CN106389879A - Application of Dendrobium nobile total alkaloids - Google Patents
Application of Dendrobium nobile total alkaloids Download PDFInfo
- Publication number
- CN106389879A CN106389879A CN201611044766.5A CN201611044766A CN106389879A CN 106389879 A CN106389879 A CN 106389879A CN 201611044766 A CN201611044766 A CN 201611044766A CN 106389879 A CN106389879 A CN 106389879A
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- herba dendrobii
- total alkaloid
- liver
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- dendrobii total
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- 239000012498 ultrapure water Substances 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 201000001862 viral hepatitis Diseases 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K2236/30—Extraction of the material
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- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
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Abstract
The invention discloses an application of Dendrobium nobile total alkaloids in the preparation of medicines for preventing or treating acute and chronic liver injury diseases. The Dendrobium nobile total alkaloids have prevention and treatment effects on CCL4, alcohol or D-galactose induced acute liver injuries and CCL4 induced chronic liver injuries. The Dendrobium nobile total alkaloids are used to obviously reduce the activity of ALT and AST in serum, substantially reduce the content of MDA in liver homogenate and improve the expression of Nrf2, Nqo1 and Ho-1mRNA. A result of liver tissue slice observation shows that after the Dendrobium nobile total alkaloids are used, the liver tissue lesion necrosis disappears, the liver lobule structure is basically complete, and the inflammatory cell infiltration is obviously mitigated.
Description
Technical field
The present invention relates to a kind of application of Herba Dendrobii total alkaloid, particularly belong to pharmaceutical technology field.
Background technology
Hepatic disease is the disease of a class serious harm human health, and sickness rate is high, including viral hepatitis, liver cirrhosis,
Fatty liver, alcoholic liver, drug-induced liver disease, genetic liver etc., these hepatopathys are easy in Acute onset, the chronic acute outbreak course of disease
Hepatic injury, especially acute liver damage occur.There is no the unified definition of acute liver damage at present, in the broadest sense, any reason is led
Cause liver function index abnormal, all prompting hepatic injury (American National clinic can be recommended, ALT, AST be higher than normal limit it is contemplated that
Acute liver damage).The common cause causing acute liver damage has：1st, infect：Common with each Hepatitis virus, secondly non-hepatitis
Poison；2nd, drug intoxication：Acetaminophen, carbon tetrachloride, ethanol etc.；3rd, ischemia or congested circulatory disturbance：Pericardial effusion, the heart
Decline, suffer a shock；4th, immune dysfunction：Common autoimmune hepatitiss；5th, metabolic disease：Acute fatty liver of pregnancy, liver bean
Shape core becomes core, hereditary tyrosinemia etc.；Other such as liver neoplasm, liver cirrhosis etc..China is a hepatopathy big country, hepatopathy
Sickness rate occupy the 3rd, the world.
Acute liver damage is clinical common hepatopathy, and the state of an illness is in progress further and may occur in which hepatic insufficiency, hepatic encephalopathy, sternly
Severe one may occur in which death.Therefore, acute liver damage early diagnosiss, early treatment are successful keys.Damage currently for hepatopathy
Treatment be mainly Drug therapy, clinically hepatic has motassium magnessium aspartate, reductive glutathione, tiopronin, sweet
Oxalic acid diamidogen etc., hepatoprotective effect mechanism is relevant with antiinflammatory, protection liver plasma membrane and metabolism, during said medicine uses, there is individuality
Difference is big, the gastrointestinal untoward reaction such as blood pressure change, abdominal distention, erythra, heating, Nausea and vomiting occurs using rear some patients.With
The use of some novel antivirals, immunoregulation agent, cytokine, the treatment of hepatopathy obtains certain development in recent years, but still
There is the problems such as expensive, individual variation is big, limit its application in clinical liver disease.China's Chinese Traditional Medicine
Rich experience is had to accumulate in terms for the treatment of hepatic disease.Substantial amounts of research has shown that Chinese herbal medicine in anti-hepatocyte injury is treated
There is good effect and unique advantage.
China's Chinese herbal medicine resource enriches, and the advantages of untoward reaction is few, and has too many levels, multi-level, Mutiple Targets comprehensively to make
Feature, possesses the advantage of uniqueness in terms for the treatment of hepatopathy.Efficient, low toxicity treatment liver is found therefore from traditional Chinese herbal medicine
The newtype drug of dirty disease has become the focus of research.
Dendrobium nobile (Deudrobium nobile Lindl) is orchid family Dendrobium Sw, is China's multi-section medical book simply
On rare Chinese medicine all on the books, enjoy the good reputation of " Herba mesonae chinensis ".Its chemical composition mainly has alkaloidss, phenols, polysaccharide, phenanthrene
Class etc., alkaloid and polysaccharide are main pharmacological components.Modern pharmacological research shows, Herba Dendrobii has lipid-loweringing, reduces blood pressure,
Adjust the effect such as immunity.In prior art, have no that Herba Dendrobii total alkaloid is used for preparing Prevention or treats acute and chronic liver
The relevant report of damage disease medicine.
Content of the invention
For solving the deficiencies in the prior art, it is an object of the invention to provide a kind of application of Herba Dendrobii total alkaloid,
Herba Dendrobii total alkaloid will be applied to preparation prevention or treat in acute and chronic liver damage disease medicine.
In order to realize above-mentioned target, the present invention adopts the following technical scheme that：
Herba Dendrobii total alkaloid in preparation prevention or treats the application in acute and chronic liver damage disease medicine.
