沙漠研究 19-1, 383- 386 (2009 )Journal of Arid Land Studies“Desert Technology IX ”
Refereed Paper

Anti-allergic Effect of Acteoside Derived fromArid Land Plant Cistanche tubulosa
Rieko IIJIMA1, Parida YAMADA1, Junkyu HAN1, Hideyuki SHIGEMORI1, Hiroko ISODA1*
Abstract: The immediate-type allergic reaction is involved in many allergic diseases such asasthma, allergic rhinitis, and sinusitis. The discovery of drugs for the treatment ofimmediate-type allergic disease is a very important subject in human health. The three kindsof phenylethanoid glycoside, acteoside, echinacoside and cistanoside A, were extracted fromCistanche tubulosa which thrives in arid land. In this report, we investigated the effect ofacteoside, echinacoside and cistanoside A on the immediate-type allergic reaction, focusing on
the basophilic cell-mediated allergic reaction. Acteoside, echinacoside and cistanoside A
decreased β-hexosaminidase release from IgE-sensitized BSA-stimulated RBL-2H3 cells at the
antigen–antibody binding stage.
Keywords: β-hexosaminidase, Basophil cells, Cistanche tubulosa, Phenylethanoid glycosides

1. Introduction
Cistanche tubulosa (Schrenk) R. Wight is a perennial parasitic plant growing on the roots ofSalvadora or Calotropis species, and distributed in North Africa, Arabia, and Asian countries (Kobayashiet al., 1987). Cistanche tubulosa thrives in arid land where environment is too harsh for the most plants.
However, Cistanche tubulosa can thrive in such environment, so they are thought to have some potent
protective mechanisms against UV stress, lack of water and some environmental stress. Cistanchetubulosa cannot do photosynthesis, because they do not have roots and reefs, so they depend on nutrient
elements as the products of their host plants to grow up themselves. People living near the Takla Makan
Desert have who used Cistanche tubulosa as a traditional medical food are noted for the longer life span.
Cistanche tubulosa seems to have effects on cerebral nerve, immunity and skin aging.
Type I allergy is induced by certain types of antigens such as foods, dust, mites, medicines, cosmetics,moldspores, and pollen. This class of antigens induces the production of antigen-specific IgE antibodies
that bind to receptors on mast cells or basophilic cells. The early phase reaction in type I allergy occurs
within minutes and then mediators such as histamine and serotonin are released from the cell. Thesemediators induce vasodilation, mucous secretion, and bronchoconstriction. Histamine, which is released
from mast cells stimulated by antigen or degranulation inducers, is usually determined by using adegranulation marker in experiments on the immediate allergic reaction.
RBL-2H3 cells, a tumor analog of mast cells, display characteristics of mucosal-type mast cells andexpress several hundred thousand IgE receptors on the membrane surface. After sensitization with IgE,
the cells respond to the antigen and release histamine. β-hexosaminidase, which is stored in the secretory
granules of mast cells, is released concomitantly with histamine when mast cells are immunologicallyactivated (Schiwartz et al., 1981). Thus, the β-hexosaminidase activity in the medium is used as a markerof mast cell degranulation (Mastuda et al., 2002). RBL-2H3 cells are therefore considered as good toolfor studying the effect of unknown compounds on histamine release and β-hexosaminidase release activity.
In the course of our search for anti-allergic compounds against BSA-induced β-hexosaminidase
release from RBL-2H3 cells, we found that the BuOH extract of Cistanche tubulosa has an inhibitory
effect. By bio-activity-guided fractionation, we isolated three kinds of phenylethanoid glycosides, such asacteoside, echinacoside, and cistanoside A. Some of these phenylethanoid glycosides appear to havevarious biological activities, such as anti-inflammatory and anti-oxidant activity (Xiong et al., 2000,Sahpaz et al., 2002). And, acteoside as one of these compounds is contained in olive fruits.
There are no reports about anti-allergic effect of the three phenylethanoid glycosides derived from
Cistanche tubulosa on IgE sensitized BSA-stimulated RBL-2H3 cells and A23187 plus PMA stimulated
KU812 cells as type I allergy model.
1* Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki, Japan*
Corresponding author. Fax: +81 29 853 5776; E-mail address: isoda@sakura.cc.tsukuba.ac.jp (H. Isoda).

