CN104644624A - Application of chlorogenic acid in preparation of medicines for treating parkinson disease - Google Patents

Abstract

The invention discloses an application of chlorogenic acid in preparation of medicines for treating a parkinson disease. The chlorogenic acid is beneficial to reduction of the toxicity of substantia nigra and striatum neurons and activation of the autophagy activity of substantia nigra and striatum neurons in a midbrain, beneficial to adjustment of blood ketone content of a parkinson disease mice model, and beneficial to inhibition of apoptosis of substantia nigra cells in the parkinson disease mice model, and plays positive improvement and treatment roles in symptoms, pathology and the like of the parkinson disease.

Description

The purposes of chlorogenic acid in the Parkinsonian medicine of preparation treatment

Technical field

The invention belongs to the new pharmaceutical usage of chlorogenic acid, particularly relate to the purposes of chlorogenic acid in the medicine of preparation treatment parkinson.

Background technology

Parkinson disease (PD) have another name called Parkinsonism, are one of modal neurodegenerative diseases.Main pathological change is clinically substantia nigra of midbrain compact part (SNc) dopamine (DA) serotonergic neuron optionally dead, disappearance, cause striatum DA not enough, thus cause the disorder of Basal ganglia dysfunction of nervous regulation, the cause of disease of its morbidity, not yet clear so far.But research is verified, this disease and oxidative stress and mitochondrial function exception etc. have substantial connection, and therefore, alleviating oxidativestress damage can as drug target to treat parkinson disease.In addition, up-to-date research display, ketoboidies can improve the clinical symptoms of the nervous system degenerative diseases such as parkinson disease, demonstrates clear and definite anti-oxidation protection effect, and in neuroprotective, has certain effect.The black substance dense area apoptosis of Parkinsonian, is the important pathologic basis of this disease, suppresses this kind of apoptosis, also can play a positive role to the treatment of this disease.

Chlorogenic acid (chlorogenic acid CGA) has another name called caffeotannic acid, the depside be made up of caffeic acid (caffeic acid CA) and quinic acid (quinic acid QA), its chemistry 3-o-caffeoyl guinic acid (3-o-caffeoylquinic acid CGA) by name.

CAS No.：327-97-9

CAS Name：

[1S-(1a,3b,4a,5a)]-3-[[3-(3,4-Dihydroxyphenyl)-1-oxo-2-propenyl]oxy]-1,4,5-trihydroxycyclohexanecarboxylic acid

System name: 1,3,4,5-tetrahydroxy cyclohexane-carboxylic acid-(Caffeic acid ester)

Chemical structural formula:

Chlorogenic acid be plant in the process of carrying out aerobic respiration, through phosphopentose pathway intermediate product synthesis a kind of Phenylpropanoid Glycosides class material.It extracts (usually extracting from plant), synthetic technology is ripe, chlorogenic acid by open applications in food, health product, multiple field such as cosmetics and medicine.Because it is present in common various vegetable and fruits widely; there is multiple biological activity, as: cardiovascular protective effect, antioxidation, uvioresistant and radiation resistance, antimutagenic and antitumaous effect, antibacterial action, antivirus action, blood lipid-reducing blood sugar-decreasing effect, immunoregulation effect etc.All have a wide range of applications in the field such as medication chemistry and food.

Summary of the invention

The object of the invention is to: the new pharmaceutical usage that chlorogenic acid is provided, especially the purposes of chlorogenic acid in the medicine of preparation treatment parkinson.

The object of the invention is realized by following technical proposals:

The purposes of chlorogenic acid in the Parkinsonian medicine of preparation treatment.

Wherein, described medicine is the medicine alleviating oxidativestress damage.

In such scheme, described oxidativestress damage refers to the causes for pathological of body due to self, causes free radical, active oxygen or active nitrogen to produce too much and antioxidant ability of organism weakens, and destroys the normal equilibrium of body oxidation-reduction, causes unbalance phenomenon.The enzyme that body now shows a series of Antioxidative Defense System reduces, as the minimizing of antioxidase SOD, glutathion peroxidase (GSH-Px) and catalase (CAT) etc.And the free radical produced etc. are by oxidation reaction, produce more malonaldehyde (MDA), thus when oxidativestress damage, the MDA value of body can obviously raise again.

