KR20030068640A - Antioxidant from pinus densiflora and process for isolation thereof - Google Patents

Abstract

PURPOSE: Provided are an antioxidant from the pine needle and an isolation process thereof. The isolated antioxidant has excellent activity as a OH scavenger. CONSTITUTION: An antioxidant from the pine needle is isolated by the steps of: extracting 8.5kg of pine needles with methanol for 3 hours; suspending the extract with distilled water and fractioning it with ethylacetate; purifying the ethylacetate fraction by silica gel column chromatography; and purifying a fraction having antioxidation activity by silica gel chromatography using a mixture of dichloromethane and methanol. The antioxidant includes kaempferol 3-O-galactoside 6'-acetate, kaempferol 3-O-galactoside, and isolariciresinol xyloside.

Description

Antioxidants from pine needles and its separation method {ANTIOXIDANT FROM PINUS DENSIFLORA AND PROCESS FOR ISOLATION THEREOF}

The present invention relates to a natural compound having antioxidant activity isolated from pine needles. More specifically, the present invention relates to an antioxidant having excellent radical scavenging ability separated from the ethyl acetate fraction obtained from the methanol extract of pine needles.

In addition to hydroxyl radicals, free radicals and reactive oxygen species or reactive nitrogen species and peroxynitrite are well known to play an important role in causing a wide variety of human diseases. 1,1-diphenyl-2-picrylhydrazyl (DPPH) is a free radical compound and has been widely used to test the free radical scavenging ability of various chemicals. Superoxide (superoxide) and generated as a result of the reaction of nitric oxide (nitric oxide) peroxy nitrite (ONOO ^-) is a species which destroys the cell by oxidation of some cellular components such as proteins, lipids and DNA. It is known to be associated with diseases such as Alzheimer's disease, rheumatoid arthritis, cancer and atherosclerosis. In addition, reactive oxygen species (ROS) are closely associated with various degenerative disorders associated with aging. Reactive oxygen species (ROS), such as hydrogen peroxide, superoxide anion, hydroxyl radicals, and nitric oxide, all form aerobic metabolism and all intracellular components such as nucleic acids, proteins, and lipids. As a result of this damage is produced in the body. Various efforts have been made to prevent oxidative damage caused by the various radicals, and there is a growing interest in natural compounds having antioxidant activity. Among the antioxidants, antioxidants that inhibit lipid peroxidation play an important role in not only preserving food but also protecting oxidative damage of living cells. Many natural products found in plants are known to retard the oxidation process in natural environments as scavengers of free radicals and free radicals, and in experiments as reducing agents. The present inventors are studying the antioxidant activity of the above-mentioned natural compounds, and have been filed with Korean Patent Application No. 2001-55753 by separating novel natural compounds having antioxidant activity from cherry leaves.

Song or red pines grow spontaneously, but in Korea, Japan, and China, they were raised in mountainous areas. The leaves of red pine have long been used as a nourishing tonic in Korean folk medicine. In Korea, pine needles have been used as tea. The present inventors have published that the methanol extract of pine needles has an effect of scavenging radicals against 1,1-diphenyl-2-picrylhydrazyl radicals. In addition, the camp-dihydro Ferrol (dihydrokaempferol) and 1- O, which are not the active compound from the methanol extract-catechins ((+) - - catechin) of (+) active ingredient, with benzoyl glucoside (1- O -benzoyl glucoside) Has been separated.

Accordingly, it is an object of the present invention to provide an antioxidant having excellent antioxidant activity and its separation method from pine needles.

The object of the present invention is to obtain a methanol extract from pine needles, and to distribute the concentrated powder of the methanol extract to dichloromethane, ethyl acetate, butanol, distilled water, and then to investigate the antioxidant activity of the extract, the antioxidant activity is the best Purification of ethyl acetate extract by silica gel column chromatography to obtain natural compounds and to investigate the radical scavenging ability of each compound, UV, IR spectra, ¹ H and ¹³ C-NMR analysis and COSY, HMQC and HMBC analysis Achieved by analyzing the compound.

