CN118976123A

CN118976123A - Nutritional targeting composition for releasing sulforaphane and application thereof

Info

Publication number: CN118976123A
Application number: CN202411066339.1A
Authority: CN
Inventors: 李晓龙; 文盛鑫; 蒋莉娜
Original assignee: Shenzhen Fushan Biotechnology Co ltd
Current assignee: Shenzhen Fushan Biotechnology Co ltd
Filing date: 2024-08-05
Publication date: 2024-11-19

Abstract

The present invention relates to a nutritional targeted composition for releasing sulforaphane comprising sulforaphane, myrosinase and alpha-cyclodextrin. The composition can effectively release the sulforaphane and simultaneously reduce the irritation to the oral cavity and the stomach. The invention also relates to the use of said composition for the preparation of a product for the prevention or treatment of a disease or condition that can be prevented or treated with sulforaphane.

Description

Nutritional targeting composition for releasing sulforaphane and application thereof

Technical Field

The invention belongs to the field of biological medicine, and relates to a nutrition targeting composition for releasing sulforaphane and application of the composition.

Background

With the change of dietary structure and living habit of people, the number of patients suffering from chronic non-infectious diseases in various countries is continuously increased, and the coexistence of multiple diseases is more serious. Chronic diseases are one of the main causes of morbidity and mortality in people worldwide, and pose a great threat to human life and health.

Accurate nutrition, or nutrition targeting, is an important idea for chemical prevention of chronic diseases. Nutritional targeting is a goal proposed on the basis of the theory of nutritional genomics. One main idea of nutrition targeting is to activate an Nrf2 (i.e. NF-E2 related factor 2, a transcription factor for regulating antioxidant stress reaction by cells) pathway by using active ingredients (such as glucoraphanin and glucoraphanin) in dietary supplements such as broccoli and the like, and promote the expression of phase II enzyme, thereby playing a corresponding role. Based on the Nrf2 pathway activation effect, the composition can play a role in treating and preventing gastric ulcer and helicobacter pylori infection, and treating and preventing diabetes, cardiovascular diseases, helicobacter pylori infection, autism, schizophrenia, depression, alzheimer disease and other diseases.

Sulforaphane (Sulforaphane, SFN) is 1-isothiocyanate-4-methylsulfonyl butane, also called sulforaphane, belongs to isothiocyanate, is a bioactive substance with the capability of preventing cancer, which is discovered from broccoli, and is the strongest anticancer component discovered in vegetables so far. The molecular action mechanism research and the cell experiment result aiming at the sulforaphane show that the sulforaphane plays a role similar to cancer chemoprevention by regulating and controlling the activity of phase II enzyme to activate the expression of genes related to the oxidation resistance, the metabolic detoxification and the like of a human body on phase I enzyme metabolites or foreign substances (Myzak MC, dashwood RH.cancer Lett.,2006, 233:208-18.). Sulforaphane is an extremely effective Nrf2 inducer, and the main action mechanism is to activate an Nrf2 signal path, regulate the activity of phase II enzymes (NQO 1, glutathione sulfhydryl transferase, gamma-glutamylcysteine synthetase, glucuronyl transferase and the like), regulate antioxidant response elements and the like. The prior art discloses various activities and actions of sulforaphane and its precursor compound sulforaphane, such as the prevention of colon cancer, pancreatic cancer and gastric cancer caused by helicobacter pylori infection (CN 1935003A; CN1170472C; CN 101208079B), nrf2 is a transcription factor regulating the expression of a number of detoxification and antioxidant enzymes. Raphanin and raphanin are known to be antimicrobial activity against gram-positive and gram-negative bacteria and yeasts. On the one hand, investigation on the molecular basis of the action mechanism of sulforaphane shows that sulforaphane and sulforaphane indirectly act as antioxidants by stimulating phase II detoxification enzymes, and have protective effects on Parkinson's disease and have diuretic, antianemic and laxative capabilities through a mouse model. On the other hand, sulforaphane and glucoraphanin, which are sulforaphane-based compounds, have also been demonstrated to have properties against ultraviolet radiation, thereby avoiding sunburn, degradation caused by ROS (reactive oxygen species), and the occurrence (Talalay P.;Fahey J.W.;Healy Z.R.;Wehage S.L.;Benedict A.L.;Min C.;Dinkova-Kostova A.T.PNAS,2007,104,17500-17505;CN104284885B), of skin cancer, which also protects the body from respiratory tract inflammation such as asthma, allergic rhinitis, and Chronic Obstructive Pulmonary Disease (COPD) (Riedl m.a.: saxon a.; diaz-Sanchez d.clinical Immunology,2009,130,244-251). Factor Nrf2 has been shown in recent years to play an important role in growth factor regulation, signaling and tissue repair (in particular, liver regeneration induced by oxidative stress) (Beyer t et al, the EMBO Journal,2008,27,212-223).

