Investigation of flavonoid profile of Scutellaria bacalensis Georgi by high performance liquid chromatography with diode array detection and electrospray ion trap mass spectrometry

Journal of Chromatography A, 1216 (2009) 4809–4814

Contents lists available at ScienceDirect

Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Short communication

Investigation of flavonoid profile of Scutellaria bacalensis Georgi by high performance liquid chromatography with diode array detection and electrospray ion trap mass spectrometry Guozhu Liu, Jinyu Ma, Yingzhuang Chen, Qingqing Tian, Yao Shen, Xiaosong Wang, Bo chen ∗ , Shouzhuo Yao Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research, Ministry of Education, Hunan Normal University, Changsha 410081, China

a r t i c l e

i n f o

Article history: Received 18 January 2009 Received in revised form 2 April 2009 Accepted 7 April 2009 Available online 15 April 2009 Keywords: Scutellaria baicalensis Georgi Flavonoids Profile HPLC–UV/MS

a b s t r a c t Scutellaria baicalensis Georgi is a well-known medicinal plant widely used in China and other East Asian countries. High performance liquid chromatography combined with diode array detection and electrospray ion trap mass spectrometry was used to determine the flavonoid profile of S. baicalensis. Under the optimized experiment conditions, 32 flavonoids were clearly detected. Eighteen main ones were doubtless identified by comparing their retention time, UV and MS (MSn ) data with isolated or commercial standards. The UV characteristics of these 18 known standards were studied in detail. The rules summarized provided valuable indications for the subsequent on-line identification processes. By interpreting both the MS and the UV data in detail, other 13 minor flavonoids in S. baicalensis were on-line identified successfully. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Scutellaria baicalensis Georgi (S. baicalensis) is a well-known medicinal plant widely used in China and other East Asian countries under the name “Huang Qin” in Chinese. It has been officially listed in the Chinese Pharmacopoeia for a long time [1]. Its root has been used for treatment of various ailments including fever, ulcer, bronchitis, hepatitis, tumor, inflammatory disease, etc. The active constituents in S. baicalensis are mainly flavonoids, and over 60 flavonoids including flavones, flavonols, flavanones, flavanonols, biflavones and chalcones have been isolated [2]. For analysis of flavonoids in S. baicalensis, previous studies were mainly focused on a few major flavonoids which are commercially available, such as wogonin, baicalein, wogonoside and baicalin, using simple HPLC coupled with UV detection [3,4]. In recent years, HPLC–UV/MS has been used, and more flavonoids have been qualitative and quantitative determined [5,6]. However, the results of these works are still far away from the goal of global analysis of flavonoids in S. baicalensis. Thus, Han et al. have conducted a study of on-line identification of flavonoids in S. baicalensis, and in total 26 flavonoids were tentatively characterized [7]. However, in that paper, most of the identification results were just based on the MS

∗ Corresponding author. Tel.: +86 731 8865515; fax: +86 731 8865515. E-mail address: [email protected] (B. chen). 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.04.021

data; the UV data obtained simultaneously were not well interpreted. In fact, the UV spectrum is a very essential and effective tool for characterization of the substitution patterns of flavonoids, for that the MS datum is usually helpless. For this reason, several results in that paper were inaccurate testified by the present study. Furthermore, the on-line tentative identification results should be further validated by authentic standards. The purpose of this text is to conduct a global and accurate qualitative analysis of flavonoids in the roots of S. baicalensis. Eighteen main flavonoids in the extract were doubtless identified by comparison with isolated or commercial standards. Furthermore, the UV characteristics of these 18 flavonoids were well studied. Based on the proposed UV rules and the known MS fragmentation rules, other 13 minor flavonoids were successfully identified on-line. 2. Experimental 2.1. Plant and chemical materials The root of S. baicalensis used for isolation was purchased from a local drugstore, and that used for profile analysis by HPLC–UV/MS was purchased from the National Institute for the Control of Pharmaceutical and Biological Products. Chrysin (4) and Scutellarin (29) were purchased from Shanghai Usea Biotech Company (Shanghai, China).

