CN103439451B - Extraction and thin-layer chromatographic scanning detection method for octacosanol from Antarctic krill - Google Patents

Abstract

The invention discloses a method for extracting octacosanol from Antarctic krill and thin-layer chromatography scanning detection. The specific steps are: extracting Antarctic krill shrimp oil, heating and refluxing with NaOH ethanol solution, extracting with petroleum ether and steaming to remove oil After the shrimp oil was treated with ether, octacosanol was obtained; the sample solution and standard solution were respectively prepared, and they were respectively spotted on the same high-efficiency thin-layer silica gel plate GF ₂₅₄ , dyed, and thin-layer scanning was performed to obtain octacosanol The ratio shift value and the front area integral value are used to calculate the relationship between the standard solution mass and the peak area integral value, and the quality of the sample liquid octacosanol is calculated according to the obtained relationship. The present invention can not only be used for the extraction and detection of octacosanol in Antarctic krill, more importantly, it can be used for the detection of octacosanol content in any raw material, product and by-product containing octacosanol. Octacosanol has extensive and important application value in the development.

Description

Extraction and thin-layer chromatography scanning detection method of octacosanol from Antarctic krill

technical field

The invention relates to an extraction and thin-layer chromatography scanning detection method of Antarctic krill octacosanol.

Background technique

Octacosanol is a naturally occurring monohydric higher alcohol, which is widely found in the lipids of roots, stems, leaves, shells and seeds of plants, and in waxes secreted by insects (such as white wax, shellac wax, Beeswax, etc.), widely distributed in animal skin, viscera, animal lipids (such as wool wax, spermaceti, fish egg lipids and marine crustaceans, etc.).

In 1949, Dr. Cureton of the University of Illinois in the United States used natural octacosanol and synthetic octacosanol to conduct 42 experiments on animals and 894 subjects, and made significant progress. Cureton's research results show that octacosanol has the following effects on the human body: ① Improve endurance and energy; ② Improve responsiveness; ③ Improve stress ability; ④ Promote hormone action and reduce muscle pain; Lower systolic blood pressure; ⑦Increase the body's metabolic rate. Later, some scholars studied the toxicity of octacosanol, and the acute toxicity test confirmed that the half lethal dose LD50 for white mice was 18g/kg (orally administered to white mice), and the safety was higher than that of table salt (table salt LD50=3g/kg).

Octacosanol is widely used in medicine, chemical industry, food (especially health care products) and cosmetics. The main function in cosmetics is to promote blood circulation, increase oxygen transport capacity, increase basal metabolic rate, absorb through the skin, have cell revitalizing effect, enhance skin activation, eliminate skin wrinkles, and also be used in lipstick, nail polish and hair conditioner, It has obvious medical effects on alopecia.

Antarctic krill is a species of krill that lives in Antarctic waters. It lives in colonies and is a key species in the Antarctic ecosystem. In terms of biomass, it may be the most successful biological species on Earth (about 500 million Ton). The reserves are huge, the annual allowable catch is 100-150 million tons, and the actual annual catch in the world is 100-500 thousand tons. But because of its high fluorine content (2.4g/kg), it cannot be directly used as food. After being caught, it is mainly used as fish bait and extracted and processed into health oil. If Antarctic krill is used as raw material to develop natural medicines for comprehensive utilization, it will have broad development prospects.

If it is proved that Antarctic krill contains octacosanol, and its development and utilization, its economic and social benefits will be very significant. Previously, gas chromatography and liquid chromatography were mostly used for the detection of octacosanol. For structural analysis, these two methods should be combined with infrared chromatography, ultraviolet, mass spectrometry and nuclear magnetic resonance spectroscopy. Gas and liquid chromatography detection methods are expensive and time-consuming.

Contents of the invention

Aiming at the above-mentioned prior art, the present invention provides an extraction and thin-layer chromatography scanning detection method of Antarctic krill octacosanol.

