Journal of Ethnopharmacology 152 (2014) 106–112

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Bioactive Lignans from Zanthoxylum alatum Roxb. stem bark with cytotoxic potential Minky Mukhija a,b, Kanaya Lal Dhar b, Ajudhia Nath Kalia b,n a b

Punjab Technical University, Kapurthala, India ISF College of Pharmacy, Ferozepur Road, Ghal Kalan, Moga 142001 Punjab, India

art ic l e i nf o

a b s t r a c t

Article history: Received 20 July 2013 Received in revised form 30 November 2013 Accepted 21 December 2013 Available online 8 January 2014

Ethanopharmacological relevance: Zanthoxylum alatum is used in traditional medicinal systems for number of disorders like cholera, diabetes, cough, diarrhea, fever, headache, microbial infections, toothache, inflammation and cancer. The aim of the present study was to evaluate Zanthoxylum alatum stem bark for its cytotoxic potential and to isolate the bioactive constitiuents. Material and methods: Cytotoxicity of the different extracts and isolated compounds was studied on lung carcinoma cell line (A549) and pancreatic carcinoma cell line (MIA-PaCa) using MTT assay. Isolation of compounds from most active extract (petroleum ether) was done on silica gel column. Structure elucidation was done by using various spectrophotometric techniques like UV, IR, 1H NMR, 13C NMR and mass spectroscopy. The type of cell death caused by most active compound C was explored by fluorescence microscopy using the acridine orange/ethidium bromide method. Result: Petroleum ether extract of plant has shown significant cytotoxic potential. Three lignans sesamin (A), kobusin (B), and 40 O demethyl magnolin (C) has been isolated. All lignans showed cytotoxic activities in different ranges. Compound C was the novel bioactive compound from a plant source and found to be most active. In apoptosis study, treatment caused typical apoptotic morphological changes. It enhances the apoptosis at IC50 dose (21.72 mg/mL) however showing necrotic cell death at higher dose after 24 h on MIA-PaCa cell lines. Conclusion: Petroleum ether extract (60–80 1C) of Zanthoxylum alatum has cytotoxic potential. The lignans isolated from the petroleum ether extract were responsible for the cytotoxic potential of the extract. 40 O demethyl magnolin was novel compound from Zanthoxylum alatum. Hence the Zanthoxylum alatum can be further explored for the development of anticancer drug. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Column chromatography Cytotoxicity Lignans MTT assay Zanthoxylum alatum

1. Introduction Cancer is a multistage process, in which the uncontrolled growth of the cells results into accumulation of lumps of cells in a particular tissue which may further metastasize. The reasons for this type of growth may be genetic, epigenetic or interplay of both (Hetts, 1998). It is a second leading cause of death after cardiovascular disease. In 1996 there were 10 million new cancer cases worldwide and six million deaths attributed to cancer. It is predicted that in 2020 there will be about 20 million new cases and 12 million deaths caused by cancer. Plant derived products have long been an important source of treatment for cancer, which is projected to become the major cause of death in this century (Mukherjee et al., 2001). According to World Health Organization, 80% of the world population living in rural areas depends on plant


Corresponding author. Tel.: þ 91 9915 939996; fax: þ 91 1636 239515. E-mail address: [email protected] (A. Nath Kalia).

0378-8741/$ - see front matter & 2014 Elsevier Ireland Ltd. All rights reserved.

based products for their health care needs. The synthetic anticancer remedies are beyond the reach of common man because of cost factor. Moreover, the conventional radiotherapy and chemotherapy with synthetic drugs evoke severe side effects including immunosuppression. In this context, plants are a potential alternative source of safer anticancer molecule/drug (Barh, 2008). More than 60% of cancer therapeutics in the market or in testing are based on natural products. Of 177 drugs approved worldwide for treatment of cancer; more than 70% are based on natural products (Brower, 2008). These include vinblastine, vincristine, the campothecin derivatives, topotecan and irinotecan, etoposide, derives from epipodophyllotoxin and paclitaxel (taxol). These are the most outstanding agents that has been found beneficial in the treatment of refractory ovarian, breast, lung and other cancers (Cragg and Newman, 2005). Still there are a huge number of molecules that still either remains to be explored by the medicinal chemists. Zanthoxylum alatum (ZA) also known as Zanthoxylum armatum belongs to family Rutaceae has been used traditionally as an

