CN109248202A - A kind of serviceberry and its extract are preparing the application in medicine resisting viral hepatitis - Google Patents
A kind of serviceberry and its extract are preparing the application in medicine resisting viral hepatitis Download PDFInfo
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Abstract
The present invention relates to a kind of serviceberries and its extract to prepare the application in medicine resisting viral hepatitis, belongs to the field of Chinese medicines.Serviceberry and its extract are serviceberry water extract, serviceberry ethanol extract or serviceberry methanolic extract preparing the application in medicine resisting viral hepatitis, the Sorbus alnifloria berry extract.Advantage is that the application directly solves Chronic HBV, the high virus load and serious hepatocellular injury of the virus hepatitis such as HCV, and indirectly improve body's immunity, enhance the vigor and anti-virus ability of immune cell, it can be with adjuvant clinical antiviral drugs application, reach liver protecting while reducing the double action of virus load, treatment virus hepatitis drug can be prepared using serviceberry and its extract.
Description
Technical field
The present invention relates to the field of Chinese medicines, in particular to Chinese medicine source antivirus hepatitis medicament.
Background technique
Virus hepatitis is a kind of infectious disease as caused by a variety of hepatitis virus based on hepatic disease.Clinically with food
It is intended to decline, is nausea, epigastric discomfort, pain in the hepatic region, out of strength for main performance.Some patients can have jaundice fever and hepatomegaly with liver
Functional lesion, some patients can chronicity, or even develop into cirrhosis, minority can develop as liver cancer.The cause of disease of virus hepatitis
Credit type, it is putative at present to have first, second, third, fourth, penta 5 kinds of hepatitis virus, respectively write HAV, HBV, HCV, HDV,
HEV, in addition to hepatitis type B virus is DNA virus, remaining is RNA virus.Own type hepatitis once had been reported that, but pathogen separation so far
Not successfully.In recent years report, belong to flavivirus heptan hepatovirus and the TTV of single stranded DNA and the relationship of human hepatitis still have dispute.
Oxyhepatitis does not have to antiviral therapy generally, early stage is only advocated in acute hepatitis C, interferon is applied to prevent slowly
Property, and chronic viral hepatitis needs antiviral therapy.1. interferon: recombinant DNA LeIF (IFN-α) can inhibit
The duplication of HBV.Intramuscular injection every other day, continuous 6 months, only 30%~50% patient obtained more lasting effect.The head of hepatitis C
Selecting drug is interferon, can be with ribavirin combination application.2. Lamivudine: being a kind of sweet class of dideoxy cytimidine core of synthesis
Drug has the function of Anti-HBV activity.Oral Lamivudine, level of HBV-DNA in serum can be decreased obviously, and the 12 weeks HBV-DNA that take medicine turn
Negative rate is up to 90% or more.Long-term administration can reduce ALT, improve liver inflammation, but HBeAg negative conversion rate only 16%~18%, treatment 6
A month or more, the variation of HBV can occur, but may continue to take this medicine, side effect can gently continue to take 1~4 year.3. general former times Lip river
Wei: being a kind of bird aminoglycoside, its long half time, concentration is high in the cell, can inhibit the duplication of HBV-DNA.This medicine pair
Effect gently can share raising curative effect with Lamivudine interferon etc..4. other disease-resistant drugs: such as acyclovir, adefovirdipivoxil, phosphine
Sodium formate etc. has certain inhibition HBV effect.
In addition there are immunological regulation agent therapy, there are commonly: 1. thymosin α1 has two-way immunoregulation effect, can rebuild original
It sends out, the immune function of secondary immunodeficiency patient.2. the cell that thymosin extrasin participates in body is immunoreacted, T lymph is induced
The differentiation and maturation of cell, reaction of the amplifier T cell to antigen, the balance of each subgroup of regulatory T-cell.3. immune ribonucleic acid is in body
It is interior can inducement interferon and enhance body's immunity.Targeted therapy, (such as DNA vaccination immune complex is controlled for new immunization therapy
Treat etc.) and gene therapy (antisense nucleic acid therapeutic therapeutic transgene).Hepatinica: 1. hepatocyte growth promoting factor promotes liver cell again
It is raw, there is protective effect to hepatocellular injury, and be adjustable body's immunity and anti-fibrosis effect.2. silibinin has protection
It is acted on liver plasma membrane is stablized.3. diammonium glycyrrhizinate has stronger anti-inflammatory, protection cell membrane and the effect for improving liver function, suitable
For with the raised chronic persistant hepatitis of glutamic-pyruvic transaminase and chronic active hepatitis.4. Ademetionine supplemented with exogenous
Ademetionine play the role of promote jaundice subside and liver function recovery.Traditional Chinese medicine: Coryza Treated by Syndrome Differentiation is to improvement symptom and liver
Function has good therapeutic effect, such as oriental wormwood, cape jasmine, radix paeoniae rubra, Radix Salviae Miltiorrhizae.
