IL-17A inhibitions of indole alkaloids from traditional Chinese medicine Qing Dai.

ETHNOPHARMACOLOGICAL RELEVANCE: Qing Dai, a famous traditional Chinese medicine (TCM), is prepared by a traditional fermentation process with the aerial part of Strobilanthes cusia. Currently, this TCM could treat various clinical inflammatory diseases, such as ulcerative colitis and psoriasis, however, the bioactive components of Qing Dai are unknown clearly. AIM OF THE STUDY: To isolate and identify the anti-IL-17A components of Qing Dai.
MATERIALS AND METHODS: Silica, RP-18 gels, and size exclusion resin were used for column chromatography to isolate the pure compounds. The structures of isolates were elucidated by NMR, MS, UV, IR spectra, and optical rotation. IL-17A protein and gene expressions were also evaluated in the Th17 cell model and luciferase reporter assay, respectively.
RESULTS: Two indole alkaloids, including one new indigodole D and cephalandole B, were isolated from Qing Dai. Indigodole D could inhibit IL-17A protein production during the Th17 polarization (EC50: 2.16 μg/mL) or after the polarization (EC50: 5.99 μg/mL) without cytotoxicity toward Th17 cells. Cephalandole B did not inhibit the IL-17A protein secretion. Nevertheless, both isolates notably inhibited IL-17A gene expression, especially cephalandole B, in a dose-dependent manner in Jukat cells with IL-17A luciferase reporter.
CONCLUSIONS: Indole alkaloids, indigodoles A, C, D, tryptanthrin, and indirubin could contribute to anti-IL 17A properties of Qing Dai. The possible biogenetic mechanisms of above-mentioned indoles were also speculated in this investigation for further promising anti-IL-17 lead drugs development.

Introduction

Qing Dai, a famous traditional Chinese medicine (TCM), is the pale blue to grayish-blue dried powder prepared by a traditional fermentation process (Lee et al., 2019; Lin, 2016; Pan et al., 2018). Currently, this TCM could treat various clinical inflammatory diseases, such as ulcerative colitis and psoriasis (Naganuma, 2019; Sugimoto et al., 2016). The aerial part of Strobilanthes cusia (Nees) Kuntze (Acanthaceae) recorded in Taiwan Herbal Pharmacopeia is the one of candidate materials that have been used to manufacture Qing Dai (Lee et al., 2019; Lin, 2016). The crude extracts and pure phytochemicals of S. cusia leave (Da-Ching-Yeh or Nan-Ban-Lan-Yeh) and root (Na-Ban-Lan-Gen) showed many pharmacological properties, including anti-viral (anti-herpes simplex virus type-1, anti-influenza A virus), anti-severe acute respiratory syndrome (SARS), antifungal, anti-inflammatory, antinociceptive, and antipyretic effects (Feng et al., 2016; Gu et al., 2015; Ho et al., 2003; Honda and Tabata, 1979; Tanaka et al., 2004; Zhou et al., 2017). To date many secondary metabolites, such as alkaloids, terpenoids, flavonoids, sterols, lignans, phenylethanoids, and isocoumarin have been isolated from this species (Feng et al., 2016; Gu et al., 2015; Honda and Tabata, 1979; Tanaka et al., 2004; Zhou et al., 2017). However, interleukin 17 (IL-17) inhibitions of S. cusia and its processed drug, Qing Dai are not known. Qing Dai could provide a highly effective approach for not only psoriasis (topical use), but inflammatory bowel disease (IBD) (oral use) in modern clinical therapy (Lee et al., 2019; Sugimoto et al., 2016). In addition, Qing Dai ointment could significantly reverse the IL-17A gene expression in psoriatic skin lesions in our previous studies (Cheng et al., 2017). Therefore, Qing Dai and its anti-IL-17 components are interesting for us and this TCM is needed to clarify the active phytochemicals for further clinical application. Our previous investigation indicated that indole alkaloids could contribute to anti-IL-17 properties of Qing Dai that was prepared from the aerial parts of S. cusia (Lee et al., 2019). Continuing studies on bioactive constituents of Qing Dai resulted in isolation of two compounds; besides, their IL-17 inhibitory effects and possible biogenetic pathways of active components were also discussed within.

