CN102579524A - Application of white flower loropetalum chinense in preparing healing medicine - Google Patents
Application of white flower loropetalum chinense in preparing healing medicine Download PDFInfo
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Abstract
The invention relates to application of white flower loropetalum chinense in preparing healing medicine, which adopts two internationally used internal models of an incision wound model and an excision wound model of rat skins to research healing effect of white flower loropetalum chinense coarse extractives. Tests show that healing speed of the rat skins can be obviously promoted, wound healing time is shortened, wound healing strength is enhanced, and regeneration of cells and vessels at position of wounds is promoted. The application of the white flower loropetalum chinense in preparing the healing medicine also finds that when the white flower loropetalum chinense coarse extractives are further separated and extracted, ingredients of petroleum ether layers, ethyl acetate layers and n-butyl alcohol layers are strong in wound healing effect.
Description
Technical field:
The present invention relates to spend in vain the application of Loropetalum chinense (R. Br.) Oliv. extract in preparation wound healing medicine.
Background technology:
Skin trauma is human common health problem; Roughly be divided into open and closed two types, wherein open wound comprises scratch, lacerated wound; Shoot wounded; Incised wound, excision are hindered and operation is hindered etc., and closed wound comprises contusion, crush injury, sprains, concussion injury, joint dislocation and subluxation, closed fracture, closure visceral injury etc.
The main function of skin is as the natural cover for defense, prevents that the outer liquid stream of body from losing, and the protection body is avoided external environmental interference and infringement.Skin trauma is a kind of common health problem, and it is arranged from ancient times, and incidence rate is higher, as deals with improperly, can cause serious problems even death.Therefore, seeking the medicine that promotes wound healing, is that the medicament research and development field is the most ancient, also is one of most important challenge.
Spending Loropetalum chinense (R. Br.) Oliv. (Loropetalum chinens) in vain and claim again to spend Loropetalum chinense (R. Br.) Oliv. in vain, is a kind of of Loropetalum chinense (R. Br.) Oliv., is the evergreen shrub plant that Hamamelidaceae (Hamamelidaceae) Loropetalum chinense (R. Br.) Oliv. belongs to, and has important medical value.Spend Loropetalum chinense (R. Br.) Oliv. in vain and begin to be stated from " Zhiwu Mingshi Tukao ", have the effect of " healing hemostasis, relieving diarrhea with astringents, granulation promoting, antiinflammatory, analgesia ", be applied to treat wound hemorrhage.All the Loropetalum chinense (R. Br.) Oliv. hemostasia and healing is spent in demonstration in vain, the promotion wound healing effect is remarkable although spend the historical records of Loropetalum chinense (R. Br.) Oliv., medication among the people and correlational study report in vain, and the material base and the mechanism of action of its effect it be unclear that.
Do not see the medicine that has report to promote wound healing with the extract preparation of spending Loropetalum chinense (R. Br.) Oliv. in vain.
Therefore, urge skin healing research to spending Loropetalum chinense (R. Br.) Oliv. in vain, seek effective ingredient, the skin wound treatment medicine is significant for seeking.
Summary of the invention:
The objective of the invention is to develop a kind of the use and spend the Loropetalum chinense (R. Br.) Oliv. extract has the effect that promotes wound healing as feedstock production medicine in vain.
The said Loropetalum chinense (R. Br.) Oliv. of spending in vain comprises flower, stem, leaf, the branch of spending the Loropetalum chinense (R. Br.) Oliv. plant in vain.
The said extract of spending Loropetalum chinense (R. Br.) Oliv. in vain is that the medicament acceptable solvent is to spending the crude extract that Loropetalum chinense (R. Br.) Oliv. carries out the lixiviate gained in vain.
The said preferred method for distilling of Loropetalum chinense (R. Br.) Oliv. extract of spending in vain is broken with spending Loropetalum chinense (R. Br.) Oliv. green wood material in vain, adopts combination bacterium enzyme solution to obtain to spend Loropetalum chinense (R. Br.) Oliv. biological wall breaking product in vain, adopts dynamic warm macerating method to extract 16 hours, obtains extracting extractum; Remove oil-soluble impuritieses such as verdazulene earlier with ethanol extract from water precipitation, the reuse decoction and alcohol sedimentation technique is removed water-solubility impurities such as saccharide, obtains spending in vain the Loropetalum chinense (R. Br.) Oliv. crude extract, and weight is the 5.5-6% of raw material weight.
Said life assemblage bacterium (title code name D424), the yeast powder when making for Jingde plate chicken (ZL92108674.1) derives from the Dutch strain spore of former strain that Jingde, Jiangxi plate chicken industrial corporation preserves.
