CN113559128A

CN113559128A - New use of maggot for improving sleep

Info

Publication number: CN113559128A
Application number: CN202010348119.3A
Authority: CN
Inventors: 黄玉水
Original assignee: Zhejiang Baiketang Biotechnology Co ltd
Current assignee: Zhejiang Baiketang Biotechnology Co ltd
Priority date: 2020-04-28
Filing date: 2020-04-28
Publication date: 2021-10-29
Anticipated expiration: 2040-04-28
Also published as: CN113559128B

Abstract

The present invention relates to the use of maggots or an extract thereof for improving, preventing and/or treating sleep disorders. The invention also provides a pharmaceutical composition for improving, preventing and/or treating sleep disorders, which comprises the maggot or an extract thereof and calcium levofolinate.

Description

New use of maggot for improving sleep

Technical Field

The invention relates to a new application of an anti-cancer drug, namely maggot, in particular to an application of maggot or an extract thereof in preparing a drug for improving sleep.

Background

Insomnia is a condition of difficulty falling asleep and/or of persistent feeling of insufficient sleep with no apparent cause, possibly due to a variety of emotional and physical ailments. Insomnia is the most common sleep disorder affecting millions of people as either a primary or co-morbid condition. Sleep disorders include six major classes: (1) insomnia (insomnia), (2) hypersomnia, (3) sleep abnormality (parasomnia), (4) circadian rhythm sleep-wake disorder (cyclic rhythm-wake disorder), (5) sleep-related breathing disorder (sleep-related breathing disorder), and (6) sleep movement disorder (sleep movement disorder). Sleep disorders produce significant distress and/or dysfunction in people suffering from the condition, requiring appropriate treatment.

The international sleep foundation has shown, in the research data for the elderly, that sleep disorders are positively associated with various diseases accompanied by aging of the human body, and can increase the risk of the onset of age-related chronic diseases such as congestive heart failure, renal failure, chronic obstructive pulmonary disease, stroke, parkinson's disease, diabetes, and the like. The subtypes of insomnia include insomnia with difficulty in falling asleep, insomnia with difficulty in maintaining sleep, early-awakening insomnia and psychogenic insomnia, and the insomnia with difficulty in maintaining sleep and the early-awakening insomnia are common in the elderly. According to the course of sleep disorder, insomnia can be classified into acute or short-term insomnia (course < 1 month), persistent insomnia (course > 4 weeks) and chronic insomnia (course > 6 months).

There are a number of drugs in the prior art that mitigate sleep through a variety of mechanisms. For example, benzodiazepines such as lorazepam, temazepam, etc., barbiturates such as phenobarbital, pentobarbital, etc., and z-drugs such as zaleplon, zolpidem, zopiclone, etc. Benzodiazepines enhance GABA by increasing the frequency of chloride channel opening. Barbiturates potentiate GABA by increasing the duration of chloride channel opening. The z-agent is an agonist of GABAa γ 1 subunit. However, these drugs generally have long half-lives and have various side effects such as confusion, depression, etc., and a high addiction potential is generated by long-term administration.

The Chrysomyiame gacephala (Chrysomya megacephala) is larva or pupa shell of Chrysomya megacephala or other kindred insects of the family Endorsifledae, and has flat cylindrical shape, length of 1-1.5cm, width of 2-3mm, yellow-white color, and slight transparency. The maggot is cold in nature, sweet in taste, salty and bitter in taste, and five toxic in property, enters spleen and stomach channels, and has the functions of tonifying spleen, resolving food, removing heat and eliminating malnutrition and the like. The existing research finds that the maggot extract can kill escherichia coli, staphylococcus aureus and the like, has broad-spectrum antibacterial activity, can promote wound healing, resist atherosclerosis, reduce blood fat, and has inhibitory activity on leukemia cells, lung cancer cells, HIV-1 integrase and the like. However, no report about the application of the maggot extract in treating insomnia is available.

