CN104688719A - Application of chlorogenic acid to preparing medicine for treating Hutchinson-Gilford syndrome - Google Patents
Application of chlorogenic acid to preparing medicine for treating Hutchinson-Gilford syndrome Download PDFInfo
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- CN104688719A CN104688719A CN201510072776.9A CN201510072776A CN104688719A CN 104688719 A CN104688719 A CN 104688719A CN 201510072776 A CN201510072776 A CN 201510072776A CN 104688719 A CN104688719 A CN 104688719A
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Abstract
The invention provides an application of chlorogenic acid to preparing a medicine for treating Hutchinson-Gilford syndrome. Chlorogenic acid can treat Hutchinson-Gilford syndrome effectively and has a better curative effect than a positive medicine rapamycin; and chlorogenic acid is safe and has few side effects and a favorable clinical application prospect.
Description
Technical field
The present invention relates to the purposes of chlorogenic acid in the medicine of preparation treatment senilism disease.
Background technology
Senilism disease (hutchinson-Gilford syndrome) belongs to heredopathia, fast 5 to 10 times of the process compared with normal of health aging, and patient's complexion is as old man, and organ also fails very soon, causes physiological function to decline.The sick virgin comparatively normal symptom occurred of senilism disease comprises: alopecia, more late long teeth, of short and small stature and subcutaneous fat minimizing etc.Affected children ranges generally can only be lived 7 to 20 years old, and major part all can die from diseases of aging, and as cardiovascular diseases, existing do not have effective Therapeutic Method, only leans on medicine for treatment.
Existing bibliographical information, chlorogenic acid has effectively to be removed interior free yl, has and maintains the normal 26S Proteasome Structure and Function of body cell, prevent and delay the effect of the phenomenons such as tumor, sudden change and aging.But senilism disease is different from the aging of ordinary meaning, senilism disease (Hutchinson-Gilford Syndrome), full name is old and feeble syndrome in one's early years, is a kind of inborn genetic disease, be a kind of rare, fatal genetic diseases, patient starts just aging rapidly from childhood.The research worker of senilism disease WARF is thought, the reason that senilism disease child's cellularity and function are degenerated gradually, because Lamin A protein causes the sudden change of cell function, the unstability of nuclear membrane causes, cause atrophy of epidermis, the various syndrome such as sebaceous gland atrophy, arteriosclerosis, cardiovascular disease occurs.
There is no the pertinent literature report of chlorogenic acid treatment senilism disease at present.
Summary of the invention
Technical scheme of the present invention there is provided the novelty teabag of chlorogenic acid.
The invention provides the purposes of chlorogenic acid in the medicine of preparation treatment senilism disease.
Wherein, described medicine is the medicine raising LMNA gene.
Wherein, described medicine is the medicine raising Lamin A/C (Lamin A/C).
Wherein, described medicine is effective ingredient by chlorogenic acid, adds the preparation that pharmaceutically acceptable adjuvant or complementary composition are prepared from.
Wherein, described pharmaceutical preparation is oral formulations, ejection preparation.
Wherein, described preparation per unit preparation is containing chlorogenic acid 1-1000mg; The Clinical practice dosage of described preparation is: 1-100mg/kg.
Chlorogenic acid of the present invention can effectively treat senilism disease, and therapeutic effect is better than positive drug rapamycin, and chlorogenic acid has been proved to be a kind of safe drugs, and side effect is little, and potential applicability in clinical practice is good.
Obviously, according to foregoing of the present invention, according to ordinary technical knowledge and the customary means of this area, not departing under the present invention's above-mentioned basic fundamental thought prerequisite, the amendment of other various ways, replacement or change can also be made.
Accompanying drawing explanation
Fig. 1 RT-PCR detects the expression of LMNA gene
Fig. 2 Wester-blot detects the expression of Lamin A/C albumen
Detailed description of the invention
Embodiment 1 prepares lyophilized injectable powder with chlorogenic acid
1. the extraction of chlorogenic acid:
Chlorogenic acid crude drug used in the present embodiment, be obtained by extraction, purification in Folium Eucommiae, purity is 99.56%.
2. the preparation of chlorogenic acid lyophilized injectable powder
2.1 prescriptions:
Above prescription is added water for injection, is stirred to and dissolves completely, after regulating pH, with the degerming microporous filter membrane fine straining of 0.22 μm, make 2ml injectable powder 2000 altogether according to the routine operation of aseptic freeze-dried injectable powder, often prop up containing chlorogenic acid 50mg.
