CN114432247A - A kind of bufatryptamine liposome and its preparation method and application - Google Patents

Abstract

The invention discloses a bufatryptamine liposome and a preparation method and application thereof, which are essentially the purpose of reducing the gastrointestinal toxicity of bufatryptamine to improve the therapeutic index and druggability. The present invention adopts the method of active drug loading to prepare the liposome preparation, the liposome preparation includes liposome particles, and the liposome particles include a carrier, and the carrier is a liposome membrane with a bimolecular structure, so the The bufatryptamines described include serotonin, bufaternidine, N-methyl serotonin, bufatamine, bufathietamine, and dehydrobufatryptamine. It is located in the central cavity of the liposome. The liposome is made from 0.1-10 parts of bufatryptamine, 5-20 parts of phospholipids, 1-10 parts of cholesterol, and 1-10 parts of vitamin E. The bufatryptamine liposomes can be used to treat pain, inflammation and depression. Compared with passive drug-loaded liposomes, active drug-loaded liposomes have the advantages of more stability, slower release, higher bioavailability, lower residual toxic target organs, and higher safety.

Description

A kind of bufatryptamine liposome and its preparation method and application

technical field

The application relates to the technical field of pharmaceutical preparations, in particular to a bufatryptamine liposome and a preparation method and application thereof. Essentially, it is used to improve the therapeutic index and druggability by reducing the gastrointestinal toxicity of bufatryptamine.

Background technique

With the development of modern society, the awareness of human health needs and the level of awareness of drug safety has been increasing, so that the safety of traditional Chinese medicine has been widely concerned by the society. Drug safety is the basis of patient safety, and safety and effectiveness are the basic attributes of drugs. Whether a drug can be accepted by society depends not only on its effectiveness, but also on its toxicity.

Toad venom, as one of the precious animal medicines in my country, has anti-tumor, cardiotonic, analgesic, anti-inflammatory and other effects, and is widely used in the treatment of cardiovascular, cancer, respiratory, mental or neurological diseases. Bufatryptamines are a class of water-soluble indole alkaloids in toadstools and are derivatives of serotonin. More than 20 species have been found, such as serotonin, bufatryptamine, N-methyl serotonin, and serotonin. Hydrogen bufatryptamine, bufathiene, etc. The applicant's previous research found that the intraperitoneal injection of bufatryptamine can significantly improve the thermal pain threshold of the mouse hot plate pain model, and significantly improve the biphasic pain behavior and the biphasic pain behavior of the mouse formalin plantar inflammatory pain model. Mechanically stimulated foot retraction threshold, that is, it has significant analgesic activity (a method for synthesizing bufatamine and its quaternary ammonium salt and its application in the preparation of analgesic and anti-inflammatory drugs, patent number ZL201911259232.8). The bufatryptamines also have antidepressant effects (the application of bufatryptamines in the preparation of antidepressant drugs CN202011077934.7).

However, the applicant's study found that bufatryptamines have significant gastrointestinal irritation, which seriously reduces their druggability. Considering that this toxicity problem is a key link that hinders the development of new bufatryptamine drugs, the present invention attempts to adopt formulation methods to reduce the gastrointestinal toxicity of bufatryptamine, so as to improve the therapeutic index.

Liposomes can be used as drug carriers to load drugs with different properties of fat-soluble and water-soluble. Based on the strong polarity and weak alkaline properties of bufatryptamines, the present invention adopts liposome inclusion technology to encapsulate the water-soluble bufatryptamines in liposomes, in order to reduce its toxicity, improve drugability and therapeutic index. ·

SUMMARY OF THE INVENTION

In order to prolong the release time of bufatryptamine substances, improve its medicinal activity, stability and bioavailability, and at the same time significantly reduce the toxic and side reactions to gastrointestinal tissue, the present invention provides a bufatryptamine liposome and Its preparation method and application.

In order to realize the above-mentioned technical purpose, the present invention adopts the following technical scheme:

A bufatryptamine liposome, comprising the following raw material components in parts by weight: 0.1-10 parts of bufatryptamine, 5-20 parts of phospholipids, 1-10 parts of cholesterol, and 1-10 parts of vitamin E.

