CN112107628B - Kumquat essential oil and preparation method and application thereof - Google Patents
Kumquat essential oil and preparation method and application thereof Download PDFInfo
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction
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Abstract
The invention discloses cumquat essential oil and a preparation method and application thereof. Kumquat essential oil is a pure natural substance extracted from kumquat fruits. The cumquat essential oil is obtained by dry steaming and extraction, the main active ingredients and the content of the cumquat essential oil are measured by GC-MS, and then a hypertension animal model test is established to prove that the cumquat essential oil and the active ingredients thereof can obviously reduce the vasoconstriction pressure, adjust the vasodilation factors such as angiotensin II and the like, and simultaneously reduce the target organ damage caused by hypertension; cell tests prove that the medicine can regulate the activity of HUVEC cells induced by angiotensin II. Therefore, the cumquat essential oil has good prevention and treatment effects on renal hypertension.
Description
Technical Field
The invention belongs to the field of traditional Chinese medicines, and particularly relates to cumquat essential oil as well as a preparation method and application thereof.
Background
Along with the continuous improvement of living standard of people, the dietary structure is greatly changed, the occurrence rate and the death number of cardiovascular and cerebrovascular diseases in China are continuously increased, the prevalence rate, the awareness rate and the control rate of hypertension of Chinese adults in the population of 35-75 years old are respectively 37.2%, 22.9% and 5.7%, the standard-reaching rate of hypertension is still lower than that of the hypertension in western developed countries, and the study on hypertension medicines is also a problem which needs to be paid close attention to except that the consciousness of patients on hypertension treatment is improved.
Kumquat, also known as luohao (Oujiang river will), long-life kumquat, milky mandarin orange, Gongsun tangerine, kumquat and the like is a fruit of Fortunella margarita (Lour) swing of kumquat of Rutaceae, wherein the kumquat fruit is rectangular or oval, is golden yellow after being mature, and is difficult to peel off from pulp. Kumquat contains various chemical components, and mainly comprises various compounds such as flavone, volatile oil, polysaccharide and the like. Among them, analysis of volatile components of kumquat fruits by Chen Nu et al identified 39 components, of which 24 terpenes, 8 lipids, 1 alcohol and 5 alkanes. Kumquat has various pharmacological actions, and has the functions of antioxidation, immunity regulation, blood fat and blood sugar reduction, bacteriostasis and gastrointestinal absorption.
At present, western medicines are mainly used for treating hypertension, and the types of the western medicines can be divided into alpha receptor blockers, beta receptor blockers, diuretics, calcium antagonists, angiotensin converting enzyme inhibitors, angiotensin II receptor antagonists and the like. However, hypertension treatment is a long-term process, and the side effect of target organ damage is not negligible. Modern researches show that part of the traditional Chinese medicines or extracts thereof have the characteristics and advantages of stable curative effect, less adverse reaction and irreplaceability of western medicines in the aspect of hypertension treatment. Therefore, the method has important significance for searching safe and effective antihypertensive drugs and drugs for target organ damage caused by hypertension, especially heart failure and renal failure from natural drugs. The kumquat has rich medicinal resources, low cost for obtaining the kumquat essential oil and is suitable for mass production.
Disclosure of Invention
The invention aims to provide cumquat essential oil and a preparation method and application thereof. The cumquat essential oil is obtained by taking cumquat fruits as a raw material and performing dry steaming extraction, the main active ingredients and the content in the cumquat essential oil are determined by GC-MS, and then a hypertension animal model test is established to prove that the cumquat essential oil and the active ingredients thereof can obviously reduce the vasoconstriction pressure, regulate the vasodilation factors such as angiotensin II and the like, and simultaneously reduce the target organ damage caused by hypertension; and further, cell tests prove that the medicine can regulate the activity of angiotensin II induced HUVEC cells, so that the medicine has good prevention and treatment effects on renal hypertension.
In order to achieve the purpose, the invention adopts the following technical scheme:
in one aspect, the cumquat essential oil contains active components including D-limonene and beta-myrcene.
Furthermore, the active components comprise, by mass, 70% -90% of D-limonene and 2% -5% of beta-myrcene.
On the other hand, the preparation method of the cumquat essential oil is provided, and comprises the following steps:
1) pretreatment: cleaning fresh kumquats, and crushing;
2) dry steaming and extracting: weighing the cumquats crushed in the step 1, performing dry steaming extraction in an essential oil extraction system device, and collecting an essential oil layer to obtain crude cumquats essential oil;
3) refining: and (3) refrigerating and standing the crude kumquat essential oil prepared in the step (2) in a refrigerator, centrifuging again, and collecting the supernatant to obtain the kumquat essential oil.
Further, the refrigerating temperature of the refrigerator in the step 3 is 2-4 ℃.
Further, in the step 3, the centrifugal rotating speed is 3000r/min, and the centrifugal time is 10 min.
On the other hand, the application of the cumquat essential oil in preparing the pharmaceutical preparation for preventing and treating renal hypertension is provided.
Further, the medicinal preparation is any one of oral liquid, emulsion, soft capsule, paste, dripping pill, pellicle, pellet, and microsphere.
The invention has the beneficial effects that:
the cumquat essential oil prepared by the invention is suitable for preventing and treating hypertension, especially renal hypertension, and has definite curative effect and no toxic or side effect. By establishing a hypertension animal model test, the cumquat essential oil and the active components thereof are proved to be capable of obviously reducing the vasoconstriction pressure, regulating the vasodilation factors such as angiotensin II and the like, and simultaneously reducing the target organ damage caused by hypertension; cell tests prove that the cumquat essential oil and the active components thereof can regulate the activity of angiotensin II induced HUVEC cells, and further prove that the cumquat essential oil has a synergistic treatment effect and an optimal effect when being used as a whole drug compared with the active components when being used alone.
