CN111067915A

CN111067915A - Application of geraniin in preparing medicine for treating vitamin D deficiency and/or deficiency diseases

Info

Publication number: CN111067915A
Application number: CN202010023284.1A
Authority: CN
Inventors: 陈鹏; 沈志强; 张莉; 李璠; 杨仁华; 何波
Original assignee: Kunming Medical University
Current assignee: Kunming Medical University
Priority date: 2020-01-09
Filing date: 2020-01-09
Publication date: 2020-04-28

Abstract

The invention provides application of germacraniin in preparation of a medicine for treating vitamin D deficiency and/or deficiency diseases, and belongs to the technical field of biological medicines, wherein the germacraniin can improve the serum calcium and phosphorus levels of a human body due to vitamin D deficiency and/or deficiency and reduce the level of alkaline phosphatase; moreover, the vitamin D-FGF-23-Klotho axial factor can be regulated to improve the levels of bone metabolism indexes BAP, TRAP5b and BGP, so that the expression level of VDR genes and/or proteins of osteoblasts is up-regulated, and diseases caused by insufficient and/or deficient vitamin D are improved. The invention also provides a medicament for treating vitamin D deficiency and/or deficiency diseases, which comprises geraniin. The invention also provides application of the geraniin and the hesperidin in preparation of medicines for treating osteoporosis complicated with diabetes, and the combined use of the geraniin and the hesperidin can obviously reduce blood sugar of patients.

Description

Application of geraniin in preparing medicine for treating vitamin D deficiency and/or deficiency diseases

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of geraniin in preparation of a medicine for treating vitamin D deficiency and/or deficiency diseases.

Background

Phyllanthus urinaria L (Phyllanthus urinaria L.) of Euphorbiaceae is also called Phyllanthus urinaria L, Phyllanthus niruri L, Phyllanthus urinaria L, and is an annual herb of Phyllanthus of Euphorbiaceae. Phyllanthus urinaria has biological and pharmacological activities of antivirus, protecting liver, anti-inflammatory, reducing blood sugar and cholesterol, antioxidation and antibiosis, and is used for treating diarrhea, dysentery, hepatitis, edema, infantile malnutritional stagnation, acute conjunctivitis, aphtha and antibiotic-resistant suppurative infection. The phenol substance of Phyllanthus urinaria is the main effective substance with biological and pharmacological activities, Geraniin (Ge) is one of the polyphenol compounds, the chemical structure is shown in figure 1, and recent research shows that Geraniin has various pharmacological activities: (1) antioxidation, and experiments in vivo and in vitro show that the geraniin can remove oxygen free radicals, hydroxyl free radicals, superoxide free radicals and the like; (2) and (3) tumor resistance: geraniin can inhibit activity of transforming growth factor, etc., and can scare off migration, invasion and anti-apoptosis ability of tumor, inhibit tumor growth, and even accelerate tumor cell apoptosis; (3) antiviral and antibacterial activity. The geraniin is easy to dissolve in strong polar solvents such as water, methanol, ethanol and the like, is extracted by a heating reflux method and is easily decomposed into gallic acid, hexahydroxy diphenyl dicarboxylic acid, ellagic acid, corilagin and corilagin, is unstable in light and heat and is easy to degrade in a photo-thermal environment, so the preparation process of the geraniin is to avoid light and heat as much as possible.

Disclosure of Invention

The invention aims to provide the application of the geraniin, wherein the geraniin can improve the serum calcium and phosphorus levels of a human body with insufficient vitamin D and/or lack of the human body and reduce the alkaline phosphatase level; moreover, the bone metabolism indexes BAP, TRAP5b and BGP level can be improved by adjusting vitamin D-FGF-23-Klotho axial factor, thereby improving diseases caused by vitamin D deficiency and/or deficiency.

The technical scheme adopted by the invention for realizing the purpose is as follows:

use of geraniin for the preparation of a medicament for the treatment of vitamin D insufficiency and/or deficiency diseases.

