KR20120119189A - Polyphenolic compounds from a mulberry seed and the use of the same - Google Patents

Abstract

PURPOSE: A manufacturing method of mulberry seed originated polyphenol compound is provided to enhance antioxidant function, thereby being useful for cosmestic material. CONSTITUTION: A polyphenol compound is one of compounds having chemical structure 1-11 which are originated from mulberry seed. An isolation refining method of the polyphenol compound comprises the following steps: obtaining mulberry seed oil and defatted meal by compressing the mulberry seed; organic solvent extracting degatted meal; and silica-gel, sephadex LH-20, ODS-A column chromatographing ethyl acetate fraction. The functional food for antioxidant contains mulberry seed originated polyphenol compound.

Description

Polyphenolic compounds from a Mulberry seed and the use of the same}

The present invention relates to various polyphenolic compounds derived from seeds of mulberry fruit mulberry, and more particularly, to extract the remaining skim foil after pressing the mulberry fruit mulberry seed organic solvent, and then subjected to column chromatography to separate the polyphenol compounds To refine and identify chemical formulas.

Recently, antioxidants capture or eliminate free radical species (R ?, ROO ?, RO?) And lipid peroxides (ROOH) that are produced during the automatic oxidation of fats. as physiologically active substances which prevent ^{_{- (, H 2 O 2,}} OH 1 O 2, O 2?) cancer by inhibiting the lipid peroxidation reaction by, arteriosclerosis, hypertension, diabetes, inflammation and aging, various active oxygen species are generated It is getting a lot of attention. Antioxidants include synthetic antioxidants and natural antioxidants. Recently, safety and effective natural antioxidants have been actively developed from plants as the safety and toxicity of synthetic antioxidants are considered to be problematic.

Polyphenolics are natural pigments that are widely found in nature. They exist mainly in vacuoles and cell membranes in free form, ester form or combined form. cyanidins, resveratrols, lignans and tannins. Typical polyphenol compounds present in plants include catechins of green tea, tannins of persimmon, isoflavones of beans, lignans of flax seeds, resveratrols of grapes and peanuts, anthocyanins of black foods (black rice, black beans, black sesame seeds, etc.) Chlorogenic acid, capsaicin of red pepper, curcumin of turmeric, and the like are known, and these have been found to have various physiological and pharmacological effects such as anticancer, antihypertensive, anti-inflammatory, antidiabetic, antioxidant and anti-aging.

On the other hand, Morus alba L.) Fruit mulberry has long been used as an anesthetic, hypertension, arthralgia and baldness cure, as well as a nutrient tonic that blackens white hair and benefits the intestines. Many studies on the properties, chemical composition and physiological activity of the fruit (Fruit) has been reported so far, but studies on the chemical composition and physiological activity of the seed (seed) is insufficient.

However, oil and skim milk are produced by milking seeds obtained as a by-product when processing fruits such as grapes, mangoes, and berries, and among them, a large amount of antioxidant polyphenol compounds are contained in the skim foil, which is used as a functional material. It has been attempted for use in the food and cosmetics industry. The inventors of the present invention not only contains a large amount of unsaturated fatty acids, such as linoleic acid and linolenic acid, but also contains tocopherol and phytosterol in the odyssey obtained as a byproduct in the manufacture of odyssey and odyne wine, and also anticancer, antihypertensive, and anti Prior studies have revealed that the diabetic components quercetin, resveratrol, and 4-prenylmoracin, etc., in the present invention, in addition to these three species of polyphenol compounds [(+)-dehydrokaempferol, (+)-dehydroquercetin and moracin ], And furthermore, these materials were confirmed to be excellent in antioxidant, anti-inflammatory and antioxidant activity, and completed the present invention.

Therefore, an object of the present invention is to search for and provide the main antioxidants of mulberry seed mulberry fruit mulberry by-products of the mulberry seed mulberry suitable for use as functional food and cosmetic materials.

