Evaluation of antioxidant-associated efficacy of flavonoid(1).pdf.doc

Journal of Ethnopharmacology 158 (2014) 325–330 Contents lists available at ScienceDirect Journal of Ethnopharmacology journal homepage: Ethnopharmacological communication Evaluation of antioxidant-associated efficacy of flavonoid extracts from a traditional Chinese medicine Hua Ju Hong (peels of Citrus grandis (L.) Osbeck) Jianping Jiang a,b, 1, Letian Shan b,1, Zhiyun Chen a,1, Haishun Xu c, Jianping Wang a, Yuwen Liu d, Yaokang Xiong b,n a The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310006, China b Zhejiang Chinese Medical University, Hangzhou 310053, China c Zhejiang Academy of Medical Sciences, Hangzhou 310013, China d Hangzhou Institute for Food and Drug Control, Hangzhou 310017, China a r t i c l e i n f o Article history: Received 23 March 2014 Received in revised form 23 September 2014 Accepted 20 October 2014 Available online 31 October 2014 Keywords: Citrus grandis (L.) Osbeck Hua Ju Hong Antioxidative activity Total flavonoids HPLC  a b s t r a c t Ethnopharmacological relevance: Hua Ju Hong (HJH, peels of Citrus grandis (L.) Osbeck) is a popularly used traditional Chinese medicine recorded by “Compendium of Materia Medica” (Ben Cao Gang Mu) in Ming Dynasty of China (1578 A.D.). With flavonoid components, HJH possesses hypolipidemic effect associated with antioxidation action. However, no report was found regarding the flavonoid profile and antioxidant activity of HJH. Materials and methods: Five purified flavonoid extracts (TFCA, TFCB, TFCC, TFCD and TFCE.) were obtained from HJH using Ca(OH)2 and HPD-300 macroporous resins, and their total flavonoids and representative flavonoid components were analyzed. In vitro tests of DPPH free radical scavenging activity, reducing power, and total antioxidant activity of each extract were evaluated. The most effective extract was selected for in vivo antioxidative evaluation using a rat hyperlipemia model. Results: The total flavonoid content was varying among each HJH extract and decreased in an order of TFCB4TFCD4TFCC4TFCE4TFCA. TFCB, TFCD, and TFCC contained more than 50% of total flavonoids, the highest content of which was found in TFCB (80.7%). HPLC analysis showed that the contents of three flavonoid components, narirutin, naringin and neohesperidin, displayed a similar trend as that of total flavonoids. In vitro antioxidative tests determined that TFCB at 0.24 to 1.2 mg/ml possessed the most significant antioxidant effects among other extracts and was also superior to BHT. In vivo experiment also revealed the significant antioxidant and antihyperlipidemic activities of TFCB started from 50 to 200 mg/kg after oral administration to hyperlipemia rats. These results indicate that TFCB with the highest flavonoid contents has the strongest antioxidant-associated activities. Conclusion: This is the first report regarding antioxidant-associated activities and relevant flavonoid components of HJH extracts, providing a promising candidate of traditional Chinese medicine for antioxidative treatment. & 2014 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Increasing evidences indicate that peroxidization induced bio-chemical changes are crucial etiological factors in many chronic human diseases, such as hyperlipemia and other cardiovascular diseases (Wazir et al., 2011). Hyperlipemia induces atherosclerotic lesion due to the accumulation of oxidized low-density lipoprotein (ox-LDL) in the artery wall (Navab et al., 1996). The peroxidation of n Corresponding author.Tel/fax: þ86 571 8663 3118. E-mail address: jiangjp77@ (J. Jiang). 1 The authors contributed equally to this work. http://dx.doi.org/10.1016/j.jep.2014.10.039 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.  LDL, triggered by oxygen free radicals (O2
, OH, H2O2), emerges as the initiating event in lesion formation, since highly atherogenic and vascular toxic potency of ox-LDL results in inflammatory responses and endothelial damage in the vessels (Halliwell, 2011; Apak et al., 2013). Lipid-peroxidation products of ox-LDL were found linked to such potency and inhibition of LDL oxidation should thereby limit its cytotoxicity in arterial walls (Hughes et al., 1994). Accordingly, antioxidants with potentials as radical scavengers, reducing agents, chelating agents or quenchers of singlet oxygen are of therapeutic value, which can protect human body from peroxidative stress damages (Hayat et al., 2010). However, many chemical antioxidants, e.g. BHT, possess harmful or toxic effects depending on the dosage used (Finley et al., 2011). It is urgently 326 J. Jiang et al. / Journal of Ethnopharmacology 158 (2014) 325–330 demanded to seek for a strong antioxidants with little side effects. Recently, increased interest is attracted by naturally derived anti-oxidant biocompounds (Khizhan et al., 2011). Flavonoids, including