Research article Received: 12 July 2013,
Revised: 8 September 2013,
Accepted: 11 October 2013
Published online in Wiley Online Library: 20 November 2013
(wileyonlinelibrary.com) DOI 10.1002/bmc.3082
Tissue distribution comparison between healthy and fatty liver rats after oral administration of hawthorn leaf extract Jingjing Yina, Jianguo Qub, Wenjie Zhanga, Dongrui Lua, Yucong Gaoa, Xixiang Yinga* and Tingguo Kanga ABSTRACT: Hawthorn leaves, a well-known traditional Chinese medicine, have been widely used for treating cardiovascular and fatty liver diseases. The present study aimed to investigate the therapeutic basis treating fatty liver disease by comparing the tissue distribution of six compounds of hawthorn leaf extract (HLE) in fatty liver rats and healthy rats after oral administration at first day, half month and one month, separately. Therefore, a sensitive and specific HPLC method with internal standard was developed and validated to determine chlorogenic acid, vitexin-4′′-O-glucoside, vitexin-2′′-O-rhamnoside, vitexin, rutin and hyperoside in the tissues including heart, liver, spleen, kidney, stomach and intestine. The results indicated that the six compounds in HLE presented some bioactivity in treating rat fatty liver as the concentrations of the six compounds varied significantly in inter- and intragroup comparisons (healthy and/or fatty liver group). Copyright © 2013 John Wiley & Sons, Ltd. Keywords: fatty liver; HPLC; hawthorn leaf extract (HLE); tissue distribution
Introduction The leaves of Crataegus pinnatifida Bge. var. major (hawthorn leaves) are a medicinal substance with a long tradition of use in China (The Pharmacopoeia Commission of PRC, 2010). Hawthorn leaves and their extract possess a wide range of biological and pharmacological activities, such as the treatment of chronic cardiac insufficiency, congestive heart failure (Pittler et al., 2003, 2005) and arrhythmia (Veveris et al., 2004), decreasing blood pressure (Walker et al., 2002), anti-thrombotic effects (Lan et al., 2005) and antioxidation (Bahorun et al., 2007). In addition, hawthorn leaf extract (HLE) presents a unique advantage in treating fatty liver (Wang et al., 2011); total flavones of hawthorn leaves (TFHL) have a hypolipidemic effect, reduce hepatic lipid accumulation and significantly prevent the hyperlipidemia and fatty liver (Ye et al., 2009), which can also alleviate oxidative stress to inhibit NF-κB, Iκ-αmRNA and protein expression, decrease NF-κB activation, and thus effectively prevent nonalcohol fatty liver disease (Yan et al., 2009). Moreover, TFHL can reduce lipid peroxidation and liver injuries caused by hepatic cytokine (Chen et al., 2007), and its mild pharmacological actions and safety with few side effects even at very high doses have been reported by Ammon and Händel (1981). There are many reports focused on in vitro and in vivo analyses of polyphenols in HLE, including chlorogenic acid (CHA) (Chang et al., 2001), vitexin-4′′-O-glucoside (VG) (Ma et al., 2007; Liu et al., 2012), vitexin-2′′-O-rhamnoside (VR) (Ying et al., 2007; Du et al., 2011), vitexin (VIT) (Wang et al., 2012), rutin (RUT) and hyperoside (HP) (Chang et al., 2005b; Ying et al., 2009; Ma et al., 2010). Among these, CHA can prevent oxidation (Ohnishi et al., 1994; Salvi et al. 2001) and inhibit hepatic glucose 6-phosphatase (Arion et al., 1997); VR and VG successfully protect the heart against anoxia/reoxygenation injury (Li et al., 2008b; Ying et al., 2008).
In addition, VR has a protective effect on injured cardiac myocytes and endothelial cells (Zhu et al., 2003, 2006) and has been demonstrated to strongly inhibit DNA synthesis in MCF-7 human breast cancer cells (Ninfali et al., 2007). HP presents cytoprotective properties against oxidative stress by scavenging intracellular resctive oxygen species and enhancing antioxidant enzyme activity (Piao et al., 2008) and against oxidative damage induced by TBHP (Li et al., 2008a). The purpose of this study is to elucidate the therapeutic material basis of HLE treatment for fatty liver disease by comparing the tissue distributions of HLE between healthy and fatty liver rats after oral administration following the different periods (at first day, half month and one month). Through chromatographic analysis the concentrations of the six compounds in the tissues in the healthy and fatty liver groups varied significantly, while obvious variation also occurred within the same group (healthy or fatty liver) after dosing for the three different periods. In the study, the contributions of the six compounds to the treatment of fatty liver disease were investigated but also the main sites of action of the six compounds were observed via their targets or accumulation in certain tissues. * Correspondence to: Xixiang Ying, Liaoning University of Traditional Chinese Medicine, Dalian 116600, China. Email: [email protected] a
School of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian 116600, China
b
Divison of Chemistry, Liaoning Institute for Food and Drug Control, Shenyang 110001, People’s Republic of China Abbreviations used: CHA, chlorogenic acid; HP, hyperoside; RUT, rutin; TFHL, total flavones of hawthorn leaves; VG, vitexin-4′′-O-glucoside; VIT, vitexin; VR, vitexin-2′′-O-rhamnoside
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Materials and methods Plant material The leaves of C. pinnatifida Bge. var. major were collected from Laizhou (Shandong, China) on 15 October 2011, and identified by Professor Wang Bing. Voucher specimens (no. 20111015) are maintained at Liaoning University of Traditional Chinese Medicine, China. Reagents and chemicals VG, VR, VIT and HP were all isolated from hawthorn leaves in our laboratory, and the chemical structures were confirmed by 1H and 13C. Their purities were >99% by HPLC analysis. Caffeic acid was used as the internal standard (IS). RUT and CHA were provided by the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China; Fig. 1). Methanol, acetonitrile and tetrahydrofuran (all of HPLC grade) were obtained from Damao (Chemical Reagent Plant, Tianjin, China), and the water used in all experiments was purified using a Milli-Q® Biocel Ultrapure Water System (Millipore, Bedford, MA, USA). All other chemicals were of analytical reagent grade and purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). Chromatographic system The experiment was performed on an Agilent 1100 series HPLC system (Agilent technology, Palo Alto, CA, USA) equipped with a quaternary pump (G1310A), a vacuum degasser (G1322A), a UV–vis spectrophotometric detector (G1314A) and Chemstation software (Agilent). The analytes were determined at room