In conclusion, based on the above mechanism, it has been found and confirmed that sulforaphane as an isothiocyanate has various activities and functions related to its phase II enzyme modulating action and Nrf2 activating action. With the continuous and intensive research, animal experiments and clinical experiments prove that the sulforaphane has the effect of preventing and/or treating various diseases including diabetes, cardiovascular diseases, helicobacter pylori infection, autism, schizophrenia, depression, alzheimer disease (Alzheimer disease, AD) and the like besides the effect in the field of chronic disease chemoprevention. For example, sulforaphane can reduce hepatic glucose production in type 2 diabetics, reduce blood glucose levels in patients (Axelsson AS et al. Sci tranl med.,2017,9 (394)); Can reduce vascular inflammation in mice, prevent TNF- α -induced adhesion of monocytes to primary epithelial cells (NALLASAMY P ET al., J Nutr biochem.,2014,25 (8): 824-33.); inhibit the colonization of the stomach by helicobacter pylori in mice and humans, reduce inflammation of the stomach caused by infection (Yanaka a et al. Cancer Prev Res (Phila), 2009,2 (4): 353-60); in clinical trials, can reverse abnormal symptoms associated with autism, such as improving oxidative stress, low antioxidant capacity, inhibited glutathione synthesis, attenuating mitochondrial capacity and oxidative phosphorylation and phenomena of lipid peroxidation and neuroinflammation in patients (Singh K, et al, proc NATL ACAD SCI USA,2014,111 (43): 15550-5.); Can effectively improve the cognitive function of patients with schizophrenia (Shiina A, et al, clin Psychopharmacol Neurosci.,2015,13 (1): 62-7.); dietary intake of broccoli sprouts rich in glucoraphanin has a preventive effect on depression (Zhang JC, et al, J Nutr biochem, 2017, 39:134-144); administration of sulforaphane improves cognitive function of beta-amyloid beta-protein, aβ -induced acute AD mouse models in Y maze and passive avoidance behavioral tests (kimhv, et al, amyoid, 2013,20 (1): 7-12.). compared to other direct antioxidants, where each molecule is only able to neutralize another radical molecule and they are destroyed in the process, the antioxidant capacity of sulforaphane is more durable and efficient, as its activation involves genes for protection against any oxidizing agent or carcinogen (epigenetic factor) (Young-Joon s. Science.2003,3,768) furthermore, han Li et al report the effect of sulforaphane on reducing pulmonary fibrosis through the Nrf2 pathway (Han Li, jiang Tao, journal of chinese new drugs and clinics, 2016, 12). It is known that cruciferous plants are the main source of sulforaphane and its precursor compound, glucoraphanin. In cruciferous plants, broccoli (also known as green, etc., from the genus brassica of the family brassicaceae) is the primary source of providing sulforaphane and its precursor compound sulforaphane.

Crucifers also contain other compounds, such as indole glucosinolates (indoleglucosinolates), which are detrimental to the health of the body. These compounds not only have a weak ability to activate detoxification systems, but also can biologically activate systems of certain carcinogens. Thus, broccoli was selected as a starting material while avoiding other disadvantageous compounds (CN 1935003 a) as much as possible.

