Your browser does not support the NLM PubReader view.

Go to this page to see a list of supporting browsers.

Comparative study on the contents of marker compounds and anti-inflammatory effects of Gamisoyo-san decoction according to storage temperature and periods

Article information

J Korean Med. 2018;39(1):22-34
Publication date (electronic) : 2018 March 31
doi : https://doi.org/10.13048/jkm.18003
Seong Eun Jin1orcid_icon, Chang-Seob Seo1orcid_icon, Nari Lee1orcid_icon, Hyeun-Kyoo Shin1orcid_icon, Hyekyung Ha1,orcid_icon
1Herbal Medicine Research Division, Korea Institute of Oriental Medicine
Correspondence to: 하혜경 (Hyekyung Ha), 대전 유성구 유성대로 1672 한국한의학연구원 한약연구부, Tel: +82-42-868-9513, Fax: +82-42-864-2120, E-mail: hkha@kiom.re.kr
Received 2017 December 20; Revised 2018 March 15; Accepted 2018 March 16.

Abstract

Objectives

The purpose of this study is to investigate changes of the marker compounds and anti-inflammatory effect of Gamisoyo-san decoction (GMSYS) depending on storage temperature and periods.

Methods

GMSYS was stored at room temperature or refrigeration for 12 months. According to storage temperature and periods, pH and sugar content of GMSYS were measured. To determine the marker compounds of GMSYS, high-performance liquid chromatography analysis was performed. To estimate the anti-inflammatory effect of GMSYS, LPS-induced pro-inflammatory mediators and cytokines were measured in RAW 264.7 cells.

Results

There was no change in pH and sugar content depending on storage temperature and periods of GMSYS. The contents of gallic acid and mangiferin in both of room temperature and refrigerated decoctions reduced with increasing storage periods. Chlorogenic acid was time-dependently decreased in case of stored at room temperature. GMSYS significantly inhibited the LPS-induced production of nitric oxide, prostaglandin E2 (PGE2) and IL-6 in RAW 264.7 cells. These effects equally maintained up to 3 months at both of room temperature and refrigeration. Since 4 months, the inhibitory effect of GMSYS on LPS-induced PGE2 production was time-dependently reduced, and the decrease in PGE2 inhibitory effect of decoction stored at refrigeration was lower than that of stored at room temperature.

Conclusions

Our results indicate that the anti-inflammatory effect of GMSYS are maintained up to 12 months, but it shows optimal efficacy up to 3 months. It is recommended to store in a refrigeration for short periods since some components decrease as storage periods becomes longer.

Keywords: Gamisoyo-san decoction; storage temperature; storage period; high performance liquid chromatography; anti-inflammation
Fig. 1

Chemical structures of the ten marker components in Gamisoyo-san.

Fig. 2

Typical HPLC chromatogram of standard solution (A) and Gamisoyo-san decoction (B) at UV wavelength 230 (I), 240 (II), 254 (III), 270 (IV), 275 (V), 325 (VI), 335 (VII) and 345 (VIII). Gallic acid (1), chlorogenic acid (2), mangiferin (3), geniposide (4), paeoniflorin (5), berberine chloride (6), liquiritin apioside (7), nodakenin (8), benzoic acid (9) and glycyrrhizin (10).

Fig.3

Effect of Gamisoyo-san on LPS-induced NO (A), PGE2 (B), TNF-α (C) and IL-6 (D) production in RAW 264.7 cells at the time of extraction.

NO level in supernatant was measured using Griess reagent. PGE2, TNF-α and IL-6 in supernatant were measured by ELISA. L-NMMA and indomethacin were used as positive controls for NO and PGE2, respectively. Triprolidine was used as a positive control for TNF-α and IL-6. The data are presented as mean ± SEM (n = 3). ##p < 0.01 versus negative control cells; *p < 0.05 and *p < 0.01 versus LPS-treated cells.