The technical scheme provided by the invention is:

The extraction and thin-layer chromatography scanning detection method of Antarctic krill octacosanol, the specific steps are:

1) Extract the dried Antarctic krill with n-hexane, filter and evaporate the n-hexane to obtain Antarctic krill oil.

2) The shrimp oil is first heated and refluxed in a water bath with a 10% NaOH ethanol solution, and then extracted with petroleum ether. The extracted liquid is added to water for layering, and the upper layer of petroleum ether solution is taken out, and the petroleum ether is evaporated to obtain a solid two Stearyl Alcohol Extract.

3) The octacosanol extract and the octacosanol standard (Sigma Company) were dissolved in chloroform to prepare a sample solution and a standard solution, respectively.

4) Different quality octacosanol standard solutions and a single quality sample solution are respectively spotted on the same high-efficiency thin-layer silica gel plate GF254, and the developing agent is added to pre-balance, and then the silica gel plate is put into the developing cylinder and expanded upwards. When the spread reaches 8cm, take it out and dry it, then wash and dye it with the dyeing solution, and dry it.

5) Carry out thin-layer scanning to obtain octacosanol R _f (ratio shift value) and peak area integral value; the relationship between standard liquid quality and peak area integral value is calculated by the thin-layer chromatograph management software winCATS, according to the sample liquid The mass of the sample liquid octacosanol was calculated from the peak area integral value.

Said step 1), wherein n-hexane is 6 times the mass volume of Antarctic krill, extracted 3 times

The volume ratio of the 10% NaOH ethanol solution, petroleum ether and water in the step 2) is 1:1:1.

The octacosanol standard solutions with different concentrations in the step 4) are 5 kinds of mass gradients, which are 0.375ug, 0.5ug, 0.625ug, 0.75ug, 1.125ug respectively.

The developer in the step 4) is petroleum ether: ethyl acetate: toluene, and the volume ratio is 3-5:0.5-1.5:0.05-0.1.

The staining solution in step 4) is water:bromothymol blue:sodium hydroxide, and the volume to mass ratio is 80-110ml:0.08-0.12g:0.6-0.8g.

Octacosanol is widely present in plants and animals. The present invention extracts octacosanol from Antarctic krill for the first time, and conducts thin-layer chromatography detection of octacosanol in Antarctic krill. More importantly, It can be used to detect the content of octacosanol in any kind of octacosanol-containing raw materials, products and by-products, and has extensive and important application value in the development of octacosanol.

Detailed ways

The present invention will be further described below in conjunction with embodiment.

Example 1

Weigh 60 g of dried Antarctic krill, add 360 ml of n-hexane, extract 3 times, filter the combined extracts, evaporate the n-hexane to obtain 3 g of Antarctic krill shrimp oil. Add 120ml of 10% NaOH ethanol solution to the shrimp oil, heat in a water bath at 80°C, and reflux for 2 hours; cool down to 50°C, add 120ml of petroleum ether, and extract at 70°C for 40 minutes; then add 120ml of water, stir thoroughly for 20 minutes, and reflux at 50°C Stand still and separate layers; take the petroleum ether solution in the upper layer, evaporate the petroleum ether and dissolve it with chloroform to obtain 8ml of sample solution.

