CN105963389B

CN105963389B - The separation method of anti-high altitude anoxia anti-fatigue activity ingredient and its application in cape jasmine

Info

Publication number: CN105963389B
Application number: CN201610305926.0A
Authority: CN
Inventors: 李茂星; 王先敏; 毛婷; 曹馨元; 马幸福
Original assignee: Individual
Current assignee: Ninety-Fourth O Hospital Of Joint Logistics Support Force Of Chinese People's Liberation Army
Priority date: 2016-05-10
Filing date: 2016-05-10
Publication date: 2019-05-24
Anticipated expiration: 2036-05-10
Also published as: CN105963389A

Abstract

The present invention relates to a kind of separation methods of high altitude anoxia anti-fatigue activity ingredient anti-in cape jasmine, method includes the following steps: after (1) mentioning cape jasmine fruit water, through filtering, concentration, drying to get gardenia powder；(2) gardenia powder is configured to aqueous solution；(3) by step, (2) resulting aqueous solution injects processed polyamide column, then with distilled water flushing, respectively obtains polyamide column loading efflux, water elution；(4) processed macroporous resin column is injected after merging polyamide loading efflux and water elution, and to get Gardenoside product after elution, reduced pressure, freeze-drying；First with 10 ~ 30% ethanol solution rinse polyamide column removal of impurities, then with 30 ~ 90% ethanol solution flushing polyamide column, obtain 30 ~ 90% ethanol eluate, the eluent be concentrated under reduced pressure, be freeze-dried after to get Gardenia Yellow product.Meanwhile the application of the disclosed Gardenia Yellow of the present invention.The invention is simple and feasible, easy to operate, can industrialized production.

Description

The separation method of anti-high altitude anoxia anti-fatigue activity ingredient and its application in cape jasmine

Technical field

The present invention relates to high altitude anoxia anti-fatigue activity anti-in the process of enriching of Chinese herbal medicine effective ingredients more particularly to cape jasmine at The separation method divided and its application.

Background technique

After people from Plain enters plateau, due to the environmental factors such as highlands rarefaction of air, atmosphere force down, partial pressure of oxygen is low, directly Connect the physical strength and military operation ability for influencing body.Wherein, influence of the Altitude to body is the most obvious, in high altitude anoxia ring After being engaged in certain physical strength or mental labour under border, blood lactase acid and other metabolins is easily caused to accumulate, and cause body generation Thanking property acid-base disturbance is generally reduced so that muscle tone declines and cause the generation of fatigue phenomenon to show labour capacity, Serious person loses work capacity.With the promotion of height above sea level, labour capacity is substantially reduced.Therefore, try to explore tired with resisting The medicine of work is product highly important, that screening effectively prevents and relieves fatigue, for improving altitude environment lower body Physical work capacity, relieve fatigue for quality of making the life better, improve working efficiency and military operation ability have it is positive Meaning.

Cape jasmine be madder wort cape jasmine (Gardenia jasminoidesEllis dry mature fruit), for clinic Conventional Chinese medicine.Its bitter cold in nature, it is nontoxic, enter the heart, liver, lung, tri-jiao channel, with purging intense heat relieving restlessness, reducing fever and causing diuresis, removing pattogenic heat from the blood and toxic material from the body function Effect.Pharmaceutical research show it is extensive with the pharmacological action of cape jasmine, have liver protection, cholagogue, analgesia, anti-inflammatory, antibacterial, it is antitumor, drop A variety of pharmacological activity such as blood pressure and blood lipoid are Chinese medicine compositions that is of crucial importance and having very great development utility value.Wherein Cape jasmine The main active constituent crocin of son, while being also the main active substances of Tibetan medicine west safflower, there is anti-oxidant, anti-artery congee Sample hardening and the effect for adjusting blood lipid are a kind of unique natural carotenoid pigments of nature, by crocetin (crocetin) it is combined into two molecule gentiobioses, including crocin-I and crocin-II, warp with higher Ji value, is widely used in food, medicine and cosmetic field.Some researches show that crocin and crocetin have Free radical and significant ground antioxidation are removed, and in the pharmacological mechanism of carotenoid, antioxidant activity is especially heavy It wants, and it has been reported that the resist oxygen lack energy of animal can be improved in the drug with Scavenger of ROS, free radical and antioxidation Power mitigates hypoxic insult.

Since west safflower yield is extremely low, the market price is higher, and Gardenia Yellow and west safflower it is having the same activity at Divide crocin, therefore, it is necessary to carry out science to Gardenia Yellow, effectively develop.

Summary of the invention

Technical problem to be solved by the invention is to provide anti-high altitude anoxia anti-fatigue activities in a kind of simple and easy cape jasmine The separation method of ingredient.

Technical problem to be solved by the invention is to provide the applications of high altitude anoxia anti-fatigue activity ingredient anti-in the cape jasmine.

To solve the above problems, in cape jasmine of the present invention anti-high altitude anoxia anti-fatigue activity ingredient separation method, packet Include following steps:

(1) after drying, the cape jasmine fruit distillation water boiling and extraction of 20 ~ 40 mesh being crushed to, through filtering, reduced pressure, spray Mist is dry to get gardenia powder；

(2) the gardenia powder is configured to the aqueous solution of 20 ~ 40 mg/mL with 25 DEG C of distilled water；

(3) by the step, (2) resulting aqueous solution presses 1.0 ~ 3.0 times of column volume with the flow velocity injection of 1.5 ~ 15mL/min Processed 14 ~ 30 mesh polyamide column of the cm of 8cm × 100, then 1.0 ~ 3.0 times of column volume is pressed with the stream of 5.0 ~ 10.0 mL/min Speed distilled water flushing polyamide column, respectively obtains polyamide column loading efflux, water elution；

(4) after polyamide loading efflux and water elution being merged, amalgamation liquid with the flow velocity whole injection of 10mL/min at The macroporous resin column managed, first by 1.0 ~ 3.0 times of column volume with the flow velocity of 5.0 ~ 10.0 mL/min distilled water flushing macropore Resin column, then press the ethyl alcohol that 2.0 ~ 10.0 times of column volume is 10 ~ 70% with the flow velocity volumetric concentration of 1.5 ~ 10.0 mL/min Solution rinses macroporous resin column, obtains 10 ~ 70% ethanol eluate, which is concentrated under reduced pressure, is freezed To get Gardenoside product after drying；

(5) pressing 1.0 ~ 3.0 times of column volume first with the flow velocity volumetric concentration of 5.0 ~ 10.0mL mL/min is 10 ~ 30% Ethanol solution rinses polyamide column removal of impurities, then by 2.0 ~ 10.0 times of column volume with the flow velocity volume of 1.5 ~ 10.0 mL/min The ethanol solution that concentration is 30 ~ 90% rinses the polyamide column, obtains 30 ~ 90% ethanol eluate, 30 ~ 90% ethyl alcohol Eluent be concentrated under reduced pressure, be freeze-dried after to get Gardenia Yellow product.

(1) the middle extraction conditions that decoct refer to that feed liquid mass ratio is 1:8 ~ 10 to the step, and temperature is 90 ~ 100 DEG C, number 2 ~ 3 times, each time is 1 hour.

The step is (1), (4) (5) the middle condition being concentrated under reduced pressure each means that temperature is 50 DEG C ~ 80 to the step with the step ℃。

(1) the middle condition being spray-dried refers to that temperature is 120 ~ 140 DEG C to the step, 20 ~ 30 m of unit time intake³, 0.1 ~ 0.4MPa of atomisation pressure.

(4) (5) the middle condition being freeze-dried each means that temperature is -55 DEG C ~ 20 DEG C to the step with the step, and vacuum degree is 0.2MPa~0.5 MPa。

(4) middle macroporous resin column refers to D-101, HPD100, XDA-6, DM-130, DA201 type macroreticular resin to the step In any one.

The step (3) in processed polyamide column and the step (4) in processed macroporous resin column each mean The ethanol elution for being first 95% with volumetric concentration, until ethanol eluate adds distilled water muddiness do not occur, then to distill washing De- pillar to no alcohol taste to get.

The step (3) in processed polyamide column size be the cm of 8cm × 100.

The obtained Gardenia Yellow of separation method of anti-high altitude anoxia anti-fatigue activity ingredient is made in cape jasmine as described above For the application of Substituted phenyl-lactic acid ingredient in medicine, it is characterised in that: using Gardenia Yellow as active constituent, be equipped with medicinal stone Any dosage form being prepared into pharmacy；Or using Gardenia Yellow as active constituent, anti-oxidant and/or antifatigue work is added Property is equipped with any dosage form that pharmaceutic adjuvant is prepared into pharmacy at being grouped as compound.