Composition of Gamisoyo-san

pH of Gamisoyo-san by storage temperature and periods

Sugar content of Gamisoyo-san by storage temperature and periods

Linear range, regression equation, correlation coefficients, LOD and LOQ for the ten marker compounds

The content of the five marker compounds in Gamisoyo-san by storage periods in room temperature

The content of the five marker compounds in Gamisoyo-san by storage periods in refrigeration

References

1. The co-textbook publishing committee of Korean Medicine College. Formula Science Seoul: Younglimsa; 2009.
2. Seo CS, Shin HK, Kim JH, Shin KS. Changes of Principal Components and Microbial Population in Pyungwi-san Decoction According to the Preservation Temperature and Period. J Korean Oriental Med 2011;32(5):41–9.
3. Ha H, Shin IS, Lim HS, Jeon WY, Kim JH, Seo CS, Shin HK. Changes in Anti-inflammatory Effect of Pyungwi-san Decoction According to the Preservation Temperature and Period. Formula Science 2012;20(2):29–35.
4. Seo CS, Kim JH, Lim SH, Shin HK. Establishment of Shelf-Life of Ssanghwa-tang by Long-term Storage Test. Kor J Pharmacogn 2012;43(3):257–64.
5. Seo CS, Kim JH, Kim SS, Lim SH, Shin HK. Evaluation of Shelf-life of Bojungikgi-tang by Long-term Storage Test. Kor J Pharmacogn 2013;44(2):200–8.
6. Jin SE, Kin OS, Shin HK, Jeong SJ. Comparative Study on Biological Activities of Gwakhyangjeonggi-san Decoction According to the Preservation Periods. J Korean Med 2014;35(3):60–9.
7. Jin SE, Kim OS, Seo CS, Shin HK, Jeong SJ. Comparative study on stability and efficacy of Banhasasim-tang decoction depending on the preservation temperature and periods. J Korean Med 2016;37(1):21–33.
8. Yoo SR, Ha H, Lee NR, Shin HK, Seo CS. A Study on Change of Marker Compounds and Biological Activity in Chungsimyeonja-eum Decoction Depending on A Storage Temperature and Periods. Kor J Herbol 2017;32(4):25–32.
9. Park IH, Kim YH, Choi SH, Yu SN, Kim SH, Ahn SC, Cho SI, Lee I. Effect of Preservation Conditions on the Stability of Samul-tang Decoctions. J Life Sci 2015;25(10):1124–31.
10. Do HJ, Shim YS, Lee JH, Ahn YJ, Ha IH, Lee YJ, et al. Stability of Danggwisu-san (Dangguixu-san) Water Extract, a Herbal Medicine, Under Various Storage Conditions. Journal of Oriental Rehabilitation Medicine 2016;26(4):1–8.
11. Jin SM. Taepyunghyeminhwajekukbang Inminweeseng Publications; 1985. p. 308.
12. Sim TK, Jung IC, Lee SR. The Effect of Gami soyo-san(Jiaweixiaoyaosan) on Serotonin Metabolism. Journal of Oriental Neuropsychiatry 2011;22(1):37–51.
13. Lee SL, Lee IS, Soh KS. Effects of Gamisoyosan(Jiaweixiaoyaosan 加味逍遙散) on Type II Collagen-Induced Arthritis. J Oriental Rehab Med 2001;12(4):167–76.
14. Jin SE, Kim OS, Yoo SR, Seo CS, Kim Y, Shin HK, et al. Anti-inflammatory effect and action mechanisms of traditional herbal formula Gamisoyo-san in RAW 264.7 macrophages. BMC Complement Altern Med 2016;16:219. 10.1186/s12906-016-1197-7.
15. Choi HM, Kim SJ, Kim IS, Lee JB, Kim JB, Moon SO, et al. Evaluation on Anti-oxidant Activity and Anti-inflammatory Effects for the New Formulation of Gamisoyosan. Kor J Herbol 2016;31(6):1–9.
16. Willoughby DA. Human arthritis applied to animal models. Towards a better therapy. Ann Rheum Dis 1975;34:471–8.
