TWI694833B - Method for preparing flavonoids from camellia seed which have hypoglycemic, hypolipidemic and antihypertensive effect - Google Patents

Abstract

The present invention related to a method for preparing flavonoids from Camelliaseed which have hypoglycemic, hypolipidemic and antihypertensive effect. The process is to extract the seed by reflux device first, and the extraction liquid will be freeze-dried. Then the obtained powder will be purified by semi-preparative HPLC and the eluent corresponding to the specific retention time will be collected. The collected eluent will be freeze-dried to obtain the non-catechin flavonoids powder.

Description

Method for preparing flavonoids with effects of lowering blood sugar, blood fat and blood pressure from tea seeds

The invention relates to a method for preparing flavonoids with the effects of lowering blood sugar, blood fat and blood pressure from tea seeds, and is a process technician who prepares specific ingredients from raw materials.

The current main use of tea seeds such as tea tree is to squeeze oil. The squeezed tea seed oil can be processed into food or detergent.

The tea seeds contain many ingredients, among which catechins and tea saponins have been extensively studied, and it has been confirmed that they have many health effects such as lowering blood fat, lowering blood sugar, and preventing cancer.

However, the seeds of the genus Tea are also rich in flavonoids other than catechins. These non-catechin-like flavonoids have not been studied too much, and their biological effects still need to be further understood, and there is currently no set of Reliable technology can be extracted from the seed components.

In view of the above, the inventor believes that there is a need for research, so with many years of experience in related technology and product design and manufacturing, adhering to the excellent design concept, research and creation of tea seeds, after continuous efforts, it was finally launched The invention prepares the flavonoid method with the effect of lowering blood sugar, blood fat and blood pressure from the seeds of the tea genus, so as to enhance the excellent effect of the seeds of the tea genus.

The main purpose of the method for preparing flavonoids with the effects of lowering blood sugar, blood fat and blood pressure from the seeds of the tea genus is to provide a process technician who can prepare flavonoids such as non-catechins from the seeds of the tea genus.

In order to achieve the above-mentioned purpose, the method for preparing flavonoids with the effects of lowering blood sugar, blood fat and blood pressure from tea seeds includes the following steps:

In the extraction process, the seeds of the tea genus are extracted using a reflux device, where the solvent used for extraction is an ethanol solution, and the extraction liquid obtained after the extraction is dried again to obtain an extraction powder;

Separation treatment, the extracted powder is separated by semi-preparative high performance liquid chromatography, and the eluent is acetonitrile and pure water or ethanol and pure water. Gradient extraction is used to make acetonitrile or ethanol within 30 minutes. From 0% to 100%, the extraction rate is 3.5mL/min, using a UV detector with a wavelength set to 230nm, take the eluent corresponding to the residence time of 2~8.5 minutes, and the eluent taken first Nitrogen is blown off acetonitrile or ethanol, and then dried to obtain non-catechin flavonoid (non-catechin flavonoids, NCF) powder.

Among them, Camellia sinensis or Camellia oleifera seeds can be used for the seeds of the tea genus.

Among them, the concentration of ethanol solution (ethanol/water) used in the extraction treatment step is below 90%, and the extraction solvent can also be changed to isopropanol, acetone or hexane.

Among them, the weight (g) of the tea seeds used in the extraction treatment step: the volume (ml) of the ethanol solution is 1:1 to 1:100.

After the NCF powder produced was subjected to thin film chromatography and high performance liquid chromatography tests, it was confirmed that it did not contain saponins and catechins.

After the animal test, the produced NCF powder was confirmed to have the effects of lowering blood sugar, blood fat and blood pressure.

Thus, the present invention provides a method for effectively extracting non-catechins-like flavonoids from tea tree seeds, and the non-catechins-like flavonoids have the biological effects of lowering blood sugar, blood fat and blood pressure, It is expected to be further applied to medicines or health foods, so the progress of the present invention can be seen.

