Method for producing a Petasites japonicus extract with improved antioxidant activity

Abstract

The present invention relates to a method for preparing a butterbur extract with enhanced antioxidant activity, wherein the average content of fukinolic acid or total phenolic compounds is increased compared to a previously known method for preparing butterbur extract, thereby enabling the preparation of a butterbur extract with enhanced antioxidant activity.

Description

Technology Field

[0001] The present invention relates to a method for preparing a butterbur extract with enhanced antioxidant activity. Background Technology

[0002] COVID-19 served as an opportunity to solidify the position of fever reducers and pain relievers as household medicines. Medicines presumed to have been affected by the pandemic include fever reducers, cold medicines, sore throat treatments, and antidiarrheals; among single ingredients, acetaminophen is a highly recognized component. An examination of sales volumes for non-steroidal (NSAID) pain relievers reveals a close correlation with infectious disease outbreaks. While NSAIDs are commonly used for everyday pain and are generally not significantly affected by seasonal changes, they were widely sold as substitutes during the acetaminophen shortage caused by side effects from vaccination. NSAIDs are non-steroidal anti-inflammatory drugs frequently used for various diseases and clinical symptoms, such as osteoarthritis, rheumatoid arthritis, gout, toothaches, and headaches. Although they are known for their excellent efficacy, frequent and long-term use has been reported to cause gastrointestinal bleeding, impairment of the protective effect of the gastric mucosa, peptic ulcers, nephrotoxicity, and cardiovascular side effects.

[0003] Symptoms such as heartburn, vomiting, nausea, pain, and indigestion caused by inflammation or ulcers, or the absence of specific abnormal findings, are referred to as functional gastrointestinal disorders, gastritis, or gastric ulcers. The number of patients with gastritis and gastroesophageal reflux disease (GERD) in Korea is estimated to be over 9.8 million. Cases of chronic or habitual gastrointestinal diseases are continuously increasing due to various causes, including modern lifestyle issues and accumulated stress; as chronic diseases, they primarily affect the middle-aged and elderly populations. According to the Health Insurance Review and Assessment Service, the number of patients with GERD among those aged 65 and older is confirmed to be 800,000, showing the highest incidence rate among the population in their 60s across all age groups. Functional dyspepsia is a common upper gastrointestinal symptom that is difficult to identify the cause of through upper gastrointestinal endoscopy. Although it is a chronic disease characterized by long-term symptoms that lowers an individual's quality of life and causes socioeconomic losses, awareness of its risks is relatively low, leading to a low rate of active treatment; this, in turn, contributes to the condition developing into a chronic disease. Chronic gastritis is known to increase the risk of gastric ulcers and gastric cancer, and gastric cancer was found to be the second most common cancer in men and the fifth most common in women, ranking high among cancer incidence rates in Korea.

[0004] Aggressive factors are broadly classified into exogenous and endogenous factors. While these factors are protected by the stomach's inherent defense mechanisms, an imbalance between these factors and defense mechanisms damages the gastric mucosa, leading to the development of gastritis and gastric ulcers.

[0005] The stomach is composed of four layers: the mucosa, submucosa, muscularis mucosa, and serosa. The gastric mucosa consists of a layer of cells responsible for absorption and secretion, and includes a mucus layer and bicarbonate ions (HCO3 -Although a healthy mucosal state is maintained through the action of substances such as prostaglandins, gastric mucosal damage can occur due to various factors including Helicobacter pylori infection, NSAIDs, stress, and alcohol. Gastric mucosal damage can progress to acute gastritis, chronic gastritis, and peptic ulcers, which cause problems such as indigestion and stomach discomfort. Consuming foods that inhibit factors causing gastric mucosal damage, such as Helicobacter pylori, or those that provide gastric mucosal protection, can help maintain the health of the gastric mucosa. Representative gastritis treatments prescribed include cimetitine, ranitidine, omeprazole, pantoprazole, misoprostol, rebamipide, and pesuprazan, as well as Artemisia annua 95% ethanol soft extract and cinnamon dry extract; however, despite their excellent therapeutic effects, their continuous use is discouraged due to various side effects, and they show limitations in treating some cases of chronic intractable gastritis.

