Pharmacokinetic Characteristics of Steamed Notoginseng by an

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Pharmacokinetic characteristics of steamed notoginseng by an efficient LCMS/MS method for simultaneously quantifying twenty-three triterpenoids Dina Zhu, Qile Zhou, Hong Li, Shiming Li, Zhaoqi Dong, Dong Li, and Wensheng Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03169 • Publication Date (Web): 10 Jul 2018 Downloaded from http://pubs.acs.org on July 16, 2018

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Journal of Agricultural and Food Chemistry

Pharmacokinetic characteristics of steamed notoginseng by an efficient LC-MS/MS method for simultaneously quantifying twenty-three triterpenoids Dina Zhua,b,e#, Qile Zhouc#, Hong Lia,e, Shiming Lid, Zhaoqi Donga¶, Dong Lic, and Wensheng Zhanga,e,f* a

Beijing Key Laboratory of Traditional Chinese Medicine Protection and Utilization, Faculty of Geographical Science, Beijing Normal University，Beijing 100875， China

b

Key Laboratory of Radiopharmaceuticals (Beijing Normal University), Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, China

c

Beijing Institute of Nutritional Resources, Beijing Academy of Science and Technology, Beijing 100069, China.

d

Hubei Key Laboratory of Economic Forest Germplasm Improvement and Resources, Huanggang Normal University, Huanggang, Hubei 438000, China

e

Engineering Research Center of Natural Medicine, Ministry of Education, Faculty of Geographical Science, Beijing Normal University, Beijing 100875, China

f

National and Local United Engineering Research Center for Panax Notoginseng Resources Protection and Utilization Technology, Kunming 650000, China

#

Author contributions: Dina Zhu and Qile Zhou contributed equally to this work.

*Corresponding author: Phone: +861062200669. Fax: +861062200669. E-mail: [email protected]

1

ACS Paragon Plus Environment


¶

Current address: Department of Neurosciences, School of Medicine, Case Western

Reserve University, Cleveland, OH 44106, USA.

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ABSTRACT: Steamed Panax notoginseng (SNG) has been widely used as a

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restorative medicine instead of the raw one, but its pharmacokinetic profile is entirely

3

unknown. To address this, we’ve developed an LC-MS/MS method with high

4

efficiency and sensitivity for simultaneous quantification of twenty-three triterpenoids

5

(notoginsenosides Fa, Fc, R1, 20(S)-R2, 20(R)-R2, ginsenosides F4, Rb1, Rg1, Rd, Re,

6

Rb2, 20(S)-Rh1, 20(R)-Rh1, Rh4, Rk1, Rk3, 20(S)-Rg2, 20(S)-Rg3, 20(R)-Rg3, Rg5, C-K,

7

20(S)-PPT, 20(S)-PPD) from SNG in rat plasma. This validated approach exhibits

8

great linearity, precisions, accuracy, recovery and stability for all analytes.

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Furthermore, we for the first time applied this method to the pharmacokinetic study of

10

SNG, and proposed Rb1, Fa, Rd, Rk1, Rg5, Rk3, Rh4, and 20(S)-PPD to be suitable

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pharmacokinetic markers of SNG due to their high exposure levels of systemic

12

plasma. Hence, this developed approach would be a powerful tool for future in vivo

13

investigation of various sources of notoginseng-related samples.

14

steamed

notoginseng,

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KEYWORDS:

16

pharmacokinetics, rat plasma

notoginsenoside,

17 18

3


LC-MS/MS,


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INTRODUCTION

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Notoginseng (NG, named San-Qi), the root of Panax notoginseng (Burk.) F. H.

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Chen, is a traditional herbal medicine used for centuries to eliminate blood stasis, stop

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bleeding, as well as mitigate swelling and pain.1 It is also widely regarded as

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functional foods in China and a dietary supplement for the U.S. health food market.2,3

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The commercialized San-Qi has been widely used in both the raw and steamed forms.

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The steamed Panax notoginseng (SNG) has been widely viewed as a restorative

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medicine instead of the raw one for its blood cell-increasing and nourishing functions

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which likely due to the difference of chemical constituents obtained during steaming

28

process.4 The major bioactive components of raw NG are saponins. Notoginsenoside

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R1 together with ginsenosides Rb1, Rd, Re, Rg1 are known to be the main

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components.5 Previous studies suggested that the steaming process generates a large

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number of effective constituents differed from those obtained from raw NG.6,7

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Ginsenosides Rk3, Rh4, Rk1, Rg5, F4, 20(S/R)-Rg3 and 20(S/R)-Rh1 are unique

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saponins which only existed in SNG but not in raw NG.8,9 These distinct transformed

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ginsenosides have displayed high potency for anti-tumor, enhancing immune function,

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and tonification.10-12 Although chemical content quantification and bioactivity

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analysis regarding SNG have been carried out in recent years, there is no

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pharmacokinetic (PK) investigation on SNG in vivo.

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Previous research on the PK analysis of raw NG showed limited analytical

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performance which only quantifies few numbers of notoginsenosides in rat plasma,

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mainly on the five major saponins (Rg1, Re, Rb1, Rd and R1).13,14 Importantly, these

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ginsenosides failed to represent the holistic PK behavior of SNG, a comprehensive PK

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study of SNG in vivo is still lacking. The study of PK properties of SNG would assist

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us to understand the efficacy and toxicity of SNG better and enable the prediction of 4


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its role in the clinic. Moreover, the oral absorption and bioavailability data would be

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extremely critical regarding the usage of SNG unique saponins. Unfortunately,

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previous PK studies narrowly focused on single or several prototypical ginsenosides

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such as Rb1, R1, Rg1, and Rg3.15–17 The transformed ginsenosides in SNG which

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include Rg5, Rh4, Rk1, as well as the metabolites of prototypical ginsenosides Rb1 and

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Rg1 such as protopanaxadiol (PPD) and protopanaxatriol (PPT) exhibit various

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therapeutic effect in vivo.18-22 Thus, the prototypical ginsenosides, and transformed

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ginsenosides, as well as their metabolites in vivo ought to be determined together to

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represent the PK profile of SNG. Furthermore, the PK markers might provide

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insightful information for drug-drug interaction and clinical applications of SNG in

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the future.

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The simultaneous quantification methods for multi-ginsenoside of SNG would be

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urgently needed which achieves higher throughput, smaller biosample volume, and

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lowest costs for PK analysis. However, current quantification methods used for the

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determination of SNG saponins are difficult for simultaneous detection of

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multi-ginsenoside.6,8,23,24 For HPLC method, the analytical duration time was too long

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for analyzing of more than ten ginsenosides; for UPLC, the peak resolution was not

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well to determine multi-analyte due to structural similarity of ginsenosides.

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Meanwhile, the low sensitivity, strong background noise, low content in vivo and

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potentially other factors have always challenged us for simultaneous and quantitative

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analysis of multi-ginsenoside from biological samples based on UV and ELSD

65

detector (coupled with HPLC/UPLC). Hence, the liquid chromatography–triple

66

quadrupole tandem mass spectrometry (LC-MS/MS) method, using different MRM

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channels at the same time, would theoretically be a better choice for simultaneous

68

quantification of multiple trace constituents from complex matrix, especially for PK 5



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study.25,26 A recent report has adopted this technique in application to quantifying nine

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notoginsenosides in rat plasma for PK study of raw NG.13 Herein, we developed a

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newly efficient LC-MS/MS technique to determine twenty-three triterpenoids in

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biological samples simultaneously. Our modified method is highly reliable and

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sensitive to determine the PK properties of SNG in rat plasma. More importantly, this

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developed approach could be extensively applied to the in vivo studies of more

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traditional herbal medicines including, but not limited to, Panax herbs,

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notoginseng-related samples and notoginseng-type functional foods.

