CN101249274A - Preparation of bletilla striata polyose water gelatin of promoting wound healing and uses thereof - Google Patents
Preparation of bletilla striata polyose water gelatin of promoting wound healing and uses thereof Download PDFInfo
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- CN101249274A CN101249274A CNA200810024674XA CN200810024674A CN101249274A CN 101249274 A CN101249274 A CN 101249274A CN A200810024674X A CNA200810024674X A CN A200810024674XA CN 200810024674 A CN200810024674 A CN 200810024674A CN 101249274 A CN101249274 A CN 101249274A
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- pseudobulbus bletillae
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- polysaccharose
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Abstract
The invention belongs to the biological medical technology field, and discloses a medical polysaccharides hydrogel used for surgical healing which consists of aldehydized bletilla striata, crosslinking agent and deionized pure water; the weight percentage of the polymer is 3 to 25 percent. The hydrogel is characterized in good anti-inflammatory action, good permeability, good moisture retention and the function of absorbing trauma secretion, improving obviously the speed of trauma healing in animal model, and having a wide application prospect as an ideal medical surgical healing medicine.
Description
One, technical field
The invention belongs to the biological medicine technology field, be specifically related to polysaccharide hydrogel of a class medical surgical wound healing and preparation method thereof.
Two, background technology
The elementary object of treatment skin trauma is a healing rate of accelerating wound surface, the prevention bacterial infection, alleviate the misery that hemorrhage, inflammatory reaction, wound infection even ulcer bring the patient, prevent the negative effect of wound, and more the back wound is attractive in appearance as far as possible physiological function.The skin wound healing incident is divided into the blood coagulation phase, inflammatory phase, cellular proliferative stage and tissue remodeling phase, be one and involve that blood coagulation, inflammation, blood vessel are reinvented, epithelial cell proliferation moves and all multifactor complex processes of tissue remodeling or the like, the result of wound healing also is by [the Mehrdad Kokabi that balance determined between these many factors, Mohammad Sirousazar, Zuhair Muhammad Hassan.PVA-clay nanocomposite hydrogels for wound dressing.European PolymerJournal, 2007,43:773-781].
Clinically, medical dressing commonly used covers wound surface promotion healing.Medical dressing is divided into traditional inertia dressing, biphasic effect dressing and biological active dressing three major types.The dressing of tradition inertia comprises the synthetic fibers gauze, plastic foil anti-adhesive gauze and wettability anti-adhesive gauze, belong to early stage wound healing casting product, only has the effect that absorbs secretions and covering and support wound, though make simply low price, reusable, can't keep wound surface moistening but exist, easy damaged cambium and cause pain when changing dressings does not have obvious facilitation or the like shortcoming to wound healing.Biphasic effect dressing comprises half permeability film, polyamino Ethyl formate foam and gelatin class aerogel dressing.These dressing have the function of the stronger middle and high degree sepage of absorption wound surface, and compared to traditional inertia dressing, its moisture-keeping functions is better, advantage is can be used for causing the local physiological moist environment that promotes wound healing, and still, dressing itself does not have function [the Sedlarik KM that promotes wound healing, Vacik J, Wichterle O.Modern dressings-hydrogels.RozhlChir, 1995,74:3-7].Along with the progress of science and technology, new pattern compress was expressed the great expectations with biological function afterwards.Ideal novel wound healing dressing must possess following characteristic: it is moistening that (1) keeps the wound surface physiological; prevent the wound drying; (2) the protection wound surface is not subjected to superinfection, and (3) absorb wound secretion, and (4) reduce the area of wound surface gangrene; (5) the stimulating growth factor; (6) material has elasticity, (7) excellent biological compatibility [PurnaSai K., Mary Babu.Collagen based dressings-a review.Burns; 2000,26:54-62].So biological active dressing arises at the historic moment.
In recent years, novel bioactive dressing is with the dressing of alginic acid salt, and dressing of collagen class and polymeric type bearing hydrocolloid dressing are main.The dressing of alginic acid salt has hemostatic function, have excellent waterproofness and anti-infection effect, but, dry or hard crust class wound surface should not use [Kneafsey B, Oshanghnessy M, Condon KC.Theuse of calcium alginate dressing in deep hand burns.Burns.1996,22:40-43].The dressing of collagen class has the platelet of promotion to be assembled and sticks at wound surface, and as the function that forms bridging between extracellular matrix promotion wound surface and the normal structure, uses comparatively extensive.And the polymer water colloid belongs to the dressing that absorbs the height juice, absorb the sepage amount and can reach more than 12 times of sole mass, and good biocompatibility, but biological function is not remarkable.
