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Effect of Cydonia oblonga Mill. leaf extract on serum lipids and liver function in a rat model of hyperlipidaemia
Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Research Paper
Effect of Cydonia oblonga Mill. leaf extract on serum lipids and liver function in a rat model of hyperlipidaemia Q1
Ablat Abliz a,1, Qimangul Aji a,1, Elzira Abdusalam a, Xiaowei Sun a, Adil Abdurahman a, Wenting Zhou a, Nicholas Moore a,b,nn, Anwar Umar a,b,n a b
Department of Pharmacology, Xinjiang Medical University, 393 Xinyi Road, 830011 Urumqi, PR China Department of Pharmacology, University Bordeaux Segalen, 146 rue Leo Saignat, 33076 Bordeaux, France
art ic l e i nf o Article history: Received 23 September 2013 Received in revised form 22 November 2013 Accepted 6 December 2013
Keywords: Cydonia oblonga Mill. leaf extract Hyperlipidaemia Antioxidant Hypercholesterolemia Hypolipidemic effect Traditional Uyghur medicine
a b s t r a c t Q4 Ethnopharmacological relevance: Cydonia oblonga Mill. leaves are traditionally used in Uyghur medicine to treat or prevent cardiovascular disease. Beyond a demonstrated effect on thrombosis, we tested it for an effect on dyslipidemia, in a rat model of hyperlipidemia. Methods: Seventy healthy Sprague Dawley rats were randomly divided into 6 groups: normal controls, model controls, simvastatin, and low-, medium- and high-dose Cydonia oblonga Mill. leaf extracts (COM), orally for 56 days. The normal controls were fed a normal diet, all other groups a high fat diet. Rat weights were recorded over time. Total cholesterol (TC), triglycerides (TG), low and high-density lipoproteins (LDL, HDL), as well as AST, ALT and total protein (TP) were measured in serum at the end of the study. The antioxidant capacity of glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), malondialdehyde (MDA) was measured in liver samples, along with lipoprotein lipase (LPL), and hepatic lipase (HL). Liver pathology was described. Results: COM dose-dependently reduced TC, TG, LDL-C and MDA, inhibited the activity of ALT, AST and LPS, increased HDL-C content, increased the activity of SOD, GSH-PX, LPL and HL, and reduced liver steatosis in hyperlipidaemia rats, which was significant at medium and high doses. The effect of COM was similar to that of simvastatin except for increased LPL and HL which were reduced by COM but not by simvastatin. Conclusion: Cydonia oblonga Mill. leaf extracts have hypolipidaemic and hepatoprotective effects, probably related to increasing antioxidant capacity and lipoprotein metabolism in the liver, and inhibition of lipogenesis. & 2013 Published by Elsevier Ireland Ltd.
1. Introduction The improvement in living standards worldwide, and the increasing use of poor quality food, at least from the nutritional point of view, results in increasing frequency of dyslipidaemia, a major risk factor for cardiovascular disease. Along with diabetes
Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; COM, Cydonia oblonga Mill.; GSH-PX, glutathione peroxidase; HDL-C, high density lipoprotein cholesterol; HL, hepatic lipase; LDL-C, low-density lipoprotein cholesterol; LPL, lipoprotein lipase; LPS, lipase; MDA, malondialdehyde; SOD, superoxide dismutase; TC, total cholesterol; TG, triglycerides; TP, total protein n Corresponding author at: Department of Pharmacology, Xinjiang Medical University, 393 Xinyi Road, 830011 Urumqi, PR China. Tel./fax: þ 86 9914362421. nn Corresponding author at: Department of Pharmacology, University Bordeaux Segalen, 146 rue Leo Saignat, 33076 Bordeaux, France. Tel.: þ33 557571560; fax: þ33 557574671. E-mail addresses: [email protected] (N. Moore), [email protected] (A. Umar). 1 Ablat Abliz and Qimangul Aji are equal contributors to this work.
and hypertension, it contributes to the increasing rate of cardiovascular diseases and ensuing morbi-mortality. The fight against such cardiovascular diseases passes through better eating habits and exercise, but also through corrective actions against hyperlipidaemia. Drugs are commonly used for that matter, and especially inhibitors of HMGCoA reductase (statins), but traditional medicines may also have beneficial effects (Chen et al., 2009; Ghule et al., 2009; Aslan et al., 2010; Kwak et al., 2010; Li et al., 2010; Liu et al., 2011; Tan et al., 2011; Lu et al., 2012). Such options merit being explored and perhaps diffused to other regions. Quince, Cydonia oblonga Mill. (COM) (Rosaceae), also called Kinashi, Soil papaya, Biye and so on, is a traditional Uyghur medicinal plant, commonly used in the Hotan and Kashgar regions of Xinjiang, in Western China, for the treatment or prevention of cardiovascular disease, among other. In traditional Uyghur medicine COM fruit, leaves, roots, branches and other parts are used as medicine (Sadik, 1993; Grundemann et al., 2011). Currently, there is much research being done on the chemical composition and antibacterial effects of COM, but less on the pharmacological
0378-8741/$ - see front matter & 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.jep.2013.12.010
Please cite this article as: Abliz, A., et al., Effect of Cydonia oblonga Mill. leaf extract on serum lipids and liver function in a rat model of hyperlipidaemia. Journal of Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.12.010i
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effects of COM, apart from a few papers on its effects on coagulation and blood pressure (Bayier et al., 2010a, 2010b; Grundemann et al., 2011; Abliz et al., 2012). These alone could justify its use in cardiovascular disease or prevention. An effect on lipid metabolism has also been suggested, and we therefore tested the impact of COM leaf extracts on a hyperlipidaemia model, studying serum lipids levels and liver enzyme activity. These could provide evidence for an effect that is complementary to that on coagulation and thrombosis, and provide a theoretical basis for the further development of COM.
