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Phytomedicine 21 (2014) 1549–1558

Contents lists available at ScienceDirect

Phytomedicine

jou rn al homepage: www.elsev ier .de /phymed

ffect of chito-oligosaccharide on the intestinal absorptions ofhenylethanoid glycosides in Fructus Forsythiae extract

ei Zhoua,b,c, Xiaobin Tand, Jinjun Shane, Ting Liua,b,c, Baochang Caia, Liuqing Dia,b,c,∗

College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210046, PR ChinaJiangsu Engineering Research Center for Efficient Delivery System of TCM, PR ChinaNanjing Engineering Research Center for Industrialization of Chinese Medicine Pellets, PR ChinaKey Laboratory of New Drug Delivery System of Chinese Meteria Medica, Jiangsu Provinical Academy of Chinese Medicine, PR ChinaJiangsu Key Laboratory of Pediatric Respiratory Disease, Institute of Paediatrics, Nanjing University of Chinese Medicine, Nanjing 210046, PR China

r t i c l e i n f o

rticle history:eceived 6 February 2014eceived in revised form 12 May 2014ccepted 27 June 2014

eywords:ructus Forsythiae extracthenylethanoid glycosideshito-oligosaccharideight junctionbsorption mechanismntiviral activity

a b s t r a c t

Phenylethanoid glycosides, the main active ingredients in Fructus Forsythiae extract possesses strongantibacterial, antioxidant and antiviral effects, and their contents were higher largely than that of otheringredients such as lignans and flavones, but their absolute bioavailability orally was significantly low,which influenced clinical efficacies of its oral preparations seriously. In the present study, the absorp-tion mechanism of phenylethanoid glycosides was studied using in vitro Caco-2 cell model. And theeffect of chito-oligosaccharide (COS) on the intestinal absorption of phenylethanoid glycosides in FructusForsythiae extract was investigated using in vitro, in situ and in vivo models. The pharmacological effectssuch as antiviral activity improvement by COS were verified by MDCK cell damage inhibition rate afterinfluenza virus propagation. The observations from in vitro Caco-2 cell showed that the absorption ofphenylethanoid glycosides in Fructus Forsythiae extract so with that in monomers was mainly restrictedby the tight junctions, and influenced by efflux transporters (P-gp and MRP2). Meanwhile, the absorptionof phenylethanoid glycosides in Fructus Forsythiae extract could be improved by COS. Besides, COS at thesame low, medium and high concentrations caused a significant, concentration-dependent increase inthe Papp-value for phenylethanoid glycosides compared to the control group (p < 0.05), and was all safefor the Caco-2 cells. The observations from single-pass intestinal perfusion in situ model showed that theintestinal absorption of phenylethanoid glycosides can be enhanced by COS. Meanwhile, the absorptionenhancing effect of phenylethanoid glycosides might be saturable in different intestine sites. In pharma-cokinetics study, COS at dosage of 25 mg/kg improved the bioavailability of phenylethanoid glycosidesin Fructus Forsythiae extract to the greatest extent, and was safe for gastrointestine from morphological

observation. In addition, treatment with Fructus Forsythiae extract with COS at dosage of 25 mg/kg pre-vented MDCK cell damage upon influenza virus propagation better than that of control. All findings abovesuggested that COS at dosage of 25 mg/kg might be safe and effective absorption enhancer for improvingthe bioavailability of phenylethanoid glycosides and the antiviral activity in vitro in Fructus Forsythiaeextract.

ntroduction

Forsythiae fructus, a fruit of Forsythia suspensa (Thunb) Vahlhat possesses antibacterial (Yang et al. 2003), antiviral (Liu et al.

004), antioxidant (Sato et al. 1991), anti-inflammatory (Bing et al.999), anti-obesity (Yoshida et al. 1995) and antiemetic (Liu et al.004) properties, has been widely used in traditional Chinese

∗ Corresponding author at: College of Pharmacy, Nanjing University of Chineseedicine, Nanjing 210046, PR China. Tel.: +86 25 86798226; fax: +86 25 83271038.

E-mail address: diliuqing@hotmail.com (L. Di).

ttp://dx.doi.org/10.1016/j.phymed.2014.06.016944-7113/© 2014 Elsevier GmbH. All rights reserved.

© 2014 Elsevier GmbH. All rights reserved.

medicine to treat gonorrhea, erysipelas, inflammation, pyrexia andulcer. And the cAMP and cGMP phospho-diesterases’s activities (Liuet al. 2004), and the subacute and chronic hepatic injuries inducedby carbon tetrachloride (CCl4) or alpha-naphthylisothiocyanate(ANIT) (Ishizuka et al. 1992) were also inhibited by Forsythiae fruc-tus.

Forsythiae fructus extract, extracted from Forsythiae fructus, hasbeen used in Chinese medicine preparations frequently (Zhou

et al. 2013), such as Shuang-Huang-Lian freeze-dried powders ofinjection, Shuang-Huang-Lian oral liquid, Yin-Qiao capsule, FufangQinlan oral liquid, Yin-Qiao-Jie-Du tablets, etc., which are exten-sively used for treating acute upper respiratory tract infection

1550 W. Zhou et al. / Phytomedicine 21 (2014) 1549–1558

Table 1The preliminary study on MS and MS/MS data of the identified components in rat plasma after oral administration of Fructus Forsythiae extracts in positive model.