The levofolinic acid calcium is also called levofolinic acid calcium, is usually used as an anti-tumor adjuvant, is used together with fluorouracil to enhance the curative effect, is used for treating tumors such as gastric cancer, colon cancer, rectal cancer and the like, can also be used for preventing or treating large-dose folic acid antagonist poisoning and is used for preventing or treating megaloblastic anemia caused by folic acid deficiency. Folate antagonists such as methotrexate bind to dihydrofolate reductase and block the conversion of folate to tetrahydrofolate. The calcium levofolinate can directly provide active folic acid, has the function of relieving the toxic reaction of excessive folic acid antagonists in vivo and is beneficial to the synthesis of dTMP, DNA, RNA and protein.

Through years of research, the inventor finds that the maggot extract has good effects of improving sleep disorder and treating insomnia. More particularly, through one accidental error operation, the inventor unexpectedly finds that the composition of the maggot extract and the calcium levofolinate has a synergistic effect, and can greatly improve the treatment effect on the sleep disorder, particularly the insomnia. The calcium levofolinate has no pharmacological action of improving sleep, but can be used together with the maggot extract to obviously enhance the activity of the maggot extract on improving sleep disorder and treating insomnia, and the specific pharmacological action mechanism is unknown.

Disclosure of Invention

In one aspect of the present invention, there is provided a pharmaceutical composition for improving, preventing and/or treating sleep disorders, comprising maggots or an extract thereof and calcium levofolinate.

In another aspect of the present invention, the pharmaceutical composition, wherein the maggot extract is prepared according to the following method:

(1) artificially breeding maggots, freeze-drying, crushing, sieving, and performing supercritical extraction with carbon dioxide fluid to obtain a crude product;

(2) adding 3% of distilled water into the crude product obtained in the step 1, and stirring and mixing; centrifuging to remove precipitate to obtain degummed oil;

(3) adding sodium hydroxide into the degummed grease obtained in the step 2, stirring, coagulating and centrifugally separating; adding distilled water into the supernatant, stirring, standing, separating, and repeating for 1-3 times until the lower layer water is not discolored by adding phenolphthalein; taking the supernatant for later use;

(4) decoloring the supernatant by using an activated carbon adsorbent: adding 4% of active carbon, decolorizing at 90 deg.C for 90min, and repeating for three times to obtain the Chrysomyiame gacephala extract.

In another aspect of the present invention, the pharmaceutical composition, wherein the ratio of the maggot or extract thereof to the calcium levofolinate is 0.1-50:1, preferably 20:1, 10:1 or 5:1 by mass.

In another aspect of the invention, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

In another aspect of the present invention, the pharmaceutical composition is a tablet, a capsule, a granule, an injection, a nasal drop, an inhalant, a sustained release agent, an aerosol or a film.

In another aspect of the invention, the use of the pharmaceutical composition in the preparation of a medicament for the prevention and/or treatment of sleep disorders is provided. The sleep disorder includes insomnia.

In another aspect of the present invention, there is provided a medicament for improving, preventing and/or treating sleep disorders, comprising maggots or an extract thereof.

In another aspect of the invention, the use of the maggot or its extract in preparing a medicament for preventing and/or treating sleep disorders is provided.

In another aspect of the invention, the sleep disorder is insomnia.

In another aspect of the present invention, a method for preparing a maggot extract is provided:

Detailed Description

Through years of research, the inventor finds that the maggots or the extractive thereof have effective pharmacological activity for preventing and/or treating sleep disorder (preferably insomnia). Furthermore, the maggot or the extract thereof and the calcium levofolinate are combined according to a specific proportion, so that the activity of improving sleep disorder can be further enhanced, and the synergistic effect is achieved.

Unless otherwise specified, the units of measurement described herein are mass.

In one aspect of the invention, the application of the maggot or the extractive thereof in preparing the medicine for preventing and/or treating the sleep disorder is provided.

In one aspect of the present invention, there is provided a pharmaceutical composition comprising maggot or its extract and calcium levofolinate.

In another aspect of the invention, the use of the pharmaceutical composition in the preparation of a medicament for the prevention and/or treatment of sleep disorders is provided.

The maggot extract is prepared by the following method:

1. artificially cultured maggots are freeze-dried, crushed, sieved and subjected to supercritical extraction by using carbon dioxide fluid to obtain a crude product.