Compared with the prior art, the kumquat serving as a medicine-food homology has the advantages of rich and easily-obtained resources, simple process for preparing the kumquat essential oil, low cost and good development value and medicinal value, and provides a research basis for further and deeply developing the kumquat essential oil.
Drawings
FIG. 1 is a graph of the effect of cumquat essential oil on angiotensin II-induced cell viability;
FIG. 2 is a graph showing the effect of D-limonene on angiotensin II induced cell viability;
FIG. 3 is a graph showing the effect of beta-myrcene on angiotensin II-induced cell viability;
FIG. 4 is a graph of the effect of D-limonene and beta-myrcene on angiotensin II induced cell viability;
FIG. 5 is a graph of the effect of D-limonene and beta-myrcene on angiotensin II induced cell viability;
FIG. 6 shows the effect of cumquat essential oil, D-limonene and beta-myrcene on angiotensin II induced cell viability.
Detailed Description
The present invention is further described in the following examples, which should not be construed as limiting the scope of the invention, but rather as providing the following examples which are set forth to illustrate and not limit the scope of the invention.
Example 1
Preparation of cumquat essential oil
1. Kumquat raw materials: purchased from the Tuwa planting base in Jiangxi province, and identified by Liu Yong teacher of pharmaceutical institute of Jiangxi Chinese medicinal university as the fruit of Fortunella Crassifolia Swingle of Rutaceae.
2. Preparation method 1) pretreatment: cleaning fresh kumquats, and crushing;
2) dry steaming and extracting: weighing 12kg of cumquats crushed in the step 1, performing dry steaming extraction in an essential oil extraction system device for 2h, stopping the device, after the steam is condensed, extracting layered liquid by using a 10ml injector, and collecting an essential oil layer to obtain crude cumquats essential oil;
3) refining: and (3) filling the crude cumquat essential oil prepared in the step (2) into a brown bottle, sealing the bottle cap by using a sealing film, refrigerating and standing at 4 ℃ in a refrigerator for one night, centrifuging at the rotating speed of 3000r/min for 10min, and collecting the supernatant to obtain the cumquat essential oil. The yield of the cumquat essential oil is 0.8 percent.
Example 2
Determination of active component content of cumquat essential oil
1. Procedure of experiment
Gas chromatography conditions: agilent 19091s-433:93.92873 column, 30m × 0.25mm × 0.25 μm. The injection port temperature was 250 ℃, the total flow was 19.6mL/min, the split ratio was 20:1, and the column flow was 1 mL/min. The interface temperature was 280 ℃. The temperature raising program is kept for 2min at 40 ℃, is raised to 250 ℃ at 10 ℃/min, is kept for 2min, is raised to 280 ℃ at 20 ℃/min, and is kept for 1 min. The carrier gas is high purity helium. The amount of sample was 1. mu.L.
Mass spectrum conditions: the electron energy of the EI ionization source is 70eV, the ion source temperature is 230 ℃, and the quadrupole rod temperature is 150 ℃. The measuring method is full scanning, the mass range is 35-550 u, the voltage of the multiplier is 1482V, and the emission current of the filament is 34.6 muA. The solvent delay time was 2.0 min.
Qualitative and quantitative: and (3) searching by using an NIST11.L spectrum library, analyzing mass spectrograms corresponding to all peaks one by one, and comparing and identifying the basic peaks, the mass-to-charge ratios, the relative abundance and the like with the standard spectrogram. And quantitatively calculating the relative percentage content of each chemical component in the volatile oil by using a peak area normalization method.
2. Measurement results
The D-limonene content was 83.07%, and the beta-myrcene content was 3.79%.
Example 3
Animal test for preventing and treating hypotension
A single dose of cumquat essential oil of 0.5ml/kg is taken to investigate the systolic pressure, the serum angiotensin II, the aldosterone and the vascular endothelial relaxation factor of a rat as indexes.
1. Experimental protocol
1.1 pharmaceutical formulation
The cumquat essential oil prepared in example 1 was diluted with a 2% tween 80 aqueous solution.
1.2 animals
SPF grade SD rats, male, body weight (180 + -10 g), 30, purchased from the Experimental animals center of Jiangxi university of traditional Chinese medicine [ SCXK (gan) 2018-.
The rats are bred in a barrier system (SYXK 2017) 0004 of experimental animals, the laboratory temperature is 23 +/-2 ℃, the relative humidity is 53 +/-3 percent, the noise is below 60 decibels, the sunshine lasts for 12 hours, the animals freely drink and eat water, padding is replaced every day, and when the weight of the animals is 180 +/-10 g, all 30 rats are subjected to surgery modeling.
1.3 establishment of hypertension animal model
1) Measuring and training the blood pressure of the rat: the rat tail artery blood pressure monitoring needs to carry out adaptive training of a tail artery manometry system every day by a rat one week in advance before surgical treatment so as to reduce stress caused by constraint of the rat to cause blood pressure fluctuation, and in addition, the blood pressure fluctuates day and night along with time, so that the same rat is enabled to carry out tail artery blood pressure monitoring in the same time period every day as much as possible in the adaptive training and experiment processes.
The specific operation steps of blood pressure measurement are as follows: the rat is placed in a bounding box, the rat tail is fixed, the rat tail is sleeved with a sleeve, and the temperature in the bounding box is kept at about 37 ℃ and the surrounding environment is quiet. After the rat is completely relaxed, tail artery blood pressure measurement is started, at least 10 preliminary measurements are carried out before each measurement, the purpose is to ensure that the animal is sufficiently prepared, the formal measurement times are 10 to 20 in each period of time, and the average value of data obtained after the measurement is finished is taken as the day-old systolic pressure data of the animal.