[0006] In the gastric disease market, a wide variety of synthetic drugs are available for prescription, ranging from H2RAs, PPIs, and prostaglandin preparations to P-CABs, which were recently developed and are leading global popularity. However, side effects associated with the prescription of single-ingredient synthetic drugs may include the development of neuroendocrine tumors directly linked to gastric acid suppression and adverse effects caused by intestinal microbial imbalance. Furthermore, sales of combination prescriptions, such as NSAID+PPI combinations and antacid (sodium bicarbonate)+PPI combinations, are showing an upward trend again.

[0007] Butterbur is a perennial herb belonging to the Asteraceae family, distributed in wetlands of mountainous regions in Korea, China, and Japan. In Korea, it is known by various names such as Meou, Meogu, Meogwi, Meowitdae, Meoutdae, and Baekchae. Its nature is cool, and it is sweet and spicy. The flowers and whole plant are harvested in March and April, while the roots are harvested in the autumn and used either raw or dried in the sun. It is either harvested from the wild or cultivated by selecting wild species; it propagates vegetatively via rhizomes and is in a state where variety differentiation is impossible due to its sterility and lack of mutations.

[0008] This study aims to seek a fundamental solution to gastrointestinal problems, such as gastrointestinal ulcers induced by anti-inflammatory drugs, and to establish an efficient and safe alternative to reduce NSAID gastropathy. Specifically, it seeks to secure and develop Butterbur as a natural material with anti-inflammatory and soothing effects that minimize gastrointestinal side effects. According to the Notification on Standards and Specifications for Food (No. 2024-35), the stems and leaves of Butterbur are included in the list of raw materials that can be used in food.

[0009] This study aimed to standardize the evidence for extracts from raw materials selected as functional ingredients through scientific verification and to secure research data on their functionality for improving gastric health and protecting the gastric mucosa, thereby ensuring competitive differentiation in the existing material and raw material market. This was based on the premise that if an extract possesses the primary functionalities of improving gastric health and protecting the gastric mucosa, while additionally exhibiting anti-inflammatory effects, it would be suitable as a functional ingredient capable of minimizing the side effects associated with the use of single components and functionally replacing complex formulations of such ingredients. Prior art literature

[0010] 1. Korean Published Patent No. 10-2025-0001358 (Published Jan. 06, 2025) The problem to be solved

[0011] The objective of the present invention is to provide a method for preparing a butterbur extract with enhanced antioxidant activity.

[0012] The objectives of the present invention are not limited to those mentioned above. The objectives of the present invention will become more apparent from the following description and will be realized by the means and combinations thereof described in the claims. means of solving the problem

[0013] To achieve the above objective, the present invention (mugwort) Petasites japonicus A method for preparing a butterbur extract is provided, comprising the steps of: extracting and filtering using a solvent containing distilled water or ethanol; concentrating the filtered butterbur extract under reduced pressure; and drying the butterbur extract concentrated under reduced pressure.

[0014] The above ethanol may be 30 volume% to 70 volume% ethanol.

[0015] The weight ratio of the above butterbur and solvent may be 1:10 to 20.

[0016] The above extraction can be carried out under conditions of 30°C to 70°C and 3 to 7 hours.

[0017] The above drying may be freeze-drying or spray-drying.

[0018] In addition, the present invention provides a butterbur extract prepared by the above-described manufacturing method.

[0019] The above butterbur extract may have an average content of fukinolic acid of 55 to 65 mg / g, an average content of total phenol of 200 to 400 mg / g, an average content of total flavonoid of 50 to 70 mg / g, or increase DPPH radical scavenging ability. Effects of the invention

[0020] The present invention relates to a method for preparing a butterbur extract with enhanced antioxidant activity, wherein the average content of fukinolic acid or total phenolic compounds is increased compared to a previously known method for preparing butterbur extract, thereby enabling the preparation of a butterbur extract with enhanced antioxidant activity.

