Preparation of Herbal Formulation for Inflammatory Bowel Disease Based on In Vitro Screening and In Vivo Evaluation in a Mouse Model of Experimental Colitis.

Many medicinal plants have been used traditionally in East Asia for the treatment of gastrointestinal disease and inflammation. The aim of this study was to evaluate the anti-inflammatory activity of 350 extracts (175 water extracts and 175 ethanol extracts) from 71 single plants, 97 mixtures of two plants, and seven formulations based on traditional medicine, to find herbal formulations to treat inflammatory bowel disease (IBD). In the in vitro screening, nitric oxide (NO), tumor necrosis factor (TNF)-α, and interleukin (IL)-6 levels were determined in LPS-treated RAW264.7 cells and the TNF-α induced monocyte-epithelial cell adhesion assay was used for the evaluation of the anti-inflammatory activity of the compounds. Dextran sulfate sodium (DSS)-induced colitis model and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis model were used to evaluate the therapeutic effect against IBD of the samples selected from the in vitro screening. KM1608, composed of Zingiber officinale, Terminalia chebula and Aucklandia lappa, was prepared based on the screening experiments. The oral administration of KM1608 significantly attenuated the severity of colitis symptoms, such as weight loss, diarrhea, and rectal bleeding, in TNBS-induced colitis. In addition, inflammatory mediators, such as myeloperoxidase, TNF-α, and IL-6 levels decreased in the lysate of colon tissues treated with KM1608. Collectively, KM1608 ameliorated colitis through the regulation of inflammatory responses within the colon, which indicated that KM1608 had potential for the treatment of IBD.

1. Introduction

Inflammatory bowel disease (IBD) is an idiopathic chronic inflammatory condition of the gastrointestinal tract, comprising Crohn’s disease and ulcerative colitis. The symptoms of IBD are chronic diarrhea, abdominal pain, rectal bleeding, weight loss, and shortening of the colon. Although the etiology of IBD remains uncertain, it is known that irregular immune response, gut microbial flora, and genetic and environmental factors are associated with the pathogenesis of IBD [1,2]. The present treatment regimen, including aminosalicylates, corticosteroids, biologics, and immunosuppressants, has therapeutic limits and leads to side effects [3]. Furthermore, recent failures of drug targets in IBD, such as IL-17, IL-13, interferon (IFN)-γ, and chemokine receptor (CCR)-9, have indicated that single target therapy for IBD is difficult owing to pathogenic heterogeneity [4]. Therefore, the discovery of alternative treatment options with multiple therapeutic targets is required. We expect that natural product formulations, such as medicinal plant extracts or traditional medicines, would offer excellent alternative therapies for IBD.

In recent decades, medicinal plant extracts, traditional medicines and their active components have been investigated for the development of novel anti-inflammatory drugs [5,6,7,8,9,10]. Many patients with IBD are interested in alternative treatments because they are dissatisfied with the current conventional treatment [11]. However, no successful therapeutics for IBD based on natural products have been developed. Thus, we aimed to explore potent herbal formulations for the treatment of IBD by using a large-scale screening test.

We tested the anti-inflammatory activity of 350 samples that were extracted (in water and 50% ethanol) from 71 single plants, 97 mixtures of two plants, and seven formulations based on traditional medicine. These plant and formulation samples were selected from various sources of traditional Chinese medicine literature, such as Shanghan Lun, Compendium of Materia Medica, and Traditional Chinese Medicine Formulary. In the in vitro screening, we determined NO production and the levels of pro-inflammatory cytokines (TNF-α and IL-6) in RAW264.7 cells, and assayed the monocyte (U937)-epithelial (HT-29) adhesion ability. In the in vivo screening, we examined the therapeutic efficacy of the selected samples in mouse models of DSS-induced colitis and TNBS-induced colitis. Furthermore, we also investigated the effects of KM1608, the final formulation selected, on TNBS-induced colitis.

2. Results and Discussion

2.1. NO Assay for Preliminary Screening of Plant Extracts

First, we screened both the water and the 50% ethanol extracts of 71 samples of single plants (Table 1), 97 samples of a 1:1 mixture of two plants (Table 2), and seven samples of a formulation based on traditional medicine (Table 3).

Table 1

NO assay results of single plant extracts.