Preferably, the bufatryptamine liposome includes the following raw material components in parts by weight: 1-7 parts of bufatryptamine, 10-20 parts of phospholipids, 1-5 parts of cholesterol, and 1-5 parts of vitamin E.

Preferably, the phospholipid is selected from one or both of soybean lecithin and lecithin.

Preferably, the particle size of the bufatryptamine liposome is 50-200 nm, the dispersion degree index (PDI) is 0.1-0.3, and the encapsulation efficiency is 70%.

Further, the present invention also provides the use of the bufatryptamine liposome in the preparation of analgesic and anti-inflammatory drugs.

Further, the present invention also provides the preparation method of the bufatryptamine liposome, which adopts the pH gradient method to prepare, and the specific steps are as follows:

(1) take by weighing the phospholipid, cholesterol, vitamin E of described recipe quantity, be that the chloroform-methanol solvent of 2:1 is fully dissolved with volume ratio, decompression rotary steams, makes it form honeycomb film;

(2) adding a certain volume of citric acid buffer for hydration, and ultrasonically fragmenting to obtain empty liposomes;

(3) get the empty liposome prepared above, add the dissolved bufatryptamine liquid with phosphate buffer, adjust the pH of the outer water phase with the sodium bicarbonate solution of pH 6-8, hatch in a water bath, to obtain the tea brown toad Tryptamine liposomal suspension;

(4) The bufatryptamine liposome suspension is divided into packages, and a mixed freeze-drying protective agent of 1-6% mannitol and 0.1-5% trehalose is added by weight, and after mixing, it is placed at -80°C It is pre-frozen in a refrigerator for 5-12 hours, and then placed in a freeze-drying machine at -60° C. for 24 hours to obtain bufatryptamine liposome freeze-dried powder.

Preferably, in step (4), a mixed freeze-drying protection agent of 4.5% mannitol and 1.5% trehalose is added by weight.

Further, the present invention also provides a bufatryptamine tablet, comprising the following components by weight: bufatryptamine liposome 10-30%, lactose 30-50%, microcrystalline cellulose 10-20%, Povidone K301-2%, sodium lauryl sulfate 1-30%, magnesium stearate 0.1-5%; preparation method: bufatryptamine liposome powder, lactose, microcrystalline cellulose, dodecyl The sodium alkyl sulfate is mixed evenly, and the mixture is granulated under the spraying of the povidone K30 ethanol solution. After the granules are dried, the magnesium stearate is added and mixed evenly, and the tablet is compressed in a single punch tablet machine.

Further, the present invention also provides a bufatryptamine capsule, comprising the following components by weight: 2-6% of bufatryptamine liposome, 8-14% of poloxamer, 40-60% of natural phospholipid , fatty acid glyceride 25-30%, polysorbate 805-10%; The preparation method is: weighing poloxamer, natural phospholipid, fatty acid glyceride, polysorbate 80, and stirring at 35-45 ° C until completely dissolved , the stirring speed is 350-450rpm, and the stirring time is 12-16min to obtain an oily mixed solution; an appropriate amount of bufatryptamine liposome powder is weighed, added to the oily mixed solution, and dispersed by ultrasonic waves at a temperature of 65-75 ℃ for 8- 12min, ultrasonic frequency is 25-30kHz, obtain liquid crystal gel precursor nano preparation, after freeze-drying, put it into empty capsule shell, namely obtain capsule.

Further, the present invention also provides a bufatryptamine granule, comprising the following components by weight: bufatryptamine liposome 25%, mannitol 55-72%, corn starch 1.3-20%, hydroxypropyl Cellulose 0.1-1.2%, sweetener 0.1-0.5%; The preparation method is as follows: Weigh the formula amount of bufatryptamine liposome, mannitol and corn starch, sieve and mix evenly, add hydroxypropyl cellulose and sweetener The flavoring agent is made into soft material, sieved to obtain wet granules, dried, sieved to integrate, and evenly mixed to obtain the finished product.

