Beneficial Effect of Herbal Formulation KM1608 on Inflammatory Bowl Diseases: A Preliminary Experimental Study

Aucklandia lappa DC., Terminalia chebula Retz and Zingiber officinale Roscoe have been traditionally used in east Asia to treat chronic diarrhea and abdominal pain. This study aimed to evaluated the anti-inflammatory activity of KM1608, which is composed of three natural herbs in a mouse model of dextran sodium sulfate (DSS)-induced ulcerative colitis. The anti-inflammatory activity and underlying mechanism were assessed in vitro using LPS-treated RAW264.7 cells. The in vivo effect of KM1608 on DSS-induced colitis was examined after oral administration in mice. KM1608 significantly inhibited the inflammatory mediators such as nitric oxide, interleukin (IL)-6, monocyte chemotactic protein 1 (MCP-1) and tumor necrosis factor (TNF)-α in LPS-treated RAW264.7 cells. The inhibitory effect of KM1608 was attributed to the reduction of Akt phosphorylation in the LPS-treated cells. In the mouse model, oral administration of KM1608 significantly improved DSS-induced colitis symptoms, such as disease activity index (DAI), colon length, and colon weight, as well as suppressed the expression of IL-6, TNF-α, and myeloperoxidase (MPO) in the DSS-induced colitis tissues. Taken together, KM1608 improved colitis through the regulation of inflammatory responses, suggesting that KM1608 has potential therapeutic use in the treatment of inflammatory diseases.

Inflammatory bowel disease (IBD) is a set of chronic inflammatory conditions that occur in the intestine, which includes Crohn's disease and ulcerative colitis.Genetic and environmental factors are known to play a complex role in IBD pathogenesis, although the mechanism remains unclear.In general, the main symptoms in patients with IBD include bleeding, diarrhea, urgency, and abdominal pain [5,6].Currently, medications for IBD include 5-aminosalicylic acid (5-ASA); a steroid, such as a prednisolone; immunomodulatory drugs, such as cyclosporine and tacrolimus; and neutralizing antibodies against TNF-α.However, 20-40% of patients treated with these drugs will undergo colonic resection owing to drug failure or side effects [7,8].Thus, there have been many attempts to develop more-effective drugs with minimum side effects to treat colitis.
Most earlier studies have focused on the individual extracts and active ingredients of natural products.However, through an in vitro screening of anti-inflammatory plants, we recently composed a potent herbal formulation (KM1608) containing A. lappa, T. chebula and Z. officinale to treat IBD.Therefore, the present study aimed to examine the chemical properties of KM1608 and assess its anti-inflammatory effect in vitro and DSS-induced ulcerative colitis in vivo.

Anti-Inflammatory Activity of KM1608 in LPS-Induced RAW264.7 Cells
To investigate the anti-inflammatory activity of KM1608, we first examined its cytotoxicity on RAW264.7 cells.RAW264.7 cells were treated with various concentrations of KM1608 for 2 h and further treated with LPS (250 ng/mL) for 18 h.As shown in Figure 2A, the treatment with KM1608 at 3.1-100 μg/mL did not exert cytotoxicity in RAW264.7 cells.
The macrophage, which differentiates from blood mononuclear cells, releases inflammation mediators, such as nitric oxide (NO), PGE2, TNF-α, IL-1, and IL-6.NO is produced from L-arginine by the nitric oxide synthases (NOS), neuronal NOS, endothelial NOS, and inducible NOS (iNOS) present in various tissues and cells.Because iNOS gene expression in the macrophage is induced by bacterial endotoxin or inflammatory stimuli, we first investigated whether KM1608 inhibits NO production in LPS-induced RAW264.7 cells.As shown in Figure 2B,C, treatment with KM1608 at 25-100 μg/mL significantly suppressed LPS-induced nitric oxide production and iNOS protein expression.The levels of other pro-inflammatory mediators, namely IL-6, TNF-α, and MCP-1, were also analyzed in LPS-induced RAW264.7 cells.As shown in Figure 3, KM1608 inhibited LPS-induced iNOS, IL-6, TNF-α and MCP-1 gene expression in a concentration-dependent manner.KM1608 at 30-60 μg/mL significantly decreased LPS-induced iNOS, IL-6 and TNF-α gene expression levels (Figure 3A,C), and KM1608 at 60 μg/mL decreased MCP-1 gene expression (Figure 3D).Similarly, IL-6, TNFα and MCP-1 production after LPS stimulation were significantly reduced by KM1608 treatment at at 50-100 μg/mL (Figure 4A-C).Taken together, these results indicated that KM1608 could possess anti-inflammatory activity in LPS-induced RAW264.7 cells.To investigate the anti-inflammatory activity of KM1608, we first examined its cytotoxicity on RAW264.7 cells.RAW264.7 cells were treated with various concentrations of KM1608 for 2 h and further treated with LPS (250 ng/mL) for 18 h.As shown in Figure 2A, the treatment with KM1608 at 3.1-100 µg/mL did not exert cytotoxicity in RAW264.7 cells.

