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Acta Pharm. 72 (2022) 289–302 Short communication https://doi.org/10.2478/acph-2022-0012 Benec fi ial eec ff ts of the fructus Sophorae extract on experimentally induced osteoporosis in New Zealand white rabbits LIDIJA CHAKULESKA Sophora japonica is a source of several flavonol, fla - ALEKSANDAR SHKONDROV vone and isoflavone glycosides that are reported to GEORGI POPOV 4 positively affect menopausal symptoms including NADYA ZLATEVA-PANAYOTOVA RENETA PETROVA osteoporotic complications. In the present study MARIYANA ATANASOVA fructus Sophorae extract (FSE) was administered ILINA KRASTEVA –1 orally for three months at a dose of 200 mg kg in IRINI DOYTCHINOVA 1,* ovariectomized (OVX) New Zealand rabbits. 3D RUMYANA SIMEONOVA computed tomography scans and histopathological Department of Pharmacology, Pharmacotherapy images revealed microstructural disturbances in and Toxicology, Faculty of Pharmacy, Medical the bones of the castrated animals. FSE recovered University of Sofia, 1000 Sofia, Bulgaria most of the affected parameters in bones in a man - Department of Pharmacognosy, Faculty of ner similar to zoledronic acid (ZA) used as a posi- Pharmacy, Medical University of Sofia, 1000 tive control. The aglycones of the main active com- Sofia, Bulgaria pounds of FSE, daidzin, and genistin, were docked into the alpha and beta estrogen receptors and sta- Department of Internal Non-infectious Diseases ble complexes were found. The findings of this Pathology and Pharmacology, Faculty of study provide an insight into the effects of FSE on Veterinary Medicine, University of Forestry 10 bone tissue loss and suggest that it could be further 1756 Sofia, Bulgaria developed as a potential candidate for the preven- Department of Surgery and Radiology, Faculty of tion of postmenopausal osteoporotic complica- Veterinary Medicine, University of Forestry, 10 tions. 1756 Sofia, Bulgaria Keywords: osteoporosis, Sophora japonica fruits, National Diagnostic and Research Veterinary ovariectomized rabbits, molecular docking, estro- Institute, Department: National Center of Animal gen receptors Health, Sofia, Bulgaria Department of Chemistry, Faculty of Pharmacy Medical University of Sofia, 1000 Sofia, Bulgaria Accepted April 7, 2021 Published online April 9, 2021 Osteoporosis (OP) is a common non-communicable disease, ae ff cting more than 200 million individuals worldwide and the leading cause of morbidity and death due to bone fractures (1). Oxidative stress (OS) is considered as the common pathogenic factor for ‘menopausal syndrome’ that includes several disturbances and diseases, such as post- menopausal osteoporosis. The estrogen decline associated with the menopausal transition is believed to be the ‘spark’ that triggers OS. A vast body of studies shows that bilateral ovariectomy induces a redox imbalance characterized by increased levels of lipid per- * Correspondence; e-mail address: firstname.lastname@example.org 289 L. Chakuleska et al.: Benec fi ial effects of the fructus Sophorae extract on experimentally induced osteoporosis in New Zealand white rabbits, Acta Pharm. 72 (2022) 289–302. oxidation markers and decreased activity of antioxidant enzymes (2). Some additional factors might contribute to developing osteoporosis. Metabolic syndrome is a constel- lation of medical conditions consisting of central obesity, hyperglycemia, hyperten- sion, and dyslipidemia, in which each acts on bone tissue in a dier ff ent way. It may play a key role in the pathogenesis of OP (3). This investigation is a continuation of our ongoing studies on plant-derived agents that could positively ae ff ct osteoporosis, in which we described the bone-pro - tective ee ff cts of Ruscus aculeatus extract in ovariectomized rats (4). To carry out the present experiment, an extract from fruits of Sophora japonica, which is known to con- tain isoflavones, which have been shown to provide estrogen-like ee ff cts in meno - pause, was used. Sophora japonica L. (Fabacaeae) is a source of natural isoflavones