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Free radical scavenging activities of mushroom polysaccharide extracts
Life Sciences, Vol. 60, No. 10, pp. 763-~l,l!EJ7 CQpytight0 1997 Elrevier scienec Inc. Printed in the USA. All rigb rcactved 00x-3205/97 $17.00 + .OO
PI1 soo24-3205(97)00004-0
ELSEVIER
FREE RADICAL
SCAVENGING ACTIVITIES OF MUSHROOM POLYSACCHARIDE EXTRACTS
F. Liu, V. E. C. Ooi* and S. T. Chang Department
of Biology, The Chinese University Shatin, N. T., Hong Kong
of Hong Kong,
(Received in final form December 19, 19%)
Summarv The superoxide and hydroxyl radical scavenging activities of eight mushroom antitumor polysaccharide extracts were investigated using phenazin methosulphateNADH-nitroblue tetrazolium system and ascorbic acid-Cu*+-cytochrome C system respectively. The results showed that six of eight mushroom polysaccharide extracts had superoxide and hydroxyl radical scavenging activities. The protein content of the polysaccharide extracts appeared to contribute a direct effect on free radical scavenging activity. However, none of the mushroom polysaccharide extracts had antioxidative activity as measured by detecting malondialdehyde (MDA) contents of liver microsomes. Kq W&:
polpac&~&
musbreom, free radical, ar$ondialdehydedismutase,oxidativestress
Reactive oxygen species produced by sunlight, ultraviolet, ionizing radiation, chemical reactions and metabolic processes have a wide variety of pathological effects, such as causing DNA damage, carcinogenesis and cellular degeneration related to aging (l-3). Superoxide and hydroxyl radicals are the two most representative free radicals. In cellular oxidation reactions, superoxide radical is normally formed first, and its effects can be magnified because it produces other kinds of cell-damaging free radicals and oxidizing agents. However, the damaging action of the hydroxyl radical is the strongest among free radicals. Many synthetic chemicals such as phenolic compounds are found to be strong radical scavengers, but they usually have side effects (4,5). Among various naturally occurring substances, polysaccharide extracts from mushrooms may prove to be one of the useful candidates in the search for effective, non-toxic substances with free radical scavenging activity. Polysaccharide extracts from Ganoderma lucidum were reported to have scavenging effects on superoxide and hydroxyl radicals (6). Superoxide radical could be quenched rapidly in the presence of PSK, a protein-bound Corresponding address : Dr. V. E. C. Ooi, Department of Biology, The Chinese University of Hong Kong, Shatin, N. T., Hong Kong. Tel No.: (852) 2609-6353, Fax No.: (852) 2603-5646
Free Radical scavenging Polysaccharides
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Vol. 60, No. 10, 19!37
polysaccharide from Coriolus versicolor, in a cell-free system consisting of hypoxanthinexanthine oxidase (7,8). In the present study, two commercially available antitumor polysaccharides, PSK (8) and lentinan (9), and six mushroom polysaccharide extracts which were known to have antitumor activity (10-17) were tested and compared for their free radical scavenging and antioxidative activities. Methods Mushroom nolvsaccharide
extracts
PSK and lentinan are commercially available antitumor agents isolated from cultured mycelia of Coriolus versicolor (8) and the’fiuiting body of Lentinus edodes (9) respectively. The fruiting bodies of Gunoderma fucidum (lo), Grifolu umbellatu (1 l), and the cultured mycelia of Volvariella volvacea (12) and Tremella fuciformis (13) were extracted with boiling water and precipitated with ethanol. The culture filtrates of Tricholoma lobayense (14-16) and Schizophyllum commune (17) were centrifuged and then precipitated with ethanol. Polvsaccharide
and orotein contents
Polysaccharides are usually associated with protein as complexes. The polysaccharide protein contents of mushroom polysaccharide extracts were determined by the anthrone (18) and Lowry method (19), respectively.
and test
SuDeroxide radicals Superoxide radicals were generated in 3.0 ml of Tris-HCl buffer (16 mM, pH 8.0), which contained 78 pM P-nicotinamide adenine dinucleotide (reduced form, NADH), 50 pM nitroblue tetrazolium (NBT), 10 pM phenazin methosulfate (PMS), and mushroom polysaccharide extracts of varying concentrations. The color reaction of superoxide radicals and NBT was detected at OD 560 nm using Milton Roy Spectronic 3000. Vitamin C and glucose were used as the control in this experiment (20). Hvdroxvl radicals Hydroxyl radicals were generated in 3 ml of sodium phosphate buffer (0.15 mM, pH 7.4), which contained 100 pM vitamin C, 100 pM CuSO, , 12 pM cytochrome C and mushroom polysaccharide extracts of varying concentrations,. The mixture was incubated at 25°C for 90 min. The transmittance of color change of cytochrome C was measured at 550 nm using the Milton Roy Spectronic 3000. Thiourea and glucose were ased as the control in this experiment. The inhibition rate of hydroxyl radical generation by thiourea was taken as 100% (6). Inhibition rate (%) was calculated using the following formula : T-T2 X 100% Inhibition rate (%) = T_TI where T is the transmittance of hydroxyl radical (OH’) generation transmittance of control and test sample systems, respectively.
system, Tl and T2 are the
Preparation of mouse liver microsomes Male BALB /c mice (8- 12 weeks old) were sacrificed by cervical dislocation.
