Structural characterization and osteoprotective effects of a novel oligo-glucomannan obtained from the rhizome of Cibotium barometz by alkali extraction

Industrial Crops & Products 113 (2018) 202–209

Contents lists available at ScienceDirect

Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop

Structural characterization and osteoprotective effects of a novel oligoglucomannan obtained from the rhizome of Cibotium barometz by alkali extraction Dong Huanga,b,c, Mengliu Zhanga, Pan Yia, Chunyan Yana,

T

⁎

a

School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006, PR China Key Laboratory of Digital Quality Evaluation of Chinese Materia Medica of State Administration of TCM, PR China c Engineering & Technology Research Center for Chinese Materia Medica Quality of the Universities of Guangdong Province, PR China b

A R T I C L E I N F O

A B S T R A C T

Keywords: Cibotium barometz Oligosaccharide Structural characterization Anti-osteoporosis Osteoblasts

Cibotium barometz is mainly distributed in eastern, southern, and southwest China as an important industrial export crop of great economical and medicinal value. The rhizome of C. barometz is widely used in Traditional Chinese Medicine clinics to treat conditions such as lumbago, limb-ache, rheumatism, and sciatica. In this study, the results of in vivo pharmacological experiments conclusively demonstrated that crude saccharides from C. barometz (CBB) exhibited osteoprotective effects in ovariectomized rats, which significantly increased bone mineral content (BMC) and bone mineral density (BMD), and prevented damage of the trabecular bone, consequently improving its biomechanical properties. To investigate the biological active ingredient(s), a novel oligo-glucomannan (denoted CBBP-1) was isolated and purified from CBB via anion-exchange and size-exclusion chromatography. Structural analysis indicated that CBBP-1 consisted of (1 → 4)-linked α-D-glucose, (1 → 6)linked β-D-glucose with (1 → 3, 6)-linked α-D-mannose, and a terminal α-D-glucose. Morphological analysis revealed that CBBP-1 had an irregular sheet structure. Furthermore, osteoblastic MC3T3-E1 cells treated with CBBP-1 had significantly increased mRNA expression of runt-related transcription factor 2, osterix, osteopontin, osteocalcin, and bone sialoprotein, indicating that CBBP-1 may stimulate osteoblastic differentiation. In conclusion, this study provides evidence that CBBP-1 may have potential as an anti-osteoporosis agent in the pharmaceutical industry.

1. Introduction Fractures resulting from osteoporosis are a major public health problem and are increasing in prevalence worldwide due to the rapidly aging population. Osteoporosis, characterized by low bone mass and microarchitectural deterioration, is a chronic disease that increases the brittleness of bone and its susceptibility to fracture (Fu et al., 2014). Bisphosphonate (BPH), calcitonin, selective androgen receptor, and hormone replacement therapy (HRT) are the most common and efficacious strategies for reducing the risk of osteoporosis (Thu et al., 2017). However, most of these agents have multiple side effects; for example, the long-term use of BPH has been linked to serious complications, such as osteonecrosis of the jaw and atypical femur fractures (Moro Álvarez et al., 2016). In addition, long-term HRT substantially increases the risk of endometrial cancer and other adverse events, such as thromboembolism and vaginal bleeding (Davison and Davis, 2003; Wiseman, 2004). Thus, the prolonged use of these agents is limited.

⁎

Consequently, there is a need for natural herbal medicines with relatively few side effects that can be used as alternative therapies for the treatment of osteoporosis. Cibotium barometz (Linn.) J. Sm. (Dicksoniaceae family), known as “Gou-Ji” in Chinese, is mainly distributed in eastern, southern, and southwest China. The total planting area is more than 7000 ha, and its annual production is estimated to be greater than 850 tons, making it one of the most important industrial export plants in China (Yang et al., 2015). As an ornamental plant, C. barometz has unique beauty. Due to its large economical and medicinal value, studies have been conducted on techniques for its artificial cultivation (Pu et al., 2015; Praptosuwiryo et al., 2015). According to the theory of Traditional Chinese medicine (TCM), C. barometz can strengthen bones and muscles, and its rhizomes are widely used in TCM clinics to treat conditions such as lumbago, limbache, rheumatism, and sciatica (Wu and Yang, 2009). Daily oral administration of C. barometz extract was shown to significantly contribute to the prevention or treatment of bone loss induced by ovariectomy

Corresponding author at: School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006, PR China. E-mail addresses: [email protected], [email protected] (C. Yan).

https://doi.org/10.1016/j.indcrop.2018.01.034 Received 14 August 2017; Received in revised form 21 December 2017; Accepted 14 January 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.

Industrial Crops & Products 113 (2018) 202–209

D. Huang et al.

position after anesthesia, and blood was obtained from the abdominal aorta. After the rats were sacrificed, the heart, liver, spleen, kidney, brain, and uterus of each rat were weighed and used to calculate the coefficient of the organ and uterus. The femurs were obtained and kept in 70% alcohol at −20 °C for the measurements of bone mineral content (BMC), bone mineral density (BMD), and biomechanical properties. All of the procedures for animal care and use were in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee of Guangdong Pharmaceutical University (License No: SYXK [Yue] 2017-0125).

(OVX) in rats (Zhao et al., 2011). To investigate the biological active ingredient(s) of C. barometz, a large number of small molecule compounds were isolated, including caffeic acid, protocatechuic acid, palmitic acid and 1-monopalmitin (Cheng et al., 2003), β-sitosterol, daucosterol, alternariol, protocatechuic aldehyde, and (24R)-stigmast-4-ene3-one (Wu et al., 2007). However, their effects on osteoporosis are unknown. Consequently, elucidation of its exact anti-osteoporosis active ingredient(s) would be essential for its use in future therapeutic applications and the pharmaceutical industry, which in turn could lead to the development of phytomedicines with industrial feasibility. There is a general consensus that saccharides (e.g., polysaccharides and oligasaccharides) have various biological activities, but little is known about the saccharide-containing fractions of C. barometz. Therefore, the aims of this study were to systematically evaluate the effects of CBB in OVX rats, to isolate and purify CBB to obtain the CBBP1 oligosaccharide, to determine the detailed structure of this novel oligosaccharide, and to evaluate its effects on the expression levels of five transcription factors, runt-related transcription factor 2 (Runx2), osterix (Osx), osteocalcin (Ocn), osteopontin (Opn), and bone sialoprotein (Bsp), in MC3T3-E1 cells during osteogenic differentiation.