4810

G. Liu et al. / J. Chromatogr. A 1216 (2009) 4809–4814

2.2. Sample preparation The root of S. baicalensis was powdered. 0.5 g of the powders was mixed with 50 mL of 60% ethanol aqueous and sonicated for 40 min. Then the supernatant was filtered through a 0.45 ␮m filter for HPLC analysis. 2.3. HPLC–UV/MS(MSn ) analysis Chromatographic separation was performed on a Shimadzu 20A system equipped a DAD UV detector (Tokyo, Japan), using a 5 ␮m Ultimate XB C18 column (250 mm × 4.6 mm) (Welch Materials, Ellicott, USA). The mobile phase consisted of water containing 0.06% acetic acid (A) and acetonitrile (B). A gradient program was used as follows: 20% B in the first 5 min, 20–30% B during 5–30 min, 30–55% B during 30–55 min, then B held at 55% for 10 min. The flow rate was set at 1 mL/min and the injection volume was 10 ␮L. Column temperature was controlled at 25 ◦ C. The on-line MS and MSn data were obtained based on a Finnigan LCQ ion trap mass spectrometer with an ESI source (San Jose, CA, USA). The outlet of the UV detector was split, only 0.2 mL/min of the effluent was delivered into the MS. The LCQ was used as following condition: in positive ESI mode, spray voltage, 4.5 kV, sheath gas flow rate, 40 arbitrary units, auxiliary gas flow rate, 15 units, heated capillary temperature, 300 ◦ C, capillary voltage, 15 V; in negative ESI mode, spray voltage, 3.5 kV, sheath gas flow rate, 40 units, auxiliary gas flow rate, 15 units, heated capillary temperature, 300 ◦ C, capillary voltage, 10 V. For full scan MS analysis, the spectra were recorded in the range of m/z 100–1000. The relative collision energy was adjusted to 40% of maximum to obtain MSn data. 2.4. Preparative isolation of the main flavonoids The root of S. baicalensis (1 kg) was powdered and refluxed at 80 ◦ C for two times with 2 L of 60% acetonitrile aqueous solution for 2 h each. After filtration, phase separation of combined extraction solution was obtained by adding dichloromethane (600 mL). The separated organic part was repeatedly chromatographed by silical gel column and then by preparative RP-HPLC, resulting in isolation of 11 flavonoid aglycones, i.e. tenaxin I (1), oroxylin A (2), skullcapflavon II (3), 5,7-dihydroxy-6,8-dimethoxy flavone (5), wogonin (6), skullcapflavon I (7), baicalein (9), norwogonin (10), 5,7,2 ,5 -tetrahydroxy-8,6 -dimethoxy flavone (23), 5,7,3,2 ,6 pentahydroxy flavone (27) and 5,7,3,2 ,6 -pentahydroxy flavanone (28). The pure standards of the five major flavonoid glycosides, i.e. wogonoside (13), chrysin-7-O-glucuronide (16), oroxylin A-7O-glucuronide (17), dihydroxybaicalein-7-O-glucuronide (21) and baicalin (24) were directly isolated and purified from the aqueous part by repeated preparative RP-HPLC. All these compounds with HPLC purities >95% showed spectroscopic and spectrometric parameters (UV, MS, NMR) identical with literatures. 3. Results and discussion