In the application of aforementioned Herba Dendrobii total alkaloid, one of Herba Dendrobii total alkaloid preparation method, is by following
Prepared by step：Take Dendrobium nobile pulverizing medicinal materials to coarse powder, with alcohol at normal temperature extraction, filter after obtaining lixiviating solution, decompression recycling ethanol,
Volatilize to extractum no alcohol taste, be subsequently added hydrochloric acid and fully dissolve, after petroleum ether extraction, adjust pH, then extracted with dichloromethane,
Combining extraction liquid, decompression and solvent recovery, obtain final product.
Specifically, in the application of aforementioned Herba Dendrobii total alkaloid, Herba Dendrobii total alkaloid is through the following steps that make
Standby：Take Dendrobium nobile pulverizing medicinal materials to coarse powder, the 95% alcohol at normal temperature extraction 3～4 being meal quality 10～12 times amount with quality
Secondary, 15～20 days every time, filter after merging lixiviating solution, decompression recycling ethanol, volatilize to extractum no alcohol taste, being subsequently added volume is
The mass concentration of extractum volume 3～5 times amount is that 5% hydrochloric acid fully dissolves, and after petroleum ether extraction, adjusts pH to 9～11, then
With dichloromethane extraction, combining extraction liquid, decompression and solvent recovery, obtain final product.
Preferably, in the application of aforementioned Herba Dendrobii total alkaloid, Herba Dendrobii total alkaloid is through the following steps that make
Standby：Take Dendrobium nobile pulverizing medicinal materials to coarse powder, be meal quality 10 times amount with quality 95% alcohol at normal temperature extract four times, often
Secondary 20 days, filter after merging lixiviating solution, decompression recycling ethanol, volatilize to extractum no alcohol taste, being subsequently added volume is extractum volume 3
The mass concentration of times amount is that 5% hydrochloric acid fully dissolves, and after petroleum ether extraction, adjusts pH to 10, then with dichloromethane extraction,
Combining extraction liquid, decompression and solvent recovery, obtain final product.
In the application of aforementioned Herba Dendrobii total alkaloid, the two of Herba Dendrobii total alkaloid preparation method, it is by following
Prepared by step：After depletion hairpin stem of Dendrobium coarse powder, plus ethanol immersion, boil extraction, filter, filtrate is condensed into extractum, uses hydrochloric acid
Aqueous dissolution, filter, filtrate cross cation exchange resin (732, Hydrogen, blade diameter length ratio 1:8), it is washed with deionized water removing impurities matter,
Use acidic ethanol eluting again, eluent is concentrated into no alcohol taste, plus alkali neutralization, through desalting processing, lyophilization, obtain final product purity higher
Herba Dendrobii total alkaloid extract.
Specifically, in the application of aforementioned Herba Dendrobii total alkaloid, Herba Dendrobii total alkaloid is through the following steps that make
Standby：Depletion hairpin stem of Dendrobium coarse powder, plus quality be meal quality 10～12 times amount 90% ethanol soak 12～16 hours, boil and carry
Take 2～4 times, 2～3 hours every time, filter, filtrate is merged and is condensed into extractum, the filtrate volume of merging is the 120 of extractum volume
Times；Subsequently use pH to be 3.5 aqueous hydrochloric acid solution dissolvings, filter, filtrate is crossed cation exchange resin, is washed with deionized water removing impurities matter,
Use acidic ethanol eluting again, eluent is concentrated into no alcohol taste, plus alkali neutralization, through desalting processing, lyophilization, obtain final product purity higher
Herba Dendrobii total alkaloid extract.
Preferably, in the application of aforementioned Herba Dendrobii total alkaloid, Herba Dendrobii total alkaloid is through the following steps that make
Standby：Depletion hairpin stem of Dendrobium coarse powder, plus quality be meal quality 10 times amount 90% ethanol soak 12 hours, boil extraction 3 times, often
Secondary 2 hours, filter, filtrate is condensed into extractum, be 3.5 aqueous hydrochloric acid solution dissolvings with pH, filter, filtrate crosses cation exchange tree
Fat, is washed with deionized water removing impurities matter, then uses acidic ethanol eluting, and eluent is concentrated into no alcohol taste, plus alkali neutralization, at desalination
Reason, lyophilization, obtain final product the higher Herba Dendrobii total alkaloid extract of purity.
Using two kinds of Herba Dendrobii total alkaloid preparation methoies of the present invention, with method phase in ZL200910301762.4
Gained Herba Dendrobii total alkaloid amount lifts 15-20% to ratio.
In the application of aforementioned Herba Dendrobii total alkaloid, medicine is using Herba Dendrobii total alkaloid as active component, plus
Enter tablet, capsule, granule, soft capsule, drop pill, syrup, sublingual lozenge or the note that pharmaceutical carrier or excipient are made
Penetrate agent.
In the application of aforementioned Herba Dendrobii total alkaloid, tablet is prepared by following steps：Using wet granule compression tablet, press
According to parts by weight meter, 1 part of Herba Dendrobii total alkaloid xeraphium and 3 parts of mixings of starch add the shallow lake that mass fraction is 10%
Slurry soft material, the citric acid being 1% containing mass fraction in starch slurry, it is allowed to gently to hold agglomerating, light pressure and dissipates, cross 16 mesh sieve series
Grain, by wet grain in 40 DEG C～60 DEG C dryings, with 16 mesh sieve granulate, and adds quality to be that the Pulvis Talci that granular mass 5% is obtained mixes
After even, direct compression obtains final product.
In the application of aforementioned Herba Dendrobii total alkaloid, capsule is prepared by following steps：Dendrobium stem is total
Alkali dried powder and the soluble starch that quality is 5 times of dry powder quality mix, and after crossing 100 mesh sieves, fill by hand to No. 5 capsules
Obtain final product.