Peak no. Components FructusForsythiaeextract

Rat plasma after oraladministration of FructusForsythiae extract

ESI+, m/z Ret. time Source

MS, MSn

1 Quinic acid + + 193.07066 [M+H]+ 0.96 Phenolic acids2 Forsythoside D + + 499.14124 [M+Na]+, 1.84 Phenylethanoid

glycosides3 Caffeic acid + + 181.0919 [M+H]+ 2.26 Phenolic acids4 Forsythoside E + + 485.16251 [M+Na]+, 3.96 Phenylethanoid

glycosides5 Hyperoside + + 465.10275 [M+H]+,

257.06033, 164.96709,229.02371, 285.05786

14.79 Flavones

6 Isoforsythoside + + 647.19397 [M+Na]+,347.05630, 321.12628

14.79 Phenylethanoidglycosides

7 Rutin + + 611.15985 [M+H]+,257.00757, 229.02271,285.04669, 164.82939

14.92 Flavones

8 Isoquercitin + + 465.10275 [M+H]+,257.06033, 164.96709,229.02371, 285.05786

15.13 Flavones

9 Forsythoside B + + 779.23651 [M+Na]+ 17.08 Phenylethanoidglycosides

10 Forsythoside A + + 647.19397 [M+Na]+,347.05630, 321.12628

17.24 Phenylethanoidglycosides

11 Pinoresinol-�-d-glucoside

+ + 543.18262 [M+Na]+,291.07315, 219.01880,249.04117, 142.96408

17.65 Lignans

12 A1# – + 557.16254 [M+Na]+,381.13086

19.92 Metabolites

13 Epipinoresinol-�-d-glucoside

+ + 543.18256 [M+Na]+ 20.39 Lignans

14 A2# – + 557.16254 [M+Na]+,381.13086

20.69 Metabolites

15 Pinoresinolmonomethylether-�-d-glucoside

+ – 529.16742 [M+Na]+ 21.75 Lignans

16 Phillyrin + + 557.19812 [M+Na]+,309.21106, 394.94855,184.85243, 542.32208

22.58 Lignans

17 Quercetin + + 303.04953 [M+H]+, 22.85 Flavones18 Arctiin + – 557.19812 [M+Na]+,

270.7206423.98 Lignans

19 A3# – + 571.17792 [M+Na]+,395.14651

24.52 Metabolites

20 Epipinoresinol + + 359.14847 [M+H]+ 27.09 Lignans

ckuefwppfit

nFficfasgrt

21 Pinoresinol + +

22 Arctigenin + +

23 Phillygenin + +

aused by virus or bacterial infection in clinical practice. And it isnown that the oral preparations of Forsythiae fructus extract aresually more accepted for patients than injections owing to theirlimination of pain and discomfort, and lower costs to produce oralormulations, but their clinical effects are unsatisfactory comparedith that of injections, which becomes one of the most limitedoints in the development of Chinese medicine preparations. Theresumptions that whether the low bioavailability of Forsythiae

ructus extract resulted in the poor efficacy or efficacy could bemproved as the absorption of active ingredients in Forsythiae fruc-us extract was enhanced, are all yet to be investigated.

As shown in Fig. 3 and Table 1, phenylethanoid glycosides, lig-ans, phenolic acids, flavones, not saponins were found in theorsythiae fructus extract. According to the HPLC figureprint pro-le (Fig. 2A) and UPLC–MS/MS chromatography (Fig. 2B), theontents of phenylethanoid glycosides such as forsythoside A, iso-orsythoside and forsythoside B (Fig. 1) were about 8.036, 1.356nd 0.8270 mg/ml, respectively, higher largely than that of lignans,

uch as pinoresinol-�-d-glucoside, arctiin, phillyrin and arcti-enin (Fig. 1) containing 1.959, 0.08130, 1.380 and 0.01773 mg/ml,espectively, and flavones, such as rutin and quercetin (Fig. 1) con-aining 0.8581 and 0.07905 mg/ml, respectively, but their absolute

359.14874[M+H]+ 27.09 Lignans373.16403 [M+H]+ 27.87 Lignans373.16403 [M+H]+ 28.04 Lignans

bioavailability orally was significantly low (unpublished). Thus, theproperties above of main ingredients in Forsythiae fructus extractled us to postulate that low bioavailability of phenylethanoid gly-cosides in Forsythiae fructus extract might result in the low efficacyof clinical therapy.

It was reported that phenylethanoid glycosides had strongantibacterial, antiviral and antioxidant activities in vitro and invivo (Zhang 2002; Li 2011; Wang et al. 2005; Wu et al. 2011),and phenylethanoid glycosides instead of lignans, phenolic acidsand flavones in Forsythiae fructus extract had good positive cor-relation between dose and effects of antibacterial, antioxidant andantiviral activities via pharmacokinetic/pharmacodynamic (PK/PD)model combined with partial least-squares (PLS) method, andthe pharmacological activities were improved as the contentsof phenylethanoid glycosides in Forsythiae fructus extract wereenhanced, and showing obvious correlation between dose ofphenylethanoid glycosides and pharmacological effects. Therefore,how to improve the bioavailability of phenylethanoid glycoside

would be related to the pharmacological efficacy improvement ofForsythiae fructus extract directly.