The object of the present invention is to isolate and identify various polyphenolic compounds from mulberry seeds present in the mulberry fruit (Fruit), and to measure their antioxidant activity using DPPH radicals, and then to obtain the mulberry from three mulberry varieties The antioxidant polyphenolic compounds of the seeds were achieved by quantitative comparison using HPLC.

The present invention has an effect of providing a variety of polyphenol compounds derived from mulberry fruit mulberry seed not known so far, and has an excellent effect of providing anti-cancer, anti-hyperlipidemic food and antioxidant functional cosmetic material using their antioxidant function.

Hereinafter, specific contents of the present invention are clearly shown in the following experimental results.

Figure 1 is a schematic diagram showing the extraction, separation and purification process of polyphenol compounds derived from mulberry fruit mulberry seed of the present invention.
Figure 2 is a chemical structural formula of 11 polyphenol compounds derived from mulberry fruit mulberry seed of the present invention.
Figure 3 is an HPLC chromatograph of the polyphenol compound derived from mulberry fruit odyssey methanol extract (A) of the present invention and the residue (B).

Experimental material

The mulberry tree used in this experiment was produced from Gimcheon, Gyeongbuk, Korea in June 2010.Morus alba L.) Using cultivar 'Iksu-mong' Audi to separate and dehydrate by repeatedly washing and dipping several times from Odak obtained as a by-product during the production of odyssey, hot air dried at 50 ± 5 ℃ was used as an experimental material. In addition, from the 'Daesung Fong' and 'Cheong Il Fong' Audi produced in Yeongdeok and Sangju, Gyeongbuk, respectively, the Odysium was separated and dried and used as a sample for quantitative analysis.

For reagents and chromatography Filler

1,1-Diphenyl-2-picrylhydrazyl (DPPH) and L-ascorbic acid used in this experiment were Sigma Chem. Co. (St. Louis, MO., USA), α-tocopherol, phosphoric acid is Wako Pure Chem. Ind. Ltd. Each was purchased from (Osaka, Japan) and used. And silica gel 60 (230-400 mesh, Merck, Darmstadt, Germany), ODS-A gel (12 nm, 150 μm, YMC, Inc., MA, USA) and Sephadex LH as fillers for column chromatography for purification. -20 (Pharmacia Biotech, Uppsala, Sweden) was used, and thin layer chromatography (TLC) for material analysis was performed using pre-coated silica gel 60 (0.25 mm, Merck, Darmstadt, Germany) plate. All other reagents were HPLC grade (Merck, Darmstadt, Germany) or analytical special reagents.

From Mr. Audi  Extraction, Separation and Purification of Polyphenol Compounds

Extraction, separation and purification of polyphenolic compounds from Audi seed was carried out as shown in FIG. Dry odyssey (18 kg) was milked using a continuous press expeller (Poongsan, Daegu, Korea) to obtain odyssey oil (5.1 kg) and skim foil (13.13 kg), respectively. L) was added and extracted continuously with an ultrasonic cleaner (Power Sonic 420, Hwashin Instrument, Seoul, Korea), filtered and concentrated to obtain methanol extract of Odystal Paper (550 g). Then, the methanol extract was dissolved in 80% aqueous methanol solution (4 L), followed by normal washing with twice of normal hexane (4 L), and degreased by dehydration. (3 L), ethyl acetate (3 L), and normal-butanol (1 L) were added sequentially, followed by two fractions, followed by anhydrous Na₂SO₄The reaction mixture was dehydrated and concentrated under reduced pressure to obtain a dichloromethane (10.5 g), ethyl acetate (4.8 g) and normal-butanol (20.3 g) fractions. Among the three fractions, ethyl acetate fraction (4.8 g) was subjected to silica gel column chromatography with chloroform-methanol (5: 1, v / v) mixed solvent, and the fractions having similar components were collected by TLC analysis. 1, 1.33 g; fr. 2, 2.22 g; fr. 3, 0.66 g; fr. 4, 0.39 g; fr. 5, 0.20 g). Then, each fraction was subjected to Sephadex LH-20 column chromatography with 80% methanol solution, and from fraction 1Compound 1 (Comp. 1, 13.7 mg) andCompound 2(Comp. 2, 3.4 mg) were isolated, and Sephadex LH-20 and ODS-A column chromatography were sequentially performed from Fraction 2.Compound 3(Comp. 3, 33.6 mg),Compound 4(Comp. 4, 4.1 mg), Compound 5(Comp. 5, 2.4 mg), Compound 6(Comp. 6, 2.2 mg), Compound 7(Comp. 7, 3.6 mg) were obtained, respectively. In addition, Sephadex LH-20 and ODS-A column chromatography were sequentially performed from fraction 3 separated from silica gel column chromatography.Compound 8(Comp. 8, 5.1 mg) andCompound 9(Comp. 9, 7.1 mg) was obtained, and Sephadex LH-20 and ODS-A column chromatography were performed from fractions 4 and 5.Compound 10 (Comp. 10, 4.1 mg) andCompound 11(Comp. 11, 2.2 mg) were each isolated purely.