The Pseudobulbus Bletillae (Rhizoma Bletillae) [Bletilla striata (Thunb.) Reichb.f.], have another name called Herba Microtis taiwanianae, Rhizoma Bletillae, Rhizoma Zingiberis Recens, be that the orchid family Pseudobulbus Bletillae (Rhizoma Bletillae) belongs to, herbaceos perennial, its tuber has the effect [insulting a principle Chinese materia medica, Beijing: the .2003 of scientific and technical literature publishing house the 5th edition .] of astringing to arrest bleeding, clearing away heat-damp and promoting diuresis, detumescence and promoting granulation.In recent years, this laboratory obtains the pure product of Pseudobulbus Bletillae polysaccharose extract (BSP) [Sun Jiantao, Zhang Junfeng, Wang Chunming, the Luo Yi patent No.: CN200410065732.5] by ion-exchange chromatography and gel permeation chromatography.This pure product cellular level experiment shows, have promotion VEGF (VEGF) and express and promote function [the Chunming Wang of vascular endothelial cell proliferation, Jiantao Sun, Junfeng Zhang, etal.A polysaccharideisolated from the medicinal herb Bletilla striata induces endothelialcells proliferation and vascular endothelial growth factor expression invitro.Biotechnology Letters, 2006,28:539-543].These biological functions are to promote the traditional treatment effect of wound healing to be consistent with the Pseudobulbus Bletillae (Rhizoma Bletillae).On above-mentioned research basis, we are with Pseudobulbus Bletillae polysaccharose aldehyde radical preparation becoming bletilla striata polyose water gelatin medical dressing.Bletilla striata polyose water gelatin has the biological function of the Pseudobulbus Bletillae (Rhizoma Bletillae) and the extracellular matrix environmental activity of hydrogel concurrently.As hydrogel, it has the good moistening function of wound surface physiological of keeping, and can absorb than sole mass is 4 times to 30 times wound exudate, and and wound surface in conjunction with closely, have air permeability and good, it thoroughly prevents bacterial infection as the distinctive tridimensional network of hydrogel; Simultaneously, it has the biological function of the Pseudobulbus Bletillae (Rhizoma Bletillae) concurrently, antiinflammatory, detumescence stimulates near the emiocytosis VEGF of wound, promotes the propagation of vascular endothelial cell, help accelerating wound healing, and good biocompatibility covers wound surface degraded voluntarily after a couple of days, can not cause being difficult to removing with the wound surface adhesion.Just because of bletilla striata polyose water gelatin has the biological function of the Pseudobulbus Bletillae (Rhizoma Bletillae) and the extracellular matrix environmental activity of hydrogel concurrently, dressing has a extensive future as novel bioactive to make it.
Three, summary of the invention
Technical problem to be solved by this invention is to disclose the medical polysaccharide hydrogel of a class, and it comprises the Pseudobulbus Bletillae polysaccharose and the deionization pure water of aldehyde radicalization, and cross-linking agent is an amount of polyamine compounds, comprises hexamethylene diamine, chitosan and poly-D-lysine.
The present invention relates to the preparation method of Pseudobulbus Bletillae (Rhizoma Bletillae) hydrogel.
The invention still further relates to the application of Pseudobulbus Bletillae (Rhizoma Bletillae) hydrogel in skin wound healing.
The Pseudobulbus Bletillae polysaccharose that the present invention relates to is by natural drug Pseudobulbus Bletillae (Rhizoma Bletillae) tuber, obtains by steps such as ion-exchange chromatography and gel permeation chromatographies.Specifically see patent of invention " a kind of liquid embolism agent for intracranial aneurysm and preparation method thereof " [Sun Jiantao, Zhang Junfeng, Wang Chunming, Luo Yi, the patent No.: CN200410065732.5].
