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Cytotoxicity and anti-inflammatory effects of root bark extracts of Acanthopanax henryi
Chinese Journal of Natural Medicines 2014, 12(2): 01210125
Chinese Journal of Natural Medicines
Cytotoxicity and anti-inflammatory effects of root bark extracts of Acanthopanax henryi KIM Jong-Hwan1, LIU Xiang-Qian2*, DAI Ling2, YOOK Chang-Soo3, LEE Kyung-Tae3 1
Herbal Medicine Research Division, Korea Food & Drug Administration, Seoul 363-700, Korea; Hunan Key Laboratory of Traditional Chinese Medicine modernization, School of Pharmacy, Hunan University of Chinese Medicine, Hunan Changsha 410208, China; 3 College of Pharmacy, Kyung-Hee University, Seoul 130-701, Korea 2
Available online 20 Feb. 2014
[ABSTRACT] AIM: To investigate the cytotoxicity, anti-inflammatory activity, and action mechanism of root bark extracts of Acanthopanax henryi. METHOD: The hot methanol extract of the root bark of A. henryi was subjected to XAD-4 column chromatography eluting with a gradient of methanol in water. The cytotoxicity and anti-inflammatory effects of the MeOH fractions were evaluated on the inhibition on lipopolysaccharide (LPS)-induced nitric oxide, prostaglandin E 2 , interleukin-1, and interleukin-6 production in RAW 264.7 macrophages. RESULTS: The 80% MeOH fraction was a better inhibitor of LPS-induced NO, PGE 2 , IL-1, and IL-6 production, and expression of inducible nitric oxide synthase (iNOS) at the protein levels in a concentration-dependent manner. CONCLUSION: The 80% MeOH fraction of A. henryi root bark has significant anti-inflammatory activity. This provides a pharmacological basis for clinical application for the treatment of inflammation. [KEY WORDS] Acanthopanax henryi (Oliv.) Harms; Root bark extract; Cytotoxic activity; Anti-inflammatory activity
[CLC Number] R285
[Document code] A
[Article ID] 2095-6975(2014)02-0121-05
Introduction Acanthopanax henryi (Oliv.) Harms (Araliaceae) has been used as a traditional medicine for the treatment of rheumatism, inflammation, fatigue and so on [1]. As an endemic Asian genus, over thirty-eight species are distributed mainly in northeastern Asia, including China, Korea, Japan, and far-east Russia. In the official compendium in China, Korea, and Japan, the root bark of this plant has been listed [2]. [Received on] 10-Oct.-2012 [Research funding] This project was supported by the PhD Program Open Foundation of the Food and Drug Administration of Korea; Hunan Provincial Natural Science Foundation (No. 11JJ2042), and the Science of Pharmaceutical Analysis of Twelfth Five-Year Key Discipline Projects of Hunan University of Chinese Medicine˗ Science of Chinese Materia Medica of Twelfth Five-Year Key Discipline Projects of Hunan Province [ Corresponding author] LIU Xiang-Qian: Prof., Tel: 86-73188458240, E-mail: [email protected] These authors have no conflict of interest to declare. Copyright © 2014, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved
The Korean Herbal Pharmacopoeia [3] will be revised to include Eleutherococci senticosi Radix et Rhizome originating from Eleutherococcus senticosus (Rupr. et Maxim.) Maxim. The Chinese Pharmacopoeia [4], edited in 2010, has two monographs, Radix et Rhizoma seu Caulis Acanthopanacis originating from the root, rhizome, and/or stem of A. senticosus (Rupr. et Maxim.) Harms and Cortex Acanthopanacis originating from the root bark of A. gracilistylus W.W. Sm., but A. henryi Harms is not included, and only appears as a Hunan local medicinal materials standard [5]. To date, the most studied species among the plants of Acanthopanax spp. for biological activities is A. senticosus (Rupr. et Maxim.) Harms. It was studied mainly for anti-microbial, anti-cancer, anti-inflammatory, anti-platelet, and anti-oxidant activities, cardiac protection, sperm mobility increase, and increases of bone turnover and bone mineral density [6-8]. The lignan taiwanin C, isolated from the root of A. chiisanensis Nakai, is reported to have anti-inflammatory activity through significant inhibition of COX-1 and COX-2, which mediate the PGE 2 production in a dose-dependent manner comparable to the NSAID, indomethacin [9-13]. However, the anti-inflammatory activity and the mechanism of action of the biologically active components of A.