1 shows the structural formula of Compound 1-3 isolated from pine needles.

The present invention provides a methanol extract from pine needles, and fractionating the concentrated powder of the methanol extract with dichloromethane, ethyl acetate, butanol and distilled water; Examining the antioxidant activity of the extract; Purifying ethyl acetate extract having the best antioxidant activity by silica gel column chromatography to obtain a natural compound and to investigate various radical scavenging ability of each compound; Analyzing the natural compound through UV, IR spectra, ¹ H and ¹³ C-NMR analysis and COSY, HMQC and HMBC analysis.

Hereinafter, the specific configurations and effects of the present invention will be described with reference to Examples, but the scope of the present invention is not limited only to these Examples.

EXAMPLE

Example 1 Methanol Extraction, Fractionation and Separation of Pine Needles

Plant material

Pine needles were collected in Geumjeongsan, Busan, Korea in April 1999. Evidence samples were stored in the herbarium of the Medicinal Plants, College of Pharmacy, Pusan National University.

reagent

1,1-diphenyl-2-picrylhydrazyl (DPPH), L-ascorbic acid, penicillamine were purchased from Sigma Chemical Company. 2 ', 7'-dichlorodihydrofluorescein diacetate (DCHF-DA), dihydrodamine 123 (DHR 123), and peroxynitrite were used as the high purity, respectively, the molecular probe It purchased from the company and Cayman.

Experiment apparatus

UV spectra were obtained from a Shimazu 202 UV spectrophotometer in methanol solution. LR FAB-MS data were obtained from JEOL JMS-HX110 / 110A spectrometer. Column chromatography used silica gel (Merck, 70-230 mesh). TLC was performed on a pre-coated Merck Kieselgel 60 F254 plate, and spots were identified using 50% H ₂ SO ₄ reaction solution.

NMR analysis

¹ H and ¹³ C-NMR spectra were expressed on a Varian UNITY-400 spectrometer (400 MHz for ¹ H and 100.0 MHz for ¹³ C). HMQC and HMBC spectra were recorded using pulsed field gradients rather than phase cycling for coherence pathway selection. Various ¹ H and ¹³ C-NMR signals are shown as s (singlet), d (doublet) and m (multiplet).

First compound (camperol 3-O-galactopyranoside 6 "-acetate, kaempferol 3- O -galactopyranoside 6 "-acetate):

Yellow powder, UV max (MeOH) 258, 353 nm; FABMS m / z 513 [M + Na] ⁺ ; ¹ H-NMR (400 MHz, DMSO- d ₆ ) d: 12.58 (OH), 10.88 (OH), 10.18 (OH), 8.04 (2H, d, J = 8.7, H-2 ', 6'), 6.85 (2H, d, J = 8.7, H-3 ＇, 5＇), 6.45 (1H, d, J = 2.0, H-8), 6.20 (1H, d, J = 2.0, H-6), 5.32 ( 1H, d, J = 7.6, H-1 "), 4.07 (1H, dd, J = 7.8, 11.3, H-6" b), 3.90 (1H, dd, J = 4.3, 11.3, H-6 "a ), 3.62 (1H, d, J = 4.3, H-4 "), 3.60 (1H, d, J = 4.3, H-5"), 3.56-3.51 (1H, m, H-2 "), 3.42- 3.38 (1H, m, H-3 "), 1.74 (CH ₃ ), ¹³ C-NMR (100 MHz, DMSO- d ₆ ) d: 156.4 (C-2), 133.2 (C-3), 177.5 (C -4), 161.2 (C-5), 98.7 (C-6), 164.1 (C-7), 93.7 (C-8), 156.4 (C-9), 103.8 (C-10), 120.8 (C- 1 ＇), 130.9 (C-2＇, 6 ＇), 115.0 (C-3＇, 5 ＇), 160.0 (C-4＇), 101.7 (C-1 "), 70.9 (C-2"), 72.8 (C-3 "), 68.1 (C-4"), 72.8 (C-5 "), 63.1 (C-6" b), 169.8 (COO), 20.1 (CH ₃ ).

