Preliminary Results of Determination of Chemical ...

International Journal of Medicinal Mushrooms, 14(3): 295–305 (2012)

Preliminary Results of Determination of Chemical Changes on Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (W.Curt.:Fr.)P. Karst. (Higher Basidiomycetes) Carried by Shenzhou I Spaceship with FTIR and 2D-IR Correlation Spectroscopy Yew Keong Choong,1 Xiangdong Chen,2 Jamia Azdina Jamal,3 Qiuying Wang,2 & Jin Lan2* 1

Phytochemistry Unit, Herbal Medicine Research Centre, Institute for Medical Research, 50588 Kuala Lumpur, Malaysia; 2Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, PR China; 3Drug and Herbal Research Centre, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia *Address all correspondence to: Jin Lan, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, PR China; Tel.: 00810-57833425, Fax: 00810-62899723; [email protected].

ABSTRACT: Spaceflight represents a complex environmental condition. Space mutagenesis breeding has achieved marked results over the years. The objective of this study is to determine the chemical changes in medicinal mushroom Ganoderma lucidum cultivated after spaceflight in 1999. Fourier transform infrared (FTIR) and two-dimensional infrared (2DIR) correlation spectroscopy were used in analysis. The sample Sx and its control Cx showed the least dissimilarities in one-dimensional FTIR spectra, but absorbance of Sx is twice as high as Cx. Sx presented a clear peak at 1648 cm–1 in 2nd derivative spectra, which could not be detected in the Cx. The 2DIR spectra showed the intensity of Sx in the range 1800–1400 cm–1 for protein is higher than the control. The sample Sx produced some carbohydrate peaks in the area of 889 cm–1 compared with the Cx. The spaceflight set up an extreme condition and caused changes of chemical properties in G. lucidum strain. KEY WORDS: medicinal mushrooms, Ganoderma lucidum, Lingzhi or Reishi mushroom, space flight, chemical changes, FTIR, 2D-IR ABBREVIATIONS: DTGS: deuterated triglycine sulfate detector; FTIR: Fourier transform infrared; 2D-IR: two-dimensional infrared; IR: infrared ray; KBr: potassium bromide; 1D: one dimensional; ORC: orbital research stations; psi: pounds per square inch; 3D: three dimensional; t-test: Student’s t-test

I. INTODUCTION The achievements of mankind in the 20th century were remarkable, with contributions of sophisticated science and advanced technologies. The research projects in this paper took place both on Earth and in space. Russia started theoretical and practical astronautics in the middle of the 20th century. This opened up the possibility of living in space. Further steps in this direction are impossible without creating the biotechnological systems of life support for long-term manned space missions and future space bases. According to most of the cosmic research reports, the physical factors acting upon biological objects in orbital research stations (ORC) can be derived from three basic groups: (1) rarity of the

atmosphere (vacuum), (2) ultraviolet and infrared radiation, radio and microwave emissions, and (3) ionizing radiation. In this context, weightlessness or microgravity, accelerations, vibration, and noise are characterized as dynamic “factors of flight.” In addition, the space object environment consisted of insulation, artificial atmosphere, changed biological cycle, the absence of natural electric and magnetic fields, and changed conditions of heat and mass transfer. Biological investigations onboard ORS are fundamentally different from the usual laboratory experiments. The factor of gravity is subject to variations to the least degree. At the same time, this factor can be quite substantial, while the mechanism of its influence on the growth and development of biolog-

1521-1437/12/$35.00 © 2012 Begell House, Inc. www.begellhouse.com

295

Preliminary Results of Determination of Chemical Changes on Lingzhi or Reishi Medicinal Mushroom

