Journal of Ethnopharmacology 169 (2015) 239–243
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Effects of Cydonia oblonga Miller leaf and fruit flavonoids on blood lipids and anti-oxydant potential in hyperlipidemia rats Anwar Umar a,b,n,1, Guldiyar Iskandar a,1, Ainiwaer Aikemu c, Wuliya Yiming a, Wenting Zhou a, Bénedicte Berké b, Bernard Begaud a,b, Nicholas Moore a,b,c,nn a
Department of Pharmacology, Xinjiang Medical University, 830011 Urumqi, PR China Department of Pharmacology – Université Bordeaux Segalen, F-33076 Bordeaux, France c Department of Drug Analysis, Xinjiang Medical University, 830011 Urumqi, PR China b
art ic l e i nf o
a b s t r a c t
Article history: Received 23 July 2014 Received in revised form 6 April 2015 Accepted 19 April 2015 Available online 28 April 2015
Objective: To study the effects of Cydonia oblonga Miller (COM) total flavonoids (TF) from leaves and fruit on the blood lipid and antioxidant potentials using hyperlipidaemic rat models. Methods: Hyperlipidaemic rat models were created with high-lipid emulsion. Rats were distributed into normal controls, hyperlipidaemic models, and daily high (160 mg/kg), medium (80 mg/kg) and low (40 mg/kg) TF from leaves and fruit and simvastatin (5 mg/kg) groups. After four weeks, serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL), alanine aminotransferase (ALT), aspartate aminotransferase (AST), as well as hepatic superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) were measured. Results: Compared with the hyperlipidaemic model group, TF significantly reduced serum TC, TG, LDL-C (P o0.01), ALT and AST (Po 0.01 or Po 0.05) and increased HDL-C (P o0.05 or P o0.01). TF also reduced MDA (Po 0.01 or P o0.01). Conclusion: COM total flavonoids can effectively regulate the metabolism of lipids, and remove oxygen free radicals. This confirms its potential value in the prevention and treatment of hyperlipidaemia. & 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cydonia oblonga miller Hyperlipidemic rats Blood lipids Oxygen free radicals Antioxidant effect
1. Introduction In traditional Uyghur medicine, the fruit and leaves of Quince (Cydonia oblonga Mill., COM) are used to treat or prevent hypertension and other cardiovascular diseases (Sadik, 1993; Xinjiang Uygur Autonomous Region Health Department (1991)), in addition to other uses (Aibaidula et al., 2009; Aslan et al., 2010; Yan et al., 2009). Quinces are also used in traditional Turkish medicine, which shares common cultural roots (Kultur, 2007). Quince fruit and leaves contain many components with known antioxidant activity (Silva et al., 2002a, 2002b, 2004a, 2005a, 2005b; Wojdylo et al., 2013; Xakir and Aikebaier, 2006). The South-western part of
n Corresponding author at: Department of Pharmacology, Xinjiang Medical University, 830011 Urumqi, PR China. Tel/fax: þ 86 991 43 62 421. nn Corresponding author at: Nicholas Moore, Department of pharmacology, University of Bordeaux Segalen, 33076, Bordeaux, France. Tel.: þ33 557571560; fax: þ33 557574671. E-mail addresses: [email protected] (A. Umar), [email protected] (N. Moore). 1 These two authors contributed equally.
http://dx.doi.org/10.1016/j.jep.2015.04.038 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
Xinjiang, People's Republic of China (PRC), around Hotan and Kashgar, is renowned for its large number of centenarians. Uyghur men from the region of Hotan have lower blood pressure than other central Asians (Kawamura et al., 2000; Kawamura et al., 2003; Sadik, 1993; Wufuer et al., 2004). This may be one of the reasons associated with the longevity of Uyghurs, and so we started a systematic experimental study of the effects of COM and its extracts on the main elements of cardiovascular risk: dietinduced hyperlipidemia (Abliz et al., 2014), thrombosis, (Zhou et al., 2014a), and hypertension, where the effects of COM were suggestive of an effect on the renin-angiotensin system (Zhou et al., 2014c; Zhou et al., 2014). Like most fruit and herbals, ripe COM contains many components, of which flavonoids are an important part (Silva et al., 2002b, 2004a, 2004c). Flavonoids from other fruit, including closely related apples, decrease plasma lipids in hypercholesterolaemic rats. (Aprikian et al., 2003; Jaroslawska et al., 2011; Leontowicz et al., 2003; Wang et al., 2011) We therefore tested Quince fruit and leaves for flavonoids and related effects. In the present paper we report on the effects of COM flavonoid extracts on experimental hyperlipidaemia.
