KR101685594B1 - Salt substitutes and salt composition with Zanthoxylum schinifolium extract for activation of TRPV1 - Google Patents

Abstract

The present invention relates to salt substitutes which can increase sodium sensitivity by increasing TRPV1 expression, and to a Zanthoxylum schinifolium salt composition comprising the same. The salt substitutes comprising an aqueous Zanthoxylum schinifolium ethanol solution increase sodium sensitivity by increasing the expression of TRPV1 gene, and reduce ingestion of salt.

Description

[0001] The present invention relates to a salt substitute for increasing the expression of TRPV1, and a salt substitution composition containing Zanthoxylum schinifolium extract for activation of TRPV1,

The present invention relates to a salt substitute which can increase the expression of TRPV1 and increase the sodium sensitivity, and a sancho salt composition containing it.

Salty taste in food is a very important characteristic that adds sweetness and flavor, and this salty taste is the taste of ions formed by dissociation of inorganic and organic alkali salts. Although salt is mainly used to produce such a salty taste, sodium contained in the salt component is known to cause edema and increase in blood pressure when overdosing.

In addition, a team of researchers at the University of Delaware Health Sciences University and Christie AnnerCare HealthSystems said that people who consume too much sodium do not have blood pressure, heart, or kidney function.

In particular, in Korea, foods having a high salt content such as kimchi, soy sauce, and salted fish are traditionally used, and health problems due to sodium are attracting attention. In addition, according to the World Health Organization (WHO), the recommended daily dose of sodium is 2000 mg (equivalent to 5 g of salt), while Korean people consume an average of 5000 mg.

To solve this problem, various attempts have been made to reduce the sodium content. Salt substitutes such as potassium chloride (KCl) or inorganic salt having a salty taste (NaBr, NaI, Na2SO4, NaNO3, etc.) are used as an example. However, these substances have not only salty taste but also bitter taste, It has another problem.

On the other hand, TRPV1 is a receptor that affects taste recognition by recognizing Na ⁺ with ENaC. ENaC has α, β, and γ subunits in rodents. Human has more δ in it, and the protein structure that forms a complex with δ is reduced by more than 10-fold in sensitivity. There is a difficulty. On the other hand, TRPV1 has a single receptor structure that reacts with Na ⁺ , K ⁺ , NH ₄ ⁺ , Ca ^2+, and is known to be easily used in taste recognition research.

Accordingly, the inventors of the present invention intend to develop a food-enhancing food material capable of reducing the intake of sodium by increasing TRPV1 expression without affecting the salty taste of food.

Korean Registered Patent No. 10-1250134 (registered on March 31, 2013) describes a low sodium salt substitute composition comprising potassium lactate and calcium ascorbate. Korean Patent No. 10-0987630 (registered on Oct. 10, 2010) discloses that 25 to 40 wt% of ammonium chloride, 45 to 60 wt% of sodium gluconate, 1 to 5 wt of yeast extract powder And 1 to 10% by weight of DL-alanine (DL-Alanine) and a method for producing the same.

It is an object of the present invention to provide a salt substitute which can increase the sodium sensitivity by decreasing the expression of TRPV1 gene and thereby reduce the intake of salt and a sancho salt composition containing the salt substitute.

In order to achieve the above object, the present invention provides a salt substitute characterized by containing, as a first aspect, an aqueous solution of Zanthoxylum schinifolium ethanol.

Zanthoxylum schinifolium ) is one of the most popular spices in Chinese cuisine. In Korea, it enters Chu-tang and serves to neutralize the bad taste and cold taste of loach. Recently, it has been cultivated in several areas as a major cultivated shrub. Pickled leaves are used as raw leaves, dried or peeled, and then dried or concentrated. In addition, it can be put into food by putting out powder, and it is known that it gives improvement effect by applying oil to burn, dermatitis and atopy by taking oil of sancho.

