New natural active component separated from Erigeworthia chrysantha as well as preparation method and application thereof

Abstract

The invention discloses a new natural active component separated from Erigea edulis as well as a preparation method and application thereof, the structural formula of the new natural active component is as follows: # imgabs0 #, and the component can increase the level of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in serum, liver and brain tissues of an aged mouse, reduce the content of malondialdehyde (MDA), improve the activity of the mouse in senescence and improve the activity of the mouse in senescence. The compound shows relatively strong anti-oxidation and anti-aging effects and can be applied to preparation of drugs or / and health-care products for treating or preventing and resisting aging; or can be applied to preparation of drugs for enhancing in-vivo antioxidant enzyme level, and provides a new treatment means for treatment and prevention of senescence.

Description

Technical Field

[0001] The invention relates to the field of separation and extraction of natural products, and in particular to a new natural active ingredient separated from Herba Scutellariae and a preparation method and application thereof. Background Art

[0002] Aging is a degenerative change in the metabolic function, autoimmunity and tissue structure of the body's tissues. There are many theories about aging in modern medicine, such as the free radical theory, the immune function degeneration theory, the neuroendocrine theory, the telomere theory, etc. The free radical theory believes that in the body's metabolic process, the increase in free radical levels and the decrease in antioxidant enzyme activity or level will reduce the body's antioxidant capacity, increase cytotoxicity, cause irreversible damage to cell membrane component proteins and DNA molecular structures, and accelerate the body's aging process.

[0003] Traditional Chinese medicine believes that imbalance of yin and yang is the internal cause of human aging. Deficiency of the five internal organs and disorder of qi and blood will accelerate human aging. Prevention before illness, nourishing the spleen and stomach, preserving essence and strengthening the kidney, adjusting yin and yang, and taking balance as the period are the basic methods to maintain body health and delay aging. Therefore, finding anti-aging active ingredients from tonic Chinese medicine has always been a research hotspot. The chemical structure categories of natural antioxidant active ingredients that have been discovered so far mainly include: phenolic compounds, flavonoid compounds, polysaccharides, saponins and alkaloids. With the improvement of people's living standards and the proposal of the national health development strategic goals, the demand for functional anti-aging products is increasing, and the research and development of anti-aging products has broad development prospects.

[0004] Eritrichium rupestre (Pall.) Bunge is the dried aerial part of Eritrichium rupestre (Pall.) Bunge, a plant of the Boraginaceae family. It is one of the commonly used Chinese and Mongolian medicines. It tastes bitter and sweet, is cool in nature, has the effect of clearing away heat and detoxifying, and is clinically used to treat high fever headache, epidemic fever, vasculitis, anthrax and other diseases. Eritrichium rupestre mainly contains chemical components such as flavonoid glycosides, lignans, organic acids, and sterols. The extract has anti-inflammatory and analgesic effects. Modern medicine has little research on the chemical components and corresponding anti-aging activity of Eritrichium rupestre, and the potential of biological activity of Eritrichium rupestre is not fully explored. Summary of the invention

[0005] The first object of the present invention is to provide a new natural active ingredient isolated from Scutellaria serrata, the structural formula of the new natural active ingredient is as follows:

[0006]

[0007] The second object of the present invention is to provide a method for isolating new natural active ingredients from Scutellaria baicalensis, the method comprising the following steps:

[0008] Step S1. Weigh the dried Scutellaria serrata, crush it into coarse powder, soak it in water, heat it under reflux and extract it to obtain the Scutellaria serrata water extract;

[0009] Step S2. Disperse the water extract of Scutellaria baicalensis with water, extract with petroleum ether, chloroform, ethyl acetate and n-butanol in sequence, take the ethyl acetate extraction portion, perform gradient elution by silica gel column chromatography, retain the elution portion of cyclohexane and ethyl acetate in a ratio of 1:2, then separate by ODS reverse phase silica gel column chromatography, retain the 40% methanol elution portion, and then purify by sephadex LH-20 gel column chromatography to obtain the monomer compound.

[0010] As a preferred embodiment of the present invention, the specific steps of preparing the water extract of Scutellaria serrata in step S1 are: weighing 2 kg of dry Scutellaria serrata, crushing it into coarse powder, soaking it in water for 8 hours, heating and refluxing extraction twice, each time for 2 hours, filtering, combining the extracts, reducing pressure to recover the solvent, and freeze-drying to obtain the water extract of Scutellaria serrata.

