Anti-inflammatory sesquiterpenes from Costus speciosus rhizomes

Journal of Ethnopharmacology 176 (2015) 365–374

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Anti-inflammatory sesquiterpenes from Costus speciosus rhizomes Ahmed A.M. Al-Attas a, Nagwa S. El-Shaer a,b, Gamal A. Mohamed a,c, Sabrin R.M. Ibrahim d,e,n, Ahmed Esmat f a

Department of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia Department of Pharmacognosy, Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt c Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut Branch, Assiut 71524, Egypt d Department of Pharmacognosy and Pharmaceutical Chemistry, College of Pharmacy, Taibah University, Al Madinah Al Munawwarah 30078, Saudi Arabia e Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt f Department of Pharmacology and Toxicology, Faculty of Pharmacy, Ain Shams University, Abbasia, Cairo 11566, Egypt b

art ic l e i nf o

a b s t r a c t

Article history: Received 10 May 2015 Received in revised form 4 November 2015 Accepted 12 November 2015

Ethnopharmacological relevance: Costus speciosus (Koen ex. Retz.) Sm. (crepe ginger, family Costaceae) is an ornamental plant used in traditional medicine for the treatment of inflammation, rheumatism, bronchitis, fever, headache, asthma, flatulence, constipation, helminthiasis, leprosy, skin diseases, hiccough, anemia, as well as burning sensation on urination. Aim of the study: The present study is designed to isolate and identify the active compounds from C. speciosus rhizomes and measure their anti-inflammatory activities. Materials and methods: The n-hexane–CHCl3 soluble fraction of the MeOH extract of C. speciosus rhizomes has been subjected to a repeated column chromatography, including normal silica gel and RP-18 column to give eight compounds. The structures of these compounds were established by UV, IR, 1D (1H and 13C), and 2D (1H–1H COSY, NOESY, HSQC, and HMBC) NMR experiments and HRESIMS data. In addition, the anti-inflammatory activity of compounds 1–8 was evaluated by measuring the levels IL-6, IL1β, TNF-α, COX-2, lipoxgenase-5, and PGE2 using enzyme-linked immunosorbent assay. Results: The n-hexane–CHCl3 soluble fraction afforded a new eudesmane acid, specioic acid (8), along with seven known compounds, 22,23-dihydrospinasterone (1), dehydrodihydrocostus lactone (mokko lactone) (2), dehydrocostus lactone (3), stigmasterol (4), arbusculin A (5), santamarine (douglanin) (6), and reynosin (7). Compounds 1, 4, and 5–7 were isolated for the first time C. speciosus. Compounds 1–4 displayed potent anti-inflammatory activity, while 7 and 8 showed moderate activity. Compounds 1–8 exhibited a concentration-related decrease in the levels of IL-1β, IL-6, TNF-α, PGE2, lipoxgenase-5, and COX-2. Compounds 5 and 6 did not significantly decrease levels of different cytokines, PGE2, lipoxgenase5, and COX-2 from PHA treatment at 1 mM. However, all tested compounds significantly decreased cytokines, PGE2, lipoxgenase-5, and COX-2 levels at concentration 100 mM. It is noteworthy that compounds 1–4 had the highest activity, where it lowered levels of cytokines, PGE2, lipoxgenase-5, and COX-2 to the extent that was no statistical difference from the control group. Thus, they decreased proinflammatory cytokines (IL-1β, IL-6, and TNF-α) with decreased level of the target enzymes (COX-2 and lipoxgenase-5) and subsequent reduction of its inflammatory product (PGE2). Conclusion: Good anti-inflammatory activities exhibited of the isolated compounds from C. speciosus corroborate the usefulness of this plant in the traditional treatment of inflammation and related symptoms. & 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: Costus speciosus Costaceae Sesquiterpenes Eudesmane acid Anti-inflammatory activity

Abbreviations: ANOVA, analysis of variance; COX-2, cyclooxygenase-2; CNS, central nervous system; COSY, Correlation Spectroscopy; DMEM, Dulbecco's Modified Eagle's Medium; EDTA, ethylenediaminetetraacetate; FBS, fetal bovine serum; HMBC, heteronuclear multiple bond correlation; HRESIMS, high resolution electron spray ionization mass spectroscopy; HRMS, high resolution mass; HSQC, heteronuclear single quantum coherence; iNOS, inducible nitric oxide synthase; LPS-activated RAW 264.7, lipopolysaccharide-activated RAW 264.7; IL-1β, interleukin-1β; IL-6, interleukin-6; IL-10, interleukin 10; IR, Infra-red; MAPK, mitogen-activated protein kinases; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; NMR, nuclear magnetic resonance; NO, nitric oxide; PBMCs, peripheral blood mononuclear cells; PGE2, prostaglandin E2; PHA, phytohemagglutinin; PKC, protein kinase C; S.D., standard deviation; TGF-β, transforming growth factor beta; TLC, thin layer chromatography; TNF-α, tumor necrosis factor-α; UV, ultraviolet n Correspondinge author at: Department of Pharmacognosy and Pharmaceutical Chemistry, College of Pharmacy, Taibah University, Al Madinah Al Munawwarah 30078, Saudi Arabia. E-mail address: [email protected] (S.R.M. Ibrahim). http://dx.doi.org/10.1016/j.jep.2015.11.026 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.

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1. Introduction

and antiinflammatory (Ali et al., 2010). Japanese use the rhizome extract for controlling syphilis (Jain, 1991). Stems are ground into paste and applied for blisters (Rani et al., 2012). Costus has been mentioned in Prophet's medicine for treatment of various respiratory diseases (Ibn Qayim, 2008). In Saudi folk medicine, the rhizome is used for treatment of sore throat, tonsillitis, and pleurisy (AL-Kattan, 2013). The rhizome possesses anti-diabetic, anti-oxidant (Vijayalaxmi and Saradha, 2008), hepatoprotective (Bhuyan and Zaman, 2008), antifertility, anticholinestrase, antipyretic (Bhattacharya et al., 1972; Hussain et al., 1992; Binny et al., 2010), cardiotonic, hydrochloretic, diuretic, CNS depressant, and antimicrobial activities (Bhattacharya et al., 1973; Asolkar et al., 1992; Singh and Srivastava, 1992; Ariharan et al., 2012). Saponins and sapogenins from C. speciosus exhibited antifungal activity (Singh and Srivastava, 1992). The alkaloids isolated from C. speciosus possessed papaverine like smooth muscle relaxant action, diuretic, cardiotonic, and central nervous system depressant activities (Bhattacharya et al., 1973). Previous phytochemical studies on C. speciosus resulted in the isolation of sesquiterpenes (Duraipandiyan et al., 2012), sterols (Gupta et al., 1981a; Akhila et al., 1987), saponins (Singh and Thakur, 1982a, 1982b; Inoue and Ebizuka, 1996; Ichinose et al., 1999), benzoquinones (Mahmood et al., 1984), phenolics (Chang et al., 2011, 2012), alkaloids (Bhattacharya et al., 1973), and fatty acids (Gupta et al., 1981a, 1981b, 1982, 1986). Herein, we report the isolation and identification of a new eudesmane acid (8) and seven known compounds (1–7) from the n-hexane–CHCl3 soluble fraction of C. speciosus rhizomes (Fig. 1). Their structures were elucidated through detailed spectroscopic analyses, including 1D, 2D NMR, and HRMS experiments, as well as comparisons with literature data. The stereochemistry of the isolated compounds was determined by NOESY experiment. The

