CN102212093B - Flavonoid glycoside compounds, method for preparing same and application - Google Patents
Flavonoid glycoside compounds, method for preparing same and application Download PDFInfo
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Abstract
The invention discloses two types of flavonoid glycoside compounds which are kaempferol-3-O-[alpha-L-rhamnopyranosyl-(1-3)-(2'' ', 4'' '-O-diacetyl-alpha-L-rhamnopyranosyl)-(1-6)]-beta-glucopyrose galactopyranoside and kaempferol-3-O-[alpha-L-rhamnopyranosyl-(1-3)-(4'' '-O-acetyl-alpha-L-rhamnopyranosyl)-(1-6)]-beta-D-glucopyrose galactopyranoside respectively. The invention also discloses a method for preparing the two types of flavonoid glycoside compounds. Moreover, pharmacological tests prove that the two types of flavonoid glycoside compounds have quite good effects of reducing blood sugar and blood fat and resisting oxidation and cerebral ischemia, and thus, the flavonoid glycoside compounds of the invention can be used for preparing medicaments, food and cosmetics for preventing and treating diabetes, reducing blood fat and resisting oxidation and cerebral ischemia.
Description
Technical field
The present invention relates to medical technical field, be specifically related to two kinds of flavonoid glycoside compounds; The invention still further relates to the preparation method of these two kinds of flavonoid glycoside compounds, and the purposes of these two kinds of flavonoid glycoside compounds in field of medicaments.
Background technology
Modern study shows, active oxygen (ROS) refers to be produced by redox reaction, and contains the general name of the very active material of a class chemical property of aerobic on its molecule, comprises superoxide anion free radical, hydrogen peroxide, hydroxyl radical free radical etc.Active oxygen especially free radical has the activity of height and extremely strong oxidizing reaction ability, can attack biomacromolecule in body by oxygenizement, as nucleic acid, protein, carbohydrate and lipid etc., make these material generation peroxidation sex change, crosslinked and fracture, thereby cause the destruction of cellularstructure and function, cause disorganization and the degeneration of body to change, cause the multiple organ of human body and tissue injury.Wherein comprise with the close major disease of Relationship between Free Radical: 1. skin aging: wrinkle, color spot, senile plaque, pachylosis, lax; 2. cerebral tissue change and cardiovascular and cerebrovascular diseases: senile dementia, Parkinson's disease, hypertension, atherosclerosis, coronary heart disease, thrombosis, apoplexy; 3. disease of immune system: systemic lupus erythematous, rheumatoid arthritis, scleroderma, xerosis, Immune Dysfunction; 4. internal secretion, digestive system: diabetes, alcoholic liver injury, hepatitis, liver cirrhosis, stomach ulcer, ulcerative enteritis; 5. genitourinary system: hysteromyoma, prostatitis, hyperplasia of prostate, prostate cancer; 6. respiratory system disease: rhinitis, asthma, adult type respiratory distress syndrome (ivrds), pulmonary emphysema, lung fibrosis; 7. malignant tumour: lung cancer, liver cancer, the esophageal carcinoma, cancer of the stomach, mammary cancer, cervical cancer, carcinoma of endometrium, Vulvar; 8. eyes pathology: cataract, ocular fundus pathology, eyeball are hemorrhage.
Therefore, people attempt by suppressing the active oxygen in body, and especially in body, excessive free radical prevents and treats above-mentioned disease by removing, and has obtained certain achievement.Cerebral protective agent Radicut injection as the exploitation of Mitsubishi Tokyo Co., Ltd.; a kind of exactly excessive free radicals that produces when removing cerebral ischemia re-pouring; thereby the protection brain cell alleviates the new drug of the sequela that cerebral thrombosis causes, has obtained good clinical effectiveness since listing.
Flavonoid compound is a class polyphenols, has biological activity comparatively widely, as anti-oxidant, anti-mutation, anti-ageing, antitumor, antibiotic etc.Wherein the most outstanding is the effect that it has very strong inhibition active oxygen, removes free radical, and toxic side effect, comprises and seeks new flavonoid compound so people make great efforts to seek active medicinal matter from flavonoid compound always less than chemical synthetic drug.At present more existing new drugs take flavonoid compound as effective constituent are applied clinically.Be used for the treatment of the diseases such as coronary heart disease, cerebral blood supply insufficiency and senile dementia as Folium Ginkgo total flavones, obtained significant clinical efficacy.In a word, flavonoid compound as can high-efficient cleaning except a class natural compounds of free radical, day by day come into one's own in the application aspect the prevention disease relevant with treatment and radical damage.
