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Discrimination of authentic Polyporus umbellatus and counterfeit by Fourier Transform Infrared and two dimensional infrared correlation spectroscopy
Journal of Molecular Structure 1199 (2020) 126917
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Journal of Molecular Structure journal homepage: http://www.elsevier.com/locate/molstruc
Discrimination of authentic Polyporus umbellatus and counterfeit by Fourier Transform Infrared and two dimensional infrared correlation spectroscopy Xiangdong Chen a, YewKeong Choong b, Weiwei Zhang a, Guoqiang Li a, Jin Lan a, * a
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100193, PR China Phytochemistry Unit, Herbal Medicine Research Centre, Institute for Medical Research, National Institute of Health, No. 1 Jalan Setia Murni U13/15, Seksyen U13, 40170, Shah Alam, Selangor, Malaysia b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 July 2019 Received in revised form 6 August 2019 Accepted 8 August 2019 Available online 9 August 2019
Polyporus umbellatus(PU) belongs to the family Polyporaceae in the phylum of Basidiomycota. The sclerotia is its main medicinal parts. It is reputedly used in traditional Chinese Medicine as anti-cancer agent and consequently is in high demand. A major problem on the medicinal use of this mushroom is the menace of the counterfeit product. Therefore, the detection of counterfeit PU and the confirmation of genuine PU are important. This study was done by collecting wild PU and cultivated PU and compared with a counterfeit which is commonly found in the market. Fourier Transform Infrared (FTIR) and TwoDimension Infrared (2DIR) for the qualitative analysis are often regarded as green methods because the original resources of the test sample are preserved. The different spectral patterns of the counterfeit PU compared with the genuine PU were clearly indicated in 1D FTIR spectrum. The 2DIR spectrum of counterfeit PU showed more than 70% dissimilarity compared to genuine PU. This rapid and simple sample preparation enhances the speed of qualitative analysis on PU and rapidly determines their differences in term of their raw material content and the active site of certain compounds. © 2019 Elsevier B.V. All rights reserved.
Keywords: Polyporous umbellatus Discrimination Fourier transform infrared Two-dimensional infrared
1. Introduction Polyporus umbellatus (Pers.) Fries has been used as a traditional Chinese medicinal fungus. It belongs to the family Polyporaceae in the phylum of Basidiomycota. The sclerotia of P. umbellatus is its main medicinal parts and widely utilized for treatment of edema, scanty urine, vaginal discharge, as well as jaundice and diarrhea [1]. Pharmacological research had also revealed that sclerotia of P. umbellatus possessed various diuretic, anticancer, antiinflammatory and hepatoprotective activities [2]. Steroids and polysaccharides have been reported as the dominating ingredients responsible for the activities of sclerotia of P. umbellatus [2]. Extensive attention has been paid to the polysaccharides due to their crucial physiological and biological functions, such as anticoagulant [3], cardiac protection [4], immunomodulatory and antitumor [5]. The wild P. umbellatus are extensively used and cannot meet the
* Corresponding author. E-mail address: [email protected] (J. Lan). https://doi.org/10.1016/j.molstruc.2019.126917 0022-2860/© 2019 Elsevier B.V. All rights reserved.
demands required for clinical application. In the past 10 years, the wild P. umbellatus have become increasingly scarce and the market price rose. As reported from the survey, the price of the wild P. umbellatus was higher than the cultivated P. umbellatus. Mass scale commercial cultivation of P. umbellatus could be an ideal solution of providing an amble stock for the market. However, P. umbellatus cultivation is technically difficult due to the requirement that its sclerotial growth must be involved with the rhizomorphs of Armillaria. sp. [2]. The association of P. umbellatus with rhizomorphs of Armillaria sp. accelerates its growth. This symbiosis is thought to promote the development of sclerotia of P. umbellatus where the rhizomorphs of Armillaria sp. gradually penetrate into the medulla layer. Among the Armillaria species, five species (Armillaria sinapina, Armillaria calvescens, Armillaria gallica, Armillaria cepistipes, and Armillaria nabsnona) formed a highly supported clade with the symbiont [6]. Armillaria gallica and Armillaria cepistipes were the species most frequently observed to be symbiotic with P. umbellatus in China and there is genetic diversity among the genotypes [7]. However the actual relationship of these two fungi is still unclear. To date, there is unsuccessful attempt to mass produce P. umbellatus, and as such the scarcity of this mushroom
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has led to the emergence of counterfeit product. Therefore the detection of counterfeit PU and the confirmation of genuine PU is important. The color of its sclerotium depends on the stage of the development. There are three stages of P. umbellatus sclerotial development. During the sclerotial initial stage, the white mycelium carted on the ground and accumulated to form the white fungal mass. The primodia is then developed and the surface color changes from white to gray, then light brown and black as the development proceeds further. This study was done by collecting wild PU and cultivated PU and compared with a counterfeit PU which was commonly found in the market. Samples were sourced from Heilongjiang, Shaanxi, Yunnan province in China in this study. The small number of samples was due to the difficulty in searching the wild and P. umbellatus has its specific growth area associated with geographical factors. The uses of Fourier Transform Infrared (FTIR) and Two-Dimension Infrared (2DIR) for the qualitative analysis are often regarded as green method because the original resources of the test sample are preserved. The nutrient and important substances of the dried sample with only the water lost can be explored and analyzed directly under the infrared beam. 2. Materials and methods 2.1. Sample A wild P. umbrellatus (PU1) was obtained from Heilongjiang Province, China and another wild P. umbrellatus (PU2) from Yunnan Province, China. A cultivated P. umbrellatus (PU3) was from a mushroom cultivation farm in Shaanxi Province, China and a counterfeit P. umbrellatus (PUC) was purchased from Anguo herb market, the largest herbal medicine market in North China. All collected samples were taxonomically identified by Prof. Jin Lan at Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China. 