New coumestan and coumaronochromone derivatives from Dalbergia boehmii Taub. (Fabaceae)

Phytochemistry Letters 21 (2017) 109–113

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New coumestan and coumaronochromone derivatives from Dalbergia boehmii Taub. (Fabaceae)

MARK

Jean Pierre Abdoua,b, Jean Momenic, Achyut Adhikarid, Nole Tsabangb, Alembert T. Tchindab, ⁎ Muhammad I. Choudharyd, Augustin E. Nkengfacka, a

Department of Organic Chemistry, Faculty of Science, University of Yaounde I, P.O. Box 812, Yaounde, Cameroon Centre for Research on Medicinal Plants and Traditional Medicine (CRPMT), Institute of Medical Research and Medicinal Plants Studies (IMPM), P.O. Box 13033 Yaounde, Cameroon c Department of Chemistry, Faculty of Science, University of Ngaoundere, P.O. Box 454, Ngaoundere, Cameroon d H E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences (ICCBS), University of Karachi, Karachi 75270, Pakistan b

A R T I C L E I N F O

A B S T R A C T

Keywords: Dalbergia boehmii Dalbergestan Dalbergichromone Leishmanicidal Cytotoxicity

Chemical investigation of leaves and heartwood of Dalbergia boehmii resulted in the isolation of two new phenolic compounds, designated dalbergestan (1) and dalbergichromone (2), along with eleven known compounds, carpachromene (3), proanthocyanidin A-2 (4); piceatannol (5); biochanin A (6); macckiain (7); homopterocarpin (8); angolensin (9); medicarpin (10); 2′,7-dihydroxy-4′,5′-dimethoxyisoflavone (11); 2′-methoxyformononetin (12); and genistein (13). The structures of the new compounds were elucidated on the basis of extensive spectroscopic analyses including, IR, UV, 1D and 2D – NMR as well as HRMS data. Some of the isolated compounds were evaluated for their in vitro insulin secretion activity on isolated mice islets, leishmanicidal activity against L. major (DESTO) promastigotes and in vitro cytotoxicity on MCF-7 cell lines. All tested compounds were inactive on glucose-stimulated insulin secretion at stimulatory glucose (20.0 mM) from MIN6 cells. Compounds 3 (IC50, 70.0 μg/ml), 6 (IC50, 60.3 μg/ml), 7 (IC50, 86.5 μg/ml) and 13 (IC50, 62.6 μg/ml) exhibited low leishmanicidal activity while compound 12 (IC50, 56.8 μg/ml) displayed a moderate activity. Compounds 3 and 5 were found to be active against MCF-7 at 50 μM with IC50 value 33.2 ± 3.79 μg/ml and 42.64 ± 5.05 μg/ml respectively.

1. Introduction Dalbergia is a large genus of trees, shrubs, lianas, and woody climbers belonging to the Fabaceae family and which are widely distributed in tropical and subtropical regions over the world (Saha et al., 2013). Species of this genus are well known for their deeply pigmented heartwood of varying colors, which are valued for used in wooden crafts, as well as in traditional medicine (Cheenpracha et al., 2009), where they are used for the treatment of different ailments like diarrhea, dysentery, dyspepsia, gonorrhea, haemorrhages, leprosy, malaria, rheumatism etc… (Saha et al., 2013). The plethora of traditional uses of plants belonging to Dalbergia genus prompted scientists to screen some of them for a wide range of biological activities including analgesic, antipyretic, anti-inflammatory (Vasudeva et al., 2009; Misar et al., 2005; Hajare et al., 2001; Kaviswami et al., 2002), antiplasmodial (Vasudeva et al., 2009), antiulcerogenic (Brito et al., 1997), antimicrobial (Gundidza and Gaza, 1993), antigiardial (Khan et al., 2000), antidiarrhoeal (Mujumdar et al., 2005), antioxidant (Wang et al., ⁎

Corresponding author. E-mail address: [email protected] (A.E. Nkengfack).

http://dx.doi.org/10.1016/j.phytol.2017.06.014 Received 25 February 2017; Received in revised form 31 May 2017; Accepted 16 June 2017 1874-3900/ © 2017 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.

