CN105963389A

CN105963389A - Separation method and application of high altitude anoxia-induced fatigue-resistant active ingredient in gardenia jasminoides ellis

Info

Publication number: CN105963389A
Application number: CN201610305926.0A
Authority: CN
Inventors: 李茂星; 王先敏; 毛婷; 曹馨元; 马幸福
Original assignee: 李茂星
Current assignee: Ninety-fourth O Hospital of the Joint Logistics Support Force of the Chinese People's Liberation Army
Priority date: 2016-05-10
Filing date: 2016-05-10
Publication date: 2016-09-28
Anticipated expiration: 2036-05-10
Also published as: CN105963389B

Abstract

The invention relates to a separation method for a high altitude anoxia-induced fatigue-resistant active ingredient in gardenia jasminoides ellis. The method comprises the following steps: (1) performing aqueous extraction on gardenia jasminoides ellis fruits, and performing filtration, concentration and drying to obtain gardenia jasminoides ellis powder; (2) preparing an aqueous solution from the gardenia jasminoides ellis powder; (3) injecting the aqueous solution obtained in step (2) into a treated polyamide column, and performing flushing with distilled water to obtain polyamide column sample effluent and aqueous eluent respectively; (4) mixing and injecting the polyamide column sample effluent and the aqueous eluent into a treatment macroporous resin column, and performing elution, reduced pressure concentration and freeze drying to obtain a geniposide product; (5) flushing the polyamide column to remove impurities with a 10 to 30 percent ethanol solution, flushing the polyamide column with a 30 to 90 percent ethanol solution to obtain 30 to 90 percent ethanol eluent, and performing reduced pressure concentration and freeze drying on the eluent to obtain a gardenia jasminoides ellis yellow pigment product. In addition, the invention also discloses the application of the gardenia jasminoides ellis yellow pigment product. The separation method is simple, easy and convenient to operate, and industrial production can be implemented.

Description

The separation method of anti-high altitude anoxia anti-fatigue activity composition and application thereof in Fructus Gardeniae

Technical field

The present invention relates to the process of enriching of Chinese herbal medicine effective ingredients, particularly relate to anti-high altitude anoxia anti-fatigue activity in Fructus Gardeniae and become The separation method divided and application thereof.

Background technology

After Plain people enters plateau, due to highlands rarefaction of air, the environmental factors such as air forces down, partial pressure of oxygen is low, directly Connect muscle power and the military operation ability affecting body.Wherein, Altitude is the most obvious, at high altitude anoxia ring on the impact of body After being engaged in certain muscle power or mental work under border, easily cause blood lactase acid and other metabolite to pile up, and cause body generation Thanking property acid-base disturbance, thus muscle tone declines and causes the generation of fatigue phenomenon, thus show work capacity and be generally reduced, Severe patient loses performance capacity.Along with the lifting of height above sea level, work capacity substantially reduces.Therefore, try to explore to have anti-tired The medicine of work is highly important, and the effective prevention of screening and the product of fatigue alleviating, for improving altitude environment lower body Physical work capacity, fatigue alleviating for quality of making the life better, improve work efficiency and military operation ability and have positive Meaning.

Fructus Gardeniae be Maguireothamnus speciosus Fructus Gardeniae (Gardenia jasminoidesEllis) dry mature fruit, for clinic Conventional Chinese medicine.Its bitter in the mouth cold in nature, nontoxic, enter the heart, liver, lung, tri-jiao channel, there is the merit of pathogenic fire purging relieving restlessness, clearing away heat and promoting diuresis, removing pathogenic heat from blood and toxic substance from the body Effect.Pharmaceutical research shows that the pharmacological action with Fructus Gardeniae is extensive, have protect the liver, function of gallbladder promoting, analgesia, antiinflammatory, antibacterial, antitumor, fall The multiple pharmacologically active such as blood pressure and blood lipoid is of crucial importance and has the Chinese crude drug composition of very great development value.Wherein Cape jasmine The active component crocin that son is main, is also the main active substances of Tibetan medicine Stigma Croci simultaneously, has antioxidation, anti-tremulous pulse medicated porridge Sample hardening and the effect of regulation blood fat, be the unique a kind of natural carotenoid pigment of nature, by α-crocetin (crocetin) it is combined into two molecule gentiobioses, including crocin-I and crocin-II, there is higher warp Ji is worth, and is widely used in food, medicine and cosmetic field.There are some researches show, crocin and α-crocetin have Removing free radical and significantly antioxidation, and in the pharmacological mechanism of carotenoid, its antioxidant activity is the heaviest Wanting, and have document to report, the medicine with Scavenger of ROS, free radical and antioxidation can improve the anoxia enduring energy of animal Power, alleviates hypoxic insult.

Owing to Stigma Croci yield is extremely low, the market price is higher, and Gardenia Yellow has identical activity and becomes with Stigma Croci Divide crocin, therefore, it is necessary to Gardenia Yellow is carried out science, effectively develops.

Summary of the invention

The technical problem to be solved is to provide anti-high altitude anoxia anti-fatigue activity in a kind of simple Fructus Gardeniae The separation method of composition.

The technical problem to be solved is to provide the application of anti-high altitude anoxia anti-fatigue activity composition in this Fructus Gardeniae.

For solving the problems referred to above, the separation method of anti-high altitude anoxia anti-fatigue activity composition in Fructus Gardeniae of the present invention, bag Include following steps:

(1) after the cape jasmine fruit being dried, being crushed to 20 ~ 40 mesh being distilled water boiling and extraction, through filtration, concentrating under reduced pressure, spray dried Dry, obtain gardenia powder；

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

(3) the aqueous solution of described step (2) gained is injected 8cm by the column volume of 1.0 ~ 3.0 times with the flow velocity of 1.5 ~ 15mL/min 14 ~ 30 mesh polyamide columns that × 100 cm processed, then by the column volume of 1.0 ~ 3.0 times with the flow velocity of 5.0 ~ 10.0 mL/min Use distilled water flushing polyamide column, respectively obtain polyamide column loading effluent, water elution liquid；

(4), after polyamide loading effluent and water elution liquid being merged, amalgamation liquid processed with flow velocity all injections of 10mL/min Macroporous resin column, first by the column volume of 1.0 ~ 3.0 times with the flow velocity of 5.0 ~ 10.0 mL/min distilled water flushing macroporous resin Post, then by the column volume of 2.0 ~ 10.0 times with the ethanol solution that flow velocity volumetric concentration is 10 ~ 70% of 1.5 ~ 10.0 mL/min Rinsing macroporous resin column, obtain the ethanol elution of 10 ~ 70%, this ethanol elution of 10 ~ 70% is through concentrating under reduced pressure, lyophilization After, obtain jasminoidin product；

The most first by the column volume of 1.0 ~ 3.0 times with the ethanol that flow velocity volumetric concentration is 10 ~ 30% of 5.0 ~ 10.0mL mL/min Solution rinses polyamide column remove impurity, then by the column volume of 2.0 ~ 10.0 times with the flow velocity volumetric concentration of 1.5 ~ 10.0 mL/min It is the ethanol solution described polyamide column of flushing of 30 ~ 90%, obtains the ethanol elution of 30 ~ 90%, this ethanol elution of 30 ~ 90% Liquid, after concentrating under reduced pressure, lyophilization, obtains Gardenia Yellow product.

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

Described step (1), described step (4) with described step (5) in the condition of concentrating under reduced pressure each mean that temperature is 50 DEG C ~ 80 ℃。

The described step (1) middle condition being spray-dried refers to that temperature is 120 ~ 140 DEG C, unit interval intake 20 ~ 30 m³, Atomisation pressure 0.1 ~ 0.4MPa.

