KR101895430B1 - Method of separating fukinolic acid from petasites japonicus - Google Patents

Method of separating fukinolic acid from petasites japonicus Download PDF

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KR101895430B1
KR101895430B1 KR1020170098111A KR20170098111A KR101895430B1 KR 101895430 B1 KR101895430 B1 KR 101895430B1 KR 1020170098111 A KR1020170098111 A KR 1020170098111A KR 20170098111 A KR20170098111 A KR 20170098111A KR 101895430 B1 KR101895430 B1 KR 101895430B1
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column chromatography
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extract
ethanol
present
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장대식
박상수
김해미
이자연
이진수
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주식회사 네이처바이오
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
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Abstract

The present invention relates to a method for separating fukinolic acid from a Petasites japonicus extract. According to the present invention, a large amount of high purity fukinolic acid can be obtained from the Petasites japonicus extract, and fukinolic acid can be used as a commercialized standard form. The method comprises the steps of: (a) adding water to Petasites japonicus leaves and performing extraction; (b) freeze-drying the extract; (c) adding ethanol to the extract and performing precipitation; (d) removing supernatant and freeze-drying the precipitate; (d) performing diaion column chromatography; (f) performing sephadex column chromatography; and (e) performing lichroprep column chromatography.

Description

머위 잎으로부터 푸키놀릭산을 분리하는 방법{METHOD OF SEPARATING FUKINOLIC ACID FROM PETASITES JAPONICUS}METHOD OF SEPARATING FUKINOLIC ACID FROM PETASITES JAPONICUS BACKGROUND OF THE INVENTION 1. Field of the Invention < RTI ID = 0.0 >

본 발명은 머위 잎 추출물로부터 푸키놀릭산을 분리하는 방법에 관한 것이다.The present invention relates to a method for separating fucinolic acid from a warty leaf extract.

푸키놀릭산(fukinolic acid)은 한약재 승마의 유효 성분으로 알려져 있고, 승마는 만성난소염, 자궁내막염, 월경불순, 월경통, 월경과다, 불임 등 부인과 증상 완화와 진통 효과에 효능이 있는 것으로 알려져 있다. 이에, 승마에 함유된 식물성 에스트로겐이 폐경여성의 에스트로겐 대체물로 효과를 나타낼 가능성이 있으나, 아직까지 그에 대한 구체적인 연구는 진행되고 있지 않다.Fukinolic acid is known to be an active ingredient in herbal medicines, and horse riding is known to be effective in relieving gynecological symptoms and analgesic effects such as chronic ovarian, endometritis, menstrual irregularities, menstrual cramps, menstrual overgrowth and infertility. Thus, although estrogen in rats may be effective as estrogen replacement in postmenopausal women, no specific studies have been conducted to date.

머위(일반명: Butterbur 또는 학명: Petasites japonicus)는 유럽뿐만 아니라 아시아 일부 및 북미에서 발견되는 다년생 관목이다. 머위의 뿌리 및 잎으로부터 분리한 추출물은 2,000년 이상 동안 치료제의 용도로 사용되었다. 오늘날 머위 추출물은 근육을 이완하는데 주로 사용되고, 위장내 복통, 평활근의 경련과 같은 상태에 처치하며, 통증 완화 효과가 있고 또한 두통에도 사용될 수 있다고 보고되었다(Eaton J., Townsend Lett, 2000, 202, 104-106). 또한, 머위는 천식 및 알레르기성 질환에 대해 치료 효과가 있다고 보고된 바 있다(Thomet OA, Simon HU, Int Arch Allergy Immunol, 2002, 129(2), 108-12). Butterbur (generic name: Butterbur or scientific name: Petasites japonicus) is a perennial shrub found not only in Europe but also in parts of Asia and North America. Extracts isolated from roots and leaves of wafers were used for therapeutic applications for over 2,000 years. Today, it has been reported that butterbur extracts are mainly used to relax muscles, to treat stomach intoxication, smooth muscle spasms, pain relief, and also for headache (Eaton J., Townsend Lett, 2000, 104-106). It has also been reported that the warts have therapeutic effects on asthma and allergic diseases (Thomet OA, Simon HU, Int Arch Allergy Immunol, 2002, 129 (2), 108-12).

