CN111235199A - Preparation of hesperetin derivative for strengthening bones - Google Patents

Preparation of hesperetin derivative for strengthening bones Download PDF

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CN111235199A
CN111235199A CN202010056046.0A CN202010056046A CN111235199A CN 111235199 A CN111235199 A CN 111235199A CN 202010056046 A CN202010056046 A CN 202010056046A CN 111235199 A CN111235199 A CN 111235199A
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张才来
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Abstract

The invention provides a preparation method and application of a hesperetin derivative for strengthening bones, belonging to the field of biological medicines; the hesperetin derivative is prepared by connecting a prenyl group to a parent ring of hesperetin and then connecting a glycosyl group by using a biological enzyme; the hesperetin derivative has the characteristics of high solubility and strong biological activity; has better effects than hesperetin in the aspects of controlling and treating osteoporosis and promoting blood circulation.

Description

Preparation of hesperetin derivative for strengthening bones
Technical Field
The invention relates to a preparation method of a hesperetin derivative for strengthening bones, belonging to the field of biological medicines.
Technical Field
Osteoporosis is a disease of metabolic disorders of bones throughout the body, in which the microstructure of bone tissues is damaged, bone mineral components and bone matrix are continuously reduced in equal proportion, bone is thinned, the number of trabeculae is reduced, bone fragility is increased, and the risk of fracture is increased. When the hormone level in the body is reduced, the bone absorption is increased, and the bone formation is reduced. Therefore, there is currently no effective treatment for osteoporosis.
Hesperidin in Mandarin orange has effects of resisting cancer, treating osteoporosis, resisting oxidation, promoting blood circulation, improving cardiovascular and cerebrovascular circulation and improving sleep, and has high economic and medicinal values. The hesperidin has low bioavailability and unobvious effect due to the low solubility of the hesperidin in water and a buffer solution.
Prenyl has an important role in biosynthesis. The existence of the isoamylene side chain increases the lipophilicity and biological guidance of the flavonoid compound, so that the flavonoid compound is easier to permeate cell membranes in organisms to reach an action target, and the medicinal value of the flavonoid compound is greatly improved.
Disclosure of Invention
According to the invention, the prenyl group is added to hesperetin, so that the problem that prenyl is less applied to the flavone compound is solved, and the effects of preventing and treating osteoporosis and promoting blood circulation of the hesperetin flavone compound are improved. However, the monomer is hardly soluble in water, and when it is used as a drug directly, its bioavailability in the human body is low, and it is difficult to sufficiently exert the therapeutic effect of the drug. Therefore, the hesperetin is modified, such as: the hesperetin is glycosylated, so that the solubility and the bioavailability of the hesperetin are improved.
The hesperetin derivative with high purity, high quality and high biological activity is obtained by glycosylation conversion of prenyl hesperetin. The solubility of the hesperetin derivative is improved by more than 300 times compared with hesperetin and hesperidin, and the hesperetin derivative can be widely applied to the fields of food, medicine and the like, so that the application range of the hesperetin is expanded.
The hesperetin derivative (I) provided by the invention comprises the following steps:
1) reacting 2g of hesperetin, 2-2.4 times of equivalent of acetic anhydride and 2-4 times of piperidine at 100-140 ℃ for 2-12 h to obtain a product IIa with the yield of 90-93%;
2) reacting 2g of IIa with 1-5 times of thiophenol, 0.1-1 time of imidazole and N-methylpyrrolidone as a solvent at 0 ℃ for 5-15 hours to obtain a product IIb, wherein the yield is 86-88%;
3) reacting IIb with 1-5 times of (2-methylbut-3-en-2-yl) isobutyl carbonate and triphenylphosphine in a THF solution at-20-0 ℃ for 12 hours to obtain IIc, wherein the yield is 81-84%;
4) adding 2 times of equivalent acetic anhydride and 8 times of ammonium acetate into 2g of IIc, and refluxing in methanol for 20 hours to obtain a product IId, wherein the yield is 90-95%.
