CN111939210A

CN111939210A - Tea seed cake extract and application thereof

Info

Publication number: CN111939210A
Application number: CN201910413177.7A
Authority: CN
Inventors: 刘志平; 吴素珍; 肖小妹; 田洪波; 罗晓婷
Original assignee: Gannan Medical University
Current assignee: Gannan Medical University
Priority date: 2019-05-17
Filing date: 2019-05-17
Publication date: 2020-11-17

Abstract

The invention belongs to the technical field of natural medicine preparation, and relates to a tea seed cake extract and application thereof. The preparation process comprises the following steps: taking the tea seed cake, adding 4-8 times of petroleum ether, and extracting under reflux twice, each time for 1-2 h; filtering to remove extractive solution, extracting the residue with 50-70% ethanol solution for two times, each for 1-3 hr, and mixing the ethanol extractive solutions; removing ethanol, adding acetone into ethanol extractive solution to reach content of 70-90%, filtering to remove precipitate, and drying the filtrate to obtain the final product. Compared with the prior art, the tea seed cake extract optimizes the contents of tea saponin, Camelliaside A and Camelliaside B, has good effect of promoting wound healing of burns and scalds, and can remarkably increase the content of tea saponin, Camelliaside A and Camelliaside BCOLα‑1AndCOL‑3 mRNA expression, useful for the preparation of a medicament.

Description

Tea seed cake extract and application thereof

Technical Field

The invention belongs to the technical field of natural medicine preparation, and relates to a tea seed cake extract and application thereof.

Background

The camellia oleifera is also called tea tree and camellia oleifera tree, is a plant of the camellia genus of the camellia family, is native to China, is a woody oil plant species specific to China, and is mainly distributed in tropical and subtropical regions. At present, the national oil tea resources account for more than 80 percent of the area of the national woody edible oil, and the annual oil tea seeds yield 64.5 million tons. The camellia oleifera abel cake is residue left after fatty oil is removed from camellia seeds, and mainly contains effective components such as residual oil, tea saponin, polysaccharide, flavonoid, protein and the like. About 100 ten thousand tons of oil tea dead cakes are thrown away every year in China, and great resource waste is caused.

The oil tea seed cake contains 10-20% of tea saponin. The biological activity of tea saponin is shown in many aspects, but due to the difference of the structure, the biological activity of tea saponin also has certain difference. Researches show that the tea saponin mainly has hemolysis and fish toxicity effects, antibacterial and anti-mutation effects, effects of inhibiting alcohol absorption, protecting intestines and stomach and killing sperms, has the effects of being biotin-like and resisting A-type and B-type influenza viruses and the like, and is mainly applied to the fields of aquaculture, cosmetics, pesticides and the like. Tea polysaccharide has antithrombotic, anticoagulant and hypoglycemic effects, and may also have effects of repairing glycometabolism disorder. The flavonoids have important biological activity in the aspects of enhancing immunity, preventing cardiovascular diseases, resisting oxidation activity and the like.

Burn, scald and burn are one of the most common accidents in daily life, for example, if the substances such as boiling water, flame, strong acid and alkali commonly used in industry are not used properly, the burn is easily caused. The degree of burn and scald depends on the area and depth of injury, and the larger the area, the deeper the depth, and the more serious the local and systemic effects. Once the bacteria invade the injury site causing repeated infection, it is very dangerous. In low-to-moderate developing countries, the rate of casualties due to burns (scalds) remains high. Although the antibacterial property of tea saponin and the antioxidation property of flavone are beneficial to the recovery of burn (scald), no report is provided on whether the tea saponin can promote the healing of wounds. In addition, the conventional tea seed cake extract has excessive impurities, which affects the drug effect. Moreover, at present, the action mechanism of the tea seed cake extract for treating the burns and scalds is not researched.