2.2. Apparatus and parameters The system of GX Fourier-Transform Infrared (FTIR) spectrometer (PerkinElmer) was used in the processing of spectrum. A Deuterated Triglycine Sulfate detector was attached for recording the infrared spectrum. The parameters were set up by accumulating 32 times scans of the IR with a resolution of 4 cm1 and the wavenumber of the middle infrared was performed in the range of 4000-400 cm1. For the 2DIR analysis, a portable programmable temperature controller was used for applied thermal perturbation with the temperature gradually increasing from 30 to 120 C. Each interval of 10 C was implemented for scanning a spectrum on the same disk. 2.3. Procedure of sample disk preparation The mushroom fine powder (2 mg) was transferred into a crystal mortar and mixed with IR grade KBr. The mixture was ground well using the pestle until a homogenous mixture was obtained. A die with smooth surface on top was inserted into the cavity of pellet press. The mixture was poured into the cavity, then a bolt press was inserted and rotated into the cavity to distribute the particles. When the bolt press was taken out, the second die was inserted and the bolt press was adjusted onto it. Later, the whole entity was transferred into the hydraulic press and the wheel was turned clockwise to secure it tightly. Then the level was pulled repeatedly until the meter read 10 tons and waited for a few seconds. The pressure was released by rotating the knob counter clockwise and turning the wheel to disengage the pellet press. Finally the press
entity was disassembled by inverting the upper body and pressing until the disk came out. The upper die was removed and the sample disk was ready. The disk was transferred to the sample holder using forceps and the cap was fixed to secure the disk. The FTIR spectrum of the tablet was generally acceptable when a transmission of 60e70% was achieved. Otherwise, the test had to be repeated with either a fresh sample or with more KBr added. After this, the sample tablet was put into the temperature controlled pool and the FTIR spectra were recorded in situ. These results were interpreted for 2D-IR correlation spectroscopy. 3. Result and discussion 3.1. FTIR spectra analysis The different spectral patterns of the counterfeit PU compared with the genuine PU were clearly indicated in FTIR spectrum. Region of 1800-400 cm1 which represented fingerprint of P. umbellatus was pinpointed for analysis. Absence of absorption at about 1730 cm1 of the raw sample could reveal the absence of ester group in these samples [8]. The starting fingerprint peak of all samples was around 1650 cm1. Additional two bands at 1650 cm1 and 1420 cm1 of the three genuine P. umbellatus were contributed by asymmetrical and symmetrical stretching vibration of COO- [9], which affirmed presence of uronic acid. In this case, PUC spectrum did not follow this pattern as its amid I peaks at 1648 cm1 was the same height level with 1035 cm1 and the peak 1420 cm1 was not present. Compared with the peak of 2926 cm1 (PUC&PU1) and 2924 cm1 (PU2 &PU3), the peak of the PUC at 1648 cm1 was stronger than all the genuine PU samples, indicating that there are more flavones in the counterfeit PU than the genuine PU. PUC has a single peak of 1384 cm1 assigned to symmetry deformation vibration ds CH3. The PU2 showing a sequence of 1374 cm1 and 1317 cm1 could be attributed to the bending of CeH and twisting vibration of HeCeH, respectively [10]. The sample PU1 and PU3 spectra have the similar sequence except the peak 1317 cm1 was shifted to 1325 cm1 (PU1) and 1324 cm1 (PU2). The peaks between 1200 and 1000 cm1 were considered as the absorption of a hydro-sugar ring vibration. In fact, these batches of groups overlapped with the (CeOeC) glycosidic bond vibration and stretching vibration of (CeOH) side groups [11]. The genuine and counterfeit P. umbellatus spectra showed the characteristic absorption peaks for polysaccahrides clearly [12]. The close characteristics of both types of samples needed to be speculated for interpretation. The double high peak at the region of 1100-1000 cm1 was the symbolic saccharides of macrofungi. Hence, PUC is also considered as a type of macrofungi due to its obvious double peak at 1075 cm1 and 1035 cm1. The observational increase in three of the genuine P. umbellatus spectra caused by the peak 891 cm1 was attributed to type of b-D-glucopyranose mainly assigning bending vibration of CeH axial in b-sugars [13]. In contrast, PUC spectrum lacked peak 891 cm1 but showed another peak of 781 cm1 which is the band of a-polysaccharides. PUC is considered a type of macrofungi but possesses lower content of b-D-glucan which plays a vital role in immune system. Both wild PU from two different locations showed closely identified spectra. However, only three peaks were exactly matched, namely peak 1374 cm1, 1040 cm1, and 607 cm1. The rest of the peaks showed differences in the range 1e4 cm1. However, the assignation of peak 1325 cm1 of PU1 was a rate of 8 cm1 differences with PU2 (Fig. 1). The peak 574 cm1 labeled on PU2 should be assigned the different vibration compared with the peak 607 cm1. The PU3 showed close spectrum peak compared with wild PU1. Nevertheless, the comparison of FTIR spectra of samples was unable to determine the actual result. The process of
X. Chen et al. / Journal of Molecular Structure 1199 (2020) 126917
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Fig. 1. The outlook of PU1, PU2, PU3 and PUC. All the samples characterized as outer black skin layer with inner white tissue content. All performed irregular shape and solid. There is certain level of accuracy to determine the genuine and the counterfeit of P. umbellatus when putting them together.
Fig. 2. FTIR spectrum of sample genuine and the counterfeit P. umbellatus in the range of 4000-400 cm1. (a) PU1: wild P. umbellatus found from Heilongjiang province; (b) PU2: wild P. umbellatus found from Yunnan province; (c) PU3: cultivated P. umbellatus from Shannxi province; (d) PUC: Agrocybe tuberosa as common counterfeit P. umbellatus in the market.
“compare” indicated the higher correlation (>0.98) of PU3 and PU2, while the correlation of PU3 and PU1 was considered “fail”
(Table 1). However, 1D FTIR result was sufficient to differentiate the PUC with the wild and cultivated P. umbellatus. The first box
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Table 1 Correlation of each sample using process “compare” from the software “Spectrum”. Sample A
Sample B
Correlation
Pass/fail (standard ¼ 0.98)
PU1 PU1 PU1 PU2 PU2 PU3
PU2 PU3 PUC PU3 PUC PUC
0.965,587 0.975,973 0.822,115 0.983,283 0.827,886 0.798,826
fail fail fail pass fail fail
raw showed the lowest correlation. This meant it is easier to differentiate cultivated P. umbellatus from Shaanxi Province compared to counterfeit P. umbellatus, while both wild types of P. umbellatus showed the higher correlation with the counterfeit P. umbellatus. Therefore, the counterfeit P. umbellatus could be easily mixed up with the wild P. umbellatus compared with the cultivated P. umbellatus.