The invention relates to a method for preparing flavonoids with the effects of lowering blood sugar, blood fat and blood pressure from the seeds of the tea genus, which comprises the following steps:

In the extraction process, the dried tea tree ( Camellia sinensis ) seeds are extracted using a reflux device, wherein the extraction solvent is 70% ethanol solution, and the weight of the tea tree seeds used (g): ethanol solution volume (ml) is 1:10, and the flask containing the ethanol solution was heated at 90°C in water, and the extraction time was 6 hours. After the extraction was completed, the extract was centrifuged and the supernatant was taken, and the supernatant was then lyophilized. Perform freeze-drying to obtain extract powder;

Separation treatment, the semi-preparative high performance liquid chromatography (semi-preparative HPLC) was used to separate the required components, and the chromatography tube used was carbon eighteen chromatography tube (model Lichrospher ^R 100 RP-18e) , The particle size of the stationary phase is 10μm), the extraction solution is acetonitrile and distilled pure water or ethanol and distilled pure water, and the gradient extraction method is used to make the acetonitrile or ethanol from 0% to 100% within 30 minutes. The lift rate is 3.5mL/min, and the detection part uses a UV detector and the wavelength is set to 230nm, and the component to be separated is the retention time of 2~8.5 minutes, so the collection corresponding to this period of retention time is collected. Eluent, and the extracted eluent is first blown off acetonitrile or ethanol with nitrogen, and then freeze-dried to obtain non-catechin-like flavonoid powder, that is, NCF (non-catechin flavonoids) powder.

The NCF powder obtained was analyzed by thin film chromatography (TLC), high performance liquid chromatography (HPLC) and liquid chromatography tandem quadrupole time-of-flight mass spectrometer (LC-Q-TOF-MS) to analyze the contained components.

1. Thin film chromatography (TLC):

The mixture of chloroform, methanol and distilled water is used as the developing agent. The volume ratio of chloroform: methanol: distilled water is 65:35:10. The chromatography piece is silica dioxide (SiO ₂ ) as the static phase adsorbent. The sample is taken NCF powder and saponin standard, both of which are dissolved in methanol and then spotted on the chromatographic slices. After expansion, they are sprayed with 10% ethanol sulfate for visualization. [Please refer to the first figure] and the chromatographic results are as follows As shown in the first figure, the Rf value of the NCF powder is 0.34, and the Rf value of the saponin standard is 0.56, which proves that the NCF powder prepared by the present invention does not contain saponin.

2. High performance liquid chromatography (HPLC):

The NCF powder was chromatographed with catechins and the chromatograms were compared. The catechins used included Gallocatechin (GC), Epigallocatechin (EGC), Catechin (C), and Epicatechin ( EC), Epigallocatechin gallate (EGCG), Gallocatechin gallate (GCG), Epicatechingallate (ECG) and Catechin gallate (CG), both of which were dissolved in 0.1% phosphoric acid and passed through a 0.45μm filter before chromatography. The analysis tube also uses a carbon eighteen chromatography tube, and the eluent uses two solutions, namely (A) 0.1% formic acid and 99.9% distilled water, and (B) 0.1% formic acid and 99.9% acetonitrile. (A) The solution is reduced from 100% to 0% within 30 minutes, and the extraction rate is 1.0 mL/min. [Please refer to the second figure] The chromatograms of the catechins and NCF powder are as the second As shown in (a) and (b) of the figure, it can be seen that the NCF powder does not have catechins.

3. Liquid chromatography tandem quadrupole time-of-flight mass spectrometer (LC-Q-TOF-MS):

The NCF powder is first separated by ultra high performance liquid chromatography (UPLC). The chromatography tube used is ACQUITY UPLC HSS T3 column. The eluent uses two solutions, namely (A) 5mM ammonium formate and (B) acetonitrile ，Gradient extraction, 95% (A) solution at 0min, 40% (A) solution at 5min, 20% (A) solution at 8min, 2% (A) solution at 18min, injection The amount of NCF powder is 5μl, and the temperature of the chromatographic tube is kept at 30°C. In the quadrupole time-of-flight mass spectrometer, the positive ion is generated by the electrospray free method, the capillary voltage is 1.5KV, and the cone voltage is 20V. The flow rate and temperature of the solvent gas flow rate are 1000 l/h and 500°C, respectively, and the collision energy is 6 eV. [Please refer to the third figure] The resulting mass spectrum is shown in the third figure, which shows that the NCF powder contains eight main components and Related related substances are Quercetin acetate, Naringenin-6-C-glucoside, Hesperetin, Luteoforol, Eriodictyol, Naringenin, Naringin and Proanthocyanidin A5'.