2. Adding 3% of distilled water into the crude product obtained in the step 1, and stirring and mixing; and (4) carrying out centrifugal separation to remove precipitates to obtain the degummed grease.

3. And (3) adding sodium hydroxide into the degummed grease obtained in the step (2), stirring, coagulating and centrifugally separating. Adding distilled water into the supernatant, stirring, standing, separating, and repeating for 1-3 times until the color of the lower layer water is unchanged by adding phenolphthalein. Taking the supernatant for later use.

4. Decoloring the supernatant by using an activated carbon adsorbent: adding 4% of active carbon, decolorizing at 90 deg.C for 90min, and repeating for three times to obtain the Chrysomyiame gacephala extract.

In another aspect of the invention, the content of the maggot extract in the medicine is 0.1-99.9%.

In another aspect of the present invention, the medicament comprises maggot or its extract and pharmaceutically acceptable carrier.

In another aspect of the invention, the medicament is in a pharmaceutically acceptable formulation.

In another aspect of the invention, the pharmaceutical composition is in a pharmaceutically acceptable formulation.

In another aspect of the invention, the weight ratio of the maggot or the extract thereof to the calcium levofolinate in the pharmaceutical composition is 0.1-50: 1.

In another aspect of the invention, the weight ratio of the maggot or the extract thereof to the calcium levofolinate in the pharmaceutical composition is 1-30: 1.

In another aspect of the invention, the weight ratio of the maggot or the extract thereof to the calcium levofolinate in the pharmaceutical composition is 2-25: 1.

In another aspect of the invention, the weight ratio of the maggot or the extract thereof to the calcium levofolinate in the pharmaceutical composition is 20: 1.

In another aspect of the invention, the weight ratio of the maggot or the extract thereof to the calcium levofolinate in the pharmaceutical composition is 10: 1.

In another aspect of the invention, the weight ratio of the maggot or the extract thereof to the calcium levofolinate in the pharmaceutical composition is 5: 1.

In another aspect of the present invention, the formulation forms include tablets, capsules, granules, injections, nasal drops, inhalants, sustained-release agents, aerosols, films, and the like.

In another aspect of the invention, the combination of maggot or its extract and calcium levofolinate is a "combination or pharmaceutical composition for simultaneous, separate or sequential use", especially referring to a "kit of parts", meaning that maggot or its extract and calcium levofolinate can be administered each independently or by different fixed combinations with different contents of components, i.e. at different time points or simultaneously. The components of the kit of parts may, for example, be administered simultaneously or chronologically, i.e. at different time points and with equal or different time intervals for any of the components of the kit of parts. Preferably, the time intervals are chosen such that the effect on the disease or condition to be treated in the combination of the components is higher than the effect which would be obtained by using only any of the components individually.

The term "preventing" refers to prophylactic administration of the combination to healthy patients to prevent the development of the diseases and conditions described herein. Furthermore, the term "prevention" may also refer to the prophylactic administration of the combination to a patient at a pre-stage of the disease to be treated.

A therapeutically effective amount of each of the components of the COMBINATION OF THE INVENTION may be administered simultaneously or in any order, and the components may be administered separately or as a fixed combination. The individual components of the combination may be administered separately at different times during the course of therapy or may be combined in divided or single combinations and administered. In addition, the term "use" also includes the use of prodrugs of any drug that can be converted in vivo to the selected drug. Accordingly, the invention is to be understood as embracing all such regimes of simultaneous or alternating treatment and the term "using" is to be interpreted accordingly.

The preferred route of administration of the dosage forms of the invention is enterally or preferably orally. Because of their ease of administration, tablets and capsules represent the best oral dosage unit form.

The effective dosage of each active ingredient used in the combination therapy may vary with the particular pharmaceutical composition used, the mode of use, or the severity of the condition being treated.

The pharmaceutically acceptable carriers used in the medicament of the present invention include fillers, diluents, excipients, stabilizers, antioxidants, disintegrants, binders, lubricants, preservatives, colorants, and the like.