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2. Materials and Methods
2.1. Reagents
Dinitrophenylated bovine serum albumin (DNP-BSA) was purchased from Cosmo Biotechnology
Co.; anti-DNP-IgE, ketotifen, L-glutamine were purchased from Sigma (Sigma Aldrich Co., USA). Fetal
bovine serum (FBS) was purchased from Hyclone Co. Ltd. Eagle’s Minimum Essential Medium (MEM)
was purchased from Nissui Pharmaceutical Co., Ltd. RPMI medium 1640 was purchased from Gibco(Gibco Co., USA.). Calcium ionophore A23187 and phorbol 12-myristate 13-acetate (PMA) wereobtained from Sigma Japan and dissolved in DMSO at a concentration of 10 mM.
2.2. Cells and cell culture
RBL-2H3 cells were purchased from Riken Cell Bank, Japan. The cells were maintained in MEM
supplemented with 10% FBS and 2 mM L-glutamine, and incubated at 37 ˚C in a 5% CO2 incubator, while
KU812 cells were maintained in a RPMI 1640 medium supplemented with 10 % FBS at 37 ˚C in a 5 %CO2 incubator.
2.3. Plant Material
The stems of Cistanche tubulosa (Orobanchaceae) were purchased from Dojindo Pharmacy in Beijing.
2.4. Extraction and IsolationThe stems of Cistanche tubulosa (500 g) were extracted with MeOH (1.5 L), and evaporated todryness in vacuo at 30 ˚C. The MeOH extract was partitioned between EtOAc (1.0 L × 3) and H2O (1.0L) and then the H2O layer was partitioned with n-BuOH (1.0 L × 3). The n-BuOH-soluble portion (3.1 g)
was subjected to a ODS column (Cosmosil ODS, 2.2 × 30 cm, MeOH/H2O, 3:7→1:0) to separate twelve
fractions (CT-BU-1~12) including echinacoside (CT-BU-9, 25.4 mg). CT-BU-10 (491 mg) was applied toa silica gel column (2.2 × 30 cm, CHCl3/MeOH/H2O, 80:25:3) to afford thirteen fractions (CT-BU10-1~13) including cistanoside A (CT-BU-10-11, 86.2 mg) and CT-BU-10-9 was purified by a silica gelcolumn (2.2 × 30 cm, CHCl3/MeOH/H2O, 80:25:3) to give acteoside (12.1 mg).
2.5. β-hexosaminidase inhibition assay
The β-hexosaminidase release inhibition assay with RBL-2H3 cells was performed according to themethod described by Yamada (Yamada et al., 2008). Three phenylethanoid glycosides were added at theIgE sensitization stage. RBL-2H3 cells were seeded onto 96-well plates at 5.0 ×104 cells/well in 100 μl ofmedium. The cells were incubated and sensitized for 24 h at 37 ˚C with 0.3 μg/ml anti-DNP-IgE. Afterincubating the cells at 37 ˚C for 10 min in 60 μl per well of a releasing mixture containing 5 μl of threephenylethanoid glycosides (0.1, 1.0, 10.0 μg/ml), the cells were exposed to 5 μl per well of 4 μg/ml
DNP-BSA in PBS (-), before being incubated at 37 ˚C for 1 h.
2.6. MTT assay
MTT assay was performed according to the method described by Yamada (Yamada et al., 2007).
RBL-2H3 cells and KU812 cells were seed onto 96-well plate at 5.0 × 104 cells. And, then cells weretreated with 100 μl of medium contained three phenylethanoid glycosides (0.1-100.0 μg/ml) at 37 ˚C, 5%
CO2 incubator for 48 h.
3. Results and Discussion
Cistanche tubulosa (Schrenk)
R. Wight (shown in Figs. 1A and B)
is a perennial parasitic plant in arid
land of North Africa, Arabia, andAsian countries (Kobayashi et al.,1987). The environment of arid
land is too severe to grow up for themost plans. So, Cistanche tubulosa

Fig. 1. (A) Cistanche tubulosa (Kotb et al., 1985) (B) Chemical
structure of thee kinds of phenylethanoid glycosides.

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Fig. 2. Effects of phenylethanoid glycosides on β-hexosaminidase. (A) The model of Type I allergy. (B) Inhibitory
effect of three phenylethanoid glycosides from Cistanche tublosa on β-hexosaminidase release from RBL-2H3cells at the antigen-antibody binding stage.