Costus speciosus (Koen ex.Retz.) Sm. (crepe ginger, family Costaceae) is an ornamental plant. It is native to the Malay Peninsula of Southeast Asia. In India the plant occurs through the hills of Himalayas from Himachal Pradesh to Assam, Vindhya Satpura hills in Central India, Eastern Ghats of Andra Pradesh and Western Ghats of Maharashtra, Karnataka, Tamil Nadu and Kerala (Sarin et al., 1974). It has been recommended for the treatment of various disorders in the Indian traditional medicine. Its rhizomes are used for the treatment of burning sensation on urination, flatulence, constipation, helminthiasis, leprosy, skin diseases, fever, hiccough, asthma, rheumatism, bronchitis, inflammation, and anemia (Karthikeyan et al., 2012). The rhizome juice is applied to head for cooling and relief from headache. It is used as an ingredient in a cosmetic to be used on eyelashes to increase sexual attractiveness, as mentioned in Kama Sutra (Najma et al., 2012). In Ayurveda system, the root is ascribed to be bitter, astringent, digestive, stimulant, and aphrodisiac (Nadkarni, 1998). Leaf infusion or decoction is utilized as a sudorific or in a bath for patients with high fever. Leaves are used for scabies, stomach ailments, and earache (Asolkar et al., 1992). The bruised leaves are applied topically to reduce fever as well as a decoction of stem is used to control fever and dysentery (Duraipandiyan et al., 2012). Also, the diabetic people eat one leaf daily to keep their blood glucose low in India (Vishalakshi and Asna, 2010). In Southeast Asia, it is used to treat boils, constipation, diarrhea, dizziness, headache, ear, eye, and nose pain, and to stop vomiting (Jain, 1991). Moreover, the juice of the burnt fresh stem is used as an ear drop for healing otitis. The decoction of the rhizome is used for crushing kidneys and bladder stones. It is used also as a cholagogue, antiamoebic, 29

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Fig. 1. Chemical structures of the isolated compounds 1–8.

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A.A.M. Al-Attas et al. / Journal of Ethnopharmacology 176 (2015) 365–374

anti-inflammatory effects of compounds 1–8 were assessed by measuring the levels of IL-1β, IL-6, TNF-α, PGE2, lipoxgenase-5, and COX-2 in the supernatant media of human peripheral blood mononuclear cells (PBMCs) stimulated by phytohemagglutinin (PHA) using enzyme-linked immunosorbent assay (Figs. 3–8).

2. Materials and methods 2.1. General Electrothermal 9100 Digital Melting Point apparatus (Electrothermal Engineering Ltd., Essex, England) was used for melting points measurement. The UV spectra were determined using the Perkin Elmer double beam spectrophotometer Model 550S (Perkin-Elmer, Waltham, MA, USA). Optical rotation was measured on a JASCO digital polarimeter. The IR spectra were measured on a Bruker spectrophotometer (Shimadzu, Kyoto, Japan). HRESIMS spectra were recorded on an LTQ Orbitrap (ThermoFinnigan, Bremen, Germany). 1D and 2D NMR spectra were recorded on Bruker BioSpin GmbH 600 MHz Ultrashield spectrometer (Bruker BioSpin, Massachusetts, USA). Column chromatographic separations were performed on silica gel 60 (0.04-0.063 mm, Merck, Darmstadt, Germany) and RP-18 (0.04-0.063 mm, Merck, Darmstadt, Germany). TLC was performed on pre-coated TLC plates with silica gel 60 F254 (0.2 mm, Merck, Darmstadt, Germany). The solvent systems used for TLC analyses were n-hexane–EtOAc (70:30, S1) and CHCl3–MeOH (95:5, S2). The compounds were detected by UV absorption at λmax 255 and 366 nm followed by spraying with p-anisaldehyde/H2SO4 reagent and heating at 110 °C for 1–2 min. 2.2. Plant material The rhizomes of C. speciosus were purchased in July 2012 from authorized local market, Thandarai, Kanchipuram District, Tamil Nadu, India. It was kindly identified by Prof. Dr. Abdulaziz Fayed, Prof. of Plant Taxonomy, Faculty of Science, Assiut University, Assiut, Egypt. A voucher specimen was deposited in herbarium of the Department of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia, under the registration number CS-1-2012. 2.3. Extraction and isolation The powdered rhizomes (200 g) were extracted with MeOH (3 L
4) at room temperature. The combined extract was concentrated under reduced pressure to afford a dark brown residue (26 g). The total MeOH extract was subjected to vacuum liquid chromatography (VLC) using n-hexane, CHCl3, EtOAc, and MeOH to afford six fractions; CS1 (1.75 g, n-hexane), CS2 (2.94 g, n-hexane/CHCl3, 50:50), CS3 (2.85 g, CHCl3), CS4 (4.12 g, CHCl3/EtOAc, 50:50), CS5 (1.97 g, EtOAc), and CS6 (8.67 g, MeOH). All fractions were monitored by TLC and fractions CS-2 and CS-3 were collected together to give CST. Fraction CST (5.70 g) was chromatographed over silica gel column (200 g
100 cm
3 cm) using n-hexane/EtOAc gradient to obtain eleven subfractions CST-1 to CST-11. Subfraction CST-2 (81 mg) was subjected to silica gel column chromatography (20 g
50 cm
1 cm) using n-hexane/ EtOAc gradient to give 1 (5.1 mg, white fine needles). Subfraction CST-3 (211 mg) was chromatographed over silica gel column (30 g
50 cm
2 cm) using n-hexane/EtOAc as an eluent to afford impure 2 and 3, which were further purified by repeated RP-18 column chromatography (100 g
50
2 cm, MeOH/H2O gradient) to give 2 (18 mg, yellow crystals) and 3 (3.1 mg, yellow crystals). Subfraction CST-4 (109 mg) was treated similar to subfraction CST3 to give 4 (5.2 mg, white fine needles). Repeated silica gel