The present inventors have determined that in the previous studies, the antioxidant activity of the mulberry area (fruit, seed, leaf, root) by the mulberry seed showed the strongest antioxidant activity. Therefore, in order to separate the main antioxidants of Odyssey, methanol extracts of Odyssey were additionally solvent fractionated using organic solvent dichloromethane, ethyl acetate and butanol, and the ethyl acetate fraction having the strongest antioxidant activity was silica-gel, Sephadex LH-20 and ODS-A column chromatography were sequentially performed to separate and purify 11 polyphenol compounds as shown in FIG. 1.

From Mr. Audi  Identification of Chemical Structure of Isolated Polyphenolic Compounds

For the structural analysis of 11 polyphenolic compounds isolated from OD, the UV-vis spectrophotometer (S-3100, SINCO, Seoul, Korea) was used. Maximum absorption wavelength by scanning in 600 nm area (λ_max) Was investigated. next,^OneWith H-NMR (400 MHz)¹³C-NMR (100 MHz) analysis was performed at room temperature using a Varian Unity Plus 400 spectrometer (Palo Alto, CA, USA) at which time the sample was CD₃Dissolved in OD. Tetramethylsilane (TMS) was added as an internal standard and the chemical shift of the sample was expressed as δ (ppm).

Of the 11 polyphenol compounds isolated from Odyssey, 8 polyphenol compounds ( Comp . 1 , Comp . 2 , Comp . 4 Comp . 5 and Comp . 6, Comp . 8, Comp . 10 and Comp . 11) are already Baga isolated and identified from mulberry fruit mulberry and mulberry seed Therefore, in the present invention, the structure of three other polyphenol compounds that have not been separated from Audi and Odyssey were identified by comparison with NMR spectra results obtained from NMR spectroscopy analysis and previous studies. First , the ¹ H-NMR spectrum of Compound 3 ( Comp . 3) was measured. As a result, δ 6.71 (d, J = 8.4 Hz), 6.76 (dd, J = 2.0 & 8.4 Hz), and 6.88 (d, J = 2.0 Hz)) three aromatic protons representing the ABX system of the flavonoid compound benzene B ring and two meta- coupled doublets of the benzene A ring at δ 5.79 (d, J = 2.0 Hz) and 5.83 (d, J = 2.0 Hz) protons and methine protons of H-2 and H-3 at δ 4.82 (d, J = 11.2 Hz) and δ 4.42 (d, J = 11.6 Hz), respectively, and their protons from coupling constants ( J = 11.2 Hz) We can see that they have a trans configuration relationship with each other. Next, the ¹³ C-NMR spectrum was observed to show that C-2 and C-3 are trans- configured from the carbon signals of δ 85.14 (C-2), 73.71 (C-3), and 198.43 (C-4). 3-flavanol structure was identified, carbon signal having two hydroxyl groups of benzene B ring at δ146.82 (C-3 ') and δ147.17 (C-4'), and δ 163.78 (C-9) , 164.54 (C-5) and 168.79 (C-7) were able to identify carbon signals with three hydroxyl groups in the benzene A ring, respectively, δ 96.35 (C-8), 97.38 (C-6), Carbon signal separation patterns of quercetin analogs were shown at 101.88 (C-10), 115.94 (C-2 '), 116.14 (C-5'), 120.97 (C-6 ') and 129.91 (C-1'). . like this Compiling the ¹ H- & ¹³ C-NMR spectra, Comp . 3 was found to be (+)-dihydroquercetin, so far Opuntia This compound has been isolated from several plants, including ficus - indica , but this is the first time that it has been identified in Audi and Odyssey.

