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Analysis and determination of oestrogen-active compounds in fructus amomi by the combination of high-speed counter-current chromatography and high performance liquid chromatography
Journal of Chromatography B, 958 (2014) 36–42
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Analysis and determination of oestrogen-active compounds in fructus amomi by the combination of high-speed counter-current chromatography and high performance liquid chromatography Hao Ying, Jinpeng Liu, Qizhen Du ∗ Institute of Food Science, Zhejiang A & F University, Linan 311300, Hangzhou, China
a r t i c l e
i n f o
Article history: Received 7 January 2014 Accepted 5 March 2014 Available online 15 March 2014 Keywords: Fructus amomi Oestrogenic activity Analysis Diarylheptanoids
a b s t r a c t Amomum longiligulare or Amomum villosum showed oestrogenic activity. In the present study, oestrogenactive components in fructus amomi, the seeds of A. longiligulare were separated by high-speed countercurrent chromatography (HSCCC) using stepwise elution of eight mobile phases with gradient polarity and advanced separation by high performance liquid chromatography (HPLC). The results yielded 17 compounds with the amount of 8–138 mg and a purity of 94.3–99.8% from a 3 g ethanolic extract of fructus amomi. The chemical structures of the compounds were identified by ESI-MS and NMR spectra, in which eight diarylheptanoids were demonstrated as the main oestrogen-active compounds in the fructus amomi. Determination of the diarylheptanoids in fructus amomi from various origins showed that fructus amomi contains more than 0.5% total diarylheptanoids. The results showed that fructus amomi is a diarylheptanoids-rich food resource possessing oestrogen-activity. The combination method of HSCCC and HPLC can be applied for the analysis of bioactive compounds by detecting the corresponding bioactivity in the HSCCC fractions and separating the target compounds with HPLC. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Phytoestrogens derived from food sources could be an effective supplement in treating human oestrogen deficiency [1]. Fructus amomi, the seeds of Amomum xanthioides, is a favourite condiment for food cooking because it possesses an aromatic and pungent odour [2]. We are investigating the phytoestrogenic activity of fructus amomi, because an oestrogen-activity assay of the total herbal plant of Amomum villosum var. xanthioides (A. xanthioides) (Zingiberaceae) demonstrated that the fractions that were extracted by CH2 Cl2 , EtOAc, and BuOH showed strong effect (Table 1) [3]. Though fructus amomi has functions on the treatment of diabetes [4,5] and its essential oil possesses antimicrobial activities [6], no reports have involved its oestrogen-activity. Therefore, the present study focuses on the chemical constituents with oestrogen-activity in fructus amomi and their content in fructus amomi from different origin. High-speed counter-current chromatography (HSCCC) is a liquid–liquid partition chromatographic method in which compounds are partitioned between two immiscible liquid phases without solid support or irreversible adsorption. It exhibits an
∗ Corresponding author. Tel.: +86 571 15958126861; fax: +86 571 88218710. E-mail address: [email protected] (Q. Du). http://dx.doi.org/10.1016/j.jchromb.2014.03.006 1570-0232/© 2014 Elsevier B.V. All rights reserved.
advantage of fractioning components containing all the chemicals in the crude sample because all of the chemicals remain within the mobile phase or stationary phase [7,8]. Conventional isolation methods can lead to the loss of active components during the isolation and purification phases because of irreversible adsorption or decomposition. Because of the advantages of HSCCC, we can first separate an extract into fractions, and then detect the oestrogenactivity of the fractions for further separation and purification. This procedure is useful for screening minor bioactive compounds by adjusting the amount of each fraction for the bioactive assay. MVLN cell, a human breast cancer cell, has been used successfully to screen Phytoestrogens or environmental oestrogen-like chemicals [9,10]. In this study, the MVLN cell line was used to detect and evaluate the oestrogen-activity of the fractions and chemicals isolated from the fructus amomi. The aim of the present study is to clarify phytoestrogenic chemicals in fructus amomi, and also to establish a method for screening Phytoestrogens from plant materials. 2. Materials and methods 2.1. Materials All solvents for extraction and separation were of analytical grade, purchased from Hangzhou Huadong Chemicals Inc., China. Fructus amomi, the seeds of Amomum longiligulare used for the
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Table 1 Extraction yields and estrogenic activities of solvent fractions of Zingiberaceae A. xanthioides Wall. Layers
Yield (w/w)
EC50 (g/ml)
n-Hexane CH2 Cl2 EtOAc BuOH 17-Estradiol (E2 )
0.38a 0.37 0.55 0.93
>100 0.409 0.323 0.056 0.0037
a
All data from Ref. [3].
extraction and separation of oestrogen-activity, were provided by Nanhai Farm, Hainan, China. Fructus amomi, the seeds of A. villosum and A. villosum var xanthioides used for determination of diarylheptanoids were purchased in a local Chinese medicine store in Hangzhou, China. The dried seeds were directly used for extraction after powdering. HPLC-grade methanol was of products of Merck (Merck Chemicals (Shanghai) Co., Ltd., China). 2.2. Extraction The freeze-dried sample (2 kg) was extracted twice with 12 l 90% ethanol for 2 h at 50 ◦ C. The extracts were combined and evaporated to syrup. The syrup was defatted with ether and lyophilised, yielding 297 g crude extract. A portion of the crude extract (3 g) was used for HSCCC separation to obtain fractions for the oestrogen-activity assay. 2.3. High-speed counter-current chromatographic separation The high-speed counter-current chromatograph used in the present study was constructed at the Institute of Food and Biological Engineering, Zhejiang Gongshang University, Hangzhou, China. The apparatus was equipped with a 1200-ml column with six-layer coils made from 5.0 mm i.d. polytetrafluoroethylene (PTFE) tubing. A K-1800 Wellchrom preparative HPLC pump (Knauer, Germany), a 50-ml sample loop made of 3-mm i.d. PTFE tubing, and a B-684 collector (Büchi, Switzerland) with 50-ml tube racks were utilised to constitute a HSCCC system. The sample solution was prepared by dissolving 3 g ethanolic extract in 45 ml stationary phase solvent (water saturated with n-butanol and ethyl acetate). At the beginning of the separation procedure, the column was filled with the stationary phase solvent. Then, the apparatus was rotated at 1000 rpm and the sample solution was injected into an HSCCC system through the sample loop with the mobile phase at a flow rate of 4.0 ml/min (head to tail mode). The effluent was collected as the mobile phase started to get out of the column. The stationary phase was water saturated with n-butanol and ethyl acetate. The stepwise elution was performed with the following mobile phases: n-hexane–ethyl acetate (1:1) for 60 min, n-hexane–ethyl acetate (1:2) for 60 min, n-hexane–ethyl acetate (1:4) for 60 min, ethyl acetate for 60 min, n-butanol–ethyl acetate (1:4) for 60 min, n-butanol–ethyl acetate (1:2) for 60 min, nbutanol–ethyl acetate (2:2) for 60 min, and n-butanol–ethyl acetate (2:1) for 60 min. The effluent was collected in 20-ml fractions by a fraction collector. After the stepwise elution, the solvent was drained from the column from the tail to head of the column and collected in 40-ml fractions. All fractions were assayed for oestrogen-activity. The fractions were combined into seven larger fractions (components) on the basis of their oestrogen-activity (Fig. 1). 2.4. HPLC analysis and preparation The oestrogen-activity components from the HSCCC separation were analysed by HPLC, and the main compounds were
Fig. 1. An oestrogenic activity-chromatogram of fractions obtained from HSCCC separation of 3 g of the ethanolic Fructus amomi extract. Column capacity: 1200 ml; Rotation speed: 1000 rpm; Stationary phase: water saturated with n-butanol and ethyl acetate; Stepwise elution: n-hexane–ethyl acetate (1:1) for 60 min, nhexane–ethyl acetate (1:2) for 60 min, n-hexane–ethyl acetate (1:4) for 60 min, ethyl acetate for 60 min, n-butanol–ethyl acetate (1:4) for 60 min, n-butanol–ethyl acetate (1:2) for 60 min, n-butanol–ethyl acetate (2:2) for 60 min, and n-butanol–ethyl acetate (2:1) for 60 min; Flow rate: 4 ml/min; Sample: 3 g ethanolic extract in 45 ml stationary phase; Fraction volume: 40 ml.