M. Mukhija et al. / Journal of Ethnopharmacology 152 (2014) 106–112

ethnomedicine for cancer (Gilani et al., 2010). The plant was used extensively in traditional practices in North-Eastern India and South-East Asia in the form of infusion and decoction at the dose of 1–2 oz (Kharshiing, 2012; Nadkarni, 2002). ZA is a perennial shrub or a small tree upto 6 m height with dense glabrous foliage and straight prickles on stem. It is distributed in Himalayas from Kashmir to Bhutan upto 2100 m and in Khasia hills upto 1350 m (Gupta et al., 2006). The bark of the plant is reported to contain a bitter crystalline principal identical with berberine and it also contains volatile oil, phenolic compounds and resin (Nadkarni, 2002). The fruit contains about 1.5% of an essential oil consisting chiefly of 1-α-phellandrene with small amounts of linalool. Leaves yields an essential oil which is a carbonyl compound identified as methyl n-nonyl ketone. The roots yields the alkaloids; dictamnine, magnoflorine, fagarine, skimmianine, xanthoplanine (Baquar, 1989; Kapoor, 1990). Carpals of the plant contain xanthoxylin (Nadkarni, 2002). Various reported pharmacological activities of ZA in different parts are anti-proliferative (Kumar and Muller, 1999), antibacterial, antifungal, anthelmintic (Mehta et al., 1981), anti-inflammatory (Bhatt and Upadhyaya, 2010), antioxidant (Batool et al., 2010), hepatoprotective (Ranawat et al., 2010; Verma and Khosa, 2010), larvicidal (Tiwary et al., 2007), antispasmodic, antidiarrhoeal, bronchodialator and in cardiovascular disorders (Gilani et al., 2010), antidysentric (Kar and Borthakur, 2008), piscicide (Ramanujam and Ratha, 2008), lousicidal potential (Kumar et al., 2003), cytotoxic (Barkatullah and Muhammad, 2011). Preliminary investigation of petroleum ether extract of ZA shows cytotoxic potential in cancer cell lines. So, the present study was carried out to isolate active compounds from plant extract which can be used for the treatment of cancer as cancer is second to cardiovascular disease as a cause of mortality.

2. Material and methods


mesh size). The column was eluted in ethyl acetate and toluene in gradient manner. Each fraction of 50 ml was collected, concentrated in rota evaporator and left for crystallization. Total number of fractions collected were 400. The fractions having same Rf values were pooled together in groups. Purification of the isolated compound was carried out by repeated recrystallization technique. Compound A (Fig. 1a) was isolated from fractions 47–74 in the mobile phase (ethyl acetate:toluene, 15:85). Crystals were separated from mobile phase and purification was done by recrystallization using acetone and methanol. Presence of compound was detected by spraying 5% FeCl3 solution and Dragendorff0 s reagent. Compound B (Fig. 1b) was isolated from fractions 75–118 in the mobile phase (ethyl acetate:toluene, 20:80). Crystals were separated from mobile phase and purification was done by recrystallization using acetone and methanol. Presence of compound was detected by spraying 5% FeCl3 solution and Dragendorff0 s reagent. Pooled Fractions 211–260 were rechromatographed over silica gel subcolumn using eluents ethyl acetate:toluene (20:80) followed by increase in polarity. Compound C (Fig. 1c) was isolated from fractions 6–27 of subcolumn in the mobile phase (ethyl acetate:toluene, 40:60). Purification of the compound was done with methanol. The structures of compounds A–C were determined by UV, IR, 1 H NMR, 13C NMR and Mass spectroscopy.

2.3. Cell culture, establishment and maintainance A-549 and MIA-PaCa cell lines were maintained in DMEM and nutrient mixture of Ham0 s F-12 medium supplemented with penicillin (100 Units/mL), streptomycin (100 μg/mL) and 10% (v/v) heat inactivated fetal bovine serum (FBS). Cells were maintained in 5% CO2 humidified incubator at 37 1C. Subculturing was done by trypsinization (0.25%) when they were reached 80% confluency. Growth medium was changed every three days.

