CN110376092A - A kind of compound haw thorn Content of Chlorogenic Acid and content of hesperidin measurement and integrated evaluating method - Google Patents
A kind of compound haw thorn Content of Chlorogenic Acid and content of hesperidin measurement and integrated evaluating method Download PDFInfo
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Abstract
The invention discloses a kind of compound haw thorn Content of Chlorogenic Acid and content of hesperidin measurement and integrated evaluating methods, more particularly to health food ingredients effect disquisition field, the preparation of preparation, reference substance and test solution including compound haw thorn water extract and alcohol extract, the calculating of dry cream rate, active constituent content measuring, the preparation of experimental subjects sample, acute toxicity testing, intestinal motility experiment, rat body weight, weight gain, the measurement of food ration and food utilization, the measurement of rat pepsin and data statistic analysis.The present invention establishes the content assaying method of compound haw thorn Content of Chlorogenic Acid and aurantiamarin, and it compared the active constituent content of compound haw thorn water extract and alcohol extract and promote digestion effect, using the content of high effective liquid chromatography for measuring compound haw thorn water extract and alcohol extract Content of Chlorogenic Acid and aurantiamarin, and comprehensive score is carried out, compound haw thorn water extract active constituent content and rush digestion effect are better than alcohol extract.
Description
Technical field
The present invention relates to health food ingredients effect disquisition technical fields, it is more particularly related to a kind of compound
Hawthorn Content of Chlorogenic Acid and content of hesperidin measurement and integrated evaluating method.
Background technique
Indigestion refers to the symptom of one group of chronic or recurrent upper abdominal pain or discomfort, is clinically common functionality
Gastrointestinal disease belongs to the scope of " epigastric pain ", " stagnant " in theory of traditional Chinese medical science, and dialectical point can be syndrome of spleen-deficiency and QIstagnation, liver-stomach disharmony
Card and syndrome of dampness-heat of spleen and stomach etc..There is the dyspeptic pathogenesis basis of document report to be spleen deficiency, liver depression is its inducement, and its clinical condition
Shape Producing reason is then that stomach Qi does not drop, mainly related with gastrointestinal motility, gastric acid secretion, treatment on invigorating the spleen, it is soothing the liver and
Stomach is its rules for the treatment of.As people's lives level is continuously improved, overeating, daily work and rest are irregular all to cause digestion not
It is good.Long-term indigestion has very important influence to the physiology and mental health of people, it is therefore necessary to rush digestion
Drug or functional food are studied, to improve level of human health and quality of life.As people are to health and health care
Pay attention to day by day, the type and yield of health food increase increasingly, and therefore, exploitation, which prevents dyspeptic health food, to be had very
The meaning of reality.
At present traditional Chinese medicine treat dyspeptic main prescription have bupleurum powder for relieving liver-qi, Xiangsha Liujunzi decoction, Xiaoyao San, guarantor and
Ball etc..The classics recipe Pachyma Compound Digestive Tonic Pill that wherein " danxi's experiential therapy " is recorded has unique in terms for the treatment of indigestion, eating accumulation
Advantage, clinical efficacy are significant.Pachyma Compound Digestive Tonic Pill is total to seven flavor medicine and is formed by hawthorn, Divine Comedy, the tuber of pinellia, Poria cocos, dried orange peel, fructus forsythiae, radish seed,
Middle hawthorn and dried orange peel are the main components in Pachyma Compound Digestive Tonic Pill, can increase gastrointestinal peristalsis, exclude the pneumatosis in alimentary canal.Hawthorn, which has, to disappear
Eat stomach invigorating, change turbid lipid-loweringing and other effects, gastrointestinal smooth muscle can be shunk, the activity of digestive enzyme is enhanced.Volatile oil in dried orange peel, tool
There is promoting the circulation of qi invigorating the spleen, the effect removed dampness by means of aromatics.
Different solvents it is distinct, so there is also obvious for ingredient and content of the same substance after different solvents extract
Difference, water and ethyl alcohol are as most common solvent, and there are significant differences for the polarity of the two, therefore to extract in extraction process
Ingredient and content have larger impact.
Summary of the invention
In order to overcome the drawbacks described above of the prior art, the embodiment of the present invention provides a kind of compound haw thorn Content of Chlorogenic Acid and orange
Skin glycosides assay and integrated evaluating method, by establishing the content assaying method of compound haw thorn Content of Chlorogenic Acid and aurantiamarin,
And compared the active constituent content of compound haw thorn water extract and alcohol extract and promote digestion effect, compound haw thorn water extract effectively at
Divide content and promote digestion effect and is better than alcohol extract.
