KR102291823B1 - Composition for anti-oxidant and anti-inflammation comprising bellflower sprout extract - Google Patents

Abstract

The present invention relates to a composition for antioxidant and anti-inflammatory properties containing sprouted bellflower extract as an active ingredient. It can be usefully used as an antioxidant and anti-inflammatory composition because it exhibits excellent antioxidant activity due to its high content of vonoids, and exhibits anti-inflammatory activity by inhibiting high nitric oxide production and cytokine production.

Description

Composition for anti-oxidant and anti-inflammation comprising bellflower sprout extract

The present invention relates to an antioxidant and anti-inflammatory composition comprising an extract of sprouted bellflower as an active ingredient.

In the living body, a significant amount of active oxygen is generated during the oxidation process for energy production. When free radicals are instantaneously generated in large amounts or chronically generated, they react strongly with cell components and cause damage to cells and tissues, and continuous cell damage can lead to DNA denaturation, lipid oxidation, protein degradation have.

In the human body, superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT), glutathione reductase, glutathione-S-transferase ), etc., there are antioxidant enzymes against free radicals, but due to various environmental pollutants, smoking, stress, etc., which increase with industrialization, there is a limit to defending against protein degradation and DNA damage only with the activity of antioxidant enzymes in the human body.

Synthetic substances such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have been developed as antioxidants, but they can cause various side effects on the human body. Research to find safe antioxidants is required.

On the other hand, inflammation (inflammation) is a defense mechanism to prevent damage to human tissue due to infection, and it is accompanied by symptoms such as development, fever, and pain, and it removes pathogenic substances and restores normal structure and function through tissue regeneration.

However, if the antigen is not removed or the inflammatory reaction is excessively or continuously caused by an internal substance, mucosal damage is promoted, and various chronic diseases such as neurodegenerative diseases and cancer can be caused.

In the immune response in the body, macrophages are involved in and control the production of inflammatory mediators such as nitric oxide (NO), prostaglandin (PG), and pro-inflammatory cytokines, Inflammatory mediators act as a defense mechanism in the human body by inducing an inflammatory response, but if the host's immune response does not respond appropriately, it can cause inflammatory diseases.

On the other hand, in order to eliminate inflammation, an anti-inflammatory agent that acts to remove an inflammatory source, reduce biological reactions and symptoms.

Substances used for anti-inflammatory purposes so far include flufenamic acid, ibuprofen, benzydamine, indomethacin, etc. as a non-steroidal type, and prednisolone as a steroid type. , and dexamethasone. In addition, allantoin, azene, hydrocortisone, etc. are known to be effective in anti-inflammatory, but there is a problem in that the amount of use is limited due to a problem of safety for the skin, or since the effect is insignificant, an inflammatory relief effect cannot be expected. Accordingly, efforts have been made to find ingredients having antioxidant and anti-inflammatory activities derived from natural products.

On the other hand, bellflower (Platycodon grandiforum A. DC) is a perennial herb belonging to the Campanualaceae that is native to or cultivated in East Asia, and its roots are mainly used as traditional herbal medicines and food. Carbohydrates (sugars) account for more than 90% of the main ingredients of bellflower, and there are other proteins, fats, and ash. Carbohydrates are mostly disaccharides or trisaccharides such as glucose, fructose, sugar, and kestose, and polysaccharides such as inulin and platycodinin (polysaccharide of about 10 fructose molecules) have been reported. In addition, triterpenoid saponin (24 types of platycodin A, C, D, D2, polygalacin D, D2, etc. have been reported) are present in about 2%. , these saponin components are attracting attention as active ingredients with various pharmacological effects of bellflower.

Bellflower has been mainly used as an expectorant, antitussive action, cough and bronchitis treatment. have. In particular, Platicodin D is a saponin having the main pharmacological component of Gilkyung (medicine made from the root of bellflower). Hoehne anti-obesity (anti-obesity), Republic of Korea Patent Registration No. 10-0696647, anti-inflammation (anti-inflammation) efficacy is known, it is known as a single ingredient with great pharmacological value. However, there have been no reports of research on sprouted bellflower.

Accordingly, as a result of the study on sprouted bellflower, the present inventors showed that the sprouted bellflower extract prepared according to the present invention exhibited excellent antioxidant activity due to higher vitamin C, polyphenol and flavonoid content compared to the two-year-old bellflower root and sprout ginseng extract. The present invention was completed by confirming that it exhibits anti-inflammatory activity by suppressing the high nitric oxide production inhibitory effect and cytokine production.

