Effects of Angelicae Gigantis Radix pharmacopuncture on Nitric Oxide and Prostaglandin E
2Production in Macrophage.
You Jin Choi, Jeong Du Roh*
Department of Acupuncture & Moxibustion, Semyung University College of Oriental Medicine, Jecheon, Korea
KEY WORDS:
Angelicae Gigantis Radix;
pharmacopuncture;
Anti-inflammatory;
NO; PGE2; Korean Medicine Received : Jul 21, 2011 Revised : Sep 2, 2011 Accepted : Sep 8, 2011
ABSTRACT
Objectives :Angelicae Gigantis Radix has been known traditional medicine with antimicrobial activi- ties and it has been widely used for treatment of blood and inflammatory diseases. In the present study, some studies examined anti-inflammation effects of Angelicae Gigantis Radix but they usually were performed by ethanol extracted Angelicae Gigantis Radix pharmacopuncture. So We investigat- ed the inhibitory effects of Angelicae Gigantis Radix pharmacopuncture by hot water and ethanol extract on Nitric oxide(NO) and Prostaglandin E2(PGE2) production in lipopolysaccharide(LPS) induced macrophage cell.
Methods : Angelicae Gigantis Radix was extracted by ethanol and hot water. Cell viability was deter- mined by MTT assay. To evaluate anti-inflammation effects of Angelicae Gigantis Radix pharmacop- uncture, we examined NO and PGE2production in LPS induced macrophages. The concentrations of NO and PGE2were measured by Griess assay and Enzyme Immuno-Assay.
Results :
1) The MTT assay demonstrated that cytotoxic effect of Angelicae Gigantis Radix pharmacopuncture by hot water extract and ethanol extract in RAW 264.7 macrophage cells were not appeared.
2) Angelicae Gigantis Radix pharmacopuncture by ethanol extract and hot water extract inhibited NO production in LPS induced macrophages significantly.
3) Angelicae Gigantis Radix pharmacopuncture by ethanol extract tended to inhibiting PGE2produc- tion in LPS induced macrophages. And Angelicae Gigantis Radix pharmacopuncture by hot water extract inhibited LPS induced production of PGE2in RAW 264.7 macrophage cells significantly.
Conclusions : This study suggests that Angelicae Gigantis Radix pharmacopuncture may have an anti-inflammatory property through the inhibition of NO and PGE2production in LPS induced macrophages. It may have a therapeutic potential for the treatment of various inflammatory diseases.
Original Article
당귀약침액이 대식세포에서 산화질소(`NO) 및 프로스타글란딘 (`Prostaglandin) 생성에 미치는 영향
2011 Korean Pharmacopuncture Instltute http://pharmacopuncture.co.kr
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://cre- ativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non- commercial use, distribution, and reproduction in any medium, provid- ed the original work is properly cited.
Corresponding Author
Jeong Du Roh. Department of Acupuncture & Moxibustion, Semyung University Oriental Medicine Hospital, 579 Sinwoul-dong, Jecheon 390-711, Korea
Tel : +82-649-1816
E-mail : [email protected] cc
I . 서론
Angelicae Gigantis Radix
Angelica gigas Nakai
2
Nitric oxide NO Prostaglandin E2 PGE2
II . 재료 및 방법 1.
Angelicae Gigantis Radix
2
300g 70% 2L 70 4
rotary evap-
orator 100 80
freezing dryer system
(Labconco, USA) 7
300g 2L 100
4
2.
Murine macrophage cell line RAW 264.7 American Type Culture Collection(ATCC,
USA) 10% Fetal bovine serum
(FBS) 1% Dulbecco's Modified
Eagle's Medium (DMEM) 37 , 5%
CO2
3.