Further, the present invention also provides a bufatryptamine injection, calculated with a capacity of 5mL, containing: 1-1.5mg of bufatryptamine liposome, acetate buffer with a concentration of 1-300mM, and a concentration of 0.1-9.0 g/L osmotic pressure regulator NaCl, the balance is water for injection, and the pH of the injection is 4-5.5; the preparation method is: after dissolving the bufatryptamine liposome powder with acetate buffer, use 0.1 -9.0g/LNaCl to adjust osmotic pressure, dilute with water for injection, adjust pH to 4-5.5, filter through membrane, fill, sterilize, complete inspection, and package.

Further, the bufatryptamine liposome makes the distribution of the drug in the gastrointestinal tissue lower, and has the advantage of higher safety.

Further, the bufatryptamine liposome can achieve the advantages of sustained drug release and improved drug efficacy maintenance time.

Preferably, in the above-mentioned bufatryptamine liposome, the bufatryptamine comprises: serotonin (serotonin), bufotenidine (bufotenidine), N-methyl serotonin (N-methylserotonin), (bufotenine), bufothionine and dehydrobufotenine.

Compared with the prior art, the beneficial effects of the present invention are:

(1) The active drug-loaded liposome preparation of bufatryptamine was prepared for the first time. The particle size of the prepared active drug-loaded liposome was between 50-200 nm, the PDI was between 0.1-0.3, and the encapsulation efficiency could reach 70%. The results of transmission scanning electron microscopy showed that the pharmaceutical liposomes prepared by this method were spherical. The bufatryptamine of the drug-loaded liposome is encapsulated in the central cavity of the liposome.

(2) In the preparation process of bufatryptamine liposome freeze-dried powder, 4.5% mannitol and 1.5% trehalose are selected as mixed freeze-drying protective agent, and the obtained freeze-dried powder has the best stability and uniformity sex.

(3) In the hot plate analgesia model, the bufatryptamine drugs have analgesic activity before and after liposome inclusion, and the liposome inclusion has a longer duration of efficacy. In a formalin inflammatory pain model, both free bufatryptamine drugs and bufatryptamine liposomal drugs reduced the expression levels of COX-2 and TNF-α mRNA in foot tissue.

(4) After the bufatryptamine drug was encapsulated in liposomes, there was less drug residue in the intestinal tissue.

(5) In the evaluation of gastrointestinal irritation, the bufatryptamine drug can significantly reduce the pathological damage to the stomach and small intestine tissue of mice after being enclosed in liposomes, and significantly reduce the toxic and side effects to the gastrointestinal tissue. It can further improve the therapeutic index and druggability.

Description of drawings

Figure 1 shows the appearance and transmission electron microscope images of drug-loaded liposomes.

FIG. 2 is a graph showing the measurement results of the particle size analyzer.

Figure 3 is the in vitro drug release curve of bufatryptamine liposome and bufatryptamine solution.

Figure 4 shows the in vitro dissolution of bufatryptamine solid oral preparations.

Figure 5 is a comparison chart of thermal pain thresholds of mice in each group after administration.

Figure 6 is a comparison chart of the percentage increase in the thermal pain threshold of mice in each group.

Figure 7 is a comparison diagram of the mechanical pain threshold (A) and the area under the curve (B) of the mice in each group for 15-120 min.

Figure 8 is a graph showing the relative expression levels of COX-2 (A) and TNF-α mRNA (B) in the mouse foot tissue of each group.

Fig. 9 is a graph showing the morphological observation of gastric tissue after intragastric administration of mice in each group.

Fig. 10 is a pathological view of the gastric tissue of mice in each group (HE, x 200).

Fig. 11 is the pathological observation diagram of the small intestine tissue of mice in each group (HE, x 200).

Figure 12 is a graph showing the comparison of pathological score values of stomach (A) and small intestine tissue (B) of mice in each group.

Figure 13 shows the comparison of residual amounts of bufatryptamines in the intestinal tissue of toxic target organs

Fig. 14 is A, the UHPLC chromatograms of four kinds of bufatryptamines standard products; B, the UHPLC chromatograms of the crude drug Bufonis bufas UHPLC chromatogram; E, UHPLC chromatogram of purified total bufa tryptamine. (1) serotonin; (2) bufaternidine; (3) bufatryptamine; (4) bufathiazine, (5) N-methylserotonin (N-methylserotonin).

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention. The materials, reagents, etc. used are all commercially available reagents and materials unless otherwise specified.