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henryi root bark were not studied until now. On this basis, the effects of the MeOH extract of this plant and its fractions were evaluated for the release of anti-inflammatory mediators, such as NO, PGE 2 , IL-1, and IL-6 in LPS-stimulated RAW 264.7 macrophage cells.
Materials and Methods Plant material The A. henryi root bark material was collected in August 2008 in Mt. Daewoong, Hunan province, China, and dried in the shade. The plant was identified by Emeritus Professor Chang-Soo Yook, Department of Pharmacy, Kyung Hee University. The voucher specimens have been deposited in the Pharmacognosy Laboratory, Department of Pharmacy, Kyung Hee University (KHUP-0714 / A. henryi root barks / 2008). Chemicals Dulbecco’s modified Eagle’s minimum essential medium
(DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Life Technologies Inc. (Grand Island, NY, USA). COX-2, iNOS and -actin were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). RAW 264.7 macrophage cells were supplied by Korea Cell Line Bank (Seoul, Korea). Pyrrolidine dithiocarbamate (PDTC) and L-N6-(1-iminoethyl) lysine (NIL) were purchased from Sigma Chemical Co. (St. Louis, MO, USA) Extraction The samples for activity study were prepared with hot MeOH (65 qC) extraction three times. Water, 30% MeOH, 50% MeOH, 80% MeOH, and 100% MeOH fractions were obtained by XAD-4 column chromatography, by elution with ?> ¥ # ^ _ # ` phase. Finally, the 100% MeOH fraction was mixed with the acetone fraction because of the similar TLC patterns containing some strong spots in RP-TLC analysis (Fig. 1).
Fig. 1 The TLC patterns of fractions of the MeOH extract of the root bark of A. henryi on XAD-4 column chromatography
Cytotoxicity of MeOH extract and fractions of the root bark of A. henryi Cell culture and pre-incubation of drugs The RAW 264.7 macrophage cell lines were grown at 37 qC in DMEM supplemented with 10% FBS, penicillin (100 units·mL1 #_ ^ = # ^ }? <¦ 1) in a humidified 5% CO 2 atmosphere. Cells were incubated with the 80% MeOH fraction at the indicated concentrations or with the positive controls, and then stimulated with LPS }?<¦ L1) for the indicated time. MTT assay for cell viability The RAW 264.7 viability after 24 h of continuous exposure to the tested compounds was measured with a colorimetric assay based on the ability of the mitochondria in viable cells to reduce MTT. Percentage of cell viability was calculated as the absorbance of treated cells/control cells × 100. Cell viability was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. RAW 264.7 cells (1.0 × 104 cells·mL1) were cultured in 96-well plates for 24 h, followed by treat # §}?<·mL1) in the presence of various concentrations (6.25, 12.5, 25, 50 and ? <·mL1) of the A. henryi root bark extract. After 24 h incubation, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bro _ }¥¢¢ ^ ^ # } <¨ ·mL1 in PBS) was added to the medium, and incubation as continued for 4 h. The supernatant was removed, and the obtained
formazan crystals were dissolved in dimethylsulfoxide (DMSO) (200 <'`^` # ^ ^_ # '¢ percent of cells showing cytotoxicity was determined relative to the control group. Determination of chemical mediator release in the LPS-stimulated RAW 264.7 cells Nitrite RAW 264.7 cells were plated at 4 × 105 cells/well in 24-well plates, and then incubated with or without LPS }?<¦ 1) in the absence or presence of various concentrations (6.25, 12.5, 25 and 50 <·mL1) of the 80% MeOH fraction for 24 h. Nitrite levels in the culture media were determined using the Griess reaction and presumed to reflect ^' _ }?< ^ "_ with Griess reagent [equal volumes of 1% (W/V) sulfanilamide in 5% (V/V) phosphoric acid and 0.1% (W/V) naphtylethylenediamine- _ _}?
