CN100360549C - Pyracantha extract and its preparation method and uses - Google Patents
Pyracantha extract and its preparation method and uses Download PDFInfo
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- CN100360549C CN100360549C CNB2005101000207A CN200510100020A CN100360549C CN 100360549 C CN100360549 C CN 100360549C CN B2005101000207 A CNB2005101000207 A CN B2005101000207A CN 200510100020 A CN200510100020 A CN 200510100020A CN 100360549 C CN100360549 C CN 100360549C
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Abstract
The present invention relates to a pyracantha (Pyracanthafortuneana(Maxim.)Li) extract, which comprises rutin and hyperin. The present invention has the preparation method that 1 to 50 times of water and one or more of methanol, ethanol, propyl alcohol, alcohol, butanol and acetone are added to dried and crushed pyracantha fruit, and an extracting solution can be obtained by extraction; the extracting solution is processed by filtration, centrifugal separation, concentration and freeze drying; macroporous resin, positive silica gel and reversed phase C18 filler are used for column chromatography; elution, separation and purification are carried out. The pyracantha extract can be used for preparing medicine which relieves and reduces adverse reactions such as liver damages, etc. caused by oxygen radicals in a stress reaction, or can be also used for preparing food or food additives which relieve and reduce adverse reactions such as liver damages, etc. caused by oxygen radicals in the stress reaction.
Description
Technical field
The present invention relates to Pyracantha extract and preparation method thereof, the invention still further relates to the application of medicine, food or the foodstuff additive of the untoward reactions such as liver injury that oxyradical causes in preparation is alleviated and improve by stress reaction of fiery sour jujube (Pyracantha fortuneana (Maxim.) Li) extract.
Background technology
Current social develop rapidly, people's quality of life improves year by year.But the problem that can not ignore is that in this course, the psychological pressure from different aspect and level that people experienced is also increasing thereupon.In the face of many-sided stress such as nervous work, lives, being in the sub-health state crowd increases year by year, even the various focuses of holing up in the body, and this situation has become society's one big potential crisis.
Various psychology and the physical stress that people bore on science, can be summarised as " stress ".Stress be the notion of the Salye twenties in 20th century, be meant under the stimulation of a kind of environment outside drastic change that the comprehensive response status that body occurs comprises many-sided reactions such as spirit, nerve, internal secretion and immunity with regard to having proposed.It stress be a kind of common factor of causing a disease, cause injury.Especially chronic persistence stress often cause the pathologic damage of internal organs, tissue, can cause metabolism disorder, brings out multiple disease, and serious harm people's physical and mental health is brought adverse influence to society.
Modern medicine study confirms that also emotionality stress obviously influence the functions such as nerve, internal secretion and immunity of body, stress or the generation development of the destruction of psychological defense mechanism and multiple disease confidential relation is arranged.In neural, internal secretion and this three's of immunity system mutual relationship, active oxygen (reactive oxygen species, ROS) or perhaps harmful oxidation of oxyradical be one of basic mechanism of inherent common between the three.
Under the standard state, the oxygen of body comes from the breathing of lung, is brought into each histoorgan of whole body by the alveolar exchange by blood and supplies biogenic oxygen.Metabolic process also produces certain oxyradical simultaneously, and the strong oxidation capacity of living radical has the unwanted bacteria of killing under standard state, and control is infected, the effect of environment in keeping.When body was experienced stressor, the hypothalmus-pituitary-adrenal axis excitability improved, and acth secretion glucocorticosteroid level raises, and brain alertness improves, and kinetism improves, the blood sugar increased blood pressure, and Pulse Rate increases, thereby mobilizes energy storage, conforms.At this moment, the blood of body flows in the peripheral circulation in a large number, makes internal organs be in a kind of state of hypoxic-ischemic.When the external world stress remove or alleviate, the every physiological function of body recovered normal, and the ischemia-reperfusion of internal organs can cause the burst of active oxygen radical to increase.Active oxygen is in the intravital a large amount of outbursts of machine and accumulate and just can cause DNA chain damage, and the infringement cytolemma promotes the early ageing process, causes various tissues and organ generation pathology.