[0011] The present invention is the first to separate a new natural active ingredient from Scutellaria serrata, and is the first to disclose that the new natural active ingredient can enhance the levels of multiple antioxidant enzymes in the body. Therefore, the new natural active ingredient provided by the present invention can be used in the preparation of drugs or / and health products for treating or preventing anti-aging; or in the preparation of drugs for enhancing the levels of antioxidant enzymes in the body.

[0012] As a preferred embodiment of the present invention, the aging is aging caused by a decrease in the activity or level of antioxidant enzymes.

[0013] As a preferred embodiment of the present invention, the antioxidant enzymes include superoxide dismutase and glutathione peroxidase.

[0014] Advantages and beneficial effects of the present invention:

[0015] (1) The present invention systematically separates the chemical components of the extract of Scutellaria scabra. The water extract of Scutellaria scabra is extracted with solvents of different polarities. The ethyl acetate extraction fraction is separated by normal phase, reverse phase silica gel and gel column chromatography in turn to obtain a new monomer compound containing a polycyclic lactone. The structure of the component is identified by using NMR, MS and other spectroscopy techniques. Pharmacological experiments show that the component can increase the levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the serum, liver and brain tissues of aged mice, and reduce the content of malondialdehyde (MDA), showing a strong antioxidant and anti-aging effect.

[0016] (2) The present invention is the first to isolate a new natural active ingredient from Scutellaria baicalensis, and is the first to disclose that the new natural active ingredient can enhance the levels of multiple antioxidant enzymes in the body. The new natural active ingredient can be used in the preparation of drugs and / or health products for the treatment or prevention of anti-aging; or in the preparation of drugs that enhance the levels of antioxidant enzymes in the body, providing a new therapeutic means for the treatment and prevention of aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Compound (new natural active ingredient isolated from Scutellaria baicalensis) 1 HNMR spectra;

[0018] Figure 2 Compound (new natural active ingredient isolated from Scutellaria baicalensis) 13 CNMR images

[0019] Figure 3 DEPT diagram of the compound (a new natural active ingredient isolated from Scutellaria baicalensis);

[0020] Figure 4 The effects of the compound (a new natural active ingredient isolated from Herba Scutellariae) on the organ indexes of mice; compared with the normal group: ## P<0.01; compared with the model group: *P<0.05, **P<0.01;

[0021] Figure 5 The effect of the compound (a new natural active ingredient isolated from Herba Scutellariae) on the level of SOD; A: serum; B: liver; C: brain tissue; compared with the normal group: ## P<0.01; compared with the model group: *P<0.05, **P<0.01;

[0022] Figure 6 The effect of the compound (a new natural active ingredient isolated from Herba Scutellariae) on the level of GSH-Px; A: serum; B: liver; C: brain tissue; compared with the normal group: ## P<0.01; compared with the model group: *P<0.05;

[0023] Figure 7 Effects of the compound (new natural active ingredient isolated from Herba Scutellariae) on MDA levels; A: serum; B: liver; C: brain tissue; compared with the normal group: ## P<0.01; compared with the model group: *P<0.05, **P<0.01. DETAILED DESCRIPTION

[0024] The present invention is not limited by the following embodiments, and specific implementation methods can be determined according to the technical scheme of the present invention and actual conditions. Unless otherwise specified, the various chemical reagents and chemicals mentioned in the present invention are all known and commonly used chemical reagents and chemicals in the prior art.

[0025] The present invention will be further described below in conjunction with embodiments:

[0026] 1. Isolation, preparation and structural identification of compounds:

[0027] 1. Experimental materials and instruments:

[0028] Eritrichium rupestre (Pall.) Bunge: Originated from Xing'an League, Inner Mongolia, it was identified as Eritrichium rupestre (Pall.) Bunge by Professor Li Genyou of Zhejiang Agricultural and Forestry University.

[0029] Instruments: Brucker Avance 500MHz superconducting nuclear magnetic resonance spectrometer, VGAuto Spec-3000 mass spectrometer, Shimadzu IR-8400IR spectrophotometer, UV-2100 ultraviolet analyzer, SEPA-3000 high-sensitivity polarimeter, column chromatography silica gel (Qingdao Ocean Chemical Group Co., Ltd., 100-400 mesh), ODS (63-212μm, Wako), Sephadex LH-20 (Sigma).

