CN101549007A - White lablab seed extract as well as extracting method and application thereof in preparing skin radiation injury resistant medicaments - Google Patents
White lablab seed extract as well as extracting method and application thereof in preparing skin radiation injury resistant medicaments Download PDFInfo
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Abstract
The invention discloses a white lablab seed extract as well as an extracting method and an application thereof, which aims to provide a white lablab seed extract as well as an extracting method and an application thereof in preparing skin radiation injury resistant medicaments. The white lablab seed extract is prepared by the method that after being crashed, white lablab seeds are extracted with physiological saline; then the extract is salted out with saturated ammonium sulphate; and finally, the extract is desalted and dried. Proved by experiments, the white lablab seed extract has special regulation and control effects on tissue cells and can relieve and stop injuries caused by extrinsic factors. Various dosage forms of skin radiation injury resistant medicaments prepared by using the white lablab seed extract as an active ingredient have better curative effects and have strong radiation resistant effects on skin.
Description
Technical field
The present invention relates to plant extract and extracting method thereof and application, particularly relate to Semen Lablab Album extract and extracting method thereof and its application in the preparation skin radiation injury resistant medicaments.
Background technology
Radiation treatment is one of three big treatment meanss of malignant tumor, but radiotherapy inevitably also can produce radiation injury and side effect to normal tissue cell when killing tumor cell, not only have a strong impact on patient's life quality, also limit simultaneously part patient's treatment, reduced patient's chance for survival.
The biological effect that is produced by radiation comprises direct effect (direct action) and indirect action (indirectaction).The offspring that direct effect is radiation self or its generation directly acts on the effect that molecule causes.As use the x-ray bombardment organism, after energy is given to certain secondary electron, this electronics reproduces into the free of target molecule, produce active free radical (free radicai), this free radical chemical property is very active, be bonded to the atom beyond the hydrogen atom in the organism, promptly may cause the degeneration of institute's binding molecule, and then produce biological effect.Indirect action is a radiation self or by the offspring of its generation, act on earlier on the medium molecule (for example hydrone), make it to become the active product of chemical property (for example free radical, hydrogen peroxide, aqueous electron etc.), this product continues to act on the effect that molecule forms again.But indirect action raying sensitive agent (radiosensitizer) or radiation protective (radioprotector) strengthen or weaken, and directly act on then not malleable.
In radiation therapy process, (radiation injury of skin) is particularly common for radiation skin injury, is one of most common complication in the radiotherapy.Radiation skin injury shows as irradiation field district pruritus, erythema, pigmentation, dryness decortication, foaming, erosion etc., but serious secondary ulcer and necrosis.Radiation skin injury can cause the patient suffering to aggravate, and also can have influence on simultaneously radiocurablely to carry out smoothly.Therefore, improving radiotherapeutic effect simultaneously, protection normal skin tissue has become urgent problem.
Tong such as new etc. (Tong Ruxin, Wang Pumin etc. the genesis mechanism of radiation skin injury and study on prevention overview. radiation protection communication, 1998; 18 (4): 23-27.) summarized the clinical manifestation and the mechanism of radiation injury of skin:
1, the effect characteristics of ray: the main ray that causes radiation skin injury is X line, β line and γ line.X, γ line penetration capacity are strong, except that skin, and subcutaneous tissue, even skeleton is also impaired, produces ulcer sometimes and does not heal for a long time.The β line loss is hindered shallow, and treatment is easy, as long as processing is proper, the prognosis majority is all fine.In addition, produce the required absorbed dose difference of identical skin injury.It is bigger that the γ line loss is hindered skin absorbs dosage, and the β line is then less.Because the penetration capacity of γ line is strong, ionization is little, and the energy of passing to skin is also little, and portion of energy consumption is at deep tissues.And the ionization of β line is strong, and the energy of skin absorbs is also many.There are quite a few X or gamma-rays skin injury to be associated with radiation sickness, make observation and treatment more difficult.
2, the classification of radiation skin injury: radiation skin injury can be divided into acute radiation skin injury and chronic radioactive skin injury again.
2.1 the clinical manifestation of acute radiation skin injury: the acute radiation skin injury generally was divided into for 4 phases, i.e. initial reaction phase, incubation period, fundamental reaction phase and convalescent period.
The initial reaction phase: patient's skin, mucosa are not found changes such as tangible pachylosis, hair follicle pimple, erythema.The patient stains back local skin subjective symptoms and the sign change does not have obvious positive findings.
Incubation period: II degree skin injury 10-65 days, average 28 days.III degree skin injury 8-34 days, average 21 days.
The fundamental reaction phase: initial skin has the sense of expanding, scratches where it itches, back pachylosis, or foxtail millet grain size hair follicle pimple appears being dispersed in.Then, skin rashes reddens gradually, and skin is normal between rash, and skin also reddens between rash subsequently, and that presses fades, and is the scratch sample after hands is grabbed and changes.Or the initial stage be speckle, patch shape erythema enlarges gradually, merges, color and luster deepens to be mulberry, that presses does not fade.The blister of different in kind appearred in 7-16 days after the erythema, shallow erosion of local table or formation ulcer behind the ulceration.
Convalescent period: enter after convalescent period, II degree skin injury begins desquamation, and pigmentation does not have obvious subjective symptoms.After the shallow III degree skin ulcer emaciated face crust, pigmentation, no cicatrization.Dark III degree healing back depigmentation forms graniphyric, may be due to metabolism of pigment is lacked of proper care.Serious skin injury becomes chronic radiodermatitis after acute stage.X line and γ line irradiation skin cause that canceration is confirmed by clinical and experiment, does not see clinical data as yet but can β line skin injury canceration take place.
