The Inducer of the Synthesis of Nerve Growth Factor From Lion's Mane (Hericium erinaceus)

© Copyright 2002 by Hirokazu Kawagishi,1 Ph.D., Shoei Furukawa,2 Ph.D., Cun Zhuang,3 Ph.D., and Rika Yunoki 4; Japan & USA
(Explore Issue: Volume 11, Number 4)

Abstract

Nerve growth factor (NGF) is closely related to Alzheimer's dementia, and studies have suggested that the disease may be prevented or its symptoms may be improved when NGF is given into the brain directly. However, since NGF is a protein it usually cannot pass through the blood-brain barrier. Recently, researchers have targeted on the substances that could pass through the blood-brain barrier and induce NGF synthesis in the brain. Some compounds with lower molecular weight have been found to have such bioactivity. Among these bioactive compounds, hericenones and erinacines, which were isolated from an edible mushroom called as Lion's Mane (Hericium erinaceus), showed remarkable activity of stimulating the synthesis of NGF. They could be developed as a dietary supplement or medicine to be used for treating Alzheimer's dementia. This article offers an introduction to the isolation method, bioactivity assay and chemical structure analysis of hericenones and erinacines.

Significance of the Study on the Inducer of the Synthesis of Nerve Growth Factor

It is expected that the application of an inducer of synthesis of nerve growth factor (NGF) would contribute to the medical treatment and the prevention of disorders related to the central and the peripheral nervous systems. For instance, the cause of Alzheimer's dementia is yet to be clarified and the way to its prevention or treatment is yet to be established. The patients, however, have similar damages in the brain to those with the disorder with basal forebrain cholinergic neuron (BFCN), suffering malfunction in BFCN associated with memory and learning (such as cell loss, atrophy of nerve cell, and neurite degeneration). Because NGF exerts a trophic action on BFCN, lack of NGF is considered one of the causes of this disease. Researches have been targeting on NGF as a treatment of the dementia (Thoenen & Barde, 1980; Yankner & Shooter, 1982; Furukawa et al., 1984, 1987; Furukawa & Furukawa, 1988, 1989; Furukawa & Kawagishi, 1991).

However, NGF is a protein and cannot pass through the blood-brain barrier. It needs to be injected directly into the brain to be effective. In fact, a remarkable finding was reported at an international medical conference in 1990. According to the report, a woman with Alzhehimer's dementia improved her symptoms, such as enhancing mental ability, after the administration of NGF derived from mice directly into her brain using catheter (Seiger et al, 1993). However, such treatment cannot be accepted as a therapy for the disease. If we could take a substance by oral administration or injection, which penetrates the membrane and stimulates the NGF synthesis inside the brain and then if this induced-NGF could repair the damaged nervous functions, such substance may be applied as a safer therapy to prevent this disease. Even if this substance could not go through the barrier, it would be still beneficial for disorders of the peripheral nervous system since NGF has a similar activity on neurons at the peripheral nervous system.

Assay Method

Based on the above concept, the screening for a substance that stimulates NGF synthesis has been carried out using primary astroglia derived from rat cerebral cortex in vitro. Among the primary cells that facilitate NGF synthesis, including fibroblastic L-M cells, we elected astroglial cells due to the following reasons: Within the brain, neuron and astroglia are responsible for NGF production. Neuron controls NGF synthesis to maintain the function in the matured brain, while in the brain at the growth period or with some damage, astroglia plays the role instead. It is possible that a substance stimulating NGF synthesis to maintain the function in the matured brain, while in a brain during the growth period or that with some damages, astroglia are believed to play the role instead. It is likely that a substance stimulating NGF synthesis by neuron generates some kind of unfavorable neural activity against the individual, since an excitatory neural network induces NGF production by neuron. This is why we elected to use astroglia cells in this screening. This assay is optimum for our purpose. The substance shows activity in this assay must demonstrate the similar activity in vivo.

