阅读说明：本技术 阿魏倍半萜内酯在制备抗血吸虫病药物中的应用 (Application of asafetida sesquiterpene lactone in preparation of anti-schistosomiasis drugs ) 是由 赵红梅 陈英琼 苏明杰 于 2021-07-06 设计创作，主要内容包括：本发明公开了阿魏倍半萜内酯在制备抗血吸虫病药物中的应用,其中所述阿魏倍半萜内酯的浓度为10～100mg/mL,所述血吸虫为日本血吸虫。本发明首次验证了来源于阿魏的阿魏倍半萜内酯在体内外均具有杀灭日本血吸虫的成虫和童虫的作用,在体内的最高减虫率可达61.26％,并且减雌作用明显,同时阿魏倍半萜内酯还具有高效杀灭体内日本血吸虫虫卵的作用,即所述阿魏倍半萜内酯对日本血吸虫的不同发育阶段均具有显著的杀灭效果,因此可将其制备成抗血吸虫病药物,为开发新型防控血吸虫病药物提供了重要的理论基础,具有广阔的市场应用前景和应用价值。(The invention discloses an application of asafetida sesquiterpene lactone in preparation of anti-schistosomiasis drugs, wherein the concentration of the asafetida sesquiterpene lactone is 10-100 mg/mL, and schistosoma japonicum is schistosoma japonicum. The invention verifies that the asafetida sesquiterpene lactone derived from asafetida has the effect of killing adult and schistosoma japonicum both in vivo and in vitro, the maximum in vivo reduction rate can reach 61.26%, the effect of reducing females is obvious, and the asafetida sesquiterpene lactone also has the effect of efficiently killing schistosoma japonicum eggs in vivo, namely the asafetida sesquiterpene lactone has obvious killing effect on different development stages of schistosoma japonicum, so that the asafetida sesquiterpene lactone can be prepared into the anti-schistosomiasis drug, provides an important theoretical basis for developing novel anti-schistosomiasis-controlling drugs, and has wide market application prospect and application value.)

1. Use of asafetida sesquiterpene lactone in preparing medicine for resisting schistosomiasis is provided.

2. The use according to claim 1, wherein the concentration of asafetida sesquiterpene lactones is 10-100 mg/mL.

3. The use of claim 1, wherein the schistosoma japonicum is schistosoma japonicum.

4. The use according to claim 3, characterized in that the asafetida sesquiterpene lactones have an in vivo and/or in vitro anti-schistosoma japonicum adult worms effect.

5. Use according to claim 3, characterized in that the asafetida sesquiterpene lactones have an in vivo and/or in vitro anti-schistosoma japonicum schistosomula effect.

6. The use according to claim 3, wherein the asafetida sesquiterpene lactones have a killing effect on Schistosoma japonicum eggs.

7. The use according to any one of claims 1 to 6, wherein the extraction process of asafetida sesquiterpene lactones comprises the following steps: taking ferula asafoetida roots, performing reflux extraction by using 95% ethanol, combining and concentrating the filtrates, adding water to dilute the filtrate until the alcohol concentration is 80%, extracting the filtrate by using petroleum ether, and recovering the solution until no alcohol smell exists, thereby obtaining an extract; dissolving the extract in water, extracting with ethyl acetate, and then NaHCO₃Solution extraction, washing to neutrality and concentration to obtain the asafetida sesquiterpene lactone.

8. The use of claim 1, wherein the medicament is in a dosage form comprising: injections, oral agents, buccal tablets, sprays, suspensions, capsules and/or suppositories.

9. The use of claim 8, wherein the medicament further comprises a pharmaceutically acceptable excipient.

10. The use of claim 9, wherein the medicament further comprises adjuvants: sodium carboxymethyl starch, beta-cyclodextrin, aluminum hydroxide gel, talcum powder, magnesium stearate and stevioside.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of asafetida sesquiterpene lactone in preparation of anti-schistosomiasis drugs.

Background

Schistosomiasis (schistosomiasis) is a parasitic disease which is co-suffering from human and livestock, seriously harmful and easy to neglect and is one of six major tropical diseases determined by WHO (blood fluke and blood fluke larva) caused by infecting human or other mammals. According to the statistics of the world health organization, as far as 2016, schistosomiasis is found in at least 76 countries around the world, and nearly 2 hundred million people need to be treated prophylactically. The most popular and serious schistosomiasis species are 5 species, which are schistosoma japonicum, schistosoma mansoni, schistosoma japonicum, schistosoma meibomianum and schistosomiasis interna. The etiology of the schistosomiasis prevalent in our country is schistosoma japonicum, which makes human or animals produce liver and intestinal fibrosis, thus seriously affecting the health and economic development of human beings.