2.2 the clinical manifestation of chronic radioactive skin injury: the chronic radioactive skin injury has long incubation period, and the state of an illness has characteristics such as tangible potentiality, carrying out property, repeatability and persistence.Show as xerosis cutis, follow the string, pigmentation or go down, hyperkeratosis, verruca, be that the leather sample hardens or the garlic skin sample is chapped, or epidermis attenuation, shallow table telangiectasis, desquamation, skin itching, easy impaired diabrosis.
3, the mechanism of radiation skin injury
3.1 cytobiology mechanism: the mechanism of curing dermal radio lesions mainly is the germinal layer cell of epithelium and the variation of veins beneath the skin.At first see at irradiated site blood capillary reflex dilatation, the congested reaction of local formation erythema occurs, and before skin ulcer forms, just blood vessel injury and microcirculation disturbance can take place.And the reason that causes poor wound healing is progressive blood capillary obstruction.Arrive radiation skin injury initial stage, histiocyte degeneration necrosis, extensively fibrosis by electron microscopic observation.Show endotheli ocytosis swelling, tube wall thickens, and tube chamber narrows down, obturation, even the endotheliocyte degeneration, and a large amount of cavitys form intercellular substance broadening, basement membrane local fracture in the endochylema.Occur the skin necrosis in different degree in a short time after being subjected to shine, form the acute radiation skin injury.At this moment, if can in time handle and control infection, can in several days or a few week, obtain clinical recovery.
But radiation skin injury often lapses in chronic pathology and changes.People's chronic radioactive skin ulcer Electronic Speculum shows, corium inner fiber, the slight pyknosis of cell karyon, collagen fiber degeneration, the part karyolysis, keratohyalin granule fragmentation, electron density increases, between a large amount of cavitys appear in the matter, the fracture of epidermis spine-like cell part desmosome, gap enlargement.The mitochondrion mild swelling, ridge fracture, visible intensive melanin granule in the formation cavity that has, endochylema.Above-mentioned variation can cause being subjected to organizing blood supply insufficiency according to the position, makes mass exchange limited.Also the someone thinks that the infringement of epidermal basal cell may account for important function in the chronic radioactive damage.The somebody thinks that ray directly acts on some other composition of wound such as collagen fiber etc., and the infringement of blood vessel may be accessory.The Japan scholar finds that after deliberation the local vascular behind the high-dose irradiation is narrow, the appearance in obturation and avascular area territory, be because be accompanied by the major injury, interstitial edema of endotheliocyte, significantly fibrosis lesion is conversely to due to the perstriction.This shows that it also is one of major reason of carrying out property of the blood vessel minimizing that takes place late period that postradiation fibrosis changes.
3.2 molecular biology mechanism: the molecular biology mechanism about radiation skin injury is still not fully aware of at present.It is generally acknowledged, ribonucleic acid, DNA (deoxyribonucleic acid), protein equimolecular are subjected to effects of ionizing radiation can produce a series of damages, and its action principle is the transmission and the absorption of emittance, make molecular excitation and ionization in the body, produce the existence of free radical and oxygen effect etc., cause macromolecular fracture.These a series of changes constitute the material base of the biological effects of radiation.Especially radiation causes the damage of DNA in the cell, causes double-stranded disorder and the mistake of duplicating, and is the basis of causing canceration.
In addition, the long-term chronic inflammatory disease of radiation-induced skin ulcer stimulates, and also is a kind of carcinogenic factor.The canceration rate of radiodermatitis is higher, and the skin carcinoma incidence rate is 20%-30%.The radioactive skin cancer is the most common with squamous cell carcinoma.The canceration process can be divided following 4 stages: the first phase (downright bad degeneration stage), the tissue necrosis degeneration at first takes place, and what continue is reparation, atrophy and trophism obstacle etc.; The second phase (hyperplasia of prostate phase), epidermal hyperplasia, false epithelioma forms, fibrohistiocytic's hypertrophy etc.; The third phase (atypical hyperplasia phase), organizational structure and cell occur and arrange unusual and heterocyst; The fourth phase (cancerating the phase), malignant tumor forms.But the case that has may very shortly just carry out the transition to the third phase without the second phase or this phase.
The damage that lonizing radiation are caused and the protection of injury all come into one's own all the time.ICRP (ICRP) has illustrated radiation proof purpose in the recommendation of nineteen ninety, mainly provide the human proper standard of protection, and the useful irradiation that the causes practice of restriction within reason.Therefore, in the middle of radiation proof practice, have strict and careful regulation or requirement.For example machine room and machine are used in strict accordance with regulation; Fully use various protective equipments (as lead apron, glove and protective spectacles etc.); Select suitable conditions of exposure, dwindle the irradiation visual field etc.
In last century five, the sixties, big countries such as the U.S. and Russia once dropped into the research that great amount of manpower and material resources is carried out antiradiation drug, there are many radioprotective materials to be found in succession over more than 40 year, in recent years develop, developed multiple radioprotectant, part has been used for animal experiment and clinical research.
Aspect radioprotectant, maximum to the research of sulfur-containing compound, as the ammonia mercapto derivatives; But most chemical compounds are all in the problem that has aspects such as stability, physiological compatibility in varying degrees; At present, domestic, abroad do not find that all it is suitable that prophylactic agent that a kind of antiradiation drug can be used as acute ionization radiation injury is used for human body.
Therefore, continue to seek new radioprotective chemical constitution, radioprotective material, have realistic meaning in the hope of finding comparatively ideal antiradiation drug.
Semen Lablab Album is a kind of conventional Chinese medicine, is the seed of leguminous plant Dolichos lablab; The Semen Lablab Album beginning is stated from " Mingyi Bielu ", and all there is cultivation all parts of the country, ground such as main product Zhejiang, Anhui, Hunan, Henan.