Example 1

1. Selection of lyophilized protective agent during the preparation of bufatryptamine liposome lyophilized powder

Divide the prepared bufatryptamine liposome suspension, add three groups of different types of freeze-drying protective agents (mannitol+trehalose, sorbitol+sucrose, PVP+lactose), mix well, pre-freeze, and set it Freeze in a -80°C refrigerator for 5-12 hours, and then place it in a -60°C freeze dryer and freeze-dry for 24 hours to obtain bufatryptamine liposome freeze-dried powder. The appearance, hydration state, encapsulation efficiency and drug loading capacity of bufatryptamine liposome lyophilized powder were evaluated. The experimental results are shown in Table 1.

The investigation result of table 1 liposome freeze-dried powder

As can be seen from the experimental results of Table 1, compared with the lyophilized protective agent combination of sorbitol+sucrose, PVP+lactose, the present invention takes mannitol and trehalose as the mixed lyophilized protective agent, and the prepared bufatryptamine liposome freeze-dried. The powder is relatively homogeneous and full in appearance, easy to hydrate when reconstituted, without obvious phospholipid block, and the encapsulation rate after reconstitution is 69.01±0.67%, and the drug load is 8.84±0.09%. More preferably, the bufatryptamine liposome freeze-dried powder prepared by using 4.5% mannitol and 1.5% trehalose as mixed freeze-drying protective agent, the encapsulation efficiency and drug loading are both optimal values, and have good stability. sex and uniformity.

2. Quality evaluation of bufatryptamine liposomes

The appearance, microscopic morphology, particle size/potential characterization, stability and drug release profile of the prepared bufatryptamine liposomes were evaluated.

2.1. Morphological characterization of drug-loaded liposomes

As shown in Figure 1, liposomes are prepared by the optimized prescription of the present invention and the process, the visible blank liposomes are white homogeneous suspensions by naked eye observation, and the bufatryptamine liposomes are dark brown homogeneous suspensions, and there is no separation layer, flocculation, precipitation and other phenomena. The blank liposomes and drug-loaded liposome lyophilized powders after lyophilization were plump and uniform in appearance. Transmission scanning electron microscopy showed that the bufatryptamine liposomes prepared by this method were spherical or nearly spherical, and the particle size was between 100-200 nm.

2.2. Determination of particle size, polydispersity coefficient and Zeta potential

The particle size analyzer results in Figure 2 show that the obtained bufatryptamine liposome freeze-dried powder has an average particle size of 130.1 ± 4.06nm, a polydispersity coefficient of 0.217, an average Zeta potential of -8.00 ± 0.88mV, and a negatively charged .

2.3. Stability evaluation

Take the lyophilized bufatryptamine liposomes, store them in a 4° refrigerator at a low temperature and avoid light, and take samples at 0, 0.5, 1, 2, 3, and 6 months, respectively, to evaluate the appearance of the bufatryptamine liposome lyophilized powder. Properties, hydration state, average particle size and potential for evaluating the physical stability of liposome lyophilized powder. The bufatryptamine liposome freeze-dried powder is stored in a 4° refrigerator at a low temperature and protected from light, and is sampled at 0, 0.5, 1, 2, 3, and 6 months after storage, and redissolved in water to measure the encapsulation efficiency of the sample, and The leakage rate was calculated and used to evaluate the chemical stability of liposome lyophilized powder. The experimental results are shown in Table 2 and Table 3.

n＝3)The physical stability investigation of table 2 toad total tryptamine liposome lyophilized powder (

n=3)

As can be seen from Table 2, the freeze-dried liposome is full and has no obvious change in appearance under the condition of low-temperature storage in a 4° refrigerator, and when redissolving in water, it is easily hydrated without phospholipid block; the microscopic particle size increases slightly with the prolongation of storage time. , the potential decreases. It shows that the obtained bufatryptamine liposome freeze-dried powder is relatively stable in physical properties when stored at 4° low temperature and protected from light.

n＝3)The chemical stability investigation of table 3 bufatryptamine liposome freeze-dried powder (

n=3)

time/month Encapsulation rate/% Leak rate/% 0 69.01±0.67 \ 0.5 68.93±0.64 0.12±0.92 1 68.07±0.35 1.37±0.50 2 68.08±0.63 1.35±0.92 3 67.22±3.14 2.60±0.46 6 65.68±1.16 4.81±1.69

As can be seen from Table 3, the lyophilized drug-loaded liposomes are stored under the condition of 4 ° of low temperature and dark storage, with the extension of time, the encapsulation efficiency does not change much, and the leakage rate is less than 5%. Show that gained bufatryptamine liposome freeze-dried powder is relatively stable in chemical property when stored in the dark at 4 ° of low temperature.

