To understand the material basis and underlying molecular machinery of antiosteoporosis activity of the
Osteoporosis is characterized as a metabolic bone disease, wherein the bone microarchitecture is deteriorated due to disruption of bone formation and resorption [
HSCCC is a liquid-liquid partition chromatographic technique, which has been widely utilized for the segregation of naturally occurring compounds [
The present study describes the effect of the bioactive fractions from the ethanol extract of FCI on the bone loss induced by OVX in mice. Also, the preparative separation of the four compounds from the bioactive fractions has been established using HSCCC in a stepwise elution mode with optimized operation parameters. Moreover, the effect of the four compounds on osteoblastic proliferation and differentiation was investigated
HPLC grade acetonitrile and analytical grade organic solvents, as well as ammonium acetate for sample preparation and HSCCC separation, were obtained from (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China). Ultrapure water was achieved by RU-B water system (Shanghai Tauto Biotech Co. Ltd., Shanghai, China) and filtered through 0.45
The preparative HSCCC instrument, TBE-300C (Shanghai Tauto Biotech), was serially connected with three multilayer coil separation columns comprising 1.9 mm internal tubing diameter of the tubing and 305 mL total volume as well as a 20 mL loop for sample loading. The
FCI (1000 g) was homogeneously powdered by a mill and ultrasonically solubilized in in 5 L of 80% ethanol at 60°C for 2 h (repeated thrice). The extracts were pooled, filtered, and dried under reduced pressure at 55°C, yielding 253.5 g, which was then solubilized in water. Subsequently, the extraction was carried out with
The separation scheme of
The fractions obtained from the ethanol extract were subjected to antiosteoporosis assay on OVX-induced osteoporosis in mice. Forty female C57/BL6 mice aged 8 weeks (
According to the results of the experiments described in Section
HSCCC was performed at 25°C column temperature and 850 rpm rotary speed. The flow rate and solvent gradients were altered according to the
HSCCC chromatograms of ethyl acetate fraction from
The FCI-E and HSCCC peak fractions were analyzed by HPLC at 25°C. Acetonitrile-0.1% H3PO4 aq was used as a gradient mobile phase for acetonitrile for 0–60 min, 15–80% mode at a flow rate of 1.0 mL/min. The effluent was monitored at 335 nm, and the structure of each peak fraction was resolved by 1H and 13C NMR spectra.
MC3T3-E1 cells (ATCC; Manassas, VA, USA) were cultured in phenol red-free
MC3T3-E1 cells were seeded and cultured for 24 h in 96-well plates at a density of 4 × 103 cells/well. Subsequently, the cells were treated with the test agent at concentrations of 0.1, 1, and 10
The cells were incubated with the test agent at concentration of 1
The cells were seeded and cultured in 24-well plates at a density of 1.5 × 105 cells/well. Subsequently, the cells were treated with the test agent at concentration of 1
MC3T3-E1 cells were cultured in 24-well plates for 24 h, followed by treatment with the test agent at concentrations of 1
Specific primers for RT-PCR analysis.
Gene | Forward | Reverse |
---|---|---|
COL-I | 5′-GAGCGGAGTACTGGATCG-3′ | 5′-GCTTCTTTTCCTTGGGGTT-3′ |
OPN | 5′-GATCAGGACAACAACGGAAAGG-3′ | 5′-GCTGGCTTTGGAACTTGCTT-3′ |
OCN | 5′-GAGGACCATCTTTCTGCTCACTCT-3′ | 5′-TTATTGCCCTCCTGCTTGGA-3′ |
Runx2 | 5′-GCACAAACATGGCCAGATTCA-3′ | 5′-AAGCCATGGTGCCCGTTAG-3′ |
5′-TCTGCTGGAAGGTGGACAGT-3′ | 5′-CCTCTATGCCAACACAGTGC-3′ |
MC3T3-E1 cells were incubated with different components from FCI in 6-well plates for Western blotting analysis. The soluble fraction of the cells was collected using RIPA lysis buffer (Thermo, DE), and the protein concentration was estimated as described above. 20
All the experiments were independently repeated at least thrice, and data were represented as mean ± SD. The results between different groups were compared using one-way analysis of variance (ANOVA).
