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Comparative study on stability and efficacy of Banhasasim-tang decoction depending on the preservation temperature and periods

Article information

J Korean Med. 2016;37(1):21-33
Publication date (electronic) : 2016 March 31
doi : https://doi.org/10.13048/jkm.16003
Seong Eun Jin1, Ohn Soon Kim2, Chang-Seob Seo1, Hyeun-Kyoo Shin1, Soo-Jin Jeong2,3,
1K-herb Research Center, Korea Institute of Oriental Medicine, Daejeon 34054, Republic of Korea
2KM Convergence Research Division, Korea Institute of Oriental Medicine, Daejeon 34054, Republic of Korea
3Korean Medicine Life Science, University of Science & Technology, Daejeon 34113, Republic of Korea
Correspondence to: 정수진 (Soo-Jin Jeong), 대전광역시 유성구 유성대로 1672 한국한의학연구원 한의약융합연구부, Tel: +82-42-868-9651, Fax: +82-42-864-2120, E-mail: sjijeong@kiom.re.kr
Received 2016 January 27; Revised 2016 March 14; Accepted 2016 March 17.

Abstract

Objectives:

Banhasasim-tang (BHSST) has been used for the treatment of the digestive and gastric diseases in Korea. This study aimed to investigate the stability and biological activities of BHSST decoction depending on the preservation temperature and periods.

Methods:

BHSST decoction was preserved at room temperatures (R/T, 23±1°C) or refrigeration (4°C) for 0, 30, 60 and 90 days. To evaluate the stability of BHSST decoction, pH and sugar content were estimated. In addition, high-performance liquid chromatography (HPLC) analysis was performed to determine marker compounds of BHSST decoction. To evaluate anti-inflammatory effect, nitric oxide (NO) and prostaglandin E2 (PGE2) productions were measured in LPS-stimulated RAW 264.7 macrophages. Antioxidant activity was examined using the assays for 3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) and 1-1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities.

Results:

There was no change in pH and sugar content depending on the preservation temperature and periods of BHSST decoction. Among the major components of BHSST, contents of liquiritin, baicalein and wogonin was reduced time-dependently both at R/T and 4°C. Inhibitory effects of BHSST decoction on NO and PGE2 productions were slightly decreased in a time-dependent manner by 90 days of preservation. In addition, BHSST decoction maintained ABTS and DPPH radical scavenging activities by 60 days while significantly reducing the activities in 90 days of preservation at R/T. By contrast, BHSST decoction had no significant change of ABTS and DPPH radical scavenging activities by 90 days at 4°C.

Conclusions:

Our results suggest that the stability and efficacy of BHSST decoction are maintained for 60 days at 4°C rather than R/T.

Keywords: Banhasasim-tang decoction; preservation temperature; preservation period: anti-inflammation; anti-oxidation
Fig. 1.

Chemical structure of Banhasasim-tang.

Fig. 2.

Typical HPLC chromatogram of Banhasasim-tang decoction at wavelength 254 (A) and 275 (B) nm. Liquiritin (1), baicalin (2), liquiritigenin (3), wogonoside (4), baicalein (5), glycyrrhizin (6), and wogonin (7).

Fig. 3.

Cytotocicity of Banhasasim-tang stored with different preservation conditions and periods in RAW 264.7 macrophages. BHSST: Banhasasim-tang.

Fig. 4.

Effect of Banhasasim-tang stored with different preservation conditions and periods on LPS-induced NO (A) and PGE2 (B) production in RAW 264.7 macrophages. Cells were pre-treated with Banhasasim-tang for 4 h and then co-stimulated with LPS (1 μg/mL) for 20 h. The levels of NO and PGE2 released into the culture supernatant were measured using Griess reagent and ELISA kit, respectively. The data are presented as the means ± SEM (n = 3). ##p<0.01 versus vehicle-treated control group; *p<0.05 and **p<0.01 versus LPS-treated group. BHSST: Banhasasim-tang, ID: indomethacin.

Fig. 5.