Next, the ¹ H-NMR spectrum of the compound 7 ( Comp . 7) was measured. As a result, A ₂ B of the benzene B ring of the flavonoid compound at δ 6.84 (d, J = 8.8 Hz) and 7.37 (d, J = 8.8 Hz) was measured. 1,4-disubstituted ( para -substituted) aromatic protons representing ₂ systems ( meta- coupled doublets) and benzene A rings at δ 5.89 (d, J = 2.0 Hz) and 5.93 (d, J = 2.0 Hz). Meta- coupled doublet protons and methine protons of H-2 and H-3 at δ 5.0 (d, J = 11.6 Hz) and 4.56 (d, J = 11.6 Hz), as well as dihydroquercetin. J = 11.6 Hz), it was confirmed that their protons had a trans configuration relationship with each other. Next, the ¹³ C-NMR spectrum was measured, and C-2 and C-3 mutually derived from carbon signals of δ 85.19 (C-2), 73.85 (C-3), and 198.67 (C-4). trans It was confirmed that the 3-flavanol skeleton is configured, benzene B at δ 159.44 (C-4 '), 130.58 (C-2'& C-6 '), and 116.35 (C-3'& C-5 ') Five carbon signals with para -hydroxyl groups in the ring and two signals with two hydroxyl groups in the benzene A ring at δ 164.76 (C-9), 165.55 (C-5) and 169.10 (C-7), respectively In addition, δ 96.55 (C-8), 97.58 (C-6), 102.01 (C-10), and 129.52 (C-1 ′) showed a separation pattern of kaempferol skeleton carbon signal. Thus Comp . Taken together the results of ¹ H- & ¹³ C-NMR spectra of the 7, the compound is (+) - dihydrokaempferol was found to be, so far Opuntia ficus - bar detached from several plants, including indica, but audio and audio This is the first time he has confirmed.

The ¹ H-NMR spectrum of Compound 9 ( Comp . 9) was measured, and two hydrogen signals (2H, d, J = 2.0 Hz, H-2 '&H-6') meta- coupled at δ 6.76. Single proton at δ 6.25 (1H, t, J = 2.0 Hz, H-4 ') and a single proton at δ 6.91 (1H, s, H-3) and δ 7.35 (1H, d, J) From benzene substituents representing the ABX type at = 8.4 Hz, H-4), 6.90 (1H, d, J = 2.0 Hz, H-7) and 6.74 (1H, dd, J = 2.4, 8.4 Hz, H-5) The compound was found to be an arylbenzofuran derivative. Next, the ¹³ C-NMR spectrum was measured to show hydroxyl benzene carbon at δ 160.09 (C-3 '&C-5'), 157.39 (C-8), 156.98 (C-6) and 156.27 (C-2). signal and carbon signal of the benzofuran skeleton at δ 122.14 (C-4), 113.39 (C-5), 98.60 (C-7), 123.18 (C-9) and 102.34 (C-3) and δ 133.95 (C- 1 '), 104.06 (C-2'& C-6 '), and 103.64 (C-4') from the meta- coupled arylbenzene type Comp . 9 was confirmed to be a moracin compound which is a 2-aryl-benzofuran derivative. Moracin and its derivatives are well known as anti-diabetic components in the upper lobe and epithelium, as well as audi. The chemical structure of the 11 polyphenol compounds separated from the OD as described above is shown in Figure 2, where the ^first H- & ¹³ C-NMR spectral data of the three polyphenol compounds separated for the first time 1].

