Biologically Active Withanolides from Withania coagulans - Journal of

Jan 14, 2013 - Department of Biochemistry, Quaid-i-Azam University, Islamabad, 45320, Pakistan. J. Nat. Prod. , 2013, 76 (1), pp 22–28. DOI: 10.1021...
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Biologically Active Withanolides from Withania coagulans Ihsan-ul-Haq,†,‡ Ui Joung Youn,† Xingyun Chai,† Eun-Jung Park,† Tamara P. Kondratyuk,† Charles J. Simmons,§ Robert P. Borris,† Bushra Mirza,⊥ John M. Pezzuto,† and Leng Chee Chang*,† †

Department of Pharmaceutical Sciences, College of Pharmacy, University of Hawaii at Hilo, Hilo, Hawaii 96720, United States Department of Pharmacy, Quaid-i-Azam University, Islamabad, 45320, Pakistan § Chemistry Department, University of Hawaii at Hilo, Hilo, Hawaii 96720-4091, United States ⊥ Department of Biochemistry, Quaid-i-Azam University, Islamabad, 45320, Pakistan ‡

S Supporting Information *

ABSTRACT: Bioassay-directed isolation and purification of the crude extract of Withania coagulans, using two assays for cancer chemopreventive mechanisms, led to the isolation of three new steroidal lactones, withacoagulin G (1), withacoagulin H (2), and withacoagulin I (3), along with six known derivatives (4−9). The structures and absolute stereochemistry of these compounds were determined on the basis of spectroscopic analyses, including 1D and 2D NMR, mass spectrometry, and CD analyses. The structure of 1 was confirmed using X-ray diffraction methods. Compounds 1−9 inhibited nitric oxide production in lipopolysaccharide-activated murine macrophage RAW 264.7 cells with IC50 values in the range of 1.9−38.2 μM. Compounds 1 and 2 were the most active (IC50 3.1 and 1.9 μM, respectively). Withanolides 1−9 exhibited inhibition of tumor necrosis factor-α (TNF-α)-induced nuclear factor-kappa B (NF-κB) activation with IC50 values in the range of 1.60−12.4 μM.

H

Antihyperglycemic leads have been isolated from W. coagulans, which have not yet been observed in W. somnifera.13 Furthermore, a distinctive thio-dimer of withanolide named ashwagandhnolide has been reported in W. somnifera.14,15 Withanolides containing a 14,20-epoxide bridge are specific to W. coagulans.

igher plants represent a rich source of previously unknown molecules with new pharmacological activities which may be lead compounds for the development of new anticancer and chemoprevention agents.1 Taxol, vinblastine, vincristine, resveratrol, camptothecin and its analogues, and etoposide and its analogues are examples of clinically important natural anticancer and cancer-chemopreventive agents. Withania coagulans Dunal (Solanaceae) is native to warm, temperate (Western Asia: Afghanistan) and tropical regions (Indian Subcontinent: India, Pakistan, Nepal). It is widely distributed in the relatively drier parts of India and Pakistan2 and is commonly known as Indian cheese maker, Indian rennet, or vegetable rennet (English). W. coagulans has been recommended for the treatment of various disorders in traditional medicine.3 It is used for the treatment of dyspepsia, dropsy, intestinal infections, rheumatism, ulcers, tuberculosis, and senile debility in the indigenous medicine of those areas.4 The fruit is reported to have diuretic and sedative properties. It is also employed as a “blood purifier” and for the treatment of asthma.5 The leaves are used as a vegetable for humans and as fodder for camels and sheep. The fruit and berries are used for milk coagulation. Aqueous extracts of the fruits of W. coagulans exhibited antidiabetic and antioxidant activities.6,7 W. coagulans has been reported to possess antimicrobial, anthelmintic,8 antifungal,9 antitumor,10 anti-inflammatory, cardiovascular,11 hepatoprotective,12 and wound-healing5 effects. Two species, W. somnifera and W. coagulans,3 are found in India and Pakistan. W. somnifera is known as “Indian ginseng” and “winter cherry” in English and “Ashwagandha” in Hindi. Withanolides are the major compounds found in both species. © 2013 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION In our continuing search for cancer chemopreventive agents of natural origin, W. coagulans was selected for further study.4 A chloroform−methanol crude extract of W. coagulans was suspended in H2O and extracted successively with n-hexane and ethyl acetate (EtOAc). The EtOAc-soluble extract exhibited the strongest inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine macrophage RAW 264.7 cells and the tumor necrosis factor alpha (TNF-α)-activated nuclear factor-kappa B (NF-κB) pathway in transfected human embryonic kidney cells 293. The EtOAc extract was fractionated by column chromatography (CC), eluting with a gradient of n-hexane and EtOAc to afford a series of fractions, which were further separated by preparative HPLC to yield nine compounds (1−9). Compound 1 was obtained as colorless needles with a molecular ion at m/z 493.2630 [M + Na]+ (calcd for C28H38O6Na, 493.2575) in the HRESIMS, corresponding to a molecular formula of C28H38O6. The UV spectrum showed an absorption maximum at 226 nm, suggesting the presence of an Received: August 2, 2012 Published: January 14, 2013 22