[0021] The effects of the present invention are not limited to those mentioned above. It should be understood that the effects of the present invention include all effects that can be inferred from the following description. Brief explanation of the drawing

[0022] Figure 1 shows the raw materials, butterbur and butterbur extract powder. Figure 2 shows the extraction steps and conditions of the butterbur extract. Figure 3 shows the chromatogram of the indicator components. Figure 4 shows a chromatogram of the butterbur distilled water extract. Figure 5 shows a chromatogram of the ethanol extract of butterbur. Figure 6 shows the total phenolic compound content of the butterbur extract. Figure 7 shows the total flavonoid content of the butterbur extract. Figure 8 confirms the DPPH radical scavenging activity of butterbur extract. Figure 9 shows a process for optimizing the method for preparing butterbur extract. Figure 10 shows the results of the first stage of optimization. Figure 11 shows the first-stage optimization results for CGA and FA. Figure 12 shows the result of two-step optimization (PI: Point prediction). Figure 13 shows the results of the three-step optimization. Figure 14 shows the total phenolic compound content of butterbur extract prepared under optimized conditions. Figure 15 shows the total flavonoid content of butterbur extract prepared under optimized conditions. Figure 16 shows the DPPH radical scavenging activity of butterbur extract prepared under optimized conditions. Specific details for implementing the invention

[0023] The above objects, other objects, features, and advantages of the present invention will be easily understood through the following preferred embodiments associated with the accompanying drawings. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided to ensure that the disclosed content is thorough and complete and to ensure that the spirit of the invention is sufficiently conveyed to a person skilled in the art.

[0024] In describing each drawing, similar reference numerals have been used for similar components. In the attached drawings, the dimensions of the structures are depicted enlarged compared to their actual size for the clarity of the invention.

[0025] In this specification, terms such as "comprising" or "having" are intended to specify the existence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof. Furthermore, when a part such as a layer, film, region, or plate is described as being "on" another part, this includes not only the case where it is "immediately above" the other part, but also the case where there is another part in between. Conversely, when a part such as a layer, film, region, or plate is described as being "below" another part, this includes not only the case where it is "immediately below" the other part, but also the case where there is another part in between.

[0026] Unless otherwise specified, all numbers, values, and / or expressions used herein to represent amounts of ingredients, reaction conditions, polymer compositions, and formulations should be understood to be modified by the term “approximately” in all cases, as these numbers are essentially approximations reflecting the various uncertainties of measurement that occur in obtaining these values ​​among other things. Furthermore, where numerical ranges are disclosed herein, such ranges are continuous and, unless otherwise indicated, include all values ​​from the minimum value of such range to the maximum value including said maximum value. Moreover, where such ranges refer to integers, they include all integers from the minimum value to said maximum value including said maximum value, unless otherwise indicated.

[0028] The present invention relates to a method for preparing a butterbur extract with enhanced antioxidant activity. When a butterbur extract is prepared under specific conditions using ethanol as a solvent, the average content of fukinolic acid or phenol in the butterbur extract is increased compared to a butterbur extract prepared by a method that does not satisfy specific conditions, thereby enhancing the antioxidant activity.

[0030] The present invention relates to butterbur ( Petasites japonicus A method for preparing a butterbur extract is provided, comprising the steps of: extracting and filtering using a solvent containing distilled water or ethanol; concentrating the filtered butterbur extract under reduced pressure; and drying the butterbur extract concentrated under reduced pressure.

[0031] The above butterbur may be crushed butterbur. Using crushed butterbur increases the surface area, allowing the components of the butterbur to be extracted uniformly, thereby increasing extraction efficiency and shortening the extraction time.

[0032] The above butterbur may be butterbur ground to 40 mesh. If the size of the ground butterbur exceeds 40 mesh, very fine particles may remain in the solvent, causing the butterbur extract to become cloudy and clogging or passing through the filter paper during the filtration process.

[0033] The above ethanol may be 30 volume% to 70 volume% ethanol, preferably 40 volume% to 60 volume% ethanol, and more preferably 50 volume% ethanol. When the above ethanol is ethanol within the above numerical range concentration, the content of fukinolic acid and phenol content of the butterbur extract prepared according to the above manufacturing method may increase, thereby improving antioxidant activity.

[0034] The weight ratio of the above butterbur and solvent may be 1:10 to 20, preferably 1:10.

[0035] The above extraction can be carried out under conditions of 30°C to 70°C and 3 to 7 hours, preferably under conditions of 30°C and 5 hours. If the above extraction conditions fall within the above numerical range, the antioxidant component of the butterbur extract prepared according to the above manufacturing method may increase.

