KR20090117284A - Method for macamides with recycling preparative liquid chromatography - Google Patents

Abstract

PURPOSE: A method for preparing macamides is provided to rapidly prepare N-benzyl hexadecane amide and N-benzyl-5-oxo-6E,8E-octadecadiene amide, that are a kind of macamides, by using a recycling preparative liquid chromatography. CONSTITUTION: A method for preparing macamides using a recycling preparative liquid chromatography comprises the steps of: taking a supernatant by extracting dried Lepidium meyenii powder with methanol at 70~80 °C for 12 hours; performing separation by injecting the supernatant in a recycling preparative liquid chromatography compring a reversed-phase column, wherein the mobile phase is 100% acetonitrile; and obtaining N-benzyl hexadecane amide and N-benzyl-5-oxo-6E,8E-octadecadiene amide through two-step refining process.

Description

Method for Macamides by Recycling Preparative Liquid Chromatography {Method for Macamides with Recycling Preparative Liquid Chromatography}

Maca (Lepidium meyenii) is a cold-tolerant herbaceous plant that grows only in the Peruvian Andes up to 4000 m above sea level and has been a favorite nutritious food and tonic for Peruvian indigenous people for thousands of years. Maca generally uses tuber roots for food and has a higher nutritional value than other cereals. Maca contains a lot of essential amino acids, higher minerals like iron and calcium than tomatoes, and important fatty acids such as linolenic acid, palmitic acid and oleic acid. In addition, maca contains various bioactive substances such as sterols, alkaloids, glucosinolate, and macamide, and research on these ingredients is being actively conducted.

Maca has traditionally been known for its effects on enhancing sexual function and improving fertility, but recently, new researches on hormonal secretion control, immune strengthening and memory improvement (FOOD STYLE 21 2007 11 (4): pp 60-63, Chinese journal of new drugs 2007 16 (1): pp45-48, Food and chemical toxicology 2007 45 (10): pp1882-1890) has been published continuously. . A Study on Remarkable Biological Activity of Lipid Extracts from Maca and Alcohol or Hexane Fractions (Natural product R & D 2007 19 (2): pp274-276, Andrologia 2002 34 (3): pp177-179, Urology 2000 55 (4) ): pp598-602) has been reported continuously, and these maca lipid extracts are known to contain a fatty acid and a macamide component, which is a characteristic substance of maca, which does not exist in other plants, and its structure has recently been clarified. Seven types of macamides reported up to now (J Agric Food Chem 2005 53: pp690-693) are characterized by the structure of benzyl group and hydrocarbon chain amide bond, and major macamides are N-benzylhexadecaneamide and N-benzyl-5-oxo-6E, 8E-octadecadieneamide is known (Chem Pharm Bull 2002 50 (7): pp988-991). However, since the macadam is mainly mixed in the lipid fractionation layer of maca, and the chemical properties are similar to those of the fatty acid, it is difficult to separate only the macamide by the physical method. In addition, it is possible to prepare macaamide in maca by a chemical method, but chemical fractionation method using toxic organic solvents such as hexane and petroleum ether and undergoing a complicated reaction in several stages (Phytochem Anal 2005 16: pp463-469) has a problem in that it takes a lot of time and money. Therefore, in the absence of commercially standardized macamide, there is a need to develop a method for manufacturing macamide more rapidly for further research on the efficacy of maca.

Recirculating preparative liquid chromatography used in the present invention is to be re-injected to the column immediately in the case of the sample is not separated by adjusting the automatic circulation device. Thus, for example, if a column of 500 mm length is recycled 10 times, it has a resolution of separating the column into 5 m length columns. In other words, recirculation is a principle that recycles a non-separated sample from the column until it is separated, thereby dramatically reducing the amount of organic solvent that has been raised as a problem in the existing preparative liquid chromatography and maximizing the separation ability. Preparative chromatography has been improved to allow separation and purification quickly and efficiently. Therefore, in the future, the use of recycle preparative liquid chromatography is expected to be highly useful for the study of separation of various bioactive substances from natural products.

