Naresuan University Journal: Science and Technology 2017; (25)3 Chemical Constituents of Fruits and Leaves of Cratoxylum cochinchinense and Their Cytotoxic Activities Benjamat Chailap1, Thanesuan Nuanyai1*, Songchan Puthong2 and Anumart Buakeaw2 1Faculty of Liberal Arts, Rajamangala University of Technology Rattanakosin, Wang Klai Kangwon Campus, Nongkae, Huahin, Prachuap Khiri Khan, 77110, Thailand 2The Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Wang Mai, Pathumwan, Bangkok, 10330, Thailand * Corresponding author. E-mail address: [email protected] Received: 5 August 2016; Accepted: 12 September 2016 Abstract Seven compounds; Vismiaquinone A (1), 7-geranyloxy-1,3-dihydroxyxanthone (2), Cochinchinone G (3), Fuscaxanthone E (4), -tocotrienol (5), -tocotrienol (6), and -tocopherol (7) were isolated from fruits and leaves of Cratoxylum cochinchinense. The structures of all isolated compounds were elucidated by basic NMR spectroscopy (1H, 13C, COSY, HSQC and δ HMBC) and compared with previous literatures. Interestingly, -tocotrienol (5) and -tocopherol (7) were the first time being isolated from C. cochinchinense. The chemical constituents of C. cochinchinense such as -tocotrienol (5), -tocotrienol (6), and -tocopherol (7) are vitamin E derivatives that have been used for cosmetic ingredients. Thus, this plant extract should be δ promising probability for applying to cosmetic ingredients. Furthermore, all isolated compounds were also tested for in vitro cytotoxic activity against five human cancer cell lines; breast (BT474), lung (ChaGo-K-1), liver (HepG2), gastric (KATO-III), and colon (SW-620) cancer cell lines by MTT assay method. The cytotoxic activity of compound 3 against SW-620 (colon cancer cell line, IC50 4.64 g/mL) was found to be stronger than other compounds. Keywords: Cratoxylum cochinchinense, cytotoxicity, xanthone, tocotrienol, vitamin E derivatives, structure elucidation Introduction tropical tree was wildly spread in Thailand and commonly known as “Tui Kliang”. Several parts of Plants of the genus Cratoxylum were reported to be this plant have been treated for relieving many a rich source of xanthones with interesting biological symptoms such as fever, cough, diarrhea, itches, and activities such as cytotoxic (Boonnak, et al., 2014: ulcers (Vo, 1997). From the previous reports, Rattanaburi, Daus, Watanapokasin, & Mahabusarakam, xanthones (Udomchotphruet, Phuwapraisirisan, 2014), antioxidant (Sim, Ee, Lim, & Sukari, 2011), Sichem, & Tip-Pyang, 2012; Duan et al., 2015; antimalarial (Laphookhieo, Maneerat, & Koysomboon, Duan et al., 2012, Nguyen et al., 2011, Jin, Wang, 2009; Laphookhieo, Syers, Kiattansakul, & Zhang, Dai, & Yao, 2009), phenolic (Laphookhieo, Chantrapromma, 2006), antibacterial (Raksat, Maneerat, & Koysomboon, 2009), and triterpenoids Laphookhieo, Cheenpracha, Ritthiwigrom, & (Bennett, Harrison, Sia, & Sim, 1993; Nguyen & Maneerat,2014; Boonnak, et al., 2010), anti- Harrison, 1999) compounds have been isolated from inflammatory (Boonnak, Chantrapromma, Tewtrakul, this species. Accordingly, xanthone and anthraquinone & Sudsai, 2014; Xiong, et al., 2014), and anti-HIV were isolated from twigs of C. cochinchinense in our activities (Reutrakul, et al., 2006). Cratoxylum previous study. Moreover, cytotoxicities of several cochinchinenes (Lour.) Bl. (Hypericaceae), the compounds were investigated. It was found that the 22 Naresuan University Journal: Science and Technology 2017; (25)3 Chemical Constituents of Fruits and Leaves of Cratoxylum cochinchinense isolated compounds exhibited broad cytotoxic activities leaves of C. cochinchinense were reported. All and Their Cytotoxic Activities against five human cancer cell lines (Nuanyai, Chailap, structures of isolated compounds were established by 1 1 2 2 Puthong, & Buakeaw, 2015). In this study, the 1D and 2D NMR spectroscopy and compared with Benjamat Chailap , Thanesuan Nuanyai *, Songchan Puthong and Anumart Buakeaw isolation and cytotoxic activity of seven isolated previous reports. Interestingly, -tocotrienol (5) and compounds, Vismiaquinone A (1), 7-geranyloxy- -tocopherol (7) were the first report of this species. 