ical objects has not been sufficiently studied yet.1 There are a few reports2,3 regarding the effects of microgravity, heavy ionizing radiation, subelectromagnetic fields, and other space factors on plant biological characteristics and genetic mutations at the protein and gene levels. However, only a small number of references4,5 exist on investigations of mushrooms under spaceflight conditions. Since the biology of mushrooms is totally different from plants, they may carry out dissimilar physiological and biochemical transformations. Hence, the main aim of investigations with mushrooms is reduced to studying the factors of growth, development, and productivity under the extreme conditions of spaceflight. Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (W.Curt:Fr.) P. Karst. (Ganodermataceae, higher Basidiomycetes), is a popular species that has been fully studied for more than 2000 years in China and other Asian countries.6 It is considered a remedy for promotion of health and longevity. Its efficacy in immunostimulating7 and anti-tumor8 activities add to its usefulness as a medicinal mushroom. A lot of research has been conducted on G. lucidum, but researchers still pay attention to new developments. China developed its first spaceship (Shenzhou I) in 1999. Besides other material and plants, four G. lucidum strains were chosen for 12 hours of spaceflight aboard Shenzhou I.9 In following years, continuing cultivations and experiments was done on their biochemical changes; the most common esterase band changes of G. lucidum strain Sx and its control Cx were chosen for further analysis. This paper refers to the internal chemical property changes of G. lucidum analysis with FTIR and 2DIR correlation spectroscopy. Fourier transform infrared (FTIR) and 2-dimensional infrared (2DIR) correlation spectroscopy have been used in analyzing the content of sseveral aspects of traditional Chinese medicine.10 This technique is rapid and causes minimum spoilage of the nature of the sample. One advantage is that it requires a smaller quantity of sample. The most important is the higher accuracy with lower systematic error and ability to show distinct differences. This work was carried out with minimum destruction of the raw material because most of the results were based on comparison. Therefore the addition of second derivative scanning and the 296

2DIR correlation spectroscopy enhance the interpretation of data from this experiment. II. MATERIALS AND METHODS A. Samples Four commercial cultivated Ganoderma lucidum strains were used for spaceflight. Strain 3 (CGMCC5.533) and strain 4 (CGMCC5.75) came from China General Microbiological Culture Collection Center (CGMCC); strain H (SAAS1020) and strain X (SAAS1021) came from Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences (SAAS). The payload carried into space is very limited; therefore, only the most popular commercial cultivated G. lucidum strains were chosen for spaceflight. These strains were separated into two batches and packed in cotton cloth envelopes. Batch A was taken on China’s first spaceship (Shenzhou I) for 12 hours of spaceflight. Batch B was kept at normal ambient condition to be used as the ground control. In the following years of continuing cultivation and experiments on their biochemical changes, the most common esterase band changes of G. lucidum strain Sx and its control Cx were chosen for further analysis. This paper refers to the internal chemical property changes of G. lucidum analyzed with FTIR and 2DIR correlation spectroscopy. B. Sample Preparation The whole fruiting body of the cultivated G. lucidum strain was pulverized into fine powder form. Batch A after spaceflight was labeled as Sx, and batch B on the ground was labeled as Cx (control). C. Apparatus Perkin-Elmer Spectrum GX Fourier-transform infrared (FTIR) spectrometer (Llantrisant, Wales, UK) with an attached DTGS detector was used as the main equipment for the whole experiment. The dynamic FTIR spectra were recorded with the above spectrometer combined with a portable, programmable temperature controller (Model 50886, Love Control), with a controllable range from room temperature up to 120°C. The Spectrum software V3.02 (Perkin Elmer) was tested and set up for the most ideal conditions. The first-derivative FTIR spectra were obtained after 16 scans at room temperature at the range of 4000–400 cm–1 with International Journal of Medicinal Mushrooms

Choong et al.

FIGURE 1. Comparison of FTIR spectra of Ganoderma lucidum in the range 4000–400cm–1. Sx: fruiting body of cultivated G. lucidum after 12-hour spaceflight; Cx: fruiting body of G. lucidum on the ground (control).

a resolution of 4 cm–1. The second-derivative IR spectra were acquired after 13-point smoothing of the original IR spectra. The thermal dependent 2D-IR spectra were obtained from the dynamic spectra series using 2DIR correlation analysis software developed by the Analysis Center of Tsinghua University (China). D. Procedures The experiment started with a tablet of KBr as a blank. About 0.01 g of G. lucidum powder of Sx was mixed evenly with 0.02 g of KBr crystal. The mixture was ground and pressed into a tablet with a pressure of not more than 10 psi. A similar procedure was done on sample Cx. The FTIR spectrum was generally acceptable when a transmission of 60%–70% was achieved. Otherwise, the test had to be repeated with either the sample or KBr added. In this experiment, samples Sx and Cx were repeated several times. The best transmission as the difference between the highest value and lowest value around 80 was chosen for further analysis. Later on, the sample tablet was put into the temperatureVolume 14, Number 3, 2012

