HERIPENES WITH PAIN-RELIEVING EFFECT, ACTIVE SUBSTANCES OF Hericium erinaceus MYCELIUM AND THE PREPARATION METHOD THEREOF, AND PHARMACEUTICAL COMPOSITION CONTAINING THE HERIPENES OR ACTIVE SUBSTANCES

Abstract

The present invention is related to an active substance of Hericium erinaceus having a pain-relieving effect, and a pharmaceutical composition including the active substance. The active substance is prepared using the following steps: (a) inoculating a mycelium of H. erinaceus on an agar plate and incubating at 15-32° C. for 8-16 days; (b) inoculating the incubated H. erinaceus mycelia from step (a) into a medium in a flask and incubating at 20-30° C. and pH 4.5-6.5 for 3-5 days; (c) inoculating the incubated H. erinaceus mycelia from step (b) into a medium in a fermentation tank and incubating at 24-32° C. and pH 4.5-5.5 for 8-16 days to obtain a fermented medium of the H. erinaceus mycelia; and (d) desiccating the fermented medium of the H. erinaceus mycelia from step (c) to obtain the powder of the H. erinaceus mycelia, which is further purified and isolated to obtain a novel compound of H. erinaceus.

Claims

1.-24. (canceled)

25. A preparation method for an active substance of a Hericium erinaceus for relieving a pain, comprising: (a) inoculating an H. erinaceus mycelium on an agar plate to be incubated; (b) inoculating the incubated mycelium in step (a) into a first medium on a small scale to be incubated; and (c) inoculating the incubated mycelium in step (b) into a second medium on a large scale to be incubated to obtain a fermented medium containing the active substance.

26. The preparation method according to claim 25, wherein the incubation in step (a) is performed at 15-32° C. for 8-16 days.

27. The preparation method according to claim 25, wherein the incubation in step (b) is performed at 20-30° C., pH 4.5-6.5, and a shaking rate of 100-250 rounds per minutes (rpm) for 3-5 days.

28. The preparation method according to claim 25, wherein in step (c) the second medium on the large scale is accommodated in a fermentation tank having a tank pressure of 0.8-1.2 kg/cm.sup.2 and a stirring rate of 10-150 rpm, a gas is introduced into the fermentation tank at an aeration rate of 0.5-1 volume per volume per minute (vvm), the gas is one selected from a group consisting of air, oxygen, carbon dioxide, nitrogen gas and a combination thereof, and the incubation is performed at 24-32° C. and pH 4.5-5.5 for 8-16 days.

29. The preparation method according to claim 25, wherein the first and the second media are the same.

30. The preparation method according to claim 29, wherein each of the first and the second media comprises one selected from a group consisting of a complex carbon and nitrogen source, animal or plant sources of one of a protein and a hydrolyzate thereof, an inorganic salt, a saccharide, a yeast extract, a malt extract, a defoaming agent and a combination thereof, and the complex carbon and nitrogen source is one of a grain and a legume, and the inorganic salt is one of a sulfate and a phosphate.

31. The preparation method according to claim 25, wherein the preparation method further comprises (d) desiccating the fermented medium to obtain a powder of the H. erinaceus mycelium.

32. The preparation method according to claim 31, wherein the powder is further extracted with an alcohol solution to obtain an alcohol extract of the H. erinaceus mycelium, and the alcohol solution is an ethanol solution of 30-100 volume-volume percentage (v/v %) or a methanol solution of 30-100 (v/v %).

33. The preparation method according to claim 32, wherein the alcohol extract is further extracted with water-ethyl acetate, followed by a column chromatography to obtain a heripene having a formula (I) as follows: ##STR00003## where R is one selected from a group consisting of hydrogen, hydroxyl, C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10 alkenyl and C.sub.2-C.sub.10 alkynyl, each of which is optionally substituted with a substitute bonding with one selected from a group consisting of halogen, oxygen, nitrogen, phosphorus and sulfur, and where the stereocenters at C-5, C-6, C-9, C-13, C-14, C-21, C22 and C-23 are one of an R configuration and an S configuration.

34. The preparation method according to claim 33, wherein the heripene is an erinacine S having a formula (II) as follows: ##STR00004## when R included in the formula (I) is hydrogen.

35. The preparation method according to claim 33, wherein the water-ethyl acetate has a ratio of 1:4 (v/v).

36. The preparation method according to claim 25, wherein the pain is one of a neuropathic pain and a cancer pain.

37. The preparation method according to claim 36, wherein the pain involves a signaling pathway with regard to a P2-purinoceptor (P2R).

38. An active substance of a Hericium erinaceus for relieving a pain as obtained according to claim 25.

39. The active substance according to claim 38, wherein the active substance is obtained in a form of a powder.

40. The active substance according to claim 39, wherein the powder is extracted as one of an alcohol extract and a heripene having a formula (I) as follows: ##STR00005## where R is one selected from a group consisting of hydrogen, hydroxyl, C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10 alkenyl and C.sub.2-C.sub.10 alkynyl, each of which is optionally substituted with a substitute bonding with one selected from a group consisting of halogen, oxygen, nitrogen, phosphorus and sulfur, and where the stereocenters at C-5, C-6, C-9, C-13, C-14, C-21, C22 and C-23 are one of an R configuration and an S configuration.

41. The active substance according to claim 40, wherein the heripene is an erinacine S having a formula (II) as follows: ##STR00006## when R included in the formula (I) is hydrogen.

42. A pharmaceutical composition for a pain-relieving effect, comprising an active substance of a Hericium erinaceus according to claim 36, and one selected from a group consisting of a biologically acceptable carrier, an excipient, a diluent and an adjuvant.