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culture media were quantified using EIA kits according to the manufacturer’s instructions. Western blot analysis Control and 80% MeOH fraction-treated RAW 264.7 cells were collected by centrifugation and washed once with phosphate-buffered saline (PBS). Washed cell pellets were resuspended in extraction lysis buffer [50 mmol·L1 HEPES pH 7.0, 250 mmol·L1 NaCl, 5 mmol·L1 EDTA, 0.1% Nonidet P-40, 1 mmol·L1 PMSF, 0.5 mmol·L1 DTT, 5 mmol·L1 NaF and 0.5 mmol·L1 sodium orthovanadate] containing 5 <¦ 1 of leupeptin and aprotinin, respectively, and incubated for 20 min at 4 qC. Cell debris was removed by microcentrifugation, and supernatants were rapidly frozen. Protein concentrations were determined using the Bio-Rad protein assay reagent, according to the manufacturer’s instructions. Forty micrograms of cellular proteins from treated or untreated cell extracts were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and electroblotted onto nitrocellulose membranes, which were incubated overnight with blocking solution (5% skim milk) at 4 qC, and then with primary antibody for 4 h. Blots were then washed four times with Tween 20/tris-buffered saline (TTBS), incubated with a 1 : 1 000 dilution of horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature, rewashed three times with TTBS,
and then developed by enhanced chemiluminescence. Statistical analysis The results were expressed as the x ± s of triplicate experiments. Statistically significant values were compared using ANOVA and Dunnett’s post-hoc test, and P values of less than 0.05 were considered statistically significant.
Results Cytotoxicity of MeOH fractions of MeOH extract of A. henryi root barks The cytotoxic effects of the MeOH extract fractions at the concentrations were evaluated in RAW 264.7 cells in the presence or absence of ?<·mL-1 of LPS using MTT assays. The half maximal inhibitory concentration (IC 50 <·mL1) of MeOH extract showed (22.87 ± 1.04) <·mL1, but the 100% MeOH fraction showed (7.69 ± 0.24) <·mL1, indicating the greater cytotoxicity of the 100% MeOH fraction. However, the IC 50 of 80% MeOH fraction was (147.45 ± 2.42) <·mL1, indicating that the inhibition of NO synthesis by the fraction was not simply due to cytotoxic effects. In addition, NO showed (16.23 ± 1.65) <·mL1, PGE 2 showed (8.76 ± 1.02) <·mL1, IL-6 showed (97.63 ± 3.26) <·mL1, and IL-1 showed (34.28 ± 1.75) <·mL1 for the 80% MeOH fraction as a pro-inflammation mediator (Table 1).
Table 1 The effects of the MeOH extract and its MeOH fractions from the root back of A. henryi on the cell viability of RAW 264.7 macrophage and the release of inflammatory mediators stimulated by LPS (n = 3) Drugs
Cytotoxicity (IC 50 , <¦ 1) RAW 264.7 mouse murine macrophage cells
MeOH Ex. 50 % MeOH Fr. 80 % MeOH Fr. 100%MeOH Fr.
22.87 ± 1.04 > 400 147.45 ± 2.42 7.69 ± 0.24
Inhibition of release of pro-inflammation mediators (IC 50 , <¦ 1) NO PGE 2 IL-6 IL-?ª 9.58 ± 2.86 > 400 16.23 ± 1.65 < 6.25
Inhibitory effects of the 80% MeOH fraction of MeOH extract from the root back of A. henryi on LPS-induced release of chemical mediators in RAW 264.7 cells To assess the effects of the 80% MeOH fraction on the LPS-induced NO, PGE 2 , IL-1, and IL-6 production in RAW 264.7 cells, cell culture medias were harvested and nitrite, PGE 2 , IL-1, and IL-6 levels were measured (Fig. 2). The fraction reduced NO and PGE 2 production in a dose-dependent manner, and L-N6-(1-iminoethyl) lysine (L-«}?< ·L1) was used as a positive NO production inhibitor, and PDTC (10 < ·L1) was used as a COX-2 selective inhibitor. The 80% MeOH fraction had no effect on cell viability at the concentra #^ ^_ }' ?' #_ <·mL1) to inhibit NO, PGE 2 , IL-6 and IL-1 production. Thus, the observed inhibitory effects were not attributable to cytotoxicity. Inhibitory effects of the 80% MeOH fraction of MeOH extract from the root bark of A. henryi on iNOS gene expression Western blotting was used to determine whether the inhibitory effects of the 80% MeOH fraction on the production of NO and PGE 2 were related to changes in the
8.76 ± 1.02
97.63 ± 3.26
34.28 ± 1.75
expressions of iNOS and COX-2. The protein levels of iNOS were significantly (P < 0.05) up-regulated in re^=#^ ?<·mL1 LPS, and the 80% MeOH fraction at 12.5, 25 #_ <·mL-1 markedly and dose-dependently inhibited protein expression of iNOS. However, the 80% ¥ # <·mL-1 had no significant inhibitory effect on the protein expression of COX-2, and the housekeeping gene, -actin (Fig. 3).