Described step (4) with described step (5) in cryodesiccated condition each mean that temperature is-55 DEG C ~ 20 DEG C, vacuum is 0.2MPa~0.5 MPa。

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

Described step (3) in the polyamide column processed and described step (4) in the macroporous resin column that processed each mean It is first the ethanol elution of 95% by volumetric concentration, adds till distilled water occurs without muddiness until ethanol elution, then to distill washing De-pillar, to without alcohol taste, to obtain final product.

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

In Fructus Gardeniae as above, the Gardenia Yellow obtained by the separation method of anti-high altitude anoxia anti-fatigue activity composition is made For Substituted phenyl-lactic acid composition in application pharmaceutically, it is characterised in that: with Gardenia Yellow as active component, it is equipped with medicinal stone Any dosage form being prepared as on pharmaceutics；Or with Gardenia Yellow as active component, add antioxidation and/or resisting fatigue is lived Property become to be grouped into compound recipe, and be equipped with any dosage form that pharmaceutic adjuvant is prepared as on pharmaceutics.

Described dosage form refers to hard capsule, soft capsule, ordinary tablet, coated tablet, Film coated tablets, special-shaped tablets, chewable tablet, effervescent Any one in sheet, dispersible tablet, granule, pill, solution, syrup.

In Fructus Gardeniae as above, the Gardenia Yellow obtained by the separation method of anti-high altitude anoxia anti-fatigue activity composition is made For antioxidation, the active component application in terms of preparing antioxidation or anti-fatigue health-product containing of anti-fatigue effect.

The present invention compared with prior art has the advantage that

1, the Gardenia Yellow that the present invention extracts can embody as the drug effect of Substituted phenyl-lactic acid composition from following 4 experiments:

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 grades of Animal Lab.s, room temperature 20 ~ 25 DEG C, air-conditioning constant temperature.

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

(4) medical material and instrument: Fructus Gardeniae water extract, Fructus Gardeniae polysaccharide, Gardenia Yellow, jasminoidin is made by oneself by laboratory, faces use Before make 0.1g/mL aqueous solution；4% paraformaldehyde (lot number: 20150727 Beijing Suo Laibao Science and Technology Ltd.s) sterile injection is used Water (Kelun Pharm Ind Co., Ltd., Sichuan, lot number: M15111005).

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

(5) experimental technique

1. 50 BALB/C mice (male and female half and half) are randomly divided into blank group (0.1mL/10g sterilized water for injection), Cape jasmine Sub-water extract (1.0g.kg-1.d-1), Gardenia Yellow group (1.0g.kg-1.d-1), many groups of (1.0g.kg-1.d-of Fructus Gardeniae 1), jasminoidin (1.0g.kg-1.d-1), gastric infusion, continuous 5 days.Observe mice physiological phenomenon, body weight change.

40 BALB/C mice (male and female half and half) are 2. randomly divided into blank group, and (0.1mL/10g sterile injection is used Water), jasminoidin low dose group (0.5g.kg^-1.d^-1), dosage group (1.0g.kg in jasminoidin^-1.d^-1), jasminoidin high dose group (2.0g.kg^-1.d^-1), continuous 5 days, gastric infusion.Observe mice physiological phenomenon, body weight change.Dissect mice and take half small intestinal, With normal saline flushing clean after, in 5mL EP pipe, add 4mL 4% paraformaldehyde fix small intestinal, room temperature does disease after placing 24h Reason section, carries out HE dyeing.

(6) experimental result

It is administered Gardenia Yellow group mice to act normally, there is no any intoxicating phenomenon, there is safety.

After administration, jasminoidin group mice lethargy, slow in action, shows serious phenomenon of having loose bowels, and stool color is deep Blueness, there is blue spot in tail local, and after being administered two days, jasminoidin group mice has occurred the phenomena of mortality, after being administered three days, Cape jasmine Sub-glycosides group hero Mus is the most dead, and female Mus only survives 1.Fructus Gardeniae water extract group mice has slight phenomenon of having loose bowels.Fructus Gardeniae water carries Taking thing and jasminoidin Mouse Weight alleviates, wherein, the change of jasminoidin group Mouse Weight is substantially.Other groups are all than compared with normal.

The jasminoidin impact on mouse small intestine histo pathological change: HE coloration result shows, naive mice small intestine Structure is normal, and mucous secretion is normal (Figure 1A).Jasminoidin low dose group mouse small intestine tissue change is obvious, and mucous secretion is hyperfunction (Figure 1B).The secretion of jasminoidin high dose group intestinal mucus becomes apparent from (Fig. 1 C).

(7) discuss

Gardenia Yellow main component is crocin (crocin) and the crocetin (crocetin) of carotenoids, has guarantor Protect the pharmacological actions such as myocardial cell, atherosclerosis, antioxidation, Adjust-blood lipid, anticoagulation and antithrombotic.Above-mentioned experimentation Finding that Gardenia Yellow does not has toxic and side effects, safety is good.

Jasminoidin can promote the wriggling of large intestine, has and rushes down lower effect.Fructus Gardeniae water extract and jasminoidin oral administration, to dynamic Thing all has significant discharge function.Above-mentioned test result indicate that jasminoidin has toxicity, and toxicity is quite obvious, Fructus Gardeniae water extraction Can contain part jasminoidin in thing, so mice also can show phenomenon of slightly having loose bowels, and jasminoidin group mice has loose bowels, phenomenon is tight Weight, intestinal mucus secretion increases, and body weight change is obvious, and mortality rate is high, has serious toxicity.

Test the antioxidation activity in vitro test of 2 Fructus Gardeniae the different extracted parts

(1) experiment purpose: by the oxidation-resistant active ingredient of antioxidation in vitro experiment screening Fructus Gardeniae.

(2) reagent and instrument:

1. medical material and reagent

Fructus Gardeniae, purchased from the Yellow River, Lanzhou Chinese Medicinal Materials Markets, is all accredited as madder through the Gansu university of TCM director Yang Xicang pharmacist of Chinese medicine Grass section plant Fructus Gardeniae (Gardenia jasminoides Ellis) dry mature fruit；1,1-diphenyl-2-trinitrobenzene Hydrazine (DPPH, Solarbio company, lot number 101029108), 2,2-azino-bis--(3-ethyl benzo thiazole phenanthroline-6-sulphur Acid) diamino salt (ABTS, Solarbio company, lot number 20151104), TRIS(Solarbio company, lot number 77861), anti-bad Hematic acid (VC, Solarbio company, lot number 50817), pyrogallic acid (Solarbio company, lot number 20151202), 1, 10-phenanthrene quinoline (Solarbio company, lot number 20150916), ethylenediaminetetraacetic acid (EDTA, Aladdin company, lot number 46631)；Sodium nitroprusside, aluminum nitrate, sodium hydroxide, sodium nitrite, the potassium ferricyanide, sodium dihydrogen phosphate, ammonium molybdate, sulfur Acid is ferrous, sulphuric acid, thiobarbituricacidα-, lecithin, sodium thiosulfate, potassium iodide, 30% hydrogen peroxide, ferric chloride, ethylenediamine tetraacetic Acetic acid disodium, trichloroacetic acid, sodium phosphate, potassium peroxydisulfate, glacial acetic acid etc. is commercially available analytical reagent；

2. instrument

3K15 type High speed refrigerated centrifuge (Sigma Co., USA)；The full-automatic fluorescence microplate reader of SpectraMax i3 type is (beautiful Molecular Devices company of state)；WD-9403F type ultraviolet-visible spectrophotometer (hewlette-packard)；BP210S Type electronic balance (Sartorius company of Germany)；DK-8A type electric heating constant temperature tank (above Nereid's grand experimental facilities company limited)； Freezer dryer (Spain's Telster LyoQuest-55 plus type).Experimental temperature 20 ~ 25 DEG C, air-conditioning constant temperature, relatively Humidity: 40% ~ 70%.

