Use of black corn polysaccharide in preparation of medicine for preventing or treating senile osteoporosis

Abstract

The application discloses application of black corn polysaccharide in preparation of a medicine for preventing or treating senile osteoporosis. The evaluation of the prevention or treatment effect of the black corn polysaccharide on senile osteoporosis includes improvement effects on serum bone metabolism markers, bone microstructure and bone biomechanics. The black corn polysaccharide of the application refers to a polysaccharide powder after water extraction, alcohol precipitation, Sevag method deproteinization, H2O2 method depigmentation and dialysis bag dialysis from dried black corn kernels. The black corn polysaccharide has improvement effects on imbalance of serum bone metabolism markers of senile osteoporosis, deterioration of bone microstructure and decrease of bone strength. The results show that the black corn polysaccharide has great improvement effects on bone loss in preventing senile osteoporosis. Therefore, the black corn polysaccharide has important significance in developing senile osteoporosis in clinic.

Description

Technical Field

[0001] This invention relates to the use of black corn polysaccharide in the preparation of drugs for the prevention or treatment of osteoporosis in the elderly. Background Technology

[0002] Osteoporosis is one of the most common skeletal diseases, a systemic bone disease characterized by low bone mass, damaged bone tissue structure, increased bone fragility, and susceptibility to fractures. Senile osteoporosis is a type of primary osteoporosis, most commonly seen in people over 70 years of age, and is a low-turnover osteoporosis caused by a significant decrease in bone formation rate due to aging. Currently, the most commonly used drugs for treating osteoporosis in clinical practice are estrogen, calcium supplements, active vitamin D, calcitonin, and fluoride, which have some efficacy, but usually have significant side effects and are expensive. Therefore, many scholars have shifted their research focus to natural products with safe and effective characteristics, and have achieved some success. Among these, research reports on the effects of plant polysaccharide extracts on senile osteoporosis are increasingly common.

[0003] Black corn (Zeamays L.), also known as black glutinous corn or purple corn, is a plant belonging to the monocotyledonous class of angiosperms and the genus Zea in the order Poales. Black corn is rich in nutrients, with higher levels of protein, amino acids, fat, and selenium than yellow corn. It also contains abundant functional melanin and essential trace elements such as Zn, Fe, Ca, and P. Black corn is also rich in polysaccharides. Studies have shown that polysaccharides are bioactive substances that can activate immune cells, enhance the body's immune function, and some even possess antiviral and anti-aging effects, showing great potential in the health food market. However, research reports on the effects of black corn polysaccharides on osteoporosis in the elderly are limited. Therefore, this invention uses black corn polysaccharides to study bone metabolism markers, bone morphology, and bone strength in osteoporosis in the elderly, preliminarily establishing the potential for developing products to prevent osteoporosis in the elderly using black corn polysaccharides. Summary of the Invention

[0004] The technical problem to be solved by this invention is to propose the application of black corn polysaccharide in the preparation of drugs for the prevention or treatment of osteoporosis in the elderly.

[0005] The evaluation of the effect of black corn polysaccharide of the present invention on the prevention or treatment of osteoporosis in the elderly is actually based on the improvement effect on serum bone metabolism markers, bone microstructure and bone biomechanics.

[0006] The black corn polysaccharide of the present invention refers to the polysaccharide powder obtained from dried black corn kernels through water extraction and alcohol precipitation, Sevag method deproteinization, H2O2 method depigmentation, and dialysis via dialysis bag.

[0007] The black corn polysaccharide of the present invention improves the imbalance of serum bone metabolism markers, deterioration of bone microstructure and decrease in bone strength in senile osteoporosis.

[0008] The results of this invention demonstrate that black corn polysaccharides have a significant effect on improving bone loss in the prevention of osteoporosis in the elderly. Therefore, black corn polysaccharides are of great significance for the clinical development of treatments for osteoporosis in the elderly. Attached Figure Description

[0009] Figure 1 HE staining images of the middle and upper segments of the tibia (×20). Detailed Implementation

[0010] To enable those skilled in the art to understand the present invention more clearly and intuitively, the present invention will be further described below with reference to the accompanying drawings.