In the present invention, Zanthoxylum schinifolium extract of the present invention increases the expression of TRPV1 gene, thereby increasing the sensitivity of sodium, thereby allowing the salt to be felt even with a small amount of salt. Thus, the present invention has been completed.

On the other hand, in the salt substitute of the present invention, the aqueous ethanol solution is preferably an aqueous solution of 20 to 50% (v / v) ethanol, more preferably 25 to 35% (v / v) .

On the other hand, in the salt substitute of the present invention, the aqueous ethanol solution is preferably used in an amount of 5 to 20 parts by weight relative to 1 part by weight of anthocyanins.

On the other hand, the present invention provides a sancho salt composition characterized by containing a salt substitute as the second embodiment. At this time, the sancho salt composition of the present invention preferably contains 0.001 to 1 part by weight of salt substitute relative to 1 part by weight of salt. When the above range is satisfied, the expression of TRPV1 gene is increased, so that the sodium sensitivity can be increased to an excellent level, so that even a small amount of salt can be salty.

The Zanthoxylum The salt substitute, which contains an aqueous solution of the schinifolium ethanol extract, increases the sensitivity of TRPV1 gene, thereby increasing sodium sensitivity and reducing the intake of salt.

FIG. 1 shows a process for extracting a sancho extract of the present invention.
FIG. 2 is a graph showing the effect of the acidulant extract of the present invention on the expression of TRPV1 mRNA according to the concentrations of extractive solvents (*: p <0.05, **: p <0.01, ***: p <0.001).
FIG. 3 is a graph showing the effect of the AChR extract of the present invention and other natural extracts on TRPV1, TRPV2, TRPV3, and TRPV4 mRNA expression.
FIG. 4 is a graph showing the effect of sodium reduction by increasing the sodium sensitivity of the acidulant extract. FIG.

Hereinafter, the structure and function of the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited to the following examples, but includes modifications of equivalent technical ideas.

[ Example  1 to 3: Preparation of Acanthopanax senticosus extract]

10 ml of 50% (v / v) ethanol aqueous solution, 30% (v / v) ethanol aqueous solution and 20% (v / v) ethanol aqueous solution were added to 1 g of the dried acid powder, Respectively.

After extraction, the mixture was centrifuged at 6,000 rpm for 1 hour, filtered through a 0.45 μm syringe filter, and lyophilized to obtain 50% (v / v) ethanol aqueous solution extract of Example 1 (V / v) ethanol aqueous solution extract (Example 2) and 30% (v / v) ethanol aqueous solution extract (Example 2) and 20% (v / v) FIG. 1 shows a process for extracting a sancho extract of the present invention. Each sample thus prepared was used in the following Experimental Examples.

[ Experimental Example  1: Measurement of yield of acidolytic extracts by solvent]

In this experimental example, the extraction yield of the acidolytic extract of each solvent was measured.

50 g of anthocyanin was added to 500 ml of 50% (v / v) ethanol aqueous solution, 30% (v / v) ethanol aqueous solution and 20% (v / v) ethanol aqueous solution respectively and extracted at 60 ° C for 12 hours.

After the extraction, the mixture was centrifuged at 6,000 rpm for 1 hour, filtered through a 0.45 μm syringe filter, and lyophilized to determine the weight and extraction yield. The values are shown in Table 1 below.

menstruum Solvent ratio Final weight (g) Yield (%) Purified water ethanol 50% (v / v) aqueous ethanol solution 5 5 2.9766 6.0 30% (v / v) aqueous ethanol solution 7 3 4.7417 9.5 20% (v / v) aqueous ethanol solution 8 2 3.7608 7.5

The results showed that the yield of aqueous ethanol solution of 30% (v / v) was about 9.5% and the yield of ethanol aqueous solution was about 7.5% and 50% (v / v) 6.0%, respectively.

