Chemical Structures and Changes of Extracts during Growth of Reishi (Ganoderma lucidum)^*1

Compounds from the mushroom reishi (Ganoderma lucidum (Leys.)) were examined for chemical structure and over time. So were the extracts during the growth of fruiting bodies. Isolated compounds were identified as ganoderic acid A (I). triterpene alcohols (II) and (III), triterpenealdehyde (IV), possibly related to methylester of ganoderic acid Y(V), by carbon- 13 nuclear magnetic resonance and other spectra. These triterpenes increased after the appearance of fruiting bodies, although they were not found in mycelia. Ergosterol and fatty acids were found in both mycelia and fruiting bodies but did not increase during growth of the latter.

Triterpenes were found more in the outer section than in the inner section of fruiting bodies. This infers that aging of a fruiting body increases its triterpenes because the outer section is older than the inner section.

Reishi, Ganoderma lucidum (lays.) (Polyporaceae), grows naturally in Japan and China, and often is called mannentake. Reishi has been culti­vated artificially on media of wood meal-rice bran and on wood blocks for the last ten years in Japan.The total annual production is reported to be 100 tons.

It also is reported that water extracts of reishi reduce blood pressure and platelet aggregation, ^(1,3) cholesterol in blood, ^(2,4) effect histamin-releasing inhibition ⁽⁵⁾ and promote antitumor activity. ^(6,7) The chemical structures of isolated compounds were clar­ified on branched glucan ^(8,9) and triterpenes. ^(10-15)

Medical effects are reported to depend on many factors, including the strains of reishi^5,16) and the sections of a fruiting body.¹⁶ In addition it has been stated that the growth period of fruiting bodies has some effects on the medical value of reishi, and these writers have expected that the chemical constituents of a fruiting body might make some changes during its growth. In this paper, we survey the chemical constituents of extracts and examine their changes during the growth of reishi.

2.1Chemical structures of compounds isolated from extracts

Fruiting bodies or sporophores of reishi were subjected to extraction with 70% aqueous methanol. The freeze-dried extract (yield, 7% of oven-dried sample) was extracted with hexane, chloroform, and ethylacetate. The chloroform extract (yield, 4% of oven-dried sample) was fractionated by silica-gel chromatography. Five compounds, A-E, were iso­lated.

Compound A was identified as ganoderic acid A (I), which had been isolated from this species ^10,14,15) before, by comparison of carbon-l3 nuclear magnetic resonance (¹³C-NMR) (Table 1) and mass spectra (MS) with those of an authentic sample.^t0~ Com­pound B had a ¹³C-NMR pattern similar to that of methylester of ganoderic acid Y (V), which also had been isolated previously from reishi¹¹⁾ (Table 1), and included a 26-alcohol structure (69.0 ppm) instead of the 26-acid structure (168.8 ppm) of (V). The struc­ture was inferred to be 3p-hydroxy-Stx-lanost-7, 9-dien-26-ol (II), supported by high-MS, MS, ultravio­let and visible spectra. (UV), infra red spectra (IR), and proton nuclear magnetic resonance spectra (¹H-NMR) of the original compound and its acetate (Fig.l). ¹³C-NMR of Compound C resembled that of Compound B. There was a 3-keto structure (216.8 ppm) in Compound C instead of the 3-alcohol struc­ture (78.9 ppm) in Compound B. High-MS, MS, IR, and ‘H-NMR supported this structure (III). Reduc­tion of Compound C with NaBH₄ gave Compound

B. Oxidation of Compound C with Mn0₂ gave Compound D (IV) by High-MS, IR, and UV. Com­pound D also was found in the chloroform extract.

