CN108354936B

CN108354936B - Application of sanguinarine in preparation of medicine for treating animal trichina

Info

Publication number: CN108354936B
Application number: CN201810251448.9A
Authority: CN
Inventors: 杨桂连; 黄海斌; 王春凤; 刘可
Original assignee: Jilin Agricultural University
Current assignee: Jilin Agricultural University
Priority date: 2018-03-26
Filing date: 2018-03-26
Publication date: 2020-01-03
Anticipated expiration: 2038-03-26
Also published as: CN108354936A

Abstract

The invention discloses an application of sanguinarine in preparing a medicament for treating animal trichinosis, wherein the medicament for treating animal trichinosis is a dimethyl sulfoxide solution of sanguinarine; sanguinarine has strong killing effect on trichina in-vivo and in-vitro adult stages, migration stages and cyst stages, and can reduce pathological damage of intestinal tract and muscle caused by trichina; the incubation period of trichinosis is generally 5-15 days, and the trichinosis mainly aimed at myotrichinosis caused by newly born larvae in a migration period during treatment; in the migration period in vivo, the insect reduction rate of 100ppm sanguinarine orally taken for three consecutive days is 47.2 percent, and the insect reduction effect is similar to that of albendazole; therefore, the sanguinarine can better treat trichinosis, has obvious effect, simple use method and wide application and popularization value.

Description

Application of sanguinarine in preparation of medicine for treating animal trichina

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of sanguinarine in preparation of a medicine for treating animal trichina.

Background

Trichinosis (A)Trichinellosis) Is an important zoonosis caused by trichinella spiralis and an important food-borne parasitic disease. The main mode of infection in humans and animals is caused by the ingestion of animal meat containing trichinella larvae cysts. The main clinical manifestations of the patient are general fever, eyelid edema, skin rash, muscle pain, etc., and the severe patient can die due to arrhythmia, pericardial effusion, heart failure, pulmonary infarction, peritonitis, etc., which seriously jeopardize the health of human and animals and the development of animal husbandry. The disease was first found in London in 1828. The infection of trichina swine trichina is found in 1881 in China, and the first patient is found in Yunnan in 1964. In recent years, the disease is found in many provinces and cities in China, and even a report of outbreak is reported.

At present, the first clinically preferred anti-trichina drug is albendazole also called albendazole, which can selectively and irreversibly inhibit glucose uptake by a worm body, so that endogenous glycogen of the worm body is exhausted, fumarate reductase is inhibited, adenosine triphosphate production is blocked, and the worm body gradually dies due to energy exhaustion. The medicine has the characteristics of broad spectrum, high efficiency, safety, convenience and the like. However, the medicine can cause the animals to have side effects of fever, myalgia, aggravation of edema and the like, and in severe cases, epilepsy, visual disturbance, intracranial pressure rise and even cerebral edema can occur. Therefore, the search and screening of highly active anti-nematode (including trichina) drugs has become an important issue for the development of current animal husbandry veterinarians in our country.

Sanguinarine is a compound extracted and separated from the whole herb of Chelidonium majus, the root tuber of corydalis edulis, the whole herb of Macleaya cordata or the aerial parts of Mesona chinensis, has very wide pharmacological effects, and is found to have anti-inflammatory, antibacterial and antioxidant effects, have anesthetic effects on central nerves, inhibit cholinesterase activity, enhance cardiac activity, stimulate salivary secretion, promote urination and have peripheral anti-adrenergic sympatholytic effects in early researches. Has been used by traditional Chinese medicine and North America for centuries. Known clinical uses are: (1) the composition has definite effects on resisting tumor, treating infectious diseases, treating trichomonas vaginitis and cervical erosion, treating skin diseases, treating acne rosacea, tinea of feet and hands, scabies, psoriasis and scalp eczema; (2) the veterinary drug has the functions of clearing away heat and toxic materials, resisting bacteria and diminishing inflammation, has special effects on various diseases such as yellow scour, white scour, edema disease, paratyphoid, infectious gastroenteritis of pigs and the like, has no drug resistance, residue and side effect, and is safe and reliable to use; (3) the biological pesticide has the functions of killing maggots and mites. In recent years, researches show that sanguinarine also has good and broad-spectrum anti-tumor activity and does not generate any toxic or side effect on normal tissues and cells.

