Monika
Mueller
*a,
Daniela
Weinmann
b,
Stefan
Toegel
b,
Wolfgang
Holzer
c,
Frank M.
Unger
a and
Helmut
Viernstein
a
aDepartment of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria. E-mail: monika.mueller@univie.ac.at; Fax: +43 (0)1 4277 9554; Tel: +43 (0)1 4277 55414
bKarl Chiari Lab for Orthopaedic Biology, Department of Orthopaedics, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria
cDepartment of Pharmaceutical Chemistry – Division of Drug Synthesis, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria
First published on 8th March 2016
The heartwood of Caesalpinia sappan is a traditional ingredient of food and beverages in South East Asia and has been used in traditional medicine as an analgesic and anti-inflammatory drug or to promote blood circulation. Scientific studies have confirmed different bioactivities associated with its use. Here, five fractions were isolated from the ethanolic extract of C. sappan heartwood, including episappanol (1), protosappanin C (2), brazilin (3), (iso-)protosappanin B (4) and sappanol (5) using high-performance liquid chromatography (HPLC). All compounds were tested for their anti-inflammatory effects in two different cell lines. Cytokine concentrations in the cell supernatant were determined using enzyme-linked immunosorbent assay (ELISA), and mRNA levels were measured using reverse-transcription quantitative polymerase chain reaction (RT-qPCR). In lipopolysaccharide-stimulated macrophages, all compounds significantly inhibited the secretion of the pro-inflammatory cytokines interleukin (IL-6) and tumor necrosis factor-alpha (TNF-α). Sappanol (5) increased the secretion of the anti-inflammatory IL-10. In IL-1β-stimulated chondrocytes, all fractions reduced the mRNA expression and the secretion of the pro-inflammatory cytokines IL-6 and TNF-α. The highest anti-inflammatory effect was found for brazilin (3) in both cell lines. Of note, this is the first study which shows the anti-inflammatory effect of sappanol and episappanol. This study provides evidence for the efficacy of the traditional use of C. sappan as an anti-inflammatory remedy. Given the high prevalence of inflammation-related pathologies including arthritis, and the urgent need to clinically intervene with these diseases, the anti-inflammatory activity of diverse compounds from C. sappan may be of interest for the development of complementary and alternative treatment strategies.
In addition to the major compound brazilin, several compounds have been isolated and identified including protosappanins, chalcones and homoisoflavones.8–10 Most studies have been published on the bioactivity of brazilin showing vasorelaxation in endothelial cells in vitro,2 hypoglycemic activity in diabetic mice,11 and antibacterial activity.12 More recently, it has been shown that brazilin reduces high glucose-induced vascular inflammation,13 and exhibits anti-thrombotic properties,14 anti-cancer activities as shown in liver, breast, lung and gingival cancer cells15 and anti-allergic activities in a murine asthma model.16
A limited number of studies have been conducted on other compounds from C. sappan. As such, protosappanin A, B and brazilein exhibited antioxidative activity.4,17 Brazilein, sappanchalcone, protosappanin C, D and E showed anti-inflammatory effects in lipopolysaccharide (LPS)-induced macrophages (J774.1) as indicated by a reduction of nitric oxide (NO) or prostaglandin E2 (PGE2) production.10,17 Sappanone A or protosappanin E inhibited IL-6 secretion in LPS-stimulated macrophages (RAW 264.7).18 Brazilein, protosappanin A, and sappanchalcone exerted inhibitory effects on a drug target for influenza treatment.19 Protosappanin A was found to be an immunosuppressive agent in heart-transplanted rats and exhibited anti-rejection activity.20
Inflammation plays a major role in a broad range of diseases including asthma, atherosclerosis, cancer, and arthritis. IL-1β stimulated chondrocytes serve as a joint inflammation model for arthritis. Previously, we have shown the anti-inflammatory activity of the ethanolic extract of C. sappan (CSE) in chondrocytes as indicated by the inhibited expression of the pro-inflammatory cytokines IL-1β and TNF-α and the inhibition of the synthesis of NO and COX-2 expression in primary chondrocytes.21 Furthermore, CSE inhibited IL-1β-induced overexpression of matrix metalloproteinases in human chondrocytes, and thus may attenuate the progression of osteoarthritis.22 Brazilin was suggested to be the major active compound of CSE, however, the contribution of other chemical entities to the suggested anti-arthritic activity of C. sappan is currently unknown. Furthermore, the anti-inflammatory effect of only a few compounds from C. sappan has been elucidated so far, while the effect of most compounds is still not clear. Additionally, the literature is not consistent on the biological activities of several of the isolated compounds.