The dosage form refers to hard capsule, soft capsule, ordinary tablet, sugar coated tablet, Film coated tablets, special-shaped tablets, chewable tablets, effervesce Piece, dispersible tablet, granule, pill, solution, any one in syrup.

The obtained Gardenia Yellow of separation method of anti-high altitude anoxia anti-fatigue activity ingredient is made in cape jasmine as described above For application of anti-oxidant, anti-fatigue effect the active constituent in terms of preparing anti-oxidant or anti-fatigue health-product containing.

Compared with the prior art, the present invention has the following advantages:

1, the Gardenia Yellow that extracts of the present invention as Substituted phenyl-lactic acid ingredient drug effect can from following 4 test in body It is existing:

Test 1 active component of cape jasmine acute toxicity testing

(1) experiment purpose: screening active component of cape jasmine toxic component.

(2) experiment condition: three-level Animal Lab., 20 ~ 25 DEG C of room temperature, air-conditioning constant temperature.

(3) experimental subjects: animal BalB/C mouse 90, half male and half female, 20 ± 2g of weight are always cured by Lanzhou military region Lanzhou Zoopery section, institute provides, quality certification number: curing dynamic word the 1402-0201st in Gansu Province.

(4) medicinal material and instrument: cape jasmine water extract, cape jasmine polysaccharide, Gardenia Yellow, Gardenoside are made by oneself by laboratory, face use Before 0.1g/mL aqueous solution is made；4% paraformaldehyde (lot number: 20150727 Beijing Suo Laibao Science and Technology Ltds) sterile injection is used Water (Kelun Pharm Ind Co., Ltd., Sichuan, lot number: M15111005).

Optical microscopy (Japanese Nikon company, E200)；Histotome (German Meikang company, HM340E)；TB- The automatic embedding machine of 718E type biological tissue (Hubei Tai Ya Electron Technology Co., Ltd).

(5) experimental method

1. 50 BALB/C mices (half male and half female) are randomly divided into blank control group, and (0.1mL/10g sterile injection is used Water), cape jasmine water extract (1.0g.kg-1.d-1), Gardenia Yellow group (1.0g.kg-1.d-1), cape jasmine multiple groups (1.0g.kg- 1.d-1), Gardenoside (1.0g.kg-1.d-1), gastric infusion, continuous 5 days.Observe mouse physiological phenomenon, changes of weight.

2. 40 BALB/C mices (half male and half female) are randomly divided into blank control group, and (0.1mL/10g sterile injection is used Water), Gardenoside low dose group (0.5g.kg^-1.d^-1), Gardenoside middle dose group (1.0g.kg^-1.d^-1), Gardenoside high dose group (2.0g.kg^-1.d^-1), continuous 5 days, gastric infusion.Observe mouse physiological phenomenon, changes of weight.Dissection mouse takes half small intestine, With normal saline flushing it is clean after, the fixed small intestine of 4% paraformaldehyde of 4mL is added in 5mL EP pipe, room temperature does disease afterwards for 24 hours Reason slice, carries out HE dyeing.

(6) experimental result

Administration Gardenia Yellow group mouse is acted normally, and without any intoxicating phenomenon, has safety.

Gardenoside group mouse is apathetic after administration, slow in action, shows serious phenomenon of having loose bowels, and stool color is deep Blue, locally there is blue spot in tail, and after administration two days, the phenomena of mortality has occurred in Gardenoside group mouse, after administration three days, Cape jasmine Sub- glycosides group hero mouse is all dead, and female mice is only survived 1.Cape jasmine water extract group mouse has slight phenomenon of having loose bowels.Cape jasmine water mentions Object and Gardenoside mouse weight is taken to mitigate, wherein the variation of Gardenoside group mouse weight is obvious.Other groups are all relatively more normal.

Influence of the Gardenoside to mouse small intestine histo pathological change: HE coloration result shows, naive mice small intestine Structure is normal, and mucous secretion is normal (Figure 1A).Gardenoside low dose group mouse small intestine tissue change is obvious, and mucous secretion is hyperfunction (Figure 1B).Gardenoside high dose group intestinal mucus secretes more obvious (Fig. 1 C).

(7) discuss

Gardenia Yellow main component is the crocin (crocin) and crocetin (crocetin) of carotenoids, tool There are the pharmacological actions such as protection cardiac muscle cell, antiatherosclerosis, anti-oxidant, Adjust-blood lipid, anticoagulation and antithrombotic.Above-mentioned experiment Research finds that Gardenia Yellow does not have toxic side effect, good security.

Gardenoside can promote the wriggling of large intestine, have effects that rush down lower.Cape jasmine water extract and Gardenoside oral administration, to dynamic Object has significant discharge function.Above-mentioned the experimental results showed that Gardenoside has toxicity, and toxicity is quite obvious, and cape jasmine water extracts Part Gardenoside can be contained in object, so mouse can also show phenomenon of slightly having loose bowels, and Gardenoside group mouse phenomenon of having loose bowels is tight Weight, intestinal mucus secretion increase, and changes of weight is obvious, and the death rate is high, has serious toxicity.

Test the antioxidation activity in vitro test of 2 cape jasmine the different extracted parts

(1) experiment purpose: the oxidation-resistant active ingredient of antioxidation in vitro experiment screening cape jasmine is passed through.

(2) reagent and instrument:

1. medicinal material and reagent

Cape jasmine is purchased from Lanzhou the Yellow River Chinese Medicinal Materials Markets, all identifies through the Gansu university of TCM director Yang Xicang pharmacist of traditional Chinese medicine For madder wort cape jasmine (Gardenia jasminoides Ellis dry mature fruit)；1,1- diphenyl -2-, three nitre Base phenylhydrazine (DPPH, Solarbio company, lot number 101029108), 2,2- connection nitrogen bases-it is bis--(3- ethyl benzo thiazole phenanthroline- 6- sulfonic acid) two ammonia salts (ABTS, Solarbio company, lot number 20151104), TRIS(Solarbio company, lot number 77861), Ascorbic acid (VC, Solarbio company, lot number 50817), pyrogallic acid (Solarbio company, lot number 20151202), 1,10- ferrosin (Solarbio company, lot number 20150916), ethylenediamine tetra-acetic acid (EDTA, Aladdin Company, lot number 46631)；Sodium nitroprusside, aluminum nitrate, sodium hydroxide, sodium nitrite, the potassium ferricyanide, sodium dihydrogen phosphate, Ammonium molybdate, ferrous sulfate, sulfuric acid, thiobarbituricacidα-, lecithin, sodium thiosulfate, potassium iodide, 30% hydrogen peroxide, tri-chlorination Iron, disodium ethylene diamine tetraacetate, trichloroacetic acid, sodium phosphate, potassium peroxydisulfate, glacial acetic acid etc. are ommercially available AR；

2. instrument

3K15 type high speed freezing centrifuge (Sigma Co., USA)；The full-automatic luciferase mark of SpectraMax i3 type Instrument (Molecular Devices company, the U.S.)；WD-9403F type ultraviolet-visible spectrophotometer (hewlette-packard)； BP210S type electronic balance (German Sartorius company)；(the upper macro experimental facilities of Nereid is limited for DK-8A type electric heating constant temperature sink Company)；Freeze drier (Spain's Telster LyoQuest-55 plus type).20 ~ 25 DEG C of experimental temperature, air-conditioning is permanent Temperature, relative humidity: 40% ~ 70%.

