A new natural angelica polysaccharide based colon-specific drug delivery system

A New Natural Angelica Polysaccharide BasedColon-Specific Drug Delivery System

SIYUAN ZHOU,1 BANGLE ZHANG,1 XINYOU LIU,1 ZENGHUI TENG,1 MENGLEI HUAN,1 TIEHONG YANG,2

ZHIFU YANG,1 MIN JIA,2 QIBING MEI2

1Department of Pharmaceutics, School of Pharmacy; Fourth Military Medical University, Xi’an, Shaanxi 710032, China

2Department of Pharmacology, School of Pharmacy; Fourth Military Medical University, Xi’an, Shaanxi 710032, China

Received 5 November 2008; revised 14 March 2009; accepted 20 March 2009

Published online 30 April 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21790

Bangle ZhanCorresponde

Fax: 86-2984774

Journal of Pharm

� 2009 Wiley-Liss

4756 JOURN

ABSTRACT: Colon-specific drug delivery systems are clinically necessary to treat colondiseases locally while minimizing systemic side effects. In this study, we extractedangelica polysaccharide from fresh roots of Angelica sinensis Diels and analyzed themonosaccharide components. With succinate as a cross-linker and angelica polysac-charide as a drug carrier, a dexamethasone–polysaccharide conjugate was synthesized.The amount of dexamethasone (Dex) loaded in the dexamethasone–polysaccharideconjugate was 14.13/100 mg. The newly synthesized dexamethasone–polysaccharideconjugate was found to greatly reduce systemic absorption of Dex and effectively deliverDex to the large intestine. When dexamethasone–polysaccharide conjugate was used totreat TNBS-induced ulcerative colitis in rats by gavage, the ulcerative area of the colonand the colonic myeloperoxidase (MPO) activity was reduced in a dose-dependentmanner. There was no effects on spleen weight, thymus weight, or peripheral bloodlymphocyte count (0.25 mmol kg�1 day�1). These results indicate that the dexametha-sone–polysaccharide conjugate has a therapeutic effect on TNBS-induced ulcerativecolitis in rats, while simultaneously reducing the systemic immunosuppression causedby Dex. Thus, the angelica polysaccharide was a promising colon-specific drug carrier,and the new dexamethasone–polysaccharide conjugate may yield a potential drug forthe treatment of human inflammatory bowel disease. � 2009 Wiley-Liss, Inc. and the

American Pharmacists Association J Pharm Sci 98:4756–4768, 2009

Keywords: angelica polysaccharide; col
on-specific drug delivery system; dexametha-sone; ulcerative colitis
INTRODUCTION

An ideal colon-specific drug delivery systemshould effectively release the drug upon entryinto the colon with as little drug release andabsorption in the stomach and small intestine aspossible. There has been considerable researchinto the design of colonic drug delivery systems

g contributed equally to this work.nce to: Qibing Mei (Telephone: 86-2984774552;552; E-mail: [email protected])

aceutical Sciences, Vol. 98, 4756–4768 (2009)

, Inc. and the American Pharmacists Association

AL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 12, DE

with several promising methods, such as pro-drugs,1–3 polymeric prodrugs,4–6 pH-dependentsystems,7 time-dependent systems,8 and biode-gradable systems.9 Nevertheless, pH and transittime can vary greatly among individuals, giventhe different pathological and dietary conditions.This can lead to premature and nonspecific drugdelivery in the gastrointestinal tract. Precisecolon drug delivery requires a built-in, colon-specific triggering mechanism, of which there arethree. Of these systems, microflora activateddelivery systems are considered the most promis-ing since the abrupt increase of the bacterialpopulation in the ascending colon represents a

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ANGELICA POLYSACCHARIDE BASED COLON-SPECIFIC DRUG DELIVERY SYSTEM 4757

noncontinuous event that is independent of GItransit time and pH.10 The critical component inmicroflora activated systems is a series of poly-saccharides, which evade enzymatic degradationin the small intestine and are predominantlymetabolized by colonic bacteria.

Natural polysaccharides (such as pectin, chit-osan, and dextran) are now extensively used forthe development of colon-specific drug deliverysystems because they are nontoxic, inexpensive,easy available in a variety of structures andproperties. Chemically, polysaccharides are effec-tive because the abundant hydroxyl groups canreadily conjugate with many biologically activecompounds.10–13 Once in the colon, polysacchar-ides are selectively degraded, releasing the activedrug. The challenge remains to find a polysac-charide with high degradability in the colon andbetter capability of protecting the drug fromrelease in the stomach and the small intestine.

Angelica sinensis Diels, a well-known Chineseherbal medicine, has been used to treat variousdiseases for thousands of years. There is mountingevidence from our lab and others have revealedthat polysaccharide extracted from angelica sine-nsis is protective in gastrointestinal tract,14–15

and also has anti-ulcer,16 and immunomodula-tion17,18 capabilities. In this study, we extractedthe angelica polysaccharide and used it as thedrug carrier to develop an angelica polysaccharidebased colon-specific drug delivery system. Thepharmacokinetics of this drug delivery systemwas investigated in rats. Furthermore, the effectsof this drug delivery system on an experimentalulcerative colitis model were also evaluated inrats.