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columns (30 g
50 cm
2 cm) of subfractions CST-5 (294 mg) using n-hexane/EtOAc gradient afforded impure 5. Subfraction CST-6 (327 mg) was treated on silica gel column chromatography (40 g
50
2 cm) using n-hexane/EtOAc as an eluent to afford impure 6 and 7. Silica gel column chromatography of CST-7 (79 mg) using n-hexane/EtOAc gradient yielded impure 8. The impure compounds 5, 6, 7, and 8 were separately purified on RP18 column chromatography (100 g
50
2 cm, MeOH/H2O gradient) to give 5 (7.1 mg, white crystals), 6 (11 mg, white crystals), 7 (6 mg, white crystals), and 8 (8.4 mg, white crystals). Other fractions were kept for further study. 2.4. Structure elucidation of isolated compounds Structure elucidation of the isolated compounds was carried out using NMR spectroscopic techniques: UV, IR, 1D (1H and 13C), and 2D (1H–1H COSY, NOESY, HSQC, and HMBC) NMR experiments together with HRESIMS data as well as comparison with those in the literature. The compounds were identified as: 22,23-Dihydrospinasterone (1): White fine needles, Rf 0.70 (S1). m.p. 158–160 °C. 1H NMR (CDCl3, 600 MHz): δH 5.20 (1 H, dd, J¼ 6.4, 3.0 Hz, H-7), 0.57 (3H, s, H-18), 1.01 (3H, s, H-19), 0.93 (3H, d, J ¼6.6 Hz, H-21), 0.85 (3H, d, J ¼6.6 Hz, H-26), 0.82 (3H, d, J¼ 6.6 Hz, H-27), 0.87 (3H, d, J ¼7.2 Hz, H-29). 13C NMR (CDCl3, 150 MHz): δC 39.4 (C-1), 38.8 (C-2), 212.1 (C-3), 44.3 (C-4), 54.9 (C5), 30.1 (C-6), 117.0 (C-7), 139.6 (C-8), 48.9 (C-9), 42.9 (C-10), 22.7 (C-11), 38.1 (C-12), 43.4 (C-13), 54.9 (C-14), 23.1 (C-15), 29.1 (C-16), 56.1 (C-17), 11.9 (C-18), 12.5 (C-19), 36.6 (C-20), 21.7 (C-21), 34.4 (C-22), 26.2 (C-23), 45.8 (C-24), 29.2 (C-25), 19.8 (C-26), 19.0 (C27), 24.7 (C-28), 12.0 (C-29). HRESIMS m/z: 413.3706 [MþH] þ (calcd for C29H49O, 413.3783). 22,23-Dihydrospinasterone was previously reported from Cucumis melo var. reticulatus seeds (Ibrahim, 2014) and whole plant of Arenaria kansuensis (Wu et al., 1995). Dehydrodihydrocostus lactone (Mokko lactone) (2): Yellow crystals, Rf 0.53 (S1). m.p. 36–37 °C. IR (KBr) νmax 3130, 1781, 1747, 886 cm  1. 1H NMR (CDCl3, 600 MHz): δH 2.88 (1H, m, H-1), 1.92 (1H, m, H-2A), 1.83 (1H, m, H-2B), 2.54 (2H, m, H-3), 2.81 (1H, m, H-5), 3.92 (1H, t, J ¼10.0 Hz, H-6), 1.97 (1H, m, H-7), 2.54 (2H, m, H-8), 2.48 (1H, m, H-9A), 2.11 (1H, m, H-9B), 2.21 (1H, m, H-11), 1.23 (3H, d, J¼ 6.6 Hz, H-13), 4.88 (1H, brs, H-14A), 4.78 (1H, brs, H-14B), 5.20 (1 H, d, J ¼2.4 Hz, H-15A), 5.05 (1H, d, J ¼2.4 Hz, H-15B). 13C NMR (CDCl3, 150 MHz): δC 47.1 (C-1), 30.2 (C-2), 32.5 (C-3), 151.2 (C-4), 52.0 (C-5), 85.3 (C-6), 49.9 (C-7), 32.5 (C-8), 37.7 (C-9), 151.2 (C-10), 42.1 (C-11), 178.7 (C-12), 13.2 (C-13), 111.9 (C14), 107.8 (C-15). HRESIMS m/z: 233.1546 [MþH] þ (calcd for C15H21O2, 233.1542). The structure of 2 was assigned by comparison of its spectral data with those reported for dehydrodihydrocostus lactone (mokko lactone) previously isolated from the Chinese drug Sen-mokko (Yuuya et al., 1999; Hikino et al., 1964). Dehydrocostus lactone (3): Yellow crystals, Rf 0.51 (S1). m.p. 35–36 °C. IR (KBr) νmax 1770, 3030, 1711, 762 cm  1. 1H NMR (CDCl3, 600 MHz): δH 2.91 (1H, m, H-1), 1.94 (1H, m, H-2A), 1.85 (1H, m, H-2B), 2.14 (1H, m, H-3A), 1.32 (1H, m, H-3B), 2.86 (1H, m, H-5), 3.97 (1H, t, J ¼10.0 Hz, H-6), 2.89 (1H, m, H-7), 2.25 (1H, m, H-8A), 1.42 (1H, m, H-8B), 2.50 (1H, m, H-9A), 2.18 (1H, m, H-9B), 6.22 (1H, d, J ¼3.6 Hz, H-13A), 5.49 (1H, d, J ¼3.6 Hz, H-13B), 4.90 (1H, brs, 14A), 4.82 (1H, brs, 14B), 5.27 (1H, d, J¼ 2.4 Hz, H-15A), 5.07 (1H, d, J ¼2.4 Hz, H-15B). 13C NMR (CDCl3, 150 MHz): δC 47.6 (C-1), 30.3 (C-2), 32.6 (C-3), 150.0 (C-4), 52.1 (C-5), 85.2 (C-6), 45.1 (C-7), 30.9 (C-8), 36.3 (C-9), 49.2 (C-10), 139.8 (C-11), 170.3 (C-12), 120.2 (C-13), 112.6 (C-14), 109.2 (C-15). HRESIMS m/z: 231.1381 [MþH] þ (calcd for C15H19O2, 231.1385). NMR data of 3 are in a good agreement with those reported for dehydrocostus lactone previously isolated from the Chinese drug Sen-mokko (Yuuya et al., 1999; Hikino et al., 1964), leaves and trunk barks of Magnolia

