Structural elucidation and anti-osteoporosis activities of polysaccharides obtained from Curculigo orchioides

Accepted Manuscript Title: Structural elucidation and anti-osteoporosis activities of polysaccharides obtained from Curculigo orchioides Authors: Xueqian Wang, Mengliu Zhang, Dawei Zhang, Xinluan Wang, Huijuan Cao, Qian Zhang, Chunyan Yan PII: DOI: Reference:

S0144-8617(18)31133-0 https://doi.org/10.1016/j.carbpol.2018.09.059 CARP 14102

To appear in: Received date: Revised date: Accepted date:

2-7-2018 2-9-2018 21-9-2018

Please cite this article as: Wang X, Zhang M, Zhang D, Wang X, Cao H, Zhang Q, Yan C, Structural elucidation and anti-osteoporosis activities of polysaccharides obtained from Curculigo orchioides, Carbohydrate Polymers (2018), https://doi.org/10.1016/j.carbpol.2018.09.059 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Structural elucidation and anti-osteoporosis activities of

polysaccharides

obtained

from

Curculigo

orchioides

a

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Huijuan Cao c, Qian Zhang**a, Chunyan Yan a,*

School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006,

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China

Department of Pharmacology, Guangdong Medical University, Dongguan,

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b

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Guangdong 523808, China

Translational Medicine R&D Center, Institute of Biomedical and Health Engineering,

M

c

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Xueqian Wang a, Mengliu Zhang a, Dawei Zhang b, Xinluan Wang c,

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Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen,

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China

* Corresponding author. Tel./fax: +86 20 39352052. E-mail addresses: [email protected] (C. Yan) 1

Highlights



The osteogenic activity of Curculigo orchioides polysaccharide (CO70) was evaluated. CO70 showed excellent anti-osteoporosis activity in ovariectomized rats.



A homogeneous heteropolysaccharide COP70-3 was isolated and purified

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

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from CO70.

The repetitive structural unit of COP70-3 was inferred for the first time.



COP70-3 obviously enhanced the differentiation and mineralization of

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

ABSTRACT

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MC3T3-E1 cells.

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Curculigo orchioides, is a traditional Chinese medicine, is used in strengthening

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tendons and bones. We evaluated the anti-osteoporosis activity of the crude polysaccharide (CO70) isolated from the rhizomes of C. orchioides in ovariectomized rats. CO70 showed excellent anti-osteoporosis activity comparable to that of

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17β-estradiol. To explore the constituents responsible for the anti-osteoporosis activity of CO70, a novel homogeneous heteropolysaccharide, COP70-3, was isolated and purified from CO70. COP70-3 has a main backbone chain of (1→5)-linked α-L-Araf, (1,3→5)-linked α-L-Araf, (1→6)-linked β-D-Galp, (1→4)-linked β-D-Manp, (1,2→ 2

5)-linked α-L-Araf, (1 → 3)-linked β-L-Rhap, (1, 3 → 6)-linked β-D-Manp, (1 → 3)-linked α-D-GalpA, (1,3→6)-linked β-D-Galp and (1→6)-linked α-D-Glcp residues. Furthermore, 1.87 nM COP70-3 obviously promoted the differentiation of MC3T3-E1 cells, while 0.94 and 1.87 nM COP70-3 significantly improved the osteogenic

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mineralization rate. These data indicate that COP70-3 has favorable anti-osteoporosis

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activity in vitro.

Keywords: Curculigo orchioides Gaertn; Polysaccharide; Ovariectomized rats;

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Anti-osteoporosis; Structural characterization

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1. Introduction

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Osteoporosis (OP) is a systemic metabolic disease characterized by disruption of

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the balance between bone resorption and bone formation, leading to low bone mass and high risk of fractures (Rodan & Martin, 2000). Elderly and postmenopausal

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women are a high-risk group for primary osteoporosis. Usually, according to the

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difference in functional mechanism, the clinical drugs of osteoporosis prevention and treatment can be divided into those that promote the mineralization of bone (calcium, vitamin D, etc.), stimulate the formation of bone (fluoride, parathyroid hormone, etc.),

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and restrain the absorption of bone (estrogen, bisphosphonates, etc.). These drugs have their own limitations, and long-term usage will lead to various side effects and complications. The exploration of natural products with less undesirable side effects would be the most helpful option in the search for new therapy for the treatment and 3

prevention of osteoporosis (Li et al., 2013). Natural polysaccharides that have some very desirable pharmacological activities have been investigated. For example, polysaccharides from Lycium barbarum leaves significantly improved the proliferation of splenocytes to show immunostimulating

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activity (Liu et al., 2012); guava polysaccharides may be beneficial for treating type 2 diabetes (Jiao, Zhang, Wang, & Yan, 2017); Lycium chinensis polysaccharides

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exhibited remarkable antitumor activity in cancer of the liver in rats (Cui et al., 2012);

and polysaccharide GCPB-1b isolated from Boshuzhi exhibited radical-scavenging

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properties (Jiang et al., 2016). The anti-osteoporosis activities of polysaccharides

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extracted from botanical sources have been reported in recent years, such as

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dioscoreae polysaccharides, tamarind polysaccharide, and oligosaccharide from

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Achyranthes bidentata (Huang, Liang, Li, & Hong, 2011; Sanyasi, Kumar, Goswami,

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Bandyopadhyay, & Goswami, 2014; Wang et al., 2017a). Dioscoreae polysaccharides inhibited bone degeneration, tamarind polysaccharide can be used to induce bone cell

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differentiation, and oligosaccharide ABW90-1 from A. bidentata can stimulate the

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differentiation of primary osteoblasts. Two saccharides (MOP70-1 and MOP70-2) obtained from the roots of Morinda officinalis as anti-osteoporosis agents significantly promoted the proliferation, differentiation and mineralization of

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MC3T3-E1 cells (Jiang et al., 2018). Two novel heteropolysaccharides (CBBP-2 and CBBP-3) obtained from water extraction residues of Cibotium barometz had osteogenic activities (Huang et al., 2018). A. bidentata, dioscoreae, M. officinalis and C. barometz are famous for their invigoration of livers and kidneys as well as muscle 4

and bone strengthening in China. The structures of these polysaccharides are very different in monosaccharide composition, sugar residues number and molecular weight, but we could conclude that the natural polysaccharides from traditional Chinese medicine, which have the function of invigorating livers and kidneys, as well

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as strengthening muscles and bones, may have the potential for anti-osteoporosis effects.

