Inhibitory Effects of 87 Species of Traditional Chinese Herbs on Nitric Oxide Production in RAW264.7 Macrophages, Activated with Lipopolysaccharide and Interferon-γ

ABSTRACT

In the current study, effects of 87 species of traditional Chinese herbs on nitric oxide (NO) production by a murine macrophage-like cell line, RAW 264.7, which was activated by lipopolysaccharide (LPS) and interferon-γ (IFN-γ) were investigated. NO production by macrophages preactivated with LPS and IFN-γ for 16 h was also inhibited by the 19 species. The inhibitory rate of the 19 species was more than 80%. Among these species, Adina rubella.Hance, Centipeda minima. (L.), and Gentiana loureirii. (G. Don) Griseb. exhibited strong inhibitory activity toward NO production, exhibiting IC₅₀ values of 3, 7.34, and 13.5 µg/ml, respectively. However, Aristolochia kaempferi. Wild var. heterophylla, Stephania tetrandra. S. Moore, Ardisia japonica. (Hornsted) Blume, and Helicteres angustifolia. (L.) also affected the cell viability. These results suggest that Adina rubella., Centipeda minima., and Gentiana loureirii. have the pharmacological ability to suppress NO production by activated macrophages. The inhibitory mechanisms of NO production will need to be further studied in the future.

Keywords:

• Macrophage
• nitric oxide
• RAW264.7
• traditional Chinese herbs

Introduction

Macrophages play a prominent role in the host defense by inducing cellular damage in infectious agents and tumors (Adams & Hamilton, 1992; Auger & Ross, 1992). Macrophages exert cytotoxic and cytostatic effects on target cells by releasing nitric oxide (NO) (Nathan & Hibbs, 1991; Nathan, 1992; Nathan & Xie, 1994). NO-mediated toxicity in the target cells includes growth arrest through the formation of an iron-nitrosyl complex in mitochondrial enzymes, thereby inhibiting cellular respiration (Drapier & Hibbs, 1986) energy depletion through deregulation of ATP metabolism (Szabo et al., 1996) and programmed cell death (Cui et al., 1994; Mannick et al., 1996; Gal et al., 1997). NO is a double-edged sword: excessive production of NO by macrophages is potentially toxic (Schmidt & Walter, 1994). Indeed, overproduction of NO has been implicated as a pathological factor in several forms of chronic human diseases, including arthritis (Connor et al., 1995), neurotoxicity (Zhang et al., 1994), and cancer (Ohshima & Bartsch, 1994; Liu & Hotchkiss, 1995). Glucocorticoids, representative anti-inflammatory agents, strongly inhibit NO production (Rosa et al., 1990). This, therefore, suggests that inhibition of chronic NO production in macrophages could be a target for potential anti-inflammatory drugs. The enzyme responsible for the synthesis of nitric oxide in macrophages is an inducible nitric oxide synthase (iNOS), which does not exist ordinarily but is strongly induced upon exposure to bacterial endotoxin and inflammatory cytokines (Stuehr & Marletta, 1985; 1987).

It is noteworthy that nearly all the types of NO-mediated toxicity in target cells occur in the producer macrophage cells (Drapier & Hibbs, 1988; Lancaster & Hibbs, 1990; Albina & Mastrofrancesco, 1993; Albina et al., 1993; Messmer et al., 1995; Zingarelli et al., 1996). Thus, responses in macrophages and cocultured target cells as a result of NO production are related. For this reason, we examined the possibility of using macrophages as a model system for the study of NO-mediated cellular damage. A number of macrophage cell lines have been found to produce NO at levels comparable to primary cells when appropriately stimulated (Stuehr & Marletta, 1987). Commonly used stimuli for NO production by macrophages include the cytokine interferon-γ (IFN-γ) and bacterial lipopolysaccharide (LPS) (Nathan & Xie, 1994). Among these cell lines, the mouse RAW264.7 cells have been shown to produce different levels of NO when stimulated with different combinations of IFN-γ and LPS (Stuehr & Marletta, 1987; Lorsbach et al., 1993).