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2. Materials and methods 2.1. Preparation of total flavonoids of the COM fruit and leaf The fruit and leaves of C. oblonga Mill. (Quince, COM) were collected in Kargilik, Xinjiang, and placed in a dry and wellventilated room. They were confirmed by Professor Parida Abliz from the department of bio-pharmacy of Xinjiang Medical University and deposited in the herbarium of the Traditional Chinese Medicine Ethnical Herbs Specimen Museum of Xinjiang Medical University under number NO.TCMEHSM2013_100. After being crushed into powder, fruit and leaves were extracted separately three times (one hour each time) with 65% alcohol (20 times the powder volume) under 60 1C, ultrasound. The filtered extracts were merged. The ethanol was recycled with a rotary evaporator to collect the COM extract. The extract was further enriched and purified to produce COM total flavonoid4 60%, measured by UV spectrophotometry using rutin as reference. (da Silva et al., 2015) Final concentration was 65.36% for leaf extracts and 61.23% for fruit extracts. 2.2. Experimental animals Sprague Dawley SPF rats, male, weighing 240720 g, were provided by animal centre of Xinjiang Medical University, certificate number: SCXK 2003-0001. All animal experiments were approved by the Institutional Ethics Committee (no. IACUC – 20130129016) 2.3. Reagents Cholesterol (Amresco, serial number 0391C426), hyocholic salt (Beijing Ao-box biotechnology Co. Ltd. serial number 20120408), Tween-80 (Tianjin Fuchen Chemical Reagents Factory, serial number 20120111), propylene glycol (Tianjin Fuchen Chemical Reagents Factory, serial number 20120604), propacil (Shanghai yuanye Bio-technology Co., Ltd, serial number AA0427LG13), total cholesterol (serial number 20130303), triglyceride (TG, serial number 20130302), LDL-C (serial number 20130312) (ref) and HDL-C kit (serial number 20130303), ALT (serial number 201306) and AST kit (EF, serial number 201305) were all products of Shenzhen Mindray biomedical electronic Co.,Ltd. Elisa kit were used for SOD, MDA, SGH-Px, serial number: 201306, same provider. Simvastatin was purchased from MSD pharmaceutical company, Hangzhou, serial number 121098, state medical permit no. J10980272. 2.4. Equipment BS-320 automatic biochemistry analyser (Shenzhen Mindray biomedical electronic Co., Ltd.), Coda-KM-00025-00 microplate reader, TDL-5 A centrifuge (Shanghai fulgor analytic equipment Co.,Ltd.)
3. Methods High fat emulsion was prepared according to published methods (Aslan et al., 2010; Ghule et al., 2009; Zhao et al., 2012): in brief, 20 g of lard and 10 g of cholesterol were added into a 200 ml beaker, then heated and molten (Abliz et al., 2014; Munshi et al., 2014; Zanwar et al., 2014). One gram propacil was mixed and thoroughly stirred to use as oil phase; in another 200 ml beaker, 30 ml of distilled water and 2 g of hyocholic salt were added and fully dissolved. Then 10 ml of propylene glycol and 10 ml of Tween80 were blended as water phase; the water phase was slowly
poured into the oil phase and evenly mixed before adding distilled water to 100 ml to produce the emulsion. The emulsion was preserved at 4 1C in a refrigerator. It was thawed in a water bath before use (Aslan et al., 2010; Ghule et al., 2009; Zhao et al., 2012). 3.1. Animals groups and models. Healthy adult male Sprague Dawley rats (weighing 240720 g) were randomly divided into 9 groups of 10 rats: normal control group, hyperlipidaemia model group, high (160 mg/kg), medium (80 mg/kg) and low dosage (40 mg/kg) of total leaf COM flavonoids, high (160 mg/kg), medium (80 mg/kg) and low dosage (40 mg/kg) of total fruit COM flavonoids, and Simvastatin (5 mg/ kg). In a first stage, each group except the control group was given intragastric high fat emulsion in the morning, 10 mL/kg. The control group was given the same amount of 0.9% saline, for 21 days. After the last administration, all groups were fasted for twelve hours, after which time blood was taken from vena caudalis to measure serum lipids. There was a significant difference in serum TC and TG between the control group and the model group, indicating successful establishment of hyperlipidemia. In the second stage, each group, except the control group, was given high fat emulsion in the morning (intragastric administration of 10 mL/kg). The control group was given the same amount of 0.9% saline. Every afternoon, the low, medium and high dosage groups were given 40 mg/kg, 80 mg/kg or 160 mg/kg of total flavonoids from COM fruit or 40 mg/kg, 80 mg/kg or 160 mg/kg of total flavonoids from COM leaves. The simvastatin group was given 5 mg/kg intragastric simvastatin solution. The second stage of administration lasted for 4 weeks, the rats were weighed on a weekly basis for modification of administration. 3.2. Measurement of indices General status: during the experiment, the rats were weighed on a weekly basis, and their appetite, behaviour, faeces, hair and mortality were closely observed. After 4 weeks of intragastric administration and 12 h of fasting, the rats were weighed. Following anaesthesia with pentobarbital 1% i.p., blood was taken from the rat's abdominal aorta and placed into centrifuge tubes free of anticoagulant for 10 min at 4 1C before a 15-minute centrifuge at 3000 r/min. Serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL), lowdensity lipoprotein cholesterol (LDL), alanine aminotransferase (ALT), aspartate aminotransferase (AST) were determined by an automatic biochemistry analyser. The abdominal cavity was opened and 0.5 g of tissue was removed from the right hepatic lobe. The tissue was placed in a pre-cooled PBS buffer and made into 10% homogenate at 4 1C, which was then centrifuged for 10 min at 3000 r/min. Afterwards, the clear supernatant liquid was isolated and optical density was determined with microplate reader after adjustment of wave length and shade selection. The process was handled strictly according to the instruction in the kit. Hepatic superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) were measured using an automatic biochemistry analyser.
4. Data processing and statistical analysis SPSS 16.0 was used for statistical analysis, all data of the experiment were expressed as mean 7sd. One-way ANOVA using the Least Squares method were used for comparison between groups. The bilateral P values from the comparisons were considered statistically significant when P o0.05 or P o0.01.
