Analysis of Low-polar Ginsenosides in Steamed Panax Ginseng at High-temperature by HPLC-ESI-MS/MS

CHEM. RES. CHINESE UNIVERSITIES 2012, 28(1), 31 36 Analysis of Low-polar Ginsenosides in Steamed Panax Ginseng at High-temperature by HPLC-ESI-MS/MS ZHANG Yu-chi 1,2, PI Zi-feng 1*, LIU Chun-ming 2, SONG Feng-rui 1*, LIU Zhi-qiang 1 and LIU Shu-ying 1 1. Changchun Center of Mass Spectrometry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China; 2. Central Laboratory, Changchun Normal University, Changchun 130032, P. R. China Abstract A high performance liquid chromatography coupled with electrospray ionization-tandem mass spectrometry(hplc-esi-ms/ms) method was developed for the analysis and identification of ginsenosides in the extracts of raw Panax ginseng(rpg) and steamed Panax ginseng at high temperatures(spght). A total of 25 ginsenosides were extracted include of which 10 low-polar ginsenosides, such as ginsenosides F4, Rk3, Rh4, 20S-Rg3, 20R-Rg3 and so on, were identified according to their HPLC retention time and MS/MS data. The results indicated that the low polar ginsenosides were seldom found in RPG. For the exploration of the transformation pattern of the ginsenosides in steam processing, the standards of ginsenosides Re, Rg1, Rb1, Rc, Rb2, Rb3 and Rd were selected and hydrolyzed at a temperature of 120 o C. The results show that these polar ginsenosides can be converted to low-polar ginsenosides such as Rg2, Rg6, F4, Rk3 and Rg5 by hydrolyzing the sugar chains. Keywords Ginsenoside; Hydrolysis; Steamed Panax ginseng; High performance liquid chromatography coupled with electrospray ionization-tandem mass spectrometry(hplc-esi-ms/ms) Article ID 1005-9040(2012)-01-031-06 1 Introduction Panax ginseng, the root of Panax ginseng C. A. Meyer, a famous traditional Chinese medicine(tcm) was used to treat cardiovascular diseases, cancer, different pains, and enhance immune activity [1 3]. Raw ginseng and processed ginseng can both be used in clinical and daily application. In raw Panax ginseng and different processed ginseng, ginsenosides were different in kind and content. Because ginsenosides with different structures have different biological activities [4,5], there are several differences between the raw and processed productions in the treatment of disease due to their different active components. Recently, there zhave been several reports which show that low-polar ginsenosides from processed production have strong biological activities such as radical scavenging, neuroprotective and anti-cancer activities [5 9]. Steaming ginseng will enhance the activity of the raw plant material. Until now, more than 60 ginsenosides have been found in Panax gensing and processed production. The structures of ginsenosides from Panax gensing were summarized by Zhang et al. [7,8]. Among various methods which had been applied to the analysis and identification of ginsenosides from the extract of Panax gensing, high performance liquid chromatography coupled with tandem mass spectrometry(hplc-ms/ms) technique appears to be most favorable and capable due to its high sensitivity, short analysis time and low level of sample consumption [9 12]. Some researches indicate that ginsenosides with large molecule weights can be hydrolyzed to ginsenosides with low molecule weights, such as Rg3, Rk1, Rg5, Rg2, F2 and so on [13 15]. In our previously study [16], the hydrolyses of ginsenoside standards and the crude extracts of ginseng were investigated at different ph values(2.4 11.2) by means of HPLC-ESI-MS (ESI: electrospray ionization), and the results indicate that the ph value influences the hydrolysis process dramatically. So far, there have been some studies of the hydrolysis of other saponins [17 20]. However, to our knowledge, no studies were published on the ginsenosides hydrolysis in steamed Panax gensing at high temperature. In this study, a HPLC-ESI-MS/MS method was developed to identify the ginsenosides in the crude extracts of RPG and SPGHT. The difference of the ginsenosides of RG and SPGHT was investigated by HPLC-ESI-MS/MS. The results of the study are helpful to the process and application of Panax ginseng. 2 Experimental 2.1 Reagents and Materials Acetonitrile and methanol were of HPLC grade from Fisher Chemicals(USA), and other reagents were of analytical grade from Beijing Chemicals(China). Water was purified on a Milli-Q water purification system(millipore, USA). Ginsenoside standards were purchased from Jilin University(China). *Corresponding authors. E-mail: mslab21@ciac.jl.cn; songfr@ciac.jl.cn Received April 1, 2011; accepted June 2, 2011. Supported by the Key Project of Jilin Provincial Science and Technology Department, China(No.20090908), the Project of Changchun Science and Technology Bureau, China(No.2008256) and the Special Cooperative Project for Hi-tech Industrialization of Jilin Provincial Science and Chinese Academy of Sciences, China(No.2009SYHZ0026).