The inventor is an object of study to spend Loropetalum chinense (R. Br.) Oliv. and separated product thereof in vain; Take international nitrofural commonly used as positive control; Hinder as study model expansion experiment with rat skin incised wound and excision; The short rat skin wound healing effect of Loropetalum chinense (R. Br.) Oliv. is spent in observation in vain, and active component is carried out promoting healing effect screening, establishes working foundation for spending the short skin healing material base research of Loropetalum chinense (R. Br.) Oliv. in vain.
The research method that the present invention adopts is:
1, adopt LC-MS to carry out preliminary analysis to spending the possible chemical constituent of Loropetalum chinense (R. Br.) Oliv. in vain;
2, adopt its promoting healing effect of rat skin incised wound model (incision wound model) and two kinds of worlds of excision wound model (excision wound model) body inner model research commonly used to spending the Loropetalum chinense (R. Br.) Oliv. crude extract in vain.
The incised wound model is meant with sharp knife blade skin or its hetero-organization is severed, can be with the parallel damage rapid damage tissue integrity of minimum.Incised wound back wound size is relevant with multiple factor, like subcutaneous fat, and wound tension force size and animal species etc.Make wound closure through mechanical system, wound recovers rapidly, and scar block organization generates minimum.No matter when handling wound is use binder, sews up the method for still clamping, and main purpose all is for slit between will organize is contracted to minimum, makes edge of wound can pass through granulation tissue and promoting epidermization and rapid the healing.Therefore, this trauma model is suitable for analyzing healing strength most; Because the restriction of volume or area when receiving wound healing, this model is not suitable for the assessment of histology or biochemistry and promoting epidermization.
The excision wound model, this wound refers to has the tissue of larger volume to be removed in the target area, and according to the order of severity, the excision wound can be subdivided into adhesive tape again and glue except that hindering, and bubbles, and lacerated wound is excised wound etc. entirely.Full excision model refers to epidermis and corium complete resection, through subcutaneous layer of fat, and hemorrhage and body fluid runs off even more serious, and more is prone to infected.This model has multiple advantage, and is bigger like wound volume, and the corium component is all participated in agglutination, only begins promoting epidermization at edge of wound, and can carry out biochemistry, histology and cell proliferation research etc. to wound site.In this model, the wound healing speed that is the basis with initial wound area or volume also is the index that often is studied.
3, further extract spending thick extraction of Loropetalum chinense (R. Br.) Oliv. in vain: the above-mentioned Loropetalum chinense (R. Br.) Oliv. crude extract of spending in vain is used ethanol extraction, ethanol extraction is used aqueous dispersion, more respectively with the organic solvent extraction that is selected from petroleum ether, ethyl acetate or n-butyl alcohol.And adopt rat skin incised wound model the extract of different solvents extraction back gained to be carried out the screening of promoting healing effect
The result:
1, spends the Loropetalum chinense (R. Br.) Oliv. extract in vain and can significantly promote the rat skin healing rate, shorten wound healing time, increase healing strength, promote wound cell and angiogenesis;
2, spend in vain in the Loropetalum chinense (R. Br.) Oliv. separation and Extraction component, petroleum ether layer, the short skin healing effect of ethyl acetate layer and n-butanol layer is stronger, points out the promoting healing working substance polarity of spending Loropetalum chinense (R. Br.) Oliv. in vain less.
Conclusion: spend Loropetalum chinense (R. Br.) Oliv. in vain and have obvious promoting healing effect, promoting healing working substance polarity is less, mainly is distributed in petroleum ether layer, ethyl acetate layer and n-butanol layer.
Description of drawings:
Fig. 1 spends Loropetalum chinense (R. Br.) Oliv. extract ultraviolet detection chromatogram in vain, wherein: Red 254nm, Blue 285nm, Green 360nm.;
Fig. 2 is the chromatography of ions figure that spends the Loropetalum chinense (R. Br.) Oliv. extract in vain: Black:m/z 191 (parent nucleus of quinic acid); Blue:m/z 285 (parent nucleus of kaempferol/luteolin); Red:m/z 301 (parent nucleus of quercetin); Green:m/z 316 (parent nucleus of myricetin).;
Fig. 3 ELC increases the incision model and recovers the 10th day wound surface skin tractive tension force, * P＜0.05, and compare with the blank group * * * P＜0.001; Compare with the positive drug group #P＜0.05. and data representation is means standard deviation .n=5;
Fig. 4 ELC shortens the complete healing time of wound surface of excision wound model, * P＜0.05, and compare with the blank group * * * P＜0.001; Compare with the positive drug group #P＜0.05. and data representation is means standard deviation .n=5;
Wound surface recovery process image recording is hindered in the excision of Fig. 5 rat;
The rebirth skin tissue staining section of Fig. 6 rat excision wound model, model group (A), ECL organizes (B) (40 *);
The rebirth skin of Fig. 7 rat excision wound model is organized CD34 immunohistochemical staining result, model group (A), and ECL organizes (B) (40 *);
Fig. 8 incision model recovers the tractive tension force of the 10th day wound surface skin, * P＜0.05, and compare with the blank group * * * P＜0.001. and data representation is means standard deviation .n=5;
Fig. 9 incision model recovers the 8th day wound surface skin tractive tension force, * P＜0.05, and compare with the blank group * * * P＜0.001. and data representation is means standard deviation .n=5.