temperature on an analytical Diamonsil ODS (150 × 4.6 mm, 5 μm; Dikma Technologies, Bejing, China) protected by a KR C18 guard column (5 μm, 35 × 8.0 mm, Dalian Create Science and Technology Co. Ltd, China). The mobile phase consisted of the solvents (A) acetonitrile–tetrahydrofuran (95:5, v/v) and (B) 0.1% aqueous formic acid (v/v) using a gradient elution of 12–17% A at 0–10 min, 17–20% A at 10–20 min, 20–23% A at 20–30 min, and then returned to initial condition for a 5 min re-equilibration, with total run time 30 min. The mobile phase was filtered and degassed under reduced pressure prior to use. The analysis was carried out at a flow rate of 1 mL/min with the detection wavelength of 270 nm. Preparation of HLE solution A dried 3 kg sample of hawthorn leaves was cut into small pieces and extracted twice by refluxing with 70% ethanol (1:10 and 1:8 w/v) for 2 h, and the extraction solutions were combined, filtered, concentrated under reduced pressure, and then passed through an AB-8 macro-porous resin column (10 × 120 cm, Shanghai, China). Initial elution with 45 L water was performed to eliminate impurities, followed by elution with 15 L 70% ethanol. The eluate was then evaporated under reduced pressure until dryness at 40°C. The residue was suspended with CMC-Na aqueous solution to give an extract with a concentration of 1 g raw material per milliliter. The administration solution was obtained and stored at 4°C before use. Using the external standard method for quantitative analysis, the contents of CHA, VG, VR, VIT, RUT and HP in the suspended solution of extract were 1.23, 12.6, 5.37, 0.12, 0.13 and 0.47 mg/mL, respectively.
Preparation of standards and quality control samples The stock solutions of CHA, VG, VR, VIT, RUT, HP and IS were prepared by precisely weighing the reference standards then dissolving in methanol to yield the concentrations of 203, 700, 492, 445, 369, 285 and 501 μg/mL, respectively. A series of standard mixture working solutions with concentrations 0.05–100 μg/mL for CHA, 0.02–500 μg/mL for VG, 0.01–250 μg/mL for VR, 0.05–40 μg/mL for VIT, 0.1–50 μg/mL for RUT and 0.05–40μg/mL for HP were obtained by diluting the mixture of the stock standard solutions with methanol. All solutions were stored at 4°C. Six calibrators were prepared by spiking the appropriate amount of the standard mixture working solutions (100 μL) into 200 μL drug-free rat plasma. Quality control (QC) samples were prepared at low, medium and high concentrations of 0.15, 2.5 and 80 μg/mL for CHA; 0.06, 4 and 400 μg/mL for VG; 0.03, 2 and 200 μg/mL for VR; 0.15, 1.5 and 64 μg/mL for VIT; 0.3, 2.5 and 40 μg/mL for RUT; 0.15, 1.5 and 32 μg/mL for HP in bulk. Aliquots were stored at 20°C until analysis. Animals and dosing
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Figure 1. Chemical structures of chlorogenic acid, vitexin-4′′-O-glucoside, vitexin-2′′-O-rhamnoside, vitexin, rutin, hyperoside.
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Male Wistar rats, weighing 180–220 g, were obtained from the Laboratory Animal Center of Liaoning University of Traditional Chinese Medicine (Shenyang, China). They were kept in an environmentally controlled breeding room for 1 week before the experiments and fed with standard laboratory food and water ad libitum; they were fasted overnight before the experiment. All experiments involving animals were approved by the Animal Ethics Committee of Liaoning University of Traditional
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0.05–40
0.1–50
HP 0.01–250
RUT 0.02–500
VR
VG
CHA, chlorogenic acid; HP, hyperoside; RUT, rutin; VG, vitexin-4′′-O-glucoside; VIT, vitexin; VR, vitexin-2′′-O-rhamnoside.
0.9992 0.9991 0.9991 0.9999 0.9997 0.9996 0.9992 0.9992 0.9991 0.9998 0.9995 0.9992 0.9997 0.9995 0.9995 0.9992 0.9986 0.9993 y = 33.818x + 12.106 y = 28.958x + 3.2693 y = 28.874x + 13.559 y = 32.727x + 0.4828 y = 38.231x + 3.124 y = 39.512x + 2.5646 y = 12.967x + 2.5287 y = 8.3702x + 9.4432 y = 15.935x + 2.5806 y = 9.9723x + 13.002 y = 14.359x + 16.177 y = 17.173x + 3.9388 y = 12.244x + 2.5179 y = 12.435x + 1.4328 y = 15.462x + 7.4863 y = 10.517x + 6.0407 y = 15.009x + 2.3312 y = 16.347x + 5.5326 Heart Liver Spleen Kidney Stomach Intestine Heart Liver Spleen Kidney Stomach Intestine Heart Liver Spleen Kidney Stomach Intestine VIT 0.05–100
0.9995 0.999 0.999 0.9992 0.9998 0.9996 0.9997 0.9995 0.999 0.9993 0.999 0.999 0.9991 0.999 0.9995 0.9996 0.9991 0.9997 y = 4.6525x + 1.6507 y = 3.5665x + 0.0018 y = 9.0248x + 7.4411 y = 12.445x + 11.884 y = 7.5952x + 8.2196 y = 7.4491x + 4.2321 y = 3.9334x + 11.93 y = 3.129x + 12.015 y = 3.5815x + 12.798 y = 3.573x + 12.363 y = 4.3102x + 12.806 y = 4.5143x + 15.472 y = 17.902x + 65.773 y = 14.276x + 24.589 y = 16.442x + 51.787 y = 16.455x + 55.301 y = 18.282x + 47.053 y = 18.021x + 43.171 Heart Liver Spleen Kidney Stomach Intestine Heart Liver Spleen Kidney Stomach Intestine Heart Liver Spleen Kidney Stomach Intestine CHA
Linear range (μg/mL) r2 Calibration curves Biosamples
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Figure 2. Typical chromatograms of tissue distribution (A–C) respectively obtained from blank stomach sample (A), blank stomach sample spiked with standard analyte and IS (B), and stomach samples at 0.50 h (C) following oral routes of administration of the hawthorn leaf extract (HLE). Peak 1, chlorogenic acid; peak 2, caffeic acid; peak 3, vitexin-4′′O-glucoside; peak 4, vitexin-2′′-O-rhamnoside; peak 5, vitexin; peak 6, rutin; peak 7, hyperoside.