3.3. Second derivative spectra analysis showed their differences regarding the content of fatty acids, while the second highlight box showed their differences in the content of saccharides. The peak 1324 cm1 of PUC was signally 5 times higher than that of other spectra. PUC lacked peaks 1420 cm1 and 1252 cm1 as showed in the spectra of genuine P. umbrellatus. On the other hand, the peak 1384 cm1 of PUC was another assignment compared with 1374 cm1 from the three P. umbrellatus samples, and the peak 1324 cm1 of PUC was signally 5 times higher than the same peak of other spectra. The three single peaks in the range of 900-400 cm1 of PUC exaggerated the dissimilarity with the genuine P. umbrellatus (see Fig. 2). 3.2. Result of “compare” Using the process of “compare” from the spectrum system, the correlation of the raw samples was compared in the range of 4000400 cm1 (Table 1). The correlation of PU samples was closer to 0.98 (the standard), but the correlation of PU raw with PUC was less than 0.85. The correlation of PU2 and PU3 was more than 0.98, which meant the cultivated P. umbrellatus contained close to or more than 98% of chemical content compared with the wild type P. umbrellatus from Yunnan Province, China. Both wild types of P. umbrellatus failed the correlation but with more than 0.95 value. The difference could be caused by some geographical factors, such as types of soil, water, mineral and the climate. In this case, the PU3-raw and PUC-
The purpose of derivative spectrum is to derive the spectrum which contains certain overlapping peaks due to the presence of a mixture of substances in the raw material. Therefore, it enhances the interpretation as the second order ideally derived the spectrum with 13 number of data points used for slope calculation. The derived peaks were obviously found within the range of 1800400 cm1 (Fig. 3). The main peak in all the samples obtained in 1D FTIR spectrum was resolved into several peaks. In the report of He [12], a water-soluble polysaccharide (Fig. 4a) was isolated from the sclerotia of P. umbellatus. It was showed to contain 90.1% of carbohydrate and 7.5% of protein. Its carbohydrate content contained 8.5% uronic acid with a molecular weight of 8.8 kDa. The 1D FTIR spectrum of this compound illustrated the peaks of 3399, 2923, 1640, 1413, 1377, 1319, 1157, 1072, and 1049. The 2nd derivative spectra did not show the exact matching of each peak from this reference but the existence of this compound could be assumed as the derivative peaks (highlighted in the boxes). As the raw sample was used, many functional groups are overlapping under the same peak. Therefore the second derivative spectra were appropriately used for matching peak when the sample was performed in the fraction or compound. The 2nd derivative spectra of all the P. umbellatus spectra were compatible with this compound IR spectrum peak except 1413 cm1. It could be the derivative peak of 1420 cm1 from 1D FTIR spectra derived to peak around 1425 and 1423 cm1. The assignation of IR peak, 1413 cm1 was interpreted
Fig. 3. Second derivative spectra of sample PU1, PU2, PU3 and PUC in the range of 1800-400 cm1. It was easier to differentiate the genuine of P. umbellatus (PU1, PU2, PU3) compared to counterfeit P. umbellatus (PUC) through the 2nd derivative spectrum. The obvious part of the dissimilarity were the range 1650-1600 cm1, 1400-1350 cm1, 12201100 cm1 and 900-750 cm1 respectively. Four square boxes indicated the dissimilarity 2nd derivative spectra area of all genuine P. umbellatus.
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Fig. 4. Molecular structure of compounds isolated from P. umbellatus. 4a: a water-soluble polysaccharide; 4b: Polyporoid A; 4c: Polyporoid B; 4d: Compound 3; 4e: Polyporusterone A.
as methylene adjacent to epoxy ring. Another report by Sun regarded three new anti-inflammatory ergostane-type ecdysteroids from the sclerotium of P. umbellatus [14]. The three compounds are Polyporoid A (Fig. 4b) isolated as a
white amorphous solid and possessed a molecular formula of C28H44O7. The IR spectrum at 3396, 2961, 2873, 1644, 1623, 1562, 1413, 1384, and 1054 cm1 due to hydroxyl and a,b-unsaturated ketone groups; Polyporoid B (Fig. 4 c) was assigned the molecular
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Fig. 5. 2DIR correlation spectra in the range of 1800-400 cm-1 sequence of active site under the Noda's rule by referring the synchronous and asynchronous spectrum. For PU1, the sequence declined from 1187 cm1, 976 cm1, 1398 cm1 and 1497 cm1; for PU2, the sequence declined from 976 cm1, 1187 cm1,1057 cm1, 1398 cm1 and 1508 cm1; for PU3, the sequence declined from 1191 cm1, 1275 cm1, 1135 cm1, 973 cm1,1398 cm1, 1504 cm1, 1057 cm1; for PUC, the sequence declined from 969 cm1,1307 cm1,1191 cm1,1395 cm1,1057 cm1 and 1511 cm1..
formula C28H44O7 (seven unsaturations). Its IR absorption bands at 3400, 2960, 2870, 1648, 1620, 1565, 1418, 1385, and 1062 cm1 implied the presence of hydroxyl, conjugated carbonyl, and olefinic groups, respectively; Compound 3 (C28H47O7) (Fig. 4 d) as white amorphous solid showed infrared spectrum with potassium bromide at 3398, 2959, 2872, 1645, 1621, 1561, 1415, 1382, and 1057 cm1. In addition, Ishida et al. [15] isolated a colorless plateshaped crystals namely Polyporusterone A (Fig. 4 e) from sclerotia of P. umbellatus for hair growth treatment. Its formula contains 28 carbon molecules (C28H46O6) indicating certain similarity of its
IR spectrum with other 3 compounds. In fact, Takatomi et al. [16] reported this compound plus other 6 types of polyporusterones. Besides the main peaks of 3300 cm1 (assigned for OH), 16441650 cm1 (assigned for C]O), it also consisted peak at 907 cm1, 880 cm1 (assigned for epoxy) and 803 cm1 (assigned for C]CH2). The second derivative spectra of the genuine P. umbellatus showed the main region of the fingerprint marker according to the report on the polysaccharide, ecdysteroids compounds and the polyporusterones complex at (1708-1610 cm1), (1583-1494 cm1), (1442-1359 cm1), (1100-1020 cm1), (925-870 cm1) and (820-
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790 cm1). Two vibration C]C bands appeared at 1645 and 1620 cm1 with an intensity stronger than that of isolated double bonds; whereby the peak 1565-1561 cm1 and 1385-1382 cm1 were described by vibration asymmetry and vibration symmetry of 1 CO and 1062 cm1 was assigned as 2 and the peak 1057-1054 cm vibration of primary hydroxyl. PUC 2nd derivative spectrum was observed with few peaks related to the compounds mentioned above such as peaks at 1620, 1562, 1384, 890 and 799 cm1. These observations however were unconvincing for differentiating between the genuine and the counterfeit P. umbellatus in second derivative spectra. However, the peak 1562 cm1 appeared at PUC was the unique peak for the comparison of the genuine P. umbellatus with the counterfeit. In addition, peaks 1562 cm1, 1384 cm1 and 1263 cm1 were the base peaks obtained from PUC, but none from the other PU. The other scenario was the appearance of two base peaks in the range of 1300-1240 cm1 of all P. umbrellatus except PUC. This comparison helps to clearly identify the counterfeit P. umbrellatus in fine powder form. In term of overall compatible matching, the pattern of PU3 2nd derivative spectra was close to PU2 but not PU1. It is initially assumed the cultivated P. umbellatus (PU3) showed at least 50% similarity of the chemical content compared to PU2. In contrast, PU1 showed sharp and larger peaks especially in the range of 17001600 cm1 and 1580-1450 cm1 which did not obviously match with PU3 2nd derivative spectra. 3.4. 