(3) experimental technique

1. DPPH free radical scavenging test

According to document, by sample and the V of different quality concentration (0.03 ~ 0.5mg/mL)_CSolution 125 μ L, is separately added into 125 μ L In 0.1mmol/L DPPH 95% ethanol solution, dark at room temperature reaction 30min, does blank with 95% alcohol solvent, surveys Measure its absorbance (A at wavelength 517nm_i)；Measure 125 μ L DPPH 95% ethanol solution and the mixing of 125 μ L 95% ethanol After absorbance (A at ripple 517nm₀)；Measure 125 μ L 95% ethanol solution and 125 μ L sample solution at wavelength 517nm Absorbance (A_j).Calculate its clearance rate by formula (1), and calculate its EC₅₀。

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

2. ABTS free radical scavenging test

Isopyknic 7mmol/L ABTS solution is mixed with 2.45mmol/L potassium peroxydisulfate be allowed to reaction be placed in dark place 12～ 16h, prepares ABTS free radical.With 95% ethanol, ABTS free-atom aqueous solution being diluted to its absorbance at wavelength 734nm is 0.70 ± 0.02.By 100 μ L different quality concentration (0.08～2.0mg/mL) sample and V_CIt is molten that solution adds 3.9mL ABTS free radical In liquid, room temperature places 10min, measures its absorbance (A at wavelength 734nm_i)；Measure 3.9mL ABTS free-atom aqueous solution with Absorbance (A at wavelength 734nm after 100 μ L95% ethanol mixing₀)；Measure 3.9mL 95% ethanol solution and 100 μ L sample Solution absorbance (A at wavelength 734nm_j).Calculate its clearance rate by formula (1), and calculate its EC₅₀。

3. hydroxyl radical free radical clearance test

Take phosphate buffered solution 2.0mL of orthophenanthroline 1.0 mL, pH=7.4 of 0.75 mmol/L, 40 μ g/mL, The FeSO of 0.75 mmol/L₄1.0 mL, the sample solution 1.0mL of different quality concentration, 0.01% H₂O₂1. 0 mL is (fresh Preparation), at 520 nm, measure absorbance A after reacting 60 min after mixing in 37 DEG C of water-baths_s.Blank group is steamed with 1.0 mL Distilled water replaces sample determination absorbance A₀, matched group replaces H with 1.0mL distilled water₂O₂With sample determination optical density A_c, use 4.0mL distilled water and the zeroing of 2.0mL phosphate buffered solution.Calculate its clearance rate by formula (2), and calculate its EC₅₀。

Clearance rate (%)=(A_s-A₀)/(A_c- A₀)

4. anti peroxidation of lipid test

Being dissolved in by 300 mg lecithin in the phosphate buffered solution of 30 mL 10 mmol/L, pH=7.40, ice bath shakes, system Obtain lecithin soln.Take lecithin soln 0.20 mL, add phosphate buffer solution 1.0 mL of pH 7.40, variable concentrations Sample solution 0.50 mL, 2.5 mmol of (0.08～2.0mg/mL)/L EDTA-Fe (II) 1.0 mL, in 37 after mixing Reacting 45 min in DEG C water-bath, add trichloroacetic acid 2.0 mL of 28% (m/V), the sulfur of 1% (m/V) is for barbital Acid 1.0 mL, mixing is placed in 100 DEG C of boiling water baths heats 10 min, measures absorbance A after cooling at 523nm_Sample, use phosphorus Phthalate buffer returns to zero, and blank tube phosphate buffer replaces sample densitometric A₀.Corresponding suppression is calculated according to formula (3) Rate and V_cCompare.

Suppression ratio (%)=(A₀-A_Sample)/A₀

5. the mensuration of total reducing power

By sample solution (0.25～10.0 mg/mL) 1.0 mL of different quality concentration, add 2.5 mL pH=6.6 Phosphate buffer, the potassium ferricyanide solution of 2.5 mL 1%, obtain mixed solution, be placed in 50 DEG C of water bath heat preservation 20 min, add 2. 5 mL10% solution of trichloroacetic acid, gained mixed solution is centrifugal (3 000 r min-1,10 min), incline and take supernatant, Accurate absorption 2.5 mL, add 2.5 mL distilled water and the liquor ferri trichloridi of 0.5 mL 0.1%, and at 700 nm, detection is inhaled Luminosity A and V_cCompare.

6. the mensuration of total antioxidant capacity

By sample solution (0.08～2.0 mg/mL) 0. 1 mL of different quality concentration, add 1.0 mL reagent solution (reagent Solution includes the sulphuric acid of 0.6mol/L, the sodium phosphate of 28 mmol/L, the ammonium molybdate of 4mmol/L).Mixed liquor is placed in 95 DEG C of water 90 min are hatched in bath.Let cool to room temperature, with distilled water as blank, at wavelength 695 nm, survey absorbance A and V_cCompare.

(4) Fructus Gardeniae the different extracted parts antioxidation in vitro result

1. the Fructus Gardeniae the different extracted parts scavenging action to DPPH free radical

As shown in Figure 2, there is notable difference, Vc, Gardenia Yellow in the different extracted parts of Fructus Gardeniae to the Scavenging activity of DPPH free radical Pigment and Fructus Gardeniae crude extract have the ability significantly removing free radical.Wherein, Vc and Gardenia Yellow are to DPPH free radical Scavenging action is notable, and along with the increase of concentration, the scavenging action of DPPH free radical is strengthened, at 0.03-by Gardenia Yellow In obvious dose-effect relationship in 0.5mg/mL concentration range, calculate the Gardenia Yellow IC to DPPH free radical₅₀It is 0.188 mg/mL.Jasminoidin and polysaccharide are suitable to the Scavenging activity of DPPH free radical, and activity is less.

2. the Fructus Gardeniae the different extracted parts scavenging action to ABTS free radical

From the figure 3, it may be seen that there is notable difference, Vc, Gardenia Yellow to the Scavenging activity of ABTS free radical in the different extracted parts of Fructus Gardeniae Pigment and Fructus Gardeniae crude extract have the ability significantly removing free radical.Wherein, Vc and Gardenia Yellow are to ABTS free radical Scavenging action is notable, and the Scavenging activity of Vc is significantly higher than each extract of Fructus Gardeniae.And along with the increase of concentration, Vc, Gardenia Yellow With Fructus Gardeniae crude extract, the scavenging action of ABTS free radical is strengthened, in obvious dose-effect in 0.08～2.0mg/mL concentration range Relation, calculates Vc, the Gardenia Yellow IC to ABTS free radical₅₀It is respectively 0.078 mg/mL, 0.510 mg/mL.Fructus Gardeniae Glycosides and saccharide portion are the most weak to the Scavenging activity of ABTS free radical, and along with the increase of concentration does not has significant change.

