Curculigo orchioides polysaccharides extraction, characterization, and their protective effects against femoral head necrosis

2.1 Extraction and purification of COPs

Curculigo orchioides rhizome obtained from Beijing Tong Ren Tang Chinese Medicine Co., Ltd. After washing and drying in the oven, the rhizomes were ground. Then, the polysaccharide was extracted by 4 kg of rhizomes ground in distilled water in a ratio of 1:10 (w:v, kg/L) at 90 °C for 4 h. The resulting solution was then boiled to increase the concentration at 60 °C for 4 h. For precipitation of COPs, the concentrated solution with 95% ethanol was concentrated to 50% and kept in an incubator at 23 °C for 24 h. The solution was re-concentrated to 70% anhydrous ethanol and kept in the incubator at 23 °C for 24 h. Finally, after the alcohol was determined by an alcohol meter, the precipitate was removed and dialyzed using the Sevag technique for 4 times.

For fractionation and purification of polysaccharide, 300 mg of the COPs was dissolved in 15 mL of distilled water and centrifuged at 8000g for 10 min. The supernatant (5 mL) was loaded onto the Diethylaminoethyl cellulose-52-cellulose column (Channel Size: 2 µm, Column Tube Length: 110 mm, Connectors: TC 1 in, Inner Diameter: 15 mm, Outer Diameter: 34 mm, and Recommended Operating Temperature: 4 to 40 °C.) after filtration. Then use distilled water as a mobile phase (distilled water) to move the solution through the column at a rate of 1.2 mL per min. After 5 h, the effluent was examined by phenol–sulfuric acid, and the washed portion was concentrated with sodium chloride and dialyzed again with distilled water. Then, the results were concentrated in a Sephadex G-75 gel filtration column at a flow rate of 18 mL/h with the help of purified distilled water, and finally lyophilized.

2.2 Molecular weight and chemical composition of COPs

High-performance liquid chromatography (HPLC) with TSK-G4000 PWXL column (7.8 × 300 mm) was used to assess the molecular weight of COPs. A 0.45 μm membrane was used to purify the COPs solution at a concentration of 1% (w/v). The mobile phase in the molecular weight examination was deionized water with a flow rate of 0.5 mL/min. Dextran T-series standards were applied for calibrating the columns.

Gas chromatography was applied to survey the composition of monosaccharides in COPs. For this purpose, 3 mg of COPs was hydrolyzed with 1.5 mL of trifluoroacetic acid (2 M) for 6 h at 120 °C. Afterwards, 100 μL of methanol was added to the solution to remove trifluoroacetic acid. Then, hydroxylamine hydrochloride (5 mg) and pyridine (0.35 mL) were added to the mixture and the mixture was incubated for 25 min at 95 °C. Next, acetic anhydride (0.5 mL) was added to the cold mixture and incubated again at 95 °C for 25 min. The sample was dried and then mixed with chloroform (0.5 mL). Finally, 0.2 μL of the final sample was used for gas chromatography-mass spectrometry analysis. 9 standard monosaccharide types were considered for graph analysis including rhamnose, arabinose, fructose, xylose, mannose, glucose, galactose, glucuronic acid, and galacturonic acid.

2.3 FTIR, methylation investigation and NMR

To explore the polysaccharide FTIR spectrum, 2 mg of COPs was assorted with 170 mg of ground dry KBr powder. The mixture was then entirely compacted in a 1 mm pellet. The FTIR spectra of samples were recognized by applying a Nicolet 5700 FTIR spectrometer in a wave number range of 400–4000 cm⁻¹ using the KBr way.

Methylation analysis was used to evaluate glycosidic bonds in COPs. For this purpose, based on the report of Pettolino et al. (2012), COPs was methylated. COPs’ methylation can be tracked by removing the O-H band in the range of 3200–3700 cm⁻¹. The 3 mg of methylated sample was then hydrolyzed with 1 mol per L trifluoroacetic acid (3 mL) for 2 h at 120 °C, reduced with 50 mg NaBH4, and acetylated with acetic anhydride-pyridine (1:1) for 1 h. Finally, it was analyzed by gas chromatography–mass spectrometry.

In order to evaluate the COPs by NMR method, 30 mg of the sample (for 5 days) was dried in vacuum. Then, the COPs was impregnated with 1 mL of D₂O and the sample was exchanged 3 times with D₂O in freeze drying mode. Afterwards, the COPs was dissolved in 0.5 mL of D₂O. Finally, the ¹H and ¹³C NMR spectra were recorded on a Bruker AV-500 spectrometer, respectively. Chemical changes were explained with acetone as an internal standard.

