KR20030079352A - Antioxidant from curcuma longa and process for isolation thereof - Google Patents

Abstract

PURPOSE: Provided are an antioxidant from Curcuma longa and a process for isolation thereof. The isolated antioxidant has excellent scavenging effect on peroxynitrite. CONSTITUTION: An antioxidant from Curcuma longa is isolated by the steps of: refluxing Curcuma longa with methanol for 3 hours; suspending the extract thereof with distilled water, and followed by fraction with dichloromethane and ethylacetate; fractioning the ethylacetate extract by dilution with a mixed solvent of dichloromethane-methanol using silica gel column; and purifying a fraction having excellent peroxynitrite scavenging activity by silica gel chromatography with a mixed solvent of dichloromethane-methanol to obtain diarylheptanoid.

Description

Antioxidant from turmeric and its isolation method {Antioxidant from Curcuma longa and process for isolation}

The present invention relates to an antioxidant separated from turmeric and a method for separating the same. More specifically, the present invention relates to an antioxidant having excellent peroxynitrite scavenging activity isolated from the ethyl acetate fraction obtained from methanol extract of turmeric and an isolation method thereof.

Antioxidants, such as inhibitors of lipid peroxidation, are important compounds that not only protect food but also defend against oxidative damage in living cells.

Many natural products derived from natural plant sources have been reported to retard the oxidation process in their natural environment as reactive oxygen scavengers, free radical scavengers or as reducing agents. Superoxide (O ₂ ⁻ ) and nitric oxide (NO) have been reported to be involved in inflammation and ischemic / perfusion processes. Nitric oxide reacts with peroxide anion peroxy nitrite (peroxynitrite; ONOO ^-) by generating arteriosclerosis, ischemia / perfusion. And it is reported to be involved in inflammation.

Curcuma longa L. (Zingiberaceae) has been used in humans for a long time in nature in tropical South and Southeast Asia (Scartezzini and Speroni, 2000). Turmeric roots are known to have antioxidant and cytoprotective roles, along with their main component, curcumin (Soudamini et al., 1992; Ramsewak et al. 2000). The antioxidant active ingredient in turmeric has been known as curcumin and its analogues (Toda et al., 1985). Turmeric has strong antioxidant activity not only in food but also in living organisms (Kundchundy and Rao, 1989, 1990, Huang and Ferraro, 1992, Sharma, 1976). Recently, antioxidant activity in the biological system has been noted for the defense of some peroxidation-related diseases (Ruby et al., 1995). Masuda et al. (1999) found that in the chemical study of curcumin's antioxidant mechanism, curcumin forms radicals to form its two-molecules and continues to produce poly-molecules. -breaking) as an antioxidant. Recently, Kato et al. (1997) reported that curcumin has a strong inhibitory effect on the formation of 3-nitrotyrosin in collagen converted by peroxynitrite.

Free radicals, including free radicals, free radicals, active nitrogen species, and peroxynitrite are involved in the causes of many human diseases (Pincemail, 1995). Peroxynitrite, produced by the reaction of superoxide anion (O ₂ ⁻ ) and nitric oxide, is a toxic cell species that can oxidize some cellular components such as proteins, lipids, and DNA. Alzheimer's Diseases, rheumatoid arthritis, cancer, and atherosclerosis (Squadrito and Pryor, 1998) are known to be involved.

In view of the above, the present inventors reflux the dried root of turmeric with methanol for 3 hours to obtain a methanol extract, suspend the extract with distilled water, and then fractionate it with dichloromethane and ethyl acetate to dichloromethane extract, ethyl acetate extract, and distilled water. After obtaining the residue, the ethyl acetate extract was eluted with a mixed solution of dichloromethane-methanol concentration gradient mixed through a silica gel column to obtain a fraction, and the fraction having peroxynitrite scavenging activity in the fraction was dichloromethane-methanol. Further purification by silica gel chromatography using a mixed solution to obtain the diarylheptanoids as an antioxidant compound, the methanol extract, dichloromethane extract, ethyl acetate extract, distilled water residue and the peroxyni of diarylheptanoids as an antioxidant compound By measuring the light scavenging activity and it completed the present invention.