Low Molecular Weight Compounds Identified

a) Catechol Compounds

In the peripheral system, the more sympathetic innervation increases, the more NGF synthesis is activated. It is highly active within submandibular gland, heart, and blood vessel in rats. We assumed that noradrenaline may be the neural signal to regulate NGF synthesis. Consequently, catecholamine (adrenaline, noradrenaline, and dopamine) has been found to accelerate NGF production significantly at stationary astrocyte (Furukawa et al., 1986a). Cholinergic agonist was only slightly effective on the synthesis among the various neurotransmitters in the central nervous system. The concentration of NGF secreted into the media increases depending on catecholamine levels and mRNA concentration for intercellular NGF also increases. The similar activity has been observed within the established fibroblastic cell line L-M as well as in the astrocyte. Further study on the structure-activity correlation has indicated that the activity is dependent on the catechol structure and that the compounds with two saturated carbons on the side chain has shown the highest effect. There was no significant difference in the presence or difference of functional groups on the side chain. Another important finding was that no adrenergic receptor had been required with these catechols to induce the reaction (Furukawa et al., 1986b, 1989; Furukawa & Furukawa, 1990). In other words, it goes without hormone and neurotransmitter activities mediated by an adrenergic receptor. This could lead us to a possible development of new medicine without side effects in the future.

Based on the above experiments in vitro, the induction of NGF was examined using 4-methylcatechol [1] (Fig. 1) in rats, and it is found that the concentration of NGF has been increased in the heart and the submandibular gland (Kaechi et al., 1993).

b) Hericenones Derived From Fruit Body of Lion's Mane

Obviously, it is quite important to clarify the internal substances that are originally responsible for the stimulation of NGF production for each organ. However, because such internally active substances like adrenalines are hormones, its administration from outside may lose the internal hormone balance, and their practical application for prevention or treatment of Alzheimer's disease may be harmful. It is preferable, therefore, that the substance is safe and becomes active specifically. With this background, the compounds, called hericenone C-H, were isolated from Lion's Mane after the screening test for the active stimulant of NGF synthesis targeting to constituents of mushrooms (Fig. 2) (Furukawa & Kawagishi, 1991; Kawagishi et al., 1991, 1993).

These compounds are the first active substances found in natural products, which are as effective as adrenaline. Lion's Mane has been appreciated as an herbal medicine in China, and the cultivation has recently started in Japan. Each group of hericenones, C [2] ­ E [4] and F [5] ­ H [7], contains the alcohol site peculiar to each group and each hericenone consists of one of three simple fatty acids. Hericenon D [3] demonstrated the strongest activity among these compounds. It means that the activity level varies according to the structure of fatty acids. The animal test is currently undergoing.

c) Erinacines Found in Mycelia of Lion's Mane

Active substances in vitro may be further investigated in vivo, but animal tests require a large quantity of sample. The cultivation of mushrooms (fruit body) needs well-controlled light, temperature, and humidity. So we tried to produce active compounds from mycelium, the pre-stage of a fruit body and easier to grow, in order to obtain considerable amount of hericenone. This experiment has not been completed yet, but we were able to obtain a series of diterpenoids named as erinacine A-I, which have powerful activities with different chemical structures from these of hericenones. (Fig. 3) (Kawagishi et al., 1994, 1996a, 1996b; Lee et al., 2000). These substances are so strong that they can be recognized as the most powerful ones among all identified substances at present (Fig. 4).

d) Other Compounds

As other active compounds, propentofylline [11] as a xanthine derivative, 1,4-benzoquinones [12, 13], PQQ [14], and OPQ [15] are known (Shinoda et al., 1990; Yamaguchi et al., 1993a). Additionally, phertamide A [16] has been reported as the second active substance derived from natural products since hericenones (Yamaguchi et al., 1993b). According to the comparative study on the active substances mentioned above, the findings with respect to the stimulation of NGF synthesis are as follows; 1. they possess more than one active site and mechanisms,

2. the specificity seems rather weak, 3. these compounds probably have an unknown common factor, 4. there must be other active groups of compounds yet to be found that can facilitate the function. As for 1., above, the following result is reported in the study of the NGF synthesis with fibroblastic cell L 929 in mice (Carswell et al., 1992). Under co-existence of propranolol 4-methylcatechol [1] demonstrated the stimulation of NGF synthesis, while isoproterenol [17] having catechol in its structure like 4-methylcatechol did not. This is because propranolol is an antagonist for adrenergic receptor, and isoproterenol is an agonist for it. This indicates that, although the analogous structure is recognized in catechols, some of them stimulate NGF synthesis with adrenergic receptor while the others can be active without it. The similar activity was observed on ganglioside, the main component of cell membranes, that was only at Schwann cell that forms myelin sheath in the peripheral nerve. No activity was confirmed at astrocytes or fibroblastic cells.