2.1. Material 2.4. Cytotoxicity assay The stem bark of ZA was collected from the local areas of Tehri (Garwal), Uttrakhand, India and authenticated from NISCAIR, New Delhi (Ref. NISCAIR/RHMD/Consult/2013/2233/14). Plant drug was shade dried (o40 1C), coarsely powdered and stored in air tight container. All solvents used were of analytical grade and purchaced from Rankem (Deejay Corporation, Jalandhar). Thin layer chromatography (TLC) was performed using silica gel 60F254 (E-Merck). Silica gel (60–120 mesh) used for column chromatography was purchased from CDH (Chemical Corporation, Ludhiana). 1H NMR and 13C NMR spectras were recorded on bruker 400 MHz spectrometer using TMS (Tetramethylsilane) as the internal standard and mass spectra were recorded on ESI-esquire 3000 bruker daltonics instrument. The cell lines A549 and MIA-PaCa were obtained from National Center for Cell Sciences NCCS, Pune (India). RPMI-1640, Nutrient Mixture F-12 Ham Kaighns Modification and Dulbeco minimum essential eagle medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin, MTT reagent, 0.5% Trypan Blue and acridine orange/ethidium bromide were purchaced from Himedia (Deejay Corporation, Jalandhar). 2.2. Extraction, isolation and purification of lignans The powdered drug (1 kg) was extracted by continuous hot extraction process using soxhlet apparatus with petroleum ether (60–80 1C). The extract was concentrated under reduced pressure to obtain a green semi-solid residue. The dried extract was subjected to column chromatography. Column was packed with silica gel (60–120

Growth inhibitory or inducing effects of various substrates on cell lines can be determined by the MTT-proliferation assay. The MTT assay was first described by Mosmann (1983). The chief advantage of this assay is that it requires fewer cells than other cytotoxic assays. In addition, it allows for multiple sample concentrations on a single 96 well plate which is then rapidly quantitated using an automated spectroscopic microplate reader (Edmondson et al., 1988). The cells were plated 24 h prior to testing in 96 well plates at a density of 3000 cells/well in 100 mL of the medium. After overnight incubation triplicate wells were treated with varying concentration of compounds and standard docetaxal ranging from (1–150 mg/mL) and incubated for 3 days. The cells were continuously exposed for a period of 72 h. After 3 days medium was replaced with 2 mL of MTT solution (5 mg/mL) and cells were incubated for 3 h. The relative percentage of metabolically active cells were compared with untreated control and then determined on the basis of mitochondrial conversion of 3-(4, 5-dimethylthiazol-2-yl) 2, 5 diphenyltetrazolium bromide (MTT) to Formazan crystals which were dissolved in dimethylsulfoxide (DMSO). Spectrophotometric absorbance of sample was determined by using micro plate reader (BIORAD) at 570/ 630 nm (Gerlier and Thomasset, 1986). Concentrations of sample showing 50% reduction in cell viability (i.e., IC50) were then calculated. An OD value of control cells (unexposed cells) was taken as 100% viability (0% cytotoxicity). % inhibition¼ (OD of control)  (OD of treated)/(OD of control) 100; OD¼Absorbance of each well.


M. Mukhija et al. / Journal of Ethnopharmacology 152 (2014) 106–112

Fig. 1. Structure of isolated compounds A, B and C from petroleum ether extract of Zanthoxylum alatum stem bark.

2.5. Fluorescence microscopic analysis of cell death Acridine orange/ethidium bromide (AO/EB) double staining assay was used. Acridine orange is taken up by both viable and nonviable cells and emits green fluorescence if interrelated into double stranded nucleic acid (DNA) or red fluorescence if bound to single stranded nucleic acid (RNA). Ethidium bromide is taken up only by non viable cells and emits red fluorescence emission and morphological aspect of chromatin condensation in the stained nuclei. Viable cells have uniform bright green nuclei with an organized structure. Apoptotic cells (which have started to undergo DNA cleavage) have green nuclei, but perinuclear chromatin condensation is visible as bright green patches or fragments. Necrotic cells have uniformly orange to red nuclei with a condensed structure. Approximately 5  103 cells were grown in 24 well plate. After 24 h. exposer of drug

cells were trypsinized using 0.25% trypsin solution. The amount of 20 mL of dye mixture (10 mL/ml AO and 10 mL EB in distilled water) was mixed with 100 mL suspension of cells. About 20 mL of mixture (cell suspension and dye) was immediately examined under microscope (Olympus) at 400  magnification (Milena et al., 2012).