It is known that the precursor glucoraphanin content is relatively higher in broccoli seeds and sprouts (shoots). Even so, it is not practical to ingest an effective dose of sulforaphane by eating broccoli. Therefore, it is necessary to achieve its effective biological efficacy by extracting broccoli, by means of its extract.

However, it is known in the art that sulforaphane is unstable, easily degradable, and difficult to preserve and use. Therefore, the precursor glucoraphanin is adopted in practical application. Glucoraphanin acts as a precursor substance and is not itself biologically active. When applying glucoraphanin, myrosinase contained in crucifers is required to decompose glucoraphanin to convert glucoraphanin into active glucoraphanin.

However, glucoraphanin itself has bitter taste, severely affecting the compliance of the application; whereas sulforaphane is irritating, especially to the stomach. These all seriously affect the compliance of the product application.

In particular, for the sulforaphane products used in the prior art, in particular for the mixture of myrosinase in solid form and of the glucoraphanin raw material (including the raw material in the form of broccoli and/or its extracts), it produces a greater irritation in the mouth and stomach, in particular the stomach, of the human body when used.

Accordingly, there is a need to overcome the problem of the prior art of the irritation of the human stomach by the glucoraphanin products, and there is a strong need to provide a composition that can effectively release glucoraphanin while reducing the irritation of the product.

Disclosure of Invention

The present inventors have unexpectedly found that in one failed test, no unpleasant taste and flavor sensation was detected in the aqueous solutions of glucoraphanin, myrosinase and alpha-cyclodextrin, and the presence of glucoraphanin was detected. Further studies have unexpectedly found that when the three are placed in water, the presence of sulforaphane is detected and the problem of having a pungent taste that sulforaphane is unacceptable is not present.

Without being limited by theory, the inventors hypothesized at the time that a mixture of glucoraphanin, myrosinase and alpha-cyclodextrin may undergo a glucoraphanin hydrolysis reaction to produce glucoraphanin when contacted with water, and at the same time, the produced glucoraphanin is instantaneously included by the alpha-cyclodextrin after a self-assembly reaction, thereby avoiding the problem of bitter taste of glucoraphanin and the problem of glucoraphanin irritation in the existing products, and thus perfectly solving the technical problems.

Based on this finding, in a first aspect of the present invention, there is provided a non-irritating solid nutritional targeting composition effective to release sulforaphane, the solid composition comprising the following components:

(1) Providing component 1 of glucoraphanin;

(2) Providing component 2 of myrosinase; and

(3) Alpha-cyclodextrin.

In the present invention, the component 1 providing glucoraphanin may be any substance or raw material capable of providing a source of glucoraphanin compounds. Preferably, the component 1 providing glucoraphanin is selected from the group consisting of crucifers, extracts thereof and mixtures thereof. The cruciferous plant is preferably selected from broccoli (briccoli), cauliflower (cauliflower), red cabbage (red cabbage), brussels sprouts (Brussels sprouts) or cabbage, particularly preferably broccoli. The crucifer may be all or a portion of a plant, such as a whole plant, an aerial portion thereof, a flower bulb, a bud, a seed, or a combination thereof. The component 1 providing glucoraphanin may also be an extract of a crucifer, such as a solvent extract, preferably an aqueous extract, an alcoholic extract, a hydroalcoholic extract. In addition to the plant tissue, extract and mixtures thereof of cruciferous plants, component 1 of the present invention that provides glucoraphanin may also include chemically synthesized, semi-chemically synthesized, enzymatically synthesized or biosynthesized glucoraphanin.