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C. orchioides is mainly distributed in Southwest and Southern China, Japan, and

Southeast Asian countries. In China, C. orchioides (Xian Mao) is a well-known

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traditional Chinese medicine (TCM) that has a long history and is widely used for the

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treatment of arthritis of the lumbar spine and knee joints, weakness, and other

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diseases (He et al., 2015). As a medicinal plant, C. orchioides is of great interest due

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to the pharmaceutical industry’s dependence on it for the production of the secondary

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compound, curculigoside. At present, medicinal C. orchioides is mainly derived from wild and artificial cultivation, and it has a huge economic value and market in the

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pharmaceutical and healthcare industry. However, because of the increasing demand

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and low survival rate of traditional cultivation of C. orchioides, recent studies have highlighted more efficient and convenient breeding methods for C. orchioides, such as

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plant tissue culture and artificial seed technology (Zhang et al., 2017). C. orchioides is an important ingredient of empirical TCM formulas for the

therapy of osteoporosis, such as Jian Gu Ke Li, Er Xian Decoction, and Gu Song Kang Jiao Nang (Zhang et al., 2016). Curculigoside, a phenolic glycoside of C. orchioides, was reported to have anti-osteoporotic activity. The metabolites of 5

curculigoside in rat were elucidated and identified, and an anti-osteoporosis active metabolite was inferred (Wang et al., 2017b). Polysaccharide, as one of the most important components of C. orchioides, may be effective for the prevention and treatment of osteoporosis; however, there are currently no detailed reports exploring

those by our group (Wang, Zhang, Zhang, Wang, & Yan, 2017).

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that or the structural characterization of C. orchioides polysaccharides, except for

(CO70)

was

investigated

in

ovariectomized

rats.

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In this study, the anti-osteoporosis activity of C. orchioides polysaccharides A novel

homogeneous

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polysaccharide (COP70-3) was isolated from CO70, its detailed structure was

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determined by the method of monosaccharide composition analysis, Fourier

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transform-infrared (FT-IR) analysis, gas chromatography-mass spectrometry (GC-MS)

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analysis and nuclear magnetic resonance (NMR) analysis, and its micromorphology

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was elucidated with a scanning electron microscope (SEM). Simultaneously, the effect of COP70-3 on differentiation and mineralization of murine pre-osteoblastic

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MC3T3-E1 cells was studied.

2. Materials and methods

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2.1. Materials and chemicals C. orchioides was obtained from Beijing Tong Ren Tang Chinese Medicine Co.,

Ltd. The voucher specimen was identified by Dr. Hongyan Ma, Guangdong Pharmaceutical University, China. 17β-estradiol (E2), standard monosaccharides, and trifluoroacetic acid (TFA) were supplied by Aladdin Industrial Co. (Shanghai, China). 6

Acetonitrile (HPLC grade) was purchased from Oceanpak Alexative Co., Ltd. (Sweden). MC3T3-E1 cells were supplied by the American Type Culture Collection (USA). Alizarin red S and BCA Protein Concentration Assay Kit were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). The alkaline phosphatase (ALP) assay kit

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was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). RIPA lysate solution was obtained from HEART Biological Technology Co., Ltd.

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2.2. Preparation of polysaccharide from C. orchioides

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(China). All other chemicals and solvents were analytical grade.

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The total polysaccharides were extracted from rhizomes of C. orchioides (40.0 kg)

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by distilled water 1:10 (w/v, kg/L) at 95°C for 3 h, and this operation was repeated

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three times. All extracts were concentrated at 60°C, and then the concentrated liquids

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were precipitated by adding 95% ethanol to a concentration of 50%, and incubating for 24 h at room temperature. Then, the supernatants were concentrated and

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precipitated by adding anhydrous ethanol to a concentration of 70%, and incubating

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once more for 24 h at room temperature (Hua, Zhang, Huang, Yi, & Yan, 2014). The ethanol concentration after adjustment was measured by an alcoholmeter. The precipitated matter was deproteinated by the Sevag method (Sevag, Lackman, &

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Smolens, 1938), dialyzed, and then freeze-dried to obtain C. orchioides polysaccharide CO70.

2.3. Sugar content detection of CO70 7

Sugar content of CO70 was detected based on previously reported methods (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956; Jiang, Kuang, Kong, & Yan, 2016). The absorbance of a CO70 solution (0.1 mg/mL) was detected using a phenol-sulfuric acid colorimetric method and measuring at 490 nm with an ultraviolet-visible

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spectrophotometer (UV-2550, Shimadzu, Japan). Glucose was used as the standard sample, and the absorbance of various concentrations was determined with the same

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method as that used to obtain the standard curve. The sugar content of CO70 was

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2.4. Monosaccharide component analysis of CO70

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calculated according to the standard curve (Fig S7).

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Monosaccharide composition analysis can provide evidence for the main

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component of the polysaccharide. The monosaccharide composition of CO70 was

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analyzed by the HPLC method (Yan et al., 2016). The CO70 sample (5 mg) was sealed and hydrolyzed in ampoules with 2.5 M TFA (2 mL) at 120°C for 6 h,

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concentrated up to dryness with absolute methanol to remove redundant TFA, and

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redissolved with 1 mL distilled water. Then, the sample and a mixture of monosaccharides as the standard (mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose) were derivatized with a

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methanolic solution of 1-phenyl-3-methyl-5-pyrazolone (PMP), and then analyzed with an Agilent 1260 HPLC system (Agilent, USA) that was equipped with a ZORBAX Eclipse XDB C-18 column and an ultraviolet detector. NaH2PO4-Na2HPO4 buffer solution (pH 6.7) and acetonitrile (83:17, V/V) were used as the mobile phase 8

(flow rate: 1 mL/min), and the detection wavelength was 250 nm.