[ Experimental Example  2: Sancho extract In extraction solvent  Following TRPV1  Measurement of Expression Level]

In this experiment, the amount of TRPV1 expression was determined according to the extraction solvent of the acantholyticus extract. Specific experimental conditions and methods are shown below.

(One) HepG2  cell culture

Hepatocellular carcinoma (HepG2), a hepatocellular carcinoma cell line, was purchased from Korean Cell Line Bank (KCLB).

HepG2 cells were cultured in medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a medium of minimum essential medium (MEM) at 37 ° C and 5% CO ₂ Lt; / RTI &gt;

(2) In extraction solvent  Treatment of Acanthopanax senticosus

The cultured HepG2 cells were supplemented with 50% (v / v) ethanol aqueous solution extract (Example 1), 30% (v / v) ethanol aqueous solution extract (Example 2) (v / v) ethanol aqueous solution extract (Example 3) were treated at concentrations of 0.1 mg / ml, 0.25 mg / ml, 0.5 mg / ml and 1 mg / ml, respectively.

(3) RT- PCR  Search for used gene expression

Extraction of RNA in cells

After cell culture, the medium was removed. Then, 1 ml of 'QIAzol Reagent' was added to each culture well and mixed several times with a pipette to obtain an RNA extraction sample from the cultured cells.

The RNA extraction sample was allowed to stand at room temperature for 5 minutes, and 0.2 ml of chloroform per 1 ml of 'QIAzol' solution was added. After shaking for 15 seconds, the reaction solution was further reacted at room temperature for 2 to 3 minutes. Centrifugation (12,000xg) at 4 ° C for 15 minutes was performed to separate the uppermost RNA, intermediate DNA, and protein layer.

The RNA layer was carefully separated and transferred to another tube carefully, so that the DNA was not contaminated. The RNA was precipitated by adding 0.5 ml of isopropyl alcohol and reacting for 10 minutes. After centrifugation (12,000 × g) at 4 ° C for 10 minutes, the supernatant was discarded and 1 ml of 75% DEPC-ethanol was added to the precipitate. After shaking a few times, it was again centrifuged (12,000 x g) at 4 ° C for 5 minutes.

After centrifugation, the supernatant was discarded, and the precipitate was dried at room temperature for 5 to 10 minutes, and then the precipitate was dissolved with RNase-free water or 0.1% DEPC distilled water. 5 μl of RNA was added to 995 μl of 0.1% DEPC distilled water, diluted 200 times, and absorbance was measured at 260 nm and 280 nm.

The concentration of RNA was calculated as 'absorbance value × 40 ㎍ / mL × 200 (dilution factor)' at 260 nm. When the ratio between 260 nm and 280 nm was 1.6 or more, the RNA was considered to be pure RNA and the next step was carried out.

○ cDNA synthesis

In order to produce 'first-stand cDNA', 9 μl of the quantified RNA was placed in a 'PCR tube', heated at 75 ° C for 5 minutes, and immediately cooled on ice. 1 μl of 'enzyme mix' and 10 μl buffer were mixed with the 'amfirivert platinum cDNA synthesis master mix kit' into the cooled tube. Thereafter, the mixture was heated at 25 DEG C for 5 minutes, at 42 DEG C for 60 minutes, and at 70 DEG C for 15 minutes, and then cooled to synthesize cDNA.

○ PCR

PCR was performed to confirm mRNA expression of TRPV1. ^{'GoTaq (R) Green Master Mix} ', 2X (Promega, USA) 10 ㎕, 'Forward Primer' (15μM) and 'Reverse Primer' respectively 1 ㎕ the (15μM), distilled water 7 ㎕ and synthesized before 'first-strand 1 μl of cDNA (DNA templet) 'was put into' PCR tube 'and mixed.

Each mixture was 'pre-denatured' at 95 ° C. for 5 minutes and then 30 cycles were performed at 95 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 30 seconds. Finally, extension at 72 ° C. for 5 minutes (extension) reaction.

