Phytochemistry Letters 6 (2013) 322–325

Inhibitors of a-glucosidase, a-amylase and lipase from Chrysanthemummorifolium

Nguyen Thi Luyen, Le Hoang Tram, Tran Thi Hong Hanh, Pham Thanh Binh, Nguyen Hai Dang,Chau Van Minh, Nguyen Tien Dat *

Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18-Hoang Quoc Viet, Cau Giay, Hanoi, Viet Nam

A R T I C L E I N F O

Article history:

Received 11 December 2012

Received in revised form 20 March 2013

Accepted 26 March 2013

Available online 17 April 2013

Keywords:

Chrysanthemum morifolium

Asteraceae

Endoperoxyguainolide

a-Glucosidase

a-Amylase

Lipase

A B S T R A C T

A new endoperoxysesquiterpene lactone, 10a-hydroxy-1a,4a-endoperoxy-guaia-2-en-12,6a-olide (1),

together with a flavanone, eriodictyol (2), and two flavone glycosides, acacetin-7-O-b-D-glucopyrano-

side (3) and acacetin-7-O-a-L-rhamopyranoside (4), were isolated from the methanol extract of

Chrysanthemum morifolium flowers by a bioassay-guided fractionation. Compound 1 showed strong

inhibitory effects against a-glucosidase and lipase activities, with IC50 values of 229.3 and 161.0 mM,

respectively. The flavone glycosides 3 and 4 inhibited both a-glucosidase and a-amylase, while

flavanone 2 was only effective against a-amylase.

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1. Introduction

The flowers of Chrysanthemum morifolium Ramat. (Asteraceae)have been used in Vietnam and other Asian countries for thetreatment of eye diseases, headaches, insomnia, and hyperglyce-mia and are widely consumed as a medicinal herbal tea (Do, 2004;Vo, 1999). C. morifolium contains a wide variety of chemicalcompounds, including sesquiterpenes, flavonoids, triterpenes, andessential oil (Kumar et al., 2005; Lee et al., 2003; Lin and Harnly,2010; Ukiya et al., 2002; Zhou et al., 2009). These compoundspossess various biological activities, including anti-oxidation,anticancer, cardiovascular protection, anti-inflammation, andanti-mutagenesis (Kumar et al., 2005; Lin et al., 2010; Miyazawaand Hisama, 2003; Ukiya et al., 2002). Diabetes is a group ofmetabolic diseases characterized by chronic hyperglycemiaresulting from deficiency in insulin secretion or action. Onetherapeutic approach for treating diabetes is to decrease thepostprandial glycemia by the inhibition of enzymes responsible forthe carbohydrate hydrolysis, such as a-glucosidase and a-amylase(Souza et al., 2012). Pancreatic lipase is a key enzyme for thedigestion of dietary triglycerides. The inhibition of lipase activityretards the fat absorption and therefore ameliorates obesity (de laGarza et al., 2011).

* Corresponding author. Tel.: +84 4 37917049; fax: +84 4 37917054.

E-mail addresses: ngtiend@imbc.vast.vn, ngtiend@hotmail.com (N.T. Dat).

1874-3900/$ – see front matter � 2013 Phytochemical Society of Europe. Published by

http://dx.doi.org/10.1016/j.phytol.2013.03.015

Our search for antidiabetic and antiobesity agents from naturalorigins identified a methanol extract of C. morifolium flowersthat showed significant inhibitory activity against a-glucosidase,a-amylase, and lipase. A bioassay-guided phytochemical investi-gation of the methanol extract of C. morifolium flowers led to theisolation of a new endoperoxysesquiterpene lactone, together witha flavanone and two flavone glycosides (Fig. 1). Their structureswere elucidated as 10a-hydroxy-1a,4a-endoperoxy-guaia-2-en-12,6a-olide (1), eriodictyol (2) (Garo et al., 1996), acacetin-7-O-b-D-glucopyranoside (3), and acacetin-7-O-a-L-rhamnopyranoside(4) (Agrawal, 1989). The inhibitory effects of the isolatedcompounds against a-glucosidase, a-amylase, and lipase werereevaluated.