The liver was
Vol. 60, No. 10,19!37
Free Radical Scavenging Polysaccharidw
765
rapidly homogenized in ice-cold 0.25 M sucrose and centrifuged at 12,000 g for 20 min at 4°C. The supernatant obtained was centrifuged at 105,000 g for 60 min at 4°C. The microsomes were washed using ice-cold 0.15 M KCl, and then stored at - 20°C. The protein content of microsomes was measured using the Lowry method (19). Linid neroxidation The product of microsomal lipid peroxidation was malondialdehyde (MDA). Microsomes (200-300 pg/ml) were incubated at 37°C for 60 min with mushroom polysaccharide extracts of varying concentrations, 10 uM FeS04 and 0.1 mM ascorbic acid in 1.O ml potassium phosphate buffer solution (0.2 M, pH 7.4). The reaction was stopped by 20 % (wt / vol) trichloroacetic acid (TCA, 1.O ml) and 0.67 % (wt/vol) 2- thiobarbituric acid (TBA, 1.5 ml) in succession, and the solution was then heated at 100°C for 15 min. After centrifugation of precipitated protein, the color reaction of MDA-TBA complex was detected at OD 532 nm using Milton Roy Spectronic 3000. Butylated hydroxyanisole and glucose were used as the control in this experiment (20). Results The polysaccharide to protein ratios of the polysaccharide extracts and commercially available polysaccharide products were quite different among eight mushrooms (Table 1). The content of protein was higher than that of polysaccharide in PSK and polysaccharide-protein complexes of Ganoderma and Grifola, the latter being the highest among eight mushrooms. On the other hand, lentinan contained almost no protein. The superoxide radicals were generated in a PMS-NADH system and assayed by the reduction of NBT. Table 2 shows the superoxide radical scavenging activity of eight polysaccharide samples. Polysaccharide extracts of Ganoderma and Grijbla exhibited the strongest superoxide radical scavenging activity while lentinan and polysaccharide extracts of Schizophyllum had only negligible activity. PSK and polysaccharide extracts of Tricholoma, Volvariella and Tremella exhibited moderate activity of superoxide radical scavenging. As the concentration of mushroom polysaccharide-protein complexes increased, their superoxide radical scavenging activities were also improved. Table 3 shows the superoxide radical scavenging activity determined at the same protein concentration level in all mushroom polysaccharide samples. The superoxide radical scavenging activity of polysaccharide extracts appeared to depend on both protein and polysaccharide contents. Table 4 shows the results of hydroxyl radical scavenging activity as observed in the presence of polysaccharide samples. PSK and polysaccharide extracts of Ganoderma demonstrated the strongest activity of hydroxyl radical scavenging whereas lentinan and polysaccharide extracts of Schizophyllum had the lowest scavenging activity. Polysaccharide extracts of Tricholoma, Volvariella, Tremella and Grifola exhibited moderate hydroxyl radical scavenging activity, following a marked dose-dependent pattern. Malondialdehyde (MDA), formed from the breakdown of polyunsaturated fatty acids, serves as a convenient index for determining the extent of lipid peroxidation (21). As compared to the control, none of the eight mushroom polysaccharide samples inhibited microsomal lipid peroxidation (Table 5). On the contrary, lentinan and PSK significantly increased microsomal lipid peroxidation.
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Vol. 60, No. 10, 1997
Free Radical Scavenging Polysaccharides
TABLE
I
Polysaccharide to Protein Ratios in Mushroom Polysaccharide and Commercially Available Products
Samples
Grifola umbellata
Polysaccharide
Extracts
I Protein ratios
0.45
(Fruiting body)
Ganoderma lucidum
0.81
(Fruiting body)
Coriolus versicolor (PSK)
0.84
(Mycelium)
Tricholoma lobayense
1.67
(Culture filtrate)
Volvariella volvacea
1.93
(Mycelium)
TremellaJirciformis
3.6
(Mycelium)
Schizophyllum commune (Culture filtrate)
20
Lentinus edodes (Len&ran)
1000
(Fruiting body)
In the present study, five mushroom polysaccharide extracts and a protein-bound polysaccharide (PSK) exhibited significant activities of superoxide and hydroxyl radical scavenging. The superoxide radical scavenging activity of polysaccharide extracts appeared to depend on the amount of protein (peptide) present as polysaccharide-protein complexes. For example, lentinan and schizophyllan, which contained only a trace amount of protein in the polysaccharide samples, demonstrated almost no scavenging activities. In contrast, PSK and polysaccharide extracts from Ganoderma and Grifola which had lower polysaccharide/protein ratios showed the strongest scavenging activities. The result is in agreement with the reports on the free radical scavenging activities of polysaccharides/protein-bound polysaccharides from the mushrooms Ganoderma lucidum (6) and Coriolus versicolor (7), respectively.