To achieve the above object, the invention provides the following technical scheme: a kind of compound haw thorn Content of Chlorogenic Acid and aurantiamarin contain
Fixed and integrated evaluating method is measured, wherein the step of measuring:
The preparation of S1, compound haw thorn water extract and alcohol extract, specifically include:
The preparation of S1.1, water extract: weighing dried orange peel medicine materical crude slice, with steam distillation extraction method extract volatile oil, the dregs of a decoction again and
Hawthorn, which mixes, adds 10 times amount water refluxing extraction 90 minutes, extracts 2 times, merges medical fluid three times, constant volume after reduced pressure；
The preparation of S1.2, alcohol extract: weighing dried orange peel medicine materical crude slice, extracts volatile oil using steam distillation extraction method, the dregs of a decoction are again
It extracts 90 minutes, extracts 2 times with hawthorn plus 10 times of 70% alcohol refluxs of amount, merge medical fluid three times, be centrifuged to obtain supernatant, depressurize dense
Constant volume after contracting；
The preparation of S2, reference substance and test solution: precision weighs a certain amount of chlorogenic acid reference substance and aurantiamarin respectively
Reference substance is set in same 25ml volumetric flask, and 0.1207mg/ml containing chlorogenic acid, aurantiamarin 0.1017mg/ml is made with methanol dissolution
Solution as chlorogenic acid and aurantiamarin mixed reference substance solution, solution after taking compound haw thorn water extract and alcohol extract constant volume is made
For test solution, reference substance and test sample use 0.22 μm of filtering with microporous membrane；
S3, dry cream rate calculate: it is accurate respectively to draw extracting solution 10ml, it is placed in water bath method in evaporating dish, is transferred to 105 DEG C
Dry 3h in baking oven, accurately weighed weight W after taking-up is cooling into drier calculate dry cream rate:
S4, active constituent content measuring: water extract and 6 μ l of alcohol extract test sample sample introduction are surveyed respectively in setting chromatographic condition
Determine peak area, the content of compound haw thorn water extract and alcohol extract Content of Chlorogenic Acid and aurantiamarin is calculated according to equation of linear regression；
The preparation of S5, experimental subjects sample, specifically include:
S5.1, positive drug: by digestion-promoting stomachic mortar grinder at fine powder, the suspension of 0.133g/ml is made of water；
S5.2, model drug: removing the capsule shells of Loperamide capsule, and powder is dissolved with water, and modeling dosage is 5mg/
kg；
S5.3, prepared Chinese ink: weighing Arabic gum 10g, adds water 80ml, boils transparent to solution, weighs active carbon powder 10g and adds
To being boiled 3 times in above-mentioned solution, it is settled to 100ml after room temperature is cooling, is placed in 4 DEG C of refrigerators and saves, shake up before use.
S5.4, compound haw thorn water extract and alcohol extract solution are prepared: the extract after S1.1 constant volumes is concentrated under reduced pressure
It to 1.35g/ml containing crude drug amount, will extract in the medical fluid after resulting volatile oil adds to concentration, while be added quite into medical fluid
In the Tween 80 of medical fluid total volume 2%, uniformly mixed be placed in 4 DEG C of refrigerators is saved, before use with the aqueous solution for containing 2% Tween 80
It is diluted to administration concentration；
S6, acute toxicity testing: kunming mice 40, SD rat 40 are taken respectively, half male and half female, mouse, rat are pressed
Gender is randomly divided into 2 groups, and every group of male and female each 10, adaptable fed starts acute toxicity testing after 3 days, intragastric administration on mice is given
The water extract and alcohol extract of 108g/kg (pressing crude drug meter), rat oral gavage give the water extract and alcohol extract of 45g/kg, Suo Youdong
It is observed one week after object administration, periodically claims its weight daily and observe animal state；
S7, intestinal motility experiment: taking male mice 100, is divided into 10 groups by weight, respectively blank group, model group,
Positive group, tween group, the low middle high dose group of water extract and the low middle high dose group of alcohol extract, water extract and the low middle high dose of alcohol extract
Respectively 2.7g/kg, 13.5g/kg, 27g/kg (by crude drug meter), continuous gavage 15 days, daily timing stomach-filling was primary, blank
Group and model group give distilled water, and tween group gives 2% tween, and experiment terminates the same day, and administration group gives a drug, blank and
Model group gives primary distilled water, and tween group gives a tween, and three ten minutes later, and administration group, model group and tween group are given
Loperamide modeling, blank group give same volume distilled water, and three ten minutes later, and every group is given prepared Chinese ink, put to death after 25 minutes dynamic
Object opens rapidly abdominal cavity and separates mesenterium, and small intestine of the clip from pylorus to ileocecus is placed on blank sheet of paper, and it is complete to measure small intestine respectively
It grows and from pylorus to the distance in prepared Chinese ink forward position, calculates ink progradation and ink progradation conversion value；
Propulsion rate conversion value:P: mouse small intestine propulsion rate；
The measurement of S8, rat body weight, weight gain, food ration and food utilization: taking male SD rat, is divided into blank
Group, positive group, tween group, low middle high three dosage groups of water extract and low middle high three dosage groups of alcohol extract, every group 10, water mentions
Object and the low middle high dose of alcohol extract are respectively 0.95g/kg, 1.89g/kg, 9.45g/kg (by crude drug meter), and blank group, which is given, steams
Distilled water, the positive group of digestion-promoting stomachic for giving 2g/kg, tween group give the distilled water containing 2% Tween 80, and all animals continuously fill
Stomach 30d, daily timing stomach-filling is primary, is recorded twice rat body weight and food intake weekly, and according to changes of weight adjust to
Dose calculates weight gain, food ration and food utilization at the end of experiment；
Food utilization=body weight evolution/food ration × 100%；
The measurement of S9, rat pepsin, specifically include:
The preparation of S9.1, protein pipe: it is clear to be filled Fresh Egg with glass capillary, then makes egg white solidifying in the hot water
Gu to get protein pipe；
The measurement of S9.2, pepsin activity and discharge rate: by S8 method groupings and administration, with penta bar at the end of experiment
Than appropriate sodium anesthetized rat, Banded Rats pylorus collects 3h gastric juice and measures its volume, and 15ml is added by every 1ml in the gastric juice of collection
Hydrochloric acid is placed in centrifuge tube and mixes, and the fresh protein pipe prepared 2, sealing is added, and 37 DEG C of constant-temperature incubations take out afterwards for 24 hours, uses
The length of vernier caliper measurement every radixin pipe both ends transparent part, finds out the average value at four ends, according to pepsin activity list
Position formula calculates pepsin activity, calculates pepsin discharge rate according to pepsin discharge rate formula；
Pepsin activity unit (Uml-1The end protein pipe transparent part length mean value of)=tetra-2×16
Pepsin discharge rate (Uh-1)=pepsin activity × gastric juice amount/h；
S10, data statistic analysis: experiment the data obtained is with mean ± standard deviationIt indicates, data use
The analysis of 5.0 software statistics of Graphpad prism, two comparison among groups are examined using t, and more comparison among groups are using single factor test variance point
Analysis is with statistical significance with p < 0.05.