Discussion and Conclusions In this study, the anti-inflammatory effects of the 80% MeOH fraction on LPS-induced RAW 264.7 macrophage cells were found by the observation of the significant inhibition through the decreased production of NO, PGE 2 , IL-?ª and IL-'¢ ^ ^ "~># }>n }¬ # ~> # }> # }¬ # PGE 2 , 83% # }># }¬ # «-1, and 37% [con }?># }¬#«L-6, respectively. To further explore the possible mechanism of these inhibitions by the 80% MeOH fraction, the expression levels of iNOS and COX-2 proteins were examined. Western blot analysis
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Fig. 2 The inhibitory effects of the 80% MeOH fraction of the MeOH extract of the root bark of A. henryi on NO, PGE 2, IL- and IL-6 production in LPS-stimulated RAW 264.7 cells. (A) The cells were pre-incubated with drugs for 1 h, and then stimulated ·mL1) for 24 h. NIL is L-N6-(1-iminoethyl) lysine, a positive NO production inhibitor (n = 3). (B) RAW 264.7 cells were pre- ·mL1) for 24 h. PDTC is pyrrolidine dithiocarbamate, a COX-2 inhibitor (n = 3). (C) The cells were pre-incubated with drugs for 1 h, and then stimu ·mL1) for 24 h. (D) The cells were pre-incubated with drugs for 1 h and then stimu ·mL1) for 24 h. #P < 0.05 vs control (-), **P < 0.01, ***P < 0.001 vs control (+)
good resource on treatments of inflammation like A. senticosuss. This phytomedicine may also be an important source of lead compounds for the development of novel therapeutics.
References [1] [2]
[3] [4]
Fig. 3 The effects of the 80% MeOH fraction of the MeOH extract of the root bark of A. henryi on iNOS and COX-2 gene expressions on LPS-induced expression of iNOS and COX-2 protein in RAW 264.7 macrophage cells. x ± s, n = 3. #P < 0.05 vs con ! * P < 0.05, ** P < 0.01, *** P < 0.001 vs LPS control (+)
[5]
[6]
[7]
showed significant inhibition on protein expression of iNOS in a dose-dependent manner, but did not show the inhibition of protein expression of COX-2. Further studies of the mechanism of action of the 80% MeOH fraction are in progress. The results of the present study indicate that the 80% MeOH fraction showed the significant inhibitory activity on the pro-inflammatory mediators such as NO, PGE 2 , etc. in vitro. Therefore, the A. henryi root barks is worth to use as the
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[8]
[9]
Yook CS. Medicinal herbs of Acanthopanax in Asia [M]. Korean: Kyungwon Media, 2001. Park SY. Studies on RAPD analysis and triterpenoidal constituents of Acanthopanax species [D]. Japan: Kumamoto University, 2002 KFDA. The Korean Herbal Pharmacopeia[S]. 2007. Pharmacopoeia Committee of China. Pharmacopoeia of the People’s Republic of China[S]. (Vol. I), Beijing: China Medical Science and Technology Press, 2010. Hunan Food and Drug Administration. Hunan Local Medicinal Materials Standard [S]. Changsha: Hunan Science and Technology Press, 2009. Liu XQ. Studies on the active constituents of Acanthopanax gracilistylus W. W. Smith [D]. Korea: Kyung Hee University, 2003. Lee S, Ban HS, Kim YP, et al. Lignans from Acanthopanax chiisanensis having an inhibitory activity on prostaglandin E2 production [J]. Phytother Res, 2005, 19 (2): 103-106. Won JH, Park SY, Nam SG, et al. Inhibition of lipopolysaccharide-induced expression of inducible nitric oxide and cyclooxygenase-2 by chiisanoside via suppression of nuclear factor- # # ' = s [J]. Biol Pharm Bull, 2005, 28 (10): 1919-1924. Kim JH, Park YM, Shin JS, et al. Fraxinellone inhibits lipopolysaccharide-induced inducible nitric oxide synthase and
KIM Jong-Hwan, et al. / Chin J Nat Med, 2014, 12(2): 121125 cyclooxygenase-2 expression by negatively regulating nuclear factor- # ' ophage cells [J]. Biol Pharm Bull, 2009, 32 (6): 1062-1068. [10] Ban HS, Lee S, Kim YP, et al. Inhibition of PGE 2 production by taiwanin C isolated from the root of Acanthopanax chiisanensis and the mechanism of action [J]. Biochem Pharmacol, 2002, 64 (9): 1345-1354. [11] Nhiem NX, Tung NH, Kiem PV, et al. Lupane triterpene glycosides from leaves of Acanthopanax koreanum and their
cytotoxic activity [J]. Chem Pharm Bull, 2009, 57 (9): 986-989. [12] Junghee H. Effects of the cell cultured Acanthopanax senticosus extract on antioxidative defense system and membrane fluidity in the liver of Type 2 diabetes mouse [J]. J Clin Biochem Nutr, 2009, 45: 101-109. [13] Yuan F. Anti-inflammatory mechanism of total glycosides of Acanthopanax giraldii [J]. Chin J Integr Med, 2009, 15 (3): 210-215.
