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Indigo Naturalis constituents in mice
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Comparison of the anti-colitis activities of Qing Dai/Indigo Naturalis constituents in mice Kei Ozawa a, 1, Daichi Mori a, 1, Ayumu Hatanaka a, Toshinori Sawano a, Jin Nakatani a, Yukinobu Ikeya c, Mikio Nishizawa b, Hidekazu Tanaka a, * a b c
Pharmacology Laboratory, Department of Biomedical Sciences, College of Life Sciences, Ritsumeikan University, Shiga, 525-8577, Japan Medical Chemistry Laboratory, Department of Biomedical Sciences, College of Life Sciences, Ritsumeikan University, Shiga, 525-8577, Japan Center for Supporting Pharmaceutical Education, Daiichi University of Pharmacy, Fukuoka, 815-8511, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 October 2019 Received in revised form 26 December 2019 Accepted 7 January 2020 Available online xxx
Qing Dai/Indigo Naturalis (QD) has been shown to ameliorate ulcerative colitis (UC) in clinical trials; however, its mechanism remains elusive. This study investigates the effects of QD on murine dextran sulfate sodium salt-induced colitis. Oral administration of QD protected the animals from colitis as manifested by weight loss, diarrhea, and rectal bleeding. QD was distinguishingly more effective than 5aminosalicylate. Focused microarray analysis of genes expressed in the distal colon suggested that QD influences the inflammatory pathway. Anti-inflammatory activity of QD was confirmed by the suppression of nitric oxide (NO) production in response to interleukin-1b in cultured hepatocytes. Some of the constituents in QD, such as tryptanthrin (TRYP) and indigo, suppressed NO production. TRYP maintained body weight but did not inhibit bleeding. Indigo, on the other hand, partially ameliorated bleeding, but did not maintain body weight. The combination of TRYP and indigo did not show additive ameliorating activity. The methanol extract of QD showed an anti-colitis activity like that of TRYP. In contrast, the methanol-insoluble QD fraction moderately ameliorated diarrhea and bleeding. Combining these two fractions resulted in full anti-colitis activity. Further clarification of the active constituents will help in the discovery of a safe and potent prescription for UC. © 2020 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: Ulcerative colitis Dextran sulfate sodium salt Qing Dai/Indigo Naturalis Tryptanthrin NO
1. Introduction A wide variety of medications has been used to treat ulcerative colitis (UC), including anti-tumor necrosis factor (TNF)-a antibodies, corticosteroids, and 5-aminosalicylates (5-ASA). However, UC patients often relapse, with some cases requiring colectomies. As such, a new treatment option that is effective for refractory cases is anticipated. In recent years, herbal medicines have increasingly been applied in patients with inflammatory bowel disease.1e6 Indigo Naturalis/Qing Dai (QD) is obtained from the extract of plants, such as Indigofera tinctoria, Isatis tinctoria, and Polygonum
* Corresponding author. Department of Biomedical Sciences, College of Life Sciences, Ritsumeikan University, 1-1-1 Noji Higashi, Kusatsu, Shiga, 525-8577, Japan. Fax: þ81-77-599-4326. E-mail address: [email protected] (H. Tanaka). Peer review under responsibility of Japanese Pharmacological Society. 1 These two authors contributed equally to this work.
tinctorium. QD has long been used to treat various inflammatory diseases, including psoriasis.7 QD has recently been reported to ameliorate UC in several clinical studies.3,5,8,9 In addition, the suppository of Xilei San, a mixed herbal prescription containing QD, has also been revealed as effective for ulcerative proctitis.1 The anticolitis activity of QD has been confirmed in animal models.10e12 Tryptanthrin (TRYP) and indigo are the main constituents of QD.12 Mouse experimental colitis revealed that TRYP enhances recovery.13,14 Indigo also ameliorates mouse colitis.10 QD is thereby a potential treatment option for UC; however, its mechanism of action is still unknown. This study investigates the effect of QD and its main constituents on murine dextran sulfate sodium salt (DSS)-induced colitis. First, the potency of QD was compared with those of 5-ASA drugs. Second, anti-inflammatory activities of QD and its constituents were examined in focused microarray analysis and interleukin-1b (IL-1b)-induced nitric oxide (NO) production assay. Finally, anti-colitis effects of these constituents were tested in mouse DSS models. This study suggests that
https://doi.org/10.1016/j.jphs.2020.01.003 1347-8613/© 2020 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: Ozawa K et al., Comparison of the anti-colitis activities of Qing Dai/Indigo Naturalis constituents in mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2020.01.003
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the integration of the activity of multiple constituents in QD is important to exert the strong anti-colitis activity.
2. Materials and methods 2.1. QD A QD sample was purchased from Tochimoto Tenkaido (Osaka, Japan). A voucher sample was deposited in the Ritsumeikan Herbarium of Pharmacognosy, Ritsumeikan University, under the code number RIN-QD-101.
2.2. Analyses of TRYP and indigo TRYP and indigo in QD were analyzed by the liquid chromatographyetandem mass spectrometry (LC-MS/MS) system (LCMS-8040, Shimadzu, Kyoto, Japan). The chromatographic separation was performed using a TSKgel ODS-100V (150 2.0 mm, 5 mm) column (Tosoh, Tokyo, Japan) under isocratic flow of water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid (1:1) at 35 C (0.2 mL/min). The LC eluent was introduced into a mass spectrometer for multiple reaction monitoring using positive ion mode with electrospray ionization. TRYP (Combi-Blocks, CA, USA) was dissolved in methanol to make the stock solutions of 0.1291, 1.291, and 12.91 mg/mL. Indigo (SigmaeAldrich, Tokyo, Japan) was dissolved in a mixture of dimethyl sulfoxide (DMSO) and methanol (1:1) to make the stock solutions of 1.068, 10.68, and 106.8 mg/mL. Each standard solution (2 mL) was analyzed in duplicate. Retention times of TRYP and indigo were 7.4 min and 10.5 min, respectively. Calibration curve of each standard compound was calculated by plotting of peak areas (y) against a series of injection amounts (x) and the regression equation was calculated in the form y ¼ Ax: TRYP, y ¼ 3,146,290,163 x (R2 ¼ 0.999); indigo, y ¼ 30,786,920 x (R2 ¼ 0.997). QD (15.073 mg) was extracted with DMSO (5 mL) under room temperature, 2 times. The two combined extracts were made up to 100 mL with methanol. The sample solution (3 mL) was analyzed in duplicate. The peak areas of TRYP and indigo in the sample solution were fitted to the calibration curves. The amounts of TRYP and indigo in 3 mL of the sample (0.47109 mg of QD) were calculated to be 0.000079 mg and 0.199590 mg, respectively. Therefore, the contents of TRYP and indigo in QD were 0.0168% and 42.4%, respectively.