Table 1. Calibration curves, correlation coefficients and linear of six compounds in tissue samples
Biosamples
Calibration curves
r2
Linear range (μg/mL)
Chinese Medicine and performed according to the Guidelines for Animal Experimentation of this institution. Sixty rats were randomly divided into two groups, the healthy group and the fatty liver group, and the lipid emulsion 10 mL/kg everyday was gavaged to the fatty liver rats group for 8 weeks. After the models were established, the HLE was orally administrated to the fatty liver group and healthy group continuously for one month at doses of 10 mL/kg (equal to 12.3 mg/kg of CHA, 126.3 mg/kg of VG, 53.7 mg/kg of VR, 1.2 mg/kg of VIT, 1.3 mg/kg of RUT and 4.7 mg/kg of HP). The rats in the two groups were sacrificed according to the different time points of 0.5, 1 and 1.5 h at first day, half month and one month after oral administration, and tissues including heart, liver, spleen, kidney, stomach and intestine (the contents of both were removed before rinsing), were collected, rinsed with physiological saline, blotted on filter paper and weighed. All the samples were stored at 20°C until analysis.
0.05–40
Tissue distribution of six polyphenols in hawthorn leaf by HPLC
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HP
RUT
VIT
VR
VG
CHA
0.15 2.5 80 0.06 4 400 0.03 2 200 0.15 1.5 64 0.3 2.5 40 0.15 1.5 32
Added concentration (μg/mL)
88.23 ± 0.08 90.56 ± 2.75 94.43 ± 3.55 86.34 ± 1.11 87.35 ± 0.8 90.17 ± 2.7 88.68 ± 0.26 86.52 ± 2.8 91.41 ± 0.6 95.18 ± 0.7 87.39 ± 0.8 89.63 ± 0.8 91.21 ± 3.7 94.20 ± 2.7 93.35 ± 3.55 87.04 ± 5.11 94.33 ± 3.55 84.33 ± 1.55
Extraction recovery (n = 5) 0.148 ± 0.02 2.56 ± 0.09 79.8 ± 1.1 0.06 ± 0.01 3.95 ± 0.50 403 ± 2.7 0.03 ± 0.01 1.96 ± 0.1 201 ± 4.5 0.15 ± 0.10 1.5 ± 0.23 64.1 ± 0.59 0.29 ± 0.08 2.6 ± 0.06 42 ± 2.2 0.15 ± 0.01 1.50 ± 0.10 32.67 ± 2.36
Found concentration (μg /mL) 7.02 4.05 2.42 4.78 5.25 1.62 13.6 10.6 6.2 7.28 5.3 4.15 3.45 3.16 2.4 2.51 6.57 4.68
RSD (%)
Intra-day
6.7 2.0 1.3 3.3 5.0 0.50 3.3 5.0 2.0 6.7 3.6. 3.1 3.3 3.9 5.0 3.3 6.7 3.2
RE (%)
0.153 ± 0.05 2.58 ± 0.12 80.2 ± 1.1 0.06 ± 0.05 4.03 ± 0.38 404 ± 3.2 0.29 ± 0.01 2.09 ± 0.12 202 ± 1.8 0.152 ± 0.05 1. 58 ± 0.19 63.3 ± 0.50 0.28 ± 0.15 2.58 ± 0.14 41.1 ± 1.6 0.15 ± 0.02 1.53 ± 0.03 32 ± 0.52
Found concentration (μg/mL) 4.9 3.28 2.25 2.52 4.57 1.44 10.6 10.2 2.36 5.09 1.33 2.87 2.09 8.2 5.09 2.12 3.43 3.3
2.6 0.5 1.8 1.3 2.4 1.0 5.3 5.5 0.5 3.3 3.3 1.2 3.3 4.0 5.0 3.3 6.7 3.3
RSD (%) RE (%)
Inter-day
96.17 ± 2.32 101.4 ± 1.69 96.26 ± 1.20 93.58 ± 2.42 98.38 ± 1.26 100.2 ± 2.03 94.14 ± 2.65 93.28 ± 2.42 86.88 ± 1.05 89.59 ± 4.72 93.38 ± 2.42 96.11 ± 1.20 93.80 ± 2.42 91.28 ± 1.42 88.48 ± 1.26 100.2 ± 2.03 93.88 ± 2.42 84.04 ± 2.65
Short-term stability
88.21 ± 1.65 93.24 ± 1.46 92.01 ± 1.07 91.65 ± 5.84 91.24 ± 2.25 93.22 ± 3.16 93.86 ± 3.18 93.80 ± 5.42 94.72 ± 0.947 88.46 ± 4.23 93.88 ± 2.42 92.07 ± 1.07 91.62 ± 5.84 93.88 ± 2.42 91.28 ± 2.25 93.20 ± 3.16 83.88 ± 0.49 93.07 ± 3.18
Long-term stability
90.7 ± 1.57 92.58 ± 1.27 90.32 ± 3.78 90.29 ± 3.80 91.49 ± 1.38 93.71 ± 2.28 95.55 ± 3.08 83.88 ± 1.12 91.11 ± 0.96 93.17 ± 4.88 92.88 ± 2.42 91.32 ± 3.78 84.29 ± 3.80 90.88 ± 2.42 92.49 ± 1.38 85.71 ± 2.28 95.88 ± 2.02 86.05 ± 3.08
Freeze–thaw stability
Accuracy (%, mean ± SD)
Table 2. Extraction recovery, precision, accuracy and stability of CHA, VG, VR, VIT, RUT and HP determination in liver (intra-day, n = 5; inter-day, n = 3 with five replicates per day)
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Tissue distribution of six polyphenols in hawthorn leaf by HPLC Sample preparation Each tissue was weighed at approximately 0.2 g and homogenized in 0.5 mL of saline solution. To the homogenate of each tissue successively were added 50 μL IS, 20 μL of acetic acid and 1 mL of methanol, followed by vortex mixing for 1 min. After samples were centrifuged at 890g for 15 min, the supernatant was separated and evaporated to dryness under a gentle stream of nitrogen at 50°C. The residue was diluted in 100 μL of mobile phase and again centrifuged at 15,092g for 5 min. Then an aliquot (20 μL) of clean supernatant was injected into HPLC column for analysis.