that could help alleviate postmenopausal symptoms. In a randomized double-blind placebo- -controlled clinical trial (5) it was shown that the consumption of the fructus Sophorae extract (FSE) possessed benec fi ial ee ff cts on the postmenopausal symptoms in women and may also decrease the risk of fracture in osteoporotic patients. Isoa fl vonoids have been the focus of studies on natural phytoestrogens and the complex of isoa fl vone glycosides extracted from fructus Sophorae was listed as a potential cause for the bene - c fi ial ee ff cts (6). These bioactive substances have been shown to exert potential anti- osteoporotic ee ff cts in both in vitro and in vivo models in rats (7–9), but to our know - ledge, they were not investigated in animals with bigger bones. In this case, however, we focused on another type of experimental animal – rabbits. They have some advan- tages over more commonly used rats, including bigger weight and thus, larger bones, similar to those of big mammals. For experimental investigation of postmenopausal osteoporosis, in addition to the common ovariectomy (OVX)-induced rat model of estrogen dec fi iency, the American Food and Drug Administration (FDA) recommends the use of larger species including rabbits, sheep, goats, dogs, and swine, which have the ability to remodel their bone cortices (10, 11). Rabbits are the smallest commonly employed laboratory animals with well-defined spontaneous cortical remodelling similar to humans, shorter remodel - ling period than larger animal models, and relatively rapid skeletal maturation (by six to nine months) (11). In addition, we decided to use another imaging method for the detection of bone abnormalities, a computed tomography (CT) scanner, which generates high-quality and more accurate images of the bone areas we are interested in; CT scanner focuses on the target area beer t tt han the X-ray. Thus, the aim of the present study was to investigate the ee ff cts of FSE on the skeletal system of rabbits with OVX-induced osteoporosis by dier ff ent imaging tech - niques and to compare the ee ff cts of the extract with the well-known anti-osteoporotic drug, zoledronic acid (ZA). EXPERIMENTAL Solvents and reagents Dichloromethane and methanol for extraction were purchased from Sigma-Aldrich, (Germany) and were of analytical grade. Acetonitrile (Sigma-Aldrich), o-phosphoric 290 L. Chakuleska et al.: Benec fi ial effects of the fructus Sophorae extract on experimentally induced osteoporosis in New Zealand white rabbits, Acta Pharm. 72 (2022) 289–302. acid and Milli-Q water (Merck, Germany) were of HPLC grade. Daidzin CRS and genistin CRS (purity over 95 %) were purchased from Chromadex (USA). Ketamine hydrochloride, xylazine, and zoledronic acid were purchased from a pharmacy in Soa fi , Bulgaria. Commercially available kits (see below) were used for measurement of calcium, phosphorus, osteocalcin, Beta-CrossLaps, and parathormone. Plant material Fruits of Sophora japonica (Fabaceae) were collected from the town of Bitola, R. N. Macedonia in September 2018. The species was identie fi d by Ilina Krasteva and a voucher specimen was deposited in the herbarium of the Faculty of Pharmacy, Medical University of Soa fi , Bulgaria (FF-127-19). Extraction The air-dried and pulverized fruits (500 g) were defae tt d with dichloromethane (5 × 2 L) to remove the lipophilic compounds and then exhaustively extracted with 80 % MeOH by percolation. The resulting methanol extract was evaporated to eliminate the solvent, lyophilized and marked as FSE (190 g). HPLC analysis The isoa fl vone content of FSE was analysed by a known HPLC method (12). The method was re-validated. Daidzin CRS and genistin CRS (Fig. 1) were dissolved in –1 methanol (1 mg mL ) and serial dilutions were made to make the calibration curves. A Young Lin 9100 HPLC system (Hogyedong, Korea) equipped with YL 9101 vacuum degasser, YL 9110 quaternary pump, YL 9131 column compartment, YL 9160 PDA detector, 7725i manual injector and software Clarity (v. 2) was used. Separations were performed