CN105147863B - Extraction method and application of total polysaccharide of Wuzi Yanzong prescription - Google Patents
Extraction method and application of total polysaccharide of Wuzi Yanzong prescription Download PDFInfo
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- CN105147863B CN105147863B CN201510502667.6A CN201510502667A CN105147863B CN 105147863 B CN105147863 B CN 105147863B CN 201510502667 A CN201510502667 A CN 201510502667A CN 105147863 B CN105147863 B CN 105147863B
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Abstract
The invention provides an extraction method for extracting total polysaccharide of a Wuzi Yanzong prescription and application of the total polysaccharide in treating diseases such as liver injury and the like. Soaking dried medicinal materials in 75% ethanol overnight, extracting with water under reflux for 2 times, mixing filtrates, concentrating under reduced pressure, adding 95% ethanol to final concentration of 60%, standing overnight, filtering, and washing precipitate with acetone to obtain crude extract of total polysaccharide of WUZI YANZONG prescription. Compared with the normal control group, the liver damage of the model control group is serious, and the liver damage degree caused by CTX can be reduced by the penta-derivative prescription total polysaccharide. Compared with a normal control group, the ALT and AST levels and the MDA content of the mice in the model control group are obviously increased, and the SOD activity is obviously reduced (P is less than 0.05); the total polysaccharide of the Wuzi Yanzong prescription can improve the degree of liver injury, remarkably reduce the ALT and AST levels and the MDA content and increase the SOD activity (P < 0.05).
Description
Technical Field
The invention relates to an extraction method of total polysaccharide of a formula of Wuzi Yanzong and application of the total polysaccharide in protecting liver injury, belonging to the field of biological medicine.
Background
The five-seed Yanzong prescription is used as a traditional Chinese herbal medicine formula in China, and consists of five medicines of semen plantaginis, raspberry, schisandra chinensis, wolfberry fruits and semen cuscutae, the wolfberry fruits with mild nature and sweet taste and the semen cuscutae with mild nature and sweet nature are used as monarch medicines, the liver and kidney tonifying yang is realized, the schisandra chinensis and the raspberry are used as ministerial medicines, the fructus schisandrae have the function of generating blood, the raspberry can nourish essence, the two medicines are warm and not hot and dry, the effects of warming and excreting essence and nourishing kidney are realized, the plantain seeds are the only sweet and slightly cold ingredients in the prescription and are used as adjuvant medicines, the effects of promoting urination and securing essence are realized, the deficiency fire in the kidney can be simultaneously relieved, and the slightly cold nature of the plantain seeds has a restraining effect on the warm nature of other medicines in the prescription.
The Wuzi Yanzong prescription has the functions of tonifying kidney and replenishing essence, replenishing essence and marrow, dredging kidney qi, and breeding the seeds and the Yanzong, is advocated by doctors of all ages, has better curative effect on male infertility, and is known as the first prescription of ancient and modern seeds. It is used for treating male genitourinary system diseases, gynecological diseases, fatty liver, nervous system, and immune system. Has various pharmacological effects of delaying aging, enhancing immunity, etc.
Cyclophosphamide (CTX) is a commonly used clinical antitumor drug, widely used for treating various solid tumors and blood system diseases, and after entering the body, the drug is decomposed under the catalysis of liver microsome enzymes to produce a product with strong alkylation effect, which can produce cytotoxicity to tumor cells, inhibit cell proliferation and induce tumor cell apoptosis. However, while inhibiting tumor cells, the compound can also affect the proliferation and differentiation of normal cells of a human body, thereby causing various side effects, and besides bone marrow inhibition, cardiotoxicity, reproductive system toxicity and the like, the compound also has hepatotoxicity, can cause liver injury and cause hepatocyte necrosis, and has a remarkable immunosuppressive effect.
