CN114748522A - Boswellia carterii extract containing frankincense exosomes as well as preparation method and application thereof - Google Patents
Boswellia carterii extract containing frankincense exosomes as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides an extract containing milk fragrance exosomes and a preparation method and application thereof, belonging to the technical field of medicine preparation. Compared with the prior art, the frankincense extract containing frankincense exosomes, especially the vinegar frankincense extract, can obviously improve ulcerative colitis symptoms, and the results of the embodiment show that aiming at rat ulcerative colitis symptoms induced by dextran sodium sulfate, the frankincense extract containing frankincense exosomes can reduce the influence of ulcerative colitis on disease activity indexes, colon length and colon mucosa injury indexes of mice and reduce the contents of inflammatory factors TNF-alpha, IL-1 beta and IL-6 in serum of a model rat. The frankincense extract containing frankincense exosome has a more definite drug effect and a better effect on ulcerative colitis.
Description
Technical Field
The invention relates to the technical field of medicine preparation, in particular to a frankincense extract containing frankincense exosomes and a preparation method and application thereof.
Background
Ulcerative Colitis (UC) is a chronic nonspecific disease of the colon and rectum whose etiology is not well defined. The lesion site is limited to the large intestine mucosa and submucosa. The clinical manifestations are diarrhea, abdominal pain and mucopurulent bloody stool. UC has become one of the serious health-threatening diseases due to its difficult cure and its easy development into colorectal cancer.
The Olibanum is resin derived from bark exudation of Boswellia carterii Birdw of Burseraceae and Boswellia bhaw-dajiana Birdw of congeneric species, and is a common Chinese medicine with effects of promoting blood circulation, activating qi-flowing, removing blood stasis, relieving swelling and relieving pain. Research reports that the boswellin preparation has good curative effect on UC, the illness state of 80 percent of patients is relieved after taking the boswellin preparation, and the effective rates of the boswellin preparation on II-grade and III-grade patients with chronic colitis are respectively 100 percent and 60 percent. Clinical double-blind random control experiments also show that the frankincense extract has a good effect on treating inflammatory bowel diseases, and about 36% of patients with inflammatory bowel diseases are treated by using the frankincense extract, so that a positive effect is achieved. In addition, the extract of boswellia carterii H15Gufic and the alcoholic preparation Aflapin have been marketed as anti-inflammatory treatments in europe. The conventional Olibanum preparation mainly comprises triterpenes boswellic acid component extract, such as H15Gufic and alcohol preparation Aflapin. Most of the frankincense compounds collected from the pharmacopoeia of the people's republic of China 2020 edition are used as raw powder. The basic research of the drug effect substance is not thorough, so that the drug effect accuracy is still to be improved.
Disclosure of Invention
The invention aims to provide an extract containing milk fragrance exosomes, and a preparation method and application thereof, and the extract has more definite drug effect and better effect on ulcerative colitis.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of a frankincense extract containing frankincense exosomes, which comprises the following steps:
pulverizing Olibanum at low temperature to obtain Olibanum powder; the low-temperature crushing temperature is-25 to-15 ℃;
putting the frankincense powder into PBS buffer solution, and performing first centrifugation to obtain supernatant; the conditions of the first centrifugation include: the temperature is 1-10 ℃, the centrifugal force is 3000-10000 g, and the centrifugation time is more than or equal to 5 min;
performing second centrifugation on the supernatant to obtain a sediment; the conditions of the second centrifugation include: the temperature is 1-10 ℃, the centrifugal force is more than or equal to 80000g, and the centrifugal time is more than or equal to 0.5 h;
dispersing the sediment into a PBS buffer solution to obtain a suspension;
adding the suspension into a sucrose density gradient solution, carrying out third centrifugation, and collecting and combining bands among different sucrose density gradient layers; from top to bottom, the mass concentration of the sucrose in the sucrose density gradient solution is 4-12%, 25-35%, 40-50% and 55-65% in sequence; the conditions of the third centrifugation include: the temperature is 1-10 ℃, the centrifugal force is more than or equal to 80000g, and the centrifugal time is more than or equal to 0.5 h;
Mixing the obtained band with a PBS buffer solution, carrying out fourth centrifugation, and removing sucrose to obtain a precipitate, wherein the precipitate is an extract containing the lactarius exosome; the conditions of the fourth centrifugation include: the temperature is 1-10 ℃, the centrifugal force is more than or equal to 80000g, and the centrifugal time is more than or equal to 0.5 h.
Preferably, the mastic is vinegar mastic.
Preferably, the preparation method of the vinegar-processed frankincense comprises the following steps: preheating a container to 100-150 ℃, adding frankincense, continuously stir-frying for 3-15 min, adding rice vinegar, continuously stir-frying for 3-15 min, spreading and cooling to obtain vinegar frankincense; the mass ratio of the frankincense to the rice vinegar is 1: (3-15).
Preferably, the number of times of the first centrifugation is 2.
Preferably, the centrifugal force of the second centrifugation is 80000-200000 g, and the centrifugation time is 0.5-3 h.
Preferably, the centrifugal force of the third centrifugation is 80000-200000 g, and the centrifugation time is 0.5-3 h.
Preferably, the centrifugal force of the fourth centrifugation is 80000-150000 g, the centrifugation time is 0.5-3 h, and the number of times of the fourth centrifugation is 2-4.