on a pre-packed ODS column Luna (100 Å, 250 × 4.6 mm, 5 µm, Pheno - menex, USA), coupled with Security Guard octadecylsilyl cartridge (Phenomenex). The binary solvent system consisted of o-phosphoric acid (0.1 %) and acetonitrile. The –1 o fl w rate was 1.0 mL min . The temperature was set at 40 °C. The injected volume was 20 µL. All chromatograms were recorded at 260 nm. All injections were made in tripli- cates and the mean value was calculated. Animals The studies were performed on sixteen skeletally mature, female New Zealand white rabbits, six-month-old, weighing 2.50 ± 0.34 kg. The experimental animals were Fig. 1. Structures of daidzin and genistin. 291 L. Chakuleska et al.: Benec fi ial effects of the fructus Sophorae extract on experimentally induced osteoporosis in New Zealand white rabbits, Acta Pharm. 72 (2022) 289–302. purchased from the National Breeding Center in Slivnitsa, Bulgaria, and were kept under standard conditions in metal cages, at a temperature of 20–25 °C, mode 12 h/12 h light/dark. The standard commercial pelleted feed and drinking water were available ad libitum during the entire experimental period of 90 days. Seven-day acclimatization of the animals was allowed before the beginning of the experiment, with regular mon -i toring of their health condition by a veterinarian. The rabbits were housed, maintained and euthanized in accordance with the relevant international rules and recommenda - tions as stated in the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientic fi Purposes (ETS 123). The Animal Care Ethics Commie tt e approved the study protocol and Ethics clearance (No 233 from 09/02/2019) was issued for the study by the Bulgarian Agency for Food Safety. Study design Sixteen female New Zealand rabbits were divided into individual cages at ran- th dom into four groups. After the acclimatization period, on the 7 day, blood from the ear vein of all rabbits was collected to establish the base level of all investigated bio- chemical parameters. Thereafter, an abdominal resection without removing the ova - ries was performed in four rabbits. They served as a control group. The other 12 ani- mals underwent bilateral ovariectomy (OVX) (13). One week after the surgical procedure, the animals were divided into groups (n = 4 each) as follows: group 1 – control group of rabbits (sham) with free access to drinking water, group 2 – OVX control rabbits with free access to drinking water, group 3 – OVX –1 rabbits treated with FSE (200 mg kg bm) dissolved in drinking water and adminis - tered per os for a period of 90 days, group 4 – OVX rabbits treated once subcutane- –1 ously with zoledronic acid (ZA) as a positive control (100 µg kg bm) diluted with saline (14). th th On the 7 and 45 day, after fasting, blood from the ear vein from all animals was taken to examine the biochemical markers of calcium homeostasis and bone turnover. th th From 85 to 90 day, blood was taken again for biochemical analysis. Thereafter, the animals were anaesthetized via intramuscular injection with the combination of 90 –1 –1 mg kg ketamine hydrochloride and 3 mg kg xylazine (14) and then were subjected to computed tomography of the vertebral, pelvic and long bones areas. On day 91, all animals were euthanized by CO respiration. The vertebral, pelvic and long bones were excised and prepared for histological analysis. Markers of calcium homeostasis On days 7, 45, and 85 blood from the ear vein was collected in tubes containing a clot activator. After centrifugation at 3,000×g for 10 min, the serum was separated and stored at –20 C for evaluation of investigated parameters. Calcium and phosphorus were measured using commercially available kits for biochemical analyzer Mindray – BP 120 (China). Parathyroid hormone (PTH) levels were analyzed using electrochemiluminescence immunoassay “ECLIA” with kits for immunoassay analyzer (Cobas-Roche, Switzerland). 292 L. Chakuleska et al.: Benec fi ial effects of the fructus Sophorae extract on experimentally induced osteoporosis in New Zealand white rabbits, Acta Pharm. 