CN113101311A - Rhodiola rosea active component and application thereof in preparing medicine for treating cardiac hypertrophy - Google Patents
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Abstract
The invention provides a preparation method of a rhodiola rosea active component and also provides application of the rhodiola rosea active component in preparing a medicament for preventing and treating cardiac hypertrophy. Experiments prove that the rhodiola rosea active component prepared by the method has better prevention and treatment effects on myocardial hypertrophy diseases when being used as an SGK1 protein inhibitor, and provides experimental evidence for clinical application of the rhodiola rosea active component in prevention and treatment of myocardial hypertrophy. Meanwhile, the active component of the rhodiola can be used for preparing a medicament for preventing and treating myocardial hypertrophy caused by overhigh activity of SGK1 protein, and has good application and development prospects.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to a preparation method of a rhodiola rosea active component and application of the rhodiola rosea active component in preparation of a medicine for preventing and treating myocardial hypertrophy.
Background
Myocardial hypertrophy is an adaptive compensatory response of the myocardium to pressure or volume overload, and moderate myocardial hypertrophy can improve the blood pumping function of the heart, but long-term or excessive hypertrophic stimulation can promote the occurrence and development of heart failure. During myocardial hypertrophy, the energy source mainly takes fatty acid oxidation energy supply as the main energy source and is converted into glycolysis mainly. The gene expression of fatty acid oxidation related enzymes of hypertrophic myocardium is reduced, the utilization of fatty acid is reduced, lipid metabolites are deposited, the structure and the function of the heart are damaged, and the life safety of the heart can be influenced or even threatened when the heart is serious.
A Chinese traditional and western medicine combined impurity published in 2009, 3.29, volume 3, published in article 3, is disclosed in "influence of rhodiola rosea and ganoderma lucidum spore powder on spontaneous hypertensive rat hypertrophic myocardial adiponectin signal pathway": the energy source for normal adult mammalian myocardium is primarily long chain fatty acid beta oxidation, studies in recent years have shown that: an important metabolic change in hypertrophic and failing myocardium is a decrease in the oxidative utilization of fatty acids, while the uptake utilization of glucose is increased. In a mouse cardiac hypertrophy model, a rat hereditary hypertension and heart failure model and a human failing heart, compared with a normal cardiac muscle, the gene expression of fatty acid oxidation-related enzymes is down-regulated, and the fatty acid oxidation is reduced. Although the oxygen consumption for glucose oxidation is reduced, the energy produced by glucose oxidation is much lower than that produced by fatty acid oxidation, and eventually the myocardial energy is deficient. The reduction of fatty acid utilization can cause local fatty acid deposition in myocardial tissues to cause arrhythmia and myocardial fibrosis, thereby promoting the occurrence and development of heart failure.
A Chinese medicine named "rhodiola root for treating myocardial hypertrophy and transforming growth factor beta" is published in 2010, 24 th volume and 6 th phase in traffic medicine1Expression impact, the article discloses: myocardial hypertrophy is the target organ response of the heart to chronic volume or pressure overload, with left ventricular hypertrophy being the most common and the earliest manifestation of target organ damage from hypertension. Recent studies have demonstrated that transforming growth factor (TGF-. beta.) is1) Is a growth factor with multiple potentialities, plays an important role in the pathological process of the left ventricular hypertrophy of the hypertension, and is positively correlated with the left ventricular hypertrophy. The number of hypertension patients in China is about 1.6 hundred million, so that the research on how to control and reverse the hypertension left ventricular hypertrophy has important practical significance. The rhodiola rosea is a rhodiola plant of Crassulaceae, and comprises the following effective components: salidroside, tyrosol, and benzhydryl piperidine, and the like. Early research finds that the medicine has the pharmacological effects of promoting blood circulation to remove blood stasis, resisting aging and resisting fatigue, and recently finds that the medicine also has certain pharmacological effectsResisting anoxia, and protecting endothelium function.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides the following technical scheme:
the preparation method of the rhodiola active component is characterized by comprising the following steps:
(1) slicing radix Rhodiolae, soaking, extracting with ethanol under reflux, sieving the extractive solution, and mixing filtrates;
(2) vacuum concentrating the filtrate, spray drying, and sieving to obtain radix Rhodiolae extract;
(3) weighing rhodiola rosea extract, adding methanol solution for dissolving, preparing extracting solution, centrifuging the extracting solution, and taking supernatant for elution to obtain rhodiola rosea active components;
the elution process experimental parameters were set as follows:
mobile phase: water A and methanol B with the flow rate of 8 ml/min; the elution gradient was: 25% of B in 0 min; 30% B for 10 min; 80min, 100% B; the sample volume was 1ml, collection was started at 0min and collected in one tube at 10min to give 8 fractions numbered HJT 1-8.