The invention provides the frankincense extract containing the frankincense exosome prepared by the preparation method in the scheme; the particle size of the frankincense exosome in the frankincense extract is below 400 nm.
The invention provides application of the frankincense extract containing the frankincense exosomes in the scheme in preparation of a medicine for treating ulcerative colitis.
Preferably, the medicament is in the form of pills, tablets, capsules, oral liquid or injections.
Compared with the prior art, the frankincense extract containing frankincense exosomes, especially the vinegar frankincense extract, can obviously improve ulcerative colitis symptoms, and the results of the embodiment show that aiming at rat ulcerative colitis symptoms induced by dextran sodium sulfate, the frankincense extract containing frankincense exosomes can reduce the influence of ulcerative colitis on disease activity indexes, colon length and colon mucosa injury indexes of mice and reduce the contents of inflammatory factors TNF-alpha, IL-1 beta and IL-6 in serum of a model rat.
Drawings
FIG. 1 is transmission electron microscope image of Olibanum and vinegar Olibanum exosomes; A. (ii) a frankincense exosome; B. vinegar-processed frankincense exosomes;
FIG. 2 shows the comparative detection results of the particle size and Zeta potential before and after vinegar roasting of Olibanum; A. comparing particle sizes before and after processing Olibanum with vinegar; B. zeta potential contrast chart before and after vinegar processing of frankincense.
FIG. 3 is a graph showing the variation of body weight of each group;
FIG. 4 is a DAI score chart for each group;
FIG. 5 shows colon lengths for each group;
FIG. 6 is a diagram of colon morphology and length measurements; wherein, A: a normal group; b: a model group; c: a Boswellia group; d: vinegar-processed frankincense group; e: a frankincense exosome group; f: vinegar frankincense exosome group; g: a positive drug group.
FIG. 7 is a graph of CMDI scores for various groups;
FIG. 8 shows various groups of pathological colon tissue sections; wherein, group A is normal; b, model; c, frankincense group; d, vinegar frankincense group; e Olibanum exosome group; f, vinegar frankincense exosome group; g positive drug group;
FIG. 9 is a HS score chart;
FIG. 10 shows the levels of inflammatory factors in the sera of each group; wherein, A: TNF-alpha; b: IL-1 β; c: IL-6.
Detailed Description
The invention provides a preparation method of a frankincense extract containing frankincense exosomes, which comprises the following steps:
pulverizing Olibanum at low temperature to obtain Olibanum powder; the low-temperature crushing temperature is-25 to-15 ℃;
putting the frankincense powder into PBS buffer solution, and performing first centrifugation to obtain supernatant; the conditions of the first centrifugation include: the temperature is 1-10 ℃, the centrifugal force is 3000-10000 g, and the centrifugation time is more than or equal to 5 min;
performing second centrifugation on the supernatant to obtain a sediment; the conditions of the second centrifugation include: the temperature is 1-10 ℃, the centrifugal force is more than or equal to 80000g, and the centrifugal time is more than or equal to 0.5 h;
Dispersing the sediment into a PBS buffer solution to obtain a suspension;
adding the suspension into a sucrose density gradient solution, carrying out third centrifugation, and collecting and combining bands among different sucrose density gradient layers; from top to bottom, the mass concentration of the sucrose in the sucrose density gradient solution is 4-12%, 25-35%, 40-50% and 55-65% in sequence; the conditions of the third centrifugation include: the temperature is 1-10 ℃, the centrifugal force is more than or equal to 80000g, and the centrifugal time is more than or equal to 0.5 h;
mixing the obtained band with a PBS buffer solution, carrying out fourth centrifugation, and removing sucrose to obtain a precipitate, wherein the precipitate is an extract containing the lactarius exosome; the conditions of the fourth centrifugation include: the temperature is 1-10 ℃, the centrifugal force is more than or equal to 80000g, and the centrifugal time is more than or equal to 0.5 h.
The invention pulverizes frankincense at low temperature to obtain frankincense powder.
In the invention, the temperature of the low-temperature pulverization is-25 to-15 ℃, and the particle size of the frankincense powder is preferably smaller than 100 meshes. The invention has no special requirement on the low-temperature crushing mode, and the frankincense powder meeting the particle size requirement can be obtained. The aim of the invention is to protect exosome from being influenced by temperature factors in the crushing process to the maximum extent.
In the present invention, the mastic is preferably vinegar mastic; the preparation method of the vinegar mastic preferably comprises the following steps: preheating the container to 100-150 ℃, adding frankincense, continuously stir-frying for 3-15 min, adding rice vinegar, continuously stir-frying for 3-15 min, spreading and cooling to obtain vinegar frankincense. In the present invention, the mass ratio of the mastic gum to the rice vinegar is preferably 1: (3-15), more preferably 1: 10.
After obtaining the frankincense powder, the invention puts the frankincense powder into PBS buffer solution, and carries out first centrifugation to obtain supernatant.
