CN111000891A - A Chinese medicinal composition with antibacterial, antiinflammatory, hemostatic and analgesic effects, and its preparation method - Google Patents
A Chinese medicinal composition with antibacterial, antiinflammatory, hemostatic and analgesic effects, and its preparation method Download PDFInfo
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Abstract
The invention discloses a Chinese medicinal composition with antibacterial, anti-inflammatory, hemostatic and analgesic effects and a preparation method thereof, wherein a pithecellobium clypearia extract and a lamiophlomis rotata extract are used as raw materials, polylactic acid-glycolic acid copolymer PLGA, vitamin E polyethylene glycol succinate TPGS and poloxamer are used as drug-carrying matrixes, an oil phase and an oil-in-water phase are formed by mixing the raw materials and the drug-carrying matrixes, nano-suspension is obtained by ultrasonic emulsification through a probe, and carbomer, collagen tripeptide, oat β -glucan and propylene glycol are added as gel matrixes to prepare the Chinese medicinal composition nano-gel.
Description
Technical Field
The invention belongs to the field of traditional Chinese medicines, and particularly relates to a traditional Chinese medicine composition with antibacterial, anti-inflammatory, hemostatic and analgesic effects and a preparation method thereof.
Background
In daily life, the conditions of scratch, cut, scratch and the like often occur. In addition, the surgical outpatient clinic often encounters infectious trauma such as a purulent wound, a chronic infectious sinus, infection and dehiscence of a suture incision, bedsores, untimely debridement of a wound and infection of a suture wound due to trauma, and the like. If the wounds are not treated timely and effectively, the healing speed of the wounds can be prolonged, the problems of wound infection, inflammation and the like are easy to occur, pain is brought to patients, and the healing speed of the wounds is also seriously influenced.
Anti-inflammatory drugs or hemostatic drugs are used in daily life, and are commonly used as essential drugs for every family. The medicines for stopping bleeding circulated in the market are various, and many traditional Chinese medicine medicines for stopping bleeding such as traumatic injury capsules and traumatic injury tablets have the effects of promoting blood circulation, removing blood stasis, relieving swelling and pain, and have no effects of stopping bleeding and promoting healing for traumatic bleeding. In addition, most of western hemostatic medicines have antibacterial and anti-inflammatory effects, and can stop bleeding and promote wound healing, but have the problems of high irritation to the wound, inconvenience in coating, easiness in leaving scars after wound healing and the like, and particularly, the existence of scars on relatively obvious parts brings various inconveniences to work and life of patients.
Therefore, the traditional Chinese medicine composition which has good antibacterial and anti-inflammatory effects, hemostatic and analgesic effects and no toxic or side effect has application value. The invention aims to provide a novel Chinese medicinal composition nano gel with the functions of inhibiting bacteria, diminishing inflammation, stopping bleeding and easing pain and no toxic or side effect by utilizing rich Chinese medicinal herb resources, and the nano gel has the characteristics of improving the solubility of fat-soluble medicaments, increasing the dissolution rate and improving the bioavailability by utilizing the characteristics of a nano preparation, thereby achieving the high-efficiency and long-acting treatment effects of inhibiting bacteria, diminishing inflammation, stopping bleeding and easing pain.
Disclosure of Invention
The invention provides a traditional Chinese medicine composition with the functions of bacteriostasis, anti-inflammation, hemostasis and analgesia and a preparation method thereof on the basis of overcoming the defects of the prior art, and the preparation has definite curative effect and simple, quick and controllable preparation method.
In order to achieve the purpose, the invention adopts the following technical scheme:
on the one hand, the traditional Chinese medicine composition with the effects of bacteriostasis, inflammation diminishing, hemostasis and analgesia is provided, and comprises the following components, by weight, 1-5 parts of pithecellobium clypearia extract, 1-5 parts of lamiophlomis rotate extract, 6-12 parts of polylactic acid-glycolic acid copolymer PLGA, 3-5 parts of vitamin E polyethylene glycol succinate TPGS, 5-10 parts of poloxamer, 8-12 parts of carbomer, 0.5-2 parts of collagen tripeptide, 1-2 parts of oat β -glucan, and 0.5-2 parts of propylene glycol.