Preferably, the soaking time in the step (1) is 1-3 h; the concentration of the ethanol solution is 60-80%.
Preferably, the reflux extraction in step (1) is performed 3 times, 1 st 8 times for 2 hours, 2 nd 8 times for 1.5 hours, and 3 rd 6 times for 1 hour.
Preferably, the sieving process in step (1) is performed by using a 50-300 mesh stainless steel sieve.
Preferably, the temperature during the vacuum concentration in the step (2) is set to be 70-80 ℃, the relative density is set to be 1.10-1.15, and the vacuum degree is set to be-0.06-0.08 Mpa.
Preferably, the sieving process in step (2) is performed by using a 50-300 mesh stainless steel sieve.
Preferably, the concentration of the methanol solution in the step (3) is 60-80%; the concentration of the extracting solution is 100-150 mg/ml.
Preferably, the rotation speed of the centrifugal treatment in the step (3) is 10000-12000r/min, and the treatment time is 10-20 min.
The invention also provides a rhodiola rosea active component HJT6 prepared by the method.
The invention also provides application of the prepared rhodiola rosea active component HJT6 as an SGK1 protein inhibitor.
The invention also provides application of the rhodiola rosea active component HJT6 in preparing a medicament for preventing and treating myocardial hypertrophy.
Compared with the prior art, the invention has the following beneficial effects:
the invention discloses the application of rhodiola rosea active component HJT6 as an SGK1 protein inhibitor for the first time, the symptom of myocardial hypertrophy of mice can be caused by overhigh activity of SGK1 protein, and experiments prove that the rhodiola rosea active component HJT6 has a remarkable inhibiting effect on SGK1, so that the rhodiola rosea active component HJT6 has good preventing and treating effects on myocardial hypertrophy diseases caused by overhigh activity of SGK1, and experimental evidence is provided for the clinical application of the rhodiola rosea active component HJT6 in preventing and treating myocardial hypertrophy.
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FIG. 1 and FIG. 2 are graphs showing the results of the rhodiola rosea active ingredient HJT6 resisting myocardial cell hypertrophy; in fig. 2, # # p <0.01vs Con group, # p <0.01vs Aldosterone group;
FIG. 3 is a graph showing the results of HJT6 resistance to cardiomyocyte hypertrophy in different comparative examples; wherein, # # p <0.01vs Con group, # p <0.05, and # p <0.01vs Aldosterone group.
Detailed Description
The present invention will be further explained with reference to specific examples in order to make the technical means, the technical features, the technical objectives and the effects of the present invention easier to understand, but the following examples are only preferred embodiments of the present invention, and not all embodiments of the present invention. Based on the embodiments in the implementation, other embodiments obtained by those skilled in the art without any creative efforts belong to the protection scope of the present invention.
The rhodiola rosea raw material is purchased from pharmaceutical industry Co., Ltd of Zhejiang university; aldosterone is purchased from Shanghai leaf biology, Inc., and the purity of the compound is more than or equal to 95%. Other raw materials are all common commercial products.