(3) experimental method

1. DPPH radicals scavenging is tested

According to document, by the sample and V of different quality concentration (0.03 ~ 0.5mg/mL)_C125 μ L of solution, is separately added into 125 In 95% ethanol solution of μ L 0.1mmol/L DPPH, dark at room temperature reacts 30min, does blank control with 95% alcohol solvent, Measure its absorbance (A at wavelength 517nm_i)；It measures 125 μ L DPPH, 95% ethanol solution and 125 μ L, 95% ethyl alcohol is mixed Absorbance (A after conjunction at wave 517nm₀)；125 μ L, 95% ethanol solution and 125 μ L sample solution are measured in wavelength 517nm Absorbance (the A at place_j).Its clearance rate is calculated by formula (1), and calculates its EC₅₀。

Clearance rate (%)=(1- (A_i- A_j) /A₀) × 100

2. ABTS radicals scavenging is tested

Isometric 7mmol/L ABTS solution is mixed to be allowed to react with 2.45mmol/L potassium peroxydisulfate and is placed in dark place 12~16h prepares ABTS free radical.ABTS free-atom aqueous solution, which is diluted to its absorbance at wavelength 734nm, with 95% ethyl alcohol is 0.70± 0.02.By 100 μ L different quality concentration (0.08~2.0mg/mL) samples and V_CIt is free that 3.9mL ABTS is added in solution In based sols, it is placed at room temperature for 10min, measures its absorbance (A at wavelength 734nm_i)；It is molten to measure 3.9mL ABTS free radical Absorbance (A at wavelength 734nm after liquid is mixed with 100 μ L95% ethyl alcohol₀)；Measure 95% ethanol solution of 3.9mL and 100 μ L Absorbance (A of the sample solution at wavelength 734nm_j).Its clearance rate is calculated by formula (1), and calculates its EC₅₀。

3. hydroxyl radical free radical clearance test

Take 0.75 mmol/L Phen, 1.0 mL, the phosphate buffer solution 2.0mL, 40 μ g/mL of pH=7.4 , 0.75 mmol/L FeSO₄1.0 mL, the sample solution 1.0mL, 0.01% H of different quality concentration₂O₂1. 0 mL is (new Fresh preparation), absorbance A is measured at 520 nm after reacting 60 min after mixing in 37 DEG C of water-baths_s.Blank group is with 1.0 mL Distilled water replaces sample to measure absorbance A₀, control group is with 1.0mL distilled water instead of H₂O₂Optical density A is measured with sample_c, It is returned to zero with 4.0mL distilled water and 2.0mL phosphate buffer solution.Its clearance rate is calculated by formula (2), and calculates its EC₅₀。

Clearance rate (%)=(A_s-A₀)/(A_c- A₀)

4. anti peroxidation of lipid is tested

300 mg lecithin are dissolved in the phosphate buffer solution of 30 mL 10 mmol/L, pH=7.40, ice bath shake It swings, lecithin soln is made.0.20 mL of lecithin soln is taken, 1.0 mL of phosphate buffer solution of pH 7.40 is added, it is different dense Spend (0.08~2.0mg/mL) sample solution 0.50 mL, 2.5 mmol/(II) 1.0 mL of L EDTA-Fe, after mixing in 45 min are reacted in 37 DEG C of water-baths, add trichloroacetic acid 2.0 mL of 28% (m/V), thio bar of 1% (m/V) Than appropriate sour 1.0 mL, mixing, which is placed in 100 DEG C of boiling water baths, heats 10 min, and absorbance A is measured at 523nm after cooling_Sample, It is returned to zero with phosphate buffer, the effective phosphate buffer of blank replaces sample densitometric A₀.It is calculated according to formula (3) corresponding Inhibiting rate and and V_cIt is compared.

Inhibiting rate (%)=(A₀- A_Sample)/A₀

5. the measurement of total reducing power

By sample solution (0.25~10.0 mg/mL) 1.0 mL of different quality concentration, be added 2.5 mL pH= 6.6 phosphate buffer, the potassium ferricyanide solution of 2.5 mL 1%, obtains mixed solution, is placed in 50 DEG C of water-baths and keeps the temperature 20 min, 2. 5 mL10% solution of trichloroacetic acid are added, gained mixed solution is centrifuged (3 000 rmin-1,10 min), inclines and takes Clear liquid, precision draw 2.5 mL, and the liquor ferri trichloridi of 2.5 mL distilled water and 0.5 mL 0.1% is added, and examine at 700 nm Survey absorbance A and and V_cIt is compared.

6. the measurement of total antioxidant capacity

By sample solution (0.08~2.0 mg/mL) 0. 1 mL of different quality concentration, 1.0 mL reagent solutions are added (including the sulfuric acid of 0.6mol/L, the sodium phosphate of 28 mmol/L, the ammonium molybdate of 4mmol/L in reagent solution).Mixed liquor is placed in 95 DEG C of water-baths are incubated for 90 min.It lets cool to room temperature, absorbance A and and V is surveyed using distilled water as blank, at 695 nm of Yu Bochang_cInto Row compares.

(4) cape jasmine the different extracted parts antioxidation in vitro result

1. scavenging effect of the cape jasmine the different extracted parts to DPPH free radical

As shown in Figure 2, there are notable difference, Vc, Cape jasmine for Scavenging activity of the different extracted parts of cape jasmine to DPPH free radical Sub- uranidin and cape jasmine crude extract have the apparent ability for removing free radical.Wherein, Vc and Gardenia Yellow are to DPPH freedom The scavenging effect of base is significant, and with the increase of concentration, Gardenia Yellow enhances the scavenging effect of DPPH free radical, It is in apparent dose-effect relationship in 0.03-0.5mg/mL concentration range, Gardenia Yellow is calculated to the IC of DPPH free radical₅₀For 0.188 mg/mL.Gardenoside and polysaccharide are suitable to the Scavenging activity of DPPH free radical, and activity is smaller.

2. scavenging effect of the cape jasmine the different extracted parts to ABTS free radical

From the figure 3, it may be seen that there are notable difference, Vc, Cape jasmine for Scavenging activity of the different extracted parts of cape jasmine to ABTS free radical Sub- uranidin and cape jasmine crude extract have the apparent ability for removing free radical.Wherein, Vc and Gardenia Yellow are to ABTS freedom The scavenging effect of base is significant, and the Scavenging activity of Vc is significantly higher than each extract of cape jasmine.And with the increase of concentration, Vc, Gardenia Yellow Pigment and cape jasmine crude extract enhance the scavenging effect of ABTS free radical, in apparent in 0.08~2.0mg/mL concentration range Vc, Gardenia Yellow is calculated to the IC of ABTS free radical in dose-effect relationship₅₀Respectively 0.078 mg/mL, 0.510 mg/mL. Gardenoside and saccharide portion are weaker to the Scavenging activity of ABTS free radical, and with the no significant changes of the increase of concentration.

3. scavenging effect of the cape jasmine the different extracted parts to hydroxyl radical free radical

As shown in Figure 4, the different extracted parts of cape jasmine show the different degrees of Scavenging activity to hydroxyl radical free radical, Vc, Gardenia Yellow and cape jasmine crude extract have the apparent ability for removing hydroxyl radical free radical.Wherein, Gardenia Yellow and cape jasmine Crude extract has apparent scavenging effect to hydroxyl radical free radical, and the Scavenging activity of Vc is significantly higher than each extract of cape jasmine.And with dense The increase of degree, Gardenia Yellow and cape jasmine crude extract enhance the scavenging effect of hydroxyl radical free radical, dense in 0.09~0.5mg/mL Spending in range is in apparent dose-effect relationship, and Gardenia Yellow and cape jasmine crude extract is calculated to the IC of hydroxyl radical free radical₅₀Respectively For 0.198mg/mL, 0.327mg/mL.Gardenoside and saccharide portion are weaker to the Scavenging activity of hydroxyl radical free radical, and with dense The no significant changes of the increase of degree.

4. cape jasmine the different extracted parts are to the inhibiting effect of lipid peroxidation

As shown in Figure 5, the different extracted parts of cape jasmine show the ability of different degrees of anti peroxidation of lipid, in phase With in 0.08~2.0mg/mL of concentration range, the anti peroxidation of lipid ability of Gardenia Yellow is most strong and is apparently higher than cape jasmine and slightly mentions Object, and with the increase of concentration, anti peroxidation of lipid ability gradually increases.Gardenoside and the anti-oxidant energy of the lipid of polysaccharide component Power is most weak and is significantly lower than Gardenia Yellow and cape jasmine crude extract, and when concentration increases, the anti peroxidation of lipid ability of the two is with dense Degree increases no significant change.

5. the measurement of the total reducing power of cape jasmine the different extracted parts

The reducing power of each substance has reacted its antioxidant activity to a certain extent, the potassium ferricyanide by antioxidant also Original has maximum absorption band at the substance that potassium ferrocyanide, potassium ferrocyanide and ferric ion generate at 700nm.Absorbance is got over The reducing power of big substance is stronger.Such as Fig. 6, in 0.25~10.0 mg/mL of same concentrations range, each extract part of cape jasmine with The increase absorbance of concentration improves, wherein the absorbance value of Gardenia Yellow and cape jasmine crude extract is obvious with the increase of concentration Increase, Gardenia Yellow is especially pronounced, close with Vc effect trend.

