CN104147205A - Preparation method and application for longan seed extracts - Google Patents
Preparation method and application for longan seed extracts Download PDFInfo
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- CN104147205A CN104147205A CN201410387909.7A CN201410387909A CN104147205A CN 104147205 A CN104147205 A CN 104147205A CN 201410387909 A CN201410387909 A CN 201410387909A CN 104147205 A CN104147205 A CN 104147205A
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Abstract
The invention relates to a preparation method and the application for longan seed extracts. The preparation method comprises the following steps: (1) compounding an extract solution with specific concentration; (2) heating the temperature of the extract solution to a specific temperature; (3) putting ground longan seed particles into the extract solution for extraction; (4) filtering and concentrating the solution which is extracted; (5) freezing and drying the solution which is concentrated; (6) preparing the longan seed extracts. Tests confirm that the longan seed extracts which are prepared by the preparation method provided by the invention can be applied to organisms, can generate an inflammation resistant reaction, reduce uricopoiesis, restrain bacterial activity, promote the growth of skin horny cells, and accelerate a wound healing speed, and at the same time, the longan seed extracts cannot cause visceral organs of the organisms to be damaged or increase the burden of the visceral organs.
Description
The application is the Chinese invention patent application (applying date: on July 8th, 2009; Application number: 200980160302.2; Denomination of invention: a kind of preparation method of seed of Arillus Longan extract and application) divisional application.
Technical field
The present invention relates to a kind of preparation method of seed of Arillus Longan extract, gained seed of Arillus Longan extract be applied to organism and there is anti-inflammatory, reduce uric acid in blood concentration, promote skin keratin cell growth, strengthen wound healing machine and turn and resist the multiple efficacies such as bacterial activity.
Background technology
One, inflammation (Inflammation):
Inflammation is a kind of very important phenomenon in Drug therapy, and for human body, it is the warning message generating before disease.In the time that tissue is injured, no matter caused by antibacterial, wound, chemical substance or other reason, near macrophage (Macrophage) wounded tissue can be activated and the task of engulfing foreign substance, also can discharge some factors and start more further defense reaction simultaneously; If Inflamed tissue continues to be subject to foreign substance to stimulate, its defence machine turns can reach even several years several months, therefore in the time of inflammatory status, the factor content discharging is often more.Confirm after deliberation: the described factor includes: nitric oxide (Nitric oxide, NO), neoplasm necrosis factor (Tumor necrosis factor, TNF), the white element of cell circle (Interleukin, IL), granule white blood cell clusters G-CSF (Granulocyte colony stimulating factor, G-CSF), monocyte group stimulating factor (Monocyte colony stimulating factor, M-CSF), granule leukocyte and monocyte group stimulating factor (Granulocyte-monocyte colony stimulating factor, GM-CSF), scholar is by the TNF called after TNF-α being produced by mononuclear cell and macrophage, lymphotoxin (the Lymphotoxin that T lymphocyte is produced, LT) called after TNF-β is distinguished.
Lipopolysaccharide (Lipopolysaccharides, LPS) is endotoxic Main Ingredients and Appearance, proves via zoopery: it is emptying that LPS can delay stomach, and before this effect is lower with LPS induction, inflammatory cytokines and nitric oxide production rising are relevant.Body is subject to after the stimulation of LPS, can trigger the cytokine profiles such as the synthetic TNF-α of mononuclear phagocyte system, IL-1 β, IL-6, participates in body defense reaction and reparation.The synthetic of TNF-α, these three kinds of cytokines of IL-1 β, IL-6 is orderly cascade, and synthetic, the TNF-α induction IL-1 β of LPS induction TNF-α synthetic and IL-1 β induce that IL-6's is synthetic.But excessive cytokine also can produce ill effect to body, for example excessive TNF-α can cause multiple Organ Failure, disseminated inravascular coagulation and toxic shock, even cause death, the animal of application TNF antibody can stop the generation of lethal endotoxin shock effectively.
The anti-inflammatory drug that the world of medicine is used is now of a great variety, for example antibiotic (Antibiotics), on-steroidal anti-inflammatory medicine (Non-steroidal anti-inflammation drugs; NSAIDs), anti-ly organize ammonia medicine (Anti-histamine drugs) etc., though there is quite good antiinflammation, have the side effect such as Drug resistance and damage the intestines and stomach.
Two, gout:
Gout is the caused a kind of disease of purine metabolic disturbance or underexcretion; its clinical characters is that acute single arthritis (Recurrent acute monoarthritis), the uric acid sodium salt of hyperuricemia (Hyperuricemia), outbreak repeatedly forms tophus (Tophi) deposition, chronic gout stone arthritis etc.; if without suitable treatment, conventionally finally can develop into gouty nephropathy (Gouty nephropathy).Primary disease is mainly divided into constitutional and the large class of Secondary cases two, in primary gout patient, there is nearly 1% patient for due to enzyme defect, and most of etiology unknowns are clinical taking gouty arthritis as main manifestations, and often with high fat of blood, hypertension, diabetes, arteriosclerosis and coronary heart disease etc.; Secondary gout often causes by reasons such as nephropathy, hematopathy and medicines, and gout is its complication.
Hyperuricemia is the most important biochemical basis of gout, but be not the synonym of gout, research is pointed out: approximately have 5～18.8% Patients with Hyperuricemia finally can develop into gout, but patient with gout certain one-phase in its course of disease will have the existence of hyperuricemia.