Semen Lablab Album is the double excellent thing of medicine food, nutritious, contain compositions such as fatty oil, protein, nicotinic acid, aminoacid, vitamin and saccharide, also contain trace element and calcium, phosphorus, ferrum etc., with seed, kind skin (Testa dolichoris), flower (Flos dolichoris) hyoscine or edible.Semen Lablab Album sweet in the mouth, light, property is flat; Seed, kind leatherware have the stomach function regulating removing dampness, invigorating spleen for diuresis, and functions such as clearing away summer-heat antidiarrheal cure mainly insufficiency of the spleen diarrhoea, nausea and vomiting, inappetence, the inferior disease of red and white leukorrhea; Flos dolichoris has functions such as expelling summer-heat, removing dampness, antidiarrheal, leukorrhagia stopping, cures mainly diseases such as heatstroke heating, vomiting diarrhea, leucorrhea, dysentery.
Going through in the middle of edition pharmacopeia, all having recorded is many Chinese patent medicines of active ingredient with the Semen Lablab Album, and in particular for all being principal agent with the Semen Lablab Album in the insufficiency of the spleen medicine that waits disease, clinical practice is very extensive.
Summary of the invention
The object of the invention provides the Semen Lablab Album extract with skin injury effect that radioprotective is caused.
Semen Lablab Album extract provided by the present invention is after Semen Lablab Album is pulverized, earlier it to be extracted with normal saline, extract is saltoutd with saturated ammonium sulfate, after the Semen Lablab Album extract that obtains after the desalination, drying again.
Second purpose of the present invention provides a kind of extracting method of above-mentioned Semen Lablab Album extract.
Extracting method provided by the present invention may further comprise the steps:
1) with the Semen Lablab Album mechanical activation comminution;
2) Semen Lablab Album powder that step 1) is obtained extracts with normal saline;
3) with step 2) extracting solution that obtains saltouts with saturated ammonium sulfate;
4) step 3) the is saltoutd precipitation that obtains is carried out desalination successively, and dried obtains Semen Lablab Album extract.
In the said extracted method, step 2) with normal saline the method that Semen Lablab Album powder extracts be can be in: the normal saline of Semen Lablab Album powder and 4-10 times of volume is mixed, at room temperature stir 0.5-6 hour after, filter, centrifugal, abandon precipitation, supernatant is a Semen Lablab Album extracting solution.The gauze of the available 4-8 layer of described filtration.
The mass/volume of ammonium sulfate (W/V) percentage concentration can be 30-100% in the step 3), and available one or more concentration are wherein saltoutd to it; Salting-out condition can be: saltoutd under 4 ℃ 1-4 hour.
With distilled water the step 3) precipitation that obtains of saltouing is carried out desalting processing in the step 4).
With distilled water the step 3) precipitation that obtains of saltouing is carried out desalting processing in the step 4).
Another object of the present invention provides a kind of skin radiation injury resistant medicaments.
The active component of medicine provided by the present invention is above-mentioned Semen Lablab Album extract.
The medicine of skin radiation injury resistant of the present invention can be made into multiple dosage form, as gel, tablet (Film coated tablets), oral liquid, capsule, unguentum or liniment etc.The medicine of above-mentioned various dosage forms all can be according to the conventional method preparation of pharmaceutical field.
When needing, in said medicine, can also add one or more pharmaceutically acceptable excipient.The addition of described excipient can be adjusted according to practical situation, as 0.1%-55.0% (W/V).
Specifically, the composition of raw materials of the skin radiation injury resistant medicaments of described gel dosage form can comprise following components by weight portion:
50 parts of Semen Lablab Album extracts,
5 parts of carbomers,
2 parts of sodium hydroxide,
1000 parts in water.
The composition of raw materials of the skin radiation injury resistant medicaments of described tablet (Film coated tablets) dosage form can comprise following components by weight portion (1000):
100 parts of Semen Lablab Album extracts,
75 parts of microcrystalline Cellulose,
70 parts of lactose,
25 parts of hyprolose,
25 parts of cross-linking sodium carboxymethyl celluloses,
95% ethanol 50-100 part,
3 parts of micropowder silica gels.
The composition of raw materials of the skin radiation injury resistant medicaments of described oral liquid formulation can comprise following components by weight portion (1000, every 10mL):
200 parts of Semen Lablab Album extracts,
100 parts of sucrose,
Benzoic acid 10-50 part,
Flavoring orange essence 10-50 part,
10000 parts in water.
The composition of raw materials of the skin radiation injury resistant medicaments of described capsule formulation can comprise following components by weight portion (1000):
50 parts of Semen Lablab Album extracts,
80 parts of pregelatinized Starch,
50 parts of lactose,
10 parts of carboxymethyl starch sodium.
The using method of the medicine of above-mentioned unguentum, gel and liniment dosage form is for directly being applied in the affected part, and consumption is generally 1-3 time/day, 0.2-1.0g/ time, is generally the course of treatment 10-20 days; The using method of the medicine of above-mentioned tablet (Film coated tablets), oral liquid and capsule dosage form is oral, and consumption is generally 0.2-10mg/kg body weight/day, is generally the course of treatment 10-20 days.The dosage and the course of treatment all can be according to the practical situation adjustment.
For improving curative effect, medicine of the present invention can also carry out combined therapy with antibiotic, immunostimulant etc.
The invention provides a kind of Semen Lablab Album extract and extracting method thereof.Experimental results show that, Semen Lablab Album extract of the present invention has special regulating and controlling effect to histiocyte, the damage that can alleviate or stop extrinsic factor to cause, with it is that the skin radiation injury resistant medicaments of the multiple dosage form of active component preparation all has curative effect preferably, and skin is had powerful radiation resistance.
Below in conjunction with specific embodiment the present invention is described in further details.
Description of drawings
Fig. 1 is the red cell agglutination experimental result.
Fig. 2 is the SDS-PAGE testing result of Semen Lablab Album extract.
Fig. 3 is a mouse skin morphosis observed result.
Fig. 4 is the testing result of metal matrix protease expression in the mouse skin tissue, A. normal group B. model group C. vehicle group D. administration group.
