Preparation method of a millipede extract and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a preparation method of a millipede extract and application thereof. Specifically, the preparation method of the millipede extract comprises the following steps: S1, soaking millipedes in 8-10 times of water, and then heating and refluxing extraction; S2, taking the above-mentioned extraction liquid, filtering, and then adding pepsin and trypsin in sequence for enzymolysis, and then carrying out incubation treatment at 30-40 DEG C, centrifuging to obtain supernatant and concentrating; S3, adding n-butanol to the concentrated supernatant for extraction 2-3 times, and then combining the extraction liquids; S4, purifying the extraction liquid prepared in step S3 to obtain the millipede extract. Through the improved process method, the millipede extract prepared by extraction has good analgesic and anti-inflammatory effects, and the prepared millipede extract has good stability, so as to help expand the application field and range, and therefore has good practical application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a preparation method of millipede extract and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing the understanding of the background of the present application and should not be taken as an acknowledgement or any form of suggestion that this information forms prior art that is already commonly known to a person of ordinary skill in the art.

[0003] Millipede (Diplopoda) Armadillidium vulgare Latreille), also known as tidal worm, is a traditional Chinese medicine insect in ancient China and folk, which has a long history of medicinal use and good therapeutic effect on various pains, inflammations and skin diseases. According to the Chinese Herbal Medicine, millipede has the effects of breaking blood stasis, removing tumor, dredging channels, promoting water, detoxifying and relieving pain, and is used for treating blood stasis, malaria, blood stasis, and the like. Modern studies have shown that millipede extract has good therapeutic effect on skin and mucous membrane diseases, analgesia and anti-inflammation.

[0004] Among them, the analgesic and anti-inflammatory effect of millipede extract has been widely studied in recent years, and corresponding drug preparations have been developed, but the pharmacological and clinical research is not deep. The inventors have previously applied for and obtained a patent for a preparation method of millipede extract and its application in anti-inflammatory and analgesic drugs (CN 101708190A). The experiments have proved that the capsule prepared from the millipede extract has good analgesic and anti-inflammatory effect. However, in the later research, it is found that when the millipede extract is further prepared into other dosage forms such as oral liquid, the preparation stability is poor, and impurities and precipitates are easily separated, which affects the subsequent production and marketing. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a preparation method of millipede extract and application thereof. The millipede extract prepared by improving the process method has good analgesic and anti-inflammatory effect, and has good stability, which helps to expand its application field and range, and therefore has good practical application value.

[0006] In order to achieve the above purpose, the present application relates to the following technical scheme:

[0007] In a first aspect of the present application, a preparation method of millipede extract is provided, which comprises:

[0008] S1, millipede is soaked in 8-10 times amount of water, and then heated and refluxed for extraction;

[0009] S2, taking the above extract, filtering, then adding pepsin and trypsin for enzymolysis, incubating at 30-40 DEG C, centrifuging to obtain supernatant and concentrating;

[0010] S3, adding n-butanol to the concentrated supernatant for extraction 2-3 times, and combining the extract;

[0011] S4, the extract prepared in step S3 is purified to obtain the final product.

[0012] In a second aspect of the present application, the above prepared extract of the porcellio scaber is provided.

[0013] In a third aspect of the present application, the above extract of the porcellio scaber is used for preparing analgesic and anti-inflammatory products.

[0014] The product can be a medicine.

[0015] Therefore, in a fourth aspect of the present application, an analgesic and anti-inflammatory medicine is provided, which comprises the above extract of the porcellio scaber and at least one pharmaceutically acceptable excipient.

[0016] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0017] The above technical solution optimizes the preparation process by adding enzymolysis and purification, and the prepared extract of the porcellio scaber has good analgesic and anti-inflammatory effects, and has good stability, so it can be applied to various analgesic and anti-inflammatory drug dosage forms, and has good practical application value. DETAILED DESCRIPTION

[0018] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used in the present application have the same meaning as generally understood by those skilled in the art to which the present application belongs.

[0019] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, components, and / or groups thereof, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. It will be understood that the scope of the application is not limited to the specific embodiments described below; it is also understood that the examples used herein are intended to be illustrative only and not limiting to the scope of the application.