72 (2022) 289–302. Markers of bone turnover Osteocalcin (OC), a marker of bone formation, and Beta-CrossLaps (β-CTx), a th th th marker of bone resorption, were analyzed on the 7 , 45 and 85 days using “ECLIA” with kits for immunoassay analyzer (Cobas-Roche). Computed tomography Evaluation of bone density was performed under general anaesthesia with a CT Picker CT PQ 5000 scanner. The CT scan machine was manipulated under 60 kV and 180 µA. The scanned images of each specimen were analyzed on the computer using the DICOM Viewer software. Bone density measurements were made at three points in dier ff ent segments of the caudal skeleton as follows: pelvic area (Pel) – in the dorsal middle area (left and right), in the area of the os ileum (left and right); the proximal segment of the femur (PF) – in the trochanter area, femoral neck, and proximal compact zone; vertebrae – in the corpus vertebrae area and laterodorsal parts of arcus vertebrae (left and right) of the fourth (Ver ), fifth (Ver ), sixth (Ver ) and seventh (Ver ) vertebrae. The mean bone 4 5 6 7 density values in Hounse fi ld units (HU) of the bone were recorded using a CT Picker CT PQ 5000 scanner. The results obtained are presented as mean values measured in HU (Hounse fi ld scale). Histological examination of rabbits’ bone Femurs and lumbar vertebrae of the rabbits from all groups were removed post mortem and fixed in 10 % buer ff ed formalin for 48 h. Fixed bones were placed in a decalcic fi ation solution (formic acid) for 14 days and then processed according to the classical paraffin method. The sections were stained with hematoxylin and eosin (H&E). Histological changes were observed and photo-documented with a light micro- scope with a built-in camera Euromex BioBlue (Belgium). Molecular docking of genistein and daidzein The aglycones daidzein and genistein were studied because gastrointestinal hydro- lysis of daidzin and genistin occurs after p.o. administration (4). The 3D structures of daidzein (DAI) and genistein (GEN) were retrieved from PubChem and docked into the ligand-binding site of the alpha and beta forms of the human estrogen receptor (ERα and ERβ) derived from RCSB (pdb ids: 1×7r, R = 2.0 Å and 1×7j, R = 2.3 Å, resp.) (15). Docking was performed by GOLD v. 5.2.2 (CCDC Ltd., Cambridge, UK) at the following settings: evaluation function ChemPLP, e fl xible ligand and binding site, radius 6 Å at the binding site, included a water molecule (HOH7 for ERα and HOH1 for ERβ) and 100 GA cycle. The residues from the binding site that are in close proxi- mity to the bound ligand were set to be e fl xible. For Erα, these were Met343, Leu346, Leu349, Glu353, Met388, Leu391, Arg394, Phe404, Ile424 and His524; for Erβ, these were Met295, Leu298, Leu301, Glu305, Met340, Leu343, Arg346, Phe356, Ile376 and His475. Genistein from the X-ray structures was used as a reference molecule in both ERs. 293 L. Chakuleska et al.: Benec fi ial effects of the fructus Sophorae extract on experimentally induced osteoporosis in New Zealand white rabbits, Acta Pharm. 72 (2022) 289–302. Statistical analysis Statistical analysis was performed using ‘MEDCALC’, v. 12.3 (MedCalc Software, Belgium). The signic fi ance of the experimental data was assessed using the non-para- metric Mann-Whitney U test. Values of p ≤ 0.05 were considered statistically signic fi ant. RESULTS AND DISCUSSION Phytochemical analysis of FSE HPLC analysis of FSE was performed by modie fi d method of Krenn and Pötsch (12). Calibration curves were constructed for each reference substance and the equa- tions and coec ffi ients of determination were obtained: R = 0.9996 for genistin (t = 18.853 ± 0.236 min) and R = 0.9998 for daidzin (t = 16.047 ± 0.343 min). Limits of detec - –1 –1 tion were: 0.0001 mg mL (genistin) and 0.0002 mg mL (daidzin). Peaks corresponding to daidzin and genistin were identie fi d in the chromato - gram of FSE (Fig. 2). Mean retention times, as well as UV spectra of the compounds (recorded by the PDA detector) in the sample, were consistent with those of the