controlled pool and the FTIR spectra were recorded in situ. These results were interpreted for 2DIR correlation spectroscopy. Dynamic FTIR spectra were obtained after 32 scans and were collected by varying the temperature from 50°C to 120°C by steps of 10°C. The full temperature scan took a total time of 30 min. The baseline of each spectrum was subtracted from each scan; therefore, all these spectra were automatically baseline corrected, and spectra were calculated within the range of 2000–2500 cm–1, setting their lowest points to zero. III. RESULTS AND DISCUSSION A. Comparison of the 1D-FTIR Spectra The 1D-FTIR spectra of G. lucidum samples under room temperature are shown in Fig. 1. This stage of spectra is typically considered for initial comparison. Most of the double lapping transmission was caused by the crude and not pure extracted sample. Both of the spectra share a similar pattern. This pattern was compared to The 2DIR correlation spectra in the Chinese Herbal Medicines com297


TABLE 1. Correlation FTIR Spectra of Cultivated Fruiting body of Ganoderma lucidum After 12-Hour Spaceflight with Fruiting Body of G. lucidum on Ground (Control) Compare range 4000–400 1700–1500 1450–1200 1160–1000 900–400

Correlation 0.991908 0.994825 0.987777 0.994329 0.970869

Factor 0.773104 0.821262 0.773373 0.826483 0.782645

pilation.11 In this record, the characteristic peaks for G. lucidum were assigned at 1075 cm–1 and 1043 cm–1. The overall comparison range of both spectra is indicated in Table 1. The 4000–400 cm–1 range was almost 99% related, but the factor was low. The 1700–1500 cm–1 range achieved the highest correlation among the listed ranges; this could be due to the stability of the protein contents. Another higher correlation was 1160–1000 cm–1, which contained the characteristic peak of G. lucidum and showed the highest factor. This indirectly interpreted consequence of the nutrients profile of this mushroom was continuously analyzed with infrared spectroscopy. However, the other two ranges (1450–1200 cm–1 and 900–400 cm–1) did not include higher correlations and factors. The reason could be alteration and changeability of lipid and polysaccharide contents of sample Sx after the space tour. The absorbance value of Sx was one time higher than Cx. The statistical significance was calculated for these data in selected ranges with paired t-test. All the four selected ranges for both samples were significantly different (p < 0.05). In the range of 3500–2800 cm–1, peaks at 3368 cm–1 and 3369 cm–1 were definitely assigned as the stretching vibration of O–H. The common backbone of –CH3 with C–H vibration was noted for the peak at 2924 cm–1, which is revealed in almost all of the spectra. The main overall comparison of G. lucidum powder spectra was divided into four regions (Fig. 2). Thus, the region of 1160–1000 cm–1 represented the main characteristic of Ganoderma spp., showing twin peaks with higher absorbance compared to the other regions. The region of 1700– 1500 cm–1 corresponded to the amide bands, suggesting the presence of protein components. This 298

Correlation criteria 0.98 0.98 0.98 0.98 0.98

Discrimination criteria 0.05 0.05 0.05 0.05 0.05

is confirmed by a report from Kohsuke Kino et al.12 regarding the isolation of a novel protein (LZ-8) with mitogenic activity in vitro and immunomodulating activity in vivo from the mycelial extract of G. lucidum. The strong absorption at 1648 cm–1 in Sx and 1647 cm–1 in Cx with very small absorption around 1540 cm–1 are mainly attributed to stretching vibration of C–N bond as well as bending vibration of N–H groups. The following five peaks in the region of 1450–1200 cm–1 are close to each other and most of them are of low intensity. The peak around 1374 cm–1 corresponds to the bending of C–H in CH3, while peaks at 1250 cm–1 and 1202 cm–1 belong to the lipid attenuated lipid. The region of 900–400 cm–1 indicated the content of carbohydrate in the samples. This is proven by various publications on the isolation of many polysaccharides, especially glucans, from G. lucidum.13,14 Additionally, Xu et al. report an isolated antitumor β-D-glucan (MW ~ 1,000,000) polysaccharide15 from the fruiting body of Grifola frondosa that corresponded to an IR absorption peak at 890 cm–1. The small but obvious peak at 891cm–1 for both Sx and Cx G. lucidum spectra proves the existence of the glucan. B. Comparison of the Second-Derivative 1D-FTIR Spectra Figure 3 shows the second-derivative spectra of G. lucidum in the region of 1800–1400 cm–1. The second-derivative spectrum is usually used to enhance the apparent resolution of the IR spectrum or even to separate previously overlapped peaks.16 Two sections of the spectra have been extracted for their district differences but not statistically with paired t-test (p > 0.05). Komoda et al.16 reported eight new terpenoid constituents of ganoderenic acids and luciderenic International Journal of Medicinal Mushrooms