2.5. Animal experiments Forty-eight 12-week-old virgin female specific-pathogen-free (SPF) SD rats

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weighing 265 ± 15 g were purchased from Guangzhou University of Chinese Medicine Animal Experimentation Center (Certificate: SCXK20130034). The rats

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were housed in a temperature- and humidity-controlled specific pathogen-free (SPF)

animal laboratory in Guangdong Pharmaceutical University and allowed free access

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to water and diet. The rats were randomly divided into two groups: one was the

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sham-operated group (non-ovariectomized untreated rats, Sham, n=12), and the other

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was the model group (n=36).

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After adaptive feeding for one week, the rats of the Sham group underwent

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bilateral laparotomy (n=12), and the model group underwent bilateral ovariectomy (n=36). On day 2 after surgery, the ovariectomized untreated rats were randomly

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divided into three additional groups: OVX (OVX rats treated with distilled water,

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OVX), positive control group (OVX rats treated with 17β-estradiol, E2), and CO70-administered group (OVX rats treated with CO70, CO70) (Cui et al., 2004). The rats were treated as follows: Sham group and OVX group: intragastric

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administration with distilled water (5 mL/kg body weight/day); E2 group: intragastric administration with E2 (25 μg/kg body weight/day); and CO70 group: intragastric administration with C. orchioides polysaccharide CO70 (400 mg/kg body weight/day) (Zeng et al., 2011). All rats were treated according to the schedule for 13 weeks. Rats 9

were weighed to regulate the CO70 and E2 doses, and the changes in body weight were recorded every week. Metabolism cages were used to collect urine over 24 hours for all rats at week 12; the urine was centrifuged (604 × g, 5 min) and stored at -80°C. After treatment, all rats were euthanized, and arterial blood was collected, centrifuged

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(604 × g, 5 min) and the serum was stored at -80°C. The uterus was removed and immediately weighed. The left femur of each rat was dissected free of muscle,

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wrapped with physiological saline-soaked gauze, and stored at -80°C.

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2.6. Bone mineral density and bone mineral content measurement

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Bone mineral density (BMD) and bone mineral content (BMC) of entire left femur,

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distal femur (2 cm), and proximal femur (1 cm) were determined in all rats using

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dual-energy X-ray absorptiometry (DXA, WI 85003, Hologic Discovery).

2.7. Bone biomechanical property measurement

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To assess the bone biomechanical property of the femora, a 3-point bending test

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was performed (Sakai et al., 2015). Before testing, the left femora of each group were removed from the -80°C freezer and thawed for 12 h at room temperature. The 3-point bending test of the left femora was performed with a bone strength tester

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(MTS 858 Mini Bionix, USA). The midpoint of the femur was placed on a holding device, and two supports were located 20 mm apart. The bending force was calculated at a speed of 6 mm/min until fractures occurred. According to the load versus displacement curve, the biomechanical parameters of the femora were obtained. 10

2.8. Microcomputed tomography (micro-CT) scanning The intact distal femoral specimens fractured in the 3-point bending test were examined using a desktop preclinical specimen micro-CT scanner (μCT-40, Scanco

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Medical, Bassersdorf, Switzerland) according to a previously reported method (Lin et al., 2016). The region of interest (ROI) of 3-mm thick femora containing only

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trabecular bone extracted from the cortical bone was acquired 1.0 mm from the growth plate. Three dimensional (3D) reconstructions of the ROI of the femora were

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performed, and the 3D reconstructed images were directly used to quantify the

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analysis program of the micro-CT workstation.

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microarchitecture. The morphometric parameters were calculated using the image

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2.9. Isolation and purification of CO70

Briefly, CO70, which was loaded onto a DEAE-52 cellulose column (Ø 2.5 × 45

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cm), was isolated with 0.00 M, 0.05 M, and then 0.15 M sodium chloride eluant.

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Fractions eluted with 0 M sodium chloride were lyophilized and named CO70-1. Fractions eluted with 0.05 M and 0.15 M sodium chloride were dialyzed against distilled water (MW cut-off of 100 Da), lyophilized, and named CO70-2 and CO70-3,

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respectively. The amounts of CO70-1 and CO70-2 were too low for further purification. CO70-3 was purified on a Sephadex G-75 gel filtration column (Ø 1.5 × 100 cm) using distilled water as the eluent. The eluate was sequentially collected according to the elution peak, concentrated, and then lyophilized to obtain COP70-3. 11

2.10. Homogeneity and relative molecular weight analysis The presence of protein and nucleic acids in COP70-3 was determined using an ultraviolet (UV) spectrophotometer with a wavelength coverage of 200–400 nm. The

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determination of homogeneity and molecular weight of COP70-3 was carried out by

already described (Huang, Li, Wan, Zhang, & Yan, 2015).

Monosaccharide

component

analysis

and

Fourier

transform-infrared

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2.11.

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high performance gel permeation chromatography (HPGPC) according to the method

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spectroscopy assessment

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The method of monosaccharide component analysis of COP70-3 was the same as

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that described in section 2.4 in this study. A PerkinElmer Fourier transform-infrared

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(FT-IR) spectrometer was used to analyze the structural characteristics of COP70-3 in

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the range of 4000–400 cm-1.

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2.12. Methylation analysis

According to the method reported by Pettolino, Walsh, Fincher, & Bacic (2012),

the COP70-3 sample (7 mg) dehydrated with anhydrous methanol was dissolved

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completely in DMSO (10 mL) in a round-bottom flask (25 mL). Then, the DMSO/NaOH slurry (10 mL/60 mg) was added to the flask, reacted in an ultrasonic reactor for 30 min, and then 15 mL CH3I (three times, each time 5 mL, 10 min interval time) was added and reacted for 30 min while the solution in the ultrasonic 12

reactor was protected from light. Disappearance of the O-H band (3200–3700 cm-1) in the IR spectrum indicated complete methylation. Then, the completely methylated COP70-3 sample was completely hydrolyzed, reduced, acetylated, and analyzed by gas chromatography-mass spectrometry (GC-MS) (GC-MS-QP 2010, Shimadzu,

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Kyoto, Japan). The temperature programming method was used for GC assay. Firstly, the temperature of column was kept at 150°C for 1 min, and increased from 150°C to

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180°C at a rate of 10°C /min and maintained at 180°C for 1 min, then up to 260°C at a

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rate of 15°C /min and kept at 260°C for 5 min. The injection temperature was 220°C.

