The Prebiotic Arabinogalactan of Anoectochilus Formosanus Prevents Ovariectomy-Induced

The prebiotic arabinogalactan of Anoectochilus formosanus prevents ovariectomy-induced osteoporosis in mice

Li-Chan Yanga, Ting-Jang Lua, Wen-Chuan Linb,*

aInstitute of Food Science and Technology, National Taiwan University, Taipei, Taiwan.

bSchool of Medicine, Graduate Institute of Basic Medical Science and Tsuzuki Institute for Traditional Medicine, China Medical University, Taichung, Taiwan

*Corresponding author: Department of Pharmacology, China Medical University, No. 91 Hsueh Shih Road, Taichung, Taiwan, R.O.C. Tel +886 4 22053366; fax +886 4 22053764

e-mail address: (W.C. Lin)

Keywords: Prebiotic; Arabinogalactan; Osteoporosis; Short chain fatty acids

ABSTRACT: Anoectochilus formosanus (Orchidaceae) has exhibited anti-osteoporosis and prebiotic properties in previous studies. In this study, these bioactivities were verified and associated with an isolated type II arabinogalactan (AGAF) in ovariectomized (OVX) mice model. Female ICR mice were OVX and administrated AGAF (5 and 15 mg/kg) or inulin (400 mg/kg) orally for 3 weeks. Streptomycin was used for blocking the bioactivities of AGAF. In results, AGAF increased the level of fecal bifidobacteria, cecal soluble Ca and short chain fatty acids. Comparing to OVX control group, AGAF improved bone mineral content, trabecular bone volume, and the number of trabecular significantly. In RT-PCR analysis, AGAF reduced the expression of tartrate-resistant acid phosphatase, cathepsin K, and osteocalcin. Streptomycin inhibited both anti-osteoporosis and prebiotic effects of AGAF. In vitro experiments revealed butyrate, not AGAF could activate osteoblasts and inhibit osteoclasts differentiation. In conclusion, this study showed AGAF prevented bone loss in OVX mice through prebiotic effects in vivo and in vitro.

1. Introduction

Osteoporosis is the most common skeletal problem caused by aging, especially in postmenopausal women (Pietschmann, et al., 2009). The deficiency of ovarian hormones is a major factor in postmenopausal osteoporosis. Bone loss may lead to related fractures and high medical costs. Hormone supplements and bisphosphonate are common therapies for osteoporosis; however, these therapies have several side effects. For example, hormone supplements increase the risk of cardiovascular disease and breast cancer; the risk of bisphosphate includes osteonecrosis of the jaw and atypical femur fractures (de Villiers & Stevenson, 2012; Khosla, et al., 2012). The lack of reliable and effective therapies to cure osteoporosis-related fragility fractures remains an important global issue (Datta, 2011). Previous studies have indicated that certain nutritional factors, such as fruit, prebiotics, and minerals, can increase bone mineral density in people diagnosed with osteoporosis (Devareddy, et al. 2008; Chen, et al., 2006; Stransky & Rysava, 2009). Nutrition could prevent and treat osteoporosis with fewer side effects than medicine therapies (Stransky & Rysava, 2009). Prebiotics are food component not absorbed or digested in the small intestine but are fermented by microbiota in the large intestine (Roberfroid, 2005). Several prebiotics, such as inulin, galactooligosaccharides, and fructooligosaccharides, are thought to improve bone health (Roberfroid, 2005;Weaver, 2005). The microbial fermentation products of prebiotics, such as short chain fatty acids (SCFA), are responsible for the increase of calcium (Ca) absorption in the large intestine. The high concentration of SCFA in the cecum leads to a decrease of cecal pH, which increases the concentrations of soluble Ca (Coxam, 2007). In addition, butyrate, one of the SCFA, belongs to a new class of antiosteoporotic agents that may be useful in the treatment of bone loss (Katono, et al., 2008; Rahman, et al., 2003; Schroeder & Westendorf, 2005). Furthermore, numerous reports have indicated that the ingestion of prebiotics or fermentable dietary fibers might be helpful in preventing osteoporosis (Coxam, 2007; Mitamura, et al., 2004).

