CN100457127C - Medicinal herb solvent extract for treating hepatic disease - Google Patents
Medicinal herb solvent extract for treating hepatic disease Download PDFInfo
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- CN100457127C CN100457127C CNB2003101234094A CN200310123409A CN100457127C CN 100457127 C CN100457127 C CN 100457127C CN B2003101234094 A CNB2003101234094 A CN B2003101234094A CN 200310123409 A CN200310123409 A CN 200310123409A CN 100457127 C CN100457127 C CN 100457127C
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Abstract
The invention relates to a solvent extract of uricaceous plant root or stem for hepatic disease treatment. The uricaceous plant has a ribosome intergenic sequence similarity greater than 70% with mountain ramie, ramie and longleaf ramie. In addition, The medical plant extract for hepatic disease treatment has an inhibitory effect to the wild type hepatitis B virus and the hepatitis B virus with lamivudine resistance.
Description
Technical field
The invention relates to a kind of medicinal plant extract that inhibition hepatitis virus B dna replication dna and hepatitis virus B surface antigen (HBs) and the excretory liver disease of e antigen (HBe) are used that has; This extract not only has the inhibition effect to wild type (wild type) hepatitis virus B, and also how the viral dna replication of the hepatitis virus B of Drug resistance (lamivudine-resistant) and viral surface antigen and the antigenic secretion tool of e suppress effect to liver.The medicinal plants source that this liver disease is used is for having specific ribosome intergenic sequence (ribosomal DNA internal transcribed spacer, abbreviation ITS sequence) contrayerva, and this ITS sequence can be used as the appreciation comparison of this medicine plant.
Background technology
It is the chronic hepatitis b carrier that there are 300,015,000 populations in the whole world, chronic hepatitis the infected of 1,250,000 is arranged approximately in the U.S., there are 3,000 to 50,000,000 people to infect hepatitis B approximately in the China's Mainland, 100,012,000 artificial chronic hepatitis b carriers, wherein there are every year 300000 people to die from hepatitis B approximately, then have three million peoples to infect hepatitis B in Taiwan approximately.
There are every year 14-32 ten thousand people to infect hepatitis B approximately in the U.S. according to estimates, because of hepatitis B infects at least seven hundred million dollars of caused economic loss every year.Chronic hepatopathy and liver cirrhosis patient in Taiwan surpass 5,000 people, for the sixth-largest cause of the death of compatriots, add diseases such as causing liver tumor, and be then more considerable.
At present the treatment hepatitis B is based on interferon (interferon) and liver how (lamivudine).Interferon allowed to be used in the treatment chronic hepatitis b in 1992 for FDA, but can cause very large side effect during use, and only about 20% B hepatopathy people responds to interferon, and how liver is used for treating chronic hepatitis b the permission by FDA in 1998, but also only there is the patient of 17-33% to respond, and Chinese are lower to liver reaction how, the most serious sudden change that then how can cause the B hepatovirus for the life-time service liver, with extending service time, its mutation rate also increases thereupon, 24% mutation rate is arranged approximately in 1 year in use, then increases to 42% to 1 year, the 3rd year is 52%, then high to 67% to the 4th year.So as can be known so far, the low side effect of high curative effect is not arranged yet and can not cause the treatment hepatitis B medicine of virus mutation.
According to discovering, hepatitis B carrier's e antigen is positive, and it is dangerous for 60 times of ordinary persons that the patient suffers from hepatocarcinoma, when estimating 70 years old, suffers from the hepatocarcinoma risk up to ninety percent; Therefore exploitation is more effective, the while can be suppressed hepatitis virus B surface antigen and the antigenic generation of e, can not cause virus mutation, but even the hepatitis B medicine of mutation inhibiting virus be the task of top priority.
Summary of the invention
In view of this, the invention provides a kind of medicinal plant extract that inhibition hepatitis virus B dna replication dna and hepatitis virus B surface antigen and the excretory liver disease of e antigen are used that has; And the method for utilizing sequence between ribosomal gene (ITS sequence) that this medicinal plants is identified.
Main purpose of the present invention provides a kind of medicinal plant extract that is used for liver disease, is to be ground dozen powder, made with solvent extraction by root or stem with mountain Boehmeria, Herba Catharanthi longifolii, Urtica thunbergiana sieb, SHUIMA, Boehmeria, wooden Boehmeria.
Another object of the present invention provides a kind of extracting method, to be used for making inhibition hepatitis virus B dna replication dna, the medicinal plant extract that hepatitis virus B surface antigen and the excretory hepatopathy of treatment e antigen are used is to be ground dozen powder, made with solvent extraction by root or stem with mountain Boehmeria, Herba Catharanthi longifolii, people cat, SHUIMA, Boehmeria, wooden Boehmeria.
Another purpose of the present invention provides a kind of medicinal plants identification method that is used for the medicinal plants of liver disease, be by and the ribosomal gene of mountain Boehmeria, Herba Catharanthi longifolii, Urtica thunbergiana sieb, SHUIMA, Boehmeria, wooden Boehmeria between sequence (Internal Transcribed Spacer, be called for short the ITS sequence) compare, its ITS sequence similarity degree is at least 70%, from going out to have to suppressing the hepatitis virus B dna replication dna, hepatitis virus B surface antigen and the excretory hepatopathy of treatment e antigen have the vegetable drug of preferable drug effect with single.
