Anti-osteoporotic effects of mixed compositions of extracellular polymers isolated from Aureobasidium pullulans and Textoria morbifera in ovariectomized mice

One hundred and sixty virgin female SPF/VAF (CrljOri:CD1 [ICR]; 7-weeks old) mice, purchased from OrientBio, Seungnam, Republic of Korea, were acclimatized for 8 days and then used for these experiments. Four mice were assigned per polycarbonate cage and reared in a humidity (45–55%), and temperature (20–25 °C), −controlled room. Light was provided for 12 h and the standard rodent chow diet (Purinafeed, Seungnam, Rep. of Korea) and water were provided freely. In this experiment, 150 mice were allocated as OVX-operated osteoporosis model and 10 mice were allocated as sham-operated control. After 34 days of OVX operation, 8 mice from each group were chosen depending on body weight deviations (OVX-mice: 37.38 ± 2.71 g, range of 34.2 ~ 44.4 g; sham-operated mice: 33.86 ± 2.53 g, range of 30.3 ~ 37.5 g). The experimental designs for this study are presented in Fig. 1, and the animals were assigned to the experimental groups as follows:

EAP, an extracellular polymer isolated from A. pullulans (SM-2001; marketed as Polycan™) and the standard leaf extract of T. morbifera (TM) were obtained from Glucan Corp., Busan, Rep. of Korea. EAP consisted of 40% total β-glucans including 13% β-1,3/1,6-glucans [34, 35]. EAP and TM were mixed thoroughly in distilled water to a final concentration of 20 mg/ml. A few samples of TM [Code No: TM2016Ku01], and EAP [Code No: EAP2016Ku01] were deposited in the herbarium, Medical Research Center for Globalization of Herbal Formulation, Daegu Haany University, Gyeongsangbuk-do, Gyeongsan-si, Rep. of Korea. The 9 different herbal formulations at a final concentration of 200 mg/kg/body weight (EAP:TM = 1:1, 1:3, 1:5, 1:7, 1:9, 3:1, 5:1, 7:1, and 9:1) and both single formulations of TM and EAP were supplied orally, once a day for 35 days after 35 days of OVX surgery. The EAP:TM mixed formulations (1:1, 1:3, 1:5, 1:7, 1:9, 3:1, 5:1, 7:1, and 9:1) were prepared by dissolving EAP and TM (100:100, 50:150, 33:167, 25:175, 20:180, 150:50, 167:33, 175:25, and 180:20 mg:mg) in distilled water, respectively (final concentration: 200 mg in 10 ml of distilled water). The prepared mixtures were then provided at a concentration of 200 mg/kg, by gastric gavages using a 1 ml syringe. The single formulations of EAP and TM were also prepared and administrated in the same manner as other mixed formulations of EAP and TM.

In addition, 0.25 mg risedronate sodium (RES; TEVA Tapi, Sheva, Israel) was also dissolved in 1 ml distilled water and provided orally at a concentration of 2.5 mg/kg [10, 15]. The control groups (OVX and Sham control) were provided with equal volumes of distilled water (vehicle) instead of test substance to provide the same experimental conditions and administration stress.

A mixture of 2 to 3% isoflurane [2–3% isoflurane in 28.5% O₂ and 70% N₂O], was used to anesthetize the animals using a rodent inhalation and rodent ventilator system and the animals were maintained with 1 to 1.5% isoflurane. The surgical protocol was carried out according to the previously reported methods [7‐9]. In the OVX treatment group, bilateral OVX was removed by open surgery and the incisions were stitched in two steps. The bilateral OVX surgery was not performed in the sham operated mice group (2nd group). The animals were sacrificed by cervical dislocation under anesthesia using 99.50% CO₂ gas and the stomach was excised with a median incision in the abdomen.

The changes in body weights were recorded on weekly basis by an automatic electronic balance. At the initiation and termination of sample administration as well as on the day of OVX, all trial mice were starved for approximately 18 h (only water was supplied) to decrease alterations due to feeding. Furthermore, body weight gains were estimated by Eq. [1], and Eq. [2].

