CN112189504A

CN112189504A - Artificially-cultivated phellinus igniarius extract for treating migraine and preparation method thereof

Info

Publication number: CN112189504A
Application number: CN202010889152.7A
Authority: CN
Inventors: 陈安徽; 刘恩岐; 巫永华; 邵颖; 张建萍
Original assignee: Xuzhou University of Technology
Current assignee: Xuzhou University of Technology
Priority date: 2020-08-28
Filing date: 2020-08-28
Publication date: 2021-01-08

Abstract

The invention belongs to the technical field of artificial cultivation of edible fungi, and particularly relates to an artificially-cultivated phellinus igniarius sporocarp extract for treating migraine, and further discloses a preparation method of the extract. The artificially cultured phellinus igniarius sporocarp extract for treating migraine is obtained by taking the phellinus igniarius sporocarp as a raw material and carrying out ethanol and water extraction, can effectively promote cerebral blood circulation, inhibit activities of 5-HT, pathogenic peptides, P substances and the like, has the effects of promoting blood circulation to remove blood stasis, dredging collaterals and relieving vasospasm, has a good analgesic effect, can effectively relieve and treat migraine symptoms, and has the advantages of safety, no toxic or side effect and the like.

Description

Artificially-cultivated phellinus igniarius extract for treating migraine and preparation method thereof

Technical Field

The invention belongs to the technical field of artificial cultivation of edible fungi, and particularly relates to an artificially-cultivated phellinus igniarius sporocarp extract for treating migraine, and further discloses a preparation method of the extract.

Background

Phellinus igniarius (Phellinus igniarius) belongs to Basidiomycotina (Basidiomycotina), Hymenomycetes (Hymenomycetes), Aphyllophorales (Aphyllophorales), Hymenochaeyaceae (Hymenochaeyaceae), Phellinus (Phellinus), and is the trade name of Phellinus igniarius (P.igniarius), Phellinus baumii (P.baumii) and Phellinus linteus (P.Linteus), and is generally grown on the stems of withered trees and stumps of broad-leaved trees such as poplar, mulberry, willow, white birch, zelkova, peach, and the like. Phellinus linteus is a precious medicinal fungus developed in recent years for many years and is named as forest gold in the name of America. According to the records of Chinese medicine dictionary, Phellinus can be used for treating gynecological diseases such as metrorrhagia, bloody stranguria, leukorrhagia, amenorrhea, etc.; modern medical research also shows that phellinus igniarius has remarkable effects of resisting tumor, bacteria and fibrosis, resisting oxidation, improving human immunity and the like, is the rare medicinal fungus with the first biological anti-tumor effect internationally acknowledged at present, and is widely applied to the industries of medicines, foods, daily chemicals, health care products and the like.

At present, due to the restriction of the particularity and complexity of physiological states and external environment, the formation of the fruit body of phellinus igniarius in nature is difficult, so that the natural phellinus igniarius is rare in quantity, the resource available for medicine in nature is limited, and the demand of domestic and foreign markets for phellinus igniarius is increased, so that medicine farmers in domestic places take away to collect the phellinus igniarius, the fruit body cannot be formed in large quantity, the phellinus igniarius cannot become a stable industrial product source, and the development of the phellinus igniarius industry is very unfavorable, so that the defect of wild resources is made up through artificial cultivation, and the method is very important. However, artificial cultivation of Phellinus linteus is extremely difficult, and has problems of harsh culture conditions and long growth cycle. At present, two methods are mainly used for artificially cultivating phellinus igniarius, one of the methods is mainly the cut-log cultivation of mulberry trees, poplar trees and oak trees, but the cut-log cultivation needs a large amount of wood resources, the resource waste is serious, and the large-scale cultivation is difficult; the other is bag cultivation, but has the problems of insufficient nutrient supply of the culture medium, small fruit body development, easy infectious microbe infection and the like.

For example, in the artificial high-yield cultivation method of phellinus igniarius disclosed in chinese patent CN1720785A, the scheme is to adopt bag cultivation, in order to solve the problems of insufficient nutrient supply of the culture medium, small development of fruit body, easy contamination of infectious microbes and the like, auxin is added into the culture medium, however, the problem of the culture medium is not fundamentally solved by the addition of the auxin, and the addition of the auxin has influence on the quality of phellinus igniarius and is not beneficial to the natural growth of phellinus igniarius. Also, like the culture medium for cultivating phellinus igniarius bag material disclosed in chinese patent CN102786333A and the method for cultivating phellinus igniarius sporocarp by using the culture medium, the method makes full use of resources such as ramulus mori, mulberry leaves and the like, can effectively reduce the waste of log resources, can quickly cultivate phellinus igniarius sporocarp in large scale, is similar to wild phellinus igniarius, and has stable quality; however, the problem of long culture time still remains.

