Biosci. Biotechnol. Biochem., 70 (12), 2948–2956, 2006
Polyphenolic Content and Physiological Activities of Chinese Hawthorn Extracts Tong C UI,1 Kozo N AKAMURA,2; y Su T IAN,3 Hiroshi K AYAHARA,2 and Yi-Ling T IAN1 1
College of Food Science and Technology of the Agricultural University of Hebei, Hebei 071001, China Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University, Nagano 399-4598, Japan 3 Department of Nutrition and Food Hygiene, Hebei Medical University, Hebei 050017, China 2
Received June 27, 2006; Accepted September 13, 2006; Online Publication, December 7, 2006 [doi:10.1271/bbb.60361]
Hawthorn polyphenol (HP) was prepared by ethyl acetate treatment of the ethanol extract (HE) of Chinese hawthorn fruit. The concentrations of 15 polyphenols in the HP, HE, extraction residue (HJ), and a hawthorn leaf extract (HF) were determined by HPLC. For HP, the total content of the 15 polyphenols was 21.4%, comprised of 19.7% of procyanidins, 1.21% of chlorogenic acid, and 0.48% of flavonoids, compared to 2.55% for the HE. The yields of procyanidin monomer, dimer, trimer, tetramer, and pentamer were 50.5%, 30.3%, 23.0%, 14.6%, and 12.5% respectively, and the mean degree of polymerization was reduced to 1.39 (HP) from 1.65 (HE). Seven different physiological actions of the four extracts were investigated. The HP showed strong O2 and OH scavenging capacities (IC50 values of 6.3 g/ml and 1.1 g/ml respectively), as well as selective prolyl endopeptidase inhibition (IC50 of 60 g/ml). The active constituents appeared to be procyanidins. Key words:
Crataegus pinnatifida; HPLC; procyanidin; antioxidant; free radicals
In Europe, standardized extracts of the leaves and flowers of Crataegus oxyacantha and Crataegus monogyna are used in the therapy for moderate (NYHA II) congestive heart failure.1,2) These extracts exert several pharmacological and clinical effects, such as antiischemia/reperfusion-injury, anti-arrhythmic, hypolipidemic, and hypotensive effects.1,2) Flavonoids and procyanidins are considered to be the two main active constituents.2) Species of Chinese hawthorn, particularly Crataegus pinnatifida and Crataegus pinnatifida Bge. var. major NE Br., have also been used as herbal medicines.2,3) Studies indicate that Chinese hawthorn extracts have beneficial effects, such as antioxidant,4,5) anti-inflammatory,6) and hypolipidemic effects,7) as well as protective effects on the brain and vascular endothelium8,9) and endothelium-dependent relaxation.10) Re-
cently, we analyzed 37 major cultivars of Chinese hawthorn using high performance liquid chromatography (HPLC), and found that procyanidins, which were present in high concentrations, were the main phenolic active constituents in these fruits.11) In general, the hawthorn found in China is an edible fruit that is popular for its special acidic taste. It is bigger than its European counterpart. Since the content of organic acid in Chinese hawthorn fruits can be up to 5% (fresh weight), they are used to produce various foods, such as jams, jellies, juices, tinned foods, wines, and various sweet foods. However, the high sugar content of these fruits counteracts the benefits of polyphenols for consumers of advanced age or for those who suffer from hypertension, cardiovascular disease, or diabetes. Hence, it is desirable to separate the polyphenol and sugar components. Previously, hawthorn extract (HE) for medical applications were ethanol-extracted from the dried fruit of the Chinese hawthorn.12) These crude extracts contain many undesirable substances, such as carbohydrates, organic acids, and moisture, which not only reduce the concentration of active constituents but also hasten procyanidin decomposition in an environment of moist acidity.13) Hence we developed a new extraction procedure14) that uses ethyl acetate treatment of HE to prepare a sugarless extract, which we call hawthorn polyphenol (HP). Using this procedure, the side-products of concentrated hawthorn juice (HJ) and hawthorn dietary fiber can also be prepared. The present report compares the phenolic active compositions of Chinese hawthorn extracts, including HP, HE, HJ, and hawthorn flavonoids (HF), a commercialized extract of Chinese hawthorn leaves, and investigates the physiological activities of these preparations in vitro.