In the application of aforementioned Herba Dendrobii total alkaloid, granule is prepared by following steps：In parts by weight, take
1 part of Herba Dendrobii total alkaloid xeraphium, 3 parts of cane sugar powder and 1.25 parts of mixings of dextrin, add 60% ethanol soft material, are allowed to light
Hold agglomerating, light pressure to dissipate, cross 16 mesh sieves and pelletize, 60 DEG C of -80 DEG C of dryings, with 16 mesh sieve granulate, sealing obtains final product.
In order to ensure the science, effectively of technical solution of the present invention, inventor has carried out a series of experiment.
First, the preparation method of Herba Dendrobii total alkaloid
1st, experiment material and instrument
1.1st, medical material and reagent
Medical material is purchased from Chishui Dendrobium nobile, is identified as the drying of Dendrobium nobile Dendrobium nobile Lindl.
Stem.Glacial acetic acid (analyzes pure, Shanghai Shen Bo Chemical Co., Ltd., lot number 1202101), C.I. 13020. (C15H15N3O2:Shanghai three love is thought
Reagent company limited), bromocresol green (C21H14Br4O5S：Shanghai San'aisi Reagent Co., Ltd.), dichloromethane (Chongqing Chuanjiang River
Learn chemical reagent work, lot number:20030106), related alkaloids universal visualization agent (bismuth potassium iodide, dilute bismuth potassium iodide, improvement bismuth iodide
Potassium) it is pure, the petroleum ether (30-60 DEG C) etc. of analysis.
1.2nd, key instrument
Q-Exactive high-performance desk-top level Four bar-track trap UPLC-MS/MS (HESI ion source) system (U.S.
Thermo company)；Genius 1022 liquid nitrogen generator (PEAK company of Britain)；The ultrapure water purification system of the MILLI-Q (U.S.
Millipore company)；Circulating water type vacuum pump (Yuhua Instrument Co., Ltd., Gongyi City)；(Shanghai is sub- flourish for Rotary Evaporators
Biochemical instrument factory)；Thermostat water bath (Shanghai Yarong Biochemical Instrument Plant)；Balance (ten thousand/, the limited public affairs of upper current chart level table
Department) etc..
2 methods and result
The extraction of 2.1 Herba Dendrobii total alkaloid
Weigh and Dendrobium nobile pulverizing medicinal materials are dried to coarse powder, 95% alcohol solvent being meal quality 10 times amount with quality is normal
Warm macerating carries four times, each 20d, merges lixiviating solution, filters, Rotary Evaporators decompression recycling ethanol, volatilizes to extractum no ethanol flavor.
Gained extractum adds 5% hydrochloric acid that volume is extractum volume 3 times amount volume fully to dissolve, and after petroleum ether extraction, acid solution adds carbon
Sour soda is to pH 10, then with dichloromethane extraction, combining extraction liquid, decompression and solvent recovery, obtains total alkaloidss test sample.With
When weigh, and calculate yield.First medical material 10Kg, obtains total alkaloids extract dry cream 50g；Second batch medical material 10Kg, obtains golden
Hairpin Herba Dendrobii biology total alkali extract dry cream 49.4g.
Depletion hairpin stem of Dendrobium coarse powder, plus quality be meal quality 10 times amount 90% ethanol soak 12 hours, boil extraction 3
Secondary, 2 hours every time, filter, filtrate is merged and is condensed into extractum, the filtrate volume of merging is 120 times of extractum volume；Subsequently use
PH is 3.5 aqueous hydrochloric acid solution dissolvings, filters.Filtrate cross cation exchange resin (732, Hydrogen, blade diameter length ratio 1:8), use deionization
Water elution impurity, then use acidic ethanol eluting, eluent is concentrated into no alcohol taste.Plus alkali neutralization, through desalting processing, lyophilization,
Obtain the higher Herba Dendrobii total alkaloid extract of purity.
2nd, DNLA is to CCl4The impact of the acute hepatic injury mice model of induction
1st, experiment material
1.1st, key instrument
721 type ultraviolet-visible spectrophotometers (Shanghai Shen Hua instrument automatic control company), -80 DEG C of low temperature refrigerator (SANYO GSs
Company), Milli QA pure water processor (Millipore company), 3K30 type High speed refrigerated centrifuge (German Sigma company),
BSS-110 electronic analytical balance (Beijing Sai Duolisi electronic balance company limited), SHHW electric heating constant temperature three water-bath (north
Capital bright Medical Instruments factory forever), Leica cytoflurography (German Leica Microsystems Ltd) etc..
1.2 medicines and reagent
Dendrobium nobile total alkaloidss (DNLA) are provided by the pharmacology emphasis building by province and ministry Ministry of Education of Zunyi Medical College laboratory and (contain
Measure as 89.5%), it is made into desired concn with after 1% tween 80 hydrotropy before use.CCL4(the Chongqing City Kowloon chemistry examination of analysis alcohol
Agent factory), Oleum Arachidis hypogaeae semen, Coomassie brilliant blue, bovine serum albumin (Solarbio company), TNF-a (ELISA) test kit
(Biotechnology systems), malonaldehyde (MDA), superoxide dismutase (SOD) measure examination box (biology is built up in Nanjing
Graduate School of Engineering).
1.3rd, laboratory animal
C57 mice, 6～8 week old, SPF level, male, body weight 18 ± 4g, by great Ping hospital of Third Military Medical University laboratory animal
Center provides (licence：2012-0011).Feeding environment is quiet, room temperature 22-23 DEG C, and illumination/dark time is circulated for 12/12h,
Each group experiment mice ad lib, drinking-water.