Method validation The selectivity was determined by comparing chromatograms of blank tissues obtained from rat prior to dosing with those of corresponding standard tissues spiked with CHA, VG, VR,VIT, RUT, HP and IS, and the samples from rat after administration of HLE. The six calibration curves were in the concentration ranges 0.05–100, 0.02–500, 0.01–250, 0.05–40, 0.1–50 and 0.05–40 μg/mL
for CHA, VG, VR, VIT, RUT and HP, respectively. The calibration curves for each analyte in different tissues were generated by plotting their peak area vs the nominal concentrations in the standard tissue samples. The regression equation was obtained by weighted (1/c2) least squares linear regression. The limits of detection and quantitation (LOD and LOQ) were determined by stepwise dilution of the QC sample at low concentration level using a signal-to-noise ratio of 3 and 10, respectively, giving an acceptable accuracy (relative error, RE) within ± 15% and a precision (relative standard devitation, RSD) that did not exceed 15%. For the intra-day precision and accuracy, five replicates of the QC tissues samples were analyzed on the same day, while the inter-day values were carried out over three consecutive days. The intra-day and inter-day precisions were defined as the RSD and accuracy was determined by calculating the RE. Recoveries of six compounds in tissues were calculated by comparing the peak areas of the extracted quality control samples with that of the unextracted standard solutions containing the equivalent amount of analytes (n = 5). The stabilities of tissue QC samples at low, medium and high concentration were carried out under three conditions.
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Figure 3. Concentrations of CHA (A), VG (B), VR (C), vitexin (D), RUT (E) and HP (F) in different tissues of rats (mean ± SD, n = 5) after an oral administration of HLE solution of doses of 10 ml/kg, equivalent to 12.3 mg/kg of CHA, 126.3 mg/kg of VG, 53.7 mg/kg of VR, 1.2 mg/kg of VIT, 1.3 mg/kg of RUT and 4.7 mg/kg of HP, respectively (from top to bottom is the first day, half month and one month of dosing periods, respectively). CHA, chlorogenic acid; HP, hyperoside; RUT, rutin; VG, vitexin-4′′-O-glucoside; VIT, vitexin; VR, vitexin-2′′-O-rhamnoside.
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Figure 3. (Continued)
Short-term and long-term stability were determined by analyzing QC samples kept at ambient temperature (25°C) for 4 h and stored at 20°C for 1 month. Freeze–thaw stability was investigated after three freeze ( 20°C) and thaw (room temperature) cycles. Then, the samples were processed and analyzed. The concentrations obtained were compared with the nominal values of QC samples.
Results and discussion Method development and optimization
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To obtain suitable retention times and good separation for CHA, VG, VR, VIT, RUT, HP and IS, a gradient elution for the determination of six polyphenols and IS was initially used and unsuccessful for producing a serious baseline drift and interference of endogenous plasma constituents with CHA, VG, VR, VIT, RUT, HP and IS. In addition, some unknown compounds in the HLE also interfered with these peaks for the analytes, thus the mobile phase was chosen after several trials with acetonitrile, tetrahydrofuran and water in various proportions and at different pH values. Finally, the mobile
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phase consisting of the solvents (A) acetonitrile–tetrahydrofuran (95:5, v/v) and (B) 0.1% aqueous formic acid (v/v) using a gradient elution of 12–17% A at 0–10 min, 17–20% A at 10–20 min and 20–23% A at 20–30 min, was used in order to obtain the optimum separation. The maximum absorptions of VG and VR were all at 270 and 332 nm, HP at 259 and 356 nm and CHA at 327 nm. To obtain the high sensitivities for each component and simultaneous determination of the six compounds, three different wavelengths of 270, 332 and 360 nm were chosen. The interferences from endogenous substances in the plasma were observed and were not beneficial to the determination of each analyte when the wavelength was set at 332 and 360 nm, and the detection wavelength was set at 270 nm with no interferences, and therefore was applied to the simultaneous analysis of the six compounds.