Disclosure of Invention

The invention aims to solve the problem that the existing trichina resisting medicine generates side effect in the treatment process, and provides an application of a high-activity nematocide, namely sanguinarine in preparing a medicine for treating animal trichina.

The application of sanguinarine in preparing medicine for treating animal trichina is provided;

the medicine is a dimethyl sulfoxide solution of sanguinarine;

the structural formula of the sanguinarine is as follows:

the invention provides an application of sanguinarine in preparing a medicament for treating animal trichinosis, wherein the medicament for treating animal trichinosis is a dimethyl sulfoxide solution of sanguinarine; sanguinarine has strong killing effect on trichina in-vivo and in-vitro adult stages, migration stages and cyst stages, and can reduce pathological damage of intestinal tract and muscle caused by trichina; the incubation period of trichinosis is generally 5-15 days, and the trichinosis mainly aimed at myotrichinosis caused by newly born larvae in a migration period during treatment; in the migration period in vivo, the insect reduction rate of 100ppm sanguinarine orally taken for three consecutive days is 47.2 percent, and the insect reduction effect is similar to that of albendazole; therefore, the sanguinarine can better treat trichinosis, has obvious effect, simple use method and wide application and popularization value.

Drawings

FIG. 1 is a scanning electron microscope for observing the damage result of sanguinarine to trichina adults;

FIG. 2 HE staining detection of pathological changes of duodenum of mice in adult stage;

FIG. 3 HE staining detection of tongue pathological changes in mice of each group during migration phase;

FIG. 4 HE staining detection of pathological changes in masseter muscle of mice in each group during migration phase;

FIG. 5 HE staining detection of pathological changes of tongue muscles of mice in cyst formation stage;

FIG. 6 HE staining detects pathological changes of masseter muscle of mice in each group in cyst stage.

Detailed Description

EXAMPLE 1 preparation of a drug for treatment of animal trichina with sanguinarine

1 sanguinarine extraction method

Taking 6kg of Macleaya microcarpa coarse powder, adding 75% ethanol, refluxing in water bath at 65 deg.C for 30min, vacuum evaporating at 70 deg.C, re-suspending with distilled water, and sequentially extracting with petroleum ether (boiling range of 30-60 deg.C), chloroform, ethyl acetate, n-butanol and distilled water under hot reflux in water bath. Concentrating, performing silica gel column chromatography, eluting with petroleum ether-ethyl acetate solvent system, performing column chromatography on the petroleum ether extract obtained by solvent treatment by gradient elution method, performing TLC inspection, and mixing the same or similar components to obtain A, B, C, D, E, F six-segment compounds. And D, performing two-stage and three-stage column chromatography separation to finally obtain four purer compounds with numbers I, II, III and IV. Identifying the structure of monomer by respectively measuring melting point (mp), electron ionization mass spectrometry (EI-MS), proton nuclear magnetic resonance spectrum (1H-NMR), 13C nuclear magnetic resonance spectrum (13C-NMR), DEPT, etc. of the monomer, and measuring sanguinarine content by ultraviolet spectrophotometry

2 identification of Compound III

The characteristics are as follows: the crystal is light yellow needle crystal, and is dissolved in methanol and ethanol solution.

Suggesting that the relative molecular mass of the compound should be 332.

