CN106551963B - New use of Aralia taibaiensis or its extract - Google Patents
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Abstract
The invention provides an application of aralia taibaiensis or an extract thereof in preparing a medicament for preventing and/or treating neurodegenerative diseases. The medicine belongs to a pure natural preparation, is not treated by an organic solvent, and is safe and reliable; the toxicity is low, and adverse reactions do not occur after long-term use; can be used for preventing and treating neurodegenerative diseases caused by various reasons.
Description
Technical Field
The invention relates to a new application of aralia taibaiensis or an extract thereof, belonging to the field of medicines.
Background
Alzheimer's disease (AD, also known as senile dementia), Parkinson's Disease (PD), Amyotrophic Lateral Sclerosis (ALS) are the most common neurodegenerative diseases (NDD), and are characterized by chronic progressive neurological diseases caused by irreversible degeneration of neurons at specific sites in the brain of a patient, the production of which is associated with an increase in Reactive Oxygen Species (ROS) in vivo. Brain diseases can cause brain injuries to cause various behavioral defects, such as language disorders, memory disorders and the like, and seriously affect the normal life activities of patients. The world health organization estimates that by 2040 years, neurodegenerative diseases will become the leading cause of death over cancer in industrialized countries. Therefore, the search for drugs for preventing and treating neurodegenerative diseases is a problem which needs to be solved urgently at present.
Araliaceae Aralia (Aralialin 1) plant has more than 40 species, most of which are distributed in Asia and North America, and 28 species are widely distributed in China, and 20 species of the plants can be used for medicine. Aralia plants are rich in protein, amino acid, vitamin, crude fiber and carotene, and are high-grade wild vegetables which are regarded as the reputations of "the king of wild vegetables". Currently, Aralia plants are mainly studied as Aralia elata (a. schmidtii Pojark), Aralia elata (Aralia decaprenia Hance), Aralia echinata (Aralia echinocaulis hand. mazz), Aralia taibaiensis, Aralia platyphylla (var. nuda Nakai), and the like. Chinese Aralia (Aralia chinensis) is widely distributed in China, Aralia taibaiensis is a new kind of Chinese Aralia established in recent years, is especially produced in Qiba mountainous areas and the rest veins of the western part of China, mainly comprises southern and western parts of Shaanxi, Lanzhou province, Sichuan northeast and Sichuan northeast, Shanxi Zhongshan and Ningxia six-disk mountainous areas, and particularly has abundant wild resources in Taibai, Zhou Zhi, Cheng county, Buddha lawn, linger, Fengxian, Ningshan, Ningqiang, Zheng, Xiyang, Ankang, Pingli, Pingyu, Heyang, Huashan and Gansu elm, Tianshui, Sichuan Moui and the like.
Aralia taibaiensis (Aralia taibaiensis L) is also called Aralia taibaiensis bark and Aralia cordata. It has mild property, sweet taste and slight bitter taste, and has effects of dispelling pathogenic wind, removing dampness, inducing diuresis to alleviate edema, promoting blood circulation, removing blood stasis, relieving pain and invigorating spleen. Folk therapy is commonly used for treating rheumatic arthritis, nephritic edema, ascites due to cirrhosis, acute and chronic hepatitis, stomachache, stranguria, metrorrhagia, traumatic injury, carbuncle and swelling.
Research shows that roots, stems and leaves of Chinese Aralia contain various chemical components, and bark of Chinese Aralia contains saponin, which can be hydrolyzed into Chinese Aralia sapogenin, namely oleanolic acid. It also contains tannin, choline, volatile oil, etc. The seeds are rich in oil and fat, and the content of the oil and fat reaches 26 percent. In addition, Aralia chinensis root bark sprouts contain various amino acids such as aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine and the like, and various trace elements such as calcium, manganese, iron, nickel, copper, cobalt, germanium and the like required by human bodies. The most studied aralis is saponin components. The aglycones are mainly Oleanolic acid (I) and Hederagenin (II), which are often linked to sugars at the 3-or 28-position to form glycosides. The sugar connected mainly comprises D-glucose, D-glucuronic acid, L-arabinose, D-galactose, L-rhamnose, D-xylose and derivatives thereof. A few saponins have glucuronic acid present as methyl ester at position 6. In general, glucuronic acid forms a linear or branched sugar chain with glucose, galactose, arabinose, etc. and then forms a glycoside with 3 β -OH, and the 28-carboxyl group forms a glycoside, which is mostly glucose.