Modern pharmacological research shows that the Wuzi Yanzong prescription can improve reproductive function, enhance immunologic function and resist oxygen radical injury, can be used for treating diseases such as male genitourinary system diseases, gynecological diseases, fatty liver and the like, and has various pharmacological effects of delaying aging, enhancing immunity and the like. And with the increase of the incidence rate of AD and the shortage of treatment drugs, the effective intervention of the Wuzi Yanzong prescription on MCI makes the MCI have the development prospect as a candidate drug for treating AD. The pharmacological actions of the Wuzi Yanzong pill are quite extensive, but the actions on the nervous system, the immune system, the glycometabolism and the like need to be further improved, and the pharmacological actions on the aspects of anti-inflammation, bacteriostasis, circulatory system and anti-tumor are not reported and are yet to be researched.
Disclosure of Invention
On the basis of the reports of the documents, the invention adopts the chemotherapeutics CTX for modeling, meanwhile, the total polysaccharide of the formula of.
The invention comprises an optimized process for extracting total polysaccharide in a formula of Wuzi Yanzong of a traditional Chinese medicine and a protection effect of the total polysaccharide on liver injury of mice caused by cyclophosphamide.
Drugs and reagents
The Wuzi Yanzong prescription consists of 40g of semen cuscutae, 40g of medlar, 5g of schisandra chinensis, 20g of raspberry and 10g of semen plantaginis, which are all provided by a great pharmacy of Yichanben Tang; CTX was purchased from Henry pharmaceutical, Inc., Jiangsu; alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), Superoxide dismutase (SOD), and Malondialdehyde (MDA) kits were purchased from tokyo bio-engineering corporation.
Instrument for measuring the position of a moving object
The ZNHW type intelligent constant temperature electric heating jacket is produced by ZNHW type intelligent constant temperature electric heating jacket produced by Steheny City Instrument Limited liability company; the circulating water multipurpose vacuum pump is produced by Xian Taikang biological science and technology limited company; the low-temperature cooling liquid circulating pump is produced by Zhengzhou great wall science and trade company Limited; the EYELA oil bath pan is manufactured by Shanghai Iran instruments, Inc.; an embedding machine; a centrifuge; an enzyme-labeling instrument; one in ten thousand balance.
Laboratory animal
SPF grade Balb/C male mice, 8 weeks old, supplied by the center for laboratory animals in Hubei province. Raising in cages at the ambient temperature of 20 +/-2 ℃ and the relative humidity of 55-65% under 12h illumination, feeding rodent with feed, and freely drinking water.
Method for extracting total polysaccharide of Wuzi Yanzong prescription
1. Weighing dried and pulverized fructus Lycii, semen Cuscutae, fructus Schisandrae chinensis, Rubi fructus, and semen plantaginis, adding 75% ethanol, soaking overnight, filtering with double-layer filter cloth, placing the filtered residue in a 60 deg.C oven, and volatilizing ethanol; drying, adding distilled water, reflux-extracting for 1.5h in a boiling water bath, filtering with double-layer filter cloth, repeatedly extracting filter residue once, filtering, mixing filtrates, concentrating in a water bath at 80 deg.C, repeatedly deproteinizing the concentrated solution in a mixed solution of chloroform and n-butanol at a volume ratio of 4:1, centrifuging the water layer after extraction, concentrating the supernatant in a water bath, adding 95% ethanol to a final volume fraction of 75%, precipitating with ethanol in a refrigerator at 4 deg.C overnight, centrifuging at 4800r/min for 10min, sequentially washing the filter residue with anhydrous ethanol, acetone and diethyl ether twice, and drying in a fume hood to obtain brown powder, i.e. the total polysaccharide extract of the formula of Wuzi Yanzong.
Grouping animals
Healthy male balb/c mice were taken 36 and randomly divided into 4 groups: normal control group, model control group, Wuzi Yanzong prescription total polysaccharide low dose group, total polysaccharide high dose group, each group is 9.
