CN114432247A

CN114432247A - Toad tryptamine liposome and preparation method and application thereof

Info

Publication number: CN114432247A
Application number: CN202210055494.8A
Authority: CN
Inventors: 马宏跃; 周婧
Original assignee: Nanjing University of Chinese Medicine
Current assignee: Nanjing University of Chinese Medicine
Priority date: 2022-01-18
Filing date: 2022-01-18
Publication date: 2022-05-06

Abstract

The invention discloses a toad tryptamine liposome and a preparation method and application thereof, and essentially the application of the toad tryptamine liposome in improving therapeutic index and drug property by reducing the gastrointestinal toxicity of the toad tryptamine. The invention adopts an active drug loading method to prepare a liposome preparation, the liposome preparation comprises liposome particles, the liposome particles comprise a carrier, the carrier is a liposome membrane with a bimolecular structure, and the bufotenine comprises serotonin, bufotenine, N-methyl serotonin, bufotenine, bufothionine and dehydrobufotenine. Which is located in the central cavity of the liposome. The liposome is prepared from 0.1-10 parts of toad tryptamine, 5-20 parts of phospholipid, 1-10 parts of cholesterol and 1-10 parts of vitamin E. The liposome of toad tryptamine can be used for treating pain, inflammation and depression. Compared with the passive drug-loaded liposome, the active drug-loaded liposome has the advantages of more stability, more sustained release, higher bioavailability, low toxic target organ residue and higher safety.

Description

Toad tryptamine liposome and preparation method and application thereof

Technical Field

The application relates to the technical field of pharmaceutical preparations, in particular to a bufotenine liposome and a preparation method and application thereof. Essentially, the use of toad tryptamine for improving the therapeutic index and the druggability by reducing the gastrointestinal toxicity of the toad tryptamine.

Background

Along with the development of modern society, the awareness of human health needs and the understanding level of medicine safety are increasingly improved, so that the safety problem of traditional Chinese medicines is widely concerned by society. The safety of the medicine is the basis of the safety of patients, the safety and the effectiveness are the basic attributes of the medicine, and whether a medicine can be accepted by the society depends on the strength of the effectiveness and the toxicity.

The toad venom, as one of the famous and precious animal medicines in China, has the effects of resisting tumor, strengthening heart, easing pain, resisting inflammation and the like, and is widely used for treating cardiovascular and cerebrovascular diseases, cancers, respiratory tract diseases, mental diseases or nervous diseases. Toad tryptamine is a water-soluble indolealkylamine alkaloid in toad venom, is a derivative of serotonin, and more than 20 of toad tryptamine, N-methyl serotonin, dehydrobufotamine, bufothionine and the like are found. The applicant finds that after the toad tryptamine substances are administrated by intraperitoneal injection, the hot pain threshold of a mouse hot plate pain model can be obviously improved, and the diphasic pain ethology and mechanical stimulation foot pain threshold of a mouse formalin plantar inflammatory pain model can be obviously improved, namely the toad tryptamine substances have obvious analgesic activity (a synthetic method of toad tryptamine and quaternary ammonium salt thereof and an application patent number ZL201911259232.8 in preparation of analgesic and anti-inflammatory medicaments). The bufotenine also has antidepressant effect (application of bufotenine in preparation of antidepressant medicine CN 202011077934.7).

However, the applicant researches and discovers that the toad tryptamine substances have obvious gastrointestinal irritation, and the drug potency of the toad tryptamine substances is seriously reduced. In view of the toxicity problem which is a key link hindering the development of new toad tryptamine drugs, the invention tries to adopt a pharmaceutical means to reduce the gastrointestinal toxicity of toad tryptamine so as to improve the therapeutic index.

The liposome is used as a drug carrier and can be loaded with drugs with different properties of fat solubility and water solubility. Based on the strong polarity and weak alkalinity of toad tryptamine substances, the invention adopts liposome inclusion technology to encapsulate water-soluble toad tryptamine components in liposome so as to reduce the toxicity of the toad tryptamine components and improve the drug forming property and therapeutic index of the toad tryptamine components. A

Disclosure of Invention

The invention provides a toad tryptamine liposome, a preparation method and application thereof, aiming at prolonging the release action time of toad tryptamine substances, improving the medicinal activity, stability and bioavailability of the toad tryptamine substances and obviously reducing toxic and side effects on gastrointestinal tissues.