2. Material and methods
and Cydonia oblonga Mill. leaf extract at low (80 mg/kg/d), medium (160 mg/kg/d), and high-dose (320 mg/kg/d).
2.5. Dosing regimen All rats had free food and water intake. Normal control group was fed with basic feed, the other groups were fed with high fat feed. Rats were given an intragastric administration every morning at 9:00–10:00. Normal control and hyperlipidaemia model groups were given normal saline, the other groups were given the corresponding test substance. The rats were weighed every other week, and drug dose was adjusted according to body weight.
2.1. Drugs and reagents Simvastatin tablets (Hangzhou MSD Pharmaceutical Co., Ltd. Lot number: 110761); cholesterol (Klontech); total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), malondialdehyde (MDA), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein (TP), lipoprotein lipase (LPL), lipase (LPS), and hepatic lipase (HL) kits were purchased from Covey reagents, Beijing Branch; Cydonia oblonga Mill. leaves extracts (dried and crushed fresh Cydonia oblonga Mill. leaf), were added 3 times to 60% ethanol, followed by ultrasonic extraction for 40 min, filtering, and then double extraction of residue using the same amount of ethanol. The three combined filtrates were then extracted with petroleum ether and freeze-dried into a powder. One gram powder was equivalent to about 1.6 g crude leaves. Cydonia oblonga Mill. leaves were identified by Prof Parida Abliz, and deposited in the herbarium of the Traditional Chinese Medicine Ethnical Herbs Specimen Museum of Xinjiang Medical University under number NO.TCMEHSM2013_100. 2.2. Animals and feed Basic feed was provided by Xinjiang Medical Laboratory Animal Centre. In high fat diet animals, high fat feed represented 73.2% of feed, with 1.5% cholesterol, 15% lard, 0.3% cholate, 10% egg yolk powder, to which were added an appropriate amount of water, sufficiently stirred and mixed, then made into strips, dried and preserved for ulterior use. Sixty healthy Sprague Dawley rats were studied, equally male and female, all provided by Xinjiang Medical Laboratory Animal Centre. Certificate number: SCXK(Xin) 20030001. All experiments were approved by the Xinjiang Medical University ethical committee for animal experimentation. 2.3. Instruments and reagents Benchmark plus full wavelength micro plate reader (BIO-RAD); TDL-5A type Centrifuge (On Haifee Pradesh analysis Instrument Co., Ltd.); SHB-III type circulating water with a vacuum pump (Zhenzhou Great Wall Branch Industry and Trade Co., Ltd.); KQ500DE type CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.). 2.4. Animal groups Rats were divided into different cages, each containing 5 rats. They were marked, weighed, and fed with basic feed, with adaptive feeding and observed for 14 days. The rats were then randomly divided into six groups of 10 rats, equally male and female: normal control group (normal standard chow), model control group (hyperlipidic feed), simvastatin group (4 mg/kg/d),
2.6. Animal handling and experimental procedures Rats were treated for 56 days and fasted overnight one day before the end of the experiment. On the last day, they were weighed, and received intraperitoneal anaesthesia with 30% chloral hydrate. They were then fixed, and blood was collected from the abdominal aorta. The whole blood was allowed to stand at room temperature for 0.5 h, then centrifuged at 3000 rpm for 10 min, and serum was pipetted for serum lipid concentration measures (Chen et al., 2009, 2012; Ghule et al., 2009; Giovanni et al., 2009; Liu et al., 2011). After blood collection, the heart, liver, spleen, and kidney were quickly taken, residual fat and fascia were removed, and the organs were washed with ice-cold saline, blotted on filter paper, and weighed to calculate the organs indexes (organ to body weight ratios: organ index (%) ¼organ weight (g)/animal weight (g) 100). The liver was washed and dried, and three samples of liver tissue were taken with a scalpel at the right lobe of the liver, measuring 1 cm3. These were quickly put into glass vials filled with formaldehyde fixative, cooled to 4 1C, embedded in paraffin, sliced, and stained with routine hematoxylin-Eosin (HE), for histopathological examination of the liver (Chen et al., 2009; Qiu et al., 2011). Another sample of the right lobe of the liver tissue, weighing 0.5 g, was put into cold saline, made into 10% liver homogenates at 4 1C and 3000 rpm for 10 min. The supernatant was sampled to measure liver tissue oxidant related indicators (Li et al., 2010; Liu et al., 2011; Wree et al., 2011).
2.7. Statistical methods SPSS 17.0 statistical software was used for data analysis. Student's t-test was used to compare treatment groups, bilateral Po 0.05 was considered statistically significant.
3. Results 3.1. Serum lipids (Table 1). In comparison with normal control group, TC, TG, and LDL-C increased significantly in the model control group. HDL-C decreased significantly, confirming the effect of the hyperlipidaemic diet (Giovanni et al., 2009). Compared with the model group, all treatment groups had significantly lower TC, TG and LDL-C though they did not return to normal values. HDL-C values were increased compared to model, including to values higher than normal controls. There was a dose-related response with COM for all measured parameters, the highest dose of COM having an effect that was similar to or greater than that of simvastatin.