In addition to confirming its ingredients, NCF powder also undergoes a glucose uptake test and an MTT test to understand its efficacy in improving glucose uptake and whether it is toxic to cells.

1. Glucose uptake test:

First, HepG2 cells were cultured in a cell culture plate at a density of 1x10 ⁴ per well. After 48 hours of cultivation to 80% or full, HepG2 cells were cultured with 5 μM insulin (Insulin) in the medium, except for Insulin (Insulin) co-cultivated, there are also added 30ng/ml TNF-α together with the cultivation, and added 30ng/ml TNF-α together with NCF powder cultured group, of which NCF powder is also divided into 500 , 1000, and 2000 ppm were added, and after incubation to 2, 4, and 6 hours, the culture liquid of each group was sucked out and centrifuged at 500g for 5 minutes. After that, the supernatant was taken and glucose reagent was added, and the solution was incubated at 37℃ for 10 minutes. After a minute, the absorbance was measured at a wavelength of 500 nm. The test results are shown in Table 1 below. The results show that TNF-α will make HepG2 cells resistant to insulin and reduce the ability of HepG2 cells to take up glucose, although the NCF powder failed to achieve complete recovery Effect, but can still reduce this insulin resistance.

Table I: Glucose levels(nmole) in media 2h 4h 6h Control 48.53±0.76 38.37±1.19 37.57±1.99 Insulin(5μM) 44.69±0.79 ⁺ 33.83±1.32 ⁺ 32.80±0.10 ⁺ Insulin+TNF-α(30ng/ml) 47.52±1.53 ^# 38.42±0.72 ^# 37.25±0.70 ^# Insulin+TNF-α+NCF (2000ppm) 41.28±2.08 ^* 31.31±2.16 ^* 29.76±2.15 ^* Insulin+TNF-α+NCF(1000ppm) 40.72±0.66 ^* 32.41±0.46 ^* 31.87±0.26 ^* Insulin+TNF-α+NCF (500ppm) 41.60±1.36 ^* 36.36±1.32 35.50±0.43 ⁺ P>0.05 vs control group; ^# P>0.05 vs insulin group; ^* P>0.05 vs insulin+ TNF-α group

2. MTT test:

Add NCF powder to HepG2 cells at different concentrations (up to 2000 ppm) together with HepG2 cells (5× ^{10 4} per well). After 3 days of culture and washing, HepG2 cells will be combined with 0.5 mg/ml MTT and 100 μl DMSO After culturing for 2 hours, the absorbance was measured at a wavelength of 595 nm. [Please refer to the fourth figure] The test results are shown in the fourth figure, and no cytotoxicity was found.

The NCF powder produced was also tested in animals to understand its effect on improving hyperglycemia, hyperlipidemia and hypertension. The test animals were 8-week-old male congenitally hypertensive rats (SHR). There are 4 groups of RD group, DM group, NCF 0.5 group and NCF 5 group, 8 in each group. After 1 week of SHR adaptation, DM group, NCF 0.5 group and NCF 5 group then induce type 2 diabetes , Induced by injecting Nicotamine (NA) and Streptozotocin (STZ) to destroy the spleen cells of SHR, reducing the ability to secrete insulin. Both NA and STZ injections were 50 mg/kg, and then RD group and DM group were fed normal feed Feeding with high fat feed, NCF 0.5 group and NCF 5 group added NCF powder in high fat feed at 0.5mg/kg/day and 5mg/kg/day respectively, all 4 groups were kept for 4 weeks Then measure the blood glucose, blood lipid and blood pressure and related indexes. The blood glucose is measured with the glucose oral tolerance test (OGTT). The measurement results of blood glucose, blood lipid and blood pressure are shown in Table 2, Table 3 and Table 4, respectively. It can be seen that NCF powder does have the effect of lowering blood sugar, blood fat and blood pressure.