The specific embodiment
1, spends the extraction of Loropetalum chinense (R. Br.) Oliv. crude extract in vain
It is broken to spend Loropetalum chinense (R. Br.) Oliv. green wood material in vain, adopts combination bacterium enzyme solution to obtain to spend Loropetalum chinense (R. Br.) Oliv. biological wall breaking product in vain, adopts dynamic warm macerating method to extract 16 hours, obtains extracting extractum; Remove oil-soluble impuritieses such as chlorophyll earlier with ethanol extract from water precipitation, the reuse decoction and alcohol sedimentation technique is removed water-solubility impurities such as saccharide, to spending the crude extract initial gross separation purification of Loropetalum chinense (R. Br.) Oliv. in vain, obtains crude extract, and weight is the 5.5-6% of raw material weight.
The used strain of said combination bacterium enzymolysis is Dutch strain spore, derives from Jingde plate chicken yeast powder, is produced by Jingde, Jiangxi plate chicken industrial corporation.
The Dutch strain spore of former strain is unique strain that does not produce enterotoxin, through cultivating, in the secondary metabolism, isolates D429, and process is the meat soup slant culture.Paraffin wax is sealed after 2 months separating thallus once more up for safekeeping, and the group amplification culture, and then separates, and after expand the crowd, extracts its of algae floating life, is lyophilized into active thalline powder.(bacterium culture medium is a bouillon agar etc.).
Concrete grammar is pressed the method operation of Chinese patent 200910242020.9 and 200910242037.4.
2, spend the Loropetalum chinense (R. Br.) Oliv. chemical composition analysis in vain
One, sample preparation
The smart title, slightly carried powder 1.06mg, and the lmL50% dissolve with methanol is crossed 0.45 μ m microporous filter membrane, promptly gets.
Two, chromatographic condition
High performance liquid chromatograph: Agilent 1200 series of high efficiency chromatograph of liquid (U.S. Agilent Technologies company) comprise PDAD, quaternary gradient pump, online degasser, automatic sampler.
Chromatographic column: YMC-Pack Pro C18 (150mm * 3.0mm ID, 5 μ m);
Mobile phase: A (methanol), B (0.1% formic acid H2O)
Gradient elution (0 → 35min, A:10% → 98%; 35min-40m
in，A：98％→98％；)；
Flow velocity 0.3mL/min;
UV scanning scope: 200-400nm;
Detect wavelength: 200-400nm; Column temperature: 40 ℃.
Three, mass spectrum condition
1, mass spectrograph: Q TRAPTM type quadrupole rod-linear ion hydrazine tandem mass spectrometer (AppliedBiosystems/MDS SCIEX, USA), ion source: the ESI source of adopting the anion detecting pattern.
2, LC-MS condition determination:
LC-MS detects spending the Loropetalum chinense (R. Br.) Oliv. sample in vain.
Spend the preliminary analysis of Loropetalum chinense (R. Br.) Oliv. chemical constituent in vain
According to the ultraviolet chromatogram of spending the Loropetalum chinense (R. Br.) Oliv. sample in vain with extract the daughter ion chromatogram, spend the chemical constituent that Loropetalum chinense (R. Br.) Oliv. possibly contain in vain and infer as follows:
Table 1 is spent the Loropetalum chinense (R. Br.) Oliv. main chemical compositions in vain
In following examples 2～4, experiment is an object of study to spend Loropetalum chinense (R. Br.) Oliv. and separated product thereof in vain, hinders as study model with rat skin incised wound and excision and launches experiment, establishes working foundation for spending the short skin healing material base research of Loropetalum chinense (R. Br.) Oliv. in vain.Material, equipment, laboratory animal used in the experiment are originated as follows
1. receive the reagent article
Spend the Loropetalum chinense (R. Br.) Oliv. crude extract in vain: chocolate brown powder provides (providing by embodiment 1 method) by Deyu Group Corp. Jiangxi Prov..