The PCR product was electrophoresed on a 1.2% agarose gel containing 0.002% ethidium bromide for 30 hours at 80 V, and the degree of gene expression was determined by ultraviolet light. The intensity of the band was analyzed and quantified by 'image J' software, and β-actin was used as an internal standard. The primer sequences of TRPV1 and beta-actin are shown in Table 2 below.

Gene Primers Size Forward Reverse TRPV1  CAG CCA CCT CAA GGA GTA TG GCC TGA AAC TCT GCT TGA CC 766 β-actin  CGA AAC TAC CTT CAA CTCCA GCA ACT AAG TCA TAG TCC GCC 324

(V / v) ethanol aqueous solution extract (Example 2), 'sancho 20% (v / v) ethanol aqueous solution extract' (Example 3) showed an increase in TRPV1 mRNA expression at all concentrations. However, the highest TRPV1 mRNA expression was found to be 150.61% at the concentration of 0.5 mg / mL of '20% (v / v) ethanol aqueous solution of Sancho 20% (Example 3)' (FIG. 2). FIG. 2 is a graph showing the effect of the acidulant extract of the present invention on the expression of TRPV1 mRNA according to the concentrations of extractive solvents (*: p <0.05, **: p <0.01, ***: p <0.001).

From the results of the above experiment, it can be concluded that the ethanol extract of the present invention may increase the sensitivity of sodium by increasing the expression of TRPV1 mRNA. Thus, it is considered that the acidophilus extract of the present invention can reduce the salt intake.

Meanwhile, in order to improve the commercial utilization of the sodium-reduced material using the acidophilus extract in the future, the following Experimental Example shows that the extract of '30% (v / v) ethanol aqueous solution of Sancho is effective in increasing the extraction yield and increasing the expression of TRPV1 Example 2) was used.

[ Comparative Example  1 to 4: Preparation of natural (ginger, cinnamon, mustard, red pepper) extracts]

10 g of a 30% (v / v) aqueous ethanol solution was added to 1 g of dried ginger, cinnamon, mustard, and pepper powder, and the mixture was extracted at 60 ° C for 12 hours.

After extraction, centrifugation was performed at 6,000 rpm for 1 hour. (Comparative Example 1), 'Cinnamon Extract' (Comparative Example 2), 'Mustard Extract' (Comparative Example 3) and 'Capsicum Extract' (Comparative Example 2) were obtained by filtration using a 0.45 μm 'syringe filter' (Comparative Example 4) were each prepared (see Fig. 1).

On the other hand, these samples were referred to as Comparative Example 1, Comparative Example 2, Comparative Example 3 and Comparative Example 4, respectively, and used in the following experimental examples.

[ Experimental Example  3: etc  Of natural extract TRPV1 , TRPV2 , TRPV3 , TRPV4  Comparison of expression level]

In this experimental example, TRPV1, TRPV2, TRPV3 and TRPV4 expression levels (degree of expression) of the sample of Example 2 and Comparative Examples 1 to 4 were measured. The specific experiment was carried out by the method of Experimental Example 2 above. However, the primer sequences of TRPV1, TRPV2, TRPV3 and TRPV4 are shown in Table 3 below.

Gene Primers Size Forward Reverse TRPV1  CAG CCA CCT CAA GGA GTA TG GCC TGA AAC TCT GCT TGA CC 766 TRPV2  TTT CCT GGC CAT CGA GTG G ATC CTC ATC CTC CTT GGG AGG 779 TRPV3  GTT TGC CTA CAA AGA GTA CCT C CAT TGA GAG TGG TTT TGC TGT TG 740 TRPV4  GGC AGG GAT CGA GGC CTA C CCA CCG AGG ACC AGC GAT C 778 β-actin  CGA AAC TAC CTT CAA CTCCA GCA ACT AAG TCA TAG TCC GCC 324

As a result of the experiment, it was confirmed that the 'extract of aqueous solution of 30% (v / v) ethanol aqueous solution of sancho (Example 2)' of the present invention highly enhanced the expression of TRPV1 and TRPV2 mRNAs in comparison with Comparative Examples 1 to 4 (FIG. FIG. 3 is a graph showing the effect of the acidoligosaccharide extract and other natural extracts of the present invention on the expression of TRPV1, TRPV2, TRPV3, and TRPV4 mRNA.