In a preferred embodiment, chromatographic condition is set as chromatography in the active constituent content measuring step
Column SHIMADZU Shim-pack GIST C18 (250 × 4.6mm, 5 μm)；Mobile phase A: acetonitrile, Mobile phase B: 0.1% phosphoric acid
Water；Elution requirement: 0-25min:10%A~17%A；25-26min:17%~20%A；26-50min:20%~25%A；Stream
Fast 1ml/min, 30 DEG C of column temperature, Detection wavelength: aurantiamarin 283nm, chlorogenic acid 327nm.
In a preferred embodiment, fasting 12h before all animals are administered in the acute toxicity testing step, just
Often drinking-water, 2 administrations of intragastric administration on mice point, every minor tick 6h, 3 administrations of rat oral gavage point, every minor tick 4h.
In a preferred embodiment, animal is deprived of food but not water before experiment terminates in the intestinal motility experimental procedure
16h。
In a preferred embodiment, by institute at the end of being tested in the measurement experiment step of the rat pepsin
There is Rat Fast to can't help water for 24 hours.
In a preferred embodiment in the preparation step of the protein pipe, glass capillary is set as internal diameter 1mm,
The length of 40mm is made in protein pipe by long 100mm before use.
Technical effect and advantage of the invention:
The present invention compared compound mountain by establishing the content assaying method of compound haw thorn Content of Chlorogenic Acid and aurantiamarin
The active constituent content of short, bristly hair or beard water extract and alcohol extract and rush digestion effect, are mentioned using high effective liquid chromatography for measuring compound haw thorn water
The content of object and alcohol extract Content of Chlorogenic Acid and aurantiamarin, and comprehensive score is carried out, pass through the urgency in health food Ergonomy research
Property toxicity, mouse small intestine movement, rat food ration, food utilization, pepsin measurement etc. Experimental comparisons' compound haw thorn water mention
The rush digestion effect of object and alcohol extract, the results showed that, compound haw thorn water extract active constituent content comprehensive score (93.78) is higher than
Alcohol extract (80.04), water extract and alcohol extract to rat, mouse without acute toxicity, can dramatically increase rat food ration (p <
0.05)；The middle and high dosage group of water extract and the high dose group mouse small intestine propulsion rate of alcohol extract improve 26.88%,
28.00%, 26.55% (p < 0.05, p < 0.01, p < 0.05), rat pepsin discharge rate increases 38.59%,
53.29%, 36.64% (p < 0.05, p < 0.01, p < 0.05), other dosage group Intestinal propulsive rates and rat stomach egg of alcohol extract
There was no significant difference (p >=0.05) for white enzyme discharge rate；The middle and high dosage group rat pepsin activity of water extract and alcohol extract increases
Strong 75.96%, 125.07%, 70.38%, 109.38% (p < 0.05, p < 0.01, p < 0.05, p < 0.01), compound haw thorn water
Extract active constituent content and rush digestion effect are better than alcohol extract.
Detailed description of the invention
Fig. 1 is the HPLC map of compound haw thorn water extract and alcohol extract of the invention.
Fig. 2 is compound haw thorn water extract in acute toxicity testing of the invention and alcohol extract to mouse (A) and rat (B) body
The influence diagram of quality.
Fig. 3 is the influence diagram of compound haw thorn water extract and alcohol extract of the invention to mouse small intestine motor function.