2. Results and discussion

Compound 1 was obtained as a colorless solid. Its HRESIMSspectrum showed a peak at m/z 281.1378 [M+H]+, corresponding tothe molecular formula C15H21O5. The 1H NMR spectrum of 1showed two olefinic doublets at dH 5.89 and 5.82 (each 1H, d,J = 6.0 Hz, H-2, 3), a doublet of doublets at dH 4.49 (1H, dd, J = 11.0,11.5 Hz, H-6), a doublet at dH 2.45 (1H, d, J = 11.5 Hz, H-5), a methyldoublet at dH 1.21 (3H, br d, J = 7.0 Hz, H-13), and two methylsinglets at dH 1.40 and 1.10 (each 3H, br s, H-14, 15). The 13C NMRand DEPT spectra of 1 indicated the presence of 15 signals, whichwere similar to those of tanaparthin-a-peroxide (Hewlett et al.,1996), except for the replacement of a a-methylen-g-lactone ring

Elsevier B.V. All rights reserved.

β

β

Fig. 1. Structure of compounds 1–4.

Table 1Inhibitory effects (IC50 in mMa) of compounds 1–4 for a-glucosidase, a-amylase and

lipase.

Compounds a-Glucosidase a-Amylase Lipase

1 229.3 � 16.7 na 161.0 � 21.4

2 na 318.2 � 26.9 na

3 451.8 � 36.4 337.1 � 28.2 na

4 362.5 � 35.1 112.5 � 15.7 na

Acabose 1907 � 156.0 732.4 � 41.6 –

Orlisat – – 108.3 � 22.1

a Data are means � SD from three experiments; na: not active.

N.T. Luyen et al. / Phytochemistry Letters 6 (2013) 322–325 323

by an a-methyl-g-lactone ring [dC 181.2 (C-12), 84.2 (C-6), 49.1(C-7), 42.4 (C-11), and 13.0 (C-13)]. The structure of 1 was alsoconfirmed by HMBC experiments (Fig. 1), which showed thecorrelations from H-13 (dH 1.21) to C-7, C-11, and C-12, from H-14(dH 1.10) to C-1 (dC 91.0), C-10 (dC 77.5), and C-9 (dC 37.8), and fromH-15 (dH 1.40) to C-3 (dC 140.3), C-4 (dC 83.3), and C-5 (dC 66.5).

Previous studies showed that the 1a,4a-endoperoxide guaia-nolides were more stable than the b-isomers, and therefore theformer were more abundant (Begley et al., 1989; Hewlett et al.,1996; Rucker et al., 1991; Trendafilova et al., 2006). Thus, 1 wasassumed to be the 1a,4a-endoperoxide guaianolide. The relativeconfigurations of 1 were then determined by 1H–1H couplingconstants and NOESY experiments (Fig. 2). The large couplingconstant of H-6 indicated the existence of a trans-fused g-lactone(Trendafilova et al., 2006). A strong NOE correlation between H-2and H-14 suggested the cis-fused A/B ring. The NOE effectsbetween H-14/H-15, H-14/H-6, H-15/H-6, H-6/H-8b, and H-15/H-8b indicated their syn b relationship. In contrast, a orientationsof H-5, H-7, and the methyl H-13 were confirmed by clear NOEeffects between H-5/H-7 and H-7/H-13. Thus, 1 was elucidatedas 10a-hydroxy-1a,4a-endoperoxide-guaia-2-en-12,6a-olide, or11b,13-dihydrotanaparthin-a-peroxide.

Compounds 1–4, obtained by bioassay-guided fractionation,were reevaluated for their inhibitory effects against a-glucosidase,a-amylase, and lipase (Table 1). Compound 1 showed strong a-glucosidase and lipase inhibitions, with IC50 values of 229.3 and161.0 mM, respectively. Notably, the a-glucosidase inhibition of 1was eightfold stronger than the antidiabetic acarbose. Guaiane

O

O

O

OH

O 12

45

6 7

8

10

1112 13

14

15 O

O

O

OH

O12

4 5 6 7

10

11 13

14

15

HH

H

H

HH

HH

H

Fig. 2. Key HMBC (!) and NOESY ($) correlations of 1.

