CN102526138A - Composition of active components from fresh purslane for decreasing blood sugar, and preparation method thereof - Google Patents
Composition of active components from fresh purslane for decreasing blood sugar, and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a composition of active components from purslane for decreasing blood sugar, and a preparation method thereof, belonging to the field of traditional Chinese medicine. The composition comprises a fresh purslane polysaccharide component, a fresh purslane polyphenol component and a fresh purslane alkaloid component, and is characterized in that the weight ratio of the polysaccharide component to the polyphenol component to the alkaloid component is (2-30):(1.2-20):(0.8-15). The preparation method takes fresh purslane as raw material, and comprises the steps of: cleaning, juicing by a juicer to obtain juice containing active components, further separating the juice by macroporous resin to obtain different solvent parts, performing in vitro and in vivo blood sugar lowering efficacy screening on the obtained different solvent parts, discarding ineffective components and keeping the active components, and mixing the active components to obtain the composition of active components from fresh purslane for decreasing blood sugar. The preparation method effectively removes impurities, concentrates active components, has a simple process and low cost, and is suitable for large-scale production.
Description
Technical field
The present invention relates to fresh Herba Portulacae blood-sugar-reducing effective components compositions and method for preparing, belong to the field of Chinese medicines.
Background technology
Herba Portulacae is the annual meat herbaceous plant of Portulacaceae Portulaca.Modern pharmacological research shows that Herba Portulacae has effects such as blood fat reducing, blood sugar lowering, atherosclerosis.The relevant in recent years hypoglycemic report of Herba Portulacae; Mainly carry out to the dried medical material of Herba Portulacae; Prepare extract through decocting in water or with the organic solvent high temperature extraction; Again extract is done some relevant activity researchs, and rarer extract is totally done further separation, analysis and activity research, more do not have correlational study to relate to bright medicine Herba Portulacae blood-sugar-reducing effective components and preparation of compositions and activity research.The dried medical material of the general employing of Chinese medicine raw material; But a lot of researchs show; The bright article drug effect of medical material is superior to dry product; Main cause is that dried medical material is easy to destroy some thermo-labile, easy oxidations, unsettled effective ingredient in the Chinese medicine ingredients in extracting the course of processing, even destroys the main active of original Chinese medicine fully, and herbal medicine efficacy is reduced.Simultaneously; Chinese medicine itself is as the system of a complicacy, and the performance of its drug effect is through multicomponent, many target spots synergism, and medical material is in the courses of processing such as drying, extraction; Destruction to labile element in the medical material; Medical material internal composition structure is changed, finally cause drug effect to reduce, in addition invalid.The present invention is directed to some shortcomings that the dried medical material of Chinese medicine is used, adopt fresh medical material, to the processing of squeezing the juice of bright medical material, the total active constituents that can keep medical material to greatest extent is not damaged.
Diabetes (DM) be one group by the h and E factor interaction; Cause absolute and relative deficiency of insulin secretion and cell that insulin sensitivity is descended; And causing the clinical syndrome of a series of metabolism disorders such as sugar, fat, protein, water, electrolyte, it is outstanding feature with the hyperglycemia.Along with growth in the living standard, the change of life style, whole world diabetics number grows with each passing day, the third-largest disease of become to continue malignant tumor, cardiovascular disease, serious threat human beings'health.According to the prediction of WHO, in the period of the 1995-2025, Chinese diabetes increasing degree will reach 68%, and it is the first that rate of increase occupies the whole world, and the absolute number of China's diabetics will break through 5,000 ten thousand when the time comes.Diabetes and complication thereof are carried out early diagnosis and therapy become the task of top priority.At present, western modern medicine treatment diabetes are in the performance curative effect; Also have bigger side effect, the Chinese medicine diabetes are with a long history, and are evident in efficacy; Can not only blood sugar lowering, make clinical symptom disappearance or improvement, but and blood fat reducing; Improve the high viscosity and high coagulant state of hemorheology, the generation of prevention and minimizing diabetic complication.
Summary of the invention
The present invention has at first compared bright, dried Herba Portulacae hypoglycemic drug effect difference from drug effect, finds that bright medicine drug effect obviously is superior to dried medical material.Therefore; It is raw material that the present invention adopts the bright medical material of Herba Portulacae; Purpose provides a kind of fresh Herba Portulacae active component composition and method of making the same with hypoglycemic activity; To overcome more existing Chinese medicines in leaching process such as conventional high-temperature, organic solvent, to the destruction of unstable chemical constituent, the shortcoming that causes activity of drug ingredients to reduce.Gather new fresh Herba Portulacae,, squeezeding juice is used the macroporous adsorbent resin separation and purification its processing of squeezing the juice; And the inside and outside hypoglycemic activity screening of coalition, remove partial impurities and invalid components, the active component in the bright medical material of enrichment; Mix active component and prepare the active component compositions,, prepare common dosage forms so that mixes with conventional adjuvant pharmaceutically; Facilitate patients, solved simultaneously that utilization rate of active components in the Herba Portulacae is low, the shortcoming of weak curative effect.The biological activity that preparation technology of the present invention is convenient, can keep crude drug to greatest extent, process equipment requires simple, and extraction time is short, and cost is low, and loss is little, is beneficial to safety in production.Gained Chinese medicine hypoglycemic activity component can be carried out large-scale production, the suitable blood sugar lowering new Chinese medicine that is developed to.
