| BackgroundMorinda officinalis capsule extract has been listed Chinese medicine(Approved by Z20050069), made up of Morinda officinalis, Polygonum multiflorum, Epimedium (leaves), Radix Dipsacus and other herbs (16kinds herbs in total). It can tonify the kidney and regulate menstruation, and is therefore effective for the syndrome of Kidney-Yang Deficiency, as manifested in the appearance of such symptoms as physical and mental fatigue, weak waist and knees, frequent urination at night, irregular menstruation, etc. The medicine lacks quality control standard of the effective composition, it plays a part in postmenopausal osteoporosis in the clinical application. The study was intended to verify the curative effect on postmenopausal osteoporosis, to explore the mechanism and to improve the quality standard of Morinda officinalis capsule extract.Objective1. The prevention and therapeutic effects as well as its mechanism of Morinda officinalis capsule extract were evaluated, to provide an experimental evidence for the extended clinical application. 2. A method for determination of Morinda officinal is compound was established to explore the pharmacodynamic materials basis on therapeutic effect.Methods1. The content determination of the main effective composition in Morinda officinalis capsule extractThe active components which were related to the potent anti-osteoporotic activity in Morinda officinalis capsule extract were determined by HPLC and UV, a method for determination of Morinda officinalis compound was established according to the principle of first part of Chinese Pharmacopeia2010. The major active ingredients which are responsible for osteoporosis in Morinda officinalis capsule extract probably as follows:The total polysaccharide and total anthraquinones of Morinda officinalis capsule extract, the stilbene glucoside, free emodin and physcion, the icarrin, the asperosaponin Ⅵ, the ferulic acid, the chlorogenic acid, the catapol in Morinda officinalis capsule extract.2. Effects of Morinda officinalis capsule extract on osteoporosis in ovariectomized female rats(1) Assay for biochemistryThe acclimatized female rats were induced for postmenopausal osteoporosis (PMOP) by bilateral ovariectomy and divided into seven groups as follows: sham-operated group, ovariectomized (OVX) control group, OVX treated with xianlinggubao (XLGB)(270mg·kg-1·-d-1), OVX treated with alendronate sodium (ALN)(3mg·kg-1·d-1) and OVX treated with MOP of graded doses (90,270,810mg·kg-1·-d-1) groups. Treatments were daily oral administered on the4th week after ovariectomy and last for12weeks. The body weight of every animal was measured once a week during the treating period. After laparotomy using anesthetized with chloral hydrate, blood and urine sample were collected via femoral artery puncture and metabolic cages. The calcium (Ca), phosphorus (P),1,25-dihydroxyvitamin D3(1,25(OH)2VD3), alkaline phosphatase (AKP), tartrate-resistant acid phosphatase (TRAP) levels in the serum, osteocalcin (OC), parathyroid hormone (PTH) and osteogenic growth peptide (OGP) in the plasma, pyridinoline (PYD) in the urine were determined by standard colorimetric and enzyme immunoassays methods.(2) Determination of bone inorganic mineral content (BMC)Bone tissue was dried to constant weight at60℃in drying oven with natural convection. The femoral wet weight, dry weight. The bone was burned at800℃for6hours by muffle furnace into ash, ash weight were determined by electronic analytical balance. The powder samples were divided into doses, weighing0.1g each sample. Bone tissue was mineralized by wet digestion. Bone trace elements concentrations of calcium (B-Ca), cuprum (B-Cu), zincum (B-Zn), ferrum (B-Fe), magnesium (B-Mg) were determined using atomic absorption spectrophotometer with a certain specific wavelength.(3) Measurement of bone mineral density (BMD)Two-dimensional total bone mineral density (t-BMD) of the left femur and lumbar vertebrae were measured by dual-energy X ray absorptiometry (DEXA)(GE Healthcare, USA) equipped with appropriate software for bone density assessment in small laboratory animals. BMD was calculated by BMC of the measured area.