Scholarly article on topic 'A comparative study on chemical composition of total saponins extracted from fermented and white ginseng under the effect of macrophage phagocytotic function'

A comparative study on chemical composition of total saponins extracted from fermented and white ginseng under the effect of macrophage phagocytotic function Academic research paper on "Chemical sciences"

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Abstract of research paper on Chemical sciences, author of scientific article — Dan Xiao, Xiu Yang, Hao Yue, Xiuli Sun, Huanxi Zhao, et al.

Abstract In this study, white ginseng was used as the raw material, which was fermented with Paecilomyces hepiali through solid culture medium, to produce ginsenosides with modified chemical composition. The characteristic chemical markers of the products thus produced were investigated using rapid resolution liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (RRLC–QTOF–MS). Chemical profiling data were obtained, which were then subjected to multivariate statistical analysis for the systematic comparison of active ingredients in white ginseng and fermented ginseng to understand the beneficial properties of ginsenoside metabolites. In addition, the effects of these components on biological activity were investigated to understand the improvements in the phagocytic function of macrophages in zebrafish. According to the established RRLC–QTOF–MS chemical profiling, the contents in ginsenosides of high molecular weight, especially malonylated protopanaxadiol ginsenosides, were slightly reduced due to the fermentation, which were hydrolyzed into rare and minor ginsenosides. Moreover, the facilitation of macrophage phagocytic function in zebrafish following treatment with different ginseng extracts confirmed that the fermented ginseng is superior to white ginseng. Our results prove that there is a profound change in chemical constituents of ginsenosides during the fermentation process, which has a significant effect on the biological activity of these compounds.

Academic research paper on topic "A comparative study on chemical composition of total saponins extracted from fermented and white ginseng under the effect of macrophage phagocytotic function"

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The comparative study on chemical composition of the total saponins extraction from fermented and white ginseng under the effect of macrophages phagocytotic function

Dan Xiao, Xiu Yang, Yue Hao, Xiuli Sun, Huanxi Zhao, ShuYing liu

PII: S1226-8453(16)30321-9

DOI: 10.1016/j.jgr.2017.03.009

Reference: JGR 268

To appear in: Journal of Ginseng Research

Received Date: 23 December 2016 Revised Date: 21 March 2017 Accepted Date: 23 March 2017

Please cite this article as: Xiao D, Yang X, Hao Y, Sun X, Zhao H, liu S, The comparative study on chemical composition of the total saponins extraction from fermented and white ginseng under the effect of macrophages phagocytotic function, Journal of Ginseng Research (2017), doi: 10.1016/ j.jgr.2017.03.009.

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1 Title: The comparative study on chemical composition of the total

2 saponins extraction from fermented and white ginseng under the

3 effect of macrophages phagocytotic function

4 The Running Title: Analyzed chemistry and immunity activity of

5 fermented and white ginseng

12 3 3 3 3 13*

6 Dan Xiao ' , Xiu Yang , HaoYue ,Xiuli Sun ,Huanxi Zhao , ShuYing liu '

7 1 ChangChun University of Technology, China;

8 2 Changchun Centre of Mass Spectrometry, Changchun Institute of Applied Chemistry, Chinese

9 Academy of Sciences, China;

10 3Jilin Ginseng Academy, Chang Chun University Of Chinese Medicine,China.

11 Email: syliu@ciac.jl.cn

20 21 22

29 ABSTRACT

30 In this study, we adopt the white ginseng as raw material, which was processed as fermented

31 ginseng products with Paecilomyces hepiali through solid culture. The characteristic chemical

32 markers of them were investigated by RRLC-Q-TOF-MS (rapid resolution liquid

33 chromatography coupled with quadrupole time -of-flight mass spectromtry) method combined

34 with multivariate statistical analysis.And the implications of these components on biological

35 activity were investigated for the improvement of the phagocytic function of macrophages in

36 Zebrafish.According to the established RRLC-QTOF-MS chemical profiling, the contents in

37 ginsenosides of high molecular weight especially malonylated protopanaxadiol ginsenosides

38 were slightly decrease due to the fermentation process, which were hydrolyzed into rare and

39 minor ginsenosides. And the facilitation on macrophage phagocytic function in zebra fish of

40 different ginseng extract was proved that fermented ginseng is superior to white ginseng.The

41 results proved that profound change of ginsenosides chemical constituents would take place

42 during the fermention process, which has a significant effect on the biological activity.