The object of the present invention is to provide a kind of fresh Herba Portulacae herb that utilizes; Squeeze the juice and separate the blood-sugar-reducing effective components compositions; The composition of said composition comprises: fresh Herba Portulacae polysaccharide component, fresh Herba Portulacae Polyphenols component and fresh Herba Portulacae alkaloid component, wherein the weight ratio of polysaccharide component, Polyphenols component, alkaloid component is 2-30: 1.2-20: 0.8-15.
Take from fresh Herba Portulacae hypoglycemic activity active component compositions, the combination of more optimizing is that the weight ratio of polysaccharide component, Polyphenols component, alkaloid component is 2-15: 1.2-10: 0.8-8.
Herba Portulacae blood-sugar-reducing effective components compositions provided by the invention is a raw material with new fresh Herba Portulacae herb, prepares according to the following steps:
Step 1: adopt new fresh Herba Portulacae herb, clean, put into juice extractor and squeeze the juice, medicinal residues are used distilled water immersion 6-12h, continue to squeeze the juice 2-3 time;
Step 2: squeezeding juice concentrates, separation, drying;
Step 3: get the squeezeding juice dried powder and use dissolved in distilled water, last AB-8 type macroporous resin column is used the distilled water eluting, extract I, get extract II with the ethanol elution of 10%-95% percent by volume;
Step 4: extract I is dry, redissolve with a small amount of distilled water, add the 40%-80% ethanol precipitation then, filter; The gained deposition is used dissolved in distilled water, add Savage reagent again and remove Deproteinization, centrifugal, get supernatant; With supernatant dialysis, drying, get the fresh Herba Portulacae polysaccharide component;
Step 5: with the extract II concentrate drying, last HP-20 type macroporous resin column is used the distilled water eluting; Concentrated, the dry fresh Herba Portulacae alkaloid component that gets, with the ethanol elution of 10%-30% percent by volume, concentrated, dry Herba Portulacae flavone aglycone and the phenolic acid component of getting; Ethanol elution with the 70%-95% percent by volume; Concentrated, the dry Herba Portulacae flavonoid glycoside component that gets merges flavone aglycone, flavonoid glycoside and phenolic acid component, gets fresh Herba Portulacae Polyphenols component;
Step 6: fresh Herba Portulacae polysaccharide component, Polyphenols component, alkaloid component are mixed, get active component compositions of the present invention.This active component can be mixed with conventional adjuvant on the pharmaceutical formulations, also can not add conventional adjuvant, processes the conventional medicine preparation.
Relate in the method for preparing of the present invention that separation method adopts centrifugalize, membrance separation and/or normal pressure or filtration under diminished pressure precipitates or precipitate and fluid separation applications; Method for concentration adopts concentrate under reduced pressure at low temperature or normal pressure concentration technology; Drying means adopts vacuum drying, lyophilization and/or spray drying.
The present invention has the active component preparation of compositions method of hypoglycemic activity, and the preferential method for preparing of selecting is: adopt centrifugalize and normal pressure to filter and precipitate or precipitate and fluid separation applications; Sample concentration adopts concentrate under reduced pressure at low temperature technology, thickening temperature≤45 ℃; Drying means adopts lyophilization.
Compositions of the present invention is mixed with pharmaceutic adjuvant, can process powder, capsule, tablet, pill, microcapsule, granule, liquid preparation etc.Wherein capsule comprises soft capsule and hard capsule, and hard capsule can be filled preparation by powder, granule, microplate or micropill.Tablet comprises ordinary tablet, dispersible tablet, chewable tablet, effervescent tablet, oral cavity disintegration tablet, slow releasing tablet, enteric coatel tablets, intra-gastric floating tablet, double-layer tablet etc.Pill comprises drop pill, honeyed pill, concentrated pill, the watered pill, micropill etc.Liquid preparation comprises decoction, mixture, oral liquid, sterilization preparation etc.
Compositions of the present invention, the preparation of its oral administration can contain excipient commonly used, like binding agent, filler, diluent, tablet agent, lubricant, disintegrating agent, wetting agent etc.; The filler that is suitable for comprises starch, bad dextrin, lactose, sucrose, microcrystalline Cellulose and other similar filleies; Suitable disintegrating agent comprises starch, polyvinylpyrrolidone and starch derivatives; Suitable lubricant comprises magnesium stearate or micropowder silica gel; Described wetting agent comprises: water, ethanol or sodium lauryl sulphate; Through mixing, to fill, the method that tabletting etc. are commonly used prepares solid oral composition.