From Mr. Audi  Determination of Antioxidant Activity of Isolated Polyphenol Compounds

Antioxidant activity of 11 polyphenol compounds isolated from OD was slightly modified with Tagashira's method to measure DPPH (1,1-diphenyl-2-picrylhydrhydryl) radical scavenging activity as follows. In other words, 2 mL of methanol solution containing 0.1 mM DPPH was added to 0.1 mL of sample solution, vortexed, and reacted at room temperature for 10 minutes. The absorbance was measured at 517 nm using a UV spectrophotometer to measure the decrease in absorbance due to reduction of DPPH. It was. DPPH radical scavenging activity was calculated according to the following equation. DPPH radical scavenging activity (%) = (1-A / B) × 100.A: absorbance of sample at 517 nm. B: absorbance of control at 517 nm. Here, the IC calculated by the regression analysis after measuring the relative DPPH radical scavenging activity by measuring the control without sample₅₀Value (concentration of sample that inhibits 50% of DPPH radicals).

Antioxidant activity of 11 polyphenol compounds isolated from OD was shown as DPPH radical scavenging activity, and the results are shown in [Table 2].

Of the 11 polyphenol compounds of the present invention, rutin (IC ₅₀ = 20.2 μM) showed the strongest antioxidant activity, followed by isoquercitrin (IC ₅₀ = 22.5 μM)> quercitrin (IC ₅₀ = 24.6 μM)> quercetin (IC ₅₀ = 27.8 μM)> (+)-dihydroquercetin (IC ₅₀ = 28.9 μM)> chlorogenic acid (IC ₅₀ = 30.6 μM) showed a low activity. All of the above polyphenolic compounds showed higher antioxidant activity than L-ascorbic acid (IC ₅₀ = 31.5 μM) and α-tocopherol (IC ₅₀ = 52.3 μM), which are well known as natural antioxidants. In contrast, (+)-dihydrokaempferol (IC ₅₀ = 33.8 μM), trans -resveratrol (IC ₅₀ = 36.2 μM) and moracin (IC ₅₀ = 47.6 μM) and its derivative 4-prenylmoracin (IC ₅₀ = 48.2 μM) It showed lower antioxidant activity than -ascorbic acid, but showed higher antioxidant activity than α-tocopherol. In addition, procatechuic acid (IC ₅₀ = 68.2 μM) showed lower DPPH radical scavenging activity than L-ascorbic acid and α-tocopherol.

Polyphenol compounds are well known as natural antioxidants with strong DPPH radical scavenging activity. The magnitude of radical scavenging activity depends on the number of hydroxyl groups and the presence of conjugated double bonds. Thus, quercetin and its glycosides (isoquercitrin, quercitrin, rutin) showed nearly similar radical scavenging activity, whereas chlorogenic acid was radical scavenging stronger than procatechuic acid, quercetin than (+)-dihydroquercetin, and resveratrol was stronger than moracin and its derivatives. It was found to exhibit activity. As such, the strong radical scavenging activity of the polyphenolic compound is not only a natural antioxidant that inhibits the automatic oxidation of fat caused by free radicals, but also inhibits lipid peroxidation induced by free radicals in the body, thereby inhibiting cancer, hypertension and diabetes. And bioactive substances for preventing and treating various lifestyle diseases such as dementia.