Laboratory can utilize uric acid enzyme process (Uricase differential spectrophotometric method) Accurate Measurement Level of Serum Uric Acid.Hyperuricemia can be divided into absoluteness and the large class of relative property two.Be called absoluteness hyperuricemia in limited time when uric acid concentration in blood exceedes the upper of solubility, in the time of 37 DEG C, in blood, uric acid saturation value is 7mg/dl, exceedes this saturation point, has gradually acicular crystal to separate out.General epidemiological study adds that taking normal person's blood uric acid meansigma methods two standard deviations, as the upper limit, think that the uric acid level in male's blood exceedes 7mg/dl, when women exceedes 6mg/dl, is called relative property metabolic arthritis disease.If Level of Serum Uric Acid exceedes 7mg/dl, the incidence rate of gout or renal calculus will increase.
The clinical manifestation of gout is divided into four-stage: asymptomatic hyperuricemia (Asymptomatic hyperuricemia), experimental study (Acute gouty arthritis), intermission (Inter-critical gout), chronic gout stone arthritis (Chronic tophaceous gout).
The more correct method of diagnosis gout is: in the time that experimental study shows effect, extract joint fluid, the needle-like urate crystal (Monosodium urate crystal) of being engulfed by neutrophils as found that there is, under polarizing microscope, present negativity two-fold light (Negative birefringent), be gout.Clinical sign or lab testing that other is common: break out single redness and swelling of joints severe pain, the Effects of Colchicine In Treatings such as first toe, instep, ankle have specially good effect person or hyperuricemia person, only can be used as the reference of diagnosis gout.
Because primary gout lacks etiological treatment, therefore can not effect a radical cure.The object of clinical treatment is: 1, control in time the acute attack of gouty arthritis; 2, long-term treatment hyperuricemia, to prevent uric acid sodium salt to deposit the destruction of joint and the kidney damage that cause.
Selection as for uric acid resisting medicine: normal renal function or mild damage, twenty-four-hour urine acid output during lower than 600mg, can use and promote urate excretion medicine; Be medium infringement (creatinine clearance rate < 35ml/ minute) or twenty-four-hour urine liquid uric acid while obviously raising at renal function, should use and suppress uricopoiesis thing.When blood uric acid obviously raises and tophus deposits in a large number, can share above two kinds of medicines, to prevent gradual gouty complication.
Aforesaid promotion urate excretion medicine (Uricosuric agent) mainly promotes urate excretion by suppressing near-end renal tubules to the heavily absorption of uric acid.For preventing that uric acid from causing the side effect of kidney damage and renal calculus in the time that kidney is discharged in a large number, all should be from low dose, dosage gradually in 7～10 days, and consider salinize urine.This quasi drugs has different (Probenecid) benzbromarone (Benzbromarone) of the third sulphur etc.
Aforesaid inhibition uricopoiesis medicine (Xanthine oxidase inhibitor), only there is allopurinol (Allopurinol), its similar hypoxanthine (hypoxanthine), there is the effect of stronger inhibition xanthine oxidase (xanthine oxidase), can suppress the formation of tophus and renal calculus, and promote tophaceous dissolving.While using anticarcinogen as mercaptopurine (Mercaptopurine) or azathioprine (Azathioprine), can improve the blood level of anticarcinogen simultaneously, now need to consider or pay attention to clinical side effects.
Three, wound healing (Wound healing):
Wound healing is a kind of dynamic (Dynamic) many cell reciprocal actions (Interactive) and is multiple step (Multiple steps) process, comprises the synthetic and organization restructuring (Tissue remodeling) of signaling, hyperplasia, cell differentiation, extracellular matrix etc.This process is relevant with the regeneration of wound epidermis and the reparation of subcutaneous connective tissue.In the process of skin wound healing, the horn cell meeting hypertrophy at epidermis edge, wound both sides, and form new epidermal area to middle movement of wound, these processes need within several days, even a few weeks longer just can complete.
There is the somatomedin (Growth factors) of substantial connection with wound healing, include FGF2 (Fibroblast growth factor 2, FGF2), platelet derived growth factor (Platelet-derive growth factor, PDGF), epidermal growth factor (Epidermal growth factor, EGF), keratinocyte growth factor (Keratinocyte growth factor, KGF), transforminggrowthfactor-α (Transforming growth factor-α, TGF-α), transforming growth factor-beta (Transforming growth factor-β, TGF-β) and VEGF (Vascular endothelial growth factor, VEGF).In these somatomedin (Growth factors), PDGF, EGF, TGF-β and VEGF are secreted by horn cell.Wherein, PDGF can attract macrophage (Macrophages) and fibroblast (Fibroblasts), and promotes the manufacture of stromatin (Matrix protein); EGF can promote with the form of autocrine (Autocrine) movement and the hypertrophy of self; TGF-β can promote hypertrophy, signaling and the angiogenesis of fibroblast (Fibroblasts), and will disengage in a large number at the initial stage of wound healing; VEGF can promote deposition and the angiogenesis of vascular permeability, short revascularization substrate (Proangiogenic matrix), and the movement that can stimulate mononuclear cell (Monocyte).Shown by above result of study: the somatomedin (Growth factors) that these horn cells discharge is all closely related with wound healing process.
On the other hand, record according to " national Chinese herbal medicine compilation ": seed of Arillus Longan can be used for treating that stomachache, burn and scald, knife injury are hemorrhage, infantile malnutrition pain due to disorder of QI, traumatic hemorrhage, scabies, eczema etc.Seed of Arillus Longan is used for the treatment of wound by ancients, has the effect of good hemostasis, analgesic therapy, granulation promoting." Huang Shi cures the side of destroying " recorded: " control injury by sword or axe, Arillus Longan core is regardless of how many, with burning withered sustainability, grinds into powder, mixes affected part and heals ".Record offered in ancient Chinese prose: " seed of Arillus Longan end, deposit knife injury, former times in Xiqin and the female army of Ba Li succour the more people "." blackish red toe biography side " also cloud: " control knife injury hemorrhage, fry and smash fine grinding with seed of Arillus Longan, apply it ".Record by above ancient book and pharmacopeia can be known: seed of Arillus Longan has therapeutic effect to skin knife injury and relevant disease, the healing that promotes wound is produced effect, but its effect machine turns and be unclear.
