CN103330733A

CN103330733A - Extraction technology of pongamia pinnata extract and use of the pongamia pinnata extract in treatment on liver injury

Info

Publication number: CN103330733A
Application number: CN 201310188871
Authority: CN
Inventors: 陈乃宏
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-05-21
Filing date: 2013-05-21
Publication date: 2013-10-02

Abstract

The invention relates to an extraction technology of pongamia pinnata extract and a use of the pongamia pinnata extract in treatment on the liver injury. The extraction technology is effectively used for researching a drug for treating the liver injury. Pongamia pinnata has a high medicinal value and root, leaves, fruits and seeds of pongamia pinnata have medicinal effects. Through the ages, in a civil field, pongamia pinnata is used for treating acute and chronic viral hepatitis and jaundice and has good effects. An effective ingredient is a Gea-Yea mixture separated from pongamia pinnata. The Gea and Yea extract can obviously improve a survival rate of a mice having a D-GalN/LPS-induced acute liver injury, can reduce liver coefficients of a mice having obstructive jaundice, can reduce ALT, AST, LDH and TBIL levels of serum, can reduce MDA and NO content of liver tissue, can relieve pathological symptoms such as necrosis, degeneration and inflammatory infiltration of liver tissue and has obvious prevention and control effects. The extraction technology is simple, economic and practical. The Gea-Yea mixture has obvious effects of improving all indexes of the liver injury, has good treatment effects and has a good application prospect.

Description

A kind of extraction process of Indian beech extract and the application in course of liver damage thereof

Technical field:

The invention belongs to biological pharmacy technical field, be specifically related to a kind of extraction process of from the vegetation water Clausena lansium (Lour.) Skeels, extracting Gea and Yea, and the application in course of liver damage.

Background technology:

Along with the progress of society and the change of people life style, such as viral hepatitis, alcoholic liver disease, hepatic disease such as drug induced hepatic injury and autoimmune liver disease, more and more common in daily life, become the thorny problem of serious harm human health.In the acute and chronic hepatopathy of nearly all type, the situation of the caused acute hepatic failure of hepatic injury accounts for half greatly.Therefore, the medicine for the treatment of hepatic injury more and more causes people's attention.The medicine that is used for the treatment of hepatic disease clinically mainly contains antiviral class, immunomodulator, hepatoprotective medicine etc., and it is of a great variety, and drug effect differs.

At present, in the protective effect that is mainly reflected in liver for clinical treatment and the experimentation of hepatic injury, and really can treat hepatic injury and promote the rare discovery of medicine of hepatocyte reparative regeneration effect.Based on the complexity of hepatic injury mechanism, Chinese medicine has certain advantage with characteristics such as its composition multiformity, multiaction approach and many target spots aspect the control hepatic injury.Report is arranged, such as a lot of middle pharmaceutically active ingredients such as gentiopicrin, Ganoderma triterpenoids, asiaticoside experimental hepatic injury is had significant protection effectiveness, therefore, the active substance of seeking the control hepatic injury from Chinese herbal medicine day by day becomes the research focus.

Indian beech Pongamia pinnata (L.) Merr. is half mangrove of pulse family (Leguminosae) Pongamia (Pongamia Vent.), the another name Semen Pongamiae Pinnatae, originate in China south, as Guangdong, Fujian, Guangxi, ground such as Yunnan, have higher medical value, but equal people's medicine such as its root, leaf, fruit and seed.Application Indian beech treatment acute and chronic hepatitis among the people and jaundice are of long duration, and think respond well, but do not see the scientific research report that has system deep.

Summary of the invention:

The objective of the invention is to: from a kind of from the vegetation water Clausena lansium (Lour.) Skeels extraction process of effective component extracting Gea, Yea, and research Gea, Yea are in the pharmacological action of hepatic injury.

One, [extraction process]

Extraction process of separating Gea, Yea from Indian beech of the present invention is:

1, merceration: water intaking Clausena lansium (Lour.) Skeels powder, with 95% soak with ethanol 2-3 days, get supernatant, reclaim ethanol, obtain extractum, add an amount of dissolved in distilled water, get water-soluble liquid;

2, extraction

2.1, get above-mentioned water-soluble liquid, repeatedly add chloroform on a small quantity, shake up, take off a layer chloroform layer, get chloroform extraction liquid;

2.2, get above-mentioned chloroform extraction liquid, repeatedly add ethyl acetate on a small quantity, shake up, get the upper strata ethyl acetate layer, extract repeatedly, combining extraction liquid reclaims and volatilizes ethyl acetate, gets acetic acid ethyl ester extract.