Liver tissue injury modeling and animal treatment
The low and high total polysaccharide dosage groups are administered 3 days in advance, starting on day 3, except for normal control group injected with normal saline of the same amount, all the other groups are injected with 50mg/kg of CTX in abdominal cavity for molding, and are administered for 1 time every 2 days for 10 times, and the administration group continues to administer the drug. After the last CTX injection, the mice were sacrificed 12 hours after fasting without water deprivation. Morphological observation of liver tissue
The liver tissue of the same part of each mouse is taken, fixed by formaldehyde, embedded by paraffin, sliced, stained by HE, and the morphological change of the liver tissue is observed by a microscope.
Liver index
The mice were weighed 12h after the last administration, the livers were removed and weighed after the mice were sacrificed, and the liver index was calculated by dividing the weight of the livers by the weight of the mice.
Serum biochemical index detection
Collecting blood from each group of mice orbit, standing for 30min, centrifuging, collecting supernatant, and detecting AST and ALT activity of serum according to AST and ALT kit.
Detection of antioxidant index
Cleaning liver tissues of each group of mice with physiological saline under ice bath condition, adding 4 ℃ physiological saline to homogenate to prepare 10% liver tissue homogenate, centrifuging at 3500r/min under 4 ℃, centrifuging, taking supernatant, centrifuging for 10min each time until the lower layer has no precipitate after centrifuging. And (4) performing operation according to the SOD and MDA kit instructions, and detecting the SOD activity and the MDA content of the liver tissue.
Statistical treatment
Data are presented as mean ± standard deviation (x ± S), differences between groups are measured by t-test, and P <0.05 indicates that the differences are statistically significant.
The total polysaccharide of the Wuzi Yanzong pill obtained by the technical scheme of the invention can obviously reduce the damage degree of liver cells caused by CTX. The mechanism may be related to the improvement of the activity of antioxidant enzymes and the level of free radical scavengers in vivo and the protection of the body from attack by oxygen free radicals. The total polysaccharide of WUZIYANZONG has protective effect on liver injury.
Drawings
FIG. 1 shows the effect of total polysaccharides from five-seed derived Zong on the liver index of mice, note: p < 0.05; p <0.01 (compared to normal control group); # P <0.05, # P <0.01 (compared to model control).
FIG. 2 is the morphological observation of liver tissues of mice in each group, wherein A is a normal control group, B is a model control group, C is a total polysaccharide low dose group, and D is a total polysaccharide high dose group.
Detailed Description
Example 1
Weighing 160g of dried medlar, 160g of semen cuscutae, 20g of schisandra chinensis, 80g of raspberry and 40g of semen plantaginis, putting into a large flask, adding 4L of 75% ethanol, soaking overnight, filtering with double-layer filter cloth, removing substances dissolved in alcohol in the formula of Wuzi Yanzong, and putting the filtered filter residue into an oven at 60 ℃ to volatilize the ethanol. Drying, adding 2.5L distilled water, reflux-extracting in boiling water bath for 1.5h, filtering with double-layer filter cloth, repeatedly extracting the residue once, filtering, mixing the filtrates, concentrating in 80 deg.C water bath to 1500mL, repeatedly deproteinizing by Sevag method (chloroform: n-butanol is 4: 1), centrifuging the water layer, concentrating the supernatant in water bath, slowly adding 95% ethanol (final volume fraction is 75%), precipitating with ethanol in a refrigerator at 4 deg.C overnight, centrifuging at 4800r/min for 10min, sequentially washing the residue with anhydrous ethanol, acetone and diethyl ether twice, and drying in a ventilated cabinet to obtain brown powder, i.e. the total polysaccharide extract of formula of Wuzi Yanzong. The extraction yield was calculated to be 1.95%.
The total weight of a prescription of Wuzi Yanzong is 115g, the average weight of an adult is 60kg, the drug administration amount of a human is 1.92g/kg, and the dosage of a mouse is about 10 times of that of the human, so the drug administration amount of the mouse is 19.2g/kg, the low dose of the total polysaccharide is 375mg/kg according to the extraction rate, and the high dose is 750 mg/kg.