2nd, experimental technique
2.1st, animal packet, administration and molding
C57 mice 40, is randomly divided into 4 groups, every group 10, i.e. blank group (distilled water+1% tween 80), model group
(distilled water+1% tween 80), DNLA low dosage (10mg/kg), DNLA high dose group (20mg/kg).Using gastric infusion
(0.1mL/10g, every 10g Mouse Weight is administered 0.1mL), once a day, successive administration after 7 days, in addition to blank group, note by equal abdominal cavity
Penetrate CCl4Acute liver model processed.Mice fasting 12 hours, free water before experiment.Except blank group is with solvent (Semen Maydiss
Oil) injection, remaining each group 0.1mL/10g dosage lumbar injection 0.2%CCl4Solution sets up acute hepatic injury model.In molding 24
Claim Mouse Weight after hour, take blood, cut hepatic tissue and detect for index of correlation.
2.2nd, the mensure of mouse liver weight in wet base index
Weigh before sacrifice, place after death takes out liver, weighs, liver index=liver weight (g)/Mouse Weight (g) ×
100%2.3rd, ALT and AST Activity determination in mice serum
Mouse orbit takes blood, separates serum, measures serum alt (phenylalanine ammonia group-transfer using full automatic biochemical apparatus
Enzyme), AST (aspartate amino transferase) activity.
2.4, mouse liver Histomorphological
Each mice same area hepatic tissue is taken to be fixed in 4% formalin, routine paraffin wax embedded section, hematoxylin-she
Red colouring (Hemaltoxylin and eosin, HE), light Microscopic observation is simultaneously taken pictures.
2.5th, malonaldehyde (MDA) content, the mensure of superoxide dismutase (SOD) activity in liver organization
Weigh hepatic tissue blocking 0.3～0.5g and put into the normal saline adding pre-cooling in the small beaker of 10mL, grinding is made
10% liver tissue homogenate.Low-temperature and high-speed centrifuge 3000r/min centrifugation 10min, Aspirate supernatant, to be checked.
2.6th, the mensure of hepatic tissue protein content
10% liver tissue homogenate's liquid normal saline dilution becomes 1%, draws this diluent 200 μ L with liquid-transfering gun and has in 5mL
Plug test tube in, add 0.1mg/mLBrandford developer 4.0mL, with eddy blending machine mix, with distilled water with method as sky
In vain, measure at 595nm after room temperature places 5min and absorb angle value, substitute into regression equation calculation protein content.
2.7th, in hepatic tissue mda content mensure
Principle：MDA can be combined with thiobarbituricacidα-, forms red product.This red product has maximum suction at 532nm
Receive peak.Experiment takes 10% liver tissue homogenate, is loaded by step in following table 1：
The mensure of table 1 hepatic tissue protein content
Swirl mixing device fully mixes, 95 DEG C of water-bath 40min, flowing water cooling after taking-up, then 3500r/min, centrifugation
10min, takes supernatant, at 532nm, 1cm optical path cuvette, and distilled water returns to zero, and surveys each pipe OD value.Counted according to following equation (1)
Calculate MDA value.
MDA (nmol/mgprot)=(measuring pipe OD value-standard blank tube OD value)/(standard pipe OD value-standard is blank
Pipe OD value) × standard pipe solubility (nmol/mL)/sample protein content (mg/mL) formula (1)
2.8th, the mensure of hepatic tissue total number born (SOD) activity
Principle：Superoxide dismutase has narrow spectrum inhibitory action to ultra-oxygen anion free radical, makes the Asia of formation
Nitrate reduces, and nitrite assumes aubergine under developer effect, mensuration absorbance can obtain its vigor.Say agent by examination
Box operational approach is carried out.And SOD value is calculated according to following equation (2).
SOD (U/mgprot)=(control tube OD value-measure pipe OD value)/control tube OD value/50% × reactant liquor is overall
Amass/take liquid measure (mL)/sample protein content (mg/mL) formula (2)
2.9th, Nrf2 in Real-time PCR detection hepatic tissue, Nqo1, Ho-1 gene expression
Take same area liver organization 50-100mg to be placed in 1mL Trizol, extract total serum IgE, measure its concentration, according to
Kit specification is extracted and purifying RNA, and surveys its absorbance at 260-280nm respectively, and A260/A280 ratio is in 1.8-
Represent in the range of 2.0 that purity is more satisfactory.Reverse transcription is for detecting Nrf2, Nqo1, Ho-1 gene using fluorescence quantitative PCR method after cDNA
Expression, primer sequence is shown in Table 2.
Table 2 real-time fluorescence quantitative PCR primer sequence
3rd, result
3.1st, DNLA is to CCl4The acute hepatic injury mice model liver index impact of induction
Test result indicate that：Compare with blank group, CCl4The liver wet weights index of model group mice substantially increases (P<
0.05).Compare with model group, DNLA is low, high dose group liver coefficient significantly reduces (P<0.05)；DNLA low dose group liver system
Count compared with model group, no significant difference (P ＞ 0.05).The results are shown in Table 3.
Table 3 is to CCl4The impact (Mean ± SE) of the acute hepatic injury mice liver index of induction
Note：Compare with blank group, #P ＜ 0.05；Compare with model group：* P ＜ 0.05,
3.2nd, DNLA is to CCl4The acute hepatic injury mice Serum ALT of induction, the impact of AST
Result shows, model group is compared with blank group, all significantly raised (P of ALT, AST activity in mice serum<0.05)；
Compare with model group, each administration group of DNLA all substantially reduces CCl4ALT, AST activity (P in caused hepatic injury mice serum<
0.05), the results are shown in Table 4.
Table 4 DNLA is to CCl4In caused hepatic injury mice serum ALT, AST activity impact (N=10)
Note：Compare with blank group,#P ＜ 0.05；Compare with model group：* P ＜ 0.05
3.3rd, DNLA pathological impact on acute hepatic injury murine liver tissue
Liver tissue slices, HE dyes, light Microscopic observation, and the liver structure of the visible mice of blank group is normal, and cell membrane is complete
Whole, liver rope queueing discipline.Model group is shown in central vein to the Focal in the form of sheets necrosis of surrounding, and central vein and portal area occur more
Unrestrained property cell infiltration, lobuli hepatis structure is destroyed.The Focal necrosis of hepatic tissue of DNLA administration group disappears, lobuli hepatis structure base
This is complete, and inflammatory cell infiltration substantially mitigates, the slight edema (see Fig. 1) of rarely seen central vein peripheral cell.