Please cite this article as: Abliz, A., et al., Effect of Cydonia oblonga Mill. leaf extract on serum lipids and liver function in a rat model of hyperlipidaemia. Journal of Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.12.010i
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Table 1 Effect of Cydonia oblonga Mill. leaf extracts on rat serum TC, TG, LDL-C, and HDL-C (m 7 SD, n¼ 10). Groups
Dose (mg/kg)
TC
TG
LDL-C
HDL-C
Normal control Model control Simvastatin COM-low COM-medium COM-high
– – 4 80 160 320
2.02 7 0.22 3.32 7 1.17△△ 2.277 0.26n 2.34 7 0.29n 2.357 0.40n 2.317 0.25n
0.59 7 0.07 0.707 0.14△ 0.577 0.11n 0.59 7 0.17 0.58 7 0.11n 0.56 7 0.09n
0.417 0.15 1.687 0.33△△△ 1.23 7 0.16nn 1.357 0.16n 1.317 0.22nn 1.23 7 0.28nn
1.04 70.39 0.75 70.14△ 1.17 70.37nn 1.0770.29nn 1.17 70.34nn 1.22 70.45nn
TC: total cholesterol; TG: triglycerides; LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol. △
Compared with normal control group, P o0.05. Compared with normal control group, P o0.01. △△△ Compared with normal control group, P o 0.001. n Compared with model control group, Po 0.05. nn Compared with model control group, Po 0.01. △△
Table 2 Effect of Cydonia oblonga Mill. leaf extract on the body weight of rats over time (m 7 SD, n¼ 10). Groups
Dose (mg kg 1)
Entry into study (g)
2 weeks (g)
4 weeks (g)
6 weeks (g)
8 weeks (g)
10 weeks (g)
Normal control Model control Simvastatin COM-low COM-medium COM-high
– – 4 80 160 320
247.3 752.9 254.2 780.5 270.0 760.4 279.4 738.8 252.5 745.3 269.4 749.0
281.8 764.9 317.9 781.1 320.4 780.6 327.5 747.5 297.5 756.2 313.5 764.2
312.3 770.9 355.6788.6 357.7 798.5 358.9755.2 335.6 766.2 356.1 780.7
316.8 7 72.8 376.6 7 103.1 382.5 7 108.6 382.17 60.6 354.27 78.0 379.7 7 93.9
356.6 790.9 411.7 7117.4 426.2 7129.7 407.0 759.7 389.0 777.6 410.4 7105.1
365.0 7 95.8 416.3 7 125.8 435.57 138.5 416.2 7 69.6 401.4 7 84.3 419.3 7 103.8
There was no significant statistical difference between the weights of each group of rats. (P40.05).
Table 3 Effect of Cydonia oblonga Mill. leaf extract on organ indexes (m 7 SD, n¼ 10). Groups
Normal control Model control Simvastatin COM-low COM-medium COM-high
Table 4 Effect of Cydonia oblonga Mill. leaf extract on rat serum AST, ALT and TP (mean 7 SD, n¼ 10).
Dose (mg/kg)
Cardiac index (%)
Liver index (%)
Spleen index (%)
Kidney index (%)
Groups
Dose (mg/kg)
AST
ALT
TP
– – 4 80 160 320
0.28 7 0.02 0.277 0.04 0.25 7 0.03 0.277 0.05 0.277 0.03 0.277 0.03
2.26 7 0.14 3.377 0.44 3.107 0.29 3.29 7 0.39 3.357 0.41 3.047 0.40
0.17 70.02 0.16 70.05 0.16 70.03 0.18 70.05 0.19 70.05 0.18 70.07
0.53 7 0.13 0.517 0.08 0.497 0.05 0.52 7 0.07 0.53 7 0.07 0.52 7 0.05
Normal control Model control Simvastatin COM-low COM-medium COM-high
– – 4 80 160 320
13.5 7 2.36 16.8 7 2.75△ 13.9 7 1.32n 16.4 7 3.03 13.4 7 1.94n 13.9 7 1.43n
11.17 3.26 16.6 7 1.48△△△ 13.7 7 7.16 13.17 5.06 12.17 5.37n 9.62 7 5.00nn
96.2 7 19.95 71.17 5.29△△ 92.0 7 18.94n 86.7 7 13.51nn 92.7 7 18.44nn 93.2 7 6.32nnn
There was no significant statistical difference between groups of rat. (P4 0.05).
AST: aspartate aminotransferase; ALT: alanine aminotransferase; TP: total protein. △
Compared to the normal group, Po 0.05. Compared to the normal group, Po 0.01. Compared to the normal group, Po 0.001. n Compared to the model group, P o0.05. nn Compared to the model group, P o 0.01. nnn Compared to the model group, Po 0.001. △△
3.2. Body weight (Table 2) All the hyperlipidic diet-fed rats were heavier than normal controls throughout the study, and there was no significant difference between the different treatment groups. 3.3. Organ indexes (Table 3) Organ indexes were computed for the heart, liver, spleen, and kidney. Though there was no significant difference between groups for any index, the liver seemed heavier in hyperlipidic fed rats than in normal controls. 3.4. Serum AST, ALT and TP (Table 4) Serum AST and ALT were significantly higher in the fat model group than in normal controls. It was lower in the treated groups than in the controls, significantly so for the two higher doses of COM. Serum TP was lower in model than in controls rats, and the various treatment groups had a higher total protein concentration than the model group. This reached normal values for simvastatin
△△△
and for the medium and higher doses of COM, with a dosedependent effect. 3.5. Peroxidation of rat liver and related enzymatic indicators (Table 5) Hyperlipidemic rats had increased liver lipids and MDA (P o0.01). The activity of antioxidant enzymes SOD and GSH-PX decreased (P o0.01, P o0.001). In the high-dose Cydonia oblonga Mill. leaf extract group, MDA content was lower (P o0.05) and GSH-PX and SOD were higher (P o0.01, P o0.05). Compared to the normal control group, there were significant differences in the activity of HL, LPL, and LPS in the hyperlipidaemia model group (P o0.01). Simvastatin and high-dose Cydonia oblonga Mill leaf extract groups had significantly higher activity of HL (Po0.05). High-dose Cydonia oblonga Mill. leaf extract group had significantly higher LPL (Po0.05). Medium and high-dose Cydonia oblonga Mill.