Accordingly, it is an object of the present invention to provide an antioxidant isolated from turmeric.

Another object of the present invention is to provide a method for separating antioxidants from turmeric.

The object of the present invention is to reflux the dried root of turmeric with methanol for 3 hours to obtain a methanol extract, and then the extract is suspended in distilled water and fractionated with dichloromethane and ethyl acetate to obtain a dichloromethane extract, ethyl acetate extract and the residue of distilled water Then, the ethyl acetate extract was eluted with a mixed solution of dichloromethane-methanol concentration gradient mixed through a silica gel column to obtain a fraction, and the fraction having peroxynitrite scavenging activity in the fraction was mixed with a dichloromethane-methanol mixture. Purified by silica gel chromatography to obtain diarylheptanoids as an antioxidant compound, and peroxynitrite scavenging activity of the methanol extract, dichloromethane extract, ethyl acetate extract, distilled water residue and diarylheptanoids as antioxidant compounds. By measuring achieved.

Hereinafter, the configuration of the present invention.

1 is a graph showing the peroxynitrite scavenging activity according to the concentration of the antioxidant compounds 1-3, which are diarylheptanoids isolated from turmeric.

The present invention comprises the steps of refluxing the dried root of turmeric with methanol for 3 hours; Suspending the extract with distilled water and distilling with dichloromethane and ethyl acetate to obtain a dichloromethane extract, an ethyl acetate extract, and a distilled water residue; Eluting the ethyl acetate extract with a mixed solution of dichloromethane-methanol concentration gradient mixed through a silica gel column to obtain a fraction; And further purifying the fraction having peroxynitrite scavenging activity in the fraction by silica gel chromatography using a dichloromethane-methanol mixture to obtain diarylheptanoids as antioxidant compounds. The methanol extract, dichloromethane extract, ethyl acetate extract, distilled water residue and the peroxynitrite scavenging activity of the antioxidant compounds of the diarylheptanoids obtained in the above step are measured.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example 1 Extraction, Fractionation and Separation of Turmeric

Plant material

The root diameter of C. longa in the present invention was purchased from a building material store in Busan, South Korea in April 2000. A sample (no. WP 010) is kept in the Department of Food and Biotechnology, Pukyong National University.

reagent

Penicillamine in the present invention was purchased from Sigma Chemical Company (St. Louis, MO, USA) and is dihydrorhodamine 123 (DHR 123) and peroxynitrite (peroxynitrite) Are high quality and purchased from Molecular Probes (Eugene, Oregon, USA) and Cayman (Ann Arbor, MI, USA), respectively.

Experiment apparatus

In the present invention, the ¹ H- and ¹³ C-NMR spectra were used with a Varian UNIYT-400 spectrometer, and the chemical shift was determined based on each residual solvent peak. It was recorded as δ value. Diversity of ¹ H- and ¹³ C-NMR signals were recorded as s (single line), d (double line), and m (multiline). Column chromatography was performed with silica gel (Merck, 70-230 mesh). TLC was performed on a Merck Kieselgel 60 F254 plate (0.25 mm) and spots were detected using 50% H ₂ SO ₄ .

Extraction, Fractionation, and Separation of Antioxidants from Turmeric

The dried root of turmeric (200 g) was refluxed with methanol (MeOH) for 3 hours. The total filtrate was concentrated to dryness in vacuo at 40 ° C. to give 21 g of methanol (MeOH) extract. After the extract was suspended in distilled water (H ₂ O) CH ₂ Cl ₂ and EtOAc to have a fraction continuously CH ₂ Cl ₂ extract 14.4g, EtOAc extracts to give the 2.2g and distilled water 3.5g residue.