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases containing a sprout sprout extract as an active ingredient.

Another object of the present invention is to provide a health functional food composition for the prevention or improvement of inflammatory diseases containing the extract of sprouted bellflower as an active ingredient.

It is another object of the present invention to provide a health functional food composition for antioxidants containing sprouted bellflower extract as an active ingredient.

In order to achieve the above purpose,

One aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases containing the sprout sprout extract as an active ingredient.

Another aspect of the present invention provides a health functional food composition for the prevention or improvement of inflammatory diseases containing the sprout sprout extract as an active ingredient.

Another aspect of the present invention provides a health functional food composition for antioxidants containing the extract of sprouted bellflower as an active ingredient.

The sprouted bellflower extract of the present invention exhibits excellent antioxidant activity due to high vitamin C, polyphenol and flavonoid contents compared to the 2-year-old bellflower root and sprout ginseng extract, and has a higher nitric oxide production inhibitory effect compared to the comparative group in the comparison of anti-inflammatory activity and cytokine production inhibitory effect, so it can be usefully used as an antioxidant and anti-inflammatory composition.

1 is a diagram showing a 70% ethanol extract and a water extract for each sample.
(From the left, whole sprouted bellflower extracted with 70% ethanol, sprouted bellflower root, sprouted bellflower leaves and stems, sprouted ginseng, 2-year-old bellflower and whole sprouted bellflower extracted with water, sprouted bellflower root, sprouted bellflower leaf and stem, sprouted ginseng, 2-year-old root balloon flower)
Figure 2 is a diagram showing the results of comparing the vitamin C content of each size (A) and each part (B) of sprouted bellflower.
3 is a two-year-old bellflower (BE, B), sprouted ginseng (GS-E, GS), whole sprouted bellflower (middle, BS-E, BS), sprouted bellflower root (BSR-E, BSR) and sprouted bellflower leaf ( BSL-E, BSL) is a diagram showing the results of comparing the macrophage viability of 70% ethanol extract.
4 is a two-year-old bellflower (BW, B), sprouted ginseng (GS-W, GS), whole sprouted bellflower (medium, BS-W, BS), sprouted bellflower root (BSR-W, BSR) and sprouted bellflower leaf ( BSL-W, BSL) is a diagram showing the results of comparing the macrophage survival rate of the water extract.
Figure 5 shows two-year-old bellflower (BE, B), sprouted ginseng (GS-E, GS), whole sprouted bellflower (medium, BS-E, BS), sprouted bellflower root (BSR-E, BSR) and sprouted bellflower leaves ( BSL-E, BSL) is a diagram showing the result of comparing the production amount of nitric oxide (NO) of 70% ethanol extract.
Figure 6 shows two-year-old bellflower (BW, B), sprouted ginseng (GS-W, GS), whole sprouted bellflower (medium, BS-W, BS), sprouted bellflower root (BSR-W, BSR) and sprouted bellflower leaves ( BSL-W, BSL) is a diagram showing the result of comparing the amount of nitric oxide (NO) production of the water extract.
7 shows two-year-old bellflower (BE, B), sprouted ginseng (GS-E, GS), whole sprouted bellflower (middle, BS-E, BS), sprouted bellflower root (BSR-E, BSR) and sprouted bellflower leaf ( BSL-E, BSL) is a diagram showing the result of comparing the IL-6 production amount of 70% ethanol extract.
8 is a view showing the size and weight of each part of sprouted bellflower.

Hereinafter, the present invention will be described in detail.

In the present invention, "bellflower" has the scientific name of Platycodon grandiflorum , and may be double bellflower, white bellflower, baby bellflower, red bellflower, or white bellflower, but is not limited thereto.

One aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases containing the sprout sprout extract as an active ingredient.

The sprouted bellflower may be 10 to 32cm, may be 12.1 to 29.5cm, may be 23.3 to 29.5cm, may be 17.3 to 20.9cm, and may be 12.1 to 16.9cm, based on the overall size.

The sprouted bellflower may be grown for 40 to 60 days, may be grown for 45 to 55 days, may be grown for 48 to 52 days, and preferably grown for 50 days.

The sprout bellflower extract may be prepared by a manufacturing method comprising the following steps:

1) preparing an extract by adding an extraction solvent to sprouted bellflower;

2) filtering the extract of step 1); and

3) lyophilizing the filtered extract of step 2) to powder.

The sprout bellflower extract may be prepared by extraction with water, alcohol, or a mixture thereof.