No.	Plant	% of LPS
		Water Extract(Group A)	Ethanol Extract(Group B)
	Control (0.1% DMSO without LPS)	18.9 ± 0.1	20.0 ± 0.6
1	Glycyrrhiza uralensis	100.3 ± 3.1	102.3 ± 2.4
2	Brassica juncea	99.2 ± 1.5	97.2 ± 1.0
3	Zingiber officinale	99.7 ± 3.8	68.1 ± 1.7
4	Sophora flavescens	101.5 ± 1.2	102.8 ± 1.8
5	Tussilago farfara	98.5 ± 5.6	99.6 ±2.0
6	Codonopsis pilosula	90.4 ± 2.6	100.2 ± 2.6
7	Ephedra sinica	95.8 ± 5.2	100.5 ± 0.8
8	Paeonia suffruticosa	104.1 ± 7.1	106.5 ± 5.1
9	Inula helenium	90.2 ± 3.8	86.3 ± 2.6
10	Pinellia ternata	93.1 ± 3.6	96.0 ± 3.7
11	Saposhnikovia divaricata	99.8 ± 4.5	99.7 ± 2.1
12	Pulsatilla koreana	64.2 ± 2.8	60.5 ± 2.3
13	Atractylodes macrocephala	96.3 ± 3.8	94.3 ± 2.5
14	Psoralea corylifolia	94.8 ± 2.6	98.9 ± 2.6
15	Belamcanda chinensis	95.9 ± 2.6	101.0 ± 2.8
16	Dioscorea batatas	97.9 ± 1.7	102.1 ± 2.8
17	Phytolacca esculenta	98.2 ± 35	106.1 ± 4.2
18	Acorus gramineus	95.9 ± 5.1	102.3 ± 0.9
19	Asiasarum sieboldi	103.7 ± 2.9	106.0 ± 2.7
20	Bupleurum falcatum	97.2 ± 2.6	70.1 ± 0.5
21	Magnolia kobus	114.2 ± 2.2	111.5 ± 2.7
22	Achyranthes bidentata	93.1 ± 4.9	97.5 ± 1.7
23	Daphne genkwa	95.1 ± 3.8	89.8 ± 2.5
24	Myristica fragrans	108.1 ± 2.5	109.3 ± 1.3
25	Coix lachryma-jobi	90.8 ± 7.5	104.1 ± 0.4
26	Aster tataricus	94.4 ± 2.2	99.9 ± 4.1
27	Paeonia lactiflora	96.2 ± 1.7	97.8 ± 3.1
28	Citrus unshiu	102.3 ± 3.1	108.0 ± 2.5
29	Cnidium officinale	104.1 ± 2.8	105.3 ± 2.3
30	Melia azedarach	47.8 ± 0.7	46.5 ± 1.6
31	Morinda citrifolia	102.4 ± 4.3	100.0 ± 1.8
32	Patrinia scabiosaefolia	90.2 ± 2.5	67.3 ± 2.7
33	Prunella vulgaris	76.4 ± 1.4	74.4 ± 3.3
34	Prunus armeniaca	104.7 ± 2.8	98.7 ± 2.1
35	Corydalis remota	109.3 ± 3.8	100.2 ± 2.0
36	Scutellaria baicalenesis	39.3 ± 0.2	35.1 ± 1.6
37	Astragalus membranaceus	101.9 ± 1.9	96.9 ± 2.1
38	Jeffersonia dubia	117.0 ± 5.1	70.1 ± 2.5
39	Rumex japonicus	113.8 ± 4.0	108.0 ± 2.2
40	Smilax china	107.4 ± 3.9	101.9 ± 2.0
41	Elsholtzia ciliata	93.0 ± 0.9	99.5 ± 1.6
42	Angelica gigas	104.3 ± 7.1	92.9 ± 1.8
43	Evodia officinalis	97.8 ± 3.0	101.0 ± 10.9
44	Aconitum carmichaeli	103.0 ± 4.8	106.1 ± 7.1
45	Machilus thunbergii	100.3 ± 5.4	28.9 ± 3.0
46	Atractylodes japonica	104.9 ± 11.2	90.4 ± 2.9
47	Terminalia chebula	58.3 ± 4.9	47.9 ± 1.9
48	Sanguisorba hakusanensis	66.4 ± 4.4	57.0 ± 2.0
49	Euryale ferox	119.3 ± 3.4	104.0 ± 5.8
50	Rheum palmatum	120.4 ± 5.5	79.0 ± 8.5
51	Rheum undulatum	108.9 ± 3.2	123.5 ± 4.7
52	Citrus aurantium	100.8 ± 1.7	107.5 ± 6.9
53	Ailanthus altissima	95.5 ± 6.0	100.1 ± 11.4
54	Pogostemon cablin	97.7 ± 2.8	74.4 ± 5.2
55	Schisandra chinensis	98.3 ± 2.8	93.5 ± 8.3
56	Nelumbo nucifera	101.2 ± 3.5	94.1 ± 5.0
57	Lindera aggregata	102.4 ± 5.8	76.5 ± 9.8
58	Aucklandia lappa	99.8 ± 3.7	14.7 ± 0.1
59	Ephedra sinica	101.7 ± 7.4	79.7 ± 7.7
60	Fritillaria thunbergii	103.7 ± 2.6	112.5 ± 7.9
61	Fritillaria cirrhosa	111.2 ± 9.4	106.5 ± 6.0
62	Carex canescens	101.6 ± 2.1	101.0 ± 10.1
63	Cinnamomum cassia	71.5 ± 1.3	32.4 ± 1.7
64	Piper nigrum	110.2 ± 4.0	98.0 ± 3.8
65	Liriope muscari	100.9 ± 5.5	91.1 ± 5.5
66	Morus alba	100.3 ± 5.9	100.2 ± 5.5
67	Phyllostachys nigra	97.2 ± 4.6	93.6 ± 2.7
68	Croton tiglium	77.4 ± 2.2	64.2 ± 2.1
69	Houttuynia cordata	102.1 ± 2.4	107.2 ± 3.3
70	Perilla frutescens	102.6 ± 3.4	96.9 ± 2.3
71	Zanthoxylum piperitum	92.2 ± 8.4	94.0 ± 7.4

Data are presented as the mean ± SEM.

Table 2

NO assay results of the extracts of 1:1 mixtures of two plants.