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2.13. Nuclear magnetic resonance (NMR) spectroscopy analysis

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The dried COP70-3 (60 mg) was completely dissolved in D2O (1000 μL), filtered

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with a 0.45 μm syringe filter, and then inspected with a Bruker AV-500 spectrometer

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(Germany) to obtain its 1H, 13C, HSQC, and HMBC NMR spectra.

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2.14. Scanning electron microscopy analysis

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The micromorphology of COP70-3 was examined with an environmental scanning electron microscope (SEM, Philips XL-30, The Netherlands), and SEM

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images under different magnification were obtained.

2.15. Effects of COP70-3 on the differentiation and mineralization of MC3T3-E1 cells MC3T3-E1 cells were cultured in 24-well plates at a density of 2.6×104 cells/well with complete medium for 24 h and then osteogenic medium for 72 h. Then, the 13

culture medium was changed to an osteogenic medium with various concentrations of COP70-3 (0.94, 1.87, and 3.74 nM) or 0.1 μM E2 as a positive control. At the same time, the normal group (Normal) was cultured only with complete medium, and the control group (Control) was cultured with osteogenic medium. Next, the MC3T3-E1

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cells were further cultured for 2 days, 4 days, 6 days, 8 days, 10 days, and 12 days. On the indicated day, the cells were lysed with 100 μL/well radioimmunoprecipitation

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assay (RIPA) buffer for 20 min. The lysate of the cells was collected and centrifuged

(12,400 × g, 5 min) at 4°C. The protein concentration of the supernatant of the cell

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lysate was measured using the BCA Protein Concentration Assay Kit, followed by an

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optical density (OD) reading at 562 nm on a microplate spectrophotometer. The

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protein supernatant was used for the assay of ALP activity with the ALP Assay Kit,

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followed by an OD reading at 405 nm on a microplate spectrophotometer. The ALP

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activity was calculated as following:

ALP activity (U/L) = protein actual molar concentration × 1000 × 100 / 45,

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where 1000 refers to the conversion of the unit of moles, 100 is the volume of the

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reaction system, and 45 is the color-changing time of the β-nitrophenol standard solution (min).

Alizarin red S staining can be used to assay the mineralization in MC3T3-E1 cells

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as previously described (Song et al., 2016). MC3T3-E1 cells were seeded in 12-well plates at a density of 5.25×104 cells/well; the volume of each well was 1 mL. Then, the cells were cultured in a 5% CO2 humidified incubator at 37°C for 72 h. Next, the cells were treated with osteogenic medium containing various concentrations of 14

COP70-3 (0.94, 1.87 and 3.74 nM) or 0.1 μM E2 as a positive control. Similarly, the normal group was cultured with complete medium, and the control group was cultured only with osteogenic medium. After incubation for 15 days, the cells were fixed with 10% neutral formalin for 30 min at room temperature, washed three times

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with phosphate buffered saline (PBS), stained with 0.1% alizarin red S in the dark for 30 min, washed again with PBS 3-5 times, observed under a microscope, and

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photographed to obtain images of the mineralized nodules. The liquid in the 12-well plates was aspirated with a pipet, and the cells were treated with 400 μL of 10%

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cetylpyridine per well for 30 min in the dark. Then, the cetylpyridine solution was

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transferred to a 96-well plate at a volume of 100 μL/well and its OD was measured at

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by the following formula:

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562 nm with a microplate spectrophotometer. The mineralization rate was calculated

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Mineralization rate (%) = (ODSample - ODNormal) / ODNormal × 100,

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where OD was the average optical density of six replicates.

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2.16. Statistical analysis

The data are presented as the mean value ± SD, and were analyzed with statistical

analysis software SPSS 16.0 for Windows (SPSS Inc., Chicago, IL, US). Probabilities

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(P) less than 0.05 were considered significant, and statistical differences among groups were obtained by analysis of variance (ANOVA) following Dunnett’s tests.

3. Results and discussion 15

3.1. Basic characteristics of CO70 The weight and sugar content of CO70 extracted from the rhizomes of C. orchioides were 288 g and 55.36%, respectively. This result demonstrated that the essential

component

of

CO70

was

polysaccharide.

Simultaneously,

the

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monosaccharide component analysis of CO70 (Fig. S8) showed that CO70 was composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose,

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galactose, xylose, and arabinose.

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3.2. Body weight and uterus coefficient

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The ovariectomized model of the female rats is a classic model for the study of

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primary osteoporosis in postmenopausal women. The ovarian absence can cause

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osteoporosis with the disequilibrium of bone resorption and bone formation, an

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increase in body weight, and a decrease in the weight of the uterus due to the estrogen deficiency in rats (Ma et al., 2015). The excessive bone resorption and insufficient

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bone formation directly causes bone loss. The effect of polysaccharide CO70 from C.

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orchioides on the body weight of OVX rats is shown in Fig. S1A. There was no statistically significant difference in the initial body weight of the four groups of rats (P > 0.05). From the fourth week (with surgery in the second week), the body weight

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of the OVX group was significantly higher than that of the Sham group (P < 0.01). Compared to the OVX group, the E2 and CO70 treatment significantly inhibited the increase in body weight from the fifth week (P < 0.01 or P < 0.05). The results showed that CO70 was effective in inhibiting the excessive weight gain that was 16

caused by estrogen secretion disorder in OVX rats. As shown in Fig. S1B, there was a significant difference in the uterus coefficient (the ratio of uterus weight to body weight, mg/g) of the OVX group versus the Sham group. This suggested that ovariectomy leads to serious metratrophia in rats, and E2

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can effectively relieve metratrophia in ovariectomized rats (Ma et al., 2011). Therefore, the E2 treatment significantly increased the uterine coefficient versus the

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OVX group (P < 0.01). Additionally, the uterus coefficient of the CO70 group was

significantly increased by 21.05% (P < 0.05) when compared with OVX group. This

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suggested that CO70 exhibits preventive effects on metratrophia in ovariectomized

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3.3. Bone mineral content and density

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rats.

