Absorption and disposition of ginsenosides after oral …dmd.aspetjournals.org/content/dmd/early/2009/09/28/dmd... · biliary excretion, inefficient metabolism, and slow renal excretion

DMD 29819

1

Absorption and disposition of ginsenosides

after oral administration of Panax notoginseng

extract to rats

Houfu Liu, Junling Yang, Feifei Du, Xiumei Gao, Xutao Ma,

Yuhong Huang, Fang Xu, Wei Niu, Fengqing Wang, Yu

Mao, Yan Sun, Tong Lu, Changxiao Liu, Boli Zhang,

Chuan Li

Shanghai Institute of Materia Medica (H.L., J.Y., F.D., F.X., W.N., F.W., Y.M.,

Y.S., T.L., Chu.L) and Graduate School (H.L., Chu.L), Chinese Academy of

Sciences, Shanghai, China; Tianjin University of Traditional Chinese Medicine,

Tianjin, China (X.G., X.M., Y.H., B.Z.); Tianjin State key Laboratory of

Pharmacodynamics and Pharmacokinetics, Tianjin Institute of Pharmaceutical

Research, Tianjin, China (Cha.L)

DMD Fast Forward. Published on September 28, 2009 as doi:10.1124/dmd.109.029819

Copyright 2009 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 September 28, 2009 as DOI: 10.1124/dmd.109.029819

at ASPE

T Journals on February 20, 2020

dmd.aspetjournals.org

Dow

nloaded from

http://dmd.aspetjournals.org/

DMD 29819

2

Running Title: ADME OF SANQI GINSENOSIDES

Address correspondence to: Dr. Chuan Li, Shanghai Center for DMPK

Research, Shanghai Institute of Materia Medica, SIBS, Chinese Academy of

Sciences, 555 Zuchongzhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203,

China. E-mail: [email protected]

Number of Text Pages: 22

Number of Tables: 0

Number of Figures: 6

Number of References: 28

Number of Words in Abstract Section: 248

Number of Words in Introduction Section: 578

Number of Words in Discussion Section: 1389

ABBREVIATIONS: AUC, area under concentration-time curve; CLB, biliary clearance;

CLR, renal clearance; CLtot,p, total plasma clearance; Cum.Ae, cumulative amount

excreted during sampling period; ESI, electrospray ionization; i.v., intravenous; LC/MS/MS,

liquid chromatography/tandem mass spectrometry; MRP2, multidrug

resistance-associated protein 2; NROTB, number of rotatable bonds; Papp, apparent

permeability coefficient; P-gp, P-glycoprotein; PK, pharmacokinetic; p.o., peroral; S,

aqueous solubility at a given pH; t1/2, elimination half-life; TPSA, topological polar surface

area.


at ASPE



Dow

nloaded from


DMD 29819

3

ABSTRACT:

Panax notoginseng (Sanqi) is a cardiovascular herb containing ginsenosides that are

believed responsible for the therapeutic effects of Sanqi. This study aimed to evaluate rat

exposure to ginsenosides following p.o. administration of Sanqi extract and to identify the

key factors affecting their absorption and disposition. Ginsenosides were administered to

rats, either in the form of Sanqi extract or as pure chemicals. The ginsenosides Ra3, Rb1,

Rd, Re, Rg1 and notoginsenoside R1 were the major saponins present in the herbal

extract. Systemic exposure to ginsenosides Ra3, Rb1, and Rd following p.o. administration

of the extract was significantly greater than that of the other compounds. Considerable

colonic deglycosylation of the ginsenosides occurred, but the plasma levels of

deglycosylated metabolites were low in rats. Poor membrane permeability and active

biliary excretion are the two primary factors limiting systemic exposure to most

ginsenosides and their deglycosylated metabolites. In contrast to other ginsenosides,

biliary excretion of ginsenosides Ra3 and Rb1 was passive. Meanwhile, the active biliary

excretion of ginsenoside Rd was significantly slower than that of other saponins. Slow

biliary excretion, inefficient metabolism, and slow renal excretion resulted in

long-circulating and thus relatively high exposure levels for these three ginsenosides. Due

to these reasons, plasma ginsenosides Ra3, Rb1 and Rd were identified as

pharmacokinetic markers for indicating rat systemic exposure to Sanqi extract. This is a

systematic investigation of the absorption and disposition of ginsenosides from an herb,

the information gained from which is important for linking Sanqi administration to its

medicinal effects.


at ASPE



Dow

nloaded from


DMD 29819

4

Introduction

Herbs have been used for medicinal purposes in China for millennia, and

traditional Chinese medicine still plays an important role in Chinese healthcare. The

dried root of Panax notoginseng (family Araliaceae) is an important Chinese

medicinal herb known as Sanqi. In traditional Chinese medicine, Sanqi is indicated

for analgesia and hemostasis (Chinese Pharmacopoeia Commission, 2005). The herb

is also used to treat patients with angina and coronary artery disease (Mashour et al.,

1998); the pharmacological mechanism may be as a selective calcium ion antagonist

that may interact with the receptor-operated calcium ion channel (Kwan, 1995). Sanqi

also exerts protective effects on the cardiovascular system (Ng, 2006).

Most of Sanqi’s bioactivities are believed to be associated with triterpene

saponins derived mainly from the tetracyclic dammarane. These compounds, known

as ginsenosides, can be classified according to their structures as

20(S)-protopanaxadiol type (ppd-type) and 20(S)-protopanaxatriol type (ppt-type).

The ppd-type ginsenosides possess sugar moieties at the C-3 and/or C-20 positions,

while ppt-type ginsenosides have a hydroxyl group at C-3 and sugar moieties at C-6

and/or C-20. The major saponins present in Sanqi include the ppd-type ginsenosides

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

These ginsenosides contain 2–5 sugars. Although ginsenosides are also prominent

constituents of other Panax species, the relative amounts of the saponin constituents

present in Sanqi differ from those in Asian ginseng (P. ginseng) and American ginseng

(P. quinquefolius). Sanqi contains approximately equivalent amounts of ginsenosides

Rb1 and Rg1, whereas ginsenoside Rb1 is often more abundant than ginsenoside Rg1 in

Asian and American ginsengs. In addition, Sanqi contains a substantial amount of

notoginsenoside GR1, which is also different from Asian and American ginsengs.


at ASPE



Dow

nloaded from


DMD 29819

5

The pharmacological effects of ginsenosides vary and can even be oppositional.

In contrast to the anti-angiogenic effects of the ppd-type ginsenoside Rb1, the ppt-type

ginsenoside Rg1 has angiogenic properties (Sengupta et al., 2004). However, the lack

of quantitative data regarding the absorption, distribution, metabolism, and excretion

of ginsenosides has hindered investigation of the pharmacological activities. Earlier

PK studies have focused on the development of bioanalytical assays for ginsenosides

and their application to preliminary PK assessments (Xu, et al., 2003; Li et al.,

2004a,b; Li et al., 2006; Li et al., 2007a,b). The PK profiles and disposition of

ppd-type and ppt-type ginsenosides after administration of Sanqi extracts remain

largely unknown. Some studies suggested low oral bioavailability for ginsenosides

Rb1 and Rg1, only 0.1% and 2%, respectively (Odani et al., 1983a,b), whereas these

values were reported to be 4% and 18%, respectively, by others (Xu et al., 2003). The

intestinal microflora can degrade ginsenosides via cleavage of the sugar moieties

(Hasegawa et al., 1996; Akao et al., 1998; Tawab et al., 2003). However, the effects of

deglycosylation on the systemic exposure to ginsenosides are inconclusive.

The objective of this study was to gain understanding about comparative

systemic exposure and PK properties of various ginsenosides from Sanqi and about

the key factors that affect their absorption and disposition. We also investigated the

influence of colonic deglycosylation on systemic exposure to the ginsenosides. The

information gained from the study is indispensable for evaluating the contributions of

the ginsenosides to the medicinal effects of Sanqi, for assessing potential herb-drug or

herb-herb interactions, and for developing Sanqi-based pharmacotherapeutics. To this

end, ginsenosides were administered to rats, either in the form of Sanqi extract or as

pure compounds. Multiple in vivo, in vitro, and in silico approaches were integrated to

determine ginsenoside exposure, absorption, and disposition.


at ASPE



Dow

nloaded from


DMD 29819

6

Materials and Methods

Chemicals and reagents. Ginsenosides Rb1 (GRb1; molecular mass: 1108), Rd

(GRd; 946), Rg3 (GRg3; 784), F2 (GF2; 784), Rh2 (GRh2; 622), Re (GRe; 946), Rg1

(GRg1; 800), Rf (GRf; 800), F1 (GF1; 638), notoginsenoside R1 (NGR1; 932),

compound-K (C-K; 622), protopanaxadiol (Ppd; 460), and protopanaxatriol (Ppt; 476)

were obtained from the National Institute for the Control of Pharmaceutical and

Biological Products (Beijing, China). Ginsenosides Ra3 (GRa3; 1240), Rc (GRc;

1078), and 20-gluco-Rf (20gRf; 962) were obtained from Fengshanjian Co., Ltd.

(Kunming, China). Ginsenoside Rh1 (GRh1; 638) was obtained from Wuhu Delta Co.,

Ltd. (Wuhu, China). Ginsenoside Rg2 (GRg2; 784) was purchased from Shanghai

Tauto Biotech Co., Ltd. (Shanghai, China). The compound purity exceeded 98%.

Hydroxypropylmethyl cellulose was obtained from Colorcon (Shanghai, China).

N,N-dimethylacetamide, Cremophor, and taurocholic acid were purchased from

Sigma-Aldrich (St Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM),

penicillin-streptomycin, and MEM non-essential amino acids were obtained from

Gibco Invitrogen Cell Culture (Grand Island, NY). Fetal bovine serum was purchased

from Hyclone (Logan, UT). Hank’s balanced salts solution (HBSS), atenolol,

rhodamine123, verapamil hydrochloride, and sulfasalazine were obtained from

Sigma-Aldrich. MK571 was obtained from Calbiochem (San Diego, CA).

HPLC-grade organic solvents, antipyrine, and sodium carboxymethylcellulose

(CMC-Na) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai,

China).

Preparation of Sanqi extract and individual ginsenosides. The dried roots of

P. notoginseng (Sanqi) were obtained from Shanghai Huayu Chinese Herbs Co., Ltd.

(Shanghai, China). For p.o. administration, 400 g of Sanqi were pulverized, mixed,


at ASPE



Dow

nloaded from


DMD 29819

7

and steeped in 2400 ml of drinking water for 0.5 h at room temperature before 1 h

sonication-enhanced extraction. The extract was separated by filtration and the residue

was re-extracted with 1600 ml of water. The pooled extract was concentrated under

reduced pressure at 40°C and was modified with hydroxypropylmethyl cellulose at

0.3% (g/ml) before adding water to 800 ml to yield Sanqi extract. The extract was

stored at −15°C pending use.