[0020] In one typical embodiment, a preparation method of a millipede extract is provided, the preparation method comprising:

[0021] S1, soaking the millipede in 8-10 times the amount of water, and then heating and refluxing to extract;

[0022] S2, taking the above extract, filtering, and then adding pepsin and trypsin in sequence to perform enzymolysis, and then incubating at 30-40°C, and then centrifuging to obtain supernatant and concentrating;

[0023] S3, adding n-butanol to the concentrated supernatant to extract 2-3 times, and then combining the extract;

[0024] S4, purifying the extract obtained in step S3 to obtain the millipede extract.

[0025] In one embodiment, the heating and refluxing in step S1 is performed 2-3 times, and the combined extract is then subjected to step S2.

[0026] In another embodiment of the present application, in step S2, the added amount of pepsin is 0.1-0.3% (preferably 0.2%) of the millipede, the enzymolysis temperature is 30-40°C (preferably 35°C), the enzymolysis pH is 2.0-4.0 (preferably pH 2.5), and the enzymolysis time is 1-3h (preferably 2h).

[0027] In another embodiment of the present application, in step S2, the added amount of trypsin is 0.2-0.4% (preferably 0.3%) of the millipede, the enzymolysis temperature is 30-40°C (preferably 35°C), the enzymolysis pH is 7.5-9.0 (preferably pH 8.0), and the enzymolysis time is 1-3h (preferably 2h).

[0028] In another embodiment of the present application, in step S2, the added amount of trypsin is 0.2-0.4% (preferably 0.3%) of the millipede, the enzymolysis temperature is 30-40°C (preferably 35°C), the enzymolysis pH is 7.5-9.0 (preferably pH 8.0), and the enzymolysis time is 1-3h (preferably 2h).

[0029] In another embodiment of the present application, after the enzymolysis treatment, a high-temperature enzyme inactivation method is used to stop the enzymolysis reaction, specifically, inactivation at 80-90°C for 3-5 minutes, and then cooling to room temperature.

[0030] The centrifugation is centrifugation at 5000-8000 rpm for 10-30 min, and the supernatant is concentrated to 40-60% (preferably 50%) of the original supernatant volume.

[0031] In another specific embodiment of the present application, in the step S3, the volume ratio of n-butanol to the concentrated supernatant is 0.5-5:1, preferably 1:1.

[0032] In another specific embodiment of the present application, in the step S4, the purification step comprises: filtering the extract solution through a dialysis membrane, and drying the filtrate to obtain the extract.

[0033] In the step S4, the dialysis membrane has a molecular weight cut-off of 30000-50000 Da, preferably 40000 Da.

[0034] Therefore, in another specific embodiment of the present application, a toadspaw extract prepared by the above preparation method is provided. The toadspaw extract obtained by the above preparation process not only has good analgesic and anti-inflammatory effects, but also has good stability, which is beneficial to the preparation of various dosage forms of products using the toadspaw extract as a raw material.

[0035] In another specific embodiment of the present application, the above toadspaw extract is provided for use in the preparation of analgesic and anti-inflammatory products.

[0036] The product can be a pharmaceutical product.

[0037] Therefore, in another specific embodiment of the present application, an analgesic and anti-inflammatory pharmaceutical product is provided, which comprises the above toadspaw extract and at least one pharmaceutically acceptable excipient.

[0038] In another specific embodiment of the present application, the excipient can be in solid or liquid form. Solid forms of preparations include powders, tablets, dispersed granules, capsules, pills and suppositories. Suitable solid excipients can be magnesium carbonate, magnesium stearate, talc, sugar or lactose. Tablets, powders, pills and capsules are solid dosage forms suitable for oral administration. Liquid forms of preparations include solutions, suspensions and emulsions, examples of which are aqueous solutions or water-propylene glycol solutions for parenteral injection, or oral solutions with added sweeteners and contrast agents. In addition, small water injections for injection, lyophilized powder injections for injection, large infusions or small infusions can also be prepared. No specific limitation is made here.

[0039] The present application is further explained and described by the following examples, but the present application is not limited by the examples. It should be understood that the examples are only used to illustrate the present application and do not limit the scope of the present application.