Figure 2.
Effect of KM1608 on nitric oxide production and iNOS protein expression in lipopolysaccharide (LPS)-induced RAW264.7 cells.RAW264.7 cells (1 × 10 5 cells/well, 96-well plate) were treated with various concentrations of KM1608 for 2 h and subsequently with LPS (250 ng/mL) for 18 h.The cytotoxicity was determined using the CCK-8-based colorimetric assay (A).After incubation, cell supernatants were collected and assayed for nitric oxide using the Griess reagent (B).RAW264.7 cells (2 × 10 6 cells/well, 6-well plate) were treated with various concentrations of KM1608 for 2 h and subsequently with LPS (250 ng/mL) for 18 h (C).Control group treated with DMSO at 0.1%.iNOS protein level was measured by immunoblotting with the specific antibodies.The bar chart displays the intensity of immunoblot bands visualized using the Image J software.Data are presented as the mean ± SD of three independent experiments.# p < 0.05 vs. the LPS group.Effect of KM1608 on nitric oxide production and iNOS protein expression in lipopolysaccharide (LPS)-induced RAW264.7 cells.RAW264.7 cells (1 × 10 5 cells/well, 96-well plate) were treated with various concentrations of KM1608 for 2 h and subsequently with LPS (250 ng/mL) for 18 h.The cytotoxicity was determined using the CCK-8-based colorimetric assay (A).After incubation, cell supernatants were collected and assayed for nitric oxide using the Griess reagent (B).RAW264.7 cells (2 × 10 6 cells/well, 6-well plate) were treated with various concentrations of KM1608 for 2 h and subsequently with LPS (250 ng/mL) for 18 h (C).Control group treated with DMSO at 0.1%.iNOS protein level was measured by immunoblotting with the specific antibodies.The bar chart displays the intensity of immunoblot bands visualized using the Image J software.Data are presented as the mean ± SD of three independent experiments.# p < 0.05 vs. the LPS group.