Vitamin D is a hormone other than a vitamin and is called the vitamin D endocrine system. The Vitamin D system comprises Vitamin D, enzymes involved in Vitamin D metabolism and receptors for Vitamin D action (VDR); when vitamin D in the organism is insufficient and/or deficient, various diseases can be caused, such as rickets, osteomalacia, osteoporosis, hand and foot spasm and the like. The geraniin can not only improve the serum calcium and phosphorus levels of insufficient vitamin D and/or deficient organisms, but also reduce the alkaline phosphatase level; and the abnormal metabolism of the vitamin D can be improved by regulating the levels of the vitamin D-FGF-23-Klotho axial factor 25(OH) VD3, 1,25(OH) VD3, FGF-23 and Klotho, so that the bone metabolism indexes of bone specific alkaline phosphatase (BAP), tartrate-resistant acid phosphatase 5b (TRAP5b) and osteocalcin (BGP) are improved, and the diseases caused by vitamin D deficiency and/or deficiency are improved. And the body has small adverse reaction to the geraniin, the invention fully explores the important application of the traditional medicine in preventing and treating diseases, and finds a new way for treating vitamin D deficiency and/or deficiency diseases.

According to one embodiment of the present invention, the geraniin is a geraniin monomer and/or a geraniin salt.

According to one embodiment of the invention, the geraniin can improve the abnormal metabolism of vitamin D by adjusting the levels of vitamin D-FGF-23-Klotho axial factor 25(OH) VD3, 1,25(OH) VD3, FGF-23 and Klotho.

According to one embodiment of the present invention, geraniin increases bone density.

According to one embodiment of the invention, geraniin can up-regulate the expression level of VDR gene and/or protein of osteoblast. Geraniin can up-regulate the expression level of Vitamin D Receptor (VDR) gene and/or protein of osteoblast, activate VDR signal path, so as to activate VDR signal path, promote proliferation and differentiation of osteoblast, inhibit bone absorption of osteoclast or release of bone calcium, and promote bone formation. That is, geraniin may improve vitamin D deficiency by modulating the vitamin D axis to promote VDR protein expression.

According to one embodiment of the invention, the vitamin D insufficiency and/or deficiency disease is a bone disease. Preferably, the bone disorders include: osteoporosis, rickets, osteomalacia, bone injury, hand and foot spasm, bone calcium loss, bone hypoplasia and skeletal abnormalities.

The invention also aims to provide a medicament for treating vitamin D deficiency and/or deficiency diseases, which comprises geraniin.

According to an embodiment of the invention, the medicament further comprises hesperidin. The combined use of the geraniin and the hesperidin can further improve the action effect of the geraniin.

Still another object of the present invention is to provide the use of geraniin and hesperidin in the preparation of a medicament for the treatment of vitamin D deficiency and/or deficiency diseases.

The invention also aims to provide application of the geraniin and the hesperidin in preparing medicines for treating osteoporosis and diabetes mellitus. The osteoporosis is difficult to cure, the health of a patient is seriously damaged after the complication diabetes mellitus, the blood sugar of the patient can be obviously reduced by using the geraniin and the hesperidin together, the treatment effective rate is improved, and the pharmaceutical composition is worthy of popularization and application.

The invention has the beneficial effects that: according to the invention, the geraniin can not only improve the serum calcium and phosphorus levels of the body due to insufficient vitamin D and/or lack of the body, but also reduce the alkaline phosphatase level; and can improve the levels of BAP, TRAP5b and BGP through regulating the level of vitamin D-FGF-23-Klotho axial factor, thereby improving diseases caused by vitamin D deficiency and/or deficiency. The geraniin can up-regulate the expression level of a Vitamin D Receptor (VDR) gene and/or protein of osteoblasts, activate a VDR signal path, and fulfill the aim of activating the VDR signal path, so that the proliferation and differentiation of osteoblasts are promoted, the bone resorption of osteoclasts is inhibited, the release of bone calcium is inhibited, and the formation of bones is promoted. The combined use of the geraniin and the hesperidin can further improve the action effect of the geraniin, can also obviously reduce the blood sugar of a patient, improves the treatment effective rate of osteoporosis combined with diabetes, and is worthy of popularization and application.

Drawings

FIG. 1 is a chemical structural formula of Geraniin of the present invention;

FIG. 2 shows the expression level of VDR gene of osteoblasts in example 4 of the present invention;

FIG. 3 shows the expression level of VDR protein in osteoblasts in example 4 of the present invention;

FIG. 4 shows the expression level of VDR protein in osteoblasts in example 4 of the present invention;

FIG. 5 is a graph of fasting plasma glucose after treatment of a patient in accordance with example 8 of the present invention;

FIG. 6 is a graph of bone density after treatment of a patient according to example 8 of the present invention.

Detailed Description

While the invention is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and are herein described in detail. It is not intended to limit the invention to the particular form disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.

These examples are provided only for more specifically illustrating the present invention, and it is apparent to those skilled in the art that the scope of the present invention is not limited to these examples according to the gist of the present invention.

One embodiment of the invention provides an application of geraniin in preparing a medicament for treating vitamin D deficiency and/or deficiency diseases.

