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Abstract [Objectives] This study was conducted to study the protective effect of the Zhuang medicine Cryptolepis buchananii Roem. et Schult. on Dgalactosamine （DGalN）induced liver injury in mice.
[Methods] A mouse model of chemical liver injury was established by DGalN， and the mice with DGalNinduced liver injury were randomly divided into the blank group， model group， positive control group and drugadministered groups. The levels of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） in the serum of the mice in each group and the levels of superoxide dismutase （SOD） and malondialdehyde （MDA） in the liver tissue of the mice were determined， and the liver pathological changes were observed.
[Results] The extracts of the Zhuang medicinal herb C. buchananii could reduce the serum ALT and AST levels （P<0.05）， increase the SOD content in the liver tissue of mice （P<0.05）， and decrease the MDA level （P<0.05）.
[Conclusions] The Zhuang medicinal herb C. buchananii has a good protective effect on DGalNinduced liver injury in mice.
Key words Cryptolepis buchananii Roem. et Schult.; Dgalactosamine; Liver injury
Gugouteng refers to the rhizome of Cryptolepis buchananii Roem.et Schult. in Cryptolepis of Asclepiadaceae[1]. C. buchananii is also known as Baiyeteng， Baimalianan， Niujiaomo， Duanchangcao[2]， Banjianiu， Baiduzong and Baijiangteng[3]. It has the functions of relaxing tendons and activating collaterals， relieving swelling and pain， and relieving internal heat or fever， and is used to treat lumbago， abdominal pain， bruises， fractures， hemorrhoids， tinea and other diseases[4]. It is a common medicinal material used by the people of Guangxi Zhuang nationality[5]. Modern pharmacological studies have found that C. buchananii has pharmacological effects such as analgesic and antiinflammatory[6]， blood sugarlowering[7-8]， hypolipidemic[9]， and liver protection effects[10]. At present， there is no public report on the pharmacodynamic study of C. buchananii on chemical liver injury. In this study， a mouse model of DGalNinduced acute liver injury was established and used to explore the pharmacological effects of the Zhuang medicinal herb C. buchananii in protecting the liver. This study provides a scientific basis and theoretical guidance for the resource development and clinical application of the Zhuang medicinal herb C. buchananii. Materials and Methods
Medicinal materials
C. buchananii materials were collected from Shiwan Mountain， Guangxi， and were identified as the aboveground part of C. buchananii Roem. et Schult by pharmacist Ma from Guangxi Yixin Medical Co.， Ltd.
Drugs and reagents
Bifendate Pills （WanBangDe Pharmaceutical Group Co.， Ltd.， lot number： A02150317）; DGalN （Shanghai Ryon Biotechnology Co.， Ltd.， lot number： RT1705U508）; saline （Guizhou Tiandi Pharmaceutical Co.， Ltd.， lot number： G16071303）; kit： ALT （lot number： 20170307）， AST （lot number： 20170306）， TP （lot number： 20170426）， SOD （lot number： 20170307）， MDA （lot number： 20170306）， all purchased from Nanjing Jiancheng Bioengineering Institute.
Instruments
Infinite M200PRO fullwavelength microplate reader （TECAN， Switzerland）; Fresco 17type refrigerated highspeed centrifugator （Thermo Fisher， US）; SQP electronic analytical balance （Sartorius Scientific Instruments （Beijing） Co.， Ltd.）; RE52A rotary evaporator （Shanghai Yarong biochemical instrument factory）.
Experiment animals
Kunming mice， 18-22 g， half male and half female， SPF level， purchased from the Experimental Animal Center of Guangxi University of Chinese Medicine， license number： SCXK Gui 20090003.
Experiment methods
Preparation of test drugs
Preparation of positive drug
A certain amount of Bifendate Pills （2.7 g） was weighed and ground into fine powder， which was dissolved in water， and mixed into a suspension having a concentration of 30 mg/ml， which was stored in a refrigerator.
Preparation of C. buchananii water extract
A certain amount of the medicinal material （0.5 kg） was weighed， added with 10 times of water， and refluxextracted with heating. The extraction was performed for two times， 40 min each time. The filtrates were combined， and the solvent was evaporated to give an extract （1 g of extract equivalent to 5.68 g of crude drug）， which was stored in a refrigerator.
Preparation of C. buchananii ethanol extract
The extraction was performed for two times， 40 min each time. The filtrates were combined， degreased with petroleum ether and evaporated to give an extract （1 g of extract equivalent to 7.35 g of crude drug）， which was stored in a refrigerator.