However, few studies have been carried out whether the lowpermeability of phenylethanoid glycoside in Forsythiae fructus

W. Zhou et al. / Phytomedicine 21 (2014) 1549–1558 1551

Fig. 1. Structural formulae of analyte standards.

Fig. 2. (A): 3D HPLC-profile of MeOH (50%) soluble portion of Fructus Forsythiae extract. The MeOH soluble portion was filtered through a 0.45 �m membrane filter and theresulting filtrate were subjected for HPLC analysis. The analyses were performed using a Waters 2695 Alliance HPLC system (Waters Corp., Milford, MA, USA), consisting of aquaternary pump solvent management system, an on-line degasser, and an autosampler. The raw data were detected by 2998 PDA, acquired, and processed with EmpowerTM

software. A Hypersil ODS C18 column (250 × 4.6 mm, 5 �m) (Thermo Scientific, Waltham, MA, USA) was applied for all analyses. The mobile phase was composed of A(methanol) and solvent B (0.2% aqueous phosphoric acid, v/v) with a linear gradient elution: 0–5 min, 15–20% A; 5–30 min, 20–25% A; 30–50 min, 25–30% A; 50–75 min,30–55% A; 75–80 min, 55–15%; 80–85 min, 15% A. The mobile phase flow rate was 1 ml min−1, the column temperature was controlled at 30 ◦C and sample injection volumewas 10 �l (tR isoforsythoside = 34.445 min, tR forsythoside A = 55.690 min, tR forsythoside B = 52.029 min, tR pinoresinol-glucoside = 46.788 min, tR phillyrin = 66.609 min). (B):UPLC–ESI-MS/MS profile of determination most of the ingredients in Fructus Forsythiae extract simultaneously.

1552 W. Zhou et al. / Phytomedicine 21 (2014) 1549–1558

Fig. 3. The preparation methods including solid-phase extraction (SPE), methanol precipitation, acetonitrile precipitation, liquid–liquid extraction with ethyl acetate andliquid–liquid extraction with the mixture of ethyl acetate and methanol were all tried. As a result, the acetonitrile precipitation was selected considering the numbero atrix

t extraB

etm

bidcb

tacmW

f chromatography peaks and the interference from the co-eluted endogenous mrolled rat plasma, (C) rat plasma after oral administration of Fructus Forsythiae

= 17.06 min).

xtract in the intestinal mucosa led to the poor bioavailability orhe bioavailability could be improved by suitable pharmaceutical

ethods.It was reported that absorption enhancers including surfactants,

ile salts and chelating agents etc. was one of the most promis-ng methods to improve the bioavailability of poorly absorbablerugs orally (Uchiyama et al. 1999), but some absorption enhancersan cause damage and irritate the intestinal mucosal mem-ranes.

Chito-oligosaccharide (COS) shown in Fig. 4, a new type of chi-osan molecular, has been suggested as promising excipients for

bsorption enhancement based on tight junctions of GI drug inases in which additional physicochemical properties in the poly-er structure were desirable (Gao et al. 2008; Zhou et al. 2014).e found that the contents of d-glucosamine, chitosan dimer,

Fig. 4. Chemical stru

for samples from the hepatic portal vein. (A) Fructus Forsythiae extract, (B) con-ct); (tR isoforsythoside = 14.74 min, tR forsythoside A = 17.17 min, tR forsythoside

chitosan trimer, chitosan tetramer, chitosan pentamer and chitosanhexamer in COS were 0.42%, 9.19%, 18.78%, 0.69%, 14.48% and 2.56%of totally, respectively (Zhou et al. 2014).

Therefore, the current study aims to demonstrate a sys-temic biopharmaceutics and pharmacokinetics characterization ofphenylethanoid glycosides in Forsythiae fructus extract. The specificobjectives of the current study include: (1) to study the absorp-tion mechanism of phenylethanoid glycosides, and find the mainfactors influencing the low bioavailability of phenylethanoid gly-cosides in Forsythiae fructus extract using in vitro Caco-2 cell. (2) Toinvestigate the effect of COS as absorption enhancer on the absorp-

tion of phenylethanoid glycosides in Forsythiae fructus extract. (3)To evaluate whether COS could be suitable absorption enhancerin Forsythiae fructus extract using comparison of pharmacologicaleffect−antiviral activity.

cture of COS.