In the present invention, the mass of the PBS buffer is preferably 3 to 9 times, and more preferably 6 times, of the mass of the mastic powder. Before the first centrifugation, the PBS buffer solution of the frankincense powder is preferably shaken for 5-100 s. In the present invention, the conditions of the first centrifugation include: the temperature is 1-10 ℃, the centrifugal force is 3000-10000 g, the centrifugation time is more than or equal to 5min, preferably, the temperature is 3-6 ℃, the centrifugal force is 5000-7000 g, and the centrifugation time is 5-40 min; more preferably, the temperature is 4 ℃, the centrifugal force is 5500g, and the time for centrifugation is 20 min. In the present invention, the number of times of the first centrifugation is preferably 2 times. The invention dissolves exosomes into PBS buffer solution through first centrifugation, and separates the exosomes from frankincense powder.
After the supernatant is obtained, the invention carries out second centrifugation on the supernatant to obtain a sediment.
In the present invention, the conditions of the second centrifugation include: the temperature is 1-10 ℃, the centrifugal force is more than or equal to 80000g, and the centrifugal time is more than or equal to 0.5 h; preferably, the temperature is 3-6 ℃, the centrifugal force is 80000-200000 g, and the centrifugation time is 0.5-3 h; more preferably, the temperature is 4 ℃, the centrifugal force is 150000g, and the time of centrifugation is 2 h. The exosomes are primarily extracted and separated by a second centrifuge.
After the sediment is obtained, the invention disperses the sediment into PBS buffer solution to obtain suspension. The invention has no special requirement on the dosage of the PBS buffer solution, and can disperse the precipitate uniformly. The invention has no special requirement on the dispersion mode, and the dispersion mode well known in the field can be adopted.
After the suspension is obtained, adding the suspension into a sucrose density gradient solution, carrying out third centrifugation, and collecting and combining bands among different sucrose density gradient layers; from top to bottom, the mass concentration of the sucrose in the sucrose density gradient solution is 4-12%, 25-35%, 40-50% and 55-65% in sequence; the conditions of the third centrifugation include: the temperature is 1-10 ℃, the centrifugal force is more than or equal to 80000g, and the centrifugal time is more than or equal to 0.5 h.
Preferably, the mass concentration of the sucrose in the sucrose density gradient solution is 8%, 30%, 45% and 60% in sequence; the temperature of the third centrifugation is preferably 3-6 ℃, the centrifugal force is preferably 80000-200000 g, and the centrifugation time is preferably 0.5-3 h; more preferably, the temperature is 4 ℃, the centrifugal force is 150000g, and the time of centrifugation is 2 h. The exosome can be purified in a sucrose gradient solution by performing third centrifugation.
After obtaining a band, mixing the obtained band with a PBS buffer solution, carrying out fourth centrifugation, and removing sucrose to obtain a precipitate, wherein the precipitate is an extract containing the lactarius exosome; the conditions of the fourth centrifugation include: the temperature is 1-10 ℃, the centrifugal force is more than or equal to 80000g, and the centrifugal time is more than or equal to 0.5 h.
In the invention, the temperature of the fourth centrifugation is preferably 3-6 ℃, the centrifugal force is preferably 80000-150000 g, and the centrifugation time is preferably 0.5-3 h; more preferably, the temperature is 4 ℃, the centrifugal force is 100000g, and the centrifugal time is 2 h.
In the present invention, the number of times of the fourth centrifugation is preferably 2 to 4 times. The invention removes the sucrose to the maximum extent by multiple centrifugations.
After obtaining the precipitate, the present invention preferably resuspends the precipitate with PBS and stores the same in a refrigerator at-80 ℃ for further use.
The invention provides the frankincense extract containing the frankincense exosome, which is prepared by the preparation method in the scheme; the particle size of the Olibanum exosome in the Olibanum extract is below 400 nm. In the invention, when the frankincense is vinegar-processed frankincense, the particle size of the frankincense exosome is below 400nm, and when the frankincense is not stir-fried with rice vinegar, the particle size of the frankincense exosome is below 200 nm.
The invention provides application of the frankincense extract containing the frankincense exosome in the scheme in preparation of a medicine for treating ulcerative colitis. In the present invention, the dosage form of the drug is preferably a pill, tablet, capsule, oral liquid or injection.
The boswellia extract containing boswellia exosomes and the preparation method and use thereof provided in the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Pulverizing Olibanum at-20 deg.C, adding 6 times of PBS, shaking for 30s, centrifuging at 5500g at 4 deg.C for 20min, and collecting supernatant. The supernatant was again centrifuged at 5500g at 4 ℃ for 20 min. Centrifuging for 2 hours at 4 ℃ of 150000g by adopting a low-temperature high-speed centrifuge, discarding the supernatant, dispersing the obtained sediment into a PBS buffer solution to obtain a suspension, sequentially adding 8%, 30%, 45% and 60% of sucrose solutions into the centrifuge tube from top to bottom, adding the suspension into the centrifuge tube, continuously performing sucrose density gradient centrifugation for 2 hours at 4 ℃ of 150000g by using the low-temperature high-speed centrifuge, separating different strips after centrifugation, collecting and combining the strips among different sucrose density gradient layers, adding PBS, continuously centrifuging for 2 hours at 100000g for removing sugar, repeating the steps twice, resuspending the obtained precipitate with PBS, and storing the precipitate in a refrigerator at-80 ℃ for later use.
Example 2
The difference from example 1 is only that the frankincense is replaced by vinegar frankincense, and the preparation method of the vinegar frankincense comprises the following steps: preheating the cleaning container to 130 deg.C, adding Olibanum, parching for 9min, spraying rice vinegar at a ratio of 10:1, parching for 11min, spreading, and cooling to obtain vinegar-processed Olibanum.