Summary of the invention
Main purpose of the present invention is to provide a kind of preparation method of seed of Arillus Longan extract and the application in organism thereof.
First the present invention provides a kind of preparation method of seed of Arillus Longan extract, comprises the following step:
(1) extraction solution of preparation certain concentration;
(2) extraction solution temperature is heated to specified temp;
(3) in extraction solution, put into the seed of Arillus Longan granule of smashing, extract;
(4) by the solution filter after extraction, concentrated;
(5) again the solution after concentrated is carried out freezing and dry;
(6) prepare seed of Arillus Longan extract.
The present invention also provides the preferred version of above-mentioned preparation method:
Extraction solution is aqueous solution or alcoholic solution;
Alcoholic solution is alcoholic solution;
The concentration of alcoholic solution is 20%～95%;
In step (2), described extraction solution is heated to 70 DEG C～90 DEG C;
In step (3), the extraction temperature of described extraction is 70 DEG C～90 DEG C;
In step (3), the extraction time of described extraction is 1～3 hour.
The present invention also provides the seed of Arillus Longan of being prepared by the method for stating extract, and its composition includes corilagin (Corilagin), ellagic acid (Ellagic acid), gallic acid (Gallic acid).
The present invention also further provides the main application of above-mentioned seed of Arillus Longan extract:
This seed of Arillus Longan extract is applied to organism anti-inflammatory;
This seed of Arillus Longan extract is applied to the uric acid person who reduces organism;
This seed of Arillus Longan extract is applied to and promotes organism wound healing;
This seed of Arillus Longan extract is applied to Antibacterial activity.
The invention still further relates to following aspect:
The purposes of 1. 1 kinds of seed of Arillus Longan extracts of item, described purposes is as the medicine of preparing Antibacterial activity, and wherein said seed of Arillus Longan extract is prepared by a preparation method, and described preparation method comprises the following step:
(1) extraction solution of preparation certain concentration;
(2) extraction solution temperature is heated to specified temp;
(3) in extraction solution, put into the seed of Arillus Longan granule of smashing, extract;
(4) by the solution filter after extraction, concentrated;
(5) again the solution after concentrated is carried out freezing and dry; And
(6) prepare seed of Arillus Longan extract.
Item 2. is according to the purposes described in item 1, and wherein this extraction solution is hydrophilic solution or lipotropy solution.
Item 3. is according to the purposes described in item 2, and wherein said lipotropy solution is ethanol water.
Item 4. is according to the purposes described in item 3, and the concentration of wherein said alcoholic solution is 20%～95%.
Item 5. is according to the purposes described in item 1, and wherein the described extraction solution of step (2) is heated to 70 DEG C～90 DEG C.
Item 6. is according to the purposes described in item 1, and wherein the extraction temperature of the described extraction of step (3) is 70 DEG C～90 DEG C.
Item 7. is according to the purposes described in item 1, and wherein the extraction time of the described extraction of step (3) is 1～3 hour.
Purposes described in item 8. foundation items 1, wherein said seed of Arillus Longan extract, its composition includes corilagin, ellagic acid and gallic acid.
9. according to 1 to 8 the purposes described in any one, wherein said antibacterial is the group that selects free escherichia coli, staphylococcus aureus, Propiobacterium and trichophyton to form.
10. according to 1 to 8 the purposes described in any one, wherein said purposes is the medicine as preparation treatment comedo and/or tinea pedis.
The seed of Arillus Longan extract being made by preparation method provided by the invention has the following advantages:
Can be applied to organism, can produce anti-inflammatory reaction, reduce uricopoiesis, Antibacterial activity, and can promote skin keratin Growth of Cells, increase speed of wound healing, can not cause biological internal organs of the body impaired or increase internal organs burden simultaneously.
Brief description of the drawings
Fig. 1: the high-performance liquid chromatograph analysis chart (HPLC figure) of the standard solution that comprises gallic acid, tanning material brevifolin carboxylic acid, ellagic acid.
Fig. 2: the high-performance liquid chromatograph analysis chart (HPLC figure) of seed of Arillus Longan extract provided by the invention.
Fig. 3: the broken line graph of table 3 construction.
Fig. 4: the broken line graph of table 4 construction.
Fig. 5: the bar chart of the meansigma methods institute construction of table 7.
Fig. 6: the bar chart of the meansigma methods institute construction of table 8.
Fig. 7: the bar chart of the meansigma methods institute construction of table 9.
Fig. 8: the broken line graph of table 10 construction.
Fig. 9: the broken line graph of table 11 construction.
Detailed description of the invention
Further illustrate technical scheme of the present invention below by specific embodiment, the present invention includes but be not limited to following embodiment.The change that does not depart from flesh and blood of the present invention of the present invention being carried out according to prior art, still belongs to protection scope of the present invention.
Embodiment 1
(1) for example, using hydrophilic solution (water) or lipotropy solution as extraction solution; The present embodiment is that compound concentration is that 20～95% alcoholic solution is as extraction solution;
(2) extraction solution temperature is heated to 70～90 DEG C;
(3) seed of Arillus Longan after smashing is put into extraction solution, make extraction temperature maintain 70～90 DEG C, extraction time is about 1～3 hour, extracts;
(4) by the solution after extraction after filtration, concentrated;
(5) carry out again the lyophilization of low-temp low-pressure;
(6) prepare seed of Arillus Longan extract.