(4) Histomorphometric analysisThe bilateral tibial and lumbar vertebrae for morphology were excised, fixed with4%paraformaldehyde in0.1M phosphate buffer system (PBS, pH7.4) for3days at room temperature, and decalcified with6%HNO3in0.1M PBS at room temperature for1week. The tissue samples were then dehydrated in a vacuum desiccators through a graded ethanol series, then defatted in xylene and embedded in paraffin, and sectioned in5μm-thicknesses slides. The slides were processed for hematoxylin-eosin (H-E) staining. The specimens were subjected to histomorphometric analysis under a light microscope with a micrometer using Image-Pro Plus6.0image analyzer system (Media Cybernetics Inc. USA). The static parameters for the cortical and cancellous bone were measured.(5) Biomechanical testing The isolated right femurs were assessed with three-point bending test using the Instron1101universal material testing machine. The isolated lumbar vertebrae (L1-4) were assessed for structural strength test (compression test) by using tensile strength testing apparatus. Prior to mechanical testing, the right femurs were slowly thawed and held at room temperature on the day of test, the length of the femurs (distance from intermalleolar to intercondylar region) was measured with a micrometer and the middle of the diaphysis was determined. The intact femur then was placed in the material testing machine on two supports separated by a distance of20mm and load was applied to the middle of the diaphysis, thus creating a three-point bending test. The biomechanical quality of the right femoral diaphysis were determined using Instron1101universal material testing machine at a speed of2mm·min-1. The central loading point was displaced, and the load was recorded until the specimen was broken. The load-displacement curve and stress-strain curve were obtained.3. The study on Mechanisms of Morinda officinalis capsule extract on Osteoporosis in Ovariectomized RatsThe expression levels of osteoprotegerin (OPG) and receptor activator of nuclear factor-KB ligand (RANKL) protein in rats bone tissue were detected by immunohistochemistry assay, to explore the mechanism of Morinda of ficinalis capsule extract on postmenopausal osteoporosis.Results1. The content determination of the main effective composition in Morinda officinalis capsule extractThe HPLC and UV method of the effective composition in Morinda of ficinalis capsule extract were established according to the principle of first part of Chinese Pharmacopeia2010. The content of total polysaccharide and total anthraquinones in Morinda officinalis capsule extract is188.99mg·g-1and1.62mg·g-1respectively, the content of stilbene glucoside, free emodin and physcion from Morinda officinalis capsule extract is33.85mg·g-1,80.45μg·g-1and41.17ug·g-1respectively, the content of icarrin is3.23mg·g-1, the content of asperosaponin VI is5.04mg·g-1, the content of ferulic acid is0.55mg·g-1, the content of chlorogenic acid is0.95mg·g-1, the content of catapol is0.34mg·g-1in Morinda officinalis capsule extract.2. Effects of Morinda officinalis capsule extract on osteoporosis in ovariectomized female rats(1) Serum, plasma and urinary chemistryCompared with Sham-Operated Group, a significant decrease(P<0.05) in serum1,25(OH)2VD3and OGP, a significant increase(P<0.05) in serum PTH, a highly significant decrease(P<0.01) in serum OC, a highly significant increase(P<0.01) in serum AKP and TRAP as well as in urinary PYD were observed in the Model Group, indicating that it was a reliable model. Compared with Model Group, a significant increase(P<0.05) in plasma OC was observed in all MOP Groups (0.81g·kg-1,0.27g·kg-1and0.09g·kg-1), and a significant decrease(P<0.05) in serum AKP and TRAP was observed in MOP High-Dose(0.81g·kg-1) and Medium-Dose(0.27g·kg-1) Group. All of these groups(0.81g·kg-1,0.27g·kg-1and0.09g·kg-1) showed a increase in serum1,25(OH)2VD3, OGP, Ca, P and a decrease in plasma PTH and urine PYD, which, however, was not statistically significant(.P>0.05).(2) Bone trace elements contentsCompared with Sham-Operated Group, a significant decrease(P<0.05) in the length, the diameter, the dry weight and the iron content of the femur, and a highly significant decrease(P<0.01) in femoral wet weight and ash weightn and copper, zinc, calcium and magnesium contents,were observed in Model Group, indicating that it was a reliable model. Compared with Model Group, a significant increase (P<0.05) in zinc content was observed in all of MOP Groups(0.81g·kg-1,0.27g·kg-1and0.09g·kg-1) and in femoral ash weight was observed in both MOP Groups(0.81g·kg-1and0.27g·kg-1), and an increase in the length and diameter of the femur, the diameter of the vertebra, femoral dry weight, wet weight and ash content, as well as in copper, iron, calcium and magnesium contents, which, however, didn’t represent a statistically significant difference for P>0.05, was observed in all of these groups.