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watsonii (Ito et al., 1984), and whole plant of Diaspananthus uniflorus (Adegawa et al., 1987). Stigmasterol (4): White fine needles, Rf 0.44 (S1). m.p. 175-176 °C. IR (KBr) νmax 3492, 2936 cm  1. 1H NMR (CDCl3, 600 MHz): δH 3.53 (1H, m, H-3), 5.35 (1H, dd, J ¼5.4, 3.6 Hz, H-6), 0.68 (3H, s, H-18), 1.01 (3H, s, H-19), 0.93 (3H, d, J ¼6.6 Hz, H-21), 5.17 (1H, dd, J ¼15.6, 8.4 Hz, H-22), 5.01 (1H, dd, J ¼15.6, 8.4 Hz, H-23), 0.81 (3H, d, J ¼7.8 Hz, H-26), 0.79 (3H, d, J¼ 7.8 Hz, H-27), 0.85 (3H, t, J ¼7.2 Hz, H-29). 13C NMR (CDCl3, 150 MHz): δC 37.3 (C1), 31.9 (C-2), 71.8 (C-3), 42.1 (C-4), 140.6 (C-5), 121.7 (C-6), 33.9 (C7), 31.7 (C-8), 50.1 (C-9), 36.5 (C-10), 21.1 (C-11), 39.8 (C-12), 42.3 (C-13), 56.9 (C-14), 24.3 (C-15), 28.3 (C-16), 56.0 (C-17), 11.9 (C-18), 18.8 (C-19), 40.5 (C-20), 23.1 (C-21), 138.3 (C-22), 129.3 (C-23), 45.8 (C-24), 29.1 (C-25), 19.8 (C-26), 19.4 (C-27), 26.1 (C-28), 12.1 (C-29). HRESIMS m/z: 413.3706 [MþH] þ (calcd for C29H49O, 413.3783). NMR data of 4 are in a good agreement with those reported for stigmasterol previously isolated from the bark of Eucalyptus globulus Labill (Mohamed and Ibrahim, 2007) and leaves of Rubus suavissimus S. Lee (Chaturvedula and Prakash, 2012). Arbusculin A (5): White crystals, Rf 0.30 (S1). [α]D þ26.7 (c 0.5, CHCl3). m.p. 76–77 °C. UV (MeOH) λmax (log ε) 203 (4.36) nm. IR (KBr) νmax 3310, 1762 cm  1. 1H NMR (CDCl3, 600 MHz): δH 1.53 (1H, m, H-1A), 1.38 (1H, m, H-1B), 1.43 (1H, m, H-2A), 1.25 (1H, m, H-2B), 1.79 (1H, m, H-3A), 1.47 (1H, m, H-3B), 1.85 (1H, d, J ¼10.8 Hz, H-5), 4.04 (1H, t, J ¼10.8 Hz, H-6), 2.62 (1H, ddd, J ¼10.8, 3.3, 3.1 Hz, H-7), 2.02 (1H, m, H-8A), 1.60 (1H, m, H-8B), 1.63 (1H, m, H-9A), 1.55 (1H, m, H-9B), 6.11 (1 H, brs, H-13A), 5.44 (1H, brs, H-13B), 0.98 (3H, brs, H-14), 1.34 (3H, brs, H-15). 13C NMR (CDCl3, 150 MHz): δC 42.9 (C-1), 41.0 (C-2), 40.0 (C-3), 71.6 (C-4), 57.9 (C-5), 81.6 (C-6), 50.8 (C-7), 22.0 (C-8), 19.4 (C-9), 37.6 (C-10), 138.5 (C-11), 170.0 (C-12), 117.8 (C-13), 19.8 (C-14), 24.2 (C-15). HRESIMS m/z: 251.1645 [MþH] þ (calcd for C15H23O3, 251.1647). NMR data of 5 are in a good agreement with those of arbusculin A, previously isolated from the root wood of formosan Michelia compressa (Liu et al., 2008). Santamarine (douglanin) (6): White crystals, Rf 0.42 (S2). [α]D þ 97.4 (c 0.5, CHCl3). m.p. 142–143 °C. UV (MeOH) λmax (log ε) 210 (4.25) nm. IR (KBr) νmax 3480, 3145, 1792, 1751, 855 cm  1. 1H NMR (CDCl3, 600 MHz): δH 3.68 (1H, dd, J ¼10.2, 6.6 Hz, H-1), 2.40 (1H, m, H-2A), 2.07 (1H, m, H-2B), 5.35 (1H, m, H-3), 2.34 (1H, brd, J ¼11.4 Hz, H-5), 3.95 (1H, t, J¼ 11.4 Hz, H-6), 2.50 (1H, ddd, J ¼11.4, 3.6, 2.2 Hz, H-7), 2.11 (1H, m, H-8A), 1.66 (1H, dt, J ¼12.6, 3.6 Hz, H-8B), 2.09 (1H, m, H-9A), 1.32 (1H, ddd, J ¼12.6, 6.6, 3.6 Hz, H-9B), 6.08 (1H, d, J¼ 3.6 Hz, H-13A), 5.41 (1H, d, J¼ 3.6 Hz, H-13B), 0.88 (3H, s, H-14), 1.84 (3H, d, J ¼1.2 Hz, H-15). 13C NMR (CDCl3, 150 MHz): δC 75.2 (C-1), 32.8 (C-2), 121.3 (C-3), 133.4 (C-4), 51.1 (C-5), 81.6 (C-6), 51.0 (C-7), 21.2 (C-8), 34.2 (C-9), 40.9 (C-10), 139.0 (C-11), 170.9 (C-12), 116.9 (C-13), 11.1 (C-14), 23.4 (C-15). HRESIMS m/z: 249.1493 [Mþ H] þ (calcd for C15H21O3, 249.1491). NMR data of 6 are in a good agreement with those reported for santamarine previously isolated from the leaves, flowers, and fruits of Laurus nobilis L. (Barla et al., 2007; Fang et al., 2005) and flowers of Tanacetum vulgare ssp. Siculum (Rosselli et al., 2012). Reynosin (7): White crystals, Rf 0.38 (S2). [α]D þ 178 (c 0.5, CHCl3). m.p. 144–145 °C. UV (MeOH) λmax (log ε) 212 (4.11) nm. IR (KBr) νmax 3492, 3170, 1767, 1646, 853 cm  1. 1H NMR (CDCl3, 600 MHz): δH 3.53 (1H, dd, J ¼11.4, 4.2 Hz, H-1), 1.85 (1H, m, 2A), 1.56 (1H, m, 2B), 2.34 (1H, ddd, J ¼13.8, 8.4, 1.2 Hz, H-3A), 2.15 (1H, brdd, J ¼13.8, 5.4 Hz, H-3B), 2.18 (1H, brd, J¼ 10.8 Hz, H-5), 4.03 (1H, t, J¼ 10.8 Hz, H-6), 2.55 (1H, dt, J ¼10.8, 5.8 Hz, H-7), 2.08 (1H, m, 8A), 1.62 (1H, m, 8B), 2.10 (1H, m, 9A), 1.36 (1H, dt, J ¼13.8, 4.2 Hz, H-9B), 6.09 (1H, d, J ¼3.0 Hz, H-14A), 5.42 (1H, d, J¼ 3.0 Hz, H-14B), 0.82 (3H, s, H-14), 5.00 (1H, d, J ¼1.2 Hz, H-15A), 4.86 (1H, d, J ¼1.2 Hz, H-15B). 13C NMR (CDCl3, 150 MHz): δC 78.2 (C-1), 31.3 (C-2), 33.5 (C-3), 142.5 (C-4), 52.9 (C-5), 79.6 (C-6), 49.6 (C-7), 21.4 (C-8), 35.7 (C-9), 43.0 (C-10), 139.2 (C-11), 170.7 (C-12), 117.1 (C-

13), 11.6 (C-14), 110.6 (C-15). HRESIMS m/z: 249.1488 [MþH] þ (calcd for C15H21O3, 249.1491) NMR data of 7 are in a good agreement with those reported for reynosin previously isolated from the leaves, flowers, and fruits of Laurus nobilis L. (Barla et al., 2007; Fang et al., 2005), whole plant of Diaspananthus uniflorus (Adegawa et al., 1987), and leaves and trunk barks of Magnolia watsonii (Ito et al., 1984). Specioic acid (8): White crystals, Rf 0.32 (S2). [α]D þ 67.4 (c 0.5, CHCl3). m.p. 135–136 °C. UV (MeOH) λmax (log ε) 218 (4.09), 268 (3.36) nm. IR (KBr) νmax 3450, 3070, 1730, 1646, 862 cm  1. NMR data (CDCl3, 600 and 150 MHz), see Table 1. HRESIMS m/z 285.1706 (calcd for C15H25O5, 285.1702). 2.5. Anti-inflammatory activity The inflammatory interleukins Il-6, Il-1β, and TNF-α were assessed using enzyme-linked immunosorbent assay kits (Orgenium Laboratories, Vantaa, Finland). PGE2, COX-2, and lipoxgenase-5 were quantified using enzyme-linked immunosorbent assay kit (Wuhan ElAab Science Co., Wuhan, China). 2.5.1. Isolation of human PBMCs PBMCs were isolated from whole blood obtained from healthy donors of whom informed consent was obtained that their donated blood might be used for scientific purposes. Separation of blood cells was performed using density centrifugation (Lymphocyte Separation Medium, Lonza, Basel, Switzerland). After isolation, cells were washed three times in phosphate buffered saline containing 1 mM EDTA. Then, they were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heatinactivated Fetal Bovine Serum (FBS) (Lonza, Basel, Switzerland), 2 mM L-glutamine, and 0.1% of penicillin/streptomycin (SigmaAldrich Co., St Louis, MO, USA) in a humidified atmosphere containing 5% CO2 for 48 h. 2.5.2. Stimulation of PBMCs Isolated PBMCs were seeded in 6-well plates at a density of 1.5
106 cells/mL in supplemented DMEM. Then, cells were Table 1 NMR spectral data of compound 8 (CDCl3, 600 and 150 MHz). No. δH [Mult., J (Hz)]

δC (Mult.)

