Protective Effect Of Total Glycoside From Cistanche Salsa On Experimental Liver Injury

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 audrey.hu@wecistanche.com

Wang Yanfang, Zhao Jijun, Hai Pengli, Xue Peifeng, Li Hui, Li Minhui

Abstract

In this experiment, a model of alcoholic liver injury in mice was established, and biochemical indicators of liver function and liver histopathological changes were used as the inspection indicators. Investigate the protective effect of the total glycoside of cistanche salsa on experimental alcoholic liver injury. Give total glycoside of cistanche salsa high-dose (130 mg/kg) and medium-dose by gavage (100 mg/kg), low-dose (65 mg/kg), and bidentate(150 mg/kg) as the positive control drug. The results showed that the serum ALT and AST are significantly increased; SOD activity in the liver is reduced, and MDA content is increased; lipidosis of liver cells appears. The total glycoside of the cistanche salsa high dose group is effective in Improving liver function indexes and reducing lipid degeneration of liver cells. On this basis, the preliminary determination of echinacoside and acteoside content in the total glycoside of cistanche salsa was 29.1%. The above experiments show that the total glycoside of cistanche salsa has a protective effect on alcoholic liver injury in mice, and it is also confirmed that echinacoside and acteoside are the main active ingredients of the total glycoside of cistanche salsa.

Keywords: total glycoside of cistanche salsa; high-performance liquid chromatography; acteoside; echinacoside; alcoholic liver injury

Citanche salsa is a Cistanche plant of the Orobanchaceae family (Orobanchaceae), also known as salina, a perennial herbaceous parasitic plant, mainly distributed in deserts, deserts, and other dry areas. The main producing areas are in Xinjiang and Inner Mongolia. 1]. Due to the fact that Herba Cistanche of the same family and the same genus have been excavated in large quantities, and the parasitic plants are difficult to reproduce, the medicinal resources are declining day by day, and it is difficult to meet the market demand. In our country, folk cistanche salsa is used as a substitute for cistanche deserticola, but its research reports are few. In order to further clarify its medicinal value, the total glycoside of cistanche salsa (total glycosides of Cistanche salsa), a class of substances containing phenylethanoid glycosides, have been pharmacologically studied. The total glycoside of cistanche salsa has a protective effect on nerves [2] and can protect DNA from oxidative damage caused by hydroxyl (OH·) [3]. Cistanche salsa phenylethanoid glycosides can significantly increase the superoxide dismutase (SOD) activity in mouse heart, liver, and brain tissues [4]. The above research suggests that the total glycoside of cistanche salsa has an antioxidant effect.

The incidence of alcoholic liver disease (ALD) is increasing year by year in my country. Alcohol has become the second leading cause of liver damage after viral hepatitis [5]. For the treatment of alcoholic liver injury, there is a lack of safe and effective drugs. How to use plant resources to develop anti-alcohol and liver-protecting drugs and health products has attracted wide attention in the pharmaceutical and food industries. After extensive literature, it has been found that alcoholic liver injury is related to oxidative stress, and the total glycoside of cistanche salsa has an effect on alcoholic liver injury. The research on the protective effect is still blank. The research in this experiment provides a scientific basis for the development and utilization of the total glycoside of cistanche salsa.

1 Material and equipment

1. 1 Medicine and reagents

Cistanche salsa was provided by the Key Laboratory of Mongolian Medicinal Material Identification and Standardization, Baotou Medical College; Bifendate dropping pills (Zhejiang Wanbang Pharmaceutical Co., Ltd., batch number A02121139); Alanine Aminotransferase (ALT) Kit, Aspartame Acid aminotransferase (AST) kit, malondialdehyde (MDA) test kit, superoxide dismutase (SOD) kit, Coomassie brilliant blue protein determination kit (Nanjing Jiancheng Institute of Bioengineering, batch number 20110422); 56 ° Liquor (Beijing Red Star Co., Ltd.); other reagents are domestically pure.

1. 2 Animals

72 male mice of Kunming species, weighing 17-22 g (Inner Mongolia University Laboratory Animal Center, Animal Qualification Certificate No.:6 (Mongolia 2002-0001)).