No.	Plants		% of LPS
			Water Extract(Group mA)	Ethanol Extract(Group mB)
	Control (0.1% DMSO without LPS)		17.0 ± 0.7	16.5 ± 1.0
1	Glycyrrhiza uralensis	Atractylodes macrocephala	92.7 ± 3.4	85.9 ± 2.0
2	Glycyrrhiza uralensis	Codonopsis pilosula	92.3 ± 1.8	83.6 ± 2.2
3	Glycyrrhiza uralensis	Citrus unshiu	92.8 ± 3.1	82.6 ± 4.6
4	Glycyrrhiza uralensis	Paeonia lactiflora	96.0 ± 2.6	71.6 ± 4.4
5	Atractylodes macrocephala	Paeonia lactiflora	91.9 ± 2.6	92.7 ± 4.2
6	Atractylodes macrocephala	Codonopsis pilosula	90.5 ± 2.1	100.6 ± 2.7
7	Atractylodes macrocephala	Citrus unshiu	93.7 ± 3.4	90.7 ± 2.6
8	Paeonia lactiflora	Aucklandia lappa	88.1 ± 5.1	14.8 ± 0.1
9	Paeonia lactiflora	Codonopsis pilosula	97.0 ± 3.5	92.1 ± 2.8
10	Paeonia lactiflora	Citrus unshiu	97.2 ± 5.4	93.7 ± 5.1
11	Astragalus membranaceus	Magnolia kobus	96.7 ± 3.6	77.7 ± 5.8
12	Astragalus membranaceus	Jeffersonia dubia	75.1 ± 4.4	71.7 ± 2.0
13	Astragalus membranaceus	Aster tataricus	96.7 ± 4.2	95.2 ± 4.8
14	Jeffersonia dubia	Glycyrrhiza uralensis	82.8 ± 2.9	71.5 ± 4.6
15	Jeffersonia dubia	Aucklandia lappa	59.6 ± 2.9	15.9 ± 0.8
16	Smilax china	Rumex japonicus	104.2 ± 6.2	100.9 ± 6.6
17	Brassica juncea	Pinellia ternata	93.5 ± 2.5	94.9 ± 4.1
18	Brassica juncea	Zingiber officinale	94.6 ± 3.0	85.6 ± 3.1
19	Paeonia lactiflora	Jeffersonia dubia	85.3 ± 2.1	60.7 ± 2.8
20	Myristica fragrans	Aconitum carmichaeli	92.4 ± 6.2	101.0 ± 1.9
21	Myristica fragrans	Sanguisorba hakusanensis	68.0 ± 5.8	71.2 ± 1.9
22	Myristica fragrans	Evodia officinalis	90.9 ± 5.1	91.7 ± 0.8
23	Myristica fragrans	Jeffersonia dubia	73.2 ± 4.3	69.6 ± 2.4
24	Myristica fragrans	Rheum palmatum	95.5 ± 7.4	111.9 ± 5.7
25	Myristica fragrans	Psoralea corylifolia	97.0 ± 6.8	102.8 ± 8.7
26	Myristica fragrans	Zingiber officinale	118.0 ± 3.0	110.2 ± 4.8
27	Myristica fragrans	Terminalia chebula	58.4 ± 3.8	51.4 ± 2.4
28	Myristica fragrans	Euryale ferox	87.2 ± 2.4	88.8 ± 4.4
29	Myristica fragrans	Citrus aurantium	91.8 ± 11.1	97.4 ± 4.4
30	Myristica fragrans	Machilus thunbergii	85.9 ± 5.2	92.1 ± 1.9
31	Myristica fragrans	Aucklandia lappa	79.6 ± 4.5	35.6 ± 1.4
32	Aconitum carmichaeli	Sanguisorba hakusanensis	66.7 ± 10.2	72.3 ± 6.8
33	Aconitum carmichaeli	Evodia officinalis	80.1 ± 9.1	84.6 ± 0.0
34	Aconitum carmichaeli	Jeffersonia dubia	66.5 ± 13.7	68.9 ± 3.1
35	Aconitum carmichaeli	Rheum palmatum	91.3 ± 12.7	114.7 ± 8.0
36	Aconitum carmichaeli	Psoralea corylifolia	90.7 ± 0.1	106.4 ± 7.0
37	Aconitum carmichaeli	Zingiber officinale	94.7 ± 4.4	109.3 ± 1.1
38	Aconitum carmichaeli	Terminalia chebula	72.5 ± 4.4	49.6 ± 0.9
39	Aconitum carmichaeli	Euryale ferox	92.3 ± 5.1	101.2 ± 2.6
40	Aconitum carmichaeli	Citrus aurantium	92.5 ± 6.3	101.2 ± 4.6
41	Aconitum carmichaeli	Machilus thunbergii	95.6 ± 2.4	101.7 ± 2.4
42	Aconitum carmichaeli	Aucklandia lappa	85.9 ± 5.9	16.7 ± 1.8
43	Sanguisorba hakusanensis	Evodia officinalis	82.5 ± 6.9	86.6 ± 2.7
44	Sanguisorba hakusanensis	Jeffersonia dubia	113.7 ± 3.5	94.2 ± 1.7
45	Sanguisorba hakusanensis	Rheum palmatum	85.2 ± 12.6	84.4 ± 2.2
46	Sanguisorba hakusanensis	Psoralea corylifolia	70.4 ± 0.7	76.3 ± 2.7
47	Sanguisorba hakusanensis	Zingiber officinale	71.7 ± 4.0	63.1 ± 0.9