Choong et al.

FIGURE 2. Comparison of FTIR spectra of Ganoderma lucidum in the range 1800–400cm–1. Sx: fruiting body of cultivated G. lucidum after 12-hour spaceflight; Cx: fruiting body of G. lucidum on the ground (control).

FIGURE 3. Comparison of second-derivative spectra of Ganoderma lucidum in the range 1800–1400 cm–1. Sx: fruiting body of cultivated G. lucidum after 12-hour spaceflight; Cx: fruiting body of G. lucidum on the ground (control).

Volume 14, Number 3, 2012

299



acids with IR V KBr around 1750 cm–1 to 1610 cm–1. However, absorption of these peaks was not obviously shown in the two current spectra. This could be due to the crude extract used in the experiment. Spectrum Sx showed a clear peak at 1648 cm–1, which was also observed in Figs. 1 and 2. This peak is not as sharp as reported in spectrum Cx under 2nd derivative. In the section around 1500 cm–1, a few peaks are distinct despite their small intensity in Sx. This scenario did not exist in Cx. In other words, the peaks 1518 cm–1, 1490 cm–1, 1471 cm–1 were hardly clear in Cx. These three peaks were assigned to 0 –substituted phenyl.18 Figure 4 shows giant peaks at the middle of the region 1400–800 cm–1. These peaks corresponded to the main characteristic of G. lucidum crude extract and seem to be generally identical in both spectra. Figure 5 reveals the second-derivative spectrum of ganoderic acid A that is present in G. lucidum. Ganoderic acid and its derivatives such as triterpenoid were isolated and studied by Hirotani et al.18 The tiny peak at 1290 cm–1 appeared in Sx 300

but not Cx, and is defined as the peak for in-plane –CH of terminal vinyl when combined with an unnoticeable peak at 1420 cm–1 in Fig. 3.17 Also, the peaks around 900 cm–1 have been reported as a region of carbohydrate content. This was proven in the article published by Mizuno’s14 research group in 1986 and supported by Y. Wang et al.19 The difference between these two spectra is that sample Sx has some small peaks beside peak 889 cm–1 which are not obvious in Cx. The second-derivative spectra of both samples in the range of 800–400 cm–1 are shown in Fig. 6. The overall region exhibited not much IR absorption. Spectrum Sx gave lower peaks than that of Cx, except for a peak at 672 cm–1 that is assigned for bending of –CH. The overtone should appear around 1250 cm–1 but the diagnostic value is limited. Referring to Fig. 4, the 1250 cm–1 band is not too strong for an overtone for –CHarom (in-plane). Nevertheless, spectrum Cx might not demonstrate proper absorption, probably due to human error and most likely noise. International Journal of Medicinal Mushrooms

Choong et al.

FIGURE 5. Second-derivative spectrum of ganoderic acid A in the range 1400–800 cm–1.