32 CHEM. RES. CHINESE UNIVERSITIES Vol.28 Raw materials of Panax ginseng were cultivated in Fusong, Jilin Province(China). 2.2 Preparation of Samples Firstly, 10 ml of 70%(volume fraction) methanol in aqueous solution was added to 1 g of the powdered sample. The suspension was ultrasonically extracted for 40 min and filtered. This extraction was repeated twice additionally. The combined filtrate was evaporated to dryness at low pressure. Then, the residue was dissolved in 5 ml of 70% methanol in aqueous solution and filtered through a 0.45 μm filter membrane prior to HPLC analysis. 2.3 HPLC-ESI-MS Analysis structures. The preliminary researches of ginsenoside Re in this lab also confirmed that negative ion mode was more sensitive, and could provide real structural information of saponins. The total ion chromatograms of the crude extracts from RPG and SPGHT are shown in Fig.1. In the extract of RPG, the contents of the polar ginsenosides were more than those of the low polar ginsenosides relatively[fig.1(a)]. But the contents of low polar ginsenosides significantly increased in the extract of SPGHT[Fig.1(B)], more than 25 HPLC peaks were detected and identified. An HPLC system consisting of a Waters 2695 pump equipped with a gradient controller, an automatic sample injector(waters 2695 Series). The separation was performed on a Capcell Pak C 18 column(250 mm 4.6 mm i.d., 5 μm, Shiseido, Japan). The column temperature was kept at 35 C. A binary mobile phase consisted of 0.2%(volume fraction) acetic acid in water (A) and acetonitrile(b) was used for the separation. All the solvents were filtered through a 0.45 μm filter prior to use. The flow-rate was kept constantly at 0.5 ml/min for a total run time of 150 min. The system was run with a gradient program: 25% B to 25% B in 2 min, 25% B to 50% B in 90 min, 50% B to 100% B in 60 min. The sample injection volume was 10 μl. A Thermo-Finngan LCQ TM ion-trap mass spectrometer (SanJose, USA) with an electrospray ion source was coupled to the above HPLC system. The mass spectrometer conditions were optimized for ginsenoside Rb1 prior to sample analysis in order to achieve maximum sensitivity. The LCQ TM ion trap mass spectrometer was operated under the following parameters: negative ion mode, the spray voltage 4.5 kv, capillary voltage 10 V, capillary temperature 250 C. High-purity nitrogen(n 2 ) was used as sheath gas and the flow rate was 10 L/min. Full scan of m/z ranging was carried out from m/z 50 to 2000. 2.4 Steaming Ginseng at High Temperature Samples of the powdered raw ginseng were steamed at 120 C in an autoclave(yxq-ls-50sii, China) for 2 h. The powder was then dried in a vacuum oven at about 80 C until constant mass. The powder of SPGHT(1 g) was immersed in 50 ml of methanol and ultrasonically extracted for 1 h at room temperature. The mixture was filtered and the filtrate was evaporated to dryness below 70 C. The residue was then dissolved in 10 ml of methanol and filtered through a 0.45 μm membrane for the HPLC-ESI-MS/MS analysis. 