Anoectochilus formosanus (Orchidaceae) is an important ethnomedicinal plant in Taiwan. It has been popularly used as a nutraceutical herbal tea in Taiwan and other Asian countries (Du, et al., 2008). In Taiwan, the aqueous extracts of A. formosanus have been certified as health food for hepatoprotection bioactivity and showed safety in the 13 week oral toxicity study in rats. In additional, Chang, et al. have indicated that A. formosamus plants cultivated by artificial are safe for use as an herbal medicine (Chang, et al., 2007). Several reports have shown that crude extracts of A. formosanus could ameliorate osteoporosis in the ovariectomized (OVX) rat model (Shih, et al.,2001; Masuda, et al., 2008; Yang, et al., in press). Masuda et al. shows that aqueous extracts of A. formosanus suppress bone loss caused by estrogen deficiency by inhibiting osteoclast formation (Masuda, et al., 2008). Our previous study also shows that water extracts of A. formosanus prevent bone loss in rats caused by OVX (Yang, et al., in press). In our previous studies, an indigestible polysaccharide isolated from A. formosanus was shown to be a potent prebiotic (Yang, et al., 2012). the indigestible polysaccharide of A. formosanus was mainly composed of type II arabinogalactan (AGAF). Water extracts of A. formosanus could enhance the level of cecal SCFAs and increase the number of fecal bifidobacteria in rats (Yang, et al., 2012). Therefore, it was suggested that the anti-osteoporosis activity of A. formosanus may be regulated by its prebiotic effect. This study examines the anti-osteoporosis effects of AGAF in OVX mice, and investigates the relationship of prebiotic properties and anti-osteoporosis activity.

1. Materials and methods

2.1. Arabinogalactan preparation

Cultured A. formosanus was purchased from Yu-Jung Farm (Pu-Li, Taiwan). Fresh plants were homogenized with distilled water, and then partitioned with ethyl acetate (Tedia Company, OH, USA). The aqueous extracts of A. formosanus were added with a 4-fold volume of 95% ethanol to precipitate crude polysaccharides, and then the crude polysaccharide was treated with α-amylase, protease and protease (Megazyme, Wicklow, Ireland) to remove starches and proteins. After enzymic treatment, AGAF was preserved in ethanol until use.

The identity and content of type II arabinogalactan in AGAF (> 80%) were analyzed by precipitation with β-glucosyl yariv reagent according to a previous study (Yang et al., 2012). For an in vivo experiment, AGAF was dissolved in distilled water, and concentrations of 0.5 and 1.5 mg/mL were prepared.

The yield rate of AGAF was 0.15% from fresh plants. Chemical analyses showed that AGAF contained 95.5% carbohydrates and 1.0% protein. AGAF is mainly composed of a (1→3)-β-D-galactan backbone with a (1→6)-β-D-galactan side chain, and is a type II arabinogalactan with an average molecular weight of 29 kDa. The monosaccharide composition of AGAF was arabinose, galactose, glucose, and mannose with a ratio of 22.4:56.5:15.4:5.4 (Yang, et al., 2012).

2.2. Animals

Eight-week-old female ICR mice were purchased from BioLASCO Co., Ltd. (Yi-Lan, Taiwan). The experimental animals received humane care, and the study protocols complied with the institutional guidelines of China Medical University for the use of laboratory animals. The animals were housed in an air-conditioned room (21–24 °C) under 12 h of light (8:00 a.m. – 8:00 p.m.), and were allowed free access to food pellets and water throughout the study.

2.3. Anti-osteoporosis effects of AGAF on OVX mice.

The experiments were performed on 40 female ICR mice. Bilateral OVX operation was performed under pentobarbital anesthesia (50 mg/kg) on the mice according to the procedure described before (Idris, 2012). Briefly, the mice were laparotomized to excise both ovaries clearly. The mice in the sham-operated group received anesthesia and a laparotomy as OVX operation, and were then sutured without removing their ovaries (Idris, 2012). After 3 days of adaptation after the surgery, the OVX mice were randomly divided into four groups, and were orally administered H2O, AGAF (5, 15 mg/kg), or inulin (Alfa Aesar, Heysham, UK) for the positive control for 3 weeks. The sham-operated group was orally treated with H2O. Each group contained 8 mice. The body weight of each animal was measured once a week until the final day of administration.