3. the Fructus Gardeniae the different extracted parts scavenging action to hydroxyl radical free radical

As shown in Figure 4, the different extracted parts of Fructus Gardeniae all shows the Scavenging activity in various degree to hydroxyl radical free radical, Vc, Gardenia Yellow and Fructus Gardeniae crude extract have the ability significantly removing hydroxyl radical free radical.Wherein, Gardenia Yellow and Fructus Gardeniae are thick Extract has obvious scavenging action to hydroxyl radical free radical, and the Scavenging activity of Vc is significantly higher than each extract of Fructus Gardeniae.And along with concentration Increase, the scavenging action of hydroxyl radical free radical is strengthened, in 0.09～0.5mg/mL concentration by Gardenia Yellow and Fructus Gardeniae crude extract In the range of in obvious dose-effect relationship, calculate Gardenia Yellow and the Fructus Gardeniae crude extract IC to hydroxyl radical free radical₅₀It is respectively 0.198mg/mL、0.327mg/mL.Jasminoidin and saccharide portion are the most weak to the Scavenging activity of hydroxyl radical free radical, and along with concentration Increase do not have significant change.

4. the Fructus Gardeniae the different extracted parts inhibitory action to lipid peroxidation

As shown in Figure 5, the different extracted parts of Fructus Gardeniae all shows the ability of anti peroxidation of lipid in various degree, identical dense In degree scope 0.08～2.0mg/mL, the anti peroxidation of lipid ability of Gardenia Yellow is the strongest and apparently higher than Fructus Gardeniae crude extract, And with the increase of concentration, its anti peroxidation of lipid ability gradually strengthens.The lipid oxidation resistance of jasminoidin and polysaccharide composition The most weak and significantly lower than Gardenia Yellow and Fructus Gardeniae crude extract, when concentration increases, the anti peroxidation of lipid ability of the two is with concentration Increase and there is no significant change.

5. the mensuration of the total reducing power of Fructus Gardeniae the different extracted parts

The reducing power of each material has reacted its antioxidant activity to a certain extent, and the potassium ferricyanide is reduced into by antioxidant Potassium ferrocyanide, the material that potassium ferrocyanide generates with ferric ion has maximum absorption band at 700nm.The biggest thing of absorbance The reducing power of matter is the strongest.Such as Fig. 6, in same concentrations scope 0.25～10.0 mg/mL, each extract part of Fructus Gardeniae is with concentration Increase absorbance improve, wherein, the absorbance of Gardenia Yellow and Fructus Gardeniae crude extract significantly increases with the increase of concentration, Gardenia Yellow is especially pronounced, close with Vc effect trend.

6. the mensuration of Fructus Gardeniae the different extracted parts total antioxidant capacity

If Fig. 7, each extract part of Fructus Gardeniae and Vc are in same concentrations scope 0.08～2.0 mg/mL, measure total anti-under 695nm The absorbance that oxidability obtains raises along with the increase of concentration.Wherein, the effect trend comparison of Gardenia Yellow is obvious, Cape jasmine The absorbance of sub-glycosides and polysaccharide composition changes little with the increase of concentration.

(5) conclusion:

This experiment extract each to Fructus Gardeniae external removing DPPH free radical, ABTS free radical, the ability of hydroxyl radical free radical and lipid Peroxidating rejection ability, total reducing power, total antioxidant capacity are studied, and result shows, each extract of Fructus Gardeniae is to DPPH Free radical, ABTS free radical, hydroxyl radical free radical and lipid peroxidation rejection ability, total reducing power, the equal table of total antioxidant capacity Reveal certain oxidation resistance, and dependency be basically identical, wherein Gardenia Yellow to DPPH free radical, ABTS free radical, The IC of hydroxyl radical free radical₅₀It is respectively 0.188 mg/mL, 0.510 mg/mL, 0.198mg/mL, and there is certain lipid mistake Oxidizing and depressing ability, reducing power and total antioxidant capacity, in the range of finite concentration, along with the increasing of Gardenia Yellow concentration Adding, its antioxidant activity strengthens.Therefore Gardenia Yellow can remove free radical effectively, has good antioxygen also ability.

Test 3 mice atmospheric closed experiments

(1) experiment purpose: by mice atmospheric closed experiment screening active component of cape jasmine Substituted phenyl-lactic acid composition.

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

(3) experimental subject: animal BalB/C mice 60, body weight 20 ± 2g, real by Lanzhou General Hospital of Lanzhou Military Command animal Test section to provide, the quality certification number: the dynamic word the 1402-0201st of Gansu Province doctor.

(4) medical material and instrument: Fructus Gardeniae water extract, Fructus Gardeniae polysaccharide, Gardenia Yellow, jasminoidin is made by oneself by laboratory, faces use Before make 0.05g/mL aqueous solution.Rhodiola rosea capsules (Radix Rhodiolae development center, Chinese People's Liberation Army 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 experimental apparatus factory)；(Germany Sartorius is public for BP210S electronic balance Department).

(5) experimental technique

1. 60 BALB/C mice (male and female half and half) are randomly divided into blank group (0.1mL/10g distilled water), Radix Rhodiolae group (0.5g.kg^-1.d^-1) Fructus Gardeniae ethanol extraction group (0.5g.kg^-1.d^-1), Gardenia Yellow group (0.5g.kg^-1.d^-1), Fructus Gardeniae is many Sugar group (0.5g.kg^-1.d^-1), jasminoidin group (0.5g.kg^-1.d^-1), gastric infusion, continuous 5 days.By mice after last administration 1h Put into the 200 mL ground wide mouthed bottles filling 5 g sodica calx (absorbing carbon dioxide and water), put 1 mice, and use for every bottle Bottleneck smeared by vaseline, covers tightly and prevents gas leakage, timing immediately.(the mice chest does not rises and falls) time is stopped for referring to mouse breathing Mark, observes the time of mice death because of anoxia.

2. 60 BALB/C mice (male and female half and half) are randomly divided into blank group (0.1mL/10g distilled water), model Group (0.1mL/10g distilled water), Radix Rhodiolae group (1.0g.kg^-1.d^-1), Gardenia Yellow low dose group ((0.25g.kg^-1.d^-1), Dosage group ((0.5g.kg in Gardenia Yellow^-1.d^-1), Gardenia Yellow high dose group (1.0g.kg^-1.d^-1), gastric infusion, even Continuous 5 days.After last administration 1h, mice is put into the 200 mL ground wide mouthed bottles filling 5 g sodica calx, puts 1 mice for every bottle, Smear bottleneck with vaseline, cover tightly and prevent gas leakage, timing immediately.(the mice chest does not rises and falls) time is stopped for referring to mouse breathing Mark, observes the time of mice death because of anoxia.Take cerebral tissue and lung tissue and measure wet quality, and with 55 DEG C of electric heating forced air dryings Case is dried 3 days, surveys cerebral tissue and the dry mass of lung tissue, calculated by formula (wet mass-dry mass)/wet quality × 100 Brain lung tissue water content.

(6) experimental result

1. the impact of atmospheric closed mice anti-hypoxia time is compared by active component of cape jasmine with blank group (NG), and the anti-hypoxia time increases Long, Gardenia Yellow group (Crocin) the mice anti-hypoxia time substantially increases (P < 0.01).With positive drug Radix Rhodiolae group (Rhodiola) comparing, Gardenia Yellow group (crocin) the mice anti-hypoxia time substantially increases.The results are shown in Table 1:

Table 1 active component of cape jasmine respectively organize the mice atmospheric closed anti-hypoxia time (, 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 < 0.05, * * p < 0.01, administration group vs blank group.