2) Preoperative preparation: adaptive feeding is given for 1 week before operation, during which period the tail arterial systolic pressure of each rat is measured by BP-2000 non-invasive animal blood pressure determination analysis system, and the measurement is repeated for 10 times, and the average value is taken. Rats were fasted for 12h before surgery without water deprivation.
3) Modeling of hypertension by two kidneys and one clamp: the male SD rat in the model group is subjected to intraperitoneal injection of 3% pentobarbital sodium (50mg/kg) for anesthesia, the animal is in an anesthesia state, a rubber band is arranged at a supine position and fixed on an operating table, a heating lamp is turned on to irradiate the whole operation area, four limbs and teeth are used for fixing the rubber band, abdominal hair of the rat is cut by curved scissors, the abdomen is disinfected by 5% iodophors, 75% alcohol is deiodinated, an abdominal muscle layer is cut by about 3cm along a median abdominal white line by using an operating knife, subcutaneous superficial fascia and muscles at two sides are slightly separated by using a cotton swab to expose viscera, the left kidney of the animal is exposed by using tweezers and a cotton swab, the left renal vein is parallel to the left renal artery (the renal vein is dark red, the renal artery is light pink), and the left renal artery is slowly and obtusedly separated. Then, blunt dissection (about 0.5cm in length) is carried out on the left renal artery at a position close to the abdominal aorta, and a U-shaped silver clip with the thickness of 0.2mm is horizontally sleeved on the renal artery so that the silver clip can completely slide into the left renal artery which is well dissected. After the stenosis was completed, the viscera was gently pushed to the abdominal cavity with a cotton swab and forceps while 0.3ml (20 ten thousand units) of gentamicin was pipetted into the abdominal cavity with a syringe, and the abdominal muscle layer was sutured with a surgical suture needle and a number 3-0 surgical suture. The skin layers were sutured with surgical suture needle 3-0 surgical suture. After the suture was completed, the post-operative animals were placed under a heating lamp until the rats were awake. The sham-operated group only isolated the left renal artery and did not stenose, and the rest of the procedure was the same as the model group. The whole operation process is carried out strictly according to aseptic operation. After the rats were awakened, they were kept under normal feeding conditions. After the operation is finished for 3 days, the rats are injected with 0.3ml (20 ten thousand units) of gentamicin in the abdominal cavity every day to avoid wound infection, skin incision, mental state and feeding and water feeding conditions are closely observed, and the feed amount within 3 days after the operation is finished needs to be strictly controlled, is as little as possible and is not suitable to be too much. During the experiment, all the feed was fed with pellet feed and water diet was freely drunk.
4) And (4) judging the standard: the tail artery systolic pressure is measured periodically every week after the double-kidney one-clip operation, when the blood pressure of the animal is obviously increased and is more than 140mmHg, and the value is stable, so that the model is successfully copied. If the blood pressure of the rat is not obviously increased after the operation, or the dead rat is the molding failure.
1.4 grouping and administration
Measuring blood pressure after rat model making operation until 8 weeks after operation, screening 14 hypertensive rats successfully molded, randomly dividing the rats into a model group and kumquat essential oil group, and carrying out intragastric administration once a day in a dosing mode for 8 weeks.
Model group: an equivalent amount of purified water;
cumquat essential oil group: 0.5ml/kg (corresponding to 10.4g/kg of cumquat crude drug).
1.5 investigating the influence of cumquat essential oil on the systolic pressure of hypertensive rats
After the first dose until the end of the dose, systolic blood pressure was measured periodically weekly in both groups of rats.
1.6 investigation of the effects of Fortunella margarita essential oil on the serum angiotensin II, aldosterone and vasodilating factor of hypertensive rats
After the last dose, both groups of rats were fasted for 12h, allowed free access to water and weighed. Rats were anesthetized with 3% sodium pentobarbital (50mg/kg) by intraperitoneal injection. A5 ml syringe is used, a needle is fixed, and blood is slowly drawn from the abdominal aorta and injected into a common vacuum blood collection tube. After the blood is placed for 2h, the blood is centrifuged at the rotating speed of 3500r/min at the temperature of 4 ℃ for 20min, the serum is sucked into an EP tube, and the angiotensin II (Ang II), Aldosterone (ALD), endothelin 1(ET-1), Nitric Oxide (NO) and Prostacyclin (PGI) are measured by a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) at the temperature of-80 ℃ for storage in a refrigerator2) And the content of Endothelial Derived Hyperpolarizing Factor (EDHF).
2. Results of the experiment
2.1 Effect of cumquat essential oil on systolic blood pressure of hypertensive rat
Following dosing, systolic blood pressure was measured weekly in both groups of rats, and the results are shown in table 1.
TABLE 1 rat systolic blood pressure results (mmHg)
| Time (week) | Model set | Cumquat essential oil |
| First week | 148.1±6.60 | 148.8±26.83 |
| Second week | 148.9±5.76 | 140.9±4.45* |
| The third week | 148.7±18.29 | 118.0±16.52** |
| The fourth side | 148.1±5.24 | 123.2±15.84** |
| The fifth week | 139.7±11.53 | 121.3±14.51* |
| The sixth week | 145.1±11.71 | 109.7±23.99** |
| The seventh week | 136.7±19.89 | 102.7±16.42** |
Represents P < 0.05 compared to model group, and represents P < 0.01 compared to model group
As can be seen from table 1, the first week of administration showed no significant difference in the reduction of systolic blood pressure compared to the model group, while the second week to the seventh week showed significant difference in the reduction of systolic blood pressure compared to the model group. The result shows that the cumquat essential oil has obvious effect of reducing the blood pressure.