The present study aimed to assess the anti-inflammatory activity of episappanol, protosappanin C, brazilin, (iso-)protosappanin B and sappanol isolated from CSE in macrophages and chondrocytes in vitro. Three cytokines indicating the inflammatory response were chosen, two cytokines which enhance inflammation (IL-6 and TNF-α) and one which counteracts inflammation (IL-10).
Due to the instability of fractions 1 and 5 under acidic conditions, the preparative separation of these fractions was performed using solvent A (5% acetonitrile in water) and solvent B (acetonitrile) without TFA, but with the same gradient as described before.
The secretion of TNF-α, IL-6, and IL-10 in the cell supernatant was analyzed using ELISA kits according to the manufacturer's protocol. The optical density at 450 nm, corrected by the reference wavelength 570 nm, was measured using an Infinite M200 microplate reader (Tecan, Crailsheim, Germany).
The viability of the cells was tested using a MTT assay as previously described.23 If the viability was 25% below the control, a compound was considered cytotoxic. The ELISA results indicating the cytokine secretion were normalized to the MTT values to reduce any variation arising from the differences in the cell density. Cells treated with only LPS served as a positive control and the amount of secreted cytokines was defined as 100%. All the results from the tested compounds were then calculated as a percent of this value. Dexamethasone was used as the reference drug. The assay was performed in triplicate on separate days. The mean and standard deviation are presented in the figures.
Total RNA extraction was carried out using the NucleoSpin RNA II Kit (Macherey-Nagel, Dueren, Germany) according to the manufacturer's instructions. Each sample was run on the Agilent 2100 Bioanalyzer Nano LabChip (Santa Clara, CA, USA) for quality control and quantification of total RNA prior to reverse transcription into cDNA using the high capacity cDNA reverse transcription kit (Life Technologies). RNA integrity numbers were between 9.3 and 9.9.
SYBR-green-based RT-qPCR assays for IL6, TNFA and IL10 expression were used as previously described21,22 and with respect to the MIQE guidelines.24 The mRNA levels of the target genes were calculated as quantities relative to the untreated control group, considering both amplification efficiencies and normalization to succinate dehydrogenase complex, subunit A (SDHA). Details on primers are given in Table 1.
Gene | Species | Accession number | Forward primer | Reverse primer | Efficiency (%) |
---|---|---|---|---|---|
IL6 | Human | NM_000600 | ATAGGACTGGAGATGTCTGAGG | AGGCAACTGGACCCGAAGG | 93.5 |
SDHA | Human | NM_004168 | TGGGAACAAGAGGGCATCTG | CCACCACTGCATCAAATTCATG | 94.6 |
TNFA | Human | NM_000594 | TCAGCAAGGACAGCAGAGG | CAGTATGTGAGAGGAAGAGAACC | 100 |
IL6 | Mouse | NM_031168 | CTGTCTATACCACTTCAC | CATCATCGTTGTTCATAC | 90.6 |
SDHA | Mouse | NM_023281 | CCAGGACTTAGAATTTGT | TTGACTGTTGATGAGAAT | 94.5 |
TNFA | Mouse | NM_013693 | TTCTGTCTACTGAACTTC | CCATAGAACTGATGAGAG | 83.8 |
Prior to treatment, SW1353 cells were starved overnight in serum-free DMEM supplemented with gentamicin. Then, cells were pretreated for 1 h with the isolated fractions in serum-free medium followed by the addition of 10 ng per ml IL-1β and further incubation for 24 h. As a negative control, cells were left untreated, and as a positive control, cells were treated only with IL-1β. Afterwards, cell supernatants were collected, centrifuged and stored at −80 °C. All treatments were performed in duplicate and were repeated three times. ELISA assays were conducted according to the manufacturer's protocol (Ready-SET-Go! ELISA, eBioscience). SYBR-green-based RT-qPCR assays for IL6 and TNFA expression were conducted as described above (section 2.7). Details on primers are given in Table 1. Concomitantly, MTT assays were performed as indicated above (section 2.7) to determine cell viability.
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Fig. 1 Chromatogram of the preparative HPLC used for separation of the 20% ethanolic extract of C. sappan. |
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Fig. 2 Structure of the five isolated fractions: (1) episappanol, (2) protosappanin C, (3) brazilin, (4) (i) protosappanin B, (ii) isoprotosappanin B and (5) sappanol. |
13 C-NMR (100 MHz, acetone-d6): δ 158.9 (C-7), 155.7 (C-8a), 145.2 (C-3′), 144.3 (C-4′), 132.2 (C-5), 129.0 (C-1′), 123.2 (C-6′), 119.0 (C-2′), 117.0 (C-4a), 115.5 (C-5′), 108.9 (C-6), 103.0 (C-8), 71.2 (C-3), 70.0 (C-4), 69.2 (C-2), 39.0 (C-9).