Effect of Wuzi Yanzong prescription Total polysaccharide on mouse liver index
Compared with the normal control group, the mouse liver index of the model control group is obviously increased (P < 0.01), compared with the model control group, the liver index of the pentazozone total polysaccharide administration group is reduced but still higher than that of the normal control group, wherein the effect of reducing the liver index of the total polysaccharide high-dose group is extremely obvious (P < 0.01), and the effect of the total polysaccharide low-dose group is slight (P < 0.05). (results are shown in FIG. 1)
Influence of total polysaccharide of Wuzi Yanzong prescription on mouse liver histomorphology
The hepatic lobule structure of the normal control group mouse is complete and clear, and the hepatic cells are radially arranged by taking the central vein as the center and have no abnormal shape; hemorrhage, congestion and edema of central vein and hepatic sinus are not seen; fibroplasia and inflammatory cell infiltration were not seen in the hepatic venule area, and no significant hepatocyte degeneration was seen (see fig. 2A). The liver lobules and liver chordae of the mice in the model control group are disordered and difficult to distinguish; the liver cells were swollen significantly, the cytoplasm was loose, the cell structure was poorly recognized, and there was liver cell necrosis (see fig. 2B). Lesions were observed in the lobules of the livers of the total polysaccharide low dose group mice, but a portion of the lobules were well-aligned, and the hepatocytes were infiltrated with inflammatory cells, swollen hepatocytes, and loose cytoplasm (see fig. 2C). The liver lobules of the total polysaccharide high-dose group are arranged neatly, the liver cell swelling and the cytoplasm loosening degree are obviously reduced compared with those of a model control group, the liver cells are uniform in shape and regular in arrangement, and no obvious difference is generated compared with a normal control group (as shown in figure 2D).
Influence of total polysaccharides of Wuzi Yanzong prescription on serum total ALT and AST levels
Compared with the normal control group mice, the serum AST and ALT activity of the other groups of mice are obviously improved, which indicates that the liver injury model is successfully established; compared with a model control group, the activity of the serum ALT and AST of the mice in the groups with low total polysaccharide and high dose is remarkably reduced, and the result shows that the five-seed derivative formula total polysaccharide can inhibit the increase of the activity of the serum ALT and AST of the mice caused by CTX. (results are shown in Table 1)
TABLE 1 Effect of the total polysaccharides of the Wuzi Yanzong prescription on serum Total ALT and AST levels (x. + -. s)
Note: p < 0.05; p <0.01 (compared to control); # P <0.05, # P <0.01 (compared to model control).
Influence of total polysaccharide of Wuzi Yanzong prescription on SOD activity and MDA content in liver tissue of mouse
Compared with a normal control group, the liver SOD activity of the mouse of the model control group is obviously reduced, and the MDA level is obviously increased. After the total polysaccharide is given, compared with a model control group, the SOD activity of the total polysaccharide in a low-dose group and a high-dose group is obviously improved, and the MDA content is reduced, which indicates that the liver injury can be obviously improved by the total polysaccharide of the Wuzi Yanzong prescription. (results are shown in Table 2)
TABLE 2 Effect of the total polysaccharides of the Wuzi Yanzong prescription on SOD activity and MDA content in liver tissue of mice (x + -s)
Note: p < 0.05; p <0.01 (compared to control); # P <0.05, # P <0.01 (compared to model control).
CTX is a broad-spectrum anti-tumor drug, and has the function of damaging normal tissue cells in addition to killing tumor cells. Research reports that CTX can cause liver injury, increase the activity of AST and ALT in serum, increase the MDA content of liver tissues, reduce the activity of SOD, and reduce the extensive moderate-to-severe water denaturation of liver cells under a liver HE stained mirror, extrusion and unclear structure of liver sinuses, liver tissue focal necrosis and a large amount of inflammatory cell infiltration. The research reports that the Wuzi Yanzong pill can improve or protect the liver, but the effective components and the action mechanism are unknown. The research result shows that the total polysaccharide of the Wuzi Yanzong pill can obviously reduce the damage degree of the liver cells caused by CTX.