The research on the drugs for treating schistosomiasis begins with 1918 years of antimony potassium tartrate, and dozens of non-antimony drugs such as nitrofuran, nicardipinazole and the like are developed later, but are abandoned due to long curative effect, poor effect, great side effect and the like. Praziquantel which is researched and developed abroad in the early 70 th of the 20 th century is a first choice medicine for treating schistosomiasis because of good insecticidal effect, small side effect, short treatment course and low price, and has good killing effect on 5 kinds of schistosomiasis which infect human bodies, and is widely applied to clinic. In the early 80 s of the 20 th century, researchers found that artemisinin has the activity of killing schistosoma, and later found that various derivatives of artemisinin such as artemether, arteether, artemether, artephenyl ester, dihydroartemether, reduced artemisinin and the like all have a certain inhibition effect on schistosoma, but artemisinin and derivatives thereof have a certain toxicity on early embryos. Meanwhile, treatment of schistosomiasis with artemisinin and its derivatives can be troublesome. Therefore, it is very important to develop new anti-schistosomiasis drugs to replace or combine with praziquantel for schistosomiasis treatment.

The resina Ferulae is resin of Umbelliferae Ferula sinkiangensis K.M.Shen or Fukang Ferula furcana.fukanensis K.M.Shen. The asafetida has high medicinal value, is bitter, pungent and warm in taste, enters spleen and stomach channels, and has the effects of helping digestion, eliminating mass, dispersing distention and killing parasites. The asafetida sesquiterpene lactone is one of asafetida extracts, and provides theoretical data and material basis for developing environment-friendly schistosomiasis prevention and control drugs by researching the in-vivo and in-vitro killing and immunoregulation effects of the asafetida sesquiterpene lactone on schistosoma japonicum.

Disclosure of Invention

The invention aims to provide application of asafetida sesquiterpene lactone in preparation of anti-schistosomiasis drugs, and researches show that asafetida sesquiterpene lactone, one of asafetida extracts, has excellent killing effect on both in-vitro cultured and in-vivo cultured schistosomiasis japonica, can effectively kill adults and schistosomiasis japonica, and can also effectively kill ova of schistosomiasis japonica, so that the asafetida sesquiterpene lactone can be prepared into the anti-schistosomiasis drugs, provides an important theoretical basis for development of novel anti-schistosomiasis drugs, and has wide application value.

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

the invention provides an application of asafetida sesquiterpene lactone in preparation of anti-schistosomiasis drugs.

Further, the concentration of the asafetida sesquiterpene lactone is 10-100 mg/mL.

Further, the schistosoma japonicum is schistosoma japonicum.

Further, the asafetida sesquiterpene lactone has the function of resisting Schistosoma japonicum adults in vivo and/or in vitro.

Further, the asafetida sesquiterpene lactone has the effect of resisting schistosoma japonicum schistosomulum in vivo and/or in vitro.

Further, the asafetida sesquiterpene lactone has the function of killing schistosoma japonicum eggs.

Further, the extraction method of the asafetida sesquiterpene lactone comprises the following steps: taking ferula asafoetida roots, performing reflux extraction by using 95% ethanol, combining and concentrating the filtrates, adding water to dilute the filtrate until the alcohol concentration is 80%, extracting the filtrate by using petroleum ether, and recovering the solution until no alcohol smell exists, thereby obtaining an extract; dissolving the extract in water, extracting with ethyl acetate, and then NaHCO₃Solution extraction, washing to neutrality and concentration to obtain the asafetida sesquiterpene lactone.

Further, the dosage form of the medicament comprises: injections, oral agents, buccal tablets, sprays, suspensions, capsules and/or suppositories.

Furthermore, the medicine also comprises pharmaceutically acceptable auxiliary materials.

Further, the medicine also comprises auxiliary materials: sodium carboxymethyl starch, beta-cyclodextrin, aluminum hydroxide gel, talcum powder, magnesium stearate and stevioside.

Compared with the prior art, the invention has the beneficial effects that: the invention verifies that the asafetida sesquiterpene lactone derived from asafetida has the effect of killing adult and schistosoma japonicum both in vivo and in vitro, the maximum in vivo reduction rate can reach 61.26%, the effect of reducing females is obvious, and the asafetida sesquiterpene lactone also has the effect of efficiently killing schistosoma japonicum eggs in vivo, namely the asafetida sesquiterpene lactone has obvious killing effect on different development stages of schistosoma japonicum, so that the asafetida sesquiterpene lactone can be prepared into the anti-schistosomiasis drug, provides an important theoretical basis for developing novel anti-schistosomiasis-controlling drugs, and has wide market application prospect and application value.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the 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.