In order to achieve the technical purpose, the invention adopts the following technical scheme:

a toad tryptamine liposome comprises 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.

Preferably, the toad tryptamine liposome comprises 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.

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

Preferably, the toad tryptamine liposome has a particle size of 50-200nm, a dispersity index (PDI) of 0.1-0.3 and an entrapment rate of 70%.

Furthermore, the invention also provides application of the bufotenine liposome in preparation of analgesic and anti-inflammatory drugs.

Further, the invention also provides a preparation method of the toad tryptamine liposome, which adopts a pH gradient method for preparation and comprises the following specific steps:

(1) weighing the phospholipid, cholesterol and vitamin E according to the prescription amount, fully dissolving the phospholipid, the cholesterol and the vitamin E by using 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 1-6% of mannitol and 0.1-5% of trehalose according to the weight ratio, uniformly mixing, pre-freezing in a refrigerator at-80 ℃ for 5-12h, then placing in a freeze dryer at-60 ℃ for freeze-drying for 24h to obtain the toad tryptamine liposome freeze-dried powder.

Preferably, a mixed lyoprotectant of 4.5% mannitol and 1.5% trehalose is added in step (4) by weight.

Further, the invention also provides a toad tryptamine tablet which comprises the following components in percentage by weight: 10-30% of toad tryptamine liposome, 30-50% of lactose, 10-20% of microcrystalline cellulose, 301-2% of povidone K, 1-30% of sodium dodecyl 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 press to obtain the toad tryptamine liposome tablet.

Further, the invention also provides a toad tryptamine capsule which comprises the following components in percentage by weight: 2-6% of toad tryptamine liposome, 8-14% of poloxamer, 40-60% of natural phospholipid, 25-30% of fatty glyceride and 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 in empty capsule shell to obtain capsule.

Further, the invention also provides toad tryptamine granules which comprise the following components in percentage by weight: 25% of toad tryptamine liposome, 55-72% of mannitol, 1.3-20% of corn starch, 0.1-1.2% of hydroxypropyl cellulose and 0.1-0.5% of sweetening agent; the preparation method comprises the following steps: weighing toad tryptamine liposome, mannitol and corn starch in a 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.

Further, the invention also provides a toad tryptamine injection, which contains the following components in a volume of 5 mL: 1-1.5mg of toad tryptamine liposome, 1-300mM acetate buffer solution, 0.1-9.0g/L osmotic pressure regulator NaCl, and the balance of water for injection, wherein the pH value of the injection is 4-5.5; 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.

Furthermore, the toad tryptamine liposome has lower distribution amount of the medicine in gastrointestinal tissues and higher safety and advantages.

Furthermore, the toad tryptamine liposome can achieve the advantages of slow release of the drug and improvement of the duration of the drug effect.

Preferably, the bufotenine liposome comprises: serotonin (serotonin), toad bufonid toad tenidine (bufotenine), N-methylpentaserotonin (N-methylerotonin), bufotenine (bufotenine), bufothionine (bufothionine), and dehydrobufotenine (dehydrobufotenine).

Compared with the prior art, the invention has the beneficial effects that:

(1) the bufotenine active drug-carrying liposome preparation is prepared for the first time, the particle size of the prepared active drug-carrying liposome is 50-200nm, PDI is 0.1-0.3, and the entrapment rate can reach 70%. The result of a transmission scanning electron microscope shows that the pharmaceutical liposome prepared by the method is in a sphere-like shape. The toad tryptamine of the medicine-carrying liposome is encapsulated in the central cavity of the liposome.

(2) In the preparation process of the toad tryptamine liposome freeze-dried powder, 4.5 percent of mannitol and 1.5 percent of trehalose are selected as a mixed freeze-drying protective agent, and the prepared freeze-dried powder has optimal stability and uniformity.

(3) In the hot plate method analgesic model, the toad tryptamine drug has analgesic activity before and after inclusion by liposome, and the duration of drug effect of the liposome inclusion compound is longer. Both free buformin drugs and buformin liposome drugs reduced the expression levels of COX-2 and TNF-alpha mRNA in the tissues of the feet in the formalin inflammatory pain model.

(4) The toad tryptamine drug has less drug residue in intestinal tract tissues after being coated by liposome.

(5) In the evaluation of gastrointestinal irritation, the toad tryptamine medicament is included by the liposome, so that pathological damage to the tissues of the stomach and the small intestine of a mouse can be obviously relieved, and toxic and side effects to the tissues of the stomach and the intestine are obviously reduced. Can further improve the therapeutic index and the application of the drug forming property.