Aralia taibaiensis has various pharmacological effects of protecting liver, reducing blood sugar, reducing blood lipid, etc. At present, the Chinese aralia taibaiensis has more reports in documents, such as: the research on antidiabetic saponin components in aralia elata and aralia taibaiensis is carried out by the fourth university of military science in 2012, 10 compounds are separated from aralia taibaiensis roots by comprehensively using separation means such as silica gel column chromatography, reversed-phase ODS column chromatography, SephadexLH-20 column chromatography, preparative high performance liquid chromatography and the like, 5 compounds are separated from aralia taibaiensis roots, the structures of the 15 compounds are identified by chemical methods (including acid hydrolysis, sugar derivation and the like) and spectral analysis (including nuclear magnetic resonance spectrum, mass spectrum and the like), the 15 compounds are triterpene saponins, the anti-oxidation and anti-glycosylation activity tests are carried out on the 5 saponins separated from aralia taibaiensis, the compounds are found to show the anti-oxidation and anti-glycosylation activities, the saponin antidiabetic activity relation is analyzed, a clear conclusion is obtained, and the antidiabetic effect of aralia taibaiensis total saponins is probably generated by the synergism of aralia taibaiensis monomers through different action mechanisms. Guo Dong Yan, et al, preliminary research on the basis of aralia taibaiensis anti-liver injury drug substances, journal of medical research, volume 41, 10, 2012, sequentially extracting aralia taibaiensis extract by a classical system solvent method (polarity from small to large: petroleum ether-chloroform-ethyl acetate-n-butanol), respectively intragastrically administering each extract to a carbon tetrachloride (CCl4) acute liver injury model mouse, detecting the activities of alanine Aminotransferase (ALT), aspartate Aminotransferase (AST) and superoxide dismutase (SOD) and the levels of Malonaldehyde (MDA) and glutathione peroxidase (GSH-Px) in serum and liver tissues, and comparing the anti-liver injury effects of each part; separating and purifying the screened active sites with macroporous adsorption resin, performing gradient elution with ethanol of different concentrations, respectively intragastrically administering the eluates to a liver injury model mouse, detecting ALT, AST, and SOD activity and MDA, GSH-Px levels in serum and liver tissue, and comparing the anti-liver injury effects of the eluted sites. Compared with the model control group, the n-butanol fraction can obviously reduce the elevation of ALT, AST and MDA caused by CCl4, and increase the activity of SOD and the level of GSH-Px in liver tissues; the n-butanol extract eluted by 70% ethanol can obviously reduce ALT, AST and MDA elevation caused by CCl4, increase SOD activity and GSH-Px level in liver tissue, and the eluent dosage has obvious positive correlation with liver protection effect. Conclusion that the pharmacodynamic active substances with the liver protection effect of the aralia taibaiensis exist in the n-butanol part eluted by 70% ethanol mostly, and the active ingredients with the liver injury resistance effect of the aralia taibaiensis are probably related to the saponins which are main ingredients in the part. The method comprises the steps of (1) measuring the level of TGF beta 1 in serum of a rat, and the like, wherein the total saponin of aralia taibaiensis is influenced on the expression of Smads4 genes of a hepatic fibrosis rat, the liver fibrosis of the rat is induced by CCl4 in the journal of No. 2 of volume 23 in 2013, the total saponin aqueous solution of the aralia taibaiensis is subjected to interventional therapy, the collagen deposition of the liver tissue is observed by Masson staining, the level of TGF beta 1 in the serum of the rat is detected by an ELISA method, and the expression condition of Smads4 in the liver tissue of the rat. The result is that the level of TGF beta 1 in the serum of a rat with hepatic fibrosis is obviously reduced by the intervention treatment of aralia taibaiensis total saponins, and the proportion of liver collagen in the area of liver tissues is reduced; the positive rate of Smads4 protein expression in the liver of the treated rat is reduced, and the positive rate of Smads4mRNA is reduced. The result shows that the aralia taibaiensis total saponin can obviously reduce the level of TGF beta 1 in serum of a liver fibrosis rat, reduce liver collagen deposition and inhibit the expression of liver Smads4, thereby weakening a liver fibrosis signal mediated by TGF beta 1 and possibly being one of molecular mechanisms for playing the anti-liver fibrosis role.
At present, there is no related report that aralia taibaiensis is used for preventing and treating neurodegenerative diseases.
Disclosure of Invention
The technical scheme of the invention is to provide a new application of aralia taibaiensis or an extract thereof.
The invention provides an application of aralia taibaiensis or an extract thereof in preparing a medicament for preventing and/or treating neurodegenerative diseases.
Wherein the medicament is a medicament for preventing and/or treating Alzheimer disease, Parkinson disease and/or amyotrophic lateral sclerosis.
Wherein the weight percentage of the total saponins in the extract is 4.68-83.9%;
wherein, the weight percentage content of the araloside A in the extract is 0.52 to 16.23 percent.
Wherein the Aralia taibaiensis is root bark of Aralia taibaiensis L belonging to Aralia of Araliaceae.
Wherein the extract is prepared by the following method:
weighing raw materials, adding water or organic solvent for extraction, filtering the extractive solution, and concentrating; or
Weighing raw materials, adding water or organic solvent for extraction, filtering the extract, concentrating into extract, passing the extract through macroporous resin, eluting with organic solvent solution or water first, then eluting with organic solvent, collecting the eluate, concentrating, and drying to obtain the final product.