In vivo dual-energy X-ray absorptiometry was used to estimate the changes in mean BMD of the right femur and the total body, 24 h after the final administration of the test materials.

After 35 days continuous treatment with the test material, right side of the femur was collected and the bone absolute wet-weights were recorded after weighting the bones; however, dry-weights were measured after drying the bones at 200 °C for 8 h. In addition, the bone absolute ash weights were measured after carbonizing the dried bones in a furnace at 800 °C for 6 h [8]. The individual body weight variances were minimized by calculating the relative weights (% body weights) using the Eq. [3].

Bone strengths [Failure load (FL); unit: Newton (N)] of the mid-shaft regions of femurs (dried right femur) were measured through a three-point bending failure stress test by a computerized testing machine as reported previously [8, 50].

At sacrifice, whole blood (approximately 1 ml) was collected in a serum collection tube and the serum was separated by centrifugation at 15,000 rpm for 10 min under 4 °C. All collected samples were stored at − 150 °C using an Ultra-deep freezer (Sanyo, Tokyo, Japan) until further analysis. Mouse Osteocalcin ELIZA Kit and Mouse bALP ELIZA Kit, purchased from MyBioSource (CA, USA), were used for measuring the serum osteocalcin and bALP levels, respectively.

The ground bone ash powder was dissolved in nitric acid (HNO₃) and the inorganic phosphate (IP) and calcium (Ca) contents were measured by the previously reported o-cresolphthalein complexone [7], and enzyme [8] methods, respectively. Furthermore, Ca/IP ratios were determined by Eq. [4].

The separated left femur of each mouse was fixed in 10% neutral buffered formalin and decalcified for 3 days in a decalcifying solution [24.4% formic acid and 0.5 N sodium hydroxide]. Following decalcification, femur trochlea head regions were longitudinally trimmed, embedded in paraffin, sectioned in 3–4 μm, and stained with hematoxylin and eosin (HE). In each prepared histological sample, the histological profiles were interpreted. The prepared histological samples were coded for blind histopathological analysis. Bone histomorphometry was performed by an automated image analyzer and the bone structure, resorption, and mass were measured in the cortical or epiphyseal regions of the femur. Thickness of the cortical bone was measured in the femurs’ mid-shaft region. Trabecular bone thickness (Tbt), length (Tbl), number (Tbn) and volume (TBV), and thickness of cortical bone (Cbt) for structure and mass, and osteoclast cell number (Ocn) and osteoclast cell ratios (OS/BS) were estimated for bone resorption [7‐9].

The experimental data is stated as mean ± standard deviation (S.D.) of eight animals. Different doses of test material were compared by the multiple comparison tests. Levene’s test was used to examine the variance homogeneity [51]. If no significant deviation was observed by the Levene’s test, then the obtained data was evaluated by one-way ANOVA by least-significant differences multi-comparison (LSD) test. If a significant deviation from variance was observed by the Levene’s test, groups were compared by a non-parametric comparison test (Kruskal-Wallis H test). If the Kruskal-Wallis H test showed a significant difference, the Mann-Whitney U test was performed to estimate the significantly different specific pairs of groups. Differences were considered significant at p < 0.05 and/or p < 0.01. SPSS ver. 14 (IBM-SPSS Inc., Chicago, IL, USA) was used for the statistical analyses [52]. To estimate the effectiveness of the test material, the percent fluctuations between treated-mice and the OVX-control were estimated respectively by Eq. [5], and Eq. [6] as demonstrated previously [53].

