CN104042647B - The application in preparing the liver protecting medicine of the Herba Ardisiae Japonicae total flavones - Google Patents
The application in preparing the liver protecting medicine of the Herba Ardisiae Japonicae total flavones Download PDFInfo
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- CN104042647B CN104042647B CN201410266578.1A CN201410266578A CN104042647B CN 104042647 B CN104042647 B CN 104042647B CN 201410266578 A CN201410266578 A CN 201410266578A CN 104042647 B CN104042647 B CN 104042647B
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Abstract
The invention belongs to tcm field, disclose and go out Herba Ardisiae Japonicae total flavones with Herba Ardisiae Japonicae root for raw material extraction purification, and as the application in preparing the liver protecting medicine.Tested by acute liver, find that it can reduce ALT, AST, TNF α, IL 1 β and IL 6 in hepatic injury mice serum or tissue active (P < 0.01, p ＜ 0.05)；The experiment of In Vitro Anti hepatitis B finds that Herba Ardisiae Japonicae total flavones all has obvious inhibiting effect (P < 0.01 to HepG2.2.15 emiocytosis HBsAg, HBeAg, p ＜ 0.05), visible Herba Ardisiae Japonicae total flavones has and preferably protects the liver and anti-HBV effect, this new application is that its medical value has opened up frontier, has important using value and economic benefit.
Description
Technical field
The invention belongs to tcm field, relate to Herba Ardisiae Japonicae root for raw material extraction purification go out Herba Ardisiae Japonicae total flavones and
Prepare the application in the liver protecting medicine.
Background technology
Herba Ardisiae JaponicaeArdisa japonica(Hornsted) BL. is Myrsinacea Ardisa plant, calls Zijin
Cattle, Herba Ardisiae Japonicae wind, four leaf tea, Herba Ardisiae Japonicae etc., be distributed mainly on each provinces and regions on the south the Yangtze river basin, and also there are distribution in Korea, Japan.Have
Heat clearing away, dampness removing, silt tissue regeneration promoting of dispelling, promoting blood circulation and hemostasis, removing toxic substances, relieving cough and expelling phlegm, dehumidifying detumescence, effect of diuresis；Soreness of waist pain, dysentery can be controlled
Disease, fracture, lung scabies hemoptysis, impairment caused by overstrain, muscles and bones are ached, the disease such as toxic swelling, chronic tracheitis, hepatitis, acute and chronic nephritis, hypertension.Short
Herba Ardisiae Japonicae is among the people is used that to control hepatic disease aspect with a long history, and protracted experience medication shows, its medical value is higher, not yet finds
Obvious toxic-side effects.Modern experimental research shows, containing compounds such as abundant flavonoids in Herba Ardisiae Japonicae.Through retrieval, there is not yet short
Herba Ardisiae Japonicae experimentation report in terms of hepatic injury, therefore, the present invention, using Herba Ardisiae Japonicae total flavones as effective substance, passes through mice
Internal protecting the liver is tested with In Vitro Anti hepatitis B, studies its application prospect in preparing the liver protecting medicine.
Summary of the invention
It is an object of the invention to go out Herba Ardisiae Japonicae total flavones with Herba Ardisiae Japonicae root for raw material extraction purification, and by this effective site
Being prepared as Chinese medicine preparation after remove impurity, as the application in preparing the liver protecting medicine, this there is not yet document at home
Reported.
The technical scheme realizing the object of the invention is:
The new application of a kind of Herba Ardisiae Japonicae total flavones, is used for preparing liver-protective medicine.
Described Herba Ardisiae Japonicae total flavones is to take Herba Ardisiae Japonicae root, is ground into coarse powder, adds 30%-95% alcohol reflux 1-3 time, closes
And extracting solution filtering, the ethanol in recovered under reduced pressure filtrate, it is concentrated to give Herba Ardisiae Japonicae extractum；Extractum adds appropriate water, fully mixes
Become suspension, add hydrochloric acid and adjust pH=3-5, use D102 type macroporous adsorbent resin to be purified, loading flow velocity 1-5 mL min-1,
Static 6-15 h after loading, with the 35%-95% ethanol elution of 1-5 times of resin volume, eluent is through aluminum nitrate complexation spectrophotometric
Method measures total flavones purity and reaches 48.68%-78.68%.