Second Compound ((+)-Isolarisyreninol Xylopyranoside, (+)-isolarisiresinol xylopyranoside):

[a] _D + 4.02 ° (c, 0.005, MeOH); HREIMS: m / z 492.1995 (C ₂₅ H ₃₂ O ₁₀ ); EIMS ( m / z ,%): 492 ([M] ⁺ , 27.3), 359 ([M-xylose + H] ⁺ , 65.4); ¹ H-NMR (400 MHz, DMSO- d ₆ ) d: 7.29 (1H, d, J = 1.6, H-2 ＇), 7.17 (1H, d, J = 8.1, H-5＇), 6.99 (1H , d, J = 1.6, 8.1, H-6 ＇), 6.84 (1H, s, H-5), 6.81 (1H, s, H-2), 4.61 (1H, d, J = 7.5, H-1 "), 4.59 (1H, dd, J = 1.9, 10.4, H-9'b), 4.53 (1H, brd, J = 10.8, H-7 '), 4.27 (1H, d, J = 4, 10, H-9b), 4.23 (1H, d, J = 5, 10, H-9a), 4.20 (1H, m, H-4 "), 4.20 (1H, dd, J = 2.2, 12, H-5" b), 4.11 (1H, d, J = 10.7, H-3 "), 4.03 (1H, d, J = 7.9, H-2"), 3.72 (OMe, C-3), 3.69 (OMe, C- 3 ＇), 3.61 (1H, dd, J = 4.1, 10.4, H-9＇a), 3.57 (1H, dd, J = 6, 12, H-5" a), 3.37 (1H, m, H- 8 ′), 3.32 (1H, doublet of doublets, J = 15.6, 11.3, H-7b), 3.12 (1H, doublet of doublets, J = 15.6, 4.4, H-7a), 2.47 (1H, m, H-8); ¹³ C-NMR (100 MHz, DMSO- d ₆ ) d: 128.05 (C-1), 112.49 (C-2), 146.89 (C-3), 145.98 (C-4), 117.89 (C-5), 134.03 (C-6), 33,75 (C-7), 39.16 (C-8), 64.19 (C-9), 137.81 (C-1 ＇), 114.33 (C-2＇), 148.46 (C- 3 ＇), 146.36 (C-4＇), 116.5 (C-5 ＇), 122.53 (C-6＇), 47.33 (C-7 ＇), 45.41 (C-8＇), 68.48 (C-9 ＇) ), 106.0 (C-1 "), 75.09 (C-2"), 78.44 (C-3 "), 71.11 (C-4"), 67.14 (C-5 "), 55.84 (OMe, C-3" ), 55.99 (OMe, C-3 ′).

A third compound (camp Ferrol 3-O- galacto-pyrano side, kaempferol 3- O -galactopyranoside):

¹ H-NMR (400 MHz, DMSO- d ₆ ) d: 12.62 (1H, brs, OH), 8.06 (2H, d, J = 8.05, H-2 ', 6'), 6.85 (2H, d, J = 8.05, H-3 ＇, 5＇), 6.43 (1H, d, J = 1.8, H-8), 6.20 (1H, d, J = 1.8, H-6), 5.39 (1H, d, J = 7.6, H-1 "), ¹³ C-NMR (100 MHz, DMSO- d ₆ ) d: 156.38 (C-2), 133.24 (C-3), 177.53 (C-4), 161.30 (C-5) , 98.71 (C-6), 164.17 (C-7), 93.67 (C-8), 156.38 (C-9), 103.94 (C-10), 121.89 (C-1 ＇), 130.88 (C-2＇ , 6 ''), 115.06 (C-3 '', 5 ''), 159.95 (C-4 ''), 101.67 (C-1 "), 71.2 (C-2"), 73.10 (C-3 "), 67.88 ( C-4 "), 75.77 (C-5"), 60.20 (C-6 ").