Effect intentionally of the present invention is the pharmaceutical composition with hypoglycemic activity that the present invention is prepared; Through detecting the influence of said composition on the cellular level, can obviously increase the glucose consumption amount of insulin resistant cell to insulin resistant grape cell sugar consumption; Show that in the body animal test results fresh Herba Portulacae active component compositions of the present invention's preparation can obviously reduce the blood glucose of diabetic mice.
Description of drawings
Fig. 1 is bright, dried Herba portulacae extract Polyphenols composition HPLC diversity ratio: the A. Herba Portulacae is dried medical material water extract; B. the bright medical material squeezeding juice of Herba Portulacae
Bright, the dried Herba portulacae extract alkaloids component difference of Fig. 2 compares: the A. Herba Portulacae is dried medical material water extract; B. hybrid standard article; C. the bright medical material squeezeding juice of Herba Portulacae
Fig. 3 Herba Portulacae squeezeding juice HP-20 washing position HPLC analysis chart: A.HP-20 washes the position; B. hybrid standard article; 1. norepinephrine; 2. dopamine
Fig. 4 Herba Portulacae squeezeding juice HP-2010% ethanol elution position HPLC analysis chart: A.HP-20 washes the position; B. standard substance: 1. caffeic acid
Fig. 5 Herba Portulacae squeezeding juice HP-2030% ethanol elution position HPLC analysis chart: A.30% alcohol is washed the position; B. hybrid standard article: 1. ferulic acid; 2. Hesperidin
Fig. 6 Herba Portulacae squeezeding juice HP-2095% ethanol elution position HPLC analysis chart: A.HP-2095% alcohol is washed the position; B. hybrid standard article: 1 Quercetin; 2 luteolins
Fig. 7 extraction separation flow chart of the present invention
The specific embodiment
Below in conjunction with the embodiment and the test of pesticide effectiveness the present invention is described further.
Get new fresh Herba Portulacae herb 5kg, put into juice extractor after cleaning and squeeze the juice, filtration, centrifugalize squeezeding juice and medicinal residues, medicinal residues continue to squeeze the juice 2-3 time with 2 times of amount distilled water immersion 6h, merge squeezeding juice, and concentrate under reduced pressure at low temperature, lyophilization get lyophilized powder.The distilled water of lyophilized powder with 5 times of volumes redissolved, and last AB-8 type macroporous resin is with distilled water and 10%-50% ethanol elution, eluent concentrating under reduced pressure, lyophilization.AB-8 washing position lyophilized powder adds a small amount of distilled water and redissolves and 50% ethanol precipitate with ethanol; The centrifugalize deposition is dissolved in a small amount of distilled water; Add Savage reagent deproteinization; Small-molecule substance is removed in dialysis, and concentrate under reduced pressure at low temperature, lyophilizing get the Polysaccharide from Portulaca oleracea component: it is positive that a-naphthols-sulfuric acid test and sugared osazone form the test qualification result, and recording total polysaccharides content through ultraviolet spectrophotometry is 81.9%; AB-8 alcohol is washed the position lyophilizing, goes up HP-20 type macroporous resin after distilled water redissolves, and uses the distilled water eluting, and the concentrated freeze-dried Herba Portulacae alkaloid component that gets: the bismuth potassium iodide result of the test is positive, and through HPLC assay result, total alkaloid content is 53.4%; 10-30%, 60%-90% volume ratio ethanol elution, concentrate, lyophilizing gets Herba Portulacae Polyphenols component: hydrochloric acid-zinc powder test, aluminum chloride test qualification result are positive, and through the HPLC assay, Polyphenols content is 57.6%; Polysaccharide from Portulaca oleracea, alkaloid, Polyphenols component are mixed, promptly get fresh Herba Portulacae blood-sugar-reducing effective components compositions, the weight ratio that wherein contains polysaccharide component, Polyphenols component, alkaloid component is 2.8: 2.2: 1.3.