Vol. 60, No. 10, 1997
Free Radical ScavengingPolysacctuuides
TABLE Effect of Mushroom Polysaccharide on Superoxide Radical Generation
767
II
Extracts and Commercially
Available Products
Conc.ug/ml
Superoxide radical generated (OD 560 nm)
5
0.682 k 0.003
5
0.560 + 0.019 0.342 + 0.010 0.118 k 0.004
17.89 49.85 82.70
20 80
0.569 k 0.001 0.340 + 0.005 0.172 k 0.006
16.57 50.15 74.78
5 20 80
0.572 f 0.013 0.448 f 0.001 0.394 f 0.004
16.13 34.3 1 42.23
Tricholoma lobayense (Culture filtrate)
5 20 80
0.562 If:0.001 0.545 * 0.003 0.514 z!z0.001
17.60 20.09 24.63
Volvariella volvacea (Mycelium)
5 20 80
0.618 + 0.004 0.553 f 0.016 0.5 19 * 0.030
9.38 18.91 23.90
Tremellaficijormis (Mycelium)
5 20 80
0.574 k 0.008 0.577 ik 0.016 0.578 + 0.008
15.84 15.39 15.25
Lentinus edodes (Lentinan) (Fruiting body)
5 20 80
0.662 k 0.010 0.648 f 0.007 0.676 k 0.007
2.93 4.99 0.88
Schizophyllum commune
5 20 80
0.696 f 0.010 0.662 k 0.006 0.672 k 0.007
- 2.05 2.93 1.47
300 pM / ml 300 pM / ml
0.225 + 0.013 0.663 + 0.005
67.01 2.79
Samples Control (Buffer) Ganoderma lucidum ( Fruiting body)
Grifola umbellata (Fruiting body)
Coriolus versicolor (PSK) (Mycelium)
(Culture filtrate)
Vitamin C Glucose
20 80 5
Inhibition %
The values of superoxide radical generation are mean + SD (n = 3).
Polysaccharide extracts from the alga Sargassum thunbergii (22) and the plant Saussurea involucrata (23) were also reported to have free radical scavenging effects. However, mechanism of free radical scavenging of polysaccharides is still not fully understood. It is known that phenolic compounds from the plant Pedicularis alashanica, such as phenylpropaniod glycosides, may react with superoxide radical by an one-electron transfer mechanism or hydrogen abstraction mechanism to form the semiquinones (24). Therefore, the
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Free Radical Scavenging Polysaccharides
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TABLE
III
Effect of Mushroom Polysaccharide Extracts and Commercially Products (10 ug proteinlmi) on Superoxide Radical Generation
Samples
Superoxide radical generated (OD 560 nm)
Available
Inhibition %
Control (Buffer)
0.798 f 0.014
Ganoderma lucidum
0.377 + 0.011
52.76
Grifola umbellata (Fruiting body)
0.477 i 0.049
40.23
Coriolus versicolor (PSK)
0.505 If 0.071
36.72
Tricholoma lobayense (Culture filtrate)
0.605 + 0.007
24.19
Tremellajixiformis
0.649 k 0.008
18.67
0.662 * 0.002
17.04
(Fruiting body)
(Mycelium)
(Mycelium) Volvariella volvacea
(Mycelium) The values of superoxide radical generation are mean + SD (n = 3). scavenging activity of phenylpropaniod glycosides for superoxide radical may be due to their reduction activities, which may be related to the number of phenolic hydroxyl groups and the conjugated system. However, it was not clear whether the mechanism of superoxide and hydroxyl radical scavenging by polysaccharide-protein complexes from mushrooms were similar to that of plant phenolic compounds. All mushroom polysaccharide extracts examined in this study did not show any antioxidative activity because they could not inhibit microsomal lipid peroxidation. Lipid peroxidation is a complex process. It involves the formation and propagation of lipid radicals, the uptake of oxygen, a rearrangement of the double bonds in unsaturated lipid, and the eventual destruction of membrane lipids, producing the breakdown products such as malondialdehyde (MDA) in microsomes (21). It was possible that in the present in vitro experiment, although Fe”/vitamin C system induced the production of microsomal peroxyl radical (25) the mushroom polysaccharide extracts appeared to have no ability in scavenging peroxyl radicals. PSK (8) and lentinan (9) are two well-known antitumor polysaccharide products from mushrooms. The other polysaccharide extracts from six mushrooms were all shown to have antitumor activity. However, the relationship among antitumor, free radical scavenging and antioxidative activities of mushroom polysaccharide extracts and products cannot be directly correlated. Further research is needed as in vitro experiment of the present study used high
Vol. 60, No. 10, 1997
Free Radical Scavenging Polysaccbarides
769
concentration of Fe2+/vitamin C and hence could not adequately reproduce the same result for in vivo situations (26). Notwithstanding, mushroom polysaccharide-protein complexes may provide a promising source for research and development of potential drugs for combating free radical pathogenesis. TABLE Effect of Mushroom Polysaccharide on Hydroxyl Radical Generation