5) Treating IId with biological enzyme to prepare the hesperetin derivative (I):
Figure BDA0002372433760000021
wherein: n is 1-5; r1、R2＝OH、OCH3、OCH2CH3；R3＝Glu；R4、R5
Figure BDA0002372433760000022
The hesperetin derivative and the formation step equation of the invention are as follows:
Figure BDA0002372433760000023
wherein: n is 1-5; r1、R2＝OH、OCH3、OCH2CH3；R3＝Glu。
The enzymatic method of the present invention comprises: one or a combination of glucosyltransferase and rhamnosyltransferase; the added glycosyl is glucose, UDP-glucose, rhamnose, UDP-rhamnose.
The reaction conditions of the invention are as follows: the reaction buffer solution is 0.05-1% by mass of dipotassium hydrogen phosphate-potassium citrate buffer solution or 0.9% by mass of NaCl buffer solution; and (3) controlling the pH value of the buffer solution to be 8-10, controlling the reaction temperature to be 35-75 ℃, controlling the reaction time to be 1-24 hours, and after the reaction is finished, performing suction filtration and drying to obtain the hesperetin derivative.
The synthesis steps of the hesperetin derivative are preferably as follows:
1) reacting 2g of hesperetin, 2-2.4 times of acetic anhydride equivalent and 2-4 times of piperidine at 120-130 ℃ for 6-8 h to obtain a product IIa.
2) 2g of IIa, 2-4 times of thiophenol, 0.2-0.5 time of imidazole and N-methylpyrrolidone as a solvent react for 8-10 hours at 0 ℃, and the product is IIb.
3) And reacting IIb with 2-3 times of (2-methylbut-3-en-2-yl) isobutyl carbonate and triphenylphosphine in a THF solution at-5-0 ℃ for 12 hours to obtain IIc.
4) Adding 2 times of equivalent of acetic anhydride and 8 times of ammonium acetate into IIc 2g, and refluxing in methanol for 20h to obtain a product IId.
5) Treating IId with biological enzyme to prepare the hesperetin derivative (I).
The structural formula of the hesperetin derivative (I-1) is as follows:
Figure BDA0002372433760000031
wherein: n is 1 to 5. R4、R5Is composed of
Figure BDA0002372433760000032
The invention aims to provide a preparation method of a hesperetin derivative.
The invention also aims to provide the application of the hesperetin derivative in preventing and treating osteoporosis.
Another object of the present invention is to provide a use of a hesperetin-based derivative for promoting blood circulation.
The raw materials mentioned in the preparation method of hesperetin of the invention are not limited to plant extracts or/and transformation products such as hesperetin, hesperidin and neohesperetin which are sold in the market.
The invention provides a pharmaceutical composition, which comprises the compound and pharmaceutically acceptable salts, carriers, excipients, diluents, vehicles or the combination thereof.
Has the advantages that: according to the hesperetin derivative disclosed by the invention, 1-5 glycosyl groups are added on a flavone parent body, so that the solubility of hesperetin/glycoside is greatly improved, the problem of poor solubility of hesperetin/glycoside is solved, the problem of decomposition and absorption of hesperetin/glycoside in a human body is also solved, and the bioavailability of hesperetin/glycoside in a medium is greatly improved.
Drawings
Fig. 1HPLC method for determining the solubility of hesperetin derivative in water.
FIG. 2 Effect of hesperetin derivatives on ALP activity.
FIG. 3 HPLC chart of hesperetin derivative.
The present disclosure is specifically described with reference to specific embodiments, but the scope of the present disclosure is not limited to the following embodiments.
