J Nat Med DOI 10.1007/s11418-014-0841-0
NATURAL RESOURCE LETTER
Inhibitory effect of selected medicinal plants on the release of pro-inflammatory cytokines in lipopolysaccharide-stimulated human peripheral blood mononuclear cells Emil Salim • Endang Kumolosasi • Ibrahim Jantan
Received: 27 February 2014 / Accepted: 11 April 2014 Ó The Japanese Society of Pharmacognosy and Springer Japan 2014
Abstract The inhibitory activities of the methanol extracts from 20 selected medicinal plants on the release of pro-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs) were evaluated. The major compound from the most active plant extract was also investigated. The inhibitory effect of the methanol extracts on the release of pro-inflammatory cytokines was tested by incubating PBMCs with the sample and then stimulating by lipopolysaccharide at 0.1 lg/ml. The level of cytokines was determined using enzyme-linked immunosorbent assay. Among the extracts tested, Andrographis paniculata extract demonstrated the strongest inhibition of interleukin (IL)-1b, IL-1a, and IL-6 release, with IC50 values of 1.54, 1.06, and 0.74 lg/ml, respectively. The IC50 value of A. paniculata extract was significantly higher than that of andrographolide on IL-1a, IL-1b, and IL-6 (p \ 0.001) release. The IC50 values of andrographolide for IL-1a, IL1b, and IL-6 were significantly higher (p \ 0.001) than that of dexamethasone. Cymbopogon citratus and Zingiber officinale strongly inhibited the release of IL-1b, with IC50 values of 3.22 and 3.17 lg/ml, respectively. To our knowledge, this is the first report that A. paniculata extract and its major compound andrographolide strongly inhibited the release of IL-1a, whereas previous studies only showed their inhibitory effect on the release of another IL-1 family member, IL-1b. The results show that these extracts and this compound have potential effects as anti-inflammatory agents by inhibiting the release of pro-inflammatory cytokines.
E. Salim E. Kumolosasi (&) I. Jantan Faculty of Pharmacy, Drug and Herbal Research Center, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia e-mail: [email protected]
Keywords Tumor necrosis factor-a Interleukin-1a Interleukin-1b Interleukin-6 Interleukin-8
Introduction Inflammation is a process of the complex biological response of various tissues to harmful stimuli and ultimately leads to the restoration of normal tissue structure and function [1]. Acute inflammation is an initial response of the body characterized by redness, swelling, pain, and heat and is one of the most important host defense mechanisms against invading pathogens, whereas chronic inflammation is an unwanted persistent state that can induce the development of inflammatory diseases [2]. Lipopolysaccharide (LPS) from Gram-negative bacteria is well known to cause bacterial sepsis mediated through the activation of monocytes, neutrophils, and macrophages [3]. The stimulation of these cells may induce oversecretion of various pro-inflammatory cytokines, eicosanoids, and nitric oxide (NO) [4, 5]. Tumor necrosis factor-a (TNF-a), interleukin (IL)-1b, IL-1a, IL-6, and IL-8 are pro-inflammatory cytokines which, despite being different proteins transcribed from diverse genes, share essential roles in acute inflammation [6]. TNF-a plays a key role in many autoimmune diseases and stimulates secretion of other inflammatory cytokines [7, 8]. IL-1 plays important roles in cell proliferation and differentiation and in pyrexia. Meanwhile, IL-6 has a role in the acute response and induces increasing antibody production through activation of lymphocytes, cell differentiation into plasma cells, and immunoglobulin production, and IL-8 recruits and retains neutrophils at the site of inflammation [9]. However, excessive and prolonged inflammatory response contributes to many inflammatory diseases, such as rheumatoid arthritis [10], cancer [11], metabolic disease [12], atherosclerosis
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[13], obesity, and cardiovascular disease [14]. Thus, blocking pro-inflammatory cytokines which mediate inflammation is a useful target in the development of new antiinflammatory drugs. Anti-inflammatory drugs such as steroids or NSAIDs have a number of adverse side effects, such as gastrointestinal discomfort, inhibition of platelet aggregation, and kidney toxicity [15], and so novel anti-inflammatory agents with fewer side effects are needed. There is considerable research interest in the identification of new anti-inflammatory agents from plants used in traditional medicine which have been reported to have anti-inflammatory activity. This study was carried out to investigate the inhibitory effect of 20 plant extracts and the major compound from the most active plant extract on the release of proinflammatory cytokines. For this purpose, human peripheral blood mononuclear cells (PBMCs) were incubated with plant extracts and then stimulated by LPS from Salmonella enteritica. The results demonstrated that methanol extracts of some plants and andrographolide, the major compound of the most active extract, have inhibitory effects on the release of pro-inflammatory cytokines.
Materials and methods Chemicals Methanol and andrographolide were purchased from Merck (Darmstadt, Germany), Lymphoprep from Axis-Shield PoC AS (Oslo, Norway), Trypan blue solution (0.4 %), RPMI1640 medium with L-glutamine, newborn calf serum, HEPES, and LPS of S. enteritica from Sigma-Aldrich Chemicals (St. Louis, MO, USA), and penicillin streptomycin solution from PAA (Pasching, Austria). Dexamethasone phosphate was obtained from Duopharma (M) Sdn Bhd (Selangor, Malaysia). Human TNF-a, IL-1a, IL-1b, and IL-6 ELISA kits were purchased from Cayman Chemicals (Ann Arbor, MI, USA) and that for IL-8 from Abnova (Taipei, Taiwan). Cytokine level measurements were carried out on a microplate reader (BioTek Instruments, Winooski, VT, USA). Plant materials The plants were collected from different parts of peninsular Malaysia between December 2010 and June 2011 and were identified, and the voucher specimens are kept in the Herbarium of Universiti Kebangsaan Malaysia, Bangi, Malaysia (Table 1).
methanol (3 9 500 ml, 24 h each) until exhaustion. The methanol extracts were concentrated by rotary evaporation to obtain various yields of crude extracts, calculated based on dry weight (Table 1). Phytochemical study Phytochemical study was carried out on extracts which had the strongest inhibition of the release of pro-inflammatory cytokines. HPLC analysis was performed on a LC-20 AT (Shimadzu) using DiscoveryÒ HSC18 (Supelco), column 5 lm, 2.5 cm 9 4.6 mm. Peaks were analyzed at 223 nm using a SPD-M20A Diode Array Detector (Shimadzu). The samples were run using an Autosampler SIL-20A (Shimadzu) with a UV–visible-light detector (SPD-M10AVP, Shimadzu) with isocratic mobile phases: acetonitrile:0.1 % phosphoric acid in water (4:6, v/v); flow rate: 1.0 ml/min. The repeatability of the method was evaluated by injecting the solution of extract and standard solution three times. Study subjects The healthy volunteers (n = 3, C18 years old) who participated in this study fulfilled the following inclusion criteria: non-smoker, fasted overnight, and did not take any medicine or supplements. Written informed consent to participate in the study was obtained from the participants. The experimental protocol for studies in humans was approved by the Human Ethical Committee of Universiti Kebangsaan Malaysia (approval No. UKM 1.5.3.5/244/NF040-2011) and followed the principles outlined in the Declaration of Helsinki [16]. Isolation of PBMCs Venous blood was collected in heparinized tubes and processed immediately. PBMCs were isolated by centrifugation on Lymphoprep at 600g for 20 min at 18–20 °C. Cells were then washed twice in RPMI-1640 at 300g for 10 min at 4 °C. PBMCs were resuspended in RPMI-1640 media with L-glutamine supplemented with 10 % heat inactivated newborn calf serum, 10 mM HEPES, and 100 U/ml penicillin and 100 lg/ml streptomycin in culture tubes. The cells were adjusted to 5 9 105 cells/ml, counted using a hemocytometer and observed under light microscopy. Cell viability
Preparation of plant extracts One hundred grams of each plant material were air-dried at room temperature (26 ± 2 °C), ground, and extracted with
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Cell viability was determined by the standard Trypan blue exclusion method. The PBMCs (5 9 105/ml) were incubated with plant extracts, each in triplicate, at 37 °C with
J Nat Med Table 1 Samples used in this study Sample no.
Scientific names
Family
Part used
Voucher no.
