KR20070060714A - A mass production method of brazilein from caesalpinia sappan l - Google Patents

Abstract

A mass production method of brazilein from Caesalpinia sappan L. is provided to reduce the time and costs for production of brazilein and obtain high purity of brazilein by eliminating a process of column chromatography and using no expensive chromatography devices, so that the brazilein is produced in the industrial scale. The mass production method of brazilein from Caesalpinia sappan L. comprises the steps of: (1) extracting Caesalpinia sappan L. with water, alcohol or a mixture thereof; (2) concentrating the extract of Caesalpinia sappan L. to obtain crude crystals of Caesalpinia sappan L.; (3) dissolving the crude crystals in alcohol; (4) concentrating the solution of step(3) in a water bath at 30-40 deg.C and 40-180 rpm; and (5) recrystallizing the concentrated solution by evaporating the solution at 15-30 deg.C for 6-72 hours, wherein the alcohol of the steps(1) and (3) is C1-C3 lower alcohol such as methanol; and the alcohol temperature of step(3) is the of 40-64 deg.C.

Description

A mass production method of brazilein from Caesalpinia sappan L.}

1 is a schematic diagram showing a method of separating brazilian lane from the existing joiner,

Figure 2 is a schematic diagram showing a separation method of brazilian lane from joiner according to the present invention,

3 is a TLC test result of the brazilian lane isolated by the present invention.

The object of the present invention is a method for mass production of brazilian lane from joiner.

Caesalpinia sappan L. is a deciduous arborescent or shrub belonging to the legume (Legminosae), which is distributed in tropical Asia such as India, Malaysia Peninsula and southern China. The pharmacological dictionary records the antimicrobial activity, central nervous system suppression, and cardiovascular activity as a pharmacological action of joiner (Kim Chang-min et al., Chinese medicinal metabolism, Jeongdam Publishing Co., pp.3130, 1998).

Joiner studies include cytotoxic effects on gastric cancer cell lines (Park, KJ, et al. , Kor. J. Pharmacogn. 28 (4), pp233-238, 1997), antioxidant effects (Lim, DK, et al. , Kor. J. Food Sci.Technol. 28 (1), pp77, 1996; Badami Shrishailappa et al. , Bio. Pharm. Bull. , 26 (11), pp1534-1537, 2003), anti-inflammatory and antibacterial deodorant effects (Kim, YS et al. , Kor. J. Pharmacogn. 26 (3), pp265-272, 1995; Hikino HT et al. , Planta Med. , 31 (3), pp214-220, 1977; Kim, KJ et al. , J. ethnopharmacology , 91 (1), pp81-87, 2004; Xu, HX and Lee, S. F, phytotheraphy Research , 18 (8), pp647-651, 2004), anticonvulsant effect (Baek, NI et al. , Arch. Pharm. Res. 23 (4), pp344-348, 2004) and anticomplement activity (Oh, SR et al. , Planta Med. , 64 (5), pp456-458, 1998) and topo Reports on tooisomerase I inhibitory activity and the like (Jeon, WK et al. , Kor. J. Pharmacogn. 30 (1), pp 1-6, 1999), and the like. In another specification the inventors Has been reported to inhibit angiogenesis.

The main components of the joiner are colorless brasins with hematoxylin and flavonoid structures.Brazilians are oxidized in air and brazilian (Moon, CK et al. , Arch. Pharm. Res. 11 (2) , pp149 ) . -154, 1988). Joiner's main ingredient, brazilin, is effective in hypertension (Moon, CK et al . , Drug Chem . Toxicol . 15 (1) , pp81, 1992), reports that brazilin controls calcium levels in platelets (Hwang, GS et. al . , Arch . Pharm . Res . 21 (6) , pp774-778, 1998) reported the results of blood glucose lowering effect of braziline in the blood (Kim, SG et al . , Arch . Pharm . Res . 21 (2) , pp 140-146, 1998). Sappanchalcone, one of the components of joiner, has been reported to inhibit platelet coagulation (Morota, T. et al. , Jpn . Kokai Tokkyo Koho , pp 6, 1990).