Samples Control OH‘ Tbiourea
IV
Extracts and Commercially
Hydroxyl radical generated (OD 550 nm)
Cont. &ml Buffer, cytochrome Buffer
Available Products
Inhibition %
51.94 k 0.71 C 70.52 f 0.85
OH’ + 760
SO.72 k 0.91
100.00
OH +200
63.55 k 0.72
37.51
(Mycelium)
OH’ +400
60.46 + 0.48
54.14
Ganoderma lucidum
OH +200
65.33 k 0.19
27.93
( Fruiting body)
OH’ +400
62.02 k 0.21
45.75
Tremellaficiformis
OH +200
65.46 + 0.59
27.23
(Mycelium)
OH +400
63.94 f 0.09
35.41
Tricholoma lobayense
Coriolus versicolor (PW
OH’ +200
68.02 + 0.33
13.46
(Culture filtrate)
OH’ +400
66.01 f 0.21
24.27
Volvariella volvacea
OH’ +200
69.60 f 0.17
4.95
(Mycelium)
OH’ +400
67.10 k 0.88
18.41
Gr$ola umbellata
OH’ +200
69.37kO.11
6.19
(Fruiting body)
OH’ +400
68.57 zk0.07
10.50
Lentinus edodes
OH’ +400
70.12 f 0.05
OH’ +400
69.98 f 0.07
OH +400
70.65 + 0.03
(Lentinan) (Fruiting body) Schizophyllum commune (Culture filtrate) Glucose
The values of hydroxyl radical generation are mean f SD (n = 3). OH’ : Hydroxyl radical generation system
Free Radical Scavenging Polysaccharides
770
Vol. 60, No. 10, 1997
TABLE V Effect of Mushroom Polysaccharide on Microsomal Lipid Peroxidation
Extracts and Commercially
Available Products
Conc.pg /ml
MDA formation (OD at 532 nm)
Control (Buffer)
30
0.486 + 0.006
Ganoderma lucidum (Fruiting body)
30 60 120
0.467k 0.009 0.501~ 0.021 0.481f 0.011
0.04 - 0.03 0.01
Grifota umbellata (Fruiting body)
30 60 120
0.488f 0.017 0.497 f 0.025 0.477 f 0.022
-0.02 0.02
Coriolus versicolor (PSK) (Mycelium)
30 60 120
0.489 k 0.008 0.492 * 0.022 0.617 + 0.017
- 0.01 - 26.95
Tricholoma lobayense (Culture filtrate)
30 60 120
0.460 k 0.024 0.468 k 0.036 0.48 1 * 0.009
0.05 0.04 0.01
Volvariella volvacea (Mycelium)
30 60 120
0.477 f 0.02 1 0.467 f 0.010 0.487 k 0.012
0.02 0.04 0
Tremellafucijormis
30 60 120
0.478 f 0.018 0.485 f 0.027 0.493 k 0.026
0.02 0 - 0.01
Lentinus edodes (Lentinan) (Fruiting body)
30 60 120
0.463 k 0.027 0.498 k 0.02 1 0.607 k 0.028
0.05 - 0.03 - 24.90
Schizophyllum commune (Culture filtrate)
30 60 120
0.474 5 0.012 0.484 + 0.026 0.494 k 0.027
0.02 0 - 0.02
Butylated hydroxyanisole Glucose
1.5 PM/ml 300 pM /ml
0.268 + 0.023 0.478 f. 0.015
44.86 1.65
Samples
(Mycelium)
MAD : malondialdehyde;
Inhibition %
0
- 0.01
the values are mean f SD (n = 3).
Acknowledements This study was partially Council), Hong Kong.
supported
by an Earmarked
Grant from RGC (Research
Grant
Vol. 60, No. 10, 1997
Free Radical Scavenging Polysaccharides
771
References 1. 2. 3. 4. 5. 6.
J. L. MARX, Science 235 529-531 (1987). R. L. ZHANG, S. A. LESKO and P. 0. P. TS’O, Scientia Sinica x676-686 (1988). T. A. SARAFIAN and D. E. BREDESEN, Free Rad. Res. 2 l-8 (1994). Y. C. ZHOU and R. L. ZHENG, Biochem. Pharmacol. 42 1177-l 179 (1991). J. HEILMANN, I. MERFORT and M. EWISS, Planta Med. 61126-129 (1995). T. X. HU, J. W. CHEN, J. Y. XU, J. Y. LU and Q. Y. YANG, Acta Biochimica and Biophysics Sinica 24 465-470 (1992) (in Chinese) 7. H. SAKAGAMI, S. KOHNO, M. TAKEDA, K. NAKAMURA, K. NOMOTO, I. UENO, S. KANEGASAKI, T. NAOE and Y. KAWAZOE, Anticancer Res. 12 19952000 (1992). 8. H. SAKAGAMI, T. AOKI, A. SIMPSON and S. I. TANUMA. Anticancer Res. 11 9931000 (1991). 9. G. CHIHARA, J. HAMURO, Y. Y. MAEDA, Y. ARAI and F. FUKUOKA, Cancer Res. 30 2776-278 1 (1970). 10. J. W. HYUN, E. C. CHOI and B. K. KIM, Korean J. Mycol. B 58-69 (1990). 11. T. MIYAZAKI, N. OIKAWA, H. YAMADA and T. YADOMAE, Carbohydr. Res. 65 235243 (1978). 12. E. KISHIDA, Y. SONE and A. MISAKI, Carbohydr. Res. 193 227-239 (1989). 13. S. UKAI, H. KIRIKI, K. NAGAI and T. KIHO, Yakugaku Zasshi 112 663-668 (1992) (in Japanese). 14. F. LIU, V. E. C. 001 and S. T. CHANG, World J. Microbial. Biotech. 1486-490 (1995). 15. F. LIU, M. C. FUNG, V. E. C. 001, and S. T. CHANG, Life Sci. 58 1795-l 803 (1996). 16. F. LIU, V. E. C. 001, W. K. LIU and S. T. CHANG, Gen. Pharmacol. 27 621-624 17. K. TABATA, W. ITO, T. KOJIMA, S. KAWABATA and A. MISAKI, Carbohydr. Res. @ 121-135 (1981). 18. H. KAWAGISHI, A. NOMURA, T. MIZUNO, A. KIMURA and S. CHIBA, Biochim. Biophys. Acta. 1034 247-252 (1988). 19. 0. M. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem. 193265-275 (1951). 