CN104758325A - Extraction method and application of moringa oleifera leaf total flavones - Google Patents
Extraction method and application of moringa oleifera leaf total flavones Download PDFInfo
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Abstract
The invention belongs to the chemical field of natural products and in particular relates to an extraction method and an application of moringa oleifera leaf total flavones. The extraction method adopts microwave and ultrasound to synergistically extract the moringa oleifera leaf total flavones, and has the advantages of high efficiency, time saving, energy conservation, low energy consumption, simple operation and good repeatability. Besides, an animal test proves that the extracted moringa oleifera leaf total flavones have a significant treatment effect on hyperuricosuria and Alzheimer's disease. The total flavones provided by the invention can reduce uric acid, and can also reduce cholesterol, triacylglycerol, urea nitrogen, creatinine and xanthine oxidase, reduce the generation of uric acid, directly decompose uric acid, improve the renal function, promote uric acid excretion, and protect multiple levels of blood vessels to reduce uric acid, so that the moringa oleifera leaf total flavones are beneficial for healing of lithemia patients and have a broad medical application prospect.
Description
Technical field
The invention belongs to field of natural product chemistry, be specifically related to extracting method of a kind of total flavones of Moringa oleifera leaf and uses thereof.
Background technology
Uric acid is a kind of containing carbon, nitrogen, oxygen, the heterocyclic compound of hydrogen, it is the end product of mankind's purine metabolism, usually generated by xanthine oxidase catalysis, under normal circumstances, in human body, the production of uric acid and excretion remain dynamic equilibrium, if the not enough growing amount and the excretion imbalance that cause uric acid in body are produced too much or discharged to uric acid, thus cause Uric Acid Content to raise, cause a series of complication, such as there is gouty arthritis, gouty nephropathy, gouty renal calculus, gouty heart disease, the severe complications such as gouty hypertension, serious affects the healthy of the mankind.
Along with people's living standard improves and dietary structure change, the prevalence of hyperuricemia rises day by day, and serious threat is to the health of the mankind.Reduce uric acid and mainly contain three kinds of approach: one is the oxidation Decomposition preventing intracellular nucleic acid, the Therapeutic Method of this approach is the material of some anti-nucleic acid oxidations of long-term taking, as astaxanthin, anthocyanidin, but such material is mainly used in treatment, and uric acid raises the gout caused, and the complications Treatment effect caused other metabolic arthritis is not remarkable; Two is block the continuation generation of uric acid or the formation of newborn uric acid, the treatment of this approach mainly adopts allopurinol to suppress the activity of xanthine oxidase, block the continuation generation of uric acid or the formation of newborn uric acid, but this medicine is inoperative to the uric acid produced, and due to the activity of xanthine oxidase suppressed, can cause uric acid precursor substance xanthine and hypoxanthic accumulation, and xanthine and hypoxanthine also there is nephrotoxicity.Three is the excretions promoting uric acid, and edible base-forming food or the appropriate alkalized urine of uric acid resisting tea energy, to promote urate excretion, but this approach uric acid resisting effect is comparatively slow, cannot reach the effect for the treatment of dialuric acid.So research and develop a kind of toxic and side effects little, the natural uric acid resisting medicine of uric acid resisting successful is the difficult problem needing solution at present badly.
At present, along with modern society's growth in the living standard, the average life of people rises year by year, and population in the world senescence phenomenon is severe.The same with hyperuricemia, the sickness rate of senile dementia constantly rises, and becomes the number one killer of harm senior health and fitness.Senile dementia is the nervous system degenerative disease of the Progressive symmetric erythrokeratodermia development of a kind of onset concealment.Clinical signs is dysmnesia, aphasia, apraxia, agnosia, the infringement of visual space technical ability, n-back test obstacle and the feature such as personality and behavior change, along with advancing of disease, patient can lose self care ability gradually, brings white elephant to the individual family of patient and society.In view of the western medicine result for the treatment of senile dementia is at present not obvious, people start the research and development more paying close attention to Chinese medicine, many Chinese medicine ingredients are improving the memory ability of senile dementia, cognitive function and slow down aging aspect have good effect, and Chinese medicine ingredients to have toxic and side effects low, to the health of patient, there is certain regulating action, be more conducive to the rehabilitation of patient.Therefore, Chinese medicine ingredients has potential advantage and wide DEVELOPMENT PROSPECT at treatment senile dementia disease.