Experiments

a) Assay In Vivo

(Fig. 5, Furukawa et al., 1983; Saito & Furukawa, 1987; Matsui et al., 1990)

Incubation and Assay for Astroglial Cell

Rats from Wistar on the fifteenth day of gestation have been obtained. The cerebrum was taken on a clean bench after removing the cerebellum and lower parts of cerebrum from the infant rats (within 3 days from the birth). Meninges were removed using forceps (the thin membrane would be peeled off the surface of the cerebrum) to collect a pure sample since even a little meninges, if remained, would make the glial cells lose the purity being mixed with fibroblastic cells that have high proliferation potency. Also, it should be avoided to use the subculture at low cell density to keep the sample purity high. The obtained cerebrum was put in a disposable centrifuge tube (15 ml), minced finely with a dissector knife (or dissector scissors or forceps), and washed in PBS (10mM phosphate-buffered saline, pH 7.4) by pipetting. Then this solution was centrifuged (3,000 x g, 10 min) and the supernatant was discarded. Repeated these steps twice. Added about 2 ml portion of 0.25% trypsin and incubated it at 37°C for 15 ~ 30 min. Added 5 ml of PBS to stop the reaction, centrifuged (3,000 x g, 10 min), and removed the supernatant. Rinsed the pellet with PBS by pipetting, centrifuged (3,000 x g, 10 min), and discarded the supernatant. Repeated this procedure one more time. After pipetting the pellet in 5 ml FCS-MEM (minimum essential media containing 10% fetal calf serum), poured another 5 ml of FCS-MEM, transferred it together with the pellet into a 10 cm laboratory dish, and incubated it in 5% CO₂ at 37°C for 2 ~ 3 days (one cerebrum / dish). It is easy to distinguish between astrocytes and meninges ­ derived cells, since the former has a nice stonewall shape and the latter has a stalky shape. Changed FCS-MEM and continued the incubation. Continuously changed the media once in every three days until the confluent was accomplished. Then, removed the media, performed trypsin treatment, and loaded the cells in each well on a 96-well plate. After switching the FCS-MEM three times in nine days at about three-day intervals, the media was converted to serum-free media [containing 0.5% bovine serum albumin (BSA)] and it was repeated three times in nine days once in every three days. During this procedure, the astrocyte was in a stationary phase and NGF production slowed down. Thymidine may be added to confirm the halt of the propagation. Also, it would be even better to make sure the decrease of NGF level in nutrient media over time. The media was exchanged with 100 µl of new one on the previous day of the sample addition. Dissolved the water insoluble samples in ethanol, methanol, or dimethyl sulfoxide (DMSO) and diluted them with the media so as not to exceed 1% of the solvent volume. Other samples had been prepared on another plate beforehand with a series of dilutions, using a multichannel pipette and incubated it for 24 hours after adding 5 µl of the experiments. Measured the NGF levels by an enzymatic immunoassay.

Storage of Astroglia cells

1. Washed the cells treated with trypsin in FCS-MEM and centrifuged it (3,000 x g, 10 min).
2. After adding 0.6ml FSC-MEM to the precipitate while cooling, put 0.4ml of 50% DMSO (in FSC-MEM).
3. After refrigerating it for 2 ­ 4 hours in an ultra-deep freezer (-80°C), stored it in liquid nitrogen.

Resuspension of the cell

1. Let the frozen cells back to the room temperature rapidly. Put it in a centrifuge tube, added about 9ml of FCS-MEM, and incubated it at 37°C for 20 ~ 30 min.
2. After centrifugation, incubated the pellet again in the FCS-MEM.

b) Enzyme immunoassay for NGF

Preparation of Antibody Plate

Anti-NGF antibody, IgG (produced by purification of anti-NGF, rabbit antiserum, by protein A column) was dilubed in 0.1 M tris-hydrochloric acid buffer (pH 7.6) till its concentration of 100 µg/ml was obtained. Put 5 µl of this solution in a drop at a time in the center of each well on a 96-well ELISA plate (U-bottom). Sealed the plate with parafilm and let it for an hour at room temperature. Removed the antibody (reusable) and filled each well with 150 µl buffer A (0.1 M tris-HCl buffer, pH 7.6 containing 0.1% BSA, 0.4 M NaCl, 1 mM MgCl₂, and 0.02% NaN₃). After approximately 30 min, the buffer was removed by an aspiration with 8 channel manifold, add 150 µl of buffer A containing 1% milk was added in order to block each well, and left it for an hour. The plate was carefully handled not to be evaporated during the procedures.

