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Screening of plants for treating osteoporosis based

on ALP activity from endemic species of Korea

a, b
Soyeon Hong , Da Seul Jung , Jae-Eok Park , Jung-Hye Choi , Chu Won Nho , Gyhye Yoo a,*
b
a
a
a
a Smart Farm Research Center, Gangneung Institute of Natural Products Korea Institute of Science and Technology (KIST), Gangneung, Gangwon-do 25451, Korea
b KHU-KIST Department of Converging Science and Technology, Kyung Hee University, Seoul 130-701, Republic of Korea
ABSTRACT RESULTS
Osteoporosis is a bone disorder with low bone density and its incidence ranges from No. Code name Extraction Method ALP activity No. Code name Extraction Method ALP activity
one in 2 women after 50. Osteoporosis is caused by an imbalance in the bone 1 FS1 EtOH extract 92.445 34 PL3 BuOH fraction 49.751
2 FV1 EtOH extract 42.667 35 PL4 Water fraction 92.244
remodeling between bone formation and destruction. In the present study, we screened
3 JD1 EtOH extract 65.83 36 AP1 EtOH extract 108.96
endemic Species of Korea having osteogenic activity to find a cure for osteoporosis.
4 CT1 EtOH extract 75.592 37 AP2 Hexane fraction 154.788
ALP (alkaline phosphatase) activity in MC3T3-E1 preosteoblasts was used to estimate 5 RL1 EtOH extract 49.922 38 AP3 BuOH fraction 110.68
osteogenic activity for screening. Ethanol extracts of 20 species of plants and their 6 RL2 Hexane fraction 143.091 39 AP4 Water fraction 101.276
fractions were prepared and utilized for screening. During 6 days of osteoblast 7 RL3 BuOH fraction 60.666 40 FV1 EtOH extract 23.971
8 LS1 EtOH extract 42.675 41 FV2 Hexane fraction 14.824
differentiation of MC3T3-E1 cells, extracts or fractions were treated and ALP activity
9 LS2 Hexane fraction 57.108 42 FV3 BuOH fraction 74.782
were measured. Among 20 species of plant extracts, three extracts, RL and AP hexane
10 LS3 BuOH fraction 16.994 43 FV4 Water fraction 86.415
fraction elevated ALP activity significantly. Among fractions of 2 selected plants, their 11 LS4 Water fraction 72.557 44 LH1 EtOH extract 64.344
hexane fractions were most effective on ALP activation. Those activities were 12 AS1 EtOH extract 64.222 45 LH2 Hexane fraction 75.134
confirmed by Western blot: RL hexane fraction elevated the level of RUNX2, Osterix 13 AS2 Hexane fraction 91.326 46 LH3 BuOH fraction 56.907
14 AS3 BuOH fraction 67.068 47 LH4 Water fraction 49.376
and type 1 collagen alpha 1 (Col1a1). AP hexane fraction elevated the level of RUNX2
15 AS4 Water fraction 81.637 48 VN1 EtOH extract 42.861
and Col1a1. In conclusion, we found 2 novel endemic species in Korea for
16 AM1 EtOH extract 82.856 49 VN2 Hexane fraction 42.891
osteoporosis, and plan to conduct animal experiments using ovariectomized mice and
17 AM2 Hexane fraction 87.193 50 VN3 BuOH fraction 48.485
find active compounds from RL Hexane fraction. 18 AM3 BuOH fraction 46.063 51 VN4 Water fraction 47.901
19 AM4 Water fraction 71.066 52 HM1 EtOH extract 89.764
INTRODUCTION 20 TQ1 EtOH extract 92.866 53 HM2 Hexane fraction 19.407
21 TQ2 Hexane fraction 97.425 54 HM3 BuOH fraction 83.863
Osteoblasts are cells responsible for bone formation to maintain bone tissue. It have 22 TQ3 BuOH fraction 97.221 55 HM4 Water fraction 68.617
23 TQ4 Water fraction 112.807 56 AA1 EtOH extract 110.623
been reported current therapies for osteoporosis such as hormone therapy and bone
24 VD1 EtOH extract 71.1913 57 AA2 Hexane fraction 113.428
resorptive inhibitory drugs have various side effects such as breast cancer, esophagitis,