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Hz, H-2) and 6.93 (m, H-3)]. An olefinic proton δH 5.59 (d, J = 6.0 Hz, H-6) showed 1H−1H COSY correlation peaks with H7, which were assigned to the vinylic protons (H-6). The 1 H−1H COSY spectrum showed correlation peaks from δH 6.93 (m, H-3) to δH 5.79 (dd, H-2) and to δH 2.86 (dd, H-4α) and 3.30 (dd, H-4β), respectively, which supported a steroidal 2-en-1-one moiety.16 The 13C NMR spectrum of 1 revealed 28 carbons: an α,β-unsaturated ketone [δC 204.0 (C-1), 127.3 (C2), 147.3 (C-3)], an α,β-unsaturated δ-lactone [δC 155.4 (C24), 124.3 (C-25), 165.7 (C-26)], an olefinic carbon [δC 125.6 (C-6)], one oxygenated methine [δC 80.1 (C-22)], one oxygenated methylene [δC 55.0 (C-27)], two oxygenated quaternary carbons [δC 86.9 (C-17) and 77.0 (C-20)], and four methyls, six methylenes, two methines, and four additional quaternary carbons. These NMR data were closely related to those of a known ergosterol-type lactone, suggesting that compound 1 was a withanolide.16 In addition, compound 1 also contains two OH groups (δH 4.10 and 4.30 s) at C-17 and C-20, respectively, confirmed by extensive HMBC analysis (Figure 1). Furthermore, an oxygenated methine proton (H-22) showed correlation peaks with C-20 (δC 77.0), C-17 (δC 86.9), C-24

unsaturated lactone. In the IR spectrum of 1, absorption bands for OH, α,β-unsaturated δ-lactone, and carbonyl groups were observed at 3402, 1720, and 1687 cm−1, respectively. The 1H NMR spectrum (Table 1) displayed characteristic signals for four tertiary methyl groups (δH 0.92, 1.16, 1.21, and 2.00) and two α,β-unsaturated olefinic protons [δH 5.79 (dd, J = 10.5, 2.4

Table 1. 1H and 13C NMR Spectroscopic Data of Compounds 1−3a 1 no.

δC mult.

1 2

204.0 q 127.3 CH

3 4α 4β 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 OH-14 OH-17 OH-20

147.3 CH 33.2 CH2 136.4 q 125.6 CH 31.1 CH2 32.9 CH 41.3 CH 50.1 q 23.5 CH2 32.0 CH2 48.0 q 52.0 CH 23.9 CH2 33.0 CH2 86.9 q 15.9 CH3 19.0 CH3 77.0 q 20.0 CH3 80.1 CH 31.9 CH2 155.4 q 124.3 q 165.7 q 55.0 CH2 20.5 CH3

2 δH (J in Hz)

5.79 (dd, 10.5, 2.4) 6.93 (m) 2.86 (dd 2.1, 21.0) 3.33 (dd 4.8, 21.0) 5.59 (d, 6.0) 1.52−1.92 (m) 1.90 (m) 1.80 (m) 1.45−2.05 (m) 1.39−1.70 (m) 1.75 (m) 1.06−1.44 (m) 1.38−1.82 (m) 0.92 (s) 1.16 (s) 1.21 (s) 4.56 (dd, 13.0, 3.5) 2.45 (m)

4.18 (d, 11.7), 4.56 (d, 11.8) 2.00 (s)

δC mult.