2000), anti-fertility (Uchendu et al., 2000), cancer chemopreventive activities (Ito et al., 2003), larvicidal and mosquito repellent (Ansari et al., 2000; Mutai et al., 2013). Previous phytochemical investigations have been so far carried out on various species of Dalbergia genus and resulted in the isolation of many isoflavonoids, neoflavonoids, steroids, cinnamylphenols, furans, quinones, glycosides and other miscellaneous compounds (Vasudeva et al., 2009; Saha et al., 2013). Dalbergia boehmii, named “Ngalayhi” in Fufulde in Northern Cameroon, is a large shrub or a small to medium-sized deciduous tree, unarmed. The bark is grey or brown, rough and flaking in older trees. It is used in some African countries, and particularly in Cameroonian folk medicine for the treatment of digestive tract diseases and against fever (Burkill, 1995). However, as part of our ongoing program of searching bioactive natural products from Cameroonian medicinal plants, the leaves and heartwood of D. boehmii were investigated. As results, two new compounds along with eleven known compounds were obtained. This paper describes the isolation and structural elucidation of those compounds as well as the evaluation of in vitro insulin secretion, leishmanicidal and

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groups and the methoxyl substituent on coumestan ring was deduced by HMBC correlations (Fig. 2). And also, the fact that no correlation peak was observed in NOESY spectrum neither between the methoxyl signal and pair of ortho aromatic protons on one hand, nor methoxyl group and pair of para aromatic protons on the other hand, led clearly to the conclusion that the methoxyl group is located at C-8, whereas one hydroxyl group occupied position C-7 and the two remaining others at C4′ et C-5′. Thus, based on these observations, structure of (1) was assigned to 3,8,9-trihydroxy-4-methoxycoumestan, a new coumestan and given the trivial name dalbergestan (Fig. 1). Compound 2, was isolated as colorless amorphous powder. Its molecular formula was deduced as C17H12O7 from high resolution electronic impact mass spectrum (HREI-MS) which showed the [M+] at m/z 328.0583 (calcd. for C17H12O7: 328.0583) revealing 12 ° of unsaturation. The IR spectrum exhibited vibration bands due to the presence of hydroxyl group (3415 cm−1); conjugated carbonyl (1627 cm−1) and C]C band of aromatic and olefinic (1506, 1442 cm−1). Its UV (MeOH) spectrum showed a prominent absorption band at λmax 253 nm, associated with other bands of much lower intensity at λmax 212; 280; 307 and 325 nm, indicating that compound 2 was an isoflavonoid type compound (Tahara et al., 1985; Mizuno et al., 1992). The 1H NMR spectrum of (2) (Table 1) displayed signals, one chelated hydroxyl group at δ 10.84 (1H; s; 5-OH) and one free hydroxyl group at δ 9.43 (1H; s; 4′-OH). Some signals which were also observed included a pair of singlets at δ 7.41 (1H; s) and 7.13 (1H; s) corresponding to a pair of para aromatic protons of ring B at C-6′ and C-3′, respectively; a pair of doublets at δ 6.49 (1H; d; J = 2.1 Hz) and 6.59 (1H; d; J = 2.1 Hz) attributed to a pair of meta aromatic protons of ring A at C-6 and C-8, respectively and two signals of singlets of 3 protons each, supporting the presence of two methoxyl groups. This was confirmed by 13C NMR broad band and DEPT spectra which showed signals for two primary carbons at δ 56.1 each corresponding to two methoxyl groups; 4 sp2 methine signals (δ 97.2; 95.8; 102.9 and 99.2). Thus, compound 2 has 11 sp2 quaternary carbons among which one conjugated carbonyl (δ 172.1); seven oxygenated (δ 161.5; 162.1; 162.1; 156.4; 145.3; 146.4 and 142.9) and 3 sp2 carbons (δ 98.7; 106.6 and 113.8). Aforementioned functionalities account for 11 ° of unsaturation. The left over C]C double bound equivalent required the presence of one ring. The absence on the 1H NMR spectrum of signal around δ 7.8–7.9 characteristic of H-2 proton of isoflavone skeleton, led to the conclusion that the additional ring was established between C-2 carbon of ring C and C2′ of ring B, leading to the coumaronochromone skeleton. This was confirmed by comparison of UV and NMR spectral data of compound 2 with those of desmoxyphyllin A, a coumaronochromone isolated from leaves of Desmodium oxyphyllum reported in the literature (Mizuno et al., 1992). Additional proof come from HMBC spectrum which showed long range correlations as depicted on Fig. 2. From the above analysis, compound 2 was deduced as 5,4′-dihydroxy-7,5′-dimethoxycoumaronochromone, a new coumaronochromone named dalbergichromone (Fig. 1). Some of isolated compounds were investigated for various bioassays such as in vitro insulin secretion, in vitro leishmanicidal activities and in vitro anticancer activity against MCF-7 cell line. Because of the small amount of some isolated compounds, not all compounds were subjected to bioassays. Compounds 1-2, 5-7, 12 were evaluated for their insulin secretory activity on freshly isolated mice islets to evaluate their biological function and compared with Arginine, a standard insulin secretagogue taken as control. The effects of tested compounds on glucose-stimulated secretion from MIN6 cells are summarized in Table 2. All tested compounds are inactive on glucose-stimulated insulin secretion at stimulatory glucose (20.0 mM) from MIN6 cells. This result did not corroborate with those of Pinent et al. (2008) who showed that flavonoids in general showed promising results on insulin secretion. Some researchers support the view that the form in which some flavonoids are found in plants is not their bioactive form (Manach et al., 2005). They