2.4 MTT assay

Human primary osteoblast (HPOS) cells were cultured at a density of 3 × 10⁴ cells per well in 96-well plate with DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin and then incubated for 12 h at 37 °C with 5% CO₂. Afterwards, the HPOS cells were incubated for 24 h at concentrations of 0.5, 1, 2 and 4 μM dexamethasone and concentrations of 50, 100, 150 and 200 μg/mL COPs, separately. To determine the effect of dexamethasone and COPs on HPOS cells, the old culture medium containing 1 μM dexamethasone was transformed to new medium containing 50, 100, 150 and 200 μg/mL COPs and incubated for 24 h at 37 °C with 5% CO₂. Subsequently, all media were replaced with PBS and 0.5 mg/mL MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was distributed in their medium. The samples were then incubated again for 4 h (50 μL per well). Then, the culture medium was drained from the wells and the crystals formed in each well were dissolved by 100 μL of dimethyl sulfoxide by shaking for 2 min. Ultimately, the cell absorbance at λ = 490 nm was calculated

2.5 Apoptosis and reactive oxygen species (ROS) assays

Flow-cytometry was used to estimate the level of COPs-induced apoptosis. After culturing HPOS cells (3 × 10⁵ cells per well) in a 6-well plate, the cells were treated with 1 µM of dexamethasone and 150 µg/mL of COPs and incubated for 24 h at 37 °C. The HPOS cells were then centrifuged at 3000g for 3 min at 4 °C. The samples were then washed with PBS. Afterwards, the HPOS cells were distributed in 100 μL of Annexin V binding buffer (HEPES buffer containing 0.1 M, NaCl 1.4 M, CaCl₂ 25 mm, pH 7.4). Next, 2 μL of Alexa Fluor 488 conjugated with Annexin V was augmented to the solution and saved in the dark at 21 °C for 15 min. Subsequently, 400 μL of the binding buffer and 5 μL of 50 μg/mL propidium iodide were added to the solution and saved in ice. Ultimately, samples were considered by FACscan (BD Bioscience).

In order to characterize the level of intracellular ROS at concentrations of 100 μg/mL COPs and 1 µM of dexamethasone, 3 × 10⁵ cells per well were cultured for 24 h in 6 well plates. The culture medium was replaced with phenol red free DMEM containing H2DCFDA (10 µM) 30 min. Then, cells were supplemented by GYY4137 (100 µM) for 30 min. Next being washed-out twice, cells were incubated with H₂O₂ (400 µM) for 2 h. The ROS generation was evaluated with a fluorescence reader (Safire2, Tecan Group Ltd.).

2.6 Lactate dehydrogenase release

Lactate dehydrogenase (LDH) release method was applied to evaluate Dexamethasone-induced cytotoxicity. HPOS were supplemented with dexamethasone (1 μM) and then incubated with concentrations of 50, 100, 150 and 200 μg/ml COPs for 24 h in an incubator maintained (at 37 °C with 5% CO₂). The cell culture was harvested and LDH release was examined in accordance with the manufacturer’s instructions (Beyotime, China).

2.7 Alkaline phosphatase (ALP) activity assay and collagen content

In order to investigate the ALP activity in the HPOS cells, the HPOS cells (3 × 10⁴ each well) were cultured into 6-well plates which were supplemented with different COPs concentrations (50, 100, 150 and 200 μg/ml) and 1 μM dexamethasone. After 3 days, the cells were washed and harvested, then the HPOS cells were lysed with 100 μL lysis buffer (Beyotime Institute of Biotechnology). An ALP reagent kit (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China) was applied to evaluate the ALP activity.

The culture medium of the HPOS cells treated with concentrations of 50, 100, 150 and 200 μg/ml COPs, and 1 μM of dexamethasone was removed after 72 h and the HPOS cells were washed twice with PBS. Then fixed with Bouin’s fluid for 1.30 h. The fixation fluid was then removed and all the HPOS cells were washed three times with distilled water. The well was stained with 120 μL of 0.1% Sirius red dye (weight/ volume) for 1.30 h at 37 °C with gentle shaking. The plates were washed again three times with 200 μL of 10 mM HCl solution. Then, 0.1 N NaOH was added to the plate to dissolve the collagen for 30 min. Ultimately, UV absorption was evaluated at 550 nm using an ELISA reader.

