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Immunomodulatory Effects of Gynostemma pentaphyllum Makino on Human Immune Cells

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Abstract

Use of herbs as health and/or dietary supplements has increased worldwide. The most common usage of botanicals is to improve the immune system. Water extract of Gynostemma pentaphyllum Makino was studied for its effects on lymphocyte proliferation and natural killer (NK) cell activity. Lymphocyte proliferation of normal peripheral blood mononuclear cells (PBMC) in response to G. pentaphyllum extract was increased at concentrations of (in ng/mL) 1, 10, 100, and (in µg/mL) 1, 5, 10 and 100. Aqueous extract of G. pentaphyllum reduced lymphocyte proliferation at 1 µg/mL, suggesting immunomodulating activities on human immunocompetent PBMC. INTRODUCTION Jiaogulan (Gynostemma pentaphyllum Makino; Cucurbitaceae) is a perennial climber, widely growing in China, Japan, Korea and Southeast Asia. It is used for treatment of inflammation, cough, hyperviscosity of sputums and chronic bronchitis (Jiang-Xu, 1979; Lin et al., 1993). Gypenosides, the total saponins with a dammarane-type basic structure (Piacente et al., 1995; Hu et al., 1996) isolated from jiaogulan, have been reported to decrease blood cholesterol, inhibit growth of tumor cells, heal peptic ulcer, relieve inflammation and pain and prevent platelet aggregation (Kimura et al, 1983; Li and Jin, 1989). Jiaogulan is also taken as tonic to improve the immune system. The effects and mechanisms of the activities on the immune system have not been well-defined. The objectives of this study were to investigate its capabilities on cell-mediated immune response (CMIR) by studying their effects on lymphocyte proliferation and natural killer (NK) cell activity of human peripheral blood mononuclear cells (PBMC).
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Immunomodulatory Effects of Gynostemma pentaphyllum Makino on
Human Immune Cells
Busarawan Sriwanthana, Weena Threesangsri
and Walailuk Wanavichet
National Institute of Health
Department of Medical Sciences
88/7 Soi Bamrasnaradura, Tivanond Rd.
Nonthaburi 11000
Thailand
Pranee Chavalittumrong, Jaree Bansiddhi
and Yenjit Techadamrongsin
Medicinal Plant Research Institute
Department of Medical Sciences
88/7 Soi Bamrasnaradura, Tivanond Rd.
Nonthaburi 11000
Thailand
Keywords: immunomodulator, jiaogulan, lymphocyte proliferation, NK cell activity
Abstract
Use of herbs as health and/or dietary supplements has increased worldwide.
The most common usage of botanicals is to improve the immune system. Water
extract of Gynostemma pentaphyllum Makino was studied for its effects on
lymphocyte proliferation and natural killer (NK) cell activity. Lymphocyte
proliferation of normal peripheral blood mononuclear cells (PBMC) in response to
G. pentaphyllum extract was increased at concentrations of (in ng/mL) 1, 10, 100,
and (in µg/mL) 1, 5, 10 and 100. Aqueous extract of G. pentaphyllum reduced
lymphocyte proliferation at 1 µg/mL, suggesting immunomodulating activities on
human immunocompetent PBMC.
INTRODUCTION
Jiaogulan (Gynostemma pentaphyllum Makino; Cucurbitaceae) is a perennial
climber, widely growing in China, Japan, Korea and Southeast Asia. It is used for
treatment of inflammation, cough, hyperviscosity of sputums and chronic bronchitis
(Jiang-Xu, 1979; Lin et al., 1993). Gypenosides, the total saponins with a dammarane-
type basic structure (Piacente et al., 1995; Hu et al., 1996) isolated from jiaogulan, have
been reported to decrease blood cholesterol, inhibit growth of tumor cells, heal peptic
ulcer, relieve inflammation and pain and prevent platelet aggregation (Kimura et al, 1983;
Li and Jin, 1989). Jiaogulan is also taken as tonic to improve the immune system. The
effects and mechanisms of the activities on the immune system have not been well-
defined. The objectives of this study were to investigate its capabilities on cell-mediated
immune response (CMIR) by studying their effects on lymphocyte proliferation and
natural killer (NK) cell activity of human peripheral blood mononuclear cells (PBMC).
MATERIALS AND METHODS
Preparation of Jiaogulan Aqueous Extract
Aerial parts of jiaogulan were collected from Chiang Mai, northern Thailand.