2DIR correlation spectra analysis 2DIR spectra, the third step to analyze the results, enhanced the differentiation and stressed on the correlation of each autopeak and crosspeak (Fig. 5). PUC showed the dissimilarity of the 2DIR spectra with other genuine P. umbellatus. The 2DIR easily revealed the differentiation of samples. By comparing the synchronous spectrum and the auto-peak, PU3 as cultivated P. umbrellatus has shown closer pattern and color with PU1. This scenario was opposite to the interpretation from the second derivative spectra, where PU3 showed closer pattern with PU2. In fact, the 1D FTIR demonstrated the similar scenario with 2DIR correlation spectra. The different scenario of interpretation occurred was due to the compatible match of the cultivated and the wild P. umbrellatus. The main autopeaks of PU1 (as labeled) created six correlation squares with the coordinated crosspeaks. They were [1497 cm1, (1398, 1497), 1398 cm1, (1497, 1398) ]; [ 1497 cm1, (1187,1497), 1187 cm1, (1497, 1187)]; [ 1497 cm1, (976,1497), 976 cm1, (1497,976)]; [ 1398 cm1, (1187,1398), 1187 cm1, (1398, 1187)]; [1398 cm1, (976,1398), 976 cm1, (1398,976)]; [ 1187 cm1, (976,1187), 976 cm1, (1187,976)]. The active auto-peak illustrated as dark red was considered the most active point and reacted faster than others, while the dark blue was considered the most passive point. The synchronous and asynchronous spectra were used to refer sequence of active site under the Noda's rule. For PU1, the sequence declined from 1187 cm1, 976 cm1, 1398 cm1 and 1497 cm1, for PU2, from 976 cm1, 1187 cm1,1057 cm1, 1398 cm1 and 1508 cm1, for PU3, from 1191 cm1, 1275 cm1, 1135 cm1, 973 cm1,1398 cm1, 1504 cm1, and 1057 cm1, while for PUC, and from 969 cm1,1307 cm1,1191 cm1,1395 cm1,1057 cm1 and 1511 cm1. Based on the sequence of active sites, PU3 showed similar sequence with PU1 where the decline started from the area of peak of 1191 cm1, followed by 973 cm1, 1398 cm1 and 1504 cm1. However, the sequence of active site of PU2 started from the area of 976 cm1. In term of crosspeak, the sequence of the active site (x,y) was dependent on their autopeak circumstance. This showed that whether x or y would first react under the thermal perturbation basically relied on the sequence of their autopeak
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active site. The analysis of 2DIR correlation spectrum showed that sequence active site represented the correlation of which compound could react first before the rest. This is important to show that when both autopeaks are actively reacted, the priority of the active site turned up at their crosspeak simultaneously in the chemical process. For example in PU1 in the correlation square of two autopeaks 1187 cm1 and 976 cm1, their crosspeaks are (976, 1187) and (1187,976). With the Noda's rule, the synchronous spectrum of the crosspeak was positive. However, it was negative in the asynchronous spectrum. Hence the 1187 cm1 is the point of reaction prior to 976 cm1. 4. Conclusion This study was an important determinant of small number of samples analysis and the proper interpretation of raw material using FTIR and 2DIR correlation spectroscopy. Therefore small number of samples is not an obstacle in the conduct of research and in obtaining the outcomes. In this study, the emphasis was elaborated on the association of the spectrum peak with the specific molecular bond vibration fingerprint of genuine P. umbellatus and counterfeit P. umbellatus. The differences and similarities of spectra of these samples have shown the ability of the FTIR techniques application without lots of samples. This rapid and simple sample preparation method enhances the speed of qualitative analysis of PU and rapidly determines their differences in term of their raw material content and the active site of certain compounds. Using “compare” as the reference tool with small number of samples is the ideal choice for the correlation coefficient was determined directly by a reference spectrum within the whole range of wavenumber. The correlation of PU samples were closer to 0.98 (the standard), but correlation of PU with PUC were less than 0.85. The correlation of PU2 and PU3 was more than 0.98, indicating that the cultivated P. umbrellatus contained close to or more than 98% of chemical contents compared to wild P. umbrellatus from Yunnan Province. Declarations of interest None. Acknowledgements The authors are grateful to the funds from National Natural Science Foundation of China (NSFC, No. 81573703) for financial support. The authors wish to thank Dr. Lee Han Lim (IMR) for editing this manuscript. References [1] Chinese Pharmacopoeia Commission, Pharmacopoeia of the People's Republic of China 2015. Set of 1, vol. 318, China Medical Science and Technology Press, Beijing, 2015. [2] Y.Y. Zhao, Traditional uses, phytochemistry, pharmacology, pharmacokinetics and quality control of Polyporus umbellatus (Pers.) Fries: a review, J. Ethnopharmacol. 149 (2013) 35e48. [3] W.R. Cai, L.L. Xie, Y. Chen, H. Zhang, Purification, characterization and anticoagulant activity of the polysaccharides from green tea, Carbohydr. Polym. 92 (2013) 1086e1090. [4] B.J. Yan, L.Y. Jing, J. Wang, A polysaccharide (PNPA) from Pleurotus nebrodensis offers cardiac protection against ischemia-reperfusion injury in rats, Carbohydr. Polym. 133 (2015) 1e7. [5] G.H. Mao, Y. Ren, W.W. Feng, Q. Li, H.Y. Wu, D. Jin, T. Zhao, C.Q. Xu, L.Q. Yang, X.Y. Wu, Antitumor and immunomodulatory activity of a water-soluble polysaccharide from Grifola frondosa, Carbohydr. Polym. 134 (2015) 406e412. [6] G. Kikuchi, H. Yamaji, Identification of Armillaria species associated with Polyporus umbellatus using ITS sequences of nuclear ribosomal DNA, Mycoscience 51 (2010) 366e372. [7] M.M. Liu, Y.M. Xing, X. Zeng, D.W. Zhang, S.X. Gu, Genetic diversity of
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Discrimination of authentic Polyporus umbellatus and counterfeit by Fourier Transform Infrared and two dimensional infrared correlation spectroscopy Xiangdong Chen a, YewKeong Choong b, Weiwei Zhang a, Guoqiang Li a, Jin Lan a, * a
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100193, PR China Phytochemistry Unit, Herbal Medicine Research Centre, Institute for Medical Research, National Institute of Health, No. 1 Jalan Setia Murni U13/15, Seksyen U13, 40170, Shah Alam, Selangor, Malaysia b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 July 2019 Received in revised form 6 August 2019 Accepted 8 August 2019 Available online 9 August 2019
Polyporus umbellatus(PU) belongs to the family Polyporaceae in the phylum of Basidiomycota. The sclerotia is its main medicinal parts. It is reputedly used in traditional Chinese Medicine as anti-cancer agent and consequently is in high demand. A major problem on the medicinal use of this mushroom is the menace of the counterfeit product. Therefore, the detection of counterfeit PU and the confirmation of genuine PU are important. This study was done by collecting wild PU and cultivated PU and compared with a counterfeit which is commonly found in the market. Fourier Transform Infrared (FTIR) and TwoDimension Infrared (2DIR) for the qualitative analysis are often regarded as green methods because the original resources of the test sample are preserved. The different spectral patterns of the counterfeit PU compared with the genuine PU were clearly indicated in 1D FTIR spectrum. The 2DIR spectrum of counterfeit PU showed more than 70% dissimilarity compared to genuine PU. This rapid and simple sample preparation enhances the speed of qualitative analysis on PU and rapidly determines their differences in term of their raw material content and the active site of certain compounds. © 2019 Elsevier B.V. All rights reserved.