2.2 Effect of cumquat essential oil on serum angiotensin II, aldosterone and vasodilating factor of blood vessel endothelium of hypertensive rat
After the last dose, a series of characterizations were made of angiotensin ii and aldosterone, vasodilator factors in the serum of both groups of rats, and the results are shown in table 2.
TABLE 2 determination of the intravascular indices of rats
Represents P < 0.05 compared to model group, and represents P < 0.01 compared to model group
As can be seen from Table 2 above, compared with the model group, the angiotensin II (Ang II), Aldosterone (ALD) and endothelin 1(ET-1) in the cumquat oil group did not have significant difference, but had a tendency of decreasing, serum Nitric Oxide (NO), Prostacyclin (PGI)2) And endothelial hyperpolarizing factor (EDHF), obviously increased. The result shows that the cumquat essential oil has the inhibition effect on serum angiotensin II (Ang II), Aldosterone (ALD) and endothelin 1(ET-1) of hypertension, and can inhibit Nitric Oxide (NO) and Prostacyclin (PGI)2) And endothelial hyperpolarizing factor (EDHF).
Example 4
Animal test for treating hypertension
According to the research result of example 3, the cumquat essential oil 0.5ml/kg has the effect of reducing blood pressure in a rat model with hypertension in two kidneys and one clip, so that the cumquat essential oil concentration is taken as a medium dose to be divided into a high dose group and a low dose group which are increased by 3 times and decreased by 3 times, and the high dose group, the medium dose group and the low dose group are respectively diluted to be 1.5ml/kg, 0.5ml/kg and 0.17ml/kg by 2% Tween 80 solution.
In addition, the hypertensive drug captopril is set as a control drug group.
By setting the administration group, blood pressure, cardiac index and renal index of rats were examined.
1.1 pharmaceutical formulation
Kumquat essential oil solution: the cumquat essential oil prepared in the example 1 is diluted by a 2% Tween 80 solution to obtain high, medium and low doses of the cumquat essential oil solution.
Captopril solution: grinding captopril tablets into fine powder, and dissolving in double distilled water to obtain the captopril tablet.
1.2 animals
SPF grade SD rats, male, with body weight (180 + -10 g) 100, were purchased from the university of Chinese medicine in Jiangxi laboratory animal center (SCXK 2018) 0003).
The feeding conditions were the same as in example 3.
1.3 establishment of hypertension animal model
The same as in example 3.
1.4 grouping and administration
60 blank model groups of hypertension rats successfully molded are screened out, and the groups are randomly divided into 6 groups of 10 rats. The administration mode is intragastric administration once a day, the administration time is 8 weeks, and the specific groups are as the following table 3:
TABLE 3 grouping and dosing concentrations
| Grouping | Number of rats | Concentration of drug administration |
| |
10 pieces of | Equal amount of purified water |
| Model set | 10 pieces of | Equal amount of purified water |
| Kumquat essential oil |
10 pieces of | 0.17ml/kg (corresponding to the crude drug 202.5g of kumquat for adults) |
| Kumquat essential oil medium- |
10 pieces of | 0.5ml/kg (corresponding to the crude drug amount 607.5g of kumquat for adults) |
| Kumquat essential oil high- |
10 pieces of | 1.5ml/kg (corresponding to the crude drug amount 1822.5g of kumquat for adults) |
| |
10 pieces of | 10.28mg/kg (equivalent to one day for adults) |
1.5 investigating the influence of different doses of cumquat essential oil on the systolic blood pressure of hypertensive rats
After the molding was successful, the systolic blood pressure of each group of rats was measured periodically every week during the 8-week period from the first administration to the completion of the administration.
1.6 investigating the influence of different doses of cumquat essential oil on the heart of hypertensive rat
After the last administration, the rats in each group were fasted for 12h, allowed free access to water and weighed. 3% sodium pentobarbital (50mg/kg) is injected into the abdominal cavity to anaesthetize the rat, the abdominal cavity is opened rapidly, the heart is taken out after full perfusion with precooled phosphate buffer saline solution, the filter paper is sucked dry, and the heart mass is weighed by an electronic balance. Calculating the heart quality index, wherein the heart quality index is the ratio of the mass to the body mass.
1.7 investigation of the Effect of different doses of cumquat essential oil on the kidney of hypertensive rat
After the last administration, the rats in each group were fasted for 12h, allowed free access to water and weighed. 3% sodium pentobarbital (50mg/kg) is injected into the abdominal cavity to anaesthetize the rat, the thoracic cavity is opened rapidly, the kidney is taken out after the pre-cooled phosphate buffer saline solution is fully filled, the filter paper is sucked dry, and the kidney mass is weighed by an electronic balance. And calculating the kidney quality index which is the ratio of the mass to the body mass.
2. Results of the experiment
2.1 investigating the influence of different doses of cumquat essential oil on the systolic blood pressure of hypertensive rats
After the molding was successful, the systolic blood pressure of each group of rats was measured within 8 weeks from the first administration to the completion of the administration, and the results are shown in Table 4.
TABLE 4 Change in systolic blood pressure (mmHg) during preventive treatment of rats in each group
# indicates P < 0.05 compared to the blank control group, P < 0.05 compared to the model group, and P < 0.01 compared to the model group.
As can be seen from Table 4 above, after 1 week of administration, the systolic blood pressure of the model group was significantly increased and significantly different from that of the blank control group; the systolic pressure of the captopril group is compared with that of the model group, and the systolic pressure is reduced and has obvious difference; the rest of the administration groups showed a tendency to decrease in systolic pressure compared to the model group, but did not show any significant difference.
After 2 weeks of administration, the systolic pressure of the model group was still the highest and significantly different compared to the blank control group; the systolic pressure of the captopril group is compared with that of the model group, and the systolic pressure is reduced and has obvious difference; the rest of the administration groups showed a tendency to decrease in systolic pressure compared to the model group, but did not show any significant difference.
