WO2007066926A1

WO2007066926A1 - A mass production method of brazilein from caesalpinia sappan l

Info

Publication number: WO2007066926A1
Application number: PCT/KR2006/005082
Authority: WO
Inventors: Hyun Ok Yang; Yeon Hee Choi
Original assignee: Korea Institute Of Science And Technology
Priority date: 2005-12-09
Filing date: 2006-11-29
Publication date: 2007-06-14
Also published as: KR20070060714A; KR100742265B1

Abstract

The present invention relates to a mass -production method of brazilein from Caesalpinia sappan L. , more precisely a mass-production method of brazilein from Caesalpinia sappan L. which comprises the steps of 1) preparing an extract from Caesalpinia sappan L. by using water, alcohol or a mixture of the two; 2) preparing a crude crystal by concentrating the extract of step 1) ; 3) dissolving the crude crystal of step 2) in alcohol; 4) concentrating the solution of step 3) ; and 5) re- crystallizing the concentrated solution of step 4) . According to the method of the invention, brazilein can be mass-produced from Caesalpinia sappan L. with high purity without column chromatography which has been necessarily performed according to the conventional methods, and thus the method of the invention does not need expensive equipments such as column chromatography, supercritical fluid extractor, HPLC, etc., suggesting that the method is efficient for mass -production of brazilein and industrially applicable.

Description

A MASS PRODUCTION METHOD OF BRAZILEIN FROM

CAESALPINIA SAPPAN L

Technical Field

The present invention relates to a mass production method of brazilein from Caesalpinia sappan L.

Background Art

Caesalpinia sappan L. is a small deciduous tree or a shrub belonging to Legminosae, which is widely distributed in tropic Asia such as India, Malaysian peninsula, southern

China, etc. According to the encyclopedia of pharmacy,

Caesalpinia sappan L. is alleged to have such pharmacological activities as antimicrobial activity, inhibitory activity to central nervous system and cardiovascular system associated activity, etc. (Kim, et al . , "JungYakDaeSaJeon" , "JeongDamChulPanSa" , pp .3130 ,

1998) . It has already been reported that Caesalpinia sappan L. has cytotoxic effect on stomach cancer cells (Park, K. J., et al., Kor. J. Pharmacogn. 28(4), pp233-238, 1997), antioxidant effect (Lim, D. K., et al . , Kor. J. Food Sci . Technol. 28(1), pp77, 1996 ; Badami Shrishailappa et al . , Bio. Pharm. Bull., 26(11), ppl534-1537, 2003), anti- inflammatory effect, antibacterial and deodorant effect

(Kim, Y. S. et al . , Kor. J. Pharmacogn. 26(3), pp265-272,

1995 ; Hikino H Tet al . , Planta Med., 31(3), pp214-220,

1977 ; Kim, K. J. et al . , J^". ethnopharmacology, 91(1), pp81-87, 2004 ; Xu, H. X. and Lee, S. F, phytotϊveraphy

Research, 18(8), pp647-651, 2004), anticonvulsant effect

(Baek, N. I. et al . , Arch. Pharm. Res. 23(4), pp344-348,

2004), anticomplementary activity (Oh, S. R. et al . , Planta

Med., 64(5), pp456-458, 1998) and topoisomerase-I inhibitory activity (Jeon, W. K. et al., Kor. J. Pharmacogn.

30(1), ppl-6, 1999), etc. In addition, a novel angiogenesis inhibition effect of Caesalpinia sappan L. has been reported in the patent description applied by the present inventors on the same date with this patent application.

The major ingredient of Caesalpinia sappan L is alleged to be the colorless brazilin having the hematoxylin/flavonoid structure. The brazilin is oxidized in the air to be brazilein (Moon, C. K. et al . , Arch. Pharm. Res. 11(2), ppl49-154, 1988). Previous reports said that the major ingredient of Caesalpinia sappan L., the brazilin, is effective for the treatment of hypertension (Moon, C. K. et al., Drug Chem. Toxicol. 15(1), pp81, 1992), regulates calcium content in platelets (Hwang, G. S. et al., Arch. Pharm. Res. 21(6), pp774-778, 1998), and lowers blood sugar (Kim, S. G. et al . , Arch. Pharm. Res. 21(2), ppl40-146, 1998) . Another ingredient of Caesalpinia sappan L. is sappanchalcone, which inhibits platelet coagulation (Morota, T. et al., Jpn. Kokai Tokkyo Koho, pp6, 1990).