48	Sanguisorba hakusanensis	Terminalia chebula	53.1 ± 3.0	52.2 ± 2.4
49	Sanguisorba hakusanensis	Euryale ferox	60.8 ± 1.0	62.8 ± 1.2
50	Sanguisorba hakusanensis	Citrus aurantium	72.7 ± 1.3	79.8 ± 2.7
51	Sanguisorba hakusanensis	Machilus thunbergii	57.2 ± 2.3	79.3 ± 5.2
52	Sanguisorba hakusanensis	Aucklandia lappa	55.9 ± 2.7	15.4 ± 0.8
53	Evodia officinalis	Jeffersonia dubia	81.7 ± 3.8	80.3 ± 3.2
54	Evodia officinalis	Rheum palmatum	112.3 ± 4.7	118.2 ± 3.0
55	Evodia officinalis	Psoralea corylifolia	102.3 ± 3.2	98.9 ± 1.4
56	Evodia officinalis	Zingiber officinale	100.8 ± 1.8	98.7 ± 4.1
57	Evodia officinalis	Terminalia chebula	74.7 ± 1.6	69.5 ± 2.1
58	Evodia officinalis	Euryale ferox	102.3 ± 2.5	95.6 ± 2.9
59	Evodia officinalis	Citrus aurantium	100.7 ± 3.0	101.3 ± 3.8
60	Evodia officinalis	Machilus thunbergii	100.8 ± 3.2	99.1 ± 3.1
61	Evodia officinalis	Aucklandia lappa	93.6 ± 7.8	34.1 ± 2.3
62	Jeffersonia dubia	Rheum palmatum	120.1 ± 2.0	106.9 ± 2.3
63	Jeffersonia dubia	Psoralea corylifolia	100.6 ± 12.6	111.8 ± 1.7
64	Jeffersonia dubia	Zingiber officinale	92.3 ± 6.1	106.2 ± 4.9
65	Jeffersonia dubia	Terminalia chebula	86.3 ± 4.7	70.1 ± 2.1
66	Jeffersonia dubia	Euryale ferox	92.3 ± 3.3	93.5 ± 6.8
67	Jeffersonia dubia	Citrus aurantium	100.2 ± 4.8	105.3 ± 3.8
68	Jeffersonia dubia	Machilus thunbergii	101.5 ± 2.1	105.9 ± 4.3
69	Jeffersonia dubia	Aucklandia lappa	80.4 ± 6.0	25.8 ± 1.7
70	Rheum palmatum	Psoralea corylifolia	110.5 ± 3.5	118.2 ± 8.7
71	Rheum palmatum	Zingiber officinale	61.8 ± 8.5	107.6 ± 0.9
72	Rheum palmatum	Terminalia chebula	105.0 ± 11.4	70.4 ± 5.7
73	Rheum palmatum	Euryale ferox	113.6 ± 8.8	117.3 ± 4.7
74	Rheum palmatum	Citrus aurantium	104.0 ± 13.9	107.1 ± 0.2
75	Rheum palmatum	Machilus thunbergii	109.7 ± 10.9	110.6 ± 2.7
76	Rheum palmatum	Aucklandia lappa	84.7 ± 5.6	25.3 ± 1.4
77	Psoralea corylifolia	Zingiber officinale	112.7 ± 16.3	113.2 ± 3.1
78	Psoralea corylifolia	Terminalia chebula	59.8 ± 4.1	70.0 ± 7.1
79	Psoralea corylifolia	Euryale ferox	98.3 ± 9.8	114.5 ± 5.2
80	Psoralea corylifolia	Citrus aurantium	102.9 ± 6.8	109.5 ± 5.2
81	Psoralea corylifolia	Machilus thunbergii	105.3 ± 2.1	114.5 ± 2.8
82	Psoralea corylifolia	Aucklandia lappa	93.7 ± 10.1	55.6 ± 2.5
83	Zingiber officinale	Terminalia chebula	70.2 ± 5.6	62.9 ± 1.3
84	Zingiber officinale	Euryale ferox	102.6 ± 7.8	107.8 ± 3.6
85	Zingiber officinale	Citrus aurantium	99.9 ± 4.9	112.2 ± 3.6
86	Zingiber officinale	Machilus thunbergii	112.6 ± 10.1	109.8 ± 3.7
87	Zingiber officinale	Aucklandia lappa	99.7 ± 8.4	25.9 ± 2.2
88	Terminalia chebula	Euryale ferox	59.2 ± 2.5	51.4 ± 2.8
89	Terminalia chebula	Citrus aurantium	68.5 ± 2.8	60.3 ± 0.9
90	Terminalia chebula	Machilus thunbergii	59.1 ± 5.5	62.0 ± 6.7
91	Terminalia chebula	Aucklandia lappa	45.8 ± 2.7	28.0 ± 8.2
92	Euryale ferox	Citrus aurantium	113.9 ± 14.3	101.7 ± 2.4
93	Euryale ferox	Machilus thunbergii	118.8 ± 15.1	102.3 ± 7.6
94	Euryale ferox	Aucklandia lappa	86.2 ± 4.0	24.4 ± 1.4
95	Citrus aurantium	Machilus thunbergii	108.4 ± 5.6	109.0 ± 7.6
96	Citrus aurantium	Aucklandia lappa	85.1 ± 3.7	30.1 ± 1.9
97	Machilus thunbergii	Aucklandia lappa	97.2 ± 5.5	23.9 ± 1.8