242-243 °C ;

EI-MS (70eV) m/z: 334.3 (M+2), 333.3(M+1), 332.3 (M+), 331.3 (M-1), 315.3 (M-CH3), 304.3, 274.3, 260.3;

¹H NMR (CD3OD, TMS, 400MHz):

8.6 (1H, d, H-4), 8.5(1H, d, H-5), 7.9(1H，d，H-8), 8.2(1H, d, H-9), 7.56 (1H, d, H-11), 6.3 (2H, s, -OCH2O-13), 8.1(1H, d, H-15), 4.9(3H, s, N-Mc), 6.3(2H, s, -OCH2O-20);

¹³C NMR (CD3OD, TMS, 400MHz):

104.3(C-1), 148.2(C-2), 149.5(C-3), 105.0 (C-4), 127.6 (C-5), 129.1 (C-6), 133.2 (C-7), 132.9 (C-8), 111.3 (C-9), 121.9 (C-10), 107.0 (C-11), 150.9 (C-12), 119.6 (C-13), 150.8 (C-14), 106.6 (C-15), 121.2 (C-16), 134.2(C-17), 52.8 (N-Me), 150.8(C-19), 118.3(C-20)。

From the above information it was concluded that compound III is sanguinarine, having the formula C20H14NO 4.

3 sanguinarine purity determination results

Performing spectrum scanning on sanguinarine reference substance solution by ultraviolet-visible spectrophotometry, and selecting 323.0nm as sanguinarine spectrophotometry to obtain detection wavelength. At the wavelength, the absorbance of the pure sanguinarine (Nanjing Guangrun biological products, Ltd., purity > 98%) solution with different concentrations is measured to draw a standard curve. The regression equation Y = -0.02285+4.40771X, r =0.99905 was calculated. The sample has an absorbance mean value of 0.073 at 323.0nm, and the concentration is calculated to be 0.01138mg/mL by substituting the sample into a standard curve regression equation, and the sanguinarine content is 99.48% multiplied by 98% -97.4904%.

Example 2 in vitro insecticidal assay of sanguinarine against Trichinella spiralis

1. Trichina separation

1) Trichina muscle larva separation

The BALB/c mouse is infected with 300 trichina muscle larvae through mouth, the mouse is killed by a cervical vertebra breaking method after 40-60 days, fur and internal organs are removed, muscles are ground, the mouse is placed in a beaker, an artificial digestive juice containing 1% of HCI and 1% of pepsin is added for digestion (the ratio of the muscles to the digestive juice is 1:7), a glass rod is used for stirring for 30min, the mouse is placed in an incubator at 37 ℃ for 16 h, stirring is not carried out regularly, residues are removed by a 60-mesh filter screen after meat residues are completely digested, filtrate is washed by physiological saline at 37 ℃ and 0.9%, the supernatant is poured out after the worm bodies are precipitated for 15-20 min, a proper amount of physiological saline is added for precipitation and washing, the operation is repeated for 3 times, finally, the supernatant is sucked out, the bottom muscle larvae on the cup are recovered, and the vitality is observed under a mirror and counted.

2) Separation of trichina adults

Each adult SD rat is subjected to oral gavage for l0000 trichinella larvae, fasting is performed for 6d, then the rat is dissected under ether anesthesia, the rat is taken out and longitudinally dissected open small intestines, then the small intestines are cut into 2-3 cm sections, the small intestine sections are subjected to swinging washing in sterile physiological saline for 3 times, and residual intestinal contents are removed. Then flatly paving the small intestine section on a filter cloth of a separation sieve of a nematode larva separator, adding physiological saline which is preheated at 37 ℃ until the small intestine is just immersed, placing the small intestine section in an incubator at 37 ℃ for incubation for 4 hours, wherein the adult worms are drilled out from the intestinal wall and enter the physiological saline through the filter cloth; washing the incubation solution containing adult worm with sterile physiological saline containing cyan and streptomycin (I000U/mL) for 2 times, and finally with RPMI-1640 containing cyan, streptomycin (I000U/mL) and 20% calf serum; the culture solution is washed for 1 time to obtain pure trichina imagoes, and the vitality is observed and counted under a microscope.