2.3. Induction of colitis Five week-old male C57BL/6JJmsSlc mice (SLC, Hamamatsu, Japan) were habituated under controlled conditions (21 C23 C) and a 12-h light/dark cycle for one week. Mice were fed a powdered rodent diet and had free access to water. All animal care and experimental procedures were approved by the Animal Care Committee of Ritsumeikan University, BiwakoKusatsu Campus. Colitis was induced by oral administration of 1.0%e1.3% (w/v) DSS (MP Biomedicals, Tokyo, Japan) dissolved in drinking water for 10 days. Isatin (Tokyo Chemical Industry, Tokyo, Japan), anthranilic acid, salicylic acid, 5-ASA (Wako Pure Chemical, Osaka, Japan), indigo, salazosulfapyridine (SigmaeAldrich), QD, and TRYP were mixed with powdered food (Table 1). The amount of each compound was adjusted to 5% of whole food weight by combining with gum arabic (Yamazen Pharmaceutical, Osaka, Japan). The average dietary intake of a normal mouse (20 g) was approximately 2.5 g/day. Thus, the food containing 5% compound is estimated to yield an intake of 7.5 g/kg/day of the compound. Weight loss greater than 20% was set as the humane endpoint by euthanization. 2.4. Scoring the diarrhea and rectal bleeding Diarrhea was scored after the observation of stool as follows: normal 0, sticky 2, highly wet 4, and liquid 6. Rectal bleeding was scored as follows: normal 0, blood is contaminated in stool 2, and gross bleeding from the anus 4. Blinded examiners performed the scorings. 2.5. Histology The distal colon was excised, immersed in 4.0% paraformaldehyde for 24 h, embedded in paraffin, sliced, and stained with hematoxylin and eosin. Histological score was determined by summing scores for epithelial damage (normal 0, crypt or goblet cell damage 1, epithelial loss < 10% 2, epithelial loss S 10% 3) and cell infiltration (normal 0, mucosa 1, submucosa 2, muscularis 3). 2.6. Focused microarray analysis TRIzol (Thermo Fischer Scientific, Tokyo, Japan) was used to purify total RNA from colon tissues. The resultant RNA samples were pooled in each group and subjected to microarray analyses
Table 1 Drugs administered through drinking water and powdered food. Group
Drinking water
Powdered food
Control DSS QD SASP 5-ASA methanol-soluble methanol-insoluble methanol-soluble þ insoluble TRYP Indigo TRYP þ Indigo
tap water 1.0%e1.3% 1.0%e1.3% 1.0%e1.3% 1.0%e1.3% 1.0%e1.3% 1.0%e1.3% 1.0%e1.3% 1.0%e1.3% 1.0%e1.3% 1.0%e1.3%
5.0% gum arabica 5.0% gum arabic 5.0% QD 4.84% gum arabic þ0.16% SASP 4.84% gum arabic þ0.16% 5-ASA gum arabic þ methanol-soluble fraction of QD (5%)c gum arabic þ methanol-insoluble fraction of QD (5%)c Methanol-soluble þ -insoluble fractions of QD (5%)d 4.92% gum arabic þ 0.08% TRYP 4.2% gum arabic þ 0.8% Indigo 4.12% gum arabic þ 0.08% TRYP þ 0.8% Indigo
DSSb DSS DSS DSS DSS DSS DSS DSS DSS DSS
a
0.75 g of gum arabic was combined with 14.25 g of powdered food. The optimum concentration of DSS varies between lots of DSS. In each set of experiment, the identical concentrations of DSS were adopted so that comparison was possible between experimental groups. c QD powder (0.75 g) was extracted with methanol, and centrifuged. The dried supernatant and insoluble pellet were combined with gum arabic to constitute 0.75 g of mixtures. The resultant mixtures were further combined with 14.25 g of powdered food. d QD powder (0.75 g) was extracted with methanol, and centrifuged. The dried supernatant and insoluble pellet were combined again, and the resultant mixture was further combined with 14.25 g of powdered food. b
Please cite this article as: Ozawa K et al., Comparison of the anti-colitis activities of Qing Dai/Indigo Naturalis constituents in mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2020.01.003
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using GENOPAL chips to monitor the mRNA expression of 209 genes involved in inflammation (Mitsubishi Rayon, Tokyo, Japan). 2.7. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) RNeasy Mini Kit (Qiagen, Tokyo, Japan) was used to extract total RNA from distal colon. The ReverTra Ace qPCR RT Kit (Toyobo Life Science, Osaka, Japan) was used to synthesize the complementary DNA. qRT-PCRs were performed in triplicate using primers (iNOS, 50 -AAGGCCACATCGGATTTCACT-30 , 50 -GATGGACCCCAAGCAAGACT T-30 ; b-actin, 50 -TCGTTGCCGGTCCACACCCG-30 , 50 -TCTGGGCCT CGTCACCCACA-30 ), THUNDERBIRD SYBR qPCR Mix (Toyobo Life Science), and a StepOnePlus Real-Time PCR System (Thermo Fischer Scientific), and semi-quantified by the DDCt method. 2.8. Estimation of NO production in hepatocytes Collagenase perfusion was used to isolate hepatocytes from the livers of five week-old male Wistar rats (Charles River Laboratories, Yokohama, Japan).15 The cells (1.2 106 cells/dish) cultured overnight in Williams’ E medium were treated with 1 nM rat IL-1b (PeproTech, NJ, USA) in the presence of QD or its constituent for 8 h. The medium (150 mL) was mixed with 150 mL of the Griess reagent [0.5% sulfanilamide, 0.05% N-(1-naphthyl) ethylenediamine, and 2.5% phosphoric acid].16 Absorbance at 540 nm was measured after incubation at room temperature for 5 min. A lactate dehydrogenase (LDH) cytotoxicity detection kit (Takara Bio, Shiga, Japan) was used to monitor cytotoxicity. 3. Results 3.1. QD shows outstanding anti-colitis activity Orally administered DSS causes chemical injury to the intestinal epithelium, resulting in exposure of the lamina propria and submucosal compartment to luminal antigens and enteric bacteria; thereby, triggering inflammation.17 Male mice are more susceptible to DSS than female.18 It was found that oral administration of 1.0%e 1.3% DSS in drinking water led to a gradual loss of body weight, as well as deteriorating diarrhea and bleeding (Fig. 1A, C, D). Thirtynine percent of the DSS-treated mice were euthanized because of body weight loss of over 20% (Fig. 1B). The optimum concentrations of DSS vary between 1.0% and 1.3%, depending on the lot of DSS. The colon tissues in the DSS-treated mice showed severe epithelial injury and inflammatory cell infiltration (Fig. 1E and F). It was found that body weight was maintained, and diarrhea and bleeding were prevented when mice were fed QD-containing food during the DSS treatment (Fig. 1AeD). On day 10, the body weights of DSS þ QD-treated mice were significantly higher than those of DSS-treated mice (p < 0.001). None of the DSS þ QD-treated mice showed over 20% body weight loss (Fig. 1B). The colon histology of DSS þ QD-treated mice was better than that of DSS-treated mice and indistinguishable from normal mice (Fig. 1E and F). QD showed outstanding anti-colitis activity compared to the conventional therapies, such as 5-ASA and salazosulfapyridine (Fig. 1AeD). QD is also effective during the recovery phase after the termination of DSS administration, and for the 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis.10 This indicates that the direct chemical interference of DSS by QD if it exists, does not completely explain the ameliorating activity of QD.
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3.2. QD suppresses inflammatory gene expression We isolated RNA from the distal colon of DSS-treated mice and DSS þ QD-treated mice in order to gain insight into the biological responses upon treatment with QD. Microarrays that were focused on inflammatory genes were used to analyze the RNA. We first pooled the genes that were induced more than 8 (¼ 23) times by DSS-treatment. Among them, we listed the genes whose expressions were strongly suppressed by co-administration of QD (Table 2). For example, matrix metalloproteinase-3 (MMP-3) in DSS-treated mice was expressed 498 (¼28.96) times higher than the control but remained as low as 2.46 (¼21.30) times by coadministration of QD. MMP-3 releases TNF-a, and induces prostaglandin-endoperoxide synthase 2 (PTGS2), also known as cyclooxygenase-2 (COX-2).19 In fact, DSS treatment induced PTGS2/ COX-2 (72.5 [ ¼ 26.18] times induction), which was strongly suppressed by QD (1.61 [ ¼ 20.69] times induction) (Table 2). The proinflammatory cytokine IL-1b and IL-1 receptor type 1 (IL-1R1) were also induced by DSS and suppressed by QD (Table 2). The ameliorating activity of QD against DSS-induced colitis may at least, in part, involve an anti-inflammatory effect. 3.3. QD suppresses IL-1b-induced NO production Suppression of inflammatory gene expressions does not necessarily indicate the direct anti-inflammatory activity of QD. We, therefore, examined if QD directly suppresses the inflammatory pathway in vitro. IL-1b (37.5 [ ¼ 25.23] times induction with DSS, Table 2) has a wide spectrum of inflammatory, metabolic, haematopoietic, and immunological properties. Some of these properties are mediated by NO.20 In fact, qRT-PCR analysis of DSS-treated mouse colon revealed 29.2 times induction of inducible nitric oxide synthase (iNOS) (p < 0.005 vs. control, n ¼ 8 mice); DSS þ QDtreated colon expressed only 5.95 times (p < 0.05 vs. DSS, n ¼ 7). The IL-1b-induced NO production can be reproduced in ratcultured intestinal epithelial cells.21 Furthermore, a similar experimental system with more profound response has been established using rat hepatocytes and is used as one of the most sensitive model systems to characterize various anti-inflammatory drugs.22 QD and IL-1b were applied to the culture medium of hepatocytes, and NO2 (a stable metabolite of NO) levels were measured in the medium after 8 h of incubation. QD showed a dose-dependent inhibition of NO production (Fig. 2A). QD was not toxic to hepatocytes because LDH activity in the medium was low (Fig. 2B). The results suggest that QD contained active constituents that directly suppress NO production. The constituents in QD have been identified.12 They include isatin, TRYP, indigo, indican, indirubin, anthranilic acid, and salicylic acid. Some of the constituents have been shown as effective regarding experimental colitis or inflammation.10,13,14,23e27 The anti-inflammatory activities of TRYP, indigo, anthranilic acid, and salicylic acid were then tested in the IL-1b-induced NO production assay. TRYP (IC50 ¼ 34.5 ± 10.5 mM [mean ± S.D.]) and indigo (IC50 ¼ 23.1 ± 11.2 mM) significantly suppressed NO production (Fig. 2), whereas anthranilic acid did not (data not shown). Salicylic acid suppressed NO production with low potency as reported previously.28 We performed LC-MS/MS analyses and found that TRYP and indigo comprised 0.0168% and 42.4% of QD, respectively. Thus, the estimated in vivo level of TRYP by orally administered QD was less than 15% of IC50 against NO production. By contrast, the estimated in vivo level of indigo was apparently higher than IC50, although indigo may not be absorbed because of its poor solubility.