The composition further comprises, by weight, 2-4 parts of pithecellobium clypearia extract, 2-4 parts of lamiophlomis rotata extract, 8-10 parts of polylactic acid-glycolic acid copolymer PLGA, 3-5 parts of vitamin E polyethylene glycol succinate TPGS, 6-9 parts of poloxamer, 9-11 parts of carbomer, 1-2 parts of collagen tripeptide, 1-2 parts of oat β -glucan and 1-2 parts of propylene glycol.
The composition further comprises, by weight, 3 parts of pithecellobium clypearia extract, 3 parts of lamiophlomis rotata extract, 9 parts of polylactic-co-glycolic acid (PLGA), 4 parts of vitamin E polyethylene glycol succinate (TPGS), 8 parts of poloxamer, 10 parts of carbomer, 1 part of collagen tripeptide, 1 part of oat β -glucan and 1 part of propylene glycol.
On the other hand, the preparation method of the traditional Chinese medicine composition with the effects of inhibiting bacteria, diminishing inflammation, stopping bleeding and easing pain is provided, and comprises the following steps:
1) preparation of crude drug extract
Crushing and sieving the pithecellobium clypearia, performing subcritical extraction by using ethyl acetate as a solvent, wherein the feeding ratio of the pithecellobium clypearia to the ethyl acetate is 1g:1 mL-1 g:5mL, the subcritical extraction temperature is 35-50 ℃, the extraction time is 60-80 min, the extraction pressure is 18-22 MPa, and removing the ethyl acetate by reduced pressure concentration to obtain the pithecellobium clypearia extract;
crushing and sieving the lamiophlomis rotata, carrying out subcritical extraction by using ethyl acetate as a solvent, carrying out subcritical extraction on the lamiophlomis rotata and the ethyl acetate according to a feeding ratio of 1g to 1 mL-1 g to 3mL, wherein the subcritical extraction temperature is 25-35 ℃, the extraction time is 60-80 min, the extraction pressure is 18-22 MPa, and carrying out reduced pressure concentration to remove the ethyl acetate to obtain a lamiophlomis rotata extract;
2) dissolving polylactic acid-glycolic acid copolymer PLGA and vitamin E polyethylene glycol succinate TPGS in ethyl acetate, adding extract of Pithecellobium clypearia and radix Lamiophlomidis Rotatae, and dissolving by ultrasonic to obtain Chinese medicinal composition oil phase;
3) adding the oil phase prepared in the step 2 into a water phase containing poloxamer, magnetically stirring for 1-2 h under the condition of water bath at the temperature of 60-80 ℃, then performing ultrasonic treatment for 30-50 min by using an ultrasonic cell disruptor probe, removing ethyl acetate, cooling, and filtering to obtain a traditional Chinese medicine composition nano suspension;
4) and (3) adding carbomer and collagen tripeptide into the Chinese medicinal composition nano suspension prepared in the step (3), sealing, placing until the matrix is completely swelled, uniformly stirring, sequentially adding oat β -dextran, poloxamer and propylene glycol, and continuously stirring for 2-3 h to obtain the Chinese medicinal composition nano gel.
Further, the volume of the water phase in the step 2 is 4-10 times that of the oil phase.
Further, the ultrasonic time of the probe of the ultrasonic cell crusher in the step 3 is 30min to 50 min.
Further, the rotation speed of stirring in the step 4 is 200rpm to 600 rpm.
The invention has the following positive effects:
1. the active substances of the raw materials of the invention are the pithecellobium clypearia extract and the lamiophlomis rotata ethyl acetate extract, the pithecellobium clypearia extract has stronger antibacterial and anti-inflammatory effects, the lamiophlomis rotata ethyl acetate extract has good hemostatic and analgesic effects, and the two active ingredients act together, so that the traditional Chinese medicine composition can realize the quick, high-efficiency and long-acting antibacterial and anti-inflammatory, hemostatic and analgesic therapeutic effects.
2. The nano-particle drug-loaded matrix used in the invention, polylactic acid-glycolic acid copolymer PLGA, shows more favorable performance compared with most other types of delivery systems; vitamin E polyethylene glycol succinate TPGS is another well-known biocompatible and amphiphilic polymer, is widely applied to the surface coating of the nanoparticles to improve the encapsulation efficiency, can improve the solubility of fat-soluble ingredients of an extract in an oil phase, enables the oil phase and a water phase to have certain intersolubility, and can form an emulsion film on the surface of the nanoparticles.