43 Keywords: Ginseng; Ginsenosides; Chemical variation; content; fermented.

44 1. Introduction

45 Ginseng is a perennial herb that contains various bioactive substances, belonging to the genus

46 panax of the family Araliaceae [1]. It is considered to be an adaptogenic herb which is used to

47 enhance immunity, radioresistance as well as the capability to cope with tumor and many

48 other diseases [2-3]. Under the most circumstances, ginsenosides is treated as the main active

49 ingredient to be used generally during the treatment by oral administration [4]. Whereas poor

50 water solubility and low bioavailability are having impacts on the pharmacological action

51 tremendously [5]. Hence, scientists have done some researches on finding better compound

52 with activity and bioavailability by preceding the structural modifications of ginsenosides,

53 during which we employ the method of modified ginsenosides particularly, and that, for

54 instance, would affect the composition of ginsenosides in every relevant respect, namely,

55 chemical hydrolysis, biological conversion, hot working, semisynthesis, etc [6-8]. Here is an

56 example, the hot processing of red ginseng to produce the special ingredient of ginsenosides

57 (Rg3, Rh2, CK) implies a large development value and a bright future for widely clinical

application which involves the functions in reducing blood fat, anti-tumour, enhancing immune system as well as protecting heart, liver and blood vessel [9]. Fermented ginseng leavened by microbial actions has become a hot spot recently, Moreover, it is employed extensively in functional food like distiller's yeast fermented ginseng wine, probiotics fermented ginseng beverage, etc [10-11]. Fermented ginseng is usually processed with several methods as follows, mild acid hydrolysis, microbial conversion, and enzyme conversion, through which major ginsenosides would be transformed into minor ones [12]. It has been proved in many studies that the utilization of multiple probiotics in fermented ginseng has changed the chemical compositions of rare ginsenosides through biological transformation. Rae SH et al. [13] confirmed that the fermented Korean ginseng with mushroom mycelium breeded by solid culture contained significantly more crude fat (4.66-12.02%) than Korean ginseng does (WG; 1.61%). Nevertheless, the effects of fermented ginseng on mitogenic activity were much greater than those of white ginseng .

Additionally, some researches has indicated that the concentrations of total ginsenosides in red ginseng leavened by Phellinns linteus, Cordyceps militaris, and Grifola frondosa were particularly at higher levels in comparison with those in red ginseng [14-15]. However, neither the effect brought by the biotransformation of ginsenosides involved in the fermented ginseng nor the difference between fermented ginseng and white ginseng has been specified. Accordingly, it is crucial to identify the chemical components presented in ginseng fermentation through different forms of microorganism as well as take the differences between fermented ginseng and white ginseng in respect of "chemical markers" components into full consideration so as to provide the chemical basis for their various pharmacological activities.Ginsenosides has an important pharmacological activity by enhancing immunity to against potential pathogens or harmful bacteria [16]. Most of the current methods were confined to the determination of a few marker constituents and there is no systematical studies on the immunity effects interfere with ginsenosides content of P.ginseng processed by different method, especially in fermented ginseng and white ginseng.