The Actinidiaceae actinidia, belongs to more than 70 kinds to evergreen liana entirely for fallen leaves, half fallen leaves, and main product is in the Asia, and China is the advantage producing region, has more than 50 to plant, the concentrated place of production be on the south the Qinling Mountains and Hengduan mountain range to the east of Continental Area.Wherein, Actinidia valvata (have another name called tweezer and close Kiwifruit) (Actinidia valvata Dunn) mainly is distributed in the provinces and regions such as Zhejiang, Jiangxi, Jiangsu, and its root is used as medicine and is the Root of Valvate Actinidia, and the clinical malignant tumour that is used for the treatment of is East China anti-tumor Chinese medicine commonly used.At present, the research of the rarely seen chemical composition about the Root of Valvate Actinidia, pharmacological action seldom report [Ding Lili, Wang Shunchun, Wang Zhengtao. the research of Root of Valvate Actinidia's chemical composition. CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2007,32 (18): 1893-1895; Zhang Yani, Liu Ling, Ling Changquan. restraining effect and the Mechanism Discussion of Root of Valvate Actinidia's efficient part to mice transplanted tumor H22. CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2006,36 (11): 918-920; Lu Yin, Chen Shaoyuan, Wu Ping, etc. medicinal plant Root of Valvate Actinidia effective constituent research (I)-Elements. journal of Zhejiang university, 2004,30 (2): 185-188; Hot magnanimity, Wu Yingchun, Xu Yanfeng, etc. the comparative studies of Actinidia valvata root and stem. The 2nd Army Medical College journal, 2008,29 (3): 298-302], report without medicinal record and research for the Actinidia valvata leaf.in recent years, we are anti-oxidant, hypoglycemic, reducing blood-fat, the Chinese medicine of anti-cerebral ischemia, in the researches on natural drugs process, find that the Actinidia valvata leaf has stronger anti-oxidant, hypoglycemic, reducing blood-fat, the effect of anti-cerebral ischemia, the chemical composition of employing system is separated, identification of means, therefrom separate and obtain two new flavonoid glycoside compounds, show that through pharmacological evaluation they have stronger anti-oxidant, reducing blood-fat, hypoglycemic, treating cerebral ischemia, thereby can be used for preparing particularly hyperlipidemia of the control disease relevant with radical damage, diabetes, the medicine of cerebral ischemia, food or makeup.
Summary of the invention
Technical problem to be solved by this invention is to provide two kinds of new flavonoid glycoside compounds; And provide from natural plant material the method for separating the above-mentioned two kinds of new flavonoid glycoside compounds of preparation; The purposes of these two kinds of new flavonoid glycoside compounds in field of medicaments proposed simultaneously.
The inventor is devoted for years in seek safer and more effective natural antioxidants from natural drug, Chinese medicine.Finally separate through unremitting effort and acquire two kinds of New Flavonoid Glycoside compounds, show to have anti-oxidant, hypoglycemic, reducing blood-fat significantly, treating cerebral ischemia through pharmacological evaluation, thereby can be used for preparing the control disease relevant with radical damage particularly medicine, food or the makeup of hyperlipidemia, diabetes, cerebral ischemia.
Two kinds of flavonoid glycoside compounds provided by the present invention, chemical name is respectively:
Kaempferol-O-[α-L-rhamnopyranosyl-(1-3)-(2 ' ' ', 4 ' ' '-O-diacetyl-α-L-rhamnopyranosyl)-(1-6)]-β-D-galactopyranoside, referred to as compound 1;
Kaempferol-O-[α-L-rhamnopyranosyl-(1-3)-(4 ' ' '-O-ethanoyl-α-L-rhamnopyranosyl-(1-6)]-β-D-galactopyranoside, referred to as compound 2.
The chemical structural formula of above-mentioned two kinds of flavonoid glycoside compounds is as the formula (1):
Wherein, compound 1:R=CH
3CO-; Compound 2:R=H.