EtOAc extract (2 g) was chromatographed using a solvent of CH ₂ Cl ₂ -MeOH (100: 1 to 10: 1) through a silica gel column (12 × 60, Silica gel 60, Merck, 200 g). 8 fractions were obtained (fraction 1-fraction 8; Fr.1-Fr.8).

Fraction 1 (500 mg) was again chromatographed with CH ₂ Cl ₂ to obtain the first compound (compound 1) (80 mg, curcumin I), and 400 mg of fractions 2 and 3 were combined to form CH ₂ Cl ₂ -MeOH (100: 1). The silica gel column was again used as a solvent to separate 20 mg of the first compound and 60 mg of the second compound (curcumin II), and 50 mg of the third compound (curcumin III) was separated from fraction 4, and 250 mg of the filtrate was added with EtOAc. Gel column chromatography was carried out to obtain 120 mg of the third compound.

Spectral data of the first compound Curcumin I: ¹ H-NMR (400 MHz, acetone- d ₆ ); 3.86 (6H, s, 2 x OCH ₃ ), 5.92 (1H, s, H-4), 6.65 (2H, d, J = 16.0 Hz, H-2, 7), 6.83 (2H, d, J = 8.3 Hz, H-5 ', 5''), 7.12 (2H, d, J = 2.0 & 8.3 Hz, H-6', 6 ''), 7.28 (2H, d, J = 2.0, H-2 ', 2 ''), 7.55 (2H, d, J = 16.0 Hz, H-1, 6), ¹³ C-NMR (100.0 MHz, acetone- d ₆ ); 184.96 (C-3, 5), 150.45 (C-4 ', 4''), 149.21 (C-3', 3 ''), 141.86 (C-1, 7), 128.64 (C-1 ', 1 ''), 124.27 (C-6 ', 6''), 122.77 (C-2, 6), 116.69 (C-5', 5 ''), 112.02 (C-2 ', 2''), 102.13 (C-4), 56.77 (2 x OCH ₃ )

Spectral data of the second compound (Curcumin II): ¹ H-NMR (400 MHz, acetone- d ₆ ); 3.86 (3H, s, OCH ₃ ), 5.92 (1H, s, H-4), 6.60 (1H, d, J = 16.0 Hz, H-7), 6.66 (1H, d, J = 16.0 Hz, H- 2), 6.83 (1H, d, J = 8.5 Hz, H-5 '), 6.85 (2H, d, J = 8.5 Hz, H-3``, 5''), 7.11 (1H, d, J = 1.7 & 8.5 Hz, H-6 ′), 7.28 (1H, d, J = 1.7 Hz, H-2 ′), 7.51 (2H, d, J = 8.5 Hz, H-2``, 6 ''), 7.55 (1H, d, J = 16.0 Hz, H-6), 7.56 (1H, d, J = 16.0 Hz, H-1), ¹³ C-NMR (100.0 MHz, acetone- d ₆ ); 185.01 (C-3), 184.92 (C-5), 160.96 (C-4 ''), 150.47 (C-4 '), 149.23, (C-3'), 141.85 (C-1), 141.50 (C -7), 131.42 (C-2``, 6 ''), 128.63 (C-1 '), 128.16 (C-1''), 124.32 (C-6'), 122.73 (C-2), 122.51 (C-6), 117.25 (C-3``, 5 ''), 116.68 (C-5 '), 111.96 (C-2'), 102.17 (C-4), 56.77 (OCH ₃ )

Spectral data of the third compound (Curcumin III): ¹ H-NMR (400 MHz, acetone- d ⁶ ); 5.92 (1H, s, H-4), 6.61 (2H, d, J = 16.0 Hz, H-2, 6), 6.85 (2H, d, J = 8.5 Hz, H-3 ', 4'), 6.85 (2H, d, J = 8.5, H-3``, 5 ''), 7.51 (2H, d, J = 8.5Hz, H-2 ', 6'), 7.51 (2H, d, J = 8.5, H-2 ", 6 "), 7.56 (2H, d, J = 16.0 Hz, H-1, 7), ¹³ C-NMR (100.0 MHz, acetone- d ₆ ); 184.96 (C-3, 5), 160.91 (C-4 ', 4''), 141.50 (C-1, 7), 131.42 (C-2', 6 ', 2'',6''), 128.16 (C-1 ', 1''), 122.49 (C-2, 6), 117.24 (C-3', 5 ', 3'',5''), 102.22 (C-4)

