CN102068535B - Immature bitter orange or bitter orange total flavonoids extract prepared by ethanol reflux and extraction and application thereof - Google Patents
Immature bitter orange or bitter orange total flavonoids extract prepared by ethanol reflux and extraction and application thereof Download PDFInfo
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Abstract
The invention relates to an immature bitter orange or bitter orange total flavonoids extract, which is prepared by the following steps of: cutting bitter orange or bitter orange into slices or crushing bitter orange or bitter orange, adding 30 to 90 percent ethanol of 5 to 16 times volume for reflux and extraction for 1 to 3 times and 1 to 3 hours every time, and filtering; and combining the filtrate, reclaiming the ethanol from the filtrate, filling the aqueous solution into the treated non-polar macroporous resin, diluting in turn by using water and less than 20 percent ethanol to remove impurities, diluting by using 25 to 90 percent ethanol and 0.5 to 2 percent sodium hydroxide, collecting ethanol eluent, concentrating the ethanol eluent under reduced pressure to obtain fluid extract with relative density of about 1.20 to 1.40 (50 to 70 DEG C), drying the fluid extract under the reduced pressure at the temperature of between 60 and 80 DEG C, and crushing to obtain the immature bitter orange or bitter orange total flavonoids extract. The extract can be used for preparing medicaments for gastrointestinal motility disorder type functional indigestion, epigastric pain, abdominal distention, asitia, belching, nausea, sickness and chemotherapy or sickness caused by other reasons.
Description
Technical field
The present invention relates to a kind of Fructus Aurantii Immaturus of alcohol reflux or Fructus Aurantii extractive of general flavone and uses thereof.
Background technology
Functional dyspepsia (FD) is a kind of common, frequently-occurring disease.Be called again gastric dynamic dysfunction, or gastric motility disorder.Be a kind of common clinical syndrome, the foreign statistic data shows, 1/3 crowd suffered from FD, accounted for 20%～30% of gop number.Domestic Digestive out-patient 50% left and right that accounts for betides 20%～40% normal population.In addition, can cause vomiting after the Chemotherapy of Tumor Patients more than 80%, preventing or arresting vomiting is mainly take medicines such as paspertin metoclopramide, ondansetrons as main, but the chemical medicine preventing or arresting vomiting nape muscle spasm symptom even might occur with asthenia, dizziness, headache.
Western medical treatment is mainly take motilium, cisapride, mosapride etc. as main at present; But with its a large amount of uses, find to have obvious Cardiotoxity, many patients Q-T interval prolongation and arrhythmia occur after using cisapride, and particularly torsades de pointes type chamber speed waits serious adverse reaction, even causes death.For this reason, FDA Food and Drug Administration is decided by that on July 23rd, 2000 rose and stops using this medicine, and China Drug Administration has also made restriction to the use of cisapride, but still can use under the doctor instructs.
Chinese traditional treatment is with the dispelling the stagnated QI removing food stagnancy, and loose painful abdominal mass class Chinese medicine reduces phlegm; Stop in treatment is stagnant, feeling of fullness distending pain etc., Fructus Aurantii Immaturus is for representing one of medicine.Fructus Aurantii Immaturus, Fructus Aurantii are dry young fruit or the immature fruit of rutaceae Citrus aurantium Linn. Citrus aurantium L. and variety or Fructus Citri sinensis Citrus sinensis Osbeckr.Fructus Aurantii Immaturus, Fructus Aurantii are from Shennong's Herbal, " Mingyi Bielu " record: " except breast side of body accumulation of phlegm in the hypochondrium; by cutting off the water; broken solid; that relieving distension is full ", from Jin Dynasty Lee physician Dong Yuan " differentiation on endogenous " pioneering zhishi daozhi pills to make by hand, clinical formulation application and modern Chinese medicine large-scale production also are used for digestive tract disease up till now, and there is the history of nearly 2,000 years front and back; Fructus Aurantii Immaturus, Fructus Aurantii main component are flavone etc., and its decocting liquid and Hesperidin etc. can significantly strengthen the ahead running of the small intestinal of normal mouse and atropine generation mice inhibition, have the effect of short digestive tract power.But Fructus Aurantii Immaturus, Fructus Aurantii only are used for clinical formulation and Chinese patent medicine preparation on a small quantity, and ample resources is not used widely, and its natural dispelling the stagnated QI removing food stagnancy effect may promote that the effect class same sex of medicine for stomach dynamic is not fully excavated with the modern times.
Summary of the invention
The Fructus Aurantii Immaturus or the Fructus Aurantii extractive of general flavone that the purpose of this invention is to provide a kind of alcohol reflux are used for gastrointestinal motility obstruction type functional dyspepsia; Upper abdominal pain, abdominal distention, anorexia, belch, feel sick, vomiting that vomiting and chemotherapeutics or other reasons cause.
Fructus Aurantii Immaturus provided by the invention or Fructus Aurantii extractive of general flavone, by Fructus Aurantii Immaturus or Fructus Aurantii are obtained with alcohol reflux, wherein the alcohol reflux method is: get Fructus Aurantii Immaturus or Fructus Aurantii, be cut into decoction pieces or pulverizing, 30%～90% alcohol reflux 1～3 time that adds 5～16 times of volumes, each reflux, extract, 1～3 hour filters; Merging filtrate, filtrate recycling ethanol, the non-polar macroporous resin of aqueous solution by having handled well, water, the ethanol elution below 20% are removed impurity successively, then use 25%～90% ethanol, 0.5%～2% sodium hydroxide eluting, collect ethanol elution, be evaporated to the approximately fluid extract of 1.20～1.40 (50 ℃～70 ℃) of relative density, the temperature of drying under reduced pressure drying is 60～80 ℃, pulverizes, and get final product.
Preferably, concrete alcohol reflux method is as follows: get Fructus Aurantii Immaturus or Fructus Aurantii, be cut into decoction pieces or pulverizing, add 40%～85% alcohol reflux 2～3 times of 6～14 times of volumes, each reflux, extract, 1～2.5 hour filters; Merging filtrate, filtrate recycling ethanol, the non-polar macroporous resin of aqueous solution by having handled well, water, the ethanol elution below 20% are removed impurity successively, then use 30%～85% ethanol, 0.5%～1.8% sodium hydroxide eluting, collect ethanol elution, be evaporated to the approximately fluid extract of 1.25～1.40 (55 ℃～70 ℃) of relative density, drying under reduced pressure, dry temperature is 65～80 ℃, pulverize, and get final product.
Preferably, concrete alcohol reflux method is as follows: get Fructus Aurantii Immaturus or Fructus Aurantii, be cut into decoction pieces or pulverizing, add 45%～80% alcohol reflux 2～3 times of 7～12 times of volumes, each reflux, extract, 1.5～2.5 hours filters; Merging filtrate, filtrate recycling ethanol, the non-polar macroporous resin of aqueous solution by having handled well, water, the ethanol elution below 20% are removed impurity successively, then use 35%～75% ethanol, 0.5～1.6% sodium hydroxide eluting, collect ethanol elution, be evaporated to the approximately fluid extract of 1.25～1.35 (55 ℃～65 ℃) of relative density, drying under reduced pressure, dry temperature is 70～80 ℃, pulverize, and get final product.
Preferably, concrete alcohol reflux method is as follows: get Fructus Aurantii Immaturus or Fructus Aurantii, be cut into decoction pieces or pulverizing, add 55%～75% alcohol reflux 2～3 times of 7～11 times of volumes, each reflux, extract, 1.5～2 hours filters; Merging filtrate, filtrate recycling ethanol, the non-polar macroporous resin of aqueous solution by having handled well, water, the ethanol elution below 20% are removed impurity successively, then use 40%～65% ethanol, 0.5～1.3% sodium hydroxide eluting, collect ethanol elution, be evaporated to the approximately fluid extract of 1.30～1.35 (60 ℃～65 ℃) of relative density, drying under reduced pressure, dry temperature is 70～75 ℃, pulverize, and get final product.