3. The gel auxiliary material used in the invention takes carbomer as a gel forming matrix, so that the adhesion of carbomer on the surface of skin is strong, the retention time of the medicine on the surface of skin can be prolonged, the combination of oat β -glucan and collagen peptide is beneficial to the effect of active substances, a layer of protective film is formed on the surface of skin, poloxamer is a natural emulsifier, so that the medicine can form uniform and tiny emulsion, and a gel preparation with stable quality is formed, and glycerin is taken as a humectant, and has good skin moistening and moisturizing effects.
4. The nano gel of the traditional Chinese medicine composition prepared by the invention can promote the percutaneous absorption of the medicine and improve the absorption efficiency as a novel administration form; the Chinese medicinal composition nanogel can directly act on the administration part, reduces adverse reaction, is convenient to use, and can avoid the first pass effect of the liver and the side effect of the gastrointestinal tract.
Drawings
FIG. 1 is an SEM transmission electron micrograph of the nanogel of the traditional Chinese medicine composition of example 1.
FIG. 2 is a graph showing the particle size distribution of the nanogel of the Chinese medicinal composition of example 1.
FIG. 3 is an in vitro release profile of the nanogel of the Chinese medicinal composition of example 1.
Detailed Description
The present invention is further described in the following examples, which should not be construed as limiting the scope of the invention, but rather as providing the following examples which are set forth to illustrate and not limit the scope of the invention.
Example 1
A traditional Chinese medicine composition with antibacterial, anti-inflammatory, hemostatic and analgesic effects comprises, by weight, 3 parts of pithecellobium clypearia extract, 3 parts of lamiophlomis rotata extract, 9 parts of polylactic acid-glycolic acid copolymer PLGA, 4 parts of vitamin E polyethylene glycol succinate TPGS, 8 parts of poloxamer, 10 parts of carbomer, 1 part of collagen tripeptide, 1 part of oat β -glucan and 1 part of propylene glycol.
The preparation method of the traditional Chinese medicine composition comprises the following steps:
1) preparation of crude drug extract
1kg of pithecellobium clypearia is taken, crushed, sieved by a 150-mesh sieve, and subcritical extraction is carried out by taking ethyl acetate as a solvent, wherein the liquid-material ratio is 1g: 2mL, extracting at 40 ℃ for 70min at 20MPa under subcritical extraction temperature, and concentrating under reduced pressure to remove ethyl acetate to obtain extract of Pithecellobium clypearia;
1kg of lamiophlomis rotata is taken, crushed, sieved by a 150-mesh sieve, and subjected to subcritical extraction by using ethyl acetate as a solvent, wherein the liquid-material ratio is 1g: 2mL, subcritical extraction temperature of 30 ℃, extraction time of 80min, extraction pressure of 20MPa, reduced pressure concentration to remove ethyl acetate, and obtaining Lamiophlomis rotata extract;
2) weighing 0.90g of polylactic-co-glycolic acid (PLGA) and 0.40g of vitamin E polyethylene glycol succinate (TPGS), dissolving in 500mL of ethyl acetate, adding 0.30g of pithecellobium clypearia extract and 0.30g of lamiophlomis rotata extract, and dissolving by ultrasonic to obtain a traditional Chinese medicine composition oil phase;
3) adding the oil phase prepared in the step 2 into 1500mL of water phase containing 0.40g of poloxamer, magnetically stirring for 2h under the condition of water bath at the temperature of 80 ℃, performing ultrasonic treatment for 40min by using an ultrasonic cell disruptor probe, removing ethyl acetate, cooling, and filtering to obtain a traditional Chinese medicine composition nano suspension;
4) and (3) weighing 1.00g of carbomer and 0.10g of collagen tripeptide, adding the carbomer and the collagen tripeptide into the Chinese medicinal composition nanosuspension prepared in the step (3), sealing, placing until the matrix is completely swelled, sequentially adding 0.10g of oat β -dextran, 0.40g of poloxamer and 0.10g of propylene glycol under electric stirring at 400rpm, and continuously stirring for 2 hours to obtain the Chinese medicinal composition nanogel.
