Combinatorial Metabolism Notably Affects Human...

DMD #51391

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Combinatorial Metabolism Notably Affects

Human Systemic Exposure to Ginsenosides

from Orally Administered Extract of Panax

notoginseng Roots (Sanqi)

Zheyi Hu, Junling Yang, Chen Cheng, Yuhong Huang,

Feifei Du, Fengqing Wang, Wei Niu, Fang Xu, Rongrong

Jiang, Xiumei Gao, Chuan Li

Shanghai Institute of Materia Medica, Chinese Academy of Sciences,

Shanghai, China (Z.H., J.Y., C.C., F.D., F.W., W.N., F.X., R.J., C.L.); Tianjin

University of Traditional Chinese Medicine, Tianjin, China (Y.H., X.G.);

Institute of Chinese Materia Medica, China Academy of Chinese Medical

Sciences, Beijing, China (C.L.).

DMD Fast Forward. Published on May 6, 2013 as doi:10.1124/dmd.113.051391

Copyright 2013 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on May 6, 2013 as DOI: 10.1124/dmd.113.051391

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Running Title: PHARMACOKINETICS AND METABOLISM OF GINSENOSIDES IN

HUMANS

Address correspondence to: Dr. Chuan Li, Laboratory for DMPK Research

of Herbal Medicines, Shanghai Institute of Materia Medica, Chinese Academy

of Sciences, 501 Haike Road, Zhangjiang Hi-Tech Park, Shanghai 201203,

China. E-mail: [email protected]

Number of Text Pages: 22

Number of Figures: 9

Number of References: 24

Number of Words in Abstract Section: 235

Number of Words in Introduction Section: 714

Number of Words in Discussion Section: 1499

ABBREVIATIONS: AUC0-t, area under concentration-time curve to the last measured

time point; CLR, renal clearance; CLtot,P, total plasma clearance; Cmax, maximum plasma

concentration; Cum.Ae, cumulative amount excreted; CYP, cytochrome P450; HLM,

human liver microsomes; MRT, mean residence time; ppd-type, 20(S)-protopanaxadiol

type; ppt-type, 20(S)-protopanaxatriol type; PK, pharmacokinetic; Tmax, time taken to

achieve the maximum concentration


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ABSTRACT

Ginsenosides are medicinal ingredients of the cardiovascular herb Panax notoginseng

roots (Sanqi). Here, we implemented a human study (ChiCTR-ONC-09000603;

www.chictr.org) to characterize pharmacokinetics and metabolism of ginsenosides from

an orally ingested Sanqi extract (a 1:10 water extract of Sanqi) and the human plasma

and urine samples were analyzed by liquid chromatography-mass spectrometry. Plasma

and urinary compounds derived from ginsenosides included (1) intestinally absorbed

ginsenosides Ra3, Rb1, Rd, F2, Rg1, and notoginsenoside R1 and (2) the deglycosylated

products compound-K, 20(S)-protopanaxadiol, 20(S)-protopanaxatriol, and their oxidized

metabolites. The systemic exposure levels of the first group compounds increased as

the Sanqi extract dose increased, but those of the second group compounds were

dose-independent. The oxidized metabolites of 20(S)-protopanaxadiol and

20(S)-protopanaxatriol represented the major circulating forms of ginsenosides in the

bloodstream, despite their large interindividual differences in exposure level. The

metabolites were formed via combinatorial metabolism that consisted of a rate-limiting

step of ginsenoside deglycosylation by the colonic microflora and a subsequent step of

sapogenin oxidation by the enterohepatic cytochrome P450 enzymes. Significant

accumulation of plasma ginsenosides and the metabolites occurred in the human

subjects receiving three-week subchronic treatment with the Sanqi extract. Plasma

20(S)-protopanaxadiol and 20(S)-protopanaxatriol could be used as pharmacokinetic

markers to reflect the subject’s microbial activities, as well as the timely-changes and

interindividual differences in plasma levels of their respective oxidized metabolites. The

information gained from the current study is relevant to pharmacology and therapeutics

of Sanqi.


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Introduction

In China, the tradition of herbal medicine has prospered over millennia and is

still accepted by the physicians, the patients, and the regulatory authority. However,

the scientific standards for accepting the value of an herbal product as a medicine

fall below the existing requirements for establishing the value of synthetic drugs.

China has announced an ambitious attempt to bring the practice of traditional

medicine into line with modern standards (Qiu, 2007).

Phytochemists traditionally work in concert with ethnopharmacologists in

herbal medicine research, where whole herb extracts, as well as the individual

compounds isolated and purified from the herbs, are applied to in vivo and/or in

vitro pharmacological assessments. However, the studies often fail to address which

chemical ingredients present are the medicinal principles responsible for the

pharmacological effects of the investigative herbal medicine. The most abundant

constituents present in an herbal medicine do not necessarily produce the highest in

vivo concentrations (such as the plasma concentrations, the tissue concentrations, or

the like) after dosing, while in vivo measured herbal metabolites are usually absent

in the dosed medicine (Lu et al., 2008; Liu et al., 2009; Li et al., 2012; Yan et al.,

2012; Li, 2012). In other words, there can be significant differences in nature and

relative levels between the chemical ingredients present in an herbal medicine and

the circulating botanical compounds after dosing. The accurate and complete

information of pharmacokinetics and metabolism is vital to the pharmacological

research for identification of the medicinal principles. Accordingly, pharmacokinetic


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(PK) scientists are needed to participate in the herbal medicine research by working

with ethnopharmacologists and phytochemists in a manner similar to that for drug

discovery. Different from the early practice that was based only on pharmacological

assessment of a range of chemicals, the current drug discovery is normally achieved

by combining medicinal chemistry and pharmacology together with

pharmacokinetics, which has resulted in improved candidate selection for preclinical

and clinical development (Pritchard et al., 2003). PK and metabolism study of an

herbal medicine tells the ethnopharmacologists which herb-derived compounds are

worth further evaluation.

Sanqi (the dried root of Panax notoginseng, family Araliaceae) has been

commonly used in the Chinese traditional medicine to eliminate blood stasis, to

arrest bleeding, to cause subsidence of swelling, and to alleviate pain (Chinese

Pharmacopoeia Commission, 2010). The medicinal herb is usually administered

orally in such forms as powders, aqueous extracts, and teas, in daily dose ranging

from 2 g to 30 g of root but averaging 3–9 g. In addition, injections prepared from

Sanqi extract have also been approved for clinical use in China. The triterpene

saponins ginsenosides are believed to be the pharmacologically active ingredients of

Sanqi and can be classified according to their structures as 20(S)-protopanaxadiol

type (ppd-type) and 20(S)-protopanaxatriol type (ppt-type) (Christensen, 2009; Lv et

al., 2009). The major saponins present in Sanqi include the ppd-type ginsenosides

Ra3, Rb1, and Rd and the ppt-type ginsenosides Re, Rg1, and notoginsenoside R1.

Recently, we investigated intestinal absorption and disposition of ginsenosides in


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rats receiving an orally (p.o.) administered Sanqi extract and found that poor

membrane permeability, rapid biliary excretion, and colonic microflora-induced

deglycosylation were the key factors affecting the rat systemic exposure to

ginsenosides (Liu et al., 2009). The major ginsenosides measured in rat plasma were

long-circulating ppd-type ginsenosides Ra3, Rb1, and Rd, which can be used as

pharmacokinetic markers (PK markers) to substantiate rat systemic exposure to the

Sanqi extract. Meanwhile, ppt-type ginsenosides Re, Rg1, and notoginsenoside R1, as

well as the deglycosylated products of both the types, were subject to rapid biliary

excretion that limited their systemic exposure levels. Interspecies differences in

biliary excretion (Mahmood and Sahajwalla, 2002; Lai, 2009) and colonic

microflora-induced metabolism (Nicholson et al., 2005; Sousa et al., 2008)

complicate the extrapolation of human absorption and disposition of ginsenosides

from the animal data. Accordingly, we implemented a human study to characterize

pharmacokinetics and metabolism of ginsenosides from an orally ingested Sanqi

extract. One of our important findings is that several metabolites derived from

ginsenosides were measured as major circulating compounds after dosing. These

metabolites, absent in the administered herbal extract, were formed via

combinatorial metabolism that consisted of a rate-limiting step of ginsenoside

deglycosylation induced by the colonic microflora and a subsequent step of

sapogenin oxidation mediated by the enterohepatic cytochrome P450 enzymes (CYP

enzymes).


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Materials and Methods

A detailed description of materials and experimental procedures is provided in

the Supplemental Materials and Methods.

Materials. Sanqi-extract, for p.o. administration to human subjects, was a 1:10

water extract, i.e., 2 kg of dried roots of Panax notoginseng (Sanqi) was used to

make 20 l of the final extract. The prepared Sanqi-extract contained ginsenosides

Rg1 (2680 μM), Rb1 (1377 μM), Rd (366 μM), Re (254 μM), Rh1 (103 μM), Ra3 (76

μM), Rg2 (60 μM), F1 (48 μM), F2 (18 μM), Rg3 (17 μM), Rb2 (14 μM), Rf (13 μM),

Rc (3 μM), and Rh2 (1 μM), as well as notoginsenosides R1 (360 μM), R3/R6 (69

μM), and 20-gluco-ginsenoside Rf (33 μM). Only one batch of Sanqi-extract was

prepared and used for the whole study. The content levels of ginsenosides in

Sanqi-extract were measured immediately after preparation and after storage at 4ºC

for 1 and 3 months. Chemical stability was defined as the retention of ≥85% of the

initial levels. The herb extract was used after shaking well.

Purified ginsenosides, 20(S)-protopanaxadiol, and 20(S)-protopanaxatriol were

obtained from the National Institutes for Food and Drug Control (Beijing, China) or

other commercial sources, the purity of which exceeded 98%. Pooled human liver

microsomes (HLM) of Chinese origin were obtained from the Research Institute for

Liver Diseases (Shanghai, China). Single cDNA-expressed human cytochrome P450

(CYP) enzymes were obtained from BD Gentest (Woburn, MA).

Human Study. The protocol for human study was approved by the Ethics

Committee of Clinical Investigation at the Second Affiliated Hospital of Tianjin


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University of Traditional Chinese Medicine (Tianjin, China). The study was

registered on Chinese Clinical Trials Registry (www.chictr.org) with a registration

number of ChiCTR-ONC-09000603. The informed consent was obtained from each

of twelve male and twelve female healthy volunteers (22–26 yr).