Preferably, concrete alcohol reflux method is as follows: get Fructus Aurantii Immaturus or Fructus Aurantii, be cut into decoction pieces or pulverizing, add 65% alcohol reflux 3 times of 9 times of volumes, each reflux, extract, 1.5 hours filters; Merging filtrate, filtrate recycling ethanol, the non-polar macroporous resin of aqueous solution by having handled well, water, the ethanol elution below 20% are removed impurity successively, then use 40%～55% ethanol, 0.8～1.2% sodium hydroxide eluting, collect ethanol elution, be evaporated to the approximately fluid extract of 1.30 (60 ℃) of relative density, drying under reduced pressure, dry temperature is 75 ℃, pulverize, and get final product.
Preferably, concrete alcohol reflux method is as follows: get Fructus Aurantii Immaturus or Fructus Aurantii, be cut into decoction pieces or pulverizing, add 60%～70% alcohol reflux 2 times of 7～9 times of volumes, each reflux, extract, 1.5 hours filters; Merging filtrate, filtrate recycling ethanol, the non-polar macroporous resin of aqueous solution by having handled well, water, the ethanol elution below 20% are removed impurity successively, then use 40%～55% ethanol, 0.7%～1.3% sodium hydroxide eluting, collect ethanol elution, be evaporated to the approximately fluid extract of 1.30 (60 ℃) of relative density, drying under reduced pressure, dry temperature is 75 ℃, pulverize, and get final product.
Preferably, concrete alcohol reflux method is as follows: get Fructus Aurantii Immaturus or Fructus Aurantii, be cut into decoction pieces or pulverizing, add 70% alcohol reflux 2 times of 8 times of volumes, each reflux, extract, 2 hours filters; Merging filtrate, filtrate recycling ethanol, the non-polar macroporous resin of aqueous solution by having handled well, water, the ethanol elution below 20% are removed impurity successively, then use 40%～50% ethanol, 0.9%～1.1% sodium hydroxide eluting, collect ethanol elution, be evaporated to the approximately fluid extract of 1.30 (60 ℃) of relative density, drying under reduced pressure, dry temperature is 75 ℃, pulverize, and get final product.
Preferably, concrete alcohol reflux method is as follows: get Fructus Aurantii Immaturus or Fructus Aurantii, be cut into decoction pieces, add 65% alcohol reflux 2 times of 7 times of volumes, each reflux, extract, 2 hours filters; Merging filtrate, filtrate recycling ethanol, the D101 macroporous resin column of aqueous solution by having handled well, water, 15% ethanol, 45% ethanol, 1% sodium hydroxide eluting, collect 45% ethanol elution successively, is evaporated to the approximately fluid extract of 1.30 (60 ℃) of relative density, drying under reduced pressure, dry temperature is 75 ℃, pulverizes, and get final product.
Preferably, wherein 15% ethanol part elution volume is every 1ml resin 3ml eluting, selects every 1ml resin 4ml eluting during 45% ethanol elution.
Preferably, wherein Fructus Aurantii Immaturus or Fructus Aurantii are pulverized by 8 mesh sieves.
Preferably, general flavone content more than 50% wherein.
Preferably, general flavone content more than 80% wherein.
Preferably, wherein also comprise flavonoids and acceptable liposoluble ingredient medically thereof, flavonoids contains one or more in naringin, neohesperidin, Hesperidin, new naringin, Radix seu Folium Tosicodendri Delavayi glucoside and aglycon thereof.
Preferably, wherein naringin content, neohesperidin content reach more than 25% respectively.
Preferably, wherein the macroporous resin applied sample amount by resin volume (ml): crude drug quality (g) is 5: 1.
Preferably, wherein the loading flow velocity of every 1ml resin between 1.25～1.55ml/h.
Preferably, wherein the elution flow rate of every 1ml resin is between 1.25～1.45ml/h, and described non-polar macroporous resin blade diameter length ratio is 1: 10, and after loading, the static adsorption time of medicinal liquid is 1 hour.
Preferably, wherein said non-polar macroporous resin repeats 8 times afterwards with 5% sodium hydroxide solution immersion of 10 times of amounts 2 hours, washes with water to neutrality; 3% hydrochloric acid solution with 10 times of amounts soaked 2 hours again, washed with water to neutrality with the rear use of regenerating.
On the one hand, the invention provides Fructus Aurantii Immaturus or Fructus Aurantii extractive of general flavone for the preparation of the purposes for the treatment of gastrointestinal motility obstruction type functional dyspepsia disease medicament.
On the one hand, the invention provides the purposes of the vomiting disease medicament that Fructus Aurantii Immaturus or Fructus Aurantii extractive of general flavone cause for the preparation for the treatment of chemotherapeutics or other reasons.
On the one hand, the invention provides Fructus Aurantii Immaturus or Fructus Aurantii extractive of general flavone for the preparation of the purposes for the treatment of Upper abdominal pain disease medicament.
On the one hand, the invention provides Fructus Aurantii Immaturus or Fructus Aurantii extractive of general flavone for the preparation of the purposes for the treatment of abdominal distention, anorexia, belch disease medicament.
On the one hand, the invention provides Fructus Aurantii Immaturus or Fructus Aurantii extractive of general flavone for the preparation of the purposes of the nauseating disease medicament for the treatment of.
On the other hand, the present invention also provides a kind of Fructus Aurantii Immaturus or Fructus Aurantii extractive of general flavone preparation, and said preparation is take described Fructus Aurantii Immaturus or Fructus Aurantii extractive of general flavone as main active.
Experimental data proves, Fructus Aurantii Immaturus or Fructus Aurantii extractive of general flavone that alcohol reflux of the present invention obtains can be used for treating gastrointestinal motility obstruction type functional dyspepsia; Upper abdominal pain, abdominal distention, anorexia, belch, feel sick, vomiting that vomiting and chemotherapeutics or other reasons cause.
More importantly, the Fructus Aurantii Immaturus that alcohol reflux of the present invention obtains or Fructus Aurantii extractive of general flavone are except having the effect of significant promotion gastric motility, and be also extremely effective to the vomiting of the drug-induceds such as cisplatin, has the unexistent effect of motilium.The Cardiotoxity that there is no again simultaneously cisapride, mosapride, the cardiac safety risk that can thoroughly avoid cisapride, mosapride to cause.
And medical science and study of pharmacy personnel can't in advance under the prerequisite of not doing experiment, learn that in advance Fructus Aurantii Immaturus or Fructus Aurantii extractive of general flavone that alcohol reflux of the present invention obtains have above-mentioned good effect.
In sum, the Fructus Aurantii Immaturus of alcohol reflux acquisition provided by the invention or Fructus Aurantii extractive of general flavone and uses thereof bring significant technique effect.
The specific embodiment
Embodiment 1
Preparation method: get Fructus Aurantii Immaturus or Fructus Aurantii, be cut into decoction pieces, add 35% alcohol reflux 3 times of 10 times of volumes, each reflux, extract, 1.5 hours filters; Merging filtrate, filtrate recycling ethanol, the AB-8 macroporous resin of aqueous solution by having handled well, water, 18% ethanol elution are removed impurity successively, then use 37% ethanol, 0.5% sodium hydroxide eluting, collect 37% ethanol elution, be evaporated to the approximately fluid extract of 1.22 (50 ℃) of relative density, drying under reduced pressure (63 ℃) is pulverized, and be get final product.
Embodiment 2
Preparation method: get Fructus Aurantii Immaturus or Fructus Aurantii, be cut into decoction pieces, add 56% alcohol reflux 3 times of 6 times of volumes, each reflux, extract, 1 hour filters; Merging filtrate, filtrate recycling ethanol, the D101 macroporous resin of aqueous solution by having handled well, water, 16% ethanol elution are removed impurity successively, then use 35% ethanol, 0.7% sodium hydroxide eluting, collect 35% ethanol elution, be evaporated to the approximately fluid extract of 1.20 (55 ℃) of relative density, drying under reduced pressure (60 ℃) is pulverized, and be get final product.