Utilize high-performance liquid chromatograph (High Performance liquid Chromatography, HPLC) analyze the seed of Arillus Longan extract of preparing, can show that its chief component composition includes gallic acid (Gallic acid) corilagin (Corilagin) ellagic acid (Ellagic acid), structural formula is as follows respectively:
Corilagin (Corilagin):
Gallic acid (Gallic acid):
Ellagic acid (Ellagic acid):
Wherein, the analysis condition of high-performance liquid chromatograph is as shown in table 1.
The analysis condition of table 1, high-performance liquid chromatograph
Taking the standard solution that comprises gallic acid, corilagin, ellagic acid as reference solution, utilize the analysis condition of above-mentioned high-performance liquid chromatograph to analyze, obtain result shown in Fig. 1, this result shows: the retention time (Retention Time) of gallic acid (42.42 μ g/ml), corilagin (52.72 μ g/ml) and ellagic acid (22.4 μ g/ml) is respectively 14.409,43.304,63.489 minutes; After utilizing high-performance liquid chromatograph to analyze aforementioned seed of Arillus Longan extract, obtain result shown in Fig. 2, show: in seed of Arillus Longan extract, the retention time of peak value composition is respectively 14.461,43.302,63.476 minutes, therefore, the main component of this seed of Arillus Longan extract is identical with standard solution, and this seed of Arillus Longan extract includes gallic acid, corilagin and ellagic acid.
In order to disclose the curative effect of seed of Arillus Longan extract of aforementioned preparation, carry out all kinds of in vivo tests and in vitro tests:
One, the in vivo test of anti-inflammatory:
(1) prepare material:
Seed of Arillus Longan extract (extraction solution is 50% alcoholic solution), seed of Arillus Longan extract A (extraction solution is pure water) and seed of Arillus Longan extract B (extraction solution is 20% alcoholic solution).
(2) in vivo test (feeding test):
1, get 24 of male SD mouses (Sprague-dawley rats), every body weight is about 200g～250g, controls 23 DEG C of receptacle indoor temperatures, and light is bright, dark each 12 hours, uses SD Mus special feed, and drinking water is through reverse osmosis processing.
2, be divided at random nine groups, 6 every group, feeding respectively:
(1) control group, only mouthful throwing reverse osmosis water;
(2) mouthful throwing seed of Arillus Longan extract (0.5g/Kg, heavy mouthful of throwing 0.5g seed of Arillus Longan extract of every Kg Mus) after the week, lumbar injection e. coli lipopolysaccharide (Lipopolysaccharides, LPS, 2.5mg/Kg, every Kg Mus re-injection is penetrated 2.5mg e. coli lipopolysaccharide) after, treat 24 hours;
(3) after mouthful throwing seed of Arillus Longan extract (0.5g/Kg) week, lumbar injection LPS (2.5mg/Kg) treats 48 hours;
(4) after lumbar injection LPS (2.5mg/Kg), treat 24 hours mouthful throwing seed of Arillus Longan extract A (0.5g/Kg);
(5) after lumbar injection LPS (2.5mg/Kg), treat 24 hours mouthful throwing seed of Arillus Longan extract B (0.5g/Kg);
(6) after lumbar injection LPS (2.5mg/Kg), treat 24 hours mouthful throwing seed of Arillus Longan extract (0.5g/Kg);
(7) after lumbar injection LPS (2.5mg/Kg), treat 48 hours mouthful throwing seed of Arillus Longan extract (0.5g/Kg);
(8) after lumbar injection LPS (2.5mg/Kg), treat 24 hours separately;
(9) mouth is thrown seed of Arillus Longan extract (0.5g/Kg) separately.
Dispensing finishes, and after going on a hunger strike an evening, under etherization, is taken a blood sample by SD Mus coeliac artery, and detection of plasma inspection is provided.
3, detection of plasma inspection:
Use enzyme-linked immunosorbent assay (ELISA) to detect neoplasm necrosis factor (TNF-α) cell interleukin (IL-1 β).
4, statistical method:
This experiment gained data, all with single-factor analysis of variance (One-way analysis of variance, ANOVA).
5, experimental result:
As can be seen from Table 2: in the part of IL-1 β, mouthful throwing seed of Arillus Longan extract and lumbar injection LPS all can cause immunoreation to SD Mus; Can be found out with Fig. 3 by table 3: in the part of TNF-α, throw in advance mouthful seed of Arillus Longan extract lumbar injection LPS and mouthful throw seed of Arillus Longan extract separately and all have the effect of anti-inflammatory again, and LPS adverse circumstance is thrown again mouthful seed of Arillus Longan extract after processing, SD Mus do not there is the effect of anti-inflammatory.
The IL-1 β value of experiment contrast in table 2, anti-inflammatory body
The TNF-α value of experiment contrast in table 3, anti-inflammatory body
Two, the in vivo test of gout and in vitro tests:
(1) prepare material: seed of Arillus Longan extract (extraction solution is 50% alcoholic solution).
(2) in vivo test (feeding test):
1, get 24 of male SD mouses (Sprague-Dawley rats), every body weight is about 200g～250g, controls approximately 23 DEG C of receptacle indoor temperatures, and light is bright, dark each 12 hours, uses SD Mus special feed, and drinking water is through reverse osmosis processing.
