CN109847044B

CN109847044B - Application of Sishen water extract in preparation of drug for resisting Parkinson's disease

Info

Publication number: CN109847044B
Application number: CN201711239470.3A
Authority: CN
Inventors: 李红玉; 朱澍芊; 支德娟; 李洋; 戴治娟
Original assignee: Lanzhou University
Current assignee: Lanzhou University
Priority date: 2017-11-30
Filing date: 2017-11-30
Publication date: 2021-11-30
Anticipated expiration: 2037-11-30
Also published as: CN109847044A

Abstract

The invention belongs to the field of biological medicines, provides a new application of a traditional Chinese medicine compound Sishen powder aqueous extract, and particularly relates to an application of the Sishen powder aqueous extract in preparation of an anti-Parkinson medicine. The in vivo experiment result of the model organism caenorhabditis elegans shows that the four-nerve water extract can obviously inhibit the expression of alpha-synuclein, and the four-nerve water extract provided by the invention can be applied to the preparation of the anti-Parkinson's disease medicine.

Description

Application of Sishen water extract in preparation of drug for resisting Parkinson's disease

Technical Field

The invention belongs to the field of biological medicines, relates to a new application of a traditional Chinese medicine compound Sishen powder, and particularly provides an application of a Sishen powder aqueous extract in preparation of an anti-Parkinson medicine.

Background

Parkinson's Disease (PD) is the second major neurodegenerative disease, manifested by decreased motor and cognitive functions. Motor ability is manifested by muscle stiffness, bradykinesia and tremor; behavioural and cognitive abilities are manifested as dementia, depression, anxiety and sleep disorders. The incidence of this disease is rapidly increasing in people older than 60 years of age. The life quality of patients is continuously reduced along with the development of diseases until the families are burdened, and a heavy burden is brought to the society and the families.

The main pathological feature of parkinson's disease is the accumulation of alpha-synuclein in brain tissue. To date, there is no effective treatment.

The traditional Parkinsonian mammal pathological model is used for screening the anti-Parkinsonia medicament, the period is long, the cost is high, and the model organism Caenorhabditis elegans (Caenorhabditis elegans) is adopted, so that the screening period can be reduced from month unit to day unit, and the Caenorhabditis elegans becomes a very useful multifunctional medicament screening and medicament action mechanism research platform. It is cheap and easy to culture; the generation period is short, the number of offspring is large, a large number of individuals with consistent backgrounds can be obtained, the experimental repeatability is ensured, and the experiment is carried out by adopting a large sample amount, so that the influence of individual difference is eliminated; is highly conserved in genes and molecular pathways with higher organisms (Kaletta and Hengartner, 2006). It is more and more favored by pharmacologists as a bridge from the primary screening of cell in vitro horizontal drugs to the secondary screening of mouse in vivo horizontal drugs. The American devGen company has been FDA approved for clinical study using humanized nematode screening for antiarrhythmic drugs.

After being fused with YFP yellow fluorescent protein, the human alpha-synuclein is placed under a nematode muscle specific promoter, so that the human alpha-synuclein can be specifically expressed in nematode muscle tissues. Because the whole body of the nematode is transparent, yellow fluorescence can be clearly observed by using a fluorescence microscope, and the stronger the fluorescence is, the higher the expression of the humanized alpha-synuclein is. The drug with the treatment effect on PD can obviously inhibit fluorescence generated by alpha-synuclein expression, and the stronger the fluorescence inhibition capability of the drug to be detected is, the better the anti-PD activity is (Fu et al, 2014).

The four-god powder is an ancient prescription, is recorded in Taiping Huimin He Ji Ju Fang, mainly comprises four traditional Chinese medicines of Chinese angelica, Szechuan lovage rhizome, dried ginger and red paeony root, and is widely applied to treating postpartum blood retention, accumulation and agglomeration, acute pain, as if a hidden corpse, angina pectoris and diarrhea.

The invention discloses that a Sishen water extract can obviously weaken the fluorescence intensity of yellow fluorescent protein fused with human alpha-synuclein in caenorhabditis elegans muscle tissues, and shows that the Sishen water extract has the activity of obviously down-regulating the expression of the human alpha-synuclein, so that the Sishen water extract can be applied to the preparation of medicines for resisting Parkinson's disease.