However, Brasillane has reports on its structural analysis (Moon, CK et al . , Arch . Pharm . Res . 11 (2), pp149-154, 1988; Kim, DS et. al . , Phytochemistry, 46 (1), pp177-178, 1997) No efficacy has been reported at all, and in another specification of the inventors filed on the same page, the angiogenesis inhibitory effect is the first activity report. In general, brachiane showed little cytotoxicity against HUVEC cells or other cancer cells, whereas compounds with angiogenesis inhibitory effects showed high cytotoxicity. In addition, the ability to inhibit angiogenesis is excellent in vitro as well as in vivo, and thus highly applicable as an anticancer agent or an anticancer adjuvant therapy, it can be said that the development value of medicines using the same is very high. The bulk separation is essential when developing the brazilian medicinal product, and in particular, the bulk separation in natural products is incomparable or almost impossible compared to other synthetic materials, so the mass separation method presented herein is very important.

Conventional brazilian lanes have generally been separated in the following manner. The seedlings were cooled in methanol at room temperature to obtain a seedling extract, followed by filtration. The filtrate was concentrated under reduced pressure to obtain methanol extract. To the methanol extract was added water: methanol (3: 1) and layered with ethyl acetate to give an ethyl acetate fraction. Subsequently, the ethyl acetate fraction is subjected to silica gel column chromatography using a chloroform: methanol (15: 1 → 5: 1) mixed solvent to obtain a fraction containing brazilian abundance. The fractions were again subjected to silica gel column chromatography with chloroform: methanol: water (10: 3: 1) mixed solvent to obtain crude braille, which was completely dissolved in hot methanol, concentrated and left at room temperature. To obtain pure brazilian lanes (Kim, DS et al. , Phytochemistry, 46 (1), pp177-178, 1997) ( FIG. 1 ).

Since the method of silica gel column chromatography is carried out at least twice to obtain the brazilian lane of the crude crystal and recrystallize it again, there is a problem that a number of steps must be taken. In addition, the column chromatography method requires scale-up for mass separation, but there are technical limitations, and therefore, a large cost is required because a large number of columns must be prepared for mass separation.

Therefore, there is a need for a method for mass separation of brazilian lanes with economical and simple separation methods.

Accordingly, the present inventors have devised a method for separating brazilian at low cost, and have devised a method that does not go through the column chromatography process, which is essentially performed in the existing separation process. The present invention was completed by confirming that expensive equipment such as column chromatography, supercritical extractor, HPLC, etc. required for the chromatography process was not necessary, and that high purity brazilians could be separated.

It is an object of the present invention to provide a method for mass separation and purification of brazilian from joinery in a low cost and short time by simple separation process steps.

The present invention

1) obtaining the extract from the joiner using water, alcohol or a mixture thereof;

2) concentrating the extract of step 1) to obtain crude crystals;

3) dissolving the crude crystals of step 2) with alcohol;

4) concentrating the solution of step 3); And

5) It provides a mass production method of brazilian from joiner comprising the step of recrystallizing the concentrate of step 4).

Hereinafter, the present invention will be described in detail.

The parts of the joiner used in the present invention are the leaves, roots and stems of the joiner, preferably the heartwood from which bark and sapwood of the joiner are removed. In addition, the joiner may be cut to an appropriate size to facilitate extraction.

The alcohol of steps 1) and 3) is preferably a lower alcohol of C ₁ to C ₃ , more preferably methanol. In addition, as an extraction method for obtaining the extract Although common methods, such as cold needle, warm needle, and reflux, are all possible, Preferably it is cold needle. In step 1), the method of extracting the seedlings is cold for about 12 hours to 72 hours, preferably 24 hours to 48 hours, and the number of extraction is 1 to 5 times, preferably 3 times.

The joiner extract extracted by the method is filtered to remove the joiner and the remaining residues are removed. In this case, a filter cloth or a filter paper may be used as the filtration method, but is not necessarily limited thereto.

The concentration method in step 2) can be any of the usual concentration methods, in step 3) the crude crystals are dissolved in a ratio of crude crystals: alcohol = 1 g: 0.1 L to 1 L, preferably 3 g: 1 l. At this time, the temperature of the alcohol is preferably 40 ℃ ~ 64 ℃.