Preferably, the extract is prepared by the following method:
decocting cortex Araliae Elatae Radicis with 12 times of water for 0.5-2 hr twice, filtering, and concentrating under reduced pressure to obtain extract; or
Extracting root bark of Aralia taibaiensis with 12 times of 50-90% ethanol under reflux twice for 0.5-2 hr each time, filtering, and concentrating under reduced pressure to obtain extract; or
Extracting root bark of Aralia taibaiensis with 12 times of 70% ethanol under reflux twice, each for 0.5-2 hr, filtering, concentrating under reduced pressure to obtain extract, loading onto HPD100 macroporous resin, eluting with 50-70% ethanol or eluting with 2-4BV water, eluting with 2-4BV 70% ethanol, collecting eluate, and drying.
The medicine is a preparation prepared by taking an aralia taibaiensis total saponin extract as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.
Wherein, the preparation is an oral preparation.
The invention also provides a pharmaceutical composition for preventing and/or treating neurodegenerative diseases, which is characterized in that: the preparation is prepared by taking an effective amount of aralia taibaiensis total saponin extract as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.
The medicine is used for preventing and/or treating neurodegenerative diseases, and has definite efficacy. The medicine belongs to a pure natural preparation, is not treated by an organic solvent, and is safe and reliable; the toxicity is low, and adverse reactions do not occur after long-term use; can be used for preventing and treating neurodegenerative diseases caused by various reasons.
Drawings
FIG. 1 shows the effect of total saponin of Aralia taibaiensis on nerve cell injury caused by hydrogen peroxide.
FIG. 2 shows the total saponin pair H of Aralia taibaiensis2O2Influence of NF-. kappa.B expression in damaged SH-SY5Y cells.
The present invention is described in further detail below by way of specific embodiments, but the present invention is not limited thereto, and various changes and substitutions may be made by those skilled in the art without departing from the spirit of the present invention within the scope of the appended claims.
Detailed Description
Example 1 preparation of the extract of Aralia taibaiensis of the invention
The root bark of Aralia taibaiensis L is taken, added with 12 times of water for decoction and extraction twice, each time lasts for 0.5-2 hours, and filtered and decompressed and concentrated into extract. The total saponin content is 4.68%, and the araloside A content is 0.52%.
Example 2 preparation of the extract of Aralia taibaiensis of the invention
Extracting root bark of Aralia taibaiensis L with 12 times of 50% ethanol under reflux for 0.5-2 hr twice, filtering, and concentrating under reduced pressure to obtain extract. The total saponin content is 11.56%, and the araloside A content is 1.54%.
Example 3 preparation of the extract of Aralia taibaiensis of the invention
Extracting root bark of Aralia taibaiensis L with 12 times of 70% ethanol under reflux for 0.5-2 hr twice, filtering, and concentrating under reduced pressure to obtain extract. The total saponin content is 16.82%, and the araloside A content is 2.31%.
Example 4 preparation of the extract of Aralia taibaiensis of the present invention
Extracting root bark of Aralia taibaiensis L with 12 times of 90% ethanol under reflux for 0.5-2 hr twice, filtering, and concentrating under reduced pressure to obtain extract. The total saponin content is 12.38%, and the araloside A content is 1.43%.
Example 5 preparation of the extract of Aralia taibaiensis of the invention
Extracting root bark of Aralia taibaiensis L with 12 times of 70% ethanol under reflux twice for 0.5-2 hr each time, filtering, concentrating under reduced pressure to obtain extract, loading onto HPD100 macroporous resin, eluting with 50% ethanol, collecting eluate, and drying. The total saponin content is 53.26%, and the araloside A content is 8.32%.
Example 6 preparation of the extract of Aralia taibaiensis of the present invention
Extracting root bark of Aralia taibaiensis L with 12 times of 70% ethanol under reflux twice, each for 0.5-2 hr, filtering, concentrating under reduced pressure to obtain extract, loading onto HPD100 macroporous resin, eluting with 70% ethanol, collecting eluate, and drying. The content of total saponin is 83.9%, and the content of araloside A is 16.23%.