In recent years, the model of zebra fish has been drawn to the behavior, immunology, toxicology and other fields [17]. Additionally, it has been one of the best animal models to study the immune function of medicine on macrophage. Because macrophages are important

cells in immunological system, having many functions such as presenting antigen, eliminating foreign body and secreting various cytokines, etc [18].It play an important role on specific cellular immunity and the non-specific immunity. Therefore, in order to keep evaluating the potential of components as properties influences on enhance immunity, screened for the improvement of the phagocytic function of macrophages in Zebrafish. In this study, we adopt the white ginseng as raw material, which was processed as fermented ginseng products with Paecilomyces hepiali through solid culture. A RRLC-Q-TOF-MS method has been developing for the identification of ginsenosides, chemical profiling of white ginseng and fermented ginseng in virtue of the establishment of chemical profiling combined with multivariate statistical analysis, is used for the systematic comparison of active ingredients in white ginseng and the fermented in order to utilize the beneficial properties of ginsenoside metabolites. Therefore, the former steps would provide a research basis for further study as well as promote the development of ginseng product with the emphasis on two different products interfere with the improvement of the phagocytic function of macrophages in Zebrafish.

2 Experimental

2.1 Chemicals and reagents

The ginsenosides standards, among which, the eighteen of them were obtained from Jilin University, Changchun, China listed as follows, the Rb1, Rb2, Rb3, Rc, Re, Rg1, Rf, Rd, Ro, 20(S)-Rg3, 20(R)-Rg3, 20(S)-Rg2, 20(R)-Rg2,20(S)-Rh1, 20(R)-Rh1, Rh2, Rk1, and Rg5. All the other chemicals are of reagent grade and obtained from local suppliers. The standard stock solutions of ginsenosides were prepared independently by dissolving 1 mg of the standard into 5mL aqueous where the purification of methanol was about 80% (methanol:water, 80:20, v/v) to achieve a concentration of 0.2 mg/mL. The preparation of mixed standard solutions was prepared through combining the aliquots of each set of individual stock solutions and diluting them to the appropriate concentration.

2.2. Plant material and sample preparation

All of the root samples from Ji'an (Jilin province) were harvested in fall and collected at the fifth year of their growth phase. White ginseng was dried at 42 °C to reach at a constant

117 weight. The strains of Paecilomyces hepiali maintained on potato dextrose agar slants were

118 received from China Center of Industrial Culture Collection (CICC), and the seed liquid was

119 cultivated based on previous studies [19]. The optimal conditions for fermentation, presented

120 as follows, were created through single factors and orthogonal experiments. The optimal

121 acetic fermentation was set with ginseng power (45 mesh) at 10%, the ratio of solid to liquid

122 is 2 to 3. Paecilomyces hepiali was achieved when the seed liquid was cultivated for 14 hours

123 and the inoculation amount was 10% , and the culture temperature is at around 26 °C. The first

124 batch of samples was collected and analyzed after 24 hours of fermentation, and the process

125 was to be proceeded up to 96 hours after every 24 hour's collection under the condition of a

126 natural pH value. Sequently, when fermentation finished, the culture was dried.

127 Samples for RRLC-Q-TOF-MS analysis of white ginseng and fermented ginseng were

128 grinded into powder. Each replicate contained 10 ginseng individuals. Every1.0 g powdered

129 ginseng sample was weighed accurately and immersed in aqueous methanol at a concentration

130 of 80% (methanol:water, 80:20, v/v) to be extracted in an ultrasonic bath at 20C for 30 min

131 and the extraction was to be repeated three times by using fresh aliquots of the solvent. When

132 the combination of the three aliquots was achieved, the solution would be centrifuged at

133 13,000 X g for 5 min, after which the supernatant would passed through a 0.45^m filter and

134 the process would be subsequently followed by an analysis through RRLC-Q-TOF-MS. All of

135 the solutions mentioned above were kept at 4C for storage and restored at room temperature

136 before utilization.

137 2.3 Detection conditions of RRLC-Q-TOF-MS

138 The extracts have been qualified through the agilent 1260 LC system using a Thermo

139 Scientific Hypersil Gold C18 column (3.0* 100 mm x 1.8 ^m), and the mobile phase

140 consisted of water with 0.1% formic acid (A) and 100% acetonitrile (B) were to be carried

141 through a program as: 0.00-3min, 25-30% B;3-5min, 30-36% B; 5-9min,36-40% B;

142 9-12min, 40-45% B; 12-13min, 45-50% B; 13-14min, 50-65% B; 14-15min 65-90% B as

143 well as the flow rate at 0.3mL/min and the volume of injected sample of 10 ^L. The column

144 and sample managers were respectively maintained at 30 C, and the peak was monitored at

145 203 nm.