MATERIALS AND METHODS

Materials

Dexamethasone (Dex) and prednisolone werepurchased from Tianjin Pharmacy Ltd, Tianjin,China. 1-phenyl-3-methyl-5-prazolone (PMP) andsuccinate anhydride were purchased from BeijingMedicine Corporation, Beijing, China. 4-dimethy-laminopyridine (4-DMPA), 1,10-carbonyldiimidazole,2,4,6-trinitrobenzenesulfonic acid, D-mannose,L-rhamnose, D-glucuronic acid, D-galacturonicacid, D-glucose, D-galactose, D-xylose, D-arabinose,and standard T-series dextran (T-18, T-50, T-80,T-150, T-270, and T-410) were obtained fromSigma Chemical Co. (St. Louis, MO) Acetonitrile

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and methanol were HPLC grade. Sprague–Dawleyrats (200� 20 g) were purchased from the ex-perimental animal research center, FourthMilitary Medical University, Xi’an, China. Theanimals were allowed water and laboratory chowad libitum. The rats were maintained in a 12 hlight-dark circle. All animal procedures wereperformed in accordance with protocols approvedby University Animal Care and Use Committee.

Methods

Isolation and Purification of thePolysaccharides from Angelica Sinensis Diels

Fresh roots of Angelica sinensis Diels werecollected in autumn from the Minxian County,in the Gansu province, China. The fresh root ofangelica sinensis was defatted with ethanol, andthen extracted with boiling distilled water. Thesupernatant was concentrated under reducedpressure and then centrifuged (3000g, 15 min,48C). The supernatant was collected, and pre-cipitated with ethanol to obtain polysaccharidepellets. The pellet was completely dissolved indistilled water and extensively dialyzed againstdistilled water with a dialysis bag (exclusion limit8 kDa). The remaining portion was concentratedand deproteinated by five cycles of freezing(�208C, 24 h storage) and thawing (room tem-perature for 3 h) to obtain crude polysaccharide.The crude polysaccharide was purified on aSaphacry S400 HR (Pharmacia AB, Uppsala,Sweden) chromatography column (100 cm� 3.5cm) and eluted with 0.1 M sodium chloride. Thepolysaccharide fraction was collected according tothe elution curve. After the polysaccharide-con-taining fraction was dialyzed and lyophilized, awhite product (angelica polysaccharide) wasobtained. The yield was 1.1% relative to the freshmaterial. Optical rotations of angelica polysac-charide wasþ33.6 at 208C (measured by a Perkin–Elmer 343 polarimeter).

Determination of Homogeneity and MolecularWeight of Angelica Polysaccharide

The molecular weight (Mw) and homogeneity ofthe angelica polysaccharide were determined andevaluated by high-performance size-exclusionchromatography (HPSEC). Briefly, polysacchar-ide was passed through a Waters Alliance 2695quaternary pump equipped with a tandem

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4758 ZHOU ET AL.

arrangement of a Shodex (OHpak SB-804 HQ,Munich, Germany, 7.8 mm� 300 mm) columnand a Biosep SEC-S3000 (Phenomenex, Torrance,CA, 7.8 mm� 300 mm) column and eluted with0.05 M NaCl at a flow rate of 0.5 mL min�1. Theelution was monitored by a Waters Alliance 2414RI detector. Glucose (20 mL) (Mw¼ 180 Da)solution (1%, w/v) and dextran (Mw¼ 2000 kDa)solution (1%, w/v) were passed through toevaluate the void and total volumes. StandardT-dextrans (T-18, T-50, T-80, T-150, T-270, and T-410) were passed through the column, and then theelution volumes were plotted against the loga-rithms of their respective molecular weight. Theelution volume of the angelica polysaccharide wasthen plotted in the same graph, and the molecularweight of angelica polysaccharide was deter-mined.

Protein and Carbohydrate Analysis inAngelica Polysaccharide

The protein present in the angelica polysacchar-ide was estimated by the method of Lowry et al.19

Bovine serum albumin was used as the standard.Carbohydrate content was measured by thephenol–sulfuric acid method20 using D-glucoseas the standard.

