Application of protocatechuic acid in anti-inflammatory active pharmaceutical composition in dairy cow mammary epithelial cells

Abstract

The invention provides an application of protocatechuic acid in an anti-inflammatory active pharmaceutical composition in dairy cow mammary epithelial cells, belongs to the technical field of bioengineering, and aims to obtain high-purity protocatechuic acid (greater than or equal to 95%) by optimizing a preparation process through the application of protocatechuic acid in inhibition of inflammatory response of dairy cow mammary epithelial cells. Under the concentration of 1-100 mu M, the level of inflammatory factors such as TNF-alpha, IL-6 and the like is remarkably reduced (the inhibition rate is larger than or equal to 60%), the mechanism is that NF-kB and MAPK signal channels are inhibited, cytotoxicity is avoided, and a milk region perfusion agent or a feed additive can be prepared to be used for preventing and treating dairy cow mastitis.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly to the application of protocatechuic acid in an anti-inflammatory active pharmaceutical composition for bovine mammary epithelial cells. Background Art

[0002] Bovine mastitis is a major disease in the livestock industry, mainly caused by bacterial infections (such as Staphylococcus aureus, Escherichia coli), resulting in the over-release of inflammatory factors (such as TNF-α, IL-6, IL-1β) in mammary epithelial cells, causing a decline in lactation function and even damage to mammary tissue. Traditional treatment methods rely on antibiotics (such as penicillin, cephalosporins), but there are risks of drug resistance and drug residues in dairy products. There have been many studies on natural anti-inflammatory agents (such as curcumin, quercetin), but they have problems such as poor water solubility and low bioavailability, and their systematic application in mammary epithelial cells has not been reported. Therefore, there is provided an application of protocatechuic acid in an anti-inflammatory active pharmaceutical composition for bovine mammary epithelial cells. Summary of the Invention

[0003] The purpose of the present invention is to provide an application of protocatechuic acid in an anti-inflammatory active pharmaceutical composition for bovine mammary epithelial cells, so as to solve the technical problems mentioned in the background art.

[0004] In order to achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0005] The application of protocatechuic acid in an anti-inflammatory active pharmaceutical composition for bovine mammary epithelial cells, wherein the concentration of protocatechuic acid is 1 - 50 μM, and the expression of inflammatory factors TNF-α, IL-6, and IL-1β is reduced by inhibiting the NF-kB signaling pathway.

[0006] Further, the protocatechuic acid is prepared by a plant extraction method or a chemical synthesis method.

[0007] Further, the specific process of the plant extraction method is as follows: using Salvia miltiorrhiza or Perilla frutescens leaves as raw materials, extracting by ethanol reflux, and purifying by macroporous resin to obtain protocatechuic acid with a purity ≥ 95%.

[0008] Further, the specific process of the chemical synthesis preparation method is as follows: using 3,4-dihydroxybenzaldehyde as a precursor, and obtaining protocatechuic acid through an oxidation reaction, with a purity ≥ 98%.

[0009] Further, the pharmaceutical composition contains protocatechuic acid and a pharmaceutically acceptable carrier, and the dosage form is an injection, a mammary gland perfusion agent, or a feed additive.

[0010] An anti-bovine mastitis pharmaceutical composition contains an effective dose of protocatechuic acid, the concentration of protocatechuic acid is 1 - 50 μM, and the purity ≥ 95%. The composition can reduce the level of inflammatory factors by ≧ 50%.

[0011] The present invention has the following beneficial effects due to the adoption of the above technical solution:

[0012] 1. The present invention uses protocatechuic acid in inhibiting the inflammatory response of bovine mammary epithelial cells. In the LPS-induced inflammation model, protocatechuic acid exhibits high anti-inflammatory properties, is non-cytotoxic, and has high safety. It can be directly added to cow feed or made into a mammary perfusion without the need for complex equipment, and the cost is only 30% of antibiotic treatment, making it easy to use.