푸키놀릭산은 승마의 지표 성분으로 알려져 있으나 머위 잎에서 순도 높은 푸키놀릭산을 분리하여 상용화한 예는 없었다. 본 발명자들은 머위 잎 추출물에서 상용화되지 않은 푸키놀릭산을 분리하는 데 성공하였고 이를 상용화하여 표준 지표로 사용하거나 지표 성분을 확인하는 용도로 사용하고자 본 발명을 완성하였다.Although puchinolic acid is known to be an index component of horse riding, there is no commercialization of puchinolic acid, which has high purity, in leaves. The present inventors succeeded in isolating fucinolic acid, which is not commercialized in the butterfly leaf extract, and completed the present invention in order to commercialize it as a standard index or to use as an indicator component.

본 발명의 목적은 (a) 머위 잎에 물을 첨가하여 추출하는 단계; (b) 상기 머위 잎 추출물을 동결 건조하는 단계; (c) 상기 머위 잎 추출물의 동결 건조물에 에탄올을 첨가한 후 침전시키는 단계; (d) 상기 침전물의 상층액을 제거하고 침전물을 동결 건조하는 단계를 포함하는 머위 잎 추출물을 수득하는 단계; (e) 상기 추출물을 다이아이온 컬럼 크로마토그래피하는 단계; (f) 상기 다이아이온 컬럼 크로마토그래피하여 얻은 용출액을 세파덱스 컬럼 크로마토그래피하는 단계; 및 (g) 상기 세파덱스 컬럼 크로마토그래피하여 얻은 용출액을 라이크로프렙 컬럼 크로마토그래피하는 단계를 포함하는 푸키놀릭산을 수득하는 방법을 제공하는 것이다.The object of the present invention is (a) a method of extracting wooly leaves by adding water to extract leaves of wooly leaves; (b) freeze-drying the wooly leaf extract; (c) adding ethanol to the lyophilized product of the woolly leaf extract and precipitating the lyophilized product; (d) removing the supernatant of the precipitate and lyophilizing the precipitate; (e) Diaion column chromatography of the extract; (f) Sephadex column chromatography of the eluate obtained by the above diionic column chromatography; And (g) subjecting the eluate obtained by the Sephadex column chromatography to leucoprene column chromatography to obtain a picolinic acid.

본 발명은 상기 목적을 달성하기 위하여, (a) 머위 잎에 물을 첨가하여 추출하는 단계; (b) 상기 머위 잎 추출물을 동결 건조하는 단계; (c) 상기 머위 잎 추출물의 동결 건조물에 에탄올을 첨가한 후 침전시키는 단계; (d) 상기 침전물의 상층액을 제거하고 침전물을 동결 건조하는 단계를 포함하는 머위 잎 추출물을 수득하는 단계; (e) 상기 추출물을 다이아이온 컬럼 크로마토그래피하는 단계; (f) 상기 다이아이온 컬럼 크로마토그래피하여 얻은 용출액을 세파덱스 컬럼 크로마토그래피하는 단계; 및 (g) 상기 세파덱스 컬럼 크로마토그래피하여 얻은 용출액을 라이크로프렙 컬럼 크로마토그래피하는 단계를 포함하는 푸키놀릭산을 수득하는 방법을 제공한다. In order to achieve the above-mentioned object, the present invention provides a method of extracting waxy leaves, comprising the steps of: (a) (b) freeze-drying the wooly leaf extract; (c) adding ethanol to the lyophilized product of the woolly leaf extract and precipitating the lyophilized product; (d) removing the supernatant of the precipitate and lyophilizing the precipitate; (e) Diaion column chromatography of the extract; (f) Sephadex column chromatography of the eluate obtained by the above diionic column chromatography; And (g) subjecting the eluate obtained by the Sephadex column chromatography to cyclohexane column chromatography to obtain picinolic acid.

상기 (c) 단계에서 에탄올은 50% 내지 80% 에탄올인 것을 특징으로 하고, 상온에서 20 내지 30 시간 동안 침전시키는 것을 특징으로 한다. In the step (c), the ethanol is 50% to 80% ethanol. The ethanol is precipitated at room temperature for 20 to 30 hours.