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and fruit had substantially lower serum AST (P o0.05). All dosage groups of total flavonoids had lower ALT, and the effects of high dosage group of leaves and medium and high dosage group of fruit were especially noticeable. (P o0.01)
5. Results 5.1. General status There was no noticeable difference in weight or habitus between treatment groups. The model group exhibited signs of diarrhoea and appetite loss. 5.2. Changes of lipid contents on the 20th day 6 rats from the control group and 2 rats from each of the treatment groups were randomly picked after 20 days and the changes of TC and TG were determined. Compared with the control group, levels of TC and TG rose substantially (P o0.01). The result indicated that the establishment of a rat models was successful (see Table 1). 5.3. Serum TC, TG, HDL-C and LDL-C concentrations Compared with the control group, the serum TC, TG and LDL-C levels of model rats increased considerably (P o0.01), while level of HDL-C significantly lowered (P o0.01); after treatment, the high dosage group of COM leaves and all three dosage groups of COM fruit had significantly lower levels of TC; all dosages groups of COM leaves and high dosage group of the fruit had considerably lower levels of TG; levels of LDL-C were reduced by high dosage group of COM leaves and medium and high dosage group of COM fruit (P o0.01), while levels of HDL-C were increased in all treatment groups. (P o0.05 or P o0.01) (see Table 2). 5.4. Effects on rat serum ALT and AST As shown in Table 3, ALT and AST were significantly higher in the model group than in the control group. Compared to the model group, high total flavonoids dosage groups of COM leaves Table 1 Serum lipids in rats during model establishment. Group
n
TC (mmol/L)
TG (mmol/L)
Control group Model group
6 6
1.157 0.46 5.157 0.78n
0.34 70.19 0.49 70.05n
TC: total cholesterol TG: triglycerides n
241
Po 0.01 compared with the control group.
5.5. Effects on the metabolism of oxygen free radicals compared to the control group, serum SOD and GSH-Px in the hyperlipidemic model group were significantly lower (P o0.01), while MDA was considerably higher (P o0.01); Compared with the hyperlipidemic model group, serum SOD and GSH-Px in medium and high dose total flavonoids rose significantly (P o0.05 or Po 0.01), while levels of MDA in all dosage groups dropped considerably. (P o0.01) (see Table 4). 6. Discussion Hyperlipidemia is an important cause of cerebral and cardiovascular disease (Fert-Bober and Sokolove, 2014; Gao et al., 2014; Kim et al., 2014). Early prevention and treatment of hyperlipidemia is important to reduce the occurrence of cerebral and cardiovascular diseases (Montori et al., 2014; Sniderman et al., 2014). Hyperlipidemia is also involved in non-alcoholic steatohepatitis (NASH), coronary heart disease, hypertension, diabetes, etc., all common life-restricting diseases. Research has indicated that every 10% drop in the serum cholesterol can lower the mortality rate of coronary heart disease patients by 13%, and total case fatality rate by 10% (Dhoble et al., 2014; Iversen et al., 2009; Lee et al., 2014; Qiu et al., 2011). Regular use of a lipid-lowering agent can have health benefits at the population level. C. oblonga is commonly used in Xinjiang, both as a cooking ingredient, and as a traditional medicine. In the latter situation, it is used to prevent and treat cardiovascular disease. We have already shown that COM can lower blood pressure, presumably through an interaction with the renin-angiotensin system (Zhou et al., 2014c; Zhou et al., 2014), and that it interacts with blood platelets and thrombotic events. (Zhou et al., 2014a) The third aspect of the exploration of a traditional medicine used for cardiovascular prevention is evidently the effect on lipids. We have shown that crude COM leaf extracts reduced lipid concentrations in a rat model of hyperlipidemia (Abliz et al., 2014). In the present paper we wished to study the effects of the flavonoids from the leaves and fruit, on the same rat model. The experimental hyperlipidaemic was induced by daily adminsitration of high fat emulsion, using chleolate into the emulsion to
Table 2 Total Cholesterol (TC), HDL-C and LDL-C after 4 weeks (Mean 7 sd, n ¼10). Groups
Dose (mg/kg)
TC (mmol/L)
TG (mmol/L)
LDL-C (mmol/L)
HDL-C (mmol/L)
Control Hyperlipidaemia Simvastatin LCTF-L MCTF-L HCTF-L LCTF-F MCTF-F HCTF-F
– – 5 40 80 160 40 80 160
1.29 7 0.15 6.127 1.96 3.917 0.54nn 5.50 7 1.47 5.767 1.53 3.59 7 1.58nn 4.747 0.94nn 2.767 0.44nn 2.577 0.38nn
0.39 70.04 0.52 70.07 0.22 70.07nn 0.23 70.08nn 0.21 70.03nn 0.18 70.03 0.47 70.07 0.45 70.09 0.39 70.11nn
0.48 70.10 6.25 72.93 5.59 71.88 6.26 72.08 5.68 72.04 2.26 70.67nn 5.15 71.58 2.41 70.57nn 2.42 70.55nn
4.29 7 0.54 2.40 7 0.22 4.417 0.84nn 4.377 0.79nn 4.617 0.56nn 4.157 0.49nn 3.107 0.28n 3.39 7 0.57nn 3.247 0.59nn
LCTF: low dosage of Cydonia oblonga Mill. total flavonoids. MCTF: medium dosage of Cydonia oblonga Mill. total flavonoids. HCTF: high dosage of Cydonia oblonga Mill. total flavonoids. L: leaves. F: fruit. n
Po 0.05 compared with the hyperlipidaemia model group. Po 0.01 compared with the hyperlipidaemia model group.