3, separate:

Get above-mentioned acetic acid ethyl ester extract, add silica gel (200-300 order) and mix sample, getting silica gel (200-300 order) adds chloroform and adorns post, to carry out a plate behind the chloroform-methanol gradient elution, get the clear bigger stream part of speckle behind the last sample, add silica gel (200-300 order) and mix sample, get silica gel (200-300 order), add chloroform and adorn post, behind the last sample with the chloroform-methanol gradient elution, separate activity extract Gea, Yea mixture.

Two, [pharmacological action]

Give mice above-mentioned through extracting activity extract Gea, the Yea mixture that separates by irritating stomach, adopt the chmice acute hepatic injury that D-GalN/LPS induces and set up the common bile duct ligation and cause two kinds of liver injury models of obstructive jaundice, by the serum liver functional parameter, the hepatic tissue biochemical indicator, activity extract Gea, Yea are observed to the influence of above-mentioned two kinds of hepatic injury in aspects such as histopathologic slide.(description of drawings: Fig. 1---Figure 12)

The result of above-mentioned experimental program proves:

1, extract Gea, Yea can significantly improve the survival rate of the chmice acute hepatic injury that D-GalN/LPS induces, reduce the liver coefficient, reduce Serum ALT, AST, LDH, TBIL level, reduce the content of hepatic tissue MDA, NO, alleviate pathological states such as necrosis of liver tissue, degeneration and inflammatory infiltration, prove that Gea, Yea have significant hepatoprotective effect.

2, extract Gea, Yea can significantly reduce obstructive jaundice mouse liver coefficient, reduce Serum ALT, AST, LDH, TBIL, ALP, TBA level, reduce the content of hepatic tissue MDA, NO, alleviate the hepatic tissue edema, pathological state such as bile duct proliferation and cell infiltration proves that Gea, Yea have significant preventive and therapeutic effect.

The mice form of medication of extract Gea of the present invention, Yea is gastric infusion.

Three, [beneficial effect]

The D-GalN/LPS of Fig. 1 Gea, Yea induces the influence of acute liver damage mice serum ALT, AST.(compare with the blank group, ^###P＜0.001, ^##P＜0.01; Compare * * * P＜0.001, * * P＜0.01, * P＜0.05 with model group; Mean ± SD, n=6)

The influence that the D-GalN/LPS of Fig. 2 Gea, Yea induces acute liver damage mice serum LDH.(compare with the blank group, ^###P＜0.001, ^##P＜0.01; Compare * * P＜0.01, * P＜0.05 with model group; Mean ± SD, n=6)

The influence that the D-GalN/LPS of Fig. 3 Gea, Yea induces acute liver damage mice serum TBIL.(compare with the blank group, ^###P＜0.001, ^##P＜0.01; Compare * * * P＜0.001, * * P＜0.01, * P＜0.05 with model group; Mean ± SD, n=6)

The influence that the D-GalN/LPS of Fig. 4 Gea, Yea induces acute liver damage murine liver tissue MDA.(compare with the blank group, ^##P＜0.01, ^#P＜0.05; Compare * * P＜0.01, * P＜0.05 with model group; Mean ± SD, n=6)

The influence that the D-GalN/LPS of Fig. 5 Gea, Yea induces acute liver damage murine liver tissue NO.(compare with the normal control group, ^##P＜0.01; Compare * * P＜0.01, * P＜0.05 with model group; Mean ± SD, n=6)

The D-GalN/LPS of Fig. 6 Gea, Yea induces the influence (HE, 100 *) of acute liver damage murine liver tissue pathomorphism

A: normal control group; The B:D-GalN/LPS model group; C: bicyclol group; D:Gea, Yea low dose group; Dosage group among E:Gea, the Yea; F:Gea, Yea high dose group

The D-GalN/LPS of Fig. 7 Gea, Yea induces the influence (HE, 100 *) of acute liver damage murine liver tissue pathomorphism

The influence of the obstructive jaundice hepatic injury of Fig. 8 Gea, Yea murine liver tissue outward appearance

A: sham operated rats; The B:BDL model group; C: Yinzhihuang oral liquid group; The D:Yea300mg/kg group; The E:Yea600mg/kg group; The F:Yea1200mg/kg group