6. the measurement of cape jasmine the different extracted parts total antioxidant capacity

Such as Fig. 7, each extract part of cape jasmine and Vc are measured at 695nm in 0.08~2.0 mg/mL of same concentrations range The absorbance that total antioxidant capacity obtains is increased with the increase of concentration.Wherein, the effect trend comparison of Gardenia Yellow is bright Aobvious, the absorbance value of Gardenoside and polysaccharide component hardly changes with the increase of concentration.

(5) conclusion:

This experiment to each extract of cape jasmine remove in vitro DPPH free radical, ABTS free radical, hydroxyl radical free radical ability and Lipid peroxidation rejection ability, total reducing power, total antioxidant capacity are studied, the results showed that, each extract pair of cape jasmine DPPH free radical, ABTS free radical, hydroxyl radical free radical and lipid peroxidation rejection ability, total reducing power, total antioxidant capacity Certain oxidation resistance is shown, and correlation is almost the same, wherein Gardenia Yellow is free to DPPH free radical, ABTS The IC of base, hydroxyl radical free radical₅₀Respectively 0.188 mg/mL, 0.510 mg/mL, 0.198mg/mL, and there is certain lipid Peroxidating rejection ability, reducing power and total antioxidant capacity, within the scope of a certain concentration, with the increasing of Gardenia Yellow concentration Add, antioxidant activity enhancing.Therefore Gardenia Yellow can effectively remove free radical, have good antioxygen also ability.

Test the experiment of 3 mouse atmospheric closeds

(1) experiment purpose: mouse atmospheric closed experiment screening active component of cape jasmine Substituted phenyl-lactic acid ingredient is passed through.

(2) experiment condition: three-level Animal Lab., 20~25 DEG C of room temperature, air-conditioning constant temperature.

(3) experimental subjects: animal BalB/C mouse 60,20 ± 2g of weight, real by Lanzhou General Hospital of Lanzhou Military Command animal Section's offer is tested, quality certification number: curing dynamic word the 1402-0201st in Gansu Province.

(4) medicinal material and instrument: cape jasmine water extract, cape jasmine polysaccharide, Gardenia Yellow, Gardenoside are made by oneself by laboratory, face use Before 0.05g/mL aqueous solution is made.Rhodiola rosea capsules (Chinese People's Liberation Army, root of kirilow rhodiola development center, Tibet Military Area Command, lot number: 130604) 0.05g/mL is made before use.Sterilized water for injection (Kelun Pharm Ind Co., Ltd., Sichuan, lot number: M15111005).

Electric drying oven with forced convection (101A, Shanghai laboratory apparatus factory)；(German Sartorius is public for BP210S electronic balance Department).

(5) experimental method

1. 60 BALB/C mices (half male and half female) are randomly divided into blank control group (0.1mL/10g distilled water), red scape Its group (0.5g.kg^-1.d^-1) cape jasmine ethanol extract group (0.5g.kg^-1.d^-1), Gardenia Yellow group (0.5g.kg^-1.d^-1), Cape jasmine Sub- polysaccharide group (0.5g.kg^-1.d^-1), Gardenoside group (0.5g.kg^-1.d^-1), gastric infusion, continuous 5 days.It will after last dose 1h Mouse is put into the 200 mL ground wide-mouth bottles for filling 5 g soda limes (absorbing carbon dioxide and water), and every bottle puts 1 mouse, and And bottleneck is smeared with vaseline, covering tightly prevents gas leakage, immediately timing.It is with mouse breathing stopping (the mouse chest does not rise and fall) time Index observes the mouse time dead due to anoxic.

2. 60 BALB/C mices (half male and half female) are randomly divided into blank control group (0.1mL/10g distilled water), model Group (0.1mL/10g distilled water), root of kirilow rhodiola group (1.0g.kg^-1.d^-1), Gardenia Yellow low dose group ((0.25g.kg^-1.d^-1), Gardenia Yellow middle dose group ((0.5g.kg^-1.d^-1), Gardenia Yellow high dose group (1.0g.kg^-1.d^-1), gastric infusion, even It is 5 days continuous.Mouse is put into after last dose 1h in the 200 mL ground wide-mouth bottles for filling 5 g soda limes, every bottle puts 1 mouse, Bottleneck is smeared with vaseline, covering tightly prevents gas leakage, immediately timing.Stop (the mouse chest does not rise and fall) time with mouse breathing to refer to Mark observes the mouse time dead due to anoxic.Take brain tissue and lung tissue to measure wet quality, and with 55 DEG C of electric heating forced air dryings It is 3 days dry in case, the dry mass of brain tissue and lung tissue is surveyed, is calculated by formula (wet mass-dry mass)/wet quality × 100 Brain lung tissue water content.

(6) experimental result

1. influence of the active component of cape jasmine to the atmospheric closed mouse anti anoxia time is compared with blank group (NG), when anti anoxia Between increase, Gardenia Yellow group (Crocin) mouse anti anoxia time obviously increases (P < 0.01).With positive drug root of kirilow rhodiola group (Rhodiola) compare, Gardenia Yellow group (crocin) mouse anti anoxia time obviously increases.It the results are shown in Table 1:

The 1 active component of cape jasmine each group mouse atmospheric closed anti anoxia time of table (, n=10)

Tab. 1 The time of mice sustaining anoxia at common atmosphere in Each group of the effective-part of Gardenia(,n=10)

Note: p < 0.01 * p < 0.05, * *, administration group vs blank group.

Note: *p<0.05,**p<0.01,medicated group compared with NG

2. low middle each dosage group of height (Crocin-L, Crocin-M, Crocin-H) the mouse atmospheric closed of Gardenia Yellow is anti- Hypoxic exposure increased compared with model group, when Gardenia Yellow high dose group (Crocin-H) is with model group (MG) anti anoxia Between compared to have conspicuousness (P < 0.05).It the results are shown in Table 2:

The 2 Gardenia Yellow each group mouse atmospheric closed anti anoxia time of table (,n=10)

Tab. 2 The time of mice sustaining anoxia at common atmosphere in each group of crocin

(,n=10)

Note: p < 0.01 * p < 0.05, * *, administration group vs model group.

Note:Note: *p<0.05,**p<0.01,medicated group compared with NG

3. influence of the Gardenia Yellow (Crocin) to mouse lung tissue and brain water content: compared with blank group, mould Type group (MG) mouse lung tissue water content obviously increases (P < 0.01), and brain water content also obviously increases (P < 0.05).Red scape Its group (Rhodiola) and low middle high each group (Crocin-L, Crocin-M, the Crocin-H) mouse lung tissue of Gardenia Yellow and Brain water content significantly reduces (P < 0.05 or P < 0.01) compared with model group, without significant change compared with blank group (NG). It the results are shown in Table 3:

3 Gardenia Yellow of table to mouse lung tissue and brain water content influence (,n=10)

Tab. 3 Effect of Crocin on the water content of lung and brain in mice (x±s, n=10)

Note:^**P < 0.01,^*0.05 model group vs blank control group of P <；^##P < 0.01,^#P < 0.05, administration group vs Model group.

Note:^**P < 0.01,^*P < 0.05, MG compared with NG group;^##P < 0.01,^#P < 0.05, medicated group compared with NG group

(7) discuss

Tissue ischemia anoxic is mainly shown as that cellular oxidation process obstacle, energy generate insufficient, anaerobic metabolism product accumulation , cell functional disorders, even eucaryotic cell structure change.Normobaric hypoxia is non-specific anoxic, and mouse is in closed container by anoxic Factor damage, is mainly reflected in the heart and cerebral anoxia.Studies have reported that reduce acute hypoxic rat dead for trans-crocetin sodium salt Rate is died, shows that crocetin can be improved the hypoxia-bearing capability of animal.Root of kirilow rhodiola plays the role of anti anoxia, can significantly improve small The hypoxia-bearing capability of mouse cardiac muscle.This experiment studies Gardenia Yellow (Crocin) Substituted phenyl-lactic acid using root of kirilow rhodiola as positive drug, The result shows that: Gardenia Yellow (Crocin) has significant extension to act on the time-to-live of mouse normobaric hypoxia condition.Anoxic Pulmonary arterial pressure is caused to increase the change with haemodynamics；Inflammatory mediator will lead to impaired vascular endothelium, and permeability increases, largely Liquid enters interstitial lung, and the liquid for entering interstitial lung can enter alveolar after cannot being efficiently absorbed, to form alveolar lung Oedema.Anoxic can cause blood-brain barrier (BBB) permeability to increase, and BBB permeability increases and can should send out vascular brain edema.This Experimental studies have found that Gardenia Yellow (Crocin) can significantly mitigate atmospheric closed mouse lung tissue and brain water content, it can Mitigate brain edema and pulmonary edema.