% Yielda
1
Ageratum conyzoides L.
Compositae
Leaf
UKM-B-29871
16.07
2
Andrographis paniculata Nees
Acanthaceae
Whole plant
UKM-B-29795
19.70
3
Averrhoa bilimbi L.
Oxalidaceae
Fruit
UKM-B-29792
19.15
4
Boesenbergia pandurata Schltr.
Zingiberaceae
Rhizome
UKM-B-29963
10.98
5
Carica papaya L.
Caricaceae
Leaf
UKM-B-29964
15.79
6
Centella asiatica (L.) Urb.
Umbelliferae
Whole plant
UKM-B-29965
10.00
7
Curcuma domestica Valeton
Zingiberaceae
Rhizome
UKM-B-29966
9.39
8
Curcuma mangga Valeton & Zijp
Zingiberaceae
Rhizome
UKM-B-29796
15.00
9
Curcuma xanthorrhiza D.Dietr.
Zingiberaceae
Rhizome
UKM-B-29967
13.66
10
Cymbopogon citratus Stapf
Graminae
Leaf
UKM-B-29968
15.15
11 12
Euphorbia hirta L. Kaempferia galanga L.
Euphorbiaceae Zingiberaceae
Whole plant Rhizome
UKM-B-29969 UKM-B-29970
9.40 16.90
13
Leucaena glauca Benth.
Leguminosae
Leaf
UKM-B-29971
13.25
14
Morinda citrifolia L.
Rubiaceae
Fruit
UKM-B-29972
24.00
15
Orthosiphon aristatus (Blume) Miq.
Lamiaceae
Whole plant
UKM-B-29770
10.20
16
Peperomia pellucida (L.) Kunth
Piperaceae
Whole plant
UKM-B-29973
2.90
17
Phyllanthus amarus Schumach.
Euphorbiaceae
Leaf
UKM-B-29872
18.64
18
Psidium guajava L.
Myrtaceae
Leaf
UKM-B-29974
17.31
19
Tinospora crispa (L.) Hook.f. & Thomson
Menispermaceae
Stem
UKM-B-29975
7.78
20
Zingiber officinale Roscoe
Zingiberaceae
Rhizome
UKM-B-29976
11.56
21
Andrographolide
–
–
–
22
Dexamethasone
–
–
–
a
Based on dry weight
Immunoassay for TNF-a, IL-1a, IL-1b, IL-6, and IL-8
5 % CO2 for 27 h. The final concentrations of the samples in the mixtures were 10 and 5 lg/ml. The blue dye uptake was an indication of cell death. The percentage viability was calculated from the total cell count.
ELISA was used for measuring TNF-a, IL-1a, IL-1b, IL-6, and IL-8 according to the manufacturer’s instructions.
Cytokine assay
Statistical analysis
PBMCs were pre-incubated with control or test compounds for 3 h at 37 °C with 5 % CO2. Dexamethasone and complete medium were used as positive and negative control, respectively. After the pre-incubation period, samples were induced by 0.1 lg/ml LPS to release cytokines. The final concentrations of the samples in the mixtures were 5, 2.5, 1.25, and 0.625 lg/ml. The final concentration of DMSO in the mixture was fixed at 0.5 % to avoid interference with the cells. The final concentrations of dexamethasone and the active compound from the most active plant in the mixtures were 5, 0.5, 0.05, and 0.005 lg/ml. Samples were harvested at different time intervals (12 h for TNF-a and IL-1b; 20 h for IL-6; and 24 h for IL-1a and IL-8) after stimulation by LPS. Cellfree supernatants were obtained by centrifugation at 300g and 4 °C for 10 min and stored at -80 °C prior to use.
All statistical analyses were performed using SPSSÒ Statistic 20. One-way analysis of variance followed by a posthoc test (Tukey’s multiple comparison) were used when significant differences at p \ 0.05 were present. Cytokine determination by ELISA was performed in duplicate, and data were obtained from three different donors. Results are expressed as mean ± standard error of the mean of three experiments. The IC50 values were calculated using GraphPad Prism v.6.01 software.
Results and discussion The cell viability test was carried out to evaluate the cytotoxicity of plant extracts (Table 1) on PBMCs. All extracts tested at concentrations of 5 lg/ml were viable ([90 %) after 27 h incubation. This concentration was
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J Nat Med Table 2 Percentage inhibition (%) of selected medicinal plant extracts, andrographolide, and dexamethasone at concentrations of 5 lg/ml on the release of cytokines
Percentage inhibition values are presented as mean ± SEM of three independent experiments performed in duplicate. Percentage inhibition [8 % was significant at p \ 0.05 when compared with negative control #
p [ 0.05 was considered not significant compared with dexamethasone
a
Dexamethasone as a positive control
Sample no.
TNF-a
IL-1b
IL-6
IL-8
1
8.2 ± 1.1
34.3 ± 3.01
14.1 ± 2.1
20.9 ± 4.7
4.1 ± 0.5
2
36.4 ± 0.9
78.2 ± 1.41#
91.9 ± 3.8#
85.1 ± 2.6#
14.8 ± 0.5
3
5.0 ± 2.4
16.2 ± 3.84
20.6 ± 4.6
6.6 ± 1.2
4.4 ± 2.3
4
8.4 ± 1.1
6.9 ± 0.69
10.5 ± 1.1
32.9 ± 1.5
10.7 ± 0.9
5
10.8 ± 1.1
12.5 ± 3.94
27.4 ± 1.2
42.9 ± 1.1
8.4 ± 2.4
6
5.5 ± 1.3
23.7 ± 4.57
23.9 ± 1.0
19.7 ± 3.0
6.0 ± 1.9
7
16.5 ± 0.7
37.3 ± 3.34
41.4 ± 3.8
9.7 ± 2.8
11.9 ± 1.5
8
10.0 ± 0.5
15.4 ± 1.58
6.4 ± 1.0
13.5 ± 2.5
12.2 ± 0.6
9
13.4 ± 1.3
38.2 ± 2.70
24.9 ± 5.0
18.3 ± 3.8
11.5 ± 2.1
10
17.3 ± 0.6
42.4 ± 3.37
63.2 ± 3.4
18.1 ± 1.2
6.7 ± 0.6
11
17.0 ± 1.5
14.7 ± 3.34
32.2 ± 1.0
3.8 ± 0.8
4.9 ± 0.5
12
12.1 ± 1.3
34.5 ± 6.54
29.3 ± 0.9
9.8 ± 1.1
10.6 ± 1.2
13
1.8 ± 0.6
34.2 ± 5.12
2.6 ± 0.9
23.0 ± 3.2
7.7 ± 2.7
14
3.0 ± 0.6
7.0 ± 1.79
5.6 ± 0.9
6.8 ± 0.4
4.4 ± 1.4
15 16
1.3 ± 0.3 3.9 ± 0.8
36.3 ± 3.15 29.5 ± 2.31
6.5 ± 1.7 18.5 ± 5.4
6.1 ± 0.5 19.6 ± 1.2
10.0 ± 1.0 6.4 ± 1.0
17
2.6 ± 0.3
31.7 ± 2.84
7.2 ± 1.4
27.1 ± 1.7
9.9 ± 3.9
18
5.7 ± 0.3
14.8 ± 0.99
6.7 ± 0.9
30.1 ± 3.0
5.5 ± 1.0
19
4.1 ± 0.5
27.0 ± 6.91
12.9 ± 3.4
30.0 ± 0.6
6.7 ± 2.3
20
5.5 ± 0.7
43.3 ± 4.11
63.5 ± 3.9
32.4 ± 0.8
9.7 ± 3.2
21
28.0 ± 4.1
82.3 ± 1.0#
86.4 ± 1.0#
54.8 ± 2.2
18.4 ± 1.2
57.6 ± 3.9
83.3 ± 3.56
90.5 ± 1.3
85.1 ± 0.6
32.5 ± 1.4
a
22
used as the highest concentration in this experiment. Among the samples tested, A. paniculata extract (no. 2) and its major compound andrographolide (no. 21) at 5 lg/ ml were the strongest inhibitors of the release of TNF-a, with percentage inhibition values of 36.4 ± 0.9 and 28.0 ± 4.1 % (Table 2), significantly lower than dexamethasone (no. 22) (p \ 0.001) with a percentage inhibition value of 57.6 ± 3.9 %. These results were in agreement with those found by Wangchuck et al. [17]. It was reported that dexamethasone at 1 lg/ml inhibited the release of TNF-a with percentage inhibition of 58 % in LPS-activated THP-1 monocytic cells. The results also showed that A. paniculata extract (2) and andrographolide (21) at 5 lg/ml strongly inhibited the release of IL-1a, with percentage inhibition values of 78.2 ± 1.41 and 82.3 ± 1.00 %, respectively (Table 2). The results also showed that A. paniculata extract and andrographolide have no significant difference when compared to dexamethasone (22) (p [ 0.1). The data indicated that A. paniculata (2), C. citratus (10), Z. officinale (20), and andrographolide (21) at 5 lg/ml strongly inhibited the release of IL-1b, with percentage inhibition values of 91.88 ± 3.79, 63.15 ± 3.45, 63.48 ± 3.95, and 86.4 ± 1.0 %, respectively (Table 2). Among these plants, A. paniculata extract and its major compound andrographolide have no significant difference from dexamethasone (22), with a percentage inhibition value of 90.5 ± 1.3 %
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IL-1a