Effect of KM1608 on Akt Phosphorylation in LPS-Induced RAW264.7 Cells
To elucidate the underlying anti-inflammatory mechanisms of KM1608, we investigated several intracellular signaling pathways in LPS-induced RAW264.7 cells.As shown in Figure 5A, LPS strongly induced the phosphorylation of MAPKs (ERK1/2, JNK1/2, p38), NF-κB p65, and c-Jun for 1 h.However, pretreatment of KM1608 for 2 h did not affect LPS-induced ERK, JNK and p38 phosphorylation.Similarly, p65 and c-Jun phosphorylation were also not altered by KM1608 in the LPS-stimulated RAW264.7 cells.It is well-known that PI3K/Akt signaling is involved in the regulation of pro-inflammatory genes and that Akt is a downstream molecule of PI3K in macrophages [26,27].Following the treatment of cells with LPS for 1 h, Akt was strongly phosphorylated.However, LPS-induced Akt phosphorylation was diminished by pre-treatment with KM1608 in a concentration-dependent manner (Figure 5B).These findings suggested that KM1608 inhibited pro-inflammatory mediators (NO, IL-6, and MCP-1) by regulating the Akt signaling pathway.Also, we confirmed inhibitory effects of KM1608 on LPS-induced MAPKs and p65 phosphorylation in short term point.When the cells were stimulated by LPS for 15 min or 30 min, MAPKs and p65 phosphorylation increased by time.KM1608 treatment at 50 µg/mL or 25 µg/mL did not affect LPS signaling at 15 min, however, LPS-induced ERK phosphorylation was slightly decreased by KM1608 at 30 min (Figure 5C).This inhibited ERK signaling by KM1608 may involve a reduction of Akt phosphorylation, and more detailed studies should be continued in the future.

Effect of KM1608 on Clinical Signs of DSS-Induced Colitis in Mice
To investigate the in vivo efficacy of KM1608 in IBD model, we established a mouse model of DSS-induced colitis.Clinical assessment of colitis was conducted using the DAI, which represents the sum of the highest score of each criterion (weight loss, stool consistency and rectal bleeding) during DSS treatment for 7 days (Figure 6A).KM1608 administration significantly ameliorated the severity of colitis in a dose-dependent manner (Figure 5A).DSS-treated mice showed a significant shortening of colon length (Figure 6B,C), which was significantly attenuated by KM1608 (600 mg/kg) (Figure 6B).As shown in Figure 6D, the colon weight/length ratio was significantly elevated in the DSS-induced group because of submucosal edema in the inflamed colon.The KM1608 200 and 600 mg/kg groups showed significantly decreased colon weight/length ratios (Figure 6D).5-ASA (an aminosalicylate anti-inflammatory drug) and prednisolone (a steroid drug) were used as reference

Effect of KM1608 on Clinical Signs of DSS-Induced Colitis in Mice
To investigate the in vivo efficacy of KM1608 in IBD model, we established a mouse model of DSS-induced colitis.Clinical assessment of colitis was conducted using the DAI, which represents the sum of the highest score of each criterion (weight loss, stool consistency and rectal bleeding) during DSS treatment for 7 days (Figure 6A).KM1608 administration significantly ameliorated the severity of colitis in a dose-dependent manner (Figure 5A).DSS-treated mice showed a significant shortening of colon length (Figure 6B,C), which was significantly attenuated by KM1608 (600 mg/kg) (Figure 6B).As shown in Figure 6D, the colon weight/length ratio was significantly elevated in the DSS-induced group because of submucosal edema in the inflamed colon.The KM1608 200 and 600 mg/kg groups showed significantly decreased colon weight/length ratios (Figure 6D).5-ASA (an aminosalicylate anti-inflammatory drug) and prednisolone (a steroid drug) were used as reference drugs.However, the 5-ASA-and prednisolone-treated groups did not show any significant improvement of colitis in this study.Furthermore, we measured MPO activity and pro-inflammatory cytokine levels (IL-6 and TNF-α) to determine the degree of inflammation in the colitis-induced colon.MPO is a peroxidase enzyme mainly found in the neutrophils and is used as an index of neutrophil infiltration.As shown in Figure 7A, MPO activity was decreased in all drug-treated groups, with the DSS-treated group showing a significantly higher MPO activity than that in the normal group.Particularly, MPO activity in the prednisolone-and KM1608 (400 and 600 mg/kg)-treated groups were significantly lower than that in the DSS group (Figure 7A).On the other hand, pro-inflammatory cytokines such as TNF-α and IL-6 levels were elevated in the inflamed tissues, causing colon tissue damage and exacerbating colitis.As shown in Figure 7B,C, TNF-α and IL-6 levels in the colon tissue were higher in the DSS group than in the normal group.In contrast, the levels of these inflammatory cytokines were lower in the KM1608 group than in the DSS group and the reference groups (5-ASA-and prednisolone-treated groups).In addition, KM1608 at 400 and 600 mg/kg inhibited the production of TNF-α and IL-6 more than the reference drug.Based on these results, we speculate KM1608 could be a candidate for a novel therapeutic agent against colitis.
Molecules 2018, 23, x FOR PEER REVIEW 8 of 13 drugs.However, the 5-ASA-and prednisolone-treated groups did not show any significant improvement of colitis in this study.Furthermore, we measured MPO activity and pro-inflammatory cytokine levels (IL-6 and TNF-α) to determine the degree of inflammation in the colitis-induced colon.MPO is a peroxidase enzyme mainly found in the neutrophils and is used as an index of neutrophil infiltration.As shown in Figure 7A, MPO activity was decreased in all drug-treated groups, with the DSS-treated group showing a significantly higher MPO activity than that in the normal group.
Particularly, MPO activity in the prednisolone-and KM1608 (400 and 600 mg/kg)-treated groups were significantly lower than that in the DSS group (Figure 7A).On the other hand, pro-inflammatory cytokines such as TNF-α and IL-6 levels were elevated in the inflamed tissues, causing colon tissue damage and exacerbating colitis.As shown in Figure 7B,C, TNF-α and IL-6 levels in the colon tissue were higher in the DSS group than in the normal group.In contrast, the levels of these inflammatory cytokines were lower in the KM1608 group than in the DSS group and the reference groups (5-ASAand prednisolone-treated groups).In addition, KM1608 at 400 and 600 mg/kg inhibited the production of TNF-α and IL-6 more than the reference drug.Based on these results, we speculate KM1608 could be a candidate for a novel therapeutic agent against colitis.