[0040] Example 1

[0041] A preparation method of a toadspaw extract, the preparation method comprising:

[0042] Take dry toad 100 g, add 1000 g water soak 1 hour, heating reflux extraction 2 times; combined above the extract, filter to which sequentially add pepsin and trypsin for enzymolysis, first add pepsin added quality is toad 0.2%, enzymolysis temperature is 35 ℃, enzymolysis pH 2.5, enzymolysis time is 2 h; Trypsin added quality is toad 0.3%, enzymolysis temperature is 35 ℃, enzymolysis pH 8.0, enzymolysis time is 2 h. Enzymolysis treatment in 85 ℃ inactivation 5 minutes, then cool to room temperature, then the enzymolysis liquid is centrifuged, centrifuged at 6000 rpm for 20 min, the supernatant is concentrated to 50% of the original volume, add equal volume of n-butanol, extract 2 times, combine the extract liquid, dialysis membrane filtration (cut-off molecular weight 40000 Da), concentrated and dried to obtain the extract.

[0043] Example 2

[0044] A preparation method of a toad extract, the preparation method comprising:

[0045] Take dry toad 100 g, add 800 g water soak 1 hour, heating reflux extraction 2 times; combined above the extract, filter to which sequentially add pepsin and trypsin for enzymolysis, first add pepsin added quality is toad 0.2%, enzymolysis temperature is 35 ℃, enzymolysis pH 2.5, enzymolysis time is 2 h; Trypsin added quality is toad 0.3%, enzymolysis temperature is 35 ℃, enzymolysis pH 8.0, enzymolysis time is 2 h. Enzymolysis treatment in 85 ℃ inactivation 5 minutes, then cool to room temperature, then the enzymolysis liquid is centrifuged, centrifuged at 8000 rpm for 20 min, the supernatant is concentrated to 50% of the original volume, add equal volume of n-butanol, extract 2 times, combine the extract liquid, dialysis membrane filtration (cut-off molecular weight 40000 Da), concentrated and dried to obtain the extract.

[0046] Example 3

[0047] A preparation method of a toad extract, the preparation method comprising:

[0048] Take 100 g of dry toad, add 1000 g of water and soak for 1 hour, then heat and reflux extract twice; combine the above extract, filter and then add pepsin and trypsin to the extract for enzymatic hydrolysis, first add pepsin at a mass of 0.2% of the toad, enzymatic hydrolysis temperature is 35°C, enzymatic hydrolysis pH is 2.5, and enzymatic hydrolysis time is 2 h; add trypsin at a mass of 0.3% of the toad, enzymatic hydrolysis temperature is 35°C, enzymatic hydrolysis pH is 8.0, and enzymatic hydrolysis time is 2 h. After enzymatic hydrolysis, inactivate at 85°C for 5 minutes, then cool to room temperature, then centrifuge the enzymatic hydrolysis liquid at 6000 rpm for 20 min, concentrate the supernatant to 60% of the original volume, add an equal volume of n-butanol, extract twice, combine the extract, filter through a dialysis membrane (cut-off molecular weight 40000 Da), concentrate and dry to obtain the extract.

[0049] Effect verification

[0050] 1.1 Pain relief effect determination

[0051] Determine the pain relief effect based on the mouse acetic acid writhing test, take 50 healthy Kunming mice, half male and half female, and randomly divide them into a control group and experimental groups 1-3, wherein the control group of Kunming mice is given 5 ml / kg of body weight of normal saline by gavage, and the experimental groups 1-4 are given toad extract obtained from the application examples 1-3 and CN 101708190A example 1 (24 mg of crude drug / kg) by gavage, respectively. Oral gavage is performed twice a day (at 9 am and 5 pm), and the administration is continued for 3 days. 1.5 h after the last administration, 0.1 mL / 10 g of 0.9% acetic acid solution is injected intraperitoneally into the mice of each group, and the number of writhing times of the mice within 20 min after the injection of the acetic acid solution is observed. The test results are shown in Table 1.

[0052] Table 1

[0053]

[0054] Note: compared with the control group, P < 0.05.

[0055] The results show that the experimental groups 1-4 can inhibit the writhing response of mice, and further, the product prepared in example 1 has the best effect.

[0056] 1.2 Mouse ear swelling experiment

[0057] Take 50 healthy Kunming mice, half male and half female, and divide them into groups and administer them according to the method of 1.1. 0.5 h after the last administration, apply 30 μl of xylene to each of the right ears of the mice to cause inflammation. 2 h after the inflammation, kill the rats, use a 10 mm diameter puncher to take the left and right ear pieces from the symmetrical parts of the ears, weigh them, and calculate the weight difference between the left and right ear pieces as the degree of swelling. The results are shown in Table 2.

