KR20000055727A - tannin isolated from plant and process for making the derivative thereof - Google Patents

Abstract

The present invention relates to the structural analysis of tannin components extracted from jujuk, persimmon, pine needles and hops and to synthesis of the derivatives of the tannins, and extract and separate tannin and polyphenols from jujupi, persimmon, pine needles and hops. Analyze the structure of the tannin components of the isolated skin whitening, bacterial toxin removal, free radical scavenging of free radicals, tumor suppression and DNA cleavage effect effective in synthesizing and providing elagic acid derivatives, gallic acid derivatives, catechin derivatives There is.

Description

Tannin isolated from plant and process for making the derivative

The present invention relates to a tannin component and a polyphenol component and derivatives thereof isolated from plants. More specifically, tannins such as gallic acid, ellagic acid, and catechin, and polyphenols such as moline and flavonol, are separated from plants such as yulpi, persimmon, pine needles, and hops. It relates to a method for preparing a double tannin derivative after structural analysis.

Chestnut (Castanea crenata Sieb. Et. Zucc.) Is a nut that has been used for a long time because of its excellent taste. In recent years, as the production of chestnut has increased, measures for consuming it have been made at various angles, and research on chestnut peeling, canning, baking, storage and lipids has been actively conducted. Yulpi was an inner skin of chestnut, a kind of industrial by-product that was removed and disposed of in the manufacture of processed foods using chestnuts. Recently, however, Yulpi is widely used as a component of skin care packs in the private sector, and its astringent and skin whitening effects are greatly recognized.

Persimmon tree is Diospyros kaki Thunberg. Persimmon tree is native to Korea, China and Japan. This persimmon has a strong growth, less damage from pests, easy to cultivate and manage, and has the advantage of not using drugs such as pesticides. Persimmon fruit is an alkaline food rich in sugars such as glucose and fructose. It is known that folk remedies are effective in stopping bowel contraction and coughing. Persimmon fruit causes a strong astringent taste due to tannins, which is due to the concentration of tannin present in the tannin cells of persimmon. These tannins are known to be synthesized in leaves, migrated to fruit, and then accumulated in fruit tannin cells. Persimmon matures and loses its astringent taste, which decreases from early June to mid-July and then increases until late August. Then decrease from early September. Research on tannin contained in persimmon leaves has been actively conducted, and the persimmon leaf differential using this has been developed as a product. Tannins in persimmon leaves are heat-stable and contain many phenolic hydroxy groups, making them easy to gel at high concentrations and soluble in water, methanol, and ethanol. In general, persimmon leaf tannin is a high molecular material and forms a strong hydrogen bond, making it difficult to measure molecular weight. Persimmon leaf tannins inhibit lipid peroxidation, similar to (-)-epigallocatechin, the main component of green tea tannins. It is estimated to be effective. Persimmon leaf tannins have been reported to have a detoxifying effect. According to the report, in general tannic acid, the binding to snake toxin is weak and is separated by centrifugation. However, persimmon leaf tannin is not easily separated by centrifugation because the binding reaction with toxin is irreversible. More effective. In addition, persimmon leaf tannin has been reported to have detoxifying effects on diphtheria, staphylococcus, tetanus and pertussis toxin. In the preclinical experiments using rats, catechins from the ether fraction of persimmon leaves showed anti-cancer and cholesterol-inhibiting effects.

Pine is scientific name Pinus densiflora Sieb. et Zucc, a cedar tree that grows on any terrain in Korea. Pine needles contain large amounts of vitamin C, which is good for scurvy and malnutrition in children. Chlorophyll, which is abundant in the leaves, can be used as a form of plaster for skin diseases. Essential oils from pine needles are used to make air purifiers, and pine needles are dried to make pine needle tea and used to treat neuralgia, arthritis and atherosclerosis. The components of fresh pine needles include ascorbic acid, carotene, vitamin K, flavonoids, anthocyanins, resins, tannins, kinasan, shikimic acid, and essential oils. Pine needles are not characterized by alkaloids, which are often toxic substances.

