阅读说明：本技术 骆驼刺糖聚合物及其制备方法和应用 (Camel thorn sugar polymer and preparation method and application thereof ) 是由 严春艳 叶振泉 李天钰 于 2020-03-13 设计创作，主要内容包括：本发明公开了骆驼刺糖聚合物AP3-2、AP3-3及其提取方法和用途,该方法以骆驼刺地上部分为原料,通过水提醇沉、Sevag法脱蛋白、DEAE纤维素柱层析、凝胶柱层析等制备骆驼刺糖聚合物。其中,AP3-2是由甘露糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖、阿拉伯糖和木糖组成的糖聚物；AP3-3是由阿拉伯糖组成的糖聚物。同时,AP3-2在抗骨质疏松实验中显示其具有显著的促成骨活性。这些研究可为其将来在制备防治骨质疏松药物或保健品或功能食品中的应用提供依据。(The invention discloses camel thorn sugar polymers AP3-2 and AP3-3 as well as an extraction method and application thereof. Wherein AP3-2 is a glycomer consisting of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, arabinose, and xylose; AP3-3 is a glycomer consisting of arabinose. Meanwhile, AP3-2 has been shown to have significant contribution to bone activity in anti-osteoporosis experiments. The researches can provide a basis for the future application of the traditional Chinese medicine composition in preparing medicines or health products or functional foods for preventing and treating osteoporosis.)

1. A camel thorn sugar polymer comprises AP3-2 and AP3-3, wherein AP3-2 is a sugar polymer composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose, and the structural formula is shown in formula (I):

wherein a and b range from 1 to 1000;

AP3-2 is a sugar polymer composed of arabinose, and the structural formula is shown in formula (II):

wherein a ranges from 1 to 1000.

2. Use of a camelid spinulose polymer according to claim 1 in the manufacture of a medicament for the prevention and/or treatment of osteoporosis.

3. A process for the preparation of a camelid tag polymer according to claim 1 or 2, comprising the steps of:

s1, soaking: soaking aerial parts of Alhagi sparsifolia to obtain soaking solution A;

s2, water extraction: heating and extracting the soak solution A, and filtering to obtain an extract B and a residue C;

s3, grading alcohol precipitation: concentrating the extracting solution B under reduced pressure, adding ethanol to make the volume concentration of the ethanol be a%, standing, and collecting precipitate and supernatant to obtain crude sugar polymer AP1 and supernatant D; concentrating the supernatant D again, adding ethanol to make the volume concentration of the ethanol be b%, standing, and collecting precipitate and supernatant to obtain crude sugar polymer AP2 and supernatant E; concentrating the supernatant E again, adding ethanol to make the volume concentration of the ethanol be c%, standing, collecting the precipitate to obtain a crude sugar polymer AP3, wherein a is more than or equal to 10 and less than b and less than c and less than 100;

s4, purification: purifying the crude sugar polymers AP1, AP2 and AP3 to obtain purified crude sugar polymers AP1, AP2 and AP 3;

s5, ion exchange column chromatography: performing ion exchange column chromatography on the purified crude sugar polymer AP3 obtained in the step S4, performing gradient elution by using a NaCl solution with the concentration of 0-2M, tracking an elution curve by using a phenol-sulfuric acid method, respectively collecting sugar parts according to the elution curve, then concentrating, dialyzing, freeze-drying, respectively dissolving by using water, and obtaining a supernatant H after centrifugal separation;

s6, molecular sieve gel column chromatography: subjecting the supernatant H obtained in the step S5 to molecular sieve gel column chromatography, eluting with water, tracking an elution curve by using a phenol-sulfuric acid method, collecting sugar parts according to the elution curve, concentrating, and freeze-drying to obtain the camel thorn sugar polymers AP3-2 and AP3-3 in the claim 1.

4. The method of claim 3, wherein the water extraction in step S2 comprises the following steps: extracting the overground part of the alhagi sparsifolia with hot water of 60-100 ℃ in a volume of 5-15 times for 1-10 hours.

5. The method according to claim 3 or 4, wherein the above parts of the alhagi sparsifolia in step S2 are stems, branches, leaves, inflorescences of alhagi sparsifolia.

6. The method according to claim 3, wherein 10. ltoreq. a < 60, 60. ltoreq. b < 80, 80. ltoreq. c < 100 in step S3.

7. The method according to claim 3, wherein the specific operations of purifying in step S4 are: removing proteins from the crude sugar polymers AP1, AP2 and AP3 by a Sevag method, dialyzing by a dialysis bag after removing proteins, and freeze-drying, wherein the cut-off molecular weight of the dialysis bag is 1000 Da.

8. The method of claim 3, wherein the ion exchange column in step S5 is ion exchange cellulose or ion exchange gel, and the cut-off molecular weight of the dialysis bag is 100 Da.