Please cite this article as: Ozawa K et al., Comparison of the anti-colitis activities of Qing Dai/Indigo Naturalis constituents in mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2020.01.003
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Fig. 1. QD shows outstanding anti-colitis activity. Mice were given tap water in the presence (DSS, DSS þ QD, DSS þ SASP, DSS þ 5-ASA) or absence (Control) of DSS. DSS-treated mice were simultaneously fed powdered food containing QD, SASP, 5-ASA, or gum arabic (DSS) for 10 days. Body weight (A), survival rate (B), diarrhea score (C), and bleeding score (D) were measured daily. On day 10, mice were euthanized to analyze the histology of the distal colon (H&E staining) (E, F). Mean ± standard error (S.E.M.) (A) and mean (C, D, F) are plotted. E, the representative images are shown. DSS-treated colon showed epithelial damage and cell infiltration, which were ameliorated by co-administration of QD. The numbers of samples are 12 (Control), 18 (DSS), 15 (DSS þ QD), 6 (DSS þ SASP), and 6 (DSS þ 5-ASA). ***p < 0.001 compared with DSS. The data were analyzed using one-way or two-way repeated ANOVA followed by Dunnett's post hoc test (A), or Wilcoxon signed-rank test followed by Bonferroni correction (C, D, F), versus DSS alone group.
3.4. TRYP and indigo exert complementary anti-colitis activities Because TRYP showed potent anti-inflammatory activity (Fig. 2), we examined whether TRYP is responsible for the colitisameliorating activity of QD. The estimated amount of TRYP included in the QD mixed in powdered food (5%) was 1.26 mg/kg/ day. We fed mice with food containing 0.08% TRYP, the amount of which is approximately 95 times higher than that included in 5%
QD and is predicted to yield plasma TRYP concentration higher than the IC50 against NO production. DSS þ TRYP-treated mice maintained their body weights (Fig. 3A and B). On day 10, the body weights of DSS þ TRYP-treated mice were significantly higher than those of DSS-treated mice (p < 0.001). However, diarrhea or bleeding was not strongly suppressed (Fig. 3C and D). The colon tissues in DSS þ TRYP-treated mice showed severe epithelial injury and inflammatory cell infiltration (Fig. 3E and F).
Please cite this article as: Ozawa K et al., Comparison of the anti-colitis activities of Qing Dai/Indigo Naturalis constituents in mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2020.01.003
K. Ozawa et al. / Journal of Pharmacological Sciences xxx (xxxx) xxx Table 2 Genes induced by DSS and suppressed by QD in focused microarray.
Mmp 3 Il6 Cxcl2 Ptgs2 Timp1 Mmp 9 Csf3 Lox Ccl7 Ccl11 Hspb1 Ier 3 Spp1 Il1b Il1r1
DSS
DSS þ QD
DSS þ TRYP
8.96 6.11 7.59 6.18 6.30 5.04 3.93 5.11 5.44 4.11 3.57 4.55 3.48 5.23 3.57
1.30 0.28 1.37 0.69 1.12 0.51 0.45 0.93 1.34 0.31 0.07 0.98 0.07 1.69 0.27
4.71 1.19 3.78 1.75 1.98 1.94 0.29 1.37 1.89 0.45 0.39 1.05 0.27 4.29 0.99
Fold induction of each gene compared with control are shown as log2 values.
Consistent with above observations, focused microarrays revealed that TRYP suppressed only a subset of the DSS-induced genes that were suppressed by QD (Table 2). For example, IL-6, PTGS2/COX-2, TIMP-1, CSF3, LOX, CCL7, and CCL11 expressions were
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suppressed by TRYP. By contrast, MMP-3 or MMP-9 was not so strongly suppressed. Failure to suppress MMPs may cause insufficient protection of basement membranes, resulting in diarrhea, bleeding, and epithelial injury. Indigo (0.8% of food, corresponded to 38% of indigo contained in 5% QD) suppressed bleeding, but it did not strongly suppress body weight loss or diarrhea (Fig. 3AeD). Indigo improved colon histology (Fig. 3E and F), but 11% of the indigo-treated mice were euthanized because of their body weight loss of over 20% (Fig. 3B). Other constituents, such as isatin, anthranilic acid, and salicylic acid did not strongly inhibit the colitis induced by DSS (Fig. 3, data not shown). TRYP and indigo exerted apparently complementary anti-colitis activities. However, the combination of TRYP and indigo did not show additive activity but instead showed a weaker effect than single treatments. TRYP þ indigo partially ameliorated diarrhea, bleeding, and colon histology (Fig. 3CeF) but did not improve body weight loss (Fig. 3A). Seventeen percent of the TRYP þ indigotreated mice were euthanized because of their body weight loss of more than 20% (Fig. 3B). Thus, TRYP and indigo show strong antiinflammatory activities, maintaining the homeostasis and the local injury of DSS-treated mice, respectively; however, they do not completely explain the anti-colitis activity of QD.
Fig. 2. QD suppresses IL-1b induced nitric oxide production. Rat hepatocytes were treated with 1 nM IL-1b in the presence or absence of QD, TRYP, or indigo (INDG) for 8 h. A, the levels of NO (as nitrite) in the medium were measured. B, the LDH activities in the medium were measured. The total LDH activity of the whole-cell extract (WCE) was defined as 100%. The data represent mean ± standard deviation (n ¼ 3).
Please cite this article as: Ozawa K et al., Comparison of the anti-colitis activities of Qing Dai/Indigo Naturalis constituents in mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2020.01.003
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Fig. 3. TRYP and indigo exert complementary anti-colitis activities. Mice were given tap water in the presence (DSS, DSS þ TRYP, DSS þ indigo, DSS þ TRYP þ indigo, DSS þ isatin) or absence (Control) of DSS. DSS treated mice were simultaneously fed powdered food containing TRYP, indigo, TRYP þ indigo, isatin or gum arabic (DSS) for 10 days. Body weight (A), survival rate (B), diarrhea score (C), and bleeding score (D) were measured daily. On day 10, mice were euthanized to analyze the histology of the distal colon (H&E staining) (E, F). Mean ± S.E.M. (A) and mean (C, D, F) are plotted. E, the representative images are shown. DSS þ TRYP-treated colon showed epithelial damage and cell infiltration. DSS þ indigo and DSS þ TRYP þ indigo-treated colon did not show significant epithelial damage or cell infiltration. The numbers of samples are 7 (control), 20 (DSS), 15 (DSS þ TRYP), 18 (DSS þ indigo), 18 (DSS þ TRYP þ indigo), and 6 (DSS þ isatin). *p < 0.05, **p < 0.01, ***p < 0.001 compared with DSS. The data were analyzed using one-way or two-way repeated ANOVA followed by Dunnett's post hoc test (A), or Wilcoxon signed-rank test followed by Bonferroni correction (C, D, F), versus DSS alone group.
3.5. TRYP-depleted QD fraction protect the colon from diarrhea and bleeding We attempted to deplete TRYP from QD to investigate the activity of QD that is not explained by TRYP. Because TRYP is very soluble in methanol,29 QD was extracted by using methanol. Mice
treated with DSS and methanol-soluble QD extract (TRYP 1.26 mg/ kg/day, indigo 0) maintained their body weights (Fig. 4A and B). On day 10, the body weights of DSS þ methanol-soluble QD extracttreated mice were significantly higher than those of mice treated with DSS alone (p < 0.05). However, diarrhea and bleeding were severer compared with mice treated with DSS þ whole QD (Fig. 4C
Please cite this article as: Ozawa K et al., Comparison of the anti-colitis activities of Qing Dai/Indigo Naturalis constituents in mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2020.01.003
K. Ozawa et al. / Journal of Pharmacological Sciences xxx (xxxx) xxx
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Fig. 4. Methanol-insoluble QD fraction protect the colon from local injuries. Mice were given tap water in the presence (DSS, DSS þ methanol-soluble QD fraction, DSS þ methanolinsoluble QD fraction, DSS þ methanol-soluble QD fraction þ methanol-insoluble QD fraction) or absence (Control) of DSS. DSS-treated mice were simultaneously fed powdered food containing the methanol-soluble QD fraction, methanol-insoluble QD fraction, the combination of methanol-soluble and -insoluble QD fractions, or gum arabic (DSS) for 10 days. Body weight (A), survival rate (B), diarrhea score (C), and bleeding score (D) were measured daily. On day 10, mice were euthanized to observe the histology of the distal colon (E, F). Mean ± S.E.M. (A), and mean (C, D, F) are plotted. E, the representative images are shown. DSS þ methanol-soluble QD fraction treated colon showed epithelial damage and cell infiltration. DSS þ methanol-insoluble QD fraction-treated colon showed little epithelial damage or cell infiltration. DSS þ the combination-treated colon showed almost no epithelial damage or cell infiltration. The numbers of samples are 9 (control), 13 (DSS), 12 (QD), 12 (methanol-soluble QD fraction), 12 (methanol-insoluble QD fraction), and 6 (combination of methanol-soluble and -insoluble fractions). *p < 0.05, **p < 0.01, ***p < 0.001 compared with DSS. The data were analyzed using one-way or two-way repeated ANOVA followed by Dunnett's post hoc test (A), or Wilcoxon signed-rank test followed by Bonferroni correction (C, D, F), versus DSS alone group.