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W. Zhou et al. / Phytom

aterials

Fructus Forsythiae extract was purchased from Jiangyin Tianjiangharmaceutics Co., Ltd. To assure the homogeneity of the formu-ation and to prepare consistent hatched, the HPLC figureprintrofile Fig. 2(A) of the Fructus Forsythiae extract was analyzed, andhe chromatographic analysis was carried out under the previoustudy (Wagner et al. 2011). A reliable UHPLC-Orbitrap MS systemas established for detecting the prototype compounds in Fruc-

us Forsythiae extract and dosed plasma, respectively (Fig. 3). Alloucher specimens were deposited in our laboratory for future ref-rence. Forsythoside A (98% pure) was purchased from Shanghaiature Standard Co., Ltd. Tinidazole (using as internal standard, IS)ere purchased from National Institute for the Control of Phar-aceutical and Biological Products. Forsythoside B (98% pure) was

urchased from Sichuan Weikeqi Bio-tech Co., Ltd. Isoforsythoside98% pure), rutin, quercetin, pinoresinol-�-d-glucoside, arctiin andhillyrin were purchased from Chengdu Herbpurify Co., Ltd. Hep-rin sodium injection was purchased from Changzhou Qianhongio-pharma Co., Ltd. Methanol and acetonitrile (HPLC grade) wereurchased from Merck (Merck, Germany), and water was puri-ed by a Milli-Q water purification system (Millipore, Bedford, MA,SA). COS (Molecular weight ≈ 800 Da) (Zhou et al. 2014; Liu et al.006) was purchased from Qingdao Honghai Bio-tech Co., Ltd. Allther chemicals and reagents were of analytical grade.

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovineerum (FBS), 0.05% trypsin–EDTA, penicillin–streptomycin andon-essential amino acids were obtained from GibcoBRI, Lifend Technologies, USA. Collagen type I, sodium pyruvate, MTT3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide)nd trypsin TPCK (tosylamide phenylethyl chloromethyl ketone-reated trypsin), cytochalasin D, EDTA, sodium caprate, verapamilVER), MK571 and fumitremorgin C (FTC) were purchased fromigma Chemical Co. (St. Louis, MO, USA). HBSS (Hank’s balancedalt solution) and PBS (phosphate buffered saline) were purchasedrom Sigma Chemical Co. (St. Louis, MO, USA). Culture cell insertsor 6 well plates (CCI, 137435) were purchased from Nalge Nuncnternational (Roskilde, Denmark).

The human colorectal cancer cell lines (Caco-2, HCT116) wereought from cell bank (Chinese Academy of Sciences). Madin-Darbyanine kidney cell lines (MDCK cell, KG067) were purchased fromeygen Biotech Co., Ltd. The influenza virus strain, A/PR8/34 (H1N1)as purchased from Chinese Academy of Preventive Medicine.

Male Sprague-Dawley (SD) rats (∼250 g) were supplied byhe Experimental Animal Center of Nanjing University of Chinese

edicine (Certificate no. SCXK2008-0033). The experimental pro-edures were in compliance with the animal ethics committee ofhe Nanjing University of Chinese Medicine.

ethods

bsorption mechanism by in vitro Caco-2 cell model

Hank’s balanced salt solution (HBSS) was used as the transportuffer for the transport study in Caco-2 cell monolayer model. It wasrepared by dissolving 9.5 g of commercial available HBSS powder

n 1000 ml water. The pH value of the buffer was adjusted to pH 6.0y 85% of phosphoric acid.

Caco-2 cells were cultured in high glucose DMEM with 10% fetalovine serum, 1% nonessential amino acids. Cells were cultured in

humidified atmosphere of 5% CO2 at 37 ◦C. After reaching 80%onfluence, Caco-2 cells were harvested with 0.05% trypsin–EDTA

olution and seeded on top of CC inserts in 6-well plates, whichas a surface area of 4.2 cm2, at a density of 1.0 × 105 cells/cm2. Therotocols for cell culture in Transwell inserts were similar to thoseescribed previously (Zhou et al. 2013).

21 (2014) 1549–1558 1553

Briefly, after culture medium was aspirated, the cell monolayerswere washed three times with blank HBSS. The transepithelial elec-trical resistance (TEER) values of cell monolayers were measured,which were more than 250 � cm2. The monolayers were incubatedwith the blank HBSS for 1 h with 37 ◦C. Thereafter the incubationmedium was aspirated. Afterwards, a solution containing the com-pound was loaded onto the apical side. Donor samples (400 �l)(apical side) and receiver samples (400 �l) (basolateral side) weretaken at different times (typically 1 h), followed by the additionof 400 �l drug donor solution to the donor side (AP) and 400 �l ofblank buffer to the receiver side (BL). The samples were taken at 0, 1,2, 3 and 4 h after incubation. At the end of the transport experiment,integrity of the monolayer was monitored by TEER value. Besides,the effects of the paracellular permeability enhancers (PPEs) (ETDAand sodium caprate) (Deli 2009; Zornoza et al. 2004) and the selec-tive inhibitor of P-gp, MRP2 and BCRP (VER, MK571 and FTC) (Bansalet al. 2009; Weiss et al. 2007; Xia et al. 2005) on the absorption ofphenylethanoid glycosides were investigated.

Effect of COS on the intestinal absorption of forsythoside A,isoforsythoside and forsythoside B in Fructus Forsythiae extract

In vitro Caco-2 cell monolayer modelCell culture experiments were described above and the

effect of COS of different concentrations on the absorption ofphenylethanoid glycosides was investigated.

