PHYTOTHERAPY RESEARCH Phytother. Res. (2015) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5281
The Anti-Osteoporosis and Antioxidant Activities of Chemical Constituents from Chrysanthemum indicum Flowers Bui Thi Thuy Luyen,1 Bui Huu Tai,1,2 Nguyen Phuong Thao,1,2 Young Mi Lee,3 Sang Hyun Lee,4 Hae Dong Jang4* and Young Ho Kim1* 1
College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea Institute of Marine Biochemistry (IMBC), Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam 3 Department of Oriental Pharmacy, College of Pharmacy, Wonkwang University and Wonkwang Oriental Medicines Research Institute, Iksan, Jeonbuk 570-749, Republic of Korea 4 Department of Food and Nutrition, Hannam National University, Daejeon 305-811, Korea 2
Two new compounds, chrysinoneside A (1) and ()-trans-chrysanthenol-6-O-β-D-glucopyranoside (2), along with 17 known compounds (3–19) were isolated from Chrysanthemum indicum flowers. The total phenolic and flavonoid contents of various fractions were determined. The EtOAC fraction had the highest total phenolic content (525.84 ± 23.51 mg GAE/g DR) and the total flavonoid content (63.49 ± 3.32 mg QE/g DR). The EtOAc and water fractions showed the greatest peroxyl radical-scavenging capacity and the ability to reduce Cu(I) ions, with ORAC and CUPRAC values ranging from 24.00 ± 0.44 to 28.06 ± 1.35 and 16.90 ± 0.51 to 49.77 ± 0.97 μM, respectively. Compounds 5–11, 18, and 19 displayed strong effects in both peroxyl radical-scavenging and reducing capacity assays at a concentration of 10 μM. The anti-osteoporosis activity of these compounds was also evaluated. Compounds 10, 13, and 19 exhibited the most potent tartrate-resistant acid phosphatase activity in receptor activator of nuclear factor-κB ligand-induced osteoclastic RAW 264.7 cells with values of 105.95 ± 1.18, 110.32 ± 3.95, and 112.58 ± 6.42%, respectively. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: Chrysanthemum indicum; chrysinoneside A; ()-trans-Chrysanthenol-6-O-β-D-glucopyranoside; antioxidant activity; antiosteoporosis activity.
Supporting information may be found in the online version of this article (Supplementary Material)
INTRODUCTION Chrysanthemum indicum (Compositae) is a well-known herb and medicinal plant with small yellow flowers that is widely distributed in Korea. Its flowers have a long history of use in traditional Korea and Chinese medicine for the treatment of infectious diseases, including pneumonia and pertussis, and in the treatment of colitis, stomatitis, cancer, fever, sores, vertigo, inflammation, and hypertension. Its flowers have also been used in herbal teas to treat/prevent some eye diseases. The presence of flavonoids, terpenoids, and phenolic compounds has been detected in C. indicum flowers (CIF). Several phenolic and flavonoid compounds were isolated as active components, including luteolin, luteoloside, luteolin 7-Oβ-D-glucopyranosiduronic acid, acacetin 7-O-(6″-α-Lrhamnopyranosyl)-β-D-glucopyranoside, (2S)-eriodictyol 7-O-β-D-glucopyranosiduronic acid, (2R)-eriodictyol 7-O-β-D-glucopyranosiduronic acid, and eriodictyol chlorogenic acid (Yoshikawa et al., 1999, Matsuda et al., 2002). Quercitrin, myricetin which exhibited antioxidant activity were found as the most abundant flavonoid of CIF (Wu et al., 2010). Some of isolated compounds have * Correspondence to: Young Ho Kim, College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea; Hae Dong Jang, Department of Food and Nutrition, Hannam National University, Daejeon 305-811, Republic of Korea. [email protected] (Young Ho Kim); [email protected] (Hae Dong Jang) E-mail: [email protected]
Copyright © 2015 John Wiley & Sons, Ltd.
been shown to exhibit inhibitory activity against nitric oxide (NO) in lipopolysaccharide-activated macrophages and inhibitory activity against rat lens aldose reductase (Yoshikawa et al., 1999, Yoshikawa et al., 2000, Matsuda et al., 2002). Furthermore, several pharmacological effects of CIF extracts have been demonstrated, including central and peripheral analgesic properties, blood pressure lowering effect, acetylcholinesterase inhibitory activity, anti-bacterial and anti-viral effects, and antiinflammatory and immunomodulatory activities (Cheng et al., 2005, Zhu et al., 2005a, Cheon et al., 2009). The antioxidant activities of CIF extracts and herbal teas containing CIF have been widely investigated by various methods in vitro, including DPPH, hydroxyl, superoxide radical and nitrite scavenging, reducing power, and free radicalinduced DNA damage prevention activities. These data suggest that CIF extracts are a potent source of natural antioxidants. Despite the antioxidant activity exhibited by CIF extracts, published accounts of the antioxidant components of CIF are lacking. Osteoporosis is a family of disorders in which the systemic bone mass is reduced and the bone microarchitecture deteriorates, increasing the risk of bone fragility and fracture. Osteoblasts and osteoclasts are specialized cells responsible for bone formation and bone resorption, respectively (Zeng et al., 2014). Bone resorption is facilitated by osteoclasts, which are multinucleated cells derived from monocyte/macrophage lineage precursors in the presence of the polypeptide growth Received 06 August 2014 Revised 28 November 2014 Accepted 02 December 2014
B. T. T. LUYEN ET AL.
factor CSF-1 (colony-stimulating factor-1) and receptor activator of nuclear factor κB ligand (RANKL). CSF-1 and RANKL are required to induce the expression of genes involved in osteoclast maturation, including tartrate-resistant acid phosphatase (TRAP), cathepsin K (CATK), calcitonin receptor, and β3-integrin, leading to the development of mature osteoclasts (Boyle et al., 2003). Osteoclasts not only play a critical role in the physiological bone remodeling process, they are also engaged in pathological bone resorption in bone lytic diseases, such as rheumatoid arthritis, osteomyelitis, osteoporosis, and periodontal disease (Franco et al., 2011). Therefore, as part of our ongoing effort to find potent biological agents from natural sources, we report herein 2 new and 17 known constituents of CIF. The total phenolic and flavonoid contents of methanol, ethyl acetate, and water fractions were determined. The antioxidant effects of these fractions and isolated compounds were evaluated using oxygen radical absorbance capacity (ORAC) and cupric ion reducing antioxidant capacity (CUPRAC) assays. In addition, the anti-osteoporosis activities of these compounds were evaluated through their inhibitory effects on osteoclast differentiation.