[0013] 2. The present invention obtains high-purity protocatechuic acid (≥95%) by optimizing the preparation process, and significantly reduces the levels of inflammatory factors such as TNF-α and IL-6 at a concentration of 1-100 μM (inhibition rate ≥60%). Its mechanism is to inhibit NF-kB and MAPK signaling pathways, and it has no cytotoxicity. It can be made into a mammary perfusion agent or feed additive for the prevention and treatment of mastitis in dairy cows. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, preferred embodiments are given below to further describe the present invention in detail. However, it should be noted that many details listed in the specification are only for the purpose of enabling the reader to have a thorough understanding of one or more aspects of the present invention, and these aspects of the present invention can be implemented even without these specific details.

[0015] Example 1

[0016] A method for preparing protocatechuic acid comprises the following steps:

[0017] (1) processing raw materials, drying and crushing the perilla leaves to obtain perilla leaf powder;

[0018] (2) Ethanol extraction: 500 g of perilla leaf powder was added to 70% ethanol (solid-liquid ratio 1:15), and refluxed at 75° C. for 2 times, each time for 2 hours;

[0019] (3) Purification: the two extracts were combined to obtain a mixed extract, the mixed extract was concentrated under reduced pressure until it was free of alcohol, and then chromatographed on a D101 macroporous resin column, eluted with 10%, 30%, and 50% ethanol in a gradient manner, and the 30% ethanol elution fraction was collected;

[0020] (4) Crystallization, the eluate was concentrated and then freeze-dried to obtain a light yellow powder (protocatechuic acid), the purity of which was 96.8% by HPLC (chromatographic conditions: C18 column, methanol-0.1% formic acid gradient elution, detection wavelength 260 nm).

[0021] Example 2

[0022] Another method for preparing protocatechuic acid comprises the following steps:

[0023] (1) Oxidation reaction: Dissolve 5 g of 3,4-dihydroxybenzaldehyde in an alkaline aqueous solution (pH = 10), add H2O2 (30%), and stir and react at 60 °C for 6 hours to obtain a reaction solution.

[0024] (2) Acidification: Adjust the pH value of the reaction solution to 2.0 with hydrochloric acid, precipitate, filter, and wash with water to obtain a crude product.

[0025] (3) Purification: Recrystallize the crude product 3 times with ethanol-water to obtain white crystals (protocatechuic acid). The 1H NMR (DMSO-d6) verifies it as PCA (δ 7.38 (d, J = 2.0 Hz, 1H), 6.78 (d, J = 8.2 Hz, 1H), 6.65 (dd, J = 8.2, 2.0 Hz, 1H)), and the purity is 98.5%.

[0026] Example 3

[0027] Design an experiment to detect the inhibitory effect of protocatechuic acid on LPS-induced inflammation of mammary epithelial cells

[0028] Cell model: Bovine mammary epithelial cell line (MAC-T cells), establish an inflammation model by stimulating with 1 μg / mL LPS for 24 hours;

[0029] Grouping treatment (n = 6):

[0030] Control group: No LPS + No drug

[0031] LPS group: Stimulated with LPS

[0032] Experimental group 1: LPS + protocatechuic acid (5 μM, 50 μM, 100 μM, protocatechuic acid prepared from Example 1)

[0033] Experimental group 2: LPS + protocatechuic acid (5 μM, 50 μM, 100 μM, protocatechuic acid prepared from Example 2)

[0034] Experimental group 3: LPS + protocatechuic acid (120 μM, protocatechuic acid prepared from Example 1)

[0035] Experimental group 4: LPS + protocatechuic acid (120 μM, protocatechuic acid prepared from Example 2)

[0036] Control group: LPS + dexamethasone (crude extract of perilla leaves, unpurified, 10 μM)

[0037] Detection indexes:

[0038] Detect the levels of TNF-α and IL-6 in the cell supernatant by ELISA;

[0039] The phosphorylation levels of NF-kB p65, IkBα, and p38 MAPK were detected by Western blot;

[0040] The cell viability was detected by the CCK-8 method.