Example 1: preparation of rhodiola rosea active component HJT6
(1) The rhodiola rosea extract process flow comprises the following steps: slicing radix Rhodiolae, soaking for 1h, reflux-extracting with 70% ethanol for 3 times, extracting 1 time with 8 times of ethanol for 2h, 2 time with 8 times of ethanol for 1.5h, and 3 time with 6 times of ethanol for 1h, sieving the extractive solution with 200 mesh stainless steel screen, and mixing the filtrates for 3 times;
(2) vacuum concentrating to relative density of 1.10-1.15(70-80 deg.C), vacuum degree of-0.06-0.08 Mpa; spray drying, and sieving with 100 mesh sieve to obtain radix Rhodiolae extract.
(3) Preparation of active component HJT 6: weighing 1g of rhodiola rosea extract, adding 10ml of 75% methanol for dissolving to prepare 100mg/ml extract, centrifuging for 10min at the rotating speed of 12000r/min, and taking supernatant for subsequent preparation;
the preparation conditions were as follows: ZORBAX SB-C18PrepHT (21.2X 250mm,7-Micron, Agilent); mobile phase: water (A) and methanol (B) at a flow rate of 8ml/min and at a column temperature of room temperature; the elution gradient was: 0min, 25% B; 10min, 30% B; 80min, 100% B; sampling volume is 1ml, collection is started at 0min, and collection is finished in one tube at 10min to obtain 8 components which are respectively numbered as HJT1-HJT 8; and (4) carrying out rotary evaporation and concentration on the sample, freezing the sample in a refrigerator at the temperature of-80 ℃, and then carrying out freeze drying for 24h for later use.
The rhodiola rosea active component provided by the invention is numbered as HJT 6.
Comparative example 1
The only difference from example 1 is that: replacing step (1) with the following process:
slicing radix Rhodiolae, soaking for 1h, reflux-extracting with 70% ethanol for 3 times, extracting 1 time 6 times for 2h, 2 time 6 times for 1.5h, and 3 time 8 times for 1h, sieving the extractive solution with 200 mesh stainless steel screen, and mixing the filtrates for 3 times.
Comparative example 2
The only difference from example 1 is that: replacing step (1) with the following process:
slicing radix Rhodiolae, soaking for 1h, reflux-extracting with 70% ethanol for 3 times, extracting 1 time 8 times for 1h, 2 times 8 times for 1.5h, and 3 times 5 times for 1h, sieving the extractive solution with 200 mesh stainless steel screen, and mixing the filtrates for 3 times.
Comparative example 3
The only difference from example 1 is that: replacing step (1) with the following process:
slicing radix Rhodiolae, soaking for 1h, reflux-extracting with 70% ethanol for 2 times, extracting 1 time with 8 times of ethanol for 2h, extracting 2 times with 8 times of ethanol for 1.5h, sieving the extractive solution with 200 mesh stainless steel screen, and mixing the filtrates for 2 times.
Comparative example 4
The only difference from example 1 is that: replacing step (1) with the following process:
slicing radix Rhodiolae, soaking for 1h, reflux-extracting with 70% ethanol for 3 times, 1 st extracting 7 times for 2h, 2 nd extracting 8 times for 1.5h, and 3 rd extracting 5 times for 1h, sieving the extractive solution with 200 mesh stainless steel screen, and mixing the filtrates for 3 times.
Comparative example 5
The only difference from example 1 is that: replacing the step (3) with the following process:
weighing 1g of rhodiola rosea extract, adding 10ml of 75% methanol for dissolving to prepare 100mg/ml extract, centrifuging for 10min at the rotating speed of 12000r/min, and taking supernatant for subsequent preparation;
the preparation conditions were as follows: ZORBAX SB-C18PrepHT (21.2X 250mm,7-Micron, Agilent); mobile phase: water (A) and methanol (B) at a flow rate of 8ml/min and at a column temperature of room temperature; the elution gradient was: 0min, 25% B; 30min, 30% B; 80min, 80% B; sampling volume is 1ml, collection is started at 0min, and collection is finished in one tube at 10min to obtain 8 components which are respectively numbered as HJT1-HJT 8; and (4) carrying out rotary evaporation and concentration on the sample, freezing the sample in a refrigerator at the temperature of-80 ℃, and then carrying out freeze drying for 24h for later use.