Disclosure of Invention

Through process optimization, the invention provides a tea seed cake extract. Pharmacological experiment results show that the tea seed cake extract can achieve the effect of repairing burn (scald) wounds by regulating the expression of skin healing related proteins, and the action mechanisms can also be used for preparing other medicines.

The preparation process of the tea seed cake extract comprises the following steps: taking the tea seed cake, adding 4-8 times of petroleum ether, and extracting under reflux twice, each time for 1-2 h; filtering to remove extractive solution, extracting the residue with 50-70% ethanol solution for two times, each for 1-3 hr, and mixing the ethanol extractive solutions; removing ethanol, adding acetone into ethanol extractive solution to reach content of 70-90%, filtering to remove precipitate, and drying the filtrate to obtain the final product.

The preparation process of the tea seed cake extract comprises the following steps: taking the tea seed cake, adding 6 times of petroleum ether, and performing reflux extraction twice, wherein each time lasts for 1.5 h; filtering to remove extractive solution, extracting the residue with 60% ethanol solution twice, each for 2 hr, and mixing the ethanol extractive solutions; removing ethanol, adding acetone into the ethanol extractive solution until the content is 80%, filtering to remove precipitate, and drying the filtrate to obtain the final product.

The tea seed cake extract has the advantages that the content of tea saponin is 6.0-8.1%, the content of Camelliaside A is 1.2-1.9%, and the content of Camelliaside B is 1.2-2.0%.

Application of the tea seed cake extract of the invention in increasingCOLα-1 Preparation of mRNA expressed drugs.

Application of the tea seed cake extract of the invention in increasingCOL-3 Preparation of mRNA expressed drugs.

The application of the tea seed cake extract is used for preparing a collagen enhancing medicament.

The application of the tea seed cake extract is used for preparing the medicine for treating burns and scalds.

Compared with the prior art, the tea seed cake extract optimizes the contents of tea saponin, Camelliaside A and Camelliaside B, has good effect of promoting wound healing of burns and scalds, and can remarkably increase the content of tea saponin, Camelliaside A and Camelliaside BCOL α-1AndCOL-3 mRNA expression, useful for the preparation of a medicament.

Drawings

FIG. 1: the effects of different experimental groups on the appearance of scald and wound healing of C57BL/6 mice skin; FIG. 2: the pathological changes of the skin tissues of the mice after the treatment of different experimental groups.

Detailed Description

The tea cake extract and the use thereof according to the present invention will be described below with reference to specific examples, but the scope of the present invention is not limited thereto.

Example 1

The preparation process of the tea seed cake extract comprises the following steps: taking the tea seed cake, adding 8 times of petroleum ether, and performing reflux extraction twice for 1h each time; filtering to remove extractive solution, extracting the residue with 70% ethanol solution twice, each for 1 hr, and mixing the ethanol extractive solutions; removing ethanol, adding acetone into the ethanol extractive solution until the content is 90%, filtering to remove precipitate, and drying the filtrate to obtain the final product.

Example 2

The preparation process of the tea seed cake extract comprises the following steps: taking the tea seed cake, adding petroleum ether of which the amount is 4 times that of the tea seed cake, and performing reflux extraction twice for 2 hours each time; filtering to remove extractive solution, extracting the residue with 50% ethanol solution for 3 hr twice, and mixing the ethanol extractive solutions; removing ethanol, adding acetone into the ethanol extractive solution until the content is 70%, filtering to remove precipitate, and drying the filtrate to obtain the final product.

Example 3

Taking the tea seed cake, adding 6 times of petroleum ether, and performing reflux extraction twice, wherein each time lasts for 1.5 h; filtering to remove extractive solution, extracting the residue with 60% ethanol solution twice, each for 2 hr, and mixing the ethanol extractive solutions; removing ethanol, adding acetone into the ethanol extractive solution until the content is 80%, filtering to remove precipitate, and drying the filtrate to obtain the final product.

