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Comparative Study on Biological Activities of Gwakhyangjeonggi-san Decoction According to the Preservation Periods

Article information

J Korean Med. 2014;35(3):60-69
Publication date (electronic) : 2014 September 30
doi : https://doi.org/10.13048/jkm.14031
Seong Eun Jin, Ohn Soon Kim, Hyeun-Kyoo Shin, Soo-Jin Jeong
Herbal Medicine Formulation Research Group, Korea Institute of Oriental Medicine
Correspondence to: 정수진 (Soo-Jin Jeong), 대전광역시 유성구 유성대로 1672 한국한의학연구원 한약연구본부 한약방제연구그룹, Tel: +82-42-868-9651, Fax: +82-42-864-2120, E-mail: sjijeong@kiom.re.kr
Received 2014 July 14; Revised 2014 September 11; Accepted 2014 September 16.

Abstract

Background:

Herbal formulas are generally served as a type of decoction. However, there is no scientific evidence for determining preservation and circulation period of herbal medicine decoctions. Thus, we investigated anti-inflammatory and anti-oxidative effects of the Gwakhyangjeonggi-san decoction according to its preservation period.

Methods:

Gwakhyangjeonggi-san decoction was stored for 0, 1, 2 or 3 months at room temperature. To evaluate anti-inflammatory effects, enzyme-linked immunosorbent assays (ELISAs) for tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), and nitric oxide (NO) assay were conducted using the culture supernatant from RAW 264.7 cells stimulated with lipopolysaccharide (LPS). The antioxidant activities were studied by measuring free radical scavenging activities on ABTS and DPPH.

Results:

Gwakhyangjeonggi-san decoction maintained the inhibitory effects on TNF-α, IL-6 and NO productions after up to 2 months of storage in LPS-treated RAW 264.7 cells. No inflammatory response was observed in 3 months of storage. In addition, the scavenging activities on ABTS and DPPH of Gwakhyangjeonggi-san decoction were reduced time-dependently and showed less than 50% inhibition after 3 months of storage.

Conclusions:

Our results suggest that preservation period of Gwakhyangjeonggi-san decoction is recommended within 2 months after storage.

Keywords: Gwakhyangjeonggi-san decoction; preservation period; anti-inflammation; anti-oxidation
Fig. 1.

Effect of Gwakhyangjeonggi-san stored with different period on LPS-induced NO production in RAW 264.7 cells. (A) Gwakhyangjeonggi-san of 0 month storage; (B) Gwakhyangjeonggi-san of 1 month storage; (C) Gwakhyangjeonggi-san of 2 month storage; (D) Gwakhyangjeonggi-san of 3 month storage. Cells were pre-treated with Gwakhyangjeonggi-san or l-NMMA (100 M) for 4 h and then stimulated with LPS (1 g/mL) for 20 h. NO production was measured in the culture medium with the Griess reaction. The data are mean values of three experiments ± SEM (n=3). # p<0.01 versus vehicle-treated control group; * p<0.05 and ** p<0.01 versus LPS-treated cells.

Fig. 2.

Effect of Gwakhyangjeonggi-san stored with different period on LPS-induced TNF-α production in RAW 264.7 cells. (A) Gwakhyangjeonggi-san of 0 month storage; (B) Gwakhyangjeonggi-san of 1 month storage; (C) Gwakhyangjeonggi-san of 2 month storage; (D) Gwakhyangjeonggi-san of 3 month storage. Cells were pre-treated with Gwakhyangjeonggi-san for 4 h and then stimulated with LPS (1 g/mL) for 20 h. The levels of TNF-α released into the culture medium were assessed using commercially available ELISA kit. The data are mean values of three experiments ± SEM (n=3). # p<0.01 versus vehicle-treated control group; ** p<0.01 versus LPS-treated cells.

Fig. 3.

Effect of Gwakhyangjeonggi-san stored with different period on LPS-induced IL-6 production in RAW 264.7 cells. (A) Gwakhyangjeonggi-san of 0 month storage; (B) Gwakhyangjeonggi-san of 1 month storage; (C) Gwakhyangjeonggi-san of 2 month storage; (D) Gwakhyangjeonggi-san of 3 month storage. Cells were pre-treated with Gwakhyangjeonggi-san for 4 h and then stimulated with LPS (1 g/mL) for 20 h. The levels of IL-6 released into the culture medium were assessed using commercially available ELISA kit. The data are mean values of three experiments ± SEM (n=3). # p<0.01 versus vehicle-treated control group; ** p<0.01 versus LPS-treated cells.

Fig. 4.

Effect of Gwakhyangjeonggi-san stored with different period on ABTS radical scavenging activity. ABTS radical cation was produced by reacting 7 mM ABTS solution with 2.45 mM potassium persulfate store in the dark at room temperature for 16 h. Then, ABTS radical solution was added to a 96-well plate containing of samples or ascorbic acid (AA, 5 g/mL). After 30 min of incubation, the absorbance was measured at 743 nm using a microplate reader. The data are mean values of three experiments ± SEM (n=3). * p<0.1, ** p<0.01 versus 0 month.

Fig. 5.

Effect of Gwakhyangjeonggi-san stored with different period on DPPH radical scavenging activity. DPPH radical solution (0.15 mM in ethanol) was added to a 96-well plate containing of samples or ascorbic acid (AA, 20 g/mL). After 30 min of incubation, the absorbance was measured at 517 nm using a microplate reader. The data are mean values of three experiments ± SEM (n=3). ** p<0.01 versus 0 month.