2.8 Mineralization

Staining density was used to evaluate the mineralization of osteoblast cells. In this regard, the amount of mineralization was determined using the Alizarin Red S method after 24 h of HPOS cell treatment with COPs and dexamethasone. After incubation of the cells according to section 3, the cells were collected and washed with a cold frozen PBS buffer. Then, the HPOS cells were fixed in 70% cold frozen ethanol at 4 °C for 30 min and stained with 0.1% Alizarin Red S-Tris-HCl solution with pH 8.3. After 20 min of staining, the dye medium was deleted and the plates were washed four times with distilled water. Ultimately, 10% (w/w) cetylpyridinium chloride solution was used to dissolve the remaining dye in the HPOS cells and was measured by UV absorption at 560 nm using an ELISA reader.

2.9 Analysis of BAX, Bcl-2, Caspase-3 and Caspase-9

The quantitative real-time PCR (qPCR) method was used to evaluate the expression level of BAX, Bcl-2, Caspase-3 and Caspase-9 genes. Trizol reagent was used to isolate the total RNA according to the manufacturer's protocol in the control and COPs at concentrations of 50, 100, 150 and 150 μg/mL. To remove the contaminant genome, RNA samples were treated with DNAse I, and a cDNA of 1 μg RNA was generated using revert Aid First Strand cDNA Synthesis Kit (Fermentas). The primers include of BAX: forward 5′-ATGGACGGGTCCGGGGAG-3′, reverse 5′-ATCCAGCCCAACAGCCGC-3′; Caspase-9: forward 5′-GTTTGAGGACCTTCGACCAGCT-3′, reverse 5′-CAACGTACCAGGAGCCACTCTT-3′; Bcl-2: forward 5′-ATCGCCCTGTGGATGACTGAGT-3′, reverse 5′-GCCAGGAGAAATCAAACAGAGGC-3′; Caspase-3: forward 5′-GGTATTGAGACAGACAGTGG-3′, reverse 5′-CATGGGATCTGTTTCTTTGC-3′; and β-actin: forward 5′-AATTCCATCATGAAGTGTGA-3′, reverse 5′-ACTCCTGCTTGCTGATCCAC-3′.

The qPCR was done by an ABI 7500 real-time PCR system (ABI, USA) using a SYBR Premix Ex Taq Reagent Kit (Takara) according to the manufacturers’ protocols. The β-actin amplification was used as a housekeeping gene.

2.10 Statistical analysis

The data were investigated with statistical analysis software SPSS 16.0 (SPSS Inc., Chicago, IL, US). Probabilities less than 0.05, 0.01 and 0.001 were considered significant, and statistical differences among groups were gained by analysis of variance (ANOVA) following Dunnett’s tests.

3.1 Isolation, molecular weight and composition of COPs

The extraction results show a 15.98% efficiency of the extraction method (water extraction, ethanol precipitation and deproteinization) in the production of COPs from the prototypes. Also, Fig. 1A exposes that the highest concentrations of COPs extracted in the tubes are 0.73, 0.45 and 0.35 mg/mL, respectively. The molecular weight of the COPs was determined to be approximately 4756 Da based on the standard dextran curve. Finally, Fig. 1B and 1C illustrated the presence of Rha, Ara, Fru, Xyl, Man, Glc, Gal, Glu A and Gal A in the extracted samples, of which the most abundant among monosaccharide were Man and Gal.

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Fig. 1

(A) The elution profile of the Curculigo orchioides polysaccharide (COPs) on a cellulose DEAE sepharose fast flow chromatography eluted with stepwise gradient of NaCl aqueous solutions (0, 0.1, 0.2 and 0.3 M) at a flow rate of 1.0 mL/min. GC–MS analysis of standard (B) monosaccharide (a: Rha, b: Ara, c: Fru, d: Xyl, e: Man, f: Glc, g: Gal, h: Glu A and i: Gal A) and (C) Curculigo orchioides polysaccharide. (D) Infrared Spectrum of Curculigo orchioides polysaccharide. (E) ¹H NMR spectrum of Curculigo orchioides polysaccharide. (F) ¹³C NMR spectrum of Curculigo orchioides polysaccharide.

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3.2 Characterization of COPs

Investigations of the properties of COPs extracted by FTIR in Fig. 1D show that the higher the tensile peak at 3350 cm⁻¹, which refers to the vibration of the O-H bond. While vibrations in the range of 2900 cm⁻¹ indicate the C-H band, in the range of 1700 cm⁻¹ and 1420 cm⁻¹ infer the C=O and the symmetric C-H bands, in the range of 1200 cm⁻¹ and 1060 cm⁻¹, respectively indicate the C-H band and pyranoside, and in the range of 850 cm⁻¹ represents the mannose pyranose ring. After COPs methylation, it was revealed that the O-H band was removed in the range of 3200–3700 cm⁻¹, indicating the success of the methylation process. The methylation outcomes, in order to determine the main and lateral structure of COPs according to retention time and ions fragments, demonstrates the presence of 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl-mannitol, 1,3,5,6-tetra-O-acetyl-2,4-di-O-methyl-mannitol, 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl-glucitol, and 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-glucitol in the COPs. These outcomes are consistent with the NMR consequences, which demonstrated the main residue linkage kinds of COPs were → 4)-D-Manp-(1→, →4)-D-Glcp-(1→, →3,6)-D-Manp-(1 → and D-Galp-(1 → ).