The specific implementation example is as follows:
example 16, 8-Diprenyl substituted Hesperetin derivative (II)d) Preparation of
1) Reacting 2g of hesperetin, 2.2 times of acetic anhydride equivalent and 2-4 times of piperidine at 100-140 ℃ for 8h to obtain a product IIaThe yield is 91.0%;
2) will IIa2g of thiophenol accounting for 1-5 times of the total weight of the raw materials, imidazole accounting for 0.4 time of the total weight of the raw materials, reacting for 5-10 hours at 0 ℃ by taking N-methyl pyrrolidone as a solvent to obtain a product IIbThe yield is 85.9%;
3) will IIbReacting with 4-5 times of (2-methylbut-3-en-2-yl) isobutyl carbonate and triphenylphosphine serving as a catalyst in a THF solution at the temperature of-4-0 ℃ for 12 hours to obtain IIcThe yield is 83.0%;
4) will IIc2g of the mixture is added with 2 times of equivalent of acetic anhydride and 8 times of ammonium acetate, and the mixture is refluxed for 20 hours in methanol to obtain a product, namely the 6, 8-diisoprenyl substituted hesperetin derivative IIdThe yield thereof was found to be 91.3%.
The structural formula of the reaction steps is as follows:
Figure BDA0002372433760000051
example 2 glycosylation of hesperetin derivatives
The method comprises the steps of taking 6, 8-diisoprenyl substituted hesperetin derivatives as raw materials, taking a reaction buffer solution as a dipotassium hydrogen phosphate-potassium citrate buffer solution with the mass fraction of 0.05%, controlling the pH value of the buffer solution to be 8, controlling the reaction temperature to be 60 ℃ and controlling the reaction time to be 18-24 hours. And adding glucosyltransferase into a reaction system, wherein the mass fraction of the glucosyltransferase is 25-30% of that of the 6, 8-diisoprenyl substituted hesperetin derivative to obtain the hesperetin derivative, the molar yield of the hesperetin derivative in the reaction system is 94.1%, after the reaction is finished, performing suction filtration and drying to obtain the hesperetin derivative, and the HPLC of the hesperetin derivative is shown in figure 3.
Example 3 hesperetin derivative solubility determination
And then adding excessive hesperetin derivative into 2mL of water phase, placing the mixture on a constant temperature oscillator at 25 +/-1 ℃ for continuous oscillation for 72h, taking out the mixture, transferring the mixture into a centrifuge tube, centrifuging the mixture for 15min at 8000r/min, taking supernatant, filtering the supernatant by using a 0.45-micrometer microporous membrane, diluting the supernatant to be within a linear range by using methanol, and measuring the solubility of the hesperetin derivative in water by using an HPLC method. The results are shown in FIG. 1.
The results show that: the solubility of the hesperetin derivative is about 230 times of that of hesperetin.
Example 4 use of Hesperetin derivatives in drugs for the prevention, treatment of bone fractures and osteoporosis
The principle is as follows: mononuclear osteoclast precursor cells can gradually fuse and differentiate into multinucleated mature osteoclasts under the induction of differentiation cytokines (such as RANKL, M-CSF). Osteoclast precursor cells do not have the capacity to dissolve bone, and only mature osteoclasts have the capacity to dissolve bone, and therefore, the level of differentiation of osteoclasts can reflect their bone-dissolving capacity.
The method comprises the following steps: osteoclast precursor cell line or primary isolated cultured mouse osteoclast precursor cells were seeded into 12-well culture plates at 10000 cells/well. A control group is arranged, a single drug adding group 1 (hesperetin derivative), a single drug adding group 2(RANKL) and a double drug adding group (hesperetin derivative + RANKL). After the cells adhere to the wall overnight, adding different doses of the compound and/or RANKL with the concentration of 50ng/ml, and continuously culturing for 3-5 days. After the cells with the RANKL alone are completely fused, the cells are washed once by distilled water preheated at 37 ℃, methanol is added for fixation for 30 seconds, the cells are washed three times by distilled water preheated at 37 ℃, then TRAP staining is carried out, and the number of TRAP positive multinucleated cells is counted under a microscope.