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BMD and BMC are the most direct and important indicators of diagnosis of osteoporosis. Fig. 1 shows that the BMD and BMC of the entire femur, distal femur,

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and proximal femur were significantly decreased in the OVX rats compared with the

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Sham group, which indicates that the model of primary osteoporosis was successfully established in this study. Compared with the OVX group, the BMD of the entire femur of the CO70 group and E2 group was markedly increased by 7.57% (P < 0.01)

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and 5.98% (P < 0.01), respectively, and the BMD of the distal femur and proximal femur was also significantly increased after CO70 and E2 treatment (P < 0.01) (Fig. 1A). Furthermore, the BMC of the entire femur of the E2 group was significantly increased by 7.02% (P < 0.05), and that of the CO70 group was significantly 17

increased by 8.52% (P < 0.01), versus the OVX group (Fig. 1B). These results suggest that CO70 can increase the BMD and BMC of femurs in OVX rats, show

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significant inhibition of bone loss, and the overall effect is comparable to that of E2.

Fig. 1. Effects of CO70 on (A) left femur bone mineral density (BMD) and (B) bone mineral content (BMC). All values are expressed as the mean ± SD. ##P < 0.01 versus Sham; *P < 0.05 versus OVX, **P < 0.01 versus OVX. 18

3.4. Bone biomechanical property Bone biomechanical parameters mainly reflect the structural mechanical properties of bone, and provide more direct information for the comprehensive evaluation of bone strength and bone toughness (Peng et al., 2013). Bone

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biomechanics is divided into structural mechanics and material mechanics. The elastic load, maximum load, and fracture load are the representative indexes of the structural

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mechanics, which reflect the changes in the bone microstructure. The stiffness, maximum strength, fracture stress, and fracture strain are the representative indicators

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of the material mechanics, which reflect the inherent quality and property of bone.

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The bone biomechanical parameters of the femur are shown in Table 1. The elastic

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stroke, elastic load, maximum stroke, maximum load, fracture load, maximum stress,

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fracture stress, and fracture strain of femur in the OVX group were significantly lower

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than that in the Sham group throughout the course of the experiment (P < 0.05). Simultaneously, the toughness coefficient and bending energy of femur in

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ovariectomized rats were significantly reduced (P < 0.01). These results demonstrated

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that ovariectomy leads to changes in the biomechanical properties of the femur, significant reduction in both bone strength and toughness, and increase in the fracture risk, which also suggested that the ovariectomized rat osteoporosis model was

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successfully built. Table 1. Effects of CO70 on bone biomechanical parameters in the left femur of ovariectomized (OVX) rats.

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Sham

OVX

E2

CO70

Elastic stroke (mm)

0.16±0.03

0.14±0.02#

0.17±0.01**

0.15±0.02*

Elastic load (N)

115.71±15.72

101.87±5.02#

120.52±8.30**

114.72±15.23*

Maximum stroke (mm)

0.58±0.09

0.51±0.04#

0.58±0.10

0.58±0.09*

Maximum load (N)

202.28±22.68

184.62±10.82#

209.22±9.52**

203.83±12.16**

Fracture stroke (mm)

0.66±0.12

0.60±0.05

0.58±0.05

Fracture load (N)

200.41±18.24

183.18±11.61#

197.42±15.26* 203.73±17.59**

Stiffness (N mm-1)

692.94±62.16

651.40±60.51

658.53±40.99

696.03±81.31

Maximum stress (MPa)

223.22±51.16

179.32±11.09#

201.93±31.26

199.05±19.25*

Fracture stress (MPa)

192.69±19.76

176.28±9.75#

187.86±32.33

189.24±16.95

Fracture strain (%)

5.83±1.15

4.72±0.48#

5.75±1.40

5.79±1.15*

Toughness coefficient

0.005±0.001

0.003±0.002##

0.005±0.001*

0.005±0.001**

Bending energy (N mm)

9.39±2.27

6.64±0.40##

10.15±1.21**

9.19±1.81**

0.60±0.06

SC R

U

N

A

M

IP T

Parameter

ED

All values are expressed as the mean ± S.D. #P < 0.05 versus Sham, ##P < 0.01 versus Sham; *P <

PT

0.05 versus OVX, **P < 0.01 versus OVX.

CC E

Compared with the OVX group, the elastic stroke, elastic load, and fracture load of femora in the E2 group were significantly increased by 21.43% (P < 0.01), 18.31% (P < 0.01), and 7.77% (P < 0.05), respectively, and in the CO70 group, they were also

A

markedly increased by 7.14% (P < 0.05), 12.61% (P < 0.05), and 11.22% (P < 0.01), respectively (Table 1). In addition, the maximum stroke, maximum stress, and fracture strain of femora in the CO70 group were significantly increased by 13.72% (P < 0.05), 11.00% (P < 0.05), and 22.67% (P < 0.05), respectively, compared to the 20

OVX group; however, we did not find any significant improvement of these properties after treatment with E2 (Table 1). Furthermore, the maximum load, toughness coefficient, and bending energy of femora in the CO70 group and E2 group were not significantly different compared to the Sham group (Table 1), reflecting that

IP T

those properties returned to normal levels after treatment with CO70 and E2. From the perspective of improving the biomechanical properties of bone, the above results

SC R

illustrate that CO70 can effectively improve the biomechanical properties of the femur

of ovariectomized rats, increase bone strength and bone toughness, reduce the risk of

N

U

fracture, and produce effects comparable to those obtained after E2 treatment.

A

3.5. Microarchitecture of trabecular bone

M

The quantitative results of the microarchitecture of trabecular bone of femora

ED

measured by micro-CT are separately shown in Table S1. Compared with the Sham group, the structure model index (SMI), bone surface/bone volume (BS/BV), and

PT

trabecular separation (Tb.Sp) of femora in the OVX group were significantly

CC E

increased (P < 0.01); the bone connection density (Conn.Dens), bone volume/total volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) of the femora in the OVX group were significantly decreased (P < 0.01). These results show

A

that the microarchitecture of the trabecular bone of the femur was damaged in ovariectomized rats, and the pathological features of osteoporosis appeared. The results also indicated that the ovariectomized rat model of osteoporosis was successfully built. 21

The SMI values of femora in the CO70 group were significantly decreased (P < 0.01) compared to the OVX group, exhibiting the same effect as E2. The Tb.Sp values of femora in the CO70 group were significantly decreased by 17.24% (P < 0.05) as compared to the OVX group. Furthermore, the Conn.Dens values of femora in the

IP T

CO70 group were significantly increased by 12.70% (P < 0.05) as compared to the OVX group, and were not significant different compared to the E2 group. The BV/TV

SC R

of femora in the CO70 group was significantly increased by 33.33% (P < 0.01) versus

the OVX group. Additionally, compared with the OVX group, significant changes in

U

Tb.N and Tb.Th were found in the femora of the CO70 group (P < 0.05). The

N

difference in the microarchitecture of trabecular bone of the femur in each group is

A

intuitively shown by the representative 3D images in Fig. 2. Compared with the Sham

M

group, the trabecular bone of the femur in the OVX group was thin-walled and

ED

broadly separated, but CO70 and E2 treatment led to the restoration of trabecular microarchitecture and promoted new bone formation. Above all, CO70 also showed

PT

significant anti-osteoporotic activity in improving trabecular bone microarchitecture

A

CC E

and preventing bone loss.