In the present invention, the myrosinase-providing component 2 may be any suitable substance or material capable of providing a source of myrosinase. Preferably, the myrosinase-providing component 2 is selected from the group consisting of crucifers, extracts thereof, and mixtures thereof. Preferably, the myrosinase-providing component 2 is selected from horseradish, radish and cabbage. In some preferred embodiments, the myrosinase-providing component 2 is selected from the group consisting of extracts of horseradish, radish, and cabbage; in other preferred embodiments, the myrosinase-providing component 2 is selected from the group consisting of a juice or slurry of horseradish, radish and cabbage, or a powder obtained by drying the juice or slurry.

In the present invention, the broccoli means all or a part of the broccoli plant. Preferably, the broccoli is selected from the edible parts in the general sense; more preferably, the broccoli is selected from the group consisting of broccoli bulbs, broccoli seeds, and broccoli sprouts, and combinations thereof.

In the present invention, the broccoli extract means an extract of all or a part of the broccoli plant, including, but not limited to, an extract of the broccoli, broccoli ball, broccoli seed, and/or broccoli sprout. In another aspect, the extract is an extract obtained by extraction with a solvent, preferably an aqueous extract, an alcoholic extract or a hydroalcoholic extract, particularly preferably an aqueous extract.

In the present invention, the component 1 providing glucoraphanin is preferably selected from the group consisting of broccoli bulbs, broccoli seeds, broccoli sprouts, broccoli extracts and mixtures thereof, and chemically or biologically synthesized forms of glucoraphanin.

In the invention, the mass ratio of the component 1, the component 2 and the alpha-cyclodextrin is (10-80): (1-80): (5-60); preferably (10-40): (1-40): (5-30), more preferably (10-30): (1-10): (5-15).

The solid compositions of the present invention may be in the form of powder, granules, capsules or tablets. More preferably, in the composition of the invention, the component 1, component 2 and alpha-cyclodextrin are all present in solid form. For example, the component 1 may be in the form of a powder (including lyophilized powder) of an extract, sprout or seed; the component 2 may be in the form of a dry powder, a dry extract (including lyophilized powder).

In some preferred embodiments, component 1 of the present invention is selected from the group consisting of broccoli seed extract, broccoli sprout powder, broccoli bulb lyophilized powder, and mixtures thereof.

In another aspect, the present application provides the use of a nutritional targeted composition according to the present application for the preparation of a product for the prevention and/or treatment of a disease or condition that can be prevented and/or treated with sulforaphane. The product may be a pharmaceutical or food product. Preferably, the product is a pharmaceutical product. In some preferred embodiments, the disease or condition associated with the prophylaxis and/or treatment of the available sulforaphane is selected from the group consisting of cancer, diabetes, cardiovascular disease, helicobacter pylori infection, autism, schizophrenia, depression, alzheimer's disease (Alzheimer disease, AD), and pulmonary fibrosis.

In another aspect, the present application provides a method for in vitro conversion of glucoraphanin to glucoraphanin, comprising the steps of:

1) There is provided a solid composition according to the present invention,

2) The composition is mixed with water or an aqueous solution.

In another aspect, the application also provides a method of supplementing a subject in need thereof with sulforaphane comprising administering to the subject a composition according to the application.

In yet another aspect, the present application provides a method of preparing a product that releases sulforaphane and reduces oral and gastric irritation when dissolved in water for use, comprising the step of mixing component 1 providing glucoraphanin, component 2 providing myrosinase, and alpha-cyclodextrin.

The inventors have unexpectedly found that by adding alpha-cyclodextrin to the composition, successful release of sulforaphane can be effectively achieved when administered in admixture with water or an aqueous solution, while reducing its gastric irritation without affecting the release of sulforaphane.

Detailed Description

Example 1: preparation of the inventive composition (powder) and control composition (powder)

Preparation of the composition of the invention: 600g of broccoli seed aqueous extract (containing 13.0% of glucoraphanin from U.S. Brassica Protection Products LLC, the same below), 60g of horseradish powder (myrosinase 30u/mg, the same below) and 300g of alpha-cyclodextrin were mixed uniformly to obtain 0.96kg of composition 1, wherein glucoraphanin accounts for 8.13%. The above composition was packaged in small bags at a weight of 5g per bag to obtain the corresponding powder product (composition of the present invention).

