TW201702382A - Hericium erinaceus having an effect of pain relief, Hericium erinaceus mycelium active substance, preparation method thereof, and pharmaceutical composition containing the same to obtain a novel compound of heripene having an effect of pain relief - Google Patents

Abstract

The present invention relates to a Hericium erinaceus active substance having an effect of pain relief and a pharmaceutical composition containing the active substance. The active substance is made by the following steps: (a) inoculating a Hericium erinaceus mycelium on a flat plate, and culturing at a temperature of 15-32DEG C for 8-16 days; (b) inoculating the Hericium erinaceus mycelium cultured in step (a) in a flask culture medium, and culturing at a temperature of 20-30DEG C and a pH of 4.5-6.5 for 3-5 days; (c) inoculating the Hericium erinaceus mycelium cultured in step (b) in a fermenter culture medium, and culturing at a temperature of 24-32DEG C and a pH of 4.5-5.5 for 8-16 days to obtain a Hericium erinaceus mycelium fermentation broth; (d) drying the Hericium erinaceus mycelium fermentation broth obtained in step (c) to obtain a Hericium erinaceus mycelium powder. The Hericium erinaceus mycelium powder can be further extracted with an alcohol solvent to obtain an alcohol extract, and further purified and separated to obtain a novel compound of heripene.

Description

Hericium erinaceus active material for reducing pain, Hericium erinaceus mycelium active substance, preparation method thereof, and pharmaceutical composition containing same

The present invention relates to an active substance having a pain-reducing effect, and more particularly to an active substance derived from Hericium erinaceus.

Pain neural transmission pathway and P2R

The generation of pain requires a pain signal to be transmitted through a series of nerve paths, and finally the message is transmitted to the brain, which can make people feel pain. The initiation of the neural transmission pathway of pain is the stimulation. Then the pain receptor converts the stimulus into a nerve signal and transmits the nerve signal to the dorsal root ganglia (DRG). This signal passes through the spinal cord and finally arrives. The hypothalamic area of the brain. In previous studies, it was pointed out that the distribution of P2-purinoceptors (P2R) can be seen in the series of nerve conduction pathways mentioned above, such as sensory nerves, sympathetic nerves, parasympathetic nerves, and central nervous tissues. The P2R on the nerve is mainly P2X ₃ R and P2X _2/3 R.

The prior art on the neurotransmission pathway of P2R and pain is listed as follows:

1. The gene shaving mouse model confirmed that P2X ₃ R, P2X ₄ R and P2X ₇ R were associated with pain receptor transmission, inflammatory pain and neuropathic pain. (Collier et al., 1966; Cockayne et al., 2000; Souslova et al., 2000; Jarvis et al., 2002; Tsuda et al., 2003; Khakh and North, 2006; Burnstock, 2008; Beggs et al. , 2012)

2. P2X ₃ R gene shaving ATP did not cause neuronal transmission on the dorsal root ganglia (DRG) and blocked peripheral pain transmission into the central nervous system. (Cockayne et al., 2000)

3. After removing P2X ₃ R, the amplitude of inflammatory pain caused by formalin decreased. (Souslova et al., 2000)

4. Switch to P2X ₃ R antagonists to block immediate pain, inflammatory pain, and visceral pain associated with neuropathic pain. (Jarvis et al., 2002)

5. In nerve-damaged mice, the amount of P2X ₄ R increased, resulting in long-term pain sensitivity. (Tsuda et al., 2003)

6. In the gene-dissected P2X ₄ R mice, the mice were found to have no pain sensitivity caused by physical damage, and a nerve signal path that was transmitted to the spine due to lack of peripheral nerve damage. (Ulmann et al., 2008)

7. In the gene shaving P2X ₇ R mice, it was found that there was almost no sensitization of inflammation and neuropathy. (Chessell et al., 2005)

8. P2X ₇ R antagonists inhibit the effects of neuropathic pain and inflammatory pain. (Honore et al., 2006)

P2R

P2R can be divided into two groups according to its receiver structure and signal transmission path (Yang and Liang, 2012), which are P2X _1-7 receiver and P2Y _1-14 receiver, respectively. The P2X _1-7 receptor has two transmembrane domains, and the C-terminal and N-terminal regions of the protein are located in the cell. In addition, there is a protein ring structure outside the cell. ). Among P2X _1-7 R, the smallest P2X ₄ R has 388 amino acids; the largest P2X ₇ R has 595 amino acids. P2XR is a cation pipe type receptor that allows Na ⁺ and Ca ^{2+ to} pass. After the receptor is stimulated, Ca ²⁺ will flow through the P2XR term, causing the intracellular free calcium concentration to rise ([Ca ²⁺ ]c↑) and increase the intracellular free calcium concentration ([Ca ²⁺ ]c↑) As a secondary signal, it causes a subsequent physiological response, and the nerve signal is transmitted back to the cerebral ventricle through the spinal cord, causing pain. At the same time, the increase of intracellular free calcium ion concentration ([Ca ²⁺ ]c↑) also affects the secretion of cells, specifically, it can cause the release of cytokine by neuroimmune cells. On the other hand, the P2Y _1-14 receptor is a G-protein coupled receptor (GPCR). Except for the P2Y _11-14 receptor, the other P2Y subtypes are associated with the G protein (G-protein, Gq). ) There is a link. When Gq is activated, it activates phospholipase C-β (PLC-β) to cleave phospholipid 4,5-diphosphoinositol (PIP2), which in turn produces inositol 1,4,5-triphosphate (IP3) Then, IP3 will bind to the IP3 receptor on the endoplasmic reticulum, and the calcium ions stored in the endoplasmic reticulum will be released, causing the intracellular free calcium concentration to rise ([Ca ²⁺ ]c↑). Accordingly, both P2XR and P2YR (except for the P2Y _11-14 receptor) can use the change in intracellular free calcium concentration as an indicator of the function of the receptor.