The alcohol may be a lower alcohol of C _{1-3, preferably ethanol.}

The alcohol may be 30% to 99% ethanol, 40% to 90% ethanol, 50% to 80% ethanol, and 60% to 75% ethanol.

The extraction solvent may be added in an amount of 1 to 50 ml per 1 g of the weight of sprouted bellflower used for extraction, and may be added in an amount of 5 to 40 ml per 1 g of the weight of sprouted bellflower used for extraction, It may be added in an amount of 10 to 30 ml per 1 g of the weight of the sprouted bellflower used, and it may be added in an amount of 15 to 25 ml per 1 g of the weight of the sprouted bellflower used for extraction.

The extraction method may be immersion, shaking extraction, Soxhlet extraction, reflux extraction or ultrasonic extraction. In this case, the extraction time may be 10 minutes to 60 minutes, 20 minutes to 50 minutes, 25 minutes to 40 minutes. The extraction may be repeated 1 to 5 times.

The sprouted bellflower may be any one or more selected from the group consisting of whole, root, leaf and stem.

Sprout bellflower extract of the present invention has strong antioxidant activity.

In one aspect of the present invention, the antioxidant activity, oxygen that enters the body through human respiration, also has beneficial effects such as making energy necessary for the human body, but in this process, free radicals, which are extra oxygen that are not good for the body, are It is produced, but free radicals attack normal cells in the body and act as a cause of aging and various diseases. Removing these free radicals is a way to prevent cell oxidation (aging), and inhibiting oxidation of these cells is antioxidant activity.

In the present invention, in order to confirm the antioxidant activity, the antioxidant components of the sprout sprouts extract were analyzed, and the DPPH radical scavenging ability and ABTS radical scavenging ability were confirmed.

The sprout bellflower extract may be an extract prepared by extraction with water, alcohol, or a mixture thereof, and preferably may be an ethanol extract of bellflower sprouts or a water extract. In addition, the ethanol extract may be an extract using 30% to 99% ethanol, 40% to 90% ethanol, 50% to 80% ethanol, or 60% to 75% ethanol.

On the other hand, the antioxidant component includes vitamin C, polyphenols and flavonoids, and the DPPH radical scavenging ability measurement is a common experimental method used to measure the antioxidant activity, and the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical is Although it has a purple color, it is a method using a light yellow color when reacted with an antioxidant, and the ABST radical scavenging ability is measured with a cyan ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) radical It is a method using what becomes transparent when it reacts with this antioxidant.

In a specific embodiment of the present invention, the present inventors divided the sprouted bellflower into whole sprouted bellflower, leaves and stems, and roots by size and part to determine the vitamin C content, which is an antioxidant component, from the sprouted bellflower extract, and the respective vitamin C content As a result of the analysis, a high content of vitamin C was confirmed in sprouted bellflower (see FIG. 2).

In addition, the present inventors analyzed polyphenol and flavonoid contents with extracts by size and part of sprouted bellflower and extracts of sprouted ginseng and two-year-old bellflower as a comparison group to confirm the content of polyphenols and flavonoids, which are another antioxidant component. It was confirmed that the total polyphenol content was superior to that of the two-year-old bellflower root and sprout ginseng extract in the sprout bellflower extract without was confirmed (see Table 2 and Table 3).

In addition, the present inventors conducted an experiment to measure the DPPH radical scavenging ability and ABTS radical scavenging ability in order to confirm the antioxidant activity of the sprouted bellflower extract. It was confirmed that the DPPH radical scavenging ability was superior to that of the extract, and the ABTS radical scavenging ability was also confirmed that the ethanol extract of sprouted bellflower, regardless of size, was superior to the two-year-old bellflower root and sprout ginseng extracts of the comparative group (see Tables 4 and 5).

Sprout bellflower extract of the present invention has an anti-inflammatory effect.

In one aspect of the present invention, anti-inflammatory is meant to include amelioration (relief of symptoms), treatment, and suppression or delay of the onset of inflammatory diseases as defined below.

In the present invention, in order to confirm the anti-inflammatory activity, the inhibitory activity of nitric oxide (NO) production and the inhibitory activity of inflammation-inducing cytokines of sprouted bellflower extract were evaluated.

The sprout bellflower extract may be an extract prepared by extraction with water, alcohol, or a mixture thereof, and preferably may be an ethanol extract of bellflower sprouts or a water extract. In addition, the ethanol extract may be an extract using 30% to 99% ethanol, 40% to 90% ethanol, 50% to 80% ethanol, or 60% to 75% ethanol.