1 H–1H COSY

HMBC

1

2.34 m

32.9 CH

2, 14

2

1.82 m 1.77 dd (13.8, 7.2) 1.60 dd (13.6, 7.2) 1.54 m – – 2.38 m 1.87 m 2.69 brdd (12.0, 11.4) 1.56 m 1.40 m 1.85 m 1.47 m – – – 6.27 brs 5.64 brs 1.09 d (6.6) 1.17 s

27.9 CH2

1, 3

2, 3, 4, 6, 10, 7 14 3, 4, 5, 10 – 14, 15

35.8 CH2

2

1, 2, 4, 5, 15

72.3 C 80.1 C 29.2 CH2

– – 7

– – 4, 7, 8, 11

37.2 CH

6, 8, 13

11, 12, 13

29.7 CH2

7, 9

30.7 CH2

8

73.8 C 145.7 C 170.6 C 125.2 CH2

– – – 7

5, 6, 7, 10, 11 6A, 7 6B, 14, 15 1, 6, 7, 8, 10 7 14 – – – – – – 6, 7, 11, 12 –

15.9 CH3 22.6 CH3

1 –

1, 5, 9, 10 3, 4, 5, 10

3 4 5 6 7 8 9 10 11 12 13 14 15

NOESY

7 14, 15 – – 1, 7, 8A 8B 1, 3A, 6A, 8A

2B, 3B, 8B, 15 2B, 3B, 6B, 8B, 14

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369

Fig. 2. Significant 1H–1H-COSY, HMBC (A), and NOESY (B) correlations of 8.

pretreated with the tested compounds at 100, 10, and 1 mM. After 24 h, they were stimulated with 10 mg/mL of mitogen PHA. Cells treated with PHA were used as a positive control group. Negative control group was treated with DMSO in a final concentration equal to test wells. DMSO concentration never exceeded 0.1%. Indomethacin (10 mM) (Sigma-Aldrich Co., St Louis, MO, USA) was used as a reference standard. After 3 days of incubation, supernatant media were used for measuring the levels of IL-6, IL-1β, TNF-α, and PGE2 (Virella, 1998).

Tukey's test for post hoc analysis. Statistical significance was acceptable to a level of p o0.05. All statistical analyses were performed using GraphPad InStat software, version 3.05 (GraphPad Software, Inc. La Jolla, CA, USA). Graphs were plotted using GraphPad Prism software, version 5.00 (GraphPad Software, Inc. La Jolla, CA, USA).

2.5.3. Assessment of COX-2, IL-1β, IL-6, and TNF-α levels The assay of these mediators was based on a quantitative sandwich enzyme immunoassay technique using enzyme-linked immunosorbent assay kits. Briefly, a specific monoclonal antibody was pre-coated onto a microplate. The analyte in standards and samples was sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for it, which was recognized by a streptavidin–peroxidase conjugate. All unbound material was then washed away and a peroxidase enzyme substrate was added. The color development was stopped and the color intensity was measured at 450 nm using a microplate reader.

Investigation of the n-hexane–CHCl3 soluble fraction of the MeOH extract of C. speciosus rhizomes using a combination of chromatographic techniques, including normal silica gel and RP-18 column chromatography afforded a new eudesmane acid (8) and seven known compounds (1–7) (Fig. 1). The relative configuration at the chiral centers was deduced by comparison of 13C chemical shifts and coupling constant values with those of related sesquiterpenes as well as NOESY spectral data. The isolated compounds were evaluated for their anti-inflammatory activity. Compound 8 was obtained as white crystals. The IR spectrum displayed absorptions at 3450 and 1730 cm  1 for hydroxyl and acid carbonyl groups, respectively (Silverstein and Webster, 1998). Its molecular formula was deduced to be C15H24O5 by HRESIMS quasi-molecular ion peak at m/z 285.1706 [M þH] þ (calcd for C15H25O5, 285.1702), requiring four degrees of unsaturation. The UV, IR, and NMR spectral data suggested that 8 had a trihydroxy eudesmane acid skeleton (Wang et al., 2013; Xiao et al., 2003; Zhao et al., 1997). The 13C NMR spectrum displayed resonances for 15 carbons, including two methyls, six methylenes, two methines, and five quaternary carbons one of them for acid carbonyl (δC 170.6) and three for oxygen-bonded carbons at δC 72.3 (C-4), 80.1 (C-5), and 73.8 (C-10) (Table 1). The 1H NMR spectrum revealed two methine protons at δH 2.34 (m, H-1) and 2.69 (brdd, J ¼12.0, 11.4 Hz, H-7). The HSQC spectrum correlated these signals to the carbons resonating at δC 32.9 (C-1) and 37.2 (C-7), respectively. Two methyl signals were detected at δH 1.17 (s, H-15)/δC 22.6 and 1.09 (d, J ¼6.6 Hz, H-14)/δC 15.9. In the 1H–1H COSY spectrum two spin systems were observed (Fig. 2). The first spin system started from H-14 to H-3 (substructure A). The HMBC correlations of H-1 to C-2, C-3, and C-14 and H-14 to C-1, and H-3 to C-1 and C-4 confirmed this spin system (Table 1, Fig. 2). The second spin system extended from H-6 to H-9 to give CH2–CH–CH2–CH2 moiety (substructure B). The singlet signal at δH 1.17 correlated with the carbon signal at δC 22.6 in HSQC spectrum assignable to H-15/C-15. Its attachment at C-4 was confirmed by HMBC cross peaks of H-15 to C-3 and C-4. The connectivity of substructures A and B at C-5 and C-10 was established by the HMBC correlations of H-2 and H-14 to C-5 and C-10, H-1 and H-9 to C-10, H-9 to C-1, H-6 to C-4,

2.5.4. Assessment of PGE2 level PGE2 was quantified using enzyme-linked immunosorbent assay kit. The assay was based on the forward sequential competitive binding technique in which PGE2 in a sample competes with horseradish peroxidase-labeled PGE2 for a limited number of binding sites on monoclonal antibodies. PGE2 in the sample was allowed to bind to the antibody in the first incubation. During the second incubation, horseradish peroxidase-labeled PGE2 binds to the remaining antibody sites. Following a wash to remove unbound materials, a substrate solution was added to the wells to determine the bound enzyme activity. The color development was stopped and the absorbance was read at 450 nm. 2.5.5. Assessment of lipoxgenase-5 level The inhibition of lipoxygenase activity was determined using enzyme-linked immunosorbent assay kits as previously outlined (yawer et al., 2007). The reaction mixture, containing tested compound solution and lipoxygenase solution in 0.1 M phosphate buffer (pH 8.8) was incubated for 5 min at 25 °C. Then, the reaction was initiated by addition of a solution substrate. After 6 min, the absorbance of the resulting mixture was measured at 234 nm. Indomethacin was used as reference standard. 2.5.6. Statistical analysis Data are presented as mean 7S.D. Comparisons were carried out using one way analysis of variance (ANOVA) followed by

3. Results and discussion

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Fig. 3. Effect of compounds 1–8 on IL-1β release from PHA-stimulated PBMCs at 100 (Panel A), 10 (Panel B), and 1 mM (Panel C). Data are presented as mean 7 S.D, n¼ 3, a statistically different from the corresponding control group at p o 0.05, b statistically different from the corresponding PHA-treated group at p o0.05.