43. A heripene compound for a pain-relieving effect, including a formula (I) as follows: ##STR00007## where R is one selected from a group consisting of hydrogen, hydroxyl, C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10 alkenyl and C.sub.2-C.sub.10 alkynyl, each of which is optionally substituted with a substitute bonding with one selected from a group consisting of halogen, oxygen, nitrogen, phosphorus and sulfur; and where the stereocenters at C-5, C-6, C-9, C-13, C-14, C-21, C22 and C-23 are one of an R configuration and an S configuration.

44. The heripene compound according to claim 43, wherein the heripene compound is an erinacine S having a formula (II) as follows: ##STR00008## when R included in the formula (I) is hydrogen.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0056] FIG. 1 illustrates an HPLC analytic spectrum of the erinacine S according to Embodiment 4.

[0057] FIGS. 2(A), 2(B), 2(C), 2(D), 2(E) and 2(F) show the gene expression of the P2R receptors in the cells, which are monitored using the RT-PCR according to Item 2 of Embodiment 5.

[0058] FIGS. 3(A), 3(B) and 3(C) show the calcium signals of the receptor in the cells, which are monitored after cells are filled with a fluorescent agent according to Item 1 of Embodiment 6, wherein (A) human osteosarcoma HOS cells, (B) rat pheochromocytoma PC12 cells, and (C) human neuroblastoma SH-SY5Y cells are stimulated with ATP and then with carbachol.

[0059] FIG. 4 shows the calcium signals correlated with the P2XR subtypes in human osteosarcoma HOS cells according to Item 2 in Embodiment 2.

[0060] FIGS. 5(A) and 5(B) show the calcium signals correlated with the P2YR subtypes in human osteosarcoma HOS cells according to Item 2 in Embodiment 2, wherein (A) the calcium signals are induced by ATP stimulation in a calcium free buffer, and (B) the calcium signals are induced by uridine triphosphate (UTP) stimulation.

[0061] FIG. 6 shows the effect of the H. erinaceus mycelia alcohol extract on the ATP-stimulated P2R's calcium signals in the human osteosarcoma HOS cells according to Item 3 in Embodiment 6.

[0062] FIGS. 7(A) and 7(B) show the inhibition effect of the calcium signals induced by (A) the alcohol extract and (B) the erinacine S treatments, followed by the ATP stimulation in the human osteosarcoma HOS cells according to Item 4 in Embodiment 6.

[0063] FIG. 8 shows the comparisons between groups in the tail-flick test on delaying the short-term pain-relieving effect after administering the H. erinaceus alcohol extraction mixture or alcohol extract according to Item 1 in Embodiment 7.

[0064] FIG. 9 shows the comparisons between groups in the tail-flick test on delaying the short-term pain-relieving effect before/after administering the H. erinaceus alcohol extraction mixture or alcohol extract according to Item 2 in Embodiment 7.

[0065] FIG. 10 shows the comparisons of the maximum possible effect calculated from the differences in the tail-flick test on delaying the short-term pain-relieving effect before/after administering the H. erinaceus alcohol extraction mixture or alcohol extract according to Item 3 in Embodiment 7.

[0066] FIG. 11 shows the comparisons between groups in the hot-plate test on delaying the long-term pain-relieving effect after administering the H. erinaceus alcohol extraction mixture or alcohol extract according to Item 4 in Embodiment 7.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0067] The purpose of the present invention is to provide a preparation method to prepare the miscellaneous types of active substances from H. erinaceus. Furthermore, novel heripene compounds are isolated and purified, and the heripenes and the active substances can further be prepared as a pharmaceutical composition with the pain-relieving effect.

[0068] Principles of the Experiments

[0069] The cellular model for P2R is established to detect the P2R's functions. At first, the P2R expression in the cell line is detected by the reverse transcription-polymerase chain reaction (RT-PCR). The receptor's functions are detected by the stimulation of the stimulator. The intracellular free calcium-ion concentration is used as the detection target of the receptor's functions because P2XR and P2YR can both be correlated with calcium-ion signaling. Although nociception can be generated in the central nervous system by the P2R transmission, it still needs to be confirmed via animal behavior. The pain-relieving effect of the active substances in the present invention can be identified by determining the generation of pain. Therefore, the level of nociception in live animals is determined by two behavioral experiments, the tail flick test and the hot-plate test. In the present invention, the potential analgesic active components are prepared from the naturally edible mushroom, analyzed via the nociception mechanism and the experiments on animals' behavior, and further prepared as a pharmaceutical composition including the erinacine S or the active substances.

[0070] Experimental Method

[0071] The Source of the Species:

[0072] The species, Hericium erinaceus, used in the embodiments of the present invention has been deposited in the China General Microbiological Culture Collection Center (CGMCC), Beijing, China with the Deposit No. CGMCC 10905. Originally, this species was purchased from the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute, Taiwan, with the Accession No. BCRC 35669. However, the active substances of H. erinaceus described in the present invention are not limited to one from which this species is obtained.