Data are presented as the mean ± SEM.

Table 3

NO assay results of prescription plant extracts.

No.	Plants		% of LPS
			Water Extract(Group PA)	Ethanol Extract(Group PB)
	Control (0.1% DMSO without LPS)		22.9 ± 4.8	19.2 ± 0.6
1	Psoralea corylifolia	Myristica fragrans	100.5 ± 7.3	104.6 ± 3.0
	Evodia officinalis	Schisandra chinensis
2	Paeonia japonica	Aucklandia lappa	95.7 ± 2.3	23.1 ± 1.4
	Cinnamomum loureirii	Cimicifuga heracleifolia
3	Evodia officinalis	Paeonia japonica	95.3 ± 3.3	25.6 ± 1.1
	Chaenomeles sinensis	Aucklandia lappa
	Coix lachryma-jobi
4	Bupleurum falcatum	Paeonia japonica	96.8 ± 2.9	23.2 ± 1.7
	Cimicifuga heracleifolia	Aucklandia lappa
5	Coix lachryma-jobi	Amomum villosum	95.7 ± 8.0	29.6 ±1.7
	Atractylodes macrocephala	Aucklandia lappa
6	Bupleurum falcatum	Cimicifuga heracleifolia	103.8 ± 11.1	106.1 ± 2.1
	Zingiber officinale	Paeonia japonica
	Atractylodes macrocephala
7	Evodia officinalis	Aucklandia lappa	99.1 ± 6.0	46.7 ± 3.2
	Amomum cardamomum	Jeffersonia dubia
	Myristica fragrans

Data are presented as the mean ± SEM.

Nitric oxide (NO) is synthesized from l-arginine by nitric oxide synthases (NOSs), such as endothelial NOS (eNOS), constitutive NOS (cNOS), and inducible NOS (iNOS), in various cells. In the patients with IBD, NO production and iNOS activity were increased in the inflamed colonic mucosa [12]. Thus, to evaluate anti-inflammatory effect of each sample (100 μg/mL), we performed the NO assay on RAW264.7 cells stimulated with LPS (1 μg/mL). Samples that were extracted with 50% ethanol tended to inhibit NO production more than samples extracted with water (Table 1, Table 2 and Table 3). In this assay, 72 extracts, which resulted in a decrease of more than 80% in NO production, were selected for the subsequent monocyte adhesion assay.

2.2. TNF-α Induced Monocyte-Epithelial Cell Adhesion Assay

The adhesion of inflammatory cells to colonic epithelial cells is an important event in colonic inflammation. In the colonic mucosa, immune cells, such as macrophages and T lymphocytes, infiltrate the mucosal layer and are located in close proximity to the epithelial layer in inflammatory conditions such as IBD. The interaction between immune cells and epithelial cells releases inflammatory mediators, such as NO, TNF-α, and IL-6, and ultimately results in the disruption of the barrier function of the intestinal epithelium [13]. Therefore, we performed a TNF-α-induced monocyte-epithelial cell adhesion assay to screen the 72 selected extracts (at 100 μg/mL) using HT-29 as the epithelial cell line and U937 as the monocyte cell line, which are precursors of macrophages. U937 cells were prelabeled with 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein/acetoxy-methyl ester (BCECF/AM) for 30 min before co-incubation with HT-29 cells. After co-incubation for 30 min, the wells were washed to remove unadhered cells, and the BCECF fluorescence was measured to evaluate cell-to-cell adhesion. 5-aminosalicylic acid (5-ASA, 20 mM) was used as the reference drug. In this assay, we selected 27 extracts that resulted in 80% lower BCECF fluorescence than TNF-α (100 ng/mL) alone (Table 4).

Table 4

Inhibitory effects of the selected 72 samples against TNF-α induced adhesion.

Samples	% of TNF-α	Samples	% of TNF-α
Control	45.8 ± 2.8	mA90	103.9 ± 12.7
5-ASA	76.2 ± 1.7	mA91	98.4 ± 5.2
A12	74.8 ± 1.6	mB8	76.6 ± 4.5
A30	61.8 ± 5.9	mB15	94.0 ± 13.1
A36	109.4 ± 8.9	mB19	105.9 ± 3.5
A47	101.4 ± 3.5	mB23	87.2 ± 12.3
A48	106.9 ± 10.8	mB27	103.5 ± 4.1
A63	71.4 ± 5.9	mB31	81.3 ± 9.6
A68	94.1 ± 7.1	mB34	93.1 ± 10.5
B3	74.3 ± 1.5	mB38	96.1 ± 8.3
B12	105.1 ± 6.7	mB42	86.4 ± 5.9
B20	56.5 ± 5.6	mB47	73.2 ± 4.2
B30	53.2 ± 2.4	mB48	98.3 ± 11.7
B32	68.1 ± 5.1	mB49	74.6 ± 7.6
B36	96.7 ± 52	mB52	85.3 ± 6.3
B38	62.0 ± 6.6	mB57	102.9 ± 7.8
B45	74.7 ± 6.5	mB61	88.3 ± 10.9
B47	71.3 ± 6.1	mB65	98.9 ± 9.3
B48	103.9 ± 14.5	mB69	107.8 ± 6.5
B58	53.9 ± 6.2	mB72	95.7 ± 12.6
B63	86.3 ± 11.9	mB76	64.8 ± 4.3
B68	103.7 ± 2.1	mB78	103.5 ± 4.4
mA15	83.0 ± 7.2	mB82	100.3 ± 8.4
mA21	75.0 ± 3.7	mB83	105.5 ± 10.5
mA27	101.3 ± 5.0	mB87	76.2 ± 2.2
mA32	87.2 ± 4.5	mB88	97.6 ± 7.9
mA34	102.3 ± 13.2	mB89	104.1 ± 9.9
mA46	83.7 ± 7.2	mB90	75.4 ± 2.4
mA48	101.9 ± 5.1	mB91	68.9 ± 6.8
mA49	97.3 ± 11.8	mB94	72.5 ± 5.0
mA51	97.6 ± 11.6	mB96	96.6 ± 7.7
mA52	97.9 ± 9.3	mB97	67.4 ± 2.8
mA71	69.9 ± 4.6	PB2	66.5 ± 8.6
mA78	92.5 ± 11.2	PB3	78.9 ± 2.8
mA83	88.9 ± 8.7	PB4	76.2 ± 8.5
mA88	107.5 ± 8.6	PB5	72.9 ± 4.1
mA89	106.7 ± 10.9	PB7	78.7 ± 3.5

Data are presented as the mean ± SEM.