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

2. the low middle height of Gardenia Yellow each dosage group (Crocin-L, Crocin-M, Crocin-H) mice atmospheric closed anti-hypoxia Time increased compared with model group, Gardenia Yellow high dose group (Crocin-H) and model group (MG) anti-hypoxia time phase Ratio has significance (P < 0.05).The results are shown in Table 2:

Table 2 Gardenia Yellow respectively organize the mice atmospheric closed anti-hypoxia time (,n=10)

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

(,n=10)

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

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

3. Gardenia Yellow (Crocin) is on mouse lung tissue and the impact of brain water content: compare with blank group, model group (MG) mouse lung tissue water content substantially increases (P < 0.01), and brain water content the most substantially increases (P < 0.05).Radix Rhodiolae group And Gardenia Yellow each group of (Crocin-L, Crocin-M, Crocin-H) mouse lung tissue of low middle height and brain group (Rhodiola) Knit water content and significantly reduce (P < 0.05 or P < 0.01) compared with model group, without significant change compared with blank group (NG).Result It is shown in Table 3:

Table 3 Gardenia Yellow on the impact of mouse lung tissue and brain water content (,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,^*P ＜ 0.05 model group vs blank group；^##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 anoxia mainly shows as cellular oxidation process obstacle, energy generates deficiency, anaerobic metabolism product is accumulated, carefully Born of the same parents' dysfunction, even cellularity change.Normobaric hypoxia is non-specific anoxia, mice in hermetic container by anoxia factor Infringement, is mainly reflected in the heart and cerebral anoxia.Studies have reported that trans-crocetin sodium salt can reduce acute hypoxic rat mortality rate, Show that α-crocetin can improve the hypoxia-bearing capability of animal.Radix Rhodiolae has the effect of anti-hypoxia, can significantly improve mouse core The hypoxia-bearing capability of flesh.This experiment, using Radix Rhodiolae as positive drug, studies Gardenia Yellow (Crocin) Substituted phenyl-lactic acid, result Show: Gardenia Yellow (Crocin) has significant prolongation effect to the time-to-live of mice normobaric hypoxia condition.Anoxia causes Pulmonary artery pressure increases and hemodynamic change；Inflammatory mediator can cause impaired vascular endothelium, and permeability raises, big quantity of fluid Enter interstitial lung, and the liquid entering interstitial lung can enter alveolar after can not being efficiently absorbed, thus form alveolar edema. Anoxia can cause blood brain barrier (BBB) permeability to increase, and BBB permeability increase can should send out vascular cerebral edema.This experiment Research finds that Gardenia Yellow (Crocin) can significantly alleviate atmospheric closed mouse lung tissue and brain water content, can alleviate Cerebral edema and pulmonary edema.

Mice power under 4 hypobaric hypoxia environments of testing exhausts swimming test

(1) experiment purpose: by the anti-fatigue active of simulation hypobaric hypoxia environment research Gardenia Yellow.

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

(4) reagent and instrument:

1. reagent: Rhodiola rosea capsules (Radix Rhodiolae development center, Chinese People's Liberation Army Tibet Military Area Command, lot number: 130604)；Macropore Adsorbent resin (D101 clean product type, Tianjin sea light Chemical Co., Ltd., lot number: 070305)；75% ethanol (Shandong rel health Sterilization Science and Technology Co., Ltd., lot number: 130619)；CK testing cassete (product batch number: 20151201), liver/muscle glycogen test kit (product batch number: 20151202), ultramicron ATP (Na+K+) testing cassete (product batch number: 20151203), ultramicron ATP (produce Lot number: Ca²⁺Mg²⁺), testing cassete (product batch number: 20151203), succinate dehydrogenase (product batch number: SDH) testing cassete (20151203), pyruvate kinase (PK) test kit (product batch number: 20151203), malonaldehyde (product batch number: MDA) testing cassete (20151202), reduced glutathion (GSH) test kit (product batch number: 20151203), pyruvate reagent box (produce and criticize Number: 20151203), Coomassie brilliant blue (product batch number: 20151218), SOD test kit (survey total) (product batch number: 20151130), BCA measure test kit (product batch number: 20151203), lactic acid dehydrogenase (LDH) testing cassete (product batch number: 20151006), lactic acid (LD) testing cassete (product batch number: 20151014) is built up biotechnology research by Nanjing is provided；10 × PBS(lot number: 20140807) purchased from Beijing Suo Laibao Science and Technology Ltd..Electric drying oven with forced convection (101A, Shanghai experiment instrument Device factory)；BP210S electronic balance (Sartorius company of Germany).

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

(5) experimental technique

1. mice swimming time detection

72 BALB/C mice, are randomly divided into 6 groups, i.e. often oxygen matched group, model group, Rhodiola rosea capsules positive controls and Fructus Gardeniae Flavochrome basic, normal, high dosage group, often group 12.Often oxygen matched group and model group gavage sterilized water for injection (0.2mL/20g), Remain each group respectively gavage give corresponding reagent.In addition to normal oxygen matched group, remaining each group is placed in large-scale low-pressure oxygen cabin simulation sea Pulling out 8000 for m altitude environments (in cabin pressure: 35.9 kPa, partial pressure of oxygen: 7.4 kPa), every morning 9:00 is with 10 m s^-1Flow velocity Drop to pressure in 4000 m(cabins: 62.1 kPa, partial pressure of oxygen: 13.8 kPa), experimenter enters cabin, gastric infusion in cabin, Changing water food and bedding and padding, continuous 5 d, after every day is administered, by cabin inner height with 10 m s^-1Flow velocity at the uniform velocity rise to pre-Dinghai Dialling 8000 m, animal freely ingests and intakes during this period.Often oxygen control rats is raised in Animal House simultaneously.Last is administered 1 After h, test at height above sea level 4000 m, lie at 1/3 tail with the load (galvanized wire) of Mice Body quality 7%, put into the depth of water 18 cm's In tank (50 cm × 30, cm × 40 cm), water temperature controls in (25 ± 1) DEG C, carries out swimming with a load attached to the body, and record mice is from putting into water Groove to mice sinks to the time that water 7 s fails to float on the surface, and is mice swimming time.

2. mice serum horizontal detection

After power exhausts swimming, taking out immediately, pluck eyeball and take blood, after standing 1 h, 3500 r/min are centrifuged 10 min, isolated blood Clearly.

3. routine blood test

Take 20 μ l EDTA anticoagulations, after adding 180 μ l blood dilution liquids, animal's whole blood cytoanalyze detects blood normal Rule index.

4. mouse muscle and hepatic tissue Activity determination

, take quadriceps femoris and the liver of two hind legs at cervical dislocation after death, clean with ice normal saline and dry, weigh 140- 150mg organizes, and cold PBS makes 10% homogenate, and 3500 r/min are centrifuged 10 min, take supernatant.

5. statistical procedures

Application SPSS 17.0 statistical software carries out statistical procedures.Result withRepresent, compare employing variance between group and divide Analysis and t check.Statistically significant for difference with P ＜ 0.05.

(6) results of animal

Respectively group mice swimming power exhausts time detecting

Comparing with normal oxygen matched group, Rhodiola rosea capsules group and Gardenia Yellow basic, normal, high dosage group can significantly extend mice power Exhausting swimming time, difference has statistical significance (P ＜ 0.01 or P ＜ 0.05).Each group mice swimming time is shown in Table 4.

Table 4 mice swimming time result (,n=12)

Note:^**P ＜ 0.01,^*P ＜ 0.05 model group vs blank group；^##P ＜ 0.01,^#P ＜ 0.05, model group vs is administered Group.