ATP

ATP is a well-known energy-carrying metastatic biomolecule. In 1929, Szent-Gyorgyi discovered that ATP also has biological functions outside the cell. In 1996, ATP was officially regarded as a neurotransmitter in the neuroscience field. In the P2R-related neural message transmission pathway, ATP is used as a neurotransmitter of P2R. When the nerve is stimulated, ATP releases from the synaptic vesicles into the synaptic cleft and stimulates P2R on the postsynaptic neurons, which are then rapidly broken down by the enzyme at the synaptic cleft. The above model of neurotransmission is also supported in the previous literature, as follows:

1. ATP mediators transmit signals to the presynaptic and postsynaptic nerves and act as neurotransmitters (Burnstock, 2006a; Burnstock, 2008)

2. ATP will be released when the tissue is injured, stimulating P2R media pain on the pain receptor.

3. ATP was found to stimulate P2R mediators on human skin to cause pain (Collier et al., 1966).

4. ATP plays an important role in pain transmission (Burnstock, 2006b; Burnstock, 2013).

Pain medication needs

With the aging of the social population and the gradual improvement of the quality of life, the market for painkillers is becoming more and more popular. Among the many types of pain, the more difficult pain to resolve is neurophathic pain and cancer pain caused by neuropathy, and the other is because there are few effective drugs that can completely suppress pain. Long-term pain symptoms require long-term use of analgesics. At present, there are mainly two types of painkillers in the clinic, one is a morphine analgesic and the other is a non-narcotic analgesic. If morphine analgesics are administered to patients for a long period of time, there will be problems of tolerance and dependence. Tolerance means that the efficacy of the drug will decrease with the number of times the drug is administered, and the dose must be administered. Aggravation can achieve a certain analgesic effect. Non-narcotic analgesics do not have the adverse effects of anesthetics, such as respiratory depression, habituation, etc., but also have analgesic and anti-inflammatory effects, which can reduce the rising body temperature (antipyretic effect) to slow the symptoms of inflammation. These drugs are commonly used to relieve mild to moderate pain caused by diseases such as rheumatism, arthritis, sciatica, and degenerative arthritis. These drugs are in turn non-steroidal anti-inflammatory agents (NSAIDs) with the side effect of inhibiting both COX-1 and COX-2. When COX-2 is inhibited, PGI2 is reduced to produce anti-inflammatory and analgesic effects, but it also promotes platelet aggregation. When COX-1 is inhibited, it will destroy the integrity of the gastrointestinal mucosa and affect the renal blood flow, which may cause side effects such as stomach injury and kidney, and inhibit platelet aggregation and become more likely to bleed. Therefore, the safety, side effects, addiction and tolerance of analgesics have become problems to be solved in order to develop better analgesics.

Hericium

Anesthesiologist Allen W. Burton said that "a new analgesic drug is not enough to meet the clinical needs of cancer, and we are often at a loss for many patients with refractory pain (Burton et al., 2007)." So with the new biological pathway as the target, Developing safe and effective analgesics is important and urgently needed for current clinical medicine. Based on this, from the safety considerations, looking for various actives from natural ingredients Quality, and the preparation of drugs for long-term use is a trend in the current industry.

According to the "Chinese medicinal fungi" records: "Herbal-headed mushrooms are sweet, flat, can benefit the five internal organs, help digestion, nourish, have good effects on indigestion, neurodegenerative and duodenal ulcers and gastric ulcers." It is known in ancient medicine that the Hericium erinaceus has the effect of treating diseases and is a dual-use medicinal diet. Hericium erinaceus , which is classified under the fungus (Fungi), Eumycota, Basidiomycotina, Basidiomycetes, Aphyllophorales, Hydnaceae ), Hericium in the family Hericioideae. On the appearance of Hericium erinaceus, the shape of its fruit body is composed of long and rough protrusions, which are soft round shape, white when fresh, and turn yellow brown after drying. The extracts from the fruiting bodies or mycelia of Hericium erinaceus contain sugars (Wang et al., 2001; Yang et al., 2003) and Erinacines (Saito et al., 1998; Kenmoku et al. , 2002; Kenmoku et al., 2004; Watanabe et al., 2007; Watanabe and Nakada, 2008; Lee et al., 2014; Li et al., 2014), dilinoleoyl-phosphatidylethanolamine ; DLPE) (Nagai et al., 2006), amino acids, proteins and trace elements (Jia et al., 2004). In the past literature, there is no shortage of the effect of the polysaccharide of Hericium erinaceus on immune mediation, hypolipidemic, hypoglycemic or inhibition of gastric inflammation and gastric cancer. So far, Japanese scholars have isolated and purified 13 kinds of cephalosporins (A, B, C, D, E, F, G, H, I, J, K, P, Q, R) from liquid fermentation products. And cephalosporin stimulates mouse stellate cells to secrete nerve growth factor (NGF) to treat diseases such as mental decline and neurasthenia. The inventors isolated, purified and identified the new compound heripene from the mycelium extract of Hericium erinaceus, and confirmed the experimental results to confirm the analgesic effect of the monkey head, as detailed later.

The object of the present invention is to provide a preparation of active substances of Hericium erinaceus and Hericium erinaceus A method which can be used to produce a monkey head mites having an effect of reducing pain, an active substance, and a pharmaceutical composition comprising the monkey head lice or the active substance.

In order to achieve the foregoing object, the present invention provides a method for preparing an active substance of Hericium erinaceus with reduced pain effect, comprising the following steps: (a) inoculating a Mycelium of Hericium erinaceus on a plate at a temperature of 15-32 ° C. The culture is carried out for 8-16 days; (b) the mycelium of the Hericium erinaceus cultured in the step (a) is inoculated into a flask medium, and cultured at a temperature of 20-30 ° C and a pH of 4.5-6.5 for 3-5 days; (c) inoculating the mycelium of Hericium erinaceus cultured in step (b) in a fermentation tank medium, and cultivating for 8-16 days at a temperature of 24-32 ° C and a pH of 4.5-5.5 to obtain a monkey mushroom Mycelium broth; (d) The step (c) of the Hericium erinaceus mycelium broth is dried to obtain a mycelium mycelium powder.

Preferably, the culture of the step (b) in the above preparation method is an oscillating culture, and the oscillation rate is 100 to 250 rpm.

Preferably, in the step (c) of the above preparation method, the tank pressure of the fermentation tank is 0.8-1.2 kg/cm ² , the stirring rate is 10-120 rpm, and a gas is introduced into the gas at aeration rate of 0.5-1 vvm. Fermentation tank.