The structure of brazilein has been analyzed (Moon, C. K. et al., Arch. Pharm. Res. 11(2), ppl49-154, 1988; Kim, D. S. et al., Phytochemistry, 46(1), ppl77-178, 1997) but the efficacy of the brazilein has not been reported. In fact, the report of angiogenesis inhibition effect of the brazilein in the other patent description coincidently applied by the present inventors with this invention seems to be the first report made in relation to the brazilein activity. In general, those compounds having angiogenesis inhibition effect exhibit cytotoxicity, whereas brazilein hardly exhibits cytotoxicity in HUVEC or other cancer cells. The angiogenesis inhibition effect is clearly detected not only in vitro but also in vivo, suggesting that the brazilein is a promising target compound for the development of an anticancer agent or an adjuvant of an anticancer agent, which means it is highly valuable in the field of medicine. To develop as a drug, the brazilein has to be mass-produced. However, it is much more difficult or even impossible to mass-produce the brazilein from a natural substance than from a synthetic compound, so the mass-production method proposed in the present invention is very important .

The conventional method for isolating brazilein is as follows. Caesalpinia sappan L. is macerated in methanol at room temperature to give Caesalpinia sappan L. extract. The extract is filtered and the filtrate is concentrated under the reduced pressure to give methanol extract . A mixture of water and methanol (3:1) is added to the methanol extract, followed by phase separation using ethyl acetate to give ethyl acetate fractions. The ethyl acetate fractions proceed to silica gel column chromatography with the mixed solvent of chloroform:methanol (15:1 → 5:1) to obtain the brazilein abundant fractions. The fractions proceed again to silica gel column chromatography with the mixed solvent of chloroform: methanol : water (10:3:1), resulting in crude brazilein. The crude brazilein is completely dissolved in hot methanol, concentrated and then left at room temperature to obtain pure brazilein (Kim, D. S. et al., Phytochemistry, 46(1), ppl77-178, 1997) (see Fig. D•

At this time, the silica gel column chromatography has to be repeated at least twice to obtain the crude brazilein and re-crystallization process is also required, making the production process complicated. Besides, column chromatography needs to be scale-up for mass-production, which is technically limited. So, multiple columns are necessarily prepared for the mass-production, raising production costs.

Therefore, it has been required to develop a mass- production method of brazilein which is simple and economical . Disclosure

Technical Problem

It is an object of the present invention to provide a mass-production and purification method of brazilein from Caesalpinia sappan L., which is characterized by simple processes done in a short time and low production costs.

Technical Solution

As an effort to develop a novel method for producing brazilein with low production costs, the present inventors designed a method excluding column chromatography which had been necessarily performed so far, and further completed this invention by confirming that the method of the present invention produces highly purified brazilein without using high cost equipments such as column chromatography, supercritical fluid extractor, HPLC, etc. The present invention provides a mass-production method for brazilein from Caesalpinia sappan L. comprising the following steps:

1) Preparing an extract from Caesalpinia sappan L. by using water, alcohol or a mixture of the two;

2) Preparing a crude crystal by concentrating the extract of step 1) ;

3) Dissolving the crude crystal of step 2) in alcohol;

4) Concentrating the solution of step 3) ; and

5) Re-crystallizing the concentrated solution of step 4) . Hereinafter, the present invention is described in detail.

The preferable parts of Caesalpinia sappan L. for the invention are leaves, roots and stems and bark and sap wood are eliminated from them to give heartwood. Caesalpinia sappan L. can be cut into pieces for better extraction.