17. Lee ES, Ju HK, Moon TC, Lee E, Jahng Y, Lee SH, et al. Inhibition of nitric oxide and tumor necrosis factor α (TNF-α) production by propenone compound through blockade of nuclear factor (NF)-κB activation in cultured murine macrophages. Biol Pharm Bull 2004;27:617–20.
18. Wink DA. Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic Biol Med 1998;25(4–5):434–56.
19. Ryu JH, Ahn H, Kim JY, Kim YK. Inhibitory activity of plant extracts on nitric oxide synthesis in LPS-activated macrophages. Phytother Res 2003;17(5):485–9.
20. Funk CD, Fitzgerald GA. Human platelet, erythroleukemia cell prostaglandin G, H synthase: cDNA cloning, expression, and gene chromosomal assignment. FASEB J 1991;5:2304–12.
21. Bishop-Bailey D, Calatayud S, Warner TD, Hla T, Michell A. Prostaglandins and the regulation of tumor growth. J Environ Pathol Tox Oncol 2002;21:93–101.
22. Feldmann M. Role of cytokines in rheumatoid arthritis. Annu Rev Immunol 1996;14:397–440.
23. Mann JR, Backlund MG, DuBois RN. Mechanism of disease: Inflammatory mediators and cancer prevention. Nat Clin Pract Oncol 2005;2:202–10.
24. Guzik TJ, Korbut R, Adamek-Guzik T. Nitric oxide and superoxide in inflammation and immune regulation. J Physiol Phamacol 2003;43:469–87.
25. Chiu CS, Deng JS, Chang HY, Chen YC, Lee MM, Hou WC, et al. Antioxidant and anti-inflammatory properties of Taiwanese yam (Dioscorea japonica Thunb. var. pseudojaponica (Hayata) Yamam.) and its reference compounds. Food Chem 2013;141:1087–96.
26. Hwang SJ, Kim YW, Park Y, Lee HJ, Kim KW. Anti-inflammatory effects of chlorogenic acid in lipopolysaccharide-stimulated RAW 264.7 cells. Inflamm Res 2014;63:81–90.
27. Garrido G, Delgado R, Lemus Y, Rodríguez J, García D, Núñez-Sellés AJ. Protection against septic shock and suppression of tumor necrosis factor alpha and nitric oxide production on macrophages and microglia by a standard aqueous extract of Mangifera indica L. (VIMANG). Role of mangiferin isolated from the extract. Pharmacol Res 2004;50:165–72.
28. Shi Q, Cao J, Fang L, Zhao H, Liu Z, Ran J, et al. Geniposide suppresses LPS-induced nitric oxide, PGE2 and inflammatory cytokine by downregulating NF-kB, MAPK and AP-1 signaling pathways in macrophages. Int Immunopharmacol 2014;20:298–306.
29. Wang QS, Gao T, Cui YL, Gao LN, Jiang HL. Comparative studies of paeoniflorin and albiflorin from Paeonia lactiflora on anti-inflammatory activities. Pharm Biol 2014;52(9):1189–95.
30. Jeong HW, Hsu KC, Lee JW, Ham M, Huh JY, Shin HJ, et al. Berberine suppresses proinflammatory responses through AMPK activation in macrophages. Am J Physiol Endocrinol Metab 2009;296:E955–64.
31. Guan Y, Li FF, Hong L, Yan XF, Tan GL, He JS, et al. Protective effects of liquiritin apioside on cigarette smoke-induced lung epithelial cell injury. Fundam Clin Pharmacol 2012;26(4):473–83.
32. Rim HK, Cho W, Sung SH, Lee KT. Nodakenin suppresses lipopolysaccharide-induced inflammatory responses in macrophage cells by inhibiting tumor necrosis factor receptor-associated factor 6 and nuclear factor-kB pathways and protects mice from lethal endotoxin shock. J Pharmacol Exp Ther 2012;342:654–64.
33. Fu Y, Zhou E, Wei Z, Liang D, Wang W, Wang T, et al. Glycyrrhizin inhibits the inflammatory response in mouse mammary epithelial cells and a mouse mastitis model. FEBS J 2014;281:2543–57.

