White tea polypeptide, white tea extract and preparation method and use thereof
By extracting peptides and white tea extracts from white tea, drugs with anti-inflammatory activity are prepared, which solves the problem of large side effects of existing anti-inflammatory drugs and achieves safe and effective anti-inflammatory effects.
Patent Information
- Application Number
- CN202211606545.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-12-12
- Publication Date
- 2025-09-26
- Estimated Expiration
- 2042-12-12
AI Technical Summary
Although existing anti-inflammatory drugs are effective, they have significant side effects, and there is a lack of safe and effective natural anti-inflammatory products.
Peptides and white tea extracts are extracted from white tea by a specific method, especially from silver needle, Gongmei, Shoumei and white peony teas, and anti-inflammatory drugs are prepared using polypeptide sequences such as LLLSKKL, VVGNYPL, ECDSCG, EVFAG and LCAY and their derivatives.
White tea peptides and extracts exhibit excellent anti-inflammatory effects, significantly inhibiting the release of nitric oxide (NO) and the secretion of the pro-inflammatory cytokine IL-6, providing a new and safe approach for the development of anti-inflammatory drugs.
Smart Images
Figure CN115806590B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of active extracts and active peptides, and particularly relates to a white tea polypeptide, a white tea extract, and a preparation method and use thereof. Background Art
[0002] Inflammation is a complex and ubiquitous condition in the body that plays a vital role in protecting the body from harmful internal or external stimuli such as pathogens, chemicals, and radiation, thereby restoring bodily functions and tissue homeostasis. Excessive inflammation is a key factor in the development of many diseases. Current methods for preventing and treating inflammation include inhibiting cellular immune responses and reducing the secretion of pro-inflammatory factors. The main drugs include glucocorticoids and non-specific immunosuppressants. However, while these anti-inflammatory drugs have good anti-inflammatory effects, they also come with a number of side effects, such as allergic reactions and dependence. Therefore, the research and development of new, safer and more effective natural anti-inflammatory products or derivatives of natural products has become an increasing focus of researchers.
[0003] Bioactive peptides have always been one of the research hotspots in the fields of life and food sciences. Many studies have found that some food-derived peptides show high anti-inflammatory activity, which provides a good direction and approach for the research and development of new anti-inflammatory drugs.
[0004] As one of my country's six traditional teas, white tea boasts a long history, with its origins dating back to the Song Dynasty. Its unique production method, which involves neither frying nor rolling, leaves the finished tea with a white, fuzzy appearance, hence the name. Due to differences among tea plant varieties and the varying tenderness of fresh leaves, tea varieties are generally categorized into: Silver Needle Baihao, White Peony, Gongmei, and Shoumei.
[0005] Because white tea is produced without high-temperature withering or vigorous rolling, and only undergoes moderate natural oxidation, it develops unique health-promoting qualities. Modern scientific research has found that white tea contains a variety of key ingredients with beneficial effects. For example, tea polyphenols have antibacterial and antimicrobial properties, can improve arrhythmias, and prevent atherosclerosis; theanine can penetrate the human brain barrier and synergize with caffeine to significantly enhance the body's excitement and coordination, prevent Parkinson's disease, and effectively enhance memory; tea pigments such as theaflavins can prevent cancer, cardiovascular and cerebrovascular diseases, and obesity; thearubigins have anti-inflammatory and antibacterial properties; and theabrownins are highly effective in weight loss and lowering blood pressure, earning them the nickname "the green gold of medicine."
[0006] In addition to its antibacterial and cardiovascular protective effects, white tea also possesses unique anti-inflammatory activity. However, there are currently few reports on the anti-inflammatory active components of white tea, and further research is needed. Summary of the Invention
[0007] The purpose of the present invention is to provide a white tea polypeptide, a white tea extract, and a preparation method and use thereof.
[0008] The present invention provides a polypeptide, which is a polypeptide of any one of the following sequences or a derivative thereof, or a combination of polypeptides of any multiple of the following sequences or derivatives thereof: LLLSKKL, VVGNYPL, ECDSCG, EVFAG.
[0009] The present invention also provides a white tea extract containing the above-mentioned polypeptide and / or a polypeptide with a sequence of LCAY or a derivative thereof.
[0010] Furthermore, the white tea extract is an extract of Yinzhen white tea, Gongmei white tea, Shoumei white tea and / or White Peony tea.
[0011] Furthermore, the white tea extract is an extract of Shoumei white tea and / or white peony tea.
[0012] Furthermore, the white tea extract is obtained by extracting according to a method comprising the following steps:
[0013] (1) After the white tea is crushed, add n-pentane to defatted and take the filter residue;
[0014] (2) adding anhydrous ethanol, heating under reflux to extract, and obtaining an extract;
[0015] (3) The extract was sequentially extracted with the following organic solvents: dichloromethane, methyl acetate, ethyl acetate, and butyl acetate, and the aqueous phase was collected;
[0016] (4) After further removing the organic solvent, the ultrafiltration membrane is used to intercept the substance with a molecular weight greater than 500 Da.