It is an object of the present invention to provide N-benzylhexadecaneamide and N-benzyl-5-oxo-6E, 8E-octadecadieneamide. In the process of separating and identifying various bioactive substances of maca from maca, the present inventors used recycled preparative liquid chromatography to form N-benzylhexadecaneamide and N-benzyl-5-oxo-6E, 8E. It was confirmed that octadecadiene amide can be produced simply and quickly, and the present invention was completed.

The present invention is a high-purity N-benzyl hexadecane amide and N- through a two-step preparative and purification process by injecting the dried maca powder extracted with an organic solvent such as methanol to the recycle preparative liquid chromatography Benzyl-5-oxo-6E, 8E-octadecadieneamide.

Maca used in the present invention is a yellow-colored Amarillo species among several maca species called Lepidium meyenii in the scientific name, and dried maca powder, which was cultivated in Junin province, Peru, dried and pulverized.

An example of a method for preparing N-benzylhexadecaneamide and N-benzyl-5-oxo-6E, 8E-octadecadieneamide using recycle preparative liquid chromatography according to the present invention is as follows.

The dried maca powder was 10-fold hydrolyzed with 100% methanol, extracted at 70-80 ° C. for 12 hours, and only the supernatant was poured into a recirculating preparative liquid chromatography system consisting of a preparative reverse phase column. It is composed of a method of obtaining N-benzyl hexadecane amide and N-benzyl-5-oxo-6E, 8E-octadecadiene amide through two steps of preparative and purification process by separating into a certain gradient composition in lilo. In the first step of the preparative process as a step of removing the sugar or fatty acid components mixed in the methanol extract of maca is fractionated each of the two kinds of the desired macaamide as shown in FIG. In the two-stage purification process, two kinds of aliquots of macamide are injected into the liquid chromatography and recycled several times, thereby producing high purity N-benzylhexadecaneamide and N-benzyl-5-oxo-6E, 8E-octadecadieneamide. It will be prepared.

The two kinds of macamides prepared by the present invention exhibited the following physical and chemical properties, and were identified as N-benzylhexadecaneamide and N-benzyl-5-oxo-6E, 8E-octadecadieneamide.

Physical and chemical properties of N-benzylhexadecane amide are as follows.

1) Electron impact mass spectrum (m / z [M + H] ⁺ ): calcd C ₂₃ H ₃₉ NO 346.3104, analytical C ₂₃ H ₃₉ NO 346; 5

2) Hydrogen nuclear magnetic resonance spectrum ( ¹ H-NMR (δ)): 7.23-7.32 (m, 5H, phenyl-5H), 5.89 (br, 1H, NH), 4.41 (d, J = 6.0 Hz, 2H, C H ₂ NH), 2.19 (t, J = 7.2 Hz, 2H, COC H ₂ ), 1.64 (m, 2H, COCH ₂ C H ₂ ), 1.34-1.20 (br, 24H, carbon chain), 0.87 (t , J = 7.2 Hz, 3H, CH ₃ )

3) Carbon nuclear magnetic resonance spectra ( ¹³ C-NMR (ppm)): 172.76, 138.24, 128.44, 127.56, 127.20, 116.26, 43.47, 36.77, 31.94, 29.72, 29.68, 29.64, 29.53, 29.39, 29.34, 27.24, 25.82 , 22.74, 14.19, 1.98

Physical and chemical properties of N-benzyl-5-oxo-6E, 8E-octadecadieneamide are as follows.

1) Electron impact mass spectrum (m / z [M + H] ⁺ ): calcd C ₂₅ H ₃₇ NO ₂ : 384.2903, analytical C ₂₅ H ₃₇ NO ₂ 384; 6

2) Hydrogen nuclear magnetic resonance spectrum ( ¹ H-NMR (δ)): 7.26-7.34 (m, 5H, phenyl-5H), 7.08-7.16 (m, 1H, COCHC H ), 6.16-6.20 (m, 2H, COCHCHC H C H , 6.06 (d, J = 15.4 Hz, 1H, COC H ), 5.77 (br, 1H, NH), 4.44 (d, J = 6.0 Hz, 2H, NHC H ₂ ), 2.53 (t, J = 7.6 Hz, 2H, C H ₂ COCH), 2.40 (t, J = 7.6 Hz, 2H, COC H ₂ CH ₂ ), 1.62 (br, 2H, COCH ₂ C H ₂ ), 1.21-1.49 (br, 16H , carbon chain) 0.90 (t, J = 7.6 Hz, 3H, CH ₃ )