MATERIALS AND METHODS General experimental procedures. Optical rotation was recorded on a JASCO DIP-370 automatic digital polarimeter. The nuclear magnetic resonance (NMR) spectra were measured using a JEOL ECA 600 spectrometer (JEOL, Tokyo, Japan) with TMS as the internal standard. The electrospray ionization (ESI) mass spectra were performed on an AGILENT 1100 LC-MSD trap spectrometer (Agilent Technologies, Palo Alto, CA, USA). The high-resolution electrospray ionization mass spectra (HR-ESI-MS) were obtained from an Agilent 6530 Accurate-Mass Q-TOF LC/MS system. Gas chromatography (GC) spectra were recorded on a Shidmazu-2010 spectrometer (Shimadzu, Kyoto, Japan). Silica gel (70– 230, 230–400 mesh, Merck, Whitehouse Station, NJ), YMC RP-18 resins (75 μm, Fuji Silysia Chemical Ltd., Kasugai, Japan), and Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden) were used as absorbents in the column chromatography. Thin layer chromatography (TLC) plates (silica gel 60 F254 and RP-18 F254, 0.25 μm, Merck) were purchased from Merck KGaA (Darmstadt, Germany). Spots were detected under UV radiation (254 and 365 nm) and by spraying the plates with 10% H2SO4 followed by heating with a heat gun.
Plant material. The flowers of C. indicum were collected in Jeju, Korea in 2011 and taxonomically identified by Prof Jae Hyun Lee at the Dongkuk University. A voucher specimen (CNU-11102) was deposited at the Herbarium of College of Pharmacy, Chungnam National University.
Extraction and isolation. The dried flowers of C. indicum (3.0 kg) were extracted with methanol (10 L × 3 times) under reflux condition. Evaporation of the solvent under reduced pressure gave MeOH extract Copyright © 2015 John Wiley & Sons, Ltd.
(400 g). The MeOH extract was suspended in H2O and successively separated with CH2Cl2 and EtOAc to yield CH2Cl2 extract (200 g), EtOAc extract (110 g), and water layer, respectively. Ethyl acetate extract was fractionated on a silica gel column chromatography (CC) eluting with gradient solvent systems of CH2Cl2/MeOH (0–100% MeOH, step-wise) to obtain eight fractions (C.1 through C.8). From fraction C.3, compound 13 (100 mg) was isolated by YMC reverse-phase (RP-18) CC using MeOH/H2O (1/1, v/v) as eluent, and further purified by silica gel CC eluting with CH2Cl2/MeOH (3/1, v/v). Using YMC RP-18 CC with solvent system MeOH/H2O (1/1. v/v) and silica gel CC with CH2Cl2/ acetone (3/1. v/v) as eluent at C.4 fraction, compounds 1 (5 mg), 8 (150 mg), and 10 (10 mg) were obtained. Fraction C.5 was separated on YMC RP-18 CC with MeOH/H2O (1/1, v/v) as eluent to give two fractions (C.5.1 and C.5.2). Compounds 7 (100 mg), 9 (34 mg), and 17 (10 mg) were obtained from fraction C.5.1 by Sephadex LH-20 CC eluting with MeOH/H2O (2/3, v/v), and purified by YMC RP-18 CC with MeOH/H2O (1/2, v/v) as eluent. Fraction C.5.2 was isolated on silica gel CC eluting with CH2Cl2/H2O (4/4, v/v) to afford compounds 12 (36 mg), 14 (7 mg), and 15 (50 mg). The water layer was fractionated on a Diaion HP-20 CC eluting with gradient solvent systems of MeOH/H2O (0–100% MeOH, step-wise) to obtain three fractions (D.1 through D.3). Fraction D.2 was isolated by CC over silica gel, eluting with gradient solvent systems of CH2Cl2/MeOH (10/1-1/1, step by step) to obtain four fractions (D.2.1 through D.2.4). Compounds 18 (13 mg) and 19 (25 mg) were afforded from fraction D.2.2 by Sephadex LH-20 CC eluting with MeOH/H2O (1/1, v/v), and purified by YMC RP-18 CC using MeOH/H2O (1/3, v/v) as eluent. Fraction D.2.3 was fractioned on YMC RP-18 CC eluting with MeOH/H2O (1/2, v/v), and further isolated by silica gel CC using solvent system CH2Cl2/MeOH (4/1, v/v) to give compounds 5 (72 mg) and 6 (5 mg). Compound 4 (20 mg) was obtained from fraction D.2.4 by YMC RP18 CC eluting with MeOH/H2O (1/3, v/v) and silica gel CC using CH2Cl2/MeOH (3/1, v/v) as eluent. Fraction D.3 was fractioned on Sephadex LH-20 CC eluting with MeOH/H2O (1/2, v/v) to give three fractions (D.3.1 through D.3.3). Fraction D.3.2 was subjected by YMC RP-18 CC eluting with MeOH/H2O (1/1. v/v) and further isolated by silica gel CC using solvent system CH2Cl2/ MeOH (4/1, v/v) to give compounds 2 (10 mg), 3 (5 mg), and 16 (40 mg). Compound 11 (7 mg) was obtained from fraction D.3.3 by YMC RP-18 CC eluting with MeOH/H2O (1/3, v/v) and silica gel CC using CH2Cl2/ MeOH (3/1, v/v) as eluent. Chrysinoneside A (1) Colorless oil; ½α26 D +4.32(c 0.08, MeOH); UV (MeOH) λmax (log ε): 211 (3.48) nm; IR (KBr) νmax: 3380, 2930, 1760, 1076, 1039 cm1; HR-ESIMS m/z: 447.1792 [M + Cl] (Calcd. C21H32O8Cl for 447.1786); 1H NMR (600 MHz, CD3OD) and 13C NMR (150 MHz, CD3OD) are given in Table 2. ()-trans-chrysanthenol-6-O-β-D-glucopyranoside (2) Colorless oil; ½α26 D 33.96 (c 0.23, MeOH); UV (MeOH) λmax (log ε): 213 (3.51) nm; IR (KBr) νmax: 3364, 2928, 1075, 1041 cm1; HR-ESI-MS m/z: 337.1636 [M + Na]+ (Calcd. C16H26O6Na for 337.1627); 1H NMR (600 MHz, CD3OD) and 13C NMR (150 MHz, CD3OD) are given in Table 2. Phytother. Res. (2015)
ANTI-OSTEOPOROSIS AND ANTIOXIDANT COMPONENTS OF C. INDICUM FLOWERS
Acid hydrolysis and sugar identification. Each compound (2.0 mg) was dissolved in 1.0 N HCl (dioxane/H2O, 1:1, v/v, 1.0 mL) and then heated to 80 °C in a water bath for 3 h. The acidic solution was neutralized with silver carbonate and the solvent thoroughly driven out under N2 gas overnight. After extraction with ethyl acetate, the aqueous layer was first TLC analysized (individual and co-analysis with standard sample: Glucose Rf: 0.29; CHCl3/MeOH/H2O, 3:2:0.3) and then concentrated to dryness using N2 gas. The residue was dissolved in 0.1 mL of dry pyridine, and then L-cysteine methyl ester hydrochloride in pyridine (0.06 M, 0.1 mL) was added to the solution. The reaction mixture was heated at 60 °C for 2 h, and 0.1 mL of trimethylsilylimidazole solution was added, followed by heating at 60 °C for 1.5 h. The dried product was partitioned with n-hexane and H2O (0.1 mL, each), and the organic layer was analyzed by gas liquid chromatography (GC): Column: column SPB-1 (0.25 mm × 30 m); detector FID, column temp 210 °C, injector temperature 270 °C, detector temperature 300 °C, carrier gas He. The absolute configuration of the monosaccharide was confirmed to be D-glucose by comparison of the retention time of the monosaccharide derivative (tR 14.11 min) with that of authentic sugar derivative samples prepared in the same manner (D-glucose derivative tR 14.11 