Fig. 4 is compound haw thorn water extract and alcohol extract of the invention to rat pepsin activity stomach function regulating protease discharge rate
Influence diagram.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
The present invention provides a kind of compound haw thorn Content of Chlorogenic Acid and content of hesperidin measurement and integrated evaluating methods, wherein surveying
Fixed step:
The preparation of S1, compound haw thorn water extract and alcohol extract, specifically include:
The preparation of S1.1, water extract: the preparation for preparing water extract of compound haw thorn water extract and alcohol extract: dried orange peel drink is weighed
Piece extracts volatile oil with steam distillation extraction method, and the dregs of a decoction are mixed with hawthorn again adds 10 times amount water refluxing extraction 90 minutes, extracts
2 times, merge medical fluid three times, constant volume after reduced pressure；
The preparation of S1.2, alcohol extract: the preparation of alcohol extract: weighing dried orange peel medicine materical crude slice, is extracted using steam distillation extraction method
Volatile oil, the dregs of a decoction add 10 times of 70% alcohol refluxs of amount to extract 90 minutes with hawthorn again, extract 2 times, merge medical fluid three times, are centrifuged
Supernatant, constant volume after reduced pressure；
The preparation of S2, reference substance and test solution: precision weighs a certain amount of chlorogenic acid reference substance and aurantiamarin respectively
Reference substance is set in same 25ml volumetric flask, and 0.1207mg/ml containing chlorogenic acid, aurantiamarin 0.1017mg/ml is made with methanol dissolution
Solution as chlorogenic acid and aurantiamarin mixed reference substance solution, solution after taking compound haw thorn water extract and alcohol extract constant volume is made
For test solution, reference substance and test sample use 0.22 μm of filtering with microporous membrane；
S3, dry cream rate calculate: it is accurate respectively to draw extracting solution 10ml, it is placed in water bath method in evaporating dish, is transferred to 105 DEG C
Dry 3h in baking oven, accurately weighed weight W after taking-up is cooling into drier calculate dry cream rate:
S4, active constituent content measuring: water extract and 6 μ l of alcohol extract test sample sample introduction are surveyed respectively in setting chromatographic condition
Determine peak area (analysis the result is shown in Figure 1), reference substance and test sample HPLC map reach baseline and be kept completely separate, and each chromatographic peak does not occur
Obvious overlapping or hangover, the good (high-efficient liquid phase chromatogram of A, B chlorogenic acid and aurantiamarin mixing reference substance of separating degree；C, D compound
The high-efficient liquid phase chromatogram of hawthorn water extract test sample；E, the high-efficient liquid phase chromatogram of F compound haw thorn alcohol extract test sample；1: green
Ortho acid, 2: aurantiamarin；A, C, E 326nm, B, D, F 283nm), according to equation of linear regression calculate compound haw thorn water extract and
The content of alcohol extract Content of Chlorogenic Acid and aurantiamarin sets chromatographic condition as chromatographic column SHIMADZU Shim-pack GIST C18
(250 × 4.6mm, 5 μm)；Mobile phase A: acetonitrile, Mobile phase B: 0.1% phosphoric acid water；Elution requirement: 0-25min:10%A~
17%A；25-26min:17%~20%A；26-50min:20%~25%A；Flow velocity 1ml/min, detects wave by 30 DEG C of column temperature
It is long: aurantiamarin 283nm, chlorogenic acid 327nm；
By S3 method measurement dry cream rates and by S4 content assaying methods respectively to compound haw thorn water extract and alcohol extract
Content of Chlorogenic Acid and aurantiamarin carry out assay, and acquired results are shown in Table 1:
The assay of table 1 compound haw thorn water extract and alcohol extract Content of Chlorogenic Acid and aurantiamarin
The result shows that water extract Content of Chlorogenic Acid and content of hesperidin are respectively 0.039% and 1.537%, dry cream rate is
48.48%；Alcohol extract Content of Chlorogenic Acid and content of hesperidin are respectively 0.037% and 2.048%, and dry cream rate is 21.44%；To water
The chlorogenic acid of extract and alcohol extract, content of hesperidin and dry cream rate carry out comprehensive score, the results showed that water extract comprehensive score
(93.78) higher by 17.2% than alcohol extract (80.04).
The preparation of S5, experimental subjects sample, specifically include:
S5.1, positive drug: by digestion-promoting stomachic mortar grinder at fine powder, the suspension of 0.133g/ml is made of water；
S5.2, model drug: removing the capsule shells of Loperamide capsule, and powder is dissolved with water, and modeling dosage is 5mg/
kg；
S5.3, prepared Chinese ink: weighing Arabic gum 10g, adds water 80ml, boils transparent to solution, weighs active carbon powder 10g and adds
To being boiled 3 times in above-mentioned solution, it is settled to 100ml after room temperature is cooling, is placed in 4 DEG C of refrigerators and saves, shake up before use.