2.4. BMC and BMD analyses The BMC of the entire left femur, distal left femur (2 cm), and proximal left femur (1 cm) was measured by dual-energy X-ray absorptiometry (WI 85003, Hologic Discovery, Marlborough, MA, USA) according to a previously reported method (Li et al., 2011). BMD was determined using the BMC of the measured area, and all values were automatically calculated using a software package (Encore 2006, GE Healthcare, Madison, WI, USA).

2. Materials and methods 2.5. Three-point bending test 2.1. Materials After the femora thawed at room temperature for 5 h, each left femur was placed at a distance of two points on a 20-mm fixing device, after which the femoral diaphysis was loaded to the breaking point using a MTS Mini Bionix Tabletop Test System (MTS 858, MTS Systems, Eden Prairie, NJ, USA) at a speed of 6 mm/min. All force and displacement data were recorded for later evaluation. Calculations of biomechanical parameters were based on previously published formulas (Zhang et al., 2009).

C. barometz was purchased from the Tong Ren Tang medicinal store and identified by Dr. Hongyan Ma of Guangdong Pharmaceutical University (Guangzhou, China). Cellulose DEAE-52 and Sephadex G-75 were purchased from GE Healthcare (Chicago, IL, USA). A total of 32 3month-old female Sprague–Dawley rats were purchased from the Chinese Medicine Animal Experimentation Center of Guangzhou University (Certificate: SCXK2013-0020). All of the other chemicals and reagents were of analytical grade.

2.6. Micro-computed tomography analysis 2.2. Extraction and purification of crude saccharide The left femur of rats was scanned using the Explore Locus SP PreClinical Specimen Micro-Computed Tomography (MicroCT) Scanner (GE Healthcare) to estimate the effects of CBB on trabecular microarchitecture. The distal part of the femur, which is richer in trabecular bone than the proximal and middle regions, was scanned from the proximal growth plate at an isotropic voxel resolution of 22 μm3. The volume of interest, which included the relative bone volume (BV/TV), trabecula number (Tb.N), trabecula separation (Tb.Sp), trabecula thickness (Tb.Th), connectivity density (Conn.D), and structure model index (SMI), were chosen for subsequent architectural parameter analyses (Cai and Zhang, 2016). All specimens were scanned in the dark.

The rhizome of C. barometz (15.0 kg) was ripped into chunks, soaked overnight in deionized water 1: 10 (w/v), and extracted at 80 °C for 3 h; this procedure was repeated three times. The residues of C. barometz were extracted with sodium hydroxide ([NaOH] 0.3 mol/L) 1:10 (w/v) at room temperature for 3 h, repeated twice, and pooled. Then the extracts were filtered, neutralized with hydrochloric acid ([HCl] 0.3 mol/L), concentrated (up to 1/30 initial volume), and centrifuged. Next, 95% ethanol was added to the supernatants at a final concentration of 70%, and they were incubated for 24 h at room temperature to obtain crude saccharides, which were deproteinized with Sevag reagent (1-butanol/chloroform, v/v = 1:4). Finally, the supernatants were dialyzed and lyophilized.

2.7. Isolation and purification of the CBBP-1 saccharide CBB has shown significant osteoprotective effects in vivo in pharmacological studies. Therefore, it was selected for further isolation and purification by dissolution in deionized water and centrifugation, after which the supernatants were separated and purified on a DEAE-52 cellulose chromatography column (Ø 2.6 × 40 cm). A constant NaCl gradient from 0 to 1 M (400 mL each step) was used as the elution solvent, and the eluents were determined with the phenol-sulfuric acid colorimetric method. Eluents with 0 M NaCl were collected, concentrated, and lyophilized to obtain the CBB-1 saccharide. CBB-1 was further purified using a Sephadex G-75 gel permeation chromatography column (Ø 1.6 × 100 cm) and eluted with distilled water at a flow rate of 0.3 mL/min to obtain the novel purified saccharide denoted CBBP-1.

2.3. Animals and treatments The experiments were completed in a specific pathogen-free animal laboratory at a relatively constant temperature of 25 ± 2 °C, 12 h light and 12 h dark cycles, and relative humidity of 40%–50%. Female Sprague–Dawley rats (235 ± 15 g) were adjusted to laboratory conditions for 1 week. Water and food were freely available during the experimental period. After anesthesia by intraperitoneal injection of pentobarbital sodium, rats in the ovariectomized group (OVX, n = 21) were treated with bilateral ovariectomy, and those in the sham-operated group (sham, n = 7) were only given laparotomy to cut off fat but not the ovaries. After a 1-week recovery from surgery, the OVX rats were randomly divided into three sub-groups: OVX group, 17β-estradiol group (25 μg/kg E2 body weight/day), and CBB group (400 mg/kg) (Liu et al., 2009). Distilled water, E2, and CBB were all administered orally for 12 weeks, and the rats were weighed every week to adjust the doses of E2 and CBB. After 12 weeks, the rats were secured in the supine

2.8. Homogeneity and average molecular weight determination of CBBP-1 CBBP-1 was analyzed using an ultraviolet-visible spectrophotometer to detect the presence of nucleic acids and proteins. High-performance 203

Industrial Crops & Products 113 (2018) 202–209

D. Huang et al.

NMR, respectively. Correlated spectroscopy (1H–1H COSY), heteronuclear multiple-quantum correlation spectroscopy (HSQC), and heteronuclear multiple bond correlation (HMBC) were performed using standard Bruker software.

gel permeation chromatography (HPGPC) was used to identify the homogeneous distribution of CBBP-1 and to calculate its relative molecular weight (Hua et al., 2014). The columns, eluted with 0.02 M monopotassium phosphate at a flow rate of 0.6 mL/min, were calibrated with a series of Dextran T-series standards of different molecular weights (Dextran T1000, T500, T70, T40, T10, and T5) at 35 °C. Then, the relative molecular weight of CBBP-1 was calculated from the calibration curve.