Materials and methods

Generals

1D and 2D NMR spectra were taken on Bruker Avance III 500 MHz. The chemical shift (δ) values are reported in ppm with CDCl3 used as the internal standard, and coupling constants (J) are in Hz. Low- and high-resolution ESIMS and EIMS were measured on a Bruker Daltonics Esquire HCT ultra high capacity trap mass spectrometer, and an Orbitrap mass spectrometer (LTQ Orbitrap XL and Q Exactive Plus, Thermo Fisher Scientific), respectively. Optical rotations, UV, and IR spectra were measured on a JASCO-P-2000 polarimeter (cell length 10 mm), a PerkinElmer#Lambda265, and a Shimadzu model IR Prestige-21 Fourier-transform infrared spectrophotometer, respectively. TLC was performed on Kieselgel 60 F254 (0.25 mm, Merck) and RP-18 F254S (0.25 mm, Merck) coated plates, spots were recognized under ultraviolet light at 254 and 356 nm, and then stained by spraying with 5% H2SO4 in MeOH before heating on a hot plate. Silica gel (Silicycle 70–230 and 230–400 mesh), RP-18 (LiChroprep® 40–63 μm, Merck), and TOYOPEARL® HW-40F (Tosoh, Japan) were used for column chromatography.

Plant material

The aerial parts of Strobilanthes cusia were collected in Putian City, Fujian Province, China and processed to TCM named Qing Dai by a local GMP pharmaceutical factory in April, 2014. The aforementioned TCM powders were imported and analyzed (Lot. No. BR0308980) by Sheng Chang Pharmaceutical Co., Ltd. in Zhongli District, Taoyuan City, Taiwan, and then was offered for this study in March, 2016. A voucher specimen (IN, 201603) was also stored at the CMRDC, CMUH, Taiwan (Lee et al., 2019).

Extraction and isolation

Qing Dai dry powders (10.0 kg) were extracted with MeOH (36 L × 4) at room temperature to obtain a crude extract (175.2 g) which was separated into an EtOAc–soluble fraction and an aqueous phase with EtOAc and H2O (1:1, v/v), respectively. The former one was further partitioned between n-hexane and 90% MeOH(aq) (1:1, v/v) to give the n-hexane- (INH, 74.8 g) and 90% MeOH(aq)- (INEA, 39.5 g) soluble crude fractions, individually.

Fractionation of INEA fraction was conducted by open column chromatography on silica gel (column: 8.0 × 27 cm, diameter × length) using gradients of n-hexane/EtOAc/MeOH (10:1:0–0:1:1) to give eight subfractions (INEA-A~INEA-H). Subfraction INEA-F (3.8 g) was subjected to a silica gel column (3.5 × 37.5 cm; n-hexane/CH2Cl2/EtOAc, 2:1:0–0:0:1) to afford seven subfractions. Subfraction INEA-F-6 (1.4 g) was further separated by silica gel chromatography (column: 4.0 × 23.5 cm; n-hexane/EtOAc, 5:1–1:1) into nine fractions (INEA-F-6-1~INEA-F-6-9). The INEA-F-6-7 (340.3 mg) was purified by RP-18 column (3.0 × 22 cm; MeOH/H2O, 65:35 to 100:0) to obtain six subfractions (INEA-F-6-7-1~INEA-F-6-7-6). Subfraction INEA-F-6-7-2 (114.3 mg) was subjected to RP-18 gel chromatography (column: 3.0 × 22 cm; MeOH/H2O, 3:1) to obtain four subfractions. INEA-F-6-7-2-3 (64.4 mg) was further purified by silica gel chromatography (column: 2.5 × 25 cm; CH2Cl2 and column: 2.5 × 25.5 cm; n-hexane/EtOAc, 3:1) to give compound 2 (3.3 mg). Subfraction INEA-F-6-7-4 (29.6 mg) was further separated by HW-40F gel chromatography (column: 2.5 × 26.5 cm; CH2Cl2/MeOH, 1:1), and its subfraction INEA-F-6-7-4-2 (10.9 mg) was isolated by silica gel chromatography (column: 2.5 × 26 cm; n-hexane/EtOAc, 3:1) to give compound 1 (8.3 mg).

Indigodole D (1)

Yellow powders; [α]D 24 –10.3 (c 1, MeOH); UV (MeOH) λ max nm (log ε): 260 (4.18), 398 (3.74) (Fig. S8); IR (neat) ν max 3319, 2961, 2922, 2853, 2359, 2342, 1697, 1682, 1605, 1487, 1462, 1319, 1159, 1099, 1018, 1005, 988, 918, 885, 752, 667 cm−1 (Fig. S9); For 1H and 13C NMR spectroscopic data, see Table 1 ; HRESIMS m/z 472.1629 [M+Na]+ (calcd for C28H23O3N3Na, 472.1632).