(p [ 0.1). Among the samples tested, A. paniculata (2) at 5 lg/ml was the only extract which strongly inhibited the release of IL-6, with a percentage inhibition value of 85.08 ± 2.55 % (Table 2). This extract had no significant difference from dexamethasone (22) (p = 1), whereas andrographolide (21) showed lower inhibition than dexamethasone (p \ 0.001. The results showed that no extract could inhibit the release of IL-8 by more than 50 % at 5 lg/ml (Table 2). Dexamethasone (22) as positive control was the strongest inhibitor, but still inhibited IL-8 by less than 50 %. The results showed that the inhibitory effect of dexamethasone on IL-6 (85.06 %) was greater than that of IL-8 (32.54 %). This was similar to a previous study carried out by Lu et al. [18] in corneal fibroblasts, which reported that the inhibitory effect of dexamethasone on IL6 was greater than that of IL-8, with percentage inhibition values of 83 and 50 %, respectively. The difficulty of these samples in inhibiting IL-8 may be due to the fact that in spite of stimulation by LPS, IL-8 was also stimulated by TNF-a, IL-1a, and IL-1b which are produced by LPSstimulated PBMCs [19, 20]. This may have caused the abundant and continuous release of IL-8. Furthermore, the extracts which have inhibition greater than 50 % were applied to the cells in different concentrations. A. paniculata extract strongly inhibited the release of IL-1a, IL-1b, and IL-6 in a dose-dependent manner, with IC50 values of 1.54, 1.06, and 0.74 lg/ml, respectively
J Nat Med Table 3 IC50 values (lg/ml) of samples which have percentage inhibition [50 % on cytokine release Plant name
TNF-a
IL-1a
IL-1b
IL-6
Andrographis paniculata Nees
nd
1.54 ± 0.07*
1.06 ± 0.04*
0.74 ± 0.07*
Curcuma domestica Valeton
nd
nd
4.91 ± 0.11
nd
Cymbopogon citratus Stapf
nd
nd
3.22 ± 0.33
nd
Zingiber officinale Roscoe
nd
nd
3.17 ± 0.26
nd
Andrographolide
nd
0.12 ± 0.06
0.53 ± 0.06
0.20 ± 0.06
Dexamethasone
0.87 ± 0.20
0.0004 ± 0.00004
0.12 ± 0.01
0.05 ± 0.01
Andrographolide (lM)
nd
0.34 ± 0.17#
1.52 ± 0.18#
0.56 ± 0.15#
Dexamethasone (lM)
1.69 ± 0.38
0.0008 ± 0.00007
0.24 ± 0.02
0.09 ± 0.02
* Significantly different (p \ 0.05) compared to andrographolide #
Significantly different (p \ 0.001) compared to dexamethasone
nd not determined (none of the tested doses exceeded 50 % inhibition). IC50 for IL-8 could not be determined
(Table 3). Some extracts such as C. citratus and Z. officinale strongly inhibited the release of IL-1b but not TNF-a, IL-1a, and IL-6. A previous study has shown that regulation of IL-1b production is through mitogen-activated protein kinase kinase 3 (MKK3) which in turn activates p38 mitogen-activated protein kinase (MAPK), but this did not occur with other cytokines, such as TNF-a [21]. Thus, the differential inhibition effect on these cytokines may be related to differential activity of the extracts on this pathway. It may indicate that these extracts inhibited these cytokines by different mechanisms. The results also showed that C. citratus inhibited the release of IL-1b in a dose-dependent manner, with an IC50 value of 3.22 lg/ml (Table 3). Francisco et al. [22] demonstrated that a C. citratus leaf fraction strongly inhibited iNOS expression, NO production, p38 MAPK, c-jun-Nterminal kinase (JNK) 1/2, and nuclear factor-kappaB (NF-jB) signaling pathways in murine macrophages, suggesting its anti-inflammatory activity. Regarding its antiinflammatory effect, Lee et al. [23] reported that C. citratus inhibited the LPS-induced activation of NF-jB in RAW 264.7 cells by inhibiting the phosphorylation of inhibitory jB (IjB) kinase-b (IKKb). Taken together, C. citratus inhibition of IL-1b may be related to the inhibition of p38 MAPK and IKKb. The results indicated that Z. officinale extract inhibited the release of IL-1b in a dose-dependent manner, with an IC50 value of 3.17 lg/ml (Table 3). The results were supported by previous studies which reported the inhibition effect of Z. officinale hexane fraction on IL-1b in LPSstimulated BV2 microglial cells [24] and its suppressive effect on the expression of pro-inflammatory genes [25]. Additionally, an in vitro study showed that Z. officinale extract suppresses pro-inflammatory cytokines and chemokines produced by synoviocytes, chondrocytes, and leukocytes due to arthritis [26]. Phytochemical studies indicated that the plant is rich in a large number of
substances, including gingerols and shogaols. These compounds show various biological activities such as antioxidant, anti-inflammatory, and anticarcinogenic properties [27]. A phytochemical study was carried out on A. paniculata extract, which had the strongest inhibition of the release of pro-inflammatory cytokines. Figure 1 shows that the methanol extract of A. paniculata contained andrographolide with a retention time of 5.004 min. This is very close to the retention time of standard andrographolide, with a value of 5.125 min. The content of andrographolide in the extract was 304 ± 9.13 mg/g extract. Andrographolide, shown in the HPLC chromatogram to be the major compound, was also investigated and the results indicated that andrographolide strongly inhibited the release of IL-1a, IL-1b, and IL-6 in a dose-dependent manner, with IC50 values of 0.12, 0.53, and 0.20 lg/ml, respectively (Table 3). These results are in agreement with those of Sheeja et al. [28], who found that the levels of proinflammatory cytokines such as IL-1b and IL-6 were effectively reduced by the administration of extract and andrographolide in metastatic tumor-bearing mice. Furthermore, Bao et al. [29] reported that andrographolide inhibited NF-jB activity at the level of IKKb activation in ovalbumin-induced allergic lung disease and human bronchial epithelial cells. Specifically, andrographolide inhibited phosphorylation of IKKb, which in turn inhibited p65 and p50 translocation to the nucleus, an initial process for gene expression of several cytokines [29]. NF-jB is known as a major transcription factor for regulating the expressions of pro-inflammatory cytokines [30]. Taken together, inhibition of pro-inflammatory cytokines in the results may have the same mechanism. The results also showed that the IC50 of A. paniculata extract was significantly higher than that of andrographolide on IL-1a, IL-1b, and IL-6 (p \ 0.02) (Table 2). To our knowledge, this is the first report that A. paniculata extract and its major compound
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J Nat Med Fig. 1 HPLC chromatograms of a A. paniculata extract and b andrographolide (Rt = 5.125 min)
andrographolide strongly inhibited the release of IL-1a. Previous studies have only showed their inhibitory effect on the release of another member of the IL-1 family, IL-1b [28, 31, 32]. Although IL-1a and IL-1b have identical activities, they are different in several ways. For example, studies with knockout mice indicate that IL-1a is essential for priming T cells during contact hypersensitivity and for the stimulation of high levels of serum immunoglobulin E following immunization with ovalbumin, whereas IL-1b, which can circulate to the brain, is essential for the induction of fever [33]. In conclusion, A. paniculata extract and its major compound andrographolide strongly inhibited the release of IL-1a, IL-1b, and IL-6, while C. citratus and Z. officinale strongly inhibited the release of IL-1b. The research findings provide evidence that A. paniculata, C. citratus, and Z. officinale extracts and andrographolide have the potential to inhibit pro-inflammatory cytokines. Inhibition of pro-inflammatory cytokines by these extracts and this compound indicate their ability to reduce inflammation
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reactions and provide further evidence that these plants may have potent anti-inflammatory properties. Acknowledgments This study was supported by the grant from Science Fund, Ministry of Agriculture Malaysia, No. 05-01-02SF1022.