Table 1. HPLC Gradient Condition

Time(min)

Flow Rate(mL min�1) A (%) B (%) Curve

0 1.00 0.0 100.0 110 1.00 8.0 92.0 635 1.00 30.0 70.0 640 1.00 30.0 70.0 175 1.00 0.0 100.0 1

Monosaccharide Identification inAngelica Polysaccharide

Angelica polysaccharide was dissolved in 2 mL ofa trifluoroacetic acid (TFA, 4 M) solution in a 5 mLampoule. The ampoule was sealed and heated inan oil bath at 1108C for 8 h. After being cooledto room temperature, the reaction mixture wascentrifuged at 2000g for 10 min. The supernatantwas collected and neutralized to pH 7 with 0.3 MNaOH. Preliminary studies have shown thatunder this hydrolysis condition, uronic acid andneutral monosaccharides were completely releasedwithout degradation.21

Monosaccharides were labeled with PMPaccording to the previous method.22 Briefly,50 mL methanolic solution of PMP (0.5 M) and50 mL aqueous sodium hydroxide (0.3 M) wereadded to a mixture of hydrolyzed monosaccharide(10–500 pmol each) in a small tube (6 cm length,5 mm i.d.), and the tube was kept at 708C for30 min. After the reaction mixture was cooledto room temperature, an equivalent amount of0.1 M HCl was added for neutralization, and the

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resultant solution was evaporated to dryness.Water (200 mL) and chloroform (200 mL) wereadded to the residue, and the mixture was shakenvigorously. The chloroform layer was discarded,and the aqueous layer was evaporated to dryness.The residue was dissolved in 50 mL of eluantfor HPLC, and 20 mL was injected to the HPLCsystem.

The chromatographic system (Waters, Milford,MA) consisted of a quaternary pump (2695), aphotodiode array detector (2996), and a LC-Workstation (Empower, Milford, MA). Chromato-graphy was performed on a Diamonsil C18 column(250 mm� 4.6 mm, 5 mm, Dikma, Beijing, China)with a flow rate of 1.0 mL min�1. The detectorwavelength was set at 320 nm and the columntemperature was kept at 258C. Mobile phase Awas 40% (v/v) acetonitrileþ 25 mM KH2PO4

buffer (pH 6.9), and mobile phase B was 15% (v/v)acetonitrileþ 25 mM KH2PO4 buffer (pH 6.9). Thegradient was run as shown in Table 1.

Synthesis of Dexamethasone–PolysaccharideConjugate

Dexamethasone–polysaccharide conjugate wassynthesized according to the previously reportedmethod.23 Dexamethasone (3 mmol), succinateanhydride (6 mmol), and 4-DMPA (1.5 mmol) weredissolved in 100 mL anhydrous acetone (treatedwith five Angstrom molecular sieve for 48 h). Thereaction mixture was run at 258C for 30 min,and the resulting solution was evaporated with arotary evaporator to produce a light yellow solid.The solid was dissolved in 5 mL anhydrousethanol and 15 mL distilled water was added.The solution was kept at 48C for 24 h to crystallize,and the crystal was filtered under reducedpressure. The crystals were dried in a P2O5 dryingpistol with refluxing of 95% ethanol undervacuum (10 mmHg) for 36 h to produce dexa-methasone succinate hemiester (DSH). The melt-ing point of DSH was 209–2128C. The yield was97%.

9 DOI 10.1002/jps


DSH (2 mmol) and 1,10-carbonyldiimidazole(3 mmol) were dissolved in 40 mL of anhydrousdimethyl sulfoxide (DMSO, treated with fiveAngstrom molecular sieve for 48 h). The reactionmixture was stirred at 258C for 30 min. Then asolution of angelica polysaccharide (1 g polysac-charide in 80 mL anhydrous DMSO) and triethy-lamine (4.5 mmol) was added, and the reactionmixture was stirred at 258C for 24 h. The poly-saccharide conjugate was precipitated by adding250 mL of ethanol/ether (v/v¼ 1:1) to the reactionmixture with stirring. The resulting polymer wasredissolved and precipitated twice to completelyisolate the unconjugated DSH from the polymer.The polymer was dispersed in ethanol andcollected by filtration under reduced pressure. Itwas then washed with anhydrous ether threetimes to produce a white powder of dexametha-sone–polysaccharide. The yield was 91.5%.

Detection of the Amount of Dexamethasone Loadedin Dexamethasone–Polysaccharide Conjugate

The content of dexamethasone in dexametha-sone–polysaccharide conjugate was measured byHPLC after alkaline hydrolysis. Briefly, dexa-methasone–polysaccharide conjugate (2 mg), and3 mL NaOH solution (0.025 M) were added to atube and the mixture was stirred at 258C for40 min. Then 0.5 mL of the reaction mixture wastaken out and neutralized to pH 7 with 1 M HCl.The amount of dexamethasone in this solutionwas detected by HPLC. Preliminary studies haveshown that under this hydrolysis condition, dexa-methasone was completely released from the poly-mer without degradation. The result indicatedthat 100 mg dexamethasone–polysaccharide con-jugate contained 14.13� 0.25 (n¼ 3) mg dexa-methasone.