After 4 weeks of administration, the systolic pressure of the model group was still the highest and significantly different compared to the blank control group; the captopril group and the model group are compared, the systolic pressure is reduced and has extremely obvious difference; compared with the model group, the essential oil low-dose group has a downward trend of contraction pressure, but has no obvious difference; compared with the model group, the dose group in the essential oil has reduced systolic pressure and has obvious difference; compared with the model group, the essential oil high-dose group has reduced systolic pressure and obvious difference, and has similar pressure reduction effect with the captopril group.
After 6 weeks of administration, the systolic pressure of the model group was still the highest and significantly different compared to the blank control group; the captopril group and the model group are compared, the systolic pressure is reduced and has extremely obvious difference; compared with the model group, the essential oil low dose group has reduced systolic pressure and has obvious difference; compared with the model group, the dosage group in the cumquat essential oil has reduced systolic pressure with extremely obvious difference, and the pressure reduction trend is equivalent to that of the Capdolapril group; compared with the model group, the cumquat essential oil high-dose group has reduced systolic pressure with extremely obvious difference, and the blood pressure reduction trend is equivalent to that of the captopril group.
After 8 weeks of administration, the systolic pressure of the model group was still the highest and significantly different compared to the blank control group; the captopril group is compared with the model group, and the systolic pressure are reduced and have extremely obvious difference; compared with the model group, the essential oil low dose group has reduced systolic pressure and has obvious difference; compared with the model group, the dosage group in the cumquat essential oil has reduced systolic pressure with extremely obvious difference, and the pressure reduction trend is equivalent to that of the Capdolapril group; compared with the model group, the cumquat essential oil high-dose group has reduced systolic pressure with extremely obvious difference, and has the same pressure reduction trend as the Capdolapril group;
the result shows that the cumquat essential oil has obvious blood pressure reducing effect on hypertension, particularly the high-dose group in the essential oil has the same blood pressure reducing tendency as that of captopril serving as a blood pressure reducing medicine, and has dosage effect.
2.2 investigating the influence of different doses of cumquat essential oil on the heart of hypertensive rat
After the last dose, rat body weight and heart wet weight were measured and rat heart index was calculated, and the results are shown in table 5.
TABLE 5 rat Heart index results (g)
Represents P < 0.05 compared to model group, and represents P < 0.01 compared to model group
As can be seen from table 5 above, 8 weeks after administration, the cardiac index of the model group was increased, but there was no significant difference, compared with that of the blank control group; compared with the model group, the cardiac index of each administration group is reduced, but no significant difference exists. The results show that the cumquat essential oil has a protective effect on the heart under the condition of hypertension.
2.3 investigating the influence of different doses of cumquat essential oil on the kidney of hypertensive rat
Animals were kidney-harvested after the last dose and two kidney tissues were observed in rats. The kidney on both sides of the blank control group has no obvious change; in the model group, only the left renal artery is narrowed, so that the left kidney is narrowed, and particularly, the left kidney is shrunk and becomes smaller and more severely at the artery side; the other administration groups also showed the occurrence of left renal stenosis, but the degree was significantly improved compared to the model group.
After the last dose, rat body weight and kidney wet weight were measured and rat kidney index was calculated, and the results are shown in table 6.
TABLE 6 rat Kidney index results (g)
Represents P < 0.05 compared to model group, and represents P < 0.01 compared to model group
As can be seen from table 6 above, after 8 weeks of administration, the renal index of the model group tended to decrease, but there was no significant difference, compared with the blank control group; compared with the model group, the kidney index of the cumquat essential oil high-dose group is increased and has obvious difference, the kidney index of the captopril group is reduced but has no obvious difference due to side effect on the kidney, and the kidney index of the other administration groups has an increasing trend but has no obvious difference.
The results show that the cumquat essential oil has a protective effect on the target organ damage kidney caused by hypertension under the condition of hypertension.
Example 5
Animal test for preventing and treating hypertension by drug administration
According to the research result of example 3, the cumquat essential oil 0.5ml/kg has the effect of reducing blood pressure in a rat model with hypertension in two kidneys and one clip, so that the cumquat essential oil concentration is taken as a medium dose to be divided into a high dose group and a low dose group which are increased by 3 times and decreased by 3 times, and the high dose group, the medium dose group and the low dose group are respectively diluted to be 1.5ml/kg, 0.5ml/kg and 0.17ml/kg by 2% Tween 80 solution.
By setting the administration group, the blood pressure of the rat was examined.
1.1 pharmaceutical formulation
Kumquat essential oil solution: the cumquat essential oil prepared in the example 1 is diluted by a 2% Tween 80 solution to obtain high, medium and low doses of the cumquat essential oil solution.
1.2 animals
SPF grade SD rats, male, with body weight (180 + -10 g) 40, were purchased from the university of Chinese medicine in Jiangxi laboratory animal center (SCXK 2018) and 0003).
The feeding conditions were the same as in example 3.
1.3 grouping and administration
The 40 rats were randomly divided into four groups of 10 rats each, and the administration was performed by intragastric administration once a day for 19 weeks, and specifically grouped as shown in table 7 below:
TABLE 7 grouping and dosing concentrations
1.4 establishment of hypertension animal model
The rats of each administration group in 1.3 were molded according to the same method as that of 1.3 in example 4.
1.5 investigating the influence of different doses of cumquat essential oil on the systolic blood pressure of hypertensive rats
The systolic blood pressure of rats in the blank model group and the high, medium and low cumquat essential oil group is measured periodically every week within 19 weeks after the molding is finished.