The alcohol of step 1) and step 3) is preferably Ci ~ C₃ lower alcohol and more preferably methanol . A method for extraction can be selected from a group consisting of enfleurage, maceration, reflux, etc, but enfleurage is more preferred. In step 1) , an extract is obtained from Caesalpinia sappan L. by enfleurage for approximately 12 ~ 72 hours and more preferably 24 ~ 48 hours. The extraction is repeated 1 - 5 times and more preferably 3 times.

The extract obtained from Caesalpinia sappan L. is filtered to eliminate the remaining Caesalpinia sappan L. and other residues. The filtration is performed by using a filter cloth or a filter paper but not always limited thereto.

In step 2, the concentration can be performed by any conventional concentration method, and in step 3, the crude crystal is dissolved in alcohol at the ratio of 1 g of crude crystal to 0.1 ( ~ 1 I alcohol, and more preferably 3 g:l i. At this time, the temperature of the alcohol is preferably 40^°C ~ 64^°C.

In step 4, the temperature of the water bath for the concentration and swirling speed are important factors for re-crystallization. So, to determine the optimum condition, experiments were performed at different temperatures with different swirling speeds. As shown in Table 1, the optimum temperature of the water bath for the mass- production of brazilein from Caesalpinia sappan L. was approximately 30^°C ~ 40^°C and the optimum swirling speed was determined to be 40 rpm ~ 180 rpm for producing a target crystal .

^ o: crystal produced, Δ : a little slurry, crystal not produced

Thus, in step 4, the preferable temperature of water bath for concentration is 30^°C ~ 40^°C, and preferable swirling speed is 40 rpm ~ 180 rpm. In step 5, recrystallization is preferably performed for 6 - 72 hours at 15^°C ~ 30^°C more preferably for 24 hours at room temperature (25 ^°C ) (see Fig. 2) . The volume of the concentrate was preferably 1/3 ~ 1/30 of the initial volume and more preferably 1/10 ~ 1/20.

The molecular weight of the re-crystallized compound was measured by MS (mass spectrometry, Micromass Quattro

Micro™ (triple quadruple mass spectrometer) ) and the molecular structure was assayed by NMR (¹H-NMR, DEPT 135,

13 C-NMR etc., Bruker Advance 500, Bruker AXS GMBH, Germany) . The results are shown below and the separated compound from Caesalpinia sappan L. was identified as brazilein represented by the following formula 1.

Structure of brazilein

1) Molecular weight: 284

2) Molecular formula: Ci₆Hi₂O₅

3) ¹H-NMR (500 MHz, DMSO-d₆) : δ 7.80 (IH, d, J=S .1 Hz, H-I) , 7.10 (IH, s, H-Il) , 6.56 (IH, d, J=8.5 Hz, H-2) , 6.35

(IH, s, H-4) , 6.32 (IH, s, H-8) , 4.45 (IH, d, J=Il.7 Hz, H- 6a) , 4.00 (IH, d, J=Il.7 Hz, H-6b) , 2.85 (2H, d, J=3.7 Hz, H-7)

4) ¹³C-NMR (125 MHz, DMSO-d₆) : δ 39 (C-7) , 72 (C-6) , 74 (C-6a) , 102 (C-4), 104 (C-Il), 110 (C-Ia), 110 (C-2), 117 (C-8) , 126 (C-IIa) , 130 (C-I) , 151 (C-12) , 152 (C-10) , 157 (C-4a) , 158 (C-7a) , 162 (C-3) , 179 (C-9) The crude crystal isolated from Caesalpinia sappan L. as indicated above and the crystal obtained from re- crystallization were analyzed by TLC (thin layer chromatography). As shown in Fig. 3, impurities were included and thus different colored layers were observed in the crude crystal (Fig. 3A) , whereas only white color was detected in the final crystal obtained from re- crystallization (Fig. 3B) , suggesting that brazilein with high purity was prepared from re-crystallization.