References 1. Kumar V, Abbas AK, Fausto N, Mitchell R (2007) Robbins basic pathology, 8th edn. Elsevier Saunders, Philadelphia 2. Shailasree S, Ruma K, Kini KR, Niranjana SR, Prakash HS (2012) Potential anti-inflammatory bioactives from medicinal plants of Western Ghats, India. Pharmacogn Commun 2(2):2–12 3. Aderem A, Ulevitch RJ (2000) Toll-like receptors in the induction of the innate immune response. Nature 406(6797):782–787 4. Saad B, Abouatta BS, Basha W, Hmade A, Kmail A, Khasib S, Said O (2011) Hypericum triquetrifolium-derived factors downregulate the production levels of LPS-induced nitric oxide and tumor necrosis factor-a in THP-1 cells. Evid Based Complement Alternat Med 2011:1–7 5. Nantel F, Denis D, Gordon R, Northey A, Cirino M, Metters KM, Chan CC (1999) Distribution and regulation of cyclooxygenase-2
J Nat Med
6. 7.
8.
9. 10.
11.
12. 13.
14. 15.
16.
17.
18.
19.
20. 21.
in carrageenan-induced inflammation. Br J Pharmacol 128(4): 853–859 Feghali CA, Wright TM (1997) Cytokines in acute and chronic inflammation. Front Biosci 2:12–26 Beutler B, Cerami A (1989) The biology of cachectin/TNF–a primary mediator of the host response. Annu Rev Immunol 7:625–655 Delgado AV, McManus AT, Chambers JP (2003) Production of tumor necrosis factor-alpha, interleukin-1beta, interleukin-2, and interleukin-6 by rat leukocyte subpopulations after exposure to substance P. Neuropeptides 37(6):355–361 Tayal V, Kalra BS (2008) Cytokines and anti-cytokines as therapeutics: an update. Eur J Pharmacol 579:1–12 Metsios GS, Stavropoulos-Kalinoglou A, Sandoo A, van Zanten JJ, Toms TE, John H, Kitas GD (2010) Vascular function and inflammation in rheumatoid arthritis: the role of physical activity. Open Cardiovasc Med J 4(1):89–96 Mantovani A, Garlanda C, Allavena P (2010) Molecular pathways and targets in cancer-related inflammation. Ann Med 42(3):161–170 Hotamisligil GS (2010) Endoplasmic reticulum stress and the inflammatory basis of metabolic disease. Cell 140(6):900–917 Frostegard J (2010) Rheumatic diseases: insights into inflammation and atherosclerosis. Arterioscler Thromb Vasc Biol 30(5):892–893 Mathieu P, Lemieux I, Despres JP (2010) Obesity, inflammation, and cardiovascular risk. Clin Pharmacol Ther 87(4):407–416 Batlouni M (2010) Nonsteroidal anti-inflammatory drugs: cardiovascular, cerebrovascular and renal effects. Arq Bras Cardiol 94(4):522–563 World Medical Association (2008) Declaration of Helsinki: ethical principles for medical research involving human subjects (http://www.wma.net/en/30publications/10policies/b3/index.html) Wangchuk P, Keller PA, Pyne SG, Taweechotipatr M (2013) Inhibition of TNF-a production in LPS-activated THP-1 monocytic cells by the crude extracts of seven Bhutanese medicinal plants. J Ethnopharmacol 148(3):1013–1017 Lu Y, Liu Y, Fukuda K, Nakamura Y, Kumagai N, Nishida, T (2006) Inhibition by triptolide of chemokine, proinflammatory cytokine, and adhesion molecule expression induced by lipopolysaccharide in corneal fibroblasts. Invest Ophthalmol Vis Sci 47(9):3796–3800 Arbabi S, Rosengart MR, Garcia I, Jelacic S, Maier RV (1999) Priming interleukin 8 production: role of platelet-activating factor and p38. Arch Surg 134:1348–1353 Baggiolini M, Clark-Lewis I (1992) Interleukin-8, a chemotactic and inflammatory cytokine. FEBS 307(1):97–101 Wysk M, Yang DD, Lu HT, Flavell RA, Davis RJ (1999) Requirement of mitogen-activated protein kinase kinase 3 (MKK3) for tumor necrosis factor-induced cytokine expression. Proc Natl Acad Sci USA 96:3763–3768
22. Francisco V, Figuerinha A, Neves BM, Rodriguez CG, Lopes MC, Cruz MT, Batista MT (2011) Cymbopogon citratus as source of new and safe anti-inflammatory drugs: bio-guided assay using lipopolysaccharide-stimulated macrophages. J Ethnopharmacol 133:818–827 23. Lee HJ, Jeong HS, Kim DJ, Noh YH, Yuk DY, Hong JT (2007) Inhibitory effect of citral on NO production by suppression of iNOS expression and NF-jB activation in RAW 264.7 cells. Arch Pharm Res 31:342–349 24. Jung HW, Yoon CH, Park KM, Han HS, Park YK (2009) Hexane fraction of Zingiberis Rhizoma Crudus extract inhibits the production of nitric oxide and pro-inflammatory cytokines in LPSstimulated BV2 microglial cells via the NF-jB pathway. Food Chem Toxicol 47:1190–1197 25. Kim HW, Murakami A, Abe M, Ozawa Y, Morimitsu Y, Williams MV, Ohigashi H (2005) Suppressive effects of mioga ginger and ginger constituents on reactive oxygen and nitrogen species generation, and the expression of inducible pro-inflammatory genes in macrophages. Antioxid Redox Signal 7:1621– 1629 26. Phan PV, Sohrabi A, Polotsky A, Hungerford DS, Lindmark L, Frondoza CG (2005) Ginger extract components suppress induction of chemokine expression in human synoviocytes. J Altern Complement Med 11:149–154 27. Aggarwal BB, Prasad S, Reuter S, Kannapan R, Yadev VR, Park B, Kim JH, Gupta SC, Phromnoi K, Sundaram C, Prasad S, Chaturvedi MM, Sung B (2011) Identification of novel antiinflammatory agents from ayurvedic medicine for prevention of chronic diseases: ‘‘reverse pharmacology’’ and ‘‘bedside to bench’’ approach. Curr Drug Targets 12(11):1595–1653 28. Sheeja K, Kuttan G (2010) Andrographis paniculata downregulates pro-inflammatory cytokine production and augments cell mediated immune response in metastatic tumor-bearing mice. Asian Pac J Cancer Prev 11:723–729 29. Bao Z, Guan S, Cheng C, Wu S, Wong SH, Kemeny DM, Leung BP, Wong WS (2009) A novel antiinflammatory role for andrographolide in asthma via inhibition of the nuclear factor-kB pathway. Am J Respir Crit Care Med 179:657–665 30. Huang SS, Chiu CS, Chen HJ, Hou WC, Sheu MJ, Lin YC, Shie PH, Huang GJ (2011) Antinociceptive activities and the mechanisms of anti-inflammation of Asiatic acid in mice. Evid Based Complement Alternat Med 2011:1–10 31. Zhu T (2013) Andrographolide protects against LPS-induced acute lung injury by inactivation of NF-jB. PLoS One 8(2):1–12 32. Shen T (2013) AP-1/IRF-3 targeted anti-inflammatory activity of andrographolide isolated from Andrographis paniculata. Evid Based Complement Alternat Med 2013:1–16 33. Sims JE, Smith DE (2010) The IL-1 family: regulators of immunity. Nat Rev Immunol 10(2):89–102
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NATURAL RESOURCE LETTER
Inhibitory effect of selected medicinal plants on the release of pro-inflammatory cytokines in lipopolysaccharide-stimulated human peripheral blood mononuclear cells Emil Salim • Endang Kumolosasi • Ibrahim Jantan
Received: 27 February 2014 / Accepted: 11 April 2014 Ó The Japanese Society of Pharmacognosy and Springer Japan 2014
Abstract The inhibitory activities of the methanol extracts from 20 selected medicinal plants on the release of pro-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs) were evaluated. The major compound from the most active plant extract was also investigated. The inhibitory effect of the methanol extracts on the release of pro-inflammatory cytokines was tested by incubating PBMCs with the sample and then stimulating by lipopolysaccharide at 0.1 lg/ml. The level of cytokines was determined using enzyme-linked immunosorbent assay. Among the extracts tested, Andrographis paniculata extract demonstrated the strongest inhibition of interleukin (IL)-1b, IL-1a, and IL-6 release, with IC50 values of 1.54, 1.06, and 0.74 lg/ml, respectively. The IC50 value of A. paniculata extract was significantly higher than that of andrographolide on IL-1a, IL-1b, and IL-6 (p \ 0.001) release. The IC50 values of andrographolide for IL-1a, IL1b, and IL-6 were significantly higher (p \ 0.001) than that of dexamethasone. Cymbopogon citratus and Zingiber officinale strongly inhibited the release of IL-1b, with IC50 values of 3.22 and 3.17 lg/ml, respectively. To our knowledge, this is the first report that A. paniculata extract and its major compound andrographolide strongly inhibited the release of IL-1a, whereas previous studies only showed their inhibitory effect on the release of another IL-1 family member, IL-1b. The results show that these extracts and this compound have potential effects as anti-inflammatory agents by inhibiting the release of pro-inflammatory cytokines.