RAW 264.7 96 well plate 1 x 105cell
/well 16 37 , 5% CO2
1, 5, 25, 125, 625 3125
/mL 16
MTT 2
formazan crystals DMSO Enzyme- Linked Immuno-Sorbant Assay ELISA read- er(Bio-Tek, USA) 570nm
control cell ratio [i.e. viability(% control) = 100 (absorb ance of treated sample)/(absorbance of control)]
4.
lipopolysacharide LPS 1, 5, 25, 125, 625 3125 /mL
16 100
Griess (0.1% naphthylethylenediamine dihy- drochloride 50 1% sulfanilamide in 5% phos- phoric acid 50 100 ELISA reader (Bio-Tek, USA)
5.
Prostaglandin E2(PGE2)
(Enzyme Immuno-Assay; EIA) RAW 264.7
10 / LPS 18
PGE2 goat anti-mouse coating
96 well plate 100 loading
primary antibody solution 50 PGE2
conjugate 50 4 overnight
200 5-20 50
stop solution 450nm
6.
SPSS Window program(Ver. 10.0)
Student's t-
test p 0.05
III . 결과 1.
MTT assay
, 5, 25, 125,
625 3125 /mL
97.80 1.07 99.21 2.77 96.61 2.84% 95.74 2.28, 98.88 1.23 97.00 2.41%
(Table 1, Fig. 1)
1, 5, 25, 125,
625 3125 /mL
100.61 2.26, 101.10 1.20, 101.67 3.01, 96.52 1.58, 97.77 3.99 90.66 2.36%
(Table 2, Fig. 2)
Group ( /mL) Cell Viability (%) Control
AGR 1
100 1.43 97.80 1.07
AGR 625 AGR 3125
98.88 1.23 97.00 2.41 AGR 5
AGR 25
99.21 2.77 96.61 2.84
AGR 125 95.74 2.28
Table 1. The effect of Angelicae Gigantis Radix pharma- copuncture by ethanol extract on cell viability of RAW 264.7 macrophage cells by MTT assay.
Fig 1. This graph describes the effect of Angelicae Gigantis Radix pharmacopuncture by ethanol extract on cell viability of RAW 264.7 macrophage cells by MTT assay.
Control : 0 /mL Angelicae Gigantis Radix pharma- copuncture treated group
AGR : Angelicae Gigantis Radix pharmacopuncture by ethanol extract
(1, 5, 25, 125, 625 & 3125 /mL)
Values are represented as mean Standard Deviation (S.D.)
2. NO
LPS RAW 264.7
NO Griess assay
NO LPS 10
/mL 78.8 1.6 M/mL
1, 5, 25, 125, 625 3125
/mL 75.5 4.2, 71.8
2.0, 67.9 1.7, 69.0 2.3, 70.7 3.3 67.2
2.0 M/mL 25, 125, 625 3125
/mL NO
(Table 3, Fig. 3)
NO LPS 10
/mL 52.8 2.2 M/mL
1, 5, 25, 125, 625 3125
/mL 50.8 1.5, 49.9
0.8, 47.8 2.1, 46.2 1.5, 46.0 1.6 20.8
1.4 M/mL 125, 625 3125
/mL NO
(Table 4, Fig. 4)
Group ( /mL) Cell Viability (%) Control
AGR 1
100 6.68 100.61 2.26
AGR 625 AGR 3125
97.77 3.99 90.66 2.36 AGR 5
AGR 25
101.10 1.20 101.67 3.01
AGR 125 96.52 1.58
Table 2. The effect of Angelicae Gigantis Radix pharmacop- uncture by hot water extract on cell viability of RAW 264.7 macrophage cells by MTT assay.
Fig 2. This graph describes the effect of Angelicae Gigantis Radix pharmacopuncture by hot water extract on cell viability of RAW 264.7 macrophage cells by MTT assay.
Control : 0 /mL Angelicae Gigantis Radix treated group
AGR : Angelicae Gigantis Radix pharmacopuncture by hot water extract
(1, 5, 25, 125, 625 & 3125 /mL) Values are represented as mean S.D.