Example 7 preparation of monomeric Compound from n-butanol fraction of Aralia taibaiensis
Extracting Aralia taibaiensis L with 12 times of 70% ethanol under reflux twice for 0.5-2 hr each time, filtering, and concentrating under reduced pressure to obtain extract. Passing through HPD100 macroporous resin, eluting with 2-4BV water to remove impurities, eluting with 2-4BV 70% ethanol, collecting eluate, concentrating under reduced pressure, and drying. Then, semi-preparative liquid phase separation is adopted to obtain araloside A. The molecular formula of araloside A is as follows: c47H74O18Molecular weight 927.1, having the formula:
the compound is white amorphous powder, and has a positive Molish reaction and a positive libermann-Burchard reaction. After acid hydrolysis, thin-layer inspection is carried out, and arabinose, glucose, glucuronic acid and aglycone oleanolic acid are detected.1H-NMR6.30(1H，d，J＝8.0Hz), 5.39(1H, s), 4.96(1H, D, J ═ 8.0Hz) show that the aglycone has three sugars, the terminal proton signals of β -D-glucopyranose, a-L-arabinofuranose and β -D-glucuronic acid, respectively.13C-NMR122.8, 144.1 shows that the aglycone is oleanolic acid type pentacyclic triterpene, and the aglycone is13Compared with oleanolic acid, C3 shifts to a low field by about 10, C28 shifts to a high field by about 4, which shows that the two bits are glycosidated and are disaccharide saponin, three carbon signals of 108.6, 107.0 and 95.7 exist in an anomeric carbon region, which shows that aglycone is connected with 3 sugars, the carbon signals of 108.6 to 62.2 are comprehensively analyzed, the three sugars are further determined to be a-L-arabinofuranose, β -D-glucuronic acid and β -D-glucopyranose, the terminal carbon of the glucose shifts to a high field by about 2, so the glucose is connected to the C28 position of the aglycone, and the compound has the advantages of high-purity, high-purity and low-purity13The C-NMR data are substantially identical to the control of araloside A (Araloside A) which is a known compound[1]Thus, the compound was determined to be araloside a (aralosidea). (1. Studies of chemical compositions of Aralia elata (C. Liaodong) Tao, Xuseiwu, Chenyingjie, et al [ J ]]Shenyang academy of drugs, 1991, 8: 265-268.)
Table 1 The13C-NMR data of compound(，0＝TMS，in C5D5N)
The beneficial effects of the present invention are demonstrated by specific pharmacodynamic tests below.
Experimental example 1 Aralia taibaiensis extract p.H2O2Study on protective effect of induced oxidative stress injury of human neuroblastoma strain (SH-SY5Y cell)
1 materials of the experiment
1.1 drugs, reagents and cells
Aralia taibaiensis total saponins (prepared from example 5, total saponin content is 53.26%, aralia saponin a content is 8.32%); SH-SY5Y cells (purchased from Shanghai cell Bank of Chinese academy of sciences), trypsin (Huamei bioengineering Co.); DMEM complete medium, newborn calf serum (hangzhou sijiqing biomaterial limited). The detection kit for the IL-6 and TNF-alpha enzyme-linked immunosorbent assay (ELISA) is purchased from Dake, Biotechnology Limited.
1.2 instruments
Enzyme linked immunosorbent assay (Genios Tecan); fluorescent enzyme-linked immunosorbent assay (Thermo Electron corporation Multiskan Spectrum); clean bench (suzhou purification plant); mini bench top high speed centrifuge (5415D) (eppendorf, germany); CO 22Incubators (sanyo corporation, japan); LD4-2A centrifuge (Beijing medical centrifuge Mill); 96-well plates (Cornning, usa); XDS-1B inverted microscope (Chongqing optical Instrument factory); a DK-8A type electric heating constant temperature water bath (Shanghai sperm macro experimental facilities, Co., Ltd.); numerical control ultrasonic cleaner (Shanghai sperm macro experimental facilities Co., Ltd.).
2 methods and results
2.1 Effect of Total saponins of Aralia taibaiensis on SH-SY5Y cell growth
2.1.1 Experimental methods:
the influence of total aralia taibaiensis saponin on the growth of SH-SY5Y cells is determined by using tetramethyl azoazolate trace enzyme reaction colorimetry (MTT), the SH-SY5Y cells (1 × 10) in logarithmic growth phase and in good growth state are obtained5one/mL) of the total saponin is inoculated in a 96-well cell culture plate (100 mu L/well), cells are attached to the wall after 24 hours of continuous culture, a normal control group and an experimental group are respectively arranged, only complete culture medium with the same volume is added in the normal control group, aralia taibaiensis total saponin with different concentrations is added in the experimental group, the final concentration of the aralia taibaiensis total saponin is 10-100 mu g/mL (prepared in example 5, the total saponin content is 53.26%, and the aralia saponin A content is 8.32%), each group is provided with 6 multiple wells, after 8 hours of culture, 20 mu L of MTT (5mg/mL) is added in each well, the continuous culture is carried out for 4 hours, 100 mu L of DMSO is added to terminate the reaction, the optical density value (A value) of each well is measured at 490nm wavelength by an enzyme linked immunosorbent assay detector, and the inhibition rate is calculated, namely × 100% of the inhibition rate (1-drug-added group A490.
2.1.2 results of the experiment: the total saponin of aralia taibaiensis is acted for 8 hours at 50 mu g/mL, the growth of SH-SY5Y cells is not obviously influenced, the inhibition rate is less than 10%, and the maximum measurement concentration of the total saponin of aralia taibaiensis can be determined to be not more than 50 mu g/mL.