[0058] Table 2

[0059]

[0060] Note: compared with the control group, P < 0.05.

[0061] The results show that the experimental groups 1-4 can significantly reduce the swelling of the rat ears, and similarly, the product prepared in Example 1 has the best effect.

[0062] 1.3 Safety study

[0063] The extracts obtained in Examples 1-3 were administered in an amount 20 times that of 1.1 for 3 weeks, and during the administration period, 10 Kunming mice were observed daily for food and water intake, voluntary activity, stool shape, and fur color; and physiological signals such as respiration, blood pressure, and electrocardiogram were monitored. No obvious toxic reactions were observed. 24 hours after the last administration, the mice in each group were sacrificed, and histopathological observation was performed, and the results showed that the food and water intake, voluntary activity, stool shape, respiration, blood pressure, electrocardiogram, and fur color of the mice were all normal, and no obvious pathological changes were observed in the heart, liver, spleen, lung, and kidney under a microscope.

[0064] 1.4 Stability test

[0065] The samples prepared in the above examples and Example 1 of CN 101708190A were subjected to an accelerated test (temperature 40℃, relative humidity 75±5%), and the results are shown in Table 3.

[0066] Table 3

[0067]

[0068] The results show that the pH of the product prepared in Example 1 of CN 101708190A decreases significantly, the content decreases significantly, and the related substances increase after 3 months of acceleration, while the samples prepared in Examples 1-3 of the present application all have good stability within 12 months, indicating that the present application has good performance in improving properties, pH, etc., thereby expanding its application field.

[0069] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit them. Although the present application has been described in detail with reference to the examples given, those skilled in the art can modify or equivalently replace the technical solutions of the present application according to their needs without departing from the spirit and scope of the technical solutions of the present application.

Claims

1. A method of preparing an extract of a millipede, characterized by, The preparation method comprises: S1, soaking the earwigs in 8-10 times the amount of water, and then heating and refluxing for extraction; S2, taking the extraction liquid, filtering, and then adding pepsin and trypsin for enzymolysis, and then incubating at 30-40℃, and then centrifuging to obtain supernatant and concentrating; S3, adding n-butanol to the concentrated supernatant for extraction 2-3 times, and then combining the extraction liquids; S4, purifying the extraction liquid prepared in step S3; In step S4, the purification step comprises: filtering the extraction liquid through a dialysis membrane, and then drying the filtrate after concentration to obtain the extract; wherein the molecular weight cut-off of the dialysis membrane is 30000-50000 Da; In step S2, the addition amount of pepsin is 0.1-0.3% of the earwigs, the enzymolysis temperature is 30-40℃, the enzymolysis pH is 2.0-4.0, and the enzymolysis time is 1-3h; The addition amount of trypsin is 0.2-0.4% of the earwigs, the enzymolysis temperature is 30-40℃, the enzymolysis pH is 7.5-9.0, and the enzymolysis time is 1-3h.

2. The production method according to claim 1, wherein In step S1, the heating and refluxing extraction is performed 2-3 times, and the combined extraction liquid continues to step S2.

3. The production method according to claim 1, wherein In step S2, the addition amount of pepsin is 0.2% of the earwigs, the enzymolysis temperature is 35℃, the enzymolysis pH is 2.5, and the enzymolysis time is 2h.

4. The production method according to claim 1, wherein The addition amount of trypsin is 0.3% of the earwigs, the enzymolysis temperature is 35℃, the enzymolysis pH is 8.0, and the enzymolysis time is 2h.

5. The production method according to claim 1, wherein After the enzymolysis treatment, high-temperature enzyme inactivation is performed to stop the enzymolysis reaction, specifically inactivating at 80-90℃ for 3-5 minutes, and then cooling to room temperature; The centrifugation is performed at 5000-8000 rpm for 10-30 minutes, and the supernatant is concentrated to 40-60% of the original supernatant volume.

6. The production method according to claim 1, wherein In step S3, the volume ratio of n-butanol to the concentrated supernatant is 0.5-5:

1.

7. The earwig extract prepared by the preparation method of any one of claims 1-6.

8. Use of the extract of the species Porcellio scaber as claimed in claim 7 for the preparation of analgesic anti-inflammatory products; wherein, The product is a drug.

9. An analgesic anti-inflammatory pharmaceutical product, characterized by, The drug comprises the earwig extract of claim 7 and at least one pharmaceutically acceptable excipient.

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