Fig. 2. Representative 3D images of the left femur from each group of rats analyzed by micro-CT.

22

3.6. Separation and purification of CO70 Crude polysaccharide CO70 from C. orchioides was isolated and purified on DEAE-52 cellulose and Sephadex G-75 columns. The elution curves of CO70 on DEAE-52 and CO70-3 on Sephadex G-75 were shown in Fig. S2, and then a

IP T

homogeneous polysaccharide COP70-3 was obtained. Its relative molecular weight was 5.3 ×104 Da on the basis of HPGPC analysis (Fig. S3). Simultaneously, the

SC R

absence of protein and nucleic acid in COP70-3 was certified by the result of no

U

absorption at 260 nm and 280 nm in the UV spectra (Cai, Xie, Chen, & Zhang, 2013).

N

3.7. Monosaccharide composition of COP70-3

A

According to the HPLC analysis of PMP derivatives (Fig. S4), COP70-3 consists

M

of rhamnose, arabinose, glucose, galactose, mannose, glucuronic acid, and

ED

galacturonic acid.

PT

3.8. FT-IR spectrum analysis

CC E

The FT-IR spectrum of carbohydrates can provide data regarding the formation of COP70-3 structural features. The FT-IR spectrum of COP70-3 was shown in Fig. S5, the strong absorption peaks at 3400 cm-1 and 2936 cm-1 were assigned to O-H

A

stretching vibrations and C-H stretching vibrations, respectively. The relatively strong absorption peaks at 1628 cm-1 and 1421 cm-1 were attributed to the presence of uronic acid in COP70-3 (Hu, Liang, & Wu, 2015; Wei et al., 2016), and this was in conformity with the results of the monosaccharide composition analysis. The 23

absorption at 1069 cm-1 and 1044 cm-1 indicates the presence of pyran-glycosides (Zhang, Nie, Yin, Wang, & Xie, 2014).

3.9. Methylation and GC-MS analysis

IP T

Methylation and GC-MS analysis can provide information regarding primary residues in polysaccharides. Methylation analysis by GC-MS of COP70-3 was shown

SC R

in Fig. S6. According to the methylated alditol acetates of sugar residues detected by GC-MS, we can draw the conclusion that COP70-3 was composed by at least nine

→6)-D-Glcp-(1→,

U

different residues: →4)-D-Manp-(1→, →3,6)-D-Manp-(1→, →3)-L-Rhap-(1→, →6)-D-Galp-(1→,

→3,6)-D-Galp-(1→,

N

D-Glcp-(1→,

A

→5)-L-Araf-(1→, and →3,5)-L-Araf-(1→. It is necessary to confirm the detailed

3.10. NMR analysis 13

C-NMR, HSQC, and HMBC spectra of COP70-3 are shown in

PT

The 1H-NMR,

ED

M

information regarding COP70-3 structure by NMR analysis.

CC E

Fig. 3A–3B and Fig. 3C–3D. Combining the NMR spectra with the results of the monosaccharide composition, FT-IR spectroscopy, and GC-MS analyses, COP70-3 contained

13

types

of

residues,

and

they

are

→5)-α-L-Araf-(1→,

A

→3,5)-α-L-Araf-(1→, →2,5)-α-L-Araf-(1→, α-L-Araf-(1→, →3)-α-L-Rhap-(1→, →4)-β-D-Manp-(1→,

→3,6)-β-D-Manp-(1→,

→6)-β-D-Galp-(1→,

→3,6)-β-D-Galp-(1→,

→3)-β-D-GalpA-(1→,

→6)-α-D-Glcp-(1→,

→3)-α-D-GlcpA-(1→, and α-D-GlcpA-(1→ and are named with A, B, D, E, F, G, I, J, 24

K, L, M, N, and P, respectively. According to the cross-peak in the HMBC spectrum and previously reported data (Jones, Vinogradov, Nomellini, & Smit, 2015; Hu, Liang, & Wu, 2015; Wang et al., 2015), chemical shifts of each proton and carbon of every residue were assigned and are shown in Table 2. However, the HMBC spectrum of

IP T

COP70-3 was used to analyze the linkage sites and sequence among residues, as

A

CC E

PT

ED

M

A

N

U

SC R

shown in Fig. 3B.

25

IP T SC R U N A M ED PT CC E

Fig. 3. (A) 1H, (B) 13C, (C) HSQC and (D) HMBC spectra of COP70-3.