Extraction, Fractionation, and Separation of Bioactive Substances from Pine Needles

8.5 kg of dry powder of pine needles were refluxed with methanol for 3 hours. The total filtrate was concentrated under vacuum at 40 ° C. to obtain 2.1 kg of methanol extract. The extract was suspended in distilled water, followed by dichloromethane (CH ₂ Cl ₂ , 600 g), ethyl acetate (EtOAc, 200 g), butanol (n-BuOH, 450 g), and distilled water (H ₂ O, 750 g). Fractionated. In the experimental model system of the present invention, 100 g of the ethyl acetate extract showing strong antioxidant activity was eluted by concentration gradient of ethyl acetate-methanol on a silica gel column to obtain 9 fractions (fractions 1-9). . Fraction 3 (9.5 g) was purified by column chromatography on a silica gel column using a solution of dichloromethane-methanol in a ratio of 5: 1 to obtain a first compound (40 mg, camphorol-3- O -galactoside-). 6 "-acetate (kaempferol 3- O -galactoside 6" -acetate) and a second compound (74 mg, isolarisyrrezinol-xyloside) were obtained. Fraction 4 (6 g) was mixed with dichloromethane-methanol in a 5: 2 solution, and then chromatographed on a silica gel column by treating with pure methanol to obtain a third compound (22 mg, camphorol-3- O -galactoside). (kaempferol 3- O -galactoside)). The chemical structure of the compound is shown in FIG.

Example 2: Radical Scavenging Capacity of Pine Needle Extract

The radical scavenging effect of each solvent extract and the natural compound obtained in Example 1 was performed by the following method.

Determination of scavenging effects on DPPH radicals

The scavenging effect of DPPH radicals was measured according to the method of Blois. 4 mL of methanol solution of various sample concentrations was added to 1.0 mL of DPPH methanol solution (1.5 × 10 ⁻¹ M). After mixing slowly and leaving at room temperature for 30 minutes, the optical density was measured at 520 nm with a spectrophotometer. Antioxidant activity of each sample was expressed as IC ₅₀ (μg / mL or μ M needed to inhibit 50% DPPH radical formation) and was calculated with a log-dose inhibition curve.

Hydroxyl radical generation analysis

The extract (final concentration: 0.5 mg / mL) was added to 1 mM H ₂ O ₂ and 0.2 mM FeSO ₄ and incubated at 37 ° C. for 5 minutes. 2 Then, raise the treated ester 2 μ M ', 7'- dichloro-dihydro-fluorescein diacetate (2' was added, 7'-dichlorodihydrofluorescein diacetate (DCHF- DA)), changes in fluorescence are an excitation wavelength It measured for 30 minutes at 485 nm and emission wavelength 530 nm.

Free Radical Generation Analysis

A mixture without a slaughtered just before the experiment Wistar male rats of 150-200 g body weight is then crushed between the extract is added to the suspension of the extract or compound, or added, for 30 minutes at 37 ℃ 12.5 μ M DCHF-DA Incubated at. 50 mM phosphate buffer pH7.4 was used. The fluorescence intensity of oxidized 2 ', 7'-dichlorodihydrofluorescein (DCF) was measured using a spectrofluorometer (Bio-Tek Instruments, Inc., Winooski, VT with excitation wavelength 460 nm and emission wavelength 530 nm). Measured in

Determination of Peroxynitrite Scavenging Activity

The scavenging activity of peroxynitrite was measured by measuring the oxidation of DHR 123 by modifying Kooy et al. DHR 123 (5 mM) dissolved in dimethylformamide in a state where impurities were removed by nitrogen treatment was stored at -80 ° C as a stock solution. The solution was then placed on ice to block light before the experiment. 90 mM sodium chloride, 50 mM phosphate buffer solution consisting of sodium, 5 mM calcium chloride (pH 7.4) and 100 μ M diethylenetriamine pentaacetic acid (DTPA) was prepared to be deionized is high purity, respectively, removal of impurities by treatment nitrogen It was. The final concentration of DHR 123 was 5 μΜ . Background fluorescence intensity and final fluorescence intensity were measured for 5 minutes with or without peroxynitrite. DHR 123 was rapidly oxidized by peroxynitrite and its final fluorescence intensity remained unchanged over time. The fluorescence intensity of the oxidized DHR 123 was measured in a microplate fluorescence reader (FL 500, Bio-Tek Instruments) with an excitation wavelength of 480 nm and emission wavelength of 530 nm. The fluorescence intensity was taken as the mean value ( n = 3) of the final fluorescence intensity minus the background fluorescence. Scavenging effect was expressed as percent inhibition of DHR 123.