2.3. TNF-α and IL-6 Production in RAW264.7 Cells

We determined the levels of inflammatory cytokines (TNF-α and IL-6) in LPS-stimulated RAW264.7 cells to evaluate the anti-inflammatory profile of the selected samples. 5-ASA was used as the reference drug. First, RAW264.7 cells were treated with LPS (1 μg/mL) for 1 h, and then samples (at 100 μg/mL) or 5-ASA (20 mM) were added for 24 h. In this assay, we selected seven extract samples (B58, mB8, mB76, mB87, mB91, mB94, and mB97; Figure 1) that resulted in significant inhibition of the production of both cytokines. Therefore, we screened these seven samples in vivo.

Figure 1

Effect of selected 27 samples on TNF-α and IL-6 production in LPS-stimulated RAW264.7 cells. RAW264.7 cells (3 × 105 cells/well, 24-well plate) were first treated with LPS (1 μg/mL) for 1 h and then treated with 5-ASA (20 mM) or sample (100 μg/mL) for 24 h. TNF-α and IL-6 concentrations were measured by using ELISA (A and B). Sample labels were following: A12 (Pulsatilla koreana), A30 (Melia azedarach), A63 (Cinnamomum cassia), mA21(Myristica fragrans and Sanguisorba hakusanensis), mA71 (Rheum palmatum and Zingiber officinale), B3 (Zingiber officinale), B20 (Bupleurum falcatum), B30 (Melia azedarach), B32 (Patrinia scabiosaefolia), B38 (Jeffersonia dubia), B45 (Machilus thunbergii), B47 (Terminalia chebula), B58 (Aucklandia lappa), mB8 (Paeonia lactiflora and Aucklandia lappa), mB47 (Sanguisorba hakusanensis and Zingiber officinale), mB49 (Sanguisorba hakusanensis and Euryale ferox), mB76 (Rheum palmatum and Aucklandia lappa), mB87 (Zingiber officinale and Aucklandia lappa), mB90 (Terminalia chebula and Machilus thunbergii), mB91 (Terminalia chebula and Aucklandia lappa), mB94 (Euryale ferox and Aucklandia lappa), mB97 (Machilus thunbergii and Aucklandia lappa), PBs (prescriptions, see Table 3). The control group was treated with 0.1% DMSO. The data are presented as the mean ± SEM of three independent experiments. * p < 0.05 vs. LPS.

2.4. In Vivo Screening of Seven Samples in Mouse Model of Experimental Colitis

In the in vivo screening, we employed C57BL/6 mice with DSS-induced colitis for the seven selected samples (B58, mB8, mB76, mB87, mB91, mB94, and mB97). 5-ASA (200 mg/kg) and all samples (200 mg/kg) were orally administered once per day during the DSS-administration period. In the DSS-induced colitis model, we used the indices of disease activity, colon length, and myeloperoxidase (MPO) activity to evaluate the efficacy of samples. Samples B58, mB87, and mB91 significantly ameliorated the symptoms of colitis, and 5-ASA and other samples tended to ameliorate the disease activity index (DAI) (Figure 2A). MPO, abundantly expressed in neutrophils, was measured to determine the levels of inflammation. 5-ASA and all samples except for mB8, slightly decreased MPO activity in the colon tissue (Figure 2C). Samples mB87, mB91, mB94, and mB97 tended to improve DSS-induced colitis; therefore, we further investigated the efficacy of these samples in ICR mice with TNBS-induced colitis. Although a single plant sample, B58, exerted an ameliorative effect of colitis, it was omitted from the next step because it was included in all of the mixture samples. In the TNBS-induced colitis model, we used the indices of disease activity, colon weight/length ratio, and MPO activity to evaluate efficacy. Sample mB91 resulted in a significant improvement in colitis and other samples led to a slight improvement in colitis in the DAI (Figure 3A). The colon weight/length ratio was measured as an index of edema in the inflamed colon. Samples mB87 and mB91 slightly decreased the colon weight/length ratio (Figure 3B). 5-ASA and all samples slightly decreased MPO activity in the colon tissue (Figure 3C). Sample mB87 (Zingiber officinale and Aucklandia lappa) and mB91 (Terminalia chebula and Aucklandia lappa) resulted in a significant improvement in both types of experimental colitis. From these results, we decided to prepare a formulation of the three medicinal plants based on mB87 and mB91, called KM1608 (a 1:2:2 mixture of Zingiber officinale, Terminalia chebula, and Aucklandia lappa).

Figure 2

Effect of seven selected samples on DSS-induced colitis in mice. Colitis was induced in C57BL/6 mice by the administration of 1.7% DSS in the drinking water for 7 days. The animals were orally administered the extract samples (200 mg/kg) or 5-ASA (200 mg/kg), the reference drug, once per day. The disease activity index was scored during the experiment (A). Colon length was measured at necropsy (B). MPO in the colon lysate was measured by using an ELISA kit (C). The data are presented as the mean ± SEM. n = 6–8, * p < 0.05 vs. control.