Comparative example 6
The only difference from example 1 is that: replacing the step (3) with the following process:
weighing 1g of rhodiola rosea extract, adding 10ml of 75% methanol for dissolving to prepare 100mg/ml extract, centrifuging for 10min at the rotating speed of 12000r/min, and taking supernatant for subsequent preparation;
the preparation conditions were as follows: ZORBAX SB-C18PrepHT (21.2X 250mm,7-Micron, Agilent); flow ofMoving phase: water (A) and methanol (B) at a flow rate of 8ml/min and at a column temperature of room temperature; the elution gradient was: 0min, 20% B; 10min, 40% B; 80min, 100% B; sampling volume is 1ml, collection is started at 0min, and collection is finished in one tube at 10min to obtain 8 components which are respectively numbered as HJT1-HJT 8; and (4) carrying out rotary evaporation and concentration on the sample, freezing the sample in a refrigerator at the temperature of-80 ℃, and then carrying out freeze drying for 24h for later use.
Comparative example 7
The only difference from example 1 is that: replacing the step (3) with the following process:
weighing 1g of rhodiola rosea extract, adding 10ml of 75% methanol for dissolving to prepare 100mg/ml extract, centrifuging for 10min at the rotating speed of 12000r/min, and taking supernatant for subsequent preparation;
the preparation conditions were as follows: ZORBAX SB-C18PrepHT (21.2X 250mm,7-Micron, Agilent); mobile phase: water (A) and methanol (B) at a flow rate of 12ml/min and at a column temperature of room temperature; the elution gradient was: 0min, 25% B; 10min, 30% B; 80min, 100% B; sampling volume is 1ml, collection is started at 0min, and collection is finished in one tube at 10min to obtain 8 components which are respectively numbered as HJT1-HJT 8; and (4) carrying out rotary evaporation and concentration on the sample, freezing the sample in a refrigerator at the temperature of-80 ℃, and then carrying out freeze drying for 24h for later use.
A total of 8 HJT6 sets prepared separately in example 1 and comparative examples 1-7 above were tested as follows:
1. the influence of the HJT6 concentration on a primary suckling mouse myocardial cell hypertrophy model is explored
The aldosterone is utilized to promote the activity of SGK1 and induce a mouse myocardial cell hypertrophy model. Separating primary suckling mouse cardiac muscle cells, inoculating the primary suckling mouse cardiac muscle cells into a 96-hole black transparent plate/6-hole plate, adhering to the wall for 24 hours, then replacing the primary suckling mouse cardiac muscle cells with serum-free basic culture solution, and starving for 24 hours for later use. The culture medium was discarded, the control group (Con) was supplemented with fresh culture medium, the model group (Aldosterone) was supplemented with 50. mu.M Aldosterone, and the administration group was supplemented with varying concentrations of rhodiola rosea fraction HJT6 (12.5. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL) prepared in example 1 and 50. mu.M Aldosterone for a combined intervention of 48h, to investigate the anti-myocardial hypertrophy effect of HJT 6.
As shown in fig. 1 and fig. 2, HJT6 can significantly reduce cardiomyocyte hypertrophy induced by SGK1 hyperactivity. And when the concentration of HJT6 is 50 mug/mL, compared with the concentrations of HJT6 of 12.5 mug/mL and 25 mug/mL, the effect of reducing myocardial cell hypertrophy induced by high activity of SGK1 is more obvious.
2. Researches the influence of HJT6 prepared by different preparation parameters on primary suckling mouse myocardial cell hypertrophy model
The aldosterone is utilized to promote the activity of SGK1 and induce a mouse myocardial cell hypertrophy model. Separating primary suckling mouse cardiac muscle cells, inoculating the primary suckling mouse cardiac muscle cells into a 96-hole black transparent plate/6-hole plate, adhering to the wall for 24 hours, then replacing the primary suckling mouse cardiac muscle cells with serum-free basic culture solution, and starving for 24 hours for later use. The culture solution was discarded, a new culture solution was added to the control group (Con), 50. mu.M Aldosterone was added to the model group (Aldosterone), and co-intervention of 50. mu.M Aldosterone with HJT6 (concentration 50. mu.g/mL) of rhodiola rosea fraction prepared in example 1 and comparative examples 1 to 7 was added to the administration group for 48 hours, and the anti-myocardial hypertrophy activity of HJT6 was investigated (10 groups in total).