Drawings

FIG. 1 is an appearance view and a transmission electron microscope image of a drug-loaded liposome.

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

Figure 3 is the in vitro release curve of toad tryptamine liposome and toad tryptamine solution.

Figure 4 shows the in vitro dissolution rate of the solid oral preparation of bufotenine.

FIG. 5 is a graph comparing the thermal pain threshold after administration to groups of mice.

FIG. 6 is a graph comparing the percent increase in the thermal pain threshold for each group of mice.

FIG. 7 is a graph comparing the mechanical pain threshold (A) and the area under the curve (B) of 15-120 min for each group of mice.

FIG. 8 is a graph of the relative expression levels of COX-2(A) and TNF- α mRNA (B) in the tissues of the feet of various groups of mice.

FIG. 9 is a diagram showing the morphological observation of the gastric tissues after the gavage administration of the mice in each group.

FIG. 10 is a pathological observation (HE, x 200) of gastric tissue of each group of mice.

FIG. 11 is a pathological observation (HE, x 200) of intestinal tissues of mice in each group.

FIG. 12 is a graph showing a comparison of pathological scores of stomach (A) and small intestine (B) tissues in each group of mice.

FIG. 13 is a graph showing the comparison of the residual amount of bufotamine in the intestinal tissues of toxic target organs

FIG. 14 is a UHPLC chromatogram of four toad tryptamine standards A; b, UHPLC chromatogram of toad venom crude drugs; c, UHPLC chromatogram of toad venom water extract; d, primarily separating UHPLC chromatogram of toad total tryptamine by macroporous resin; e, UHPLC chromatogram of purified toad total tryptamine. (1) Serotonin; (2) bufotenidine; (3) bufotenine; (4) bufothionine, (5) N-methylpentatryptamine (N-methylerotonin).

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The materials, reagents and the like used are all commercially available reagents and materials unless otherwise specified.

Example 1

1. Selection of freeze-drying protective agent in preparation process of toad tryptamine liposome freeze-dried powder

Subpackaging the prepared bufotenine liposome suspension, adding three groups of different freeze-drying protective agents (mannitol, trehalose, sorbitol, sucrose, PVP and lactose), mixing, pre-freezing, namely placing in a refrigerator at-80 ℃ for freezing for 5-12h, then placing in a freeze dryer at-60 ℃ for freeze-drying for 24h, and obtaining the bufotenine liposome freeze-dried powder. And evaluating the appearance, hydration state, encapsulation rate and drug loading capacity of the toad tryptamine liposome freeze-dried powder. The results of the experiment are shown in table 1.

TABLE 1 examination of Liposome lyophilized powders

The experimental results in table 1 show that compared with the combination of sorbitol, sucrose and PVP and lactose, the bufotenine liposome freeze-dried powder prepared by using mannitol and trehalose as the mixed freeze-drying protective agent has relatively uniform and full appearance, is easy to hydrate during redissolution, has no obvious phospholipid block, and has an encapsulation rate of 69.01 +/-0.67% and a drug loading rate of 8.84 +/-0.09% after redissolution. More preferably, the toad tryptamine liposome freeze-dried powder prepared by taking 4.5% mannitol and 1.5% trehalose as a mixed freeze-drying protective agent has optimal encapsulation efficiency and drug loading capacity, and has good stability and uniformity.

2. Quality evaluation of toad tryptamine liposome

And (3) evaluating the appearance, microscopic morphology, particle size/potential characterization, stability and drug release curve of the prepared bufotenine liposome.

2.1 morphological characterization of drug-loaded liposomes

As shown in figure 1, when the liposome is prepared according to the optimized prescription and process, the blank liposome is white uniform suspension observed by naked eyes, and the toad tryptamine liposome is dark brown uniform suspension, and the phenomena of layering, flocculation, precipitation and the like are avoided. The blank liposome and the freeze-dried powder of the drug-loaded liposome after freeze-drying treatment are full and uniform in appearance. The transmission scanning electron microscope shows that the toad tryptamine liposome prepared by the method is spherical or nearly spherical, and the particle size is between 100 and 200 nm.

2.2 measurement of particle diameter, polydispersity and Zeta potential

As shown in figure 2, the obtained lyophilized powder of bufotenine liposome has average particle diameter of 130.1 + -4.06 nm, polydispersity of 0.217, average Zeta potential of-8.00 + -0.88 mV, and negative charge.