Preferably, the filler is selected from at least one of starch, pre-crosslinked starch, dextrin, chitosan, mannose, microcrystalline cellulose, polyanhydrides, polyphosphoesters, polyurethanes, fibrin, liposomes, polyethylene glycol, galactose, and povidone.

Preferably, the diluent is selected from at least one of mannitol, microcrystalline cellulose, lactose, starch, saccharin.

Preferably, the stabilizer is at least one selected from cyclodextrin and its derivatives, polyethylene glycol, tween, span, dextran, and mannitol.

Preferably, the antioxidant is at least one selected from the group consisting of ascorbic acid, sodium metabisulfite, benzoic acid, citrate, sorbic acid, sodium sulfite, sodium bisulfite and sodium thiosulfate.

Preferably, the disintegrant is selected from croscarmellose sodium, crospovidone, hydroxypropyl cellulose, hydroxypropyl starch, polysorbate 80, sodium lauryl sulfate, and the like.

Preferably, the binder is selected from hydroxypropyl methylcellulose, povidone, starch, dextrin, microcrystalline cellulose, sodium alginate, methylcellulose, and the like.

Preferably, the lubricant is selected from stearic acid, magnesium stearate, talc, polyethylene glycol, sodium lauryl sulfate, and the like.

Preferably, the preservative is selected from benzoic acid, paraben, sorbic acid, ethanol and the like.

Detailed Description

Example 1: preparation of maggot extract

The maggot extract is prepared according to the following method:

1. freeze drying artificially cultured maggot, pulverizing, sieving with 60 mesh sieve, performing supercritical extraction with carbon dioxide fluid at 50 deg.C under 25MPA pressure and CO₂The flow rate is 25L/h, and the extraction is carried out for 2 hours to obtain a crude product.

2. Heating the crude product obtained in the step 1 to 80 ℃, adding distilled water with the temperature of 80 ℃ and the volume of the crude product being 3 percent, stirring for 30 minutes, and fully mixing; and (4) carrying out centrifugal separation at the speed of 5000r/min, and removing precipitates to obtain the degummed grease.

3. And (3) heating the degummed grease obtained in the step (2) to 60 ℃, adding 0.3% (w/w) of sodium hydroxide, stirring for 1 hour to obtain a fine saponified substance, heating to 80 ℃, obtaining a floccule, coagulating, and carrying out centrifugal separation at 5000 r/min. Heating the supernatant to 80 deg.C, adding 80-85 deg.C distilled water 30% of the supernatant volume, stirring for 30min, standing, separating with separating funnel, heating the supernatant again, adding water, stirring, standing, measuring pH of the separated lower layer water, repeating for 3 times, and adding phenolphthalein to the lower layer water without changing color. Taking the supernatant for later use.

4. Decoloring with an adsorbent: adding activated carbon with a volume of 4% of the clear liquid, decolorizing at 90 deg.C for 90min, and repeating for three times to obtain maggot extract.

Example 2: pharmacological test for improving sleep disorder

1. Test method

60 ICR mice are male, 18-22 g in weight and 6-8 weeks old. Animals are placed at room temperature (22 +/-1) DEG C, relative humidity of 65 percent and normal day and night rhythm regulation illumination time environment, and eat standard feed and freely drink water.

In the experiment, a consumptive disease insomnia model is caused by combining running bench exhaustion training and intraperitoneal injection of PCPA (p-chlorophenylalanine), and the sleep function of a mice with the consumptive disease insomnia model improved by the maggot extract is researched.

Except for the blank group, mice in the other groups were subjected to a 30min running out training (25m/min) 1 time per day for 7 consecutive days. And injecting PCPA (250mg/kg) suspension into the abdominal cavity on the 8 th day, wherein after the injection of the medicine, the mouse has contracture pain and the symptom is relieved after the medicine lasts for 30-60 min. And evaluating the success of the model according to the change of the mouse sign and the measurement index. After 2 days of modeling, the mice have sleep disorder, easy fright and slow movement, which indicates that the consumptive disease insomnia model is successfully modeled.