3 Results and Discussion 3.1 HPLC-ESI-MS Analysis of the Crude Extract of RPG and SPGHT Cui et al. [21] reported that mass spectrum data obtained in negative ion mode gave more useful information on saponin Fig.1 Total ion chromatograms(tic) of RPG(A) and SPGHT(B) 3.2 HPLC-ESI-MS/MS Analysis of Polar Ginsenosides in RPG HPLC and MS data provided useful information to the identification of the components in ginseng. A total of 25 HPLC peaks were identified according to the information of retention time(t R ), deprotonated molecules([m H] ), MS/MS fragment ion characteristics compared with the authentic standards reported from the literature [8,16 19] (Table 1). Further identification of the structures of the 25 compounds by HPLC-ESI-MS and MS/MS data are shown in Table 1. The compounds exhibited intense deprotonated molecular ions [M H], [2M H] and their respective adduct ions [M+AcOH H] in negative ion mode, which agreed with the results of Fuzzati et al. [22] and Zhang et al. [23]. The adduct ions [M+AcOH H] were formed from acetic acid used in the mobile phase. The MS spectrum of peak 1(Table 1) is shown in Fig.2(A). Two ions were found in the spectrum, the deprotonated molecular ion [M H] at m/z 945 and its adduct ion [M+AcOH H] at m/z 1005. The MS/MS spectrum of the ion at m/z 945 is shown in Fig.2(B). All the fragment ions in the MS/MS spectrum were produced directly from the parent ion. Five main fragment ions at m/z 799, 783, 765 and 475 were observed in Fig.2(B). The mass differences between the parent ion and the fragment ion at m/z 799 and 783 were 146 and 162, respectively, corresponding to the neutral loss of a deoxyhexose unit and a hexose unit. The loss of the two sugar units indicated that two different terminal residues were in the glycosidic moieties of

No.1 ZHANG Yu-chi et al. 33 the saponin structure, one residue was a deoxyhexose and the other was a hexose. The fragment ion at m/z 765 corresponded Peak No. Identified compound to the loss of a hexose unit and one molecule of water. The fragment ion at m/z 637 was directly produced from the parent Table 1 Compounds identified by HPLC-ESI-MS/MS in negative ion mode * Retention time/min M w MS/MS, m/z 1 Re 11.34 946 945[M H], 1005[M+AcOH H], 799[M Rha H], 783[M Glc H], 765[M Glc H 2 O H], 637[M Rha Glc H] 2 Rg1 11.85 800 799[M H], 637[M Glc H], 475[M 2Glc H] 3 Rf 36.48 800 859[M+AcOH H], 799[M H], 637[M Glc H], 475[M 2Glc H] 4 Rg2 38.97 784 783[M H], 637[M Rha H], 475[M Rha Glc H] 5 Rb1 40.50 1108 1107[M H], 945[M Glc H], 783[M 2Glc H], 621[M 3Glc H], 459[M 4Glc H] 6 Acetylated Rb1 41.59 1150 1107[M Ac H], 945[M Ac Glc H], 783[M Ac 2Glc H], 621[M Ac 3Glc H], 459[M Ac 4Glc H] 7 Rc 43.71 1078 1077[M H], 945[M Ara(f) H], 783[M Ara(f) Glc H], 621[M Ara(f) 2Glc H], 459[M Ara(f) 3Glc H] 8 Rb2 44.17 1078 1077[M H], 945[M Ara(p) H], 783[M Ara(p) Glc H], 621[M-Ara(p) 2Glc H], 459[M Ara(p) 3Glc H] 9 Rd 48.61 946 945[M H], 783[M Glc H], 621[M 2Glc H], 459[M 3Glc H] 10 Rb3 49.02 1078 1077[M H], 945[M Ara(p) H], 783[M Xyl Glc H], 621[M Xyl 2Glc H], 459[M Xyl 3Glc H] 11 Ro 54.32 956 955[M H], 1911[2M H] 12 Rs1 57.34 1120 1119[M H], 1179[M+AcOH H], 945[M Ac Ara(p) H], 783[M Ac Ara(p) Glc H], 621[M Ac Ara(p) 2Glc H], 459[M Ac Ara(p) 3Glc H] 13 Rs2 58.61 1120 1119[M H], 1179[M+AcOH H], 945[M Ac Ara(p) H], 783[M Ac Ara(p) Glc H], 621[M Ac Ara(p) 2Glc H], 459[M Ac Ara(p) 3Glc H] 14 Rg6 70.89 766 765[M H], 825[M+AcOH H], 