2. positive control drug
Nitrofural crude drug: available from maple Eurya plant experimental article brand shop.
3. main agents, medicine
4. capital equipment instrument
5. reagent preparation
2% sodium alginate: take by weighing the 20g sodium alginate, gradation adds in the 1000ml distilled water and heating, treats to be stirred to complete dissolving after its swelling;
1% nitrofural: take by weighing the 0.5g nitrofural and be dissolved in 2% sodium alginate of 50ml, fully stir, every preparation again at a distance from 4 days;
1% is tried thing: take by weighing 0.5g and tried thing (as spending Loropetalum chinense (R. Br.) Oliv. in vain, No. 1 component, No. 2 components etc.) and be dissolved in 2% sodium alginate of 50ml, fully stir, whenever prepared again at a distance from 4 days.
Phosphate buffer (0.01mol/L PBS): accurate weighing 8.00g NaCl, 0.20g KCl, 1.56g Na
2HPO
4H
2O, 0.20g KH
2PO
4, being dissolved in one by one successively in the aseptic tri-distilled water of 800ml, magnetic agitation makes it to dissolve fully; Adjust pH is 7.2～7.4, adds aseptic tri-distilled water to the 1000ml mixing; In the packing saline bottle, 8 pounds of 15～20min disinfection with high pressure steam sterilizations, 4 ℃ of storages are subsequent use.
0.01mol/L citrate buffer (CB, pH6.0): take by weighing trisodium citrate 3g, citric acid 0.4g is dissolved in the 1000mL distilled water, is stirred to complete dissolving, and hydrochloric acid is transferred pH to 6.0.
6 animals
The Wistar rat, male, 150g-200g, available from laboratory animal resource center of Nat'l Pharmaceutical & Biological Products Control Institute, credit number is: SCXK-(capital) 2009-0017.
The body inner model that short skin healing effect is estimated in common in the world being used at present mainly contains rat skin incised wound model, excision wound model, four kinds on dead space wound surface model (dead space wound model) and burn model
The present invention is an object of study to spend Loropetalum chinense (R. Br.) Oliv. and separated product thereof in vain, hinders as study model with rat skin incised wound and excision and launches experiment, establishes working foundation for spending the short skin healing material base research of Loropetalum chinense (R. Br.) Oliv. in vain.
Experimental technique:
1) 15 rats are divided into 3 groups at random, are respectively blank control group, the nitrofural group with spend the Loropetalum chinense (R. Br.) Oliv. group in vain, every single cage of rat is raised, in case it baits wound mutually;
2) adaptability was raised after three days, the anesthesia of 3% pentobarbital sodium, and the back QUMAO is scratched the wound of a long 6cm with scalpel apart from the about 1.5cm of center line place at every rat back, reach subcutaneous (not damaging its fascia layer) deeply, and every separated 1cm surgical thread stitching is in case wound dehiscence;
3) to the wound coating of each group: the blank control group wound is coated with 2% sodium alginate; Nitrofural matched group wound is coated with 1% nitrofural; Spending Loropetalum chinense (R. Br.) Oliv. group wound in vain is coated with 1% and spends Loropetalum chinense (R. Br.) Oliv. in vain; Every animal all puts homemade necklace (similar with Elizabethan's necklace of house pet) at head, licks wound in case it is licked;
4) every day coating under 3% isoflurane gas anesthesia state; Postoperative was removed stitching thread on the 8th day; Postoperative the 10th day is put to death rat, and near the skin of the 1.5cm scope wound is cut, and is partitioned into 3 parts of the skins of the long 1cm of wound in middle; Survey healing of wound is drawn back required lowest pull with strain gauge (the band peak value is preserved), this 3 piece of data is averaged as the final pulling force of this animal wound.
Experimental result: spend Loropetalum chinense (R. Br.) Oliv. in vain and promote rat skin incised wound model wound healing
In the incised wound model, we adopt wound to tear this index of tension force and estimate and respectively organize the wound recovery situation.The result shows that the nitrofural group is as positive control, and the 10th day after surgery wound torn tension force and compared obvious lifting with blank control group, proves this modelling success; Spending Loropetalum chinense (R. Br.) Oliv. group (ELC) wound in vain tears tension force and has compared extremely significance with blank control group and increase; Compare with the nitrofural group simultaneously; Obvious increase (Fig. 3) is also arranged, be illustrated in identical time Diplopterygium glaucum (Thunb ex Houtt) Nakai flower Loropetalum chinense (R. Br.) Oliv. and have the wound healing promoting effect, and ratio is with the nitrofural better effects if under the concentration.