min, L-glucose derivative tR 14.26 min). Total phenolic content. The total phenolic content (TPC) was determined using the Folin–Ciocalteau (FC) colorimetry method (Waterhouse, 2002). A total of 50 μL of sample, a gallic acid calibration standard, or blank was mixed with 4.2 mL of FC reagent (6%) and 0.75 mL of Na2CO3 (20%). The tubes were incubated for 90 min at room temperature in the dark, after which the absorbance was measured at 760 nm in a spectrophotometer. The analyses were performed in triplicate. The TPC of each sample, expressed as the gallic acid equivalent (GAE) in milligrams per gram of dry residue (mg GAE/g DR), was calculated using a gallic acid standard curve (concentration range: 20–200 μg/mL). Total flavonoid content. The total flavonoid content was determined using a modified colorimetric assay based on a previously described method with quercetin (SigmaAldrich Co.) as the standard (Swieca et al., 2014). A total of 1 mL of extract was mixed with 1 mL of 2% AlCl3·6H2O in ethanol and incubated at room temperature for 10 min. The absorbance of the solutions at 430 nm was measured after incubation for 15 min at room temperature. The total flavonoid content of each sample, expressed as mg quercetin equivalent per g of dry residue (mgQE/g DR), was calculated using quercetin calibration curve (concentration range: 50–500 μg/mL). Oxygen radical absorbance capacity (ORAC) assay. The assay was carried out on a Tecan GENios multifunctional plate reader (Tecan, Salzburg, Austria) with fluorescent filters using excitation and emission wavelengths of 485 nm and 535 nm, respectively. In the final assay mixture, 40-nM fluorescein was used as a target of free radical attack with 20 mM 2,2-azobis dihydrochloride (AAPH) as a peroxyl radical generator in the presence of different concentrations of sample or positive control (Kurihara et al., 2004). Trolox (1.0 μM) was used as a positive control and prepared fresh daily. The analyzer Copyright © 2015 John Wiley & Sons, Ltd.
was programmed to record the fluorescence of fluorescein every 2 min during 200-min incubation at 37 °C after addition of AAPH. All fluorescence measurements were expressed relative to the initial reading. Final results were calculated based on the difference in the area under the fluorescence decay curve between the blank and each sample. ORACROO· was expressed as micromoles of Trolox equivalents (TE). One ORAC unit is equivalent to the net protection area provided by 1 μM of Trolox.
Determination of reduction capacity. The reducing abilities of isolated compounds were determined according to cupric ion reducing antioxidant capacity (CUPRAC) method (Aruoma et al., 1998). In brief, 40 μL of different concentrations of the compounds was mixed with 160 μL of the mixture containing 0.5 mM CuCl2 and 0.75 mM neocuproine in 10.0 mM phosphate buffer, pH 7.4. Reaction mixture was incubated at room temperature for 1 h, and then the absorbance was measured with a micro-plate reader at 454 nm. Increased absorbance of the reaction mixture indicates increased reducing power and was converted into Cu(I) ion concentrations. The CUPRAC values were expressed as μM of Cu(I) ion reduced from Cu (II) by antioxidant. TRAP activity. RAW 264.7 (macrophages (preosteoclasts) from BALB/c mice) cells were cultured in 96-well plates (1 × 104 cells/mL) containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) FBS for 2 days. Medium was then replaced with test samples in differentiation medium containing 50 ng/mL RANKL. The differentiation medium was replaced every 2 days. After differentiating the RAW 264.7 cells into osteoclasts for 5 days, the medium was removed, and the cell monolayer was gently washed twice using ice-cold PBS. The cells were fixed in 3.5% formaldehyde for 10 min and ethanol– acetone (1:1) for 1 min. Subsequently, the dried cells were incubated in 50.0 mM citrate buffer (pH 4.5) containing 10 mM sodium tartrate and 6 mM p-nitrophenylphosphate (PNPP). After 1 h incubation, the reaction mixtures were transferred to new well plates containing an equal volume of 0.1 N NaOH. Absorbance was measured at 408 nm using an ELISA reader. TRAP activity was expressed as a percentage of the control. Statistical analysis. All data represent the mean ± S.D. of at least three independent experiments performed in triplicates. Statistical significance is determined by oneway ANOVA followed by Dunnett’s multiple comparison test, P < 0.05, using GraphPad Prism 6 program (GraphPad Software Inc., San Diego, CA, USA).
RESULTS AND DISCUSSION Antioxidant activity of the extracts from C. indicum flowers A methanol extract of CIF was suspended in H2O and then partitioned sequentially with CH2Cl2 and EtOAc Phytother. Res. (2015)
B. T. T. LUYEN ET AL.
to give corresponding CH2Cl2, EtOAc, and H2O fractions. These fractions were evaluated for their antioxidant effects at concentrations of 1.0 and 5.0 μg/mL (see Table 1). Of these, the EtOAc and H2O fractions showed significant activity against peroxyl radicals. At a concentration of 5.0 μg/mL, the total antioxidant capacities in the ORAC assay were determined to be 27.39 ± 0.20, 28.06 ± 1.35 times higher than the protection provided by 1.0 μM Trolox, respectively. The small difference in their ORAC values at a concentration of 1.0 μg/mL (26.81 ± 0.21, 24.00 ± 0.44, respectively) suggests the potent antioxidant activity of these fractions. Moreover, the EtOAc and H2O fractions displayed good reducing power with CUPRAC values of 43.61 ± 1.30, 45.07 ± 1.63 and 16.90 ± 0.51, 49.77 ± 0.97, at concentrations of 1.0 and 5.0 μg/mL, respectively. Total phenolic, total flavonoid contents of methanol extract, and its soluble fractions from C. indicum flowers Phenolic compounds, as secondary metabolites, are widely distributed in fruits and vegetables. They are formed via the shikimate pathway and are considered to be the main contributors to the antioxidant capacity of plants and to protect organisms from damage caused by free radical-induced oxidative stress. The antioxidant activity of phenolics is mainly due to their redox properties such as reducing agents, hydrogen donors, and free radical scavengers (Ozsoy et al., 2009). The correlation between antioxidant activity and TPC has been reported in numerous studies (Ozyurek et al., 2014). Thus, the TPC was determined using standard gallic acid as a standard, and the results were expressed as mg GAE/g DR. Table 1 shows the TPC of the crude extract and its soluble fractions. Accordingly, the TPC of the EtOAc fraction (525.84 ± 23.51 mg GAE/g DR) was higher than that of the methanol extract (68.59 ± 0.59 mg GAE/g DR) and water fraction (38.58 ± 0.64 mg GAE/g DR). Flavonoids, one of the most diverse and widespread groups of natural products, possess a wide variety of pharmacological and biochemical properties, including antioxidative, anti-microbial, anti-allergenic, anti-viral, antiinflammatory, and vasodilatory actions. The antioxidant activity of flavonoids is of great interest because of their ability to inhibit free radical formation and to scavenge free radicals (Pietta, 2000). Our results show that the highest flavonoid content was detected in the EtOAc fraction (63.49 ± 3.32 mg QE/g DR) while the lowest level was detected in the methanol extract (9.27 ± 0.97 mg QE/g DR) (see Table 1). These results indicate that the TPC and total flavonoid content of the