Figure 3

Effect of four selected samples on TNBS-induced colitis in mice. Colitis was induced in ICR mice by the intrarectal injection of TNBS. The animals were treated with extract samples (200 mg/kg) or 5-ASA (200 mg/kg), used as the reference drug, once per day. Disease activity index was scored during the experiment (A). Colon weight and length were measured at necropsy (B). MPO in the colon tissue lysate was measured by using an ELISA kit (C). The data are presented as the mean ± SEM. n = 6–8, * p < 0.05 vs. control.

2.5. Effect of KM1608 on Mice with TNBS-Induced Colitis

We conducted a more detailed investigation of the in vivo efficacy of KM1608 in TNBS-induced colitis. KM1608 (200, 400, and 600 mg/kg), 5-ASA (200 mg/kg), and prednisolone (5 mg/kg) were orally administered once per day. 5-ASA and prednisolone were used as the reference drugs. We used the following indices to evaluate the efficacy of KM1608: DAI, colon length, colon weight/length ratio, MPO activity, TNF-α, and IL-6. KM1608 significantly decreased the DAI and the colon weight/length ratio in a dose-dependent manner (Figure 4A,C). KM1608 resulted in a slight improvement in the colon length (Figure 4B). We determined the MPO activity and pro-inflammatory cytokines (TNF-α and IL-6) as a marker of inflammation in the colon tissue of colitis-induced mice. KM1608 (600 mg/kg) administration resulted in a significant decrease in MPO activity and TNF-α level (Figure 5A,B), and slightly decreased IL-6 level in the colon tissue lysate (Figure 5C). In addition, KM1608 (600 mg/kg) resulted in better parameters for many of the indices used for colitis evaluation than 5-ASA and prednisolone.

Figure 4

Effect of KM1608 on TNBS-induced colitis in mice. Colitis was induced in ICR mice by the intrarectal injection of TNBS. The animals were treated with KM1608 (200, 400, and 600 mg/kg), 5-ASA (200 mg/kg), and prednisolone (5 mg/kg) orally once per day. 5-ASA and prednisolone were used as the reference drugs. The disease activity index was scored during the experiment (A). Colon length and weight were measured at necropsy (B,C). The data are presented as the mean ± SEM. n = 6–8, * p < 0.05 vs. control.

Figure 5

KM1608 inhibits the inflammatory factors involved in TNBS-induced colitis. Colitis was induced in ICR mice by the intrarectal injection of TNBS. The animals were treated with KM1608 (200, 400, and 600 mg/kg), 5-ASA (200 mg/kg), and prednisolone (5 mg/kg) orally once per day. 5-ASA and prednisolone were used as the reference drugs. The MPO, TNF-α, and IL-6 levels in the colon tissue lysates were measured by using ELISA kits (A–C). The data are presented as the mean ± SEM of three independent experiments. * p < 0.05 vs. control.

Medicinal plants generally contain multiple bioactive compounds that are responsible for the beneficial effects on human diseases through synergistic actions [14]. Synergistic anti-inflammatory effects of the compound combination of the herbal formula GuGe FengTong, prepared from three herbs, Spatholobus suberectus (Leguminosae), Dioscorea nipponica (Dioscoreaceae), and Zingiber officinale (Zingiberaceae), were recently reported [15]. Compared with the single compounds, the combination of two compounds, biochanin A and 6-gingerol, could synergistically inhibit the production of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and the activation of MAPK signaling pathway in LPS-stimulated RAW264.7 cells [15]. In addition, a combination effect was also evident on the antioxidant and anti-inflammatory effects of honokiol and modified citrus pectin in mouse monocytes [16]. In other research areas, synergistic antidiabetic activity of Vernonia amygdalina and Azadirachta indica has also been reported [17]. Synergism has also supported and been identified in drug-target interaction studies, in which drugs with multiple target network mechanisms are believed to have greater efficacy for the treatment of disease [18].

Several studies have reported the anti-inflammatory effect of plant extracts; for example, Terminalia chebula and Zingiber officinale ameliorated acetic acid-induced colitis in rats via the inhibition of MPO activity [19] and the inhibition of MPO activity, TNF-α, prostaglandin E2 [20], respectively, and Aucklandia lappa extract ameliorated DSS-induced colitis in mice via the inhibition of IFN-γ and IL-6 [21]. Moreover, 6-gingerol, active component of Zingiber officinale, has anti-inflammatory activity via inhibiting NO production, iNOS expression and cyclooxygenase activity [22], ellagic acid, active component of Terminalia chebula, inhibits cyclooxygenase activity and reduces paw edema in the carrageenan-induced edema [23], and also dehydrocostus lactone, active component of Aucklandia lappa, inhibits NO and TNF-α production in LPS-activated RAW264.7 cells [24]. In the present study, the administration of KM1608, a mixture of the extracts from three plants, exerted more potent therapeutic effects than the administration of each plant individually and the mixture of the extracts of two plants at the same dose in DSS-induced colitis. These results indicate that KM1608 has therapeutic potential for the treatment of IBD.

3. Materials and Methods

3.1. Plant Material

All medicinal plant samples were purchased from Songrim Muyak (Seoul, Korea). The samples were extracted twice with water or 50% ethanol (v/v) at 80 °C for 3 h, and the extracted solutions were filtered and evaporated. The samples that consisted of a mixture of two plants were mixed at a 1:1 (w/w) ratio, traditional medicine formulations were mixed at the ratios based on prescription, and KM1608 was mixed in a 1:2:2 ratio (Zingiber officinale: Terminalia chebula: Aucklandia lappa) before the extraction process. The extracts were then freeze dried to obtain the powders used as the test extract samples.

