Research Note
J Ginseng Res Vol. 36, No. 4, 369-374 (2012)
http://dx.doi.org/10.5142/jgr.2012.36.4.369

Inhibitory Effects of Ginsenoside Re Isolated from Ginseng Berry onHistamine and Cytokine Release in Human Mast Cells and Human
Alveolar Epithelial Cells
Hye Min Bae1, Ok Sun Cho1, Shin Jung Kim2, Byung Ok Im3, Soon Hyun Cho4, Sena Lee5,Myung-Gyou Kim5, Kyung Tack Kim6, Kang Hyun Leem5*, and Sung Kwon Ko1*
1

Department of Oriental Medical Food & Nutrition, Semyung University, Jecheon 390-711, Korea
College of Pharmacy, Sookmyung Women’s University, Seoul 140-742, Korea
3Department of Natural Medicine Resources, Semyung University, Jecheon 390-711, Korea
4Department of Pharmacy & Food, Daewon University College, Jecheon 390-702, Korea
5College of Oriental Medicine, Semyung University, Jecheon 390-711, Korea
6Processing Technology Research Group, Korea Food Research Institute, Seongnam 463-746, Korea
2The berry of Panax ginseng significantly inhibited the histamine releases at the concentration of 30 μg/mL (p<0.05) and 10μg/mL (p<0.01). The ginsenoside Re from ginseng berry was found out to have a potent effect in the experiment of histamin andcytokine release.
Keywords: Panax ginseng, Ginseng berry, Ginsenoside Re, Histamine, Cytokine

The root of ginseng (Panax ginseng Meyer) has been
traditionally used for medicine and food stuff. The primary physiologically active substances of ginseng are
ginsenosides, polyacetylenes, ginseng proteins, polysaccharides, and phenolic compounds, etc. [1-4]. Ginsenosides, in particular, have been noticed as the principal
effective component of ginseng showing various biochemical and pharmacological efficacies. A number ofresearchers have studied the components of ginseng since
the late 1960s starting with the research of Shibata [5,6],
whose research group identified the chemical structuresof ginsenoside. The physiological activities of these ginsenosides have been reported to show anti-cancer effects[7], anti-diabetic effects [8], protection of the central

nervous system [9], anti-arteriosclerotic and hypertensive
effects [10,11], improvement of liver function and clearing of hangovers [12], anti-fatigue and anti-stress effects[13,14], anti-oxidative qualities [15], anti-inflammatory
effects [16], promotion of protein synthesis [17], andstrengthening the immune system [18]. To examine
and identify the efficacies of ginsenosides as mentioned
above, a number of biochemical and pharmacologicalstudies have been conducted. Many researchers are still
eagerly trying to discover new efficacies of ginsenoside.
Until recently, most of the ginseng research has focused on the components of the ginseng root [19-21].
However, in addition to the ginseng root, the componentsof the ginseng berry and their pharmacological activities

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial
use, distribution, and reproduction in any medium, provided the original
work is properly cited.

Received 04 Jul. 2012, Revised 24 Aug. 2012, Accepted 24 Aug. 2012
*Corresponding authors

E-mail: lkh@semyung.ac.kr
Tel: +82-43-649-1341, Fax: +82-43-649-1341
E-mail: skko@semyung.ac.kr
Tel: +82-43-649-1433, Fax: +82-43-649-1759ⓒ The Korean Society of Ginseng369