Cite this article as: KIM Jong-Hwan, LIU Xiang-Qian, DAI Ling, YOOK Chang-Soo, LEE Kyung-Tae. Cytotoxicity and anti-inflammatory effects of root bark extracts of Acanthopanax henryi [J]. Chinese Journal of Natural Medicines, 2014, 12(2): 121-125
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Chinese Journal of Natural Medicines
Cytotoxicity and anti-inflammatory effects of root bark extracts of Acanthopanax henryi KIM Jong-Hwan1, LIU Xiang-Qian2*, DAI Ling2, YOOK Chang-Soo3, LEE Kyung-Tae3 1
Herbal Medicine Research Division, Korea Food & Drug Administration, Seoul 363-700, Korea; Hunan Key Laboratory of Traditional Chinese Medicine modernization, School of Pharmacy, Hunan University of Chinese Medicine, Hunan Changsha 410208, China; 3 College of Pharmacy, Kyung-Hee University, Seoul 130-701, Korea 2
Available online 20 Feb. 2014
[ABSTRACT] AIM: To investigate the cytotoxicity, anti-inflammatory activity, and action mechanism of root bark extracts of Acanthopanax henryi. METHOD: The hot methanol extract of the root bark of A. henryi was subjected to XAD-4 column chromatography eluting with a gradient of methanol in water. The cytotoxicity and anti-inflammatory effects of the MeOH fractions were evaluated on the inhibition on lipopolysaccharide (LPS)-induced nitric oxide, prostaglandin E 2 , interleukin-1, and interleukin-6 production in RAW 264.7 macrophages. RESULTS: The 80% MeOH fraction was a better inhibitor of LPS-induced NO, PGE 2 , IL-1, and IL-6 production, and expression of inducible nitric oxide synthase (iNOS) at the protein levels in a concentration-dependent manner. CONCLUSION: The 80% MeOH fraction of A. henryi root bark has significant anti-inflammatory activity. This provides a pharmacological basis for clinical application for the treatment of inflammation. [KEY WORDS] Acanthopanax henryi (Oliv.) Harms; Root bark extract; Cytotoxic activity; Anti-inflammatory activity
[CLC Number] R285
[Document code] A
[Article ID] 2095-6975(2014)02-0121-05
Introduction Acanthopanax henryi (Oliv.) Harms (Araliaceae) has been used as a traditional medicine for the treatment of rheumatism, inflammation, fatigue and so on [1]. As an endemic Asian genus, over thirty-eight species are distributed mainly in northeastern Asia, including China, Korea, Japan, and far-east Russia. In the official compendium in China, Korea, and Japan, the root bark of this plant has been listed [2]. [Received on] 10-Oct.-2012 [Research funding] This project was supported by the PhD Program Open Foundation of the Food and Drug Administration of Korea; Hunan Provincial Natural Science Foundation (No. 11JJ2042), and the Science of Pharmaceutical Analysis of Twelfth Five-Year Key Discipline Projects of Hunan University of Chinese Medicine˗ Science of Chinese Materia Medica of Twelfth Five-Year Key Discipline Projects of Hunan Province [ Corresponding author] LIU Xiang-Qian: Prof., Tel: 86-73188458240, E-mail: [email protected] These authors have no conflict of interest to declare. Copyright © 2014, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved
The Korean Herbal Pharmacopoeia [3] will be revised to include Eleutherococci senticosi Radix et Rhizome originating from Eleutherococcus senticosus (Rupr. et Maxim.) Maxim. The Chinese Pharmacopoeia [4], edited in 2010, has two monographs, Radix et Rhizoma seu Caulis Acanthopanacis originating from the root, rhizome, and/or stem of A. senticosus (Rupr. et Maxim.) Harms and Cortex Acanthopanacis originating from the root bark of A. gracilistylus W.W. Sm., but A. henryi Harms is not included, and only appears as a Hunan local medicinal materials standard [5]. To date, the most studied species among the plants of Acanthopanax spp. for biological activities is A. senticosus (Rupr. et Maxim.) Harms. It was studied mainly for anti-microbial, anti-cancer, anti-inflammatory, anti-platelet, and anti-oxidant activities, cardiac protection, sperm mobility increase, and increases of bone turnover and bone mineral density [6-8]. The lignan taiwanin C, isolated from the root of A. chiisanensis Nakai, is reported to have anti-inflammatory activity through significant inhibition of COX-1 and COX-2, which mediate the PGE 2 production in a dose-dependent manner comparable to the NSAID, indomethacin [9-13]. However, the anti-inflammatory activity and the mechanism of action of the biologically active components of A.