2, be divided at random three groups, every group each 8, wherein one group is matched group, this group oral administration reverse osmosis water; One group is experimental group, and this group is thrown the heavy hypoxanthine (Hypoxathine) of 300mg/Kg Mus and added the oxonic acid (Oxonic acid, a kind of uricase inhibitor) that 250mg/Kg Mus is heavy; Another group is seed of Arillus Longan experimental group, and this group mouth is thrown heavy oxonic acid (Oxonic acid) and the seed of Arillus Longan 0.1% (wt%) of heavy hypoxanthine (Hypoxathine) the 250mg/Kg Mus of 300mg/Kg Mus; Add the main purpose of oxonic acid (Oxonic acid), be to induce height of formation uric acid person in SD mouse body; SD Mus can drink water arbitrarily, during dispensing, observes animal twice every day, and weigh once every day.Dispensing finishes, and after going on a hunger strike an evening, under etherization, is taken a blood sample by SD Mus afterbody, provides serum biochemistry to learn and checks.
(1) matched group: mouthful throwing reverse osmosis water.
(2) experimental group: mouthful heavy hypoxanthine (Hypoxathine) of throwing 300mg/Kg Mus, adds the oxonic acid (Oxonic acid) that 250mg/Kg Mus is heavy.
(3) seed of Arillus Longan group: cast the heavy hypoxanthine (Hypoxanthine) of 300mg/Kg Mus, add the oxonic acid (Oxonic acid) that 250mg/Kg Mus is heavy, add 0.1% (Wt%) seed of Arillus Longan.
3, serum biochemistry is learned and is checked:
Use automatic biochemical analyzer (Ciba-cornint 550) to measure, detect SD Mus serum uric acid concentration.
4, statistical method:
This experiment gained data, all with single-factor analysis of variance (One-way analysis of variance, ANOVA).
5, experimental result:
Can be found out by form 4 and Fig. 4: seed of Arillus Longan extract (extraction solution is 50% alcoholic solution) can effectively reduce SD Mus serum uric acid, reduce effect up to 32%.
The uric acid concentration of experiment contrast in table 4, gout body
| Time | Matched group uric acid concentration | Experimental group uric acid concentration | Seed of Arillus Longan group uric acid concentration |
| 0 hour | 7.3±1.5mg/dl | 8.8±1.2mg/dl | 8.2±1.7mg/dl |
| 1 hour | 7.6±1.6mg/dl | 40.6±12.1mg/dl | 27.7±7.3mg/dl |
(3) experiment in vitro (xanthine oxidase (Xanthine oxidase) suppresses experiment):
1, make the xanthine (Xanthine) of 50mmol/L with phosphate buffer solution (PBS).
2, xanthine oxidase (Xanthine oxidase) is mixed with to 0.1～0.2unit/ml with phosphate buffer solution (PBS).
3, prepare the sample of various seed of Arillus Longan extract:
(1) preparation pure water, extraction seed of Arillus Longan.
(2) preparation 20% alcoholic solution, extraction seed of Arillus Longan.
(3) preparation 50% alcoholic solution, extraction seed of Arillus Longan.
(4) preparation 95% alcoholic solution, extraction seed of Arillus Longan.
4, get a positive controls---clinical application allopurinol (Allopurinol), in positive controls, add xanthine oxidase (Xanthine oxidase), react after 5 minutes, add xanthine (Xanthine).
5, get a blank group, this group is only added pure water.
6, in various seed of Arillus Longan extract samples, add xanthine oxidase (Xanthine oxidase), react 5 minutes, then add xanthine (Xanthine).
7, utilize spectrophotometer, choose wavelength 290nm, the light absorption value of detecting various seed of Arillus Longan extract samples and matched group changes, and detecting in every 20 seconds once, is detected altogether 5 minutes, then calculated its enzyme activity through instrument.
8, maximum inhibition=[1-(experimental group activity/matched group activity)]
9, experimental result:
Can be found out by form 5: various seed of Arillus Longan extracts all can suppress the activity of xanthine oxidase (Xanthine oxidase), and maximum inhibition reaches as high as 60%.
The result contrast of table 5, gout experiment in vitro (xanthine oxidase suppresses experiment)
Three, gout toxicity test:
(1) prepare material: seed of Arillus Longan extract.
(2) acute toxicity test: SD Mus is divided into 2 groups, 8～10 every group, before experiment, go on a hunger strike an evening, but water without cease, oral administration seed of Arillus Longan extract (450mg/ml), observes poisoning symptom and records body weight change and dead situation in 14 days; One half lethal dose (LD
50) and 95% believable limit calculate according to the method for Litctchfield and Wilcoxon Er Shi.
Feeding acute toxicity test on the (three) 28: be divided into 2 groups, every group each 10, mouthful throwing seed of Arillus Longan extract 1g/kg, 3g/kg and deionized water (1ml/100g) respectively, continuous 28 days.During dispensing, observe animal twice every day, weigh weekly once.Dispensing finishes, and after going on a hunger strike an evening, is under etherization taken a blood sample by SD Mus coeliac artery, provides hematology and serum biochemistry to learn and checks.
(4) serum biochemistry is learned and is checked:
Measure with automatic biochemical analyzer (Ciba-cornint 550), test item comprises bran propylhomoserin oxalacetic acid transaminase (GOT), the amino transaminase of bran propylhomoserin third (GPT), albumin (Albumin), globulin (Globulin), kreatinin (Greatinine).
(5) statistical method:
This experiment the data obtained, all with single-factor analysis of variance (One-way analysis of variance, ANOVA), and carries out Dunnett inspection, is less than 0.01 thinks that there were significant differences with P value.