Transmission electron microscopy of exosomes: dissolving 4g of uranyl acetate powder in 100mL of preheated (50-60 ℃) filtered water to prepare 4% uranyl acetate stock solution. 1mL of 4% uranyl acetate stock solution was diluted into 3mL of filtered water. A drop of the sample was deposited onto the surface of the coated grid. Add 1% uranyl acetate on top of the sample and wait 15 seconds. After the sample was well absorbed, the uranyl acetate was gently blotted with a paper towel without contacting the surface. The sample was dried at room temperature for 5 minutes. The image of the sample was scanned by a transmission electron microscope and the result is shown in FIG. 1 (scale: 100 nm). In fig. 1, a is the boswellia exosomes extracted in example 1; b is vinegar frankincense exosome extracted in example 2.
Detecting the particle size concentration of the exosome: exosome particle size distribution, particle concentration and zeta potential were determined using a ZetaView PMX 110 nanoparticle tracking analyzer. The sample cell was washed with deionized water and calibrated with polystyrene microspheres (110 nm). The sample is dissolved by PBS buffer solution, and the detection result is shown in figure 2, wherein in figure 2, A is a comparison graph of particle sizes before and after the frankincense vinegar roasting, and B is a comparison graph of Zeta potential before and after the frankincense vinegar roasting. Through the detection comparison research of the particle size and the Zeta potential, the particle size of exosomes after the frankincense vinegar roasting is increased, the particle size before the vinegar roasting (example 1) is below 200nm, and the particle size after the vinegar roasting (example 2) is below 400 nm. Meanwhile, after the frankincense is roasted with vinegar, the zeta potential distribution of the exosome is increased by a distribution area of +/-20 mV. It can be seen that the stability of the frankincense exosomes is reduced through vinegar roasting treatment, so that the exosomes are easy to aggregate, and the phenomenon is proved in transmission electron microscope observation of the exosomes (figure 1).
Pharmacological experiment
1. Weight management method
Effect on the change in body weight of mice with Dextran Sulfate Sodium Salt (DSS) induced Ulcerative Colitis (UC): self-adaptively feeding 70 SPF (specific pathogen free) C57BL/6J mice for one week, and using DSS (DSS) solution with the mass fraction of 2% to replace drinking water for simulating the course of disease to mold the molded animals at 1-6 d, 20-25 d and 39-44 d in sequence, wherein the normal animals can freely drink water. The 60 molded animals are randomly divided into a frankincense group (RF, 1.5g/kg), an vinegar frankincense group (PF, 1.5g/kg), a frankincense exosome group (ERF, 1.5g/kg), an vinegar frankincense exosome group (EPF, 1.5g/kg), a positive drug sulfasalazine group (Y, 1.5g/kg) and a model group (M), 10 animals in each group are subjected to intragastric administration intervention once a day and 36 days continuously from 13 days according to a therapeutic administration principle, and a sodium carboxymethylcellulose (CMC-Na) solution with the mass fraction of 0.3 percent is intragastric administered to a normal group and the model group. The body weights of the mice were weighed and data were recorded before the start of molding and after each cycle, respectively, and the results are shown in fig. 3 and table 1.
Body weight results
As can be seen from FIG. 3 and Table 1, the body weights of the normal group increased gradually and the body weights of the model group and the administration groups increased in a fluctuating manner within 1-49 d of the experiment. At 49d, the model group significantly decreased body weight compared to the normal group (P < 0.01); compared with the model group, the weight average of the frankincense group, the vinegar frankincense group, the frankincense exosome group, the vinegar frankincense exosome group and the positive medicine group shows an increasing trend, wherein the positive medicine group has very significant difference (P is less than 0.01); compared with the frankincense group, the vinegar frankincense group has more obvious weight increase, and shows that the vinegar frankincense has stronger effect of promoting weight increase; compared with the frankincense exosome group, the weight of the vinegar frankincense exosome group is increased more obviously, and the effect of the vinegar frankincense exosome in promoting weight gain is stronger.
TABLE 1 results of body weight measurement of mice in each group
^ P < 0.01 compared with normal group; p < 0.01 compared to model group.
DAI method
Effect on DSS-induced UC mouse Disease Activity Index (DAI): self-adaptively feeding 70 SPF (specific pathogen free) C57BL/6J mice for one week, and using DSS (DSS) solution with the mass fraction of 2% to replace drinking water for simulating the course of disease to mold the molded animals at 1-6 d, 20-25 d and 39-44 d in sequence, wherein the normal animals can freely drink water. The 60 molded animals are randomly divided into a frankincense group (RF, 1.5g/kg), an vinegar frankincense group (PF, 1.5g/kg), a frankincense exosome group (ERF, 1.5g/kg), an vinegar frankincense exosome group (EPF, 1.5g/kg), a positive drug sulfasalazine group (Y, 1.5g/kg) and a model group (M), 10 animals in each group are subjected to intragastric administration intervention once a day and continuously for 36 days from 13 days according to a therapeutic administration principle, and a CMC-Na solution with the mass fraction of 0.3 percent is intragastric administered to a normal group and the model group. DAI scoring and data recording were performed on mice before the start of molding and after each cycle, respectively. DAI scoring criteria were: the weight is not reduced, the stool is normal, the stool is not occult blood, and the naked eye bloody stool is scored as 0; the weight is reduced by 1-5%, the excrement is loose, and the excrement is occult with blood and is scored as 1; the weight is reduced by 5-10%, the excrement is loose, the excrement is occult with blood, and the score is 2; the weight is reduced by 10-15%, the excrement is loose stool, and the visual blood stool is scored for 3 points; weight loss > 15%, loose stools, watery stools which were not adhered to the anus, loose stools, which were not adhered to the anus, and visual bloody stools, which were rated 4. The test results are shown in fig. 4 and table 2.