The influence of the obstructive jaundice hepatic injury of Fig. 9 Gea, Yea mice serum ALT, AST.(compare with sham operated rats, ^###P＜0.001; Compare * * * P＜0.001, * * P＜0.01, * P＜0.05 with the BDL group; Mean ± SD, n=6)

The influence of the obstructive jaundice hepatic injury of Figure 10 Gea, Yea mice serum LDH, ALP, TBA, TBIL.(compare with sham operated rats, ^###P＜0.001; Compare * * * P＜0.001, * * P＜0.01, * P＜0.05 with the BDL group; Mean ± SD, n=6)

The influence of the obstructive jaundice hepatic injury of Figure 11 Gea, Yea murine liver tissue MDA, NO.(compare with sham operated rats, ^##P＜0.05; Compare * * P＜0.01, * P＜0.05 with the BDL model group; Mean ± SD, n=6)

The influence of the obstructive jaundice hepatic injury of Figure 12 Gea, Yea murine liver tissue pathomorphism (HE, 100 *)

A: sham operated rats; The B:BDL model group; C: Yinzhihuang oral liquid group; D:Gea, Yea300mg/kg group; E:Gea, Yea600mg/kg group; F:Gea, Yea1200mg/kg group

The specific embodiment:

Embodiment 1: the chmice acute liver injury model that adopts D-GalN/LPS to induce, and by the serum liver functional parameter, the hepatic tissue biochemical indicator, the influence of activity extract Gea, the hepatic injury of Yea is observed in aspects such as histopathologic slide.

Experimental technique

1. materials and methods

1.1 medicine

Extract Gea, Yea, hospital provides by Xuan Wu, Beijing; Bicyclol (Bicyclol) is available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute, lot number: 101044-201001; D-Gal (D-GalN), the precious biochemical equipment company limited of moral provides lot number: 20110406; Lipopolysaccharide (LPS), SIGMA company provides, lot number: 20110523.

1.2 laboratory animal

The C57BL/6 mice, male, body weight 18-22g, the SPF level, the Gea experimental section is available from Beijing Vital River Experimental Animals Technology Co., Ltd., certification of fitness SCXK (capital) 2009-0017; The Yea experimental section is available from Si Beifu (Beijing) laboratory animal Science and Technology Ltd., certification of fitness SCXK (capital) 2011-0004.Animal feeding is in cleaning level Animal House, and ambient temperature and relative humidity control replace the feed of freely drinking water at 224-2 ° of C and 55 ± 5%, 12h light and shade.Animal conforms and experimentizes behind the 5d, and all experimental implementation all meet the laboratory animal working specification.

1.3 key instrument and reagent

The main experimental apparatus of table 1

The main experiment reagent of table 2

1.4 laboratory animal grouping, administration and modeling

According to the preliminary experiment result, adjust Gea, Yea dose gradient, get 36 C57BL/6 mices and be divided into dosage group (1800mg/kg), Gea, Yea high dose group (2400mg/kg) and bicyclol group (300mg/kg) among normal control group, D-GalN/LPS model group, Gea, Yea low dose group (1200mg/kg), Gea, the Yea at random.Press 0.15ml/10g body weight gastric infusion 7d before the modeling of administration group, other organizes the parallel equal-volume drug solvent PEG400 that gives, 1h after the last administration, cause the acute liver damage model by the disposable injection D-GaIN in 0.15ml/10g body weight abdominal cavity (700mg/kg)/LPS (10 μ g/kg), normal control group lumbar injection equal-volume normal saline, contamination 6h handles animal.Water was can't help in the 12h fasting before animal was handled.

According to the preliminary experiment result, reduce Gea, Yea dose gradient, get 36 C57BL/6 mices and be divided into dosage group (600mg/kg), Gea, Yea high dose group (1200mg/kg) and bicyclol group (300mg/kg) among normal control group, D-GalN/LPS model group, Gea, Yea low dose group (300mg/kg), Gea, the Yea at random.Administration, modeling, the time of drawing materials are the same.

1.5 draw materials and index observing, detection

1.5.1 serum liver functional parameter

Behind the D-GalN/LPS poisoning 6h, mice is extractd eyeball get blood, blood collecting is to 1.5ml EP pipe, and room temperature leaves standstill 1h, then with the centrifugal 10min of 825g/min, and separation of serum ,-20 ° of C refrigerators are preserved.Automatic clinical chemistry analyzer is measured mice serum alanine aminotransferase (ALT), aspartate amino transferase (AST), total bilirubin (TBIL) and lactic acid dehydrogenase (LDH) level.