9. The method of claim 3, wherein the molecular sieve gel chromatography in step S6 is performed using Sephadex G or Sephacryl S series molecular sieve chromatography column.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a camel thorn sugar polymer, a preparation method of a seminal sugar polymer, and an application of the camel thorn sugar polymer in preventing and treating osteoporosis.

Background

Osteoporosis (0P) is a systemic bone disease in which bone density and bone quality are reduced due to various causes, bone microarchitecture is destroyed, bone brittleness is increased, and thus fracture is likely to occur. The incidence of osteoporosis is rapidly increasing due to the aging of the population. The postmenopausal women have a sharp decline of estrogen level, which leads to an increase of bone conversion rate and is easy to induce osteoporosis, and the osteoporosis becomes a serious public health problem facing the current society. The key to treating this common metabolic bone disease is maintaining a balance between bone resorption and bone formation. At present, hormone replacement therapy, bisphosphonates, selective hormone receptor modulators and calcitonin are used for reducing the risk of primary and recurrent fractures in clinic, but these drugs have certain side effects in the long-term use process, so the development of novel anti-osteoporosis drugs with high efficiency and low toxicity is imperative. The natural medicine has the advantages of long history, small side effect and the like in the aspect of treating osteoporosis, and is widely concerned by people.

Alhagi pseudoalhagi Desv belonging to the genus Camellia of the family Leguminosae and distributed in inner Mongolia, Gansu, Qinghai and Xinjiang in China. Hasakstein, wuzbecstein, tukulmastan, gilgisten and taggesteins are also distributed. The alhagi sparsifolia has high nutritive value and is the most common feed for ruminants. In addition, the alhagi sparsifolia has high medicinal value. The alhagi sparsifolia contains various bioactive substances, such as flavonoids, alkaloids, polysaccharides, vitamins, fatty acids, steroids, coumarins, tannins and the like. They have good performance in anti-asthma, yang-strengthening, antipyretic, appetite-stimulating, antirheumatic, diuretic, sedative, expectorant, antidiarrheal, anti-inflammatory, antiviral, antibacterial, antihypertensive, hepatoprotective, immunomodulating and dermatological treatments.

At present, the research on the alhagi sparsifolia at home and abroad mainly focuses on the extraction, separation and identification of small molecular substances of the alhagi sparsifolia and the pharmacological activity of crude extracts. Although the camel thorn coarse sugar polymer is reported, the structural characteristics and the physicochemical properties of the camel thorn coarse sugar polymer are not clear, and the structure of the camel thorn sugar polymer is not reported. In addition, the research on the camel thorn sugar polymer in the aspect of preventing and treating the osteoporosis at home and abroad is not reported yet.

Disclosure of Invention

In a first aspect, the invention aims to provide a camel thorn sugar polymer.

The second aspect of the invention aims to provide a preparation method of the camel thorn sugar polymer.

The third aspect of the invention aims to provide the application of the camel thorn sugar polymer in preparing the medicine for preventing and/or treating osteoporosis.

The technical scheme adopted by the invention is as follows:

in a first aspect of the invention, a camel thorn sugar polymer is provided, which comprises AP3-2 and AP3-3, wherein AP3-2 is a glycopolymer composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose, and the structural formula is shown as the formula (I):

wherein a and b range from 1 to 1000;

AP3-2 is a sugar polymer composed of arabinose, and the structural formula is shown in formula (II):

wherein a ranges from 1 to 1000.

In a second aspect of the invention, there is provided a use of the camelid thorn sugar polymer of the first aspect of the invention in the preparation of a medicament for the prevention and/or treatment of osteoporosis.

In a third aspect of the invention, there is provided a process for preparing a camel chewing gum polymer according to the first or second aspect of the invention, comprising the steps of:

s1, soaking: soaking aerial parts of Alhagi sparsifolia to obtain soaking solution A;

s2, water extraction: heating and extracting the soak solution A, and filtering to obtain an extract B and a residue C;

s3, grading alcohol precipitation: concentrating the extracting solution B under reduced pressure, adding ethanol to make the volume concentration of the ethanol be a%, standing, and collecting precipitate and supernatant to obtain crude sugar polymer AP1 and supernatant D; concentrating the supernatant D again, adding ethanol to make the volume concentration of the ethanol be b%, standing, and collecting precipitate and supernatant to obtain crude sugar polymer AP2 and supernatant E; concentrating the supernatant E again, adding ethanol to make the volume concentration of the ethanol be c%, standing, collecting the precipitate to obtain a crude sugar polymer AP3, wherein a is more than or equal to 10 and less than b and less than c and less than 100;

s4, purification: purifying the crude sugar polymers AP1, AP2, AP3 and AP4 to obtain purified crude sugar polymers AP1, AP2, AP3 and AP 4;

s5, ion exchange column chromatography: performing ion exchange column chromatography on the purified crude sugar polymer AP3 obtained in the step S4, performing gradient elution by using a NaCl solution with the concentration of 0-2M, tracking an elution curve by using a phenol-sulfuric acid method, respectively collecting sugar parts according to the elution curve, then concentrating, dialyzing, freeze-drying, respectively dissolving by using water, and obtaining a supernatant H after centrifugal separation;

s6, molecular sieve gel column chromatography: subjecting the supernatant H obtained in the step S5 to molecular sieve gel column chromatography, eluting with water, tracking an elution curve by using a phenol-sulfuric acid method, collecting sugar parts according to the elution curve, concentrating, and freeze-drying to obtain the camel thorn sugar polymers AP3-2 and AP3-3 in the claim 1.