Respectively the detection of group mouse metabolism product is compared with normal oxygen group, and the content of anoxia model group Mouse Blood blood urea nitrogen is notable Rising, difference has statistical significance (P ＜ 0.05).Rhodiola rosea capsules group and Gardenia Yellow is compared with anoxia model group Dosage group high, middle can significantly reduce the content of blood urea nitrogen, and difference has statistical significance (P ＜ 0.05)；Compare 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). Comparing 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 acetone acid, in Gardenia Yellow, dosage group also can improve the content of acetone acid in serum, and difference has statistics meaning Justice (P ＜ 0.05)；Comparing with normal oxygen group, in anoxia model group mouse liver, muscle and serum, lactic acid content significantly raises, difference There is statistical significance (P ＜ 0.01 or P ＜ 0.05).Comparing with anoxia model group, Rhodiola rosea capsules group and Gardenia Yellow are high Dosage group can significantly reduce lactic acid content in mouse liver, muscle and serum, and difference has statistical significance (P ＜ 0.01 or P ＜ 0.05) in Gardenia Yellow, low dose group also can reduce the content of lactic acid in serum, difference has statistical significance (P ＜ 0.01).Respectively organize the testing result of BUN, acetone acid and LD in mouse liver, muscle, serum and be shown in Table 5:

Table 5 respectively organize BUN, acetone acid and LD in mouse liver, muscle, serum detection (,n=12)

Respectively the detection of group mice oxidation index is compared with normal oxygen group, and in anoxia model group mouse liver, muscle, GSH is notable Reducing, difference has statistical significance (P ＜ 0.01 or P ＜ 0.05).Compare with anoxia model group, Rhodiola rosea capsules group and Fructus Gardeniae Flavochrome high dose group can significantly improve GSH content, and difference has statistical significance (P ＜ 0.01 or P ＜ 0.05), Gardenia Yellow In pigment, dosage group also can improve the content of GSH in liver, and difference has statistical significance (P ＜ 0.05)；Compare with normal oxygen group, In anoxia model group mouse liver, muscle, MDA content significantly raises, and difference has statistical significance (P ＜ 0.01).With anoxia mould Type group compares, and Rhodiola rosea capsules group and Gardenia Yellow middle and high dosage group can significantly reduce in mouse liver and muscular tissue MDA content, difference has statistical significance (P ＜ 0.01 or P ＜ 0.05)；Compare with normal oxygen group, anoxia model group mouse liver, In muscle and serum, SOD activity significantly reduces, and difference has statistical significance (P ＜ 0.01).Compare with anoxia group, Radix Rhodiolae glue Wafer Gardenia Yellow high dose group can significantly improve SOD activity in mouse liver, muscle and serum, and difference has statistics Meaning (P ＜ 0.01 or P ＜ 0.05), and in Gardenia Yellow, low dose group also can improve the content of SOD in liver and muscle, Difference has statistical significance (P ＜ 0.01 or P ＜ 0.05).Respectively organize GSH, MDA, SOD inspection in mouse liver, muscle, serum Survey the results are shown in Table 6.

GSH, MDA, SOD testing result in mouse liver, muscle, serum respectively organized by table 6

Respectively group mouse metabolism regulatory enzyme detection

Comparing with normal oxygen group, in anoxia model group mouse liver, muscle, serum, LDH activity weakens, and difference has statistical significance (P ＜ 0.01 or P ＜ 0.05)；Compare with anoxia model group, Rhodiola rosea capsules group and Gardenia Yellow group high dose group Mouse Liver In dirty, muscle, serum, LDH activity strengthens, difference statistically significant (P ＜ 0.01 or P ＜ 0.05), dosage in Gardenia Yellow Group also can improve the content of LDH in serum, and difference has statistical significance (P ＜ 0.01).Respectively organize mouse liver, muscle, serum Middle LDH testing result is shown in Table 7.

LDH testing result in mouse liver, muscle, serum respectively organized by table 7

Respectively group mice Energy Metabolism Enzyme detection

Compare with normal oxygen group, MDH, SDH, PK, Ca-Mg-ATP enzyme and Na-K-in anoxia model group mouse liver, muscle and serum Atpase activity significantly reduces, and difference has statistical significance (P ＜ 0.01).Compare with anoxia model group, Rhodiola rosea capsules group and Gardenia Yellow middle and high dosage group can significantly strengthen MDH, SDH, PK, Ca-Mg-ATP enzyme and Na-K-in Muscle Tissue Atpase activity, difference has statistical significance (P ＜ 0.01 or P ＜ 0.05), and Gardenia Yellow low dose group also can improve muscle Na-K-ATP enzymatic activity in PK and liver in middle SDH, serum, difference has statistical significance (P ＜ 0.01).The results are shown in Table 8, table 9:

Table 8 respectively organizes MDH, SDH, PK, Ca-Mg-ATP enzyme and Na-K-ATP enzyme testing result in mouse liver, muscle, serum

Table 9 respectively organizes mice Ca-Mg-ATP enzyme, Na-K-ATP and CK enzyme testing result

Respectively group mice ergastic substances detection

Comparing with normal oxygen group, anoxia model group Glycogen content significantly reduces, and difference has statistical significance (P ＜ 0.01 or P ＜ 0.05).Comparing with anoxia model group, Rhodiola rosea capsules group and Gardenia Yellow each dosage group can dramatically increase glycogen deposit Amount, difference has statistical significance (P ＜ 0.01 or P ＜ 0.05).Each group Glycogen testing result is shown in Table 10.

Glycogen testing result respectively organized by table 10

(7) discuss:

Result shows, Gardenia Yellow has good resisting fatigue effect.Gardenia Yellow can strengthen the work of lactic acid dehydrogenase Power, reduces lactic acid and piles up, reduce the concentration of BUN, it is seen that Gardenia Yellow can alleviate muscle tissue damage, thus reduces the energy The consumption of material, delay fatigue；Hepatic glycogen amount of storage can be passed through as the evaluation index of internal energy substance, Gardenia Yellow Raise Body development and liver glycogen level, during motion, increase the release of ATP, promote energy metabolism；By reducing because of strenuous exercise The membrane structure damage caused, reduces CK enzymatic activity, maintains homeostasis, and then increases exercise intensity and movement time, it is achieved Its anti-exercise fatigue effect；SOD is widely present in body cell, the superoxide anion of the excess that can produce in purged body from By base, protection DNA, protein and cell membrane exempt from the destruction of superoxide radical, and MDA can directly reflect Free Radical Level, its Content height is the important symbol of tissue cell insult, it is known that Gardenia Yellow effect in terms of removing free radical, antioxidation shows Write, thus reach the effect of resisting fatigue；The change of SDH activity can reflect cellular energy metabolism situation, and Gardenia Yellow can strengthen SDH and MDH activity, promotes body aerobic oxidation metabolism；PK is the rate-limiting enzyme of glycolytic pathway, plays during zymolysis Decisive role, Gardenia Yellow can increase PK content, promotes glycolysis, increases ATP, thus reaches the effect of resisting fatigue.

2, the present invention can efficiently separate enrichment gardenia yellow pigment with high color value, jasminoidin has the most also carried out enrichment with pure Change, thus improve the utilization rate of medical material, for by Gardenia Yellow science, effectively develop, and then be applied to sport nutrition food Lay a good foundation in product field.

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

Accompanying drawing explanation

Below in conjunction with the accompanying drawings the detailed description of the invention of the present invention is described in further detail.

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

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

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

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

Fig. 5 is the lipid oxidation resistance of Fructus Gardeniae the different extracted parts of the present invention.

Fig. 6 is total reducing power of Fructus Gardeniae the different extracted parts of the present invention.

Fig. 7 is the total antioxidant capacity of Fructus Gardeniae the different extracted parts of the present invention.