The preparation of Pseudobulbus Bletillae (Rhizoma Bletillae) hydrogel of the present invention may further comprise the steps:
1, Pseudobulbus Bletillae polysaccharose is dissolved in the deionization pure water, adds an amount of sodium metaperiodate, stirring reaction spends the night, and removes sodium metaperiodate through the deionized water dialysis, obtains the Pseudobulbus Bletillae polysaccharose of aldehyde radicalization;
2, the mass ratio of the Pseudobulbus Bletillae polysaccharose of aldehyde radicalization and polyamine crosslinking agent is 20: 1,8: 1, and 4: 1,2: 1,1: 1,1: 2, mix post-heating evaporation water solvent, obtain the Pseudobulbus Bletillae (Rhizoma Bletillae) hydrogel of different proportionings.
The bletilla striata polyose water gelatin healing skin wound effect that the present invention relates to is by setting up the mouse skin trauma model, the antiinflammatory in wound healing process of research bletilla striata polyose water gelatin, detumescence, and the effect of quickening wound healing.
The advantage of bletilla striata polyose water gelatin of the present invention has been to suppress effectively the inflammatory episode of wound formation initial stage (2-3 days), has reduced inflammatory episode to wound surface and wound surface normal surrounding tissue and cells injury effect.Wound surface and not red all around pneumonedema thereof, incrustation is at skin surface, and crust was fallen in 8 days, and wound closure is rapid, and comparison is shortened according to kpetrolatum gauze and iodine fluorine gauze healing time, and whole process does not have bacterial infection; Because the effect of absorbed sepage, wound surface do not have sepage to retain the influence healing; Because the hydrogel air permeability and good, wound is not subjected to the negative effect of anaerobic environment; Because Pseudobulbus Bletillae (Rhizoma Bletillae) hydrogel is natural component, cover wound surface degraded voluntarily after a couple of days, so there is not the problem that is difficult to remove after causing more with the site of injury adhesion.Bletilla striata polyose water gelatin of the present invention satisfies the condition that the dressing of a ideal skin wound healing hydrogel class should possess, and has certain biologic activity, and safe, easy to use.
Four, description of drawings
Fig. 1 a is the pictorial diagram of the different proportioning Pseudobulbus Bletillae (Rhizoma Bletillae)-aquagels.
Fig. 1 b is the pictorial diagram of the different proportioning Pseudobulbus Bletillae (Rhizoma Bletillae)s-poly-D-lysine hydrogel.
Fig. 2 is the one-tenth glue mark of different proportioning bletilla striata polyose water gelatins, and white column is the Pseudobulbus Bletillae (Rhizoma Bletillae)-aquagel group, and tiltedly the frame column is the Pseudobulbus Bletillae (Rhizoma Bletillae)-poly-D-lysine hydrogel group.
Fig. 3 a is the swelling ratio of different proportioning bletilla striata polyose water gelatins, and white column is the Pseudobulbus Bletillae (Rhizoma Bletillae)-aquagel group, and tiltedly the frame column is the Pseudobulbus Bletillae (Rhizoma Bletillae)-poly-D-lysine hydrogel group.
Fig. 3 b (1) is the preceding pictorial diagram of the Pseudobulbus Bletillae (Rhizoma Bletillae)-aquagel swelling of 4: 1 proportionings.
Fig. 3 b (2) is the pictorial diagram after the Pseudobulbus Bletillae (Rhizoma Bletillae)-aquagel swelling of 4: 1 proportionings.
Fig. 3 c (1) is the preceding pictorial diagram of the Pseudobulbus Bletillae (Rhizoma Bletillae)-poly-D-lysine swelling behavior of 8: 1 proportionings.
Fig. 3 c (2) is the pictorial diagram behind the Pseudobulbus Bletillae (Rhizoma Bletillae)-poly-D-lysine swelling behavior of 8: 1 proportionings.
Fig. 4 is the breathability of different proportioning bletilla striata polyose water gelatins, the white block diagram Pseudobulbus Bletillae (Rhizoma Bletillae)-aquagel group, and tiltedly the frame column is the Pseudobulbus Bletillae (Rhizoma Bletillae)-poly-D-lysine hydrogel group.