2.6. Statistical analysis All experiments were carried out three times, independently. The data obtained were expressed in terms of mean, standard deviation values. Wherever appropriate, the data were also subjected to unpaired two tailed student0 s t test. The p o0.05 was considered to be statistically significant.

M. Mukhija et al. / Journal of Ethnopharmacology 152 (2014) 106–112

3. Result and discussion In the present study, petroleum ether extract of the dried stem bark of ZA was chromatographed on a silica gel column and thus isolated and purified 3 lignans; sesamin, kobusin and 40 O demethyl magnolin. Compound A was isolated as colorless crystals, 40 mg (0.5% w/w yield); Rf 0.74 (ethylacetate:toluene, 30:70); m.p. 120–122 1C. Compound gave positive FeCl3 test for phenolics (Hawker et al., 1972) and Dragendorff0 s reagent test; as some lignans give false positive Dragendorff0 s test (Joshi and Aeri, 2009). UV λmax (chloroform): 278 nm; IR (KBr) spectrum showed stretching for C–O at 1239.30 cm  1 for ethers, The stretching for C–H and CQC at 2922.5 and 1609, 1502.16, 1488.29 cm  1 respectively. 1H NMR showed signals which indicates tetrahydrofuran type of lignan. Since, the molecule appears to be symmetrical, the signals for only nine protons instead of eighteen protons was observed in 1H NMR (CDCl3, 400 MHz, δ with TMS¼0). δ 6.84 (dd, 2H, H-60 and H-6″ of benzene ring in benzo-1,3-dioxole moieties, J¼1.24, 7.85), 6.79 (d, 4H, H-20 , H-2″, H- 50 and H-5″ of benzene ring in benzo-1,3-dioxole moieties, J¼7.85), 5.95 (s, 4H, H-30 b and H-3″b of O-CH2-O in benzo-1,3dioxole moieties), 4.7 (d, 2H, H-2 and H-6 of tetrahydrofuran moieties, J¼4.36), 4.23 (m, 2H, H-4 and H-8 of tetrahydrofuran moieties), 3.86 (dd, 2H, J¼3.64, 9.24, H-4 and H-8 of tetrahydrofuran moieties), 3.04 (m, 2H, H-1 and H-5 of tetrahydrofuran moieties). The structure was further confirmed by the study of 13C NMR (CDCl3,100 MHZ, δ with TMS¼0) having C-4″,40 (δ 147.98), C-3″,30 (δ 147.12), C-1″,10 (δ 135.04), C-6″, 60 (δ 119.39), C-5″,50 (δ 108.21), C-2″,20 (δ 106.51), C-3″b,30 b (δ 101.10), C-2,6 (δ 85.80), C-4,8 (δ 71.72), C-1,5 (δ 54.33). The DEPT was performed to find out methine, methylene and methyl carbons. MS ESþ: m/z¼378 [M þ þ1þNa] þ . Thus on the basis of above spectral data compound A showed symmetrical nature and was characterized as tetrahydrofuran type of lignan named as sesamin, having molecular formula C20H18O6. Compound B was isolated as colorless crystals; 50 mg (0.6% w/w yield); Rf 0.54 (ethylacetate:toluene, 30:70); m.p. 116–118 1C. Compound gave positive FeCl3 test and false positive test with Dragendorff0 s reagent; UV λmax (methanol): 281 nm; IR (KBr) showed stretching for C–H at 2854.88 cm  1 for methoxy, C–O stretching at 1137.06 cm  1, 1238.93 cm  1 for ethers, CQC