Disclosure of Invention

The invention aims to provide a new application of a herba tetrastigmae aqueous extract in preparation of a drug for resisting Parkinson's disease, and particularly relates to an application of the herba tetrastigmae aqueous extract in preparation of the drug for resisting Parkinson's disease.

The four-god powder comprises the following raw material medicines: chinese angelica root, Chuan-xiong rhizome, dried ginger and red peony root.

Preferably, the four-god powder comprises the following raw material medicines in parts by weight: 1 part of angelica, 1 part of ligusticum wallichii, 1 part of rhizoma zingiberis and 1 part of radix paeoniae rubra.

The preparation method of the four-drug powder comprises the following steps: weighing 6g of angelica sinensis, 6g of ligusticum wallichii, 6g of rhizoma zingiberis and 6g of red paeony root, adding 200mL of distilled water, soaking for 20min, heating and boiling for 30 min, primarily filtering out medicine residues by gauze, collecting medicine liquid for later use, soaking the medicine residues for 5-10min by 150mL of distilled water again, heating and boiling for 25min, filtering out the medicine residues by the gauze again to obtain supernatant, combining the medicine liquids of the two times, concentrating and fixing the volume to 50mL, centrifuging for 2 times at 12000rpm for 10min, discarding insoluble precipitates, filtering and sterilizing by a 0.22um water system filter head, subpackaging and storing at-20 ℃ for later use.

The invention relates to an application of 'Sishen san' in preparing anti-Parkinson disease drugs, which can be added with pharmaceutically acceptable carriers such as filler, lubricant, wetting agent, absorption promoting agent, adsorption carrier, diluent, excipient and the like, and can be added with sweetening agent, flavoring agent and the like if necessary to prepare various dosage forms of injection, powder, granules, powder, pills, oral liquid, tablets and other drugs, and the preparation method can be understood by those skilled in the art.

Specific examples are provided below to realize the application of the aqueous extract of tetrapack in the preparation of anti-parkinson drugs, but the scope of protection of the present invention is not limited to the following.

Detailed Description

Example preparation of aqueous extract of Sishen san

Decocting to prepare Sishen powder: weighing 6g of angelica, 6g of ligusticum wallichii, 6g of rhizoma zingiberis and 6g of red paeony root, adding 200mL of distilled water, soaking for 20min, heating and boiling for 30 min, primarily filtering out medicine residues by gauze, collecting medicine liquid for later use, soaking the medicine residues for 5-10min by 150mL of distilled water again, heating and boiling for 25min, filtering out the medicine residues by the gauze again to obtain supernatant, combining the two medicine liquids, concentrating and fixing the volume to 50mL, centrifuging for 2 times at 12000rpm for 10min, discarding insoluble precipitates, filtering and sterilizing by a 0.22um water system filter head, subpackaging and storing at-20 ℃ for later use.

EXAMPLES preparation of comparative aqueous extracts

Preparation of a comparative preparation by decoction: weighing 6g of angelica, 6g of ligusticum wallichii and 6g of dried ginger, adding 200mL of distilled water, soaking for 20min, heating and boiling, keeping for 30 min, primarily filtering out medicine residues by using gauze, collecting medicine liquid for later use, soaking the medicine residues for 5-10min by using 150mL of distilled water again, heating and boiling, keeping for 25min, filtering out the medicine residues by using the gauze again to obtain supernatant, combining the medicine liquids of the two times, concentrating, fixing the volume to 50mL, centrifuging for 2 times at 12000rpm for 10min, discarding insoluble precipitates, filtering and sterilizing by using a 0.22um water system filter head, subpackaging, and keeping at-20 ℃ for later use.

Example preparation of aqueous extract of Ligusticum wallichii

Decocting to prepare rhizoma Ligustici Chuanxiong: weighing 6g of ligusticum wallichii, adding 200mL of distilled water, soaking for 20min, heating and boiling, keeping for 30 min, primarily filtering medicine residues by using gauze, collecting medicine liquid for standby, soaking the medicine residues for 5-10min by using 150mL of distilled water again, heating and boiling, keeping for 25min, filtering the medicine residues by using the gauze again to obtain supernatant, combining the two medicine liquids, concentrating, fixing the volume to 50mL, centrifuging for 2 times at 12000rpm for 10min, removing insoluble precipitate, filtering and sterilizing by using a 0.22um water system filter head, subpackaging and keeping at-20 ℃ for standby.