prepared by preparative HPLC. The analytical HPLC system consisted of an Alliance 2695 separations module, an ODS AQ column (150 mm × 3.9 mm i.d., 3 m) and a low temperature evaporative light scattering detector (ELSD-LTII, Shimadzu, Japan). The gradient elution was carried out as follows: water-formic acid (99.5:0.5, v/v) 100% to methanol 100% from 0 to 40 min at a flow rate of 0.8 ml/min. For preparative HPLC, a preparative ODS AQ column (250 mm × 20 mm i.d., 15 m) was employed. The preparative HPLC conditions were determined based on the analytical HPLC results. 2.5. ESI-MS and NMR All ESI-MS experiments were performed on a Bruker Esquire LC–MS ion trap multiple mass spectrometer (Bremen, Germany) in positive and negative ionisation modes analysing ions up to m/z 2200. 1 H-, 13 C-, and DEPT 90/135-NMR spectra were recorded in DMSO-d 6 on a Bruker 400 (Karlsruhe, Germany) with 400 MHz for 1H-, and 100 MHz for 13 C-measurements. 2.6. Luciferase assay for detection and evaluation of oestrogen-activity An assay based on the MVLN cell line was used to detect the fractions from HSCCC separation and evaluate the oestrogenactivity of the isolated compounds [11]. One ml solution of each HSCCC tube fraction (40 ml) was evaporated to dryness under vacuum. The residue was dissolved with 2 ml of cell culture solution for assay of oestrogen-activity. The cells were routinely maintained as monolayer cultures at 37 ◦ C in a 5% CO2 -enriched humidified air atmosphere. The cultures were grown in 25 cm2 plastic cell culture flasks in Eagle’s minimal essential medium containing 5% foetal calf serum supplemented with penicillin (100 IU/ml), streptomycin (100 mg/ml), 4-(2-hydroxyethyl)-1piperazi-neethanesulfonic acid (10 mM) and NaHCO3 (0.5 g/l). Cells were grown to confluence and passaged with trypsin–EDTA (0.05/0.02%). For the luciferase assay, the MVLN cells were plated in 96-well plates at a density of 3 × 104 cells/well in embryo freezing medium containing 5% hormone-reduced dextran-coated charcoal-treated foetal bovine serum. The cells were cultivated for 3 days, and
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Fig. 2. HPLC-ELSD analysis of the fractions obtained from the HSCCC separation. Number over peak represents of the compound No. in Table 2. Column: ODS AQ (150 × 3.9 mm i.d., 3 m); Gradient elution: water-formic acid (99.5:0.5, v/v) 100% to methanol 100% from 0 to 40 min; flow rate: 0.8 ml/min; Detector: a low temperature evaporative light scattering detector.
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Fig. 3. The structure of the compounds obtained from the separation and purification by HSCCC and preparative HPLC.
then incubated with various compounds for 24 h. At the end of the treatment, luciferase activity was measured using a commercial Luciferase Assay Kit (Progema, Madison, USA) according to the manufacturer’s protocol. The resultant chemiluminescence was measured using a Molecular Devices Gemini XPS plate reader (Sunnyvale, CA). For each tested sample, we determined the EC50 (concentration required to reach 50% of the maximum chemiluminescence intensity). In brief, the luciferase activity of each sample was assayed at four concentrations 5000, 10,000, 15,000 and 20,000 nM. The EC50 was obtained through a plotting luciferase activity vs sample concentration. All measurements were carried out in triplicate. 3. Results and discussion It is difficult to separate the components in the ethanolic extract of fructus amomi because the polarities of the components range from low to high. A stepwise elution with mobile phases of gradient polarity can yield better separation than a single mobile phase. We employed a wide range of polarities by adjusting the ratio and composition of the solvents that comprised the mobile phase. The low polar mobile phases (n-hexane–ethyl acetate at 1:1, 1:2, and 1:4) were used to elute the low-polarity components, followed by
increasingly polar mobile phases (ethyl acetate; n-butanol–ethyl acetate, 1:4; n-butanol–ethyl acetate, 1:2; n-butanol–ethyl acetate, 2:2; and n-butanol–ethyl acetate, 2:1) to elute medium- and highpolarity components. The performance of the elution with these mobile phases was satisfied because the stationary phase in the column occupied 41% at the end of the separation. The entire run, including the mobile phase elution and the stationary phase extrusion, resulted in 86 fractions (each 40-ml). All the fractions were detected for their oestrogen-activity. The advantage of this method is that the entire sample can be recovered. An oestrogen-activity-chromatogram based on the assay values of all the fractions from the HSCCC separation is shown in Fig. 1. Six of the oestrogen-active components (FrA–FrE) were eluted by the mobile phases, and one oestrogen-active component (FrG) was drained from the stationary phase in the column. The polarity of components FrA–FrG increased from low to high. The high performance liquid chromatography (HPLC) chromatograms (Fig. 2) of components FrA–FrG were compared with the crude extract using an evaporative light scattering detector (ELSD) to afford a relatively complete detection of the compounds in the components. After evaporating the solvents and freeze-drying the residues, FrA–FrG were obtained with the amounts 65, 87, 151, 282, 75, 38, and 16 mg, respectively.
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Table 2 The NMR data of the compounds 5, 7 and 10 which were the major diarylheptanoids in the fructus amomi. Position
1 2 3 4 5 6 7 1 2 3 4 5 6 1 2 3 4 5 6
5
7
10
ıC
ıH
ıC
ıH
ıC
ıH
81.10 d 34.24 t 25.11 t 32.45 t 78.77 d 39.68 t 31.79 t 136.34 s 115.94 d 146.04 s 145.57 s 114.65 d 118.76 d 134.44 s 130.37 d 116.03 d 156.28 s 116.03 d 130.37 d
4.20 (dd, 11.2, 1.8, 1H) 1.51 (m, 2H) 1.91 (m, 2H) 1.30 (m, 2H) 3.43 (m, 1H) 1.78 (m, 2H) 2.64 (m, 2H)
30.65 t 42.69 t 202.83 s 131.59 d 149.25 d 35.68 t 34.52 t 133.16 s 130.30 d 116.15 d 156.57 s 116.15 d 130.30 d 132.99 s 130.37 d 116.15 d 156.61 s 116.15 d 130.37 d
2.65 (t, 7.5, 2H) 2.46 (m, 2H)
30.69 t 42.75 t 202.83 s 131.56 d 149.30 d 35.65 t 34.80 t 133.79 s 116.50 d 146.21 s 144.53 s 116.30 d 120.66 d 133.18 s 133.30 d 116.15 d 156.61 s 116.15 d 133.30 d
2.76 (m, 2H) 2.81 (m, 2H)
6.85 (d, 1.9, 1H)
6.73 (m, 1H) 6.69 (d, 1.8, 1H) 7.00 (d, 8.4, 1H) 6.68 (d, 8.4, 1H) 6.68 (d, 8.4, 1H) 7.00 (d, 8.4, 1H)
3.1. Purification and identification of the compounds in the fractions Compounds corresponding to major HPLC peaks in components FrA–FrE were targeted for preparative HPLC separations. Compounds 1–17 were obtained from the components. The amount of the isolated compounds ranged from 8 to 138 mg. The purities of the compounds ranged from 94.3% to 99.8%, as determined from the HPLC peak area percentage. The structural identification of the compounds was carried out by comparison of ESI-MS, 1 H NMR, and 13 C NMR spectra with those reported in the literature [12–25]. The ESI-MS of compounds 1–17 yielded ions with m/z ESI-MS (m/z) as follows: ESI-MS (m/z): 313 [M−H]− , 329 [M−H]− , 299 [M−H]− , 299 [M−H]− , 295 [M−H]− , 361 [M−H]− , 311 [M−H]− , 297 [M−H]− , 331 [M−H]− , 313 [M−H]− , 431 [M−H]− , 151 [M−H]− , 163 [M−H]− , 431[M−H]− , 463 [M−H]− , 417 [M−H]− , and 593 [M−H]− , respectively. An analysis of 1 H NMR and 13 C NMR spectra combined with the ESI-MS data identified compounds 1–17 (Fig. 3) as 3,5-dihydroxy-7,4 -dimethoxyflavone (1), 3,5,3 -trihydroxy-7,4 dimethoxyflavone (2), 3,5,7-trihydroxy-4 -methoxyflavone (3), 3-hydroxy-7-(4 -hydroxyphenyl)-1-(4 -hydroxyphenyl)heptane (4), 1,5-epoxy-7-(3 ,4 -dihydroxyphenyl)1-(4 -hydroxyphenyl) heptane (5), 3,5-dihydroxy-7-(4 -hydroxy-3 -methoxyphenyl) 1-(3 ,4 -dihydroxyphenyl)heptane (6), 7-(4 -hydroxyphenyl)-1(4 -hydroxyphenyl)-5-hepten-3-one (7), 7-(4 -hydroxyphenyl)1-(4 -hydroxyphenyl)-3-heptanone (8), 3,5-dihydroxy-7(4 -hydroxyphenyl)-1-(3 ,4 -dihydroxyphenyl)heptane (9), 7-(3 ,4 -dihydroxyphenyl)-1-(4 -hydroxyphenyl)-5-hepten3-one (10), 3,5-diacetoxy-7-(3 ,4 -dihydroxyphenyl)-1-(3 ,4 dihydroxyphenyl) heptane (11), 4-methoxybenzoic acid (12), 4-(4-hydroxyphenyl)-2-butanone (13), genistein-7-O-d-glucoside (14), quercetin-3-O--d-glucopyranoside (15), luteolin-8-C-␣-l-arabinoside (16) and kaempferol-3-O-␣-lrhamnopyranosyl-(1-4)--d-glucopyranoside (17). Compounds 5, 7, and 10 (see the 1 H NMR and 13 C NMR data in Table 2, and the spectra in the Supplementary material) are the major diarylheptanoid compounds in the fructus amomi, according to the amounts obtained from the separation of the crude extracts. The majority of these compounds were obtained for the first time from fructus amomi.