anticancer activities of some isolated compounds. 2. Results and discussion Air-dried and powdered leaves and heartwood of D. boehmii were successively extracted by maceration at room temperature with a mixture of dichoromethane and methanol (1:1 v/v) to give crude extracts. Repeated column chromatography of the crude extract of the leaves over silica gel and sephadex LH20 yielded two new compounds (1) and (2) along with two known compounds identified as carpachromene (3) (Zheng et al., 2008) and proanthocyanidin A-2 (4) (Lou et al., 1999). The same procedure applied to the heartwood extract led to the isolation of nine compounds identified as piceatannol (5) (Brinker and Seigler, 1991); biochanin A (6) (Dos Santos and De Carvalho, 1995); macckiain (7) (Park et al., 2003); homopterocarpin (8) (Agrawal, 1989); angolensin (9) (Tringali, 1995); medicarpin (10) (Duddeck et al., 1987); 2′,7-dihydroxy-4′,5′-dimethoxyisoflavone (11) (Taechowisan et al., 2014); 2′-methoxyformononetin (12) (Jain et al., 1996) and genistein (13) (Almahy and Alhassan, 2011). The eleven known compounds were identified by their spectral data and by comparison of these data with those published in the literature. Compound 1, was obtained as colorless amorphous powder. Its molecular formula C16H10O7, corresponding to eleven degrees of unsaturation, was deduced from the high resolution electronic impact mass spectrum (HREI-MS), which showed the [M+] at m/z 314.0427 (calcd. for C16H10O7: 314.0426). The IR spectrum suggested the presence of hydroxyl (3309 cm−1); carbonyl (1718 cm−1); aromatic C]C groups (1608, 1435 cm−1) and ether function (1288, 1172 cm−1). Its UV spectrum showed absorption band at λmax (MeOH): 218, 248, 310, 352 nm, characteristic of coumestan chromophore (Yadav et al., 2004). The 1H NMR (Table 1) spectrum of compound 1 showed a pair of doublets at δ 7.66 (1H, d, J = 9.0 Hz) and 7.23 (1H, d, J = 9.0 Hz) due to ortho aromatic protons H-5 and H-6, respectively. A pair of singlets at δ 8.08 (s) and 7.61 (s) was attributed to para aromatic protons at positions H-3′ and H-6′, respectively, while the signal of 3 protons singlet at δ 3.97 was due to one methoxyl group. The 13C NMR (Table 1) broad band and DEPT spectra displayed resonances for one methoxyl group, four methine and eleven quaternary carbons. The 13C NMR spectrum displayed signal for lactone carbonyl (δ 158.5), seven oxygenated quaternary carbons at δ 159.9 (C-4); 154.9 (C-7); 136.3 (C-8); 147.9 (C8a); 148.3 (C-1′); 146.3 (C-4′); 150.3 (C-5′). Position of the 3 hydroxyl Table 1 1 H- (600 MHz; 500 MHz) and 13C- (125 MHz) NMR spectral data of dalbergestan (1) in C5D5N and dalbergichromone (2) in DMSO-d6. No