Take Herba Ardisiae Japonicae total flavones, add pharmaceutic adjuvant, be prepared as various pharmaceutical preparation according to a conventional method, including tablet,
Granule, oral liquid, injection, capsule, pill；Herba Ardisiae Japonicae general flavone content 0.01%～99.9% in preparation, surplus is that medicament becomes
Type adjuvant.
The extracting method of Herba Ardisiae Japonicae the liver protecting and ALT lowering effective ingredient total flavones, there is not yet document report.Inventor is by real
Testing research to find, Herba Ardisiae Japonicae effective ingredient total flavones is preferably dissolved in the ethanol of more than 30%, therefore, uses 30% in invention
～the alcohol reflux of 95%.But because containing substantial amounts of impurity in alcohol extract, the present invention filters out from multiple macroporous resin
D102 type remove impurity, obtains effective site Herba Ardisiae Japonicae total flavones after purification, and it can be prepared as, for oral Chinese medicine preparation, making sheet
Agent, granule, oral liquid, injection, capsule, pill etc., the preparation method of the corresponding dosage form that its preparation method is recorded to document is general.
Compared with prior art, present invention firstly discloses and go out Herba Ardisiae Japonicae total flavones with Herba Ardisiae Japonicae root for raw material extraction purification
Method, and this effective site is prepared as tablet, granule, oral liquid, injection, capsule, pill etc. and effective to this
Position carries out internal protecting the liver and the research of In Vitro Anti hepatitis B experiment, is experimentally confirmed Herba Ardisiae Japonicae total flavones and has hepatoprotective
Effect, can be used for preparing liver-protective medicine.
Accompanying drawing explanation
Fig. 1 be Herba Ardisiae Japonicae total flavones anti-mouse acute liver damage of the present invention experiment blank group pathological section figure (HE dye,
×200)；
Fig. 2 be Herba Ardisiae Japonicae total flavones anti-mouse acute liver damage experimental model group of the present invention pathological section figure (HE dye,
×200)；
Fig. 3 be Herba Ardisiae Japonicae total flavones anti-mouse acute liver damage of the present invention experiment the positive group pathological section figure (HE dye,
×200)；
Fig. 4 is pathological section figure (the HE dye of Herba Ardisiae Japonicae total flavones anti-mouse acute liver damage of the present invention experiment high dose group
Color, × 200)；
Fig. 5 is pathological section figure (the HE dye of dosage group in Herba Ardisiae Japonicae total flavones anti-mouse acute liver damage of the present invention experiment
Color, × 200)；
Fig. 6 is pathological section figure (the HE dye of Herba Ardisiae Japonicae total flavones anti-mouse acute liver damage of the present invention experiment low dose group
Color, × 200).
Detailed description of the invention
Below in conjunction with embodiment, present disclosure is further described, but is not limitation of the invention.
One, the extraction of Herba Ardisiae Japonicae total flavones and purification:
Taking Herba Ardisiae Japonicae root and break into coarse powder, take coarse powder 1kg, add 60% ethanol 6 L reflux, extract, 2 times, each 1.5 h, merging carries
Take liquid and filter, the ethanol in recovered under reduced pressure filtrate, it is concentrated to give Herba Ardisiae Japonicae extractum；Extractum adds appropriate water, is fully mixed into mixed
Suspension, adds hydrochloric acid and adjusts pH=5, uses D102 type macroporous adsorbent resin to be purified, loading flow velocity 3 mL min-1, quiet after loading
Only 12 h, first with 35% ethanol elution of 2 times of resin volumes, then with 4 times of resin volume 70% ethanol elutions, flow velocity 2.5 mL
min-1, collect 70% ethanol elution, 70% ethanol elution measures total flavones purity through aluminum nitrate chelating reaction-spectrophotometry and reaches
71.20%, concentrating under reduced pressure eluent, dried the dry cream of Herba Ardisiae Japonicae total flavones；Dry for Herba Ardisiae Japonicae total flavones cream is broken into fine powder, can
Add corresponding adjuvant, make tablet, granule, oral liquid, injection, capsule, pill etc..