Wherein, described medicine is the medicine raising HMGCS2 gene.

In such scheme, described HMGCS2 gene is the gene of coding trihydroxy trimethyl CoA synthase 2, and its expression becomes positive correlation with corresponding enzymatic activity.Also be proportionate with the ketoboidies generation of body.

Wherein, described medicine is the medicine activating trihydroxy trimethyl CoA synthase 2.

In such scheme, trihydroxy trimethyl CoA synthase 2 is ketoplastic key enzymes in body, and it is present in various internal organs widely.Its generation level that is active and body ketoboidies is proportionate.

Wherein, described medicine promotes ketoplastic medicine.

In such scheme; ketoboidies is the product that fatty acid incomplete oxidation produces; up-to-date result of study display; ketoboidies is for neurodegenerative diseases; as having energy supplement and anti-oxidation protection effect in parkinson disease, wherein mechanism it be unclear that, but research is verified; increase the ketoboidies content in this type of Disease body, certain control action can be produced to the state of an illness.

Wherein, described medicine is effective ingredient by chlorogenic acid, adds the preparation that one or more pharmaceutically acceptable pharmaceutical excipients are prepared from.

In such scheme, chlorogenic acid both can as sole active ingredient, also can as one of effective ingredient.

Wherein, described preparation is oral formulations or ejection preparation.

In such scheme, concrete dosage form can be the various pharmaceutical dosage form of powder, pill, oral solution, tablet, capsule, granule, lyophilized injectable powder etc.

Wherein, the unit formulation of described preparation is containing chlorogenic acid 1-1000mg.

Wherein, the Clinical practice dosage of described preparation is: 1-100mg/kg.

Aforementioned main formula case of the present invention and each further selection scheme thereof can independent assortment to form multiple scheme; be the present invention can adopt and claimed scheme; those skilled in the art can understand there is multiple combination according to prior art and common practise after understanding the present invention program; be the claimed technical scheme of the present invention, do not do exhaustive at this.

Beneficial effect of the present invention: the present invention's test shows, chlorogenic acid has certain therapeutic effect for parkinson disease, the result display of embodiment, chlorogenic acid, by alleviating oxidativestress damage, activating key enzyme trihydroxy trimethyl CoA synthase 2 activity of ketoboidies synthesis, regulating ketoboidies biosynthesis and suppress the aspects such as nigral cell apoptosis, improves Parkinsonian symptom and concrete pathological state and treats.

Specifically:

1. chlorogenic acid can reduce the oxidativestress damage degree of disturbances in patients with Parkinson disease;

2. can raise HMGCS2 gene, this gene is the gene of coding synthesis downstream enzyme;

3. by raising HMGCS2 gene, the enzyme (trihydroxy trimethyl CoA synthase 2) activated corresponding to it is active, and this enzyme makes body synthesize the key enzyme of ketoboidies, and its activity becomes positive correlation with content with the biosynthesis of ketoboidies.

4. increase body ketone body levels, have good curative effect for Parkinsonian, chlorogenic acid can regulate the ketone body levels raising rat model.

5. chlorogenic acid can suppress the apoptosis of patient's substantia nigra neuron cell.In brief, the pathological characters of parkinson morbidity comprises oxidativestress damage and substantia nigra neuron apoptosis etc., embodiment proves that chlorogenic acid can reduce oxidative stress, suppress nigral cell apoptosis, in addition chlorogenic acid can also raise gene corresponding to ketoboidies synthesis key enzyme and enzymatic activity, regulates the generation of ketoboidies.

To sum up, chlorogenic acid is of value to the toxicity reducing black substance and striatal neuron, the autophagy activating substantia nigra of midbrain and striatal neuron is active, the blood ketone content being of value to parkinson disease mouse model regulates, be of value to the apoptosis suppressing parkinson disease mouse model substantia nigra cell, for Parkinsonian disease and pathology etc., there is positive improvement and therapeutical effect.

Accompanying drawing explanation

Fig. 1 is the relative expression quantity of HMGCS2 gene in each group of mouse kidney of the embodiment of the present invention 2;

Fig. 2 is relative value's (100%) of trihydroxy trimethyl CoA synthase 2 activity of each group of mice of the embodiment of the present invention 2;

Fig. 3 is the apoptosis flow cytometer detection datagram of each group of mice nigral cell of the embodiment of the present invention 2.

