Permeation-enhancing effect of aloe-emodin anthrone on water-soluble and poorly permeable compounds in rat colonic mucosa

Article abstract of DOI:10.1248/bpb.25.1608

The aims of this study were to examine the enhancing effects of aloe-emodin enthrone (AEA) on the colonic membrane permeability of water-soluble and poorly permeable compounds and to clarify the mechanism of the permeation-enhancing activity of AEA. The permeation-enhancing activity of AEA was estimated from changes in the permeability coefficient of 5(6)-carboxyfluorescein (CF) in rat colonic mucosa using a Ussing-type chamber. Various inhibitors were used to investigate the mechanism of action of AEA. The structural change in the membrane and the cytotoxicity of AEA in the intestinal mucosa were evaluated by measuring the electrical resistance of the membrane (R_m) and lactate dehydrogenase (LDH) activity, respectively. AEA significantly increased the permeation of CF in a dose-dependent manner. The enhanced permeability was significantly suppressed by a histamine H₁ receptor antagonist, pyrilamine, and a mast cell stabilizer, ketotifen, but not by a histamine H ₂ receptor antagonist, cimetidine. The enhancing effect was also inhibited by an inhibitor of protein kinase C (PKC). Potential difference and short-circuit current values decreased, while R_m values remained constant throughout the experiment. The addition of AEA to the mucosal solution decreased R_m to 30%, but then remained constant. LDH activity with AEA was not significantly different from that of the control. In conclusion, AEA is a candidate for effective absorption enhancers without damage of the membrane and cytotoxicity. We propose that AEA stimulates mast cells within the colonie mucosa to release histamine, which probably bind to the H₁ receptor. The intracellular PKC route activated by H₁ receptor activation enhances the permeability of water-soluble and poorly permeable drugs via opening of tight junctions in rat colonic membrane.

Full text of DOI:10.1248/bpb.25.1608

1608
Biol. Pharm. Bull. 25(12) 1608—1613 (2002)
Vol. 25, No. 12
Permeation-Enhancing Effect of Aloe-emodin Anthrone on
Water-Soluble and Poorly Permeable Compounds in Rat Colonic Mucosa
Mamiko KAI, Kazutaka HAYASHI, Ippei KAIDA, Hatsumi AKI,* and Magobei YAMAMOTO
Department of Pharmaceutical Care and Health Sciences, Faculty of Pharmaceutical Sciences, Fukuoka University;
8–19–1 Nanakuma, Jonan-ku, Fukuoka 814–0180, Japan. Received May 13, 2002; accepted September 12, 2002
The aims of this study were to examine the enhancing effects of aloe-emodin anthrone (AEA) on the colonic
membrane permeability of water-soluble and poorly permeable compounds and to clarify the mechanism of the
permeation-enhancing activity of AEA. The permeation-enhancing activity of AEA was estimated from changes
in the permeability coefficient of 5(6)-carboxyfluorescein (CF) in rat colonic mucosa using a Ussing-type cham-
ber. Various inhibitors were used to investigate the mechanism of action of AEA. The structural change in the
membrane and the cytotoxicity of AEA in the intestinal mucosa were evaluated by measuring the electrical resis-
tance of the membrane (Rm) and lactate dehydrogenase (LDH) activity, respectively. AEA significantly increased
the permeation of CF in a dose-dependent manner. The enhanced permeability was significantly suppressed by a
histamine H1 receptor antagonist, pyrilamine, and a mast cell stabilizer, ketotifen, but not by a histamine H2 re-
ceptor antagonist, cimetidine. The enhancing effect was also inhibited by an inhibitor of protein kinase C (PKC).
Potential difference and short-circuit current values decreased, while Rm values remained constant throughout
the experiment. The addition of AEA to the mucosal solution decreased Rm to 30%, but then remained constant.
LDH activity with AEA was not significantly different from that of the control. In conclusion, AEA is a candidate
for effective absorption enhancers without damage of the membrane and cytotoxicity. We propose that AEA
stimulates mast cells within the colonic mucosa to release histamine, which probably bind to the H1 receptor. The
intracellular PKC route activated by H1 receptor activation enhances the permeability of water-soluble and
poorly permeable drugs via opening of tight junctions in rat colonic membrane.