For achieving the above object, the contrayerva root of liver disease or the solvent extractable matter of stem of being used for provided by the invention, extraction is from a contrayerva, and this contrayerva itself and mountain Boehmeria (Boehmeria frutescens), sequence (Internal Transcribed Spacer) similarity is greater than more than 70% between the ribosomal gene of Herba Catharanthi longifolii (Boehmeria zollingeriana), Urtica thunbergiana sieb (Urticathunbergiana), SHUIMA (Pilea spp), Boehmeria (Boehmeria nivea) and wooden Boehmeria (Boehmeriadensiflora).
Wherein between the ribosomal gene of this mountain Boehmeria sequence shown in sequence recognition number 1,2 and 3.
Wherein between the ribosomal gene of this Herba Catharanthi longifolii sequence shown in sequence recognition number 4,5 and 6.
Wherein between the ribosomal gene of this Urtica thunbergiana sieb sequence shown in sequence recognition number 7,8 and 9.
Wherein between the ribosomal gene of this SHUIMA sequence shown in sequence recognition number 10,11 and 12.
Wherein between the ribosomal gene of this Boehmeria sequence shown in sequence recognition number 13,14 and 15.
Sequence is shown in sequence recognition number 16,17 and 18 between wherein should the ribosomal gene of wood Boehmeria.
Wherein the medicinal plants that sequence can be used for the medicinal plant extract of liver disease between the ribosomal gene of one of mountain Boehmeria, Herba Catharanthi longifolii, Urtica thunbergiana sieb, SHUIMA, Boehmeria, wooden Boehmeria is identified.
Wherein plant extract is that contrayerva DNA to be measured, chain polymerization enzyme reaction amplification amplify through extracting, sequence between the ribosomal gene of sequence fragment sequencing, analyses and comparison mountain Boehmeria, Herba Catharanthi longifolii, Urtica thunbergiana sieb, SHUIMA, Boehmeria, wooden Boehmeria between ribosomal gene, the step of setting up sequence data storehouse between the contrayerva ribosomal gene identify.
The extracting method of this solvent extractable matter comprises the following steps:
(a) these contrayerva roots or stem are ground dozen powder, obtain a powder;
(b) with gained powder in a high polar solvent or its solvent mixture treatment step (a), obtain extracting solution; Through evaporating dried solid extraction thing; And
(c) again with the aqueous layer of gained in low polar solvent and this step of aqueous solvent mixture process (b), through evaporating dried solid extraction thing.
Wherein this high polar solvent is methanol, ethanol or its mixture.
Wherein this low polar solvent is selected from a group and comprises: ethyl acetate, dichloromethane, chloroform, tetrachloromethane, cyclohexane extraction, normal hexane, n-butyl alcohol ether, benzene, and above-mentioned mixture.
Wherein this low polar solvent is an ethyl acetate.
Wherein liver disease is the hepatitis B treatment.
And can be used for the hepatitis B treatment of anti-sweet how medicine.
Wherein the solvent extractable matter of the contrayerva root of liver disease or stem cooperates other liver disease medicines to use.
Contrayerva medical material authentication method provided by the invention may further comprise the steps:
(a) provide sequence (ITS) information bank between the ribosomal gene of a known contrayerva medical material;
(b) provide the root or the stem of a contrayerva medical material, extract its DNA;
(c) design one introduction is as follows to (Primer),
5’-CACACCGCCCGTCCTACCGA-3’
5’-ACTCGCCGTTACTAGGGGAA-3’
And carry out polymerase chain reaction at the DNA that is extracted in this step (b), obtain one the one ITS sequence;
(d) a resulting ITS sequence in the step (c) is carried out sequencing (sequencing); And
(e) the ITS sequence data in an ITS sequence and this information bank is carried out the similarity comparison, when similarity is above greater than 70%, be the contrayerva medical material in this step (b).
Wherein this step (b) DNA extraction method is to handle with CTAB (cetyltrimethylammoniumbromide), PVPP (polyvinyl polypyrrolidone) and different salinity earlier, centrifugal through precipitation again, Chloroform extraction and alcohol precipitation and get.
Wherein the contrayerva medical material in this step (b) is selected from a group and comprises: mountain Boehmeria (Boehmeria frutescens), Herba Catharanthi longifolii (Boehmeria zollingeriana), Urtica thunbergiana sieb (Urticathunbergiana), SHUIMA (Pilea spp), Boehmeria (Boehmeria nivea) and wooden Boehmeria (Boehmeriadensiflora).
In one embodiment, the present invention with the medicinal plant extract of liver disease as the application that in vitro suppresses virus: (1) suppresses the secretion of wild type (wild-type) hepatitis virus B dna replication dna and viral surface antigen (HBs) and e antigen (HBe); (2) suppress anti-liver how hepatitis virus B dna replication dna and the viral surface antigen and the antigenic secretion of e of the property of medicine (lamivudine-resistant).
In another specific embodiment, the present invention is with the application as the medicinal plants identification method of plant extract of the ITS sequence of mountain Boehmeria, Herba Catharanthi longifolii, Urtica thunbergiana sieb, SHUIMA, Boehmeria, wooden Boehmeria.
Description of drawings
Fig. 1 suppresses hepatitis virus B s antigen and the antigenic effect of e in the zooblast for the contrayerva extract.