Animal grouping
110 mice were randomly divided into 11 groups， 10 in each group， including the blank group， model group， positive control group （bifendate 600 mg/kg） and administration groups. According to the preexperiment results， it was determined that the high， medium and low dose administration groups of the water extract and ethanol extracts were 20， 10， and 5 g/kg， respectively. The blank group and the model group were intragastrically administered with the same dose of physiological saline every day， and the administration groups were intragastrically administered once a day for 10 consecutive days. Establishment of DGalNinducted liver injury model
One hour after the last administration of each group of mice， the blank group was intraperitoneally injected with 0.9% saline at 10 ml/kg， and other groups were intraperitoneally injected with DGalNsolution （500 mg/kg） at 10 ml/kg. After modeling， the mice were fasted but provided with water. After 18 h， the eyeballs were taken from the mice， and the serum was separated to detect the contents of ALT and AST in the serum; and after the mice were sacrificed， the liver was quickly dissected and the liver index was calculated. Part of the liver tissue was taken， added with 9 times of ice physiological saline， and centrifuged at 3 000 r/min for 10 min. The supernatant was taken to detect various indexes of ALT， AST， TP， SOD and MDA. The liver tissue on the large lobe of the liver was fixed with 4% paraformaldehyde and subjected to HE staining， and the degree of liver tissue damage was observed under a microscope.
Data processing
Statistical analysis was performed using SPSS 23.0 statistical software. Data results were expressed as x ±s. Oneway analysis of variance was adopted， and data of different groups were compared using t test， in which P value was calculated， and the difference was considered to be significant at P<0.05 and extremely significant at P<0.01.
Results and Analysis
Effect on liver index in mice with DGalNinduced liver injury
Compared with the blank control group， the liver index of the model group was significantly higher （P<0.01）. Compared with the model group， the liver index of the drugadministered groups （high， medium and low doses） and the positive control group were significantly lower （P<0.05）， as shown in Table 1.
Liver index=Liver weight/Mouse weight×100%
Effects on serum ALT and AST levels in the serum of mice with DGalNinduced liver injury
Compared with the model group， the serum ALT and AST levels in the drugadministered groups （high， medium and low doses）and the positive control group decreased significantly （P<0.01 in the water extract administration groups and P<0.05 in the ethanol extract administration group）. The results are shown in Table 2.
Effects on SOD and MDA in liver homogenate of DGalNinduced liver injury mice
Compared with the model group， the activity of SOD in the liver homogenate of the mice in the drugadministered groups （high， medium and low doses） and the positive control group increased significantly （P<0.01）; and the activity of MDA in the liver homogenate of the mice in the drugadministered groups （high， medium and low doses） and the positive group decreased significantly （P<0.01）. The results are shown in Table 3. Effects on pathological changes in the liver of mice with DGalNinducedliver injury
The pathological sections showed that compared with the model group， with drugadministered groups （high， medium and low doses） improved the mice with DGalNinduced liver injury to different degrees， and their liver tissue degeneration， necrosis and inflammatory cell infiltration were alleviated （P<0.05）. The results are shown in Table 4.
Agricultural Biotechnology 2020
Discussion
Dgalactosamine （DGalN） is a hepatocellular phosphate uridine interfering agent， which can cause diffuse necrosis and inflammation of the liver. The liver pathological changes caused by it are similar to clinical viral hepatitis， so it is often used in liver protection screening experiments for clinical drugs[11]. Therefore， this study established a DGalNinduced liver injury model， which was used for exploring the hepatoprotective effect of the Zhuang medicinal herb C. buchananii.
This study showed that the high， middle and low dose groups of the water and ethanol extracts of the Zhuang medicinal herb C. buchananii all could reduce the liver index， reduce the serum ALT， AS and TMDA activity in mice with liver injury， and improve SOD activity. The pathological section showed that the degree of inflammatory lesions in the liver tissue of mice was improved. This study confirms that the extract of C. buchananii can enhance the antiinjury ability of hepatocytes and has the effects of protecting liver and reducing enzyme. Its role may be to reduce the synthesis of lipid peroxides and inhibit the synthesis of plasma membrane structural proteins， thereby achieving liver protection.