Normally, AST is mainly present in tissue cells, is present in a large amount in the liver, is present in a small amount in the serum, and causes a high concentration of AST in the serum when severe necrosis or destruction of the liver occurs. ALT is mainly present in liver cytoplasm, the content of ALT in serum is low, and the ALT content in serum is greatly increased if liver cells are necrotic or injured, so the injury degree of the liver can be judged by measuring the AST and ALT content in serum. The experimental result shows that the total polysaccharide of the Wuzi Yanzong prescription can obviously reduce the activity of AST and ALT in serum. The result shows that the total polysaccharide of the Wuzi Yanzong prescription can reduce the damage degree of liver cells. The suggestion shows that the total polysaccharide of the Wuzi Yanzong prescription has protective effect on liver injury.
SOD is an important antioxidant, can remove oxygen free radicals, can judge liver peroxidation damage by measuring SOD activity, and MDA is a product of oxidation reaction of free radicals acting on lipid, so that the measurement of MDA amount can often reflect the lipid peroxidation degree in an organism and indirectly reflects the cell damage degree. According to the experiment, under the experimental condition that CTX causes liver injury to mice, the result shows that the total polysaccharide of the Wuzi Yanzong prescription can obviously improve the liver SOD activity and reduce the MDA content of liver tissues, and the Wuzi Yanzong prescription total polysaccharide can reduce the damage degree of liver cells. The suggestion shows that the total polysaccharide of the Wuzi Yanzong prescription has protective effect on liver injury.
In conclusion, the chemotherapy drug CTX can damage liver tissues, cause pathological damage of liver cells, increase the activity of AST and ALT in serum, increase the content of MDA and reduce the activity of SOD.
Claims (1)
1. An application of total polysaccharide of Wuzi Yanzong prescription in preparing medicine for preventing and treating liver cell injury caused by cyclophosphamide, wherein in the treatment process, the dosage of the total polysaccharide of Wuzi Yanzong prescription is 750 mg/kg; the extraction method of the total polysaccharide of the Wuzi Yanzong prescription comprises the following steps: weighing dried and pulverized fructus Lycii, semen Cuscutae, fructus Schisandrae chinensis, Rubi fructus, and semen plantaginis, adding 75% ethanol, soaking overnight, filtering with double-layer filter cloth, placing the filtered residue in a 60 deg.C oven, and volatilizing ethanol; drying, adding distilled water, reflux-extracting for 1.5h in a boiling water bath, filtering with double-layer filter cloth, repeatedly extracting filter residue once, filtering, mixing filtrates, concentrating in a water bath at 80 deg.C, repeatedly deproteinizing the concentrated solution in a mixed solution of chloroform and n-butanol at a volume ratio of 4:1, centrifuging the water layer after extraction, concentrating the supernatant in a water bath, adding 95% ethanol to a final volume fraction of 75%, precipitating with ethanol in a refrigerator at 4 deg.C overnight, centrifuging at 4800r/min for 10min, sequentially washing the filter residue with anhydrous ethanol, acetone and diethyl ether twice, and drying in a fume hood to obtain brown powder, i.e. the total polysaccharide extract of the formula of Wuzi Yanzong.
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| "五子衍宗方对环磷酰胺致小鼠肝细胞DNA损伤的保护作用";黄威峰、刘苗苗、袁丁、彭奔、张长城;《中成药》;20150531;第37卷(第5期);第1093-1096页 * |
| "加味五子衍宗方及其有效部位对H2O2损伤PC12细胞的保护作用";王斌、王学美;《中国实验方剂学杂志》;20090831;第15卷(第8期);第57-60页 * |
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