Fig. 2 suppresses in the zooblast wild type and anti-liver how mutant hepatitis virus B s antigen and the antigenic effect of e for the liver disease plant extract.
Fig. 3 suppresses the how effect of mutant hepatitis virus B dna replication dna of wild type and anti-liver in the zooblast for the liver disease plant extract.
Fig. 4 is contrayerva ribosome ITS sequence alignment result.
The specific embodiment
In the explanation of the present invention, the mountain Boehmeria is meant Urticaceae Boehmeria platymiscium (Boehmeriafrutescens); Herba Catharanthi longifolii is meant Urticaceae Boehmeria platymiscium (Boehmeria zollingeriana); Urtica thunbergiana sieb is meant that contrayerva (Urtica thunbergiana), SHUIMA are meant that contrayerva (Pileaspp), Boehmeria are meant that Urticaceae Boehmeria platymiscium (Boehmeria nivea) and wooden Boehmeria are meant Urticaceae Boehmeria platymiscium (Boehmeria densiflora).
The present invention includes a kind of medicinal plant extract that is used for liver disease, grind dozen powder, make with solvent extraction by root or stem with mountain Boehmeria, Herba Catharanthi longifolii, Urtica thunbergiana sieb, SHUIMA, Boehmeria, wooden Boehmeria.
And powder is beaten in grinding of aforementioned medicinal plant extract, be to smash to pieces, to grind with the root of mountain Boehmeria, Herba Catharanthi longifolii, Urtica thunbergiana sieb, SHUIMA, Boehmeria, wooden Boehmeria or stem, physics modes such as chopping are handled and are made it become single granule, be preferably be milled to Powdered, in order to follow-up solvent extraction.
Above-mentioned medical material treatment step is known by the personage in this technology, teaching especially, and also be covered by in the category of the present invention.
Aforementioned solvents is extracted, and is the active ingredient of extracting medicinal plants with solvent extraction,, as water, methanol, ethanol or its solvent mixture, but is not limited thereto the extract that extracts as solvent earlier with high polar solvent; The extract of gained or extract with low polar solvent again with extract, it is below 10 that the present invention defines its dielectric constant, as ethyl acetate, dichloromethane, chloroform, tetrachloromethane, cyclohexane extraction, normal hexane, n-butyl alcohol ether, benzene or its mixture, but be not limited thereto, extract.
In an embodiment of the present invention, earlier with high polar solvent such as alcohol reflux heating, again with low polar solvent such as ethyl acetate or extract the active ingredient of medicinal plants with different water and ethanol mixed proportion solution.
Above-mentioned medicinal plants medical material grind beat powder after, extraction step also can be optionally carried out preceding extraction step with methanol or ethanol or its mixture.As mentioned above, though methanol and ethanol are to belong to hydrophilic, high polar solvent (dielectric constant is about 26 to 31), but because except protein, oils and fats and wax, other compositions in the Chinese herbal medicine cell all have dissolubility to a certain degree in methanol or ethanol, therefore, use methanol or ethanol, or its mixture extracts and can help the extraction of follow-up the present invention with low polar solvent.
Another kenel of the present invention provides a kind of extracting method of the medicinal plant extract with the sick treatment of hepatoprotective, is to make by the root of mountain Boehmeria, Herba Catharanthi longifolii, Urtica thunbergiana sieb, SHUIMA, Boehmeria, wooden Boehmeria or its stem being ground the extraction of beating powder, handling, handling with low polar solvent with high polar solvent earlier.Optionally, high polar solvent treatment step can methanol or ethanol or its mixture and carry out preceding extraction step, and this can help the extraction of follow-up the present invention with low polar solvent.
Yet,, can after extraction step of the present invention, carry out various purification steps for the purity that makes active ingredient in the extract improves.The method that extract is carried out purification is teaching especially, and is known by the personage in general this technology, and spendable method comprises, for example, chromatography, crystallization process, Filtration, the sedimentation method etc., should look the purpose desiring to reach and determining.
Another kind of the present invention is used for the medicinal plant extract of liver disease, is to beat powder by the root of mountain Boehmeria, Herba Catharanthi longifolii, Urtica thunbergiana sieb, SHUIMA, Boehmeria, wooden Boehmeria or stem are ground, make with solvent extraction and have the how extract of (lamivudine-resistant) hepatitis virus B of inhibition wild type (wild-type) hepatitis virus B and anti-liver.
Aforementioned medicinal plant extract suppresses how hepatitis virus B effect of wild type hepatitis virus B and anti-liver, by suppressing the secretion of hepatitis virus B dna replication dna and hepatitis virus B surface antigen (HBs) and e antigen (HBe).
In one embodiment, the present invention with the medicinal plant extract of liver disease as the application that in vitro suppresses virus: (1) suppresses wild type hepatitis virus B dna replication dna and viral surface antigen and the antigenic secretion of e; (2) suppress anti-the liver how sick longevity dna replication dna of hepatitis B and viral surface antigen and the antigenic secretion of e.
The medicinal plant extract of liver disease of the present invention can cooperate other materials and use, and for example, medicine or its mixture or the nutrition etc. of other antagonism viruses are to reach the synergistic activity that strengthens antiviral effect.
The present invention can combine individually or with pharmaceutically acceptable carrier or excipient, offers medicine with the form of single dose or multiple dose.Medically acceptable carrier or diluent and any other known adjuvant and excipient can be allocated according to traditional technology, for example, referring to Remington ' sPharmaceutical Sciences, the 19th edition, Gennaro edits, Mack publishing company, Easton, PA (1995).