Effect of Banhasasim-tang stored with different preservation conditions and periods on ABTS (A) and DPPH (B) radical scavenging activity. ABTS or DPPH radical solution was added to a 96-well plate containing of several concentrations (50, 100, 200, 400 μg/mL) of Banhasasim-tang. After 30 min of incubation, the absorbance (ABTS; 734 nm, DPPH; 517nm) was measured using a microplate reader. RC50 is concentration of the sample which is required to scavenge 50% of radicals. The data are presented as the means ± SEM (n = 3). **p<0.01 versus 0 days.

Composition of Banhasasim-tang

pH and sugar content of Banhasasim-tang by preservation temperature and periods

Content of the seven marker compounds in Banhasasim-tang by preservation periods in room temperature

Content of the seven marker compounds in Banhasasim-tang by preservation periods in refrigeration

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Article information Continued

Copyright © 2016 The Society of Korean Medicine all rights reserved

Fig. 1.

Fig. 1.

Chemical structure of Banhasasim-tang.

Fig. 2.

Fig. 2.

Typical HPLC chromatogram of Banhasasim-tang decoction at wavelength 254 (A) and 275 (B) nm. Liquiritin (1), baicalin (2), liquiritigenin (3), wogonoside (4), baicalein (5), glycyrrhizin (6), and wogonin (7).

Fig. 3.

Fig. 3.

Cytotocicity of Banhasasim-tang stored with different preservation conditions and periods in RAW 264.7 macrophages. BHSST: Banhasasim-tang.

Fig. 4.

Fig. 4.

Effect of Banhasasim-tang stored with different preservation conditions and periods on LPS-induced NO (A) and PGE2 (B) production in RAW 264.7 macrophages. Cells were pre-treated with Banhasasim-tang for 4 h and then co-stimulated with LPS (1 μg/mL) for 20 h. The levels of NO and PGE2 released into the culture supernatant were measured using Griess reagent and ELISA kit, respectively. The data are presented as the means ± SEM (n = 3). ##p<0.01 versus vehicle-treated control group; *p<0.05 and **p<0.01 versus LPS-treated group. BHSST: Banhasasim-tang, ID: indomethacin.

Fig. 5.

Fig. 5.

Effect of Banhasasim-tang stored with different preservation conditions and periods on ABTS (A) and DPPH (B) radical scavenging activity. ABTS or DPPH radical solution was added to a 96-well plate containing of several concentrations (50, 100, 200, 400 μg/mL) of Banhasasim-tang. After 30 min of incubation, the absorbance (ABTS; 734 nm, DPPH; 517nm) was measured using a microplate reader. RC50 is concentration of the sample which is required to scavenge 50% of radicals. The data are presented as the means ± SEM (n = 3). **p<0.01 versus 0 days.

Table 1.

Composition of Banhasasim-tang

Herbal medicine Scientific name Amount (g) Origin
Pinelliae Tuber Pinellia ternata 7.500 China
Scutellariae Radix Scutellaria baicalensis 5.625 Gurye, Korea
Ginseng Radix Panax ginseng 5.625 Yeongju, Korea
Glycyrrhizae Radix et Rhizoma Glycyrrhiza uralensis 5.625 China
Zingiberis Rhizoma Zingiber officinale 3.750 Taean, Korea
Coptidis Rhizoma Coptis japonica 1.875 China
Zingiberis Rhizoma Crudus Zingiber officinale 3.750 Ulsan, Korea
Zizyphi Fructus Zizyphus jujuba 3.750 Yeongcheon, Korea
Total amount 37.50

Table 2.

pH and sugar content of Banhasasim-tang by preservation temperature and periods

Storage method Lot pH Sugar content (Brix)
0* 30 60 90 0* 30 60 90
Room temperature 1 4.70 4.65 4.63 4.61 3.13 3.10 2.90 2.87
2 4.70 4.65 4.62 4.61 3.13 3.10 2.93 2.77
3 4.70 4.65 4.62 4.60 3.13 3.10 2.90 2.83
4 4.70 4.65 4.62 4.60 3.13 3.07 2.90 2.87
Refrigeration 1 4.70 4.71 4.70 4.70 3.13 2.87 2.90 2.87
2 4.70 4.71 4.71 4.70 3.13 2.83 2.87 2.90
3 4.70 4.70 4.70 4.70 3.13 2.77 2.87 2.87
4 4.70 4.70 4.70 4.69 3.13 2.93 2.87 2.87
*

day

Table 3.