Embodiment 2 prepares pill with chlorogenic acid
1. the extraction of chlorogenic acid
The chlorogenic acid used in the present embodiment, be obtained by extraction, purification in Flos Lonicerae, purity is 98.22%.
2. the preparation of chlorogenic acid pill
2.1 prescription
2.2. method for making:
Get appropriate PVP K30, solution is mixed with dehydrated alcohol, get chlorogenic acid and the starch of recipe quantity again, after adopting equivalent dilution method mix homogeneously, add in the alcoholic solution of PVP K30, abundant stirring is obtained soft material afterwards, and adopt stranding ball legal system to obtain chlorogenic acid pill 1000, every pill is containing chlorogenic acid 1mg.
Embodiment 3 prepares oral solution with chlorogenic acid
1. the extraction of chlorogenic acid
The chlorogenic acid used in the present embodiment, be obtained by extraction, purification in Folium Eucommiae, purity is 99.88%.
2. the preparation of chlorogenic acid oral solution
2.1 prescription
2.2 method for making
Get chlorogenic acid and the sodium pyrosulfite of recipe quantity, be dissolved in 10L water for injection, according to the conventional fabrication process of oral liquid, after filtration, sterile filling becomes 1000 oral liquids, and often propping up oral liquid is 10mL, containing chlorogenic acid 300mg.
Embodiment 4 prepares tablet with chlorogenic acid
1. the extraction of chlorogenic acid:
The chlorogenic acid used in the present embodiment, be obtained by extraction, purification in Flos Lonicerae, purity is 98.02%.
2. the preparation of chlorogenic acid tablet
2.1 prescriptions:
2.2 method for makings:
The present embodiment adopts wet granular compression produces chlorogenic acid tablet processed.(1) measure hypromellose by prescription and make aqueous solution; (2), after getting the chlorogenic acid of recipe quantity, starch and mannitol mix homogeneously, add hypromellose aqueous solution, after stirring, make soft material; (3) rule of operation of soft material wet granulation routinely will prepared, sieves, obtains uniform granule after dry and granulate; (4) tabletting after being mixed homogeneously with magnesium stearate by obtained granule, makes 1000 tablets altogether, and every sheet is containing chlorogenic acid 100mg.
Embodiment 5 prepares capsule with chlorogenic acid
1. the extraction of chlorogenic acid:
The chlorogenic acid used in the present embodiment, be obtained by extraction, purification in Flos Lonicerae, purity is 99.27%.
2. the preparation of chlorogenic acid capsule:
2.1 prescriptions:
2.2 method for makings:
Get chlorogenic acid and the Icing Sugar of recipe quantity, mix homogeneously, add 80% alcoholic solution and make soft material, dry, prepare 2000 capsules according to the conventional fabrication process of capsule after granulate, every capsules is containing chlorogenic acid 50mg.
Embodiment 6 prepares granule with chlorogenic acid
1. the extraction of chlorogenic acid
The chlorogenic acid used in the present embodiment, be obtained by extraction, purification in Folium Eucommiae, purity is 98.74%.
2. the preparation of chlorogenic acid granule
2.1 prescriptions:
2.2 method for makings:
Get PVP K30, add water for injection, make solution.After getting the chlorogenic acid of recipe quantity, mannitol and lactose mix homogeneously, add PVP K30 solution, make soft material.According to the conventional fabrication process of granule, soft material is sieved, after dry and granulate, obtains granule.Aseptically subpackage granule, prepares 400 bags of granules, and every bag of granule is containing chlorogenic acid 500mg.
Embodiment 7 prepares powder with chlorogenic acid
1. the extraction of chlorogenic acid:
The chlorogenic acid crude drug that the present embodiment is used, be obtained by extraction, purification in Folium Eucommiae, purity is 98.61%.
2. the preparation of chlorogenic acid powder:
2.1 prescription
Purity is the chlorogenic acid 1000g of 98.61%
2.2 method for making
Get after recipe quantity chlorogenic acid sieves, according to the conventional fabrication process of powder, aseptic subpackaged one-tenth is containing 1000 bottle/bag powders, and every bottle/bag powder is containing chlorogenic acid 1000mg.