The test results of the nanogel of the traditional Chinese medicine composition prepared in example 1 are as follows:
1. particle size morphology and particle size
The nanogel of the traditional Chinese medicine composition prepared in example 1 is observed for particle size morphology by adopting an SEM transmission electron microscope, and the result is shown in figure 1, and figure 1 shows that the nanogel of the traditional Chinese medicine composition is in a quasi-circular shape and has regular distribution morphology; the particle size is measured by a laser particle size analyzer, the result is shown in figure 2, and the particle size distribution of the Chinese medicinal composition nanogel is 120nm to 260nm as can be seen from figure 2.
2. In vitro Release behavior Studies
The in vitro release degree of the nanogel of the traditional Chinese medicine composition prepared in example 1 was determined by a basket method. Precisely weighing 0.3g, placing in a dialysis bag, fastening two ends at a certain distance with thread, removing air bubbles in the bag, placing in a rotary basket, and placing in a small cup. The release medium is 200mL of ultrasonic distilled water, the temperature of the circulating water is controlled at 37.0 +/-1 ℃, the speed of a rotary basket is 75rpm, 5mL of the release medium is sampled after 5min, 10min, 15min, 20min, 30min, 45min, 60min, 90min and 120min of release, 5mL of the release medium is supplemented, the content of the sample is measured after the sample is filtered by a 0.22-micrometer microporous filter membrane, and the measurement result is shown in figure 3 by using shanzhiside methyl ester.
As shown in FIG. 3, the release rate of the Chinese medicinal composition nanogel reaches 81.2% in 30min, the release rate reaches 85.7% in 45min, and the release reaches balance.
3. Stability survey
The accelerated stability test of the Chinese medicinal composition nanogel is carried out under the conditions that the temperature is 40 ℃ and the relative humidity is 75%, samples are taken in 1 month, 2 months, 3 months and 6 months respectively, and the appearance, the particle size, the content of the unique liposoluble constituent gardenia glucoside methyl ester and the content of the pithecellobium clypearia liposoluble constituent kaempferol of the Chinese medicinal composition nanogel are measured in the standing process, and the results are shown in the following table 1.
TABLE 1 stability test results
Note: p < 0.05 compared to day 0, P < 0.01 compared to day 0.
As can be seen from table 1 above, under the conditions of a temperature of 40 ℃ and a relative humidity of 75%, the traditional Chinese medicine composition nanogel has no difference in appearance during a standing process for 6 months, and no statistical difference exists in changes of particle size, content of shanzhiside methylester and content of kaempferol, which indicates that the traditional Chinese medicine composition nanogel has high stability.
Example 2
1. Bacteriostatic action
Drug control group: taking the extract of the pithecellobium clypearia and the extract of the lamiophlomis rotata, adding purified water to prepare 0.2 percent solution containing the extract of the pithecellobium clypearia and the extract of the lamiophlomis rotata respectively.
Precisely sucking 300 mu L of test bacteria liquid of staphylococcus aureus, escherichia coli, pseudomonas aeruginosa and candida albicans, sucking the test bacteria liquid into a sterile culture dish, pouring 10-16 mL of nutrient broth agar culture medium, continuously shaking to enable the bacteria liquid to be uniform, cooling and solidifying the culture medium, punching holes on the culture dish at equal intervals, and marking. Adding 100 μ L of control and the above test group samples, gently shaking and mixing, drying, reversing the plate, culturing in an aerobic constant temperature incubator at 37 deg.C, observing results after 1h, 2h, 4h, 8h, 12h and 24h, measuring the size of the zone of inhibition, measuring for 3 times, and taking the average value, wherein the experimental results of the zone of inhibition are shown in Table 2.
Antibacterial zone experiment judgment standard: the diameter of the inhibition zone is more than 20mm, and the drug is extremely sensitive: 15-20 mm, high sensitivity: 10-15 mm, medium sensitivity: 7-9 mm, low sensitivity: less than 7mm, insensitive.
TABLE 2 measurement results of the zone of inhibition
Note: p < 0.05 compared to control, P < 0.01 compared to control.