[0073] Submerged Cultivation:

[0074] The liquid culture of the H. erinaceus mycelia is described as follows. The mycelium of H. erinaceus was inoculated on an agar plate to incubate at an adequate temperature of 15-32° C. for about 14 days. Subsequently, the mycelium was inoculated into a medium (where the ingredients are listed below) in a flask, and incubated to the early log phase with shakes at 20-30° C., pH 4.5-6.5 at the shaking rate of 100-250 rounds per minute (rpm) for 3-5 days. Finally, the culture in the flask was inoculated into the medium (which has the same ingredients as in the flask) of the fermentation tank, and cultured at 24-32° C., a tank pressure of 0.8-1.2 kg/cm.sup.2, a pH value of about 4.5-5.5, an aeration rate of a gas of 0.5-1 volume per volume per minute (vvm) and a stirring rate of 10-150 rpm for 8-16 days to obtain a fermentation medium of H. erinaceus mycelia including the mycelia and the supernatant, where the gas is air, a mixture of air, oxygen, carbon dioxide or nitrogen, and the preferred gas is air.

[0075] The formula of the medium is listed as follows.

TABLE-US-00001 Ingredients Amount (wt. %) Complex carbon and nitrogen source 0.01-10 Animal or plant sources of protein or its hydrolyzate 0.01-5  Yeast or melt extract thereof (powder or cream) 0.001-2  Inorganic salt 0.0001-2   Saccharide 0.01-20 Defoaming agent  0.01-0.5 Water Add to 100 wt. %

[0076] The complex carbon and nitrogen source may be grain (e.g. wheat powder or wheat bran) or legume (e.g. soybean flour, mung bean flour and so on), the inorganic salt may be magnesium sulfate, potassium hydrogen phosphate, potassium dihydrogen phosphate, ferric sulfate, zinc sulfate and so on, the saccharide may be glucose, fructose, maltose, sucrose and so on, and the remainder is water in addition to the ingredients above.

[0077] A defoaming agent may be additionally supplemented into the medium in the fermentation tank to prevent the generation of too much foam during the cultivation, and may be commercially common ones, such as a 0.01% water-based defoaming agent including silicon oil and silicones. The cultivation method in the embodiment is described below in detail.

[0078] The Detection of Calcium Signals Using Fluorescence:

[0079] Cells were filled with a fluorescent dye-based calcium indicator, “Fura-2, AM cell permeant” (ThermoFisher Scientific, Waltham, Mass., U.S.A.), and the variation of the fluorescence level at an emission wavelength of 505 nm was determined at two excitation wavelengths of 340 nm and 380 nm using a fluorescence spectrometer (SPEX® 1681, Spec Industries, Inc., New Jersey, U.S.A.). During the experiment, ATP was added to cells to stimulate the P2R expression, and the variation of the fluorescence level was observed. Finally, 1 wt. % digitonin was added to enable the cells to become transparent, and the maximum fluorescence ratio (Rmax) was obtained at a concentration of 2 mM Ca.sup.2+. Ethyleneglycol bis(2-aminoethylether)tetraacetic acid (EGTA) then was added to chelate with the calcium ions to obtain a minimum fluorescence ratio (Rmin). The dissociation coefficient (Kd) of fura-2 to Ca.sup.2+ is 225 nM. The intracellular calcium-ion concentration was calculated using formulas (Grynkiewicz et al., J. Biol. Chem. 1985. 260(6):3440-3450).

[0080] Tail Flick Test:

[0081] The tail flick test (also called the thermosensitive tail-flicking assay) (Fukui et a., Eur. J. Pharmacol. 2001. 419(1):25-31; Okada et al., J. Pharmacol. Exp. Ther. 2002. 303(1):66-73) is mainly used to measure the threshold of nociception when a mouse's tail is stimulated by infrared radiation. During the experiment, mice were not anesthetized, and the mouse tail (1 to 2 cm away from the end) was disposed on a specific groove of a heating panel. Next, the infrared radiation heater from an infrared light source calibrator was turned on, and the emitted infrared concentrated on the mouse tail via a paraboloid reflector. When the experimental animal felt pain, its tail gently flicked the panel, and thus this reaction is called the tail flick test. When the tail flick reaction occurred, the built-in sensor detected this reaction and recorded the tail-flick reaction time. The threshold of nociception may be expressed as the seconds of the tail flick, also may be expressed as the variation of seconds before/after administration. Even the variation of the seconds of tail flick can be further calculated as a maximum possible effect (MPE). The formula is: (% MPE)=(Postdrug latency−Predrug latency)/(Cutoff time−Predrug latency)×100%.

[0082] Hot-Plate Test:

[0083] In the mouse hot-plate test (Yaksh and Tyce, Brain Res. 1979. 171(1):176-181), the heater was first pre-heated to a specific temperature, and then the experimental animal was subjected to the heat stimulation from the metal plate of the heater. The reaction that the experimental animal felt pain and jumped due to heat is called the paw withdrawal reaction. The required time that each experimental animal jumps after being disposed on the heater is called the paw withdrawal reaction time. Finally, the paw withdrawal reaction time refers to the threshold of nociception for the experimental animal.

Embodiment 1: The Incubation of H. erinaceus Mycelium and the Preparation of its Active Substances

[0084] The Incubation on the Agar Plate:

[0085] The H. erinaceus mycelium was inoculated on a potato dextrose agar (PDA) plate and incubated at 25° C. for about 7 days.

[0086] The Incubation in the Flask:

[0087] The H. erinaceus mycelium was aseptically scraped from the agar plate above to inoculate into the medium (see below) in the flask, followed by the shaking incubation in the orbital incubator at a shaking rate of 120 rounds per minute (rpm), at about 26° C., pH 5.0 for 5 days.

[0088] The formula of the medium is listed as follows.