In a specific embodiment of the present invention, the present inventors analyzed macrophage viability to determine whether the sprout sprout extract was cytotoxic, and as a result, it was confirmed that there was no cytotoxicity in the ethanol extract (see FIG. 3), and in the case of the water extract Also, it was confirmed that no cytotoxicity was observed (see FIG. 4 ).

In addition, the present inventors analyzed the nitric oxide (NO) production amount of the sprout bellflower extract to confirm the anti-inflammatory effect of the sprout bellflower extract. It was confirmed that the amount of nitric oxide production decreased, and it was confirmed that a high nitric oxide production inhibitory effect was exhibited (see FIG. 5 ). It was confirmed that the amount of nitric oxide production decreased even in the case of the water extract (see FIG. 6 ).

In addition, the present inventors analyzed the cytokine production amount of the sprout bellflower extract to confirm the anti-inflammatory effect of the sprout bellflower extract, and as a result, it was confirmed that the cytokine significantly decreased according to the sample treatment concentration, and the cytokines of the sprout bellflower extract It was confirmed that the inhibition of Cain production was most effective in leaves + stems (see FIG. 7 ).

Therefore, the extract of sprouted bellflower of the present invention can be usefully used as a pharmaceutical composition for preventing or treating inflammatory diseases.

The inflammatory disease may be selected from the group consisting of inflammatory bowel disease, glomerulonephritis, inflammatory skin disease, retinitis, gastritis, hepatitis, enteritis, arthritis, tonsillitis, pharyngitis, bronchitis, pneumonia, pancreatitis and nephritis.

The pharmaceutical composition for the prevention or treatment of inflammatory diseases containing the sprout sprout extract of the present invention as an active ingredient may further include additional components, for example, may further include at least one of a carrier, an excipient, and a diluent. For example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, It may include at least one of polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

The pharmaceutical composition for the prevention or treatment of inflammatory diseases may have various formulations, and according to conventional methods, external preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., suppositories for sterile injection solutions It can be formulated and used in the form. In the case of formulation, it can be prepared by using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. commonly used. emulsions, lyophilized formulations and suppositories are included. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin, glycerogelatin, etc. may be used.

Another aspect of the present invention provides a health functional food composition for the prevention or improvement of inflammatory diseases containing the sprout sprout extract as an active ingredient.

The health functional food composition for the prevention or improvement of inflammatory diseases containing the sprout bellflower extract as an active ingredient may additionally contain one or more active ingredients exhibiting the same or similar function. For administration, it may be prepared by additionally including one or more food-acceptable carriers.

The health functional food composition for the prevention or improvement of inflammatory diseases containing the sprout sprout extract of the present invention as an active ingredient is preferably in any one formulation selected from beverages, pills, tablets, capsules, and powders. However, the present invention is not limited thereto.

Another aspect of the present invention provides a health functional food composition for antioxidants containing the extract of sprouted bellflower as an active ingredient.

The health functional food composition for antioxidants containing the sprout bellflower extract as an active ingredient may additionally contain one or more active ingredients exhibiting the same or similar function. For administration, it may be prepared by additionally including one or more food-acceptable carriers.

The health functional food composition for antioxidants containing the sprout sprout extract of the present invention as an active ingredient is preferably in any one formulation selected from beverages, pills, tablets, capsules, and powders, but is not limited thereto.

Hereinafter, the present invention will be described in detail through Examples and Experimental Examples.

However, the Examples and Experimental Examples described below are merely illustrative of the present invention in detail in one aspect, and the present invention is not limited thereto.

<Example 1> Preparation of 70% ethanol extract of sprouted bellflower

For the measurement of antioxidant components and antioxidant activity, as described in <Experimental Example 1>, 40 mL of 70% ethanol was added to 2 g of powder of lyophilized sprouted bellflower (Yeongdong-gun, Chungcheongbuk-do) for each size and part, followed by ultrasonic extraction for 30 minutes, followed by 3000 The supernatant was recovered by centrifugation at rpm for 10 minutes (Union 55R, Hanil Science industrial Co., Inchen, Korea). This process was repeated twice and the recovered supernatant was used as Whatman No. 2 After filtration with filter paper, it was added to 100 mL and used to measure antioxidant components and antioxidant activity. As a comparison group, two-year-old bellflower root and sprouted ginseng were prepared and used in the same manner as in the preparation of the freeze-dried sprouted bellflower extract sample.

<Experimental Example 1> Analysis of antioxidant components of sprouted bellflower

<1-1> Vitamin C content analysis

To analyze the vitamin C content of sprouted bellflower, the following experiment was performed.