As can be seen from Table 2, the overall diameter of the inhibition zone of the drug control group is small, which indicates that the inhibition effect is not obvious. And observing the change of the antibacterial effect of the drug control group for 24 hours, wherein the diameter of the antibacterial ring is the largest at 1 hour, which shows that the release of the antibacterial active ingredients is the highest peak at 1 hour, the diameter of the antibacterial ring is slowly reduced for 2 to 4 hours, and the diameter is rapidly reduced for 12 to 24 hours. The result shows that the antibacterial active ingredients of the drug control group are difficult to release, so the antibacterial effect is not obvious, the peak value of drug release within 1h is the maximum, and the drug effect is more obviously reduced within 8 h.
Compared with the control group, the diameter of the example group is obviously larger than that of the control group (P is less than 0.01) at each time point, which shows that the antibacterial active ingredients in the control drug can obviously increase the release of the active ingredients when the traditional Chinese medicine composition nanogel is prepared, and has obvious inhibition effect on staphylococcus aureus, escherichia coli and pseudomonas aeruginosa. By observing the change of the antibacterial effect for 24 hours in the group of the embodiment 1, the antibacterial effect for 1 hour is higher, the peak is reached in 2 hours, and then the antibacterial effect slowly decreases, however, the antibacterial effect is still continuous in 12-24 hours, so that the traditional Chinese medicine composition nanogel can achieve the aim of slow-release, efficient and durable sterilization.
Example 3
Animal pharmacodynamic experiment
1. Anti-inflammatory action
1) Effect on mouse capillary Permeability
Taking 50 qualified mice with half male and female and 18-22 g weight, randomly dividing the mice into 5 groups, wherein the groups are as follows:
blank control group: normal saline with the same volume and the medicine application area is 2cm2；
Positive drug control group: compound dexamethasone gel 0.15g/kg, and the medicine application area is 2cm2；
The traditional Chinese medicine composition nanogel low-dose group: 0.15g/kg, the area of the medicine application is 1cm2；
The traditional Chinese medicine composition nanometer gel medium dose group comprises: 0.20g/kg, the area of the medicine application is 2cm2；
The traditional Chinese medicine composition nanogel high-dose group comprises: 0.25g/kg, the area of the medicine coating is 4cm2；
Carrying out depilation treatment on the same position on the left side of the spinal column of each group of mice according to the skin area of different dosage groups, uniformly coating each administration group, fixing by using medical adhesive plaster, externally applying the administration for 1 time every day for 3 consecutive days, carrying out intravenous injection on each mouse by 0.10mL of physiological saline solution with the mass fraction of 0.3% 30min after the last administration, immediately administering 0.2mL of acetic acid physiological saline with the mass fraction of 0.6% for each, carrying out cervical vertebra dislocation for 15min, cutting skin area slices of the skin part treated by depilation administration, placing the cut skin slices into a glass test tube, adding 5mL of acetone saline solution (acetone: physiological saline solution) into the glass test tube, placing the glass test tube in the dark for 48h at room temperature, rotating/separating the heart at 3000 r for 10min, taking supernatant, carrying out color comparison at the wavelength of 590nm, and reading and recording the OD value (absorbance). The capillary permeability test uses the read OD value as an evaluation index.
TABLE 3 results of OD values of capillary permeability experiments
Note: denotes P < 0.01 compared to the blank group, and P < 0.05 compared to the blank group.
As can be seen from table 3, each administration group had an inhibitory effect on the mouse capillary permeability caused by glacial acetic acid compared to the blank control group, and particularly, the compound dexamethasone gel group, the traditional Chinese medicine composition nanogel medium dose group and the compound dexamethasone gel high dose group had significant differences compared to the blank control group. The high dosage of the nanometer gel of the Chinese medicinal composition is better than that of the compound dexamethasone gel group. The results show that the Chinese medicinal composition nanogel has good anti-inflammatory effect.