Vitamin D is a fat-soluble substance generated by the skin after photolysis of ultraviolet rays in sunlight, and belongs to a steroid hormone in a human body; when vitamin D in the organism is insufficient and/or deficient, various diseases can be caused, such as rickets, osteomalacia, osteoporosis, hand and foot spasm and the like. The geraniin can not only improve the serum calcium and phosphorus levels of insufficient vitamin D and/or deficient organisms, but also reduce the alkaline phosphatase level; and the abnormal metabolism of the vitamin D can be improved by regulating the levels of the vitamin D-FGF-23-Klotho axial factor 25(OH) VD3, 1,25(OH) VD3, FGF-23 and Klotho, so that the bone metabolism indexes of bone specific alkaline phosphatase (BAP), tartrate-resistant acid phosphatase 5b (TRAP5b) and osteocalcin (BGP) are improved, and the diseases caused by vitamin D deficiency and/or deficiency are improved. And the body has small adverse reaction to the geraniin, the invention fully explores the important application of the traditional medicine in preventing and treating diseases, and finds a new way for treating vitamin D deficiency and/or deficiency diseases.

In one embodiment of the present invention, the geraniin is a geraniin monomer and/or a geraniin salt.

In one embodiment of the present invention, the geraniin is derived from an active extract of Phyllanthus urinaria L, a plant of Euphorbiaceae.

In one embodiment of the present invention, the geraniin is obtained by the following method:

1) adding the phyllanthus urinaria powder into an extracting solution containing 0.3-1.5% of lactic acid, 20-25% of ammonium sulfate and 17-23% of ethanol according to the mass concentration of 1:10-20, extracting for 40-50min under the ultrasonic wave of 150 plus 180W, centrifuging for 5-12min at 8000 plus 15000r/min, standing and layering the supernatant in a separating funnel, and filtering through a 0.45 mu m microporous filter membrane to obtain the extracting solution; the geraniin is hydrolysable tannin, is extracted by a heating reflux method and is easily decomposed into gallic acid, hexahydroxy diphenyl dicarboxylic acid, ellagic acid, corilagin and corilagin, and the active ingredients in the plant materials can be accelerated to enter a solvent by utilizing strong vibration, high acceleration, strong cavitation effect, stirring effect and the like generated by ultrasonic waves, so that the extraction rate of the active ingredients is increased, the extraction time is shortened, and the influence of high temperature on the extracted ingredients can be avoided; meanwhile, the extracting solution can better destroy the increase of the acting force of the phenolic substances and the matrix under the lower ethanol concentration, is beneficial to the dissolution of the phenolic substances, has strong selectivity on the phenolic substances, is beneficial to the extraction of the phenolic substances in the phyllanthus urinaria powder, and ensures that the extraction rate of the phenolic substances in the phyllanthus urinaria powder can reach at least 8.4 percent;

2) preparing a solvent system from petroleum ether, ethyl acetate, methanol, ammonium citrate and water according to a volume ratio of 0.3-0.7:5-15:0.1-0.3:0.04-0.0.08:18-25, placing the solvent system into a separating funnel, shaking uniformly, standing for layering, after balancing for 1-4h, separating an upper phase from a lower phase, taking the upper phase as a stationary phase and the lower phase as a mobile phase;

3) filling the fixed phase into a column of a high-speed counter-current chromatograph at the flow rate of 10-15mL/min, then operating a main machine of the high-speed counter-current chromatograph in the forward direction at the rotating speed of 700 plus 800r/min, simultaneously pumping the mobile phase at the flow rate of 0.5-1.5mL/min, when the mobile phase begins to flow out of the chromatographic column, injecting an extracting solution which is dissolved into the mobile phase according to the mass concentration of 1-10mg/mL through an injection valve, entering a sample separation state, simultaneously setting the ultraviolet detection wavelength to be 280nm, and then collecting the geraniin fraction according to the ultraviolet spectrogram of a detector. The high-speed countercurrent chromatography (HSCCC) is a better method for separating compounds which are similar in property and easy to denature at present, the distribution coefficient of the geraniin in the solvent system used in the embodiment is proper, the geraniin can be better separated, the purity and the recovery rate of the geraniin fraction are improved, and the purity of the geraniin fraction can reach at least 94% and the recovery rate can reach at least 90% through HPLC detection. Further purifying the geraniin fraction to obtain more than 99% of pure geraniin.

