A method for the enzymatic preparation of an antineoplastic composition from beta-carotene and bovine liver powder

Abstract

The application discloses a method for preparing an antitumor composition by enzymatic preparation of beta-carotene and ox liver powder, and belongs to the technical field of biological agents. The application utilizes an endogenous complex enzyme system in ox liver powder to catalyze the directional cleavage of beta-carotene under mild conditions in vitro, and generates abscisic acid in situ; the abscisic acid inhibits the elongation growth of tumor cells and blocks tumor proliferation. Residual carotenoids in the reaction system form a complex system with abscisic acid, and have a synergistic antitumor effect, and the activity is better than that of single abscisic acid with the same dose. The application does not need high-purity purified abscisic acid, raw materials are easy to obtain, the process is simple, and the production cost is low, and therefore the application has a good application prospect in the field of antitumor preparations.

Description

manual Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to a process for preparing an antitumor composition by in vitro enzymatic conversion. Background Technology

[0002] Malignant tumors continue to threaten human health. The typical biological characteristics of tumor cells are their uncontrolled elongation and unlimited proliferation. Abscisic acid (ABA), a carotenoid derivative, has been shown in current research to inhibit cell elongation and growth, and thus possesses certain inhibitory activity against tumor cell proliferation.

[0003] Current protocols involving abscisic acid (ABA) in antitumor treatment generally use artificially purified ABA raw materials for experiments and formulation development. This approach has significant drawbacks: First, the separation and purification process for high-purity ABA is complex and costly; second, simply adding ABA exogenously results in a limited mode of action and limited tumor-suppressing effect; and third, there is a lack of low-cost, in-situ ABA-generating composite preparation systems.

[0004] β-carotene is a widely distributed natural carotenoid. Physiological studies have shown that the conventional metabolic pathway of β-carotene in animals mainly produces vitamin A-like substances such as retinol and retinal; it is difficult to break down into abscisic acid under normal physiological conditions. Currently, there are no reports on the use of endogenous enzymes from bovine liver powder to catalyze the directed conversion of β-carotene into abscisic acid, and its application in the preparation of antitumor compositions. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies that directly use purified abscisic acid, such as high cost and limited tumor-suppressing effect, and to provide a method for the enzymatic preparation of a tumor-suppressing composition from β-carotene and bovine liver powder. This invention relies on an endogenous complex enzyme system from bovine liver powder to catalyze the directional cleavage of β-carotene and the in-situ generation of abscisic acid under controlled in vitro conditions. The residual carotenoids, enzyme metabolic derivatives, and abscisic acid in the system work synergistically to achieve an antitumor effect superior to that of the same dose of pure abscisic acid.

[0006] This invention provides a method for the enzymatic preparation of an antitumor composition from β-carotene and bovine liver powder, comprising the following steps:

[0007] S1. Raw material pretreatment: Pass the beef liver powder through an 80-120 mesh sieve; take β-carotene powder, mix it with a buffer solution, and stir to prepare a β-carotene suspension;

[0008] S2. Enzymatic conversion reaction: Add pretreated bovine liver powder to the β-carotene suspension and adjust the pH of the system to 5.5-7.0; control the reaction temperature at 28-38℃ and stir continuously in the dark for 18-36 hours.

[0009] S3. Reaction termination: Heat to 55-65℃ and hold for 15-30 minutes to inactivate the active enzymes in the bovine liver powder and terminate the lysis reaction.

[0010] S4. Solid-liquid separation: Centrifuge to remove solid residue, collect the supernatant to obtain a tumor-suppressing composition rich in abscisic acid.

[0011] Furthermore, the raw material ratio is as follows: 1 part β-carotene and 3 to 8 parts beef liver powder.

[0012] Furthermore, the buffer solution is a phosphate buffer solution.

[0013] The tumor-suppressing mechanism of this invention is explained as follows: The core physiological function of abscisic acid (ABA) is to inhibit cell elongation. The fundamental characteristic of malignant proliferation of tumor cells is continuous cell elongation and uncontrolled, unlimited cell division. The ABA produced by the system prepared in this invention, when applied to tumor cells, can significantly inhibit the elongation process of tumor cells, halt the continuous operation of the tumor cell cycle, inhibit tumor cell proliferation, and induce apoptosis in tumor cells, thereby producing a tumor-suppressing effect.