Example 1

1. Extraction of asafetida sesquiterpene lactone

Extracting resina Ferulae with 95% ethanol under reflux for three times, mixing the three filtrates, concentrating, diluting with water to ethanol concentration of 80%, extracting with petroleum ether for three times, recovering the solution to obtain extract, dissolving the extract with water, extracting with ethyl acetate to obtain extractive solution, inoculating with 5% NaHCO₃Extracting with water solution for three times, washing with water to neutrality, concentrating, drying, grinding, and sieving to obtain powdered asafetida sesquiterpene lactone.

2. Killing effect of asafetida sesquiterpene lactone on in-vitro culture of adult schistosoma japonicum and schistosomulum

The method comprises the following steps: after the young mouse is infected with Schistosoma japonicum cercaria for 18 days, the schistosoma japonicum schistosomula is collected by means of hepatic portal vein perfusion, and after 35 days, the adult schistosoma japonicum katomula is collected by means of the same method. Respectively culturing the trichlorfon and the adults in vitro in DMEM culture solution, respectively adding asafetida sesquiterpene lactone with the concentration of 10mg/ml, 20mg/ml and 30mg/ml and praziquantel solution with the concentration of 30mg/ml, continuously culturing for 3 days, and setting up a blank control group only added with distilled water. Observing the activity and death condition of the worm, calculating the death rate and activity reduction rate of the schistosome, scanning the sample by using a scanning electron microscope, and observing the epidermis of the worm.

As a result: (1) the results on the adults showed that the adults survived well in the media without asafetida sesquiterpene lactones (placebo); in the culture solution containing asafetida sesquiterpene lactone with different concentrations, the polypide has spasm in different degrees, and the death rate and activity reducing rate of schistosome are obviously raised with the time. The asafetida sesquiterpene lactone with the concentration of 10mg/ml, 20mg/ml and 30mg/ml has the effect of killing imagoes.

Wherein after 30mg/ml of asafetida sesquiterpene lactone acts for 24 hours, the death rate and activity reduction rate of the polypide respectively reach 30.0% and 67.78%, and the activity score of the polypide has statistical significance (p is less than 0.05) compared with the difference of a blank control group; after 48 hours of action, the death rate and the activity reduction rate of the worm bodies reach 46.7 percent and 66.67 percent, and the activity value has statistical significance (p is less than 0.05) compared with the difference of a control group. The mortality rate and the activity reduction rate of the worms after 24 hours of action of the praziquantel solution with the same concentration are respectively 36.7 percent and 68.89 percent, and the mortality rate and the activity reduction rate of adults after 48 hours of action are respectively 53.3 percent and 71.11 percent, which indicates that the action effect of the asafetida sesquiterpene lactone is similar to that of the praziquantel which is a common medicament in the field. And the death rate and the activity reduction rate of the polypide after the ferulic sesquiterpene lactone acts for 72 hours are respectively 60.0 percent and 85.56 percent, compared with the fact that the death rate and the activity reduction rate of the polypide after the praziquantel solution acts for 72 hours are only 56.7 percent and 86.67 percent, namely after the ferulic sesquiterpene lactone acts for 72 hours, the insecticidal effect of the ferulic sesquiterpene lactone is slightly better than that of the praziquantel. The above results demonstrate that asafetida sesquiterpene lactone has the effect of resisting Schistosoma japonicum adult in vitro.

(2) The results on the juvenile insects show that 10mg/ml, 20mg/ml and 30mg/ml asafetida sesquiterpene lactone reagents have excellent killing effect on the juvenile insects within 24h, 48h and 72h, and the activity scores have statistical significance (p is less than 0.05) compared with the differences of a control group. Wherein the death rate and the activity reduction rate of the polypide after the culture solution of asafetida sesquiterpene lactone with the concentration of 30mg/ml acts for 24 hours are respectively 46.7 percent and 75.56 percent, and the effect is similar to that of a praziquantel reagent with the same concentration, namely the asafetida sesquiterpene lactone has the effect of schistosoma japonicum trichurian cultured in vitro by an antibody.

3. Killing effect of asafetida sesquiterpene lactone on schistosoma japonicum cultured in mouse body

The method comprises the following steps: the young mice are infected with Schistosoma japonicum cercaria and administered at 18d and 35d after infection, respectively. The experimental components are asafetida sesquiterpene lactone groups with the concentration of 10mg/ml, 20mg/ml and 30mg/ml and praziquantel solution groups with the concentration of 30mg/ml, and a blank control group is additionally arranged. The mice were dissected 7 weeks after administration, the number of worms was collected from the hepatic portal vein, the average total number of worms, the average number of female worms and the average number of worm eggs in different treatment groups after administration for 18 days and 35 days were counted, the statistical results are shown in tables 1 and 2, the reduction rate and the reduction rate of female worms in the experimental group compared with the blank control group were calculated, and the reduction rate of eggs in the liver digestion was calculated, and the results are shown in table 3. Statistical analysis of the groups using the sps statistical analysis software, analysis of statistical significance of differences between groups using the ANOVA test (II)^*P＜0.05，^**P＜0.01)。