3 δH (J in Hz)

203.8 q 127.3 CH 147.3 CH 33.2 CH2 135.8 q 125.6 CH 30.8 CH2 31.9 CH 41.3 CH 50.3 q 24.3 CH2 28.7 CH2 54.9 q 150.5 q 117.6 CH 41.8 CH2 86.7 q 18.7 CH3 21.5 CH3 76.7 q 19.5 CH3 80.1 CH 31.9 CH2 155.4 q 124.3 q 165.7 q 54.0 CH2 20.6 CH3

4.10 (s) 4.30 (s)

δC mult. 210.6 q 40.0 CH2

5.79 (dd, 10.5, 2.4) 6.93 (m) 2.85 (dd, 21.0, 3.2) 3.30 (dd, 21.0, 3.2)

120.0 CH 129.0 CH

5.60 (d, 6.0) 1.80−2.50 (m) 1.9 (m) 1.8 (m) 1.54−2.07 (m) 2.00−2.30 (m)

5.15 (m) 2.3−3.0 (m) 1.21 (s) 1.19 (s) 1.17 (s) 4.56 (dd, 13.0, 3.5) 2.38−2.49 (m)

4.15 (d, 11.7), 4.56 (d, 11.8) 2.01 (s) 4.20 (s) 4.35 (s)

140.1 q 127.9 CH 21.7 CH2 35.9 CH 36.0 CH 51.7 q 22.0 CH2 32.2 CH2 54.0 q 83.3 q 26.0 CH2 33.7 CH2 87.3 q 17.3 CH3 19.9 CH3 77.4 q 17.5 CH3 82.3 CH 31.8 CH2 151.1 q 122.0 q 166.0 q 12.1 CH3 20.2 CH3

δH (J in Hz) 2.64 3.39 5.65 6.08

(dd 20.2, 2.1) (dd 20.2, 2.1) (m) (dd, 9.8, 1.8)

5.71 2.31 1.68 1.82

(br d, 6.0) (m) (m) (m)

1.20−1.51 (m) 1.5 (m)

1.60 (m) 1.41−1.56 (m) 1.16 (s) 1.28 (s) 1.30 (s) 4.46 (dd, 13.0, 3.5) 2.38−2.54 (m)

1.77 1.89 5.53 4.42 6.32

(s) (s) (s) (s) (s)

a

Spectra recorded at 1H (400 MHz) and 13C NMR (100 MHz) in DMSO-d6. Chemical shifts (δ) are in ppm, and coupling constants (J in Hz) are given in parentheses. The assignments were based on DEPT, COSY, NOESY, HSQC, and HMBC experiments. 23

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Figure 1. Key HMBC and COSY correlations of compounds 1−3.

(δC 155.4), and C-26 (δC 165.7) in the HMBC spectrum, indicating that the δ-lactone ring is attached at C-20. Pyridine is known to form weak hydrogen bonds with OH groups and can influence the chemical shift of neighboring protons.17 These observed chemical shifts are useful in determining the position and orientation of OH groups.17 Differences of the 1H NMR chemical shifts (Δ = δ CDCl3 − δ C5D5N) (Table 2) of the Table 2. Pyridine-Induced Shifts of H-18, H-21, and H-22 in Compounds 1 and 2 (400 MHz) compound

H

1

H18 H21 H22 H18 H21 H22

2

δH C5D5N (J in Hz)

δH CDCl3 (J in Hz)

Figure 3. ORTEP drawing of compound 1 using 15% probability ellipsoids at room temperature.

δH CDCl3 − δH C5D5N (J in Hz)

1.24 s

0.87 s

−0.37

1.58 s

1.22 s

−0.36

5.10 dd (13.0, 3.2) 1.24 s

4.59 dd (3.56, 12.8) 1.21 s

−0.53

1.58 s

1.26 s

−0.32

5.21 dd (3.6, 13)

4.70 dd (3.56, 13.5)

−0.53

lactone system, exhibited a positive Cotton effect near 245 nm, supporting the 22R configuration. This inference was supported by study of another withanolide (withametelin) that has a positive Cotton effect near 250 nm and was shown to have the C-22R configuration.20 NOESY correlations between OH-17/ CH3-21 and CH3-21/H-22 support OH-17 having the αorientation (Figure 2). The H that is bonded to C22 is on the same side as the OH bonded to C17, supporting the 22R configuration. These data led to the structure (20S,22R)17,20,22,27-tetrahydroxy-1-oxo-ergosta-2,5,14,24-tetraen-26-oic acid-22,26-lactone, and it was named withacoagulin G. Compound 2 had a molecular ion at m/z 491.2469 [M + Na]+ (calcd for C28H36O6Na, 491.2431) in the HRESIMS. The IR spectrum showed the presence of OH (3370 cm−1), α,βunsaturated δ-lactone (1715 cm−1), and carbonyl (1690 cm−1) groups. The 1H and 13C NMR data of 2 (Table 1) exhibited characteristic signals for a withanolide skeleton. The 1H NMR data for 1 and 2 were almost identical, with the following exceptions: signals for a pair of methylene protons at δH 1.06− 1.44 (2H, m, H-15) in 1 were absent; instead, the methylene protons were replaced with an olefinic proton for H-15 of ring D in 2. The 13C NMR spectrum of 2 exhibited signals for an olefinic carbon at δC 117.6 (C-15) and a quaternary carbon at δC 150.5 (C-14) but lacked resonances for a methylene group at δC 23.9 (C-15) and a methine at δC 52.0 (C-14) as compared