Voucher specimen of jiaogulan (Bandsiddhi 43-21) was deposited at the Botanical
section, Medicinal plant research institute, Department of medical sciences. Botanical
identification was confirmed (Backer et al., 1963; Wu et al., 1983) and compared with
authentic specimens at two herbaria, the Bangkok Herbarium (BK), Department of
agriculture and the forest herbarium (BKF), Royal forest department, Ministry of
agriculture and cooperative, Thailand. The plant was washed thoroughly, cut into
segments, oven-dried at 40°C and ground. Fifteen grams of dried and ground aerial parts
of jiaogulan were extracted with distilled water for 2 h using a reflux method. Filtrate was
collected and residues further extracted with distilled water for 2 h. Filtrates collected
from both extractions were pooled and dried under vacuum in a rotary evaporator. The
amount of dried extract obtained was 3.79 g. The dried extract was dissolved in distilled
water to make a stock concentration of 10 mg/mL, filtered and refrigerated until use.
Proc. WOCMAP III, Vol.6: Traditional Medicine & Nutraceuticals
Eds. U.R. Palaniswamy, L.E. Craker and Z.E. Gardner
Acta Hort. 680, ISHS 2005
166
Subjects
A total of 52 healthy Thai donors of the National blood bank, The Thai Red Cross
society were recruited in this study. They were 20-50 y old, and none had a history of
hepatitis B infection, nor had a risk for HIV-1 exposure.
Preparation of Mononuclear Cells
Mononuclear cells were separated from heparinized blood using Ficoll-Hypaque
density gradient (Boyum, 1968). The mononuclear cells were counted and adjusted to an
appropriate concentration in complete RPMI 1640 (RPMI 1640 medium supplemented
with 2 mM glutamine, 10 mM HEPES, 100 U/mL penicillin G, and 100 µg/mL
streptomycin) containing 10% fetal bovine serum (FBS; Grand Island Biological
Company, Grand Island, NY, USA) for further assays.
Lymphoproliferation Assay
Lymphocyte proliferative response was done as described earlier (Sriwanthana
and Chavalittumrong, 2001). Purified mononuclear cells (2x106 cells/mL) were cultured
in triplicates in 96-well microtiter plates (Costar, Cambridge, MA, USA) with the extract
at final concentrations of (in ng/mL) 1, 10, 100, and (in µg/mL) 1, 5, 10 and 100 in
complete RPMI 1640 containing 10% FBS. The cultures were incubated at 37°C with 5%
CO2 for 72 h. Lymphocyte proliferation was determined by uptaking of 3H-thymidine at
18 h before harvesting. The radioactivity was measured by a liquid scintillation counter
(Topcount Microplate Scintillation and Luminescence Counter, Packard Instrumental Co.,
CT, USA). The degree of activation was expressed as a stimulation index [S.I., the ratio
of the 3H-thymidine uptake in count per minute (CPM) of samples with extract to those
without extract]. Phytohemagglutinin HA16/17 (Murex Diagnostics Limited, Dartford,
England) at 2 µg/mL was added to the culture system to check for cell survival.
NK Cell Activity Assay
PBMC (2x106 cells/mL) were incubated in the presence or the absence of
jiaogulan extract at the final concentrations of 10 ng/mL, 100 ng/mL, 1 µg/mL, 5 µg/mL,
and 10 µg/mL at 37°C for 18 h. After incubation, the cultures were washed and then used
as effector cells for the assay of NK cell activity. K 562 cells were used as target cells and
were grown in complete RPMI 1640 containing 10% FBS. The target cells (2x106 cells)
were labeled with 100 µCi of Na251CrO4 (specific activity 37.0 MBq/µg: Amersham,
Buckinghamshire, UK) at 37°C, 5% CO2 for 60 min, washed 3 times with RPMI 1640
containing 10% FBS.
Cytotoxicity assay was performed as described earlier (Sriwanthana and
Chavalittumrong, 2001). In brief, 2x103 target cells/well and a PBMC effector-to-target
cell ratios (E:T) of 90:1, 30:1, 10:1, and 3:1 were set up in triplicate in 96-well round-
bottom microtiter plates (Corning Incorporated, Corning, NY, USA). The plates were
incubated for 4 h at 37°C with 5% CO2. After incubation, supernatants from each well
(100 µL) were transferred into tubes and counted in a Gamma counter (Cobra Series
Gamma Counter Systems, Packard Instrumental Co., CT, USA). The percentage of
cytolysis was calculated as: %cytolysis = (experimental release - spontaneous release) /
(maximal release - spontaneous release). Spontaneous release was measured by
incubation of target cells with medium alone, while maximal release was measured by
lysis of target cells with 5% Triton X-100. NK cell activity was expressed as lytic units
(LU)/107 PBMCs as determined by least squares analysis derived from the percentage of
specific lysis of all E:T ratios. One LU was defined as the number of effector cells
required for 20% specific lysis of 1x104 target cells.
Statistical Analysis
Data were expressed as mean ± SE and compared using Student’s paired t-test.