Above-mentioned two kinds of flavonoid glycoside compounds have following physico-chemical property and Wave Spectrum feature:
Compound 1: pale yellow powder, the molish reacting positive, mp:181-183 ℃,
ESI-MS m/z:825.28[M+H]
+, 847.28[M+Na]
+, 823.28[M-H]
-HR-ESI-MS：847.2279([M+Na]
+，C
37H
44O
21Na
+，cal.847.2273)。
1H-NMR and
13The C-NMR data see Table 1; in conjunction with 2D-NMR data (seeing accompanying drawing 11-13) be accredited as Kaempferol-O-[α-L-rhamnopyranosyl-(1-3)-(2 ' ' ', 4 ' ' '-O-diacetyl-α-L-rhamnopyranosyl)-(1-6)]-β-D-galactopyranoside.
Compound 2: pale yellow powder, the molish reacting positive, mp:203-205 ℃,
ESI-MS m/z:783.28[M+H]
+, 805.28[M+Na]
+, 781.28[M-H]
-HR-ESI-MS：805.2166([M+Na]
+，C
35H
42O
20Na
+，cal.805.2167)。
1H-NMR and
13The C-NMR data see Table 1, in conjunction with 2D-NMR data (seeing accompanying drawing 20-22) be accredited as Kaempferol-O-[α-L-rhamnopyranosyl-(1-3)-(4 ' ' '-O-ethanoyl-α-L-rhamnopyranosyl-(1-6)]-β-D-galactopyranoside.
Table 1. compound 1,2
1H-,
13C-NMR(d
6-DMSO) data
Annotate: (1)
aMagneticstrength 500MHz used; (2)
bMagneticstrength 125MHz used is with the multiplicity of the definite C of DEPT spectrum; (3)
cThe ownership of chemical shift can be exchanged; (4)
dThe ownership of chemical shift can be exchanged
The relevant Spectral Characteristic of above-mentioned two kinds of compounds is respectively as shown in accompanying drawing 5～22.
Above-mentioned two kinds of flavonoid glycoside compounds mainly separate preparation, wherein preferred seed plant, or Actinidiaceae actinidia, most preferably Actinidiaceae actinidia Actinidia valvata from higher plant.About scientific definition and the category of Actinidiaceae Actinidia, referring to " Chinese Higher plant section belong to dictionary " (revised edition) (Hou Kuanzhao compiles, Beijing: Science Press, 1982) and Chinese Plants will the 49th volume (Beijing: Science Press, 1984).Be generally stem branch and the leaf of these plants for the preparation of the vegetable material of these two kinds of New Flavonoid Glycoside compounds, preferred leaf is as extracting raw material.
The preparation method of these two compounds provided by the invention comprises the steps:
(1) the higher plant raw material extracts with the suitable solvent of 5～100 times, gets extracting solution.
This step solvent used can be preferably 50%～95% ethanol or methanol aqueous solution for methyl alcohol, ethanol or the aqueous acetone solution of water or various concentration, more preferably 70%～95% ethanol or methanol aqueous solution, most preferably 80% ethanol or methanol aqueous solution.Certainly, consider safety factors and cost factor in possible organic solvent residual, production, aqueous ethanolic solution is more commonly used.
Extracting mode can select conventional thermal backflow extraction, diacolation extract or soak and extract, and other pharmaceutically acceptable extracting modes.Extract as thermal backflow and can select 12 times, 10 times, 10 times, 1.5 hours, 1 hour, 1 hour, also can select other acceptable extraction solvent loads, extraction time, extraction time.Diacolation extracts, soaks to extract and can select 5～100 times of solvents, although select too much or very few solvent, also can partly or entirely realize the described flavonoid glycoside compound of this patent, can not consist of material alterations to the present invention.
Higher plant of the present invention is selected from spermatophyte, preferred Actinidiaceae actinidia, and Actinidiaceae actinidia Actinidia valvata most preferably, agents area is selected leaf.
(2) concentrated
Be 1.02～1.15 thick medicinal extract with said extracted liquid simmer down to proportion.
The concentrated mode of this step extracting solution can adopt 40～100 ℃ of concentrating under reduced pressure, oven dry is concentrated and other pharmaceutically acceptable concentrated modes.Certainly the too high temperature of scruple may impact the stability of target component, selects 50～80 ℃ of temperature concentrated more feasible.