The plant that Moringa (MoringaoleiferaLam.) is Moringaceae Moringa is perennial tropical deciduous tree, is extensively planted in Asia and African subtropical and tropical zones, has very strong adaptability to edaphic condition and rainfall.Also there is cultivation on the ground such as China Guangdong, Taiwan, and Moringa both can be used for be viewed and admired, Er Qiegen, leaf and tender fruit edible; Seed can extract oil, oil-containing about 30%, has very high economic worth.Find after deliberation, Moringa is except having abundant nutritive value, also good activity is shown in antioxidation, blood sugar lowering, blood fat reducing, antifungal, and flavone compound in leaf of Moringa and polysaccharide compound have the effects such as blood sugar lowering, blood fat reducing, blood pressure lowering, antitumor, antioxidation, antiviral, treatment cardiovascular diseases, improving water flood, it is the plant with greatly research developing potentiality.
Food and biotechnology journal disclose the extracting method of total flavones of Moringa oleifera leaf and through testing, hypoglycemic effect (Vol.26 No.4 Jul.2007) proves that the total flavones of leaf of Moringa has significant effect to blood sugar lowering, but in leaf of Moringa total flavones to the effect of uric acid resisting and senile dementia, there is not been reported.
Summary of the invention
Not remarkable for the medication effect of hyperuricemia and senile dementia in order to solve prior art, the large defect of toxic and side effects, the object of the present invention is to provide extracting method of a kind of total flavones of Moringa oleifera leaf and uses thereof, to solve above defect.
The extracting method of a kind of total flavones of Moringa oleifera leaf provided by the invention, comprises the following steps:
(1) get the raw materials ready: take 1kg leaf of Moringa in 50 DEG C of dry 2-3 h, pulverize, cross 40-50 mesh sieve, obtain leaf of Moringa powder for subsequent use;
(2) defat: get step (1) leaf of Moringa powder, add the petroleum ether of leaf of Moringa opaque amount 2-4 times of volume, reflux defat 3 times at 68 DEG C, each 5-7 h, crosses elimination filtrate, by filter residue and drying, obtain dry filtering residue for subsequent use;
(3) extract: the 65%-75% ethanol adding filtering residue quality 10-15 times volume in the dry filtering residue that step (2) obtains, 6-8min is processed under microwave power 500-600W, to put in 75 DEG C of-80 DEG C of tepidariums supersound extraction 2 times, each extraction 20-30min, filter to obtain extracting solution, merge extractive liquid, obtains total flavones of Moringa oleifera leaf crude extract;
(4) purification: the total flavones of Moringa oleifera leaf crude extract that step (3) obtains is added in adsorption resin column, use distilled water 2 times of column volumes successively, the each 3 times of volumes of volume fraction 30% ethanol, 50% ethanol, 70% ethanol carry out gradient elution, adjusted volume flow is 2mL/min, often 15min collected by pipe, with NaNO
2-Al (NO
3)
3-NaOH system color developing detection, under wavelength is 510nm, measure absorbance inspection know, collect the ethanol elution of 50% and 70%, merge eluent, put 1/5 of original volume by also concentrated for eluent decompression recycling ethanol, obtain concentrated solution, concentrated solution is placed in the extractum of 60 DEG C of water bath methods to relative density 0.16-0.25,50 DEG C of cold drying to constant weight, obtain total flavones of Moringa oleifera leaf powder.
In the extracting method of total flavones of Moringa oleifera leaf of the present invention, the boiling range of step (2) PetroChina Company Limited. ether is 60 ~ 90 DEG C.Adsorption resin column wherein in step of the present invention (3) is AB-8 type macroporous adsorptive resins.
The extracting method of total flavones of Moringa oleifera leaf of the present invention is preferably:
(1) get the raw materials ready: take 1kg leaf of Moringa in 50 DEG C of drying 2.5 h, pulverize, cross 45 mesh sieves, obtain leaf of Moringa powder for subsequent use;
(2) defat: get step (1) leaf of Moringa powder, adds the petroleum ether of leaf of Moringa opaque amount 3 times of volumes, and reflux defat 3 times at 68 DEG C, each 6 h, crosses elimination filtrate, by filter residue and drying, obtains dry filtering residue for subsequent use;
(3) extract: 70% ethanol adding filtering residue quality 12 times of volumes in the dry filtering residue that step (2) obtains, 7min is processed under microwave power 600W, to put in 80 DEG C of tepidariums supersound extraction 2 times, each extraction 25min, filter to obtain extracting solution, merge extractive liquid, obtains total flavones of Moringa oleifera leaf crude extract;
(4) purification: the total flavones of Moringa oleifera leaf crude extract that step (3) obtains is added in AB-8 type macroporous adsorptive resins, use distilled water 2 times of column volumes successively, the each 3 times of volumes of volume fraction 30% ethanol, 50% ethanol, 70% ethanol carry out gradient elution, adjusted volume flow is 2mL/min, often 15min collected by pipe, with NaNO
2-Al (NO
3)
3-NaOH system color developing detection, under wavelength is 510nm, measure absorbance inspection know, collect the ethanol elution of 50% and 70%, merge eluent, put 1/5 of original volume by also concentrated for eluent decompression recycling ethanol, obtain concentrated solution, concentrated solution is placed in the extractum of 60 DEG C of water bath methods to relative density 0.20,50 DEG C of cold drying to constant weight, obtain total flavones of Moringa oleifera leaf powder.