E. Salim E. Kumolosasi (&) I. Jantan Faculty of Pharmacy, Drug and Herbal Research Center, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia e-mail: [email protected]
Keywords Tumor necrosis factor-a Interleukin-1a Interleukin-1b Interleukin-6 Interleukin-8
Introduction Inflammation is a process of the complex biological response of various tissues to harmful stimuli and ultimately leads to the restoration of normal tissue structure and function [1]. Acute inflammation is an initial response of the body characterized by redness, swelling, pain, and heat and is one of the most important host defense mechanisms against invading pathogens, whereas chronic inflammation is an unwanted persistent state that can induce the development of inflammatory diseases [2]. Lipopolysaccharide (LPS) from Gram-negative bacteria is well known to cause bacterial sepsis mediated through the activation of monocytes, neutrophils, and macrophages [3]. The stimulation of these cells may induce oversecretion of various pro-inflammatory cytokines, eicosanoids, and nitric oxide (NO) [4, 5]. Tumor necrosis factor-a (TNF-a), interleukin (IL)-1b, IL-1a, IL-6, and IL-8 are pro-inflammatory cytokines which, despite being different proteins transcribed from diverse genes, share essential roles in acute inflammation [6]. TNF-a plays a key role in many autoimmune diseases and stimulates secretion of other inflammatory cytokines [7, 8]. IL-1 plays important roles in cell proliferation and differentiation and in pyrexia. Meanwhile, IL-6 has a role in the acute response and induces increasing antibody production through activation of lymphocytes, cell differentiation into plasma cells, and immunoglobulin production, and IL-8 recruits and retains neutrophils at the site of inflammation [9]. However, excessive and prolonged inflammatory response contributes to many inflammatory diseases, such as rheumatoid arthritis [10], cancer [11], metabolic disease [12], atherosclerosis
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[13], obesity, and cardiovascular disease [14]. Thus, blocking pro-inflammatory cytokines which mediate inflammation is a useful target in the development of new antiinflammatory drugs. Anti-inflammatory drugs such as steroids or NSAIDs have a number of adverse side effects, such as gastrointestinal discomfort, inhibition of platelet aggregation, and kidney toxicity [15], and so novel anti-inflammatory agents with fewer side effects are needed. There is considerable research interest in the identification of new anti-inflammatory agents from plants used in traditional medicine which have been reported to have anti-inflammatory activity. This study was carried out to investigate the inhibitory effect of 20 plant extracts and the major compound from the most active plant extract on the release of proinflammatory cytokines. For this purpose, human peripheral blood mononuclear cells (PBMCs) were incubated with plant extracts and then stimulated by LPS from Salmonella enteritica. The results demonstrated that methanol extracts of some plants and andrographolide, the major compound of the most active extract, have inhibitory effects on the release of pro-inflammatory cytokines.
Materials and methods Chemicals Methanol and andrographolide were purchased from Merck (Darmstadt, Germany), Lymphoprep from Axis-Shield PoC AS (Oslo, Norway), Trypan blue solution (0.4 %), RPMI1640 medium with L-glutamine, newborn calf serum, HEPES, and LPS of S. enteritica from Sigma-Aldrich Chemicals (St. Louis, MO, USA), and penicillin streptomycin solution from PAA (Pasching, Austria). Dexamethasone phosphate was obtained from Duopharma (M) Sdn Bhd (Selangor, Malaysia). Human TNF-a, IL-1a, IL-1b, and IL-6 ELISA kits were purchased from Cayman Chemicals (Ann Arbor, MI, USA) and that for IL-8 from Abnova (Taipei, Taiwan). Cytokine level measurements were carried out on a microplate reader (BioTek Instruments, Winooski, VT, USA). Plant materials The plants were collected from different parts of peninsular Malaysia between December 2010 and June 2011 and were identified, and the voucher specimens are kept in the Herbarium of Universiti Kebangsaan Malaysia, Bangi, Malaysia (Table 1).
methanol (3 9 500 ml, 24 h each) until exhaustion. The methanol extracts were concentrated by rotary evaporation to obtain various yields of crude extracts, calculated based on dry weight (Table 1). Phytochemical study Phytochemical study was carried out on extracts which had the strongest inhibition of the release of pro-inflammatory cytokines. HPLC analysis was performed on a LC-20 AT (Shimadzu) using DiscoveryÒ HSC18 (Supelco), column 5 lm, 2.5 cm 9 4.6 mm. Peaks were analyzed at 223 nm using a SPD-M20A Diode Array Detector (Shimadzu). The samples were run using an Autosampler SIL-20A (Shimadzu) with a UV–visible-light detector (SPD-M10AVP, Shimadzu) with isocratic mobile phases: acetonitrile:0.1 % phosphoric acid in water (4:6, v/v); flow rate: 1.0 ml/min. The repeatability of the method was evaluated by injecting the solution of extract and standard solution three times. Study subjects The healthy volunteers (n = 3, C18 years old) who participated in this study fulfilled the following inclusion criteria: non-smoker, fasted overnight, and did not take any medicine or supplements. Written informed consent to participate in the study was obtained from the participants. The experimental protocol for studies in humans was approved by the Human Ethical Committee of Universiti Kebangsaan Malaysia (approval No. UKM 1.5.3.5/244/NF040-2011) and followed the principles outlined in the Declaration of Helsinki [16]. Isolation of PBMCs Venous blood was collected in heparinized tubes and processed immediately. PBMCs were isolated by centrifugation on Lymphoprep at 600g for 20 min at 18–20 °C. Cells were then washed twice in RPMI-1640 at 300g for 10 min at 4 °C. PBMCs were resuspended in RPMI-1640 media with L-glutamine supplemented with 10 % heat inactivated newborn calf serum, 10 mM HEPES, and 100 U/ml penicillin and 100 lg/ml streptomycin in culture tubes. The cells were adjusted to 5 9 105 cells/ml, counted using a hemocytometer and observed under light microscopy. Cell viability
Preparation of plant extracts One hundred grams of each plant material were air-dried at room temperature (26 ± 2 °C), ground, and extracted with
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Cell viability was determined by the standard Trypan blue exclusion method. The PBMCs (5 9 105/ml) were incubated with plant extracts, each in triplicate, at 37 °C with
J Nat Med Table 1 Samples used in this study Sample no.
Scientific names
Family
Part used
Voucher no.
% Yielda
1
Ageratum conyzoides L.