Please cite this article as: Abliz, A., et al., Effect of Cydonia oblonga Mill. leaf extract on serum lipids and liver function in a rat model of hyperlipidaemia. Journal of Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.12.010i
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Table 5 Effect of Cydonia oblonga Mill. leaf extract on GSH-PX, SOD, MDA, HL, LPS in the liver of the rats (m 7 SD, n¼ 10). Groups
Dose (mg kg 1)
GSH-PX
MDA
SOD
HL
LPL
LPS
Saline control Model control Simvastatin COM-low COM-medium COM-high
– – 4 80 160 320
46.9 7 3.89 40.0 7 3.302;△△△ 45.3 7 7.21 42.3 7 4.36 43.4 7 6.56 45.8 7 3.464;nn
6.56 7 0.39 7.217 0.461;△△ 6.81 7 0.40 6.82 7 0.63 6.707 0.62 6.577 0.593;n
127.5 7 7.11 115.5 7 7.801;△△ 123.9 7 8.913;n 116.0 7 8.12 118.17 7.80 125.9 7 9.283;n
291.2 7 24.8 245.5 7 27.51;△△ 279.9 7 28.93;n 248.4 7 35.2 266.37 44.7 275.5 7 31.23;n
460.0 7 27.6 411.4 7 26.71;△△ 426.3 7 36.6 435.7 7 31.8 438.5 7 39.3 458.0 7 46.63;n
70.7 7 4.12 79.17 6.021;△△ 76.3 7 4.92 74.4 7 6.82 71.8 7 3.954;nn 70.8 7 3.214;nn
GSH-Px: Glutathion peroxidase; MDA: Manodialdehyde; SOD: superoxide dismutase; HL hepatic lipase, LPL: lipoprotein lipase; LPS: lipase. △△
Compared to the normal group, Po 0.01. Compared to the normal group, Po 0.001. n Compared to the model group, P o 0.05. nn Compared to the model group, Po 0.01. △△△
Fig. 1. Liver pathology slices in hyperlipidaemic rats. A: normal control group. B: model control group. C: simvastatin control group. D: low-dose COM group. E: mediumdose COM group. F: high-dose COM group.
leaf extract groups were associated with lower LPS activity (Po0.01) compared to model . 3.6. Rat liver pathology (Fig. 1) By visual appraisal, the liver colour of normal group looked red with smooth surface, the liver volume was moderate, accompanied with normal tissue elasticity. Rats in the model group had a white and pink liver, and a tense capsule with swelling tissue, which was less flexible than in the normal group, and had a greasy feeling when touched. Compared to the model group, the treated groups' livers had a rosy colour in varying degrees, with greater tissue elasticity. After HE staining of liver tissue, in the normal control group the structure of liver tissue was normal, the liver cells had polygonal edges with clear cell borders, and the nucleus was round and clear, located in the centre of the cell with abundant cytoplasm. Fatty degeneration was not found in the liver (Ghule et al., 2009). In the model control group; the liver volume was significantly increased compared with the normal group, and there appeared to be more fat vacuoles, some cell nuclei apparently with a typical fatty degeneration. Compared to the high fat model group, most of the cells in the simvastatin group had been restored to normal levels, liver cell cords arranged normally, and the overall cell degeneration had been significantly improved. Among the Cydonia oblonga Mill. leaf extract groups, medium-dose and high-dose groups had a better preventive effect and the overall cell degeneration showed a significant improvement, as seen in Fig. 1.
with amounts of leaf extracts that are consistent with traditional use of the plant. The exact mechanism is uncertain. It could be related to its known antioxidant effects which are non specific, but also on a more specific effect on lipoprotein lipase, which contrary to simvastatin is restored to normal values by the higher doses of COM, or on lipase, which also reverts to normal values. Other enzymes and markers of oxidative stress, such as SOD, GSH-PX and MDA are also normalized to the same extent as with simvastatin. The dose-dependency of the effects pleads for a pharmacological mechanism, even if the nature of the effect is unknown.
5. Conclusion Our study confirms the dose-dependent effect of COM on dyslipidemia, which along with other effects of the plant on cardiovascular risk factors such as coagulation and thrombosis, may explain or comfort its value in cardiovascular prevention as used in traditional Uyghur medicine. These results also warrant further research in the mechanism of action of COM, and on its effects in man.
