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

Seong Eun Jin; Chang-Seob Seo; Nari Lee; Hyeun-Kyoo Shin; Hyekyung Ha; Jin, Seong Eun; Seo, Chang-Seob; Lee, Nari; Shin, Hyeun-Kyoo; Ha, Hyekyung

doi:10.13048/jkm.18003

JKM > Volume 39(1); 2018 > Article

Jin, Seo, Lee, Shin, and Ha: Comparative study on the contents of marker compounds and anti-inflammatory effects of Gamisoyo-san decoction according to storage temperature and periods

Original Article

The Journal of Korean Medicine 2018; 39(1): 22-34.

Published online: March 31, 2018

DOI: https://doi.org/10.13048/jkm.18003

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

Seong Eun Jin¹

, Chang-Seob Seo¹

, Nari Lee¹

, Hyeun-Kyoo Shin¹

, Hyekyung Ha¹^{, *}

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 December 20, 2017 Revised March 15, 2018 Accepted March 16, 2018

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 PGE_2, 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
1	Refrigeration	4.72±0.02

2	Room temperature	4.50±0.00
2	Refrigeration	4.58±0.02

3	Room temperature	5.03±0.01
3	Refrigeration	5.13±0.01

4	Room temperature	5.01±0.01
4	Refrigeration	5.12±0.01

5	Room temperature	5.00±0.01
5	Refrigeration	5.09±0.02

6	Room temperature	4.96±0.02
6	Refrigeration	5.11±0.01

12	Room temperature	4.97±0.01
12	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
1	Refrigeration	4.00±0.02

2	Room temperature	3.91±0.01
2	Refrigeration	3.92±0.03

3	Room temperature	3.92±0.01
3	Refrigeration	3.89±0.01

4	Room temperature	3.99±0.01
4	Refrigeration	3.93±0.02

5	Room temperature	3.98±0.03
5	Refrigeration	3.98±0.01

6	Room temperature	3.91±0.03
6	Refrigeration	3.87±0.00

12	Room temperature	4.08±0.01
12	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

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