Extracting method of the present invention obtains total flavones of Moringa oleifera leaf through defat with petroleum ether, Combined microwave-ultrasound extraction and the remove impurity of AB-8 type macroporous adsorptive resins, in order to ensure the quality of total flavones, the each 3 times of volumes of the present invention's volume fraction 30 % ethanol, 50% ethanol, 70% ethanol carry out gradient elution, and warp is with NaNO
2-Al (NO
3)
3-NaOH system color developing detection, under wavelength is 510nm, measure absorbance inspection know, find that the content of total flavones of Moringa oleifera leaf in 50% ethanol and 70% ethanol is higher, namely the eluent through 50% ethanol and 70% ethanol elution is collected, greatly improve purification efficiency, therefore, the present invention to have the peak of very large absorption as leaf of Moringa flavone compound characteristic absorption peak under the determined wavelength of 510nm, and total flavones of Moringa oleifera leaf content is 18%-35%.
Combined microwave-ultrasound extraction total flavones of Moringa oleifera leaf of the present invention utilizes microwave energy to make the temperature of cell interior increase rapidly, and cell interior pressure increases makes cell rupture, and in medical material, effective ingredient flows out, and is dissolved in extract at a lower temperature.The method homogeneous heating, the thermal efficiency is higher, and extraction rate is fast.And have efficiently than traditional reflux, extract, save time, energy-conservation advantage, have a good application prospect in Chinese medicine extraction.
In addition, present invention also offers the purposes of total flavones of Moringa oleifera leaf in preparation treatment antihyperuricemic disease drug.The main path of uric acid resisting is the oxidation Decomposition preventing intracellular nucleic acid, and total flavones has scavenging free radicals, antiinflammatory, antibacterial, antiviral, antioxidation, hypoglycemic multiple effect in leaf of Moringa, the Oxidation of total flavones of Moringa oleifera leaf effectively can prevent the oxidation Decomposition of intracellular nucleic acid, thus reaches the effect of uric acid.
Confirm through test example of the present invention, leaf of Moringa flavone of the present invention is high, in and the uric acid resisting effect of low dose group be better than allopurinol, and in certain scope, the effect of leaf of Moringa flavonoid dose more uric acid resistings is more obvious, leaf of Moringa flavone of the present invention is except reducing except uric acid, to cholesterol, triacylglycerol, blood urea nitrogen, creatinine, xanthine oxidase also has significant reducing effect, by reducing cholesterol, triacylglycerol, blood urea nitrogen, creatinine, xanthine oxidases etc. reduce uricopoiesis, Direct Resolution uric acid, improve renal function and promote urate excretion, the many levels such as protection blood vessel reduce uric acid, more be conducive to the rehabilitation of hyperuricemia patient.
Present invention also offers the purposes of total flavones of Moringa oleifera leaf in preparation treatment senile dementia.Prove through animal experiment, total flavones of Moringa oleifera leaf of the present invention low, in compared with model group, there is significant difference with the mice escape latency of high dose group, all significantly shorten the escape latency of mice, wherein the 20th day, the mice escape latency of high dose group of the present invention has pole significant difference compared with model group, proves that total flavones of Moringa oleifera leaf is to A β
1-42induced mice senile dementia disease model has significant therapeutic effect, flavone high dose group most pronounced effects especially of the present invention.
Compared with prior art, leaf of Moringa extracting method of the present invention adopts Combined microwave-ultrasound extraction total flavones of Moringa oleifera leaf, with have efficiently than traditional reflux, extract, save time, energy-conservation advantage, have a good application prospect in Chinese medicine extraction.
detailed description of the invention:
Below by way of the description of detailed description of the invention, the invention will be further described, but this is not limitation of the present invention, those skilled in the art are according to basic thought of the present invention, various amendment or improvement can be made, but only otherwise depart from basic thought of the present invention, all within the scope of the present invention.