References
[1] CUI YL， RAN XD， et al. Zhonghua Yaohai[M]. Harbin： Haerbin Press， 1993： 459. （in Chinese）
[2] Guangxi Institute of Chinese Medicine & Pharmaceutical Science. Guangxi medicinal plants directory （Guangxi Yaoyongzhiwu Minglu）[M]. Guangxi： Guangxi Peoples Publishing House， 1986. （in Chinese）
[3] Editorial Group of Yunnan Chinese Herbal Medicine. Yunnan Chinese herbal medicine （Yunnan Zhongcaoyao）[M]. Yunnan： Yunnan Peoples Publishing House， 2011. （in Chinese）
[4] Editorial Group of Collection of Traditional Chinese Medicine in China. Collection of traditional Chinese medicine in China[M]. Beijing： Peoples Medical Publishing House， 1976： 170. （in Chinese）. （in Chinese）
[5] HUANG RS. Zhuangyao Xianbian[M]. Nanning： Guangxi Science and Technology Press，2015： 305. （in Chinese） [6] ZHANG XS， WEN LY， QIN HL， et al. Experimental study on analgesic and antiinflammatory pharmacological effects of alcohol extract of Cryptolepis buchananii Roem. et Schult.[J]. Lishizhen Medicine and Materia Medica Research， 2009， 20（11）： 2735-2736. （in Chinese）
[7] ZHANG XS， HUANG X， PAN NY. Experimental study on the effect of Cryptolepis buchananii Roem. et Schult. extract on blood glucose in normal mice[J]. Lishizhen Medicine and Materia Medica Research， 2010， 21（10）： 2699-2700. （in Chinese）
[8] LU CL， LIANG XN， CHEN Y. effect of aqueous extracts from Cryptolepis buchananii Roem. et Schult on blood glucose in mice with experiment hyperglycemia[J]. Guangxi Medical Journal，2013，35（6）：702-704. （in Chinese）
[9] ZHANG XS， ZHANG Z， ZHOU YQ. Pharmacological experimental research of aquatic extractant from Cryptolepis buchananii on the fat lipids of mice[J]. Lishizhen Medicine and Materia Medica Research， 2013， 24（10）： 2356-2357. （in Chinese）
[10] DONG ML， PAN QD， RUAN YE， et al. Protective effects of Cryptolepis buchananii extract on acute liver injury in mice[J]. Journal of Youjiang Medical University for Nationalities， 2014， 36（3）： 358-360. （in Chinese）
[11] XU ZN， WU XM， WANG P， et al. 18α glycyrrhizic acid in the treatment of acute live injury induced by Dgalactosamine in rats[J]. Journal of Clinical Hepatology， 2012， 28（1）： 63-65. （in Chinese）
[Methods] A mouse model of chemical liver injury was established by DGalN， and the mice with DGalNinduced liver injury were randomly divided into the blank group， model group， positive control group and drugadministered groups. The levels of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） in the serum of the mice in each group and the levels of superoxide dismutase （SOD） and malondialdehyde （MDA） in the liver tissue of the mice were determined， and the liver pathological changes were observed.
[Results] The extracts of the Zhuang medicinal herb C. buchananii could reduce the serum ALT and AST levels （P<0.05）， increase the SOD content in the liver tissue of mice （P<0.05）， and decrease the MDA level （P<0.05）.
[Conclusions] The Zhuang medicinal herb C. buchananii has a good protective effect on DGalNinduced liver injury in mice.
Key words Cryptolepis buchananii Roem. et Schult.; Dgalactosamine; Liver injury
Gugouteng refers to the rhizome of Cryptolepis buchananii Roem.et Schult. in Cryptolepis of Asclepiadaceae[1]. C. buchananii is also known as Baiyeteng， Baimalianan， Niujiaomo， Duanchangcao[2]， Banjianiu， Baiduzong and Baijiangteng[3]. It has the functions of relaxing tendons and activating collaterals， relieving swelling and pain， and relieving internal heat or fever， and is used to treat lumbago， abdominal pain， bruises， fractures， hemorrhoids， tinea and other diseases[4]. It is a common medicinal material used by the people of Guangxi Zhuang nationality[5]. Modern pharmacological studies have found that C. buchananii has pharmacological effects such as analgesic and antiinflammatory[6]， blood sugarlowering[7-8]， hypolipidemic[9]， and liver protection effects[10]. At present， there is no public report on the pharmacodynamic study of C. buchananii on chemical liver injury. In this study， a mouse model of DGalNinduced acute liver injury was established and used to explore the pharmacological effects of the Zhuang medicinal herb C. buchananii in protecting the liver. This study provides a scientific basis and theoretical guidance for the resource development and clinical application of the Zhuang medicinal herb C. buchananii. Materials and Methods
Medicinal materials
C. buchananii materials were collected from Shiwan Mountain， Guangxi， and were identified as the aboveground part of C. buchananii Roem. et Schult by pharmacist Ma from Guangxi Yixin Medical Co.， Ltd.