Accurately weigh 1 mg of octacosanol standard substance (Sigma Company), add chloroform to dilute to 4 ml, and prepare a standard solution with a concentration of 0.25 mg/ml. Precisely pipette 1.5, 2, 2.5, 3, 4.5ul octacosanol standard solution and 1.5ul sample solution on the same high-efficiency thin-layer silica gel plate GF ₂₅₄ (10cm×10cm), add 10ml developing agent, the developing agent is Petroleum ether: ethyl acetate: toluene=4:1:0.05, pre-balance for 20 minutes, put the plate into the expansion cylinder (12cm×15cm×10cm), unfold upward, the expansion distance is 8cm, take it out and dry it, and use 100ml of staining solution Rinse and dye for 1 minute, the formula of the staining solution is water: bromothymol blue: sodium hydroxide = 100ml: 0.1g: 0.7g, and dry at 50°C. It can be seen that the two points of the standard and the sample are aligned on the same level and are both Dye blue, use Camag3 type thin-layer scanner to perform thin-layer tomography scanning at 615nm absorption wavelength, scanning speed is 20mm/s, data resolution is 50um/step, and R _f =0.45, and the irradiation lamp is tungsten lamp. For detection and calculation, take the mass (ng) of octacosanol standard substance as the abscissa (X), and the peak area integral value as the ordinate (Y), and the standard substance and peak area integral are directly obtained by the thin-layer chromatography management software winCATS The regression equation of value relationship Y=20.5552+10.8837X, r=0.99962, RSD=1.35%. The results show that the linearity of the sample solution is good in the range of 0.375-1.125ug, the content of octacosanol in 1.5ul sample solution is 0.59666ug, and the calculated 8ml extract contains 3.182mg of octacosanol, which is 60g of dry Antarctic phosphorus The content of octacosanol in shrimp is 3.182mg.

Example 2

The difference from Example 1 is that the developing agent is 15ml, the developing agent is petroleum ether: ethyl acetate: toluene=5:1.5:0.1, the dyeing agent is 80ml, and the staining liquid formula is water: bromothymol blue: sodium hydroxide =80ml: 0.08g: 0.6g, other steps are the same as in Example 1, according to the linear regression equation in Example 1, the average content of octacosanol in 8ml of sample solution (ie 60g of dried Antarctic krill) is 3.181mg.

Example 3

The difference from Example 1 is that the developing agent is 12ml, the developing agent is petroleum ether: ethyl acetate: toluene=3:0.5:0.05, the dyeing agent is 110ml, and the staining liquid formula is water: bromothymol blue: sodium hydroxide =110ml: 0.12g: 0.8g, other steps are the same as in Example 1, according to the linear regression equation in Example 1, the average content of octacosanol in 8ml of sample solution (ie 60g of dried Antarctic krill) is 3.183mg.

Claims (3)

1. The extraction of Antarctic krill octacosanol and the thin-layer chromatography scanning detection method are characterized in that the specific steps are: 1) Extract the dried Antarctic krill with n-hexane, filter and steam the n-hexane to obtain Antarctic krill oil; 2) The shrimp oil is first heated and refluxed in a water bath with a 10% NaOH ethanol solution, and then extracted with petroleum ether. The extracted liquid is added to water for standing and layering, and the upper layer of petroleum ether solution is taken out, and the petroleum ether is evaporated to obtain Solid octacosanol extract; 3) using chloroform to dissolve the octacosanol extract and the octacosanol standard substance to prepare a sample solution and a standard solution respectively; 4) Different quality octacosanol standard solutions and single quality sample solution were respectively spotted on the same high-efficiency thin-layer silica gel plate GF ₂₅₄ , adding developing agent, pre-balanced, and then putting the silica gel plate into the developing cylinder, and developing upward , when the spread reaches 8cm, take it out and dry it, then wash and dye it with the dyeing solution, and dry it; 5) Carry out thin-layer scanning, obtain octacosanol ratio shift value R _f and peak area integral value; Calculate the standard liquid quality and peak area integral value relation by thin-layer chromatograph management software winCATS, according to the peak area of sample liquid The integral value calculates the quality of sample liquid octacosanol; The developer in the step 4) is petroleum ether: ethyl acetate: toluene, the volume ratio is 3-5:0.5-1.5:0.05-0.1; The dyeing liquid in the step 4) is water:bromothymol blue:sodium hydroxide, and the volume to mass ratio is 80-110ml:0.08-0.12g:0.6-0.8g. 2. The method according to claim 1, characterized in that: in said step 1), wherein n-hexane is 6 times the mass volume of Antarctic krill, and is extracted 3 times. 3. The method according to claim 1, characterized in that: the volume ratio of the concentration 10% NaOH ethanol solution, sherwood oil and water in the step 2) is 1:1:1.