We are during nearly hundred kinds of medicinal plant screening active ingredients to Changbaishan area, it was found that several to have relatively strong resist
The plant of hepatitis virus, wherein just including serviceberry.
Sorbus alnifloria is commonly called as: Ma Jiamu (northeast popular name), alias: smelly Chinese scholartree, Sorbus alnifloria, water paulownia, river Chinese catalpa, chrysanthemum Chinese catalpa, Latin
Name: Sorbus pohuashanensis, rosaceae, Sorbus arbor.Compound leaf class Chinese scholartree leaf, therefore have the smelly Chinese scholartree of alias again.It is distributed in
China Heilungkiang, Jilin, Liaoning, the Inner Mongol, Hebei, Shanxi, Gansu, Shandong, the Yangtze river basin and northern area；Japan also has.
Tender leaf is edible；Leaf or bark can make pesticide, can kill rice borer, planthopper；Fruit medicine has significant diuresis, can make diuretics,
Control kidney trouble, kidney qi cystitis, cirrhosis, ascites.
Summary of the invention
The present invention provides a kind of serviceberry and its extract is preparing the application in medicine resisting viral hepatitis, and the application is straight
It connects and solves Chronic HBV, the high virus load and serious hepatocellular injury of the virus hepatitis such as HCV, and indirectly improve machine
Body immunity function enhances the vigor and anti-virus ability of immune cell, can be reached with adjuvant clinical antiviral drugs application
Liver protecting reduces the double action of virus load simultaneously.
The technical solution adopted by the present invention is that: serviceberry and its extract are preparing antiviral property hepatitis as active component
Application in drug, wherein the serviceberry and its extract include the serviceberry original plant and its solvent extractable matter, wherein
The solvent extractable matter is water, ethyl alcohol, methanolic extract.
The serviceberry and its extract is used to prepare the application in medicine resisting viral hepatitis, can pass through following sides
Method preparation:
It 1. taking serviceberry, dry, pulverize, add pharmacy acceptable auxiliary material, tablet, capsule, powder, pill is made
Deng pharmaceutically acceptable dosage form.
2. taking serviceberry, with the extraction of water, ethyl alcohol or methanol equal solvent, it is concentrated to dryness, obtains Sorbus alnifloria berry extract, mentioned with this
Taking object is raw material, adds pharmacy acceptable auxiliary material, tablet, capsule, powder, pill, soft capsule etc. is made and pharmaceutically may be used
With the dosage form of receiving.
3. the ethanol extract that further preferred serviceberry is extracted through ethyl alcohol, ethanol extract is again through silica gel column chromatography with stone
Oily ether: acetone=(100~1): 1 gradient elution, collect petroleum ether: acetone=5:1 component is further tied through ethyl acetate again
Crystalline substance, obtained active component are raw material, add pharmacy acceptable auxiliary material, tablet, capsule, powder, pill, flexible glue is made
The pharmaceutically acceptable dosage form such as capsule.
The invention has the advantages that the application directly solves Chronic HBV, the high virus load of the virus hepatitis such as HCV and serious
Hepatocellular injury, and indirectly improve body's immunity, enhance the vigor and anti-virus ability of immune cell, can be with
Adjuvant clinical antiviral drugs application reaches liver protecting while reducing the double action of virus load, using serviceberry and its
Extract can prepare treatment virus hepatitis drug.