DAI results
As can be seen from fig. 4 and table 2, the DAI score of the normal group was 0 at each time point of administration measured at 1 to 49d, and the DAI of the model group and each administration group was fluctuated. The DAI was significantly increased in the model group compared to the normal group. The DAI was reduced in each group compared to the model group. At 49d, the DAI in the model group was significantly higher (P < 0.01) compared to the normal group; compared with the model group, the frankincense group, the vinegar frankincense exosome group and the positive medicine group are obviously reduced in DAI (P is less than 0.05 or less than 0.01), and the frankincense exosome group shows a reduction trend; compared with the frankincense group, the DAI score of the vinegar frankincense group is lower (P is less than 0.05), which shows that the vinegar frankincense has better effect of relieving the symptoms of DSS-induced UC than the frankincense; compared with the frankincense exosome group, the vinegar frankincense exosome group has lower DAI, and shows that the vinegar frankincense exosome has better effect of reducing DAI.
TABLE 2DAI scoring results
^ P < 0.01 compared with normal group; p < 0.05, P < 0.01, compared to model group; compared with the frankincense group, the preparation method has the advantages that,#P＜0.05。
3. colon length methods
Effect on DSS-induced UC mouse colon length: self-adaptively feeding 70 SPF (specific pathogen free) C57BL/6J mice for one week, and using DSS (DSS) solution with the mass fraction of 2% to replace drinking water for simulating the course of disease to mold the molded animals at 1-6 d, 20-25 d and 39-44 d in sequence, wherein the normal animals can freely drink water. The 60 molded animals are randomly divided into a frankincense group (RF, 1.5g/kg), an vinegar frankincense group (PF, 1.5g/kg), a frankincense exosome group (ERF, 1.5g/kg), an vinegar frankincense exosome group (EPF, 1.5g/kg), a positive drug sulfasalazine group (Y, 1.5g/kg) and a model group (M), 10 animals in each group are subjected to intragastric administration intervention once a day and continuously for 36 days from 13 days according to a therapeutic administration principle, and a CMC-Na solution with the mass fraction of 0.3 percent is intragastric administered to a normal group and the model group. At 49d, the mice were anesthetized and then bled from the orbit, and after death by removing the neck, the colon tissue was peeled off and the length of the colon was measured. The test results are shown in fig. 5, fig. 6 and table 3.
Results of colon Length
As shown in fig. 5, fig. 6 and table 3, the colon length of the model group mouse is significantly shortened (P < 0.01) compared with the colon of the normal group mouse, indicating that DSS can significantly inhibit the growth of the colon of the mouse; compared with the model group, the colon length of the mouse of each administration group is longer, which shows that the drug relieves the inhibition effect of DSS on the growth of the colon of the mouse; compared with the frankincense group, the length of the colon of the vinegar frankincense group mouse is longer, which suggests that the effect of the vinegar frankincense on improving the length of the colon of the UC mouse is better than that of the frankincense; compared with the frankincense exosome group, the colon length of the mice in the vinegar frankincense exosome group is longer, and the effect of the vinegar frankincense exosome on improving the colon length of the UC mice is better than that of the frankincense exosome.
TABLE 3 mouse Colon Length results
^ P < 0.01 compared with the normal group.
CMDI Process
Effect on DSS-induced UC mouse Colonic mucosal lesion index (CMDI): self-adaptively feeding 70 SPF (specific pathogen free) C57BL/6J mice for one week, and using DSS (DSS) solution with the mass fraction of 2% to replace drinking water for simulating the course of disease to mold the molded animals at 1-6 d, 20-25 d and 39-44 d in sequence, wherein the normal animals can freely drink water. The 60 molded animals are randomly divided into a frankincense group (RF, 1.5g/kg), an vinegar frankincense group (PF, 1.5g/kg), a frankincense exosome group (ERF, 1.5g/kg), an vinegar frankincense exosome group (EPF, 1.5g/kg), a positive drug sulfasalazine group (Y, 1.5g/kg) and a model group (M), 10 animals in each group are subjected to intragastric administration intervention once a day and continuously for 36 days from 13 days according to a therapeutic administration principle, and a CMC-Na solution with the mass fraction of 0.3 percent is intragastric administered to a normal group and the model group. At 49d, mice were anesthetized and then bled from the orbit, and after death by removal of the neck, the colon tissue was peeled off, the length of the colon was measured, washed with ice saline, and visually evaluated for CMDI scoring. The CMDI rating scale was: normal, no congestion edema, smooth surface, no erosion or ulcer, score 0; mild hyperemia and edema, smooth mucosal surface, no ulcer or erosion, grade 1; moderate congestion and edema, unsmooth and granular mucosal surface, ulcer, erosion or intestinal adhesion, rated 2; high degree of hyperemia and edema, erosion and ulcer, longitudinal channel of ulcer is less than 1.0cm, and the score is 3; high degree of hyperemia and edema, erosion or ulcer, ulcer longitudinal channel >1.0cm, and score 4. The results are shown in FIG. 7 and Table 4.