Get new fresh Herba Portulacae herb 5kg, put into juice extractor after cleaning and squeeze the juice, filtration, centrifugalize squeezeding juice and medicinal residues, medicinal residues continue to squeeze the juice 2-3 time with 3 times of amount distilled water immersion 8h, merge squeezeding juice, and concentrate under reduced pressure at low temperature, lyophilization get lyophilized powder.In the distilled water of lyophilized powder redissolution with 5 times of volumes, last AB-8 type macroporous resin is with distilled water and 10%-60% ethanol elution; Eluent concentrating under reduced pressure, lyophilization; AB-8 washing position lyophilized powder adds a small amount of distilled water and redissolves, 60% ethanol precipitate with ethanol, and the centrifugalize deposition is dissolved in a small amount of distilled water; Add savage reagent deproteinization; Small-molecule substance is removed in dialysis, and concentrate under reduced pressure at low temperature, lyophilizing get the Polysaccharide from Portulaca oleracea component: it is positive that a-naphthols-sulfuric acid test and sugared osazone form the test qualification result, and recording total polysaccharides content through ultraviolet spectrophotometry is 52.1%; AB-8 alcohol is washed the position lyophilizing, goes up HP-20 type macroporous resin after distilled water redissolves, and uses the distilled water eluting, the concentrated freeze-dried Herba Portulacae alkaloid component that gets: positive through the bismuth potassium iodide result of the test, and HPLC assay result, total alkaloid content is 66.2%; 10-35%, 65%-90% volume ratio ethanol elution, concentrated freeze-dried Herba Portulacae Polyphenols component: positive through hydrochloric acid-zinc powder test, aluminum chloride test qualification result, through the HPLC assay, Polyphenols content is 58.7%; Polysaccharide from Portulaca oleracea, alkaloid, Polyphenols component are mixed, promptly get fresh Herba Portulacae blood-sugar-reducing effective components compositions, the weight ratio that wherein contains polysaccharide component, Polyphenols component, alkaloid component is 2.9: 2.6: 1.5.
Embodiment 3
Get new fresh Herba Portulacae herb 5kg, put into juice extractor after cleaning and squeeze the juice, filtration, centrifugalize squeezeding juice and medicinal residues, medicinal residues continue to squeeze the juice 2-3 time with 4 times of amount distilled water immersion 10h, merge squeezeding juice, and concentrate under reduced pressure at low temperature, lyophilization get lyophilized powder.In the distilled water of lyophilized powder redissolution with 5 times of volumes, last AB-8 type macroporous resin is with distilled water and 10%-65% ethanol elution; Eluent concentrating under reduced pressure, lyophilization; AB-8 washing position lyophilized powder adds a small amount of distilled water and redissolves, 70% ethanol precipitate with ethanol, and the centrifugalize deposition is dissolved in a small amount of distilled water; Add savage reagent deproteinization; Small-molecule substance is removed in dialysis, and concentrate under reduced pressure at low temperature, lyophilizing get the Polysaccharide from Portulaca oleracea component: it is positive that a-naphthols-sulfuric acid test and sugared osazone form the test qualification result, and recording total polysaccharides content through ultraviolet spectrophotometry is 84.7%; AB-8 alcohol is washed the position lyophilizing, goes up HP-20 type macroporous resin after distilled water redissolves, and uses the distilled water eluting, and the concentrated freeze-dried Herba Portulacae alkaloid component that gets: the bismuth potassium iodide result of the test is positive, and through HPLC assay result, total alkaloid content is 54.4%; 15-35%, 70%-90% volume ratio ethanol elution, the concentrated freeze-dried Herba Portulacae Polyphenols component that gets: hydrochloric acid-zinc powder is tested, aluminum chloride test qualification result is positive, and through the HPLC assay, Polyphenols content is 59.1%; Polysaccharide from Portulaca oleracea, alkaloid, Polyphenols component are mixed, promptly get fresh Herba Portulacae blood-sugar-reducing effective components compositions, the weight ratio that wherein contains polysaccharide component, Polyphenols component, alkaloid component is 3.1: 2.3: 1.8.
Embodiment 4
Get new fresh Herba Portulacae herb 5kg, put into juice extractor after cleaning and squeeze the juice, filtration, centrifugalize squeezeding juice and medicinal residues, medicinal residues continue to squeeze the juice 2-3 time with 5 times of amount distilled water immersion 12h, merge squeezeding juice, and concentrate under reduced pressure at low temperature, lyophilization get lyophilized powder.In the distilled water with 5 times of volumes of lyophilized powder redissolution, last AB-8 type macroporous resin is with distilled water and 10%-70% ethanol elution; Eluent concentrating under reduced pressure, lyophilization; AB-8 washing position lyophilized powder adds a small amount of distilled water and redissolves, 80% ethanol precipitate with ethanol, and the centrifugalize deposition is dissolved in a small amount of distilled water; Add savage reagent deproteinization; Small-molecule substance is removed in dialysis, and concentrate under reduced pressure at low temperature, lyophilizing get the Polysaccharide from Portulaca oleracea component: it is positive that a-naphthols-sulfuric acid test and sugared osazone form the test qualification result, and recording total polysaccharides content through ultraviolet spectrophotometry is 50.9%; AB-8 alcohol is washed the position lyophilizing, goes up HP-20 type macroporous resin after distilled water redissolves, and uses the distilled water eluting, and the concentrated freeze-dried Herba Portulacae alkaloid component that gets: the bismuth potassium iodide result of the test is positive, and through HPLC assay result, total alkaloid content is 58.9%; 10-30%, 70%-95% volume ratio ethanol elution, the concentrated freeze-dried Herba Portulacae Polyphenols component that gets: hydrochloric acid-zinc powder is tested, aluminum chloride test qualification result is positive, and through the HPLC assay, Polyphenols content is 77.6%; Polysaccharide from Portulaca oleracea, alkaloid, Polyphenols component are mixed, promptly get fresh Herba Portulacae blood-sugar-reducing effective components compositions, the weight ratio that wherein contains polysaccharide component, Polyphenols component, alkaloid component is 3.5: 2.6: 1.9.