The mass spectrometer was operated in the negative ion mode and the total ion chromatogram (TIC) mode. In accordance with the requirement of MS analysis conditions as: desolvation gas flow=600L/h, desolvation temperature=250C, capillary voltage=2500V, cone voltage=35 V and scan range=m/z 100-1500, the fragment ions were obtained using collision energy of 35% through both MS2 and MS3 experiments.

2.4 Multivariate statistical analysis

With the Agilent Mass Hunter Workstation software-Qualitative Analysis and Q-TOF Quantitative Analysis (Version B.04.00, Build 4.0.479.5, Service Pack 3, and Agilent Technologies, Inc. 2011), all the HPLC-QTOF/MS/MS data were fully recorded to assess the quality comprehensively and explore the discriminated variables of fermented ginseng and white ginseng. Peak integration was calculated automatically and supplemented with manual operation. Components of different samples appeared as the same when they showed similar retention times with a tolerance of 0.2 min, accurate mass weights with the tolerance of 0.05 Da, and ion fragments. No specific mass or adduct was excluded except reagent. The ion intensity would give sample codes aligned in a table and would be corrected manually, and the table was imported into SIMCA-P Software (Version13.0.3) afterward. Peak intensities were treated as X variables, while the sums of the peak intensities were taken as Y variables. All the variables were normalized before principle component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA).

2.5 The ascertainment of maximum tolerable concentration (MTC) of drug

Place the sample of zebra fish in six pore plates with the concentration of 62.5, 125, 250, 500 and 1000 ^g/mL ginseng extract respectively given to each. Meanwhile, set the control group in which the zebra fish was to be treated with water for fish. Each control group would be allotted 30 samples. The zebra fish was observed daily and the dead ones were removed each day as well. Sequently, the number and toxicity of zebra fish in each control group were statistically analyzed after disposal to determine the maximum tolerable concentration (MTC) of ginseng extract.

174 2.6The establishment of animal models

175 Inject nano-activated carbon (PM2.5) in concentration of 2.3 mg/mL into the veins of 2 dpf

176 zebra fish (equivalent to human I.V.) with 10nLfor each, which means to establish zebra fish

177 PM 2.5 phagocytosis model at the dose of 23ng per zebra fish.

178 2.7The facilitation on macrophage phagocytic function from

179 different ginseng extract

180 Confirm the concentration of phagocytosis function test, according to the experimental results

181 of maximum tolerance concentration, was set at the medium value (166.7 ^g/mL) with the

182 normal control group and the modeling control group adapted at the same time. After all these

183 procedure, the zebra fish would be stained with neutral red solution, sequently, the

184 macrophage phagocytosis facilitative rate determined by the calculation of the number of

185 phagocytosed macrophages (N) would be used to evaluate the effects of different ginseng

186 extracts on the macrophages.

187 3 Results and Discussion

188 3.1RRLC-QTOF-MS analysis of fermented ginseng processing

189 product

190 All extracts from the samples were obtained with RRLC-Q/TOF/MS method. The typical

191 profiles of different kinds of ginseng are shown in Figure.1, according to which, the

192 fermented ginseng contains more diverse ginsenoside compounds than the white ginseng does,

193 with 40 ginsenosides detected in fermented ginseng approximately. Among the ginsenosides

194 founded in fermented ginseng, Eighteen components were unambiguously authenticated as

195 ginsenosides Rb1, Rb2, Rb3, Rc, Re, Rg1, Rf, Rd, Ro, 20(S)-Rg3, 20(R)-Rg3, 20(S)-Rg2,