nn
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Table 3 Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and AST/ ALT after 4 weeks (mean7 sd, n¼ 10). Group
Control Hyperlipidaemia Simvastatin LCTF-L MCTF-L HCTF-L LCTF-F MCTF-F HCTF-F
Dose (mg/kg)
AST (U/L)
– – 5 40 80 160 40 80 160
88.03 7 6.42 111.26 7 9.12 108.55 7 8.40 114.88 7 8.43 112.687 9.46 99.28 7 12.56n 117.08 7 11.47 114.28 7 9.33 99.767 4.74n
ALT (U/L) 34.56 73.26 55.3375.22 51.43 79.11 47.36 74.18n 47.56 76.05n 43.18 77.84nn 38.56 75.79nn 36.73 74.21nn 36.73 74.31nn
ALT/AST
0.38 7 0.01 0.50 7 0.07 0.47 7 0.08 0.41 7 0.04n 0.39 7 0.06n 0.44 7 0.08n 0.32 7 0.05nn 0.34 7 0.05nn 0.36 7 0.04nn
LCTF: low dosage of Cydonia oblonga Mill. total flavonoids MCTF: medium dosage of Cydonia oblonga Mill. total flavonoids HCTF: high dosage of Cydonia oblonga Mill. total flavonoids L: leaves F: fruit
Superoxide dismutase (SOD) and GSH-Px are two essential antioxidants, which can remove the free oxygen radicals produced by metabolism in physiological states and protect cells. The activity of SOD and GSH-Px indirectly reflects the abilities to eliminate free oxygen radicals. MDA is a product of the oxidation of unsaturated fatty acid induced by free radicals, the contents of which could indirectly reflect the extent of oxidation of body fat (Liu and Ng, 2000). In this study as expected COM total flavonoids improved the activity of SOD and GSH-Px in hepatic tissues, inhibited the generation of MDA and improved the antioxygenic potentials of the hepatic tissue, which is consistent with hepatic protection. In conclusion, Cydonia total flavonoids (fruit and leaves) could regulate the blood lipid metabolism in rats while removing the oxygen free radicals and improving the antioxygenic potentials.
Acknowledgement
n
Po 0.05 compared with the hyperlipidaemia model group. nn Po 0.01 compared with the hyperlipidaemia model group.
This research work was supported by the National Natural Science Foundation of China (No. 81260490).
Table 4 Markers of oxidative stress after 4 weeks (mean 7 sd, n¼ 10). Group
Control Hyperlipidaemia Simvastatin LCTF-L MCTF-L HCTF-L LCTF-F MCTF-F HCTF-F
References
Dose (mg/kg)
SOD (U/L)
MDA (nmol/L)
GSH-PX (pmol/L)
– – 5 40 80 160 40 80 160
228.0 7 17.1 199.0 7 8.99 213.9 7 13.5n 204.27 10.8 212.5 7 15.0n 223.4 7 7.72nn 201.0 7 14.9 208.8 7 10.7n 222.5 7 7.39nn
4.32 7 0.38 6.85 7 0.98 4.46 7 0.35nn 4.42 7 0.38nn 3.95 7 0.45nn 3.487 0.69nn 4.82 7 0.56nn 3.32 7 0.54nn 3.56 7 0.56nn
41.7 7 3.53 35.17 1.28 38.2 7 3.49n 37.0 7 2.39 38.9 7 1.43nn 42.4 7 3.13nn 36.9 7 3.25 38.17 1.99n 39.7 7 2.97nn
SOD: superoxide dismutase, MDA: malondialdehyde, GSH-Px: glutathion peroxidase. LCTF: low dosage of Cydonia oblonga Mill. total flavonoids. MCTF: medium dosage of Cydonia oblonga Mill. total flavonoids. HCTF: high dosage of Cydonia oblonga Mill. total flavonoids. L: leaves. F: fruit. n
Po 0.05 compared with the model group. Po 0.01 compared with the model group.
nn
increase absorption of cholesterol, and an antithyroid drug (propylthiouracil). As a result of the intervention, serum TC, TG and LDLC were significantly higher in the model group than in control group (Po0.05), and HDL-C noticeably lower (Po0.05), confirming the establishment of the model. After administration of COM flavonoids, serum TC, TG and LDL-C dropped to different extents, while HDL-C went up, indicating that total flavonoids of COM fruit and leaves could regulate the lipid in hyperlipidemic rats. When there is damage, necrosis or degeneration of hepatic cells, ALT is released into the bloodstream, which causes an increase in serum ALT. Serum ALT is one of the most sensitive indexes in examining hepatic injury. AST Concentrations are highest in cardiac muscles, followed by liver. When the liver is compromised, AST concentrations in serum increase significantly (Hu et al., 2011). Concentrations of serum ALT and AST rose significantly, indicating liver damage in hyperlipaemic rats. Comparing with the model groups, medium and high dosage of total flavonoids in COM leaves and high dosage of total flavonoids in fruit lowered AST, and all dosage groups ALT, especially for high doses of leavf flavonoids and medium and high doses of fruit flavonoids, indicating reduced lipid induced liver injury. The effect, especially on ALT, was noticeably greater than that of simvastatin.