Fig. 4. Effect of compounds 1–8 on IL6 release from PHA-stimulated PBMCs at 100 (Panel A), 10 (Panel B), and 1 mM (Panel C). Data are presented as mean7 S.D, n ¼3, a statistically different from the corresponding control group at p o 0.05, b statistically different from the corresponding PHA-treated group at po 0.05.

and H-15 to C-5 and C-10. The presence of an exomethylene group was confirmed by the signals at δH 6.27 and 5.64/δC 125.2 (C-13) and 145.7 (C-11). Its situation at C-7 was proved by the HMBC correlations of H-13 with C-6 and C-7 (Fig. 2). The carbon signal at δC 170.6 (C-12) in the 13C NMR spectrum indicated the presence of acid carbonyl in 8, which was confirmed by the IR absorption band at 1710 cm  1. The attachment of the carbonyl group at C-11 was secured based on the HMBC cross peaks of H-7 and H-13 to C-12. The configuration at the stereogenic centers was suggested in part by comparison of the coupling constants and 13C chemical shifts with those of related sesquiterpens (Wang et al., 2013; Xiao et al., 2003; Zheng et al., 2003; Zdero et al., 1990) and confirmed by NOESY experiment. The NOESY cross correlations of H-1 to H-7 suggested that these protons present on the same side of the

molecule. Similarly, the NOESY cross peaks of H-14 to H-15 placed these protons were on the other side of the molecule (Fig. 2). Furthermore, the observed cross peaks of H-15 and H-14 with H-6B, H-6A with H-1, H-7, and H-8A suggested that A/B rings were trans-fused. Finally, full assignment of 8 was achieved from concrete interpretation of 1D and different 2D NMR spectroscopic data as depicted. On reviewing the literature, 8 was found to be a new natural product and named specioic acid. Binny et al. (2010) reported that the ethanolic extract of C. speciosus rhizome possessed anti-inflammatory activities at doses of 400 and 800 mg/kg using carrageenan induced paw edema and cotton pellet induced granuloma formation methods (Binny et al., 2010). Moreover, the methanol extract of aerial parts of C. speciosus at doses 400 and 800 mg/kg showed significant anti-

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Fig. 5. Effect of compounds 1–8 on PGE2 release from PHA-stimulated PBMCs at 100 (Panel A), 10 (Panel B), and 1 mM (Panel C). Data are presented as mean7 S.D, n¼ 3, a statistically different from the corresponding control group at p o 0.05, b statistically different from the corresponding PHA-treated group at p o 0.05.

inflammatory activity (19.36 and 40.05% reduction) at 5 h compared to diclofenac (10 mg/kg, 63.0% reduction) (Srivastava et al., 2013). It is noteworthy to mention that sesquiterpene lactones have shown anti-inflammatory activities in both in-vitro and in-vivo studies. However, their mechanisms were not fully understood. In fact, the anti-inflammatory activity could be explained by many proposed mechanisms of action. The most important one was the inhibition of enzymes like cyclooxygenases and lipoxygenases responsible for eicosanoid generation. This in turn reduces the concentrations of inflammatory mediators such as prostaglandins and leukotrienes. Also, they prevent the activation of NF-κB target genes, such as those for the cytokines IL-1α, IL-1β, IL-6, IL-8, and TNF (Kreuger et al., 2012). Interestingly, it has been shown that some sesquiterpene lactones (e.g., anhydroverlotorin, costunolide, 5-deoxy-5-hydroperoxyepitelekin, 5-deoxy-5-hydroperoxytelekin, and umbellifolide) inhibited both TNF-α and PMA-induced NF-κB

Fig. 6. Effect of compounds 1–8 on TNF-α release from PHA-stimulated PBMCs at 100 (Panel A), 10 (Panel B), and 1 mM (Panel C). Data are presented as mean 7 S.D, n¼ 3, a statistically different from the corresponding control group at p o0.05, b statistically different from the corresponding PHA-treated group at po 0.05.

with an IC50 r 20 mM (Fischedick et al., 2012). Cho et al. (1998) reported that santamarine (6) and reynosin (7) exhibited anti-inflammatory activities by inhibiting TNF-α production in LPS-activated RAW 264.7 cells with IC50 values of 105 and 87.4 μM, respectively (Cho et al. 1998). Furthermore, it was reported that costunolide inhibited NO production by down-regulating iNOS expression, through the inhibition of IkBs' phosphorylation and degradation, which are essential for the activation of NF-κB (Koo et al., 2001). Thus, the anti-inflammatory effects of 1–8 were assessed by measuring the levels of IL-1β, IL-6, TNF-α, PGE2, lipoxgenase-5, and COX-2 in supernatant media of PHA-stimulated PBMCs (Virella, 1998). The cells treated with PHA showed significant increase in

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Fig. 7. Effect of compounds 1–8 on COX-2 release from PHA-stimulated PBMCs at 100 (Panel A), 10 (Panel B), and 1 mM (Panel C). Data are presented as mean 7 S.D, n¼ 3, a statistically different from the corresponding control group at p o 0.05, b statistically different from the corresponding PHA-treated group at p o0.05.

IL-1β level compared with that of the untreated control cells (Fig. 3). Treatment with 1–8 resulted in a concentration-related decrease in IL-1β level. Compounds 5 and 6 did not significantly decrease IL-1β level from PHA treatment at 1 mM. However, all compounds significantly decreased IL-1β compared to PHA treatment at 100 mM. Moreover, 1–4 decreased IL-1β level to the extent that was not statistically different from the untreated group. The same pattern of activity was observed while assessing IL-6 level as shown in Fig. 4. Only compounds 5 and 6 did not significantly decrease IL-6 level at 1 mM compared to PHA treatment. While, all the tested compounds significantly decreased IL-6 at 100 mM. Compound 1–4 had the highest activity, where it lowered IL-6 level to control value. Similarly, cell treatment with compounds 1– 8 showed a concentration-related decrease in the level of PGE2

Fig. 8. Effect of compounds 1–8 on lipoxygenase-5 release from PHA-stimulated PBMCs at 100 (Panel A), 10 (Panel B), and 1 mM (Panel C). Data are presented as mean7 S.D, n¼ 3, a statistically different from the corresponding control group at po 0.05, b statistically different from the corresponding PHA-treated group at po 0.05.

(Fig. 5). Notably, most compounds at 100 mM significantly decreased PGE2 levels from PHA-treated group without any statistical difference from the untreated group, except 5 and 6. Compounds 1–4 at 100 mM reduced TNF-α level to the extent that was no statistical difference from the control group (Fig. 6). The tested samples at 10 mM caused a concentration-related decrease in TNFα level but failed to reach that of the control values. Compounds 5 and 7 failed to significantly decrease TNF-α level at 1 mM when compared to PHA-treated group. COX-2 and lipoxgenase-5 levels were assessed as shown in Figs. 7 and 8, respectively. The PHAtreated cells showed a significant increase of COX-2 and lipoxgenase-5 levels compared with the untreated control cells. Compounds 5 and 6 did not significantly decrease COX-2 and lipoxgenase-5 levels at 1 mM, compared to PHA treatment. However, all compounds significantly decreased COX-2 and lipoxgenase-5

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levels at 100 mM concentration compared to PHA treatment. In addition, compounds 1–4 reduced COX-2 and lipoxgenase-5 levels to the extent that was not statistically different from the control group (Figs. 7 and 8). It is worthy noted that inflammatory response is regulated by a precisely-modulated reaction between inflammatory mediatorsalso known as cytokines-and cells (Sacca et al., 1997). Generally, cytokines could be categorized as pro- or anti-inflammatory, depending on their roles in inflammation (Hopkins, 2003). Proinflammatory cytokines such as IL-1β, IL-6 and TNF-α are involved in the initiation and amplification of inflammation, while anti-inflammatory cytokines (e.g., IL-10 and TGF-β) negatively modulate these events (Dinarello, 2000; Opal and DePalo, 2000). It has been shown that proinflammatory cytokines act by activation of transcription factors (e.g., NF-κB and AP-1) and protein kinases (MAPK and PKC). In turn, the expression of many target genes is increased to maintain the inflammatory state. For example, cytokines might be responsible for the induction of several enzymes (e.g., iNOS and COX-2) that are crucial to initiate and maintain the inflammatory process (Kracht and Saklatvala, 2002). The current results are in agreement with these mechanistic studies, where the tested sesquiterpene lactone compounds caused a concentration-dependant decrease in the proinflammatory cytokines (IL-1β, IL-6, and TNF-α) with decreased level of the target enzymes (COX-2 and lipoxgenase-5) and subsequent reduction of its inflammatory product (PGE2).