2) Effect of ear swelling in mice by Paraxylene
Taking 50 qualified mice with half male and female and 18-22 g weight, randomly dividing the mice into 5 groups, wherein the groups are as follows:
blank control group: normal saline with the same volume and the medicine application area is 2cm2；
Positive drug control group: compound dexamethasone gel 0.15g/kg, and the medicine application area is 2cm2；
The traditional Chinese medicine composition nanogel low-dose group: 0.15g/kg, the area of the medicine application is 1cm2；
Chinese medicinal compositionNanogel medium dose groups: 0.20g/kg, the area of the medicine application is 2cm2；
The traditional Chinese medicine composition nanogel high-dose group comprises: 0.25g/kg, the area of the medicine coating is 4cm2；
The method comprises the steps of conducting depilation treatment on the same position on the left side of the spinal column of the back of each group of mice according to the skin area of different dosage groups, evenly coating each administration group, fixing the administration groups by using medical adhesive tapes, externally applying the administration groups for 1 time every day for 3 continuous days, dripping 0.05mL of dimethylbenzene on the right ear of each mouse after the last administration, using the left ear as a control, killing animals after 15min, cutting off the same area of two ears on a puncher with the diameter of 6mm, weighing the ears on a precision analysis balance with the sensing quantity of 1mg, calculating the swelling degree of each group according to the weight difference of the left ear and the right ear, calculating the swelling inhibition rate (%), comparing the difference between the groups with the control group, and obtaining the result as shown in the following table 4.
TABLE 4 results of rate of ear swelling in mice by xylene
Note: denotes P < 0.01 compared to the blank group, and P < 0.05 compared to the blank group.
As can be seen from table 4, each administration group can inhibit mouse ear swelling caused by xylene compared with the blank control group, and particularly, the compound dexamethasone gel group and the traditional Chinese medicine composition nano-gel medium-dose group and the compound dexamethasone gel high-dose group have significant difference compared with the blank control group. The effect of the nanometer gel of the traditional Chinese medicine composition is equivalent to that of the compound dexamethasone gel group. The results show that the traditional Chinese medicine composition nanogel can well inhibit mouse ear swelling caused by xylene, so that the traditional Chinese medicine composition nanogel has a good anti-inflammatory effect.
2. Analgesic effect (twisting method)
Taking 50 qualified mice with half male and female and 18-22 g weight, randomly dividing the mice into 5 groups, wherein the groups are as follows:
blank control group: physiological salt with same volumeWater, the area of the applied medicine is 2cm2；
Positive drug control group: 0.14g/kg of Yunnan Baiyao spray, and the application area is 2cm2；
The traditional Chinese medicine composition nanogel low-dose group: 0.15g/kg, the area of the medicine application is 1cm2；
The traditional Chinese medicine composition nanometer gel medium dose group comprises: 0.20g/kg, the area of the medicine application is 2cm2；
The traditional Chinese medicine composition nanogel high-dose group comprises: 0.25g/kg, the area of the medicine coating is 4cm2；
Depilating at the same position of the left side of spinal column of each group of mice according to skin area of different dosage groups, uniformly smearing each administration group, fixing with medical adhesive plaster, externally applying for 1 time every day for 3 consecutive days, and performing intraperitoneal injection of 0.6% glacial acetic acid 0.1mL/10g to relieve pain. The number of twists of the small rats in each group within 20min of the injection of the analgesic agent was recorded and the results are shown in table 5 below.
TABLE 5 mouse writhing frequency results
Note: denotes P < 0.01 compared to the blank group, and P < 0.05 compared to the blank group.
As can be seen from Table 5 above, each administration group was compared with the blank control group
The animal writhing reaction times are reduced, the medium dosage is obviously different, and the high dosage group and the Yunnan white drug powder spray group are extremely obviously different; compared with Yunnan white drug spray, the high-dose group is equivalent to the Yunnan white drug spray group, and the frequency of writhing reaction is the least. The results show that: the Chinese medicinal composition nanogel has good analgesic effect, and the low dose is less than the medium dose and less than the high dose.
3. Hemostatic action
Taking 50 rats with the weight of 180-220 g, randomly dividing the rats into 5 groups, wherein the groups are as follows: blank control group:
normal saline with the same volume and the medicine application area is 4cm2；
Positive drug control group: 0.14g/kg of Yunnan Baiyao spray,the area of the medicine coating is 4cm2；
The traditional Chinese medicine composition nanogel low-dose group: 0.15g/kg, the area of the medicine coating is 2cm2；
The traditional Chinese medicine composition nanometer gel medium dose group comprises: 0.20g/kg, the area of the medicine coating is 4cm2；
The traditional Chinese medicine composition nanogel high-dose group comprises: 0.25g/kg, the area of the medicine coating is 6cm2；
Depilating at the same position of the left side of spinal column of the back of each group of rats according to skin area of different dosage groups, uniformly smearing each administration group, fixing with medical adhesive plaster, and externally applying for 1 time daily.