The specific embodiment
Method therefor is conventional method if no special instructions among the following embodiment, and concrete steps can be referring to " molecular cloning experiment guide " (J. Sa nurse Brooker D.W. Russell work, Huang Peitang etc. translate, the third edition, 2002, Science Press).
Described percent concentration is mass/volume (W/V) percent concentration or volume/volume (V/V) percent concentration if no special instructions.
The preparation of embodiment 1, Semen Lablab Album extract
The preparation method of Semen Lablab Album extract of the present invention may further comprise the steps:
1) with 90g Semen Lablab Album mechanical activation comminution powdered.
2) Semen Lablab Album powder that step 1) is obtained extracts with normal saline, method is: Semen Lablab Album powder is mixed with the normal saline of 6 and 8 times of volumes (4-10 times volume all can) respectively, after at room temperature stirring 3 hours (0.5-6 hour all can) separately, with 4-8 layer filtered through gauze, merge two batches of filtrates, at 4 ℃ of centrifugal 15min of following 20000rpm, abandon precipitation then, supernatant is a Semen Lablab Album extracting solution.
3) to step 2) add solid (NH in the extracting solution that obtains
4)
2SO
4, respectively to (NH
4)
2SO
430%, saltouts after 40%, 50%, 60%, 70%, 80%, 90%, 100% (W/V) saturated concentration, in 4 ℃ of refrigerators, leave standstill 2 hours (1-4 hour all can), at 4 ℃ of centrifugal 15min of following 20000rpm, collect each autoprecipitation then.
4) with step 3) through the saturated (NH of variable concentrations
4)
2SO
4The precipitation that obtains of saltouing is carried out desalting processing with distill water dialysis earlier respectively, and lyophilization then obtains Semen Lablab Album extract.Merge Semen Lablab Album extract separately, weigh, obtain Semen Lablab Album extract 1.46g altogether.
The preparation of embodiment 2, Semen Lablab Album extract
The preparation method of Semen Lablab Album extract of the present invention may further comprise the steps:
1) with 180g Semen Lablab Album mechanical activation comminution to powder.
2) Semen Lablab Album powder that step 1) is obtained extracts with normal saline, method is: Semen Lablab Album powder is mixed with the normal saline of 4,7 and 10 times of volumes (4-10 times volume all can) respectively, after at room temperature stirring 6 hours (0.5-6 hour all can) separately, with 4-8 layer filtered through gauze, merge three batches of filtrates, at 4 ℃ of centrifugal 25min of following 20000rpm, abandon precipitation then, supernatant is a Semen Lablab Album extracting solution.
3) to step 2) add solid (NH in the extracting solution that obtains
4)
2SO
4, to 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% (W/V) saturated concentration, saltout respectively, in 4 ℃ of refrigerators, leave standstill 4 hours (1-4 hour all can), at 4 ℃ of centrifugal 15min of following 25000rpm, collect each autoprecipitation then.
4) with step 3) through the saturated (NH of variable concentrations
4)
2SO
4The precipitation that obtains of saltouing is carried out desalting processing with distill water dialysis earlier, and lyophilization then obtains Semen Lablab Album extract.Merge, weigh, obtain Semen Lablab Album extract 4.88g altogether.
The preparation of embodiment 3, Semen Lablab Album extract
The preparation method of Semen Lablab Album extract of the present invention may further comprise the steps:
1) with 360g Semen Lablab Album mechanical activation comminution.
2) Semen Lablab Album powder that step 1) is obtained extracts with normal saline, method is: Semen Lablab Album powder is mixed with the normal saline of 4,6,8 and 10 times of volumes (4-10 times volume all can) respectively, after at room temperature stirring 0.5 hour (0.5-6 hour all can) separately, with 4-8 layer filtered through gauze, merge four batches of filtrates, at 4 ℃ of centrifugal 25min of following 30000rpm, abandon precipitation then, supernatant is a Semen Lablab Album extracting solution.
3) to step 2) add solid (NH in the extracting solution that obtains
4)
2SO
4, to 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% (W/V) saturated concentration, saltout respectively, in 4 ℃ of refrigerators, leave standstill 1 hour (1-4 hour all can), at 4 ℃ of centrifugal 10min of following 30000rpm, collect each autoprecipitation then.
4) with step 3) through the saturated (NH of variable concentrations
4)
2SO
4The precipitation that obtains of saltouing is carried out desalting processing with distill water dialysis earlier, and lyophilization then obtains Semen Lablab Album extract.Merging is also weighed, and obtains Semen Lablab Album extract 8.68g altogether.
Embodiment 4, red cell agglutination experiment
Detect Semen Lablab Album extract of the present invention to erythrocytic agglutination with following experiment, method is as follows:
1, preparation red cell suspension: mice is extractd eyeball and gets the about 1mL of blood, add the anticoagulant of 0.2mL 2%EDTA-Na solution, the centrifugal 10min of back 2000rpm is mixed, abandon supernatant, to precipitate the flushing with 10mL PBS (0.01M), the centrifugal 10min of 2000rpm abandons supernatant, wash 3 times, it is standby that precipitation is mixed with 2% resuspended liquid with PBS.
2, red cell agglutination experiment: on 96 hole blood coagulation plates, after every hole adds 50 μ l PBS buffer, what add embodiment 1-3 acquisition respectively precipitates the Semen Lablab Album extract that obtains through the different ammonium sulfate saturations of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% (W/V): whenever ranked first the hole and add 50 μ l extracts, mixing; Get 50 μ l solution again from first hole and add second hole, mixing; Get 50 μ l solution again from second hole and add the 3rd hole, mixing; The rest may be inferred, does doubling dilution, adds the 12 hole until get 50 μ l solution from 11-holes, abandons 50 μ l solution from the suction of the 12 hole behind the mixing.Then, in each hole, add the resuspended liquid of 50 μ l erythrocyte that step 1 obtains, all add back jolting 10min on shaking table, blood coagulation plate room temperature is left standstill 1-2h, observe agglutination.
