Effects of Inhalable Microparticles of  on Chronic Obstructive Pulmonary Disease in a Mouse Model

Eung-Seok Lee; Jong-min Han; Min-Hee Kim; Uk Namgung; Yoon Yeo; Yang-Chun Park; Lee, Eung-Seok; Han, Jong-min; Kim, Min-Hee; Namgung, Uk; Yeo, Yoon; Park, Yang-Chun

doi:10.13048/jkm.13012

JKM > Volume 34(3); 2013 > Article

Lee, Han, Kim, Namgung, Yeo, and Park: Effects of Inhalable Microparticles of Socheongryong-tang on Chronic Obstructive Pulmonary Disease in a Mouse Model

Original Article

Journal of Korean Medicine 2013; 34(3): 54-68.

Published online: September 30, 2013

DOI: https://doi.org/10.13048/jkm.13012

Effects of Inhalable Microparticles of Socheongryong-tang on Chronic Obstructive Pulmonary Disease in a Mouse Model

Eung-Seok Lee¹, Jong-min Han¹, Min-Hee Kim², Uk Namgung², Yoon Yeo⁴, Yang-Chun Park¹

¹Division of Respiratory System, Dept. of Internal Medicine, College of Oriental Medicine, Daejeon University

²Dept. of Neurophysiology, College of Oriental Medicine, Daejeon University

³Institute of Traditional Medicine and Bioscience, Daejeon University

⁴College of Pharmacy, Purdue University

Correspondence to: 박양춘 (Yang-Chun Park), 대전광역시 중구 대흥동 22-5 대전대학교대전한방병원 한방내과, Tel : +82-42-229-6919, Fax : +82-42-254-3403, E-mail : omdpyc@dju.kr

Received April 23, 2013 Revised May 21, 2013 Accepted May 21, 2013

Abstract

Objectives:

This study aimed to evaluate the effects of microparticles of Socheongryong-tang (SCRT) on chronic obstructive pulmonary disease (COPD) in a mouse model.

Methods:

The inhalable microparticles containing SCRT were produced by spray-drying with leucine as an excipient, and evaluated with respect to the aerodynamic properties of the powder by Andersen cascade impactor (ACI). Its equivalence to SCRT extract was evaluated using lipopolysaccharide (LPS) and a cigarette-smoking (CS)-induced murine COPD model.

Results:

SCRT microparticles provided desirable aerodynamic properties (fine particle fraction of 49.6±5.5% and mass median aerodynamic diameter of 4.8±0.3 μm). SCRT microparticles did not show mortality or clinical signs over 14 days. Also there were no significant differences in body weight, organ weights or serum chemical parameters between SCRT microparticle-treated and non-treated groups. After 14 days the platelet count significantly increased compared with the non-treated group, but the values were within the normal range. Inhalation of SCRT microparticles decreased the rate of neutrophils in blood, granulocytes in peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage fluid (BALF) and level of TNF-α and IL-6 in BALF on COPD mouse model induced by LPS plus CS. This effect was verified by histological findings including immunofluorescence staining of elastin, collagen, and caspase 3 protein in lung tissue.

Conclusions:

These data demonstrate that SCRT microparticles are equivalent to SCRT extract in pharmaceutical properties for COPD. This study suggests that SCRT microparticles would be a potential agent of inhalation therapy for the treatment of COPD.

Keywords: Socheongryong-tang, inhalable microparticles, chronic obstructive pulmonary disease, spray drying, cigarette smoking

Fig. 1.

Experimental plan of repeated LPS+CS exposure.

Fig. 2.

Effect of SCRT-MP on neutrophil % of blood in COPD mice. Mice were challenged by aspiration of LPS+CS, and then treated with dexamethasone (PC: positive control, 0.5 mg/kg), SCRT (160 mg/kg, p.o), SCRT microparticle (50, 100 mg/kg) for 21 days (n=4).

*: p<0.05 †: p<0.01 compared to COPD-CT by T-test.

‡: p<0.05 compared to Intact by T-test.

Fig. 3.

Effect of SCRT-MP on CD11b/Gr-1 cell rate of PBMC (A) and BALF (B) in COPD mice. Mice were challenged by aspiration of LPS+CS, and then treated with dexamethasone (PC: positive control, 0.5 mg/kg), SCRT (160 mg/kg, p.o), SCRT microparticle (50, 100 mg/kg) for 21 days.

Fig. 4.

Effects of SCRT-MP on TNF-α and IL-6 production in COPD mice. Mice were challenged by aspiration of LPS+CS, and then treated with dexamethasone (PC: positive control, 0.5 mg/kg), SCRT (160 mg/kg, p.o), SCRT microparticle(50, 100 mg/kg) for 21 days (n=4). (A) TNF-α, (B) IL-6.

*: p<0.05 †: p<0.01 ‡: p<0.001 compared to COPD_CT by T-test.

∮ : p<0.001 compared to Intact by T-test.

Fig. 5.

Histological analysis(A) of lung tissues. Mice were challenged by aspiration of LPS+CS, and then treated with dexamethasone (PC: positive control, 0.5 mg/kg), SCRT (160 mg/kg, p.o), SCRT microparticle (50, 100 mg/kg) for 21 days. Lung tissues were examined under microscope (200 × magnification) after H&E staining. Asterisks denote alveoli. Immunofluorescence analysis(B) for caspase 3, elastin, collagen in lung tissue. Mice were challenged by aspiration of LPS+CS, and then treated with dexamethasone (PC: positive control, 0.5 mg/kg), SCRT (160 mg/kg, p.o), SCRT microparticle(50, 100 mg/kg) for 21 days. Lung tissues were examined under microscope (200 × magnification) after immunofluorescence staining against caspase 3, elastin, collagen. Asterisks denote alveoli.