The
Bioassay of different fractions from FCI for ovariectomized (OVX) mice. OVX mice were orally given 100 mg/kg
The two-phase solvent systems comprising various ratio volumes of
The
Solvent system | | |||
---|---|---|---|---|
Compound 1 | Compound 2 | Compound 3 | Compound 4 | |
(1) Chloroform-methanol-water (4 : 2 : 2) | 0.21 | 0.29 | 1.08 | 2.15 |
(2) Chloroform-methanol-water (4 : 3 : 3) | 0.25 | 0.31 | 1.11 | 2.06 |
(3) Chloroform-methanol-water (4 : 3 : 2) | 0.55 | 0.86 | 1.27 | 3.38 |
(4) | 0.62 | 1.13 | 2.29 | 9.05 |
Compound 1: acacetin; Compound 2: apigenin; Compound 3: luteolin; Compound 4: linarin.
Therefore, a stepwise HSCCC elution mode simultaneously separated the compounds with large
Similarly, a number of HSCCC separation experiments were performed for optimizing the operational parameters, including the column temperature, resolution speed, and the mobile phase flow rate for preparative separations. Initially, the optimal column temperature, revolution speed, and the solvent’s flow rate were 25°C, 850 rpm, and 5.0 mL/min, respectively. The sample size encountered an enormous limitation with the destruction of hydrodynamic equilibrium caused by the emulsification between the two phases. About 100 mL of the stationary phase was eluted upon 300 mg sample injection, leading to the decline in the retention rate of the stationary phase from 69.6% to 37.0%. Thus, the sample loading capacity was limited, and it reduced the peaks’ resolution on HSCCC. The addition of electrolytes was the appropriate strategy to enhance the coagulation of droplets. Ultimately, 0.5% saturated ammonium acetate, a volatile salt, of the total volume was added to eliminate emulsification to avoid the time-consuming desalting process and increase the sample loading capacity from 300 mg to 500 mg.
One-step HSCCC preparative separation of 500 mg crude sample resulted in 6.7 mg compound 1, 13.7 mg compound 2, 33.4 mg compound 3, and 32.8 mg compound 4 with the purities of 95.1%, 98.0%, 98.5%, and 98.6%, respectively, as revealed by HPLC analysis (Figure
HPLC chromatograms of ethyl acetate fraction from
1H and 13C NMR spectra determined the chemical structures of the peak fractions.
Compared with the data in the literature [
Chemical structures of the four flavonoids from
Skeleton is composed of several cell types that are continuously evolved for the integration of the structure in a global mineral and nutrient homeostasis [
Effect of the four compounds on cell proliferation of MC3T3-E1 cells. The cell viability was measured by CCK-8 assay. All the compounds significantly affected the cell proliferation after 48 h incubation. Data are represented as the means ± SD of three independent experiments.
ALP is a key osteoblastic phenotype marker contributing towards a high phosphate concentration for mineral deposition [
Effect of the four bioactive compounds on alkaline phosphatase (ALP) activity of MC3T3-E1 cells. The ALP activity was found to be significantly increased by the four bioactive compounds at a concentration of 1
The degree of mineralized matrix in cells is an important marker for the analysis of the osteogenic potential of the four compounds. The formation of calcified deposition was found to be significantly increased by the four bioactive compounds at a concentration of 1
Effect of the four compounds on mineralization of MC3T3-E1 cells. (a) After treatment with test agent at concentration of 1
Osteoblast differentiation is regulated by a large number of osteogenic differentiation molecular markers such as COL-I, OPN, and OCN. COL-I is a vital organic component in the extracellular matrix (ECM) of the bone that regulates the early osteoblast differentiation and consociates the cell surface integrins with other ECM proteins [
Effect of four compounds on osteogenesis-related gene and protein expression. (a) MC3T3-E1 cells were pretreated with 1
AKT signaling pathway is pivotal for the regulation of osteogenic differentiation and bone remolding [
In the present study, a competent fractionation method was developed for the first time for the identification and purification of bioactive compounds from the ethanol extract of
Ovariectomy
High-speed countercurrent chromatography
Cell counting kit-8
Distribution constants
Nuclear magnetic resonance
Alkaline phosphatase
Osteopontin
Osteocalcin
Type I collagen
Runt-related transcription factor 2
Bone mineral density
Bone volume/tissue volume
Trabecular space
Trabecular number.
The authors declare that they have no competing interests.
This work was supported by Natural Science Foundation of China (Grants nos. 81371949 and 11402175), Science and Technology commission of Shanghai, China (Grant no. 16411960900), China Postdoctoral Science Foundation (Grant no. 2016M591717), and the Shanghai Municipal Commission of Health and Family Planning (Grant no. 2014065).