Method validation The selectivity of different tissues elucidated using the chromatograms of stomach are shown in Fig. 2(A–C), indicating that there were no interfering peaks in the region of the
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Tissue distribution of six polyphenols in hawthorn leaf by HPLC
Figure 3. (Continued)
peak of the analyte and IS. The calibration curves, correlation coefficients and linear ranges of the six compounds in each tissue are listed in Table 1. The LOD (S/N = 3) and the LOQ (S/N = 10) were respectively 0.0053 and 0.016 μg/mL for CHA, 0.0023 and 0.007 μg/mL for VG, 0.0013 and 0.004 μg/mL for VR, 0.0044 and 0.013 μg/mL for VIT, 0.001 and 0.003 μg/mL for RUT, and 0.0053 and 0.0016 μg/mL for HP in different tissues. The RSDs of the six compounds were all
Revised: 8 September 2013,
Accepted: 11 October 2013
Published online in Wiley Online Library: 20 November 2013
(wileyonlinelibrary.com) DOI 10.1002/bmc.3082
Tissue distribution comparison between healthy and fatty liver rats after oral administration of hawthorn leaf extract Jingjing Yina, Jianguo Qub, Wenjie Zhanga, Dongrui Lua, Yucong Gaoa, Xixiang Yinga* and Tingguo Kanga ABSTRACT: Hawthorn leaves, a well-known traditional Chinese medicine, have been widely used for treating cardiovascular and fatty liver diseases. The present study aimed to investigate the therapeutic basis treating fatty liver disease by comparing the tissue distribution of six compounds of hawthorn leaf extract (HLE) in fatty liver rats and healthy rats after oral administration at first day, half month and one month, separately. Therefore, a sensitive and specific HPLC method with internal standard was developed and validated to determine chlorogenic acid, vitexin-4′′-O-glucoside, vitexin-2′′-O-rhamnoside, vitexin, rutin and hyperoside in the tissues including heart, liver, spleen, kidney, stomach and intestine. The results indicated that the six compounds in HLE presented some bioactivity in treating rat fatty liver as the concentrations of the six compounds varied significantly in inter- and intragroup comparisons (healthy and/or fatty liver group). Copyright © 2013 John Wiley & Sons, Ltd. Keywords: fatty liver; HPLC; hawthorn leaf extract (HLE); tissue distribution
Introduction The leaves of Crataegus pinnatifida Bge. var. major (hawthorn leaves) are a medicinal substance with a long tradition of use in China (The Pharmacopoeia Commission of PRC, 2010). Hawthorn leaves and their extract possess a wide range of biological and pharmacological activities, such as the treatment of chronic cardiac insufficiency, congestive heart failure (Pittler et al., 2003, 2005) and arrhythmia (Veveris et al., 2004), decreasing blood pressure (Walker et al., 2002), anti-thrombotic effects (Lan et al., 2005) and antioxidation (Bahorun et al., 2007). In addition, hawthorn leaf extract (HLE) presents a unique advantage in treating fatty liver (Wang et al., 2011); total flavones of hawthorn leaves (TFHL) have a hypolipidemic effect, reduce hepatic lipid accumulation and significantly prevent the hyperlipidemia and fatty liver (Ye et al., 2009), which can also alleviate oxidative stress to inhibit NF-κB, Iκ-αmRNA and protein expression, decrease NF-κB activation, and thus effectively prevent nonalcohol fatty liver disease (Yan et al., 2009). Moreover, TFHL can reduce lipid peroxidation and liver injuries caused by hepatic cytokine (Chen et al., 2007), and its mild pharmacological actions and safety with few side effects even at very high doses have been reported by Ammon and Händel (1981). There are many reports focused on in vitro and in vivo analyses of polyphenols in HLE, including chlorogenic acid (CHA) (Chang et al., 2001), vitexin-4′′-O-glucoside (VG) (Ma et al., 2007; Liu et al., 2012), vitexin-2′′-O-rhamnoside (VR) (Ying et al., 2007; Du et al., 2011), vitexin (VIT) (Wang et al., 2012), rutin (RUT) and hyperoside (HP) (Chang et al., 2005b; Ying et al., 2009; Ma et al., 2010). Among these, CHA can prevent oxidation (Ohnishi et al., 1994; Salvi et al. 2001) and inhibit hepatic glucose 6-phosphatase (Arion et al., 1997); VR and VG successfully protect the heart against anoxia/reoxygenation injury (Li et al., 2008b; Ying et al., 2008).
In addition, VR has a protective effect on injured cardiac myocytes and endothelial cells (Zhu et al., 2003, 2006) and has been demonstrated to strongly inhibit DNA synthesis in MCF-7 human breast cancer cells (Ninfali et al., 2007). HP presents cytoprotective properties against oxidative stress by scavenging intracellular resctive oxygen species and enhancing antioxidant enzyme activity (Piao et al., 2008) and against oxidative damage induced by TBHP (Li et al., 2008a). The purpose of this study is to elucidate the therapeutic material basis of HLE treatment for fatty liver disease by comparing the tissue distributions of HLE between healthy and fatty liver rats after oral administration following the different periods (at first day, half month and one month). Through chromatographic analysis the concentrations of the six compounds in the tissues in the healthy and fatty liver groups varied significantly, while obvious variation also occurred within the same group (healthy or fatty liver) after dosing for the three different periods. In the study, the contributions of the six compounds to the treatment of fatty liver disease were investigated but also the main sites of action of the six compounds were observed via their targets or accumulation in certain tissues. * Correspondence to: Xixiang Ying, Liaoning University of Traditional Chinese Medicine, Dalian 116600, China. Email: [email protected] a
School of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian 116600, China
b
Divison of Chemistry, Liaoning Institute for Food and Drug Control, Shenyang 110001, People’s Republic of China Abbreviations used: CHA, chlorogenic acid; HP, hyperoside; RUT, rutin; TFHL, total flavones of hawthorn leaves; VG, vitexin-4′′-O-glucoside; VIT, vitexin; VR, vitexin-2′′-O-rhamnoside