It tests mouse power under 4 hypobaric hypoxia environments and exhausts swimming test

(1) experiment purpose: the anti-fatigue active that simulation hypobaric hypoxia environment studies Gardenia Yellow is passed through.

(2) experiment condition: three-level Animal Lab., 20~25 DEG C of room temperature, air-conditioning constant temperature, relative humidity: 40%~70%.

(4) reagent and instrument:

1. reagent: Rhodiola rosea capsules (Chinese People's Liberation Army, root of kirilow rhodiola development center, Tibet Military Area Command, lot number: 130604)； Macroporous absorbent resin (the net product type of D101, Tianjin sea light Chemical Co., Ltd., lot number: 070305)；75% ethyl alcohol (Shandong benefit Er Kang sterilizes Science and Technology Co., Ltd., lot number: 130619)；CK testing cassete (product batch number: 20151201), liver/muscle glycogen examination Agent box (product batch number: 20151202), ultramicron ATP (Na+K+) testing cassete (product batch number: 20151203), ultramicron ATP (product batch number: Ca²⁺Mg²⁺), testing cassete (product batch number: 20151203), succinate dehydrogenase (product batch number: SDH) test Box (20151203), pyruvate kinase (PK) kit (product batch number: 20151203), malonaldehyde (product batch number: MDA) test Box (20151202), reduced glutathione (GSH) kit (product batch number: 20151203), pyruvate reagent box (production Lot number: 20151203), Coomassie brilliant blue (product batch number: 20151218), SOD kit (survey total) (product batch number: 20151130), BCA assay kit (product batch number: 20151203), lactic dehydrogenase (LDH) testing cassete (product batch number: 20151006), lactic acid (LD) testing cassete (product batch number: 20151014) builds up biotechnology research institute by Nanjing and provides；10 × PBS(lot number: 20140807) it is purchased from Beijing Suo Laibao Science and Technology Ltd.Electric drying oven with forced convection (101A, Shanghai experiment instrument Device factory)；BP210S electronic balance (German Sartorius company).

2. instrument: DYC-9070 type simulated high altitude hypobaric hypoxia animal experimental chamber (Guizhou wind and thunder aerial armament Limited Liability Company)；3K15 type high speed freezing centrifuge (Sigma Co., USA)；The full-automatic luciferase mark of SpectraMax i3 type Instrument (Molecular Devices company, the U.S.)；Animal's whole blood cytoanalyze (XT-2000i, Japanese Sysmex)； WD-9403F type ultraviolet-visible spectrophotometer (hewlette-packard)；BP210S type electronic balance (German Sartorius Company)；DK-8A type electric heating constant temperature sink (the upper macro experimental facilities Co., Ltd of Nereid)；TB-718E type biological tissue wraps automatically Bury machine (Hubei Tai Ya Electron Technology Co., Ltd)；E200 type optical microscopy (Japanese Nikon company)；HM340E type group Knit slicer (German Meikang company)；Electric homogenizer (Polytron@PT 1200E, Kinematica AG company), it is cold Lyophilizer (Spain's Telster LyoQuest-55 plus type).

(5) experimental method

1. mouse swimming time detects

72 BALB/C mices, are randomly divided into 6 groups, i.e., normal oxygen control group, model group, Rhodiola rosea capsules positive controls and Gardenia Yellow low, middle and high dose groups, every group 12.Normal oxygen control group and model group stomach-filling sterilized water for injection (0.2mL/ 20g), corresponding reagent is given in stomach-filling to remaining each group respectively.Remaining each group is placed in large-scale low-pressure oxygen cabin in addition to normal oxygen control group 8000 m altitude environment of altitude simulating (in cabin pressure: 35.9 kPa, partial pressure of oxygen: 7.4 kPa), every morning 9:00 is with 10 m s^-1Flow velocity drops to pressure in 4000 cabins m(: 62.1 kPa, partial pressure of oxygen: 13.8 kPa), experimenter is into cabin, the stomach-filling in cabin Water food and padding, continuous 5 d, after daily administration, by cabin inner height with 10 m s are changed in administration^-1Flow velocity at the uniform velocity rise to Predetermined 8000 m of elevation, animal freely ingests and intakes during this period.Normal oxygen control rats are raised simultaneously in animal house.Last After 1 h is administered, is tested in 4000 m of height above sea level, lain at 1/3 tail with the load (galvanized wire) of mouse weight 7%, be put into the depth of water 18 In the sink (cm × 30 of 50 cm × 40 cm) of cm, water temperature control carries out swimming with a load attached to the body in (25 ± 1) DEG C, and record mouse is from putting Enter sink to mouse and sink to 7 s of water to fail the time to float on the surface, as mouse swimming time.

2. mice serum level detects

It after power exhausts swimming, takes out immediately, plucks eyeball and take blood, 3500 r/min are centrifuged 10 min after standing 1 h, isolated Serum.

3. blood routine

20 μ l EDTA anticoagulations are taken, after 180 μ l blood dilution liquids are added, are detected on animal's whole blood cytoanalyze Routine blood indexes.

4. mouse muscle and hepatic tissue Activity determination

After cervical dislocation is put to death, the quadriceps muscle of thigh and liver of two hind legs are taken, is cleaned and is dried with ice physiological saline, weigh 140- 10% homogenate is made in 150mg tissue, cold PBS, and 3500 r/min are centrifuged 10 min, take supernatant.

5. statistical procedures

Statistical procedures are carried out using 17.0 statistical software of SPSS.As a result withIt indicates, comparison among groups use variance Analysis and t are examined.It is that difference is statistically significant with P < 0.05.

(6) results of animal

1. each group mouse swimming power exhausts time detection

Compared with normal oxygen control group, Rhodiola rosea capsules group and Gardenia Yellow low, middle and high dose groups can significantly extend small Mouse swimming time, difference have statistical significance (P < 0.01 or P < 0.05).Each group mouse swimming time is shown in Table 4。

4 mouse swimming time result of table (,n=12)

Note:^**P < 0.01,^*0.05 model group vs blank control group of P <；^##P < 0.01,^#P < 0.05, model group vs Administration group.

2. each group mouse metabolism product detection, compared with normal oxygen group, the content of anoxia model group mouse blood urea nitrogen is significant Raising, difference have statistical significance (P < 0.05).Rhodiola rosea capsules group and Gardenia Yellow compared with anoxia model group High, middle dose group can significantly reduce the content of blood urea nitrogen, and difference has statistical significance (P < 0.05)；Compared with normal oxygen group, Acetone acid content in model group mouse liver, muscle and serum significantly reduces, and difference has statistical significance (P < 0.05). Compared with anoxia model group, Rhodiola rosea capsules group and Gardenia Yellow high dose group can significantly improve liver, muscle and serum The content of middle pyruvic acid, Gardenia Yellow middle dose group can also improve the content of pyruvic acid in serum, and difference is anticipated with statistics Adopted (P < 0.05)；Compared with normal oxygen group, lactic acid content is significantly increased in anoxia model group mouse liver, muscle and serum, difference With statistical significance (P < 0.01 or P < 0.05).Compared with anoxia model group, Rhodiola rosea capsules group and Gardenia Yellow are high Dosage group can significantly reduce mouse liver, lactic acid content in muscle and serum, and difference has statistical significance (P < 0.01 or P < 0.05) in Gardenia Yellow, low dose group can also reduce the content of lactic acid in serum, and difference has statistical significance (P < 0.01).Each group mouse liver, muscle, the testing result of BUN, pyruvic acid and LD are shown in Table 5 in serum:

5 each group mouse liver of table, muscle, in serum BUN, pyruvic acid and LD detection (,n=12)

3. each group mouse oxidation index detects compared with normal oxygen group, GSH is significant in anoxia model group mouse liver, muscle It reduces, difference has statistical significance (P < 0.01 or P < 0.05).Compared with anoxia model group, Rhodiola rosea capsules group and cape jasmine Uranidin high dose group can significantly improve GSH content, and difference has statistical significance (P < 0.01 or P < 0.05), Gardenia Yellow Pigment middle dose group can also improve the content of GSH in liver, and difference has statistical significance (P < 0.05)；Compared with normal oxygen group, MDA content significantly increases in anoxia model group mouse liver, muscle, and difference has statistical significance (P < 0.01).With anoxic mould Type group compares, and Rhodiola rosea capsules group and the middle and high dosage group of Gardenia Yellow can significantly reduce in mouse liver and musculature MDA content, difference have statistical significance (P < 0.01 or P < 0.05)；Compared with normal oxygen group, anoxia model group mouse liver, SOD activity significantly reduces in muscle and serum, and difference has statistical significance (P < 0.01).Compared with anoxic group, root of kirilow rhodiola glue Wafer Gardenia Yellow high dose group can significantly improve mouse liver, SOD activity in muscle and serum, and difference has statistics Meaning (P < 0.01 or P < 0.05), and in Gardenia Yellow, low dose group can also improve the content of SOD in liver and muscle, Difference has statistical significance (P < 0.01 or P < 0.05).Each group mouse liver, muscle, GSH, MDA, SOD inspection in serum Survey the results are shown in Table 6.