Group ( /mL) NO synthesis ( M)
Control AGR 1
78.8 1.6 75.5 4.2
AGR 625 AGR 3125
70.7 3.3*
67.2 2.0*
AGR 5 AGR 25
71.8 2.0 67.9 1.7*
AGR 125 69.0 2.3*
Table 3. The effect of Angelicae Gigantis Radix pharmacop- uncture by ethanol extract on the Nitric Oxide(NO) production of LPS induced RAW 264.7 macroph- age cells by NO synthesis assay.
Fig 3. This graph describes the effect of Angelicae Gigantis Radix pharmacopuncture by ethanol extract on the Nitric Oxide(NO) production of LPS induced RAW 264.7 macrophage cells by NO synthesis assay.
NO : Nitric oxide
Control : LPS 10 /mL treated group
LPS : Lipopolysaccharide (+ : treated with LPS) AGR : LPS 10 /mL +Angelicae Gigantis Radix phar- macopuncture by ethanol extract
(1, 5, 25, 125, 625 & 3125 /mL)
All of the groups are treated with LPS 10 /mL Values are represented as mean S.D.
* Statistically significant difference from the Control group, as determined by Student's t-test as p 0.05
3. PGE2
LPS RAW 264.7
PGE2
PGE2
4.15 0.02pg/mL LPS
821.24 1.63pg/mL
50, 100, 150 200 /mL
824.81 9.82, 817.94 18.25, 802.45 19.86 799.64 14.25pg/mL PGE2
(Table 5, Fig. 5)
50, 100, 150 200 /mL
839.35 13.90, 837.14 9.91, 739.77 2.62 726.47 9.45pg/mL
150 200 /mL PGE2
Group ( /mL) NO synthesis ( M)
Control AGR 1
52.8 2.2 50.8 1.5
AGR 625 AGR 3125
46.0 1.6*
20.8 1.4*
AGR 5 AGR 25
49.9 0.8 47.8 2.1
AGR 125 46.2 1.5*
Table 4. The effect of Angelicae Gigantis Radix pharma- copuncture by hot water extract on the Nitric Oxide(NO) production of LPS induced RAW 264.7 macrophage cells by NO synthesis assay.
Fig 4. This graph describes the effect of Angelicae Gigantis Radix pharmacopuncture by hot water extract on the NO production of LPS induced RAW 264.7 macrophage cells by Nitric Oxide(NO) synthesis assay.
NO : Nitric oxide
Control : LPS 10 /mL treated group
LPS : Lipopolysaccharide (+ : treated with LPS) AGR : LPS 10 /mL +Angelicae Gigantis Radix phar- macopuncture by hot water extract
(1, 5, 25, 125, 625 & 3125 /mL) Values are represented as mean S.D.
All of the groups are treated with LPS 10 /mL
* Statistically significant difference from the Control group, as determined by Student's t-test as p 0.05
Group ( /mL) PGE2synthesis (pg) Normal
Control
4.15 0.02 821.24 1.63
AGR 200 799.64 14.25
AGR 50 AGR 100
824.81 9.82 817.94 18.25
AGR 150 802.45 19.86
Table 5. The effect of Angelicae Gigantis Radix pharma- copuncture by ethanol extract on the Prostagla- ndin E2(PGE2) production of RAW 264.7 macrop- hage cells by PGE2synthesis assay.
Fig 5. This graph describes the effect of Angelicae Gigantis Radix pharmacopuncture by ethanol extract on the Prostaglandin E2(PGE2) production of RAW 264.7 macrophage cells by PGE2synthesis assay.
PGE2: Prostaglandin E2
Normal : LPS 0 /mL treated group Control : LPS 10 /mL treated group
LPS : Lipopolysaccharide (- : not treated with LPS, + : treated with LPS)
AGR : LPS 10 /mL and Angelicae Gigantis Radix pharmacopuncture by ethanol extract
(50, 100, 150 & 200 /mL)
Values are represented as mean S.D.