A two-period, open-label, single center study was performed at the National

Clinical Research Center of the hospital (Tianjin, China; Fig. 1). In period 1, all the

subjects were randomly assigned to one of three dosage groups, with four male and

four female subjects per group. Each subject was asked to fast overnight before

receiving a single p.o. dose of Sanqi-extract (90, 180, or 270 ml/subject) in the next

morning. The low dose level was derived from the dose level recommended by the

Pharmacopoeia of the People’s Republic of China. The intermediate and high dose

levels were also used clinically. Serial blood samples (~1 ml collected in heparinized

tubes) were taken from an antecubital vein catheter at 0, 0.5, 1.5, 3, 4.5, 8, 12, 15, 24,

30, 38, 48, and 56 h after dosing. The blood samples were then centrifuged at 3000×

g for 5 min, and the resulting plasma fractions were frozen at −70°C until analysis.

Meanwhile, serial urine samples were also collected predose and at 0–3, 3–6, 6–10,

10–14, 14–24, 24–32, 32–40, 40–48, and 48–72 h postdose, which were weighed

prior to storage at −70°C without use of any preservative. In period 2 (after three-day

wash-out period following the first acute p.o. treatment with Sanqi-extract), the four

male subjects of the low-dose group continued to receive a subchronic p.o. treatment

with Sanqi-extract at 90 ml/subject/day for three weeks. On days 11, 18, and 24 of

period 2, the blood and urine samples were collected according to the preceding time


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schedules, except for the sampling only performed 0–24 h after the dosing on days

11 and 18. The hepatic and renal function of subjects was monitored before period 1

and 72 h after a single dose of Sanqi-extract, as well as on days 4, 12, 15, 19, 20, 21,

22, 23, and 25 (before the daily dose of Sanqi-extract) of period 2. Serum alanine

aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP),

albumin/globulin (A/G) ratio, total bilirubin (TBiL), and direct bilirubin (DBiL)

were monitored as liver function markers for the human subjects, while serum

creatinine (CRE) and blood urea nitrogen (BUN) were also assessed to monitor their

renal function. The reference ranges, indicating normal liver or kidney function, of

ALT, AST, TP, A/G ratio, TBiL, DBiL, CRE, and BUN were 0–40 U/l, 0–40 U/l,

64–87 g/l, 1–2.5, 0–17.1 μM, 0–8.0 μM, 22–106 μM, and 1.7–8.3 mM, respectively.

In Vitro Metabolism Studies. Pure 20(S)-protopanaxadiol,

20(S)-protopanaxatriol, and compound-K at 2 μM was incubated with HLM in

presence of the cofactor(s) NADPH, UDPGA, or the both to assess metabolic stability

and to support metabolite characterization. A variety of cDNA-expressed human CYP

enzymes (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6,

CYP2E1, CYP3A4, and CYP3A5) were used for the enzyme identification. To

determine the enzyme kinetics, 20(S)-protopanaxadiol (1.56–200 μM) or

20(S)-protopanaxatriol (0.08–200 μM) was incubated with NADPH-fortified HLM

under linear metabolite formation conditions, which were performed in duplicate in

96-well plates.

Liquid Chromatography-Mass Spectrometry Analyses. An AB Sciex API


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4000 Q Trap mass spectrometer (Toronto, Canada) interfaced via a Turbo V ion

source with a Waters Acquity UPLC separation module (Milford, MA, USA) was

used for metabolite profiling and quantification for study purposes. The human

plasma and urine samples (50 μl) were precipitated with 150 μl of methanol before

measurement. Meanwhile, in vitro metabolism study samples (100 μl) were treated

with an equal volume of methanol to stop the reaction before analysis. Validation of

the quantification assays with the reference standards available were implemented

according to the US Food and Drug Administration guide on bioanalytical method

validation (http://www.fda.gov/cder/guidance/index.htm) to demonstrate that their

performance characteristics were reliable for the intended use.

Data Analysis. PK parameters were determined by non-compartmental method

using the Kinetica 5.0 (Thermo Fisher Scientific; Philadelphia, PA), which were

shown as means ± standard deviation. Dose proportionality assessment was conducted

by the regression of log-transformed data (the Power Model) with the criterion that

was calculated according to the method by Smith et al. (2000). Michaelis constant (Km)

and maximum velocity (Vmax) values were determined by nonlinear regression

analysis using the Michaelis-Menten equation (rate of metabolite formation as a

function of substrate concentration) using GraFit software (version 5; Erithacus

Software Ltd., Surrey, UK). The ratio of Vmax and Km was used to calculate in vitro

intrinsic clearance (CLint). The Shapiro-Wilk test of normality was used to check the

distribution shape of systemic exposure levels of ginsenoside-derived compounds in

AUC0-t. The Kendall’s tau-b coefficients were calculated using PASW statistics 18


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software (SPSS Inc., Chicago, IL) to estimate whether the systemic exposure levels of

a certain ginsenoside-derived compound were positively or negatively correlated to

those of another ginsenoside-derived compound. Correlation was statistically

significant at P < 0.01 level (two-tailed).


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Results

Safety of Sanqi-extract. No serious adverse event was observed during the

human study. After a p.o. dose of Sanqi-extract at 90, 180, or 270 ml/subject, none

of liver or kidney function indicators exceeded the upper normal levels. During the

subsequent multiple dose treatment, the four male subjects (m1–m4; receiving

Sanqi-extract at 90 ml/subject/day) had the hepatic or renal function indicators

within the normal ranges during the study period, except for some transient elevation

of alanine aminotransferase (ALT) for subjects m2–m4. Subject m3 had elevated

ALT (54–79 U/l) on days 15–22. This subject withdrew from the study after day 18

to let his elevated ALT return to the normal level (<40 U/l).

Appearance of Plasma and Urine Ginsenosides and Metabolites after p.o.

Administration of Sanqi-extract. Ginsenosides and their deglycosylated products

occurred in human plasma and urine after a p.o. dose of Sanqi-extract (270

ml/subject), which were compared with the profiles of the control plasma and urine

samples before dosing. As shown in Fig. 2B, Ppd-type ginsenoside Rb1 was the most

abundant plasma Sanqi saponin absorbed, while other saponins measured in plasma

were ppd-type ginsenosides Ra3, Rd, F2, Rg3, and Rh2, as well as ppt-type

ginsenosides Re, Rg1, F1, Rh1, and notoginsenoside R1. These plasma ginsenosides

were also excreted into urine (Fig. 2C), except ginsenosides Rg3 and Rh2.

Meanwhile, 20-gluco-ginsenoside Rf and notoginsenoside R3/6 were measured in

urine but not in plasma. Although ppt-type ginsenoside Rg1 had a higher content

level in the administered Sanqi-extract than ginsenoside Rb1, its plasma abundance


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was significantly lower than that of the ppd-type ginsenoside (Fig. 2B). However,

urinary ginsenoside Rg1 was the most abundant of all the ginsenosides excreted into

urine (Fig. 2C). Notably, the deglycosylated products compound-K,

20(S)-protopanaxadiol, and 20(S)-protopanaxatriol, absent in the ingested

Sanqi-extract (Fig. 2A), also occurred substantially in plasma with

20(S)-protopanaxatriol being ~3-times more abundant than plasma ginsenoside Rb1

(Fig. 2B).

Although 20(S)-protopanaxadiol and 20(S)-protopanaxatriol circulated

significantly in the bloodstream, they are not measured in the urine samples. To

understand the elimination of the deglycosylated products, their metabolism was

further assessed. A total of 29 metabolites of the deglycosylated products were

detected in the plasma samples (Fig. 2D). Plasma M8, M11, and M12 had their

abundances 1.2–11 times higher than 20(S)-protopanaxatriol, while the abundances of

plasma M3–M7, M9, M10, and M13–M22 were 7–72% of that of the sapogenin.

Meanwhile, a total of 29 urinary metabolites were measured (Fig. 2E). The plasma

metabolites M17–M22 were absent in urine, while the urinary metabolites M30 and

M31–M35 were not measured in plasma. The most abundant metabolites in urine

were M8 and M6, i.e., 131% and 90% of urinary ginsenoside Rg1, respectively, while

urinary M3, M5, M7, M11–M13, M23–M25, M27, M29, and M35 were only

10%–43%. It is worth mentioning that the preceding plasma and urine metabolites

(M1–M35) were absent in the administered Sanqi-extract. As to the ginsenosides

present in Sanqi-extract, their oxidized or conjugated metabolites were not measured


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in the human samples.

In Vitro Metabolism of 20(S)-Protopanaxadiol, 20(S)-Protopanaxatriol,

and Compound-K. To help characterize the preceding in vivo metabolites

(M1–M35), in vitro metabolism studies were implemented with HLM.

20(S)-Protopanaxadiol or 20(S)-protopanaxatriol was labile when incubating with

NADPH-fortified HLM (in vitro t1/2, 6 and 4 min, respectively; Supplemental Table

1). Furthermore, both the sapogenins were quite stable with UDPGA-fortified HLM

(>120 min). These data suggested that the oxidation of the sapogenins was rapid in

the liver, while the glucuronidation was relatively slow. Compound-K was slowly

metabolized with the human microsomal enzymes (>120 min). According to the in

vitro data (Supplemental Table 1), M16–M22, M34, and M35 were characterized as

the oxidized metabolites of 20(S)-protopanaxadiol, while M3–M15, M23–M29, and

M31–M33 were derived from 20(S)-protopanaxatriol. M1, M2, and M30 were the

metabolites of compound-K. Figure 3 depicts the proposed metabolic pathways of

the deglycosylated products in the humans.

Human CYP3A4 and CYP3A5 were the major enzymes responsible for the

oxidation of 20(S)-protopanaxadiol or 20(S)-protopanaxatriol (Fig. 4). In addition,

CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and

CYP2E1 could also mediate the oxidation of 20(S)-protopanaxadiol. The Michaelis

constants of HLM towards the formation of M20/M21/M22 from

20(S)-protopanaxadiol and those for M8/M10/M11/M12 from 20(S)-protopanaxatriol

were 4.0–6.6 and 0.2–1.1 μM, respectively (Supplemental Table 2).


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Plasma Pharmacokinetics and Urinary Excretion of Ginsenosides and their

Major Metabolites after p.o. Administration of Sanqi-extract. Figure 5 shows

plasma level-time profiles of the major ginsenosides after a p.o. dose of

Sanqi-extract to the human subjects, as well as those of the deglycosylated products

[compound-K, 20(S)-protopanaxadiol, and 20(S)-protopanaxatriol] and the oxidized

metabolites of the sapogenins, while the plasma PK parameters are summarized in

Supplemental Tables 3–5. After dosing at 270 ml/subject, the plasma concentrations

of ppd-type ginsenosides Ra3, Rb1, and Rd increased up to Cmax (3.1, 27.9, and 9.3

nM, respectively) with Tmax of 8–12 h and then slowly decreased with t1/2 of 33–57 h.