Preparation of control composition: 600g of broccoli seed aqueous extract (containing 13.0% of glucoraphanin), 60g of horseradish powder and 300g of starch are uniformly mixed to obtain 0.96kg of composition 2, wherein the content of glucoraphanin is 8.13%. The above composition was packaged in small bags at a weight of 5g per bag to obtain the corresponding powder product (control composition).

1G of each of the two powder products is taken and dissolved in 40ml of water at 37 ℃ to react to generate the sulforaphane, sampling is carried out at 60min, the content of the sulforaphane in the water solution is measured by HPLC, and the generation rate of the sulforaphane is calculated.

HPLC determination method of sulforaphane: the sample solution was filtered through a 0.45 μm filter and analyzed by HPLC. HPLC conditions: chromatographic column: FIG. Unitary C (4.6 mm. Times.250 mm,5 μm); column temperature: 30 ℃; mobile phase: 70% water-30% acetonitrile; flow rate: 0.8mL/min; sample injection amount: 10. Mu.L; ultraviolet detection wavelength: 245nm.

And freeze-drying the two reaction solutions, and collecting the powder containing the sulforaphane and the sulforaphane alpha-cyclodextrin inclusion compound.

Taking 0.100g of the two powders, respectively washing with 50ml of dichloromethane to remove non-inclusion sulforaphane on the surfaces of the powders, and detecting the non-inclusion sulforaphane content in the washing liquid by HPLC. And respectively dissolving 0.100g of the two powders in 50ml of water, detecting the content of the sulforaphane in the water solution by HPLC, calculating the content of the sulforaphane in the powder of 0.100g, and further calculating the inclusion rate of the sulforaphane (the inclusion rate of the sulforaphane = (1-the content of the sulforaphane in the washing solution/the content of the sulforaphane in the sampling powder) ×100%).

The experimental results are shown in tables 1 and 2 below.

Table 1 comparison of the release of sulforaphane from powder products of the compositions of the invention and of the control compositions

TABLE 2 comparison of the inclusion rate of sulforaphane in powder products of the inventive and control compositions

Powder group	Inclusion rate of sulforaphane (%)
		Inventive compositions	98.1％
Control composition	7.8％

Example 2: preparation of the inventive composition (tablet) and control composition (tablet)

Preparation of the inventive composition: 300g of broccoli seed water extract, 30g of horseradish powder, 150g of alpha-cyclodextrin and 450g of other tabletting auxiliary materials are uniformly mixed, and the tablet is pressed according to the tablet weight of 1 g/tablet, and is coated with a film coating, so that about 0.93kg of the tablet is obtained, wherein the glucoraphanin accounts for 4.19%. The above composition was packaged in 60 tablets per bottle and the bottle mouth was sealed after adding a desiccant to obtain the corresponding tablet product (composition of the present invention).

Preparation of control composition: 300g of broccoli seed water extract, 30g of horseradish powder, 150g of starch and 450g of other tabletting auxiliary materials are uniformly mixed, and the mixture is compressed according to the tablet weight of 1 g/tablet, and is coated with a film coating, so that about 0.93kg of control tablet is obtained, wherein the glucoraphanin accounts for 4.19%. The above composition was packaged in 60 bottles, and after adding a desiccant, the bottle mouth was sealed to obtain a corresponding tablet product (control composition).

2G of each of the two tablet products is taken and dissolved in 40ml of water at 37 ℃ to react to generate the sulforaphane, sampling is carried out at 60min, the content of the sulforaphane in the water solution is measured by HPLC, and the generation rate of the sulforaphane is calculated.

The experimental results are shown in tables 3 and 4 below.

