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Transport and bioavailability studies of astragaloside iv, an active ingredient in radix astragali

C Basic & Clinical Pharmacology & Toxicology 2004, 95, 295–298.
Printed in Denmark . All rights reserved
Copyright C ISSN 1742-7835 Transport and Bioavailability Studies of Astragaloside IV,
an Active Ingredient in Radix Astragali
Yongchuan Gu1, Guangji Wang1, Guoyu Pan1, J. Paul Fawcett2, Jiye A1 and Jianguo Sun1
1Center of Pharmacokinetics, China Pharmaceutical University, Nanjing, China and 2School of Pharmacy, University of Otago, Dunedin, New Zealand (Received April 13, 2004; Accepted August 23, 2004) Abstract: Astragaloside IV is an important constituent of Radix Astragali, a herbal remedy widely used in traditionalChinese medicine. Radix Astragali is administered orally but little is known about the transport properties and bioavail-ability of astragaloside IV. In this paper we report studies of the absorption of astragaloside IV in the perfused ratintestinal model, transport and uptake in Caco-2 cell monolayers and in vivo bioavailability in rat after an oral dose. Inthe perfused rat intestinal model, absorption of astragaloside IV was low from an aqueous solution but was significantlyhigher from a solution of Radix Astragali. Absorption was not affected by bile duct ligation. Transport through Caco-2cells gave a very low permeability value (mean Papp of 6.7∫1.0¿10ª8 cm/sec.) and uptake was unaffected by P-glycoproteininhibitors. The absolute bioavailability of astragaloside IV in rat was 2.2%. The correlation between low permeability invitro and poor bioavailability in vivo indicates in vitro absorption studies are useful in the evaluation of traditional Chinesemedicines.
Radix Astragali had been widely used as an effective herbal China) and housed six to a plastic walled cage with unlimited access remedy in traditional Chinese medicine for its antiinflam- to food and water except for 12 hr before and during an experiment.
The protocols for animal experiments were reviewed and approved matory, antimicrobial and immune stimulating activities by the China Pharmaceutical University Animal Ethics Committee.
(Rios & Waterman 1997; Pharmacopoeia of P.R. China2000). Astragaloside IV (fig. 1), a cycloartane-type triter- Cell culture materials. Dulbecco's modified Eagle's medium and pene glycoside, is regarded as a characteristic and active non-essential amino acids were obtained from GIBCO Invitrogen constituent of Radix Astragali and the content of astragalo- Co. (CA, USA). Foetal calf serum, L-glutamine and penicillin/strep- side IV is the main criterion of its quality assurance and tomycin (10,000 U/ml, 10,000 mg/ml) were obtained from TBD Co.
(Tianjin, China). Caco-2 cells were obtained from ATCC (Rock- control. Although astragaloside IV has relatively high lipo- ville, MD, USA) and used between passages 40–65. All other cell philicity (ClogP of 2.0), the high molecular weight (785) and culture materials were obtained from Costar (Cambridge, MA, low solubility predict it would be poorly absorbed and have low bioavailability. We were interested to confirm this andestablish whether in vitro methods of drug absorption Absorption in the in situ perfused rat intestinal model. Experiments would correctly predict bioavailability in vivo. Accordingly, were carried out as described previously (Ikumi et al. 1997; Stewartet al. 1997). Rats were divided into 4 groups (nΩ6), two of which we examined transport properties of astragaloside IV in the were subjected to bile duct ligation before an experiment. After an in situ perfused rat intestinal model, uptake and transport overnight fast, they were anaesthetized with urethane (1 g/kg by in monolayers of the human colon carcinoma cell line Caco- intraperitoneal injection) and affixed supine on a surface under suit- 2 and absolute bioavailability in rat.
able lighting to maintain body temperature. The small intestine wasthen exposed by a midline abdominal incision, and two polyethylenecannulae (outer diameter, 5 mm; inner diameter, 3 mm) were in- Materials and Methods
serted through small slits at the proximal and distal ends (about 90cm apart) and secured by ligation with silk suture. The intestine Compounds and animals. Astragaloside IV, cyclosporin A and vera- was returned to the abdominal cavity to aid in maintaining its integ- pamil were purchased from the National Institute for the Control rity. Using a syringe, saline at 37 æ was slowly flushed through the of Pharmaceutical and Biological Products (Beijing, China). Radix gut until the effluent solution was clear after which the remaining Astragali Oral Solution (containing 0.5 mg/ml astragaloside IV) was solution was carefully expelled by means of air pumped from the obtained from the Jiangsu Provincial Hospital (Nanjing, China).