Statistical analysis

Numbers are expressed as experimental standard error ± mean of 3 or 5 times.

As shown in Table 1, the scavenging activity of the dichloromethane-, ethyl acetate-, butanol-, and distilled water-fractions of the pine needles of the present invention against DPPH was in the order of ethyl acetate fraction> butanol fraction> distilled water fraction> methanol fraction> dichloromethane fraction. The IC ₅₀ of the extracts was 13.2, 24.3, 25.1, 32.5 and 45.4 μg / mL, respectively. Among the five extracts, it was found that the ethyl acetate soluble fraction of the methanol extract had the most excellent effect on eliminating free radicals. In addition, the ethyl acetate-fractions contained hydroxyl radicals, total free radical species (ROS) and peroxynitrite (ONOO ⁻ ) at concentrations of 40, 40 and 10 μg / mL, respectively, 82.13 ± 5.31, 59.15 ± 3.4 and 95.6 ± 0.09 Significantly inhibited in%. The ethyl acetate-fractions of pine needles from the above measurement results showed that the scavenger of active oxygen species including free radicals, peroxynitrite and hydroxyl radicals. Ethyl acetate fractions had high scavenging activity, while dichloromethane and water fractions both showed weak activity in the experimental model system.

DPPH of the extract from pine needle extract, · OH, ONOO ^-, and antioxidant activity investigation of ROS extract DPPH ^a ONOO ^-b OH ^c ROS ^d Methanoldichloromethaneethyl acetate n-butanol distilled water L-phenyl ascorbate 32.545.413.224.325.19.1 80.38 ± 1.4421.36 ± 1.0495.60 ± 0.0982.28 ± 1.8969.02 ± 1.2993.14 ± 0.52 -29.79 ± 5.18-357.45 ± 10.482.13 ± 5.3161.70 ± 4.4227.66 ± 0.43 -392.80 ± 21.3-907.36 ± 50.059.15 ± 3.450.55 ± 3.740.38 ± 3.2014.7 ± 1.5

^a DPPH shows free radical scavenging activity (IC ₅₀ : μg / mL). ^b ONOO ⁻ represents the percent inhibition of peroxynitrite at an experimental concentration of 10 μg / mL. ^c · OH is from 40 ㎍ / mL in concentration in the experiment with _{1.0 mM H 2 O 2 0.2 mM} FeSO 4 shows the percent inhibition of the hydroxyl radical generation. ^d ROS represents the percent inhibition of total free radical generation in the microsome fraction of liver at concentrations of 40 μg / mL.

In general, flavonoids and phenolic acids have received a great deal of attention because of their radical scavenging ability, so the present inventors believe that these compounds may also be present in the ethyl acetate fraction of pine needles of the present invention, thereby chemically analyzing ethyl acetate soluble fractions having antioxidant activity. It was. The fractions were separated by continuous column chromatography to give two known flavonoids (first and third compound) and one lignan (second compound).

The above compounds are each (+)-isolarysirinol 9'- O - b -D- xylopyranoside (second compound), camperol-3- O - b -D-galactopyranoside- With 6 "-acetate (first compound) and camphorol 3- O - b -D-galactopyranoside (third compound), the data recorded on the spectrometer were analyzed and finally confirmed by comparison with a standard sample. Positioning of the ¹ H and ¹³ C NMR spectra was confirmed by the HMQC and HMBC spectra. Among the above compounds, camperol-3- O - b -D-galactopyranoside-6 "-acetate was found by the inventors to be natural. Esau splits second.