Beneficial effect of the present invention is proved below by way of concrete pharmacodynamics test.
Test example 1: chlorogenic acid is on the impact of the expression of the LMNA gene in senilism disease mouse model myocardial cell and Lamin A/C albumen thereof.
1. experiment material
1.1 animal
Zmpste24 mice (Zmpste24 gene knockout) 30, normal mouse 10.
1.2 Experimental agents and instrument
Rapamycin, PCR instrument, total RNA extraction reagent box, cDNA first chain synthetic agent box, electrophresis apparatus, electrophoresis tank, gel imaging instrument;
2. experimental technique
2.1 experiment groupings
By senilism disease model Zmpste24 mice 30, be divided into 4 groups at random, often organize 10 mices.Be set to model control group (control group, n=10), rapamycin treatment group (RAPA group, n=10), chlorogenic acid treatment group (LYS group, n=10) respectively.Meanwhile, the common mice 10 that the do not carry out gene knockout Normal group (NC group, n=10) as experiment is got.
2.2 dosage regimen
This experiment adopts the mode of gastric infusion to carry out drug treatment to each group of mice, and its concrete dosage regimen is as shown in table 1:
Table 1. tests grouping, administering mode and dosage (note: the administration volume of all administration groups is identical)
2.3 test experience
2.3.1RT-PCR method detects the expression of the LMNA gene in each group of mouse cardiac myocytes.
(1) extraction of myocardial cell total serum IgE
RNAprep Pure cultured cell/antibacterial total RNA extraction reagent box (centrifugal column type) is utilized to extract intracellular total serum IgE, should pollute by prevention RNase in the middle of whole leaching process, centrifuge tubes used etc. all do without ferment treatment, timely replacing glove, all operations all carries out on super-clean bench.Operating process by specification carries out, and concrete extraction step is summarized as follows:
After experiment in 1.28 days terminates, adopt strength vertebra dislocation method to put to death, open rapidly thoracic cavity and win heart, packet numbering.The heart of every mouse is shredded uniformly, and afterwards, from every part of heart fragment, take 20mg sample, remaining sample is frozen in-70 DEG C of refrigerators, for subsequent use;
2. in Mei Fen cardiac muscular tissue, add the lysate RL being added with beta-mercaptoethanol in advance of 300 μ l, with electric homogenizer tissue abrasion thoroughly.The RNase-free ddH of 600 μ l is added in homogenate
2the E.C. 3.4.21.64 of O and 10 μ l, mixes rear 56 DEG C of process 20min;
3., by aforesaid liquid centrifugal 5min under 12,000rpm condition, careful Aspirate supernatant uses;
4. in supernatant, slowly add the dehydrated alcohol of 0.5 times of volume, be transferred in adsorption column CR3 after mixing, the centrifugal 1min of 12,000rpm, discards waste liquid, stays adsorption column;
5. add protein liquid removal RW1 in adsorption column, remove albumen, centrifugal 1min, discards waste liquid afterwards;
6. add DNase I working solution, remove the DNA on cylinder;
7. after cleaning adsorption column respectively with protein liquid removal and rinsing liquid, adsorption column is put in collecting pipe, fully volatilizes residual liquid above;
8. in adsorption column, add 60 μ lRNase free ddH2O, after placing 2min, the centrifugal 2min of 12,000rpm, obtains mRNA sample;
(2) reverse transcription of mRNA
The first chain cDNA that the RNA solution synthesis utilizing cDNA first chain synthetic agent box and said extracted to obtain is corresponding.Concrete process of reverse-transcription operates to specifications, is summarized as follows:
1. get being placed on ice bath without enzyme centrifuge tube of 200 μ l, and in wherein adding following solutions:
2., after centrifugal, centrifuge tube is positioned in PCR instrument, 60 DEG C hatch 10min after, brief collect liquid after, add following reagent after being transferred to rapidly cooled on ice 3min:
3. centrifuge tube mixed gently and after brief centrifugation, PCR instrument 45 DEG C be set and hatch 60min, afterwards 95 DEG C of heating 10min.30ul RNase-Free ddH is added in the most backward cDNA solution obtained
2o is diluted to 50ul, remembers the first chain cDNA.
Preserve under the product obtained is placed in-20 DEG C of conditions.