1.5.2 after getting blood, mice is put to death in cervical vertebra dislocation, observes liver outward appearance situation, cut open get and the weighing liver heavy, calculate the liver coefficient.

1.5.3 liver organization biochemical indicator

Get fritter hepatic tissue rinsing to the ice-cold normal saline, clean residual blood, filter paper blots, weigh, add PBS and make 10% liver tissue homogenate with automatic homogenizer, measure malonaldehyde (MDA) content and nitric oxide (NO) level in liver tissue homogenate's liquid, concrete operations are according to the test kit description.

1.5.4 liver organization PATHOMORPHOLOGICAL OBSERVATION OF PULLORUM

Get right lobe of liver, place 4% paraformaldehyde fixing rapidly, behind the fixation of tissue 24h, each group is got same position and is drawn materials, and the routine paraffin wax embedded section carries out hematoxylin-eosin (HE) dyeing, observes the hepatic tissue pathology form and changes.

The HE dyeing flow:

Dewaxing: white sheet → dimethylbenzene (I) 15min → dimethylbenzene (II) 15min

Aquation: soak 100% ethanol 5min * 2 time → 95% ethanol 5min * 1 time → 85% ethanol 5min * 1 time → 75% ethanol 5min * 1 time → dH2O5min * 2 time after the dewaxing successively

Dyeing: the rapid flush away loose colour → dH2O of Harris Lignum Sappan seminal fluid 5min → tap water washes slightly → and 75% acidic alcohol differentiation (observing under the mirror) → tap water flushing section washes slightly to returning indigo plant → dH2O → Yihong ethanol liquid 30s.

Mounting: 1 of 70% ethanol 3min * 2 time → 80% ethanol 3min * 2 time → 90% ethanol 3min * 1 time → 100% ethanol 3min * 1 time → dimethylbenzene 2min * 1 time → mountant (resinene)

1.6 statistical procedures

Adopt SPSS13.0 software to add up, relatively adopt independent T-test, result to represent with Mean ± SD between group, P＜0.05 is thought and is had significant difference.

2. experimental result

2.1 liver outward appearance situation and liver coefficient

Normal control group liver is bronzing, and lustrous surface is even, the quality softness, and marginal area is sharp keen; D-GalN/LPS model group liver presents atropurpureus, lose former glossyly, quality becomes fragile, the edge passivation, obviously enlargement and congestion Gea, the visible liver situation of Yea pretreated group are the improvement of dose dependent, with the liver outward appearance of high dose group near the normal control group; Gea, each dosage group of Yea pretreatment all present and improve effect preferably, and all close to the normal control liver, naked eyes are difficult to conclude the difference between each group; Positive drug bicyclol group mouse liver is compared no significant difference with the normal control group.

The liver coefficient is the situation that can objectively react liver damage, as can be seen from Table 3, the liver coefficient of D-GalN/LPS model group mice obviously increases, compare with the normal control group, has significant difference (P＜0.001), hepatic injury takes place in prompting, and Gea, Yea pretreated group liver coefficient are the reduction of dose dependent, and middle and high dosage group contrast model group has significant difference; Gea, each dosage group of Yea pretreatment all can reduce the liver coefficient, and no significant difference between each dosage group.

Table 3 extract Gea, the D-GalN/LPS of Yea induce the influence of acute liver damage mouse liver coefficient

Compare ##P＜0.01 with normal group; Compare * * * P＜0.001, * * P＜0.01, * P＜0.05 with model group; Mean ± SD, n=6

2.2 serum liver functional parameter

2.2.1Gea, the D-GalN/LPS of the Yea influence of inducing acute liver damage mice serum ALT, AST

ALT and AST are two important indicators estimating liver function.As shown in Figure 2, D-GalN/LPS model group mice serum ALT, AST compare with the normal control group, significantly raise, and the prompting liver damages; But the reduction ALT of Gea, Yea pretreated group dose dependent, AST level, significantly (Fig. 1 A), points out the hepatocyte of Gea, Yea to have protective effect with middle and high dosage group effect; Gea, each dosage group of Yea pretreatment all can reduce ALT, AST level, but do not present dose-dependence, between each group and no significant difference (Fig. 1, B), prompting Gea, Yea hepatocyte plays a protective role.