Reaction With the Samples

The milk solution was aspirated and then 20 µl of sample solutions was immediately placed in each well. It must be performed quickly to avoid evaporation of the samples and dryness of wells. It is recommended that the sample solutions were prepared beforehand on another plate and filled the wells using a multichannel pipette all at once. Sealed it tight with parafilm and let it react in a shaker with mild movement for 2 hours at room temperature. After aspirating the sample solutions, 150 ml of buffer A was poured and then removed by aspiration. This procedure was repeated twice to wash the plate.

Addition of Labeled Antibody

Biotinylated anti-NGF antibody (biotinylated after the purification using an affinity chromatography, 10 µg/ml) was diluted 1,000-fold in buffer A (with 1% serum of a healthy rabbit) and 20 µl of the solutions were added to each well. Put lids with parafilm tightly and let them stand for 12 ~ 18 hours at 4°C.

Treatment with Streptoavidin-ß-D-Galactosidase

Streptoavidin-ß-D-glactosidase was purchased from the market and diluted 6,000-fold with buffer A. Took 20 µl to each well. Sealed it tight with parafilm and incubated for an hour at room temperature. After the incubation, rinsed each well three times with buffer A and added 20 ml of 4-methylumbelleferyl-ß-D-galactoside (10 µg/ml in buffer A). Sealed with parafilm and allowed it to react for 5 hours at room temperature shutting the light in the dark. Terminated the reaction by pouring 100 µl of 0.1 M glycine-NaOH buffer solution (pH 10.3). Measured the fluorescence intensity with an excitation wavelength of 360nm and a detector wavelength of 450nm. The concentration of NGF was calculated from the fluorescence intensity of the samples by plotting the intensity of standard NGF on a loglog graph. A fluorophotometer with flow cells is employed for more accurate evaluation, instead of a micro plate reader, which is used in the regular assays but not reliable due to inconsistent data.

c) Assay In Vivo

(Determination of the NGF Concentration at Each Organ By Adding Samples)

Administration to Animals

Wistar rats (male) were employed. Applied the appropriate amount of the samples so as to obtain its blood concentrations of 100 µg/ml, 10 µg/ml, 1 µg/ml, and 0.1 µg/ml respectively (the average weight of Wistar rats is 131g at 6 weeks of age and 160.6g at 7 weeks, and the blood weighs 5.2 ~ 6.2% of the body weight). Administered 0.5 ml of each concentration of the samples intraperitoneally (The water-soluble samples were dissolved in PBS. The fat-soluble samples were dissolved in ethanol, then, PBS was added to dilute them to make 20% ethanol solution.) into the abdominal cavity twice a day at 12-hour intervals. The rats were sacrificed with chloroform 4 hours after the fifth injection and removed the heart, submandibular gland (right and left), sciatic nerve (right and left), and cerebrum.

Extraction Process

1) Heart, Submandibular Gland, and Cerebrum

Homogenized them in the extract buffer (1 M NaCl, 2% BSA, 2mM EDTA, 1 M tris-HCl containing 0.08 unit/ml aprotinin, pH 7.6. The final pH needs to be exact.) to make 2 or 5% (w/v) solution. Ultracentrifuged the homogenate (300,000 x g, 15 min) and froze the supernatant for the enzyme immunoassay.

2) Sciatic Nerve

Cut the sciatic nerve (2 cm) that had been deep-frozen immediately after its removal into 15 pieces with approximately 1.3 mm length each. Put each piece in a 96-well microplate and poured 100 µl extract buffer. The extraction was carried out by repeating the freezing and thawing method. The extract was centrifuged (3,000 x g, 30 min), and each supernatant was transferred to a 96-well microplate, then frozen and stored for the enzyme immunoassay.

d) Hericenones Isolated From a Fruit Body of Lion's Mane (Fig. 6)

The fruit body of the mushroom was crashed in acetone by a blender and left for 1 ~ 2 days for the extraction. The liquid extract was processed with vacuum filtration and the mushroom fruit body was further extracted by acetone twice. The extract was concentrated using an evaporator till 2 l of the volume is obtained and fractionated with chloroform. Ethyl acetate was added to the aqueous phase for more extraction. The fractionation of the extract would be an essential step for applying the compounds to the assay, because there is an optimum concentration for the activation of NGF synthesis and also most of the fractions at this stage exhibit cytotoxic activity. Therefore, silica gel chromatography and preparative thin layer chromatography (TLC) were employed and obtained two types of fractions, one with hericenones C-E and the other with hericenones F-H. Both fractions were spotted at almost the same distance on the silica gel TLC and the separation was only possible by high performance liquid chromatography (HPLC) using ODS column.

