Determination of Squalene in Licorice by High Performance Liquid Chromatography

Abstract: Squalene is widely distributed in plants, but the content is not high, much lower than 5% of the unsaponifiables in vegetable oils, only a few are more, such as olive oil containing squalene 150 ~ 700mg / 100g, rice bran oil The content is 332mg / 100g.

Objective To investigate the presence of squalene in licorice. Methods Soxhlet extraction with petroleum ether as solvent and quantitative analysis of squalene in licorice seeds and roots by high performance liquid chromatography (HPLC). Results Squalene was present in licorice seeds and licorice roots, but the content was low. The HPLC method used in this experiment is feasible for determination of squalene. The chromatographic conditions determined are: Waters Corporation C18, mobile phase: acetonitrile: methanol = 60:40, detection wavelength is 210 nm, flow rate is 2.0 ml / min, column temperature It is 30 ℃. Conclusion Squalene can be considered as a new substance with high development and utilization value in licorice for further development and utilization.

[Keywords] licorice; squalene; extraction; high performance liquid chromatography

Abstract: ObjectiveTodeterminesqualeneinGlycyrrhizauralensisFischbyHPLC.

MethodsTheoilinrootandseedofGlycyrrhizauralensisFisch.wasextractedbySoxhletsextractorwithpetrolether, squalenewasseparatedbyTLCandHPLC.

ResultsThecontentofsqualeneinGlycyrrhizauralensisFisch.waslow (inoliveandpalmabout1.16%). HPLCestablishedinthisthesiswassuitabletoanalyzesqualene, theHPLCconditionswereasfollows: column: WatersCorporationC18column; mobilephase, V (acetonitrile) flow: V, m ,;

ConclusionThedeterminationmethodofaqualeneinGlycyrrhizauralensisFishwasestablished.

Keywords: GlycyrrhizauralensisFisch .; Squalene; Extraction; HPLC

Glycyrrhizauralensis is a commonly used bulk medicinal material. It is used as root and rhizome. It is known as "Ten Fang Jiu Cao". The previous researches mostly focused on the physiology of licorice, chemical composition analysis, and standardized cultivation. In the related research on the biosynthesis of glycyrrhizic acid, the main pharmacological component in licorice, licorice was initially found to contain squalene. Squalene was originally discovered from shark liver oil and was named Squalene in 1914. Its chemical name is 2, 6, 10, 15, 19, 23-hexamethyl-2, 6, 10, 14, 18, 22-tetracosahexaene, an open-chain triterpene, also known as cod liver oil terpene, also known as squalene [1], has a physical fitness and anti-fatigue effect, can be used for liver disease treatment, and has certain anti-cancer, anti-cancer And moisturizing beauty effect [2 ~ 4], so it is widely used in health care products and cosmetics. At present, the analysis and detection of squalene mostly use gas chromatography [5 ~ 11], which requires methylation for derivatization pretreatment. In this paper, based on previous researches, squalene in licorice root oil was tested by high performance liquid chromatography. Now reported as follows.

1 Materials and instruments

1.1 Materials Artificial cultivation of Ural Glycyrrhiza uralensis (2 years old) was collected from Hui'anbao Town, Yanchi County, Ningxia on 2008-06-24, and was identified as Ural Glycyrrhiza by Professor Wang Jun of our hospital. At the same time the seeds of Ural licorice were collected in November of that year.

1.2 Reagents methanol and acetonitrile are chromatographically pure, petroleum ether (60 ~ 90 ℃), chloroform are analytically pure. Squalene standard (Sigma), purity 98%.

1.3 Instrument Agilent (USA) 1100 high-performance liquid chromatograph, VWD ultraviolet detector, 10μl micro sampler, chromatographic column: Waters Corporation C18; RE-52B rotary evaporator (Shanghai Yarong Biochemical Instrument Factory).

2 Methods and results

2.1 Preparation of standard solution Pipette 6.25μl of squalene standard stock solution in a 25ml volumetric flask and dilute to the mark with chloroform to obtain a standard solution with a concentration of 0.214mg / ml.

2.2 Preparation of samples Extraction of licorice root by cable extraction method: Weigh 20g of licorice root powder through a 40 mesh sieve, wrap it with filter paper and put it in a cable extraction device, while controlling the temperature of the water bath to be constant at 75 ℃, adding 200ml of petroleum ether Extraction under reflux until the oil is completely dissolved in petroleum ether. The petroleum ether extract was rotavaporized with a rotary evaporator until there was no petroleum ether, and 0.5 g of yellow licorice root oil was obtained, and the volume was adjusted to 10 ml with chloroform. 0.45μm microporous filter membrane was filtered to obtain licorice root oil as test solution. In the same way, licorice seeds are used as extraction materials, divided into peeled and non-peeled, to obtain golden yellow licorice seed oil, constant volume, through 0.45μm microporous filter membrane, that is, licorice seed oil test solution.