S5.4, compound haw thorn water extract and alcohol extract solution are prepared: the extract after S1.1 constant volumes is concentrated under reduced pressure
It to 1.35g/ml containing crude drug amount, will extract in the medical fluid after resulting volatile oil adds to concentration, while be added quite into medical fluid
In the Tween 80 of medical fluid total volume 2%, uniformly mixed be placed in 4 DEG C of refrigerators is saved, before use with the aqueous solution for containing 2% Tween 80
It is diluted to administration concentration；
S6, acute toxicity testing: kunming mice 40, SD rat 40 are taken respectively, half male and half female, mouse, rat are pressed
Gender is randomly divided into 2 groups, and every group of male and female each 10, preceding fasting 12h, normal water, after adaptable fed 3 days is administered in all animals
Start acute toxicity testing, intragastric administration on mice gives the 108g/kg water extract and alcohol extract of (by crude drug meter), intragastric administration on mice point 2
Secondary administration, every minor tick 6h, rat oral gavage give the water extract and alcohol extract of 45g/kg, 3 administrations of rat oral gavage point, between each
It every 4h, is observed one week after all animals administrations, periodically claim its weight daily and observes animal state (acute toxicity testing result is shown in
Fig. 2, whereinN=20)；
The acute toxicity testing of compound haw thorn water extract and alcohol extract is as the result is shown: mouse, rat be not in 7 days after administration
See poisoning manifestations, the growth of mouse weight is had no significant effect, the behavior of animal and appearance are showed no exception, without small in the observation period
Mouse, rats death illustrate compound haw thorn water extract and alcohol extract to mouse maximal tolerance dose > 108g/kg, to rat MTD > 45g/
Kg, according to acute toxicity dose grading standard, the water extract and alcohol extract of the sample belong to nontoxic grade；
S7, intestinal motility experiment: taking male mice 100, is divided into 10 groups by weight, respectively blank group, model group,
Positive group, tween group, the low middle high dose group of water extract and the low middle high dose group of alcohol extract, water extract and the low middle high dose of alcohol extract
Respectively 2.7g/kg, 13.5g/kg, 27g/kg (by crude drug meter), continuous gavage 15 days, daily timing stomach-filling was primary, blank
Group and model group give distilled water, and tween group gives 2% tween, and animal is deprived of food but not water 16h before experiment terminates, and experiment terminates to work as
It, administration group gives a drug, and blank and model group give primary distilled water, and tween group gives a tween, and 30 minutes
Afterwards, administration group, model group and tween group give Loperamide modeling, and blank group gives same volume distilled water, and three ten minutes later, often
Group gives prepared Chinese ink, puts to death animal after 25 minutes, opens abdominal cavity rapidly and separates mesenterium, clip is small from pylorus to ileocecus
Intestines are placed on blank sheet of paper, are measured small intestine overall length respectively and from pylorus to the distance in prepared Chinese ink forward position, are calculated ink progradation and prepared Chinese ink pushes away
Into rate conversion value；
Propulsion rate conversion value:P: mouse small intestine propulsion rate；
Indigestion is related with gastric emptying and enterocinesia, therefore can be by measuring intestinal motility functional evaluation digestive function shape
State, Loperamide are clinical common antidiarrheal agent, and it is right that excessive use, which then can lead to constipation, can be used for constructing indigestion model,
Influence than compound haw thorn water extract and alcohol extract to mouse small intestine motor function uses Lip river after 15d is administered to mouse continuous gavage
Piperazine butylamine constructs indigestion model, digests effect by the rush that measurement ink progradation evaluates compound haw thorn water extract and alcohol extract
Fruit (result such as Fig. 3, wherein### indicates p < 0.001 compared with blank group；* the p compared with model group is indicated
<0.05；* indicates p < 0.01 compared with model group), compared with blank group (59.27 ± 5.09) %, model group Intestinal propulsive rate
(39.25 ± 5.92) % reduces 33.78%, has statistical significance (p < 0.001), shows the indigestion model of this experiment
It constructs successfully；Compared with model group, tween group (40.82 ± 4.76) % is had no notable difference (p >=0.05), positive group (56.55
± 5.53) %, middle dose group (49.8 ± 3.88) % of compound haw thorn water extract, high dose group (50.24 ± 8.19) % are significant
It improves Intestinal propulsive rate (p < 0.05), has been respectively increased 44.08%, 26.88%, 28.00% relative to model group, and alcohol extract
Intestinal propulsive rate (p < 0.05) only is significantly improved in high dose (49.67 ± 4.19) %, is improved relative to model group
26.55%, and water extract middle dose group has statistical significance (p < 0.05) compared with alcohol extract middle dose group；
After Intestinal propulsive rate is converted, compared with blank group (0.88 ± 0.05), model group (0.68 ± 0.06) small intestine turns
Changing value reduces 22.72%, has statistical significance (p < 0.001), shows this experiment Constipation Model modeling success；With model group
Comparison, tween group (0.69 ± 0.05) small intestine conversion value no significant difference (p >=0.05), positive group of (0.85 ± 0.06), compound
Hawthorn water extract middle dose group (0.78 ± 0.04), water extract high dose group (0.80 ± 0.08), alcohol extract high dose group (0.82
± 0.07) have statistical difference (p < 0.05) with model group, Intestinal propulsive rate (43.88 ± 5.10) % of alcohol extract middle dose group