Two, dry for Herba Ardisiae Japonicae total flavones cream is prepared as the embodiment of medicine:
Pharmaceutical preparation embodiment one: take Herba Ardisiae Japonicae total flavones dry cream fine powder, nearly weigh 20g, adds starch to total amount 100g, system
Grain, is pressed into tablet, element tablet weight 0.3g, the every total flavones 0.5g Han Herba Ardisiae Japonicae.
Pharmaceutical preparation embodiment two: take Herba Ardisiae Japonicae total flavones dry cream fine powder, nearly weigh 20g, add cane sugar powder and dextrin (sucrose:
Dextrin=3:1) mixture to total amount 500g, pelletize, obtain granule, subpackage, every parcel 5g, the least comprise Herba Ardisiae Japonicae total flavones
0.2g。
Pharmaceutical preparation embodiment three: take Herba Ardisiae Japonicae total flavones dry cream fine powder, nearly weigh 20g, with white sugar 100g, edible distillate spirit
60mL, heating for dissolving, finally add water 500ml, filters, obtains oral liquid, subpackage, every 5ml, every total flavones Han Herba Ardisiae Japonicae
0.2g。
Pharmaceutical preparation embodiment four: take Herba Ardisiae Japonicae total flavones dry cream fine powder, nearly weigh 10 g, inject water to 500ml, add
Heat, filters, and filtrate, filters to 6.5-7.0, cold preservation with 1% sodium hydroxide solution regulation pH value, and filtrate is with mannitol, and fills
Penetrate with water to ormal weight, filter, embedding, sterilizing, to obtain final product.
Pharmaceutical preparation embodiment five: take Herba Ardisiae Japonicae total flavones dry cream fine powder, nearly weighs 20g, adds capsule and makes 200 altogether, often
Grain total flavones 0.1g Han Herba Ardisiae Japonicae.
Pharmaceutical preparation embodiment six: take Herba Ardisiae Japonicae total flavones dry cream fine powder, nearly weighs 20g, adds refined honey and reaches 500g to weight
, 5g is distributed into 1, every total flavones 0.2g Han Herba Ardisiae Japonicae.
Three, protect the liver in Herba Ardisiae Japonicae total flavones body experiment and In Vitro Anti hepatitis B experiment method and result:
Experiment one, Herba Ardisiae Japonicae total flavones the liver protecting are tested as follows:
(1) preparation of Herba Ardisiae Japonicae total flavones
Take Herba Ardisiae Japonicae root coarse powder 1kg, add 60% ethanol 6 L reflux, extract, 2 times, each 1.5 h, united extraction liquid also filters,
Ethanol in recovered under reduced pressure filtrate, is concentrated to give Herba Ardisiae Japonicae extractum；Extractum adds suitable quantity of water, is fully mixed into suspension, adds hydrochloric acid and adjusts
PH=5, uses D102 type macroporous adsorbent resin to be purified, loading flow velocity 3 mL min-1, static 12 h after loading, first with 2 times
35% ethanol elution of resin volume, then with 4 times of resin volume 70% ethanol elutions, flow velocity 2.5 mL min-1, collect 70% second
Alcohol eluen, 70% ethanol elution measures total flavones purity through aluminum nitrate chelating reaction-spectrophotometry and reaches 71.20%.Reclaim second
Alcohol, concentrating under reduced pressure, the most i.e. obtain Herba Ardisiae Japonicae total flavones.Herba Ardisiae Japonicae total flavones adds corresponding adjuvant, makes tablet, granule
Agent, oral liquid, injection, capsule, pill etc..
(2) experiment is protected the liver
1.1 instruments, medicine and animal
TGL-16K table-type high-speed refrigerated centrifuge, Hunan Xiang Yi centrifuge factory；AU600 full-automatic biochemical detector, difficult to understand
Woods Bath produces；01ymPus BX51 microscope, Olympus produces；BS1105 electronic balance, sartorius company of Germany.
Thermostat water bath XMTD 6000, Shanghai Chang Feng instrument plant；THZ-C-1 Desk type refrigeration constant-temperatureoscillator oscillator, Taicang experimental facilities
Factory.