In one embodiment of the invention, the geraniin can improve the abnormal metabolism of vitamin D by adjusting the levels of vitamin D-FGF-23-Klotho axial factor 25(OH) VD3, 1,25(OH) VD3, FGF-23, and Klotho.

According to one embodiment of the invention, geraniin and hesperidin up-regulate the expression level of VDR genes and/or proteins of osteoblasts, so as to activate VDR signal pathways, promote proliferation and differentiation of osteoblasts, inhibit bone resorption of osteoclasts, or inhibit bone calcium release. Preferably, the geraniin and the hesperidin enable the expression level of the VDR gene and/or the VDR protein to be up-regulated by at least 2.5 times.

In one embodiment of the present invention, geraniin increases bone density.

In one embodiment of the present invention, geraniin can up-regulate the expression level of VDR gene and/or protein of osteoblast. Geraniin can up-regulate the expression level of Vitamin D Receptor (VDR) gene and/or protein of osteoblast, activate VDR signal path, so as to activate VDR signal path, promote proliferation and differentiation of osteoblast, inhibit bone absorption of osteoclast or release of bone calcium, and promote bone formation. That is, geraniin may improve vitamin D deficiency by modulating the vitamin D axis to promote VDR protein expression.

In one embodiment of the invention, the vitamin D insufficiency and/or deficiency disease is a bone disease. Preferably, the bone disorders include: osteoporosis, rickets, osteomalacia, bone injury, hand and foot spasm, bone calcium loss, bone hypoplasia and skeletal abnormalities.

The invention also provides a medicine for treating vitamin D deficiency and/or deficiency diseases, which comprises geraniin.

According to one embodiment of the invention, the medicament is for modulating vitamin D-FGF-23-Klotho Axis factor 25(OH) VD3, 1,25(OH) VD3, FGF-23, Klotho levels.

According to an embodiment of the invention, the medicament further comprises hesperidin. The combined use of the geraniin and the hesperidin can further improve the action effect of the geraniin. Preferably, the weight ratio of the geraniin to the hesperidin is 1: 0.01-0.5.

In one embodiment of the present invention, the medicament further comprises one or more agents selected from the group consisting of: vitamin C, vitamin A, vitamin D, vitamin B1, vitamin B2, vitamin B6, pantothenic acid, folic acid, calcium lactate, calcium and magnesium. Preferably, the medicament further comprises vitamin D and calcium lactate.

In one embodiment of the present invention, the medicament further comprises a pharmaceutically acceptable carrier and/or excipient and/or diluent. Among the carriers, excipients, and diluents that may be included are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

In one embodiment of the invention, the medicament is an oral composition. The medicine is in the form of powder, pill, granule, tablet, capsule, suspension, liquid, emulsion or syrup. The solid drug is produced by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. Furthermore, in addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used. The liquid medicine may contain various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc., in addition to conventional simple diluents such as water and liquid paraffin.

The invention also provides a use method of the medicine, and the medicine is applied according to the amount of 1-200mg of geraniin per day. Preferably, administration is 1 or divided into 2-4 administrations per day.

In one embodiment of the invention, the drug is administered by ingestion for 9-12 weeks or more.

The invention also provides application of the geraniin and the hesperidin in preparation of medicines for treating vitamin D deficiency and/or deficiency diseases.

The embodiment of the invention also provides application of the geraniin and the hesperidin in preparation of medicines for treating osteoporosis and diabetes. The osteoporosis is difficult to cure, the health of a patient is seriously damaged after the complication diabetes mellitus, the blood sugar of the patient can be obviously reduced by using the geraniin and the hesperidin together, the treatment effective rate is improved, and the pharmaceutical composition is worthy of popularization and application. Preferably, the diabetes is type II diabetes.

The present invention is further described in detail with reference to the following examples:

example 1:

the geraniin is obtained by the following method:

1) adding cacumen Securinegae Suffruticosae powder into extractive solution containing 22% ammonium sulfate and 29% ethanol at a ratio of 1:14, extracting under 160W ultrasonic wave for 45min, centrifuging at 10000r/min for 8min, standing supernatant in separating funnel for layering, and filtering with 0.45 μm microporous membrane to obtain extractive solution;

2) preparing a solvent system from petroleum ether, ethyl acetate, methanol, ammonium citrate and water according to a volume ratio of 0.5:10:0.2:0.05:20, placing the solvent system in a separating funnel, shaking uniformly, standing for layering, after balancing for 2 hours, separating an upper phase from a lower phase, taking the upper phase as a stationary phase, and taking the lower phase as a mobile phase;

3) filling a column of a high-speed counter-current chromatograph with the stationary phase at a flow rate of 12mL/min, then operating a main machine of the high-speed counter-current chromatograph in a forward direction at a rotating speed of 750r/min, simultaneously pumping the mobile phase at a flow rate of 1.0mL/min, when the mobile phase begins to flow out of the chromatographic column, injecting an extracting solution dissolved in the mobile phase at a mass concentration of 3mg/mL through an injection valve, entering a sample separation state, simultaneously setting an ultraviolet detection wavelength to be 280nm, and then collecting geraniin fraction according to an ultraviolet spectrogram of a detector. HPLC detection shows that the purity of the geraniin fraction is 95.2%, and the recovery rate is 90.8%.