Key words aloe-emodin anthrone; absorption enhancer; histamine; Ussing chamber; epithelial permeability; paracellular path-
way
Aloe (Cape aloes from Aloe ferox. or A. arborescens
In this study, the effects of AEA on the permeability of
MILL., Socotrine aloes from Aloe perryi BAK. and Curacao water-soluble and poorly permeable compounds to the
aloes from Aloe vera LINN.) is widely used in the manufac- colonic epithelial cell membrane and the permeation-enhanc-
ture of food products and beverages, pharmaceuticals, and ing mechanism of AEA, particularly with regard to the role
cosmetics because of its aromatic properties, bitter taste, of histamine released from mast cells in the colonic mucosa,
cathartic activity derived from anthraquinones, and other were investigated to elucidate the effectiveness of AEA as a
pharmacological activities (such as emolliency, reduction of new class of absorption enhancer for water-soluble and
inflammation, and acceleration of wound healing, although it poorly permeable compounds.
is not yet well understood which activity is related to which
component).1) R- and S-barbaloins (10-glucopyranosyl-1,8- MATERIALS AND METHODS
dihydroxy-3-(hydroxymethyl)-9(10H)-anthracenone: BAs),
the main components of aloe, are two diastereoisomeric C-
Materials Histamine, cimetidine, ketotifen, pyrilamine,
glucosides differing in the configuration of C-10 in the aloe- 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine dihydrochlo-
emodin anthrone (AEA) moiety.2) It is generally accepted ride (H7), and bovine serum albumin (BSA) were purchased
that BAs are the bitter and purgative principle of the aloe from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Com-
product.1)
pound 48/80 was an oligomeric mixture of condensation
Ishii et al. reported that BAs were transformed into AEA products of N-methyl-p-methoxyphenethylamine and
(1,8-dihydroxy-3-(hydroxymethyl)-9(10H)-anthracenone) formaldehyde (Purity: Ͼ98% by TLC)8) and obtained from
under anaerobic conditions and aloe-emodin (1,8-dihydroxy- Sigma Chemical Co. 5(6)-Carboxyfluorescein (CF, MW
3-(hydroxymethyl)-9,10-anthracenedione, AE) under aerobic 376.2) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfon-
conditions through an enzymatic redox reaction involving rat amide (W7) were purchased from Seikagaku Kogyo Co.
intestinal microflora.3) Che et al. have shown the transforma- (Tokyo, Japan). Other reagents were of analytical grade or
tion of BA to AEA by human intestinal bacteria.4)
higher.
Animals Male Wistar rats (250—300 g), at least 72 d
Previously, we studied the antiinflammatory effects of BAs
and related compounds and found that AEA induced the re- old, were used in this study. The rats had free access to food
lease of histamine from isolated rat mast cells, whereas AE and water prior to killing. All procedures involving experi-
neither induced nor inhibited it.5) Histamine, an inflamma- mental animals were carried out according to the guidelines
tory mediator derived from mast cells, was reported to in- of the Experimental Animal Care and Use Committee of
crease intestinal epithelial permeability.6,7) Therefore we hy- Fukuoka University.
pothesize that AEA enhances mucosal permeability, resulting
Preparation of AEA BA was prepared from commer-
in the release of histamine from mast cells in the colonic mu- cial aloin (Sigma Chemical Co.) by recrystallization from
cosa.