Migraine is a pulsating headache with repeated attacks, belongs to a large household in many headache types, is often accompanied by aura such as flashing, blurred vision, limb numbness and the like before the attacks, and is also accompanied by neurological and mental dysfunction. Migraine is a disease that can worsen gradually, and the frequency of onset is generally higher and higher. Migraine mainly includes three major types of migraine, including typical migraine, common migraine and cluster migraine, and also includes familial hemiplegic migraine, abdominal migraine, neuropsychiatric migraine, basilar migraine, retinal migraine and menstrual migraine. At present, the main common drugs for treating and/or preventing migraine are aspirin, ibuprofen, ergotamine preparation, sumatriptan, propranolol, pizotifen, mecceripine, nimodipine, flunarizine, sodium valproate, amitriptyline, cola and other chemicals, however, new methods for treating and/or preventing migraine, especially for treating and/or preventing migraine by using natural products, are still expected.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to provide an artificially cultured phellinus linteus sporocarp extract for treating migraine;

the second technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned phellinus igniarius sporophore extract for treating migraine.

In order to solve the technical problems, the preparation method of the artificially cultured phellinus igniarius sporocarp extract for treating migraine is characterized by comprising the steps of artificially culturing the phellinus igniarius sporocarp and extracting the phellinus igniarius sporocarp, and specifically comprises the following steps of:

(1) preparing a cultivation bag material: taking 20-30 parts by weight of mulberry twig sawdust, 30-50 parts by weight of brewing waste residue, 10-30 parts by weight of molasses, 3-8 parts by weight of activated sludge particles, 1-5 parts by weight of quick lime and KH₂PO₄0.2-0.5 weight part, and uniformly mixing to obtain the required culture medium; adding water into the culture medium, mixing uniformly, and bagging for later use;

(2) mycelium culture: inoculating a phellinus igniarius mother seed into the cultivation bag material, and carrying out constant-temperature cultivation on the inoculated cultivation bag material until the cultivation bag material is full of mycelia;

(3) and (3) fruiting body culture: cutting the bag material full of mycelia to produce mushrooms, continuously carrying out constant-temperature culture until the sporocarp is mature, and harvesting;

(4) crushing the phellinus igniarius sporocarp to obtain powder, adding an ethanol water solution to carry out immersion reflux extraction, and respectively collecting an ethanol extracting solution and residues;

(5) adding water into the residues for decoction and extraction, and collecting decoction;

(6) and mixing the ethanol extract and the decoction, concentrating under reduced pressure until no alcohol smell exists, and vacuum drying to obtain the required artificially cultured phellinus igniarius sporocarp extract.

Specifically, in the step (1), the activated sludge particles are aerobic activated sludge particles.

Specifically, in the step (1), the mass ratio of the culture medium to water is 40-50%: 50 to 60 percent.

Specifically, in the step (2), the inoculation amount of the phellinus igniarius mother seeds is 5-10 wt% of the culture medium.

Specifically, in the step (2), the process conditions of the mycelium culturing step include: the culture temperature is controlled to be 25-30 ℃, the air humidity is 50-80%, and the illumination intensity is 10-20 lux.

Specifically, in the step (3), the process conditions of the fruiting body cultivation step include: the culture temperature is controlled to be 25-30 ℃, the air humidity is 70-80%, and the illumination intensity is 30-100 lux.

Specifically, in the step (4):

the volume percentage content of the ethanol water solution is 60-80 v/v%;

the dosage ratio of the phellinus igniarius sporophore powder to the ethanol water solution is 10 g: 200 and 300 mL.

Specifically, in the step (5), the ratio of the phellinus igniarius sporophore powder to the water is 10 g: 200 and 300 mL.

The invention also discloses an artificially cultured phellinus igniarius sporocarp extract for treating migraine, which is prepared by the method.

The invention also discloses application of the phellinus igniarius sporocarp extract in preparing a medicine for treating migraine.