http://ginsengres.org pISSN: 1226-8453 eISSN: 2093-4947

J Ginseng Res Vol. 36, No. 4, 369-374 (2012)15 min, histamine levels of the supernatants were measured. Specific processes of histamine assay were as follows. NaOH (1M) and o-phthaldialdehyde (1 mg/mL inEtOH) were added to all wells. After 4 min, fluorescentmeasurements were made using a Synergy HT (Bio-Tek
Instruments Inc., Winooski, VT, USA).
In order to determine release of inflammatory cytokines on human alveolar basal epithelial cells (A549) bytreatment with ginseng berry, cytokines were measuredusing the RayBio Human Cytokine Antibody Array kit(RayBiotech, Norcross, GA, USA) according to theprotocol of the manufacturer. In brief, A549 cells wasseeded into a 6 well plate at a density of 2×105 cells/welland incubated at 37°C for 24 h. After then, ginseng berryor ginsenoside Re with lipopolysaccharide (LPS) wastreated and incubated for 24 h, and the cellular proteins
were extracted from the cells using a protein extract buffer. Array membranes were incubated with blocking buffer at room temperature for 30 min to block membranes
and blocking buffer was decanted and 1 mL of each protein was added each membrane. After 2 h, each protein
was decanted and membranes were washed 5 times withwash buffer I and II. And then, biotin-conjugated anticytokines were diluted with blocking buffer and wereadded and incubated to each membrane for 2 h. Membranes were washed 5 times and incubated with 1,000
fold diluted horseradish peroxidase-conjugated streptavidin for 2 h and then washed 5 times. Enhanced chemiluminescence buffer was added to each membrane and thesignals were detected using Fusion chemiluminescenceimaging system and analyzed by Bio1D software (VilberLourmat, Marne La Vallee, France).
The results were expressed as mean±SD. The data

have also been reported such as: anti-diabetic [22-24],anti-cancer [25,26], anti-oxidant, anti-aging [27-30], andanti-stress [31], etc. As the interest in the ginseng berry
has increased recently, many studies have been carriedout. However, the research on the anti-allergic effects ofginseng berries has not been performed. Therefore, thepresent study investigated the influence on anti-histamine
releasing activity of the ginseng berry (except the seeds
from the berry), and the active components were purified
and identified as ginsenoside Re.
The berry of a 4-year cultivated ginseng plant wascollected at Kimjae (cultivated by Gil Kim) in Korea on
July 14, 2007. Images of the ginseng berry are shown inFig. 1. These specimens were stored at the Oriental Medical Food Research Laboratory of Semyung University.
Dried ginseng berries (5 kg) were ground to powder andextracted twice with 1 L of 95% ethyl alcohol for 2 h ina water bath (60°C). The extracts were concentrated by avacuum evaporator (Eyela Co., Tokyo, Japan).
In order to determine the effects of ginseng berries onhistamine release, the human leukemic mast cells (HMC1) were used. The HMC-1 was cultured in Iscove’s
modified Dulbecco’s media containing 10% bovine serum albumin, 2 mM L-glutamine, 100 IU/mL penicillin,
and 50 μg/mL streptomycin. The cells were cultured at
37°C in a 5% CO2 with 95% humidity and were passaged every 2 to 3 d. HMC-1 was subcultured in a 24
well plate and stabilized for 24 h. They were divided
into six groups; vehicle-treated blank, control, 30, 10, 3,and 1 μg/mL ginseng berry-treated groups. After 30 min,every well except the blank group was treated with compound 48/80 (1 mg/mL). The same volume of distilledwater was treated in the blank wells. After incubation forFig. 1. Photograph of ginseng berry (A) and histamine content of cultured supernatants of human mast cells (B). Blank group was not incubatedwith compound 48/80, and other experimental groups were incubated with compound 48/80 (*p<0.05, **p<0.01 vs. control).

http://dx.doi.org/10.5142/jgr.2012.36.4.369

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Fig. 2. Microphotographs of degranulation in human mast cells. (A) An unstimulated mast cell. (B) The cell has been activated to secrete its
stored histamine by compound 48/80. (C) The extracts of ginseng berry (30 μg/mL) prevented the histamine secretion induced by compound48/80.

Fig. 3. Chemical structures of compound I (A) and histamine content of cultured supernatants of human mast cells (B). Blank group was not incubated with compound 48/80, however, other experimental groups were incubated with compound 48/80 (*p<0.05, **p<0.01 vs. control).

was analyzed by one-way ANOVA, followed by Tukey’s
post-hoc analysis using SPSS (SPSS Inc., Chicago, IL,USA). Differences were considered significant at p<0.05.
Toluidine blue (pH 5.0) staining was performed toobserve the cytotoxicity of tested materials, because cell
death including apoptosis and necrosis could increase
histamine concentration. The degranulations in HMC-1
stimulated with compound 48/80 were observed morphologically after 0.05% toluidine staining (Fig. 2B) whereas
not stimulated HMC-1 showed the darker staining ofgranules in cytoplasm (Fig. 2A). The extract of ginseng
berry prevented the degranulation of HMC-1 (Fig. 2C).
As shown in Fig. 1B, the histamine secretion in blankgroup was 3.0±0.2 μg/mL and it was increased by compound 48/80 treatment to 19.9±3.1 μg/mL in the control
group. The compound 48/80-induced histamine secretions were reduced by the ginseng berry treatment ina dose-dependent manner. The histamine secretions at
the concentrations of 30, 1, 3, 10, and 30 μg/mL were13.6±0.3, 15.2±1.4, 11.1±1.7, and 6.6±0.7 μg/mL, respectively (Fig. 1B), with the statistical significances at
30 μg/mL (p<0.01) and 10 μg/mL (p<0.05).