(1) bright, dried medicinal ingredient HPLC relatively
1. Polyphenols composition HPLC diversity ratio is than chromatographic condition: Agilent1100 type high performance liquid chromatograph (DAD detector); Chromatographic column: Alltima C18 post (250mm * 4.6mm, 5 μ m); Column temperature is 35 ℃; Flow velocity: 1.0mL/min; Detect wavelength: 360nm; Sample size: 10 μ L; Acetonitrile-0.2% phosphate aqueous solution gradient elution, the eluent gradient elution program is seen table 1, chromatogram is seen Fig. 1.
Each eluent of table 1 phase gradient elution program that flows
2. alkaloids component difference comparison colours spectral condition: Agilent1100 type high performance liquid chromatograph (UV-detector); Chromatographic column: Alltima C18 post (250mm * 4.6mm, 5 μ m); Column temperature is 25 ℃; Flow velocity: 0.8mL/min; Detect wavelength: 280nm; Sample size: 10 μ L; Mobile phase: contain 70% methanol, 30mmol/L SDS, the 0.02mol/L potassium dihydrogen phosphate is used H
3PO
4Transferring PH is 3.2, crosses the 0.45um filter membrane.Chromatogram is seen Fig. 2.
Show by HPLC result, bright, dried Herba Portulacae medicinal substances extract, not only variant on the composition, also variant on some component content.
(2) fresh Herba Portulacae different solvents position, the active component assay
1. HP-20 washing alkaloid constituent content is measured chromatographic condition: Agilent1100 type high performance liquid chromatograph (UV-detector); Chromatographic column: Alltima C18 post (250mm * 4.6mm, 5 μ m); Column temperature is 25 ℃; Flow velocity: 0.8mL/min; Detect wavelength: 280nm; Sample size: 10 μ L; Mobile phase: contain 70% methanol, 30mmol/L SDS, 0.02mol/L potassium dihydrogen phosphate, and use H
3PO
4Transferring PH is 3.2, crosses the 0.45um filter membrane.Chromatogram is seen Fig. 3.
Measuring the result with index property composition norepinephrine and DOPAMINE CONTENT IN RABBIT is foundation; Accumulate similar ultraviolet absorption peak peak area; According to the relation of peak area and content, calculate that the total alkaloids constituent content accounts for 53.3% of this solvent position whole content among the present invention.
2. the Polyphenols constituent content is measured chromatographic condition: Agilent1100 type high performance liquid chromatograph (DAD detector); Chromatographic column: Alltima C18 post (250mm * 4.6mm, 5 μ m); Column temperature is 35 ℃; Flow velocity: 1.0mL/min; Detect wavelength: 360nm; Sample size: 10 μ L; Acetonitrile-0.2% phosphate aqueous solution gradient elution, the eluent gradient elution program is seen table 2, chromatogram is seen Fig. 4, Fig. 5, Fig. 6.
Each eluent of table 2 phase gradient elution program that flows
Caffeic acid, ferulic acid and the Quercetin of measuring according to the present invention, content of luteolin are measured the result; Have similar ultraviolet in the accumulation liquid phase collection of illustrative plates and inhale the peak-to-peak area, calculate that total polyphenols class constituent content accounts for 59.6% of this solvent position whole content among the present invention.
Embodiment 6 tests of pesticide effectiveness
Fresh Herba Portulacae blood-sugar-reducing effective components compositions provided by the invention has good hypoglycemic activity, and through animal experiment checking in cell in vitro test and the body, the test of pesticide effectiveness is reported as follows:
(1) test cell line
1, cell strain: HePG 2 cell strains are available from Shanghai RESEARCH ON CELL-BIOLOGY institute of Chinese Academy of Sciences cell bank.
2, the foundation of HepG2 cell insulin resistant model
Investigated the influence to model stability of insulin action concentration and action time, blank group, the not blank culture medium of inoculating cell are established in experiment; The normal control group contains the RPMI-1640 culture fluid of 2% NBCS, and model group, and the insulin concentration that contains of new preparation is respectively 10
-5Mol/L, 10
-6Mol/L, 10
-7Mol/L, 10
-8The RPMI-1640 culture fluid of 2% NBCS of mol/L, according to the glucose consumption amount of cell, result of the test is selected normal control group and the maximum insulin action concentration (10 of model group grape cell sugar consumption amount difference
-6Mol/L) be the insulin suitable concentration; According to each group grape cell sugar consumption amount, selecting the maximum insulin action time of normal control group and model group grape cell sugar consumption amount difference is the suitable time of insulin action.Can know that by experimental result data the maximum insulin action concentration of normal control group and model group grape cell sugar consumption amount difference is 10
-6Mol/L, be 24h action time.