1) Effect of clotting time
The rats in the above groups were administered continuously for 3 days, and 60min after administration, the clotting time of the rats was measured. Blood was collected from the retrobulbar venous plexus of rats using a capillary glass tube having an inner diameter of 1mm and a length of 10cm, and the timing was started when the glass tube was filled with blood, and then the capillary glass tubes at both ends (about 0.5cm) were broken every 30 seconds and slowly pulled left and right, and the presence or absence of the presence of blood coagulation streaks at the broken portion was observed, and the time elapsed until the blood coagulation streaks appeared was the blood coagulation time, and the results are shown in table 6 below.
TABLE 6 clotting time results in rats
Note: denotes P < 0.01 compared to the blank group, and P < 0.05 compared to the blank group.
As can be seen from table 6, the administration groups were significantly different from the blank control group in that they were able to reduce the clotting time of rats. The results show that the Chinese medicinal composition nanogel can reduce the blood coagulation time of rats and has good hemostatic effect.
2) Four experiments of hemagglutination
Above-mentioned each group of rat is administered 7 days in succession, and 1 hour postadministration is intraperitoneal injection in proper order and is hydrated chloric acid solution anesthesia, and is fixed, and the abdominal aorta is got blood and is injected into the disposable vacuum blood taking vessel that contains the sodium citrate anticoagulant, turns upside down gently and shakes evenly, places centrifuge in immediately, and the centrifugation is got the upper serum, adopts full-automatic hemagglutination analysis appearance, determines four parameters of hemagglutination respectively, include: thrombin Time (TT), Activated Partial Thromboplastin Time (APTT), Prothrombin Time (PT), fibrinogen recording test results (FIB), which are reported in table 7 below.
TABLE 7 hemagglutination four test results
Note: denotes P < 0.01 compared to the blank group, and P < 0.05 compared to the blank group.
From table 7 above, the results are as follows:
thrombin Time (TT): compared with a blank control group, each administration group has significant difference, particularly a high-dose group and a Yunnan white drug spray group have significant difference, so that rapid hemostasis is facilitated; compared with the Yunnan white drug spray control group, the high-dose group is equivalent to the Yunnan white drug spray group.
Partially activated thrombin time (APTT): compared with a blank control group, the dosage groups are reduced, and the high-dosage group and the Yunnan white drug powder spray group have significant difference; compared with the Yunnan white drug spray control group, the high-dose group is equivalent to the Yunnan white drug spray group; compared with the high-dose group and the medium-dose group, the high-dose group and the medium-dose group have no significant difference, which shows that even if the dosage of the Chinese medicinal composition nanogel is continuously increased, the APTT is not reduced any more, and the Chinese medicinal composition nanogel has a quick hemostasis effect and is not accompanied with a thrombosis factor.
Prothrombin Time (PT): compared with a blank control group, each administration group has significant difference, particularly a high-dose group and a Yunnan white drug spray group have significant difference, so that rapid hemostasis is facilitated; compared with the Yunnan white drug spray control group, the high-dose group is equivalent to the Yunnan white drug spray group.
Fibrinogen (FIB): compared with a blank control group, the administration groups are reduced, and particularly, the middle and high dose groups have significant difference. Compared with the Yunnan white drug spray group, the high-dose group is equivalent to the Yunnan white drug spray group; compared with the high-dose group and the medium-dose group, the high-dose group and the medium-dose group have no significant difference, which shows that even if the dosage of the Chinese medicinal composition nanogel is continuously increased, the APTT is not reduced any more, and the Chinese medicinal composition nanogel has a quick hemostasis effect and is not accompanied with a thrombosis factor.
The results show that: the Chinese medicinal composition nanogel has obvious hemostatic effect, the low dose of the hemostatic effect is less than the medium dose and less than the high dose, and the dosage is increased from the medium dose to the high dose without accompanying thrombosis factors.
Claims (7)
1. A traditional Chinese medicine composition with antibacterial, anti-inflammatory, hemostatic and analgesic effects is characterized by comprising, by weight, 1-5 parts of pithecellobium clypearia extract, 1-5 parts of lamiophlomis rotate extract, 6-12 parts of polylactic acid-glycolic acid copolymer PLGA, 3-5 parts of vitamin E polyethylene glycol succinate TPGS, 5-10 parts of poloxamer, 8-12 parts of carbomer, 0.5-2 parts of collagen tripeptide, 1-2 parts of oat β -glucan, and 0.5-2 parts of propylene glycol.