Example 2 Measurement of Peroxynitrite Scavenging Activity

The scavenging activity of peroxynitrite was measured by measuring the oxidation of DHR123 by modifying Kooy et al.

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

For statistical analysis, the numerical values are expressed as the mean ± 3 experimental standard errors.

The experimental results are shown in Table 1. As can be seen from Table 1, the scavenging activity of methanol extract of turmeric and the peroxynitrite scavenging activity of solvent fractions such as CH ₂ Cl _2- , EtOAc-, and H ₂ O- soluble fractions of methanol extract were > CH ₂ Cl ₂ >MeOH> H ₂ O in order of increasing IC ₅₀ of the solvent fractions were 0.3 ± 0.08, 1.3 ± 0.09, 1.7 ± 0.08, and 55.0 ± 7.67 μg / ml, respectively.

Peroxynitrite Scavenging Activity of Extracts Isolated from Turmeric extract ONOO ^-a Methanol 1.7 ± 0.08 Dichloromethane 1.3 ± 0.09 Ethyl acetate 0.3 ± 0.08 Distilled water 55.0 ± 7.67 Penicillamine 13.14 ± 0.52

ONOO ^-a : Inhibitory activity of peroxynitrite (IC 50: µg / ml)

The ethyl acetate (EtOAc) aqueous fraction of methanol extract of turmeric showed the most significant peroxynitrites scavenging effect. The scavenging effect of the EtOAc-fraction surpassed the scavenging activity of penicillamine, a well-known peroxynitrite scavenger.

The dichloromethane (CH ₂ Cl ₂ ) fraction also showed strong scavenging activity but slightly lower than the EtOAc fraction. On the other hand, distilled water (H ₂ O) water soluble fraction showed the weakest activity among them.

These results indicate that CH ₂ Cl ₂ -and EtOAc fractions of methanol extracts, as well as methanol extracts of turmeric with relatively weak polarity, exhibited the scavenging effect of peroxynitrite, and their peroxynitrite scavenging activity was due to antioxidant activity of turmeric. It seems to contribute to the activity.

Both the CH ₂ Cl ₂ -and EtOAc fractions had high scavenging activities, while the H ₂ O fraction showed weak activity.

Significant scavenging effects of fractions derived from methanol extract of turmeric on peroxynitrite can be explained by the compositional differences of the components contained therein. Scavengers of the fractions were diarylheptanoids. This is because they are widely known in turmerics (Jitoe et al., 1992) and have polar properties that dissolve in dichlormethane and ethyl acetate. Therefore, the most active EtOAc-soluble fractions were purified by continuous column chromatography to separately identify well-known diarylheptanoids 1-3 having the following structural formulas. The compounds appeared in curcumin I, II and III and their structure was determined by comparison with well-known spectrophotometric data and with standards (Roughley & Whiting, 1973).

Peroxynitrite scavenging activity of the separated diarylheptanoids (diarylhepatnoids) is shown in Table 2 and FIG. These results indicate that the aromatic 3-methoxy 4-hydroxy cinnamoyl (feruroyl) group {4-hydroxy cinnamoyl (feruloyl)-} is a potent scavenger of peroxynitrite. Curcumin III with coumaroyl group had a low scavenging effect. In particular, curcumin I containing two Peruroyl groups showed the strongest scavenging activity. The scavenging activity of curcumin I was compared with penicillamine and both of these compounds effectively scavenged peroxynitrite. The concentrations of 50% authentic peroxynitrite for curcumin I and penicillamine were 4.0 ± 0.04 μM and 2.38 ± 0.34 μM, indicating that curcumin I was effective.