619[M Rha H], 457[M Rha Glc H] 15 F4 77.85 766 765[M H], 825[M+AcOH H], 619[M Rha H], 457[M Rha Glc H] 16 Rk3 81.18 620 619[M H], 679[M+AcOH H], 457[M Glc H] 17 Rh4 90.48 620 619[M H], 679[M+AcOH H], 457[M Glc H] 18 20S-Rg3 100.48 784 783[M H], 843[M+AcOH H], 1567[2M H], 621[M Glc H], 459[M 2Glc H] 19 20R-Rg3 103.27 784 783[M H], 843[M+AcOH H], 1567[2M H], 621[M Glc H], 459[M 2Glc H] 20 20S-Rs3 117.51 826 825[M H], 783[M Ac H], 621[M Ac Glc H], 459[M Ac Glc H] 21 20R-Rs3 119.10 826 825[M H], 783[M Ac H], 621[M Ac Glc H], 459[M Ac Glc H] 22 Rk1 124.44 766 765[M H], 825[M+AcOH H], 603[M Glc H], 441[M 2Glc H] 23 Rg5 126.16 766 765[M H], 825[M+AcOH H], 603[M Glc H], 441[M 2Glc H] 24 Rs5 134.87 808 807[M H], 867[M+AcOH H], 1615[2M H], 765[M Ac H], 602[M Ac Glc H], 441[M Ac 2Glc H] 25 Rs4 136.12 808 807[M H], 867[M+AcOH H], 1615[2M H], 765[M Ac H], 602[M Ac Glc H], 441[M Ac 2Glc H] * Glc: β-d-glucose; Xyl: β-d-xylose; Rha: α-l-rhamnose; Ara(f): α-l-arabinose(furanose); Ara(p): α-l-arabinose(pyranose); Ac: acetyl. Fig.2 HPLC-ESI-MS spectrum of peak 1 obtained in negative ion mode(a) and MS/MS spectrum of the parent ion m/z 945(B) ion at m/z 945, corresponding to the loss of a disaccharide consisting of a deoxyhexose and a hexose, which indicated that the hexose in the disaccharide was directly attached to the saponin aglycone. Similarly, the fragment ion at m/z 475 was also a daughter ion produced from the parent ion at m/z 945, corresponding to the loss of all three sugar units. Based on the MS/MS data and HPLC retention time compared with the authentic standard, the compound represented by peak 1 was identified as ginsenoside Re [24]. The deprotonated molecular ion [M H] of peak 2(Table 1) was at m/z 799, which confirmed the molecular weight to be 800. Two main fragment ions at m/z 637 and 475 were found in its MS/MS spectrum, corresponding to the neutral losses of one hexose molecule and two hexose molecules, respectively. The compound represented by peak 2 was therefore identified as ginsenoside Rg1 by comparing its retention time and mass spectral data with those of the authentic standard. The MS spectrum of peak 3(Table 1) also gave the deprotonated molecular ion [M H] at m/z 799 and its adduct ion [M+AcOH H] at m/z 859. The fragment ions at m/z 637 and 475 could be detected in the MS/MS spectrum of ion m/z 799. The compound represented by peak 3 was therefore identified

34 CHEM. RES. CHINESE UNIVERSITIES Vol.28 as ginsenoside Rf by comparing the retention time and MS/MS cleavage pattern with those of the authentic standard. The deprotonated molecular ion [M H] of peak 4 at m/z 783 gave similar fragment ions of [M deoxyhexose H] and [M deoxyhexose hexose H] at m/z 637 and 475 in MS/MS spectrum. The compound corresponding to peak 4 was therefore identified as ginsenoside Rg2 by comparing the retention time and MS data with those of the authentic standard. With the same method, we confirmed the compounds represented from peak 5 to peak 13 as ginsenoside Rb1, acetylated ginsenosides Rb1, Rc, Rb2, Rd, Rb3, Ro, Rs1 and Rs2, respectively. Peaks 7, 8 and 10 had the same deprotonated molecular ion [M H] at m/z 1077, and these three compounds were identified as ginsenoside Rc, Rb2 and Rb3, respectively. 