3) Trichinella spiralis neonatal larva (NBL) isolation

Adding 15 mL of RPMI-1640 culture solution containing penicillin, streptomycin (each L000U/mL) and 20% calf serum into a 75 mL tissue culture bottle, filtering the culture solution containing NBI by using a 280-mesh sterile mesh screen, culturing adults in the screen for 1 time by the same method as the method, pouring the filtered culture solution containing NBL into a sterile centrifuge tube, centrifuging for 5 min at 2000r/min, sucking out supernatant, reserving 1mL of culture solution, mixing uniformly, sucking 10 mu L of culture solution, observing the activity of NBL under a lens, and counting.

2. Sanguinarine in vitro insecticidal action

200 collected worms in three stages are inoculated into a cell culture plate of a prepared culture medium (RPMI-1640 containing 1 percent of double antibody and 20 percent of fetal bovine serum); dissolving sanguinarine with dimethyl sulfoxide (DMSO), and adding sanguinarine solution with final concentration of 3, 5, 7, 9, 11, 13, 15ppm into each pore of imago; adding sanguinarine solution with final concentration of 12, 14, 16, 18, 20, 22, 24, 26, 28, 30ppm into each hole of muscle larva; the neonatal larvae are added with sanguinarine solution with final concentration of 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8 and 3.0ppm respectively per well. Blank control and DMSO control are respectively carried out, and each concentration is carried out in 3 duplicate wells; 37 ℃ and 5% CO₂Culturing for 24h in the environment of (1), and observing the survival condition of each group of trichina under a microscope; worm mortality = (dead worms/total number of worms) × 100%; fixing each group of adult trichina with 3.5% glutaraldehyde, fixing with 1% osmic acid, dehydrating with gradient ethanol, drying at critical point, spraying gold with ion sputtering instrument, and observing injury of sanguinarine to adult trichina with scanning electron microscope.

Data were processed with SPSS 17.0 and presented as mean ± standard deviation, and analysis of significance of differences was performed using analysis of variance.

The external killing effect of sanguinarine on trichinella spiralis adults is shown in table 1, the sanguinarine has a strong killing effect on trichinella spiralis adults, the mortality rate of adults and the sanguinarine concentration are in positive correlation when the sanguinarine concentration is 3-15 ppm, the mortality rate of the trichinella spiralis 100% when the sanguinarine concentration is 15ppm, the mortality rate of 24h LC50 and LC90 are 6.69ppm and 12.89ppm respectively, scanning electron microscopy results are shown in fig. 1, blank control group results are shown in fig. 1A ~ C, sanguine concentration 15ppm group results are shown in fig. 1D ~ F, blank control group worms have uniform epidermal folds (fig. 1A), bell-shaped mating adnexa at the tail end of male worms are composed of a pair of leaf-shaped small pieces, two pairs of mastoids and a genital pore, the leaf-shaped small pieces of the mating adnexa are all extended outwards, erect, mastoid and fully exposed (fig. 1B), longitudinal striata longitudinal striation group worm surface is arranged between the male worms, 15ppm of the mating adnexa cross striata cross-shaped male worm is covered, and a cross striation is covered by a striae fold (fig. 1) and a serious striae mating cross fold and a cross striation fold is covered by a transverse striation graph (1E).

The in vitro killing effect of sanguinarine on trichina newborn larvae is shown in table 2. When the concentration of sanguinarine is 1.4-3 ppm, the death rate of the newly born larva of the trichina rises along with the rise of the concentration of the sanguinarine. The mortality rates for the parasites in the 1.2ppm and 1.4ppm groups were significantly lower than the blank control group, which may be related to the growth promoting effect of low doses of sanguinarine. At a concentration of 3ppm, the mortality rate of the newly born larvae of trichinella was 100%, and the 24-hour LC50 and LC90 were 1.86ppm and 2.67ppm, respectively.