Peroxynitrite Scavenging Activity of Compounds Isolated from Turmeric compound ONOO ^-a Cumin I 10.88 ± 0.11 Curcumin II 18.90 ± 0.90 Curcumin III 96.53 ± 4.20 Penicillamine 15.94 ± 2.31

The relative scavenging effects of turmeric methanol extract, curcumin I, II and III on peroxynitrite scavenging activity can be explained by structural differences. Structurally, curcumin I consists of two ortho-methoxylated phenols and one β-diketone, all of which are conjugated and Kirker Min III consists of two parahydroxylated phenols and one β-diketone, whereas cumin II is ortho-methoxylated phenols, respectively. -methoxylated phenols), para-hydroxylated phenols and β-diketones.

From the above results, it was found that ortho-methoxylated phenol (feruloyl group) is necessary to exhibit the highest peroxynitrite scavenging activity.

These results are in good agreement with recent studies that inhibit peroxitrite-dependent tyrosine nitration (ferulic acid is more than p-coumaric acid). Stronger (Pannala et al., 1998)}.

Phenolic acids associated with hydroxycinnamate are potent antioxidants due to their hydrogen-donating properties (Rice-Evans et al., 1996) and metal-chelating properties (Salah et al., 1995). It has been reported to function.

Some studies have shown that these compounds can prevent protein-binding and nitration caused by free tyrosine peroxynitrite, as well as dihydrorhodamine 123 and DNA (Oshima et al., 1998). Has been reported to inhibit oxidation due to peroxynitrite. The results indicate that peroxynitrite scavenging activity is effected by changes in chemical structure and thus can also be applied to the scavenging activities of several different kinds of compounds.

The mechanism by which phenolic acids compounds eliminate peroxynitrite has not yet been demonstrated. The basic structure and other structural elements of phenolic acid have been found to be important in the elimination mechanism. Pannala et al. (1998) suggested that there are two possible mechanisms for eliminating peroxynitrite. The first is nitrification and the second is electron donation. Phenolic acid compounds are nitrified by peroxynitrite as in the case of monohydroxylated structures such as p-coumaric acid and ferulic acid. Nitrogen from tyrosine peroxynitrite by reduction of active nitrogen species that act as a substrate for nitration or have been demonstrated for catechol structures such as caffeic acid. Nitration can be suppressed [Pannala et al., 1997; 1998; Kerry and Rice-Evans, 1999].

The results of the present study showed that methanol extracts of turmeric and its various fractions and components isolated from them inhibited the atherosclerosis, ischemia / perfusion, and inflammation caused by peroxynitrite. It is believed that these natural compounds would be even more meaningful if studies were conducted to prevent damage caused by various peroxynitrites in the pathological state of in vivo.

As described above, the diarylhep, an antioxidant compound obtained by separating the fraction obtained by distilling the methanol extract of turmeric of the present invention with ethyl acetate, using a dichloromethane-methanol as a solvent, was chromatographed on a silica gel column. Tanoids have excellent peroxynitrite scavenging activity and are very useful inventions in the manufacture of antioxidants and bioactive substances.

Claims (2)

Refluxing the dry root of turmeric with methanol for 3 hours; Suspending the extract with distilled water and distilling with dichloromethane and ethyl acetate to obtain a dichloromethane extract, an ethyl acetate extract, and a distilled water residue; Eluting the ethyl acetate extract with a mixed solution of dichloromethane-methanol in a concentration gradient through a silica gel column to obtain a fraction; And purifying the fraction having peroxynitrite scavenging activity in the fraction by silica gel chromatography using dichloromethane-methanol mixture to obtain diarylheptanoids. A turmeric-derived antioxidant, which is obtained by the separation method according to claim 1, and contains, as an active ingredient, diarylheptanoids of the following structural formula having peroxynitrite scavenging activity.