and D). These effects of the methanol extract of QD were reminiscent of TRYP (see Fig. 3). The methanol-insoluble QD fraction (TRYP 0, indigo 3.2 g/kg/ day) exhibited distinct ameliorating activity. The fraction strongly suppressed diarrhea and bleeding, and exerted strong weight-
maintaining effect (Fig. 4AeD). This activity of the methanolinsoluble QD fraction was stronger than indigo. Full QD activity was observed with the combination of the methanol extract and methanol-insoluble fraction. The combination-treated mice maintained their body weight, histology,
Please cite this article as: Ozawa K et al., Comparison of the anti-colitis activities of Qing Dai/Indigo Naturalis constituents in mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2020.01.003
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and were free from diarrhea or bleeding (Fig. 4). On day 10, the body weights of DSS þ methanol-soluble fraction þ methanolinsoluble fraction-treated mice were significantly higher than mice treated with DSS alone (p < 0.001). The data suggest that methanolinsoluble fraction likely contains constituents, which are responsible for suppressing diarrhea and bleeding, other than indigo.
4. Discussion QD is highly effective in such intractable cases of UC when standard medications do not yield satisfactory results.8 This is consistent with the outstanding anti-colitis activity, which is by far more potent than 5-ASA and salazosulfapyridine (Fig. 1). Several reports demonstrate the anti-colitis activity of QD in animal models.10e12 However, this is the first study that directly compares the anti-colitis activity of QD with other drugs used to treat the same condition. Because QD is a crude plant extract, the action of each constituent must be analyzed individually. The safety of each constituent must also be examined in-depth because orally administering QD causes pulmonary hypertension.3 Since QD was first applied to UC patients,8 its constituents have been examined regarding their effectiveness for experimental colitis in animals. For example, indirubin, indigo, isatin, and TRYP have been tested in DSS- or TNBS-induced colitis models.10,14,23e26 A study also tested TRYP before the initial trial of the QD.13 However, there are no previous studies that directly compare the anti-colitis activities of these constituents in the same condition. To our knowledge, the present study is the first to directly compare their potencies. The effects of TRYP on DSS-induced colitis were mysterious. TRYP efficiently maintained body weight and the survival rate. However, TRYP was entirely ineffective against bleeding (Fig. 3). The same pattern of effect was reproduced with the methanol extract of QD, which is supposed to contain TRYP (Fig. 4). The maintenance of body weight may be explained by the antiinflammatory activity of TRYP, as shown in our study (Fig. 2) and previous reports.30,31 Failure to control rectal bleeding may partially be explained by anti-angiogenic activity and inhibition of hepatocyte growth factor induction of TRYP.32e34 Unidentified constituents that are included in the methanol-insoluble fraction of QD potentially exert anti-diarrhea and hemostatic effects. The maintenance of epithelial integrity, including the barrier function, is clearly involved in the protection or recovery from colitis. QD likely contains unidentified factors responsible for these functions. Orally administered TRYP is absorbed rapidly and targets plasma, liver, kidneys, and lungs.35 This suggests that the colon epithelium is not exposed to high concentrations of TRYP from the luminal side. QD is a powder largely insoluble in various solvents. Administering TRYP as part of the QD powder might result in the efficient delivery to the distal colon, where it might exert ameliorating activity to the epithelium directly from the intestinal lumen. In vitro and animal model experiments have confirmed that QD is a strong candidate prescription to apply for intractable UC cases. It has also become clear that QD has outstanding anti-colitis activity, which is much stronger than that of 5-ASA drugs. Some of the active constituents, including TRYP and indigo, exhibit antiinflammatory activity; however, this does not completely explain the outstanding colitis-ameliorating activity of whole QD. The high insolubility of whole QD may help with the delivery of the active constituents to the colon. Also, the additional function of unidentified constituents may involve the maintenance of intestinal barrier function. Further clarification of the active constituents, their optimum combinations, and their delivery to the affected loci will aid in the discovery of a safe and potent prescription for UC.
Declaration of Competing Interest The authors declare no competing financial interests. Acknowledgement We thank T. Okuyama, Y. Yamauchi, A. Yamamoto, N. Tanaka, R. Aoshiba, and R. Inui for discussion and technical assistance. This work was supported by MEXT-Supported Program for the Strategic Research Foundation at Private Universities (H.T.) and the AsiaJapan Research Institute of Ritsumeikan Asia-Japan Research Organization, Ritsumeikan University (Y.I. and M.N.). Abbreviations 5-ASA COX-2 DMSO DSS IFN IL-1b IL-1R1 IL-6 iNOS LC-MS/MS LDH MMP PTGS2 QD TNBS TNF-a TRYP UC
5-aminosalicylate cyclooxygenase-2 dimethyl sulfoxide dextran sulfate sodium salt interferon interleukin-1b interleukin-1 receptor type 1 interleukin-6 inducible nitric oxide synthase liquid chromatographyetandem mass spectrometry lactate dehydrogenase matrix metalloproteinase prostaglandin-endoperoxide synthase 2 Qing Dai (Indigo Naturalis) 2,4,6-trinitrobenzenesulfonic acid tumor necrosis factor-a tryptanthrin ulcerative colitis
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K. Ozawa et al. / Journal of Pharmacological Sciences xxx (xxxx) xxx 13. Micallef MJ, Iwaki K, Ishihara T, et al. The natural plant product tryptanthrin ameliorates dextran sodium sulfate-induced colitis in mice. Int Immunopharm. 2002;2(4):565e578. 14. Wang Z, Wu X, Wang CL, et al. Tryptanthrin protects mice against dextran sulfate sodium-induced colitis through inhibition of TNF-alpha/NF-kappaB and IL-6/STAT3 pathways. Molecules. 2018;23(5). 15. Kanemaki T, Kitade H, Hiramatsu Y, Kamiyama Y, Okumura T. Stimulation of glycogen degradation by prostaglandin E2 in primary cultured rat hepatocytes. Prostaglandins. 1993;45(5):459e474. 16. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem. 1982;126(1):131e138. 17. Cooper HS, Murthy SN, Shah RS, Sedergran DJ. Clinicopathologic study of dextran sulfate sodium experimental murine colitis. Lab Invest. 1993;69(2): 238e249. 18. Babickova J, Tothova L, Lengyelova E, et al. Sex differences in experimentally induced colitis in mice: a role for estrogens. Inflammation. 2015;38(5): 1996e2006. 19. Steenport M, Khan KM, Du B, Barnhard SE, Dannenberg AJ, Falcone DJ. Matrix metalloproteinase (MMP)-1 and MMP-3 induce macrophage MMP-9: evidence for the role of TNF-alpha and cyclooxygenase-2. J Immunol. 2009;183(12): 8119e8127. 20. Palmi M, Meini A. Role of the nitric oxide/cyclic GMP/Ca2þ signaling pathway in the pyrogenic effect of interleukin-1beta. Mol Neurobiol. 2002;25(2):133e147. 21. Kitagawa K, Hamada Y, Kato Y, et al. Epidermal growth factor and interleukin-1b synergistically stimulate the production of nitric oxide in rat intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol. 2004;287(6):G1188eG1193. 22. Nakajima A, Yamamoto Y, Yoshinaka N, et al. A new flavanone and other flavonoids from green perilla leaf extract inhibit nitric oxide production in interleukin 1b-treated hepatocytes. Biosci Biotechnol Biochem. 2015;79(1):138e146. 23. Gao W, Guo Y, Wang C, et al. Indirubin ameliorates dextran sulfate sodiuminduced ulcerative colitis in mice through the inhibition of inflammation and the induction of Foxp3-expressing regulatory T cells. Acta Histochem. 2016;118(6):606e614. 24. Gao W, Zhang L, Wang X, Yu L, Wang C, Gong Y. The combination of indirubin and isatin attenuates dextran sodium sulfate induced ulcerative colitis in mice. Biochem Cell Biol. 2018;96(5):636e645.