sesquiterpenes are known as potential antitumor and anti-inflammatory agents (Repetto and Boveris, 2010; Zhang et al.,2012a); however, their a-glucosidase and lipase inhibitoryactivities have not been reported. Recently, a new compound,1b,5a-guaiane-4b,10a-diol-6-one, isolated from Acorus calamus

showed promising antidiabetic activity in an insulin-mediatedglucose consumption model of HepG2 cells (Zhou et al., 2012). Thiscompound may also be involved in the a-glucosidase inhibition ofthe ethyl acetate fraction of A. calamus (Si et al., 2010). Theflavanone eriodictyol (2), which has previously been found in thisplant (Lin and Harnly, 2010), only showed inhibitory effectsagainst a-amylase activity. Although this compound has beenshown to be an attractive candidate as a glucose-lowering andinsulin-resistance-improving agent for the treatment of diabetes(Zhang et al., 2012b), this is the first report on its a-amylaseinhibitory activity. Two flavone glycosides, 3 and 4, showed stronginhibition against a-glucosidase and a-amylase in comparisonwith the positive control acarbose. Note that these flavones are themain compounds described in the extraction and isolation section.Thus, these compounds may be important contributors to thehyperglycemia-lowering property of C. morifolium. Flavonoids areknown to be the main constituents in the Chrysanthemum species,and acacetin glucosides, including 3, have previously been found inC. morifolium (Lai et al., 2007; Lin and Harnly, 2010; Wang et al.,2010). However, acacetin-7-O-a-L-rhamnopyranoside (4) wasisolated for the first time from this plant.

3. Materials and methods

3.1. General experimental procedures

Optical rotation values were recorded a JASCO P-2000 digitalpolarimeter. The IR spectrum was obtained from a Tensor 37 FT-IRspectrometer. NMR experiments were carried out on a BrukerAM500 FT-NMR spectrometer using tetramethylsilane (TMS) asinternal standard. The ESI-MS were recorded on an Agilent 1200series LC-MSD Ion Trap. The HR-ESI-MS were recorded on an FT-ICR mass spectrometer. Absorbance of bioassay solution was readusing an xMark microplate spectrophotometer.

3.2. Plant materials

The flowers of Chrysanthemum morifolium Ramat. werecollected in Son Tay, Hanoi, Vietnam in August 2011, and identifiedby Dr. Tran Huy Thai, Institute of Ecology and Biological Resources,Vietnam Academy of Science and Technology. The voucherspecimens were deposited at the herbarium of the Institute ofEcology and Biological Resources.

3.3. Extraction and isolation

The air-dried and powdered flowers of C. morifolium (2.3 kg)were extracted with methanol (5 L � 3 times) in a sonic bath. Thecombined extracts were concentrated to give 245.0 g of crude

N.T. Luyen et al. / Phytochemistry Letters 6 (2013) 322–325324

extract (57%, 77%, and 64% inhibition of a-glucosidase, a-amylase,and lipase, respectively, at 500 mg/mL), which was then resus-pended in water (1.5 L) and successively partitioned with hexaneand ethyl acetate (each 0.5 L � 3 times) to obtain 70.4 and 6.1 g ofhexane and ethyl acetate residues, respectively. The ethyl acetateresidue (72%, 75%, and 54% inhibition of a-glucosidase, a-amylase,and lipase, respectively, at 500 mg/mL) was chromatographed on asilica gel column eluted with a gradient of 1–100% methanol inchloroform to afford five fractions, E1–5. Fraction E1 (68%inhibition of a-amylase at 500 mg/mL) was passed through aSephadex LH-20 column eluted by methanol–water (3:2, v/v) toobtain 2 (5.7 mg). Fraction E2 (84% and 79% inhibition of a-glucosidase and lipase, respectively, at 500 mg/mL) was fraction-ated on a silica gel column eluted with hexane–acetone (2:1, v/v) togive four subfractions, E2.1–4. Compound 1 (5.1 mg) was purifiedfrom E2.3 (88% and 85% inhibition of a-glucosidase and lipase,respectively, at 500 mg/mL) using a reverse-phase C18 columneluted with methanol–water (3:2, v/v). The water residue wasfiltered through a diaion HP-20 column eluted by 0%, 50%, and100% methanol in water to obtain three fractions, W1–3. FractionW3 (61% and 91% inhibition of a-glucosidase and a-amylase,respectively, at 500 mg/mL) was chromatographed on a silica gelcolumn eluted with 20% methanol in chloroform to afford threefractions, A3.1–3. Fraction A3.1 (76% and 100% inhibition ofa-glucosidase and a-amylase, respectively, at 500 mg/mL) waspurified by a silica gel column eluted with chloroform–methanol–water (50:10:1, v/v/v) to give 3 (4.1 g). Compound 4 (2.8 g) wasseparated from A3.3 (96% and 100% inhibition of a-glucosidase anda-amylase, respectively, at 500 mg/mL) using a Sephadex LH-20column eluted by methanol–water (1:1, v/v).