2. Results of the experiment
The systolic blood pressure of rats was measured by low, medium and high doses of cumquat oil at the start of molding until one week after molding, for a total of 19 weeks, and the results are shown in Table 8.
TABLE 8 Change in systolic blood pressure (mmHg) during prophylactic administration in the rats of each group
As can be seen from table 9 above, there was no significant increase from the first week of surgery to the nineteen weeks of surgery, compared with the blank control group. The result shows that the cumquat essential oil has the effect of preventing hypertension.
Example 6
Cell test of cumquat essential oil for preventing and treating hypertension
Based on the results of the studies in examples 3-5, 1.5ml/kg, 0.5ml/kg and 0.17ml/kg of cumquat essential oil in the high, medium and low dose groups have the effect of lowering blood pressure in a rat model with hypertension in two kidneys and one clamp, and the results of the test in example 2, which show that the content of D-limonene is 83.07% and the content of beta-myrcene is 3.79%, were used as the background to study the pharmacodynamic effect of the essential oil and effective components in the hypertension model caused by the damage of Human Umbilical Vein Endothelial Cells (HUVEC). The essential oil was set to have a concentration gradient of 7 concentration groups of 1:1000, 1:10000, 1:20000, 1:40000, 1:80000, 1:160000, 1:320000, etc., a model group (angiotensin II, AngII, concentration of 10 μmol/L), a positive drug group (captopril, concentration of 10 μmol/L), a control group (cells, drug dissolution medium of 500 μ M concentration, culture solution, MTT, Formazan dissolution solution), and a zero-adjustment group (culture medium, MTT, Formazan dissolution solution).
Through the arrangement, the drug effect of the essential oil on a hypertension model caused by damage of Human Umbilical Vein Endothelial Cells (HUVEC) is investigated.
1.1 pharmaceutical formulation
Kumquat essential oil solution: the cumquat essential oil prepared in example 1 was collected, 20. mu.l of the filtered essential oil was sucked by a pipette gun, and the filtered solution (20. mu.l of DMSO and 1960. mu.l of water) was added to prepare a sample stock solution (i.e., dilution of the stock solution in multiples on a 96-well plate).
Preparation of complete medium: 90% DMEM medium + 10% serum + 1% amino acids + 1% double antibody.
1.2 Experimental procedures
(1) HUVEC cells were digested from culture flasks with 0.25% trypsin;
(2) collecting logarithmic phase cells, adjusting cell suspension concentration to 1 × 104Each 100 μ L, divided into 96-well plates, each well 100 μ L, 1 × 104Individual cells/well;
(3) put 5% CO2Incubating at 37 ℃ in an incubator to allow cells to adhere to the wall, incubating for 14 hours until cell monolayers are paved on the bottom of a hole (a 96-hole flat bottom plate), adding a drug with a concentration gradient, discarding a culture solution of the 96-hole plate before adding the drug, and adding 100 mu l of cumquat essential oil mother solution diluted by each concentration gradient into each hole, wherein the specific operation is to dilute 1.1 of the sample mother solution by using a culture medium to 1:3200, 1:1600, 1:800, 1:400, 1:200, 1:100 and 1:10 concentration gradients); then adding culture solution containing 1 mu mol/L Ang II serum to act, and adding 9 rows of wells.
The preparation method of the Ang II serum culture solution comprises the following steps: 0.00052g of Ang II is weighed and dissolved in 50ml of culture solution to prepare 10 mu M of inducing solution, 700 mu L of inducing solution is added with 6300 mu L of culture solution to prepare 10 mu mol/L of Ang II solution.
(4) Add 100. mu.l of solvent (column 2) dissolving the drug to the drug control well, add 200. mu.l of medium to the zero setting well;
(5) put 5% CO2Incubating the cells in a cell incubator for 24 hours at 37 ℃, and observing the cells under an inverted microscope;
(6) adding 10 μ l MTT solution (5mg/ml, namely 0.5% MTT) into each well, mixing uniformly, and continuing culturing for 5 h;
(7) the culture was terminated and the well medium was carefully aspirated (slowly with a syringe, without working on a clean bench). 150 μ L of DMSO (protected from light) was added to each well and the formazan crystals were fully dissolved by shaking on a shaker at low speed of 200rpm for 10 min. The absorbance of each well was measured at 490nm to 590nm in an enzyme linked immunosorbent assay.
(8) Calculation of cell viability
The cell survival rate was [ (additivated group) - (zero adjusted group) ]/[ (negative control group) - (zero adjusted group) ].
2. Results of the experiment
Compared with the control group, the results are shown in fig. 1, the survival rate of the cells in the model group is remarkably reduced (P is less than 0.01), the cell morphology is remarkably changed, the cell arrangement is irregular, the shrinkage degeneration is realized, the number is remarkably reduced, compared with the model group, the cell activity is remarkably improved (P is less than 0.05) after the treatment of each kumquat essential oil concentration, a certain dosage effect exists, the gradient of the original essential oil after being diluted by 100 times is in the range of 1:10, 1:100, 1:200 and 1:400, the effect is gradually increased, the gradient is in the range of 1:800, 1:1600 and 1:3200, compared with the positive medicine captopril, the effect of each kumquat essential oil concentration group is superior to that of the positive medicine (P is less than 0.05) except for the three groups of concentrations of 1:10, 1:1600 and 1: 3200.