2.14. Morphological analysis The micro-morphological characteristics of CBBP-1 were determined using a scanning electron microscope (JSM-7001, Japan Electron Optic Laboratory Co. Ltd., Tokyo, Japan). The dried powder of CBBP-1 was distributed on the specimen stage of the SEM with doublesided adhesive tape and then sprayed with gold powder. The sample was observed under the SEM at 500-, 1000-, 1500- and 3000-x magnifications under a high vacuum.

2.9. Fourier transform-infrared spectroscopy analysis CBBP-1 (1 mg) and potassium bromide (150 mg) were mixed, milled to powder, dried in a vacuum oven, and pressed into a pellet. The infrared spectrum was recorded using a Perkin-Elmer FT-IR Spectrometer in the infrared region.

2.15. Cell culture and treatment 2.10. Monosaccharide identification and quantification of CBBP-1 Complete α-MEM (Cat. No. 11095-080; Gibco) containing 10% FBS, 100 U/mL penicillin G, and 100 mg/mL of streptomycin was used. The MC3T3-E1 cells were seeded onto 24-well culture plates at a density of 25,000 cells/mL, and maintained in a 5% CO2 humidified incubator at 37 °C for 72 h. Then the cells were induced to differentiate in osteogenic medium supplemented with complete α-MEM, 10 mM β-glycerophosphate, and 50 μg/mL ascorbic acid. The control group was induced to differentiate in osteogenic medium; positive control cells were treated with E2 (0.1 μM) and the CBBP-1 group was treated with different concentrations of CBBP-1 (34.6 μM, 69.2 μM, 138.4 μM). The medium was changed every 3 days.

To identify and quantify the monosaccharide composition of CBBP1, the PMP-pre-column HPLC method was adopted (Huang et al., 2015). Briefly, CBBP-1 (5 mg) was dissolved in 2 mL of 3 M trifluoroacetic acid (TFA) and hydrolyzed in a sealed tube at 120 °C in an oil bath for 6 h. Methanol was added to the hydrolysates to remove excess TFA, and this process was repeated numerous times. Then, a mixture of hydrolysates (100 μL), 0.3 M NaOH (100 μL), and a 0.5-M methanolic solution of PMP (100 μL) were allowed to react for 30 min at 70 °C and neutralized with 0.3 M HCl (105 μL). The aqueous solution of derivatization was extracted with equivalent volumes of chloroform and repeated three times until the excess PMP had been mostly removed. The sample was passed through an Agilent ZORBAX Eclipse XDB-C18 column (5 μm, Ø 4.6 × 250 mm) at 25 °C and eluted with a mixture of 0.05 M phosphate buffer solution (pH 6.7) and acetonitrile (83:17, v/v) at a flow rate of 1 mL/min.

2.16. Quantitative real-time PCR The extraction of total RNA was performed on days 6, 8, 10, and 12 from MC3T3-E1 cell cultures using the RNA Extraction Kit (Cat. No. APMN-RNA-250; Axygen), according to the manufacturer’s instructions. RNA concentration was analyzed with an ultraviolet-visible spectrophotometer. The mRNA (600 ng) was reverse transcribed with the RT Kit (Cat. No. RR036A; Takara, Tokyo, Japan) according to the manufacturer’s instructions to obtain 15 μL cDNA, and stored at −40 °C. Then 1 μL cDNA was amplified with quantitative PCR (qPCR) using SYBR Premix Ex Taq™ II (Takara). mRNA expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and determined using the △△Ct method (sense and antisense primers used for PCR are listed in Table S1).

2.11. Determination of the absolute configuration of monosaccharide from CBBP-1 The absolute configuration of the monosaccharide was determined according to previous studies (Patra et al., 2012). Briefly, the hydrolysates of CBBP-1 (1 mg) were allowed to react with a 0.625 M R-(−)-2butanol solution of HCl (300 μL) at 80 °C for 12 h in a sealed tube. After evaporation, the residue was dried for 12 h in a vacuum over P2O5 and then treated with a mixture of bis (trimethylsilyl) trifluoroacetamide (BSTFA) and DMF (300 μL, 1:1) for 30 min at 75 °C. Acetone (2 mL) was added to the residue, which was then filtered through a 0.45-μm pore membrane filter for gas chromatographic analysis using an Agilent 6820 GC System (Agilent, Santa Clara, CA, USA). Similarly, the standards were treated and analyzed accordingly.

2.17. Statistical analysis All data from these experiments are expressed as the mean ± standard error of the mean (SEM) for each group, and analyzes were performed with SPSS statistical analysis software (Chicago, IL, USA). Differences among the OVX, sham, CBB, and E2 groups were analyzed using one-way analysis of variance following Dunnett’s tests. In all of the analyses, P values less than 0.05 were considered statistically significant.

2.12. Methylation analysis of CBBP-1 Methylation of CBBP-1 was performed according to the method of Needs and Selvendran (Wang et al., 2015) with slight revisions. The methylated products were hydrolyzed and reduced with sodium borohydride, followed by acetylation with an equivalent volume of acetic anhydride-pyridine for 1 h at 95 °C. The final samples of methylated alditol acetates were analyzed by gas chromatography-mass spectrometry (GC–MS-QP 2010, Shimadzu, Kyoto, Japan).

