Bioactive Compounds from Carissa spinarum

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PHYTOTHERAPY RESEARCH Phytother. Res. (2012) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.4607

Bioactive Compounds from Carissa spinarum Ruchira Wangteeraprasert,1 Vimolmas Lipipun,2 Mekala Gunaratnam,3 Stephen Neidle,3 Simon Gibbons4 and Kittisak Likhitwitayawuid1* 1

Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand 2 Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand 3 CRUK Biomolecular Structure Group, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, UK 4 Department of Pharmaceutical and Biological Chemistry, The School of Pharmacy, University of London, London WC1N 1AX, UK

In our continuing efforts to find new antiherpetic agents from plants, an extract prepared from the stems of Carissa spinarum L. was found to possess appreciable activity against herpes simplex viruses (HSV I and II). A chemical study of this plant was then initiated, and this led to the isolation of 12 compounds, including a coumarin, two cardiac glycosides and nine lignans. These isolated compounds were evaluated for several biological activities, including antiherpetic, cytotoxic, antioxidant and antibacterial effects. The cardiac glycoside evomonoside was found to be the only antiherpetic principle, showing moderate activity against herpes simplex virus types I and II in the inactivation method. The lignans ( )-carinol, ( )-carissanol and ( )-nortrachelogenin exhibited cytotoxicity against breast (MCF7) and lung (A549) cancer cells. Moderate anti-DPPH free radical activity was observed for all the lignans. None of the isolates showed antibacterial activity. Copyright © 2012 John Wiley & Sons, Ltd. Keywords: carissa; lignan; cardiac glycoside; herpes simplex; cytotoxic; antioxidant.

INTRODUCTION Carissa spinarum L. (Apocynaceae) has been used in folkloric medicine in several countries. In Thailand, the stem of this plant is used as a restorative agent and a stimulant (Wutthamawech, 1997), whereas in India its roots are used as a purgative and as an antidote to snake-bite (Pakrashi et al., 1968). In Uganda, the bark is reputed to be a veterinary drug for the treatment of chicken pox and skin diseases (Gradé et al., 2009). Previous phytochemical investigation revealed the presence of sterols, terpenes and uncharacterized cardiac glycosides from the roots and bark (Pakrashi et al., 1968), whereas sesquiterpenes, phenolic compounds and lignans were isolated from the stems (Rao et al., 2005). Two lignans including ( )-carinol and ( )-carissanol and the chloroform extract from the stems of this plant were evaluated and showed free radical scavenging activity with DPPH (Rao et al., 2005). In this study, from the stems of C. spinarum, 12 compounds were isolated, and examined for antioxidative, antiherpetic, antibacterial and cytotoxic effects.

MATERIALS AND METHODS Plant material. The stems of Carissa spinarum L. were collected from the botanical garden of the Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand, in June 2008. Authentication was performed by * Correspondence to: Kittisak Likhitwitayawuid, Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand. E-mail: [email protected]

Copyright © 2012 John Wiley & Sons, Ltd.

comparison with herbarium specimens at the Museum of Natural Medicine, Faculty of Pharmaceutical Sciences, Chulalongkorn University. A voucher specimen (RW 062551) has been deposited at the Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University. Instruments. The UV spectra were obtained on a Shimadzu UV-160A UV/Vis spectrophotometer. The ESI-mass spectra were measured with a Micromass Q-TOF Global Tandem mass spectrometer or a Thermo Navigator mass spectrometer. 1H NMR (500 MHz) and 13 C NMR (125 MHz) spectra were obtained with a Bruker AV-500 NMR spectrometer. Optical rotations were measured on a Perkin Elmer Polarimeter 343. Extraction and isolation. The dried stems of C. spinarum (2 kg) were macerated with methanol to give a methanol extract (123 g). Then 55 g of this extract was then divided into 13 portions and each portion was separated on a flash column (C-18; MeOH/H2O 4:6) to yield 5 fractions (A–E). The cumulative fraction A was bulked (37.8 g) and was re-fractionated on a Sephadex LH-20 (MeOH) column to afford six fractions (A1–A6). Fraction A3 (16 g) was further chromatographed on SiO2 (CH2Cl2/MeOH gradient) to give eight fractions (A3A–A3H). Fraction A3C (326 mg) was purified by preparative-TLC (CH2Cl2-MeOH = 9.8:0.2) to give scopoletin (1) (15 mg) and ( )-nortrachelogenin (2) (173 mg). Fraction A3D (396.6 mg) was further purified on a SiO2 column (CH2Cl2/MeOH gradient) to give ( )-carissanol (3) (81 mg). Fraction A3E (191 mg) was separated by preparative-TLC (SiO2; CH2Cl2/MeOH 9.6:0.4) to give ( )-carinol (4) (103 mg), while fraction A3F (243 mg) Received 14 October 2011 Revised 08 December 2011 Accepted 16 December 2011