6.05 (dt, 15.9, 1.4, 1H) 6.86 (dt, 15.9, 6.9, 1H) 2.77 (m, 2H) 2.77 (m, 2H) 6.98 (dd, 8.5, 4.6, 1H) 6.69 (dd, 8.5, 5.6, 1H) 6.69 (dd, 8.5, 5.6, 1H) 6.98 (dd, 8.5, 4.6, 1H) 6.98 (dd, 8.5, 4.6, 1H) 6.69 (dd, 8.5, 5.6, 1H) 6.69 (dd, 8.5, 5.6, 1H) 6.98 (dd, 8.5, 4.6, 1H)
6.07 (d, 15.8, 1H) 6.87 (m, 1H) 2.46 (m, 2H) 2.63 (m, 2H) 6.62 (d, 2.1, 1H)
6.66 (d, 3.7, 1H) 6.49 (dd, 8.0, 2.1, 1H) 6.98 (d, 8.5, 1H) 6.68 (m, 1H) 6.68 (m, 1H) 6.98 (d, 8.5, 1H)
Table 3 Estrogenicity of compounds in luciferase induction assay with MVLN cells. Sample
EC50 (M)
Crude extract 3,5-Dihydroxy-7,4 -dimethoxyflavone (1) 3,5,3 -Trihydroxy-7,4 -dimethoxyflavone (2) 3,5,7-Trihydroxy-4 -methoxyflavone (3) 3-Hydroxy-7-(4 -hydroxyphenyl)-1-(4 -hydroxyphenyl) heptane (4) 1,5-Epoxy-7-(3 ,4 -dihydroxyphenyl) 1-(4 -hydroxyphenyl) heptane (5) 3,5-Dihydroxy-7-(4 -hydroxy-3 -methoxyphenyl)-1-(3 ,4 dihydroxyphenyl) heptane (6) 7-(4 -Hydroxyphenyl)-1-(4 -hydroxyphenyl)-5-hepten-3-one (7) 7-(4 -Hydroxyphenyl)-1-(4 -hydroxyphenyl)-3-heptanone (8) 3,5-Dihydroxy-7-(4 -hydroxyphenyl)-1-(3 ,4 dihydroxyphenyl) heptane (9) 7-(3 ,4 -Dihydroxyphenyl)-1-(4 -hydroxyphenyl)-5-hepten3-one (10) 3,5-Diacetoxy-7-(3 ,4 -dihydroxyphenyl)-1-(3 ,4 dihydroxyphenyl) heptane (11) 4-Methoxybenzoic acid (12) 4-(4-Hydroxyphenyl)-2-butanone (13) Genistein-7-O--d-glucoside (14) Quercetin-3-O--d-glucopyranoside (15) Luteolin-8-C-␣-l-arabinoside (16) Kaempferol-3-O-␣-l-rhamnopyranosyl-(1-4)--dglucopyranoside (17) 17--Estradiol (E2)b
82.3 × 10−7 a 62.7 × 10−8 53.2 × 10−8 43.6 × 10−8 76.4 × 10−7 18.3 × 10−8 50.6 × 10−7
62.5 × 10−8 30.3 × 10−7 44.7 × 10−7
52.6 × 10−8
27.2 × 10−8
>20 × 10−6 >20 × 10−6 23.1 × 10−8 11.9 × 10−6 13.3 × 10−6 >20 × 10−6
39.7 × 10−12
a
Mole concentration of ethanolic extract was calculated using the molecular weight of genistein-7-O--d-glucoside. b Used as positive control.
3.2. Oestrogen-activity of the compounds The oestrogen-activity of the compounds isolated from the fructus amomi was assayed based on the MVLN cell line using 17--estradiol as the standard. As shown in Table 3, the compounds showed various degrees of oestrogen-activity in MVLN cells. The compounds 1, 2, 3, 5, 7, 10, 11, and 14 exhibited an
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Table 4 The content of diarylheptanoids in fructus amomi from different origin (mg/g, average value, n = 3). Diarylheptanoids