The in vitro killing effect of sanguinarine on trichina muscle larvae is shown in table 3. The result shows that when the concentration of sanguinarine is 12-30 ppm, the death rate of the trichina muscle larvae is increased along with the increase of the concentration of the sanguinarine; compared with a blank control group, the 24-hour mortality rate of the trichina muscle larvae is obviously increased by 18-30 ppm sanguinarine; at a concentration of 30ppm, the rate of trichina larvae mortality was 100% and for 24h LC50 and LC90 was 18.49ppm and 26.42ppm, respectively.

Example 3 Sanguinarine in vivo insecticidal assay against Trichinella spiralis

6 weeks old female BALB/C mice, 18-20g, purchased from Beijing Huafukang Biotech GmbH.

Trichina, which was bred by female Kunming mouse, was maintained by animal parasite laboratory of animal science and technology institute of Jilin agriculture university.

1. Sanguinarine antitrichter trichinesis in vivo experiment

60 female BALB/C mice of 6 weeks old were randomly selected 10 as blank control, and the remaining 50 mice were infected with 300 trichinella larvae each, and were randomly divided into 5 groups (attack group, DMSO control group, albendazole group, 70ppm group, 80ppm group). Mice in each group were orally administered with physiological saline, DMSO in an amount equivalent to that in the 80ppm group, 50mg/kg albendazole, 70ppm and 80ppm sanguinarine, respectively, 24 hours after infection. On day 7 after infection, mice were sacrificed and the adult trichinella was isolated according to the adult worm isolation method in example 1, and the reduction rate was counted and calculated, the reduction rate = (attack worm group worm load-experimental group worm load)/attack worm group worm load x 100%. The mouse duodenum was isolated, sections were fixed in paraffin embedded in 10% formaldehyde solution, HE stained, and pathological changes were observed under light microscopy.

2. In vivo experiment of sanguinarine on larvae in migratory stage of trichina

60 female BALB/C mice of 6 weeks old were randomly selected 10 as blank control, and the remaining 50 mice were infected with 300 trichinella larvae each, and were randomly divided into 5 groups (attack group, DMSO control group, albendazole group, 80ppm group, 100ppm group). Mice in each group were orally administered with physiological saline, DMSO in an amount equivalent to that in the 100ppm group, 50mg/kg albendazole, 80ppm and 100ppm sanguinarine once a day on days 13, 14 and 15 after infection, respectively. Mice sacrificed on day 30 post infection trichinella myolarvae were isolated according to the myolarva isolation method in example 1, and the number of parasites per gram of muscle and the reduction rate = (attack group per gram of muscle and insect-experimental group per gram of muscle and insect) per attack group per gram of muscle and insect and x 100% were counted and calculated. The mouse tongue muscle and the mouse masseter muscle are separated, fixed by 10 percent formaldehyde solution, cut into sections by paraffin embedding, and stained by HE, and pathological changes are observed under a light microscope.

3. Sanguinarine anti-trichina cyst stage larva in vivo experiment

60 female BALB/C mice of 6 weeks old are randomly selected as blank control, and the other 50 female BALB/C mice are infected with 300 trichina muscle larvae each and are randomly divided into 5 groups (attack group, DMSO control group, albendazole group, 150ppm group and 200ppm group); mice in each group were orally administered with physiological saline, DMSO in an amount equivalent to that in the 200ppm group, 50mg/kg albendazole, 150ppm and 200ppm sanguinarine once a day on days 34, 35 and 36 after infection, respectively. Mice sacrificed at 46 days post infection trichinella muscle larvae were isolated according to the muscle larvae isolation procedure of example 1, and the number of parasites per gram of muscle and the reduction rate were counted and calculated, the reduction rate = (attack group per gram of muscle worm charge-experimental group per gram of muscle worm charge)/attack group per gram of muscle worm charge x 100%. The mouse tongue muscle and the mouse masseter muscle are separated, fixed by 10 percent formaldehyde solution, cut into sections by paraffin embedding, and stained by HE, and pathological changes are observed under a light microscope.