reference substances. It was found that 1 mg of FSE contained 0.057 mg daidzin and 0.085 mg genistin. The content of the isoa fl vonoid glycosides, calculated as per 1 g dry plant material was 0.03 g daidzin and 0.05 g genistin. Molecular docking of genistein (GEN) and daidzein (DAI) into the estrogen receptors The best-scored docking poses of GEN and DAI into ERα and ERβ are presented in Fig. 3. According to the ChemPLP scores, both molecules bind beer tt to the ERβ –1 Fig. 2. HPLC chromatograms of: a) standard substances daidzin and genistin (a mixture, 0.1 mg mL of each) and b) of FSE, with peaks corresponding to daidzin (15.8 min) and genistin (18.8 min). Other peaks were not identie fi d. 294 L. Chakuleska et al.: Benec fi ial effects of the fructus Sophorae extract on experimentally induced osteoporosis in New Zealand white rabbits, Acta Pharm. 72 (2022) 289–302. a) b) c) d) Fig. 3. The best-scored docking poses and interactions of GEN and DAI with ERα (a and c) and ERβ (b and d), resp. Hydrogen bonds are shown as green dots. The structural water molecule is presented in a “ball and stick” style. receptor than to the ERα (Table I). The ER binding site is an oval hydrophobic pocket with polar residues at both ends. The two molecules bind in a similar way to both receptors. The isoa fl vone moiety lies tangentially on the hydrophobic pocket, whereas the OH groups at both ends form hydrogen bonds with the polar residues. The phenolic Table I. ChemPLP fitness scores, piecewise linear potential scores S(PLP) and root mean square deviations (RMSD) for genistеin (GEN) and daidzеin (DAI) docked into estrogen receptors ERα ERβ Compound ChemPLP RMSD (Å) S(PLP) ChemPLP RMSD (Å) S(PLP) GEN 87.70 1.342 –75.74 93.49 1.299 –85.26 DAI 88.28 1.233 –76.35 93.63 1.299 –82.26 The bound GEN from the X-ray structures was used as a reference ligand. ERα – estrogen receptor alpha form; ERβ – estrogen receptor beta form; ChemPLP – GOLD ChemPLP t fi ness score (ChemPLP is the name of the function; GOLD is the protein-ligand docking software), RMSD – a root mean square deviation of the docked pose according to the crystallographic structure, S(PLP) – a piecewise linear potential score included in ChemPLP fitness score 295 L. Chakuleska et al.: Benec fi ial effects of the fructus Sophorae extract on experimentally induced osteoporosis in New Zealand white rabbits, Acta Pharm. 72 (2022) 289–302. OH (4’-OH) forms three hydrogen bonds with Glu353 and Arg394 from ERα and with Glu305 and Arg346 from ERβ. Additionally, it binds to the structural water molecule that forms a dense network of hydrogen bonds between the ligand and ER. The iso- a fl vone 7-OH group forms a hydrogen bond with His524 from ERα and with His475 from ERβ. The same group in GEN makes an additional hydrogen bond with Leu476 on ERβ. The isoa fl vone 5-OH group of GEN does not form any hydrogen bonds. The higher ChemPLP scores for ERβ are coming from the higher S(PLP) values. The term S(PLP) in ChemPLP evaluates the steric complementarity between ligand and receptor (16). More negative values correspond to beer tt complementarity. The results are in good agreement with the 40 times higher selectivity of GEN to ERβ (15). The estrogenic potency of phytoestrogens is associated mainly with ERβ activation triggering many of the biological responses, including osteogenic ee ff cts (17). On the other side, most of the side-ee ff cts of estrogens are associated with the activation of ERα (18). The beer b tt inding to ERβ could be a benec fi ial property of GEN and DAI. Body mass changes Throughout the experiment, all animals gained weight. OVX rabbits gained 47 % (p < 0.05) compared with the initial value at the beginning of the experiment. At the end of the experimental period, the highest body mass gain (BMG) was observed in OVX rabbits by 29 % (p < 0.05), compared to the BMG of the control group. When com - pared to the OVX group at the end of the experiment, the groups treated with FSE and ZA showed signic fi antly lower BWG of 24 and 22 %, resp. The decreased level