Origin Chinese Guangdonga
Chinese Hainanb
Burmac
Thailandc
Malaysiac
4 5 6 7 8 9 10 11
0.31 0.42 0.06 1.78 0.48 0.03 3.21 0.07
0.21 0.95 0.26 1.33 0.34 0.17 2.42 0.19
0.55 0.76 0.00 2.21 0.35 0.00 4.51 0.00
0.43 1.57 0.00 1.73 0.59 0.00 3.97 0.0
0.39 0.98 0.00 1.52 0.38 0.00 4.23 0.00
Total
6.36
5.87
8.38
8.29
7.50
a b c
A. villosum var. xanthioides. A. villosum. A. longiligulare.
oestrogen-activity at an EC50 level of ×10−8 (18.3 × 10−8 to 62.7 × 10−8 M). The crude extract and compounds 4, 6, 8, and 9 showed an activity at an EC50 level of ×10−7 . Compounds 12, 13, 15, 16, and 17 showed a weak activity (EC50 > level of ×10−6 ). It is clear that flavonoids and diarylheptanoids were active compounds in the fructus amomi. Among the flavonoid compounds, flavones (1, 2, and 3) and isoflavonoid (14) were the main active components. Other glycosyl flavonoids showed low activity. The structure–activity relationship agreed with the regular rules, i.e. glycosylation affects the oestrogen-activity of flavonoids [26], and the substitutions on the B-ring played key roles in oestrogen-activity [27]. Among the diarylheptanoids, the order of oestrogen-activity was 5 > 11 > 10 > 7 > 8 > 6 > 4, which suggested that diarylheptanoid (5) with a similar structure to the B-C ring of flavones was more potent than the others. More than tri- and quad-substituted phenolic hydroxyl groups such as 5, 9, 10, and 11 were stronger than their di-hydroxyl analogues 7, 6, 4, and 8. Unsaturation of the alkyl chain as in 7 and 10 increased the activity relative to their saturated analogue 8. The structure–activity relationship agreed with the results in the literature [28,29]. Compounds 5, 7, and 10 possessed strong oestrogen activity and were major constituents among the diarylheptanoids, on the basis of the amounts obtained from the separation and HPLC analysis of the crude extract (Fig. 1). Therefore, it can be concluded that diarylheptanoids were the oestrogenic-active compounds in the fructus amomi. 3.3. Diarylheptanoids in fructus amomi from different origin
Alnus formosana showed better anti-inflammatory activities than oregonin [30], diarylheptanoids from Alnus nepalensis leaves exhibited antifilarial activity [31], diarylheptanoids from Schrankia leptocarpa possessed trypanocidal and antiplasmodial activities in the sub-micromolar to the micromolar ranges against Trypanosoma brucei rhodesiense and Plasmodium falciparum K1 multiresistant strain [32], diarylheptanoids from Betula platyphylla bark had the potential to prevent the progression of neurodegenerative pathologies and to promote cognitive performance [33], and diarylheptanoids from Aframomum melegueta showed antioestrogen-activity in a receptor cofactor assay system for Era [28]. The content of the total diarylheptanoids in fructus amomi was relatively high (more than 0.55%). Therefore, fructus amomi is a diarylheptanoids-rich resource, and it may supply some diarylheptanoids for advanced research of bioactivities. If detection of other bioactivity is applied to assay the HSCCC fractions, a similar method can be used for the analysis of other bioactive components. The combination method of HSCCC and HPLC will be useful for screening target compounds. 4. Conclusion The combination of HSCCC and HPLC was applied to isolate the oestrogen-active compounds in fructus amomi. Eight diarylheptanoids were obtained and identified as the main oestrogen-active compounds in the fructus amomi of A. longiligulare. The fructus amomi of A. longiligulare and A. villosum contained total diarylheptanoids of more than 0.55%. The results demonstrated that fructus amomi is a diarylheptanoids-rich food phytoestrogen resource, and the combination method of HSCCC and HPLC can be applied for the screening of bioactive compounds.
The fructus amomi used in China is mainly from Burma, Thailand, Malaysia, Chinese Guangdong, and Chinese Hainan. The composition of fructus amomi from different locations varies because they belong to different spices or subspices. Table 4 shows the content of the eight diarylheptanoids in the samples of different origin. Samples from Burma, Thailand, and Malaysia (A. villosum) exhibited similar diarylheptanoid composition, which was different from the sample from Chinese Hainan (A. longiligulare). A. villosum did not contain compounds 6, 9, and 11, which were contained in A. longiligulare. The sample of A. villosum var. xanthioides showed similar diarylheptanoid composition to A. villosum Lour. The total content of diarylheptanoids in the subspices was lower than in the spices. These data suggested that the fructus amomi of A. villosum possessed stronger oestrogen-activity than A. longiligulare. The content of the total diarylheptanoids in all the fructus amomi was 5.5 mg/g dry weight (Table 3).
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jchromb. 2014.03.006.
3.4. Discussion
References
The bioactivities of diarylheptanoids have being studied recently. For example, the diarylheptanoids isolated from
Acknowledgments This research was supported by a fund of the National Natural Science Foundation of China (Grant No. 31270724), and a fund of Natural Science Foundation of Zhejiang Province (Grant No. LZ12C16004). Appendix A. Supplementary data
[1] J.L. Limer, V. Speirs, Breast Cancer Res. 6 (2004) 119. [2] M. Wu, P. Guo, S.W. Tsui, H. Chen, Z. Zhao, Food Res. Int. 48 (2012) 226.
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[3] I.G. Kim, S.C. Kang, K.C. Kim, E.S. Choung, O.P. Zee, Environ. Toxicol. Pharmacol. 25 (2008) 75. [4] H. Rho, J. Lee, B. Koo, Z. Zhao, J. Park, H. Kim, J. Korean Diabetes 26 (2002) 126. [5] Y. Kang, H.Y. Kim, J. Ethnopharmacol. 92 (2004) 103. [6] S. Zhang, Z. Wang, T. Wang, M. Li, J. Lin, Nat. Prod. Res. 23 (2011) 464. [7] Q. Du, H. Chen, Food Chem. 119 (2010) 567. [8] Q. Du, Z. Li, Y. Ito, J. Chromatogr. A 923 (2001) 271. [9] E. Demirpence, M.J. Duchesne, E. Badia, D. Gagne, M. Pons, J. Steroid Biochem. Mol. Biol. 46 (1993) 355. [10] A. Freyberger, G. Schmuck, Toxicol. Lett. 155 (2005) 1. [11] J.C. Le Bail, F. Varnat, J.C. Nicolas, G. Habrioux, Cancer Lett. 130 (1998) 209. [12] K. Zumbanse, A. Dohring, Adv. Synth. Catal. 352 (2010) 1135. [13] L.Q. Cui, K. Liu, C. Zhang, Org. Biomol. Chem. 9 (2011) 2258. [14] H. Kikuzaki, M. Kobayashi, N. Nakatani, Phytochemistry 30 (1991) 3647. [15] J. Chen, J.J. Karchesy, R.F. González-Laredo, Planta Med. 64 (1998) 74. [16] K.S. Sjöberg, P.G. Knutsson, J. Chem. Ecol. 21 (1995) 1339. [17] C.A.C. Araujo, L.V. Alegrio, L.L. Leon, Phyochemistry 49 (1998) 751. [18] J. Li, C.R. Liao, J.Q. Wei, L.X. Chen, F. Zhao, Bioorg. Med. Chem. Lett. 21 (2011) 5363. [19] A. Yokosuka, Y. Mimaki, H. Sakagami, Y. Sashida, J. Nat. Prod. 65 (2002) 283. [20] M.G. Phan, T.T.C. Truong, T.S. Phan, K. Matsunami, H. Otsuka, Chem. Nat. Compd. 47 (2011) 735.
[21] E. Lee, B. Moon, Y. Park, S. Hong, S. Lee, Y. Lee, Y. Lim, Bull. Korean Chem. Soc. 29 (2008) 507. [22] M.H. Rossia, M. Yoshidab, J.G.S. Maiac, Phytochemistry 45 (1997) 1263. [23] D.T. Huong, D.V. Luong, T.P. Thao, T.V. Sung, Pharmazie 60 (2005) 627. [24] M. Nomura, T. Tokoroyama, T. Kubota, Phytochemistry 20 (1981) 1097. [25] S. Sang, K. Lapsley, W. Jeong, P.A. Lachance, C. Ho, R.T. Rosen, J. Agric. Food Chem. 50 (2002) 2459. [26] G. Innocenti, E. Vegetob, S. Dall’Acqua, P. Ciana, M. Giorgetti, E. Agradi, A. Sozzi, G. Fico, F. Tome, Phytomedicine 14 (2007) 147. [27] Z. Huang, F. Fang, J. Wang, C.W. Wong, FEBS Lett. 584 (2010) 22. [28] A.M. El-Halawany, M. Hattori, Food Chem. 134 (2012) 219. [29] A. Suksamrarn, M. Ponglikitmongkol, K. Wongkrajang, A. Chindaduang, S. Kittidanairak, A. Jankam, B. Yingyongnarongkul, N. Kittipanumat, R.C.P. Khetkam, P. Piyachaturawat, Bioorg. Med. Chem. 16 (2008) 6891. [30] Y. Lai, C. Chen, W. Lin, S. Lee, Phytochemistry 73 (2012) 84. [31] D. Yadav, S.C. Singh, R.K. Verma, K. Saxena, R. Verma, P.K. Murthy, M.M. Gupta, Phytomedicine 20 (2013) 124. [32] L. Lagnika, B. Weniger, B. Attioua, O. Jensen, C. Anthaume, A. Sanni, M. Kaiser, A. Lobstein, C. Vonthron-Senecheau, S. Afr. J. Bot. 83 (2012) 92. [33] K.Y. Lee, E.J. Jeong, J. Huh, M. Cho, T.B. Kim, B.J. Jeon, S.H. Kim, H.P. Kim, S.H. Sung, Phytomedicine 19 (2012) 1315.