water-methanol. The synthesis of AEA from BA was based
* To whom correspondence should be addressed. e-mail: akih@fukuoka-u.ac.jp
© 2002 Pharmaceutical Society of Japan

December 2002
1609
on the method of Hay and Haynes.9) An aqueous solution
(100 ml) of purified BA (5 g), sodium borate (10 g), and
phenylhydrazine hydrochloride (2 g) was refluxed for 3 h
under an atmosphere of nitrogen. The dark red solution was
acidified with diluted hydrochloric acid, and the precipitated
yellow solid was extracted using ether (ca. 500 ml). Evapora-
tion of the washed and dried (Na2SO4) ether extract gave a
reddish solid, which on purification with a prepacked column
(Lobor LiChroprep RP-18, 40—60 mm, 310ϫ25 mm i.d.,
Merk Japan Ltd., Tokyo, Japan) gave a yellow crystal identi-
fied as AEA by FAB-MS (JEOL JMS-HX 110 controlled by
a JEOL DA 7000 data system, Tokyo, Japan). Positive-ion
FAB-MS: m/z 257 ([MϩH]ϩ). Yield, 0.073 g, 2.4%, mp
199 °C. The purity of the synthesized AEA was determined
by HPLC. The HPLC system (Shimadzu LC10AD, Shi-
madzu Co., Kyoto, Japan) consisted of a UV spectrometric
detector (Shimadzu SPD-6AV), and chromatopac (Shimadzu
C-R6A). The column used was a Wakosil II 5C 18HG
(Wako, Osaka, Japan, 4.6 mm i.d.ϫ25 cm) and the mobile
phase was acetonitrile/water (2 : 1, v/v). The UV detector was
set at 295 nm and the flow rate was 1.0 ml/min at 40 °C. The
retention time of AEA was 21.6 min. The purity of AEA was
found to be more than 98%.
Histamine Release from Rat Peritoneal Mast Cells
The composition of the Tyrode HEPES solution (THS, pH
7.4) was as follows (g/l); NaCl (8.01), KCl (0.20),
NaH2PO4·2H2O (0.06), HEPES (4.76), CaCl4·2H2O (0.26),
MgCl2·6H2O (0.20), glucose (1.01), and gelatin (1.0). The
preparation of peritoneal mast cells and the assay of hista-
mine release from mast cells were carried out by a modified
version of the method described by Nakagomi et al.10) The
mast cells were collected from Wistar rat peritoneal cavities
after injection of 20 ml of THS, and centrifuged in a 38% so-
lution of BSA (dϭ1.068) for purification. The purity of the
mast cells was about 88—92% as observed under an Olym-
pus phase-contrast microscope. Various concentrations of
sample solution (AEA or compound 48/80) was prepared
with 0.1% BSA in THS. AEA was first dissolved in dimethyl
sulfoxide (DMSO) and diluted by the solution. The final con-
centration of DMSO in the assay media was less than 1%
(v/v), at which DMSO did not affect the cell viability and the
subsequent histamine assay. An aliquot (20 ml) of the cell
suspension (1—2ϫ106 cells/ml, 0.1% BSA in THS) was
warmed at 37 °C for 10 min, and then 20 ml of sample solu-
tion was added and incubated at 37 °C for 10 min. After the
incubation, the cells were cooled on ice, then centrifuged at
1500 g for 15 min. Next, 0.5 ml of 0.8 M perchloric acid was
added to 50 ml of the supernatant and centrifuged at 1500 g
for 15 min. The supernatant (150 ml) was then used for the
measurement of histamine release from the cells by fluoro-
metric analysis with o-phthalaldehyde. After the addition of
2 ml of 0.1 M hydrochloric acid, 0.4 ml of 1 M sodium hydro-
xide, and 0.1 ml of 1% o-phthalaldehyde in ethanol to the
supernatant, the solution was allowed to stand for 4 min. The
reaction was stopped by adding 0.2 ml of 3 M hydrochloric
acid. The concentration of histamine was determined fluoro-
metrically using a Shimadzu model RF-500 spectrofluorome-
ter (Shimadzu) at 355-nm excitation and 450-nm emission.
The amount of histamine released was expressed relative to
that from the 0.05% Triton X-100-treated cell supernatant.
Thus the percentage of release was defined as 100
[(SϪB)/(CϪB)], where B is the value of the control without
sample, and S and C are the amount of histamine released by
the sample and 0.05% Triton X-100, respectively.
Permeability Experiments Using Ussing-type Cham-
bers The composition of Krebs-Hensleit bicarbonate
Ringer’s solution (KBR solution, pH 7.4) was as follows
(g/l): NaCl (6.9), KCl (0.354), CaCl2 (0.282), NaHCO3 (2.1),
KH2PO4 (0.162), MgSO4·7H2O (0.294), and glucose (1.8).
CF was used as a model of water-soluble and poorly perme-
able compounds.