Hof, whose scientific name is Humulus Lupulus, is mainly used as a material for beer. This hop contains polyphenol compounds and tannin compounds. In beer production, the tannins in the hops combine with proteins to form precipitates that degrade the quality of beer.

As described above, tannins and polyphenols contained in yeolpi, persimmon, pine needles and hops are effective in skin whitening, removing toxins from bacteria, free radical scavenging, tumor suppression, and DNA cleavage. Recently, the necessity of producing tannins and polyphenol components is increasing.

Therefore, the present inventors are contained in the representative tannin-containing resource plants of our country, Yulepi (Castanea crenata Sieb. Et Zucc.), Persimmon (Diospyros kaki Thunb.), Pine needles (Pinus densiflora Sieb. Et Zucc.) The present invention was completed by extracting and purifying the tannin and polyphenol components present, and analyzing the components and preparing tannin derivative components.

An object of the present invention is to provide an extract and separation of tannin components from yulpi, persimmon, pine needles and hops. Another object of the present invention is to synthesize and provide a derivative of the extracted tannin component.

The above object of the present invention is to extract and separate and purify the tannin and polyphenol components contained in Yulpi, persimmon, pine needles, hops by solvent extraction method, and then the structure is analyzed by HPLC and ¹ H-NMR, ¹³ C-NMR, molecular weight spectrophotometer. This was accomplished by analyzing and synthesizing tannin derivatives such as gallic acid, catechin, and ellagic acid.

Hereinafter, the configuration and operation of the present invention will be described in detail.

FIG. 1 shows a flowchart in which yulp is extracted in a low order from an organic solvent having a low polarity.

Figure 2 shows the results of analyzing the organic solvent extract of Yulpi by HPLC.

3 shows a flowchart in which persimmons are extracted in a high order from an organic solvent having a low polarity.

Figure 4 shows the result of analyzing the organic solvent extract of persimmon by HPLC.

5 shows a flow chart of pine needles extracted in a low order from a low polar organic solvent.

Figure 6 shows the results of analyzing the organic solvent extract of pine needles by HPLC.

7 shows a flowchart in which hops are extracted in a low order from an organic solvent having a low polarity.

8 shows the results of HPLC analysis of the organic solvent extract of Hope.

In the present invention, the tannin and polyphenol components of Yulpi are extracted and separated using an organic solvent and water, purified by column chromatography, and analyzed by HPLC, ¹ H NMR, ¹³ C NMR, and molecular weight spectrum. ; Extracting, separating, and purifying tannin components of persimmon in the same manner as said yulpi; Extracting, separating, and purifying tannin and polyphenol components of pine needles in the same manner as in Yulpi and persimmon, followed by structural analysis; Extracting, separating and purifying the tannin component and the polyphenol component of the hops in the same manner as the yulpi, persimmon and pine needles, and then analyzing the structure; Synthesis of tannin derivatives such as gallic acid derivatives, catechin derivatives, and ellagic acid derivatives among tannins isolated from the yeast, persimmon, pine needles, and hops.

Yulpi, persimmon, pine needles, and hop meal of the experimental materials used in the present invention were domestically purchased from the Kyungdong Herbal Medicines market and TLC (Thin Layer Chromatography) was used by Kieselgel 60F ₂₅₄ plate (Merck), and n-BuOH: Acetic acid: H ₂ O (4: 1: 1), ethyl acetate: hexane (1: 1), methylene chloride: methanol MeOH (9: 1), methylene chloride: methanol (3: 1), chloroform: Methanol (3: 1), Chloroform: Methanol (9: 1), Chloroform: Methanol (20: 1), Chloroform: Methanol (2: 1), Chloroform: Methanol (4: 1) And the like were used. Detection of developed components was performed using 10% Phosphomolybdic acid-EtOH and 1% FeCl ₃ -EtOH reagents, and in the case of Phosphomolybdic acid-EtOH reagents. The TLC plate was immersed in the detection solution, taken out, and baked in an oven. In the case of 1% FeCl ₃ -EtOH reagent, it was sprayed with a spray and dried. HPLC was performed using Dionex-system and analyzed by gradient conditions. Each peak isolated was identified by comparison with the retention time of the standard, and the analysis conditions were as follows.