Specifically, the phellinus igniarius sporocarp extract can be added with conventional auxiliary materials according to a conventional method to prepare a clinically acceptable oral preparation.

The artificially cultured phellinus igniarius sporocarp extract for treating migraine is obtained by taking the phellinus igniarius sporocarp as a raw material and carrying out ethanol and water extraction, can effectively promote cerebral blood circulation, inhibit activities of 5-HT, pathogenic peptides, P substances and the like, has the effects of promoting blood circulation to remove blood stasis, dredging collaterals and relieving vasospasm, has a good analgesic effect, can effectively relieve and treat migraine symptoms, and has the advantages of safety, no toxic or side effect and the like.

According to the method, the phellinus igniarius sporocarp raw material selected by the extract is efficiently cultivated by adopting artificial bag materials, the cultivation of the phellinus igniarius sporocarp is tried based on the traditional artificial bag material type cultivation method, mulberry twig wood chips, brewing waste residues and molasses are selected as main cultivation raw materials, and the recycling of industrial waste materials such as the brewing waste residues and the molasses is realized under the condition that the nutrition components of the phellinus igniarius cultivation are met; the activated sludge particles further added depend on the components of microorganisms, organic matters and the like contained in the activated sludge particles, so that the absorption of nutrient components of the cultivation raw materials and the forming of phellinus igniarius mycelia and sporocarp are greatly promoted, the cultivation period of phellinus igniarius is greatly shortened, the cultivation efficiency of phellinus igniarius sporocarp is effectively improved, and the yield and quality guarantee is provided for the downstream application of phellinus igniarius.

Detailed Description

In the following examples of the present invention, the phellinus linteus mother strain is obtained by separation and purification by a conventional method, activated by a conventional PDA plate medium, and inoculated into a conventional PD mother strain medium for culture, thereby obtaining the phellinus linteus mother strain.

In the following examples of the present invention, the aerobic activated sludge granules are commercially available conventional products in the prior art.

In the following embodiments of the present invention, the brewing waste residue is waste residue generated in a general liquor brewing process, such as waste residue obtained in a general kaoliang spirit brewing process, for example, the method includes the following steps:

(1) taking 90kg of sorghum, crushing the sorghum into 4-8 pieces/granule of the sorghum, uniformly mixing, adding 55kg of water (75 ℃) for grain moistening for 18 hours, and then filling into a steamer and steaming for 80min to obtain grain grains;

(2) taking the grain stillage out of the retort in the step S1, adding 28kg of cold water for raising the cold, adding 10kg of Daqu and 0.03kg of aroma-producing active dry yeast after raising the cold to obtain large residues, and putting the large residues into a pool at 12 ℃ for large residue fermentation for 28 days;

(3) taking the fermented large residues, adding 15kg of rice hulls, uniformly mixing, filling into a steamer, and distilling for 50min to obtain large residue wine and grain lees for later use;

(4) taking the grain stillage obtained in the step S3 out of a steamer, adding 30kg of water, cooling, adding 8kg of Daqu and 0.02kg of aroma-producing active dry yeast to obtain secondary slag, and putting the secondary slag into a jar at 15 ℃ for secondary slag fermentation for 28 days;

(5) taking the fermented second residue, adding rice hulls accounting for 10 wt% of the weight of the second residue, uniformly mixing, filling into a steamer, and distilling for 50min to obtain second residue wine;

(6) blending the obtained big dreg wine and the second dreg wine according to the mass ratio of 45:55, mixing and blending the two dregs wine, and then reducing the mixture to 54 ℃ to obtain the wine.

And mixing the waste residues generated by the solid-liquid separation in the steps to obtain the brewing waste residues of the common brewing process, and carrying out solid-liquid separation and drying treatment on the brewing waste residues until the water content is lower than 10%.

Example 1

The preparation method of the artificially cultured phellinus igniarius sporocarp extract for treating migraine comprises the following steps of:

(1) preparing a cultivation bag material: taking 20 parts by weight of mulberry twig sawdust, 50 parts by weight of wine-making waste residue, 10 parts by weight of molasses, 8 parts by weight of aerobic activated sludge particles, 1 part by weight of quicklime and KH₂PO₄0.5 part by weight, and uniformly mixing to obtain the required culture medium; according to the proportion of 50%: adding water into the culture medium in a mass ratio of 50%, uniformly mixing, and bagging for later use;