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MATERIALS AND METHODS

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Chemicals. Reference standards of notoginsenosides Fa, Fc, R1, 20(S)-R2, 20(R)-R2,

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ginsenosides F4, Rb1, Rg1, Rd, Re, Rb2, 20(S)-Rh1, 20(R)-Rh1, Rh4, Rk1, Rk3,

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20(S)-Rg2, 20(S)-Rg3, 20(R)-Rg3, Rg5, 20(S)-PPT, 20(S)-PPD, C-K (ginseng saponin

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compound K) were used (Push Bio-Technology, Chengdu, China). The internal

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standard of digoxin (IS) was obtained from National Institutes for Food and Drug

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Control (Beijing, China). Purities of all standards were above 98.0% and their

84

structures were presented in Figure 1. Acetonitrile and methanol (Fisher Scientific,

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Fair Lawn, NJ, USA) were LC-MS grade. Ammonium acetate (Sigma-Aldrich, St.

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Louis, MO, USA) was HPLC grade. Water was purified using a Millipore pure water

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system (Millipore, Bedford, MA, USA).

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Preparation of Standards and Samples. Each stock solution of these 23 authentic

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standards above in MeOH was diluted to appropriate concentrations of working

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solutions. The blank rat plasma was used for dilutions to calibrate Rb1, Rg5, Rd, and

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Rh4 at the concentrations of 1000, 500, 200, 100, 50, 20, 8, 2 ng/mL; F4, Fa, R1, Rb2,

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Rg1, Rk1, Rk3 and 20(S)-PPD at the concentrations of 500, 200, 100, 50, 20, 5, 2, 1

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ng/mL; those of C-K, Fc, Re, 20(S)-Rg3, 20(R)-Rg3, 20(S)-R2, 20(R)-R2, 20(S)-Rg2, 6


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20(S)-Rh1, 20(R)-Rh1 and 20(S)-PPT at the concentrations of 200, 100, 50, 20, 5, 2, 1,

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0.5 ng/mL. The primary stock solution of IS was diluted to yield working solution of

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1 µg/mL. All prepared working solutions were maintained at 4 °C before use.

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Sample solutions were prepared as previously described.26 In brief, protein

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precipitation method was used to extract plasma samples from rats. Rat plasma

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sample (100 µL) was mixed with 1 mL of 4:1(v/v) MeOH-ACN containing 10 µL of

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1.0 µg/mL digoxin. The mixture was subsequently vibrated to make itself

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homogeneous. After centrifuging (12000×g, 10 min), the obtained supernatants were

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relocated into a new tube followed by drying under N2 gas flow at 45 °C. 200 µL

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MeOH was used to reconstitute the residue, then mix it thoroughly by vortexing until

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completely dissolved. After centrifuging, an aliquot of 2 µL obtained supernatant was

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prepared to analyze using the LC-MS/MS system.

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Instrumentation and Chromatographic Analysis. The LC-MS/MS system

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contained a liquid chromatography (LC) -30AD system and a triple quadrupole mass

108

spectrometer (MS)-8050 system (Shimadzu, Shimadzu Corporation, Kyoto, Japan).

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The LC configurated a LC-30A binary pump, a CTO-20AC column oven together

110

with a SIL-30AC autosampler, while MS instrument equipped with an electrospray

111

ionization (ESI) source. The results acquisition and analysis were complied by

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LabSolutions LCMS Ver. 5.6 software.

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Liquid chromatographic separation was performed on a 2.1 mm × 100 mm, 1.7 µm

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ACQUITY UPLC® BEH Shield RP-C18 column (Waters Corp., Milford, MA, USA),

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combined with a RP-C18 VanGuardTM pre-column at 30 °C column temperature. The

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eluent A was water with 0.1 mM ammonium acetate, and B was ACN. An optimized

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gradient elution condition was set as 20 to 30% B (0–3 min), 30 to 33% B (3–5 min),

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33 to 35% B (5–6 min), 35 to 42% B (6–8 min), 42 to 47% B (8–16 min), 47 to 52% 7



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B (16–17 min), 52 to 80% B (17–19 min), 80 to 95% B (19–20 min), 95 to 20% B

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(20–21 min), and 20 to 20% B (21–24 min) under constant flow rate at 0.4 mL/min.

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Meanwhile, the autosampler was maintained at 4 °C with 2 µL injection volume.

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The mass spectrometer was realized on a negative mode of ESI source. The

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optimized MS conditions were set at: drying gas flow, 10 L/min; nebulizer gas flow, 3

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L/min; interface voltage, 3 KV; interface temperature, 300 °C; detector voltage, 1.8

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KV; heat block temperature, 400 °C; heating gas flow, 10 L/min; and desolvation

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temperature, 250 °C. The detection of analytes was performed by multiple reaction

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monitoring (MRM). The optimized parameters of MRM transition, Dwell time,

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collision energy, Q1 and Q3 Pre Bias for the twenty-three triterpenoid analytes are

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presented in Table 1.

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Method Validation for Quantitation. The validation of our LC-MS/MS method was

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carried out by evaluating its linearity, specificity, the lower limit of detection (LLOD)

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and lower limit of quantification (LLOQ), precision, accuracy, matrix effect, recovery

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and stability of analytes.

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Specificity was tested using six different rat blank plasma samples. It was

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determined by excluding any endogenous interference present at or near the retention

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time of analytes and IS. Eight different calibration solutions of 23 analytes were

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prepared in rat plasma. The calibration curve was constructed by plotting a linear

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regression analysis depending on each peak area ratio of the analyte and IS to the

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analyte content. The response equivalent to a signal-to-noise (S/N) ratio of 3 and 10

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times were defined as LLOD and LLOQ, respectively, which represents the sensitivity

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of this LC-MS/MS detection.

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To test the intraday precision, six replicates of 23 analytes were analyzed on the

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same day, whereas duplicate samples were determined on three consecutive days for 8


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interday precision. They were required to show a relative standard deviation (RSD) of

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no more than 15% at low, medium and high levels (20% for LLOQ). In addition,

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accuracy depended on the amount of an analyte recovered. The nominal concentration

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(Cnom) and the average value of observed concentration (Cobs) were used to calculate

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the accuracy of this method. Accuracy (relative error, RE within ±15%) was assessed

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using the percentage of subtracting Cobs from Cnom.

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Three quality control (QC) levels (low, medium, high concentrations) of samples

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were prepared (n=6). The comparison between the average peak area of the QC

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sample in MeOH and the average peak area of the extracted QC sample dissolved in

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pre-extracted blank plasma using 4:1 (v/v) of MeOH-ACN was used to determine the

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matrix effect of the 23 analytes from rat plasma. Six replicates above at three QC

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levels were used to determine recoveries of all 23 analytes in rat blank plasma based

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on the comparison between the average peak area of the QC sample in MeOH and the

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QC sample in blank plasma followed by extracting from 4:1 (v/v) of MeOH-ACN.

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The stabilities of the 23 constituents in rat plasma were estimated by exposing with

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short-term storage (24 h, 4 ºC), three times of freeze-thaw cycles as well as long-term

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storage (30 days, -20 ºC) at three concentrations (n=6). The result of which would be

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considered as satisfactory stability when the accuracy deviation reached within ±15%

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of the nominal values.

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Pharmacokinetic Study in Rats. All animal studies were approved by Beijing

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Normal

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SYXK20150038) complied by the guide for the care and use of laboratory animals.

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Sprague-Dawley (SD) male rats (180~220 g) were obtained from the Beijing Vital

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River Laboratory Animal Technology Co., Ltd. (Beijing, China). Rats were housed in

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groups and maintained on a controlled standard condition with free access to normal

University

Laboratory

Animals

Care

and

9


Use

Committee

(No.


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chow for one week before experiments were performed.