Table 1

1H and 13C NMR spectroscopic data (500 and 125 MHz, CDCl3) for 1.

position	1
	δH (J in Hz)	δC
1
2		82.9
3		199.9
4	7.53 (d, 7.5)	124.3 (e)
5	6.92 (t, 7.5)	121.2
6	7.33 (t, 7.5)	136.5
7	6.31 (d, 7.5)	114.6
8		159.0
9		125.9
1′	5.43 brs
2′		85.9
3′		200.3
4′	7.53 (d, 7.5)	124.5 (e)
5′	6.88 (t, 7.5)	119.7
6′	7.59 (t, 7.5)	139.6
7′	6.94 (d, 7.5)	112.1
8′		160.2
9′		119.8
1″	6.82 s
2″		74.2
3″		197.4
4″	7.60 (d, 7.5)	124.7
5″	6.74 (t, 7.5)	118.1
6″	7.39 (t, 7.5)	137.8
7″	6.75 (d, 7.5)	111.9
8″		160.1
9″		121.4
1‴	2.15 (m)2.38 (m)	27.7
2‴	0.74 (3H, t, 7.5)	7.6
1′′′′	3.64 (m)	52.1
2′′′′	0.67 (3H, d, 7.0)	7.3

e: exchanged; m: multiple.

HPLC system

A SHIMADZU LC-20AT (Kyoto, Japan) equipped with a degasser, a binary pump, a DAD detector SPD–M20A, and a liquid handler SIL–20A autosampler was applied to analyze the chemical profile of Qing Dai. The mobile phase consisted of water containing 0.1% formic acid (eluent A) and acetonitrile (eluent B). The gradient program was used as following: 10%–76% B (22 min), 76%–85% (10 min), 85% isocratic elution (5 min), 85%–100% (5 min), 100% isocratic elution (8 min) with a HPLC column, COSMOSIL® 5C18–MS–II (5 μm, 4.6 × 250 mm I.D.) performed at 25 °C. Before each analysis run, an equilibration time of 20 min was allowed. The flow rate was 1.0 mL/min and the injection volume was 20 μL. The pure compounds and crude extract concentrations were 0.1 and 1.0 mg/mL, respectively. UV/Vis spectra were recorded in the range of 200–800 nm.

T helper 17 (Th17) cell polarization and intracellular staining

Naïve CD4 T cells were isolated from spleens and peripheral lymph nodes harvested from C57BL/6 mice via EasySep™ Mouse T Cell Isolation Kit (STEMCELL Technologies Inc., Vancouver, Canada) according to the manufacturer's protocol as previously described (Ben-Shaanan et al., 2018). The kit is designed to isolate T cells from single-cell suspensions of splenocytes and lymphocytes by negative selection. Unwanted cells are targeted for removal with biotinylated antibodies directed against non-T cells and streptavidin-coated magnetic particles. Desired CD4+ T cells are isolated. 1 × 105 cells/well CD4+ T cells were cultured in 96 well plate coated with 5 μg/mL anti-CD3 Ab for 5 days in the following polarization condition: 1 μg/mL anti-CD28 Ab (Biolegend), 20 ng/mL IL-6 (PeroTech), 1.25 ng/mL TGF-β (PeroTech), 20 ng/mL IL-1β (PeroTech), 20 ng/mL IL-23 (R&D, USA), 20 μg/mL anti–IFN–γ Ab (Biolegend), and 20 μg/mL anti-IL-4 Ab (Biolegend). Culture medium used was IMDM (Gibco) supplemented with 1.0 mM sodium pyruvate (Gibco), 0.1 mM nonessential amino acids (Gibco), 100 IU/mL penicillin, 100 μg/mL streptomycin (Gibco), and 5% heat-inactivated FBS (Hyclone) (Harris et al., 2007; Yen et al., 2009).

For evaluation of the effect of Th17 polarization (“co-treated” experiment), naïve CD4+ T cells were cultured with or without indicated compounds in the medium during the Th17 polarization for five days. For investigation of the level of IL-17A production after polarization (“post-treated” experiment), the polarized Th17 cells were treated with or without indicated compounds for 16 h (Lee et al., 2019).