methanol extract were effectively enriched in the EtOAc soluble fraction. Structural determination Based on these data, we selected the EtOAc and aqueous fractions for an analysis of their chemical constituents. Using a combination of various chromatographic steps over silica gel and YMC reversed-phase C18 resin, compounds 1–19 were isolated (see Fig. 1). Of these, two new compounds (1 and 2) were obtained. Compound 1 was obtained as a colorless oil. The HRESI-MS of 1 exhibited a pseudo-molecular ion peak at m/z 447.1792 [M + Cl] (Calcd. C21H32O8Cl for 447.1786), which is consistent with the molecular formula of C21H32O8. The IR spectrum displayed a strong band at 1760 cm1, indicating the presence of a lactone functional group. The 1H NMR and 13C NMR spectra of 1 showed signals assignable to three methyls at δH 1.44 (d, J = 7.2 Hz, H-13), 1.49 (s, H-15), and 1.70 (s, H14); two oxygenated methines at δH 4.60 (ddd, J = 1.8; 10.5; 10.5 Hz, H-6) and 4.72 (t, J = 9.6 Hz, H-8); and two olefinic protons at δH 4.97 (d, J = 7.8 Hz, H-3) and 4.53 (d, J = 10.2 Hz, H-9) (see Table 2). The 13C NMR and DEPT spectra revealed 21 carbon signals, including three methyl carbons, four methylene carbons, ten methine carbons (including six oxygenated, two olefinic, and two aliphatic methines), and three quaternary carbons with one carbonyl carbon. Among them, an anomeric (δC 98.7) and five other oxygenated carbon signals (δC 78.3, 78.2, 74.9, 72.1, and 63.1) were assigned to the sugar moiety. Assignment of the protons and carbons in the aglycone moiety was done based on twodimensional (2D) spectra. The correlation spectroscopy (COSY) spectra of 1 indicated two individual correlation systems (see Fig. 2): fragment A [H2-1 (δH 2.03, 2.31)/H2-2 (δH 2.16, 2.33)/H-3 (δH 4.97)] and fragment B [H-5α (δH 2.44)/H-6 (δH 4.60)/H-7 (δH 2.55)/H-8 (δH 4.72)/H-9 (δH 4.53), H-7/H-11 (δH 2.86)/H-13 (δH 1.44)]. The connectivity of the two fragments was determined through HMBC correlations. Diagnostic HMBC correlations were observed between a tertiary methyl H3-14 (δH 1.70) and carbons C-1 (δC 40.0), C-9 (δC 130.1), and C-10 (δC 140.3). Another tertiary methyl H3-15 (δH 1.49) correlated with carbons C-3 (δC 131.4), C-4 (δC 133.0), and C-5 (δC 47.6). These data suggested that fragments A and B connected with each other via C-4 and C-10 (see Fig. 2). The above mentioned evidences suggested a germacranolide sesquiterpene lactone glycoside, bearing oxygen functions (hydroxyl and lactone) at C-6, C-8 and two trisubstituted double bonds at C-3/C-4 and C-9/C-10 (Rustaiyan et al., 1990, Sanz
Table 1. Peroxyl radical-scavenging capacity, reducing capacity, total phenolic contents (TPC), and total flavonoid contents (TFC) of the methanol extracts of C. indicum flowers and its soluble fractions. (ME: methanol extract, DMF: dichloromethane fraction, EAF: ethyl acetate fraction, WF: water fraction) ORACRCOO (TE, μM) Sample ME DMF EAF WF
Reduction power (Copper(I) ion, μM)
1 μg/mL
5 μg/mL
1 μg/mL
5 μg/mL
5.47 ± 0.28 4.70 ± 0.25 26.81 ± 0.21 24.00 ± 0.44
21.22 ± 0.19 19.53 ± 0.38 27.39 ± 0.20 28.06 ± 1.35
2.68 ± 0.13 3.56 ± 0.23 43.61 ± 1.30 16.90 ± 0.51
9.43 ± 0.30 14.34 ± 0.85 45.70 ± 1.63 49.77 ± 0.97
Copyright © 2015 John Wiley & Sons, Ltd.
TPC (mg GAE/g DR)
TFC (mg QE/g DR)
68.59 ± 0.59 NT 525.84 ± 23.51 38.58 ± 0.64
9.27 ± 0.97 NT 63.49 ± 3.32 12.47 ± 0.58 Phytother. Res. (2015)
ANTI-OSTEOPOROSIS AND ANTIOXIDANT COMPONENTS OF C. INDICUM FLOWERS
Figure 1. The chemical structures of compounds 1–19 from the flowers of C. indicum.
et al., 1990). A δ-lactone ring was formed between C-12 and C-6, which was supported by the downfield shift of C-6 (δC 81.4); HMBC correlations between H3-13/C-7 (δC 54.6), C-11 (δC 41.5), and C-12 (δC 182.2); and a weak HMBC correlation between H-6 and C-12. Moreover, glucosidic bonding at C-8 was indicated by both HMBC correlations of an anomeric proton H-1′ (δH 4.28) with C-8 (δC 70.6), and an oxymethine proton H-8 (δH 4.72) with C-1′ (δC 98.7). Furthermore, acid
hydrolysis of 1 followed by TLC, GC analysis, as well as a comparison with authentic D-glucose, confirmed the presence of a D-glucose moiety in 1. Next, the stereogenic centers of 1 were confirmed by Rotating frame nuclear Overhauser effect spectroscopy (ROESY) experiments (see Fig. 2). Steric interactions were observed for H3-13 (δH 1.44) with both H-6 (δH 4.60) and H-8 (δH 4.72) in the ROESY spectra of 1, indicating β-orientations at C-7, C-11 and α-orientations at C-6, C-8. The α/β/α configuration system of C-6/C7/C-8 was also confirmed by large values for both JH7/H-6 and JH-7/H-8 (J = 10.5 Hz), as well as in agreement with previous literatures (Rustaiyan et al., 1990). Moreover, the circular dichroism (CD) spectra of 1 exhibited a negative Cotton effect at 200 nm (not a maximum, lowest recorded value) and a positive band at 216 nm, which were assigned to the trans, trans-cyclodecadiens (Takeda and Horibe, 1975). In addition, the βconfiguration of C-11 was also supported by ROESY correlations of H-7 (δH 2.55)/H-11 (δH 2.86), H-7/H-5α (δH 2.44) and by the absence of a ROESY correlation between H-7 and H3-13 (δH 1.44). The olefinic geometries were identified to be both 3E and 9E according to the ROESY interactions of H-3 (δH 4.97)/H-5α, H3-15 (δH 1.49)/H-2β (δH 2.33), H-9 (δH 4.53)/H-1α (δH 2.03), and H3-14 (δH 1.70)/H-8 (δH 4.72), as well as the up-field chemical shifts of C-14 and C-15 (