HPLC analysis conditions

Analyzer: Dionex Dual Pump DX-500

Detector: Dionex UV Detector

Column: Cosmosil packed column (4.6 × 150 ㎜, 5ph Type)

μ-Bondapak C18 reverse-phase column (4.6 × 150 mm)

Mobile phase: Water (0.025% Phosphoric acid),

Methanol (0.025% Phosphoric acid)

Flow rate: 1 ml / min

Record system: Shimadzu integrator recorder (C-R7A)

Resins used for Gel Filtration Chromatography were Sephadex LH-20 and MCI-gel CHP-20P. The eluted mobile phases were distilled water and methanol.

Example 1 Extraction, Separation, Purification and Structural Analysis of Tannin and Polyphenol Components of Yulpi

Step 1: Extract and Separate

In this step, 7 kg of Yulpi were extracted four times for 3 weeks (3 weeks) at room temperature with 70% acetone solution and then filtered through filter paper. Acetone in the extract was removed under reduced pressure, and the components of the crude extract were examined by TLC. The mobile phase used was n-BuOH: acetic acid: H ₂ O (4: 1: 1) and ethyl acetate: hexane (1: 1). The aqueous layer was placed in a round buttom flask and the water was carefully removed so as not to exceed 40 ° C. to obtain a solid substance (500 g). This solid material was taken up in distilled water and placed in a separatory funnel and extracted with an organic solvent (Petroleum ether, Ether, EtOAc, BuOH). As shown in FIG. 1, the organic solvents were extracted in the order of high solvent (Pet. Ether → Ether → EtOAc → BuOH) in the low polar organic solvent, and the components were examined by TLC after extraction separation (Chloroform: MeOH = 9: 1) . The extraction was repeated using an organic solvent until the color of the extracted solution became clear. Each fraction solution was blown off under reduced pressure, and the obtained fractions were filled with nitrogen gas and stored frozen.

Second Step: Purification by Column Chromatography

In this step, the yulp ether fraction was weighed and subjected to column chromatography. The Sehpadex LH-20 resin to be filled in the column was previously swelled in MeOH solvent for 1 day and then swollen, and the column was filled with methanol solvent. The sample was completely dissolved in a small amount of methanol, filtered through filter paper and then loaded. After loading, the mixture was eluted under isocratic conditions using methanol solvent. The components of the eluted solution were identified by TLC (Chloroform: MeOH = 3: 1, Chloroform: MeOH = 20: 1), and then collected by fractions to remove all solvents under reduced pressure, and weighed. Sephadex LH-20 was swelled in MeOH for one day in order to perform column chromatography with gradient, then packed into a column under concentration gradient (100% MeOH → 80% MeOH → 60% MeOH → 40% MeOH → 20% MeOH → distilled water). The fractions separated under the same conditions were dissolved in methanol again, placed in a column, and eluted under a concentration gradient (distilled water → 20% MeOH → 40% MeOH → 60% MeOH → 80% MeOH → 100% MeOH). After each fraction was collected by an automatic fractional collector, separation of each component was confirmed by TLC (Chloroform: MeOH = 3: 1, Chloroform: MeOH = 20: 1). When it was confirmed by TLC and the separation of spots was insufficient, chromatography was repeatedly performed under a single solvent condition and gradient condition using MCI-gel CHP-20P. The other fractions ethyl acetate and butanol fractions were also subjected to column chromatography in the same manner.