6 each group mouse liver of table, muscle, GSH, MDA, SOD testing result in serum

4. each group mouse metabolism adjusts enzyme detection

Compared with normal oxygen group, anoxia model group mouse liver, muscle, LDH activity weakens in serum, and difference has statistics Meaning (P < 0.01 or P < 0.05)；Compared with anoxia model group, Rhodiola rosea capsules group and Gardenia Yellow group high dose group are small Mouse liver, muscle, LDH activity enhancing in serum, difference is statistically significant (P < 0.01 or P < 0.05), in Gardenia Yellow Dosage group can also improve the content of LDH in serum, and difference has statistical significance (P < 0.01).Each group mouse liver, muscle, LDH testing result is shown in Table 7 in serum.

7 each group mouse liver of table, muscle, LDH testing result in serum

5. each group mouse Energy Metabolism Enzyme detects

Compared with normal oxygen group, in anoxia model group mouse liver, muscle and serum MDH, SDH, PK, Ca-Mg-ATP enzyme and Na-K-ATP enzymatic activity significantly reduces, and difference has statistical significance (P < 0.01).Compared with anoxia model group, root of kirilow rhodiola glue Capsule group and the middle and high dosage group of Gardenia Yellow can significantly enhance in Muscle Tissue MDH, SDH, PK, Ca-Mg-ATP enzyme and Na-K-ATP enzymatic activity, difference have statistical significance (P < 0.01 or P < 0.05), and Gardenia Yellow low dose group can also mention SDH in high muscle, Na-K-ATP enzymatic activity, difference have statistical significance (P < 0.01) in PK and liver in serum.As a result see Table 8, table 9:

8 each group mouse liver of table, muscle, MDH, SDH, PK, Ca-Mg-ATP enzyme and Na-K-ATP enzyme detection knot in serum Fruit

9 each group mouse Ca-Mg-ATP enzyme of table, Na-K-ATP and CK enzyme testing result

6. each group mouse ergastic substances detect

Compared with normal oxygen group, anoxia model group Glycogen content is significantly reduced, and difference has statistical significance (P < 0.01 or P < 0.05).Compared with anoxia model group, Rhodiola rosea capsules group and each dosage group of Gardenia Yellow can dramatically increase sugar Former storage level, difference have statistical significance (P < 0.01 or P < 0.05).Each group Glycogen testing result is shown in Table 10.

10 each group Glycogen testing result of table

(7) discuss:

The result shows that Gardenia Yellow has good antifatigue effect.Lactic dehydrogenase can be enhanced in Gardenia Yellow Vigor, reduce lactic acid accumulation, reduce the concentration of BUN, it is seen that Gardenia Yellow can reduce muscle tissue damage, to reduce The consumption of energy substance, delay fatigue；Evaluation index of the hepatic glycogen amount of storage as internal energy substance, Gardenia Yellow can Body development and liver glycogen are horizontal by increasing, and when movement increases the release of ATP, promote energetic supersession；By reducing because acutely The damage of membrane structure caused by moving, reduces CK enzymatic activity, maintains homeostasis, and then increase exercise intensity and run duration, Realize its anti-sports fatigue effect；SOD is widely present in body cell, can remove the excessive super oxygen yin that generates in vivo from Sub- free radical, protects DNA, protein and cell membrane from the destruction of superoxide radical, and MDA can directly reflect free radical water Flat, content height is the important symbol of tissue cell insult, it is known that Gardenia Yellow is removing free radical, anti-oxidant aspect Significant effect, to achieve the effect that antifatigue；The active variation of SDH can reflect cellular energy metabolism situation, Gardenia Yellow SDH and MDH activity can be enhanced, promote body aerobic oxidation metabolism；PK is the rate-limiting enzyme of glycolytic pathway, in glycolysis process In play decisive role, Gardenia Yellow can increase PK content, promote glycolysis, increase ATP, to reach antifatigue effect Fruit.

2, the present invention can efficiently separate enrichment gardenia yellow pigment with high color value, at the same also by Gardenoside carried out enrichment and it is pure Change, to improve the utilization rate of medicinal material, for by Gardenia Yellow science, effectively develop, and then is applied to sport nutrition and eats It lays a good foundation in product field.

3, the invention is simple and feasible, easy to operate, safe, reproducible, can industrialized production.

Detailed description of the invention

Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.

Fig. 1 is mouse small intestine pathological section figure of the invention.

Fig. 2 is clearance rate of the cape jasmine the different extracted parts of the present invention to DPPH free radical.

Fig. 3 is clearance rate of the cape jasmine the different extracted parts of the present invention to ABTS free radical.

Fig. 4 is clearance rate of the cape jasmine the different extracted parts of the present invention to hydroxyl radical free radical.

Fig. 5 is the lipid oxidation resistance of cape jasmine the different extracted parts of the present invention.

Fig. 6 is total reducing power of cape jasmine the different extracted parts of the present invention.

Fig. 7 is the total antioxidant capacity of cape jasmine the different extracted parts of the present invention.

Specific embodiment

The separation method of anti-high altitude anoxia anti-fatigue activity ingredient in 1 cape jasmine of embodiment, comprising the following steps:

Will drying, be crushed to the cape jasmine fruits of 20 mesh distillation water boiling and extraction after, through filtering, reduceds pressure, be sprayed Drying is to get gardenia powder.

Wherein: decocting extraction conditions and refer to that feed liquid mass ratio (g/g) is 1:8, temperature is 90 DEG C, and number is 2 times, when each Between be 1 hour.

The dry part of the item of reduced pressure refers to that temperature is 50 DEG C.

The condition of spray drying refers to that temperature is 120 DEG C, 20 m of unit time intake³, atomisation pressure 0.1MPa.

(2) gardenia powder is configured to the aqueous solution of 20 mg/mL with 25 DEG C of distilled water.

(3) by step, (2) resulting aqueous solution presses 1.0 times of column volume with the flow velocity injection processed 14 of 1.5mL/min Mesh polyamide column, then the column volume for pressing 1.0 times are respectively obtained poly- with the flow velocity of 5.0 mL/min distilled water flushing polyamide column Amide column loading efflux, water elution.

Wherein: processed polyamide column refer to by having a size of 8cm × polyamide column of 100 cm is first with volumetric concentration 95% ethanol elution, until ethanol eluate adds distilled water muddiness do not occur, then to distill water elution pillar to no alcohol Taste to get.

(4) after polyamide loading efflux and water elution being merged, amalgamation liquid with the flow velocity whole injection of 10mL/min at The macroporous resin column managed, first by 1.0 times of column volume with the flow velocity of 5.0 mL/min distilled water flushing macroporous resin column, then Macroporous resin column is rinsed with the ethanol solution that the flow velocity volumetric concentration of 1.5 mL/min is 30% by 2.0 times of column volume, is obtained 30% ethanol eluate, 30% ethanol eluate be concentrated under reduced pressure, be freeze-dried after to get Gardenoside product.

Wherein: macroporous resin column refers to D-101 type macroreticular resin.

The condition of reduced pressure refers to that temperature is 50 DEG C.

The condition of freeze-drying refers to that temperature is -55 DEG C, vacuum degree 0.5MPa.

Processed macroporous resin column refers to the ethanol elution for first using macroreticular resin volumetric concentration to be 95%, until ethyl alcohol Until eluent adds distilled water muddiness do not occur, then with distill water elution pillar to no alcohol taste to get.

(5) first rinsed by 1.0 times of column volume with the ethanol solution that the flow velocity volumetric concentration of 5.0mL mL/min is 10% Polyamide column removal of impurities, then rinsed by 2.0 times of column volume with the ethanol solution that the flow velocity volumetric concentration of 1.5 mL/min is 75% The polyamide column obtains 75% ethanol eluate, 75% ethanol eluate be concentrated under reduced pressure, be freeze-dried after to get Gardenia Yellow product.

Wherein: the condition of reduced pressure refers to that temperature is 50 DEG C.