embodiment 1,the preparation of total flavones of Moringa oleifera leaf of the present invention
(1) get the raw materials ready: take 1kg leaf of Moringa in 50 DEG C of dry 2h, pulverize, cross 40 mesh sieves, obtain leaf of Moringa powder for subsequent use;
(2) extract: get step (1) leaf of Moringa powder, the boiling range adding leaf of Moringa opaque amount 2 times of volumes is the petroleum ether of 60 ~ 90 DEG C, and reflux defat 3 times at 68 DEG C, each 5 h, cross elimination filtrate, by filter residue and drying, obtain dry filtering residue for subsequent use;
(3) extract: 65% ethanol adding filtering residue quality 10 times of volumes in the dry filtering residue that step (2) obtains, 8min is heated under microwave power 500W, to put in 75 DEG C of tepidariums supersound extraction 2 times, each extraction 30min, filter to obtain extracting solution, merge extractive liquid, obtains total flavones of Moringa oleifera leaf crude extract;
(4) purification: the total flavones of Moringa oleifera leaf crude extract that step (3) obtains is added in AB-8 type macroporous adsorptive resins, use distilled water 2 times of column volumes successively, the each 3 times of volumes of volume fraction 30% ethanol, 50% ethanol, 70% ethanol carry out gradient elution, adjusted volume flow is 2mL/min, often 15min collected by pipe, with NaNO
2-Al (NO
3)
3-NaOH system color developing detection, under wavelength is 510nm, measure absorbance inspection know, collect the ethanol elution of 50% and 70%, merge eluent, put 1/5 of original volume by also concentrated for eluent decompression recycling ethanol, obtain concentrated solution, concentrated solution is placed in the extractum of 60 DEG C of water bath methods to relative density 0.16,50 DEG C of cold drying to constant weight, obtain total flavones of Moringa oleifera leaf powder.
embodiment 2,the preparation of total flavones of Moringa oleifera leaf of the present invention
(1) get the raw materials ready: take 1kg leaf of Moringa in 50 DEG C of dry 2h, pulverize, cross 45 mesh sieves, obtain leaf of Moringa powder for subsequent use;
(2) extract: get step (1) leaf of Moringa powder, the boiling range adding leaf of Moringa opaque amount 3 times of volumes is the petroleum ether of 60 ~ 90 DEG C, and reflux defat 3 times at 68 DEG C, each 6 h, cross elimination filtrate, by filter residue and drying, obtain dry filtering residue for subsequent use;
(3) extract: 70% ethanol adding filtering residue quality 12 times of volumes in the dry filtering residue that step (2) obtains, 7 min are heated under microwave power 600W, to put in 80 DEG C of tepidariums supersound extraction 2 times, each extraction 25 min, filter to obtain extracting solution, merge extractive liquid, obtains total flavones of Moringa oleifera leaf crude extract;
(4) purification: the total flavones of Moringa oleifera leaf crude extract that step (3) obtains is added in AB-8 type macroporous adsorptive resins, use distilled water 2 times of column volumes successively, the each 3 times of volumes of volume fraction 30% ethanol, 50% ethanol, 70% ethanol carry out gradient elution, adjusted volume flow is 2mL/min, often 15min collected by pipe, with NaNO
2-Al (NO
3)
3-NaOH system color developing detection, under wavelength is 510nm, measure absorbance inspection know, collect the ethanol elution of 50% and 70%, merge eluent, put 1/5 of original volume by also concentrated for eluent decompression recycling ethanol, obtain concentrated solution, concentrated solution is placed in the extractum of 60 DEG C of water bath methods to relative density 0.20,50 DEG C of cold drying to constant weight, obtain total flavones of Moringa oleifera leaf powder.
embodiment 3,the preparation of total flavones of Moringa oleifera leaf of the present invention
(1) get the raw materials ready: take 1kg leaf of Moringa in 50 DEG C of drying 3 h, pulverize, cross 50 mesh sieves, obtain leaf of Moringa powder for subsequent use;
(2) extract: get step (1) leaf of Moringa powder, the boiling range adding leaf of Moringa opaque amount 4 times of volumes is the petroleum ether of 60 ~ 90 DEG C, and reflux defat 3 times at 68 DEG C, each 7 h, cross elimination filtrate, by filter residue and drying, obtain dry filtering residue for subsequent use;
(3) extract: 75% ethanol adding filtering residue quality 15 times of volumes in the dry filtering residue that step (2) obtains, 8 min are heated under microwave power 550W, to put in 78 DEG C of tepidariums supersound extraction 3 times, each extraction 20 min, filter to obtain extracting solution, merge extractive liquid, obtains total flavones of Moringa oleifera leaf crude extract;
(4) purification: the total flavones of Moringa oleifera leaf crude extract that step (3) obtains is added in AB-8 type macroporous adsorptive resins, use distilled water 2 times of column volumes successively, the each 3 times of volumes of volume fraction 30% ethanol, 50% ethanol, 70% ethanol carry out gradient elution, adjusted volume flow is 2mL/min, often 15min collected by pipe, with NaNO
2-Al (NO
3)
3-NaOH system color developing detection, under wavelength is 510nm, measure absorbance inspection know, collect the ethanol elution of 50% and 70%, merge eluent, put 1/5 of original volume by also concentrated for eluent decompression recycling ethanol, obtain concentrated solution, concentrated solution is placed in the extractum of 60 DEG C of water bath methods to relative density 0.25,50 DEG C of cold drying to constant weight, obtain total flavones of Moringa oleifera leaf powder.