Drugs and reagents
Bifendate Pills （WanBangDe Pharmaceutical Group Co.， Ltd.， lot number： A02150317）; DGalN （Shanghai Ryon Biotechnology Co.， Ltd.， lot number： RT1705U508）; saline （Guizhou Tiandi Pharmaceutical Co.， Ltd.， lot number： G16071303）; kit： ALT （lot number： 20170307）， AST （lot number： 20170306）， TP （lot number： 20170426）， SOD （lot number： 20170307）， MDA （lot number： 20170306）， all purchased from Nanjing Jiancheng Bioengineering Institute.
Instruments
Infinite M200PRO fullwavelength microplate reader （TECAN， Switzerland）; Fresco 17type refrigerated highspeed centrifugator （Thermo Fisher， US）; SQP electronic analytical balance （Sartorius Scientific Instruments （Beijing） Co.， Ltd.）; RE52A rotary evaporator （Shanghai Yarong biochemical instrument factory）.
Experiment animals
Kunming mice， 18-22 g， half male and half female， SPF level， purchased from the Experimental Animal Center of Guangxi University of Chinese Medicine， license number： SCXK Gui 20090003.
Experiment methods
Preparation of test drugs
Preparation of positive drug
A certain amount of Bifendate Pills （2.7 g） was weighed and ground into fine powder， which was dissolved in water， and mixed into a suspension having a concentration of 30 mg/ml， which was stored in a refrigerator.
Preparation of C. buchananii water extract
A certain amount of the medicinal material （0.5 kg） was weighed， added with 10 times of water， and refluxextracted with heating. The extraction was performed for two times， 40 min each time. The filtrates were combined， and the solvent was evaporated to give an extract （1 g of extract equivalent to 5.68 g of crude drug）， which was stored in a refrigerator.
Preparation of C. buchananii ethanol extract
The extraction was performed for two times， 40 min each time. The filtrates were combined， degreased with petroleum ether and evaporated to give an extract （1 g of extract equivalent to 7.35 g of crude drug）， which was stored in a refrigerator.
Animal grouping
110 mice were randomly divided into 11 groups， 10 in each group， including the blank group， model group， positive control group （bifendate 600 mg/kg） and administration groups. According to the preexperiment results， it was determined that the high， medium and low dose administration groups of the water extract and ethanol extracts were 20， 10， and 5 g/kg， respectively. The blank group and the model group were intragastrically administered with the same dose of physiological saline every day， and the administration groups were intragastrically administered once a day for 10 consecutive days. Establishment of DGalNinducted liver injury model
One hour after the last administration of each group of mice， the blank group was intraperitoneally injected with 0.9% saline at 10 ml/kg， and other groups were intraperitoneally injected with DGalNsolution （500 mg/kg） at 10 ml/kg. After modeling， the mice were fasted but provided with water. After 18 h， the eyeballs were taken from the mice， and the serum was separated to detect the contents of ALT and AST in the serum; and after the mice were sacrificed， the liver was quickly dissected and the liver index was calculated. Part of the liver tissue was taken， added with 9 times of ice physiological saline， and centrifuged at 3 000 r/min for 10 min. The supernatant was taken to detect various indexes of ALT， AST， TP， SOD and MDA. The liver tissue on the large lobe of the liver was fixed with 4% paraformaldehyde and subjected to HE staining， and the degree of liver tissue damage was observed under a microscope.
Data processing
Statistical analysis was performed using SPSS 23.0 statistical software. Data results were expressed as x ±s. Oneway analysis of variance was adopted， and data of different groups were compared using t test， in which P value was calculated， and the difference was considered to be significant at P<0.05 and extremely significant at P<0.01.
Results and Analysis
Effect on liver index in mice with DGalNinduced liver injury
Compared with the blank control group， the liver index of the model group was significantly higher （P<0.01）. Compared with the model group， the liver index of the drugadministered groups （high， medium and low doses） and the positive control group were significantly lower （P<0.05）， as shown in Table 1.
Liver index=Liver weight/Mouse weight×100%
Effects on serum ALT and AST levels in the serum of mice with DGalNinduced liver injury
Compared with the model group， the serum ALT and AST levels in the drugadministered groups （high， medium and low doses）and the positive control group decreased significantly （P<0.01 in the water extract administration groups and P<0.05 in the ethanol extract administration group）. The results are shown in Table 2.