Traditional Chinese herbs have been used as traditional remedies for thousands of years. Many efforts are directed to the finding of natural compounds that may influence NO production in macrophages. In the current study, we collect 87 species of traditional Chinese herbs used for the treatment of inflamatory disease. We investigated the effects of the 87 species of traditional Chinese herbs on nitric oxide production in RAW264.7 macrophages, activated with LPS and INF-γ. Cytotoxicity of these 87 species of traditional Chinese herbs was measured by using the MTT assay method (Osmann, 1983).

Materials and Methods

Materials

The traditional Chinese herbs belonging to 42 families were collected from various regions of Taiwan and China and were identified by Technician Nien-yung Chiu (Graduate Institute of Chinese Pharmaceutical Sciences, Chine Medical University, Taichung, Taiwan) and Prof. Weichun Wu (Division of Pharmacognosy, Shenyang Pharmaceutical University, Shenyang, China), where voucher specimens are deposited. All voucher specimens are given a number (No. 1–No. 87 in Table 1), correspondingly.

Table 1 Inhibitory activity of 87 species of traditional Chinese herbs on NO production by a murine macrophage-like cell line, RAW264.7, activated with lipopolysaccharide and interferon-γ and the viability of macrophage by the MTT assay.

No.	Plant	Family	Plant part	NO^a.	IC50(µg/ml)	MTT^b.
1	Gendarussa ventricosa. (Wall.) Nees	Acanthaceae	Whole plant	22.02	—	107.53
2	Mananthus patentiflora. (Hemsl.) Bremek.	Acanthaceae	Whole plant	48.53	—	97.21
3	Actinidia chinensis. Planch. var. setosa. Li	Actinicliaceae	Stem	12.60	—	101.60
4	Actinidia collosa. Lindl.	Actinicliaceae	Stem	20.33	—	100.28
5	Actinidia latifolia. (Gardn. & Champ.) Merr.	Actinicliaceae	Stem	14.43	—	109.03
6	Actinidia rubricaulis. Dunn	Actinicliaceae	Stem	20.33	—	92.74
7	Achyranthes aspera. L.	Amaranthaceae	Root	72.41	—	97.39
8	Aristolochia fangchi. Y.C. Wu, L.D. Chow & S.M. Hwang	Aristolochiaceae	Root	48.46	—	110.76
9	Aristolochia kaempferi. Willd. f. heterophylla. (Hemsl.) S.M. Hwang	Aristolochiaceae	Root	103.37	—	38.47
10	Aristolochia moupinensis. Franch.	Aristolochiaceae	Stem	67.89	—	114.6
11	Mahonia bealei. (Fort.) Carr.	Berberidaceae	Stem	62.45	—	106.56
12	Ananas comosus. (L.) Merr.	Bromeliaceae	Peel	36.72	—	99.36
13	Lobelia chinensis. Lour. [L. radicans. Thunb.]	Campanulaceae	Whole plant	24.59	—	110.51
14	Lonicera hypoglauca. Miq.	Caprifoliaceae	Dry flower bud	66.18	—	91.27
15	Drymaria cordata. (L.) Willd. ex Roem. & Schult.	Caryophyllaceae	Whole plant	28.42	—	96.3
16	Euonymus fortunei. (Turcz.) Hand. Mazz.	Celastraceae	Twig with leaf	36.72	—	107.1