Step 3: HPLC Analysis

The HPLC analyzer was a DIONEX system and the detector chose 254nm because most tannins had aromatic rings. The solvent condition of the mobile phase was a concentration gradient, and the sample to be separated was completely dissolved in methanol and injected. The concentration gradient of the mobile phase solvent increased the methanol content of 0.025% H ₃ PO ₄ to 64% from 10% to 18 minutes using the μ-Bondapak C ₁₈ reverse-phase column. The condition was set to increase to 100% until 22 minutes and to decrease to 10% of the initial concentration again by 30 minutes. When using a Cosmosil packed column, methanol containing 0.025% H ₃ PO ₄ was increased from 10% to 18% to 64%, up to 22 minutes to 100% and then to 27 minutes Only methanol which was the same solvent was sent out. When analyzing the aqueous phase of Yulpi, mobile phase conditions were performed by applying a μ-Bondapak C ₁₈ reversed phase column. Cosmosil packed column conditions were analyzed by the analysis of the sample fractionated with an organic solvent and the fraction after column chromatography treatment. As a result, as shown in Fig. 2, tannins, such as gallic acid, ellagic acid and catechin, were identified, and polyphenol compounds flavonol, naringenin, and quercetin were also identified. That is, the standard and HPLC data obtained after diluting the fractions from each fraction in methanol solvent were found to match the peaks and indirectly confirmed the tannin components and polyphenol compounds by fractions. First of all, pet. In ether extracts, ellagic acid and quercetin were identified, and other polyphenol compounds such as flavonol and morin were found. In the ether extract, excess ellagic acid was identified and other polyphenol compounds could be found. In ethyl acetate extract, the amount of gallic acid was dominant, and polyphenol compounds such as ellagic acid and catechin were found. Butanol extract, which has the highest polarity, confirmed the presence of gallic acid and ellagic acid, especially naringenin was higher than other substances. Yulpi had a lot of garlic and ellagic acid. Among them, jutanin was found to be ellagic acid, and as shown in Table 1, 500g of yelp contained eligic acid (82.5g), and other polyphenols were quercetin (32.5g) and moline. There were 21.3 g of morin, 13.8 g of naringenin, 5 g of gallic acid, and 3.8 g of catechin. Therefore, the yield of each tannin component of Yulpi extracted with the organic solvent (Pet. Ether, Ether, Ethyl acetate, BuOH) in the first step was as shown in Table 2.

The amount of tannins and other polyphenolic compounds in yulpi (g / 500g) Ellagic acid 82.50 g Quercetin 32.50 g Maureen 21.30 g Naringenin 13.80 g Garlic acid 5.00 g Catechin 3.80 g

Yield Yield of Organic Solvents Sample Yield (g / 0.5L) PET ether fraction 7.0 g Ether fraction 3.8 g Ethyl acetate fraction 4.3 g Butanol fraction 3.5 g

Step 4: Structural Analysis

Of the components analyzed by the HPLC of step 3, ellagic acid and quercetin were separated into single components by repeated gel filtration chromatography and recrystallization, ¹ H NMR, ¹³ C NMR, and molecular weight spectroscopy. The structure was confirmed with a photometer. As a result, quercetin was obtained as a yellow powder, green in the FeCl ₃ test, and red in the Zn-HCl test. Δ 6.10 (1H, d, J = 1 Hz, H-6), δ 6.40 (1H, d, J = 1 Hz, H-8), δ 6.9 (1H, d, in ¹ H-NMR (DMSO-d ₆ ) J = 4.3 Hz, H-6 '), δ 7.55 (1H, dd, J = 4.2, 1.1 Hz, H-5'), δ 7.6 (1H, d, J = 1.0 Hz, H-2 '), δ 9.35 (1H, d, J = 6.6 Hz, 4'-OH), δ 12.5 (1H, s, 5-OH) were observed. Elacic acid (4,4 ', 5,5', 6,6'-Hexahydroxydiphenic acid dilactone) was found to be 7.4 (4H, S, 4,4 ', 5,5'-OH) in ¹ H NMR. 112.8 δ112.8 (C-1), δ115.4 (C-5), δ117.5 (C-6), δ144.7 (C-2), δ152.9 (C-4), at ¹³ C NMR, δ 153.2 (C-3) and δ 164.3 (C-7) were observed.