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Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Effects of Cydonia oblonga Miller leaf and fruit flavonoids on blood lipids and anti-oxydant potential in hyperlipidemia rats Anwar Umar a,b,n,1, Guldiyar Iskandar a,1, Ainiwaer Aikemu c, Wuliya Yiming a, Wenting Zhou a, Bénedicte Berké b, Bernard Begaud a,b, Nicholas Moore a,b,c,nn a
Department of Pharmacology, Xinjiang Medical University, 830011 Urumqi, PR China Department of Pharmacology – Université Bordeaux Segalen, F-33076 Bordeaux, France c Department of Drug Analysis, Xinjiang Medical University, 830011 Urumqi, PR China b
art ic l e i nf o
a b s t r a c t
Article history: Received 23 July 2014 Received in revised form 6 April 2015 Accepted 19 April 2015 Available online 28 April 2015
Objective: To study the effects of Cydonia oblonga Miller (COM) total flavonoids (TF) from leaves and fruit on the blood lipid and antioxidant potentials using hyperlipidaemic rat models. Methods: Hyperlipidaemic rat models were created with high-lipid emulsion. Rats were distributed into normal controls, hyperlipidaemic models, and daily high (160 mg/kg), medium (80 mg/kg) and low (40 mg/kg) TF from leaves and fruit and simvastatin (5 mg/kg) groups. After four weeks, serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL), alanine aminotransferase (ALT), aspartate aminotransferase (AST), as well as hepatic superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) were measured. Results: Compared with the hyperlipidaemic model group, TF significantly reduced serum TC, TG, LDL-C (P o0.01), ALT and AST (Po 0.01 or Po 0.05) and increased HDL-C (P o0.05 or P o0.01). TF also reduced MDA (Po 0.01 or P o0.01). Conclusion: COM total flavonoids can effectively regulate the metabolism of lipids, and remove oxygen free radicals. This confirms its potential value in the prevention and treatment of hyperlipidaemia. & 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cydonia oblonga miller Hyperlipidemic rats Blood lipids Oxygen free radicals Antioxidant effect
1. Introduction In traditional Uyghur medicine, the fruit and leaves of Quince (Cydonia oblonga Mill., COM) are used to treat or prevent hypertension and other cardiovascular diseases (Sadik, 1993; Xinjiang Uygur Autonomous Region Health Department (1991)), in addition to other uses (Aibaidula et al., 2009; Aslan et al., 2010; Yan et al., 2009). Quinces are also used in traditional Turkish medicine, which shares common cultural roots (Kultur, 2007). Quince fruit and leaves contain many components with known antioxidant activity (Silva et al., 2002a, 2002b, 2004a, 2005a, 2005b; Wojdylo et al., 2013; Xakir and Aikebaier, 2006). The South-western part of
n Corresponding author at: Department of Pharmacology, Xinjiang Medical University, 830011 Urumqi, PR China. Tel/fax: þ 86 991 43 62 421. nn Corresponding author at: Nicholas Moore, Department of pharmacology, University of Bordeaux Segalen, 33076, Bordeaux, France. Tel.: þ33 557571560; fax: þ33 557574671. E-mail addresses: [email protected] (A. Umar), [email protected] (N. Moore). 1 These two authors contributed equally.
http://dx.doi.org/10.1016/j.jep.2015.04.038 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
Xinjiang, People's Republic of China (PRC), around Hotan and Kashgar, is renowned for its large number of centenarians. Uyghur men from the region of Hotan have lower blood pressure than other central Asians (Kawamura et al., 2000; Kawamura et al., 2003; Sadik, 1993; Wufuer et al., 2004). This may be one of the reasons associated with the longevity of Uyghurs, and so we started a systematic experimental study of the effects of COM and its extracts on the main elements of cardiovascular risk: dietinduced hyperlipidemia (Abliz et al., 2014), thrombosis, (Zhou et al., 2014a), and hypertension, where the effects of COM were suggestive of an effect on the renin-angiotensin system (Zhou et al., 2014c; Zhou et al., 2014). Like most fruit and herbals, ripe COM contains many components, of which flavonoids are an important part (Silva et al., 2002b, 2004a, 2004c). Flavonoids from other fruit, including closely related apples, decrease plasma lipids in hypercholesterolaemic rats. (Aprikian et al., 2003; Jaroslawska et al., 2011; Leontowicz et al., 2003; Wang et al., 2011) We therefore tested Quince fruit and leaves for flavonoids and related effects. In the present paper we report on the effects of COM flavonoid extracts on experimental hyperlipidaemia.
