International Journal of Food Sciences and Nutrition, Volume 55, Number 1 (February 2004) 75 /83

Ganoderma lucidum (Lingzhi); acute and short-term biomarker response to supplementation

Sissi Wachtel-Galor,1 Yim-Tong Szeto,1 Brian Tomlinson2 and Iris FF Benzie1
1

Ageing & Health Group, School of Nursing, The Hong Kong Polytechnic University, Kowloon, Hong Kong SAR, China; 2Department of Clinical Pharmacology, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China

Ganoderma lucidum (Lingzhi) is a popular Chinese herb with an impressive array of reputed health benefits, including antioxidant properties. However, these require scientific validation. The aim of this study was to investigate in vitro antioxidant capacity of Lingzhi, absorption and systemic distribution of Lingzhi antioxidants, and effects of short-term (10 days) supplementation on biomarkers of antioxidant status, coronary heart disease (CHD) risk and DNA damage. In this double-blinded, placebo-controlled, cross-over intervention study, blood and urine samples were collected from 10 healthy volunteers at 0 (fasting) and 45, 90, 135 and 180 min post-ingestion of a single dose (1.1g) of Lingzhi. Repeat fasting samples were collected after 10 days supplementation with 0.72g/d Lingzhi. The acute response (up to 3 hours) was also investigated with a larger dose (3.3g) of Lingzhi (n 0/7). Results showed that the total antioxidant capacity (as the FRAP value) of an aqueous suspension of Lingzhi was 360 mmol/g. Ingestion of Lingzhi caused a significant post-ingestion increase (mean9/SEM 239/3 mmol/L; P B/0.05) in plasma antioxidant capacity, with peak response at 90min. Average increase of 299/11% (P B/0.05) in urine antioxidant capacity was seen within 3 hours of ingestion. After 10 days supplementation with 0.72g per day of Lingzhi, fasting plasma lipid standardised a-tocopherol concentration and urine antioxidant capacity increased (P B/0.05). Fasting plasma ascorbic acid and total a-tocopherol concentrations and erythrocyte SOD and GPx activities increased slightly but non-significantly with supplementation. Plasma lipids and uric acid tended to decrease, but changes were not statistically significant. No discernable differences were seen in other variables measured. Results indicate that Lingzhi intake causes an acute increase in plasma antioxidant capacity. No deleterious effects on measured variables were seen. The pattern of biomarker response after supplementation indicated possible benefit in terms of antioxidant status and CHD risk, but further study is needed to elucidate the nature and longer-term effects of the absorbable antioxidants from Lingzhi.

Correspondence to: Dr Iris Benzie, Ageing & Health Group, School of Nursing, The Hong Kong Polytechnic University, Kowloon, Hong Kong SAR, China. Fax (0)852 23649663 e-mail: hsbenzie@inet.polyu.edu.hk

ISSN 0963-7486 printed/ISSN 1465-3478 online # 2004 Taylor & Francis Ltd DOI: 10.1080/09637480310001642510