Preferably, the gas in the aforementioned preparation method is air, oxygen, carbon dioxide, nitrogen, or a combination thereof.

Preferably, the same medium is used in the step (b) and the step (c) in the above preparation method.

Preferably, the medium in the above preparation method comprises a comprehensive carbon and nitrogen source, an animal or plant derived protein or a hydrolyzate thereof, an inorganic salt, a saccharide, a yeast or malt extract, and an antifoaming agent.

Preferably, the comprehensive carbon and nitrogen source in the preparation method is cereal or legume; inorganic salt It is a sulfate or a phosphate.

Preferably, the mycelium of Hericium erinaceus in step (d) is further extracted with an alcohol solvent to obtain a mycelial extract of Hericium erinaceus.

Preferably, the alcohol solvent is 30-100 v/v% ethanol or 30-100 v/v% methanol.

Preferably, the mycelium alcohol extract of Hericium erinaceus is further extracted with water and ethyl acetate, and then analyzed by column chromatography to obtain a heripene having the following structural formula (I):

Wherein R is hydrogen, hydroxyl, C ₁ -C ₁₀ alkyl, C ₁ -C ₁₀ alkenyl, or C ₁ -C ₁₀ alkynyl, wherein the aforementioned functional group is selectively halogen, oxygen, nitrogen Substituted by a substituent of phosphorus, or sulfur; wherein the steric structure of the 5th, 6th, 9th, 13th, 14th, 21st, 22nd, and 23th carbon atoms is R or S.

The aforementioned functional group is optionally substituted by the aforementioned substituent, and the functional groups of the above two R may be substituted individually or simultaneously by a substituent containing a halogen, oxygen, nitrogen, phosphorus, or sulfur.

Preferably, when R of the formula (I) of the genus Hericium is hydrogen, it is a erinacine S having the following structural formula (II):

Preferably, the ratio of water to ethyl acetate is 1:4.

Preferably, the pain in the aforementioned preparation method is neuropathic pain or cancer pain.

Preferably, the painful signal path in the aforementioned preparation method comprises P2R.

The present invention further provides a monkey head active material having a pain-reducing effect, which is prepared by any one of the aforementioned preparation methods.

Preferably, the active substance is prepared by a partial preparation method as described above, wherein the active material of the Hericium erinaceus is in the form of a powder.

Preferably, the aforementioned active substance is prepared by a partial preparation method as described above, wherein the active material of the Hericium erinaceus is an alcohol extract.

Preferably, the aforementioned active substance is prepared by the partial preparation method as described above, wherein the active material of the Hericium erinaceus is the monkey head carp having the structural formula (I).

Preferably, when R of the formula (I) of the genus Hericium is hydrogen, it is a cephalosporin S having the formula (II).

The present invention further provides a compound of the formula, having the structural formula (I).

Preferably, when R of the formula (I) of the genus Hericium is hydrogen, it is a cephalosporin S having the formula (II).

Preferably, the aforementioned Hericium erinaceus compound has the effect of reducing pain.

The invention further provides a pharmaceutical composition for reducing pain comprising the aforementioned Hericium erinaceus active, and a biologically acceptable carrier, excipient, diluent or adjuvant.

The invention further provides a pharmaceutical composition for reducing pain comprising the aforementioned Hericium erinaceus, and a biologically acceptable carrier, excipient, diluent or adjuvant.

The first panel shows the HPLC analysis profile of the cephalosporin S in Example 4.

The second panel shows the gene expression of the P2R receptor on the cells by RT-PCR in the second point of Example 5.

The third figure shows that in the first point of the sixth embodiment, the cells are filled with a fluorescent agent, and the calcium signal of the receptor on the cell is detected. Among them, (A) human osteosarcoma HOS cells, (B) rat nerve PC12 cells, (C) human neuroma SH-SY5Y cells measured by ATP stimulation; (D) human neuroma SH-SY5Y cells The result of carbon choline stimulation.

The fourth panel shows the calcium signal linked to the P2XR subtype on human osteosarcoma HOS cells in the second point of Example 6.

The fifth panel shows the calcium signal linked to the P2YR subtype on human osteosarcoma HOS cells in the second point of Example 6. Among them, (A) calcium signal caused by ATP stimulation in calcium-free buffer; (B) calcium signal caused by UTP stimulation.

The sixth panel shows the effect of the mycelium extract of Hericium erinaceus on the ATP-stimulated P2R calcium signal in human osteosarcoma HOS cells in the third point of Example 6.

Figure 7 shows the inhibitory effect of (A) alcohol extract and (B) Hericium S on the calcium signal of P2R stimulated by ATP in human osteosarcoma HOS cells in the fourth point of Example 6.

Figure 8 shows the inter-group ratio of the flash tail experiment performed on the alcohol extract of Hericium erinaceus and the alcohol extract of Hericium erinaceus in the first point of Example 7 for delaying short-term pain. Compared.

The ninth panel shows the comparison between the groups before and after the administration of the alcoholic extract of the Hericium erinaceus and the alcohol extract of Hericium erinaceus in the second point of Example 7 for the short-term pain effect.

The tenth graph shows the maximum possible calculation of the difference between the pre- and post-dose of the flash tail experiment performed on the alcohol extract of Hericium erinaceus and the alcohol extract of Hericium erinaceus in the third point of Example 7 Comparison between effects of groups.

Fig. 11 is a view showing the comparison between the groups of the seventh embodiment of the seventh embodiment, the fermented mixture of the genus Hericium erinaceus and the alcohol extract of Hericium erinaceus, and the hot plate experiment for delaying the long-term pain effect.

The object of the present invention is to provide a preparation method by which various active substances derived from Hericium erinaceus can be prepared, and the novel compound Hericium erinaceus can be further purified and isolated, and the active material can be further Further prepared into a pharmaceutical composition having the effect of reducing pain.

