CN114588137B

CN114588137B - Application of triclosan in preparation of medicines for killing parasitic nematodes

Info

Publication number: CN114588137B
Application number: CN202210284583.XA
Authority: CN
Inventors: 张念章; 徐媛滨; 付宝权; 李文卉; 王萌; 李婷婷; 曲自刚; 王金磊
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2022-03-22
Filing date: 2022-03-22
Publication date: 2023-04-21
Anticipated expiration: 2042-03-22
Also published as: CN114588137A

Abstract

The invention provides an application of triclosan in preparing a medicine for killing parasitic nematodes, belongs to the technical field of medicinal chemistry, and has the advantages of obvious lethal effect on the nematode in each period, low dosage, high safety, and important reference value for developing a new medicine for killing the nematodes, wherein triclosan can be used as one of candidate molecules for treating the nematode diseases.

Description

Application of triclosan in preparation of medicines for killing parasitic nematodes

Technical Field

The invention relates to the technical field of pharmaceutical chemistry, in particular to application of triclosan in preparation of a medicine for killing parasitic nematodes.

Background

Nematodes belong to the class of nematoda of the phylum linear, and are parasitic to humans, mostly intestinal parasites, such as hookworms, roundworms, whipworms, pinworms, and the like, and sometimes intestinal and tissue parasites, such as trichina, adults are parasitic to the intestinal tract and larvae are parasitic to muscles. Different nematode parasitic human bodies cause different symptoms, such as trichinosis is a parasitic disease of both human and animals, is also a wide-range food-borne parasitic disease, can infect more than 150 mammals including human, has main symptoms of systemic fever, abdominal pain, eyelid edema, myocarditis, systemic muscle pain and the like, and serious patients can even die due to complications, thus causing great harm to human health; for another example, ascariasis affects the digestive and absorptive functions of the small intestine and can also cause mucositis, thereby causing a series of digestive tract symptoms, the patient may exhibit anorexia, dyspepsia, abdominal pain, diarrhea or constipation, and the severely infected child may develop dysplasia.

The current clinical drugs for treating nematodiasis are mainly albendazole and benzimidazole. Albendazole is the preferred anti-trichina drug recommended by the world health organization and the ministry of health of China, and due to the low bioavailability, the drugs can not completely and effectively inhibit and kill the packages of the newly-grown larvae and myolarvae in a host body, if the dosage of the drugs is increased, obvious side effects can occur, and symptoms such as blurred vision, increased intracranial pressure and the like can occur after the patients take the drugs. Currently, albendazole and benzimidazole also have a tendency to develop resistance. Up to now, research has found that various plant extracts have different degrees of killing effect on nematodes, resveratrol has insect repellent effect on new larva and adults of trichina, but has no obvious killing effect on muscle larva in vitro. Therefore, the clinical urgent need is to find safer and more effective therapeutic drugs, find and screen safe and efficient nematicides, which has important significance in both clinical aspects and in the aspect of social and economic development of China.

Disclosure of Invention

The invention aims to provide an application of triclosan in preparing a medicine for killing parasitic nematodes.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an application of triclosan in preparing a medicine for killing parasitic nematodes.

Preferably, the nematodes include one or more of the group consisting of trichina, pig roundworm, horse auxiliary roundworm, dog toxoplasma, shi Shibei roundworm, chicken roundworm, heterodera, ma Jianwei nematoda, heterodera, rabbit bolt tail nematoda, mouse enterobia, trichostrongylus, strongyloides, net tail nematoda, strongyloides guanariensis, haemonchus contortus, haemonchus prandii, ora spinosa, caenorhabditis elegans, rochanteria, sucking nematodes, meloidogyne incognita, meloidogyne, ma Sizhuang nematodes, deer filarial, ceros, filarial, heartworm and serous filarial.

Preferably, the nematode is a parasitic nematode of humans and animals.

Preferably, the trichina is one or more of myolarvae, novacells or adults.

Preferably, the effective action concentration of triclosan is 0.02 to 0.2mM.

Preferably, the medicament comprises triclosan as an active ingredient and pharmaceutically acceptable excipients.

Preferably, the active ingredient of the medicament further comprises other compounds which kill parasitic nematodes.