[0036] The above extraction may be a reflux extraction. The above reflux extraction may be an extraction method that repeats the process of heating a liquid solvent to evaporate it, cooling it again to condense it back into the original container, and returning it.

[0037] The above vacuum concentration can be performed at 45°C to 55°C.

[0038] The above drying may be freeze-drying or spray-drying, and preferably spray-drying. If the above drying is spray-drying, the content of chlorogenic acid and fukinolic acid in the butterbur extract prepared according to the above manufacturing method may increase.

[0040] In addition, the present invention provides a butterbur extract with enhanced antioxidant activity prepared by the above-described manufacturing method.

[0041] The above butterbur extract may have an average content of fukinolic acid of 55 to 65 mg / g, an average content of total phenol of 200 to 400 mg / g, an average content of total flavonoid of 50 to 70 mg / g, or increase DPPH radical scavenging ability.

[0043] The present invention will be explained in detail below through examples and comparative examples. However, the following examples and comparative examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples.

[0045] Example 1 . Confirmation of indicator components of butterbur extract

[0046] 1. Steps and Conditions for Butterbur Extraction

[0047] Dried stems and leaves of butterbur were purchased from Cheongmyeong Herb (2023 domestic production, Chungbuk, Korea) and used (Fig. 1).

[0048] Dried butterbur was ground to 40 mesh, and 10 times its weight (w / v) of distilled water or 50% ethanol was added and refluxed at 50°C for 5 hours. After filtration through 100 mesh, the filtered extract was concentrated under reduced pressure in a 50°C water bath and then freeze-dried to prepare an extract powder (Fig. 2).

[0049] division amount used Solvent ratio extraction solvent extraction temperature Extraction time extraction method 1 10g 1:10 W E 50℃ 5h Reflux extraction 1: Meowi, W: Water, E: 50%EtOH

[0050] 2. Confirmation of indicator components

[0051] The indicator components of the butterbur extract were identified using HPLC, and the equipment analysis conditions were as shown in Table 2 below. The indicator components were identified by purchasing a standard (reagent, Sigma Aldrich).

[0052] equipment Waters HPLC e2695 Slope condition column Dikma C18 250*4.6mm 5um Time(min) A(%) B(%) Infusion amount 10 uL 0 90 10 detector UV 278nm 13 90 10 column temperature 30℃ 35 71 29 flux 1.0 mL / min 37 71 29 Mobile phase (A) 0.3% Phosphoric Acid / DW 42 75 25 Mobile phase (B) Acetonitrile 42.1 90 10 diluent 50% MeOH 52 90 10

[0053] Table 3 below shows the indicator substances of butterbur, and the chromatogram results for them were compared with the chromatogram results of the butterbur extract of Example 1-1 above.

[0054] The major indicator substances were found to be five types of Fukinolic acid (FA), Chlorogenic acid (CGA), 3,5-Dicaffeoylquinic acid, 4,5-Dicaffeoylquinic acid, and 3,4-Dicaffeoylquinic acid (Fig. 3).

[0056] Compounds RT MW Formula CGA 13.393 354.31 g / mol C 16 H 18 O9 FA 31.398 434.35 g / mol C 20 H 18 O 11 3,4 31.639 516.45 g / mol C 25 H 24 O 12 3,5 32.722 516.45 g / mol C 25 H 24 O 12 4,5 34.111 516.45 g / mol C 25 H 24 O 12 CGA; Chlorogenic acid, FA; Fukinolic acid,3,4; 3,4-Dicaffeoylquinic acid, 3,5; 3,5-Dicaffeoylquinic acid, 4,5; 4,5-Dicaffeoylquinic acid

[0057] As a result, according to Table 4 below, CGA was 13.02 mg / g and 13.53 mg / g and 3,4-Dicaffeoylquinic acid was 1.64 mg / g and 1.13 mg / g in W1 (butterbur distilled water extract) and E1 (butterbur 50% EtOH extract), respectively, and were similar; however, compared to W1, FA was 25.35 mg / g and 42.48 mg / g in E1, 3,5-Dicaffeoylquinic acid was 8.02 mg / g and 14.68 mg / g, and 4,5-Dicaffeoylquinic acid was 5.28 mg / g and 8.40 mg / g, with the content being higher in E1 than in W1 (Figs. 4 and 5).