2.4. In vitro drug release research

As shown in Figure 3, the release rate of free bufatryptamine in phosphate buffer is faster, and the release rate of the drug has reached 80.56±1.18% at 5h; while the release rate of bufatryptamine after liposome inclusion is relatively Slow, the drug release rate did not exceed 50% at 5h, which was 42.29±1.01%, and the release rate reached 80.10±0.34% at 12h, that is, after liposome encapsulation, the release rate of bufatryptamine was significantly slower than that of free bufatryptamine drugs.

Example 2 Pharmacological experiment

1. Hot plate analgesia experiment

The qualified female ICR strain mice were screened by the hot plate and randomly divided into groups according to the basic heat pain threshold, and the mice were administered with a volume of 10 mL/kg. Except for the positive drug morphine hydrochloride, which was administered by intraperitoneal injection, the rest of the groups were administered by gavage. medicine. The blank control group was given 1.4g/kg blank liposome by gavage, and its total lipid dose was equivalent to the total lipid adjuvant mass in the 45mg/kg bufatryptamine liposome freeze-dried powder given by gavage. Before drug intervention, the hot and cold plate pain meter was pre-controlled to a stable 55±0.1℃. In each group of animals, the hotpaw withdrawal latency (HPWL) was measured at 30min, 60min, 90min, 120min, and 150min after administration, and the increase rate of pain threshold in each group was calculated.

As shown in Figure 4, there was no significant difference in the basal thermal pain threshold of each administration group (P>0.05); the morphine hydrochloride group had the highest thermal pain threshold at 30 minutes, which was relatively the same as the blank control group and the basal thermal pain threshold before administration in this group. Very significant difference (P<0.01); the thermal pain threshold of Free-SA (non-liposome inclusion bufatryptamine) 15mg/kg and Lipo-SA (liposome inclusion bufatryptamine) 15mg/kg in the administration group was lower than There was no significant change within the measurement range; compared with the positive drug morphine hydrochloride group, the thermal pain thresholds of Free-SA45 mg/kg and Lipo-SA45 mg/kg in the administration group were the highest at 60min and 90min, respectively, and were similar to the pre-administration baseline in this group. The thermal pain threshold was significantly different (P<0.01). At 150 minutes, the thermal pain thresholds in each group basically returned to normal levels.

As shown in Figure 5, there was no significant change in the percentage increase in the average thermal pain threshold in the blank group; after intraperitoneal injection of morphine hydrochloride, the thermal pain threshold rate increased by 81.58% at 30 minutes, and the analgesic activity was better at this time; the Free-SA45 mg/kg group averaged at 60 minutes. The percentage of thermal pain threshold was the highest, up to 50.40%, while the thermal pain threshold was increased by 56.78% at 90min in the equal dose of bufatryptamine liposome group.

Based on the results of this experiment, it is shown that in the hot plate analgesia model, the bufatryptamine drugs have analgesic activity before and after liposome inclusion, but the onset time lags after inclusion.

2. Formalin inflammatory pain test

Select healthy adult ICR strain mice, half male and half male, fasted for 12 hours before the experiment, and randomly grouped according to body weight. The blank control group and the formalin model group were given 1.4g/kg blank liposome by intragastric administration, and the total lipid dose was equivalent to the total lipid excipient mass in the 45mg/kg bufatryptamine liposome administered by intragastric administration . Except for the morphine hydrochloride positive drug group, which was administered by intraperitoneal injection at a volume of 10 mL/kg, the mice in the other groups were administered by intragastric administration at a dose of 10 mL/kg. Before the experiment, the baseline mechanical pain threshold (baseline) of the mice in each group was measured. After 15 minutes of administration in each group, 20 μL of 2.5% formalin solution was injected subcutaneously along the right hind foot of the mice with a 1 mL sterile syringe. Model was established, and the pain threshold of mechanical stimulation of the right hind paw of mice at different times after modeling was measured.