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All animals were fasted overnight but accessed water freely. After oral gavage of a

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single dose of SNG extract at 2 g/kg, heparinized blood was collected from

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ophthalmic veins for 200 µL at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 48 and 72 h. After

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centrifuging (3000×g, 10 min, 4 ºC), the harvested supernatants were stored at -20 °C

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before use. Plasma concentration-time (C-T) profile was plotted by using a DAS 2.0

175

version software, and PK parameters were calculated by noncompartmental model.

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RESULTS AND DISCUSSION

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Modification of Chromatography and MS Conditions. LC and MS conditions were

178

optimized for achieving good chromatographic behavior including the best

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chromatographic separation, peak symmetry, short run time and maximum MS

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sensitivity.25,26 For optimization of chromatographic condition, acetonitrile was

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chosen instead of methanol, since a better peak shape as well as shortened analytical

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time could be obtained. The different aqueous phases of water, water containing

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acetic acid, water containing ammonium acetate (0.01−1.0 mM) were investigated,

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and the optimal eluent of ammonium acetate at a concentration of 0.1 mM was

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chosen, for showing the best sensitivity of MRM response and better reproducibility

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of all analytes. For optimization of MS condition, the quasi-molecular ion peaks and

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MRM transitions of all compounds were investigated in both of the ESI+ and ESI–

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mode. We found that higher relative intensity of ion response was shown in negative

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ionization mode. Most of the analytes elicited plenty of deprotonated molecular ions

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in ESI– mode. However, F4, Rh4 and Rk3 are three unique ginsenosides that their

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precursor ions of MRM transition have been selected as [M+CH3COOH–H]–.

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Subsequently, the Labsolutions software was used to automatically optimize the

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parameters of MRM transition. For each of the analytes, we’ve selected the two most 10


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abundant and interference-free product ions, one MRM ion transition was selected for

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quantification, while the other was chosen for qualification. The optimized MRM

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transition and parameters of all 23 analytes with IS are displayed in Table 1.

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Method Validation. We’ve compared the MRM chromatograms of all 23 analytes

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and digoxin in the rat plasma as well as the blank rat plasma to validate the specificity

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of the method we developed. The typical MRM chromatograms for the blank rat

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plasma (A), the blank rat plasma together with 23 reference standards and digoxin (B),

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and sample plasma of 8h with oral gavage of 2.0 g/kg SNG extract (C) were displayed

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in Figure 2. There were no obvious endogenous peaks interfered with determination

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of the 23 analytes and IS. Besides, there were 5 pairs of isomers which are

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20(S/R)-Rg3, 20(S/R)-R2, 20(S/R)-Rh1, Rk1/Rg5, and Rk3/Rh4. Each pair of isomer

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shared the same MRM transition of 769.5→475.4, 783.5→621.5, 637.5→475.4,

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765.5→603.5, 679.5→619.5 respectively. However, we could identify each of

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isomers by comparing their retention time of single reference standard.

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A lower LLOQs was obtained with this method as it laid within a concentration

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range of 0.18 to 7.69 ng/mL for all 23 analytes in triplicate analyses, and the LLODs

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ranged from 0.06 to 2.56 ng/mL. All of calibration curves of the analytes showed

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satisfactory linearity (r2, a range of 0.9974-0.9998). All values regarding individual

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analytes’ calibration curve, linear range, r2, LLOD and LLOQ were summarized and

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provided in Table 2.

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As shown in Table 3, the intraday precision and accuracy of all analytes showed a

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range of 3.13% to 14.77% (n=6, RSD%), and −14.17% to 13.91% (n=6, RE%)

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respectively. Interday precision and accuracy ranged from 2.15% to 14.67% (n=6,

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RSD%) and from –13.29% to 13.73% (n=6, RE%) respectively. These observations

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indicate a good inter- and intra-day precision and accuracy that fulfils the acceptance 11



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criteria of bioanalytical method validation as suggested by the guidelines of FDA and

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EMA.27,28

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In order to acquire a higher recovery for the 23 analytes, some extraction reagents

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such as ethyl acetate, n-butanol, and acetonitrile were investigated. We’ve also

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compared the ratio of MeOH-ACN from 5:1 to 1:1 (v/v), and finally selected the

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optimal ratio of 4:1 (v/v) for extracting the plasma samples, which achieved

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satisfactory recovery and deducted matrix effects from rat plasma sample. The

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extraction recovery ranged from (74.02±8.47) % to (94.19±12.21) % within the

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acceptable limits (Table 4), suggesting that this protein precipitation process enabled

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consistent data acquisition. Meanwhile, the detailed matrix effects derived from QC

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samples at three different concentrations were between (85.09±14.03) % and

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(95.87±8.50) %. Moreover, while we were detecting all 23 analytes under present

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MRM conditions, no disturbance by matrix effect was observed.

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As summarized in Table 5, all tested analytes exhibited good stabilities in plasma

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samples regardless of any concentrations (low, medium and high) under the indicated

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storage conditions. We observed an RE of -10.11 to 14.65% for samples under the

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condition of 24 h short-term storage, an RE of -14.33 to 14.54% for samples

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underwent three freeze-thaw cycles, and an RE of -9.58 to 14.24% for samples

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purposed for long-term tests.

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Pharmacokinetics Study. We utilized this efficient and validated LC–ESI–MS/MS

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approach to study the PK profile of SNG in vivo after oral gavage of a single dose

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SNG at 2.0 g/kg in rats. The results were described in Figure 3. The mean plasma C-T

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profiles of all 23 analytes were plotted and displayed accordingly (Figure 3A). We

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further calculated the PK parameters using a DAS 2.0 software given the

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noncompartmental model (Table 6). 12


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We found that the values of Cmax and AUC were higher in three PPD-type

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ginsenosides Fa, Rb1 and Rd than other prototype ginsenosides (consistent with PK

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behavior of NG13). Also, a PPD-type metabolite, 20(S)-PPD, exhibited the highest

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Cmax and AUC compared to other ginsenoside metabolites. Whereas, the PPT-type

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ginsenosides such as Rg1, Re and metabolite 20(S)-PPT showed lower Cmax and AUC,

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which possibly attributed to the interference challenged by low intestinal absorption

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rate and fast biliary excretion as previously reported.29 These results indicated a better

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absorption of PPD-type ginsenosides in rat gastrointestinal systems than that of

252

PPT-types (Figure 3B). However, the required time for absorptions of PPT-types (Rg1:

253

Tmax –0.83h, R1: Tmax –1.17h, 20(S)-Rh1: Tmax –0.63h, 20(R)-Rh1: Tmax –0.79h) were

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overall shorter than that of PPD-types (Rb2, Rd, Rb1: Tmax –8~9.33h).

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Moreover, we observed that the amount of sugar substituent groups was found to

256

influence the absorption rate of ginsenosides. As presented, a Tmax of 0.63h–2.83h

257

was shown in monosaccharide-ginsenoside (20S/R-Rh1, Rk3 and Rh4), a Tmax of 1h–4h

258

was shown in disaccharide-ginsenoside (R2, Rk1, Rg2, Rg3, and Rg5), and a Tmax of

259

1.17h–9.33h was shown in trisaccharide- and tetrasaccharide- as well as

260

pentasaccharide-ginsenoside (Fa, R1, Rb1, Rb2 and Rd). These observations suggested

261

that sugar moieties indeed slowed the absorption and elimination of different types of

262

ginsenosides (Figure 3C). In addition, 3 pairs of 20(S/R) epimers were also studied,

263

including R2, Rg3 and Rh1 (Figure 3D). We found that 20(S) configurations had much

264

higher values of AUC and Cmax than corresponding 20(R) configurations.17 Although

265

several point-in-time of 20(R)-Rg2, Rb3, Fe, Rg6, Rh2 could be detected in rat plasma,

266

they fail to reach the standard quantifiable limit and therefore excluded from our PK

267

study, due to their limited amount in SNG and potentially low bioavailability. Notably,

268

some ginsenosides exhibited double peaks in the C–T figures, and the mechanisms of 13



269

which remains to be determined.