After incubation, cells were restimulated for 5 h in the presence of PMA (50 ng/mL), ionomycin (500 ng/mL), and GolgiStop (BD Biosciences). The IL-17A secreting cells were stained with anti-CD4 Ab (BD Biosciences) and anti-IL-17A Ab (BioLegend) and analyzed by a BD FACSVerse flow cytometer (Lee et al., 2019).

IL-17 luciferase reporter assay

The resultant IL-17 promoter-luciferase gene was cloned and constructed as described previously (Lee et al., 2019). Jurkat cells (4 × 106) were transfected with 10 μg IL-17 promoter-luciferase gene construct (pGL4-hIL-17prom) or control vector (pGL4.18) by electroporation according the manufacturer's protocol (Neon transfection system, Invitrogen). Stable expression of IL-17 luciferase reporter from a clone of Jurkat cells were selected with G418 (800 μg/mL, GIBCO) for neomycin resistance. IL-17 luciferase reporter stably transfected cells (IL-17Luc cells) were seeded at the density of 1 × 105 cells/well in 96-well plate followed by treatment with PMA (50 ng/mL), ionomycin (500 ng/mL) and indicating compounds for 5 h. Assay medium was renewed and added a volume of Steady-Glo® Reagent equal to the volume of culture medium in the well for 30 min. Then the activity of luciferase in the transfected cells was measured with the BioTek Synergy microplate reader.

Results and discussion

Structure elucidation of compound 1

The MeOH extract of Qing Dai powders was partitioned into EtOAc- and H2O-soluble extracts, and then the former one was further separated into n-hexane- and 90% MeOH-soluble fractions. Chromatographic fractionation of the active 90% MeOH-soluble one afforded new indigodole D (1) and known cephalandole B (2) (1D & 2D NMR spectra in supporting information) (Wu et al., 2006) (see Fig. 1).

Fig. 1

Structures of compounds 1 and 2.

The molecular formula of 1 was deduced as C28H23O3N3 due to the appearance of an [M+Na]+ ion at m/z 472.1629 in the HRESIMS. IR absorptions at 3319, 1697, and 1682 cm−1 supported the presence of NH and carbonyl groups, respectively. Other IR absorptions at 1605, 1487, and 1462 cm−1 indicated an aromatic system. The UV spectra showed absorption maxima at 260 and 398 nm. In 1D NMR spectra (Table 1), twenty-eight carbon signals were observed, which agreed with two methyls, one methylene, thirteen methines, and twelve quaternary carbons. Among the twelve quaternary carbons, three were identified as carbonyl carbons on the basis of chemical shifts at δ 197.4, 199.9, and 200.3.1D NMR, HSQC, and COSY data also indicated the presence of three sets of o-disubstituted benzene rings, one at δH/δC 6.31 (d, J = 7.5 Hz)/114.6, 6.92 (t, J = 7.5 Hz)/121.2, 7.33 (t, J = 7.5 Hz)/136.5, 7.53 (d, J = 7.5 Hz)/124.3, another at δH/δC 6.88 (t, J = 7.5 Hz)/119.7, 6.94 (d, J = 7.5 Hz)/112.1, 7.53 (d, J = 7.5 Hz)/124.5, 7.59 (t, J = 7.5 Hz)/139.6, and the other at δ 6.74 (t, J = 7.5 Hz)/118.1, 6.75 (d, J = 7.5 Hz)/111.9, 7.39 (t, J = 7.5 Hz)/137.8 and 7.60 (d, J = 7.5 Hz)/124.7. The MS and NMR data of 1 were similar to those of indigodole A (Lee et al., 2019), except that 1 has an ethyl moiety instead of methyl group in indigodole A. On the basis of HMBC [δH 0.74 (H3-2‴)/δC 27.7 (C-1‴) and 82.9 (C-2), δH 2.15, 2.38 (H2-1‴)/δC 7.6 (C-2‴), 74.2 (C-2″), 82.9 (C-2), and 199.9 (C-3)] and COSY correlations [δ 2.15, 2.38 (H2-1‴)/0.74 (H3-2‴)], the ethyl group was assigned at C-2 (δ 82.9) (Fig. 2 ). In the HMBC spectrum, the methyl protons at δ 0.67 (3H, d) exhibited 2 J interactions with C-1‴′ (δ 52.1) as well as 3 J interactions with C-2″ (δ 74.2) and C-2′ (δ 85.9). Additionally, the methine proton δ 3.64 had HMBC correlations with C-2′′′′ (δ 7.3), C-2″ (δ 74.2), C-2′ (δ 85.9), C-3″ (δ 197.4), and C-3′ (δ 200.3). These and other key HMBC connections are shown in Fig. 2. The NOESY spectrum showed the correlations of NH-1″/H3-2′′′′ and H-1″′′/NH-1′, H2-1‴ (Fig. 3 ) and the relative configurations of 1 were temporarily determined as β–NH–1′, α–NH–1″, β–CH2–1‴, and α–CH3–2′′′′. Compound 1 has been named indigodole D, a constituent isomer of indigodole A.