(6) experimental result:
1, acute toxicity:
Acute toxicity test main purpose is the dosage that the medicine of seeking once to cast high dose causes the death of experimental animal half, the every 100g weight of SD Mus empty stomach maximum can cast the dosage of 1.0ml, drug level is the concentration of 450mg/ml, therefore the dosage of acute toxicity test is taking 15g/kg as benchmark, if SD pindone does not have death entirely, no longer carry out.
10 of SD Mus, it is benchmark that per os once casts seed of Arillus Longan extract 15g/kg, observes 14 days, there is no dead situation, i.e. a half lethal dose (LD for SD Mus
50) be greater than 15g/kg.Cast after seed of Arillus Longan extract to the 14th day SD Mus body weight and control group and relatively there is no difference.
2, continuous feeding toxicity test on the 28th:
Use maximum dose level taking a half lethal dose 1/5 as principle, therefore selecting maximum dose level is 3g/kg, and 1g/kg.
(1) 8～10 of every group of SD Mus, within continuous 28 days, mouth is thrown seed of Arillus Longan extract 1g/kg and 3g/kg, without dead situation, also there is no significant change with control group comparison body weight.
(2) serum biochemistry is learned and is checked:
As shown in table 6, continuous 28 days oral administration seed of Arillus Longan extracts, the Biochemical Indices In Serum of SD Mus 1g/kg and 3g/kg extract group and Normal group does not have difference.
(3) main organs weight:
Continuous 28 days oral administration seed of Arillus Longan extracts, the liver of SD Mus 1g/kg and 3g/kg group and the weight of kidney and control group there is no difference, show: seed of Arillus Longan extract does not affect main organs weight.
(4) pathological examination:
Continuous 28 days oral administration seed of Arillus Longan extract 1g/kg and 3g/kg group are carried out check pathological section, and the heart of SD Mus, liver, kidney, spleen, adrenal gland, seminal vesicle, testis etc. are all unchanged.
3, experimental result:
Seed of Arillus Longan extract can not cause biological internal organs of the body impaired or increase internal organs burdens.
Table 6, continuous feeding toxicity test result
| ? | Control group | 14 days | 28 days | 28 days |
| SD Mus quantity (Number of mice) | 8 | 10 | 10 | 10 |
| Body weight (Original weight) before experiment | 19.2±1.8 | 19.6±1.1 | 19.1±1.5 | 19.5±0.8 |
| Body weight (Final weight) after experiment | 19.2±1.8 | 19.2±1.8 | 19.2±1.8 | 25.9±1.07 |
| Mouth dosage (Oral Sample) | 0 | 15g/kg | 1g/kg | 3g/kg |
| Kreatinin (Creatinine) | 0.395±0.15 | 0.435±0.07 | 0.51±0.084 | 29.9±14.8 |
| Bran propylhomoserin oxalacetic acid transaminase (GOT) | 30.2±15.1 | 32.88±15.6 | 38.9±6.2 | 29.9±14.8 |
| The amino transaminase of bran propylhomoserin third (GPT) | 8.27±5.54 | 14.95±10.1 | 7.8±3.8 | 9.44±3.29 |
| Albumin (Albumin) | 2.8±1.37 | 2.75±1.08 | 2.63±1.64 | 2.57±0.8 |
| Globulin (Globulin) | 2.5±1.12 | 2.55±1.5 | 3.3±1.79 | 2.84±0.68 |
Four, bacteriostatic test:
(1) prepare material:
Get taking seed of Arillus Longan extract as nucleus 2.5mg/ml " P bean gel (containing the ProductName of seed of Arillus Longan extract) " of (in every ml " P bean gel ", containing 2.5mg seed of Arillus Longan extract), the grade that makes one times with reverse osmosis water is opened phosphoric acid liquid (1 × PBS, isotonic solution refers to the solution that does not cause erythrocyte membrane distortion), again prepared grade is opened to phosphoric acid liquid and utilize aseptic filter membrane (Mini pore) to filter, prepare phosphoric acid liquid such as " P bean gel " for aseptic condition.
(2) test strain:
Escherichia coli (Escherichia coli) and staphylococcus aureus (Staphylococcus aureus):
1, by the antibacterial such as escherichia coli, staphylococcus aureus, use respectively LB broth fluid medium, 37 DEG C of continuous culture 16 hours, cultured bacterium liquid is opened to phosphoric acid liquid (1 × PBS) with the grade of a times to be cleaned three times, after each cleaning with 3000rpm centrifugal 10 minutes, remove supernatant.Utilize spectrogrph to test its absorbance (OD value) the bacterium liquid after cleaning, finally open phosphoric acid liquid dilution with the grade of a times again, preparing absorbance is the bacterium liquid of 0.3 (OD=0.3), to test.
2, by a times of preparing etc. a phosphoric acid liquid (1 × PBS) add respectively the bacterium such as escherichia coli, the staphylococcus aureus liquid of 10 times of serial dilutions, at 37 DEG C, process 1 hour continuously.
3, get 5,10,20,50,100 μ l and be applied in LB culture medium processing bacterium liquid after 1 hour, at 37 DEG C, continuous culture is after 18 hours, count the bacterium number of every dish culture medium, bacterium liquid is taking phosphoric acid liquid processors such as P bean gels as experimental group, and bacterium liquid is taking phosphoric acid liquid processors such as reverse osmosis water as matched group.
4, above step is carried out respectively three times, last statistical result.
5, experimental result:
If table 7 is with as shown in Fig. 5, compared with matched group, experimental group has obviously reduced the number of bacteria of escherichia coli and staphylococcus aureus, thereby confirm: " the P bean gel " taking seed of Arillus Longan extract as nucleus has the function that suppresses escherichia coli and staphylococcus aureus really, can be applied to the generation that suppresses comedo.