Compositae
Leaf
UKM-B-29871
16.07
2
Andrographis paniculata Nees
Acanthaceae
Whole plant
UKM-B-29795
19.70
3
Averrhoa bilimbi L.
Oxalidaceae
Fruit
UKM-B-29792
19.15
4
Boesenbergia pandurata Schltr.
Zingiberaceae
Rhizome
UKM-B-29963
10.98
5
Carica papaya L.
Caricaceae
Leaf
UKM-B-29964
15.79
6
Centella asiatica (L.) Urb.
Umbelliferae
Whole plant
UKM-B-29965
10.00
7
Curcuma domestica Valeton
Zingiberaceae
Rhizome
UKM-B-29966
9.39
8
Curcuma mangga Valeton & Zijp
Zingiberaceae
Rhizome
UKM-B-29796
15.00
9
Curcuma xanthorrhiza D.Dietr.
Zingiberaceae
Rhizome
UKM-B-29967
13.66
10
Cymbopogon citratus Stapf
Graminae
Leaf
UKM-B-29968
15.15
11 12
Euphorbia hirta L. Kaempferia galanga L.
Euphorbiaceae Zingiberaceae
Whole plant Rhizome
UKM-B-29969 UKM-B-29970
9.40 16.90
13
Leucaena glauca Benth.
Leguminosae
Leaf
UKM-B-29971
13.25
14
Morinda citrifolia L.
Rubiaceae
Fruit
UKM-B-29972
24.00
15
Orthosiphon aristatus (Blume) Miq.
Lamiaceae
Whole plant
UKM-B-29770
10.20
16
Peperomia pellucida (L.) Kunth
Piperaceae
Whole plant
UKM-B-29973
2.90
17
Phyllanthus amarus Schumach.
Euphorbiaceae
Leaf
UKM-B-29872
18.64
18
Psidium guajava L.
Myrtaceae
Leaf
UKM-B-29974
17.31
19
Tinospora crispa (L.) Hook.f. & Thomson
Menispermaceae
Stem
UKM-B-29975
7.78
20
Zingiber officinale Roscoe
Zingiberaceae
Rhizome
UKM-B-29976
11.56
21
Andrographolide
–
–
–
22
Dexamethasone
–
–
–
a
Based on dry weight
Immunoassay for TNF-a, IL-1a, IL-1b, IL-6, and IL-8
5 % CO2 for 27 h. The final concentrations of the samples in the mixtures were 10 and 5 lg/ml. The blue dye uptake was an indication of cell death. The percentage viability was calculated from the total cell count.
ELISA was used for measuring TNF-a, IL-1a, IL-1b, IL-6, and IL-8 according to the manufacturer’s instructions.
Cytokine assay
Statistical analysis
PBMCs were pre-incubated with control or test compounds for 3 h at 37 °C with 5 % CO2. Dexamethasone and complete medium were used as positive and negative control, respectively. After the pre-incubation period, samples were induced by 0.1 lg/ml LPS to release cytokines. The final concentrations of the samples in the mixtures were 5, 2.5, 1.25, and 0.625 lg/ml. The final concentration of DMSO in the mixture was fixed at 0.5 % to avoid interference with the cells. The final concentrations of dexamethasone and the active compound from the most active plant in the mixtures were 5, 0.5, 0.05, and 0.005 lg/ml. Samples were harvested at different time intervals (12 h for TNF-a and IL-1b; 20 h for IL-6; and 24 h for IL-1a and IL-8) after stimulation by LPS. Cellfree supernatants were obtained by centrifugation at 300g and 4 °C for 10 min and stored at -80 °C prior to use.
All statistical analyses were performed using SPSSÒ Statistic 20. One-way analysis of variance followed by a posthoc test (Tukey’s multiple comparison) were used when significant differences at p \ 0.05 were present. Cytokine determination by ELISA was performed in duplicate, and data were obtained from three different donors. Results are expressed as mean ± standard error of the mean of three experiments. The IC50 values were calculated using GraphPad Prism v.6.01 software.
Results and discussion The cell viability test was carried out to evaluate the cytotoxicity of plant extracts (Table 1) on PBMCs. All extracts tested at concentrations of 5 lg/ml were viable ([90 %) after 27 h incubation. This concentration was
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J Nat Med Table 2 Percentage inhibition (%) of selected medicinal plant extracts, andrographolide, and dexamethasone at concentrations of 5 lg/ml on the release of cytokines
Percentage inhibition values are presented as mean ± SEM of three independent experiments performed in duplicate. Percentage inhibition [8 % was significant at p \ 0.05 when compared with negative control #
p [ 0.05 was considered not significant compared with dexamethasone
a
Dexamethasone as a positive control
Sample no.
TNF-a
IL-1b
IL-6
IL-8
1
8.2 ± 1.1
34.3 ± 3.01
14.1 ± 2.1
20.9 ± 4.7
4.1 ± 0.5
2
36.4 ± 0.9
78.2 ± 1.41#
91.9 ± 3.8#
85.1 ± 2.6#
14.8 ± 0.5
3
5.0 ± 2.4
16.2 ± 3.84
20.6 ± 4.6
6.6 ± 1.2
4.4 ± 2.3
4
8.4 ± 1.1
6.9 ± 0.69
10.5 ± 1.1
32.9 ± 1.5
10.7 ± 0.9
5
10.8 ± 1.1
12.5 ± 3.94
27.4 ± 1.2
42.9 ± 1.1
8.4 ± 2.4
6
5.5 ± 1.3
23.7 ± 4.57
23.9 ± 1.0
19.7 ± 3.0
6.0 ± 1.9
7
16.5 ± 0.7
37.3 ± 3.34
41.4 ± 3.8
9.7 ± 2.8
11.9 ± 1.5
8
10.0 ± 0.5
15.4 ± 1.58
6.4 ± 1.0
13.5 ± 2.5
12.2 ± 0.6
9
13.4 ± 1.3
38.2 ± 2.70
24.9 ± 5.0
18.3 ± 3.8
11.5 ± 2.1
10
17.3 ± 0.6
42.4 ± 3.37
63.2 ± 3.4
18.1 ± 1.2
6.7 ± 0.6
11
17.0 ± 1.5
14.7 ± 3.34
32.2 ± 1.0
3.8 ± 0.8
4.9 ± 0.5
12
12.1 ± 1.3
34.5 ± 6.54
29.3 ± 0.9
9.8 ± 1.1
10.6 ± 1.2
13
1.8 ± 0.6
34.2 ± 5.12
2.6 ± 0.9
23.0 ± 3.2
7.7 ± 2.7
14
3.0 ± 0.6
7.0 ± 1.79
5.6 ± 0.9
6.8 ± 0.4
4.4 ± 1.4
15 16
1.3 ± 0.3 3.9 ± 0.8
36.3 ± 3.15 29.5 ± 2.31
6.5 ± 1.7 18.5 ± 5.4
6.1 ± 0.5 19.6 ± 1.2
10.0 ± 1.0 6.4 ± 1.0
17
2.6 ± 0.3
31.7 ± 2.84
7.2 ± 1.4
27.1 ± 1.7
9.9 ± 3.9
18
5.7 ± 0.3
14.8 ± 0.99
6.7 ± 0.9
30.1 ± 3.0
5.5 ± 1.0
19
4.1 ± 0.5
27.0 ± 6.91
12.9 ± 3.4
30.0 ± 0.6
6.7 ± 2.3
20
5.5 ± 0.7
43.3 ± 4.11
63.5 ± 3.9
32.4 ± 0.8
9.7 ± 3.2
21
28.0 ± 4.1
82.3 ± 1.0#
86.4 ± 1.0#
54.8 ± 2.2
18.4 ± 1.2
57.6 ± 3.9
83.3 ± 3.56
90.5 ± 1.3
85.1 ± 0.6
32.5 ± 1.4
a
22
used as the highest concentration in this experiment. Among the samples tested, A. paniculata extract (no. 2) and its major compound andrographolide (no. 21) at 5 lg/ ml were the strongest inhibitors of the release of TNF-a, with percentage inhibition values of 36.4 ± 0.9 and 28.0 ± 4.1 % (Table 2), significantly lower than dexamethasone (no. 22) (p \ 0.001) with a percentage inhibition value of 57.6 ± 3.9 %. These results were in agreement with those found by Wangchuck et al. [17]. It was reported that dexamethasone at 1 lg/ml inhibited the release of TNF-a with percentage inhibition of 58 % in LPS-activated THP-1 monocytic cells. The results also showed that A. paniculata extract (2) and andrographolide (21) at 5 lg/ml strongly inhibited the release of IL-1a, with percentage inhibition values of 78.2 ± 1.41 and 82.3 ± 1.00 %, respectively (Table 2). The results also showed that A. paniculata extract and andrographolide have no significant difference when compared to dexamethasone (22) (p [ 0.1). The data indicated that A. paniculata (2), C. citratus (10), Z. officinale (20), and andrographolide (21) at 5 lg/ml strongly inhibited the release of IL-1b, with percentage inhibition values of 91.88 ± 3.79, 63.15 ± 3.45, 63.48 ± 3.95, and 86.4 ± 1.0 %, respectively (Table 2). Among these plants, A. paniculata extract and its major compound andrographolide have no significant difference from dexamethasone (22), with a percentage inhibition value of 90.5 ± 1.3 %
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IL-1a