3, administration
After will being in the cell dissociation of exponential phase, using the RPMI-1640 culture fluid adjustment cell density that contains 10% NBCS is 5 * 10
4Individual/mL, be inoculated in 96 well culture plates every hole 100 μ L cell suspension.Blank group, normal control group, model group and administration group are established in experiment, in 37 ℃, 5%CO
2After hatching 24h in the incubator, the normal control group is changed 90ul serum-free medium and 10ul PBS, and model group is changed 90 μ L serum-free mediums and 10ul PBS, and drug group adds 90ul serum-free medium and corresponding medicine respectively, and dosage is 10ul.In 37 ℃, 5%CO
2After hatching 24h in the incubator, discard culture fluid,, change that to contain concentration of glucose be 26.4mmolL with PBS washing 2 times
-1The culture fluid of serum-free, hatch 24h after, detect the residue glucose content in the culture fluid with the glucose assays test kit.After the glucose consumption experiment finishes, discard the culture fluid in 96 orifice plates, with PBS washing 2 times; Every hole adds 10ul MTT and 100ul serum-free medium, behind the cultivation 4h, discards culture fluid; Every hole adds 100ulDMSO, shakes 15min on the microoscillator, and ELIASA is measured each administration group absorbance.Getting rid of the influence of cell proliferation to glucose consumption, is contrast with the blank control group, calculates the glucose consumption amount of respectively organizing cell (glucose consumption amount (glucose surplus in culture medium empty group glucose-drug group culture medium)/respectively organize MTTOD value).
4, experimental result
Table 3 aquatic foods, the overall external hypoglycemic drug effect of dried Herba portulacae extract are relatively
Annotate: compare ^P＜0.01 with the normal control group; Compare with model control group,
*P＜0.05,
*P＜0.01; Bright medicine and dried medicine group are relatively
#P＜0.05,
##P＜0.01.
The outer blood sugar decreasing effect of the Herba Portulacae blood-sugar-reducing effective components composition of table 4 different instances
(respectively organizing grape cell sugar consumption amount (mmol/L) after getting rid of the cell proliferation influence)
Annotate: compare with the diabetes model matched group,
*P＜0.05,
*P＜0.01; Self compare before the administration with after the administration,
#P＜0.05,
##P＜0.01
Result of the test shows: several kinds of Herba Portulacae blood-sugar-reducing effective components compositionss all have the obvious functions of blood sugar effect.
Blood glucose decline percentage rate=(a-b)/a * 100%
A: blood glucose value before the administration, b: blood glucose value after the administration.
(2) animal experiment
1, sample: Herba Portulacae aquatic foods, dried medicinal substances extract and active component compositions.
2, experimental animal: C57BL/6 mice: male, body weight (22 ± 2) g is provided by Chinese Academy of Medical Sciences's Experimental Animal Center.
3, test method:
(1) preparation of diabetes model: healthy male C57BL/6 mice, freely to ingest, drink water, after a week was fed in adaptation, fasting can't help water 12 hours, the STZ solution of lumbar injection 150mg/kg.Detect the variation of fasting glucose on the 7th day, and chose the diabetes model that is of blood glucose value >=14.9mmol/L.
(2) mice of formation diabetes model is one group by 8, and gastric infusion removes to learn survey blood glucose in the 8th day tail vein respectively.
(3) blood-sugar level measuring: the GT-1810 blood glucose meter of using love section to come international trade (Shanghai) Co., Ltd. to produce is measured.
4, test result:
The Herba Portulacae blood-sugar-reducing effective components compositions blood sugar lowering result of the test that adopts different process to obtain is seen table 6.
Hypoglycemic drug effect relatively in table 6 aquatic foods, the overall body of dried Herba portulacae extract
Annotate: compare ^P＜0.001 with the normal control group; Compare with model control group,
*P＜0.05,
*P＜0.01; Bright medicine and dried medicine group are relatively
#P＜0.05,
##P＜0.01.
Blood sugar decreasing effect in the Herba Portulacae blood-sugar-reducing effective components composition of table 7 different instances
Annotate: compare with the diabetes model matched group,
*P＜0.05,
*P＜0.01; Self compare before the administration with after the administration,
#P＜0.05,
##P＜0.01
Result of the test shows: several kinds of Herba Portulacae blood-sugar-reducing effective components compositionss all have the obvious functions of blood sugar effect.
Blood glucose decline percentage rate=(a-b)/a * 100%
A: blood glucose value before the administration, b: blood glucose value after the administration.