2.3 evaluation of stability

And (3) taking the freeze-dried toad tryptamine liposome, storing the freeze-dried toad tryptamine liposome in a refrigerator at a low temperature of 4 ℃ in the dark, sampling at 0, 0.5, 1, 2, 3 and 6 months respectively, and evaluating the appearance, hydration state, average particle size and potential of the freeze-dried toad tryptamine liposome powder for evaluating the physical stability of the freeze-dried powder of the liposome. Storing the toad tryptamine liposome freeze-dried powder in a 4-degree refrigerator in a low-temperature dark place, respectively sampling at 0, 0.5, 1, 2, 3 and 6 months after storage, adding water for redissolving, measuring the encapsulation rate of the sample, and calculating the leakage rate for evaluating the chemical stability of the liposome freeze-dried powder. The results of the experiment are shown in tables 2 and 3.

TABLE 2 physical stability Studies of lyophilized powder of toad Total tryptamine liposomes (

n＝3)

As can be seen from Table 2, the appearance of the freeze-dried liposome is full and has no obvious change under the condition of low-temperature storage in a 4-degree refrigerator, and the freeze-dried liposome is easy to hydrate and has no phospholipid blocks when being redissolved by adding water; the microscopic particle size slightly increases with the storage time, and the potential decreases. The physical properties of the obtained toad tryptamine lipidosome freeze-dried powder are relatively stable when the toad tryptamine lipidosome freeze-dried powder is stored at a low temperature of 4 ℃ in a dark place.

TABLE 3 investigation of chemical stability of lyophilized powder of bufotenine liposomes: (

n＝3)

Time/month	Encapsulation efficiency/%	Leakage 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 freeze-dried drug-loaded liposome has little change in encapsulation efficiency with the time extension under the condition of low-temperature and dark storage at 4 degrees, and the leakage rate is less than 5 percent. The obtained toad tryptamine liposome freeze-dried powder is relatively stable in chemical property when stored at low temperature of 4 ℃ in a dark place.

2.4 in vitro drug Release study

As shown in figure 3, the release rate of free toad tryptamine in phosphate buffer is relatively fast, and the release of the free toad tryptamine in phosphate buffer reaches 80.56 +/-1.18% at 5 h; the release rate of the liposome-encapsulated toad tryptamine is relatively slow, the drug release rate is not more than 50% at 5h and is 42.29 +/-1.01%, and the release rate is 80.10 +/-0.34% at 12h, namely the release rate of the toad tryptamine drug is obviously slower than that of the free toad tryptamine drug after the toad tryptamine is encapsulated by the liposome.

EXAMPLE 2 pharmacological experiments

1. Hot plate analgesic test

And screening qualified female ICR strain mice on a hot plate, randomly grouping according to a basic thermal pain threshold value, and administrating by using a volume of 10mL/kg, wherein except for the positive medicament morphine hydrochloride which is used for intraperitoneal injection, the other groups are intragastric administration. The blank control group is administrated with 1.4g/kg of blank liposome by intragastric administration, and the total lipid dose is equivalent to the total lipid adjuvant mass in 45mg/kg of toad tryptamine liposome freeze-dried powder administrated by intragastric administration. Before the intervention of the medicine, the cold and hot plate pain measuring instrument is controlled to be stable 55 +/-0.1 ℃. Experiment each group of animals respectively determine the hot pain threshold (HPWL) at 30min, 60min, 90min, 120min and 150min after administration, and calculate the pain threshold increase rate of each group of experiment.

As shown in fig. 4, there was no significant difference in basal thermal pain threshold (P >0.05) for each administration group; the maximal thermal pain threshold value of the morphine hydrochloride group is 30min, and the relative significant difference (P is less than 0.01) between the maximal thermal pain threshold value and the basal thermal pain threshold value of the blank control group and the group before administration is very obvious; the pain threshold of Free-SA (non-liposome inclusion bufotamine) 15mg/kg and Lipo-SA (liposome inclusion bufotamine) 15mg/kg in the administration group is not obviously changed in the measurement range; compared with the morphine hydrochloride group, Free-SA45 mg/kg and Lipo-SA45 mg/kg of the administration group have the highest thermal pain threshold values of 60min and 90min respectively, and have very significant difference (P <0.01) compared with the basal thermal pain threshold value before administration of the group. By 150min, the thermal pain threshold returned to essentially normal levels in each group.

