KR102120005B1 - A composition having anti-oxidation or anti-bacterial activity comprising Glehnia littoralis extracts - Google Patents

Abstract

The present invention relates to an antimicrobial and antioxidant composition containing a Glehnia littoralis extract. More specifically, the present invention relates to a composition in which an ethyl acetate fraction of a Glehnia littoralis leaf ethanol extract containing Kaempferol 3-O-β-rutinoside, Cynarin, Spiraeoside, and Purpurin has an antibacterial and antioxidant effect.

Description

A composition having anti-oxidation or anti-bacterial activity comprising Glehnia littoralis extracts}

The present invention relates to an antimicrobial and antioxidant composition comprising an ethyl acetate fraction of ethanol extract of Gaetbangpung leaf as an active ingredient.

Oxygen is an essential element in various metabolic reactions to maintain life, and it is also necessary for detoxifying poisons in the human body, but oxygen is not only beneficial to the human body, so various pollutants such as enzymes, reducing metabolism, chemicals, and photochemical reactions in the body, and physical and chemical When it is converted into free radicals with very high reactivity such as superoxide radical, hydroxyl radical, hydrogen peroxide, and singlet oxygen due to environmental factors, etc. It has a double-sided effect that causes fatal oxygen toxicity in the body ( Korea J. Biotechnol . Bioeng . , 2001, 16(6), 592-602). Synthetic antioxidants such as BHT (butylated hydroxy toluene) and BHA (butylated hydroxy anisole), which are synthetic antioxidants for removing these free radicals, have been widely used so far due to their excellent antioxidant effect and economic efficiency. Due to the issue of, the allowed food or consumption is strictly limited.

In addition, in order to maintain the quality of products such as food, cosmetics, textiles, and medicines for a long time, a preservative to prevent spoilage by microorganisms is essential, and especially in cosmetics, the shelf life is relatively long and in the case of cosmetics with many microbial nutrients, the preservative is more Is required. The risk factor is always latent compared to the economics of synthetic raw materials, as raised through the recent'oxygen incident', and the problem caused when the risk factor is realized is fatal to evaluate profits based on simple arithmetic statistics.

As a conventional preservative, paraben-based preservatives, which are most commonly used in cosmetics, food, and pharmaceuticals, have been suggested to cause skin allergies and resistant bacteria. To solve these problems, research on natural preservatives with excellent product safety and economic efficiency Is going on. Natural preservatives (preservatives) are additives used for long-term preservation and extended shelf life in food and cosmetics, and refer to bio-derived materials used to prevent spoilage by microorganisms, and chemical synthetic preservatives are used and used due to the risk of continuous use. Despite the fact that the scope is strictly limited, there are constant concerns about various side effects such as chronic and acute toxicity, mutation, and carcinogenesis.

Meanwhile, Glehnia littoralis Fr. Schm.) is a perennial plant belonging to the mountain type (Umbelliferae). It is a medicinal plant that grows on sandy beaches of Korea and is distributed in Japan, Manchuria, and China. Its roots are called Buksasam, Binbangpung or Liberation Wind, and the effects of Yangeum, Chungcheong, Geodam, and Jihae Therefore, it has been traditionally used as an anti-inflammatory, antitussive agent and digestive contract. In the case of a number of studies previously published as an academic study of Gaetbangpung, components mainly belonging to coumarin, polyacetylnene, lignan, and monoterpenoids have been reported from the roots, which are the main medicinal sites of Gaetbangpung, and have major pharmacological activities such as antioxidant, anti-inflammatory, and anti-inflammatory properties. Microbial activity has been reported. On the other hand, in other sites, the recent studies of Gaetbang wind fruit and outpost have reported the presence of monoterpenoid, alkyl glycoside, aromatic glycoside, and pyranocoumarins.

Gaetbangpung contains a lot of beneficial ingredients for the human body in the outpost, and it is preferable to use the root part because it contains more medicinal ingredients in the root, but the root has a stronger bitter taste than foliage, so it can be consumed as it is or added to food. In this case, it may cause a feeling of rejection of the ingested person, and also, when extracting the useful component of the root with water, it takes a long time and the extraction amount of the useful component is small, and the useful component of the root has a problem of low absorption rate to the human body when ingested.

