Natural Product Research

Formerly Natural Product Letters

ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20

Hepatoprotective effect against CCl4-induced

acute liver damage in mice and High-performance
liquid chromatography mass spectrometric
method for analysis of the constituents of extract
of Rubus crataegifolius

Yongjiao Sun, Lingyun Jia, Zhanbo Huang, Jing Wang, Jincai Lu & Jing Li

To cite this article: Yongjiao Sun, Lingyun Jia, Zhanbo Huang, Jing Wang, Jincai Lu & Jing
Li (2017) Hepatoprotective effect against CCl4-induced acute liver damage in mice and
High-performance liquid chromatography mass spectrometric method for analysis of the
constituents of extract of Rubus crataegifolius, Natural Product Research, 31:22, 2695-2699, DOI:
10.1080/14786419.2017.1292264

To link to this article: http://dx.doi.org/10.1080/14786419.2017.1292264

View supplementary material Published online: 21 Mar 2017.

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Download by: [UNIVERSITY OF ADELAIDE LIBRARIES] Date: 13 October 2017, At: 01:22
 Natural Product Research, 2017
VOL. 31, NO. 22, 2695–2699
https://doi.org/10.1080/14786419.2017.1292264

SHORT COMMUNICATION

Hepatoprotective effect against CCl4-induced acute

liver damage in mice and High-performance liquid
chromatography mass spectrometric method for analysis of
the constituents of extract of Rubus crataegifolius
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Yongjiao Suna, Lingyun Jiaa, Zhanbo Huangb, Jing Wanga, Jincai Lua and Jing Lia
a
School of  Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, China;
b
School of Biological Medicine and Chemical Engineering, Liaoning Institute of Science and Technology,
Benxi, PR China

ABSTRACT ARTICLE HISTORY

This study is an attempt to evaluate the hepatoprotective activity Received 25 July 2016
of Rubus Crataegifolius against carbon tetrachloride-induced liver Accepted 29 January 2017
injury in mice. 70% ethanolic, ethyl acetate and n-BuOH extract of
KEYWORDS
R. crataegifolius were administered daily for 14 days in experimental Hepatoprotective effect;
animals before they were treated with CCl4. The hepatoprotective Rubus crataegifolius; HPLC-
activity of the extracts in this study was compared with the reference EIS-MS/MS
drug silymarin. A high-performance liquid chromatography mass
spectrometric (HPLC-EIS-MS/MS) method was developed for the
determination of the constituents of the extracts. According to
the data of HPLC-EIS-MS/MS, the chemical structures of the largely
14 constituents of R. crataegifolius were identified online without
time-consuming isolation. Ethyl acetate extracts of R. crataegifolius
showed strong antioxidant activities and significant protective effect
against acute hepatotoxicity induced by CCl4. According to the data
of HPLC-EIS-MS/MS, Oleanic acid, Phlorizin dehydrate and Quercetin-
3-rhamnoside are considered as the main hepatoprotective factor in
ethyl acetate extract.

CONTACT  Lingyun Jia  jialingyun2003@126.com

 Supplemental data for this article can be accessed at http://dx.doi.org/10.1080/14786419.2017.1292264.
© 2017 Informa UK Limited, trading as Taylor & Francis Group
 2696   Y. SUN ET AL.

1. Introduction
Hepatic disease is one of the most common diseases to affect human health. Carbon tetra-
chloride (CCl4) is an extensively used hepatotoxic agent in preclinical animal studies. (Han
et al. 2016) Rubus crataegifolius is also known as Niu diedu, Rosaceae Rubus, widely distributed
in north-east, Inner Mongolia, Hebei, Shanxi, Henan and other provinces (regions) of China.
Its roots are always as traditional Chinese medicine. Root portion has dispelling wind,
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Figure 1. Effects of all extracts on ALT activities in serum of mice with CCl4- induced liver injury.
Notes: Data were expressed as the mean  ±  SD (n  =  8).(### p  <  0.001,## p  <  0.01,compared with the normal group;
***p < 0.001,**p < 0.01,*p < 0.05,compared with the model group).

Figure 2. Effects of all extracts on AST activities in serum of mice with CCl4- induced liver injury.
Notes: Data were expressed as the mean  ±  SD (n=8).(### p  <  0.001,## p  <  0.01,compared with the normal group;
***p < 0.001,**p < 0.01,*p < 0.05,compared with the model group).
 NATURAL PRODUCT RESEARCH   2697
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Figure 3. Effects of all extracts on SOD activities in serum of mice with CCl4- induced liver injury.
Notes: Data were expressed as the mean  ±  SD (n=8).(### p  <  0.001,## p  <  0.01,compared with the normal group;
***p < 0.001,**p < 0.01,*p < 0.05,compared with the model group).

Figure 4. Effects of all extracts on GSH activities in serum of mice with CCl4- induced liver injury.
Notes: Data were expressed as the mean  ±  SD (n=8).(### p  <  0.001,## p  <  0.01,compared with the normal group;
***p < 0.001,**p < 0.01,*p < 0.05,compared with the model group).

removing dampness and other functions, for the treatment of chronic hepatitis, rheumatoid
arthritis. Modern pharmacological studies have shown that its root has a variety of pharma-
cological effects, such as anti-organisations peroxide, anti-tumour, anti-inflammatory and
antibacterial.
 2698   Y. SUN ET AL.
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Figure 5. Effects of all extracts on MDA activities in serum of mice with CCl4- induced liver injury.
Notes: Data were expressed as the mean  ±  SD (n=8).(### p  <  0.001,## p  <  0.01,compared with the normal group;
***p < 0.001,**p < 0.01,*p < 0.05,compared with the model group).