3. Results and discussion 3.1. Body and organ weights Previous studies have shown that body weight increase in OVX rats as a mechanism to provide an additional stimulus for bone neoformation (Notomi et al., 2003). In addition, fat deposition caused by estrogen deficiency can also lead to weight gain (Hertrampf et al., 2008). The initial body weights of the rats in the four groups were similar and slowly increased in the first 4 weeks (Fig. S1A). From the tenth week until the end of the experiment, the body weight of the OVX group was much higher that of the sham group (P < 0.01). However, E2 and CBB

2.13. Nuclear magnetic spectroscopy analyses of CBBP-1 CBBP-1 (50 mg) was dissolved in D2O (700 μL) using an ultrasonic method and centrifuged at 10,000 rpm for 10 min. Then the supernatant was filtered through a 0.8-μm membrane filter and analyzed on a Bruker AV-500 spectrometer (Bremen, Germany) operating at 500 MHz and 125 MHz for proton nuclear magnetic resonance (1H NMR) and 13C 204

Industrial Crops & Products 113 (2018) 202–209

D. Huang et al.

treatment significantly inhibited body weight increase compared to the OVX group (P < 0.05 or 0.01). The uterus index of the OVX group was significantly lower than that of the sham group (P < 0.01), whereas E2 and CBB significantly increased the uterus index compared to the OVX group (P < 0.01 or 0.05) (Fig. S1B). These results show that CBB significantly inhibited the body weight gain and atrophy of uterine tissue in the OVX rats, both of which are induced by estrogen deficiency. Furthermore, there were no significant differences in the index of heart, liver, spleen, kidney, and brain between the CBB and OVX groups after 12 weeks of treatments, suggesting that treatment with CBB on a prolonged basis causes almost no side effects or toxicity. 3.2. BMC and BMD of the femur The reduction of bone mass is one of the key factors that endanger the integrity of the bone, causing increased bone brittleness and a high risk of bone fracture. Bone mass was evaluated by measuring the BMD in a previous study (Li et al., 2014). The BMC and BMD of the left femur are shown in Fig. 1A and 1B, respectively. Compared with the sham group, the BMC and BMD in the OVX group were markedly decreased (P < 0.01). CBB significantly increased the BMC of the whole femur, distal femur, and proximal femur by 10.89%, 13.68%, and 17.84%, respectively, compared to the OVX group, and E2 increased it by 8.21%, 12.96%, and 16.85%, respectively (P < 0.05 or 0.01). Compared with the OVX group, the BMD of the whole femur, distal femur, and proximal femur in the CBB group was significantly increased by 7.93%, 9.28%, and 8.86%, respectively (P < 0.05), whereas there were no significant differences in BMD between the OVX and E2 groups. These results indicate that treatment with CBB for 12 weeks significantly increased the BMC and BMD in rats with OVX-induced osteoporosis. 3.3. Biomechanical quality of the femur Fig. 1. Effects of CBB on the bone mineral content (BMC) (A) and bone mineral density (BMD) (B) in the left femur of OVX rats, as evaluated by dual-energy X-ray absorptiometry. ##P < 0.01 vs. Sham group; *P < 0.05, **P < 0.01 vs. OVX group.

The biomechanical quality of the femur, which includes structural biomechanical parameters and material biomechanical parameters, was obtained to determine the actual effect of the samples on biomechanical competence (Ederveen et al., 2001). Bone strength is connected to bone density, architecture, connectivity, and mineralization (Einhorn, 1992). The three-point bending test was adopted to evaluate the biomechanical properties of bone in this paper (Cheng et al., 2011). The results are presented in Table 1. OVX resulted in a significant decrease in the maximum load, fracture load, maximum stress, fracture stress, fracture strain, Young’s modulus, and energy (P < 0.05 or 0.01) compared to the sham group, indicating that the model of OVX-induced osteoporotic rat was successfully developed. Compared with the OVX group, treatment with E2 significantly increased the maximum load, fracture load, maximum stress, fracture strain, energy, and stiffness (P < 0.05 or 0.01). CBB increased the maximum load, maximum stroke, fracture load, fracture stroke, maximum stress, fracture stress, fracture strain, Young’s modulus, and energy by 15.05%, 24.53%, 13.49%, 26.72%,

24.62%, 21.59%, 17.93%, 19.17%, and 51.82% respectively, compared with the OVX group (P < 0.05 or 0.01). These results show that treatment with CBB can dramatically improve the femoral diaphysis biomechanical competence of rats with OVX-induced osteoporosis, which is consistent with the overall results of BMC and BMD in this animal model.

3.4. MicroCT evaluation The geometry properties, material properties, BMC, and BMD of the femur were obtained to evaluate bone fragility from the macro perspective, but Micro-CT analysis of the bone was conducted to provide

Table 1 Effects of CBB on bone biomechanical parameters in the femoral diaphysis of ovariectomized (OVX) rats. Parameters Maximum load (N) Maximum stroke Fracture load (N) Fracture stroke (mm) Maximum stress Fracture stress (MPa) Fracture strain (%) Youngs’ modulus Energy (N × mm) Stiffness (N/mm)

OVX

Sham ##

201. 63 ± 4.95 0.54 ± 0.027 188.61 ± 6.32 0.66 ± 0.038 213.86 ± 8.31 206.51 ± 8.62 16. 79 ± 0.29 10790 ± 877 11.720 ± 0.35 658.27 ± 24.60

179.28 ± 4.27 0.53 ± 0.019 171.95 ± 3.70# 0.58 ± 0.021 176.49 ± 5.58## 169.21 ± 4.89## 13.72 ± 0.48## 8092 ± 128# 7.670 ± 0.49## 607.74 ± 25.99

Values are mean ± SEM, n = 7 in each group. #P < 0.05 vs. Sham group.

##

CBB 206.26 ± 2.51 0.66 ± 0.040** 195.14 ± 2.85** 0.735 ± 0.025** 201.11 ± 9.78* 205.74 ± 8.69** 16.18 ± 0.56* 9643 ± 540* 11.645 ± 1.21* 645.75 ± 12.85

P < 0.01 vs. Sham group. *P < 0.05 vs. OVX group.

205

E2 **

**

P < 0.01 vs. OVX group.