Content of the seven marker compounds in Banhasasim-tang by preservation periods in room temperature

Lot Compound Content (mg/g)
0* 30 60 90
Mean SD RSD (%) Mean SD RSD (%) Mean SD RSD (%) Mean SD RSD (%)
1 Liquiritin 3.74 0.01 0.19 3.50 0.03 0.79 3.32 0.12 3.51 3.18 0.06 1.75
Baicalin 51.82 0.22 0.43 51.52 0.05 0.10 50.39 0.12 0.24 51.34 1.24 2.41
Liquiritigenin 3.94 0.01 0.29 3.94 0.04 0.91 3.90 0.01 0.16 3.70 0.02 0.61
Wogonoside 14.42 0.05 0.35 15.15 0.06 0.39 14.70 0.03 0.22 15.77 0.03 0.22
Baicalein 0.80 0.00 0.63 0.12 0.00 3.94 0.06 0.00 3.08 0.05 0.00 8.34
Glycyrrhizin 5.61 0.06 1.06 5.97 0.08 1.35 5.38 0.02 0.42 5.32 0.08 1.50
Wogonin 0.77 0.00 0.32 0.62 0.01 2.36 0.11 0.00 0.90 0.03 0.00 7.04
2 Liquiritin 3.74 0.01 0.19 3.46 0.01 0.36 3.47 0.08 2.42 3.18 0.13 4.03
Baicalin 51.82 0.22 0.43 52.13 0.04 0.08 49.66 0.04 0.07 47.51 0.35 0.74
Liquiritigenin 3.94 0.01 0.29 3.94 0.00 0.04 3.89 0.05 1.28 3.69 0.01 0.36
Wogonoside 14.42 0.05 0.35 15.30 0.05 0.30 14.53 0.01 0.08 14.60 0.03 0.21
Baicalein 0.80 0.00 0.63 0.14 0.00 1.82 0.05 0.00 7.22 0.05 0.01 16.27
Glycyrrhizin 5.61 0.06 1.06 5.93 0.07 1.18 5.57 0.03 0.63 5.28 0.05 0.88
Wogonin 0.77 0.00 0.32 0.58 0.02 2.70 0.22 0.00 0.01 0.06 0.00 1.71
3 Liquiritin 3.74 0.01 0.19 3.51 0.01 0.23 3.43 0.04 1.05 3.24 0.02 0.54
Baicalin 51.82 0.22 0.43 52.62 0.14 0.26 49.55 0.08 0.17 51.12 0.87 1.70
Liquiritigenin 3.94 0.01 0.29 3.95 0.00 0.12 3.97 0.06 1.46 3.70 0.04 1.11
Wogonoside 14.42 0.05 0.35 15.57 0.07 0.44 14.44 0.09 0.61 15.45 0.02 0.11
Baicalein 0.80 0.00 0.63 0.14 0.00 1.10 0.06 0.00 6.43 0.04 0.00 8.17
Glycyrrhizin 5.61 0.06 1.06 5.94 0.06 0.98 5.52 0.05 0.97 5.36 0.02 0.38
Wogonin 0.77 0.00 0.32 0.59 0.01 2.22 0.19 0.00 0.79 0.06 0.00 0.19
4 Liquiritin 3.74 0.01 0.19 3.48 0.02 0.66 3.42 0.04 1.26 3.26 0.12 3.71
Baicalin 51.82 0.22 0.43 49.84 0.06 0.12 49.50 0.23 0.46 51.36 0.97 1.89
Liquiritigenin 3.94 0.01 0.29 3.93 0.01 0.26 3.90 0.01 0.34 3.79 0.03 0.82
Wogonoside 14.42 0.05 0.35 14.95 0.01 0.09 14.38 0.04 0.29 15.60 0.04 0.23
Baicalein 0.80 0.00 0.63 0.15 0.00 0.40 0.05 0.00 1.43 0.05 0.00 3.87
Glycyrrhizin 5.61 0.06 1.06 5.99 0.06 1.04 5.46 0.02 0.46 5.26 0.08 1.43
Wogonin 0.77 0.00 0.32 0.60 0.02 2.75 0.18 0.00 1.41 0.10 0.00 0.08
*

day

Table 4.