A

Table 2. 1H and 13C NMR chemical shifts of COP70-3 were recorded in D2O at 27°C

→5)-α-L-Araf-(1→

C1 H1 110.3

C2 H2 84.4

C3 H3 79.4

C4 H4 86.6

C5 H5 69.6

C6 H6 --

A

5.13

4.34

3.98

4.06

3.92

--

→3,5)-α-L-Araf-(1→ B

110.0 5.17

84.0 4.17

86.3 4.11

79.4 4.05

69.4 3.84

---

Residue

26

77.3 3.73 80.0 4.45 75.5 3.79 75.5 3.86 79.4 4.16 79.4 3.98 79.5 4.05

→3)-β-D-GalpA-(1→ L →6)-α-D-Glcp-(1→ M →3)-α-D-GlcpA-(1→ N α-D-GlcpA-(1→

106.7 4.67 100.6 5.08 100.9 5.18 101.2

78.6 3.50 75.5 3.70 72.4 3.80 72.1

79.4 4.16 76.4 3.95 78.3 3.95 72.0

P

5.31

N

A

M

69.4 3.84 69.4 3.70 71.8 3.44 73.3 3.69 73.4 3.69 72.0 4.16 72.5 4.28

----19.3 1.29 63.4 3.94 64.0 4.03 63.9 3.84 64.0 3.75

72.1 4.08 73.0 3.87 71.8 3.68 76.9

72.6 3.46 68.0 3.90 73.7 3.73 69.3

177.2 -63.9 3.27 177.8 -177.6

3.88

3.71

3.75

--

ED

4.08

82.1 3.61 86.6 3.98 71.3 3.94 80.6 3.91 75.4 3.85 75.1 3.69 76.0 3.39

IP T

87.2 4.20 83.1 3.76 72.7 3.46 72.1 4.00 72.4 4.00 77.8 3.73 76.3 3.81

SC R

111.2 5.46 112.0 5.28 101.0 4.93 103.8 4.95 103.6 4.77 106.1 4.54 106.7 4.63

U

→2,5)-α-L-Araf-(1→ D α-L-Araf-(1→ E →3)-α-L-Rhap-(1→ F →4)-β-D-Manp-(1→ G →3,6)-β-D-Manp-(1→ I →6)-β-D-Galp-(1→ J →3,6)-β-D-Galp-(1→ K

PT

The peaks at δ3.92/86.3 ppm (AH5/BC3) and δ5.31/110.0 ppm (PH1/BC1)

CC E

suggested that O-5 of residue A was linked to the C-3 of residue B, and O-1 of residue P was linked to the C-1 of residue B. Likewise, the linkages of residue B O-5 with residue J C-6, residue J O-1 with residue G C-4, and residue G O-1 with residue D

A

C-5 were deduced by the signals at δ3.84/63.9 ppm (BH5/JC6), δ4.54/80.6 ppm (JH1/GC4), and δ4.95/69.4 ppm (GH1/DC5), respectively. In addition, signals at δ5.28/111.2 ppm (EH1/DC1), δ4.20/75.5 ppm (DH2/FC3), and δ4.93/103.6 ppm (FH1/IC1) separately illustrate that O-1 of residue E was linked to the C-1 of residue 27

D, O-2 of residue D was linked to the C-3 of residue F, and O-1 of residue F was linked to the C-1 of residue I. Peaks at δ4.16/112.0 ppm (LH3/EC1), δ3.75/106.7 ppm (KH6/LC1), and δ3.92/106.7 ppm (AH5/KC1) show the linkages of residue L O-3 with residue E C-1, residue K O-6 with residue L C-1, and residue A O-5 with residue

IP T

K C-1. Additionally, the signals at δ5.28/110.3 ppm (EH1/AC1), δ4.05/79.4 ppm (KH3/IC3), δ4.03/69.6 ppm (IH6/AC5), and δ3.84/110.3 ppm (BH5/AC1) suggested

SC R

that O-1 of residue E was linked to the C-1 of residue A, O-3 of residue K was linked

to the C-3 of residue I, O-6 of residue I was linked to the C-5 of residue A, and O-5 of

U

residue B was linked to the C-1 of residue A. The linkages of O-1 of residue N with

N

C-3 of residue B, O-1 of residue J with C-3 of residue N, and O-1 of residue E with

A

C-3 of residue B were deduced by the peaks at δ5.18/86.3 ppm (NH1/BC3),

M

δ4.54/78.3 ppm (JH1/NC3), and δ5.28/86.3 ppm (EH1/BC3), respectively. Parallel

ED

results consist of the signals at δ3.27/86.3 ppm (MH6/BC3) and δ5.08/110.0 ppm (MH1/BC1), which reveal that O-6 of residue M was linked to the C-3 of residue B

PT

and O-1 of residue M was linked to the C-1 of residue B. Furthermore, the signals at

CC E

δ3.84/106.1 ppm (JH6/JC1), δ4.95/80.6 ppm (GH1/GC4), δ4.93/75.5 ppm (FH1/FC3), δ3.92/110.3 ppm (AH5/AC1), and δ5.18/78.3 ppm (NH1/NC3) illustrate that their corresponding residues were internal repetitive linkages. The possible repetitive

A

structure unit of COP70-3 was inferred and is shown in Fig. 4.

28

IP T SC R U N A M PT

ED

Fig. 4. Predicted repetitive structural unit of COP70-3.

3.11. Micromorphology analysis

CC E

As shown in Fig. 5A–5B, the SEM images of COP70-3 at magnifications of

1000× and 5000× revealed that the micromorphology of COP70-3 consists of an

A

irregular laminar structure with various branches. This indicates that the distribution of COP70-3 with a complicated structure was variable and chaotic.

29

IP T SC R

Fig. 5. SEM images of COP70-3 (A: 1000×, B: 5000×).

U

3.12. Effects of COP70-3 on the differentiation and mineralization of MC3T3-E1 cells

N

MC3T3E1 cells have been reported to retain their capacity to differentiate into

A

osteoblasts, and these cells undergo a temporal pattern of osteoblast development

M

similar to that of in vivo bone formation (Quarles, Yohay, Lever, Caton, & Wenstrup,

CO70

from

C.

ED

1992; Kahai, Lee, Seth, & Yang, 2010). Based on the anti-osteoporosis activity of orchioides

in

ovariectomized

rats,

we

assessed

the

PT

differentiation-inducing activity of COP70-3 isolated and purified from CO70 on

CC E

MC3T3-E1 cells by evaluating ALP activity. ALP activity is a phenotypic marker for osteoblast differentiation (Wennberg et al., 2000). As shown in Fig. 6, different concentrations of COP70-3 affected the differentiation of MC3T3-E1 cells at different

A

time points. The ALP activity of COP70-3 at 1.87 nM was remarkably higher than that of the control group (P < 0.05) and even the E2 group (P < 0.05) after MC3T3-E1 cells were cultured in osteogenic medium for 10 days. The effect of COP70-3 on the ALP activity in MC3T3-E1 cells was related to its concentration and culture time. In 30

this study, COP70-3 with a concentration of 1.87 nM was the most effective in promoting MC3T3-E1 cell differentiation when the cells were cultured in

A

N

U

SC R

IP T

osteoblast-inducing medium for 10 days.