C. Comparison of the 2D-IR Spectra The temperature-dependent 2D-IR spectra for the range 1800–1400 cm–1 are shown in Fig. 7a. The noticeable similarities in the synchronous 2D-IR spectra of the two samples in this range mainly ex-

hibit the characteristic absorption of carbonyls and olefinic bonds. Both samples exhibit a very strong autopeak at 1647 cm–1, assigned to the carbonyl absorption of ligustilides with the skeleton of glactone, demonstrating that such carbonyl groups



301


FIGURE 7. Comparison of synchronous 2D-IR spectra of Ganoderma lucidum in the range 1800–1400 cm–1. Sx: fruiting body of cultivated G. lucidum after 12-hour spaceflight; Cx: fruiting body of G. lucidum on the ground (control).

are very susceptible to thermal perturbation. Besides the strong peak, five areas of dissimilarity were observed between the spectra. These were the autopeaks around 1450 cm–1, 1500 cm–1, and 1750 cm–1, and crosspeaks around (1770, 1650) and (1650, 1500). Figure 7b shows very slight difference of both autopeaks in this region, except the intensity of spectrum Sx is 619 more than that of spectrum Cx and is further enhanced by the 3D spectra (Fig. 7c). Such a phenomenon suggests the possibility of higher protein content in the Sx compared to that of Cx. The overall intensity of synchronous 2D-IR spectra in Sx was stronger than that of Cx, especially in the cluster region between 1175 cm–1 and 1325 cm–1 (Fig. 8a). In addition, the blue crosspeak of Sx that is coordinated between 980 to 1100 along the x-axis and y-axis has darker color than that of Cx. This region of peaks is important because it is the main diagnostic characteration 302

for Ganoderma spp. The 4 × 4 peak cluster can be clearly divided into three parts. The part starting from 1025 to 1175 cm–1 has autopeak (Fig. 8b) with higher intensity, but the intensity of the subsequent autopeaks was reduced evenly at the rate of 5%–7%, respectively. A similar scenario was observed for the part started at the autopeak 1190 cm–1, which is considered the highest peak for the whole region. Intensity of the subsequent peaks was reduced at the rate of 25% each. In terms of correlation, the weak crosspeaks (1083, 1038) were correlated positively with the autopeak at 1038 cm–1 and 1083 cm–1. The former is attributed to vibration C–C and the latter partly belongs to the absorption of 0-substituted phenyl. The other strongly and positively correlated crosspeaks are (1220, 1188), (1219, 1188), (1283, 1219), (1188, 1137), (1188, 980), and (1219, 980). Figure 8c shows the 3D spectra in this range to aim for clearer visual comparison. Figure 9a shows a precise peak at the right top International Journal of Medicinal Mushrooms

Choong et al.


of the spectra in the range of 800–400 cm–1. Spectrum Sx has a rounded up and centralized autopeak at 678 cm–1 (Fig. 9b), and the color of 2D-IR has more contrast than that of spectrum Cx. This finding is very consistent with 1D-FTIR spectra. The colorful spectra of 2D-IR supports the confidence of detection and determination of the bending of – CH. Figure 9c showed the spectrum in 3D throughout the range. IV. CONCLUSIONS For the first time it has been possible to determine the chemical composition of G. lucidum after spaceflight using FTIR and 2D-IR correlation spectroscopy. The expected change in G. lucidum under the extreme of weightlessness conditions was generally correlated with their efficacy in the medicinal field. The results are still at an analytical stage, and sufficient trials still need to be carried out. Most of the comparisons are highly pinpointed on second-derivative and 2D-IR spectra. The peaks Volume 14, Number 3, 2012

assigned to the protein content in Sx are more stable than in Cx. However, the peaks attributed to the carbohydrate content in Sx seem unstable, and certain peaks were lost, especially in the range around 900 cm–1. The second-derivative spectrum of ganoderic acid A in the range 1400–800 cm–1 supported this phenomenon even through the Cx spectrum was interrupted by noise. ACKNOWLEDGMENTS We would like to thank Noor Azlina Ahmad Jamil for collaboration during the experiment at Universiti Kebangsaan Malaysia. We wish to take this opportunity to express appreciation to Chemistry Department, Tsinghua University, especially Prof. Sun Su-Qin and Dr. Zhou Qun, for their guidance in application of FTIR and 2DIR techniques. Last but not least, the authors are grateful to the National Natural Science Foundation of China (NSFC, NO.81173660) for their financial support, and express our deepest gratitude to the Islamic Devel303



opment Bank for their support during the Merit Scholarship Programme 2008. REFERENCES 1.

2.

3. 4.

5.

6.

7.