TABLE-US-00002 Ingredients Amount (wt. %) Glucose 2.0 Yeast extract 0.1 Animal or plant sources of 0.1 protein and its hydrolyzate Magnesium sulfate 0.001 soybean flour 0.1 Water Add to 100 wt. %

[0089] The Incubation in the Fermentation Tank:

[0090] The medium used in the fermentation tank is the same as the incubation step for the flask. The incubated mycelia in the flask were inoculated into the medium in the fermentation tank. At 26° C., a tank pressure of 0.5-1.0 kg/cm.sup.2, pH 5.0 and with or without (air lift) a stirring rate of 10-150 rpm, air was introduced at an aeration rate of 0.5-1 volume per volume per minute (vvm), and the inoculated mycelia were incubated for 12 days. The fermented medium containing the mycelia, the supernatant and the active substances having the pain-relieving effect was obtained after 12 days. The fermented medium was freeze-dried to obtain freeze-dried powder of the H. erinaceus mycelia (abbreviated as “the freeze-dried powder”). A 20-metric ton fermented medium was freeze-dried to obtain about 80 kg freeze-dried powder.

Embodiment 2: The Alcohol Extraction of the Active Substances of H. erinaceus Myceha

[0091] The freeze-dried powder of the H. erinaceus mycelia was added to a 95 v/v % ethanol solution (1:25 w/w) to perform a first extraction, followed by the ultra-sonication at a sonication rate of 120 rpm for 1 hour. The suspension was centrifuged to obtain the supernatant. A second extraction was performed on the supernatant using 85 v/v % ethanol solution, and the ultra-sonication and centrifugation steps above were repeated to obtain a supernatant. Finally, the supernatant was concentrated under reduced pressure to obtain a cream of the H. erinaceus mycelia alcohol extract (hereinafter abbreviated as “the alcohol extract”).

Embodiment 3: The Preparation of the Alcohol Extract Mixture

[0092] The freeze-dried powder and an equal amount of the alcohol extract were mixed, and then centrifuged to obtain the supernatant. The supernatant was freeze-dried to obtain a mixture of the H. erinaceus mycelia and the alcohol extract (hereinafter abbreviated as “the alcohol extraction mixture”).

Embodiment 4: The Preparation and Analysis of the Erinacine S Standard

[0093] The preparation method for the erinacine S standard is described as follows. The H. erinaceus mycelia alcohol extract was subjected to liquid-liquid partition (H.sub.2O:ethyl acetate (EA)=1:4 (v/v)), and the obtained ethyl acetate extract was further subjected to column chromatography on silica gel and Sephadex® LH-20 silica gel, followed by the gradient elution of n-hexane:EA (10:1, 3:1, 3:2, 1:1, 1:2, 0:1; v/v), to obtain 7 sub-partitions. The sub-partition 3 (which was eluted with n-hexane:EA=3:2 (v/v)) was further subjected to column chromatography on the Sephadex LH-20 silica gel to obtain a new compound, erinacine S (i.e. formula (I)), whose structural formula was identified via chemical analysis, and characteristic analysis was performed via high performance liquid chromatography (HPLC). The erinacine S was obtained using a reverse chromatography column Cosmosil 5C18-AR-II (Nacalai USA, California, U.S.A.) at 40° C. and eluted using acetonitrile starting from 60 volume % and reaching to 65 volume % within 20 minutes at 1 ml/min of flow rate and 290 nm of the UV detection wavelength. The retention time of the erinacine S is 14 minutes. The HPLC analytic result is shown in FIG. 1 (where the label is the erinacine S standard). In FIG. 1, the upper curve indicates the freeze-dried powder of the H. erinaceus mycelia alcohol extract, and the lower curve indicates the erinacine S standard. The erinacine S standard is a standard prepared using the method above by the Applicant, and acts as a quantification basis for the erinacine S in the components of the extract.

[0094] The chromatography plot of the prepared alcohol extract in Embodiment 2 shows that peaks appear at the retention times of 2, 7 and 14 minutes. Comparing the erinacine S standard with the one peak at 14 minutes, it can be determined that the peak indicates erinacine S. The erinacine S in the alcohol extract is quantified as 59 ppm.

[0095] The .sup.1H and .sup.12C nuclear magnetic resonance (NMR) of the erinacine S are described in the following table.

TABLE-US-00003 position δ.sub.H (multiplicity, J in Hz) δ.sub.C HMBC (H.fwdarw.C) 1 1.54 (1H, dt, 13.2, 7.8) 38.2 (t) C2, C-3, C-4, C-8, C-9, C-17 1.61 (1H, dt, 13.2, 7.8) 2 2.28 (2H, t, 7.2) 28.4 (t) C-1, C-3, C-4, C-9 3 138.6 (s) 4 139.3 (s) 5 2.64 (1H, d, 9.6) 46.5 (d) C-3, C-4, C-6, C-9, C-10, C-11, C-16 6 42.5 (s) 7 1.80 (2H, m) 28.9 (t) C-5, C-6, C-8, C-9, C-16 8 1.40 (1H, m) 36.8 (t) C-1, C-7, C-9, C-17 1.51 (1H, dd, 12.6, 6.0) 9 49.6 (s) 10 1.69 (1H, m) 25.1 (t) C-4, C-5, C-6, C-11, C-12 2.02 (1H, m) 11 1.99 (1H, m) 32.3 (t) C-12, C-13, C-15 3.76 (1H, m) 12 129.4 (s) 13 3.80 (1H, dd, 10.8, 1.8) 49.1 (d) C-6, C-12, C-14, C-15, C-22, C-23 14 4.40 (1H, d, 10.8) 91.3 (d) C-6, C-7, C-12, C-16 15 142.7 (s) 16 1.11 (3H, s) 19.4 (q) C-5, C-6, C-7, C-14 17 1.07 (3H, s) 25.0 (q) C-1, C-4, C-8, C-9 18 2.77 (1H, septet, 6.6) 27.3 (d) C-2, C-3, C-4, C-19, C-20 19 0.97 (3H, d, 6.6) 21.4 (q) C-3, C-18, C-20 20 0.98 (3H, d, 6.6) 21.9 (q) C-3, C-18, C-19 21 6.13 (1H, s) 113.3 (s) C-13, C-14, C-25 22 93.0 (s) 23 83.2 (s) 24 193.6 (s) 25 4.48 (1H, d, 10.2) 80.8 (t) C-21, C-22, C-23, C-24 4.60 (1H, d, 10.2) 15-OH 10.76 (1H, s) C-12, C-15, C-24