4. Conclusion Investigation of the n-hexane–CHCl3 soluble fraction of C. speciosus rhizomes afforded a new eudesmane acid, specioic acid (8), along with seven known compounds. Compounds 1, 4, and 5–7 were reported for the first time C. speciosus. Compounds 1–4 displayed potent anti-inflammatory activity, followed by 7 and 8. Anti-inflammatory activity results supported the rationale behind the traditional use of this plant in inflammatory conditions.

Conflict of interest The authors have no conflict of interest to declare.

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2015.11.026.

References Adegawa, S., Miyase, T., Ueno, A., 1987. Sesquiterpene lactones from Diaspananthus uniflorus (SCH. BIP.) KITAM. Chem. Pharm. Bull. 35, 1479–1485. Akhila, A., Gupta, M.M., Thakur, R.S., 1987. Direct cyclisation of squalene to 5αstigmast-9(11)-en-3β-ol via Δ9(11) lanosterol in Costus speciosus: a unique finding in sterol biosynthesis. Tetrahedron Lett. 28, 4085–4088. Ali, R.M., Abu Samah, Z., Mustapha, N.M., Hussein, N., 2010. ASEAN Herbal and Medicinal Plants. The Association of Southeast Asian Nations (ASEAN), Jakarta, Indonesia, pp. 121–122. AL-Kattan, M.O., 2013. Anti-bacterial effect of Indian costus and sea-qust and their water extracts on some pathogenic bacteria of the human respiratory system. J. Med. Plants Res. 7, 1418–1423. Ariharan, V.N., Meena Devi, V.N., Rajakokhila, M., Prasad, P.N., 2012. Antibacterial activity of Costus speciosus rhizome extract on some pathogenic bacteria. Int. J. Adv. Life Sci. 4, 24–27. Asolkar, L.V., Kakkar, K.K., Chakre, O.J., 1992. Second Supplement to Glossary of Indian Medicinal Plants with Active Principles Part I (A–K). (1965–1981). Publications and Informations Directorate (CSIR), New Delhi, p. 414. Barla, A., Topçu, G., Öksüz, S., Tümen, G., Kingston., D.G.I., 2007. Identification of

373

cytotoxic sesquiterpenes from Laurus nobilis L. Food Chem. 104, 1478–1484. Bhattacharya, S.K., Parik, A.K., Debnath, P.K., Pandey, V.B., Neogy, N.C., 1972. Anticholinesterase activity activity of Costus speciosus alkaloids. Indian J. Pharmacol. 4, 178 178. Bhattacharya, S.K., Parikh, A.K., Debnath, P.K., Pandey, V.B., Neogy, N.C., 1973. Pharmacological studies with the alkaloids of Costus speciosus. J. Res. Indian Med. 8, 10–19. Bhuyan, B., Zaman, K., 2008. Evaluation of hepatoprotactive activity of rhizomes of Costus speciosus (J. Konig.) Smith. Pharmacologyonline 4, 119–126. Binny, K., Sunil, K.G., Thomas, D., 2010. Antiinflammatory and antipyretic properties of the rhizome of Costus speciosus (Koen.) Sm. J. Basic Clin. Pharm. 1, 177–181. Chang, Y.Q., Tan, S.N., Yong, J.W.H., Ge, L., 2011. Surfactant-assisted pressurized liquid extraction for determination of flavonoids from Costus speciosus by micellar electrokinetic chromatography. J. Sep. Sci. 34, 462–468. Chang, Y.Q., Tan, S.N., Yong, J.W.H., Ge, L., 2012. Determination of flavonoids in Costus speciosus and Etlingera elatior by liquid chromatography–mass spectrometry. Anal. Lett. 45, 345–355. Chaturvedula, V.S.P., Prakash, I., 2012. Isolation of stigmasterol and β-sitosterol from the dichloromethane extract of Rubus suavissimus. Int. Curr. Pharm. J. 1, 239–242. Cho, J.Y., Park, J., Yoo, E.S., Baik, K.U., Jung, J.H., Lee, J., Park, M.H., 1998. Inhibitory effect of sesquiterpene lactones from Saussurea lappa on tumor necrosis factoralpha production in murine macrophage-like cells. Planta Medica 64, 594–597. Dinarello, C.A., 2000. Pro-inflammatory cytokines. Chest 118, 503–508. Duraipandiyan, V., Al-Harbi, N.A., Ignacimuthu, S., Muthukumar, C., 2012. Antimicrobial activity of sesquiterpene lactones isolated from traditional medicinal plant, Costus speciosus (Koen ex.Retz.) Sm. BMC Complement. Altern. Med. 12, 13–18. Fang, F., Sang, S., Chen, K.Y., Gosslau, A., Ho, C., Rosen, R.T., 2005. Isolation and identification of cytotoxic compounds from Bay leaf (Laurus nobilis). Food Chem. 93, 497–501. Fischedick, J.T., Standiford, M., Johnson, D.A., De Vos, R.C.H., Todorović, S., Banjanac, T., Verpoorte, R., Johnson, J.A., 2012. Activation of antioxidant response element in mouse primary cortical cultures with sesquiterpene lactones isolated from Tanacetum parthenium. Planta Med. 78, 1725–1730. Gupta, M.M., Lal, R.N., Shukla, Y.N., 1981a. 5α-Stigmast-9(11)-en-3β-ol from Costus speciosus roots. Phytochemistry 20, 2557–2559. Gupta, M.M., Lal, R.N., Shukla, Y.N., 1981b. Aliphatic hydroxyketones and diosgenin from Costus speciosus roots. Phytochemistry 20, 2553–2555. Gupta, M.M., Lal, R.N., Shukla, Y.N., 1982. Aliphatic compounds from Costus speciosus roots. Phytochemistry 21, 230–231. Gupta, M.M., Verma, R.K., Akhila, A., 1986. Oxo acids and branched fatty acid esters from rhizomes of Costus speciosus. Phytochemistry 25, 1899–1902. Hikino, H., Meguro, K., Kusano, G., Yakemoto, T., 1964. Structure of mokko lactone and dehydrocostus lactone. Chem. Pharm. Bull. 12, 632–634. Hopkins, S.J., 2003. The pathophysiological role of cytokines. Leg. Med. 5, S45–S57. Hussain, A., Virmani, O.P., Popli, S.P., Misra, L.N., Gupta, M.M., Srivastava, G.N., Abraham, Z., Singh, A.K., 1992. Dictionary of Indian Medicinal Plants. CIMAP, Lucknow, India, p. 546. Ibn Qayim, A., 2008. The Prophetic Medicine. Translation: Khalil, M. Pub. Dar AlManarah. Egypt, pp. 91–102. Ibrahim, S.R.M., 2014. New chromone and triglyceride from Cucumis melo seeds. Nat. Prod. Commun. 9, 205–208. Ichinose, K., You, S., Kawano, N., Hayashi, K., Yao, X., Ebizuka, Y., 1999. Heterologous expression of furostanol glycoside 26-O-β-glucosidase of Costus speciosus in Nicotiana tabacum. Phytochemistry 51, 599–603. Inoue, K., Ebizuka, Y., 1996. Purification and characterization of furostanol glycoside 26-O-β-glucosidase from Costus speciosus rhizomes. FEBS Lett. 378, 157–160. Ito, K., Iida, T., Kobayashi, T., 1984. Guaiane sesquiterpenes from Magnolia watsonii. Phytochemistry 23, 188–190. Jain, S.K., 1991. Dictionary of Indian folk Medicine and Ethno Botany. Deep Publications, New Delhi. Karthikeyan, J., Reka, V., Giftson, R.V., 2012. Characterisation of bioactive compounds in Costus speciosus (koen) by reversed phase HPLC. Int. J. Pharm. Sci. Res. 3, 1461–1465. Koo, T.H., Lee, J., Park, Y.J., Hong, Y., Kim, H.S., Kim, K., Lee, J.J., 2001. A sesquiterpene lactone, costunolide, from Magnolia grandiflora inhibits NF-kB by targeting IkB phosphorylation. Planta Med. 67, 103–107. Kracht, M., Saklatvala, J., 2002. Transcriptional and post-transcriptional control of gene expression in inflammation. Cytokine 20, 91–106. Kreuger, M.R.O., Grootjans, S., Biavatti, M.W., Vandenabeele, P., D’Herde, K., 2012. Sesquiterpene lactones as drugs with multiple targets in cancer treatment: focus on parthenolide. Anticancer Drugs 23, 883–896. Liu, C., Chen, Y., Cheng, M., Lee, S., Abd Elrazek, M.H., Chang, W., Chen, Y., Chen, I., 2008. Cytotoxic constituents from the root wood of formosan Michelia compressa. J. Chil. Chem. Soc. 53, 1523–1524. Mahmood, A., Shukla, Y.N., Thakur, R.S., 1984. Benzoquinones from Costus speciosus seeds. Phytochemistry 23, 1725–1727. Mohamed, G.A., Ibrahim, S.R.M., 2007. Eucalyptone G, a new phloroglucinol derivative and other constituents from Eucalyptus globulus Labill. ARKIVOC xv, 281–291. Nadkarni, K.M., 1998. Indian Medicinal Plants and Drugs-with their Medicinal Properties and Uses. Asiatic Publishing House, New Delhi, p. 450. Najma, C., Chandra, K.J., Ansarul, H., 2012. Effect of Costus specious Koen on reproductive organs of female albino mice. Int. Res. J. Pharm. 3, 200–202. Opal, S.M., DePalo, V.A., 2000. Anti-inflammatory cytokines. Chest 117, 1162–1172.