Detailed description of the invention

Following non-limiting examples is for illustration of the present invention.

Embodiment 1. chlorogenic acid is investigated pharmacodynamics in the body of mouse model of Parkinson's disease

Experiment material

1.1 experiment reagents and instrument

Chlorogenic acid (chapter bio tech ltd, Sichuan nine) rotenone (SIGMA), dimethyl sulfoxide (DMSO, SIGMA), Semen Maydis oil (Luhua Group Co., Ltd., Shandong), Mus α-SYN monoclonal antibody, SABC immunohistochemical reagents box are purchased (Wuhan doctor's moral company), Mus tyrosine hydroxylase monoclonal antibody, rabbit microtubule-associated protein light chain 3 polyclonal antibody (the green skies), mda content and superoxide dismutase, glutathion peroxidase and hydroperoxidase reagent box (biological engineering company limited is built up in Nanjing).All the other reagent are domestic commercially available analytical pure.

1.2 animal models and grouping

This experiment adopts the method for subcutaneous injection rotenone to build Parkinsonian mouse model.Buy bull C57BL/6 mice 30, body weight 25-28g, is provided by Chinese Academy of Sciences's Shanghai Experimental Animal Center.Mice is divided into Normal group (NC group, n=10), rotenone (PD model group, n=10), chlorogenic acid treatment group (LYS group, n=10) by body weight stochastic averagina.In experiment, rotenone is first dissolved in the DMSO of extremely low volume, is then dissolved in Semen Maydis oil (being made into 2mg/ml oil solution).Rotenone group (PD model group) every day is in neck dorsal sc injection rotenone oil solution (3mg/kg Mouse Weight), Normal group (NC group) is in the isopyknic Semen Maydis oil of neck dorsal sc injection (DMSO containing respective volume), chlorogenic acid treatment group (LYS group) every day in neck dorsal sc injection rotenone, simultaneously according to dosage 30mg/kg/d gavage chlorogenic acid-.The free diet of all mices of experimental session and drinking-water, temperature 22 ± 2 DEG C, relative humidity (60 ± 15) %.Observe animal general state every day show and record animal behavior change, continue 5 weeks.

2. test Testing index

2.1 WMT-200 water are got lost

WMT-200 water video analytic system (Chengdu TME Technology Co., Ltd.) of getting lost adopts computer vision related algorithm, the behavior state of real-time tracking animal, and analysis and record animal search for the time needed for target after entering water: before experiment, mice is placed on platform and adapts to 20s, being faced the wall and meditated from different quadrant at random by mice subsequently inserts in pond, mice stops record after climbing up platform 5s, the longest writing time is that 30s is (if can not appear on the stage in 30s, guide mice to climb up platform and adapt to 10s, finally mice is dried and put people's mouse cage).So mice is inserted swimming pool, be 1 group for 5 times, measure four groups of average slip time of mice respectively, to evaluate its Spatial learning ability.Within 2nd, 3,4 and 5 week, test by above-mentioned steps after experiment process.

2.2 oxidative stress Indexs measure

Oxidative stress refers to that body is when suffering various destructive stimulus, and in body, high activity molecule such as reactive oxygen free radical and active nitrogen free radical produces too much, and degree of oxidation exceeds the removing of oxide, oxidative system and antioxidant system unbalance, thus cause tissue injury.The enzyme that body now shows a series of Antioxidative Defense System reduces, as the minimizing of antioxidase SOD, glutathion peroxidase (GSH-Px) and catalase (CAT) etc.And produce more malonaldehyde (MDA).SOD, GSH-Px, CAT and MDA are detected, the degree of body oxidativestress damage can be reflected, for Parkinsonian disease etc., there is directive significance.

After 5 weeks behavioral value, adopt de-cervical approach to break end rapidly and get brain, substantia nigra of midbrain and striatum are isolated rapidly in ice face, and glass homogenizer makes 10% tissue homogenate ,-80 DEG C of Refrigerator stores.With BCA standard measure albumen during experiment, according to test kit description, chemical colorimetry is adopted to detect SOD, GSH-Px, CAT activity and MDA content in cerebral tissue.