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Analysis and determination of oestrogen-active compounds in fructus amomi by the combination of high-speed counter-current chromatography and high performance liquid chromatography Hao Ying, Jinpeng Liu, Qizhen Du ∗ Institute of Food Science, Zhejiang A & F University, Linan 311300, Hangzhou, China
a r t i c l e
i n f o
Article history: Received 7 January 2014 Accepted 5 March 2014 Available online 15 March 2014 Keywords: Fructus amomi Oestrogenic activity Analysis Diarylheptanoids
a b s t r a c t Amomum longiligulare or Amomum villosum showed oestrogenic activity. In the present study, oestrogenactive components in fructus amomi, the seeds of A. longiligulare were separated by high-speed countercurrent chromatography (HSCCC) using stepwise elution of eight mobile phases with gradient polarity and advanced separation by high performance liquid chromatography (HPLC). The results yielded 17 compounds with the amount of 8–138 mg and a purity of 94.3–99.8% from a 3 g ethanolic extract of fructus amomi. The chemical structures of the compounds were identified by ESI-MS and NMR spectra, in which eight diarylheptanoids were demonstrated as the main oestrogen-active compounds in the fructus amomi. Determination of the diarylheptanoids in fructus amomi from various origins showed that fructus amomi contains more than 0.5% total diarylheptanoids. The results showed that fructus amomi is a diarylheptanoids-rich food resource possessing oestrogen-activity. The combination method of HSCCC and HPLC can be applied for the analysis of bioactive compounds by detecting the corresponding bioactivity in the HSCCC fractions and separating the target compounds with HPLC. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Phytoestrogens derived from food sources could be an effective supplement in treating human oestrogen deficiency [1]. Fructus amomi, the seeds of Amomum xanthioides, is a favourite condiment for food cooking because it possesses an aromatic and pungent odour [2]. We are investigating the phytoestrogenic activity of fructus amomi, because an oestrogen-activity assay of the total herbal plant of Amomum villosum var. xanthioides (A. xanthioides) (Zingiberaceae) demonstrated that the fractions that were extracted by CH2 Cl2 , EtOAc, and BuOH showed strong effect (Table 1) [3]. Though fructus amomi has functions on the treatment of diabetes [4,5] and its essential oil possesses antimicrobial activities [6], no reports have involved its oestrogen-activity. Therefore, the present study focuses on the chemical constituents with oestrogen-activity in fructus amomi and their content in fructus amomi from different origin. High-speed counter-current chromatography (HSCCC) is a liquid–liquid partition chromatographic method in which compounds are partitioned between two immiscible liquid phases without solid support or irreversible adsorption. It exhibits an
∗ Corresponding author. Tel.: +86 571 15958126861; fax: +86 571 88218710. E-mail address: [email protected] (Q. Du). http://dx.doi.org/10.1016/j.jchromb.2014.03.006 1570-0232/© 2014 Elsevier B.V. All rights reserved.
advantage of fractioning components containing all the chemicals in the crude sample because all of the chemicals remain within the mobile phase or stationary phase [7,8]. Conventional isolation methods can lead to the loss of active components during the isolation and purification phases because of irreversible adsorption or decomposition. Because of the advantages of HSCCC, we can first separate an extract into fractions, and then detect the oestrogenactivity of the fractions for further separation and purification. This procedure is useful for screening minor bioactive compounds by adjusting the amount of each fraction for the bioactive assay. MVLN cell, a human breast cancer cell, has been used successfully to screen Phytoestrogens or environmental oestrogen-like chemicals [9,10]. In this study, the MVLN cell line was used to detect and evaluate the oestrogen-activity of the fractions and chemicals isolated from the fructus amomi. The aim of the present study is to clarify phytoestrogenic chemicals in fructus amomi, and also to establish a method for screening Phytoestrogens from plant materials. 2. Materials and methods 2.1. Materials All solvents for extraction and separation were of analytical grade, purchased from Hangzhou Huadong Chemicals Inc., China. Fructus amomi, the seeds of Amomum longiligulare used for the
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Table 1 Extraction yields and estrogenic activities of solvent fractions of Zingiberaceae A. xanthioides Wall. Layers
Yield (w/w)
EC50 (g/ml)
n-Hexane CH2 Cl2 EtOAc BuOH 17-Estradiol (E2 )
0.38a 0.37 0.55 0.93
>100 0.409 0.323 0.056 0.0037
a
All data from Ref. [3].
extraction and separation of oestrogen-activity, were provided by Nanhai Farm, Hainan, China. Fructus amomi, the seeds of A. villosum and A. villosum var xanthioides used for determination of diarylheptanoids were purchased in a local Chinese medicine store in Hangzhou, China. The dried seeds were directly used for extraction after powdering. HPLC-grade methanol was of products of Merck (Merck Chemicals (Shanghai) Co., Ltd., China). 2.2. Extraction The freeze-dried sample (2 kg) was extracted twice with 12 l 90% ethanol for 2 h at 50 ◦ C. The extracts were combined and evaporated to syrup. The syrup was defatted with ether and lyophilised, yielding 297 g crude extract. A portion of the crude extract (3 g) was used for HSCCC separation to obtain fractions for the oestrogen-activity assay. 2.3. High-speed counter-current chromatographic separation The high-speed counter-current chromatograph used in the present study was constructed at the Institute of Food and Biological Engineering, Zhejiang Gongshang University, Hangzhou, China. The apparatus was equipped with a 1200-ml column with six-layer coils made from 5.0 mm i.d. polytetrafluoroethylene (PTFE) tubing. A K-1800 Wellchrom preparative HPLC pump (Knauer, Germany), a 50-ml sample loop made of 3-mm i.d. PTFE tubing, and a B-684 collector (Büchi, Switzerland) with 50-ml tube racks were utilised to constitute a HSCCC system. The sample solution was prepared by dissolving 3 g ethanolic extract in 45 ml stationary phase solvent (water saturated with n-butanol and ethyl acetate). At the beginning of the separation procedure, the column was filled with the stationary phase solvent. Then, the apparatus was rotated at 1000 rpm and the sample solution was injected into an HSCCC system through the sample loop with the mobile phase at a flow rate of 4.0 ml/min (head to tail mode). The effluent was collected as the mobile phase started to get out of the column. The stationary phase was water saturated with n-butanol and ethyl acetate. The stepwise elution was performed with the following mobile phases: n-hexane–ethyl acetate (1:1) for 60 min, n-hexane–ethyl acetate (1:2) for 60 min, n-hexane–ethyl acetate (1:4) for 60 min, ethyl acetate for 60 min, n-butanol–ethyl acetate (1:4) for 60 min, n-butanol–ethyl acetate (1:2) for 60 min, nbutanol–ethyl acetate (2:2) for 60 min, and n-butanol–ethyl acetate (2:1) for 60 min. The effluent was collected in 20-ml fractions by a fraction collector. After the stepwise elution, the solvent was drained from the column from the tail to head of the column and collected in 40-ml fractions. All fractions were assayed for oestrogen-activity. The fractions were combined into seven larger fractions (components) on the basis of their oestrogen-activity (Fig. 1). 2.4. HPLC analysis and preparation The oestrogen-activity components from the HSCCC separation were analysed by HPLC, and the main compounds were
Fig. 1. An oestrogenic activity-chromatogram of fractions obtained from HSCCC separation of 3 g of the ethanolic Fructus amomi extract. Column capacity: 1200 ml; Rotation speed: 1000 rpm; Stationary phase: water saturated with n-butanol and ethyl acetate; Stepwise elution: n-hexane–ethyl acetate (1:1) for 60 min, nhexane–ethyl acetate (1:2) for 60 min, n-hexane–ethyl acetate (1:4) for 60 min, ethyl acetate for 60 min, n-butanol–ethyl acetate (1:4) for 60 min, n-butanol–ethyl acetate (1:2) for 60 min, n-butanol–ethyl acetate (2:2) for 60 min, and n-butanol–ethyl acetate (2:1) for 60 min; Flow rate: 4 ml/min; Sample: 3 g ethanolic extract in 45 ml stationary phase; Fraction volume: 40 ml.