20. J. LI, R. L. ZHENG, Z. M. LIU and Z. J. JIA, Acta Pharmacologica Sinica 13 427-430 (1992) 21. H. ESTERBAUER, in: Free Radical in Liver Injure (edited by G. POLL K. H. CHEESEMAN, M. U. DIANZANI and T. F. SLATER), IRL Press, Oxford, p. 29-48 (1985). 22. E. X. ZHANG, L. J. YU and X. XIAO, Zhongguo Haiyang Yaowu 14 l-4 (1995) (in Chinese) 23. R. L. ZHENG, G. S. LIU, G. X. XING, Z. J. JIA, M. DU and L. Q. Quan, Acra Pharmacologica Sinica 14 47-49 (1993) (in Chinese) 24. P. F. WANG, J. H. KANG, R. L. ZHENG, Z. H. YANG, J. F. LU, J. J. GAO and Z. J. JIA, Biochem. Pharmacol. ig 687-691 (1996). 25. B. A. IGOR, I. D. ANATOLII, V. B. ALEKSANDER, A. K. VLADIMIR and I. P. ALLA, Biochem. Pharmacol. 38 1763-I 769 (1989). 26. G. MINOTTI, M. D. GENNARO, D. DUG0 and P. GRANONE, Free Rad. Res. Comms. 1_299-106 (1991).
PI1 soo24-3205(97)00004-0
ELSEVIER
FREE RADICAL
SCAVENGING ACTIVITIES OF MUSHROOM POLYSACCHARIDE EXTRACTS
F. Liu, V. E. C. Ooi* and S. T. Chang Department
of Biology, The Chinese University Shatin, N. T., Hong Kong
of Hong Kong,
(Received in final form December 19, 19%)
Summarv The superoxide and hydroxyl radical scavenging activities of eight mushroom antitumor polysaccharide extracts were investigated using phenazin methosulphateNADH-nitroblue tetrazolium system and ascorbic acid-Cu*+-cytochrome C system respectively. The results showed that six of eight mushroom polysaccharide extracts had superoxide and hydroxyl radical scavenging activities. The protein content of the polysaccharide extracts appeared to contribute a direct effect on free radical scavenging activity. However, none of the mushroom polysaccharide extracts had antioxidative activity as measured by detecting malondialdehyde (MDA) contents of liver microsomes. Kq W&:
polpac&~&
musbreom, free radical, ar$ondialdehydedismutase,oxidativestress
Reactive oxygen species produced by sunlight, ultraviolet, ionizing radiation, chemical reactions and metabolic processes have a wide variety of pathological effects, such as causing DNA damage, carcinogenesis and cellular degeneration related to aging (l-3). Superoxide and hydroxyl radicals are the two most representative free radicals. In cellular oxidation reactions, superoxide radical is normally formed first, and its effects can be magnified because it produces other kinds of cell-damaging free radicals and oxidizing agents. However, the damaging action of the hydroxyl radical is the strongest among free radicals. Many synthetic chemicals such as phenolic compounds are found to be strong radical scavengers, but they usually have side effects (4,5). Among various naturally occurring substances, polysaccharide extracts from mushrooms may prove to be one of the useful candidates in the search for effective, non-toxic substances with free radical scavenging activity. Polysaccharide extracts from Ganoderma lucidum were reported to have scavenging effects on superoxide and hydroxyl radicals (6). Superoxide radical could be quenched rapidly in the presence of PSK, a protein-bound Corresponding address : Dr. V. E. C. Ooi, Department of Biology, The Chinese University of Hong Kong, Shatin, N. T., Hong Kong. Tel No.: (852) 2609-6353, Fax No.: (852) 2603-5646
Free Radical scavenging Polysaccharides
764
Vol. 60, No. 10, 19!37
polysaccharide from Coriolus versicolor, in a cell-free system consisting of hypoxanthinexanthine oxidase (7,8). In the present study, two commercially available antitumor polysaccharides, PSK (8) and lentinan (9), and six mushroom polysaccharide extracts which were known to have antitumor activity (10-17) were tested and compared for their free radical scavenging and antioxidative activities. Methods Mushroom nolvsaccharide
extracts
PSK and lentinan are commercially available antitumor agents isolated from cultured mycelia of Coriolus versicolor (8) and the’fiuiting body of Lentinus edodes (9) respectively. The fruiting bodies of Gunoderma fucidum (lo), Grifolu umbellatu (1 l), and the cultured mycelia of Volvariella volvacea (12) and Tremella fuciformis (13) were extracted with boiling water and precipitated with ethanol. The culture filtrates of Tricholoma lobayense (14-16) and Schizophyllum commune (17) were centrifuged and then precipitated with ethanol. Polvsaccharide
and orotein contents
Polysaccharides are usually associated with protein as complexes. The polysaccharide protein contents of mushroom polysaccharide extracts were determined by the anthrone (18) and Lowry method (19), respectively.