CMDI results
As shown in fig. 7 and table 4, the normal group CMDI was 0, and the model group mouse CMDI score was significantly increased (P < 0.01) compared to the normal group, indicating that DSS caused damage to the colonic mucosa; the CMDI score was lower in each dosing group compared to the model group; compared with the frankincense group, the vinegar frankincense group is lower in CMDI score, and the vinegar frankincense group is better than frankincense in relieving the UC mouse colon mucosa injury; compared with the frankincense exosome group, the vinegar frankincense exosome group has lower CMDI score, and the vinegar frankincense exosome is prompted to have stronger effect of relieving the UC mouse colon mucosa injury than the frankincense exosome.
TABLE 4CMDI scoring results
^ P < 0.01 compared with the normal group.
5. Colon histological observation method
Effect on DSS-induced colonic histopathological changes in UC mice: self-adaptively feeding 70 SPF-grade C57BL/6J mice for one week, sequentially performing modeling on the modeled animals at 1-6 d, 20-25 d and 39-44 d by using DSS solution with the mass fraction of 2% to replace drinking water to simulate the repeated process of the course of disease, and allowing the normal animals to freely drink water. The 60 molded animals are randomly divided into a frankincense group (RF, 1.5g/kg), an vinegar frankincense group (PF, 1.5g/kg), a frankincense exosome group (ERF, 1.5g/kg), an vinegar frankincense exosome group (EPF, 1.5g/kg), a positive drug sulfasalazine group (Y, 1.5g/kg) and a model group (M), 10 animals in each group are subjected to intragastric administration intervention once a day and continuously for 36 days from 13 days according to a therapeutic administration principle, and a CMC-Na solution with the mass fraction of 0.3 percent is intragastric administered to a normal group and the model group. At 49d, the mouse is anesthetized, blood is taken from the orbit, after the neck is removed and the mouse is died, the colon tissue is stripped, a section of the colon tissue with the most serious pathological changes of the mouse is taken and immersed into 4% tissue fixing solution for fixation, the colon tissue is embedded by conventional paraffin, a section is prepared, the section is stained by Hematoxylin-eosin (HE) staining method, and the pathological changes of the colon tissue are observed by a microscope.
Histological observation of the colon
The colon tissue is observed by HE staining, as shown in figure 8, the normal group of mice has thick and clear colon (A) intestinal wall, complete epithelium, regular gland arrangement, normal crypt, abundant goblet cells and no inflammatory cell infiltration. Compared with the normal group, the colon wall of the colon (B) of the mouse in the model group is thinned, the epithelium is incomplete, the epithelial cells are necrotic and shed, the ulcer symptoms are serious, and a large number of inflammatory cells infiltrate the mucosal muscularis; the colon (C) of the frankincense group mouse is slightly thick in intestinal wall, ulcer is formed in a part of areas, glands are arranged regularly, crypts are normal, goblet cells are rich, and a small amount of inflammatory cells infiltrate; compared with the frankincense group, the colon (D) of the mice in the vinegar frankincense group is slightly thick in intestinal wall, complete in epithelium, less in ulcer part, orderly in glandular arrangement, expanded in crypt, rich in goblet cells, less in inflammatory cell infiltration and obviously improved in ulcer symptom, which shows that the vinegar frankincense has better effect of improving DSS (DSS) to cause pathological change of UC mouse colon tissues than the frankincense; the colon (E) of the mice in the frankincense exosome group has thick intestinal wall, few ulcer parts, regularly arranged glands, normal crypts, rich goblet cells and small amount of inflammatory cell infiltration; compared with the frankincense exosome group, the colon (F) intestinal wall of the mice in the vinegar frankincense exosome group is thick, the epithelium is complete, the ulcer parts are few, the glands are arranged regularly, the crypt is normal, the goblet cells are rich, the inflammatory cell infiltration is few, and the ulcer symptom is obviously improved; the vinegar frankincense exosome has better effect of improving pathological change of UC mouse colon tissue caused by DSS than frankincense exosome. The positive drug group mouse has clear colon (G) intestinal wall, complete epithelium, orderly arranged glands, normal crypt, rich goblet cells and no inflammatory cell infiltration, which shows that the ulcer symptoms are obviously improved after the drug is administered.