[0058] In the case of the total amount of indicator substances, it was 53.31 mg / g in W1 and 80.21 mg / g in E1, indicating that the content of indicator substances was higher in the 50% ethanol extract.

[0059] Solvent CGA FA 3,4 3,5 4,5 Total W1 13.02 25.35 1.64 8.02 5.28 53.31 E1 13.53 42.48 1.13 14.68 8.40 80.21 W1; Water, E1; 50%EtOH, Unit: mg / g CGA; Chlorogenic acid, FA; Fukinolic acid,3,4; 3,4-Dicaffeoylquinic acid, 3,5; 3,5-Dicaffeoylquinic acid, 4,5; 4,5-Dicaffeoylquinic acid

[0060] 3. Check for antioxidant components

[0061] 1) Total phenolic compound content

[0062] The total phenolic compound content was determined by mixing 0.1 mL of 2N Folin-Ciocalteu reagent and 1.5 mL of distilled water with 200 μL of the sample and letting it stand at room temperature for 5 minutes, then adding 200 μL of 10% sodium carbonate (Na2CO3), blocking light, and letting it stand at room temperature for 30 minutes, after which the absorbance was measured at 760 nm using a spectrophotometer. Tannic acid was used as the standard reagent.

[0063] As a result, W1 was found to be 91.11±4.03 (mg Tannic acid / g) and E1 was 107.03±6.85 (mg Tannic acid / g) (Fig. 6).

[0065] 2) Total flavonoid content

[0066] The total flavonoid content was determined by adding 2 mL of diethylene glycol to 200 μL of the sample, adding 200 μL of 1 N NaOH, incubating at 37°C for 1 hour, and then measuring the absorbance at 420 nm using a spectrophotometer. Kaempferol was used as the standard reagent.

[0067] As a result, W1 was found to be 39.44±1.55 (mg Kaempferol / g) and E1 was 59.29±0.82 (mg Kaempferol / g) (Fig. 7).

[0069] 3) DPPH radical scavenging activity

[0070] For DPPH radical scavenging activity, samples were diluted to various concentrations using ethanol, 500 μL of each was taken, 1 mL of 200 μM DPPH solution dissolved in ethanol was added, and the mixture was reacted at room temperature (dark room) for 15 minutes, after which the absorbance was measured at 517 nm.

[0071] The DPPH radical scavenging rate is calculated from the ratio of the reduced absorbance in the sample-treated group compared to the absorbance of the control group without the sample, and the sample concentration corresponding to a 50% scavenging rate (IC) is determined using the relationship between concentration and scavenging rate. 50 ) was calculated. Ascorbic acid (Vitamin C) was used as a positive control reagent.

[0072] Vitamin C IC 50 The IC50 of fukinolic acid is 24.3 mcg / g, according to the literature. 50 The IC50 of chlorogenic acid is 7.6 mcg / g. 50 It was 34.2 mcg / g (Jahn, A., & Petersen, M., (2022)).

[0073] As a result, W1 was 48.86±13.77 (%), 108.21±31.73 (IC 50 It was found to be , mcg / g), and E1 was 49.43±9.68 (%), 103.84±20.73 (IC 50 It was found to be , mcg / g) (Fig. 8).

[0075] Example 2 Optimization design of the manufacturing method for butterbur extract

[0076] 1. Optimization Design of the Method for Manufacturing Butterbur Extract

[0077] To design a method for preparing a butterbur extract with enhanced antioxidant activity, the preparation steps and conditions of the butterbur extract were carried out as shown in Fig. 9.

[0079] 2. Confirmation of raw material indicator components

[0080] Distilled water equivalent to 20 times the weight (w / v) was added to the raw material (butterbur) and extracted once at 80°C for 4 hours. After filtration, the filtered extract was concentrated under reduced pressure in a 50°C water bath, freeze-dried, and then the indicator components were analyzed.

[0081] As a result, as shown in Table 5 below, the average content of FA, the indicator substance in the raw material, was found to be 41.21 ± 3.41 mg / g. The classifications in Table 5 are as follows: 1-1: 1st batch No. 1 production, 1-2: 1st batch No. 2 production, 2-1: 2nd batch No. 1 production, 2-2: 2nd batch No. 2 production, 3-1: 3rd batch No. 1 production, 3-2: 3rd batch No. 2 production.