1. 3 Instruments

Ultraviolet-visible spectrophotometer [Unico (Shanghai) Instrument Co., Ltd., model UV-2000]; Microplate reader (Beckman Coulter, USA, model AD340); High-speed centrifuge (Hangzhou Aipu Instrument Equipment Co., Ltd., model TG18-WS); High-Performance Liquid Chromatograph (Thermo Fisher Scientific, Model Ultimate 3000); Analytical Balance (METTLER TOLEDO Instruments Co., Ltd. Model AR153O); Ultrasonic Oscillator (Shanghai Branson, Model SB3200); Freeze desiccant (Beijing Boyikang Experimental Instrument Co., Ltd., model FD-1-50).

2 Methods and results

2. 1 Study on total glycoside of cistanche salsa activity

2. 1. 1 Total glycoside of cistanche salsa extraction

2. 1. 1. 1 Extraction of crude extract

Weigh 100 g of cistanche salsa coarse powder, add 600 mL of 70% ethanol and soak for 3 h, ultrasonically extract for 30 min, filter, and repeat the above steps twice on the filter residue, combine the filtrate and concentrate to recover ethanol until it has no alcohol taste, collect the extract and freeze-dry it to obtain cistanche salsa crude extract 19.46 g.

2. 1. 1. 2 Purification process

Place 10 g of the crude extract of cistanche salsa in a beaker, add 20 mL of water to dissolve in ultrasound, add 80 mL of absolute ethanol, shake well and place it for 12 hrs and then do filtration, the filtrate was concentrated to recover ethanol until no alcohol taste, and freeze-dried to obtain 8.408 g of the total glycoside of cistanche salsa extract.

2. 1. 2 Grouping and administration of animals

72 mice were fed adaptively for 2 days before the experiment and were randomly divided into 6 groups, each with 12 mice, namely a blank control group, an alcoholic liver injury model group, and a positive control group (150 mg/kg bidentate), Total glycoside of cistanche salsa high-dose group (130 mg/kg), the total glycoside of cistanche salsa medium-dose group (100 mg/kg) and total glycoside of cistanche salsa low-dose group (65 mg/kg). The animals in each group were given intragastric administration (ig) twice a day, and the ig volume was 0.2 mL/20 g body weight. The blank control group and the model group were given equal volumes of saline. Two hours after each administration, except for the blank control group, the other groups were given 56° liquor at 0.4 mL/20 g body weight ig for 10 consecutive days to cause an acute alcoholic liver injury model. On the 9th day of the model building, fasting was not allowed. On the 10th day, 56° white wine was given for 3 hrs. The eyeballs were taken blood and the serum was separated. After blood collection, the mice were sacrificed. Weigh, take the right lobe of the liver and place it in a 10% formaldehyde fixative solution, and place the rest of the liver in a refrigerator at -20 ℃.

2. 1. 3 Statistical processing

Use SPSS 11.5 statistical software to process data, analysis of variance, P <0.05 is statistically significant.

2. 1. 4 The effect of the total glycoside of cistanche salsa on body weight, liver weight, and liver index of acute alcoholic liver injury in mice

As shown in Table 1, the liver weight and liver index of mice in the model group were significantly higher than those in the blank control group (P<0.05), indicating liver damage. Compared with the model group, the liver weight and liver index of mice in the total glycoside of the cistanche salsa high-dose group and the positive control group were significantly reduced (P <0.01). There was no significant difference between the total glycoside of cistanche salsa medium and low-dose groups ( P>0.05).

2. 1. 5 The effect of total glycoside of cistanche salsa on serum AST and ALT

The collected blood was centrifuged at 3.5×103 r/min for 10 min to prepare serum. Using Lai's method, UV-Vis spectrophotometer 505 nm readings were used to determine the activity of ALT and AST in serum. As shown in Table 2, serum ALT and AST in the model group were significantly higher than those in the blank control group (P<0.05), indicating that the model group was successfully modeled. The serum ALT and AST of mice in the positive control group were significantly lower than those in the model group (P<0.01). Compared with the model group, the serum ALT and AST of mice in the total glycoside of the cistanche salsa high-dose group were significantly lower (P<0.01). There was no significant difference between the total glycoside of the cistanche salsa middle and low dose group and the model group (P>0.05).