Table 7, antibacterial (escherichia coli and staphylococcus aureus) result of the test
(3) test strain: Propiobacterium (Propionibacterium acne):
1, Propiobacterium is utilized at anBAP broth fluid medium, 37 DEG C to continuous culture 48 hours, cultured bacterium liquid is opened to phosphoric acid liquid (1 × PBS) with the grade of a times to be cleaned three times, after each cleaning with 3000rpm centrifugal 10 minutes, remove supernatant.Utilize spectrogrph to test its absorbance (OD value) the bacterium liquid after cleaning, finally open phosphoric acid liquid dilution with the grade of a times again, making absorbance is the bacterium liquid of 0.3 (OD=0.3), to test.
2, by a times of preparing etc. a phosphoric acid liquid (1 × PBS) add respectively the Propiobacterium of 10 times of serial dilutions, process 1 hour continuously at 30 DEG C.
3, get 5,10,20,50,100 μ l and be applied in LB culture medium processing bacterium liquid after 1 hour, at 30 DEG C, continuous culture is after 48 hours, count the bacterium number of every dish culture medium, bacterium liquid is taking phosphoric acid liquid processors such as " P bean gels " as experimental group, and bacterium liquid is taking phosphoric acid liquid processors such as reverse osmosis water as matched group.
4, above step is carried out respectively three times, last statistical result.
5, experimental result:
If table 8 is with as shown in Fig. 6, compare with matched group, experimental group has obviously reduced the number of Propiobacterium, thereby confirms: " the P bean gel " taking seed of Arillus Longan extract as nucleus has the function that suppresses Propiobacterium really, can be applied to the generation that suppresses comedo.
Table 8, antibacterial (Propiobacterium) result of the test
(4) test strain: trichophyton (Trichophyton rubrum):
1, trichophyton is utilized at IMA plate (Inhibit mold Agar) culture medium, 30 DEG C to continuous culture 96 hours, cultured bacterium liquid is opened to phosphoric acid liquid (1 × PBS) with the grade of a times to be cleaned three times, after each cleaning with 3000rpm centrifugal 10 minutes, remove supernatant.Utilize spectrogrph to test its absorbance (OD value) the bacterium liquid after cleaning, finally open phosphoric acid liquid dilution taking the grade of a times and prepare the bacterium liquid of absorbance as 0.1 (OD=0.1), to test.
2, by a times of preparing etc. a phosphoric acid liquid (1 × PBS) add respectively the trichophyton of 10 times of serial dilutions, process 1 hour continuously at 30 DEG C.
3, the bacterium liquid of processing after 1 hour is got 5,10,20,50,100 μ l and is applied to LB culture medium, at 130 DEG C, continuous culture is after 96 hours, count the bacterium number of every dish culture medium, bacterium liquid is taking phosphoric acid liquid processors such as " P bean gels " as experimental group, and bacterium liquid is taking phosphoric acid liquid processors such as reverse osmosis water as matched group.
4, above step is carried out respectively three times, last statistical result.
5, experimental result: if table 9 is with as shown in Fig. 7, compare with matched group, experimental group has obviously reduced the number of bacteria (approximately 30%) of trichophyton, show: " the P bean gel " taking seed of Arillus Longan extract as nucleus has the function that suppresses the mycetes such as trichophyton really, can be applied to the generation that suppresses tinea pedis.
Table 9, antibacterial (trichophyton) result of the test
Five, promote wound healing machine to turn test:
(1) prepare material:
1, get seed of Arillus Longan extract 5g and be dissolved in 250ml intermediate water, with No. 2 filter paper filterings, then filter rear for subsequent use via 0.45 μ m, 0.22 μ m filter membrane.
2, preparation 0%, 0.25%, 2.5%, 5.0% and 10.0% (Wt%) seed of Arillus Longan extract, as horn cell culture fluid.
3, human keratinocyte (Human epidermal keratinocytes; HEKa-C005-5C).
(2) cell culture: get 1 × 104cells/ml human keratinocyte (HEKa-C005-5C), with horn cell culture medium (the Cascade biologics that is added with keratinocyte growth factor (KC supplements) and penicillin-streptomycin (Penicillin-Streptomycin), USA), in 37 DEG C, containing 5%CO
2incubator in cultivate, this batch of cell is the first generation; Change every three days a subculture, until eight points, first generation cell full after, remake successive transfer culture.Add 0.25% pancreatin cell dissociation buffer (Trypsin-EDTA solution, pancreatin containing 0.25% and 0.02% EDTA), in 37 DEG C effect 5 minutes after, the horn cell suspending is rinsed after neutralization with 10% supernatant, insert in centrifuge tube centrifugal 10 minutes of 1500rpm, remove supernatant, with horn cell culture medium Eddy diffusion cell, with 1:3 dilution, and continue at containing concentration 5%CO again
2incubator in cultivate; Getting its third generation tests.
(3) hyperplasia aptitude tests: carry out with crystal violet (Crystal violet) staining, human keratinocyte (HEKa-C005-5C) is through 24, 48, after within 72 hours, cultivating, the actual state of first growing with inverted microscope observing cell, clean twice, cell with 200 μ l supernatant, with cell fixative fixed cell 20 minutes, clean cell twice with 200 μ l PBST again, under room temperature, dye 30 minutes with 100 μ l crystal violet (Crystal violet) solution, clean cell three times with 200 μ l supernatant again, with 1%SDS dissolved cell, under room temperature, convolution concussion is cultivated 1 hour, to catch nuclear Crystal Violet Dye stripping, measure the absorbance (OD value) under wavelength 595nm, while is with the reference wavelength correction absorbance (OD value) of 650nm.