Acknowledgements This research work was supported by the National Natural Science Foundation of China (No. 81260490). References
4. Discussion In this experimental exploratory study, we found that hyperlipidic food in rats resulted in hyperlipidaemia with hepatic consequences, validating the model. The administration of COM to these hyperlipidaemic rats reduced serum lipids and hepatic parameters approximately to the same extent as simvastatin, at least at the higher COM doses chosen here, both on the serum lipid parameters, and on the liver consequences of excessive lipid intake, especially on lipid accumulation in hepatocytes. This occurs
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Please cite this article as: Abliz, A., et al., Effect of Cydonia oblonga Mill. leaf extract on serum lipids and liver function in a rat model of hyperlipidaemia. Journal of Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.12.010i
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Please cite this article as: Abliz, A., et al., Effect of Cydonia oblonga Mill. leaf extract on serum lipids and liver function in a rat model of hyperlipidaemia. Journal of Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.12.010i
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Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Research Paper
Effect of Cydonia oblonga Mill. leaf extract on serum lipids and liver function in a rat model of hyperlipidaemia Q1
Ablat Abliz a,1, Qimangul Aji a,1, Elzira Abdusalam a, Xiaowei Sun a, Adil Abdurahman a, Wenting Zhou a, Nicholas Moore a,b,nn, Anwar Umar a,b,n a b
Department of Pharmacology, Xinjiang Medical University, 393 Xinyi Road, 830011 Urumqi, PR China Department of Pharmacology, University Bordeaux Segalen, 146 rue Leo Saignat, 33076 Bordeaux, France
art ic l e i nf o Article history: Received 23 September 2013 Received in revised form 22 November 2013 Accepted 6 December 2013
Keywords: Cydonia oblonga Mill. leaf extract Hyperlipidaemia Antioxidant Hypercholesterolemia Hypolipidemic effect Traditional Uyghur medicine
a b s t r a c t Q4 Ethnopharmacological relevance: Cydonia oblonga Mill. leaves are traditionally used in Uyghur medicine to treat or prevent cardiovascular disease. Beyond a demonstrated effect on thrombosis, we tested it for an effect on dyslipidemia, in a rat model of hyperlipidemia. Methods: Seventy healthy Sprague Dawley rats were randomly divided into 6 groups: normal controls, model controls, simvastatin, and low-, medium- and high-dose Cydonia oblonga Mill. leaf extracts (COM), orally for 56 days. The normal controls were fed a normal diet, all other groups a high fat diet. Rat weights were recorded over time. Total cholesterol (TC), triglycerides (TG), low and high-density lipoproteins (LDL, HDL), as well as AST, ALT and total protein (TP) were measured in serum at the end of the study. The antioxidant capacity of glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), malondialdehyde (MDA) was measured in liver samples, along with lipoprotein lipase (LPL), and hepatic lipase (HL). Liver pathology was described. Results: COM dose-dependently reduced TC, TG, LDL-C and MDA, inhibited the activity of ALT, AST and LPS, increased HDL-C content, increased the activity of SOD, GSH-PX, LPL and HL, and reduced liver steatosis in hyperlipidaemia rats, which was significant at medium and high doses. The effect of COM was similar to that of simvastatin except for increased LPL and HL which were reduced by COM but not by simvastatin. Conclusion: Cydonia oblonga Mill. leaf extracts have hypolipidaemic and hepatoprotective effects, probably related to increasing antioxidant capacity and lipoprotein metabolism in the liver, and inhibition of lipogenesis. & 2013 Published by Elsevier Ireland Ltd.
1. Introduction The improvement in living standards worldwide, and the increasing use of poor quality food, at least from the nutritional point of view, results in increasing frequency of dyslipidaemia, a major risk factor for cardiovascular disease. Along with diabetes
Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; COM, Cydonia oblonga Mill.; GSH-PX, glutathione peroxidase; HDL-C, high density lipoprotein cholesterol; HL, hepatic lipase; LDL-C, low-density lipoprotein cholesterol; LPL, lipoprotein lipase; LPS, lipase; MDA, malondialdehyde; SOD, superoxide dismutase; TC, total cholesterol; TG, triglycerides; TP, total protein n Corresponding author at: Department of Pharmacology, Xinjiang Medical University, 393 Xinyi Road, 830011 Urumqi, PR China. Tel./fax: þ 86 9914362421. nn Corresponding author at: Department of Pharmacology, University Bordeaux Segalen, 146 rue Leo Saignat, 33076 Bordeaux, France. Tel.: þ33 557571560; fax: þ33 557574671. E-mail addresses: [email protected] (N. Moore), [email protected] (A. Umar). 1 Ablat Abliz and Qimangul Aji are equal contributors to this work.
and hypertension, it contributes to the increasing rate of cardiovascular diseases and ensuing morbi-mortality. The fight against such cardiovascular diseases passes through better eating habits and exercise, but also through corrective actions against hyperlipidaemia. Drugs are commonly used for that matter, and especially inhibitors of HMGCoA reductase (statins), but traditional medicines may also have beneficial effects (Chen et al., 2009; Ghule et al., 2009; Aslan et al., 2010; Kwak et al., 2010; Li et al., 2010; Liu et al., 2011; Tan et al., 2011; Lu et al., 2012). Such options merit being explored and perhaps diffused to other regions. Quince, Cydonia oblonga Mill. (COM) (Rosaceae), also called Kinashi, Soil papaya, Biye and so on, is a traditional Uyghur medicinal plant, commonly used in the Hotan and Kashgar regions of Xinjiang, in Western China, for the treatment or prevention of cardiovascular disease, among other. In traditional Uyghur medicine COM fruit, leaves, roots, branches and other parts are used as medicine (Sadik, 1993; Grundemann et al., 2011). Currently, there is much research being done on the chemical composition and antibacterial effects of COM, but less on the pharmacological
0378-8741/$ - see front matter & 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.jep.2013.12.010
Please cite this article as: Abliz, A., et al., Effect of Cydonia oblonga Mill. leaf extract on serum lipids and liver function in a rat model of hyperlipidaemia. Journal of Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.12.010i
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effects of COM, apart from a few papers on its effects on coagulation and blood pressure (Bayier et al., 2010a, 2010b; Grundemann et al., 2011; Abliz et al., 2012). These alone could justify its use in cardiovascular disease or prevention. An effect on lipid metabolism has also been suggested, and we therefore tested the impact of COM leaf extracts on a hyperlipidaemia model, studying serum lipids levels and liver enzyme activity. These could provide evidence for an effect that is complementary to that on coagulation and thrombosis, and provide a theoretical basis for the further development of COM.