207.79 ± 3.29** 0.56 ± 0.024 191.68 ± 6.79* 0.68 ± 0.040 193.77 ± 3.02* 183.45 ± 5.63 16.80 ± 0.58** 8402 ± 171 10.594 ± 0.62* 681.42 ± 11.51*

Industrial Crops & Products 113 (2018) 202–209

D. Huang et al.

Table 2 Effects of CBB on bone parameters, as measured by MicroCT at the distal femur region of OVX rats. Parameters

OVX

Sham

CBB

E2

BV/TV(%) Conn.D.(1·mm−3) SMI Tb.N(1·mm−1) Tb.Th(mm) Tb.Sp(mm)

0.11 ± 0.03## 26.31 ± 9.02## 2.19 ± 0.31## 1.45 ± 0.21## 0.08 ± 0.01# 0.84 ± 0.23##

0.28 ± 0.08 81.99 ± 31.42 1.22 ± 0.62 4.23 ± 1.07 0.09 ± 0.01 0.25 ± 0.07

0.19 ± 0.03** 38.75 ± 6.16* 1.63 ± 0.14** 1.86 ± 0.22** 0.10 ± 0.00** 0.59 ± 0.07*

0.14 ± 0.11 57.61 ± 52.57 2.65 ± 0.80 3.23 ± 1.11* 0.07 ± 0.01 0.45 ± 0.27*

Values are mean ± SEM, n = 6 in each group. #P < 0.05 vs. Sham group.

##

P < 0.01 vs. Sham group. *P < 0.05 vs. OVX group.

**

P < 0.01 vs. OVX group.

Fig. 2. Three dimensional architecture of trabecular bone within the distal metaphyseal femur region.

Fig. 3. The predicted structure of CBBP-1.

structures from rod to plate form. The representative samples are shown in Fig. 2.

more information on trabecular bone architecture from a micro perspective (Genant et al., 2007). Measuring such microarchitectural parameters, including the BV/TV, SMI, Tb.Th and Tb.Sp, with a MicroCT scanner may improve our ability to estimate bone strength (Laib et al., 2001; Kazakia and Majumdar, 2006). In addition, SMI is an estimation of the plate-rod characteristics of the structure. SMI 0 and 3 represent bone that consists purely of plate- or rod-like structures (Zhang et al., 2009). The results of our analysis are shown in Table 2. Compared with the sham group, the OVX group had significantly lower BV/TV, Conn.D, Tb.N, and Tb.Th values, and higher SMI and Tb.Sp values by the end of experiment (P < 0.05 or 0.01), indicating that the OVX group had fewer, thinner, and more broken trabecula, and that the trabecular bone structures changed from plate to rod. These data confirmed that the OVX-induced osteoporosis rat model was successfully established. Compared to the OVX group, Tb.N and Tb.Th of CBB were significantly increased by 28.27%, 25.00%, while Tb.Sp and SMI of CBB were significantly decreased by 29.76%, 25.57%. Furthermore, there were no significant differences between the CBB and sham groups with regard to the Tb.Th and SMI values. Thus, CBB not only prevent damage to the trabecula, but it significantly increased the Tb.N, decreased the Tb.Sp, and even restored trabecular thickness, returning the trabecular

3.5. Isolation, purification, homogeneity, and molecular weight of CBBP-1 The crude saccharide, denoted as CBB, was extracted from the water extract residues of C. barometz with 0.3 M NaOH solution, and its yield was approximately 8.25%. After ethanol precipitation, deproteinization and dialysis, CBB was isolated and purified by using DEAE-Cellulose 52 and G-75 gel filtration chromatogram to obtain CBBP-1. The yield of CBBP-1 was about 0.425%. The CBBP-1 was confirmed as having no nucleic acids and protein by UV spectrometry. A single narrow peak was shown in HPGPC implying that CBBP-1 was a homogeneous saccharide, and the molecular weight of CBBP-1 was found to be 1445 Da. Thus, CBBP-1 was estimated to a degree of polymerization (DP) of 9. 3.6. Chemical analysis of CBBP-1 In the analysis of monosaccharide composition, the standard monosaccharides were tagged with PMP residue, followed by HPLC analysis. Similarly, CBBP-1 was hydrolyzed into individual 206

Industrial Crops & Products 113 (2018) 202–209

D. Huang et al.

Fig. 4. SEM images of CBBP-1 (A: 500×, B: 1000×,C: 1500×, D: 5000×).

analyzed with the 1H–1H COSY spectrum (Fig. S3C), there are four anomeric signals in the 1H–1H COSY spectrum, which correlated to the four anomeric carbon signals in HSQC spectra of CBBP-1 (Fig. S3D). This result confirms that there are four sugar residues in CBBP-1, which was consistent with the results of GC–MS analysis. The four anomeric signals of sugar residues were labeled A, B, D, and E in spectra. The α/β configuration of each sugar residue was judged according to the chemical shift of the anomeric H. Then it could be easily concluded that residues A, B, D, E were α, α, β, α configuration, respectively. The above result is in accord with previous reports. According to the data from the methylation analysis, (1 → 4) linked glucose was the most abundant residue. The downfield shifts of C-4 (76.7 ppm) and according to the previously reported data, implying that residue A was expected to be the (1 → 4) linked glucose, which was confirmed in the HMBC spectra of CBBP-1 (Fig. S3E) (Huang et al., 2015). Accordingly, combining the information from the 1H, 13C, 2D NMR techniques (1H–1H COSY, HSQC, and HMBC spectra), methylation analysis data and previous literature data, a complete description of residues A, B, D, and E was proposed in Table S3 (Mondal et al., 2006; Pei et al., 2015; Li et al., 2013; Yi et al., 2015). Finally, the HMBC spectrum of CBBP-1 was analyzed for the linkage sites and sequence among the sugar residues. That a cross peak was observed between H-4 (3.59 ppm) of residue A and C-1 (99.6 ppm) of residue A (AC1/AH4) suggested that C-1 of residue A was linked to O-4 of residue A, while the cross peak at 76.7/5.34 ppm (AC4/AH1) indicated that C-4 of residue A was linked to O-1 of residue A. The combination of above results and the methylation analysis result which (1 → 4) linked glucose account for 48.96% of the total sugar residues can be easily obtained the existence of repetitive unit of (1 → 4) linked glucose. Similarly, the cross peaks at 71.1/5.34 ppm (DC6/AH1), 75.5/ 4.58 ppm (EC3/DH1), 99.7/3.59 ppm (BC1/AH4), 99.7/5.16 ppm (BC1/EH1) and 71.7/5.31 ppm (EC6/BH1) suggested that C-6 of residue D was linked to O-1 of residue A, C-3 of residue E to O-1 of residue D, C-1 of residue B to O-4 of residue A, C-1 of residue B to O-1 of residue E and C-6 of residue E to O-1 of residue B. Finally, combined with relative molecular weight, monosaccharides analysis, IR, methylation analysis and NMR data, CBBP-1 was identified a novel oligo-glucomannan (Liu et al., 2015). Its structure was shown in Fig. 3. Up to now, the structure of CBBP-1 has not been reported in the literature.