R. WANGTEERAPRASERT ET AL.

was purified on a SiO2 column (CH2Cl2/MeOH gradient) to yield (+)-cycloolivil (5) (28 mg). Fraction B (2.8 g) was fractionated on Sephadex LH20 (MeOH) to afford seven fractions (B1–B7). Fraction B3 (1.3 g) was re-chromatographed on a solid phase extraction (SPE) column (SiO2; CH2Cl2/MeOH gradient) to give eight fractions (B3A–B3H). Fraction B3C (320 mg) was further separated by preparative-TLC (SiO2; CH2Cl2/MeOH 9.6:0.4) to afford (+)-8-hydroxypinoresinol (6) (6 mg). Fraction B3G (193 mg) was purified by preparative-TLC (SiO2, CH2Cl2/acetone/MeOH 10:1:0.2) to afford ( )-olivil (7) (18 mg). Separation of fraction C (1.1 g) on Sephadex LH-20 (MeOH) yielded seven fractions (C1–C7). Fraction C3 (120 mg) was further separated on an SPE column (SiO2, CH2Cl2/MeOH gradient) to give six fractions (C3A–C3F). Separation of fraction C3C (25 mg) by preparative-TLC (SiO2; CH2Cl2/MeOH 9.4:0.6) yielded ( )-secoisolariciresinol (8) (2 mg). Fraction D (800 mg) was fractionated by vacuum-liquid column chromatography (VLC) on SiO2, (CH2Cl2/ MeOH gradient) to give 13 factions (D1–D13). Fraction D4 (63 mg) was further purified by preparative-TLC (SiO2; CH2Cl2/MeOH 9.6:0.4) to give (+)-pinoresinol (9) (9 mg). Finally, fraction E (9 g) was fractionated by VLC on SiO2, (CH2Cl2/MeOH gradient) to give eight fractions (E1–E8). Fraction E2 (928 mg) was re-chromatographed on an SPE column (SiO2; hexane/CH2Cl2/acetone gradient) to afford eight fractions (E2A–E2H). Fraction E2F (271 mg) was further purified by preparative-TLC (SiO2; CH2Cl2/MeOH 9.8:0.2) to give carissone (10) (76 mg). Fraction E3 (1.2 g) was fractionated on Sephadex LH-20 (CHCl3/MeOH gradient) to give nine fractions (E3A–E3I). Fraction E3D (212 mg) was further separated on an SPE column (SiO2, hexane/EtOAc gradient) to yield digitoxigenin-3-O-b-D-digitalopyranoside (11) (7 mg). Separation of fraction E5 (229 mg) on Sephadex LH-20 (CH2Cl2/MeOH gradient) gave evomonoside (12) (12 mg). Determination of antiherpes simplex activity. Anti-HSV activity of the isolated compounds was determined by the modified plaque reduction assay (Chuanasa et al., 2008; Lipipun et al., 2003). Determination of free radical scavenging activity. Assays for free radical scavenging activity were conducted in duplicate using 96-well microtiter plates according to a previously described assay (Likhitwitayawuid et al., 2006). Determination of antibacterial activity. In this study, six strains of Staphylococcus aureus were used, including ATCC 25923 (S. aureus standard strain), SA1199B (S. aureus that over-expresses the norA gene encoding the NorA MDR efflux pump), XU212 (S. aureus tetracyclineresistant strain), RN4220 (S. aureus that possesses the MsrA macrolide efflux protein), and EMRSA-15 and EMRSA-16 (S. aureus methicillin-resistant strains) (Shiu and Gibbons, 2006). Determination of cytotoxic activity. Three cell lines were used in this study, including two human cancer cell lines, breast (MCF7) and lung (A549), and a normal Copyright © 2012 John Wiley & Sons, Ltd.

human cell line (WI-38, lung fibroblast) (Gunaratnam et al., 2007).