(Table 6, Fig. 6)
IV . 고찰
argi- nine O2 nitric oxide synthase
radical NO septic shock
PGE2 NO
COX-2
PGE2
stress
13) 14)
NO
2
Fig 6. This graph describes the effect of Angelicae Gigantis Radix pharmacopuncture by hot water extract on the Prostaglandin E2(PGE2) production of RAW 264.7 macrophage cells by PGE2synthesis assay.
PGE2: Prostaglandin E2
Normal : LPS 0 /mL treated group Control : LPS 10 /mL treated group
LPS : Lipopolysaccharide (- : not treated with LPS, + : treated with LPS)
AGR : LPS 10 /mL +Angelicae Gigantis Radix phar- macopuncture by hot water extract
(50, 100, 150 & 200 /mL)
Values are represented as mean S.D.
* Statistically significant difference from the Control group, as determined by Student's t-test as p 0.05
Group ( /mL) PGE2synthesis (pg) Normal
Control
4.15 0.02 821.24 1.63
AGR 200 726.47 9.45*
AGR 50 AGR 100
823.35 13.90 837.14 9.91
AGR 150 739.77 2.62*
Table 6. The effect of Angelicae Gigantis Radix pharma- copuncture by hot water extract on the Prostagl- andin E2(PGE2) production of RAW 264.7 macrop- hage cells by PGE2synthesis assay.
RAW 264.7 MTT assay
1, 5, 25, 125, 625 3125 /mL
LPS RAW 264.7
NO
25, 125, 625
3125 /mL LPS
125, 625 3125
/mL LPS
NO
LPS RAW 264.7
PGE2 LPS PGE2
PGE2
PGE2 150 200 /mL
NO PGE2
NO
PGE2
V . 결론
LPS RAW 264.7
NO PGE2
1. MTT assay
2. NO
25, 125, 625 3125
/mL LPS
125, 625 3125 /mL LPS
3. PGE2
PGE2
150 200 /mL LPS
LPS RAW
264.7 NO PGE2
VI. References
1.
2004:109-12.
2. Abul K.Abbas, Andrew H.Lichtman, Shiv Pill- ai. Cellular and Molecular Immunology.
6th edition, Saunders Elsevier. 2006:75-97.
3. Guo LY, Hung TM, Bae KH, Shin EM, Zhou HY, Hong YN, Kang SS, Kim HP, Kim YS.
Anti-inflammatory effects of schisandrin iso- lated from the fruit of Schisandra chinensis Baill. Eur J Pharmacol. 2008;591:293-9.
4. Kim DH, Park SJ, Jung JY, Kim SC, Byun SH.
Anti-inflammatory Effects of the Aqueous Extract of Hwangnyeonhaedok-tang in LPS- activated Macrophage Cells. The Korea Association of Herbology. 2009;24(4):39-47.
5. Bae GS, Park HJ, Kim DY, Seo SW, Park KB,
Kim BJ, Song JM, Lee KY, Na C, Shin BC, Park SJ, Song HJ, Hwang SY. Inhibitory Effect of Extract of Teucrium veronicoides on the Production of Inflammatory Cytokines. The Korea Association of Herbology.
2008;23(3):119-125.
6. Yun YG, Kim GM, Lee SJ, Ryu SH, Jang SI.
Inhibitory Efeect of Aqueous Extract from Lonicera japonica Flower on LPS-induced Inflammatory Mediators in RAW 264.7 Macr- ophages. The Korea Association of Herbology.
2007;23(3):117-25.
7. Park SJ, Song HJ. Anti-inflammatory effect of extract of Pulsatilla koreana NAKAI in LPS- stimulated Murine peritoneal macrophage. The Korea Association of Herbology.
2007;22(1):111-7.
8.
2005:631-4 9.