Keywords: Polyporous umbellatus Discrimination Fourier transform infrared Two-dimensional infrared
1. Introduction Polyporus umbellatus (Pers.) Fries has been used as a traditional Chinese medicinal fungus. It belongs to the family Polyporaceae in the phylum of Basidiomycota. The sclerotia of P. umbellatus is its main medicinal parts and widely utilized for treatment of edema, scanty urine, vaginal discharge, as well as jaundice and diarrhea [1]. Pharmacological research had also revealed that sclerotia of P. umbellatus possessed various diuretic, anticancer, antiinflammatory and hepatoprotective activities [2]. Steroids and polysaccharides have been reported as the dominating ingredients responsible for the activities of sclerotia of P. umbellatus [2]. Extensive attention has been paid to the polysaccharides due to their crucial physiological and biological functions, such as anticoagulant [3], cardiac protection [4], immunomodulatory and antitumor [5]. The wild P. umbellatus are extensively used and cannot meet the
* Corresponding author. E-mail address: [email protected] (J. Lan). https://doi.org/10.1016/j.molstruc.2019.126917 0022-2860/© 2019 Elsevier B.V. All rights reserved.
demands required for clinical application. In the past 10 years, the wild P. umbellatus have become increasingly scarce and the market price rose. As reported from the survey, the price of the wild P. umbellatus was higher than the cultivated P. umbellatus. Mass scale commercial cultivation of P. umbellatus could be an ideal solution of providing an amble stock for the market. However, P. umbellatus cultivation is technically difficult due to the requirement that its sclerotial growth must be involved with the rhizomorphs of Armillaria. sp. [2]. The association of P. umbellatus with rhizomorphs of Armillaria sp. accelerates its growth. This symbiosis is thought to promote the development of sclerotia of P. umbellatus where the rhizomorphs of Armillaria sp. gradually penetrate into the medulla layer. Among the Armillaria species, five species (Armillaria sinapina, Armillaria calvescens, Armillaria gallica, Armillaria cepistipes, and Armillaria nabsnona) formed a highly supported clade with the symbiont [6]. Armillaria gallica and Armillaria cepistipes were the species most frequently observed to be symbiotic with P. umbellatus in China and there is genetic diversity among the genotypes [7]. However the actual relationship of these two fungi is still unclear. To date, there is unsuccessful attempt to mass produce P. umbellatus, and as such the scarcity of this mushroom
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has led to the emergence of counterfeit product. Therefore the detection of counterfeit PU and the confirmation of genuine PU is important. The color of its sclerotium depends on the stage of the development. There are three stages of P. umbellatus sclerotial development. During the sclerotial initial stage, the white mycelium carted on the ground and accumulated to form the white fungal mass. The primodia is then developed and the surface color changes from white to gray, then light brown and black as the development proceeds further. This study was done by collecting wild PU and cultivated PU and compared with a counterfeit PU which was commonly found in the market. Samples were sourced from Heilongjiang, Shaanxi, Yunnan province in China in this study. The small number of samples was due to the difficulty in searching the wild and P. umbellatus has its specific growth area associated with geographical factors. The uses of Fourier Transform Infrared (FTIR) and Two-Dimension Infrared (2DIR) for the qualitative analysis are often regarded as green method because the original resources of the test sample are preserved. The nutrient and important substances of the dried sample with only the water lost can be explored and analyzed directly under the infrared beam. 2. Materials and methods 2.1. Sample A wild P. umbrellatus (PU1) was obtained from Heilongjiang Province, China and another wild P. umbrellatus (PU2) from Yunnan Province, China. A cultivated P. umbrellatus (PU3) was from a mushroom cultivation farm in Shaanxi Province, China and a counterfeit P. umbrellatus (PUC) was purchased from Anguo herb market, the largest herbal medicine market in North China. All collected samples were taxonomically identified by Prof. Jin Lan at Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China. 2.2. Apparatus and parameters The system of GX Fourier-Transform Infrared (FTIR) spectrometer (PerkinElmer) was used in the processing of spectrum. A Deuterated Triglycine Sulfate detector was attached for recording the infrared spectrum. The parameters were set up by accumulating 32 times scans of the IR with a resolution of 4 cm1 and the wavenumber of the middle infrared was performed in the range of 4000-400 cm1. For the 2DIR analysis, a portable programmable temperature controller was used for applied thermal perturbation with the temperature gradually increasing from 30 to 120 C. Each interval of 10 C was implemented for scanning a spectrum on the same disk. 2.3. Procedure of sample disk preparation The mushroom fine powder (2 mg) was transferred into a crystal mortar and mixed with IR grade KBr. The mixture was ground well using the pestle until a homogenous mixture was obtained. A die with smooth surface on top was inserted into the cavity of pellet press. The mixture was poured into the cavity, then a bolt press was inserted and rotated into the cavity to distribute the particles. When the bolt press was taken out, the second die was inserted and the bolt press was adjusted onto it. Later, the whole entity was transferred into the hydraulic press and the wheel was turned clockwise to secure it tightly. Then the level was pulled repeatedly until the meter read 10 tons and waited for a few seconds. The pressure was released by rotating the knob counter clockwise and turning the wheel to disengage the pellet press. Finally the press