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Materials and methods Plant material The leaves of C. pinnatifida Bge. var. major were collected from Laizhou (Shandong, China) on 15 October 2011, and identified by Professor Wang Bing. Voucher specimens (no. 20111015) are maintained at Liaoning University of Traditional Chinese Medicine, China. Reagents and chemicals VG, VR, VIT and HP were all isolated from hawthorn leaves in our laboratory, and the chemical structures were confirmed by 1H and 13C. Their purities were >99% by HPLC analysis. Caffeic acid was used as the internal standard (IS). RUT and CHA were provided by the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China; Fig. 1). Methanol, acetonitrile and tetrahydrofuran (all of HPLC grade) were obtained from Damao (Chemical Reagent Plant, Tianjin, China), and the water used in all experiments was purified using a Milli-Q® Biocel Ultrapure Water System (Millipore, Bedford, MA, USA). All other chemicals were of analytical reagent grade and purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). Chromatographic system The experiment was performed on an Agilent 1100 series HPLC system (Agilent technology, Palo Alto, CA, USA) equipped with a quaternary pump (G1310A), a vacuum degasser (G1322A), a UV–vis spectrophotometric detector (G1314A) and Chemstation software (Agilent). The analytes were determined at room
temperature on an analytical Diamonsil ODS (150 × 4.6 mm, 5 μm; Dikma Technologies, Bejing, China) protected by a KR C18 guard column (5 μm, 35 × 8.0 mm, Dalian Create Science and Technology Co. Ltd, China). The mobile phase consisted of the solvents (A) acetonitrile–tetrahydrofuran (95:5, v/v) and (B) 0.1% aqueous formic acid (v/v) using a gradient elution of 12–17% A at 0–10 min, 17–20% A at 10–20 min, 20–23% A at 20–30 min, and then returned to initial condition for a 5 min re-equilibration, with total run time 30 min. The mobile phase was filtered and degassed under reduced pressure prior to use. The analysis was carried out at a flow rate of 1 mL/min with the detection wavelength of 270 nm. Preparation of HLE solution A dried 3 kg sample of hawthorn leaves was cut into small pieces and extracted twice by refluxing with 70% ethanol (1:10 and 1:8 w/v) for 2 h, and the extraction solutions were combined, filtered, concentrated under reduced pressure, and then passed through an AB-8 macro-porous resin column (10 × 120 cm, Shanghai, China). Initial elution with 45 L water was performed to eliminate impurities, followed by elution with 15 L 70% ethanol. The eluate was then evaporated under reduced pressure until dryness at 40°C. The residue was suspended with CMC-Na aqueous solution to give an extract with a concentration of 1 g raw material per milliliter. The administration solution was obtained and stored at 4°C before use. Using the external standard method for quantitative analysis, the contents of CHA, VG, VR, VIT, RUT and HP in the suspended solution of extract were 1.23, 12.6, 5.37, 0.12, 0.13 and 0.47 mg/mL, respectively.
Preparation of standards and quality control samples The stock solutions of CHA, VG, VR, VIT, RUT, HP and IS were prepared by precisely weighing the reference standards then dissolving in methanol to yield the concentrations of 203, 700, 492, 445, 369, 285 and 501 μg/mL, respectively. A series of standard mixture working solutions with concentrations 0.05–100 μg/mL for CHA, 0.02–500 μg/mL for VG, 0.01–250 μg/mL for VR, 0.05–40 μg/mL for VIT, 0.1–50 μg/mL for RUT and 0.05–40μg/mL for HP were obtained by diluting the mixture of the stock standard solutions with methanol. All solutions were stored at 4°C. Six calibrators were prepared by spiking the appropriate amount of the standard mixture working solutions (100 μL) into 200 μL drug-free rat plasma. Quality control (QC) samples were prepared at low, medium and high concentrations of 0.15, 2.5 and 80 μg/mL for CHA; 0.06, 4 and 400 μg/mL for VG; 0.03, 2 and 200 μg/mL for VR; 0.15, 1.5 and 64 μg/mL for VIT; 0.3, 2.5 and 40 μg/mL for RUT; 0.15, 1.5 and 32 μg/mL for HP in bulk. Aliquots were stored at 20°C until analysis. Animals and dosing
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Figure 1. Chemical structures of chlorogenic acid, vitexin-4′′-O-glucoside, vitexin-2′′-O-rhamnoside, vitexin, rutin, hyperoside.
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Male Wistar rats, weighing 180–220 g, were obtained from the Laboratory Animal Center of Liaoning University of Traditional Chinese Medicine (Shenyang, China). They were kept in an environmentally controlled breeding room for 1 week before the experiments and fed with standard laboratory food and water ad libitum; they were fasted overnight before the experiment. All experiments involving animals were approved by the Animal Ethics Committee of Liaoning University of Traditional
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Biomed. Chromatogr. 2014; 28: 637–647
Biomed. Chromatogr. 2014; 28: 637–647
0.05–40
0.1–50
HP 0.01–250
RUT 0.02–500
VR
VG
CHA, chlorogenic acid; HP, hyperoside; RUT, rutin; VG, vitexin-4′′-O-glucoside; VIT, vitexin; VR, vitexin-2′′-O-rhamnoside.
0.9992 0.9991 0.9991 0.9999 0.9997 0.9996 0.9992 0.9992 0.9991 0.9998 0.9995 0.9992 0.9997 0.9995 0.9995 0.9992 0.9986 0.9993 y = 33.818x + 12.106 y = 28.958x + 3.2693 y = 28.874x + 13.559 y = 32.727x + 0.4828 y = 38.231x + 3.124 y = 39.512x + 2.5646 y = 12.967x + 2.5287 y = 8.3702x + 9.4432 y = 15.935x + 2.5806 y = 9.9723x + 13.002 y = 14.359x + 16.177 y = 17.173x + 3.9388 y = 12.244x + 2.5179 y = 12.435x + 1.4328 y = 15.462x + 7.4863 y = 10.517x + 6.0407 y = 15.009x + 2.3312 y = 16.347x + 5.5326 Heart Liver Spleen Kidney Stomach Intestine Heart Liver Spleen Kidney Stomach Intestine Heart Liver Spleen Kidney Stomach Intestine VIT 0.05–100
0.9995 0.999 0.999 0.9992 0.9998 0.9996 0.9997 0.9995 0.999 0.9993 0.999 0.999 0.9991 0.999 0.9995 0.9996 0.9991 0.9997 y = 4.6525x + 1.6507 y = 3.5665x + 0.0018 y = 9.0248x + 7.4411 y = 12.445x + 11.884 y = 7.5952x + 8.2196 y = 7.4491x + 4.2321 y = 3.9334x + 11.93 y = 3.129x + 12.015 y = 3.5815x + 12.798 y = 3.573x + 12.363 y = 4.3102x + 12.806 y = 4.5143x + 15.472 y = 17.902x + 65.773 y = 14.276x + 24.589 y = 16.442x + 51.787 y = 16.455x + 55.301 y = 18.282x + 47.053 y = 18.021x + 43.171 Heart Liver Spleen Kidney Stomach Intestine Heart Liver Spleen Kidney Stomach Intestine Heart Liver Spleen Kidney Stomach Intestine CHA
Linear range (μg/mL) r2 Calibration curves Biosamples
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Figure 2. Typical chromatograms of tissue distribution (A–C) respectively obtained from blank stomach sample (A), blank stomach sample spiked with standard analyte and IS (B), and stomach samples at 0.50 h (C) following oral routes of administration of the hawthorn leaf extract (HLE). Peak 1, chlorogenic acid; peak 2, caffeic acid; peak 3, vitexin-4′′O-glucoside; peak 4, vitexin-2′′-O-rhamnoside; peak 5, vitexin; peak 6, rutin; peak 7, hyperoside.