Results and evaluation: as shown in table 1, the number of osteoclasts induced to differentiate by RANKL was significantly reduced in the osteoclast precursor cell line or primary isolated cultured mouse osteoclast precursor cells after adding the compound of the present invention, compared to the control group, and this result confirmed that: the compound can effectively inhibit the differentiation of osteoclast induced by RANKL.
TABLE 1 Effect of hesperetin derivatives on differentiation of Primary cultured bone marrow BMMs cells into osteoclasts
Concentration of the Compound Concentration of Increment rate (%)
Blank control -/- 0.00
Single drug adding group 1 1.0μM 18.12％
Single drug adding group 2 1.0μM 12.21％
Double drug adding set 1 1.0μM 10.72％
As can be seen from table 1, after adding the hesperetin derivative, the number of osteoclasts induced to differentiate by RANKL was significantly reduced in either the osteoclast precursor cell line or the primary isolated cultured mouse osteoclast precursor cells, as compared to the control group, and this result demonstrates that: the hesperetin derivative can effectively inhibit the differentiation of osteoclast induced by RANKL.
Example 5 urine and blood sample Collection of Hesperetin derivatives
7-8 week-old female NIH mice were housed at 22 deg.C under light and dark conditions (12 h: 12h) and fed a normal calcium level (0.6% Ca) control diet for 2 days, after 12 week-old mice were sham or Ovariectomy (OVX) restored for 2 weeks, mice were randomly selected and divided into five groups, one group consisting of sham vehicle (2% ethanol) and the remainder OVX + vehicle, OVX +17 β -estradiol (E2; 2 mg. g.)-1·day-1) And two groups of OVX + hesperetin derivatives (0.2 and 0.4 mg. multidot.g)-1Day-1). Five groups of animals were given orally for 6 weeks. Animals were exposed to a diet containing 0.6% Ca and 0.65% P during which time the mice were allowed free access to water and urine was collected, and then the mice were anesthetized and sacrificed. Blood samples were taken from the inferior vena cava and mouse sera were stored at-80 ℃ until analysis.
Example 6 Hesperetin derivatives analysis of serum and urine Biochemical indicators
The weight gain of OVX mice treated with E2 and the hesperetin derivative, respectively, was significantly reduced (P)<0.05 vs OVX + 2% ethanol). Treatment with E2, but without the hesperetin derivative, significantly increased uterine weight (P) in OVX mice<0.05). The results indicate that hesperetin derivatives are unable to mimic estrogens to exert the effect of uterine hormones in mice. E2 changed serum P, but not serum Ca (P) in OVX mice<0.05 vs OVX + 2% ethanol). Using a monohesperetin derivative (0.2 mg. g)-1·day-1Or 1.4mg g-1·day-1) Treated OVX mice had no altered serum P or Ca. OVX itself increases mouse urinary Ca excretion (P)<0.001. Sham surgery + 2% ethanol). High dose hesperetin derivative (0.4mg g)-1·day-1) Or E2 inhibited OVX-induced reductions in urinary Ca excretion by 19% and 24%, respectively, in OVX mice (p1.5.OVX + 2% ethanol), respectively. Treatment of hesperetin derivatives at lower dosesThe OVX mice had reduced urinary Ca excretion of 38% and 46% (P)<0.05). Urinary deoxypyridine (DPD) levels are biochemical markers for assessing bone resorption. The results show that OVX causes a significant increase in urine DPD levels (P)<0.05 sham surgery + 2% ethanol), whereas treatment of OVX mice with E2 inhibited urinary DPD levels by 48% (P)<0.01 vs OVX + 2% ethanol). The hesperetin derivative did not alter the DPD level in urine of OVX mice.
The hesperetin derivative is shown to reduce the discharge of Ca in urine of mice and increase the level of DPD, so the hesperetin derivative can play a strong role in strengthening bones.