6. Colon Histopathological Scoring (HS) method
Effect on DSS-induced colonic histopathological changes in UC mice: self-adaptively feeding 70 SPF (specific pathogen free) C57BL/6J mice for one week, and using DSS (DSS) solution with the mass fraction of 2% to replace drinking water for simulating the course of disease to mold the molded animals at 1-6 d, 20-25 d and 39-44 d in sequence, wherein the normal animals can freely drink water. The 60 molded animals are randomly divided into a frankincense group (RF, 1.5g/kg), an vinegar frankincense group (PF, 1.5g/kg), a frankincense exosome group (ERF, 1.5g/kg), an vinegar frankincense exosome group (EPF, 1.5g/kg), a positive drug sulfasalazine group (Y, 1.5g/kg) and a model group (M), 10 animals in each group are subjected to intragastric administration intervention once a day and continuously for 36 days from 13 days according to a therapeutic administration principle, and a CMC-Na solution with the mass fraction of 0.3 percent is intragastric administered to a normal group and the model group. At 49d, after the mouse is anesthetized, blood is taken from the orbit, after the neck is removed and the mouse is died, the colon tissue is stripped, a section of the colon tissue with the most serious pathological changes of the mouse is taken and immersed into 4% tissue fixing solution for fixation, the colon tissue is embedded by conventional paraffin, a section is prepared, HE staining is carried out, pathological changes of the colon tissue are observed by a microscope for HS grading, and the grading grade is as follows: normal, no edema and ulcer, histological lesions, score 0; congestion edema, few inflammatory cells, score 1; superficial erosion, congestion and edema of mucosa and submucosa, rated 2; most inflammatory cells, crypt abscesses, necrosis of the mucosal layer and formation of ulcers, disease deepened to submucosa or muscularis, scored 3.
Colon Histopathological Score (HS) results
As shown in fig. 9 and table 5, the HS score of the normal group is 0, and the HS score of the model group mice is significantly increased (P < 0.01) compared with the normal group, which indicates that DSS causes severe edema of colon, necrosis of large area of mucosa, change or even disappearance of crypt structure and severe inflammatory cell infiltration; compared with the model group, the HS score of each administration group is obviously reduced (P is less than 0.01), and the pathological condition of colon tissues is improved; compared with the frankincense group, the vinegar-milk smoked chicken prognosis mice have clearer colon crypts, lighter inflammatory cell infiltration degree and lower HS score of the vinegar-frankincense group; compared with the frankincense exosome group, the vinegar frankincense exosome group is low in HS score, and the vinegar frankincense exosome is prompted to have a stronger effect of relieving colon pathological changes caused by DSS than the frankincense exosome.
TABLE 5HS score results
^ P < 0.01 compared with the normal group; p < 0.01, compared to model group.
7. Method for detecting contents of Tumor necrosis factor (Tumor necrosis factor-alpha, TNF-alpha), interleukin-1 beta (Interlenkin-1 beta, IL-1 beta) and interleukin-6 (Interlenkin-6, IL-6) in serum
Effect on DSS-induced changes in UC mouse inflammatory factor levels: self-adaptively feeding 70 SPF (specific pathogen free) C57BL/6J mice for one week, and using DSS (DSS) solution with the mass fraction of 2% to replace drinking water for simulating the course of disease to mold the molded animals at 1-6 d, 20-25 d and 39-44 d in sequence, wherein the normal animals can freely drink water. The 60 molded animals are randomly divided into a frankincense group (RF, 1.5g/kg), an vinegar frankincense group (PF, 1.5g/kg), a frankincense exosome group (ERF, 1.5g/kg), an vinegar frankincense exosome group (EPF, 1.5g/kg), a positive drug sulfasalazine group (Y, 1.5g/kg) and a model group (M), 10 animals in each group are subjected to intragastric administration intervention once a day and continuously for 36 days from 13 days according to a therapeutic administration principle, and a CMC-Na solution with the mass fraction of 0.3 percent is intragastric administered to a normal group and the model group. At 49d, mice were bled from the orbit after anesthesia and stored in 1.5mL EP tubes. Standing for 1h, centrifuging at 4 deg.C and 3500r/min for 15min, collecting supernatant, packaging, and storing at-80 deg.C. The levels of inflammatory factors TNF-alpha, IL-1 beta and IL-6 in the serum of each group of mice are respectively measured by an ELISA method. The operation is carried out according to the kit instructions.
Detection result of contents of inflammatory factors TNF-alpha, IL-1 beta and IL-6 in serum
As shown in fig. 10 and table 6: compared with the normal group, the contents of inflammatory factors TNF-alpha, IL-1 beta and IL-6 in the model group are obviously increased (P <0.01), which indicates that DSS induces the inflammatory response of mice; compared with the model group, the content of TNF-alpha, IL-1 beta and IL-6 in the partial administration group is obviously reduced (P <0.05 or P < 0.01); compared with the frankincense group, the contents of inflammatory factors TNF-alpha, IL-1 beta and IL-6 in the vinegar frankincense group are lower, and TNF-alpha has very significant difference (P <0.01), which indicates that the anti-inflammatory effect is enhanced after the frankincense is stir-fried with vinegar; compared with the frankincense exosome group, the vinegar frankincense exosome group is lower in content of inflammatory factors TNF-alpha, IL-1 beta and IL-6, and the TNF-alpha has significant difference (P is less than 0.05), which indicates that the vinegar frankincense exosome has better effect of reducing serum inflammatory factors.