2.2 Total Aralia taibaiensisSaponin pair H2O2Effect of induced oxidative stress injury of SH-SY5Y cells
2.2.1 methods of experiment:
the cell density of SH-SY5Y cells with good logarithmic growth phase and growth state is adjusted to 1 × 105one/mL, seeded in 96-well plates at 100. mu.l per well. After the cells adhere to the wall overnight, removing the culture medium in each hole, and respectively setting a normal control group, a model group and an experimental group, wherein the normal control group is only added with complete culture medium with the same volume, the model group and the experimental group are added with fresh medicine-containing culture medium, each hole is 100 mul, and the final concentrations of the medicines are respectively: 5. mu.g/ml, 10. mu.g/ml, 20. mu.g/m. (test drugs were dissolved in DMSO at a final DMSO concentration of less than 0.05%). Incubation was continued in the 37 ℃ incubator for 24 h. Discarding the culture medium in each well, adding the complete culture medium with the same volume into a normal control combined model group, adding the culture medium containing hydrogen peroxide into an experimental group, wherein each well is 100 mu l, and the hydrogen peroxide concentration is respectively as follows: 5. mu.g/ml, 10. mu.g/ml, 20. mu.g/ml. After further culturing for 6 hours, 20ul of MTT (5mg/mL) was added, and after further culturing for 4 hours, the supernatant was discarded, and 100ul of DMSO was added to terminate the reaction. The absorbance (A value) of each well was measured at a wavelength of 492nm using a fully automatic enzyme-linked immunoassay instrument.
2.2.2 the results of the experiment are shown in Table 1 and FIG. 1.
TABLE 1 Effect of Aralia taibaiensis Total saponins on Hydrogen peroxide-induced nerve cell injury: (
n＝6)
Comparison with model group of P <0.05, P <0.01
The results show that: aralia taibaiensis total saponin pair H2O2The nerve cell injury has a protection effect, and compared with a model group, the two dose groups of 10 mug/ml and 20 mug/ml have significant difference (P is less than 0.01); of these, 10. mu.g/ml is the most potent, and the anti-lesion effect is reduced when the dosage is increased to 20. mu.g/ml.
2.3 Aralia taibaiensisTotal saponins to H2O2Effect of Interleukin-6 (IL-6), tumor necrosis factor (TNF-a), prostaglandin E2(PGE2) levels in damaged SH-SY5Y cells
2.3.1 methods of experiment:
take 2.2H2O2Induced SH-SY5Y cell oxidative stress injury experiment cell culture supernatant, an ELISA kit detects the content of IL-6 and calculates the inhibition rate, wherein the inhibition rate is × 100 percent (model group concentration-concentration of a test group/model group concentration).
2.3.2 results of the experiment, see table 2.
TABLE 2 influence of Aralia taibaiensis total saponins on IL-6, TNF-a, PGE2 content in nerve cell injury cell induced by hydrogen peroxide: (
n＝6)
Note: p <0.05, P <0.01 compared to model groups.
The results show that: the aralia taibaiensis total saponin can reduce the content of IL-6, TNF-a and PGE2 in nerve cells damaged by hydrogen peroxide, and the ratio of two dosage groups of 10 mu g/ml and 20 mu g/ml to a model group is obvious in difference (P is less than 0.01), so that the aralia taibaiensis total saponin can reduce secondary inflammation caused by hydrogen peroxide damage and protect nerve cells.
2.4 Aralia taibaiensis Total saponins Pair H2O2Effect of NF-. kappa.B expression in damaged SH-SY5Y cells
2.4.1 Collection of protein samples
Collecting H2O2After the injured SH-SY5Y cells are lysed, supernatant is taken, and the total protein content in the sample is determined by using a BCA protein concentration determination kit. Subpackaging the total protein, and placing the subpackaged total protein into a refrigerator at the temperature of 80 ℃ below zero for later use.
2.4.2 WestBlatting method for detecting expression of NF- κ B
Preparing 12% concentrated gel and 6% separating gel, respectively filling the concentrated gel and the 6% separating gel between two glass plates, placing the gel into an electrophoresis tank filled with electrophoresis buffer solution, loading the histones in a lane in equal volume, performing electrophoresis, membrane transfer and 5% skim milk sealing, incubating with NF-kB primary antibody, shaking gently at 4 ℃ overnight, and incubating with HRP-labeled secondary antibody at room temperature for 1 h. The film was scanned after exposure in the dark room.
2.4.3 results of the experiment, see FIG. 2.
The results show that: aralia taibaiensis total saponin pair H2O2The expression of NF-kB in the damaged SH-SY5Y cells has inhibition effect, the dosage is increased, the inhibition effect is enhanced, and the high dosage is equivalent to that of a normal group.
Experimental example 2 study on improving action of aralia taibaiensis total saponins on learning and memory disorder model mice
1 materials of the experiment
1.1 Experimental animals
A clean-grade male Kunming mouse is selected, and the weight of the mouse is 18-22 g. Randomly raising the chickens in cages with 8 chickens per cage, and freely drinking water and taking food. The room temperature was maintained at 24 ℃ and 2 ℃ and the relative humidity was 55-65%.