Embodiment 3
Preparation method: get Fructus Aurantii Immaturus or Fructus Aurantii, be cut into decoction pieces, add 40% alcohol reflux 2 times of 11 times of volumes, each reflux, extract, 1 hour filters; Merging filtrate, filtrate recycling ethanol, the D101 macroporous resin of aqueous solution by having handled well, water, 14% ethanol elution are removed impurity successively, then use 40% ethanol, 0.8% sodium hydroxide eluting, collect 40% ethanol elution, be evaporated to the approximately fluid extract of 1.23 (55 ℃) of relative density, drying under reduced pressure (65 ℃) is pulverized, and be get final product.
Embodiment 4
Preparation method: get Fructus Aurantii Immaturus or Fructus Aurantii, be cut into decoction pieces, add 45% alcohol reflux 3 times of 5 times of volumes, each reflux, extract, 1 hour filters; Merging filtrate, filtrate recycling ethanol, the D101 macroporous resin of aqueous solution by having handled well, water, 15% ethanol elution are removed impurity successively, then use 35% ethanol, 0.6% sodium hydroxide eluting, collect 35% ethanol elution, be evaporated to the approximately fluid extract of 1.20 (60 ℃) of relative density, drying under reduced pressure (70 ℃) is pulverized, and be get final product.
Embodiment 5
Preparation method: get Fructus Aurantii Immaturus or Fructus Aurantii, be cut into decoction pieces, add 55% alcohol reflux 2 times of 12 times of volumes, each reflux, extract, 2 hours filters; Merging filtrate, filtrate recycling ethanol, the AB-8 macroporous resin of aqueous solution by having handled well, water, 10% ethanol elution are removed impurity successively, then use 45% ethanol, 0.8% sodium hydroxide eluting, collect 45% ethanol elution, be evaporated to the approximately fluid extract of 1.22 (58 ℃) of relative density, drying under reduced pressure (65 ℃) is pulverized, and be get final product.
Embodiment 6
Preparation method: get Fructus Aurantii Immaturus or Fructus Aurantii, be cut into decoction pieces, add 50% alcohol reflux 2 times of 7 times of volumes, each reflux, extract, 2 hours filters; Merging filtrate, filtrate recycling ethanol, the D101 macroporous resin of aqueous solution by having handled well, water, 15% ethanol elution are removed impurity successively, then use 45% ethanol, 1.1% sodium hydroxide eluting, collect 45% ethanol elution, be evaporated to the approximately fluid extract of 1.25 (55 ℃) of relative density, drying under reduced pressure (65 ℃) is pulverized, and be get final product.
Embodiment 7
Preparation method: get Fructus Aurantii Immaturus or Fructus Aurantii, pulverize, add 60% alcohol reflux 2 times of 8 times of volumes, each reflux, extract, 2.5 hours filters; Merging filtrate, filtrate recycling ethanol, the AB-8 macroporous resin of aqueous solution by having handled well, water, 9% ethanol elution are removed impurity successively, then use 42% ethanol, 0.8% sodium hydroxide eluting, collect 42% ethanol elution, be evaporated to the approximately fluid extract of 1.30 (50 ℃) of relative density, drying under reduced pressure (65 ℃) is pulverized, and be get final product.
Embodiment 8
Preparation method: get Fructus Aurantii Immaturus or Fructus Aurantii, pulverize, add 60% alcohol reflux 2 times of 8 times of volumes, each reflux, extract, 2.5 hours filters; Merging filtrate, filtrate recycling ethanol, the AB-8 macroporous resin of aqueous solution by having handled well, water, 14% ethanol elution are removed impurity successively, then use 50% ethanol, 0.9% sodium hydroxide eluting, collect 50% ethanol elution, be evaporated to the approximately fluid extract of 1.30 (55 ℃) of relative density, drying under reduced pressure (65 ℃) is pulverized, and be get final product.
Embodiment 9
Preparation method: get Fructus Aurantii Immaturus or Fructus Aurantii, pulverize, add 70% alcohol reflux 2 times of 9 times of volumes, each reflux, extract, 2 hours filters; Merging filtrate, filtrate recycling ethanol, the AB-8 macroporous resin of aqueous solution by having handled well, water, 8% ethanol elution are removed impurity successively, then use 45% ethanol, 0.9% sodium hydroxide eluting, collect 45% ethanol elution, be evaporated to the approximately fluid extract of 1.25 (55 ℃) of relative density, drying under reduced pressure (70 ℃) is pulverized, and be get final product.
Embodiment 10
Preparation method: get Fructus Aurantii Immaturus or Fructus Aurantii, pulverize, add 65% alcohol reflux 2 times of 9 times of volumes, each reflux, extract, 2 hours filters; Merging filtrate, filtrate recycling ethanol, the AB-8 macroporous resin of aqueous solution by having handled well, water, 15% ethanol elution are removed impurity successively, then use 45% ethanol, 0.9% sodium hydroxide eluting, collect 45% ethanol elution, be evaporated to the approximately fluid extract of 1.30 (60 ℃) of relative density, drying under reduced pressure (70 ℃) is pulverized, and be get final product.
Embodiment 11
Get Fructus Aurantii Immaturus or Fructus Aurantii, be cut into decoction pieces, add 65% alcohol reflux 3 times of 7 times of volumes, each reflux, extract, 1.5 hours filters; Merging filtrate, filtrate recycling ethanol, the D101 macroporous resin column of aqueous solution by having handled well, water, 15% ethanol, 40% ethanol, 1% sodium hydroxide eluting successively, collect 40% ethanol elution, be evaporated to the approximately fluid extract of 1.30 (60 ℃) of relative density, drying under reduced pressure (75 ℃), pulverize, and get final product.
Embodiment 12
Get Fructus Aurantii Immaturus or Fructus Aurantii, be cut into decoction pieces, add 65% alcohol reflux 2 times of 7 times of volumes, each reflux, extract, 2 hours filters; Merging filtrate, filtrate recycling ethanol, the D101 macroporous resin column of aqueous solution by having handled well, water, 15% ethanol, 45% ethanol, 1% sodium hydroxide eluting successively, collect 45% ethanol elution, be evaporated to the approximately fluid extract of 1.30 (60 ℃) of relative density, drying under reduced pressure (75 ℃), pulverize, and get final product.
Experimental data 1: extraction process optimization research
Flavonoid glycoside compound generally is soluble in hot water, methanol, ethanol, pyridine and dilute alkaline soln, and indissoluble or be insoluble in the organic solvents such as benzene, ether, chloroform, petroleum ether.Main active in Fructus Aurantii Immaturus is the flavanone constituents take naringin and neohesperidin as representative, and the polarity of these flavanones is all larger than general flavones ingredient, in water soluble and low-concentration ethanol, is soluble in the Diluted Alcohol solution of hot water or heat.
According to the physicochemical property of contained main chemical compositions in medical material, with reference to related documents, simultaneously in conjunction with the trial test result, adopt alcohol extraction, the method for purification by macroporous resin on extracting solution.
Investigate the rate of transform that index is respectively the rate of extract, general flavone content and total flavones
1. the extraction process condition is preferred
1.1 extract the selection of solvent
Fructus Aurantii Immaturus was pulverized 8 mesh sieves, get 12 parts, and be divided into 4 groups, every group average three parts, water, 65% ethanol, 95% ethanol and saturated limewater extract separately.The results are shown in Table 1.
Table 1 extracts the selection of solvent
Conclusion: as shown in Table 1, three kinds of extraction solvents are best with 65% ethanol extraction effect, therefore take 65% ethanol as extracting solvent.
1.2 the selection of medical material granularity
The Fructus Aurantii Immaturus that crushes is sieved, respectively drink sheet, be broken into the Semen Glycines size particles, three parts, 8 orders, 20 orders, 40 order, totally 15 parts, add respectively alcohol reflux.The results are shown in Table 2.