본 발명에서 수득한 푸키놀릭산은 순도 95% 내지 98%인 것을 특징으로 할 수 있다. The puincinolic acid obtained in the present invention may be characterized by a purity of 95% to 98%.

본 발명에 있어서, 에탄올은 50% 내지 80% 에탄올인 경우에 불필요한 성분이 잘 녹아나게 되고 특히 에탄올 70%에서 머위 잎 물 추출 동결건조품의 불필요한 성분이 잘 녹아나게 되어, 불필요한 성분이 녹아 있는 상층부를 제거하고 침전물만 회수하여 유용한 머위 잎 추출물을 획득할 수 있다. 본 발명에 따르면, 머위 잎을 물 추출하는 단계, 동결 건조하는 단계, 에탄올 추출하는 단계를 순차적으로 거침으로써, 머위 잎의 불필요한 성분을 효과적으로 제거하고 유용한 성분만을 수득할 수 있게 된다.In the present invention, when 50% to 80% ethanol of ethanol is used, unnecessary components are well melted. Especially, unnecessary components of freeze-dried product of water-extracted freeze-dried water from 70% ethanol are melted well. And only the precipitate can be recovered to obtain a useful curd leaf extract. According to the present invention, it is possible to effectively remove unnecessary components from the wooly leaves and to obtain only useful components by sequentially performing the step of water-extracting the wooly leaves, the step of freeze-drying and the step of extracting ethanol.

본 발명에 따르면 머위 잎 추출물을 다이아이온 컬럼 크로마토그래피(7.0 × 44.8 cm, Acetone/H2O=0/10→ 2/8 → 4/6 → 6/4 → 8/2 → 10/0), 세파덱스 컬럼크로마토그래피(4.5 × 56.2 cm, Acetone/H2O=2/8), 라이크로프렙 컬럼크로마토그래피(4.9 × 45.0 cm, Acetone/H2O=2/8)를 순차적으로 실시함으로써 분리 단계를 줄이고 이를 통해 푸키놀릭산을 수득할 수 있다.According to the present invention, the wooly leaf extract was purified by Diaion column chromatography (7.0 × 44.8 cm, Acetone / H 2 O = 0/10 → 2/8 → 4/6 → 6/4 → 8/2 → 10/0) Separation was carried out by sequential sequential column chromatography on Sephadex (4.5 x 56.2 cm, Acetone / H 2 O = 2/8) and by recrystallization column chromatography (4.9 x 45.0 cm, Acetone / H 2 O = 2/8) Step can be reduced and puccinolic acid can be obtained.

본 발명에 따르면 머위 잎 추출물로부터 순도 높은 푸키놀릭산을 대량 수득할 수 있고, 이를 상용화된 표준폼으로 사용할 수 있다.According to the present invention, it is possible to obtain a large amount of high purity pucolinic acid from a wooly leaf extract and use it as a commercialized standard foam.

도 1은 본 발명에 따라 수득한 푸키놀릭산의 NMR 실험 결과를 나타낸 것이다.
도 2는 본 발명에 따라 수득한 푸키놀릭산의 HPLC 크로마토그램 결과를 나타낸 것이다.
도 3 및 4는 본 발명에 따라 수득한 푸키놀릭산의 qNMR 실험 결과를 나타낸 것이다.Fig. 1 shows NMR results of puincinolic acid obtained according to the present invention.
2 shows HPLC chromatogram results of puincinolic acid obtained according to the present invention.
Figures 3 and 4 show the results of the qNMR experiment of fucinolic acid obtained according to the present invention.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당 업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

시약 및 기기Reagents and devices

다이아이온(Diaion HP-20), 세파덱스(Sephadex LH-20; Amersham Pharmacia Biotech) 및 라이크로프렙(LiChroprep RP-18; Merck, 40-63 m)을 컬럼크로마토그래피에 사용하였다. TLC 분석은 Silica gel 60 glass plates (Merck), Silica gel 60 RP 18 F254sglassplates(Merck)를 이용하였으며, 20% (v/v) H2SO4시약 (Aldrich)에 담근 후 110℃에 10분간 발색시켰다. 실험에 사용된 모든 유기용매는 증류한 후 사용하였다. NMR 실험은 JEOL 500 MHz NMR spectrometer로 수행하였으며, 화학식 이동값(chemical shifts)은 잔류 NMR 용매나 TMS를 기준으로 측정하였다.Diaion HP-20, Sephadex LH-20 (Amersham Pharmacia Biotech) and Lychroprep RP-18 (Merck, 40-63 m) were used for column chromatography. TLC analysis was performed using silica gel 60 glass plates (Merck) and silica gel 60 RP 18 F 254s glassplates (Merck), immersed in 20% (v / v) H 2 SO 4 reagent (Aldrich) It was developed. All organic solvents used in the experiment were used after distillation. NMR experiments were performed with a JEOL 500 MHz NMR spectrometer and chemical shifts were measured based on residual NMR solvent or TMS.