As described above, Gaetbangpung, which contains a large amount of natural human useful ingredients, has been mainly used as a medicinal herb with strong medicinal roots, and stems and leaves are used in a form that is simply consumed without special processing. In the current state of registration of raw materials from the Ministry of Food and Drug Safety, Gaetbangpung permits young petiole for edible use, so it is necessary to identify the components and functional properties of the leaves, but little research has been done on this.

Korean Registered Patent No. 1,413,363 Korean Registered Patent No. 1,867,480 Korean Registered Patent No. 1,610,267 Korean Registered Patent No. 1,787,566

Therefore, the present invention was devised to solve the above problems, and the problem to be solved of the present invention is that the ethyl acetate fraction of Gaetbangpung leaf ethanol extract, which is a natural product having excellent antioxidant or antibacterial activity, has no side effects on the human body. It is to provide a composition comprising.

Specifically, the problem to be solved of the present invention is that the present invention is excellent in antimicrobial activity against food poisoning bacteria including gram-positive and negative bacteria, and is safe and excellent without side effects on the human body. It is to provide a composition comprising an ethyl acetate fraction as an active ingredient.

However, the technical problems to be achieved in the present invention are not limited to the technical problems mentioned above, and other technical problems that are not mentioned are clearly apparent to those skilled in the art from the following description. Will be understandable.

In order to achieve the above technical problem, the present invention provides an antibacterial and antioxidant composition comprising an ethyl acetate fraction of ethanol extract of Gaetbangpung leaf as an active ingredient.

In one embodiment, the fraction contains camperol 3-O-β-rutinoside, Cynarin, Spiraeoside and Purpurin can do.

In one embodiment, the ethanol extract of Gaetbangpung leaf can be extracted with a 70% by weight ethanol solvent.

In one embodiment, the ethyl acetate fraction is Bacillus cereus ( B. cereus ), Staphylococcus aureus ( S. aureus ), Listeria ( L. monocytogenes ), Clostridium perfringens ( C. perfringens ), Salmonella typhimurium It may have an inhibitory activity of S. Typhimurium, E. coli , V. parahaemolyticus , C. jejuni , and C. coli .

In one embodiment, the ethyl acetate fraction may have a total polyphenol content of 102.15±0.53 μg GAE/mg and a total flavonoid content of 219.73±0.76 μg CE/mg.

In one embodiment, the ethyl acetate fraction has a reducing power (Oyaizu method) of 0.14±0.00 EC ₅₀ mg/mL, DPPH radical scavenging activity (Riaz Blois method) of 95.75±0.15%, and TAC (Harisa method) of 936.37. ±18.33 CRE, FRAP (Boufadi method) may be 89839.08±283.46 μM.

The antimicrobial and antioxidant composition according to the present invention has excellent anti-oxidant effect, and has excellent characteristics of growth inhibitory activity against causative bacteria such as food poisoning, and thus prevents, improves or prevents diseases caused by oxidative action and microbial infection or It can be usefully used for food and medicine for treatment.

In addition, the antimicrobial and antioxidant composition according to the present invention has the effect of extending the storage period of various foods due to its antioxidant and antibacterial activity, helping to maintain the health of the intake, as well as increasing the preference of various foods.

However, the effects obtainable in the present invention are not limited to the above-mentioned effects, and other effects not mentioned will be clearly understood by those skilled in the art from the following description. Will be able to.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art to which the present invention pertains may easily practice. However, since the description of the present invention is only an example for structural or functional description, the scope of the present invention should not be interpreted as being limited by the examples described in the text. That is, since the embodiments can be variously changed and have various forms, it should be understood that the scope of the present invention includes equivalents capable of realizing technical ideas. In addition, the purpose or effect presented in the present invention does not mean that a specific embodiment should include all of them or only such an effect, and the scope of the present invention should not be understood as being limited thereby.

The meaning of the terms described in the present invention should be understood as follows.

Terms such as "first" and "second" are for distinguishing one component from other components, and the scope of rights should not be limited by these terms. For example, the first component may be referred to as the second component, and similarly, the second component may also be referred to as the first component. When a component is said to be "connected" to another component, it should be understood that other components may exist in the middle, although they may be directly connected to the other component. On the other hand, when a component is said to be "directly connected" to another component, it should be understood that no other component exists in the middle. On the other hand, other expressions describing the relationship between the components, that is, "between" and "immediately between" or "neighboring to" and "directly neighboring to" should be interpreted similarly.