[0011] Example 1: Preparation of black corn polysaccharide

[0012] Weigh an appropriate amount of dried black corn kernels, soak them overnight in 95% ethanol, dry them at 50℃ to constant weight, and pulverize them through a 60-mesh sieve. Add deionized water at a material-to-liquid ratio of 1:20 (g / ml), extract with ultrasound at 70℃ and 100W for 1 hour, centrifuge at 3000 r / min and 4℃ for 10 minutes, collect the supernatant, and extract the precipitate again with ultrasound-assisted water extraction to obtain a supernatant. Combine the supernatants and rotary evaporate to 1 / 10 of the supernatant volume to obtain a concentrated solution. Then add three times the volume of 95% ethanol to the concentrated solution to precipitate the precipitate, stir continuously, let it stand overnight at 4℃, centrifuge at 3000 r / min and 4℃ for 10 minutes, collect the precipitate, dissolve the precipitate in deionized water, rotary evaporate, and freeze-dry to obtain crude polysaccharide from black corn kernels. Prepare a solution of the crude polysaccharide, remove protein using the Sevag method, remove pigment using the H2O2 method, dialyze through a dialysis bag (3500 kDa), freeze-dry to obtain black corn kernel polysaccharide, and store for later use.

[0013] Example 2: Experiment on the Inhibition of Senile Osteoporosis by Black Corn Polysaccharide

[0014] Test equipment: Microtome (Leica GmbH, Germany), paraffin embedding machine (Leica GmbH, Germany), Z005 universal testing machine (Zwick / Roll GmbH, Germany).

[0015] Experimental methods: Enzyme-linked immunosorbent assay (ELISA), H&E staining, and biomechanical measurements were used for the investigation.

[0016] (1) Experimental design:

[0017] ① Solution preparation:

[0018] Low-dose black corn polysaccharide solution (200 mg / kg·d): Weigh 20 mg of black corn polysaccharide and add 1 ml of deionized water and stir thoroughly. Vortex the mixed black corn polysaccharide solution and then place it in an ultrasonic machine at 100 W and 50 °C for 20 min to obtain a 20 mg / ml black corn polysaccharide solution. The intervention is carried out daily at a dosage of 10 ml / kg.

[0019] High-dose black corn polysaccharide solution (400 mg / kg·d): Weigh 40 mg of black corn polysaccharide and prepare it in the same way as the low-dose black corn polysaccharide solution to obtain a 40 mg / ml black corn polysaccharide solution. The intervention is carried out at a dosage of 10 ml / kg per day.

[0020] ② Modeling and drug administration:

[0021] Twenty SD rats were randomly divided into four groups: a normal control group, a D-galactose model group, a low-dose black corn polysaccharide intervention group, and a high-dose black corn polysaccharide intervention group, with five rats in each group. The D-galactose model group, the low-dose black corn polysaccharide group, and the high-dose black corn polysaccharide group were subcutaneously injected with D-galactose solution at a dose of 100 mg / kg / day. The control group received an equal volume of physiological saline intraperitoneally. Simultaneously, the low-dose and high-dose black corn polysaccharide groups received drug intervention, with the low-dose group receiving 200 mg / kg / day and the high-dose group receiving 400 mg / kg / day. The control group and the D-galactose model group received 10 ml / kg of pure water. After 12 weeks of intervention, the rats were sacrificed, and blood was collected from the abdominal aorta and stored at -80℃ for later use. Femurs and tibias were also harvested for later use.

[0022] (2) Enzyme-linked adsorption method (ELISA): Bone resorption markers: type I collagen carboxy-terminal peptide (CTX), tartrate-resistant acid phosphatase 5b (TRACP5b), bone formation markers: bone alkaline phosphatase (BALP), type I procollagen amino-terminal peptide (PINP), and bone turnover regulators: osteoprotegerin (OPG) and nuclear factor κB receptor activator ligand (RANKL) were detected according to the kit instructions. The results are shown in Table 1.

[0023] Table 1: Effects of black corn polysaccharide on serum bone metabolism markers in aged osteoporotic rats

[0024]

[0025]

[0026] Note: Compared with the blank group, * P < 0.05 ** P < 0.01, compared with the D-galactose model group, # P < 0.05 ##P < 0.01

[0027] Bone turnover markers are products of the bone remodeling process, including bone formation markers and bone resorption markers. They reflect the rate of bone turnover and are used to assess the effectiveness of osteoporosis treatment and predict fracture risk. Compared with the normal group, the model group rats showed decreased serum BALP and PINP, and increased serum TRACP-5b and CTX, all with statistically significant differences. After intervention with 400 mg / kg black corn polysaccharide, the serum BALP and PINP of rats were significantly increased, while the serum TRACP-5b and CTX were significantly decreased. These results suggest that black corn polysaccharide can regulate bone metabolism balance in aged osteoporotic rats by promoting bone formation and inhibiting bone resorption. OPG and RANKL are important factors in regulating bone turnover, and the ratio of OPG to RANKL is used to measure the balance of bone turnover in vivo. Compared with the normal group, the model group rats showed decreased serum OPG and OPG / RANKL, and increased serum RANKL, both with statistically significant differences, suggesting that senile osteoporosis leads to bone metabolism imbalance by promoting bone resorption and inhibiting bone formation. After intervention with black corn polysaccharide, the serum OPG and OPG / RANKL of rats increased significantly, while serum RANKL decreased, but the difference was not statistically significant. These results indicate that black corn polysaccharide can effectively regulate the serum bone metabolism balance in senile osteoporotic rats.