Example therapeutic Effect of Site Shensan on caenorhabditis Parkinsonii

1. Biological material

(1) Caenorhabditis elegans OW13 was purchased from Caenorhabditis Genetics Center (CGC); for transgenic strains, fusion of human alpha-synuclein and YFP yellow fluorescent protein is inserted under a muscle-specific promoter, so that the muscle tissue of the strain nematode specifically expresses the human alpha-synuclein, and the stronger the fluorescence is observed by a fluorescence microscope, the higher the alpha-synuclein is expressed. The medicine with the treatment effect on PD can obviously inhibit fluorescence generated by alpha-synuclein expression, and the stronger the fluorescence inhibition capacity of the medicine to be detected is, the better the anti-PD activity is. This example adopts

The strain of caenorhabditis elegans OW13 is used as a pathological model for screening anti-PD drugs.

(2) Escherichia coli OP50 (uracil leaky mutant), purchased from Caenorhabditis Genetics Center (CGC), was used as feed for C.elegans.

2. Reagent

(1) Solid NGM (Newatode Growth Medium) medium composition and preparation (taking 1 liter as an example):

composition (I)	Content (wt.)
		NaCl	3.00g
K₂HPO₄	2.34g
		KH₂PO₄	17.23g
Peptone	2.50g
		Agar-agar	17.00g
Supplement H₂O to	1000mL

After preparing the solid NGM culture medium, sterilizing at 121 deg.C under high pressure and constant temperature for 20min, adding 5mg/mL cholesterol 1mL, 1M MgSO₄1mL,1M CaCl₂1mL was shaken well and poured hot into a sterilized 9cm plate, approximately 20 mL/plate. Standing for solidification of the culture medium.

(2) M9 liquid formula

Composition (I)	Content (wt.)
		Na₂HPO₄	6.00g
KH₂PO₄	3.00g
		NaCl	5.00g
1MMgSO₄	1.00mL
		Supplement H₂O to	1000mL

(3) Preparation of lysate: a6.4% NaClO solution and a 1M NaOH solution were mixed in a volume ratio of 1: 1.

3. Preparing NGM plate containing four-god powder water extract and other water extracts

The aqueous extract of Sishen prepared in example one was diluted with sterile water, added to NGM medium at final concentrations of 2.4mg/mL, 9.6mg/mL and 48mg/mL, poured onto each plate, and left to stand for solidification of the medium. Coli OP50 was spread evenly on the medium as feed for nematodes.

The comparative aqueous extract prepared in example two was diluted with sterile water, added to NGM medium at final concentrations of 2.4mg/mL, 9.6mg/mL and 48mg/mL, respectively, and NGM was poured onto each plate and allowed to stand for solidification of the medium. Coli OP50 was spread evenly on the medium as feed for nematodes.

Diluting the rhizoma Ligustici Chuanxiong aqueous extract prepared in example three with sterile water, adding NGM culture medium with final concentration of 2.4mg/mL, 9.6mg/mL and 48mg/mL, respectively, pouring NGM into each plate, and standing for solidification of the culture medium. Coli OP50 was spread evenly on the medium as feed for nematodes.

4. Carrying out the step

(1) And (3) culturing nematodes:

the nematodes were plated on solid NGM plates coated with E.coli OP50 and then incubated in an incubator at 20 ℃ and synchronized when they reached adult growth.

(2) Nematode synchronization:

selecting NGM culture medium containing a large amount of imagoes and part of nematode eggs which have been hatched, flushing the nematodes from the culture medium by using M9 liquid, transferring the nematodes to a centrifuge tube, standing the nematode to enable the nematodes to freely settle to the bottom of the centrifuge tube, and discarding supernatant. And adding the lysate into the centrifuge tube according to the quantity of the nematode, oscillating the centrifuge tube on a vortex mixer for 5 to 7 minutes, stopping vortex when the nematode is completely broken, subpackaging the centrifuge tube with 1.5mL, and washing the nematode eggs with M9 solution for three times.