17	Laggera alata. (D. Don) Sch. Bip. ex Oliv.	Compositae	Whole plant	30.75	—	113.29
18	Blumea balsamifera. (L.) DC.	Compositae	Whole plant	105.81	30.03	80.52
19	Centipeda minima. (L.) A. Br. & Ascher.	Compositae	Whole plant	104.56	7.34	78.65
20	Elephantopus scaber. L.	Compositae	Whole plant	34.02	—	111.39
21	Kalimeris indica. (L.) Sch. Bip.	Compositae	Whole plant	91.91	32.37	93.34
22	Senecio scandens. Buch. Ham.	Compositae	Whole plant	58.09	—	101.92
23	Taraxacum mongolicum. Hand. Mazz.	Compositae	Whole plant	76.14	—	106.36
24	Xanthium sibiricum. Patr.	Compositae	Fruit	46.06	—	108.83
25	Erycibe subspicata. Wall. ex G. Don	Convolvulaceae	Stem	35.12	—	94.92
26	Porana racemosa. Roxb. var. tomen tella. Wu	Convolvulaceae	Whole plant	70.04	—	86.67
27	Gynostemma pentaphyllum. (Thunb.) Makino	Cucurbitaceae	Whole plant	34.55	—	113.65
28	Cibotium barometz. (L.) J. Smith [Polypodium barometz. L.]	Dicksoniaceae	Rhizome	36.84	—	102.6
29	Antidesma montanum. Bl.	Euphorbiaceae	Leaf	53.97	—	106.91
30	Glochidion eriocarpum. Champ. ex Benth.	Euphorbiaceae	Leaf	57.88	—	97.93
31	Mallotus apelta. (Lour.) Muell. Arg.	Euphorbiaceae	Leaf	77.59	—	111.64
32	Securinega suffruticosa. (Pall.) Rehd.	Euphorbiaceae	Twig with leaf	90.67	26.12	103.79
33	Gentiana loureirii. (G. Don) Griseb.	Gentianaceae	Whole plant	98.81	13.5	97.03
34	Imperata cylindrica. (L.) Beauv. var. major. (Nees) C.E. Hubb.	Gramineae	Rhizome	23.98	—	99.17
35	Hypericum japonicum. Thunb.	Guttiferae	Whole plant	77.18	—	111.34
36	Salacia polysperma. Hu	Hippocrateaceae	Root	52.38	—	109.06
37	Scutellaria barbata. D. Don [S. rivularis. Wall.]	Labiatae	Whole plant	92.86	38.28	95.59
38	Clerodendranthus spicatus. (Thunb.) C.Y. Wu ex H.W. Li	Labiatae	Whole plant	52.28	—	109.71
39	Leonurus artemisia. (Lour.) S.Y. Hu	Labiatae	Whole plant	81.12	37.46	109.57
40	Rabdosia nervosa. (Hemsl.) C.Y. Wu & H.W. Li	Labiatae	Whole plant	74.27	—	111.98
41	Rabdosia ternifolia. (D. Don) Hara	Labiatae	Whole plant	33.53	—	111.18
42	Sargentodoxa cuneata. (Oliv.) Rehd. & Wils. [Holboellia cuneata. Oliv.]	Lardizabalaceae	Stem	17.68	—	108.09
43	Abrus cantoniensis. Hance	Leguminosae	Whole plant	95.64	68.31	111.59
44	Bauhinia championii. (Benth.) Benth.	Leguminosae	Root or stem	85.68	40.47	104.19
45	Bauhinia variegata. L.	Leguminosae	Root	30.56	—	111.37
46	Derris robusta. (DC.) Benth.	Leguminosae	Stem with root	46.83	—	103.12
47	Lespedeza cuneata. (Dum. Cours.) G. Don	Leguminosae	Whole plant	39.42	—	103.45
48	Sophora tonkinensis. Gagnep.	Leguminosae	Root	−161.84	—	102.92
49	Zornia gibbosa Spanoghe. [Zornia diphylla. (L.) Pers.]	Leguminosae	Whole plant	89.21	40.12	109.81
50	Strychnos wallichiana. Steud. ex DC.	Loganiaceae	Seed	27.58	—	107.05
51	Oxyspora vagans. (Roxb.) Wall.	Melastomataceae	Twig with leaf	96.03	19.79	101.10