−0.03

most diagnostic signals for compound 1 were found for H-22 (Δ = −0.53), CH3-18 (Δ = −0.37), and CH3-21 (Δ = −0.36). A larger pyridine-induced shift of H-22 and a moderate induced shift of CH3-21 were also observed.18 The OH group configurations were inferred by a comparison with philadelphicalactones A and B18 and iso-withanone,19 which have pyridineinduced shifts of H-22 and CH3-21, which were shown to have the OH-17 configuration18,19 consistent with the α-orientation of OH-17 in 1. X-ray diffraction analysis of compound 1 confirmed its structure and relative configuration. The OH bonded to C17 is on the opposite side of the ring to the methyl C-18 group, as shown by the ORTEP drawing in Figure 3. Compound 1, a withanolide bearing an α,β-unsaturated δ-

Figure 2. Key NOESY correlations of compounds 1 and 3. 24

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Table 3. Results of Cancer Chemopreventive Assays of Purified Compounds from W. coagulansa nitrite assay (NO) compound

% inhib.b

1 2 3 4 5 6 7 8 9 e L-NMMA f TPCK BAY-11f

100 100 69.8 95.1 84.6 69.1 76.3 31.4 88.5

± ± ± ± ± ± ± ± ±

6.1 1.1 0.4 5.7 1.7 1.3 3.4 4.8 1.4

IC50 (μM) 1.9 3.1 29.0 6.9 10.9 10.4 38.2

± ± ± ± ± ± ±

0.1 0.3 0.7 1.5 2.8 2.1 1.4

37.5 ± 1.1 25.1 ± 2.3

NF-κB % surv.c 96.7 70.4 100 100 100 100 100 100 100

± ± ± ± ± ± ± ± ±

% inhib.d

6.5 7.9 5.6 5.2 6.1 4.8 11.6 13.5 10.3

90.1 98.3 89.9 90.1 90.8 90.2 88.6 92.8 86.9

± ± ± ± ± ± ± ± ±

2.9 6.2 2.8 3.4 5.3 4.3 10.3 8.0 9.02

IC50 (μM) 11.8 5.0 8.8 1.6 5.7 5.7 12.4 7.0 12.3

± ± ± ± ± ± ± ± ±

2.9 1.2 3.1 1.0 0.92 0.29 1.8 0.95 2.5

% surv.c 93.2 90.3 89.4 88.6 100 100 48.3 100 71.9

± ± ± ± ± ± ± ± ±

5.5 11 9 2.2 9 16 4.3 3.8 8.3

10.8 ± 1.7 5.0 ± 0.9

a The NF-κB assay was performed two times in duplicate. To determine IC50 for NF-κB inhibition, dose dependence experiments were performed at the highest compound concentration of 20 μM and then diluted 7-fold and tested three times each. IC50 values were calculated by using Table Curve 2D Windows version 4.07 (SPSS Inc., Chicago, IL, USA). b% inhibition of NO production at 20 μg/mL. c% cell survival at a concentration of 20 μM. d% inhibition of NF-κB production at 20 μM. ePositive control for NO. fPositive control for NF-κB.