Singular expressions are to be understood to include plural expressions unless the context clearly indicates otherwise, and terms such as "comprises" or "have" include the features, numbers, steps, actions, components, parts or components described. It is to be understood that a combination is intended to be present, and should not be understood as pre-excluding the existence or addition possibility of one or more other features or numbers, steps, actions, components, parts or combinations thereof.

All terms used herein have the same meaning as generally understood by a person skilled in the art to which the present invention pertains, unless otherwise defined. The terms defined in the commonly used dictionary should be interpreted to be consistent with meanings in the context of related technologies, and cannot be interpreted as having ideal or excessively formal meanings unless explicitly defined in the present invention.

The antimicrobial and antioxidant composition of the present invention includes an ethyl acetate fraction of ethanol extract of Gaetbangpung leaf as an active ingredient.

As the extraction method using ethanol, it is preferable to use shaking extraction, Soxhlet extraction, or reflux extraction, but is not limited thereto. It is preferable to extract the methanol by adding 1 to 10 times to the amount of washed, dried, and powdered leaves. The extraction temperature is preferably 20°C to 100°C, more preferably 20°C to 60°C, and most preferably 50°C, but is not limited thereto. In addition, the extraction time is preferably 4 to 24 hours, more preferably 5 to 12 hours, and most preferably 6 hours, but is not limited thereto. In addition, the number of extractions is preferably 1 to 5 times, more preferably 2 to 4 repetitions, and 3 times is most preferred, but is not limited thereto. At this time, ethanol is preferably 70% by weight.

The ethanol extract may further include a step of filtration, concentration under reduced pressure, and drying. It is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator, but it is not limited to this. In addition, the drying is preferably dried under reduced pressure, vacuum drying, boiling drying, spray drying or freeze drying, but is not limited thereto.

After the ethanol extract was suspended in water, a fraction extracted with ethyl acetate is obtained. The fractionation process may be obtained by repeating 1 to 5 times, preferably 3 times, and it is preferable to concentrate under reduced pressure after fractionation, but is not limited thereto.

The ethylacetate fraction of the ethanol extract of Gaetbangpung leaf according to the present invention was separated to identify the contained substance, and as a result, camperol 3-O-β-rutinoside (Kaempferol 3-O-β-rutinoside), cinnarin (Cynarin) , It was confirmed that it contains Spiraeoside and Purpurin.

The term "antioxidant" of the present invention refers to an action of inhibiting oxidation, and the human body has a balance of an oxidizing accelerator (prooxidant) and an antioxidant (antioxidant). When it is achieved and tilted toward the oxidative acceleration, oxidative stress is induced, causing potential cell damage and pathological diseases. Reactive oxygen species (ROS), which are the direct cause of these oxidative stresses, are unstable and highly reactive, so they can easily react with various biological materials, attack polymers in the body, and irreversibly damage or mutate cells and tissues. It causes cytotoxicity and carcinogenesis. Reactive nitrogen species (RNS) such as NO, HNO ₂ and ONOO ^- are produced in large quantities due to the immune response of macrophage neutrophils and other immune cells during an inflammatory reaction, and ROS is also generated. The free radicals as described above oxidize and destroy cells in the body, and thus are exposed to various diseases.

Reducing power, DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity measurement, TAC (total antioxidant capacity) and to confirm the antioxidant effect of ethyl acetate fraction of ethanol extract of Gaetbangpung leaf in a specific embodiment of the present invention As a result of measuring the FRAP (ferric reducing antioxidant potential) value, it was confirmed that it shows a strong antioxidant effect. In addition, it was confirmed that the total polyphenol content and the total flavonoid content are also excellent.

In addition, the term "antibacterial" of the present invention refers to the action of inhibiting or controlling the growth and proliferation of microorganisms such as bacteria and fungi, the microorganism of the present invention is not limited thereto, but Bacillus cereus ( B. cereus ) , Staphylococcus aureus (S. aureus), Listeria (L. monocytogenes), Clostridium perfringens (C. perfringens), S. typhimurium (S. typhimurium), Escherichia coli (E. coli), Vibrio (V there can be parahaemolyticus), Campylobacter your Jeju (C. jejuni), and Campylobacter coli (C. coli).