52	Stephania tetrandra. S. Moore	Menispermaceae	Root tuber	102.98	—	22.97
53	Tinospora sinensis. (Lour.) Merr.	Menispermaceae	Stem	25.61	—	99.23
54	Ficus pumila. L.	Moraceae	Stem with leaf	74.07	—	99.56
55	Ficus tikoua. Bur.	Moraceae	Syconium	100	17.51	79.98
56	Psidium guajava. L.	Myrtaceae	Leaf	17.28	—	112.82
57	Ardisia japonica. (Hornsted) Blume	Myrsinaceae	Whole plant	103.53	—	44.82
58	Ardisia solanacea. Roxb.	Myrsinaceae	Whole plant	53.97	—	108.2
59	Embelia laeta.(L.) Mez	Myrsinaceae	Whole plant	53.94	—	103.4
60	Nervilia aragoana. Gaud.	Orchidaceae	Whole plant	33.74	—	97.36
61	Nervilia purpurea. (Hayata) Schltr.	Orchidaceae	Whole plant	18.29	—	99.34
62	Polygala fallax. Hemsl.	Polygalaceae	Whole plant	55.39	—	105.97
63	Polygala glomerata. Lour. var. villosa. C.Y. Wu	Polygalaceae	Whole plant	25.52	—	104.54
64	Drynaria fortunei. (Kunze) J. Smith [Polypodium fortunei. Kunze]	Polypodiaceae	Rhizome	34.96	—	112.66
65	Phellinus linteus. (Berk. & Curt.) Teng	Polyporaceae	Sporophore	33.54	—	95.65
66	Clematis argentilucida. (Levl. & Vant.) W.T. Wang	Ranunculaceae	Stem	37.4	—	110.13
67	Agrimonia pilosa. Ledeb.	Rosaceae	Whole plant	98.81	27.44	88.82
68	Rosa laevigata. Michx.	Rosaceae	Fruit	17.37	—	97.13
69	Adina rubella. Hance	Rubiaceae	Whole plant	103.53	3	79.64
70	Damnacanthus indicus. (L.) Gaertn. f.	Rubiaceae	Whole plant	33.40	—	98.18
71	Gardenia jasminoides. Ellis	Rubiaceae	Fruit	46.03	—	105.32
72	Gardenia jasminoides. Ellis var. grandiflora. Nakai	Rubiaceae	Fruit	45.02	—	109.42
73	Hedyotis diffusa. Willd. [Oldenlandia diffusa. (Willd.) Roxb.]	Rubiaceae	Whole plant	25.61	—	109.63
74	Mussaenda pubescens. Ait.f.	Rubiaceae	Stem with leaf	51.24	—	97.34
75	Neonauclea griffithi. (Hook. f.) Merr.	Rubiaceae	Twig	43.85	—	109.45
76	Psychotria yunnanensis. Hutch.	Rubiaceae	Whole plant	53.57	—	97.12
77	Schizomussaenda dehiscens. (Craib) Li	Rubiaceae	Stem with root	62.70	—	101.20
78	Selaginella doederleinii. Hieron.	Selaginellaceae	Whole plant	83.61	47.11	98.87
79	Picrasma quassioides. (D. Don) Benn.	Simarubaceae	Bark	28.63	—	87.43
80	Helicteres angustifolia. L.	Sterculiaceae	Root	102.07	—	54.88
81	Hydrocotyle sibthorpoides. Lam.	Umbelliferae	Whole plant	43.36	—	99.06
82	Verbena officinalis. L.	Verbenaceae	Whole plant	38.17	—	94.67
83	Viola philippica. Cav. (Viola yedoensis. Makino)	Violaceae	Whole plant	49.39	—	116.73
84	Ampelopsis japonica. (Thunb.) Makino [Paullinia japonica. Thunb.]	Vitaceae	Root tuber	48.42	—	128.5
85	Cissus hastata. (Miq.) Planch.	Vitaceae	Whole plant	34.23	—	96.75
86	Tetrastigma crucistum. Craib & Gag.	Vitaceae	Whole plant	52.78	—	113.76
87	Tetrastigma membranaceum. Wu & Wang	Vitaceae	Whole plant	75.20	—	104.84