Composition of Herbal Medicine in Extract of Gwakhyangjeonggi-san

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Article information Continued

Copyright © 2014 The Society of Korean Medicine all rights reserved

Fig. 1.

Fig. 1.

Effect of Gwakhyangjeonggi-san stored with different period on LPS-induced NO production in RAW 264.7 cells. (A) Gwakhyangjeonggi-san of 0 month storage; (B) Gwakhyangjeonggi-san of 1 month storage; (C) Gwakhyangjeonggi-san of 2 month storage; (D) Gwakhyangjeonggi-san of 3 month storage. Cells were pre-treated with Gwakhyangjeonggi-san or l-NMMA (100 M) for 4 h and then stimulated with LPS (1 g/mL) for 20 h. NO production was measured in the culture medium with the Griess reaction. The data are mean values of three experiments ± SEM (n=3). # p<0.01 versus vehicle-treated control group; * p<0.05 and ** p<0.01 versus LPS-treated cells.

Fig. 2.

Fig. 2.

Effect of Gwakhyangjeonggi-san stored with different period on LPS-induced TNF-α production in RAW 264.7 cells. (A) Gwakhyangjeonggi-san of 0 month storage; (B) Gwakhyangjeonggi-san of 1 month storage; (C) Gwakhyangjeonggi-san of 2 month storage; (D) Gwakhyangjeonggi-san of 3 month storage. Cells were pre-treated with Gwakhyangjeonggi-san for 4 h and then stimulated with LPS (1 g/mL) for 20 h. The levels of TNF-α released into the culture medium were assessed using commercially available ELISA kit. The data are mean values of three experiments ± SEM (n=3). # p<0.01 versus vehicle-treated control group; ** p<0.01 versus LPS-treated cells.

Fig. 3.

Fig. 3.

Effect of Gwakhyangjeonggi-san stored with different period on LPS-induced IL-6 production in RAW 264.7 cells. (A) Gwakhyangjeonggi-san of 0 month storage; (B) Gwakhyangjeonggi-san of 1 month storage; (C) Gwakhyangjeonggi-san of 2 month storage; (D) Gwakhyangjeonggi-san of 3 month storage. Cells were pre-treated with Gwakhyangjeonggi-san for 4 h and then stimulated with LPS (1 g/mL) for 20 h. The levels of IL-6 released into the culture medium were assessed using commercially available ELISA kit. The data are mean values of three experiments ± SEM (n=3). # p<0.01 versus vehicle-treated control group; ** p<0.01 versus LPS-treated cells.

Fig. 4.

Fig. 4.

Effect of Gwakhyangjeonggi-san stored with different period on ABTS radical scavenging activity. ABTS radical cation was produced by reacting 7 mM ABTS solution with 2.45 mM potassium persulfate store in the dark at room temperature for 16 h. Then, ABTS radical solution was added to a 96-well plate containing of samples or ascorbic acid (AA, 5 g/mL). After 30 min of incubation, the absorbance was measured at 743 nm using a microplate reader. The data are mean values of three experiments ± SEM (n=3). * p<0.1, ** p<0.01 versus 0 month.

Fig. 5.

Fig. 5.

Effect of Gwakhyangjeonggi-san stored with different period on DPPH radical scavenging activity. DPPH radical solution (0.15 mM in ethanol) was added to a 96-well plate containing of samples or ascorbic acid (AA, 20 g/mL). After 30 min of incubation, the absorbance was measured at 517 nm using a microplate reader. The data are mean values of three experiments ± SEM (n=3). ** p<0.01 versus 0 month.

Table 1.

Composition of Herbal Medicine in Extract of Gwakhyangjeonggi-san

Herbal medicine Latin name Original region Company Amount (g)
Agastache rugosa (Fisch. et Meyer) O. Kuntze Agastachis Herba Andong, Gyeongbuk, Korea Gwangmyungdang Pharm 5.63
Perilla frutescens var. crispa (Thunb.) H. Deane Perillae Folium Yeongcheon, Gyeongbuk, Korea Gwangmyungdang Pharm 3.75
Angelica dahurica Benth. et Hook. f. Angelicae Dahuricae Radix Uljin, Gyeongbuk, Korea Gwangmyungdang Pharm 1.88
Areca catechu L. Aracae Pericarpium China Gwangmyungdang Pharm 1.88
Poria cocos F. A. Wolf Poria Sclerotium Pyeongchang, Gangwon, Korea Gwangmyungdang Pharm 1.88
Magnolia officinalis Rehd. et E. H. Wils. Magnoliae Cortex China Gwangmyungdang Pharm 1.88
Atractylodes macrocephala Koidz. Atractylodes Rhizoma Alba China Gwangmyungdang Pharm 1.88
Citrus reticulata Blanco Citrus Unshius Pericarpium Jeju, Korea Gwangmyungdang Pharm 1.88
Pinellia ternata Breit. Pinelliae Tuber China Gwangmyungdang Pharm 1.88
Platycodon grandiflorum A. DC. Platycodonis Radix Andong, Gyeongbuk, Korea Gwangmyungdang Pharm 1.88
Glycyrrhiza uralensis Fisch. Glycyrrhizae Radix et Rhizoma China Gwangmyungdang Pharm 1.88
Ziziphus jujuba var. inermis (Bunge) Rehder Zizyphi Fructus Yeongcheon, Gyeongbuk, Korea Gwangmyungdang Pharm 3.75
Zingiber officinale Rosc. Zingiberis Rhizoma Recens Ulsan, Korea Gwangmyungdang Pharm 3.75