecules, nuclear morphology studies were done by DAPI staining and intracellular ROS accumulation detection. The derivative 2b showed an increase in the generation of cellular and mitochondrial levels of ROS in a concentration-dependent manner, ultimately leading to apoptosis. This was caused by ROS through the involvement of the mitochondrial pathway in SW-620 colon cancer cells. The inactivation of Bcl-2 is carried out by caspase–3 which acts downstream of Bax/Bcl-2, enabling its anti-apoptotic capacity to promote cell death.[24] It may be concluded that 2b significantly induces an apoptotic effect on colorectal cancer cells in vitro, through its mechanism associated with the Bcl-2/Bax/caspase-3 pathway. To the best of our knowledge, the present study is the first report on the semisynthesis of neoandrographolide derivatives and their anticancer studies. Therefore, 4′,6′-benzylidene neoandrographolide is a novel candidate for antitumor therapy in patients with colon cancer.