2.2.2 the influence that the D-GalN/LPS of extract Gea, Yea induces acute liver damage mice serum LDH

As shown in Figure 2, Serum LDH raises rapidly behind modeling 6h, and the prompting liver damage discharges a large amount of LDH and goes into blood.But the reduction Serum LDH level of Gea, Yea pretreated group dose dependent, significantly (Fig. 2 A), points out the hepatocyte of Gea, Yea to play a protective role with middle and high dosage group effect; Gea, the middle and high dosage group of Yea pretreatment can significantly reduce the Serum LDH level, and (Fig. 2, B), the hepatocyte of prompting Gea, Yea plays a protective role.

2.2.3Gea, the D-GalN/LPS of the Yea influence of inducing acute liver damage mice serum TBIL

Clinically, TBIL is mainly used in the diagnosis of jaundice and the discriminating of jaundice degree.As shown in Figure 3, behind the lumbar injection D-GalN/LPS6h, serum T BIL namely raises and is more than 3 times of normal control group, and the prompting hepatocyte is acute impaired, bilirubin metabolism generation obstacle.But the reduction serum T BIL level of Gea, Yea pretreated group dose dependent, significantly (Fig. 3 A), points out the hepatocyte of Gea, Yea to have protective effect with middle and high dosage group effect; Gea, each dosage group of Yea pretreatment all can significantly reduce serum T BIL level, and (Fig. 3, B), the hepatocyte of prompting Gea, Yea has protective effect.

2.3 hepatic tissue MDA, NO level

2.3.1Gea, the D-GalN/LPS of the Yea influence of inducing acute liver damage murine liver tissue MDA

MDA is the index of measuring for lipid peroxidation.As shown in Figure 4, get MDA showed increased in the D-GalN/LPS model group murine liver tissue, prompting liver generation lipid peroxidation.But the level of MDA in the reduction hepatic tissue of Gea, Yea pretreated group dose dependent, significantly (Fig. 4 A), points out Gea, Yea to have lipoid peroxidization resistant with middle and high dosage group effect; Gea, each dosage group of Yea pretreatment all can significantly reduce hepatic tissue MDA level, and (Fig. 4, B), prompting Gea, Yea have lipoid peroxidization resistant.

2.3.2 the influence that the D-GalN/LPS of extract Gea, Yea induces acute liver damage murine liver tissue NO

As shown in Figure 5, after D-GalN/LPS handled 6h, the NO level obviously raise in the murine liver tissue; The NO level of Gea, Yea processed group presents the reduction of dose dependent, with the reduction effect of middle and high dosage group significantly (Fig. 5, A); The NO level of Gea, Yea processed group all shows decline, and no significant difference between three dosage groups (Fig. 5, B).

2.4 liver organization PATHOMORPHOLOGICAL OBSERVATION OF PULLORUM

2.4.1 the D-GalN/LPS of extract Gea, Yea induces the morphologic influence of acute liver damage mouse liver histopathology

Liver section HE coloration result as shown in Figure 6, normal control group Mouse Liver leaflet structure is clear, liver rope marshalling is radial centered by central vein, hepatocyte is normal, the portal area does not have inflammatory cell infiltration, hepatic sinusoid does not normally have hyperemia; The disorder of D-GalN/LPS model group Mouse Liver leaflet structure distinguishes unclear, and liver is sought the meaning from, hepatocyte diffuse necrosis, visible dark painted eosinophilic body, and portal area cell infiltration, hepatic sinusoid are obviously expanded and a large amount of erythrocyte alluvials are arranged.Gea, Yea pretreated group murine liver tissue form present the improvement of dose dependent, Gea, Yea low dose group effect are undesirable, the visible hepar damnification situation of dosage group makes moderate progress among Gea, the Yea, the lobules of liver clear in structure, hepatic necrosis is rare, the hepatic sinusoid expansion alleviates the erythrocyte alluvial and reduces accidental cell infiltration; Gea, the visible hepar damnification situation of Yea high dose group are significantly improved, the lobules of liver clear in structure, and liver rope marshalling, hepatocyte does not have necrosis, the basic normal no erythrocyte alluvial of hepatic sinusoid.Equally matched with bicyclol group protection effect.Prompting extract Gea, the variation of the D-GalN/LPS inducing mouse of Yea acute liver damage pathomorphism have significant improvement effect.