Embodiment 5: The Establishment and Analysis of the Cell Model for P2R

[0096] 1. The incubation of cells:

[0097] Three cell lines, i.e. rat pheochromocytoma cell line PC12, human neuroblastoma cell line SH-SY5Y and human osteosarcoma cell line HOS, were used. PC12 cells were incubated in a Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (v/v) horse serum (HS) and 5% (v/v) fetal bovine serum (FBS) (Kao et al., Toxicol. Sci., 2012, 125(2):462-472). Human neuroblastoma SH-SY5Y cells were incubated in a DMEM/F-12 medium supplemented with 10% (v/v) FBS (Liu et al., J. Toxicol. Sci., 2009, 34(3):255-263). HOS cells were incubated in a minimum essential medium (MEM) (containing Earle's salts, 2 mM L-glutamine and 0.1 mM non-essential amino acids) supplemented with 1.5 g/L sodium bicarbonate and 1 mM pyruvate sodium, and an extra 5% (v/v) FBS was added during use. Cells were grown in a 5% CO.sub.2 humidified incubator at 37° C. The medium was replaced every 2-3 days, and cells were sub-cultured using trypsin-ethylenediaminetetraacetic acid (EDTA) (Liu et al., Eur. J. Pharmacol., 2011, 650(1):34-40).

[0098] 2. The Detection of P2R mRNA:

[0099] Messenger RNA (mRNA) was extracted from cells to prepare complementary DNA (cDNA). Specific primers for individual P2R subtype receptors were designed, wherein SEQ ID NOs: 1˜2, 3˜4, 5˜6, 7˜8, 9˜10, 11˜12 and 13˜14 respectively are primers for human P2X.sub.1, P2X.sub.2, P2X.sub.3, P2X.sub.4, P2X.sub.5, P2X.sub.6 and P2X.sub.7 subtype receptors, SEQ ID NOs: 15˜16, 17˜18, 19˜20, 21˜22, 23˜24, 25˜26, 27˜28 and 29˜30 respectively are primers for human P2Y.sub.1, P2Y.sub.2, P2Y.sub.4, P2Y.sub.5, P2Y.sub.6, P2Y.sub.9, P2Y.sub.10 and P2Y.sub.11 subtype receptors, SEQ ID NOs: 31˜32 are primers for human β-actin, SEQ ID NOs: 33˜34, 35˜36, 37˜38, 39˜40, 41˜42, 43˜44 and 45˜46 respectively are primers for rat P2X.sub.1, P2X.sub.2, P2X.sub.3, P2X.sub.4, P2X.sub.5, P2X.sub.6 and P2X.sub.7 subtype receptors, SEQ ID NOs: 47˜48, 49˜50, 51˜52, 53˜54, 55˜56, 57˜58 and 59˜60 respectively are primers for rat P2Y.sub.1, P2Y.sub.2, P2Y.sub.4, P2Y.sub.6, P2Y.sub.12, P2Y.sub.13 and P2Y.sub.14 subtype receptors, and SEQ ID NOs: 61˜62 are primers for rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the mRNA expressions of the P2R subtype receptors were amplified using polymerase chain reaction (PCR) and monitored. At first, cells were washed with phosphate buffered saline (PBS), and mRNA was obtained in the column using a NucleoSpin® RNA II kit (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany), and the concentration of RNA was determined using an ACTGene ASP-2680 spectrophotometer (CellTAGen, Seoul, Republic of Korea). Furthermore, the reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using SuperScript® III Reverse Transcriptase (Invitrogen™, ThermoFisher Scientific, Waltham, Mass., U.S.A.). mRNA was reverse-transcribed as cDNA, and then PCR was performed on this cDNA using a Q-Amp™ 2× HotStart PCR master mix (containing Taq DNA polymerase) (Bio-Genesis Technologies, Inc., Taiwan) and the P2R primers. The obtained PCR product was electrophoresed in 1.5% (w/v) agarose gel in the buffer (containing 0.5×Tris-borate EDTA (TBE)) in an electrophoresis tank. The voltage during the electrophoresis was set at 50 V, and the voltage was increased to 100 V for 15 minutes. After electrophoresis, the agarose gel was stained with ethidium bromide (EtBr), and then was transferred to a gel image system to be illuminated by ultraviolet (UV) radiation. The gel image was recorded, and the P2R expressed by the cells was observed.