Experimental principle

The P2R cell model was established to detect P2R function. Firstly, the P2R expressed on the cell strain was detected by RT-PCR, and then the stimulant was used to stimulate the function of the receptor. Since both P2XR and P2YR can be linked to the calcium ion signal, The intracellular free calcium ion concentration serves as a detection item for the receptor function. Although pain sensation can be transmitted to the central nervous system through P2R, it is still necessary to determine the effect of pain on the pain reduction by confirming the behavior of the animal to confirm the occurrence of pain. Therefore, the two kinds of behavioral tests of painful flash tail and thermal pain were used to determine the pain intensity of the living body of the animal. In this case, through the analysis of the above pain mechanism and the in vivo analysis of animal behavior, the potential analgesic active ingredient is searched for from natural edible mites, and further developed to contain the serotonin S or the active ingredient. Pharmaceutical composition.

experimental method

Source of bacteria:

The Hericium erinaceus strain used in the examples of the present invention was purchased from the Food Industry Research (BCRC 35669), but the active material of the Hericium erinaceus described in the present invention is not limited to the strain.

Liquid culture:

The liquid culture method of the mycelium of Hericium erinaceus is as follows, which comprises inoculating the mycelium on a plate and culturing at a suitable temperature such as 15-32 ° C for about 14 days. Next, the hyphae were scraped off and inoculated into the flask, and cultured under shaking at 20-30 ° C, pH 4.5-6.5, and shaking rate of 100-250 rpm until the beginning of the log period, 3-5 days, using the following medium. Finally, the flask culture is inoculated into the fermentation tank medium (with the flask medium) at a pressure of 0.5-1.2 v/cm 2 at 24-32 ° C, and a pH of about 4.5-5.5, at a vapour rate of 0.5-1 vvm. Air, or a mixture of air and oxygen, carbon dioxide or nitrogen, preferably air, for 8-16 days at a stirring rate of 10-150 rpm, to obtain a mycelium fermentation mycelium, including mycelium and clarification liquid.

The medium formula is as follows:

The comprehensive carbon and nitrogen source may be cereals (such as wheat flour, bran) or beans (such as soybean powder, mung bean powder, soy flour, etc.); wherein the inorganic salt may be magnesium sulfate or dipotassium hydrogen phosphate. , potassium dihydrogen phosphate, iron sulfate, zinc sulfate, etc.; wherein the sugar may be glucose, Fructose, maltose, sucrose, etc.; wherein, in addition to the above ingredients, the remainder is water.

An antifoaming agent may be additionally added to the fermentation tank medium to inhibit the formation of a large amount of foam during the cultivation, wherein the defoaming agent may be a commercially available conventional defoaming agent, such as 0.01% antifoaming agent such as eucalyptus oil or enamel resin. An aqueous defoamer. The culture method of the specific examples is as described later.

Fluorescence detection of calcium signals:

The cells were filled with calcium fluoride Fura-2/AM, and the fluorescence intensity change produced by the emission wavelength of 505 nm was measured by a fluorescence spectrum analyzer (SPEX 1681) at a double excitation wavelength of 340 nm and 380 nm. During the experiment, ATP was added to stimulate P2R, and the fluorescence change was observed. Finally, 1% digitonin was added to make the cells transparent, and the maximum fluorescence ratio (Rmax) was obtained under 2 mM Ca ²⁺ . Ethylene glycol bis(2-aminoethylether) tetraacetic acid (EGTA) is combined with calcium ions to obtain the minimum fluorescence ratio (Rmin), and the dissociation constant (Kd) of fura-2 and Ca ²⁺ It is 225nM. The intracellular calcium ion concentration was determined by the formula (Grynkiewicz et al., 1985).

Pain flash tail test:

The tail flick test (Fukui et al., 2001; Okada et al., 2002) is primarily used to measure the pain threshold of infrared heat stimulation in the tail of a rat. During the experiment, the mouse tail (1 to 2 cm from the trailing end) was placed on a specific groove of the heating panel without anesthesia. Next, the infrared radiant heat source of the infrared light source calibration instrument is turned on, and the emitted infrared light is focused on the tail of the experimental animal through a parabolic mirror. When the experimental animal feels pain, the tail will tap the table. A reaction is called a flash tail reaction. When the flash tail reaction occurs, the built-in sensor can detect this reaction and simultaneously record the flash tail reaction time. The pain threshold can be displayed by the number of flash tail seconds, or by the change value of the flash tail seconds before and after administration, and even the change value of the flash tail seconds can be further calculated. The data calculation expression is the maximum possible impact percentage (Maximal Possible Effect). Calculation formula As follows: (% MPE) = (Postdrug latency - Predrug latency) / (Cutoff time - Predrug latency) * 100.

Mouse thermal pain test:

The mouse Hot-Plate Test (Yaksh and Tyce, 1979) first preheated the heating plate to a certain temperature, then placed the experimental animals on a metal panel and applied heat to stimulate the experimental animals. When the experimental animal feels pain due to heat, it will produce a jumping reaction called a contraction-reaction reaction, and the time required for each experimental animal to be placed on the hot plate until the jump occurs is called the contraction reaction time. Finally, the time of the contraction reaction is represented as the pain threshold of the experimental animal.

Example 1: Culture of Mycelium of Hericium erinaceus and Preparation of Active Substance

Plate culture:

The mycelium was inoculated on a plate medium, and cultured at 25 ° C for about 7 days using potato dextrose Agar (PDA).

Flask culture:

The hyphae on the plate were scraped and inoculated into the flask, and shaken on a shaker at about 26 ° C, pH 5.0, and a rotation speed of 120 rpm for 5 days;

Medium formula:

Fermentation tank culture:

The culture medium used in the fermentation tank culture is the same as the flask culture step, and the flask culture is inoculated into the fermentation tank medium at a stirring rate of 26-1.0 kg/cm 2 , pH 5.0, 10-150 rpm at 26 ° C or not. With air lift, air was introduced at a rate of 0.5-1.0 VVM and cultured for 12 days. After 12 days, the mashing solution was obtained, which included mycelium and clear liquid, and contained the active substance of Hericium erinaceus having a pain-reducing effect. After the lyophilized solution is freeze-dried, the Hericium erinaceus mycelium lyophilized powder (abbreviated as lyophilized powder) can be obtained, and 20 metric tons of the lyophilized broth can be lyophilized after being freeze-dried to obtain about 80 kg. powder.