1.2 Experimental instruments
Mouse diving tower instrument (institute of Chinese medical science and technology, Pharmacopeia); stat Fax-2100 enzyme linked immunosorbent assay (Awareness, USA); high speed refrigerated centrifuge (eppendorf, germany); flow cytometry (COULTZER EPICSXL2 MCL); DIONEX ultimate 3000 semi-preparative high performance chromatograph (Daian liquid chromatography Co., Ltd., USA)
1.3 drugs and reagents
Aralia taibaiensis total saponins (prepared in example 5, the total saponin content is 53.26%, aralia saponin a content is 8.32%), scopolamine (manufactured by shanghai setiforme pharmaceutical co., ltd.), sodium nitrite and other reagents are domestic analytical pure.
2 experimental methods and results
2.1 administration by groups
Male mice of Kunming species are divided into 8 groups (four groups for experiments of memory acquisition disorder and memory consolidation disorder), which are respectively a normal control group, a model control group and a high and low dosage group of aralia taibaiensis total saponins. The total saponin of Aralia taibaiensis (prepared from example 5, total saponin content of 53.26%, araloside A content of 8.32%) was administered at volume of 10 ml/kg. The normal control group and the model control group were gazed with physiological saline. The preparation is administered 1 time daily for 3 weeks.
2.2 model preparation
A memory acquisition disorder model (5 mg/kg intraperitoneal injection of 0.3mg/ml scopolamine) is prepared by using scopolamine, and the model is tested after training for 24 hours after injection. A memory consolidation disorder model was prepared with sodium nitrite (i.e., sodium nitrite was injected intraperitoneally at l00 mg/kg) and tested 24 hours later. Test after 24 hours of training after injection.
2.3 results
2.3.1 diving platform experiment
The mouse is placed in a box to adapt to the environment for 30min, then the 36V alternating current is conducted, after the mouse is shocked, the mouse jumps to a rubber platform to avoid injurious stimulation, and if the mouse jumps down from the platform, the mouse returns to the platform again after being stimulated. So training for 5min, the mouse can form memory and stay on the platform. The test is carried out at an interval of 24 hours after training, the mouse is placed on the platform, the latency period of the mouse jumping off the platform for the first time and the frequency (namely error frequency) of the mouse jumping off the platform by electric shock within 5min are recorded.
The results show that after the model is made, the model group has obviously poor spirit, slow behavior, reduced spontaneous activity and reduced sensitivity to the outside, and the mice with the aralia taibaiensis total saponin group have good mental state, quick action and sensitive response to the outside stimulus and have no difference with the normal group.
2.3.1.1 effects on learning memory acquisition disorders, see Table 3.
TABLE 3 improving action of Aralia taibaiensis total saponins on learning and memory impairment: (
n＝8)
Note: p <0.05, P <0.01 compared to model groups.
The results show that: in training and testing of the model group, the error frequency is obviously increased compared with that of the normal group, the latency is obviously shortened compared with that of the normal group, and the difference with the normal group is obvious (P is less than 0.01). The training performance and the testing performance of the aralia taibaiensis total saponin group are both obviously better than those of a model group (P < 0.01). The Aralia taibaiensis total saponins have obvious improvement effect on mouse memory acquisition disorder models.
2.3.1.2 effects on learning memory consolidation disorder, see Table 4.
TABLE 4 improvement of Aralia taibaiensis Total saponins on learning consolidation disorder: (
n＝8)
Note: p <0.05, P <0.01 compared to model groups.
The results show that: the number of errors of the test results of the model group is obviously increased, the latency period is shortened, and the difference with the normal group is obvious (P < 0.01). The test result of the aralia taibaiensis total saponin group is better than that of the model group, and the aralia taibaiensis total saponin group has an inhibiting effect on the memory consolidation disorder caused by sodium nitrite.
2.3.1.3 Effect on the apoptosis Rate of nerve cells
The mouse is killed by cutting off the head, the left brain is taken on an ice bench (the cerebellum is removed), the brain tissue is cut into pieces by washing with normal saline, and 0.5 percent of pancreatin is added to digest for 30min in a constant temperature water bath at 37 ℃. Filtering with 400 mesh cell sieve for 2 times at 1200r/min, centrifuging for 10min, discarding supernatant, adding PBS solution 1.5ml, rinsing for 2 times (1200r/min, centrifuging for 5min, discarding supernatant); resuspending the cells in 0.3m1PBS to obtain mouse brain tissue single cell suspension. And detecting the apoptosis rate of the brain nerve cells by using a flow cytometer. The percentage of apoptotic cells was calculated. The results are shown in Table 5.
TABLE 5 Effect of Aralia taibaiensis Total saponins on apoptosis of nerve cells: (
n＝8)
Note: p <0.05, P <0.01 compared to model groups.
The results show that: the apoptosis rate of the nerve cells of the model group is obviously increased, the difference is significant (P is less than 0.01) compared with that of the normal group, the apoptosis rate of the nerve cells of the aralia taibaiensis total saponin group is reduced compared with that of the model group, and the aralia taibaiensis total saponin group has the function of obviously inhibiting the apoptosis of the nerve cells in the brain of the mouse and is expressed by the reduction of the number of apoptotic cells and the reduction of the apoptosis rate.