Their MRT values were 50–82 h. The Tmax and MRT of ppd-type ginsenoside F2

were 13 h and 22–29 h, respectively. The renal excretion of ginsenosides Ra3, Rb1,

Rd, and F2 was slow with CLR of 0.01–0.04 l/h and their urinary Cum.Ae(0→72h)

accounted for only ~0.01% of the compound doses from the p.o. ingested

Sanqi-extract. The AUC0-56h of ginsenosides Ra3, Rb1, Rd, and F2 increased in

Sanqi-extract dose-related manners (90–270 ml/subject), but the dose proportionality

was inconclusive (Fig. 6). Compound-K exhibited a significantly higher mean

AUC0-56h than ginsenoside Rb1, demonstrating 4749 versus 749 nM·h after dosing at

270 ml/subject, while the former’s interindividual difference in AUC0-56h was

significantly larger than that of the latter. Meanwhile, compound-K had a Tmax of

10–30 h and an MRT of 23–26 h. The average CLR value of compound-K was very

low (0.01 l/h). Significant interindividual difference in plasma occurrence was also

observed with 20(S)-protopanaxadiol. The AUC0-56h value of compound-K or


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20(S)-protopanaxadiol correlated poorly with the dose of Sanqi-extract (Fig. 6). In

addition, compound-K or 20(S)-protopanaxadiol was correlated poorly with

ginsenosides Ra3, Rb1, Rd, and F2 in AUC0-56h for the individual subjects (Fig. 7).

Compound-K was also correlated poorly with 20(S)-protopanaxadiol.

At the Sanqi-extract dose 270 ml/subject, ppt-type ginsenoside Rg1 and

notoginsenoside R1 had Tmax of 6–7 h and Cmax values of 10.2 and 1.4 nM,

respectively. Their MRT values were 7–13 h. The renal excretion of these ppt-type

ginsenosides was rapid with CLR of 5.6 and 4.0 l/h, respectively, while their urinary

Cum.Ae(0-24h) accounted for ~0.06% of the compound doses from Sanqi-extract. The

AUC0-38h values of ginsenoside Rg1 and notoginsenoside R1 increased in Sanqi-extract

dose-related manners, but lack of proportionality was concluded (Fig. 6). Ppt-type

ginsenoside F1 had a Tmax of 14–19 h and a Cmax of 4.3 nM. Its CLR was 0.7 l/h.

Although 20(S)-protopanaxatriol had higher systemic exposure as compared with the

major Sanqi ginsenosides, the interindividual difference was larger. The AUC0-56h of

20(S)-protopanaxatriol was independent of the Sanqi-extract dose (Fig. 6), which was

also not correlated with AUC0-38h of ginsenosides Rg1, F1, or notoginsenoside R1 for

the individual subjects (Fig. 7). There appeared to be correlation between

20(S)-protopanaxatriol and 20(S)-protopanaxadiol in AUC0-56h (P < 0.01).

As shown in Fig. 5, the plasma concentrations of M16, M17, and M19–M22

increased and then decreased concurrently as the concentration of their precursor

20(S)-protopanaxadiol changed with time. In addition, there were significantly

positive correlations in AUC0-56h between 20(S)-protopanaxadiol and these


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metabolites for the individual subjects (P < 0.01; Fig. 7). Meanwhile, the plasma

concentrations of M4–M8 and M10–M15 also changed concomitantly with that of

20(S)-protopanaxatriol and significant correlations existed between the sapogenin and

its oxidized metabolites (P < 0.01). Similar to the situations of their parent sapogenins,

the AUC0-56h value of the preceding oxidized metabolites appeared to be Sanqi-extract

dose-independent (Fig. 6). With respect to the renal excretion, the ppd-derived

metabolites were not or negligibly recovered in the human urine after oral

administration of San qi-extract. In contrast, many of the ppt-derived metabolites (in

the ranking order of M6>M7>M5≈M13>M8) were notably excreted into urine with

CLR of 0.1–1.3 l/h.

Figure 8 depicts AUC0-24h changes of ginsenosides and the metabolites for the

subjects m1, m2, and m4 who received Sanqi-extract for additional 21 days. The

AUC0-24h of ginsenosides Ra3, Rb1, and Rd increased significantly during the

multiple-dose treatment, indicative of 1.9–11.3-fold increases on day 11 as compared

with the data on day 1 and 1.3–10.3-fold increases on day 18. However, the AUC0-24h

values on day 24 tended to decrease for these subjects. The AUC0-24h values of

compound-K and ginsenosides F1 and F2 did not exhibit a clear initial increase and

eventual decrease. Subjects m1 and m2 had notable increases in AUC0-24h values of

ginsenoside Rg1 and notoginsenoside R1 on day 18 (3.5–7.4-fold of the day 1 data)

and decreases on day 24 (22%–55% of the day 18 data). 20(S)-Protopanaxadiol and

20(S)-protopanaxatriol had significantly increased AUC0-24h values on days 11, 18,

and 24 as compared with the data on day1. Notably, the AUC0-24h values of


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ppd-derived M16, M17, and M19–M22 increased and decreased as the AUC0-24h

value of 20(S)-protopanaxadiol changed with time (Fig. 8). Similar situation occurred

with 20(S)-protopanaxatriol and its metabolites M4–M8 and M10–M15 (Fig. 8). In

addition, there were significantly positive correlations in AUC0-24h between

20(S)-protopanaxadiol and its metabolites M16, M17, and M19–M22 and between

20(S)-protopanaxatriol and the metabolites M4–M8 and M10–M15 (P < 0.01; Fig. 9).


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Discussion

Unlike synthetic drugs, herbal medicines are complex chemical mixtures.

Benefits of herb therapy are normally based on a range of activities of compounds

working together. Accordingly, ADME/PK study of an herb medicine involves

investigation of multiple botanical compounds to link the medicine administration and

the medicinal benefits. An earlier systematic ADME/PK study of a Sanqi extract was

performed in rats (Liu et al., 2009), which provided the indispensable guidance for us

to implement strategically this follow-up multi-compound human PK study.

Similar to the situation in rats, the ppd-type ginsenosides Ra3, Rb1, and Rd from

p.o. dosed Sanqi-extract had long elimination half-lives (>30 h) in the human subjects

and significant accumulation in AUC0-24h during the multiple-dose treatment. Their

systemic exposure levels increased in a Sanqi-extract dose-dependent manner (90–270

ml/subject). These plasma ginsenosides were considered to be Sanqi-extract

dose-dependent PK markers to substantiate human systemic exposure to the oral herb

extract. Unlike the situation in rats, the human systemic exposure levels of the

ppt-type ginsenoside Rg1 and notoginsenoside R1 and the ppd-type ginsenoside F2

also increased in Sanqi-extract dose-related manners. Poorer biliary excretion in

humans than in rats (Mahmood and Sahajwalla, 2002; Lai, 2009) likely accounted for

the interspecies differences. Accordingly, plasma ginsenosides Rg1, F2 and

notoginsenoside R1 could also be used as Sanqi-extract dose-dependent PK markers in

humans.

The most important finding of this human study was the substantially measured


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plasma metabolites that resulted from the combinatorial metabolism of ginsenosides

(consisting of the saponin deglycosylation by the colonic microflora and the

subsequent sapogenin oxidation by the enterohepatic CYP enzymes). Deglycosylation

of ginsenosides via action of the colonic microflora has been demonstrated in vitro

and in vivo for years (Hasegawa et al., 1996; Tawab et al., 2003; Yang et al., 2006)

and the human CYP-mediated oxidation of the sapogenins has also been assessed in

vitro recently (Kasai et al., 2000; Hao et al., 2010; Li et al., 2011). Unlike the situation

in humans, these metabolites were poorly detected in rat plasma after p.o. dose of

Sanqi extract (Liu et al., 2009). The human colon is heavily bacterial-colonized as

compared with the rat colon (Sousa et al., 2008), which likely accounts for the

observed metabolism interspecies difference. The amount of variation in the colonic

microflora is considerable between human individuals (Nicholson et al., 2005). In this

study, we observed marked interindividual differences in formation of the

deglycosylated compound-K, 20(S)-protopanaxadiol, and 20(S)-protopanaxatriol.

Notably, the systemic exposure levels of oxidized metabolites of the sapogenins,

regardless of the sex of subject or the Sanqi-extract dose level, were well correlated

with those of the sapogenins, rather than the levels of the ginsenosides absorbed (Fig.

7 and 9). The colonic microflora-mediated deglycosylation appeared to be the

rate-limiting step in the combinatorial metabolism of ginsenosides. As shown in Fig. 5,

although the Tmax values of compound-K, 20(S)-protopanaxadiol, and

20(S)-protopanaxatriol were delayed ~20 h as compared with those of the

ginsenosides, these deglycosylated products and their oxidized metabolites were


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almost identical in Tmax. Even if the gastrointestinal transit time (normally 4–5 h) to

colon is taken into consideration, these data still suggested that the deglycosylations

were significantly slower than the enterohepatic oxidations. In addition, the

deglycosylation activities of the colonic microflora appeared to be induced by the

unabsorbed ginsenosides during the subchronic treatment with Sanqi-extract. Despite

the structural similarity, multiple CYP enzymes mediated the oxidation of

20(S)-protopanaxadiol, whereas only CYP3A4 and CYP3A5 were implicated in the

metabolism of 20(S)-protopanaxatriol. This finding suggests that the

20(S)-protopanaxadiol oxidation is relatively refractory to the effects of

CYP-inhibitors. The oxidized metabolites from the sapogenins were likely cleared via

renal excretion and/or glucuronidation-associated biliary excretion (Supplemental

Figure 1).

Besides the preceding dose-dependent PK markers, we further proposed two

other concepts of dose-independent PK markers for Sanqi-extract. The colonic

microflora mediating the ginsenoside deglycosylation played a key role in the

combinatorial metabolism. The plasma compound-K, 20(S)-protopanaxadiol, and

20(S)-protopanaxatriol could be used as PK markers to reflect the interindividual

differences in deglycosylation activities of the colonic microflora. This proposal is

also supported by in vitro formation of these deglycosylated products from

ginsenosides via action of the human fecal microflora under anaerobic conditions

(Hasegawa et al., 1996). In addition, the plasma concentration of

20(S)-protopanaxadiol and the plasma levels of oxidized metabolites (M16–M22)


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concomitantly increased up to maxima and then decreased with almost the same Tmax

and MRT values. The same situation also occurred with 20(S)-protopanaxatriol and its

oxidized metabolites (M4–M8 and M10–M15). Significant correlations in AUC (Fig.