Liver is one of organ that blood flow enriches the most in the human body, and is very responsive to hypoxic-ischemic, is the major objective that various active oxygens are attacked, and the toxic action of oxyradical plays key effect in the hepatic injury pathogenic process.The experiment confirm liver acute ischemia again under the stress situation of perfusion the oxyradical burst increase.The oxyradical that body produces by enzyme system and non-enzyme system, can attack polyunsaturated fatty acid (the polyunsaturated fatty acid in the microbial film, PUFA) cause peroxidatic reaction of lipid, generate lipid peroxidation product as mda (MDA), ketone group, hydroxyl or hydroperoxy, and new oxyradical etc.Lipid peroxidation not only changes into the lipolysis product that the activity chemistry agent is a non-free radical to active oxygen, and props up the chain type reaction by chain type or chain type and amplify the active oxygen effect.Liver plasma membrane produces a large amount of new active oxy groups in damaged, inspire the chain reaction of lipid peroxidation, therefore and the various cells of the lipid peroxide that produces in can liver injury, so one of main mechanism the when lipid peroxidation of liver plasma membrane is hepatocellular injury.Thereby test that topmost lipid peroxide---the content of MDA can reflect the snperoxiaized degree of liver inner lipid, reflects the degree of liver cell damaged indirectly.The lipid peroxidation macro manifestations that oxyradical causes is: the flowability of liver plasma membrane is descended, and permeability increases, and gpt is released into blood, and enzyme activity increases in the blood.Thereby the vigor of measuring gpt in blood plasma or the serum can be used as an important indicator of liver injury diagnosis.
In view of the development trend of present modern society, people are in increasing, various stress in, body descends to the defense function of active oxygen, internal organs are in sub-health state for a long time.Therefore, from healthy and living habit angle, have anti-stress effect, medicine, food and the foodstuff additive that can improve and alleviate the various untoward reactions such as liver injury that stress cause become the people's attention focus and press for.
Fire sour jujube (Pyracantha fortuneana (Maxim.) Li) is a kind of evergreen wild bush, belong to Rosaceae apple subfamily Pyracantha, its fruit has another name called Fruit of Fortune Firethorn, red Miss, rescues army provisions etc., is widely distributed in China southeast, southwest and each province, northwest, and resource is very abundant.
In recent years, fiery sour jujube has caused the extensive attention of Chinese scholars as the natural anti-oxidation material, and it is to more existing reports such as resistant function of oxyradical.Thereby but up to now about the anti allergic reaction of fiery sour jujube or fiery sour jujube effective constituent rutin and Quercetin 3-galactoside, improve effect that oxyradical burst that stress reaction causes increases the various clinical symptom that cause report as yet.
Summary of the invention
The object of the present invention is to provide a kind of is the Pyracantha extract of main component with rutin and Quercetin 3-galactoside.
The present invention also aims to provide the preparation method of described Pyracantha extract.
The present invention also aims to provide the application in the medicine of the untoward reactions such as liver injury that oxyradical causes in preparation is alleviated and improve by stress reaction of described Pyracantha extract.
The present invention also aims to provide the food of the untoward reactions such as liver injury that oxyradical causes in preparation is alleviated and improve by stress reaction of described Pyracantha extract or the application in the foodstuff additive.
The components by weight percent of Pyracantha extract of the present invention is as follows:
Rutin 1%-50%
Quercetin 3-galactoside 0.3%-50%
Other component surpluses;
Described other components mainly are other flavones, phenolic acid and the glucoside compounds that obtains in the leaching process; The structural formula of rutin and Quercetin 3-galactoside is as follows:
Rutin (rutin) Quercetin 3-galactoside (hyperoside);
Pyracantha extract of the present invention is to adopt one or more any mixed solvent of lower alcohols such as water or water and methyl alcohol, ethanol, acetone isopolarity solvent to extract.More specifically, the preparation method of Pyracantha extract of the present invention comprises the steps:
(1) adds in the water that is equivalent to its 1～50 times of weight, methyl alcohol, ethanol, propyl alcohol, butanols, the acetone one or more in the dry and firethorn fruit pulverized, extract and obtain extracting solution;
(2) extracting solution that obtains of step (1) after filtration, centrifugation concentrates lyophilize;
(3) through macroporous resin, purification on normal-phase silica gel and anti-phase C18 filled column chromatography, wash-out separation and purification.
Extraction described in the step (1) adopts one or more methods in cold soaking, diafiltration, reflux, ultrasonic, the microwave to carry out.
The optimum extraction time in the step (1) is 30 seconds to 24 hours.
Because be rich in rutin and Quercetin 3-galactoside in the firethorn fruit, with class nutritive ingredients such as rich in amino acid, lipid acid, polysaccharide, trace elements, high polar compound in all belonging to, the lower alcohols of general water or different concns, acetone isopolarity solvent or any mixed solvent extract.When extracting, suitable determining alcohol can improve the rate of recovery of activeconstituents in the fiery sour jujube.But for extraction of active ingredients in the fiery sour jujube, said extracted method and arbitrary combination thereof all can reach extract of the present invention.