270

We are the first to investigate the PK behaviors of SNG originated transformed

271

ginsenosides F4, Rg5, Rk1, Rk3, and Rh4. Interestingly, these ginsenosides were

272

unabundant in SNG, but the systemic exposure of which was surprisingly high (Figure

273

3A). Among which, Rg5 exhibited quite a number of therapeutic effects including

274

anti-inflammation, improves cognitive dysfunction, promotes angiogenesis and

275

vasorelaxation.30–32 Therefore, the PK investigation of the transformed compounds of

276

SNG is of great necessity for knowing the mechanisms of how SNG became

277

therapeutically effective as well as for understanding the difference between each

278

component extracted from Panax notoginseng in its role of chemical properties.

279

Usually, the constituents displayed therapeutic efficacy in vivo were the metabolites

280

than instead of prototypes.33 In this study, we characterized 3 types of metabolites

281

20(S)-PPT, 20(S)-PPD and C-K which we found could be detected as early as 2 h to 4

282

h from rat plasma, and the Cmax of which could be reached at 10.33–12 h. Notably, we

283

found that 20(S)-PPD, a metabolite from Fa, Rb1, Rb2 and Rd (the most abundant

284

prototype ginsenosides in SNG) exhibited very high systemic exposure level (Figure

285

3E). Moreover, in recent studies, it has been demonstrated with decent

286

pharmacological activities.3,12 Our result might explain how 20(S)-PPD became an

287

effective constituent of SNG in vivo.

288

We reason the good systemic exposure of PPD-type ginsenosides might attribute to

289

great solubility, the long t1/2 and abundant content in SNG. However, other high

290

content ginsenosides such as R1 failed to correlate with high systemic exposure. We

291

think this is due to its fast biliary excretion or other reasons.14 Rg3 and PPD have been

292

reported to be metabolites of Rb1,34 and Rg2, Rh1, PPT were the metabolites of Rg1.35

293

Small molecule PPD and PPT, as polar metabolites, were shown with much higher 14


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intestinal absorption rates in vivo. The metabolism process of these two metabolites

295

improved the bioavailability of big-polar ginsenosides for the high systemic exposure

296

of their metabolites.

297

It is widely accepted that an individual effective constituent cannot represent a

298

complex herb medicine. Considering the complexity and variety of different

299

compounds in a herb system, the PK properties can be rather diverse and complicated.

300

Hence, multi-components PK analysis is necessary for unraveling the mechanisms of

301

pharmacological effects of an herb. Usually, a good PK property could be viewed as a

302

PK marker that correlates with good bioactivity and satisfied systemic exposure of a

303

constituent from herb medicine.36 The identification of PK markers is of great help for

304

evaluating critical properties of the drug such as the interactions of drug-drug and the

305

toxicity of clinical practice. In this study, the systemic exposure of 23 triterpenes was

306

carefully analyzed, and we found several components including Rb1, Fa, Rd, Rk1, Rg5,

307

Rk3, Rh4, and 20(S)-PPD in plasma could be used as PK markers for SNG in vivo due

308

to high systemic exposure levels (Figure 3A). Notably, we identified eight

309

ginsenosides with integrated PK behaviors suitable as markers representing

310

comprehensive PK behavior of SNG. Among which, these transformed ginsenosides

311

Rk1, Rg5, Rk3 and Rh4 represent the characteristic PK markers for SNG, which might

312

explain the differences in clinical practice between SNG and NG.

313

In conclusion, our work represents the first comprehensive PK study for SNG in

314

rats by using an efficient and validated LC-MS/MS method. This optimized method

315

exhibited great linearity, higher sensitivity, satisfied precision and accuracy of

316

intraday and interday, preeminent recovery, matrix effect, as well as stability.

317

Importantly, a lower limit of quantification was obtained with this method for

318

twenty-three triterpenoids of SNG in rat plasma. Furthermore, the analytical method 15



319

we developed enabled our attempt for tracking the PK features of SNG in rats. We for

320

the first time proposed that Rb1, Fa, Rd, Rk1, Rg5, Rk3, Rh4, and 20(S)-PPD could be

321

viewed as PK markers of SNG in rats which was attributed to constantly obtained

322

high levels of systemic exposure and potent bioactivity. Our study on PK properties of

323

SNG assisted us to better understand the efficacy and toxicity of SNG and enable the

324

prediction of its role in the clinic. This promising approach could be extensively

325

applied to future pharmacokinetic studies of other Panax herbs, notoginseng-related

326

herbal medicines or formulas and notoginseng-type functional foods in the field of

327

agricultural and food chemistry.

328

ABBREVIATIONS AND NOMENCLATURE

329

F4, ginsenoside F4; Rb1, ginsenoside Rb1; Rb2, ginsenoside Rb2; Rd, ginsenoside Rd;

330

Re, ginsenoside Re; Rg1, ginsenoside Rg1; Rg5, ginsenoside Rg5; Rh4, ginsenoside

331

Rh4; Rk1, ginsenoside Rk1; Rk3, ginsenoside Rk3; 20(S)-Rg2, 20(S)-ginsenoside Rg2;

332

20(S)-Rg3, 20(S)-ginsenoside Rg3; 20(R)-Rg3, 20(R)-ginsenoside Rg3; 20(S)-Rh1,

333

20(S)-ginsenoside Rh1; 20(R)-Rh1, 20(R)-ginsenoside Rh1; Fa, notoginsenoside Fa;

334

Fc, notoginsenoside Fc; R1, notoginsenoside R1; 20(S)-R2, 20(S)-notoginsenoside R2;

335

20(R)-R2, 20(R)-notoginsenoside R2; 20(S)-PPT, 20(S)-protopanaxatriol; 20(S)-PPD,

336

20(S)-protopanaxadiol; C-K, ginseng saponin compound K; Cnom, nominal

337

concentration; Cobs, observed concentration; ESI, electrospray ionization; IS, internal

338

standard; LLOD, lower limits of detection; LLOQ, lower limits of quantification;

339

MRM, multiple reaction monitoring; PK, pharmacokinetic; QC, quality control; RSD,

340

relative standard deviation; S/N, signal-to-noise ratio; SNG, steamed panax

341

notoginseng; LC–MS/MS, liquid chromatography–triple quadrupole tandem mass

342

spectrometry.

343

Funding 16


Page 16 of 37

Page 17 of 37


344

This work was supported by National Key R&D Plan (No. 2017YFC1702500), the

345

Beijing Joint Project for the Central-Affiliated University (2017-01) and the National

346

Nature Science Foundation of China (81771152).

347

Notes

348

The authors declare no conflict of interest.

349

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350

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351

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ginsenoside Rg1, Re and notoginsenoside R1 in human plasma by LC-MS/MS and

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chromatography-electrospray ionization mass spectrometry. J. Pharm. Biomed.

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in rat plasma by LC-MS/MS: application to a pharmacokinetic study. J. Sep. Sci.

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2013, 36 (12), 1904-1912.

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(18) Jaeschke, H. Comments on caspase-mediated anti-apoptotic effect of ginsenoside

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Rg5, a main rare ginsenoside, on acetaminophen-induced hepatotoxicity in mice. J.

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Agric. Food Chem. 2018, 66 (7), 1732-1733.

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(19) Khamessi, O.; Ben, M. H.; ElFessi-Magouri, R.; Kharrat, R. RK1, the first very

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short peptide from Buthus occitanus tunetanus inhibits tumor cell migration,

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proliferation and angiogenesis. Biochem. Biophys. Res. Commun. 2018, 499 (1),

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protective effect of 20(S)-protopanaxadiol (PPD) against chronic sleep deprivation

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(CSD)-induced memory impairments in mice. Brain Res. Bull. 2018, 137,

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249-256.

