CN110229204B

CN110229204B - Preparation method of compound PGHG in Parkinson disease protection medicine

Info

Publication number: CN110229204B
Application number: CN201910458658.XA
Authority: CN
Inventors: 唐琳; 孙意冉; 贺丽波; 陈放
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2019-05-29
Filing date: 2019-05-29
Publication date: 2020-04-17
Anticipated expiration: 2039-05-29
Also published as: CN110229204A

Abstract

The invention discloses a preparation method of a compound PGHG in a medicament for treating Parkinson's disease, which comprises the following steps: ultrasonic extracting with 70% ethanol (1:25), sequentially extracting with petroleum ether and ethyl acetate, concentrating the extractive solution under reduced pressure, and freeze drying to obtain herba Penthori chinensis stem polyphenol fragment (PSE); separating by high-speed countercurrent chromatography (HSCCC) to obtain A, B fraction, further purifying fraction A by preparative HPLC, manually collecting eluate according to chromatographic peak, concentrating under reduced pressure to obtain monomer compound with purity of 99%, and identifying as PGHG by mass spectrum and nuclear magnetism. PSE and PGHG can obviously inhibit the reduction of the ATP level of SH-SY5Y cells induced by 6-hydroxydopamine, and improve the cell activity. The PGHG can regulate the expression of apoptosis protein and inflammatory protein by activating Nrf2/HO-1 antioxidant pathway. The neuroprotection has a significant effect in the prevention and treatment of parkinson's disease.

Description

Preparation method of compound PGHG in Parkinson disease protection medicine

Technical Field

The invention relates to the technical field of medicines, in particular to a preparation method of a compound PGHG in a Parkinson disease protection medicine.

Background

Parkinson's Disease (PD) is the second chronic progressive central nervous system disease of the world after alzheimer's disease, threatening the health of over 600 million people in the world. The clinical manifestations of PD include bradykinesia, postural instability, resting tremor and rigidity, and increased susceptibility to memory impairment and behavioral changes. The onset of PD is closely related to aging of the population. With the further aggravation of aging in China, the incidence of PD is continuously increased, and serious harm is brought to the society and the economy. However, no specific effective intervention or treatment is available at present. Therefore, in the face of the health threat posed by PD, the development of its treatments and drugs has become one of the major and hot spots of research in the world's medical field.

Penthorum chinense Pursh (PenthorumchinensePursh), also called Eupatorium japonicum, Salix babylonica, penthorum chinense Pursh and the like, is the overground part of penthorum chinense Pursh of Saxifragaceae (Saxifragaceae) Geranium (Penthorumgoronov. exL.) and is a common medicinal material of Michthysana gorskimminuta in Ming dynasty, belonging to medicinal and edible plants in Ming dynasty, mainly distributed in northern China, east China, Central China, Shaxi, Sichuan and Guizhou and the like, mainly produced in Sichuan suria gorgiensis, and belonging to medicinal and edible plants in China, and prepared by penthorum chinense Pursh to prevent liver diseases, the penthorum chinense Pursh is warm in nature, sweet in taste, nontoxic, has the effects of clearing heat and removing toxicity, promoting urination, activating blood, calming liver, invigorating spleen, dispelling yellow, and the effect of removing yellow toxin, has the effects of clearing heat and reducing swelling, promoting urination, activating blood, calming liver, tonifying liver, strengthening spleen, and removing yellow scar, and has the excellent effects of inducing the liver damage of the Pubescen-5-g-6 polysaccharide and the early-5-p and the same as well as the test and the effective of.

Disclosure of Invention

The present invention aims to solve the above problems and to provide a method for preparing PGHG, a compound used in drugs for protecting parkinson's disease.

The invention realizes the purpose through the following technical scheme:

a first aspect of the present invention relates to a process for the preparation of PGHG: PSE was separated by high-speed countercurrent chromatography (HSCCC) in forward inversion to give A, B fractions, which were concentrated under reduced pressure and then dissolved in appropriate amounts of methanol and passed through 0.22. mu. mPTE filters. Further purification was performed by preparative HPLC under the following chromatographic conditions: the chromatographic column is C18 column (250mm × 10mm, 5 μm), the column temperature is set at 28 deg.C, 100 μ L of sample is injected at 5mL/min, A, B fraction is isocratically eluted with 60% methanol and 55% methanol respectively, the effluent is manually collected according to chromatographic peak with 280nm and 335nm as detection wavelength, the effluent is concentrated under reduced pressure, and compound PGHG is obtained from fraction A.

The second aspect of the invention relates to a new application of the penthorum chinense pursh stem polyphenol fragment and PGHG in the protection of Parkinson disease.