3. experimental result

The behavior state of 3.1 each group mices and body weight

Within 10th day, lethargy is there is in PD model group mice from experiment, slowly movable, hair is loose and matt, appetite obviously goes down, in succession there is health flexing, hypokinesia in beginning in the 16th day, time and with tetanic, performance of trembling, the typical behaviour feature of display parkinson, shows parkinsonian mouse modeling success.There is not health flexing, hypokinesia in all the other 2 groups of mices, tetanic, performance of trembling.Each group of mice measured body weight every day is found, PD model group Mouse Weight increase comparatively other respectively group slowly, but the difference that compares between group that there are no significant.

3.2 respectively organize mouse brain black substance oxidative stress index

The measurement result of the black substance oxidative stress index of each group of mice is shown, the mice of PD model group, the value that the mice that its determined every biochemical indicator all departs from NC group measures.The every oxidative stress index of mice of LYS treatment group all obtains to be recovered largely.The result measured is as shown in table 1:

Table 1 mice substantia nigra of midbrain oxidative stress index ( n=10)

* p < 0.05 is compared with NC group; △ p < 0.05 is compared with PD model group

MDA, GSH-Px, SOD, CAT of PD model group are all significantly higher than NC group (P<0.05), significant difference (P<0.05) is all had between each group of data of LYS treatment group and PD model group, and and between NC group, there is no significant difference (p>0.05).(3) right side of mice striatum oxidative stress index

The measurement result of right side of mice striatum oxidative stress index is as shown in table 2:

Table 2 right side of mice striatum oxidative stress index ( n=10)

* p < 0.05 is compared with NC group; △ p < 0.05 is compared with PD model group

MDA, GSH-Px, SOD, CAT of PD model group are all significantly higher than NC group (P<0.05), each group of data of chlorogenic acid treatment group all have significant difference (P<0.05) compared with PD model group, and and between NC group, there is no significant difference (p>0.05).(4) water is got lost neurobehavioral analysis

2nd, 3,4 and 5 weekends after experiment process, measure the average slip time of mice respectively, result shows, PD model group mice the 3rd weekend average slip time be significantly higher than NC group (P<0.05), the 4th and 5 weekend differences extremely significantly (P<0.01).And LYS treatment group mice the 3rd weekend average slip time significantly lower than PD model group (P<0.05), the 4th and 5 weekend differences extremely significantly (P<0.01), experiment concrete outcome is as shown in table 3:

Table 3 mice experiment process after each weekend average slip time ( n=5)

* p < 0.05, * * p < 0.01 is compared with NC group; △ p < 0.05, △ △ p < 0.01 is compared with PD model group

(5) TH, α-SYN and the reaction of LC-3B immuno positive

Gather image statistics analysis to find, PD model group mice black substance TH positive cell number significantly shines and LYS group (P<0.01) lower than NC, α-SYN, LC3-B positive cell number is significantly higher than NC group and LYS group (P<0.01).LYS treatment group compares with PD model group, and TH positive neuron digital display work increases, and α-SYN, LC3-B positive neuron digital display work declines (P<0.05), and specific experiment result is as shown in table 4:

Table 4 mice substantia nigra of midbrain dense area TH, α-SYN and LC3-B cell number ( n=10)

* * p < 0.01 is compared with NC group; △ p < 0.05 is compared with PD model group

Gather image statistics analysis to find, PD model group mouse striaturn TH immuning positive cell digital display work shines and LYS treatment group (P<0.01) lower than NC, α-SYN, LC3-B positive cell number is significantly higher than NC and LYS group (P<0.01).LYS treatment group and PD model group compare, and TH immuning positive cell number and α-SYN, LC3-B positive neuron number are significantly higher than PD model group, and have significance difference (P<0.05), specific experiment result is as shown in table 5:

Brain striatum TH, α-SYN and LC3-B cell number in table 5 mice ( n=10)

* * p < 0.01 is compared with NC group; △ p < 0.05 is compared with PD model group,

4 statistical procedures

The experimental data of continuous variable form with represent, comparison two sample t-test between 2 groups of data.Adopt SPSS13.0 (SolutionsStatistical Package for the Social Sciences/Statistical Productand Service Solutions) statistical software, P<0.05 is for there being statistical significance.