[0100] The gene expressions of P2R in the rat pheochromocytoma cell line PC12, human neuroblastoma cell line SH-SY5Y and human osteosarcoma cell line HOS were determined using RT-PCR. FIGS. 2(A), 2(C) and 2(E) show the mRNA expressions of the P2X subtype receptors, FIGS. 2(B), 2(D) and 2(F) show the mRNA expressions of the P2Y subtype receptors, and HOS, SH-SY5Y or PC12 in the lower left corner in each panel is the cell line's name. It can be seen that the P2XR subtypes (i.e. P2X.sub.1R, P2X.sub.4R, P2X.sub.5R, P2X.sub.6R and P2X.sub.7R) and the P2YR subtypes (i.e. P2Y.sub.1R, P2Y.sub.2R, P2Y.sub.4R, P2Y.sub.6R and P2Y.sub.11R) were detected in the human osteosarcoma cell line HOS, the P2XR subtypes (i.e. P2X.sub.3R, P2X.sub.4R, P2X.sub.5R, P2X.sub.6R and P2X.sub.7R) and the P2YR subtypes (i.e. P2Y.sub.1R, P2Y.sub.2R, P2Y.sub.4R and P2Y.sub.11R) were detected in the human neuroblastoma cell line SH-SY5Y, and the P2XR subtypes (i.e. P2X.sub.2R, P2X.sub.3R and P2X.sub.4R) and the P2YR subtype (i.e. P2Y.sub.12R) were detected in the rat neuronal cell line PC12.

Embodiment 6: The Inhibition Effect of the Active Substances on Calcium Signals

[0101] 1. The P2R-Correlated Calcium Signals Detected in the Cell Lines:

[0102] The rat pheochromocytoma cell line PC12, human neuroblastoma cell line SH-SY5Y and human osteosarcoma cell line HOS were filled with Fura-2 calcium ion-sensitive fluorescent dye to detect the intracellular free calcium-ion concentration. Furthermore, the P2R on the cell membrane was stimulated using ATP to observe the variations of the intracellular free calcium-ion concentration. FIGS. 3(A), 3(B) and 3(C) show the variations of the calcium signals of the receptors in (A) human osteosarcoma HOS cells, (B) rat pheochromocytoma PC12 cells and (C) human neuroblastoma SH-SY5Y cells after being stimulated with 0.1 mM ATP. The intracellular free calcium-ion concentration in human neuroblastoma SH-SY5Y cells did not vary. To exclude the possibility that cells do not generate bio-molecules which are correlated to calcium-ion signals, the acetylcholine receptor on the human neuroblastoma SH-SY5Y cells was further stimulated with 0.1 mM carbachol to confirm that the cells do have molecules which are correlated to calcium-ion signals, and the results are shown in FIG. 3(C).

[0103] 2. Calcium Signals Correlated to the Individual P2R Subtypes:

[0104] Comparing the P2R-correlated calcium signals in three cell lines stimulated with ATP, the variation of the calcium-ion concentration in human osteosarcoma HOS cells was the most significant, and thus the influence of the agent on P2R can clearly be determined. Therefore, human osteosarcoma HOS cells were selected as the sample to further detect the P2R subtypes which may be correlated with calcium signals in human osteosarcoma HOS cells. The 2-methylene ATP (2-MeATP, the selective stimulator for the P2X.sub.1R and P2X.sub.5R subtypes), α,β-methylene ATP (α,β-MeATP, the selective stimulator for the P2X.sub.7 subtype), 2-(methylthio)adenosine 5′-triphosphate (2-MeSATP, the selective stimulator for the P2X.sub.1 subtype) and cytosine 5′-triphosphate (CTP, the selective stimulator for the P2X.sub.4 subtype) were used to determine the P2R subtypes in human osteosarcoma HOS cells. The curves from left to right in FIG. 4 show the calcium-ion signals induced by the specific stimulators (CTP, 2-MeATP, α,β-MeATP and 2-MeSATP) for the subtype receptors, indicating that at least the calcium signals correlated with P2X.sub.1, P2X.sub.4, P2X.sub.5 and P2Y.sub.7 can be detected in human osteosarcoma HOS cells, and the reaction for P2X.sub.4 is the most significant.

[0105] The P2Y receptor is correlated with the G-protein (Gq). The Gq can be activated after stimulating the receptor, to further activate phospholipase C so as to generate inositol 1,4,5-trisphosphate (IP3) and release calcium ions from the IP3-sensitive calcium pool in the cell. Therefore, the remaining calcium ions in the buffer in the experiment can be chelated when using a calcium ion-free buffer supplemented with an adequate amount of EGTA, so that no extracellular calcium ions can flow into the cells. On this condition, the intracellular free calcium-ion concentration was determined to indicate that the calcium ions are released from the calcium pool in the cell. FIGS. 5(A) and 5(B) show the calcium signals correlated with the P2Y receptor, wherein FIG. 5(A) shows that the calcium signals are induced by using ATP to stimulate the P2R in a calcium ion free buffer, and this is a response for the P2R receptor-correlated calcium signals because no extracellular calcium ions flow into the cells; and FIG. 5(B) shows the calcium signals induced using the uridine triphosphate (UTP) to stimulate cells. It is shown in FIGS. 5(A) and 5(B) that at least the P2Y.sub.2R- and P2Y.sub.4R-correlated calcium signals can be detected in human osteosarcoma HOS cells.

[0106] 3. The P2R-Correlated Calcium Signals Inhibited by the Active Substances of H. erinaceus:

[0107] ATP is a stimulator for P2R in the body, and thus 0.1 mM ATP is used to stimulate the P2R receptor. The H. erinaceus alcohol extract was added before the calcium signals were induced using ATP to stimulate the P2R. A secondary signal is equivalent to a physiological reaction after a receptor is stimulated. Therefore, it can be seen in FIG. 6 that the P2R's calcium signals induced by ATP are completed inhibited, and it is proved that P2R's functions can be inhibited by the H. erinaceus alcohol extract. It is proved in the above experiments that there are a variety of P2XRs and P2YRs on the HOS cells and the functions of the P2R receptors can be completely inhibited by the H. erinaceus alcohol extract, and it is determined that the H. erinaceus alcohol extract has an inhibition effect on receptors such as P2X.sub.1, P2X.sub.4, P2X.sub.5, P2Y.sub.7, P2Y.sub.2, P2Y.sub.4 receptors and so on.