(p [ 0.1). Among the samples tested, A. paniculata (2) at 5 lg/ml was the only extract which strongly inhibited the release of IL-6, with a percentage inhibition value of 85.08 ± 2.55 % (Table 2). This extract had no significant difference from dexamethasone (22) (p = 1), whereas andrographolide (21) showed lower inhibition than dexamethasone (p \ 0.001. The results showed that no extract could inhibit the release of IL-8 by more than 50 % at 5 lg/ml (Table 2). Dexamethasone (22) as positive control was the strongest inhibitor, but still inhibited IL-8 by less than 50 %. The results showed that the inhibitory effect of dexamethasone on IL-6 (85.06 %) was greater than that of IL-8 (32.54 %). This was similar to a previous study carried out by Lu et al. [18] in corneal fibroblasts, which reported that the inhibitory effect of dexamethasone on IL6 was greater than that of IL-8, with percentage inhibition values of 83 and 50 %, respectively. The difficulty of these samples in inhibiting IL-8 may be due to the fact that in spite of stimulation by LPS, IL-8 was also stimulated by TNF-a, IL-1a, and IL-1b which are produced by LPSstimulated PBMCs [19, 20]. This may have caused the abundant and continuous release of IL-8. Furthermore, the extracts which have inhibition greater than 50 % were applied to the cells in different concentrations. A. paniculata extract strongly inhibited the release of IL-1a, IL-1b, and IL-6 in a dose-dependent manner, with IC50 values of 1.54, 1.06, and 0.74 lg/ml, respectively
J Nat Med Table 3 IC50 values (lg/ml) of samples which have percentage inhibition [50 % on cytokine release Plant name
TNF-a
IL-1a
IL-1b
IL-6
Andrographis paniculata Nees
nd
1.54 ± 0.07*
1.06 ± 0.04*
0.74 ± 0.07*
Curcuma domestica Valeton
nd
nd
4.91 ± 0.11
nd
Cymbopogon citratus Stapf
nd
nd
3.22 ± 0.33
nd
Zingiber officinale Roscoe
nd
nd
3.17 ± 0.26
nd
Andrographolide
nd
0.12 ± 0.06
0.53 ± 0.06
0.20 ± 0.06
Dexamethasone
0.87 ± 0.20
0.0004 ± 0.00004
0.12 ± 0.01
0.05 ± 0.01
Andrographolide (lM)
nd
0.34 ± 0.17#
1.52 ± 0.18#
0.56 ± 0.15#
Dexamethasone (lM)
1.69 ± 0.38
0.0008 ± 0.00007
0.24 ± 0.02
0.09 ± 0.02
* Significantly different (p \ 0.05) compared to andrographolide #
Significantly different (p \ 0.001) compared to dexamethasone
nd not determined (none of the tested doses exceeded 50 % inhibition). IC50 for IL-8 could not be determined
(Table 3). Some extracts such as C. citratus and Z. officinale strongly inhibited the release of IL-1b but not TNF-a, IL-1a, and IL-6. A previous study has shown that regulation of IL-1b production is through mitogen-activated protein kinase kinase 3 (MKK3) which in turn activates p38 mitogen-activated protein kinase (MAPK), but this did not occur with other cytokines, such as TNF-a [21]. Thus, the differential inhibition effect on these cytokines may be related to differential activity of the extracts on this pathway. It may indicate that these extracts inhibited these cytokines by different mechanisms. The results also showed that C. citratus inhibited the release of IL-1b in a dose-dependent manner, with an IC50 value of 3.22 lg/ml (Table 3). Francisco et al. [22] demonstrated that a C. citratus leaf fraction strongly inhibited iNOS expression, NO production, p38 MAPK, c-jun-Nterminal kinase (JNK) 1/2, and nuclear factor-kappaB (NF-jB) signaling pathways in murine macrophages, suggesting its anti-inflammatory activity. Regarding its antiinflammatory effect, Lee et al. [23] reported that C. citratus inhibited the LPS-induced activation of NF-jB in RAW 264.7 cells by inhibiting the phosphorylation of inhibitory jB (IjB) kinase-b (IKKb). Taken together, C. citratus inhibition of IL-1b may be related to the inhibition of p38 MAPK and IKKb. The results indicated that Z. officinale extract inhibited the release of IL-1b in a dose-dependent manner, with an IC50 value of 3.17 lg/ml (Table 3). The results were supported by previous studies which reported the inhibition effect of Z. officinale hexane fraction on IL-1b in LPSstimulated BV2 microglial cells [24] and its suppressive effect on the expression of pro-inflammatory genes [25]. Additionally, an in vitro study showed that Z. officinale extract suppresses pro-inflammatory cytokines and chemokines produced by synoviocytes, chondrocytes, and leukocytes due to arthritis [26]. Phytochemical studies indicated that the plant is rich in a large number of
substances, including gingerols and shogaols. These compounds show various biological activities such as antioxidant, anti-inflammatory, and anticarcinogenic properties [27]. A phytochemical study was carried out on A. paniculata extract, which had the strongest inhibition of the release of pro-inflammatory cytokines. Figure 1 shows that the methanol extract of A. paniculata contained andrographolide with a retention time of 5.004 min. This is very close to the retention time of standard andrographolide, with a value of 5.125 min. The content of andrographolide in the extract was 304 ± 9.13 mg/g extract. Andrographolide, shown in the HPLC chromatogram to be the major compound, was also investigated and the results indicated that andrographolide strongly inhibited the release of IL-1a, IL-1b, and IL-6 in a dose-dependent manner, with IC50 values of 0.12, 0.53, and 0.20 lg/ml, respectively (Table 3). These results are in agreement with those of Sheeja et al. [28], who found that the levels of proinflammatory cytokines such as IL-1b and IL-6 were effectively reduced by the administration of extract and andrographolide in metastatic tumor-bearing mice. Furthermore, Bao et al. [29] reported that andrographolide inhibited NF-jB activity at the level of IKKb activation in ovalbumin-induced allergic lung disease and human bronchial epithelial cells. Specifically, andrographolide inhibited phosphorylation of IKKb, which in turn inhibited p65 and p50 translocation to the nucleus, an initial process for gene expression of several cytokines [29]. NF-jB is known as a major transcription factor for regulating the expressions of pro-inflammatory cytokines [30]. Taken together, inhibition of pro-inflammatory cytokines in the results may have the same mechanism. The results also showed that the IC50 of A. paniculata extract was significantly higher than that of andrographolide on IL-1a, IL-1b, and IL-6 (p \ 0.02) (Table 2). To our knowledge, this is the first report that A. paniculata extract and its major compound
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J Nat Med Fig. 1 HPLC chromatograms of a A. paniculata extract and b andrographolide (Rt = 5.125 min)
andrographolide strongly inhibited the release of IL-1a. Previous studies have only showed their inhibitory effect on the release of another member of the IL-1 family, IL-1b [28, 31, 32]. Although IL-1a and IL-1b have identical activities, they are different in several ways. For example, studies with knockout mice indicate that IL-1a is essential for priming T cells during contact hypersensitivity and for the stimulation of high levels of serum immunoglobulin E following immunization with ovalbumin, whereas IL-1b, which can circulate to the brain, is essential for the induction of fever [33]. In conclusion, A. paniculata extract and its major compound andrographolide strongly inhibited the release of IL-1a, IL-1b, and IL-6, while C. citratus and Z. officinale strongly inhibited the release of IL-1b. The research findings provide evidence that A. paniculata, C. citratus, and Z. officinale extracts and andrographolide have the potential to inhibit pro-inflammatory cytokines. Inhibition of pro-inflammatory cytokines by these extracts and this compound indicate their ability to reduce inflammation
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reactions and provide further evidence that these plants may have potent anti-inflammatory properties. Acknowledgments This study was supported by the grant from Science Fund, Ministry of Agriculture Malaysia, No. 05-01-02SF1022.