prepared by preparative HPLC. The analytical HPLC system consisted of an Alliance 2695 separations module, an ODS AQ column (150 mm × 3.9 mm i.d., 3 m) and a low temperature evaporative light scattering detector (ELSD-LTII, Shimadzu, Japan). The gradient elution was carried out as follows: water-formic acid (99.5:0.5, v/v) 100% to methanol 100% from 0 to 40 min at a flow rate of 0.8 ml/min. For preparative HPLC, a preparative ODS AQ column (250 mm × 20 mm i.d., 15 m) was employed. The preparative HPLC conditions were determined based on the analytical HPLC results. 2.5. ESI-MS and NMR All ESI-MS experiments were performed on a Bruker Esquire LC–MS ion trap multiple mass spectrometer (Bremen, Germany) in positive and negative ionisation modes analysing ions up to m/z 2200. 1 H-, 13 C-, and DEPT 90/135-NMR spectra were recorded in DMSO-d 6 on a Bruker 400 (Karlsruhe, Germany) with 400 MHz for 1H-, and 100 MHz for 13 C-measurements. 2.6. Luciferase assay for detection and evaluation of oestrogen-activity An assay based on the MVLN cell line was used to detect the fractions from HSCCC separation and evaluate the oestrogenactivity of the isolated compounds [11]. One ml solution of each HSCCC tube fraction (40 ml) was evaporated to dryness under vacuum. The residue was dissolved with 2 ml of cell culture solution for assay of oestrogen-activity. The cells were routinely maintained as monolayer cultures at 37 ◦ C in a 5% CO2 -enriched humidified air atmosphere. The cultures were grown in 25 cm2 plastic cell culture flasks in Eagle’s minimal essential medium containing 5% foetal calf serum supplemented with penicillin (100 IU/ml), streptomycin (100 mg/ml), 4-(2-hydroxyethyl)-1piperazi-neethanesulfonic acid (10 mM) and NaHCO3 (0.5 g/l). Cells were grown to confluence and passaged with trypsin–EDTA (0.05/0.02%). For the luciferase assay, the MVLN cells were plated in 96-well plates at a density of 3 × 104 cells/well in embryo freezing medium containing 5% hormone-reduced dextran-coated charcoal-treated foetal bovine serum. The cells were cultivated for 3 days, and
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Fig. 2. HPLC-ELSD analysis of the fractions obtained from the HSCCC separation. Number over peak represents of the compound No. in Table 2. Column: ODS AQ (150 × 3.9 mm i.d., 3 m); Gradient elution: water-formic acid (99.5:0.5, v/v) 100% to methanol 100% from 0 to 40 min; flow rate: 0.8 ml/min; Detector: a low temperature evaporative light scattering detector.
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Fig. 3. The structure of the compounds obtained from the separation and purification by HSCCC and preparative HPLC.
then incubated with various compounds for 24 h. At the end of the treatment, luciferase activity was measured using a commercial Luciferase Assay Kit (Progema, Madison, USA) according to the manufacturer’s protocol. The resultant chemiluminescence was measured using a Molecular Devices Gemini XPS plate reader (Sunnyvale, CA). For each tested sample, we determined the EC50 (concentration required to reach 50% of the maximum chemiluminescence intensity). In brief, the luciferase activity of each sample was assayed at four concentrations 5000, 10,000, 15,000 and 20,000 nM. The EC50 was obtained through a plotting luciferase activity vs sample concentration. All measurements were carried out in triplicate. 3. Results and discussion It is difficult to separate the components in the ethanolic extract of fructus amomi because the polarities of the components range from low to high. A stepwise elution with mobile phases of gradient polarity can yield better separation than a single mobile phase. We employed a wide range of polarities by adjusting the ratio and composition of the solvents that comprised the mobile phase. The low polar mobile phases (n-hexane–ethyl acetate at 1:1, 1:2, and 1:4) were used to elute the low-polarity components, followed by
increasingly polar mobile phases (ethyl acetate; n-butanol–ethyl acetate, 1:4; n-butanol–ethyl acetate, 1:2; n-butanol–ethyl acetate, 2:2; and n-butanol–ethyl acetate, 2:1) to elute medium- and highpolarity components. The performance of the elution with these mobile phases was satisfied because the stationary phase in the column occupied 41% at the end of the separation. The entire run, including the mobile phase elution and the stationary phase extrusion, resulted in 86 fractions (each 40-ml). All the fractions were detected for their oestrogen-activity. The advantage of this method is that the entire sample can be recovered. An oestrogen-activity-chromatogram based on the assay values of all the fractions from the HSCCC separation is shown in Fig. 1. Six of the oestrogen-active components (FrA–FrE) were eluted by the mobile phases, and one oestrogen-active component (FrG) was drained from the stationary phase in the column. The polarity of components FrA–FrG increased from low to high. The high performance liquid chromatography (HPLC) chromatograms (Fig. 2) of components FrA–FrG were compared with the crude extract using an evaporative light scattering detector (ELSD) to afford a relatively complete detection of the compounds in the components. After evaporating the solvents and freeze-drying the residues, FrA–FrG were obtained with the amounts 65, 87, 151, 282, 75, 38, and 16 mg, respectively.
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Table 2 The NMR data of the compounds 5, 7 and 10 which were the major diarylheptanoids in the fructus amomi. Position
1 2 3 4 5 6 7 1 2 3 4 5 6 1 2 3 4 5 6
5
7
10
ıC
ıH
ıC
ıH
ıC
ıH
81.10 d 34.24 t 25.11 t 32.45 t 78.77 d 39.68 t 31.79 t 136.34 s 115.94 d 146.04 s 145.57 s 114.65 d 118.76 d 134.44 s 130.37 d 116.03 d 156.28 s 116.03 d 130.37 d
4.20 (dd, 11.2, 1.8, 1H) 1.51 (m, 2H) 1.91 (m, 2H) 1.30 (m, 2H) 3.43 (m, 1H) 1.78 (m, 2H) 2.64 (m, 2H)
30.65 t 42.69 t 202.83 s 131.59 d 149.25 d 35.68 t 34.52 t 133.16 s 130.30 d 116.15 d 156.57 s 116.15 d 130.30 d 132.99 s 130.37 d 116.15 d 156.61 s 116.15 d 130.37 d
2.65 (t, 7.5, 2H) 2.46 (m, 2H)
30.69 t 42.75 t 202.83 s 131.56 d 149.30 d 35.65 t 34.80 t 133.79 s 116.50 d 146.21 s 144.53 s 116.30 d 120.66 d 133.18 s 133.30 d 116.15 d 156.61 s 116.15 d 133.30 d
2.76 (m, 2H) 2.81 (m, 2H)
6.85 (d, 1.9, 1H)
6.73 (m, 1H) 6.69 (d, 1.8, 1H) 7.00 (d, 8.4, 1H) 6.68 (d, 8.4, 1H) 6.68 (d, 8.4, 1H) 7.00 (d, 8.4, 1H)
3.1. Purification and identification of the compounds in the fractions Compounds corresponding to major HPLC peaks in components FrA–FrE were targeted for preparative HPLC separations. Compounds 1–17 were obtained from the components. The amount of the isolated compounds ranged from 8 to 138 mg. The purities of the compounds ranged from 94.3% to 99.8%, as determined from the HPLC peak area percentage. The structural identification of the compounds was carried out by comparison of ESI-MS, 1 H NMR, and 13 C NMR spectra with those reported in the literature [12–25]. The ESI-MS of compounds 1–17 yielded ions with m/z ESI-MS (m/z) as follows: ESI-MS (m/z): 313 [M−H]− , 329 [M−H]− , 299 [M−H]− , 299 [M−H]− , 295 [M−H]− , 361 [M−H]− , 311 [M−H]− , 297 [M−H]− , 331 [M−H]− , 313 [M−H]− , 431 [M−H]− , 151 [M−H]− , 163 [M−H]− , 431[M−H]− , 463 [M−H]− , 417 [M−H]− , and 593 [M−H]− , respectively. An analysis of 1 H NMR and 13 C NMR spectra combined with the ESI-MS data identified compounds 1–17 (Fig. 3) as 3,5-dihydroxy-7,4 -dimethoxyflavone (1), 3,5,3 -trihydroxy-7,4 dimethoxyflavone (2), 3,5,7-trihydroxy-4 -methoxyflavone (3), 3-hydroxy-7-(4 -hydroxyphenyl)-1-(4 -hydroxyphenyl)heptane (4), 1,5-epoxy-7-(3 ,4 -dihydroxyphenyl)1-(4 -hydroxyphenyl) heptane (5), 3,5-dihydroxy-7-(4 -hydroxy-3 -methoxyphenyl) 1-(3 ,4 -dihydroxyphenyl)heptane (6), 7-(4 -hydroxyphenyl)-1(4 -hydroxyphenyl)-5-hepten-3-one (7), 7-(4 -hydroxyphenyl)1-(4 -hydroxyphenyl)-3-heptanone (8), 3,5-dihydroxy-7(4 -hydroxyphenyl)-1-(3 ,4 -dihydroxyphenyl)heptane (9), 7-(3 ,4 -dihydroxyphenyl)-1-(4 -hydroxyphenyl)-5-hepten3-one (10), 3,5-diacetoxy-7-(3 ,4 -dihydroxyphenyl)-1-(3 ,4 dihydroxyphenyl) heptane (11), 4-methoxybenzoic acid (12), 4-(4-hydroxyphenyl)-2-butanone (13), genistein-7-O-d-glucoside (14), quercetin-3-O--d-glucopyranoside (15), luteolin-8-C-␣-l-arabinoside (16) and kaempferol-3-O-␣-lrhamnopyranosyl-(1-4)--d-glucopyranoside (17). Compounds 5, 7, and 10 (see the 1 H NMR and 13 C NMR data in Table 2, and the spectra in the Supplementary material) are the major diarylheptanoid compounds in the fructus amomi, according to the amounts obtained from the separation of the crude extracts. The majority of these compounds were obtained for the first time from fructus amomi.