Specifically, the sprouted bellflower obtained from Yeongdong-gun, Chungcheongbuk-do was classified into large, medium, and small by size as shown in Table 1, classified into whole sprouted bellflower by part, leaves, stems, and roots. Put in an mL centrifuge tube, 25 mL of 5% MPA solution containing 5 mM TCEP was added, and homogenized for 2 minutes with a homogenizer (Polytron RT 2500 E, Kinematica AG, Luzern, Switzerland) at 2,000 rpm.

로 유지하였다. 이동상은 0.1% TFA를 0.6 mL/min 속도로 흘려주었고, 시료 10 ㎕를 주입하여 UV detector를 사용하여 254 nm에서 분석하였다.Then, centrifugation was performed at 3,000 rpm for 10 minutes (Union 55R, Hanil Science industrial Co., Inchen, Korea), and the supernatant was adjusted to a final 25 mL. The extract was filtered through a 0.2 μm syringe filter and used as a sample for HPLC analysis. For analysis conditions by HPLC (Waters 2695, Milford, MA, USA), Mightysil RP-18 GP column (4.6Х250 mm, 5 μm, Kanto chemical, Tokyo, Japan) was used as the column, and the temperature was 20

was maintained as The mobile phase was flowed with 0.1% TFA at a rate of 0.6 mL/min, and 10 μl of the sample was injected and analyzed at 254 nm using a UV detector.

As a result, as shown in FIG. 2 , as a result of analyzing the vitamin C content by size of sprouted bellflower, it was found that the large size was 3.24 mg/g, the medium size was 2.92 mg/g, and the small size was 2.64 mg/g (FIG. 2). A). The vitamin C content for each part was 2.92 mg/g in total, 3.49 mg/g in the leaf + stem, and 1.31 mg/g in the root, showing the highest content in the leaf + stem (FIG. 2B).

culture
Way sample
division part Length (cm) Weight (g)
*100 root average facility
House















large
(Big)



all
(Whole) 26.4±3.1 88.67±4.43 Leaf + Stem (Net)
(Leaf+Stem) 17.0±2.8 _ root
(Root) 9.4±2.4 _ China
(Middle)



all
(Whole) 19.1±1.8 46.04±2.68 Leaf + Stem (Net)
(Leaf+Stem) 11.4±2.1 _ root
(Root) 7.6±1.4 _ small
(Small)



all
(Whole) 14.5±2.4 20.30±0.73 Leaf + Stem (Net)
(Leaf+Stem) 7.5±1.2 _ root
(Root) 7.0±0.1 _

<1-2> Measurement of total polyphenol content

_{2 mL of 2% Na 2} CO ₃ solution was added to 0.1 mL of each extraction sample of sprouted bellflower by size and part prepared in <Example 1>, left at room temperature for 3 minutes, and then 50% Folin-Siocalto reagent (Folin- 0.1 mL of Ciocalteu's reagent) was added, mixed, and left at room temperature for 30 minutes, and then the absorbance value at 750 nm was measured using a Microplate Reader (Infinite 200 PRO, TECAN, Austria). The total polyphenol content was expressed in mg garlic acid per g of the sample after preparing a standard calibration curve obtained by experimenting in the same manner as the sample using gallic acid as a standard material.

As a result, as shown in Tables 2 and 3, it was confirmed that the sprouted bellflower extract had better total polyphenol content than the two-year-old bellflower root and sprout ginseng extracts of the comparative group, regardless of the size. The total polyphenol content of the sprouted bellflower extract by size showed the highest content of the medium-sized sprouted bellflower extract, which was about 5.4 times higher than that of the two-year-old bellflower root extract and about 2.4 times higher than that of the sprouted ginseng extract. The total polyphenol content of sprouted bellflower extract for each part was highest in the leaf + stem.

Total polyphenol content (mg/g) Total flavonoid content (mg/g) 2 year old bellflower
(root) 7.71±0.18 ^e1) 0.44±0.01 ^e Sprout ginseng (medium size) 17.59±1.05 ^d 2.03±0.04 ^d Sprout bellflower (large size) 38.51±0.47 ^b 22.04±0.43 ^c Sprout bellflower (medium size) 41.62±0.19 ^a 24.74±0.59 ^a Sprout bellflower (small size) 37.35±0.34 ^c 23.76±0.33 ^b There is a significant difference between the different superscripts (p<0.05, Duncan multi-range test).