For i.v. administration, GRa3, GRb1, GRc, GRd, GF2, GRg3, C-K, GRh2, Ppd,

20gRf, GRe, NGR1, GRg1, GRf, GRg2, GRh1, GF1, and Ppt were prepared

individually as injectable 1.0-mM solutions in 0.5% N,N-dimethylacetamide, 9.5%

Cremophor, and 90% saline.

Experimental animals. Rat studies were conducted according to protocols

approved by the Review Committee of Animal Care and Use at the Shanghai Institute

of Materia Medica (Shanghai, China). Male Sprague-Dawley rats (220–250 g;

Shanghai SLAC Laboratory Animal Co., Shanghai, China) were housed in rat cages

(48 × 29 × 18 cm3) in a unidirectional airflow room under controlled temperature

(20–24°C), relative humidity (40–70%), and a 12-h light-dark cycle. Filtered tap

water was available ad libitum, and the rodents were given commercial rat chow ad

libitum except for the overnight period prior to dosing. Rats were acclimated to the

facilities and environment for 7 days prior to the experiments.

Plasma pharmacokinetic studies in rats. Rats were randomly assigned to

different groups (3 rats/group) to receive a p.o. dose of Sanqi extract at 4, 8, or 16

ml/kg (via gavage). In addition, bolus i.v. doses of the injectable solutions of

individual ginsenosides, including GRa3, GRb1, GRc, GRd, GF2, GRg3, C-K, GRh2,

Ppd, 20gRf, GRe, NGR1, GRg1, GRf, GRg2, GRh1, GF1, and Ppt, at 5 μmol/kg were

given to rats (2 or 3 rats/compound) through the tail vein. Serial blood samples [~60


at ASPE



Dow

nloaded from


DMD 29819

8

µl; 0, 5, 15 (p.o. only), 30 min, and 1 (i.v. only), 2, 4 (i.v. only), 6, 8 (p.o. only), 10, 12

(p.o. only), 15, 24, 36, and 48 h] were collected in heparinized tubes from the orbital

sinus under light ether anesthesia. The blood samples were centrifuged at 3000g for 5

min and the plasma fractions were decanted and frozen at −70°C until analysis.

In a multiple-dose study, rats received Sanqi extract at a daily p.o. dose of 4

ml/rat. Four rats per time points were chosen randomly for blood sampling on days 1,

8, 15, or 22. Serial blood samples (~60 µl; 0, 15, 30 min, and 2, 6, 8, 10, 12, 15, and

24 h post dose) were collected and centrifuged to yield plasma fractions.

Excretion studies in rats. Rats were housed in Nalgene metabolic cages (1

rat/cage) and received p.o. Sanqi extract at 8 ml/kg (via gavage). Urine and feces

samples were collected from four rats at 0–4, 4–10, and 10–24 h on day 1 and every

24 h over the next 3 days. The urine and feces collection tubes were frozen at −15°C

to keep collected samples stable. All excretory samples were weighed before storage

at −70°C. The thawed feces samples were homogenized in 9 volumes of saline for

analysis. Similar urinary/fecal excretion studies were performed for individual

ginsenosides administered in i.v. boluses of 5 μmol/kg.

For bile sampling, rats were anesthetized with ether, the bile duct was cannulated

using polyethylene tubing, and bile was collected at 0–1, 1–3, 3–6, 6–12, 12–18,

18–24, 24–30, 30–36, 36–42, and 42–48 h after a p.o. dose of Sanqi extract at 8 ml/kg

(vial gavage; 3 rats/collection interval). Sodium taurocholate solution (pH 7.4) was

infused into the duodenal cannula (~1 ml/h) during bile collection. The bile samples

were weighed and stored at −70°C. Similar biliary excretion studies were performed

for individual ginsenosides administered in i.v. boluses of 5 μmol/kg.

Tissue distribution study in rats. Rats under ether anesthesia were sacrificed

by bleeding (~10 ml blood; the resulting plasma samples used for analysis) the


at ASPE



Dow

nloaded from


DMD 29819

9

abdominal aorta at 0, 5, 15 min, 1, 2, 4, 24, or 48 h (3 rats/time point) post i.v. bolus

of GRb1, GRg1, or C-K at 5 µmol/kg (via tail vein). Samples of abdominal adipose,

testicle, bladder, spleen, stomach, small intestine, large intestine, kidney, liver, lung,

heart, and brain were excised and then rinsed in ice-cold saline before gently blotting

on absorbent paper and weighing. The large tissues were homogenized in 4 volumes

of ice-cold saline, whereas the small ones were prepared in 9 volumes. The

homogenates were stored at −70°C.

Pharmacokinetic data processing. Plasma PK parameters were determined

by non-compartmental and compartmental methods using the Kinetica 2000 software

package (version 3.0; Philadelphia, PA). The Cmax and Tmax were observed values

without interpolation. The area under concentration-time curve up to the last

measured time point (AUC0→t) was calculated using the trapezoidal rule. The value of

AUC0→∞ was generated by extrapolating AUC0→t to infinity using the elimination rate

constant and the last measured concentration. The biliary clearance (CLB) and the

renal clearance (CLR) were calculated from the cumulative amount excreted (Cum.Ae)

in the bile and in the urine, respectively, divided by the plasma AUC. Dose

proportionality studies on AUC0→∞ and Cmax were conducted by the regression of

log-transformed data (power model) (Smith et al., 2000).

Cell culture and transport study in Caco-2 monolayers. Caco-2 cells

(American Type Culture Collection, Manassas, VA) were cultured as described

previously (Dai et al., 2008). Bidirectional transport experiments were conducted in

triplicate at 2–50 μM for the individual ginsenosides in HBSS. Dimethyl sulfoxide

was used as cosolvent to improve the solubility of one- or two-sugar containing

ginsenosides, Ppd, and Ppt, but its final concentration did not exceed 1% (v/v). After

incubation for 2 h at 37°C, 50-μl samples were collected from both the receiver and


at ASPE



Dow

nloaded from


DMD 29819

10

donor compartments, and the compound recovery was determined. The apparent

permeability coefficient (Papp) was expressed in cm/s and the efflux ratio

(Papp(basolateral→apical)/Papp(apical→basolateral)) >3 was considered to be a positive result,

suggesting that the tested compound was an in vitro substrate for the efflux

transporter(s). The P-gp substrate rhodamine 123 exhibited substantial directional

preference with the efflux ratio of 51.6, which was significantly reduced to 2.3 in the

presence of the P-gp inhibitor verapamil. For sulfasalazine, a MRP2 substrate, the

presence of the MRP2 inhibitor MK571 caused reduction of efflux ratios from 137 to

4.8. These data suggested the normal presence of the efflux transporters in the Caco-2

cell monolayers used.

In silico assessment of permeability and solubility. Chemoinformatic

assessment of the physicochemical properties governing intestinal absorption was

performed for ginsenosides and their aglycones. Aqueous solubility (S) was calculated

with ACD/aqueous solubility v8.02 via the ACD/I-Lab service (Advanced Chemistry

Development Inc., Toronto, Canada). The octanol-water partition coefficient (LogP)

was calculated using Pallas software (Pallas 3.6, CompuDrug International, Sedona,

AZ). The total hydrogen bond count (donors and acceptors), topological polar surface

area (TPSA), and number of rotatable bonds (NROTB) were determined using

Molinspiration Property Calculator (Molinspiration Cheminformatics, Bratislava,

Slovak Republic). Lipinski’s Rule of 5 (Lipinski et al., 1997) and the molecular

surface properties (total hydrogen bond count, TPSA, and NROTB) (Veber et al.,

2002) were used to predict the permeability of the tested compounds.

LC/MS/MS bioassays. Validated bioanalytical methods were used to measure

ginsenosides and their metabolites. Biological samples (20 μL), including plasma,

tissue homogenate, excretory, and cell culture medium samples, were precipitated


at ASPE



Dow

nloaded from


DMD 29819

11

with 80 μL of methanol followed by centrifugation at 21885g for 5 min before

LC/MS/MS analysis. TSQ Quantum mass spectrometer (Thermo Fisher, San Jose, CA)

was interfaced via an ESI probe with a liquid chromatograph (Agilent, Waldbronn,

Germany). The LC separation was achieved on a Phenomenex® Synergi 5 μm C18

column (Torrance, CA). The LC mobile phases were CH3OH/H2O (2:98, v/v,

containing 0.08 mM HCOONH4) for A and CH3OH/H2O (98:2, v/v, containing 0.08

mM HCOONH4) for B, which were used for pulse gradient chromatography (Wang et

al., 2007). The gradient parameters including the start proportion, the elution

proportion, the elution proportion segment, and the column equilibrium segment were

15% B, 100% B, 5.4 min, and 4.4 min, respectively, except for excretory samples

requiring a 5-min start proportion segment. ESI and collision energy were optimized

to maximize generation of the sodiated or protonated molecules and to produce the

characteristic product ions, respectively. The precursor-to-product ion pairs used for

selected reaction monitoring of GRa3, GRb1, GRc, GRd, GF2, GRg3, C-K, GRh2, Ppd,

20gRf, GRe, NGR1, GRg1, GRf, GRg2, GRh1, GF1, and Ppt were m/z 1263→497,

1131→365, 1101→335, 969→789, 807→627, 807→365, 645→203, 623→407,

461→443, 985→365, 969→789, 955→775, 823→643, 823→365, 807→349,

639→621, 661→203, and 477→109, respectively. An online motorized six-port

divert valve was used to introduce the LC eluent flow to the ion source for data

acquisition over the period of 3–9 min, except for excretory samples (8–14 min).

Matrix-matched calibration curves were constructed for the analytes (1.37 or

12.3–3000 ng/ml) using weighted linear regressions of the analyte peak area against

the corresponding nominal concentrations of the analyte (ng/ml), which showed good

linearity (r2 >0.99).


at ASPE



Dow

nloaded from


DMD 29819

12

Results

Rat systemic exposure to ginsenosides from p.o. Sanqi extract. Major

ginsenosides in Sanqi extract were the ppd-type GRa3 (4.6 mM), GRb1 (23.2 mM),

and GRd (6.9 mM) and the ppt-type GRe (5.2 mM), NGR1 (7.9 mM), and GRg1 (33.1

mM). 20gRf and GRh1 were present at lower levels (2.2 mM for both). In addition,

several minor ginsenosides, including GRc, GF2, GRg3, GRf, GRg2, and GF1 were at

the levels of 0.1–0.5 mM. No other ginsenosides or aglycones were detected in Sanqi

extract.