syringe. Then a solution (50 ml) containing astragaloside IV (50 mg/ All other reagents were analytical grade. Unless otherwise indicated, ml) or Radix Astragali Oral Solution (5 ml) in Krebs-Ringer solu- astragaloside IV was dissolved in dimethyl sulfoxide (DMSO) and tion was circulated at 2.0 ml/min using a peristaltic pump from a diluted to the desired concentration with water (final concentration reservoir maintained at 37 æ with bubbling O2/CO2 (95/5). At half of DMSO⬍0.1%). Sprague-Dawley rats (male, 190–210 g) were pur- hourly intervals up to 6 hr, aliquots of perfusion solution (1 ml) chased from Sino-British Sippr/BK Lab Animal Ltd (Shanghai, were removed from the reservoir and replaced with 5 ml Krebs-Ringer solution. Solutions were filtered (0.22 mm) and analysed forastragaloside IV. The concentrations were corrected for the change Author for correspondence: J. Paul Fawcett, School of Pharmacy, in volume of circulating fluid on the basis of the change in concen- University of Otago, P.O. Box 913, Dunedin, New Zealand (fax tration of phenolsulfophthalein in the perfusate (initial concen- 64 3 4797034, e-mail njguyc/
tration 100 mM) as determined by UV spectroscopy. After an ex- YONGCHUAN GU ET AL.
Absorption parameters of astragaloside IV in the perfused rat intestinal model with and without bile duct ligation (data are mean∫S.D. for6 rats).
Astragaloside IV aqueous solution Radix Astragali Oral Solution Absorption rate* (mg/g/hr) * Balanced ANOVA shows this is significantly lower from aqueous solution than fromRadix Astragali Oral Solution (PΩ0.02); no interaction between solution and bile duct ligation.
periment, the part of the small intestine under study was removed, 1.5, 2, 3, 4, 6, 8 and 12 hr after intravenous injection and 0.25, 0.5, dried at 40 æ and weighed. Absorption rate constant (ka), percent 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12 and 24 hr after oral administration.
astragaloside IV absorbed in 6 hr (F) and absorption rate (mg/gintestine/hr) were calculated from the linear log concentration ver- Assay of astragaloside IV. Astragaloside IV was analysed as pre- sus time plots and corrected for differences in dry weight of the viously described (Gu et al. 2004). Plasma samples were subjected to solid phase extraction before analysis. HPLC was carried outon a reversed phase C18 column with mass spectrometric detection Uptake and transport in Caco-2 cell monolayers. Uptake of astra- (selective ion monitoring at m/z 807.5).
galoside IV by cultured monolayers of Caco-2 cells was examinedas previously described (Pan et al. 2002). Experiments were per- Data analysis. All data are given as mean∫standard deviation formed with confluent epithelial cell monolayers grown on 6-well (S.D.). The effects of solution and ligation in the in situ perfused plates. The culture medium was renewed and 3 hr later cells were rat intestinal model were analyzed using balanced ANOVA with washed three times with 1 ml aliquots of HBSS at 37 æ and then solution, ligation and their interaction included in the model. Dif- preincubated in HBSS for 20 min. Uptake was initiated by adding ferences were considered to be statistically significant when P⬍0.05.
2 ml HBSS containing astragaloside IV (50 mg/ml) alone or with P- Pharmacokinetic parameters (maximum plasma concentration glycoprotein inhibitors (cyclosporin A 5 mM, verapamil 200 mM) at (Cmax) and the corresponding time (tmax), absorption rate constant 37 æ. After various times (up to 40 min.), solutions were removed (ka), elimination half-life (t1/2), clearance (CL), volume of distri- and the monolayers washed three times with 1 ml aliquots of ice- bution (Vd), area under the curve (AUC0»¬) and absolute bioavail- cold HBSS and scraped into 1 ml HBSS solution. Cells were rup- ability) were obtained from concentration-time curves by non-linear tured by three freeze-thaw cycles and, after centrifugation, astra- least-squares regression using the program PKAnalystA for Win- galoside IV was assayed in the supernatant and expressed per milli- dows (MicroMath, USA).
gram of protein. Protein concentration was determined by the Coo-massie Brilliant Blue method.