A kenel more of the present invention provides a kind of medicinal plants identification method that is used for the medicinal plant extract of liver disease, by with mountain Boehmeria, Herba Catharanthi longifolii, Urtica thunbergiana sieb, (the Internal Transcribed Spacer of sequence between the ribosomal gene of SHUIMA, Boehmeria, wooden Boehmeria, be called for short the ITS sequence) to compare, its ITS sequence similarity degree is at least 70%.
The aforementioned medicinal plants identification method that is used for the medicinal plant extract of liver disease, by carrying out the ITS sequence alignment with the ITS sequence of the mountain Boehmeria with inhibition hepatitis virus B activity extract, Herba Catharanthi longifolii, Urtica thunbergiana sieb, SHUIMA, Boehmeria, wooden Boehmeria, the similarity of its ITS sequence is at least 70%.
The aforementioned medicinal plants identification method that is used for the medicinal plant extract of liver disease, comprise the following steps: to extract total DNA of contrayerva to be measured, extract the ITS fragment among total DNA with chain polymerization enzyme reaction (PolymeraseChain Reaction, abbreviation PCR) big this institute of amplification event then; Now carries out sequencing at this ITS fragment, obtains the ribosomal ITS sequence of contrayerva; Can set up after the ITS sequence via analyses and comparison mountain Boehmeria, Herba Catharanthi longifolii, Urtica thunbergiana sieb, SHUIMA, Boehmeria, wooden Boehmeria and have the ribosomal ITS sequence data of liver disease usefulness contrayerva storehouse.
The aforementioned medicinal plants identification method that is used for the medicinal plant extract of liver disease, by with the mountain Boehmeria, Herba Catharanthi longifolii, Urtica thunbergiana sieb, SHUIMA, Boehmeria, the ITS sequence alignment of wood Boehmeria, the ITS1 of mountain Boehmeria wherein, 5.8S and the sequence recognition number of ITS2 is respectively: 1,2, nucleotide sequence shown in 3, the ITS1 of Herba Catharanthi longifolii, 5.8S and the sequence recognition number of ITS2 is respectively: 4,5, nucleotide sequence shown in 6, the ITS1 of Urtica thunbergiana sieb, 5.8S and the sequence recognition number of ITS2 is respectively: 7,8, nucleotide sequence shown in 9, the ITS1 of SHUIMA, 5.8S and the sequence recognition number of ITS2 is respectively: 10,11, nucleotide sequence shown in 12; The sequence recognition number of the ITS1 of Boehmeria, 5.8S and ITS2 is respectively: 13, the nucleotide sequence shown in 14,15, the sequence recognition number of the ITS1 of wooden Boehmeria, 5.8S and ITS2 is respectively: 16, the nucleotide sequence shown in 17,18.
The aforementioned medicinal plants identification method that is used for the employing plant extract of liver disease, by with the ITS sequence alignment of mountain Boehmeria, Herba Catharanthi longifolii, people cat, SHUIMA, the ITS similarity of its comparison is at least 70%.
In one embodiment, the present invention is with the application as the medicinal plants identification method of plant extract of the ITS sequence of mountain Boehmeria, Herba Catharanthi longifolii, Urtica thunbergiana sieb, SHUIMA, Boehmeria, wooden Boehmeria.
The present invention will be further described by following examples, but these embodiment only are used to illustrate, but not be used to limit claim of the present invention.
Embodiment 1: the extraction of medicinal plant extract
The extracting method of medicinal plant extract is as follows:
(1) chooses the contrayerva of mountain Boehmeria, Herba Catharanthi longifolii, Urtica thunbergiana sieb, SHUIMA, Boehmeria, wooden Boehmeria.
(2) respectively root or stem's position process of step (1) Herba Urticae Cannabinae tannin plant medical material are cleaned, scrape off crust and then chop up sheet, pulverize.
(3) get powder 600g in the step (2) respectively, carried out reflux 10 hours with 95% ethanol 3000ml, liquid is got in leaching purely, and concentrating under reduced pressure is removed ethanol, adds ethyl ester ethyl ester and each 1000ml of distilled water, stirs after 30 minutes, leaves standstill 1 hour.
(4) with the water layer product of step (3), the water layer lyophilizing is dry concentrated with the ethyl acetate phase-splitting extraction of 500ml, be extract.
Embodiment 2: the test cell line of medicinal plant extract
The test cell line of medicinal plant extract is as follows:
(1) selects the cell strain HepG2.2.15 that contains hepatitis virus B for use, cultivate with the DMEM culture medium.
(2) cultivated five days in extract 500 μ g/ml adding step (1) cell strain with embodiment 1.
(3) respectively at first and third, five day, the cell culture fluid of step (2) is collected, carry out centrifugal (2000xg 2 minutes).