[0083] division FA(mg / g) Mean SD 1-1 36.26 1.08 1-2 44.00 0.60 2-1 40.87 1.23 2-2 39.05 0.39 3-1 40.91 1.62 3-2 46.15 0.91 Aver. 41.21 3.41

[0084] 3. Verify optimal extraction conditions

[0085] 1) Requirements for Creating an Optimized Model

[0086] Optimization was performed randomly using the Central Composite Design (CCD) of the Response Surface Methodology, and the requirements for generating the optimization model are as shown in Table 6 below.

[0088] Build information Study type Response surface Design type Central composite Subtype Randomized Design model Quadratic Runs 17 Blocks No Factors Factor Name Units Minimum Maximum Coded low Coded high A Solvent % 30.00 70.00 -1↔30.00 +1↔70.00 B Temperature ℃ 30.00 70.00 -1↔30.00 +1↔70.00 C Time h 3.00 7.00 -1↔3.00 +1↔7.00

[0089] In addition, the optimization step was carried out in three stages as shown in Table 7 below, and the optimization criteria were set from 3.5σ (0.67 cpk / 97.72%) to 6.0σ (1.50 cpk / 99.99966%).

[0091] Variable Step 1 Step 2 Step 3 A B C D E F Manipulated EtOH(%) 30, 50, 70 3, 5, 7 1:10, 1:20 1, 2 FD, SD 30, 50, 70 Controlled D(1:10), E(1) 1 step set 2 step set - Solvent(%, A), Temperature(℃, B), Time(h, C), Ratio(D), Repeat(E), Dry(F)

[0092] The specific optimization steps generated accordingly are as shown in Table 8 below.

[0094] Run Step 1 Run Step 2 Run Step 3 Factor 1-1 Factor 1-2 Factor 1-3 Factor 2-1 Factor 2-2 Factor 3-1 Solvent Temperature Time Ratio Repeat Dry % ℃ h 1 70 70 7 1 1:10 2 1 FD 2 70 30 3 2 1:20 1 2 SD 3 30 70 7 3 1:20 2 4 50 50 5 5 50 50 5 6 30 30 7 7 50 30 5 8 70 70 3 9 50 70 5 10 70 50 5 11 30 50 5 12 50 50 3 13 50 50 5 14 50 50 7 15 30 70 3 16 70 30 7 17 30 30 3

[0095] 2) Step 1: Establish Optimization Conditions

[0096] According to Table 9, Figures 10 and 11 below, the first stage optimization result was selected at 50% EtOH, 30℃, and 5h.

[0097] It was confirmed that the Solvent(%, A) condition yielded a value of 50.581, indicating that 50% EtOH is the optimal solvent concentration, the Temperature(°C, B) condition yielded a value of 30.634, indicating that 30°C is the optimal temperature, and the Time(h, C) condition yielded a value of 4.664, indicating that 5h is the optimal time condition.

[0099] p-value A B C σ Yield(%) CGA * - - 3.5 97.72 FA * *** - 5.5 99.9968 *: P<0.05, **: P<0.01, ***: P<0.001

[0100] 3) Establishment of Step 2 optimization conditions

[0101] According to Fig. 12, the second stage optimization result was selected at 1:10, 50% EtOH, 30℃, and 5h.

[0102] The average CGA content was 0.0632±0.0019 mg / mL, there was no difference in the conditions for the highest value, the highest value was 0.0653 mg / mL, and the cpk value was 0.0511(0.0457-0.0564).

[0103] The average FA content was 0.1803±0.0137 mg / mL, the conditions for the highest value were 10 times the solvent and 5h, the highest value was 0.1962 mg / mL, and the cpk value was 0.1562 (0.1281-0.1843).

[0105] 4) Establishment of 3-Step Optimization Conditions

[0106] According to Fig. 13, the 3-step optimization result was selected as 1:10, 50% EtOH, 30℃, 5h, SD.

[0107] Regarding drying conditions, FD took 5 days and SD took 1 day, so SD was selected as the optimal condition.