Example 2 Extraction, Separation, Purification and Structural Analysis of Tannin and Polyphenol Components of Persimmon

Step 1: Extract and Separate

Persimmon used in this step (Diospyros kaki Thunberg) was collected in September before maturation was confirmed to taste directly, and stored in -70 ℃ freezer was used. After cutting 5 kg of persimmon and extracting twice at room temperature for 3 weeks each with 70% acetone and filtered with filter paper. At this time, since the extract of persimmon contains a large amount of sugar (sugar), the high viscosity took a long time to filter. Acetone was removed under reduced pressure, and the crude extracts were examined by TLC (n-BuOH: Acetic acid: H ₂ O = 4: 1: 1, Ethyl acetate: Hexane = 1: 1). The aqueous layer was placed in a round bottom flask to remove all the water to obtain a solid material, which was transformed into an insoluble material that was insoluble in any solvent and could not be blown out anymore. The extract was extracted and separated as an organic solvent. This is shown in FIG. 3.

Second Step: Purification by Column Chromatography

The organic solvent extract of persimmon was purified by column chromatography in the same manner as in step 2 of Example 1.

Step 3: HPLC Analysis

In the same manner as in step 3 of Example 1, the organic solvent extract of persimmon was analyzed by HPLC. As a result, as shown in Figure 4 it was found that the jutannin component of persimmon is gallic acid. In particular, a large amount of gallic acid was found in the ethyl acetate layer among the various organic solvents of the second step, and also a large amount of moline was found. Comparison of the standard with C ₁₈ -reversed phase HPLC showed that 7.6 g of gallic acid and 4.5 g of murine were detected per 5 kg of persimmon. This is summarized in Table 3. Therefore, the tannin component and the polyphenol component of the persimmons extracted with the organic solvent in the first step were confirmed. The yields of the fractions obtained by sequential fractionation with ether, Ether, EtOAc, and BuOH were summarized, which shows the amount of fractions fractionated per liter of aqueous layer remaining after acetone was blown off. Among the fractions, the ethyl acetate layer contained a large amount of tannic gallic acid, while a small amount of polyphenolic compounds. Although small in the ether fraction, a number of polyphenol compounds such as flavonol, morphine, naringenin, and quercetin, and tannins such as gallic acid, erragic acid, and catechin were detected.

The amount of tannins and other polyphenol compounds in persimmon (g / 500g) Garlic acid 7.60 g Maureen 4.50 g Flavonol 3.72g Naringenin 2.84 g Ellagic acid 2.44 g Quercetin 2.40 g Catechin 1.12 g

Organic Solvent Extraction Yield of Persimmon Sample Yield (g / L) Pet. Ether fraction 0.6g Ether fraction 1.6 g Ethyl acetate fraction 3.0 g Butanol fraction 1.0 g

Step 4: Structural Analysis

Among the components analyzed by HPLC in the same manner as in step 4 of Example 1, gallic acid was separated into single components by repeated gel filtration chromatography and recrystallization, IR spectrum, ¹ H NMR, and ¹³ The structure was confirmed by C NMR. Experimental results, gallic acid showed a blue color in the FeCl ₃ Test as colorless needle-like crystals, from 3496cm ^-1 in the IR spectrum was found phenol sanim show the absorption band due to OH groups in the COO, 1668cm ^{-1 1} H-NMR spectrum in Singlet signal by galloyl corresponding to hydrogen integration value 2H at δ7.05 ppm was shown.In ¹³ C-NMR spectrum, C-3 at δ110.3, C-4 at δ146.4, δ170.4 Carbonyl signal by COOH group was observed at.

Example 3: Extraction, Separation, Purification and Structural Analysis of Tannin and Polyphenol Components of Pine Needles

Step 1: Extract and Separate

2 Kg of pine needles (Pinus densiflora Sieb. Et Zucc.) Were extracted twice with 70% acetone for 3 weeks at room temperature, and then filtered through a filter paper. After filtration, fractionation with an organic solvent was carried out in the same manner as in the first step of Example 1. This is shown in FIG. 5.

Second Step: Purification by Column Chromatography

The organic solvent extract of pine needles was purified by column chromatography in the same manner as in step 2 of Example 1.