2. Material and methods
and Cydonia oblonga Mill. leaf extract at low (80 mg/kg/d), medium (160 mg/kg/d), and high-dose (320 mg/kg/d).
2.5. Dosing regimen All rats had free food and water intake. Normal control group was fed with basic feed, the other groups were fed with high fat feed. Rats were given an intragastric administration every morning at 9:00–10:00. Normal control and hyperlipidaemia model groups were given normal saline, the other groups were given the corresponding test substance. The rats were weighed every other week, and drug dose was adjusted according to body weight.
2.1. Drugs and reagents Simvastatin tablets (Hangzhou MSD Pharmaceutical Co., Ltd. Lot number: 110761); cholesterol (Klontech); total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), malondialdehyde (MDA), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein (TP), lipoprotein lipase (LPL), lipase (LPS), and hepatic lipase (HL) kits were purchased from Covey reagents, Beijing Branch; Cydonia oblonga Mill. leaves extracts (dried and crushed fresh Cydonia oblonga Mill. leaf), were added 3 times to 60% ethanol, followed by ultrasonic extraction for 40 min, filtering, and then double extraction of residue using the same amount of ethanol. The three combined filtrates were then extracted with petroleum ether and freeze-dried into a powder. One gram powder was equivalent to about 1.6 g crude leaves. Cydonia oblonga Mill. leaves were identified by Prof Parida Abliz, and deposited in the herbarium of the Traditional Chinese Medicine Ethnical Herbs Specimen Museum of Xinjiang Medical University under number NO.TCMEHSM2013_100. 2.2. Animals and feed Basic feed was provided by Xinjiang Medical Laboratory Animal Centre. In high fat diet animals, high fat feed represented 73.2% of feed, with 1.5% cholesterol, 15% lard, 0.3% cholate, 10% egg yolk powder, to which were added an appropriate amount of water, sufficiently stirred and mixed, then made into strips, dried and preserved for ulterior use. Sixty healthy Sprague Dawley rats were studied, equally male and female, all provided by Xinjiang Medical Laboratory Animal Centre. Certificate number: SCXK(Xin) 20030001. All experiments were approved by the Xinjiang Medical University ethical committee for animal experimentation. 2.3. Instruments and reagents Benchmark plus full wavelength micro plate reader (BIO-RAD); TDL-5A type Centrifuge (On Haifee Pradesh analysis Instrument Co., Ltd.); SHB-III type circulating water with a vacuum pump (Zhenzhou Great Wall Branch Industry and Trade Co., Ltd.); KQ500DE type CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.). 2.4. Animal groups Rats were divided into different cages, each containing 5 rats. They were marked, weighed, and fed with basic feed, with adaptive feeding and observed for 14 days. The rats were then randomly divided into six groups of 10 rats, equally male and female: normal control group (normal standard chow), model control group (hyperlipidic feed), simvastatin group (4 mg/kg/d),
2.6. Animal handling and experimental procedures Rats were treated for 56 days and fasted overnight one day before the end of the experiment. On the last day, they were weighed, and received intraperitoneal anaesthesia with 30% chloral hydrate. They were then fixed, and blood was collected from the abdominal aorta. The whole blood was allowed to stand at room temperature for 0.5 h, then centrifuged at 3000 rpm for 10 min, and serum was pipetted for serum lipid concentration measures (Chen et al., 2009, 2012; Ghule et al., 2009; Giovanni et al., 2009; Liu et al., 2011). After blood collection, the heart, liver, spleen, and kidney were quickly taken, residual fat and fascia were removed, and the organs were washed with ice-cold saline, blotted on filter paper, and weighed to calculate the organs indexes (organ to body weight ratios: organ index (%) ¼organ weight (g)/animal weight (g) 100). The liver was washed and dried, and three samples of liver tissue were taken with a scalpel at the right lobe of the liver, measuring 1 cm3. These were quickly put into glass vials filled with formaldehyde fixative, cooled to 4 1C, embedded in paraffin, sliced, and stained with routine hematoxylin-Eosin (HE), for histopathological examination of the liver (Chen et al., 2009; Qiu et al., 2011). Another sample of the right lobe of the liver tissue, weighing 0.5 g, was put into cold saline, made into 10% liver homogenates at 4 1C and 3000 rpm for 10 min. The supernatant was sampled to measure liver tissue oxidant related indicators (Li et al., 2010; Liu et al., 2011; Wree et al., 2011).
2.7. Statistical methods SPSS 17.0 statistical software was used for data analysis. Student's t-test was used to compare treatment groups, bilateral Po 0.05 was considered statistically significant.
3. Results 3.1. Serum lipids (Table 1). In comparison with normal control group, TC, TG, and LDL-C increased significantly in the model control group. HDL-C decreased significantly, confirming the effect of the hyperlipidaemic diet (Giovanni et al., 2009). Compared with the model group, all treatment groups had significantly lower TC, TG and LDL-C though they did not return to normal values. HDL-C values were increased compared to model, including to values higher than normal controls. There was a dose-related response with COM for all measured parameters, the highest dose of COM having an effect that was similar to or greater than that of simvastatin.