Data were processed with SPSS 17 and presented as mean ± standard deviation, and analysis of significance of differences was performed using analysis of variance.

4. Influence of sanguinarine on adult stage, migration stage and cyst stage of trichina

1) Results of the leaf load and the insect reduction rate of mice in three stages of sanguinarine to trichina

Results of the trichinemia load and the reduction rate of the trichinemia of mice in each group of three stages of the trichinemia by sanguinarine are shown in table 2, 70ppm of sanguinarine and 80ppm of the group trichinemia load in the adult stage are obviously reduced compared with the group of the attacking worms, and the reduction rates are respectively 37.2 percent and 36.9 percent. The insect reduction rate of albendazole group (50 mg/kg) reaches 97.7 percent; the insect charge of each treatment group in the migration period is obviously lower than that of an attack group, the insect reduction rate of 80 and 100ppm sanguinarine groups is 26.4 percent and 47.5 percent, the insect reduction rate of an albendazole group is 54.2 percent, and the insect reduction effect of the trichinella trichine; the sanguinarine 150 and 200ppm of the histidinoid load in the cyst formation period are obviously reduced compared with the attack group, and the insect reduction rate is 31.7 percent and 41.2 percent respectively.

TABLE 4 results of trichinella load and reduction rate in three periods for each group of mice

2) Influences of sanguinarine on pathological changes of small intestine, masseter muscle and tongue muscle of mice in imago stage, migration stage and cyst stage of trichinella spiralis

At 7d after infection, taking inflammatory small intestine as pathological section, and observing pathological influence of sanguinarine on the small intestine in the adult stage of trichina under a light microscope after HE dyeing; as shown in figure 2, the small intestine of the attack group and the DMSO group presents the typical pathological characteristics of trichina infection, such as intestinal wall fiber hyperplasia, edema, massive inflammatory cell infiltration, intestinal villus shortening and villus length-to-crypt depth ratio reduction. The pathological changes of the small intestine of the treatment group (70 ppm, 80ppm, albendazole) are obviously improved, infiltration inflammatory cells are reduced, and the intestinal villus form is complete; pathological observation of massonian in migration and cyst formation periods of mice shows that the number of the massonian cysts in attack groups and DMSO groups is large, obvious inflammatory cell infiltration including eosinophilic granulocyte, neutrophil, monocyte and lymphocyte appears around the cysts, and myocyte degeneration, necrosis and striation disappear; the periencysted inflammatory cell infiltration of each group treated by sanguinarine and albendazole is obviously reduced, and the pathological injury of muscle cells is obviously lighter than that of the former group.

From the above results, it can be seen that: sanguinarine has strong killing effect on trichina in-vivo and in-vitro adult stages, migration stages and cyst stages, and can reduce pathological damage of intestinal tract and muscle caused by trichina; the incubation period of trichinosis is generally 5-15 days, so that the trichinosis mainly aimed at the myotrichinosis caused by newly grown larvae in the migration period during treatment; the test result of the in vivo migration period shows that the insect reduction rate of 100ppm sanguinarine orally taken for three consecutive days is 47.2 percent, and the insect reduction effect is similar to that of albendazole. Therefore, the sanguinarine can better treat trichinosis, has obvious effect, simple use method and wide application and popularization value.

Claims

1. Application of sanguinarine as sole activity in preparing medicine for treating animal trichina is provided.

2. The use of sanguinarine as sole activity in the manufacture of a medicament for the treatment of trichina in an animal according to claim 1, wherein: the medicine is a dimethyl sulfoxide solution of sanguinarine.

3. Use of sanguinarine as sole activity in the preparation of a medicament for the treatment of trichinella in an animal according to claim 1 or 2, characterized in that: the structural formula of the sanguinarine is as follows:

。