In a specific embodiment of the present invention, in order to confirm the antibacterial effect of the ethyl acetate fraction of the ethanol extract of Gaetbangpung leaf, it was confirmed that the antibacterial activity against the strains was excellent, and the growth inhibition activity in the strain was excellent.

Hereinafter, preferred examples are provided to help understanding of the present invention, but the following examples are merely illustrative of the present invention, and it is apparent to those skilled in the art that various changes and modifications are possible within the scope and technical scope of the present invention. It is also natural that such modifications and modifications fall within the scope of the appended claims.

Example 1: Preparation of the fraction of Gaetbangpung leaf extract

Freeze-dried young leaves of Gaetbangpung, then extract and filter with 70% ethanol (EE), n-hexane (HF), ethylacetate (EAF), butanol (BF) , Fractions were prepared by sequentially sequencing while increasing the polarity of the solvent with water (H ₂ O, WF).

Experimental Example 1: Food poisoning bacteria inhibitory activity

Antibacterial activity was measured using the Disc diffusion method. At this time, a disc of 6 mm was used, and the fraction concentration of each extract was performed at 100 μg/disc.

Indicator
microorganism Inhibition zone (mm ± SD) EE HF EAF BF WF Gram (+) B. cereus 6.81±0.32 ND ¹ 7.27±0.27 ND ND S. aureus ND ND 7.80±0.41 ND ND L. monocytogenes 8.22±0.08 10.45±0.35 9.80±0.02 ND ND C. perfringens 6.81±0.01 7.79±0.03 7.91±0.11 8.24±0.05 7.11±0.01 Gram (-) S. Typhimurium ND ND 7.83±0.31 ND ND E. coli ND 7.18±0.52 7.53±0.14 ND ND V. parahaemolyticus ND 7.00±0.15 7.97±0.31 7.26±0.10 ND C. jejuni 6.82±0.01 7.79±0.13 7.91±0.01 8.24±0.05 7.11±0.01 C. coli 7.15±0.06 8.05±0.11 8.55±0.43 8.96±0.46 7.57±0.23 Data are mean ± SD ( n = 3).
¹ ND, not detected (inhibition zones with values ≤7 mm were assumed as being absent of antimicrobial activity).

Table 1 shows the results of confirming the antimicrobial activity of nine food poisoning bacteria of each solvent fraction of the ethanol extract of Gaetbangpung leaf. It can be seen that the fractions of each solvent show activity against both Gram-positive and negative bacteria, and in particular, showed the strongest activity against L. monocytogenes .

Among them, the ethyl acetate fraction of ethanol extract from Gaetbangpung leaves showed strong activity against all Gram positive and negative bacteria.

Experimental Example 2: Antioxidant activity

1) Measurement of antioxidant content

The total polyphenolic content and total flavonoid content of the fractions of the ethanol extract of Gaetbangpung leaf were measured.

Total polyphenol content was analyzed using the Folin-Ciocalteu method (Dewanto, Wu, Adom, and Liu, 2002). After oxidation by adding 5 mL Folin-Ciocalteu's reagent (Sigma Co., St. Louis, MO) to 200 μL of the sample. Neutralize by adding 3 mL of 5% sodium carbonate. Thereafter, after 30 minutes of incubation at room temperature in the cow, the absorbance of each sample was measured at 725 nm. The standard curve was used to calculate the content after preparing a calibration curve using gallic acid (Sigma Co.).

Total flavonoid content was analyzed using a colorimetric method (Dewanto, Wu, Adom, and Liu, 2002). 500 μL of sample is mixed with 75 μL of 5% NaNO ₂ and reacted for 5 minutes at room temperature. Then, 150 μL of 10% AlCl ₃ ·6H ₂ O was added to the mixture, followed by 0.5 mL of 1 M NaOH and 275 μL of deionized water. The absorbance of the sample was measured at 510 nm. Total flavonoid content was expressed in μg catechin equivalent (mg) per mg sample.