1984
10. Hwang HS, Ahn BC, Park DS. The effects of Angelicae gigantis Radix Aqua-acupuncture by Density on immune response induced by radiation in Mice. The Journal of Korean Acupuncture and Moxibustion Society.
1994;11(4):113-29.
11. Moon JY, Lim JK. A Study of the Effects of Moxibustion and A.G.R aqua-acupuncture at Ganshu(BL19) and Qimen(LR14) on Hyperlip- idemia Induced by Cholesterol in Rats. The Journal of Korean Acupuncture and Moxibust- ion Society. 1995;12(2):119-34.
12. Sung IW, Chae WS. The effects of Aqua- acupuncture of Hyunsoshang and Dangghuimi on Thrombosis Induced by Endotoxin in Rats.
The Journal of Korean Acupuncture and Moxibustion Society. 1994;11(1):391-404.
13.
2008
14. LPS RAW 264.7
2003
15. Inhibitory Effect of Angelica gigas Nakai Extract on Nitric Oxide Production in RAW 264.7 Cells. The Journal of Korean Oriental Medicine Society. 2007;28(2):138-48.
16. The Korean Society of Pathologists. Text of Pathology. Seoul;Komoonsa 1995:83-9.
17. Mori M. Regulation of nitric oxide synthesis and apoptosis by arginine recycling. J Nutr.
2007;137:1616-20.
18. Bae SC, Kim DY, Kim TH, Jun JB, Jung SS, Lee IH, Yoo DH, Kim SY, Lee EY, Chang SY.
Nitric oxide(NO) in Inflammatory Arthritis.
The Journal of Korean Orienatal Internal Medicine Society. 1997;52(1):32-41.
19. The Role of Spinal NO on the Mechanical Allodynia in the Inflammatioa Model by Freund`s Complete Adjuvant. The Journal of the Korean Society of Anesthesiologists.
2000;38(6):1068-74.
20. Kuo CL, Chi CW, Liu TY. The anti-inflamma- tory potential of berberine in vitro and in vivo.
Cancer Lett. 2004;203:127-37.
21. Lee DU, Kang YJ, Park MK, Lee YS, Seo HG, Kim TS, Kim CH, Chang KC. Effects of 13- alkyl-substituted berberine alkaroids on the expression of COX- , TNF-alpha, iNOS, and IL-12 production in LPS-stimulated macro- phages. Life Sci. 2003;73:1401-12.
22. Kim KW, Ha KT, Park CS, Jin UH, Chang HW, Lee IS, Kim CH. Folygonum cuspidatum, compared with baicalin and berberine, inhibits inducible nitric oxide synthase and cyclooxygenase-2 gene expressions in RAW 264.7 macrophages. Vascul Pharmacol.
2007; 47:99-107.
23. Hong JW, Woo WH, Jeon BH. Study on the Anti-inflammatory and Anti-thrombotic Effects of Bibangtalmyungsan in the experi- mental animal. The Korean Journal of Oriental Medical Pathology. 1998;12(2):63-72.
24. Kim CH, Youn HM, Jang KJ, Song CH, Ahn CB. Inhibitory Effect on NO, Scavenging Effect on DPPH Radical in Whallak-tang. The Journal of Korean Acupuncture and Moxibus- tion Society. 2004;21(5):69-78.
25. Jung WS, Yoo HM, Seo SW, Cho JK, Son JW, Park MC, Choi CM, Yeom SR, Hwang SW, Kim YW, Song DS, Chae YS, Park SJ, Shin MK, Song HJ. Anti-inflammatory effect of extract of Asarum sieboldii in LPS-stimulated Murine peritoneal macrophage. The Korea Association of Herbology.
2006;21(2):189-195.
26. Park HJ, Bae GS, Kim DY, Seo SW, Park KB, Kim BJ, Somg JM, Lee KY, Na C, Shin BC, Park SJ, Song HJ, Hwang SY. Inhibitory Effect of Extract from Ostericum koreanum on LPS-induced Proinflammatory Cytokines Production in RAW 264.7 Cells. The Korea Association of Herbology. 2008;23(3):127-34.