The on-line UV and MS data of these components were obtained and were summarized in Table 1. 3.2. Directly identification of the main constituents by comparison with isolated and commercial standards To obtain a doubtless identification of constituents in S. baicalensis, authentic standards are needed. Sixteen flavonoids including eleven aglycones and five glucuronides were successfully isolated by solvent grouping and repeated silica gel column chromatography and preparative RP-HPLC, and their structure were elucidated by their spectroscopic and spectrometric data (UV, MS and NMR). Furthermore, two commercial standards (4 and 29) were available. Thus, a total of 18 main flavonoids in the extract of S. baicalensis could be doubtless recognized by comparing their retention time, UV and MS (MSn ) data with standards. 3.3. UV characteristics of the 18 flavonoid standards Analysis of UV patterns of the known flavonoids and conclusion of the rules will be quite valuable for subsequent on-line identification of structurally related unknown ones. Thus, the UV characteristics of the 18 flavonoid standards were studied in detail, and several rules were proposed as follow: (1) in the UV spectra of flavanones (21 and 28), band I, associated with the absorption ascribed to the B-ring cinnamoyl system which is not existed in flavanones, is absent as expected, while band II, associated with absorption involving the A-ring benzoyl system, shows great bathochromic shift (above 15 nm) with respect to values for flavone analogues (e.g. 21/24 and 28/4). (2) In the spectra of the four unusual 2 ,6 -dioxygenated flavones (3, 7, 23 and 27), band I is also absent or observed as only several slight shoulders because of the destruction of the resonance structure of the B-ring cinnamoyl system due to the serious steric hindrance formed by the 2 ,6 -disubstitute group, while band II shows great hypsochromic shift (5–10 nm) compared to the common ones with similar A-ring (e.g. 3/1, 7/6 and 23/6). (3) For flavones without both 6-oxygenation and B-ring oxygenation (4, 6, 10, 13 and 16), band II is the major UV absorption peak while band I is usually observed as only a small shoulder. However, a clear band I absorption can be observed for 6-oxygenated analogues (2, 5, 9 and 24), although band II is still the major peak. Furthermore, both 6-oxygenation and 8oxygenation lead to bathochromic shift of band II, but the shift due to 8-oxygenation is much greater (e.g. 6/2, 10/9 and 24/16), and hydroxylation is much more effective than methoxylation (e.g. 9/2 and 10/6). (4) As exampled by scutellarin (29) as shown in Fig. 2A, 4 -hydroxylation shifts both band I and band II towards longer wavelength, and the intensity of band I is higher than band II. However, for the 2 -hydroxylated flavone (tenaxin I (1) as shown in Fig. 2B), band I is a minor peak though bathochromic shift is also observed. (5) All 7-O-glucuronides show essentially identical UV spectra with their corresponding aglycones (e.g. 2/17, 4/16, 6/13 and 9/24), indicating the glycosylation of 7-OH has little effect on the UV spectrum.

3.1. General aspects 3.4. On-line identification of unisolated constituents Flavonoid aglycones showed better ionization efficiencies in positive ESI mode, but their corresponding glycosides, especially glucuronides, exhibited better result in negative ESI mode. Thus, for performing a high-quality profile analysis of S. baicalensis, the components with retention time shorter than 37 min (most of them are flavonoid glucuronides) were analyzed in negative ESI ion mode, while constituents eluted after 37 min (all of them are aglycones) were analyzed in positive ESI mode. By this means, 32 components, numbered as 1–32, were clearly detected in both DAD UV and MS TIC chromatograms of the extract of S. baicalensis as shown in Fig. 1.

Positive ESI analysis of 20 gave the [M+H]+ ion at m/z 447 and the aglycone cation at m/z 271. Further CAD of m/z 271 yielded the same fragmentation patterns as the MS2 of the protonated norwogonin (10), thus identifying 20 as a glucuronide of norwogonin. Furthermore, the UV spectrum of 20 was identical to that of norwogonin, revealing the glycosylation of 7-OH for 20. Thus, 20 was positively determined to be norwogonin-7-O-glucuronide which is different from the result proposed by Han et al., who assigned this component to be galangin (erroneously written as galengin)-

G. Liu et al. / J. Chromatogr. A 1216 (2009) 4809–4814

4811

Fig. 1. LC–UV/MS chromatograms for the flavonoid standards and the extract of S. baicalensis. (A) UV chromatogram at 276 nm and (B) MS total ion current (TIC) chromatogram of the 18 flavonoid standards; (C) UV chromatogram at 276 nm and (D) MS TIC chromatogram of the S. baicalensis extract.

7-O-glucuronide [7]. By the same identification procedure, other four 7-O glycosides, i.e. 12, 18, 22 and 25 were positively identified as 5,7-dihydroxy-6,8-dimethoxy flavone-7-O-glucuronide, wogonin-7-O-glucoside, oroxylin A-7-O-glucoside and baicalein-7O-glucoside, respectively. Component 14 exhibited the same MS behavior as baicalin (24) in both negative and positive ESI ion mode, indicating 14 is also a glucuronide of baicalein. However, the UV spectrum of 14 was quite

different from that of baicalin and baicalein—band II was located at 272 nm with a hypsochromic shift of 4 nm. The shift associated with the glycosylation of 5-OH is usually of 5–15 nm for both band I and band II and the intensity of band II relative to band I will become low [8,9]. Thus, we assigned 14 to be baicalein-6-O-glucuronide. Another proof for this identification is that the UV spectrum of 14 is quite similar to 2 (oroxylin A) as they share similar substitution patterns.