The selection of table 2 medical material granularity
Conclusion: by relevant data in table 2 as can be known, add alcohol reflux, the granularity of medical material has larger difference, be not that the medical material granule is more thin better, wherein the highest with the rate of transform that is broken into Semen Glycines size particles and 8 order medical material the rate of extract and total flavonoid glycoside, actual in conjunction with producing, select the Semen Glycines size particles as the final particle size after cracking of medical material.
1.3 the preferred extraction process of orthogonal experiment
According to above experimental result, existing with extraction time in leaching process (A), extraction time (B), add 65% amount of alcohol (C), extract before four of soak times (D) as the investigation factor, each factor is established three levels, presses L9 (3
4) orthogonal table carries out orthogonal (table 3, table 4), investigating index is the rate of extract and the total flavonoid glycoside rate of transform.
Table 3 factor level table
Get 27 parts of Fructus Aurantii Immaturus granules, every group average three parts, totally nine groups, press table 4 L9 (34) orthogonal test table design experiment.The results are shown in Table 4.
Table 4 L9 (3
4) orthogonal test table
(1) intuitive analysis:
The rate of extract optimised process: B
3C
3A
3D
1(B, C are principal element)
Total flavonoid glycoside rate of transform optimised process: B
3C
3A
1D
1(B, C are principal element)
(2) variance analysis
The results are shown in Table 5.
The variance analysis of table 5 take the total flavonoid glycoside rate of transform as index
*F
1-0.05(2，2)＝19.0
**F
1-0.01(2，2)＝99.0
(3) conclusion: intuitive analysis as can be known, extraction time (B) and solvent for use volume (C) are larger on extraction effect impact, extraction time (A) and soak time (D) are less on the extraction effect impact.Carry out variance analysis take the rate of transform as index, table 5 analysis result has further been verified the intuitive analysis conclusion, and namely set factor B, C have considerable influence to extraction effect.Because factor A is less to decocting influential effect, determine tentatively that thus extraction conditions is that 18 times of amount 65% ethanol (with the medical material weight ratio) are carried (8 times, 5 times, 5 times) 3 times, each extraction time 1h, medical material does not soak.
2. demonstration test
Get 3 parts of Fructus Aurantii Immaturus (Semen Glycines size particles), the optimal extract process of choosing by orthogonal test carries out demonstration test, that is: (8 times/1h, 5 times/1h, 5 times/1h) extract respectively 3 times, medical material does not soak 18 times of amount 65% ethanol.The results are shown in Table 6.
Table 6 demonstration test measurement result
Conclusion: as shown in Table 6, press selection process 65% ethanol extraction 3 times, total flavonoid glycoside is existing to be suggested more than 95%, and three sample total flavonoid glycoside rates of transform more consistent (RSD is 1.88%), and result shows that the last optimum extraction process of determining is reasonable, stable.
Experimental data 2: the purification research of citrus aurantium total flavone extract
2.1 separation purifying technique is investigated
Fructus Aurantii Immaturus proposes a large amount of water-solubility impurities after with 65% ethanol extraction, must be further purified processing to 65% ethanol extract, to improve the content of flavonoids effective constituent, water-solubility impurity mostly is inorganic salt, saccharide, pectin and protein-based, and the active component in Fructus Aurantii Immaturus is flavone compound, according to flavonoid compound characteristic and present China isolation and purification method commonly used, therefore select to come this flavones ingredient of enriching and purifying with macroporous resin.
Investigate by applied sample amount, determine that finally the macroporous resin adsorption 200g crude drug of 1000ml is as final applied sample amount (being 0.2g crude drug/ml resin).
Afterwards, by the investigation of elution volume, determine that 15% ethanol part elution volume is defined as 3000ml (being every 1ml resin 3ml 15% ethanol elution), selects during 45% ethanol elution with 4000ml (1ml resin 4ml 45% ethanol elution).
Again further, by the investigation of loading flow velocity, determine that the loading flow velocity should be chosen in better (being that the loading flow velocity of every 1ml resin is between 1.25～1.55ml/h) between 1250ml/h～1550ml/h.
2.2 orthogonal optimum seeking macroporous resin Optimization Technology
According to above experimental result, existing with elution flow rate, macroporous resin blade diameter length ratio and loading after each factor is established three levels as the investigation factor three of static adsorption times of medicinal liquid, press L 9 (3
4) orthogonal table carries out orthogonal (table 7, table 8), the investigation index is content and the total flavonoid glycoside rate of transform of the rate of extract, total flavonoid glycoside.
Table 7 factor level table
Get and be equivalent to 9 parts of crude drug 200g aqueous solutions, by table 8L9 (3
4) on the macroporous resin (every resin dress 1000ml macroporous resin) of anticipating, use respectively the water of 4000ml, ethanol, 4000ml 45% ethanol and 3000ml 95% ethanol elution of 3000ml 15%.The results are shown in Table 8.
Table 8 L9 (3
4) orthogonal test table
(1) intuitive analysis
Analyze the rate of extract optimised process: A according to the R value
1B
1C
3(A is principal element)
Analyze total flavonoid glycoside rate of transform optimised process: A according to the R value
1B
1C
3(A is principal element)
(2) variance analysis
The results are shown in Table 9, table 10.
The variance analysis of table 9 take the rate of extract as index
The variance analysis of table 10 take the total flavonoid glycoside rate of transform as index
*F
1-0.05(2，2)＝19.0
**F
1-0.01(2，2)＝99.0
(3) conclusion: intuitive analysis as can be known, the elution flow rate of resin (A) is principal element, after the blade diameter length ratio of macroporous resin (C) and loading, the static adsorption time (B) of medicinal liquid impact is less.The rate of transform take the rate of extract and total flavonoid glycoside is carried out variance analysis as index, and in table 9,10, analysis result has further been verified the intuitive analysis conclusion, and namely set factor A has considerable influence to elute effect.Be the leading indicator of this project due to general flavone content, column chromatographic column footpath is actual than suitability for industrialized production, determines that tentatively optimum condition is A
1B
1C
2That is: elution flow rate is 1250～1450ml/h (elution flow rate that is every 1ml resin is between 1.25～1.45ml/h), and blade diameter length ratio is 1: 10, and after loading, the static adsorption time of medicinal liquid is 1 hour.
2.3 demonstration test and the test of resin access times
Get Fructus Aurantii Immaturus (Semen Glycines size particles) and add alcohol reflux, reclaim ethanol, aqueous solution is 13 parts, and every part is equivalent to the 200g crude drug, standby.
A. get 1 part and be splined on 1000ml macroporous resin (the resin column blade diameter length ratio is 1: 10), the loading flow velocity is 1400ml/h, static adsorption 1 hour.Use successively ethanol, 4000ml 45% ethanol and 3000ml 95% ethanol elution of water, 3000ml 15% of 4000ml with the flow velocity eluting of 1400ml/h.Collect 45% ethanol elution and carry out assay.The results are shown in Table 11.
B. with a with after resin to be washed to without alcohol flavor, separately get this resin on the sample of a refrigerator cold-storage, the step reprocessing of pressing a.Resin carries out absorption next time (the same a.b of following steps) successively.
Table 11 demonstration test measurement result
Conclusion: the demonstration test of being undertaken by optimised process as can be seen from Table 11, the sample total flavonoid glycoside content and the total flavonoid glycoside rate of transform that repeat 8 gained all increase.And the indices homogeneous of sample, stable, illustrate that this technique is best, stable.Need later on resin is carried out Regeneration Treatment for 8 times in use simultaneously.
2.4 service test after resin regeneration
5% sodium hydroxide solution of resin with 10 times of amounts soaked 2 hours, wash with water to neutrality; 3% hydrochloric acid solution with 10 times of amounts soaked 2 hours again, washed with water to neutrality.The resin that regeneration is good by demonstration test and a resin access times test lower method absorb-elute successively 3 times, calculates the rate of extract, Liquid Detection total flavonoid glycoside content and the rate of transform.The results are shown in Table 12.
Service test after table 12 resin regeneration
After result shows resin regeneration, operating position is good.