monosaccharides and tagged with PMP residue for HPLC analysis. The results revealed that CBBP-1 was composed of glucose and mannose (Fig. S2A, B). According to the monosaccharide composition, the analysis of its absolute configuration of only needs D/L-configurations of glucose and mannose as standards. Compared with the standards, GC analysis revealed that the mannose and glucose of CBBP-1 were all in the D-configuration. As shown in the IR spectrum of CBBP-1 (Fig. S2C), a broad and intense band at 3390.82 cm−1 and a band at 2925.97 cm−1 of CBBP-1 corresponded to hydroxyl stretching vibrations and CeH stretching vibrations, respectively. A weak band appeared at 1637.92 cm−1 was due to the presence of bending vibration of eOH. The absorption peak at 1026.97 cm−1, 1077.84 cm−1, and 1151.99 cm−1 was confirmed to be a pyranose (Zhu et al., 2011). Two signals appearing at 934.30 cm−1 and 763.07 cm−1 were the results of asymmetrical stretching vibration and symmetrical stretching vibration of pyranose, respectively (Zhang et al., 2016). Analysis of the IR spectrum of CBBP-1 implied that only pyranose was present in the oligasaccharide. Furthermore, to obtain information on the linkage sites of monosaccharide and the corresponding percentage, methylation analysis of CBBP-1 was conducted. GC–MS results showed four methylated alditol acetates of sugar residues, including 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl-D-glucitol, 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-D-glucitol, 1,3,5,6tetra-O-acetyl-2,4-di-O-methyl-D-mannitol and 1,5,6-tri-O-acetyl-2,3,4tri-O-methyl-D-glucitol. Combining the molecular weight and relative molar ratio of the sugar residues, the CBBP-1 was composed of (1 → 4), (1 → ), (1 → 6) linked glucose, and (1 → 3,6) linked mannose at a ratio of 4: 3: 1: 1 (see Table S2).

3.7. NMR spectroscopy analysis of CBBP-1 NMR spectroscopy plays a paramount important role in the structural analysis of saccharides. The 1H NMR spectrum of CBBP-1 (Fig. S3A) exhibited three peaks in the anomeric region: an overlapping peaks signal appeared at about 5.3 ppm, a signal at 5.16 ppm and a signal appeared at 4.58 ppm. In the 13C NMR spectrum of CBBP-1 (Fig. S3B), four anomeric carbon signals appeared at 99.7 ppm, 99.6 ppm, 95.7 ppm, and 91.8 ppm. The anomeric H of CBBP-1 was further 207

Industrial Crops & Products 113 (2018) 202–209

D. Huang et al.

3.8. Morphological analysis The micro-morphological characteristics of CBBP-1 were analyzed by SEM at magnifications of 500, 1000, 1500 and 5000-fold (Fig. 4A-D). The results showed that CBBP-1 exhibited an irregular sheet. In addition, the SEM image of CBBP-1 at magnification of 5000-fold showed that the surface of irregular sheet has globular process which may be formed by branched structures of the oligo-glucomannan. 3.9. qPCR analysis Runx2 and Osx are key transcription factors in initiating and regulating osteoblast differentiation, and regulating the expression of bone-related genes such as Opn, Bsp, and Ocn (Chiu et al., 2010). Meanwhile, Ocn, Opn, and Bsp genes are essential for terminal osteoblast differentiation and bone mineralization (Yang et al., 2013). The effects of CBBP-1 on the expression levels of the studied transcription factors (Runx2, Osx, Ocn, Opn, Bsp) in MC3T3-E1 cells during osteogenic differentiation were quantified. As shown in Fig. 5A, MC3T3-E1 cells treated with CBBP-1 showed a significant dose-dependent increase in the mRNA expression of Runx2 (P < 0.05 or 0.01) compared with both the control group and the E2 group after 8 days. On day 12, Runx2 expression with treatment of 69.2 μM and 138.4 μM CBBP-1 significantly increased to 201.74% and 445.37%, respectively, compared to the E2 group (P < 0.01). Osx mRNA expression was also significantly upregulated in CBBP-1-treated cultures compared to both the control and E2 groups on day 8 (P < 0.01) (Fig. 5B). These data suggested that CBBP-1 induced the high expression of Osx and Runx2 during osteogenic differentiation. As shown in Fig. 5C–E, the expression levels of Ocn and Bsp in CBBP-1 groups were significantly upregulated compared to the control group from days 8–12 (P < 0.05 or 0.01). Furthermore, Bsp expression with 69.2 μM CBBP-1 significantly increased to 333.67%, 217.85%, and 178.08% compared to the E2 group (P < 0.05 or 0.01) on days 8, 10 and 12, respectively. The aforementioned results suggested that CBBP-1 induced the high expression of Ocn and Bsp during late osteogenic differentiation. Meantime, compared with the control group, Opn expression in the CBBP-1 group was statistically upregulated on day 6 (P < 0.01 or P < 0.05). In conclusion, MC3T3-E1 cells treated with CBBP-1, especially at concentrations of 69.2 μM and 138.4 μM, led to a significant increase in the expression of osteogenic differentiation-related genes during osteogenic differentiation, which demonstrates that CBBP-1 may promote osteoblastic differentiation. Thus, CBBP-1 may be an active ingredient that is responsible for the osteoprotective effects of CBB. 4. Conclusions Based on TCM theory, the oral administration of the C. barometz decoction is widely used to ameliorate lumbago, limb-ache, sciatica as well as other arthralgias. However, in this study, the water extraction residues of C. barometz were extracted with 0.3 M sodium hydroxide solution to obtain crude saccharides (CBB). First, the osteoprotective effects of CBB were systematically evaluated in ovariectomized rats, and the results showed that the daily oral administration of CBB for 12 weeks could significantly increase the BMC and BMD of the whole femur by 10.89% and 7.93%, respectively, compared to the OVX group, and prevent bone loss in OVX rats. According to micro-CT analysis, treatment with CBB prevented the deterioration of trabecular bone microarchitecture in OVX rats by increasing the Tb.N, decreasing the Tb.Sp, and restoring the Tb.Th. The maximum load, maximum stroke, fracture load, fracture stroke, maximum stress, fracture stress and fracture strain of the CBB group also increased by 15.05%, 24.53%, 13.49%, 26.72%, 24.62%, 21.59%, and 17.93%, respectively compared to the OVX group, indicating that CBB improved biomechanical quality. Taken together, these results provide strong evidence that the oral administration of CBB can treat or prevent osteoporosis. In addition,