and test
SuDeroxide radicals Superoxide radicals were generated in 3.0 ml of Tris-HCl buffer (16 mM, pH 8.0), which contained 78 pM P-nicotinamide adenine dinucleotide (reduced form, NADH), 50 pM nitroblue tetrazolium (NBT), 10 pM phenazin methosulfate (PMS), and mushroom polysaccharide extracts of varying concentrations. The color reaction of superoxide radicals and NBT was detected at OD 560 nm using Milton Roy Spectronic 3000. Vitamin C and glucose were used as the control in this experiment (20). Hvdroxvl radicals Hydroxyl radicals were generated in 3 ml of sodium phosphate buffer (0.15 mM, pH 7.4), which contained 100 pM vitamin C, 100 pM CuSO, , 12 pM cytochrome C and mushroom polysaccharide extracts of varying concentrations,. The mixture was incubated at 25°C for 90 min. The transmittance of color change of cytochrome C was measured at 550 nm using the Milton Roy Spectronic 3000. Thiourea and glucose were ased as the control in this experiment. The inhibition rate of hydroxyl radical generation by thiourea was taken as 100% (6). Inhibition rate (%) was calculated using the following formula : T-T2 X 100% Inhibition rate (%) = T_TI where T is the transmittance of hydroxyl radical (OH’) generation transmittance of control and test sample systems, respectively.
system, Tl and T2 are the
Preparation of mouse liver microsomes Male BALB /c mice (8- 12 weeks old) were sacrificed by cervical dislocation.
The liver was
Vol. 60, No. 10,19!37
Free Radical Scavenging Polysaccharidw
765
rapidly homogenized in ice-cold 0.25 M sucrose and centrifuged at 12,000 g for 20 min at 4°C. The supernatant obtained was centrifuged at 105,000 g for 60 min at 4°C. The microsomes were washed using ice-cold 0.15 M KCl, and then stored at - 20°C. The protein content of microsomes was measured using the Lowry method (19). Linid neroxidation The product of microsomal lipid peroxidation was malondialdehyde (MDA). Microsomes (200-300 pg/ml) were incubated at 37°C for 60 min with mushroom polysaccharide extracts of varying concentrations, 10 uM FeS04 and 0.1 mM ascorbic acid in 1.O ml potassium phosphate buffer solution (0.2 M, pH 7.4). The reaction was stopped by 20 % (wt / vol) trichloroacetic acid (TCA, 1.O ml) and 0.67 % (wt/vol) 2- thiobarbituric acid (TBA, 1.5 ml) in succession, and the solution was then heated at 100°C for 15 min. After centrifugation of precipitated protein, the color reaction of MDA-TBA complex was detected at OD 532 nm using Milton Roy Spectronic 3000. Butylated hydroxyanisole and glucose were used as the control in this experiment (20). Results The polysaccharide to protein ratios of the polysaccharide extracts and commercially available polysaccharide products were quite different among eight mushrooms (Table 1). The content of protein was higher than that of polysaccharide in PSK and polysaccharide-protein complexes of Ganoderma and Grifola, the latter being the highest among eight mushrooms. On the other hand, lentinan contained almost no protein. The superoxide radicals were generated in a PMS-NADH system and assayed by the reduction of NBT. Table 2 shows the superoxide radical scavenging activity of eight polysaccharide samples. Polysaccharide extracts of Ganoderma and Grijbla exhibited the strongest superoxide radical scavenging activity while lentinan and polysaccharide extracts of Schizophyllum had only negligible activity. PSK and polysaccharide extracts of Tricholoma, Volvariella and Tremella exhibited moderate activity of superoxide radical scavenging. As the concentration of mushroom polysaccharide-protein complexes increased, their superoxide radical scavenging activities were also improved. Table 3 shows the superoxide radical scavenging activity determined at the same protein concentration level in all mushroom polysaccharide samples. The superoxide radical scavenging activity of polysaccharide extracts appeared to depend on both protein and polysaccharide contents. Table 4 shows the results of hydroxyl radical scavenging activity as observed in the presence of polysaccharide samples. PSK and polysaccharide extracts of Ganoderma demonstrated the strongest activity of hydroxyl radical scavenging whereas lentinan and polysaccharide extracts of Schizophyllum had the lowest scavenging activity. Polysaccharide extracts of Tricholoma, Volvariella, Tremella and Grifola exhibited moderate hydroxyl radical scavenging activity, following a marked dose-dependent pattern. Malondialdehyde (MDA), formed from the breakdown of polyunsaturated fatty acids, serves as a convenient index for determining the extent of lipid peroxidation (21). As compared to the control, none of the eight mushroom polysaccharide samples inhibited microsomal lipid peroxidation (Table 5). On the contrary, lentinan and PSK significantly increased microsomal lipid peroxidation.