Total polyphenolic content
(μg GAE ¹ /mg) Total flavonoid content
(μg CE ² /mg) EE 24.91 ± 0.63 ^d 35.85 ± 0.44 ^d HF 50.62 ± 1.08 ^c 80.54 ± 1.07 ^c EAF 102.15 ± 0.53 ^a 219.73 ± 0.76 ^a BF 54.99 ± 0.21 ^b 122.75 ± 2.46 ^b WF 10.47 ± 0.21 ^e 4.75 ± 0.11 ^e Data are shown as the mean ± SD (n = 3).
^ae Different letters within a column differ significantly, based on Duncan's multiple-range test (P <0.05).
¹ : Presented as the number of μg GAE (gallic acid equivalent).
² : Presented as the number of μg CE (catechin equivalent).

As shown in Table 2 above, it can be seen that the total polyphenol content of the ethyl acetate fraction of the ethanol extract of Gaetbangpung leaf is 2 to 10 times higher than that of other solvent fractions. In addition, it was confirmed that the total flavonoid content of the ethyl acetate fraction was also superior to other solvent fractions.

2) Antioxidant activity measurement

The antioxidant activity was measured using the following four methods.

[How to measure]

① Reducing power: Oyaizu method

② DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity measurement: Blois method

③ TAC (total antioxidant capacity): Harisa method

④ FRAP (ferric reducing antioxidant potential) value measurement: Boufadi method

Reducing power (EC ₅₀ mg/mL) DPPH
(%) TAC
CRE ¹ (μM) FRAP
FIE ² (μM) EE 0.74±0.05 ^c 91.39±0.32 ^ab 87.77±0.87 ^c 7637.07±39.23 ^c HF 1.46±0.04 ^a 34.75±1.47 ^d 15.57±0.11 ^d 2164.76±28.71 ^d EAF 0.14±0.00 ^e 95.75±0.15 ^a 936.37±18.33 ^a 89839.08±283.46 ^a BF 0.27±0.01 ^d 88.77±7.52 ^b 626.77±5.90 ^b 31945.98±523.88 ^b WF 1.37±0.08 ^b 63.64±2.45 ^c 20.40±0.03 ^d 1881.52±16.93 ^d Ascorbic acid 0.10±0.00 90.16±0.13 - - Data are shown as the mean ± SD (n = 3).
^ae Different letters within a column differ significantly, based on Duncan's multiple-range test (P <0.05).
¹ Copper reducing equivalent.
² Fe ^{2 +} iron equivalent.

As shown in Table 3, it was confirmed that the four antioxidant activities of ethyl acetate fraction of Gaetbangpung leaf ethanol extract are superior to other solvent fractions, and the reducing power and DPPH activity are superior even when compared with commercially available ascorbic acid.

Experimental Example 3: Cytotoxicity evaluation (in vitro)

Cytotoxicity was evaluated by performing a CCK-8 assay using HepG2 cell, a liver cancer cell. At this time, the cell density of the cell treated for 24 hours is 5×10 ⁴ and the sample (a fraction of each solvent) is treated at a concentration of 50, 100, 200, 400 μg/mL for 24 hours, and the CCK-8 reagent is 4 Treated for hours.

Cytoxicity (%) 50 μg/mL 100 μg/mL 200 μg/mL 400 μg/mL EE 0.96 7.20 9.23 41.69 HF 0.00 2.53 26.81 44.20 EAF 2.49 5.50 25.85 29.87 BF 3.08 14.06 23.63 33.87 WF 0.00 1.23 26.06 56.15

As shown in Table 4, the cytotoxicity of the ethyl acetate extract of the ethanol extract of Gaetbangpung leaf showed a survival rate of 70% or more at a concentration of 400 μg/mL, and a cell survival rate of 90% or more at a concentration of 100 μg/mL of food poisoning control. Did.

Example 2: Ethyl Acetate of Gaetbangpung Leaf Ethanol Extract Fraction Identification of 4 major compounds

Dry gyebangpung leaves (500 g) were finely ground with a blender and extracted three times for 24 hours in 70% ethanol. Then, the 70% ethanol extract was concentrated under reduced pressure to obtain a 70% ethanol concentrate. After dissolving the concentrate in 500 mL of distilled water, liquid-liquid extraction (3 times, 1:1 (v:v)) was performed with a low-polarity solvent (n-Hexane, Ethyl acetate, Butanol, Water). Each extracted solvent layer was collected, concentrated under reduced pressure, and analyzed using Ethyl acetate fraction.

