Original Article Mahidol University Journal of Pharmaceutical Sciences 2013; 40 (3), 1-7 Research and Development of Ganoderma lucidum Cultivated in Thailand P. Poomsing1, K. Pattanapanyasat2, P. Wongsinkongman3, and N. Soonthornchareonnon1* 1 Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University 2 Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University 3 Medicinal Plant Research Institute, Department of Medical Sciences, Ministry of Public Health Abstract Polysaccharides and triterpenes are the major pharmacologically active components in Ganoderma lucidum or Lingzhi, a medicinal mushroom well known for its immunomodulating activity. The Ganoderma lucidum used for this experiment were cultivars (MG1, MG2, and MG5) cultivated in Thailand collected from Muang Ngai Special Agricultural Project under the Patronage of Her Majesty Queen Sirikit, Chiang Mai province. The objectives of this study were to select the appropriate Lingzhi cultivars for cultivation, to investigate suitable wood logs as well as to study the appropriate harvest time. Quantitative determinations were performed using colorimetry and high performance liquid chromatography (HPLC). The results of this study indicated that the optimal harvest time of Lingzhi was 110 days after cultivation, the wood logs suitable for Lingzhi growth were paper mulberry, and longan wood logs, and the appropriate Lingzhi cultivar for cultivation in Thailand was MG2 since it produced the highest total polysaccharide content. The broken spore walls provide the highest percentage yield of major chemical compounds, especially triterpenoids. Keyword: Ganoderma lucidum, Lingzhi, Triterpenes, Polysaccharides, Qualitative and quantitative analyses. INTRODUCTION for a long time, but the information for their activities and chemical components were Ganoderma lucidum (Fr.) Karst. insufficient. So it is interesting to study in (belonging to Polyporaceae family)1 known terms of qualitative and quantitative analyses as Lingzhi in China, one of the most famous for determining the chemical compounds mushroom, has been widely used for promoting from Lingzhi to select the Lingzhi cultivars, health and longevity in China and other examine the suitable wood logs for cultivation Asian countries for over 2,000 years. Evidence and determine the appropriate harvest time. has accumulated concerning the medical The samples that used for these application of Lingzhi in the treatment of experiments including Muang Ngai-1 (MG1), various kinds of ailments such as bronchitis, Muang Ngai-2 (MG2), and Muang Ngai-5 anorexia, gastritis, hepatitis, nephritis, (MG5). From the scientific reports, it was haemorrhoid, dysmenorrhoea, constipation, found that triterpenes and polysaccharides lupus erythematosis, and chronic diseases were the major pharmacologically active such as migraine, hypertension, arthritis, components in Lingzhi. asthma, diabetes, hypercholesterolemia, The triterpenes were bitter components cardiovascular problems including cancers1-7. that caused G. lucidum had remarkably strong In Thailand, Lingzhi was cultivated bitter taste. Since ganoderic acids A and B *Corresponding author: Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University. 447 Sri Ayuthaya Road, Rajthevi, Bankok 10400, Thailand. E-mail address: [email protected] Tel.: +662644-8677 ext. 5527; Fax: +662644-8701 2 P. Poomsing et al. were firstly discovered, more than 130 methods for analyzing chemical compounds types of triterpenes had been isolated from both qualitatively and quantitatively by using the fruity bodies, mycelia, cultured media colorimetric and high performance liquid and spores of G. lucidum and they had been chromatographic (HPLC) techniques. The received considerable attention owing to their objective of this study was to study the conspicuous pharmacological activities. The condition for cultivation Lingzhi in Thailand activities of their triterpenes were reported such as the appropriate harvest time of spore as anticholesterol, anti-complement, anti- and fruity body, the suitable wood logs for hepatitis B, anti-histamine, anti-HIV-1, cultivation and the appropriate Lingzhi cultivar. anti-HIV-1 protease, anti-hypertension, antinociceptive, and cytotoxic effect1,8-12. MATERIALS AND METHODS The polysaccharides also show an Plant materials: important role as functional food ingredients, because the alternative and complementary The Lingzhi cultivars including medicines are now paid increasing attention. Muang Ngai-1 (MG1), Muang Ngai-2 (MG2), The scientific elucidation of the role of the and Muang Ngai-5 (MG5) were collected polysaccharides in phytomedicines is very from Muang Ngai Special Agricultural important. The activities of polysaccharides Project under the