As shown in Fig. 1, the ppd-type GRa3, GRb1, and GRd were measurable in rat

plasma up to 48 h following p.o. administration of Sanqi extract (4–16 ml/kg),

whereas the ppt-type GRe, NGR1, and GRg1 were measurable only within 24 h. Of the

other ginsenosides, the ppd-type GRc, GF2, and GRg3 were only measured in

nanomolar plasma levels at 6–10 h after dosing at 8 and/or 16 ml/kg. The ppt-type

GRf, GRg2, GRh1, and GF1, except for 20gRf, were not detected in plasma.

Compound-K (C-K; ppd-type) was also measured in rat plasma with delayed

occurrence, i.e., 6 h after dosing. C-K was not contained in the administered Sanqi

extract; it was a colonic deglycosylated product of the bulky ppd-type ginsenosides

(Hasegawa et al., 1996; Akao et al., 1998). We found that C-K could be measured in

rat feces following p.o. administration of purified GRb1 or GRd.

(Insert Figure 1 Here)

The maximal plasma concentrations of the ppd-type GRa3, GRb1, or GRd

occurred 6–10 h after dosing. The exposure levels of GRa3, GRb1, and GRd in

AUC0→∞ and Cmax increased directly with Sanqi extract dose from 4 to 16 ml/kg but

nonlinearly. However, no significant dose-exposure relationship was observed for

C-K. In addition, we also observed a strong linear relationship from a scatter plot of


at ASPE



Dow

nloaded from


DMD 29819

13

logAUC0→∞ of GRa3, GRb1, and GRd versus their corresponding logarithmic

compound doses from the ingested extract (r2 = 0.96; n = 27; Fig. 1). The mean oral

bioavailability of GRa3, GRb1, and GRd after administration of the extract was quite

low: 0.1–0.2%. Due to their long t1/2, i.e., 13, 18, and 13 h for GRa3, GRb1, and GRd,

respectively, significant increases in systemic exposure were observed in the

multiple-dose study (Fig. 1). The plasma AUC0→24h and Cmax of these ppd-type

ginsenosides increased from day 1 to day 15 and the values (corrected for rat body

weight) on day 15 were 2.1–2.6-times higher than those on day 1. However, the

systemic exposure levels on day 22 increased only 1.3–1.9-fold compared with those

on day 1. The reason for these decreases remains to be explored.

Double or triple peaks occurred in the plasma-concentration time courses of the

ppt-type GRe, NGR1, and GRg1 following p.o. dose of Sanqi extract (Fig. 1). Despite

the interanimal variability, the peak appearance patterns for GRe, NGR1, and GRg1 for

a given rat were almost identical. The F values of these ppt-type ginsenosides ranged

from 0.2 to 0.6%. Dislike the preceding ppd-type compounds, the systemic exposure

levels of GRe, NGR1, and GRg1 were poorly correlated with the p.o. extract dosage.

In addition, the AUC0→t values of GRe, NGR1, and GRg1, normalized according to the

corresponding compound doses from the extract, were about 1/10 of those of the

ppd-type compounds.

Collectively, although their total amounts present in Sanqi extract were

comparable, the systemic exposure levels of the ppd-type ginsenosides GRa3, GRb1,

and GRd were significantly greater than those of the ppt-type ginsenosides GRe,

NGR1, and GRg1. The long t1/2 of these ppd-type compounds counteracted the

unfavorable effects of their poor F on the systemic exposure levels.

Permeability and solubility of ginsenosides and their deglycosylated


at ASPE



Dow

nloaded from


DMD 29819

14

products. Because of the poor F, the permeability and solubility of individual

ginsenosides were assessed in vitro and in silico to gain understanding of the

mechanisms governing intestinal absorption of ginsenosides. Because it had been

reported that ginsenosides could be stripped of their sugar moieties by the colonic

microflora (Hasegawa et al., 1996; Akao et al., 1998; Tawab et al., 2003), some

monoglycosides and aglycones were also included in the assessment. As shown in Fig.

2, the Papp values of ginsenosides measured in Caco-2 monolayers suggested that the

membrane permeability of most ginsenosides was poor with the Papp values <3×10−7

cm/s. GF2, C-K, GRh2, GRh1, and GF1 possessed slightly increased Papp values

(around 1×10−6 cm/s); the Papp values of Ppd and Ppt were about 2×10−6 cm/s. The

comparable bidirectional Papp values of most the ginsenosides and their aglycones

suggested passive diffusion as the transport mechanism, except for GF2 and GF1,

which exhibited efflux ratios 10.0 and 48.7, respectively. The efflux ratio of GF2 was

significantly reduced to 2.1 and 2.2, in the presence of verapamil and MK571,

respectively, suggesting that this ginsenoside might be the substrate of both P-gp and

MRP2. Similar situation occurred for GF1, i.e., the efflux ratio was reduced to 12.0

and 9.6 with verapamil and MK571, respectively.


In silico assessment suggested that many ginsenosides could be defined as

soluble (Fig. 2). S values ranged from 160×10−6 to 17 000×10−6 M, which were

significantly greater than the highest initial concentrations in the Caco-2 study

(50×10−6 M). However, the solubility significantly decreased for compounds with

fewer or no sugar moieties attached. The S values of GF1 and GRh1 were 86×10−6 and

75×10−6 M, respectively, whereas those of GRg3, C-K, GRh2, Ppd, GRg2, and Ppt

ranged from 0.08×10−6 to 9×10−6 M. The S values did not vary significantly as pH


at ASPE



Dow

nloaded from


DMD 29819

15

changed. On the other hand, most the ginsenosides, except C-K, GRh2, Ppd, GF1,

GRh1, and Ppt, had unfavorable traits underlying poor membrane permeability,

including high total hydrogen bond counts ranging from 22 to 44 (donors and

acceptors; favorable ≤12), high TPSA from 219 Å to 436 Å (favorable value ≤140 Å),

and high flexibility ranging from 10 to 18 (favorable value ≤10). Reducing the number

of sugar moieties decreased hydrogen-bonding capacity, molecular flexibility, and

molecular mass, but also reduced solubility significantly (Fig. 2). In addition, the

lipophilicity of the Ppd and Ppt aglycones was significantly higher (LogP, 5.4 and 4.4,

respectively) than that of the ginsenosides (LogP, −0.1–3.1).

Collectively, our in vitro observations and in silico calculations suggested that

the poor intestinal absorption of the ginsenosides could be attributed to poor

membrane permeability, which was influenced by the increased sugar number.

Although these traits were improved for the deglycosylated products, significant

increases in intestinal absorption appeared to be limited by their lowered solubility.

Structure-dependent disposition of i.v. administered ginsenosides in rats.

In order to understand the variations in the systemic exposure to ginsenosides

following p.o. administration of Sanqi extract, pure ginsenosides and their aglycones

were administered as i.v. boluses. As shown in Fig. 3, the systemic exposure levels of

the ginsenosides were directly related to their t1/2. GRa3, GRb1, GRc, and GRd had

significantly longer t1/2 values (7.5–19.8 h) than the other ginsenosides (0.2–3.2 h).

The wide variation in CLtot.p was the primary reason for the considerable differences

in t1/2. The tested ginsenosides could be divided into two groups according to CLtot.p:

(1) the 3–5 sugar-containing ppd-type ginsenosides GRa3, GRb1, GRc, and GRd

(CLtot.p, 4–20 ml/h/kg) and (2) the ppt-type (352–2718 ml/h/kg) and the minor

ppd-type ginsenosides (505–2296 ml/h/kg, except for GF2 being 66 ml/h/kg). In


at ASPE



Dow

nloaded from


DMD 29819

16

contrast to CLtot.p, the Vss values for most ginsenosides fell within the range of 93

ml/kg for GRg3 to 533 ml/kg for GRg1, which was between the rat plasma volume (31

ml/kg) and the rat total body water volume (670 ml/kg) (Davies and Morris, 1993).

The Vss value of GF1 was 808 ml/kg. Ppd and Ppt had short t1/2 values of 1.5 and 0.2 h,

respectively, as well as high CLtot.p (1482 and 6960 ml/h/kg, respectively) and large

Vss (2016 and 1716 ml/kg, respectively).

Collectively, the notable differences in systemic exposure among ginsenosides

were primarily related to their variations in CLtot.p, and the clearance mechanisms

were most likely subject to structure-specific recognition. Analysis of the structural

features of the compounds suggested that the attachment of 4 for more sugar moieties

significantly reduced the rate of elimination. In addition, the sugar attachment site

appeared to be also relevant as demonstrated by the significant difference in CLtot.p

between the ppd-type GRd and the ppt-type GRe, NGR1, and 20gRf (Fig. 3).


Elimination pathways of ginsenosides in rats receiving Sanqi extract. To

identify the primary elimination pathways that influenced systemic exposure to the

ginsenosides, we evaluated the biliary and renal excretion profiles of the ginsenosides,

as well as and the metabolism, in rats. In addition, fecal samples were also analyzed to

monitor intestinal nonabsorption, biliary excretion, and/or colonic deglycosylation of

ginsenosides. As shown in Fig. 4, the ppd-type ginsenosides (GRa3, GRb1, and GRd)

and the ppt-type ginsenosides (NGR1, GRe, and GRg1) exhibited significant

differences in excretion profiles. In contrast to the rat systemic exposure levels (as per

AUC), the cumulative amounts of the ppt-type ginsenosides excreted intact into bile

and urine, corrected according to the corresponding compound doses from Sanqi

extract, were significantly greater than those of the ppd-type ginsenosides.


at ASPE



Dow

nloaded from


DMD 29819

17


As shown in Fig. 4, the most abundant fecal compounds in rats were GRg1

(Cum.Ae, 39.1 μmol/kg), C-K (20.9 μmol/kg), and GF1 (7.1 μmol/kg). We speculated

that the high levels of these compounds in feces resulted from the colonic microflora

stripping the sugar moieties from the ginsenosides in the extract. Their absence (C-K)

or presence at a very low level (GF1) in Sanqi extract supports this hypothesis. The

contribution of colonic deglycosylation of major ppt-type ginsenosides to the fecal

GRg1 was supported by two lines of evidence: (1) fecal recovery of GRg1 following

p.o. administration of pure GRg1 was significantly lower than that following p.o.

administration of Sanqi extract (at the same GRg1 dose) in the same rats, and (2) p.o.

administration of pure 20gRf, GRe, and NGR1 generated fecal GRg1. Meanwhile,

poor intestinal absorption and rapid biliary excretion accounted for the relatively low

plasma levels of GRg1, C-K, and GF1. Although both of the aglycones Ppd and Ppt

were detected in fecal samples following p.o. administration of Sanqi extract, their

fecal Cum.Ae values were quite low: 0.4 and 0.2 μmol/kg, respectively. Ppd and Ppt

were not detected in plasma, bile, or urine, suggesting that they were two minor

metabolites in negligible quantities.