Herba Ardisiae Japonicae is accredited as Ardisa plant Herba Ardisiae Japonicae through Medical Colleges Of Guilin Du Ze township professorArdisa japonica
(Hornsted) root of BL..Glutamate pyruvate transaminase (ALT), glutamic oxaloacetic transaminase, GOT (AST), tumor necrosis factor-alpha (TNF-α), Bai Jie
Element-6(IL-6) be all purchased from Nanjing and build up Bioengineering Research Institute, lot number is respectively as follows: 20130212,20130414,20130418,
20130316,20130612, carbon tetrachloride (CCl4): the north of the Changjiang River, Wuhan City chemical reagent factory, lot number: 20070908；Bifendate
Drop pill: Zhejiang Prov WanBang Pharmaceutical Co., Ltd, lot number: 140112.
Kunming kind SPF level mice 60, body weight 19-22g, male and female half and half, Medical Colleges Of Guilin's SPF Experimental Animal Center carry
For (credit number SCXK2007-0001).
1.2 method
60 mices be randomly divided into blank group, model group, positive drug bifendate group and high for reagent thing, in,
Low dose group.Positive controls presses 150 mg kg-1, for reagent thing group by 600,400,200 mg kg-1Dosage gavage
It is administered, every day 1 time, totally 7 days.After last is administered, in addition to blank group, remaining each treated animal equal lumbar injection 0.12% CCl4
Oleum Arachidis hypogaeae semen (0.01 mL g-1), water 24h is can't help in fasting, and eyeball takes blood, centrifugal, isolates serum, biochemical process
The activity of detection ALT, AST；Enzyme linked immunosorbent assay (ELISA) method detection TNF-α, IL-1 Β and IL-6 biochemical indicator.Blood sampling
After, dissect rapidly, take out liver, same position lobules of liver takes hepatic tissue, is fixed in 4% paraformaldehyde solution, stone
Wax embedding, section, HE dyeing, observe liver histopathology change.Application SPSS13.0 software carry out statistical analysis, data all with± s represents (the results are shown in Table 1, table 2).
1.3 result
From Tables 1 and 2 result, model group compared with blank group, ALT, AST, TNF-α, IL-1 β and IL-
6 activity have pole significant difference (P < 0.01), and this experiment modeling success is described.Compare with model group, Mouse oral positive drug and
Herba Ardisiae Japonicae flavone all can significantly suppress ALT, AST, TNF-α, IL-1 β and IL-6 activity (P < 0.01, P < 0.05), and becomes
Dose-effect relationship.Therefore can determine whether that Herba Ardisiae Japonicae total flavones has the effect of hepatoprotective, its mechanism of action may be by suppression liver
Inflammatory reaction play hepatoprotective effect.Dyeing through HE, light Microscopic observation, normal mouse hepatocyte is evenly distributed, structural integrity.
Model group liver tissue injury is serious, hepatocyte arrangement disorder, and quantity reduces, and edema in various degree occurs, occurs downright bad and becomes silted up
Blood, pathological changes is distributed as main with vein.And the hepatic necrosis degree relatively model group of Herba Ardisiae Japonicae total flavones medication group has and substantially changes
Kind, prompting Herba Ardisiae Japonicae total flavones can be effectively improved acute liver (Fig. 1).
Table 1 Herba Ardisiae Japonicae total flavones is to CCL4Cause the impact of ALT and AS in hepatic injury mice serum( ±s, n=10)
Compare with model group:*P< 0.05, * *P<0.01。
Table 2 Herba Ardisiae Japonicae total flavones is to CCL4Cause TNF-α, the impact of IL-1 β and IL-6 in hepatic injury mice serum( ± s, n=10)
Compare with model group:*P< 0.05, * *P<0.01。
This experimental result display Herba Ardisiae Japonicae flavone in treating group significantly reduces hepatic injury mice serum ALT, AST, TNF-
α, IL-1 β and IL-6 content, compares variant (P < 0.5, P < 0.01) with model group；It is impaired that pathology shows that it can obviously improve
Liver, prompting Herba Ardisiae Japonicae total flavones has liver protection function, and mechanism of action may have and is situated between by TNF-α, IL-1 β and IL-6
The hepatocyte inflammatory reaction led, protects hepatocyte effectively.