In step 4), the conditions of water bath temperature and rotation speed for concentration have a significant effect on recrystallization, and the experiment was performed by changing them to find an optimization condition. As a result, as shown in Table 1, in the mass separation method of brazilian lane from the joiner according to the present invention, when the temperature of the water bath is about 30 ℃ ~ 40 ℃, the rotation speed is 40 rpm ~ 180 rpm crystals are produced, It can be seen that it is the most optimal condition for generating.

Water bath temperature (℃) Rotational Speed (RPM) 20 30 40 80 100 120 140 160 180 200 220 20 × × × × × × × × × × 25 × × × × × × × × × × 30 × △ ○ ○ ○ ○ ○ ○ ○ △ × 35 × △ ○ ○ ○ ○ ○ ○ ○ △ × 40 × △ ○ ○ ○ ○ ○ ○ ○ △ × 45 × × × × × × × × × × 50 × × × × × × × × × × 55 × × × × × × × × × ×

※ ○: crystal formation, △: slightly slurry state, ×: no crystal formation

Therefore, the temperature of the water bath (concentration) in the concentration in step 4) is preferably 30 ° C ~ 40 ° C, the rotational speed 40 rpm ~ 180 rpm, then in step 5) for 6 hours to 72 hours, preferably It is preferable to recrystallize by leaving evaporated for 24 hours at 15 ℃ to 30 ℃, preferably room temperature (25 ℃) (see Fig. 2 ). In addition, the amount of concentrate by the method is preferably about 1/3 to 1/30 of the initial volume, more preferably 1/10 to 1/20.

In addition, MS (mass spectrometry, Micromass Quattro Micro ^TM (triple quadrupole mass spectrometer)) method was performed to measure the molecular weight of the compound separated by the recrystallization, and NMR (1H-NMR, DEPT 135, 13C-) for molecular structure analysis. NMR et al., Bruker Advance 500, Bruker AXS GMBH, Germany). The measurement results thereof are as follows, whereby it was confirmed that the material separated from the joiner is brazil lane of the formula (1 ).

<Formula 1>

Brazilian structure

1) Molecular Weight: 284

2) Molecular formula: C ₁₆ H ₁₂ O ₅

3) 1H-NMR (500MHz, DMSO- d ₆ ): δ 7.80 (1H, d, J = 8.7 Hz, H-1), 7.10 (1H, s, H-11), 6.56 (1H, d, J = 8.5 Hz, H-2), 6.35 (1H, s, H-4), 6.32 (1H, s, H-8), 4.45 (1H, d, J = 11.7 Hz, H-6a), 4.00 (1H, d, J = 11.7 Hz, H-6b), 2.85 (2H, d, J = 3.7 Hz, H-7)

4) ¹³ C-NMR (125MHz, DMSO- d ₆ ): δ 39 (C-7), 72 (C-6), 74 (C-6a), 102 (C-4), 104 (C-11) , 110 (C-1a), 110 (C-2), 117 (C-8), 126 (C-11a), 130 (C-1), 151 (C-12), 152 (C-10), 157 (C-4a), 158 (C-7a), 162 (C-3), 179 (C-9)

In addition, crude crystals isolated from the above-described method and crystals obtained after recrystallization were analyzed by thin layer chromatography (TLC). As a result, as shown in Fig . 3 , in the crude crystals ( A in Fig. 3 ), impurities were mixed and separated into various colors (layers), but only one color was shown in the crystals ( B in Fig. 3 ) after recrystallization. As a result, it was confirmed that the purity of brazilian lane was increased due to recrystallization.

As shown in Fig . 1 , in the conventional method for extracting brazilian from the joiner, the joiner is cooled in methanol at room temperature to obtain a joiner extract, followed by filtration. The filtrate is reduced and concentrated to obtain methanol X. To the methanol extract was added water: methanol (3: 1) and layered with ethyl acetate to give an ethyl acetate fraction. Thereafter, the ethyl acetate fraction is purified by silica gel column chromatography using a chloroform: methanol (15: 1 → 5: 1) mixed solvent to obtain a fraction containing a lot of brazane. The fractions were again subjected to silica gel column chromatography with chloroform: methanol: water (10: 3: 1) mixed solvent to obtain crude braille, which was completely dissolved in hot methanol, concentrated and left at room temperature. To obtain pure brazilian (Kim, DS et al. , Phytochemistry, 46 (1), pp177-178, 1997) ( FIG. 1 ). In this way, 470 mg (0.47%) of brazilian lane is obtained from 100 g of seedlings when extracting brazilian lane. However, the method is not only troublesome to go through several steps of column chromatography, but also has a considerable cost in the mixed solvent, column, and expensive equipment required to perform column chromatography when separating at a large capacity. .