Fig. 2. UV spectra (A) of scutellarin (29)–flavone with 4 -hydroxylation and (B) of tenaxin I (2)–flavone with 2 -hydroxylation.

4812

Table 1 Identification of flavonoids in S. baicalensis by HPLC–UV/MS. Rt (min)

UV max (nm)

ESI-MS (MSn ) m/z (abundance)

Identification results

1 2 3 4

54.22 52.42 51.52 51.52

275, 336 272, 313 269 268, 313(sh)

5,2 -Dihydroxy-6,7,8-trimethoxy flavone (Tenaxin I)a 5,7-Dihydroxy-6-methoxy flavone (Oroxylin A)a 5,2 -Dihydroxy-6,7,8,6 -tetramethoxy flavone (Skullcapflavon II)a 5,7-Dihydroxy flavone (Chrysin)a

5 6 7 8 9 10

50.96 50.45 49.78 49.07 41.15 39.61

277, 320 274 263 276, 323 276, 323 279

11 12

33.38 32.62

287 278, 318

13 14

32.27 31.33

274, 349(sh) 272, 316

15 16 17 18 19 20

30.11 29.65 28.96 28.34 27.80 27.55

279, 316 267, 304(sh) 272, 312 274, 317(sh) 283, 322 280, 350(sh)

21

27.02

293, 350(sh)

22 23 24 25 26 27 28 29 30 31 32

26.12 23.72 22.40 21.76 19.25 15.35 11.60 11.53 11.04 10.34 9.92

272, 325 264, 306(sh) 276, 315 276, 316 272, 332 253, 302(sh) 289 282, 333 272, 315 291(sh), 331 272 315

ESI+ -MS: 345, MS2 [345]: 330(100), 315(33), 312(39), 297(9) ESI+ -MS: 285, MS2 [285]: 270(100) ESI+ -MS: 375, MS2 [375]: 360(100), 342(34), 345(46), 327(10) ESI+ -MS: 255, MS2 [255]: 231(38), 213(15), 209(47), 187(18), 171(39), 153(100), 129(20) ESI+ -MS: 315, MS2 [315]: 300(100), 285(13), 267(19) ESI+ -MS: 285, MS2 [285]: 270(100) ESI+ -MS: 345, MS2 [345]: 300(100), 312(23), 284(30) ESI+ -MS: 539, MS2 [539]: 419(100), 393(25), 269(13) ESI+ -MS: 271, MS2 [271]: 253(100), 229(12), 225(48), 197(7), 169(26), 123(20) ESI+ -MS: 271, MS2 [271]: 253(27), 229(9), 225(27), 215(17), 201(20), 197(7), 187(9), 173(16), 169(100), 123(7) ESI− -MS: 461,285, MS2 [461]: 285(100); ESI+ -MS: 463,287, MS2 [287]:153(100) ESI− -MS: 489, 313, MS2 [489]: 313(100); ESI+ -MS: 591, 315, MS2 [315]: 300(100), 285(17), 267(15) ESI− -MS: 459, 283, MS2 [459]: 283(100) ESI− -MS: 445, 269, MS2 [445]: 269(100), ESI+ -MS: 447, 271, MS2 [271]: 253(100), 229(17), 225(54), 197(5), 169(31), 123(17) ESI− -MS: 475, 299, MS2 [475]: 299(100), MS2 [299]: 284(100) ESI− -MS: 429, 253, MS2 [429]: 253(100) ESI− -MS: 459,283, MS2 [459]: 283(100) ESI− -MS: 445,283, MS2 [445]: 283(100) ESI− -MS: 475, 299, MS2 [475]: 299(100), MS3 [299]: 284(100) ESI− -MS: 445, 269, MS2 [445]: 269(100) ESI+ -MS: 447,271, MS2 [271]: 253(27), 229(9), 225(27), 215(17), 201(20), 197(7), 187(9), 173(16), 169(100), 123(7) ESI− -MS: 447, 271, MS2 [447]: 271(100) ESI+ -MS: 449, 273, MS3 [273]: 255(7), 187(16), 169(100), 131(39) ESI− -MS: 445, 283, MS2 [445]: 283(100) ESI− -MS: 345, MS2 [345]: 330(100), 315(53) ESI− -MS: 445, 269, MS2 [445]: 269(100) ESI− -MS: 431, 269, MS2 [431]: 269(100) ESI− -MS: 475, 299, MS2 [475]: 299(100) MS3 [299]: 284(100) ESI− -MS: 301 ESI− -MS: 303 ESI− -MS: 461, 285, MS2 [461]: 285(100) ESI− -MS: 547, MS2 [547]: 529(6), 487(4), 457(56), 427(100), 367(15), 337(26) ESI− -MS: 623, MS2 [623]: 461(100), MS3 [461]: 315(100) ESI− -MS: 547, MS2 [547]: 529(18), 487(70), 457(100), 427(53), 367(52), 337(59)

