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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

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.

Fig. 1
Chemical structures of the ten marker components in Gamisoyo-san.
jkm-39-1-22f1.gif
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).
jkm-39-1-22f2.gif
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.
jkm-39-1-22f3.gif
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

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