Table 3 comparison of the release of sulforaphane from tablet products of the inventive and control compositions

Powder group	60Min release rate of sulforaphane (%)
		Inventive compositions	61.9％
Control composition	61.1％

Table 4 comparison of inclusion rates of sulforaphane in tablet products of the inventive and control compositions

Powder group	Inclusion rate of sulforaphane (%)
		Inventive compositions	98.5％
Control composition	8.0％

Examples 3-4 in another set of examples, compositions of the invention (powders and tablets) were prepared as in example 1, except that the ratio of the components was varied (300 g broccoli seed aqueous extract: 100g horseradish powder: 150g alpha-cyclodextrin). Similar results were obtained by examining the release rate and inclusion rate of sulforaphane using the same method and conditions as in example 1, i.e., inclusion rate significantly better than the control composition without α -cyclodextrin, while the release rate of sulforaphane was not affected.

Compared with a control composition without alpha-cyclodextrin, the inclusion rate of the sulforaphane of the composition containing the alpha-cyclodextrin is obviously improved by more than 10 times; on the other hand, the released sulforaphane content is close, that is, the releasing rate of the sulforaphane is not affected. The composition of the invention containing the alpha-cyclodextrin and the control composition without the alpha-cyclodextrin are taken by adding water respectively, and the irritation of the composition of the invention containing the alpha-cyclodextrin to the oral cavity and the stomach is obviously reduced, which shows that the alpha-cyclodextrin can include the sulforaphane released by the composition containing the glucoraphanin and the myrosinase in water, so that the irritation of the composition to the oral cavity and the stomach of a human body is reduced, and the release of the sulforaphane is not influenced. Such results are unexpected.

It should be understood that while the present invention has been specifically disclosed by preferred embodiments, those skilled in the art may now make use of the optional features, modifications, improvements and variations of the invention presented herein, and that these modifications, improvements and variations are considered to be within the scope of the invention. The materials, methods, and embodiments provided herein as exemplary preferred example schemes are exemplary and are not intended to limit the scope of the invention.

Claims

1. A solid composition comprising the following components:

1) Providing component 1 of glucoraphanin;

2) Providing component 2 of myrosinase; and

3) Alpha-cyclodextrin.

2. The composition of claim 1, wherein said component 1 is selected from the group consisting of crucifers, extracts thereof, and mixtures thereof.

3. The composition of claim 2, wherein the cruciferous plant is broccoli.

4. A composition according to any one of claims 2 to 3, wherein the cruciferous plant is selected from the group consisting of a flower ball, seed, sprout, and mixtures thereof.

5. The composition according to any one of claims 1 to 4, wherein the mass ratio of component 1, component 2 and alpha-cyclodextrin is (10-80): 1-80): 5-60.

6. The composition of any one of claims 1 to 5 in the form of a powder, granule, capsule or tablet formulation.

7. The composition of any one of claims 1 to 6, wherein the component 1 is selected from the group consisting of broccoli seed extract, broccoli sprout powder, broccoli bulb lyophilized powder, and mixtures thereof, and chemically or biologically synthesized forms of glucoraphanin.

8. The composition of any one of claims 1 to 7, wherein the component 2 is selected from horseradish, radish, cabbage; juice or slurry thereof; and extracts thereof.

9. Use of a composition according to any one of claims 1 to 8 for the preparation of a product for the prevention or treatment of a disease or condition that can be prevented or treated with sulforaphane.

10. The use according to claim 9, wherein the disease or condition which can be prevented or treated with sulforaphane is selected from the group consisting of cancer, diabetes, cardiovascular disease, helicobacter pylori infection, autism, schizophrenia, depression, alzheimer's disease and pulmonary fibrosis.

11. A method for in vitro conversion of glucoraphanin to glucoraphanin, comprising the steps of:

1) The composition according to claim 1 to 8,

2) The composition is mixed with water or an aqueous solution.

12. A method of supplementing a subject in need thereof with a sulforaphane comprising administering to the subject a composition of any one of claims 1 to 8.

13. A method of preparing a product that releases sulforaphane and reduces irritation to the oral cavity and stomach when dissolved in water application comprising the step of mixing component 1 providing glucoraphanin, component 2 providing myrosinase, and alpha-cyclodextrin.