As shown in Fig. 1, the conventional method for extracting brazilein from Caesalpinia sappan L. is as follows. Caesalpinia sappan L. is macerated in methanol at room temperature to give Caesalpinia sappan L. extract. The extract is filtered and the filtrate is concentrated under the reduced pressure to give methanol extract. The mixture of water and methanol (3:1) is added to the methanol extract, followed by phase separation using ethyl acetate to give ethyl acetate fractions. The ethyl acetate fractions proceed to silica gel column chromatography with the mixed solvent of chloroform:methanol (15:1 → 5:1) to obtain the brazilein abundant fractions. The fractions proceed again to silica gel column chromatography with the mixed solvent of chloroform:methanol :water (10:3:1), resulting in crude brazilein. The crude brazilein is completely dissolved in hot methanol, concentrated and then left at room temperature to obtain pure brazilein (Kim, D. S. et al., Phytochemistry, 46(1), ppl77-178, 1997) (Fig. 1) . According to this method, 470 mg (0.47%) of brazilein is obtained from 100 g of Caesalpinia sappan L. However, this conventional method requires complicated processes including repeated column chromatography and thereby high costs for performing column chromatography repeatedly which needs a mixed solvent, column, and expensive equipments for mass-production using it.

On the contrary, according to the mass-production method of the present invention, approximately 30 g (0.34%) ~ 45 g (0.5%) of brazilein can be extracted from 9 kg of Caesalpinia sappan L. and high cost column chromatography is excluded, suggesting that the production costs are significantly reduced for the mass-production of brazilein.

According to the conventional method, high yield (8%) of brazilein is possible from making the crude crystal solution stand at room temperature for one month (Kim, D. S. et al., Phytochemistry, 46(1), ppl77-178, 1997), whereas according to the method of the invention much more brazilein is obtained by various^' batches during the same period of time.

Therefore, the method of the invention is more suitable for the mass-production of brazilein. Description of Drawings

The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

Fig. 1 is a schematic diagram illustrating the conventional method for separation of brazilein from Caesalpinia sappan L.

Fig. 2 is a schematic diagram illustrating the separation of brazilein from Caesalpinia sappan L. according to the method of the present invention.

Fig. 3 is a photograph showing the result of TLC with the isolated brazilein.

Mode for Invention

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention. Example 1: Isolation and identification of brazilein from

Caesalpinia sappan L.

<1-1> Extract of Caesalpinia sappan L.

9 kg of Caesalpinia sappan L. sections were put in an extraction bag, which was dipped in 18 L of 100% methanol, followed by extraction for 48 hours at room temperature.

This extraction was repeated three times and the extraction solution was filtered with a filter cloth. <l-2> Preparation of the crude crystal of Caesalpinia sappan L .

The extract filtered in the above Example <1-1> was concentrated under the reduced pressure until the liquid was completely evaporated, resulting in a dark red crude crystal.

3 g of the crude crystal was completely dissolved in 1 L of hot methanol (50"C) .

<l-3> Preparation of the final crystal of Caesalpinia sappan L.

The crude crystal containing solution prepared in Example 2 was concentrated under the reduced pressure in a rotary evaporator (EYELA N-1000, EYELA, Japan) in a 40°C water bath with 100 rpm until the initial volume was reduced to 1/20. The concentrated solution was left at room temperature for 24 hours, leading to re- crystallization. Thus, pure brazilein was obtained from Caesalpinia sappan L. (Fig. 2) .

According to the method of the invention, 45 g of brazilein was obtained from 9 kg of Caesalpinia sappan L.

Examples 2 ~ 10: Isolation of brazilein from Caesalpinia sappan L.

Some of the experimental conditions of Example 1 were modified, by which brazilein was isolated from Caesalpinia sappan L. And the results are shown in Table 2.

* : completely evaporated

Experimental Example 1 : Analysis of the purity of brazilein The crude crystal of Caesalpinia sappan L. extract isolated in Example 1 and the final crystal obtained from re-crystallization were analyzed by TLC (thin layer chromatography) (Fig. 3) .

The solid phase for TLC was Ci₈ reverse phase and the moving phase was methanol : water = 1:1. The TLC plate used herein was purchased from Merck (no.1.15389.0001, RP-18, F254S, Germany) . The sample was developed in the developing solvent (methanol :water = 1:1) in a TLC chamber at room temperature and then the solvent was dried and color development was induced with 5% sulfuric acid solution. As shown in Fig. 3, a yellow dot was detected by the naked eye (Rf, 0.46). As shown in Fig. 3, the crude crystal separated from

Caesalpinia sappan L. (Fig. 3A) was compared with the final crystal obtained after re-crystallization (Fig. 3B) . As a result, the crude crystal had impurities, whereas brazilein obtained from the re-crystallization was highly purified.