The separation method of anti-high altitude anoxia anti-fatigue activity ingredient in 2 cape jasmine of embodiment, comprising the following steps:

Will drying, be crushed to the cape jasmine fruits of 30 mesh distillation water boiling and extraction after, through filtering, reduceds pressure, be sprayed Drying is to get gardenia powder.

Wherein: decocting extraction conditions and refer to that feed liquid mass ratio (g/g) is 1:10, temperature is 100 DEG C, and number is 3 times, every time Time is 1 hour.

The condition of reduced pressure refers to that temperature is 80 DEG C.

The condition of spray drying refers to that temperature is 140 DEG C, 30 m of unit time intake³, atomisation pressure 0.4MPa.

(2) gardenia powder is configured to the aqueous solution of 30 mg/mL with 25 DEG C of distilled water.

(3) by step, (2) resulting aqueous solution presses 3.0 times of column volume with processed 30 mesh of flow velocity injection of 15mL/min Polyamide column, then the column volume for pressing 3.0 times are respectively obtained poly- with the flow velocity of 10.0 mL/min distilled water flushing polyamide column Amide column loading efflux, water elution.

Wherein: processed polyamide column is the same as embodiment 1.

(4) after polyamide loading efflux and water elution being merged, amalgamation liquid with the flow velocity whole injection of 10mL/min at The macroporous resin column managed, first by 3.0 times of column volume with the flow velocity of 10.0 mL/min distilled water flushing macroporous resin column, Macroporous resin column is rinsed with the ethanol solution that the flow velocity volumetric concentration of 10.0 mL/min is 10% by 10.0 times of column volume again, Obtain 10% ethanol eluate, 10% ethanol eluate be concentrated under reduced pressure, be freeze-dried after to get Gardenoside product.

Wherein: macroporous resin column refers to HPD100 type macroreticular resin.

The condition of reduced pressure refers to that temperature is 80 DEG C.

The condition of freeze-drying refers to that temperature is 20 DEG C, and vacuum degree is 0.2 MPa.

Processed macroporous resin column is the same as embodiment 1.

(5) first rinsed by 3.0 times of column volume with the ethanol solution that the flow velocity volumetric concentration of 10.0mL mL/min is 30% Polyamide column removal of impurities, then rushed by 10.0 times of column volume with the ethanol solution that the flow velocity volumetric concentration of 10.0 mL/min is 30% The polyamide column is washed, 30% ethanol eluate is obtained, after which is concentrated under reduced pressure, is freeze-dried, i.e., Obtain Gardenia Yellow product.

Wherein: the condition of reduced pressure refers to that temperature is 80 DEG C.

The separation method of anti-high altitude anoxia anti-fatigue activity ingredient in 3 cape jasmine of embodiment, comprising the following steps:

Will drying, be crushed to the cape jasmine fruits of 40 mesh distillation water boiling and extraction after, through filtering, reduceds pressure, be sprayed Drying is to get gardenia powder.

Wherein: decocting extraction conditions and refer to that feed liquid mass ratio (g/g) is 1:9, temperature is 95 DEG C, and number is 2 times, when each Between be 1 hour.

The condition of reduced pressure refers to that temperature is 60 DEG C.

The condition of spray drying refers to that temperature is 130 DEG C, 25 m of unit time intake³, atomisation pressure 0.2MPa.

(2) gardenia powder is configured to the aqueous solution of 40 mg/mL with 25 DEG C of distilled water.

(3) by step, (2) resulting aqueous solution presses 2.0 times of column volume with processed 20 mesh of flow velocity injection of 5mL/min Polyamide column, then the column volume for pressing 2.0 times respectively obtain polyamides with the flow velocity of 8.0 mL/min distilled water flushing polyamide column Amine column loading efflux, water elution.

Wherein: processed polyamide column is the same as embodiment 1.

(4) after polyamide loading efflux and water elution being merged, amalgamation liquid with the flow velocity whole injection of 10mL/min at The macroporous resin column managed, first by 2.0 times of column volume with the flow velocity of 8.0 mL/min distilled water flushing macroporous resin column, then Macroporous resin column is rinsed with the ethanol solution that the flow velocity volumetric concentration of 5.0 mL/min is 30% by 6.0 times of column volume, is obtained 30% ethanol eluate, 30% ethanol eluate be concentrated under reduced pressure, be freeze-dried after to get Gardenoside product.

Wherein: macroporous resin column refers to XDA-6 type macroreticular resin.

The condition of reduced pressure refers to that temperature is 60 DEG C.

The condition of freeze-drying refers to that temperature is -30 DEG C, and vacuum degree is 0.3 MPa.

Processed macroporous resin column is the same as embodiment 1.

(5) first rinsed by 2.0 times of column volume with the ethanol solution that the flow velocity volumetric concentration of 8.0mL mL/min is 15% Polyamide column removal of impurities, then rinsed by 6.0 times of column volume with the ethanol solution that the flow velocity volumetric concentration of 5.0 mL/min is 50% The polyamide column obtains 50% ethanol eluate, 50% ethanol eluate be concentrated under reduced pressure, be freeze-dried after to get Gardenia Yellow product.

Wherein: the condition of reduced pressure refers to that temperature is 60 DEG C.

The separation method of anti-high altitude anoxia anti-fatigue activity ingredient in 4 cape jasmine of embodiment, comprising the following steps:

Wherein: decocting extraction conditions and refer to that feed liquid mass ratio (g/g) is 1:8.5, temperature is 92 DEG C, and number is 3 times, every time Time is 1 hour.

The condition of reduced pressure refers to that temperature is 70 DEG C.

The condition of spray drying refers to that temperature is 125 DEG C, 20 m of unit time intake³, atomisation pressure 0.3MPa.

(3) by step, (2) resulting aqueous solution presses 1.5 times of column volume with processed 25 mesh of flow velocity injection of 10mL/min Polyamide column, then the column volume for pressing 1.5 times respectively obtain polyamides with the flow velocity of 6.0 mL/min distilled water flushing polyamide column Amine column loading efflux, water elution.

Wherein: processed polyamide column is the same as embodiment 1.

(4) after polyamide loading efflux and water elution being merged, amalgamation liquid with the flow velocity whole injection of 10mL/min at The macroporous resin column managed, first by 1.5 times of column volume with the flow velocity of 6.0 mL/min distilled water flushing macroporous resin column, then Macroporous resin column is rinsed with the ethanol solution that the flow velocity volumetric concentration of 3.0 mL/min is 50% by 4.0 times of column volume, is obtained 50% ethanol eluate, 50% ethanol eluate be concentrated under reduced pressure, be freeze-dried after to get Gardenoside product.

Wherein: macroporous resin column refers to DM-130 type macroreticular resin.

The condition of reduced pressure refers to that temperature is 70 DEG C.

The condition of freeze-drying refers to that temperature is -10 DEG C, and vacuum degree is 0.4 MPa.

Processed macroporous resin column is the same as embodiment 1.

(5) first rinsed by 1.5 times of column volume with the ethanol solution that the flow velocity volumetric concentration of 6.0mL mL/min is 20% Polyamide column removal of impurities, then rinsed by 4.0 times of column volume with the ethanol solution that the flow velocity volumetric concentration of 3.0 mL/min is 75% The polyamide column obtains 75% ethanol eluate, 75% ethanol eluate be concentrated under reduced pressure, be freeze-dried after to get Gardenia Yellow product.

Wherein: the condition of reduced pressure refers to that temperature is 70 DEG C.

The separation method of anti-high altitude anoxia anti-fatigue activity ingredient in 5 cape jasmine of embodiment, comprising the following steps:

Wherein: decocting extraction conditions and refer to that feed liquid mass ratio (g/g) is 1:9.5, temperature is 98 DEG C, and number is 2 times, every time Time is 1 hour.

The condition of reduced pressure refers to that temperature is 80 DEG C.

The condition of spray drying refers to that temperature is 140 DEG C, 25 m of unit time intake³, atomisation pressure 0.1MPa.

(3) by step, (2) resulting aqueous solution presses 2.5 times of column volume with processed 30 mesh of flow velocity injection of 12mL/min Polyamide column, then the column volume for pressing 2.5 times respectively obtain polyamides with the flow velocity of 9.0 mL/min distilled water flushing polyamide column Amine column loading efflux, water elution.

Wherein: processed polyamide column is the same as embodiment 1.