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2. Materials and methods 2.1. Preparation of total flavonoids of the COM fruit and leaf The fruit and leaves of C. oblonga Mill. (Quince, COM) were collected in Kargilik, Xinjiang, and placed in a dry and wellventilated room. They were confirmed by Professor Parida Abliz from the department of bio-pharmacy of Xinjiang Medical University and deposited in the herbarium of the Traditional Chinese Medicine Ethnical Herbs Specimen Museum of Xinjiang Medical University under number NO.TCMEHSM2013_100. After being crushed into powder, fruit and leaves were extracted separately three times (one hour each time) with 65% alcohol (20 times the powder volume) under 60 1C, ultrasound. The filtered extracts were merged. The ethanol was recycled with a rotary evaporator to collect the COM extract. The extract was further enriched and purified to produce COM total flavonoid4 60%, measured by UV spectrophotometry using rutin as reference. (da Silva et al., 2015) Final concentration was 65.36% for leaf extracts and 61.23% for fruit extracts. 2.2. Experimental animals Sprague Dawley SPF rats, male, weighing 240720 g, were provided by animal centre of Xinjiang Medical University, certificate number: SCXK 2003-0001. All animal experiments were approved by the Institutional Ethics Committee (no. IACUC – 20130129016) 2.3. Reagents Cholesterol (Amresco, serial number 0391C426), hyocholic salt (Beijing Ao-box biotechnology Co. Ltd. serial number 20120408), Tween-80 (Tianjin Fuchen Chemical Reagents Factory, serial number 20120111), propylene glycol (Tianjin Fuchen Chemical Reagents Factory, serial number 20120604), propacil (Shanghai yuanye Bio-technology Co., Ltd, serial number AA0427LG13), total cholesterol (serial number 20130303), triglyceride (TG, serial number 20130302), LDL-C (serial number 20130312) (ref) and HDL-C kit (serial number 20130303), ALT (serial number 201306) and AST kit (EF, serial number 201305) were all products of Shenzhen Mindray biomedical electronic Co.,Ltd. Elisa kit were used for SOD, MDA, SGH-Px, serial number: 201306, same provider. Simvastatin was purchased from MSD pharmaceutical company, Hangzhou, serial number 121098, state medical permit no. J10980272. 2.4. Equipment BS-320 automatic biochemistry analyser (Shenzhen Mindray biomedical electronic Co., Ltd.), Coda-KM-00025-00 microplate reader, TDL-5 A centrifuge (Shanghai fulgor analytic equipment Co.,Ltd.)
3. Methods High fat emulsion was prepared according to published methods (Aslan et al., 2010; Ghule et al., 2009; Zhao et al., 2012): in brief, 20 g of lard and 10 g of cholesterol were added into a 200 ml beaker, then heated and molten (Abliz et al., 2014; Munshi et al., 2014; Zanwar et al., 2014). One gram propacil was mixed and thoroughly stirred to use as oil phase; in another 200 ml beaker, 30 ml of distilled water and 2 g of hyocholic salt were added and fully dissolved. Then 10 ml of propylene glycol and 10 ml of Tween80 were blended as water phase; the water phase was slowly
poured into the oil phase and evenly mixed before adding distilled water to 100 ml to produce the emulsion. The emulsion was preserved at 4 1C in a refrigerator. It was thawed in a water bath before use (Aslan et al., 2010; Ghule et al., 2009; Zhao et al., 2012). 3.1. Animals groups and models. Healthy adult male Sprague Dawley rats (weighing 240720 g) were randomly divided into 9 groups of 10 rats: normal control group, hyperlipidaemia model group, high (160 mg/kg), medium (80 mg/kg) and low dosage (40 mg/kg) of total leaf COM flavonoids, high (160 mg/kg), medium (80 mg/kg) and low dosage (40 mg/kg) of total fruit COM flavonoids, and Simvastatin (5 mg/ kg). In a first stage, each group except the control group was given intragastric high fat emulsion in the morning, 10 mL/kg. The control group was given the same amount of 0.9% saline, for 21 days. After the last administration, all groups were fasted for twelve hours, after which time blood was taken from vena caudalis to measure serum lipids. There was a significant difference in serum TC and TG between the control group and the model group, indicating successful establishment of hyperlipidemia. In the second stage, each group, except the control group, was given high fat emulsion in the morning (intragastric administration of 10 mL/kg). The control group was given the same amount of 0.9% saline. Every afternoon, the low, medium and high dosage groups were given 40 mg/kg, 80 mg/kg or 160 mg/kg of total flavonoids from COM fruit or 40 mg/kg, 80 mg/kg or 160 mg/kg of total flavonoids from COM leaves. The simvastatin group was given 5 mg/kg intragastric simvastatin solution. The second stage of administration lasted for 4 weeks, the rats were weighed on a weekly basis for modification of administration. 3.2. Measurement of indices General status: during the experiment, the rats were weighed on a weekly basis, and their appetite, behaviour, faeces, hair and mortality were closely observed. After 4 weeks of intragastric administration and 12 h of fasting, the rats were weighed. Following anaesthesia with pentobarbital 1% i.p., blood was taken from the rat's abdominal aorta and placed into centrifuge tubes free of anticoagulant for 10 min at 4 1C before a 15-minute centrifuge at 3000 r/min. Serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL), lowdensity lipoprotein cholesterol (LDL), alanine aminotransferase (ALT), aspartate aminotransferase (AST) were determined by an automatic biochemistry analyser. The abdominal cavity was opened and 0.5 g of tissue was removed from the right hepatic lobe. The tissue was placed in a pre-cooled PBS buffer and made into 10% homogenate at 4 1C, which was then centrifuged for 10 min at 3000 r/min. Afterwards, the clear supernatant liquid was isolated and optical density was determined with microplate reader after adjustment of wave length and shade selection. The process was handled strictly according to the instruction in the kit. Hepatic superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) were measured using an automatic biochemistry analyser.
4. Data processing and statistical analysis SPSS 16.0 was used for statistical analysis, all data of the experiment were expressed as mean 7sd. One-way ANOVA using the Least Squares method were used for comparison between groups. The bilateral P values from the comparisons were considered statistically significant when P o0.05 or P o0.01.