80 mice are divided into 8 groups according to a random principle, and each group comprises 10 mice, namely a blank group, a model group, a maggot extract group, a calcium levofolinate group, a maggot extract and calcium levofolinate group 1-3 and a positive control group. Respectively performing intragastric administration on a maggot extract group (40mg/kg), a calcium levofolinate group (4mg/kg), a maggot extract and calcium levofolinate 1 group (20mg/kg +1mg/kg), a maggot extract and calcium levofolinate 2 group (20mg/kg +2mg/kg) and a maggot extract and calcium levofolinate 3 group (20mg/kg +4 mg/kg). The blank group and the model group are separately intragastrically infused with distilled water of the same volume, the positive control drug is diazepam, and the administration dose of the mice is 0.75 mg/kg. The above groups are administered by intragastric administration 1 time daily for 15 days.

Each group of mice was observed for food intake/d, body weight, mental, activity, posture, hair and serum 5-HT, DA and ACTH levels. Treatment 15d, mice in each group were fasted for 12h and blood was taken decapitatively. Standing blood at 4 deg.C for 90min, centrifuging at 3500r/min for 10min, collecting serum, and storing in refrigerator at-20 deg.C. Serum 5-HT, DA and ACTH levels were determined by enzyme-linked immunosorbent assay (ELISA) using serum.

2. And (5) experimental results.

1. Mouse signs change.

After administration, after the administration, the appetite of the mice begins to recover to normal after the gavage of the maggot extract group, the maggot extract and the calcium levofolinate 1-3 groups and the positive control group, the weight increases in different degrees, the action is gradually normal, the bodies are stretched, the hair color is glossy, the mucous membrane of the skin is pink, no difference is generated compared with the normal mice, and the sleep rhythm of the mice recovers to normal.

The mice in the model group and the calcium levofolinate group still have the defects of sleep disorder, withered hair color and slow movement without obvious improvement.

2. Effect on serum 5-HT, DA and ACTH levels

TABLE 1 Effect on serum 5-HT, DA and ACTH levels

As can be seen from Table 1, the serum 5-HT, DA and ACTH levels of the model group mice are significantly reduced (P <0.01) compared with the blank group, indicating that the modeling is successful; compared with the model group, the maggot extract and calcium levofolinate 1-3 group and the positive control group can obviously increase 5-HT, DA and ACTH (P is less than 0.01), and the effect on treating the sleep disorder of the mice is shown.

Example 3 evaluation of sleep improvement function Using test method for improving sleep function

1. Experimental methods

120 ICR mice are male, 18-22 g in weight and 6-8 weeks old. Animals are placed at room temperature (22 +/-1) DEG C, relative humidity of 65 percent and normal day and night rhythm regulation illumination time environment, and eat standard feed and freely drink water.

60 mice were used for the direct sleep experiment and 60 mice for the barbiturate sodium sleep latency experiment. Animals were randomly divided into a maggot extract group, a calcium levofolinate group, a maggot extract + calcium levofolinate 1-3 groups and a negative control group, each group consisting of 10 animals. The dosage of each group of drugs is as in example 2, and the oral gavage is performed, and the negative control group is given with equal amount of distilled water, and the gavage is performed for 1 time per day and continuously for 30 days.

The method is carried out according to the technical Specification for health food inspection and evaluation of Ministry of health. The test for improving sleep function comprises: direct sleep experiment and barbital sodium sleep latency experiment.

(1) Direct sleep experiments: and observing whether the animals in the administration group have sleep phenomenon after being administered with the test sample and the negative control group is administered with distilled water with the same volume. The disappearance of the righting reflex is used as an index for sleeping. If the patient can not be righted for more than 30-60s, the righting reflex is considered to disappear, and the patient enters sleep. The recovery of the righting reflex is arousal, and the time from disappearance of the righting reflex to recovery is the sleeping time of the animal. The number of animals falling asleep and the sleeping time of the negative control group and the administration group were recorded.

(2) Barbiturate sodium sleep latency experiments: before formal experiments, the prior experiments are carried out, the dosage of the barbital sodium which enables the animals to fall asleep for 100 percent but does not lead to overlong sleep time is determined, and the formal experiments are carried out by using the dosage. After the animals are administrated for 15min for the last time, barbital sodium (the experimental dosage is 280mg/kgBW) is injected into the abdominal cavity of each group of animals, the injection amount is 0.1mL/10g BW, the influence of the tested sample on the sleep latency of the barbital sodium is observed by taking the disappearance of the righting reflex as an index.