RESULTS AND DISCUSSION From the stems of C. spinarum, 12 compounds namely scopoletin (1) (Vasconcelos et al., 1998), ( )-nortrachelogenin (2) (Achenbach et al., 1983; Lin et al., 1999), ( )carissanol (3) (Khamlach et al., 1990), ( )-carinol (4) (Khamlach et al., 1990), (+)-cycloolivil (5) (Ghogomu-Tih et al., 1985), (+)-8-hydroxypinoresinol (6) (Yeo et al., 2004), ( )-olivil (7) (Yeo et al., 2004), ( )-secoisolariciresinol (8) (Xie et al., 2003), (+)-pinoresinol (9) (Xie et al., 2003), carissone (10) (Achenbach et al., 1985), digitoxigenin-3-Ob-D-digitalopyranoside (11) (Cabrera et al., 1993) and evomonoside (12) (Hyun et al., 1995) were isolated and identified by comparison of their spectral data with reported values (Fig. 1). Compounds 1, 5, 6 and 10–12 were not found previously in this plant (Pakrashi et al., 1968; Rao et al., 2005). All compounds were evaluated for their activity in the anti H. simplex assay, and only evomonoside (12) was active, exhibiting more than 50% inhibition at 100 mg/mL without toxicity against the Vero cell line. In the inactivation method, the IC50 values against HSV-1 and HSV-2 were calculated to be 120.2 and 168.3 mM, respectively, and the IC50 value for acyclovir against HSV-1 was 2.8 mM. This is the first report for the antiherpetic activity of 12. The antiherpetic activity observed in this study correlates with the ethnoveterinary use of this plant for treating skin diseases (Pakrashi et al., 1968). Compounds were also evaluated for their ability to scavenge DPPH free radicals at an initial concentration of 100 mg/mL. Compounds causing more than 50% inhibition were further analysed for their IC50 values, and the results are summarized in Table 1. The lignans ( )nortrachelogenin (2), ( )-carissanol (3), ( )-carinol (4), (+)-cycloolivil (5), (+)-8-hydroxypinoresinol (6), ( )-olivil (7), ( )-secoisolariciresinol (8) and (+)-pinoresinol (9) showed moderate activity, in agreement with earlier reports (Rao et al., 2005; Tiwari et al., 2001; Pérez-Bonilla et al., 2006; Lee et al., 1998; Hu et al., 2007; Kuo et al., 2011). It is possible that these lignans are responsible for the restorative effects of the plant via their antioxidant properties, whereas the glycosides of digitoxigenin (11 and 12) act as stimulating agents through cardiostimulant effects. Due to the limited amount of each sample, only five compounds namely scopoletin (1), ( )-nortrachelogenin (2), ( )-carissanol (3), ( )-carinol (4) and carissone (10) were investigated for their activity against the S. aureus strains. However, none of these showed antibacterial activity at a concentration of 128 mg/mL. As for the cytotoxicity of the isolates, the most active compound was ( )-carinol (4) (IC50 value 100 mg/mL against A549, MCF7 and WI-38 cells, respectively), and this might imply that the absence Phytother. Res. (2012)

BIOACTIVE COMPOUNDS FROM CARISSA SPINARUM

Figure 1. Structures of compounds isolated from C. spinarum.

Table 1. IC50 values for free radical scavenging activity of compounds from C. spinarum Compound Scopoletin (1) ( )-Nortrachelogenin (2) ( )-Carissanol (3) ( )-Carinol (4) (+)-Cycloolivil (5) (+)-8-Hydroxypinoresinol (6) ( )-Olivil (7) ( )-Secoisolariciresinol (8) (+)-Pinoresinol (9) Carissone (10) Digitoxigenin 3-o-b-D-digitalopyranoside (11) Evomonoside (12) Quercetin

IC50 (mM) nd 35.8 33.4 20.2 33.2 69.5 18.1 26.2 43.4 nd nd nd 4.6

nd, not determined, due to weak activity (< 50% inhibition at 100 mg/mL).

of the hydroxyl group at C-9′ decreases the cytotoxic activity. It should be mentioned that the cytotoxicity data for these lignans were obtained for the first time in this investigation, and they help to support a recent finding Copyright © 2012 John Wiley & Sons, Ltd.

on the cytotoxic potential of C. spinarum stem extract against human leukaemia HL-60 cells (Sehar et al., 2011). In addition, the presence of several lignans in this plant explains its claimed use as a purgative since some lignans are known to have purgative activity, for example, podophyllotoxin from Podophyllum, and elenoside from Justicia hyssopifolia (Navarro et al., 2006). However, the effects of each of these isolates on the intestinal mobility remain to be investigated before any conclusion can be drawn. To conclude, 12 known compounds were isolated from the stems of Carissa spinarum, and some of the isolated compounds displayed biological activities. Eight lignans showed moderate free radical scavenging activity, whereas the cardiac glycoside evomonoside showed antiherpes simplex virus activity. The free radical scavenging activity observed for the isolates appears to lend supporting evidence to its traditional use as a restorative agent, whereas the antiherpetic activity of evomonoside agrees with the use of the stem of this plant for treating skin diseases in animals. It could be speculated that the ethnomedical use of C. spinarum as a stimulant may have come from the pharmacological properties of the cardiac glycosides present in the stems. Finally, some lignans were found Phytother. Res. (2012)

R. WANGTEERAPRASERT ET AL.

to exhibit moderate cytotoxic activity, and this is consistent with the results obtained from a recent study on an extract prepared from C. spinarum stems.

Acknowledgements

University for partial financial support through the 90th Anniversary Chulalongkorn University (Ratchadaphiseksomphot) Endowment Fund.

Conflict of Interest

R.W. is grateful to the Thailand Research Fund for a 2005 Royal Golden Jubilee Scholarship (PHD/0032/2548). We thank Chulalongkorn

The authors have declared that there is no conflict of interest.

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