Fig. 2

Key COSY and HMBC correlations for compound 1.

Fig. 3

Key NOESY correlations for compound 1.

Effects of isolates on IL-17A inhibition

Primary mouse CD4+ lymphocytes were polarized intoTh17 cells to investigate the effect of two indole alkaloids 1 and 2 on Th17 polarization (co-treated experiment) and IL-17A secretion after Th17 polarization (post-treated experiment). In Fig. 4, Fig. 5 B, compounds 1 and 2 were co-cultured with CD4+ lymphocytes in the skewing medium for five days to evaluate the effect of isolates on Th17 polarization (co-treated experiment). Intracellular staining with flow cytometric analysis (Fig. S17A) showed that 1 reduced the polarization of IL-17A secretion cells (EC50 value = 2.16 μg/mL) without significant cytotoxicity to T cells (Fig. 4A and B). However, compound 2 co-treatment did not significantly inhibit IL-17A polarization in a dose-dependent manner (Fig. 5A and B and Fig. S17B). In Fig. 5B, although it seemed that compound 2 could inhibit IL-17A protein production at concentrations from 0.2 to 1.56 μg/mL in a dose-dependent manner; however, this dose-dependent inhibitory effect was not seen at concentrations from 3.125 to 12.5 μg/mL.

Fig. 4

Th17 cells cytotoxicity (A, C) and IL-17 protein expression (B, D) data of compound 1. For the co-treated experiment, CD4+ T cells were cultured for five days with or without compound 1 in the skewing medium during Th17 cell polarization. After five days, cell viability was measured by staining with 1 μg/mL propidium iodide (A). The IL-17 secreting cells were stained with anti-CD4 Ab and anti-IL-17 Ab and then analyzed by flow cytometry (B). For the post-treated experiment, the polarized Th17 cells were treated with or without compound 1 for 16 h. After incubation, cells were restimulated with PMA and ionomycin for 5 h, and then cell viability was measured by staining with 1 μg/mL propidium iodide (C). The IL-17 secreting cells were stained with anti-CD4 Ab and anti-IL-17 Ab and then analyzed by flow cytometry (D). The data shown are representative of three independent experiments. All statistical tests were performed by the student's t-test at the two-tailed significance level of 0.05. **p < 0.01, and ***p < 0.001 compared with the DMSO group.

Fig. 5

Th17 cells cytotoxicity (A, C) and IL-17 protein expression (B, D) data of compound 2. For the co-treated experiment, CD4+ T cells were cultured for five days with or without compound 2 in the skewing medium during Th17 cell polarization. After five days, cell viability was measured by staining with 1 μg/mL propidium iodide (A). The IL-17 secreting cells were stained with anti-CD4 Ab and anti-IL-17 Ab and then analyzed by flow cytometry (B). For the post-treated experiment, the polarized Th17 cells were treated with or without compound 2 for 16 h. After incubation, cells were restimulated with PMA and ionomycin for 5 h, and then cell viability was measured by staining with 1 μg/mL propidium iodide (C). The IL-17 secreting cells were stained with anti-CD4 Ab and anti-IL-17 Ab and then analyzed by flow cytometry (D). The data shown are representative of three independent experiments. All statistical tests were performed by the student's t-test at the two-tailed significance level of 0.05. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the DMSO group.