6.05 (dt, 15.9, 1.4, 1H) 6.86 (dt, 15.9, 6.9, 1H) 2.77 (m, 2H) 2.77 (m, 2H) 6.98 (dd, 8.5, 4.6, 1H) 6.69 (dd, 8.5, 5.6, 1H) 6.69 (dd, 8.5, 5.6, 1H) 6.98 (dd, 8.5, 4.6, 1H) 6.98 (dd, 8.5, 4.6, 1H) 6.69 (dd, 8.5, 5.6, 1H) 6.69 (dd, 8.5, 5.6, 1H) 6.98 (dd, 8.5, 4.6, 1H)
6.07 (d, 15.8, 1H) 6.87 (m, 1H) 2.46 (m, 2H) 2.63 (m, 2H) 6.62 (d, 2.1, 1H)
6.66 (d, 3.7, 1H) 6.49 (dd, 8.0, 2.1, 1H) 6.98 (d, 8.5, 1H) 6.68 (m, 1H) 6.68 (m, 1H) 6.98 (d, 8.5, 1H)
Table 3 Estrogenicity of compounds in luciferase induction assay with MVLN cells. Sample
EC50 (M)
Crude extract 3,5-Dihydroxy-7,4 -dimethoxyflavone (1) 3,5,3 -Trihydroxy-7,4 -dimethoxyflavone (2) 3,5,7-Trihydroxy-4 -methoxyflavone (3) 3-Hydroxy-7-(4 -hydroxyphenyl)-1-(4 -hydroxyphenyl) heptane (4) 1,5-Epoxy-7-(3 ,4 -dihydroxyphenyl) 1-(4 -hydroxyphenyl) heptane (5) 3,5-Dihydroxy-7-(4 -hydroxy-3 -methoxyphenyl)-1-(3 ,4 dihydroxyphenyl) heptane (6) 7-(4 -Hydroxyphenyl)-1-(4 -hydroxyphenyl)-5-hepten-3-one (7) 7-(4 -Hydroxyphenyl)-1-(4 -hydroxyphenyl)-3-heptanone (8) 3,5-Dihydroxy-7-(4 -hydroxyphenyl)-1-(3 ,4 dihydroxyphenyl) heptane (9) 7-(3 ,4 -Dihydroxyphenyl)-1-(4 -hydroxyphenyl)-5-hepten3-one (10) 3,5-Diacetoxy-7-(3 ,4 -dihydroxyphenyl)-1-(3 ,4 dihydroxyphenyl) heptane (11) 4-Methoxybenzoic acid (12) 4-(4-Hydroxyphenyl)-2-butanone (13) Genistein-7-O--d-glucoside (14) Quercetin-3-O--d-glucopyranoside (15) Luteolin-8-C-␣-l-arabinoside (16) Kaempferol-3-O-␣-l-rhamnopyranosyl-(1-4)--dglucopyranoside (17) 17--Estradiol (E2)b
82.3 × 10−7 a 62.7 × 10−8 53.2 × 10−8 43.6 × 10−8 76.4 × 10−7 18.3 × 10−8 50.6 × 10−7
62.5 × 10−8 30.3 × 10−7 44.7 × 10−7
52.6 × 10−8
27.2 × 10−8
>20 × 10−6 >20 × 10−6 23.1 × 10−8 11.9 × 10−6 13.3 × 10−6 >20 × 10−6
39.7 × 10−12
a
Mole concentration of ethanolic extract was calculated using the molecular weight of genistein-7-O--d-glucoside. b Used as positive control.
3.2. Oestrogen-activity of the compounds The oestrogen-activity of the compounds isolated from the fructus amomi was assayed based on the MVLN cell line using 17--estradiol as the standard. As shown in Table 3, the compounds showed various degrees of oestrogen-activity in MVLN cells. The compounds 1, 2, 3, 5, 7, 10, 11, and 14 exhibited an
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Table 4 The content of diarylheptanoids in fructus amomi from different origin (mg/g, average value, n = 3). Diarylheptanoids