The results of Fig. 1E shows the greater accumulation of ¹H NMR and ¹³C NMR spectra in the region of 3.2–5.8 and 58–112 ppm, respectively. In addition, the presence of the O-acetyl group in COPs is confirmed based on carbon signals at 20 ppm and proton signals at 2.1 ppm. Analysis of ¹H NMR and ¹³C NMR spectra indicates the presence of seven peaks in confirmation of FTIR outcomes, which indicate the bands and chemical components of monosaccharides in the COPs structure. The provided signals demonstrate the presence of β-D-Manp (4.70 to 4.80 ppm and 60.5 to 61.1 ppm in the ¹H and ¹³C NMR spectrum, respectively), β-D-Galp (4.82 to 4.93 ppm and 99.54 to 100.43 ppm in the ¹H and ¹³C NMR spectrum, respectively), α-L-Rhap (3.94 to 4.16 ppm and 71.3 to 71.8 ppm in the ¹H and ¹³C NMR spectrum, respectively), β-D-GalpA (3.46 to 3.50 ppm and 73.4 to 74.7 ppm in the ¹H and ¹³C NMR spectrum, respectively), α-D-GlcpA (3.68 to 3.80 ppm and 76.4 to 77.1 ppm in the ¹H and ¹³C NMR spectrum, respectively), and α-L-Araf (5.12 to 5.20 ppm and 69.4 to 71.2 ppm in the ¹H ¹³C NMR spectrum, respectively) (Fig. 1F). Definitely, the chemical shifts of anomeric protons below 4.77 ppm and carbon resonance above 100.45 ppm display β-linked-anomeric of the Galp residues, which is in line with the methylation results.

3.3 Cell viability, apoptosis and ROS analysis

The results of Fig. 2A displays that the use of dexamethasone at concentrations of 0.5 to 4 μM significantly reduces the viability of HPOS cells compared to the control group. Moreover, concentrations of COPs 50, 100, and 150 μg/mL increased cell viability by 9.9%, 12.2%, and 11.4%, respectively, compared with control (Fig. 2B). Whereas the concentration of 200 μg/mL did not show a significant difference in the survival of HPOS cells compared to the control. Although increasing dexamethasone concentration decreases cell viability, concomitant use of dexamethasone with COPs significantly reduces the effect of dexamethasone on cell viability (Fig. 2C). The results illustrate that the use of 100 μg/mL COPs is appropriate to enhance cell viability in the presence of dexamethasone.

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Fig. 2

(A) Effects of dexamethasone on cell viability in vitro. (B) Effects of Curculigo orchioides polysaccharide (COPs) on cell viability in vitro. (C) Effect of COPs on cell viability pre-treated with dexamethasone in vitro. Two-dimensional contour density plots of osteoblasts cells determined by flow cytometry assay. (D) Control (Q1: 5.21%, Q2: 1.02%, Q3: 2.85% and Q4: 90.92%), (E) Dexamethasone (1 µM) (Q1: 15.11%, Q2: 22.60%, Q3: 29.02% and Q4: 32.81%), (F) Curculigo orchioides polysaccharide (150 µg/mL) + dexamethasone (1 µM) (Q1: 10.37%, Q2: 19.46%, Q3: 20.15% and Q4: 50.02%). Cell death measured using propidium iodide (PI) and Annexin V-FITC staining, (G) the effects of dexamethasone and Curculigo orchioides polysaccharide + dexamethasone on the ROS production. Statistical differences were measured at level of *P < 0.05, ^**P < 0.01 and ^***P < 0.001.

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As shown in Fig. 2D and 2E, the use of dexamethasone increases necrosis and apoptosis in HPOS cells significantly compared to the control group by increasing the level of necrosis (15.11% vs 5.21%), and apoptosis in Q2 (1.02% vs. 25.60%) or Q3 (2.85% vs. 32.02%). Conversely, the application of 150 μg/mL COPs simultaneously with dexamethasone reduced necrosis (10.37% vs. 15.11%) and apoptosis by 42 and 31% in Q2 (19.46%) and Q3 (20.15%), respectively (Fig. 2F). Based on the results of apoptosis, as expected, the use of COPs in the presence of dexamethasone significantly reduced intracellular ROS levels from 499 to 274 (Fig. 2G). Nonetheless, compared with the control group, the use of COPs in the presence of dexamethasone could not stop intracellular ROS levels optimally. Thus, the use of COPs can have an inhibitory effect on the generation of intracellular ROS and prevent the development of femoral necrosis activity.