Article information Continued

Copyright © 2018 The Society of Korean Medicine all rights reserved

Fig. 1

Fig. 1

Chemical structures of the ten marker components in Gamisoyo-san.

Fig. 2

Fig. 2

Typical HPLC chromatogram of standard solution (A) and Gamisoyo-san decoction (B) at UV wavelength 230 (I), 240 (II), 254 (III), 270 (IV), 275 (V), 325 (VI), 335 (VII) and 345 (VIII). Gallic acid (1), chlorogenic acid (2), mangiferin (3), geniposide (4), paeoniflorin (5), berberine chloride (6), liquiritin apioside (7), nodakenin (8), benzoic acid (9) and glycyrrhizin (10).

Fig.3

Fig.3

Effect of Gamisoyo-san on LPS-induced NO (A), PGE2 (B), TNF-α (C) and IL-6 (D) production in RAW 264.7 cells at the time of extraction.

NO level in supernatant was measured using Griess reagent. PGE2, TNF-α and IL-6 in supernatant were measured by ELISA. L-NMMA and indomethacin were used as positive controls for NO and PGE2, respectively. Triprolidine was used as a positive control for TNF-α and IL-6. The data are presented as mean ± SEM (n = 3). ##p < 0.01 versus negative control cells; *p < 0.05 and *p < 0.01 versus LPS-treated cells.

Table 1

Composition of Gamisoyo-san

Latin name Scientific name Amount (g) Origin
Paeoniae Radix Paeonia lactiflora Pallas 4.500 Uiseong, Korea
Atractylodis Rhizoma Alba Atractylodes macrocephala Koidzumi 4.500 China
Anemarrhenae Rhizoma Anemarrhena asphodeloides Bunge 3.750 Kangjin, Korea
Lycii Radicis Cortex Lycium chinense Miller 3.750 China
Angelicae Gigantis Radix Angelica gigas Nakai 3.750 Bonghwa, Korea
Poria Sclerotium Poria cocos Wolf 3.000 Pyeongchang, Korea
Liriope Tuber Liriope platyphylla Wang et Tang 3.000 Miryang, Korea
Rehmanniae Radix Recens Rehmannia glutinosa Liboschitz var. purpurea Makino 3.000 Gunwi, Korea
Gardeniae Fructus Gardenia jasminoides Ellis 1.875 Gurye, Korea
Phellodendri Cortex Phellodendron amurense Ruprecht 1.875 China
Platycodonis Radix Platycodon grandiflorum A. De Candolle 1.125 Muju, Korea
Glycyrrhizae Radix et Rhizoma Glycyrrhiza uralensis Fischer 1.125 China
Total 35.250

Table 2

pH of Gamisoyo-san by storage temperature and periods

Storage period (months) Storage method pH
0 5.18±0.01
1 Room temperature 4.65±0.00
Refrigeration 4.72±0.02
2 Room temperature 4.50±0.00
Refrigeration 4.58±0.02
3 Room temperature 5.03±0.01
Refrigeration 5.13±0.01
4 Room temperature 5.01±0.01
Refrigeration 5.12±0.01
5 Room temperature 5.00±0.01
Refrigeration 5.09±0.02
6 Room temperature 4.96±0.02
Refrigeration 5.11±0.01
12 Room temperature 4.97±0.01
Refrigeration 5.07±0.01

Data are presented as mean ± SEM (n = 3).

Table 3

Sugar content of Gamisoyo-san by storage temperature and periods

Storage period (months) Storage method Brix
0 3.77±0.03
1 Room temperature 3.83±0.02
Refrigeration 4.00±0.02
2 Room temperature 3.91±0.01
Refrigeration 3.92±0.03
3 Room temperature 3.92±0.01
Refrigeration 3.89±0.01
4 Room temperature 3.99±0.01
Refrigeration 3.93±0.02
5 Room temperature 3.98±0.03
Refrigeration 3.98±0.01
6 Room temperature 3.91±0.03
Refrigeration 3.87±0.00
12 Room temperature 4.08±0.01
Refrigeration 3.90±0.00

Data are presented as mean ± SEM (n = 3).

Table 4

Linear range, regression equation, correlation coefficients, LOD and LOQ for the ten marker compounds

Compound Linear range (ng/mL) Regression equationa Correlation coefficient LODb (ng/mL) LOQc (ng/mL)
Gallic acid 0.63–40.00 y=38259.49x−10572.16 0.9999 0.05 0.15
Chlorogenic acid 0.63–40.00 y=41318.61x−24432.53 0.9996 0.01 0.03
Mangiferin 0.63–40.00 y=50951.35x−13475.92 0.9999 0.01 0.04
Geniposide 0.63–40.00 y=16710.90x−141.25 1.0000 0.04 0.11
Paeoniflorin 0.63–40.00 y=10839.56x+774.96 0.9997 0.08 0.23
Berberine 1.56–100.00 y=60688.22x−23759.65 1.0000 0.00 0.01
Liquiritin apioside 0.63–40.00 y=15209.48x−2448.89 1.0000 0.05 0.16
Nodakenin 1.56–100.00 y=32877.31x−14561.30 1.0000 0.01 0.03
Benzoic acid 0.31–20.00 y=35372.59x−5310.55 0.9999 0.02 0.07
Glycyrrhizin 0.63–40.00 y=8282.39x−1435.76 1.0000 0.09 0.27
a

y: peak area (mAU) of compounds; x: concentration (mg/mL) of compounds.

b

LOD = 3.3σ × S.

c

LOQ = 10σ × S.