Determination of content

(1) Tea saponin content determination

Precisely weighing 20.00 mg of tea saponin standard substance (Camelliasenin B1), placing in a 10 mL volumetric flask, dissolving with appropriate amount of 80% ethanol solution, metering to the scale mark, shaking, and storing in a refrigerator for use. Transferring 2 mg/mL standard use solution 0.0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 mL into 8 colorimetric tubes, accurately diluting with 80% ethanol to 1.0 mL, adding 0.5 mL 8% vanillin ethanol solution and 5.0 mL 77% sulfuric acid solution, mixing well, and placing in 60Keeping the temperature in a constant temperature water bath kettle for 15 min, taking out, cooling in ice water bath for 10 min, and standing at room temperature. The reagent was used as a blank reference, and the absorbance of the solution was measured at 550 nm using a 1 cm cuvette to obtain the regression equation A =0.8978C-0.0221, R²= 0.9991. The extracts of the tea seed cakes of examples 1 to 3 were prepared into solutions having a concentration of about 4mg/ml, and then measured by adding vanillin concentrated sulfuric acid for color development according to the above method, and each sample was measured in parallel 3 times. The results are shown in table 1 below:

TABLE 1 measurement results of tea saponin content in examples 1-3

Sample (I)	A mean value	Content (%)
			Example 1	0.197	6.10
Example 2	0.181	5.64
			Example 3	0.268	8.06

(2) Measurement of flavone content

The content of Camellia sinensis cake extract, i.e., Camellia sinensis A and Camellia sinensis B, was measured by HPLC. A chromatographic column: c18 (ODS, 250 mm. times.4.6 mm, 5.0 um); mobile phase: acetonitrile 0.1% H3PO4=20: 80; detection wavelength: 260 nm; column temperature: 38 ℃; flow rate: 1.0 mL/min, and the sample size is 10 ul. Methodology when qualified, the tea cake extracts of examples 1-3 were each prepared as solutions at a concentration of about 4mg/ml and each sample was assayed in parallel 3 times. The results are shown in table 2 below:

TABLE 2 results of the content of Camelliaside A and Camelliaside B in examples 1 to 3

Sample (I)	Camelliaside A	Camelliaside B
			Example 1	1.24%	1.26%
Example 2	1.82%	1.91%
			Example 3	1.65%	1.71%

Experiment of drug effect

The method comprises the steps of selecting female C57BL/6 mice with the size of about 6-8 weeks, wherein the female C57BL/6 mice are female and have the weight of 20-25 g, randomly dividing the mice into 5 groups (a pure water group, a Meibao group, an example 1 group, an example 2 group and an example 3 group), preparing 10 mice per group, anesthetizing, preparing skins, and continuously contacting boiling water with the skin of the back of the mouse for 25 s to cause a circular deep II-degree scald wound with the diameter of the back of the mouse being about 1.5 cm. The experiment was divided into three time points according to the observed scald recovery time: day 3, day 7 and day 14. The medicine is administered separately in groups, and each mouse is applied to the scald by 0.05mL, and examples 1-3 are applied by 30 mg/mL.

The mice were housed in the animal house of the research center of Gannan institute of medicine, and were fed with water in normal diet, and each group of mice was given pure water or each drug every afternoon every day. Measuring the scald area of the back of the mouse at different time periods, namely the third day, the 7 th day and the 14 th day, killing the mouse, collecting the serum of the mouse, taking the skin with the size of 2.0 cm multiplied by 2.0 cm in the scald area of the back and the peripheral area, evenly dividing the skin into 4 equal parts, fixing one part of the blood with 4 percent paraformaldehyde neutral solution for 1 night, dehydrating, transparentizing, waxing and embedding the blood, manufacturing a paraffin section with the size of 4 mu m, and performing hematoxylin-eosin (HE) staining microscopic examination. Another part is taken, qRT-PCR detects related genes of skin tissue wound healingCOLα- 1、COL-3mRNA expression levels and collagen staining. The experimental results are shown in the attached figures 1-2 and tables 3-4.