Rat in situ single pass intestinal perfusion studyRat in situ single pass intestinal perfusion model was set at pre-

viously described by us (Zhou et al. 2012). Briefly, SD rats (bodyweight: 250–300 g) were fasted overnight with free access to water.The rats were anesthetized with 20% urethane solution (6 mg/kg).A midline abdominal incision was made and the small intestinewas exposed. The bile duct was ligated in order to avoid bile secre-tion into the perfusate. For the regional absorption of drugs, threeintestinal sections were isolated and cannulated (all were 10 cmlong): duodenum, jejunum and ileum. Each segment was rinsedwith normal saline at 37 ◦C for 20 min until the washing appearedclear. After that, the perfusion solution of drugs as solvent was con-nected to the each segment and perfusing through each part ofthe three intestine sections. At the beginning of 30 min, the cir-culation rate was 0.2 ml min−1 controlled by a peristaltic pumpto pre-balance, then, perfusate samples were collected. Solutionscontaining 64.3 �M for forsythoside A, 10.9 �M for isoforsythosideand 5.47 �M for forsythoside B with or without COS were perfusedthrough the intestinal lumen to investigate the effect of COS on thepermeabilities of the three studied compounds. The perfusate sam-ples were collected at 30–60, 60–90, 90–120 and 120–150 min, andstored at −80 ◦C refrigerator until analysis.

Rat in vivo pharmacokinetics studyProduct Fructus Forsythiae extracts was dissolved in saline with

or without COS to give the concentration of 80% (v/v) immedi-ately prior to drug administration. The contents of forsythoside A,isoforsythoside and forsythoside B were determined to be 8.036,1.356, 0.8270 mg/ml of extracts, respectively, respectively. Male SDrats (∼250 g) were kept in an environmentally controlled breed-ing room (temperature: 20 ± 2 ◦C, relative humidity: 60 ± 5%) for1 week. The animals were fasted for 12 h prior to drug adminis-tration of product Fructus Forsythiae extracts with or without COS

prepared with a dose of 12.5 ml/kg. After dosing for 0, 5, 10, 15,20, 30, 40, 55, 70, 100, 160, 250, 480 min, blood was collected fromthe pre-intubated catheter and put into tubes with heparin sodiuminjection (10 �l) and ascorbic acid (2 �g) at predetermined time

1554 W. Zhou et al. / Phytomedicine 21 (2014) 1549–1558

Fig. 5. (A) Bidirectional permeation of phenylethanoid glycosides across Caco-2 cell monolayers, (B) correlation between TER and the Papp-values. TER values are indicatedas follows: high, 800, and low, 300, (C) effect of paracellular permeability enhancers – EDTA on absorption parameters of phenylethanoid glycosides, (D) effect of paracellularp oid gl( ect of

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ermeability enhancers – sodium caprate on absorption parameters of phenylethanF) effect of MK571 on absorption parameters of phenylethanoid glycosides, (G) effs the mean ± S.D. of at least three experiments. (*) p < 0.05, (**) p < 0.01, (n.s.) no sig

oints. Subsequently, plasma was prepared by centrifugation at816 × g for 7 min and stored at −80 ◦C for further analysis.

ample preparations and analysisSample treatment and UPLC–MS/MS analysis for samples col-

ected from in vitro, in situ and in vivo models, respectively wereescribed previously (unpublished).

ffect of Fructus Forsythiae extracts with or without COS onnfluenza virus

MDCK cells were grown in DMEM as described previouslyMehrbod et al. 2009), supplemented with 10% FBS and 1%en/Strep at 37 ◦C in a humidified incubator. The media washanged two to three times per week. The influenza virus was prop-gated in MDCK cells in the presence of 1 �g/ml of Trypsin TPCK toreate the working stock. During antiviral evaluations, media sup-lemented with FBS was sucked out and the cell washed with PBSnd then it was treated as needed. The media supplemented withrypsin TPCK was added.

Serum after administration orally into Fructus Forsythiae

xtracts with or without COS was added to the MDCK cells afterhe propagation with influenza virus. The cells were incubated at7 ◦C for 48 h before viability testing by measuring the conversionf MTT as described previously (Zhou et al. 2013).

ycosides, (E) effect of VER on absorption parameters of phenylethanoid glycosides,FTC on absorption parameters of phenylethanoid glycosides. Results are expressednt difference, compared with the control group.

CalculationFor Caco-2 monolayer model, the apparent permeability coef-

ficient (Papp) was calculated as Papp = [(dQ/dt)]/[A × C], dQ/dt(�g/S) was the flux rate, A was the effective surface area ofthe cell monolayer (4.2 cm2), and C0 (�g/ml) was the initialdrug concentration in the donor chamber. For rat single-passintestinal perfusion in situ model, the concentration of per-fusion fluid was calculated as Cout(corrected) = CoutPRin/PRout andthe effective permeability coefficient (Peff) was calculated asPeff = Qln[Cin/Cout(corrected)]/2�rL. Cout(corrected) was effluent drugconcentration with correction; Cout was effluent drug concentra-tion without correction; Cin was influent drug concentration; PRinwas influent phenol red concentration; PRout were effluent phe-nol red concentration; Q was perfusate flow rate; r was radius ofintestinal segment and l was intestinal segment length. Inhibitionrate = [OD(drug) − OD(model)]/[OD(control) − OD(model)].