196 20(R)-Rg2,20(S)-Rh1, 20(R)-Rh1, Rh2, Rk1, and Rg5 by comparing the retention times, m/z

197 values and fragment ions with those of the reference compounds, and twenty-four constituents

198 were tentatively identified by analyzing specific MS/MS fragment ions, accurate mass, and

199 isotopic ratio patterns. Additionally, in MS/MS spectra, the glycoside bond of saponin

200 components was breaking down through Collision Induced Dissociation (CID) technology to

201 generate characteristic ions for further confirmation of the molecular structure. Figure 2

shows the MS/MS spectra of representative types of ginsenoside. For example, Peak 1 under first mass spectrometry data showed the result of analysis as;[M-H]- m/z for 799.4799, adduct ions [M+CH3COO]- m/z for 845.6031, its characteristic of MS/ MS containing characteristic fragment ion m/z 475.3787,which indicated that this chemical compound belonged to the PPT group. The corresponding fragment ion originated from the break of the glycoside bond involves,[M-H-Glc]-, [M-H-GlcGlc]-, [M-H-GlcGlc-C6H 12]-, [GlcGlc-H2O-H]-, [Glc-H2O]-, [2.5A1p], m/z 391.2866 for [M-H-XlyGlc-C6H 12]-, and m/z 161.0354 for[Glc-H2O]-, with the Figure 2 A from which the result showed. Its elementary composition (C42H72O14) has depended on the mass lynx 4.2 software, enrolling at 4.4pm. Built on the MS fragment pathways and structural composition of saponin, it could be preliminaries inferred that Peak 1 was 20(Glc) -Rf. We could also deduce that Peak 2 was NotoR1 to form characteristic fragment ion such as [2GlcXly-H]- (m/z 475.3588), [Glc-H]-(m/z 179.0504) and others (see Figure 2B). In this processed ginseng experiment, the variational properties of ginsenosides in processing fermented ginseng could be identified as markers by LC-MS scan mode and selective ion mode.

3.2RRLC-Q-TOF-MS-based chemical profiling coupled with Multivariate statistical analysis of ginseng

The related data were to be processed by unsupervised PCA and supervised OPLS-DA with SIMCA-P Software to study the differences between fermented ginseng and white ginseng, besides, to further explore their potential characteristic markers. From the result of analytical methods, we could observe that white ginseng and fermented ginseng were clustered into two groups (see Figure 3), which indicated that the metabolite profiles of the two materials were distinct, and different samples of each material were consistent with each other. The following S-plot generated by OPLS-DA presented in no S-form indicated that the synactic nuliticomponents existed in the chemical differences between the two groups rather than only a few characteristic constitunents (see Figure 4). Some components, namely, malonyl-Rb1/Rc (peak 13), malonyl-Rc (peak 14), malonyl-Rb2/Rb3 (peak 17 and 18), malonyl-Rd isomer (peak 21and 22), Ra1/Ra2 (peak 11) and malonyl-Rg1 (peak 5) were all presented in an abundant way in white ginseng, whereas the same components could hardly be found in

231 fermented ones. At the same time, some components like ginsenosides F1 isomer (peak 6),

232 20(S)-Rh1/20(R)-Rh1isomer (peak 9 and 10), Rg9 (peak 21), Rs2/Rg8 (peak 22), Rg6 isomer

233 (peak 23), Rk3 isomer (peak 24), 20 (S)-Rg3/20(R)-Rg3 (peaks 25and26), Rh4 (peak27), Rs3

234 (peak28), Rk1 (peak29), Rg5 (peak30) and the other ten components (Rs7,F2, 20(S)-Rh2,

235 20(R)-Rh2/CK, Rs5, Rs4, Rk2, Rh3, Ra7/Ra8/Ra9 and R1) were prominent in the fermented

236 ginsengs rather than in the white ones. The concentrations of these core components were

237 obtained by calculating the peak area ratios of individual ginsenoside and were totally relied

238 on the RRLC-Q-TOF-MS chemical profiling data of white ginseng and fermented ginseng

239 mentioned above.