Preferably, the compound comprises one or more of octyl gallate, ethyl 4-aminobutyrate hydrochloride, pyrrole-2-carboxylic acid, indole-3-acetamide, estradiol benzoate, o-hydroxyphenylacetic acid, spermidine hydrochloride, cinnamyl alcohol, 1, 5-pentanediamine.

Preferably, the dosage form of the medicament comprises granules, water-dispersible granules, capsules, tablets or emulsions.

The beneficial effects of the invention are as follows:

the invention provides an application of triclosan in preparing a medicine for killing parasitic nematodes, which has remarkable killing effect on the nematodes in different development periods, low dosage and high safety, can be used as one of candidate molecules for treating nematode diseases, and has important reference value for developing new medicines for killing the nematodes.

Drawings

FIG. 1 is a graph of the effect of 0.2mM triclosan on the morphology of the trichina muscle larvae for different times (40X), A: adding triclosan anterior muscle larva morphology; b: muscle larva morphology at 24 h; c: muscle larva morphology at 48 h; d: muscle larva morphology at 72h;

FIG. 2 is a graph of the effect (40X) of 0.2mM triclosan on the morphology of adult triclosan before and after in vitro action, A: the morphology of adult triclosan added; b: adding triclosan and then forming the adult trichina;

FIG. 3 is a graph of the effect of 0.2mM triclosan on the morphology of the newly grown larvae before and after in vitro action (40X), A: the morphology of the newly grown larvae before addition of triclosan; b: the morphology of the new-born larvae after triclosan addition.

Detailed Description

In the present invention, the nematodes preferably include one or more of the group consisting of trichina, pig roundworm, horse-bound roundworm, dog toxoplasma, shi Shibei roundworm, chicken roundworm, heterodera, ma Jianwei, heterodera, rabbit-tailed nematode, mouse-pinworm, trichostrongylus, strongyloides, net-tailed nematode, filiform net-tailed nematode, guand-strongylus, haemonchus bovinsis, haemonchus prandii, ora-spinosa, praecox, sheep-mouth nematode, dog-mouth nematode, pig-tail nematode, rochanter, kokumi sucking nematode, meloidogyne, ma Sizhuang nematode, deer-tail nematode, cercosis, filarial, canine heartworm, and pig serous silk worm. The invention selects 10 compounds: triclosan, octyl gallate, 4-aminobutyric acid ethyl ester hydrochloride, pyrrole-2-carboxylic acid, indole-3-acetamide, estradiol benzoate, o-hydroxyphenylacetic acid, spermidine hydrochloride, cinnamyl alcohol and 1, 5-pentanediamine, and the results show that triclosan has killing effect on trichina in different development periods. The nematode is preferably a human and animal parasitic nematode. In the present invention, the effective action concentration of triclosan is preferably 0.02 to 0.2mM; the trichina is preferably one or more of myolarvae, neolarvae or adults. In the present invention, the medicament preferably comprises triclosan as an active ingredient and pharmaceutically acceptable excipients, and the active ingredient of the medicament preferably further comprises other compounds for killing parasitic nematodes, and the compounds preferably comprise one or more of octyl gallate, ethyl 4-aminobutyrate hydrochloride, pyrrole-2-carboxylic acid, indole-3-acetamide, estradiol benzoate, o-hydroxyphenylacetic acid, spermidine hydrochloride, cinnamyl alcohol and 1, 5-pentanediamine. In the present invention, the dosage form of the drug preferably includes granules, water-dispersing agents, capsules, tablets or emulsions. The auxiliary materials can comprise one or more of diluents, colorants, sweeteners, coating agents, binders, absorbents, disintegrants, release agents, dispersants, wetting agents, cosolvents, buffering agents and surfactants.

In the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise.

The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

1 materials and methods

1.1 laboratory animals, trichina species, materials

1.1.1 laboratory animals: rats, kunming rats, were purchased from animal centers of the animal institute of Lanzhou veterinary research, national academy of agricultural sciences.

1.1.2 Trichinella spiralis species: the trichina used in this study was trichina Henan strain (Trichinella spiralis, T1), and Kunming mice were passaged for seed protection.