Fig. 5 a is in the C57BL/6 mice wound healing experimentation, uses the Pseudobulbus Bletillae (Rhizoma Bletillae)-poly-D-lysine hydrogel flap coverage of 8: 1 proportionings, and each time point wound surface is in kind to be shone.
Fig. 5 b is in the C57BL/6 mice wound healing experimentation, uses the kpetrolatum gauze flap coverage, and each time point wound surface is in kind to be shone.
Fig. 5 c is in the C57BL/6 mice wound healing experimentation, uses iodine fluorine gauze flap coverage, and each time point wound surface is in kind to be shone.
Fig. 6 is in the C57BL/6 mice wound healing experimentation, and each organizes each time point wound surface area integral, and ■ is the Pseudobulbus Bletillae (Rhizoma Bletillae)-poly-D-lysine hydrogel group of 8: 1 proportionings, the 0th, and the kpetrolatum gauze group, △ is an iodine fluorine gauze group.
Five, the specific embodiment
1, the preparation of hydrogel ()
The pure product of 2g Pseudobulbus Bletillae polysaccharose are dissolved in the deionization pure water, add 0.3 gram-1.0 gram sodium metaperiodates, stir, and react 24 hours, obtain the Pseudobulbus Bletillae polysaccharose of aldehyde radicalization after the dialysis of deionization pure water, 4 ℃ of preservations.
Mass ratio by aldehyde radical Pseudobulbus Bletillae polysaccharose and cross-linking agent hexamethylene diamine is 20: 1 or 8: 1 or 4: 1 or 2: 1 or 1: 1 or 1: 2, mixes post-heating evaporation water solvent, obtains the Pseudobulbus Bletillae (Rhizoma Bletillae)-hexamethylene diamine hydrogel of different proportionings, and sealing is placed on 4 ℃ of refrigerators and preserves.The different proportioning Pseudobulbus Bletillae (Rhizoma Bletillae)s-hexamethylene diamine hydrogel all is the slide stratiform.At 20: 1,8: 1,4: 1, under 2: 1 proportionings, hydrogel was crisp frangible, and mechanical strength is relatively poor.At 1: 1, under 1: 2 proportioning, hydrogel had certain toughness and elasticity, and mechanical strength is better.
2, the preparation of hydrogel (two)
Aldehyde radical Pseudobulbus Bletillae polysaccharose building-up process is with embodiment 1.
Mass ratio by aldehyde radical Pseudobulbus Bletillae polysaccharose and cross-linking agent chitosan is 20: 1 or 8: 1 or 4: 1 or 2: 1 or 1: 1 or 1: 2, mixes post-heating evaporation water solvent, obtains the Pseudobulbus Bletillae (Rhizoma Bletillae)-aquagel of different proportionings, and sealing is placed on 4 ℃ of refrigerators and preserves.At 20: 1, under 8: 1 proportionings, hydrogel was hyaloid, the slide stratiform, and more crisp frangible, mechanical strength is relatively poor.At 4: 1, under 2: 1 proportionings, hydrogel was resilient sheet, and mechanical strength is better, and certain toughness is arranged.At 1: 1, under 1: 2 proportioning, hydrogel was the fruit jelly shape, good springiness, and mechanical strength is good.The different proportioning Pseudobulbus Bletillae (Rhizoma Bletillae)s-aquagel pictorial diagram is seen Fig. 1 a.
3, the preparation of hydrogel (three)
Aldehyde radical Pseudobulbus Bletillae polysaccharose building-up process is with embodiment 1.
Mass ratio by aldehyde radical Pseudobulbus Bletillae polysaccharose and cross-linking agent poly-D-lysine is 20: 1 or 8: 1 or 4: 1 or 2: 1 or 1: 1 or 1: 2, mix post-heating evaporation water solvent, obtain the Pseudobulbus Bletillae (Rhizoma Bletillae)-poly-D-lysine hydrogel of different proportionings, sealing is placed on 4 ℃ of refrigerators and preserves standby.Under 20: 1 proportionings, hydrogel is hyaloid, and the slide stratiform has certain toughness and mechanical strength.At 8: 1,4: 1, under 2: 1 proportionings, hydrogel was the fruit jelly shape, good springiness, and mechanical strength is good.At 1: 1, under 1: 2 proportioning, hydrogel was brown opaque flakes stratiform, does not have elasticity, easily splits, and mechanical strength is medium.The different proportioning Pseudobulbus Bletillae (Rhizoma Bletillae)s-poly-D-lysine hydrogel pictorial diagram is seen Fig. 1 b.