(4) the centrifugal supernatant of step (3) is partly carried out the test that hepatitis virus B surface antigen and e antigen suppress; Centrifugal confluent monolayer cells down partly then carries out the test of cytotoxicity or hepatitis virus B dna content.Cell is after twice of buffer solution for cleaning, adding 50 μ l prepares MTT in the DMEM culture fluid in advance again and puts into 37 ℃ of culture fluid reactions 30 minutes to 2 hours, add 150 μ lDMSO again and make the reaction colour generation, 590nm reads light absorption value with spectrogrph, compare with the cell light absorption value that does not add medicine, as the toxic foundation of medicine pair cell.Cell is after twice of buffer solution for cleaning.Add the buffer contain Triton-100, place and react 15 minutes on ice, draw together and get collection reacted solution cell to centrifuge tube, put give react 15 minutes more on ice after, with 1300xg centrifugal 1 minute, collect upper strata liquid and precipitate; Upper strata liquid extracts hepatitis virus B DNA in the Cytoplasm with automatic nucleic acid abstraction instrument.
The following explanation of test cell line result:
Fig. 1 suppresses hepatitis virus B s antigen and the antigenic effect of e in the zooblast for the contrayerva extract.Contrayerva extract (500 μ g/ml) adds among the cell HepG2.2.15, respectively at first and third, five days collecting cell culture fluid, analyze hepatitis virus B s antigen and the antigenic content of e respectively with ferment immunoreation method (ELISA), with the viral s antigen in the Cell sap that does not add medicine and e antigen relatively, detect hepatitis virus B s antigen and e antigen in the different contrayerva extract pair cells in various degree inhibition effect is arranged.
Fig. 2 is that the liver disease plant extract suppresses in the zooblast wild type and anti-liver how mutant hepatitis virus B s antigen and the antigenic effect of e.How the commentaries on classics of saltant hepatitis B disease poison DNA plastid is planted to hepatocyte HepG2 will to contain wild type and anti-liver, how (200 μ g/ml) plants hepatocyte in the difference commentaries on classics to add plant extract (500 μ g/ml) or liver, through cultivating two days later, collect cell culture fluid, analyze hepatitis virus B s antigen and the antigenic content of e respectively with ferment immunoreation method (ELISA), find that the plant extract added can suppress wild type and anti-liver how saltant hepatitis virus B s antigen and e antigen simultaneously; And how liver only can suppress wild type hepatitis virus B s antigen and e antigen.
Fig. 3 is that the liver disease plant extract suppresses the how effect of mutant hepatitis virus B dna replication dna of wild type and anti-liver in the zooblast.To contain wild type and anti-liver how saltant hepatitis virus B DNA plastid will change respectively and plant to HepG2 and HuH7 hepatocyte, how (200 μ g/ml) plants hepatocyte in the difference commentaries on classics to add plant extract (500 μ g/ml) or liver, after cultivating four days, collect the cell training, behind the separating and extracting viral DNA, with the agar electrophoresis with DNA separately after, change the special film (HybondN) that stagnates, warp is to be hybridized by Dig labeled virus DNA, behind the tabletting, obtaining different cells changes in the plant different pharmaceutical to the different inhibitory action of B hepatovirus dna replication dna.C is not for adding the controlled delivery of pharmaceutical agents group; L is for handling liver experimental group how, and it only has inhibitory action to wild type B hepatovirus dna replication dna; B is for handling the test group of plant extract, shows how saltant hepatitis virus B dna replication dna all has inhibitory action to wild type and anti-liver for it.
Embodiment 3: the contrayerva ITS Sequence Identification of medicinal plants
(1) root of contrayerva medical material and stem scrape off crust to reduce microbial contamination through cleaning, and then chop up sheet, freeze with liquid nitrogen and pulverize after crisp;
(2) the DNA abstracting method mainly adopts CTAB, PVPP and different salinity to handle, and gets through precipitation, centrifugal, Chloroform extraction and alcohol precipitation;
(3) introduction of design PCR amplification amplification ITS sequence is as follows to (primer):
5’-CACACCGCCCGTCGCTCCTACCGA-3’
5’-ACTCGCCGTTACTAGGGGAA-3’
Carry out polymerase chain reaction with Tm=60 ℃;
(4) duplicated dna fragmentation after automatic sequence analyser (ABI 3100) analyzes sequence with DNAMAN 8. software carry out sequence similarity degree comparison;
Wherein the extraction step of this step (2) is as follows:
(2-1) extraction buffer:
100mM?Tris.HCl
20mM?EDTA
1M?NaCl
1％CTAB(cetyltrimethylammonium?bromide)
1%PVPP (Polyvinyl Polypyrrolidone adds before use)
(2-2) 3-100mg sample+0.5ml extracts buffer;
(2-3) 70 ℃ of reactions 30 minutes;
(2-4) with chloroform: IAA=24: 1 extraction, with 10, centrifugal 5 minutes of 000g;
(2-5) get supernatant, add the long-pending precipitation buffering liquid of diploid;
50mM?Tris.HCl
10mM?EDTA
40mM NaCl
1％CTAB
Reversed 2 minutes;
(2-6) centrifugal 15 minutes with 13000g;
(2-7) with the 1.2M NaCl of 350 μ l precipitate dissolving with step (2-6);
(2-8) reacted 30 minutes at 37 ℃ with Rnase A;
(2-9) with chloroform: IAA=24: 1 extraction, with 10, centrifugal 5 minutes of 000g;
(2-10) isopropanol of 0.6 times of volume of adding left standstill 15 minutes at 20 ℃;
(2-11) under 4 ℃ centrifugal 20 minutes with 13000g;
(2-12) with the EtOH washing and precipitating thing of 1ml 70%; And
(2-13) precipitate is dissolved in (starting material of every 20mg uses the buffer of 10-25 μ l) in the TE buffer.