Pharmacokinetics of Dexamethasone–Polysaccharide Conjugate in Rats

Rats were randomly divided into 18 groups. Therewere six rats in each group. They were fastedfor 12 h before the experiments. As the darkcycle ended at approximately 7 am, dexametha-sone (Dex) or dexamethasone–polysaccharideconjugate, suspended in 0.5% carboxymethylcel-lulose sodium solution, was administered to ratsby gavage at a dose of 5 mmol kg�1 (2.5 mL kg�1)between 8 and 10 am. The rats were anesthetizedwith diethyl ether, and 2–3 mL of blood was drawn


from the rats’ abdominal aorta 1, 2.5, 3.5, 4.5, 6, 9,10.5, 12, and 15 h after administration. (1) Theblood was heparinized, and the plasma wasseparated by centrifugation (2000g, 48C, and7 min). Plasma (1.0 mL) and 200 mL of 1 mg L�1

prednisolone (internal standard) were added toeach tube. The mixture was extracted twice byvortexing with 2.0 mL of a mixture of methyl tert-tubtyl ether and pentane (v/v¼ 6/4). The organicphase was removed into clear tubes and evapo-rated to dryness at 258C under a gentle stream ofnitrogen in a ventilation cabinet. The residue wasdissolved in 50 mL of methanol and then 20 mL ofthis solution was analyzed by HPLC. (2) Imme-diately after the collection of blood, the contentsand the mucosa of the stomach, the proximal ofthe small intestine (PSI), the distal of the smallintestine (DSI), the cecum and the colon wereremoved. The contents and the mucosa werediluted to 100 g L�1 with chilled (48C) phosphatebuffer (pH 6.8, 0.1 M). Diluted contents (0.7 mL)(or the mucosa) of the stomach, PSI, DSI, thececum and the colon were added into differenttubes, respectively. Into each of these tubes,200 mL of saturated aqueous sodium chlorideand 100 mL of prednisolone solution (6 mg L�1,internal standard) were added. The samples wereextracted as above.

Determination of Dex in Plasma and Contentsof GI Tract

The amounts of Dex in the mucosa, the contents ofGI tract and the plasma were detected by HPLC(Waters), which consisted of a quaternary pump(2695), a photodiode array detector (2996), anda LC-Workstation (Empower). The mobile phaseconsisted of 35% acetonitrile and 65% 50 mMtrisodium citrate adjusted to pH 4.1 with phos-phoric acid. A phenomenex C18 analytical column(250 mm� 4.6 mm, 5 mm) was used. The columntemperature was kept at 258C. The flow rate was1 mL min�1 and a detection wavelength of 242 nmwas used.

Trinitrobenzenesulfonic Acid (TNBS)-InducedExperimental Ulcerative Colitis Model andthe Efficacy of Dexamethasone–PolysaccharideConjugate

The TNBS-induced experimental colitis modelwas selected to study the ameliorating effectsof the dexamethasone–polysaccharide conjugate.


4760 ZHOU ET AL.

This is the most relevant model for chronicinflammation of colon.24,25 The animals were foodfasted 48 h before the experiments, and allowedfood and water ad libitum after the administra-tion of TNBS. To induce inflammation, all thegroups except the healthy control group weretreated by a procedure discussed below. The ratswere randomized into six groups, six rats for eachgroup. The rats (except for the healthy controlgroup) were anesthetized with ether. A rubbercatheter (OD, 2 mm) was inserted rectally intothe colon such that the tip was 8 cm proximal tothe anus. 2,4,6-Trinitrobenzenesulfonic acid dis-solved in 38% ethanol (v/v) was instilled into thelumen of the colon through the rubber catheter atthe dose of 125 mg kg�1. Dex or dexamethasone–polysaccharide conjugate were administered bygavage 12 h after the induction of ulcerative colitis.The doses of the dexamethasone–polysaccharideconjugate were 0.05, 0.25, and 1.25 mmol kg�1

day�1, and the dose of Dex was 0.25 mmol kg�1

day�1. The colitis rats were treated for 7 days.The rats were sacrificed by CO2 after measuring

the lymphocyte count in the peripheral blood.(1) The thymus and the spleen were removed andweighed. (2) A piece of the colon was also takenfrom the region of only administration (8 cmproximal to the anus). The colon was opened alongits mesenteric border and gently rinsed of itscontents with iced normal saline. The colon wasthen layed flat, mucosal surface upwards, on aplate chilled at 48C. A transparent film was placed5 mm above the mucosal surface, and the area ofulceration and total surface area were traced bya marker-pencil. The colon tissue was weighed.(3) The colonic MPO activity was determined aspreviously reported.26 (4) Representative sectionsfrom the colon were fixed in 10% formaldehyde.Formaldehyde-fixed, paraffin-embedded sectionswere stained with H&E for further histology study.

Statistical Methods

The statistical analysis of the data was performedaccording to one-way analysis of variance(ANOVA).

RESULTS

Homogeneity and Molecular Weight ofAngelica Polysaccharide

The HPSEC showed a single and symmetricalsharp peak, indicating that the extracted angelica


polysaccharide was homogeneous. The weight-average molecular weight of the angelica poly-saccharide was determined from a calibrationcurve prepared with standard dextrans and foundto be 50 kDa.