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Introduction Mushrooms are considered a special kind of food, and as items of food delicacy because of their characteristic texture and flavour. The chemical, biological, and biochemical properties of the fruiting bodies of mushrooms are numerous, and higher basidiomycetes mushrooms are used in folk medicine throughout the world (Borchers et al., 1999; Wasser & Weiss, 1999). Ganoderma lucidum (Figure 1) is a woody mushroom which is used widely in the promotion of health and longevity in Asia and, increasingly, worldwide. In China the mushroom is known as Lingzhi and in Japan as Reishi (Jong & Birmingham, 1992; Mizuno et al., 1995; Chang & Buswell, 1999). The wide consumption of this mushroom and its medicinal benefits have been reported since ancient times, and reference to Lingzhi can be found in the Shen Nong Ben Cao, which is the earliest written Chinese Classic of the Materia Medica composed in the Eastern Han Dynasty (25 /220A.D). To date, numerous studies have identified a wide variety of constituents within Lingzhi, including triterpenes and polysaccharides, which have been hypothesized to contribute to the antioxidant, lipid lowering, anticancer, antiviral, to name but a few, of its reputed properties. Various antioxidants from plants and mushrooms are reported to prevent oxidative damage, and hence, may help reduce cancer and heart diseases. Both aqueous and lipophilic extracts of Lingzhi have shown antioxidant properties in in-vitro systems (Zhu et al., 1999; Lee et al., 2001). These findings on the antioxidant properties of the Lingzhi mushroom raise questions regarding absorption of these antioxidants and whether they can be utilised by the human body to lower risk of chronic disease. Lingzhi has no reported toxic effects, even at very high doses (Chang, 1994). This apparent lack of toxicity and reputed widespread health benefits form an attractive combination that has resulted in this herb becoming increasingly popular as a healthfood supplement and panacea. However, published human studies with this herb are few, and the health claims and effects of Lingzhi require scientific validation. The aim of this study, therefore, was to explore the potential cardioprotective effect of Lingzhi in vivo and to examine the antioxidant properties of the Lingzhi mushroom. Specifically, we measured the in vitro antioxidant capacity of Lingzhi, investigated the bioavailability (absorption and systemic distribution) of antioxidants in Lingzhi, and determined the effect of short-term (10 days) Lingzhi supplementation on biomarkers of oxidant/antioxidant balance and coronary heart disease (CHD) risk. Subjects and methods A total of 14 subjects consented to take part in this crossover intervention study. These were apparently healthy, normotensive adults, with no history of chronic disease, and not on any regular medication. The age range of the subjects was 22 to 56 years (mean(SD) 35.2 (10.4)) and BMI of 19 to 27 Kg/m2 (mean (SD) 22.6 (2.6)). In the acute post-ingestion study to assess absorption and systemic distribution (bioavailability) of antioxidants, 10 subjects ingested 1.1g and, in a separate experiment, seven subjects ingested 3.3g of a commercially available Lingzhi powder preparation. Three subjects participated in both the low and high dose trials, with at least two weeks between these experiments. Lingzhi and visually identical placebo capsules were supplied by Vitagreen Co Ltd Hong Kong. For the 1.1g dose the

Figure 1. The Lingzhi mushroom (Ganoderma lucidum )

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Lingzhi (and placebo) was taken in the form of capsules, while for the 3.3g dose the Lingzhi was taken in the form of powder released from the capsules. Ten subjects (4 men, 6 women) participated in the 10-day double-blinded placebo controlled crossover supplementation study in which 2 capsules/ day of Lingzhi (total of 0.72 g, which is the equivalent of 6.6g of fresh mushroom) or placebo were taken. There was at least a twoweek washout period between the active and placebo treatments. Five subjects were allocated, on a non-selective basis, to begin with placebo, and five to begin with Lingzhi. In the acute post-ingestion experiments, a fasting heparinised venous blood sample (4 ml) from the antecubital vein and a urine sample were collected from each subject, after which Lingzhi or placebo was ingested with 200/400 ml of warm water. Additional venous blood samples were collected into heparinised blood collection tubes at 45, 90, 135 and 180 minutes post-ingestion. Urine samples were collected at 90 minutes intervals into clean glass containers containing no preservative for 3 hours post-ingestion. Subjects remained fasting, except for sips of water, for the entire 3 hour period of sample collection. In the short-term (10 day) supplementation study, fasting blood and urine samples were taken on days 1 and 11. Compliance was checked by regular telephone contact with volunteers during each 10-day treatment and by counting returned capsules at the end of each treatment period. Blood samples were stored at 48C until separation, which was within three hours of collection. Plasma was used immediately thereafter for measurement of ascorbic acid (vitamin C) concentration and total antioxidant capacity (as the FRAP value). White cells were harvested and used on the day of collection for measurement of DNA damage. Washed erythrocytes and aliquots of plasma were stored at (/708C for other biochemical variables, all of which were measured within 4 months. Urine was tested on the day of collection. Filtered suspensions of Lingzhi powder in water were prepared and measured for total antioxidant capacity immediately after preparation.