embodiment 4,the determination test of total flavones of Moringa oleifera leaf content of the present invention
1, liquid is contrasted: precision takes rutin 10mg, is settled in 50ml bottle the contrast liquid shaken up as 0.2g/L with 30% ethanol.
2, the foundation of standard curve regression equation: accurate absorption contrasts liquid 0, 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6ml, put in 25ml volumetric flask respectively, each family 30% ethanol is to 10ml, shake up, add 5% sodium nitrite 1ml, shake up, leave standstill 6min, add 10% aluminum nitrate 1ml, shake up, leave standstill 6min, add 1mol/L sodium hydroxide also 10ml again, shake up with after 30% ethanol standardize solution, place 15min, using 0 pipe as blank, absorbance A value is measured at 510nm place, with absorbance A value for abscissa, concentration C is vertical coordinate, drawing standard curve, calculate equation of linear regression.
3, measure: the total flavones of Moringa oleifera leaf powder 50mg taking embodiment 1, embodiment 2, embodiment 3 preparation, be settled in 50ml bottle with 30% ethanol and shake up, the method prepared according to standard curve measures absorbance A value, A value is substituted into equation of linear regression, calculate the content of total flavones, result is as shown in table 1.
The determination test data of table 1 total flavones of Moringa oleifera leaf content of the present invention
| Embodiment 1 | Embodiment 2 | Embodiment 3 | |
| The content (%) of total flavones | 19.32% | 32.06% | 20.02% |
As shown in Table 1, the extracting method of total flavones of Moringa oleifera leaf of the present invention is simple to operate, and purification rate is high, with the backflow of prior art carry have save time, energy-conservation advantage, be beneficial to large-scale promotion and the application of this extracting method.
test example 5,total flavones of Moringa oleifera leaf of the present invention is tested the impact of hyperuricemia
1, subjects: male mouse of kunming 72.
2, hyperuricemia Animal Model: adopt lumbar injection oxonic acid potassium salt method, at the Oteracil Potassium of the abdominal cavity disposable celiac injection 300mg/kg of kunming mice, normal mouse is compared, and serum uric acid level significantly improves, there is significant difference, show modeling success.
3, medication:
72 kunming mices are divided into 6 groups at random, stay one group as a control group, all the other are all at the oxonic acid potassium salt of the abdominal cavity disposable celiac injection 300mg/kg of kunming mice, be masked as dosage group and flavone low dose group in model group, allopurinol group, flavone high dose group, flavone respectively, administering mode is as follows:
Matched group: gavage gives the normal saline of same volume;
Model group; Gavage gives the normal saline of same volume;
Allopurinol group: gavage gives the allopurinol of 40mg/kg;
Flavone high dose group; Gavage gives total flavones of Moringa oleifera leaf prepared by 300mg/kg embodiment 2;
Dosage group in flavone; Gavage gives total flavones of Moringa oleifera leaf prepared by 200mg/kg embodiment 2;
Flavone low dose group; Gavage gives total flavones of Moringa oleifera leaf prepared by 100mg/kg embodiment 2.
4, result of the test:
Continuous gavage 7 days, after last administration 1h, gets blood, separation of serum, adopts ELISA (ELISA method) to measure Mouse Blood uric acid, cholesterol, triacylglycerol, blood urea nitrogen, creatinine; Get the activity that liver organization detects xanthine oxidase activity, result is as shown in table 2.
Table 2 total flavones of Moringa oleifera leaf of the present invention affects test data to hyperuricemia
| Group | Blood uric acid (μm ol/L) | Cholesterol (μm ol/L) | Triacylglycerol (μm ol/L) | Blood urea nitrogen (μm ol/L) | Creatinine (μm ol/L) | Xanthine oxidase (μm ol/L) |
| Matched group | 89.4±9.2 | 1.50±0.10 | 0.32±0.12 | 1.34±0.18 | 35.6±17.2 | 50.6±20.8 |
| Model group | 260.2±38.4 | 2.32±0.12 | 0.84±0.13 | 4.26±0.09 | 105.8±15.4 | 95.7±19.6 |
| Allopurinol group | 212.2±36.4* | 2.17±0.13* | 0.66±0.11* | 3.13±0.14* | 90.5±16.8* | 73.1±20.2* |
| Flavone high dose group | 180.4±34.8** | 1.94±0.18** | 0.52±0.18** | 2.82±0.12** | 72.8±16.4** | 59.9±21.4** |
| Dosage group in flavone | 199.6±35.2* | 2.02±0.16* | 0.58±0.18* | 3.06±0.11* | 80.7±16.6* | 66.8±20.6* |
| Flavone low dose group | 206.8±36.2* | 2.15±0.12* | 0.66±0.14* | 3.10±0.14* | 89.6±16.2* | 71.6±20.2* |
Compared with model group: * P<0.05, * P<0.01.