Monosaccharide Components of AngelicaPolysaccharide

The chromatogram of PMP-labeled monosacchar-ides in the angelica polysaccharide is shown inFigure 1. The results indicate that angelicapolysaccharide is composed of rhamnose, galac-turonic acid, glucose, galactose, and arabinose in amolar ratio of 0.05:0.26:14.47:1.00:1.17. Glucose isthe predominant monosaccharide in the angelicapolysaccharide.

Content of Carbohydrate and Protein inAngelica Polysaccharide

The angelica polysaccharide contained 2.0%protein and 98.0% carbohydrate (w/w).

Fourier Transform Infrared Spectroscopy (FTIR)Spectra of Angelica Polysaccharide

FTIR spectra (KBr disc) were recorded with aShzmadzu FTIR-8400S Infrared Spectrophot-ometers for detecting functional groups. The FTIRspectrum of the angelica polysaccharide is shownin Figure 2. The peak at 3417 cm�1 was assignedto the O–H stretching vibration, the two peaks at2937 and 1380 cm�1 were the C–H stretchingvibrations, the peaks at 1743 and 1253 cm�1 werethe C––O and C–O stretching vibrations, the peakat 1024 cm�1 was the C–O of the C–O–Cstretching vibration.

The Linearity and Precision of Analytical Method

Standard curves were prepared to determine theamounts of Dex in the plasma, the mucosa, andthe contents of GI tract of the rats. The linearregression equation are y¼ 0.000650��0.00943,y¼ 0.0031��0.0191, and y¼ 0.0043��0.0214,respectively. The precision of the analyticalmethod, expressed as the intraday and interdayrelative standard deviations (RSD), was below3.4% for quality control samples. The accuracy,expressed as the relative error (RE), was within�4.1% for all the analytes. The recovery of

9 DOI 10.1002/jps

Figure 1. HPLC analysis of monosaccharides in angelica polysaccharide. (A) Stan-dard monsaccharide, (B) angelica polysaccharide. 1-mannose, 2-rhamnose, 3-glucuronicacid, 4-galacturonic acid, 5-glucose, 6-galactose, 7-arabinose, and 8-xylose.

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Figure 2. FTIR spectra of angelica polysaccharide.

4762 ZHOU ET AL.

dexamethasone with this analytical methodvaried from 92% to 109%. The lower limit ofquantification (LLOQ) is 0.01 mg mL�1.

Dex Distribution in Rat GI Tract afterOral Administration of Dex andDexamethasone–Polysaccharide Conjugate

After oral administration, Dex was mainly distri-buted in the contents and the mucosa of stomachand proximal intestine. Very little amount ofDex was detected in the contents and mucosa ofthe cecum and the colon (Tabs. 2 and 3). However,no Dex could be detected in the contents andthe mucosa of the stomach or the proximalintestine after dexamethasone–polysaccharideconjugate was orally administered. Dex was

Table 2. Distribution of Dex in Contents of Different SectioDose of 5 mmol kg�1

Time (h)

Dex

Stomach PSI

1 8.67� 1.32 3.42� 0.132.5 5.93� 0.80 1.44� 0.253.5 4.22� 0.56 1.43� 0.414.5 2.61� 0.30 1.16� 0.226 2.27� 0.61 0.96� 0.209 1.50� 0.60 0.73� 0.0910.5 0.95� 0.47 0.56� 0.1812 0.73� 0.08 0.41� 0.0815 0.32� 0.05 /

PSI, proximal small intestine; DSI, distal small intestine; ‘‘/’’, noData are mean�SD, n¼ 6.


detected primarily in the contents of the cecumand the colon. A large amount of DSH wasfound in the contents of the cecum and the colon.A considerable amount of Dex was released intothe contents of the colon between 6 and 10.5 hafter dosing (Tabs. 4–6).

Concentration of Dex in Plasma afterOral Administration of Dex andDexamethasone–Polysaccharide Conjugate

After oral administration of Dex and the dexa-methasone–polysaccharide conjugate, the plasmadrug concentration over time was obtained(Tab. 7). The area under the curve (AUC0–t) wascalculated by the linear trapezoidal rule fromzero to the last plasma drug concentration. These

ns of Rat GI Tract after Oral Administration of Dex at a

(mg g�1 Contents)

DSI Cecum Colon

1.59� 0.40 0.32� 0.16 /2.29� 0.45 1.34� 0.20 1.13� 0.211.78� 0.37 1.70� 0.41 1.60� 0.431.26� 0.37 1.11� 0.20 1.13� 0.160.84� 0.19 0.96� 0.08 0.98� 0.200.68� 0.21 0.47� 0.04 0.84� 0.200.40� 0.14 0.35� 0.17 0.56� 0.230.25� 0.07 0.20� 0.06 0.46� 0.10

/ / /

ndetectable.