Detailed description of the invention
Fig. 1 is action diagram of the Sorbus alnifloria berry extract to HepG2.2.15 cell line virus load, wherein A control group, B rummy
Husband is fixed, C Sorbus alnifloria fruit powder, D decocting Stewed Dish, E ethanol extract, and F ethyl acetate crystallizes position；
Fig. 2 is action diagram of the Sorbus alnifloria berry extract to HepG2.2.15 cell line HBsAg, wherein A control group, B rummy husband
It is fixed, C Sorbus alnifloria fruit powder, D decocting Stewed Dish, E ethanol extract, F ethyl acetate crystallization position；
Fig. 3 is action diagram of the Sorbus alnifloria berry extract to JFH-Huh7 cell line virus load, wherein A control group, B Te Lapu
Wei, C Sorbus alnifloria fruit powder, D decocting Stewed Dish, E ethanol extract, F ethyl acetate crystallize position；
Fig. 4 is action diagram of the Sorbus alnifloria berry extract to JFH-Huh7 cell line HCVcore, wherein A control group, B Te Lapu
Wei, C Sorbus alnifloria fruit powder, D decocting Stewed Dish, E ethanol extract, F ethyl acetate crystallize position.
Specific embodiment
Embodiment 1: serviceberry prepares the application of medicine resisting viral hepatitis
1, serviceberry antiviral property hepatitis capsule (Sorbus pohuashanensis capsule, SPC): Sorbus alnifloria is collected
Fruit is dried in the shade, and crushes, hard capsule preparation is made.
2, serviceberry antiviral property hepatitis piece (Sorbus pohuashanensis tablet, SPT): collecting serviceberry,
It dries in the shade, crushes, extracted three times with 8~10 times of 70%~80% EtOH Sonicates of amount, 25~35 minutes every time, filtering, merging filtrate,
It is evaporated, crushes, add starch appropriate, tabletting.
3, it serviceberry antiviral property hepatitis oral solution (Sorbus pohuashanensis oral liquid, SPOL): takes
Serviceberry, 10 times of amount water decoct 30~50 minutes, and filtering, filtrate is concentrated to give liquid extract, adds auxiliary material, and oral solution is made.
Embodiment 2: serviceberry medicine resisting viral hepatitis activity research
Sorbus alnifloria fruit powder, decocting Stewed Dish, ethanol extract are prepared, ethyl acetate crystallizes position, and (serviceberry is extracted through ethyl alcohol
Ethanol extract, ethanol extract is again through silica gel column chromatography with petroleum ether: acetone=(100~1): 1 gradient elution, collects petroleum
Ether: acetone=5:1 component, further through re-crystallizing in ethyl acetate, obtained active component.
1 material
1.1 medicinal materials and reagent
Serviceberry, the fruit of rosaceae Sorbus plant flowers Chinese catalpa Sorbus pohuashanensis, picks up from Changbai Mountain arteries and veins
Regional near a river, hepatitis B replicating cell system (HepG2.2.15), hepatitis C replicating cell system (JFH-Huh7) are purchased from ATCC
Cell bank, DMEM cell culture medium, fetal calf serum are purchased from GIBCO company；
1.2 instrument
Thermo cell incubator (the silent winged generation that science and technology of match)；II grades of Biohazard Safety Equipments (Thermo company of the U.S.)；More function
It can enzyme mark (German Bio-Tek company)；Real-time fluorescence quantitative PCR instrument (American AB I company)；KQ-250B type numerical control supersonic is clear
Wash device (Kunshan Ultrasonic Instruments Co., Ltd.)；RE-52AA rotary evaporator (Shanghai Yarong Biochemical Instrument Plant)；DZF6020 is true
Empty drier (Shanghai Yiheng Scientific Instruments Co., Ltd)；
1.3 reagent
Physiological saline (Sichuan degree Cologne Pharmaceutical Co)；Fetal calf serum (FBS, Corning, the U.S.)；Phosphorus
Phthalate buffer (PBS, BI, Israel)；Trypan blue cell dye (Trypan blue Solution, Sigma, the U.S.)；Carefully
Cellular lysate liquid (Lysing Buffer, BD, the U.S.)；Dimethyl sulfoxide (DMSO, Beijing biology Co., Ltd of ancient cooking vessel state, China)；
RNAlater (Sigma, the U.S.)；Extract cell/tissue total serum IgE related reagent: RNA extracts kit (Quan Shijin, China)；Instead
Transcript reagent: reverse transcription reagent box (Quan Shijin, China)；Real-time quantitative PCR reagent: real-time quantitative PCR kit (Quan Shijin,
China)；RNase-Free water (Sigma, the U.S.)；HBsAg and HCVcore Concentration Testing ELSIA kit (China of section, in
State)；
2 methods
To document [Yang D, et al.Complete replication of hepatitis B virus and
hepatitis C virus in a newly developed hepatomacell line.Proc Natl Acad Sci U
S A.2014,111 (13): E1264-73] in the measurement of HBV virus load, the measurement of HCV virus carrying capacity, HBsAg and HCVcore
Method for measurement of concentration is improved.