M

Fig. 6. Effects of COP70-3 on the ALP activity of MC3T3-E1 cells. C1: COP70-3 at 0.94 nM; C2: COP70-3 at 1.87 nM; and C3: COP70-3 at 3.74 nM. All values are expressed as the mean ± SD. < 0.05 versus Normal, ##P < 0.01 versus Normal, *P < 0.05 versus Control,

ED

#P

P < 0.05 versus

PT

E2.

△

CC E

The formation of mineralized nodules is an important marker during osteoblastic

maturation (Song et al., 2016). As shown in Fig. 7, MC3T3-E1 cells were cultured for

A

15 days, and the image of the control group shows calcium nodules, indicating that an osteogenic differentiation inducer can successfully induce the mineralization of MC3T3-E1 cells. The numbers of calcium nodules of various concentrations in the COP70-3 and E2 group were higher than those in the Control group, and this 31

demonstrated that COP70-3 and E2 can promote MC3T3-E1 cell mineralization. The quantitative effect of COP70-3 on the mineralization rate of MC3T3-E1 cells is presented in Fig. 8. COP70-3 at 0.94 and 1.87 nM can significantly improve the mineralization rate of MC3T3-E1 cells (P < 0.01), and there was no significant

IP T

difference compared with the E2 group (P > 0.05). However, a higher concentration of polysaccharide COP70-3 cannot improve the mineralization rate of MC3T3-E1

SC R

cells compared with the Control group (P > 0.05). These results proved that the effect

of COP70-3 on the mineralization of MC3T3-E1 cells was also related to its

CC E

PT

ED

M

A

N

U

concentration and culture time.

Fig. 7. Images at 4× showing the effect of COP70-3 on the mineralized nodules of MC3T3-E1

A

cells after being cultured in osteogenic medium for 15 days.

32

IP T SC R

Fig. 8. The quantitative effect of COP70-3 on the mineralization rate of MC3T3-E1 cells after being cultured in osteogenic medium for 15 days. C1: COP70-3 at 0.94 nM; C2: COP70-3 at 1.87

N

U

nM; and C3: COP70-3 at 3.74 nM. All values are expressed as the mean ± SD. **P < 0.01 versus

M

A

Control.

Overall, we can preliminarily illustrate that with the proper concentration of

ED

COP70-3, anti-osteoporosis activity in vitro is observed upon promoting MC3T3-E1

PT

cell differentiation and mineralization under the premise of suitable culture time.

CC E

4. Conclusions

As a famous kidney-tonifying traditional medicine, C. orchioides has been widely

A

used for the treatment of arthritis of the lumbar spine and knee joints, weakness, and other conditions. Polysaccharide is one of the most important components of C. orchioides and may be effective for the prevention and treatment of osteoporosis. However, no systematic research regarding this herb or its components has been 33

reported thus far. Herein, we first report the anti-osteoporosis activity of C. orchioides polysaccharide on ovariectomized rats. The polysaccharide content of CO70 was 55.36%, and this significantly inhibited the increase in body weight and metratrophia in ovariectomized rats, with no obvious toxicity or detrimental side effects over

IP T

long-term administration. Compared with the OVX group, the BMD of the entire femur of the CO70 group was markedly increased by 7.57% (P < 0.01), and the BMC

SC R

was significantly increased by 8.52% (P < 0.01). The elastic stroke, elastic load, and

fracture load of the femora in the CO70 group were also clearly increased by 7.14%

U

(P < 0.05), 12.61% (P < 0.05), and 11.22% (P < 0.01), respectively. Bone strength

N

and bone toughness were increased, and the risk of fracture was reduced. The Tb.Sp

A

of femora in the CO70 group was significantly decreased by 17.24% (P < 0.05) as

M

compared to the OVX group. The CO70 treatment also effectively increased the Tb.N,

ED

Tb.Th, Conn.Den, and BV/TV, and improved the microarchitecture of the trabecular bone of femora in ovariectomized rats. The overall effect of CO70 in ovariectomized

PT

rats was comparable to that of E2.

CC E

The characterization of polysaccharides contained in CO70 had great significance for the further structure-function relationship study, and the development and application of the C. orchioides polysaccharide. Therefore, to reveal the active

A

ingredients responsible for the effects of CO70, we isolated and purified the constituents from CO70 with DEAE-52 cellulose and Sephadex G-75 columns, and a novel homogeneous heteropolysaccharide COP70-3 with a molecular weight of 5.3 × 104 Da was obtained. The results of monosaccharide composition, FT-IR spectroscopy, 34

GC-MS and NMR analyses showed that COP70-3 is composed of →5)-α-L-Araf-(1→, →3,5)-α-L-Araf-(1→, →2,5)-α-L-Araf-(1→, α-L-Araf-(1→, →3)-α-L-Rhap-(1→, →4)-β-D-Manp-(1→,

→3,6)-β-D-Manp-(1→,

→6)-β-D-Galp-(1→,

→3,6)-β-D-Galp-(1→,

→3)-β-D-GalpA-(1→,

→6)-α-D-Glcp-(1→,

IP T

→3)-α-D-GlcpA-(1→, and α-D-GlcpA-(1→. The possible repetitive structural unit of COP70-3 was inferred. Furthermore, the SEM images revealed that COP70-3

SC R

possessed an irregular laminar structure with various branches.

The most effective concentration of COP70-3 for promoting MC3T3-E1 cell

U

differentiation was 1.87 nM, and 0.94 nM and 1.87 nM can also significantly improve

N

the mineralization rate of MC3T3-E1 cells. The current study examined the effect of

A

COP70-3 on the differentiation and mineralization of MC3T3-E1 cells and indicates

M

that COP70-3 promotes anti-osteoporosis activity in vitro. In conclusion, this study

ED

reports the systematical purification, structural identification, and anti-osteoporosis activity of COP70-3 and notes its potential as a natural anti-osteoporotic agent that

PT

can be utilized in the pharmaceutical and healthcare industries. Further study to

CC E

examine the mechanisms of anti-osteoporosis activity of COP70-3 in vitro is ongoing.

A

Acknowledgments This study was funded by the National Natural Science Foundation of China (Nos.

81673557 and U1703110), the Science and Technology Program of Guangdong Province (Nos. 2014A050503067 and 2015A020211032) and the Education Program of Guangdong Province (No. 2017KZDXM048). 35

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IP T

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