[0017] The present invention also provides a method for preparing the above-mentioned white tea extract, comprising the following steps:
[0018] (a) After the white tea is crushed, n-pentane is added for defatting, and the filter residue is collected;
[0019] (b) adding anhydrous ethanol, heating under reflux to extract, and obtaining an extract;
[0020] (c) extracting the extract with the following organic solvents in sequence: dichloromethane, methyl acetate, ethyl acetate, and butyl acetate, and then taking the aqueous phase;
[0021] (d) After further removing the organic solvent, the product with a molecular weight greater than 500 Da was retained by an ultrafiltration membrane.
[0022] Furthermore, the number of times of n-pentane degreasing in step (a) is 3 to 7 times;
[0023] And / or, the reflux extraction in step (b) is performed 3 to 7 times;
[0024] And / or, each organic solvent in step (c) is extracted 3 to 7 times.
[0025] The present invention also provides use of the above polypeptide or the above white tea extract in preparing anti-inflammatory drugs.
[0026] The present invention also provides the use of a polypeptide with a sequence of LCAY or a derivative thereof in the preparation of anti-inflammatory drugs.
[0027] The present invention also provides an anti-inflammatory drug, which is a preparation prepared with the above-mentioned white tea polypeptide, polypeptide with the sequence LCAY or its derivatives, and / or the above-mentioned white tea extract as active ingredients, and pharmaceutically acceptable excipients.
[0028] The present invention provides a white tea extract, obtained by a specific extraction method, that exhibits excellent anti-inflammatory effects. Further sequencing and screening revealed peptide sequences that frequently occur in the extract, confirming that white tea peptides with these sequences also exhibit excellent anti-inflammatory effects, providing a new avenue for the development of anti-inflammatory drugs.
[0029] The term "derivative" in the present invention refers to a substance formed by replacing atoms or atomic groups in the chemical structure of a polypeptide molecule by other atoms or atomic groups.
[0030] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.
[0031] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Figure 1 The cytotoxicity results of Shoumei oligopeptide, Mudan oligopeptide and dexamethasone on RAW264.7 cells (n=3).
[0033] Figure 2 The cytotoxicity results of white tea peptide on RAW264.7 cells (n=3).
[0034] Figure 3 The effect of LPS on the morphology of RAW264.7 cells.
[0035] Figure 4Effects of Shoumei Paeonia lactiflora oligopeptide on NO release induced by LPS in RAW264.7 cells (n=3); Note: a=50 μg·mL -1 ; b = 100 μg·mL -1 ; c = 200 μg·mL -1 ; Compared with the model group, * indicates significant difference (P<0.05); ** indicates extremely significant difference (P<0.01).
[0036] Figure 5 Effects of white tea synthetic peptides on LPS-induced NO release in RAW264.7 cells (n=3); Note: a=50 μg·mL -1 ; b = 100 μg·mL -1 ; c = 200 μg·mL -1 ; Compared with the model group, * indicates significant difference (P<0.05); ** indicates extremely significant difference (P<0.01).
[0037] Figure 6 Effects of Shoumei Paeonia lactiflora oligopeptides on IL-6 secretion in RAW264.7 cells induced by LPS (n=3); Note: a=50 μg·mL -1 ; b = 100 μg·mL -1 ; c = 200 μg·mL -1 ; Compared with the model group, * indicates significant difference (P<0.05); ** indicates extremely significant difference (P<0.01).
[0038] Figure 7 Effects of white tea synthetic peptides on IL-6 secretion in RAW264.7 cells induced by LPS (n=3); Note: a=50 μg·mL -1 ; b = 100 μg·mL -1 ; c = 200 μg·mL -1 ; Compared with the model group, * indicates significant difference (P<0.05); ** indicates extremely significant difference (P<0.01). DETAILED DESCRIPTION
[0039] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.
[0040] It should be noted that although the examples of the present invention only list two varieties of Shoumei white tea and Bai Mudan tea, the extraction method of the present invention is also applicable to the extraction of other varieties of white tea, and the obtained extracts and the determination of polypeptide sequences have results comparable to those of the examples provided by the present invention.