Materials and Methods Chemicals. Xanthine oxidase (EC 1.1.3.22), cyto-
y To whom correspondence should be addressed. Tel/Fax: +81-265-77-1638; E-mail:
[email protected] Abbreviations: HE, hawthorn ethanol extract; HF, hawthorn leaf flavonoids; HJ, concentrated hawthorn juice; HP, hawthorn polyphenols; ACE, angiotensin-(I)-converting enzyme; S, responding value of the sample; B, responding value of the blank; mDP, mean degree of polymerization; IC50 , concentration for inhibitory effect at 50%
Polyphenol and Physiological Activities of Hawthorn Extracts
chrome C, tyrosinase (EC 1.14.18.1, from mushroom), lipoxygenase (EC 1.13.11.12, from soybean), elastase (EC 3.4.21.36, from porcine pancreas), angiotensinconverting enzyme (ACE, EC 3.4.15.1, from rabbit lung), xanthine, tyrosine, linoleic acid, N-succinyl-AlaAla-Ala-p-nitroanilide, epicatechin, chlorogenic acid, rutin, hippuric acid, and 1-methyl-2-pyrrolidinone were purchased from Sigma Chemical (St. Louis, MO). Procyanidin B2, vitexin, vitexin-200 -O-rhamnoside, hyperoside, and isoquercitrin were purchased from Funakoshi (Tokyo). Prolyl endopeptidase (EC 3.4.21.26) was from Toyobo (Osaka). Z-Gly-Pro-pNA and Hippuyl-His-Leu were from Wako (Tokyo). For the HPLC mobile phase, HPLC-grade methanol and acetonitrile were purchased from Kanto Kagaku (Tokyo), and tetrahydrofuran was from Wako. Purified water was obtained using the Sartorius purification system (Sartorius AG, Gottingen, Germany). Other reagents used in the experimental procedure were of analytical-grade. Procyanidins B5 and C1 were isolated and purified from the fruits of C. pinnatifida Bge. var. major NE Br., and were identified by electrospray ionization-mass spectrometry (ESI-MS) and the thiolysis method.15,16) The vitexinglucoside used for peak identification was isolated from the leaves of C. pinnatifida Bge. var. major NE Br. and confirmed by MS and photometric methods. Preparation of hawthorn extracts and sample solutions. Hawthorn (C. pinnatifida Bge. var. major NE Br.) fruits were collected in Xinglong County of Hebei Province, China in October, 2000. The fresh fruits (9.3 kg) with seeds removed were cut into small pieces and extracted three times with 12 liter of ethanol (95%, 80%, and 80%, v/v) at room temperature overnight. The residue was used to produce hawthorn dietary fiber of high pectin content. The ethanol extracts were combined and evaporated at 40 C under reduced pressure, to produce 1.86 kg of HE (70%, w/w), which had the appearance of a brown mucus-like liquid. The HE (200 g) was dissolved in 250 ml of water and extracted twice with 250 ml of petroleum ether to remove the lipid components, and then extracted twice with 450 ml of ethyl acetate. The ethyl acetate layer was evaporated under reduced pressure to produce 5.6 g of HP, which had the appearance of a brown solid. The water phase was evaporated under reduced pressure to give 185 g of HJ as a 70% (w/w) concentrated solution. The 80% hawthorn leaf extract, HF (batch number, 200208006), was purchased from Zhongjin Medicine (Jincheng, Shanxi Province, China). The extracts were diluted to 10 mg (dry weight)/ml using 70% methanol, and the standards were diluted to 4 mg/ml in methanol. These storage solutions were diluted with water to appropriate concentrations for the analysis of physiological activities, and were filtered through a 0.45-mm membrane before HPLC. Instruments. For determination of polyphenols in the
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hawthorn extracts and for estimation of the angiotensinconverting enzyme activity, the Shimadzu LC-10AVP HPLC system equipped with a photodiode array detector (Shimadzu, Tokyo) was employed. For estimation of free-radical scavenger efficacy and enzyme activity, a V530 spectrophotometer equipped with an HMC-358 constant temperature system (Jasco, Osaka, Japan) and a FP 750 fluorescence spectrophotometer (Jasco) were employed. HPLC/MS analysis was performed using the Agilent 1100 Series LC/MSD Trap System (Agilent Technologies, Palo Alto, CA). Quantitative analysis. Determination of the organic acids in the hawthorn extracts was performed by the titration method, and the acid content