(3) decolouring removal of impurities
Above-mentioned thick medicinal extract with proper method decolouring, removal of impurities, is got the decolouring after product.
The decolouring of the described thick medicinal extract of this step, impurity-removing method can adopt resin absorption decolouring, removal of impurities, specifically can select the model resins such as D101, AB-8, MCI-Gel, with the water of 3～10 times with the abundant agitation and dilution of thick medicinal extract after, cross resin column absorption, eluent wash-out with 2～10 times of column volumes, collect elutriant, concentrated, be the decolouring after product.Eluent can be 10%～75% ethanol, methyl alcohol, aqueous acetone solution, preferred 30%～50% ethanol, methanol aqueous solution, most preferably 30%～50% aqueous ethanolic solution.
The decolouring of the described thick medicinal extract of this step, impurity-removing method also can select with the water of 3～10 times with the abundant agitation and dilution of thick medicinal extract after successively with ethers, ester class, n-butanol extraction, n-butanol portion is merged concentrated, get the decolouring after product.Described ether solvent can be ether, sherwood oil, preferred sherwood oil.Described esters solvent ethyl acetate.
(4) chromatographic separation
The after product that will decolour adopts that chromatographic process is separated, purifying, must these two kinds of flavonoid glycoside compounds.
Chromatographic process of the present invention can adopt purification on normal-phase silica gel, reverse phase silica gel, polymeric amide, dextrane gel Sephadex LH-20 as filler.If adopt the normal phase silica gel chromatography method, can adopt column chromatography methods, can select chloroform: methyl alcohol, methylene dichloride: methyl alcohol, chloroform: ethanol, chloroform: methyl alcohol: water, chloroform: ethanol: the water gradient elution.If adopt reverse phase silica gel, polymeric amide, dextrane gel Sephadex LH-20 chromatographic process, can adopt column chromatography methods, eluent can be selected methyl alcohol: water, ethanol: water, acetonitrile: water, acetone: the water gradient elution.In above-mentioned chromatographic process, sectional is accepted the wash-out fraction, follows the tracks of with TLC and detects, and identical fraction merges, reclaims.
In concrete chromatographic separation process, above-mentioned chromatographic process can be used alone or in combination, for separate, purification effect is better.For example, fraction for normal phase silica gel column chromatography institute wash-out, reception, merging, still contain impurity if wherein contain the fraction of target component, can process through dextrane gel Sephadex LH-20 column chromatography methods again, removal of impurities and separating effect can be improved greatly.
In a word, according to polarity order, the molecular size of molecule, those skilled in the art use above-mentioned chromatographic process alone or in combination, can separate to obtain compound 1,2 from the decolouring after product.
Embodiment subsequently will prove, two kinds of new flavonoid glycoside compounds of the present invention have stronger anti-oxidant, reducing blood-fat, hypoglycemic, treating cerebral ischemia, can be used as the application of pharmaceutical composition, be prepared into conventional pharmaceutical dosage form with pharmaceutically acceptable pharmaceutical excipient, as tablet, pill, paste, capsule, solution, granule and injection.The food of conventionally form can also be prepared into acceptable supplementary material, additive on bromatology, as beverage, jelly, cake etc.Also can be equipped with acceptable makeup auxiliary material, be prepared into common makeup form.
Description of drawings
Fig. 1 is compound 1,2 removing DPPH Free Radical design sketch.
Fig. 2 is compound 1,2 removing hydroxyl radical free radical action effect figure.
Fig. 3 is compound 1,2 removing superoxide anion radical effect design sketch.
Fig. 4 is compound 1,2 inhibition mouse liver even slurry lipid peroxidation schematic diagram.
Fig. 5 is the HR-ESI-MS collection of illustrative plates of compound 1.
Fig. 6 a, 6b are the ESI-MS collection of illustrative plates to compound 1.
Fig. 7 is the IR collection of illustrative plates of compound 1.
Fig. 8 is compound 1
1The H-NMR collection of illustrative plates.
Fig. 9 is compound 1
13The C-NMR collection of illustrative plates.
Figure 10 is the DEPT collection of illustrative plates of compound 1.
Figure 11 is the HH-COSY collection of illustrative plates of compound 1.
Figure 12 is the HMQC collection of illustrative plates of compound 1.
Figure 13 is the HMBC collection of illustrative plates of compound 1.