Please cite this article as: Abliz, A., et al., Effect of Cydonia oblonga Mill. leaf extract on serum lipids and liver function in a rat model of hyperlipidaemia. Journal of Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.12.010i
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Table 1 Effect of Cydonia oblonga Mill. leaf extracts on rat serum TC, TG, LDL-C, and HDL-C (m 7 SD, n¼ 10). Groups
Dose (mg/kg)
TC
TG
LDL-C
HDL-C
Normal control Model control Simvastatin COM-low COM-medium COM-high
– – 4 80 160 320
2.02 7 0.22 3.32 7 1.17△△ 2.277 0.26n 2.34 7 0.29n 2.357 0.40n 2.317 0.25n
0.59 7 0.07 0.707 0.14△ 0.577 0.11n 0.59 7 0.17 0.58 7 0.11n 0.56 7 0.09n
0.417 0.15 1.687 0.33△△△ 1.23 7 0.16nn 1.357 0.16n 1.317 0.22nn 1.23 7 0.28nn
1.04 70.39 0.75 70.14△ 1.17 70.37nn 1.0770.29nn 1.17 70.34nn 1.22 70.45nn
TC: total cholesterol; TG: triglycerides; LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol. △
Compared with normal control group, P o0.05. Compared with normal control group, P o0.01. △△△ Compared with normal control group, P o 0.001. n Compared with model control group, Po 0.05. nn Compared with model control group, Po 0.01. △△
Table 2 Effect of Cydonia oblonga Mill. leaf extract on the body weight of rats over time (m 7 SD, n¼ 10). Groups
Dose (mg kg 1)
Entry into study (g)
2 weeks (g)
4 weeks (g)
6 weeks (g)
8 weeks (g)
10 weeks (g)
Normal control Model control Simvastatin COM-low COM-medium COM-high
– – 4 80 160 320
247.3 752.9 254.2 780.5 270.0 760.4 279.4 738.8 252.5 745.3 269.4 749.0
281.8 764.9 317.9 781.1 320.4 780.6 327.5 747.5 297.5 756.2 313.5 764.2
312.3 770.9 355.6788.6 357.7 798.5 358.9755.2 335.6 766.2 356.1 780.7
316.8 7 72.8 376.6 7 103.1 382.5 7 108.6 382.17 60.6 354.27 78.0 379.7 7 93.9
356.6 790.9 411.7 7117.4 426.2 7129.7 407.0 759.7 389.0 777.6 410.4 7105.1
365.0 7 95.8 416.3 7 125.8 435.57 138.5 416.2 7 69.6 401.4 7 84.3 419.3 7 103.8
There was no significant statistical difference between the weights of each group of rats. (P40.05).
Table 3 Effect of Cydonia oblonga Mill. leaf extract on organ indexes (m 7 SD, n¼ 10). Groups
Normal control Model control Simvastatin COM-low COM-medium COM-high
Table 4 Effect of Cydonia oblonga Mill. leaf extract on rat serum AST, ALT and TP (mean 7 SD, n¼ 10).
Dose (mg/kg)
Cardiac index (%)
Liver index (%)
Spleen index (%)
Kidney index (%)
Groups
Dose (mg/kg)
AST
ALT
TP
– – 4 80 160 320
0.28 7 0.02 0.277 0.04 0.25 7 0.03 0.277 0.05 0.277 0.03 0.277 0.03
2.26 7 0.14 3.377 0.44 3.107 0.29 3.29 7 0.39 3.357 0.41 3.047 0.40
0.17 70.02 0.16 70.05 0.16 70.03 0.18 70.05 0.19 70.05 0.18 70.07
0.53 7 0.13 0.517 0.08 0.497 0.05 0.52 7 0.07 0.53 7 0.07 0.52 7 0.05
Normal control Model control Simvastatin COM-low COM-medium COM-high
– – 4 80 160 320
13.5 7 2.36 16.8 7 2.75△ 13.9 7 1.32n 16.4 7 3.03 13.4 7 1.94n 13.9 7 1.43n
11.17 3.26 16.6 7 1.48△△△ 13.7 7 7.16 13.17 5.06 12.17 5.37n 9.62 7 5.00nn
96.2 7 19.95 71.17 5.29△△ 92.0 7 18.94n 86.7 7 13.51nn 92.7 7 18.44nn 93.2 7 6.32nnn
There was no significant statistical difference between groups of rat. (P4 0.05).
AST: aspartate aminotransferase; ALT: alanine aminotransferase; TP: total protein. △
Compared to the normal group, Po 0.05. Compared to the normal group, Po 0.01. Compared to the normal group, Po 0.001. n Compared to the model group, P o0.05. nn Compared to the model group, P o 0.01. nnn Compared to the model group, Po 0.001. △△
3.2. Body weight (Table 2) All the hyperlipidic diet-fed rats were heavier than normal controls throughout the study, and there was no significant difference between the different treatment groups. 3.3. Organ indexes (Table 3) Organ indexes were computed for the heart, liver, spleen, and kidney. Though there was no significant difference between groups for any index, the liver seemed heavier in hyperlipidic fed rats than in normal controls. 3.4. Serum AST, ALT and TP (Table 4) Serum AST and ALT were significantly higher in the fat model group than in normal controls. It was lower in the treated groups than in the controls, significantly so for the two higher doses of COM. Serum TP was lower in model than in controls rats, and the various treatment groups had a higher total protein concentration than the model group. This reached normal values for simvastatin
△△△
and for the medium and higher doses of COM, with a dosedependent effect. 3.5. Peroxidation of rat liver and related enzymatic indicators (Table 5) Hyperlipidemic rats had increased liver lipids and MDA (P o0.01). The activity of antioxidant enzymes SOD and GSH-PX decreased (P o0.01, P o0.001). In the high-dose Cydonia oblonga Mill. leaf extract group, MDA content was lower (P o0.05) and GSH-PX and SOD were higher (P o0.01, P o0.05). Compared to the normal control group, there were significant differences in the activity of HL, LPL, and LPS in the hyperlipidaemia model group (P o0.01). Simvastatin and high-dose Cydonia oblonga Mill leaf extract groups had significantly higher activity of HL (Po0.05). High-dose Cydonia oblonga Mill. leaf extract group had significantly higher LPL (Po0.05). Medium and high-dose Cydonia oblonga Mill.