The coagulation design sketch as shown in Figure 1, the Semen Lablab Album extract that obtains through the different ammonium sulfate saturations precipitations of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% (W/V) that embodiment 1-3 obtains all has agglutination to erythrocyte.
The SDS-PAGE of embodiment 5, Semen Lablab Album extract (polyacrylamide gel electrophoresis) detects
The Semen Lablab Album extract that obtains through the different ammonium sulfate saturation precipitations of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% (W/V) that embodiment 1-3 is obtained carries out the SDS-PAGE detection, and detection method may further comprise the steps:
1, glass plate is installed;
2, preparation 5mL 15% separation gel;
3, carefully separation gel is injected ready two glass gaps, stay certain space, add water gently on the separation gel upper strata and cover, to stop the inhibitory action of air oxygen to gel polymerisation for the upper strata concentrates glue;
When 4, treating between gel and the water to occur the very strong line of refractivity, illustrate that gel polymerisation finishes, blot upper water with absorbent paper;
5, preparation 2mL concentrates glue;
6, will concentrate glue and pour into the separation gel upper strata, insert comb, carefully not produce bubble;
7, when concentrated glue polymerization, get each 10 μ l of extract that different ammonium sulfate saturations precipitations obtain, add 2.5 μ l sample-loading buffers, boiling water bath boils 5min;
8, glass plate is put into electrophoresis tank, add 1 * Tris-glycine electrophoretic buffer, take out comb;
9, use micro sample-adding rifle application of sample in order;
10, electrophoresis: 80V voltage, arrive at separation gel top to dyestuff, change 130V voltage, arrive at the bottom of separation gel, deenergization until the dyestuff forward position;
11, take off gel, cut concentrated glue, separation gel is put into a plate, add 0.25% coomassie brilliant blue R250 dyeing 1h;
12, reclaim dye liquor,, change twice with the destaining solution decolouring.
(from left to right: swimming lane 1 is the Semen Lablab Album extract that 100% ammonium sulfate saturation precipitation obtains to electrophoresis result as shown in Figure 2, swimming lane 2 is the Semen Lablab Album extract that 90% ammonium sulfate saturation precipitation obtains, swimming lane 3 is the Semen Lablab Album extract that 80% ammonium sulfate saturation precipitation obtains, swimming lane 4 is Mark, swimming lane 5 is the Semen Lablab Album extract that 70% ammonium sulfate saturation precipitation obtains, swimming lane 6 is the Semen Lablab Album extract that 60% ammonium sulfate saturation precipitation obtains, swimming lane 7 is the Semen Lablab Album extract that 50% ammonium sulfate saturation precipitation obtains, swimming lane 8 is the Semen Lablab Album extract that 40% ammonium sulfate saturation precipitation obtains, swimming lane 9 is the Semen Lablab Album extract that 30% ammonium sulfate saturation precipitation obtains), show 30%, 40%, 50%, 60%, 70%, 80%, 90%, all contain identical composition in the Semen Lablab Album extract that the different ammonium sulfate saturation precipitations of 100% (W/V) obtain, just the content height is variant.Above-mentioned red cell agglutination experimental result (embodiment 4) and SDS-PAGE testing result (embodiment 5) show, the present invention all is effective extracts through the Semen Lablab Album extract that the different ammonium sulfate saturation precipitations of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% (W/V) obtain, therefore decision is directly saltoutd with 100% saturated ammonium sulfate, and the result can obtain the 5.2g extract with extracting method of the present invention from the 200g Semen Lablab Album.
Embodiment 6, anti-
60Co gamma-rays radiation damage experiment
Laboratory animal: 40 of Kunming kind white mice, female, hero half and half, quality (20 ± 2) g.
Experiment reagent: ether, formaldehyde, glacial acetic acid, hydrochloric acid, hydroxyproline standard substance, chloramine-T, para diaminobenzene formaldehyde, citrate buffer solution, perchloric acid, NaOH, pentobarbital sodium, superoxide dismutase (SOD) and BCA protein determination kit all build up bio-engineering research institute available from Nanjing.
One, preparation skin radiation injury resistant medicaments
1, the preparation of skin radiation injury resistant gel
The prescription of skin radiation injury resistant gel that with Semen Lablab Album extract of the present invention is effective ingredient is as follows:
Semen Lablab Album extract 50g,
Carbomer 5g,
Sodium hydroxide 2g,
Water 1000mL.
The excipient prescription:
Carbomer 5g,
Sodium hydroxide 2g,
Water 1000mL.
The preparation of excipient: with 1000mL water 5g carbomer swelling is spent the night (12-24 hour), add the 2g sodium hydroxide again and regulate pH value to 7, stir promptly, keep in Dark Place.
The excipient character: the semisolid that white is fine and smooth, quality is even, denseness is suitable, and stretchability is good, nonirritant, no greasy feeling.
The preparation of skin radiation injury resistant gel: 5g carbomer swelling is spent the night with 500mL water, add the 2g sodium hydroxide again and regulate pH value to 7, reuse 500mL water is with 50g Semen Lablab Album extract suspendible, at last with both mix homogeneously, obtain the skin radiation injury resistant gel, keep in Dark Place.
The medicine character: this gel is the semisolid of brownish black exquisiteness, and quality is even, denseness is suitable, and stretchability is good, nonirritant, no greasy feeling.
2, the preparation of skin radiation injury resistant tablet (Film coated tablets)
The prescription of skin radiation injury resistant tablet that with Semen Lablab Album extract of the present invention is effective ingredient is as follows:
Semen Lablab Album extract 100g,
Microcrystalline Cellulose 75g,
Lactose 70g,
Hyprolose 25g,
Cross-linking sodium carboxymethyl cellulose 25g,
95% ethanol 50g,
Micropowder silica gel 3g,
Make 1000.