To measure plasma and Lingzhi antioxidant capacity, and to assess absorption and systemic distribution (which we term bioavailability) of Lingzhi antioxidants, we used a sensitive and well-validated biomonitoring tool, the Ferric Reducing /Antioxidant Power (FRAP) assay (US patented; Benzie & Strain, 1996a). Conceptually, if dietary antioxidants are absorbed and enter the systemic circulation there will be an increase in plasma antioxidant capacity, the magnitude of increase reflecting the amount of antioxidants absorbed. If these antioxidants are also excreted, there will be an increase in urinary antioxidant capacity. The FRAP assay enables objective and highly reproducible results to be obtained on complex biological samples, it is simple and rapid enough to be performed on freshly collected specimens, and has been used successfully in the past to monitor absorption and excretion of tea antioxidants (Benzie et al., 1999). In this study, we also used a variation of the FRAP assay, known as ferric reducing (antioxidant) power and ascorbic acid concentrations (FRASC). This enables the measurement of both the total antioxidant capacity (as the FRAP value) and the ascorbic acid concentration simultaneously (Benzie & Strain, 1997). FRASC was performed as described previously in detail (Benzie & Strain, 1999). Several other biomarkers of oxidant:antioxidant balance were measured pre- and post- 10 days supplementation. These included: plasma a-tocopherol (vitamin E; total and lipid standardised); malondialdehyde (MDA; a lipid peroxidation degradation product); erythrocyte superoxide dismutase (SOD); glutathione peroxidase (GPx); baseline DNA damage, and the resistance of DNA to a standard oxidant challenge. Plasma MDA and a-tocopherol concentrations were measured by HPLC according to published methods and following our standard protocols (Chirico et al., 1993; Brandt et al., 1996; Jentzsch et al., 1996). SOD and GPx were measured using commercial kit methods (Randox, Co Antrim, Northern Ireland) and enzyme activities were standardised to haemoglobin content, which was measured using a standard spectrophotometric technique. DNA

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damage and resistance to a standard oxidant challenge (induced by H2O2) in white blood cells were measured, as previously published (Collins et al., 1997; Szeto & Benzie, 2002), by the comet assay, a well-validated tool for the detection of single strand breaks in DNA in individual cells. Results are presented as the %DNA in the comet tail, which correlates directly with DNA damage. Biomarkers of CHD risk measured included plasma lipids and uric acid. Plasma uric acid, a putative antioxidant but also an independent risk factor for CHD (Benzie & Strain, 1996b), was measured using a commercial kit method (Roche, Basel, Switzerland). Plasma total cholesterol (TC) and triacylglycerol (Tg) were measured using commercial test kit methods (Roche) as biomarkers of CHD risk and also to permit lipid standardisation of plasma a-tocopherol concentrations (Thurnham et al., 1986). Plasma high density lipoprotein cholesterol (HDL-C) was measured using a phosphotungstate precipitation method (Lopes-Virella et al., 1977) and a commercial kit for cholesterol (Roche). Plasma low density lipoprotein cholesterol (LDL-C) concentration was calculated using Friedewald equation (Friedewald et al., 1972). Urine creatinine was measured by the Jaffe reaction using a commercial kit (Roche) and results used to concentration-standardise the urine FRAP values. Ethical approval for this study was granted by the Ethics Human Subcommittee of The Hong Kong Polytechnic University, and all procedures involving human subjects complied with the Declaration of Helsinki, as revised in 2000. For statistical analysis, Graphpad software was used (version 2.01, San Diego, USA). For the acute post-ingestion study, the Kruskal-Wallis test (non parametric analysis of variance) followed by Dunns multiple comparison test was used to investigate timed post-ingestion changes in plasma antioxidant power (FRAP values) in relation to baseline levels, and the Mann Whitney test was used to compare responses to Lingzhi and placebo. For the short term (10 days) supplementation study, the Wilcoxon matched pairs test was used to compare