Effects on analgesic activity and duration of drug effect

As shown in Figure 6, there was no significant difference in the basal pain thresholds of the experimental groups before modeling; except for the blank control group, after subcutaneous injection of 2.5% formalin solution on the plantar of the right hind foot of each experimental group, the mechanical contraction measured 15 minutes later. Foot pain threshold was significantly decreased. Compared with the formalin inflammatory pain model group, the mechanical foot pain threshold of the positive drug morphine hydrochloride group was significantly increased within 15-40 minutes (P<0.05); The pain threshold was greater than that of the model group, and the comparison between the groups was statistically significant (P<0.05). Compared with the blank group, the Free-SA45 mg/kg administration group had no statistical difference at 75 minutes (P>0.05). Compared with the model group, the mechanical stimulation threshold of foot retraction in the -SA administration group increased from 45 to 120 minutes, and the differences between the groups were statistically significant (P<0.05).

Compared with the blank control group, the area under the curve in the formalin model group was significantly reduced within 15-120 minutes, with a very significant difference (P<0.01). The area under the curve of the bufatryptamine drug treatment group had extremely significant statistical difference (P<0.01). Compared with the same dose of Free-SA administration group and Lipo-SA administration group, under the curve after liposome inclusion The area is higher.

Based on the results of this experiment, it is shown that in the formalin plantar inflammatory pain model, compared with the bufatryptamine drug group, the analgesic activity after liposome inclusion is comparable to it, and the drug effect time can be prolonged.

3. Effects on the expression levels of COX-2 and TNF-αmRNA in foot tissue

As shown in Figure 7(A), compared with the blank group, the expression of COX-2 mRNA in the foot tissue of the formalin inflammatory pain model group was significantly up-regulated (P<0.01); compared with the model group, intraperitoneal injection of morphine hydrochloride positive drug could reduce COX The expression of -2 mRNA has extremely significant statistical difference (P<0.01); as above, the four groups of bufatryptamine drug groups can significantly reduce the expression of COX-2 mRNA in the foot tissue after treatment, and the Free-SA and The expression levels of COX-2 mRNA in Lipo-SA administration groups were almost the same.

As shown in Figure 7(B), compared with the blank group, the expression of TNF-α mRNA in the foot tissue of the formalin inflammatory pain model group was significantly up-regulated (P<0.01); The expression of TNF-α mRNA in the foot tissue could be down-regulated in the drug treatment group; however, compared with the model group, only the TNF-α mRNA expression in the Free-SA45 mg/kg and Lipo-SA45 mg/kg groups was significantly different (P<0.01) .

Based on the above experimental results, it shows that in the formalin inflammatory pain model, both free bufatryptamine drugs and bufatryptamine liposome drugs can reduce the expression levels of COX-2 and TNF-α mRNA in foot tissue.

4. Evaluation of gastrointestinal irritation

During the experiment, the homemade bufatryptamine drug was prepared with pure water at concentrations of 13.5mg/mL and 27mg/mL, bufatryptamine liposome lyophilized powder was prepared at concentrations of 424mg/mL and 848mg/mL, and blank liposomes were used. The lyophilized powder was prepared at a concentration of 820 mg/mL, and each sample test solution was administered in a single dose according to the maximum administration dose of 0.4 mL/10 g in mice, and a normal saline solution was used as a blank control to evaluate each test to be tested. Irritation of the gastrointestinal tract in mice by high-dose intragastric administration of the drug.

On the basis of the previous pre-experiment, this experiment set the exposure period of gastrointestinal irritation after oral administration to 6h after administration. Immediately after each administration, the activity of each group of experimental animals within 2 h was recorded. During the observation period, the activities of each animal were observed and recorded in detail, and whether there was decreased activity, vomiting, diarrhea, or even death were counted. After the observation period was over, the following results were evaluated.