Pharmacokinetic analysisThe peak concentrations (Cmax) and the time to reach the peak

concentrations (Tmax) were determined directly from the plasmaconcentration–time profiles. The area under the curve (AUC) wascalculated by the trapezoidal method from time zero to the finalsampling. The absorption enhancement ratios of drugs with or

edicine 21 (2014) 1549–1558 1555

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Fig. 6. (A) Bidirectional permeation of phenylethanoid glycosides in Fruc-tus Forsythiae extract across Caco-2 cell monolayers, (B) the efflux ratios ofphenylethanoid glycosides in Fructus Forsythiae extract and in monomers.

W. Zhou et al. / Phytom

ithout enhancers were calculated as Absorption enhancementatio = AUCwith enhancer/AUCcontrol (without enhancer).

tatistical analysisStatistical significance in the Papp, Peff values, pharmacokinetic

arameters and inhibition rate index obtained from various treat-ent groups was estimated by the analysis of variance (Student

-test) or one-way ANOVA. A p value of less than 0.05 was con-idered to be significantly different. All data were expressed asean ± SD.

esults

ntestinal absorption mechanism of phenylethanoid glycosides inructus Forsythiae extract using in vitro Caco-2 cell model

Bidirectional permeation of phenylethanoid glycosides acrossaco-2 cell monolayers was examined (Fig. 5A). The permeationf phenylethanoid glycosides in the apical-to-basolateral direc-ion was high significantly than that in the basolateral-to-apicalirection in the presence of middle concentration, and all of thefflux ratios were less than 1.0, which indicated that the absorptionf phenylethanoid glycosides might not be affected by transportirections.

As illustrated in Fig. 5C and D, the Papp-values of phenylethanoidlycosides were enhanced as the concentration of paracellular per-eability enhancers – EDTA and sodium caprate – were increased

xhibiting a clearly concentration-dependent effect. Also in Fig. 5B,he Papp-values of phenylethanoid glycosides were inversely cor-elated with the TER, suggesting that they mainly permeatedcross Caco-2 cell monolayers via the paracellular pathways, com-ining with the results (Fig. 5A, C and D). This finding alsouggested that the intestinal absorption of phenylethanoid glyco-ides was restricted when the epithelial tight junction was tightnough.

Fig. 5(E–G) summarized the Papp values of the phenylethanoidlycosides in the presence of inhibitors. The effects of expo-ure to VER (50 �M and 100 �M) and MK571 (50 �M) did notncrease the Papp-values for forsythoside A, isoforsythoside andorsythoside B significantly, but the Papp-values (Fig. 5E) fororsythoside A, isoforsythoside and forsythoside B were increasedignificantly to 183.91% (14.6 ± 0.332) × 10−7 cm/s, 206.59%13.1 ± 0.222) × 10−7 cm/s and 188.60% (12.4 ± 0.581) × 10−7 cm/s,espectively in the presence of 150 �M of VER. Besides, theapp-values (Fig. 5F) were increased significantly to 249.45%19.8 ± 4.19) × 10−7 cm/s, 267.01% (17.7 ± 2.81) × 10−7 cm/s and59.39% (16.8 ± 7.34) × 10−7 cm/s in the presence of 150 �M ofK571 for forsythoside A, isoforsythoside and forsythoside B,

espectively. In addition, Papp-values (Fig. 5G) in the presence of 1,, 4 �M of FTC changed little in forsythoside A, isoforsythoside andorsythoside B groups. The results above indicated that the intesti-al absorption of phenylethanoid glycosides such as forsythoside, isoforsythoside and forsythoside B were influenced by efflux

ransporters such as P-gp, MRP2, not BCRP.As shown in Fig. 6A, the permeation of phenylethanoid gly-

osides in Fructus Forsythiae extract in the apical-to-basolateralirection was also high than that in the basolateral-to-apical direc-ion in the presence of middle concentration (some of them hadignificance) and the efflux ratios of phenylethanoid glycosidesuch as isoforsythoside and forsythoside B in Fructus Forsythiae

xtract was higher significantly than that of monomers (Fig. 6B),hich might be attributed to the influence of other ingredients

n Fructus Forsythiae extract on the efflux transporters (P-gp andRP2), though the effect was weak (efflux ratios < 1.0).

Fig. 7. Effect of COS on absorption parameters of phenylethanoid glycosides in Caco-2 cell in vitro model. Results are expressed as the mean ± S.D. (*) p < 0.05 and (**)p < 0.01 compared with the control group.

All data from Figs. 5 and 6 indicated that the permeability ofphenylethanoid glycosides in Fructus Forsythiae extract so with thatin monomers was mainly restricted by the tight junctions.