[0108] 4. the Correlation Between the Inhibition Functions of the H. erinaceus Active Substances and the Erinacine S and their Concentrations:

[0109] FIGS. 7(A) and 7(B) show the inhibition effect of (A) the H. erinaceus alcohol extract of 2.5, 5 and 50 μg/ml and (B) the erinacine S of 5, 10 and 15 μg/ml on the variation of the intracellular calcium-ion concentration in human osteosarcoma HOS cells induced using the ATP to stimulate the P2R receptor, and the inhibition effect is more significant in a dosage-dependent manner, indicating that the dosage given is correlated with the inhibition effect.

Embodiment 7: The Determination of the Effect that the H. erinaceus Active Substances Reduce Animals' Pain

[0110] This embodiment was performed to observe the mice's pain reaction to the extrinsic stimulation before/after administration via the tail flick test and the hot-plate test, after the mice were administered with the H. erinaceus active substances (i.e. the alcohol extract obtained in Embodiment 2 and the alcohol extraction mixture obtained in Embodiment 3) for a short term or a long term.

[0111] 1. The Tail Flick Test after the H. erinaceus Active Substances were Administered for a Short Term:

[0112] Mice in this experiment were divided into three groups, i.e. the control group, the H. erinaceus mycelia/alcohol extract (the alcohol extraction mixture group), and the H. erinaceus alcohol extract (the alcohol extract group). Each group had five male C57BL/6 Narl mice which were older than eight weeks old. The mice were orally administered with 2500 mg/kg of drugs via a soft feeding tube, and the mice in the control group were fed with an equal amount of water. The mouse's nociception was determined by the tail flick test before administration, and 30 and 60 minutes after administration. The experimental results were expressed as mean±standard deviation, and the significance of statistics among the alcohol extraction mixture group, the alcohol extract group and the control group was analyzed using Student's t-test. Therefore, the pain-relieving effect of administering the active substances for a short term can be observed, and the results are shown in FIG. 8.

[0113] The tail-flick basic values were examined before administration, the tail-flick reaction time in three groups was 4.1±1.5 seconds (s) in the control group, 4.3±1.9 s in the alcohol extraction mixture group, and 4.5±1.2 s in the alcohol extract group. The p-value for Student's t-test between the alcohol extraction mixture group and the control group was 0.8, the p-value for Student's t-test between the alcohol extract group and the control group was 0.6, and the p-value for Student's t-test between the alcohol extraction mixture group and the alcohol extract group was 0.8, indicating that there was no significant difference between groups before administration.

[0114] The mouse's nociception reaction was determined by the tail flick test 30 minutes after administration. The experimental results show that the tail flick reaction time was 2.0±0.5 s in the control group, 6.2±1.6 s in the alcohol extraction mixture group, and 8.4±1.1 s in the alcohol extract group. The p-value for Student's t-test between the alcohol extraction mixture group and the control group was 0.0008 with a significant difference, the p-value for Student's t-test between the alcohol extract group and the control group was 0.000009 with a significant difference, and the p-value for Student's t-test between the alcohol extraction mixture group and the alcohol extract group was 0.07 without a significant difference.

[0115] The mouse's nociception reaction was determined by the tail flick test 60 minutes after administration. The experimental results showed that the tail-flick reaction time was 2.1±0.57 s in the control group, 5.7±2.46 s in the alcohol extraction mixture group, and 7.2±1.9 s in the alcohol extract group. The p-value for Student's t-test between the alcohol extraction mixture group and the control group was 0.0256 with a significant difference, the p-value for Student's t-test between the alcohol extract group and the control group was 0.0016 with a significant difference, and the p-value for Student's t-test between the alcohol extraction mixture group and the alcohol extract group was 0.33 without a significant difference.

[0116] 2. The Difference in the Tail Flick Test Before/after Administration with the H. erinaceus Active Substances for a Short Term:

[0117] The experimental conditions are similar to the condition in the subsection “1. The tail flick test after the H. erinaceus active substances was administered for a short term”, and the difference of the tail-flick reaction times among the alcohol extract group, the alcohol extraction mixture group and the control group was compared. Therefore, the pain-relieving effect of administering the active substances for a short term can be observed, and the results are shown in FIG. 9.

[0118] The mouse's nociception reaction was determined by the tail flick test 30 minutes after administration. The experimental results show that the tail-flick reaction time in the control group was shorter compared to the tail-flick basic value before administration, with a difference of −2.1±1.7 s; the tail-flick reaction time in the alcohol extraction mixture group was longer compared to that before administration, with a difference of 2.6±2.18 s; and the tail-flick reaction time in the alcohol extract group was longer compared to that before administration, with a difference of 3.9±1.61 s. The p-value for Student's t-test between the alcohol extraction mixture group and the control group was 0.008 with a significant difference, the p-value for Student's t-test between the alcohol extract group and the control group was 0.00108 with a significant difference, and the p-value for Student's t-test between the alcohol extraction mixture group and the alcohol extract group was 0.35 without a significant difference.