a b

Identification result obtained by comparing the retention time, UV spectra and MS data with authentic standard. On-line identification result.

5,7-Dihydroxy-6,8-dimethoxy flavonea 5,7-Dihydroxy-8-methoxy flavone (Wogonin)a 5,2 -Dihydroxy-7,8,6 -trimethoxy flavone (Skullcapflavon I)a 5,5 ,6,6 ,7,7 -Hexahydroxy-8,8 -biflavone (8,8”-bibaicalein)b 5,6,7-Trihydroxy flavone (Baicalein)a 5,7,8-Trihydroxy flavone (Norwogonin)a 5,7-Trihydroxy-2 -methoxy flavanone-7-O-glucuronideb 5,7-Dihydroxy-6,8-dimethoxy flavone-7-O-glucuronideb Wogonin-7-O-glucuronide (Wogonoside)a Baicalein-6-O-glucuronideb 5,6,7-Trihydroxy-8-methoxy flavone-7-O-glucuronideb Chrysin-7-O-glucuronidea Oroxylin A-7-O-glucuronidea Wogonin-7-O-glucosideb 5,7,8-Trihydroxy-6-methoxy flavone-7-O-glucuronideb Norwogonin-7-O-glucuronideb 5,6,7-Trihydroxy flavanone (Dihydroxybaicalein)-7-O-glucuronidea Oroxylin A-7-O-glucosidea 5,7,2 ,5 -Tetrahydroxy-8,6 -dimethoxy flavonea Baicalein-7-O-glucuronide (Baicalin)a Baicalein-7-O-glucosideb 5,7,2 -Trihydroxy-6-methoxy flavone-7-O-glucuronideb 5,7,3,2 ,6 -Pentahydroxy flavonea 5,7,3,2 ,6 -pentahydroxy flavanonea Scutellarein-7-O-glucuronide (Scutellarin)a Chrysin-6-C-glucosyl-8-C-arabonosideb Unidentified Chrysin-6-C-arabinosyl-8-C-glucosideb

G. Liu et al. / J. Chromatogr. A 1216 (2009) 4809–4814

Peak number

G. Liu et al. / J. Chromatogr. A 1216 (2009) 4809–4814

4813

Fig. 3. Chemical structures of flavonoids identified.

Components 15, 19 and 26 are three isomers as they all exhibited the [M−H]− ion at m/z 475 and the aglycone anion at m/z 299. MS2 of the three m/z 299 ions all yielded the product at m/z 284 due to the loss of a radical CH3 • fragment. This reveals the presence of a methoxyl and three hydroxyls located on each aglycone of these three glucuronides. The UV spectra of these three flavonoids are different. Both 19 and 15 showed a low-intensity but clear band I absorption at 319 and 316 nm respectively, which is the characteristic of flavones with 6-oxygenation but without B-ring oxygenation. 19 presented the band II peak at 283 nm rather than that of 15 (278 nm). Thus, 19 was

identified as 5,7,8-trihydroxy-6-methoxyflavone-7-O-glucuronide while 15 was assigned to be 5,6,7-trihydroxy-8-methoxyflavone7-O-glucuronide. 26 exhibited a low-intensity but clear band I absorption at 334 nm, which is typical for 2 -hydroxylation. Furthermore, the band II at 272 nm is the characteristic of a flavone with a 5,7-dihydroxylated and 6-methoxylated A-ring. Thus, 26 was identified as 5,7,2 -trihydroxy-6-methoxy flavone-7-O-glucuronide. The UV spectrum of 11 was typical for flavanone—band II at 287 nm while band I disappeared. Positive ESI analysis of 11 showed the [M+H]+ ion at m/z 463 and the aglycone cation at m/z 287. MS2 fragmentation of m/z 287 gave the only clean ion at m/z 153 corre-