27. Kim SY, Park YK. Ethanol Extract of Forsythiae Fructus Inhibits the Production of Inflammatory Mediators in LPS-stimulated BV-2 Microglial Cells. The Korea Association of Herbology. 2008;23(3):93-102.
28. Jung HW, Park YK. Inhibitory effects of Coptidis Rhizoma on the LPS-induced produc- tion of nitric oxide and TNF- in mouse macr- ophage cells. The Korea Association of Herbo- logy. 2006;21(2):165-73.
29. Lee HJ, Cho HS, Hwang MS, Jung CY, Lee DG, Kim EJ, Kim KS, Kim KH. Effect of Cervi Pantotrichum Cornu Pharmacopuncture on Suppressing the Expression of iNOS and Production of NO in Type Collagen-induced Arthritis Mice. The Journal of Korean Acupu- ncture and Moxibustion Society.
2008;25(5):105-16.
30. Min JY, Park YK. Effect of Dipasaci Radix Water Extract in LPS-induced Inflammatory Response in RAW 264.7 Mouse Macrophage.
The Korea Association of Herbology.
2009;24(4):189-95.
31. Kim DH, Seo JC, Lim SC, Jung TY, Han SW.
The Scavenging Effect on NO, DPPH and Inhibitory Effect on IL-4 in Radix Cynanchi Wilfordii Herbal-Acupuncture Solution. The Journal of Korean Acupuncture and Moxibust- ion Society. 2003;20(4):42-52.
32. Lee KM, Seo JC, Han SW. Scavenging Effect on NO, DPPH and Inhibitory Effect on IL-4 in Astragali Radix Herbal-Acupuncture Solution.
The Journal of Korean Acupuncture and Moxibustion Society. 2003;20(2):184-94.
33. Park YK. Inhibitory effects of Zanthoxylum piperitum on the LPS-induced production of nitric oxide and proinflammatory cytokines in RAW 264.7 cells. The Korea Association of Herbology. 2006;21(4):69-76.
34. Ryu EJ, Kim CH, Kim YS, Park YB, Koh HK, Kang SK. Effects of Angelica gigas Nakai Aqua-acupuncture on the Ovarian Function.
The Journal of Korean Acupuncture and Moxibustion Society. 1998;15(1):305-15.
35. Ahn JC, Moon JY, Lim JK. Antioxidant effects of A.G.R.(Angelicae gigantis Radix) Aqua- acupuncture solution in vitro. The Journal of Korean Acupuncture and Moxibustion Society.
1996;13(2):254-62.
36. Han SG, Lee BR. Effects of Angelica gigas Nakai herbal acupuncture into Hyolhae(SP10) of brain ischemic injury induced by Intralum- inal Filament insertion in the rats. The Journal of Korean Acupuncture and Moxibustion Society. 2004;21(2):1-20.
37. Chang SY, Kim EH, Lee EY. Effect of Postnatal Angelicae Gigantis Radix Herb- acupuncture on Cell Proliferation in Offspring Rats with Prenatal Noise Stress during Pregna- ncy. The Journal of Korean Acupun-cture and Moxibustion Society.
2006;23(3):47-56.
38. Ryu MS, Yun YC, Kim JH. The Effect of Angelicae gigas NAKAI Pharmacopuncture at
ST36 and BL23 on Freund's Adjuvant Arthritis in Rats. The Journal of Korean Acupuncture and Moxibustion Society. 2010;27(5):25-34.
39. Kim KS, Song YJ. Effects of Aqua-Acupunc- ture with RADIX ANGELICAE GIGANTIS and RADIX ASTRAGALI on the Blood in the Liver Damage Rats with CCl4. The Journal of Korean Acupuncture and Moxibustion Society.
1996;13(1):1-10.