9 DOI 10.1002/jps

Table 4. Amount of Dex Released in the Contents of Different Sections of Rat GI Tract after Oral Administration ofthe Dexamethasone–Polysaccharide Conjugate at a Dose of 5 mmol kg�1

Time (h)

Dex (mg g�1 Contents)

Stomach PSI DSI Cecum Colon

1 / / / / /2.5 / / / / /3.5 / / 0.26� 0.04 0.59� 0.20 /4.5 / / 0.12� 0.05 1.13� 0.26 0.67� 0.226 / / 0.08� 0.04 1.83� 0.35 2.83� 0.299 / / / 0.21� 0.08 1.41� 0.1510.5 / / / 0.15� 0.06 0.92� 0.0712 / / / / 0.20� 0.1015 / / / / /

PSI, proximal small intestine; DSI, distal small intestine; ‘‘/’’, nondetectable.Data are mean�SD, n¼6.

Table 3. Dex in Mucosa from Different Sections of Rat GI Tract after Oral Administration of Dex at a Dose of5 mmol kg�1

Time (h)

Dex (mg g�1 Mucosa)


1 2.82� 0.20 1.04� 0.53 0.27� 0.10 / /2.5 1.27� 0.11 0.49� 0.22 0.33� 0.05 0.29� 0.04 0.15� 0.033.5 0.97� 0.23 0.38� 0.15 0.42� 0.18 0.53� 0.19 0.36� 0.114.5 0.70� 0.25 0.32� 0.12 0.20� 0.06 0.50� 0.20 0.45� 0.186 0.48� 0.21 0.12� 0.12 0.09� 0.03 0.30� 0.10 0.20� 0.109 0.25� 0.06 0.08� 0.02 0.03� 0.01 0.06� 0.02 0.10� 0.0410.5 0.13� 0.05 0.03� 0.02 0.03� 0.02 0.04� 0.01 0.08� 0.0412 0.08� 0.03 / 0.02� 0.01 0.03� 0.01 0.05� 0.0215 / / / /


Table 5. Amount of Dex in Different Mucosal Sections of Rat GI Tract after Oral Administration of theDexamethasone–Polysaccharide Conjugate at a Dose of 5 mmol kg�1

Time (h)

Dex (mg g�1 Mucosa)


1 / / / / /2.5 / / / / /3.5 / / 0.06� 0.03 / /4.5 / / 0.09� 0.04 0.17� 0.05 0.05� 0.026 / / 0.07� 0.03 0.20� 0.08 0.28� 0.039 / / / 0.30� 0.11 0.65� 0.1410.5 / / / 0.13� 0.05 0.22� 0.0912 / / / / 0.10� 0.0315 / / / / /


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Table 6. Amount of DSH in the Contents of Different Sections of the Rat GI Tractafter Oral Administration of the Dexamethasone–Polysaccharide Conjugate at a Dose of5 mmol kg�1

Time (h)

DSH (mg g�1 mucosa)


1 / / / / /2.5 / / / / /3.5 / / 0.18� 0.07 / /4.5 / / 0.11� 0.05 0.83� 0.30 1.02� 0.366 / / 0.08� 0.04 2.44� 0.40 4.16� 1.359 / / / 0.30� 0.08 1.54� 0.1410.5 / / / 0.21� 0.06 0.27� 0.1012 / / / / 0.06� 0.0215 / / / / /

PSI, proximal small intestine; DSI, distal small intestine; ‘‘/’’, nondetectable.Data are mean�SD, n¼ 6.

4764 ZHOU ET AL.

values are 11875 and 334 mg h L�1, respectively.When Dex is administered orally, the Cmax issignificantly higher than that for orally adminis-tered dexamethasone–polysaccharide conjugate(p< 0.01).

Efficacy of Dexamethasone–PolysaccharideConjugate

When dexamethasone–polysaccharide conjugatewas used to treat ulcerative colitis in rats by oraladministration, the ulcerative area of colon andthe colonic MPO activity was reduced in a dose-dependent manner (Fig. 3, Tab. 8). Lymphocytecount in peripheral blood, spleen weight, andthymus weight of rats were markedly reducedby oral administration of free Dex at a dose of0.25 mmol kg�1 day�1 (p< 0.05 vs. control). How-

Table 7. Concentration of Dex in Rats PlasmDexamethasone–Polysaccharide Conjugate at

Time (h)

Dex Co

Free Dex De

1 1641� 1512.5 1881� 1193.5 1778� 2424.5 1502� 1786 991� 1129 433� 10210.5 286� 7912 150� 3115 50� 19

‘‘/’’, nondetectable.Data are mean�SD, n¼ 6.


ever, dexamethasone–polysaccharide conjugatehad no effect on these parameters at the samedose (p> 0.05 vs. control) (Tab. 9).