240 As it was shown in Table 1, the content of 4 protopanaxadiol ginsenosides (Rb1, Rc ,Rb2and

241 Rb3) in fermented ginseng were higher than those in white ginseng on average.Besides, as

242 same as the average content of 5 malonylated protopanaxadiol ginsenosides (mRb1, mRc,

243 mRb2/mRb3, and mRd), the content of protopanaxatriol ginsenosides (Rg1,Re and Rf) were

244 much lower than those in white ginseng on average. It seems that malonylated protopanaxadi-

245 -ol ginsenosides (mRb1, mRc, mRb2, and mRd) which is abundant in white ginseng are more

246 likely to be transformed into small molecular weight and rare ginsenosides. Such as a few rare

247 ginsenosides (Rg8,Rg9,Rh3 et al) was only found in fermented ginseng.In this study we also

248 observed. The changes in ginsenosides during fermentation process was similar with red

249 ginseng processing. For example, Hydrolysis of glycosidic bond at C-(20) and isomerous

250 reaction of C-(20) were the main reaction of dammarane type ginsenosides. Transformation

251 way of PPD group maybe that ginsenosides Rb1,Rc, Rb2 hydrolase could only hydrolyze the

252 glycosidic bond at the C-20 position into ginsenoside-Rd. But may further hydrolysis ginsen-

253 -oside-Rd to Rg3, at same time Rg3 removed C-(20) hydroxyl group to form double bond to

254 form ginsenoside-Rk1 or Rg5.But there are special changes in our study,such as the content of

255 ginsenoside Rd in fermented ginseng was 1.5 times higher than white ginseng.All literatures

256 basically states that the ginsenoside Rd in red ginseng processing was reduced [20].The

257 transformation mechanism need further exploration and research.

258 3.3 Comparison of the facilitation on macrophage phagocytic

259 function from different ginseng extract

In order to keeply evaluate the potential of these components as properties influences on enhancing immunity, the current study screened the facilitation on macrophage phagocytic function from different ginseng extract. According to the model characteristics, the Zebrafish brain is used as typified part to observe the function of macrophage phagocytosis.To fulfill this, total ginsenosides was calculated as the sum of ginsenoside fractions. No zebra fish died in different control groups at the concentrations of 62.5, 125, 250 and 500 ^g/mL, whereas all the zebra fish died in the group where the set point of concentration was 1000 ^g/mL. Hence, the MTC for zebra fish from the ginseng extract was determined to be 500 ^g/mLbased on the results mentioned above.

The numbers of phagocytosed macrophages for activated carbon nano-particles are 13, 15, and 20 in the control group with the concentration of white ginseng at 55.6, 166.7 and 500 ^g/mL, and compared the aforesaid numeric with those in the modeling control group (p <0.001), the rates of facilitated function for phagocytosis of zebra fish macrophages are 85.7%, 114.3% and 185.7%, respectively. The numbers of phagocytosed macrophages for activated carbon nano-particles are 14, 18, and 23 in the control group with the concentration of fermented ginseng at 55.6, 115.7 and 500 ^g/mL, which describes a contrasting example with those in modeling control group (p< 0.001). Furthermore, the rates of facilitated function for phagocytosis of zebra fish macrophages are 100%, 157.1% and 228.7%, which is in contrast to those of the low concentration group (55.6 ^g/mL, p<0.001) where the white ginseng is adopted as material (Table 2). Besides, the differences exist significantly in both high (500 ^g/mL) and medium concentration groups (166.7 ^g/mL) (Figure 5).Different kinds of ginseng extract could effectively facilitate the macrophage phagocytosis of zebra fish and shows a concentration-correlation, however, the facilitated function of fermented ginseng is better than that of the white ginseng.It is further confirmed that the rare ginsenoside generated during the fermented process help make the phagocytosis of macrophage in fermented ginseng more efficacious compared with that in white ginseng.