However, the method according to the present invention was able to obtain about 30 g (0.34%) to 45 g (0.5%) at 9 kg of joiner, depending on the conditions, and does not go through the column chromatography method. It can be seen that lanes can be obtained.

In addition, the conventional method increased the yield of brazilian lane (8%) from the joiner by leaving the joiner crude crystal solution for one month (Kim, DS et al.). al . , Phytochemistry, 46 (1), pp177-178, 1997), The present invention can obtain a larger amount of brazilian lane during the same period when obtaining brazilian lanes in different batches.

Therefore, it can be seen from the above that the method of the present invention is a suitable method for mass separation (manufacturing) of brazilian lane.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

Example 1 Isolation and Identification of Brazilian Lane from Joiner

<1-1> joiner extract

9Kg of chopped saplings were put in the extraction bag and immersed in 18L of 100% methanol and extracted for 48 hours at room temperature. The process was repeated three times, and the extract was filtered using a filter cloth.

<1-2> Obtaining joining crude crystals

The filtered extract of Example 1-1 was concentrated using a concentrator under reduced pressure until the solution was completely evaporated, thereby obtaining a dark red crude crystal.

3 g of the crude crystals were completely dissolved in 1 L of hot (50 ° C.) methanol.

<1-3> Joiner Crystals Obtained

The filtrate was poured into a 40 ° C water bath using a rotary evaporator (EYELA N-1000, EYELA, Japan) until the solution in which the crude crystal was dissolved in Example 2 was about 1/20 of the initial volume. The mixture was heated and decompressed and concentrated at a rotation speed of 100 rpm. The concentrated solution under reduced pressure was left at room temperature for 24 hours to recrystallize to obtain pure brazilian from joiner ( FIG. 2 ).

As a result of using the method of the present invention it was possible to isolate 45 g of brazilian lane from 9 kg of joiner.

Example 2-10 Separation of Brazilian Lane from Joiner

The Brazilian lane was separated from the joiner by changing the conditions of Example 1 of the present invention, which is shown in Table 2 .

Example Step 1 Step 2 Step 3 Step 4 Step 5 Yield Alcohol(%) Extraction Method (Time × Times) Concentration Solvent temperature (℃) Concentration ratio, temperature (℃) and rotation speed (rpm) Neglect time 2 100% methanol 48 × 3 1/10 50 ℃ 1/10, 40 ℃, 100 rpm 24 35.8 g 3 95% methanol 36 × 4 1/30 50 ℃ 1/20, 40 ℃, 120 rpm 24 30.6 g 4 90% methanol 24 × 3 1/20 45 ℃ 1/20, 40 ℃, 100 rpm 36 27.7 g 5 90% methanol 18 × 3 1/3 55 ℃ 1/10, 40 ° C, 140 rpm 60 22.2 g 6 85% methanol 36 × 3 * 60 ℃ 1/10, 40 ℃, 100 rpm 48 23.9 g 7 100% methanol 18 × 4 1/15 40 ℃ 1/30, 40 ℃, 100 rpm 6 31.4 g 8 80% methanol 24 × 4 1/10 45 ℃ 1/30, 40 ℃, 160 rpm 72 18.3 g 9 99% ethanol 24 × 3 * 64 ℃ 1/20, 40 ℃, 100 rpm 12 20.2 g 10 95% ethanol 24 × 3 1/20 50 ℃ 1/10, 40 ° C, 180 rpm 24 16.6 g

*: Completely evaporate

Experimental Example 1 Purity Analysis of Brazilian Lane

The crude crystals and the crystals obtained after the recrystallization of the seedling extract isolated in Example 1 were analyzed by TLC (Thin layer chromatography) and the like ( Fig. 3 ).