4.1. General situation observation

During the observation period of gastrointestinal irritation, all the test animals used in each group survived. In the blank control group and the blank liposome group, there were no abnormalities in the mental state, behavioral activities, and secretions of the animals. However, the animals in the low and high dose groups of free bufatryptamine administered by a single gavage occasionally experienced nausea and vomiting reflex behavior, accompanied by hypokinesis, sluggishness, and decreased activity. This phenomenon was mainly maintained for half an hour after administration. Almost back to normal after hours. As can be seen from Table 4, the gag reflex rates of the animals in the low and high dose groups of free bufatryptamine were 25.0% and 62.5%, respectively. There was no gag reflex in the two groups of bufatryptamine liposome group, and there was no obvious abnormality in general behavior. The results showed that a single high-dose intragastric administration of bufatryptamine after liposome inclusion could reduce the gag reflex behavior in mice.

Table 4 Behavioral evaluation of mice in each group by gavage

group N/only Dosage (mg/kg) gag reflex Control 8 \ 0/8 (0%) Empty-Lipo 8 \ 0/8 (0%) Free-SA540mg/kg 8 540 2/8 (25.0%) Free-SA1080mg/kg 8 1080 5/8 (62.5%) Lipo-SA540mg/kg 8 540 0/8 (0%) Lipo-SA1080mg/kg 8 1080 0/8 (0%)

4.2 Histopathological examination

After the observation period, the anatomical examination found that only the gastric tissue of the two Free-SA administration groups was relatively large in volume, with gastric flatulence and superficial capillary microcongestion, and there was a certain dose-dependence; See obvious abnormalities, the results are shown in Figure 8. The rest of the organs of the tested animals showed no obvious abnormality.

According to the pathological examination results in Figure 9 and Figure 10, there were no obvious lesions in the stomach and small intestine tissue of the blank control group and blank liposome group; while the free bufatryptamine high-dose group observed mild atypia of gastric mucosal epithelial cells, and muscle layer. The gap became wider, and the histopathological score was higher than that of the blank control group (Fig. 11). In the small intestinal mucosa of the Free-SA administration group, obvious inflammatory cell infiltration, erosion and shedding of epithelial cells, structural disorder, and mild muscle cells were observed in the Free-SA administration group. Fibrosis, the degree of injury has a significant dose-dependent relationship, and the pathological score is significantly higher than that of the blank group (P<0.01).

4.3 Comparison of residues of bufatryptamines in intestinal tissue

As shown in Figure 13, after administration, bufatryptamine remained in the intestinal tissue of mice, causing toxic and side effects, while the residual amount of the drug in the intestinal tissue was significantly reduced after liposome inclusion (Lipo-SA administration group). It can be seen that the liposome encapsulates and changes the tissue distribution of the drug, reduces the accumulation or residue in toxic organs, and improves the safety of the drug.

Based on the above results, it is shown that the bufatryptamine drug can significantly reduce the pathological damage to the stomach and small intestine tissue of mice after inclusion in liposomes.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, some modifications or equivalent replacements can be made to the technical solutions. Modifications or equivalent replacements should also be regarded as the protection scope of the present invention.

Claims (11)

1. The toad tryptamine liposome is characterized by comprising the following raw material components in parts by weight: 0.1-10 parts of toad tryptamine, 5-20 parts of phospholipid, 1-10 parts of cholesterol and 1-10 parts of vitamin E.

2. The bufotenine liposome of claim 1, which is characterized by comprising the following raw materials in parts by weight: 1-7 parts of toad tryptamine, 10-20 parts of phospholipid, 1-5 parts of cholesterol and 1-5 parts of vitamin E.

3. The bufotenine liposome according to claim 2, wherein the phospholipid is selected from one or both of soybean lecithin and lecithin; the particle diameter of the toad tryptamine liposome is 50-200nm, the dispersity index is 0.1-0.3, and the entrapment rate is 70%.

4. Use of the bufotenine liposome as claimed in any one of claims 1-3 in preparation of analgesic and anti-inflammatory drugs.