2.4. Assessment of prebiotic effect of AGAF

On day 7 after AGAF treatment, fresh feces were collected for analysis for bifidobacteria. Fresh feces were homogenized with 0.1% peptone diluent, followed by serial decimal dilutions. The number of bifidobacteria was counted on Bifidobacteria iodoacetate medium-25 agar after incubation at 37 ℃ for 48 h under anaerobic conditions (95% N2, 3% CO2, and 2% H2) (Munoa & Pares, 1988). After incubation, a single colony was counted, and the results were expressed as the log values of the CFUs per gram of wet weight of feces.

2.5. Ca concentrations and SCFA

After the mice were sacrificed, the ceca were removed and weighed immediately. The cecal contents were collected. The cecal walls were then flushed with 0.9% saline, blotted dry with filter paper, and weighed, and were then stored at -80 ℃ for reverse transcription-polymerase chain reaction (RT-PCR) analysis. The cecal contents were stored at -80 ℃ until SCFA determination using HPLC analysis (Niven, et al., 2004). For SCFA analysis, the cecal content samples were defrosted on ice, and were diluted with 0.0085N sulfuric acid. The cecum samples were shaken and centrifuged at 12000 × g for 20 min. The supernatant was diluted to proper concentrations for HPLC analysis. The SCFAs were analyzed by a Transgenomic ICSep Transgenomic (300 × 7.8 mm, Omaha, NE, USA) at 65 °C, and eluted with 0.0085N sulfuric acid at a flow rate of 0.4 mL min-1. The peaks were detected by a Shodex RI-71 detector (Showa Denko, Tokyo, Japan).

2.6. Bone Ca content

Soft tissues were removed from the lumbar vertebra, and were immersed in a mixed solvent (chloroform: methanol = 2:1, Showa, Tokyo, Japan) to remove bone lipids subcutaneously (Honda, 2001). De-fatty lumbar vertebrae were incinerated at 1000 °C for 12 h for ashing. Bone ash was weighed and solved in 6N HCl (Wako, Osaka, Japan) for determination of Ca with the o-cresolphthalein complexone method with commercial kits (Randox, Crumlin, UK). Values were expressed as milligrams of Ca of bone dry weight.

2.7. Microcomputed tomography (microCT) analyses

The right femurs of the mice were preserved in 75% alcohol until scanning. The bone microarchitecture of the distal right femoral metaphysis was measured using a microtomography scanner (SkyScan 1076, Kontizh, Belgium), with an isotropic resolution of 17 m in all 3 spatial dimensions. To analyze the interest volume of trabecular, 100 slices were selected from the edge of distant direction to the proximal direction. The region of interest volume was analyzed without the cortical bone. The bone and tissue volumes were measured directly from the original 3-dimensional images, and the trabecular volume fraction (bone volume/tissue volume, %) was normalized to compare samples of different sizes. The other examined parameters of the trabecular structure were trabecular thickness, trabecular number, and trabecular separation, which were calculated directly from the 3-dimensional images.

2.8. RT-PCR analysis

Total RNA was extracted with Trizol (Invitrogen, CA, USA) from the tibia and cecal walls, followed by acid guanidinium thiocyanate-phenol-chloroform extraction, as described by Chomczynski and Sacchi (1987). A 3 g sample of total RNA was subjected to reverse transcription with moloney murine leukemia virus reverse transcriptase in a 50 L reaction volume. The cDNA was amplified by PCR. The primers used for cecal wall RNA extract were CaBP and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal standard and were 5’- CAGAACCGAAGACTAGCGCA-3’ and 5’-GCACAAAACAAAGTGGGTGC -3’(product size, 283 bp),and 5’-TGTGTCCGTCGTGGATCTGA-3’ and 5’-CCTGCTTCACCACCTTCTTGA-3’(product size, 76 bp), respectively. The primers for mice tibia RNA extract were TRAP, 5’-CCAATGCCAAAGAGATCGCC -3’ and 5’- TCTGTGCAGAGACGTTGCCAAG-3’(product size, 216 bp), Cathepsin K , 5’- CTGCCCATAACCTGGAGG-3’ and 5’- GCCCTGGTTCTTGACTGG-3’(product size, 230 bp), OCN, 5’- AGACCGCCTACAAACGCATC-‘3’ and 5’-ACAGGGAGGATCAAGTCCCG -3’(product size, 113 bp), Runx2, 5’-CCGAGAAGTGGTTCCCGGTCCTG-3’ and 5’- CGACAGATCTGGAGCCTGCGGA-3’(product size, 173 bp), and GAPDH as internal standard. . Electrophoresis was performed on 2% agarose gels in a 0.5X TBE buffer (Amersco, OH, USA) for PCR products, and each lane was loaded with a fixed volume of the sample. PCR products were visualized using ethidium bromide staining.