Embodiment 3 spends the short rat skin excision of Loropetalum chinense (R. Br.) Oliv. wound model healing evaluation of effect in vain
Operational approach:
1) 15 rats are divided into 3 groups at random, are respectively blank control group, the nitrofural group with spend the Loropetalum chinense (R. Br.) Oliv. group in vain, every single cage of rat is raised, in case it baits wound mutually;
2) adaptability was raised after three days; The anesthesia of 3% pentobarbital sodium; The back QUMAO, one-sided slightly by the whole bark skin excision (not damaging fascia layer) of the about 400mm2 size in top at every rat back spinal column, a ruler is put with the object of reference as actual size in the place on the same plane of wound; Digital camera is taken, record initial wound area size;
3) postoperative wound place coating: the blank control group wound is coated with 2% sodium alginate; Nitrofural group wound is coated with 1% nitrofural; Spend Loropetalum chinense (R. Br.) Oliv. group wound in vain and be coated with 1% and spend Loropetalum chinense (R. Br.) Oliv. in vain, every animal all puts self-control necklace (similar with Elizabethan's necklace of house pet) at head, licks wound in case it is licked;
4) every day coating under 3% isoflurane gas anesthesia state, every as stated above wound was taken following the tracks of its healing degree at a distance from three days, until postoperative 15 days, adhere to that coating did not have until the wound and anyly scabbed residually every day, write down its wound healing natural law;
5) the rebirth skin tissue was cut in the 18th day after surgery, 4% paraformaldehyde is fixed, and stays and does dyeing usefulness.
The short rat skin excision of the Loropetalum chinense (R. Br.) Oliv. wound model healing effect of spending in vain is observed in HE dyeing
1) dehydration: distillation washing 3 times; 70% ethanol 2h or spend the night; 80% ethanol 2h or spend the night; 95% ethanol 1h; 100% ethanol 1h; Xylene 20min;
2) waxdip 20min;
3) section is to 8 μ m;
4) dewaxing: xylene 10min; 100% ethanol 5min; 95% ethanol 5min; 80% ethanol 5min; 70% ethanol 5min; Distilled water 5min;
5) haematoxylin dyeing 5-6min distilled water is gone over; Salt sour water (100ml water adds 4-5 and drips dense HCl) is gone over; More than the tap water flushing 10min; Distilled water is gone over; 70% ethanol 5-10min; 80% ethanol 5-10min;
6) Yihong dyeing 12min (preparation of 0.5% usefulness, 80% ethanol) 95% ethanol 5min * 2; 100% ethanol 5min * 2; Xylene 10min * 2;
7) neutral gum mounting;
8) just putting microscopically observes.
The short rat skin excision of the Loropetalum chinense (R. Br.) Oliv. wound model healing effect of spending in vain is observed in IHC dyeing
1) dewaxing: 60 ℃ of 20min; Xylene I10min, xylene II10min.
2) gradient alcohol dehydration: 100% ethanol I 5min, 100% ethanol II 5min, 95% ethanol 3min, 80% ethanol 3min, 70% ethanol 3min.
3) deactivating endogenous peroxydase: 3%H
2O
2Lucifuge is hatched 10min under the room temperature, and PBS washes 3 times * 3min;
4) antigen retrieval: put in the 0.01M citrate buffer (PH6.0) and boil, more than the natural cooling 30min, 3 times * 3min of PBS flushing to the room temperature.
5) sealing: with confining liquid (generally consistent with two anti-sources, like the normal sheep serum working solution) sealing, 10～30min under the room temperature inclines and does not wash.
6) anti-hatching: drip anti-4 ℃ of refrigerator incubated overnight of suitable concentration, 37 ℃ of rewarming 45min, PBS washes 5 times * 3min;
7) two anti-hatching: drip biotin labeling two and resist, hatch 30min for 37 ℃, PBS washes 5 times * 3min;
8) SP reaction: drip the Streptavidin working solution of horseradish peroxidase-labeled, hatch 30min for 37 ℃, PBS washes 5 times * 3min;
9) DAB colour developing: configuration DAB/H
2O
2Reactant liquor is hatched tissue slice, mirror control dyeing down, and general 3～10min, tap water fully wash;
10) conventional dehydration: 50% ethanol 1-2min, 70% ethanol 1-2min, 95% ethanol 1-2min, 95% ethanol 1-2min, 100% ethanol: 1-2min, 100% ethanol: 1-2min;
11) transparent: xylene 1 * (1-2) min → xylene 2 * (1-2) min;
12) mounting: neutral gum;
13) just putting microscopically observes.