TABLE 1 influence of different treatment groups on Schistosoma japonicum (18d)

Note: p < 0.05 in comparison with the control blank, P < 0.01 in comparison with the control blank

TABLE 2 influence of different treatment groups on Schistosoma japonicum (35d)

Note: p < 0.05 in comparison with the control blank, P < 0.01 in comparison with the control blank

TABLE 3 reduction of insect, female and egg reduction rates of the different treatment groups

And (4) analyzing results:

(1) influence of asafetida sesquiterpene lactone on total number of schistosoma japonicum cultured in mouse

The results of acting on young mice infected with schistosoma japonicum 18d show that the reduction rates of the asafetida sesquiterpene lactone group and the praziquantel group with different concentrations in mice are 22.59%, 43.41%, 55.20% and 55.48% respectively. The results of acting on young mice infected with schistosoma japonicum 35d show that the reduction rates of the asafetida sesquiterpene lactone group and the praziquantel group with different concentrations in the schistosoma japonicum in mice are 36.59%, 61.26%, 69.87% and 71.85% respectively. Namely, the asafetida sesquiterpene lactone has high insect-reducing effect similar to that of praziquantel.

Respectively counting the data of adult and schistosomulum periods of schistosoma japonicum in mice treated by each group, wherein the results show that the asafetida sesquiterpene lactone has excellent killing effect on the schistosomulum periods and schistosomulum periods of the mice in comparison with a blank control group, the higher the dosage is, the higher the insect reduction rate is, the more obvious the effect is, and the insect reduction rate is relatively higher after the asafetida sesquiterpene lactone is used in the adult period.

(2) Influence of asafetida sesquiterpene lactone on female schistosoma japonicum cultured in mouse

The results of acting on young mice infected with schistosoma japonicum 18d showed that the female-reducing rate of the asafetida sesquiterpene lactone group and the praziquantel group with different concentrations were 15.05%, 42.09%, 58.02% and 59.65%, respectively. The results of acting on young mice infected with schistosoma japonicum 35d showed that the female-reducing rate of the asafetida sesquiterpene lactone group and the praziquantel group with different concentrations were 40.62%, 64.85%, 72.86% and 74.59%, respectively. Namely, the asafetida sesquiterpene lactone has high-efficiency hypoestrogenic effect, and the effect is similar to that of praziquantel.

Respectively counting the data of adult and schistosomulum periods of schistosoma japonicum in mice treated by each group, wherein the results show that the asafetida sesquiterpene lactone has excellent hypoestrogenic effect on the adult and schistosomulum periods of schistosomulum in mice compared with a blank control group, and the hypoestrogenic effect is more obvious when the dosage is higher and the hypoestrogenic effect is higher, wherein the hypoestrogenic effect is relatively higher when the asafetida sesquiterpene lactone is used in the adult period.

(3) Action of asafetida sesquiterpene lactone on schistosoma japonicum eggs cultured in mice

The results of acting on young mice infected with schistosoma japonicum 18d showed that the oviposition reduction rates of the asafetida sesquiterpene lactone group and the praziquantel group at different concentrations were 17.32%, 35.32%, 47.95% and 49.99%, respectively. The results of acting on young mice infected with schistosoma japonicum 35d showed that the oviposition reduction rates of the asafetida sesquiterpene lactone group and the praziquantel group at different concentrations were 23.38%, 37.83%, 58.34% and 60.76%, respectively. Namely, the asafetida sesquiterpene lactone has excellent effect of killing schistosome ovum, and the effect is similar to that of praziquantel.

Respectively counting the data of adult and schistosomulum periods of schistosoma japonicum in mice treated by each group, wherein the results show that the asafetida sesquiterpene lactone has excellent effect of killing ova in the adult period and the schistosomulum period of schistosomulum in mice compared with a blank control group, the ova reduction rate is higher when the dosage is higher, the effect is more obvious, and the ova reduction rate is relatively higher when the asafetida sesquiterpene lactone is used in the adult period.

Example 2

According to the experimental results, the asafetida sesquiterpene lactone has high-efficiency and excellent killing effect on the schistosoma japonicum and can directly act on the inside and outside of a body, so that the asafetida sesquiterpene lactone can be prepared into the schistosomiasis resistance medicament, and the specific steps are as follows:

grinding and drying the asafetida sesquiterpene lactone obtained by extraction, sieving, dispersing into uniform powder, adding auxiliary materials such as sodium carboxymethyl starch (CMS-Na), beta-cyclodextrin, aluminum hydroxide gel, talcum powder, magnesium stearate and stevioside according to a proportion, mixing uniformly, and directly tabletting to prepare tablets.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.