was expressed as the amount of citric acid. HPLC quantitative analysis of the polyphenols in the hawthorn extracts was performed on a Mightysil RP-18 GP Aqua (5-mm; 250 mm 4.6 mm i.d.) column (Kanto Kagaku, Tokyo). The flow rate was 0.8 ml/min and the injection volume was 5 ml. Formic acid (0.04% v/v) was employed as mobile phase A, and methanol:acetonitrile:tetrahydrofuran (52:37:11, v/v/v) was employed as mobile phase B. The gradient procedure was 0–8 min with 16% B, 8–20 min with 16– 18% B, 20–30 min with 18–24% B, 30–33 min with 24% B, 33–38 min with 24–50% B, 38–45 min with 50% B, 45–48 min with 50–16% B, and 48–55 min with 16% B. The column temperature was 40 C and the wavelength used for detection was 280 nm for the procyanidins, 325 nm for chlorogenic acid, and 350 nm for the flavonoids. The concentrations of epicatechin, procyanidin B2, B5, C1, chlorogenic acid, hyperoside, isoquercitrin, vitexin-200 -rhamnoside, and vitexin-glycoside were determined by the external standard method. The remaining six unidentified oligomeric procyanidins, viz., trimer II, trimer III, tetramer I, tetramer II, pentamer I, and pentamer II, were calculated using the peak areas and calibration curve of epicatechin. For vitexin-glycoside in the HF, the content was calculated using vitexin-200 -Orhamnoside as the reference. Determination of hippuric acid was performed using the isocratic HPLC method. Briefly, a TSK-Gel ODS 120A (5-mm; 150 mm 4.6 mm i.d.) column (Tosoh, Tokyo) was set up at 30 C and used under the following conditions: the mobile phase was acetonitrile:0.04% (v/v) phosphoric acid (12:88, v/v), the flow rate was 0.8 ml/min, the UV detector was set at 228 nm, and the injection volume was 10 ml. HPLC-MS analysis. HPLC separations were performed as described above for the polyphenol determinations. The mass spectra were acquired in ESI mode using nitrogen gas at 300 C, with a flow rate of 10 l/ min, a nebulizer pressure of 50 psi, a quadrupole temperature of 30 C, and a capillary voltage of 3,500 V. The mass spectrometer was operated in positive mode at 80 eV; the target was 1,200 m=z and the scanning range was 100–2,000 m=z.
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T. C UI et al.
Superoxide radical scavenger efficacy assay. The cytochrome C method17) was employed to monitor superoxide radical (O2 ) scavenging efficacy. Thus, 1.5 ml of 0.2 mM phosphate buffer (pH 7.8) that contained 0.1 mM EDTA Na2 , 0.5 ml of 0.3 mM xanthine in phosphate buffer solution, 0.5 ml of 0.06 mM cytochrome C, and 0.3 ml of each sample or blank solution were mixed. After incubation at 25 C for 5 min, the reaction was started by the addition of 0.2 ml of 0.06 U/ ml of xanthine oxidase, and absorbance was monitored continuously for 30 s with the kinetic mode at 550 nm. The variation rates of the absorbance (A/min) of the samples (S) and the blank (B) were measured, and the scavenging efficacies (E %) of the samples are calculated according to the following equation (eq. 1):
.
E % ¼ 100 ðB SÞ=B
ð1Þ
S and B are the responding values of the sample and blank in the tests of scavenging and inhibitory action respectively. This equation was used to find scavenging and inhibitory activities in this study. Hydroxyl radical scavenging efficacy assay. A fluorescence quenching method18) was employed to measure hydroxyl radical ( OH) scavenging efficacy. To 50 ml of 20 mM H2 O2 solution, 0.5 ml of 0.85 M benzoic acid solution and 0.5 ml of each sample or blank solution were added. After exposure for 20 min under a UV lamp (30 W, 280 nm), the fluorescence intensity of each sample (S) or blank (B) was determined immediately with an excitation wavelength of 302 nm and an emission wavelength of 411 nm. The scavenging efficacy (E %) of each sample was calculated according to eq. 1. Tyrosinase activity assay. Tyrosinase activity was measured19) by mixing 0.9 ml of 0.2 M Mcllvaine buffer (pH 6.8), 1 ml of 0.03% tyrosine in Mcllvaine buffer, and 1 ml of each sample solution or water (as the blank) and incubating the mixture for 5 min at 30 C. To start the reaction, 0.1 ml of 1,500 U/ml tyrosinase was added. After 20 min, the reaction was terminated by the