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KIM Jong-Hwan, et al. / Chin J Nat Med, 2014, 12(2): 121125
henryi root bark were not studied until now. On this basis, the effects of the MeOH extract of this plant and its fractions were evaluated for the release of anti-inflammatory mediators, such as NO, PGE 2 , IL-1, and IL-6 in LPS-stimulated RAW 264.7 macrophage cells.
Materials and Methods Plant material The A. henryi root bark material was collected in August 2008 in Mt. Daewoong, Hunan province, China, and dried in the shade. The plant was identified by Emeritus Professor Chang-Soo Yook, Department of Pharmacy, Kyung Hee University. The voucher specimens have been deposited in the Pharmacognosy Laboratory, Department of Pharmacy, Kyung Hee University (KHUP-0714 / A. henryi root barks / 2008). Chemicals Dulbecco’s modified Eagle’s minimum essential medium
(DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Life Technologies Inc. (Grand Island, NY, USA). COX-2, iNOS and -actin were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). RAW 264.7 macrophage cells were supplied by Korea Cell Line Bank (Seoul, Korea). Pyrrolidine dithiocarbamate (PDTC) and L-N6-(1-iminoethyl) lysine (NIL) were purchased from Sigma Chemical Co. (St. Louis, MO, USA) Extraction The samples for activity study were prepared with hot MeOH (65 qC) extraction three times. Water, 30% MeOH, 50% MeOH, 80% MeOH, and 100% MeOH fractions were obtained by XAD-4 column chromatography, by elution with ?> ¥ # ^ _ # ` phase. Finally, the 100% MeOH fraction was mixed with the acetone fraction because of the similar TLC patterns containing some strong spots in RP-TLC analysis (Fig. 1).
Fig. 1 The TLC patterns of fractions of the MeOH extract of the root bark of A. henryi on XAD-4 column chromatography
Cytotoxicity of MeOH extract and fractions of the root bark of A. henryi Cell culture and pre-incubation of drugs The RAW 264.7 macrophage cell lines were grown at 37 qC in DMEM supplemented with 10% FBS, penicillin (100 units·mL1 #_ ^ = # ^ }? <¦ 1) in a humidified 5% CO 2 atmosphere. Cells were incubated with the 80% MeOH fraction at the indicated concentrations or with the positive controls, and then stimulated with LPS }?<¦ L1) for the indicated time. MTT assay for cell viability The RAW 264.7 viability after 24 h of continuous exposure to the tested compounds was measured with a colorimetric assay based on the ability of the mitochondria in viable cells to reduce MTT. Percentage of cell viability was calculated as the absorbance of treated cells/control cells × 100. Cell viability was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. RAW 264.7 cells (1.0 × 104 cells·mL1) were cultured in 96-well plates for 24 h, followed by treat # §}?<·mL1) in the presence of various concentrations (6.25, 12.5, 25, 50 and ? <·mL1) of the A. henryi root bark extract. After 24 h incubation, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bro _ }¥¢¢ ^ ^ # } <¨ ·mL1 in PBS) was added to the medium, and incubation as continued for 4 h. The supernatant was removed, and the obtained
formazan crystals were dissolved in dimethylsulfoxide (DMSO) (200 <'`^` # ^ ^_ # '¢ percent of cells showing cytotoxicity was determined relative to the control group. Determination of chemical mediator release in the LPS-stimulated RAW 264.7 cells Nitrite RAW 264.7 cells were plated at 4 × 105 cells/well in 24-well plates, and then incubated with or without LPS }?<¦ 1) in the absence or presence of various concentrations (6.25, 12.5, 25 and 50 <·mL1) of the 80% MeOH fraction for 24 h. Nitrite levels in the culture media were determined using the Griess reaction and presumed to reflect ^' _ }?< ^ "_ with Griess reagent [equal volumes of 1% (W/V) sulfanilamide in 5% (V/V) phosphoric acid and 0.1% (W/V) naphtylethylenediamine- _ _}?