Figure 14 is the HR-ESI-MS collection of illustrative plates of compound 2.
Figure 15 a, 15b are the ESI-MS collection of illustrative plates of compound 2.
Figure 16 is the IR collection of illustrative plates of compound 2.
Figure 17 is compound 2
1The H-NMR collection of illustrative plates.
Figure 18 is compound 2
13The C-NMR collection of illustrative plates.
Figure 19 is the DEPT collection of illustrative plates of compound 2.
Figure 20 is the HH-COSY collection of illustrative plates of compound 2.
Figure 21 is the HMQC collection of illustrative plates of compound 2.
Figure 22 is the HMBC collection of illustrative plates of compound 2.
Embodiment
Below in conjunction with the drawings and specific embodiments, further set forth the present invention.These embodiment are interpreted as only being used for explanation the present invention and are not used in restriction protection scope of the present invention.After the content of having read the present invention's record, those skilled in the art can make various changes or modifications the present invention, and these equivalences change and modification falls into claim limited range of the present invention equally.
Embodiment 1: compound 1,2 extraction separate
Actinidia valvata leaf (gather from mountain area, Quzhou, Zhejiang in September, 2006) medicinal material 15.3Kg, with 80% ethanol, 12 times, 10 times, 10 times, 1.5 hour, 1 hour, 1 hour, refluxing extraction, the extracting solution filtered while hot, united extraction liquid, decompression and solvent recovery is to distinguishing the flavor of without alcohol, distilled water is added to 15L, 5000rpm, centrifugal 5 minutes, supernatant liquor 5L crosses MCI-Gel resin column absorption (diameter 10cm * height 120cm), respectively with the water of 50L, 10% ethanol, 40% ethanol is as the eluent wash-out, 1000ml/ part receives, follow the tracks of with TLC and detect fraction (the silica GF254 thin layer plate that receives, developping agent: propyl carbinol-Acetic Acid-Water=4:1:5 upper strata, coloration method: visual under ultraviolet lamp 254nm), the fraction (concentrating on 40% ethanol elution part) that contains target component merges, recovery is concentrated into dried, after product must decolour.The decolouring after product is with dissolve with methanol, filtration, get part filtrate and cross Sephadex LH-20 post, with 40% methanol-eluted fractions, 50ml/ part receives, follow the tracks of to detect fraction (silica GF254 thin layer plate, developping agent: propyl carbinol-Acetic Acid-Water=4:1:5 upper strata, coloration method: visual under ultraviolet lamp 254nm) with TLC, to contain the less fraction of target component, impurity merge, concentrated, reclaim solvent to doing, respectively compound 1,2.Above-mentioned supernatant liquor and resin decolorization after product filtrate after centrifugal is repeatedly repeated above step, finally obtains compound 1(15.2g), compound 2(36.7g).
Embodiment 2: compound 1,2 extraction separate
Actinidia valvata leaf (gather from mountain area, Quzhou, Zhejiang in June, 2007) medicinal material 1Kg, with the 50L80% ethanol percolate extraction, united extraction liquid, decompression and solvent recovery is to 1L, successively with sherwood oil, ethyl acetate, water-saturated n-butanol extraction, n-butanol portion is merged, concentrates, get the decolouring after product.The decolouring after product is with dissolve with methanol, filtration, get part filtrate and cross Sephadex LH-20 post, with 10%～50% methanol aqueous solution gradient elution, 50ml/ part receives, and follows the tracks of with TLC and detects the fraction that receives (silica GF254 thin layer plate, developping agent: propyl carbinol-Acetic Acid-Water=4:1:5 upper strata, coloration method: visual under ultraviolet lamp 254nm), to contain the less fraction of target component, impurity merge, concentrated, reclaim solvent to doing, respectively compound 1,2.Above-mentioned decolouring after product filtrate is repeatedly repeated above step, finally obtains compound 1(1.8g), compound 2(3.9g).