Histological Evaluation

Seven days after dexamethasone–polysaccharideconjugate (1.25mmol kg�1 day�1) was used to treatulcerative colitis in rats by oral administration,the damaged colonic mucosa was repaired, withlittle evidence of injury (Fig. 4).

DISCUSSION

In the preparation of colon-specific drug deliverysystems, the crucial step is to select a proper drugcarrier. Due to the toxicity associated with

a after Oral Administration of Dex anda Dose of 5 mmol kg�1

ncentration of (mg L�1)

xamethasone–Polysaccharide Conjugate

///

9� 620� 831� 1126� 1019� 99� 7

9 DOI 10.1002/jps

Figure 3. Effect of the dexamethasone–polysacchar-ide conjugate on colonic MPO activity of ulcerativecolitis rat. Data are mean�SD, n¼ 6. ##p< 0.01,#p< 0.05 versus model. ��p< 0.01 versus normal.


synthetic polymer systems,27,28 a wide variety ofnatural polymers are being used for the designand development of colon-targeted delivery sys-tems. Most of these systems are based on theknowledge that anaerobic bacteria in the colon areable to recognize the various polymers as sub-strates and degrade them with enzymes. Most ofthe recent research uses polysaccharides, espe-cially from plant origin, to create degradablecolon-specific substrates.

Ulcerative colitis, which is a common diseaseof the gastrointestinal (GI) tract, has a high-incidence rate in western countries. This diseasecan easily develop into a colonic tumor. Thepresent treatments available are not satisfactory.

Table 8. Effects of Dexamethasone–Polysaccharide ConjuWeight of Ulcerative Colitis Rats

Group Dose (mmol kg�1 day�1) Percent of Ulcera

Normal 0Model 27.0� 8Dex 0.25 17.7� 8Conjugate 0.05 9.1� 6.0Conjugate 0.25 4.9� 3.0Conjugate 1.25 2.5� 1.0

Percent of ulcerative area¼ [colon ulcerative area/total colon areData are mean�SD, n¼6.��p<0.01 versus normal.##p<0.01.#p< 0.05 versus model.þþp<0.01.þp<0.05 versus Dex.


Although the administration of glucocorticoidshas generally been effective, it is normally limitedto treat the disease in its acute form and can causesystemic side effects, such as adrenosuppressionand immunosuppression with long-term usage.In theory, selective delivery of a drug to the coloncould lower the required dose, reduce systemicside effects, and improve curative effects. There-fore, we set up and evaluated an angelicapolysaccharide based colon-specific drug deliverysystem of dexamethasone in vivo.

Our studies indicate that the angelica poly-saccharide is composed of rhamnose, galacturonicacid, glucose, galactose, and arabinose. Thisindicates that the angelica polysaccharide isdifferent from other natural colon-specific degrad-able polysaccharides. For example, dextran iscomposed of glucose, chitosan is composed ofglucosamine, and pectin is composed of galac-turonic acid and rhamnose. So, it is reasoned thatthe backbone of the angelica polysaccharide maybe different from other polysaccharides, thusexerting an effect on colon-specific drug deliveryas a result of its five different kinds of mono-saccharide components.

When Dex alone was orally administered torats, it was mainly distributed in the contents andthe mucosa of the stomach and the small intestine,leading to high levels of Dex in the plasma.However, when the dexamethasone–polysacchar-ide conjugate was orally administered to rats, aconsiderable amount of Dex was released from theconjugate in the cecum and the colon. Cmax andAUC0–t after oral administration of the conjugatewas less than 5% of that of free Dex at the samedose. This implies that the absorption of Dex inconjugate with the polysaccharide was greatly

gate on Colonic Macroscopic Ulceration and Colon

tive Area (%) Colon Weight/Body Weight (mg g�1)

2.5� 0.2.0�� 3.9� 0.7��

.1## 3.8� 0.6##,þþ 3.3� 0.5#

##,þþ 3.2� 0.6#,þ

##,þþ 3.1� 0.5#,þ

a]�100%.


Table 9. Effects of Dexamethasone–Polysaccharide Conjugate on Lymphocyte Count in Peripheral Blood, Weight ofThymus and Spleen in Ulcerative Colitis Rats

Group Dose (mmol kg�1 day�1) Thymus Index (mg g�1) Spleen Index (mg g�1) Lymphocyte (�109 L�1)

Control 2.48� 0.48 3.06� 0.69 11.3� 3.0Model 2.21� 0.38 3.11� 0.76 13.9� 3.5Dex 0.25 0.93� 0.23�� 2.10� 0.58�� 2.3� 1.0��

Conjugate 0.05 2.40� 0.32 3.23� 0.64 13.0� 3.5Conjugate 0.25 2.13� 0.36 2.88� 0.63 12.9� 3.4Conjugate 1.25 1.43� 0.26� 2.26� 0.41� 4.0� 1.0��

Thymus index¼ thymus weight/body weight; Spleen index¼ spleen weight/body weight.Data are mean�SD, n¼ 6.�p<0.05 versus normal.��p< 0.01.