Audi varieties Mr. Audi  Content measurement of polyphenolic compound

After grinding the dried Odyssie seeds (10 g), which were obtained by cultivar varieties, they were degreased by stirring at room temperature with a chloroform: methanol (2: 1, v / v) mixed solvent (200mL), followed by drying the degreasing foil obtained by filtration. Odysbacbac was obtained, and the methanol extract obtained by ultrasonic extraction with 90% aqueous methanol solution (200 mL) for 2 hours was filtered and concentrated to obtain a methanol extract. Then dissolve it in 90% methanol solution (15 mL), take some stock solution and pass it through a membrane filter (0.45 μm, 13 mm, Pall Gelman Lab., Michigan, USA). The content of the polyphenol compound was measured. In this case, the separated polyphenol compounds were identified by comparing with the retention time of the purely separated polyphenol compounds. The maximum absorption wavelength of each compound was measured using a photodiode array detector, and then 290, 310, 330 and 350 nm. The calibration curve of each compound was prepared by using the regression graph, and then the content of each polyphenol compound was calculated. The recovery rates of 11 polyphenol compounds were determined by procatechuic acid (95%), chlorogenic acid (95%), rutin (95%), isoquercitrin (93%), quercitrin (92%) and dihydroquercetin (90%). , dihydrokaempferol (90%), t -resveratrol (93%), quercetin (90%), moracin and 4-prenylmoracin (90%).

The HPLC chromatograms of 11 standard polyphenolic compounds and degreasing CBC methanol extract for quantitative analysis of polyphenolic compounds contained in Odyssey are shown in FIG. The result of measuring the content of the compound is shown in [Table 3].

First of all, the content of polyphenolic compounds (mg / 100 g, dry odyssey) of three odyssey seeds showed the highest rutin of 31.1-60.0 mg, followed by quercitrin (7.2? 34.2 mg)> (+)-dihydroquercetin (13.2? 33.1 mg)> quercetin (15.8? 19.5 mg)> 4-prenylmoracin (10.5? 43.3 mg)> isoquercitrin (5.8? 15.4 mg)> chlorogenic acid (0.0? 15.3 mg)> moracin (4.7? 7.2 mg)> Procatechuic acid (0.0-11.6 mg) was the lowest. On the other hand, the contents of (+)-dihydrokaempferol and trans -resveratrol were traces (<0.1 mg). On the other hand, the contents of polyphenolic compounds of the mulberry varieties of three mulberry varieties showed that the Daesung mulberry Odysium is high in rutin and quercetin derivatives, which are anti-cancer, antihypertensive and antioxidant flavonoids. The contents of antioxidant phenolic acid such as procatechuic and chlorogenic acid were high, and the antidiabetic moracin and 4-prenylmoracin were 1.5 ~ 2.5 times higher than other cultivars. On the other hand, Cheongil Mulberry Odys had the lowest content of polyphenol compounds among the three varieties, and especially procatechuic and chlorogenic acid were not detected.

Statistical processing

The results of the experiments were measured three times and expressed as mean and standard deviation.The significance test between the mean values of each treatment was analyzed using ANOVA (Statistical Package for Social Sciences, SPSS Inc., Chicago, IL, USA) software. Was carried out. For items with significant differences between means, use Duncan's multiple range test.pSignificant difference test was performed at <0.05 level.

Polyphenol compounds derived from ODD of the present invention can be used as a main material in antioxidant functional foods, anti-aging functional cosmetics and pharmaceutical compositions such as anti-cancer, antihypertensive, anti-diabetic, so it is a very useful invention in the biopharmaceutical industry.

Claims (3)

Polyphenol compound of any one of the following structural formulas 1-11 derived from mulberry fruit mulberry seed.

Pressing mulberry fruit mulberry seed to obtain the mulberry seed oil and skim foil, and then extracted the organic solvent of the skim foil, and then ethyl acetate fraction was subjected to Silica-gel, sephadex LH-20, ODS-A column chromatography in sequence A separation and purification method of a polyphenol compound according to claim 1.
Functional food for antioxidant containing polyphenol compound derived from Odyssie obtained by the manufacturing method of Claim 2.

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