3.3 HPLC-ESI-MS/MS Analysis of Low Polar Ginsenosides in SPGHT From Fig.1, it can be seen that the mass spectrum relative abundances of ginsenosides from peak 14 to peak 25 in SPGHT are much higher than those in RPG. These ginsenosides with low polarity were called as rare ginsenosides, since they were seldom found in RPG. In this study, most of the polar ginsenosides were hydrolyzed to low polar ginsenosides by steaming at a high temperature. Therefore, these low content ginsenosides could be obtained. The HPLC peaks 14, 15, 22 and 23 showed the same deprotonated molecular ion [M H] at m/z 765 in MS spectrum [Fig.3(A)], corresponded to the ginsenosides Rg5, Rg6, Rk1 and F4. Two main fragment ions at m/z 603 and m/z 441 were observed in MS/MS spectra of the parent ion peaks 22 and 23[Fig.3(B)], the fragment ion at m/z 603 corresponded to the loss of a hexose unit, the fragment ion at m/z 441 corresponded to the loss of two hexose units. So ginsenosides Rg6 and F4 were excluded from peaks 22 and 23 because of the only one hexose in their structures. The MS fragment pathways of ginsenosides Rg5 and Rk1 were similar. Peak 23 was identified as ginsenoside Rg5 by the standard while peak 22 corresponded to ginsenoside Rk1. The difference between ginsenosides Rg5 and Rk1 was the double bond at the 20th site, that is, the 20th site double bond of ginsenoside Rg5 was between C-20 and C-22, while the 20th site double bond of Rk1 was between C-20 and C-21. The polarity of ginsenoside Rg5 is less than that of ginsenoside Rk1 according to their retention order on C 18 column. With the same inference, peaks 14 and 15 have the same deprotonated molecular ion [M H] at m/z 765 and its adduct ion [M+AcOH H] at m/z 825[Fig.4(A)]. Fragment ions of [M deoxyhexose H] at m/z 619 and [M deoxyhexose hexose H] at m/z 457 were detected in the MS/MS spectra [Fig.4(B)]. The compounds corresponding to peaks 14 and 15 were identified as ginsenosides F4 and Rg6 by comparing the retention time and MS/MS data. The polarity of ginsenoside Rg6 is also less than that of ginsenoside F4. Based on the analysis method above mentioned, the retention time of ginsenoside Rg5 is longer than that of ginsenoside Rg6 in reverse chromatography because of less hydroxyl at the aglycone skeleton of Rg5. Similarly, peaks 16 21, 24 and 25 were identified as ginsenosides Rk3, Rh4, 20R-Rg3, 20S-Rg3, 20R-Rs3, 20S-Rs3, Rs5 and Rs4, respectively. Fig.4 HPLC-ESI-MS spectrum of peaks 14 and 15 obtained in the negative ion mode(a) and MS/MS spectrum of the parent ion m/z 765(B) 3.4 Hydrolysis Process of Ginsenosides Fig.3 HPLC-ESI-MS spectrum of peaks 22 and 23 obtained in negative ion mode(a) and MS/MS spectrum of the parent ion m/z 765(B) The above results indicate that the polar ginsenosides such as acetylated ginsenoside Rb1, ginsenosides Re, Rg1, Rc, Rb2, Rb3 and Rd could hydrolyze to form low polar ginsenosides, such as ginsenosides Rg6, F4, Rk3, Rh4, 20S-Rg3, 20R-Rg3, Rk1, Rg5, Rs4 and Rs5. For the exploration of transform mechanisms of ginsenosides in steaming processing, ginsenosides standards Re, Rg1, Rb1, Rc, Rb2, Rb3, Rd(2 mg of each standard) were steamed under the same conditions as those for processing SPGHT.