Animal and Cell Culture
Female C57BL/6 mice were purchased from Daehan Bio Link (Seoul, Korea) at 7 weeks of age and acclimatized for 7 days in a specific pathogen-free (SPF) environment under constant conditions (temperature: 23 ± 2 • C; humidity: 50 ± 5%; light/dark cycle: 12 h) at a facility in Kolmar Korea Co., Ltd (Sejong, Korea).All animal studies were performed according to the instructions of the Ethics Committee for Use of Experimental Animals at Kolmar Korea Co., Ltd.(confirmation number: 16-NP-IBD-011-P).The macrophage cell line RAW 264.7 was purchased from the Korean Cell Line Bank (KCLB) (Seoul, Korea) and seeded in DMEM containing 10% heat-inactivated FBS and 1% penicillin-streptomycin obtained from Life Technologies (Waltham, MA, USA).The cells were maintained at 37 • C in a humidified atmosphere with 5% CO 2 .

Induction of Colitis by DSS
Acute colitis was induced in mice for 7 days by adding 1.7% (w/v) DSS to the drinking water.The mice were measured daily for body weight, stool consistency and rectal bleeding.The normal group received only water without DSS.The DSS group received only drinking water containing 1.7% of DSS.The KM1608 groups received DSS-containing drinking water and KM1608 (200, 400, or 600 mg/kg).The 5-ASA group received DSS-containing drinking water and 5-ASA (200 mg/kg).The prednisolone group received DSS-containing drinking water and prednisolone (5 mg/kg).Carboxymethylcellulose (CMC) solution (0.5%) was used to dissolve KM1608, 5-ASA and prednisolone for in vivo experiments.All drugs were orally administered once daily during the experiment.Animals were sacrificed after 7 days of DSS treatment.

Disease Activity Index (DAI)
Intestinal disease activity was assessed based on body weight loss, diarrhea accompanied by blood and mucus, and colonic shortening.DAIs were assessed using a scoring system (Table 2) as described by Murthy et al. [28] with little modification, and were calculated using the following formula: DAI = (weight loss score) + (stool consistency score) + (rectal bleeding score).