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25. Socca EA, Luiz-Ferreira A, de Faria FM, et al. Inhibition of tumor necrosis factoralpha and cyclooxigenase-2 by Isatin: a molecular mechanism of protection against TNBS-induced colitis in rats. Chem Biol Interact. 2014;209:48e55. 26. Tokuyasu N, Shomori K, Amano K, et al. Indirubin, a constituent of the Chinese herbal medicine qing-dai, attenuates dextran sulfate sodium-induced murine colitis. Yonago Acta Med. 2018;61(2):128e136. 27. Wen Q, Zhou L, Chen H, Zhang Y. N-(3', 4'-dimethoxycinnamonyl) anthranilic acid alleviated experimental colitis by inhibiting autoimmune response and inducing CD4þ CD25þ regulatory T cells production. J Gastroenterol Hepatol. 2013;28(8):1330e1338. 28. Sakitani K, Kitade H, Inoue K, et al. The anti-inflammatory drug sodium salicylate inhibits nitric oxide formation induced by interleukin-1b at a translational step, but not at a transcriptional step, in hepatocytes. Hepatology. 1997;25(2):416e420. 29. Liau BC, Jong TT, Lee MR, Chen SS. LC-APCI-MS method for detection and analysis of tryptanthrin, indigo, and indirubin in daqingye and banlangen. J Pharmaceut Biomed Anal. 2007;43(1):346e351. 30. Ishihara T, Kohno K, Ushio S, Iwaki K, Ikeda M, Kurimoto M. Tryptanthrin inhibits nitric oxide and prostaglandin E(2) synthesis by murine macrophages. Eur J Pharmacol. 2000;407(1e2):197e204. 31. Recio MC, Cerda-Nicolas M, Potterat O, Hamburger M, Rios JL. Anti-inflammatory and antiallergic activity in vivo of lipophilic Isatis tinctoria extracts and tryptanthrin. Planta Med. 2006;72(6):539e546. 32. Chang HN, Huang ST, Yeh YC, et al. Indigo naturalis and its component tryptanthrin exert anti-angiogenic effect by arresting cell cycle and inhibiting Akt and FAK signaling in human vascular endothelial cells. J Ethnopharmacol. 2015;174:474e481. 33. Liao X, Zhou X, Mak NK, Leung KN. Tryptanthrin inhibits angiogenesis by targeting the VEGFR2-mediated ERK1/2 signalling pathway. PloS One. 2013;8(12), e82294. 34. Motoki T, Takami Y, Yagi Y, Tai A, Yamamoto I, Gohda E. Inhibition of hepatocyte growth factor induction in human dermal fibroblasts by tryptanthrin. Biol Pharm Bull. 2005;28(2):260e266. 35. Zhang X, Xia J, Zhang W, Luo Y, Sun W, Zhou W. Study on pharmacokinetics and tissue distribution of single dose oral tryptanthrin in Kunming mice by validated reversed-phase high-performance liquid chromatography with ultraviolet detection. Integr Med Res. 2017;6(3):269e279.
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Comparison of the anti-colitis activities of Qing Dai/Indigo Naturalis constituents in mice Kei Ozawa a, 1, Daichi Mori a, 1, Ayumu Hatanaka a, Toshinori Sawano a, Jin Nakatani a, Yukinobu Ikeya c, Mikio Nishizawa b, Hidekazu Tanaka a, * a b c
Pharmacology Laboratory, Department of Biomedical Sciences, College of Life Sciences, Ritsumeikan University, Shiga, 525-8577, Japan Medical Chemistry Laboratory, Department of Biomedical Sciences, College of Life Sciences, Ritsumeikan University, Shiga, 525-8577, Japan Center for Supporting Pharmaceutical Education, Daiichi University of Pharmacy, Fukuoka, 815-8511, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 October 2019 Received in revised form 26 December 2019 Accepted 7 January 2020 Available online xxx
Qing Dai/Indigo Naturalis (QD) has been shown to ameliorate ulcerative colitis (UC) in clinical trials; however, its mechanism remains elusive. This study investigates the effects of QD on murine dextran sulfate sodium salt-induced colitis. Oral administration of QD protected the animals from colitis as manifested by weight loss, diarrhea, and rectal bleeding. QD was distinguishingly more effective than 5aminosalicylate. Focused microarray analysis of genes expressed in the distal colon suggested that QD influences the inflammatory pathway. Anti-inflammatory activity of QD was confirmed by the suppression of nitric oxide (NO) production in response to interleukin-1b in cultured hepatocytes. Some of the constituents in QD, such as tryptanthrin (TRYP) and indigo, suppressed NO production. TRYP maintained body weight but did not inhibit bleeding. Indigo, on the other hand, partially ameliorated bleeding, but did not maintain body weight. The combination of TRYP and indigo did not show additive ameliorating activity. The methanol extract of QD showed an anti-colitis activity like that of TRYP. In contrast, the methanol-insoluble QD fraction moderately ameliorated diarrhea and bleeding. Combining these two fractions resulted in full anti-colitis activity. Further clarification of the active constituents will help in the discovery of a safe and potent prescription for UC. © 2020 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: Ulcerative colitis Dextran sulfate sodium salt Qing Dai/Indigo Naturalis Tryptanthrin NO
1. Introduction A wide variety of medications has been used to treat ulcerative colitis (UC), including anti-tumor necrosis factor (TNF)-a antibodies, corticosteroids, and 5-aminosalicylates (5-ASA). However, UC patients often relapse, with some cases requiring colectomies. As such, a new treatment option that is effective for refractory cases is anticipated. In recent years, herbal medicines have increasingly been applied in patients with inflammatory bowel disease.1e6 Indigo Naturalis/Qing Dai (QD) is obtained from the extract of plants, such as Indigofera tinctoria, Isatis tinctoria, and Polygonum
* Corresponding author. Department of Biomedical Sciences, College of Life Sciences, Ritsumeikan University, 1-1-1 Noji Higashi, Kusatsu, Shiga, 525-8577, Japan. Fax: þ81-77-599-4326. E-mail address: [email protected] (H. Tanaka). Peer review under responsibility of Japanese Pharmacological Society. 1 These two authors contributed equally to this work.
tinctorium. QD has long been used to treat various inflammatory diseases, including psoriasis.7 QD has recently been reported to ameliorate UC in several clinical studies.3,5,8,9 In addition, the suppository of Xilei San, a mixed herbal prescription containing QD, has also been revealed as effective for ulcerative proctitis.1 The anticolitis activity of QD has been confirmed in animal models.10e12 Tryptanthrin (TRYP) and indigo are the main constituents of QD.12 Mouse experimental colitis revealed that TRYP enhances recovery.13,14 Indigo also ameliorates mouse colitis.10 QD is thereby a potential treatment option for UC; however, its mechanism of action is still unknown. This study investigates the effect of QD and its main constituents on murine dextran sulfate sodium salt (DSS)-induced colitis. First, the potency of QD was compared with those of 5-ASA drugs. Second, anti-inflammatory activities of QD and its constituents were examined in focused microarray analysis and interleukin-1b (IL-1b)-induced nitric oxide (NO) production assay. Finally, anti-colitis effects of these constituents were tested in mouse DSS models. This study suggests that
https://doi.org/10.1016/j.jphs.2020.01.003 1347-8613/© 2020 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: Ozawa K et al., Comparison of the anti-colitis activities of Qing Dai/Indigo Naturalis constituents in mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2020.01.003
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the integration of the activity of multiple constituents in QD is important to exert the strong anti-colitis activity.
2. Materials and methods 2.1. QD A QD sample was purchased from Tochimoto Tenkaido (Osaka, Japan). A voucher sample was deposited in the Ritsumeikan Herbarium of Pharmacognosy, Ritsumeikan University, under the code number RIN-QD-101.
2.2. Analyses of TRYP and indigo TRYP and indigo in QD were analyzed by the liquid chromatographyetandem mass spectrometry (LC-MS/MS) system (LCMS-8040, Shimadzu, Kyoto, Japan). The chromatographic separation was performed using a TSKgel ODS-100V (150 2.0 mm, 5 mm) column (Tosoh, Tokyo, Japan) under isocratic flow of water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid (1:1) at 35 C (0.2 mL/min). The LC eluent was introduced into a mass spectrometer for multiple reaction monitoring using positive ion mode with electrospray ionization. TRYP (Combi-Blocks, CA, USA) was dissolved in methanol to make the stock solutions of 0.1291, 1.291, and 12.91 mg/mL. Indigo (SigmaeAldrich, Tokyo, Japan) was dissolved in a mixture of dimethyl sulfoxide (DMSO) and methanol (1:1) to make the stock solutions of 1.068, 10.68, and 106.8 mg/mL. Each standard solution (2 mL) was analyzed in duplicate. Retention times of TRYP and indigo were 7.4 min and 10.5 min, respectively. Calibration curve of each standard compound was calculated by plotting of peak areas (y) against a series of injection amounts (x) and the regression equation was calculated in the form y ¼ Ax: TRYP, y ¼ 3,146,290,163 x (R2 ¼ 0.999); indigo, y ¼ 30,786,920 x (R2 ¼ 0.997). QD (15.073 mg) was extracted with DMSO (5 mL) under room temperature, 2 times. The two combined extracts were made up to 100 mL with methanol. The sample solution (3 mL) was analyzed in duplicate. The peak areas of TRYP and indigo in the sample solution were fitted to the calibration curves. The amounts of TRYP and indigo in 3 mL of the sample (0.47109 mg of QD) were calculated to be 0.000079 mg and 0.199590 mg, respectively. Therefore, the contents of TRYP and indigo in QD were 0.0168% and 42.4%, respectively.