entity was disassembled by inverting the upper body and pressing until the disk came out. The upper die was removed and the sample disk was ready. The disk was transferred to the sample holder using forceps and the cap was fixed to secure the disk. The FTIR spectrum of the tablet was generally acceptable when a transmission of 60e70% was achieved. Otherwise, the test had to be repeated with either a fresh sample or with more KBr added. After this, the sample tablet was put into the temperature controlled pool and the FTIR spectra were recorded in situ. These results were interpreted for 2D-IR correlation spectroscopy. 3. Result and discussion 3.1. FTIR spectra analysis The different spectral patterns of the counterfeit PU compared with the genuine PU were clearly indicated in FTIR spectrum. Region of 1800-400 cm1 which represented fingerprint of P. umbellatus was pinpointed for analysis. Absence of absorption at about 1730 cm1 of the raw sample could reveal the absence of ester group in these samples [8]. The starting fingerprint peak of all samples was around 1650 cm1. Additional two bands at 1650 cm1 and 1420 cm1 of the three genuine P. umbellatus were contributed by asymmetrical and symmetrical stretching vibration of COO- [9], which affirmed presence of uronic acid. In this case, PUC spectrum did not follow this pattern as its amid I peaks at 1648 cm1 was the same height level with 1035 cm1 and the peak 1420 cm1 was not present. Compared with the peak of 2926 cm1 (PUC&PU1) and 2924 cm1 (PU2 &PU3), the peak of the PUC at 1648 cm1 was stronger than all the genuine PU samples, indicating that there are more flavones in the counterfeit PU than the genuine PU. PUC has a single peak of 1384 cm1 assigned to symmetry deformation vibration ds CH3. The PU2 showing a sequence of 1374 cm1 and 1317 cm1 could be attributed to the bending of CeH and twisting vibration of HeCeH, respectively [10]. The sample PU1 and PU3 spectra have the similar sequence except the peak 1317 cm1 was shifted to 1325 cm1 (PU1) and 1324 cm1 (PU2). The peaks between 1200 and 1000 cm1 were considered as the absorption of a hydro-sugar ring vibration. In fact, these batches of groups overlapped with the (CeOeC) glycosidic bond vibration and stretching vibration of (CeOH) side groups [11]. The genuine and counterfeit P. umbellatus spectra showed the characteristic absorption peaks for polysaccahrides clearly [12]. The close characteristics of both types of samples needed to be speculated for interpretation. The double high peak at the region of 1100-1000 cm1 was the symbolic saccharides of macrofungi. Hence, PUC is also considered as a type of macrofungi due to its obvious double peak at 1075 cm1 and 1035 cm1. The observational increase in three of the genuine P. umbellatus spectra caused by the peak 891 cm1 was attributed to type of b-D-glucopyranose mainly assigning bending vibration of CeH axial in b-sugars [13]. In contrast, PUC spectrum lacked peak 891 cm1 but showed another peak of 781 cm1 which is the band of a-polysaccharides. PUC is considered a type of macrofungi but possesses lower content of b-D-glucan which plays a vital role in immune system. Both wild PU from two different locations showed closely identified spectra. However, only three peaks were exactly matched, namely peak 1374 cm1, 1040 cm1, and 607 cm1. The rest of the peaks showed differences in the range 1e4 cm1. However, the assignation of peak 1325 cm1 of PU1 was a rate of 8 cm1 differences with PU2 (Fig. 1). The peak 574 cm1 labeled on PU2 should be assigned the different vibration compared with the peak 607 cm1. The PU3 showed close spectrum peak compared with wild PU1. Nevertheless, the comparison of FTIR spectra of samples was unable to determine the actual result. The process of
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Fig. 1. The outlook of PU1, PU2, PU3 and PUC. All the samples characterized as outer black skin layer with inner white tissue content. All performed irregular shape and solid. There is certain level of accuracy to determine the genuine and the counterfeit of P. umbellatus when putting them together.
Fig. 2. FTIR spectrum of sample genuine and the counterfeit P. umbellatus in the range of 4000-400 cm1. (a) PU1: wild P. umbellatus found from Heilongjiang province; (b) PU2: wild P. umbellatus found from Yunnan province; (c) PU3: cultivated P. umbellatus from Shannxi province; (d) PUC: Agrocybe tuberosa as common counterfeit P. umbellatus in the market.
“compare” indicated the higher correlation (>0.98) of PU3 and PU2, while the correlation of PU3 and PU1 was considered “fail”
(Table 1). However, 1D FTIR result was sufficient to differentiate the PUC with the wild and cultivated P. umbellatus. The first box
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Table 1 Correlation of each sample using process “compare” from the software “Spectrum”. Sample A
Sample B
Correlation
Pass/fail (standard ¼ 0.98)
PU1 PU1 PU1 PU2 PU2 PU3
PU2 PU3 PUC PU3 PUC PUC
0.965,587 0.975,973 0.822,115 0.983,283 0.827,886 0.798,826
fail fail fail pass fail fail
raw showed the lowest correlation. This meant it is easier to differentiate cultivated P. umbellatus from Shaanxi Province compared to counterfeit P. umbellatus, while both wild types of P. umbellatus showed the higher correlation with the counterfeit P. umbellatus. Therefore, the counterfeit P. umbellatus could be easily mixed up with the wild P. umbellatus compared with the cultivated P. umbellatus.