2.1 cell culture
Hepatitis B replicating cell system (HepG2.2.15) and hepatitis C replicating cell are housed by being stored in liquid nitrogen
The cryopreservation tube of system (JFH-Huh7) is immediately placed in 37 DEG C of water-baths, jiggles repeatedly and cell is caused to melt completely.Melt in cell
Prepare a 15mL centrifuge tube in the process to be placed in Biohazard Safety Equipment, and 5mL is added containing 10% fetal calf serum into centrifuge tube
DMEM culture solution, 37 DEG C water-bath 5 minutes；Under aseptic technique carefully draw cell suspension, drop in preheated 15mL from
In heart pipe, centrifuge tube is slowly shaken during instillation.It is centrifuged after cell shifts completely, 1500rpm is centrifuged 10min；
After discarding supernatant, 1mL DMEM complete culture solution is added into centrifuge tube, is repeatedly resuspended after pressure-vaccum cell uniform.Again to centrifugation
For supplemented medium to 10mL, 1500rpm is centrifuged 10min in pipe；Cell transfer is seeded in 25T culture bottle after abandoning supernatant, to
The DMEM culture solution that 6mL contains 10% fetal calf serum is wherein added, is placed in 37 DEG C, saturated humidity, contains 5%CO2Incubator in often
Rule culture.The had digestive transfer culture after cytotostatic, and be incubated in 96 well culture plates, every hole cell number is 1 × 105, cell is adherent
It is spare that each group drug is added after culture 24 hours；
The preparation of the sample solution of 2.2 serviceberry antiviral property hepatitis products
Serviceberry antiviral property hepatitis product is shown in " embodiment 1 ", takes corresponding product, it is thin that capsule, which takes content, tablet to grind,
Powder, oral solution are directly drawn, and respectively plus phosphate buffer solution is made into the sample solution of respective concentration；
2.3 products are to HBV inhibiting effect in cell line
HepG2.2.15 cell line is incubated in 96 well culture plates, every hole cell number is 1 × 105, cell adhere-wall culture
Each group drug is added after 24 hours, drug to be added sucks cells and supernatant after 72 hours, freezes HBsAg concentration to be measured, pastes
Parietal cell is washed 3 times with the PBS buffer solution for being warmed to 37 DEG C, blows afloat collection cell precipitation, carries out qRT-PCR experiment detection HBV disease
Malicious copy number；
ELSIA method detects the concentration of HBsAg in cells and supernatant；
Lamivudine is as positive control；
2.4 products are to HCV inhibiting effect in cell line
JFH-Huh7 cell line is incubated in 96 well culture plates, every hole cell number is 1 × 105.Cell adhere-wall culture 24
Each group drug is added after hour.Drug to be added sucked cells and supernatant after 72 hours, froze HCVcore concentration to be measured, patch
Parietal cell is washed 3 times with the PBS buffer solution for being warmed to 37 DEG C, blows afloat collection cell precipitation, carries out qRT-PCR experiment detection HCV disease
Malicious copy number；
ELSIA method detects the concentration of HCVcore in cells and supernatant；
Te Lapuwei is as positive control；
3 results and analysis
3.1 products are to HBV inhibiting effect
Sorbus alnifloria fruit powder (C), decocting Stewed Dish (D), ethanol extract (E), ethyl acetate crystallize position (F) thermosol in 1640 trainings
It supports in base, concentration 2.0mg/mL, sterile filters filtration sterilization, 1640 complete medium groups (A) are used as blank control group, rummy
Husband fixed (B) is used as positive control, and concentration is 10 μM；
The result shows that the DNA copy number of HBV is 6.8 × 10 after the culture of HepG2.2.15 cell line 72 hours5, it is added 10
μM Lamivudine after, DNA copy number is remarkably decreased, and is down to 4.8 × 103, there is significant difference (p < compared with the control group
0.01), Sorbus alnifloria fruit powder (C), decocting Stewed Dish (D) cannot significantly lower the DNA copy of HBV in HepG2.2.15 cells and supernatant
Number (p > 0.05), ethanol extract (E) and ethyl acetate crystallization position (F) can significantly lower DNA copy number (the p < of HBV
0.01), ethyl acetate crystallization position effect is better than ethanol extract, as shown in Figure 1.