With small intestine push conversion value (0.75 ± 0.07) no difference of science of statistics (p >=0.05), it is small to show that compound haw thorn water extract promotes
The effect of bowel movement is better than alcohol extract；
The measurement of S8, rat body weight, weight gain, food ration and food utilization: taking male SD rat, is divided into blank
Group, positive group, tween group, low middle high three dosage groups of water extract and low middle high three dosage groups of alcohol extract, every group 10, water mentions
Object and the low middle high dose of alcohol extract are respectively 0.95g/kg, 1.89g/kg, 9.45g/kg (by crude drug meter), and blank group, which is given, steams
Distilled water, the positive group of digestion-promoting stomachic for giving 2g/kg, tween group give the distilled water containing 2% Tween 80, and all animals continuously fill
Stomach 30d, daily timing stomach-filling is primary, is recorded twice rat body weight and food intake weekly, and according to changes of weight adjust to
Dose calculates weight gain, food ration and food utilization at the end of experiment；
Food utilization=body weight evolution/food ration × 100%；
Gastric emptying time extension is dyspeptic another classical symptom, and indigestion causes appetite stimulator, food ration bright
It is aobvious to reduce, thus lead to weight loss, food utilization decline；Conversely, the more good then food ration of digestive function, weight gain more
Greatly, food utilization is higher, and continuous gavage gives rat compound haw thorn water extract or alcohol extract 30 days, by measurement food ration,
Weight gain, food utilization evaluation compound haw thorn water extract and alcohol extract rush digestion effect (result such as table 5, wherein
N=10), positive group, compound haw thorn water extract and alcohol extract increase weight to rat body weight and food utilization all has no significant effect (p
>=0.05), but can dramatically increase food ration (p<0.05), compared to the blank group, tween group food ration without significant changes (p>=
0.05), positive group food ration increases 10.23% (p < 0.05), and water extract low, middle and high dose groups food ration increases separately
9.53%, 11.93%, 12.54%, alcohol extract low, middle and high dose groups food ration increased separately 10.22%, 13.4%,
14.97%, water extract equal no difference of science of statistics (p >=0.05) compared between alcohol extract same dose group, show water extract with
The effect that alcohol extract increases food ration is suitable；
Note: * indicates p < 0.05 compared with blank group；* indicates p < 0.01 compared with blank group；
The measurement of S9, rat pepsin, specifically include:
The preparation of S9.1, protein pipe: internal diameter 1mm, it is clear that the glass capillary of long 100mm is filled Fresh Egg, then in heat
It solidifies egg white to get protein pipe, is put into 4 DEG C of refrigerators and saves, the length of 40mm is made before use；
The measurement of S9.2, pepsin activity and discharge rate: by S8 method groupings and administration, with penta bar at the end of experiment
Than appropriate sodium anesthetized rat, Banded Rats pylorus collects 3h gastric juice and measures its volume, and 15ml is added by every 1ml in the gastric juice of collection
Hydrochloric acid is placed in centrifuge tube and mixes, and the fresh protein pipe prepared 2, sealing is added, and 37 DEG C of constant-temperature incubations take out afterwards for 24 hours, uses
The length of vernier caliper measurement every radixin pipe both ends transparent part, finds out the average value at four ends, according to pepsin activity list
Position formula calculates pepsin activity, calculates pepsin discharge rate according to pepsin discharge rate formula；
Pepsin activity unit (Uml-1The end protein pipe transparent part length mean value of)=tetra-2×16
Pepsin discharge rate (Uh-1)=pepsin activity × gastric juice amount/h；
The rush of compound haw thorn water extract and alcohol extract is compared by measurement pepsin activity and pepsin discharge rate
(result is as shown in figure 4, wherein for digestion effectN=10, * indicate p < 0.05 compared with blank group；* is indicated and blank group
Compare p < 0.01): pepsin activity (167.2 ± 51.62) Uml with blank group-1With pepsin discharge rate (76.97
±47.66)U·h-1It compares, pepsin activity (157.18 ± 41.26) Uml of tween group-1With pepsin discharge rate
(71.63±39.93)U·h-1It has no significant effect (p >=0.05), positive group of (311.91 ± 78.02) Uml-1, compound haw thorn
Water extract and alcohol extract middle and high dosage (294.21 ± 48.07,376.31 ± 76.76,284.87 ± 45.18,350.08 ±
64.89)U·ml-1Pepsin activity significantly increases (p < 0.05), pepsin activity enhances 86.55% respectively,
75.96%, 125.07%, 70.38%, 109.38%；Positive group of (106.48 ± 42.17) Uh-1, the middle and high dosage of water extract
Group and alcohol extract high dose group (106.67 ± 35.19,117.99 ± 42.08,105.17 ± 45.09) Uh-1Pepsin row
Output dramatically increases (p < 0.05), and pepsin discharge rate has increased separately 38.34%, 38.59%, 53.29%, 36.64%,
Show that compound haw thorn water extract and alcohol extract can significantly increase pepsin activity, the two effect is suitable；Water extract increases stomach
The minimum effective dose of protease discharge rate is the medium dosage of this experiment, and the minimum effective dose of alcohol extract is that it is high
Dosage, therefore water extract increases the effect of pepsin discharge rate better than alcohol extract；
S10, data statistic analysis: experiment the data obtained is with mean ± standard deviationIt indicates, data use
The analysis of 5.0 software statistics of Graphpad prism, two comparison among groups are examined using t, and more comparison among groups are using single factor test variance point
Analysis is with statistical significance with p < 0.05.