Drawn by table 2, leaf of Moringa flavone of the present invention be high, in the uric acid resisting effect of low dose group, there is compared with model group significant effect, and in certain scope, the effect of total flavones of Moringa oleifera leaf dosage more uric acid resistings is more obvious.Compared with positive controls allopurinol group; the uric acid resisting effect of total flavones of Moringa oleifera leaf of the present invention is better than allopurinol group; and total flavones of Moringa oleifera leaf of the present invention is except reducing except uric acid; also significant reducing effect is had to cholesterol, triacylglycerol, blood urea nitrogen, creatinine, xanthine oxidase; reducing uricopoiesis, Direct Resolution uric acid by reducing cholesterol, triacylglycerol, blood urea nitrogen, creatinine, xanthine oxidase etc., improving the many levels reduction uric acid such as renal function promotion urate excretion, protection blood vessel, be more conducive to the rehabilitation of hyperuricemia patient.
test example 6,total flavones of Moringa oleifera leaf of the present invention is to A β
1-42the therapeutical effect of induced mice senile dementia model
1 materials and methods
1.1 test material
Experimental animal is mice, 9 weeks ages of Mus, and body weight 36-38g, is provided by experimental animal center, Nanjing, A β
1-42purchased from American Sigma company, Morris water maze is purchased from Shanghai Ji Liang company.
The foundation of 1.2 mice senile dementia models and evaluation
1.2.1 intracerebral ventricle injection A β
1-42the preparation of solution: by A β
1-42be dissolved in physiological saline solution, make A β concentration be 10 mmol/L, put in 37 DEG C of calorstats to hatch and carry out aging in 3 days.
1.2.2 the making of animal model: raise under standard environment, is divided into 2 groups at random: matched group and model group, often organizes 10.2 groups of there was no significant differences on Mus age and body weight.Animal gives after adaptability feeds 1 week, by mice with 2% pentobarbital sodium intraperitoneal anesthesia (40 ~ 50 mg/kg weight), be fixed on stereo brain orienting instrument, cut off head hair, skin is cut after iodine tincture disinfection, select right side tricorn for injection target area with reference to " mouse brain stereotaxic atlas ", in bregma 1.0 mm backward, center line is other opens 1.6 mm places, bore with three edged needle and open skull, expose cerebral dura mater, use microsyringe again with the speed of 12 μm/s from vertical inserting needle 4.0 mm in brain surface, 10 mmol/L A β 1-42 solution 5 μ l are slowly injected, slowly pin is removed after let the acupuncture needle remain at a certain point 2 min, sew up the incision.Matched group injects equal-volume physiological saline solution.
1.2.3 Morris water maze behavioristics measures: 2 groups of mices started to carry out the test of Morris water maze respectively at postoperative 10th day.Test program is orientation navigation test: last 5 days, first 2 days is the training adaptation phase, and latter 3 days record achievements, if mice finds platform in 1 min, record its actual escape latency; If find platform not yet in 1 min, then drawn upper mounting plate by experimenter and stop 20 S, escape latency is recorded as 1 min.
1.2.4 the evaluation of animal model
As can be seen from following table, escape latency matched group from the 1st day of test data sheet of model group just obviously extends (P < 0.05 or P < 0.01), and escape latency between model group 3 days and escape latency no significant difference between matched group 3 days, show that the senile dementia type adopting the method to set up is reliably accurate, may be used for the evaluating drug effect of curing senile dementia medicine.
Table 3 is for the test data of the evaluating drug effect of curing senile dementia medicine
| Group | First day | Second day | 3rd day |
| Matched group | 20.14±9.84 | 20.20±6.60 | 20.68±5.54 |
| Model group | 40.98±10.86 | 40.26±6.36 | 41.04±7.30 |
2 animal models and grouping administration
Be set to model group according to above-mentioned modeling method modeling, and dosage group and flavone high dose group in matched group, flavone low dose group, flavone are set, often organize 10, raise under standard environment.Each group of administering mode is as described below:
Matched group: gavage gives the normal saline of same volume;
Model group: gavage gives the normal saline of same volume;
Flavone low dose group: gavage gives total flavones of Moringa oleifera leaf prepared by 100mg/kg embodiment 2;
Dosage group in flavone: gavage gives total flavones of Moringa oleifera leaf prepared by 200mg/kg embodiment 2;
Flavone high dose group: gavage gives total flavones of Moringa oleifera leaf prepared by 300mg/kg embodiment 2.