Example 2:

the geraniin is obtained by the following method:

2) preparing a solvent system from petroleum ether, ethyl acetate, methanol and water according to the volume ratio of 0.5:10:0.2:20, placing the solvent system into a separating funnel, shaking uniformly, standing for layering, after balancing for 2 hours, separating an upper phase from a lower phase, taking the upper phase as a stationary phase, and taking the lower phase as a mobile phase;

3) filling a column of a high-speed counter-current chromatograph with the stationary phase at a flow rate of 12mL/min, then operating a main machine of the high-speed counter-current chromatograph in a forward direction at a rotating speed of 750r/min, simultaneously pumping the mobile phase at a flow rate of 1.0mL/min, when the mobile phase begins to flow out of the chromatographic column, injecting an extracting solution dissolved in the mobile phase at a mass concentration of 3mg/mL through an injection valve, entering a sample separation state, simultaneously setting an ultraviolet detection wavelength to be 280nm, and then collecting geraniin fraction according to an ultraviolet spectrogram of a detector. HPLC detection shows that the purity of the geraniin fraction is 89.7%, and the recovery rate is 82.5%.

Comparing example 1 with example 2, it can be seen that the distribution coefficient of geraniin in the solvent system used in example 1 is appropriate, so that the geraniin can be well separated, and the purity and recovery rate of the geraniin fraction can be improved.

Example 3:

effect of Geraniin on bone metabolism of vitamin D Japanese Long-ear rat

1. Experimental animals:

40 male SD female rats of 3 months old, clean grade, body mass (180. + -.20 g), purchased from Shanghai Si Laike laboratory animals Co., Ltd. [ laboratory animal certification no: SCXK (Shanghai) 2003-0003 ].

2. Moulding and grouping

After 40 rats were adaptively bred for 7 days, the rats were randomly divided into 3 groups of a normal control group, a vitamin D-deficient model group and a vitamin D +10 mg/kg-D-geraniin-deficient treatment group, and 10 rats were taken in each group. Feeding the vitamin D-deficient model group, the vitamin D +10 mg/kg-D geraniin-deficient treatment group 1, the vitamin D +10 mg/kg-D geraniin +2.5 mg/kg-D hesperidin-deficient treatment group 2 and the vitamin D +2.5 mg/kg-D hesperidin-deficient treatment group 3 by using the feed lacking vitamin D, freely drinking clean filtered water, placing the filtered water in a dark room for feeding, and establishing a vitamin D-deficient rat model. The normal control group rats were fed with normal feed and were freely drunk clean filtered water, and were normally illuminated for 12h daily without any treatment, and were normal healthy rats. The breeding environment is clean. The pharmaceutical intervention and the molding are carried out simultaneously. And (4) carrying out serum biochemical index detection after feeding for 10 weeks.

3. Analysis of results

3.1 serum alkaline phosphatase, serum calcium and phosphorus concentration detection

The concentrations of alkaline phosphatase, serum calcium and phosphorus in the serum of the rat are measured by adopting a full-automatic biochemical analyzer, and the results are shown in table 1, so that compared with a normal control group, the serum calcium and phosphorus content in the serum of the rat lacking the vitamin D model group is lower, and the alkaline phosphatase content is higher; compared with the model group, the rats in the treatment 1 group and the treatment 3 group have higher serum calcium and phosphorus contents and lower alkaline phosphatase contents, and the difference has statistical significance; compared with the model group, the serum calcium and phosphorus content and the alkaline phosphatase content in the blood serum of the rats in the 2 groups are not greatly changed; in addition, compared with the model group, the serum calcium and phosphorus content in the serum of the rat of the treatment 1 group is higher, the alkaline phosphatase content is lower, and the difference has statistical significance. The results show that the geraniin can improve the serum calcium and phosphorus levels of the rats with vitamin D deficiency, reduce the alkaline phosphatase level, effectively improve the serum calcium and phosphorus contents in the serum of the rats with vitamin D deficiency, reduce the alkaline phosphatase content, regulate and control the steady state of the serum calcium, phosphorus and alkaline phosphatase, and finally play a role in increasing the vitamin D content; the hesperidin hardly influences the serum calcium and phosphorus levels and the phosphatase level of rats with vitamin D deficiency; the addition of hesperidin in geraniin can further enhance the effect of geraniin on serum calcium, phosphorus and alkaline phosphatase in the serum of rats with vitamin D deficiency.