Eight mice from each group showing significant (p < 0.05) increases in body weights were selected for OVX models 34-days post-surgery [OVX mice: 37.38 ± 2.71 g, weight range of 34.2 g to 44.4 g; sham-operated mice: 33.86 ± 2.53 g, weight range 30.3 g to 37.5 g]. All OVX-control groups revealed significant increases in body weights. Furthermore, significantly (p < 0.01; p < 0.05) higher body weight gains were observed in OVX-mice during the 5-weeks of OVX recovery/induction period. Although, obvious decreases in body weights were noticed during some periods of treatment with EAP:TM 1:1, 1:5, 3:1, and 7:1 as compared with the OVX-mice; animals treated with all EAP and TM formulations showed significant (p < 0.01) decreases in body weight gains. Specifically, EAP:TM (3:1) treated OVX mice revealed significant (p < 0.05) reductions in body weight gains as compared with mice treated with other formulations. RES (2.5 mg/kg)-treated mice showed no significant variations in body weights and body weight gains throughout the entire experimental period (Table 1; Additional file 1).

OVX control groups showed significant decreases in absolute and relative wet, dry, and ash weights of femur as compared with that in sham control groups. Conversely, noticeable increases in wet, dry, and ash weights of femur were demonstrated by all mice administered with test substances, including RES as compared with OVX controls. In particular, EAP:TM 3:1-treated groups exhibited significantly (p < 0.01; p < 0.05) higher femur relative wet-weights, and absolute and relative dry and ash weights as compared with the mice administered with EAP or TM single formulations (Table 2; Additional file 2).

OVX control groups displayed a significant (p < 0.01) decline in serum bALP activity and a noteworthy rise in serum osteocalcin. However, all formulations of EAP and TM significantly (p < 0.01) reduced the serum osteocalcin levels and significantly (p < 0.01) increased the bALP activity. Particularly, EAP:TM (3:1) treated mice revealed significant (p < 0.01; p < 0.05) decreases in serum osteocalcin content and increases in serum bALP activity. RES (2.5 mg/kg) supplied OVX mice also showed significantly (p < 0.01) decreases in serum osteocalcin levels; yet, both groups demonstrated similar serum bALP activity compared to the OVX control group (Figs. 2 and 3; Additional file 3).

A significantly decreased total body and femur mean BMD were observed in OVX mice. However, the test substances-administered groups showed significantly increased femur mean BMD and total body, including the EAP single formulation, compared with that in OVX control mice. In particular, EAP:TM (3:1)-treated mice showed significant (p < 0.01) increased femur mean BMD and total body as compared to mice supplied with the single formulations of EAP or TM (Table 3; Fig. 4; Additional file 4).

The strength (FL) of the mid-shaft region of the right dry femur in OVX mice showed noteworthy decreases; while, significant (p < 0.01; p < 0.05) increases in the femur FL were detected in all groups treated with test substances, including TM single formulations, compared with that in OVX controls. Specifically, EAP:TM (3:1)-treated mice consistently showed significant (p < 0.01) increases in the femur FL compared to mice treated with single formulations of EAP or TM (Table 3; Additional file 5).

OVX mice showed significant (p < 0.01) decreases in femur Ca and IP contents. While, a significantly (p < 0.01; p < 0.05) increased trend in femur Ca and IP contents was observed in all groups treated with the test substances, especially in mice treated with EAP:TM (3:1). No noteworthy variation in femur Ca/IP ratio was observed in OVX mice compared with that in sham control. Furthermore, no significant differences in femur Ca/IP ratios were detected in mice administered with the test substances, including RES (2.5 mg/kg), compared with that in the OVX control group (Table 3; Additional file 6).

OVX control mice showed a typical osteoporotic histological profile, whereas left femur of the sham-control mice showed relatively well-developed cortical and trabecular bones. The periosteum of cortical bones showed increased connective tissues, however, a dramatic decrease in cortical and trabecular bone mass was observed. Although, RES reduced the trabecular bone loss, no changes in cortical bone masses were observed. However, a dramatic increase in bone structure and mass of cortical and trabecular bones was observed for all the tested formulations; especially, EAP:TM (3:1) treated groups showed a visible inhibition in the bone mass loss of femur and the histopathological activation of osteoclast cells than the other treatment groups (Tables 4 and 5; Fig. 5).