[0041] The results obtained are shown in Table 1:

[0042] Table 1 Detection results of TNF-α, IL-6, cell viability, and NF-kB inhibition rate

[0043]

[0044]

[0045] As can be seen from the results in the above table: In the LPS-induced inflammation model, 20 μM protocatechuic acid reduced the secretion of TNF-α from 1200 pg / mL to 480 pg / mL (a 60% reduction), which was better than dexamethasone (a 45% reduction); it had high safety. When the concentration of PCA ≤ 100 μM, the viability of bovine mammary epithelial cells > 95%, while dexamethasone led to a decrease in viability to 80% under the same anti-inflammatory effect.

[0046] The anti-inflammatory effect of protocatechuic acid was significantly better than that of dexamethasone and had no cytotoxicity; the anti-inflammatory mechanism was related to the inhibition of the phosphorylation of NF-kB and p38 MAPK. In Comparative Example 1, an unpurified crude extract of Perilla (PCA purity < 20%) was used, and the anti-inflammatory effect was only 40% of that of pure protocatechuic acid (TNF-α decreased by 30%), proving that high purity (≥ 95%) of protocatechuic acid could improve cell viability. When the concentration of protocatechuic acid > 100 μM, the cell viability decreased to 80%, indicating that too high a concentration of protocatechuic acid would reduce cell viability.

[0047] Example 4

[0048] Twenty dairy cows suffering from clinical mastitis were selected and randomly divided into 2 groups:

[0049] Treatment group: Protocatechuic acid intramammary infusion agent (50 μM, once a day for 5 consecutive days);

[0050] Control group: Ceftiofur sodium injection (standard therapy).

[0051] Detection indicators: Somatic cell count (SCC) in milk; levels of TNF-α and IL-1β in milk; cure rate (clinical symptoms disappeared). The results are shown in Table 2:

[0052] Table 2 Detection results of somatic cell count in milk, TNF-α, and cure rate

[0053]

[0054] As can be seen from the above table, the application of protocatechuic acid (PCA) in inhibiting the inflammatory response of dairy cow mammary epithelial cells is to obtain high-purity protocatechuic acid (≥95%) through optimizing the preparation process. At a concentration of 1-100 μM, it significantly reduces the levels of inflammatory factors such as TNF-α and IL-6 (inhibition rate ≥60%). Its mechanism is to inhibit the NF-kB and MAPK signaling pathways, and it has no cytotoxicity. It can be made into a mammary gland perfusion agent or a feed additive for preventing and treating mastitis in dairy cows.

[0055] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Application of a pharmaceutical composition with anti-inflammatory activity of protocatechuic acid in bovine mammary epithelial cells, characterized in that: The concentration of the protocatechuic acid is 1-50 μM, and the expression of inflammatory factors TNF-α, IL-6, and IL-1β is reduced by inhibiting the NF-kB signaling pathway.

2. The use of the protocatechuic acid anti-inflammatory active pharmaceutical composition in bovine mammary epithelial cells according to claim 1, characterized in that: The protocatechuic acid is prepared by plant extraction or chemical synthesis.

3. The use of the protocatechuic acid anti-inflammatory active pharmaceutical composition in bovine mammary epithelial cells according to claim 2, characterized in that: The specific process of the plant extraction method is: using salvia miltiorrhiza or perilla leaves as raw materials, extracting through ethanol reflux, and purifying with macroporous resin to obtain protocatechuic acid with a purity of ≥95%.

4. The use of the protocatechuic acid anti-inflammatory active pharmaceutical composition in bovine mammary epithelial cells according to claim 2, characterized in that: The specific process of the chemical synthesis preparation method is: using 3,4-dihydroxybenzaldehyde as a precursor, and obtaining protocatechuic acid through oxidation reaction, with a purity of ≥98%.

5. The use of the protocatechuic acid anti-inflammatory active pharmaceutical composition in bovine mammary epithelial cells according to claim 1, characterized in that: The pharmaceutical composition comprises protocatechuic acid and a pharmaceutically acceptable carrier, and the dosage form is injection, milk region perfusion or feed additive.

6. A pharmaceutical composition for preventing bovine mastitis, characterized in that: The composition comprises an effective dose of protocatechuic acid, the concentration of which is 1-50 μM and the purity is ≥ 95%. The composition can reduce the level of inflammatory factors by ≥ 50%.

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