response to each treatment (difference between days 1 and 11, Lingzhi vs placebo), with each subject acting as his or her own control. Results The Lingzhi powder showed antioxidant capacity, with FRAP value of 360 mmol/g. For reference, the FRAP value of pure ascorbic acid is 11,364 mmol/g. In the acute post ingestion bioavailability study, no increase in plasma FRAP was seen with the low dose (1.1 g) of Lingzhi over the 3 hour monitoring period, however, the postLingzhi FRAP values were higher in all subjects at all time points post-ingestion when compared to the matching values after placebo, owing to a sustained decrease in plasma FRAP during extended fasting (Figure 2). This suggested absorption and systemic distribution of antioxidants from Lingzhi. This was confirmed in the high dose (3.3 g) bioavailability study, in which a significant post-ingestion increase (mean9/ SEM of 239/3 mmol/L; P B/0.05) in plasma FRAP was seen, with a peak at 90 min postingestion (Figure 2). This increase was significant when post-ingestion FRAP values

Figure 2. Acute change in plasma antioxidant capacity, expressed as the ferric reducing/antioxidant power (FRAP) value, after intake of Lingzhi extract [1.1g (triangles), 3.3g (squares) or placebo (circles)]; Mean9/SEM; n 0/10; *signicantly (P B/0.05) different from baseline (Kruskal-Wallis analysis of variance followed by Dunns test); # signicantly different (P B/0.01) from corresponding result after ingestion of placebo (Mann Whitney test).

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were compared to baseline (fasting) levels and to matching post-placebo levels. An average increase of 299/11% (mean9/SEM; P B/0.05) in urine FRAP values was also seen at 90 min post Lingzhi ingestion (3.3g). This increase was not seen post-placebo (results not shown). Changes in fasting plasma and urine variables after 10 days of Lingzhi supplementation are shown in Table 1. It can be seen that the FRAP values in urine, but not in plasma, were significantly (P B/0.05) higher after Lingzhi supplementation. Plasma ascorbic acid concentration and erythrocyte SOD and GPx activities tended to be higher after Lingzhi supplementation, but changes were not statistically significant. No significant changes in lipids or total a-tocopherol were seen, but, owing to small decreases in lipid levels accompanied by a small increase in total a-tocopherol, lipid standardized a-tocopherol increased (P B/0.05) after Lingzhi supplementation. No significant changes were seen in MDA, baseline DNA damage, or DNA damage induced by a standard oxidant challenge. Plasma uric acid levels were slightly lower after Lingzhi supplementation, but again, this did not reach statistical significance. Discussion Ganoderma lucidum (Lingzhi) has long been used for the promotion of health and longevity in China and many parts of Asia. Among cultivated mushrooms, Lingzhi is unique in being consumed for its medicinal, rather than nutritional, value and a variety of commercial Lingzhi products is available in the forms of tea, powder, and dietary supplements (Chiu et al., 2000). Recent reports give credibility to some of the ancient claims of the health benefits of this dietary supplement (Chen & Miles, 1996; Soo, 1996). These claims include extending lifespan and increasing youthful vigor and vitality, and specific biomedical applications and reported effects in animal and in vitro test systems of Lingzhi include control of hypertension, lowering blood lipid levels, modulating the immune system, and the prevention and treatment of cancer (Komoda et al., 1989;