In order to detect the protective effect of the penthorum chinense Pursh stem polyphenol fragments and PGHG on the Parkinson disease, the penthorum chinense Pursh stem polyphenol fragments and PGHG are prepared into liquid medicines with different concentrations by using culture media respectively and are used for treating human glioma cells SH-SY5Y cells, and the protective effect of the liquid medicines on the SH-SY5Y cell Parkinson disease induced by 6-hydroxydopamine (6-OHDA) under different doses is observed respectively. The result shows that the penthorum chinense pursh stem polyphenol fragment and PGHG can remarkably relieve the SH-SY5Y cell activity and ATP level reduction caused by 6-OHDA, and the effect is better than that of positive control Baicalein (Baicalein). The penthorum chinense pursh stem polyphenol fragment and PGHG have good potential in preparing the medicine for treating the Parkinson disease.

In the preparation of the medicine for treating the Parkinson's disease, the protective effect of the PGHG on the Parkinson's disease comprises the activation of an Nrf2/HO-1 antioxidant pathway and the down-regulation of the expression of an apoptosis protein Caspase-3 and an inflammatory protein NF kappa B, COX-2.

The invention has the beneficial effects that:

the invention relates to a preparation method of a compound PGHG in a Parkinson disease protection medicament. PSE and PGHG can obviously inhibit the reduction of the ATP level of SH-SY5Y cells induced by 6-hydroxydopamine, and improve the cell activity. The PGHG can regulate the expression of apoptosis protein and inflammatory protein by activating Nrf2/HO-1 antioxidant pathway. The neuroprotection has a significant effect in the prevention and treatment of parkinson's disease.

Drawings

FIG. 1 is a diagram of HSCCC separation for a PSE;

FIG. 2 is an ESI-MS profile of PGHG

FIG. 3 is a structural formula of PGHG

FIG. 4 is a graph showing the effects of PSE and PGHG on 6-OHDA-induced SH-SY5Y cells

In fig. 4: (A) measuring the cell viability; (B) ATP level determination

FIG. 5 is a graph of the effects of PSE and PGHG on 6-OHDA-induced SH-SY5Y cell oxidation, apoptosis and inflammatory protein expression

In fig. 5: (A) antioxidant-related proteins; (B) apoptosis and inflammatory proteins

Detailed Description

The invention will be further described with reference to the accompanying drawings in which:

example 1: PGHG separation method

1.1 establishment of the separation method

Carrying out ultrasonic extraction on the penthorum chinense pursh stem dry powder by using 25 times volume of 70% ethanol (material-liquid ratio is 1:25) at 55 ℃ and 59kHz, repeating the ultrasonic extraction twice for 40 min/time, combining the filtrates obtained by suction filtration, carrying out reduced pressure concentration by using a rotary evaporator, then adding 200mL of ultrapure water for heavy suspension, extracting by using ethyl acetate (10 times) according to the volume ratio of 1:1, carrying out reduced pressure concentration, and carrying out freeze drying to obtain the penthorum chinense pursh stem polyphenol fragment (PSE).

An n-hexane-ethyl acetate-methanol-water (2:5:2:5, v/v/v) solvent system is selected, the upper phase is used as a mobile phase for elution, the flow rate is 5.0mL/min, the rotation speed is 800rpm, the forward reverse mode is adopted, the detection wavelength is 280nm, the sample amount is 200mg (200mg of a sample is dissolved in 20mL of the upper phase), the separation temperature of equipment is set to be 28 ℃, and under the separation condition, the retention rate of the stationary phase can reach 73.3%. A, B fractions were collected manually according to the HSCCC profile (FIG. 1).

The A, B fraction from the HSCCC separation was concentrated to dryness under reduced pressure, dissolved in a small amount of methanol, filtered through a 0.22 μm PTFE membrane and further purified by preparative HPLC using the following chromatographic conditions: the chromatographic column is a C18 column (250mm multiplied by 10mm, 5 mu m), the column temperature is set to 25 ℃, 100 mu L of sample is injected at 5mL/min, A, B fractions are respectively isocratically eluted by 60% methanol and 55% methanol, the effluent is manually collected according to chromatographic peaks by taking 280nm and 335nm as detection wavelengths, and the effluent is concentrated under reduced pressure to obtain the monomeric compound I from the A fraction.

1.2 structural characterization of Compounds

The mass spectrum conditions are as follows: electrospray ion source (ESI), taper hole voltage 30V, capillary voltage 0.8kV, probe temperature 600 ℃, ion source temperature 126 ℃, liquid flow 0.4mL/min, and scanning mode of negative ions with a scanning range of m/z150-900 and a collection rate of 10 points/second.