2 3 4 4a 5 6 7 8 8a 1′ 2′ 3′ 4′ 5′ 6′ 8-OCH3 5-OH 4′-OH 7-OCH3 5′-OCH3

1

2

δH (m, J, Hz)

δC

δH (m, J, Hz)

δC

– – – – 7.66 d (9.0) 7.23 d (9.0) – – – – – 8.08 s – – 7.61 s 3.97 s – – – –

158.5 103.5 159.9 103.5 117.0 114.9 154.9 136.3 147.9 148.3 115.6 106.0 146.3 150.3 99.9 60.9 – – – –

– – – – – 6.49 d (2.1) – 6.59 d (2.1) – – – 7.13 s – – 7.41 s – 10.84 s 9.43 s 3.84 s 3.86 s

161.5 98.7 172.1 106.6 162.1 97.2 162.1 95.8 156.4 113.8 145.3 102.9 146.4 142.9 99.2 – – – 56.1 56.1

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Fig. 1. Chemical structures of compounds 1 and 2.

Fig. 2. Key HMBC correlations of compounds 1 and 2.

ring B, the greater the antileishmanial activity (Tasdemir et al., 2006). The comparison of IC50 of compounds 6 and 13 suggested that methylation of hydroxyl group on ring B did not have notable influence on leishmanicidal activity. Compounds 3-6 and 12 were investigated for their anticancer activity against MCF-7 cell lines. Compounds 4, 6 and 12 did not exhibit any anticancer activity (IC50 ˃ 50.0 μg/ml) whereas compounds 3 and 5 were found to be active against MCF-7 at 50 μM with IC50 value 33.2 ± 3.79 μg/ml and 42.64 ± 5.05 μg/ml respectively (Table 2). The anticancer activity observed with compound 5 is in accordance several studies demonstrating that many stilbenes are also considered as potential chemopreventive agents (Fresco et al., 2006). Some studies on the anticancer activities of flavonoids have shown that those with a structure in which ring B is linked to ring C at position C-2 would

have to be modified by some enzymes of the body to thus have an activity (Pinent et al., 2008). Compounds 3-7, 12-13 were investigated for antileishmanial activity against L. major (DESTO) promastigotes and the results are illustrated in Table 2. Compounds 4 and 5 did not show any leishmanicidal activity. Compounds 3 (IC50, 70.0 μg/ml), 6 (IC50, 60.3 μg/ml), 7 (IC50, 86.5 μg/ml) and 13 (IC50, 62.6 μg/ml) exhibited low leishmanicidal activity while compound 12 (IC50, 56.8 μg/ml) displayed a moderate activity. Their IC50s were compared to those of Amphotericin B and Pentamidine taken as positive control, with IC50 values respectively 0.29 μg/ml and 5.09 μg/ml. This result shows that biflavonoid (4) and stilbene (5) did not display antileishmanial activities. However, flavone (3), pterocarpan (7) and isoflavones (6, 12, and 13) have low or moderate activities. As to regards isoflavones, the more substituted the

Table 2 Insulin secretion, leishmanicidal and anticancer activities data of compounds 1-7, 12-13. Compounds

1 2 3 4 5 6 7 12 13 Arginine Amphotericin B Pentamidine Doxorubicin

Insulin Secretion activity

Leishmanicidal activity

Anticancer activity

% increase insulin secretions

IC50 (μg/ml) ± S.D

% Inhibition/Stimulation

IC50 (μg/ml) ± SD

42.1 34.2 – – 39.5 76.3 42.1 41.1 – 299.5 – – –

– – 70 ± 2.1 ˃100.0 ˃100.0 60.3 ± 2.9 86.5 ± 2.6 56.8 ± 2.9 62.6 ± 2.8 – 0.29 ± 0.05 5.09 ± 0.09 –

– – 75.22 1.02 52.98 3.62 – 14.18 – – – – 89.19

– – 33.2 ± 3.79 ˃50.0 42.64 ± 5.05 ˃50.0 – ˃50.0 – – – – 0.92 ± 0.1

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3.4. Dalbergestan (1)

exhibit good activity against breast cancer cells as the case with baicalein and quercetin (So et al., 1996).