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(22) Lee, S. Y.; Jeong, J. J.; Eun, S. H.; Kim, D. H. Anti-inflammatory effects of

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ginsenoside Rg1 and its metabolites ginsenoside Rh1 and 20(S)-protopanaxatriol in

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mice with TNBS-induced colitis. Eur. J. Pharmacol. 2015, 762, 333-343.

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(23)Wu, S.; Guo, C. L.; Cui, X. M.; Yang, X. Y. Simultaneous determination of ten

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kinds of saponins in raw and steamed Panax notoginseng root and rhizome by

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HPLC. J. Chin. Med. Mater. 2015, 38 (8), 1622-1625.

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determination of thirteen saponins in Shusanqi Powder by HPLC. Chin. Tradit.

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quantification of twenty-one ginsenosides and their three aglycones in rat plasma

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by a developed UFLC-MS/MS assay: Application to a pharmacokinetic study of

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of G-Re in volunteers. J. Chromatogr. B. 2011, 879 (22), 2011-2017.

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(30) Kim, T. W.; Joh, E. H.; Kim, B.; Kim, D. H. Ginsenoside Rg5 ameliorates lung

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inflammation in mice by inhibiting the binding of LPS to toll-like receptor-4 on

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macrophages. Int. Immunopharmacol. 2012, 12 (1), 110-116.

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Ginsenoside Rg5 improves cognitive dysfunction and beta-amyloid deposition in

455

STZ-induced memory impaired rats via attenuating neuroinflammatory responses.

456

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457

(32) Cho, Y. L.; Hur, S. M.; Kim, J. Y.; Kim, J. H.; Lee, D. K.; Choe, J.; Won, M. H.;

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H.; Kim, Y. M. Specific activation of insulin-like growth factor-1 receptor by

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ginsenoside Rg5 promotes angiogenesis and vasorelaxation. J. Biol. Chem. 2015,

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(33) Yang, X. W. Pharmacokinetic studies of chemical constituents of ginseng, Mod. Chin. Med. 2016, 18, 16–35.

464

(34) Xie, H. T.; Wang, G. J.; Sun, J. G.; Tucker, I.; Zhao, X. C.; Xie, Y. Y.; Li, H.;

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Jiang, X. L.; Wang, R.; Xu, M. J.; Wang, W. High performance liquid

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469

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(35) Sun, J.; Wang, G.; Haitang, X.; Hao, L.; Guoyu, P.; Tucker, I. Simultaneous rapid

470

quantification of ginsenoside Rg1 and its secondary glycoside

Rh1 and aglycone

471

protopanaxatriol in rat plasma by liquid chromatography-mass spectrometry after

472

solid-phase extraction. J. Pharm. Biomed. Anal. 2005, 38 (1), 126-132.

473

(36) Lu, T.; Yang, J.; Gao, X.; Chen, P.; Du F; Sun, Y.; Wang, F.; Xu, F.; Shang, H.;

474

Huang, Y.; Wang, Y.; Wan, R.; Liu, C.; Zhang, B.; Li, C., Plasma and urinary

475

tanshinol from Salvia miltiorrhiza (Danshen) can be used as pharmacokinetic

476

markers for cardiotonic pills, a cardiovascular herbal medicine. Drug Metab.

477

Dispos. 2008, 36 (8), 1578-1586.

478

22


Page 23 of 37


479

Figure captions

480

Figure 1. Chemical structures of the 23 triterpenoids and digoxin (internal standard).

481

Figure 2. The representative MRM chromatograms of the 23 analytes and digoxin. (A)

482

Blank rat plasma, (B) blank rat plasma spiked with the 23 analytes and digoxin, (C)

483

rat plasma after oral gavage of 2.0 g/kg SNG extract at 8h.

484

Figure 3. Mean plasma concentration-time (C-T) curves of (A) the 23 triterpenoids,

485

(B) the PPD-type and PPT-type ginsenosides, (C) the ginsenosides with different

486

amounts of sugar substituent groups, (D) 3 pairs of 20(S/R) epimers, and (E) 3 types

487

of metabolites from the most abundant prototype ginsenosides in rats after oral gavage

488

of 2.0 g/kg SNG extract.

489

23



Page 24 of 37

490

Table 1. Optimized MS Conditions of the 23 Analytes and IS.

Analyte

tR (min)

MRM transition (m/z) Precursor ion→product ion

Dwell time (msec)

Collision energy (V)

Q1 Pre Bias (V)

Q3 Pre Bias (V)

R1

2.658

931.5→637.5

147

39

22

32

Rg1

2.990

799.5→637.5

147

25

25

30

Re

3.033

945.6→637.5

147

40

22

34

Fa

5.316

1239.6→1107.5

82

51

30

40

Rb1

5.849

1107.6→945.5

82

45

10

35

20(S)-R2

5.877

769.5→475.4

82

36

10

33

Fc

6.238

1209.6→1077.5

82

49

30

40

20(R)-R2

6.240

769.5→475.4

82

36

10

33

20(S)-Rg2

6.352

783.5→475.4

82

38

10

34

20(S)-Rh1

6.433

637.5→475.4

82

25

20

32

20(R)-Rh1

6.710

637.5→475.4

82

25

20

32

Rb2

6.858

1077.6→783.5

82

46

30

39

Rd

7.503

945.5→621.5

117

39

15

30

F4

9.451

825.5→765.5

197

22

19

37

Rk3

9.525

679.5→619.5

197

19

23

35

Rh4

9.865

679.5→619.5

197

19

23

35

20(S)-Rg3

11.10

783.5→621.5

197

32

25

30

20(R)-Rg3

9 11.40

783.5→621.5

197

32

25

30

20(S)-PPT

8 12.67

475.4→391.5

297

30

10

45

C-K

3 14.90

621.5→161.3

297

23

28

31

Rk1

5 15.66

765.5→603.5

297

30

25

32

Rg5

0 16.14

765.5→603.5

297

30

25

32

20(S)-PPD

5 20.34

459.4→375.4

597

30

5

40

Digoxin

0 4.783

779.5→649.4

147

33

10

32

24


Page 25 of 37


Table 2. The Linear Regression Data, LLOD and LLOQ of the 23 Analytes. Analyte

Standard curve

r2

Linear range (ng/mL)

LLOD (ng/mL)

LLOQ (ng/mL)