Step 3: HPLC Analysis

In the same manner as in Example 3, Example 1, the organic solvent extract of pine needles was analyzed by HPLC. As a result, as shown in Figure 6, catechin, ellagic acid, quercetin, flavonol and the like were confirmed. In addition, many peaks that did not match the standard were found at elution time 3.5 minutes, 8 minutes, 12 minutes, 13 minutes, 13.5 minutes, 16.5 minutes, and 18.5 minutes. After extracting 2 Kg of pine needles with 70% aqueous acetone, 65 g of crude extract was obtained. The catechin, morine, and flavonol could be separated into single components through repeated chromatography and recrystallization. Tannins and polyphenol compounds contained in pine needles are summarized in Table 5 and the yields are shown in Table 6.

The amount of tannin and other polyphenol compounds in pine needles (g / 1kg) Flavonol 12.40 g Ellagic acid 3.20 g Catechin 1.80 g Quercetin 1.70 g Maureen 650 mg

Yield of Organic Solvents from Pine Needles Sample Yield (g / L) PET, ether fraction 0.75 g Ether fraction 0.45 g Etel acetate fraction 1.08g Butanol fraction 0.35 g

Step 4: Structural Analysis

Among the components analyzed in HPLC in the same manner as in step 4 of Example 1, the morphine and flavonol were separated into single components by repeated gel filtration chromatography and recrystallization, ¹ H NMR and ¹³ C. The structure was confirmed by NMR. Experimental results, Maureen ¹ H - δ 6.15 in the NMR (1H, d, J = 1Hz, H-6), δ 6.35 (1H, d, J = 1Hz, H-8), δ 6.45 (1H, m, H -3 '), δ 6.5 (1H, d, J = 1.1 Hz, H-5'), δ 7.45 (1H, d, J = 4.3 Hz, H-6 '). ¹³ C-NMR, δ94.6 (C-8), δ99.3 (C-6), δ104.6 (C-10), δ105.1 (C-5 '), δ109.3 (C-3' ), δ111.4 (C-6 '), δ132.2 (C-1'), δ136.4 (C-3 '), δ150 (C-2'), δ157.5 (C-4 '), δ 159.0 (C-9), δ 162.5 (C-2), δ 162.8 (C-5), δ 165.5 (C-7), and δ 177.7 (C-4) were observed. In ¹ H-NMR of flavonols, δ 7.55 (4H, m, H-5, H-6, H-7, H-8), δ 7.70 (2H, m, H-3 ', H-4'), δ 8.10 (1H, dd, J = 4.0, 0.7 Hz, H-5 '), δ 8.25 (2H, dd, J = 5.6, 1.4 Hz, H-2', H-6 '), δ 9.65 (1H, S, 2-OH) and ¹³ C-NMR showed δ118.6 (C-8), δ121.5 (C-7), δ124.8 (C-6), δ125.0 (C-5), δ126.5 (C-10), δ127.4 (C-4 '), δ127.9 (C-3'), δ128.7 (C-5 '), δ130.1 (C-2'), δ131 .5 (C-6 '), δ 134.0 (C-1'), δ 139.3 (C-3), δ 145.4 (C-9), δ 154.8 (C-2), δ 173.2 ( C-4) was observed.

Example 4 Extraction, Separation, Purification and Structural Analysis of Hope and Tannin and Polyphenol Components

Step 1: Extract and Separate

Hopbun (Humulus Lupulus) 2 Kg with 70% aqueous acetone was repeatedly extracted twice at room temperature for 3 weeks, and then filtered and completely removed acetone under reduced pressure. When removing acetone and water by using a rotary evaporator, it was difficult to generate a large amount of bubbles in the flask. Thus, a small amount of butanol was added thereto to remove solvent while removing bubbles. The aqueous extract obtained here was filtered to filter out insoluble matters such as chlorophyll and the filtrate was used as it was. After filtration, fractionation with an organic solvent was carried out in accordance with the yeolpi of Example 1 and the method of persimmon of Example 2. This is shown in FIG.

Second Step: Purification by Column Chromatography

The organic solvent extract of Hope was purified by column chromatography in the same manner as in Yule of Example 1.