^a.NO production inhibitory activity, inhibition (%) by (0.1 mg/ml).

^b.Viability (%) = absorbance of sample/absorbance of control × 100%.

Preparation of extracts

All the traditional Chinese herbs were twice refluxed with 50% aqueous EtOH for 1 h at 90°C. After filtration, the filtrates were combined and concentrated at 40°C with reduced vacuum pumps and freeze-dried for assay.

Sample preparation

Accurately weighed 1 mg of 50% aqueous EtOH extract dissolved in 20 μl DMSO served as sample solution.

Cell culture

RAW 264.7 cells, a mouse macrophage-like cell line transformed with the Abelson's leukemia virus, were obtained from the American Type Culture Collection (Rockville, MD, USA). The cells were maintained by culturing in Ham's F12 medium supplemented 10% heat-inactivated fetal bovine serum in a CO₂ incubator (5% CO₂–95% humidified air) at 37°C. The cells were seeded onto plastic Petri dishes (Falcon, no. 1001; Becton Dickinson, Franklin Lakes, NJ, USA) and passaged twice a week. (Ishii et al., 2001).

Agents

Nutrient Mixture F-12 Ham medium, LPS (Escherichia coli., serotype O55:B5), L-glutamine, N.-1-naphthylethylenediamine dihydrochloride, sulfanilamide, phosphoric acid (H₃PO₄), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), sodium nitrite (NaNO₂), and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemicals Company (St. Louis, MO, USA). Fetal bovine serum was from Gibco, kanamycin sulfate was from Wako (Osaka, Japan), and recombinant mouse IFN-γ was from Genzyme (MA, USA).

Nitrite assay

The first and last columns were preserved for blank and negative control (LPS, and IFN-γ only, without sample solution) respectively. The cells were seeded 200 µl (1.2 × 10⁶ cells/ml) onto each well of 96-well flat-bottom plate and then incubated at 37°C and 5% CO₂ for 1 h. Next, the sampling solution 0.4 µl was added to the culture simultaneously with both Escherichia coli. LPS (100 ng/ml) and recombinant mouse IFN-γ (100 U/ml). Then, cells were incubated at 37°C and 5% CO₂ for approximately 16 h and subsequently chilled on ice. Culture supernatant (100 µl) was placed in the same well of another 96-well flat-bottom plate. (The residues of culture were left to the cell viability assay.) A standard solution of NaNO₂ was placed in alternate wells on the same plate. To quantity nitrite, 100 µl of Griess reagent (1% sulfanilamide in 5% H₃PO₄ and 0.1% N.-1-naphthylethylenediamine dihydrochloride) were individually added to each well. After 10 min, the absorbance of the mixture at 570 nm was determined with a microplate reader (model 3550, Bio-Rad, Muenchen, Germany) and the background absorbance (655 nm) was subtracted (Kitanaka et al., 1998; Daikonya et al., 2002). The inhibition of NO production inhibitory activity in the presence of each plant extract was calculated from the following formula. where A._c is the absorbance of control, A._s is the absorbance of sample, and A._b is the absorbance of blank.

Cell viability assay

MTT was used as an indicator of cell viability as determined by the mitochondria-dependent reduction to formazone. The MTT reagent only reacts with living cells and can be read on a microplate reader. After the 100 µl of culture supernatant was removed to quantity nitrite, 25 µl of MTT reagent (2 mg/ml) were added to the residue medium and incubated for 4 h. The supernatant was removed, and 150 µl DMSO was added to each well and mixed thoroughly to dissolve the dark blue crystals, waiting for a few minutes to ensure that all formazone crystals were dissolved, the reaction products were colorimetrically quantified at 570 nm using a microplate reader, and the background absorbance (655 nm) was subtracted. Plates were normally read within 1 h of adding the DMSO (Mosmann, 1983). The viability of macrophage cells was calculated from the following formula:

Statistics

Results were analyzed for statistical significance by Dunnett's test for multiple comparisons. A p value < 0.05 was regarded as indicating a significant difference.