Table 1. Calibration curves, correlation coefficients and linear of six compounds in tissue samples
Biosamples
Calibration curves
r2
Linear range (μg/mL)
Chinese Medicine and performed according to the Guidelines for Animal Experimentation of this institution. Sixty rats were randomly divided into two groups, the healthy group and the fatty liver group, and the lipid emulsion 10 mL/kg everyday was gavaged to the fatty liver rats group for 8 weeks. After the models were established, the HLE was orally administrated to the fatty liver group and healthy group continuously for one month at doses of 10 mL/kg (equal to 12.3 mg/kg of CHA, 126.3 mg/kg of VG, 53.7 mg/kg of VR, 1.2 mg/kg of VIT, 1.3 mg/kg of RUT and 4.7 mg/kg of HP). The rats in the two groups were sacrificed according to the different time points of 0.5, 1 and 1.5 h at first day, half month and one month after oral administration, and tissues including heart, liver, spleen, kidney, stomach and intestine (the contents of both were removed before rinsing), were collected, rinsed with physiological saline, blotted on filter paper and weighed. All the samples were stored at 20°C until analysis.
0.05–40
Tissue distribution of six polyphenols in hawthorn leaf by HPLC
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Copyright © 2013 John Wiley & Sons, Ltd.
HP
RUT
VIT
VR
VG
CHA
0.15 2.5 80 0.06 4 400 0.03 2 200 0.15 1.5 64 0.3 2.5 40 0.15 1.5 32
Added concentration (μg/mL)
88.23 ± 0.08 90.56 ± 2.75 94.43 ± 3.55 86.34 ± 1.11 87.35 ± 0.8 90.17 ± 2.7 88.68 ± 0.26 86.52 ± 2.8 91.41 ± 0.6 95.18 ± 0.7 87.39 ± 0.8 89.63 ± 0.8 91.21 ± 3.7 94.20 ± 2.7 93.35 ± 3.55 87.04 ± 5.11 94.33 ± 3.55 84.33 ± 1.55
Extraction recovery (n = 5) 0.148 ± 0.02 2.56 ± 0.09 79.8 ± 1.1 0.06 ± 0.01 3.95 ± 0.50 403 ± 2.7 0.03 ± 0.01 1.96 ± 0.1 201 ± 4.5 0.15 ± 0.10 1.5 ± 0.23 64.1 ± 0.59 0.29 ± 0.08 2.6 ± 0.06 42 ± 2.2 0.15 ± 0.01 1.50 ± 0.10 32.67 ± 2.36
Found concentration (μg /mL) 7.02 4.05 2.42 4.78 5.25 1.62 13.6 10.6 6.2 7.28 5.3 4.15 3.45 3.16 2.4 2.51 6.57 4.68
RSD (%)
Intra-day
6.7 2.0 1.3 3.3 5.0 0.50 3.3 5.0 2.0 6.7 3.6. 3.1 3.3 3.9 5.0 3.3 6.7 3.2
RE (%)
0.153 ± 0.05 2.58 ± 0.12 80.2 ± 1.1 0.06 ± 0.05 4.03 ± 0.38 404 ± 3.2 0.29 ± 0.01 2.09 ± 0.12 202 ± 1.8 0.152 ± 0.05 1. 58 ± 0.19 63.3 ± 0.50 0.28 ± 0.15 2.58 ± 0.14 41.1 ± 1.6 0.15 ± 0.02 1.53 ± 0.03 32 ± 0.52
Found concentration (μg/mL) 4.9 3.28 2.25 2.52 4.57 1.44 10.6 10.2 2.36 5.09 1.33 2.87 2.09 8.2 5.09 2.12 3.43 3.3
2.6 0.5 1.8 1.3 2.4 1.0 5.3 5.5 0.5 3.3 3.3 1.2 3.3 4.0 5.0 3.3 6.7 3.3
RSD (%) RE (%)
Inter-day
96.17 ± 2.32 101.4 ± 1.69 96.26 ± 1.20 93.58 ± 2.42 98.38 ± 1.26 100.2 ± 2.03 94.14 ± 2.65 93.28 ± 2.42 86.88 ± 1.05 89.59 ± 4.72 93.38 ± 2.42 96.11 ± 1.20 93.80 ± 2.42 91.28 ± 1.42 88.48 ± 1.26 100.2 ± 2.03 93.88 ± 2.42 84.04 ± 2.65
Short-term stability
88.21 ± 1.65 93.24 ± 1.46 92.01 ± 1.07 91.65 ± 5.84 91.24 ± 2.25 93.22 ± 3.16 93.86 ± 3.18 93.80 ± 5.42 94.72 ± 0.947 88.46 ± 4.23 93.88 ± 2.42 92.07 ± 1.07 91.62 ± 5.84 93.88 ± 2.42 91.28 ± 2.25 93.20 ± 3.16 83.88 ± 0.49 93.07 ± 3.18
Long-term stability
90.7 ± 1.57 92.58 ± 1.27 90.32 ± 3.78 90.29 ± 3.80 91.49 ± 1.38 93.71 ± 2.28 95.55 ± 3.08 83.88 ± 1.12 91.11 ± 0.96 93.17 ± 4.88 92.88 ± 2.42 91.32 ± 3.78 84.29 ± 3.80 90.88 ± 2.42 92.49 ± 1.38 85.71 ± 2.28 95.88 ± 2.02 86.05 ± 3.08
Freeze–thaw stability
Accuracy (%, mean ± SD)
Table 2. Extraction recovery, precision, accuracy and stability of CHA, VG, VR, VIT, RUT and HP determination in liver (intra-day, n = 5; inter-day, n = 3 with five replicates per day)
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Tissue distribution of six polyphenols in hawthorn leaf by HPLC Sample preparation Each tissue was weighed at approximately 0.2 g and homogenized in 0.5 mL of saline solution. To the homogenate of each tissue successively were added 50 μL IS, 20 μL of acetic acid and 1 mL of methanol, followed by vortex mixing for 1 min. After samples were centrifuged at 890g for 15 min, the supernatant was separated and evaporated to dryness under a gentle stream of nitrogen at 50°C. The residue was diluted in 100 μL of mobile phase and again centrifuged at 15,092g for 5 min. Then an aliquot (20 μL) of clean supernatant was injected into HPLC column for analysis.