To further elucidate the excretion profiles of ginsenosides, individual pure

compounds were i.v. administered to rats at 5 μmol/kg. We confirmed that biliary

excretion was the major elimination route for most ginsenosides, demonstrating the

percentage of i.v. dose excreted intact into bile 43–100%. The ginsenosides were

excreted rapidly via active biliary transport, indicative of high bile-to-plasma

distribution ratios (AUCbile/AUCplasma, 22–1907). The AUCbile/AUCplasma ratio of GRd

was 4.4. However, the biliary excretion of GRa3, GRb1, and GRc were slow and their

AUCbile/AUCplasma ratios ranged from 0.2 to 0.4. As to renal excretion, most


at ASPE



Dow

nloaded from


DMD 29819

18

ginsenosides were slow and passively excreted into rat urine. GRg3 and

monoglycosides were detected in negligible amounts in urine. However, when

corrected for plasma protein binding, the unbound CLR of NRG1, 20gRf, and GRg1,

497–1007 ml/h/kg, exceeded the rat glomerular filtration rate of 314 ml/h/kg (Davies

and Morris, 1993), suggesting that some active tubular secretion occurred in rats.

Figure 5 shows the excretion profiles of GRb1 and GRg1, which could represent the

two types of typical situations among the tested ginsenosides. About 10% and 40% of

i.v. administered GRb1 were excreted intact into rat bile and urine, respectively,

whereas the corresponding fractions for GRg1 were around 73% or 18%, respectively.

The CLB and CLR were 1840 and 427 ml/h/kg, respectively, for GRg1, but only 1.0

and 3.5 ml/h/kg, respectively, for GRb1.

As to the metabolism of i.v. administered GRb1, the monooxidized metabolite,

the deglycosylated metabolite (GRd), and the monooxidized metabolite of GRd were

measured in rat bile and urine (Fig. 5). These metabolic reactions were catalyzed by

enzymes in rat tissues such as the liver. About 1.8% and 1.5% of i.v. administered

GRb1 were eliminated very slowly as the tissue-deglycosylated metabolite GRd via

bile and urine, respectively, and the values would continue to increase beyond 48 h for

bile and 96 h for urine. The tissue-oxidation of GRb1 appeared to take place faster

than the tissue-deglycosylation, but to lesser extent. We estimated that in rats about

10–15% of i.v. administered GRb1 might be ultimately eliminated via these metabolic

pathways. As for i.v. administered GRg1, only one monooxidized metabolite was

detected in rat bile and urine, which accounted for about 0.2% and 0.1% of the dose,

respectively. The significantly minimal tissue-metabolism observed for GRg1 could

have resulted from, at least in part, the rapid biliary and renal excretions. Recently,

Qian et al. also detected the oxidation and deglycosylation products of GRb1 in rat


at ASPE



Dow

nloaded from


DMD 29819

19

urine (2006).


Collectively, rapid and extensive biliary excretion was a key factor limiting the

systemic exposure to most ginsenosides from Sanqi extract, which appeared to

involve active secretion mechanisms. However, the ppd-type GRa3, GRb1, GRc, and

GRd circumvented or significantly reduced the active secretion into bile. Renal

excretion also contributed considerably to the elimination of some ginsenosides

including GRa3, GRb1, GRc, GRd, NRG1, 20gRf, and GRg1, but via different

transportation mechanisms. There was considerable deglycosylation of unabsorbed

ginsenosides by the colonic microflora, but the colonic metabolism did not

significantly improve overall systemic exposure to ginsenosides from orally

administered Sanqi extract. The relative contributions of tissue-deglycosylation and

tissue-oxidation to overall elimination of ginsenosides appeared to be poor.

Comparative tissue exposure to i.v. administered GRb1, GRg1, and C-K.

To address whether the observed differences in plasma concentration among

ginsenosides reflect well the situation in tissues, a comparative study of tissue

distribution was performed with GRb1, C-K, and GRg1, which was based on

measurements in whole rat tissue. These three compounds were selected as prototype

compounds for the study, because GRb1 and GRg1 were the ppd-type and the ppt-type

ginsenosides, respectively, present in the greatest amounts in Sanqi extract and

because their GRb1, C-K, and GRg1 were the compounds measured in large amounts

in rat plasma or excretory samples (Fig. 4). To ease comparisons, each investigational

compound was administered as an i.v. bolus of 5 μmol/kg. Following dosing, all

compounds transferred rapidly from blood to tissues with Tmax values of 0.1–2.7 h for

different tested tissues. Plasma and tissue concentrations of GRb1 at 5 min post dose


at ASPE



Dow

nloaded from


DMD 29819

20

(C5min) were in the rank order of plasma (50 μM) > liver (22 μM) > kidney (19 μM) >

heart (16 μM) > lung (12 μM) > bladder (5.1 μM) > spleen (4.7 μM) > testicle (3.8

μM) > large intestine (3.2 μM) > small intestine (2.7 μM) > stomach (2.2 μM) ≈

adipose (1.9 μM) > brain (0.8 μM). The tissue AUC values of GRb1 versus the

corresponding plasma value are shown in Fig. 6.

Consistent with the differences observed for plasma, both the C5min and AUC of

GRb1 in the tissues were much greater than the corresponding levels for GRg1 or C-K.

The tissue t1/2 values of GRb1 (14–31 h) were comparable with the corresponding

plasma data (20 h), which were significantly longer than those of GRg1 (0.2–1 h;

except for the kidney 22 h) or C-K (0.3–6 h). Despite its generally low levels in tissue,

GRg1 exhibited a relatively high C5min and AUC in the liver (70 μM and 28 μM·h,

respectively) compared with the corresponding plasma data (11 μM and 2 μM·h,

respectively). Similar phenomena were observed for C-K: 276 μM and 242 μM·h,

respectively, in the liver versus 29 μM and 11 μM·h, respectively, in plasma. The

hepatic uptake of GRg1 and C-K could be associated with the active biliary secretion

mediated by the hepatic transporters. In addition, renal uptake of GRg1 was also

extensive, demonstrating a relatively high C5min (86 μM) and AUC (99 μM·h) as

compared with the corresponding plasma data. In agreement with their unfavorable

properties regarding membrane permeability, GRb1, GRg1, and C-K were poorly

delivered to the CNS.


Collectively, the significant differences in the plasma levels among ginsenosides

could reflect the situations in the tissues. In addition, the tissue distribution data for

GRg1 and C-K support the possible involvement of active secretion mechanisms in

the hepatic and/or renal elimination of the compounds.


at ASPE



Dow

nloaded from


DMD 29819

21

Discussion

Bioactive constituents with favorable PK properties and existing in adequate

abundance in a medicinal herb are most likely to account for the pharmacological

effects of the herb and to form the basis of its therapeutic efficacy. Our current and

recent studies (Lu et al., 2008) indicate that both animals and humans are exposed

significantly to some but not all constituents of an herbal medicine after dosing and

that the PK profiles, like the pharmacological activities, can be used as a “sieve” to

assess the importance and usefulness of the individual herbal constituents. Such PK

studies are essential for understanding the link between the herb consumption and the

pharmacological effects and for identifying the medicinal principles from the

chemical constituents in an herbal medicine.

In contrast to Western medicines that normally contain only one active ingredient,

herbal medicines contain numerous chemical constituents. Multi-component PK

studies of an herbal medicine can be challenging. Such studies are complicated by the

great diversity of the constituents with regard to both chemical structure and content.

Variations in chemical structures result in different physiochemical properties and PK

profiles among the constituents, whereas the variations in content result in

dissimilarities in constituent dosages. Some important goals for PK studies of an

herbal medicine are to (1) measure systemic exposure to the administered medicine,

(2) identify suitable “PK markers” indicative of exposure to the medicine, (3)

compare the PK properties of the constituents, (4) understand the pathways that

influence their systemic exposure, (5) analyze the quantitative structure-PK

relationships for the constituents and metabolites, and (6) assess differences in PK

properties between the animal species and humans. It is worth mentioning that the

constituents in an herbal medicine are often structurally related, which may be divided


at ASPE



Dow

nloaded from


DMD 29819

22

into more than one class. Analysis of quantitative structure-PK relationships (QSPKR)

of herbal chemicals may gain insight into the role of molecular properties and/or

functional group presentation in the PK of compounds, and may help understand the

PK trends within the compound series and anticipate which homologues have

favorable PK properties compatible with the pharmacological activities. The key role

that structure-based differentiation can play in the absorption and disposition of herbal

constituents highlights that the QSPKR analysis is a vital component of the

multi-component PK study. In the current study, QSPKR analysis helped us identify

key factors affecting the systemic exposure to different ginsenosides and their

deglycosylated products following administration of Sanqi extract.

The PK properties of constituents contained in an herbal medicine can be quite

different. When bioactive constituents from an herb are measurable in a biosample,

such as plasma or urine, and have favorable PK properties that could be used to

substantiate systemic exposure to the herb, they are referred to as “PK markers” of the

herb (Lu et al., 2008). Identification of PK markers for herbs could be helpful in the

design and interpretation of toxicity and clinical studies, as well as in the evaluation of

potential herb-drug or herb-herb interactions. Here, we measured rat systemic

exposure to ginsenosides and their deglycosylated products following p.o.

administration of Sanqi extract and compared their PK properties. We found that the

ppd-type GRa3, GRb1, and GRd measured in plasma were suitable PK markers of p.o.

administered Sanqi extract in rats. The conclusion was made on the basis of the

dose-dependent systemic exposures and PK properties of these ginsenosides. In

contrast, the other plasma ginsenosides, including the ppt-type GRe, NGR1, and GRg1

and the metabolite ppd-type C-K, were not the PK markers for Sanqi extract because

of their low exposure levels and poor dose proportionality. Although the ppt-type


at ASPE



Dow

nloaded from


DMD 29819

23

ginsenosides were excreted into the rat urine in substantial amounts, large inter-animal

variation prevented the use of the urinary compounds as surrogate PK markers.