Table 1.

The Composition of Socheongryong-tang (SCRT)

Herbal name	Pharmacognostic name	Lot No.	Country of origin	Amount (g)
Mahwang	Ephedrae Herba	M011005	China	6.0
Baekjakyak	Paeoniae Radix	J031008	Gyeongbuk, Korea	6.0
Omija	Schizandrae Fructus	A151008	Gyeongbuk, Korea	6.0
Banha	Pinelliae Tuber	B031003	China	6.0
Sesin	Asiasari Radix et Rhizoma	S381004	China	4.0
Geongang	Zingiberis Rhizoma	K021008	Chungnam, Korea	4.0
Gyeji	Cinnamomi Cortex	K031006	Vietnam	4.0
Gamcho	Glycyrrhizae Radix et Rhizoma	K370910	China	4.0
Total amount				40.0

Table 2.

Aerodynamic Properties of Microparticle of SCRT

ED^* (%)	FPF^† of CD^‡ (%)	FPF of ED (%)	FPF of ND^∮ (%)	MMAD‖ (μm)
89.0±18.0	49.6±5.5	27.8±6.5	25.4±9.7	4.8±0.3

* ED: Emitted dose,

† FPF: fine particle fraction,

‡ CD: collection dose,

∮ ND: nominal dose (11.1±1.7 mg),

‖ MMAD: mass median aerodynamic diameter.

Table 3.

Body Weight Changes of Male Mice on 14 Day Single Dose Toxicity Study of SCRT Microparticle

	Body weight (g)
	Intact^*	Mock^†	SCRT-MP^‡
0 day	22.54±0.27	21.69±0.28	22.14±0.20
5 day	23.29±0.46	22.00±0.31	22.57±0.30
10 day	24.43±0.37	23.36±0.39	23.64±0.34
14 day	24.58±0.49	23.64±0.53	24.00±0.61

Values are expressed as mean±S.D. (n=7).

* Intact: negative, not inhaled;

† Mock: inhaled air;

‡ SCRT-MP: inhaled SCRT microparticle (240 mg/kg).

Table 4.

Organ Weight of Male Mice Inhaled SCRT Microparticle on 14 Day Single Dose Toxicity Study

	Intact^*	Mock^†	SCRT-MP^‡
Liver (g)	1.35±0.06	1.26±0.11	1.12±0.06
Kidney (g)	0.35±0.02	0.32±0.01	0.31±0.01

Values are expressed as mean±S.D. (n=7). Intact: negative, not inhaled;

† Mock: inhaled air;

‡ SCRT-MP: inhaled SCRT microparticle (240 mg/kg).

Table 5.

Hematological Values of Male Mice Inhaled SCRT Microparticle on 1 and 14 Day in Single Dose Toxicity Study

	Intact^*		Mock^†		SCRT-MP^‡
	1 Day	14 Day	1 Day	14 Day	1 Day	14 Day
WBC (10³/mm³)	3.93±2.23	5.99±2.14	4.06±2.33	4.55±1.55	6.02±1.03	6.49±1.84
RBC (10⁶/mm³)	7.96±1.52	9.02±0.84	8.75±0.06	8.03±2.46	7.19±4.20	9.02±1.30
HGB (g/dl)	9.83±2.11	10.80±0.93	10.47±0.86	9.66±3.11	8.40±5.29	10.28±2.46
HCT (%)	41.50±7.62	45.49±4.11	44.23±0.68	40.52±12.39	35.37±21.10	44.28±7.44
PLT (10³/mm³)	467.00±139.86	598.14±83.23	524.00±128.64	604.20±204.95	411.67±285.63	1,015.20±142.25^*

Values are expressed as mean±S.D. (n=7).

* Intact: negative, not inhaled;

† Mock: inhaled air;

‡ SCRT-MP: inhaled SCRT microparticle (240 mg/kg).

*: p<0.01 compared to Intact by T-test.

Table 6.

Clinical Chemistry Values of Male Mice Inhaled SCRT Microparticle on 1 Day Single Dose Toxicity Study

	Intact^*		Mock^†		SCRT-MP^‡
	1 Day	14 Day	1 Day	14 Day	1 Day	14 Day
GOT (IU/L)	10.00±3.43	19.29±6.66	7.57±1.78	14.86±2.48	11.29±2.78	10.00±5.08
GPT (IU/L)	0.33±0.56	8.86±3.93	0.00±0.00	2.00±0.38	0.14±0.14	1.17±0.48
ALP (IU/L)	9.17±1.05	7.29±1.29	10.29±1.04	6.71±0.89	8.57±1.15	6.17±0.60
Creatinine (IU/L)	0.05±0.02	0.00±0.00	0.04±0.02	0.00±0.00	0.04±0.02	0.00±0.00

Values are expressed as mean±S.D. (n=7).

* Intact: negative, not inhaled;

† Mock: inhaled air;

‡ SCRT-MP: inhaled SCRT microparticle (240 mg/kg).

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