Embodiment 3: compound 1,2 pharmacological evaluation
1, hypoglycemic experiment
Get blood glucose value greater than the spontaneous diabetes B db/db mouse (Nanjing University's model animal center) of 20mmol/L, random packet.Administration group mouse is pressed table 2 dosage gastric infusion; Positive drug group mouse stomach gives Glyburide; The model group mouse stomach gives the equal-volume blank solvent.Each treated animal administration every day 1 time, 1 week of successive administration.Respectively at after administration in 1,3,7 day 3 hours, Mouse Tail-tip was got blood, measures its blood sugar concentration with luxuriant brilliant blood glucose meter, investigated after administration spontaneous diabetes B db/db mouse blood sugar concentration as influencing factor, and experimental result sees Table 2.But result shows the blood sugar concentration of compound 1,2 gastric infusions significance reduction mouse after 3 days.
Table 2. compound 1,2 impacts on Spontaneous type Ⅱ diabetes db/db mouse blood sugar (mmol/L)
With model group ratio, * p＜0.05
2, lipid-lowering test
Get 20～22g male ICR mouse, random packet, adaptability are fed grouping afterwards in 5 days.Administration group mouse is pressed table 3 dosed administration, the atorvastatin of positive controls gastric infusion 10mg/kg, hyperlipidemia model group give the blank solution of equal volume, 1 times/day, successive administration 14 days, water is can't help in fasting after administration in the 14th day, and the last medication is after 2 hours, except the isopyknic physiological saline of blank group abdominal injection, all the other respectively organize equal abdominal injection 75% yolk emulsion, and 0.5ml/ only causes Experimental Hyperlipemia disease.Inject after 20 hours, get blood from the mouse orbit venous plexus, detect and respectively organize mice serum cholesterol and triglyceride level, experimental result sees Table 3.But result shows compound 1,2 significancees and reduces triglyceride level and the total cholesterol level of hyperlipidemia mouse.
Table 3. compound 1,2 impacts on acute hyperlipidemia model mouse cholesterol and triglyceride level
With model group ratio, * p＜0.05
3, anti-oxidant experiment
(1) remove DPPH(1,1-phenylbenzene-2-picryl hydrazine) the free radical experiment
Take compound 1,2 appropriate, respectively with methyl alcohol be formulated as 0.005,0.025,0.125,0.5,1.0,2.5, the solution of 5.0mg/ml concentration, getting 2ml need testing solution and 2ml0.1mg/ml DPPH solution puts in tool plug centrifuge tube, shake up, placed 20 minutes, survey its absorbance as blank in 517nm take 80% ethanol, replicate(determination) 3 times is calculated as follows medicine to free radical scavenging activity:
DPPH clearance rate (%)=[A
0-(A
1-A
2)]/A
0* 100%
A
0: the absorbance of 2mlDPPH solution+2ml blank solvent
A
1: the absorbance of 2mlDPPH solution+2ml sample solution
A
2: the absorbance of 2ml blank solvent+2ml sample solution
Result shows that compound 1,2 all has the effect (Fig. 1) of removing more by force the DPPH free radical.
(2) remove the hydroxyl radical free radical experiment
The hydroxyl radical free radical kit method that adopts Nanjing to build up Graduate School of Engineering is measured, and reagent preparation and operation steps are undertaken by the test kit specification sheets.Result shows the effect (Fig. 2) that compound 1,2 is had stronger removing hydroxyl radical free radical.
(3) remove the superoxide radical experiment
Adopting Nanjing to build up Graduate School of Engineering superoxide anion kit method measures, add successively reagent one 1.0ml, reagent two 0.1ml, reagent three 0.1ml, reagent four 0.1ml, sample 1ml in reaction system, abundant mixing, put 37 ℃ of waters bath with thermostatic control 40 minutes, add developer 2.0ml, mixing is with the distilled water zeroing, at the 550nm mensuration A of place
SampleControl group does not add sample, supplies volume with distilled water, measures A with method
ContrastApplication of sample group and control group do not add developer, supply volume with distilled water respectively, measure A with method
i0And A
0Calculate clearance rate with following formula:
Superoxide radical clearance rate (%)=[(A
Contrast-A
0)-(A
Sample-A
i0)]/(A
Contrast-A
0) * 100%
Result shows that compound 1,2 has the effect (Fig. 3) of stronger removing ultra-oxygen anion free radical.