In view of Pyracantha extract is to develop as the finished product with medicine or food, should select aqueous ethanol to be advisable so consider to extract solvent from safety perspective as far as possible.
The ratio of firethorn fruit and solvent does not have specific limited during extraction, and the ratio of fiery sour jujube and solvent is advisable with 1: 1～50 weight ranges.
The present invention does not have specific limited to extracting temperature, all is suitable for to the solvent boiling point temperature from room temperature.Certainly room temperature, normal pressure and in the solvent boiling point scope, extract ideal.
Extraction time was best from 30 seconds to 24 hours.
When extracting Pyracantha extract, also can in solvent, add materials such as sodium bicarbonate, SODIUM ASCORBATE as required.These extracting method can not influence the biological activity that fiery sour jujube had originally.
Described Pyracantha extract can be used for preparing alleviates and improves the liver injury that caused by oxyradical in the stress reaction and the medicine of clinical symptom thereof.
Described Pyracantha extract also can be used for preparing alleviates and improves the liver injury that caused by oxyradical in the stress reaction and the medicine of clinical symptom thereof.Drug prepared can be taken for a long time, and is safe, has no side effect.
Pyracantha extract of the present invention also can be used for preparing food or the foodstuff additive of alleviating and improving the untoward reactions such as liver injury that caused by oxyradical in the stress reaction.Prepared food or foodstuff additive can be taken for a long time, and are safe, have no side effect.
Pyracantha extract solid agent usage quantity provided by the invention is generally at 0.1% to 99% (W/W), and oral dosage form interpolation concentration is generally comparatively suitable in 0.1% to 50% (W/W) scope usually.In this concentration range, Pyracantha extract is suitable for directly taking, and is accepted by the human consumer easily.Certainly, as the increase along with add-on of the Pyracantha extract of additive, pained sensation may appear.Its terms of settlement can require according to market demand and human consumer's psychology to change formulation, for example uses formulations such as tablet, granule, capsule.Lower concentration Pyracantha extract solution can be used as daily beverage and offers the human consumer.
Pyracantha extract provided by the invention and fiery sour jujube activeconstituents can be used for various oral dosage forms and put on market, are used to prevent and improve by the fatigue that stress cause and liver injury and clinical symptom thereof.Certainly, Pyracantha extract also can combine with pharmaceutical carriers such as vehicle commonly used and make preparation.In case of necessity, can also add preparation additives such as some sanitass, antioxidant, pigment, sweeting agent.
Relatively Shi Yi fiery sour jujube vehicle generally considers to have lactose, sucrose, N.F,USP MANNITOL, starch, crystalline cellulose etc.Lubricant generally should adopt Magnesium Stearate, calcium stearate, talcum powder etc.Solvent uses Purified Water, ethanol, propylene glycol etc. usually.Sanitas generally adopts butylene-chlorohydrin, benzylalcohol, dihydroxy acetic acid etc.Antioxidant is more suitable with sulphite, xitix etc. usually.
Pyracantha extract provided by the invention, can its solution or powder add to be used in the goods oral.Pyracantha extract also can combine with the Other Drinks raw material and make certain beverage product, as fiery sour jujube beverage, soda pop, nectar, lactobacillus drink, sports beverages, soya-bean milk etc.Pyracantha extract it is also conceivable that daily breads such as making biscuit, chocolate, candy, chewing gum, snack, jelly dessert, bread, bean curd, yoghourt.
Pyracantha extract provided by the invention and contained activeconstituents thereof can be used as medicine, heath food, food or foodstuff additive, are used to prevent and improve by the fatigue that stress cause and liver injury and clinical symptom thereof.Among the present invention, the dosage of Pyracantha extract can suitably be selected according to user's conditions such as relative state of health, age, sex and body weight.Adult's mean body weight is with 60 kilograms of calculating, and oral Pyracantha extract generally can be considered at 1 gram to 5 gram scopes in one day, part vic.There are not problems such as side effect more than certain 5 grams yet.