addition of 0.1 ml of 6.5% NaN3 and the absorbance of each sample (S) and blank (B) was determined at 475 nm. The tyrosinase inhibitory efficacies (E %) were calculated according to eq. 1. Lipoxygenase activity assay. The reported method20) was modified to measure lipoxygenase activities. Briefly, 2 ml of 0.2 M borate buffer (pH 9.0), 20 ml of 30,000 U/ml lipoxygenase-borate buffer, and 20 ml of each sample solution in methanol or methanol alone (as the blank) were mixed. After 5 min of incubation at 25 C, the reaction was started by the addition of 50 ml of linoleic acid solution (0.6% in EtOH, v/v). The absorbance was monitored continuously for 5 min at 234 nm. The lipoxygenase activity of the sample (S) or blank (B) was expressed as the variation rate of the
absorbance (A/min), and the inhibitory efficacies (E %) were calculated according to eq. 1. Prolyl endopeptidase activity assay. The previously described method21) was modified to assay prolyl endopeptidase activity. Thus, 1.8 ml of 0.1 M phosphate buffer (pH 7.0), 0.1 ml of 3.5 mM Z-Gly-Pro-pNA in 40% (v/v) dioxane, and 0.2 ml of each sample solution or water (as the blank) were mixed and pre-incubated for 5 min at 30 C. The reaction was started by adding 0.1 ml of 0.3 U/ml prolyl endopeptidase in phosphate buffer, and absorbance was monitored continuously for 1 min at 410 nm. The prolyl endopeptidase activities were expressed as the variation rates of absorbance (A/ min), and the inhibitory efficacies (E %) were calculated according to eq. 1. Elastase activity assay. To measure elastase activity,22) 2.5 ml of 0.2 M Tris–HCl buffer, 0.1 ml of 3 mM SA3 N-MP solution, and 0.3 ml of each sample solution or water (as the blank) were mixed and incubated at 25 C for 5 min. The reaction was started by the addition of 0.1 ml of 3 U/ml of elastase in Tris buffer, and the absorbance at 410 nm was monitored for 5 min. The elastase activities were expressed as the variation rates of absorbance (A/min), and the inhibitory efficacies (E %) were calculated according to eq. 1. All of the IC50 (concentration in mg/ml required to inhibitory activity by 50%) values were calculated from the log doseinhibition (scavenging) curves. ACE activity assay. ACE activity was measured by a HPLC method that employed Hippuyl-His-Leu as the substrate.23) For this assay, 60 ml of 1 M NaCl–0.2 mM boric acid buffer (pH 8.3), 50 ml of 12.5 mM HippuylHis-Leu in the boric acid buffer, and 50 ml of each sample solution or water (as the blank) were mixed. The sample solutions were at a concentration of 4,000 mg/ ml. The mixture and 40 ml of 50 mU/ml of ACE in the boric acid buffer were co-incubated for 120 min at 37 C, and the reaction was terminated by the addition of 200 ml of 0.1 M HCl. Ethyl acetate (0.8 ml) was added to extract the released hippuric acid, and the samples were stored at 4 C for 30 min. Subsequently, 0.4 ml of the supernatant was removed and evaporated to dryness, and the residue was dissolved in 100 ml of methanol. A 10 ml aliquot of the solution was used in the determination of hippuric acid, which was measured by the HPLC method described above. The ACE activities were expressed as the concentration of hippuric acid, and the inhibitory efficacy (E %) was calculated according to eq. 1.
Results and Discussion Preparation of HP Procyanidins, which are the main phenolic constituents in Chinese hawthorn,11) can easily be extracted
Polyphenol and Physiological Activities of Hawthorn Extracts
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Table 1. Concentrations of Organic Acid and Polyphenols in Chinese Hawthorn Extracts (expressed as g/100 g dry-weight; average SD; n ¼ 3) Peak 1 4 2 14 6 3 8 5 7 9 10 13 15 12 11
Component
HE
HP
HJ
HF
Organic acid Chlorogenic acid Epicatechin Procyanidin B2 Procyanidin B5 Procyanidin C1 Procyanidin trimer II Procyanidin trimer III Procyanidin tetramer I Procyanidin tetramer II Procyanidin pentamer I Procyanidin pentamer II Hyperoside Isoquercitrin Vitexin-200 -rhamnoside Vitexin-glucoside Total polyphenols PC mDP
20:62 0:11 0:15 0:03 0:78 0:04 0:78 0:03 0:08 0:01 0:28 0:01 0:09 0:01 0:03 0:01 0:09 0:01 0:11 0:01 0:03 0:01 0:05 0:01 0:03 0:03 0:02 0:01