실험 재료Experimental material

분리에 사용된 머위 잎(Petasites japonicus)은 물을 첨가하여 환류 추출하고 동결 건조 한 뒤 생기는 파우더를 70% 에탄올에 녹여서 생기는 침전물을 KP-1 라 명칭하고 사용하였다.The leaves used for separation ( Petasites japonicus ) was refluxed by adding water, lyophilized, and the resulting powder was dissolved in 70% ethanol and the resulting precipitate was named KP-1.

머위 잎 추출물(KP-1)의 제조 및 성분 분리 Preparation and component separation of butterfly leaf extract (KP-1)

4 배수의 물을 첨가하여 4시간 동안 100℃ 에서 추출한 뒤 추출물을 동결 건조시켜 동결 건조품(250 g)을 수득하였다(머위 잎 대비 25%). 그 다음, 파우더 형태의 머위 잎 추출물 동결 건조품에 70% 에탄올을 첨가하여 24 시간 동안 상온에서 용출시켰다. 불필요한 물질이 녹아있는 상층부를 제거하고 머위 잎 추출물만 남아있는 침전물을 회수하여 동결 건조하여 동결건조품(200g)을 수득하였다(물 추출 동결건조품 대비 80%). 침전물(KP-1) 100g에 대하여 Diaion HP-20 컬럼 크로마토그래피(7.0 × 44.8 cm, Acetone/H2O=0/10→ 2/8 → 4/6 → 6/4 → 8/2 → 10/0)를 실시하여 15개의 분획물(F1~F15)로 분리하였다. 이 중 분획물 F6(6.9 g)에 대하여 세파덱스 컬럼크로마토그래피(4.5 × 56.2 cm, Acetone/H2O=2/8)를 실시하여 9개의 소분획물(F6-1~F6-9)로 나누었다. 소분획물 F6-5(1.7 g)을 라이크로프렙 컬럼크로마토그래피(4.9 × 45.0 cm, Acetone/H2O=2/8)를 실시하여 화합물 1(510 mg)을 분리하였다. Four times more water was added and the mixture was extracted at 100 ° C for 4 hours. The extract was lyophilized to obtain 250 g of a lyophilized product (25% as compared to a curd leaf). Then, 70% ethanol was added to the lyophilized powder of waxy leaf extract in powder form and eluted at room temperature for 24 hours. The supernatant from which the unnecessary substances were dissolved was removed, and the remaining precipitate with only the wooly leaf extract was recovered and lyophilized to obtain 200 g of a lyophilized product (80% of the water-extracted freeze-dried product). Diaion HP-20 column chromatography (7.0 × 44.8 cm, Acetone / H 2 O = 0/10 → 2/8 → 4/6 → 6/4 → 8/2 → 10/10) was carried out on 100 g of the precipitate (KP- 0) was performed to separate 15 fractions (F1 to F15). The fraction F6 (6.9 g) was fractionated into nine small fractions (F6-1 to F6-9) by Sephadex column chromatography (4.5 × 56.2 cm, Acetone / H 2 O = 2/8). The fraction F6-5 (1.7 g) was subjected to recrystallization by column chromatography (4.9 × 45.0 cm, Acetone / H 2 O = 2/8) to separate Compound 1 (510 mg).

NMR 실험을 통한 화합물 1의 구조 확인Identification of Structure of Compound 1 by NMR Experiment

화합물 1을 NMR(Varian 500 MHz)을 통해 화합물 1의 구조를 확인하였다(도 1).Compound 1 was confirmed by NMR (Varian 500 MHz) to have the structure of Compound 1 (FIG. 1).