Total polyphenol content (mg/g) Total flavonoid content (mg/g) Sprout bellflower
(all) 41.62±0.19 ^b1) 24.74±0.59 ^b Sprout bellflower (leaf + stem) 51.35±0.99 ^a 33.31±1.56 ^a Sprout bellflower (root) 9.43±0.07 ^c 0.76±0.02 ^c There is a significant difference between the different superscripts (p<0.05, Duncan multiple range test).

<1-3> Total flavonoid content measurement

_{1 mL of distilled water and 75 μl of 5% NaNO 2} were added to 250 μl of each extraction sample of sprouted bellflower by size and region prepared in <Example 1>, and then left at room temperature for 5 minutes. Then, 150 μl of 10% AlCl _3· 6H 2 O was added and left for 6 minutes, and then 500 μl of 1M NaOH was added. 11 minutes after NaOH was added, the absorbance value of the reaction solution at 510 nm was measured using a Microplate Reader (Infinite 200 PRO, TECAN, Austria). After a calibration curve was prepared using (+)-catechin hydrate as a standard material, the total flavonoid content was expressed in mg per g of the sample.

As a result, as shown in Tables 2 and 3, it was confirmed that the sprouted bellflower extract had better total flavonoid content than the two-year-old bellflower root and sprout ginseng extracts of the comparative group, regardless of the size. The total flavonoid content of the sprouted bellflower extract by size showed the highest content of the medium-sized sprouted bellflower extract, which was about 56.2 times higher than that of the two-year-old bellflower root extract and about 12.2 times higher than that of the sprouted ginseng extract. The total flavonoid content of sprouted bellflower extract for each part was highest in the leaf + stem.

<Experimental Example 1> Evaluation of antioxidant activity of ethanol extract of sprouted bellflower

<1-1> Measurement of DPPH radical scavenging ability

The following experiment was performed to measure the DPPH radical scavenging activity of the ethanol extract of sprouted bellflower.

Specifically, DPPH (2,2-Diphenyl-1-picrylhydrazyl) was sufficiently dissolved in 70% ethanol for 60 minutes to prepare a DPPH solution. To 1 mL of DPPH solution, 50 μl of 70% ethanol extract of sprouted bellflower for each size and region prepared by the method described in <Example 1> was added and left at room temperature for 30 minutes. Then, the decrease in absorbance was measured at 520 nm using a Microplate Reader (Infinite 200 PRO, TECAN, Austria) and expressed as L-ascorbic acid equivalent capacity (AEAC).

As a result, as shown in Tables 4 and 5, it was confirmed that, regardless of the size, the ethanol extract of sprouted bellflower was superior to the two-year-old bellflower root and sprout ginseng extracts of the comparative group in scavenging DPPH radicals. The DPPH radical scavenging ability of the ethanol extract of sprouted bellflower by size was the best in the medium-sized sprouted bellflower extract, which was about 28.7 times higher than that of the two-year-old bellflower root extract and about 23.5 times higher than that of the sprouted ginseng extract. The DPPH radical scavenging ability of the ethanol extract of sprouted bellflower by part was measured to be the highest in the leaf + stem.

DPPH radical scavenging ability ABTS radical scavenging ability (mg AA eq/g) (mg AA eq/g) 2 year old bellflower
(root) 2.51±0.04 ^b 2.93±0.00 ^e Sprout ginseng (medium size) 3.07±0.41 ^b 5.30±0.11 ^d Sprout bellflower
(Large size) 70.79±5.48 ^a 28.46±0.58 ^c Sprout bellflower (medium size) 72.01±5.76 ^a 30.76±0.28 ^a Sprout bellflower (small size) 66.04±3.35 ^a 29.27±0.34 ^b There is a significant difference between the different superscripts (p<0.05, Duncan multiple range test).

DPPH radical scavenging ability ABTS radical scavenging ability (mg AA eq/g) (mg AA eq/g) Sprout bellflower
(all) 72.01±5.76 ^b 30.76±0.28 ^b Sprout bellflower (leaf + stem) 85.18±4.27 ^a 37.74±0.49 ^a Sprout bellflower (root) 2.74±0.09 ^c 4.51±0.06 ^c There is a significant difference between the different superscripts (p<0.05,
Duncan multi-range test)

<1-2> Measurement of ABTS radical scavenging ability

In order to measure the ABTS radical scavenging activity of the ethanol extract of sprouted bellflower, the following experiment was performed.