10a-Hydroxy-1a,4a-peroxide-2-guaien-12,6a-olide (1): Color-less solid; [a]D

24 = +60.2 (c 0.05, MeOH); IR nmax (KBr): 3421, 2360,1653, 1558 cm�1; 1H NMR (500 MHz, CD3OD): d 1.10 (3H, br s, H-14), 1.21 (3H, d, J = 7.0 Hz, H-13), 1.40 (3H, br s, H-15), 1.50 (1H, m,H-8b), 1.55 (1H, m, H-9b), 1.93 (1H, m, H-8a), 2.08 (1H, m, H-7),2.38 (2H, m, H-9a and H-11), 2.45 (1H, d, J = 11.5 Hz, H-5), 4.49(1H, dd, J = 11.0, 11.5 Hz, H-6), 5.82 (1H, d, J = 6.0 Hz, H-3), 5.89(1H, d, J = 6.0 Hz, H-2); 13C NMR (125 MHz, CD3OD): d 91.0 (C, C-1),135.8 (CH, C-2), 140.3 (CH, C-3), 83.3 (C, C-4), 66.5 (CH, C-5), 84.2(CH, C-6), 49.1 (CH, C-7), 27.7 (CH2, C-8), 37.8 (CH2, C-9), 77.5 (C, C-10), 42.4 (CH, C-11), 181.2 (C, C-12), 13.0 (CH3, C-13), 23.4 (CH3, C-14, 15); ESI-MS m/z: 281.4 [M+H]+; HR-ESI-MS m/z: 281.1378[M+H]+ (calcd. 281.1389 for C15H21O5).

3.4. Assay for a-glucosidase inhibition

The a-glucosidase (G0660, Sigma–Aldrich) enzyme inhibitionassay was performed according to the previously describedmethod (Ali et al., 2002). The sample solution (2 mL dissolved inDMSO) and 0.5 U/mL a-glucosidase (40 mL) were mixed in 120 mLof 0.1 M phosphate buffer (pH 7.0). After 5 min pre-incubation,5 mM p-nitrophenyl-a-D-glucopyranoside solution (40 mL) wasadded and the solution was incubated at 37 8C for 30 min. Theabsorbance of released p-nitrophenol was measured at 405 nm.The inhibitory activity was expressed as relative absorbancedifference (%) of test sample to the absorbance change of thecontrol where test solution was replaced by neat DMSO. Acarbosewas used as positive control.

3.5. Assay for a-amylase inhibition

The a-amylase (A8220, Sigma–Aldrich) enzyme inhibitoryactivity was measured using the method reported by Kusano et al.(2011) with slight modifications. Substrate was prepared byboiling 100 mg potato starch in 5 mL phosphate buffer (pH 7.0)for 5 min, then cooling to room temperature. The sample

(2 mL dissolved in DMSO) and substrate (50 mL) were mixed in30 mL of 0.1 M phosphate buffer (pH 7.0). After 5 min pre-incubation, 5 mg/mL a-amylase solution (20 mL) was added andthe solution was incubated at 37 8C for 15 min. The reaction wasstopped by adding 50 mL 1 M HCl, and then 50 mL iodine solutionwas added. The absorbances were measured at 650 nm by amicroplate reader. Acarbose was used as positive control.

3.6. Assay for lipase inhibition

The assay was performed according to the method described byMcDougall et al. (2009) with slight modification. Lipase fromporcine pancreas (Type II, L3126, Sigma–Aldrich) was dissolved indistillated water at 10 mg/mL and centrifuged at 4500 rpm for10 min to remove insolubles. Five microliters of samples dissolvedin DMSO were preincubated with 150 mL lipase for 5 min, then855 mL of 100 mM Tris buffer (pH 7.5) containing 0.1 mM p-nitrophenyl laurate were added and incubated for 30 min at 37 8C.The reacted solutions were centrifuged at 4500 rpm for 5 min andthe absorbance of the supernatants was read at 405 nm. Inhibitionof lipase activity was expressed as the percentage decrease in theabsorbance when porcine pancreatic lipase was incubated with thetest compounds. Orlistat was used as positive control.

Acknowledgments

This work is supported by a grant from the Ministry of Scienceand Technology (NCCBDHUD/2011–2014). We thank the Instituteof Chemistry, Vietnam Academy of Science and Technology for theNMR and HRMS measurements.

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