Effect of COS on the intestinal absorption of forsythoside A,isoforsythoside and forsythoside B in Fructus Forsythiae extractin vitro Caco-2 cell model

As shown in Fig. 7, COS at the low, medium and high concen-

trations caused a significant, concentration-dependent increase inthe Papp-value for forsythoside A, isoforsythoside and forsytho-side B compared to the control group (p < 0.05). The highestPapp-value for forsythoside A, isoforsythoside and forsythoside

1556 W. Zhou et al. / Phytomedicine 21 (2014) 1549–1558

Fig. 8. Effect of COS on absorption parameters of phenylethanoid glycosides in rat single pass intestinal perfusion via duodenum (A), jejunum (B) and ileum (C) in situ model.Results are expressed as the mean ± S.D. (*) p < 0.05 and (**) p < 0.01 compared with the control group.

F orsyths troint

B(w

Eii

aBteps

Ei

stati(att

ig. 9. Plasma concentration–time profiles of phenylethanoid glycosides in Fructus Fide and 0.82 mg/kg for forsythoside B) with COS after administration to the rat gas

was increased to 561.0% (44.62 ± 9.755) × 10−7 cm/s, 989.6%70.33 ± 7.210) × 10−7 cm/s and 1282% (86.33 ± 3.316) × 10−7 cm/sith addition of 0.1% (w/v) of COS.

ffect of COS on the intestinal absorption of forsythoside A,soforsythoside and forsythoside B in Fructus Forsythiae extractn situ single pass intestinal perfusion model

As shown in Fig. 8, COS at middle dose yielded maximumbsorption for forsythoside A, isoforsythoside and forsythoside

in duodenum, jejunum and ileum. The results indicated thathe intestinal absorption of phenylethanoid glycosides can benhanced by COS. Meanwhile, the absorption enhancing effect ofhenylethanoid glycosides might be saturable in different intestineites.

ffect of COS on the bioavailability of forsythoside A,soforsythoside and forsythoside B in vivo pharmacokinetics study

As shown in Fig. 9 and Table 2, the Cmax of forsytho-ide A, isoforsythoside and forsythoside B was increasedo 161% (267.6 ± 92.32) ng/ml, 214% (172.8 ± 39.28) ng/mlnd 185.4% (142.6 ± 51.91) ng/ml, respectively comparedo the control groups. Besides, the AUC of forsythoside A,soforsythoside and forsythoside B was increased to 290%

12,378 ± 2752.4) ng min/ml, 252% (10,251 ± 3538.5) ng min/mlnd 214% (8264.8 ± 2973.5) ng min/ml, respectively with the addi-ion of COS at the dosage of 25 mg/kg, although we found that inhe previous study that COS at the dosage of 25 mg/kg could result

iae extract (equivalent to 8.03 mg/kg for forsythoside A, 1.35 mg/kg for isoforsytho-estine by in vivo pharmacokinetics study.

in the maximum enhancing absorption for phenylethanoid gly-cosides components like forsythoside A (unpublished). Therefore,these findings indicated that 25 mg/kg COS would be the promis-ing enhancer for improving the bioavailability of phenylethanoidglycosides in Fructus Forsythiae extract in rats.

Effect of Fructus Forsythiae extract with or without COS oninfluenza virus

As shown in Fig. 10, the inhibition rate of COS group wasno significant compared with that of PBS group, although therewas a remarkable increase in inhibition rate value after adminis-trating orally 20 mg/kg ribavirin as a positive control. However, thedifference of antiviral activity between Fructus Forsythiae extractwith or without COS was significant. The inhibition rate of FructusForsythiae extract with COS at dosage of 25 mg/kg was higher thanthat of without COS (most of them had significance), which indi-cated that the pharmacological effects such as antiviral effect ofFructus Forsythiae extract could be significantly improved by addi-tion of COS.

Discussion

In the present study, we found from Fig. 3 and Table 1 thatabout 21 peaks were detected in dosed plasma. Among these peaks,

phenylethanoid glycosides, phenolic acids, lignans and flavonescontaining 18 peaks also appeared in the MS spectra of Fruc-tus Forsythiae extract, indicating that these components might beabsorbed into the rat plasma in the original form, and possibly

W. Zhou et al. / Phytomedicine 21 (2014) 1549–1558 1557

Table 2Pharmacokinetic parameter of forsythoside A, isoforsythoside and forsythoside B in rats after oral administration of Fructus Forsythiae extract with or without the additionof COS (mean ± S.D., n = 6).

Cmax (ng/ml) Tmax (min) AUC0–480min (ng min/ml) Enhancement ratio

Forsythoside A Control 165.2 ± 86.25 8.571 ± 2.440 4262.6 ± 1167.0 –COS 267.6 ± 92.32* 6.667 ± 2.582 12,378 ± 2752.4** 2.904

Isoforsythoside Control 80.66 ± 35.78 8.571 ± 2.440 4073.6 ± 1366.5 –COS 172.8 ± 39.28** 7.500 ± 2.739 10,251 ± 3538.5** 2.517

Forsythoside B Control 76.89 ± 39.28 8.571 ± 2.440 3850.2 ± 1562.7 –COS 142.6 ± 51.91* 6.667 ± 2.582 8264.8 ± 2973.5** 2.147

n.s. – no significant difference, compared with the control.* p < 0.05.