Twenty triterpenes already have been isolated from reishi. All were acids except for two C₂₇ ketones. C₃₀ ketones and C₃₀ aldehyde were isolated for the first time. Residual Compound E was identified as ergosterol by UV, IR, and MS, but had been isolated previously from reishi.⁵⁾

2.2 Changes of extracts during growth of fruiting bodies

To obtain more information on chemical con­stituents, extracts from growing fruiting bodies were compared with those of vegetative mycelia (“V.M.”) and two other types of mycelia, one being extruded in air (“M.A.”) and the other growing in a medium (“M.M.”). Fruiting bodies, “M.A.”, and “M.M.” were sampled every five days after the appearance of the fruiting bodies, and extracts from these samples were quantified. The results are shown in Fig. 2-1. Fruit­ing bodies were found to have smaller amounts of extract than “M.A.” and “M.M.” after five days, and amounts decreased with time. On the contrary, triterpenes increased after fruiting body appearance. Results are shown in Fig. 2-2. No triterpenes were found in “V.M.”, “M.A.” and “M.M.”. Thus triterpenes are very characteristic of fruiting bodies.

It has not been determined whether the increase of triterpenes depends on the increasing metabolism of old sections or on the accelerated metabolism of newly formed sections in a fruiting body, because the fruiting bodies analyzed contained both new and old sections as shown in Fig. 2-3. This is discussed in a later section.
 

2.3 Amounts of extracts in sections of a fruiting body

     Each section of a fruiting body has distinctive secondary metabolites. Both a stipe and a pileus were observed separately. in addition, we focused atten­tion on the newly-produced mycelia of a fruiting body extended from the center of a stipe or between a gill and the upper side of a pileus, as shown in Fig. 3. Therefore, a stipe was divided into inner and outer sections, and a pileus was divided into a gill and inner and outer sections. This enabled us to obtain data for distinguishing extracts of newly produced mycelia from those of existing mycelia.

Amounts of 70% aqueous methanol extract (Table 3-1), triterpenes (Table 3-2), ergosterol, fatty acids, and phenols (Table 3-3) were measured. A gill was found to have less triterpenes and phenols but more fatty acids than other sections. A pileus had more ergosterol than a stipe. Further it is shown in Tables 3-1, 3-2, and 3-3 that old sections (outer parts) have more chloroform soluble substances, more triterpenes, and more phenols than new sections (inner parts), but all have similar amounts of ergosterol and fatty acids. These findings and the preceding discussion on increases of triterpenes during growth of a fruiting body. indicate some relationship between the age of a fruiting body and the content of triterpenes. That is. the content of triterpenes and phenols in­creased when a fruiting body was kept in a medium longer.
 
 

Six strains of commercial reishi were investigated for growth, and a strain from Nakano City, Nagano Prefecture, was selected as the best one for growing tests in this research. The fruiting bodies of this strain also were used for chemical analysis. Chemical analy­sis was made by ‘³C-NMR, JEOL JNM-FX-lOO; high resolution MS, JEOL DX-300; UV, Shimazu UV-200; IR, Shimadzu IR-400; and ‘H-NMR, JEOL JNM-4H-l00. HPLC (high performance liquid chromatography analysis was by a Shimadzu LC3A. 

3.1 Isolation and identification of compounds

One kg of air-dried fruiting bodies ~vas extracted with 8 1 of~70% aqueous methanol three times, and the extract was freeze-dried to become powder. The powder, suspended in 11 of water, was extracted successively with hexane (0.6 g), chloroform (43.9 g), and ethylacetate (6.3 g). (Yields are in parentheses.) The chloroform extract was chromatographed by silica gel with a hexane-ethylacetate-acetic acid sys­tem. Compounds A, B, C, D, and E were fractionated. Compounds A and E were identified as ganoderic acid A and ergosterol, respectively, as shown in the preceding section. Compound B: White crystals, mp l86-I8TC, High MS; Calculated for C₃₀H₄₈O₂: 440.3828, observed: 440.3662. MS m/z: 440 (M⁺), 425 (M⁺-CH₃), 422 (M⁺-H₂O), and 407 (M⁺-CH₃-H₂O). Uv? ^MeOH_max mm (log ?): 236 (4.12), 243 (4.17), and 252 (4.01). IR_? ^KBr_max cm^-1 3370, 2930, 1450, 1375, 1040, and 990. ¹H-NMR (in CDCI₃) ?: 0.58 (5, 6H), 0.85 (S, 12H), 0.98 (S, 6H), 1.02 (S, 6H), 1.69 (S, 6H), 4.01 (S, 4H), and 5.40 (m, 6H).