TABLE 1 serum alkaline phosphatase, serum calcium and phosphorus concentrations in the animals of each group

Note: a represents a statistical difference from the normal control group (P <0.05), b represents a statistical difference from the model control group (P <0.05), and c represents a statistical difference from the model control group (P < 0.05).

3.2 serum D-FGF-23-Klotho Axis factor level assay

The levels of 25(OH) VD3, 1,25(OH) VD3, FGF-23 and Klotho in rat serum vitamin D-FGF-23-Klotho axial factor are detected by using an electrochemiluminescence immunoassay method. The results of the detection of 25(OH) VD3, 1,25(OH) VD3, FGF-23 and Klotho in the serum are shown in Table 2, and it can be seen that compared with the normal control group, the serum of the rat in the vitamin D-deficient model group has lower contents of 25(OH) VD3, 1,25(OH) VD3 and Klotho and higher content of FGF-23; compared with the model group, the rats in the treatment 1 group and the treatment 2 group have higher serum 25(OH) VD3, 1,25(OH) VD3 and Klotho content and lower FGF-23 content, and the difference has statistical significance; compared with the model group, the rats in the 3 groups treated have smaller changes in serum content of 25(OH) VD3, 1,25(OH) VD3, Klotho and FGF-23; compared with the treatment group 1, the treatment group 2 has higher serum 25(OH) VD3, 1,25(OH) VD3 and Klotho content and lower FGF-23 content in rats, and the difference has statistical significance. The Geraniin can regulate the levels of vitamin D-FGF-23-Klotho axial factor 25(OH) VD3, 1,25(OH) VD3, FGF-23 and Klotho in the serum of a vitamin D deficient rat, and metabolic hesperidin for improving abnormal vitamin D has no obvious influence on the levels of vitamin D-FGF-23-Klotho axial factor 25(OH) VD3, 1,25(OH) VD3, FGF-23 and Klotho in the serum of the vitamin D deficient rat; however, the addition of hesperidin in geraniin can further promote the effect of geraniin on vitamin D-FGF-23-Klotho axial factor in serum of rats with vitamin D deficiency.

TABLE 2 serum D-FGF-23-Klotho axial factor levels in various groups of animals

3.3 rat bone metabolism index detection

The levels of the rat bone metabolism indicators BAP, TRAP5b and BGP are detected by an electrochemiluminescence immunoassay method. The rat is killed by adopting a head breaking method, the femur on the left side of the rat is picked up, and the bone density of the rat is measured by adopting a dual-energy X-ray bone density measuring instrument: comprises a greater trochanter, a cadre part and an epiphyseal part, and the average value of the greater trochanter, the cadre part and the epiphyseal part is taken to calculate the average bone density. The results of the measurements of the bone metabolism indexes BAP, TRAP5b, BGP and bone density are shown in Table 3, and it can be seen that the levels of BAP and TRAP5b are increased and the levels of BGP and bone density are decreased in rats lacking the vitamin D model group compared with the normal control group; compared with the model group, the rats BAP and TRAP5b in the treatment 1 group and the treatment 2 group are reduced in level, the BGP and bone density levels are increased, and the difference has statistical significance; the levels of BAP and TRAP5b and BGP and bone density were not significantly changed in the treated 3 rats compared to the model group; compared with the treatment group 1, the treatment group 2 rats slightly increase the BAP and TRAP5b levels in serum, slightly decrease the BGP and bone density levels, and the difference has statistical significance. The results show that the geraniin can regulate bone metabolism indexes BAP, TRAP5b, BGP and bone density levels of rats with vitamin D deficiency, so as to improve diseases caused by vitamin D deficiency and/or deficiency, and hesperidin has little influence on the bone metabolism indexes BAP, TRAP5b, BGP and bone density levels of rats with vitamin D deficiency; however, the addition of hesperidin in geraniin can further promote the effect of geraniin on the bone metabolism index of rats with vitamin D deficiency.