Patronage of Her Majesty were reported as anti-tumor, anti-hypertension, Queen Sirikit, Chiang Mai province (Fig. 1). hypoglycemic in diabetes, and immune The samples were identified and kept at modulating1,13-18. the herbarium at the Faculty of Pharmacy, This experiment was composed of the Mahidol University. Figure 1. The sample of Lingzhi cultivars; MG1 = Muang Ngai-1, MG2 = Muang Ngai-2, MG5 = Muang Ngai-5 Extraction procedures: hour and drying by lyophilization to give a Extraction method for the determi- water extract of Lingzhi. nation of total triterpenes was performed by sonicating 1 g of powder of Lingzhi with 20 Quantitative analysis by using colorimetric ml of 95% methanol in an ultrasonic bath method at 60ºC for 30 minutes, then filtered, the Total polysaccharides and uronic residue was washed with methanol for 3 acids contents were determined by forming 19 times and adjusted volume with methanol. color with phenol-H2SO4 reagent and For the determination of polysaccharides m-hydroxybiphenyl reagent20, respectively, and uronic acid was prepared by extraction and examined under colorimeter, using glucose with hot water (95-100ºC) three times for 1 and glucuronic acid as reference standards. Research and Development of Ganoderma lucidum Cultivated in Thailand 3 Triterpenes content was measured by Preparation of ganoderic acids colorimetric method by reacting with 5% reference standard (GA, GF and GH) for vanillin/glacial acetic acid and perchloric HPLC: Every standard compound was acid, using ursolic acid as reference standard21. accurately weight and dissolved in 95% methanol to prepare a concentration of 2.5 mg/5 ml. The internal standard (cortisone Quantitative analysis of ganoderic acids 21-acetate) solution was prepared at a final 22 using HPLC method concentration of 20 µg/ml in 95% methanol Condition for HPLC analysis: The Preparation of samples for HPLC: ® A 50.0 mg of dried powder was extracted method uses a Inertsil ODS-3 (250×4.0 mm, 5 µm) column as the stationary phase, with 1.0 ml 95% methanol in ultrasonic a gradient mobile phase of acetonitrile bath for 1 hr. This solution was centrifuged (CH CN), perchloric acid (HClO ), ammonium at 14,000 rpm. The supernatant liquid was 3 4 acetate (CH3COONH4), deionized water, collected, filtered with nylon filter and kept and methanol, and UV detection at 252 nm. in a well-closed injection vial. as described in Table 1. Table 1. HPLC conditions for the analysis of ganoderic acids content Analytical column : Inertsil® ODS-3 (250×4.0 mm, 5 µm) Guard column : Inertsil® ODS-3 (10×4.0 mm, 5 µm) Flow rate : 1 ml/min Temperature : 55 °C Detector : UV 252 nm Injection volume : 10 µl Mobile phase : CH3CN HClO4 CH3COONH4 DI-H2O CH3OH Mobile phase (ml) (M) (M) (ml) (ml) A 150 0.019 0.020 250 1.50 B 60 0.019 0.020 250 1.50 RESULTS AND DISCUSSION content of polysaccharides (% w/w) in broken spores, non-broken spores, fruity 1. Selection of Lingzhi cultivars bodies and stalk in MG2 were 4.77, 4.21, The Lingzhi cultivars including 3.06 and 1.72 %w/w, respectively, and Muang Ngai-1 (MG1), Muang Ngai-2 (MG2), the stalks of MG2 contained the highest and Muang Ngai-5 (MG5) were determined content of uronic acid (% w/w). The highest the contents of total polysaccharides, total content of uronic acid in Chinese cultivars uronic acids and total triterpenes by using was broken spore of G5 (Table 4). colorimetric method and the content of total ganoderic acids was performed by using 2. Studying of suitable wood logs for Lingzhi high performance liquid chromatographic cultivation (HPLC) method compared with Chinese To evaluate the triterpenes content cultivars (G5 and G9). The result in Table 2 and composition among the fruity bodies showed that Chinese cultivars composed of cultivated on different mushroom-growing total triterpenes content more than cultivars logs, a single strain of G. lucidum (MG5) cultivated in Thailand. Comparing to Thai was cultivated on Rain tree, Paper mulberry, cultivars, MG5 (stalk) contained the highest Tamarind, Mango, Longan, and Neem content of total triterpenes and total ganoderic wood logs. The result showed that MG5 acids. The result in Table 3 showed that the cultivated on the paper mulberry wood log 4 P. Poomsing et al. contained the highest triterpenes content, showed that the contents of total triterpenes and longan wood log was suitable for the and total polysaccharides were the highest production of polysaccharides (Table 5). in fruity bodies at 110 days, the content of polysaccharides in spores was higher than 3. Determination of optimal harvest fruity bodies about two times. Therefore, time The fruity bodies and spores of MG5 the optimal harvest time for fruity bodies were studied at different period of harvest