The following explanation of contrayerva ITS Sequence Identification result of medicinal plants:
Fig. 4 is contrayerva ribosome ITS sequence alignment result.
The foregoing description only is to give an example for convenience of description, and the interest field that the present invention advocated should be as the criterion so that claim is described certainly, but not only limits to the foregoing description.
SEQUENCE?LISTING
<110>Industrial?Technology?Research?Institute
＜120〉liver disease is with medicinal plant solvent extractable matter
<130>
<160>18
<170>PatentIn?version?3.2
<210>1
<211>219
<212>DNA
<213>Boehmeria?frutescens
<400>1
tcgtaacctg?ccatgcagaa?caacccgcga?acatgtttat?taatctcttg?gcgcgtttgt 60
ggcccttcgg?ggagacaaac?tcgccttgtg?ttgggggccc?ccgactttaa?aacaaatcgg 120
gcgcggtatg?cgccaaggaa?acaataaaag?atcgagccgc?aacctcgagg?caaggacctt 180
ggcgtgttgc?ggtcggtcgc?taaaatgaaa?tgtcgtaac 219
<210>2
<211>155
<212>DNA
<213>Boehmeria?frutescens
<400>2
cgactctcgg?caacggatat?ctcggctctc?gcatcgatga?agaacgtagc?gaaatgcgat 60
acttggtgtg?aattgcagaa?tcccgtgaac?catcgagtct?ttgaacgcaa?gttgcgcccg 120
aagccgttag?gccgagggca?cgtctgcctg?ggctc 155
<210>3
<211>238
<212>DNA
<213>Boehmeria?frutescens
<400>3
acgcaccgtc?gccccctccc?caaaccagtc?tttttgacgg?gattgggtgg?ggcggatatt 60
ggcctcccgt?gcgaatgcgt?gcggctggcc?caaaatcgag?tccccggctt?tgtttgccgc 120
gacattcggt?ggttgtcgat?ctttcggtgc?cctgtcgcgc?gcaaagtagc?ttagccgagg 180
gactgtgagc?aaagacccta?acgcgcgctt?tgatggaccc?attgaggcgc?cctcgacg 238
<210>4
<211>219
<212>DNA
<213>Boehmeria?zoll?ingeriana
<400>4
tcgtaacctg?ccatgcagaa?caacccgcga?acatgtttat?taatctcttg?gcgcgtttgt 60
ggcccttcgg?ggagacaaac?tcgccttgtg?ttgggggccc?ccgactttaa?aacaaatcgg 120
gcgcggtatg?cgccaaggaa?acaataaaag?atcgagccgc?aacctcgagg?caaggacctt 180
ggcgtgttgc?ggtcggtcgc?taaaatgaaa?tgtcgtaac 219
<210>5
<211>155
<212>DNA
<213>Boehmeria?zoll?ingeriana
<400>5
cgactctcgg?caacggatat?ctcggctctc?gcatcgatga?agaacgtagc gaaatgcgat 60
acttggtgtg?aattgcagaa?tcccgtgaac?catcgagtct?ttgaacgcaa?gttgcgcccg 120
aagccgttag?gccgagggca?cgtctgcctg?ggcgt 155
<210>6
<211>237
<212>DNA
<213>Boehmeria?zoll?ingeriana
<400>6
acgcaccgtc?gccccctccc?caaaccagtc?tttttgacgg?gattgggtgg?ggcggatatt 60
ggcctcccgt?gcgaatgcgt?gcggctggcc?caaaatcgag?tccccggctt?tgtttgccgc 120
gacattcggt?ggttgtcgat?ctttcggtgc?cctgtcgcgc?gcaaagtagc?ttagccgagg 180
gactgtgagc?aaagacccta?acgcgcgttt?gatggaccca?ttgaggcgcc?ctcgacg 237
<210>7
<211>194
<212>DNA
<213>Urlica?thunbergiana
<400>7
tcgaacctgc?ttcatgcaaa?aataacccgt?gaataagttc?ttacgttttg?gagcgagtat 60
ggcccgtaca?cgaccattct?tgtcccgctt?ctaacaacca?aaggcgcggg?agcgccaagg 120
aaaatcaaaa?acgaatttga?ctcctgcctc?ggtgcattgc?atcgtgtcag?cgagtgtatt 180
cgataagtca?taac 194
<210>8
<211>156
<212>DNA
<213>Urlica?thunbergiana
<400>8
cgactctcgg?caacggatat?ctcggctctc?gcatcgatga?agaacgtagc?aaaatgcgat 60
acgtggtgtg?aattgcagga?tcccgtgaac?catcgagttt?ttgaacgcaa?gttgcgcccg 120
aagcctttag?gctgagggca?cgtctgcctg?ggcgtc 156
<210>9
<211>239
<212>DNA
<213>Urlica?thunbergiana
<400>9
acgcaccgtt?gccccccaaa?tttcgcagtc?cactacggat?tgttgaggtg?cgtgggggcg 60
taaagtggct?tcccgtcggc?tttgtccggc?ggttggccta?aaaatgaatc?cctagccgcg 120
gtgcgcgcgg?catttggtgg?tcatcaatat?ttcgaacacc?gccgtgcgct?cccgtgtcgc 180
gaaggatgtc?actaataaaa?acccgatgcc?tcgctttgtg?aagagcggag?cttacaacg 239
<210>10
<211>219
<212>DNA
<213>Pilea?spp.