Origin Chinese Guangdonga
Chinese Hainanb
Burmac
Thailandc
Malaysiac
4 5 6 7 8 9 10 11
0.31 0.42 0.06 1.78 0.48 0.03 3.21 0.07
0.21 0.95 0.26 1.33 0.34 0.17 2.42 0.19
0.55 0.76 0.00 2.21 0.35 0.00 4.51 0.00
0.43 1.57 0.00 1.73 0.59 0.00 3.97 0.0
0.39 0.98 0.00 1.52 0.38 0.00 4.23 0.00
Total
6.36
5.87
8.38
8.29
7.50
a b c
A. villosum var. xanthioides. A. villosum. A. longiligulare.
oestrogen-activity at an EC50 level of ×10−8 (18.3 × 10−8 to 62.7 × 10−8 M). The crude extract and compounds 4, 6, 8, and 9 showed an activity at an EC50 level of ×10−7 . Compounds 12, 13, 15, 16, and 17 showed a weak activity (EC50 > level of ×10−6 ). It is clear that flavonoids and diarylheptanoids were active compounds in the fructus amomi. Among the flavonoid compounds, flavones (1, 2, and 3) and isoflavonoid (14) were the main active components. Other glycosyl flavonoids showed low activity. The structure–activity relationship agreed with the regular rules, i.e. glycosylation affects the oestrogen-activity of flavonoids [26], and the substitutions on the B-ring played key roles in oestrogen-activity [27]. Among the diarylheptanoids, the order of oestrogen-activity was 5 > 11 > 10 > 7 > 8 > 6 > 4, which suggested that diarylheptanoid (5) with a similar structure to the B-C ring of flavones was more potent than the others. More than tri- and quad-substituted phenolic hydroxyl groups such as 5, 9, 10, and 11 were stronger than their di-hydroxyl analogues 7, 6, 4, and 8. Unsaturation of the alkyl chain as in 7 and 10 increased the activity relative to their saturated analogue 8. The structure–activity relationship agreed with the results in the literature [28,29]. Compounds 5, 7, and 10 possessed strong oestrogen activity and were major constituents among the diarylheptanoids, on the basis of the amounts obtained from the separation and HPLC analysis of the crude extract (Fig. 1). Therefore, it can be concluded that diarylheptanoids were the oestrogenic-active compounds in the fructus amomi. 3.3. Diarylheptanoids in fructus amomi from different origin
Alnus formosana showed better anti-inflammatory activities than oregonin [30], diarylheptanoids from Alnus nepalensis leaves exhibited antifilarial activity [31], diarylheptanoids from Schrankia leptocarpa possessed trypanocidal and antiplasmodial activities in the sub-micromolar to the micromolar ranges against Trypanosoma brucei rhodesiense and Plasmodium falciparum K1 multiresistant strain [32], diarylheptanoids from Betula platyphylla bark had the potential to prevent the progression of neurodegenerative pathologies and to promote cognitive performance [33], and diarylheptanoids from Aframomum melegueta showed antioestrogen-activity in a receptor cofactor assay system for Era [28]. The content of the total diarylheptanoids in fructus amomi was relatively high (more than 0.55%). Therefore, fructus amomi is a diarylheptanoids-rich resource, and it may supply some diarylheptanoids for advanced research of bioactivities. If detection of other bioactivity is applied to assay the HSCCC fractions, a similar method can be used for the analysis of other bioactive components. The combination method of HSCCC and HPLC will be useful for screening target compounds. 4. Conclusion The combination of HSCCC and HPLC was applied to isolate the oestrogen-active compounds in fructus amomi. Eight diarylheptanoids were obtained and identified as the main oestrogen-active compounds in the fructus amomi of A. longiligulare. The fructus amomi of A. longiligulare and A. villosum contained total diarylheptanoids of more than 0.55%. The results demonstrated that fructus amomi is a diarylheptanoids-rich food phytoestrogen resource, and the combination method of HSCCC and HPLC can be applied for the screening of bioactive compounds.
The fructus amomi used in China is mainly from Burma, Thailand, Malaysia, Chinese Guangdong, and Chinese Hainan. The composition of fructus amomi from different locations varies because they belong to different spices or subspices. Table 4 shows the content of the eight diarylheptanoids in the samples of different origin. Samples from Burma, Thailand, and Malaysia (A. villosum) exhibited similar diarylheptanoid composition, which was different from the sample from Chinese Hainan (A. longiligulare). A. villosum did not contain compounds 6, 9, and 11, which were contained in A. longiligulare. The sample of A. villosum var. xanthioides showed similar diarylheptanoid composition to A. villosum Lour. The total content of diarylheptanoids in the subspices was lower than in the spices. These data suggested that the fructus amomi of A. villosum possessed stronger oestrogen-activity than A. longiligulare. The content of the total diarylheptanoids in all the fructus amomi was 5.5 mg/g dry weight (Table 3).
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jchromb. 2014.03.006.
3.4. Discussion
References
The bioactivities of diarylheptanoids have being studied recently. For example, the diarylheptanoids isolated from
Acknowledgments This research was supported by a fund of the National Natural Science Foundation of China (Grant No. 31270724), and a fund of Natural Science Foundation of Zhejiang Province (Grant No. LZ12C16004). Appendix A. Supplementary data
[1] J.L. Limer, V. Speirs, Breast Cancer Res. 6 (2004) 119. [2] M. Wu, P. Guo, S.W. Tsui, H. Chen, Z. Zhao, Food Res. Int. 48 (2012) 226.
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H. Ying et al. / J. Chromatogr. B 958 (2014) 36–42
[3] I.G. Kim, S.C. Kang, K.C. Kim, E.S. Choung, O.P. Zee, Environ. Toxicol. Pharmacol. 25 (2008) 75. [4] H. Rho, J. Lee, B. Koo, Z. Zhao, J. Park, H. Kim, J. Korean Diabetes 26 (2002) 126. [5] Y. Kang, H.Y. Kim, J. Ethnopharmacol. 92 (2004) 103. [6] S. Zhang, Z. Wang, T. Wang, M. Li, J. Lin, Nat. Prod. Res. 23 (2011) 464. [7] Q. Du, H. Chen, Food Chem. 119 (2010) 567. [8] Q. Du, Z. Li, Y. Ito, J. Chromatogr. A 923 (2001) 271. [9] E. Demirpence, M.J. Duchesne, E. Badia, D. Gagne, M. Pons, J. Steroid Biochem. Mol. Biol. 46 (1993) 355. [10] A. Freyberger, G. Schmuck, Toxicol. Lett. 155 (2005) 1. [11] J.C. Le Bail, F. Varnat, J.C. Nicolas, G. Habrioux, Cancer Lett. 130 (1998) 209. [12] K. Zumbanse, A. Dohring, Adv. Synth. Catal. 352 (2010) 1135. [13] L.Q. Cui, K. Liu, C. Zhang, Org. Biomol. Chem. 9 (2011) 2258. [14] H. Kikuzaki, M. Kobayashi, N. Nakatani, Phytochemistry 30 (1991) 3647. [15] J. Chen, J.J. Karchesy, R.F. González-Laredo, Planta Med. 64 (1998) 74. [16] K.S. Sjöberg, P.G. Knutsson, J. Chem. Ecol. 21 (1995) 1339. [17] C.A.C. Araujo, L.V. Alegrio, L.L. Leon, Phyochemistry 49 (1998) 751. [18] J. Li, C.R. Liao, J.Q. Wei, L.X. Chen, F. Zhao, Bioorg. Med. Chem. Lett. 21 (2011) 5363. [19] A. Yokosuka, Y. Mimaki, H. Sakagami, Y. Sashida, J. Nat. Prod. 65 (2002) 283. [20] M.G. Phan, T.T.C. Truong, T.S. Phan, K. Matsunami, H. Otsuka, Chem. Nat. Compd. 47 (2011) 735.
[21] E. Lee, B. Moon, Y. Park, S. Hong, S. Lee, Y. Lee, Y. Lim, Bull. Korean Chem. Soc. 29 (2008) 507. [22] M.H. Rossia, M. Yoshidab, J.G.S. Maiac, Phytochemistry 45 (1997) 1263. [23] D.T. Huong, D.V. Luong, T.P. Thao, T.V. Sung, Pharmazie 60 (2005) 627. [24] M. Nomura, T. Tokoroyama, T. Kubota, Phytochemistry 20 (1981) 1097. [25] S. Sang, K. Lapsley, W. Jeong, P.A. Lachance, C. Ho, R.T. Rosen, J. Agric. Food Chem. 50 (2002) 2459. [26] G. Innocenti, E. Vegetob, S. Dall’Acqua, P. Ciana, M. Giorgetti, E. Agradi, A. Sozzi, G. Fico, F. Tome, Phytomedicine 14 (2007) 147. [27] Z. Huang, F. Fang, J. Wang, C.W. Wong, FEBS Lett. 584 (2010) 22. [28] A.M. El-Halawany, M. Hattori, Food Chem. 134 (2012) 219. [29] A. Suksamrarn, M. Ponglikitmongkol, K. Wongkrajang, A. Chindaduang, S. Kittidanairak, A. Jankam, B. Yingyongnarongkul, N. Kittipanumat, R.C.P. Khetkam, P. Piyachaturawat, Bioorg. Med. Chem. 16 (2008) 6891. [30] Y. Lai, C. Chen, W. Lin, S. Lee, Phytochemistry 73 (2012) 84. [31] D. Yadav, S.C. Singh, R.K. Verma, K. Saxena, R. Verma, P.K. Murthy, M.M. Gupta, Phytomedicine 20 (2013) 124. [32] L. Lagnika, B. Weniger, B. Attioua, O. Jensen, C. Anthaume, A. Sanni, M. Kaiser, A. Lobstein, C. Vonthron-Senecheau, S. Afr. J. Bot. 83 (2012) 92. [33] K.Y. Lee, E.J. Jeong, J. Huh, M. Cho, T.B. Kim, B.J. Jeon, S.H. Kim, H.P. Kim, S.H. Sung, Phytomedicine 19 (2012) 1315.