5. try to make a comment

In sum, chlorogenic acid effectively can improve the pathological symptom of the parkinson mouse model that rotenone manufactures, reduce the toxicity of black substance and striatal neuron, the autophagy activating substantia nigra of midbrain and striatal neuron is active, parkinson disease is had to the effect of prevention and therapy.

Embodiment 2. chlorogenic acid is active on mouse model of Parkinson's disease kidney HMGCS2 gene and corresponding trihydroxy trimethyl CoA synthase 2, in body the impact of ketoboidies content and nigral cell apoptosis situation investigate

1. experiment material

1.1 experiment reagents and instrument

Chlorogenic acid, madopar (Benserazide sheet, Shanghai company limited of Roche Group), microplate reader (the vigorous mk3 microplate reader of Finland's thunder), real-time fluorescence quantitative PCR instrument (Applied biosystems; ABI), total RNA extraction reagent box (Tian Gen biochemical technology company limited), TIANScriptcDNA first chain synthetic agent box (Tian Gen biochemical technology company limited), Annexin-FITC-PI two dye apoptosis detection kit (the green skies), mice ketoboidies detect ELISA kit (Sigma), flow cytometer (Beckman) etc.

1.2 animal models and grouping

BALB/C mice 30, random taking-up 10, as Normal group (NC group), injects the contrast liquid (dimethyl sulfoxide: 10% Polyethylene Glycol=1:1) of equivalent every day.Other 30 mices, press the dosage of 1mg/kg every day, lumbar injection rotenone, successive administration builds up PD model mice in 28 days, PD model mice stochastic averagina is divided into 3 groups, called after respectively: madopar treatment group (positive drug treatment group), chlorogenic acid treatment group (LYS group) and Parkinson disease model matched group (PD model group), the 28th day after injecting rotenone continuously, observe mice, find mouse body flexing, hypokinesia, time and with tetanic, to tremble performance, the typical behaviour feature of display parkinson, show parkinsonian mouse modeling success.In the 29th day to the 36th day chlorogenic acid treatment group and madopar treatment group, press the dosage of 60mg/kg and 125mg/kg respectively, gastric infusion.PD model group and Normal group, then inject isopyknic normal saline.

2. measuring index

The blood ketone content detection of 2.1 each group mices

Extractd eyeball in the 36th day that tests to each group of mice and get blood, be placed in by EDTA rinse in vitro by got blood, number respectively, the centrifugal 15min of 2000g/min, gets supernatant.The blood ketone content that ELISA kit measures each group of mice is detected with mice ketoboidies.

The detection of HMGCS2 gene and corresponding enzymatic activity in 2.2 mouse kidneys

Cervical dislocation method is got mouse kidney, is divided into A and B two parts to do following experiment respectively after putting to death mice:

(1) in mouse kidney, the expression of HMGCS2 gene detects

1. the extraction of the total mRNA of mouse kidney cell

Treating excess syndrome tests mouse kidney A part tissue homogenate, adds the lysate being added with beta-mercaptoethanol of 500 μ l wherein, mixing 5min.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; Strict concrete steps of pressing total RNA extraction reagent box extract mRNA, frozen for subsequent use in-80 DEG C of refrigerators.

2. the synthesis of cDNA first chain

The mRNA solution that the present embodiment adopts cDNA first chain synthetic agent box and above-mentioned steps to extract and obtains synthesizes the first corresponding chain cDNA.Concrete process of reverse-transcription operates to specifications, cDNA first chain obtained, frozen in-20 DEG C carry out preserving, for subsequent use.

3. RT-PCR is quantitative

SYBER GREEN fluorescent dye is combined with the DNA of double-strand can produce very strong fluorescence, by detecting and record final fluorescence intensity, can obtain the total amount generating DNA.CDNA product, HMGCS2 primer (forward primer: GTATGGGCTTCTGTTCG synthesized in fluorescent dye, (1) (2) step is added in test tube; Downstream primer: GGCGTTGGTGGTATCTA) carry out RT-PCR after mix homogeneously and detect body series and use RNase-free water to replace cDNA products group to contrast for NTC.Each sample all sets β-actin primer sets as relative value, carries out relative quantification.