Moreover, we investigated the level of IL-17A production from Th17 cells by adding compounds 1 and 2 for 16 h after Th17 polarization (post-treated experiment) (Fig. 4, Fig. 5D). Compound 1 could significantly inhibit IL-17 production (EC50 value = 5.99 μg/mL) without cytotoxicity to Th17 cells (Fig. 4C and D). However, the decrease of IL-17A protein production of compound 2 in the post-treated experiment (Fig. 5D) might be due to the cytotoxicity to Th17 cells at 25 and 50 μg/mL concentrations (Fig. 5C). Besides, it has to be mentioned that in the IL-17 luciferase reporter assay, both compounds 1 and 2 could notably inhibit the IL-17 gene expression in Jukat cells (immortalized T lymphocytes) which were transfected with IL-17 luciferase reporters (Fig. 6 ).

Fig. 6

Compounds 1 and 2 inhibited the IL-17 gene expression in the IL-17 luciferase reporter assay. IL-17Luc cells were stimulated with PMA and ionomycin, and co-treated with DMSO or indicating compounds for 5 h. Then the activity of luciferase in the transfected cells was measured with the microplate reader. Vector was control vector (pGL4.18) transfected cells. IL 17A was IL-17 promoter-luciferase gene (pGL4-hIL-17prom) transfected cells. P + I: PMA and ionomycin. The data shown are representative of three independent experiments. All statistical tests were performed by the student's t-test at the two-tailed significance level of 0.05. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the DMSO group.

In our previous study, indole alkaloids, including indigodole A (3), indigodole C (4), tryptanthrin (5), and indirubin (6) were also suggested as anti-IL-17 contributors of Qing Dai (Lee et al., 2019). Those active components were applied to chemical profile analyses using HPLC/PDA chromatograms. As shown in Fig. 7 , the HPLC chemical analysis of this TCM methanolic extract indicated active compounds 1–6 at retention times of 21–27 min (Table S1).

Fig. 7

HPLC/UV chemical profile of Qing Dai methanolic crude extract with active indole alkaloids. The detection wavelength is at 254 nm.

Plausible biogenetic pathways for active indole alkaloids of Qing Dai

Qing Dai is manufactured by a traditional process from indigo plants such as S. cusia. The fresh botanical aerial portion containing leaves with stems are immersed in water to rot by the enzymes from itself or microorganisms. After that, the tissue residues are taken out and lime (CaCO3) is added. The mixture is constantly stirred that makes oxygen in air to take part in. Consequently, a lot of foam on the liquid surface will be generated, collected, and further dried to prepare the pale blue to grayish-blue powder that is used as TCM named Qing Dai (Lee et al., 2019; Lin, 2016; Pan et al., 2018).

It is possible to describe the biosynthetic origins of indole alkaloids of Qing Dai on the basis of three major factors, precursor indole components from S. cusia, oxygen in air, and alkali condition. In the proposed mechanisms (Fig. 8, Fig. 9, S18), indole will be oxidized to indolone, cytochrome P-450 (Cyt P-450) is a monooxygenase for oxidation, acetyl-CoA could offer ethyl group, and NADPH (nicotinamide adenine dinucleotide phosphate) will give hydrogen for reduction reaction. The plausible biogenetic mechanisms of active indigodoles A, C, and D are shown in Fig. 8, Fig. 9 , respectively, in addition, those of indirubin, tryptanthrin, and indigodole B, that a tryptanthrin derivative without anti-IL-17 property in our previous investigation, are shown in Fig. S18.

Fig. 8

Plausible biogenetic pathways for indigodoles A (3) and D (1).

Fig. 9

Plausible biogenetic pathway for indigodole C (4).

Conclusions

Qing Dai, a processed drug from indigo plants, has been used in modern clinical therapy for many inflammation diseases. But their active components were reported only rarely and we believe they are worthy of more investigations. In this study, two indole alkaloids, including new indigodole D (1), were obtained from Qing Dai and both isolates showed inhibition against IL-17A gene expression, especially 1 also showed dose-dependent inhibition on IL-17A protein production. Besides, indole alkaloids indigodole A, indigodole C, tryptanthrin, and indirubin could contribute to the anti-IL-17A properties of Qing Dai as well. The possible biogenetic mechanisms of above-mentioned indoles were also speculated by us and could provide valuable information for medicinal chemists to make more active Qing Dai indole analogues. Structure-activity relationships and IL-17 mechanism of action will be needed to further evaluate and clearly clarify for further clinical application.

Author contributions

Conceived and designed the experiments: CLL and HRY. Performed the experiments: CLL, CMW, YCS, YLC, CJC. Analyzed the data: CLL, YHK, HRY, CJC. Wrote the paper: CLL, YHK, HRY, YCS.

Declaration of competing interest

The authors declare no conflict of interest.