The Anti-Osteoporosis and Antioxidant Activities of Chemical Constituents from Chrysanthemum indicum Flowers Bui Thi Thuy Luyen,1 Bui Huu Tai,1,2 Nguyen Phuong Thao,1,2 Young Mi Lee,3 Sang Hyun Lee,4 Hae Dong Jang4* and Young Ho Kim1* 1
College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea Institute of Marine Biochemistry (IMBC), Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam 3 Department of Oriental Pharmacy, College of Pharmacy, Wonkwang University and Wonkwang Oriental Medicines Research Institute, Iksan, Jeonbuk 570-749, Republic of Korea 4 Department of Food and Nutrition, Hannam National University, Daejeon 305-811, Korea 2
Two new compounds, chrysinoneside A (1) and ()-trans-chrysanthenol-6-O-β-D-glucopyranoside (2), along with 17 known compounds (3–19) were isolated from Chrysanthemum indicum flowers. The total phenolic and flavonoid contents of various fractions were determined. The EtOAC fraction had the highest total phenolic content (525.84 ± 23.51 mg GAE/g DR) and the total flavonoid content (63.49 ± 3.32 mg QE/g DR). The EtOAc and water fractions showed the greatest peroxyl radical-scavenging capacity and the ability to reduce Cu(I) ions, with ORAC and CUPRAC values ranging from 24.00 ± 0.44 to 28.06 ± 1.35 and 16.90 ± 0.51 to 49.77 ± 0.97 μM, respectively. Compounds 5–11, 18, and 19 displayed strong effects in both peroxyl radical-scavenging and reducing capacity assays at a concentration of 10 μM. The anti-osteoporosis activity of these compounds was also evaluated. Compounds 10, 13, and 19 exhibited the most potent tartrate-resistant acid phosphatase activity in receptor activator of nuclear factor-κB ligand-induced osteoclastic RAW 264.7 cells with values of 105.95 ± 1.18, 110.32 ± 3.95, and 112.58 ± 6.42%, respectively. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: Chrysanthemum indicum; chrysinoneside A; ()-trans-Chrysanthenol-6-O-β-D-glucopyranoside; antioxidant activity; antiosteoporosis activity.
Supporting information may be found in the online version of this article (Supplementary Material)
INTRODUCTION Chrysanthemum indicum (Compositae) is a well-known herb and medicinal plant with small yellow flowers that is widely distributed in Korea. Its flowers have a long history of use in traditional Korea and Chinese medicine for the treatment of infectious diseases, including pneumonia and pertussis, and in the treatment of colitis, stomatitis, cancer, fever, sores, vertigo, inflammation, and hypertension. Its flowers have also been used in herbal teas to treat/prevent some eye diseases. The presence of flavonoids, terpenoids, and phenolic compounds has been detected in C. indicum flowers (CIF). Several phenolic and flavonoid compounds were isolated as active components, including luteolin, luteoloside, luteolin 7-Oβ-D-glucopyranosiduronic acid, acacetin 7-O-(6″-α-Lrhamnopyranosyl)-β-D-glucopyranoside, (2S)-eriodictyol 7-O-β-D-glucopyranosiduronic acid, (2R)-eriodictyol 7-O-β-D-glucopyranosiduronic acid, and eriodictyol chlorogenic acid (Yoshikawa et al., 1999, Matsuda et al., 2002). Quercitrin, myricetin which exhibited antioxidant activity were found as the most abundant flavonoid of CIF (Wu et al., 2010). Some of isolated compounds have * Correspondence to: Young Ho Kim, College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea; Hae Dong Jang, Department of Food and Nutrition, Hannam National University, Daejeon 305-811, Republic of Korea. [email protected] (Young Ho Kim); [email protected] (Hae Dong Jang) E-mail: [email protected]
Copyright © 2015 John Wiley & Sons, Ltd.
been shown to exhibit inhibitory activity against nitric oxide (NO) in lipopolysaccharide-activated macrophages and inhibitory activity against rat lens aldose reductase (Yoshikawa et al., 1999, Yoshikawa et al., 2000, Matsuda et al., 2002). Furthermore, several pharmacological effects of CIF extracts have been demonstrated, including central and peripheral analgesic properties, blood pressure lowering effect, acetylcholinesterase inhibitory activity, anti-bacterial and anti-viral effects, and antiinflammatory and immunomodulatory activities (Cheng et al., 2005, Zhu et al., 2005a, Cheon et al., 2009). The antioxidant activities of CIF extracts and herbal teas containing CIF have been widely investigated by various methods in vitro, including DPPH, hydroxyl, superoxide radical and nitrite scavenging, reducing power, and free radicalinduced DNA damage prevention activities. These data suggest that CIF extracts are a potent source of natural antioxidants. Despite the antioxidant activity exhibited by CIF extracts, published accounts of the antioxidant components of CIF are lacking. Osteoporosis is a family of disorders in which the systemic bone mass is reduced and the bone microarchitecture deteriorates, increasing the risk of bone fragility and fracture. Osteoblasts and osteoclasts are specialized cells responsible for bone formation and bone resorption, respectively (Zeng et al., 2014). Bone resorption is facilitated by osteoclasts, which are multinucleated cells derived from monocyte/macrophage lineage precursors in the presence of the polypeptide growth Received 06 August 2014 Revised 28 November 2014 Accepted 02 December 2014
B. T. T. LUYEN ET AL.
factor CSF-1 (colony-stimulating factor-1) and receptor activator of nuclear factor κB ligand (RANKL). CSF-1 and RANKL are required to induce the expression of genes involved in osteoclast maturation, including tartrate-resistant acid phosphatase (TRAP), cathepsin K (CATK), calcitonin receptor, and β3-integrin, leading to the development of mature osteoclasts (Boyle et al., 2003). Osteoclasts not only play a critical role in the physiological bone remodeling process, they are also engaged in pathological bone resorption in bone lytic diseases, such as rheumatoid arthritis, osteomyelitis, osteoporosis, and periodontal disease (Franco et al., 2011). Therefore, as part of our ongoing effort to find potent biological agents from natural sources, we report herein 2 new and 17 known constituents of CIF. The total phenolic and flavonoid contents of methanol, ethyl acetate, and water fractions were determined. The antioxidant effects of these fractions and isolated compounds were evaluated using oxygen radical absorbance capacity (ORAC) and cupric ion reducing antioxidant capacity (CUPRAC) assays. In addition, the anti-osteoporosis activities of these compounds were evaluated through their inhibitory effects on osteoclast differentiation.