2.3.1.4 high performance liquid chromatography for detecting Dopamine (DA)
The experimental animal is killed by cutting off the head, the right brain tissue is taken out on an ice platform quickly, the right brain tissue is weighed by an analytical balance, then the right brain tissue is put into liquid nitrogen for freezing, and the right brain tissue is transferred into a refrigerator at the temperature of minus 80 ℃ for storage to be tested. When the test is carried out, 100mg of brain tissue is taken out, placed in lmlPBs for ice bath homogenization, added with 0.4mol/1 perchloric acid according to the volume ratio of 3:1 to remove protein, centrifuged at 4 ℃, and 20 mu l of supernatant is sampled. And detecting the DA content by adopting high performance liquid chromatography, wherein the detection wavelength is 280 nm. The DA content per g brain tissue was calculated [ 20. mu.l DA content (ng)/20. mu.l total sample (. mu.l) ]/hippocampal wet weight (g), see Table 6.
TABLE 6 influence of Aralia taibaiensis Total saponins on DA content in mouse brain: (
n＝8)
Note: p <0.05, P <0.01 compared to model groups.
The results show that: the brain of the mouse in the model group has obviously reduced DA content, and compared with the mouse in the normal group, the difference has significance P <0.01), and the aralia taibaiensis total saponin group can obviously inhibit the reduction of DA content.
And (3) knotting: 1. aralia taibaiensis total saponin pair H2O2The nerve cell injury has a protective effect, and is specifically represented as follows: the total saponin of Aralia taibaiensis can reduce the content of IL-6, TNF-a and PGE2 in nerve cells damaged by hydrogen peroxide and the content of IL-6, TNF-a and PGE2 in nerve cells damaged by hydrogen peroxideSecondary inflammation of (1), and to H2O2The expression of NF-kB in damaged SH-SY5Y cells has an inhibiting effect.
2. The aralia taibaiensis total saponins have obvious improvement effect on a mouse learning and memory disorder model, and are specifically represented as follows: the aralia taibaiensis total saponin has obvious improvement effect on a mouse memory acquisition disorder model, has inhibition effect on memory consolidation disorder caused by sodium nitrite, and has the effects of obviously inhibiting nerve cell apoptosis in a mouse brain, reducing the number of apoptotic cells, reducing the apoptosis rate and inhibiting the reduction of DA content.
Alzheimer's disease (AD, also called senile dementia), Parkinson's Disease (PD), Amyotrophic Lateral Sclerosis (ALS) are the most common neurodegenerative diseases (NDD), and the exact pathophysiological mechanism leading to NDD is not yet defined, and there are various causes and hypotheses, among which the representative hypothesis is the neurotransmitter imbalance hypothesis, the oxidative stress hypothesis, the mitochondrial dysfunction theory, the excitotoxicity theory, the abnormal protein accumulation theory, the gene mutation, the inflammatory process, the immune abnormality, and the apoptosis hypothesis. Recent increasing data has demonstrated that mitochondrial dysfunction and oxidative stress interact with several other mechanisms, and that oxidative stress and mitochondrial involvement may be major triggers leading to NDD. Reactive Oxygen Species (ROS) are continuously produced and eliminated in cells, and oxidative stress occurs when the clearance is reduced due to increased ROS production or reduced macromolecular repair for oxidative modification. ROS play an important role in cell signaling, and are involved in a variety of physiological and pathological processes.
Hydrogen peroxide (H)2O2) Is a product of in vivo metabolism and is also a ROS, and plays a key role in inducing damage and even death of neuronal cells. H2O2The resulting oxidative damage of cells has become one of the important tools for studying the oxidative damage of nerve cells. H2O2Easily penetrate cell membrane, react with reduced iron ion in cell to generate highly toxic hydroxyl radical, and can be used for treating various cellsCausing toxicity, particularly brain tissue neuronal cells, because the neuronal membrane contains a large amount of oxidation substrates such as polyunsaturated fatty acids, and the catalase activity is low, and the cells are more susceptible to oxidative damage than other parts.
SH-SY5Y cell is a subline (SK-N-SH → SH-SY → SH-SY5 → SH-SY5Y) derived from a metastatic bone tumor focus cell SK-N-SH of a neuroblastoma patient established in 1970 after three cloning, shows medium-level dopamine- β -hydroxylase activity, has low degree of cell differentiation, is similar to normal nerve cell in cell morphology, physiological and biochemical functions, and has obvious axon, and SH-SY5Y cell is widely used for an experiment for researching nerve cell injury2O2Induction of oxidative stress is highly sensitive, therefore H2O2The SH-SY5Y cell oxidative damage induction model is widely applied to the research of oxidative stress induced nerve cell degeneration and apoptosis.