It is obtained in summary as drawn a conclusion:
Assay result and comprehensive score show water extract Content of Chlorogenic Acid and content of hesperidin is respectively 0.039% He
1.537%, dry cream rate is 48.48%；Alcohol extract Content of Chlorogenic Acid and content of hesperidin are respectively 0.037% and 2.048%, dry cream
Rate is 21.44%；The content height difference of chlorogenic acid and aurantiamarin in water extract and alcohol extract may be due to water and ethyl alcohol
Polarity it is different, show that compound haw thorn water extract score (93.78) is higher than by the comprehensive score of active constituent content and medicinal extract rate
Alcohol extract (80.04)；
Acute toxicity testing shows compound haw thorn water extract and alcohol extract is nontoxic rank, to mouse, Growth in Rats without
It influences, can successfully construct mouse indigestion model (p < 0.001) using Loperamide, compound haw thorn water extract improves mouse
The effect of gastrointestinal motility function is better than alcohol extract (p < 0.05), by rat food ration, gastric secretion stomach function regulating protease activity
The measurement of property, the results showed that the compound haw thorn water extract and alcohol extract of basic, normal, high dosage can dramatically increase rat food ration
(p < 0.05, p < 0.01, p < 0.01, p < 0.05, p < 0.01, p < 0.01), and the two effect is suitable；The water extract of middle and high dosage
It is dramatically increased pepsin activity (p < 0.05, p < 0.01) with alcohol extract, water extract, which increases the minimum of pepsin discharge rate, to be had
It imitates dosage and is lower than alcohol extract, therefore, in terms of comprehensive, compound haw thorn water extract promotees digestion effect and is better than alcohol extract；
Compound haw thorn preparation is because it promotees the characteristics of digestive function is defined, had no toxic side effect, using wide in clinical and life
It is general, it is mentioned by comparing compound haw thorn water and the active constituent content of two kinds of extracts of alcohol extracting and promotees digestion effect, to be related
Novel product exploitation provides more preferably extracting method and pharmacodynamics foundation.
Finally, it should be noted that the foregoing is only a preferred embodiment of the present invention, it is not intended to restrict the invention,
All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in of the invention
Within protection scope.
Claims (6)
1. a kind of compound haw thorn Content of Chlorogenic Acid and content of hesperidin measurement and integrated evaluating method, it is characterised in that: wherein measure
The step of:
The preparation of S1, compound haw thorn water extract and alcohol extract, specifically include:
The preparation of S1.1, water extract: weighing dried orange peel medicine materical crude slice, extracts volatile oil with steam distillation extraction method, the dregs of a decoction are again and hawthorn
It mixes and adds 10 times amount water refluxing extraction 90 minutes, extract 2 times, merge medical fluid three times, constant volume after reduced pressure；
The preparation of S1.2, alcohol extract: weighing dried orange peel medicine materical crude slice, extracts volatile oil using steam distillation extraction method, the dregs of a decoction are again and mountain
Short, bristly hair or beard adds 10 times of 70% alcohol refluxs of amount to extract 90 minutes, extracts 2 times, merges medical fluid three times, supernatant is centrifuged to obtain, after reduced pressure
Constant volume；
The preparation of S2, reference substance and test solution: precision weighs a certain amount of chlorogenic acid reference substance and aurantiamarin control respectively
Product are set in same 25ml volumetric flask, 0.1207mg/ml containing chlorogenic acid are made with methanol dissolution, aurantiamarin 0.1017mg/ml's is molten
Liquid is as chlorogenic acid and aurantiamarin mixed reference substance solution, and the solution after taking compound haw thorn water extract and alcohol extract constant volume is as confession
Test sample solution, reference substance and test sample use 0.22 μm of filtering with microporous membrane；
S3, dry cream rate calculate: it is accurate respectively to draw extracting solution 10ml, it is placed in water bath method in evaporating dish, is transferred to 105 DEG C of baking ovens
Middle dry 3h, accurately weighed weight W after taking-up is cooling into drier, calculates dry cream rate:
S4, active constituent content measuring: peak is measured to water extract and 6 μ l of alcohol extract test sample sample introduction respectively in setting chromatographic condition
Area calculates the content of compound haw thorn water extract and alcohol extract Content of Chlorogenic Acid and aurantiamarin according to equation of linear regression；
The preparation of S5, experimental subjects sample, specifically include:
S5.1, positive drug: by digestion-promoting stomachic mortar grinder at fine powder, the suspension of 0.133g/ml is made of water；
S5.2, model drug: removing the capsule shells of Loperamide capsule, and powder is dissolved with water, and modeling dosage is 5mg/kg；
S5.3, prepared Chinese ink: weighing Arabic gum 10g, adds water 80ml, boils transparent to solution, weighs active carbon powder 10g and adds to
It states and is boiled in solution 3 times, be settled to 100ml after room temperature is cooling, be placed in 4 DEG C of refrigerators and save, shake up before use；
S5.4, compound haw thorn water extract and alcohol extract solution are prepared: the extract after S1.1 constant volumes being concentrated under reduced pressure into and is contained
Crude drug amount 1.35g/ml will be extracted in the medical fluid after resulting volatile oil adds to concentration, while is added into medical fluid and be equivalent to medicine
The Tween 80 of liquid total volume 2%, uniformly mixed be placed in 4 DEG C of refrigerators save, are diluted before use with the aqueous solution containing 2% Tween 80
To administration concentration；
S6, acute toxicity testing: kunming mice 40, SD rat 40 are taken respectively, half male and half female, mouse, rat press gender