Acetate of Compound B: MS m/z: 524 (M⁺), 464 (M⁺-AcOH), 449 (M⁺-AcOH-CH₃), 408 (M⁺-2 AcOH) and 393 (M⁺-2 AcOH-CH₃). IR_v ^KBr_maxcm^-1: 2960, 1740, 1495, 1375, 1260, 1040, and 990. ¹H-NMR (in CDCI₃) ?: 0.57 (S. 6H), 0.88 (S, 12H), 1.02 (S, 6H), 1.67 (S, 6H), 2.02 (S, 12H), 4.48 (S, 4H), and 5.40 (m, 6H).

Compound C: mp. 94-95ºC, High-MS; Calculated for C₃₀H₄₆0₂: 438.368, observed: 438.347. MS m/z: 438 (M⁺), 420 (M⁺-H₂O) and 405 (M⁺-CH₃-H₂0). IR_? ^KBr_max cm^-1 : 3400, 2940, 1710, 1450, 1375, and 1110. ¹H-NMR (in CDCJ₃) ?: 0.59 (S, 6H), 0.89 (S. 6H), 1.09 (S, 6H), 1.13 (S, 6H), 1.20 (S, 6H), 1.65 (S, 6H), 3.97 (S, 4H), and 5.40 (M, 6H).

Compound D: mp. 143ºC, High-MS; Calculated for C₃₀H₄₄0₂: 436.352, observed: 436.334. MS m/ z: 436 (M⁺) and 421 (M⁺-CH₃). IR_v ^KBr_max 2970, 1710, 1680, 1450, 1375, 1110, and 1000.Uv?^hexane_max nm (log ?): 227 (4.15), 234 (4.22), 2.41 (4.19), and 2.51 (3.96).

Reduction of Compound C to Compound B by NaBH₄: Five mg of Compound C was dissolved in dioxane (1 ml) and NaBH₄ (29 rng in 70% aqueous EtOH, 1 ml) was added in a dioxane solution and kept for 1 hr at room temperature. Compound B was obtained after the decomposition of NaBH₄ with acetic acid.

Oxidation of Compound C to Compound D by activated MnO₂: MnSO₄ (1.2 g) was dissolved in 40% aqueous NaOH (15 ml). This solution was added slowly to a hot-water solution of KMnO₄ (60 ml: 9.6 g). The resulting precipitate, Mn0₂ was filtrated off, washed with water, dried at l2OC, and powdered. Forty-five mg of Compound C was dis­solved in CH₂C1₂ (11.5 ml), added the above-mentioned activated Mn0₂ (46.0 mg), and vibrated for 1 hr at room temperature. This mixture was filter­ed, and the filtrate was evaporated to yield Com­pound D.

3.2Extracts from a fruiting body grown in an experimental chamber

Reishi was incubated in a sterilized medium, a 5 X 10 cm bottle having wood meal-rice bran (3: 1, v/v), at 25ºC for 2 or 5weeks. When the mycelia prevailed in the medium, vegetative mycelia (“V.M.”) were collected from mycelia sticking on the inside wall of the bottle. Then the bottle was placed in a chamber at 27C. Thereafter, three samples were taken every five days, as follows: “M.A.” was sampled from mycelia extending into the air over the medium. “M.M.” was sampled from mycelia growing between the wall of the bottle and the medium because it was impossible for mycelia to be taken from the wood meal-rice bran medium. A fruiting body was taken from the surface of the medium in a bottle. Four samples were taken successively from separately in­cubated media.

Samples of fruiting bodies for investigations over time were taken every five days as a cluster of mycelia was produced on the surface of the medium and grew to become yellow on the 5th day when a new white cluster of mycelia was produced on top of the old yellow cluster (Fig. 2-3).

Each sample was extracted independently with MeOH: CHC1₃: H₂O (2: 1: 0.8, v/v/v). Each extract was freeze-dried, extracted with chloroform, and weighed.

Amounts of triterpenes, ergosterol, and free fatty-acids were measured by HPLC. Phenols were treated with Folin-Denis reagent.