Example 7 cell proliferation assay and ALP Activity of Hesperetin derivatives
Dose-dependent effects of hesperetin derivative on proliferation of rat osteoblast-like UMR-106 cells (culture method same as in example 10) and ALP activity. At 24h, hesperetin derivatives at 10nM to 1mM increased UMR-106 cell proliferation (P <0.05 vs vehicle). All concentrations (0.1nM to 10mM) of hesperetin derivative increased osteoblast proliferation when the cells were cultured for 48 h. In particular, 0.1-10nM hesperetin derivatives are effective in increasing the osteoblast number by 33% to 40% (P <0.01 and P <0.001 relative to vehicle). ALP is a common marker for assessing osteoblast differentiation. As observed with E2, hesperetin derivative at a certain concentration range (10nM-1mM) significantly increased ALP activity in UMR-106 cells (FIG. 2). Low concentrations of hesperetin derivative (1nM) significantly increased osteoblast differentiation by 14% (P <0.001), whereas 10nM E2 increased ALP activity by 15% (P <0.001)
Therefore, it is known that the hesperetin derivative of 10nM to 1mM can enhance ALP activity, remarkably promote differentiation of osteoblasts, and effectively prevent osteoporosis symptoms.
Example 10 Effect of hesperetin derivatives on Cold-exposed rat Tail skin temperature
1. Experimental animals: wistar rat, male, weight 180-220 g, provided by Guangdong medical laboratory animal center
2. Main drugs and reagents: prazosin (Pra), hesperetin derivatives (n ═ 1-5), tolazoline (Tol), and sodium chloride injection (physiological saline).
3. Determination of rat tail skin temperature in cold exposure environment
A male Wistar rat with the weight of 180-220 g at SPF level is placed in a low-temperature chamber (0 +/-2 ℃) after 1h of preventive intragastric administration of a tested drug, and at different time points in the cold exposure process, an infrared camera is used for shooting at 1/3 positions from the tail root of the rat, and the change of skin temperature is recorded.
4. Statistical methods and data analysis
Data are expressed as mean standard deviation (± s). SPSS13.0 statistical software is used, variance analysis is adopted in the multi-group mean difference test, the LSD method or Dunnett's method is further used for pairwise comparison, and the difference is more than 0.05, so that the statistical significance is achieved.
5. Results of the experiment
In the second part of research, 2.0mg/kg of hesperetin derivative with the best antithrombotic effect and 0.6mg/kg of prazosin which are used as the medium dosage of the novel anti-freezing prescription are formulated into high and low dosages according to the equal ratio of 3 times (the ratio of the two medicines is 10:3 unchanged) for preventive intragastric administration of cold-exposed rats. The results show (table 3) that the tail skin temperature of rats in each group is greatly reduced after cold exposure for 120min without significant difference; after the cold exposure for 240min, the tail skin temperature of the ani2.5+ Tol 12.5 group is obviously improved (P is less than 0.05) compared with that of a blank control group, and the tail skin temperature of the rats in the medium and high dose groups which are formed by compounding the prenyl hesperetin derivative and the prazosin is obviously higher than that of other groups (P is less than 0.05), which shows that the tail skin temperature of the rats in the three dose groups of the hesperetin derivative and the prazosin is in a rising trend compared with that of the blank group.
Table 3: effect of Hesperetin derivative (G-hes) and prazosin (Pra) on rat Tail skin temperature Exposure rats to Cold
Figure BDA0002372433760000093
(n＝6)
Figure BDA0002372433760000092
*P＜0.05vs control ▲P＜0.05vs Ani 2.5+Tol 12.5 #P＜0.05vs 120min
The experiment result shows that the novel anti-freezing composition consisting of the hesperetin derivative and the prazosin can improve the skin temperature of a part which is easy to freeze (represented by a rat tail part in the experiment) in a cold exposure environment, and the effect can reach 2-4 hours. The vasodilatation function, the blood circulation condition and the skin temperature of the patient are coordinated, the tolazoline has a synergistic effect, and the skin temperature is the most intuitive and direct index for evaluating the anti-freezing effect of the medicine, so that the vasodilatation can be promoted, and the epidermis temperature can be increased.