The colon was enucleated from male Wistar rats under
ether anesthesia. The colon mucosa stripped of the underly-
ing muscle was mounted as a flat sheet in an Ussing-type
chamber (WPI, Inc., U.S.A., 0.636 cm2 exposed surface area).
Briefly, KBR solution was added to both the mucosal and
serosal sides of the Ussing chamber, and the specimen was
incubated for 20—30 min at 37 °C under bubbling with 95%
O2 and 5% CO2. Then the mucosal solution was replaced
with 10 ml of KBR solution containing CF (100 mg/ml) to-
gether with AEA (0.5 mM), histamine (0.01 mM), and/or com-
pound 48/80 (0.1 mM) and incubated for 1 h at 37 °C. An
aliquot of 0.2 ml was taken from the serosal side and 3 ml of
1 M sodium hydroxide was added. The concentration of CF
was determined fluorometrically at 490-nm excitation and
520-nm emission. The apparent permeability coefficient
(Papp) was calculated using the following equation:
PappϭdQ/dtϫ1/(ACo), where dQ/dt represents the steady-
state flux of CF on the serosal side, Co the initial concentra-
tion of CF on the mucosal side, and A the gross surface area
of the membrane.11) The short-circuit current (Isc) and trans-
mucosal potential difference (PD) were measured and trans-
mucosal electrical resistance (Rm) was obtained according to
Ohm’s law as the electrophysiological parameters.
Treatment with Various Inhibitors: Pyrilamine (20 mg/kg)
or cimetidine (100 mg/kg) was intraperitoneally administered
to the rats 10 min prior to excision of the colonic segment as
described by Murakami et al.12) Ketotifen (3.125 mg/kg) was
intraperitoneally given 30 min prior to the preparation of the
colon.12) The colonic segment pretreated with various in-
hibitors was subjected to permeability experiments. Com-
pound 48/80 (2.5 mg/ml), W7 (50 mM), or H7 (50 mM) was ap-
plied on the mucosal side of the chamber and incubated for
20 min at 37 °C as described previously.13)
Measurment of Lactate Dehydrogenase Activity Male
Wistar rats (250—300 g) were intraperitoneally anesthetized
with sodium pentobarbital. The intestine was exposed
through a midline incision, and a closed loop was prepared
using approximately 5 cm of the colon by ligation. One milli-
liter of KBR solution containing 0.5 mM AEA was intro-
duced into the loop via a vinyl catheter immediately after lig-
ation. After 1 h, the loop was removed to measure lactate de-
hydrogenase (LDH) release in the loop using a commercial
enzymatic assay kit (LDH-10; Shimadzu Chemical Co.).
Statistical Analysis Statistical analysis was performed
using the unpaired Student’s t-test or ANOVA followed by
Scheffe’s F test. A value of pϽ0.05 was considered signifi-
cant. The intraobserver or interobserver variation was Ͻ5%
in each experiment.

1610
Vol. 25, No. 12
RESULTS
AEA-Induced Histamine Release Figure 1 shows the
histamine release induced from rat peritoneal mast cells by
AEA or compound 48/80. The release was rapid and reached
a maximum within 1 min of the initiation of incubation. Ten
minutes of incubation was sufficiently long to measure the
release. Although AEA was less effective than compound
48/80, AEA induced the release of histamine in a dose-de-
pendent manner at concentrations of 2ϫ10Ϫ4—10Ϫ2 M. The
threshold concentration was around 1ϫ10Ϫ4 M. At concentra-
tions above 1ϫ10Ϫ2 M, the percentage of histamine released
by AEA reached a plateau (55—60%).
Fig. 1. Histamine Release from Rat Peritoneal Mast Cells Induced by
AEA or Compound 48/80
Effect of AEA on the Permeation of CF in Rat Colonic
Mucosa The permeation of CF in rat colonic mucosa was
studied using the Ussing chamber method. Table 1 lists the
permeability coefficient (Papp) values for CF when various
concentrations of AEA were added to the mucosal solution.
Since CF is water soluble, CF could not readily permeate
through the isolated colonic mucosa in the absence of AEA.