stretching at 1608 cm  1, 1504.15 cm  1, 1443.17 cm  1 and C–H stretching at 2925.38 cm  1 for aromatic ring. Compound B showed 1H NMR signals at 6.93 (s, 2H, H-20 and H-2″ in benzene rings of benzo-1,3-dioxole and 3,4- dimethoxybenzene), 6.85 (s, 2H, H-5″ and H-6″ in benzene rings of benzo-1,3-dioxole), 6.81 (d, 1H, H-60 in 30 ,40 -dimethoxybenzene, J¼ 1.52), 6.77 (d, 1H, H-50 in 30 ,40 -dimethoxybenzene, J ¼7.92), 5.95 (s, 2H, H-3″b of O-CH2-O in benzo-1,3-dioxole moieties), 4.86 (d, 2H, H-6 and H-2 in tetrahydrofuran moieties, J ¼5.48), 4.42 (d, 2H, H-4 and H-8 axial protons in tetrahydrofuran moieties, J ¼9.6), 4.1 (d, 2H, H-4 and H-8 equitorial protons in tetrahydrofuran moieties), 3.91 (s, 3H, H-40 a –OCH3 in 30 ,40 -dimethoxybenzene), 3.88 (s, 3H, H-30 a –OCH3 in 3,4-dimethoxybenzene), 2.87 (m, 2H, H-1 and H-5 in tetrahydrofuran moieties,). The two H0 s H, H (δ 6.85) are chemically non equivalent but magnetically equivalent so appears as 2H singlet. The 1HNMR therefore indicates that dimethoxy phenyl is axial while methylene dioxyphenyl is equatorial. 13C NMR displayed signals at C-30 (δ 148.85), C-4″ (δ 148.02), C-40 (δ 147.96), C-3″ (δ 147.21), C-1″ (δ 135.17), C-6″ (δ 119.57), C-60 (δ 117.71), C-50 (δ 108.96), C-20 (δ 108.16), C-3″b (δ 101.05), C-6 (δ 87.68), C-8 (δ 71.01), C-4 (δ 69.76), C-40 a (δ 55.94), C-30 a (δ 55.91), C-1,5 (δ 50.16). MS ES þ: m/z¼ 393 [M þNa] þ . So the mass of the compound was 370. Thus on the basis of above spectral data compound B was characterized as tetrahydrofuran type of lignan named as kobusin having molecular formula C21H22O6. Compound C was isolated as resinous yellow colored mass; 45 mg (0.56% w/w yield); Rf 0.41 (ethylacetate:toluene, 30:70); Compound gave FeCl3 test and false positive test with Dragendorff0 s reagent. Various efforts were made to recrystallize but it fails and compound was found to be hygroscopic as well as phenolic; UV λmax (methanol): 279.8 nm, 241.6 nm; IR (KBr) showed O–H stretching at 3424.03 cm  1 for hydroxyl group, C–H stretching at 2847.67 cm  1 for methoxy, C–O stretching at 1234.75 cm  1, 1127.85 cm  1 for ethers, CQC stretching at 1591.10 cm  1 and C–H streching at 2924.88 cm  1 for aromatic ring. 1H NMR showed signals at 6.7–6.9 (m, 3H, H-2″, H-5″ and H-6″ in benzene ring of 3,4-dimethoxybenzene), 6.5 (s, 2H, H-20 and H-60 of benzene ring 4 hydroxy-3,5-dimethoxybenzene), 4.7 (dd, 2H, H-60 and H-20 of tetrahydrofuran moieties, J ¼6.96),

Fig. 2. Effects of pet ether extract, sesamin, kobusin, 40 O demethyl magnolin and standard drug docetaxal on A549 human lung cancer cell lines. The cells were treated with various concentrations and cytotoxicity was measured by MTT assay. Values were expressed as mean 7 SD.


M. Mukhija et al. / Journal of Ethnopharmacology 152 (2014) 106–112

Fig. 3. Effect of petroleum ether extract, sesamin, kobusin, 40 O demethyl magnolin and standard drug docetaxal on MIA-PaCa human pancreatic cancer cell lines. The cells were treated with various concentrations and cytotoxicity were measured by MTT assay. Values were expressed as mean 7 SD.

Table 1 IC50 values of extract and isolated compounds on A549 cancer cell line and MIA-PaCa cancer cell line. S. No.