[0108] The average CGA content was 14.72±0.05 mg / g, with values ​​of 14.76 mg / g under SD (Spray-Dried) conditions and 14.68 mg / g under FD (Freeze-Dried) conditions.

[0109] The FA content averaged 44.30±1.62 mg / g, with values ​​of 43.16 mg / g under SD conditions and 45.45 mg / g under FD conditions.

[0111] 4. Confirmation of Indicator Components of Butterbur Extract According to Optimization Conditions

[0112] 1) Check FA content

[0113] According to the optimization conditions established in Examples 2-3 above, 50% ethanol equivalent to 10 times the weight (w / v) was added to the raw material (butterbur) and extracted once at 30°C for 5 hours. After filtration, the filtered extract was concentrated under reduced pressure in a 50°C water bath, spray-dried, and then the indicator components were analyzed.

[0114] As a result, according to Table 10 below, the average content of FA, the indicator substance of the optimized extract powder, was found to be 60.36±2.25 mg / g. This result was confirmed to be 1.42 times higher than the average content of FA, the indicator substance, before optimization, which was 42.48 mg / g (E1).

[0116] LOT FA(mg / g) Mean SD 1 62.89 0.75 2 58.01 0.64 3 60.18 1.18 Aver. 60.36 2.25

[0117] The above results are superior to the results obtained by hot-air drying butterbur leaves at 50°C for 24 hours, adding 15 times the amount of distilled water to the dried leaves for reflux extraction at 100°C for 4 hours and freeze-drying, adding 4 times the amount of 70% EtOH to the prepared powder, shaking at room temperature for 24 hours, and then freeze-drying, in which the fuguinolic acid content was 26.57±2.32 mg / g (Eun Jin Kim et al. (2020)). It was also confirmed that these results are superior to the results obtained by freezing edible parts (leaves, stems) of butterbur at -70°C and freeze-drying them, extracting the prepared powder with 80% EtOH for 24 hours and then freeze-drying, in which the fuguinolic acid content was 235.0±4.8 mg / 100g DW (Eun Mi Ahn et al. (2020)).

[0119] 2) Verification of Indicator Component Test Method

[0120] Fukinolic acid method validation involves reviewing specificity, linearity, accuracy, precision, limit of quantification (LOQ), and limit of detection (LOD) to verify the validity of the analytical method for quantifying Fukinolic acid in butterbur extract using an established method. The set values ​​according to the evaluation method for all items were verified as shown in Table 11 below.

[0122] item Evaluation method setting value Specificity · Detection time during HPLC analysis (Review of retention time) · Fukinolkic acid detection time: Peak detected at the 24-minute mark Linearity standard · Confirmed linearity with 3 replications at 5 concentrations · Verified at standard concentrations of 6.25–100 mg / L · R^2 = 0.999 or higher Sample · Confirmed linearity with 3 replications at 5 concentrations · Verified at 40-160% of the standard concentration · R^2 = 0.999 or higher Accuracy · Review recovery rate using standard substances at 3 concentrations · Verified at 50-150% of standard concentration · Average recovery rate (%): 97.15-101.95 Precision · Evaluation of repeatability and daily / inter-instrument / inter-examiner precision by two examiners using two types of instruments over 6 days · RSD(%): 0.66-1.18 Limit of Quantity (LOQ) · Limit of Quantification (LOQ) = (10 × standard deviation of the y-intercept) / average value of the calibration curve slope · Limit of quantification (mg / L): 71.5862 Limit of Detect (LOD) · Limit of Detection (LOD) = (3.3 × standard deviation of y-intercept) / average value of calibration curve slope · Detection limit (mg / L): 23.6234

[0123] 5. Confirmation of Antioxidant Components in Butterbur Extract According to Optimization Conditions

[0124] 1) Total phenolic compound content

[0125] The total phenolic compound content was determined by mixing 0.1 mL of 2N Folin-Ciocalteu reagent and 1.5 mL of distilled water with 200 μL of the sample and letting it stand at room temperature for 5 minutes, then adding 200 μL of 10% sodium carbonate (Na2CO3), blocking light, and letting it stand at room temperature for 30 minutes, after which the absorbance was measured at 760 nm using a spectrophotometer. Tannic acid was used as the standard reagent.