Experimental result: spend Loropetalum chinense (R. Br.) Oliv. in vain and promote rat skin excision wound model wound healing
Visible by table 2, to spend Loropetalum chinense (R. Br.) Oliv. in vain and can significantly quicken excision and hinder speed of wound healing, coating spends Loropetalum chinense (R. Br.) Oliv. group wound in vain and healed 32%, and the blank control group wound of the same period only healed 14.5% after 3 days; In administration time subsequently, spend Loropetalum chinense (R. Br.) Oliv. group speed of wound healing in vain always faster than blank control group, healing basically in the time of the 15th day finishes, and the healing of the blank control group of the same period reaches 94%.Nitrofural also shows tangible wound healing promoting effect as positive control, but effect slightly is inferior to and spends the Loropetalum chinense (R. Br.) Oliv. group in vain.We also can find simultaneously, and wound is very fast at wound healing rate in early stage, reaches after 90% in healing, and healing rate just obviously descends.
Table 2 rat skin excision wound model wound healing percentage percentage rate
*P＜0.05，
***P＜0.001，vs?control.Data?are?expressed?as?mean±SD.n＝5.
Scabbing and come off fully in the wound, can be considered to wound healing fully, and the natural law that comes off of therefore scabbing is the important indicator of wound recovery situation.Spend the Loropetalum chinense (R. Br.) Oliv. group in vain and first rat in the time of the 13rd day, occur and scab and come off fully, all coming off in the time of the 15th day finishes, and coating is described after 15 days, and the rat wound heals fully; The nitrofural group first rat occurred and scabs and come off fully in the time of the 14th day, all coming off during by the 16th day finishes; The healing of blank control group wound just obviously lags behind, and first rat wound of appearance scabs and come off fully in the time of the 17th day, and last rat is just scabbed and comes off during by the 20th day, and Loropetalum chinense (R. Br.) Oliv. and the true obviously accelerating wound healing speed (Fig. 4) of nitrofural spent in vain is described.
Visible by Fig. 5, blank control group, the nitrofural group is basic identical with the wound area of spending Loropetalum chinense (R. Br.) Oliv. group operation manufacturing in vain; Along with the time lengthening of coating, obvious difference has appearred in wound recovery situation of each group: in the time of the 8th day, the nitrofural group with spend Loropetalum chinense (R. Br.) Oliv. group wound area in vain and compare with blank control group to have and obviously dwindle; In the time of the 15th day, nitrofural group wound heals basically, spends Loropetalum chinense (R. Br.) Oliv. group wound in vain and heals fully; And that the blank control group wound has only healed is about 90%, and healing still takes time fully.
Rat wound rebirth skin organizes HE coloration result (Fig. 6) to show, blank control group cambium cell is arranged more loose, and collagen fiber generate not obvious, and epithelium generates not exclusively, shows not healing fully of wound; Spend Loropetalum chinense (R. Br.) Oliv. group cambium cell in vain and arrange densification, collagen fiber deposition showed increased, epithelium generates fully, and fibroblast also has obvious increase, and it is more complete to explain that tissue generates, the wound healing better off.
IHC coloration result (Fig. 7) shows that the new vessels of blank control group rebirth skin is less, and inflammatory cell still has bulk deposition, and it is more to spend Loropetalum chinense (R. Br.) Oliv. group skin new vessels in vain, and inflammatory cell obviously reduces, and shows that its wound recovery situation is better than blank control group.
Experiment conclusion
Spend Loropetalum chinense (R. Br.) Oliv. in vain and slightly be extracted in the model experiment of rat incised wound, coating is after 10 days:
1, wound is torn tension force has obvious increase (p＜0.001) than blank control group, and the healing mechanical strength becomes big, and the collagen tissue hypertrophy is described, crosslinked increasing between the collagen fiber, and wound healing is more abundant;
2, compare with positive control nitrofural group, spending the Loropetalum chinense (R. Br.) Oliv. antibacterial activity in vain has significance to increase, and shows that spending the effect of Loropetalum chinense (R. Br.) Oliv. promoting healing under the equal conditions in vain is better than nitrofural;
Spending Loropetalum chinense (R. Br.) Oliv. in vain slightly is extracted in the experiment of rat excision wound model:
1, speed of wound healing is obviously accelerated, and the scar piece time that comes off obviously reduces, and shows that wound healing is faster;
2, synkaingenesis skin HE dyeing and CD34 new vessels IHC coloration result show, spend Loropetalum chinense (R. Br.) Oliv. group collagen fiber deposition in vain and increase, and epithelium generates fully, and fibroblast obviously increases, and new vessels increases, and inflammatory cell reduces, and wound healing is more abundant.