(3) Bone mineral density analysisCompared with Sham-Operated Group, a highly significant decrease in femoral and vertebral BMD(P<0.01)was observed in Model Group, indicating that it was a reliable model. Compared with Model Group, a highly significant increase(P<0.01) in vertebral BMD was observed in all of MOP Groups(0.81g·kg-1,0.27g·kg-1and0.09g·kg-1), and a highly significant increase(P<0.01) in femoral BMD in High-Dose Group (0.81g·kg-1) was also found, which showed that MOP, to some extent, improved BMD of femur and vertebrae and decreased bone loss induced by ovariectomy.(4) Static parameters of Cancellous bone in Lumbar vertebra and tibial cortical boneCompared with Sham-Operated Group, a highly significant decrease(P<0.01) in vertebra%Tb.Ar, Tb. N., Tb.Th. and tibial%Ct.Ar, a highly significant increase (P<0.01) in Tb. Sp.of the cancellous bone in the lumbar vertebra and%Ma.Ar of the cortical bone in the tibial, as well as a significant decrease in tibial Ct. Ar.(P<0.05), were observed in Model Group, indicating that it was a reliable model. Compared with Model Group, a highly significant increase in%Ct.Ar and decrease in%Ma.Ar. was observed in all of MOP Groups(0.81g·kg-1,0.27g·kg-1and0.09g·kg-1) with P<0.01. All of these groups (0.81g·kg-1,0.27g·kg-1and0.09g·kg-1) showed an increase in%Tb.Ar, Tb.Th, Tb.N and Ct.Ar and a decrease in Tb.Sp, which, however, was not statistically significant(P>0.05).(5) Femur and vertebrae biomechanical measurementsCompared with Sham-Operated Group, a highly significant decrease in the maximum load, maximum displacement of femur and the maximum displacement, modulus of vertebra were observed in Model Group with P<0.01, indicating that it was a reliable model. Compared with Model Group, a significant increase(P<0.05) in the maximum load of the femur was observed in MOP High-and Medium-Dose Groups (0.81g·kg-1and0.27g·kg-1), a significant increase(P<0.05) in the maximum load of vertebra was observed in all MOP Groups (0.81g·kg-1,0.27g·kg-1and0.09g·kg-1), an increase in the maximum load of the femur was observed in MOP Low-Dose Group (0.09g-kg-1), as well as in the maximum displacement and modulus of the femur and vertebra was observed in all MOP Groups (0.81g-kg-1,0.27g-kg-1and0.09g-kg-1), which, however, didn’t represent a statistically significant difference for P>0.05.3. The study on mechanisms of Morinda officinalis capsule extract on osteoporosis in Ovariectomized ratsCompared with Sham-Operated Group, a highly significant decrease in OPG and increase in RANKL was observed in Model Group with P<0.01, indicating that it was a reliable model. Compared with Model Group, a highly significant increase in OPG and decrease in RANKL was observed in all of MOP Groups (0.81g·kg-1,0.27g·kg-1and0.09g·kg-1) in OB and MSC with P<0.01.Conclusions1. The content of total polysaccharide and total anthraquinones in Morinda officinalis capsule extract is188.99mg·g-1and1.62mg·g-1respectively, the content of stilbene glucoside, free emodin and physcion from Morinda officinalis capsule extract is33.85mg·g-1,80.45μg·g-1and41.17μg·g-1respectively, the content of icarrin is3.23mg·g-1, the content of asperosaponin Ⅵ is5.04mg·g-1, the content of ferulic acid is0.55mg-g-1, the content of chlorogenic acid is0.95mg·g-1, the content of catapol is0.34mg·g-1in Morinda officinalis capsule extract.2. Morinda officinalis capsule extract can regulate and improve bone metabolism, the physical, biomechanical and morphologic properties of the bones and increase bone mineral density and contents in the ovariectomized rats, and is thereby therapeutic and curative on osteoporosis.3. Morinda officinalis capsule extract increases OPG and reduce RANKL expression in OB and MSC, has better effect in regulating OPG/RANKL system, may be one of the important mechanisms of treatment on postmenopausal osteoporosis. |
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