Meanwhile, renal excretion of GRa3, GRb1, GRd, and C-K was very slow.

Poor membrane permeability was a major factor limiting intestinal absorption of

ginsenosides, which was attributed mainly to the sugar moieties increasing the

hydrogen bond count, polar surface area, and molecular flexibility of the molecules to

unfavorable levels. Furthermore, reducing the sugar moiety content appeared not to

improve significantly intestinal absorption, because of the associated decrease in

solubility. This is supported by our rat data that the fecal Cum.Ae values of the

deglycosylated products exceeded substantially the corresponding sum of biliary and

urinary Cum.Ae. In addition, the poor permeability could also partially explain our

observations in the tissue distribution study. In most cases, the tissue concentrations of

ginsenosides were lower than the corresponding plasma levels, and the compounds

persisted in the organ capillaries and the organ interstitial fluid, rather than the organ

cells. However, the liver concentrations of most ginsenosides were significantly

higher than the plasma levels. This can most likely be attributed to some uptake

transporters mediating active transport of the ginsenosides into the hepatic cells.

Most of the ginsenosides and their deglycosylated products were subject to rapid

extensive biliary excretion through active transport, resulting in their short t1/2 values

and low systemic exposure levels. Meanwhile, renal excretion of most ginsenosides is

slow and its role in the overall elimination could be minimized by rapid extensive

biliary excretion. There were two kinds of exception, i.e., (1) slow but extensive renal

excretion because of slow biliary excretion for GRa3, GRb1, and GRc and (2) rapid

renal excretion involving active tubular secretion for NRG1, 20gRf, and GRg1. In

addition, the contribution of ginsenoside metabolism to their overall elimination was


at ASPE



Dow

nloaded from


DMD 29819

24

also limited. The metabolism occurring in rat tissues, such as the liver, included

deglycosylation and oxidation. The tissue-deglycosylation was found to be quite slow,

while the tissue-oxidation was rapid. Compared with the minor tissue-deglycosylation,

the pre-absorption deglycosylation of ginsenosides mediated by the gut microflora

appeared to be relatively extensive. Our data (Fig. 4) suggested that the colonic

deglycosylation of the ppd-type ginsenosides in rats occurred at C-20 until only one

glucose remained, i.e., GRd, and then deglycosylation preferentially took place at C-3,

followed by stripping of the last glucose moiety at C-20 to yield the aglycone Ppd.

Meanwhile, the initial colonic deglycosylation of the ppt-type ginsenosides appeared

to occur preferentially at C-6 to yield GRg1. Further deglycosylation occurred at either

C-6 or C-20 to form GF1 or GRh1, respectively, which was followed by stripping of

the last glucose moiety at C-20 or C-6, respectively, to yield the aglycone Ppt.

The attachment of four and five sugar moieties in the ppd-type GRa3 and GRb1,

respectively, appeared to play a key role in blocking access to the biliary transporters

responsible, resulting in slow biliary excretion of the two compounds (CLB: 1.5 and

1.0 ml/h/kg, respectively) in rats. GRd had a significantly slower CLB value (19

ml/h/kg) than the other three-sugar containing ginsenosides, 20gRf, GRe, and NGR1

(258–775 ml/h/kg), suggesting that the sugar attachment sites also influenced the

active biliary excretion. In addition, GRa3, GRb1, and GRd underwent limited

metabolism and slow renal excretion. Slow elimination of these bulky ppd-type

ginsenosides made them long-circulating, which counteracted the unfavorable effect

of poor intestinal absorption on their systemic exposure.

GRb1 and GRd have been reported to possess activities associated with the

putative cardiovascular effects of Sanqi. GRb1 has estrogenic activity (Lee et al., 2003)

via a mechanism independent of estrogen receptor-binding (Cho et al., 2004). GRb1


at ASPE



Dow

nloaded from


DMD 29819

25

also has antioxidant activity (Liu et al., 2003). GRd can dilate vascular muscle by

blocking Ca2+ influx through receptor- and store-operated Ca2+ channels in the muscle

cells (Guan et al., 2006). The pharmacological activities of GRa3 remain to be

understood.

Drug exposure is a crucial determinant of drug response, and therefore its

efficacy and safety. In the current study, we compared the systemic exposure to

putatively active ginsenosides from p.o. administered Sanqi extract and investigated

the relevant mechanisms governing exposure to ginsenosides. In summary, our data

indicate that poor membrane permeability and rapid and extensive active biliary

excretion are two primary factors limiting systemic exposure to most Sanqi

ginsenosides and their deglycosylated metabolites. The major ginsenosides measured

in the plasma of rats were long-circulating GRa3, GRb1, and GRd, because of their

slow biliary excretion. These plasma ginsenoside can be used as suitable PK markers

for Sanqi extract. Considerable colonic deglycosylation occurred but the systemic

exposure to the metabolites was low. The PK profiles of the Sanqi ginsenosides in

humans were comparable to those found in rats. Slight interspecies differences could

have resulted from humans being relatively poor biliary excretors compared with rats.

The details of the human study will be reported elsewhere.


at ASPE



Dow

nloaded from


DMD 29819

26

References

Akao T, Kanaoka M, Kobashi K (1998) Appearance of compound K, a major

metabolite of ginsenoside Rb1 by intestinal bacteria, in rat plasma after oral

administration — measurement of compound K by enzyme immunoassay. Biol

Pharm Bull 21: 245–249.

Chinese Pharmacopoeia Commission (2005) Pharmacopoeia of the People’s Republic

of China (English Edition, Volume I). Beijing: People’s Medical Publishing

House. pp. 10–11.

Cho J, Park W, Lee S, Ahn W, Lee Y (2004) Ginsenosides-Rb1 from Panax ginseng

C.A. Meyer activates estrogen receptor-α and -β, independent of ligand binding.

J Clin Endocrinol Metab 89: 3510–3515.

Dai J-Y, Yang J-L, Li C (2008) Transport and metabolism of flavonoids from Chinese

herbal remedy Xiaochaihu-tang across human intestinal Caco-2 cell monolayers.

Acta Pharmacol Sin 29: 1086–1093.

Davies B, Morris T (1993) Physiological parameters in laboratory animals and

humans. Pharm Res 10: 1093–1095.

Guan Y-Y, Zhou J-G, Zhang Z, Wang G-L, Cai B-X, Hong L, Qiu Q-Y, He H (2006)

Ginsenoside-Rd from panax notoginseng blocks Ca2+ influx through receptor-

and store-operated Ca2+ channels in vascular smooth muscle cells. Eur J

Pharmacol 548: 129–136.

Hasegawa H, Sung JH, Matsumiya S, Uchiyama M (1996) Main ginseng saponin

metabolites formed by intestinal bacteria. Planta Med 62: 453–457.

Kwan CY (1995) Vascular effects of selected antihypertensive drugs derived from

traditional medicinal herbs. Clin Exp Pharmacol Physiol 22: s297–s299.

Lee Y, Jin Y, Lim W, Park W, Cho J, Jang S, Lee SK (2003) Ginsenoside-Rb1 acts as a


at ASPE



Dow

nloaded from


DMD 29819

27

weak phytoestrogen in MCF-7 human breast cancer cells. Arch Pharm Res 26:

58–63.

Li L, Sheng Y-X, Zhang J-L, Wang C-S, Guo D-A (2004a) HPLC determination of

four active saponins from Panax notoginseng in rat serum and its application to

pharmacokinetic studies. Biomed Chromatogr 18: 849–856.

Li L, Zhang J-L, Sheng Y-X, Ye G, Guo H-Z, Guo D-A (2004b) Liquid

chromatographic method for determination of four active saponins from Panax

notoginseng in rat urine using solid-phase extraction. J Chromatogr B 808:

177–183.

Li L, Sheng Y-X, Zhang J-L, Wang S-S, Guo D-A (2006) High-performance liquid

chromatographic assay for the active saponins from Panax notoginseng in rat

tissues. Biomed Chromatogr 20: 327–335.

Li X-Y, Sun J-G, Wang G-J, Hao H-P, Liang Y, Zheng Y-T, Yan B, Sheng L-S (2007a)

Simultaneous determination of Panax notoginsenoside R1, ginsenoside Rg1, Rd,

Re and Rb1 in rat plasma by HPLC/ESI/MS: platform for the pharmacokinetic

evaluation of total panax notoginsenoside, a typical kind of multiple constituent

traditional Chinese medicine. Biomed Chromatogr 21: 735–746.

Li X-Y, Wang G-J, Sun J-G, Hao H-P, Xiong Y-Q, Yan B, Zheng Y-T, Sheng L-S

(2007b) Pharmacokinetic and absolute bioavailability study of total Panax

notoginseng, a typical multiple constituent traditional Chinese medicine (TCM)

in rats. Biol Pharm Bull 30: 847–851.

Lipinski CA, Lombardo F, Dominy BW, Feeney PJ (1997) Experimental and

computational approaches to estimate solubility and permeability in drug

discovery and development settings. Adv Drug Deliv Rev 23: 3–25.

Liu Z-Q, Luo X-Y, Liu G-Z, Chen Y-P, Wang Z-C, Sun Y-X (2003) In vitro study of


at ASPE



Dow

nloaded from


DMD 29819

28

the relationship between the structure of ginsenoside and its antioxidative or

prooxidative activity in free radical induced hemolysis of human erythrocytes. J

Agric Food Chem 51: 2555–2558.

Lu T, Yang J-L, Gao X-M, Chen P, Du F-F, Sun Y, Wang F-Q, Xu F, Shang H-C,

Huang Y-H, Wang Y, Wan R-Z, Liu C-X, Zhang B-L, Li C (2008) Plasma and

urinary tanshinol from Salvia miltiorrhiza (Danshen), can be used as

pharmacokinetic markers for cardiotonic pills, a cardiovascular herbal medicine.

Drug Metab Dispos 36: 1578–1586.

Mashour NH, Lin GI, Frishman WH (1998) Herbal medicine for the treatment of

cardiovascular disease. Arch Intern Med 158: 2225–2234.

Ng TB (2006) Pharmacological activity of Sanqi ginseng (Panax notoginseng). J

Pharm Pharmacol 58: 1007–1019.

Odani T, Tanizawa H, Takino Y (1983a) Studies on the absorption, distribution,

excretion and metabolism of ginseng saponins. II. The absorption, distribution,

and excretion of ginsenoside Rg1 in the rat. Chem Pharm Bull 31: 292–298.

Odani T, Tanizawa H, Takino Y (1983b) Studies on the absorption, distribution,

excretion and metabolism of ginseng saponins. III. The absorption, distribution,

and excretion of ginsenoside Rb1 in the rat. Chem Pharm Bull 31: 1059–1066.