The present invention loads mouse as the oxidative stress experimental model with Restraint Stress (restraint stress), estimate Pyracantha extract to (the oxygen radical absorbance capacity of ORAC in the mice plasma, ORAC), mda in the liver (MDA) content, the active influence of gpt in the blood plasma (ALT).The result shows that the anti-oxidative ability of mice that Pyracantha extract brings out loading is low, the blood plasma Glutamate pyruvate transaminase rises shows the significant effect of improving on the statistics.In the antioxidation in vitro experiment, activeconstituents rutin and Quercetin 3-galactoside that Pyracantha extract is contained also demonstrate stronger antioxygenation.Based on The above results, confirm that of the present invention is the establishment of new purposes of the Pyracantha extract of effective constituent with rutin and Quercetin 3-galactoside.
Can prove that according to above-mentioned experiment the invention provides with rutin and Quercetin 3-galactoside is that the Pyracantha extract of activeconstituents is a kind of safe, from the natural antioxygenation of passing through, thereby reach and alleviate and improve the untoward reactions such as liver injury that cause by oxyradical in the stress reaction, be used to prepare can take for a long time, medicine, food or the foodstuff additive of safe without toxic side effect.
In addition, it is that the Pyracantha extract of activeconstituents also can be used as antioxidant or anti-oxidant constituent raw material that the present invention also points out with rutin and Quercetin 3-galactoside, is used to alleviate and improve the untoward reactions such as liver injury by causing.Because Pyracantha extract provided by the invention is the material of natural origin, and among the people edible experience arranged for a long time, be applicable to the people of each age level and healthy state.Therefore, be that the Pyracantha extract of activeconstituents can be used as heath food, foodstuff additive with rutin and Quercetin 3-galactoside, multiple use is provided.Have, although rutin and Quercetin 3-galactoside are known compounds, it confirms by the present invention is first by the anti-oxidant activity that the ORAC method demonstrates again.
Description of drawings
Fig. 1 is that the HPLC of Pyracantha extract of the present invention analyzes collection of illustrative plates;
Fig. 2 be the described Pyracantha extract of Fig. 1 to restrain stress mice plasma ORAC (ORAC) impact analysis figure;
Fig. 3 be the described Pyracantha extract of Fig. 1 to restrain stress mice plasma in Vitamin C content influence analysis chart;
Fig. 4 be the described Pyracantha extract of Fig. 1 to restrain stress the mouse liver tissue in Vitamin C content influence analysis chart;
Fig. 5 is antioxidation in vitro Capability index (ORAC) the measurement result figure of the described Pyracantha extract of Fig. 1;
Fig. 6 is the described Pyracantha extract effective constituent of Fig. 1---antioxidation in vitro Capability index (ORAC) the measurement result figure of rutin;
Fig. 7 is the described Pyracantha extract effective constituent of Fig. 1---antioxidation in vitro Capability index (ORAC) the measurement result figure of Quercetin 3-galactoside;
Fig. 8 be the described Pyracantha extract of Fig. 1 to restrain stress mice plasma mda (MDA) content impact analysis figure;
Fig. 9 is the described Pyracantha extract of Fig. 1 to impact analysis figure that stress mouse liver mda (MDA) content;
Figure 10 is the described Pyracantha extract of Fig. 1 to stress the active impact analysis figure of mice plasma gpt (ALT).
Embodiment
Embodiment 1: the preparation of Pyracantha extract
The sour jujube dry fruit 100 of getting fire restrains, and suitably pulverizes, and adds 2000 milliliters of refluxing extraction of 60% aqueous ethanolic solution 1 hour, and extracting solution separates through centrifugal (1000 rev/mins, 5 minutes) after gauze filters again, and the lyophilize of recovery supernatant liquor promptly gets Pyracantha extract.This extract adds starch according to a certain percentage, the Magnesium Stearate pharmaceutical adjunct can be made tablet, also can make film coated tablet through the dressing operation.
Embodiment 2: the preparation of Pyracantha extract
The sour jujube dry fruit 200 of getting fire restrains, and suitably pulverizes, and adds distilled water and soaks 3 hours for 2000 milliliters 90 ℃, extracting solution is after gauze filters, separate through centrifugal (1500 rev/mins, 5 minutes) again, obtain fiery sour jujube granule with auxiliary material by spray-drying process after the recovery supernatant concentration.
Embodiment 3: the preparation of Pyracantha extract
The sour jujube dry fruit 50 of getting fire restrains, and suitably pulverizes, and adds 500 milliliters of supersound extraction of acetone soln 0.5 hour, and extracting solution again through centrifugal (800 rev/mins, 3 minutes) separation, reclaims the supernatant liquor lyophilize and promptly gets Pyracantha extract after gauze filters.Lyophilize promptly gets Pyracantha extract.This Pyracantha extract adds a certain proportion of sweeting agent, pigment, antioxidant etc. after the dissolving of water equal solvent, make fiery sour jujube oral liquid or beverage.