(2) mycelium culture: inoculating a phellinus igniarius mother seed liquid material accounting for 10 wt% of the culture medium into the culture bag material, placing the inoculated culture bag material in a culture space with the temperature of 26-27 ℃ and the air humidity of 60-65% for constant-temperature culture, controlling the illumination condition of a culture room to be 15lux in the period, and keeping the interval ventilation state of the culture room; observing the culture condition of the bag material at any time, and recording the culture time of the mycelium overgrowing with the culture bag material;

(3) and (3) fruiting body culture: cutting the bag material full of mycelia to grow mushrooms, continuously controlling the temperature to be 27-28 ℃, the air humidity to be 70-75% and the illumination intensity to be 50lux to carry out constant-temperature culture, keeping the interval ventilation state among the cultures, observing the fruiting condition of the bag material at any time, harvesting in time and recording the mature time of the fruiting bodies;

(4) crushing the collected phellinus igniarius sporocarp into medium powder in a swing type crusher and fully and uniformly mixing; putting another 20g of powder into a round-bottom flask, adding 500mL of ethanol water solution with volume content of 70 v/v%, soaking for 1h, performing reflux extraction for 2h, filtering, and collecting filtrate and residues respectively;

(5) adding 500mL of water into the ethanol extraction residue, and decocting and extracting for 2 h;

(6) mixing the above ethanol extractive filtrate and decoction, placing in rotary evaporator, recovering ethanol until no ethanol smell is observed, and vacuum drying to obtain Phellinus linteus fruiting body extract.

Example 2

(1) preparing a cultivation bag material: taking mulberry twig wood chips30 parts by weight of brewing waste residue, 30 parts by weight of molasses, 3 parts by weight of aerobic activated sludge particles, 5 parts by weight of quicklime and KH₂PO₄0.2 part by weight, and uniformly mixing to obtain the required culture medium; according to the proportion of 50%: adding water into the culture medium in a mass ratio of 50%, uniformly mixing, and bagging for later use;

Example 3

(1) preparing a cultivation bag material: 25 parts of mulberry twig sawdust, 40 parts of brewing waste residue, 20 parts of molasses, 5 parts of activated sludge particles, 3 parts of quick lime and KH₂PO₄0.3 part by weight, and uniformly mixing to obtain the required culture medium; according to the proportion of 50%: adding water into the culture medium in a mass ratio of 50%, uniformly mixing, and bagging for later use;

Comparative example 1

The method for preparing the artificially cultured phellinus linteus fruiting body extract of the present comparative example is the same as example 3, except that the aerobic activated sludge granules are not added to the culture substrate.

Examples of the experiments

1. Phellinus igniarius sporophore character

The time for culturing mycelium and fruiting body of Phellinus linteus in the above examples 1-3 and comparative example 1 was recorded, respectively, and the weight and quality of harvested Phellinus linteus fruiting body were measured, and the recorded results are shown in Table 1 below.

TABLE 1 Phellinus linteus fruiting body culture

As can be seen from the above results, the method for cultivating phellinus igniarius sporocarp based on artificial bag material of the invention can effectively shorten the cultivation time of phellinus igniarius sporocarp and improve the cultivation quality of phellinus igniarius sporocarp.

2. Effect of drugs on nitroglycerin-induced rat migraine model

Nitroglycerin is a fat-soluble compound, easily penetrates the blood brain barrier, and is a Nitric Oxide (NO) donor. Migraine-like headache can appear in migraine patients after nitroglycerin is infused for a period of time, and the NO synthase inhibitor can stop acute attack of migraine, which shows that NO plays an important role in pathogenesis of migraine.

Taking 40 SD rats (with the weight of 200 +/-20 g) and dividing the rats into 4 groups of 10 rats according to weight hierarchy, wherein the groups are respectively a model group, a positive group, an experimental group (an extract of example 3) and a control group (an extract of comparative example 1); wherein the content of the first and second substances,

model group: nasal drip normal saline with the same volume as the experimental group;

a positive drug group: 5 mg/capsule of flunarizine hydrochloride, which is a commercial product, and the administration dosage is 2mg/kg (twice of the clinical equivalent), and the flunarizine hydrochloride is administrated by intragastric administration;

experimental groups: the administration preparation is the extract prepared in the example 3, suspension is prepared for nasal drop administration, and the administration dosage is 0.5mg/kg body weight according to the amount of the extract;

control group: the administration preparation was the extract prepared in comparative example 1, and a suspension was prepared for nasal administration at an administration dose of 0.5mg/kg body weight in terms of the amount of the extract.