of estrogens induced by OVX is linked to body mass increase because estrogens and estrogen receptors regulate various aspects of glucose and lipid metabolism (19). The results of this study are in line with the meta-analysis, performed by Akhlaghi and co-workers (20) showing that isoflavones may reduce body mass index (BMI) in women. Parameters of calcium homeostasis and bone turnover markers The changes in parameters of calcium homeostasis and bone turnover markers th are shown in Table II. On the 7 day, all measured parameters were similar to the th control levels. No die ff rences were observed between the groups. On the 45 day, the levels of calcium were higher (p < 0.05) in untreated and all treated OVX rabbits than in the sham control group. In the OVX, FSE and ZA groups calcium level was higher by 35, 26 and 20 %, resp., compared to the controls (p < 0.05). Calcium concentrations in FSE and ZA-treated groups were not statistically dier ff ent from the OVX group on th the 45 day. At the end of the experiment, calcium concentrations in OVX, FSE and ZA-treated groups were still signic fi antly higher – 42, 33 and 31 % resp., compared with the sham-controls and similar to the OVX group. The increased bone turnover following estrogen deprivation after OVX is one of the reasons for higher calcium levels. Probably the observed hypercalcemia suppressed the secretion of the parathy- roid hormone (PTH) which was not detectable in this study (Table II). Phosphorus level, measured as phosphates, was higher (p < 0.05) by 58 and 50 % in OVX and FSE-treated group, resp., compared to the control group. The bone turn- over rate was probably higher than in the control and ZA group, leading to an increased 296 L. Chakuleska et al.: Benec fi ial effects of the fructus Sophorae extract on experimentally induced osteoporosis in New Zealand white rabbits, Acta Pharm. 72 (2022) 289–302. ff ff fi ff fi Table II. Biomarkers of calcium homeostasis and bone turnover markers th th th 7 day 45 day 85 day Biomarker Control OVX FSE ZA Control OVX FSE ZA Control OVX FSE ZA Calcium a a a,b a a a 2.48 ± 0.12 2.32 ± 0.16 2.21 ± 0.11 2.42 ± 0.12 2.81 ± 0.22 3.79 ± 0.18 3.55 ± 0.10 3.36 ± 0.08 2.54 ± 0.20 3.61 ± 0.31 3.34 ± 0.18 3.41 ± 0.25 –1 (mmol L ) Phosphorus a a b a a b 1.59 ± 0.12 1.61 ± 0.15 1.66 ± 0.21 1.62 ± 0.26 1.53 ± 0.18 2.55 ± 0.30 2.42 ± 0.25 1.76 ± 0.15 1.52 ± 0.20 2.56 ± 0.33 2.40 ± 0.13 1.81 ± 0.12 –1 (mmol L ) OC a a a,b a a a,b 6.8 ± 1.41 6.2 ± 1.02 6.4 ± 1.22 7.2 ± 1.44 5.8 ± 1.14 11.2 ± 0.56 11.4 ± 1.72 17.7 ± 1.67 6.15 ± 070 9.0 ± 0.78 10.4 ± 1.03 14.2 ± 0.95 –1 (ng mL ) β-CTx < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 –1 (ng mL ) PTH < 4 < 4 < 4 < 4 < 4 < 4 < 4 < 4 < 4 < 4 < 4 < 4 –1 (pg mL ) Results are expressed as mean ± SD (n = 4). a b Statistically signicant dierence: p < 0.05 vs. control; p < 0.05 vs. OVX. Table III. Values of CT of dierent segments in HU Measurements in the region of Group Proximal femur th th th th Pelvis – Pel 4 vertebra – Ver 5 vertebra – Ver 6 vertebra – Ver 7 vertebra – Ver 4 5 6 7 – PF Control 1233.25 ± 67.28 1121.12 ± 46.12 911.15 ± 56.79 878.78 ± 31.23 856.8 ± 37.58 874.55 ± 46.46 a a a a a a OVX 941.5 ± 14.85 649.84 ± 39.24 816.23 ± 13.2 778.13 ± 22.41 720.04 ± 16.19 615.33 ± 56.33 b b b b b FSE 1060.08 ± 36.52 1022.66 ± 68.78 877.63 ± 20.94 911.08 ± 37.49 832.58 ± 63.28 696.33 ± 39.12 b b b b b b ZA 1141.67 ± 47.05 936.58 ± 21.93 919.59 ± 39.12 1059.25 ± 22.28 916 ± 15.66 788.23 ± 28.36 a b Results are expressed as mean ± SD (n = 4). Statistically signicant dierence: p < 0.05 vs. control; p < 0 .05 vs. OVX. L. Chakuleska et al.: Benec fi ial effects of the fructus Sophorae extract on experimentally induced osteoporosis in New Zealand white rabbits, Acta Pharm. 