MATERIALS AND METHODS General experimental procedures. Optical rotation was recorded on a JASCO DIP-370 automatic digital polarimeter. The nuclear magnetic resonance (NMR) spectra were measured using a JEOL ECA 600 spectrometer (JEOL, Tokyo, Japan) with TMS as the internal standard. The electrospray ionization (ESI) mass spectra were performed on an AGILENT 1100 LC-MSD trap spectrometer (Agilent Technologies, Palo Alto, CA, USA). The high-resolution electrospray ionization mass spectra (HR-ESI-MS) were obtained from an Agilent 6530 Accurate-Mass Q-TOF LC/MS system. Gas chromatography (GC) spectra were recorded on a Shidmazu-2010 spectrometer (Shimadzu, Kyoto, Japan). Silica gel (70– 230, 230–400 mesh, Merck, Whitehouse Station, NJ), YMC RP-18 resins (75 μm, Fuji Silysia Chemical Ltd., Kasugai, Japan), and Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden) were used as absorbents in the column chromatography. Thin layer chromatography (TLC) plates (silica gel 60 F254 and RP-18 F254, 0.25 μm, Merck) were purchased from Merck KGaA (Darmstadt, Germany). Spots were detected under UV radiation (254 and 365 nm) and by spraying the plates with 10% H2SO4 followed by heating with a heat gun.
Plant material. The flowers of C. indicum were collected in Jeju, Korea in 2011 and taxonomically identified by Prof Jae Hyun Lee at the Dongkuk University. A voucher specimen (CNU-11102) was deposited at the Herbarium of College of Pharmacy, Chungnam National University.
Extraction and isolation. The dried flowers of C. indicum (3.0 kg) were extracted with methanol (10 L × 3 times) under reflux condition. Evaporation of the solvent under reduced pressure gave MeOH extract Copyright © 2015 John Wiley & Sons, Ltd.
(400 g). The MeOH extract was suspended in H2O and successively separated with CH2Cl2 and EtOAc to yield CH2Cl2 extract (200 g), EtOAc extract (110 g), and water layer, respectively. Ethyl acetate extract was fractionated on a silica gel column chromatography (CC) eluting with gradient solvent systems of CH2Cl2/MeOH (0–100% MeOH, step-wise) to obtain eight fractions (C.1 through C.8). From fraction C.3, compound 13 (100 mg) was isolated by YMC reverse-phase (RP-18) CC using MeOH/H2O (1/1, v/v) as eluent, and further purified by silica gel CC eluting with CH2Cl2/MeOH (3/1, v/v). Using YMC RP-18 CC with solvent system MeOH/H2O (1/1. v/v) and silica gel CC with CH2Cl2/ acetone (3/1. v/v) as eluent at C.4 fraction, compounds 1 (5 mg), 8 (150 mg), and 10 (10 mg) were obtained. Fraction C.5 was separated on YMC RP-18 CC with MeOH/H2O (1/1, v/v) as eluent to give two fractions (C.5.1 and C.5.2). Compounds 7 (100 mg), 9 (34 mg), and 17 (10 mg) were obtained from fraction C.5.1 by Sephadex LH-20 CC eluting with MeOH/H2O (2/3, v/v), and purified by YMC RP-18 CC with MeOH/H2O (1/2, v/v) as eluent. Fraction C.5.2 was isolated on silica gel CC eluting with CH2Cl2/H2O (4/4, v/v) to afford compounds 12 (36 mg), 14 (7 mg), and 15 (50 mg). The water layer was fractionated on a Diaion HP-20 CC eluting with gradient solvent systems of MeOH/H2O (0–100% MeOH, step-wise) to obtain three fractions (D.1 through D.3). Fraction D.2 was isolated by CC over silica gel, eluting with gradient solvent systems of CH2Cl2/MeOH (10/1-1/1, step by step) to obtain four fractions (D.2.1 through D.2.4). Compounds 18 (13 mg) and 19 (25 mg) were afforded from fraction D.2.2 by Sephadex LH-20 CC eluting with MeOH/H2O (1/1, v/v), and purified by YMC RP-18 CC using MeOH/H2O (1/3, v/v) as eluent. Fraction D.2.3 was fractioned on YMC RP-18 CC eluting with MeOH/H2O (1/2, v/v), and further isolated by silica gel CC using solvent system CH2Cl2/MeOH (4/1, v/v) to give compounds 5 (72 mg) and 6 (5 mg). Compound 4 (20 mg) was obtained from fraction D.2.4 by YMC RP18 CC eluting with MeOH/H2O (1/3, v/v) and silica gel CC using CH2Cl2/MeOH (3/1, v/v) as eluent. Fraction D.3 was fractioned on Sephadex LH-20 CC eluting with MeOH/H2O (1/2, v/v) to give three fractions (D.3.1 through D.3.3). Fraction D.3.2 was subjected by YMC RP-18 CC eluting with MeOH/H2O (1/1. v/v) and further isolated by silica gel CC using solvent system CH2Cl2/ MeOH (4/1, v/v) to give compounds 2 (10 mg), 3 (5 mg), and 16 (40 mg). Compound 11 (7 mg) was obtained from fraction D.3.3 by YMC RP-18 CC eluting with MeOH/H2O (1/3, v/v) and silica gel CC using CH2Cl2/ MeOH (3/1, v/v) as eluent. Chrysinoneside A (1) Colorless oil; ½α26 D +4.32(c 0.08, MeOH); UV (MeOH) λmax (log ε): 211 (3.48) nm; IR (KBr) νmax: 3380, 2930, 1760, 1076, 1039 cm1; HR-ESIMS m/z: 447.1792 [M + Cl] (Calcd. C21H32O8Cl for 447.1786); 1H NMR (600 MHz, CD3OD) and 13C NMR (150 MHz, CD3OD) are given in Table 2. ()-trans-chrysanthenol-6-O-β-D-glucopyranoside (2) Colorless oil; ½α26 D 33.96 (c 0.23, MeOH); UV (MeOH) λmax (log ε): 213 (3.51) nm; IR (KBr) νmax: 3364, 2928, 1075, 1041 cm1; HR-ESI-MS m/z: 337.1636 [M + Na]+ (Calcd. C16H26O6Na for 337.1627); 1H NMR (600 MHz, CD3OD) and 13C NMR (150 MHz, CD3OD) are given in Table 2. Phytother. Res. (2015)
ANTI-OSTEOPOROSIS AND ANTIOXIDANT COMPONENTS OF C. INDICUM FLOWERS
Acid hydrolysis and sugar identification. Each compound (2.0 mg) was dissolved in 1.0 N HCl (dioxane/H2O, 1:1, v/v, 1.0 mL) and then heated to 80 °C in a water bath for 3 h. The acidic solution was neutralized with silver carbonate and the solvent thoroughly driven out under N2 gas overnight. After extraction with ethyl acetate, the aqueous layer was first TLC analysized (individual and co-analysis with standard sample: Glucose Rf: 0.29; CHCl3/MeOH/H2O, 3:2:0.3) and then concentrated to dryness using N2 gas. The residue was dissolved in 0.1 mL of dry pyridine, and then L-cysteine methyl ester hydrochloride in pyridine (0.06 M, 0.1 mL) was added to the solution. The reaction mixture was heated at 60 °C for 2 h, and 0.1 mL of trimethylsilylimidazole solution was added, followed by heating at 60 °C for 1.5 h. The dried product was partitioned with n-hexane and H2O (0.1 mL, each), and the organic layer was analyzed by gas liquid chromatography (GC): Column: column SPB-1 (0.25 mm × 30 m); detector FID, column temp 210 °C, injector temperature 270 °C, detector temperature 300 °C, carrier gas He. The absolute configuration of the monosaccharide was confirmed to be D-glucose by comparison of the retention time of the monosaccharide derivative (tR 14.11 min) with that of authentic sugar derivative samples prepared in the same manner (D-glucose