to 1. The configuration at C-22 was determined to be R based on Cotton effects in the circular dichroism spectrum. Compound 2, a withanolide bearing an α,β-unsaturated δlactone system, exhibited a positive Cotton effect near 250 nm, supporting the 22R configuration. This inference was supported by study of another withanolide (withametelin) that has a positive Cotton effect near 250 nm and was shown to have the C-22R configuration.20 Using the pyridine effect on OH signals in the proton NMR described previously, we found the following shift differences (Δ = δ CDCl3 − δ C5D5N) for the most diagnostic signals of compound 2 (Table 2) to be at H-22 (Δ = −0.53), CH3-18 (Δ = −0.03), and CH3-21 (Δ = −0.32) for compound 2. There was also a large pyridine-induced shift of H-22 and a moderate induced shift of CH3-21.18 The minor chemical induced shift of CH3-18 might be due to the presence of the olefinic proton at C-15, thus supporting the α-orientation for OH-17 in 2. Thus, the structure of 2 was (20S,22R)17,20,22,27-tetrahydroxy-1-oxo-ergosta-2,5,14,24-tetraen-26-oic acid-22,26-lactone, and it was named withacoagulin H. Compound 3 had the molecular formula C 28 H 38 O 6 (HRESIMS). The UV spectrum showed an absorption maximum at 225 nm, typical of an unsaturated lactone. In the IR spectrum of compound 3, absorption bands for OH group(s) and a carbonyl function were observed at 3370 and 1730 cm−1, respectively. The 1H and 13C NMR spectra of 3 (Table 1) displayed the characteristic proton and carbon signals for five methyl groups and three olefinic groups. The 13C NMR and DEPT spectra of 3 disclosed seven methylenes, three methines, and three oxygenated quaternary carbons at δC 87.3 (C-17), 77.4 (C-20), and 83.3 (C-14), a downfield shifted carbonyl at δC 210.6 (C-1), and a lactone carbonyl carbon at δC 166.0 (C-26). In the HMBC spectrum of 3, olefinic protons at δH 5.65 (m, H-3) and 6.08 (d, H-4) exhibited correlation peaks with a carbonyl carbon at δC 210.6 (C-1) and an olefinic carbon at δC 127.9 (C-6), respectively, which were assigned to the C3−C-4 vinylic protons. A methyl proton that resonated at δH 1.77 (H-27) showed correlation peaks with the C-26 lactone carbon and an olefinic quaternary carbon (C-23), indicating that a methyl group was at the C-25 olefinic carbon instead of the oxygenated methylene group of 2. In addition, three OH groups resonated at δH 5.53, 4.42, and 6.32 in the 1H NMR and are attached to C-14, C-17, and C-20, respectively. These

assignments were confirmed by extensive HMBC analysis (Figure 1). The configurations of the C-14 and C-17 OH groups were deduced from a NOESY spectrum. The OH-14 was β-oriented, as confirmed by NOEs correlations of OH-14 with CH3-18, and OH-20 with CH3-18, while the OH-17 configuration was indicated by NOE correlations of OH-20 with CH3-18, and CH3-21 with CH3-18 (Figure 2). Thus, compound 3 was (14S,17R,20S,22R)-14,17,20,22-tetrahydroxy1-oxo-ergosta-2,5,24-trien-26-oic acid-22,26-lactone, and given the trivial name withacoagulin I. The absolute configurations of the eight stereogenic C atoms of 1 could not be determined directly from the X-ray crystal structure analysis. However, the absolute configuration of C22 for these compounds is known to be R, as discussed previously, which happens to be the same for the enantiomeric structure refined in the crystal structure analysis and shown in Figure 3. Thus, in accordance with the ORTEP drawing in Figure 3, the absolute configuration of C20, which is opposite to that of C22, must be S for compounds 1−3. Compounds 1−9 were evaluated for their inhibitory activities in two cancer chemoprevention assays (Table 3), with percent inhibition at a concentration of 20 μg/mL and the results reported as IC50 values. The inhibition of NO production in LPS-activated murine macrophage RAW 264.7 cells was determined as an indirect marker to monitor iNOS activity. Nitric oxide, the molecular messenger and component of the inflammatory response, is synthesized endogenously from Larginine by three isoforms of NO synthase (NOS). These three isoforms are endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). Inducible nitric oxide synthase is the isoform most consistently associated with chronic inflammation and tumor production.21 Hussain et al.22 gave genetic and mechanistic evidence to prove that an inflammatory microenvironment and an increased level of NO can accelerate tumor development. A consistent relationship between up-regulation of iNOS and cancers of the prostate, bladder, ovary, oral cavity, and esophagus has been observed. Moreover, overexpression appears to occur during early tumor development in these organs, suggesting the role of iNOS inhibitors as cancer chemopreventive agents.21,23 Animal studies have also shown the role of iNOS in the promotion of colon carcinogenesis and the chemopreventive effects of 25