[0014] Meanwhile, incompletely converted β-carotene and various enzymatic hydrolysis intermediates in the reaction system can produce a synergistic effect with abscisic acid. Compared with the same dose of purified abscisic acid alone, the composition prepared in this invention has a stronger inhibitory effect on tumor cells, producing unexpected synergistic anti-tumor activity.

[0015] The present invention has the following advantages: 1. The present invention uses in vitro enzymatic in-situ generation of abscisic acid, which eliminates the need for high-purity extraction of abscisic acid in advance, simplifies the production process, and significantly reduces the cost of raw material preparation; 2. The composition is a multi-component complex system, in which multiple active ingredients work synergistically, and the antitumor activity is superior to that of abscisic acid alone; 3. The preparation conditions are mild, with low-temperature enzymatic reaction throughout the process, so the active substances are not easily destroyed, and the process is easy to scale up for production; 4. The raw materials β-carotene and bovine liver powder are widely available and easy to obtain. Detailed Implementation

[0016] The present invention will be further described in detail below with reference to specific embodiments. Example 1 A method for enzymatically preparing an antitumor composition from β-carotene and bovine liver powder: S1, Raw material pretreatment: Bovine liver powder is sieved through a 100-mesh sieve; 1g of β-carotene is added to a phosphate buffer solution and stirred evenly to form a suspension; S2, Enzymatic conversion: 5g of sieved bovine liver powder is added, and the pH is adjusted to 6.2; the temperature is controlled at 32℃, and the reaction is carried out with stirring in the dark for 24h; S3, Inactivation termination: the temperature is raised to 60℃ and kept at 20min to inactivate the enzyme activity; S4, centrifugation at 3500r / min for 20min is performed to remove the solid precipitate, and the supernatant is collected to obtain the antitumor composition. Example 2 A method for enzymatically preparing an antitumor composition from β-carotene and bovine liver powder: S1, raw material pretreatment: bovine liver powder is passed through an 80-mesh sieve; 1g of β-carotene is used to prepare a buffer suspension; S2, enzymatic conversion: 3g of bovine liver powder is added, and the pH is adjusted to 5.6; the temperature is 29℃, and the reaction is carried out with stirring in the dark for 36h; S3, the temperature is raised to 55℃ and kept at that temperature for 30min to inactivate the β-carotene; S4, the supernatant antitumor composition is obtained by centrifugation. Example 3 A method for enzymatically preparing an antitumor composition from β-carotene and bovine liver powder: S1, raw material pretreatment: bovine liver powder is passed through a 120-mesh sieve; 1g of β-carotene is used to prepare a buffer suspension; S2, enzymatic conversion: 8g of bovine liver powder is added, and the pH is adjusted to 6.8; the temperature is 37℃, and the reaction is carried out with stirring in the dark for 18h; S3, the temperature is raised to 65℃ and kept at 15min for inactivation; S4, the supernatant is collected by centrifugation.

[0017] The efficacy verification will be conducted by Shanxi Cancer Hospital in subsequent in vitro tumor cell inhibition experiments. A blank control group, a pure abscisic acid control group, and the experimental group containing the composition of this invention will be set up to detect the tumor cell proliferation inhibition rate in each group and verify the synergistic tumor-suppressing effect of the composite system. The relevant experimental data will serve as supporting material in response to the examination opinions during the substantive examination stage.

[0018] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Those skilled in the art should understand that any modifications or equivalent substitutions made to the present invention without departing from its principles fall within the protection scope of the present invention.

Claims

1. A method for enzymatically preparing an antitumor composition from β-carotene and bovine liver powder, characterized in that, Includes the following steps: S1. Raw material pretreatment: Pass the bovine liver powder through an 80-120 mesh sieve; take β-carotene powder, mix it with a buffer solution, and stir to prepare a β-carotene suspension; S2. Enzymatic conversion reaction: Add the pretreated bovine liver powder to the β-carotene suspension, adjust the pH of the system to 5.5-7.0; control the reaction temperature at 28-38℃, and stir continuously in the dark for 18-36 hours; S3. Reaction termination: Heat to 55-65℃ and keep warm for 15-30 minutes to inactivate the active enzymes in the bovine liver powder and terminate the lysis reaction; S4. Solid-liquid separation: Centrifuge to remove solid residue, collect the supernatant, and obtain the tumor-inhibiting composition rich in abscisic acid.

2. The preparation method according to claim 1, characterized in that, Raw material ratio: 1 part β-carotene, 3-8 parts beef liver powder.

3. The preparation method according to claim 1, characterized in that, The buffer solution is a phosphate buffer solution.