Fig. 5. Effects of CBBP-1 on the expression levels of Runx2 (A), Osx (B), Ocn (C), Opn (D) and Bsp (E) after 6, 8, 10, and 12 days of MC3T3-E1 cell culture during osteogenic differentiation. *P < 0.05, **P < 0.01 vs. control group; #P < 0.05, ##P < 0.01 vs. E2 group.

208

Industrial Crops & Products 113 (2018) 202–209

D. Huang et al.

there were no significant differences in the indexes of the heart, liver, spleen, kidney and brain between the CBB and OVX groups, implying that prolonged treatment with CBB had no obvious side effects or toxicity. To determine the active components responsible for the effects of CBB, a novel oligo-glucomannan (CBBP-1) with a molecular weight of 1445 Da (DP = 9) was obtained using a DEAE-52 cellulose chromatography column and Sephadex G-75 gel filtration. Structural analysis showed that the backbone of CBBP-1 consisted of (1 → 4)-linked α-Dglucose, (1 → 6)-linked β-D-glucose with (1 → 3,6)-linked α-D-mannose, and a terminal α-D-glucose. More importantly, MC3T3-E1 cells treated with CBBP-1 showed a significant increase in the mRNA expression of Runx2, Osx, Ocn, Opn, and Bsp, suggesting that CBBP-1 may promote osteoblastic differentiation to play a role in anti-osteoporosis. Thus, CBBP-1 was a good potential anti-osteoporotic agent for use in the pharmaceutical industry. The structure-activity relationship of CBBP-1 requires further elucidation.

osteoporosis. Rev. Endocr. Metab. Dis. 7, 67–74. Laib, A., Kumer, J.L., Majumdar, S., Lane, N.E., 2001. The temporal changes of trabecular architecture in ovariectomized rats assessed by MicroCT. Osteoporos. Int. 12, 936–941. Li, X., Song, Q.-S., Wang, J.-Y., Leng, H.-J., Chen, Z.-Q., Liu, Z.-J., Dang, G.-T., Song, C.-L., 2011. Simvastatin induces estrogen receptor-alpha expression in bone, restores bone loss, and decreases ERα expression and uterine wet weight in ovariectomized rats. J. Bone Miner. Metab. 29, 396–403. Li, N., Yan, C., Hua, D., Zhang, D., 2013. Isolation, purification, and structural characterization of a novel polysaccharide from Ganoderma capense. Int. J. Biol. Macromol. 57, 285–290. Li, T., Wu, S.-M., Xu, Z.-Y., Ou-Yang, S., 2014. Rabbiteye blueberry prevents osteoporosis in ovariectomized rats. J. Orthop. Surg. Res. 9, 56–63. Liu, Z.-G., Zhang, R., Li, C., Ma, X., Liu, L., Wang, J.-P., Mei, Q.-B., 2009. The osteoprotective effect of Radix Dipsaci extract in ovariectomized rats. J. Ethnopharmacol. 123, 74–81. Liu, J., Xu, Q., Zhang, J., Zhou, X., Lyu, F., Zhao, P., Ding, Y., 2015. Preparation, composition analysis and antioxidant activities of konjac oligo-glucomannan. Carbohydr. Polym. 130, 398–404. Mondal, S., Chakraborty, I., Rout, D., Islam, S.S., 2006. Isolation and structural elucidation of a water-soluble polysaccharide (PS-I) of a wild edible mushroom, Termitomyces striatus. Carbohydr. Res. 341, 878–886. Moro Álvarez, M.J., Neyro, J.L., Castañeda, S., 2016. Therapeutic holidays in osteoporosis: long-term strategy of treatment with bisphosphonates. Med. Clin. (Barc) 146, 24–29. Notomi, T., Okimoto, N., Okazaki, Y., Nakamura, T., Suzuki, M., 2003. Tower climbing exercise started 3 months after ovariectomy recovers bone strength of the femur and lumbar vertebrae in aged osteopenic rats. J. Bone Miner. Res. 18, 140–149. Patra, P., Das, D., Behera, B., Maiti, T.K., Islam, S.S., 2012. Structure elucidation of an immunoenhancing pectic polysaccharide isolated from aqueous extract of pods of green bean (Phaseolus vulgaris L.). Carbohydr. Polym. 87, 2169–2175. Pei, J.-J., Wang, Z.-B., Ma, H.-L., Yan, J.-K., 2015. Structural features and antitumor activity of a novel polysaccharide from alkaline extract of Phellinus linteus mycelia. Carbohydr. Polym. 115, 472–477. Praptosuwiryo, T.N., Pribadi, D.O., Rugayah, 2015. Growth, development and morphology of gametophytes of golden chicken fern (Cibotium barometz (L.) J. Sm.) in natural media. Biodiversitas 16, 303–330. Pu, L., Huang, H., Fu, Q., 2015. Study on the influencing factors of Cibotium barometz spore germination. Seed 34, 84–86. Thu, H.E., Mohamed, I.N., Hussain, Z., Shuid, A.N., 2017. Eurycoma longifolia as a potential alternative to testosterone for the treatment of osteoporosis: exploring timemannered proliferative, differentiative and morphogenic modulation in osteoblasts. J. Ethnopharmacol. 195, 143–158. Wang, C., Hua, D., Yan, C., 2015. Structural characterization and antioxidant activities of a novel fructan from Achyranthes bidentata Blume, a famous medicinal plant in China. Ind. Crop. Prod. 70, 427–434. Wiseman, R.A., 2004. Breast cancer: critical data analysis concludes that estrogens are not the cause, however lifestyle changes can alter risk rapidly. J. Clin. Epidemiol. 57, 766–772. Wu, Q., Yang, X.-W., 2009. The constituents of Cibotium barometz and their permeability in the human Caco-2 monolayer cell model. J. Ethnopharmacol. 125, 417–422. Wu, Q., Yang, X.-W., Yang, S.-H., Zou, L., Yan, J., 2007. Chemical constituents of Cibotium barometz. Nat. Prod. Res. Dev. 19, 240–243. Yang, D., Okamura, H., Nakashima, Y., Haneji, T., 2013. Histone demethylase Jmjd3 regulates osteoblast differentiation via transcription factors Runx2 and Osterix. J. Biol. Chem. 288, 33530–33541. Yang, C.-Z., Liu, X.-F., Cai, D.-L., Fan, S.-M., 2015. Investigation on resource and quality assessment of Cibotii Rhizoma. Chin. J. Chin. Mater. Med. 40, 1919–1924. Yi, P., Li, N., Wan, J.-B., Zhang, D., Li, M., Yan, C., 2015. Structural characterization and antioxidant activity of a heteropolysaccharide from Ganoderma capense. Carbohydr. Polym. 121, 183–189. Zhang, R., Liu, Z.G., Li, C., Hu, S.J., Liu, L., Wang, J.P., Mei, Q.B., 2009. Du-Zhong (Eucommia ulmoides Oliv.) cortex extract prevent OVX-induced osteoporosis in rats. Bone 45, 553–559. Zhang, Z., Kong, F., Ni, H., Mo, Z., Wan, J.-B., Hua, D., Yan, C., 2016. Structural characterization, α-glucosidase inhibitory and DPPH• scavenging activities of polysaccharides from guava. Carbohydr. Polym. 144, 106–114. Zhao, X., Wu, Z.-X., Zhang, Y., Yan, Y.-B., He, Q., Cao, P.-C., Lei, W., 2011. Anti-osteoporosis activity of Cibotium barometz extract on ovariectomy-induced bone loss in rats. J. Ethnopharmacol. 137, 1083–1088. Zhu, Z.-Y., Liu, R.-Q., Si, C.-L., Zhou, F., Wang, Y.-X., Ding, L.-N., Jing, C., Liu, A.-J., Zhang, Y.-M., 2011. Structural analysis and anti-tumor activity comparison of polysaccharides from Astragalus. Carbohydr. Polym. 85, 895–902.