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Free Radical Scavenging Polysaccharides
TABLE
I
Polysaccharide to Protein Ratios in Mushroom Polysaccharide and Commercially Available Products
Samples
Grifola umbellata
Polysaccharide
Extracts
I Protein ratios
0.45
(Fruiting body)
Ganoderma lucidum
0.81
(Fruiting body)
Coriolus versicolor (PSK)
0.84
(Mycelium)
Tricholoma lobayense
1.67
(Culture filtrate)
Volvariella volvacea
1.93
(Mycelium)
TremellaJirciformis
3.6
(Mycelium)
Schizophyllum commune (Culture filtrate)
20
Lentinus edodes (Len&ran)
1000
(Fruiting body)
In the present study, five mushroom polysaccharide extracts and a protein-bound polysaccharide (PSK) exhibited significant activities of superoxide and hydroxyl radical scavenging. The superoxide radical scavenging activity of polysaccharide extracts appeared to depend on the amount of protein (peptide) present as polysaccharide-protein complexes. For example, lentinan and schizophyllan, which contained only a trace amount of protein in the polysaccharide samples, demonstrated almost no scavenging activities. In contrast, PSK and polysaccharide extracts from Ganoderma and Grifola which had lower polysaccharide/protein ratios showed the strongest scavenging activities. The result is in agreement with the reports on the free radical scavenging activities of polysaccharides/protein-bound polysaccharides from the mushrooms Ganoderma lucidum (6) and Coriolus versicolor (7), respectively.
Vol. 60, No. 10, 1997
Free Radical ScavengingPolysacctuuides
TABLE Effect of Mushroom Polysaccharide on Superoxide Radical Generation
767
II
Extracts and Commercially
Available Products
Conc.ug/ml
Superoxide radical generated (OD 560 nm)
5
0.682 k 0.003
5
0.560 + 0.019 0.342 + 0.010 0.118 k 0.004
17.89 49.85 82.70
20 80
0.569 k 0.001 0.340 + 0.005 0.172 k 0.006
16.57 50.15 74.78
5 20 80
0.572 f 0.013 0.448 f 0.001 0.394 f 0.004
16.13 34.3 1 42.23
Tricholoma lobayense (Culture filtrate)
5 20 80
0.562 If:0.001 0.545 * 0.003 0.514 z!z0.001
17.60 20.09 24.63
Volvariella volvacea (Mycelium)
5 20 80
0.618 + 0.004 0.553 f 0.016 0.5 19 * 0.030
9.38 18.91 23.90
Tremellaficijormis (Mycelium)
5 20 80
0.574 k 0.008 0.577 ik 0.016 0.578 + 0.008
15.84 15.39 15.25
Lentinus edodes (Lentinan) (Fruiting body)
5 20 80
0.662 k 0.010 0.648 f 0.007 0.676 k 0.007
2.93 4.99 0.88
Schizophyllum commune
5 20 80
0.696 f 0.010 0.662 k 0.006 0.672 k 0.007
- 2.05 2.93 1.47
300 pM / ml 300 pM / ml
0.225 + 0.013 0.663 + 0.005
67.01 2.79
Samples Control (Buffer) Ganoderma lucidum ( Fruiting body)
Grifola umbellata (Fruiting body)
Coriolus versicolor (PSK) (Mycelium)
(Culture filtrate)
Vitamin C Glucose
20 80 5
Inhibition %
The values of superoxide radical generation are mean + SD (n = 3).
Polysaccharide extracts from the alga Sargassum thunbergii (22) and the plant Saussurea involucrata (23) were also reported to have free radical scavenging effects. However, mechanism of free radical scavenging of polysaccharides is still not fully understood. It is known that phenolic compounds from the plant Pedicularis alashanica, such as phenylpropaniod glycosides, may react with superoxide radical by an one-electron transfer mechanism or hydrogen abstraction mechanism to form the semiquinones (24). Therefore, the
Vol. 60, No. 10, 1997
Free Radical Scavenging Polysaccharides
768
TABLE
III
Effect of Mushroom Polysaccharide Extracts and Commercially Products (10 ug proteinlmi) on Superoxide Radical Generation
Samples
Superoxide radical generated (OD 560 nm)
Available
Inhibition %
Control (Buffer)
0.798 f 0.014
Ganoderma lucidum
0.377 + 0.011
52.76
Grifola umbellata (Fruiting body)
0.477 i 0.049
40.23
Coriolus versicolor (PSK)
0.505 If 0.071
36.72
Tricholoma lobayense (Culture filtrate)
0.605 + 0.007
24.19
Tremellajixiformis
0.649 k 0.008
18.67
0.662 * 0.002
17.04
(Fruiting body)
(Mycelium)
(Mycelium) Volvariella volvacea