Embodiment 7
Get new fresh Herba Portulacae herb 5kg, put into juice extractor after cleaning and squeeze the juice, filtration, centrifugalize squeezeding juice and medicinal residues, medicinal residues continue to squeeze the juice 2-3 time with 4 times of amount distilled water immersion 10h, merge squeezeding juice, and concentrate under reduced pressure at low temperature, lyophilization get lyophilized powder.In the distilled water of lyophilized powder redissolution with 5 times of volumes, last AB-8 type macroporous resin is with distilled water and 10%-65% ethanol elution; Eluent concentrating under reduced pressure, lyophilization; AB-8 washing position lyophilized powder adds a small amount of distilled water and redissolves, 70% ethanol precipitate with ethanol, and the centrifugalize deposition is dissolved in a small amount of distilled water; Add savage reagent deproteinization; Small-molecule substance is removed in dialysis, and concentrate under reduced pressure at low temperature, lyophilizing get the Polysaccharide from Portulaca oleracea component: it is positive that a-naphthols-sulfuric acid test and sugared osazone form the test qualification result, and recording total polysaccharides content through ultraviolet spectrophotometry is 84.7%; AB-8 alcohol is washed the position lyophilizing, goes up HP-20 type macroporous resin after distilled water redissolves, and uses the distilled water eluting, and the concentrated freeze-dried Herba Portulacae alkaloid component that gets: the bismuth potassium iodide result of the test is positive, and through HPLC assay result, total alkaloid content is 54.4%; 15-35%, 70%-90% volume ratio ethanol elution, the concentrated freeze-dried Herba Portulacae Polyphenols component that gets: hydrochloric acid-zinc powder is tested, aluminum chloride test qualification result is positive, and through the HPLC assay, Polyphenols content is 59.1%; With Polysaccharide from Portulaca oleracea, alkaloid, Polyphenols combination of components, promptly get fresh Herba Portulacae blood-sugar-reducing effective components compositions, the weight ratio that wherein contains polysaccharide component, Polyphenols component, alkaloid component is 3.1: 2.3: 1.8.
Get polysaccharide component and mix, be prepared into the polysaccharide component micropill with microcrystalline Cellulose, cross-linking sodium carboxymethyl cellulose; Get the Polyphenols component and mix, be prepared into Polyphenols component micropill with microcrystalline Cellulose, tween 80; Get the alkaloid component and mix, be prepared into alkaloid component micropill with microcrystalline Cellulose, chitosan; The above-mentioned micropill for preparing is filled in the capsule, promptly obtains fresh Herba Portulacae blood-sugar-reducing effective components compositions pellt capsule.
Get polysaccharide component and microcrystalline Cellulose, lactose, micropowder silica gel, Pulvis Talci mix homogeneously, full powder compaction becomes microplate; Get the Polyphenols component and mix with microcrystalline Cellulose, tween 80, be prepared into granule, add micropowder silica gel, Pulvis Talci, magnesium stearate, mix homogeneously is pressed into microplate; Get the alkaloid component and mix with microcrystalline Cellulose, lactose, micropowder silica gel, Pulvis Talci, full powder compaction becomes microplate; The above-mentioned microplate for preparing is filled in the capsule, promptly obtains fresh Herba Portulacae blood-sugar-reducing effective components compositions microplate capsule.
Get polysaccharide component, Polyphenols component, alkaloid component, polyethylene glycol 6000, stevioside and be dissolved in the distilled water, regulate pH to 5.5-7.0, filter, fill is in oral liquid bottle, and sterilization promptly gets fresh Herba Portulacae blood-sugar-reducing effective components oral liquid.
Get polysaccharide component, Polyphenols component, alkaloid component and mix with microcrystalline Cellulose, tween 80, be prepared into granule, add micropowder silica gel, Pulvis Talci, magnesium stearate, mix homogeneously is pressed into tablet.
Get the Macrogol 4000 heating mix homogeneously of polysaccharide component, Polyphenols component, alkaloid component and 2 times of amounts, drip and process drop pill, promptly get fresh Herba Portulacae blood-sugar-reducing effective components drop pill.
Get the dextrin mix homogeneously of polysaccharide component, Polyphenols component, alkaloid component and 2 times of amounts, process granule, promptly get fresh Herba Portulacae blood-sugar-reducing effective components granule.
Claims (9)
1. take from fresh Herba Portulacae blood-sugar-reducing effective components compositions for one kind; The composition that it is characterized in that said composition comprises fresh Herba Portulacae polysaccharide component, fresh Herba Portulacae alkaloid component and fresh Herba Portulacae Polyphenols component, and wherein the weight ratio of polysaccharide component, Polyphenols component, alkaloid component is 2-30: 1.2-20: 0.8-15.
2. a kind of fresh Herba Portulacae hypoglycemic activity active component compositions of taking from according to claim 1, the weight ratio that it is characterized in that polysaccharide component, Polyphenols component, alkaloid component is 2-15: 1.2-10: 0.8-8.
3. a kind of fresh Herba Portulacae blood-sugar-reducing effective components compositions of taking from according to claim 1 is characterized in that the assay through HPLC, is that the Polyphenols constituent content of representative is greater than 55% with Quercetin, luteolin, caffeic acid, ferulic acid.
4. a kind of fresh Herba Portulacae blood-sugar-reducing effective components compositions of taking from according to claim 1 is characterized in that the assay through HPLC, is that the alkaloid constituent content of representative is greater than 50% with norepinephrine and dopamine.