Example 7
Cell test for preventing and treating hypotension by using D-limonene
According to the research result of the example 6, the concentration gradient of 7 kumquat essential oil concentrations, such as 1:1000, 1:10000, 1:20000, 1:40000, 1:80000, 1:160000 and 1:320000, has a significant effect of enhancing the HUVEC cell activity induced by angiotensin II (AngII) (P is less than 0.05), the content of D-limonene in the essential oil is 83%, the relative density of D-limonene is 0.840-0.880 mg/mu l, the concentration gradient of the kumquat essential oil concentration, which has the strongest effect on the HUVEC cell activity, of the essential oil concentration 1:40000 is calculated, and the content of D-limonene is 11 mu g/ml. Therefore, the D-limonene was set at a concentration gradient of 1. mu.g/ml, 10. mu.g/ml, 20. mu.g/ml, 40. mu.g/ml, 80. mu.g/ml, 160. mu.g/ml, 320. mu.g/ml, 640. mu.g/ml, and the like in 8 concentration groups to examine the pharmacodynamic action in the hypertension model caused by the damage to Human Umbilical Vein Endothelial Cells (HUVEC), and the settings and concentrations in the other groups were the same as those in example 6.
1.1 pharmaceutical formulation
D-limonene solution: after filtration, 7.62. mu.l of D-limonene was pipetted by a pipette, the filtered solution (10. mu.l DMSO and 982. mu.l water) was added to prepare a sample stock solution (dilution of the stock solution in multiples on a 96-well plate), and other reagents were prepared as in example 6.
1.2 Experimental procedures
The same as in example 6.
2. Results of the experiment
Compared with the control group, the results are shown in figure 2, the survival rate of the cells in the model group is obviously reduced (P < 0.01), the morphology of the cells is obviously changed in a damaging way, the arrangement of the cells is irregular, the cells are shrunk and denatured, and the number of the cells is obviously reduced, compared with the model group, the activity of the cells is obviously improved (P < 0.05) after the treatment of each concentration of the D-limonene, and a certain dosage effect exists, the D-limonene concentration ranges of 1 mu g/ml, 10 mu g/ml, 20 mu g/ml and 40 mu g/ml are gradually increased, and then the D-limonene concentration ranges are gradually reduced to 640 mu g/ml, compared with the positive medicine captopril, the effects of each concentration group of the D-limonene are better than that of the positive medicine (P < 0.05) except that of the concentration of 1 mu g/ml.
Example 8
Cell test for preventing and treating blood pressure reduction by beta-myrcene
According to the research results of example 6, 7 concentrations of cumquat essential oil, such as concentration gradient of 1:1000, 1:10000, 1:20000, 1:40000, 1:80000, 1:160000, 1:320000, and the like, have significant enhancement effect (P < 0.05) on the activity of HUVEC cells induced by angiotensin II (Ang II), the content of the bound beta-myrcene in the essential oil is 4%, the content of the bound D-limonene in the essential oil is 83%, the concentration of the D-limonene with the strongest effect on the activity of HUVEC cells is calculated to be about 2 mug/ml, therefore, the concentration gradient of the beta-myrcene is set to be 8 concentration groups, such as 0.5 mug/ml, 1 mug/ml, 2 mug/ml, 5 mug/ml, 20 mug/ml, 40 mug/ml, 80 mug/ml, 160 mug/ml, and the like, so as to examine the effect of hypertension model of Human Umbilical Vascular Endothelial Cells (HUVEC) on damage, the remaining set-up and concentrations were the same as in example 6.
1.1 pharmaceutical formulation
Beta-myrcene solution: after filtration, 10. mu.l of beta-myrcene was aspirated by a pipette, and then a solution (50. mu.l of DMSO and 960. mu.l of water) was added to prepare a sample stock solution (i.e., dilution of the stock solution in multiples on a 96-well plate), the stock solution was diluted 50-fold to obtain a maximum concentration of 160. mu.g/ml, and other reagents were prepared as in example 5.
1.2 Experimental procedures
The same as in example 6.
2. Results of the experiment
Compared with the control group, the results are shown in figure 3, the survival rate of the cells in the model group is obviously reduced (P is less than 0.01), the morphology of the cells is obviously changed in a damaging way, the arrangement of the cells is irregular, the cells are shrunk and denatured, and the number of the cells is obviously reduced, compared with the model group, the activity of the cells is obviously improved (P is less than 0.05) after being treated by each concentration of the beta-myrcene, and a certain dosage effect exists, the effect is gradually increased within the range of 0.5 mu g/ml, 1 mu g/ml, 2 mu g/ml, 5 mu g/ml and 20 mu g/ml of the beta-myrcene, and then gradually reduced to the range of 160 mu g/ml, compared with captopril serving as a positive drug, the effect of each concentration group of the beta-myrcene is better than that of the positive drug (P is less than 0.05).
Example 9
Cell test for preventing and treating blood pressure by using mixture of D-limonene and beta-myrcene
According to the research results of examples 7 and 8, the significant enhancement effect (P < 0.05) of each concentration of D-limonene and beta-myrcene on the activity of angiotensin II (Ang II) induced HUVEC cells is obtained, the concentrations of D-limonene and beta-myrcene on the activity of angiotensin II (Ang II) induced HUVEC cells are respectively 40 mug/ml and 20 mug/ml, therefore, the ratio of the concentration of D-limonene (40 mug/ml) to the concentration of beta-myrcene (20 mug/ml) is used for examining the pharmacodynamic effect of the D-limonene and beta-myrcene composition gradient 1:5 mass mixing, 1:3 mass mixing, 1:2 mass mixing, 1:1 mass mixing, 2:1 mass mixing and 5:1 mass mixing on a hypertension model caused by damage of Human Umbilical Vein Endothelial Cells (HUVEC), the remaining sets of concentrations were set as in example 5 and were divided into two experiments, the first with gradient 1:5, 1:3, 1:2, of D-limonene and beta-myrcene compositions, with the mixture concentrations set at 20. mu.g/ml and 40. mu.g/ml for each set of mixing ratios, and the second with gradient 1:1, 2:1, 5:1, of D-limonene and beta-myrcene compositions, with the mixture concentrations set at 20. mu.g/ml and 40. mu.g/ml for each set of mixing ratios.