The 1H-NMR data are in agreement with Namikoshi et al. (1987).8
13
C-NMR (100 MHz, acetone-d6): δ 204.0 (7-HO), 158.9 (C-4a), 158.6 (C-3), 145.0 (C-10), 144.7 (C-11), 133.4 (C-1), 132.4 (C-12a), 125.3 (C-8a), 123.0 (C-12b), 119.9 (C-9), 117.2 (C-12), 111.5 (C-2), 108.0 (C-4), 75.1 (C-7), 74.4 (C-6), 37.1 (C-8).
13 C-NMR (100 MHz, acetone-d6): δ 157.6 (C-7), 155.5 (C-8a), 145.1 (C-3′), 144.8 (C-4′), 137.4 (C-6′), 132.0 (C-5), 131.5 (C-1′), 115.6 (C-4a), 112.6 (C-2′), 112.3 (C-5′), 109.6 (C-6), 103.9 (C-8), 77.7 (C-3), 70.8 (C-2), 51.1 (C-4), 42.9 (C-9).
The numbering of the brazilin ring system used here (Fig. 3C) is the one introduced by Fu et al. (2008).9 Fuke et al. and Kim et al. used a different numbering system.25,26
13 C-NMR (100 MHz, acetone-d6): δ 159.2 (C-4a), 158.7 (C-3), 144.4 (C-10 and C-11), 133.3 (C-1), 132.3 (C-12a), 127.3 (C-8a), 123.3 (C-12b), 119.6 (C-9), 117.2 (C-12), 111.0 (C-2), 108.0 (C-4), 75.8 (C-6), 72.1 (C-7), 67.9 (C-7a), 39.8 (C-8).
Minor isomer (protosappanin B): 1 H-NMR (400 MHz, acetone-d6): δ 7.00 (d, 1 H, 3J1,2 = 8.1 Hz, H-1), 6.85 (br s, 1 H, H-9), 6.71 (br s, 1 H, H-12), 6.62 (m, 1 H, H-2), 6.56 (m, 1 H, H-4), 4.35 (d, 1 H, 2J6,6′ = 11.7 Hz, H-6), 3.57 (m, 2 H, H-6′ and H-7a), 3.38 (d, 1 H, 2J7a′,7a = 10.9 Hz, H-7a′), 2.74 (A-part of an AB-system, 1 H, 2J8,8′ = 13.5 Hz, H-8), 2.67 (B-part of an AB-system, 1 H, 2J8′,8 = 13.5 Hz, 1 H, H-8′).
13 C-NMR (100 MHz, acetone-d6): δ 160.6 (C-4a), 158.7 (C-3), 144.4 (C-10 and C-11), 132.3 (C-1), 131.7 (C-12a), 128.3 (C-8a), 125.2 (C-12b), 118.9 (C-9), 117.4 (C-12), 111.7 (C-2), 108.8 (C-4), 77.2 (C-6), 72.6 (C-7), 65.6 (C-7a), 42.5 (C-8).
The 1H and 13C-NMR data are in agreement with Fu et al. (2008).9
13 C-NMR (100 MHz, acetone-d6): δ 159.3 (C-7), 155.6 (C-8a), 145.2 (C-3′), 144.5 (C-4′), 132.7 (C-5), 129.0 (C-1′), 122.9 (C-6′), 118.7 (C-2′), 116.4 (C-4a), 115.4 (C-5′), 109.4 (C-6), 103.1 (C-8), 70.2 (C-3), 69.4 (C-4), 67.8 (C-2), 40.7 (C-9).
The 1H-NMR data are in agreement with Namikoshi et al. (1987).8
Fraction | Compound | [M + H] | MW [g] |
---|---|---|---|
1 | Episappanol | 305.1020 | 304.0941 |
2 | Protosappanin C | 303.0863 | 302.0784 |
3 | Brazilin | 287.0914 | 286.0835 |
4 | Protosappanin B + isoprotosappanin B | 305.1020 | 304.0941 |
5 | Sappanol | 305.1020 | 304.0941 |
The absolute level of IL-6 secretion was up to 5000 pg ml−1 for the positive control and up to 100 pg ml−1 for the negative control. The values for the TNF-α secretion were up to 2500 pg ml−1 for the positive control and up to 300 pg ml−1 for the negative control, respectively. CSE significantly exerted anti-inflammatory effects down to a concentration of 10 μg ml−1 as indicated by a reduction of the IL-6 and TNF-α secretion with an IC50 of 8 and 36 μg ml−1, respectively (Fig. 3). No significant effect on IL-10 was observed.