3.4th, MDA content, the impact of SOD activity in the acute hepatic injury mice hepatic tissue that DNLA induces to CCL4
Compare with blank control group, the significantly raised (P of liver injury model group MDA content<0.01), SOD activity substantially reduces
(P<0.01)；Compare with model group, DNLA high and low dose group all can significantly reduce liver homogenate MDA content (P<0.01)；SOD lives
All significantly raised (the P of property<0.01), the results are shown in Table 5.
Table 5 DNLA is to CCl4Impact (the Mean of MDA content and SOD activity in the acute hepatic injury mice hepatic tissue of induction
±SE)
Note：Compare with blank group, #P ＜ 0.05；Compare with model group：* P ＜ 0.05, * * P<0.01
3.5th, DNLA is to Nrf2 in acute hepatic injury mice hepatic tissue, the impact of Nqo1, Ho-1mRNA expression
Result shows, model group Nrf2, and the expression of Nqo1, Ho-1mRNA is significantly lower than blank group (P<0.05), DNLA gives
Medicine group Nrf2, Nqo1, Ho-1mRNA expression is significantly higher than model group (P<0.05), result is shown in Fig. 2-4.
3rd, the acute hepatic injury mice model impact that DNLA induces on D-galactosamine
1st, material and method
Experiment material, packet and the same Section 2 of medication.The method of modeling is that lumbar injection 6.5%D- galactosamine is molten
Liquid 10mL/kg, modeling 24h posterior orbit takes blood, separates serum, measures ALT, AST and measures, calculates liver coefficient.
2nd, experimental result
Result shows, compares with blank group, and model group serum alt, AST are significantly higher than blank group (P<0.01)；With mould
Type group compares, and DNLA is low, high dose group ALT, AST value is significantly lower than liver injury model group (P<0.05, (P<0.01).Prompting
Mice serum ALT, AST that DNLA causes to D-galactosamine hepatic injury raises has obvious inhibitory action.Liver coefficient is each
No significant difference (P between group>0.05).The results are shown in Table 6.
The impact (n=10, χ ± s) to D-galactosamine hepatic injury mice serum ALT, AST and liver coefficient for table 6 DNLA
Note：Compare with blank group:**P<0.01；Compare with model group:#P<0.01.
4th, the impact to the acute hepatic injury mice model of ethanol induction
1st, material and method
Packet and the same Section 2 of medication, the method for modeling be every afternoon gavage give 56 degree of Beijing strong, colourless liquor distilled from sorghum
0.1mL/10g, continuous 7d.Modeling 24h posterior orbit takes blood, separates serum, measures ALT, AST and calculates liver coefficient.
2nd, experimental result
Result shows, compares with Normal group, and model group mice serum ALT, AST are significantly higher than blank group (P<
0.05)；Compare with model group, DNLA administration group mice serum ALT, AST value is all significantly lower than liver injury model group (P<0.05).
Prompting DNLA raises to hepatic injury mice serum transaminase caused by ethanol has obvious inhibitory action, but between liver coefficient each group
No significant difference (P>0.05), the results are shown in Table 7.
The impact (n=10, χ ± s) to alcoholic hepatic injury mice serum ALT, AST activity and liver coefficient for table 7 DNLA
Note：Compare with blank group：*P<0.05；Compare #P with model group<0.05
5th, DNLA is to CCl4The impact of caused chronic hepatic injury rat model
1st, experiment material
1.1st, laboratory animal：Male SD (Spragye-Dawley) rat, cleaning grade, the month at monthly age 3-4, body weight about 160-
180g, provides (licence by great Ping hospital of Third Military Medical University Experimental Animal Center：SCXK- army 2007-017).
1.2nd, medicine and reagent and the same Section 2 of equipment.
2nd, experimental technique
2.1st, experiment packet and administration：
Healthy SD rat 80, male and female dual-purpose, body weight 180 ± 10g, 18～25 DEG C of raising temperature, feed under experimental situation
After supporting one week, rat is randomly divided into 6 groups, every group 10, i.e. blank control group, model group, Herba Dendrobii total alkaloid is low,
Middle and high dosage group, positive controls.Every morning, gavage concentration was 10mg/kg, 20mg/kg, 40mg/kg respectively, blank
Group and model group equal gavage distilled water daily, positive controls daily gavage bifendate 100mg/kg.
2.2nd, modelling：
Test the 1st day, remaining each group equal subcutaneous injection 40%CCl in addition to blank control group4Vegetable oil solution 5ml/kg, with
Afterwards every 3-4 days subcutaneous injection 3mL/kg.Each group all starts simultaneously in modeling, continuous 45 days.In the 45th day with 2% penta bar ratios
Animal, Testing index is put to death after appropriate sodium (45mg/kg) intraperitoneal injection of anesthesia.
2.3rd, Biochemical Indexes
Serum AST, ALT activity are measured on automatic clinical chemistry analyzer；Measure tissue SOD with xanthine oxidase to live
Property, measure MDA content in hepatic tissue with thiobarbituricacidα- method, in hepatic tissue, protein content is measured with Coomassie Brilliant Blue, institute
Some assay methods are carried out by test kit operating instruction.
2.4th, histopathologic slide is observed
Take rat same area hepatic tissue, fixed with 10% formalin solution, specimens paraffin embedding slices, conventional H E contaminates
Color, light Microscopic observation liver histopathology changes.