R1

y = 0.2156 x + 0.0016

0.9996

1–500

0.26

0.78

Rg1

y = 0.2498 x + 0.0041

0.9991

1–500

0.21

0.63

Re

y = 0.1563 x + 0.0019

0.9995

0.5–200

0.06

0.18

Fa

y = 0.0863 x – 0.0004

0.9992

2–500

0.47

1.40

Rb1

y = 0.0238 x + 0.0003

0.9993

2–1000

0.35

1.06

20(S)-R2

y = 0.5847 x + 0.0104

0.9988

0.5–200

0.09

0.27

Fc

y = 0.1831 x + 0.0004

0.9998

1–200

0.33

0.98

20(R)-R2

y = 0.3879 x + 0.0073

0.9989

0.5–200

0.07

0.21

20(S)-Rg2

y = 0.6326 x + 0.0109

0.9986

0.5–200

0.14

0.42

20(S)-Rh1

y = 0.6635 x + 0.0183

0.9976

1–200

0.20

0.60

20(R)-Rh1

y = 0.6919 x + 0.0201

0.9974

0.5–200

0.13

0.40

Rb2

y = 0.0334 x + 0.0005

0.9997

2–500

0.36

1.09

Rd

y = 0.1622 x + 0.0023

0.9994

2–1000

0.31

0.94

F4

y = 0.0780 x + 0.0009

0.9998

5–500

1.53

4.58

Rk3

y = 0.3460 x + 0.0006

0.9998

2–500

0.49

1.46

Rh4

y = 0.0765 x + 0.0005

0.9998

8–1000

1.68

5.03

20(S)-Rg3

y = 0.7317 x + 0.0089

0.9992

1–200

0.28

0.85

20(R)-Rg3

y = 0.6748 x + 0.0133

0.9981

1–200

0.24

0.72

20(S)-PPT

y = 0.4229 x – 0.0027

0.9992

2–200

0.45

1.35

C-K

y = 0.2719 x + 0.0030

0.9997

2–200

0.65

1.94

Rk1

y = 0.0495 x – 0.0004

0.9992

5–500

0.92

2.75

Rg5

y = 0.0168 x – 0.0002

0.9977

8–1000

2.56

7.69

20(S)-PPD

y = 0.0012 x + 0.0001

0.9978

5–500

1.40

4.21

25



Page 26 of 37

Table 3. Precision and Accuracy of the 23 Analytes at Three Different Levels in Rat Plasma.

Analyte R1

Rg1

Re

Fa

Rb1

20(S)-R2

Fc

20(R)-R2

20(S)-Rg2

20(S)-Rh1

20(R)-Rh1

Rb2

Rd

F4

200 20

Intra-day (n=6) Measured Precision Accuracy (ng/mL) (RSD %) (RE%) 189.63 6.25 5.19 18.80 8.56 6.02

Inter-day (n=6) Measured Precision Accuracy (ng/mL) (RSD %) (RE%) 181.53 4.12 9.23 19.01 10.11 4.93

1 200

0.90 191.29

14.42 5.23

9.87 4.35

1.09 208.99

14.54 6.36

-8.95 -4.49

20

18.65

9.48

6.74

20.43

8.21

-2.13

1 50

1.04 45.46

13.12 8.33

-3.88 9.08

1.10 43.40

8.85 4.82

-9.99 13.19

5 0.5

4.77 0.44

10.23 13.91

4.69 11.23

4.71 0.45

11.53 12.26

5.84 9.04

Content (ng/mL)

200

204.41

8.36

-2.21

186.96

8.93

6.52

20

21.26

11.55

-6.30

22.50

6.49

-12.48

2

2.20

14.65

-10.10

2.23

14.16

-11.48

500

470.76

5.30

5.85

473.08

6.71

5.38

50

46.20

8.15

7.60

47.93

5.28

4.13

2

1.79

13.97

10.26

1.73

10.34

13.73

50 5

50.78 5.20

8.05 6.32

-1.55 -4.08

45.87 5.29

5.20 7.81

8.26 -5.82

0.5 50

0.53 48.09

7.07 7.79

-6.49 3.83

0.55 44.62

9.51 6.31

-10.17 10.76

5 1

4.87 0.88

12.12 14.24

2.63 12.41

4.70 0.90

9.65 12.97

5.92 10.38

50

47.72

6.75

4.55

43.51

7.21

12.97

5

4.52

10.85

9.58

4.52

10.17

9.67

0.5

0.43

11.23

13.45

0.51

12.19

-2.82

50 5

51.23 5.24

8.60 8.05

-2.45 -4.74

47.63 5.12

8.47 4.42

4.74 -2.37

0.5 50

0.47 46.94

11.83 6.67

5.59 6.13

0.55 53.66

14.67 6.75

-9.66 -7.31

5

4.48

5.03

10.43

5.31

8.28

-6.16

1

0.91

14.21

9.48

0.89

12.73

11.48

50

47.73

9.09

4.53

46.64

8.70

6.71

5

4.81

6.08

3.71

4.63

10.43

7.44

0.5

0.46

12.74

7.17

0.45

13.93

10.24

200

189.43

6.97

5.28

187.85

7.42

6.08

20 2

18.21 1.77

10.91 14.63

8.97 11.34

18.21 1.81

12.39 12.88

8.95 9.66

500 50

482.09 52.52

5.15 3.13

3.58 -5.04

471.79 54.65

7.06 12.16

5.64 -9.29

2 200

2.28 183.87

7.97 5.82

-14.17 8.07

2.14 185.81

10.34 6.41

-6.95 7.10

26


Page 27 of 37


Rk3

Rh4

20(S)-Rg3

20(R)-Rg3

20(S)-PPT

C-K

Rk1

Rg5

20(S)-PPD

50 5

55.34 5.47

3.88 4.29

-10.68 -9.32

45.32 4.59

6.25 10.25

9.35 8.26

200 20

193.29 18.11

4.19 7.30

3.36 9.45

213.22 22.15

7.75 5.43

-6.61 -10.73

2

1.80

8.09

10.07

1.85

10.92

7.47

500

471.79

3.25

5.64

453.88

8.84

9.22

50

47.57

9.11

4.86

46.33

3.14

7.34

8

7.36

8.57

7.95

7.33

13.28

8.36

50

50.55

7.11

-1.10

50.62

4.10

-1.24

5 1

4.87 0.94

12.19 11.86

2.63 6.38

5.04 0.87

9.27 12.90

-0.82 13.42

50

50.41

8.96

-0.83

48.34

10.07

3.33

5

5.21

12.74

-4.27

4.72

10.92

5.64

1

1.07

12.27

-6.54

0.86

14.44

13.71

100

92.53

8.22

7.47

105.19

4.17

-5.19

20 2

18.99 1.82

5.61 14.58

5.05 8.86

21.15 2.26

7.28 8.14

-5.74 -12.76

100 20

91.52 18.52

8.57 4.11

8.48 7.38

98.74 20.56

4.01 4.34

1.26 -2.82

2 200

1.79 183.53

12.32 3.15

10.69 8.23

2.06 190.48

5.61 3.05

-3.16 4.76

50 5

51.91 5.57

5.33 14.38

-3.81 -11.43

49.32 5.62

4.67 8.56

1.37 -12.31

500

468.33

8.13

6.33

471.99

6.22

5.60

50

44.70

12.60

10.61

48.46

11.14

3.07

8

6.89

14.77

13.91

7.07

7.05

11.67

200

204.47

5.98

-2.24

209.46

5.90

-4.73

50 5

51.75 5.46

7.04 9.57

-3.49 -9.11

53.72 5.66

2.15 13.78

-7.44 -13.29

27



Page 28 of 37

Table 4. Recovery and Matrix Effect for Assay of 23 Analytes at Three Different Levels in Rat Plasma. Recovery (n=6) Analyte

Spiked (ng/mL)

Determined (%)

RSD

(ng/mL) R1

Rg1

Re

Fa

Rb1

20(S)-R2

Fc

20(R)-R2

20(S)-Rg2

20(S)-Rh1

20(R)-Rh1

Rb2

Rd

F4

200 20 1 200 20 1 50 5 0.5 200 20 2 500 50 2 50 5 0.5 50 5 1 50 5 0.5 50 5 0.5 50 5 1 50 5 0.5 200 20 2 500 50 2 200 50

157.58 16.94 0.82 147.62 16.85 0.88 44.32 4.28 0.40 160.52 16.85 1.72 409.45 39.49 1.80 44.51 3.84 0.41 43.84 4.44 0.76 40.15 4.14 0.37 39.52 4.34 0.41 40.03 4.35 0.91 37.57 4.46 0.43 169.96 16.54 1.84 426.50 41.91 1.88 155.62 40.87

Matrix effects (n=6)

78.79 84.70 82.00 73.81 84.25 88.00 88.64 85.60 80.00 80.26 84.25 86.00 81.89 78.98 90.00 89.02 76.80 82.00 87.68 88.80 76.00 80.30 82.80 74.00 79.04 86.80 82.00 80.06 87.00 91.00 75.14 89.20 86.00 84.98 82.70 92.00 85.30 83.82 94.00 77.81 81.74