The Papp values increased from the AEA concentration of
0.3 mM, but became saturated as the concentration increased
over 0.5 mM. Thus AEA caused the release of histamine from
mast cells (м0.4 mM) and enhanced the permeability of CF
in the colon mucosa (Ͼ0.1 mM) as shown in Fig. 1 and Table
1, respectively. Therefore the concentration of AEA was
fixed at 0.5 mM in all the following experiments.
Histamine release was expressed as a percentage of the release induced by 0.05%
Triton X-100. Each point represents the meanϮS.D. of five experiments. ᭺, AEA; ᭹,
compound 48/80 (comp 48/80).
Table 1. Effect of AEA on the Permeability of CF
Papp
(ϫ10Ϫ6 cm/s)
AEA Concentration
(mM)
0 (control)
0.05
0.1
0.3
0.5
3.691Ϯ0.992
3.922Ϯ3.501
5.100Ϯ2.561
7.925Ϯ2.035a)
11.593Ϯ3.308a)
11.950Ϯ2.503a)
12.073Ϯ3.020a)
0.7
1.0
Role of Histamine in the Permeation-Enhancing Effect
of AEA To clarify whether the permeation-enhancing ac-
tivity of AEA was related to the histamine stored in mucosal
mast cells, the influence of exogenous and endogenous hista-
mine on the CF permeability was studied in the presence of
0.01 mM of histamine and 0.1 mM of compound 48/80, re-
spectively. The results are shown in Fig. 2. The CF perme-
ability was increased approximately two-fold compared with
the control by the addition of exogenous histamine and com-
pound 48/80. Compound 48/80 at 0.1 mM released about
57% of the histamine stored in mucosal mast cells (Fig. 1)
and 0.01 mM of histamine added to the mucosal solution was
sufficiently in excess. However, Papp values following both
additions were almost equal to that upon the addition of
0.5 mM AEA, where only 13% of histamine was released
from mast cells (Fig. 1). The permeability of CF significantly
decreased in the colonic mucosa of rats pretreated with pyril-
amine as an antagonist of the H1 histamine receptor, with a
significant difference of pϽ0.001, even if histamine existed
in excess.
Each value represents the meanϮS.D. of five to seven rats. a) pϽ0.001 (vs. control).
Fig. 2. Effects of Exogenous and Endogenous Histamine on CF Perme-
ability in Isolated Rat Colonic Mucosa with and without in Vivo Pretreat-
ment with Pyrilamine
Pyrilamine (20 mg/kg) was administered intraperitoneally to the rats 10 min prior to
excision of the colonic segment. Black or white column represents CF permeability co-
efficients in colonic mucosa with or without pretreatment by pyrilamine, respectively.
* pϽ0.001, Papp with vs. without pretreatment with pyrilamine.
Figure 3 shows the enhancing effect of AEA on the CF
permeability in the isolated colonic mucosa of rats pretreated
with pyrilamine as an H1 receptor antagonist, cimetidine as
an H2 receptor antagonist, or ketotifen as a mast cell stabi-
lizer. These histamine inhibitors were administered intraperi-
toneally to the rats before excision of the colonic segment
and had no effect on the permeation of CF. In the colonic
mucosa of rats pretreated with pyrilamine and ketotifen, Papp
values in the presence of 0.5 mM AEA were significantly re-
duced to the levels of the control without AEA (pϽ0.001),
indicating that the permeation-enhancing activity of AEA
was inhibited by pyrilamine and ketotifen. On the other hand,
the enhancing activity of AEA was not influenced by cimeti-
dine, resulting in a much higher Papp in the presence vs. the
absence of 0.5 mM AEA (pϽ0.001). Thus the histamine re-
leased by AEA and bound to the H1 receptor plays an impor-
tant role in the permeation-enhancing actions of AEA.
Inhibition of Permeation-Enhancing Activity of AEA
To clarify the mechanism of action of AEA, the effects of
several inhibitors such as W7, H7, and compound 48/80 on
CF permeation were investigated. W7 is a calmodulin antag-
onist and H7 a protein kinase C (PKC) inhibitor. Compound
48/80 was used as a specific inhibitor of phospholipase C
(PLC) at low concentrations.8) Figure 4 shows the permeation
coefficients for CF when inhibitors were added to the mu-
cosal solution in the absence and presence of 0.5 mM AEA.