TABLE 3 groups of experimentsCOLα-1Results of mRNA expression levels summarized (x. + -. s, n = 10)

Time of treatment	Model set	Positive control group	Example 1	Example 2	Example 3
						Day 3	0.014±0.0044	0.087±0.0445*	0.045±0.0163*	0.098±0.0706**	0.159±0.0604**
Day 7	0.119±0.0347	0.162±0.0435*	0.103±0.0277	0.189±0.0587**	0.267±0.0838**
						Day 14	0.214±0.0463	0.422±0.0887*	0.494±0.1052*	0.522±0.1167**	0.621±0.0942**

Denotes p <0.05 relative to the same fold model group; denotes p <0.05 relative to the same day model group

TABLE 4 summary of expression level results of COL-3 mRNA in each experimental group (x. + -. s, n = 10)

Time of treatment	Model set	Positive control group	Example 1	Example 2	Example 3
						Day 3	0.047±0.0133	0.084±0.0248*	0.082±0.0340*	0.075±0.0221*	0.268±0.0906**
Day 7	0.136±0.0425	0.123±0.0302	0.115±0.0342	0.145±0.0380	0.307±0.0668**
						Day 14	0.138±0.0655	0.334±0.1033*	0.337±0.0700*	0.378±0.0604**	0.521±0.0841**

Denotes p <0.05 relative to the simultaneous model group; denotes p <0.05 relative to the same day model group

The effect on healing of the scald wound surface is as follows: the wound diameters of the mice on the 3 rd, 7 th and 14 th days after the administration were measured to calculate the wound healing rates, and the graphs showed that the healing rates of the groups of examples 1 to 3 were significantly higher than those of the model group and the positive controlGroup (A)P＜0.05）。

The results of HE staining of skin histopathology after scald are as follows: in the three example groups on days 3, 7 and 14, the number of inflammatory cells infiltrating and necrotic tissue was significantly reduced compared to the model group (P < 0.05), and the formation of granulation tissue was observed, especially on day 14. The inflammatory cell infiltration and tissue necrosis of the positive control group are between the model group and the three example groups.

To pairCOLα-1、COL-3Effect of expression level of mRNA: compared with the model group, the three example groups can be remarkably increased at 3 rd, 7 th and 14 th daysCOLα-1AndCOL-3 mRNA expression, but significant levels vary greatly.

Claims

1. The tea seed cake extract is characterized by comprising the following preparation processes: taking the tea seed cake, adding 4-8 times of petroleum ether, and extracting under reflux twice, each time for 1-2 h; filtering to remove extractive solution, extracting the residue with 50-70% ethanol solution for two times, each for 1-3 hr, and mixing the ethanol extractive solutions; removing ethanol, adding acetone into ethanol extractive solution to reach content of 70-90%, filtering to remove precipitate, and drying the filtrate to obtain the final product.

2. The tea cake extract according to claim 1, which is prepared by a process comprising: taking the tea seed cake, adding 6 times of petroleum ether, and performing reflux extraction twice, wherein each time lasts for 1.5 h; filtering to remove extractive solution, extracting the residue with 60% ethanol solution twice, each for 2 hr, and mixing the ethanol extractive solutions; removing ethanol, adding acetone into the ethanol extractive solution until the content is 80%, filtering to remove precipitate, and drying the filtrate to obtain the final product.

3. The tea seed cake extract as claimed in claim 1, wherein the content of tea saponin is 6.0-8.1%, the content of Camelliaside a is 1.2-1.9%, and the content of Camelliaside B is 1.2-2.0%.

4. The tea cake extract as claimed in claim 1, for increasingCOLα-1 Preparation of mRNA expressed drugs.

5. The tea cake extract as claimed in claim 1, for increasingCOL-3 Preparation of mRNA expressed drugs.

6. The tea cake extract according to claim 1, wherein the preparation of a medicament for enhancing collagen.

7. The tea cake extract according to claim 1, wherein the preparation of a medicament for treating burns and scalds.