Lee & Rhee, 1990; Willard, 1990; Furusawa et al., 1992; Shiao et al., 1994; Wang et al., 1997; Min et al., 2000; Lee et al., 2001). Chemical studies have found that polysaccharides and triterpenes are two major physiologically active constituents of Lingzhi. Plant polysaccharides are reported to confer anti-inflammatory, hypoglycemic, anti-viral, anti-ulcer and anti-cancer effects (Mizuno, 1995; Shiao et al., 1994; Kim et al., 1999). Triterpenes are a class of naturally occurring compounds whose carbon skeletons are composed of isoprene C5 units (e.g menthol, vitamin A). Terpenes were found in Ginko biloba, rosemary and ginseng and were found to contribute to their antioxidant and cardioprotective effects (Mashour et al, 1998; Haralampidis et al., 2002). The problem facing consumers, nutritionists and other healthcare professionals now is that there are insufficient scientific data available with regard to the efficacy, safety and mode of action of Lingzhi, and other traditional herbal medicines. This underlines the urgent need for extensive research and controlled trials to support or control their use as food supplements for health promotion and successful ageing. In this study we investigated in vitro antioxidant power and bioavailability of Lingzhi antioxidants, and performed a small, short-term human intervention trial to explore the potential cardioprotective effects of Lingzhi. Our data confirm that Lingzhi has antioxidant capacity. Comparing the antioxidant content of teas, fruits and vegetables (based on our published data: Benzie & Szeto, 1999; Szeto et al, 2002), the FRAP value of black tea ranges between 132/654 and green tea between 272 /1144 mmol/g of dry tea leaves, the FRAP value (recalculated as mmol/g dry weight) of fruits and vegetables is 81 mmol/g for orange, 70 mmol/g for green apple, 39 mmol/g for onion, 38 mmol/g for cauliflower. The FRAP value of Lingzhi was 360 mmol/g, indicating a fairly high antioxidant content. Previous studies (Zhu et al., 1999; Lee et al., 2001) have shown that Lingzhi contains compounds with antioxidant properties. However, what is interesting and which has not been previously studied is

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Table 1. Values of test results [mean (SEM)] in fasting samples after 10 days supplementation with Lingzhi and placebo capsules
Placebo Day 0 Plasma FRAP (mmol/L) Urine FRAP (mmol/mmol creatinine) Plasma ascorbic acid (mmol/L) Plasma a-tocopherol (mmol/L) / Lipid Standardised a-tocopherol (mmol/mmol TC'Tg) Plasma total cholesterol (mmol/L) Plasma triacylglycerol (mmol/L) Plasma HDL (mmol/L) Plasma LDL (mmol/L) Plasma LDL/HDL (mmol/mmol) Plasma uric acid (mmol/L) Erythrocyte SOD (units/g Hb.) Erythrocyte GPx (units/g Hb.) Malondialdehyde (mmol/L) % DNA damage in comet base (baseline) % DNA damage in comet tail (after standard oxidant challenge) 1206 (53) 0.40 (0.03) 72.6 (7) 23.9 (1.5) 4.77 (0.2) 4.33 (0.2) 0.80 (0.1) 1.10 (0.08) 2.87 (0.2) 2.74 (0.3) 330 (33) 1294 (105) 50.3 (7) 0.59 (0.04) 7.76 (0.4) 21.1 (1.7) Day 10 1170 (58) 0.36 (0.06) 61.6 (5) 24.3 (1.6) 4.43 (0.2) 4.62 (0.2) 0.97 (0.1) 1.08 (0.14) 3.10 (0.2) 3.71 (0.9) 322 (33) 1292 (103) 50.1 (7) 0.55 (0.05) 8.1 (0.2) 23.3 (1.1) Response to placebo (/36 (38) (/0.04 (0.04) (/11 (5) 0.4 (0.7) (/0.34 (0.1) 0.29 (0.2) 0.17 (0.1) (/0.02 (0.08) 0.23 (0.13) 0.96 (0.74) (/8 (11) (/2 (28) (/0.1 (1.6) (/0.04 (0.0) 0.3 (0.5) 2.2 (1.4) Day 0 1188 (74) 0.36 (0.07) 67.0 (5) 24.2 (1.9) 4.52 (0.2) 4.45 (0.3) 0.96 (0.1) 1.13 (0.09) 2.89 (0.2) 2.67 (0.2) 326 (34) 1305 (108) 48.9 (7) 0.62 (0.06) 8.16 (0.8) 24.6 (3.0) Lingzhi Day 10 1177 (63) 0.48 (0.08) 72.4 (6) 24.9 (2.0) 4.79 (0.3) 4.39 (0.2) 0.85 (0.1) 1.13 (0.08) 2.87 (0.17) 2.63 (0.21) 297 (34) 1347 (126) 51.0 (7) 0.60 (0.05) 8.13 (0.5) 24.8 (2.0) Response to Lingzhi (/11 (39) 0.12 (0.08) * 5.4 (5.2) 0.7 (0.3) 0.27 (0.2)* (/0.06 (0.3) (/0.11 (0.1) 0.00 (0.07) (/0.01 (0.23) (/0.04 (0.14) (/29 (24) 42 (30) 2.1 (1.4) (/0.02 (0.0) (/0.03 (1.3) 0.2 (3.3)

*Significantly different from response to placebo, PB/0.05 (Wilcoxon matched pairs test).