The total fatty-acids were measured in methanolysis products of samples. That is, samples were hydrolysed by 1 N methanolic NaOH for 2 hrs at 90ºC. After neutralization, the chloroform-soluble part was obtained. Fifty gram of the dried chloroform-part, containing ?-cholestan as an internal standard, was refluxed in 2.5% methanolic HCI (2 ml). The reaction mixture was fractionated by ethylacetate, and the ethylacetate layer was measured in methylester of fatty acids by gel-permeation chromatography. 

3.3 Quantitative analysis of chemical constituents in sections of a fruiting body

The stipe and pileus of a fruiting body were separated. The stipe was divided into inner and outer sections, and the pileus was divided into gill and outer sections. The weights of each sections were shown in Fig. 4. The division was based on color. That is, only a thin layer of the surface was more dark-red than most of the brown outer layer which was more light in color than the inner layer.

A fruiting body was extracted with 70% aqueous methanol. The extract was divided into a chloroform-soluble part and an insoluble part.

The amounts of chemical constituents were deter­mined by the methods previously described. 

Acknowledgement: We thank Mr. A. Nakazawa, Nagano City, Nagano Prefecture, for kindly supplying the vegetative mycelia and fruiting bodies.

1)Kubo, M.; Matsuda, H.; Nogami, M.; An­chi, S.; Takahashi, T.: Yakugaku Zasshi, 103, 871-877 (1983).

2)Kanmatsuse, K.; Kajiwara, N.; Hayashi, K.; Shimogaichi, S.; Fukinbara, I.; Lshikawa, H.; Tarnura, T.: ibid, 105, 942-947 (1985).

3)Shimizu, A.; Yano, T.; Saito, Y.; Ishida, Y.: Chem. Pharm. Bull. 33, 3012-3013 (1985).

4)Kubo, M.; Matsuda, H.; Tanaka, M. Kimura, Y.; Tani, T.; Arichi, S.; Okuda, I.; Kirigaya, N.: Kiso to Rinsho, 13, 1367-1374 (1979).

5)Kohda, H.; Tokumoto. W.; Sakamoto, K.; Fujii, M. ; Hirai. Y. ; Yamasaki, K. Komoda. Y.: Nakamura, H.; Ishihara, S.; Uchida. M.: Chem: Pharm Bull. 33, 1367-1374 (1985).

6)Mizuno, I.; Usui, T.; Tomoda, M.; Shin­kai, K.; Shimizu, M.; Araka~va, M.; Tanaka, M.: Bull. Fac Agric Shizuoka Univ, 30, 41-50 (1980).

7)Mivazaki, T.; Nishiziina, M.: Chem Pharm Bull, 29. 3611-3616 (1981).

8)Miyazaki. T.: Carbohydr Res, 109, 290-294 (1982).

9)Mizuno, I.; Kato, N.; Totsuka, A.; Takena­ka, K.: Shinkai, K.; Shimizu, M.: Nippon Nogeikagaku Kaishi, 58, 871-880 (1984).

10)Kubota, T.; Asaka, Y.; Miura, I.; Mori, H.: Helv Chin: Acta, 65, 611-619 (1982).

11)Toth, 5. 0.: Luu, B.; Beck, 5.-P.; Ourisson, 0: J Chem Res (M), 299, 2722-2787 (S) (1983).

12)Nishitoba, T.; Sato, H.; Kasai, T.; Kawagi­shi, H.; Sakamura, S.: Agric Biol Chem, 48, 2905-2907 (1984).

13)Kikuchi, I.; Matsuda, 5; Kadota, S.; Murai, Y.: Ogita, Z.: Chem Pharm Bull, 33, 2624-2627 (1985).

14)Kikuchi, I.; Matsuda, S.; Murai, Y.; Ogita, Z.: ibid, 33, 2628-2631 (1985).

15)Komoda, Y.; Nakamura, H.; Ishihara, S.; Uchida, M.; Kohda, H.; Yamasaki, K.: Chem Pharm Bull, 33, 4829-4835 (1985).
Arichi, S.; Tani, T.; Kubo, M.; Matsuda, H.; Yoshimuna, N.; Kinigaya, N.: Kiso to Rinsho, 13, 4239-4244 (1979)