According to above-mentioned separation, purification research, merge extractive liquid, after medicinal material extract filters, the D101 macroporous resin column of filtrate by having handled well, and water, 15% ethanol, 45% ethanol, 1% sodium hydroxide eluting, collect 45% ethanol elution successively.
Experimental data 3: the research of citrus aurantium total flavone extract drying means and baking temperature
Get Fructus Aurantii Immaturus 1kg, totally 10 parts, according to above-mentioned definite extraction, purifying process, after extraction, purification, collect 45% ethanol elution, decompression recycling ethanol also is concentrated into the approximately extractum of 1.30 (60 ℃) of relative density, adopts respectively oven drying method, boulton process to compare research to above-mentioned extractum.Oven drying method and boulton process carry out respectively the comparison of 5 kinds of different temperatures (50 ℃, 65 ℃, 75 ℃, 85 ℃, 95 ℃).Investigate the content of total flavonoid glycoside.The results are shown in Table 13 and table 14.
The measurement result of table 13 oven drying method different temperatures
The measurement result of table 14 boulton process different temperatures
From table 13,14 as can be known, oven drying method is long than the dry required time of vacuum on the same temperature level, and effect is not as vacuum drying.The sample color and luster of oven drying method oven dry is relatively poor, be difficult for pulverizing, and along with the rising of temperature, oven drying method easily causes carbonization because of hot-spot, thereby causes the color sample blackout, and effective ingredient is lost.Easy-to-drawly during vacuum drying driedly become cellular, be conducive to pulverize, and color and luster is better, vacuum drying temperature is controlled at 75 ℃ of left and right and is advisable simultaneously, saves time and can not cause the loss of effective ingredient.
Below all select embodiment 12 Fructus Aurantii Immaturus total flavones glucoside extracts as experimental group
Experimental data 4: effect is told in Fructus Aurantii Immaturus total flavones glucoside extract town
4.1 the Fructus Aurantii Immaturus total flavones glucoside extract causes the impact of pigeon vomiting on copper sulfate
60 of pigeons, body weight 280～350g, male and female half and half.Be divided at random 6 groups: matched group, embodiment 12 Fructus Aurantii Immaturus total flavones glucoside extracts (20,40,80,160mgkg
-1) 4 dosage group and positive drug group (fourth beautiful jade 40mgkg
-1), 10 every group, ♀ ♂ half and half.Each is organized the pigeon fasting and can't help water 16 hours, next day single administration, matched group gives the distilled water gavage, other group gavage relative medicines, 10mlkg
-1Respectively organize pigeon after 1 hour and give 3.5% copper-bath 10mgkg
-1The gavage modeling.Record is respectively organized pigeon vomiting latent time and is vomitted number of times in 2 hours, and result of the test shows, the Fructus Aurantii Immaturus total flavones glucoside extract (20,40,80,160mgkg
-1) single administration all can not extend the vomiting latent time of pigeon due to copper sulfate, but the Fructus Aurantii Immaturus total flavones glucoside extract (40,80mgkg
-1) single administration can reduce the vomiting number of times of pigeon due to copper sulfate, sees Table 15.
Table 15 Fructus Aurantii Immaturus total flavones glucoside extract causes the impact of pigeon vomiting on copper sulfate
Annotate: compare with matched group,
*P＜0.05,
*P＜0.01
4.2 the Fructus Aurantii Immaturus total flavones glucoside extract causes the impact of pigeon vomiting on cisplatin
80 of pigeons, body weight 280～350g, male and female half and half.Be divided at random 8 groups: matched group, embodiment 12 Fructus Aurantii Immaturus total flavones glucoside extracts (20,40,80,160mgkg
-1) 4 dosage group and positive drug 3 groups of (fourth beautiful jade 80,160mgkg
-1With Ondansetron Hydrochloride injection 5ugkg
-1) 10 every group, ♀ ♂ half and half.Each is organized the pigeon fasting and can't help water 16 hours, next day single administration, matched group gives the distilled water gavage, other groups give relative medicine, the injection of 1 hour pneumoretroperitoneum gives 13mgkg
-1Cisplatin for inj.Record is respectively organized pigeon vomiting latent time and is vomitted number of times in 4 hours, and result of the test shows, Fructus Aurantii Immaturus total flavones glucoside extract 80mgkg
-1Single administration can extend the vomiting time of pigeon due to cisplatin, but 20,40 and 160mgkg
-1The time all can not extend the vomiting time of pigeon due to cisplatin; The Fructus Aurantii Immaturus total flavones glucoside extract (80,160mgkg
-1) single administration can reduce the vomiting number of times of pigeon due to cisplatin, sees Table 16
Table 16 Fructus Aurantii Immaturus total flavones glucoside extract causes the impact of pigeon vomiting on cisplatin
Annotate: compare with matched group,
*P＜0.05,
*P＜0.01
4.3 the Fructus Aurantii Immaturus total flavones glucoside extract causes the impact of hybrid dog vomiting on apomorphine
56 of hybrid dogs, body weight 8.0～10.0kg, male and female half and half.Be divided at random 7 groups: matched group, embodiment 12 Fructus Aurantii Immaturus total flavones glucoside extracts (15,30,60,120mgkg
-1) 4 dosage group and 2 groups of (motilium 30mgkg of positive drug
-1With metoclopramide injection 1mgkg
-1), 8 every group, ♀ ♂ half and half.Each is organized the fasting of hybrid dog and can't help water 16 hours, next day single administration, matched group gives the distilled water gavage, other groups give relative medicine gavage, 5mlkg
-1Give 500ugkg after 1 hour
-1The apomorphine subcutaneous injection.Record is respectively organized hybrid dog vomiting latent time and is vomitted number of times in 1 hour.Result of the test shows, the Fructus Aurantii Immaturus total flavones glucoside extract (15,30,60,120mgkg
-1) single administration all can not reduce apomorphine and cause hybrid dog vomiting number of times; The Fructus Aurantii Immaturus total flavones glucoside extract (15,30,120mgkg
-1) also can not extend the vomiting latent time that apomorphine causes the hybrid dog, but Fructus Aurantii Immaturus total flavones glucoside extract 60mgkg
-1The time can extend the vomiting latent time of hybrid dog, see Table 17.
Table 17 Fructus Aurantii Immaturus total flavones glucoside extract causes the impact of hybrid dog vomiting on apomorphine
Annotate: compare with matched group,
*P＜0.05
Experimental data 5: the Fructus Aurantii Immaturus total flavones glucoside extract is to the intestinal progradation
5.1 the Fructus Aurantii Immaturus total flavones glucoside extract causes the impact of the low mice of intestinal function on dopamine
70 of mices, body weight 19～22g is divided into 7 groups at random: matched group, model group, embodiment 12 Fructus Aurantii Immaturus total flavones glucoside extracts (25,50,100,200mgkg
-1) 4 dosage group and positive drug group (motilium 50mgkg
-1), 10 every group, ♀ ♂ half and half.Each is organized the mice fasting and can't help water 16 hours, next day single administration, matched group gavage distilled water, other groups give the relative medicine gavage.After 30 minutes, except matched group, all the other mices give 1.7mgkg to each mouse stomach
-1The modeling of dopamine hydrochloride inj subcutaneous injection, after 20 minutes, 70 mices gavage respectively 2% sodium carboxymethyl cellulose carbon powder suspension 0.2ml10g
-1, after 20 minutes, mice takes off neck execution, takes out immediately gastrointestinal, and tiling is measured the carbon powder front end to distance and the small intestinal total length of pylorus, calculates ink propulsive rate with ratio between two.Result of the test shows, subcutaneous injection 1.7mgkg
-1The dopamine intestinal that can obviously suppress mice advance, the Fructus Aurantii Immaturus total flavones glucoside extract (25,50,100mgkg
-1) can improve the mice intestinal propelling activity that is suppressed by dopamine, see Table 18.