Cell Viability
RAW 264.7 macrophages were treated with the indicated concentration of KM1608 for 2 h.The cells were subsequently treated with LPS (250 ng/mL) for 18 h.Following treatment, 20 µL of CCK-8 solution was added to the cells and the cells were further incubated at 37 • C, 5% CO 2 for the evaluation of cytotoxic effects.KM1608 (herbal formulation) was dissolved in dimethyl sulfoxide (DMSO) prior to use for in vitro experiments, and the final concentration was kept at <0.1%.
3.8.Determination of Nitrite, IL-6, MCP-1, TNF-a, and MPO Production RAW 264.7 cells (1 × 10 5 cells/well, 96-well plate) were treated with KM1608 at various concentrations for 2 h and were subsequently treated with LPS (250 ng/mL).After 18 h of incubation, nitrite production was estimated by using Griess reagent and a standard curve previously prepared using sodium nitrite purchased from Promega (Promega, Fitchburg, WI, USA).The cell supernatants (50 µL) were mixed with the same volume of Griess reagent and the absorbance was measured at 520 nm using a microplate reader (Molecular Devices Co., San Jose, CA, USA).IL-6 and MCP-1 levels in the cell supernatants were evaluated using a sandwich enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instruction.For the analysis of IL-6, TNF-α, and MPO in colitis-induced colon tissue, colon tissue samples were suspended in lysis buffer (Intron, Seoul, Korea) and ground using a homogenizer (Scilogex, Rocky hill, CT, USA).The supernatant was collected by centrifugation (10,000 rpm, 20 min, 4 • C).The IL-6, TNF-α, and MPO levels in the supernatant were measured using ELISA kits according to the manufacturer's instructions.

Cell Lysate Preparation and Immunoblotting
RAW 264.7 cells were treated with the indicated concentrations of KM1608 for 2 h and subsequently treated with LPS for 1 h or 18 h.Following treatment, the cells were washed with cold PBS and lysed with cold RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.25% deoxycholate, 1% NP-40, and 1 mM EDTA) containing 1 mM DTT, 1 mM PMSF, 1 mM sodium orthovanadate, and 10 mM β-glycerophosphate.Cell lysis, supernatant collection, protein quantification, protein electrophoresis, protein transfer, and membrane development were all performed as described previously [29].The relative protein expression compared to controls was quantified using the Image J (National Institutes of Health, Bethesda, MD, USA).

Figure 2 .
Figure 2.Effect of KM1608 on nitric oxide production and iNOS protein expression in lipopolysaccharide (LPS)-induced RAW264.7 cells.RAW264.7 cells (1 × 10 5 cells/well, 96-well plate) were treated with various concentrations of KM1608 for 2 h and subsequently with LPS (250 ng/mL) for 18 h.The cytotoxicity was determined using the CCK-8-based colorimetric assay (A).After incubation, cell supernatants were collected and assayed for nitric oxide using the Griess reagent (B).RAW264.7 cells (2 × 10 6 cells/well, 6-well plate) were treated with various concentrations of KM1608 for 2 h and subsequently with LPS (250 ng/mL) for 18 h (C).Control group treated with DMSO at 0.1%.iNOS protein level was measured by immunoblotting with the specific antibodies.The bar chart displays the intensity of immunoblot bands visualized using the Image J software.Data are presented as the mean ± SD of three independent experiments.# p < 0.05 vs. the LPS group.

Figure 5 .
Figure 5.Effect of KM1608 on the phosphorylation of MAPKs and Akt in RAW264.7 cells.RAW264.7 cells (2 × 10 6 cells/well, 6-well plate) were treated with various concentrations of KM1608 for 2 h and subsequently with LPS (250 ng/mL) for 1 h (A,B).RAW264.7 cells (2 × 10 6 cells/well, 6-well plate) were treated with various concentration of KM1608 for 2 h and subsequently with LPS (250 ng/mL) for 15 min or 30 min (C).Control group was treated with DMSO at 0.05%.Whole cell-lysates were immunoblotted with the indicated specific-antibodies. β-Actin served as an internal loading control.Whole Western blot membranes of Figure 5C; See Figure S1.The bar chart displays the intensity of immunoblot bands visualized using the Image J software.Data are presented as the mean ± SD of three independent experiments.