TABLE 6 results of inflammatory factor content in each group of sera
^ P <0.01 compared with normal group; comparing to the model group, P <0.05, P < 0.01; compared with the frankincense group, the preparation method has the advantages that,##p is less than 0.01; compared with the frankincense exosome group,aP＜0.01。
example 3
Pulverizing Olibanum at low temperature, adding 3 times of PBS, shaking for 5s, centrifuging at 2 deg.C under 3000g for 5min, and collecting supernatant. Centrifuging the supernatant again at 2 deg.C 3000g for 5 min; centrifuging for 0.5h at 2 ℃ of 80000g by adopting a low-temperature high-speed centrifuge, discarding the supernatant, dispersing the obtained sediment into a PBS buffer solution to obtain a suspension, sequentially adding 4%, 25%, 40% and 55% of sucrose solutions into a centrifuge tube from top to bottom, adding the suspension into the centrifuge tube, continuously performing sucrose density gradient centrifugation for 0.5h at 2 ℃ of 80000g by using the low-temperature high-speed centrifuge, separating different strips after centrifugation, collecting and combining the strips among different sucrose density gradient layers, adding PBS, continuously centrifuging for 0.5h at 80000g for removing sugar, repeating for 2 times, re-suspending the obtained precipitate by using PBS, and storing in a refrigerator at-80 ℃ for later use.
Example 4
Preheating clean container to 150 deg.C, adding Olibanum, parching for 12min, spraying rice vinegar at a ratio of 8:1, parching for 15min, spreading, and cooling to obtain vinegar-processed Olibanum. Pulverizing Olibanum (Vinegar Olibanum), adding 9 times of PBS, shaking for 100s, centrifuging at 10 deg.C 10000g for 40min, and collecting supernatant. Centrifuging the supernatant at 10 deg.C 10000g for 30 min; centrifuging for 3 hours at 10 ℃ 200000g by adopting a low-temperature high-speed centrifuge, discarding the supernatant, dispersing the obtained precipitate into a PBS buffer solution to obtain a suspension, sequentially adding 12%, 35%, 50% and 65% of sucrose solutions into the centrifuge tube from top to bottom, adding the suspension into the centrifuge tube, continuously performing sucrose density gradient centrifugation for 3 hours at 10 ℃ 200000g by using the low-temperature high-speed centrifuge, separating different strips after centrifugation, collecting and combining the strips among different sucrose density gradient layers, adding PBS, continuously centrifuging for 3 hours at 150000g for sugar removal, repeating for 3 times, resuspending the obtained precipitate with PBS, and storing in a refrigerator at-80 ℃ for later use.
The results show that the extract containing the milk fragrance exosome, and the preparation method and the application thereof provided by the invention have the advantages of more definite drug effect and better effect on ulcerative colitis.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A preparation method of a frankincense extract containing frankincense exosomes is characterized by comprising the following steps:
pulverizing Olibanum at low temperature to obtain Olibanum powder; the temperature of the low-temperature crushing is-25 to-15 ℃;
putting the frankincense powder into a PBS buffer solution, and performing first centrifugation to obtain a supernatant; the conditions of the first centrifugation include: the temperature is 1-10 ℃, the centrifugal force is 3000-10000 g, and the centrifugation time is more than or equal to 5 min;
performing second centrifugation on the supernatant to obtain a precipitate; the conditions of the second centrifugation include: the temperature is 1-10 ℃, the centrifugal force is more than or equal to 80000g, and the centrifugal time is more than or equal to 0.5 h;
dispersing the sediment into a PBS buffer solution to obtain a suspension;
adding the suspension into a sucrose density gradient solution, performing third centrifugation, and collecting and combining bands among different sucrose density gradient layers; from top to bottom, the mass concentration of the sucrose in the sucrose density gradient solution is 4-12%, 25-35%, 40-50% and 55-65% in sequence; the conditions of the third centrifugation include: the temperature is 1-10 ℃, the centrifugal force is more than or equal to 80000g, and the centrifugal time is more than or equal to 0.5 h;
Mixing the obtained band with a PBS buffer solution, carrying out fourth centrifugation, and removing sucrose to obtain a precipitate, wherein the precipitate is an extract containing the lactarius exosome; the conditions of the fourth centrifugation include: the temperature is 1-10 ℃, the centrifugal force is more than or equal to 80000g, and the centrifugal time is more than or equal to 0.5 h.
2. The method of claim 1, wherein the boswellia is vinegar boswellia.
3. The method as claimed in claim 2, wherein the method for preparing vinegar mastic comprises: preheating a container to 100-150 ℃, adding frankincense, continuously stir-frying for 3-15 min, adding rice vinegar, continuously stir-frying for 3-15 min, spreading and cooling to obtain vinegar frankincense; the mass ratio of the frankincense to the rice vinegar is 1: (3-15).
4. The method of claim 1, wherein the number of times of the first centrifugation is 2.
5. The method according to claim 1, wherein the second centrifugation is performed at a centrifugal force of 80000 to 200000g for 0.5 to 3 hours.
6. The method according to claim 1, wherein the third centrifugation is performed at a centrifugal force of 80000 to 200000g for 0.5 to 3 hours.
7. The method for preparing the compound of claim 1, wherein the centrifugal force of the fourth centrifugation is 80000-150000 g, the centrifugation time is 0.5-3 h, and the number of times of the fourth centrifugation is 2-4.
8. A mastic extract containing mastic exosomes prepared by the preparation method as claimed in any one of claims 1 to 7; the particle size of the Olibanum exosome in the Olibanum extract is below 400 nm.
9. Use of boswellia extract containing boswellia exosomes of claim 8 in the preparation of a medicament for treating ulcerative colitis.
10. The use according to claim 9, wherein the medicament is in the form of a pill, tablet, capsule, oral liquid or injection.
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