Embodiment 4: the preparation of Pyracantha extract
The sour jujube dry fruit 150 of getting fire restrains, and suitably pulverizes, and adds 1500 milliliters of microwave extraction of 30% aqueous ethanolic solution 40 minutes, and extracting solution separates through centrifugal (1200 rev/mins, 5 minutes) after gauze filters again, and the lyophilize of recovery supernatant liquor promptly gets Pyracantha extract.This Pyracantha extract, can its solution or powder add in the food such as biscuit, bread, chocolate, candy, jelly, milk preparation.
The lyophilize extract of the fiery sour jujube of embodiment 1 to 4 gained is used for following experiment, verifies its effect.
Embodiment 5: the separation of fiery sour jujube activeconstituents and analysis
Above-mentioned Pyracantha extract through macroporous resin, purification on normal-phase silica gel and anti-phase C18 filled column chromatography wash-out, is carried out separation and purification.Therefrom separate obtaining component, confirm that two kinds of main components are respectively rutin and Quercetin 3-galactoside through nucleus magnetic resonance method of spectroscopy such as (NMR) mensuration chemical structure.Fig. 1 is the high-efficient liquid phase analysis collection of illustrative plates of Pyracantha extract, and analysis condition is as follows:
Chromatographic column: Shiseido pak C-18 (5 μ m 4.6*250mm);
Flow velocity: 1ml/min;
Detect wavelength: 280nm;
Column temperature: 35 ℃;
Moving phase:
| Time (minute) | 0 | 30 | 45 | 60 |
| Moving phase (acetonitrile-water) | 5％ | 25％ | 100％ | 100％ |
Since with ethanol-extracted, with other polar solvents and use water extraction, perhaps hybrid system extraction, in every gram Pyracantha extract, the content of above-mentioned activeconstituents rutin is 10 milligrams, the content of Quercetin 3-galactoside is 3 milligrams.
Embodiment 6: Pyracantha extract is to the influence of Restraint Stress load mice plasma ORAC level
The experiment of stress loading use animal be 7 age in week male Kunming kind small white mouse, buy (credit number is 2004A019) by Guangdong Medical Lab Animal Center.23 ± 2 ℃ of raising temperatures, lighting hours 12 hours/day (7:00～19:00 turns on light).After raising a week, experimentize.In the Restraint Stress experiment, give the Pyracantha extract aqueous solution by 500mg/kg, 250mg/kg and 125mg/kg dosage oral administration mode.Control group is given and equal-volume distilled water.7 every group of mouse, every mouse fasting forces to put into contained device, the only fasting of blank group.Restrain after 12 hours and under etherization puts into the centrifuge tube that heparin was handled, through 5000 rev/mins of separated plasmas after centrifugal 5 minutes by the heart blood sampling.Be after 3% perchloric acid removes albumen through final concentration again, reclaimed non-albumen supernatant liquor in centrifugal 15 minutes by 15000 rev/mins and be used for ORAC and analyze.Blood plasma ORAC analyzes and uses multi-functional fluorescence analyser (Tecan Genios, Austria), excite with emission wavelength and be respectively 485nm and 525nm, the oxygenant of attacking fluorescein (disodium fluorescein) uses 2,2 '-azobis-2-amidinopropane-dihydrochloride (AAPH; Wako Pure Chemical Industries, Ltd.Osaka, Japan).Potency unit is with reference to watermiscible vitamin E (6-hydro-2,5,7,8-tetramethylchroman-2-carboxylic acid; Trolox, Wako PureChemical Industries, Ltd.), detailed method is with reference to paper (J Agric Food Chem., 51:3273-3279,2003).Experimental result is done statistical procedures with the t check, and the result as shown in Figure 2.Among Fig. 2, (A is the blank group of fasting, i.e. normal control group; B stress blank organize, promptly stress control group; C is the Pyracantha extract high dose group; Dosage group in the D Pyracantha extract; E is the Pyracantha extract low dose group.Experimental result shows: with stress blank organize (distilled water control group) and compare, be that the ability of activity resistent oxyradical obviously strengthens to blood plasma ORAC activity with the contained mouse of Pyracantha extract, wherein high dose group compare with the Stress model group and have statistical significance (
*And have dose-effect relationship between the various dose p＜0.05).Pyracantha extract does not demonstrate any toxic side effect and detrimentally affect in this experiment.