1Faculty of Liberal Arts, Rajamangala University of Technology Rattanakosin, Wang Klai Kangwon Campus, Nongkae, Huahin, 1,3-dihydroxyxanthone (2), Cochinchinone G (3), In addition, the cytotoxicities against five human Prachuap Khiri Khan, 77110, Thailand 2The Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Wang Mai, Pathumwan, Bangkok, 10330, Fuscaxanthone E (4), -tocotrienol (5), - cancer cell lines of these compounds were also Thailand tocotrienol (6), and -tocopherol (7) from fruits and reported. δ * Corresponding author. E-mail address: [email protected] Received: 5 August 2016; Accepted: 12 September 2016 Abstract Seven compounds; Vismiaquinone A (1), 7-geranyloxy-1,3-dihydroxyxanthone (2), Cochinchinone G (3), Fuscaxanthone E (4), -tocotrienol (5), -tocotrienol (6), and -tocopherol (7) were isolated from fruits and leaves of Cratoxylum cochinchinense. The structures of all isolated compounds were elucidated by basic NMR spectroscopy (1H, 13C, COSY, HSQC and δ HMBC) and compared with previous literatures. Interestingly, -tocotrienol (5) and -tocopherol (7) were the first time being isolated from C. cochinchinense. The chemical constituents of C. cochinchinense such as -tocotrienol (5), -tocotrienol (6), and -tocopherol (7) are vitamin E derivatives that have been used for cosmetic ingredients. Thus, this plant extract should be δ promising probability for applying to cosmetic ingredients. Furthermore, all isolated compounds were also tested for in vitro cytotoxic activity against five human cancer cell lines; breast (BT474), lung (ChaGo-K-1), liver (HepG2), gastric (KATO-III), and colon (SW-620) cancer cell lines by MTT assay method. The cytotoxic activity of compound 3 against SW-620 (colon cancer cell line, IC50 4.64 g/mL) was found to be stronger than other compounds. Keywords: Cratoxylum cochinchinense, cytotoxicity, xanthone, tocotrienol, vitamin E derivatives, structure elucidation Introduction tropical tree was wildly spread in Thailand and commonly known as “Tui Kliang”. Several parts of Plants of the genus Cratoxylum were reported to be this plant have been treated for relieving many Figure 1 Isolated compounds from fruits and leaves of C. cochinchinense. a rich source of xanthones with interesting biological symptoms such as fever, cough, diarrhea, itches, and activities such as cytotoxic (Boonnak, et al., 2014: ulcers (Vo, 1997). From the previous reports, Methods and Materials Plant materials Rattanaburi, Daus, Watanapokasin, & Mahabusarakam, xanthones (Udomchotphruet, Phuwapraisirisan, The fruits and leaves of C. cochinchinense were 2014), antioxidant (Sim, Ee, Lim, & Sukari, 2011), Sichem, & Tip-Pyang, 2012; Duan et al., 2015; General experiment procedures collected in October 2013 from Nongkae, Huahin antimalarial (Laphookhieo, Maneerat, & Koysomboon, Duan et al., 2012, Nguyen et al., 2011, Jin, Wang, NMR spectra were recorded in CDCl3 and DMSO- Prachuap Khiri Khan, Thailand and stored at room Zhang, Dai, & Yao, 2009), phenolic (Laphookhieo, d6 on Bruker AV400 spectrometers at 400 MHz for temperature. The voucher specimen (No. 190330) has 2009; Laphookhieo, Syers, Kiattansakul, & 1 13 Chantrapromma, 2006), antibacterial (Raksat, Maneerat, & Koysomboon, 2009), and triterpenoids H NMR and 100 MHz for C NMR using TMS been deposited at the Forest Herbarium, Royal Forest Laphookhieo, Cheenpracha, Ritthiwigrom, & (Bennett, Harrison, Sia, & Sim, 1993; Nguyen & (tetramethylsilane) as internal standard. Merck’s silica Department, Bangkok, Thailand. Maneerat,2014; Boonnak, et al., 2010), anti- Harrison, 1999) compounds have been isolated from gel 60 No.7734 was used as adsorbents for column Extraction and isolation inflammatory (Boonnak, Chantrapromma, Tewtrakul, this species. Accordingly, xanthone and anthraquinone chromatography. Merck’s thin layer chromatography Fresh fruits of C. cochinchinense (0.5 kg) were & Sudsai, 2014; Xiong, et al., 2014), and anti-HIV were isolated from twigs of C. cochinchinense in our (TLC) aluminum, silica gel 60 F254 precoated, 20x20 ground and macerated with methanol (2.0 L for 3 activities (Reutrakul, et al., 2006). Cratoxylum previous study. Moreover, cytotoxicities of several cm, layer thickness 0.2 mm were used for TLC days) at ambient temperature. The methanol extract cochinchinenes (Lour.) Bl. (Hypericaceae), the compounds were investigated. It was found that the analysis. The detection was visualized under ultraviolet solution was dried in vacuo, and extracted with ethyl light at the wavelength of 254 nm and dipped with acetate and water. The crude extract of ethyl acetate (NH4)6Mo7O24 in 5% H2SO4 solution. (7.15 g) were separated on a silica gel column 23 Naresuan University Journal: Science and Technology 2017; (25)3 chromatography (CC) and eluted with a gradient of 8), 162.7 (C-1), 162.0 (C-3), 133.7 (C-10a), hexane-ethyl acetate (9:1 to 0:10) to afford four 133.5x2 (C-4a, C-6), 126.4 (C-3ʹ), 124.8 (C- fractions (A-D). Fraction A (500.0 mg) was 2), 124.4 (C-7), 121.6 (C-2ʹ), 121.1 (C-5), chromatographed over silica gel CC and eluted with 114.0 (C-8a), 111.3 (C-9a), 103.7 (C-10a), gradient hexane-acetone (9:1 to 8:2) to afford 56.2 (OCH3-3), 22.2 (C-1ʹ), 22.1x2 (C-4´, C- compound 1 (50.0 mg) and 5 (125.0 mg). Fraction 5´), 17.8 (CH3-6). B (440.0 mg) was separated by silica gel CC and 7-geranyloxy-1,3-dihydroxyxanthone(2).Yellow