2.4.2 the D-GalN/LPS of extract Gea, Yea induces the morphologic influence of acute liver damage mouse liver histopathology

Liver section HE coloration result as shown in Figure 7, normal control group Mouse Liver leaflet structure is clear, liver rope marshalling is radial centered by central vein, hepatocyte is normal, the portal area does not have inflammatory cell infiltration, hepatic sinusoid does not normally have hyperemia; The disorder of D-GalN/LPS model group Mouse Liver leaflet structure distinguishes unclear, and liver is sought the meaning from, hepatocyte diffuse necrosis, visible dark painted eosinophilic body, and portal area cell infiltration, hepatic sinusoid are obviously expanded and a large amount of erythrocyte alluvials are arranged.Pretreated each the dosage group of Gea, Yea all shows good improvement effect, the lobules of liver clear in structure as seen, the hepatic sinusoid expansion alleviates, no erythrocyte alluvial in the blood sinus, the portal area cell infiltration obviously reduces, and prompting extract Gea, the variation of the D-GalN/LPS inducing mouse of Yea acute liver damage pathomorphism have significant improvement effect.

Embodiment 2 comes the hepatic injury of simulating human obstructive jaundice by preparation common bile duct ligation mouse model, give extract Gea, Yea and irritate stomach processing mice, then execute the common bile duct ligation, inquire into the influence of extract Gea, the common bile duct ligation of Yea mice serum biochemical indicator and liver organization pathologic structure.

Experimental technique:

1. materials and methods

1.1 medicine

Extract Gea, Yea, hospital provides by Xuan Wu, Beijing; Yinzhihuang oral liquid, Shuanghe High-Tech. Natural Medicine Co., Ltd., Beijing, lot number: 272451.

1.2 laboratory animal

The C57BL/6 mice, male, body weight 18-22g, the SPF level, available from Si Beifu (Beijing) laboratory animal Science and Technology Ltd., certification of fitness SCXK (capital) 2011-0004.Animal feeding is in cleaning level Animal House, and ambient temperature and relative humidity control replace the feed of freely drinking water at 224 ± 2 ° of C and 55 ± 5%, 12h light and shade.Animal conforms and experimentizes behind the 7d.

1.3 instrument and reagent

Table 4 experimental apparatus

Table 5 experiment reagent

1.4 the foundation of laboratory animal grouping, administration and obstructive jaundice liver injury model

Get 20 C57BL/6 mices and be divided into the normal control group at random, BDL-12h, 24h, 48h, 72h group are implemented the common bile duct ligation, and subsequent experimental best model condition is determined in the detection of drawing materials when each organizes corresponding time point.The 12h fasting be can't help water, the postoperative routine feeding before the art.

1.4.1 extract Gea, the grouping of Yea laboratory animal, administration

Get 36 C57BL/6 mices and be divided into dosage group (600mg/kg), Gea, Yea high dose group (1200mg/kg) and Yinzhihuang oral liquid group (150mg/kg) among BDL sham operated rats, BDL model group, Yea low dose group (300mg/kg), Gea, the Yea at random.Press 0.15ml/10g body weight gastric infusion 5d before the modeling of administration group, 1 time/d, other organizes the parallel equal-volume drug solvent Polyethylene Glycol (PEG400) that gives, and water is can't help in the 6h fasting before the 5d administration, execute the common bile duct ligation behind the administration 2h, the BDL sham operated rats is opened abdomen dissociate common bile duct but not ligation.The conventional feed drinking-water of postoperative continued administration in per 24 hours, and totally 2 times, the 6h fasting be can't help water, the processing of drawing materials behind the administration 2h before the last administration.

1.5 draw materials and index observing, detection

1.5.1 serum liver functional parameter

When organizing corresponding time point, mice is extractd eyeball get blood, blood collecting is to 1.5ml EP pipe to each, and room temperature leaves standstill 1h, then with the centrifugal 10min of 3000r/min, and separation of serum ,-20 ° of C refrigerators are preserved.Automatic clinical chemistry analyzer is measured mice serum alanine aminotransferase (ALT), aspartate amino transferase (AST), total bilirubin (TBIL), lactic acid dehydrogenase (LDH), alkali phosphatase (ALP) and TOTAL BILE ACID TBA (TBA) level.

1.5.2 after animal is got blood, open abdomen record liver cosmetic variation, get liver, weigh, calculate the liver coefficient.

1.5.3 liver organization biochemical indicator

After getting blood, mice is put to death in the cervical vertebra dislocation, advancing abdomen from former operative incision cuts open and gets liver and weigh, get fritter hepatic tissue rinsing to the ice-cold normal saline, clean residual blood, filter paper blots, weigh, add PBS and make 10% liver tissue homogenate with automatic homogenizer, measure malonaldehyde (MDA) content and nitric oxide (NO) level in liver tissue homogenate's liquid, concrete operations are according to the test kit description.