Comparative Example 1: Comparison of the yields of brazilein

The yield of brazilein according to the method of the present invention was compared with that of the conventional extraction method of Kim, et al . (Kim, D. S. et al., Phytochemistry, 46(1), ppl77-178, 1997) (Fig. 1).

<!-!> Extract of Caesalpinia sappan L.

100 g of Caesalpinia sappan L. sections was dipped in 200 mi of 100% methanol, followed by extraction at room temperature for three days. This extraction was repeated three times and the extraction solution was filtered with a filter cloth. The filtrate was concentrated under the reduced pressure to give methanol extract. 200 mi of a mixture of water and methanol (3:1) was added to the methanol extract, followed by phase separation using 100 mi of ethyl acetate to give ethyl acetate fractions. <l-2> Preparation of crude brazilein The ethyl acetate fractions proceeded to silica gel (Kieselgel 60 (70-230 mesh) , Merck) column chromatography with the mixed solvent of chloroform:methanol (15:1 → 5:1) to obtain brazilein abundant fractions. The obtained fractions proceeded again to silica gel column chromatography with the mixed solvent of chloroform: methanol :water (10:3:1) to give crude brazilein.

<l-3> Preparation of brazilein

The prepared crude brazilein was completely dissolved in 200 m& of hot methanol and concentrated until the initial volume was reduced to 1/5, which was then left at room temperature for 12 hours (overnight) . The solution was then filtered to give 470 mg of pure brazilein.

From the above method, , 470 mg (0.47%) of brazilein was obtained from 100 g of Caesalpinia sappan L. In the meantime, 30 g (0.34%) ~ 45 g (0.5%) of brazilein can be obtained from 9 kg of Caesalpinia sappan L. according to the method of the present invention.

Industrial Applicability

Unlike the conventional extraction method of brazilein, the method of the present invention produces highly purified brazilein without expensive equipments such as column chromatography, supercritical fluid extractor, HPLC, etc. Thus, the method of the present invention is economical and efficient to mass-produce or industrially isolate brazilein.

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims .

Claims

[Claim l]

A mass-production method of brazilein from Caesalpinia sappan L. comprising the following steps:

1) Preparing an extract from Caesalpinia sappan L. by- using water, alcohol or a mixture of the two;

2) Preparing a crude crystal by concentrating the extract of step 1) ;

3) Dissolving the crude crystal of step 2) in alcohol;

4) Concentrating the solution of step 3); and

5) Re-crystallizing the concentrated solution of step 4) . [Claim 2]

The mass-production method of brazilein from Caesalpinia sappan L. according to claim 1, wherein the alcohol used in step 1) and step 3) is Ci ~ C₃ lower alcohol. [Claim 3]

The mass-production method of brazilein according to claim 2, wherein the alcohol is methanol.

[Claim 4] The mass-production method of brazilein according to claim 1, wherein the temperature of the alcohol used in step 3) is 40^°C ~ 64 ^°C .

[Claim 5]

The mass-production method of brazilein according to claim 1, wherein the temperature of water bath for concentration in step 4) is 30^°C ~ 40^°C and the swirling speed is 40 rpm ~ 180 rpm.

[Claim 6]

The mass-production method of brazilein according to claim 1, wherein the re-crystallization of step 5) is performed by evaporating the solution of step 4) for 6 - 72 hours.

[Claim 7]

The mass-production method of brazilein according to claim 6, wherein the re-crystallization is performed by evaporating the solution for 24 hours.

[Claim 8]

The mass-production method of brazilein according to claim 1, wherein the re-crystallization of step 5) is performed by evaporating the solution of step 4) at 15°C ~ 30^°C.

[Claim 9]

The mass-production method of brazilein according to claim 8, wherein the re-crystallization is performed by evaporating the solution at 25°C.