5. The method for preparing bufotenine liposome according to any one of claims 1-3, wherein the preparation is carried out by pH gradient method, comprising the following steps:

(1) weighing phospholipid, cholesterol and vitamin E according to the prescription amount, fully dissolving the phospholipid, the cholesterol and the vitamin E in a chloroform-methanol solvent with the volume ratio of 2:1, and carrying out rotary evaporation under reduced pressure to form a honeycomb-shaped film;

(2) adding a certain volume of citric acid buffer solution for hydration, and carrying out ultrasonic crushing to prepare empty liposome;

(3) adding dissolved bufotenine liquid medicine with phosphate buffer solution into the prepared empty liposome, adjusting pH of external water phase with sodium bicarbonate solution with pH of 6-8, and incubating in water bath to obtain dark brown bufotenine liposome suspension;

(4) subpackaging the toad tryptamine liposome suspension, adding a mixed freeze-drying protective agent of 0.1-6% of mannitol and 0.1-5% of trehalose according to the weight ratio, uniformly mixing, placing in a pre-freezing machine, then placing in a freeze-drying machine, and freeze-drying to obtain the toad tryptamine liposome freeze-dried powder.

6. The method for preparing bufotenine liposome according to claim 5, wherein a mixed lyoprotectant of 4.5% mannitol and 1.5% trehalose is added in step (4) by weight.

7. The toad tryptamine tablet is characterized by comprising the following components in percentage by weight: 10-30% of bufotenine liposome as claimed in any one of claims 1-3, 30-50% of lactose, 10-20% of microcrystalline cellulose, 301-2% of povidone K, 1-30% of sodium lauryl sulfate, and 0.1-5% of magnesium stearate; the preparation method comprises the following steps: mixing toad tryptamine liposome powder, lactose, microcrystalline cellulose and sodium dodecyl sulfate uniformly, granulating the mixture under the spraying of povidone K30 ethanol solution, drying the granules, adding magnesium stearate, mixing uniformly, and tabletting in a single-punch tablet machine to obtain the toad tryptamine liposome tablet.

8. The toad tryptamine capsule is characterized by comprising the following components in percentage by weight: 2-6% of bufotenine liposome as claimed in any one of claims 1-3, 8-14% of poloxamer, 40-60% of natural phospholipid, 25-30% of fatty acid glyceride, 805-10% of polysorbate;

the preparation method comprises the following steps: weighing poloxamer, natural phospholipid, fatty glyceride and polysorbate 80, stirring at 35-45 ℃ until the components are completely dissolved, wherein the stirring speed is 350-; weighing appropriate amount of bufotenine liposome powder, adding into the oily mixed solution, ultrasonically dispersing at 65-75 deg.C for 8-12min with ultrasonic frequency of 25-30kHz to obtain liquid crystal gel precursor nanometer preparation, lyophilizing, and encapsulating to obtain capsule.

9. The toad tryptamine granules are characterized by comprising the following components in percentage by weight: 25% of bufotenine liposome as claimed in any one of claims 1-3, 55-72% of mannitol, 1.3-20% of corn starch, 0.1-1.2% of hydroxypropyl cellulose, 0.1-0.5% of sweetener;

the preparation method comprises the following steps: weighing toad tryptamine liposome, mannitol and corn starch according to the formula ratio, sieving, mixing uniformly, adding hydroxypropyl cellulose and a sweetening agent to prepare a soft material, sieving to obtain wet granules, drying, sieving, integrating and mixing uniformly to obtain the toad tryptamine liposome powder.

10. A bufotenine injection, which is characterized by comprising the following components in a volume of 5 mL: the bufotenine liposome according to any one of claims 1 to 3, wherein 1 to 1.5mg of bufotenine liposome is prepared from 1 to 300mM of acetate buffer, 0.1 to 9.0g/L of osmotic pressure regulator NaCl, and the balance is water for injection, and the pH of the injection is 4 to 5.5; the preparation method comprises the following steps: dissolving bufotenine liposome powder in acetate buffer solution, adjusting osmotic pressure with 0.1-9.0g/LNaCl, diluting with water for injection, adjusting pH to 4-5.5, filtering with filter membrane, bottling, sterilizing, inspecting, and packaging.

11. The bufotenine liposome according to any one of claims 1-3, wherein the bufotenine comprises: serotonin, bufotenine, N-methyl serotonin, bufotenine, bufothionine, and dehydrobufotenine.

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