2 groups are randomly divided into, every group of male and female each 10, adaptable fed starts acute toxicity testing after 3 days, intragastric administration on mice gives 108g/
The water extract and alcohol extract of kg (pressing crude drug meter), rat oral gavage give the water extract and alcohol extract of 45g/kg, and all animals are given
It is observed one week after medicine, periodically claim its weight daily and observes animal state；
S7, intestinal motility experiment: male mice 100 are taken, is divided into 10 groups, respectively blank group, model group, the positive by weight
The low middle high dose group of group, tween group, water extract and the low middle high dose group of alcohol extract, water extract and the low middle high dose difference of alcohol extract
For (press crude drug meter) 2.7g/kg, 13.5g/kg, 27g/kg, continuous gavage 15 days, daily timing stomach-filling was primary, blank group and
Model group gives distilled water, and tween group gives 2% tween, and experiment terminates the same day, and administration group gives a drug, blank and model
Group gives primary distilled water, and tween group gives a tween, and three ten minutes later, and administration group, model group and tween group give Lip river piperazine
Butylamine modeling, blank group give same volume distilled water, and three ten minutes later, and every group is given prepared Chinese ink, and animal is put to death after 25 minutes, fast
Speed opens abdominal cavity and separates mesenterium, and small intestine of the clip from pylorus to ileocecus be placed on blank sheet of paper, measure respectively small intestine overall length and from
Pylorus calculates ink progradation and ink progradation conversion value to the distance in prepared Chinese ink forward position；
Propulsion rate conversion value:P: mouse small intestine propulsion rate；
The measurement of S8, rat body weight, weight gain, food ration and food utilization: taking male SD rat, is divided into blank group, sun
Property group, tween group, low middle high three dosage groups of water extract and low middle high three dosage groups of alcohol extract, every group 10, water extract and
The low middle high dose of alcohol extract is respectively 0.95g/kg, 1.89g/kg, 9.45g/kg (by crude drug meter), and blank group, which is given, distills
Water, the positive group of digestion-promoting stomachic for giving 2g/kg, tween group give the distilled water containing 2% Tween 80, all animal continuous gavages
30d, daily timing stomach-filling is primary, is recorded twice rat body weight and food intake weekly, and adjust and be administered according to changes of weight
Amount calculates weight gain, food ration and food utilization at the end of experiment；
Food utilization=body weight evolution/food ration × 100%；
The measurement of S9, rat pepsin, specifically include:
The preparation of S9.1, protein pipe: it is clear that Fresh Egg is filled with glass capillary, then solidifies egg white in the hot water, i.e.,
Obtain protein pipe；
The measurement of S9.2, pepsin activity and discharge rate: by S8 method groupings and administration, amobarbital is used at the end of experiment
Sodium anesthetized rat, Banded Rats pylorus collect 3h gastric juice and measure its volume, and 15ml hydrochloric acid is added by every 1ml in the gastric juice of collection
It is placed in centrifuge tube and mixes, the fresh protein pipe prepared 2, sealing is added, 37 DEG C of constant-temperature incubations take out afterwards for 24 hours, use vernier
The length of calliper to measure every radixin pipe both ends transparent part, finds out the average value at four ends, public according to pepsin activity unit
Formula calculates pepsin activity, calculates pepsin discharge rate according to pepsin discharge rate formula；
Pepsin activity unit (Uml-1The end protein pipe transparent part length mean value of)=tetra-2×16
Pepsin discharge rate (Uh-1)=pepsin activity × gastric juice amount/h；
S10, data statistic analysis: experiment the data obtained is with mean ± standard deviationIt indicates, data use Graphpad
The analysis of 5.0 software statistics of prism, two comparison among groups are examined using t, more comparison among groups using one-way analysis of variance, with p <
0.05 is with statistical significance.
2. a kind of compound haw thorn Content of Chlorogenic Acid according to claim 1 and content of hesperidin measurement and integrated evaluating method,
It is characterized by: setting chromatographic condition as chromatographic column SHIMADZU Shim-pack in the active constituent content measuring step
GIST C18 (250 × 4.6mm, 5 μm)；Mobile phase A: acetonitrile, Mobile phase B: 0.1% phosphoric acid water；Elution requirement: 0-25min:
10%A~17%A；25-26min:17%~20%A；26-50min:20%~25%A；Flow velocity 1ml/min, 30 DEG C of column temperature,
Detection wavelength: aurantiamarin 283nm, chlorogenic acid 327nm.
3. a kind of compound haw thorn Content of Chlorogenic Acid according to claim 1 and content of hesperidin measurement and integrated evaluating method,
It is characterized by: fasting 12h before all animals are administered in the acute toxicity testing step, normal water, intragastric administration on mice point 2 times
Administration, every minor tick 6h, 3 administrations of rat oral gavage point, every minor tick 4h.
4. a kind of compound haw thorn Content of Chlorogenic Acid according to claim 1 and content of hesperidin measurement and integrated evaluating method,
It is characterized by: animal is deprived of food but not water 16h before experiment terminates in the intestinal motility experimental procedure.
5. a kind of compound haw thorn Content of Chlorogenic Acid according to claim 1 and content of hesperidin measurement and integrated evaluating method,
It is characterized by: all Rat Fasts can't help water for 24 hours at the end of testing in the measurement experiment step of the rat pepsin.
6. a kind of compound haw thorn Content of Chlorogenic Acid according to claim 1 and content of hesperidin measurement and integrated evaluating method,
It is characterized by: glass capillary is set as internal diameter 1mm, long 100mm, before use by egg in the preparation step of the protein pipe
The length of 40mm is made in white pipe.
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