Qian T-X, Jiang Z-H and Cai Z-W (2006) High-performance liquid chromatography

coupled with tandem mass spectrometry applied for metabolic study of

ginsenoside Rb1 on rat. Anal Biochem 352: 87–96.

Sengupta S, Toh S-A, Sellers LA, Skepper JN, Koolwijk P, Leung HW, Yeung H-W,

Wong RNS, Sasisekharan R, Fan T-PD (2004) Modulating angiogenesis: the Yin

and the Yang in ginseng. Circulation 110: 1219–1225.

Smith BP, Vandenhende FR, DeSante KA, Farid NA, Welch PA, Callaghan JT, Forgue


at ASPE



Dow

nloaded from


DMD 29819

29

ST (2000) Confidence interval criteria for assessment of dose proportionality.

Pharm Res 17: 1278–1283.

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

Degradation of ginsenosides in humans after oral administration. Drug Metab

Dispos 31: 1065–1071.

Veber DF, Johnson SR, Cheng H-Y, Smith BR, Ward KW, Kopple KD (2002)

Molecular properties that influence the oral bioavailability of drug candidates. J

Med Chem 45: 2615–2623.

Wang L, Sun Y, Du F-F, Niu W, Lu T, Kan J-M, Xu F, Yuan K-H, Qin T, Liu C-X, Li

C (2007) ‘LC-electrolyte effects’ improve the bioanalytical performance of

liquid chromatography/tandem mass spectrometric assays in supporting

pharmacokinetic study for drug discovery. Rapid Commun Mass Spectrom 21:

2573–2584.

Xu Q-F, Fang X-L, Chen D-F (2003) Pharmacokinetics and bioavailability of

ginsenoside Rb1 and Rg1 from Panax notoginseng in rats. J Ethnopharmcol 84:

187–192.


at ASPE



Dow

nloaded from


DMD 29819

30

Footnotes

This work was supported by Grant 2005CB523403 from the Chinese

Ministry of Science and Technology, Grant 2009ZX09304-002 from the

Chinese Ministry of Health, Grant 90209044 from the National Natural Science

Foundation of China, and Grant 08DZ1980200 from the Science & Technology

Commission of Shanghai Municipality.


at ASPE



Dow

nloaded from


DMD 29819

31

FIG. 1. Plasma concentration-time profiles of ginsenosides in rats following p.o.

administration of Sanqi extract. In the single-dose study, rats received Sanqi extract at

4, 8, or 16 ml/kg. In the multiple-dose study, rats received Sanqi extract at 4

ml/rat/day for 22 days. The right upper panel shows good logarithmic compound

dose-exposure relationship among the bulky ppd-type ginsenosides GRa3, GRb1, and

GRd, but the doses of other ginsenosides did not correlate with their exposures.

FIG. 2. Relationship between the sugar-substitution in ginsenosides and their PK

properties that limit intestinal absorption. GF2 and GF1 exhibited efflux ratios greater

than 8 in Caco-2 monolayers.

FIG 3. Relationship between the sugar-substitution in ginsenosides and their

disposition profiles in rats following i.v. administrations. The i.v. dose of each

individual compound was 5 μmol/kg. The PK parameters were calculated from the

plasma concentration-time curves.

FIG. 4. Rat excretion profiles of ginsenosides and their deglycosylated products

following p.o. administration of Sanqi extract. The p.o. dose of Sanqi extract was 8

ml/kg. The red numbers 3, 6, and 20 in parentheses indicate the deglycosylation sites

in the ginsenosides. Note that the measured urinary Cum.Ae values of C-K, GF1 and

GRh1 could be greater than the actual values due to fecal contamination (which

contained substantially high amounts of the compounds) during sample collection.

FIG. 5. Rat excretion profiles of i.v. administered GRb1, GRg1, and their metabolites.

The i.v. dose of each individual compound was 5 μmol/kg. Both the oxidized (Ox-)

and deglycosylated metabolites were measured. GRd was the only measurable

deglycosylated metabolite of i.v. administered GRb1, whereas no deglycosylated

metabolite of GRg1 was detected in urine or bile following i.v. administration of the


at ASPE



Dow

nloaded from


DMD 29819

32

compound.

FIG. 6. Tissue distribution profiles of i.v. administered GRb1, GRg1, and C-K in rats.

The i.v. dose of each individual compound was 5 μmol/kg. The plasma Vss values for

i.v. administered GRb1, GRg1, and C-K in rats were 220, 533, and 441 ml/kg,

respectively.


at ASPE



Dow

nloaded from


Pla

sma

conc

entr

atio

n (n

M)

Time (h)

0

1.5

3.0

1 8 15 22

AU

C(µ

M·h

)

0

0.8

1.6

Cm

ax(µ

M)

1 8 15 220

15

30

AU

C(µ

M·h

)

1 8 15 22

Duration of administration (day)

0.001

0.01

0.1

1

10

100

1 10 100 1000

AU

C (

µM

·h)

Dose (µmol/kg)

0

70

140

210

280

350 GRa3

0

300

600

900

1200

1500 GRb1

0

200

400

600

8001000

300 6 12 2418 4236 48

GRd

0

40

80

120

160

200

0 6 12 2418

GRe

0

40

80

120

160

200

0 6 12 2418

NGR1

0

150

300

450

600

750

0 6 12 2418

GRg1

GRa3GRb1GRd

C-K

GRc

GF2

GRg3

16 ml/kg 8 ml/kg 4 ml/kg

GRe

NGR1

GRg1

20gRf

0

40

80

120

160200

0 6 12 2418

C-K

R2 = 0.90

30

20

10

00 4 8 16

R2 = 0.94

0 4 8 16

R2 = 0.92

0 4 8 16

AU

C(µ

M·h

)

Dose of Sanqi extract (ml/kg)

0.6

0.4

0.2

00 4 8 16

3.6

2.4

1.2

00 4 8 16 0 4 8 16 0 4 8 16

R2 = 0.23

R2 = 0.00 R2 = 0.05R2 = 0.22

GRe NGR1 GRg1GRa3 GRb1 GRd C-K

Pla

sma

conc

entr

atio

n (n

M)

Time (h)

0

1.5

3.0

1 8 15 22

AU

C(µ

M·h

)

0

0.8

1.6

Cm

ax(µ

M)

1 8 15 220

15

30

AU

C(µ

M·h

)

1 8 15 22


0

1.5

3.0

1 8 15 22

AU

C(µ

M·h

)

0

0.8

1.6

Cm

ax(µ

M)

1 8 15 220

0.8

1.6

Cm

ax(µ

M)

1 8 15 221 8 15 220

15

30

AU

C(µ

M·h

)

1 8 15 221 8 15 22


0.001

0.01

0.1

1

10

100

1 10 100 1000

AU

C (

µM

·h)

Dose (µmol/kg)

0.001

0.01

0.1

1

10

100

1 10 100 1000

AU

C (

µM

·h)

Dose (µmol/kg)

0

70

140

210

280

350 GRa3

0

70

140

210

280

350 GRa3

0

300

600

900

1200

1500 GRb1

0

300

600

900

1200

1500 GRb1

0

200

400

600

8001000

300 6 12 2418 4236 48

GRd

0

200

400

600

8001000

300 6 12 2418 4236 48300 6 12 2418 4236 48

GRd

0

40

80

120

160

200

0 6 12 2418

GRe

0

40

80

120

160

200

0

40

80

120

160

200

0 6 12 24180 6 12 2418

GRe

0

40

80

120

160

200

0 6 12 2418

NGR1

0

40

80

120

160

200

0

40

80

120

160

200

0 6 12 24180 6 12 2418

NGR1

0

150

300

450

600

750

0 6 12 2418

GRg1

0

150

300

450

600

750

0 6 12 24180 6 12 2418

GRg1

GRa3GRb1GRd

C-K

GRc

GF2

GRg3


GRe

NGR1

GRg1

20gRf

GRa3GRb1GRd

C-K

GRc

GF2

GRg3


GRe

NGR1

GRg1

20gRf

0

40

80

120

160200

0 6 12 2418

C-K

0

40

80

120

160200

0 6 12 24180 6 12 2418

C-K

R2 = 0.90

30

20

10

00 4 8 16

R2 = 0.94

0 4 8 16

R2 = 0.92

0 4 8 16

AU

C(µ

M·h

)


0.6

0.4

0.2

00 4 8 16

3.6

2.4

1.2

00 4 8 16 0 4 8 16 0 4 8 16

R2 = 0.23

R2 = 0.00 R2 = 0.05R2 = 0.22


R2 = 0.90

30

20

10

00 4 8 16

R2 = 0.94

0 4 8 16

R2 = 0.92

0 4 8 16

R2 = 0.90

30

20

10

00 4 8 16

R2 = 0.94

0 4 8 16

R2 = 0.92

0 4 8 16

AU

C(µ

M·h

)


0.6

0.4

0.2

00 4 8 16

3.6

2.4

1.2

00 4 8 16 0 4 8 16 0 4 8 16

R2 = 0.23

R2 = 0.00 R2 = 0.05R2 = 0.22

0.6

0.4

0.2

00 4 8 16

0.6

0.4

0.2

00 4 8 16

3.6

2.4

1.2

00 4 8 16

3.6

2.4

1.2

00 4 8 160 4 8 16 0 4 8 160 4 8 16 0 4 8 160 4 8 16

R2 = 0.23

R2 = 0.00 R2 = 0.05R2 = 0.22


Figure 1


at ASPE



Dow

nloaded from


Figure 2

GRa3

GRc

GRb1

GRd

GF2

GRg3

GRg2GRg1 NGR1

20gRf

GRe

C-K

GRh1

GRh2

PptPpd

GRf

GF1

GF1 GF2

0.5x10-6

10-5

GRa3

GRc

GRb1

GRd

GF2

GRg3

GRg2GRg1 NGR1

20gRf

GRe

C-K

GRh1

GRh2

PptPpd

GRf

GF1

GF1 GF2

0.5x10-6

10-5

1

S(M

)P

app

(cm

/s)

Sugar count

0 4 5Sugar count

1 2 3

7

4

1

-2

LogP

200

300

400

100

0

TP

SA

(Å

)