1.1 pharmaceutical formulation
Composition solution: according to the proportion, 10ul of beta-myrcene after filtration is sucked by a pipette, and then a solution (50ul of DMSO and 960 ul of water) is added to prepare a sample mother solution (namely, the sample mother solution is mixed with D-limonene as a stock solution for initial multiple dilution). The sample stock (i.e., dilution mixed with β -myrcene as a starting multiple of the stock solution) was prepared by pipetting 7.62 μ l of D-limonene after filtration using a pipette and adding the filtered solution (10 μ l DMSO and 982 μ l water).
Other reagents were formulated as in example 6.
1.2 Experimental procedures
The same as in example 6.
2. Results of the experiment
Compared with the control group, the results are shown in fig. 4 and fig. 5, the survival rate of the cells in the model group is obviously reduced (P is less than 0.01), the cell morphology is obviously changed in a damaging way, the arrangement of the cells is irregular, the cells are shrunk and denatured, and the number of the cells is obviously reduced, compared with the model group, the cell viability is obviously improved after the D-limonene and beta-myrcene composition is subjected to gradient 1:5 mass mixing, 1:3 mass mixing and 1:2 mass mixing concentration treatment (P is less than 0.05, shown in fig. 4), the effect is equivalent to that of the positive drug captopril, no difference exists between groups (P is more than 0.05, shown in fig. 4), but the effect is better when the mixing ratio is 40 mu g/ml; compared with a model group, after the D-limonene and beta-myrcene composition gradient 1:1 mass mixing, 2:1 mass mixing and 5:1 mass mixing concentration treatment, the cell viability is obviously improved (P is less than 0.05, see figure 5), compared with a positive drug captopril, except that the 2:1 mass mixing (20 mu g/ml) and the 5:1 mass mixing groups have no difference, other groups are obviously better than the positive drug captopril (P is less than 0.01, see figure 5), the effect is better when the mixing ratio is 40 mu g/ml, and the D-limonene and beta-myrcene composition gradient 1:1 mass mixing, 2:1 mass mixing and 5:1 mass mixing concentration show a decline trend on the pharmacodynamic action of a hypertension model caused by damage of Human Umbilical Vein Endothelial Cells (HUVEC), namely the D-limonene and beta-myrcene composition gradient 1:1 mass mixing effect is optimal, and the effect was the best one in each mixing group at a mixing ratio of 40. mu.g/ml.
Example 10
Cellular test for different ingredients to prevent and treat hypotension
The results of the studies according to examples 6-9 gave the strongest concentrations of cumquat essential oil, D-limonene, beta-myrcene and the composition of D-limonene and beta-myrcene for inducing the activity of HUVEC cells by angiotensin II (Ang II), namely 40. mu.g/ml mixed by mass at 400-fold dilution (1:400), 40. mu.g/ml of D-limonene, 20. mu.g/ml of beta-myrcene and the composition of D-limonene and beta-myrcene, respectively, after 100-fold dilution of the original essential oil. To further clarify the significant effect of the essential oils on the angiotensin ii (ang ii) inducing HUVEC cell viability, experiments were again conducted at the strongest concentrations of the above kumquat essential oil, D-limonene, β -myrcene, and D-limonene and β -myrcene combination on the angiotensin ii (ang ii) inducing HUVEC cell viability.
1.1 pharmaceutical formulation
The same as in examples 6 to 9.
1.2 Experimental procedures
The same as in examples 6 to 9.
2. Results of the experiment
Compared with the control group, the results are shown in FIG. 6, the cell survival rate of the model group is obviously reduced (P < 0.01), and the cell morphology is obviously changed in an injurious way, the cell arrangement is out of regularity, and the number is obviously reduced due to shrinkage denaturation, compared with the model group, the cell activity of each group treated by the cumquat essential oil (1:400), the D-limonene (40), the beta-myrcene (20) and the composition of the D-limonene and the beta-myrcene (1:1-40) is obviously improved (P is less than 0.05), the effect is better than that of the positive drug captopril (P is less than 0.05), the effect of diluting the original essential oil by 100 times is strongest when the original essential oil is diluted by 400 times, which shows that the effect of the cumquat essential oil on the activity of angiotensin II (Ang II) induced HUVEC cells is still better, and the result also supports the previous whole animal experiment result.
Claims (5)
1. The application of kumquat essential oil in preparing a pharmaceutical preparation for preventing and treating renal hypertension is characterized in that the active components of the kumquat essential oil comprise D-limonene and beta-myrcene; the active components comprise, by mass, 70-90% of D-limonene and 2-5% of beta-myrcene.
2. The application of kumquat essential oil in preparing a pharmaceutical preparation for preventing and treating renal hypertension according to claim 1, wherein the preparation method of the kumquat essential oil comprises the following steps:
1) pretreatment: cleaning fresh kumquats, and crushing;
2) dry steaming and extracting: weighing the cumquats crushed in the step 1) and performing dry steaming extraction in an essential oil extraction system device, and collecting an essential oil layer to obtain crude cumquats essential oil;
3) refining: refrigerating and standing the crude kumquat essential oil prepared in the step 2) in a refrigerator, centrifuging again, and collecting the supernatant to obtain the kumquat essential oil.
3. The use according to claim 2, wherein the preparation method of cumquat essential oil in step 3) is characterized in that the refrigerating temperature of the refrigerator is 2-4 ℃.
4. The use according to claim 2, wherein in step 3), the centrifugation speed is 3000r/min and the centrifugation time is 10 min.
5. Use according to claim 1, characterized in that: the medicinal preparation is any one of oral liquid, emulsion, soft capsule, paste, dripping pill, pellicle, pellet, and microsphere.
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