Effect of Safflower and Safflower Seed Extract on Osteogenic Differentiation of MC3T3E1 Cells

Yu, Sung-ryul; Shin, Seon-mi

The Journal of Internal Korean Medicine > Volume 36(4); 2015 > Article

Article

The Journal of Internal Korean Medicine 2015;36(4): 518-526.

홍화, 홍화씨 추출물이 MC3T3E1 세포의 골분화 과정에 미치는 영향

유성률¹, 신선미²

¹세명대학교 임상병리학과
²세명대학교 한의과대학 내과학교실

Effect of Safflower and Safflower Seed Extract on Osteogenic Differentiation of MC3T3E1 Cells

Sung-ryul Yu¹, Seon-mi Shin²

¹Dept. of Clinical Laboratory Science, Se-Myung University
²Dept. of Internal Medicine, College of Oriental Medicine, Se-Myung University

Correspondence

Seon-mi Shin ,Tel: 043-649-1814, Fax: 043-645-1382, Email: bunggujy21@hanmail.net

Received: November 20, 2015, Accepted: December 28, 2015, Published online: December 31, 2015.

ABSTRACT

Objectives:
This study investigated the effect of purified safflower (Carthamus tinctorius Linne) and safflower seed (Carthamus tinctorius L. seed; CS) extract, using hot water and ethanol extract methods , on the osteogenic differentiation of MC3T3E1 cells.

Methods:
The safflower and safflower seed were extracted with hot water and ethanol. The samples were concentrated by a rotary evaporator and then freeze-dried using a freeze-dryer. The MC3T3E1 cells were propagated and maintained in DMEM (Gibco) containing 10% FBS and a 1% antibiotic antimycotic solution. To induce osteogenic differentiation, the cells were treated for 14 days with DMEM with 10 mM β-glycerophosphate and 50 μM ascorbic acid. Extract doses were confirmed by the results of an MTT assay, and treatment of the extracts was performed in a differentiation medium every two days. The ALP staining and activity were tested after osteogenic differentiation for five days, and after 14 days, osteogenic differentiation was determined by alizarin red S staining. The mRNA expressions of osteogenic-related genes were quantified using quantitative real-time PCR.

Results:
In the results of the MTT assay, all concentrations of safflower extracts had no toxicity in the MC3T3El cells. But in the groups of 100 ng/ml and 200 ng/ml concentrations of safflower seed extracts, the cell viability was significantly reduced by up to 40-50%. So we fixed the treatment concentration of the extract at 50 ng/ml. In the ALP and alizarin red S staining, all extract groups increased osteogenic differentiation compared with the control group. The water-safflower extract group showed the highest mRNA level of Alp, Runx2, and Dlx5 genes. The mRNA level of Ocn, an osteogenic gene related to late-stage differentiation, in the ethanol-safflower extract group increased the mineralization more significantly than in other groups.

Conclusions:
These data suggest that the extract of safflower increases the osteoblastic differentiation activates of MC3T3E1 cells like the extract of safflower seed. The water-extract and ethanol-extract of safflower have effects on different stages of osteogenesis in MC3T3El. Not only safflower seed but also safflower will be useful therapeutic reagents for age-associated chronic diseases such as osteoporosis.

Key words: osteoblastic differentiation, safflower, safflower seed, osteoporosis