Colorless amorphous powder; m.p. 292–299 °C; UV λmax (MeOH) nm: 218, 248, 310, 352; IR υmax (KBr) cm−1: 3309 (OH), 1718 (C]O), 1608, 1435 (C]C); 1H- and 13C NMR (See Table 1); EI-MS m/z (rel.int.) 314 [M+] (100), 299 (78), 284 (11), 271 (15), 243 (14), 215 (15), 187 (63), 159 (5); HREI-MS m/z 314.0430 (calcd. for C16H10O7: 314.0427).

3. Experimental section 3.1. General experimental procedures Melting points were determined on a Büchi M-560 melting point apparatus and were uncorrected. The infrared (IR) spectra were recorded on VECTOR22 while Ultraviolet (UV) spectra were recorded on THERMO ELECTRON ∼ VISIONpro SOFTWARE V4.10. HREI-MS spectra were recorded on JEOL JMS-600H mass spectrometer. Electrospray ionization mass spectrometry were recorded on Q STAR XL of AB Sciex Company.1H NMR experiments were performed on a Bruker AM 400 (600) and AMX 500 NMR (Avance) instruments using the UNIX data system at 400 (600) and 500 MHz respectively. The 13C NMR spectrum was recorded at 75, 100, 125 MHz using CD3OD, DMSO, and C5D5N as solvents. Chromatographic columns were performed on silica gel and Sephadex LH20. Fractions were monitored by TLC using Merck pre-coated silica gel sheets (60F254), and spots were visualized by using fluorescence (254 and 386 nm) and by heating silica gel plates sprayed with ceric sulphate solution.

3.5. Dalbergichromone (2) Colorless amorphous powder; m.p. 314–321 °C; UV λmax (MeOH) nm: 212, 253, 280, 307, 325; IR υmax (KBr) cm−1: 3415 (OH), 1627 (C]O), 1506, 1442 (C]C); 1H- and 13C NMR (See Table 1); EI-MS m/z (rel. int.) 328 [M+] (58), 311 (15), 299 (10), 283(19), 255 (2), 148 (3), 129 (2), 111 (3); HREI-MS m/z 328.0585 (calcd. for C17H12O7: 328.0583). 3.6. Bioassays 3.6.1. In vito insulin secretion assay Insulin secretion assay were carried out according to a recent developed protocol by Siddiqui et al. (2014). In brief, mice were anaesthetized with pentobarbitone sodium (30.0 mg/kg) and the pancreas was distended with 3 ml collagenase solution (1 mg/ml) via the common bile duct. The whole pancreas was removed from each mouse and digested in collagenase solution at 37° C for 15 min. The digested islets were further purified by centrifugation at 1000 rpm at 4° C for 1 min followed by filtration using a pre- wetted 70.0 μm cell strainer. Finally, islets were manually hand- picked with a siliconized Pasteur pipette under a NIKON SMZ745 stereomicroscope. The isolation and purification medium used was Hank’s Balanced Salt Solution (HBSS) without calcium, magnesium and phenol red. Immediately after isolation, the islets were pre-incubated for 30 min at 37° C in Krebs-Ringer bicarbonate (KRB) buffer solution containing 0.1% BSA and 3.0 mM glucose. Thereafter, batches of five size-matched islets were incubated for 60 min in KRB with 3.0 mM (basal) or 16.7 mM (stimulatory) glucose, both in the absence and presence of test compounds. At the end of the incubation, 200.0 μl aliquots were immediately frozen until insulin assay. Insulin was measured using an Ultra-Sensitive Mouse Insulin ELISA kit according to the manufacturer’s protocol and normalized or the number of islets. Data (Table 2) are shown as means ± SEM from three independent experiments each in triplicates. **P < 0.01 compared to control. Arg, Arginine (50.0 mM). Concentration and insulin secretion activities by 20.0 mM glucose were taken as 100%. All tested compounds were solubilized in DMSO and used at the dose of 50.0 mM.