3.3. Second derivative spectra analysis showed their differences regarding the content of fatty acids, while the second highlight box showed their differences in the content of saccharides. The peak 1324 cm1 of PUC was signally 5 times higher than that of other spectra. PUC lacked peaks 1420 cm1 and 1252 cm1 as showed in the spectra of genuine P. umbrellatus. On the other hand, the peak 1384 cm1 of PUC was another assignment compared with 1374 cm1 from the three P. umbrellatus samples, and the peak 1324 cm1 of PUC was signally 5 times higher than the same peak of other spectra. The three single peaks in the range of 900-400 cm1 of PUC exaggerated the dissimilarity with the genuine P. umbrellatus (see Fig. 2). 3.2. Result of “compare” Using the process of “compare” from the spectrum system, the correlation of the raw samples was compared in the range of 4000400 cm1 (Table 1). The correlation of PU samples was closer to 0.98 (the standard), but the correlation of PU raw with PUC was less than 0.85. The correlation of PU2 and PU3 was more than 0.98, which meant the cultivated P. umbrellatus contained close to or more than 98% of chemical content compared with the wild type P. umbrellatus from Yunnan Province, China. Both wild types of P. umbrellatus failed the correlation but with more than 0.95 value. The difference could be caused by some geographical factors, such as types of soil, water, mineral and the climate. In this case, the PU3-raw and PUC-
The purpose of derivative spectrum is to derive the spectrum which contains certain overlapping peaks due to the presence of a mixture of substances in the raw material. Therefore, it enhances the interpretation as the second order ideally derived the spectrum with 13 number of data points used for slope calculation. The derived peaks were obviously found within the range of 1800400 cm1 (Fig. 3). The main peak in all the samples obtained in 1D FTIR spectrum was resolved into several peaks. In the report of He [12], a water-soluble polysaccharide (Fig. 4a) was isolated from the sclerotia of P. umbellatus. It was showed to contain 90.1% of carbohydrate and 7.5% of protein. Its carbohydrate content contained 8.5% uronic acid with a molecular weight of 8.8 kDa. The 1D FTIR spectrum of this compound illustrated the peaks of 3399, 2923, 1640, 1413, 1377, 1319, 1157, 1072, and 1049. The 2nd derivative spectra did not show the exact matching of each peak from this reference but the existence of this compound could be assumed as the derivative peaks (highlighted in the boxes). As the raw sample was used, many functional groups are overlapping under the same peak. Therefore the second derivative spectra were appropriately used for matching peak when the sample was performed in the fraction or compound. The 2nd derivative spectra of all the P. umbellatus spectra were compatible with this compound IR spectrum peak except 1413 cm1. It could be the derivative peak of 1420 cm1 from 1D FTIR spectra derived to peak around 1425 and 1423 cm1. The assignation of IR peak, 1413 cm1 was interpreted
Fig. 3. Second derivative spectra of sample PU1, PU2, PU3 and PUC in the range of 1800-400 cm1. It was easier to differentiate the genuine of P. umbellatus (PU1, PU2, PU3) compared to counterfeit P. umbellatus (PUC) through the 2nd derivative spectrum. The obvious part of the dissimilarity were the range 1650-1600 cm1, 1400-1350 cm1, 12201100 cm1 and 900-750 cm1 respectively. Four square boxes indicated the dissimilarity 2nd derivative spectra area of all genuine P. umbellatus.
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Fig. 4. Molecular structure of compounds isolated from P. umbellatus. 4a: a water-soluble polysaccharide; 4b: Polyporoid A; 4c: Polyporoid B; 4d: Compound 3; 4e: Polyporusterone A.
as methylene adjacent to epoxy ring. Another report by Sun regarded three new anti-inflammatory ergostane-type ecdysteroids from the sclerotium of P. umbellatus [14]. The three compounds are Polyporoid A (Fig. 4b) isolated as a
white amorphous solid and possessed a molecular formula of C28H44O7. The IR spectrum at 3396, 2961, 2873, 1644, 1623, 1562, 1413, 1384, and 1054 cm1 due to hydroxyl and a,b-unsaturated ketone groups; Polyporoid B (Fig. 4 c) was assigned the molecular
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Fig. 5. 2DIR correlation spectra in the range of 1800-400 cm-1 sequence of active site under the Noda's rule by referring the synchronous and asynchronous spectrum. For PU1, the sequence declined from 1187 cm1, 976 cm1, 1398 cm1 and 1497 cm1; for PU2, the sequence declined from 976 cm1, 1187 cm1,1057 cm1, 1398 cm1 and 1508 cm1; for PU3, the sequence declined from 1191 cm1, 1275 cm1, 1135 cm1, 973 cm1,1398 cm1, 1504 cm1, 1057 cm1; for PUC, the sequence declined from 969 cm1,1307 cm1,1191 cm1,1395 cm1,1057 cm1 and 1511 cm1..
formula C28H44O7 (seven unsaturations). Its IR absorption bands at 3400, 2960, 2870, 1648, 1620, 1565, 1418, 1385, and 1062 cm1 implied the presence of hydroxyl, conjugated carbonyl, and olefinic groups, respectively; Compound 3 (C28H47O7) (Fig. 4 d) as white amorphous solid showed infrared spectrum with potassium bromide at 3398, 2959, 2872, 1645, 1621, 1561, 1415, 1382, and 1057 cm1. In addition, Ishida et al. [15] isolated a colorless plateshaped crystals namely Polyporusterone A (Fig. 4 e) from sclerotia of P. umbellatus for hair growth treatment. Its formula contains 28 carbon molecules (C28H46O6) indicating certain similarity of its
IR spectrum with other 3 compounds. In fact, Takatomi et al. [16] reported this compound plus other 6 types of polyporusterones. Besides the main peaks of 3300 cm1 (assigned for OH), 16441650 cm1 (assigned for C]O), it also consisted peak at 907 cm1, 880 cm1 (assigned for epoxy) and 803 cm1 (assigned for C]CH2). The second derivative spectra of the genuine P. umbellatus showed the main region of the fingerprint marker according to the report on the polysaccharide, ecdysteroids compounds and the polyporusterones complex at (1708-1610 cm1), (1583-1494 cm1), (1442-1359 cm1), (1100-1020 cm1), (925-870 cm1) and (820-