[0028] (3) HE staining: After fixing the proximal and distal ends of the tibia for 24 hours, the specimens were rinsed with running water to remove residual fixative. The tissues were dehydrated with ethanol of varying concentrations, cleared with xylene I-xylene II, infiltrated with paraffin I-paraffin II, and embedded in paraffin. The embedded bone specimens were sectioned, with continuous coronal sections from the proximal end and continuous sagittal sections from the distal end, with a thickness of 4 μm. The sections were floated on a slide at 45°C to flatten the tissues, retrieved with a glass slide, and placed in a 37°C oven overnight before being stored at room temperature for later use. The bone sections were baked in a 65°C oven for 2 hours, then soaked in xylene I-xylene II, ethanol of varying concentrations, and washed with distilled water for dewaxing. After hematoxylin staining, blueing, differentiation, eosin staining, dehydration with ethanol of varying concentrations, and clearing with xylene, the sections were mounted and observed, photographed, and morphological parameters were measured under a microscope. HE staining images of the mid-shaft and upper tibia are shown below. Figure 1 As shown.

[0029] like Figure 1HE staining of the proximal tibia revealed that, compared to the normal group, the model group rats had a reduced number of trabeculae, a looser arrangement, and thinner, narrower trabeculae with larger inter-stretched spaces, exhibiting structural fractures and incompleteness. HE staining of rats treated with black corn polysaccharide showed that different concentrations of black corn polysaccharide could maintain the trabecular structure to varying degrees, manifested as an increase in trabecular area and number, and a decrease in separation. Corresponding morphological measurements indicated that, compared to the normal group, the model group rats showed a significant decrease in %Tb.Ar and Tb.N, while a significant increase in Tb.Sp, suggesting trabecular structure destruction in the aging osteoporosis rat model. After high-dose black corn polysaccharide intervention, %Tb.Ar and Tb.N significantly increased, while Tb.Sp significantly increased and decreased, suggesting that 400 mg / kg black corn polysaccharide can effectively prevent trabecular structure destruction in aging osteoporosis rats.

[0030] ① HE staining was used to observe changes in bone structure. Combined with bone morphometrics, the percentage of trabecular area (%Tb.Ar), trabecular width (Tb.Wi), number of trabecular bones (Tb.N), and trabecular separation (Tb.Sp) of cancellous bone were measured. The results are shown in Table 2.

[0031] Table 2: Effects of black corn polysaccharide on morphological parameters of the proximal tibia in aged osteoporotic rats

[0032]

[0033] Note: Compared with the blank group, * P < 0.05 ** P < 0.01, compared with the D-galactose model group, # P < 0.05 ## P < 0.01

[0034] ② The morphological parameters of the mid-tibia of aged osteoporotic rats were measured, and the total cortical bone area (T.Ar), cortical bone area percentage (%Ct.Ar), medullary cavity area percentage (%Ma.Ar), and cortical bone thickness (Ct.Th) were obtained. The results are shown in Table 3.

[0035] Table 3: Effects of black corn polysaccharide on morphological parameters of the midshaft of the tibia in aged osteoporotic rats

[0036]

[0037] Note: Compared with the blank group, * P < 0.05 ** P < 0.01, compared with the D-galactose model group, # P < 0.05 ## P < 0.01

[0038] Compared with the blank group, the T.Ar of the D-galactose model group rats was significantly reduced, while %Ct.Ar, Ct.Th and %Ma.Ar showed no significant differences. However, after black corn polysaccharide intervention, there were no significant differences in %Ct.Ar, %Ct.Ar, %Ma.Ar and Ct.Th, indicating that the changes in the mid-tibia of the model mice were not significant, and black corn polysaccharide intervention had no significant effect on the mid-tibia.