374

A.A.M. Al-Attas et al. / Journal of Ethnopharmacology 176 (2015) 365–374

Rani, A.S., Sulakshana, G., Patnaik, S., 2012. Costus speciosus, an antidiabetic plantreview. Fons Sci. J. Pharm. Res. 1, 50–53. Rosselli, S., Bruno, M., Raimondo, F.M., Spadaro, V., Varol, M., Koparal, A.T., Maggio, A., 2012. Cytotoxic effect of eudesmanolides isolated from flowers of Tanacetum vulgare ssp. Siculum. Molecules 17, 8186–8195. Sacca, R., Cuff, C.A., Ruddle, N.H., 1997. Mediators of inflammation. Curr. Opin. Immunol. 9, 851–857. Sarin, Y.K., Bedi, K.L., Atal, C.K., 1974. Costus speciosus rhizome as source of diosgenin. Curr. Sci. 43, 569–570. Silverstein, R.M., Webster, F.X., 1998. Spectrometric Identification of Organic Compounds. John Wiley, New York. Singh, S.B., Thakur, R.S., 1982a. Costusoside-I and costusoside-J, two new furostanol saponins from the seeds of Costus speciosus. Phytochemistry 21, 911–915. Singh, S.B., Thakur, R.S., 1982b. Saponins from the seeds of Costus speciosus. J. Nat. Prod. 45, 667–671. Singh, U.P., Srivastava, B.P., 1992. Antifungal activity of steroid saponins and sapogenins from Avena sativa and Costus speciosus. Nat. Rio Claro 17, 71–77. Srivastava, S., Singh, P., Jha, K.K., Mishra, G., Srivastava, S., Khosa, R.L., 2013. Antiinflammatory, analgesic and antipyretic activities of aerial parts of Costus speciosus Koen. Indian J. Pharm. Sci. 75, 83–88. Vijayalaxmi, M.A., Saradha, N.C., 2008. Screening of Costus speciosus extracts for antioxidant activity. Fitoterapia 79, 197–198. Virella, G., 1998. Immunoserology. In: Virella, G. (Ed.), Medical Immunology, 4th ed. Marcel Dekker, New York, pp. 259–281.

Vishalakshi, D.D., Asna, U., 2010. Nutrient profile and antioxidant components of Costus specious Sm. and Costus igneus. Indian J. Nat. Prod. Resour. 1, 116–118. Wang, G., Li, G.G., Li, T., Xu, J., Ma, F., Wu, X., Ye, W., Li, Y., 2013. Eudesmane-type sesquiterpene derivatives from Laggera alata. Phytochemistry 96, 201–207. Wu, F., Koike, K., Nikaido, T., Ishii, K., Ohmoto, T., Ikeda, K., 1995. Terpenoids and flavonoids from Arenaria kansuensis. Chem. Pharm. Bull. 38, 2281–2282. Xiao, Y., Zheng, Q., Zhang, Q., Sun, H., Gueritte, F., Zhao, Y., 2003. Eudesmane derivatives from Laggera pterodonta. Fitoterapia 74, 459–463. Yawer, M.A., Ahmed, E., Malik, A., Ashraf, M., Rasool, M.A., Afza, N., 2007. New lipoxygenase-inhibiting constituents from Calligonum polygonoids. Chem. Biodivers. 4, 1578–1585. Yuuya, S., Hagiwara, H., Suzuki, T., Ando, M., Yamada, A., Suda, K., Kataoka, T., Nagai, K., 1999. Guaianolides as immunomodulators. synthesis and biological activities of dehydrocostus lactone, mokko lactone, eremanthin, and their derivatives. J. Nat. Prod. 62, 22–30. Zdero, C., Bohlmannand, F., King, R.M., 1990. Eudesmane derivatives and other constituents from Apalochlamys spectabilis and Cassinia species. Phytochemistry 29, 3206 3206. Zhao, Y., Yue, J., He, Y., Lin, Z., Sun, H., 1997. Eleven new eudesmane derivatives from Laggera pterodonta. J. Nat. Prod. 60, 545–549. Zheng, Q.X., Xu, Z.J., Sun, X.F., Gueritte, F., Cesario, M., Cheng, C.H.K., Sun, H.D., Zhao, Y., 2003. New eudesmane and eremophilane derivatives from Laggera Alata. Chin. Chem. Lett. 14, 393–396.