Detailed description of the invention

The separation method of anti-high altitude anoxia anti-fatigue activity composition in embodiment 1 Fructus Gardeniae, comprises the following steps:

(1) after the cape jasmine fruit being dried, being crushed to 20 mesh being distilled water boiling and extraction, through filtration, concentrating under reduced pressure, spray drying, Obtain 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 of times is 2 times, time each Between be 1 hour.

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

The condition being spray-dried refers to that temperature is 120 DEG C, unit interval intake 20 m³, atomisation pressure 0.1MPa.

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

(3) 14 aqueous solution of step (2) gained processed with the flow velocity injection of 1.5mL/min by the column volume of 1.0 times Mesh polyamide column, then by the column volume of 1.0 times with the flow velocity of 5.0 mL/min distilled water flushing polyamide column, respectively obtain poly- Amide post loading effluent, water elution liquid.

Wherein: the polyamide column processed refer to by the polyamide column of a size of 8cm × 100 cm by volumetric concentration be first The ethanol elution of 95%, adds till distilled water occurs without muddiness until ethanol elution, then with distilled water eluting pillar to without alcohol Taste, to obtain final product.

(4) after polyamide loading effluent and water elution liquid being merged, amalgamation liquid with flow velocity all injections of 10mL/min at The macroporous resin column managed, first by the column volume of 1.0 times with the flow velocity of 5.0 mL/min distilled water flushing macroporous resin column, then Rinse macroporous resin column by the column volumes of 2.0 times with the ethanol solution that flow velocity volumetric concentration is 30% of 1.5 mL/min, obtain The ethanol elution of 30%, this ethanol elution of 30%, after concentrating under reduced pressure, lyophilization, obtains jasminoidin product.

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

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

Cryodesiccated condition refers to that temperature is-55 DEG C, and vacuum is 0.5MPa.

The macroporous resin column processed refers to macroporous resin be first the ethanol elution of 95% by volumetric concentration, until ethanol Eluent adds till distilled water occurs without muddiness, then with distilled water eluting pillar to without alcohol taste, to obtain final product.

The most first rinse with the ethanol solution that flow velocity volumetric concentration is 10% of 5.0mL mL/min by the column volume of 1.0 times Polyamide column remove impurity, then rinse with the ethanol solution that flow velocity volumetric concentration is 75% of 1.5 mL/min by the column volume of 2.0 times Described polyamide column, obtains the ethanol elution of 75%, and this ethanol elution of 75%, after concentrating under reduced pressure, lyophilization, to obtain final product Gardenia Yellow product.

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

The separation method of anti-high altitude anoxia anti-fatigue activity composition in embodiment 2 Fructus Gardeniae, comprises the following steps:

(1) after the cape jasmine fruit being dried, being crushed to 30 mesh being distilled water boiling and extraction, through filtration, concentrating under reduced pressure, spray drying, Obtain 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 of times is 3 times, every time Time is 1 hour.

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

The condition being spray-dried refers to that temperature is 140 DEG C, unit interval intake 30 m³, atomisation pressure 0.4MPa.

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

(3) 30 mesh aqueous solution of step (2) gained processed with the flow velocity injection of 15mL/min by the column volume of 3.0 times Polyamide column, then by the column volume of 3.0 times with the flow velocity of 10.0 mL/min distilled water flushing polyamide column, respectively obtain poly- Amide post loading effluent, water elution liquid.

Wherein: the polyamide column processed is with embodiment 1.

(4) after polyamide loading effluent and water elution liquid being merged, amalgamation liquid with flow velocity all injections of 10mL/min at The macroporous resin column managed, first by the column volume of 3.0 times 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 flow velocity volumetric concentration is 10% of 10.0 mL/min again by the column volume of 10.0 times, Obtaining the ethanol elution of 10%, this ethanol elution of 10%, after concentrating under reduced pressure, lyophilization, obtains jasminoidin product.

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

Cryodesiccated condition refers to that temperature is 20 DEG C, and vacuum is 0.2 MPa.

The macroporous resin column processed is with embodiment 1.

The most first rinse with the ethanol solution that flow velocity volumetric concentration is 30% of 10.0mL mL/min by the column volume of 3.0 times Polyamide column remove impurity, then rush with the ethanol solution that flow velocity volumetric concentration is 30% of 10.0 mL/min by the column volume of 10.0 times Washing described polyamide column, obtain the ethanol elution of 30%, this ethanol elution of 30% is after concentrating under reduced pressure, lyophilization, i.e. Obtain Gardenia Yellow product.

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

The separation method of anti-high altitude anoxia anti-fatigue activity composition in embodiment 3 Fructus Gardeniae, comprises the following steps:

(1) after the cape jasmine fruit being dried, being crushed to 40 mesh being distilled water boiling and extraction, through filtration, concentrating under reduced pressure, spray drying, Obtain 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 of times is 2 times, time each Between be 1 hour.

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

The condition being spray-dried refers to that temperature is 130 DEG C, unit interval intake 25 m³, atomisation pressure 0.2MPa.

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

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

Wherein: the polyamide column processed is with embodiment 1.

(4) after polyamide loading effluent and water elution liquid being merged, amalgamation liquid with flow velocity all injections of 10mL/min at The macroporous resin column managed, first by the column volume of 2.0 times with the flow velocity of 8.0 mL/min distilled water flushing macroporous resin column, then Rinse macroporous resin column by the column volumes of 6.0 times with the ethanol solution that flow velocity volumetric concentration is 30% of 5.0 mL/min, obtain The ethanol elution of 30%, this ethanol elution of 30%, after concentrating under reduced pressure, lyophilization, obtains jasminoidin product.

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

Cryodesiccated condition refers to that temperature is-30 DEG C, and vacuum is 0.3 MPa.

The macroporous resin column processed is with embodiment 1.

The most first rinse with the ethanol solution that flow velocity volumetric concentration is 15% of 8.0mL mL/min by the column volume of 2.0 times Polyamide column remove impurity, then rinse with the ethanol solution that flow velocity volumetric concentration is 50% of 5.0 mL/min by the column volume of 6.0 times Described polyamide column, obtains the ethanol elution of 50%, and this ethanol elution of 50%, after concentrating under reduced pressure, lyophilization, to obtain final product Gardenia Yellow product.

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

The separation method of anti-high altitude anoxia anti-fatigue activity composition in embodiment 4 Fructus Gardeniae, comprises 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 of times is 3 times, every time Time is 1 hour.

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

The condition being spray-dried refers to that temperature is 125 DEG C, unit interval intake 20 m³, atomisation pressure 0.3MPa.

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

Wherein: the polyamide column processed is with embodiment 1.

(4) after polyamide loading effluent and water elution liquid being merged, amalgamation liquid with flow velocity all injections of 10mL/min at The macroporous resin column managed, first by the column volume of 1.5 times with the flow velocity of 6.0 mL/min distilled water flushing macroporous resin column, then Rinse macroporous resin column by the column volumes of 4.0 times with the ethanol solution that flow velocity volumetric concentration is 50% of 3.0 mL/min, obtain The ethanol elution of 50%, this ethanol elution of 50%, after concentrating under reduced pressure, lyophilization, obtains jasminoidin product.

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

Cryodesiccated condition refers to that temperature is-10 DEG C, and vacuum is 0.4 MPa.

The macroporous resin column processed is with embodiment 1.