As shown in fig. 5, there was no significant change in the percentage increase in the mean hot pain threshold for the blank group; the thermal pain threshold rate is improved by 81.58% in 30min after morphine hydrochloride is injected into the abdominal cavity, and the analgesic activity is better at the moment; the Free-SA45 mg/kg group showed the highest percentage increase in the mean thermal pain threshold at 60min, which reached 50.40%, while the equal dose bufotamine liposome group showed the increase in the thermal pain threshold at 90min of 56.78%.

The experiment result shows that in the hot plate analgesic model, the toad tryptamine medicine has analgesic activity before and after being included by the liposome, but the onset time is delayed after inclusion.

2. Formalin inflammatory pain test

Healthy adult ICR strain mice are selected, half of the mice are female and half of the mice, the mice are fasted and not forbidden for 12 hours before the experiment, and the mice are grouped according to a weight random method. The blank liposome of 1.4g/kg is administrated by intragastric administration to both the hollow white control group and the formalin model group, and the total lipid dose is equivalent to the total lipid adjuvant mass in the toad tryptamine liposome of 45mg/kg administrated by intragastric administration. Except for the morphine hydrochloride positive drug group which is administrated by abdominal cavity injection with the volume of 10mL/kg, the other groups of mice are administrated by gastric gavage with the dose of 10 mL/kg. Before the experiment, the basal mechanical pain threshold (baseline) of each group of mice is measured, after each group is administrated for 15min, 20 mu L of 2.5% formalin solution is administrated by a 1mL sterile syringe along the subcutaneous injection of the right hind foot of the mice for molding, and the mechanical stimulation and foot contraction reaction pain threshold of the right hind foot of the mice at different moments after molding is measured.

Influence on analgesic Activity and duration of drug action

As shown in fig. 6, there was no significant difference in the basal pain threshold before each group of the experiment was modeled; except for the blank control group, the mechanical foot-contracting pain threshold value measured at 15min is obviously reduced after 2.5 percent formalin solution is injected subcutaneously into the sole of the right hind foot of each experimental group. Compared with a formalin inflammatory pain model group, the mechanical foot pain reduction threshold of the positive drug morphine hydrochloride group is obviously increased within 15-40 min (P is less than 0.05); in the two Free-SA administration groups, the mechanical pain threshold value is greater than that of the model group within 60-90 min, and the comparison between the groups has statistical significance (P is less than 0.05); compared with the blank group, the Free-SA45 mg/kg administration group has no statistical difference at 75min (P > 0.05); the mechanical stimulation foot pain threshold of the two Lipo-SA administration groups is increased at 45-120 min relative to that of the model group, and the difference between the two groups has statistical significance (P < 0.05).

The formalin model group is opposite to the blank control group, the area under the curve is obviously reduced within 15-120 min, and the difference is very obvious (P is less than 0.01); compared with a blank control group and a model group, the areas under the curves of the morphine hydrochloride yang medicine group and the four toad tryptamine medicine treatment groups have extremely significant statistical difference (P is less than 0.01); the Free-SA administration group and the Lipo-SA administration group at equal doses were relatively higher in area under the curve after inclusion with liposomes.

The experimental result is combined, and the result shows that in the formalin plantar inflammatory pain model, compared with a toad tryptamine drug group, the analgesic activity of the drug after liposome inclusion is equivalent to that of the drug, and the drug effect time can be prolonged.

3. Effect on expression levels of COX-2 and TNF-alpha mRNA in foot tissue

As shown in fig. 7(a), COX-2mRNA expression was significantly up-regulated in the foot tissues of the formalin inflammatory pain model group (P <0.01) as compared to the blank group; compared with a model group, the morphine hydrochloride positive drug injected into the abdominal cavity can reduce the expression of COX-2mRNA, and has extremely obvious statistical difference (P < 0.01); as above, after four groups of toad tryptamine drug groups are treated, COX-2mRNA expression in foot tissues can be obviously reduced, and the COX-2mRNA expression levels of Free-SA and Lipo-SA administration groups under the same dose are almost consistent.