The animals of the above groups were injected with nitroglycerin injection 10mg/kg, 0.2ml/100g subcutaneously, 2 minutes after molding, the experimental group was administered by nasal drip for 1 time, and migraine reaction of rats was continuously observed for 180 minutes after administration.

From the beginning of modeling, taking every 30min as a time interval, adopting a duration segmentation method, respectively recording the times of head scratching and cage climbing of the rat in each time interval, calculating the total times of cage climbing and the total times of head scratching of all the rats, and showing the test result in table 2; the time of the first appearance of head-bending and the incidence of ear-red were also recorded and the results are shown in Table 3.

TABLE 2 Total cage climbing times and Total head bending cumulative times

TABLE 3 first flex time and ear Red Rate

As can be seen from the data in tables 2-3 above, the Phellinus linteus fruiting body extract of the present invention has the effect and effect of treating migraine.

3. Fluorometric determination of 5-HT, NA and DA content in brain tissue and serum

After the behavioral observation of the whole body signs of the animals in the above experimental example 2 was completed, the brain tissue was taken out from each group of rats by decapitation, the brain was removed on an ice bench, the cerebellum was removed, the meninges and blood vessels were removed, the brain was washed with ice-cold physiological saline, the blood stain was removed, the filter paper was wiped, the hypothalamic part was excised, and ice-cold physiological saline was added to prepare a brain tissue homogenate.

Measuring the content of 5-HT, DA and NE in brain tissue of each group of animals, taking a blank group of rats which are not processed by the model as a blank group, and directly taking the brain tissue to measure the content of 5-HT, DA and NE. The test results are shown in Table 4.

TABLE 4 brain 5-HT, NA and DA content (ng/g)

Group of	5-HT	NA	DA
				Model set	655	495	1735
Positive group	1345	609	2252
				Experimental group	1319	598	2261
Control group	1308	595	2240
				Normal group	1382	615	2430

As can be seen from the results in table 4, the phellinus linteus fruit body extract of the present invention has the effect and effect of treating migraine.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims

1. The preparation method of the artificially cultured phellinus igniarius sporocarp extract for treating migraine is characterized by comprising the steps of artificially culturing the phellinus igniarius sporocarp and extracting the phellinus igniarius sporocarp, and specifically comprises the following steps:

2. The method for preparing the fruiting body extract of Phellinus linteus cultured artificially for treating migraine according to claim 1, wherein in the step (1), the activated sludge granules are aerobic activated sludge granules.

3. The method for preparing the fruiting body extract of Phellinus linteus artificially cultured for treating migraine according to claim 1 or 2, wherein in the step (1), the mass ratio of the culture medium to water is 40-50%: 50 to 60 percent.

4. The method for preparing fruiting body extract of artificially cultivated Phellinus linteus for treating migraine according to any one of claims 1 to 3, wherein in the step (2), the inoculation amount of Phellinus linteus mother species is 5-10 wt% of the cultivation medium.

5. The method for preparing the fruiting body extract of Phellinus linteus cultured artificially for the treatment of migraine according to any one of claims 1 to 4, wherein the process conditions of the mycelium culturing step in the step (2) include: the culture temperature is controlled to be 25-30 ℃, the air humidity is 50-80%, and the illumination intensity is 10-20 lux.

6. The method for preparing the fruiting body extract of Phellinus linteus cultured artificially for treating migraine according to any one of claims 1 to 5, wherein in the step (3), the process conditions of the fruiting body culturing step include: the culture temperature is controlled to be 25-30 ℃, the air humidity is 70-80%, and the illumination intensity is 30-100 lux.

7. The method for preparing the fruiting body extract of Phellinus linteus cultured artificially for the treatment of migraine according to any one of claims 1 to 6, wherein in the step (4):

the volume percentage content of the ethanol water solution is 60-80 v/v%;

8. The method for preparing the cultured Phellinus linteus fruiting body extract for the treatment of migraine according to any one of claims 1 to 7, wherein in the step (5), the ratio of the Phellinus linteus fruiting body powder to the water is 10 g: 200 and 300 mL.

9. An artificially cultivated phellinus linteus fruit body extract for treating migraine prepared by the method of any one of claims 1 to 8.

10. Use of Phellinus linteus fruiting body extract as claimed in claim 9 for the preparation of a medicament for the treatment of migraine.