Double Dan oral cyclophosphamide in rat femur biomechanics and biochemical indexes
【Abstract】 Objective To observe the double-Dan oral
cyclophosphamide in rats caused by osteoporosis models. Methods 4.5 mg * kg-1 * d-1 of cyclophosphamide gavage to
rats, continuous 15 d, caused by osteoporosis model; double-
Dan preventive oral administration, and with the positive drug carbonate Calcium D-dimensional comparison. After the end of the experiment, take the right rat femur bone biomechanical
testing, followed by detection of bone calcium, phosphorus, magnesium, and hydroxyproline content. The results clearly lead to cyclophosphamide in rat femur bone loss of organic matter and minerals, bio-mechanical performance degradation,
double-Dan oral liquid can effectively antagonize these effects, and the effect of dimension D and calcium carbonate rather. Conclusion Shuangdan Oral caused by cyclophosphamide can be effective in preventing bone loss in rat femur and bone quality.
Key words double-Dan oral cyclophosphamide in rat bone
mineral calcium carbonate, D-dimensional bone biomechanics
Abstract: ObjectiveTo study osteoporosis of
cyclophosphamide and investigate the preventive effects of Shuangdan oral liquid on rats.MethodsCyclophosphamide at the
dose of 4.5 mg * kg-1 * d-1 was given to the rats orally for
15 days while Shuangdan oral liquid and calcium and vitamin D3 were given to cyclophosphamide-treated rats orally. At the
experiment, bone biomechanics analysis of the right femur were
performed in fresh sections. Then the hydroxyproline, Ca, P, Mg of bone were tested.ResultsBiomechanics property and bone mineral on femur of rats decreased
significantly.ConclusionShuangdan oral liquid can prevent
osteopathologic change from cyclophosphamide-induced on rats.
Key words: Shuangdan oral liquid; Cyclophosphamide; Calcium and vitamin D3; Rats; Bone mineral; Bone biomechanics
Oral pairs recorded in the 2005 edition of Dan "Chinese Pharmacopoeia", by the two Chinese herbal medicines Salvia and
moutan composed of well-known traditional Chinese medicine
prescriptions. Dan oral liquid has been reported pairs of scavenging oxygen free radicals, improve blood circulation, enhance immunity, as well as anti-inflammatory, anti-tumor and
so on. Of its active ingredients, including Danshensu, Tan, paeonol and paeoniflorin and so on, the research group reported Danshensu can enhance osteoblast activity, prevention and treatment of glucocorticoid-induced osteoporosis in rats
 , also reported cyclophosphamide can lead to osteoporosis in rats and mice to produce toxicity [2,3], found that cyclophosphamide inhibit the activity of osteoblast bone loss caused by the role of the effects on bone are similar to the type of osteoporosis in older pathological changes. This study observed pairs of Dan oral liquid on cyclophosphamide-mediated
osteoporosis, the impact of femur, and with the positive drug calcium carbonate dimension D for comparison. Are reported below.
1 Materials and methods
1.1 Drugs and reagents
1.1.1 Drug Salvia, medicinal herbs such as moutan purchased from a local pharmacy, origin of Sichuan. Cyclophosphamide: Jiangsu Hengrui Medicine Co., Ltd., batch number: 403,501. Dimension D of calcium carbonate tablets:
U.S. oz pharmaceutical companies, batch number: 5B16656, the equivalent of calcium per tablet 300mg, VitD100 unit.
1.1.2 Reagent Danshensu Standard: China's Pharmaceutical and Biological Products Institution, batch 110855-200304;
methanol (HPLC pure, Tianjin Chemical Reagent Company of Friends 4); water as a triple-distilled water; glacial acetic
acid (AR, Longxi Chemical Plant, Shantou); hydroxyproline Kit (alkaline solution, Nanjing Jiancheng Bio-Technology Co.,
1.2 Instrument AE240 electronic balance (Mettler - Toledo
Instruments, Shanghai Branch production), high-performance
liquid chromatography: Agilent1100 Series quaternary gradient pump, Agilent1100 manual injector, Agilent1100 series of a diode array detector , Agilent1100 Workstation (Agilent
Corporation); ultrasonic cleaner, 858 Mini Bionix test system-
type material (MTS, USA); inductively coupled plasma emission spectrometer (ICP, the United States TJA Corporation); UV-752
UV spectrophotometer (Shanghai No. 3 Medical Instrument
Oral drug preparation 1.3.1 Preparation of double-Dan:
Based on the 2005 edition of "Chinese Pharmacopoeia" method of extraction, taking Salvia 600 g, Cortex Moutan 300 g, Cortex Moutan distillation, collecting distillates. Dregs and Dan
participated in boiling water twice, the first 1 2 h, the first two times 1 h, the merger fried liquid filtration, the filtrate concentrated to the relative density of 1.15 to clear paste, add ethanol to contain alcohol reached 60%,
refrigerated 24 h, filtration, filtrate recovery of ethanol to
the relative density of 1.15 to clear paste, adding moutan distillates, and finally concentrated extract per 100 ml containing crude drug 191.7 g, sealed sterilization, 4 ?