1.1.3 reagents: the 10 active compounds used in the experiment were respectively: octyl gallate (mr 282.33); ethyl 4-aminobutyrate hydrochloride (mr 153.61); pyrrole-2-carboxylic acid (mr 111.1); indole-3-acetamide (mr 174.2); estradiol benzoate (mr 376.49): purity was 97.74%; triclosan (mr289.54): purity > 97%; o-hydroxyphenylacetic acid (mr 152.15); 1, 5-pentanediamine (Mr175.1); spermidine hydrochloride (mr 254.63); cinnamyl alcohol (mr 134.18). All active compounds were purchased from Selleck biotechnology limited, usa. Stock solutions of 10 active compounds were prepared in dimethyl sulfoxide (DMSO), the initial concentrations of the 10 active compounds were 10mM, and stored in a-80 ℃ refrigerator for use. All working concentrations were diluted by RPMI-1640 medium.

1.2 separation of the adult, newborn and myolarvae of the trichina

Collecting laboratory seed-protecting Kunming mice, removing neck, killing, removing limbs, head, tail, viscera and adipose tissue, separating muscle, cutting, and digesting muscle by pepsin to obtain muscle larva. 1 ten thousand trichina infected by each SD rat by oral drenchingMyolarvae were sacrificed 5d after infection, small intestines were dissected rapidly, and the wales were dissected and cut into 2cm sections. The intestinal segment is washed 3 times by using sterile physiological saline, then the small intestinal segment is laid on filter cloth, and the sterile physiological saline is added to submerge the small intestine. After incubation of the small intestine in an incubator at 37 ℃ for 4 hours, the supernatant was slowly decanted and the adults were collected. The collected adults were placed in a medium containing an antibiotic (200U/mL penicillin, 200. Mu.g/mL streptomycin) and 20% fetal bovine serum in a culture medium of Roswell Park Memorial Institute-1640 (RPMI-1640), 5% CO at 37 ℃C ₂ Incubating for 12h, filtering with 200 mesh sieve to separate new larva, centrifuging for 5min at 2000r/min, and discarding supernatant to obtain new larva.

1.3 method

The collected adult trichina, newborn larvae and myolarvae were cultured in RPMI-1640 medium (containing 200U/mL penicillin, 200 μg/mL streptomycin and 20% fetal bovine serum) using 96-well microtiter plates, and 100 trichina and dilutions of each drug were added to each well. The specific method comprises the following steps: each drug was diluted in a gradient of 0.2mM, 0.1mM, 0.05mM, 0.01mM, and a control blank, RPMI-1640 medium (containing 200U/mL penicillin, 200. Mu.g/mL streptomycin, and 20% fetal bovine serum), was set up as the final concentration of DMSO in the maximum drug concentration of the test group in the same test, in triplicate. At 37℃5% CO ₂ The medium-incubation muscle larvae are 24, 48 and 72 hours; adults and new larvae were incubated for 24h, and trichina in each well was observed and counted with an inverted microscope. Mortality = average number of dead insects/average total number of insects x 100%. The insect body is C-shaped or linear, or observed for more than 30s, and has no movement signs, so that death of the insect body can be judged.

1.4 statistical analysis

Data were treated with SPSS 26.0 and differential significance analysis was performed using analysis of variance, expressed as mean ± Standard Deviation (SD). And after statistical treatment, if the P value is smaller than 0.05, the difference is considered to be obvious, and the statistical significance is realized.

2 results

2.1 In vitro assay of nematicidal Activity of 10 active Compounds against myolarvae

The in vitro anti-nematode activity of 10 active compounds against myolarvae is shown in table 1.

TABLE 1 lethality of 10 active compounds at different concentrations to muscle larva in vitro for different times

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Delta mean ± standard deviation, +p < 0.05 compared to the blank control, "-" represents control.