Ethyl acetate fraction analysis of Gaetbangpung was performed using an ultra high performance liquid chromatography (UPLC) system. Separation was performed using an Acquity UPLC HSS T3 column (100 mm×2.1 mm, 1.8 μm, Waters) at a column temperature of 40° C. and a flow rate of 0.5 mL/min. As the mobile phase, solvent A (water + 0.1% formic acid) and solvent B (acetonitrile + 0.1% formic acid) were used. The solvent composition ratio is 0 to 5 min (solvent A 97%), 5 to 16 min (solvent B, 3 to 100% liner gradient), 16 to 17 min (solvent B 100%), 17 to 19 min (3 to 100%) reverse liner gradient solvent B), 19-25 min (97% solvent A) and injected 5 μL of each sample. The material eluted from the column was analyzed in positive and negative ion mode of a high-resolution tandem mass spectrometer SYNAPT G2 Si HDMS QTOF (Waters) instrument. In positive mode, the capillary voltage was 2 kV and the cone voltage was 40 V, and in the negative mode, the capillary voltage was 1 kV and the cone voltage was 40 V. The scan range was 50 to 1200 Da and the scan time was 0.2 s.

All analyzed mass spectrometry data were subjected to chromatogram sorting, peak picking, peak area extraction, and normalization using commercial software UNIFI (version 1.71; Waters Corporation, Milford, MA). For accurate mass spectrometry, Leucine enkephalin was used as an external standard to perform real-time mass value correction.

As a result of the analysis, the following four main compounds were identified, and their structural confirmation data are shown in Table 5 below.

① Camperol 3-O-β-rutinoside

② Cynarin

③ Spieoside

④ Purpurin

Substance name Molecular formula constitutional formula Molecular Weight
(g/mol) Neutral
mass (Da) Observed neutral mass (Da) Mass error (mDa) Observed
RT (min) Observed m/z Adducts Kaempferol
3-O-β-
rutinoside C ₂₇ H ₃₀ O ₁₅

594.522 594.15847 594.1575 -1.6 4.79 593.1503 -H Cynarin C ₂₅ H ₂₄ O ₁₂

516.455 516.12678 516.1264 -0.8 5.1 515.1191 -H Spiraeoside C ₂₁ H ₂₀ O ₁₂

464.379 464.09548 464.0952 -0.7 4.64 463.0879 -H Purpurin C ₁₄ H ₈ O ₅

256.213 256.03717 256.0371 -0.3 6.1 301.0353 +HCOO, -H

Experimental Example 4: Antibacterial activity of four major compounds of ethyl acetate fraction

Growth inhibition was measured by treating L. monocytogenes , C. perfringens , and C. coli food poisoning bacteria as sensitive and treating each of the four compounds at concentrations of 10 mM and 50 mM.

Inhibition zone (mm ± SD) L. monocytogenes C. perfringens C. coli Kaempferol 3-O-β-rutinoside 10 mM 6.69±0.10 7.08±0.07 7.91±0.36 50 mM 7.01±0.12 7.10±0.06 8.08±0.18 Cynarin 10 mM 6.89±0.11 7.19±0.06 8.19±0.10 50 mM 7.07±0.08 7.70±0.18 8.28±0.09 Spiraeoside 10 mM 7.70±0.08 7.22±0.04 7.86±0.44 50 mM 7.82±0.12 7.83±0.06 7.86±0.44 Purpurin 10 mM 8.14±0.23 12.41±0.08 11.29±0.24 50 mM 9.18±0.04 17.62±0.29 14.07±0.33

(Data are shown as the mean ± SD (n = 3))

As shown in Table 6, the four flavonoid derivative compounds have antibacterial activity against Gram positive and negative food poisoning bacteria, among which purpurin has the highest activity, followed by spiraeoside, cynarin, and kaempferol 3-O-β-rutinoside It can be confirmed that appears.

In addition, the antimicrobial activity according to the concentration of the compound is three types (spiraeoside, cynarin, kaempferol 3-O-β-rutinoside), there is little activity change according to the concentration, purpurin is 50 mM C. perfringens and C. coli It showed high activity against.