Table 18 Fructus Aurantii Immaturus total flavones glucoside extract causes on dopamine the impact that the mice intestinal advances
Annotate: compare with matched group,
△Compare with model group P＜0.05,
*P＜0.05,
*P＜0.01
5.2 the Fructus Aurantii Immaturus total flavones glucoside extract causes the impact of the low mice of intestinal function on atropine
70 of mices, body weight 19～22g is divided into 7 groups at random: matched group, model group, embodiment 12 Fructus Aurantii Immaturus total flavones glucoside extracts (25,50,100,200mgkg
-1) 4 dosage group and positive drug group (motilium 50mgkg
-1), 10 every group, ♀ ♂ half and half.Each is organized the mice fasting and can't help water 16 hours, next day single administration, matched group gavage distilled water, other groups give the relative medicine gavage.After 30 minutes, except matched group, all the other mices give 2.5mgkg to each mouse stomach
-1The modeling of atropine subcutaneous injection, after 20 minutes, 70 mices gavage respectively 2% sodium carboxymethyl cellulose carbon powder suspension 0.2ml10g
-1, after 20 minutes, mice takes off neck execution, takes out immediately gastrointestinal, and tiling is measured the carbon powder front end to distance and the small intestinal total length of pylorus, calculates ink propulsive rate with ratio between two.Result of the test shows, subcutaneous injection 2.5mgkg
-1The atropine intestinal that can obviously suppress mice advance, Fructus Aurantii Immaturus total flavones glucoside extract 50mgkg
-1Can improve the mice intestinal propelling activity that is suppressed by atropine, see Table 19.
Table 19 Fructus Aurantii Immaturus total flavones glucoside extract causes on atropine the impact that the mice intestinal advances
Annotate: compare with matched group,
△P＜0.05; Compare with model group,
*P＜0.05
5.3 the Fructus Aurantii Immaturus total flavones glucoside extract causes the impact of the low mice of intestinal function on epinephrine
70 of mices, body weight 19～22g, be divided at random 7 groups: matched group, model group, embodiment 12 Fructus Aurantii Immaturus total flavones glucoside extracts (25,50,100,200mgkg-1) 4 dosage groups and positive drug groups (motilium 50mgkg-1), 10 every group, ♀ ♂ half and half.Each is organized the mice fasting and can't help water 16 hours, next day single administration, matched group gavage distilled water, other groups give the relative medicine gavage.After 30 minutes, except matched group, all the other mices give 0.5mgkg to each mouse stomach
-1The modeling of adrenalin hydrochloride injection subcutaneous injection, after 20 minutes, 70 mices gavage respectively 2% sodium carboxymethyl cellulose carbon powder suspension 0.2ml10g-1, after 20 minutes, mice takes off neck execution, take out immediately gastrointestinal, tiling, measure the carbon powder front end to distance and the small intestinal total length of pylorus, calculate ink propulsive rate with ratio between two.Result of the test shows, subcutaneous injection 0.5mgkg
-1The epinephrine intestinal that can obviously suppress mice advance, the Fructus Aurantii Immaturus total flavones glucoside extract (25,50,100,200mgkg
-1) all can not improve the mice intestinal propelling activity by epinephrine inhibited, see Table 20.
Table 20 Fructus Aurantii Immaturus total flavones glucoside extract causes on epinephrine the impact that the mice intestinal advances
Annotate: compare with matched group,
△ △P＜0.01
Experimental data 6: the Fructus Aurantii Immaturus total flavones glucoside extract is on gastric secretion, gastric acid and pepsic impact
6.1 the impact on gastric secretion, gastric acid
48 of rats, body weight 230～270g is divided into 6 groups at random: matched group, embodiment 12 Fructus Aurantii Immaturus total flavones glucoside extracts (20,40,80,160mgkg
-1) 4 dosage group and positive drug group (motilium 40mgkg
-1), 8 every group, ♀ ♂ half and half.Before test, the rat fasting be can't help water 36 hours, gastric infusion of rat, and matched group gives distilled water, and other groups give relative medicine.The administration while, each rat is opened the abdominal cavity under the ether light anaesthesia, the ligation pylorus, and the rat gastric juice of 5 hours after the collection pylorus ligation, with the centrifugal 5min of 3000r/min, the gastric contents such as place to go food debris are with the accurate weighing gastric juice of graduated cylinder amount.Measure gastric juice 1ml, be placed in conical flask and add the equivalent distilled water, mix homogeneously adds each 2 of 0.5% pair of dimethyl azoaniline alcoholic solution of free acid indicator and total acid indicator 0.5% phenolphthalein alcoholic solution, if mix in gastric juice and contain hydrochloric acid, namely be cherry red.Drip the NaOH of 0.01mol/L with burette,, limit edged shaking flasks disappears to red, till beginning orange colour to occur.Be the terminal point of free hydrochloric acid.Write down the amount that spends till NaOH no longer deepens to redness, be the terminal point of total acidity.Write down twice titration and spend NaOH solution total amount.Calculate concentration and total secretory volume of free hydrochloric acid and total acid.Result of the test shows, the Fructus Aurantii Immaturus total flavones glucoside extract (20,40,80mgkg
-1) gastric secretion of pylorus ligation rat is had no significant effect, but Fructus Aurantii Immaturus total flavones glucoside extract 160mgkg
-1Can reduce the gastric secretion of rat; The Fructus Aurantii Immaturus total flavones glucoside extract (80,160mgkg
-1) concentration of can raise pylorus ligation rat free hydrochloric acid and total acid, but each dosage of Fructus Aurantii Immaturus total flavones glucoside extract (20,40,80,160mgkg
-1) on the secretory volume of free hydrochloric acid and total acid without impact, see Table 21.
Table 21 Fructus Aurantii Immaturus total flavones glucoside extract is to rat gastric secretion, free hydrochloric acid and total acid concentration as influencing factor
Annotate: compare * P＜0.05, * * P＜0.01 with matched group
6.2 on pepsic impact
The capillary glass-tube that internal diameter 1～2mm thickness is well-balanced is cleaned and is dried, and gets appropriate Ovum Gallus domesticus album and fully beats the even rear filtered through gauze of using, and above-mentioned glass capillary is utilized siphonage, fills Ovum Gallus domesticus album (the pipe planted agent sneaks into without bubble).Then be positioned in 85 ℃ of heat steams and make protein coagulating.After cooling, take out paraffin with protein pipe two ends sealing, standby in the ice storage case.Get the conical flask that gastric juice 1ml puts into 50ml during experiment, add 0.05N hydrochloric acid solution 15ml, shake up, put two of approximately long 2.5cm protein pipe into.Filled in bottleneck, be put in 37 ℃ of calorstats temperature and incubate 24h.Measure the length (mm) of protein pipe two ends transparent part with chi and ask its meansigma methods with the value of four ends, with the pepsic unit=meansigma methods of following formula calculating
2* 16.Result of the test shows, each dosage of Fructus Aurantii Immaturus total flavones glucoside extract (20,40,80,160mgkg
-1) pepsin activity of pylorus ligation rat is had no significant effect, see Table 22.