1.5.4 liver organization PATHOMORPHOLOGICAL OBSERVATION OF PULLORUM

Get right lobe of liver, place 4% paraformaldehyde fixing rapidly, behind the fixation of tissue 24h, each group is got same position and is drawn materials, and the routine paraffin wax embedded section carries out hematoxylin-eosin (HE) dyeing, observes the hepatic tissue pathology form and changes.HE flow process concrete grammar is induced the acute liver damage experiment with D-GalN/LPS.

1.6 statistical procedures

2, the influence of the obstructive jaundice hepatic injury of extract Gea, Yea, by following some proved:

2.1 body surface feature

BDL sham operated rats mice active situation is good, and hair color is shiny black; BDL model group mice active situation is good substantially, weight loss, and activity is poor slightly, and urine yellow and Excreta are thinning, and hair color tarnishes, and ear edge, ball portion, tail point place begin jaundice, and the prompting jaundice symptom occurs; Contrast BDL model group, each dosage group of Gea, Yea all can to a certain degree be improved the mice state, better with the middle and high dosage group of Gea, Yea effect, the mice vigor is near sham operated rats, ear edge, ball portion and afterbody xanthochromia are not obvious, perusal and Yinzhihuang oral liquid group do not have significant difference, and showing the biliary obstruction of extract Gea, Yea and causing jaundice symptom has certain improvement effect.

Observe and the liver coefficient 2.2 liver is directly perceived

Liver outward appearance situation as shown in Figure 8, BDL sham operated rats mouse liver is bronzing, lustrous surface is even, the quality softness, marginal area is sharp keen, it is transparent light yellow that the gallbladder size normally is; A large amount of yellow specklees appear in BDL model group mouse liver surface, and surface irregularity is smooth, present the edema sample and change, and liver edge is blunt thick, and gallbladder expands visible a large amount of cholestasis, and the hepatic injury of prompting obstructive jaundice occurs; Perusal, each dosage group of Gea, Yea is all improved the liver situation, remarkable with dosage group effect among Gea, the Yea, liver increases but color is tending towards normal, the edge is blunt thick, surface red color visible [sample point but do not have yellow speckle and occur, effect and Yinzhihuang oral liquid group do not have significant difference, point out the obstructive jaundice hepatic injury of Gea, Yea tool to have some improvement.

The liver coefficient is the situation that can objectively react liver damage, as can be seen from Table 6, the liver coefficient 6.04 ± 0.67% of BDL model group mice, with the apparent in view increase of sham operated rats, has significant difference (P＜0.001), damage modeling success takes place in the prompting hepatic injury, each dosage group of Gea, Yea all can reduce the liver coefficient, obvious with dosage group effect among Gea, the Yea, drop to 5.24 ± 0.68% (P＜0.05), near the Yinzhihuang oral liquid group, prompting extract Gea, Yea are, and the liver damage situation has some improvement.

The influence of table 6 extract Gea, Yea BDL mice body weight and liver

Compare ###P＜0.001 with sham operated rats; Compare * * P＜0.01, * P＜0.05 with the BDL group; Mean ± SD, n=6

2.3 the influence of the obstructive jaundice hepatic injury of extract Gea, Yea mice serum liver function index

As shown in Figure 9, BDL model group mice serum ALT, AST level significantly raise, show BDL after liver cell sustain damage; Compare with the BDL model group, each dosage group of Gea, Yea all can reduce Serum ALT, AST level, but does not present dose-dependence, with dosage group effect among Gea, the Yea remarkable (P＜0.001, P＜0.05)

As Figure 10, shown in the A, BDL model group mice serum LDH significantly raises, and the prompting hepatocyte sustains damage, and each dosage group of Gea, Yea all can reduce the Serum LDH level, with the middle and high dosage group of Gea, Yea effect remarkable (P＜0.01, P＜0.05).

As Figure 10, shown in the B, BDL model group mice serum ALP significantly raises, and shows that the cholestasis sexually transmitted disease (STD) has taken place to be become, and the middle and high dosage group of Gea, Yea can significantly reduce ALP level (P＜0.05, P＜0.05).

As Figure 10, shown in the C, compare with the BDL sham operated rats, BDL model group mice serum TBA raises nearly 160 times, shows that the cholestasis sexually transmitted disease (STD) has taken place to be become, and liver function is obviously impaired after the prompting obstructive jaundice, and bile acid concentration significantly raises.The middle and high dosage group of Gea, Yea can significantly reduce serum T BA level (P＜0.01, P＜0.01).