derivative tR 14.11 min, L-glucose derivative tR 14.26 min). Total phenolic content. The total phenolic content (TPC) was determined using the Folin–Ciocalteau (FC) colorimetry method (Waterhouse, 2002). A total of 50 μL of sample, a gallic acid calibration standard, or blank was mixed with 4.2 mL of FC reagent (6%) and 0.75 mL of Na2CO3 (20%). The tubes were incubated for 90 min at room temperature in the dark, after which the absorbance was measured at 760 nm in a spectrophotometer. The analyses were performed in triplicate. The TPC of each sample, expressed as the gallic acid equivalent (GAE) in milligrams per gram of dry residue (mg GAE/g DR), was calculated using a gallic acid standard curve (concentration range: 20–200 μg/mL). Total flavonoid content. The total flavonoid content was determined using a modified colorimetric assay based on a previously described method with quercetin (SigmaAldrich Co.) as the standard (Swieca et al., 2014). A total of 1 mL of extract was mixed with 1 mL of 2% AlCl3·6H2O in ethanol and incubated at room temperature for 10 min. The absorbance of the solutions at 430 nm was measured after incubation for 15 min at room temperature. The total flavonoid content of each sample, expressed as mg quercetin equivalent per g of dry residue (mgQE/g DR), was calculated using quercetin calibration curve (concentration range: 50–500 μg/mL). Oxygen radical absorbance capacity (ORAC) assay. The assay was carried out on a Tecan GENios multifunctional plate reader (Tecan, Salzburg, Austria) with fluorescent filters using excitation and emission wavelengths of 485 nm and 535 nm, respectively. In the final assay mixture, 40-nM fluorescein was used as a target of free radical attack with 20 mM 2,2-azobis dihydrochloride (AAPH) as a peroxyl radical generator in the presence of different concentrations of sample or positive control (Kurihara et al., 2004). Trolox (1.0 μM) was used as a positive control and prepared fresh daily. The analyzer Copyright © 2015 John Wiley & Sons, Ltd.
was programmed to record the fluorescence of fluorescein every 2 min during 200-min incubation at 37 °C after addition of AAPH. All fluorescence measurements were expressed relative to the initial reading. Final results were calculated based on the difference in the area under the fluorescence decay curve between the blank and each sample. ORACROO· was expressed as micromoles of Trolox equivalents (TE). One ORAC unit is equivalent to the net protection area provided by 1 μM of Trolox.
Determination of reduction capacity. The reducing abilities of isolated compounds were determined according to cupric ion reducing antioxidant capacity (CUPRAC) method (Aruoma et al., 1998). In brief, 40 μL of different concentrations of the compounds was mixed with 160 μL of the mixture containing 0.5 mM CuCl2 and 0.75 mM neocuproine in 10.0 mM phosphate buffer, pH 7.4. Reaction mixture was incubated at room temperature for 1 h, and then the absorbance was measured with a micro-plate reader at 454 nm. Increased absorbance of the reaction mixture indicates increased reducing power and was converted into Cu(I) ion concentrations. The CUPRAC values were expressed as μM of Cu(I) ion reduced from Cu (II) by antioxidant. TRAP activity. RAW 264.7 (macrophages (preosteoclasts) from BALB/c mice) cells were cultured in 96-well plates (1 × 104 cells/mL) containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) FBS for 2 days. Medium was then replaced with test samples in differentiation medium containing 50 ng/mL RANKL. The differentiation medium was replaced every 2 days. After differentiating the RAW 264.7 cells into osteoclasts for 5 days, the medium was removed, and the cell monolayer was gently washed twice using ice-cold PBS. The cells were fixed in 3.5% formaldehyde for 10 min and ethanol– acetone (1:1) for 1 min. Subsequently, the dried cells were incubated in 50.0 mM citrate buffer (pH 4.5) containing 10 mM sodium tartrate and 6 mM p-nitrophenylphosphate (PNPP). After 1 h incubation, the reaction mixtures were transferred to new well plates containing an equal volume of 0.1 N NaOH. Absorbance was measured at 408 nm using an ELISA reader. TRAP activity was expressed as a percentage of the control. Statistical analysis. All data represent the mean ± S.D. of at least three independent experiments performed in triplicates. Statistical significance is determined by oneway ANOVA followed by Dunnett’s multiple comparison test, P < 0.05, using GraphPad Prism 6 program (GraphPad Software Inc., San Diego, CA, USA).
RESULTS AND DISCUSSION Antioxidant activity of the extracts from C. indicum flowers A methanol extract of CIF was suspended in H2O and then partitioned sequentially with CH2Cl2 and EtOAc Phytother. Res. (2015)
B. T. T. LUYEN ET AL.
to give corresponding CH2Cl2, EtOAc, and H2O fractions. These fractions were evaluated for their antioxidant effects at concentrations of 1.0 and 5.0 μg/mL (see Table 1). Of these, the EtOAc and H2O fractions showed significant activity against peroxyl radicals. At a concentration of 5.0 μg/mL, the total antioxidant capacities in the ORAC assay were determined to be 27.39 ± 0.20, 28.06 ± 1.35 times higher than the protection provided by 1.0 μM Trolox, respectively. The small difference in their ORAC values at a concentration of 1.0 μg/mL (26.81 ± 0.21, 24.00 ± 0.44, respectively) suggests the potent antioxidant activity of these fractions. Moreover, the EtOAc and H2O fractions displayed good reducing power with CUPRAC values of 43.61 ± 1.30, 45.07 ± 1.63 and 16.90 ± 0.51, 49.77 ± 0.97, at concentrations of 1.0 and 5.0 μg/mL, respectively. Total phenolic, total flavonoid contents of methanol extract, and its soluble fractions from C. indicum flowers Phenolic compounds, as secondary metabolites, are widely distributed in fruits and vegetables. They are formed via the shikimate pathway and are considered to be the main contributors to the antioxidant capacity of plants and to protect organisms from damage caused by free radical-induced oxidative stress. The antioxidant activity of phenolics is mainly due to their redox properties such as reducing agents, hydrogen donors, and free radical scavengers (Ozsoy et al., 2009). The correlation between antioxidant activity and TPC has been reported in numerous studies (Ozyurek et al., 2014). Thus, the TPC was determined using standard gallic acid as a standard, and the results