<400>10
tcgtaacctg?ccatgcagaa?caacccgcga?acatgtttat?taatctcttg?gcgtgtttgt 60
ggcctttcgg?ggagacaaac?tcgccttgtg?ttgggggccc?ccgactttaa?aacaactcgg 120
gcgcggtatg?cgccaaggaa?acaaaaaaag?atcgagccgc?aacctcgagg?cgaaaacctc 180
ggcgtgttgc?ggtcgttcgc?tacaattaaa?tgtcgtaac 219
<210>11
<211>155
<212>DNA
<213>Pilea?spp.
<400>11
cgactctcgg?caacggatat?ctcggctctc?gcatctatga?aaaacgtaac?gaaatgcgat 60
acttggtgtg?aattgcagaa?tcccgtgaac?catcgagtct?ttgaacgcaa?gttgcgcccg 120
aagccgttag?gccgagggca?cgtctgcctg?ggctc 155
<210>12
<211>223
<212>DNA
<213>Pilea?spp.
<400>12
acgcaccgtc?gccccctttc?caaacgcatt?gggtggagcg?gatattggcc?tcccgtgcga 60
atgcgtgcgg?ttggcctaaa?atcgagtccc?cggctttgtt?tgccgcgaca?ttcggtggtt 120
gtcgatcttt?cggtgccctg?tcgcgcgcaa?agcagtatag?ccgagggctt?tgagcgaaga 180
tcctgacgcg?cgctttgatg?gacccattga?ggcgccctcg?acg 223
<210>13
<211>219
<212>DNA
<213>Boehmeria?nivea
<400>13
tcgtaacctg?ccatgcagaa?caacccgcga?acatgtttat?taatctcttg?gcgcgtttgt 60
ggcccttcgg?ggagacaaac?tcgccttgtg?ttgggggccc?ccgactttaa?aacaaatcgg 120
gcgcggtatg?cgccaaggaa?acaataaaag?atcgagccgc?aacctcgagg?caaggacctt 180
ggcgtgttgc?ggtcggtcgc?taaaatgaaa?tgtcgtaac 219
<210>14
<211>154
<212>DNA
<213>Boehmeria?nivea
<400>14
gactctcggc?aacggatatc?tcggctctcg?catcgatgaa?gaacgtagcg?aaatgcgata 60
cttggtgtga?attgcagaat?cccgtgaacc?atcgagtctt?tgaacgcaag?ttgcgcccga 120
agccgttagg?ccgagggcac?gtctgcctgg?gcgt 154
<210>15
<211>237
<212>DNA
<213>Boehmeria?nivea
<400>15
acgcaccgtc?gccccctccc?caaaccagtc?tttttgacgg?gattgggtgg?ggcggatatt 60
ggcctcccgt?gcgaatgcgt?gcggctggcc?caaaatcgag?tccccggctt?tgtttgccgc 120
gacattcggt?ggttgtcgat?ctttcggtgc?cctgtcgcgc?gcaaagtagc?ttagccgagg 180
gactgtgagc?aaagacccta?acgcgcgttt?gatggaccca?ttgaggcgcc?ctcgacg 237
<210>16
<211>190
<212>DNA
<213>Boehmeria?densiflora
<400>16
tcgtaacctg?cccagcagaa?tgacccgtga?acaagtgctt?ctatcaaact?cggggcgtgg 60
tccgggtccc?ttataccaaa?gggacccgag?acccgcctcg?cgtcggggcc?ccccgactat 120
aaaccaaaac?tcgggcgcgg?tatgcgccaa?ggaaagtgag?aaggtcgcta?ttttattaga 180
gcgtcgcaat 190
<210>17
<211>155
<212>DNA
<213>Boehmeria?densiflora
<400>17
gactctcggc?aacggatatc?tcggctctcg?catcgatgaa?gaacgtagcg?aaatgcgata 60
cttggtgtga?attgcagaat?cccgtgaacc?atcgagtctt?tgaacgcaag?ttgcgcccga 120
agcctttcgg?ccgagggcac?gtctgcctgg?gcgtc 155
<210>18
<211>235
<212>DNA
<213>Boehmeria?densi?flora
<400>18
acgcatcgtc?gcccccactc?ctcccaggtt?cttctgggcc?gttggtgtgg?ggcggataat 60
ggcctcccgt?acgcttgcgg?tgcggatggc?cgaaaattga?gtccccggct?tcgtttgccg 120
cgacattcgg?tggtcgtcga?ttactcggtg?tcccgtcgtg?cgcgcggccg?gagggctcgc 180
tggaatgacc?cacgcggccc?gttgctggac?ccccagtgat?gcgcgccctt?ctaag 235
Claims (17)
1. one kind is used for the contrayerva root of liver disease or the solvent extractable matter of stem, extraction is from a contrayerva, and this contrayerva itself and mountain Boehmeria (Boehmeria frutescens), the sequence similarity degree is greater than more than 70% between the ribosomal gene of Herba Catharanthi longifolii (Boehmeria zollingeriana), Urtica thunbergiana sieb (Urtica thunbergiana), SHUIMA (Pileaspp), Boehmeria (Boehmeria nivea) or wooden Boehmeria (Boehmeria densiflora);
Wherein the extracting method of this solvent extractable matter comprises the following steps:
(a) this contrayerva root or stem are ground dozen powder, obtain a powder;
(b) with gained powder in a high polar solvent or its solvent mixture treatment step (a), obtain extracting solution, wherein this high polar solvent is water, methanol, ethanol or its mixture; Through evaporating dried solid extraction thing; And
(c) again with the aqueous layer of gained in low polar solvent and this step of aqueous solvent mixture process (b), through evaporating dried solid extraction thing; Wherein this low polar solvent is selected from a group and comprises: ethyl acetate, dichloromethane, chloroform, tetrachloromethane, cyclohexane extraction, normal hexane, n-butyl alcohol ether, benzene, and above-mentioned mixture.
