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

Seong Eun Jin; Ohn Soon Kim; Chang-Seob Seo; Hyeun-Kyoo Shin; Soo-Jin Jeong; Jin, Seong Eun; Kim, Ohn Soon; Seo, Chang-Seob; Shin, Hyeun-Kyoo; Jeong, Soo-Jin

doi:10.13048/jkm.16003

JKM > Volume 37(1); 2016 > Article

Jin, Kim, Seo, Shin, and Jeong: Comparative study on stability and efficacy of Banhasasim-tang decoction depending on the preservation temperature and periods

Original Article

The Journal of Korean Oriental Medicine 2016; 37(1): 21-33.

Published online: March 31, 2016

DOI: https://doi.org/10.13048/jkm.16003

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

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 January 27, 2016 Revised March 14, 2016 Accepted March 17, 2016

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.

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

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