4, the one-tenth glue mark property research of hydrogel
The one-tenth glue mark of each proportioning bletilla striata polyose water gelatin calculates as follows:
5, the swelling property of hydrogel research
The swelling ratio of each proportioning bletilla striata polyose water gelatin calculates as follows:
6, the microorganism permeability of hydrogel research
Different proportioning bletilla striata polyose water gelatins are made becomes diameter 4cm, and the thin layer of thick 2mm is placed into TSA (Trypsin soy sugars) surface, sterilization, then with escherichia coli with 10
8The concentration of/ml is added drop-wise to different proportioning bletilla striata polyose water gelatins surface, observes the area that TSA goes up antibacterial every day.Each proportioning bletilla striata polyose water gelatin all has the character that prevents that fully antibacterial from seeing through.
7, the air permeability studies of hydrogel
The breathability of bletilla striata polyose water gelatin is calculated as follows:
8, the animal model experiment of bletilla striata polyose water gelatin skin wound healing ()
Choose 72 of male C57BL/6 mices, body weight 22～25g is divided into three groups at random, is respectively iodine fluorine gauze wound healing matched group, kpetrolatum gauze wound healing matched group, the Pseudobulbus Bletillae (Rhizoma Bletillae) of 8: 1 proportionings-poly-D-lysine hydrogel wound healing group.All male C57BL/6 mices are all anaesthetized with ketamine, and the anesthesia back by hair 2cm * 3cm, makes circular skin excision wound surface two places of diameter 6mm at the back at quilt hair place.Each is organized mice and uses one deck iodine fluorine gauze respectively, and one deck kpetrolatum gauze is perhaps smeared one deck Pseudobulbus Bletillae (Rhizoma Bletillae)-complete flap coverage of poly-D-lysine hydrogel, and the two-layer sterile gauze of reuse covers gauze containing or above the hydrogel, uses immobilization with adhesive tape.
Take pictures to the wound surface of each group mice every day.Carry out integration with wound surface area in the photoshop software comparison film, make and respectively form the curve that the face area dwindles.
| Relatively | The Pseudobulbus Bletillae (Rhizoma Bletillae)-poly-D-lysine hydrogel | Kpetrolatum gauze | Iodine fluorine gauze |
| 2 days | Wound surface is smooth, circular low inflammation | The wound surface depression, circle has swelling, inflammation | The wound surface depression, red and swollen around the irregular wound surface, |
| 5 days | Subcrustal healing does not have inflammation | The incrustation healing still has inflammation | The incrustation healing still has inflammation |
| 8-11 days | Crust is fallen, and wound surface closes up fast | Crust is arranged, and wound surface closes up slow | Crust is arranged, and wound surface closes up slow |
| 12-13 days | Wound surface all calms down | ||
| 14 days | Wound surface calms down | Wound surface calms down |
It is in kind according to seeing Fig. 5 that each organizes each time point wound surface.
Each is organized each time point wound surface area integral figure and sees Fig. 6.
Experimental result shows the Pseudobulbus Bletillae (Rhizoma Bletillae)-poly-D-lysine hydrogel that uses 8: 1 proportionings, and is smooth at wound healing process initial stage (2-3 days) wound, do not continue red and swollen symptom, kept suitable level of inflammation, and antiinflammatory rapidly afterwards.By contrast, kpetrolatum gauze group and iodine fluorine gauze group, in wound healing process initial stage wound redness, level of inflammation still remained on higher level at the 5th day, the just thoroughly detumescence later on up to 7 days has influenced wound surface and has entered and organize the formation phase to carry out that epithelium is reinvented and tissue regeneration.So, the Pseudobulbus Bletillae (Rhizoma Bletillae) of 8: 1 proportionings-poly-D-lysine hydrogel has reduced lasting inflammatory episode epithelium has been reinvented inhibitory action with tissue regeneration, wound surface and not red all around pneumonedema thereof, incrustation is mainly at skin surface, crust was fallen in 8 days, wound closure is rapid, and comparison is shortened according to kpetrolatum gauze group and iodine fluorine gauze group healing time.