7 and 9) between 20(S)-protopanaxadiol and the ppd-derived metabolites and those

between 20(S)-protopanaxatriol and the ppt-derived metabolites suggested that the

plasma sapogenins could be used as PK markers to reflect the timely-changes and

interindividual differences in plasma levels of their respective oxidized metabolites. In

pharmacological studies of herbal medicines, the intensity of pharmacological

response was often found to be correlated poorly with the dose. Unlike the absorbed

ginsenosides, the plasma levels of the ppd-derived or ppt-derived metabolites were

dependent on the human individual’s deglycosylation activities of colonic microflora

rather than the Sanqi-extract dose. The information gained and the associated PK

markers identified may be implicated when the dose-response relationship is assessed

for Sanqi in humans.

Poor oral bioavailability of many herbal constituents is often associated with

their low levels of systemic exposure, which impedes translation of many in vitro

pharmacological activities of the compounds to the therapeutic effects of herbal

medicines (Zhang et al., 2012). However, we found that ppd-type ginsenosides Ra3,

Rb1, and Rd from a p.o. ingested Sanqi extract had considerably high plasma levels in

rats despite their <1% oral bioavailability (Liu et al., 2009). These ppd-type

ginsenosides were long-circulating because their elimination kinetics was associated

with high metabolic stability and slow biliary and urinary excretion. In this human


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study, we found that the deglycosylated products compound-K, 20(S)-protopanaxadiol,

20(S)-protopanaxatriol, and their many oxidized metabolites had high plasma levels

and accumulated substantially with repeated doses of Sanqi-extract. Assessing the

pharmacological activities of these herbal compounds with significant exposure levels

after dosing is important for evaluation of oral Sanqi’s therapeutic values.

Accordingly, additional studies are planned to make pharmacological assessments of

the herbal metabolites found in this study.

Considerable hepatotoxicity and nephrotoxicity were observed in rats receiving

multiple p.o. doses of “notoginseng total saponin” (containing ginsenosides Rg1, Rb1,

Rd, Re, and notoginsenoside R1) at 1600 mg/day/kg for thirteen weeks (Ma and Dai,

2011). Our earlier rat Sanqi study indicated that the major ppt-type ginsenosides Rg1,

Re, and notoginsenoside R1, as well as the ppd-type compound-K, were subject to

rapid biliary excretion via an active transport mechanism and had high liver levels

(Liu et al., 2009). In this study, no significant hepatotoxicity and nephrotoxicity were

observed for the human subjects receiving oral Sanqi-extract, except for the subject

m3. Although this subject had obviously increased ALT during the multiple-dose

treatment, his plasma levels of ginsenosides and the metabolites were not high as

compared with the other subjects. According to dose normalization by body surface

area (Reagan-Shaw et al., 2007), the daily doses (800–1600 mg/kg) in the rat study by

Ma and Dai (2011) were about 20–40 times greater than the daily dose for our

subchronic treatment of the human subjects m1–m4 with Sanqi-extract (90

ml/subject). Although the potential effects of ginsenosides and their metabolites on


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the hepatic and renal function need to be confirmed by more studies, it is nonetheless

wise to exercise caution when high dose levels of Sanqi extracts or the associated

pharmaceutical products are administered for a long period.

In pharmacological assessment of an herbal medicine, it is important to

understand not only its chemical content but also which herb-derived compounds

circulate considerably in the bloodstream after dosing. In this study, notable

differences were found between the circulating compounds derived from ginsenosides

after p.o. ingestion of Sanqi-extract and the ginsenosides present in the extract. Our

study provides information for the ethnopharmacologists on which Sanqi-derived

compounds are worth further evaluation, while it also tells the phytochemists which

Sanqi-derived compounds should be prepared for the pharmacological and safety

assessments. In summary, 20(S)-protopanaxadiol, 20(S)-protopanaxatriol, and their

oxidized metabolites, as well as compound-K, represent major circulating forms of

ginsenosides in the human bloodstream after p.o. ingestion of Sanqi-extract. However,

the systemic exposure levels of these metabolites are of large interindividual variance,

which depend on the deglycosylation activities of the colonic microflora of individual

subjects, rather than the Sanqi-extract dosage size. Compound-K,

20(S)-protopanaxadiol, and 20(S)-protopanaxatriol measured in human plasma can be

used as PK markers to reflect the microbial activities of human individuals. In

addition, plasma 20(S)-protopanaxadiol and 20(S)-protopanaxatriol can also serve as

PK markers to indicate the timely-changes and interindividual differences in plasma

levels of their respective oxidized metabolites. The ppd-type ginsenosides Ra3, Rb1


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and Rd, as well as the ppt-type ginsenosides Rg1, F2, and notoginsenoside R1, can be

used as Sanqi-extract dose-dependent PK markers to substantiate human systemic

exposure to the orally ingested extract. Most plasma ginsenosides, as well as their

metabolites, accumulate substantially in the systemic circulation during multi-dose

treatment with oral Sanqi-extract.


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Authorship Contributions

Participated in research design: Li, Yang, Huang, and Gao

Conducted experiments: Hu, Yang, Cheng, Du, Huang, Wang, Niu, and Xu

Performed data analysis: Li, Hu, Yang, Cheng, and Jiang

Wrote or contributed to the writing of the manuscript: Li, Hu, Yang, and Cheng


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gastrointestinal microbiota as a site for the biotransformation of drugs. Int J Pharm 363:1–25.

Tawab MA, Bahr U, Karas M, Wurglics M, and Schubert-Zsilavecz M (2003) Degradation of

ginsenosides in humans after oral administration. Drug Metab Dispos 31:1065–1071.

Yan R, Ko NL, Ma B, Tam YK, and Lin G (2012) Metabolic conversion from co-existing

ingredient leading to significant systemic exposure of Z-butylidenephthalide, a minor ingredient

in Chuanxiong Rhizoma in rats. Curr Drug Metab 13:524–534.

Yang L, Liu Y, and Liu C-X (2006) Metabolism and pharmacokinetics of ginsenosides. Asian J

Pharmacodyn Pharmacokinet 6:103–120.

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Footnotes

Zheyi Hu, Junling Yang, and Chen Cheng contributed equally to the study. This work was

supported by the National Science Fund of China for Distinguished Young Scholars [Grant

30925044], the National Basic Research Program of China [Grant 2012CB518403], the National

Science and Technology Major Project of China “Key New Drug Creation and Manufacturing

Program” [Grants 2009ZX09304-002, 2012ZX09304007, and 2012ZX09303010], and the

Knowledge Innovation Program of the Chinese Academy of Sciences [Grant

KSCX2-YW-R-191].


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Legends for Figures

Fig. 1. Flowchart for the two-period, open-label, single center human study. Twenty four subjects

were randomly assigned to one of three dosage groups (four males and four females per group). After

the random assignment, the subjects were renumbered before receiving a p.o. dose of Sanqi-extract at

90 (the male subjects m1–m4 and the female subjects f1–f4), 180 (m5–m8 and f5–f8), or 270

ml/subject (m9–m12 and f9–f12). In addition, the subjects m1–m4, after three-day wash-out period

following the first acute p.o. treatment with Sanqi-extract, continued to receive multiple p.o. doses of

the herb extract at 90 ml/subject/day for three weeks. Their blood and urine samples were collected

on days 11, 18, and 24. “HF/RF” denotes hepatic function and renal function.

Fig. 2. Ginsenosides present in Sanqi-extract (panel A), as well as plasma (panels B and D) and

urinary ginsenosides and their metabolites measured (panels C and E) after p.o. administration of

Sanqi-extract (270 ml/subject). The blood was sampled from the human subjects 8 and 24 h after

dosing, which normally had high levels of the ginsenosides absorbed and those of the metabolites

formed, respectively. After centrifugation, all the plasma fractions (10 μl from each) were pooled

before analysis. Meanwhile, the urine samples (0–24 h after dosing) were also collected from these

subjects and the sample combining (20 μl from each) was also performed for analysis. Compound-K,

20(S)-protopanaxadiol, and 20(S)-protopanaxatriol were not present in Sanqi-extract; they were

ginsenosides’ metabolites measured in plasma. The symbol “×” (in red) denotes the ginsenoside or

metabolite not measured in human or urine. Blue, red, and black bars are used to show the content

levels of ginsenosides in Sanqi-extract, their plasma levels, and their urine levels, respectively, while

pink and grey bars exhibit the plasma and the urine levels of ginsenosides’ metabolites, respectively.

The response was determined as compound peak area corrected with the intensity ratio of the

fragment ion to the precursor ion. Relative abundance measures are expressed in percentile, with 100

being 20(S)-protopanaxatriol in the plasma data set and with 100 being ginsenoside Rg1 in the urinary

data set.

Fig. 3. Proposed metabolism pathways of compound-K, 20(S)-protopanaxadiol, and

20(S)-protopanaxatriol in humans. The metabolites in the brackets are proposed intermediates, which

were not measured in the samples of human plasma (HP) or human urine (HU). The metabolites M36,


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M37, and M38 were the glucuronides of M20, M21, and M22, respectively, which were measured in

the samples of rat bile (RB) after i.v. administration of 20(S)-protopanaxadiol.

Fig. 4. Human CYP enzymes mediating the oxidation of 20(S)-protopanaxadiol or that of

20(S)-protopanaxatriol. The substrate and enzyme concentration used were 2 μM and 50 pmol

P450/ml, respectively, for all the test cDNA-expressed CYP enzymes and the incubation time was 5

min (panels A and B) or 30 min (panels C and D). The response was determined as compound peak

area corrected with the intensity ratio of the fragment ion to the precursor ion. The relative abundance

was expressed as percentile, with 100, in the data sets, being the most abundant M8 that was formed

after incubation with CYP3A4 for 30 min.

Fig. 5. Mean plasma concentration-time profiles of ginsenosides and their major metabolites after a

single p.o. dose of Sanqi-extract at 90 (green solid lines), 180 (blue solid lines), or 270 ml/subject (red

solid lines). The subject f12 (receiving Sanqi-extract at 270 ml/subject; red dashed lines) exhibited

marked extensive but delayed formation of 20(S)-protopanaxadiol and 20(S)-protopanaxatriol, and

their oxidized metabolites.