H2O2Is a well-known cytotoxin, has strong membrane permeability and high oxygen availability of the brain, so that the central nervous system neurons are particularly easy to be damaged by oxidation. But in H2O2In the established nerve cell oxidative stress injury model experiment, the action mechanism of the medicine is mostly discussed from the antioxidation action. The research finds that the aralia taibaiensis total saponin pair H2O2The induced SH-SY5Y cell oxidative stress injury has a good protection effect, but the anti-oxidation effect is weak, so that the protection effect mechanism of the aralia taibaiensis total saponins on the nerve cell oxidative stress injury is proved, and the protection effect mechanism of the aralia taibaiensis total saponins on the nerve cell oxidative stress injury is researched from an inflammatory reaction. The result shows that the inhibition of the NF-kB inflammatory signal pathway activity is one of the action mechanisms of the aralia taibaiensis total saponin on the protection of nerve cells.
NF-kB is transcription factor closely related to inflammation mechanism, and multiple factors such as oxidant, cell factor, etc. can activate NF-kB, and the activation of NF-kB can promote the gene expression of adhesion molecule, cell surface receptor and cell factor, further aggravate inflammatory reaction. Recent researches show that local excessive inflammatory reaction of brain tissues is one of main causes of oxidative stress injury of the brain tissues, and a new way for protecting the brain tissues and reducing the injury is possibly opened up by discussing the change rule of NF-kB in nerve cell stress injury by medicaments.
The aralia taibaiensis saponin can inhibit NF-kB activation and reduce NF-kB nuclear translocation, thereby inhibiting transcription and expression of COX-2, IL-6 and the like, inhibiting inflammation cascade reaction, reducing the content of inflammatory factors, reducing the accumulation of ROS, thereby reducing cell damage and playing a role in protecting cells.
NF- κ B is closely related to apoptosis and can induce apoptosis through death receptor pathway and mitochondria-dependent pathway. Research results show that the aralia taibaiensis saponin and the apoptosis promoter can inhibit nerve cell apoptosis when used together, and the effect is probably related to the inhibition of NF-kB activation.
The invention respectively causes mouse acquired and consolidated memory disorder models through scopolamine and sodium nitrite, observes the influence of aralia taibaiensis total saponins on different learning and memory links by adopting a diving platform experiment, detects the apoptosis rate of monoamine neurotransmitters dopamine and brain nerve cells, and discusses the influence of the aralia taibaiensis total saponins on learning and memory functions. The scopolamine and the sodium nitrite respectively block different memory links to cause a dysmnesia animal model, better simulates the characteristic of hypomnesis, and is a mature and reliable dysmnesia model. The aralia taibaiensis total saponins can obviously improve learning memory acquisition of dysmnesia model mice and improve memory consolidation disorder. The total saponin of Aralia taibaiensis can inhibit apoptosis of brain nerve cells by increasing dopamine content in brain tissue, and can prevent and improve learning and memory. Hypomnesia is a sign symptom of mild cognitive impairment, and is the earliest symptom and essential characteristic of senile dementia. Therefore, the total saponin of Aralia taibaiensis can be used for preventing and treating neurodegenerative diseases such as senile dementia.
In conclusion, the medicine is used for preventing and/or treating neurodegenerative diseases, and has clear efficacy. The medicine belongs to a pure natural preparation, is not treated by an organic solvent, and is safe and reliable; the toxicity is low, and adverse reactions do not occur after long-term use; can be used for preventing and treating neurodegenerative diseases caused by various reasons.
Claims (6)
1. The application of the aralia taibaiensis extract in preparing the medicine for relieving nerve cell injury is characterized in that the total saponin content in the extract is 4.68-83.9 wt%, and the medicine is a preparation prepared by taking the aralia taibaiensis total saponin extract as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.
2. Use according to claim 1, characterized in that: the weight percentage of the araloside A in the extract is 0.52-16.23%.
3. Use according to claim 1, characterized in that: the Aralia taibaiensis is derived from root bark of Aralia taibaiensis L belonging to Aralia of Araliaceae.
4. Use according to claim 1, characterized in that: the extract is prepared by the following method:
weighing raw materials, adding water or organic solvent for extraction, filtering the extractive solution, and concentrating; or
Weighing raw materials, adding water or organic solvent for extraction, filtering the extract, concentrating into extract, passing the extract through macroporous resin, eluting with organic solvent or water first, then eluting with organic solvent, collecting the eluate, concentrating, and drying to obtain the final product.
5. Use according to claim 4, characterized in that: the extract is prepared by the following method:
decocting cortex Araliae Elatae Radicis with 12 times of water for 0.5-2 hr twice, filtering, and concentrating under reduced pressure to obtain extract; or
Extracting root bark of Aralia taibaiensis with 12 times of 50-90% ethanol under reflux twice for 0.5-2 hr each time, filtering, and concentrating under reduced pressure to obtain extract; or
Extracting root bark of Aralia taibaiensis with 12 times of 70% ethanol under reflux twice, each for 0.5-2 hr, filtering, concentrating under reduced pressure to obtain extract, loading onto HPD100 macroporous resin, eluting with 50-70% ethanol or eluting with 2-4BV water, eluting with 2-4BV 70% ethanol, collecting eluate, and drying.
6. Use according to claim 1, characterized in that: the preparation is an oral preparation.
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