Please cite this article as: Abliz, A., et al., Effect of Cydonia oblonga Mill. leaf extract on serum lipids and liver function in a rat model of hyperlipidaemia. Journal of Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.12.010i
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Table 5 Effect of Cydonia oblonga Mill. leaf extract on GSH-PX, SOD, MDA, HL, LPS in the liver of the rats (m 7 SD, n¼ 10). Groups
Dose (mg kg 1)
GSH-PX
MDA
SOD
HL
LPL
LPS
Saline control Model control Simvastatin COM-low COM-medium COM-high
– – 4 80 160 320
46.9 7 3.89 40.0 7 3.302;△△△ 45.3 7 7.21 42.3 7 4.36 43.4 7 6.56 45.8 7 3.464;nn
6.56 7 0.39 7.217 0.461;△△ 6.81 7 0.40 6.82 7 0.63 6.707 0.62 6.577 0.593;n
127.5 7 7.11 115.5 7 7.801;△△ 123.9 7 8.913;n 116.0 7 8.12 118.17 7.80 125.9 7 9.283;n
291.2 7 24.8 245.5 7 27.51;△△ 279.9 7 28.93;n 248.4 7 35.2 266.37 44.7 275.5 7 31.23;n
460.0 7 27.6 411.4 7 26.71;△△ 426.3 7 36.6 435.7 7 31.8 438.5 7 39.3 458.0 7 46.63;n
70.7 7 4.12 79.17 6.021;△△ 76.3 7 4.92 74.4 7 6.82 71.8 7 3.954;nn 70.8 7 3.214;nn
GSH-Px: Glutathion peroxidase; MDA: Manodialdehyde; SOD: superoxide dismutase; HL hepatic lipase, LPL: lipoprotein lipase; LPS: lipase. △△
Compared to the normal group, Po 0.01. Compared to the normal group, Po 0.001. n Compared to the model group, P o 0.05. nn Compared to the model group, Po 0.01. △△△
Fig. 1. Liver pathology slices in hyperlipidaemic rats. A: normal control group. B: model control group. C: simvastatin control group. D: low-dose COM group. E: mediumdose COM group. F: high-dose COM group.
leaf extract groups were associated with lower LPS activity (Po0.01) compared to model . 3.6. Rat liver pathology (Fig. 1) By visual appraisal, the liver colour of normal group looked red with smooth surface, the liver volume was moderate, accompanied with normal tissue elasticity. Rats in the model group had a white and pink liver, and a tense capsule with swelling tissue, which was less flexible than in the normal group, and had a greasy feeling when touched. Compared to the model group, the treated groups' livers had a rosy colour in varying degrees, with greater tissue elasticity. After HE staining of liver tissue, in the normal control group the structure of liver tissue was normal, the liver cells had polygonal edges with clear cell borders, and the nucleus was round and clear, located in the centre of the cell with abundant cytoplasm. Fatty degeneration was not found in the liver (Ghule et al., 2009). In the model control group; the liver volume was significantly increased compared with the normal group, and there appeared to be more fat vacuoles, some cell nuclei apparently with a typical fatty degeneration. Compared to the high fat model group, most of the cells in the simvastatin group had been restored to normal levels, liver cell cords arranged normally, and the overall cell degeneration had been significantly improved. Among the Cydonia oblonga Mill. leaf extract groups, medium-dose and high-dose groups had a better preventive effect and the overall cell degeneration showed a significant improvement, as seen in Fig. 1.
with amounts of leaf extracts that are consistent with traditional use of the plant. The exact mechanism is uncertain. It could be related to its known antioxidant effects which are non specific, but also on a more specific effect on lipoprotein lipase, which contrary to simvastatin is restored to normal values by the higher doses of COM, or on lipase, which also reverts to normal values. Other enzymes and markers of oxidative stress, such as SOD, GSH-PX and MDA are also normalized to the same extent as with simvastatin. The dose-dependency of the effects pleads for a pharmacological mechanism, even if the nature of the effect is unknown.
5. Conclusion Our study confirms the dose-dependent effect of COM on dyslipidemia, which along with other effects of the plant on cardiovascular risk factors such as coagulation and thrombosis, may explain or comfort its value in cardiovascular prevention as used in traditional Uyghur medicine. These results also warrant further research in the mechanism of action of COM, and on its effects in man.
Acknowledgements This research work was supported by the National Natural Science Foundation of China (No. 81260490). References
4. Discussion In this experimental exploratory study, we found that hyperlipidic food in rats resulted in hyperlipidaemia with hepatic consequences, validating the model. The administration of COM to these hyperlipidaemic rats reduced serum lipids and hepatic parameters approximately to the same extent as simvastatin, at least at the higher COM doses chosen here, both on the serum lipid parameters, and on the liver consequences of excessive lipid intake, especially on lipid accumulation in hepatocytes. This occurs
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Please cite this article as: Abliz, A., et al., Effect of Cydonia oblonga Mill. leaf extract on serum lipids and liver function in a rat model of hyperlipidaemia. Journal of Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.12.010i
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Please cite this article as: Abliz, A., et al., Effect of Cydonia oblonga Mill. leaf extract on serum lipids and liver function in a rat model of hyperlipidaemia. Journal of Ethnopharmacology (2013), http://dx.doi.org/10.1016/j.jep.2013.12.010i