2. And (5) experimental results.

(1) Has direct sleep effect.

After administration, each group of mice shows symptoms such as quiet and reduced autonomic activity, and individual mice enter a sleeping state, particularly more mice in the maggot extract and calcium levofolinate combined administration group, which shows that the combined administration has the effect of promoting sleeping.

TABLE 2 Effect on direct sleep in mice

(2) Effect on sleep time of sodium pentobarbital.

Under the hypnotic effect of the sodium pentobarbital, the sleep time of mice in each administration group is prolonged compared with that of a negative control group, wherein the combined administration group and the negative control group have a significant difference (P <0.01), and the sample has the effect of prolonging the sleep time of the sodium pentobarbital.

TABLE 3 Effect on sleep time of sodium pentobarbital

Grouping	Number of mice (only)	Sleep time (min)
			Negative control group	10	19
Maggot extract group	10	28
			Calcium levofolinate group	10	18
Maggot extract and calcium levofolinate 1 group	10	37
			Maggot extract and calcium levofolinate 2 groups	10	45
Maggot extract and calcium levofolinate 3 groups	10	46

3. Conclusion

(1) The maggot extract, the maggot extract and the calcium levofolinate composition can effectively promote the sleep of mice and prolong the sleep time of the sodium pentobarbital. Particularly, the maggot extract and calcium levofolinate combined administration group can remarkably promote and prolong the sleep of mice, and has a synergistic effect compared with the maggot extract used alone.

(2) The single use of the calcium levofolinate has no sleep promoting effect, but the calcium levofolinate can obviously enhance the sleep promoting effect of the maggot extract.

(3) The sleep-promoting synergistic effect of the composition of the maggot extract and the calcium levofolinate is not dose-dependent.

Example 3: preparation of tablets

Maggot extract 60g

Calcium levofolinate 3g

Mixing the above active ingredients with appropriate amount of conventional adjuvants for preparing tablet, and making into 1000 tablets by conventional tabletting machine.

Example 4: preparation of tablets

Maggot extract 50g

Levofolinic acid calcium 5g

Example 5: preparation of capsules

Maggot extract 50g

Calcium levofolinate 10g

Mixing the above active ingredients with appropriate amount of conventional adjuvants for preparing capsule, mixing, and making into 1000 tablets.

Claims

1. A pharmaceutical composition for improving, preventing and/or treating sleep disorders, comprising maggot or its extract and calcium levofolinate.

2. The pharmaceutical composition as set forth in claim 1, wherein the maggot extract is prepared according to the following method:

(3) adding sodium hydroxide into the degummed grease obtained in the step 2, stirring, coagulating and centrifugally separating; adding distilled water into the supernatant, stirring, standing, separating, and repeating for 1-3 times until the lower layer water is unchanged by adding phenolphthalein; taking the supernatant for later use;

3. The pharmaceutical composition according to claim 1 or 2, wherein the ratio of the maggot or extract thereof to the calcium levofolinate is 0.1-50:1, preferably 20:1, 10:1 or 5:1 by mass.

4. The pharmaceutical composition of any one of claims 1-3, comprising a pharmaceutically acceptable carrier.

5. The pharmaceutical composition according to any one of claims 1 to 4, which is a tablet, capsule, granule, injection, nasal drop, inhalant, sustained-release agent, aerosol or film.

6. Use of a pharmaceutical composition according to any one of claims 1 to 5 for the preparation of a medicament for the improvement, prevention and/or treatment of sleep disorders.

7. A medicament for improving, preventing and/or treating sleep disorders, which comprises maggot or its extract.

8. The drug as claimed in claim 7, wherein the maggot extract is prepared according to the following method:

9. Use of maggot or its extract in preparing medicine for improving, preventing and/or treating sleep disorder is provided.

10. Use as claimed in claim 6 or 9, wherein the sleep disorder is insomnia.