2.3. Induction of colitis Five week-old male C57BL/6JJmsSlc mice (SLC, Hamamatsu, Japan) were habituated under controlled conditions (21 C23 C) and a 12-h light/dark cycle for one week. Mice were fed a powdered rodent diet and had free access to water. All animal care and experimental procedures were approved by the Animal Care Committee of Ritsumeikan University, BiwakoKusatsu Campus. Colitis was induced by oral administration of 1.0%e1.3% (w/v) DSS (MP Biomedicals, Tokyo, Japan) dissolved in drinking water for 10 days. Isatin (Tokyo Chemical Industry, Tokyo, Japan), anthranilic acid, salicylic acid, 5-ASA (Wako Pure Chemical, Osaka, Japan), indigo, salazosulfapyridine (SigmaeAldrich), QD, and TRYP were mixed with powdered food (Table 1). The amount of each compound was adjusted to 5% of whole food weight by combining with gum arabic (Yamazen Pharmaceutical, Osaka, Japan). The average dietary intake of a normal mouse (20 g) was approximately 2.5 g/day. Thus, the food containing 5% compound is estimated to yield an intake of 7.5 g/kg/day of the compound. Weight loss greater than 20% was set as the humane endpoint by euthanization. 2.4. Scoring the diarrhea and rectal bleeding Diarrhea was scored after the observation of stool as follows: normal 0, sticky 2, highly wet 4, and liquid 6. Rectal bleeding was scored as follows: normal 0, blood is contaminated in stool 2, and gross bleeding from the anus 4. Blinded examiners performed the scorings. 2.5. Histology The distal colon was excised, immersed in 4.0% paraformaldehyde for 24 h, embedded in paraffin, sliced, and stained with hematoxylin and eosin. Histological score was determined by summing scores for epithelial damage (normal 0, crypt or goblet cell damage 1, epithelial loss < 10% 2, epithelial loss S 10% 3) and cell infiltration (normal 0, mucosa 1, submucosa 2, muscularis 3). 2.6. Focused microarray analysis TRIzol (Thermo Fischer Scientific, Tokyo, Japan) was used to purify total RNA from colon tissues. The resultant RNA samples were pooled in each group and subjected to microarray analyses
Table 1 Drugs administered through drinking water and powdered food. Group
Drinking water
Powdered food
Control DSS QD SASP 5-ASA methanol-soluble methanol-insoluble methanol-soluble þ insoluble TRYP Indigo TRYP þ Indigo
tap water 1.0%e1.3% 1.0%e1.3% 1.0%e1.3% 1.0%e1.3% 1.0%e1.3% 1.0%e1.3% 1.0%e1.3% 1.0%e1.3% 1.0%e1.3% 1.0%e1.3%
5.0% gum arabica 5.0% gum arabic 5.0% QD 4.84% gum arabic þ0.16% SASP 4.84% gum arabic þ0.16% 5-ASA gum arabic þ methanol-soluble fraction of QD (5%)c gum arabic þ methanol-insoluble fraction of QD (5%)c Methanol-soluble þ -insoluble fractions of QD (5%)d 4.92% gum arabic þ 0.08% TRYP 4.2% gum arabic þ 0.8% Indigo 4.12% gum arabic þ 0.08% TRYP þ 0.8% Indigo
DSSb DSS DSS DSS DSS DSS DSS DSS DSS DSS
a
0.75 g of gum arabic was combined with 14.25 g of powdered food. The optimum concentration of DSS varies between lots of DSS. In each set of experiment, the identical concentrations of DSS were adopted so that comparison was possible between experimental groups. c QD powder (0.75 g) was extracted with methanol, and centrifuged. The dried supernatant and insoluble pellet were combined with gum arabic to constitute 0.75 g of mixtures. The resultant mixtures were further combined with 14.25 g of powdered food. d QD powder (0.75 g) was extracted with methanol, and centrifuged. The dried supernatant and insoluble pellet were combined again, and the resultant mixture was further combined with 14.25 g of powdered food. b
Please cite this article as: Ozawa K et al., Comparison of the anti-colitis activities of Qing Dai/Indigo Naturalis constituents in mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2020.01.003
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using GENOPAL chips to monitor the mRNA expression of 209 genes involved in inflammation (Mitsubishi Rayon, Tokyo, Japan). 2.7. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) RNeasy Mini Kit (Qiagen, Tokyo, Japan) was used to extract total RNA from distal colon. The ReverTra Ace qPCR RT Kit (Toyobo Life Science, Osaka, Japan) was used to synthesize the complementary DNA. qRT-PCRs were performed in triplicate using primers (iNOS, 50 -AAGGCCACATCGGATTTCACT-30 , 50 -GATGGACCCCAAGCAAGACT T-30 ; b-actin, 50 -TCGTTGCCGGTCCACACCCG-30 , 50 -TCTGGGCCT CGTCACCCACA-30 ), THUNDERBIRD SYBR qPCR Mix (Toyobo Life Science), and a StepOnePlus Real-Time PCR System (Thermo Fischer Scientific), and semi-quantified by the DDCt method. 2.8. Estimation of NO production in hepatocytes Collagenase perfusion was used to isolate hepatocytes from the livers of five week-old male Wistar rats (Charles River Laboratories, Yokohama, Japan).15 The cells (1.2 106 cells/dish) cultured overnight in Williams’ E medium were treated with 1 nM rat IL-1b (PeproTech, NJ, USA) in the presence of QD or its constituent for 8 h. The medium (150 mL) was mixed with 150 mL of the Griess reagent [0.5% sulfanilamide, 0.05% N-(1-naphthyl) ethylenediamine, and 2.5% phosphoric acid].16 Absorbance at 540 nm was measured after incubation at room temperature for 5 min. A lactate dehydrogenase (LDH) cytotoxicity detection kit (Takara Bio, Shiga, Japan) was used to monitor cytotoxicity. 3. Results 3.1. QD shows outstanding anti-colitis activity Orally administered DSS causes chemical injury to the intestinal epithelium, resulting in exposure of the lamina propria and submucosal compartment to luminal antigens and enteric bacteria; thereby, triggering inflammation.17 Male mice are more susceptible to DSS than female.18 It was found that oral administration of 1.0%e 1.3% DSS in drinking water led to a gradual loss of body weight, as well as deteriorating diarrhea and bleeding (Fig. 1A, C, D). Thirtynine percent of the DSS-treated mice were euthanized because of body weight loss of over 20% (Fig. 1B). The optimum concentrations of DSS vary between 1.0% and 1.3%, depending on the lot of DSS. The colon tissues in the DSS-treated mice showed severe epithelial injury and inflammatory cell infiltration (Fig. 1E and F). It was found that body weight was maintained, and diarrhea and bleeding were prevented when mice were fed QD-containing food during the DSS treatment (Fig. 1AeD). On day 10, the body weights of DSS þ QD-treated mice were significantly higher than those of DSS-treated mice (p < 0.001). None of the DSS þ QD-treated mice showed over 20% body weight loss (Fig. 1B). The colon histology of DSS þ QD-treated mice was better than that of DSS-treated mice and indistinguishable from normal mice (Fig. 1E and F). QD showed outstanding anti-colitis activity compared to the conventional therapies, such as 5-ASA and salazosulfapyridine (Fig. 1AeD). QD is also effective during the recovery phase after the termination of DSS administration, and for the 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis.10 This indicates that the direct chemical interference of DSS by QD if it exists, does not completely explain the ameliorating activity of QD.