Investigation of Antioxidant Activity against DPPH, ONOO-, · OH and ROS of Compounds Isolated from Ethyl Acetate Fraction of Pine Needle compound DPPH ^a ONOO ^-b OH ^c ROS ^d One 92.34 24.42 ± 1.24 NS NS 2 7.42 29.86 ± 0.20 NS NS 3 74.72 16.99 ± 3.99 NS NS L-ascorbic acid 7.98 13.67 ± 1.73 55.06 ± 8.96 Phenylsilamine 3.20 ± 0.36

^a DPPH shows free radical scavenging activity (IC ₅₀ : μ M). ^b ONOO ⁻ shows inhibitory activity of peroxynitrite (IC ₅₀ : μ M). ^c · OH shows the inhibitory activity of hydroxyl radical generation in 1.0 mM H ₂ O ₂ and 0.2 mM FeSO ₄ (IC ₅₀ : μ M). ^d ROS shows the inhibitory activity of total free radical development in the simplified microsome fraction (IC ₅₀ : μ M). NS indicates that no no effect up to a concentration of 80 μ M concentration.

As shown in Table 2, the antioxidant activity of the three compounds isolated from the ethyl acetate fractions was examined. As a result, the flavonoid camphorol 3- O - b -D-galactopyranoside and its 6 "-acetate ( 1,3 compounds) and the lignan isolarisyrrezinol xyloxide (second compound) showed high antioxidant activity in the DPPH and peroxynitrite model system.Lignan (second compound) was IC ₅₀ for DPPH radicals. Has a strong radical scavenging activity of about 7.42 μ M. Lignan (second compound) showed higher radical scavenging activity than L-ascorbic acid, which is well known as an antioxidant. ₅₀ is shown a scavenging medium to 29.86 ± 0.20 μ M. flavonoids (first, third compounds) is the IC ₅₀ for each 24.42 peroxy nitrite 1.24 μ M and 16.99 exhibited a ± 3.99 moderate activity as μ M, showed a high scavenging activity is IC _50, respectively 92.34 μ M and 72.74 μ M for the DPPH radical. Generally, the lignans are antineoplastic (antitumor ), Antistress, antimitotic, antiviral, antihepatotoxic, and cardiovascular activity. Menites and related phenolic acids are known to function as potent antioxidants because of their hydrogen donor properties and metal chelating properties. It can be said that it provided the possibility of use as an antioxidant for treating them.

As described above through the examples, the present invention is a camphorol which is a flavonoid obtained by separating the fraction obtained by fractionating the methanol extract of pine needles with ethyl acetate using a silica gel column using ethyl acetate-methanol as the elution solvent. 3- O - b -D-galactopyranoside and its 6 "-acetate (compounds 1 and 3) and the lignan isolarisyresinol xyloxide (compound 2) are DPPH free radicals, peroxynites Light, hydroxyl radicals, total active oxygen species removal ability is excellent because it is an effective invention in the manufacture of bioactive substances.

Claims (2)

8.5 kg of pine needles were refluxed with methanol for 3 hours, the extract was suspended in distilled water, fractionated with ethyl acetate, and the ethyl acetate extract was eluted with a mixed solution of ethyl acetate-methanol in concentration gradient in refining the silica gel column. To obtain a fraction, and the fraction having antioxidant activity in the fraction was further purified by silica gel column chromatography using dichloromethane-methanol mixture to prepare a flavonoid-based camphorol 3- O -galactoside-6 "-acetate (kaempferol 3- O -galactoside 6 "-acetate) and camp Ferrol -3- O - galactosyl side (the antioxidant derived from pine needles made to obtain a side (isolariciresinol xyloside) with kaempferol-3- O -galactoside) and lignans which Ra receiver register play Giles Method of separation of substances. Isolarisyrrezinol xylosides, camphorol-3- O -galacto, which are obtained by the separation method of claim 1 and have an scavenging effect on peroxynitrite, DPPH radicals, free radicals and reactive oxygen species. A red pine-derived antioxidant characterized by containing side-6 "-acetate and camphorol-3- O -galactoside as an active ingredient.