3.2. Plant materials Leaves and hearthwood of Dalbergia boehmii Taub. were collected in Touboro in the North region of Cameroon in September 2012. The plant material was identified by Dr Tsabang Nole and a Voucher specimen (66887/HNC) has been deposited in the Cameroon National Herbarium in Yaounde.

3.3. Extraction and isolation Chopped, air-dried and ground leaves (3.0 kg) and hearthwood (4.0 kg) of Dalbergia boehmii were macerated each in a (1:1, v/v) mixture of dichoromethane and methanol (3 × 9l, 6 days) at room temperature. The resulting solution was filtered and concentrated on a rotatory evaporator under reduced pressure to give an amorphous green material (490.8 g) for leaves, and a brownish and gummy crude extract (136.0 g) for hearthwood. A portion (150.0 g) of the crude extract of leaves was fractionated by silica gel column chromatography using a gradient elution with successively Hexane-EtOAc and EtOAcMeOH. From this, 10 fractions (F1–F10) were obtained according to the chromatographic profiles on TLC. Purification of F5 (5.0 g) on silica gel column chromatography (Hexane-EtOAc 4:1) followed with purification on Sephadex LH20 (Hexane-CH2Cl2-MeOH 7-4-0.5) furnished 10.1 mg of compound 3. Compound 1 (2.9 mg) was obtained from F8 (9.0 g) (Hexane-EtOAc 3–7). F9 (7.0 g) was applied to silica gel column chromatography to yield compound 2 (3.4 mg) and compound 4 (37.2 mg). The crude extract of hearthwood was fractionated by silica gel column chromatography using a gradient elution with successively Hexane-EtOAc and EtOAc-MeOH, resulting to eight fractions (F'1-F'8). The fraction (F'2) eluted with Hexane-EtOAc (9:1) was subjected to column chromatography on silica gel (eluent: Hexane-EtOAc 9:1) and further purification on preparative TLC (eluent: Hexane- CH2Cl2) yielded compound 8 (3.0 mg). The fraction (F'4) eluted using HexaneEtOAc (4:1) was purified by column chromatography with silica gel (eluent: Hexane-EtOAc 8:2) followed by preparative TLC (eluent: CHCl3-MeOH 25:1) to yield compounds 9 (2.6 mg), 10 (3.0 mg), 7 (10.4 mg) and 12 (50.5 mg). Repeated column chromatography with CH2Cl2-EtOAc of fraction 5 (eluted with Hexane-EtOAc 3:2) yielded compound 6 (15.2 mg), 11 (2.1 mg) and 13 (5.4 mg). While fraction F'6 eluted with Hexane- EtOAc (2:3) furnished compound 5 (50.6 mg).

3.6.2. In vitro leishmanicidal assay In vitro leishmanicidal assay were carried out according to the protocol developed by Choudhary et al. (2005) and Habtemariam (2003). Leishmania major (DESTO) promastigotes were grown in bulk early in modified NNN biphasic medium using normal physiological saline. Leishmania parasite promastigotes were cultured with RPMI 1640 medium (Sigma, St. Louis, USA) supplemented with 10% heat inactivated fetal Calf serum (FCS) (PAA Laboratories GmbH, Austria) Parasites at log phase were centrifuged at 2000 rpm for 10 min, and washed three times with saline at same speed and time. Parasites were diluted with fresh culture medium to a final density of 1 × 106 cells/ ml. In a 96-well micro titer plate, medium was added in different wells; 20.0 μl of the experimental compound was added in medium and serially diluted. 100.0 μl of parasite culture was added in all wells. Two rows were left for negative and positive control. Negative controls received only medium while the positive control contained varying concentrations of standard antileishmanial compounds e.g. Amphotericin B (MP Biomedical Inc.) and Pentamidine (ICN Biomedical Inc). The plate was incubated between 22 and 25 °C for 72 h. The culture was examined microscopically on an improved Neubaure counting chamber 112