Fig. 6. AUC0-56h values of compound-K, 20(S)-protopanaxadiol, 20(S)-protopanaxatriol, the

ppd-derived metabolites (M16, M17, and M19–M22), and the ppt-derived metabolites (M4–M8 and

M10–M15) being Sanqi-extract dose-independent. The exposure values were measured in human

subjects (solid circles, the male subjects; open circles, the female subjects) receiving a p.o. dose of

Sanqi-extract at 90 (green circles), 180 (blue circles), or 270 ml/subject (red circles). Unlike the

metabolites’ situations, AUC0-56h values of ginsenoside Ra3, Rb1, Rd, F2, Rg1, and notoginsenoside R1

increased in Sanqi-extract dose-dependent manners. The term “r” denotes the correlation coefficient.

The ratio of dose-normalized geometric mean AUC values is given before the parentheses, while the

numbers in the parentheses are the statistically derived 90% confidence interval. The critical interval

was 0.797–1.203.

Fig. 7. Strong positive correlations existing in AUC0-56h between 20(S)-protopanaxadiol and its

oxidized metabolite (M16, M17, M19, M20, M21, or M22), between 20(S)-protopanaxatriol and its

oxidized metabolite (M4, M5, M6, M7, M8, M10, M11, M12, M13, M14, or M15), and between

20(S)-protopanaxadiol and 20(S)-protopanaxatriol in the human subjects (solid circles, the male


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subjects; open circles, the female subjects) receiving a single p.o. dose of Sanqi-extract at 90 (green

circles), 180 (blue circles) or 270 ml/subject (red circles). Meanwhile, no significant correlation occur

in AUC0-56h between compound K and ginsenoside Ra3, Rb1, Rd, or F2, or 20(S)-protopanaxadiol,

between 20(S)-protopanaxadiol and ginsenoside Ra3, Rb1, Rd, or F2, between 20(S)-protopanaxatriol

and notoginsenoside R1, ginsenoside Rg1, or F1.

Fig. 8. Plasma AUC0-24h of ginsenosides and their major metabolites changing with time in the

subjects m1–m4 who received Sanqi-extract at 90 ml/subject/day (green solid lines) for three weeks.

Fig. 9. Strong positive correlations existing in AUC0-24h between 20(S)-protopanaxadiol and its

oxidized metabolite (M16, M17, M19, M20, M21, or M22), between 20(S)-protopanaxatriol and its

oxidized metabolite (M4, M5, M6, M7, M8, M10, M11, M12, M13, M14, or M15), and between

20(S)-protopanaxadiol and 20(S)-protopanaxatriol in the human subjects (m1–m4) who participated in

the multiple dose study. Meanwhile, no significant correlation occurred in AUC0-24h between

compound K and Ra3, Rb1, Rd, or F2, or 20(S)-protopanaxadiol, between 20(S)-protopanaxadiol and

ginsenoside Ra3, Rb1, Rd, or F2, between and notoginsenoside R1, ginsenoside Rg1, or F1. The

exposure values were measured on days 1, 11, 18 and 24 (for m1, m2 and m4 only) after initiation of

the multiple dose study.


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Figure 1

11 12 13 14

Blood/urinesampling

for PK study

HF/RFassessmentof all the 24 subjects

7 8 9 103 4 5 60 1 2 15 16 17 18 19 20 21 22 23 24 25

Period 1 Period 2

HF/RFassessmentof m1–m4 & f1–f4

HF/RFassessmentof m1–m4

HF/RFassessmentof m3 & m4


HF/RFassessment

of m4

Blood/urine sampling

for PK study

Blood/urinesampling

for PK study


for PK study

Daily dose at90 ml/subject to

m1–m4

The subject m3withdrew from the study.

Single dose at90 ml/subject to

m1–m4 & f1–f4


m5–m8 & f5–f8

Single dose at270 ml/subject tom9–m12 & f9–f12


Study day

HF/RFassessmentof m3m9–m12& f9–f12


HF/RFassessmentof m5–m8 &f5–f8

HF/RFassessmentof m3

11 12 13 14

Blood/urinesampling

for PK study

HF/RFassessmentof all the 24 subjects

7 8 9 103 4 5 60 1 2 15 16 17 18 19 20 21 22 23 24 25

Period 1 Period 2

HF/RFassessmentof m1–m4 & f1–f4


HF/RFassessmentof m3 & m4


HF/RFassessment

of m4


for PK study

Blood/urinesampling

for PK study

Blood/urinesampling

for PK study


for PK study


m1–m4


m1–m4




m1–m4 & f1–f4


m1–m4 & f1–f4


m5–m8 & f5–f8

Single dose at270 ml/subject tom9–m12 & f9–f12


Study day

HF/RFassessmentof m3m9–m12& f9–f12


HF/RFassessmentof m5–m8 &f5–f8

HF/RFassessmentof m3


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Figure 2

100

0

0

Rel

ativ

e ab

unda

nce

(%)

100300

600

900

X X X X X X

Com

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Gin

seno

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Rg 1 M2

M3

M4

M5

M6

M7

M8

M9

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M25

M26

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M28

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M32

M33

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M35

X X X X X X

D (Human plasma)

E (Human urine)

M1

20(S

)-pro

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naxa

triol

X X

Compound

A (Sanqi-extract)

0

2000

4000

Con

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n (µ

M)

Gin

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Ra 3

Gin

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Rb 1

Gin

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Rb 2

Gin

seno

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Rc

Gin

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Rd

Gin

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Rg 3

Gin

seno

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F2

Gin

seno

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Rh 2

20-G

luco

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seno

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Rf

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Re

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R1

Not

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6G

inse

nosi

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inse

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inse

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inse

nosi

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17 18 1 3325

4 360

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3

B (Human plasma)

C (Human urine)

50

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100

Rel

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(%)

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Ra 3

Gin

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Rb 1

Gin

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Rd

Gin

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F2

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Rg 3

Com

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(S)-p

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100

0

0

Rel

ativ

e ab

unda

nce

(%)

100300

600

900

X X X X X X

Com

poun

d-K

Gin

seno

side

Rg 1 M2

M3

M4

M5

M6

M7

M8

M9

M10

M11

M12

M13

M14

M15

M16

M17

M18

M19

M20

M21

M22

M23

M24

M25

M26

M27

M28

M29

M30

M31

M32

M33

M34

M35

X X X X X X

D (Human plasma)

E (Human urine)

M1

20(S

)-pro

topa

naxa

triol

X X

Compound

A (Sanqi-extract)

0

2000

4000

Con

cent

ratio

n (µ

M)

Gin

seno

side

Ra 3

Gin

seno

side

Rb 1

Gin

seno

side

Rb 2

Gin

seno

side

Rc

Gin

seno

side

Rd

Gin

seno

side

Rg 3

Gin

seno

side

F2

Gin

seno

side

Rh 2

20-G

luco

-gin

seno

side

Rf

Gin

seno

side

Re

Not

ogin

seno

side

R1

Not

ogin

seno

side

R3/

6G

inse

nosi

de R

g 1G

inse

nosi

de R

g 2G

inse

nosi

de R

fG

inse

nosi

de F

1G

inse

nosi

de R

h 1

7613

7714 3

366

17 18 1 3325

4 360

6926

8060 13 48 10

3

B (Human plasma)

C (Human urine)

50

0

50

100

Rel

ativ

e ab

unda

nce

(%)

100

0

Gin

seno

side

Ra 3

Gin

seno

side

Rb 1

Gin

seno

side

Rd

Gin

seno

side

F2

Gin

seno

side

Rg 3

Com

poun

d-K

Gin

seno

side

Rh 2

20(S

)-pro

topa

naxa

diol

20-G

luco

-gin

seno

side

Rf

Gin

seno

side

Re

Not

ogin

seno

side

R1

Not

ogin

seno

side

R3/

6G

inse

nosi

de R

g 1G

inse

nosi

de F

1G

inse

nosi

de R

h 120

(S)-p

roto

pana

xtrio

l

X X

X XXX

20(S

)-pr

otop

anax

adio

l


at ASPE



Dow

nloaded from


Figure 3

M1[HP,HU]

M2[HP,HU]

M3/M6[HP,HU]

M3/M6[HP,HU]

Glu

Metabolism of compound-K Metabolism of 20(S)-protopanaxatriol

HO

OH

O

Glc

O

HO

OH

O

Glc

HO

OH

O

Glc

O

HO

OH

OH

HO

OH

OH

HO

OH

OH

HO

HO

OH

OH

HO

OH

OH

M30[HU]

O

M10[HP,HU]

M21[HP]

O x 2

O

O

M19[HP]

O

O

M16[HP,HU]

M20[HP]

M22[HP]

Metabolism of 20(S)-protopanaxadiol

20(S)-protopanaxadiol[HP]

M38[RB]

M36[RB]

M37[RB]

HO

OH

O

Glc

HO

OH

OH

HOO

HO

OHHO

HO

OH

OH

HOO

HO

OHHO

OH

OH

HO

OH

O

HO

O

OH

O

HO

HO

OH

O

HO

O

OHO

HO

HO

OH

O

HO

HO

OH

OH

HOO

HO

OH

OH

HO

HO

OH

OH

HOO

M12[HP,HU]

M14[HP,HU]

M15[HP,HU]

M4/M5/M8[HP,HU]

M11[HP,HU]

M7[HP,HU]

M13[HP,HU]

O

Glu

O

O O

OGlu

20(S)-protopanaxatriol[HP]

O

OHO

HO

OH

HO

OH

O

HO

OH

HO

OH

O

HO

OH

O

OH

O

HO

OH

HO

OHO

HO

OH

HO

OHO

HO

OH

O

OHO

HO

OH

HO

OH

O

HO

OH

HO

OHO

HO

OH

O

OHO

HO

OH

Compound-K[HP,HU]

HO

OH

O

HO

M35[HU]M35[HU]

M34[HU]M34[HU]

M17[HP]M17[HP]

M29[HP,HU]

M29[HP,HU]

M25/M27[HP,HU]

M25/M27[HP,HU]

M23/M24[HP,HU]

M23/M24[HP,HU]

M1[HP,HU]

M2[HP,HU]

M3/M6[HP,HU]

M3/M6[HP,HU]

Glu

Metabolism of compound-K Metabolism of 20(S)-protopanaxatriol

HO

OH

O

Glc

O

HO

OH

O

Glc

O

HO

OH

O

Glc

HO

OH

O

Glc

O

HO

OH

O

Glc

O

HO

OH

OH

HO

OH

OH

HO

OH

OH

HO

OH

OH

HO

OH

OH

HO

HO

OH

OH

HO

HO

OH

OH

HO

OH

OH

HO

OH

OH

HO

OH

OH

M30[HU]