Determined (%)

(%)

(ng/mL)

7.63 10.85 5.15 8.87 12.31 14.35 5.20 9.82 14.15 11.60 13.37 13.96 12.32 7.49 11.19 8.03 12.90 10.12 7.10 10.80 14.01 4.84 11.79 8.26 8.12 7.60 9.20 12.62 9.51 13.10 7.12 10.44 13.97 5.81 11.69 8.57 7.55 10.67 12.21 5.99 9.47

176.44 17.97 0.87 170.32 17.31 0.89 47.52 4.61 0.45 179.48 17.14 1.77 461.05 43.89 1.86 45.13 4.34 0.43 46.91 4.75 0.90 42.89 4.27 0.45 43.41 4.39 0.43 42.79 4.36 0.85 43.42 4.57 0.45 177.32 17.85 1.73 447.25 44.95 1.90 172.46 44.73

28


88.22 89.85 87.00 85.16 86.55 89.00 95.04 92.20 90.00 89.74 85.70 88.50 92.21 87.78 93.00 90.26 86.80 86.00 93.82 95.00 90.00 85.78 85.40 90.00 86.82 87.80 86.00 85.58 87.20 85.00 86.84 91.40 90.00 88.66 89.25 86.50 89.45 89.90 95.00 86.23 89.46

RSD (%) 7.35 11.37 8.89 8.09 10.62 12.85 8.13 7.18 12.70 10.27 12.95 9.51 10.02 8.62 12.36 5.36 10.59 14.86 8.90 7.80 13.62 9.41 12.20 13.42 10.66 10.19 14.03 13.00 10.73 14.34 8.91 11.78 14.12 6.46 9.19 11.70 8.15 7.64 12.65 8.66 6.13

Page 29 of 37


Rk3

Rh4

20(S)-Rg3

20(R)-Rg3

20(S)-PPT

C-K

Rk1

Rg5

20(S)-PPD

5 200 20 2 500 50 8 50 5 1 50 5 1 100 20 2 100 20 2 200 50 5 500 50 8 200 50 5

4.60 170.08 18.63 1.81 446.55 42.11 6.07 37.39 4.11 0.89 39.02 4.06 0.94 87.97 16.74 1.84 75.09 18.43 1.80 165.80 37.01 3.88 399.05 43.03 6.85 162.30 41.82 4.51

92.00 85.04 93.15 90.50 89.31 84.22 75.88 74.78 82.20 89.00 78.04 81.20 94.00 87.97 83.70 92.00 75.09 92.15 90.00 82.90 74.02 77.60 79.81 86.06 85.63 81.15 83.64 90.20

14.67 6.94 11.53 10.89 8.72 4.94 7.12 9.14 5.61 12.60 8.03 13.15 11.00 6.57 12.02 11.40 9.78 12.49 14.21 5.00 8.47 10.70 10.09 8.91 6.74 7.47 11.24 8.12

4.56 180.30 19.06 1.82 471.10 44.43 6.88 43.98 4.58 0.93 44.45 4.34 0.92 95.87 18.17 1.88 85.33 17.54 1.70 172.56 42.85 4.31 434.25 44.21 7.12 172.56 43.56 4.67

29


91.20 90.15 95.30 91.00 94.22 88.86 86.00 87.96 91.60 93.00 88.90 86.80 92.00 95.87 90.85 94.00 85.33 87.70 85.00 86.28 85.70 86.20 86.85 88.42 89.00 86.28 87.12 93.40

12.90 9.02 8.63 14.24 7.35 10.89 9.49 10.86 7.20 14.53 7.51 12.24 9.59 8.50 10.24 12.48 6.67 8.13 14.19 6.67 11.15 9.61 11.90 10.78 9.84 10.50 13.18 11.58


Page 30 of 37

Table 5. Stability Profiles of the 23 Analytes at Three Different Levels in Rat Plasma. Analyte R1

Rg1

Re

Fa

Rb1

20(S)-R2

Fc

20(R)-R2

20(S)-Rg2

Spiked (ng/mL) 200 20 1 200 20 1 50 5 0.5 200 20 2 500 50 2 50 5 0.5 50 5 1 50 5 0.5 50

Post-preparation (24h, 4 °C) (n=6) Measured (ng/mL) Accuracy (%) 190.04±11.03 4.98 18.49±0.46 7.57 0.88±0.07 11.63 195.61±11.89 2.19 20.92±2.14 -4.61 1.09±0.11 -9.21 46.72±3.91 6.57 4.52±0.14 9.62 0.47±0.06 5.32 195.28±10.76 2.36 18.71±1.35 6.45 1.79±0.15 10.62 491.7±29.9 1.66 48.11±2.16 3.77 1.71±0.19 14.65 45.94±2.69 8.12 4.92±0.50 1.62 0.46±0.02 7.38 49.54±2.11 0.92 4.78±0.45 4.48 1.05±0.13 -5.28 46.38±3.79 7.24 4.76±0.53 4.82 0.43±0.06 13.31 52.49±2.54 -4.98

Freeze/thaw stability (n=6) Measured (ng/mL) Accuracy (%) 185.89±16.41 7.05 17.41±1.67 12.93 0.91±0.13 8.67 186.9±12.52 6.55 22.05±2.54 -10.26 1.06±0.15 -5.95 45.37±4.38 9.26 4.68±0.29 6.40 0.46±0.07 8.30 185.38±17.69 7.31 18.15±1.43 9.24 1.73±0.17 13.70 481.72±37.62 3.66 48.61±5.04 2.78 1.76±0.26 12.01 47.30±3.89 5.40 4.75±0.69 5.08 0.43±0.04 13.39 52.07±3.74 -4.15 4.63±0.53 7.30 1.12±0.16 -11.86 45.34±4.13 9.32 4.80±0.66 4.06 0.46±0.07 8.27 51.18±3.81 -2.36 30


Long-term stability (n=6) Measured (ng/mL) Accuracy (%) 188.44±12.91 5.78 17.88±0.83 10.62 0.90±0.12 10.23 189.75±14.4 5.13 21.05±2.03 -5.27 1.07±0.14 -7.45 46.47±3.93 7.06 4.59±0.23 8.15 0.47±0.06 6.27 192.47±14.7 3.76 18.49±1.67 7.57 1.72±0.14 14.06 473.38±26.37 5.32 46.17±3.94 7.66 1.73±0.17 13.39 46.49±3.22 7.02 4.81±0.63 3.70 0.44±0.03 11.10 51.79±1.84 -3.59 4.69±0.25 6.19 1.09±0.13 -9.10 46.09±3.29 7.82 4.68±0.61 6.40 0.45±0.06 10.40 49.59±3.05 0.82

Page 31 of 37


20(S)-Rh1

20(R)-Rh1

Rb2

Rd

F4

Rk3

Rh4

20(S)-Rg3

20(R)-Rg3

5 0.5 50 5 1 50 5 0.5 200 20 2 500 50 2 200 50 5 200 20 2 500 50 8 50 5 1 50 5 1

4.84±0.29 0.55±0.03 47.35±1.43 5.08±0.24 0.92±0.07 47.73±2.86 4.54±0.14 0.44±0.06 181.96±11.21 18.56±1.49 2.08±0.16 472.14±38.34 49.07±2.72 1.86±0.27 181.74±7.65 44.79±4.68 4.68±0.43 183.5±11.87 19.97±1.93 1.74±0.20 481.48±38.71 43.62±4.64 7.01±0.97 52.11±2.55 5.09±0.61 1.09±0.08 45.37±2.29 5.04±0.25 0.94±0.06