Step 3: HPLC Analysis

In the same manner as in step 3 of Example 1, the organic solvent extract isolate of Hope was analyzed by HPLC. As a result of the experiment, it was found that ellagic acid, naringenin, and quercetin were contained as shown in FIG. 8. After re-extracting the aqueous acetone extract with BuOH, Sephadex LH-220 chromatography and MCI gel CHP20 chromatography were repeatedly performed to isolate and confirm quercetin and quercetin-3-rhamnoside. The amounts of tannins and other polyphenol compounds contained in the hops are shown in Table 7 and the organic solvent extraction yields are shown in Table 8. Tannins and polyphenol components of hops contained ellagic acid, morine, quercetin, marinegenin, and flavonol in 0.5 g of hexane extraction fraction, and ellagic acid, naringenine, and flabo in 1.2 g ethyl acetate extract fraction. Among the 2.3g butanol extract fractions, it was found that there were gallic acid, ellagic acid, maline, quercetin, naringenin, and flavonol.

The amount of tannin and other polyphenolic compounds in hops (g / 1kg) Ellagic acid 3.21 g Naringenin 2.90 g Quercetin 2.63 g Maureen 1.63 g Flavonol 1.56 g Garlic acid 1.12 g

Hope of Organic Solvent Extraction Sample Yield (g / L) PET ether ether fraction 1.65 g Ether fraction 3.73 g Ethyl acetate fraction 3.96 g Butanol fraction 7.59 g

Step 4: Structural Analysis

Among the components analyzed by HPLC in the same manner as in step 4 of Example 1, quercetin and quercetin-3-lammoside were separated into single components by repeated gel filtration chromatography and recrystallization. The structure was confirmed by ¹ H NMR and ¹³ C NMR. As a result, quercetin and quercetin-3-rhamnoside were obtained as yellow powder, and both components were green in the FeCl ₃ test and red in the Zn-HCl test. Quercetin is δ: 6.10 (1H, d, J = 1 Hz, H-6), 6.40 (1H, d, J = 1 Hz, H-8), 6.9 (1H) in ¹ H-NMR (DMSO-d ₆ ). , d, J = 4.3 Hz, H-6 '), 7.55 (1H, dd, J = 4.2, 1.1 Hz, H-5'), 7.6 (1H, d, J = 1.0 Hz, H-2 '), δ9.35 (1H, d, J = 6.6 Hz, 4′-OH), 12.5 (1H, s, 5-OH) and ¹³ C-NMR (DMSO-d ₆ ) at δ: 93.5 (C-8), 98.4 (C-6), 103.2 (C-10), 115.2 (C-2 '), 115.8 (C-5'), 120.2 (C-6 '), 122.1 (C-1'), 135.9 (C- 3), 145.2 (C-3 '), 147.8 (C-4'), 147.9 (C-9), 156.3 (C-2), 160.9 (C-5), 164.1 (C-7), 176.0 (C -4) was observed and quercetin-3-rhamnoside was found at ¹ H-NMR (acetone-d ₆ + D ₂ O) at δ: 0.81 (3H, d, J = 6 Hz), 5.25 (1H, s), 6.19 (1H, d, J = 2 Hz), 6.38 (1H, d = 2 Hz), 6.86 (1H, d, J = 8 Hz), 7.22 (1H, d, J = 2 Hz), 7.28 (1H, dd, J = 2, 8 Hz), 12.63 (1H, s): ¹³ C-NMR (acetone-d ₆ ) δ: 17.6, 70.1, 70.4, 70.7, 71.3, 93.8, 98.8, 101.9, 104.1, 115.6, 115.7, 120.8, 121.0, 134.3, 145.4, 148.6, 156.5, 157.4, 161.4, 164.5, 177.8.

Example 5: Synthesis of Gallic Acid Derivatives

In the present Example, a lipid-soluble derivative was designed and synthesized by molecular formula targeting gallic acid, which is relatively stable and simple in structure, and converts functional groups by extracting separation, purification and structural analysis from persimmon in Example 2. That is, the novel gallic acid derivative represented by following formula (I) was manufactured.

