Graduate School of Bioresource Sciences, Akita Prefectural University: Shimoshinjo-nakano, Akita , Japan
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1 Mycosystema 菌物学报 15 November 2011, 30(6): ISSN CN Q 2011 Institute of Microbiology, CAS, all rights reserved. Analysis of lichen substances including triterpenoids by high performance liquid chromatography with a differential refractive index detector and a photodiode array detector Hikari SATO Kojiro HARA Masashi KOMINE Yoshikazu YAMAMOTO * Graduate School of Bioresource Sciences, Akita Prefectural University: Shimoshinjo-nakano, Akita , Japan Abstract: A new method for analysis of lichen triterpenoids was established using high performance liquid chromatography with the combination of a differential refractive index detector (RID) and a photodiode array detector (PDA). It is proved that this method was convenient to detect and identify aromatic and aliphatic lichen substances; it enabled quantitative analysis of substances having no or less absorption of ultraviolet rays such as triterpenoids. In addition, they can be measured in high accuracy compared with the TLC method. Key words: extract, thallus, zeorin, aliphatic, Myelochroa, Lecanora INTRODUCTION Because lichens produce characteristic secondary metabolites (lichen substances), they have been used as medicines, dyes and cosmetics since ancient times. Culberson & Kristinsson (1970) applied thin layer chromatography (TLC) for identification of lichen substances, and Culberson and her group has developed this method (Culberson 1972a; Culberson & Johnson 1982). Culberson (1972b) next introduced another chromatographic method, high performance liquid chromatography (HPLC) to analyze lichen substances quantitatively. Further study on HPLC was promoted by Yoshimura et al. (1994). They used HPLC with a photodiode array detector (PDA) and analyzed many aromatic lichen substances by the combination of retention time and UV spectrum. However, quantitative analysis of aliphatic lichen substances having no or less UV absorption such as triterpenoids has been only performed by gas-liquid chromatography (Shibata et al. 1965). HPLC with a differential refractive index detector (RID) has been used in analyses of sugars and sugar alcohols, but not done in that of lichen triterpenoids. Triterpenoids as well as aromatic metabolites of lichens play roles as taxonomical markers; therefore, it is necessary to develop the method that can analyze both of aromatic and aliphatic * Corresponding author. yyamamoto@akita-pu.ac.jp Received: , accepted:
2 Vol.30 No compounds in lichen thalli. We investigate analytical method of lichen substances including triterpenoids by HPLC with the combination of PDA and RID. 1 MATERIALS AND METHODS 1.1 Chemicals Lupeol was purchased from Sigma-Aldrich (WGK Germany). Zeorin and 16-O-acetylleucotylic acid (ALA) were isolated from Lecanora argopholis (Ach.) Ach. by Prof. Takahashi of Meiji College of Pharmacy and Myelochroa aurulenta (Tuck.) Elix & Hale by us, respectively. 1.2 Lichen materials We used lichen materials of Heterodermia japonica (M.Sato) Swinscow & Krog (Yamamoto collected at Mt. Nyugasa, Nagano Pref.), Lecanora megalocheila (Hue) H.Miyaw. (Yamamoto at Rishiri Is., Hokkaido Pref.), L. muralis (Schreb.) Rabenh. (Yamamoto at Nyudo Point, Akita Pref. and Yamamoto at Jeju Is., Korea), L. subimmergens Vain. (Yamamoto at Takao and Yamamoto at Hozukyo, Kyoto Pref.), Myelochroa aurulenta (Yamamoto at Ohara, Kyoto Pref.), M. entotheiochroa (Hue) Elix & Hale (Yamamoto at Okutama Lake, Tokyo Pref.), M. irrugans (Nyl.) Elix & Hale (Yamamoto at Akkeshi-cho, Hokkaido Pref.), M. leucotyliza (Nyl.) Elix & Hale (Yamamoto at Ohara, Kyoto Pref.), Nephroma arcticum (L.) Torss. (Yamamoto at Koosamo, Finland), Nephromopsis ornate (Müll.Arg.) Hue (Yamamoto at Mt. Nyugasa, Nagano Pref.), Peltigera aphthosa (L.) Willd. (Yamamoto at