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790 cm1). Two vibration C]C bands appeared at 1645 and 1620 cm1 with an intensity stronger than that of isolated double bonds; whereby the peak 1565-1561 cm1 and 1385-1382 cm1 were described by vibration asymmetry and vibration symmetry of 1 CO and 1062 cm1 was assigned as 2 and the peak 1057-1054 cm vibration of primary hydroxyl. PUC 2nd derivative spectrum was observed with few peaks related to the compounds mentioned above such as peaks at 1620, 1562, 1384, 890 and 799 cm1. These observations however were unconvincing for differentiating between the genuine and the counterfeit P. umbellatus in second derivative spectra. However, the peak 1562 cm1 appeared at PUC was the unique peak for the comparison of the genuine P. umbellatus with the counterfeit. In addition, peaks 1562 cm1, 1384 cm1 and 1263 cm1 were the base peaks obtained from PUC, but none from the other PU. The other scenario was the appearance of two base peaks in the range of 1300-1240 cm1 of all P. umbrellatus except PUC. This comparison helps to clearly identify the counterfeit P. umbrellatus in fine powder form. In term of overall compatible matching, the pattern of PU3 2nd derivative spectra was close to PU2 but not PU1. It is initially assumed the cultivated P. umbellatus (PU3) showed at least 50% similarity of the chemical content compared to PU2. In contrast, PU1 showed sharp and larger peaks especially in the range of 17001600 cm1 and 1580-1450 cm1 which did not obviously match with PU3 2nd derivative spectra. 3.4. 2DIR correlation spectra analysis 2DIR spectra, the third step to analyze the results, enhanced the differentiation and stressed on the correlation of each autopeak and crosspeak (Fig. 5). PUC showed the dissimilarity of the 2DIR spectra with other genuine P. umbellatus. The 2DIR easily revealed the differentiation of samples. By comparing the synchronous spectrum and the auto-peak, PU3 as cultivated P. umbrellatus has shown closer pattern and color with PU1. This scenario was opposite to the interpretation from the second derivative spectra, where PU3 showed closer pattern with PU2. In fact, the 1D FTIR demonstrated the similar scenario with 2DIR correlation spectra. The different scenario of interpretation occurred was due to the compatible match of the cultivated and the wild P. umbrellatus. The main autopeaks of PU1 (as labeled) created six correlation squares with the coordinated crosspeaks. They were [1497 cm1, (1398, 1497), 1398 cm1, (1497, 1398) ]; [ 1497 cm1, (1187,1497), 1187 cm1, (1497, 1187)]; [ 1497 cm1, (976,1497), 976 cm1, (1497,976)]; [ 1398 cm1, (1187,1398), 1187 cm1, (1398, 1187)]; [1398 cm1, (976,1398), 976 cm1, (1398,976)]; [ 1187 cm1, (976,1187), 976 cm1, (1187,976)]. The active auto-peak illustrated as dark red was considered the most active point and reacted faster than others, while the dark blue was considered the most passive point. The synchronous and asynchronous spectra were used to refer sequence of active site under the Noda's rule. For PU1, the sequence declined from 1187 cm1, 976 cm1, 1398 cm1 and 1497 cm1, for PU2, from 976 cm1, 1187 cm1,1057 cm1, 1398 cm1 and 1508 cm1, for PU3, from 1191 cm1, 1275 cm1, 1135 cm1, 973 cm1,1398 cm1, 1504 cm1, and 1057 cm1, while for PUC, and from 969 cm1,1307 cm1,1191 cm1,1395 cm1,1057 cm1 and 1511 cm1. Based on the sequence of active sites, PU3 showed similar sequence with PU1 where the decline started from the area of peak of 1191 cm1, followed by 973 cm1, 1398 cm1 and 1504 cm1. However, the sequence of active site of PU2 started from the area of 976 cm1. In term of crosspeak, the sequence of the active site (x,y) was dependent on their autopeak circumstance. This showed that whether x or y would first react under the thermal perturbation basically relied on the sequence of their autopeak
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active site. The analysis of 2DIR correlation spectrum showed that sequence active site represented the correlation of which compound could react first before the rest. This is important to show that when both autopeaks are actively reacted, the priority of the active site turned up at their crosspeak simultaneously in the chemical process. For example in PU1 in the correlation square of two autopeaks 1187 cm1 and 976 cm1, their crosspeaks are (976, 1187) and (1187,976). With the Noda's rule, the synchronous spectrum of the crosspeak was positive. However, it was negative in the asynchronous spectrum. Hence the 1187 cm1 is the point of reaction prior to 976 cm1. 4. Conclusion This study was an important determinant of small number of samples analysis and the proper interpretation of raw material using FTIR and 2DIR correlation spectroscopy. Therefore small number of samples is not an obstacle in the conduct of research and in obtaining the outcomes. In this study, the emphasis was elaborated on the association of the spectrum peak with the specific molecular bond vibration fingerprint of genuine P. umbellatus and counterfeit P. umbellatus. The differences and similarities of spectra of these samples have shown the ability of the FTIR techniques application without lots of samples. This rapid and simple sample preparation method enhances the speed of qualitative analysis of PU and rapidly determines their differences in term of their raw material content and the active site of certain compounds. Using “compare” as the reference tool with small number of samples is the ideal choice for the correlation coefficient was determined directly by a reference spectrum within the whole range of wavenumber. The correlation of PU samples were closer to 0.98 (the standard), but correlation of PU with PUC were less than 0.85. The correlation of PU2 and PU3 was more than 0.98, indicating that the cultivated P. umbrellatus contained close to or more than 98% of chemical contents compared to wild P. umbrellatus from Yunnan Province. Declarations of interest None. Acknowledgements The authors are grateful to the funds from National Natural Science Foundation of China (NSFC, No. 81573703) for financial support. The authors wish to thank Dr. Lee Han Lim (IMR) for editing this manuscript. References [1] Chinese Pharmacopoeia Commission, Pharmacopoeia of the People's Republic of China 2015. Set of 1, vol. 318, China Medical Science and Technology Press, Beijing, 2015. [2] Y.Y. Zhao, Traditional uses, phytochemistry, pharmacology, pharmacokinetics and quality control of Polyporus umbellatus (Pers.) Fries: a review, J. Ethnopharmacol. 149 (2013) 35e48. [3] W.R. Cai, L.L. Xie, Y. Chen, H. Zhang, Purification, characterization and anticoagulant activity of the polysaccharides from green tea, Carbohydr. Polym. 92 (2013) 1086e1090. [4] B.J. Yan, L.Y. Jing, J. Wang, A polysaccharide (PNPA) from Pleurotus nebrodensis offers cardiac protection against ischemia-reperfusion injury in rats, Carbohydr. Polym. 133 (2015) 1e7. [5] G.H. Mao, Y. Ren, W.W. Feng, Q. Li, H.Y. Wu, D. Jin, T. Zhao, C.Q. Xu, L.Q. Yang, X.Y. Wu, Antitumor and immunomodulatory activity of a water-soluble polysaccharide from Grifola frondosa, Carbohydr. Polym. 134 (2015) 406e412. [6] G. Kikuchi, H. Yamaji, Identification of Armillaria species associated with Polyporus umbellatus using ITS sequences of nuclear ribosomal DNA, Mycoscience 51 (2010) 366e372. [7] M.M. Liu, Y.M. Xing, X. Zeng, D.W. Zhang, S.X. Gu, Genetic diversity of
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