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extracted with n-hexane (3 × 4 L), EtOAc (3 × 4 L), and n-butanol (3 × 4 L) successively. The EtOAc extracts (125 g) were subjected to silica gel column chromatography (CC; ⌀ 15 cm; 230−400 mesh, 2.5 kg) using a gradient of hexane−acetone (8:2 to 1:1), to afford 19 fractions (E1−E19). Fractions E6, E9, E13, and E18 were the most active in both cancer chemopreventive mechanism assays. Fraction E13 (7 g) was subjected to silica gel CC (⌀ 6 cm; 230−400 mesh, 800 g), with hexane−EtOAc (5:1 to 0:1), yielding 15 subfractions (E13.1 to E13.15). Compounds 1 (5 mg) and 2 (6 mg) were crystallized in MeOH−CHCl3 (1:1) from the subfractions E13.7 and E13.10, respectively. Subfraction E13.8 (0.5 g) was subjected to preparative HPLC (MeOH−H2O = 30:70 to 100:0) to yield 7 (3 mg). Fraction E9 (7 g) was subjected to silica gel CC (⌀ 6 cm; 230−400 mesh, 500 g), with hexane−EtOAc (5:1 to 0:1), yielding 12 subfractions (E9.1 to E9.12). Compound 3 (3 mg) was crystallized from MeOH−CHCl3 (1:1) from the subfraction E9.5. Fraction E6 (5 g) was subjected to silica gel CC (⌀ 6 cm; 230−400 mesh, 500 g), with hexane−EtOAc (9:1 to 2:1), yielding 10 subfractions (E6.1 to E6.10). Compound 4 (3 mg) was crystallized in MeOH−CHCl3 (1:1) from the subfraction E9.5. Subfraction E6.7 (0.3 g) was subjected to preparative HPLC (MeOH−H2O = 30:70 to 100: 0) to yield 6 (10 mg) and 8 (9.5 mg). Fraction E18 (10 g) was subjected to silica gel CC (⌀ 10 cm; 230−400 mesh, 1000 g), with hexane−EtOAc (5:1 to 0:1) as the solvent system, yielding 15 subfractions (E18.1 to E18.15). Subfraction E18.5 (0.8 g) was subjected to preparative HPLC (MeOH−H2O = 20:80 to 100:0) to yield 5 (6 mg) and 9 (11 mg). Withacoagulin G (1): colorless needles; UV (MeOH) λmax (log ε) 226 (3.7) nm; IR νmax (KBr) 3402, 1720, 1687 cm−1; CD (c 0.01, MeOH) 245 (+37.3); 1H (400 MHz) and 13C NMR (100 MHz) data, see Table 1; HRESIMS m/z 493.2630 [M + Na]+ (calcd for C28H38O6Na, 493.2575). Withacoagulin H (2): white, amorphous powder; UV (MeOH) λmax (log ε) 225 (3.9) nm; IR νmax (KBr) 3370, 1715, 1690 cm−1; CD (c 0.01, MeOH) 250 (+35.2); 1H (400 MHz) and 13C NMR (100 MHz) data, see Table 1; HRESIMS m/z 491.2469 [M + Na]+ (calcd for C28H36O6Na, 491.2431). Withacoagulin I (3): white, amorphous powder; UV (MeOH) λmax (log ε) 225 (4.2) nm; IR νmax (KBr) 3370, 1730 cm−1; CD (c 0.01, MeOH) 254 (+36.1); 1H (400 MHz) and 13C NMR (100 MHz) data, see Table 1; HRESIMS m/z 493.2563 [M + Na]+ (calcd for C28H38O6Na, 493.2563). X-ray Crystal Structure Determination of Withacoagulin G (1). A crystal of compound 1 in the form of a flat plate of dimensions 0.35 × 0.35 × 0.10 mm was mounted on a glass fiber. X-ray data were collected at 298 K with a Nonius Kappa CCD diffractometer to a maximum 2θ value of 58.3° using monochromatized Mo Kα radiation; 5798 reflections were collected and 3425 were unique. Crystal data for 1: C28H38O6, Mr = 470.6, orthorhombic space group P212121, a = 5.917(5) Å, b = 18.7995(14) Å, c = 22.8056(16) Å, V = 2536.8 Å3, Z = 4, ρ = 1.232 g/cm3. The structure was solved by direct methods (SHELXD). All non-H atoms were refined anisotropically, and all H atoms were refined using the riding model. The final cycle of fullmatrix least-squares refinement on F converged at R1 = 0.057, wR2 = 0.051, and GOF = 1.28 for 921 reflections with I > 3σ(I); a robustresistant weighting scheme was used in the least-squares refinements. The final Fourier difference map was featureless; the max./min. peaks were 0.23/−0.25 e−/Å3. Crystallographic data for the structure reported in this paper have been deposited with the Cambridge Crystallographic Data Centre. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-(0)1223-336033 or e-mail: [email protected]); deposition number 905905. Inhibition of Nitric Oxide Production in LipopolysaccharideActivated Murine Macrophage RAW 264.7 Cells (iNOS) Assay. The inhibitory effects of samples on NO production were evaluated in LPS-activated murine macrophage RAW 264.7 cells using a method described previously.30 Briefly, RAW 264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with penicillin G sodium (100 units/mL), streptomycin sulfate (100 μg/ mL), amphotericin B (0.25 μg/mL), and 10% fetal bovine serum