72 (2022) 289–302. –1 blood phosphorus level in both groups. Treatment of OVX rats with ZA (100 µg kg ) decreased signic fi antly the phosphorus level by 31 %, compared to the OVX group and restored this marker to near the control level, which could mean that the drug more ee ff ctively incorporates phosphorus into bones. At the end of the experimental period, th the results for phosphorus levels were similar to those of the 45 day. OVX and FSE- -treated groups had signic fi antly higher phosphorus level with 68 and 37 %, resp., th than the control. On the 85 day of the experiment, ZA decreased signic fi antly the phosphorus level by 36 %, compared to the OVX group (Table II). This decrease was not seen in the FSE-treated group. th On the 45 day the osteocalcin levels were higher by 93, 97 and 205 % in OVX, FSE, and ZA group, resp., compared to the sham control rabbits (p < 0.05). This trend persisted until day 85, when this marker was 46, 68 and 130 %, higher in the OVX, FSE and ZA groups resp., compared to the control group. These results are in accordance with the fact that osteocalcin levels are increased in metabolic bone diseases with incre ased bone or osteoid formation, including osteoporosis (21). It is the most abundant non- -collagen protein in the bone matrix, where it regulates mineralization and is produced by mature osteoblasts during bone formation (22). In ZA treated group, as opposed to the FSE group, osteocalcin level was higher for 58 % (p < 0.05) than in the OVX only th th group on the 45 as well as the 85 day. This probably means that the ZA stimulates bone re-mineralization in osteoporotic animals more ec ffi iently than FSE. The levels of the parathyroid hormone (PTH) and Beta-CrossLaps (β-CTx) were below the detection limit of the method used in all experimental groups (Table II). It might be speculated that this is due to the hypercalcemia from one side, and the pre- dominance of the process of bone formation than the process of resorption from another side. Further investigations are needed in order to explain why this happened also in the control animals. Computed tomography The dier ff ences in the bone structure of the three main regions of interest [pelvic area (Pel), the proximal segment of the femur (PF) and vertebrae (Ver)] are presented in Table III. Data show that the highest (p < 0.05) bone density in all studied segments was found in the group treated with ZA (Pel –1141.67 HU; PF – 936.58 HU; Ver – 1069.32 4,5,6,7 HU). These findings are consistent with the results obtained by Öztürk et al. (14) who found that ZA stimulated bone formation and decreased the relapse ratio after expan- sion in the sagia tt l suture of rats. Nevertheless, FSE also presented similar well-defined protective bone microstructural ee ff cts that were visible on the histomorphological photomicrographs and CT scanner (Figs. S1-S3). The most prominent ee ff ct of FSE on bone mass density (BMD) was seen in the proximal region of the femur (Fig. S2), where BMD was 9 % higher than in the ZA group. Generally, the most pronounced ee ff cts on BMD were seen on the proximal region of the femur (Table III, Fig. S2). OVX decreased BMD signic fi antly by 42 %, compared to the control group, whereas FSE and ZA increased BMD by 57 and 44 %, resp., compared to the OVX group. Most of the fractures in post- menopausal women appear in the region of the hip (1, 4), so FSE could be a good candidate for the prevention of fractures in this bone region. 298 L. Chakuleska et al.: Benec fi ial effects of the fructus Sophorae extract on experimentally induced osteoporosis in New Zealand white rabbits, Acta Pharm. 72 (2022) 289–302. The weakest ee ff ct on bone density was seen in the region of Ver4. BMD decreased by 10 % only in castrated animals, compared with the controls. FSE and ZA increased th BMD in the 4 vertebrae by 8 and 13 %, compared to OVX animals (Table III, Fig. S3). From the computed tomography examinations, it was found that FSE showed bone- protective ee ff cts, although less pronounced than ZA. Compared to non-treated cas - trated rabbits, FSE treatment signic fi antly increased bone density as follows: in the pelvic th th region – by 13 %, in the proximal femur by 57 %, in the 4 vertebra – by 8 %, in the 5 th th vertebra – by 17 %, in the 6 vertebra by 16 %, and in the 7 vertebra by 13 % (Table