[0119] The mouse's nociception reaction was determined by the tail flick test 60 minutes after administration. The experimental results show that the tail-flick reaction time in the control group was shorter compared to the tail-flick basic value before administration, with a difference of −1.0±0.71 s; the tail-flick reaction time in the alcohol extraction mixture group was longer compared to that before administration, with a difference of 2.0±2.53 s; and the tail-flick reaction time in the alcohol extract group was longer compared to that before administration, with a difference of 2.6±1.77 s. The p-value for Student's t-test between the alcohol extraction mixture group and the control group was 0.033 with a significant difference, the p-value for Student's t-test between the alcohol extract group and the control group was 0.0028 with a significant difference, and the p-value for Student's t-test between the alcohol extraction mixture group and the alcohol extract group was 0.66 without a significant difference.

[0120] 3. The Maximum Possible Effects in the Tail Flick Test after Administration of the H. erinaceus Active Substances for a Short Term

[0121] The conditions of this experiment are similar to those in the subsection “1. The tail flick test after the H. erinaceus active substances was administered for a short term” above, and the maximum possible effects (MPE) are calculated as follows. The tail-flick reaction time after administration minus the tail-flick reaction time before administration (a tail-flick basic value for a single mouse) have a difference, and then the difference divided by the tail-flick basic value for this single mouse is the MPE. Therefore, the pain-relieving effect of administering the active substances for a short term can be observed, and the results are shown in FIG. 10.

[0122] The mouse's nociception reaction was determined by the tail flick test 30 minutes after administration. The experimental results show that the tail-flick reaction time in the control group was shorter (about half) compared to the tail-flick basic value before administration; the tail-flick reaction time in the alcohol extraction mixture group was longer compared to that before administration; and the tail-flick reaction time in the alcohol extract group was longer by about one time compared to that before administration. The p-value for Student's t-test between the alcohol extraction mixture group and the control group was 0.03 with a significant difference, the p-value for Student's t-test between the alcohol extract group and the control group was 0.0134 with a significant difference, and the p-value for Student's t-test between the alcohol extraction mixture group and the alcohol extract group was 0.31 without a significant difference.

[0123] The mouse's nociception reaction was determined by the tail flick test 60 minutes after administration. The experimental results show that the tail-flick reaction time in the control group was shorter compared to the tail-flick basic value before administration; the tail-flick reaction time in the alcohol extraction mixture group was longer by about one time compared to that before administration; and the tail-flick reaction time in the alcohol extract group was longer compared to that before administration. The p-value for Student's t-test between the alcohol extraction mixture group and the control group was 0.05 with a significant difference, the p-value for Student's t-test between the alcohol extract group and the control group was 0.004 with a significant difference, and the p-value for Student's t-test between the alcohol extraction mixture group and the alcohol extract group was 0.61 without a significant difference. It is predicted that the H. erinaceus active substances have an immediate pain-relieving effect which can at least continue for about 60 minutes.

[0124] 4. The Hot-Plate Test after Administration of the H. erinaceus Active Substances for a Long Term:

[0125] This assay is used to determine whether the administration of the H. erinaceus active substances for a long term can delay in response caused by the hot plate. Each mouse was fed with 2500 mg/kg active substance for three days, and the interval between two feeds was 24 hours. Next, the hot-plate test was performed after feeding the active substances for three days, the amounts of paw lifting, licking or jumping were determined and the results are shown in FIG. 11.

[0126] After administration for three days, the experimental results show that the paw withdrawal reaction time was 6.4±1.8 s in the control group, 11.7±2.5 s in the alcohol extraction mixture group, and was 11.5±1.8 s in the alcohol extract group. The p-value for Student's t-test between the alcohol extraction mixture group and the control group was 0.005 with a significant difference, the p-value for Student's t-test between the alcohol extract group and the control group was 0.0025 with a significant difference, and the p-value for Student's t-test between the alcohol extraction mixture group and the alcohol extract group was 0.09 without a significant difference.

[0127] 30 minutes after performing the first-time hot-plate test on administration day 3, the paw withdrawal reaction time was determined again with the same experimental method. The experimental results show that the paw withdrawal reaction time was 6.3±0.8 s in the control group, 11.8±2.5 s in the alcohol extraction mixture group, and was 8.9±1.2 s in the alcohol extract group. The p-value for Student's t-test between the alcohol extraction mixture group and the control group was 0.007 with a significant difference, the p-value for Student's t-test between the alcohol extract group and the control group was 0.010 with a significant difference, and the p-value for Student's t-test between the alcohol extraction mixture group and the alcohol extract group was 0.09 without significant difference.

[0128] After calculating the first-determination data after feeding for 3 days and the data from the after-30-minutes determination, the experimental results show that the paw withdrawal reaction time was 5.7±2.4 s in the control group, 11.7±2.3 s in the alcohol extraction mixture group, and was 10.3±2.0 s in the alcohol extract group. The p-value for Student's t-test between the alcohol extraction mixture group and the control group was 0.00006 with a significant difference, the p-value for Student's t-test between the alcohol extract group and the control group was 0.0003 with a significant difference, and the p-value for Student's t-test between the alcohol extraction mixture group and the alcohol extract group was 0.81 without a significant difference.

[0129] According to this animal experiment, it can be seen that the alcohol extraction mixture and the alcohol extract have significant pain-relieving effects on a short-term reaction after a 30-minute administration, and the pain-relieving effect tended to decline after a 60-minute administration. In the experiment where the alcohol extraction mixture and alcohol extract were fed for a long term, deallergization occurs and the pain-relieving effect tends to stabilize.

[0130] In conclusion, the H. erinaceus active substances including the H. erinaceus mycelia, the H. erinaceus alcohol extract, the H. erinaceus alcohol extraction mixture and the erinacine S disclosed in the present invention have been proven to have the pain-relieving effect, and can be widely used for pain relief.