2.9. Plasma bone markers

At the end of the experiment, the mice were sacrified, and their blood was drawn with heparin. The plasma was separated from the blood samples by centrifugation at 2000 × g for 10 min. The plasma was stored at -30 °C until assay. The plasma osteocalcin (OCN) were measured by commercial enzyme-linked immunosorbent assay (ELISA) kits (Biomedical Technologies Inc., Stoughton, MA, USA), and the plasma carboxy-terminal collagen cross-links (CTx) were analyzed using ELISA kits (Immunegiagnostic Systems, AZ, USA).

2.10. Effects of AGAF on OVX mice with streptomycin supplement

40 female ICR mice were treated with sham operations or OVX, and OVX mice were allocated into groups that were H2O, AGAF (15 mg/kg po. daily) with or without streptomycin sulfate (SM; Sigma Aldrich, MO, USA) treatment for 3 weeks. Each group contained eight mice. SM was dissolved in the drinking water at a concentration of 2 mg/mL (Asahara, 2001). The sham-operated group was orally administrated H2O. The body weight of each mouse was recorded every week until the end of the experiment. The mice were assessed for prebiotic activity in vivo. At the end of the experiment, mice femurs were removed for microCT analyses.

2.11. In vitro assay of alkaline phosphatase activity on murine osteoblast MC3T3-E1 cells

The cells of murine osteoblast MC3T3-E1 were grown in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, UT, USA) supplemented with 10% (v/v) FBS (Hyclone, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin. Incubation was conducted in a CO2 incubator (5% CO2, 95% air) at 37 °C. The cells were subcultured every 2 or 3 days by 0.25% (w/v) trypsin plus a 0.02% (w/v) ethylenediaminetetraacetic acid tetrasodium salt solution (Gibco, NY, USA).

For the alkaline phosphatase (ALP) activity assay, the MC3T3-E1 cells were seeded in 48-well plates (104 cells/well) containing DMEM in addition to 10% FBS (Iwami & Moriyama, 1993). After the cells attached to the bottom of the wells, the culture medium was changed to DMEM + 10% FBS medium containing 10 mM disodium β-glycerophosphate (Sigma Aldrich), 0.15 mM ascorbic acid (Sigma Aldrich), and 10-8 M dexamethasone (Sigma Aldrich) (Isama & Tsuchiya, 2003).

Simultaneously, different concentrations of sodium butyrate or AGAF were added to the culture medium in the well. On day 6 after cultivation, the cells were washed twice with phosphate buffered saline and harvested in a 200 μL/well of a lysis buffer (pH 8.2, 10 mM Tris-HCl, 2 mM MgCl2, and 0.05% Triton X-100). Cells were lysed through an ultrasonic processor (Vibra-Cell, Sonics & Materials, CT, USA) with 30 J in energy. Aliquots were reserved for protein analysis. A total of 300 μL of 8 mM p-nitrophenyl phosphate (Sigma Aldrich) in a 0.1 M sodium carbonate buffer (pH 10) containing 1 mM MgCl2 were added to the reaction mixture, which was incubated at 37 °C for 30 min. The reaction was stopped by adding 50 μL of 1.0 N NaOH/well.26 The yellow sample solutions containing p-nitrophenol for the reaction product were measured at 405 nm using a microplate reader. A standard curve was prepared using p-nitrophenol phosphate. The total protein contents of cell lysates was measured by a Bradford reagent (Sigma Aldrich) using albumin for the standard (Bradford, 1976).