Claims (5)

1. A preparation method of a hesperetin derivative for strengthening bones is characterized by comprising the following steps: the structural formula of the hesperetin derivative is shown as the formula (I):
Figure FDA0002372433750000011
wherein: n is 1-5; r1、R2＝OH、OCH3、OCH2CH3；R3＝Glu；R4、R5
Figure FDA0002372433750000012
The hesperetin derivative is prepared from hesperidin, hesperetin and hesperetin-7-O-glucoside as raw materials;
the preparation method of the hesperetin derivative comprises the following steps:
1) reacting 2g of hesperetin, 1-2.4 times of equivalent of acetic anhydride and 2-5 times of piperidine at 100-140 ℃ for 2-12 hours to obtain a product IIa;
2) reacting 2g of IIa with 1-5 times of thiophenol, 0.1-1 time of imidazole and N-methylpyrrolidone as a solvent at 0 ℃ for 5-15 hours to obtain a product IIb;
3) reacting IIb with 1-5 times of (2-methylbut-3-en-2-yl) isobutyl carbonate and triphenylphosphine serving as a catalyst in a THF solution at-20-0 ℃ for 12 hours to obtain IIc;
4) adding 2 times of equivalent of acetic anhydride and 8 times of ammonium acetate into IIc 2g, and refluxing in methanol for 20h to obtain a product IId.
5) Treating IId with biological enzyme to prepare hesperetin derivative (I);
the biological enzyme comprises: glucosidase, glucoside transferase.
2. The preparation of the hesperetin derivative for bone strengthening according to claim 1, which is characterized in that: the synthesis steps of the hesperetin derivative are preferably as follows:
1) reacting 2g of hesperetin, 2-2.4 times of equivalent of acetic anhydride and 2-4 times of piperidine at 120-130 ℃ for 6-8 h to obtain a product IIa;
2) reacting 2g of IIa with 2-4 times of thiophenol and 0.2-0.5 time of imidazole for 8-10 hours at 0 ℃ by taking N-methylpyrrolidone as a solvent to obtain a product IIb;
3) reacting IIb with 2-3 times of (2-methylbut-3-en-2-yl) isobutyl carbonate and triphenylphosphine serving as catalysts in a THF solution at the temperature of-5-0 ℃ for 12 hours to obtain IIc;
4) adding 2g of IIc into 2 times of equivalent of acetic anhydride and 8 times of ammonium acetate, and refluxing in methanol for 20 hours to obtain a product IId;
5) treating IId with biological enzyme to prepare hesperetin derivative (I);
the structural formula of the hesperetin derivative (I-1) is as follows:
Figure FDA0002372433750000021
wherein: n is 1-5; r4、R5Is composed of
Figure FDA0002372433750000022
3. The preparation of a hesperetin derivative for bone strengthening according to claim 1 or 2, characterized in that: the conditions for treating IId with the biological enzyme are as follows: the reaction buffer solution is 0.05-1% by mass of dipotassium hydrogen phosphate-potassium citrate buffer solution or 0.9% by mass of NaCl buffer solution; and (3) controlling the pH value of the buffer solution to be 8-10, controlling the reaction temperature to be 35-75 ℃, controlling the reaction time to be 1-24 h, and after the reaction is finished, performing suction filtration and drying to obtain the hesperetin derivative.
4. The preparation of a hesperetin derivative for bone strengthening according to claim 1 or 2, characterized in that: the hesperetin derivative can be used for preventing and treating osteoporosis, promoting blood circulation, reducing blood lipid and lowering blood pressure.
5. The preparation of a hesperetin derivative for bone strengthening according to claim 1 or 2, characterized in that: the hesperetin derivative can be used for pharmaceutically acceptable salts, carriers, excipients, diluents, vectors or pharmaceutical compositions thereof.
CN202010056046.0A 2020-01-17 2020-01-17 Preparation of hesperetin derivative for strengthening bones Withdrawn CN111235199A (en)

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