4766 ZHOU ET AL.

reduced in the small intestine. Most importantly,the newly synthesized dexamethasone–polysac-charide conjugate can selectively deliver Dexto the colon, which resulted in reduced Dexconcentration in the rat’s blood. Moreover, theseresults also indicated that the ester bond inthe dexamethasone–polysaccharide conjugatewas efficiently protected from hydrolysis bycarboxyesterase. This occurs in the upper GItract by the angelica polysaccharide backbone.The backbone of the angelica polysaccharide waspresumed to be broken down by microorganismsin the cecum and the colon, and significant

Figure 4. Histology of colon of rats subjected toTNBS. (A) Healthy control, (B) colitis control showingmucosal injury characterized by absence of epitheliumand a massive mucosal/submucosal infiltration ofinflammatory cells, and (C) the recovery of rat mucosalafter treatment with dexamethasone–polysaccharide.


amounts of Dex released in these parts of theGI tract.

When the dexamethasone–polysaccharide con-jugate was administered to rats, large amounts ofDSH were found in the contents of the cecum andthe colon. No detectable DSH was found in themucosa or the plasma. This indicated that therewere two ways for the dexamethasone–polysac-charide conjugate to release Dex into the GI tractof rats. First, the dexamethasone–polysaccharideconjugate can be hydrolyzed to release Dexdirectly. Alternatively, the conjugate can behydrolyzed to release DSH, and then DSH canbe hydrolyzed further to release Dex. The latter isthe main route for the conjugate to release Dex.29

Colonic MPO activity, which reflects the quan-tity of neutrophils in tissue, is an objective indexfor evaluating inflammatory bowel disease(IBD).30 Another metric of IBD is edema of thecolon, which is directly related to the weight ofthe tissue. Healing of TNBS-induced colitis wasassessed by measuring the colonic surface area ofulceration, colon weight, and colonic MPO activ-ity. When the dexamethasone–polysaccharideconjugate and Dex (0.25 mmol kg�1 day�1) wereused to treat ulcerative colitis in rat by oraladministration, the ulcerative area of the colonwas reduced by 81.5% and 34.4%, respectively.Colon weight was reduced by 17.9% and 2.6%,respectively. Finally, colonic MPO activity wasreduced by 70.6% and 56.6%, respectively (Tab. 8,Fig. 3). This implies that the dexamethasone–polysaccharide conjugate was able to facilitate therepair of the damaged colonic mucosa, and thiswas more effective than the same dose of Dex. Itsuggests that the dexamethasone–polysaccharideconjugate has a satisfactory therapeutic effect onulcerative colitis in rats.

9 DOI 10.1002/jps


Cho et al.14 found that the angelica poly-saccharide possess an anti-inflammatory actionthrough inhibiting neutrophil infiltration ingastrointestinal damage induced by ethanol orindomethacin in rats. This implies that angelicapolysaccharide could potentially be useful toprevent neutrophil-dependent mucosal injury inthe gastrointestinal tract. Recently, it is reportedin rats that angelica polysaccharide has protectiveeffects in an ulcerative colitis model induced by2,4-dinitrobenzene sulphonic acid (DNBS). Theprotective effects of angelica sinensis polysacchar-ide are closely related to the prevention ofoxidative stress, which may occur during neu-trophil infiltration in the pathological processof ulcerative colitis.31 Accordingly, these resultssuggest that angelica polysaccharide in dexa-methasone–polysaccharide conjugate play a rolein anti-inflammatory action in TNBS-inducedulcerative colitis rats.

Moreover, the dexamethasone–polysaccharideconjugate did not decrease the lymphocyte countin the peripheral blood, nor did it decrease theweights of the spleen or the thymus in rats at thedose of 0.25 mmol kg�1 day�1 (p> 0.05 vs. control,Tab. 9). Conversely, the oral administration of freeDex at the same dose did greatly reduce lympho-cyte count in peripheral blood and did reduce theweights of the spleen and the thymus (p< 0.05vs. control). This indicates that the systemicimmunosuppression caused by Dex was reducedthrough oral administration of the dexametha-sone–polysaccharide conjugate. This is becausethe angelica polysaccharide based drug deliverysystem limits the overall amount of drug absorbedand thus, the impact on side effects.

In summary, our data demonstrates that thedexamethasone–polysaccharide conjugate greatlyreduces systemic absorption of Dex and effectivelydelivers Dex to the large intestine. Furthermore,the dexamethasone–polysaccharide conjugate hada satisfactory therapeutic effect on TNBS-inducedulcerative colitis in rats in a dose-dependentmanner. Angelica polysaccharide is a promisingcolon-specific drug carrier, and the development ofthis conjugate may yield a new and potentiallybetter way to treat human inflammatory boweldisease.

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9 DOI 10.1002/jps

A new natural angelica polysaccharide based colon-specific drug delivery system

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