TLC was carried out under the conditions of stationary phase C ₁₈ reversed phase, mobile phase methanol: water = 1: 1. The TLC plate used in the experiment was manufactured by Merck (Merck, no.1.15389.0001, RP-18, F254S, Germany), using a TLC chamber at room temperature using a developing solvent (methanol: water = 1: 1). Was developed and the solvent was dried and then developed with 5% sulfuric acid solution. At this time , yellow spots were observed when visually observed as in FIG. 3 (Rf, 0.46).

Therefore, the result of comparing the crude crystals (A in Fig. 3) and recrystallized crystals (B in Fig. 3) after separation from the joiner, crude crystals, as shown in Figure 3. In the case of Brazilian lane one impurity but recrystallization mixture of It was confirmed that the purity was increased.

Comparative Example 1 Comparison of Brazilian Lane Yield

Kim et al. (Kim, DS et. al . , Phytochemistry, 46 (1), pp177-178, 1997) was isolated and purified according to the extraction method ( Fig. 1 ).

<1-1> joiner extract

100 g of finely chopped saplings were immersed in 200 ml of 100% methanol and extracted for 3 days at room temperature. This process was repeated three times, and then filtered using a filter cloth. To the methanol extract were added 200 ml of water: methanol (3: 1) and layered three times with 100 ml of ethyl acetate to give an ethyl acetate fraction.

<1-2> Crude Brazilian Lane

The ethyl acetate fractions were eluted with silica gel (Kieselgel 60 (70-230 mesh), Merck) column chromatography using a chloroform: methanol (15: 1 → 5: 1) mixed solvent to obtain a fraction containing brazilian lane. Get The fractions were again subjected to silica gel column chromatography with a chloroform: methanol: water (10: 3: 1) mixed solvent to obtain crude brazilian.

<1-2> Brazilian lane acquisition

The crude brarolein obtained above was completely dissolved in 200 ml of hot methanol, concentrated to 1/5 of the initial volume, and left at room temperature for about 12 hours (overnight). It was then filtered to obtain 470 mg of pure brazilian lane.

In this way, 470 mg (0.47%) of brazilian lane was obtained from 100 g seedlings upon brazilian extraction. On the other hand, by the method of the present invention, about 30 g (0.34%) to 45 g (0.5%) could be obtained at 9 kg of joiner depending on the conditions.

As described above, the separation method of brazilian lane from the joiner according to the present invention can be separated from the high purity brazilian lane without undergoing the column chromatography process that is essentially performed in the existing separation process, and also in the column chromatography process It does not require expensive equipment such as column chromatography, supercritical extractor, HPLC, etc., which is efficient for separating large amounts of brazil, enabling commercial production of brazil.

Claims (9)

1) obtaining the extract from the joiner using water, alcohol or a mixture thereof; 2) concentrating the extract of step 1) to obtain crude crystals; 3) dissolving the crude crystals of step 2) with alcohol; 4) concentrating the solution of step 3); And 5) A mass production method of brazilian from joiner comprising recrystallizing the concentrate of step 4). The method of claim 1 wherein the alcohol of steps 1) and 3) is a lower alcohol of C ₁ to C ₃ . The method of claim 2, wherein the alcohol is methanol. The method of claim 1, wherein the alcohol of step 3) is a mass production method of brazilian, characterized in that 40 ℃ ~ 64 ℃. The method of claim 1, wherein the concentration of the water bath during the concentration of step 4) is 30 ℃ ~ 40 ℃, rotation speed 40 rpm ~ 180 rpm, characterized in that the mass production method of brazilian lane. The method of claim 1, wherein the recrystallization of step 5) is characterized in that the solution of step 4) is evaporated for 6 to 72 hours. 7. The method of claim 6, wherein the recrystallization is evaporated for 24 hours. The method of claim 1, wherein the recrystallization of step 5) is a mass production method of brazilian lane characterized in that the solution of step 4) is left to evaporate at 15 ℃ to 30 ℃. 10. The method of claim 8, wherein the recrystallization is left to evaporate at 25 ℃.

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