푸키놀릭산(Fukinolic acid)- Brownish yellow powder; Fukinolic acid - Brownish yellow powder;

1H-NMR(500MHz,D2O)δ 2.84/2.88 (2H, d, J = 14.0 Hz, H-3), 3.36 (1H, s, H-2), 6.22 (1H, d, J = 16.0, H-2’’), 6.62 (1H, dd, J = 8.0, 2.0 Hz, H-6’), 6.72 (1H, d, J = 8.0 Hz, H-5’), 6.74 (1H, d, J = 1.5 Hz, H-2’), 6.77 (1H, d, J = 8.0 Hz, H-5’’’), 6.93 (1H, dd, J = 8.5, 1.5 Hz, H-6’’’), 7.01 (1H, d, J = 2.0 Hz, H-2’’’), 7.53 (1H, d, J = 16.0, H-3’’); 1 H-NMR (500MHz, D 2 O) δ 2.84 / 2.88 (2H, d, J = 14.0 Hz, H-3), 3.36 (1H, s, H-2), 6.22 (1H, d, J = 16.0 , 6.72 (1H, d, J = 8.0 Hz, H-5 '), 6.74 (1H, d, J = 8.0, 2.0 Hz, H- J = 1.5 Hz, H-2 '), 6.77 (1H, d, J = 8.0 Hz, H-5''), 6.93 (1H, dd, J = 8.5, 1.5 Hz, H- , 7.01 (1H, d, J = 2.0 Hz, H-2 '''), 7.53 (1H, d, J = 16.0, H-3'');

13C-NMR(125MHz,D2O)δ 40.3 (C-4), 79.3 (C-2), 80.4 (C-3), 114.1 (C-2’’), 115.0 (C-2’’’), 115.7 (C-5’), 116.0 (C-5’’’), 118.2 (C-2’), 122.7 (C-6’’’), 122.8 (C-6’), 126.9 (C-1’’’), 129.1 (C-1’), 142.5 (C-4’), 143.2 (C-3’), 144.1 (C-3’’’), 146.4 (C-3’’), 147.0 (C-4'''), 168.9 (C-1’’), 174.0 (C-1), 177.3 (C-5). 13 C-NMR (125MHz, D 2 O) δ 40.3 (C-4), 79.3 (C-2), 80.4 (C-3), 114.1 (C-2 ''), 115.0 (C-2 ''' ), 115.7 (C-5 '), 116.0 (C-5'''), 118.2 (C-2 '), 122.7 1 ''), 129.1 (C-1 '), 142.5 (C-4'), 143.2 (C-3 '), 144.1 (C-3' (C-4 '''), 168.9 (C-1''), 174.0 (C-1), 177.3 (C-5).

[화합물 1][Compound 1]

Figure 112017074689994-pat00001
Figure 112017074689994-pat00001

HPLC 실험 HPLC Experiment

표준물질 5mg을 정밀하게 달아 25mL 정용 플라스크에 증류수를 넣어 용해한 후 적정 농도로 희석하여 표준용액으로 사용하였다. 시험용액의 제조를 위해 시료를 적절히 취한 후 100mL 정용 플라스크에 넣고 증류수를 넣은 후 30분 동안 초음파 추출하였다. 초음파 추출물을 방냉한 후 증류수로 표선까지 맞추고 0.45um PTFE 멤브레인 필터로 여과하여 시험용액으로 사용하였다. 기기분석 조건은 다음과 같다:5 mg of the standard substance was precisely weighed, dissolved in distilled water in a 25 mL constant volume flask, diluted to the appropriate concentration, and used as the standard solution. For the preparation of the test solution, the sample was appropriately taken, placed in a 100 mL constant volume flask, and distilled water was added thereto, followed by ultrasonic extraction for 30 minutes. The supernatant extract was allowed to cool, then mixed with distilled water to the same level, and filtered through a 0.45um PTFE membrane filter. Instrumental analysis conditions are as follows:

기기(Instrument)Instrument HPLCHPLC 검출기(Detector)Detector UV (DAD)UV (DAD) 파장(Wavelength)Wavelength 330nm330nm 컬럼(Column)Column Supelco C18 (5um, 4.6*250mm)Supelco C18 (5 [mu] m, 4.6 * 250 mm) 이동상(Mobile Phase)Mobile Phase A: 0.1% Formic acid in DW
B: 0.1% Formic acid in Acetonitrile