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and fruit had substantially lower serum AST (P o0.05). All dosage groups of total flavonoids had lower ALT, and the effects of high dosage group of leaves and medium and high dosage group of fruit were especially noticeable. (P o0.01)
5. Results 5.1. General status There was no noticeable difference in weight or habitus between treatment groups. The model group exhibited signs of diarrhoea and appetite loss. 5.2. Changes of lipid contents on the 20th day 6 rats from the control group and 2 rats from each of the treatment groups were randomly picked after 20 days and the changes of TC and TG were determined. Compared with the control group, levels of TC and TG rose substantially (P o0.01). The result indicated that the establishment of a rat models was successful (see Table 1). 5.3. Serum TC, TG, HDL-C and LDL-C concentrations Compared with the control group, the serum TC, TG and LDL-C levels of model rats increased considerably (P o0.01), while level of HDL-C significantly lowered (P o0.01); after treatment, the high dosage group of COM leaves and all three dosage groups of COM fruit had significantly lower levels of TC; all dosages groups of COM leaves and high dosage group of the fruit had considerably lower levels of TG; levels of LDL-C were reduced by high dosage group of COM leaves and medium and high dosage group of COM fruit (P o0.01), while levels of HDL-C were increased in all treatment groups. (P o0.05 or P o0.01) (see Table 2). 5.4. Effects on rat serum ALT and AST As shown in Table 3, ALT and AST were significantly higher in the model group than in the control group. Compared to the model group, high total flavonoids dosage groups of COM leaves Table 1 Serum lipids in rats during model establishment. Group
n
TC (mmol/L)
TG (mmol/L)
Control group Model group
6 6
1.157 0.46 5.157 0.78n
0.34 70.19 0.49 70.05n
TC: total cholesterol TG: triglycerides n
241
Po 0.01 compared with the control group.
5.5. Effects on the metabolism of oxygen free radicals compared to the control group, serum SOD and GSH-Px in the hyperlipidemic model group were significantly lower (P o0.01), while MDA was considerably higher (P o0.01); Compared with the hyperlipidemic model group, serum SOD and GSH-Px in medium and high dose total flavonoids rose significantly (P o0.05 or Po 0.01), while levels of MDA in all dosage groups dropped considerably. (P o0.01) (see Table 4). 6. Discussion Hyperlipidemia is an important cause of cerebral and cardiovascular disease (Fert-Bober and Sokolove, 2014; Gao et al., 2014; Kim et al., 2014). Early prevention and treatment of hyperlipidemia is important to reduce the occurrence of cerebral and cardiovascular diseases (Montori et al., 2014; Sniderman et al., 2014). Hyperlipidemia is also involved in non-alcoholic steatohepatitis (NASH), coronary heart disease, hypertension, diabetes, etc., all common life-restricting diseases. Research has indicated that every 10% drop in the serum cholesterol can lower the mortality rate of coronary heart disease patients by 13%, and total case fatality rate by 10% (Dhoble et al., 2014; Iversen et al., 2009; Lee et al., 2014; Qiu et al., 2011). Regular use of a lipid-lowering agent can have health benefits at the population level. C. oblonga is commonly used in Xinjiang, both as a cooking ingredient, and as a traditional medicine. In the latter situation, it is used to prevent and treat cardiovascular disease. We have already shown that COM can lower blood pressure, presumably through an interaction with the renin-angiotensin system (Zhou et al., 2014c; Zhou et al., 2014), and that it interacts with blood platelets and thrombotic events. (Zhou et al., 2014a) The third aspect of the exploration of a traditional medicine used for cardiovascular prevention is evidently the effect on lipids. We have shown that crude COM leaf extracts reduced lipid concentrations in a rat model of hyperlipidemia (Abliz et al., 2014). In the present paper we wished to study the effects of the flavonoids from the leaves and fruit, on the same rat model. The experimental hyperlipidaemic was induced by daily adminsitration of high fat emulsion, using chleolate into the emulsion to
Table 2 Total Cholesterol (TC), HDL-C and LDL-C after 4 weeks (Mean 7 sd, n ¼10). Groups
Dose (mg/kg)
TC (mmol/L)
TG (mmol/L)
LDL-C (mmol/L)
HDL-C (mmol/L)
Control Hyperlipidaemia Simvastatin LCTF-L MCTF-L HCTF-L LCTF-F MCTF-F HCTF-F
– – 5 40 80 160 40 80 160
1.29 7 0.15 6.127 1.96 3.917 0.54nn 5.50 7 1.47 5.767 1.53 3.59 7 1.58nn 4.747 0.94nn 2.767 0.44nn 2.577 0.38nn
0.39 70.04 0.52 70.07 0.22 70.07nn 0.23 70.08nn 0.21 70.03nn 0.18 70.03 0.47 70.07 0.45 70.09 0.39 70.11nn
0.48 70.10 6.25 72.93 5.59 71.88 6.26 72.08 5.68 72.04 2.26 70.67nn 5.15 71.58 2.41 70.57nn 2.42 70.55nn
4.29 7 0.54 2.40 7 0.22 4.417 0.84nn 4.377 0.79nn 4.617 0.56nn 4.157 0.49nn 3.107 0.28n 3.39 7 0.57nn 3.247 0.59nn
LCTF: low dosage of Cydonia oblonga Mill. total flavonoids. MCTF: medium dosage of Cydonia oblonga Mill. total flavonoids. HCTF: high dosage of Cydonia oblonga Mill. total flavonoids. L: leaves. F: fruit. n
Po 0.05 compared with the hyperlipidaemia model group. Po 0.01 compared with the hyperlipidaemia model group.