Transport experiments were performed with confluent epithelial cell monolayers grown on Collagen coated Transwells as previously described (Bartrakova et al. 1998). Before an experiment, conflu- Absorption in the in situ perfused rat intestinal model. ence was established by ensuring transepithelial electrical resistancewas ⬎180 W cm2. Culture medium was renewed and 3 hr later cells The results of this study are shown in table 1. The absorp- were washed three times with 1 ml aliquots of Hank's balanced salt tion rate of astragaloside IV from aqueous solutions was solution (HBSS) at 37 æ. Transport was initiated by adding 1.5 ml significantly lower than from Radix Astragali Oral Solution HBSS containing different concentrations of astragaloside IV (10, (PΩ0.02). Absorption rate was also lower in ligated rats 20, 30 mg/ml) to the apical chamber of the Transwell and 2.6 mldrug-free HBSS to the basolateral chamber. Aliquots (7¿100 ml) than in non-ligated rats but the effect was not significant were removed from the basolateral chamber between 15 and 120 (PΩ0.053). There was no interaction between solution and min. and replaced with drug-free HBSS. Solutions were centrifuged bile duct ligation.
and analysed for astragaloside IV. Apparent permeability coef-ficients (Papp, cm/s) were calculated using the equation PappΩ(dQ/ Uptake and transport in Caco-2 cell monolayers. dt)C0/A where C0 is the initial concentration on the apical side andA is the surface area of the Transwell (Hilgers et al. 1990).
Uptake of astragaloside IV was time-dependent and ap-peared to be approaching steady state at 40 min. (fig. 2A).
Pharmacokinetics in rat. Groups of rats (nΩ6) were administered Using astragaloside IV uptake in 40 min., increasing initial either an intravenous (2.5 mg/kg via the tail vein) or oral dose (20.0 concentration of astragaloside IV (1–100 mg/ml) was ac- mg/kg) of astragaloside IV (1 mg/ml) dissolved in ethanol and di-luted with normal saline. Blood samples were obtained for analysis companied by an increase in uptake approaching saturation of astragaloside IV from the femoral vein at 0.03, 0.17, 0.3, 0.5, 1, at 50 mg/ml (fig. 2B). The effect of P-glycoprotein inhibitors Pharmacokinetic parameters of astragaloside IV after single intravenous (2.5 mg/kg) and oral (20.0 mg/kg) doses of astragaloside IV in rat.
AUC0»¬ (mg ¡ hr/ml)

ABSORPTION OF ASTRAGALOSIDE IV (cyclosporin A and verapamil) on the uptake of astragalo-side IV was not significant. Apical to basolateral transportof astragaloside IV was linear between 15 and 120 min. (fig.
3) and Papp values at concentrations of 10, 20 and 30 mg/mlwere 7.82, 6.19 and 5.95¿10ª8 cm/sec. respectively.
Pharmacokinetics in rat. The concentration-time profiles for plasma astragaloside IVfollowing administration of 2.5 mg/kg intravenous and 20.0mg/kg oral doses are shown in fig. 4. Pharmacokinetic par-ameters are given in table 2. The disposition of astragalo-side IV was adequately described by a three-compartmentmodel for the intravenous dose and a two-compartmentmodel for the oral dose. The absolute bioavailability of as-tragaloside IV in rat was found to be 2.2%.
The absorption of astragaloside IV in the in vitro models oftransport studied here was found to be low. This is probablydue to its large molecular weight and poor solubility bothin water (⬍1 mg/ml) and in lipid. The trend of decreasingPapp for transport across Caco-2 cell monolayers with con-centration suggests astragaloside IV may inhibit its owntransport and that the transport is predominantly via theparacellular route (Lee & Thakker 1999). Uptake by Caco-2cells in the presence of cyclosporin A or verapamil indicatesastragaloside IV is not a substrate for P-glycoprotein andthat its poor absorption is not due to the action of thisefflux protein.
Fig. 2 A. Effect of time on uptake of astragaloside IV (initial con- The low bioavailability of astragaloside IV in rat is con- centration 50 mg/ml) by Caco-2 cells alone (S) and in the presence sistent with the extremely low permeability constant for of 5 mM cyclosporin A (O) or 200 mM verapamil (g). B. Effect ofinitial concentration of astragaloside IV on its uptake over 40 min.
alone (S) and in the presence of 5 mM cyclosporin A (O) (Data aremean∫S.D. for 3 determinations).
transport through Caco-2 cells (Artursson & Karlsson1991). Although there is no simple relationship between Fig. 3. Transport of astragaloside IV through Caco-2 cell mono-layers at three concentrations of 30 mg/ml (g), 20 mg/ml (H) and 10 Fig. 1. Chemical structure of astragaloside IV.
mg/ml (O) (Data are means of 3 determinations).

The authors wish to thank Miss Jue Song, Ms Haitang Xie and Ms Zhengzhong Cao. We are also grateful to Jiang-su Pharmaceutical Research Institute for financial assist-ance.
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