Figure 112017074689994-pat00002
A: 0.1% Formic acid in DW
B: 0.1% Formic acid in Acetonitrile

Figure 112017074689994-pat00002
유량(Flow rate)Flow rate 0.8mL/min0.8 mL / min 주입용량(Injection volume)Injection volume 10㎕10 μl 실행 시간(Run Time)Run Time 53min53min 컬럼 온도(Column temperature)Column temperature 25℃25 ℃

계산식:formula:

Figure 112017074689994-pat00003
Figure 112017074689994-pat00003

그 결과, 도 2에 나타난 바와 같이 피크(peak)가 1개로 검출되는 것으로 보아, 불순물이 함유되지 않은 채로 푸키놀릭산이 순수하게 분리된 것을 확인할 수 있었다.As a result, as shown in FIG. 2, one peak was detected, and it was confirmed that the puchinolic acid was purely separated without containing any impurities.

[실시예 4][Example 4]

qNMR 실험qNMR experiment

푸키놀릭산의 순도를 확인하기 위하여 qNMR(Varian NMR system 500MHz, VnmrJ 2.2 for acquisition, mNova 9.0 for processing) 실험을 수행하였다. 그 결과, 도 3 및 도 4에 나타난 바와 같이 순도(97.37%) 높은 푸키놀릭산을 수득할 수 있었다.To confirm the purity of puchinolic acid, qNMR (Varian NMR system 500 MHz, VnmrJ 2.2 for acquisition, mNova 9.0 for processing) was performed. As a result, puincinolic acid having a high purity (97.37%) as shown in Fig. 3 and Fig. 4 could be obtained.

Claims (4)

(a) 머위 잎에 물을 첨가하여 추출하는 단계;
(b) 상기 머위 잎 추출물을 동결 건조하는 단계;
(c) 상기 머위 잎 추출물의 동결 건조물에 에탄올을 첨가한 후 침전시키는 단계;
(d) 상기 침전물의 상층액을 제거하고 침전물을 동결 건조하는 단계를 포함하는 머위 잎 추출물을 수득하는 단계;
(e) 상기 추출물을 다이아이온 컬럼 크로마토그래피하는 단계;
(f) 상기 다이아이온 컬럼 크로마토그래피하여 얻은 용출액을 세파덱스 컬럼 크로마토그래피하는 단계; 및
(g) 상기 세파덱스 컬럼 크로마토그래피하여 얻은 용출액을 라이크로프렙 컬럼 크로마토그래피하는 단계를 포함하는 푸키놀릭산을 수득하는 방법.
(a) adding water to the leaves of waxy leaves;
(b) freeze-drying the wooly leaf extract;
(c) adding ethanol to the lyophilized product of the woolly leaf extract and precipitating the lyophilized product;
(d) removing the supernatant of the precipitate and lyophilizing the precipitate;
(e) Diaion column chromatography of the extract;
(f) Sephadex column chromatography of the eluate obtained by the above diionic column chromatography; And
(g) subjecting the eluate obtained by the Sephadex column chromatography to cyclohexane column chromatography.
제 1항에 있어서,
상기 에탄올은 50% 내지 80% 에탄올인 것을 특징으로 하는 머위 잎 추출물로부터 푸키놀릭산을 수득하는 방법.
The method according to claim 1,
Wherein the ethanol is 50% to 80% ethanol. ≪ Desc / Clms Page number 20 >
제 1항에 있어서,
상기 (c) 단계는 상온에서 20 시간 내지 30 시간 동안 침전시키는 것을 특징으로 하는 머위 잎 추출물로부터 푸키놀릭산을 수득하는 방법.
The method according to claim 1,
Wherein the step (c) is carried out at room temperature for 20 to 30 hours.
제 1항에 있어서,
상기 수득한 푸키놀릭산은 순도 95% 내지 98%인 것을 특징으로 하는 머위 잎 추출물로부터 푸키놀릭산을 수득하는 방법.
The method according to claim 1,
Wherein the puchinolic acid obtained is a purity of 95% to 98%.
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