2.  Results and discussion

2.1.  Effect of the extracts on CCl4-induced acute liver damage in mice
2.1.1.  Body weight and liver weights
There was no significant alteration in the body weights or food intake among the mice of
various treatment groups. The relative liver index in both RC-pretreated mice and the CCl4-
treated animals were similar (data not shown) (Singh et al. 2008).

2.1.2.  Effects of RC on serum AST and ALT activities of mice with CCl4-induced liver
injury
The results are shown in the figure 1 and figure 2.

2.1.3.  Effects of RC on serum SOD, GSH and MDA activities of mice with CCl4-induced
liver injury
The results are shown in the figure 3, figure 4 and figure 5.

2.2.  HPLC-EIS-MS/MS analysis

The ESI-MS/MS system consisted of a High-performance liquid chromatograph and a mass
spectrometer (Bruker Company, USA). Specific analysis show in supplementary materials.

2.3. Discussion
CCl4 has been widely used in animal models to investigate chemical toxin-induced liver dam-
age and as an excellent model to evaluate the efficacy of hepatoprotectants (Lin et al. 2014).
Serum hepatobiliary enzymes such as AST and ALT are present in high concentrations in the
 NATURAL PRODUCT RESEARCH   2699

liver under normal conditions. When there is hepatocyte necrosis or membrane damage,
these enzymes will be released into the circulation, as indicated by elevated serum enzyme
levels (Vuda et al. 2012). When liver cells are attacked by free radicals, SOD and GSH-Px can
be reduced because of the excessive consumption, so that it can be understood that the
higher the activity of SOD and GSH-Px in the body, the faster the velocity of radical scavenging
is MDA, (Minor et al. 1993; Cigremis et al. 2009)the final product generated in the metabolism
of lipid peroxides when lipids are attacked by free radicals(Lu et al. 2012; Zhang et al.2013)
can further damage the cells, which can not only reflect the degree of sensitivity of lipid
peroxidation but also indirectly reflect the degree of cell injury (Pirinccioglu et al. 2010).
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4. Conclusions
From the biochemical indicators, it was showed ethyl acetate extracts were having the
strongest antioxidant activities and significant protective effect against acute hepatotoxicity
induced by CCl4, the order of hepatotoxicity effects of these extracts was ethyl acetate
extract > total extract > n-BuOH extract. According to the results of HPLC-EIS-MS/MS, Oleanic
acid, Phlorizin dihydrate and quercetin-3-rhamnoside may be considered as the main hepato-
protective factor in ethyl acetate extract.

Acknowledgements
The authors are thankful to Prof. Lu and Prof. Jia for their encouragement and support during the
course of study.

References
Cigremis Y, Turel H, Adiguzel K, Akgoz M, Kart A, Karaman M, Ozen H. 2009. The effects of acute
acetaminophen toxicity on hepatic mRNA expression of SOD, CAT, GSH-Px, and levels of peroxynitrite
nitric oxide, reduced glutathione, and malondialdehyde in rabbit. Mol Cell Biochem. 323:31–38.
Han B, Gao Y, Wang YL, Wang L, Shang ZH, Wang S, Pei J. 2016. Protective effect of a polysaccharide from
Rhizoma Atractylodis Macrocephalae on acute liver injury in mice. Int J Biol Macromolec. 87:85–91.
Lin YC, Cheng KM, Huang HY, Chao PY, Hwang JM, Lee HH, Lu CY, Chiu YW, Liu JY. 2014. Hepatoprotective
activity of Chhit-Chan-Than extract powder against carbon tetrachloride-induced liver injury in rats.
J Food Drug Anal. 22:220–229.
Lu BN, Xu YS, Xu LN, Cong XN, Yin LH, Li H, Peng JY. 2012. Mechanism investigation of dioscin against
CCl4-induced acute liver damage in mice. Environ Toxicol Pharmacol. 34:127–135.
Minor T, Isselhard W, Yamamoto Y, Obara M, Saad S. 1993. The effects of allopurinol and SOD on lipid
peroxidation and energy metabolism in the liver after ischemia in an aerobic/anaerobic persufflation.
Surg Today. 23:728–732.
Pirinccioglu AG, Gökalp D, Pirinccioglu M, Kizil G, Kizil M. 2010. Malondialdehyde (MDA) and protein
carbonyl (PCO) levels as biomarkers of oxidative stress in subjects with familial hypercholesterolemia.
Clin Biochem. 43:1220–1224.
Singh N, Kamath V, Narasimhamurthy K, Rajini PS. 2008. Protective effect of potato peel extract against
carbon tetrachloride-induced liver injury in rats. Environ Toxicol and Pharmacol. 26:241–246.
Vuda M, D’Souza R, Upadhya S. 2012. Hepatoprotective and antioxidant activity of aqueous extract of
Hybanthus enneaspermus against CCl4-induced liver injury in rats. Exp Toxicol Pathol. 64:855–859.
Zhang S, Lu BN, Han X, Xu LN, Qi Y, Yin LH, Xu YW, Zhao YY, Liu KX, Peng JY. 2013. Protection of the
flavonoid fraction from Rosa laevigata Michx fruit against carbon tetrachloride-induced acute liver
injury in mice. Food Chem Toxicol. 55:60–69.