e) Isolation and Structural Determination of Erinacines Derived from Mycelia of Lion's Mane Isolation (Fig. 7)

Centrifuged the mycelia following the 4 weeks of shake culture and separated the mycelia from the culture filtrate. The culture filtrate was concentrated by an evaporator and fractionated with ethyl acetate and water. The mycelia was put in 85% ethanol for extraction, concentrated by an evaporator, and then fractionated with ethyl acetate and water. Since the fractions in the culture filtrate did not demonstrate any activity, we repeated extraction of mycelia using silica gel column chromatography and preparative TLC and obtained the purified erinacines.

Structural Determination

1) Erinacine A

Erinacine A [8] has the molecular formula of C₂₅H₃₆O₆ detected by a high-resolution fast-atom bombardment mass spectrometry (FAB-MS) and is a pentose glycoside in diterpene identified using ¹H-NMR and ¹³C-NMR. The existence of a conjugated aldehyde group (* 9.31, * 194.3) is indicated in this substance. The acetylation by acetic anhydride in pyridine was carried out to determine its sugar type and the linkage of the sugar and aglycone, as the signals for the sugars were overlapped by ¹H-NMR and could not be identified. All signals for the sugars were analyzed by the triacetyl compounds that had been produced during this acetylation [ * 4.56 (d, J = 6.23, H-1'), 4.89 (dd, J = 6.23, 8.06, H-2'), 5.07 (dd, J = 8.06, 8.06, H-3'), 4.86 (m, H-4'), 3.95 (dd, J = 12.09, 4.76, H-5'), 3.31 (dd, J = 12.09, 7.70, H-5') ]. This result suggests that its sugar type is xylose and the linkage is made by ß-bond. Also, the compound 8 was proven to have ß-D-xylosido due to the fact that it was hydrolyzed by ß-glucosidase. The structure of this isolated aglycone has been identical with Allocyathine B2 in every data including its specific rotation, whose absolute configuration had been previously determined by Ayer et al. after its isolation from Cyathus earlei. This fact determined the structure of the compound 8 with its specific rotation (Ayer & Lee, 1979).

2) Erinacine B

We presumed that erinacine B [9] was ß-xyloside just like compound 8 because of the molecular formula of C₂₅H₃₆O₆ as in 8 as well as from analysis by NMR spectrum. However, since compound 9 has less double bond by one compared to that of compound 8 and it gave diacetate after its acetylation, we concluded that one more bond exists between sugar and aglycone in 9 forming a ring. The analysis of the heteronuclear multiple bond correlation (HMBC) spectrum has confirmed the location of the bond (Fig. 8). Its stereochemistry has determined the aspects that the ¹H-NMR spectrum provided the coupling constant between H-13 and H-14 9.71 Hz suggesting the configuration of trans-diaxial, and that the cross peaks appeared from H-14 to H-16, H-5 to H-17, and H-5 to H-13, respectively, on the NOESY spectrum.

3) Erinacine C

The results provided by the NMR erinacine C [10] were very similar to those by 9. However, compound 10 did not have the formyl group, which had observed in 9, and it still had the molecular formula of C₂₅H₃₈O_{6 .}, Therefore, the presence of hydroxymethyl group is suggested instead of formyl group. This chemical structure has been confirmed by the fact that the oxidization of compound 10 with 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) produced erinacine B [9].

About the Authors

Hirokazu Kawagishi, Ph.D., is a professor in the department of applied biological chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan. Syoei Furukawa, Ph.D., is a professor in the department of molecular biology, Gifu Pharmaceutical University, 5-6-1 Mitahora-Higashi, Gifu 502-8585, Japan. Cun Zhuang, Ph.D., is a senior scientist in Bio Research Institute, P.O. Box 1354, Paramus, N.J. 07653, USA, bioresearch@maitake.com, and Rika Yunoki is an assistant scientist at Maitake Products, Inc., 222 Bergen Turnpike, Ridgefield Park, New Jersey 07660, USA, customerservice@maitake.com, (201) 229-0101.

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