References 1. Kumar V, Abbas AK, Fausto N, Mitchell R (2007) Robbins basic pathology, 8th edn. Elsevier Saunders, Philadelphia 2. Shailasree S, Ruma K, Kini KR, Niranjana SR, Prakash HS (2012) Potential anti-inflammatory bioactives from medicinal plants of Western Ghats, India. Pharmacogn Commun 2(2):2–12 3. Aderem A, Ulevitch RJ (2000) Toll-like receptors in the induction of the innate immune response. Nature 406(6797):782–787 4. Saad B, Abouatta BS, Basha W, Hmade A, Kmail A, Khasib S, Said O (2011) Hypericum triquetrifolium-derived factors downregulate the production levels of LPS-induced nitric oxide and tumor necrosis factor-a in THP-1 cells. Evid Based Complement Alternat Med 2011:1–7 5. Nantel F, Denis D, Gordon R, Northey A, Cirino M, Metters KM, Chan CC (1999) Distribution and regulation of cyclooxygenase-2
J Nat Med
6. 7.
8.
9. 10.
11.
12. 13.
14. 15.
16.
17.
18.
19.
20. 21.
in carrageenan-induced inflammation. Br J Pharmacol 128(4): 853–859 Feghali CA, Wright TM (1997) Cytokines in acute and chronic inflammation. Front Biosci 2:12–26 Beutler B, Cerami A (1989) The biology of cachectin/TNF–a primary mediator of the host response. Annu Rev Immunol 7:625–655 Delgado AV, McManus AT, Chambers JP (2003) Production of tumor necrosis factor-alpha, interleukin-1beta, interleukin-2, and interleukin-6 by rat leukocyte subpopulations after exposure to substance P. Neuropeptides 37(6):355–361 Tayal V, Kalra BS (2008) Cytokines and anti-cytokines as therapeutics: an update. Eur J Pharmacol 579:1–12 Metsios GS, Stavropoulos-Kalinoglou A, Sandoo A, van Zanten JJ, Toms TE, John H, Kitas GD (2010) Vascular function and inflammation in rheumatoid arthritis: the role of physical activity. Open Cardiovasc Med J 4(1):89–96 Mantovani A, Garlanda C, Allavena P (2010) Molecular pathways and targets in cancer-related inflammation. Ann Med 42(3):161–170 Hotamisligil GS (2010) Endoplasmic reticulum stress and the inflammatory basis of metabolic disease. Cell 140(6):900–917 Frostegard J (2010) Rheumatic diseases: insights into inflammation and atherosclerosis. Arterioscler Thromb Vasc Biol 30(5):892–893 Mathieu P, Lemieux I, Despres JP (2010) Obesity, inflammation, and cardiovascular risk. Clin Pharmacol Ther 87(4):407–416 Batlouni M (2010) Nonsteroidal anti-inflammatory drugs: cardiovascular, cerebrovascular and renal effects. Arq Bras Cardiol 94(4):522–563 World Medical Association (2008) Declaration of Helsinki: ethical principles for medical research involving human subjects (http://www.wma.net/en/30publications/10policies/b3/index.html) Wangchuk P, Keller PA, Pyne SG, Taweechotipatr M (2013) Inhibition of TNF-a production in LPS-activated THP-1 monocytic cells by the crude extracts of seven Bhutanese medicinal plants. J Ethnopharmacol 148(3):1013–1017 Lu Y, Liu Y, Fukuda K, Nakamura Y, Kumagai N, Nishida, T (2006) Inhibition by triptolide of chemokine, proinflammatory cytokine, and adhesion molecule expression induced by lipopolysaccharide in corneal fibroblasts. Invest Ophthalmol Vis Sci 47(9):3796–3800 Arbabi S, Rosengart MR, Garcia I, Jelacic S, Maier RV (1999) Priming interleukin 8 production: role of platelet-activating factor and p38. Arch Surg 134:1348–1353 Baggiolini M, Clark-Lewis I (1992) Interleukin-8, a chemotactic and inflammatory cytokine. FEBS 307(1):97–101 Wysk M, Yang DD, Lu HT, Flavell RA, Davis RJ (1999) Requirement of mitogen-activated protein kinase kinase 3 (MKK3) for tumor necrosis factor-induced cytokine expression. Proc Natl Acad Sci USA 96:3763–3768
22. Francisco V, Figuerinha A, Neves BM, Rodriguez CG, Lopes MC, Cruz MT, Batista MT (2011) Cymbopogon citratus as source of new and safe anti-inflammatory drugs: bio-guided assay using lipopolysaccharide-stimulated macrophages. J Ethnopharmacol 133:818–827 23. Lee HJ, Jeong HS, Kim DJ, Noh YH, Yuk DY, Hong JT (2007) Inhibitory effect of citral on NO production by suppression of iNOS expression and NF-jB activation in RAW 264.7 cells. Arch Pharm Res 31:342–349 24. Jung HW, Yoon CH, Park KM, Han HS, Park YK (2009) Hexane fraction of Zingiberis Rhizoma Crudus extract inhibits the production of nitric oxide and pro-inflammatory cytokines in LPSstimulated BV2 microglial cells via the NF-jB pathway. Food Chem Toxicol 47:1190–1197 25. Kim HW, Murakami A, Abe M, Ozawa Y, Morimitsu Y, Williams MV, Ohigashi H (2005) Suppressive effects of mioga ginger and ginger constituents on reactive oxygen and nitrogen species generation, and the expression of inducible pro-inflammatory genes in macrophages. Antioxid Redox Signal 7:1621– 1629 26. Phan PV, Sohrabi A, Polotsky A, Hungerford DS, Lindmark L, Frondoza CG (2005) Ginger extract components suppress induction of chemokine expression in human synoviocytes. J Altern Complement Med 11:149–154 27. Aggarwal BB, Prasad S, Reuter S, Kannapan R, Yadev VR, Park B, Kim JH, Gupta SC, Phromnoi K, Sundaram C, Prasad S, Chaturvedi MM, Sung B (2011) Identification of novel antiinflammatory agents from ayurvedic medicine for prevention of chronic diseases: ‘‘reverse pharmacology’’ and ‘‘bedside to bench’’ approach. Curr Drug Targets 12(11):1595–1653 28. Sheeja K, Kuttan G (2010) Andrographis paniculata downregulates pro-inflammatory cytokine production and augments cell mediated immune response in metastatic tumor-bearing mice. Asian Pac J Cancer Prev 11:723–729 29. Bao Z, Guan S, Cheng C, Wu S, Wong SH, Kemeny DM, Leung BP, Wong WS (2009) A novel antiinflammatory role for andrographolide in asthma via inhibition of the nuclear factor-kB pathway. Am J Respir Crit Care Med 179:657–665 30. Huang SS, Chiu CS, Chen HJ, Hou WC, Sheu MJ, Lin YC, Shie PH, Huang GJ (2011) Antinociceptive activities and the mechanisms of anti-inflammation of Asiatic acid in mice. Evid Based Complement Alternat Med 2011:1–10 31. Zhu T (2013) Andrographolide protects against LPS-induced acute lung injury by inactivation of NF-jB. PLoS One 8(2):1–12 32. Shen T (2013) AP-1/IRF-3 targeted anti-inflammatory activity of andrographolide isolated from Andrographis paniculata. Evid Based Complement Alternat Med 2013:1–16 33. Sims JE, Smith DE (2010) The IL-1 family: regulators of immunity. Nat Rev Immunol 10(2):89–102
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