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and IC50 values of fractions possessing antileishmanial activity were calculated by Software Ezfit 5.03 Perella Scientific. DMSO was used to dissolve tested compounds such that the final concentration of DMSO in culture is 0.5%. Concentration of tested compounds was 250 μg/ml and was serially diluted from 100 to 3.125 μg/ml for IC50 values calculations. All assays were run in duplicate. 3.6.3. In vitro cytotoxicity with MCF-7 cell lines In vitro cytotoxicity activity of pure compounds was evaluated on MCF-7 cell lines using the standard MTT (3-[4,5-dimethylthiazole-2-yl]2, 5-diphenyl-tetrazolium bromide) colorimetric assay according to Scudiero et al. (1988). MCF-7cell lines were cultured in Dulbecco’s modified Eagle medium (containing 10% fetal bovine serum) in 75 cc flasks, and kept in 5% CO2 incubator at 37 °C. Upon confluency, cells were harvested and plated in 96-well tissue culture treated flat bottom plates (seeding density 8000 cells/well for MCF) in 100.0 μl medium. Next day, compounds were dissolved in DMSO and added in triplicate at 50.0 μM concentration, and incubated for 48 h. After 48 h incubation, the compounds were removed and 200.0 μl MTT at 0.5 mg/ml was added to each well and incubated at 37 °C for 3 h. Formazan crystals, formed by reduction of MTT were dissolved in 100.0 μl DMSO and absorbance was taken at 570 nm using micro-plate reader (Spectra Max plus, Molecular Devices, CA, USA). The percent inhibition or decrease in viable cells was calculated by following formula: % Inhibition = 100 − ((mean of O.D. of test compound − mean of O.D. of negative control)/(mean of O.D. of positive control − mean of O.D. of negative control) × 100) If compounds showed 50% of more inhibition, they were further processed for IC50 calculation. Twenty (20.0) mM stock concentration of compounds were diluted to working concentration of 50.0 μM, and then further serial dilutions were made in order to get less than 50% inhibition. The IC50 was then calculated by using EZ-fit5 software. Acknowledgments The authors would like to thank TWAS (The World Academy of Science), the H.E.J. Research Institute of Chemistry, and the International Center for Chemical and Biological Sciences (ICCBS) of the University of Karachi, Karachi-75270, Pakistan; for the award of TWAS-ICCBS Sandwich Postgraduate fellowship FR number: 3240275059 to J.P.A. References Agrawal, P.K., 1989. Carbon-13 NMR of Flavonoids. Elsevier, Amsterdam. Almahy, H.A., Alhassan, N.I., 2011. Studies on the chemical constituents of the leaves of Ficus bengalensis and their antimicrobial activity. J. Sci. Technol. 13, 118–124. Ansari, M.A., Razdan, R.K., Tandon, M., Vasudevan, P., 2000. Larvicidal and repellent actions of Dalbergia sissoo Roxb (F-Leguminosae) oil against mosquitoes. Bioresour. Technol. 73, 207–211. Brinker, A.M., Seigler, D.S., 1991. Isolation and identification of piceatannol as a phytoalexin from sugarcane. Phytochemistry 30, 3229–3232. Brito, A.R.M.S., Cota, R.H.S., Nunes, D.S., 1997. Gastric antiulcerogenic effects of Dalbergia monetaria L. in rats. Phytother. Res. 11, 314–316. Burkill, H.R.M., 1995. The Useful Plants of West Tropical Africa, vol. 3 Royal Botanic Gardens Kew, London. Cheenpracha, S., Karalai, C., Ponglimanont, C., Kanjana-Opas, A., 2009. Candenatenins AF, phenolic compounds from the heartwood of Dalbergia candenatensis. J. Nat. Prod. 72, 1395–1398. Choudhary, M.I., Yousuf, S., Ahmed, S., Samreen, Yasmeen K., Atta-ur-Rahma, 2005. Antileishmanial physalins from Physalis minima. Chem. Biol. 2, 1164–1173. Dos Santos, S.A., De Carvalho, G., 1995. Unambiguous 1H- and 13C NMR assignments of isoflavones from virola caducifolia. J. Braz. Chem. Soc. 6 (4), 349–352.

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