were expressed as mg GAE/g DR. Table 1 shows the TPC of the crude extract and its soluble fractions. Accordingly, the TPC of the EtOAc fraction (525.84 ± 23.51 mg GAE/g DR) was higher than that of the methanol extract (68.59 ± 0.59 mg GAE/g DR) and water fraction (38.58 ± 0.64 mg GAE/g DR). Flavonoids, one of the most diverse and widespread groups of natural products, possess a wide variety of pharmacological and biochemical properties, including antioxidative, anti-microbial, anti-allergenic, anti-viral, antiinflammatory, and vasodilatory actions. The antioxidant activity of flavonoids is of great interest because of their ability to inhibit free radical formation and to scavenge free radicals (Pietta, 2000). Our results show that the highest flavonoid content was detected in the EtOAc fraction (63.49 ± 3.32 mg QE/g DR) while the lowest level was detected in the methanol extract (9.27 ± 0.97 mg QE/g DR) (see Table 1). These results indicate that the TPC and total flavonoid content of the
methanol extract were effectively enriched in the EtOAc soluble fraction. Structural determination Based on these data, we selected the EtOAc and aqueous fractions for an analysis of their chemical constituents. Using a combination of various chromatographic steps over silica gel and YMC reversed-phase C18 resin, compounds 1–19 were isolated (see Fig. 1). Of these, two new compounds (1 and 2) were obtained. Compound 1 was obtained as a colorless oil. The HRESI-MS of 1 exhibited a pseudo-molecular ion peak at m/z 447.1792 [M + Cl] (Calcd. C21H32O8Cl for 447.1786), which is consistent with the molecular formula of C21H32O8. The IR spectrum displayed a strong band at 1760 cm1, indicating the presence of a lactone functional group. The 1H NMR and 13C NMR spectra of 1 showed signals assignable to three methyls at δH 1.44 (d, J = 7.2 Hz, H-13), 1.49 (s, H-15), and 1.70 (s, H14); two oxygenated methines at δH 4.60 (ddd, J = 1.8; 10.5; 10.5 Hz, H-6) and 4.72 (t, J = 9.6 Hz, H-8); and two olefinic protons at δH 4.97 (d, J = 7.8 Hz, H-3) and 4.53 (d, J = 10.2 Hz, H-9) (see Table 2). The 13C NMR and DEPT spectra revealed 21 carbon signals, including three methyl carbons, four methylene carbons, ten methine carbons (including six oxygenated, two olefinic, and two aliphatic methines), and three quaternary carbons with one carbonyl carbon. Among them, an anomeric (δC 98.7) and five other oxygenated carbon signals (δC 78.3, 78.2, 74.9, 72.1, and 63.1) were assigned to the sugar moiety. Assignment of the protons and carbons in the aglycone moiety was done based on twodimensional (2D) spectra. The correlation spectroscopy (COSY) spectra of 1 indicated two individual correlation systems (see Fig. 2): fragment A [H2-1 (δH 2.03, 2.31)/H2-2 (δH 2.16, 2.33)/H-3 (δH 4.97)] and fragment B [H-5α (δH 2.44)/H-6 (δH 4.60)/H-7 (δH 2.55)/H-8 (δH 4.72)/H-9 (δH 4.53), H-7/H-11 (δH 2.86)/H-13 (δH 1.44)]. The connectivity of the two fragments was determined through HMBC correlations. Diagnostic HMBC correlations were observed between a tertiary methyl H3-14 (δH 1.70) and carbons C-1 (δC 40.0), C-9 (δC 130.1), and C-10 (δC 140.3). Another tertiary methyl H3-15 (δH 1.49) correlated with carbons C-3 (δC 131.4), C-4 (δC 133.0), and C-5 (δC 47.6). These data suggested that fragments A and B connected with each other via C-4 and C-10 (see Fig. 2). The above mentioned evidences suggested a germacranolide sesquiterpene lactone glycoside, bearing oxygen functions (hydroxyl and lactone) at C-6, C-8 and two trisubstituted double bonds at C-3/C-4 and C-9/C-10 (Rustaiyan et al., 1990, Sanz
Table 1. Peroxyl radical-scavenging capacity, reducing capacity, total phenolic contents (TPC), and total flavonoid contents (TFC) of the methanol extracts of C. indicum flowers and its soluble fractions. (ME: methanol extract, DMF: dichloromethane fraction, EAF: ethyl acetate fraction, WF: water fraction) ORACRCOO (TE, μM) Sample ME DMF EAF WF
Reduction power (Copper(I) ion, μM)
1 μg/mL
5 μg/mL
1 μg/mL
5 μg/mL
5.47 ± 0.28 4.70 ± 0.25 26.81 ± 0.21 24.00 ± 0.44
21.22 ± 0.19 19.53 ± 0.38 27.39 ± 0.20 28.06 ± 1.35
2.68 ± 0.13 3.56 ± 0.23 43.61 ± 1.30 16.90 ± 0.51
9.43 ± 0.30 14.34 ± 0.85 45.70 ± 1.63 49.77 ± 0.97
Copyright © 2015 John Wiley & Sons, Ltd.
TPC (mg GAE/g DR)
TFC (mg QE/g DR)
68.59 ± 0.59 NT 525.84 ± 23.51 38.58 ± 0.64
9.27 ± 0.97 NT 63.49 ± 3.32 12.47 ± 0.58 Phytother. Res. (2015)
ANTI-OSTEOPOROSIS AND ANTIOXIDANT COMPONENTS OF C. INDICUM FLOWERS
Figure 1. The chemical structures of compounds 1–19 from the flowers of C. indicum.
et al., 1990). A δ-lactone ring was formed between C-12 and C-6, which was supported by the downfield shift of C-6 (δC 81.4); HMBC correlations between H3-13/C-7 (δC 54.6), C-11 (δC 41.5), and C-12 (δC 182.2); and a weak HMBC correlation between H-6 and C-12. Moreover, glucosidic bonding at C-8 was indicated by both HMBC correlations of an anomeric proton H-1′ (δH 4.28) with C-8 (δC 70.6), and an oxymethine proton H-8 (δH 4.72) with C-1′ (δC 98.7). Furthermore, acid
hydrolysis of 1 followed by TLC, GC analysis, as well as a comparison with authentic D-glucose, confirmed the presence of a D-glucose moiety in 1. Next, the stereogenic centers of 1 were confirmed by Rotating frame nuclear Overhauser effect spectroscopy (ROESY) experiments (see Fig. 2). Steric interactions were observed for H3-13 (δH 1.44) with both H-6 (δH 4.60) and H-8 (δH 4.72) in the ROESY spectra of 1, indicating β-orientations at C-7, C-11 and α-orientations at C-6, C-8. The α/β/α configuration system of C-6/C7/C-8 was also confirmed by large values for both JH7/H-6 and JH-7/H-8 (J = 10.5 Hz), as well as in agreement with previous literatures (Rustaiyan et al., 1990). Moreover, the circular dichroism (CD) spectra of 1 exhibited a negative Cotton effect at 200 nm (not a maximum, lowest recorded value) and a positive band at 216 nm, which were assigned to the trans, trans-cyclodecadiens (Takeda and Horibe, 1975). In addition, the βconfiguration of C-11 was also supported by ROESY correlations of H-7 (δH 2.55)/H-11 (δH 2.86), H-7/H-5α (δH 2.44) and by the absence of a ROESY correlation between H-7 and H3-13 (δH 1.44). The olefinic geometries were identified to be both 3E and 9E according to the ROESY interactions of H-3 (δH 4.97)/H-5α, H3-15 (δH 1.49)/H-2β (δH 2.33), H-9 (δH 4.53)/H-1α (δH 2.03), and H3-14 (δH 1.70)/H-8 (δH 4.72), as well as the up-field chemical shifts of C-14 and C-15 (