flavones, flavonols, and flavanones are the most common and widely distributed group of plant polyphenols possessing significant antioxidant and antihyperlipidemic activities (Harbone, 1993). Citrus, one of the most important and popularly consumed fruits, is very abundant in flavonoids. Citrus grandis (L.) Osbeck (pomelo) is such a fruit native to Southeast Asia and China with many nutritional and health benefits. The dried peel of this fruit was processed and widely applied as a traditional Chinese medicine, named Hua Ju Hong (HJH) in Chinese. It was originally recorded for the medicinal use by “Tang Materia Medica” (Tang Ben Cao) in the Tang Dynasty of China (659 A.D.), one of the earliest pharmacopoeia of the world. The modern Pharmacopoeia of China described its traditional use as a potential lipid-regulating medicine with sweet, bitter and pungent flavors as well as phlegm eliminating and digestion promoting activities (China Pharmacopeia Committee, 2010). Flavonoids have been found as the major component in Citrus grandis and may contribute to the medicinal activities of HJH, since flavonoid concentrations are high in the peel of Citrus grandis (Xi et al., 2014). Naringin, narirutin, and neohesperidin are the predominant flavonoids formed in Citrus grandis (Kawaii et al., 1999). Previous studies have primarily focused on the quantification of flavonoid compounds and the antioxidant capacity of Citrus grandis (Mokbel and Hashinaga, 2006; Tripoli et al., 2007). However, neither flavonoid composition nor antioxidant or antihyperlipidemic activity of HJH (processed peel of Citrus grandis) has yet been reported. To fill the gap, in this study, five purified flavonoid extracts of HJH (TFCA, TFCB, TFCC, TFCD and TFCE) were obtained, profiled, and evaluated for their antioxidant activity as well as antihyperlipi-demic activity by in vitro and in vivo experiments. 2. Materials and methods 2.1. Fruit material and reagents Fruits of Citrus grandis were collected from Zhejiang province, China, and identified by Prof. Xiong Yaokang at College of Medicine, Zhejiang Chinese Medical University (Voucher: Jiang J.P., 101011, ZM). Peels of the fruits were removed, cleaned, and chopped into pieces using a stainless steel knife. After sun drying, the pieces were stored for use, named HJH. The standardized prepared material of HJH was provided by Zhejiang Chinese Medical University Medical Pieces., LTD. All reagents used were of analytical grade. Synthetic antiox-idant butylated hydroxytoluene (BHT), Folin-Ciocalteureagent,2,20-diphenyl-1-picrylhydrazyl (DPPH), and trichloroacetic acid (TCA) were purchased from Sigma Chemicals Co. (St.Louis, MO, USA). Other chemicals were purchased from China National Medicine Group Shanghai Corporation (Shanghai, China). HPD-300 macro-porous resins were purchased from Hebei Cangzhou Baoen Chemical Co., Ltd. (Cangzhou, China). 2.2. Isolation and purification of HJH flavonoid extracts 100 g of HJH was soaked and extracted with 1600 ml of 0.10% Ca (OH)2 solution at 100 1C for 1.5 h. After filtration by Whatman No.1 filters (W&R Balston Ltd, London, UK), the extract was evaporated to dryness and dissolved in ten-fold volume of distilled water. The extract solution was then subjected to HPD-300 macroporous resins column (weight ratio: 1:20) for gradient elution with 2-fold bed volume of resin ethanol (0%, 30%, 50%, 70%, and 90%) at 1 ml/min. Each elution fraction was collected and concentrated to dryness, named TFCA, TFCB, TFCC, TFCD, and TFCE, respectively, as flavonoid extracts.  2.3. Determination of total flavonoids The content of total flavonoids in TFCA, TFCB, TFCC, TFCD, and TFCE were determined as previously described with minor mod-ifications (Ozsoy et al., 2008). Briefly, each flavonoid extract was dissolved in methanol (1:1, w/v) and sequentially added with 0.5 ml of 10% Al(NO3)3, 0.5 ml of 5% NaNO2, and 5 ml of 1 M NaOH at 0, 5, and 10 min. The absorbance at 510 nm for each extract was measured using a spectrophotometer (Shimadzu Corporation, Kyoto, Japan). The total flavonoids content in each extract was calculated using a standard curve prepared with rutin, and expressed in terms of mg of rutin equivalents per g solid extract of HJH. 2.4. Chemical analysis of flavonoid components A high performance liquid chromatography (HPLC) system (SCL-10Avp, Shimadzu, Kyoto, Japan) equipped with four pumps (LC-10Atvp), a UV–vis detector (SPD-10Avp), and an auto sampler (SIL-10Advp) were applied to determine the contents of three flavonoid compounds, naringin, narirutin and neohesperidin. Each 10 ml of the filtered samples was separated on a hypersil C18 column (250  4.6 mm2 i.d., Thermo Fisher Scientific, Waltham, MA, USA) at 35 1C, with a controlled flow rate of 1 ml/min and set wavelengths of 283 nm. The mobile phase was composed of (A) methanol and (B) 0.5% acetic acid (38:62, v/v). Identification of the three compounds was performed based on the retention times of the sample peaks compared to those of the authentic reference standards. The amount of each compound in the flavonoid extracts was estimated by the use of external standard calibration. 