O

M10[HP,HU]

M21[HP]

O x 2

O

O

M19[HP]

O

O

M16[HP,HU]

M20[HP]

M22[HP]

Metabolism of 20(S)-protopanaxadiol

20(S)-protopanaxadiol[HP]

M38[RB]

M36[RB]

M37[RB]

HO

OH

O

Glc

HO

OH

O

Glc

HO

OH

OH

HOO

HO

OH

OH

HOO

HO

OHHO

HO

OHHO

HO

OH

OH

HOO

HO

OH

OH

HOO

HO

OHHO

OH

OH

HO

OHHO

OH

OH

HO

OH

O

HO

HO

OH

O

HO

O

OH

O

HO

O

OH

O

HO

HO

OH

O

HO

HO

OH

O

HO

O

OHO

HO

O

OHO

HO

HO

OH

O

HO

HO

OH

O

HO

HO

OH

OH

HOO

HO

OH

OH

HOO

HO

OH

OH

HO

HO

OH

OH

HO

HO

OH

OH

HOO

HO

OH

OH

HOO

M12[HP,HU]

M14[HP,HU]

M15[HP,HU]

M4/M5/M8[HP,HU]

M11[HP,HU]

M7[HP,HU]

M13[HP,HU]

O

Glu

O

O O

OGlu

20(S)-protopanaxatriol[HP]

O

OHO

HO

OH

O

OHO

HO

OH

HO

OH

O

HO

OH

HO

OH

O

HO

OH

HO

OH

O

HO

OH

HO

OH

O

HO

OH

O

OH

O

HO

OH

O

OH

O

HO

OH

HO

OHO

HO

OH

HO

OHO

HO

OH

HO

OHO

HO

OH

HO

OHO

HO

OH

O

OHO

HO

OH

O

OHO

HO

OH

HO

OH

O

HO

OH

HO

OH

O

HO

OH

HO

OHO

HO

OH

HO

OHO

HO

OH

O

OHO

HO

OH

O

OHO

HO

OH

Compound-K[HP,HU]

HO

OH

O

HO

HO

OH

O

HO

M35[HU]M35[HU]

M34[HU]M34[HU]

M17[HP]M17[HP]

M29[HP,HU]

M29[HP,HU]

M25/M27[HP,HU]

M25/M27[HP,HU]

M23/M24[HP,HU]

M23/M24[HP,HU]


at ASPE



Dow

nloaded from


Figure 4

1A2 2A6 2C8 2C92B6 2C19 2E12D6 3A53A4

100

80

60

40

20

0

CYP-mediated metabolism of 20(S)-protopanaxadiol

CYP-mediated metabolism of 20(S)-protopanaxatriol

CYP enzyme

Rel

ativ

e ab

unda

nce

(%)

Ppd in vitrometabolites

M20M21

M22

Ppt in vitrometabolites

M8M10

M11M12

M19

3A53A4

CYP enzyme

100

80

60

40

20

0

A B

C D

1A2 2A6 2C8 2C92B6 2C19 2E12D6 3A53A4

100

80

60

40

20

0

CYP-mediated metabolism of 20(S)-protopanaxadiol

CYP-mediated metabolism of 20(S)-protopanaxatriol

CYP enzyme

Rel

ativ

e ab

unda

nce

(%)


M20M21

M22


M8M10

M11M12

M19


M20M21

M22


M8M10

M11M12

M19

3A53A4

CYP enzyme

100

80

60

40

20

0

A B

C D


at ASPE



Dow

nloaded from


Figure 5

Time (h)

Ginsenoside Ra34

2

0

Ginsenoside Rd20

10

0

Ginsenoside Rb140

20

0

Ginsenoside F220

10

0

Compound-K600

300

0

20(S)-Protopanaxadiol80

40

0

40

20

0

M16 M1780

40

0

120

60

0

M19

0 20 40 60

M2050

25

0

M2280

40

0

M21300

150

0

Ginsenoside F18

4

0

Notoginsenoside R14

2

0

Ginsenoside Rg120

10

0

M4160

80

0

20(S)-Protopanaxatriol400

200

0

M5280

140

0

M6 M7 M880

40

0

90

45

0

2000

1000

0

M10240

120

0

M11 M12800

400

0

4500

2250

0

M14M13 M15220

110

0

180

90

0

70

35

00 20 40 60 0 20 40 60 0 20 40 60

Female human subjects

0 20 40 60 0 20 40 60

Ginsenoside Ra34

2

0

Male human subjectsGinsenoside Rd

20

10

0

40

20

0

Ginsenoside Rb1

Ginsenoside F220

10

0

600

300

0

Compound-K80

40

0

20(S)-Protopanaxadiol

M16 M1740

20

0

80

40

0

M19120

60

0

50

25

0

M20 M2280

40

0

M21300

150

0

Ginsenoside F18

4

0

Notoginsenoside R14

2

0

Ginsenoside Rg120

10

0

M4160

80

0

20(S)-Protopanaxatriol 400

200

0

280

140

0

M5

90

45

0

M6 M7 M82000

1000

0

80

40

0

M10240

120

0

M124500

2250

0

M11800

400

0

M13 M14 M1570

35

0

180

90

0

220

110

00 20 40 60 0 20 40 60 0 20 40 60

Pla

sma

conc

entra

tion

(nM

)

0 20 40 600 20 40 600 20 40 60

Pla

sma

conc

entra

tion

(nM

)

Time (h)

Ginsenoside Ra34

2

0

Ginsenoside Rd20

10

0

Ginsenoside Rb140

20

0

Ginsenoside F220

10

0

Compound-K600

300

0


40

0

40

20

0

M16 M1780

40

0

120

60

0

M19

0 20 40 60

M2050

25

0

M2280

40

0

M21300

150

0

Ginsenoside F18

4

0

Notoginsenoside R14

2

0

Ginsenoside Rg120

10

0

M4160

80

0


200

0

M5280

140

0

M6 M7 M880

40

0

90

45

0

2000

1000

0

M10240

120

0

M11 M12800

400

0

4500

2250

0

M14M13 M15220

110

0

180

90

0

70

35

00 20 40 60 0 20 40 60 0 20 40 60


0 20 40 60 0 20 40 60

Ginsenoside Ra34

2

0

Ginsenoside Rd20

10

0

Ginsenoside Rb140

20

0

Ginsenoside F220

10

0

Compound-K600

300

0


40

0

40

20

0

M16 M1780

40

0

120

60

0

M19

0 20 40 60

M2050

25

0

M2280

40

0

M21300

150

0

Ginsenoside F18

4

0

Notoginsenoside R14

2

0

Ginsenoside Rg120

10

0

M4160

80

0


200

0

M5280

140

0

M6 M7 M880

40

0

90

45

0

2000

1000

0

M10240

120

0

M11 M12800

400

0

4500

2250

0

M14M13 M15220

110

0

180

90

0

70

35

00 20 40 60 0 20 40 60 0 20 40 60


0 20 40 60 0 20 40 60

Ginsenoside Ra34

2

0


20

10

0

40

20

0

Ginsenoside Rb1

Ginsenoside F220

10

0

600

300

0

Compound-K80

40

0


M16 M1740

20

0

80

40

0

M19120

60

0

50

25

0

M20 M2280

40

0

M21300

150

0

Ginsenoside F18

4

0

Notoginsenoside R14

2

0

Ginsenoside Rg120

10

0

M4160

80

0


200

0

280

140

0

M5

90

45

0

M6 M7 M82000

1000

0

80

40

0

M10240

120

0

M124500

2250

0

M11800

400

0

M13 M14 M1570

35

0

180

90

0

220

110

00 20 40 60 0 20 40 60 0 20 40 60

Pla

sma

conc

entra

tion

(nM

)

0 20 40 600 20 40 600 20 40 60

Pla

sma

conc

entra

tion

(nM

)

Ginsenoside Ra34

2

0

4

2

0


20

10

0

Ginsenoside Rd20

10

0

20

10

0

40

20

0

40

20

0

Ginsenoside Rb1

Ginsenoside F220

10

0

20

10

0

600

300

0

600

300

0

Compound-K80

40

0

80

40

0


M16 M1740

20

0

40

20

0

80

40

0

80

40

0

M19120

60

0

120

60

0

50

25

0

50

25

0

M20 M2280

40

0

80

40

0

M21300

150

0

300

150

0

Ginsenoside F18

4

0

8

4

0

Notoginsenoside R14

2

0

Notoginsenoside R14

2

0

Ginsenoside Rg120

10

0

Ginsenoside Rg120

10

0

20

10

0

M4160

80

0

160

80

0


200

0


200

0

400

200

0

280

140

0

280

140

0

M5

90

45

0

90

45

0

M6 M7 M82000

1000

0

2000

1000

0

80

40

0

80

40

0

M10240

120

0

M10240

120

0

240

120

0

M124500

2250

0

M124500

2250

0

4500

2250

0

M11800

400

0

M11800

400

0

800

400

0

M13 M14 M1570

35

0

70

35

0

180

90

0

180

90

0

220

110

0

220

110

00 20 40 600 20 40 60 0 20 40 600 20 40 60 0 20 40 600 20 40 60

Pla

sma

conc

entra

tion

(nM

)

0 20 40 600 20 40 600 20 40 600 20 40 600 20 40 600 20 40 60

Pla

sma

conc

entra

tion

(nM

)This article has not been copyedited and formatted. The final version may differ from this version.