(In the above formula, R generally represents a lower alkyl group having a C ₁ to C ₅ carbon group and a higher alkyl group having a C ₆ to C ₁₈ carbon group.)

The gallic acid derivative represented by Formula (I) was reacted with benzyl chloride and potassium carbonate to selectively protect the alcohol of gallic acid, and then carboxylic acid was prepared using sodium hydroxide. It was then reacted with methyliodine, hexyltosylate and stearyltosylate to introduce various ester groups. Benzyl groups were effectively demineralized by hydrogenation using a palladium catalyst. This is shown in the following formula (II).

(Wherein R is the same as formula (I))

To synthesize methyl gallate, methyl ester was synthesized by adding iodomethane and K ₂ CO ₃ to the tribenzyl calate. This is shown in the following formula (III).

Hexyl gallate was synthesized after the synthesis of hexyl alcohol tosylate (Hexyl alcohol tosylate) and dissolved in DMF and tribenzyl gallate (Tribenzyl gallate) and CsCO ₃ and reacted at 55 ℃. This is as shown in following formula (IV).

In the synthesis of gallic acid stearyl ester, stearyl alcohol tosylate was synthesized, dissolved in DMF, and triacetyl gallic acid and CsCO ₃ were added and reacted at 55 ° C. . This is as shown in the following formula (V).

Example 6: Synthesis of Catechin Derivatives

In this example, a catechin derivative represented by the following formula (VI) and catechin (2- (3,4-dihydroxyphenylchroman-3,5,7-triol) were prepared.

(In the formula, R generally represents a lower acyl group having a carbon group of C1 to C5 and a higher acyl group having a carbon group of C6 to C18.)

In order to protect the alcohol of catechin, the first reaction was carried out using a benzyl group, and then acetic anhydride was reacted to introduce an acyl group. Finally, the hydrogenation reaction was performed using paradium as a catalyst. This process is as shown to the following formula (i).

(Wherein R is the same as formula (VI))

Example 7: Synthesis of Ellagic Acid Derivatives

In this example, the ellagic acid derivative represented by the following formula (i), 2- (2-carboxy-4,5,6-trihydroxyphenyl) -3,4,5-trihydroxymethylbenzoate), are prepared It was.

(In the above formula, R generally represents a lower alkyl group having a C ₁ to C ₅ carbon group and a higher alkyl group having a C ₆ to C ₁₈ carbon group.)

The ellagic acid derivative was protected with alcohol using a benzyl group, and then reacted with methyl iodine after reacting base and benzyl bromide simultaneously for ring-opening and protection. This is shown in Formula (XI).

(Wherein R is the same as formula (i))

As described above with reference to the above examples, the present invention extracts, separates and purifies tannin and polyphenol components from yulpi, persimmon, pine needles, and hops to analyze their structure and remove skin whitening and bacterial toxins from the separated tannin components. It is a very useful invention in the biomass industry because it has an excellent effect of synthesizing and providing ellagic acid derivatives, gallic acid derivatives and catechin derivatives which are effective in free radical scavenging, tumor suppression and DNA cleavage.

Claims (6)

Tannin compounds extracted from jujupi, persimmon, pine needles and hops, respectively. The tannin compound according to claim 1, wherein the tannin component is gallic acid, ellagic acid, or catechin. After extracting and filtering each of Yulpi, persimmon, pine needles, and hops with acetone, all of the crude extract obtained by removing acetone was removed. Extraction and separation method of tannins from yulpi, persimmon, pine needles and hops, characterized in that the tannin component is sequentially separated in order. Gallic acid derivative represented by following formula (I). (Wherein R represents a lower alkyl group having a C ₁ to C ₅ carbon group and a higher alkyl group having a C ₆ to C ₁₈ carbon group) The catechin derivative represented by following formula (VI). (Wherein R represents a lower alkyl group having a C ₁ to C ₅ carbon group and a higher alkyl group having a C ₆ to C ₁₈ carbon group) Elagic acid derivative represented by following formula (i). (Wherein R represents a lower alkyl group having a C ₁ to C ₅ carbon group and a higher alkyl group having a C ₆ to C ₁₈ carbon group)