2. solvent extractable matter as claimed in claim 1 is characterized in that, wherein between the ribosomal gene of this mountain Boehmeria sequence shown in sequence recognition number 1,2 and 3.
3. solvent extractable matter as claimed in claim 1 is characterized in that, wherein between the ribosomal gene of this Herba Catharanthi longifolii sequence shown in sequence recognition number 4,5 and 6.
4. solvent extractable matter as claimed in claim 1 is characterized in that, wherein between the ribosomal gene of this Urtica thunbergiana sieb sequence shown in sequence recognition number 7,8 and 9.
5. solvent extractable matter as claimed in claim 1 is characterized in that, wherein between the ribosomal gene of this SHUIMA sequence shown in sequence recognition number 10,11 and 12.
6. solvent extractable matter as claimed in claim 1 is characterized in that, wherein between the ribosomal gene of this Boehmeria sequence shown in sequence recognition number 13,14 and 15.
7. solvent extractable matter as claimed in claim 1 is characterized in that, wherein should the ribosomal gene of wood Boehmeria between sequence shown in sequence recognition number 16,17 and 18.
8. solvent extractable matter as claimed in claim 1 is characterized in that, wherein the medicinal plants that sequence can be used for the medicinal plant extract of liver disease between the ribosomal gene of one of mountain Boehmeria, Herba Catharanthi longifolii, Urtica thunbergiana sieb, SHUIMA, Boehmeria, wooden Boehmeria is identified.
9. solvent extractable matter as claimed in claim 8, it is characterized in that wherein plant extract is that contrayerva DNA to be measured, chain polymerization enzyme reaction amplification amplify through extracting, sequence between the ribosomal gene of sequence fragment sequencing, analyses and comparison mountain Boehmeria, Herba Catharanthi longifolii, Urtica thunbergiana sieb, SHUIMA, Boehmeria, wooden Boehmeria between ribosomal gene, the step of setting up sequence data storehouse between the contrayerva ribosomal gene identify.
10. solvent extractable matter as claimed in claim 1 is characterized in that, wherein this low polar solvent is an ethyl acetate.
11. solvent extractable matter as claimed in claim 1 is characterized in that, wherein liver disease is the hepatitis B treatment.
12. solvent extractable matter as claimed in claim 1 is characterized in that, and can be used for the hepatitis B treatment of anti-sweet how medicine.
13. solvent extractable matter as claimed in claim 1 is characterized in that, wherein the solvent extractable matter of the contrayerva root of liver disease or stem cooperates other liver disease medicines to use.
14, a kind of medicinal plant extract that is used for the hepatitis B treatment, this medicinal plant extract grinds the root or the stem of mountain Boehmeria, Herba Catharanthi longifolii, Urtica thunbergiana sieb or SHUIMA dozen powder, makes with solvent extraction;
Wherein the extracting method of solvent extractable matter comprises the following steps: root or the stem of mountain Boehmeria, Herba Catharanthi longifolii, Urtica thunbergiana sieb, SHUIMA, Boehmeria or wooden Boehmeria are ground dozen powder, extract with high polar solvent, the extract of gained is handled extraction with low polar solvent again and is made;
This high polar solvent treatment step adopts methanol or ethanol or its mixture to carry out;
This low polar solvent treatment step adopts ethyl acetate, dichloromethane, chloroform, tetrachloromethane, cyclohexane extraction, normal hexane, n-butyl alcohol ether, benzene or its mixture to carry out.
15. medicinal plant extract as claimed in claim 14 is characterized in that, wherein the low polar solvent treatment step carries out with ethyl acetate.
16. medicinal plant extract as claimed in claim 14 is characterized in that, wherein hepatitis B treatment medicinal plant extract can further cooperate other liver disease medicines to use.
17. medicinal plant extract as claimed in claim 14 is characterized in that, wherein this hepatitis B is anti-sweet how medicine hepatitis B.
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|---|---|---|---|
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| CN102552344B (en) * | 2010-12-27 | 2014-04-16 | 财团法人工业技术研究院 | Ramie plant extract, pharmaceutical composition containing the same and use thereof in improving liver function and treating liver diseases |
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| CN1300623A (en) * | 1999-12-20 | 2001-06-27 | 张发平 | Chinese patent medicine |
| CN1368065A (en) * | 2001-02-05 | 2002-09-11 | 杨孟君 | Nano medicine 'Saixianning' and its preparing process |
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| CN1300623A (en) * | 1999-12-20 | 2001-06-27 | 张发平 | Chinese patent medicine |
| CN1368065A (en) * | 2001-02-05 | 2002-09-11 | 杨孟君 | Nano medicine 'Saixianning' and its preparing process |
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