The preparation of skin radiation injury resistant tablet: according to above-mentioned pharmaceutical formulation, take by weighing Semen Lablab Album extract of the present invention, add microcrystalline Cellulose, lactose and 15g hydroxypropyl cellulose, use 95% alcohol granulation, 60 ℃ of dryings, granulate; Add tabletting behind all the other adjuvant mix homogeneously, make 1000; The bag film-coat, packing obtains the skin radiation injury resistant tablet.
3, the preparation of skin radiation injury resistant oral liquid
The prescription of skin radiation injury resistant oral liquid that with Semen Lablab Album extract of the present invention is effective ingredient is as follows:
Semen Lablab Album extract 200g,
Sucrose 100g,
Benzoic acid 10g,
Flavoring orange essence 10g,
Water 10000mL,
Make 1000.
The preparation of skin radiation injury resistant oral liquid: take by weighing principal agent and adjuvant according to pharmaceutical formulation, add water dissolution, boil, place, filter, packing, every 10mL, sterilization obtains the skin radiation injury resistant oral liquid.
4, the preparation of skin radiation injury resistant capsule
The prescription of skin radiation injury resistant capsule that with Semen Lablab Album extract of the present invention is effective ingredient is as follows:
Semen Lablab Album extract 50g,
Pregelatinized Starch 80g,
Lactose 50g,
Carboxymethyl starch sodium 10g,
Make 1000.
The preparation of skin radiation injury resistant capsule: take by weighing principal agent and adjuvant according to above-mentioned pharmaceutical formulation, cross 80 mesh sieves, mixing, directly encapsulated, packing obtains the skin radiation injury resistant capsule.
Two, anti-
60Co gamma-rays radiation damage experiment
1, experiment grouping
Mice is in experiment prospective adaptation one week of environment, raising temperature 22-25 ℃, be divided into 4 groups at random after the depilation of back: (back is left intact the blank group, the normal nursing), (back is left intact the radiation model group, carry out radiation (method of radiating is as follows), negative control group), (1g excipient (preparation method is seen step 1) is smeared at the back to vehicle group, carry out radiation), administration group (be divided into four groups again, the antiradiation drug that four kinds of dosage forms of 1g step 1 preparation are smeared at the back respectively carries out radiation).(annotate: when the administration lowest dose level was 0.3g, experiment still can obtain similar result)
2, mice
60The radiation of Co gamma-rays
Mice is fixed on the special plank,, only exposes the back after the processing, use with lead shielding
60The Co gamma-rays shines, close rate 2.72Gy/min, irradiation dose 5.0Gy.Shine after 2 days, put to death mice, observe every index.(annotate: when exposure dose was 3.0-7.0Gy, experiment still can obtain similar result)
3, the mouse skin morphosis is observed
Place formalin to fix 48 hours the skin samples of each group mice, ethanol dewaters step by step, carries out ultrathin section and HE dyeing after the paraffin embedding.
The result as shown in Figure 3, normal group (blank group) mouse skin structural integrity, epidermis and corium boundary line are clear, epidermis the rule, skin corium is thicker, it is more even to dye, the cell number of plies is more; Radiation model group mouse skin structure disturbance, epidermis and corium boundary line still can be divided, and epidermis is owed rule, the remarkable attenuation of corium, it is inhomogeneous to dye, and the cell number of plies reduces; The same model group of vehicle group mouse skin structure; Obviously than model group and vehicle group mouse skin structural integrity, epidermis and corium boundary line are clear for administration group mouse skin structure, and epidermis is rule, and skin corium is thicker, and it is more even to dye, and the cell number of plies is more.Above-mentioned experimental result proves that Semen Lablab Album extract of the present invention can reduce the suffered radiation damage of mouse skin, all has curative effect preferably with the skin radiation injury resistant medicaments of the multiple dosage form of its preparation, and skin is had powerful radiation resistance.
Three, the mensuration of collagen content and hydroxyproline content in the mouse skin
Aging and the collagen protein of mammal and human skin have substantial connection.With advancing age, collagen content reduces gradually in the skin, therefore, and youth's dry shrinkage that seems of senile skin, the gloss that follows the string.Hydroxyproline accounted for 12%～14% during collagen protein was formed, and in other tissue, except that the tissue that mainly contains elastin laminin individually contains 1%～3%, contained hydroxyproline hardly.Content height and stable hydroxyproline composition are big characteristics of collagen protein, therefore, can or directly measure the index of hydroxyproline content as the skin aging degree by collagen content in the quantitative assay skin.Detect collagen content and the hydroxyproline content of respectively organizing the mouse skin sample in the step 2, wherein, the hydroxyproline content assay method is as follows:
1, hydroxyproline standard curve preparation
Precision takes by weighing the hydroxyproline standard substance 50.0mg that is dried to constant weight, and with 0.01M HCl dissolving and be diluted to 100mL (1mL is equivalent to 500 μ g hydroxyprolines), this is a standard reserving solution.Get above-mentioned standard reserving solution 2.0mL, thin up is standard application liquid to 100mL (1mL is equivalent to 10 μ g hydroxyprolines).
Get above-mentioned standard application liquid 0,0.2,0.4,0.6,0.8 and 1.0mL, place 10mL tool plug scale test tube respectively, each pipe volume is adjusted into 1.0mL with deionized water; Then, add citrate buffer solution 1.0mL and toluene-sodium-sulfonchloramide liquid 1.0mL successively respectively, place 6min behind the mixing; Added chloric acid 1.0mL again, and placed 5min behind the mixing, after each pipe added p-DMAB liquid 1.0mL, mixing was placed 10min in 75 ℃ of waters bath with thermostatic control, be chilled to room temperature more rapidly, and water to adjust each pipe volume be 5.0mL.
Make blank with first pipe, measure trap in 562nm wavelength place, (μ g) is abscissa with hydroxyproline content, and trap is a vertical coordinate drawing standard curve.