Example 2: Alcohol extraction of mycelium active material of Hericium erinaceus

The lyophilized powder of Hericium erinaceus mycelium was added to the lyophilized powder by 25 times by weight of 95v/v% ethanol for the first extraction, and then extracted by ultrasonic shock at an oscillation rate of 120 rpm for one hour, and the suspension was centrifuged and centrifuged. After taking the supernatant. The supernatant was subjected to a second extraction with 85 v/v% ethanol, and the above extraction step was repeated to obtain a supernatant. Finally, the supernatant was concentrated under reduced pressure to obtain a creamy mycelium extract of Hericium erinaceus (referred to as an alcohol extract).

Example 3: Preparation of an alcohol extraction mixture

The above-mentioned lyophilized powder is uniformly mixed with the alcohol extract, followed by centrifugation, and the supernatant after centrifugation is taken, and the supernatant is completely dried by freeze-drying to obtain mycelium mycelium/alcohol. Extraction mixture (referred to as alcohol extraction mixture).

Example 4: Extraction and Analysis of Hericium S

The mycelial extract of Hericium erinaceus L. was extracted by liquid-liquid partitioning with water and ethyl acetate at a ratio of 1:4, and the obtained ethyl acetate layer was analyzed by tannin gel and LH-20 gel column chromatography. The new compound, erinacine S, was chromatographed on a Cosmosil 5C ₁₈ -AR-II column at 40 ° C with acetonitrile starting at 60 ° C, gradually increasing acetonitrile to 65% in 20 minutes at a flow rate of 1 ml / Min, the wavelength is 290 nm, and the cephalosporin S appears in about 14 minutes. The results of this HPLC analysis are shown in the first figure. The upper layer curve in the first figure is the analysis result of the alcohol extract obtained by the above experimental steps, and the lower layer is the monkey head standard (the standard product of the monkey head S is prepared by the applicant himself as a standard extraction). Whether or not the substance component contains the judgment basis of the monkey head S). The curve of the alcohol extract showed a peak at 2, 7, and 14 minutes. It can be confirmed that the peak of the alcohol extract and the unique peak of the cephalosporin standard appear at 14 minutes, and the alcohol extract obtained by the above steps contains the cephalosporin S.

The hydrogen spectrum and carbon spectrum of the cephalosporin S were obtained by NMR, as shown in the following table:

Example 5: Establishment and analysis of P2R cell model

Cell culture

Three cell lines were used, namely a rat nerve PC12 cell line, a human neuroma SH-SY5Y cell line, and a human osteosarcoma HOS cell. The PC12 cell line was cultured in a high sugar medium of Dulbecco's Modified Eagle Medium (DMEM) containing 10% horse serum (HS) and 5% fetal bovine serum (FBS) (Kao et al., 2012). The human neuroma SH-SY5Y cell line (Liu et al., 2009) was cultured in DMEM/F-12 medium containing 10% FBS. HOS cells are cultured in Minimum Essential Medium (MEM) containing Earle's salts, 2 mM L-glutamine, and 0.1 mM non-essential amino acids. It was added with 1.5 g/l sodium bicarbonate and 1 mM pyruvate sodium, and 5% FBS was added at the time of use. Incubate at 37 ° C, 5% carbon dioxide, change medium every 2-3 days, and subculture with trypsin-ethylenediaminetetraacetic acid (EDTA) (Liu et al., 2011) ).

2. Detection of P2R mRNA

Self-cellular extraction of messenger RNA (mRNA) to produce complementary DNA (cDNA), designing specific primers for each P2R subtype to be amplified by polymerase chain reaction (PCR) The amount of mRNA expression of the P2R subtype receptor was detected. First, the cells were washed with Phosphate buffered saline (PBS), and the mRNA was obtained through a column using a NucleoSpin® RNA II (MACHEREY-NAGEL) kit, and the RNA concentration was measured with an ACTGene ASP-2680 machine. Then to SuperScript® Ⅲ Reverse Transcriptase (Invitrogen) anti-enzyme reverse transcription polymerase chain reaction (RT-PCR), reverse transcription of mRNA to cDNA, cDNA and then by using this ^{Q-Amp TM 2XHotstart PCR master mix} ( containing Taq DNA polymerase) plus P2R primer for PCR. The resulting PCR product was subjected to colloidal electrophoresis using a 1.5% colloid in an electrophoresis tank containing 0.5X Tris borate edta (TBE) buffer. The electrophoresis voltage was set to 50 volts, and after 10 minutes, the conversion voltage was 100 volts and another 15 minutes passed. After the electrophoresis was completed, the colloid was stained with ethidium bromide (EtBr), and the colloid was transferred to a gelatinizing system to be excited by ultraviolet light, and images were recorded to observe the P2R of the cell strain.

The results of P2R gene expression were detected by RT-PCR on rat neural PC12 cell line, human neuroma SH-SY5Y cell line and human osteosarcoma HOS cells. The left column of the second figure shows the mRNA expression of each subtype of the P2X receptor, the right column shows the mRNA expression of each subtype of the P2Y receptor, and the bottom is the name of each cell strain. It is shown that P2XR subtypes are detected in human osteosarcoma HOS cells: P2X ₁ R, P2X ₄ R, P2X ₅ R, P2X ₆ R, P2X ₇ R, and P2YR subtypes are P2Y ₁ R, P2Y ₂ R, P2Y ₄ R, P2Y ₆ R, P2Y ₁₁ R; P2XR subtypes can be detected on human neuroma SH-SY5Y cells: P2X ₃ R, P2X ₄ R, P2X ₅ R, P2X ₆ R, P2X ₇ R, P2YR subtypes P2Y ₁ R, P2Y ₂ R, P2Y ₄ R, P2Y ₁₁ R; P2XR subtypes can be detected in rat neural PC12 cells with P2X ₂ R, P2X ₃ R, P2X ₄ R subtypes, P2YR subtypes with P2Y ₁₂ R.