As shown in fig. 7(B), TNF- α mRNA expression was significantly up-regulated in the foot tissues of the formalin inflammatory pain model group (P <0.01) as compared to the blank group; after modeling, the morphine hydrochloride yang medicine group and four bufotenine medicine treatment groups can reduce the expression of TNF-alpha mRNA in the foot tissues; however, compared with the model group, TNF-alpha mRNA expression was significantly different in only Free-SA45 mg/kg and Lipo-SA45 mg/kg groups (P < 0.01).

Taken together, the above experimental results demonstrate that both free buformin drugs and buformin liposome drugs can reduce the expression levels of COX-2 and TNF-alpha mRNA in the tissues of the feet in the formalin inflammatory pain model.

4. Evaluation of gastrointestinal irritation

During experiments, the home-made toad tryptamine drugs are prepared into concentrations of 13.5mg/mL and 27mg/mL by using pure water, the toad tryptamine liposome freeze-dried powder is prepared into concentrations of 424mg/mL and 848mg/mL, the blank liposome freeze-dried powder is prepared into a concentration of 820mg/mL, each sample test solution is subjected to single administration according to a maximum administration amount of 0.4mL/10g for intragastric administration of a mouse, and physiological saline solution is used as blank control for evaluating the irritation of the high-dose intragastric administration of each drug to be tested on the gastrointestinal tract of the mouse.

On the basis of the preliminary experiments, the exposure period of gastrointestinal irritation after the gastric lavage is set as 6 hours after the administration. The activity of each group of experimental animals was photographically recorded within 2h immediately after each dose. In the observation period, the activity of each animal is observed and recorded in detail, and the existence of the decline of activity, vomiting, diarrhea, even death and the like is counted. After the observation period was completed, the following evaluation results were performed.

4.1 general observations

The animals in each group survived during the observation period of gastrointestinal irritation. The blank control group and the blank liposome group have no abnormality in mental state, behavior activity, secretion condition and the like of each animal. And the reflex behaviors of nausea and vomiting are occasionally seen in the animals with low or high doses of free toad tryptamine given by single intragastric administration, and the phenomena of little movement, dull movement and reduced motility are mainly maintained for half an hour after administration and almost return to normal after one hour. As can be seen from Table 4, the vomiting reflectivities of the free bufotenine low and high dose groups were 25.0% and 62.5%, respectively. Animals in two groups of bufotenine liposome groups had no vomiting reflex, and had no obvious abnormality in general behavior. The results show that the single high-dose intragastric administration of the toad tryptamine medicament after being included by the liposome can reduce the vomiting reflex behavior of the mice.

TABLE 4 evaluation of gavage administration behaviours in groups of mice

Group of	N/only	Dosage (mg/kg)	Vomiting reflectivity
				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 is finished, the anatomical examination finds that the stomach tissue volume of only two Free-SA administration groups is relatively large, the stomach flatulence and the surface capillary vessel micro congestion exist, and certain dose dependence exists; no obvious abnormality was observed in the stomach tissue of the other groups, and the results are shown in FIG. 8. The other organs of the tested animal have no obvious abnormality.

As can be seen from the pathological examination results in FIGS. 9 and 10, the stomach and small intestine tissues of the blank control group and the blank liposome group have no obvious lesion; in the free toad tryptamine high dose group, slight abnormal shape of epithelial cells of the gastric mucosa, widening of muscular layer gaps and increased histopathological score value compared with that of a blank control group are observed (figure 11); the small intestinal mucosa of the Free-SA administration group is obviously infiltrated by inflammatory cells, rotten and shed by epithelial cells, disordered structure and mild fibrosis of cells of a muscle layer, the damage degree has obvious dose correlation, and the pathological score value is obviously higher than that of a blank group (P < 0.01); compared with the equal dose of Lipo-SA and Free-SA, the damage degree of the small intestinal mucosa is obviously reduced.

4.3 comparison of the residual amount of bufotamine in intestinal tissue

As shown in fig. 13, after administration, bufotamine remained in intestinal tissues of mice to cause toxic and side effects, while the amount of drug remained in intestinal tissues was significantly reduced after inclusion with liposomes (Lipo-SA administration group). Therefore, the liposome wraps and changes the tissue distribution of the medicine, reduces accumulation or residue in toxic organs, and improves the safety of the medicine.

The results show that the pathological damage to the stomach and small intestine tissues of the mice can be obviously alleviated after the toad tryptamine medicament is included by the liposome.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications or equivalents may be made to the technical solution without departing from the principle of the present invention, and these modifications or equivalents should also be regarded as the protection scope of the present invention.

Claims

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;

(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.