Results

Inhibitory effects on NO production by activated macrophages

As shown in Table 1, Blumea balsamifera. (L.) DC. (IC₅₀: 30.03 µg/ml); Centipeda minima. (L.) A. Br. & Ascher. (IC₅₀: 7.34 µg/ml); Kalimeris indica. (L.) Sch. Bip. (IC₅₀: 32.37 µg/ml); Securinega suffruticosa. (Pall.) Rehd. (IC₅₀: 26.12 µg/ml); Gentiana loureirii. (G. Don) Griseb. (IC₅₀: 13.5 µg/ml); Scutellaria barbata. D. Don (IC₅₀: 38.28 µg/ml); Leonurus artemisia. (Lour.) S. Y. Hu (IC₅₀: 37.46 µg/ml); Abrus cantoniensis. Hance (IC₅₀: 68.31 µg/ml); Bauhinia championii. (Benth.) Benth. (IC₅₀: 40.47 µg/ml); Zornia gibbosa. Spanoghe (IC₅₀: 40.12 µg/ml); Oxyspora vagans. (Roxb.) Wall. (IC₅₀: 19.79 µg/ml); Ficus tikoua. Bur. (IC₅₀: 17.51 µg/ml); Agrimonia pilosa. Ledeb. (IC₅₀: 27.44 µg/ml); Adina rubella. Hance (IC₅₀: 3 µg/ml); and Selaginella doederleinii. Hieron. (IC₅₀: 47.11 µg/ml) exhibit significant NO production inhibitory activity. The inhibitory rates of these species are more than 80%.

Cell viability

In the MTT assay, we found that 83 species do not show cytotoxity with the viability of macrophage cells over 78%. Aristolochia kaempferi. Wild var. heterophylla., Stephania tetrandra S. moore., Ardisia japonica. (Hornsted) Blume, and Helicteres angustifolia. (L.) showed cytotoxicity (Table 1).

Discussion

We found that 19 species of the traditional Chinese herbs inhibited NO production in activated macrophage. The degree of inhibitory effects of these species on NO production was, in order (by numbering in Table 1), 18 > 19 > 57 = 69 > 9 > 52 > 80 > 55 > 33 = 67 > 51 > 43 > 37 > 21 > 32 > 49 > 44 > 78 > 39, at a concentration of 0.1 mg/ml. The others showed little or no inhibition at 0.1 mg/ml. Among the 19 species of traditional Chinese herbs, Adina rubella. Hance (IC₅₀: 3 µg/ml), Centipeda minima. (L.) A. Br. et Ascher. (IC₅₀: 7.34 µg/ml), Oxyspora vagans. (Roxb.) Wall. (19.79µg/ml), Ficus tikoua. Bur. (17.51 µg/ml), and Gentiana loureirii. (G. Don) Griseb. (13.5 µg/ml) showed the strongest inhibitory activity on NO production.

In the MTT assay, we found that Aristolochia kaempferi. var. heterophylla., Stephania tetrandra., Ardisia japonica., and Helicteres angustifolia. affected cell viability. These results suggest that Adina rubella, Centipeda minima, Gentiana loureirii, Ficus tikoua,. and Oxyspora vagans. have the pharmacological ability to suppress NO production by activated macrophages.

Inhibition of NO production by macrophages act mainly through two mechanisms: one is the inhibition of iNOS expression, and the other is the inhibition of enzyme activity. Most iNOS inhibitors act through either of these mechanisms. The current findings encourage further studies to clarify the signaling pathway for the inhibition of iNOS protein induction by the above-mentioned plants. Identification of other potential anti-inflammatory constituents of the active plants is also of interest for the future.

Acknowledgments

This study was supported by grants from the Interchange Association (Japan) and the National Science Council. This study was also supported by grant CMC90-GCC-07 and CMC91-GCC-09 from China Medical University, Taiwan, Republic of China.