Specifically, 7 mM ABTS (2,2'-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid, Sigma Chemical Co.)) and 2.45 mM potassium photosulfate (Sigma Chemical Co.) were reacted in the dark at room temperature for 12 to 16 hours. to generate ABTS radical cations, and then diluted with distilled water so that the absorbance value at 735 nm was 1.4 to 1.5 using a Microplate Reader (Infinite 200 PRO, TECAN, Austria). To 1.0 mL of the diluted ABTS·+ solution, 50 μl of 70% ethanol extract of sprouted bellflower was added and left at room temperature for 30 minutes. Then, the decrease in absorbance at 734 nm was measured and expressed as L-ascorbic acid equivalent capacity (AEAC).

As a result, as shown in Tables 4 and 5, it was confirmed that, regardless of the size, the ethanol extract of sprouted bellflower was superior to the two-year-old bellflower root and sprout ginseng extracts of the comparative group in scavenging ABTS radicals. The ABTS radical scavenging ability of the ethanol extract of sprouted bellflower by size was the best in the medium-sized sprouted bellflower extract, which was about 10.5 times higher than that of the two-year-old bellflower root extract and about 5.8 times higher than that of the sprouted ginseng extract. The ABTS radical scavenging ability of the ethanol extract of sprouted bellflower by part was highest in the leaf + stem.

<Experimental Example 2> Evaluation of anti-inflammatory activity of ethanol extract of sprouted bellflower

<2-1> Macrophage culture

For macrophage culture for evaluation of anti-inflammatory activity, RAW 264.7 cells were purchased from ATCC (American Type Culture Collection, TIB-71), and 10% bovine in Dulbecco's Modified Eagle's Medium (DMEM, Hyclone Laboratories, Logan, UT, USA). Fetal serum (FBS, Gibco, Grand Island, NY, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin (Hyclone Laboratories) were used in a mixed medium and cultured at _{37° C., 5% CO 2 in an incubator.}

<2-2> Preparation of sample for evaluation of anti-inflammatory activity of sprouted bellflower

Whole sprouted bellflower (middle, BS-E, BS), sprouted bellflower root (BSR-E, BSR), sprouted bellflower leaf, obtained by freeze-drying the 70% ethanol (E) extract prepared by the method described in <Example 1> (BSL-E, BSL), sprouted ginseng (GS-E, GS), and bellflower (BE, B) extract powder were dissolved in distilled water and used for the experiment after a 0.2 μm syringe filter. The water extract was used in the experiment by replacing only the 70% ethanol extract and the solvent with water and extracting in the same manner as above.

<2-3> Macrophage viability analysis of sprouted bellflower extract

After seeding Raw 264.7 cells at 5Х10 ⁵ cells/well (100 μl) in a 96-well plate, the extracts (5-500 μl/ml) prepared in <Example 4> were treated at various concentrations, and the untreated control group (control) ), treated with the same amount of endotoxin free water as the extracts, and incubated for 24 hours at _{37°C, 5% CO 2 in an incubator.} Then, 0.01 ml/well of EZ-cytox (Daeil Lab Service, Seoul, Korea) was treated using a water-soluble tetrazolium salt method and _{reacted in a CO 2} incubator for 2 hours with a microplate reader (Tecan, Infinite M200, Salzburg, Austria). Absorbance was measured at 450 nm. Cell viability (%) was calculated and expressed as a percentage compared to the negative control group that was not treated with the sample in the following way.

(absorbance of sample/average absorbance of control) Х 100

As a result of comparing the macrophage viability of the 70% ethanol extract, as shown in FIG. 3 , the ethanol extract of sprouted bellflower and two-year-old bellflower compared to the control group exhibited a survival rate of 93% or more at a treatment concentration of 400 μg/mL or less. It was confirmed that there was no cytotoxicity at a concentration of μg/mL or less. In the case of sprouted ginseng, the survival rate was 94% or higher at a treatment concentration of 200 μg/mL or less ( FIG. 3 ).

As a result of comparing the macrophage survival rate of the water extract, as shown in FIG. 4 , compared to the untreated control group, when the sprouted bellflower and sprouted ginseng water extract were treated, the survival rate was 91% or more at a concentration of 500 μg/mL or less. , in the case of 2-year-old bellflower, 89% viability at 500 μg/mL, and no cytotoxicity was observed at a concentration of 400 μg/mL or less ( FIG. 4 ).