0 4 51 2 3

500

GRa3

GRcGRb

1

GRdNGR

1

20gRf

GRe

Ppd

10

Ppt

GRh1GF

1

GRg1

GRg2

GRfC-KGRh2

GF2GRg3

GRa3

GRcGRb

1

GRdNGR

1

20gRf

GRe

Ppd

10

Ppt

GRh1GF

1

GRg1

GRg2

GRfC-KGRh2

GF2GRg3

GRa3

GRcGRb1GRd

NGR1

20gRf

GReGF2GRg

3

GRg2

GRg1GRf

GRh1GF1C-KGRh

2

Ppt

Ppd

5

0GRa

3GRcGRb1GRd

NGR1

20gRf

GReGF2GRg

3

GRg2

GRg1GRf

GRh1GF1C-KGRh

2

Ppt

Ppd

5

0

PptPpd

GRh1GF

1

C-KGRh2

GF2

GRg3

GRg2

GRg1

GRfGRdNGR

1

20gRfGRe

GRcGRb1

GRa3

140 ÅPptPpd

GRh1GF

1

C-KGRh2

GF2

GRg3

GRg2

GRg1

GRfGRdNGR

1

20gRfGRe

GRcGRb1

GRa3

140 Å

GRa3

GRcGRb1

GRd

NGR1

20gRf

GRe

GF2GRg3GRg2

GRg1GRf

GRh1GF

1C-KGRh2

PptPpd

12

GRa3

GRcGRb1

GRd

NGR1

20gRf

GRe

GF2GRg3GRg2

GRg1GRf

GRh1GF

1C-KGRh2

PptPpd

12

0

10

40

Tot

al H

-bon

d co

unt 50

20

30

4

8

16

NR

OT

B

20

12

0

0 4 51 2 310-8

10-7

10-6

10-5

10-2

10-4

10-6

10-8

GRa3

GRc

GRb1

GRdNGR1

20gRf

GReGF2

GRg3GRg2

GRg1

GRfGRh1GF1C-KGRh2

Ppt

Ppd

50x10-6

GRa3

GRc

GRb1

GRdNGR1

20gRf

GReGF2

GRg3GRg2

GRg1

GRfGRh1GF1C-KGRh2

Ppt

Ppd

50x10-6

: apical basolateral

: basolateral apical GRa3

GRc

GRb1

GRd

GF2

GRg3

GRg2GRg1 NGR1

20gRf

GRe

C-K

GRh1

GRh2

PptPpd

GRf

GF1

GF1 GF2

0.5x10-6

10-5

GRa3

GRc

GRb1

GRd

GF2

GRg3

GRg2GRg1 NGR1

20gRf

GRe

C-K

GRh1

GRh2

PptPpd

GRf

GF1

GF1 GF2

0.5x10-6

10-5

1

S(M

)P

app

(cm

/s)

Sugar count

0 4 5Sugar count

1 2 3

7

4

1

-2

LogP

200

300

400

100

0

TP

SA

(Å

)

0 4 51 2 3

500

GRa3

GRcGRb

1

GRdNGR

1

20gRf

GRe

Ppd

10

Ppt

GRh1GF

1

GRg1

GRg2

GRfC-KGRh2

GF2GRg3

GRa3

GRcGRb

1

GRdNGR

1

20gRf

GRe

Ppd

10

Ppt

GRh1GF

1

GRg1

GRg2

GRfC-KGRh2

GF2GRg3

GRa3

GRcGRb1GRd

NGR1

20gRf

GReGF2GRg

3

GRg2

GRg1GRf

GRh1GF1C-KGRh

2

Ppt

Ppd

5

0GRa

3GRcGRb1GRd

NGR1

20gRf

GReGF2GRg

3

GRg2

GRg1GRf

GRh1GF1C-KGRh

2

Ppt

Ppd

5

0

PptPpd

GRh1GF

1

C-KGRh2

GF2

GRg3

GRg2

GRg1

GRfGRdNGR

1

20gRfGRe

GRcGRb1

GRa3

140 ÅPptPpd

GRh1GF

1

C-KGRh2

GF2

GRg3

GRg2

GRg1

GRfGRdNGR

1

20gRfGRe

GRcGRb1

GRa3

140 Å

GRa3

GRcGRb1

GRd

NGR1

20gRf

GRe

GF2GRg3GRg2

GRg1GRf

GRh1GF

1C-KGRh2

PptPpd

12

GRa3

GRcGRb1

GRd

NGR1

20gRf

GRe

GF2GRg3GRg2

GRg1GRf

GRh1GF

1C-KGRh2

PptPpd

12

0

10

40

Tot

al H

-bon

d co

unt 50

20

30

4

8

16

NR

OT

B

20

12

0

0 4 51 2 310-8

10-7

10-6

10-5

10-2

10-4

10-6

10-8

GRa3

GRc

GRb1

GRdNGR1

20gRf

GReGF2

GRg3GRg2

GRg1

GRfGRh1GF1C-KGRh2

Ppt

Ppd

50x10-6

GRa3

GRc

GRb1

GRdNGR1

20gRf

GReGF2

GRg3GRg2

GRg1

GRfGRh1GF1C-KGRh2

Ppt

Ppd

50x10-6


: basolateral apical


: basolateral apical


at ASPE



Dow

nloaded from


Figure 3

10000

1000

100

10

1

CL t

ot,p

(ml/h

/kg)

0 41 2 3 5

2000

1000

0

2500

1500

500Vss

(ml/k

g)

Sugar count

0

5

10

15

20t 1

/2(h

)

GF2

GReNGR1

20gRf

GRg1

GRg3

GRg2

GRf

GF1

Ppd

Ppt

GRa3

GRc

GRd

C-K

GRh2GRh1

GRb1

GF2

GReNGR1

20gRf

GRg1

GRg3

GRg2

GRf

GF1

Ppd

Ppt

GRa3

GRc

GRd

C-K

GRh2GRh1

GRb1

GF1

C-KGRh1GRh2

GRg1

GRg2

GRg3

GRd

NGR1

GRe20gRf GRa3GRc

GRb1GF2

GRf

Rat total body water(670 ml/kg)Rat plasma

volume(31 ml/kg)

Ppd

Ppt

GF1

C-KGRh1GRh2

GRg1

GRg2

GRg3

GRd

NGR1

GRe20gRf GRa3GRc

GRb1GF2

GRf


volume(31 ml/kg)

Ppd

Ppt

R1O

R2O

HO(20)

(3)

(20)

(3)OH notexistingat C-6

Ppd-type

(20)

HO

R2O

HO

OR3(6)

(3)

Ppt-type

(20)No Sugarattachedto C-3

(6)

Glucose Xylose Rhamnose Arabinose

R1O

R2O

HO(20)

(3)

(20)


Ppd-type

(20)

HO

R2O

HO

OR3(6)

(3)

Ppt-type


(6)


(6)


R1O

R2O

HO(20)

(3)

(20)


Ppd-type

(20)

HO

R2O

HO

OR3(6)

(3)

Ppt-type


(6)


R1O

R2O

HO(20)

(3)

(20)


Ppd-type

(20)

HO

R2O

HO

OR3(6)

(3)

Ppt-type


(6)


(6)


R1O

R2O

HO(20)

(3)

(20)


Ppd-type

(20)

HO

R2O

HO

OR3(6)

(3)

Ppt-type


(6)


R1O

R2O

HO(20)

(3)

(20)


Ppd-type

(20)

HO

R2O

HO

OR3(6)

(3)

Ppt-type


(6)


(6)


R1O

R2O

HO(20)

(3)

(20)


Ppd-type

(20)

HO

R2O

HO

OR3(6)

(3)

Ppt-type


(6)


R1O

R2O

HO(20)

(3)

(20)


Ppd-type

(20)

HO

R2O

HO

OR3(6)

(3)

Ppt-type


(6)


(6)


GRa3

GRcGRb1

GRd

GF2GRg3

GRg2GRg1

NGR120gRfGRe

GRh1GF1GRh2

PptPpd

GRf

C-K

0

500

1000

1500

0 5 10 15 20

t1/2 (h)

AU

C (

µM

·h)

GRd

GRa3 GRb1

GRc

The otherginsenosides

0

500

1000

1500

0 5 10 15 20

t1/2 (h)

AU

C (

µM

·h)

GRd

GRa3 GRb1

GRc


GRa3

GRcGRb1

GRd

GF2GRg3

GRg2GRg1

NGR120gRfGRe

GRh1GF1GRh2

PptPpd

GRf

C-K

0

500

1000

1500

0 5 10 15 20

t1/2 (h)

AU

C (

µM

·h)

GRd

GRa3 GRb1

GRc


0

500

1000

1500

0 5 10 15 20

t1/2 (h)

AU

C (

µM

·h)

GRd

GRa3 GRb1

GRc


10000

1000

100

10

1

CL t

ot,p

(ml/h

/kg)

0 41 2 3 5

2000

1000

0

2500

1500

500Vss

(ml/k

g)

Sugar count

0

5

10

15

20t 1

/2(h

)

GF2

GReNGR1

20gRf

GRg1

GRg3

GRg2

GRf

GF1

Ppd

Ppt

GRa3

GRc

GRd

C-K

GRh2GRh1

GRb1

GF2

GReNGR1

20gRf

GRg1

GRg3

GRg2

GRf

GF1

Ppd

Ppt

GRa3

GRc

GRd

C-K

GRh2GRh1

GRb1

GF1

C-KGRh1GRh2

GRg1

GRg2

GRg3

GRd

NGR1

GRe20gRf GRa3GRc

GRb1GF2

GRf


volume(31 ml/kg)

Ppd

Ppt

GF1

C-KGRh1GRh2

GRg1

GRg2

GRg3

GRd

NGR1

GRe20gRf GRa3GRc

GRb1GF2

GRf


volume(31 ml/kg)

Ppd

Ppt

R1O

R2O

HO(20)

(3)

(20)


Ppd-type

(20)

HO

R2O

HO

OR3(6)

(3)

Ppt-type


(6)


R1O

R2O

HO(20)

(3)

(20)


Ppd-type

(20)

HO

R2O

HO

OR3(6)

(3)

Ppt-type


(6)


(6)


R1O

R2O

HO(20)

(3)

(20)


Ppd-type

(20)

HO

R2O

HO

OR3(6)

(3)

Ppt-type


(6)


R1O

R2O

HO(20)

(3)

(20)


Ppd-type

(20)

HO

R2O

HO

OR3(6)

(3)

Ppt-type


(6)


(6)


R1O

R2O

HO(20)

(3)

(20)


Ppd-type

(20)

HO

R2O

HO

OR3(6)

(3)

Ppt-type


(6)


R1O

R2O

HO(20)

(3)

(20)


Ppd-type

(20)

HO

R2O

HO

OR3(6)

(3)

Ppt-type


(6)


(6)


R1O

R2O

HO(20)

(3)

(20)


Ppd-type

(20)

HO

R2O

HO

OR3(6)