σ is the standard deviation of the blank response and S is the slope of the calibration curve.

Table 5

The content of the five marker compounds in Gamisoyo-san by storage periods in room temperature

Compound Content (mg/g)
0* 1 2 3 4 5 6 12
Gallic acid 0.32 0.10±0.003 0.03±0.003 0.02±0.000 0.01±0.003 0.01±0.006 0.01±0.003 0.01±0.003
Chlorogenic acid 0.27 0.25±0.000 0.23±0.000 0.21±0.003 0.17±0.003 0.18±0.000 0.18±0.000 0.14±0.000
Mangiferin 0.34 0.25±0.000 0.20±0.003 0.17±0.000 0.09±0.000 0.08±0.000 0.12±0.000 0.07±0.013
Geniposide 2.85 2.82±0.030 2.79±0.058 2.98±0.013 2.77±0.007 2.10±0.021 2.12±0.007 2.17±0.000
Paeoniflorin 2.66 2.58±0.006 2.61±0.021 2.62±0.003 2.66±0.012 2.50±0.029 2.54±0.009 2.58±0.003
Berberine 0.53 0.42±0.003 0.40±0.000 0.40±0.003 0.31±0.003 0.47±0.006 0.31±0.003 0.31±0.006
Liquiritin apioside 0.39 0.34±0.007 0.30±0.003 0.32±0.003 0.31±0.003 0.32±0.000 0.32±0.000 0.32±0.006
Nodakenin 0.32 0.31±0.003 0.32±0.000 0.33±0.000 0.33±0.003 0.33±0.000 0.33±0.003 0.34±0.000
Benzoic acid 0.27 0.29±0.003 0.28±0.003 0.27±0.000 0.27±0.000 0.27±0.000 0.27±0.003 0.29±0.000
Glycyrrhizin 0.42 0.37±0.003 0.37±0.000 0.38±0.000 0.36±0.000 0.37±0.006 0.37±0.003 0.37±0.000
*

month

Table 6

The content of the five marker compounds in Gamisoyo-san by storage periods in refrigeration

Compound Content (mg/g)
0* 1 2 3 4 5 6 12
Gallic acid 0.32 0.28±0.003 0.24±0.003 0.20±0.006 0.16±0.003 0.14±0.010 0.12±0.006 0.05±0.003
Chlorogenic acid 0.27 0.27±0.000 0.26±0.003 0.26±0.000 0.25±0.000 0.26±0.000 0.25±0.003 0.23±0.000
Mangiferin 0.34 0.28±0.003 0.29±0.003 0.27±0.003 0.20±0.000 0.24±0.006 0.24±0.000 0.19±0.003
Geniposide 2.85 2.79±0.009 2.70±0.067 2.93±0.072 2.75±0.041 2.08±0.010 2.07±0.015 2.03±0.000
Paeoniflorin 2.66 2.57±0.015 2.57±0.012 2.58±0.003 2.64±0.006 2.46±0.015 2.49±0.013 2.63±0.000
Berberine 0.53 0.40±0.003 0.45±0.003 0.40±0.015 0.32±0.007 0.52±0.015 0.30±0.003 0.31±0.006
Liquiritin apioside 0.39 0.34±0.009 0.34±0.007 0.33±0.009 0.31±0.003 0.31±0.006 0.31±0.007 0.29±0.000
Nodakenin 0.32 0.31±0.000 0.30±0.003 0.31±0.000 0.31±0.003 0.31±0.000 0.31±0.000 0.31±0.000
Benzoic acid 0.27 0.28±0.000 0.27±0.000 0.26±0.003 0.26±0.000 0.26±0.000 0.26±0.003 0.27±0.003
Glycyrrhizin 0.42 0.36±0.000 0.38±0.000 0.36±0.009 0.36±0.003 0.37±0.000 0.34±0.000 0.33±0.000
*

month