** p < 0.01.

Fig. 10. Effect of Fructus Forsythiae extract with or without COS at the dosage of2e(

bbetmFatsp

5 mg/kg on influenza virus. Inhibition rate was assayed with MTT 48 h later andxpressed as percentage of controls (data ±S.D. n = 8). (*) p < 0.05, (**) p < 0.01 andn.s.) no significant difference.

ecome the real active ingredients. And it has been illustratedy us that the phenylethanoid glycosides in Fructus Forsythiaextract were related more directly to the pharmacological effectshan phenolic acids and flavones using PK-PD combined with PLS

ethod. Meanwhile, as shown in Fig. 10, treatment with Fructusorsythiae extract with COS improved MDCK cell viability better

fter influenza virus propagation. All evidence above supportinghe hypothesis that the phenylethanoid glycosides could be con-idered as one of the important marker compounds to control theharmacology of Fructus Forsythiae extract.

Fig. 11. Possible routes for absorption

It was reported that the oral bioavailability in rats of forsytho-side A was significantly low (0.5%) (Wang et al. 2010). Surprisingly,until recently the absorption mechanism of phenylethanoid gly-cosides was completely unknown. It was reported by Lu et al.(2010) that the bioavailability of forsythoside A oily formulationorally was five fold higher than that of non-oily formulation, andZhang (2002) conducted a physicochemical study of forsytho-side A, showing it to be a highly hydrophilic compound almostcompletely dissociated in biological fluids. This physicochemicalproperty of the drug led us to postulate that the low permeabilityof the drug in the intestinal mucosa was the one of the impor-tant reasons for its reported low bioavailability, and Food andDrug Administration (FDA) also recognized that the poor perme-ation of drugs across the intestinal mucosa (usually due to theirhigh hydrophilicity) was one of the common factors leading tofailed absorption and thus to low drug bioavailability (BA). Thus,the absorption mechanism using in vitro Caco-2 cell was studies(Fig. 5), and the results indicated that P-gp and MRP2 participatedin the absorption of phenylethanoid glycosides in the intestine,and the permeability was mainly restricted by the tight junctions(Fig. 11). In order to illustrate the permeability of phenylethanoidglycosides in Fructus Forsythiae extract, it was found (Fig. 5B)that the efflux ratios isoforsythoside and forsythoside B in FructusForsythiae extract was significantly higher than that of monomers,which might be attributed to the influence of other ingredientsin Fructus Forsythiae extract on the efflux transporters (P-gp and

MRP2), but efflux effect was weak (efflux ratios < 1.0), which indi-cated the permeability of phenylethanoid glycosides in FructusForsythiae extract was also mainly restricted by the tight junc-tions.

of phenylethanoid glycosides.

1 edicine

pitaatcii(hwToep

poaehiv6ciF

C

A

uSPoIffP

R

B

B

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Caco-2 cell models. Acta Pharmacol. Sin. 33, 1069–1079.

558 W. Zhou et al. / Phytom

It was shown (Fig. 9, Table 2) that the bioavailability ofhenylethanoid glycosides in Fructus Forsythiae extract were

mproved significantly, compared with control, and the absorp-ion enhancing effect of COS was affected by their concentrations,

maximal absorption enhancing effect of COS was observed at dosage of 25 mg/kg, not higher doses. The result was consis-ent with our previous report that COS at dosage of 25 mg/kg wasonsidered as safe and effective absorption enhancer for improv-ng the bioavailability of phenolic acids and the antiviral activityn vitro in Flos Lonicerae extract (Zhou et al. 2013). Besides, Gao et al.2008) also reported the absorption enhancing effect of chitosanexamer for FD4, and found that its absorption enhancing effectas almost saturable up to 0.5% (w/v) using in situ loop method.

herefore, our present result indicated that chitosan has someptimal concentrations to show the greatest absorption enhancingffects for improving the intestinal absorption of drugs includinghenylethanoid glycosides in Fructus Forsythiae extract.

The present study demonstrated that the absorption ofhenylethanoid glycosides in Fructus Forsythiae extract can be notnly improved greatest, but also safety in gastrointestine by COSt the dosage of 25 mg/kg. Meanwhile, It was reported (Thanout al. 2001, 2007) that the AUC values of low molecular weighteparin (LMWH) were improved by 7 and 18 times by low viscos-

ty grade mono-N-carboxymethyl chitosan (LMCC) and high lowiscosity grade N-sulfonato-N,O-carboxymethyl chitosan (SNOCC-0) respectively via intraduodenal administration. Therefore, otherhitosan derivatives as absorption enhancers need to be furthernvestigated in order to improve the pharmacological effects ofructus Forsythiae extract better.

onflict of interest

There is no conflict of interest.

cknowledgements

The present study is supported financially by the National Nat-ral Science Foundation of China (81001499), the Jiangsu Naturalcience Foundation (BK2010560), “Qing Lan” Project from Jiangsurovincial Technology Innovation Team Support Scheme, the pri-rity Academic Program Development of Jiangsu Higher Educationnstitution (no. ysxk-2010), the Fourth Phase of “333” Projectrom Jiangsu Province (BRA2013201) and 2012 program sponsoredor Scientific Innovation Research of College Graduate in Jiangsurovince (623).

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