(Mycelium) The values of superoxide radical generation are mean + SD (n = 3). scavenging activity of phenylpropaniod glycosides for superoxide radical may be due to their reduction activities, which may be related to the number of phenolic hydroxyl groups and the conjugated system. However, it was not clear whether the mechanism of superoxide and hydroxyl radical scavenging by polysaccharide-protein complexes from mushrooms were similar to that of plant phenolic compounds. All mushroom polysaccharide extracts examined in this study did not show any antioxidative activity because they could not inhibit microsomal lipid peroxidation. Lipid peroxidation is a complex process. It involves the formation and propagation of lipid radicals, the uptake of oxygen, a rearrangement of the double bonds in unsaturated lipid, and the eventual destruction of membrane lipids, producing the breakdown products such as malondialdehyde (MDA) in microsomes (21). It was possible that in the present in vitro experiment, although Fe”/vitamin C system induced the production of microsomal peroxyl radical (25) the mushroom polysaccharide extracts appeared to have no ability in scavenging peroxyl radicals. PSK (8) and lentinan (9) are two well-known antitumor polysaccharide products from mushrooms. The other polysaccharide extracts from six mushrooms were all shown to have antitumor activity. However, the relationship among antitumor, free radical scavenging and antioxidative activities of mushroom polysaccharide extracts and products cannot be directly correlated. Further research is needed as in vitro experiment of the present study used high
Vol. 60, No. 10, 1997
Free Radical Scavenging Polysaccbarides
769
concentration of Fe2+/vitamin C and hence could not adequately reproduce the same result for in vivo situations (26). Notwithstanding, mushroom polysaccharide-protein complexes may provide a promising source for research and development of potential drugs for combating free radical pathogenesis. TABLE Effect of Mushroom Polysaccharide on Hydroxyl Radical Generation
Samples Control OH‘ Tbiourea
IV
Extracts and Commercially
Hydroxyl radical generated (OD 550 nm)
Cont. &ml Buffer, cytochrome Buffer
Available Products
Inhibition %
51.94 k 0.71 C 70.52 f 0.85
OH’ + 760
SO.72 k 0.91
100.00
OH +200
63.55 k 0.72
37.51
(Mycelium)
OH’ +400
60.46 + 0.48
54.14
Ganoderma lucidum
OH +200
65.33 k 0.19
27.93
( Fruiting body)
OH’ +400
62.02 k 0.21
45.75
Tremellaficiformis
OH +200
65.46 + 0.59
27.23
(Mycelium)
OH +400
63.94 f 0.09
35.41
Tricholoma lobayense
Coriolus versicolor (PW
OH’ +200
68.02 + 0.33
13.46
(Culture filtrate)
OH’ +400
66.01 f 0.21
24.27
Volvariella volvacea
OH’ +200
69.60 f 0.17
4.95
(Mycelium)
OH’ +400
67.10 k 0.88
18.41
Gr$ola umbellata
OH’ +200
69.37kO.11
6.19
(Fruiting body)
OH’ +400
68.57 zk0.07
10.50
Lentinus edodes
OH’ +400
70.12 f 0.05
OH’ +400
69.98 f 0.07
OH +400
70.65 + 0.03
(Lentinan) (Fruiting body) Schizophyllum commune (Culture filtrate) Glucose
The values of hydroxyl radical generation are mean f SD (n = 3). OH’ : Hydroxyl radical generation system
Free Radical Scavenging Polysaccharides
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Vol. 60, No. 10, 1997
TABLE V Effect of Mushroom Polysaccharide on Microsomal Lipid Peroxidation
Extracts and Commercially
Available Products
Conc.pg /ml
MDA formation (OD at 532 nm)
Control (Buffer)
30
0.486 + 0.006
Ganoderma lucidum (Fruiting body)
30 60 120
0.467k 0.009 0.501~ 0.021 0.481f 0.011
0.04 - 0.03 0.01
Grifota umbellata (Fruiting body)
30 60 120
0.488f 0.017 0.497 f 0.025 0.477 f 0.022
-0.02 0.02
Coriolus versicolor (PSK) (Mycelium)
30 60 120
0.489 k 0.008 0.492 * 0.022 0.617 + 0.017
- 0.01 - 26.95
Tricholoma lobayense (Culture filtrate)
30 60 120
0.460 k 0.024 0.468 k 0.036 0.48 1 * 0.009
0.05 0.04 0.01
Volvariella volvacea (Mycelium)
30 60 120
0.477 f 0.02 1 0.467 f 0.010 0.487 k 0.012
0.02 0.04 0
Tremellafucijormis
30 60 120
0.478 f 0.018 0.485 f 0.027 0.493 k 0.026
0.02 0 - 0.01
Lentinus edodes (Lentinan) (Fruiting body)
30 60 120
0.463 k 0.027 0.498 k 0.02 1 0.607 k 0.028
0.05 - 0.03 - 24.90
Schizophyllum commune (Culture filtrate)
30 60 120
0.474 5 0.012 0.484 + 0.026 0.494 k 0.027
0.02 0 - 0.02
Butylated hydroxyanisole Glucose
1.5 PM/ml 300 pM /ml
0.268 + 0.023 0.478 f. 0.015
44.86 1.65
Samples
(Mycelium)
MAD : malondialdehyde;
Inhibition %
0
- 0.01
the values are mean f SD (n = 3).
Acknowledements This study was partially Council), Hong Kong.
supported
by an Earmarked
Grant from RGC (Research
Grant
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Free Radical Scavenging Polysaccharides
771
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