(4) after polyamide loading efflux and water elution being merged, amalgamation liquid with the flow velocity whole injection of 10mL/min at The macroporous resin column managed, first by 2.5 times of column volume with the flow velocity of 9.0 mL/min distilled water flushing macroporous resin column, then Macroporous resin column is rinsed with the ethanol solution that the flow velocity volumetric concentration of 8.0 mL/min is 70% by 6.0 times of column volume, is obtained 70% ethanol eluate, 70% ethanol eluate be concentrated under reduced pressure, be freeze-dried after to get Gardenoside product.

Wherein: macroporous resin column refers to DA201 type macroreticular resin.

The condition of reduced pressure refers to that temperature is 80 DEG C.

The condition of freeze-drying refers to that temperature is 10 DEG C, vacuum degree 0.4MPa.

Processed macroporous resin column is the same as embodiment 1.

(5) first rinsed by 2.5 times of column volume with the ethanol solution that the flow velocity volumetric concentration of 9.0mL mL/min is 25% Polyamide column removal of impurities, then rinsed by 8.0 times of column volume with the ethanol solution that the flow velocity volumetric concentration of 8.0 mL/min is 90% The polyamide column obtains 90% ethanol eluate, 90% ethanol eluate be concentrated under reduced pressure, be freeze-dried after to get Gardenia Yellow product.

The Gardenia Yellow extracted in above-described embodiment 1 ~ 5 refers to as the application of Substituted phenyl-lactic acid ingredient in medicine: Using Gardenia Yellow as active constituent, it is equipped with any dosage form that medicinal stone is prepared into pharmacy；Or with cape jasmine yellow Element is active constituent, adds anti-oxidant and/or anti-fatigue active at being grouped as compound, and be equipped with pharmaceutic adjuvant and be prepared into pharmacy On any dosage form.Dosage form refer to hard capsule, soft capsule, ordinary tablet, sugar coated tablet, Film coated tablets, special-shaped tablets, chewable tablets, Effervescent tablet, dispersible tablet, granule, pill, solution, any one in syrup.

Embodiment 6 mixes 1 ~ 5 gained Gardenia Yellow of embodiment and cistanche extracts according to the ratio of 1:1, Capsule is made into using practice of pharmacy, for preventing and treating high altitude anoxia type fatigue.

Embodiment 7 mixes 1 ~ 5 gained Gardenia Yellow of embodiment and cistanche extracts according to the ratio of 3:1, Dispersible tablet is made into using practice of pharmacy, for preventing and treating high altitude anoxia type fatigue.

Embodiment 8 mixes 1 ~ 5 gained Gardenia Yellow of embodiment and cistanche extracts according to the ratio of 5:1, Granule is made into using practice of pharmacy, for preventing and treating high altitude anoxia type fatigue.

Embodiment 9 mixes 1 ~ 5 gained Gardenia Yellow of embodiment and cistanche extracts according to the ratio of 7:1, Pill is made into using practice of pharmacy, for preventing and treating high altitude anoxia type fatigue.

Embodiment 10 mixes 1 ~ 5 gained Gardenia Yellow of embodiment and cistanche extracts according to the ratio of 1:3 It closes, solution is made into using practice of pharmacy, for preventing and treating high altitude anoxia type fatigue.

Obtained Gardenia Yellow can be used as anti-oxidant, anti-fatigue effect active constituent and exist in above-described embodiment 1 ~ 5 Prepare the application in terms of anti-oxidant or anti-fatigue health-product containing.

Claims

1. the separation method of anti-high altitude anoxia anti-fatigue activity ingredient in cape jasmine, comprising the following steps:

Will drying, be crushed to the cape jasmine fruits of 20 ~ 40 mesh distillation water boiling and extraction after, through filtering, reduceds pressure, by spraying do It is dry to get gardenia powder；

(3) by the step, (2) resulting aqueous solution presses 1.0 ~ 3.0 times of column volume with the flow velocity injection 8cm of 1.5 ~ 15mL/min Processed 14 ~ 30 mesh polyamide column of × 100 cm, then 1.0 ~ 3.0 times of column volume is pressed with the flow velocity of 5.0 ~ 10.0 mL/min With distilled water flushing polyamide column, polyamide column loading efflux, water elution are respectively obtained；

(4) after merging polyamide loading efflux and water elution, amalgamation liquid is all injected processed with the flow velocity of 10mL/min Macroporous resin column, first by 1.0 ~ 3.0 times of column volume with the flow velocity of 5.0 ~ 10.0 mL/min distilled water flushing macroreticular resin Column, then press the ethanol solution that 2.0 ~ 10.0 times of column volume is 10 ~ 70% with the flow velocity volumetric concentration of 1.5 ~ 10.0 mL/min Macroporous resin column is rinsed, obtains 10 ~ 70% ethanol eluate, which is concentrated under reduced pressure, is freeze-dried Afterwards to get Gardenoside product；

(5) the ethyl alcohol for being first 10 ~ 30% with the flow velocity volumetric concentration of 5.0 ~ 10.0mL mL/min by 1.0 ~ 3.0 times of column volume Solution rinses polyamide column removal of impurities, then by 2.0 ~ 10.0 times of column volume with the flow velocity volumetric concentration of 1.5 ~ 10.0 mL/min The polyamide column is rinsed for 30 ~ 90% ethanol solution, obtains 30 ~ 90% ethanol eluate, 30 ~ 90% ethanol elution Liquid be concentrated under reduced pressure, be freeze-dried after to get Gardenia Yellow product.

2. the separation method of anti-high altitude anoxia anti-fatigue activity ingredient in cape jasmine as described in claim 1, it is characterised in that: described (1) the middle extraction conditions that decoct refer to that feed liquid mass ratio is 1:8 ~ 10 to step, and temperature is 90 ~ 100 DEG C, and number is 2 ~ 3 times, when each Between be 1 hour.

3. the separation method of anti-high altitude anoxia anti-fatigue activity ingredient in cape jasmine as described in claim 1, it is characterised in that: described Step is (1), (4) (5) the middle condition being concentrated under reduced pressure each means that temperature is 50 DEG C ~ 80 DEG C to the step with the step.

4. the separation method of anti-high altitude anoxia anti-fatigue activity ingredient in cape jasmine as described in claim 1, it is characterised in that: described (1) the middle condition being spray-dried refers to that temperature is 120 ~ 140 DEG C to step, 20 ~ 30 m of unit time intake³, atomisation pressure 0.1 ~ 0.4MPa。

5. the separation method of anti-high altitude anoxia anti-fatigue activity ingredient in cape jasmine as described in claim 1, it is characterised in that: described (4) (5) the middle condition being freeze-dried each means that temperature is -55 DEG C ~ 20 DEG C to step with the step, and vacuum degree is 0.2MPa ~ 0.5 MPa。

6. the separation method of anti-high altitude anoxia anti-fatigue activity ingredient in cape jasmine as described in claim 1, it is characterised in that: described Step (4) in macroporous resin column refer to any one in D-101, HPD100, XDA-6, DM-130, DA201 type macroreticular resin.

7. the separation method of anti-high altitude anoxia anti-fatigue activity ingredient in cape jasmine as described in claim 1, it is characterised in that: described Step (3) in processed polyamide column and the step (4) in processed macroporous resin column each mean and first use volumetric concentration For 95% ethanol elution, until ethanol eluate adds distilled water muddiness do not occur, then to distill water elution pillar to no alcohol Taste to get.

8. the obtained cape jasmine yellow of the separation method of anti-high altitude anoxia anti-fatigue activity ingredient in cape jasmine as described in claim 1 Application of the element as Substituted phenyl-lactic acid ingredient in medicine preparation, it is characterised in that: using Gardenia Yellow as active constituent, be equipped with Medicinal stone is prepared into any dosage form in pharmacy；Or using Gardenia Yellow as active constituent, add it is anti-oxidant and/or Anti-fatigue active is equipped with any dosage form that pharmaceutic adjuvant is prepared into pharmacy at being grouped as compound.

9. the obtained cape jasmine yellow of the separation method of anti-high altitude anoxia anti-fatigue activity ingredient in cape jasmine as claimed in claim 8 Application of the element as Substituted phenyl-lactic acid ingredient in medicine preparation, it is characterised in that: the dosage form refer to hard capsule, soft capsule, Ordinary tablet, sugar coated tablet, Film coated tablets, special-shaped tablets, chewable tablets, effervescent tablet, dispersible tablet, granule, pill, solution, syrup In any one.

10. the obtained Gardenia Yellow of separation method of anti-high altitude anoxia anti-fatigue activity ingredient in cape jasmine as described in claim 1 Application of the pigment as anti-oxidant, anti-fatigue effect active constituent in terms of preparing anti-oxidant or anti-fatigue health-product containing.