[0126] As a result, LOT1 showed values ​​of 334.35±8.64 (mg Tannic acid / g), LOT2 showed 305.44±8.02 (mg Tannic acid / g), and LOT3 showed 316.90±6.31 (mg Tannic acid / g), with a final average value of 318.90±14.27 (mg Tannic acid / g), which was confirmed to be 2.98 times higher than 107.03±6.85 (mg Tannic acid / g, E1) before optimization (Fig. 14).

[0128] 2) Total flavonoid content

[0129] The total flavonoid content was determined by adding 2 mL of diethylene glycol to 200 μL of the sample, adding 200 μL of 1 N NaOH, incubating at 37°C for 1 hour, and then measuring the absorbance at 420 nm using a spectrophotometer. Kaempferol was used as the standard reagent.

[0130] As a result, LOT1 showed values ​​of 65.03±1.54 (mg Kaempferol / g), LOT2 showed 54.40±0.58 (mg Kaempferol / g), and LOT3 showed 60.50±0.29 (mg Kaempferol / g), with a final average value of 59.98±4.69 (mg Kaempferol / g), which was confirmed to be similar to 59.29±0.82 (mg Kaempferol / g, E1) before optimization (Fig. 15).

[0132] 3) DPPH radical scavenging activity

[0133] DPPH radical scavenging activity was determined by diluting the samples to various concentrations using ethanol, taking 500 μL of each, adding 1 mL of 200 μM DPPH solution dissolved in ethanol, reacting at room temperature (in the dark) for 15 minutes, and measuring the absorbance at 517 nm. The DPPH radical scavenging rate was calculated from the ratio of the decrease in absorbance in the sample-treated group to the absorbance of the control group without the sample, and the sample concentration corresponding to a 50% scavenging rate (IC₀) was determined using the relationship between concentration and scavenging rate. 50 ) was calculated. Ascorbic acid (Vitamin C) was used as a positive control reagent.

[0134] Vitamin C IC 50 The IC50 of fukinolic acid is 24.3 mcg / g, according to the literature. 50 The IC50 of chlorogenic acid is 7.6 mcg / g. 50 It was 34.2 mcg / g.

[0135] As a result, regarding DPPH radical scavenging activity (%), LOT1 showed values ​​of 54.44±0.79 (%), LOT2 showed 48.16±2.76 (%), and LOT3 showed 50.66±0.57 (%), with a final average value of 51.08±3.10 (%), which was confirmed to be an increase compared to 49.43±9.68 (%) before optimization (E1) (Fig. 16).

[0136] As a result, DPPH radical scavenging activity IC 50 In relation to this, LOT1 is 91.86±1.33 (IC 50 , mcg / g), LOT2 is 104.04±5.80 (IC 50 , mcg / g), LOT3 was 98.71±1.12 (IC 50 It showed values ​​of , mcg / g), and the final average was 98.21±6.10 (IC 50 It had a value of , mcg / g). Before optimization (E1) 103.84±20.73 (IC 50 It had a value of , mcg / g) (Fig. 16).

[0138] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0139] The scope of the present invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

Claims

Claim 1 coltsfoot( Petasites japonicus A method for preparing a butterbur extract comprising the steps of: extracting and filtering using a solvent containing distilled water or ethanol; concentrating the filtered butterbur extract under reduced pressure; and drying the pressure-concentrated butterbur extract. Claim 2 A method for preparing a butterbur extract according to claim 1, characterized in that the ethanol is 30 volume% to 70 volume% ethanol. Claim 3 A method for preparing a butterbur extract according to claim 1, characterized in that the weight ratio of the butterbur and the solvent is 1:10 to 20. Claim 4 A method for preparing a butterbur extract according to claim 1, characterized in that the extraction is performed under conditions of 30℃ to 70℃ and 3 to 7 hours. Claim 5 A method for preparing a butterbur extract according to claim 1, characterized in that the drying is freeze-drying or spray-drying. Claim 6 Butterbur extract prepared by the method of any one of claims 1 to 5. Claim 7 The butterbur extract of claim 6, characterized in that the butterbur extract has an average content of fukinolic acid of 55 to 65 mg / g, an average content of total phenol of 200 to 400 mg / g, an average content of total flavonoid of 50 to 70 mg / g, or increases DPPH radical scavenging activity.

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