2.5th, the statistical analysiss of data
Experimental data is counted with SPSS13.0 statistical software, and all data represent with (Mean ± SE), and multigroup is compared and adopt
With ONE-WAY ANOVA process, variance is together using LSD method, heterogeneity of variance Games-Howell method, P<0.05 has statistics to anticipate
Justice.
3rd, experimental result
3.1st, DNLA causes the impact of chronic hepatic injury rat blood serum ALT, AST activity to CCl4
Result shows, with model group rats serum alt, AST activity apparently higher than normal group (P<0.05), with model group
Relatively, each dosage group of DNLA (10,20,40mg/kg) all can substantially reduce rat blood serum ALT, AST activity (P<0.05), result
It is shown in Table 8.
Table 8 DNLA is to CCl4The impact (n=8, x ± s) of caused chronic hepatic injury rat blood serum AST and ALT activity
Note：Compare with blank control group, * P<0.05, * * P<0.01；Compare #P with model group<0.05.
3.2nd, DNLA is to CCl4Cause the pathological impact of rat chronic Liver Tissue
HE dyes, and light Microscopic observation is visible, and Normal group murine liver tissue structure is normal, the no pathology such as degeneration, necrosis
Change, and CCl4Group liver tissues of rats structure visible different degrees of degeneration and necrosis under light microscopic, the obvious hypertrophy of fibrous tissue,
Wherein hepatic cell fattydegeneration, part of hepatocytes has the change of bubble sample, and part of hepatocytes core is big, deep dye, and karyokinesiss picture is clear to wait inflammation
Sexual cell infiltrates, and the hepatic necrosis of each dosage group of DNLA, degeneration and inflammatory cell infiltration degree all substantially mitigate.
The invention has benefit that：A kind of application of Herba Dendrobii total alkaloid that the present invention provides, i.e. Dendrobium nobile Lindl stone
Dry measure used in former times biology total alkali in preparation prevention or treats the application in acute and chronic liver damage disease medicine.Herba Dendrobii total alkaloid pair
CCL4, the acute liver damage that ethanol or D- galactose are induced, CCL4Caused chronic hepatic injury is respectively provided with preventive and therapeutic effect.
(1) for CCl4The acute liver damage of induction, it is bright that Herba Dendrobii total alkaloid enables to liver coefficient after using
Aobvious minimizing, hence it is evident that reducing serum alt, AST activity, significantly reduces MDA content in hepatic tissue, SOD activity is all significantly raised,
And Nrf2 in hepatic tissue can be significantly improved, Nqo1, Ho-1mRNA express.Observed by liver tissue slices, using Dendrobium nobile
After biology total alkali, the Focal necrosis of hepatic tissue disappears, and lobuli hepatis structure is substantially complete, and inflammatory cell infiltration substantially mitigates.
(2) for ethanol induction acute liver damage, Herba Dendrobii total alkaloid use after, reduce serum alt,
AST value, raises to ethanol induced Acute serum of hepatic injury transaminase and has obvious inhibitory action.
(3) for the acute liver damage of D-galactosamine induction, after Herba Dendrobii total alkaloid uses, reduce in serum
ALT, AST value, the Serum ALT that D-galactosamine hepatic injury is caused, AST raise has obvious inhibitory action.
(4) for CCl4Caused chronic hepatic injury, Herba Dendrobii total alkaloid apply after, reduce serum alt,
AST activity, to hepatic necrosis, degeneration and inflammatory cell infiltration degree all have obvious mitigation effect.
Brief description
Fig. 1 is the impact figure (HE 200 ×) to murine liver tissue morphological change for the DNLA of the present invention；
Fig. 2 be DNLA to the impact figure of Nrf2 expression in murine liver tissue ()；
Fig. 3 be DNLA to the impact figure of Nqo1 expression in murine liver tissue ()；
Fig. 4 be DNLA to the impact figure of Ho-1mRNA expression in murine liver tissue ()；
The implication of in figure reference：Fig. 1：A- blank group, B- model group, C-DNLA low dose group (DNLA-L), D-
DNLA high dose group (DNLA-H)；Fig. 2～Fig. 4：Compare with blank group, #P ＜ 0.05；Compare with model group：* P ＜ 0.05.
Specific embodiment
Below in conjunction with specific embodiment, the present invention is further introduced.
Embodiment 1
Herba Dendrobii total alkaloid in preparation prevention or treats the application in acute and chronic liver damage disease medicine.Medicine is
Using Herba Dendrobii total alkaloid as active component, add the tablet that pharmaceutical carrier or excipient are made.Tablet is by following step
Rapid preparation：Using wet granule compression tablet, in parts by weight, 1 part of Herba Dendrobii total alkaloid xeraphium is mixed for 3 parts with starch
Even, add the starch slurry soft material that mass fraction is 10%, the citric acid being 1% containing mass fraction in starch slurry, be allowed to light
Hold agglomerating, light pressure to dissipate, cross 16 mesh sieves and pelletize, by wet grain in 40 DEG C～60 DEG C dryings, with 16 mesh sieve granulate, and add the quality to be
After the Pulvis Talci of prepared granular mass 5% mixes, direct compression obtains final product.Herba Dendrobii total alkaloid is through the following steps that make
Standby：Take Dendrobium nobile pulverizing medicinal materials to coarse powder, extract 3 times with 95% alcohol at normal temperature that quality is meal quality 11 times amount, every time
18 days, filter after merging lixiviating solution, decompression recycling ethanol, volatilize to extractum no alcohol taste, being subsequently added volume is 4 times of extractum volume
The mass concentration of amount is that 5% hydrochloric acid fully dissolves, and after petroleum ether extraction, adjusts pH to 10, then with dichloromethane extraction, closes
And extract, decompression and solvent recovery, obtain final product.The usage of tablet and consumption are：Gastric infusion, in terms of Herba Dendrobii total alkaloid,
Low dosage 10mg/kg, high dose 20mg/kg.