(3)

Ppt-type


(6)


R1O

R2O

HO(20)

(3)

(20)


Ppd-type

(20)

HO

R2O

HO

OR3(6)

(3)

Ppt-type


(6)


(6)


GRa3

GRcGRb1

GRd

GF2GRg3

GRg2GRg1

NGR120gRfGRe

GRh1GF1GRh2

PptPpd

GRf

C-K

0

500

1000

1500

0 5 10 15 20

t1/2 (h)

AU

C (

µM

·h)

GRd

GRa3 GRb1

GRc


0

500

1000

1500

0 5 10 15 20

t1/2 (h)

AU

C (

µM

·h)

GRd

GRa3 GRb1

GRc


GRa3

GRcGRb1

GRd

GF2GRg3

GRg2GRg1

NGR120gRfGRe

GRh1GF1GRh2

PptPpd

GRf

C-K

0

500

1000

1500

0 5 10 15 20

t1/2 (h)

AU

C (

µM

·h)

GRd

GRa3 GRb1

GRc


0

500

1000

1500

0 5 10 15 20

t1/2 (h)

AU

C (

µM

·h)

GRd

GRa3 GRb1

GRc



at ASPE



Dow

nloaded from


Figure 4

Compd.GRa3 Glc2-Glc

R1

GRb1 Glc2-GlcGlc2-GlcGRc

GRd Glc2-Glc

GRg3 Glc2-GlcGF2 GlcCompd. R1

Ppd HC-K H

R2Glc6-Glc3-XylGlc6-GlcGlc6-AraGlcGlc

HGlc

GlcHR2

HGRh2

R1O

OHR2O

(3) (20)

Compd. R1

Compd. R1

HO

OHR2O

OR1

(20)

(6)

NGR1

20gRfGRe

Glc2-GlcGlc2-RhaGlc2-Xyl

GRg1 GlcGlc2-GlcGRf

GRg2 Glc2-Rha GRh1 GlcGF1 H

R2HGlc H

R2GlcGlcGlcGlcHHHPpt

Ppd-type Ppt-type

HO

Glc-O

HO

O-Glc2-Xyl

HO

Glc-O

HO

O-Glc

GRg1[D] NGR1

NGR1

HO

Glc-O

HO

O-Glc2-Rha

GRe

[D]

− Xyl

(6)− Rha

(6)

[P][U][B][F]

GRe [D]

[P][U][B][F]

GRh1GF1

− Glc

(6)

− Glc

(20)− Glc

(6)

− Glc

(20)

[U][B][F]

GRg1

[P][U][B][F]

20gRf [D] GRf [D] GRh1[D]

20gRf[P][U][B][F]

GRf

− Glc

(6)

− Glc

(6)

[B][F]

− Glc

(20)

[U][B][F]

GRcGlc-2Glc-O

Xyl-3Glc-6Glc-O

HO

GRa3

Glc-2Glc-O

Glc-6Glc-O

HO

GRb1

GF2

HO

Glc-O

HO

C-K GRh2

[P]

− Glc

(3)− Glc

(20)

− Glc

(20)− Glc

(3)

− Glc

(3)

− Glc

(3)

GRc [D]

[P][U][B]

[P][U][B][F]

GRg3[P][F]

[F]

GRb1[D] GRd [D]GRa3[D]

[P][U][B][F]

[P][U][B][F]

-Sanqi extract-

− Xyl

(20)

GF2 [D]

Glc-2Glc-O

Glc-O

HO

GRd

[P][U][B][F]

− Glc

(20)− Ara

(20)

GF1[D] GRg2 [D]

GRg2[U][B][F]

GRg3 [D]

− Rha

(6)

− Glc

(20)

[F]Ppt[F]Ppd

Plasma: AUC

Urine: Cum.Ae

Bile: Cum.Ae

Feces: Cum.Ae

Individual dose from Sanqi extract:

50 µmol/kg

5 µM·h

50 nmol/kg

50 nmol/kg

5 µmol/kg

[P][U][B][F]

[D]

Intestinal absorption

Deglycosylation

− Glc

(20)

HO

Glc-O

HO

O-Glc2-Xyl

HO

Glc-O

HO

O-Glc

GRg1[D] NGR1

NGR1

HO

Glc-O

HO

O-Glc2-Rha

GRe

HO

Glc-O

HO

O-Glc2-Rha

GRe

[D]

− Xyl

(6)− Rha

(6)

[P][U][B][F]

[P][U][B][F]

GRe [D]

[P][U][B][F]

[P][U][B][F]

GRh1GF1

− Glc

(6)

− Glc

(20)− Glc

(6)

− Glc

(20)

[U][B][F]

[U][B][F]

GRg1

[P][U][B][F]

20gRf [D] GRf [D] GRh1[D]

20gRf[P][U][B][F]

GRf

− Glc

(6)

− Glc

(6)

[B][F]

− Glc

(20)

[U][B][F]

[U][B][F]

GRcGlc-2Glc-O

Xyl-3Glc-6Glc-O

HO

GRa3

Glc-2Glc-O

Xyl-3Glc-6Glc-O

HO

GRa3

Glc-2Glc-O

Glc-6Glc-O

HO

GRb1

Glc-2Glc-O

Glc-6Glc-O

HO

GRb1

GF2

HO

Glc-O

HO

C-K GRh2

[P]

− Glc

(3)− Glc

(20)

− Glc

(20)− Glc

(3)

− Glc

(3)

− Glc

(3)

GRc [D]

[P][U][B]

[P][U][B]

[P][U][B][F]

[P][U][B][F]

GRg3[P][F]

GRg3[P][F][P][F]

[F][F]

GRb1[D] GRd [D]GRa3[D]

[P][U][B][F]

[P][U][B][F]

[P][U][B][F]

[P][U][B][F]

-Sanqi extract-

− Xyl

(20)

GF2 [D]

Glc-2Glc-O

Glc-O

HO

GRd

Glc-2Glc-O

Glc-O

HO

GRd

[P][U][B][F]

[P][U][B][F]

− Glc

(20)− Ara

(20)

GF1[D] GRg2 [D]

GRg2[U][B][F]

GRg2[U][B][F]

GRg3 [D]

− Rha

(6)

− Glc

(20)

[F][F]Ppt[F][F]Ppd

Plasma: AUC

Urine: Cum.Ae

Bile: Cum.Ae

Feces: Cum.Ae

Individual dose from Sanqi extract:

50 µmol/kg

5 µM·h

50 nmol/kg

50 nmol/kg

5 µmol/kg

[P][U][B][F]

[D]

Intestinal absorption

Deglycosylation

− Glc

(20)

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

DM

D Fast Forw

ard. Published on September 28, 2009 as D

OI: 10.1124/dm

d.109.029819 at ASPET Journals on February 20, 2020 dmd.aspetjournals.org Downloaded from


Figure 5

% o

f i.v

. dos

e ex

cret

ed

Time (h)

10

20

30

40

50

0

0.5

1.0

1.5

2.0

0

20

40

60

80

100

0

0 24 48 72 96

GRb1

Metabolites of GRb1

0 24

GRg1

0.1

0.2

0.3

0.4

0.5

00 24

Metabolites of GRg1

GRb1

GRg1

Biliary excretion

Urinary excretion

Ox-GRg1

Ox-GRb1

GRd Ox-GRd

% o

f i.v

. dos

e ex

cret

ed

Time (h)

10

20

30

40

50

0

0.5

1.0

1.5

2.0

0

20

40

60

80

100

0

10

20

30

40

50

0

0.5

1.0

1.5

2.0

0

20

40

60

80

100

0

0 24 48 72 96

GRb1

Metabolites of GRb1

0 24

GRg1

0 24

GRg1

0.1

0.2

0.3

0.4

0.5

0

0.1

0.2

0.3

0.4

0.5

00 24

Metabolites of GRg1

GRb1

GRg1

Biliary excretion

Urinary excretion

Ox-GRg1

Ox-GRb1

GRd Ox-GRd


at ASPE



Dow

nloaded from


Adipose

Testicle

Bladder

Kidney

Large intestine

Small intestine

Stomach

Spleen

Liver

Lung

Heart

Brain

PlasmaTis

sue

vers

us p

lasm

aAUC (µM·h)

0 200 400 600

Large intestine

Small intestine

Bladder

Kidney

Stomach

Spleen

Liver

Lung

Heart

Brain

Plasma

Adipose

Testicle

0 10 20 30

Bla

Adi

Tes

LIn

SIn

Sto

Spl

Liv

Lun

Hea

Bra

Pla

Bla

Adi

Tes

LIn

SIn

Sto

Spl

Liv

Lun

Hea

Bra

Pla

0 0.5 1 1.5

0 200 400 600 0 200 400 600

Bla

Adi

Tes

LIn

Sto

Lun

Bra

Pla

0 10 20 30

400 20

t1/2 (h)

400 20400 20

GRb1

GRg1

C-K

Adipose

Testicle

Bladder

Kidney

Large intestine

Small intestine

Stomach

Spleen

Liver

Lung

Heart

Brain

Plasma

Adipose

Testicle

Bladder

Kidney

Large intestine

Small intestine

Stomach

Spleen

Liver

Lung

Heart

Brain

PlasmaTis

sue

vers

us p

lasm

aAUC (µM·h)

0 200 400 6000 200 400 600

Large intestine

Small intestine

Bladder

Kidney

Stomach

Spleen

Liver

Lung

Heart

Brain

Plasma

Adipose

Testicle

Large intestine

Small intestine

Bladder

Kidney

Stomach

Spleen

Liver

Lung

Heart

Brain

Plasma

Adipose

Testicle

0 10 20 30

Bla

Adi

Tes

LIn

SIn

Sto

Spl

Liv

Lun

Hea

Bra

Pla

Bla

Adi

Tes

LIn

SIn

Sto

Spl

Liv

Lun

Hea

Bra

Pla

0 0.5 1 1.5

Bla

Adi

Tes

LIn

SIn

Sto

Spl

Liv

Lun

Hea

Bra

Pla

0 0.5 1 1.5

0 200 400 6000 200 400 600 0 200 400 6000 200 400 600

Bla

Adi

Tes

LIn

Sto

Lun

Bra

Pla

0 10 20 30

400 20 400 20

t1/2 (h)

400 20 400 20400 20 400 20

GRb1

GRg1

C-K

Figure 6


at ASPE



Dow

nloaded from


Documents

Absorption and disposition of ginsenosides after oral …dmd.aspetjournals.org/content/dmd/early/2009/09/28/dmd... · biliary excretion, inefficient metabolism, and slow renal excretion