2. The traditional Chinese medicine composition with antibacterial, anti-inflammatory, hemostatic and analgesic effects is characterized by comprising, by weight, 2-4 parts of pithecellobium clypearia extract, 2-4 parts of lamiophlomis rotate extract, 8-10 parts of polylactic acid-glycolic acid copolymer PLGA, 3-5 parts of vitamin E polyethylene glycol succinate TPGS, 6-9 parts of poloxamer, 9-11 parts of carbomer, 1-2 parts of collagen tripeptide, 1-2 parts of oat β -glucan, and 1-2 parts of propylene glycol.
3. The traditional Chinese medicine composition with antibacterial, anti-inflammatory, hemostatic and analgesic effects is characterized by comprising, by weight, 3 parts of pithecellobium clypearia extract, 3 parts of lamiophlomis rotata extract, 9 parts of polylactic-co-glycolic acid (PLGA), 4 parts of vitamin E polyethylene glycol succinate (TPGS), 8 parts of poloxamer, 10 parts of carbomer, 1 part of collagen tripeptide, 1 part of oat β -glucan and 1 part of propylene glycol.
4. A preparation method of a traditional Chinese medicine composition with antibacterial, anti-inflammatory, hemostatic and analgesic effects is characterized by comprising the following steps:
1) preparation of crude drug extract
Crushing and sieving the pithecellobium clypearia, performing subcritical extraction by using ethyl acetate as a solvent, wherein the feeding ratio of the pithecellobium clypearia to the ethyl acetate is 1g:1 mL-1 g:5mL, the subcritical extraction temperature is 35-50 ℃, the extraction time is 60-80 min, the extraction pressure is 18-22 MPa, and removing the ethyl acetate by reduced pressure concentration to obtain the pithecellobium clypearia extract;
crushing and sieving the lamiophlomis rotata, carrying out subcritical extraction by using ethyl acetate as a solvent, carrying out subcritical extraction on the lamiophlomis rotata and the ethyl acetate according to a feeding ratio of 1g to 1 mL-1 g to 3mL, wherein the subcritical extraction temperature is 25-35 ℃, the extraction time is 60-80 min, the extraction pressure is 18-22 MPa, and carrying out reduced pressure concentration to remove the ethyl acetate to obtain a lamiophlomis rotata extract;
2) dissolving polylactic acid-glycolic acid copolymer PLGA and vitamin E polyethylene glycol succinate TPGS in ethyl acetate, adding extract of Pithecellobium clypearia and radix Lamiophlomidis Rotatae, and dissolving by ultrasonic to obtain Chinese medicinal composition oil phase;
3) adding the oil phase prepared in the step 2 into a water phase containing poloxamer, magnetically stirring for 1-2 h under the water bath condition of the temperature of 60-80 ℃, then ultrasonically treating by using an ultrasonic cell crusher probe, removing ethyl acetate, cooling and filtering to obtain a traditional Chinese medicine composition nano suspension;
4) and (3) adding carbomer and collagen tripeptide into the Chinese medicinal composition nano suspension prepared in the step (3), sealing, placing until the matrix is completely swelled, uniformly stirring, sequentially adding oat β -dextran, poloxamer and propylene glycol, and continuously stirring for 2-3 h to obtain the Chinese medicinal composition nano gel.
5. The preparation method of the traditional Chinese medicine composition with antibacterial, anti-inflammatory, hemostatic and analgesic effects according to claim 4, wherein the volume of the water phase in the step 2 is 4-10 times that of the oil phase.
6. The method for preparing a Chinese medicinal composition with antibacterial, anti-inflammatory, hemostatic and analgesic effects of claim 4, wherein the ultrasound time of the probe of the ultrasonic cell disruptor in step 3 is 30min to 50 min.
7. The preparation method of the traditional Chinese medicine composition with antibacterial, anti-inflammatory, hemostatic and analgesic effects according to claim 4, wherein the stirring speed in the step 4 is 200rpm to 600 rpm.
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