2. 1. 6 The effect of the total glycoside of cistanche salsa on the MDA content and SOD activity in the liver

Take 0.3 g of liver tissue to prepare 10% and 1% homogenates. Centrifuge the 1% liver homogenate with 3.5 × 103 r/min for 10 min and take the supernatant. The solution, the protein in liver homogenate was determined by the Coomassie Brilliant Blue method. Take 1% liver homogenate supernatant, operate according to the SOD test box, and read at 450 nm on the microplate reader. Take 10% liver homogenate supernatant, operate according to the MDA determination kit, and measure the absorbance (A532) with a spectrophotometer at 532 nm.

As shown in Table 3, the MDA content in the liver of the model group was significantly higher than that in the blank control group (P<0.05), and the SOD activity was significantly reduced (P<0.05). Compared with the model group, the MDA content in the liver of the positive control group was significantly lower (P<0.01), and the activity of SOD was significantly increased (P<0.01). Compared with the model group, the MDA content of the total glycoside of the cistanche salsa high-dose group was significantly lower (P<0.01), and the activity of SOD was significantly increased (P<0.01). The total glycoside of cistanche salsa medium and low dose groups had no difference in MDA and SOD compared with the model group (P>0.05).

2. 1. 7 The effect of total glycoside of cistanche salsa on pathological changes of liver tissue in mice

Pathological examination: liver tissue fixed with 10% formaldehyde, conventional paraffin-embedded section (slice thickness 5 μm), HE staining, and observation of liver histopathological changes under the light microscope.

The liver of the blank control group is dark red in color and soft in texture. The structure of liver tissue is normal and the structure of liver lobules is intact. It is polygonal with clear boundaries, and the nucleus is round and clearly located in the center of the cell. The liver volume of the mice in the model group and the total glycoside of cistanche salsa medium and low dose groups increased and the color became yellow. The pathological changes under the microscope were mainly fatty degeneration of hepatocytes, with diffuse lipid vacuoles and hepatocytes in the cells. Large, the cytoplasm is filled with tiny lipid droplets and vacuoles, and the nucleus is still in the center. In the positive control group and the total glycoside of the cistanche salsa high-dose group, the lipidosis of liver cells in mice was reduced to a certain extent, and the number of lipid droplets was reduced to a certain extent.

2. 2 Principal component analysis of total glycoside of cistanche salsa

2. 2. 1 Chromatographic conditions

The chromatographic conditions were chromatographic column ZORBAX ODS (250×4.6 mm, 5μm); mobile phase: methanol 0.2% formic acid solution (27.5∶72.5), gradient elution; column temperature 30℃; flow rate 1 mL/min; detection wavelength 334 nm. Under the above chromatographic conditions, the separation of echinacoside and acteoside is good.

2. 2. 2 Preparation of reference solution

Accurately weigh out 1.3 mg and 1.2 mg of echinacoside and acteoside reference substance to prepare 0.13 mg/mL echinacoside reference substance solution and 0.12 mg/mL acteoside reference solution.

2. 2. 3 Preparation of test solution

Accurately weigh 0.1082 g of the total glycoside of cistanche salsa extract and dilute to 50 mL with the mobile phase solution.

2. 2. 4 Linear relationship

Precisely draw the reference solution 0. 06, 0. 12. 0. 18. 0. 24. 0. 30, 0. 48. 0. 54. 0. 72. 1. At 20 mg/mL, inject 5 μL into the liquid chromatography in sequence, and calculate the regression equation with the injection volume (μg) of the two reference substances as the abscissa and the corresponding peak area as the ordinate. echinacoside: Y = 11.868 X-0.0785, R = 0.9997; acteoside: Y = 18.026X-0.1913, R = 0.9997. As a result, the echinacoside reference substance has a good linear relationship in the range of 0.0635 to 0.4445 μg; the acteoside reference substance is in the range of 0.059 to 1.18 μg The inner linear relationship is good.

2. 2. 5 Precision test

Precisely draw 10 μL of the same test solution, and repeat the injection 5 times under the above chromatographic conditions. Record the chromatogram and determine the peak area. The result is the RSD of echinacoside 0.89%, RSD of acteoside is 1.87%, indicating that the precision of the instrument is good.

2. 2. 6 Stability test

The same test solution, respectively, at 0, 2, 4, 8, 16, 32, 40, 48 h, accurately draw 10 L, inject it into the liquid chromatograph, record the chromatogram, and measure its peak area. The result is the RSD of echinacoside 1. 58%, the RSD of acteoside is 0. 49%, indicating that the test solution is within 48 hours

The stability is good.