The presentation of results of these two kinds of models is spent significantly accelerating wound healing of Loropetalum chinense (R. Br.) Oliv. in vain.
To spend the Loropetalum chinense (R. Br.) Oliv. crude extract in vain and carry out separation and Extraction according to following steps:
1, spend Loropetalum chinense (R. Br.) Oliv. powder 481.3g in vain, 95% ethanol extraction 2 times, solvent load is respectively 3600ml, 3000ml, extraction time is 1h.Get 170g, yield 35.3%.This part 9.8g that keeps sample.
2,95% ethanol extraction is used aqueous dispersion, use petroleum ether, ethyl acetate, n-butanol extraction respectively.
3, residual residue continues with 80% ethanol extraction 1 time, extract obtainedly uses aqueous dispersion, uses ethyl acetate, n-butanol extraction respectively.Above-mentioned steps 2,3 is seen following diagram.
Residual residue continues with 80% ethanol extraction 1 time, solvent load 3000ml, and extraction time 1h, 9.9g keeps sample.
The promoting healing effect of selecting for use rat incised wound model to screen the extract of spending Loropetalum chinense (R. Br.) Oliv. in vain; The concrete operations step is with the method described in 3; Research is earlier tried component No. 1 and No. 6; Again according to experimental result, 2,3,4, No. 5 components of further separation and Extraction product of No. 1 component that the promoting healing effect is arranged are carried out promoting healing effect screening.
Screening technique:
According to above-mentioned separating and extracting process; No. 1 component is for spending Loropetalum chinense (R. Br.) Oliv. first step extract in vain; The 2-9 component all is extraction separation on No. 1 component basis; Wherein be for 2-5 number that No. 1 component is carried out the aqueous dispersion extraction resulting polarity in the back component different with dissolubility, residue obtained continuation purification obtains component No. 6 after this step extraction finishes, and No. 6 components separating and extracting again obtain the 7-9 component.Adopt rat skin incised wound model that separation component is carried out promoting healing effect screening, owing to be the extract of No. 1 component 2-5 number, the 7-9 component is the extract of No. 6 components, so at first No. 1, No. 6 are studied.
The selection result:
No. 1 component can obviously improve the rat wound and tear tension force, demonstrates the promoting healing effect;
No. 6 components also can improve wound and tear tension force, but DeGrain, and prompting promoting healing effective ingredient does not separate fully when extracting No. 1 component, still have part to remain in No. 6 components, but because its content is very little, so promoting healing effect not strong (Fig. 8).Therefore, the searching of spending Loropetalum chinense (R. Br.) Oliv. promoting healing action component in vain can concentrate on 2-5 number, and need not carry out follow-up screening operation 7-9 number.
Adopt rat skin incised wound model that 2,3,4, No. 5 components are carried out promoting healing effect screening, the result shows, No. 2 petroleum ether layers; No. 3 ethyl acetate layers and No. 4 n-butanol layer wounds are torn tension force has obvious lifting than blank control group, and No. 5 water layer wounds tear tension force and slightly rise, but does not have significant difference; Explain that promoting healing action component polarity is less; Mainly concentrate on petroleum ether layer, ethyl acetate layer and n-butanol layer are owing to separate not thorough; Slightly remaining at water layer, content is not enough to produce significant difference (Fig. 9).So extracting, later separation can launch around 2,3, No. 4.
Experiment conclusion
This research is main adopt two kinds of rat skin incised wound model and excision wound models in the world common model research spend the short skin healing effect of Loropetalum chinense (R. Br.) Oliv. in vain, and adopt the incised wound model to urge the skin wound healing effect and screen to spending the Loropetalum chinense (R. Br.) Oliv. separator in vain.
For the incised wound model, can survey its wound after 10 days at coating and tear tension force.Wound is torn the wound that tension force is about to part healing and is fully withdrawn required lowest pull.At the beginning of wound healing, it is less to tear tension force, and the wound both sides have only very little power to make separately to condense together; After this directly be cross-linked to form increase along with collagenation increases with collagen fiber, wound is torn tension force and increased sharply, and collagenation is many more, and is crosslinked close more, explains that wound recovers good more.