DMD Fast Forward. Published on May 6, 2013 as DOI: 10.1124/dmd.113.051391

at ASPE



Dow

nloaded from


Figure 6

Pla

sma

AU

C0-

56h

(µM

·h)

Ginsenoside Ra3r = 0.80, P <0.011.204 (0.861, 1.542)

0

0.08

0.16

0

0.8

1.6 r = 0.63, P < 0.010.849 (0.455, 1.242)

Ginsenoside Rb1

0

0.2

0.4Ginsenoside Rd

0

0.11

0.22 r = 0.63, P <0.011.447 (0.777, 2.117)

Ginsenoside F2

r = 0.29, P = 0.18

0

4.5

9.0Compound-K

0

1.0

2.020(S)-Protopanaxadiol

r = 0.10, P = 0.69

0

0.45

0.90 r = 0.01, P = 0.96M16

0

1.1

2.2 r = 0.08, P = 0.70M17

0 2 4 60

1.5

3.0 r = 0.00, P = 0.97M19

0

0.6

1.2 r = 0.18, P = 0.41

0 2 4 6

M20

0

3.0

6.0 r = 0.08, P = 0.73

0 2 4 6

M21

0

0.8

1.6 r = 0.12, P = 0.60

0 2 4 6

M22

0

0.1

0.2 r = 0.90, P <0.011.914 (1.568, 2.260)

Ginsenoside Rg1

Sanqi extract dose (ml/kg)

0

0.15

0.03 r = 0.86, P <0.012.215 (1.730, 2.700)

Notoginsenoside R1

0

0.08

0.16 r = 0.30, P = 0.16Ginsenoside F1 20(S)-Protopanaxatriol

r = 0.14, P = 0.53

0

4.0

8.0

0

2.0

4.0 r = 0.14, P = 0.53M4

0

1.0

2.0 r = 0.03, P = 0.88M6

0

4.0

8.0 r = 0.01, P = 0.95M5

0

1.1

2.2 r = 0.06, P = 0.77M7

0

30

60 r = 0.06, P = 0.79M8

0

3.0

6.0 r = 0.14, P = 0.54M10

0

11

22 r = 0.05, P = 0.84M11

0

60

120 r = 0.06, P = 0.78

0 2 4 6

M12

r = 0.03, P = 0.84

0

2.5

5.0

0 2 4 6

M13

0

2.5

5.0 r = 0.02, P = 0.93

0 2 4 6

M14

0

1.0

2.0 r = 0.03, P = 0.88

0 2 4 6

M15

Plas

ma

AU

C0-

56h

(µM

·h)

r = 0.73, P < 0.011.207 (0.783, 1.630)

270 ml/subject180 ml/subject90 ml/subject

DoseFemaleMale


DoseFemaleMale

Pla

sma

AU

C0-

56h

(µM

·h)

Ginsenoside Ra3r = 0.80, P <0.011.204 (0.861, 1.542)

0

0.08

0.16

0

0.8

1.6 r = 0.63, P < 0.010.849 (0.455, 1.242)

Ginsenoside Rb1

0

0.2

0.4Ginsenoside Rd

0

0.11

0.22 r = 0.63, P <0.011.447 (0.777, 2.117)

Ginsenoside F2

r = 0.29, P = 0.18

0

4.5

9.0Compound-K

0

1.0

2.020(S)-Protopanaxadiol

r = 0.10, P = 0.69

0

0.45

0.90 r = 0.01, P = 0.96M16

0

1.1

2.2 r = 0.08, P = 0.70M17

0 2 4 60

1.5

3.0 r = 0.00, P = 0.97M19

0

0.6

1.2 r = 0.18, P = 0.41

0 2 4 6

M20

0

3.0

6.0 r = 0.08, P = 0.73

0 2 4 6

M21

0

0.8

1.6 r = 0.12, P = 0.60

0 2 4 6

M22

0

0.1

0.2 r = 0.90, P <0.011.914 (1.568, 2.260)

Ginsenoside Rg1

Sanqi extract dose (ml/kg)

0

0.15

0.03 r = 0.86, P <0.012.215 (1.730, 2.700)

Notoginsenoside R1

0

0.08

0.16 r = 0.30, P = 0.16Ginsenoside F1 20(S)-Protopanaxatriol

r = 0.14, P = 0.53

0

4.0

8.0

0

2.0

4.0 r = 0.14, P = 0.53M4

0

1.0

2.0 r = 0.03, P = 0.88M6

0

4.0

8.0 r = 0.01, P = 0.95M5

0

1.1

2.2 r = 0.06, P = 0.77M7

0

30

60 r = 0.06, P = 0.79M8

0

3.0

6.0 r = 0.14, P = 0.54M10

0

11

22 r = 0.05, P = 0.84M11

0

60

120 r = 0.06, P = 0.78

0 2 4 6

M12

r = 0.03, P = 0.84

0

2.5

5.0

0 2 4 6

M13

0

2.5

5.0 r = 0.02, P = 0.93

0 2 4 6

M14

0

1.0

2.0 r = 0.03, P = 0.88

0 2 4 6

M15

Plas

ma

AU

C0-

56h

(µM

·h)

r = 0.73, P < 0.011.207 (0.783, 1.630)


DoseFemaleMale


DoseFemaleMale


at ASPE



Dow

nloaded from


Figure 7

Ginsenoside Ra30

3

6

9

0 0.04 0.08 0.12 0.16

Compound-Kr = 0.17, P = 0.23

Pla

sma

AU

C0-

56h

(µM

·h)

0

0.7

1.4

2.1

0 0.5 1.0 1.5 2.0

r = 0.70, P < 0.0120(S)-Protopanaxadiol

M17

0 0.04 0.08 0.12 0.160

0.7

1.4

2.1 r = 0.01, P = 0.9520(S)-Protopanaxadiol

Ginsenoside Ra3

Ginsenoside Rb1

r = 0.16, P = 0.28Compound-K

0 0.4 0.8 1.2 1.6

0 0.7 1.4 2.1 2.8


M19

Ginsenoside Rd

0 0.1 0.2 0.3 0.4

r = 0.29, P = 0.05Compound-K

0 0.3 0.6 0.9 1.2


M20

0 0.1 0.2 0.3 0.4

r = 0.09, P = 0.5920(S)-Protopanaxadiol

Ginsenoside Rd

Ginsenoside F2

r = 0.30, P = 0.04

0 0.06 0.12 0.18 0.24

Compound-K

0 1.5 3.0 4.5 6.0


M21

r = -0.15, P = 0.33

0 0.06 0.12 0.18 0.24


Ginsenoside F2

0 0.5 1.0 1.5 2.0

r = -0.14, P = 0.36Compound-K


0 0.4 0.8 1.2 1.6


M22

0

3

6

9

0 30 60 90 120

r = 0.87, P < 0.01

M12

20(S)-Protopanaxatriol

0

3

6

9

0 0.5 1.0 1.5 2.0

r = 0.75, P < 0.01

M6


r = -0.00, P = 1

0

3

6

9

0 0.006 0.012 0.018 0.024

Notoginsenoside R1


0 1.5 3.0 4.5 6.0

r = 0.91, P < 0.01

M13

20(S)-Protopanaxatriol0 0.6 1.2 1.8 2.4

r = 0.90, P < 0.01

M7


r = -0.09, P = 0.52

0 0.05 0.10 0.15 0.20

Ginsenoside Rg1


0 1.1 2.2 3.3 4.4

r = 0.88, P < 0.01

M14


Plasma AUC0-56h (µM·h)

0 15 30 45 60

r = 0.88, P < 0.01

M8


r = 0.12, P = 0.43

0 0.04 0.08 0.12 0.16

Ginsenoside F1


0 0.5 1.0 1.5 2.0

r = 0.92, P < 0.01

M15


r = 0.91, P < 0.01

M10


0 1.0 2.0 3.0 4.0

r = 0.88, P < 0.01

M4


0 0.5 1.0 1.5 2.0

r = 0.69, P < 0.01



r = 0.88, P < 0.01

M11


0 2.0 4.0 6.0 8.0

r = 0.86, P < 0.01

M5


0 0.21 0.42 0.63 0.84


M16

0 0.4 0.8 1.2 1.6

r = -0.04, P = 0.8120(S)-Protopanaxadiol

Ginsenoside Rb1

Plas

ma

AU

C0-

56h

(µM

·h)

Ginsenoside Ra30

3

6

9

0 0.04 0.08 0.12 0.16

Compound-Kr = 0.17, P = 0.23

Pla

sma

AU

C0-

56h

(µM

·h)

0

0.7

1.4

2.1

0

0.7

1.4

2.1

0 0.5 1.0 1.5 2.0


M17

0 0.04 0.08 0.12 0.160

0.7

1.4

2.1 r = 0.01, P = 0.9520(S)-Protopanaxadiol

Ginsenoside Ra3

Ginsenoside Rb1

r = 0.16, P = 0.28Compound-K

0 0.4 0.8 1.2 1.6

0 0.7 1.4 2.1 2.8


M19

Ginsenoside Rd

0 0.1 0.2 0.3 0.4

r = 0.29, P = 0.05Compound-K

0 0.3 0.6 0.9 1.2


M20

0 0.1 0.2 0.3 0.4


Ginsenoside Rd

Ginsenoside F2

r = 0.30, P = 0.04

0 0.06 0.12 0.18 0.24

Compound-K

0 1.5 3.0 4.5 6.0


M21

r = -0.15, P = 0.33

0 0.06 0.12 0.18 0.24


Ginsenoside F2

0 0.5 1.0 1.5 2.0

r = -0.14, P = 0.36Compound-K


0 0.4 0.8 1.2 1.6


M22

0

3

6

9

0 30 60 90 120

r = 0.87, P < 0.01

M12


0

3

6

9

0

3

6

9

0 0.5 1.0 1.5 2.0

r = 0.75, P < 0.01

M6


r = -0.00, P = 1

0

3

6

9

0 0.006 0.012 0.018 0.024

Notoginsenoside R1


0 1.5 3.0 4.5 6.0

r = 0.91, P < 0.01

M13


r = 0.90, P < 0.01

M7


r = -0.09, P = 0.52

0 0.05 0.10 0.15 0.20

Ginsenoside Rg1


0 1.1 2.2 3.3 4.4

r = 0.88, P < 0.01

M14



0 15 30 45 60

r = 0.88, P < 0.01

M8


r = 0.12, P = 0.43

0 0.04 0.08 0.12 0.16

Ginsenoside F1


0 0.5 1.0 1.5 2.0

r = 0.92, P < 0.01

M15


r = 0.91, P < 0.01

M10


0 1.0 2.0 3.0 4.0

r = 0.88, P < 0.01

M4


0 0.5 1.0 1.5 2.0

r = 0.69, P < 0.01



r = 0.88, P < 0.01

M11


0 2.0 4.0 6.0 8.0

r = 0.86, P < 0.01

M5


0 0.21 0.42 0.63 0.84


M16

0 0.4 0.8 1.2 1.6


Ginsenoside Rb1

Plas

ma

AU

C0-

56h

(µM

·h)


at ASPE



Dow

nloaded from


Figure 8

Study Day

M19

0.10

0.05

0

Compound-K

1.2

0.6

0

4.0

2.0

01 18 24

Ginsenoside Ra3

M132.0

1.0

0

2.4

1.2

011 18 24

M4

11

1

M20

Ginsenoside Rb10.2

0.1

0

M10

M5

0.6

0.3

0

1 18 24

M14

1 11 18 24

11

Ginsenoside Rd

M16

0.04

0

Ginsenoside F1

M6

2.4

1.2

0

1.2

0.6

0

M15

0.02

M22

Ginsenoside F2

M17

1 11 18 24

Subject m1

Subject m2

Subject m3

Subject m4

M7

Pla

sma

AU

C0-

24h

(µM

·h)