3
3.2. QD suppresses inflammatory gene expression We isolated RNA from the distal colon of DSS-treated mice and DSS þ QD-treated mice in order to gain insight into the biological responses upon treatment with QD. Microarrays that were focused on inflammatory genes were used to analyze the RNA. We first pooled the genes that were induced more than 8 (¼ 23) times by DSS-treatment. Among them, we listed the genes whose expressions were strongly suppressed by co-administration of QD (Table 2). For example, matrix metalloproteinase-3 (MMP-3) in DSS-treated mice was expressed 498 (¼28.96) times higher than the control but remained as low as 2.46 (¼21.30) times by coadministration of QD. MMP-3 releases TNF-a, and induces prostaglandin-endoperoxide synthase 2 (PTGS2), also known as cyclooxygenase-2 (COX-2).19 In fact, DSS treatment induced PTGS2/ COX-2 (72.5 [ ¼ 26.18] times induction), which was strongly suppressed by QD (1.61 [ ¼ 20.69] times induction) (Table 2). The proinflammatory cytokine IL-1b and IL-1 receptor type 1 (IL-1R1) were also induced by DSS and suppressed by QD (Table 2). The ameliorating activity of QD against DSS-induced colitis may at least, in part, involve an anti-inflammatory effect. 3.3. QD suppresses IL-1b-induced NO production Suppression of inflammatory gene expressions does not necessarily indicate the direct anti-inflammatory activity of QD. We, therefore, examined if QD directly suppresses the inflammatory pathway in vitro. IL-1b (37.5 [ ¼ 25.23] times induction with DSS, Table 2) has a wide spectrum of inflammatory, metabolic, haematopoietic, and immunological properties. Some of these properties are mediated by NO.20 In fact, qRT-PCR analysis of DSS-treated mouse colon revealed 29.2 times induction of inducible nitric oxide synthase (iNOS) (p < 0.005 vs. control, n ¼ 8 mice); DSS þ QDtreated colon expressed only 5.95 times (p < 0.05 vs. DSS, n ¼ 7). The IL-1b-induced NO production can be reproduced in ratcultured intestinal epithelial cells.21 Furthermore, a similar experimental system with more profound response has been established using rat hepatocytes and is used as one of the most sensitive model systems to characterize various anti-inflammatory drugs.22 QD and IL-1b were applied to the culture medium of hepatocytes, and NO2 (a stable metabolite of NO) levels were measured in the medium after 8 h of incubation. QD showed a dose-dependent inhibition of NO production (Fig. 2A). QD was not toxic to hepatocytes because LDH activity in the medium was low (Fig. 2B). The results suggest that QD contained active constituents that directly suppress NO production. The constituents in QD have been identified.12 They include isatin, TRYP, indigo, indican, indirubin, anthranilic acid, and salicylic acid. Some of the constituents have been shown as effective regarding experimental colitis or inflammation.10,13,14,23e27 The anti-inflammatory activities of TRYP, indigo, anthranilic acid, and salicylic acid were then tested in the IL-1b-induced NO production assay. TRYP (IC50 ¼ 34.5 ± 10.5 mM [mean ± S.D.]) and indigo (IC50 ¼ 23.1 ± 11.2 mM) significantly suppressed NO production (Fig. 2), whereas anthranilic acid did not (data not shown). Salicylic acid suppressed NO production with low potency as reported previously.28 We performed LC-MS/MS analyses and found that TRYP and indigo comprised 0.0168% and 42.4% of QD, respectively. Thus, the estimated in vivo level of TRYP by orally administered QD was less than 15% of IC50 against NO production. By contrast, the estimated in vivo level of indigo was apparently higher than IC50, although indigo may not be absorbed because of its poor solubility.
Please cite this article as: Ozawa K et al., Comparison of the anti-colitis activities of Qing Dai/Indigo Naturalis constituents in mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2020.01.003
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Fig. 1. QD shows outstanding anti-colitis activity. Mice were given tap water in the presence (DSS, DSS þ QD, DSS þ SASP, DSS þ 5-ASA) or absence (Control) of DSS. DSS-treated mice were simultaneously fed powdered food containing QD, SASP, 5-ASA, or gum arabic (DSS) for 10 days. Body weight (A), survival rate (B), diarrhea score (C), and bleeding score (D) were measured daily. On day 10, mice were euthanized to analyze the histology of the distal colon (H&E staining) (E, F). Mean ± standard error (S.E.M.) (A) and mean (C, D, F) are plotted. E, the representative images are shown. DSS-treated colon showed epithelial damage and cell infiltration, which were ameliorated by co-administration of QD. The numbers of samples are 12 (Control), 18 (DSS), 15 (DSS þ QD), 6 (DSS þ SASP), and 6 (DSS þ 5-ASA). ***p < 0.001 compared with DSS. The data were analyzed using one-way or two-way repeated ANOVA followed by Dunnett's post hoc test (A), or Wilcoxon signed-rank test followed by Bonferroni correction (C, D, F), versus DSS alone group.
3.4. TRYP and indigo exert complementary anti-colitis activities Because TRYP showed potent anti-inflammatory activity (Fig. 2), we examined whether TRYP is responsible for the colitisameliorating activity of QD. The estimated amount of TRYP included in the QD mixed in powdered food (5%) was 1.26 mg/kg/ day. We fed mice with food containing 0.08% TRYP, the amount of which is approximately 95 times higher than that included in 5%
QD and is predicted to yield plasma TRYP concentration higher than the IC50 against NO production. DSS þ TRYP-treated mice maintained their body weights (Fig. 3A and B). On day 10, the body weights of DSS þ TRYP-treated mice were significantly higher than those of DSS-treated mice (p < 0.001). However, diarrhea or bleeding was not strongly suppressed (Fig. 3C and D). The colon tissues in DSS þ TRYP-treated mice showed severe epithelial injury and inflammatory cell infiltration (Fig. 3E and F).
Please cite this article as: Ozawa K et al., Comparison of the anti-colitis activities of Qing Dai/Indigo Naturalis constituents in mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2020.01.003
K. Ozawa et al. / Journal of Pharmacological Sciences xxx (xxxx) xxx Table 2 Genes induced by DSS and suppressed by QD in focused microarray.
Mmp 3 Il6 Cxcl2 Ptgs2 Timp1 Mmp 9 Csf3 Lox Ccl7 Ccl11 Hspb1 Ier 3 Spp1 Il1b Il1r1
DSS
DSS þ QD
DSS þ TRYP
8.96 6.11 7.59 6.18 6.30 5.04 3.93 5.11 5.44 4.11 3.57 4.55 3.48 5.23 3.57
1.30 0.28 1.37 0.69 1.12 0.51 0.45 0.93 1.34 0.31 0.07 0.98 0.07 1.69 0.27
4.71 1.19 3.78 1.75 1.98 1.94 0.29 1.37 1.89 0.45 0.39 1.05 0.27 4.29 0.99
Fold induction of each gene compared with control are shown as log2 values.
Consistent with above observations, focused microarrays revealed that TRYP suppressed only a subset of the DSS-induced genes that were suppressed by QD (Table 2). For example, IL-6, PTGS2/COX-2, TIMP-1, CSF3, LOX, CCL7, and CCL11 expressions were
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suppressed by TRYP. By contrast, MMP-3 or MMP-9 was not so strongly suppressed. Failure to suppress MMPs may cause insufficient protection of basement membranes, resulting in diarrhea, bleeding, and epithelial injury. Indigo (0.8% of food, corresponded to 38% of indigo contained in 5% QD) suppressed bleeding, but it did not strongly suppress body weight loss or diarrhea (Fig. 3AeD). Indigo improved colon histology (Fig. 3E and F), but 11% of the indigo-treated mice were euthanized because of their body weight loss of over 20% (Fig. 3B). Other constituents, such as isatin, anthranilic acid, and salicylic acid did not strongly inhibit the colitis induced by DSS (Fig. 3, data not shown). TRYP and indigo exerted apparently complementary anti-colitis activities. However, the combination of TRYP and indigo did not show additive activity but instead showed a weaker effect than single treatments. TRYP þ indigo partially ameliorated diarrhea, bleeding, and colon histology (Fig. 3CeF) but did not improve body weight loss (Fig. 3A). Seventeen percent of the TRYP þ indigotreated mice were euthanized because of their body weight loss of more than 20% (Fig. 3B). Thus, TRYP and indigo show strong antiinflammatory activities, maintaining the homeostasis and the local injury of DSS-treated mice, respectively; however, they do not completely explain the anti-colitis activity of QD.
Fig. 2. QD suppresses IL-1b induced nitric oxide production. Rat hepatocytes were treated with 1 nM IL-1b in the presence or absence of QD, TRYP, or indigo (INDG) for 8 h. A, the levels of NO (as nitrite) in the medium were measured. B, the LDH activities in the medium were measured. The total LDH activity of the whole-cell extract (WCE) was defined as 100%. The data represent mean ± standard deviation (n ¼ 3).