1 2 3 4 5

Name of sample

Petroleum ether extract Sesamin Kobusin 40 O demethyl magnolin Docetaxel

IC50 value A549


68.11 71.231 37.46 71.097 34.71 72.331 26.47 71.871 12.01 72.107

63.75 7 1.0924 34.047 1.7621 32.86 7 2.0271 21.727 1.5071 11.127 2.2062

species shows antitumour activity in mice (Dymock, 1972). Other lignans with cytotoxic properties are burseran from Bursera microphylla; Burseraceae (Cole et al., 1969), liriodendrin from Penstemon deustus; Scrophulariaceae (Jolad ,1980), styraxin from Styrax officinalis; Styraxaceae (Ulubelen et al., 1978), arctigenin and trachelogenin from Ipomoea cairica; Convolvulaceae (Trums and Eich ,1989), steganacin and steganagin from Steganotaenia araliaceae; Umbelliferae (Kupchan and Britton, 1964). So, in the present study isolated lignans sesamin, kobusin and 40 O demethyl magnolin from stem bark of ZA were subjected to cytotoxic study. 3.1. Cytotoxic assay

4.2 (m, 2H, H-4 and H-8 axial protons of tetrahydrofuran moieties,), 3.9 (s, 2H, H-4, H-8 equitorial protons of tetrahydrofuran rings), 3.85 (s, 6H, H-30 a and H-50 a –OCH3 of 4-hydroxy-3,5dimethoxybenzene), 3.84 (s, 3H, H-3″ –OCH3 of 3,4-dimethoxybenzene), 3.90 (s, 3H, H-3″a, –OCH3 of 3,4-dimethoxybenzene), 3.11 (m, 2H, H-1, H-5 in tetrahydrofuran moieties). 13C NMR showed signals at -30 ,50 (δ 153.43), C-3″ (δ 149.17), C-4″ (δ 148.62), C-40 (δ 137.39), C-10 (δ 136.82), C-50 (δ 133.47), C-1″ (δ 133.38), C-6″ (δ 118.51), C-5″ (δ 110.96), C-2″ (δ 109.14), C-60 (δ 102.72), C-2,6 (δ 85.80), C-4,8 (δ 71.76), C-30 a (δ 55.95), C-50 a,3″ a (δ 55.92), C-4″a (δ 54.09). Since 1H NMR showed signs for 4 protons indicating a symmetrical nature of the molecule with two phenyl ring in equatorial as also confirmed by their appropriate chemical shifts. The DEPT was performed to find out methine, methylene and methyl carbons. MS ES  : M þ at 402.12. Thus, on the basis of above spectral data compound C was characterized as 40 O demethyl magnolin having molecular formula C22H26O7. This compound appears to be first report from a natural source. Sesamin and Kobusin was first reported by Tocher (1891) and Liu et al. (2006) respectively. Lignans are natural products which are formed by two C6–C3 units β-β0 linked. The term “lignan” was introduced by Harwoth which describes that the lignans are the dimers of phenylpropanoid (C6–C3) units linked by the central carbon atoms of their side chain (Konklugil, 1994). Lignans stands as the most outstanding class of compounds for their cytotoxic characters, e.g., podophyllotoxin, a-peltatin and 5-peltatin isolated from podophyllum

Petroleum ether extract and isolated lignans in different concentrations (1–150 mg/ml) were screened for its cytotoxic potential by using MTT assay on A-549 and MIA-PaCa cancer cell lines. This assay is a novel method of quantifying metabolically viable cells through their ability to reduce a soluble yellow tetrazolium salt to blue-purple formazan crystals. These formazan crystals are thought to be produced by the mitochondrial enzyme succinate dehydrogenase and can be dissolved and quantified by measuring the absorbance of the resultant solution. The absorbance of the solution is related to the number of live cells (Slater et al., 1963). The extract and lignans of ZA caused significant inhibition of viability of cancer cell lines. Results were expressed in IC50 values. The isolated compounds showed more cytotoxicity than petroleum ether extract (Figs. 2 and 3). In case of A549 cell line the IC50 value for petroleum ether extract was found to be 68.11 7 1.231 mg/mL whereas for sesamin, kobusin, 40 O demethyl magnolin and docetaxel IC50 value was 37.46 71.097 mg/mL, 34.71 7 2.331 mg/mL, 26.477 1.871 mg/mL, 12.01 72.107 mg/mL respectively and in case of MIA-PaCa cell line the IC50 value for petroleum ether extract was found to be 63.75 71.0924 mg/ml whereas for sesamin, kobusin, 40 O demethyl magnolin and docetaxel was 34.0471.7621 mg/mL, 32.8672.0271 mg/mL, 21.7271.5071 mg/mL, 11.12 72.2062 mg/ml respectively (Table 1). The results of MTT assay revealed that both extract and isolated compounds showed cytotoxic activity. The isolated compounds belongs to tetrahydrofuran class of lignans and tetrahydrofuran lignans from other plants such as talaumidin, acetyl talaumidin isolated from Talauma