HepG2.2.15 cell line culture supernatant is collected, the concentration of HBsAg in ELISA method detection cells and supernatant.It is empty
The HBsAg of Bai Zuzhong is 35IU/mL, and after 10 μM of Lamivudine is added, the concentration of HBsAg is remarkably decreased, and is down to 12IU/mL,
There is significant difference (p < 0.001) compared with the control group, Sorbus alnifloria fruit powder (C), decocting Stewed Dish (D) cannot significantly lower HBsAg
Concentration (p > 0.05), ethanol extract (E) and ethyl acetate crystallization position (F) can significantly lower concentration (the p < of HBsAg
0.01), ethyl acetate crystallization position effect is better than ethanol extract, as shown in Figure 2；
3.2 products are to HCV inhibiting effect
Sorbus alnifloria fruit powder (C), decocting Stewed Dish (D), ethanol extract (E), ethyl acetate crystallize position (F) thermosol in 1640 trainings
It supports in base, concentration 2.0mg/mL, sterile filters filtration sterilization, 1640 complete medium groups (A) are used as blank control group, Te La
Pu Wei (B) is used as positive control, and concentration is 5 μM；
The result shows that the RNA copy number of HCV is 2.3 × 10 after the culture of JFH-Huh7 cell line 72 hours6GE/mL adds
After entering 5 μM of Te Lapuwei, RNA copy number is remarkably decreased, and is down to 5.3 × 104GE/mL has conspicuousness compared with the control group
Difference (p < 0.001), Sorbus alnifloria fruit powder (C), decocting Stewed Dish (D) cannot significantly lower RNA in JFH-Huh7 cells and supernatant and copy
Shellfish number (p > 0.05), ethanol extract (E) and ethyl acetate crystallization position (F) can significantly lower the RNA copy number (p of HCV
< 0.01), ethyl acetate crystallization position effect is better than ethanol extract, as shown in Figure 3；
JFH-Huh7 cell line culture supernatant is collected, the concentration of HCVcore in ELISA method detection cells and supernatant is empty
The HCVcore concentration of Bai Zuzhong is 1.3 × 106Fmol/L, after 5 μM of Te Lapuwei is added, under the concentration of HCVcore is significant
Drop, is down to 3.3 × 103Fmol/L has significant difference (p < 0.001) compared with the control group, and Sorbus alnifloria fruit powder (C), water decoct
Object (D) cannot significantly lower the concentration (p > 0.05) of HCVcore, and ethanol extract (E) and ethyl acetate crystallization position (F) can
Significantly to lower the concentration (p < 0.01) of HCVcore, ethyl acetate crystallization position effect is better than ethanol extract, such as Fig. 4 institute
Show.
Claims (4)
1. serviceberry is preparing the application in medicine resisting viral hepatitis.
2. Sorbus alnifloria berry extract is preparing the application in medicine resisting viral hepatitis.
3. Sorbus alnifloria berry extract according to claim 2 exists preparing the application in medicine resisting viral hepatitis, feature
In: the Sorbus alnifloria berry extract is serviceberry water extract, serviceberry ethanol extract or serviceberry methanolic extract.
4. Sorbus alnifloria berry extract according to claim 3 exists preparing the application in medicine resisting viral hepatitis, feature
In: serviceberry ethanol extract is again through silica gel column chromatography with petroleum ether: acetone=(100~1): 1 gradient elution, collects petroleum
Ether: acetone=5:1 component, further through re-crystallizing in ethyl acetate.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115197243A (en) * | 2021-04-13 | 2022-10-18 | 中国中医科学院中药研究所 | Sulfur-containing dibenzofuran type alkaloid and preparation method and application thereof |
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| CN102421446A (en) * | 2009-05-12 | 2012-04-18 | 乌尔萨法姆药物有限责任公司 | Immune stimulating composition comprising an extract of aronia sp. in combination with selenium and/or zinc |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115197243A (en) * | 2021-04-13 | 2022-10-18 | 中国中医科学院中药研究所 | Sulfur-containing dibenzofuran type alkaloid and preparation method and application thereof |
| CN115197243B (en) * | 2021-04-13 | 2023-11-10 | 中国中医科学院中药研究所 | Sulfur-containing dibenzofuran alkaloid and preparation method and application thereof |
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Application publication date: 20190122 |