For the excision wound model, wound healing process is accompanied by granulation tissue hyperplasia and wound area dwindles, and what content was maximum in the granulation tissue is myofibroblast; Be rich in actin; These albumen produce mechanical pulling force when shrinking, to the center tractive, wound area dwindles gradually with edge of wound.In the tissue reconstruction stage, wound constantly shrinks, and then can form some surperficial scar block organizations, accomplishes if skin histology is rebuild, and scar block organization promptly comes off.Speed of wound healing, scar block organization all can be used as the evaluation index of rat skin excision wound model the time that comes off.Collagen fiber; Epithelial cell, fibroblast, endotheliocyte all plays an important role in the wound repair process; The wound revascularization can provide nutrient substance for tissue repair; Therefore adopt haematoxylin Yihong staining and CD34 new vessels Immunohistochemical Staining to observe the accumulation of collagen fiber in the cambium, epithelial cell, fibroblastic generation and angiogenesis situation, these all can reflect the recovery situation of damaged tissues.
Spend in vain Loropetalum chinense (R. Br.) Oliv. slightly extract separate with different solvents after, in the model experiment of rat incised wound, each component is screened, find:
No. 2 extracts (petroleum ether layer), No. 3 extracts (ethyl acetate layer), No. 4 extracts (n-butanol layer) are torn tension force obvious facilitation are all arranged for promoting the rat wound, and the effective ingredient that the short skin healing of Loropetalum chinense (R. Br.) Oliv. is spent in prompting in vain should be distributed in these three layers;
No. 5 (water layer) component does not then have positive effect.
Petroleum ether, ethyl acetate, n-butyl alcohol, water, this polarity is ascending, according to the similar principle that mixes, spends that the promoting healing action component should belong to the less material of polarity in the Loropetalum chinense (R. Br.) Oliv. in vain.
Claims (6)
1. spend the Loropetalum chinense (R. Br.) Oliv. extract in vain and promote the purposes in the wound healing medicine in preparation; The said Loropetalum chinense (R. Br.) Oliv. extract of spending in vain is broken with spending Loropetalum chinense (R. Br.) Oliv. green wood material in vain, adopts combination bacterium enzyme solution to obtain to spend in vain Loropetalum chinense (R. Br.) Oliv. biological wall breaking product; Adopt dynamic warm macerating method to extract 16 hours, obtain extracting extractum; Remove oil-soluble impuritieses such as chlorophyll earlier with ethanol extract from water precipitation, the reuse decoction and alcohol sedimentation technique is removed water-solubility impurities such as saccharide, obtains spending in vain the Loropetalum chinense (R. Br.) Oliv. crude extract, and weight is the 5.5-6% of raw material weight.
2. the described purposes of claim 1; The said Loropetalum chinense (R. Br.) Oliv. extract of spending in vain is that the resulting Loropetalum chinense (R. Br.) Oliv. crude extract of spending in vain of claim 1 is used ethanol extraction; Ethanol extraction is used aqueous dispersion, respectively with being selected from the resulting extract of petroleum ether, ethyl acetate or n-butanol extraction.
3. the described purposes of claim 1, the said Loropetalum chinense (R. Br.) Oliv. of spending in vain is selected from flower, stem, leaf, the branch of spending Loropetalum chinense (R. Br.) Oliv. in vain.
4. medicine that promotes wound healing, its active component is to spend the Loropetalum chinense (R. Br.) Oliv. extract in vain; The said Loropetalum chinense (R. Br.) Oliv. extract of spending in vain is broken with spending Loropetalum chinense (R. Br.) Oliv. green wood material in vain, adopts combination bacterium enzyme solution to obtain to spend Loropetalum chinense (R. Br.) Oliv. biological wall breaking product in vain, adopts dynamic warm macerating method to extract 16 hours, obtains extracting extractum; Remove oil-soluble impuritieses such as chlorophyll earlier with ethanol extract from water precipitation, the reuse decoction and alcohol sedimentation technique is removed water-solubility impurities such as saccharide, to spending the crude extract initial gross separation purification of Loropetalum chinense (R. Br.) Oliv. in vain, obtains crude extract, and weight is the 5.5-6% of raw material weight.
5. the described medicine of claim 1; The said Loropetalum chinense (R. Br.) Oliv. extract of spending in vain is that the resulting Loropetalum chinense (R. Br.) Oliv. crude extract of spending in vain of claim 1 is used ethanol extraction; Ethanol extraction is used aqueous dispersion, respectively with being selected from the resulting extract of petroleum ether, ethyl acetate or n-butanol extraction.
6. the described medicine of claim 1, the said Loropetalum chinense (R. Br.) Oliv. of spending in vain is selected from flower, stem, leaf, the branch of spending Loropetalum chinense (R. Br.) Oliv. in vain.
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