Example 6: Analysis of the inhibitory effect of active substances on calcium signals

1. Detection of P2R-linked calcium signals on cell lines

The rat nerve PC12 cell line, human neuroma SH-SY5Y cell line and human osteosarcoma HOS cells were filled with Fura-2 calcium ion sensitive fluorescent dye to detect intracellular free calcium ion concentration and stimulate cell membrane through ATP. On the P2R, the change in the concentration of free calcium ions in the cells was observed. The third panel shows changes in the receptor calcium signal measured by human osteosarcoma HOS cells (A), rat neural PC12 cells (B), and human neuroma SH-SY5Y cells (C) stimulated with 0.1 mM ATP. However, since there is no change in the concentration of free calcium ions in the human neuroma SH-SY5Y cells, it is possible to exclude the biomolecules from which the cells do not produce calcium ion signals. Then, the acetylcholine receptor on the human neuroma SH-SY5Y cells was stimulated with 0.1 mM carbachol to confirm that the cells did have a calcium ion-related molecule, and the results are shown in the third graph ( D).

2. P2R subtypes linked to calcium signals

Comparing the P2R-linked calcium signals stimulated by ATP in three cell lines, the calcium ion concentration of human osteosarcoma HOS cells was the most obvious, so it was possible to clearly discriminate the effect of the added agents on P2R. Therefore, human osteosarcoma HOS cells were selected as materials, and P2R subtypes that bind calcium signals on human osteosarcoma HOS cells were further detected. 2-MeATP (P2X ₁ R, P2X ₅ R subtype selective stimulator), α, β- MeATP (P2X ₇ subtype selective stimulator), 2-MeSAT (P2X ₁ subtype selective stimulator), and CTP (P2X ₄ subtype selective stimulator) to detect P2R subtypes on human osteosarcoma HOS cells. The fourth picture shows the calcium ion signal caused by the proprietary stimulants of CTP, 2-MeATP, α, β -MeATP, and 2-MeSATP subtype receptors from left to right, and the results show human flesh. At least the calcium signals linked to P2X ₁ , P2X ₄ , P2X ₅ and P2Y ₇ were detected on the tumor HOS cells, and the P2X ₄ reaction was the most significant.

The P2Y receptor is linked to Gq, which stimulates the receptor to activate Gq, thereby activating phospholipase C, producing IP3, releasing calcium ions in the intracellular IP3-sensitive calcium reservoir, thus using calcium-free buffer. In the experiment, adding an appropriate amount of EGTA in the experiment can chelate the residual calcium ions in the buffer, resulting in no extracellular calcium ions in the cells. Under this condition, a single cell can be observed by observing the intracellular free calcium ion concentration. Calcium ions released from the internal calcium reservoir. Figure 5 shows the calcium signal connected to the P2Y receiver. (A) shows the calcium signal linked to the P2R stimulated by ATP in a buffer without calcium ions. Because there is no calcium influx, it is P2Y. The receptor is linked to the calcium signal, and (B) is the calcium signal caused by UTP (P2Y2 and P2Y4 stimulator) stimulating cells. The results of the fifth panel show that at least the calcium signals linked to P2Y ₂ R and P2Y ₄ R can be detected on human osteosarcoma HOS cells.

3. Hericium erinaceus active substance inhibits P2R-linked calcium signal

ATP is a bulk stimulant of P2R in the body, so the P2R receptor is stimulated with 0.1 mM ATP. Before the ATP stimulates the calcium signal induced by P2R, the alcohol extract of Hericium erinaceus is added. Since the secondary signal is equivalent to the physiological response of the receptor after stimulation, it can be seen from the sixth figure that the calcium signal of P2R caused by ATP It is completely inhibited, and the alcohol extract of Hericium erinaceus can inhibit P2R function. The above experiments have confirmed that there are various P2XR and P2YR on HOS cells, and the extract of Hericium erinaceus can completely inhibit the function of P2R receptor, so it is reasonable to speculate that the extract of Hericium erinaceus is P2X ₁ , P2X ₄ , P2X ₅ and P2Y ₇ and P2Y _{2 .} And the receiver such as P2Y ₄ has the effect of suppression.

4. The dependence of the active substances of Hericium erinaceus and the inhibitory function and concentration of Hericium S

Figure 7 (A) shows the extraction of the alcohol extracts of the different concentrations of 2.5, 5, 50 μg / ml, and the seventh figure (B) shows the administration of different concentrations of 5, 10, 15 μg / ml of the monkey head S The effect of intracellular calcium ion concentration induced by ATP-stimulated P2R receptor in human osteosarcoma HOS cells has an inhibitory effect, and the inhibitory effect is more significant as the dose administered is more significant, indicating that the dose is administered. The inhibitory effect is concentration dependent.

Example 7: Determination of the effect of active substances of Hericium erinaceus on reducing pain in animals

In this example, the alcohol extract obtained in the second embodiment and the alcohol extract mixture obtained in the third embodiment were observed by a flash tail test and a hot plate test to observe the short-term and long-term administration of the active substance of the monkey mushroom, and the mouse was externally The irritated pain responds to differences before and after administration.

1. Short-term application of the flash tail test of active substances of Hericium erinaceus

The experiment was divided into three groups: control group, Hericium erinaceus mycelium/alcohol extract group (alcohol extract mixture group), and Hericium erinaceus alcohol extract group (alcohol extract group). Each group of 5 C57BL/6 Narl male rats over 8 weeks was administered with 2500 mg/kg in a hose, and the control group was given an equal amount of water before administration, 30 minutes after administration, and The pain sensation of the mice was detected by a flash tail test 60 minutes after administration. its The results of the experiment were expressed as mean ± standard deviation and were counted by Student t test to analyze the difference between the alcohol extract group, the alcohol extract mixture group and the control group. Thereby, the effect of short-term administration of the active substance on pain reduction was observed, and the results are shown in the eighth figure.

Before the administration, the basic value of the flash tail was tested. The flash tail seconds of the three groups were 4.1 ± 1.5 seconds in the control group, 4.3 ± 1.9 seconds in the alcohol extraction mixture group, and 4.5 ± 1.2 seconds in the alcohol extract group. Wherein, between the alcohol extraction mixture and control group student t between the p-value group test of 0.8; between alcohol extraction and control groups student t p-value test of 0.6; between alcohol extraction mixture components with an alcohol extraction was set student t test of p A value of 0.8 indicates no significant difference between the groups before administration.