[0041] Example 1. Preparation of crude white tea oligopeptide extract
[0042] Take 500g of Shoumei white tea or white peony tea sample, crush it, add 3.0L of n-pentane to degrease for 15min (stir every 5min), filter, repeat this process 5 times, then add 3.0L of anhydrous ethanol and reflux for 30min, repeat this process 5 times, and combine the extracts. The extract is concentrated under reduced pressure at 40°C, dissolved with an appropriate amount of ultrapure water, and then extracted with dichloromethane, methyl acetate, ethyl acetate, and butyl acetate (each solvent is extracted 5 times, 2.0L / time), take the aqueous phase, rotary evaporate the residual organic reagent at 40°C, moderately concentrate, cut the >500Da interval with a 500Da ultrafiltration membrane, freeze-dry, and obtain the finished product, which is stored at -20°C for use, namely the Shoumei oligopeptide crude extract or the peony oligopeptide crude extract for subsequent experimental verification.
[0043] Example 2: Sequence determination and preparation of white tea polypeptide
[0044] After high-throughput sequencing of the crude extract of white tea and white tea oligopeptides obtained in Example 1, peptide sequences with high sequencing result scores, high credibility and high occurrence frequency were screened: LCAY (SEQ ID NO.1), LLLSKKL (SEQ ID NO.2), VVGNYPL (SEQ ID NO.3), ECDSCG (SEQ ID NO.4), EVFAG (SEQ ID NO.5), and Suzhou Qiangyao Biotechnology Co., Ltd. was commissioned to synthesize them.
[0045] The beneficial effects of the present invention are demonstrated by experimental examples below.
[0046] Experimental Example 1: Activity Characterization of the White Tea Oligopeptide Extract and White Tea Peptide of the Present Invention
[0047] 1. Experimental methods
[0048] Cell culture
[0049] Macrophage RAW267.4 cells were cultured in DMEM medium (containing 10% FBS) in an incubator at 37° C. and 5% CO 2 . Cells were passaged when they reached 70% to 80% of their confluence.
[0050] Experimental groups
[0051] RAW264.7 cells were divided into experimental groups and treated as follows: normal group: complete culture medium containing 10% FBS was added; model group: normal RAW264.7 was induced by 1 μg / mL LPS; drug administration group: different concentrations of crude extracts of white tea oligopeptides (Shoumei oligopeptide or Peony oligopeptide) and synthetic white tea peptides (LCAY, LLLSKKL, VVGNYPL, ECDSCG, EVFAG) were pretreated for 2 hours, and then LPS with a final concentration of 1 μg / mL was added for induction for 24 hours; positive control group: dexamethasone was pretreated for 2 hours, and then LPS with a final concentration of 1 μg / mL was added for induction for 24 hours.
[0052] Cell viability assay
[0053] The density of RAW264.7 cells was adjusted to 10 5 Cells were seeded in 96-well plates at 10 μL / mL. After 12 h, the supernatant was discarded and the cells were treated according to the above experimental groups. After incubation in an incubator for 24 h, 10 μL of CCK reagent was added to each well and incubated at 37°C for 2 h. The OD value was measured at 450 nm.
[0054]
[0055] NO level determination
[0056] Adjust the cell density to 2 × 10 5 / mL was seeded in a 24-well plate. After incubation for 12 hours, RAW264.7 cells were pretreated with different concentrations of white tea oligopeptides and dexamethasone for 2 hours. Subsequently, RAW264.7 cells were stimulated with 1μg / mL LPS and incubated for 24 hours. The cell culture supernatant was taken for NO content determination. In summary, 50μL of cell culture supernatant was mixed with 50μL GriessⅠ and 50μL GriessⅡ reagents, incubated at room temperature for 15 minutes, and the absorbance was measured at 540nm using a microplate reader. The NO inhibition rate was determined according to the following formula.
[0057]
[0058] Determination of IL-6 inflammatory factor
[0059] Adjust the cell density to 2 × 10 5 / mL were seeded in 24-well plates and incubated for 12 hours. RAW264.7 cells were pretreated with different concentrations of white tea oligopeptides (50μg / mL, 100μg / mL, 200μg / mL) and dexamethasone (50μg / mL) for 2 hours. Then, RAW264.7 cells were stimulated with 1μg / mL of LPS and incubated for 24 hours. The cell culture supernatant was aspirated to determine the IL-6 inflammatory factor content, and the operation was performed according to the instructions of the Elisa kit (IL-6).
[0060] 2. Experimental results
[0061] 1. Cell viability results
[0062] like Figure 1 、 Figure 2 As shown in the results, at concentrations of 50 μg / mL to 200 μg / mL, crude extracts of white tea oligopeptides and synthetic white tea peptides were non-toxic to mouse macrophage RAW 264.7 (cell viability > 90%) and exhibited a certain growth promoting effect. This may be because white tea oligopeptides and synthetic white tea peptides have the characteristics of small molecular weight, high safety, and easy absorption, and can serve as nutrients for cells, thereby promoting cell growth. In summary, 50 μg / mL, 100 μg / mL, and 200 μg / mL were selected as subsequent experimental concentrations.