(3) RT-PCR is quantitative
EvaGreen fluorescent dye is combined with the DNA of double-strand can produce very strong fluorescence, and by detecting final fluorescence intensity, we can obtain reacting the total amount generating DNA.CDNA product, LMNA primer (forward primer 5 '-GCAAGATACACCCAAGAGCC-3 ' synthesized in fluorescent dye, (1) (2) step is added in test tube, downstream primer 5 '-ACACCTGGGTTCCCTGTTC-3 '), carry out RT-PCR after grouping mix homogeneously and detect body series and use RNase-free water to replace cDNA products group to contrast for NC.
2.3.2Lamin the analysis of A/C protein expression
(1) Western Blot detects
1. taken out by biological specimen frozen before, every increment product cut the cardiac muscular tissue of about 80mg, add appropriate PBS homogenate, supernatant discarded, add the cell pyrolysis liquid of 5 times of volumes, place 30min on ice, 10000rpm4 DEG C of centrifugal 1h, collect supernatant;
2. the protein concentration of BCA standard measure sample;
3. protein sample is made identical concentration, add sample buffer, boiling water boiling 5min;
4. electrophoresis is carried out according to a conventional method;
5. pvdf membrane is taken off, TBS soaks 10min, 5% defatted milk powder closes 1h, TBS eluting 2 times, adds primary antibodie (1:300), after hatching 2h, TBST eluting 2 times, add two anti-(1:4000) 1h, TBST eluting again 2 times, afterwards by observed result after the colour developing of Western-blue dye liquor.
3. experimental result
3.1RT-PCR method detects the expression of the LMNA gene in each group of mouse cardiac myocytes
RT-PCR experimental result is as shown in Figure 1: compared with NC group, the expression of the LMNA of control group is all obviously lowered.The LMNA level of LYS treatment group significantly raises, and compared with positive drug RAPA treatment group, its LMNA gene expression is increased more significantly.
3.2Western Blot electrophoresis tests result
Electrophoresis tests result is as shown in Figure 2: compared with NC group, the expression of the lamin A/C albumen of control group all significantly reduces.The laminA of LYS treatment group increases significantly, and compared with positive drug RAPA treatment group, the expression of its laminA albumen is significantly increased.This experimental result, the expression result of mRNA corresponding to gene aspect LMNA is basically identical.
4 statistical procedures
The experimental data of continuous variable form represents with x ± sd, comparison two sample t-test between 2 groups of data.Adopt SPSS13.0 statistical software, P<0.05 is for there being statistical significance.
5. conclusion
Above-mentioned test can be found out, from gene and albumen two aspects have detected LYS be used for the treatment of senilism disease time, the expression of LMNA gene and corresponding albumen thereof in cardiac muscular tissue.Result of the test shows, and chlorogenic acid treatment senilism disease can raise the expression of LMNA gene and albumen thereof significantly, and the treatment for senilism disease has significant curative effect, this effect, compared with positive drug rapamycin, has significant difference.Theoretical foundation is provided for chlorogenic acid clinical treatment senilism disease.
Claims (6)
1. the purposes of chlorogenic acid in the medicine of preparation treatment senilism disease.
2. want the purposes described in 1 according to right, it is characterized in that: described medicine is the medicine raising LMNA gene.
3. purposes according to claim 1, is characterized in that: described medicine is the medicine raising Lamin A/C (Lamin A/C).
4. the purposes according to claim 1-3 any one, is characterized in that: described medicine is effective ingredient by chlorogenic acid, adds the preparation that pharmaceutically acceptable adjuvant or complementary composition are prepared from.
5. purposes according to claim 4, is characterized in that: described pharmaceutical preparation is oral formulations, ejection preparation.
6. purposes according to claim 5, is characterized in that: described preparation per unit preparation is containing chlorogenic acid 1-1000mg; The Clinical practice dosage of described preparation is: 1-100mg/kg.
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| CN102300578A (en) * | 2008-12-01 | 2011-12-28 | 延寿有限责任公司 | Methods And Compositions For Altering Health, Wellbeing, And Lifespan |
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| CN102300578A (en) * | 2008-12-01 | 2011-12-28 | 延寿有限责任公司 | Methods And Compositions For Altering Health, Wellbeing, And Lifespan |
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Inventor after: Zhang Jie Inventor after: Huang Wang Inventor before: Zhang Jie Inventor before: Jia Jing Inventor before: Huang Wang |
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