<2-4> Confirmation of nitric oxide (NO) production of sprouted bellflower extract

Raw 264.7 cells prepared in <Example 3> were treated with each sample prepared in <Example 4> by concentration, and _{cultured at 37° C., 5% CO 2 in an} incubator for 2 hours. Then, it was stimulated with lipopolysaccharide (LPS) treatment (1 ug/ml) and cultured for 24 hours, and treated by dividing into an untreated control group, an LPS-treated control group, and a test group (sample by concentration + LPS). Then, 50 μl of the supernatant was transferred to a new plate, mixed with the same amount of grease reagent (Sigma, USA), and after reaction at 37° C. for 15 minutes, absorbance was measured at 540 nm using a microplate reader.

As a result of comparison of the nitric oxide (NO) production amount of the 70% ethanol extract, as shown in FIG. 5 , the nitric oxide production amount was decreased in a concentration-dependent manner according to the sample treatment concentration of the 70% ethanol extract of sprouted bellflower. When the nitric oxide production amount of the untreated LPS control group was 100%, when the 400 μg/mL sample was treated, the nitric oxide production amount was 27% for roots, 35% for total, 50% for leaves + stems, and 79% for two-year-old bellflower. Compared to two-year-old bellflower having an inhibitory effect on nitric oxide production by about 20%, it was confirmed that the ethanol extract of sprouted bellflower showed an effect of inhibiting nitric oxide production by 50%, 65% of the total, and 73% of the root in the case of leaves and stems (FIG. 5).

As a result of comparison of the nitric oxide (NO) production of the water extract, as shown in FIG. 6 , the amount of nitric oxide produced was significantly reduced according to the sample treatment concentration of the water extract of sprouted bellflower. When the nitric oxide production amount of the untreated LPS control group was 100%, when the 400 μg/ml sample was treated, the total nitric oxide production amount was 60%, the root 61%, the leaf + stem 64%, and the two-year-old bellflower 82%. Compared to 2-year-old bellflower having an inhibitory effect on nitric oxide production by about 18%, it was confirmed that the ethanol extract of sprouted bellflower exhibits 36%, 39%, root, and 40% nitric oxide production inhibitory effect in the case of leaves and stems (FIG. 6).

<2-5> Measurement of IL-6 production of sprouted bellflower extract

To measure the amount of IL-6, a cytokine, induced after lipopolysaccharide (LPS) treatment in macrophages % ethanol extract was treated with lipopolysaccharide (LPS) in the method described in <Experimental Example 4-2>. Then, using the mouse IL-6 ELISA kit (ALPCO, Salem NH, USA) in each RAW 264.7 cell culture medium, the concentration of IL-6 cytokine was measured by measuring the absorbance at 450 nm with the culture medium diluted appropriately according to the protocol presented. was confirmed.

As a result, as shown in FIG. 7 , IL-6 was significantly decreased according to the concentration of the sample. When the IL-6 production amount of the untreated LPS control was 100%, when the 400 μg/ml sample was treated, the IL-6 production amount was 66% for leaves + stems, 71% for total, 77% for roots, and 79% for two-year-old bellflower. Cain production inhibition was most effective in leaf + stem.

Therefore, 70% ethanol sprouted bellflower extract showed excellent antioxidant activity due to high polyphenol and flavonoid content compared to 2-year-old bellflower root and sprouted ginseng extract, and also showed high nitric oxide production inhibitory effect compared to the comparative group in the comparison of anti-inflammatory activity. Since it exhibits an inhibitory effect on cytokine production, it can be usefully used as an antioxidant and anti-inflammatory composition.

Claims (13)

Health functional food composition for the prevention or improvement of inflammatory diseases containing sprout bellflower extract as an active ingredient
The health functional food composition for preventing or improving inflammatory diseases according to claim 1, wherein the sprouted bellflower has a size of 12.1 to 29.5 cm.
The health functional food composition for preventing or improving inflammatory diseases according to claim 1, wherein the sprouted bellflower is a bellflower grown for 45 to 55 days.
[Claim 2] The health functional food composition for the prevention or improvement of inflammatory diseases according to claim 1, wherein the sprout bellflower extract is prepared by extraction with water, alcohol, or a mixture thereof.
The health functional food composition for preventing or improving inflammatory diseases according to claim 1, wherein the sprouted bellflower extract inhibits the production of nitric oxide (NO).
The health functional food composition for preventing or improving inflammatory diseases according to claim 1, wherein the sprouted bellflower extract inhibits the production of IL-6.
The method of claim 1, wherein the inflammatory disease is selected from the group consisting of inflammatory bowel disease, glomerulonephritis, inflammatory skin disease, retinitis, gastritis, hepatitis, enteritis, arthritis, tonsillitis, pharyngitis, bronchitis, pneumonia, pancreatitis and nephritis. to do A health functional food composition for preventing or improving inflammatory diseases.
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