Embodiment 7: Pyracantha extract is to the influence of Restraint Stress load mice plasma and liver Vitamin C level
Stress load laboratory animal, experimental technique and experiment grouping is all identical with embodiment six with dosage.Mouse is restrained and stress under etherization put into the centrifuge tube that heparin was handled by the heart blood sampling after 12 hours, through 5000 rev/mins of separated plasmas after centrifugal 5 minutes.Be 10% metaphosphoric acid solution through final concentration again, centrifugal 15 minutes of 12000rpm gets supernatant liquor and is used for ascorbic analysis.Vitamins C working sample treatment process in the hepatic tissue is to get the perchloric acid solution that 0.1g tissue adds 1ml 3%, and homogenate and under 12000rpm centrifugal 15 minutes are got 20 times of supernatant liquor dilutions and are used for HPLC and analyze.Vitamins C is measured and is used the HP1050 of Hewlett-Packard series of high efficiency liquid chromatograph (comprising column oven, de-aerator, infusion pump, VWD UV-detector); Marco workstation and C
18Chromatographic column (4.6 * 150mm; COSMOSIL Company).Moving phase is 0.01mol/l potassium phosphate buffer (phosphoric acid is transferred pH value to 3): methyl alcohol (v: v=99: 1); Flow velocity is 1.0ml/min; The ultraviolet detection wavelength is 245nm.
Experimental result is done statistical procedures with t check, blood plasma Vitamin C analytical results as shown in Figure 3, among the figure, A is the blank group of fasting, i.e. normal control group; B is the blank group of Stress model, promptly stress control group; C is the Pyracantha extract high dose group; Dosage group in the D Pyracantha extract; E is the Pyracantha extract low dose group; Ordinate zou is the Vitamin C content (microgram) in every milliliter of blood plasma.
Vitamin C analytical results is seen Fig. 4 in the liver, and among the figure, A is the blank group of fasting, i.e. normal control group; B stress blank organize, promptly stress control group; C is the Pyracantha extract high dose group; Dosage group in the D Pyracantha extract; E is the Pyracantha extract low dose group; Ordinate zou is the Vitamin C content (microgram) in every gram hepatic tissue.
Experimental result shows: with stress blank organize (distilled water control group) and compare, obviously strengthen to mice plasma Vitamin C level with Pyracantha extract, high dose group (500mg/kg) have statistical significance (
*P＜0.05), and the various dose group have certain dose-effect relationship.Give with Pyracantha extract after stress mouse liver in Vitamin C compare with model control group, level obviously strengthens, the Vitamin C level of three dosage groups all have statistically utmost point significant difference (
*And have certain dose-effect relationship p＜0.01).Pyracantha extract does not demonstrate any toxic side effect and detrimentally affect in this experiment.
Embodiment 8: the external ORAC activity of Pyracantha extract and effective constituent thereof
The antioxidation in vitro experiment is that Pyracantha extract and monomer component directly are added in the 96 hole enzyme plates, and observation sample inhibited oxidation agent AAPH is to the delayed action of the fluorescence decay of fluorescein (disodium fluorescein).As shown in Figure 5, figure is total, and the negative blank of AAPH shows only adds fluorescent agent, does not add oxyradical and produces substance A APH and antioxidant, and fluorescence intensity is unattenuated; The positive blank of AAPH shows only adds fluorescent agent and oxyradical generation substance A APH, does not add antioxidant, and fluorescence decay is the fastest; Standard antioxidant Trolox (2.5 micromole) is 2.5 micromolar standard antioxidant---the resistance of oxidation of Trolox for the reaction final concentration, and Pyracantha extract (0.78 mcg/ml) and Pyracantha extract (0.39 mcg/ml) are illustrated in the resistance of oxidation of Pyracantha extract under the specific final concentration condition; Ordinate zou is a relative intensity of fluorescence, and the result shows, Pyracantha extract by the speed of active oxygen radical cancellation, and has tangible dose-effect relationship at the external fluorescent substance that can significantly delay between the Pyracantha extract of different concns.