5. fresh Herba Portulacae blood-sugar-reducing effective components preparation of compositions method is characterized in that preparing according to the following steps:
Step 1: adopt new fresh Herba Portulacae herb, clean, squeeze the juice, medicinal residues are used distilled water immersion 6-12h, continue to squeeze the juice 2-3 time;
Step 2: squeezeding juice concentrates, separation, drying;
Step 3: get the squeezeding juice dried powder and use dissolved in distilled water, cross AB-8 type macroporous resin column, the distilled water eluting gets extract I, and the ethanol elution of 10%-95% percent by volume gets extract II;
Step 4: extract I is dry, add a small amount of distilled water redissolution and 40%-80% alcoholic solution deposition, filter; The gained deposition is used dissolved in distilled water, add Savage reagent again and remove Deproteinization, centrifugal, get supernatant, with supernatant dialysis, drying, get the fresh Herba Portulacae polysaccharide component;
Step 5: with the extract II concentrate drying, go up HP-20 type macroporous resin column after distilled water redissolves on a small quantity, distilled water eluting, concentrated, the dry fresh Herba Portulacae alkaloid component that gets; The ethanol elution of 10%-30% percent by volume, concentrated, dry fresh Herba Portulacae flavone aglycone and the phenolic acid component of getting; The ethanol elution of 70%-95% percent by volume, concentrated, the dry Herba Portulacae flavonoid glycoside component that gets merges flavone aglycone, flavonoid glycoside and phenolic acid component, gets fresh Herba Portulacae Polyphenols component;
Step 6: fresh Herba Portulacae polysaccharide component, Polyphenols component, alkaloid component are mixed, obtain compositions.
6. the active component preparation of compositions method with hypoglycemic activity according to claim 5 is characterized in that separation method adopts centrifugalize, membrance separation and/or normal pressure or filtration under diminished pressure precipitates or precipitate and fluid separation applications; Sample concentration adopts concentrate under reduced pressure at low temperature or normal pressure concentration technology; The drying means of sample adopts vacuum drying, lyophilization and/or spray drying.
7. the active component preparation of compositions method with hypoglycemic activity according to claim 6 is characterized in that adopting centrifugalize and normal pressure to filter and precipitates or precipitate and fluid separation applications; Sample concentration adopts concentrate under reduced pressure at low temperature technology, thickening temperature≤45 ℃; Drying means adopts lyophilization.
8. the active component preparation of compositions method with hypoglycemic activity according to claim 5; In step 6; Fresh Herba Portulacae polysaccharide component, Polyphenols component, alkaloid component are mixed with pharmaceutic adjuvant, process powder, capsule, tablet, pill, microcapsule, granule, liquid preparation etc.
9. be to prepare in the medicine and use according to the described hypoglycemic active component compositions of fresh Herba Portulacae of taking from of claim 1-4.
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Cited By (4)
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| CN102973619A (en) * | 2012-12-31 | 2013-03-20 | 山东大学 | Process for extracting indoline amide alkaloid from purslane and detection methods for indoline amide alkaloid |
| CN103554237A (en) * | 2013-10-14 | 2014-02-05 | 安徽农业大学 | Active glycoprotein extracted from purslane and preparation method thereof |
| CN104280499A (en) * | 2014-10-10 | 2015-01-14 | 成都三联草本生物科技有限公司 | Method for controlling quality of medicinal material, portulaca oleracea L. |
| CN105132172A (en) * | 2015-08-28 | 2015-12-09 | 安徽中烟工业有限责任公司 | Method for preparing orris root flavonoid matter for tobaccos from orris roots |
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| CN101700260A (en) * | 2009-11-13 | 2010-05-05 | 秦大伟 | Purslane seed polysaccharide granule preparation and preparation method thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102973619A (en) * | 2012-12-31 | 2013-03-20 | 山东大学 | Process for extracting indoline amide alkaloid from purslane and detection methods for indoline amide alkaloid |
| CN102973619B (en) * | 2012-12-31 | 2014-06-11 | 山东大学 | Process for extracting indoline amide alkaloid from purslane and detection methods for indoline amide alkaloid |
| CN103554237A (en) * | 2013-10-14 | 2014-02-05 | 安徽农业大学 | Active glycoprotein extracted from purslane and preparation method thereof |
| CN104280499A (en) * | 2014-10-10 | 2015-01-14 | 成都三联草本生物科技有限公司 | Method for controlling quality of medicinal material, portulaca oleracea L. |
| CN105132172A (en) * | 2015-08-28 | 2015-12-09 | 安徽中烟工业有限责任公司 | Method for preparing orris root flavonoid matter for tobaccos from orris roots |
| CN105132172B (en) * | 2015-08-28 | 2019-06-18 | 安徽中烟工业有限责任公司 | A method of preparing tobacco orrisroot Flavonoid substances from orrisroot |
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| CN102526138B (en) | 2016-02-24 |
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