Nuclear magnetic identification: the dry powder of the compound was dissolved in 0.5mL of deuterated DMSO and subjected to structural identification by nuclear magnetic resonance (1H-NMR and 13C-NMR). The specific results are as follows:

a compound I: light brown to yellow powder, ESI-MS spectrum see fig. 2, M/z871.26[ M-H ] -, which shows a molecular weight of 872.26, 1H-NMR (DMSO-d6,600mhz) δ ppm:7.53(1H, s),7.40(4H, dt, J ═ 19.6,7.0Hz),6.88(2H, s),6.36(1H, s),6.28(1H, s),6.23(2H, d, J ═ 3.1Hz),6.07(1H, d, J ═ 12.6Hz),5.31(2H, dt, J ═ 19.4,8.4Hz),5.09(1H, d, J ═ 9.8Hz),4.81(1H, t, J ═ 10.0Hz),3.63(1H, M), 13C-d 6 (hh-dl-6 mhz), hh-115 g ═ 9.8Hz),4.81(1H, t, J ═ 10.0Hz),3.63(1H, M), 13C-dl-6 (hh-29 g-29, hh ═ 9.8, 18 g — (35, 35 g —, hh 3.3.3.3.3 g —, 35, 3 g —, 5-gcd —,5'),127.17(C-2',6'),119.51(galloylC-1),109.42(galloylC-2,6),104.06(C-10),79.19(C-2),71.88(C-5 "), 71.18 (C-2"), 70.08(C-4 "), 40.13(C-3), the above data are substantially in accordance with the literature reports, so that Compound 11 was identified as Pinoceimrin-7-O- [ 3' -O-galloyl-4 ',6' - (S) -hexahy-

The structural formula of the dihydrodiphenoyl ] - β -D-glucoside (PGHG) is shown in figure 3.

Example 2: interference effect of penthorum chinense Pursh polyphenol fragment and PGHG on SH-SY5Y cell Parkinson disease induced by 6-hydroxydopamine (6-OHDA)

2.1 cell viability assay

SH-SY5Y cells are inoculated in a 96-well plate, after 12 hours, culture media are added to a control group and a model group, 1,2,5,10 mu g/mL penthorum chinense pursh polyphenol fragment (PSE), 1,2,5,10 mu MPGHG and 20 mu M baicalein (Bai) are respectively added to a drug group for treatment for 12 hours, then 200 mu M6-OHDA is added to the model group and the drug group for treatment for 12 hours, and fresh culture media are added to the control group. Finally, sucking off liquid in each well, adding 100 mu L of culture medium (containing 10 mu LCCK-8), placing in an incubator for incubation, measuring the light absorption value at 450nm by using an enzyme-labeling instrument after 40min, and calculating the cell survival rate according to the OD value: OD drug/OD control.

2.2ATP level detection

SH-SY5Y cells are inoculated in a 6-well plate, after 12 hours, culture media are added to a control group and a model group, 2,5,10 mu g/mL penthorum chinense pursh polyphenol fragment (PSE) and 2,5,10 mu MPGHG are respectively added to a drug group for treatment for 12 hours, then 200 mu M6-OHDA is added to the model group and the drug group for treatment for 12 hours, and fresh culture media are added to the control group. Sucking the culture solution, collecting the cells into a 1.5mL centrifugal tube after trypsinization, centrifuging at 2000rpm and 4 ℃ for 7min, sucking the supernatant, adding 200 mu LRIPA lysate into the cell sediment, performing vortex oscillation lysis for 15s, performing lysis for 3 times at an interval of 5min each time, finally centrifuging at 15000rpm and 4 ℃ for 20min, collecting the supernatant, performing reaction according to the instructions of the ATP content determination kit, finally determining the light absorption value at 636nm, and calculating the ATP content of each group.

2.3 results and analysis

As can be seen from A in FIG. 5, 5. mu.g/mLPSE and 5. mu.MPGHG can well inhibit 6-OHDA-induced reduction of SH-SY5Y cell viability, and the required concentration is much lower than that of positive control baicalein, indicating that PSE and PGHG have significant advantages in protecting nerves. Since 6-OHDA can decrease ATP level of nerve cells, ATP level in cells is detected. The results indicate that PSE and PGHG can significantly increase the 6-OHDA-induced reduction of SH-SY5Y cellular ATP levels (B in fig. 4), indicating that they can elevate mitochondrial energy synthesis and slow down mitochondrial disturbances caused by oxidative stress. In conclusion, the penthorum chinense pursh stem polyphenol fragments and PGHG have good potential in preparing the medicine for treating the Parkinson disease.