Experimental Example 5: ethyl acetate Fraction Antioxidant activity of 4 major compounds

[How to measure]

① Reducing power: Oyaizu method

② DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity measurement: Riaz Rajoka method

Substance name Reducing power EC ₅₀ (mg/mL) DPPH EC ₅₀
(mg/mL) Kaempferol 3-O-β-rutinoside 12.49±0.46 18.32±1.10 Cynarin 0.05±0.01 0.19±0.02 Spiraeoside 0.27±0.00 0.36±0.01 Purpurin 0.13±0.00 0.14±0.00 Ascorbic acid 0.08±0.00 0.07±0.00

(Data are shown as the mean ± SD (n = 3))

As shown in Table 7, it can be seen that all four of the flavonoid derivative compounds show antioxidant activity as a result of RP and DPPH experiments. As a result of RP, cynarin showed the highest antioxidant activity and higher activity than the positive control (ascorbic acid). In addition, as a result of DPPH, purpurin showed the highest activity, and kaempferol 3-O-β-rutinoside showed the lowest antioxidant activity among the four compounds like RP.

Experimental Example 6: Purpurin inhibitory activity of food poisoning bacteria

Antibacterial activity was measured using the Disc diffusion method. At this time, the disc was used 6 mm, it was carried out while changing the concentration of purpurin as shown in Table 8. The antibiotic gentamicin was used as a control.

Indicator
microorganism Inhibition zone (mm ± SD) Purpurin Gentamicin 10 mM 50 mM 100 mM 200 mM 100 mM Gram (+) B. cereus 11.66±0.78 14.60±0.57 16.22±0.12 17.22±0.17 16.15±0.39 S. aureus nd ¹ n.d n.d n.d 7.18±0.01 L. monocytogenes 8.14±0.23 9.18±0.04 10.56±0.19 12.45±0.57 13.23±0.18 C. perfringens 12.41±0.08 17.62±0.29 19.86±0.76 20.74±0.37 n.d. Gram (*?*) E. coli 7.90±0.06 12.14±0.50 7.90±0.06 12.14±0.50 9.68±0.04 S. Typhimurium n.d. n.d. n.d. n.d. 7.87±0.22 Y. enterocolitica n.d. n.d. n.d. n.d. 10.49±0.04 V. parahaemolyticus 7.93±0.25 8.10±0.20 8.03±0.06 8.15±0.15 12.25±0.53 C. jejuni 8.39±0.10 12.54±0.29 13.87±0.36 16.61±0.72 18.54±1.41 C. coli 11.27±0.24 14.07±0.33 13.86±0.33 14.59±0.17 13.10±0.45 Data are mean ± SD ( n = 3).
¹ nd, not detected (inhibition zones with values ≤7 mm were assumed as being absent of antimicrobial activity).

Table 8 shows the results of confirming the antibacterial activity against 10 food poisoning bacteria according to the change in purpurin concentration obtained from the ethyl acetate fraction of ethanol extract of Gaetbangpung leaf. In particular, it showed strong activity against C. perfringens . Also B. The cereus , C. perfringens , and C. coli showed stronger activity than the chemical antibiotic gentamicin.

Claims (10)

Ethyl acetate fraction of Gaetbangpung leaf ethanol extract contains as an active ingredient, the fraction is campferol 3-0-β-rutinoside (Kaempferol 3-0-β-rutinoside), cinnarin (Cynarin), spireoside Antioxidant food composition characterized by containing any one of (Spiraeoside) and Purpurin (Purpurin).
delete The antioxidant food composition of claim 1, wherein the ethanol extract of Gaetbangpung leaf is extracted with a 70% by weight ethanol solvent.
The method according to claim 1, wherein the ethyl acetate fraction is Bacillus cereus (B.cereus), Staphylococcus aureus (S. aureus), Listeria (L. monocytogenes), Clostridium perfringens (C. perfringens), Salmonella typhimurium Antioxidant food characterized by having inhibitory activity of S. Typhimurium, E. coli, V. parahaemolyticus, C. jejuni, and C. coli Composition.
The antioxidant food composition of claim 1, wherein the ethyl acetate fraction has a total polyphenol content of 102.15±0.53 μg GAE/mg and a total flavonoid content of 219.73±0.76 μg CE/mg.
The method according to claim 1, Reducing power of the ethyl acetate fraction (Oyaizu method) is 0.14±0.00 EC50 mg/mL, DPPH radical scavenging activity (Blois method) is 95.75±0.15%, and TAC (Harisa method) is 936.37±18.33. CRE, FRAP (Boufadi method) is an antioxidant food composition, characterized in that 89839.08 ± 283.46 μM. delete delete delete delete