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culture media were quantified using EIA kits according to the manufacturer’s instructions. Western blot analysis Control and 80% MeOH fraction-treated RAW 264.7 cells were collected by centrifugation and washed once with phosphate-buffered saline (PBS). Washed cell pellets were resuspended in extraction lysis buffer [50 mmol·L1 HEPES pH 7.0, 250 mmol·L1 NaCl, 5 mmol·L1 EDTA, 0.1% Nonidet P-40, 1 mmol·L1 PMSF, 0.5 mmol·L1 DTT, 5 mmol·L1 NaF and 0.5 mmol·L1 sodium orthovanadate] containing 5 <¦ 1 of leupeptin and aprotinin, respectively, and incubated for 20 min at 4 qC. Cell debris was removed by microcentrifugation, and supernatants were rapidly frozen. Protein concentrations were determined using the Bio-Rad protein assay reagent, according to the manufacturer’s instructions. Forty micrograms of cellular proteins from treated or untreated cell extracts were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and electroblotted onto nitrocellulose membranes, which were incubated overnight with blocking solution (5% skim milk) at 4 qC, and then with primary antibody for 4 h. Blots were then washed four times with Tween 20/tris-buffered saline (TTBS), incubated with a 1 : 1 000 dilution of horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature, rewashed three times with TTBS,
and then developed by enhanced chemiluminescence. Statistical analysis The results were expressed as the x ± s of triplicate experiments. Statistically significant values were compared using ANOVA and Dunnett’s post-hoc test, and P values of less than 0.05 were considered statistically significant.
Results Cytotoxicity of MeOH fractions of MeOH extract of A. henryi root barks The cytotoxic effects of the MeOH extract fractions at the concentrations were evaluated in RAW 264.7 cells in the presence or absence of ?<·mL-1 of LPS using MTT assays. The half maximal inhibitory concentration (IC 50 <·mL1) of MeOH extract showed (22.87 ± 1.04) <·mL1, but the 100% MeOH fraction showed (7.69 ± 0.24) <·mL1, indicating the greater cytotoxicity of the 100% MeOH fraction. However, the IC 50 of 80% MeOH fraction was (147.45 ± 2.42) <·mL1, indicating that the inhibition of NO synthesis by the fraction was not simply due to cytotoxic effects. In addition, NO showed (16.23 ± 1.65) <·mL1, PGE 2 showed (8.76 ± 1.02) <·mL1, IL-6 showed (97.63 ± 3.26) <·mL1, and IL-1 showed (34.28 ± 1.75) <·mL1 for the 80% MeOH fraction as a pro-inflammation mediator (Table 1).
Table 1 The effects of the MeOH extract and its MeOH fractions from the root back of A. henryi on the cell viability of RAW 264.7 macrophage and the release of inflammatory mediators stimulated by LPS (n = 3) Drugs
Cytotoxicity (IC 50 , <¦ 1) RAW 264.7 mouse murine macrophage cells
MeOH Ex. 50 % MeOH Fr. 80 % MeOH Fr. 100%MeOH Fr.