3.2. Animal and Cell Culture

Seven-week-old female C57BL/6 and ICR mice were purchased from Daehan Bio Link (Seoul, Korea) and acclimated for 7 days in a specific pathogen-free (SPF) environment under constant conditions (temperature: 23 °C ± 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 in accordance with the instructions of the Ethics Committee for Use of Experimental Animals at Kolmar Korea Co., Ltd. (confirmation number: 16-NP-IBD-011-P). The RAW264.7 mouse macrophage cell line was purchased from the ATCC (Manassas, VA, USA), and the cells were seeded in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin obtained from Life Technologies (Waltham, MA, USA). The human colorectal adenocarcinoma cell line, HT-29, and the human monocytic cell line, U937, were purchased from the ATCC (USA) and seeded in RPMI supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin. The cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2.

3.3. Determination of Nitric oxide (NO), TNF-α, IL-6, and MPO Production

RAW264.7 cells (3 × 105 cells/well in a 24-well plate) were treated LPS (1 μg/mL) for 1 h and then treated with 5-ASA (20 mM) or samples (100 μg/mL) for 24 h. After incubation for 24 h, nitrite production was estimated by using Griess reagent [25] and a standard curve previously prepared using sodium nitrite (Promega, Fitchburg, WI, USA). The cell supernatant (50 μL) was mixed with an equal volume of Griess reagent and the absorbance at 540 nm was measured by using a microplate reader (Molecular Devices Co., San Jose, CA, USA). 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 by 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 (R&D Systems, Minneapolis, MN, USA), and MPO (HK210, Hycult Biotechnology, Wayne, PA, USA) levels in the supernatant were measured by using ELISA kits in accordance with the manufacturer’s instructions.

3.4. Monocyte Adhesion Assay

U937 cells were prelabeled with BCECF/AM (10 μg/mL, Sigma, St. Louis, MO, USA) for 30 min at 37 °C. HT-29 cells (2 × 106 cells/well in 48-well plates)) were pretreated with sample (100 μg/mL) or 5-ASA (20 mM) for 1 h and then stimulated with TNF-α for 24 h [26]. Subsequently, HT-29 cells were co-incubated with BCECF/AM-prelabeled U937 cells (5 × 105 cells/well) for 30 min at 37 °C. The wells were washed twice with PBS to remove unadhered U937 cells. The cells were lysed with 0.1% Triton X-100 in 0.1 M Tris and BCECF fluorescence was analyzed by using a microplate reader (TECAN, Grödig, Austria), with excitation at 485 nm and emission at 520 nm.

3.5. DSS-Induced Colitis

Acute colitis was induced in C57BL/6 mice for 7 days by the addition of 1.7% (w/v) DSS to drinking water. Daily measurements of body weight, stool consistency, and rectal bleeding were conducted. The normal group received water without DSS. The control group received drinking water containing 1.7% DSS. The sample groups received DSS-containing drinking water and extract samples (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 the extract samples, 5-ASA, and prednisolone for in vivo experiments. All drugs were orally administered once per day during the experiment. A clinical assessment was performed to determine the DAI. DAI comprised the total score of each of the following: (weight loss: 1 = 1–5%; 2 = 5–10%; 3 = 10–20%; 4 = >20%; stool consistency: 0 = normal; 2 = loose stool; 4 = diarrhea; and rectal bleeding: 0 = negative; 2 = mild; 4 = severe). If an animal died, DAI was scored as 15. The animals were sacrificed after 7 days of DSS treatment, and the colon length was measured.

3.6. TNBS-Induced Colitis

Acute colitis was induced in ICR mice. A 100 μL aliquot of 0.5% TNBS solution dissolved in ethanol (50%, v/v) was instilled into the colon via a cannula to induce colitis. To prevent outflow of the agents from the anus, mice were held in the head-down position for 1 min after the instillation. Body weight and disease symptoms were assessed on three subsequent days. A clinical assessment was performed to determine the DAI. DAI was scored as described in Section 3.5. If an animal died, DAI was scored as 15. After 5 days of TNBS injection, the animals were sacrificed and then colon edema, length, and weight were measured.

3.7. Statistical Analysis

The results are expressed as the mean ± SEM. Statistical comparisons were performed by using one-way analysis of variance (ANOVA) followed by Tukey’s test. A value of p < 0.05 was considered to indicate significant difference.

4. Conclusions

This study was conducted to discover potent formulations of natural products with anti-inflammatory activity. In the in vitro screening experiments, we tested the effects of 350 extracted samples on NO, TNF-α, and IL-6 production in LPS-stimulated RAW264.7 cells and on TNF-α-induced monocyte (U937)-colonic epithelial (HT-29) adhesion ability. Finally, we found the formulation KM1608, composed of Zingiber officinale, Terminalia chebula, and Aucklandia lappa, through in vivo screening in a mouse model of experimental colitis. KM1608 significantly ameliorated the severity of colitis and the colon weight/length ratio in a dose-dependent manner. In addition, KM1608 inhibited MPO activity and pro-inflammatory cytokines in the colon tissue lysate of DSS-induced colitis. Moreover, the ameliorative effect of KM1608 on DSS-induced colitis was more potent than that of 5-ASA or prednisolone. Collectively, KM1608 administration improved the symptoms of colitis and the inflammatory responses. IBD such as ulcerative colitis and Crohn’s disease are very complicate disease to control pathophysiology with single target therapy. Many trials have failed to development of IBD therapeutics with single target agents. We believe that future studies are required to consider multiple target therapy and/or synergistic combination like as herbal formulation. We hope that our present study and the formulation will provide useful ways for the treatment of IBD.