4814

G. Liu et al. / J. Chromatogr. A 1216 (2009) 4809–4814

sponding to the 1,3 A+ ion, revealing two hydroxyl groups located on the A-ring and a methoxyl group on the B-ring. The previous isolated flavanones with a single methoxyl or hydroxyl group on the B-ring from S. baicalensis and other baicalensis species were almost all 2 -substituted [2]. Thus, we proposed 11 to be 5,7-dihydroxy-2 methoxy flavanone-7-O-glucuronide. Component 8 exhibited the [M+H]+ ion at m/z 539, and two UV absorptions at 276 and 323 nm identical to that of baicalein (9). Biflavones will keep the UV characteristic of their corresponding flavone counterparts [9]. Thus, 8 was assigned to be a biflavone of baicalein, 8,8 -bibaicalein, which has been often isolated from baicalensis species [10]. Furthermore, 30 and 32 were assigned to be chrysin-6-C-glucosyl-8-C-arabonoside and chrysin6-C-arabinosyl-8-C-glucoside respectively, the same as the studies by Wu et al. and Han et al. [5,7]. For component 31, Han et al. have assigned it to be isorhamnetin7-O-rhamnosyl glucoside [7]. However, the UV spectra for isorhamnetin and its 7-O-glycosides in literatures were characterized by the two absorptions at 253 and 370 nm [8,9], which are quite different from that of 31—two absorptions at 291 and 331 nm—observed by us and also by Han et al. Thus the identification of 31 as a 7-O-glycoside of isorhamnetin is unreliable. Unfortunately, the present study also failed to propose a positive identification of 31 based on the available MS and UV data. The chemical structures of all flavonoids identified were shown in (Fig. 3). 4. Conclusion In this study, a comprehensive study of flavonoid profile of S. baicalensis has been conducted by HPLC–UV/MS. Thirty-two com-

ponents were clearly detected. Eighteen major flavonoids were unambiguously identified by comparison with isolated and commercial standards, while 13 minor flavonoids were successfully on-line characterized. Furthermore, we have demonstrated that HPLC–UV is a powerful complementary tool of HPLC–MS for structural characterization of flavonoids. Acknowledgments This work was financially supported by the National “973” project (2006CB504701), the National Natural Science Foundation of China (20875028), 20080542003, 2008[890], and the Science Research Foundation of Hunan Province (2007FJ1005, 2008FJ3063). References [1] National Commission of Chinese Pharmacopoeia, Pharmacopoeia of People’s Republic of China, Chemical Industry Press, Beijing, 2005, p. 211. [2] H. Wen, S. Xiao, Y. Wang, G. Luo, Nat. Prod. Res. Dev. 16 (2004) 575. [3] Y.Y. Zhang, H.Y. Don, Y.Z. Guo, H. Ageta, Y. Harigaya, M. Onda, K. Hashimoto, Y. Ikeya, M. Okada, M. Maruno, Biomed. Chromatogr. 12 (1998) 31. [4] Z.Y. He, P.X. Cao, G.Y. Liang, Z.C. Liu, X.G. Wu, Zhongguo Zhongyao Zazhi 27 (2002) 258. [5] W. Wu, C.Y. Yan, L. Li, Z.Q. Liu, S.Y. Liu, J. Chromatogr. A 1047 (2004) 213. [6] C.R. Horvatha, P.A. Martosb, P.K. Saxena, J. Chromatogr. A 1062 (2005) 199. [7] J. Han, M. Ye, M. Xu, J.H. Sun, B.R. Wang, D.A. Guo, J. Chromatogr. B 848 (2007) 355. [8] T.J. Mabry, K.R. Markham, M.B. Thomas, The Ultraviolet Spectra of Flavones and Flavonols, Springer-Verlag, Berlin, 1970. [9] L. Huang, D.Q. Yu, The Application of Ultraviolet Spectra in Organic Chemistry, Science Press, Beijing, 2000. [10] Y. Kikuchi, Y. Miyaichi, Y. Yamaguchi, H. Kizu, T. Tomimori, K. Vetschera, Chem. Pharm. Bull. 39 (1991) 199.