The impact of table 22 Fructus Aurantii Immaturus total flavones glucoside extract on rat free hydrochloric acid, total acid secretory volume and pepsin activity
Experimental data 7: the impact of Fructus Aurantii Immaturus total flavones glucoside extract on smooth muscle
7.1 the impact on the gastrointestinal smooth muscle that exsomatizes
Select the healthy adult rat, the head of fiercelying attack causes dusk, takes out rapidly full stomach and duodenum stage casing 1-2cm, and the place cuts off along greater gastric curvature, with containing 95%O
2And 5%CO
2The Krebs liquid (composition: NaCl 117mmol/L, KCl 4.8mmol/L, CaCl of the fully saturated pH 7.35～7.45 of mist
22.5mmol/L, MgCl
21.2mmol/L, NaH
2PO
41.2mmol/L, NaHCO
325mmol/L, Glucose 11mmol/L) repeatedly rinse and exenterate, get respectively at the bottom of the gastric antrum stomach function regulating longitudinal and transverse flesh bar and duodenum stage casing and be about 1cm, wide approximately 0.5cm, the ventilation hook is fixed in the bottom, the upper end is connected with transducer (JH-2 type muscular tension sensor), and it is inserted in the bath pipe that fills oxygen-saturated Krebs liquid (pH 7.35～7.45) 20ml, the adjustment preload is 1g, change liquid once every 20min, by ventilation hook direct oxygen injection in the body lotion, use BL-420E biological function experimental system and record its tension force and frequency.Outer tube through constant current infusion pump [continue, constant temperature (37 ± 0.5 ℃), constant speed (3～4ml/min)] perfusion to keep temperature in bath.After specimen is stable, add medicine ultimate density to be measured to be respectively 0.25mg/L, 0.4mg/L, 0.55mg/L, 0.7mg/L, 0.85mg/L and 1.00mg/L, record and observe medication front and back frequency and tension change.The isolated test result shows, embodiment 12 Fructus Aurantii Immaturus total flavones glucoside extract (0.25mg.ml
-1～1.0mg.ml
-1) can reduce longitudinal and transverse shape flesh and duodenum smooth muscle tension force at the bottom of the gastric antrum stomach function regulating, suppress gastrointestinal motility, see Table 23～27.
The impact of table 23 Fructus Aurantii Immaturus total flavones glucoside extract on exsomatize gastric antrum transversal tension force and frequency
Annotate: with comparison before administration,
*P＜0.05,
*P＜0.01
The impact of table 24 Fructus Aurantii Immaturus total flavones glucoside extract on exsomatize gastric antrum longitudinal muscle tension force and frequency
Annotate: with comparison before administration,
*P＜0.05,
*P＜0.01
Table 25 Fructus Aurantii Immaturus total flavones glucoside extract on the stomach that exsomatizes at the bottom of the impact of transversal tension force and frequency
Annotate: with comparison before administration,
*P＜0.05,
*P＜0.01
Table 26 Fructus Aurantii Immaturus total flavones glucoside extract on the stomach that exsomatizes at the bottom of the impact of longitudinal muscle tension force and frequency
Annotate: with comparison before administration,
*P＜0.05,
*P＜0.01
The impact of table 27 Fructus Aurantii Immaturus total flavones glucoside extract on Isolated Duodenum stage casing tension force and frequency
Annotate: with comparison before administration,
*P＜0.05,
*P＜0.01
7.2 on the impact at body duodenum smooth muscle
56 of rats, body weight 180～230g is divided into 7 groups at random: matched group, model group, embodiment 12 Fructus Aurantii Immaturus total flavones glucoside extracts (20,40,80,160mgkg
-1) 4 dosage group and positive drug group (fourth beautiful jade 40mgkg
-1), 8 every group, ♀ ♂ half and half.The rat oral gavage administration, matched group and model group give distilled water, and other groups give relative medicine.Each is organized the rat fasting and can't help water 16 hours, next day single administration, after 30 minutes, except matched group, all the other rats give 1.5mgkg to each rat oral gavage
-1The modeling of dopamine hydrochloride inj subcutaneous injection, chloral hydrate anesthesia, open the abdominal cavity along the abdomen median line, selection duodenum stage casing is 2-3cm approximately, be fixed on intestinal segment two ends on the aperture of intestinal tube stationary pipes both sides with stitching thread, with a stitching thread, one end is sewn on intestinal wall in the middle of intestinal segment, the other end is passed by intestinal tube stationary pipes central authorities and is connected on JH-2 type muscular tension sensor, and records tension force and the frequency of each rat preduodenal by BL-420E biological function experimental system again.Result of the test shows, subcutaneous injection 1.5mgkg
-1Dopamine can obviously be suppressed at tension force and the frequency of body rat preduodenal, the Fructus Aurantii Immaturus total flavones glucoside extract (20,40,80,160mgkg
-1) can improve tension force and frequency by the rat preduodenal of dopamine inhibition, see Table 28.
Table 28 Fructus Aurantii Immaturus total flavones glucoside extract is on the impact in body duodenum stage casing tension force and frequency
Annotate: compare with matched group,
△P＜0.05; Compare with model group,
*P＜0.05,
*P＜0.01
Claims (13)
1. Fructus Aurantii Immaturus or Fructus Aurantii extractive of general flavone are for the preparation of the purposes of the vomiting disease medicament that causes for the treatment of chemotherapeutics, it is characterized in that: described Fructus Aurantii Immaturus or Fructus Aurantii extractive of general flavone are by Fructus Aurantii Immaturus or Fructus Aurantii are obtained with alcohol reflux, wherein the alcohol reflux method is: get Fructus Aurantii Immaturus or Fructus Aurantii, be cut into decoction pieces or pulverizing, 30%～90% alcohol reflux 1～3 time that adds 5～16 times of volumes, each reflux, extract, 1～3 hour filters, merging filtrate, filtrate recycling ethanol, the non-polar macroporous resin of aqueous solution by having handled well, wherein the loading flow velocity of every 1ml resin is between 1.25～1.55ml/h, water successively, ethanol elution below 20% is removed impurity, use again 25%～90% ethanol, 0.5%～2% sodium hydroxide eluting, collect ethanol elution, be evaporated to relative density and be about 1.20～1.40 fluid extract 50 ℃～70 ℃ mensuration, drying under reduced pressure, dry temperature is 60～80 ℃, pulverize, and get final product, wherein general flavone content is greater than 80%, also comprise flavonoids and acceptable liposoluble ingredient medically thereof, flavonoids contains naringin, neohesperidin, Hesperidin, new naringin, Radix seu Folium Tosicodendri Delavayi glucoside and aglycon thereof, described naringin content, neohesperidin content reaches more than 25% respectively.
2. purposes as claimed in claim 1, concrete alcohol reflux method is as follows: get Fructus Aurantii Immaturus or Fructus Aurantii, be cut into decoction pieces or pulverizing, add 40%～85% alcohol reflux 2～3 times of 6～14 times of volumes, each reflux, extract, 1～2.5 hour filters; Merging filtrate, filtrate recycling ethanol, the non-polar macroporous resin of aqueous solution by having handled well, water, the ethanol elution below 20% are removed impurity successively, then use 30%～85% ethanol, 0.5%～1.8% sodium hydroxide eluting, collect ethanol elution, be evaporated to relative density and be about 1.25～1.40 fluid extract 55 ℃～70 ℃ mensuration, drying under reduced pressure, dry temperature is 65～80 ℃, pulverize, and get final product.
3. purposes as claimed in claim 2, concrete alcohol reflux method is as follows: get Fructus Aurantii Immaturus or Fructus Aurantii, be cut into decoction pieces or pulverizing, add 45%～80% alcohol reflux 2～3 times of 7～12 times of volumes, each reflux, extract, 1.5～2.5 hours filters; Merging filtrate, filtrate recycling ethanol, the non-polar macroporous resin of aqueous solution by having handled well, water, the ethanol elution below 20% are removed impurity successively, then use 35%～75% ethanol, 0.5～1.6% sodium hydroxide eluting, collect ethanol elution, be evaporated to relative density and be about 1.25～1.35 fluid extract 55 ℃～65 ℃ mensuration, drying under reduced pressure, dry temperature is 70～80 ℃, pulverize, and get final product.
4. purposes as claimed in claim 3, concrete alcohol reflux method is as follows: get Fructus Aurantii Immaturus or Fructus Aurantii, be cut into decoction pieces or pulverizing, add 55%～75% alcohol reflux 2～3 times of 7～11 times of volumes, each reflux, extract, 1.5～2 hours filters; Merging filtrate, filtrate recycling ethanol, the non-polar macroporous resin of aqueous solution by having handled well, water, the ethanol elution below 20% are removed impurity successively, then use 40%～65% ethanol, 0.5～1.3% sodium hydroxide eluting, collect ethanol elution, be evaporated to relative density and be about 1.30～1.35 fluid extract 60 ℃～65 ℃ mensuration, drying under reduced pressure, dry temperature is 70～75 ℃, pulverize, and get final product.