[0063] from Figure 3 As can be seen, cells in the normal group appear round or nearly round, with a smooth surface and distinct cell boundaries. When stimulated with 1 μg / mL LPS, the cells differentiated, gradually becoming spindle-shaped and producing numerous pseudopodia. The cell area increased, and bubbles and particles within the cells were dispersed in the cell culture medium. Overall, they appeared to be dendritic cells, demonstrating the effectiveness of LPS modeling.
[0064] Studies have shown that when inflammation occurs, macrophages release a large amount of NO. Under normal conditions, macrophages have very weak expression of nitric oxide synthase, but during inflammation, nitric oxide synthase will be expressed in large quantities and converted into NO, leading to inflammation and cell damage. Therefore, this experiment used the Griess method to determine the NO content in the cell supernatant to indirectly show the level of inflammation between the groups. Figure 4 、 Figure 5 It can be seen that compared with the normal group, when RAW264.7 was stimulated with 1 μg / mL LPS, the NO content in the model group increased significantly (P<0.01), indicating that the cell inflammation model was successfully established. Figure 4 It can be seen that compared with the model group, the Shoumei oligopeptide and Peony oligopeptide groups could significantly inhibit NO release (P<0.01), and both showed concentration dependence. Figure 5 It can be seen that compared with the model group, the five different white tea peptides can also reduce the release of NO, and show a concentration-dependent manner.
[0065] IL-6 is an important pro-inflammatory cytokine that stimulates the growth and differentiation of immune cells and enhances the ability of macrophages to present antigens during immune and inflammatory responses. IL-6 is produced in the early stages of inflammation, so inhibiting the secretion of IL-6 can effectively treat inflammation. Figure 6 、 Figure 7Compared with the normal group, RAW 264.7 cells stimulated with 1 μg / mL LPS secreted a large amount of IL-6 pro-inflammatory cytokine (P<0.01). Compared with the model group, the Shoumei oligopeptide, Peony oligopeptide, and White Tea polypeptide administration groups all significantly inhibited the secretion of IL-6 pro-inflammatory cytokine, and all showed a concentration-dependent manner. Therefore, both crude White Tea oligopeptide extract and White Tea polypeptide can inhibit LPS-induced cellular IL-6 secretion and have certain anti-inflammatory activity.
[0066] In summary, the present invention provides a white tea oligopeptide extract and a white tea polypeptide of a specific sequence, which have anti-inflammatory activity and can exert anti-inflammatory effects by inhibiting the release of NO and regulating the level of cytokine IL-6.
Claims
1. A polypeptide, characterized in that It is a polypeptide of any one of the following sequences, or a combination of polypeptides of any multiple of the following sequences: LLLSKKL, VVGNYPL, ECDSCG, EVFAG.
2. A white tea extract, characterized in that Contains the polypeptide according to claim 1 and a polypeptide with the sequence LCAY.
3. The white tea extract according to claim 2, wherein It is an extract of Shoumei white tea and / or white peony tea.
4. The extract according to claim 2 or 3, characterized in that It is extracted according to a method comprising the following steps: (1) After the white tea is crushed, add n-pentane to defatted and take the residue; (2) Add anhydrous ethanol, heat and reflux to extract, and obtain the extract; (3) The extract was extracted with the following organic solvents in sequence: dichloromethane, methyl acetate, ethyl acetate, and butyl acetate, and the aqueous phase was collected; (4) After further removal of the organic solvent, the ultrafiltration membrane is used to intercept substances with a molecular weight greater than 500 Da.
5. The method for preparing the extract according to any one of claims 2 to 4, characterized in that: The steps include: (a) After the white tea leaves are crushed, add n-pentane to defatted tea leaves and filter the residue. (b) adding anhydrous ethanol, heating under reflux to extract, and obtaining an extract; (c) extracting the extract with the following organic solvents in sequence: dichloromethane, methyl acetate, ethyl acetate, and butyl acetate, and then taking the aqueous phase; (d) After further removing the organic solvent, the ultrafiltration membrane is used to intercept the substances with a molecular weight greater than 500 Da.
6. The preparation method according to claim 5, wherein The number of times of n-pentane degreasing in step (a) is 3 to 7 times; And / or, the number of reflux extractions in step (b) is 3 to 7 times; And / or, each organic solvent in step (c) is extracted 3 to 7 times.
7. Use of the polypeptide according to claim 1 or the white tea extract according to any one of claims 2 to 5 in the preparation of an anti-inflammatory drug.
8. Use of a polypeptide with the sequence LCAY in the preparation of anti-inflammatory drugs.
9. An anti-inflammatory drug, characterized in that It is a preparation made with any one or more of the following substances as active ingredients, plus pharmaceutically acceptable excipients: 1) The polypeptide of claim 1; 2) The white tea extract of any one of claims 2 to 5.