Figure 5 .
Figure 5.Effect of KM1608 on the phosphorylation of MAPKs and Akt in RAW264.7 cells.RAW264.7 cells (2 × 10 6 cells/well, 6-well plate) were treated with various concentrations of KM1608 for 2 h and subsequently with LPS (250 ng/mL) for 1 h (A,B).RAW264.7 cells (2 × 10 6 cells/well, 6-well plate) were treated with various concentration of KM1608 for 2 h and subsequently with LPS (250 ng/mL) for 15 min or 30 min (C).Control group was treated with DMSO at 0.05%.Whole cell-lysates were immunoblotted with the indicated specific-antibodies. β-Actin served as an internal loading control.Whole Western blot membranes of Figure 5C; See Figure S1.The bar chart displays the intensity of immunoblot bands visualized using the Image J software.Data are presented as the mean ± SD of three independent experiments.

Figure 6 .Figure 6 .
Figure 6.KM1608 improves the symptoms of DSS-induced colitis.Ulcerative colitis was induced in C57BL/6 mice by administering 1.7% DSS in the drinking water for 7 days.During this period, the mice also received KM1608 (200, 400, and 600 mg/kg), 5-ASA (200 mg/kg), or prednisolone (5 mg/kg) orally once a day.5-ASA and prednisolone were used as reference drugs.DAI was scored in all sevenFigure 6. KM1608 improves the symptoms of DSS-induced colitis.Ulcerative colitis was induced in C57BL/6 mice by administering 1.7% DSS in the drinking water for 7 days.During this period, the mice also received KM1608 (200, 400, and 600 mg/kg), 5-ASA (200 mg/kg), or prednisolone (5 mg/kg) orally once a day.5-ASA and prednisolone were used as reference drugs.DAI was scored in all seven of the study groups (A).Colons were harvested on day 7 and (B) colon length (C) and colon weight (D) were measured.* p < 0.05 or ** p < 0.01 vs. the DSS group.
of the study groups (A).Colons were harvested on day 7 and (B) colon length (C) and colon weight (D) were measured.* p < 0.05 or ** p < 0.01 vs. the DSS group.

Figure 7 .
Figure 7. KM1608 inhibits the inflammatory factors involved in DSS-induced colitis.Ulcerative colitis was induced in C57BL/6 mice by administering 1.7% DSS in the drinking water for 7 days.During the period, the mice also received KM1608 (200, 400, and 600 mg/kg), 5-ASA (200 mg/kg), or prednisolone (5 mg/kg) orally once a day.MPO, TNF-α, and IL-6 levels in the colon tissue lysates were measured using ELISA kits (A-C).Data are presented as the mean ± SD of three independent experiments.* p < 0.05 or ** p < 0.01 vs. the DSS group.

Figure 7 .
Figure 7. KM1608 inhibits the inflammatory factors involved in DSS-induced colitis.Ulcerative colitis was induced in C57BL/6 mice by administering 1.7% DSS in the drinking water for 7 days.During the period, the mice also received KM1608 (200, 400, and 600 mg/kg), 5-ASA (200 mg/kg), or prednisolone (5 mg/kg) orally once a day.MPO, TNF-α, and IL-6 levels in the colon tissue lysates were measured using ELISA kits (A-C).Data are presented as the mean ± SD of three independent experiments.* p < 0.05 or ** p < 0.01 vs. the DSS group.

Table 1 .
Contents of representative compounds in KM1608.

Table 1 .
Contents of representative compounds in KM1608.