There has actually been a lot of research have shown that Rg3 strengthens the body's response restrain cancer cell by making the immune system more effective.Rg3 was gradually used for adjunctive therapy and provides a new pathway for the therapy of cancer.This study has been proven that the added content of Rg3 hydrolyzed through fermentation.Therefore, this study

290 will provide a new idea of Rg3 production by microbial fermentation was brought forward.

291 These results have important instruction meaning and reference value of medicinal uses for

292 fermented ginseng.

293 4. Conclusions

294 In this study, we intended to confirm the fermentation process with Paecilomyces hepiali

295 obtained by solid culture, and measure ginsenosides employed RRLC-Q-TOF-MS chemical

296 profiling of white ginseng and fermented ginseng without microbial strains. As a consequence,

297 40 ginsenosides were identified and the specific chemical components of fermented ginseng

298 were elucidated approximately. The chemical compositions as well as the ratio of the

299 component of ginsenosides was more various in the fermented ginseng compared to those in

300 the white ginseng, which would considered the total amount of saponins in ginseng has

301 generated rare and small molecular weight ginsenosides through unique biological

302 fermentation. The result of comparison of the facilitation on macrophage phagocytic function

303 in zebra fish between different ginseng extract was showed that fermented ginseng is superior

304 to white ginseng.Fermented processing will become a hot point, which will open a new field

305 of ginseng development in the future as well.

306 Acknowledgements

307 This research work was supported by the National Ministry of Science and Technology of

308 China (Grant No. 2011BAI03B00); the National Science Foundation of China (Grant No.

309 21175127 and21475012) and the National Science Foundation of Jilin province education

310 department of China (Grant No. 201500005000382).

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I,20(glc)-Rf 2.notoRl 3Rgl

4 . Re

5.m-Rgt

6. m-Rc

8.F3/notoR2

9.1I1RT 10.Rg2

II.Ral/Ra2

i m c(m in)

12.Rbl

13.mRbl/Rc

14.mRc

15.Rb2

16.Rb3

17.mRb2

18. mRbJ

20.mRd

21.mRd

□J u.-r

« 0.2

I I . I I I . I . I > I . I . I . I I I I I—

10 15 20 25 30 35 40 45 50 55 60 65

Time(mln)

1.20(glc)Rf 21.Rs9

2.NuLu R1 22. Rs2/Rg8

3.Rgl 23.Rg6

4 .Re 24.Rk3

S.mRc 25.2D(S) Rg3

6.F1 26.20(R)-Rg3

7.Rf 27.24.Rt4

8.F3/notoR2 2K.hls3

».2«(S)-Rhl 29.RR1

10.20(R)-Rli2 30.Rg5

ll.Rgl 31.Rs7

12.Ral/R»2 32. F2

13.Rbl 33.20(S)-Rh2

14.Rc 34.20(R)-Rh2/CK

15.Rb2 3S.Rs5

16.Rb3 36.Rs4

17.mRb2/mRb3 37.Rk2

lS.Rd 3S.HH3

ly.mRd 39.Ra7/Ra8/Ra9

20.2 \IaRd 40. R1

Figure 1 RRLC-Q-TOF-MS TIC of the extract of different kinds of ginseng acquired in negative ion mode. (A)white ginseng; (B) fermented ginseng

Figure 2 ESI-Q-TOF-MS/MS spectra of ginsenosides . The mass spectrometer was operated in the negative ion mode and the total ion chromatogram (TIC) mode. The fragment ions were obtained using collision energy of 35% through both MS2 and MS3 experiments.(A)20(Glc) -Rf;(B) Rk1 ;

1 1 1 1 1 1 1 -300 -200 -100 0 100 200 300

X-Axis :PC1 {96.3 2% ) Y-Axis :PC2(1.82%)

Figure 3 (A) PCA scores plot of extractive respectively from fermented ginseng and white ginseng. The white ginseng as raw material, which was processed as fermented ginseng products with Paecilomyces hepiali through solid culture. The extractive of fermented ginseng is represented by triangular(A), and the extractive of whit ginseng is represented by circular(#).(B)OPLS -DA of white ginseng and fermented ginseng.