2, the preparation of sample test liquid
After respectively organizing mice execution bark fetching in the step 2, scrape off subcutaneous fat, ether blots, and is cut into fragment, and precision takes by weighing 20mg and does for test agent.Getting the skin samples that above-mentioned precision weighs puts in the 10mL tool plug scale test tube, add 2mL 6MHCl mixing, behind the close plug, place 100 ℃ of waters bath with thermostatic control 3 hours, be chilled to room temperature after the taking-up gradually, add 1.0mL10M NaOH again and transfer pH5-7, after the filtration, to 5.0mL, this is the sample test liquid with the filtrate thin up.
3, hydroxyproline content is measured in the sample
Precision is measured sample test liquid 200 μ l and is put tool plug scale in vitro, after adding water to 1.0mL, the preparation method of standard curve in 1 set by step, from " adding citrate buffer solution 1.0mL " same operation, according to the hydroxyproline content in the standard curve calculation sample of trap that records and step 1 drafting.
The result is as shown in table 1, model group and vehicle group mice are behind partial radiation, collagen content and hydroxyproline content in the skin are compared with normal group, p＜0.01 has marked difference, has all reduced certain level, and administration group mice is behind partial radiation, collagen content and hydroxyproline content in the skin are compared with normal group, and p＞0.05 does not have marked difference.Above-mentioned testing result shows; after the administration; the skin of mice has obtained protection; the radiation damage degree that is subjected to obviously reduces; thereby prove that further Semen Lablab Album extract of the present invention can reduce the suffered radiation damage of mouse skin; skin radiation injury resistant medicaments with the multiple dosage form of its preparation all has curative effect preferably, and skin is had powerful radiation resistance.
Table 1 collagen content and hydroxyproline content measurement result
| Hydroxyproline (mg/g) | Collagen (mg/g) | |
| Normal group | 4.64±0.35 | 35.71±2.69 |
| Model group | 3.32±0.50 | 25.55±3.87 |
| Vehicle group | 3.44±0.13 | 26.46±1.02 |
| The administration group | 4.15±0.13 | 31.95±1.03 |
Four, antioxidase SOD Determination on content in the mouse skin
The mechanism of radiation damage is: effects of ionizing radiation is in organism biomembrane, enzyme, nucleic acid etc., cause the injury response of a series of free radicals, lipid peroxide is body main radioactivity poisonous substance after irradiation, when irradiation back body is not found any damage as yet, it just produces, therefore, it is the product after the radiation, can increase the weight of radiation damage again.The protection system of body defence free radical lipid peroxidation injury mainly is the enzyme scavenger: comprise SOD, catalase (CAT), glutathion peroxidase (GSH-Px), the intravital antioxidase of machine is the defying age material of " endogenous ".The active reduction of antioxidant defense enzyme (as SOD) can make Skin Cell present aging state, and senile cell slows down owing to metabolism, and is poor to the repair function of damage, easier photic damage, the acceleration skin aging of causing.Therefore, the height of SOD content in the skin has determined the size of skin injury degree.
Detect the SOD content of respectively organizing the mouse skin sample in the step 2 with following method: the quality of mouse skin sample is respectively organized in weighing, adds the normal saline of pre-cooling, grinds and frozen centrifugation with glass homogenizer, is prepared into the tissue homogenate of 10% (V/V).Before measuring SOD content, measure the total protein content of skin histology homogenate earlier.With superoxide dismutase (SOD) mensuration test kit and according to the SOD content in the requirement mensuration mouse skin sample of test kit description.
The result is as shown in table 2, model group and vehicle group mice are behind partial radiation, SOD content in the skin is compared with normal group, p＜0.01, has marked difference, all reduced certain level, and administration group mice is behind partial radiation, SOD content in the skin is compared with normal group, and p＞0.05 does not have marked difference, illustrate that Semen Lablab Album extract of the present invention can reduce the radiation damage degree of mouse skin, thereby prove that further Semen Lablab Album extract of the present invention can reduce the suffered radiation damage of mouse skin, all have curative effect preferably, skin is had powerful radiation resistance with the skin radiation injury resistant medicaments of the multiple dosage form of its preparation.
Table 2 superoxide dismutase (SOD) assay
| SOD(U/mg prot) |
| Normal group | 65.62±2.50 |
| Model group | 53.38±3.74 |
| Vehicle group | 53.34±5.01 |
| The administration group | 63.01±5.65 |
Five, the expression of metal matrix protease (MMPs) and determination of activity rinsing liquid in the mouse skin tissue:
Tris 6.05g，
Calcium chloride 0.5550g,
Concentrated hydrochloric acid 3.23mL,
Zinc chloride (1mM) 1mL,
Add distilled water to 1L.
Eluent: 400mL rinsing liquid+10mL TRLTON X-100.
Incubating Solution: 200mL rinsing liquid+0.06g Brij 35 (Brij 35 detergent [Brij is the trade mark of ICI Americas company for a kind of non-ionic detergent, i.e. dodecyl polyglycol ether, or title polyoxyethylene laurel ether]).
Studies show that the generation of MMPs can be degraded/suppress and be synthesized the extracellular matrix of collagen, causes the skin repair defective, produces photoaging, so the degree that how much has also determined skin aging of MMPs content.Detect the expression of respectively organizing MMPs in the mouse skin sample in the step 2, detection method may further comprise the steps:
1) each group mouse skin is made 10% tissue homogenate, carry out 1% gelatin SDS-PAGE, testing result meets the requirements.
2) glass plate is installed.