Please cite this article as: Ozawa K et al., Comparison of the anti-colitis activities of Qing Dai/Indigo Naturalis constituents in mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2020.01.003
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Fig. 3. TRYP and indigo exert complementary anti-colitis activities. Mice were given tap water in the presence (DSS, DSS þ TRYP, DSS þ indigo, DSS þ TRYP þ indigo, DSS þ isatin) or absence (Control) of DSS. DSS treated mice were simultaneously fed powdered food containing TRYP, indigo, TRYP þ indigo, isatin or gum arabic (DSS) for 10 days. Body weight (A), survival rate (B), diarrhea score (C), and bleeding score (D) were measured daily. On day 10, mice were euthanized to analyze the histology of the distal colon (H&E staining) (E, F). Mean ± S.E.M. (A) and mean (C, D, F) are plotted. E, the representative images are shown. DSS þ TRYP-treated colon showed epithelial damage and cell infiltration. DSS þ indigo and DSS þ TRYP þ indigo-treated colon did not show significant epithelial damage or cell infiltration. The numbers of samples are 7 (control), 20 (DSS), 15 (DSS þ TRYP), 18 (DSS þ indigo), 18 (DSS þ TRYP þ indigo), and 6 (DSS þ isatin). *p < 0.05, **p < 0.01, ***p < 0.001 compared with DSS. The data were analyzed using one-way or two-way repeated ANOVA followed by Dunnett's post hoc test (A), or Wilcoxon signed-rank test followed by Bonferroni correction (C, D, F), versus DSS alone group.
3.5. TRYP-depleted QD fraction protect the colon from diarrhea and bleeding We attempted to deplete TRYP from QD to investigate the activity of QD that is not explained by TRYP. Because TRYP is very soluble in methanol,29 QD was extracted by using methanol. Mice
treated with DSS and methanol-soluble QD extract (TRYP 1.26 mg/ kg/day, indigo 0) maintained their body weights (Fig. 4A and B). On day 10, the body weights of DSS þ methanol-soluble QD extracttreated mice were significantly higher than those of mice treated with DSS alone (p < 0.05). However, diarrhea and bleeding were severer compared with mice treated with DSS þ whole QD (Fig. 4C
Please cite this article as: Ozawa K et al., Comparison of the anti-colitis activities of Qing Dai/Indigo Naturalis constituents in mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2020.01.003
K. Ozawa et al. / Journal of Pharmacological Sciences xxx (xxxx) xxx
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Fig. 4. Methanol-insoluble QD fraction protect the colon from local injuries. Mice were given tap water in the presence (DSS, DSS þ methanol-soluble QD fraction, DSS þ methanolinsoluble QD fraction, DSS þ methanol-soluble QD fraction þ methanol-insoluble QD fraction) or absence (Control) of DSS. DSS-treated mice were simultaneously fed powdered food containing the methanol-soluble QD fraction, methanol-insoluble QD fraction, the combination of methanol-soluble and -insoluble QD fractions, or gum arabic (DSS) for 10 days. Body weight (A), survival rate (B), diarrhea score (C), and bleeding score (D) were measured daily. On day 10, mice were euthanized to observe the histology of the distal colon (E, F). Mean ± S.E.M. (A), and mean (C, D, F) are plotted. E, the representative images are shown. DSS þ methanol-soluble QD fraction treated colon showed epithelial damage and cell infiltration. DSS þ methanol-insoluble QD fraction-treated colon showed little epithelial damage or cell infiltration. DSS þ the combination-treated colon showed almost no epithelial damage or cell infiltration. The numbers of samples are 9 (control), 13 (DSS), 12 (QD), 12 (methanol-soluble QD fraction), 12 (methanol-insoluble QD fraction), and 6 (combination of methanol-soluble and -insoluble fractions). *p < 0.05, **p < 0.01, ***p < 0.001 compared with DSS. The data were analyzed using one-way or two-way repeated ANOVA followed by Dunnett's post hoc test (A), or Wilcoxon signed-rank test followed by Bonferroni correction (C, D, F), versus DSS alone group.
and D). These effects of the methanol extract of QD were reminiscent of TRYP (see Fig. 3). The methanol-insoluble QD fraction (TRYP 0, indigo 3.2 g/kg/ day) exhibited distinct ameliorating activity. The fraction strongly suppressed diarrhea and bleeding, and exerted strong weight-
maintaining effect (Fig. 4AeD). This activity of the methanolinsoluble QD fraction was stronger than indigo. Full QD activity was observed with the combination of the methanol extract and methanol-insoluble fraction. The combination-treated mice maintained their body weight, histology,
Please cite this article as: Ozawa K et al., Comparison of the anti-colitis activities of Qing Dai/Indigo Naturalis constituents in mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2020.01.003
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and were free from diarrhea or bleeding (Fig. 4). On day 10, the body weights of DSS þ methanol-soluble fraction þ methanolinsoluble fraction-treated mice were significantly higher than mice treated with DSS alone (p < 0.001). The data suggest that methanolinsoluble fraction likely contains constituents, which are responsible for suppressing diarrhea and bleeding, other than indigo.
4. Discussion QD is highly effective in such intractable cases of UC when standard medications do not yield satisfactory results.8 This is consistent with the outstanding anti-colitis activity, which is by far more potent than 5-ASA and salazosulfapyridine (Fig. 1). Several reports demonstrate the anti-colitis activity of QD in animal models.10e12 However, this is the first study that directly compares the anti-colitis activity of QD with other drugs used to treat the same condition. Because QD is a crude plant extract, the action of each constituent must be analyzed individually. The safety of each constituent must also be examined in-depth because orally administering QD causes pulmonary hypertension.3 Since QD was first applied to UC patients,8 its constituents have been examined regarding their effectiveness for experimental colitis in animals. For example, indirubin, indigo, isatin, and TRYP have been tested in DSS- or TNBS-induced colitis models.10,14,23e26 A study also tested TRYP before the initial trial of the QD.13 However, there are no previous studies that directly compare the anti-colitis activities of these constituents in the same condition. To our knowledge, the present study is the first to directly compare their potencies. The effects of TRYP on DSS-induced colitis were mysterious. TRYP efficiently maintained body weight and the survival rate. However, TRYP was entirely ineffective against bleeding (Fig. 3). The same pattern of effect was reproduced with the methanol extract of QD, which is supposed to contain TRYP (Fig. 4). The maintenance of body weight may be explained by the antiinflammatory activity of TRYP, as shown in our study (Fig. 2) and previous reports.30,31 Failure to control rectal bleeding may partially be explained by anti-angiogenic activity and inhibition of hepatocyte growth factor induction of TRYP.32e34 Unidentified constituents that are included in the methanol-insoluble fraction of QD potentially exert anti-diarrhea and hemostatic effects. The maintenance of epithelial integrity, including the barrier function, is clearly involved in the protection or recovery from colitis. QD likely contains unidentified factors responsible for these functions. Orally administered TRYP is absorbed rapidly and targets plasma, liver, kidneys, and lungs.35 This suggests that the colon epithelium is not exposed to high concentrations of TRYP from the luminal side. QD is a powder largely insoluble in various solvents. Administering TRYP as part of the QD powder might result in the efficient delivery to the distal colon, where it might exert ameliorating activity to the epithelium directly from the intestinal lumen. In vitro and animal model experiments have confirmed that QD is a strong candidate prescription to apply for intractable UC cases. It has also become clear that QD has outstanding anti-colitis activity, which is much stronger than that of 5-ASA drugs. Some of the active constituents, including TRYP and indigo, exhibit antiinflammatory activity; however, this does not completely explain the outstanding colitis-ameliorating activity of whole QD. The high insolubility of whole QD may help with the delivery of the active constituents to the colon. Also, the additional function of unidentified constituents may involve the maintenance of intestinal barrier function. Further clarification of the active constituents, their optimum combinations, and their delivery to the affected loci will aid in the discovery of a safe and potent prescription for UC.
Declaration of Competing Interest The authors declare no competing financial interests. Acknowledgement We thank T. Okuyama, Y. Yamauchi, A. Yamamoto, N. Tanaka, R. Aoshiba, and R. Inui for discussion and technical assistance. This work was supported by MEXT-Supported Program for the Strategic Research Foundation at Private Universities (H.T.) and the AsiaJapan Research Institute of Ritsumeikan Asia-Japan Research Organization, Ritsumeikan University (Y.I. and M.N.). Abbreviations 5-ASA COX-2 DMSO DSS IFN IL-1b IL-1R1 IL-6 iNOS LC-MS/MS LDH MMP PTGS2 QD TNBS TNF-a TRYP UC
5-aminosalicylate cyclooxygenase-2 dimethyl sulfoxide dextran sulfate sodium salt interferon interleukin-1b interleukin-1 receptor type 1 interleukin-6 inducible nitric oxide synthase liquid chromatographyetandem mass spectrometry lactate dehydrogenase matrix metalloproteinase prostaglandin-endoperoxide synthase 2 Qing Dai (Indigo Naturalis) 2,4,6-trinitrobenzenesulfonic acid tumor necrosis factor-a tryptanthrin ulcerative colitis
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Please cite this article as: Ozawa K et al., Comparison of the anti-colitis activities of Qing Dai/Indigo Naturalis constituents in mice, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2020.01.003