iNOS inhibitors in preclinical colon cancer models. Sharma et al.24 demonstrated the role of iNOS inhibitors as chemopreventive agents in a primary rat tracheal epithelial transformation system, suggesting their use in lung cancer prevention in rodents. Among the withanolides of the current study, the most potent activity was demonstrated by compounds 1 (IC50 1.9 μM) and 2 (IC50 3.1 μM), followed by compounds 4 (IC50 6.9 μM), 6 (IC50 10.4 μM), 5 (IC50 10.9 μM), 3 (IC50 29.0 μM), 9 (IC50 37.5 μM), 7 (IC50 38.2 μM), and 8 (31.4% inhibition at 20 μg/mL). Promotion of cell growth is a necessary feature of all types of cancers and can be attained by abnormally activated or deregulated signaling pathways involved in cell cycle regulation or abnormal growth signals outside the malignant cell. TNF-α is one of the activators of nuclear factor kappa-B, and NF-κB is an inducible transcription factor that plays an important role in the regulation of apoptosis, cell differentiation, and cell migration. Its activation may promote cell proliferation and further prevent programmed cell death through transcriptional activation of genes that suppress apoptosis.26,27 NF-κB is commonly involved as a regulator of genes that control cell proliferation and cell survival. Many different types of human tumors have misregulated NF-κB that is constitutively active. Thus, inhibition of NF-κB signaling has a potential application for the prevention or treatment of cancer.25,28 In the TNF-αactivated NF-κB inhibition assay, compound 4 showed the most potent inhibition among the tested compounds, with an IC50 of 1.6 μM, followed by 2 (IC50 5.0 μM), 5 (IC50 5.7 μM), 6 (IC50 5.7 μM), 8 (IC50 7.0 μM), 3 (IC50 8.8 μM), 1 (IC50 11.8 μM), 9 (IC50 12.3 μM), and 7 (IC50 12.4 μM). Ichikawa et al.29 reported 11 withanolides isolated from W. somnifera that exhibited NF-κB inhibitory activity. To the best of our knowledge, these withanolides from W. somnifera are not found in W. coagulans. All of our isolated withanolides had significant potential in chemoprevention assays, but compounds 1 and 2 showed the best overall results in both assays. These analogues should be studied in more advanced models to establish in vivo efficacy.

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EXPERIMENTAL SECTION

General Experimental Procedures. UV spectra were recorded on a Shimadzu PharmaSpec-1700 UV−visible spectrophotometer. CD spectra were measured on a JASCO J-815 spectropolarimeter. IR spectra were measured on a Bruker Tensor-27 spectrophotometer. 1Dand 2D-NMR spectra were recorded on a Bruker AVANCE (400 MHz) spectrometer. Mass spectra and high-resolution MS spectra were performed with a BioTOF II ESI mass spectrometer. Thin-layer chromatography was performed on silica gel 60 F254 (0.25 mm, Merck, Germany). Silica gel (230−400 mesh, Merck, Germany) and RP-18 (YMC·GEL ODS-A, 12 nm, S-150 μm) were used for column chromatography. Semipreparative HPLC was conducted on a Beckman Coulter Gold-168 system equipped with a photodiode array detector using an Alltech reversed-phase Econosil C-18 column (10 μm, 10 × 250 mm) with a flow rate of 2.0 mL/min. Plant Material. W. coagulans was collected from Karapa (Banda Daood Shah) District Karak, Khyber Pakhtonkhwa, Pakistan, in July 2010. Plant identification was confirmed by Dr. Rizwana A. Qureshi, Department of Plant Sciences, Quaid-i-Azam University, Islamabad. A voucher specimen (HMP-459) was also deposited in the “Herbarium of Medicinal Plants of Pakistan”, Quaid-i-Azam University, Islamabad, Pakistan. Extraction and Isolation. The dried aerial parts of W. coagulans (10 kg) were extracted with CHCl3−MeOH (1:1, 30 L) at room temperature. The solvent was concentrated in vacuo to yield a crude extract (700 g), which was then suspended in distilled water (4 L) and 26