All the isolated fractions (1–5) induced a significant and dose-dependent change in the cytokine secretion profile as indicated by a decrease of the pro-inflammatory cytokines IL-6 (Fig. 4Ai) and TNF-α (Fig. 4Bi) (Table 3) at the cytokine level. At the mRNA level, the decrease of IL6 was significant whereas the decrease of TNFA was not significant (Fig. 4Aii and 4Bii). Fraction 5 increased the secretion of the anti-inflammatory IL-10 (Fig. 4C), whereas the other fractions had no significant effect on this cytokine. Brazilin (3) exerted the highest anti-inflammatory effect with a significant inhibition of the IL-6 secretion down to 5 μg ml−1 and a significant inhibition of the TNF-α secretion down to 10 μg ml−1. The IC50 values were 18 μM for IL-6 and 29 μM for TNF-α, respectively. Episappanol (1), protosappanin C (2), and (iso-)protosappanin B (4) significantly inhibited the IL-6 and TNF-α secretion at 50 μg ml−1. Sappanol (5) significantly reduced the IL-6 and TNF-α secretion at the highest concentration tested (100 μg ml−1). Besides brazilin, protosappanin C (2) and (iso-)protosappanin B (4) were the most effective fractions with IC50 values of 123 μM and 128 μM as indicated by IL-6 secretion (Table 3).
Macrophages | Chondrocytes | |||
---|---|---|---|---|
Sample | IL-6 | TNF-α | IL-6 | TNF-α |
1 | 155 μM | >164 μM | >33 μM | 24 μM |
2 | 123 μM | 121 μM | 21 μM | 16 μM |
3 | 18 μM | 29 μM | 5 μM | 3 μM |
4 | 128 μM | >164 μM | >33 μM | 22 μM |
5 | >164 μM | >164 μM | >33 μM | >33 μM |
CSE | 8 μg ml−1 | 36 μg ml−1 | >10 μg ml−1 | 1.6 μg ml−1 |
Dexamethasone | 2 nM | 1 μM | 65 nM | 66 nM |
All the fractions (1–5) exerted a significant reduction of the TNF-α secretion at a concentration of 10 μg ml−1 or lower (Fig. 6B, Table 3). Brazilin (3) was effective at 1 μg ml−1 also. The IC50 values for TNF-α ranged between 3 μM for brazilin and >33 μM for sappanol (Table 3). These findings are in agreement with the qPCR results, showing that the fractions (1–5) significantly inhibited IL6 and TNFA mRNA expression levels at a concentration of 10 μg ml−1 (Fig. 6ii) with the highest effect observed for brazilin.
In this study, five fractions were isolated from the ethanolic (20%) extract of C. sappan, namely episappanol (1), protosappanin C (2), brazilin (3), (iso-)protosappanin B (4) and sappanol (5). The occurrence of the isolated compounds in C. sappan has already been shown previously.8,9,27
To the best of our knowledge, this is the first study showing the anti-inflammatory effect of sappanol and episappanol. Interestingly, sappanol even increases the secretion of the anti-inflammatory, IL-10. The anti-inflammatory effect of protosappanin C and protosappanin B (2, 4) in macrophages has been described previously. Our result showing a moderate effect of protosappanin C is in agreement with Washiyama et al. (2009)10 and Sasaki et al. (2007)17 who showed the effect on PGE2 or NO reduction in J774.1 macrophage cells. Our results on the anti-inflammatory effects of protosappanin B are in contrast to those of Washiyama et al. (2009)10 who found no activity of protosappanin B in J774.1 macrophage cells. Of note, the literature is not consistent on the anti-inflammatory activity of different protosappanins. Sasaki et al.17 found a slight anti-inflammatory activity of protosappanin B and in J774.1 cells as indicated by the inhibition of NO production and iNOS expression. The compounds isolated from C. sappan are unstable which may contribute to the inconsistency of the literature.
The anti-inflammatory effect of CSE and brazilin in RAW 264.7 macrophages in this study is consistent with the literature. Hong et al. showed an inhibition of COX-2 and iNOS expression of the methanolic extract in RAW 264.7 macrophages.6 Brazilin exhibits anti-inflammatory activity via various mechanisms such as inhibition of NO production and regulation of nuclear factor kappa-B (NF)-κB and activator protein-1 in RAW 264.7 cells28–30 or via inducing heme oxygenase-1 expression and inhibition of the production of PGE2, TNF-α and IL1-β.29
Here, we also show the bioactivity of 1–5 in human SW1353 chondrocytes. To the best of our knowledge, this is the first report that demonstrates the anti-inflammatory effect of protosappanin B and C, sappanol and episappanol in chondrocytes. The in vitro and in vivo anti-inflammatory activity of CSE was shown previously.7,21 We also showed the anti-arthritic effect of brazilin previously.21,22 Thus, this study elucidates the anti-arthritic effect of other constituents besides brazilin.
Furthermore, this study confirms that C. sappan exerts an anti-inflammatory effect and might therefore provide an explanation for the analgesic effect via the reduction of swelling and inflammation according to its traditional use.1
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