5. purposes as claimed in claim 4, concrete alcohol reflux method is as follows: get Fructus Aurantii Immaturus or Fructus Aurantii, be cut into decoction pieces or pulverizing, add 65% alcohol reflux 3 times of 9 times of volumes, each reflux, extract, 1.5 hours filters; Merging filtrate, filtrate recycling ethanol, the non-polar macroporous resin of aqueous solution by having handled well, water, the ethanol elution below 20% are removed impurity successively, then use 40%～55% ethanol, 0.8～1.2% sodium hydroxide eluting, collect ethanol elution, be evaporated to relative density and be about 1.30 fluid extract 60 ℃ of mensuration, drying under reduced pressure, dry temperature is 75 ℃, pulverize, and get final product.
6. purposes as claimed in claim 4, concrete alcohol reflux method is as follows: get Fructus Aurantii Immaturus or Fructus Aurantii, be cut into decoction pieces or pulverizing, add 60%～70% alcohol reflux 2 times of 7～9 times of volumes, each reflux, extract, 1.5 hours filters; Merging filtrate, filtrate recycling ethanol, the non-polar macroporous resin of aqueous solution by having handled well, water, the ethanol elution below 20% are removed impurity successively, then use 40%～55% ethanol, 0.7%～1.3% sodium hydroxide eluting, collect ethanol elution, be evaporated to relative density and be about 1.30 fluid extract 60 ℃ of mensuration, drying under reduced pressure, dry temperature is 75 ℃, pulverize, and get final product.
7. purposes as claimed in claim 4, concrete alcohol reflux method is as follows: get Fructus Aurantii Immaturus or Fructus Aurantii, be cut into decoction pieces or pulverizing, add 70% alcohol reflux 2 times of 8 times of volumes, each reflux, extract, 2 hours filters; Merging filtrate, filtrate recycling ethanol, the non-polar macroporous resin of aqueous solution by having handled well, water, the ethanol elution below 20% are removed impurity successively, then use 40%～50% ethanol, 0.9%～1.1% sodium hydroxide eluting, collect ethanol elution, be evaporated to relative density and be about 1.30 fluid extract 60 ℃ of mensuration, drying under reduced pressure, dry temperature is 75 ℃, pulverize, and get final product.
8. purposes as claimed in claim 4, concrete alcohol reflux method is as follows: get Fructus Aurantii Immaturus or Fructus Aurantii, be cut into decoction pieces, add 65% alcohol reflux 2 times of 7 times of volumes, each reflux, extract, 2 hours filters; Merging filtrate, filtrate recycling ethanol, the D101 macroporous resin column of aqueous solution by having handled well, water, 15% ethanol, 45% ethanol, 1% sodium hydroxide eluting, collect 45% ethanol elution successively, is evaporated to relative density and is about 1.30 fluid extract 60 ℃ of mensuration, drying under reduced pressure, dry temperature is 75 ℃, pulverizes, and get final product.
9. purposes as claimed in claim 8, wherein 15% ethanol part elution volume is every 1ml resin 3ml eluting, selects every 1ml resin 4ml eluting during 45% ethanol elution.
10. as the described purposes of claim 1-9 any one, wherein Fructus Aurantii Immaturus or Fructus Aurantii are broken into the Semen Glycines size particles.
11. purposes as described in claim 1～9 any one, wherein the macroporous resin applied sample amount is by resin volume ml: crude drug quality g is 5: 1.
12. as the described purposes of claim 1-9 any one, wherein the elution flow rate of every 1ml resin is between 1.25～1.45ml/h, described non-polar macroporous resin blade diameter length ratio is 1: 10, and after loading, the static adsorption time of medicinal liquid is 1 hour.
13. as the described purposes of claim 1-9 any one, wherein said non-polar macroporous resin repeats 8 times afterwards with 5% sodium hydroxide solution immersion of 10 times of amounts 2 hours, washes with water to neutrality; 3% hydrochloric acid solution with 10 times of amounts soaked 2 hours again, washed with water to neutrality with the rear use of regenerating.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010608298 CN102068535B (en) | 2010-12-28 | 2010-12-28 | Immature bitter orange or bitter orange total flavonoids extract prepared by ethanol reflux and extraction and application thereof |
| PCT/CN2011/078014 WO2012088885A1 (en) | 2010-12-28 | 2011-08-04 | Total flavonoid extract of fructus aurantii immaturus or fructus aurantii, and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010608298 CN102068535B (en) | 2010-12-28 | 2010-12-28 | Immature bitter orange or bitter orange total flavonoids extract prepared by ethanol reflux and extraction and application thereof |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2013101153349A Division CN103191237A (en) | 2010-12-28 | 2010-12-28 | Fructus aurantii immaturus or fructus aurantii total-flavonoid extract obtained through ethanol reflux extraction and application thereof |
| CN2013101153048A Division CN103191236A (en) | 2010-12-28 | 2010-12-28 | Fructus aurantii immaturus or fructus aurantii total-flavonoid extract obtained through ethanol reflux extraction and application thereof |
| CN2013101153866A Division CN103191238A (en) | 2010-12-28 | 2010-12-28 | Fructus aurantii immaturus or fructus aurantii total-flavonoid extract obtained through ethanol reflux extraction and application thereof |
| CN2013101154093A Division CN103191239A (en) | 2010-12-28 | 2010-12-28 | Fructus aurantii immaturus or fructus aurantii total-flavonoid extract obtained through ethanol reflux extraction and application thereof |
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| CN102716233A (en) * | 2012-05-18 | 2012-10-10 | 北京师范大学 | Flavonoids extract containing naringin and application thereof |
| CN103601775A (en) * | 2013-11-27 | 2014-02-26 | 桂林益元素生物科技有限公司 | Method for extracting hesperidin from orange peels |
| CN104352609A (en) * | 2014-10-16 | 2015-02-18 | 陕西天谷生物科技集团有限公司 | Preparation method of water-soluble immature bitter orange extract with bitterness removed |
| CN107927481A (en) * | 2017-12-15 | 2018-04-20 | 佛山实瑞先导材料研究院（普通合伙） | A kind of teenager's maltose nutritious drink and preparation method thereof |
| CN109316487B (en) * | 2018-11-20 | 2021-03-19 | 河南中医药大学 | Extraction method and application of thoroughfare bitter orange ingredient for treating respiratory inflammation |
| CN109912666B (en) * | 2019-03-14 | 2023-04-11 | 重庆市铜梁区子奇药材有限公司 | Method for simultaneously extracting high-purity naringin and neohesperidin from fructus aurantii |
| CN113616744A (en) * | 2021-09-01 | 2021-11-09 | 山西大学 | Preparation method and application of millet whole grain flavone active component |
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| CN1594344A (en) * | 2004-06-28 | 2005-03-16 | 浙江大学 | Process for separating high pure neohesperidin from fruit of citron or trifoliate orange |
| CN1748740A (en) * | 2004-09-17 | 2006-03-22 | 王英锋 | Imature bitter orange total flavone and its preparing mthod for its preparation and quality control method |
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| CN1594344A (en) * | 2004-06-28 | 2005-03-16 | 浙江大学 | Process for separating high pure neohesperidin from fruit of citron or trifoliate orange |
| CN1748740A (en) * | 2004-09-17 | 2006-03-22 | 王英锋 | Imature bitter orange total flavone and its preparing mthod for its preparation and quality control method |
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Effective date of registration: 20190115 Address after: 341000 East Avenue, Shahe Industrial Park Station, Zhanggong District, Ganzhou City, Jiangxi Province Patentee after: Jiangxi Qingfeng Pharmaceutical Co., Ltd. Address before: 341000 No. 8 East Avenue, Shahe Industrial Park Station, Ganzhou City, Jiangxi Province Co-patentee before: Jiangxi Qingfeng Drugs Research Co., Ltd. Patentee before: Jiangxi Qingfeng Pharmaceutical Co., Ltd. |