December 2002
1611
Fig. 5. Effect of AEA on Rm of the Rat Colonic Mucosa at pH 7.4 and
37 °C
AEA (0.5 mM) was added to the mucosal solution 30 min after the mounting of the
colonic mucoa (indicated by an arrow). Results are expressed as the meanϮS.D. of 11
experiments. Opened or closed circles represent the control or the sample in the pres-
ence of 0.5 mM AEA, respectively.
Fig. 3. Enhancing Effect of AEA on CF Permeability through Colonic
Mucosa of Rats Pretreated with Pyrilamine, Cimetidine, or Ketotifen
Pyrilamine (20 mg/kg) or cimetidine (100 mg/kg) was administered intraperitoneally
to the rats 10 min prior to excision of the colonic segment. Ketotifen (3.124 mg/kg) was
given intraperitoneally to the rats 30 min prior to the preparation of the colon. AEA
(0.5 mM) was added to the colonic mucosal solution. Black or white column represents
CF permeability coefficients in the presence or absence of AEA, respectively. Results
are expressed as the meanϮS.D. of 5 to 7 experiments. * pϽ0.001, Papp in the presence
spectively. When 0.5 mM AEA was added to the mucosal so-
lution at 30 min after the mounting of the tissue, PD and Isc
decreased slowly: PD, 3.5 mV to 2.0 mV; and Isc, 44 mA·
cmϪ2 to 28 mA·cmϪ2 in 40 min. Figure 5 shows the time
course of Rm after mounting the colonic mucosa. Rm also re-
mained nearly constant under the control conditions. After
the addition of 0.5 mM AEA to the mucosal solution, Rm was
immediately lowered by about 20—25 ohm·cmϪ2 and then
remained at about 70 ohm·cmϪ2.
†
vs. the absence of 0.5 mM AEA; pϽ0.001, Papp vs. the corresponding control in the
presence of 0.5 mM AEA.
The cytotoxicity of AEA in the colonic mucosa was evalu-
ated using the in situ loop method by measuring the LDH
crossing the rat colonic epithelium into the lumen for 1 h.
This method could measure LDH leakage with much higher
sensitivity than the Ussing chamber method,14) and the leak-
age of LDH was shown to be a useful marker of local toxic-
ity.15) LDH activity with 0.5 mM AEA (49.4Ϯ18.0 U/l/cm)
was not significantly different from that of the control
(43.2Ϯ7.7 U/l/cm). Since AEA had little potential to cause
toxicity at this concentration, the decrease in Rm after the ad-
dition of AEA shown in Fig. 5 might indicate a reduction in
paracellular resistance. Thus a transient opening of tight
junctions would seem less damaging than a disruption of the
cell membrane structure.
Fig. 4. Inhibitory Effects of W7, H7, and Compound 48/80 on Perme-
ation-enhancing Activity of AEA
AEA (0.5 mM) was added to the colonic mucosal solutin. Compound 48/80 (comp
48/80, 2.5 mg/ml), W7 (50 mM), or H7 (50 mM) was applied to the mucosal side of the
chamber, and the preparation was incubated for 20 min at 37 °C. Each column ex-
presses the meanϮS.D. of 5 to 7 experiments. * pϽ0.001, Papp in the presence vs. the
†
absence of 0.5 mM AEA; and pϽ0.01, Papp vs. the corresponding control without in-
hibitor.
In the absence of AEA, no effect of any inhibitor on CF per-
meability was observed. The value of Papp increased three-
fold compared with the control upon the addition of 0.5 mM
AEA, but was significantly reduced by H7 (pϽ0.01), but not
by W7 and compound 48/80. Accordingly, it was suggested
that PKC contributed to the permeation-enhancing effect of
AEA in the intestinal mucosa and that the AEA activity was
not influenced by the calmodulin antagonist or PLC inhibitor.