25 VD2 Hexane fraction 83.9433 58 AA3 BuOH fraction 102.804
jaw bone necrosis, etc. Therefore, we tried to select a new therapeutic agent derived 26 VD3 BuOH fraction 65.0956 59 AA4 Water fraction 108.197
from natural products affecting the enhancement of osteoblast activity. Osteoblast 27 VD4 Water fraction 86.5124 60 AD1 EtOH extract 107.442
differentiation or osteoblast maturation expresses markers such as the figure below. 28 HY1 EtOH extract 89.0464 61 AD2 Hexane fraction 99.299
29 HY2 Hexane fraction 77.2637 62 AD3 BuOH fraction 105.824
ALP is one of the marker expressed early in osteoblast differentiation. The
30 HY3 BuOH fraction 107.007 63 AD4 Water fraction 94.068
differentiated osteoblasts induce the formation of bone tissues with expressed
31 HY4 Water fraction 101.717 64 LF1 EtOH extract 94.877
RUNX2, Osterix and Col1a1. In this study, we tried to screen plants that induce
32 PL1 EtOH extract 83.045 65 LF2 Hexane fraction 100.809
osteoblast differentiation from endemic species of Korea to cure postmenopausal 33 PL2 Hexane fraction 112.703 66 LF3 BuOH fraction 88.082
osteoporosis. 67 LF4 Water fraction 89.861
Table 1 . ALP activity of Endemic Species of Korea
5
For analysis ALP activity, MC3T3-E1 cells were seeded at 1X10 cells /ml in 24 well
plates and cultured for 6 days in osteogenic medium with various concentrations of
each extract. Osteogenic medium containing 50 μg/mL L-ascorbic acid and 10mM β-
glycerophosphate. ALP activity were measures at 405 nM after cell lysis was
incubated with pNPP. Samples were treated with 20µg/ml. ALP activity was
expressed % of differentiated cells
ALP activity ***
150
ALP activity (% of Differentiated cells) 200 ***
100
Osteoblast differentiation lineage 50
Julie C., et al. Bone remodelling at a glance. Journal of cell science, 2011, 124.7: 991-998. ***
Undiff 0 Diff RL2 TQ4 HY2 AP2 AP3 HM1 HM2
CONCLUSION
Figure1 . ALP activity of Endemic Species of Korea
*p< 0.05, **p<0.01, ***p<0.001, compared to differentiated cells
• Among 20 species and their fractions, Hex fractions of RL and AP showed the
induction ALP activity in MC3T3-E1 pre osteoblast. RL2 has been shown to
increase the expression of all major differentiation markers, RUNX2, OSX and
Cola1a1. AP increased the expression of RUNX2 and Col1a1, but showed no
change in the expression level of OSX.
• We will clarify whether osteoblast signaling pathways such as Wnt, BMP and
TGFβ signaling using MC3T3-E1 cells with both of RL2 and AP2.
• We will evaluate the degree of restoration of bone density using postmenopausal
osteoporosis animal model which induced by ovariectomy.
Figure 2. Increase of osteoblast differentiation markers by plants extract
• We will find out the active molecules in RL2 and AP2 via active guided treatment in MC3T3-E1 pre osteoblast.
fractionation and NMR analysis. In order to evaluate the differentiation activity of osteoblasts, MC3T3-E1 cells were
seeded at 1X10 cells /ml in 100mm plates and cultured for 6 days in osteogenic
5
medium with various concentrations of each extract. Proteins were extracted after 6
days of differentiation. Antibodies against β-actin, RUNX2(runt-related exprecssion
ACKNOWLEDGEMENTS factor), osterix (OSX), collagen type 1 alpha 1 (Col1a1), as well as secondary anti-
mouse were purchased from Santa Cruz Biotechnology (Dallas, TX). Each sample
This work was supported by KIST open research program (2E31300)
was treated at a concentration of 10 µg/ml for Low and 20 µg/ml for High.