Experiment two: Herba Ardisiae Japonicae total flavones In Vitro Anti hepatitis B is tested
1 medicine, reagent, cell and instrument
1.1 medicines and reagent
PBS phosphate-buffered salt (Fuzhou Maixin biotechnology Development Co., Ltd, lot number: 09030901)；Trypsin 1:
250(U.S. Amresco, lot number: 351813039)；PPM1 culture fluid (U.S. GIBCO, lot number: 810815)；The MTT(U.S.
Amresco, import subpackage)；Dimethyl sulfoxide (U.S. Sigma, lot number: 20080329)；Enzyme-linked immunosorbent assay method second
(being purchased from Shanghai Kehua Bio-technology Co., Ltd, lot number is respectively for HBsAg B, cAg diagnostic kit
It is 20080320；20081030).
1.2 cell strain
HBV-DNA clone's transfected with human hepatoma carcinoma cell 2.2.15 cell line is built by U.S. Mount Sinai medical center, warp
Pharmacology teaching and research room of Medical Colleges Of Guilin introduce after Secondary Culture voluntarily.
1.3 instrument
BT224S type electronic analytical balance: Beijing Sai Duolisi instrument system company limited product；303-0 type electric heating Wen Pei
Foster case: Jinan Yun Cheng Instrument Ltd. product；SW-CJ-1FDA type superclean bench: Jiangsu Su Jing group product；MCO-
15AC type CO2 gas incubator: Japan's SANYO product；TS100-F type inverted microscope: Japan's NIKON product；ELX800 type
Full-automatic microplate reader: U.S.'s BIO-TEK product.
2 methods
The preparation of 2.1 reagents
2.1.1 the preparation of Herba Ardisiae Japonicae total flavones
Adding dimethyl sulfoxide in Herba Ardisiae Japonicae extractum, being configured to concentration is 0.5 g mL-1Medicinal liquid, medicinal liquid is with 0.22
The micropore filter of μm filters, and is then configured to 9,4.5,2.5,1.5,0.5,0.25 mg mL with cell culture fluid-1Difference dense
The medicinal liquid of degree, is placed in 4 DEG C of refrigerators stand-by.
2.1.2 the preparation of PBS phosphate buffered solution
In PBS phosphate-buffered salt, add ultra-pure water be configured to the buffer solution (pH=7.2-7.4) of 0.01N, use diameter
The micropore filter of 0.22 μm filters in an aseptic environment, is placed in 4 DEG C of refrigerators stand-by.
2.1.3 the preparation of Digestive system
Add the PBS phosphate buffered solution prepared and be made into 0.25% trypsin solution, exist with the micropore filter of 0.22 μm
Filter under gnotobasis, be placed in 4 DEG C of refrigerators stand-by.
The recovery of 2.2 HepG2.2.15 cell strains and cultivation
Sucking-off after being melted in 37 DEG C of water-baths by the HepG2.2.15 cell of liquid nitrogen storage, adds the cultivation of 10 times amount
Liquid, shakes up, 1200 r min-1Centrifugal, abandon supernatant, then clean 1 time with culture fluid.Cell strain is seeded in culture bottle, puts
37 ℃、5 % CO2Growing in cell culture incubator, according to cell growth status, culture fluid 1-3d changes 1 time.
Treat that HepG 2.2.15 cell covers with culture bottle, abandon culture fluid, add the pancreatin of 0.25%, digest 3～4 at 37 DEG C
Min, adds culture fluid and blows and beats into unicellular, and 1:3 passes on.Cell again covers with rear and digests, and is made into 1 × 10 with culture fluid4 Individual
mL-1Cell suspension, be inoculated on 96 porocyte culture plates, every hole 180 μ L, incubator put 24 h, waits cell attachment to become
Test after monolayer.