(3) Various aqueous extracts inhibited the expression of human alpha-synuclein in caenorhabditis elegans OW13 muscle tissue

The synchronized young nematodes are subpackaged in centrifuge tubes, incubated in M9 buffer for 48 hours to become synchronized L1-stage larvae, centrifuged at 4000rpm for 5min to remove redundant M9, transferred to NGM plates coated with OP50, cultured at 20 ℃ to L3 stage, washed with M9 solution to remove redundant M9, and finally transferred to NGM culture dishes containing different kinds of water extracts with different concentrations and blank NGM dishes coated with OP50 and added with sterile water with the same volume as the water extracts, wherein all the NGM dishes contain 50 mu M5-fluorodeoxyuridine (FuDR) to inhibit the propagation of the nematodes and avoid generation overlapping. 60 nematodes per dish, three dishes per drug concentration were used in parallel and incubation was continued for 72 hours at 25 ℃. The solution was washed with M9, centrifuged at 4000rpm for 5min to remove excess M9, added with 20mM NaN3 to anesthetize nematodes, observed under a fluorescent microscope and photographed. Each group was observed randomly for 25 nematodes and all pictures were analyzed quantitatively for fluorescence intensity using Image J software. The results are shown in tables 1 and 2.

TABLE 1 therapeutic action of aqueous extract of Sishen san on PD nematode

TABLE 2 therapeutic effect of the four-drug aqueous extract and the comparative aqueous extract on PD nematodes

The fluorescence intensity in the table represents the expression level of alpha-synuclein in nematode muscle tissue due to fusion with alpha-synuclein, and the lower the value, the higher the anti-PD activity of the aqueous extract of tetrastigma, i.e., the stronger the anti-PD effect of the aqueous extract of tetrastigma. Control-represents sterile water blank. Table 1 shows the therapeutic effect of the aqueous extract of Sishen san on PD nematodes, and Table 2 shows the therapeutic effect of the aqueous extract of Sishen san and the comparative aqueous extract on PD nematodes. Different letters mean that the difference is significant p < 0.05.

The experimental result shows that the four-god water extract in the embodiment obviously inhibits the expression of the humanized alpha-synuclein in the muscle tissue of the nematode, and the four-god water extract has an obvious treatment effect on the PD.

The above examples prove that the four-god powder provided by the invention has a remarkable treatment effect on PD caenorhabditis elegans, and the four-god powder water extract provided by the invention is prompted to have a potential of PD resistance and can be applied to preparation of medicines or health care products for preventing and treating PD.

Reference to the literature

Fu R.H.,Harn H.J.,Liu S.P.,Chen C.S.,Chang W.L.,et al.n-Butylidenephthalide protects against dopaminergic neuron degeneration and a-Synuclein accumulation in Caenorhabditis elegans models ofParkinson’s disease.PLoS ONE 2014,9(1):e85305.doi:10.1371/journal.pone.0085305.

Kaletta T,Hengartner MO.Finding function in novel targets:C.elegans as a model organism.Nat Rev Drug Discov 2006,5:387e398.

Ma,L.,Qin,C.,Wang,M.,Gan,D.,Cao,L.,Ye,H.,et al.Preparation,preliminarycharacterization and inhibitory effect on human colon cancer HT-29cells ofanacidic polysaccharide fraction from Stachysfloridana Schuttl.ex Benth.Food and Chemical Toxicology 2013,60:269–276.

Fu R.H.,Wang Y.C.,Chen C.S.,Tsai R.T.,Liu S.P.,Chang W.L.,Lin H.L.,et al.Acetylcorynoline attenuates dopaminergic neuron degeneration and a-synuclein aggregation in animal models of Parkinson’s disease.Neuropharmacology 82(2014)108e120.

Claims

1. The application of the aqueous extract of the traditional Chinese medicine compound Sishen powder in preparing the anti-Parkinson's disease medicine is characterized in that the Sishen powder consists of angelica, ligusticum wallichii, dried ginger and red paeony root in a mass ratio of 1:1:1: 1.

2. The use of claim 1, wherein the preparation method of the powder comprises the following steps: weighing 6g of angelica, 6g of ligusticum wallichii, 6g of rhizoma zingiberis and 6g of red paeony root, adding 200mL of distilled water, soaking, heating and boiling, primarily filtering out dregs of a decoction with gauze, collecting liquid medicine for later use, soaking the dregs of a decoction again, heating and boiling, filtering out the dregs of a decoction with gauze again to obtain supernatant, combining the liquid medicines of the two times, concentrating, fixing the volume to 50mL, centrifuging, removing insoluble precipitate, filtering and sterilizing by using a 0.22um water system filter head, and storing at-20 ℃ for later use after subpackaging.

3. The use of claim 1, wherein the powder is prepared into injections, powders, granules, pills, oral liquids or tablets by adding pharmaceutically acceptable carriers.