In Fig. 6, AAPH feminine gender, positive blank and standard antioxidant Trolox (2.5 micromole) implication are with last identical, and rutin (0.78 micromole) and rutin (0.39 micromole) are illustrated in the resistance of oxidation of rutin under the specific final concentration condition; Ordinate zou is a relative intensity of fluorescence; Among Fig. 7, AAPH feminine gender, positive blank and standard antioxidant Trolox (2.5 micromole) implication are with last identical, and Quercetin 3-galactoside (0.78 micromole) and Quercetin 3-galactoside (0.39 micromole) are illustrated in the resistance of oxidation of Quercetin 3-galactoside under the specific final concentration condition; Ordinate zou is a relative intensity of fluorescence; Fig. 6,7 shows that monomer component rutin of fiery sour jujube and Quercetin 3-galactoside and some other flavonoid compound also have corresponding activity, promptly at the external fluorescent substance that can significantly delay by the speed of active oxygen radical cancellation.Therefore can reach a conclusion: Pyracantha extract and two kinds of monomer components thereof---rutin and Quercetin 3-galactoside all has a stronger activity resistent oxyradical effect external.It is the flavones and the flavonoid glycoside compound of representative that the activity resistent oxyradical effect that Pyracantha extract is described may come from rutin and Quercetin 3-galactoside.
Embodiment 9: Pyracantha extract is to the mensuration of mda content in blood plasma and the liver
Body produces oxyradical by enzyme system and non-enzyme system can attack polyunsaturated fatty acid in the microbial film, cause lipid peroxidation, and therefore form lipid peroxidation product---mda (MDA), and cause cellular metabolism and dysfunction, in addition dead.Thereby the test MDA amount usually can reflect body inner lipid peroxidation degree, reflect the degree of cell damaged indirectly.
Mouse after the Restraint Stress is got blood and liver under the etherization state, blood centrifugal blood plasma, liver is produced 5% liver tissue homogenate with physiological saline, 3000 rev/mins centrifugal 10 minutes, get blood plasma and the tissue homogenate supernatant liquor is measured wherein mda content.Measuring principle utilize mda to contract with thiobarbituricacid (TBA) and, form red product, in the character that there is maximum absorption band at the 532nm place, measure by colorimetry.Measure and use mda to measure test kit (Nanjing is built up), be reflected in 96 orifice plates and carry out, use microplate reader (Finland's thunder is won the MK3 microplate reader) under 532nm, to measure light absorption ratio (process of specifically measuring is referring to the test kit specification sheets).
Mda content in the computation organization needs the protein content in the known tissue.Therefore, produce 2% liver tissue homogenate with physiological saline equally, utilize the protein content (use the protein quantification test kit, Nanjing is built up, and concrete operation method is seen the test kit specification sheets) in the Coomassie brilliant blue method mensuration tissue.
MDA content in the blood plasma, Pyracantha extract height (500mg/kg), in (250mg/kg) and low (125mg/kg) dosage group all obviously reduce than the Stress model group, wherein high dosage compare with model group with the median dose group and have utmost point significant difference statistically (
*P＜0.01), have certain dose-effect relationship between the administration group, experimental result as shown in Figure 8, A is the blank group of fasting, i.e. normal control group; B is the blank group of Stress model, promptly stress control group; C is the Pyracantha extract high dose group; Dosage group in the D Pyracantha extract; E is the Pyracantha extract low dose group; Ordinate zou is the mda content (nmole/milliliter) in the blood plasma.MDA content in the liver tissue homogenate, the Pyracantha extract high dose group is compared with the Stress model group has significant difference statistically, though middle low dose group has obvious reduction not have statistical significance, experimental result as shown in Figure 9, A is the blank group of fasting, i.e. normal control group among the figure; B is the blank group of Stress model, promptly stress control group; C is the Pyracantha extract high dose group; Dosage group in the D Pyracantha extract; E is the Pyracantha extract low dose group; Ordinate zou is the mda content (nmole/gram) in the liver; Fig. 8 and Fig. 9 illustrate to Pyracantha extract all can significantly reduce stress mice plasma and liver in mda content, illustrate that Pyracantha extract has anti-oxidant in the good body, as to eliminate free radical and protection body effect.Pyracantha extract does not demonstrate any toxic side effect and detrimentally affect in this experiment.
Embodiment 10: Pyracantha extract is to the influence of blood plasma gpt (ALT) level
The vigor of blood plasma gpt is a characteristic index of hepatocellular injury degree.Stress reaction can cause that a large amount of free radicals and cytokine produce.Therefore, these phenomenon explanations, at first the scavenger cell in the activating liver cell activates various cytokines then, causes inflammatory reaction, but the cytotoxicity of induction of lymphocyte, scavenger cell, by number of ways such as hepatocellular apoptosis damage liver cell.