m1

m2

m4

m3

m1

m2

m4

m3

0.8

0.4

01 11 18 24

m1m2

m4

m3

6

3

0

m1

m2m4

m3

1.0

0.5

0

m1

m2

m4

m3

0.8

0.4

0

m1

m2

m4

m3

4.0

2.0

0

m1

m2

m4

m3

2.4

1.2

0

m1

m2

m4

m3

0.04

0.02

0

m2

m4

m1

m3

m1m2

m4

m3

m1

m2

m4

m3

m1

m2

m4

m3

m1

m2

m4

m3

m1

m2

m4

m3

m1

m4m2

m3m1m2m4

m3

m1m2

m4

m31.2

0.6

0

m1m4

m2

m3

2.8

1.4

0

m3

m2m4

m1

20(S)-Protopanaxadiol1.6

0.8

0

m1

m2

m4

m3

M217.0

3.5

0

m1m2

m4

m3

Notoginsenoside R10.010

0.005

0

m1

m4

m2

m3

Ginsenoside Rg10.08

0

0.04

m2

m1m4m3

20(S)-Protopanaxatrio4.8

2.4

0m1

m2m4m3

M828

14

0

m1

m2

m4

m3

M1112

6

0

m1m2

m4

m3

M1260

30

01 11 18 24

m1

m2

m4

m3

1 18 2411

Pla

sma

AU

C0-

24h

(µM

·h)

Study Day

M19

0.10

0.05

0

Compound-K

1.2

0.6

0

4.0

2.0

01 18 24

Ginsenoside Ra3

M132.0

1.0

0

2.4

1.2

011 18 24

M4

11

1

M20

Ginsenoside Rb10.2

0.1

0

M10

M5

0.6

0.3

0

1 18 24

M14

1 11 18 24

11

Ginsenoside Rd

M16

0.04

0

Ginsenoside F1

M6

2.4

1.2

0

1.2

0.6

0

M15

0.02

M22

Ginsenoside F2

M17

1 11 18 24

Subject m1

Subject m2

Subject m3

Subject m4

M7

Pla

sma

AU

C0-

24h

(µM

·h)

m1

m2

m4

m3

m1

m2

m4

m3

0.8

0.4

01 11 18 24

m1m2

m4

m3

0.8

0.4

01 11 18 24

m1m2

m4

m3

6

3

0

m1

m2m4

m3

6

3

0

m1

m2m4

m3

1.0

0.5

0

m1

m2

m4

m3

1.0

0.5

0

m1

m2

m4

m3

0.8

0.4

0

m1

m2

m4

m3

0.8

0.4

0

m1

m2

m4

m3

4.0

2.0

0

m1

m2

m4

m3

4.0

2.0

0

m1

m2

m4

m3

2.4

1.2

0

m1

m2

m4

m3

2.4

1.2

0

m1

m2

m4

m3

0.04

0.02

0

m2

m4

m1

m3

0.04

0.02

0

m2

m4

m1

m3

m1m2

m4

m3

m1

m2

m4

m3

m1

m2

m4

m3

m1

m2

m4

m3

m1

m2

m4

m3

m1

m4m2

m3m1m2m4

m3

m1m2

m4

m31.2

0.6

0

m1m4

m2

m3

1.2

0.6

0

m1m4

m2

m3

2.8

1.4

0

m3

m2m4

m1

2.8

1.4

0

m3

m2m4

m1

20(S)-Protopanaxadiol1.6

0.8

0

m1

m2

m4

m3

1.6

0.8

0

m1

m2

m4

m3

M217.0

3.5

0

m1m2

m4

m3

7.0

3.5

0

m1m2

m4

m3

Notoginsenoside R10.010

0.005

0

m1

m4

m2

m3

0.010

0.005

0

m1

m4

m2

m3

Ginsenoside Rg10.08

0

0.04

m2

m1m4m3

0.08

0

0.04

m2

m1m4m3

20(S)-Protopanaxatrio4.8

2.4

0m1

m2m4m3

4.8

2.4

0m1

m2m4m3

M828

14

0

m1

m2

m4

m3

28

14

0

m1

m2

m4

m3

M1112

6

0

m1m2

m4

m3

12

6

0

m1m2

m4

m3

M1260

30

01 11 18 24

m1

m2

m4

m3

60

30

01 11 18 24

m1

m2

m4

m3

1 18 2411

Pla

sma

AU

C0-

24h

(µM

·h)


at ASPE



Dow

nloaded from


Figure 9

0

1.0

2.0

3.0

0 0.025 0.050 0.075 0.100

Compound-K

Plas

ma

AU

C0-

24h

(µM

·h)

r = -0.08, P = 0.69

Ginsenoside Ra3

0

0.5

1.0

1.5

0 0.6 1.2 1.8 2.4


M17

Ginsenoside Ra3

0

0.5

1.0

1.5

0 0.025 0.05 0.075 0.100


0 0.3 0.6 0.9 1.2

Compound-Kr = -0.07, P = 0.73

Ginsenoside Rb1

r = 0.03, P = 0.88

0 0.05 0.10 0.15 0.20

Compound-K

Ginsenoside Rd

0 0.01 0.02 0.03 0.04

Compound-Kr = 0.20, P = 0.30

Ginsenoside F2

0 1.0 2.0 3.0 4.0


M19

Ginsenoside Rb1

0 0.3 0.6 0.9 1.2


0 0.25 0.50 0.75 1.00


M20

0 0.05 0.10 0.15 0.20


Ginsenoside Rd

0 1.6 3.2 4.8 6.4

M21


0 0.01 0.02 0.03 0.04


Ginsenoside F2

0 0.4 0.8 1.2 1.6

Compound-Kr = -0.21, P = 0.33


0 0.3 0.6 0.9 1.2

M22


0 0.15 0.30 0.45 0.60

M16


0 2.5 5.0 7.5 10

r = 0.83, P < 0.01


M11

0 0.4 0.8 1.2 1.6

r = 0.64, P < 0.0120(S)-Protopanaxatriol


0 1.0 2.0 3.0 4.0


M5

0

1.6

3.2

4.8

0 0.2 0.4 0.6 0.8


M6

0

1.6

3.2

4.8

0 15 30 45 60


M12

0

1.6

3.2

4.8

0 0.0025 0.005 0.0075 0.01

r = 0.18, P = 0.3520(S)-Protopanaxatriol

Notoginsenoside R1


0 0.25 0.50 0.75 1.00


M7

0 0.6 1.2 1.8 2.4

r = 0.87, P < 0.01

M13


0 0.02 0.04 0.06 0.08


Ginsenoside Rg1

0 7 14 21 28


M8

0 0.5 1.0 1.5 2.0


M14

0 0.01 0.02 0.03 0.04


Ginsenoside F1

0 1.6 3.2 4.8 6.4


M10

0 0.2 0.4 0.6 0.8


M15

0 0.6 1.2 1.8 2.4


M4

Plas

ma

AUC

0-24

h(µ

M·h

)

0

1.0

2.0

3.0

0 0.025 0.050 0.075 0.100

Compound-K

Plas

ma

AU

C0-

24h

(µM

·h)

r = -0.08, P = 0.69

Ginsenoside Ra3

0

0.5

1.0

1.5

0 0.6 1.2 1.8 2.4


M17

Ginsenoside Ra3

0

0.5

1.0

1.5

0 0.025 0.05 0.075 0.100


0 0.3 0.6 0.9 1.2

Compound-Kr = -0.07, P = 0.73

Ginsenoside Rb1

r = 0.03, P = 0.88

0 0.05 0.10 0.15 0.20

Compound-K

Ginsenoside Rd

0 0.01 0.02 0.03 0.04

Compound-Kr = 0.20, P = 0.30

Ginsenoside F2

0 1.0 2.0 3.0 4.0


M19

Ginsenoside Rb1

0 0.3 0.6 0.9 1.2


0 0.25 0.50 0.75 1.00


M20

0 0.05 0.10 0.15 0.20


Ginsenoside Rd

0 1.6 3.2 4.8 6.4

M21


0 0.01 0.02 0.03 0.04


Ginsenoside F2

0 0.4 0.8 1.2 1.6

Compound-Kr = -0.21, P = 0.33


0 0.3 0.6 0.9 1.2

M22


0 0.15 0.30 0.45 0.60

M16


0 2.5 5.0 7.5 10

r = 0.83, P < 0.01


M11

0 0.4 0.8 1.2 1.6



0 1.0 2.0 3.0 4.0


M5

0 2.5 5.0 7.5 10

r = 0.83, P < 0.01


M11

0 0.4 0.8 1.2 1.6



0 1.0 2.0 3.0 4.0


M5

0

1.6

3.2

4.8

0 0.2 0.4 0.6 0.8


M6

0

1.6

3.2

4.8

0 15 30 45 60


M12

0

1.6

3.2

4.8

0 0.0025 0.005 0.0075 0.01


Notoginsenoside R1


0 0.25 0.50 0.75 1.00


M7

0 0.6 1.2 1.8 2.4

r = 0.87, P < 0.01

M13


0 0.02 0.04 0.06 0.08


Ginsenoside Rg1

0 7 14 21 28


M8

0 0.5 1.0 1.5 2.0


M14

0 0.01 0.02 0.03 0.04


Ginsenoside F1

0 1.6 3.2 4.8 6.4


M10

0 0.2 0.4 0.6 0.8


M15

0 0.6 1.2 1.8 2.4


M4

0

1.6

3.2

4.8

0 0.2 0.4 0.6 0.8


M6

0

1.6

3.2

4.8

0 15 30 45 60


M12

0

1.6

3.2

4.8

0 0.0025 0.005 0.0075 0.01


Notoginsenoside R1


0 0.25 0.50 0.75 1.00


M7

0 0.6 1.2 1.8 2.4

r = 0.87, P < 0.01

M13


0 0.02 0.04 0.06 0.08


Ginsenoside Rg1

0 7 14 21 28


M8

0 0.5 1.0 1.5 2.0


M14

0 0.01 0.02 0.03 0.04


Ginsenoside F1

0 1.6 3.2 4.8 6.4


M10

0 0.2 0.4 0.6 0.8


M15

0 0.6 1.2 1.8 2.4


M4

Plas

ma

AUC

0-24

h(µ

M·h

)


at ASPE



Dow

nloaded from


Combinatorial Metabolism Notably Affects Human...

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