167
RESULTS
Lymphocyte Proliferation
The extract-induced proliferative responses were examined from 52 PBMC in
cultures containing jiaogulan. Similar patterns of blastogenesis of lymphocytes were
found in responses to jiaogulan. The responses were elevated with the water extract of
jiaogulan ranging from concentrations of 1 ng/mL to 100 µg/mL (P < 0.05) (Table 1).
NK Cell Activity
Reduction in NK activity was observed in PBMC treated with 1 µg/mL of
jiaogulan extract compared with untreated ones (P = 0.02) (Table 2).
DISCUSSION
Humoral and cellular immunity are defense mechanisms against foreign bodies.
Botanicals are suggested to act primarily on cellular rather than humoral immune
responses. Several medicinal plants are traditionally believed to promote health by their
immunomodulatory activities. Our studies were designed to investigate the in vitro effects
of jiaogulan on lymphocyte proliferation and NK cell activity of human PBMC.
The increase in lymphocyte proliferation, quantified as stimulation index (S.I.),
was demonstrated in a dose-dependent response at concentrations of 1 ng/mL to 1 µg/mL.
The response decreased slightly at concentrations over 1 µg/mL, which may be due to
amounts of active constituents in the extract or other factors induced during lymphocyte
blastogenesis.
NK cells are known to play an important role as one of the first lines of host
defense mechanisms against a variety of infections and cancers. We measured the effect
of the aqueous extracts of jiaogulan on NK cell activity of normal PBMC, in 13 donors.
Our result showed a reduction in the function of NK cells at 1 µg/mL. The reduction in
NK activity was not due to the toxicity of jiaogulan, because the viability of PBMC was
greater than 95% in the presence or the absence of the extract (data not shown). In
contrast, the NK activity at other concentrations revealed no difference compared to the
control. Jiaogulan extract at 1 µg/mL may induce some soluble factors that could decrease
the activity.
It has been reported that gypenosides, saponins isolated from jiaogulan, enhanced
T and B lymphocyte proliferation and interleukin-2 (IL-2) production of splenocytes in
both normal and immunosuppressed mice (Li and Xing, 1992). Gypenosides also
demonstrated their effect on humoral and cellular immunocompetence in γ-irradiated
mice (Chen et al., 1996). Moreover, it was found to possess antioxidant effect in human
and murine phagocytes (Li et al., 1993). Our findings showed similar results of aqueous
extract on human immune cells. Enhancement of lymphocyte proliferation, may be due to
the effect of gypenosides.
Our study suggested that aqueous extracts of jiaogulan possess
immunomodulatory activities on human PBMC. Chronic toxicity of the extract in Wistar
rats performed indicated no toxicity at concentrations as high as 750 mg/kg/d jiaogulan
(P. Chavalittumrong, pers. commun., 2002). Further studies to evaluate safety and
efficacy of the extract in humans are needed to use for benefits of both
immunocompetence and immunocompromised, as those with HIV-infection or cancers.
ACKNOWLEDGEMENTS
We acknowledge staffs of the National blood bank, The Thai Red Cross society
for providing blood from their regular donors. We also thank staffs in phytochemistry and
toxicology units at Medicinal plant research institute for their help.
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169
Tables
Table 1. Effect of jiaogulan (Gynostemma pentaphyllum) on lymphocyte proliferation of
normal PBMC (n=52).
Extract concentrations Stimulation Index (S.I.)
(Mean ± SE)
Control 1.00 ± 0.00 NS
1 ng/mL 1.41 ± 0.08 *
10 ng/mL 1.61 ± 0.12 *
100 ng/mL 2.01 ± 0.15 *
1 µg/mL 2.86 ± 0.20 *
5 µg/mL 2.36 ± 0.15 *
10 µg/mL 2.14 ± 0.13 *
100 µg/mL 2.45 ± 0.23 *
PHA 2 µg/mL 120.17 ± 14.1 *
NS Not significant, * Significant at p < 0.05
Table 2. NK cell activity by normal PBMC in the presence of jiaogulan (Gynostemma
pentaphyllum) extract (n=13).
Extract concentrations Lytic units
(Mean ± SE)
Control 85.16 ± 18.04 NS
10 ng/mL 84.67 ± 17.33NS
100 ng/mL 76.81 ± 14.71 NS
1 µg/mL 52.54 ± 9.89 *
5 µg/mL 66.13 ± 14.51 NS
10 µg/mL 71.63 ± 10.92 NS
NS Not significant, * Significant at p < 0.05
... The extract was prepared as previously described (Sriwanthana et al., 2005). Briefly, aerial parts of G. pentaphyllum were washed thoroughly, cut into segments of appropriate size, dried in the oven at 40 O C and ground. ...
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