(2) detection of trihydroxy trimethyl CoA synthase 2 activity

Trihydroxy trimethyl CoA synthase 2 is the enzymes corresponding to HMGCS2 gene, the present embodiment detects its activity, specific experiment step is as follows: the mouse kidney tissue getting above-mentioned B part, add after anhydrous acetonitrile leaves standstill 10min after adding PBS homogenate, with the centrifugal 20min of the rotating speed of 5000g/min, get supernatant, use mice trihydroxy trimethyl CoA synthase 2ELISA test kit, detect the activity of enzyme.Using the detected value of NC group as benchmark, all the other are respectively organized mice detected value and carry out statistics and analysis with the ratio (%) of the detected value of NC group as final data.

The apoptosis test experience of 2.3 mice substantia nigra neurons

The present embodiment adopts the apoptosis situation of Annexin-V-FITC-PI two dye apoptosis test kit to nigral cell to detect.Specific experiment operation is as follows:

1. by after sacrifice, open cranium immediately and take out full brain, then according to mouse brain collection of illustrative plates black substance location, take out fast Nigral Tissue and also put into Trizol lysate, by after the abundant homogenate of Nigral Tissue of taking off, with PBS abundant re-suspended cell counting gently;

2. 1 × 10 is got ⁵individual resuspended cell, centrifugal 5 minutes of 1000g, after abandoning supernatant, add 195 μ l Annexin V-FITC in conjunction with liquid gently with mixing with cells; Add 5 μ l Annexin V-FITC subsequently, mix gently; Room temperature lucifuge hatches 10min again; Finally use 1000g centrifugal 5 minutes, abandon supernatant, add 190 μ l Annexin V-FITC in conjunction with liquid re-suspended cell gently;

3. add 10 μ l propidium iodide stain liquid, mix gently, ice bath lucifuge is placed; FITC mono-dye group and the mono-dye group of PI are set respectively, for the adjustment compensated, these two groups difference only a kind of fluorescent dye of single dye;

4. the cell handled well is carried out flow cytomery immediately, Annexin V-FITC is green fluorescence, and PI is red fluorescence.

3. experimental result

The blood ketone content detection result of 3.1 each group mices

The blood ketone content detection result display of each group mice in embodiment, the mice of PD model group, its blood ketone content, compared with NC group, decreases drastically, and has significant difference (p<0.05) between two groups.After using chlorogenic acid treatment, its blood ketone content to some extent significantly improves, its value does not have significant difference (p>0.05) compared with NC group, and the mice of positive control medicine madopar treatment, its blood ketone content raises obvious not, and still have significant difference (p<0.05) between normal group, specific experiment result is as shown in table 1.

The measurement result of table 1 different group Mouse Blood ketone concentration (mmol/L, )

*: p<0.05 and PD model group compares #:p<0.05 compared with NC group

Compared with madopar group, ×: p<0.05

The testing result of HMGCS2 gene and corresponding enzyme (trihydroxy trimethyl CoA synthase 2) activity in 3.2 mouse kidneys

(1) each relative quantification testing result organizing HMGCS2 gene in mouse kidney

In the present embodiment, using β-actin as reference gene, using HMGCS2 gene relative to the expression of β-actin as the numerical value of final statistics, the result display obtained: the HMGCS2 gene expression dose in the mouse kidney tissue of PD model group, compared with the HMGCS2 expression in NC group mouse kidney, there is obvious downward, illustrated that in made parkinson mouse model, HMGCS2 down regulation of gene expression is a part for its pathological characters.And with chlorogenic acid treatment after mouse kidney in, the expression of its HMGCS2 gene has had significant rise, and there is significant difference (p<0.05) between model group, HMGCS2 gene level in the mouse kidney tissue of positive control drug madopar treatment group also has a small amount of increasing, and still has significant difference (p<0.05) with the HMGCS2 gene expression dose of chlorogenic acid treatment group. specific experiment result as shown in Figure 1:

(3) in each group mouse kidney, the relative activity of trihydroxy trimethyl CoA synthase 2 detects

The present embodiment adopts the ELISA kit of trihydroxy trimethyl CoA synthase 2 to detect this enzymatic activity in each group of mouse kidney, and with the measured value of NC group for benchmark, calculate all the other respectively and respectively organize the enzymatic activity of mice relative to NC group, concrete experimental result is as shown in Figure 2: it is active that LYS group can strengthen trihydroxy trimethyl CoA synthase 2 significantly, its value, compared with other organizes mice, all has significant difference (p<0.05).