Above-mentioned administration group is respectively at administration afterwards in 10 days after modeling, within 11st day, be recorded as the 1st day, every day is administered once, observe drinking water for animals and diet situation every day, respectively at administration the 1st day, administration the 5th day, administration the 10th day, administration the 15th day, administration measures the escape latency of mice for 20 days with Morris water maze behavioristics assay method.Mice is put to death after last mensuration completes.Each group of mice Morris water maze behavioristics measurement result is as shown in the table.
The each administration group of table 4 is to A β
1-42the therapeutic effect (escape latency, unit S) of induced mice senile dementia model
| Group | 1st day | 5th day | 10th day | 15th day | 20th day |
| Matched group | 20.30±10.02 | 19.66±4.40 | 21.14±5.32 | 20.10±6.28 | 19.86±5.64 |
| Model group | 41.40±10.08 | 42.14±7.02 | 41.20±9.32 | 38.96±6.30 | 40.32±7.12 |
| Flavone low dose group | 40.20±8.60 | 36.28±3.92 | 32.24±3.28 | 27.62±2.44* | 24.62±3.02* |
| Dosage group in flavone | 40.18±8.12 | 36.20±3.54 | 32.20±3.08 | 27.52±2.62* | 24.46±2.72* |
| Flavone high dose group | 40.08±7.78 | 35.80±3.62 | 31.54±4.98 | 26.78±3.32* | 22.68±2.40** |
Compared with model group, * P < 0.05, * * P < 0.01;
As can be seen from Table 4: each administration group escape latency there was no significant difference of the 1st day, but along with the prolongation of administration time, the escape latency difference of each administration group mice strengthens, 15th day and the 20th day, total flavones of Moringa oleifera leaf of the present invention low, in compared with model group, there is significant difference with the mice escape latency of high dose group, all significantly shorten the escape latency of mice, wherein the 20th day, the mice escape latency of high dose group of the present invention has pole significant difference compared with model group, proves that total flavones of Moringa oleifera leaf is to A β
1-42induced mice senile dementia disease model has significant therapeutic effect, flavone high dose group most pronounced effects especially of the present invention.
Claims (6)
1. an extracting method for total flavones of Moringa oleifera leaf, is characterized in that, comprises the following steps:
(1) get the raw materials ready: take 1kg leaf of Moringa in 50 DEG C of dry 2-3 h, pulverize, cross 40-50 mesh sieve, obtain leaf of Moringa powder for subsequent use;
(2) defat: get step (1) leaf of Moringa powder, add the petroleum ether of leaf of Moringa opaque amount 2-4 times of volume, reflux defat 3 times at 68 DEG C, each 5-7 h, crosses elimination filtrate, by filter residue and drying, obtain dry filtering residue for subsequent use;
(3) extract: the 65%-75% ethanol adding filtering residue quality 10-15 times volume in the dry filtering residue that step (2) obtains, 6-8min is processed under microwave power 500-600W, to put in 75 DEG C of-80 DEG C of tepidariums supersound extraction 2 times, each extraction 20-30min, filter to obtain extracting solution, merge extractive liquid, obtains total flavones of Moringa oleifera leaf crude extract;
(4) purification: the total flavones of Moringa oleifera leaf crude extract that step (3) obtains is added in adsorption resin column, use distilled water 2 times of column volumes successively, the each 3 times of volumes of volume fraction 30% ethanol, 50% ethanol, 70% ethanol carry out gradient elution, adjusted volume flow is 2mL/min, often 15min collected by pipe, with NaNO
2-Al (NO
3)
3-NaOH system color developing detection, under wavelength is 510nm, measure absorbance inspection know, collect the ethanol elution of 50% and 70%, merge eluent, put 1/5 of original volume by also concentrated for eluent decompression recycling ethanol, obtain concentrated solution, concentrated solution is placed in the extractum of 60 DEG C of water bath methods to relative density 0.16-0.25,50 DEG C of cold drying to constant weight, obtain total flavones of Moringa oleifera leaf powder.
2. the extracting method of leaf of Moringa as claimed in claim 1, it is characterized in that, the boiling range of step (2) PetroChina Company Limited. ether is 60 ~ 90 DEG C.