The most first rinse with the ethanol solution that flow velocity volumetric concentration is 20% of 6.0mL mL/min by the column volume of 1.5 times Polyamide column remove impurity, then rinse with the ethanol solution that flow velocity volumetric concentration is 75% of 3.0 mL/min by the column volume of 4.0 times Described polyamide column, obtains the ethanol elution of 75%, and this ethanol elution of 75%, after concentrating under reduced pressure, lyophilization, to obtain final product Gardenia Yellow product.

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

The separation method of anti-high altitude anoxia anti-fatigue activity composition in embodiment 5 Fructus Gardeniae, comprises 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 of times is 2 times, every time Time is 1 hour.

The condition being spray-dried refers to that temperature is 140 DEG C, unit interval intake 25 m³, atomisation pressure 0.1MPa.

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

Wherein: the polyamide column processed is with embodiment 1.

(4) after polyamide loading effluent and water elution liquid being merged, amalgamation liquid with flow velocity all injections of 10mL/min at The macroporous resin column managed, first by the column volume of 2.5 times with the flow velocity of 9.0 mL/min distilled water flushing macroporous resin column, then Rinse macroporous resin column by the column volumes of 6.0 times with the ethanol solution that flow velocity volumetric concentration is 70% of 8.0 mL/min, obtain The ethanol elution of 70%, this ethanol elution of 70%, after concentrating under reduced pressure, lyophilization, obtains jasminoidin product.

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

Cryodesiccated condition refers to that temperature is 10 DEG C, and vacuum is 0.4MPa.

The macroporous resin column processed is with embodiment 1.

The most first rinse with the ethanol solution that flow velocity volumetric concentration is 25% of 9.0mL mL/min by the column volume of 2.5 times Polyamide column remove impurity, then rinse with the ethanol solution that flow velocity volumetric concentration is 90% of 8.0 mL/min by the column volume of 8.0 times Described polyamide column, obtains the ethanol elution of 90%, and this ethanol elution of 90%, after concentrating under reduced pressure, lyophilization, to obtain final product Gardenia Yellow product.

The Gardenia Yellow extracted in above-described embodiment 1 ~ 5 refers in application pharmaceutically as Substituted phenyl-lactic acid composition: With Gardenia Yellow as active component, it is equipped with any dosage form that medicinal stone is prepared as on pharmaceutics；Or with Fructus Gardeniae yellow Element is active component, adds antioxidation and/or anti-fatigue active and becomes to be grouped into compound recipe, and is equipped with pharmaceutic adjuvant and is prepared as pharmaceutics On any dosage form.Dosage form refer to hard capsule, soft capsule, ordinary tablet, coated tablet, Film coated tablets, special-shaped tablets, chewable tablet, Any one in effervescent tablet, dispersible tablet, granule, pill, solution, syrup.

Embodiment 1 ~ 5 gained Gardenia Yellow is mixed according to the ratio of 1:1 by embodiment 6 with Herba Cistanches extract, Use practice of pharmacy to make capsule, be used for preventing and treat high altitude anoxia type tired.

Embodiment 1 ~ 5 gained Gardenia Yellow is mixed according to the ratio of 3:1 by embodiment 7 with Herba Cistanches extract, Use practice of pharmacy to make dispersible tablet, be used for preventing and treat high altitude anoxia type tired.

Embodiment 1 ~ 5 gained Gardenia Yellow is mixed according to the ratio of 5:1 by embodiment 8 with Herba Cistanches extract, Use practice of pharmacy to make granule, be used for preventing and treat high altitude anoxia type tired.

Embodiment 1 ~ 5 gained Gardenia Yellow is mixed according to the ratio of 7:1 by embodiment 9 with Herba Cistanches extract, Use practice of pharmacy to make pill, be used for preventing and treat high altitude anoxia type tired.

Embodiment 1 ~ 5 gained Gardenia Yellow is mixed according to the ratio of 1:3 by embodiment 10 with Herba Cistanches extract Close, use practice of pharmacy to make solution, be used for preventing and treat high altitude anoxia type tired.

Gardenia Yellow obtained by above-described embodiment 1 ~ 5 can exist as the active component of antioxidation, anti-fatigue effect Prepare the application in terms of antioxidation or anti-fatigue health-product containing.

Claims

1. the separation method of anti-high altitude anoxia anti-fatigue activity composition in Fructus Gardeniae, comprises the following steps:

2. the separation method of anti-high altitude anoxia anti-fatigue activity composition in Fructus Gardeniae as claimed in claim 1, it is characterised in that: described The step (1) middle extraction conditions that decocts refers to that feed liquid mass ratio is 1:8 ~ 10, and temperature is 90 ~ 100 DEG C, and number of times is 2 ~ 3 times, time each Between be 1 hour.

3. the separation method of anti-high altitude anoxia anti-fatigue activity composition in Fructus Gardeniae as claimed in claim 1, it is characterised in that: described Step (1), described step (4) with described step (5) in the condition of concentrating under reduced pressure each mean that temperature is 50 DEG C ~ 80 DEG C.

4. the separation method of anti-high altitude anoxia anti-fatigue activity composition in Fructus Gardeniae as claimed in claim 1, it is characterised in that: described The step (1) middle condition being spray-dried refers to that temperature is 120 ~ 140 DEG C, unit interval intake 20 ~ 30 m³, atomisation pressure 0.1 ~ 0.4MPa。

5. the separation method of anti-high altitude anoxia anti-fatigue activity composition in Fructus Gardeniae as claimed in claim 1, it is characterised in that: described Step (4) with described step (5) in cryodesiccated condition each mean that temperature is-55 DEG C ~ 20 DEG C, vacuum is 0.2MPa ~ 0.5 MPa。

6. the separation method of anti-high altitude anoxia anti-fatigue activity composition in Fructus Gardeniae as claimed in claim 1, it is characterised in that: described Step (4) middle macroporous resin column refers to any one in D-101, HPD100, XDA-6, DM-130, DA201 type macroporous resin.

7. the separation method of anti-high altitude anoxia anti-fatigue activity composition in Fructus Gardeniae as claimed in claim 1, it is characterised in that: described Step (3) in the polyamide column processed and described step (4) in the macroporous resin column that processed each mean and first use volumetric concentration It is the ethanol elution of 95%, adds till distilled water occurs without muddiness until ethanol elution, then with distilled water eluting pillar to without alcohol Taste, to obtain final product.

8. Fructus Gardeniae yellow obtained by the separation method of anti-high altitude anoxia anti-fatigue activity composition in Fructus Gardeniae as claimed in claim 1 Element as Substituted phenyl-lactic acid composition in application pharmaceutically, it is characterised in that: with Gardenia Yellow as active component, be equipped with medicinal Any dosage form that stone is prepared as on pharmaceutics；Or with Gardenia Yellow as active component, add antioxidation and/or resist tired Labor active component composition compound recipe, and it is equipped with any dosage form that pharmaceutic adjuvant is prepared as on pharmaceutics.

9. Fructus Gardeniae yellow obtained by the separation method of anti-high altitude anoxia anti-fatigue activity composition in Fructus Gardeniae as claimed in claim 8 Element as Substituted phenyl-lactic acid composition in application pharmaceutically, it is characterised in that: described dosage form refers to hard capsule, soft capsule, common In sheet, coated tablet, Film coated tablets, special-shaped tablets, chewable tablet, effervescent tablet, dispersible tablet, granule, pill, solution, syrup Any one.

10. Gardenia Yellow obtained by the separation method of anti-high altitude anoxia anti-fatigue activity composition in Fructus Gardeniae as claimed in claim 1 Pigment is as antioxidation, the active component application in terms of preparing antioxidation or anti-fatigue health-product containing of anti-fatigue effect.