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the objective measure of this antioxidant capacity, whether these antioxidant compounds are absorbed and pass into the plasma, and whether they cause any discernable effect on biomarkers of antioxidant status, oxidative stress or risk of age-related disease after a period of supplementation. According to the literature, there is no agreed recommended dosage of Lingzhi, and the reported dosage varies significantly between studies. The dosage reported to afford a cure or to alleviate symptoms has ranged between 1.5 g to 9 g a day of a dried extract of the fruiting body of G. lucidum (Soo, 1996). However, doses of up to 30g have been used with no toxic side effects reported (Chang, 1994). The dosages used in this study were single doses of 1.1g and 3.3g of extract for the acute post ingestion study, and 0.72g per day for the short-term (10 days) supplementation study. These doses are at the lower end of the accepted dosage range of Lingzhi extract, but were in line with the manufacturers recommended dose for health maintenance and were appropriate for this first supplementation trial of healthy subjects. Our acute ingestion results show that the lower dose (1.1g) maintained baseline plasma antioxidant capacity during the fasting period, and that the higher dose (3.3g) caused a significant increase in the FRAP value. It is of interest that the plasma FRAP values decreased after placebo ingestion in all subjects. We have noted this effect before (Benzie et al, 1999) and suggest that the decrease on extended fasting is due to the bodys continued consumption of antioxidants without their usual replenishment by dietary intake. Clearly, intake of dietary antioxidants is necessary to maintain defences, even in the short term. The new data presented here indicate that the Lingzhi preparation used contains antioxidants, and at least some of these antioxidant components are readily absorbed, reach the plasma quite rapidly, and cause a significant increase in the plasma FRAP value. The 2 /3% acute post-ingestion increase with Lingzhi compares well with the 4% increase previously reported in plasma FRAP following ingestion of strong green

tea (Benzie et al , 1999). Supplementation for 10 days led to increased urinary total antioxidant capacity, as FRAP, but no significant increase was observed in fasting plasma. This may reflect control of an integrated antioxidant system whereby the concentration of one or more antioxidants decreases owing to increased supply of others. No statistically significant changes were seen in any of the other variables measured, except for lipid standardised a-tocopherol concentrations. However, the pattern of change in the other variables is interesting. Plasma ascorbic acid concentrations tended to increase, and uric acid and lipid concentrations tended to decrease, with Lingzhi supplementation. It is interesting to note that the natural statins, which inhibit cholesterol synthesis and are effective lipid lowering agents, are fungal secondary metabolites (Manzoni & Rollini, 2002). It is possible that Lingzhi, a woody fungus, contains some similarly acting component, however, this remains to be confirmed by further study. Further study is also warranted in the light of our preliminary findings and in relation to the direct, independent associations between uric acid, lipids and CHD risk (Freedman et al., 1995; Verdecchia et al., 2000), the possible antiatherogenic effect of vitamin E (Traber, 2001), and the reported inverse association of ascorbic acid and mortality (Khaw et al., 2001). In conclusion, this human intervention study has shown for the first time that the Chinese herb Lingzhi contains absorbable antioxidants that enter the circulating plasma and cause a significant acute increase in plasma antioxidant capacity. These bioavailable antioxidants may contribute to the reputed, but not yet scientifically validated, health effects of this commonly consumed Chinese medicinal mushroom. No deleterious effects on any of the variables measured were seen with supplementation for 10 days, and there was a trend for the biomarker profile to improve in terms of antioxidant status and CHD risk. Further study is needed to elucidate longer-term effects of regular Lingzhi ingestion, and which specific antioxidant components are absorbed.

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Acknowledgements */The authors are grateful to The Hong Kong Polytechnic University and to VitaGreen (HK) Ltd for nancial support for this study.

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