Acknowledgments This study was funded by the National Natural Science Foundation of China (Nos. 81673557 and U1703110) and the Science and Technology Program of Guangdong Province (Nos. 2014A050503067 and 2015A020211032). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.indcrop.2018.01.034. References Cai, Y.-L., Zhang, X.-C., 2016. Protective effect of Rhizoma drynariae extract on osteoporosis in ovariectomized rat model. Trop. J. Pharm. Res. 15, 1447–1452. Cheng, Q.-H., Yang, Z.-L., Hu, Y.-M., 2003. Studies on the chemical constituents of Cibotium barometz. Prog. Pharm. Sci. 27, 298–299. Cheng, M., Wang, Q., Fan, Y., Liu, X., Wang, L., Xie, R., Ho, C.C., Sun, W., 2011. A traditional Chinese herbal preparation, Er-Zhi-Wan, prevent ovariectomy-induced osteoporosis in rats. J. Ethnopharmacol. 138, 279–285. Chiu, R., Smith, K.E., Ma, G.K., Ma, T., Smith, R.L., Goodman, S.B., 2010. Polymethylmethacrylate particles impair osteoprogenitor viability and expression of osteogenic transcription factors Runx2, Osterix, and Dlx5. J. Orthop. Res. 28, 571–577. Davison, S., Davis, S.R., 2003. Hormone replacement therapy: current controversies. Clin. Endocrinol. 58, 249–261. Ederveen, A.G.H., Spanjers, C.P.M., Quaijtaal, J.H.M., Kloosterboer, H.J., 2001. Effect of 16 months of treatment with tibolone on bone mass, turnover, and biomechanical quality in mature ovariectomized rats. J. Bone. Miner. Res. 16, 1674–1681. Einhorn, T.A., 1992. Bone strength: the bottom line. Calcified Tissue Int. 51, 333–339. Fu, S.-W., Zeng, G.-F., Zong, S.-H., Zhang, Z.-Y., Zou, B., Fang, Y., Lu, L., Xiao, D.-Q., 2014. Systematic review and meta-analysis of the bone protective effect of phytoestrogens on osteoporosis in ovariectomized rats. Nutr. Res. 34, 467–477. Genant, H.K., Delmas, P.D., Chen, P., Jiang, Y., Eriksen, E.F., Dalsky, G.P., Marcus, R., Martin, J.S., 2007. Severity of vertebral fracture reflects deterioration of bone microarchitecture. Osteoporosis. Int. 18, 69–76. Hertrampf, T., Schleipen, B., Velders, M., Laudenbach, U., Fritzemeier, K.H., Diel, P., 2008. Estrogen receptor subtype-specific effects on markers of bone homeostasis. Mol. Cell. Endocrinol. 291, 104–108. Hua, D., Zhang, D., Huang, B., Yi, P., Yan, C., 2014. Structural characterization and DPPH radical scavenging activity of a polysaccharide from guava fruits. Carbohydr. Polym. 103, 143–147. Huang, Y., Li, N., Wan, J.-B., Zhang, D., Yan, C., 2015. Structural characterization and antioxidant activity of a novel heteropolysaccharide from the submerged fermentation mycelia of Ganoderma capense. Carbohydr. Polym. 134, 752–760. Kazakia, G.J., Majumdar, S., 2006. New imaging technologies in the diagnosis of

209