2.12. In vitro Osteoclasts differentiation from murine macrophage RAW264.7 cell line

RAW264.7 cells were maintained in an α-modified Eagle’s medium (Hyclone), supplemented with 10% FBS (Gibco, CA, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin and L-glutamine (Biological industry, Kibbutz Beit-Haemek, Israel). Cells were cultured at a density of 2 × 103 cells/mL in a 24-well plate in the presence of a receptor activator of nuclear factor kappa B ligand (RANKL) for 5 days (Rahman, et al., 2003). RAW264.7 cells were incubated with different concentrations of sodium butyrate or AGAF to examine their effects on osteoclast differentiation. On day 5, cells were treated with 4% formaldehyde solution for 10 min, and then stained to obtain the tartrate-resistant acid phosphatase (TRAP) of the osteoclasts. TRAP staining was applied to measure the presence of osteoclasts, and stained with a standard kit (387A-1 kit, Sigma-Aldrich). The TRAP-positive multinucleated osteoclasts (least 3 nuclei) were counted (Parfitt, et al. 1987).

2.13. Statistical analysis

The results were expressed as mean  SD. All experimental data were analyzed using one-way analysis of variance (ANOVA) with the Duncan multiple-range test. Values of p < 0.05 were considered statistically significant.

1. Results

3.1. Assessment of prebiotic effect of AGAF

The number of fecal bifidobacteria was different between the sham and OVX-H2O group. The OVX treatment caused a 9.1% decrease in the number of bifidobacteria for the OVX mice than in the sham group. Administration of AGAF for 7 days led to a 20.7% (5 mg/kg) and 21.2% (15 mg/kg) increase in bifidobacteria in the stool compared to the OVX-H2O group (Table 1). In addition, inulin treatment also led to a 15.2% increase in the number of fecal bifidobacteria over the OVX-H2O group.

3.2. Cecal Ca concentrations and SCFA

The cecal Ca concentrations in the OVX-H2O group and sham group were equal. For the level of cecal Ca concentration, the AGAF administrated groups had 35.1% (5 mg/kg) and 42.7% (15 mg/kg) greater levels than the OVX-H2O group; administrated with inulin were 39.8% greater than OVX-H2O group significantly (Table 1).

The results of SCFA analyses are shown in Table 1. Concentrations of butyric acid in the sham group and OVX-H2O group have significant differences. The OVX-H2O group had a decrease in the level of butyric acid of 12.1% compared to the sham group. In addition, no differences in lactic acid, acetic acid, and propionic acid were observed between the sham group and the OVX-H2O group. The analysis showed significantly higher concentrations of acetic acid, propionic acid, and butyric acid in the OVX-AGAF group (15 mg/kg) compared to the OVX-H2O group (26.3%, 66.0%, and 57.7% increases, respectively). The OVX-inulin group showed increases in propionic acid and butyric acid of 53.6% and 16.4% compared to the OVX-H2O group. In this study, total SCFA is the amount of lactic acid, acetic acid, propionic acid, and butyric acid; no differences were observed between the sham and OVX-H2O groups. Moreover, the OVX mice administered AGAF led to a 21.0% (5 mg/kg) and 31.25% (15mg/kg) increase; inulin led to a significant 12.8% increase in the total SCFAs in the ceca of the OVX mice, compared to the OVX-H2O group.

3.3. Bone Ca content

The amount of Ca in the lumbar vertebrae was determined using o-cresophthalein complexone. The total Ca content and Ca ratio of lumbar vertebrae was calculated. The results were showed that OVX treatment reduced the Ca content by 11.4% (14.0 ± 1.3 mg) and the Ca ratio by 16.9% (12.3 ± 0.6%) in the lumbar vertebrae compared to sham group (15.8 ± 1.0 mg in Ca content; 14.8 ± 0.8% in Ca ratio). The results of AGAF treatment (5 mg/kg) were 13.3 ± 0.9 mg in Ca content and 13.1 ± 0.1% in the Ca ratio, and shown no significant difference compared to OVX-H2O group. Administration of AGAF (15 mg/kg) caused a significant 13.6% increase in the Ca content (15.9 ± 0.4 mg), and a 14.6% increase in the Ca ratio (14.1± 0.1%); however, administration of inulin increased the Ca ratio (13.8 ± 0.5%) only, compared to the OVX-H2O group. Bone Ca content of inulin treatment group was 14.9 ± 1.9 mg and displayed a minor increase to OVX-H2O group.