9, the animal model experiment of bletilla striata polyose water gelatin skin wound healing (two)
C57BL/6 mouse skin trauma model makes up with embodiment 4.
The Pseudobulbus Bletillae (Rhizoma Bletillae)-aquagel according to 1: 1 proportioning of embodiment 2 described method configurations carries out the experiment of C57BL/6 mouse skin wound healing.In the wound healing process, wound surface was smooth in the 2nd day, circle, and inflammation is low; Formed subcrustal healing, and do not have inflammation in the 6th day; Crust was fallen in the 9th day to the 10th day, and wound surface closes up afterwards; The 13rd day to the 14th day, wound surface all calmed down, and the healing time comparison slightly shifts to an earlier date according to kpetrolatum gauze group and iodine fluorine gauze group.The level of inflammation of the Pseudobulbus Bletillae (Rhizoma Bletillae)-aquagel group is comparatively gentle, and that contrast kpetrolatum gauze group and iodine fluorine gauze are formed face is still red and swollen at the 5th day, the just detumescence fully later on up to 7 days.The inflammatory episode of optimal level and antiinflammation make that the bletilla striata polyose water gelatin wound healing speed of 1: 1 proportioning is faster than matched group.
Claims (5)
1. medical polysaccharide hydrogel, it is characterized by: described hydrogel is made up of Pseudobulbus Bletillae polysaccharose, cross-linking agent hexamethylene diamine or the chitosan of aldehyde radicalization or poly-D-lysine and deionization pure water, the mass percent of polymer is 3-25%, and the mass percent of deionization pure water is 75-97%.
2. according to the preparation method of the described medical polysaccharide hydrogel of claim 1, it is characterized in that carrying out according to the following steps: Pseudobulbus Bletillae polysaccharose is dissolved in the deionization pure water, add 0.3 gram-1.0 gram sodium metaperiodates, stirring reaction 24 hours, with the Pseudobulbus Bletillae polysaccharose that obtains aldehyde radicalization after the dialysis of deionization pure water, mass ratio by aldehyde radical Pseudobulbus Bletillae polysaccharose and cross-linking agent hexamethylene diamine is 20: 1 or 8: 1 or 4: 1 or 2: 1 or 1: 1 or 1: 2 again, mix post-heating evaporation water solvent, obtain the Pseudobulbus Bletillae (Rhizoma Bletillae)-hexamethylene diamine hydrogel of different proportionings.
3. according to the preparation method of the described medical polysaccharide hydrogel of claim 1, it is characterized in that carrying out according to the following steps: Pseudobulbus Bletillae polysaccharose is dissolved in the deionization pure water, add 0.3 gram-1.0 gram sodium metaperiodates, stirring reaction 24 hours, with the Pseudobulbus Bletillae polysaccharose that obtains aldehyde radicalization after the dialysis of deionization pure water, mass ratio by aldehyde radical Pseudobulbus Bletillae polysaccharose and cross-linking agent chitosan is 20: 1 or 8: 1 or 4: 1 or 2: 1 or 1: 1 or 1: 2 again, mix post-heating evaporation water solvent, obtain the Pseudobulbus Bletillae (Rhizoma Bletillae)-aquagel of different proportionings.
4. according to the preparation method of the described medical polysaccharide hydrogel of claim 1, it is characterized in that carrying out according to the following steps: Pseudobulbus Bletillae polysaccharose is dissolved in the deionization pure water, add 0.3 gram-1.0 gram sodium metaperiodates, stirring reaction 24 hours, with the Pseudobulbus Bletillae polysaccharose that obtains aldehyde radicalization after the dialysis of deionization pure water, mass ratio by aldehyde radical Pseudobulbus Bletillae polysaccharose and cross-linking agent poly-D-lysine is 20: 1 or 8: 1 or 4: 1 or 2: 1 or 1: 1 or 1: 2 again, mix post-heating evaporation water solvent, obtain the Pseudobulbus Bletillae (Rhizoma Bletillae)-poly-D-lysine hydrogel of different proportionings.
5. according to the application of the described medical polysaccharide hydrogel of claim 1 in preparation treatment surgery wound healing medicine.
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