No.1 ZHANG Yu-chi et al. 35 Then the products of reaction were analyzed by ESI-MS/MS. Ginsenoside Re hydrolyzed to ginsenosides Rg2, Rg6 and F4[Fig.5(A)], while ginsenoside Rg1 hydrolyzed to ginsenosides Rk3 and Rh4[Fig.5(B)]. Protopanaxdiol ginsenosides such as Rb1, Rb2, Rc and Rd all hydrolyzed to ginsenoside Rg3. At the same time, ginsenoside Rg3 was dehydrated one molecular H 2 O at the position of C-20/22(20S) or C-20/21 (20R) and converted into ginsenosides Rk1 and Rg5[Fig.5(C)]. The structure transformation processes of these ginsenosides are summarized in Fig.6. The hydrolyzed results show that polar ginsenosides could convert to low polar ginsenosides by hydrolyzing sugar chains at a high temperature. The result is consistent with the detection result of the extract of SPGHT. Fig.5 Mass spectra of less polar ginsenosides hydrolyzed from standards (A) Mass spectrum of ginsenosides Rg2, Rg6 and F4 hydrolyzed by ginsenoside Re; (B) mass spectrum of ginsenosides Rk3 and Rh4 hydrolyzed by ginsenoside Rg1; (C) mass spectrum of ginsenosides Rg3, Rk1 and Rg5 hydrolyzed by ginsenosides Rb1, Rb2, Rc and Rd. Fig.6 Hydrolysis processes from polar ginsenosides to less polar ginsenosides (A) Hydrolysis process of ginsenoside Re to ginsenosides Rg2, F4 and Rg6; (B) hydrolysis process of ginsenosides Rb1, Rc, Rb2, Rb3 and Rd to ginsenosides Rg3, Rk1 and Rg5. 4 Conclusions In this study, the difference of ginsenosides of RPG and SPGHT was studied by HPLC-ESI-MS/MS method. Among the 25 ginsenosides extracted 10 low-polar ginsenosides, such as Rg6, F4, Rk3, Rh4, 20S-Rg3, 20R-Rg3, Rk1 and Rg5 were identified. The methods and results of the structural identification of ginsenosides in the extract of RPG and SPGHT are useful. The results show that polar ginsenosides can hydrolyze to low-polar ginsenosides during steaming at high temperature. The hydrolysis of ginsenoside standards at a high temperature confirmed the conversion of ginsenosides in SPGHT. The conclusion of the study is helpful to the process and application of Panax ginseng. References [1] Ni X. Z., Wang B. Q., Zhi Y., Wei N. N., Zhang X., Li S. S., Tai G. H., Zhou Y. F., Zhao J. M., Chem. Res. Chinese Universities, 2010, 26(2), 230 [2] Wang Y., Gao Q. P., Li G. R., Chen Y. H., Luo H. M, Gao Y., Jiang R. Z., Chem. Res. Chinese Universities, 2011, 27(1), 104 [3] Liao B., Newmark H., Zhou R., Exp. Neurol., 2002, 173, 224 [4] Li L., Tsao R., Dou J. P., Song F. R., Liu Z. Q., Liu S. Y., Anal. Chim. Acta, 2005, 536, 21 [5] Schlag E. M., McIntosh M. S., Phytochemistry, 2006, 67, 1510 [6] Ha Y. W., Lim S. S., Ha I. J., Na Y. C., Seo J. J., Shin H., Son S. H., Kim Y. S., J. Chromatogr. A, 2007, 1151, 37 [7] Zhang H. J., Wu Y. J., Cheng Y. Y., J. Pharm. Biomed. Anal., 2003, 31, 175

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