22.87 ± 1.04 > 400 147.45 ± 2.42 7.69 ± 0.24
Inhibition of release of pro-inflammation mediators (IC 50 , <¦ 1) NO PGE 2 IL-6 IL-?ª 9.58 ± 2.86 > 400 16.23 ± 1.65 < 6.25
Inhibitory effects of the 80% MeOH fraction of MeOH extract from the root back of A. henryi on LPS-induced release of chemical mediators in RAW 264.7 cells To assess the effects of the 80% MeOH fraction on the LPS-induced NO, PGE 2 , IL-1, and IL-6 production in RAW 264.7 cells, cell culture medias were harvested and nitrite, PGE 2 , IL-1, and IL-6 levels were measured (Fig. 2). The fraction reduced NO and PGE 2 production in a dose-dependent manner, and L-N6-(1-iminoethyl) lysine (L-«}?< ·L1) was used as a positive NO production inhibitor, and PDTC (10 < ·L1) was used as a COX-2 selective inhibitor. The 80% MeOH fraction had no effect on cell viability at the concentra #^ ^_ }' ?' #_ <·mL1) to inhibit NO, PGE 2 , IL-6 and IL-1 production. Thus, the observed inhibitory effects were not attributable to cytotoxicity. Inhibitory effects of the 80% MeOH fraction of MeOH extract from the root bark of A. henryi on iNOS gene expression Western blotting was used to determine whether the inhibitory effects of the 80% MeOH fraction on the production of NO and PGE 2 were related to changes in the
8.76 ± 1.02
97.63 ± 3.26
34.28 ± 1.75
expressions of iNOS and COX-2. The protein levels of iNOS were significantly (P < 0.05) up-regulated in re^=#^ ?<·mL1 LPS, and the 80% MeOH fraction at 12.5, 25 #_ <·mL-1 markedly and dose-dependently inhibited protein expression of iNOS. However, the 80% ¥ # <·mL-1 had no significant inhibitory effect on the protein expression of COX-2, and the housekeeping gene, -actin (Fig. 3).
Discussion and Conclusions In this study, the anti-inflammatory effects of the 80% MeOH fraction on LPS-induced RAW 264.7 macrophage cells were found by the observation of the significant inhibition through the decreased production of NO, PGE 2 , IL-?ª and IL-'¢ ^ ^ "~># }>n }¬ # ~> # }> # }¬ # PGE 2 , 83% # }># }¬ # «-1, and 37% [con }?># }¬#«L-6, respectively. To further explore the possible mechanism of these inhibitions by the 80% MeOH fraction, the expression levels of iNOS and COX-2 proteins were examined. Western blot analysis
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KIM Jong-Hwan, et al. / Chin J Nat Med, 2014, 12(2): 121125
Fig. 2 The inhibitory effects of the 80% MeOH fraction of the MeOH extract of the root bark of A. henryi on NO, PGE 2, IL- and IL-6 production in LPS-stimulated RAW 264.7 cells. (A) The cells were pre-incubated with drugs for 1 h, and then stimulated ·mL1) for 24 h. NIL is L-N6-(1-iminoethyl) lysine, a positive NO production inhibitor (n = 3). (B) RAW 264.7 cells were pre- ·mL1) for 24 h. PDTC is pyrrolidine dithiocarbamate, a COX-2 inhibitor (n = 3). (C) The cells were pre-incubated with drugs for 1 h, and then stimu ·mL1) for 24 h. (D) The cells were pre-incubated with drugs for 1 h and then stimu ·mL1) for 24 h. #P < 0.05 vs control (-), **P < 0.01, ***P < 0.001 vs control (+)
good resource on treatments of inflammation like A. senticosuss. This phytomedicine may also be an important source of lead compounds for the development of novel therapeutics.
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Fig. 3 The effects of the 80% MeOH fraction of the MeOH extract of the root bark of A. henryi on iNOS and COX-2 gene expressions on LPS-induced expression of iNOS and COX-2 protein in RAW 264.7 macrophage cells. x ± s, n = 3. #P < 0.05 vs con ! * P < 0.05, ** P < 0.01, *** P < 0.001 vs LPS control (+)
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showed significant inhibition on protein expression of iNOS in a dose-dependent manner, but did not show the inhibition of protein expression of COX-2. Further studies of the mechanism of action of the 80% MeOH fraction are in progress. The results of the present study indicate that the 80% MeOH fraction showed the significant inhibitory activity on the pro-inflammatory mediators such as NO, PGE 2 , etc. in vitro. Therefore, the A. henryi root barks is worth to use as the
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Cite this article as: KIM Jong-Hwan, LIU Xiang-Qian, DAI Ling, YOOK Chang-Soo, LEE Kyung-Tae. Cytotoxicity and anti-inflammatory effects of root bark extracts of Acanthopanax henryi [J]. Chinese Journal of Natural Medicines, 2014, 12(2): 121-125
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