2. 2. 7 Repeatability test

Accurately weigh about 0.2 mg of the same batch of samples, 5 parts in total, and prepare 5 parts of the solution. According to the content determination method, accurately pipet 10 μL into the liquid chromatograph, record the chromatogram, and determine the peak area. The result is echinacoside The RSD of 1.91% and the RSD of Verbascum glycosides were 2.08%, indicating that the determination method has good repeatability.

2. 2. 8 Recovery rate test

Accurately weigh 0.2 g of a total glycoside of cistanche salsa, add a certain amount of echinacoside and acteoside reference substances respectively, prepare the test solution according to the method under 2.2.3 above, and perform the determination according to the content determination method under the specified chromatographic conditions. The result The average recovery rate of echinacoside is 98. 49%, RSD 1. 06%, the average recovery rate of acteoside is 97.35%, and the RSD is 1.45%.

2. 2. 9 Determination of samples

Precisely draw 10 μL each of the reference solution and the test solution, inject the sample, determine the chromatographic conditions above, and calculate the sample content by the external standard method. The results are shown in Table 4 The average content of echinacoside is 24.5%, and the average content of acteoside is 4.6%.

3 Discussion

Alcoholic liver damage refers to liver damage and a series of pathological changes caused by long-term heavy drinking. Long-term excessive alcohol intake can cause liver cell damage, and then develop liver fibrosis, and even liver cirrhosis [6]. Compared with the model group, the total glycoside of the cistanche salsa high-dose group in this experiment significantly reduced the activity of ALT and AST in the mouse serum, significantly reduced the content of MDA in the liver, and significantly increased the activity of SOD, which reduced the lipidosis of liver cells to a certain extent. , Reduce the number of lipid droplets. There was no significant difference between the middle and low dose group and the model group. The results of this experiment are consistent with the research results of the protective effect of the total glycosides of Cistanche on alcoholic liver injury in mice [7].

The total glycoside of cistanche salsa protects liver cell damage by reducing the permeability of the cell membrane and increasing the content of ALT and AST released into the blood; it can increase the activity of SOD and eliminate a large number of free radicals produced by alcohol in the body's metabolism. ; It can reduce the formation of MDA and prevent it from damaging the membrane structure of tissue cells [8-10]. The total glycoside of cistanche salsa has a protective effect on alcoholic liver injury, and there is a quantitative effect relationship. Checking the literature, there is no quantitative analysis of the active ingredients of total glycoside of cistanche salsa. In this paper, the content of acteoside and echinacoside, the main components of the total glycoside of cistanche salsa, was quantitatively analyzed for the first time by HPLC, which accounted for 29.1% of total glycosides. Provide a scientific basis for the development and utilization of total glycoside of cistanche salsa. Modern pharmacological studies have found that echinacoside has a protective effect on PC12 cells damaged by hydrogen peroxide and oxidative stress damage in vascular dementia rats [11,12]. Both echinacoside and acteoside have inhibitory effects on the cytotoxicity of hepatocytes induced by D GalN and TNF-α [13]. Furthermore, it is speculated that the total glycoside of cistanche salsa protects mice from alcoholic liver injury may be enhanced by echinacoside and acteoside to enhance cellular antioxidant enzyme activity, improve the antioxidant capacity of liver cells, thereby maintaining the integrity of liver cell plasma membrane, so that liver cells can function normally Defensive and compensatory functions. We will further isolate the compounds in future studies and use molecular biology techniques to study the regulation of monomeric compounds on liver cell apoptosis and their relationship with the regulation of related target genes.

references

1 Zhang HW. Studies on chemical constituents and

fingerprint of Cistanche salsa (C. A. Mey.) G. Beck. Ho hot: Inner Mongolia Medical College, MSc. 2008.

2 Tian XF, Pu XP. Phenylethanoid glycosides from Cistanches salsa inhibit apoptosis induced by methyl phenyl pyridinium ion in neurons. J Ethnopharmacol, 2005, 97: 59-63.

3 Wang XW, Jiang XY, Wu LY. Scavenging effects of glycosides of Cistanche on free radicals and its protection against OH·-induced DNA damage in vitro China Pharm J, 2001, 36: 29-32.

4 Wu HH. Preparation and antioxidant activity of phenylethanoid glycosides from Cistanche salsa. Nanjing: Nanjing Agricultural University, MSc. 2008.