Effect of AEA on Electrophysiological Parameters of
Colonic Mucosa CF permeability increased in the rat
colonic mucosa as the AEA concentration increased. How-
ever, it is apparent that many, if not most, of the compounds
examined as membrane permeation enhancers in vitro often
cause cytotoxicity or membrane damage. Both the perme-
ability enhancement and cytotoxicity depended on the length
of exposure, and thus we assessed the changes in colonic mu-
cosa caused by AEA using the electrophysiological parame-
ters PD, Isc, and Rm. Under control conditions, PD and Isc
reached maximum values at 10 min after mounting the
colonic mucosa; this was followed by a slow decrease to
30 min and constant values of 3.5 mV and 38 mA·cmϪ2, re-
DISCUSSION
There are two permeation pathways for colorectal drug ab-
sorption: the transcellular pathway across the intestinal ep-
ithelial cell membrane and paracellular pathway to the lateral
intercellular space via tight junctions between epithelial
cells.16) The transcellular pathway is generally the principal
route for drugs with some degree of lipophilicity. On the
other hand, for water-soluble compounds or poorly perme-
able macromolecules such as polypeptides and proteins, the
paracellular pathway is considered an important absorption
route. The opening of the paracellular route is effective for
the absorption enhancement of such compounds. In recent
years, absorption enhancers have been actively investigated
and some of their actions and enhancing mechanisms in
transepithelial membranes have been clarified.17—19) Some
absorption enhancers have also been found in folk medicines
and health foods. It was reported that Glycyrrhizin (Grz), a
major constituent of licorice with steroid-like activity, pos-
sesses in vivo enhancing activity with respect to the nasal and

1612
Vol. 25, No. 12
rectal absorption of antibiotics, insulin, and calcitonin.20—22)
Imai et al. showed the mechanism of action of Grz in vivo
and in vitro.23)
with AEA might interact with the H1 receptor to activate
PKC, and further with cell membrane protein to widen tight
junctions, resulting in improved permeability. Further work is
in progress to measure DAG and the intracellular level of cal-
cium ions to elucidate the detailed intracellular mechanism
induced by AEA.
In conclusion, AEA is of potential use as an absorption
enhancer in colonic mucosa. The permeation-enhancing
mechanism is based on the widening of tight junctions par-
tially mediated via histamine, which binds to the H1 receptor
and activates PKC, without cytotoxicity in the colonic ep-
ithelium.
Previously, we studied the antiinflammatory effects of BA,
a component of aloes and related compounds, and found
AEA of the anaerobic product from BA to have histamine-re-
leasing activity in isolated rat mast cells.5) It has been known
that mast cell-derived inflammatory mediators such as hista-
mine, platelet-activating factor, and nitric oxide increase the
intestinal epithelial permeability of drugs and disrupt the
barrier function.6,7,24) It is also reported that histamine con-
tributes to the chemically induced hypermeability in intesti-
nal mucosa caused by absorption enhancers.25—27)
REFERENCES
First, to confirm the activity of AEA as an absorption en-
hancer, we investigated the effect of AEA on the permeabil-
ity of CF as a water-soluble and poorly permeable model
compound. AEA clearly enhanced the permeability of CF at
the low concentrations, as shown in Table 1, indicating that
AEA has an enhancing effect.
1) Okamura N., Asai M., Hine N., Yagi A., J. Chromatogr. A, 746, 225—
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2) Manitto P., Monti D., Speranza G., J. Chem. Soc. Perkin Trans. 1,
1990, 1297—1300 (1990).
3) Ishii Y., Tanizawa H., Takino Y., Yakugaku Zasshi, 108, 904—910
(1988).
Next, we investigated the role of histamine in the perme-
ability of CF produced by AEA. The enhanced permeability
of CF as a result of the treatment with AEA was inhibited by
an H1 receptor antagonist of pyrilamine and a mast cell stabi-
lizer of ketotifen, but not by an H2 receptor antagonist of
cimetidine (Figs. 2, 3). The result suggests that small
amounts of histamine released from mast cells play an im-
portant role in the permeation-enhancing effect of AEA. In
other words, AEA stimulated mast cells within the colonic
mucosa membrane to release histamine, which probably
bound to the H1 receptor, and resulted in the response.
4) Che Q.-M., Akao T., Hattori M., Tsuda Y., Kobashi K., Namba T.,
Planta Med., 57, 15—19 (1991).
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