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(FBS). The cells were seeded in 96-well culture plates with 1 × 105 cells/well and allowed to adhere for 24 h at 37 °C in a humidified atmosphere containing 5% CO2. The cells were treated with samples dissolved in phenol red-free DMEM for 30 min followed by 1 μg/mL of LPS treatment for 20 h. The concentration of NO in the cultured medium was measured by using Griess reagent [90 μL of 1% sulfanilamide and 90 μL of 0.1% N-(1-naphthyl)ethylenediamine in 2.5% H3PO4 in each well]. The standard curve was created by using known concentrations of sodium nitrite, and the absorbance was measured at 540 nm. To evaluate the cytotoxic effect of samples in RAW 264.7 cells under assay conditions, a sulforhodamine B (SRB) assay was performed. Briefly, after fixation with 10% trichloroacetic acid (TCA), cells were stained with 0.4% SRB solution in 1% acetic acid followed by dissolving bound SRB in 10 mM Tris-buffer. The optical density was determined at 515 nm. L-NG-Monomethyl arginine citrate (L-NMMA), as a positive control of this assay, showed an IC50 value of 25.1 μM. Tumor Necrosis Factor-α Activated Nuclear Factor-Kappa B Assay. Stable transfected human embryonic kidney cells 293 (Panomics, Fremont, CA, USA) were used for monitoring changes occurring along the NF-κB pathway.31 According to the product information insert, the 293/NFκB-luc cell line was obtained by cotransfection of pNFκB-luc (Panomics P/N LR0051) and pHyg into human embryonic kidney 293 cells, followed by hygromycin selection. TNF-α-induced luciferase activity was used to select clones from the hygromycin-resistant cells. These cells maintain a chromosomal integration of a luciferase reporter construct regulated by multiple copies of the NF-κB response element. These cells were seeded into 96-well plates at 2 × 104 cells per well. Cells were maintained in DMEM (Invitrogen Co.; Carlsbad, CA, USA), supplemented with 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine. After 48 h incubation, the medium was replaced and the cells were treated with various concentrations of test substances. TNF-α (human, recombinant, E. coli, Calbiochem, Gibbstown, NJ, USA) was used as an activator at a concentration of 2 ng/mL (0.14 nM). After a six-hour incubation of cells with tested samples, the reaction was stopped by adding 50 μL of Reporter Lysis Buffer (Promega, Madison, WI, USA) and frozen overnight at −80 °C. The cells were thawed, and the inhibiting activity was measured using a LUMIstar Galaxy luminometer (BMG, Offenburg, Germany) and Luc assay system from Promega (Madison, WI, USA). The gene product, luciferase enzyme, reacts with luciferase substrate, and the emitted light was detected by the luminometer: data were calculated as percent inhibition. The samples that showed more than 70% inhibition at 20 μg/mL were tested at different concentrations to find IC50. To avoid false positive results due to cytotoxic effects on samples, a cytotoxicity assay was run simultaneously. The procedure was the same as above, except that 96 transparent well plates were used instead of 96 whitewalled well plates, and after a six-hour incubation, the cells were treated with 50 μL of 20% TCA and incubated at 4 °C for 30 min. TCA was then removed and the cells were washed four times with tap water. The plates were air-dried overnight, and 100 μL of 0.4% SRB in 1% acetic acid was added to each well for 30 min at room temperature. Wells were then washed four times with 1% acetic acid, and the plates were again air-dried overnight. To each well was added 200 μL of 10 mM Tris base (pH 10), which was mixed for 10 min on a gyratory shaker to solubilize the bound SRB. The optical density was measured using a micro plate reader (Biotek) at 515 nm, and the percent survival was determined. Samples that showed more than 50% inhibition at 20 μg/mL were tested at 3-fold serial dilutions to find IC50. Nα-Tosyl-Lphenylalanine chloromethyl ketone was used as a positive control (IC50 3.8 μM).

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Article

AUTHOR INFORMATION

Corresponding Author

*Tel: 808-981-8018. Fax: 808-933-2974. E-mail: lengchee@ hawaii.edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank P. Williams and J. Dai (University of Hawai’i Manoa Chemistry Department) for assistance with mass spectrometry. This research was supported by the Higher Education Commission Pakistan under the IRSIP program (to I.H.) and NCI Program Project P01 CA48112 (to J.M.P.).

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REFERENCES

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ASSOCIATED CONTENT

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