Ninety-five percent EtOH extract of ginseng berry(1,080 g) was dissolved in distilled water and it waspartitioned 10 times with diethyl ether resulting in 45 gof diethyl ether fraction and 998 g of water fraction. The
water fraction (998 g) was suspended in distilled water
and was adsorbed in a Diaion HP-20 (Mitsubishi Chemical Co., Tokyo, Japan) ion exchange resin column. Thirty
percent MeOH fraction, 50% MeOH fraction, 70%
MeOH fraction, and 100% MeOH fraction were eluted
in the order named. The 30% MeOH fraction (10 g) was
then subjected to an octadecyl silane (12 nm S-75 um;
YMC ODS-A, YMC Co., Kyoto, Japan) gel column bygradient elution with 30% to 100% MeOH and resulted
in 4 subfractions (F1-F4). The F3 subfraction (1.2 g) thatexerted the highest inhibitory effect on histamine release
was rechromatographed on silica gel column with amixture of the solvents (CHCl3:MeOH:H2O=70:30:4 v/v) and 0.6 g of compound I was isolated and identified
as ginsenoside Re by the spectroscopic methods of 1HNMR, 13C-NMR, and FAB-MS (Fig. 3A). The isolated
ginsenoside Re treatment at concentrations of 1, 3, 10,
and 30 μM significantly reduced histamine secretion371

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Fig. 4. (A-D) Cytokine array photographs and quantitatively analyzing graphs. A549 cells were incubated lipopolysaccharide (LPS) and/or eachsample (*p<0.05 vs. LPS treated control). RANTES, regulated and normal T cell expressed and secreted.

exhibiting 49.6±4.8%, 47.2±5.3%, 51.3±4.5%, and
79.0±1.2%, respectively (p<0.05) (Fig. 3B).
The content of ginsenoside Re in crude extract was
found out via HPLC analysis (5.9%). The ginsenosidecontent in 30 μg of crude extract is 1.7 μg, accordingly.

http://dx.doi.org/10.5142/jgr.2012.36.4.369

As the molecular weight of ginsenoside Re is 947.14, themolar concentration of ginsenoside Re (1.7 μg/mL, thecontent in 30 μg/mL of crude extract) is 1.8 μM. The effect of 1 and 3 μM of ginsenoside Re could significantly
reduce the histamine secretion. Therefore ginsenoside372

Bae et al. Ginsenoside Re and Allergy

Re might be thought to be a main effective component ofginseng berry in the present histamine secreting experiment.
In cytokine assay experiments, the most effective dosages of ginseng berry and ginsenoside Re were determined to be 30 μg/mL and 30 μM, respectively. Because
ginsenoside Re is a component of ginseng berry extract(5.9%), 1.7 μg/mL ginsenoside Re treated group was also
tested to find out the role of ginsenoside Re in total crude
extract.
The IL-1α, IL-8, IL-10, and regulated and normal T
cell expressed and secreted (RANTES) secretion wereincreased by LPS treatment (Fig. 4). Ginseng berry extract could reduce the IL-1α, IL-8, IL-10, and RANTES
secretion induced by LPS treatment. Similarly, ginsenoside Re (1.7 μg/mL, the amount in 30 μg of ginseng
berry extract) could reduce the IL-1α, IL-8, IL-10, andRANTES secretion. In the experiments of IL-8 and IL10, ginsenoside Re (30 μM) showed the effects in a dosedependent manner.
As results of the present study, ginsenoside Re wasthought to be a main effective component of ginseng berry and the effects of ginsenoside Re were more powerfulin higher dosages. The health promoting product having
higher content of ginsenoside Re might be a health functional food.

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