Pairs of the quality control of oral Dan: Two Dan oral
liquid by HPLC fingerprint detection, column: Agilient C18 (250 mm × 4.65 mm, 5 μm); mobile phase: methanol (A) -1% ice
acetic acid (B), procedures for gradient elution: 10% A (5 min) -30% A (15min); flow rate: 1ml/min; temperature is room
temperature; detection wavelength: 280 nm. Precision Weigh Danshensu 0.104,0.208,0.312,0.520,0.624,0.728 mg sodium standard constant volume with triple-distilled water to 5 ml,
respectively, 20 μl precision drawing the above-mentioned
reference substance injected into liquid chromatograph, record chromatography images in order to peak area of the Danshensu concentration (μg / ml) for linear regression calculation, was the linear regression equation: Y = 11 981X 11.492 (r =
0.999 9). Then according to Danshensu fingerprint peak area to calculate pairs of Dan oral liquid content per milliliter of Danshensu.
1.3.2 Animals and Grouping three-month-old rats were 40
male and female in half, body weight (208 ? 28) g, by the
Experimental Animal Center of Guangdong Medical College, SPF grade, animal Qualified证号: 2005A023. The principle of
reciprocity according to the weight then divided into four groups of 10 rats. The normal control group: gavage for 5 ml * kg-1 * d-1 saline; cyclophosphamide Group: gavage given
cyclophosphamide 4.5 mg * kg-1 * d-1; calcium carbonate
dimension D: ig given cyclophosphamide 4.5 mg * kg-1 * d-1 and
calcium carbonate-dimensional D 0.4 mg * kg-1 * d-1; pairs of
Dan Prevention Group: ig given cyclophosphamide 4.5 mg * kg -
1 * d-1 and dual-Dan oral 5 mg * kg-1 * d-1. 4 animals were
free of water and eat standard feed. Experiment were administered 15 d, end of the experiment, take the right femur with saline gauze, wrapped in aluminum foil to save -20 ?
until biomechanical testing and bone mineral content of
organic matter and testing.
1.3.3 biomechanical testing  test, it will save the femur -20 ? thaw at room temperature, normal saline re-wet. -
Type material with the 858 Mini Bionix test system for monitoring and analyzing the right rat femur biomechanical properties. Rheometer will be placed on the femur three-point
bending test, loading speed of 0.01 mm / s, span of 15 mm. Records of the stress - strain curve, read from the curve and calculation of elastic strength, maximum strength, breaking
strength, stiffness coefficient, bending energy and fracture energy absorption were measured.
1.3.4 Bone mineral content of organic matter and mechanical testing of biological detection of bone material 80 ? after drying to constant weight, with the AE240 electronic balance weighing; then, each bone sample plus 6 mol / L HCl at 108 ? temperature digestion 16 h later, the
digestive juice were divided into two. Determined by ICP in a diluted bone Ca, P, trace elements contents in weight compared with the backbone, with the amount of bone mineral elements (mg) / core weight (g) ratio as indicators of bone mineral
content, bone mineral Element volume / weight ratio of the backbone of the bigger shows that bone mineral in the bone the
greater the proportion. After filtration another digestive juice pH, adjusted to 6, and then, after "Chinese Pharmacopoeia" Determination of hydroxyproline in the absorbance of the standard curve read out its content. As the organic matter in bone collagen accounts for more than 90% of bone collagen hydroxyproline content in relatively stable, accounting for 12.5%, therefore, according to bone
hydroxyproline content can calculate the relative content of organic matter in bone. The bone organic matter = bone hydroxyproline / 12.5% / 90%. By Bone organic matter / weight ratio of the backbone, as indicators of bone organic matter
content. The ratio is higher, indicating the higher organic matter content of bone.
1.3.5 Data processing parameter values used (? s), said
analysis of variance with SPSS12.0. With the rate of change indicates the experimental rats the changes in the relative
parameters 〔such as: changes in the rate of A = (to be more groups / A group -1) × 100%〕.
2.1 pairs of Dan oral liquid preparation and quality control items according to 1.3.1 for the experiment were prepared by double-Dan oral solution, to be detected by high performance liquid chromatography fingerprint shown in Figure 1, the map of Danshensu standard Figure 2. The detection and calculation, double-Dan oral per ml Danshensu 2.431 09 mg.
Figure 1 Double-HPLC fingerprint of Dan oral solution
Figure 2 Danshensu standard high performance liquid quality control map (omitted)
2.2 pairs of Dan oral cyclophosphamide in rat femur biomechanics influence the outcome indicators in Table 1.
Table 1 Double Dan oral cyclophosphamide in rat femur biomechanical effects (abbreviated)
n = 10; A, BC as the rate of change: A comparison with the control group; B model with the cyclophosphamide group; C dimension D with the calcium carbonate group; * P