The results in table 1 and fig. 1 show that triclosan has a significant lethal effect on muscle larvae, and that the lethal effect of triclosan on muscle larvae is positively correlated with dose and time. Compared with the control group, when the medicine is added for 24 hours, 10 active compounds have no obvious lethal effect on muscle larvae, the muscle larvae curl, the internal structure is clear, the epidermis structure is complete, and the insect vigor is good (A in figure 1); at 24h, the muscle larvae no longer spiral, the body turns straight, the vitality declines, and peristalsis is seen in the body part (B in figure 1); at 48h, only triclosan showed significant mortality, dead worms were dead in a "C" shape, no signs of activity were found in the worm observations for 30s (C in FIG. 1), and mortality to worms increased significantly from 75.6% to 90.1% when the concentration was increased from 0.02mM to 0.2mM (P < 0.05); at 72h, the mortality rate of triclosan to muscle larvae is increased from 89.3% at 0.02mM to 97.7% at 0.2mM, the larvae show obvious blackening and rigidification (D in figure 1), the mortality rate of the cinnamyl alcohol to the muscle larvae can reach 63.8% at the concentration of 0.2mM, and the concentrations of other active compounds have no obvious killing effect on the muscle larvae of the trichina at all gradients of the concentration.

2.2 In vitro assay of anti-nematode Activity of 10 active Compounds on adults

The in vitro nematicidal effect of 10 active compounds on adults is shown in Table 2.

TABLE 2 mortality after 24h of in vitro action of 10 active Compounds at different concentrations on adults

As shown by the results of Table 2 and FIG. 2, only octyl gallate and triclosan have strong lethal effects on adults, and the mortality rates after 24 hours of action of the triclosan concentrations of 0.02mM, 0.05mM, 0.1mM and 0.2mM are 93.7%, 99%, 100% and 100% respectively; the adult insects were linear after 24h of action and were stiff (fig. 2). Mortality rate reaches 100% (P < 0.05) after 24 hours of 0.1mM octyl gallate and 0.2mM octyl gallate; at concentrations of 0.1mM and 0.2mM, the adults all died, with mortality significantly higher than that of the control group (P < 0.05). While the other 8 active compounds had no obvious lethal effect on adult trichina at concentrations of 0.02mM, 0.05mM, 0.1mM, 0.2mM.

Analysis of in vitro anti-nematode Activity of 2.310 active Compounds on New-born larvae

The in vitro nematicidal effect of 10 active compounds on newborn larvae is shown in Table 3.

TABLE 3 mortality after 24 in vitro effects of 10 active Compounds at different concentrations on neonatal larvae

As shown in Table 3 and FIG. 3, 10 active compounds had obvious lethal effects on newly born larvae of triclosan after 24 hours of treatment at concentrations of 0.02mM, 0.05mM, 0.1mM and 0.2mM, the mortality rate was significantly higher than that of the control group (P < 0.05), and the larvae were stiff after 24 hours of treatment at a concentration of 0.2mM (B in FIG. 3).

The overall result shows that triclosan has obvious lethal effect on the trichina in each period. Octyl gallate has obvious lethal effect on adult and new-born larvae of artemia. 4-aminobutyric acid ethyl ester hydrochloride, pyrrole-2-carboxylic acid, indole-3-acetamide, estradiol benzoate, o-hydroxyphenylacetic acid, spermidine hydrochloride, cinnamyl alcohol and 1, 5-pentanediamine 8 compounds have obvious lethal effect on new larvae of the trichina. The invention observes that dead trichinae are in a regular C shape, dead nematodes appear in a short period, particularly octyl gallate and triclosan show remarkable lethal effect on adults and newborn larvae after 24 hours of action at high concentration (0.1 mM and 0.2 mM), and triclosan also shows remarkable lethal effect on myolarvae after 48 hours; in contrast, control parasites at the same time and concentration were very low lethal. Therefore, triclosan can be used as one of candidate molecules for treating trichinosis, and has important reference value for developing new trichina medicines.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. Application of triclosan in preparing medicines for killing trichina is provided.

2. The use according to claim 1, wherein the trichina is one or more of a myolarva, a neolarva or an adult.

3. The use according to claim 1, wherein the triclosan has an effective concentration of 0.02 to 0.2mM.

4. The use according to claim 1, wherein the medicament comprises triclosan as an active ingredient and pharmaceutically acceptable excipients.

5. The use according to claim 4, wherein the pharmaceutical dosage form comprises a granule, a water-dispersible agent, a capsule, a tablet or an emulsion.