3. the extracting method of leaf of Moringa as claimed in claim 1, it is characterized in that, the adsorption resin column in step (3) is AB-8 type macroporous adsorptive resins.
4. the extracting method of leaf of Moringa as claimed in claim 1, is characterized in that, comprise the following steps:
(1) get the raw materials ready: take 1kg leaf of Moringa in 50 DEG C of drying 2.5 h, pulverize, cross 45 mesh sieves, obtain leaf of Moringa powder for subsequent use;
(2) defat: get step (1) leaf of Moringa powder, adds the petroleum ether of leaf of Moringa opaque amount 3 times of volumes, and reflux defat 3 times at 68 DEG C, each 6 h, crosses elimination filtrate, by filter residue and drying, obtains dry filtering residue for subsequent use;
(3) extract: 70% ethanol adding filtering residue quality 12 times of volumes in the dry filtering residue that step (2) obtains, 7min is processed under microwave power 600W, to put in 80 DEG C of tepidariums supersound extraction 2 times, each extraction 25min, filter to obtain extracting solution, merge extractive liquid, obtains total flavones of Moringa oleifera leaf crude extract;
(4) purification: the total flavones of Moringa oleifera leaf crude extract that step (3) obtains is added in AB-8 type macroporous adsorptive resins, use distilled water 2 times of column volumes successively, the each 3 times of volumes of volume fraction 30% ethanol, 50% ethanol, 70% ethanol carry out gradient elution, adjusted volume flow is 2mL/min, often 15min collected by pipe, with NaNO
2-Al (NO
3)
3-NaOH system color developing detection, under wavelength is 510nm, measure absorbance inspection know, collect the ethanol elution of 50% and 70%, merge eluent, put 1/5 of original volume by also concentrated for eluent decompression recycling ethanol, obtain concentrated solution, concentrated solution is placed in the extractum of 60 DEG C of water bath methods to relative density 0.20,50 DEG C of cold drying to constant weight, obtain total flavones of Moringa oleifera leaf powder.
5. the purposes of total flavones of Moringa oleifera leaf as claimed in claim 1 in preparation treatment antihyperuricemic disease drug.
6. the purposes of total flavones of Moringa oleifera leaf as claimed in claim 1 in preparation treatment senile dementia.
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| CN104997817A (en) * | 2015-07-30 | 2015-10-28 | 太阳树（厦门）生物工程有限公司 | Extraction method of moringa oleifera leaf extractive |
| CN105126067A (en) * | 2015-09-30 | 2015-12-09 | 北京知蜂堂蜂产品有限公司 | Composition for reducing uric acid and application thereof |
| CN105998105A (en) * | 2016-08-01 | 2016-10-12 | 昆明医科大学第附属医院 | Externally applied medicine for treating gout |
| CN107184621A (en) * | 2017-05-16 | 2017-09-22 | 云南省林业科学院 | A kind of extracting method of leaf of Moringa active ingredient and its application |
| CN107307416A (en) * | 2017-05-23 | 2017-11-03 | 集美大学 | A kind of method that low temperature ultrasonic extracts Moringa polyphenol |
| CN108295102A (en) * | 2018-02-13 | 2018-07-20 | 广东药科大学 | A kind of moringa oleifera leaf extractive and extracting method with Lipid-lowering activities |
| CN113995777A (en) * | 2021-09-22 | 2022-02-01 | 福建卫生职业技术学院 | Extraction method of moringa oleifera leaf total flavonoids |
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| CN104983759A (en) * | 2015-08-10 | 2015-10-21 | 青岛蓝盛洋医药生物科技有限责任公司 | Traditional Chinese medicine composition treating diabetic nephropathy and preparing method thereof |
| CN105126067A (en) * | 2015-09-30 | 2015-12-09 | 北京知蜂堂蜂产品有限公司 | Composition for reducing uric acid and application thereof |
| CN105126067B (en) * | 2015-09-30 | 2018-05-22 | 北京知蜂堂健康科技股份有限公司 | A kind of composition of anti-trioxypurine and application thereof |
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| CN107184621A (en) * | 2017-05-16 | 2017-09-22 | 云南省林业科学院 | A kind of extracting method of leaf of Moringa active ingredient and its application |
| CN107307416A (en) * | 2017-05-23 | 2017-11-03 | 集美大学 | A kind of method that low temperature ultrasonic extracts Moringa polyphenol |
| CN108295102A (en) * | 2018-02-13 | 2018-07-20 | 广东药科大学 | A kind of moringa oleifera leaf extractive and extracting method with Lipid-lowering activities |
| CN113995777A (en) * | 2021-09-22 | 2022-02-01 | 福建卫生职业技术学院 | Extraction method of moringa oleifera leaf total flavonoids |
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