2.3 medicines are to cytotoxicity experiment
After cell attachment monolayer, add medicinal liquid 20 μ L, add final concentration of 0.9 in culture fluid makes hole, 0.6,0.3,0.1,
0.05 、0.02mg·L-1, setting cell controls group simultaneously, every concentration repeats 5 holes.MTT 20 μ L is added after medicine effect 72 h
Continue to cultivate 4 h.Remove supernatant, clean 2 times with PBS buffer solution, pat dry with absorbent paper.Add dimethyl sulfoxide 150 μ L
In each hole, vibrate 5 min, measures light absorption value (OD), calculate medicine to cell-damaging rate at 490nm.
2.4 antigen inhibitory action experiments
According to the result of Drug toxicity trails, drug level is set to 0.3,0.1,0.05,0.02 mg mL-1, each concentration
Add 3 holes, set cell controls group and culture fluid blank group simultaneously, after medicine effect 72 h, collect supernatant and put culture plate
On ,-20 DEG C of freezings are standby.Use HBsAg, HBeAg content in ELISA method detection supernatant, calculate the antagonism of Herba Ardisiae Japonicae flavone
Former inhibition percentage, and calculate medium effective concentration IC50With therapeutic index (TI).
TI= TC50/IC50
2.5 data process and statistical analysis
Experimental data all with " ±s" represent, add up with Excel 2003 statistical software.
3 results
The 3.1 Herba Ardisiae Japonicae total flavones toxic action to HepG2.2.15 cell
When liquor strength is 0.9,0.6,0.3 mg mL-1Time, the destructive rate of cell is up to 69.45% and 46.35%,
31.26%, there is overt toxicity；When liquor strength is 0.1,0.05,0.02 mg mL-1Time, destructive rate is respectively 10.36%,
8.32%, 5.33%, toxicity is inconspicuous.The Herba Ardisiae Japonicae total flavones TC to HepG2.2.15 cell strain is recorded through mtt assay50For
1.311 mg·mL-1, TC0=0.0213 mg·mL-1, combined microscopy result, test setting 0.1,0.05,0.02 mg mL-1
The impact that HepG2.2.15 cell HBsAg, HBeAg are secreted by 3 concentration studies medicines.
The impact that HepG2.2.15 cell HBsAg, HBeAg are secreted by 3.2 Herba Ardisiae Japonicae total flavones
After experimental result shows 72 h, Herba Ardisiae Japonicae total flavones to HBsAg, HBeAg of HepG2.2.15 cell strain point
Secrete and have certain inhibitory action, and in a certain amount effect relationship.The Herba Ardisiae Japonicae total flavones IC to HBsAg, HBeAg50It is respectively
0.768、0.899 mg·mL-1, TI value be respectively 1.71,1.46, it is seen that its to HepG2.2.15 emiocytosis HBsAg,
HBeAg is inhibited, is shown in Table 3.
Table 3 Herba Ardisiae Japonicae total flavones to HepG2.2.15 cell cultivate in secrete HBsAg, HBeAg inhibitory action (
± s,n=3)
* p ＜ 0.05, * * p ＜ 0.01 is compared with cell controls group.
Experimental result shows, HepG2.2.15 emiocytosis HBsAg, HBeAg are all had and substantially press down by Herba Ardisiae Japonicae total flavones
Make use, in a certain amount effect relationship.
Claims (2)
1. Herba Ardisiae Japonicae total flavones application in preparing the liver protecting medicine.
Application the most according to claim 1, is characterized in that: the extraction of described Herba Ardisiae Japonicae total flavones and purification process be take short
Herba Ardisiae Japonicae root, is ground into coarse powder, adds 30%-95% alcohol reflux 1-3 time, and united extraction liquid also filters, in recovered under reduced pressure filtrate
Ethanol, be concentrated to give Herba Ardisiae Japonicae extractum；Extractum adds suitable quantity of water, is fully mixed into suspension, adds hydrochloric acid and adjusts pH=3-5, uses D102
Type macroporous adsorbent resin is purified, loading flow velocity 1-5 mL min-1, static 6-15 h after loading, with 1-5 times of resin volume
35%-95% ethanol elution, eluent through aluminum nitrate chelating reaction-spectrophotometry measure total flavones purity reach 48.68%-
78.68%。
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