Yamabe, Hokkaido). Natural thalli were stored at -30 before extraction. Each crashed sample (0.2g) was extracted with acetone or ethanol by starring at room temperature for 24h. and filtered, and then the filtrate was evaporated in vacuo under 40. The extracts (10mg) and standard chemicals (1mg) were dissolved in acetone (1mL) for analysis by TLC and HPLC. 1.3 TLC analysis Acetone solutions were subjected to reversed-phase TLCs under following conditions: TLC, Silica gel 60 RP-18 F 254s 5 10cm, , Merck Japan, Tokyo; solvent systems, methanol (80%, 90% and 100%), acetonitrile (80% and 100%), 100% 2-propanol; detection, heating at 100 after the spray of 10% H 2 SO HPLC analysis Acetone solutions were subjected to HPLC under following conditions: instrument, SHIMADZU HPLC 10A-DP; column, YMC-Pack ODS A; column temp, 40 ; solvent system, methanol:phosphoric acid (100:1); flow rate, 1mL/min; detectors, photodiode array detector (SPD-M10A) with the range of nm and differential refractive index detector (RID-10A). 1.5 Measurement of zeorin content 1.0, 2.0, 4.0 and 8.0mg of zeorin were dissolved in 1.0mL of acetone, of which each 10μL was injected to RID-HPLC. Each peak area obtained was measured and the standard curve was made. 2 RESULTS 2.1 TLC analysis of zeorin and extracts of M. aurulenta In order to investigate optimal solvent system for HPLC analysis of acetone and ethanol extracts of M. aurulenta containing a few triterpenoids and zeorin, a lichen triterpenoid, were subjected to reversed-phase TLCs with six kinds of solvent systems (Fig. 1). It is proved that solvents of acetonitrile (80% and 100%), methanol (80%, 90% and 100%) and 100% 2-propanol were not suitable to separate its triterpenoids, but 100% methanol well developed them. 2.2 Analysis of HPLC with PDA and RID of three authentic triterpenoids and acetone extract of M. aurulenta Three authentic triterpenoids, 16-O-acetylleucotylic acid (ALA), zeorin and lupeol were subjected to HPLC with RID and PDA by using solvent system, methanol: phosphoric acid (100:1) and they eluted at retention times (Rts) of 3.8, 4.9 and 9.5min, respectively (Fig. 2). Their chemical structures are shown in Fig. 2. These triterpenoids have no or less UV absorption; although they showed no peak in their PDA chromatograms at 菌物学报
3 946 Mycosystema 254nm, their peaks were appeared separately in RID chromatograms. Compounds having stronger polarity and earlier retention time in HPLC analysis. Polarity of ALA having one hydroxyl and carboxyl groups is the strongest of these three; therefore, ALA had the earliest retention time (3.8min). On the other hand, as lupeol having a hydroxyl group showed the weakest polarity, it appeared at the latest (9.5min). Acetone extract of M. aurulenta contained atranorin, ALA and zeorin was also subjected to HPLC with RID and PDA. A PDA chromatogram at 254nm showed one peak of atranorin at 3.0min; on the other hand, a RID chromatogram did three peaks of atranorin, ALA at 4.9min, and zeorin at 9.5min. 2.3 Analysis of HPLC with PDA and RID of triterpenoids in lichen thalli It is important for lichen taxonomists to analyze triterpenoids such as zeorin. Acetone extracts of four lichen species, Heterodermia japonica, Nephroma arcticum, Nephromopsis ornata and Peltigera aphthosa Fig. 1 Reversed-phase TLCs of acetone (A) and ethanol extracts (Et) of Myelochroa aurulenta and zeorin (Z), an authentic lichen triterpenoid. Solvents were 80% acetonitrile (80% AcCN), 100% acetonitrile (100% AcCN), 80% methanol (80% MeOH), 90% methanol (90% MeOH), 100% methanol (100% MeOH) and 100% 2-propanol (100% PrOH). TLC was Silica gel 60 RP-18 F 254s 5 10cm, , Merck Japan, Tokyo. Detection was heating at 100 after the spray of 10% H 2 SO 4. Fig. 2 HPLCs with PDA (254nm) and RID of three authentic triterpenoids (ALA, zeorin and lupeol) and acetone extract of Myelochroa aurulenta. Chemical structures of ALA, zeorin and lupeol.