2.5. Animals Male SD rats (SPF II) weighing 180–220 g were purchased from the SLAC-CAS, Shanghai, China (Certificate no. SCXK2008-0016) and treated in strict accordance with the China legislation on the use and care of laboratory animals. 2.6. In vitro antioxidant test 2.6.1. DPPH free radical scavenging activity Radical scavenging activity of TFCA, TFCB, TFCC, TFCD and TFCE was determined using DPPH as a free radical with some modifica-tions (Slinkard and Singleton, 1977; Burits and Bucar, 2000). Briefly, 0.004% of DPPH radical solution in methanol was prepared and each 4 ml of the DPPH solution was mixed with 1 ml sample solution in methanol at different concentrations (0.24–1.2 mg/ml). The mixture was incubated for 30 min in the dark at room temperature. The radical scavenging activity was spectrophotome-trically evaluated by monitoring the absorbance at 517 nm and the scavenging capability against DPPH radical was calculated as follows: Scavenging activityð %Þ ¼ ð Ac–AsÞ=Ac  100% where Ac and As were absorbances at 517 nm of the control and sample, respectively. All tests were performed in triplicate, and the data are the means and standard deviations of three replicates. 2.6.2. Reducing power The reducing power of TFCA, TFCB, TFCC, TFCD and TFCE were determined according to the method of Oyaizu (1986). The sample solutions at different concentrations (0.24–1.2 mg/ml) were mixed with 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5 ml of potassium ferricyanide (1%), and then incubated at 50 1C for 20 min. Afterwards, the mixture was added with 2.5 ml of TCA J. Jiang et al. / Journal of Ethnopharmacology 158 (2014) 325–330 327 (10%) and centrifuged for 10 min at 1000 g (MSE Mistral 2000, London, UK). Approx. 2.5 ml of the supernatant was collected and mixed with distilled water (2.5 mL) and 0.5 M of ferric chloride (0.1%). The absorbance of the reaction mixture was spectrophoto-metrically read at 700 nm, which positively correlated to the reducing power level. All tests were performed in triplicate. 2.6.3. Total antioxidant activity The total antioxidant activity of TFCA, TFCB, TFCC, TFCD and TFCE was evaluated using the method described by Pan et al. (2010). Briefly, an aliquot of 1 ml of the sample solution at different concentrations (0.24–1.2 mg/ml) was mixed with 3 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) in vials. The vials were capped and incubated in a water bath at 95 1C for 30 min, followed by cooling to room temperature. The absorbance of each mixture, read at 695 nm, was associated positively with the total antiox-idant capacity. BHT was used as positive control for comparison. All tests were performed in triplicate. 2.7. In vivo antioxidant test The sixty rats were randomly divided into six groups (n¼10). Except the normal group, all others (model group, positive control group, TFCX high dose group, TFCX middle dose group, and TFCX low dose group) were fed with high fat diet (added with 10% yolk powder, 0.5% cholate, 5% lard oil, and 2% cholesterol) to establish the hyperlipemia model. The TFCX groups were daily given the strongest antioxidative extract among TFCA, TFCB, TFCC, TFCD and TFCE by oral administration. The positive control group was daily given vitamin C at 20 mg/kg. After 4 weeks treatment, the rat serums were collected and analyzed as follows: SOD (superoxide dismutase) and MDA (methane dicarboxylic aldehyde) levels were tested using ELISA kits, and TC (total cholesterol), TG (triglyceride), HDL-C (high-density lipoprotein) and LDL-C (low-density lipoprotein) levels were assessed  by the Vitalab Selectra E Chemistry Analyzer (Selectra E, Vital Scientific N.V., Dieren, the Netherlands). 2.8. Statistical analysis Data were expressed as mean7SD and subjected to one-way ANOVA, followed by Fisher's least significant difference (LSD) multiple comparisons. All analyses were performed using an updated version of DPS software (Tang and Feng, 2007). 3. Results 3.1. Contents of total flavonoids and flavonoid components in HJH extracts The total flavonoid content, determined as rutin equivalent, was varying among each HJH extract and decreased in an order of TFCB4TFCD4TFCC4TFCE4TFCA (Fig. 1). TFCB, TFCD, and TFCC contained more than 50% of total flavonoids, the highest content of which was found in TFCB (80.7%). By using HPLC, three components, narirutin, naringin and neohesperidin, were quantitatively identi-fied as the representatives of the total flavonoids in each extract. The content of each flavonoid component varied among different HJH extracts, displaying a similar trend as that of total flavonoids (TFCB4TFCD