(4) In Vitro Anti lipid peroxidation experiment
ICR mouse overnight fasting, next day, sacrificed by decapitation, took out rapidly liver, puts rinsing repeatedly in 4 ℃ of physiological saline, rejects fat and sheath, uses the filter paper suck dry moisture, weighs, and makes 10% tissue homogenate with the PBS of pH7.4 under condition of ice bath, and is standby.Get above-mentioned liver homogenate 1.0ml, add, add the test liquid of 1.0ml different concns, make final concentration be respectively 0.22mg/ml, 0.44mg/ml, 0.88mg/ml, add at last 1mmol/L FeSO
450 μ l, 1mmol/L H
2O
250 μ l, the blank group replaces test liquid with pure water, in 37 ℃ of constant temperature water bath vibrations 1.5 hours, adds 10% trichoroacetic acid(TCA) 2.0ml after taking-up after mixing, 0.67%TBA1.0ml, reaction 15 minutes in boiling water bath after mixing, after taking out, cold water is cooling, with 3000rpm centrifugal 15 minutes, get supernatant liquor in 532nm place colorimetric, with the blank tube zeroing, measure optical density A value, calculate inhibiting rate with following formula:
Lipid peroxidation inhibiting rate (%)=(A contrast-A sample)/A contrast * 100%
Result shows that compound 1,2 all has the effect (Fig. 4) of stronger inhibition mouse liver even slurry lipid peroxidation.
4, to the provide protection of focal brain ischemia-reperfusion injury in rats
Experimental animal is the Wistar rat, and is male, body weight 250～280g.Prepare the Focal Ischemia-Reperfusion in Rats model with the bolt collimation method.With rat with 10% Chloral Hydrate 3ml/Kg intraperitoneal injection of anesthesia, neck median incision, separation, ligation right carotid, external carotid artery and bifurcated artery thereof.Separate the right side internal carotid artery, separate wing jaw artery downwards along internal carotid artery, this branch of root ligation.Place bulldog clamp at the standby line of internal carotid artery near-end, far-end, arteria carotis communis crotch otch inserts the 4-0 nylon wire, and its degree of depth is 17～20mm, and the bolt line enters internal carotid artery, enters cranium to arteria cerebri anterior, and all blood flows of blocking-up arteria cerebri media are originated.Remove bulldog clamp, tighten standby line, stay the long the end of a thread of 1cm outward, skin suture.Tail vein injection medicine during ischemic.Ischemic is perfusion again after 2 hours, need again not anaesthetize and cut skin, lift gently institute's the end of a thread that stays to resistance is arranged time prompting nylon wire head end to the arteria carotis communis incision, blood flow is logical again.Sham operated rats is except plug wire not, and all the other steps are the same.Pour into again the mouse that survives after 24 hours, observe rat behavior and learn variation, carry out the neurological scoring.Behavior scoring method: with reference to 5 minutes standards of grading processed of Zea Longa: 0 minute, impassivity damage symptom; 1 minute, can not full extension offside fore paw: 2 minutes, turn-take laterally; 3 minutes, topple over to offside; 4 minutes, can not spontaneously walk, the loss of consciousness.Then broken end is got the mouse brain fast.At the crown brain sheet of the anterior commissure plane thick approximately 2mm of cutting-out, put at once in 2%TTC solution, hatched 30 minutes for 37 ℃.Infarct presents white, and non-infarct presents redness.Measure with planimeter (C63 ias) and respectively distinguish area, and calculate the per-cent (%) that infarct accounts for whole crown.Experimental result shows the infarct size of rat when compound 1,2 significantly reduces ischemical reperfusion injury, and focal brain ischemia-reperfusion injury in rats is had stronger provide protection (table 4).
The provide protection that table 4. medicine is cerebral ischemia re-pouring injured to the rats after transient focal type
5, acute toxicity test
60 of ICR mouse, male and female half and half, body weight 18～20g is provided by the The 2nd Army Medical College Experimental Animal Center.Begin test after 3 days in 20 ± 2 ℃ of laboratories adaptations of room temperature.Be divided at random 6 groups, gavage gives compound 1,2 each 500mg/Kg, 1000mg/Kg, 2000mg/Kg body weight respectively.Observe immediately the number of the response situation of each dosage group mouse and the death in 7 days, and the pathological anatomy inspection is carried out in survival and dead mouse.Result shows each dosage group mouse without death, and sick inspection shows no obvious abnormalities variation, shows that compound 1,2 orally has a higher security.