4 Vol.30 No were analyzed by HPLC with PDA and RID. Fig. 3 showed their constituents. Zeorin was found in acetone extracts of Heterodermia japonica and Nephroma arcticum as described in a reference (Yoshimura 1974). Atranorin in H. japonica, nephroartctin in N. arcticum and tenuiorin in P. aphthosa were also found by their PDA chromatograms. On the other hand, N. ornata and P. aphthosa did not produce zeorin. Our results support that N. ornata contained aliphatic acids and P. aphthosa did phlebic acids as shown by Yoshimura (1974). Fig. 4 HPLCs with RID of acetone extracts of lichen thalli of Myelochroa aurulenta, M. entotheiochroa, M. irrugans and M. leucotyliza and authentic triterpenoids (ALA and zeorin). Fig. 3 HPLCs with PDA (254nm) and RID of acetone extracts of lichen thalli of Heterodermia japonica, Nephroma arcticum, Nephromopsis ornata and Peltigera aphthosa and authentic triterpenoids (ALA and zeorin). 2.4 Analysis of HPLC with PDA and RID of ALA in Myelochroa spp. Acetone extracts of Myelochroa aurulenta, M. entotheiochroa, M. irrugans and M. leucotyliza were analyzed by HPLC with PDA and RID. RID chromatograms of these specimens were shown in Fig. 4. ALA was isolated from the acetone extract of M. aurulenta and found to exhibit the antiproliferative activity against HL-60 human leukemia cells (Tokiwano et al. 2009). Fig. 4 showed that ALA was distributed in only M. aurulenta but also M. entotheiochroa and not existed in M. irrugans and M. leucotyliza. Zeorin was found in two extracts of M. irrugans and M. leucotyliza. 2.5 Analysis of HPLC with PDA and RID of zeorin in Lecanora spp. Acetone and ethanol extracts of five specimens of Lecanora megalocheila, L. muralis and L. subimmergens were analyzed by HPLC with PDA and RID. HPLC chromatograms and zeorin contents of these specimens were shown in Fig. 5 and Table 1. Four extracts contained zeorin, a taxonomic marker of the genus Lecanora. This indicates that four specimens having zeorin belong to Lecanora. Usnic acid is a taxonomical marker of L. muralis. The specimen collected at Jeju Is. containing zeorin and usnic acid was identified to L. muralis; on the other hand, the specimen collected at Nyudo Point had not zeorin and usnic acid, and it could not be identified to Lecanora. Table 1 showed that there was a wide range of zeorin content such as 0.029mg/mg in Lecanora megalocheila, 1.32 in L. muralis and 0.83 in L. subimmergens. Table 1 Zeorin content in ethanol extracts of lichen thalli of Lecanora spp. Zeorin content Species Collection site (mg/mg of DW) L. megalocheila Rishiri Is., Hokkaido Pref L. muralis Jeju Is., Korea 1.32 L. subimmergens Takao, Kyoto Pref 菌物学报
5 948 Mycosystema Fig. 5 HPLCs with PDA and RID of acetone extracts of lichen thalli of Lecanora megalocheila, L. muralis (collected at Jeju Is, Korea and at Nyudo Point, Akita Pref.) and L. subimmergens (collected at Takao and at Hozukyo, Kyoto Pref.) and zeorin. 3 DISCUSSION As lichen substances are mainly aromatic compounds, methods detecting these compounds contained in lichen thalli have been developing; they are UV radiation, spot color tests and so on. On the other hand, aliphatic compounds, triterpenoids and fatty acid derivatives are also important as taxonomical markers. However, it is difficult to detect them in lichen thalli. Shibata et al. (1965) reported lichen triterpens having no or less UV absorption can be analyzed by gas-liquid chromatography (GLC) and also Culberson & Kristinsson (1970) applied thin layer chromatography (TLC) for identification of aliphatic lichen substances. Culberson (1972b) also introduced high performance liquid chromatography (HPLC) to analyze lichen substances and Yoshimura et al. (1994) developed HPLC with a photodiode array detector (PDA) and analyzed many aromatic lichen substances by the combination of retention time and UV spectrum. HPLC with a