Fig. 3. Cytotoxic effect of phenylethanoid glycosides from Cistanche tubulosa on KU812 cells and RBL-2H3 cells.
The percent cell viability was calculated relative to the untreated cells. (A) The cell viability of KU812 cells,(B) the cell viability of RBL-2H3 cells.

is considered to contain a lot of anti-oxidant compounds such as echinacoside, acteoside, and cistanoside A forprotection against the environmental stress. hese compounds are phenylethanoid glycosides, whose structuresare shown in Figure 1C.
3.1. Three phenylethanoid glycosides extracted from Chistanche tubulosa were screened for theirinhibitory effect on β-hexosaminidase release by RBL-2H3 cells.
We can perform to screen anti-allergic effects at three different stages. The first is antigen-antibody
binding stage (Fig. 2A), the second is antibody-receptor binding stage (Fig. 2B), and the last is calcium
influx (Fig. 2C).
This paper reports inhibitory effects of β-hexosaminidase release from IgE-sensitized BSA
stimulated RBL-2H3 cells at the antigen-antibody stage. The inhibition rate of β-hexosaminidase releasefrom RBL-2H3 as affected by three samples was shown in Figure 3B. In the present study, we compared
the effects of samples with the clinically available anti-allergic drug, ketotifen fumarate (Keto.), which is
known as a mast cell stabilizer, H1- receptor antagoist and eosinophil inhibitor (Harish et al., 2001).
The acteoside at 1.0 μg/ml concentration had the most potent inhibitory effect among otherphenylethanoid glycoside, tested followed by echinacoside and cistanoside A at the antigen–antibody
binding stage. It suggests that R1 site and R2 site should be hydrogen group to exert inhibitory effect on
β-hexosamindase release. Echinacoside has glucose group, and cistanoside A has not only glucose groupbut methyl group, instead of hydrogen group as acteoside.

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3.2 Cytotoxic effect of three phenylethanoid glycosides on RBL-2H3 and KU812 cells.
We used the MTT assay to assess the cytotoxicity of three phenylethanoid glycosides on RBL-2H3cells and KU812 cells. The cells were treated with samples for 48 h at final concentration of 0.1, 1.0,10.0, and 100.0 μg/ml. The results showed that three phenylethanoid glycosides did not have cytotoxicity
on both cells at 0.1-10.0 μg/ml. Echinacoside also did not show any cytotoxicity at 100.0 μg/ml on
KU812 cells. The other treatmented cells at 100 μg/ml showed around 90 % viability.
These results suggest that the reduction of β-hexosaminidase release in Figure 2B is not caused bycell death. Three kinds of phenylethanoid glycosides did not show cytotoxity, and then, these compounds
could inhibit β-hexosaminidase release.
4. Conclusions
In this study, we found that the three kinds of phenylethanoid glycoside extracted from Cistanchetubulosa have anti-allergic effect on BSA-stimulated RBL-2H3 cells. Although acteoside, cistanoside Aand echinacoside have inhibitory effect on β-hexosaminidase release, they have some differences in termsof effect and concentration. These differences might be caused from the difference in structure, so wewill elucidate the relationship between the structure and anti-allergic effect in the future.
Cytokines produced by basophilic cells, TNF-α, IL-4, IL-13, and IL-5 are key molecules for allergic
inflammation such as IgE production, Th2 differentiation, and allergic inflammation (Borish et al., 2003).
The reduction of pro-inflammatory cytokines from mast cells or basophil cells is one of the key indicatorsof reduced allergic symptom (Paul et al., 1993). So, the inhibitory effects of phenylethanoid glycosides
on the cytokine production should be investigated in the future.
These findings provide evidence that the three kinds of phenylethanoid glycosides extracted fromCistanche tubulosa could be a candidate as an anti-allergic agent. Mitogen-activated protein kinase(MAPKs) pathways are said to modulate cytokine production. The detailed mechanism of phenyethanoid
glycosides effects on signal pathways behind its anti-allergy effect as an anti-oxidant is the subject of afuture study.
Acknowledgement

This study was supported in part by funds from the Grant-in-Aid for Scientific Research (Grant No.
17255011).
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