nn
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Table 3 Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and AST/ ALT after 4 weeks (mean7 sd, n¼ 10). Group
Control Hyperlipidaemia Simvastatin LCTF-L MCTF-L HCTF-L LCTF-F MCTF-F HCTF-F
Dose (mg/kg)
AST (U/L)
– – 5 40 80 160 40 80 160
88.03 7 6.42 111.26 7 9.12 108.55 7 8.40 114.88 7 8.43 112.687 9.46 99.28 7 12.56n 117.08 7 11.47 114.28 7 9.33 99.767 4.74n
ALT (U/L) 34.56 73.26 55.3375.22 51.43 79.11 47.36 74.18n 47.56 76.05n 43.18 77.84nn 38.56 75.79nn 36.73 74.21nn 36.73 74.31nn
ALT/AST
0.38 7 0.01 0.50 7 0.07 0.47 7 0.08 0.41 7 0.04n 0.39 7 0.06n 0.44 7 0.08n 0.32 7 0.05nn 0.34 7 0.05nn 0.36 7 0.04nn
LCTF: low dosage of Cydonia oblonga Mill. total flavonoids MCTF: medium dosage of Cydonia oblonga Mill. total flavonoids HCTF: high dosage of Cydonia oblonga Mill. total flavonoids L: leaves F: fruit
Superoxide dismutase (SOD) and GSH-Px are two essential antioxidants, which can remove the free oxygen radicals produced by metabolism in physiological states and protect cells. The activity of SOD and GSH-Px indirectly reflects the abilities to eliminate free oxygen radicals. MDA is a product of the oxidation of unsaturated fatty acid induced by free radicals, the contents of which could indirectly reflect the extent of oxidation of body fat (Liu and Ng, 2000). In this study as expected COM total flavonoids improved the activity of SOD and GSH-Px in hepatic tissues, inhibited the generation of MDA and improved the antioxygenic potentials of the hepatic tissue, which is consistent with hepatic protection. In conclusion, Cydonia total flavonoids (fruit and leaves) could regulate the blood lipid metabolism in rats while removing the oxygen free radicals and improving the antioxygenic potentials.
Acknowledgement
n
Po 0.05 compared with the hyperlipidaemia model group. nn Po 0.01 compared with the hyperlipidaemia model group.
This research work was supported by the National Natural Science Foundation of China (No. 81260490).
Table 4 Markers of oxidative stress after 4 weeks (mean 7 sd, n¼ 10). Group
Control Hyperlipidaemia Simvastatin LCTF-L MCTF-L HCTF-L LCTF-F MCTF-F HCTF-F
References
Dose (mg/kg)
SOD (U/L)
MDA (nmol/L)
GSH-PX (pmol/L)
– – 5 40 80 160 40 80 160
228.0 7 17.1 199.0 7 8.99 213.9 7 13.5n 204.27 10.8 212.5 7 15.0n 223.4 7 7.72nn 201.0 7 14.9 208.8 7 10.7n 222.5 7 7.39nn
4.32 7 0.38 6.85 7 0.98 4.46 7 0.35nn 4.42 7 0.38nn 3.95 7 0.45nn 3.487 0.69nn 4.82 7 0.56nn 3.32 7 0.54nn 3.56 7 0.56nn
41.7 7 3.53 35.17 1.28 38.2 7 3.49n 37.0 7 2.39 38.9 7 1.43nn 42.4 7 3.13nn 36.9 7 3.25 38.17 1.99n 39.7 7 2.97nn
SOD: superoxide dismutase, MDA: malondialdehyde, GSH-Px: glutathion peroxidase. LCTF: low dosage of Cydonia oblonga Mill. total flavonoids. MCTF: medium dosage of Cydonia oblonga Mill. total flavonoids. HCTF: high dosage of Cydonia oblonga Mill. total flavonoids. L: leaves. F: fruit. n
Po 0.05 compared with the model group. Po 0.01 compared with the model group.
nn
increase absorption of cholesterol, and an antithyroid drug (propylthiouracil). As a result of the intervention, serum TC, TG and LDLC were significantly higher in the model group than in control group (Po0.05), and HDL-C noticeably lower (Po0.05), confirming the establishment of the model. After administration of COM flavonoids, serum TC, TG and LDL-C dropped to different extents, while HDL-C went up, indicating that total flavonoids of COM fruit and leaves could regulate the lipid in hyperlipidemic rats. When there is damage, necrosis or degeneration of hepatic cells, ALT is released into the bloodstream, which causes an increase in serum ALT. Serum ALT is one of the most sensitive indexes in examining hepatic injury. AST Concentrations are highest in cardiac muscles, followed by liver. When the liver is compromised, AST concentrations in serum increase significantly (Hu et al., 2011). Concentrations of serum ALT and AST rose significantly, indicating liver damage in hyperlipaemic rats. Comparing with the model groups, medium and high dosage of total flavonoids in COM leaves and high dosage of total flavonoids in fruit lowered AST, and all dosage groups ALT, especially for high doses of leavf flavonoids and medium and high doses of fruit flavonoids, indicating reduced lipid induced liver injury. The effect, especially on ALT, was noticeably greater than that of simvastatin.
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