Figure 4 The phenotype map of macrophages engulfed Nano activated carbon. The part painted with blue was used as the observation site,and the red arrows represent Macrophage which is engulfing Nano activated carbon.(A)The Whole zebra fish(B)Normal control group; (C)The control model group; (D-F) The test group with the medium dosage of extractive from fermented ginseng with the dose of 55.6, 166.7 and 500 ^g/ml,respectively.(G-I) The test group with the medium dosage of extractive from white ginseng with the dose of 55.6, 166.7 and 500p,g/ml,respectively.

ED White ginseng ■ Fermented ginseng

55.6ug/ml 166.7ug/ml 500ug/ml

Concentration *** pdl.OOl

Figure 5.The macrophage phagocytic rate of white ginseng and fermented ginseng.The macrophage phagocyticrate rate was calculated by the ratio of the number of macrophages swallowing Nano activated carbon in the test group and model control group. Data are depicted as the mean values plus the standard deviation (SD) in rate of macrophage phagocytic function

between two groups ( x + s, n=10). ***p< 0.001 Indlicate differences versus in the model

group and p values indicate differences between the group of white ginseng and fermented ginseng .

Table 1 Relative content of ginsenosides of PPD, PPT, malonyl, and transformed types in white and fermented ginsengs

Group Ginsenosides White ginseng Fermented ginseng

Ra1 1.86% 0.43%

Ra2 1.99% 0.32%

Rb1 10.23% 4.47%

PPD-group Rb2 5.96% 3.04%

Rb3 4.76% 2.43%

Rc 5.81% 1.34%

Rd 2.42% 3.63%

Rg3 1.45% 7.39%

Rg1 10.69% 4.78%

Re 5.78% 3.54%

Rf 6.14% 3.32%

PPT-group Noto-R1 1.44% 0.72%

20(glc)-Rf 0.68% 0.43%

F3/NotoR2 1.05% 1.91%

20(s)-Rg2 2.47% 6.92%

mRb1 6.31% 0

mRb2 7.43% 0

mRb3 2.23% 0

mRc 4.68% 0

Malyonyl

mRd 3.56% 0.83%

mRe 1.51% 0.3%

mRg1 2.80% 0

2MRd 3.01% 1.09%

Rg6 0 2.47%

Rh4 0 0.87%

Rs2/Rg8 0 3.47%

20(R)-Rh2/CK 0 2.24%

Transformed

20(R)Rg3 0 4.73%

20(R)Rg2 0 2.74%

Rk1 0 3.68%

Rg5 0 3.12%

Rh3 0 2.27%

Rh1 0 6.78%

F1 0 0.79%

Rs3 0 2.67%

R1 0 1.43%

Ra1/Ra8/Ra9 0 2.74%

Rs7 0 3.14%

Rk2 0 1.07%

F2 0 2.14%

Rg9 0 3.01%

Table 2 The quantitative result of the phagocytic function of macrophages in Zebrafish brain (n=10)

The number of macrophages

Concentration

Group engulfed the nano activated

(ug/mL)

carbon(Mean ±SD)

Macrophage phagocytic rate (%)

Nomol control - 0 -

Model control - 7 ± 1 -

55.6 13 ± i *** 85.7***

Fermented ginseng 166.7 15 ± 1*** 114.3***

500 20 ±1*** 185.7***

55.6 14 ± 1 *** 100***

White ginseng 166.7 18 ±1*** 157 1***

500 23 ±1*** 228.7***

Noting :***p < 0.001 (Compared with model control group)

Highlights

1.This article is attempt to compare the identification of ginsenosides, chemical profiling of white ginseng and fermented ginseng by RRLC-Q-TOF-MSn.

2.At the same time authors discuss the total saponins extraction from fermented and white ginseng effection on macrophage phagocytic function.