Effects on SOD and MDA in liver homogenate of DGalNinduced liver injury mice
Compared with the model group， the activity of SOD in the liver homogenate of the mice in the drugadministered groups （high， medium and low doses） and the positive control group increased significantly （P<0.01）; and the activity of MDA in the liver homogenate of the mice in the drugadministered groups （high， medium and low doses） and the positive group decreased significantly （P<0.01）. The results are shown in Table 3. Effects on pathological changes in the liver of mice with DGalNinducedliver injury
The pathological sections showed that compared with the model group， with drugadministered groups （high， medium and low doses） improved the mice with DGalNinduced liver injury to different degrees， and their liver tissue degeneration， necrosis and inflammatory cell infiltration were alleviated （P<0.05）. The results are shown in Table 4.
Agricultural Biotechnology 2020
Discussion
Dgalactosamine （DGalN） is a hepatocellular phosphate uridine interfering agent， which can cause diffuse necrosis and inflammation of the liver. The liver pathological changes caused by it are similar to clinical viral hepatitis， so it is often used in liver protection screening experiments for clinical drugs[11]. Therefore， this study established a DGalNinduced liver injury model， which was used for exploring the hepatoprotective effect of the Zhuang medicinal herb C. buchananii.
This study showed that the high， middle and low dose groups of the water and ethanol extracts of the Zhuang medicinal herb C. buchananii all could reduce the liver index， reduce the serum ALT， AS and TMDA activity in mice with liver injury， and improve SOD activity. The pathological section showed that the degree of inflammatory lesions in the liver tissue of mice was improved. This study confirms that the extract of C. buchananii can enhance the antiinjury ability of hepatocytes and has the effects of protecting liver and reducing enzyme. Its role may be to reduce the synthesis of lipid peroxides and inhibit the synthesis of plasma membrane structural proteins， thereby achieving liver protection.
References
[1] CUI YL， RAN XD， et al. Zhonghua Yaohai[M]. Harbin： Haerbin Press， 1993： 459. （in Chinese）
[2] Guangxi Institute of Chinese Medicine & Pharmaceutical Science. Guangxi medicinal plants directory （Guangxi Yaoyongzhiwu Minglu）[M]. Guangxi： Guangxi Peoples Publishing House， 1986. （in Chinese）
[3] Editorial Group of Yunnan Chinese Herbal Medicine. Yunnan Chinese herbal medicine （Yunnan Zhongcaoyao）[M]. Yunnan： Yunnan Peoples Publishing House， 2011. （in Chinese）
[4] Editorial Group of Collection of Traditional Chinese Medicine in China. Collection of traditional Chinese medicine in China[M]. Beijing： Peoples Medical Publishing House， 1976： 170. （in Chinese）. （in Chinese）
[5] HUANG RS. Zhuangyao Xianbian[M]. Nanning： Guangxi Science and Technology Press，2015： 305. （in Chinese） [6] ZHANG XS， WEN LY， QIN HL， et al. Experimental study on analgesic and antiinflammatory pharmacological effects of alcohol extract of Cryptolepis buchananii Roem. et Schult.[J]. Lishizhen Medicine and Materia Medica Research， 2009， 20（11）： 2735-2736. （in Chinese）
[7] ZHANG XS， HUANG X， PAN NY. Experimental study on the effect of Cryptolepis buchananii Roem. et Schult. extract on blood glucose in normal mice[J]. Lishizhen Medicine and Materia Medica Research， 2010， 21（10）： 2699-2700. （in Chinese）
[8] LU CL， LIANG XN， CHEN Y. effect of aqueous extracts from Cryptolepis buchananii Roem. et Schult on blood glucose in mice with experiment hyperglycemia[J]. Guangxi Medical Journal，2013，35（6）：702-704. （in Chinese）
[9] ZHANG XS， ZHANG Z， ZHOU YQ. Pharmacological experimental research of aquatic extractant from Cryptolepis buchananii on the fat lipids of mice[J]. Lishizhen Medicine and Materia Medica Research， 2013， 24（10）： 2356-2357. （in Chinese）
[10] DONG ML， PAN QD， RUAN YE， et al. Protective effects of Cryptolepis buchananii extract on acute liver injury in mice[J]. Journal of Youjiang Medical University for Nationalities， 2014， 36（3）： 358-360. （in Chinese）
[11] XU ZN， WU XM， WANG P， et al. 18α glycyrrhizic acid in the treatment of acute live injury induced by Dgalactosamine in rats[J]. Journal of Clinical Hepatology， 2012， 28（1）： 63-65. （in Chinese）