Content of the seven marker compounds in Banhasasim-tang by preservation periods in refrigeration

Lot Compound Content (mg/g)
0* 30 60 90
Mean SD RSD (%) Mean SD RSD (%) Mean SD RSD (%) Mean SD RSD (%)
1 Liquiritin 3.74 0.01 0.19 3.69 0.02 0.58 3.71 0.01 0.34 3.48 0.01 0.41
Baicalin 51.82 0.22 0.43 49.00 0.08 0.17 46.95 0.05 0.11 55.63 0.73 1.32
Liquiritigenin 3.94 0.01 0.29 3.92 0.02 0.39 3.94 0.01 0.32 3.68 0.04 1.11
Wogonoside 14.42 0.05 0.35 12.96 0.05 0.36 11.35 0.00 0.03 16.60 0.10 0.63
Baicalein 0.80 0.00 0.63 0.68 0.02 2.78 0.14 0.01 3.91 0.39 0.00 0.76
Glycyrrhizin 5.61 0.06 1.06 5.99 0.03 0.44 5.72 0.04 0.76 5.49 0.04 0.79
Wogonin 0.77 0.00 0.32 0.61 0.00 0.21 0.66 0.01 1.21 0.37 0.00 0.06
2 Liquiritin 3.74 0.01 0.19 3.67 0.01 0.31 3.69 0.01 0.40 3.28 0.01 0.38
Baicalin 51.82 0.22 0.43 48.56 0.10 0.21 47.06 0.08 0.17 54.82 0.80 1.45
Liquiritigenin 3.94 0.01 0.29 3.94 0.02 0.48 3.80 0.01 0.38 3.74 0.02 0.51
Wogonoside 14.42 0.05 0.35 12.43 0.04 0.32 12.29 0.00 0.02 15.50 0.01 0.09
Baicalein 0.80 0.00 0.63 0.66 0.01 2.18 0.12 0.00 3.41 0.25 0.01 3.68
Glycyrrhizin 5.61 0.06 1.06 5.97 0.02 0.28 5.66 0.04 0.71 5.39 0.03 0.47
Wogonin 0.77 0.00 0.32 0.69 0.00 0.27 0.56 0.00 0.35 0.16 0.00 1.06
3 Liquiritin 3.74 0.01 0.19 3.76 0.01 0.34 3.71 0.04 1.06 3.30 0.05 1.66
Baicalin 51.82 0.22 0.43 49.81 0.06 0.11 45.80 0.36 0.78 54.94 0.28 0.52
Liquiritigenin 3.94 0.01 0.29 3.95 0.02 0.44 3.94 0.03 0.87 3.68 0.06 1.70
Wogonoside 14.42 0.05 0.35 13.23 0.04 0.29 10.78 0.02 0.21 15.74 0.02 0.13
Baicalein 0.80 0.00 0.63 0.73 0.02 2.20 0.12 0.00 3.37 0.24 0.02 7.91
Glycyrrhizin 5.61 0.06 1.06 5.99 0.06 0.94 5.65 0.05 0.81 5.42 0.02 0.36
Wogonin 0.77 0.00 0.32 0.73 0.00 0.39 0.57 0.00 0.53 0.14 0.00 0.63
4 Liquiritin 3.74 0.01 0.19 3.68 0.01 0.28 3.77 0.10 2.64 3.32 0.07 1.99
Baicalin 51.82 0.22 0.43 46.90 0.06 0.13 46.62 0.05 0.11 55.42 0.33 0.60
Liquiritigenin 3.94 0.01 0.29 3.94 0.01 0.31 3.84 0.12 3.22 3.74 0.03 0.93
Wogonoside 14.42 0.05 0.35 11.96 0.04 0.31 11.52 0.01 0.06 15.93 0.02 0.11
Baicalein 0.80 0.00 0.63 0.66 0.02 2.46 0.14 0.00 1.29 0.26 0.00 1.40
Glycyrrhizin 5.61 0.06 1.06 5.99 0.02 0.25 5.66 0.06 1.07 5.37 0.07 1.21
Wogonin 0.77 0.00 0.32 0.73 0.00 0.35 0.60 0.00 0.24 0.17 0.00 0.32
*

day