From these results, it was confirmed that the acidosis extract of the present invention can further increase the sensitivity of sodium to ginger, cinnamon, mustard and pepper extracts.

[ Experimental Example  4: Sensory evaluation]

In this experiment, 10 sensual agents with superior ability to discriminate the taste difference were selected and analyzed. First, the descriptive terms for each sample were derived, and the standard form for the terms was presented and eight training sessions were conducted. The reference samples and sample concentrations for training are shown in Table 4 below.

division Standard form Sample concentration Salty taste NaCl 0.1%, 0.2%, 0.4%, 0.8%, 1.6% sweetness Sugar 0.1%, 0.2%, 0.4%, 0.8%, 1.6% Sour taste Citric acid 0.1%, 0.2%, 0.4%, 0.8%, 1.6% bitter Caffeine 0.1%, 0.2%, 0.4%, 0.8%, 1.6% Richness MSG 0.001%, 0.002%, 0.004%, 0.008%, 0.016%

The emotionally recognized common sense of taste terms was recalled from memory. In the course of the training, the reference score was presented based on the consensus of the personnel and the strength was measured using the 15 - point linear scale method. The standard forms and reference points used for taste evaluation are shown in Table 5 below.

division Term Definition Sample concentration (%) Benchmark Salty taste The taste of NaCl 0.1 2 0.4 6 0.8 10 sweetness Sucrose flavors 0.1 2 0.4 6 0.8 10 Sour taste Citric acid taste 0.1 2 0.4 6 0.8 10 bitter Caffeic acid taste 0.1 2 0.4 6 0.8 10 Richness MSG flavor 0.001 2 0.004 6 0.008 10

Thereafter, a 5% salt aqueous solution was prepared as a standard foam, and 0.2%, 0.4%, 0.6%, 0.8% and 1.0% of the acid extracts were mixed with a 1% salt solution as a comparative solution.

Using a '30% (v / v) aqueous solution of aqueous ethanol solution of sancho (Example 2)', descriptive analysis and sensory evaluation were carried out.

Salt is added to each salt aqueous solution (comparison solution) and salt is added until the salty taste becomes similar to the standard form. The salt added was recorded, and the amount of the added acid extract was confirmed.

On the other hand, statistical significance of the results obtained by the experiment was expressed as mean ± SD using the 'Graphpad Instat program', and the statistical significance of the mean difference of each experimental group was analyzed using 'one-way ANOVA test' And the significance between the experimental groups was verified.

As a result of the experiment, it was found that the taste of 3.5 g of salt corresponding to 5 g of salt and the taste of 0.0175 g of the acid extract (0.5 wt% based on the added salt) showed the same value. When the amount of the acid extract was constant, The intensity felt was proportional to the amount of salt. In addition, through the enhancement of taste recognition by using the sancho extract, the amount of salt was reduced by about 30% or more (FIG. 4). FIG. 4 is a graph showing the effect of sodium reduction by increasing the sodium sensitivity of the acidulant extract. FIG.

Claims (5)

delete delete delete delete (A) adding 10 parts by weight of a 30% (v / v) ethanol aqueous solution to 1 part by weight of Zanthoxylum schinifolium and extracting it at 60 ° C for 12 hours to prepare an aqueous 30% And
(B) preparing an acid-fast-acid salt composition by adding 0.005 part by weight of an aqueous solution of 30% ethanolic aqueous solution of acacia to 1 part by weight of salt,
Wherein the acidic salt composition increases gene expression of TRPV1 and TRPV2.

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