TABLE 3 rat bone metabolism index detection

Group of	BAP(U/L)	TRAP5b(U/L)	BGP(μg/L)	Bone Density (g/cm)²)
					Normal control group	58.53±2.37	4.80±0.58	54.37±5.65	1.47±0.05
Model control group	113.37±9.77	10.04±1.05	27.39±3.11	0.91±0.03
					Treatment 1 group	73.63±4.56^ab	5.47±0.77^ab	48.55±4.27^ab	1.24±0.07^ab
Treatment of 2 groups	59.43±4.14^abc	5.01±0.78^abc	44.01±3.34^abc	1.42±0.08^abc
					Treatment of 3 groups	100.12±8.57	8.96±1.11	27.55±3.27	1.02±0.04

Example 4:

influence of Geraniin on osteoblast VDR gene and protein expression

1. Osteoblasts: MC-3T3-E1 mouse embryonic osteoblasts were purchased from the Chinese academy of sciences cell bank.

2. Osteoblast culture and grouping

Osteoblast monolayer adherent growth is carried out in DMEM basal medium containing 10% fetal bovine serum and placed at 37 ℃ and 5% CO₂Subculturing under the condition. Osteoblasts in logarithmic phase were randomly divided into 5 groups, normal control group (medium was added and normal culture was performed without any other intervening factors), test 1 group (medium added contained 1. mu.g/mL geranium), test 2 group (medium added contained 1. mu.g/mL geranium and 0.25. mu.g/mL hesperidin), and test 3 group (medium added contained 0.25. mu.g/mL hesperidin), osteoblasts in logarithmic phase were inoculated in a well plate, medium was added, culture was performed for 24h, old culture solution was removed, and PBS was rinsed 3 times.

3. Analysis of results

3.1 RT-PCR method for detecting the influence of Geraniin on the VDR gene expression level of osteoblast

Extracting the rinsed osteoblasts, extracting total RNA from the cells by Trizol, adding sample according to the instructions of a kit (PrimeScript RT Master Mix (Perfect Real Time) reverse transcription kit (batch No. DRR047S, Takara, Japan)) to perform reverse transcription reaction, amplifying by an ABI 7300 type fluorescence quantitative PCR instrument under the conditions of pre-denaturation: 95 ℃ for 10min, denaturation: 95 ℃ for 15s, annealing temperature: 60 ℃ for 15s, extension: 72 ℃ for 35s, cycle times: 40, and primer sequences shown in Table 4, synthesized by Shanghai Biotechnology engineering service Limited, obtaining the Ct value of each sample and each gene amplification by using the analysis software of the instrument, taking β -actin as an internal reference gene, and obtaining the relative expression quantity RQ of the target gene as 2^-△△Ct. As shown in fig. 2, the expression level of the VDR gene in osteoblasts in test 1 and test 2 was significantly increased (the expression level of the VDR gene was up-regulated by at least 3 times) compared to the normal control group, but the expression level of the VDR gene in osteoblasts in test 3 was not significantly changed. The results show that the independent use of hesperidin has little influence on the expression level of the VDR gene of osteoblasts, the geraniin can remarkably up-regulate the expression level of the VDR gene of osteoblasts, and the common addition of the geraniin and hesperidin can further up-regulate the expression level of the VDR gene of osteoblasts.

TABLE 4 primer sequences for RT-PCR detection

Group of	Primer and method for producing the same
		β-actin-F	5’-ACTGCTGAGCTTTGGGGAGC-3’
β-actin-R	5’-GCATGCTGCTAATATCACGA-3’
		VDR-F	5’-GACCTACGTAATCGATCGAC-3’
VDR-R	5’-GCACAGTCGTACGTATAGCA-3’

3.2 Western Blot method for detecting the influence of Geraniin and hesperidin on the expression of VDR protein of osteoblast

Respectively extracting the rinsed osteoblasts, extracting osteoblast protein according to the instruction of a cell protein extraction kit, determining the protein concentration by using a BCA kit, adding the prepared protein sample into 10% gel for electrophoresis, transferring to a PVDF membrane, sealing for 2h by using 10% skimmed milk, adding a primary antibody (1:100), standing overnight at 4 ℃, washing the membrane by using TBST (10 min multiplied by 3), adding a secondary antibody (1:200), incubating for 2h at room temperature, washing the membrane by using TBST (10 min multiplied by 3), and performing ECL luminescence development. The film was scanned and the results analyzed by a gel imaging system. The relative expression level of the target protein is the expression level of the target protein per reference expression level in the same sample. The VDR protein expression levels in the cells of the groups are shown in fig. 3 and 4, and it can be seen that the VDR protein expression levels of the bone cells of the group 1 and the group 2 are significantly increased compared to the normal control group, while the VDR protein expression levels of the bone cells of the group 3 are not significantly changed, so that the VDR gene protein is at least up-regulated by 3 times. The results show that the independent use of hesperidin has little influence on the expression level of the VDR protein of the osteoblast, the geraniin can remarkably up-regulate the expression level of the VDR protein of the osteoblast, and the joint addition of the geraniin and the hesperidin can further up-regulate the expression level of the VDR protein of the osteoblast.