Using the group of not adding seed of Arillus Longan extract as control group, try to achieve growth promotion rate.
(4) with ELISA measure the secreted somatomedin of human keratinocyte (growth factors) emission:
1, human keratinocyte collects its supernatant after stimulating, and measures the amount of somatomedin (growth factors) in culture fluid with Commercial kits.
2, first get the microtitration plate (Microtitration plate) of 96 orifice plates (96well plates), prevent not binding site with bovine serum albumin (Bovine serum albumin), clean with PBS-Tween again, add the each 100 μ l of supernatant after stimulation, react after 2 hours in 37 DEG C, clean with PBS-Tween, then add the anti-somatomedin of rabbit (Rabbit-anti-growth factor Ab-HRP Chemicon, Temecula, CA), in 2 hours afterwash of 37 DEG C of reactions, add the substrate (Substrate of colour generation, include o-phenylenediamine O-phenyldiamine), after colour generation, add the 2NH of 50 μ l
2sO
4cessation reaction, and measure the absorbance under wavelength 450nm, thus measure the concentration of the vascular endothelial cell growth factor (VEGF) in somatomedin (growth factors).
(5) experimental result:
1, if table 10 is with as shown in Fig. 8, conform to the result under microscope with crystal violet (Crystal violet) Determination Staining Reproductive activity, the group of demonstration 2.5%, 5.0% and 10% it promote the multiple of Keratiocyte growth to be respectively 1.25,1.26 and 1.50 times of control group, wherein only 10% group has statistical remarkable meaning (its p value is less than 0.05), show: only must 10% seed of Arillus Longan extract, can effectively promote human keratinocyte's growth.
2, if table 11 is with as shown in Fig. 9, measure the result obtaining via Enzyme-linked Immunosorbent Assay (ELISA), applying collagen I (Collagen I, and fibronectin (Fibronectin CI), FN) group, after adding 5% and 10% seed of Arillus Longan extract to cultivate, supernatant vascular endothelial growth factor (VEGF) increases and significantly rise (p<0.05) with dosage, show: seed of Arillus Longan extract, promoting to include the mechanism that promotes blood vessel hyperplasia in wound healing process, can promote organism wound healing.
Table 10, measured by way of crystal violet dyeing Reproductive activity result
| Dosage (v/v%) | Growth multiple |
| 0 | 1±0.060 |
| 1.25 | 0.98±0.075394 |
| 2.5 | 1.26±0.059782 |
| 5 | 1.27±0.090245 |
| 10 | 1.50±0.119184 * |
Table 11, enzyme-linked immunosorbent assay result
From the in vivo test of enumerating above, in vitro tests and toxicity test: seed of Arillus Longan extract provided by the invention, can be used for organism and produce anti-inflammatory reaction, reduction uricopoiesis, Antibacterial activity, also can promote the growth of skin keratin cell, increase the speed of wound healing, this seed of Arillus Longan extract can't cause internal organs in organism impaired or increase its burden simultaneously, therefore can feel at ease to use.
Claims (10)
1. a purposes for seed of Arillus Longan extract, described purposes is as the medicine of preparing Antibacterial activity, and wherein said seed of Arillus Longan extract is prepared by a preparation method, and described preparation method comprises the following step:
(1) extraction solution of preparation certain concentration;
(2) extraction solution temperature is heated to specified temp;
(3) in extraction solution, put into the seed of Arillus Longan granule of smashing, extract;
(4) by the solution filter after extraction, concentrated;
(5) again the solution after concentrated is carried out freezing and dry; And
(6) prepare seed of Arillus Longan extract.
2. according to purposes claimed in claim 1, wherein this extraction solution is hydrophilic solution or lipotropy solution.
3. according to purposes claimed in claim 2, wherein said lipotropy solution is ethanol water.
4. according to purposes claimed in claim 3, the concentration of wherein said alcoholic solution is 20%～95%.
5. according to purposes claimed in claim 1, wherein the described extraction solution of step (2) is heated to 70 DEG C～90 DEG C.
6. according to purposes claimed in claim 1, wherein the extraction temperature of the described extraction of step (3) is 70 DEG C～90 DEG C.
7. according to purposes claimed in claim 1, wherein the extraction time of the described extraction of step (3) is 1～3 hour.
8. foundation purposes claimed in claim 1, wherein said seed of Arillus Longan extract, its composition includes corilagin, ellagic acid and gallic acid.
9. the purposes described in any one according to claim 1 to 8, wherein said antibacterial is the group that selects free escherichia coli, staphylococcus aureus, Propiobacterium and trichophyton to form.
10. the purposes described in any one according to claim 1 to 8, wherein said purposes is the medicine as preparation treatment comedo and/or tinea pedis.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107213098A (en) * | 2017-04-27 | 2017-09-29 | 钦州市圆森教育咨询有限公司 | A kind of longan seed Deacne pack and preparation method thereof |
| CN113368119A (en) * | 2021-05-28 | 2021-09-10 | 昆明医科大学 | Application of Corilagin in preparation of medicine for treating burns and scalds |
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2009
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| Title |
|---|
| 陈颖峰等，: "正交法优化龙眼核中黄酮类物质提取条件的研究", 《广东化工》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107213098A (en) * | 2017-04-27 | 2017-09-29 | 钦州市圆森教育咨询有限公司 | A kind of longan seed Deacne pack and preparation method thereof |
| CN113368119A (en) * | 2021-05-28 | 2021-09-10 | 昆明医科大学 | Application of Corilagin in preparation of medicine for treating burns and scalds |
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