304

Mashinsky AL, Nechitailo GS. Results and prospects of studying the gravitationally sensitive systems of plants under conditions of space flight. Cosmic Res. 39;4:317– 27. Translated from Kosmicheskle Issledovaniya. 2001;39(4):339–50. Chai DM, Xiang Q, Tao YS, Tang JT. Progress on the effect on space environment on Plant. Sci Technol Rev. 2007;25(1):38–42. Cyranoski D. Satellite will probe mutating seeds in space. Nature. 2001;410(6831):857. Qi JJ, Chen XD, Lan J. Studies on esterase isozymes and mycelium growth speed of Ganoderma lucidum carried by Shenzhou spaceship. Acta Agricult Nucleatae Sin. 2002;16(5):289–92. Qi JJ, Ma RC, Chen XD, Lan J. Analysis of genetic variation in Ganoderma lucidum after space flight. Adv Space Res. 2003;31(6):1617–22. Ma C, Guan SH, Yang M, Liu X, Guo DA. Differential protein expression in mouse splenic mononuclear cells treated with polysaccharides from spores of Ganoderma lucidum. Pytomed. 2008;15(4):268–76. Habijanic J, Berovic M, Wraber B, Hodzar D, Boh B. Im-

8.

9.

10.

11.

12.

13.

14.

munostimulatory effects of fungal polysaccharides from Ganoderma lucidum submerged biomass cultivation. Food Technol Biotechnol. 2001;39(4):327–31. Muller CI, Kumagai T, O’Kelly J, Seeram NP, Heber D, Koeffler HPh. Ganoderma lucidum causes apoptosis in leukemia, lymphoma and multiple myeloma cells. Leukemia Res. 2006;30(7):841–48. Chen XD, Lan J. Physiological and biological study of Ganoderma lucidum carried on spaceship. World Sci Technol-Modernization Trad Chin Med. 2005;7(2):18–21. Mei DQ, Wang JH. The development of research on Chinese drug by infrared spectroscopy. J Chongqing Uni. 2003;(11):56–59. Sun SQ, Zhou Q, Qin Z. Atlas of two-dimensional correlation infrared spectroscopy for traditional Chinese medicine identification. Beijing: Chemical Industry Press; 2003. Kino K, Yamashita A, Yamaoka K, Watanabe J, Tanaka S, Ko K, Shimizu K, Tsunoo H. Isolation and characterization of a new immunomodulatory protein Lingzhi-8 (LZ-8) from Ganoderma lucidum. J Biol Chem. 1989;264(1):472–78. Tang W, Liu JW, Zhao WM, Wei DZ, Zhong JJ. Ganoderic acid T from Ganoderma lucidum mycelia induces mitochondria mediated apoptosis in lung cancer cells. Life Sci. 2006;80(3):205–11. Mizuno T, Ohsawa K, Hagiwara N, Kuboyama R. FracInternational Journal of Medicinal Mushrooms

Choong et al.

tionation and characterization of antitumor polysaccharides from Maitake, Grifola frondosa. Agric Biol Chem. 1986;50(7):1679–88. 15. Xu CH, Sun SQ, Guo CQ, Zhou Q, Tao JX, Isao N. Multi-steps infrared macro-fingerprint analysis for thermal processing of Fructus viticis. Vib Spectrosc. 2006;41:118–25. 16. Komoda Y, Nakamura H, Ishihara S, Uchida M, Kohda H, Yamasaki K. Structures of new terpenoid constituents of Ganoderma lucidum (Fr.) Karst. (Polyporaceae). Chem Pharm Bull. 1985;33(11):4829–35.


17. Nakanishi K, Solomon PhS. Infrared absorption spectroscopy. 2nd ed. San Francisco: Holden-Day; 1977. 18. Hirotani M, Asaka I, Ino C, Furuya T, Shiro M. Ganoderic acid derivatives and ergosta-4, 7, 22-triene-3,6dione from Ganoderma lucidum. Phytochemistry. 1987;26(10):2797–2803. 19. Wang YF, Zhang M, Ruan D, Shashkov AS, Kilcoyne M, Savage AV, Zhang L. Chemical components and molecular mass of six polysaccharides isolated from the sclerotium of Poria cocos. Carbohydr Res. 2004;339(2):327–34.

305