[0030] 2. Extraction and separation:

[0031] Weigh 2kg of dried Scutellaria serrata, crush it into coarse powder, soak it in water for 8 hours, heat and reflux to extract twice, each time for 2 hours, filter, combine the extracts, recover the solvent under reduced pressure, freeze-dry, and obtain the Scutellaria serrata water extract. Disperse 200g of the water extract with water, extract it with petroleum ether, chloroform, ethyl acetate, and n-butanol in sequence. Take the ethyl acetate extraction part (60g), use silica gel column chromatography for gradient elution, the eluent is cyclohexane (Hexane) and ethyl acetate (EtOAc) in different proportions, wherein the Hexane / EtOAc (1:2) elution part (34.6g) is separated by ODS reverse phase silica gel column chromatography, and the 40% methanol elution part is further purified by sephadex LH-20 gel column chromatography for multiple times to obtain the monomer compound.

[0032] 3. Compound structure data:

[0033] The above-mentioned monomer compound separated and purified is a white powder, α 3 D 1.0 -43.5(MeOH c 1.0); UVλMeOHmax nm(logε):241(4.11)nm; IRν max (cm -1 )3332,2933,2368,1697,1502; HR-FABMS m / z289.1501[M+H] + (calculated for C 17 H 21 O 4 289.1454). 1 H NMR and13 C NMR data are shown in Table 1, and the spectrum is shown in Figures 1 to 3 As shown, after analysis, the specific structure of the monomer compound is as follows:

[0034]

[0035] 1,3,4,4a,5,6-hexahydro-7-methylcyclopenta[c]pyran-5-yl 4-methoxybenzoate Table 1. Compounds 1 H NMR and 13 C NMR data (CD 3 OD)

[0036]

[0037] 2. Anti-lipid peroxidation effects of compounds on D-galactose-induced aging mice

[0038] 1. Experimental materials:

[0039] Malondialdehyde (MDA, batch number: 20211021), superoxide dismutase (SOD, batch number: 20210918), glutathione-peroxidase (GSH-Px, batch number: 20210806), D-galactose, Nanjing Jiancheng Biotechnology Co., Ltd.

[0040] 50 clean grade Kunming (KM) male mice, weighing 18-22 g, from the Experimental Animal Center of Zhejiang Academy of Medical Sciences, license number: SCXK(Zhe)2019-0002.

[0041] 2. Animal grouping, modeling and drug administration

[0042] Fifty male Kunming mice were adaptively fed for one week and randomly divided into a normal group, a model group, a positive group, a low-dose group and a high-dose group, with 10 mice in each group. Model group: 0.2 ml D-galactose (200 mg / kg, prepared with saline) was injected intraperitoneally in the morning, and an equal volume of saline was gavaged in the afternoon. Positive control group: D-galactose (200 mg / kg) was injected intraperitoneally in the morning, and vitamin C (100 mg / kg) was gavaged in the afternoon. Low-dose group (50 mg / kg) and high-dose group (100 mg / kg): D-galactose (200 mg / kg) was injected intraperitoneally in the morning, and gavage was given in the afternoon. The administration cycle was 6 weeks. After the last administration, fasting but not water was allowed for 12 hours, blood was collected from the eye sockets, and the mice were killed by ether anesthesia by dislocation of the neck. The liver, thymus and spleen of the mice were taken and weighed; the organ index was calculated. Organ index (%) = organ mass (g) / body weight (g) × 100%.

[0043] Preparation of serum and tissue fluid: The blood was centrifuged at 4°C, 3000r / min for 10min, and serum was collected for testing. The liver and brain tissues of mice were peeled off, and physiological saline precooled at 4°C was added to prepare tissue homogenate, which was centrifuged at 4°C, 3000r / min for 10min. The supernatant was collected and the levels of SOD, GSH-Px and MDA in serum, liver homogenate and brain homogenate were tested according to the instructions of the kit.

[0044] 3. Effects of compounds on mouse organ indexes

[0045] Depend on Figure 4 It can be seen that compared with the normal group, the liver, spleen and thymus indexes of the model group mice were significantly reduced (P<0.01). After drug intervention, compared with the model group, the liver, spleen and thymus indexes of the mice in each drug group increased to varying degrees, among which the positive group and the high-dose group mice had statistical differences (P<0.05). Regarding the effect on liver index, the effect of the high-dose group was stronger than that of the positive control group. It shows that the compound can inhibit tissue and organ atrophy to a certain extent and has an anti-aging effect.