M. Mukhija et al. / Journal of Ethnopharmacology 152 (2014) 106–112


Fig. 4. Micrographs (magnification  400) of AO/EB stained MIA-PaCa (human pancreatic cells) A: unexposed (control) cells have normal nucleus indicating live cells, B: cells exposed to IC50 dose (21.72 mg/mL) for 24 h display condensed or fragmented chromatin suggesting apoptosis, and C: cells exposed to highest dose (150 mg/mL) for 24 h have a structurally normal nucleus indicating cell necrosis.

hodgsonii has previously been reported with cytotoxic potential in different tumor cell lines (Nascimento et al., 2004). So, this further supports to our recent findings. The most potent inhibition of cell proliferation was observed with lignan C i.e., 40 O demethyl magnolin in both cell lines. So, only compound C was subjected to apoptosis study. Natural bioactive substances can modify redox status and interfere with basic cellular functions such as cell cycle, apoptosis, inflammation, angiogenesis, invasion and metastatis (Kampa et al., 2007). Apoptosis plays an important role in embryological development, cell differentiation, cell proliferation, elimination of seriously damaged cells or tumor cells by chemopreventive or chemotherapeutic agents and many other physiological processes (Galati et al., 2000). Apoptotic cells and bodies are rapidly recognized by macrophages before cell lysis, and removed without inducing inflammation. Therefore induction of apoptosis is an important mechanism of chemoprevention and chemotherapy of cancer (Milena et al., 2012). To determine whether the inhibition of cell proliferation by the most potent compound C was due to the induction of apoptosis, the compound was assessed with the acridine orange/ethidium bromide method. MIA-PaCa cells were exposed to 40 O demethyl magnolin and after 24 h the cells were treated with acridine orange/ethidium bromide to determined the apoptosis. Fluorescence microscopy images showed morphological changes such as reduction in size and cell volume, cell shrinkage, membrane blebbing, chromatin condensation, nuclear fragmentation and formation of apoptotic bodies of treated cells (Fig. 4). Results of AO/EB clearly indicates that 40 O demethyl magnolin enhance the apoptosis at IC50 dose (21.72 mg/mL), however showing necrotic cell death at higher dose after 24 h. The present study was designed to investigate the potential therapeutic capabilities of ZA and isolated lignans in human cancer cell lines (A549 and MIA-PaCa) and mechanism of cell death of most potent compound. The use of natural herbal medicines or dietary agents is being increasingly utilized as an effective way for the management of many cancer treatments. Apoptosis, identified as one of the most fundamental biological processes in eukaryotes in which individuals cells die by activating intrinsic “suicide” mechanisms, suggested to play a key role in cell death, caused by a variety of insults. The results revealed that all isolated compounds has inhibitory effect on cancer cells but 40 O demethyl magnolin was found to be most potent and induced apoptosis as the mechanism of cell death.

4. Conclusion The petroleum ether extract of ZA stem bark has potential for cytotoxic activity. Thus, traditional value of bark of ZA has been scientifically proved by MTT and AO/EB assay on human carcinoma cell lines. It has been proved that cytotoxic potential of the

petroleum ether extract is due to lignans, particularly sesamin, kobusin and 40 O demethyl magnolin.

Acknowledgment We express our sincere thanks to Punjab Technical University, Kapurthala for allowing us to proceed with the research proposal. We also express our sincere thanks to the Management and Shri. Parveen Garg, Honorable Chairman for providing necessary facilities and Mr. Hemraj Heer, Assistant Professor, Biotechnology, ISF College of Pharmacy, Moga (Punjab) for his technical help in processing of cytotoxic and apoptosis assay in animal tissue culture lab.

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Bioactive Lignans from Zanthoxylum alatum Roxb. stem bark with cytotoxic potential.

Zanthoxylum alatum is used in traditional medicinal systems for number of disorders like cholera, diabetes, cough, diarrhea, fever, headache, microbia...
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