Embodiment 2
Herba Dendrobii total alkaloid in preparation prevention or treats the application in acute and chronic liver damage disease medicine.Medicine is
Using Herba Dendrobii total alkaloid as active component, add the capsule that pharmaceutical carrier or excipient are made.Capsule pass through with
Lower step preparation：Herba Dendrobii total alkaloid dried powder and quality are mixed for the soluble starch of 5 times of dry powder quality, mistake
After 100 mesh sieves, fill by hand and obtain final product to No. 5 capsules.Herba Dendrobii total alkaloid is through the following steps that prepare：Depletion hairpin stone
Dry measure used in former times pulverizing medicinal materials, to coarse powder, are extracted 3 times with 95% alcohol at normal temperature that quality is meal quality 12 times amount, 15 days every time, merge leaching
Filter after extract, decompression recycling ethanol, volatilize to extractum no alcohol taste, be subsequently added the quality that volume is extractum volume 5 times amount dense
Spend the hydrochloric acid for 5% fully to dissolve, after petroleum ether extraction, adjust pH to 9, then with dichloromethane extraction, combining extraction liquid, subtract
Pressure recycling design, obtains final product.The usage of capsule and consumption are：Gastric infusion, in terms of Herba Dendrobii total alkaloid, low dosage
10mg/kg, high dose 20mg/kg.
Embodiment 3
Herba Dendrobii total alkaloid in preparation prevention or treats the application in acute and chronic liver damage disease medicine.Medicine is
Using Herba Dendrobii total alkaloid as active component, add the granule that pharmaceutical carrier or excipient are made.Granule pass through with
Lower step preparation：In parts by weight, 1 part of Herba Dendrobii total alkaloid xeraphium, 3 parts of cane sugar powder and 1.25 parts of dextrin is taken to mix
Even, add 60% ethanol soft material, be allowed to gently to hold agglomerating, light pressure and dissipate, cross 16 mesh sieves granulations, 60 DEG C of -80 DEG C of dryings, use 16 mesh
Sieve granulate, sealing obtains final product.Wherein, Herba Dendrobii total alkaloid is through the following steps that prepare：Take Dendrobium nobile pulverizing medicinal materials extremely
Coarse powder, be meal quality 10 times amount with quality 95% alcohol at normal temperature extract four times, 20 days every time, merge lixiviating solution after mistake
Filter, decompression recycling ethanol, volatilize to extractum no alcohol taste, being subsequently added the mass concentration that volume is extractum volume 3 times amount is 5%
Hydrochloric acid fully dissolves, and after petroleum ether extraction, adjusts pH to 10, then is extracted with dichloromethane, combining extraction liquid, recovered under reduced pressure is molten
Agent, obtains final product.The usage of granule and consumption are：Gastric infusion, in terms of Herba Dendrobii total alkaloid, low dosage 10mg/kg, high agent
Amount 20mg/kg.
Embodiment 4
Herba Dendrobii total alkaloid in preparation prevention or treats the application in acute and chronic liver damage disease medicine.Medicine is
Using Herba Dendrobii total alkaloid as active component, add the soft capsule that pharmaceutical carrier or excipient are made.Wherein, Dendrobium nobile
Biology total alkali is through the following steps that prepare：Depletion hairpin stem of Dendrobium coarse powder, plus quality be meal quality 11 times amount 90% ethanol
Soak 16 hours, boil extraction 2 times, 2.5 hours every time, filter, filtrate is merged and is condensed into extractum, the filtrate volume of merging is
120 times of extractum volume；Subsequently use pH to be 3.5 aqueous hydrochloric acid solutions dissolvings, filter, filtrate crosses cation exchange resin, spend from
Sub- water elution impurity, then use acidic ethanol eluting, eluent is concentrated into no alcohol taste, plus alkali neutralization, and through desalting processing, freezing is dry
Dry, obtain final product the higher Herba Dendrobii total alkaloid extract of purity.The usage of soft capsule and consumption are：Gastric infusion, with Dendrobium nobile Lindl
Herba Dendrobii biology total alkali meter, low dosage 10mg/kg, high dose 20mg/kg.
Embodiment 5
Herba Dendrobii total alkaloid in preparation prevention or treats the application in acute and chronic liver damage disease medicine.Medicine is
Using Herba Dendrobii total alkaloid as active component, add the drop pill that pharmaceutical carrier or excipient are made.Wherein Dendrobium nobile
Biology total alkali is through the following steps that prepare：Depletion hairpin stem of Dendrobium coarse powder, plus quality be meal quality 12 times amount 90% ethanol
Soak 13 hours, boil extraction 4 times, 3 hours every time, filter, filtrate is merged and is condensed into extractum, the filtrate volume of merging is leaching
120 times of paste volume；Subsequently use pH to be 3.5 aqueous hydrochloric acid solution dissolvings, filter, filtrate is crossed cation exchange resin, used deionization
Water elution impurity, then use acidic ethanol eluting, eluent is concentrated into no alcohol taste, plus alkali neutralization, through desalting processing, lyophilization,
Obtain final product the higher Herba Dendrobii total alkaloid extract of purity.The usage of drop pill and consumption are：Gastric infusion, with Dendrobium nobile
Biology total alkali meter, consumption is 10mg/kg～20mg/kg.