3) Carbon nuclear magnetic resonance spectrum ( ¹³ C-NMR (ppm)): 201.15, 173.02, 145.99, 143.21, 138.78, 131.45, 128.99, 128.87, 128.00, 127.95, 43.95, 40.75, 37.10, 33.51, 31.76, 30.10, 29.46 , 28.78, 26.04, 25.91, 24.69, 22.67, 14.46

< Example 1 >: Manufacturing method of N-benzyl hexadecane amide

100 g of dried maca powder was extracted 10 times with 100% methanol and extracted for 12 hours at 75 ° C. to collect only the supernatant, and filtered with a 0.45 μm syringe filter to obtain 500 ml of chromatographic injection sample. Two reversal preparative columns (JAIGEL-ODS-AP, 20 × 500 mm, JAI, Japan) are used to connect each other, and the UV wavelength is 210 nm and 100 ml of the sample is injected and stayed in the primary preparative chromatogram. Only the peak of 94 minutes is aliquoted. In the two-stage purification process, the collected fractions are collected and re-injected, and the recycle process is recycled from the point of occurrence of peaks to the point of disappearance as shown in FIG. 7 in the chromatogram of 210 nm ultraviolet wavelength and removing the remaining impurities. Repeatedly, 421.3 mg of N-benzylhexadecaneamide having the structural formula of FIG. 1 was obtained.

< Example 2 >: Production method of N-benzyl-5-oxo-6E, 8E-octadecadieneamide

As in Example 1, 100 g of dried maca powder was extracted with 100% methanol to obtain 500 ml of a chromatography injection sample. Two reversal preparative columns (JAIGEL-ODS-AP, 20 × 500 mm, JAI, Japan) are used to connect each other, and the UV wavelength is 210 nm and 100 ml of the sample is injected and stayed in the primary preparative chromatogram. Only peaks with a 44 minute time are aliquoted. All the fractions were reinjected and recycled five times in a 280 nm UV wavelength, from the point of occurrence of the chromatogram peak to the point of disappearance as shown in FIG. Eggplant obtained 187.6 mg of N-benzyl-5-oxo-6E, 8E-octadecadieneamide.

Figure 1 shows the chemical structural formula of N-benzylhexadecaneamide.

Figure 2 shows the chemical formula of N-benzyl-5-oxo-6E, 8E-octadecadieneamide.

3 shows a schematic diagram of a liquid chromatography system for recycling preparative.

① mobile phase ② degasser

③ High pressure pump ④ Mobile phase mixer

⑤ Sample injector ⑥ Preparative column

⑦ UV detector ⑧ Automatic circulation device

⑨ Preparative sample collector ⑩ Circulating sample injector

Figure 4 shows the chromatogram of the first step preparative chromatogram in the recycle preparative chromatogram, # 1 is the peak of N-benzyl-5-oxo-6E, 8E-octadecadieneamide and # 2 is N-benzyl The peak of hexadecaneamide is shown.

Figure 5 shows the electron shock mass spectrum of N-benzylhexadecaneamide.

Figure 6 shows the electron shock mass spectrum of N-benzyl-5-oxo-6E, 8E-octadecadieneamide.

Figure 7 shows the chromatogram of the two-step purification of N-benzylhexadecaneamide in the recycle preparative chromatogram.

FIG. 8 shows the chromatogram of the two-step purification of N-benzyl-5-oxo-6E, 8E-octadecadieneamide in a recycle preparative chromatogram.

Claims (2)

A process for preparing N-benzylhexadecaneamide and N-benzyl-5-oxo-6E, 8E-octadecadieneamide, which are a kind of macamide, using recycled liquid chromatography from Maca (Lepidium meyenii). The method of claim 1, wherein N-benzylhexadecaneamide and N-benzyl-5-oxo-6E, 8E-octadecadieneamide are prepared by recycle preparative liquid chromatography using a reversed phase column.

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