3.4 LDH assay

The results of LDH in Fig. 3A shows that the use of dexamethasone increases LDH by 4.3 times compared to the control group. However, the results indicate a decrease in LDH levels in the use of COPs along with dexamethasone. This means that with increasing COPs levels, LDH concentration decreases significantly from 23.6% in the dexamethasone group to 22.1% (at a concentration of 50 µg/mL of COPs), 17.2% (at a concentration of 100 µg /mL of COPs), 12.3% (at a concentration of 150 µg /mL of COPs) and 10.7%. (at a concentration of 200 µg /mL of COPs). Despite the decrease in LDH level, LDH concentration is still higher than the control group in the presence of COPs and dexamethasone.

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Fig. 3

Effects of Curculigo orchioides polysaccharide on (A) lactate dehydrogenase (LDH), (B) alkaline phosphatase (ALP) activity, (C) collagen (COL: measured by a Sirius Red-based assay) and (D) mineralization of human primary osteoblasts cells in vitro. ^a,b,c,d,e Least square means with different letters in superscripts are different at *P < 0.05.

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3.5 Alkaline phosphatase (ALP) activity, collagen content and mineralization

Because ALP activity is a reliable marker for early detection of osteoblastogenesis, its evaluation is suitable for controlling femoral necrosis activity, and repairing the damaged area. As shown in Fig. 3B, ALP activity in dexamethasone-treated HPOS cells were significantly reduced compared with the control group. However, the use of COPs effectively reduces the negative effects of dexamethasone. Concentrations of 150 and 200 μg/mL of COPs will have the greatest effect on the suppression of femoral head necrosis by reducing the negative activity of dexamethasone. On the other hand, the results of the study of collagen content in HPOS cells revealed that the increase in collagen levels by COPs is similar to the increase in ALP activity (Fig. 3C).

As shown in Fig. 3D, intracellular calcium levels are reduced with the dexamethasone. Also, the use of COPs in comparison with the dexamethasone group effectively increases intracellular calcium levels. Among the different concentrations of COPs, the greatest effect is observed on concentrations of 150 µg /mL and 200 µg /mL.

3.6 Effects of COPs on genes expression

The expression of BAX (Fig. 4A), Bcl-2 (Fig. 4B), Caspase-3 (Fig. 4C), and Caspase-9 (Fig. 4D) were assessed by qPCR assay.

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Fig. 4

Effects of Curculigo orchioides polysaccharide on (A) BAX, (B) Bcl-2, (C) Caspase-3, and (D) Caspase-9 expression of human primary osteoblasts cells induced by dexamethasone (1 µM). Statistical differences were measured at level of *P < 0.05, ^**P < 0.01, and ^***P < 0.001.^a,b,c,d,e Least square means with different letters in superscripts are different at *P < 0.05.

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As shown in Fig. 4, despite a 1.39-fold reduction in Bcl-2, the production and expression of BAX, Caspase-9 and Caspase-3 genes increased 1.5-fold, 2.24-fold and 2.44-fold in the presence of dexamethasone, respectively. While, the application of COPs with different concentrations in the presence of dexamethasone significantly reduced the expression of BAX, Caspase-9 and Caspase-3 genes. Also, the use of COPs significantly improved the expression of the Bcl-2 gene. Despite a significant decrease in the expression of BAX, Caspase-9 and Caspase-3 genes at a concentration of 100 μg/mL COPs, the results show that the most effective decrease in the expression of BAX and Caspase-9 genes is related to a concentration of 150 μg/mL COPs with 40.9% and 29.7% reduction compared to dexamethasone, respectively. While the greatest decrease in the expression Caspase-3 gene refers to the concentration of 200 μg/mL of COPs with 58.1% reduction compared to dexamethasone. On the other hand, the use of tetrad with a concentration of 150 μg/mL causes a 2.46-fold and 1.66-fold increase in Bcl-2 compared to the dexamethasone and the control groups. Since increasing Bcl-2 can reduce the level of apoptosis in HPOS cells, increasing Bcl-2 will increase the hope of bone tissue regeneration. Overall, the inhibitory effect of polysaccharides on Caspase-3 gene was greater than that of BAX and Caspase-9, whereas, similar to MTT results, the best concentration of COPs to reduce apoptosis-inducing genes is 150 μg/mL.