Embodiment 4: the preparation of capsule
Get embodiment 1 or 2 and extract to separate a kind of in the compound 1,2 that obtains or their mixture, add medicinal starch appropriate, fully incapsulate after mixing, make every capsule that contains above-claimed cpd 100mg for orally using.
Embodiment 5: the preparation of injection
Get embodiment 1 or 2 and extract to separate a kind of in the compound 1,2 that obtains or their mixture, inject with pure water and Polysorbate 80 in right amount, make injection that every 25ml contains above-claimed cpd 100mg for vein or inject.
Claims (3)
1. flavonoid glycoside compound, its structural formula be as the formula (1):
Wherein:
R=CH
3CO-, its chemical name be Kaempferol-O-[α-L-rhamnopyranosyl-(1-3)-(2 ' ' ', 4 ' ' '-O-diacetyl-α-L-rhamnopyranosyl)-(1-6)]-β-D-galactopyranoside.
2. the preparation method of flavonoid glycoside compound, is characterized in that, comprises the steps:
(1) choosing the Actinidia valvata leaf is raw material, and the aqueous ethanolic solution take concentration as 80% extracts;
(2) with said extracted liquid simmer down to proportion be 1.02～1.15 thick medicinal extract;
(3) the thick medicinal extract that obtains is used ethers, ester class, n-butanol extraction successively, merge n-butanol portion, perhaps with after the abundant agitation and dilution of thick medicinal extract water that obtains, adopt nonpolar or low-pole resin absorption, then water, 10%～75% aqueous ethanolic solution wash-out successively, collect the aqueous ethanol elutriant and be concentrated into dried;
(4) above-mentioned n-butanol extraction position or resin 10%～75% ethanol water elution thing are obtained two kinds of flavonoid glycoside compounds through chromatographic separation; a kind of be Kaempferol claimed in claim 1-O-[α-L-rhamnopyranosyl-(1-3)-(2 ' ' '; 4 ' ' '-O-diacetyl-α-L-rhamnopyranosyl)-(1-6)]-β-D-galactopyranoside, another kind be Kaempferol-O-[α-L-rhamnopyranosyl-(1-3)-(4 ' ' '-O-ethanoyl-α-L-rhamnopyranosyl-(1-6)]-β-D-galactopyranoside.
3. flavonoid glycoside compound claimed in claim 1 is for the preparation of the purposes in hypoglycemic, reducing blood-fat, anti-oxidant or anti-focal brain ischemia-reperfusion injury medicine.
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| CN102617656B (en) * | 2012-02-02 | 2014-04-23 | 中国人民解放军第二军医大学 | Method for separating and preparing high purity flavonoid glycoside compounds from actinidia valvata dunn leaves |
| CN103267759B (en) * | 2013-05-17 | 2015-11-18 | 无锡中德伯尔生物技术有限公司 | A kind of detection method of thiazolidine ketone medicine and detection kit |
| CN103613623B (en) * | 2013-11-28 | 2016-05-11 | 江苏科技大学 | The application of multiple-stage solvent extraction in the synthetic isoquercitrin of enzymatic |
| CN103804443B (en) * | 2014-01-16 | 2016-03-23 | 沈阳药科大学 | Flavonoid glycoside compound and preparation method thereof |
| CN104825505B (en) * | 2015-04-29 | 2018-08-21 | 中国人民解放军第二军医大学 | The Actinidia valvata leaf composition to resist myocardial ischemia |
| CN109369750B (en) * | 2018-11-30 | 2020-12-25 | 中南林业科技大学 | Method for extracting kaempferol galactoside compounds from Nanshan tea |
| CN109705178B (en) * | 2018-12-28 | 2022-12-27 | 贵州中医药大学 | Compound extracted from caulis Sinomenii, and its extraction process and application |
| CN113150049B (en) * | 2020-04-20 | 2022-08-19 | 中南大学 | Cyclocarya paliurus extract and application thereof in resisting gout and reducing uric acid |
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Inventor after: Xin Hailiang Inventor after: Ling Changquan Inventor after: Su Yonghua Inventor after: Wu Yingchun Inventor after: Sheng Jiayu Inventor after: Zhou Jun Inventor before: Ling Changquan Inventor before: Xin Hailiang Inventor before: Su Yonghua Inventor before: Wu Yingchun Inventor before: Sheng Jiayu Inventor before: Zhou Jun |
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