According to the method of the third aspect of the invention, the above-ground part of alhagi sparsifolia in step S1 is the stem, branch, leaf, inflorescence of alhagi sparsifolia.

According to the method of the third aspect of the present invention, the water extraction in step S2 specifically comprises: extracting the overground part of the alhagi sparsifolia with hot water of 60-100 ℃ in a volume of 5-15 times for 1-10 hours.

According to the method of the third aspect of the present invention, in step S3, a is 10. ltoreq. a < 60, b is 60. ltoreq. b < 80, and c is 80. ltoreq. c < 100.

According to the method of the third aspect of the present invention, the specific operations of purifying in step S4 are: removing proteins from the crude sugar polymers AP1, AP2 and AP3 by a Sevag method, dialyzing by a dialysis bag after removing proteins, and freeze-drying, wherein the cut-off molecular weight of the dialysis bag is 1000 Da.

According to the method of the third aspect of the present invention, the ion exchange column in step S5 is ion exchange cellulose or ion exchange gel, and the molecular weight cut-off of the dialysis bag is 100 Da.

According to the method of the third aspect of the present invention, the molecular sieve gel chromatography in step S6 uses Sephadex G or Sephacryl S series molecular sieve chromatographic column.

The invention has the beneficial effects that:

1. the camel thorn sugar polymer which is not reported before is prepared by purifying the camel thorn sugar polymer by ion exchange chromatography and molecular sieve gel column chromatography, and the physicochemical properties, the molecular weight, the monosaccharide composition and the like of the camel thorn sugar polymer are systematically analyzed and confirmed, so that the characteristic structure of the camel thorn sugar polymer is successfully obtained.

2. The camel thorn coarse sugar polymer is separated and purified by column chromatography, the effect is obvious, and camel thorn sugar polymers AP3-2 and AP3-3 are prepared for the first time.

3. The invention identifies the prepared camel thorn sugar polymers AP3-2 and AP3-3, defines the physicochemical properties and the structure of the camel thorn sugar polymers, and provides a structural basis for researching the pharmacological activity mechanism of the camel thorn sugar polymers.

4. The invention provides a preparation method of a crude sugar polymer and a refined sugar polymer in alhagi sparsifolia, and an activity research of the alhagi sparsifolia polymer in the aspect of preventing and treating osteoporosis, provides a basis for the application of the alhagi sparsifolia polymer in the fields of medicines and the like, and also provides the application of the alhagifolia polymer in medicines for preventing and/or treating osteoporosis.

5. Compared with the traditional water boiling method for extracting the camel thorn sugar polymer, the invention adopts a water extraction and alcohol precipitation method, carries out graded alcohol precipitation on the camel thorn sugar polymer from low to high in alcohol concentration, and carries out primary separation on the camel thorn sugar polymer, and meanwhile, the high-concentration alcohol can separate the sugar polymer with large polarity and good water solubility from the sugar polymer with small polarity and poor water solubility, so that the difficulty of later-stage separation and purification is greatly reduced. The preparation process is simple, the operation is convenient, and the large-scale production can be realized.

Drawings

FIG. 1: an infrared spectrum of AP 3-2.

FIG. 2: of AP3-2¹³C NMR spectrum.

FIG. 3: of AP3-2¹H NMR spectrum.

FIG. 4: of AP3-2¹H-¹H COSY map.

FIG. 5: HSQC spectrum of AP 3-2.

FIG. 6: HMBC mapping of AP 3-2.

FIG. 7: effect of AP3-2 on the proliferation of MC3T3-E1 cells.

FIG. 8: effect of AP3-2 on the differentiation of MC3T3-E1 cells.

FIG. 9: effect of AP3-2 on MC3T3-E1 cell mineralization.

FIG. 10: an infrared spectrum of AP 3-3.

FIG. 11: of AP3-3¹³C NMR spectrum.

FIG. 12: of AP3-3¹H NMR spectrum.

FIG. 13: of AP3-3¹H-¹H COSY map.

FIG. 14: HSQC spectrum of AP 3-3.

FIG. 15: HMBC mapping of AP 3-3.

Detailed Description

The present invention will be described in detail with reference to specific embodiments, and the exemplary embodiments and descriptions thereof herein are provided to explain the present invention but not to limit the present invention.