[0046] 4. Effects of compounds on SOD levels in mice

[0047] Depend on Figure 5 It can be seen that compared with the normal group, the SOD levels in the serum, liver tissue and brain tissue of the model group mice were significantly reduced (P<0.01). After drug intervention, compared with the model group, the SOD levels in the serum, liver tissue and brain tissue of the drug group mice increased to varying degrees, among which the positive group and the high-dose compound group were significantly increased (P<0.05 or P<0.01), and the effect of the high-dose group was stronger than that of the positive control group.

[0048] 5. Effects of compounds on GSH-Px in serum, liver and brain tissues of aged mice

[0049] GSH-Px is one of the important peroxide decomposing enzymes in the body, which plays a role in protecting the structure and function of cell membranes. Figure 6 It can be seen that compared with the normal group, the GSH-Px levels in the serum, liver tissue and brain tissue of the mice in the model group were significantly decreased (P<0.01); after drug intervention, compared with the model group, the GSH-Px levels in the serum, liver tissue and brain tissue of the mice in the drug treatment group were increased to varying degrees, among which the positive group and the high-dose group were significantly increased (P<0.05), and the effect of the high-dose drug treatment group was stronger than that of the positive control group.

[0050] 6. Effects of compounds on MDA in serum, liver and brain tissue of aged mice

[0051] MDA is the main product of lipid peroxidation in the body, and the increase of MDA can reflect the degree of tissue oxidative damage. Figure 7 It can be seen that compared with the normal group, the MDA levels in the serum, liver tissue and brain tissue of the mice in the model group were significantly increased (P<0.01). After drug intervention, compared with the model group, the MDA levels in the serum, liver tissue and brain tissue of the mice in the drug treatment group were reduced to varying degrees, among which the positive group and the high-dose drug treatment group were significantly reduced (P<0.05 or P<0.01), and the activity of the high-dose drug treatment group was stronger than that of the positive control group.

[0052] In summary, the present invention is the first to isolate a new monomer compound 1,3,4,4a,5,6-hexahydro-7-methylcyclopenta[c]pyran-5-yl 4-methoxybenzoate from the extract of Scutellaria baicalensis. The compound has strong antioxidant and anti-aging effects, and the therapeutic effect of the high-dose group is better than that of the positive control group, which provides an effective therapeutic means for the treatment and prevention of aging.

[0053] The above technical features constitute the embodiments of the present invention, which have strong adaptability and implementation effect. Non-essential technical features can be added or reduced according to actual needs to meet the requirements of different situations.

Claims

1. A new natural active ingredient isolated from Herba Scutellariae, characterized in that: The structural formula of the new natural active ingredient is as follows:

2. The method for preparing a new natural active ingredient isolated from Scutellaria serrata according to claim 1, characterized in that: The method comprises the following steps: Step S1. Weigh the dried Scutellaria serrata, crush it into coarse powder, soak it in water, heat it under reflux and extract it to obtain the Scutellaria serrata water extract; Step S2. Disperse the water extract of Scutellaria baicalensis with water, extract with petroleum ether, chloroform, ethyl acetate and n-butanol in sequence, take the ethyl acetate extraction portion, perform gradient elution by silica gel column chromatography, retain the elution portion of cyclohexane and ethyl acetate in a ratio of 1:2, then separate by ODS reverse phase silica gel column chromatography, retain the 40% methanol elution portion, and then purify by sephadex LH-20 gel column chromatography to obtain the monomer compound.

3. The preparation method according to claim 2, characterized in that: Step S1: The specific steps of preparing the water extract of Scutellaria serrata are: weighing 2 kg of dry Scutellaria serrata, crushing it into coarse powder, soaking it in water for 8 hours, heating and refluxing extraction twice, each time for 2 hours, filtering, combining the extracts, reducing pressure to recover the solvent, and freeze-drying to obtain the water extract of Scutellaria serrata.

4. The new natural active ingredient isolated from Scutellaria serrata according to claim 1 is used in the preparation of drugs or / and health products for treating or preventing anti-aging; or in the preparation of drugs for enhancing the level of antioxidant enzymes in the body.

5. The use according to claim 4, characterized in that: The aging is caused by a decrease in the activity or level of antioxidant enzymes.

6. The use according to claim 4, characterized in that: The antioxidant enzymes include superoxide dismutase and glutathione peroxidase.

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