Example 3: effect of PGHG on 6-OHDA-induced antioxidant, apoptotic and inflammatory protein expression in SH-SY5Y cells

SH-SY5Y cells are inoculated to a 60mm culture dish, after the cell confluence reaches 70-80%, culture media are added into a control group and a model group, 2,4 and 8 mu MPGHG is added into a drug group respectively for 12h, then 200 mu M6-OHDA is added into the model group and the drug group for 12h, fresh culture media are added into the control group, the culture solution is sucked away, the cells are collected into a 1.5mL centrifugal tube after trypsinization, centrifuged at 2000rpm and 4 ℃ for 7min, the supernatant is sucked away, cell precipitates are added with 200 mu LRIPA lysate, vortex cracked for 15s and cracked for 3 times at intervals of 5min, finally centrifuged at 15000rpm and 4 ℃ for 20min, the supernatant is collected, a BCA protein concentration determination kit is used for determining the protein concentration, after leveling, 5XLOADingbuffer is added according to the ratio of 4:1, boiling, SDS-PAGE protein electrophoresis, membrane transfer, Nrf2, NF kappa-1, NF kappa B, HO-2, Caspase-3, GAActin- β, a secondary antibody gel imaging system at normal temperature and development is carried out after overnight imaging.

The results show that PGHG can up-regulate the expression of Nrf2 and HO-1 (a in fig. 5) in a concentration-dependent manner, i.e., PGHG can activate Nrf2 antioxidant pathway. In addition, 6-OHDA can up-regulate Caspase-3 expression to induce apoptosis; PGHG can inhibit apoptosis by down-regulating Caspase-3 expression and up-regulating the expression of anti-apoptotic protein Bcl-2. Moreover, PGHG can also inhibit the expression of inflammation-related proteins NF kappa B and COX-2 caused by 6-OHDA so as to relieve the inflammation (B in figure 5).

The penthorum chinense pursh stem producing area used in the embodiment is Sichuan China, and is purchased from Xiangchen Xiangcao co-culture of Osmanthus fragrans in Irish county, Sichuan province. Both drugs and reagents were purchased from the Chengdong chemical reagent plant.

The foregoing shows and describes the general principles and features of the present invention, together with the advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. A method for preparing a compound PGHG in a medicament for treating Parkinson' S disease is characterized by adopting high-speed countercurrent chromatography, selecting n-hexane-ethyl acetate-methanol-water with a volume ratio of 2:5:2:5 as a solvent system, eluting an upper phase as a mobile phase, separating a penthorum chinense pursh stem polyphenol fragment at a flow rate of 5.0mL/min and a rotation speed of 800rpm in a forward-reverse mode to obtain A, B fractions, dissolving the A, B fractions by using a proper amount of methanol respectively after vacuum concentration, passing the dissolved methanol through a 0.22 mu m PTFE filter membrane, further purifying by adopting preparative HPLC (high performance liquid chromatography), wherein the chromatographic conditions are that a C18 chromatographic column with the size of 250mm multiplied by 10mm and the size of 5 mu m is set at 25 ℃, 100 mu L is injected at 5mL/min, A, B fractions are eluted by using 60% methanol and 55% methanol respectively in an isocratic manner, 280nm and 335nm are used as detection wavelengths, manually collecting an effluent according to chromatographic peaks, carrying out vacuum concentration, and obtaining a compound PGA effluent from the PGA fraction, wherein the chemical name of the PGHG is 7-O- [3 "-O-4" -6 galloyl ] - β ";

carrying out ultrasonic extraction on the penthorum chinense pursh stem dry powder by using 25 times volume of 70% ethanol at a material-liquid ratio of 1:25 at 55 ℃ and 59kHz, repeating the ultrasonic extraction twice for 40 min/time, combining the filtrate obtained by suction filtration, carrying out reduced pressure concentration by using a rotary evaporator, then adding 200mL of ultrapure water for re-suspension, extracting by using ethyl acetate for 10 times according to the volume ratio of 1:1, carrying out reduced pressure concentration, and carrying out freeze drying to obtain the penthorum chinense pursh stem polyphenol fragment.

2. Use of the compound PGHG and penthorum chinense pursh stem polyphenol fraction as claimed in claim 1 in the manufacture of a medicament for the treatment of parkinson's disease, wherein: the therapeutic effect of the penthorum chinense pursh stem polyphenol fragments and PGHG on Parkinson's disease is to improve cell activity and ATP level.

3. Use of the compound PGHG, according to claim 2, in the manufacture of a medicament for the treatment of parkinson's disease, wherein: the PGHG has the treatment effect on the Parkinson disease and is selected from activating Nrf2/HO-1 antioxidant pathway and reducing the expression of apoptosis protein Caspase-3 and inflammatory protein NF kappa B, COX-2.