Method validation The selectivity was determined by comparing chromatograms of blank tissues obtained from rat prior to dosing with those of corresponding standard tissues spiked with CHA, VG, VR,VIT, RUT, HP and IS, and the samples from rat after administration of HLE. The six calibration curves were in the concentration ranges 0.05–100, 0.02–500, 0.01–250, 0.05–40, 0.1–50 and 0.05–40 μg/mL
for CHA, VG, VR, VIT, RUT and HP, respectively. The calibration curves for each analyte in different tissues were generated by plotting their peak area vs the nominal concentrations in the standard tissue samples. The regression equation was obtained by weighted (1/c2) least squares linear regression. The limits of detection and quantitation (LOD and LOQ) were determined by stepwise dilution of the QC sample at low concentration level using a signal-to-noise ratio of 3 and 10, respectively, giving an acceptable accuracy (relative error, RE) within ± 15% and a precision (relative standard devitation, RSD) that did not exceed 15%. For the intra-day precision and accuracy, five replicates of the QC tissues samples were analyzed on the same day, while the inter-day values were carried out over three consecutive days. The intra-day and inter-day precisions were defined as the RSD and accuracy was determined by calculating the RE. Recoveries of six compounds in tissues were calculated by comparing the peak areas of the extracted quality control samples with that of the unextracted standard solutions containing the equivalent amount of analytes (n = 5). The stabilities of tissue QC samples at low, medium and high concentration were carried out under three conditions.
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Figure 3. Concentrations of CHA (A), VG (B), VR (C), vitexin (D), RUT (E) and HP (F) in different tissues of rats (mean ± SD, n = 5) after an oral administration of HLE solution of doses of 10 ml/kg, equivalent to 12.3 mg/kg of CHA, 126.3 mg/kg of VG, 53.7 mg/kg of VR, 1.2 mg/kg of VIT, 1.3 mg/kg of RUT and 4.7 mg/kg of HP, respectively (from top to bottom is the first day, half month and one month of dosing periods, respectively). CHA, chlorogenic acid; HP, hyperoside; RUT, rutin; VG, vitexin-4′′-O-glucoside; VIT, vitexin; VR, vitexin-2′′-O-rhamnoside.
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Figure 3. (Continued)
Short-term and long-term stability were determined by analyzing QC samples kept at ambient temperature (25°C) for 4 h and stored at 20°C for 1 month. Freeze–thaw stability was investigated after three freeze ( 20°C) and thaw (room temperature) cycles. Then, the samples were processed and analyzed. The concentrations obtained were compared with the nominal values of QC samples.
Results and discussion Method development and optimization
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To obtain suitable retention times and good separation for CHA, VG, VR, VIT, RUT, HP and IS, a gradient elution for the determination of six polyphenols and IS was initially used and unsuccessful for producing a serious baseline drift and interference of endogenous plasma constituents with CHA, VG, VR, VIT, RUT, HP and IS. In addition, some unknown compounds in the HLE also interfered with these peaks for the analytes, thus the mobile phase was chosen after several trials with acetonitrile, tetrahydrofuran and water in various proportions and at different pH values. Finally, the mobile
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phase consisting of the solvents (A) acetonitrile–tetrahydrofuran (95:5, v/v) and (B) 0.1% aqueous formic acid (v/v) using a gradient elution of 12–17% A at 0–10 min, 17–20% A at 10–20 min and 20–23% A at 20–30 min, was used in order to obtain the optimum separation. The maximum absorptions of VG and VR were all at 270 and 332 nm, HP at 259 and 356 nm and CHA at 327 nm. To obtain the high sensitivities for each component and simultaneous determination of the six compounds, three different wavelengths of 270, 332 and 360 nm were chosen. The interferences from endogenous substances in the plasma were observed and were not beneficial to the determination of each analyte when the wavelength was set at 332 and 360 nm, and the detection wavelength was set at 270 nm with no interferences, and therefore was applied to the simultaneous analysis of the six compounds.
Method validation The selectivity of different tissues elucidated using the chromatograms of stomach are shown in Fig. 2(A–C), indicating that there were no interfering peaks in the region of the
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Tissue distribution of six polyphenols in hawthorn leaf by HPLC
Figure 3. (Continued)
peak of the analyte and IS. The calibration curves, correlation coefficients and linear ranges of the six compounds in each tissue are listed in Table 1. The LOD (S/N = 3) and the LOQ (S/N = 10) were respectively 0.0053 and 0.016 μg/mL for CHA, 0.0023 and 0.007 μg/mL for VG, 0.0013 and 0.004 μg/mL for VR, 0.0044 and 0.013 μg/mL for VIT, 0.001 and 0.003 μg/mL for RUT, and 0.0053 and 0.0016 μg/mL for HP in different tissues. The RSDs of the six compounds were all