differential refractive index detector (RID) used in analysis of sugars and sugar alcohols has not been applied for identification of lichen triterpenoids. We investigate to develop the method that can analyze both of aromatic and aliphatic compounds in lichen thalli. In order to investigate optimal solvent system for HPLC analysis of lichen triterpenoids, we subjected acetone extract of M. aurulenta to reversed-phase TLC and confirmed which solvent could separate its triterpenoids very well (Fig. 1). Consequently, 100% methanol was so good to separate them that we used it for HPLC analysis. Three authentic triterpenoids, ALA, zeorin and lupeol were subjected to HPLC with RID and PDA eluting methanol:phosphoric acid (100:1) (Fig. 2). We obtained good separation of three authentic triterpenoids having their Rts of 3.8, 4.9 and 9.5min, respectively and detected atranorin, ALA and zeorin in M. aurulenta thallus by the analysis of HPLC with PDA and RID. Acetone extracts of four lichen species, Heterodermia japonica, Nephroma arcticum, Nephromopsis ornata and Peltigera aphthosa containing lichen triterpenoids mainly were analyzed by HPLC with PDA and RID in order to make sure that this analytical method was worthy for identification of lichen substances. Aromatic compounds such as atranorin and nephroartctin and aliphatic compounds such as zeorin and phlebic acids were identified by HPLC with PDA and RID (Fig. 3). We also analyzed acetone extracts of Myelochroa aurulenta, M. entotheiochroa, M. irrugans and M. leucotyliza to identify ALA in M. aurulenta and M. entotheiochroa and
6 Vol.30 No zeorin in M. irrugans and M. leucotyliza (Fig. 4). Acetone extracts of five specimens of Lecanora megalocheila, L. muralis and L. subimmergens were analyzed (Fig. 5). Zeorin was found in four specimens except one; according to this result it was not belong to the genus Lecanora. Thus, it is proved that HPLC with the combination of PDA and RID is a good system for detection and identification of constituents of a lichen thallus. We analyzed zeorin content in extracts of Lecanora megalocheila, L. muralis and L. subimmergens by HPLC with RID (Table 1). It was proved that there was a wide range of zeorin content among specimens of the same genus Lecanora. This suggests that quantitative analysis indicates the chemical diversity among the same genus. In addition, they can be measured in high accuracy compared with the TLC method. Acknowledgments: We thank Prof. Kunio Takahashi of Meiji College of Pharmacy for the gift of zeorin. [REFERENCES] Culberson CF, 1972a. Improved conditions and new data for the identification of lichen products by a standardized thin-layer chromatographic method. Journal of Chromatography, 72: Culberson CF, 1972b. High-speed liquid chromatography of lichen extracts. Bryologist, 75: Culberson CF, Johnson A, Substitution of methyl t-butyl ether for diethyl ether in the standardized thin-layer chromatographic method for lichen products. Journal of Chromatography, 238: Culberson CF, Kristinsson H, Standardized method for the identification of lichen products. Journal of Chromatography, 46: Shibata S, Furuya T, Iizuka H, Gas-liquid chromatography of lichen substances. I. Studies on zeorin. Chemical & Pharmaceutical Bulletin (Tokyo), 13: Tokiwano T, Sato H, Obara T, Hirota H, Yoshizawa Y, Yamamoto Y, A lichen substance as an antiproliferative compound against HL-60 human leukemia cells: 16-O-acetylleucotylic acid isolated from Myelochroa aurulenta. Bioscience Biotechnology & Biochemistry, 73: Yoshimura I, Lichen flora of Japan in color. 48 pls. Hoikusya, Osaka. 349 Yoshimura I, Kinoshita Y, Yamamoto Y, Huneck S, Yamada Y, Analysis of secondary metabolites from lichen by high performance liquid chromatography with a photodiode array detector. Phytochemical Analysis, 5: 菌物学报
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