3) preparation separation gel and spacer gel
Separation gel
Water 2.3mL,
30% acrylamide 2.64mL,
Tris pH8.8 1.5M 2.0mL，
10％SDS 0.08mL，
1% gelatin 0.9mL,
10% ammonium persulfate 0.08mL,
TEMED 5μl。
Spacer gel
Water 2.7mL,
30% acrylamide 0.67mL,
Tris pH6.8 1.0M 0.5mL，
10％SDS 0.04mL，
10% ammonium persulfate 0.04mL,
TEMED 4μl。
4) carefully separation gel is injected ready two glass gaps,, add water then lightly on the separation gel upper strata and cover, to stop the inhibitory action of air oxygen to gel polymerisation for the upper strata spacer gel stays certain space.
When 5) treating between gel and the water to occur the very strong line of refractivity, illustrate that gel polymerisation finishes, blot upper water with absorbent paper.
6) spacer gel is poured into the separation gel upper strata, insert comb, carefully do not produce bubble.
7) glass plate is put into electrophoresis tank, add 1 * Tris-glycine electrophoretic buffer, take out comb, with electrophoretic buffer flushing comb hole.
8) get 10 μ l samples, add 10 μ l sample-loading buffers (1 *), use micro sample-adding rifle application of sample in order.
9) electrophoresis: 90V voltage, arrive at separation gel top to dyestuff, change 120V voltage, arrive at the bottom of separation gel, deenergization until the dyestuff forward position.
10) take off gel, cut spacer gel, separation gel is put into a plate, with the washing of the eluent of 4 ℃ of pre-coolings, 45min * 2 time;
11) with rinsing liquid rinsing 2 times, each 20min.
12) gel is placed Incubating Solution, under 37 ℃, hatch 24h.
13) with the dyeing of 0.25% coomassie brilliant blue R250, the bright band that whether shows MMPs on blue background is observed in the decolouring back.
The result as shown in Figure 4; MMPs content in vehicle group and the model group is apparently higher than normal group and administration group, and the administration group is compared with normal group, and the administration group is a little more than normal group; illustrate that Semen Lablab Album extract of the present invention can reduce the generation of MMPs, promptly has protective effect to radiation damage.This experimental result further proof Semen Lablab Album extract of the present invention can reduce the suffered radiation damage of mouse skin, all has curative effect preferably with the skin radiation injury resistant medicaments of the multiple dosage form of its preparation, and skin is had powerful radiation resistance.
Claims (10)
1, a kind of Semen Lablab Album extract is after Semen Lablab Album is pulverized, earlier it to be extracted with normal saline, extract is saltoutd with saturated ammonium sulfate, after the Semen Lablab Album extract that obtains after the desalination, drying again.
2, a kind of method of extracting the described Semen Lablab Album extract of claim 1 may further comprise the steps:
1) with the Semen Lablab Album mechanical activation comminution;
2) Semen Lablab Album powder that step 1) is obtained extracts with normal saline;
3) with step 2) extracting solution that obtains saltouts with saturated ammonium sulfate;
4) step 3) the is saltoutd precipitation that obtains is carried out desalination successively, and dried obtains Semen Lablab Album extract.
3, extracting method according to claim 2, it is characterized in that: described step 2) to the method that Semen Lablab Album powder extracts be: the normal saline of Semen Lablab Album powder and 4-10 times of volume is mixed with normal saline, after at room temperature stirring 0.5-6 hour, filter, centrifugal, abandon precipitation, supernatant is a Semen Lablab Album extracting solution.
4, extracting method according to claim 3 is characterized in that: filter with 4-8 layer gauze.
5, according to claim 2 or 3 described extracting method, it is characterized in that: the mass/volume of ammonium sulfate (W/V) percentage concentration is 30-100% in the described step 3); Salting-out condition is: saltoutd under 4 ℃ 1-4 hour.
6, according to claim 2 or 3 described extracting method, it is characterized in that: with distilled water the step 3) precipitation that obtains of saltouing is carried out desalting processing in the described step 4).
7, described Semen Lablab Album extract of claim 1 or the preparation-obtained Semen Lablab Album extract of the arbitrary method of claim 2 to 6 application in the preparation skin radiation injury resistant medicaments.
8, application according to claim 7 is characterized in that: the dosage form of described medicine is unguentum, gel or liniment.
9, according to claim 7 or 8 described application, it is characterized in that: also be added with one or more excipient in the described medicine; The addition of described excipient is 0.1%-55.0%.
10, application according to claim 9 is characterized in that: the composition of raw materials of the skin radiation injury resistant medicaments of described gel dosage form comprises following components by weight portion:
50 parts of Semen Lablab Album extracts,
5 parts of carbomers,
2 parts of sodium hydroxide,
1000 parts in water;
The composition of raw materials of the skin radiation injury resistant medicaments of described Tabules comprises following components by weight portion:
100 parts of Semen Lablab Album extracts,
75 parts of microcrystalline Cellulose,
70 parts of lactose,
25 parts of hyprolose,
25 parts of cross-linking sodium carboxymethyl celluloses,
95% ethanol 50-100 part,
3 parts of micropowder silica gels;
The composition of raw materials of the skin radiation injury resistant medicaments of described oral liquid formulation comprises following components by weight portion:
200 parts of Semen Lablab Album extracts,
100 parts of sucrose,
Benzoic acid 10-50 part,
Flavoring orange essence 10-50 part,
10000 parts in water;
The composition of raw materials of the skin radiation injury resistant medicaments of described capsule formulation comprises following components by weight portion:
50 parts of Semen Lablab Album extracts,
80 parts of pregelatinized Starch,
50 parts of lactose,
10 parts of carboxymethyl starch sodium.
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Application publication date: 20091007 Assignee: SOUTH CHINA CENTER FOR STEM CELL BIOLOGY AND REGENERATIVE MEDICINE OF ACADEMY OF MILITARY MEDICAL SCIENCES Assignor: Inst. of Field Blood Transfusion, Academy of Military Medical Sciences, PLA Contract record no.: 2015990000123 Denomination of invention: White lablab seed extract as well as extracting method and application thereof in preparing skin radiation injury resistant medicaments Granted publication date: 20111228 License type: Exclusive License Record date: 20150324 |
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