The blood plasma of Restraint Stress mouse is measured the vigor of gpt (ALT) wherein by reitman-frankel method.Measuring principle utilizes gpt (ALT) under 37 ℃ and PH 7.4 conditions, acts on the substrate that L-Ala and α-Tong Wuersuan are formed, and generates pyruvic acid and L-glutamic acid.After reaction for some time, add 2,4 dinitrophenyl hydrazine (DNPH) hydrochloric acid soln, i.e. termination reaction, carbonyl addition in DNPH and the ketone acid simultaneously, generation pyruvic acid phenylhydrazone.Phenylhydrazone is reddish-brown under alkaline condition, colorimetry is measured.Measure in the concrete experiment and use gpt (ALT) to measure test kit (Nanjing is built up), be reflected in 96 orifice plates and carry out, use microplate reader (Finland's thunder is won the MK3 microplate reader) under 492nm, to measure light absorption ratio.
Experimental result as shown in figure 10, among the figure, A is the blank group of fasting, i.e. normal control group; B is the blank group of Stress model, promptly stress control group; C is the Pyracantha extract high dose group; Dosage group in the D Pyracantha extract; E is the Pyracantha extract low dose group; Ordinate zou is gpt (ALT) activity in the blood plasma, and the result represents with international unit (IU/L); This figure shows: the Stress model group significantly raises than normal group ALT, and statistics relatively has significant difference, illustrates to restrain to cause the experiment mice acute liver damage.Pyracantha extract senior middle school low dose group all can significantly reduce since restrain stress due to the rising of mice plasma ALT, wherein high dosage and middle dosage group compare with the Stress model group show significant difference (
*P＜0.05).Small dose group also has the effect that reduces plasma A LT, but action effect does not have statistical significance.Pyracantha extract does not demonstrate any toxic side effect and detrimentally affect in this experiment.
Above-mentionedly experimental results show that provided by the invention is that the Pyracantha extract of activeconstituents is a kind of safe with rutin and Quercetin 3-galactoside, from natural pass through anti-oxidant, thereby reach alleviation and improve the untoward reactions such as liver injury that cause by oxyradical in the stress reaction, be used to prepare medicine that can take for a long time, safe without toxic side effect, food and foodstuff additive.
The embodiment experimental result shows that the various dose group of Pyracantha extract all can reduce plasma A LT activity, can effectively reduce the MDA content of liver tissue homogenate.Therefore infer that transaminase lowering, anti peroxidation of lipid prevent peroxide injury, the generation of minimizing lipid peroxide etc. may be the importance that the untoward reaction mechanisms of action such as liver injury that caused by oxyradical in the stress reaction are alleviated and improved to Pyracantha extract.In addition, it is that the Pyracantha extract of activeconstituents also can be used as antioxidant or anti-oxidant constituent raw material that the present invention also points out with rutin and Quercetin 3-galactoside, is used for some because the prevention and the assisting therapy of the disease that oxidation causes.Because Pyracantha extract provided by the invention is the material of natural origin, and among the people secular edible experience arranged, be suitable for the people of each age level and state of health.Therefore, be that the Pyracantha extract of activeconstituents can be used as medicine, food, foodstuff additive, healthy beverage etc. multiple use is provided with rutin and Quercetin 3-galactoside.Have again,, study its antioxygenation by the ORAC method and confirm by the present invention is first although rutin and Quercetin 3-galactoside are known compounds.
Claims (3)
1, a kind of Pyracantha extract is characterized in that components by weight percent is as follows:
Rutin 1%-50%
Quercetin 3-galactoside 0.3%-50%
Other component surpluses;
Described other components mainly are other flavones, phenolic acid and the glucosides class materials that obtains in the leaching process; The structural formula of rutin and Quercetin 3-galactoside is as follows:
Described Pyracantha extract is prepared by following method, comprises the steps:
(1) adds in the water that is equivalent to its 1～50 times of weight, methyl alcohol, ethanol, propyl alcohol, butanols, the acetone one or more in the dry and firethorn fruit pulverized, extract and obtain extracting solution;
(2) extracting solution that obtains of step (1) after filtration, centrifugation concentrates lyophilize;
(3) through macroporous resin, purification on normal-phase silica gel and anti-phase C18 filled column chromatography, wash-out separation and purification;
Extraction described in the step (1) adopts one or more methods in cold soaking, diafiltration, reflux, ultrasonic, the microwave to carry out; Extraction time is 30 seconds to 24 hours.
2, the application in the medicine of the described Pyracantha extract of the claim 1 liver injury untoward reaction that oxyradical causes in preparation is alleviated and improve by stress reaction.
3, the food of the described Pyracantha extract of the claim 1 liver injury untoward reaction that oxyradical causes in preparation is alleviated and improve by stress reaction or the application in the foodstuff additive.
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