Table 2: Measurement results of blood glucose and related indexes Measurement RD DM NCF 0.5 NCF 5 (n=8) (n=8) (n=8) (n=8) OGTT(mg/dl) 0(min) 106.8±8.8 407.2±30.3 ^# 288.0±90.6 ^* 119.8±56.3 ^{* ◎} 30(min) 107.2±20.6 547.0±36.1 ^# 512.3±59.9 357.7±117.1 ^{* ◎} 60(min) 105.4±11.4 500.7±19.6 ^# 487.8±52.8 311.7±144.3 ^{* ◎} 90(min) 122.4±6.6 452.3±45.9 ^# 380.0±53.8 ^* 296.3±129.6 ^{* ◎} 120(min) 94.8±14.3 424.7±30.3 ^# 370.8±32.7 ^* 270.5±85.4 ^{* ◎} IAUC (mg*min/dl) 13074.0±1505.1 57475.0±1411.9 ^# 51281.3±3545.2 ^* 34825.0±1295.2 ^{* ◎} FBG(mg/dl) 107.4±10.5 500.3±38.9 ^# 305.2±62.3 ^* 161.3±62.6 ^{* ◎} HbA1c(%) 4.40±0.10 9.27±0.25 ^# 9.32±0.48 ^* 6.93±1.72 ^{* ◎} Insulin(ng/ml) 3.29±0.60 1.27±0.35 ^# 2.08±0.13 ^* 1.89±0.39 ^* HOMA-IR 0.87±0.08 1.47±0.17 ^# 1.43±0.19 0.75±0.29 ^{* ◎} GLP-1(nmole) 229.3±50.2 82.1±12.6 198.9±27.8 124.4±22.9 IAUC: area under the blood glucose curve FBG: fasting blood glucose HbA1c: glycated hemoglobin HOMA-IR: insulin resistance GLP-1 (nmole): glucagon-like peptide-1 ^# p>0.05 vs RD; ^* p>0.05 vs DM; ^◎ p>0.05 vs NCF 0.5

Table 3: Blood lipid measurement results Measurement RD DM NCF 0.5 NCF 5 (n=8) (n=8) (n=8) (n=8) TG(mg/dl) 68.6±23.64 564.0±78.7 ^a 181.2±46.5 ^ab 54.2±27.0 ^bc TC(mg/dl) 68.6±18.3 208.2±28.4 ^a 82.2±13.2 ^b 56.7±10.6 ^bc LDL-C(mg/dl) 6.8±1.1 52.4±8.3 ^a 15.1±6.4 ^ab 4.6±1.7 ^bc HDL-C(mg/dl) 21.0±5.7 16.5±2.2 12.7±3.5 16.5±2.2 Non-HDL(mg/dl) 47.6±12.6 191.7±28.0 ^a 69.5±16.5 ^b 40.2±9.02 ^bc AI 2.3±0.1 11.8±2.2 ^a 6.5±3.7 ^b 2.4±0.4 ^bc TG: triglyceride TC: total cholesterol LDL-C: low-density lipoprotein cholesterol HDL-C: high-density lipoprotein cholesterol Non-HDL: non-high-density lipoprotein cholesterol AI: arteriosclerosis index (TC-HDL)/ HDL﹞ ^a p>0.05 vs RD; ^b p>0.05 vs DM; ^c p>0.05 vs NCF 0.5

Table 4: Blood pressure and heartbeat measurement results Measurement RD DM NCF 0.5 NCF 5 (n=8) (n=8) (n=8) (n=8) HR(times/sec) 432.6±31.6 404.2±11.7 340.6±30.0 ^* 346.7±17.1 ^* SBP(mmHg) 191.4±12.8 183.4±7.0 170.0±7.8 ^* 152.9±4.6 ^{* ◎} DBP(mmHg) 134.9±9.7 120.9±3.9 ^# 112.5±4.8 ^* 100.6±4.5 ^{* ◎} MBP(mmHg) 163.1±10.3 151.2±4.4 ^# 146.3±6.0 ^* 126.7±2.0 ^{* ◎} HR: heartbeat SBP: systolic blood pressure DBP: diastolic blood pressure MBP: mean blood pressure ^# p>0.05 vs RD; ^* p>0.05 vs DM; ^◎ p>0.05 vs NCF 0.5