After 30 minutes of administration, the pain response of the mice was detected by the flash tail test. The results of the experiment showed that the flash tail seconds of the control group was 2.0 ± 0.5 seconds; the flash tail seconds of the alcohol extract mixture group was 6.2 ± 1.6 seconds; The group flash tail seconds is 8.4 ± 1.1 seconds. Between the alcohol extraction mixture and control groups student t p value test of 0.0008, with a significant difference; between alcohol extract, and control groups student t p value test of 0.000009, with a significant difference; between alcohol extraction mixture components with an alcohol extract, group The p value of student t test was 0.07, with no significant difference.

After 60 minutes of administration, the pain response of the mice was detected by the flash tail test. The results of the experiment showed that the number of flash tails in the control group was 2.1 ± 0.57 seconds; the number of flash tails in the alcohol extract mixture group was 5.7 ± 2.46 seconds; The number of flash tail seconds is 7.2 ± 1.9. There was a significant difference in the p-value of 0.0256 between the alcohol-extracted group and the control group. The p-value of the student t test between the alcohol extract group and the control group was 0.0016, which was significantly different; between the alcohol extract mixture group and the alcohol extract group. The p value of student t test was 0.33, with no significant difference.

2. The difference between the short-term administration of the active tail of the active material of Hericium erinaceus before and after the administration

The experimental conditions were similar to those of the above-mentioned "short-term application to the tail-flick test of the active substance of Hericium erinaceus", which compared the difference in the difference between the alcohol extract group, the alcohol extract mixture group, and the flash tail time between the control group and the control group. Thereby, the effect of short-term administration of the active substance on pain reduction was observed, and the results are shown in the ninth figure.

After 30 minutes of administration, the pain response of the mice was detected by the flash tail test. The results showed that the number of flash tails in the control group was shorter than that of the control group before the administration, and the difference was -2.1 ± 1.7 seconds; The number of flash tails in the mixture group was longer than that before the drug, and the difference was 2.6±2.18 seconds. The number of flash tails in the alcohol extract group was longer than that before the drug, and the difference was 3.9±1.61 seconds. There was a significant difference in the p- value of student t test between the alcohol extract mixture group and the control group of 0.008, and there was a significant difference between the alcohol extract group and the control group. The p value of the student t test was 0.00108, which was significantly different between the alcohol extract mixture group and the alcohol extract group. The p value of student t test was 0.35, with no significant difference.

After 60 minutes of administration, the pain response of the mice was detected by the flash tail test. The results showed that the number of flash tails in the control group was shorter than that of the control group before the administration, and the difference was -1.0±0.71 sec. The flash tail seconds of the mixture group were longer than that of the drug, and the difference was 2.0±2.53 seconds. The number of flash tails in the alcohol extract group was longer than that before the drug, and the difference was 2.6±1.77 seconds. Between the alcohol extraction mixture and control groups student t p value test of 0.033, with a significant difference; between alcohol extract, and control groups student t p value test of 0.0028, with a significant difference; between alcohol extraction mixture components with an alcohol extract, group There was no significant difference in the p value of 0.66 for student t test.

3. The maximum possible impact percentage of short-term application to the active tail of the Hericium erinaceus active substance

The experimental conditions are similar to the conditions of the above-mentioned "short-term application to the flash tail test of the active substance of Hericium erinaceus", and the maximum possible effects (MPE) are calculated, and the number of flash tail seconds detected after administration is reduced. The number of flash tails before dosing (single mouse flash tail basis value), and this difference is divided by the single mouse flash tail basis value, that is, the maximum possible impact percentage. Thereby, the effect of short-term administration of the active substance on pain reduction was observed, and the results are shown in the tenth chart.

After 30 minutes of administration, the pain response of the mice was detected by the flash tail test. The results showed that the number of flash tails in the control group was shorter than that of the control group before the administration, which was about half of the base value; The number of flash tail seconds is longer than that before administration; the number of flash tails in the alcohol extract group is about twice as long as before the medication. Between the alcohol extraction mixture and control groups student t p value test of 0.03, with a significant difference; between alcohol extract, and control groups student t p value test of 0.0134, with a significant difference; between alcohol extraction mixture components with an alcohol extract, group Student p test p value of 0.31, no significant difference.

After 60 minutes of administration, the pain response of the mice was detected by the flash tail test. The results showed that the number of flash tails in the control group was shorter than the base value before the administration; the number of flash tails in the alcohol extract mixture group was longer than that before administration. About twice as much; the number of flash tails in the alcohol extract group is longer than before the medication. Between the alcohol extraction mixture and control groups student t p value of 0.05 test, the substantial deviation; between alcohol extract, and control groups student t p value test of 0.004, with a significant difference; between alcohol extraction mixture components with an alcohol extract, group The p value of student t test was 0.61, with no significant difference. It is speculated that the active substance of Hericium erinaceus has an immediate effect of reducing pain, and the effect of reducing pain can last at least 60 minutes.

4. Thermal pain test for long-term administration of active substances of Hericium erinaceus

In order to test whether the long-term application of the active substance of Hericium erinaceus can delay the pain response caused by the heating plate, in the long-term pain, the active substance of 2500 mg/kg is fed for 3 days, and the feeding interval is 24 hours. Then, after feeding the active material for 3 days, a hot plate test was performed, and the number of times of Paw lifting, licking, or jumping was measured, and the results are shown in Fig. 11.

After 3 days of administration, the experimental results showed that the reaction time of the control group was 6.4±1.8 seconds; the reaction time of the alcohol extraction mixture was 11.7±2.5 seconds; the reaction time of the alcohol extraction group was extended to 11.5± 1.8 seconds. There was a significant difference in the p-value of the student t test between the alcohol extract mixture group and the control group of 0.005. There was a significant difference in the p-value of the student t test between the alcohol extract group and the control group of 0.0025. There was a significant difference between the alcohol extract mixture group and the alcohol extract group. The p value of student t test was 0.09, with no significant difference.