The apoptosis test experience result of 3.3 mice nigral cells

The apoptosis of Midbrain nigra neurons is one of Parkinsonian important indication, the apoptosis test experience result of mice nigral cell as shown in Figure 3: the mouse brain nigral cell apoptosis of PD group is comparatively serious, embodies obviously parkinson pathological symptom.Many Mei Ba and chlorogenic acid treatment after mice, the percentage ratio of its apoptosis has had larger downward, and two treatment groups all demonstrate very positive anti-apoptotic ability, experimental result is as shown in Figure 3: wherein, D4 Regional Representative apoptotic cell, D3 Regional Representative normal cell, all the other are damaged or dead cell

4 statistical procedures

The experimental data of continuous variable form with represent, 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

The present embodiment has investigated chlorogenic acid affects situation to the indices of the parkinson mouse model caused by rotenone.Result shows; chlorogenic acid can improve the expression of HMGCS2 gene in parkinson mouse kidney significantly; and then the trihydroxy trimethyl CoA synthase 2 corresponding to HMGCS2 also demonstrates stronger activation; this enzyme is the crucial catalyzing enzyme of ketoboidies synthesis in body; ketoboidies has protection and antioxidation for Parkinsonian; also show the blood ketone testing result of each group of mice, chlorogenic acid effectively can regulate the blood ketone content of mice, urgees to make it restore to normal level.In addition, the apoptosis test experience of mice nigral cell is found, chlorogenic acid has the ability of certain opposing nigral cell apoptosis, for Parkinsonian process, there is certain control action, to sum up, described embodiment as the Parkinsonian drug research and development for the treatment of, provides theoretical basis for chlorogenic acid.

Embodiment 3: prepare 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 Flos Lonicerae, purity is 99.52%.

2. the preparation of chlorogenic acid lyophilized injectable powder

2.1 prescriptions:

Above prescription is dissolved in water for injection completely, after filtration, then uses the degerming microporous filter membrane fine straining of 0.22 μm, after regulating pH, make 2ml injectable powder 1000 altogether according to the routine operation of sterile powder injection, often prop up containing chlorogenic acid 40mg.

Embodiment 4: prepare pill 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 98.33%.

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 5: prepare 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 Herba Arctii leaf, purity is 99.04%.

1. the preparation of chlorogenic acid oral solution

2.1 prescription

2.2 method for making

Get chlorogenic acid and the sodium sulfite 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 200mg.

Embodiment 6: prepare 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 98.37%.

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 lactose 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 7: prepare capsule 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 99.25%.

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 8: prepare 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 sucrose 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 9: prepare powder with chlorogenic acid

2. the extraction of chlorogenic acid:

The chlorogenic acid crude drug that the present embodiment is used, be obtained by extraction, purification in Herba Arctii leaf, purity is 98.82%.

2. the preparation of chlorogenic acid powder:

2.1 prescription

Purity is the chlorogenic acid 1000g of 98.82%

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.

The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.

Claims (10)

1. the purposes of chlorogenic acid in the Parkinsonian medicine of preparation treatment.

2. purposes as claimed in claim 1, is characterized in that: described medicine is the medicine alleviating oxidativestress damage.

3. purposes as claimed in claim 1, is characterized in that: described medicine is the medicine raising HMGCS2 gene.

4. purposes as claimed in claim 1, is characterized in that: described medicine is the medicine activating trihydroxy trimethyl CoA synthase 2.

5. purposes as claimed in claim 1, is characterized in that: described medicine promotes ketoplastic medicine.

6. purposes as claimed in claim 1, is characterized in that, described medicine suppresses the apoptotic medicine of Parkinsonian's substantia nigra.

7. the purposes as described in claim arbitrary in claim 1 to 6, is characterized in that: described medicine is effective ingredient by chlorogenic acid, adds the preparation that one or more pharmaceutically acceptable pharmaceutical excipients are prepared from.

8. purposes as claimed in claim 7, is characterized in that: described preparation is oral formulations or ejection preparation.

9. purposes as claimed in claim 7 or 8, is characterized in that: the unit formulation of described preparation is containing chlorogenic acid 1-1000mg.

10. the purposes as described in claim 7,8 or 9, is characterized in that: the Clinical practice dosage of described preparation is: 1-100mg/kg.