As shown in fig. 3, the rhodiola rosea component HJT6 prepared in example 1 can significantly reduce cardiomyocyte hypertrophy induced by too high SGK1 activity, while the rhodiola rosea component HJT6 prepared in comparative examples 1 to 7 is not ideal for reducing cardiomyocyte hypertrophy induced by too high SGK1 activity, and is attenuated to a different extent than in example 1; among them, the comparative examples 5 to 7 resulted in the collected HJT6 fraction containing only a small amount of or even no rhodiola rosea active ingredient due to changes in methanol concentration, treatment time and flow rate during elution set during elution, and thus resulted in a great reduction in the effect on anti-myocardial mast cells.
3. Fraction resolution was performed on HJT6 prepared in example 1 group
Chromatographic conditions are as follows: the column was Waters ACQUITY UPLC HSS T3(50 mm. times.2.1 mm i.d.,1.8 μm); taking 0.1% formic acid solution as mobile phase A and 0.1% formic acid acetonitrile as mobile phase B, and performing linear gradient elution for 0-2min, 3-5%; 2-6min, 5-12%; 6-16min, 12-22% B; 16-30min, 22-30% B; 30-32min,30-80min, flow rate of 0.4 ml/min; the column temperature was 25 ℃; the detection wavelength is 275 nm; the amount of sample was 1. mu.l.
The retention time of the rhodiola rosea active component HJT6 corresponding to each component is shown in Table 1:
TABLE 1
Finally, it should be noted that the above-mentioned contents are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, and that the simple modifications or equivalent substitutions of the technical solutions of the present invention by those of ordinary skill in the art can be made without departing from the spirit and scope of the technical solutions of the present invention.
Claims (9)
1. The preparation method of the rhodiola active component is characterized by comprising the following steps:
(1) slicing radix Rhodiolae, soaking, extracting with ethanol under reflux, sieving the extractive solution, and mixing filtrates;
(2) concentrating the filtrate, drying, and sieving to obtain radix Rhodiolae extract;
(3) weighing rhodiola rosea extract, adding methanol solution for dissolving, preparing extracting solution, centrifuging the extracting solution, and taking supernatant for elution to obtain rhodiola rosea active components;
the elution process experimental parameters were set as follows:
mobile phase: water A and methanol B with the flow rate of 8 mL/min; the elution gradient was: 25% of B in 0 min; 30% B for 10 min; 80min, 100% B; the sample introduction volume is 1mL, collection is started at 0min, and collection is completed in one tube at 10min, so that 8 components are obtained, and the number of the components is HJT 1-8.
2. The method according to claim 1, wherein the soaking time in the step (1) is 1 to 3 hours; the concentration of the ethanol solution is 60-80%.
3. The process according to claim 1, wherein the reflux extraction in the step (1) is carried out 3 times, 1 time for 2 hours in an amount of 8 times, 2 times for 1.5 hours in an amount of 8 times, and 3 times for 1 hour in an amount of 6 times.
4. The method according to claim 1, wherein a 50-300 mesh stainless steel screen is used for the sieving in step (1).
5. The method according to claim 1, wherein the temperature during the vacuum concentration in the step (2) is set to 70 to 80 ℃, the relative density is set to 1.10 to 1.15, and the degree of vacuum is set to-0.06 to-0.08 Mpa.
6. The method according to claim 1, wherein the concentration of the methanol solution in the step (3) is 60 to 80%; the concentration of the extracting solution is 100-150 mg/mL.
7. The rhodiola rosea active component HJT6 prepared by the method of any one of claims 1 to 6.
8. The use of the rhodiola rosea active ingredient HJT6 as an SGK1 protein inhibitor according to claim 7.
9. The use of the rhodiola rosea active ingredient HJT6 in the manufacture of a medicament for the prevention and treatment of myocardial hypertrophy as claimed in claim 7.
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