[0039] (4) Bone biomechanical measurements:

[0040] The femur length was measured and recorded using vernier calipers. The bone sample was placed on a Z005 universal testing machine for a three-point bending test of the femur. The parameters were set as follows: span 20 mm, compression speed 2 mm / min. After the femur was completely fractured, the maximum and minimum inner diameter and the maximum and minimum outer diameter of the femur were measured and recorded using vernier calipers. The changes in bone strength were evaluated by combining the data obtained from the three-point bending test of the femur, such as maximum load, stress, strain, elastic load, elastic deflection, bone structure hardness, bone elastic modulus, and bone rigidity coefficient, with the femur inner and outer diameter parameters. The results are shown in Table 4.

[0041] Table 4: Effects of black corn polysaccharide on the bone biomechanics of the femur in aged osteoporotic rats

[0042]

[0043] Note: Compared with the blank group, * P < 0.05 ** P < 0.01, compared with the D-galactose model group, # P < 0.05 ## P < 0.01

[0044] Bone biomechanics studies the mechanical properties of bone tissue under external forces and its biological effects after stress, comprehensively reflecting bone mass, bone structure, and bone quality. Bone biomechanical measurements showed that, compared to the control group, the maximum load, bone structural stiffness, maximum energy absorption, and bone structural toughness of the femur in the D-galactose model group were significantly reduced, while the elastic modulus showed no statistically significant difference. Compared to the D-galactose model group, the maximum load, bone structural stiffness, maximum energy absorption, bone structural toughness, and elastic modulus of the femur in rats treated with black corn polysaccharide were significantly increased. These results suggest that black corn polysaccharide may have a role in improving bone strength in aged rats with osteoporosis.

[0045] (1) Bone metabolism markers include two major categories: bone formation markers (BALP, PINP, OPG) and bone resorption markers (TRACP5b, CTX, RANKL). The results of bone metabolite measurement in aged osteoporotic rats showed that the body upregulates bone resorption while inhibiting bone formation, thereby disrupting the balance of bone metabolism. Black corn polysaccharide intervention can effectively improve the balance of bone metabolism.

[0046] (2) Trabeculae are the supporting structures for bone hematopoiesis. Observation of morphological parameters of the proximal tibia and HE pathological section results revealed that the percentage of trabecular area (%Tb.Ar), number of trabeculae (Tb.N), and width of trabecular bone (Tb.Wi) were all decreased in aged osteoporotic rats, indicating bone loss. Black corn polysaccharide effectively protected trabecular morphology and inhibited bone loss after administration.

[0047] Biomechanics is a commonly used method for assessing bone strength. The results showed that age-related osteoporosis leads to a decrease in the femur's load-bearing capacity and reduced bone stiffness, suggesting that age-related osteoporosis may increase bone fragility. Intervention with black corn polysaccharides resulted in an increase in the maximum load on the femur, and improvements in bone structure toughness and stiffness, suggesting that black corn polysaccharides may increase bone strength.

[0048] The above description of the embodiments is provided to enable those skilled in the art to understand and apply the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the embodiments described herein, and any improvements and modifications made to the present invention by those skilled in the art based on the disclosure thereof should be within the scope of protection of the present invention.

Claims

1. Application of black corn polysaccharide in the preparation of drugs for the prevention or treatment of osteoporosis in the elderly; the black corn polysaccharide refers to the polysaccharide powder obtained from dried black corn kernels after water extraction and alcohol precipitation, Sevag method deproteinization, H2O2 method depigmentation, and dialysis using a dialysis bag. The preparation method of the black corn polysaccharide includes the following steps: (1) Weigh the dried black corn kernels, soak them in ethanol overnight, dry them to constant weight and crush them for later use; (2) After adding deionized water, ultrasonic extraction was performed, followed by centrifugation, and the supernatant was collected. The ultrasonic extraction conditions were 70℃ and 100 W. (3) The supernatant was subjected to rotary evaporation to obtain a concentrated solution; (4) Then add ethanol to the concentrate to precipitate the protein, stir constantly, and refrigerate overnight; (5) Centrifuge, collect the precipitate, dissolve the precipitate in deionized water, evaporate by rotary evaporation, and freeze dry to obtain crude polysaccharide from black corn kernels; (6) The crude polysaccharide was prepared into a solution, and the protein was removed by the Sevag method, the pigment was removed by the H2O2 method, and after dialysis by dialysis bag, it was freeze-dried to obtain black corn kernel polysaccharide.

2. The application as described in claim 1, characterized in that, In steps (2) and (5), the centrifugation conditions are 3000 r / min and 4℃.

3. The application as described in claim 1, characterized in that, In step (2), the precipitate after centrifugation is subjected to ultrasonic-assisted water extraction twice, and the resulting supernatant is combined and then processed in step (3).

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