Thirty minutes after the first detection of thermal hyperalgesia for 3 days of administration, the reduction reaction time was again measured by the same experimental method. The experimental results showed that the reaction time of the control group was 6.3±0.8 seconds; the reaction time of the alcohol extraction mixture was 11.8±2.5 seconds; the reaction time of the alcohol extraction group was 8.9±1.2 seconds. Between the alcohol extraction mixture and control groups student t p value test of 0.007, with a significant difference; between alcohol extract, and control groups student t p value test of 0.010; between alcohol extraction mixture components with an alcohol extraction was set student t test of The p value was 0.09 with no significant difference.

The data of the first measurement after 3 days of feeding and the data measured after 30 minutes were combined and calculated. The experimental results showed that the reaction time of the control group was 5.7±2.4 seconds; the reaction time of the extraction of the Hericium erinaceus group was 11.7± 2.3 seconds; the reaction time of the alcohol extract group was 10.3 ± 2.0 seconds. Between the alcohol extraction mixture and control groups student t p value test of 0.00006, there is significant difference; between alcohol extract, and control groups student t p value test of 0.0003, with a significant difference; between alcohol extraction mixture components with an alcohol extract, group The p value of student t test was 0.81, with no significant difference.

According to this animal test, the effect of reducing the pain after 30 minutes of administration was remarkable in the short-term effect of the alcohol extract and the alcohol extract, and the effect of reducing pain after 60 minutes of administration was decreased. In the long-term test of feeding the alcohol extract mixture and the alcohol extract, there is a desensitization phenomenon, and the effect of reducing the pain sensation tends to be stable.

In summary, the active substance of the Hericium erinaceus provided by the present invention comprises: mycelium of Hericium erinaceus, alcohol extract of Hericium erinaceus, alcoholic mixture of Hericium erinaceus and Hericium S have been experimentally proven to reduce pain Its efficacy can be widely used in the treatment of pain relief.

Claims (24)

A method for preparing an active substance of Hericium erinaceus with reduced pain effect comprises the following steps: (a) inoculating a Mycelium of Hericium erinaceus on a plate and culturing at a temperature of 15-32 ° C for 8-16 days; b) inoculation of the mycelium of Hericium erinaceus cultured in step (a) in a flask medium, and culturing for 3-5 days at a temperature of 20-30 ° C and a pH of 4.5-6.5; (c) step (b) The cultured mycelium of Hericium erinaceus is inoculated into a fermentation tank medium and cultured for 8-16 days at a temperature of 24-32 ° C and a pH of 4.5-5.5 to obtain a mycelium mycelium fermentation broth; d) drying the Hericium erinaceus mycelium broth in step (c) to obtain a mycelium mycelium powder. The preparation method of the first aspect of the patent application, wherein the culture of the step (b) is an oscillating culture, and the oscillation rate is 100-250 rpm. The preparation method of the first aspect of the patent application, wherein the tank pressure of the fermentation tank in the step (c) is 0.8-1.2 kg/cm ² , the stirring rate is 10-150 rpm, and the aeration rate is 0.5-1 vvm. Gas to the fermentation tank. The preparation method of claim 3, wherein the gas is air, oxygen, carbon dioxide, nitrogen, or a combination thereof. The preparation method of claim 1, wherein the same medium is used in the step (b) and the step (c). The preparation method of claim 5, wherein the medium comprises a comprehensive carbon and nitrogen source, an animal or plant derived protein or a hydrolyzate thereof, an inorganic salt, a saccharide, a yeast or a malt extract, and an antifoaming agent. The preparation method of claim 6, wherein the comprehensive carbon and nitrogen source is cereal or legume; and the inorganic salt is sulfate or phosphate. The preparation method of claim 1, wherein the mycelium mycelium powder of the step (d) is further extracted with an alcohol solvent to obtain a mycelium alcohol extract of Hericium erinaceus. The preparation method of claim 8, wherein the alcohol solvent is 30-100 v/v% ethanol or 30-100 v/v% methanol. The preparation method of claim 8 , wherein the mycelium alcohol extract of the Hericium erinaceus is further extracted with water and ethyl acetate, and then analyzed by column chromatography to obtain a structural formula (I) having the following formula (I) Heripene: Wherein R is hydrogen, hydroxyl, C ₁ -C ₁₀ alkyl, C ₁ -C ₁₀ alkenyl, or C ₁ -C ₁₀ alkynyl, wherein the aforementioned functional group is selectively halogen, oxygen, nitrogen Substituted by a substituent of phosphorus, or sulfur; wherein the steric structure of the 5th, 6th, 9th, 13th, 14th, 21st, 22nd, and 23th carbon atoms is R or S. The preparation method of claim 10, wherein when the R of the formula (I) is hydrogen, the system is a erinacine S having the following structural formula (II): The preparation method of claim 10, wherein the ratio of the water to the ethyl acetate is 1:4. The preparation method of claim 1, wherein the pain is neuropathic pain or cancer pain. The preparation method of claim 13, wherein the signal path of the pain comprises a purinergic receptor (P2-purinoceptor, P2R). An active substance of Hericium erinaceus having a pain-reducing effect, which is prepared by the method of any one of claims 1-14. The active substance of the medicinal material of claim 15 is prepared by the method of any one of claims 1 to 7, wherein the active material of the Hericium erinaceus is in the form of a powder. The active substance of claim 15 of the patent application, when prepared by the method of claim 8 or 9, wherein the active material of the Hericium erinaceus is an alcohol extract. The active substance of claim 15 is prepared by the method of claim 10 or 12, wherein the Hericium erinaceus active substance is a monkey head having the formula (I). The active substance of claim 18, wherein when the R of the formula (I) is hydrogen, it is a serotonin S having the formula (II). A compound of the genus Hericium, having the structural formula (I). The compound of claim 20, wherein when the R of the formula (I) is hydrogen, it is a serotonin S having the formula (II). The compound of the genus Hericium, as claimed in claim 20, has the effect of reducing pain. A pharmaceutical composition having a pain-reducing effect comprising the Hericium erinaceus active substance according to any one of claims 15 to 19, and a biologically acceptable carrier, excipient, diluent or adjuvant. A pharmaceutical composition having a pain-reducing effect comprising a compound of Hericium erinaceus as claimed in claim 20 or 22, and a biologically acceptable carrier, excipient, diluent or adjuvant.