As Figure 10, shown in the D, BDL model group mice serum TBIL significantly raises behind BDL48h, shows that hyperbilirubinemia has taken place mice.Each dosage group of Gea, Yea all can reduce the total bilirubin level, with the middle and high dosage group of Gea, Yea effect comparatively obviously (P＜0.01, P＜0.01).

2.4 MDA, NO content in the liver organization

As shown in figure 11, the MDA in the BDL model group murine liver tissue is showed increased, prompting liver generation lipid peroxidation.The dosage group can significantly reduce the level (P＜0.05) of MDA in the hepatic tissue among Gea, the Yea, and prompting Gea, Yea have lipoid peroxidization resistant.

As Figure 11, shown in the B, the NO in the BDL model group murine liver tissue significantly raises, and the basic, normal, high dosage group of Gea, Yea can significantly reduce the level (P＜0.05, P＜0.01, P＜0.01) of NO in the hepatic tissue, and prompting Gea, Yea can suppress the generation of NO.

2.5 liver organization pathological change

Liver section HE coloration result as shown in figure 12, BDL sham operated rats mice: the lobules of liver clear in structure, liver rope marshalling is radial centered by central vein, hepatocyte is normal, the portal area does not have inflammatory cell infiltration, hepatic sinusoid does not normally have hyperemia; BDL model group mice: the lobules of liver structure disturbance distinguishes unclear, liver seek the meaning from, as seen a large amount of cell infiltration in portal area, as seen bile duct epithelial cell hypertrophy, little bile duct proliferation appears in the portal area, swelling of liver cell, and endochylema is bright, the liver rope is because pressurized is arranged irregularity, and hepatic sinusoid and central vein expansion also have a small amount of erythrocyte alluvial; The liver structure of Gea, Yea processed group mice is clearly better, it is more undesirable that Gea, Yea low dose group hepatic tissue improve situation, significantly improve and the middle and high dosage group of Gea, Yea is visible, the lobules of liver clear in structure, liver rope marshalling, the portal area cell infiltration obviously reduces, and the little bile duct proliferation in bile duct epithelial cell hypertrophy and portal area alleviates, swelling of liver cell is rare, and hepatic sinusoid and central vein degrees of expansion are significantly improved.

Claims

1. the extraction process of an Indian beech extract and the application in course of liver damage thereof, it is characterized in that: extraction process is as follows:

1.1, merceration: water intaking Clausena lansium (Lour.) Skeels powder, with 95% soak with ethanol 2-3 days, get supernatant, recovery ethanol obtains extractum, adds an amount of dissolved in distilled water, gets water-soluble liquid;

1.2, the extraction: get above-mentioned water-soluble liquid, repeatedly add chloroform on a small quantity, shake up, take off a layer chloroform layer, get chloroform extraction liquid;

1.3, get above-mentioned chloroform extraction liquid, repeatedly add ethyl acetate on a small quantity, shake up, get the upper strata ethyl acetate layer, extract repeatedly, combining extraction liquid reclaims and volatilizes ethyl acetate, gets acetic acid ethyl ester extract;

1.4, separate: get above-mentioned acetic acid ethyl ester extract, add silica gel (200-300 order) and mix sample, getting silica gel (200-300 order) adds chloroform and adorns post, to carry out a plate behind the chloroform-methanol gradient elution, get the clear bigger stream part of speckle behind the last sample, add silica gel (200-300 order) and mix sample, get silica gel (200-300 order), add chloroform and adorn post, behind the last sample with the chloroform-methanol gradient elution, separate activity extract Gea, Yea mixture.

2. Gea as claimed in claim 1, Yea are in the pharmacological action of hepatic injury, it is characterized in that: (1) extract Gea, Yea can significantly improve the survival rate of the chmice acute hepatic injury that D-GalN/LPS induces, reduce the liver coefficient, reduce Serum ALT, AST, LDH, TBIL level, reduce the content of hepatic tissue MDA, NO, alleviate pathological states such as necrosis of liver tissue, degeneration and inflammatory infiltration, prove that Gea, Yea have significant hepatoprotective effect; (2) extract Gea, Yea can significantly reduce obstructive jaundice mouse liver coefficient, reduce Serum ALT, AST, LDH, TBIL, ALP, TBA level, reduce the content of hepatic tissue MDA, NO, alleviate the hepatic tissue edema, pathological state such as bile duct proliferation and cell infiltration proves that Gea, Yea have significant preventive and therapeutic effect.