Example 5:

a medicine for treating vitamin D deficiency and/or deficiency diseases comprises geraniin. The medicine is applied according to the amount of 30mg geraniin per day, and the application is divided into 3 times per day.

Example 6:

a medicine for treating vitamin D deficiency and/or deficiency diseases comprises geraniin and hesperidin at weight ratio of 1: 0.25. The medicine is applied according to the amount of 30mg geraniin per day, and the application is divided into 3 times per day.

Example 7:

the medicine for treating osteoporosis complicated with diabetes comprises geraniin and hesperidin in a weight ratio of 1: 0.25.

Example 8:

geraniin and hesperidin for treating osteoporosis complicated with diabetes

150 patients with osteoporosis and diabetes are randomly divided into 3 groups according to different clinical treatment methods, wherein 1 group is treated: geraniin; treatment 2 groups: hesperidin; for treatment 3 groups, geraniin and hesperidin were used. Statistical analysis is carried out on non-treatment researches such as sex, age, body mass, disease onset time, complications and the like of 3 groups of patients, the differences have no statistical significance, P is more than 0.05, and the method has comparability. All patients underwent routine basic therapy including health education, quantitative diet control, regular exercise, and administration of hypoglycemic agents or insulin injections for basic hypoglycemic therapy. Wherein 1 group of patients are treated, the geraniin is 30mg and is orally taken for 3 times per day. Treatment 2 groups: hesperidin 7.5mg, 3 times per day, and is orally taken. Treatment 3 groups: 30mg of geraniin and 7.5mg of hesperidin are used for 3 times per day. In 3 groups, 2 weeks are used as 1 treatment course, and 4 treatment courses are observed. The fasting blood glucose and the lumbar vertebrae density before and after the treatment were observed, and the results are shown in fig. 5 and fig. 6, respectively. As can be seen in FIG. 5, treatment 1 and treatment 2 patients had a slight decrease in fasting glucose, but had poor results; treatment 2 patients had a significant reduction in fasting plasma glucose after treatment. As can be seen from fig. 6, the average bone density of the lumbar vertebrae was significantly increased after the treatment of the patients in the treatment groups 1 and 2, and the effect of the treatment group 2 was significantly better than that of the treatment group 1, while the improvement of the treatment group 3 was not significant. The results show that the geraniin and the hesperidin can achieve a good effect of controlling blood sugar during the co-treatment, can improve the bone density of a patient, and can realize the auxiliary blood sugar control during the treatment of osteoporosis.

Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.

The above embodiments are merely illustrative, and not restrictive, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the protection scope of the present invention should be defined by the claims.

Claims

1. Use of geraniin for the preparation of a medicament for the treatment of vitamin D insufficiency and/or deficiency diseases.

2. Use according to claim 1, characterized in that: the Geraniin is Geraniin monomer and/or Geraniin salt.

3. Use according to claim 1, characterized in that: the geraniin can improve the abnormal metabolism of vitamin D by regulating the levels of vitamin D-FGF-23-Klotho axial factor 25(OH) VD3, 1,25(OH) VD3, FGF-23 and Klotho.

4. Use according to claim 1, characterized in that: the Geraniin increases bone density.

5. Use according to claim 1, characterized in that: the expression level of VDR genes and/or proteins of osteoblasts is up-regulated.

6. Use according to claim 1, characterized in that: the vitamin D deficiency and/or deficiency disease is a bone disease. Preferably, the bone diseases include: osteoporosis, rickets, osteomalacia, bone injury, hand and foot spasm, bone calcium loss, bone hypoplasia and skeletal abnormalities.

7. A medicament for treating vitamin D insufficiency and/or deficiency diseases comprising the geraniin as set forth in claim 1.

8. The medicament of claim 7, wherein: the medicine also comprises hesperidin.

9. Use of geraniin and hesperidin for the preparation of a medicament for the treatment of vitamin D deficiency and/or deficiency disorders.

10. Application of geraniin and hesperidin in preparing medicine for treating osteoporosis complicated with diabetes is provided.