III). Histological examination of the bones of rabbits Osteoporosis rabbit’ models hold great potential as platforms for advancing our understanding of cortical remodelling and associated bone porosity (9). Microscopic examination of the lumbar vertebrae and femurs from the control animals revealed Fig. 4. Photomicrographs of the histological specimens of the lumbar vertebrae of rabbits (H&E stain - ing). a) Control group: normal histoarchitectonics of bone structure (100×); b) rabbit with ovariectomy: thinning of the lamellar bone tissue with the presence of destructive foci (arrows) (100×); c) rabbit treated with FSE: normal histological structure of the bone with signs of the regenerative process (100×); d) rabbit treated with ZA: unaltered histological structure of the bone (100×). 299 L. Chakuleska et al.: Benec fi ial effects of the fructus Sophorae extract on experimentally induced osteoporosis in New Zealand white rabbits, Acta Pharm. 72 (2022) 289–302. normal histological parameters, expressed in unaltered spongious substance and compact tissue, as well as normal shape, size, and density (Figs. 4a and 5a). In cas- trated rabbits, thinning of the lamellar structure was found in vertebral bodies and femoral diaphysis. In some areas of the vertebrae, destructive changes were detected, expressed in the disintegration of the bone tissue (Fig. 4b). Similar changes with evi- dence of osteodystrophy were observed in the femurs (Fig. 5b). In animals treated with FSE and ZA, a preserved microscopic bone structure was observed. In some areas, recovery processes were found, expressed in the ossic fi ation of the vertebrae bodies and normal histological structure of the lamellar component (Figs. 4c, d). The same histological features were observed in the femoral diaphysis. Signic fi ant regenerative processes were found in some areas, expressed in mineralization and ossic fi ation of the femurs with properly formed lamellar structure (Figs. 5c, d). Isoa fl vonoids, such as daidzein and genistein, showed therapeutic ee ff cts in the case of osteoporosis. Yoon and collaborators (23) showed that genistein is the most Fig. 5. Photomicrographs of the histological specimens of the right femurs of the rabbits (H&E stain- ing). a) Control group: normal histological structure of the bone (100×); b) rabbit with ovariectomy: lytic and destructive changes in the bone structure (arrow) (400×); c) rabbit from FSE group: normal histological structure of the bone with signs of mineralization and ossic fi ation (100×); d) rabbit from ZA group: normal histological architecture of bone with signs of regeneration (100×). 300 L. Chakuleska et al.: Benec fi ial effects of the fructus Sophorae extract on experimentally induced osteoporosis in New Zealand white rabbits, Acta Pharm. 72 (2022) 289–302. abundant compound found in extracts from fruits of S. japonica and a key phytoestrogen with osteogenic activity. Moreover, this team studied the ee ff cts of genistein in vitro on cell lines, which supports our discussion and allows us to extrapolate their results to the present in vivo findings, especially with larger laboratory animals such as rabbits, which have advantages over rats, as we have already clarie fi d above. Some other experimental studies concluded that daidzein is even more ec ffi ient than genistein in preventing ovariectomy-induced bone loss in rats (24). Nevertheless, there is limited information on the activity of extracts, containing the glycosidic forms of these isoa fl vonoids. That is why we investigated the FSE standardized by its glycosidic content. CONCLUSIONS In conclusion, our findings suggest that FSE demonstrates a signic fi ant, but less pronounced, bone protective ee ff ct in OVX-induced osteoporotic rabbits compared to zoledronic acid. 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Acta Pharmaceutica – de Gruyter
Published: Jun 1, 2022
Keywords: osteoporosis; Sophora japonica fruits; ovariectomized rabbits; molecular docking; estrogen receptors
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