Embodiment 6
Herba Dendrobii total alkaloid in preparation prevention or treats the application in acute and chronic liver damage disease medicine.Medicine is
Using Herba Dendrobii total alkaloid as active component, add the syrup that pharmaceutical carrier or excipient are made.Wherein, Dendrobium nobile
Biology total alkali is through the following steps that prepare：Depletion hairpin stem of Dendrobium coarse powder, plus quality be meal quality 10 times amount 90% ethanol
Soak 12 hours, boil extraction 3 times, 2 hours every time, filter, filtrate is merged and is condensed into extractum, the filtrate volume of merging is leaching
120 times of paste volume；Subsequently use pH to be 3.5 aqueous hydrochloric acid solution dissolvings, filter, filtrate is crossed cation exchange resin, used deionization
Water elution impurity, then use acidic ethanol eluting, eluent is concentrated into no alcohol taste, plus alkali neutralization, through desalting processing, lyophilization,
Obtain final product the higher Herba Dendrobii total alkaloid extract of purity.The usage of syrup and consumption are：Gastric infusion, with Dendrobium nobile
Biology total alkali meter, consumption is 10mg/kg～20mg/kg.
In embodiment 1～6, medicine can also be using Herba Dendrobii total alkaloid as active component, add pharmaceutical carrier or
Sublingual lozenge or injection that excipient is made.
Claims (9)
1. Herba Dendrobii total alkaloid in preparation prevention or treats the application in acute and chronic liver damage disease medicine.
2. Herba Dendrobii total alkaloid according to claim 1 application it is characterised in that：Described dendrobium stem is total
Alkali is through the following steps that prepare：Take Dendrobium nobile pulverizing medicinal materials to coarse powder, with alcohol at normal temperature extraction, filter after obtaining lixiviating solution,
Decompression recycling ethanol, volatilizes to extractum no alcohol taste, is subsequently added hydrochloric acid and fully dissolves, and after petroleum ether extraction, adjusts pH, then uses
Dichloromethane extracts, combining extraction liquid, and decompression and solvent recovery obtains final product.
3. Herba Dendrobii total alkaloid according to claim 2 application it is characterised in that：Described dendrobium stem is total
Alkali is through the following steps that prepare：Take Dendrobium nobile pulverizing medicinal materials to coarse powder, be meal quality 10～12 times amount with quality
95% alcohol at normal temperature extracts 3～4 times, 15～20 days every time, filters, decompression recycling ethanol, volatilize to extractum after merging lixiviating solution
No alcohol taste, is subsequently added the hydrochloric acid that the mass concentration that volume is extractum volume 3～5 times amount is 5% and fully dissolves, extracted with petroleum ether
After taking, adjust pH to 9～11, then with dichloromethane extraction, combining extraction liquid, decompression and solvent recovery, obtain final product.
4. Herba Dendrobii total alkaloid according to claim 1 application it is characterised in that：Described dendrobium stem is total
Alkali is through the following steps that prepare：After depletion hairpin stem of Dendrobium coarse powder, plus ethanol immersion, boil extraction, filter, filtrate is condensed into
Extractum, with aqueous hydrochloric acid solution dissolving, filters, filtrate crosses cation exchange resin, be washed with deionized water removing impurities matter, then with acidity second
Alcohol eluting, eluent is concentrated into no alcohol taste, plus alkali neutralization, through desalting processing, lyophilization, obtains final product.
5. Herba Dendrobii total alkaloid according to claim 4 application it is characterised in that：Described dendrobium stem is total
Alkali is through the following steps that prepare：Depletion hairpin stem of Dendrobium coarse powder, plus the 90% ethanol leaching that quality is meal quality 10～12 times amount
Bubble 12～16 hours, boils extraction 2～4 times, 2～3 hours every time, filters, and filtrate is merged and is condensed into extractum, the filtrate of merging
Volume is 120 times of extractum volume；The aqueous hydrochloric acid solution dissolving being subsequently 3.5 with pH, filters, filtrate crosses cation exchange tree
Fat, is washed with deionized water removing impurities matter, then uses acidic ethanol eluting, and eluent is concentrated into no alcohol taste, plus alkali neutralization, at desalination
Reason, lyophilization, obtain final product.
6. Herba Dendrobii total alkaloid according to claim 1 application it is characterised in that：Described medicine is with Dendrobium nobile Lindl stone
Dry measure used in former times biology total alkali as active component, adds pharmaceutical carrier or tablet that excipient is made, capsule, granule, soft capsule, drips
Pill, syrup, sublingual lozenge or injection.
7. Herba Dendrobii total alkaloid according to claim 6 application it is characterised in that：Described tablet is by following step
Rapid preparation：Using wet granule compression tablet, in parts by weight, 1 part of Herba Dendrobii total alkaloid xeraphium is mixed for 3 parts with starch
Even, add the starch slurry soft material that mass fraction is 10%, the citric acid being 1% containing mass fraction in starch slurry, be allowed to light
Hold agglomerating, light pressure to dissipate, cross 16 mesh sieves and pelletize, by wet grain in 40 DEG C～60 DEG C dryings, with 16 mesh sieve granulate, and add the quality to be
After the Pulvis Talci of prepared granular mass 5% mixes, direct compression obtains final product.
8. Herba Dendrobii total alkaloid according to claim 6 application it is characterised in that：Described capsule passes through following
Prepared by step：Herba Dendrobii total alkaloid dried powder and quality are mixed for the soluble starch of 5 times of dry powder quality, crosses 100
After mesh sieve, fill by hand and obtain final product to No. 5 capsules.
9. Herba Dendrobii total alkaloid according to claim 6 application it is characterised in that：Described granule passes through following
Prepared by step：In parts by weight, 1 part of Herba Dendrobii total alkaloid xeraphium, 3 parts of cane sugar powder and 1.25 parts of dextrin is taken to mix
Even, add 60% ethanol soft material, be allowed to gently to hold agglomerating, light pressure and dissipate, cross 16 mesh sieves granulations, 60 DEG C of -80 DEG C of dryings, use 16 mesh
Sieve granulate, sealing obtains final product.
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