3.14 -10.11 5.29 -1.56 8.33 4.53 9.30 11.71 9.02 7.18 -3.81 5.57 1.87 7.15 9.13 10.42 6.33 8.25 0.17 12.90 3.70 12.75 12.40 -4.23 -1.87 -8.74 9.26 -0.83 6.11

4.75±0.47 0.51±0.05 46.08±3.85 4.77±0.45 0.90±0.13 46.84±4.01 4.42±0.19 0.43±0.06 179.35±12.36 19.13±1.79 1.93±0.20 462.31±31.67 44.35±4.51 1.78±0.22 182.69±11.60 43.77±3.69 4.47±0.63 179.62±8.21 21.91±2.83 1.78±0.27 453.82±34.08 44.74±5.37 6.89±0.94 54.97±4.47 4.83±0.63 1.14±0.16 47.36±3.43 5.18±0.36 1.05±0.12

5.06 -1.24 7.83 4.61 10.39 6.31 11.54 14.41 10.32 4.34 3.61 7.54 11.30 10.86 8.65 12.46 10.61 10.19 -9.53 11.13 9.24 10.52 13.93 -9.94 3.35 -14.33 5.28 -3.54 -5.15

31


4.69±0.32 0.53±0.05 46.53±2.93 4.95±0.38 0.89±0.12 46.45±3.51 4.58±0.25 0.44±0.06 182.93±14.87 19.64±1.25 2.02±0.14 466.17±35.99 45.28±4.04 1.85±0.21 179.51±13.21 45.3±2.40 4.34±0.62 182.63±12.53 20.76±2.13 1.79±0.24 468.68±41.24 45.2±4.38 7.10±0.75 51.61±3.22 4.92±0.55 1.10±0.13 45.96±2.46 5.42±0.47 1.01±0.09

6.21 -5.47 6.94 0.95 11.40 7.10 8.34 12.59 8.54 1.82 -0.85 6.77 9.44 7.29 10.24 9.40 13.27 8.68 -3.82 10.42 6.26 9.60 11.20 -3.21 1.52 -9.58 8.08 -8.34 -1.23


20(S)-PPT

C-K

Rk1

Rg5

20(S)-PPD

100 20 2 100 20 2 200 50 5 500 50 8 200 50 5

92.19±6.71 19.75±2.02 1.73±0.14 92.43±3.01 17.96±1.15 1.77±0.15 191.07±13.66 44.85±5.54 4.72±0.54 473.87±26.92 46.18±1.99 7.13±0.70 192.99±5.75 47.66±3.91 4.63±0.33

7.81 1.23 13.30 7.57 10.18 11.65 4.47 10.30 5.51 5.23 7.65 10.84 3.51 4.67 7.30

89.31±7.81 18.42±1.70 1.77±0.23 95.32±5.95 17.86±1.43 1.71±0.24 184.61±15.43 43.39±4.63 4.53±0.50 451.55±38.83 43.3±2.66 6.84±1.02 184.10±10.59 46.44±3.06 4.32±0.62

10.69 7.88 11.30 4.68 10.72 14.39 7.70 13.22 9.34 9.69 13.41 14.54 7.95 7.12 13.58

32


Page 32 of 37

90.25±5.68 18.87±1.61 1.73±0.25 93.63±5.19 18.17±1.29 1.79±0.16 186.62±8.30 43.83±5.25 4.64±0.64 463.45±41.94 43.76±5.31 6.82±0.75 188.6±11.84 45.73±3.91 4.44±0.56

9.75 5.67 13.34 6.37 9.13 10.62 6.69 12.34 7.15 7.31 12.49 14.24 5.70 8.55 11.19

Page 33 of 37


Table 6. Plasma PK Parameters of the 23 Analytes after Oral Gavage of 2.0 g/kg SNG in Rats. Analyte R1 Rg1 Re Fa Rb1 20(S)-R2 Fc 20(R)-R2 20(S)-Rg2 20(S)-Rh1 20(R)-Rh1 Rb2 Rd F4 Rk3 Rh4 20(S)-Rg3 20(R)-Rg3 20(S)-PPT C-K Rk1 Rg5 20(S)-PPD

Cmax (ng/mL) 71.97±17.61 78.01±11.34 17.07±3.85 180.38±31.89 2017.42±435.54 11.39±1.98 15.85±4.52 11.24±2.39 13.81±3.16 24.10±4.65 18.52±4.94 79.38±15.34 869.97±132.74 16.18±4.23 105.60±20.92 543.16±66.18 25.92±6.14 12.53±5.20 23.07±6.81 22.91±7.06 147.94±40.69 819.37±97.50 133.23±18.88

Tmax (h) 1.17±0.41 0.83±0.26 1.33±0.52 8.00 8.00 1.58±1.28 8.67±1.63 0.83±0.61 1.00±0.82 0.63±0.31 0.79±0.70 8.00 9.33±2.07 3.00±1.10 2.33±2.16 2.83±2.48 2.67±1.03 3.67±1.51 10.33±2.66 12.00 4.00±1.26 3.67±1.51 12.00

t1/2 (h) 7.64±3.69 20.94±9.83 7.48±1.85 27.89±16.33 14.71±2.29 18.51±5.32 17.13±4.45 4.61±1.84 15.17±6.80 12.26±4.28 4.47±1.77 18.19±6.18 14.83±3.28 17.46±4.17 10.17±2.79 9.05±1.40 15.54±7.07 11.99±5.21 18.8±7.57 18.54±7.13 19.71±3.83 14.75±4.25 21.35±3.48

MRT0→t (h) 9.79±2.48 14.96±2.36 11.27±2.68 26.42±2.54 22.52±3.21 14.80±2.63 24.54±2.62 6.81±1.67 12.77±4.17 13.30±3.54 6.22±1.30 23.67±3.29 22.13±3.09 15.03±2.19 14.28±2.06 13.60±1.31 17.50±2.10 15.14±2.41 27.90±5.30 19.51±2.38 18.78±2.20 16.29±1.79 28.65±1.20

MRT0→∞ (h) 12.77±4.54 32.99±11.47 14.24±5.55 43.89±9.45 26.53±3.66 28.42±5.42 31.76±5.69 8.78±3.12 20.21±8.63 25.01±10.63 7.42±2.46 34.09±11.04 30.49±9.26 25.31±7.49 16.25±2.45 14.57±1.68 22.02±4.93 19.68±7.46 62.71±25.93 29.27±8.24 26.12±5.11 20.55±3.21 38.23±3.38

33


AUC0→t (h· ng/mL) 636.70±152.98 844.34±196.80 173.29±33.17 4172.35±445.66 43459.32±6435.93 128.33±32.94 295.74±57.89 85.40±27.05 146.26±36.80 222.40±67.73 128.38±44.12 2315.7±423.28 19142.34±3078.84 234.59±62.95 1656.72±325.79 8765.08±749.69 504.18±94.56 228.56±64.52 666.92±157.98 446.05±60.15 2640.82±399.71 13167.37±1491.43 4067.3±223.56

AUC0→∞ (h· ng/mL) 653.39±156.84 1110.81±213.46 176.38±32.70 5221.28±912.00 45553.54±6171.77 151.31±27.75 336.49±63.34 97.21±38.75 160.95±56.27 280.64±76.49 135.05±53.01 2593.76±586.72 20115.62±3727.12 284.22±78.77 1679.73±350.48 8820.18±726.61 531.22±131.35 245.51±81.67 975.48±226.45 534.37±87.12 2904.28±518.18 13843.45±1898.79 4582.12±187.13


Figure 1.

34


Page 34 of 37

Page 35 of 37


Figure 2.

35



Figure 3.

36


Page 36 of 37

Page 37 of 37


Graphic for table of contents

37


Pharmacokinetic Characteristics of Steamed Notoginseng by an

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