The method for preparing flavonoids with the effects of lowering blood sugar, blood fat and blood pressure from the seeds of the tea genus has the advantage that the flavonoids other than catechin can be effectively extracted from the seeds of the genus tea and the flavonoids It has health care effects such as lowering blood sugar and blood fat, and can be further developed for application in food or medicine.

The above is only one of the preferred embodiments of the present invention, and it should not be used to limit the scope of the present invention. That is to say, all equal changes and modifications made according to the scope of the patent application should still fall within the scope of this creative patent.

In summary, when the invention is known to be novel and progressive, and the invention has not been seen in any publication, it must comply with the provisions of Article 22 of the Patent Law.

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The first figure is the result of thin film chromatography of the flavonoid powder produced in the method of preparing flavonoids with the effects of lowering blood sugar, blood fat and blood pressure from the seeds of the genus Tea of the present invention. The second figure is a chromatogram of high-performance liquid chromatography of the flavonoid powder produced in the method of preparing flavonoids with the functions of lowering blood sugar, blood fat and blood pressure from the seeds of the genus Tea of the present invention. The third figure is the mass spectrum of the flavonoid powder produced in the method for preparing flavonoids with the effects of lowering blood sugar, blood fat and blood pressure from the seeds of the genus Camellia according to the present invention for mass spectrometry analysis. The fourth figure is a graph showing the results of the cytotoxicity test (MTT) of the flavonoid powder produced in the method for preparing flavonoids with the effects of lowering blood sugar, blood fat and blood pressure from the seeds of the genus Tea of the present invention.

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Claims (5)

A method for preparing flavonoids with the effects of lowering blood sugar, blood fat and blood pressure from the seeds of the tea genus, including: extraction treatment, the tea seeds are extracted by a reflux device, and the tea genus seeds used are Camellia sinensis or Camellia oleifera Seed, the extraction solvent is ethanol solution, the extraction liquid obtained after extraction is centrifuged and the supernatant is taken, and the supernatant liquid is then freeze-dried to obtain the extraction powder; separation treatment, the extraction powder is used in a semi-preparative high-performance liquid phase Chromatography is used to separate the components. The chromatographic tube used is carbon eighteen chromatographic tube. The extraction solution is acetonitrile and pure water. The gradient extraction method is used to make acetonitrile from 0% to 100% within 30 minutes. The extraction rate 3.5mL/min, using a UV detector with a wavelength set to 230nm, take the eluent corresponding to the retention time of 2~8.5 minutes, and the eluent taken is first blown off acetonitrile with nitrogen and then freeze-dried. Those who have flavonoid powders other than catechins. The method for preparing flavonoids with the effects of lowering blood sugar, blood fat and blood pressure from the seeds of the genus Tea as described in item 1 of the patent application scope, wherein in the extraction treatment step, the concentration of the ethanol solution used is less than 90% . The method for preparing flavonoids with the effects of lowering blood sugar, blood fat, and blood pressure from the seeds of the genus Tea as described in item 1 of the scope of the patent application, wherein, in the extraction treatment step, the weight of the genus of tea seeds used (g) : The volume of ethanol solution (ml) is 1:1~1:100. The method for preparing flavonoids with the effects of lowering blood sugar, blood fat and blood pressure from the seeds of the genus Tea as described in item 1 of the patent application scope, wherein in this extraction treatment step, the ethanol solution is heated at 90°C in water, And the extraction time is 6 hours. The method of preparing flavonoids with the effects of lowering blood sugar, blood fat and blood pressure from the seeds of the genus Tea as described in item 1 of the patent application scope, in which acetonitrile can be changed to ethanol.

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