2.3 Selection of wavelength Taking chloroform as reference, the licorice root oil diluted with chloroform was scanned with a dual-beam ultraviolet spectrophotometer from 190 to 700 nm wavelength range. The ultraviolet spectrum shows that the licorice root oil has two maximum absorption peaks at 210 and 286 nm. The squalene structure is an isolated double bond, so it will not have strong absorption at 286nm, so the maximum absorption wavelength of squalene should be 210nm, so the detection wavelength is set at 210nm.

2.4 Selection of extraction solvent Because of the low polarity of oils and fats and the fact that squalene is an unsaturated hydrocarbon compound, two solvents, petroleum ether and chloroform, were selected as extraction agents. The experimental results show that the amount of oil and fat obtained when using petroleum ether as the extractant is higher than that using chloroform, therefore, the experiment uses petroleum ether as the extractant.

2.5 Selection of mobile phase Squalene is an unsaturated hydrocarbon compound, which can be separated by reverse phase chromatography. According to Chen Quanbin [12] method, acetonitrile / methanol is used as the mobile phase, and the ratio is acetonitrile / methanol = 60/40. Using the detection wavelength determined by item "2.3" as 210nm, the samples were analyzed at a flow rate of 2.0, 1.0ml / min, column temperature of 20 ℃ and 30 ℃. 60/40, the detection wavelength is 210nm, the flow rate is 2.0ml / min, and the column temperature is 30 ℃, which has a good separation effect. The chromatogram is shown in Figure 1. Take 10μl of standard solution for injection, and analyze according to the same chromatographic conditions as described above. The chromatogram is shown in Figure 2. It shows that under the above chromatographic conditions, the squalene in the standard solution and the sample to be tested is fully separated, and the squalene retention time is about 15.5 min. The retention time of squalene in the sample is the same as that in the standard solution. Fig 1 Chromatogram of squalene sampleFig 2 Chromatogram of squalene standard

2.6 Drawing the standard curve Accurately absorb the standard solution, inject 2, 4, 6, 8, 10μl in sequence, determine the peak area according to the chromatographic conditions of "2.5", and use the peak area to regress the injection volume to obtain the regression of squalene The equation is Y = 970.85X-6.336, r = 0.9998; the results show that squalene has a good linear relationship in the range of 0.428 ~ 2.14μg.

2.7 Precision experiment The above squalene standard solution was continuously injected 5 times, 10 μl / time, and the peak area was determined according to the chromatographic conditions of “2.5”. The results are shown in Table 1. Table 1 Precision Experiment Results Repeat Times Peak Area Mean RSD

2.8 Stability experiment Weigh accurately 20.000g of licorice root powder sample to make a test solution, sample every 8h, measure the peak area according to the "2.5" chromatographic conditions and calculate the squalene content. The results are shown in Table 2. Table 2 Results of stability experiments Time interval t / h Peak area Average peak area RSD

2.9 Repeatability experiment Weigh accurately 20.0000g of licorice root powder sample, 3 parts, each made into the test solution, according to the "2.5" chromatographic conditions to determine the peak area and calculate the squalene content. The results are shown in Table 3. Table 3 Repeatability experiment results Number of repetitions Sample weight m / g Injection volume V / μl Peak area Squalene content

2.10 Sample recovery rate experiment using sample recovery method, accurately weigh the licorice samples of known content, and then add 5μl of squalene standard solution, respectively, extract according to the aforementioned extraction method and dilute to 10ml with chloroform, and inject 10μl According to the "2.5" chromatographic conditions for quantitative analysis, determine the peak area and calculate the squalene content, calculate the recovery rate, the results are: 99.86%, 99.94%, 100.09%, the average recovery rate: 99.96%. It meets the requirements of 95% ~ 105% of Part I of the 2005 edition of the Chinese Pharmacopoeia.

2.11 Sample measurement results The above artificially cultivated ural licorice and licorice seeds were prepared according to the preparation method of the "2.2" sample solution, measured under the above "2.5" chromatographic conditions, and the squalene content was calculated by the external standard method. The results are shown in Table 4. Table 4 Squalene content in licorice root and licorice seeds

3 conclusions

According to the above analysis, squalene is present in licorice, but the content is low. Under the chromatographic conditions: Waters Corporation C18, mobile phase: acetonitrile: methanol = 60: 40, detection wavelength 210 nm, flow rate 2 ml / min, column temperature 30 ℃, the squalene peak and other miscellaneous peaks in licorice root and licorice seed oil are very good Good separation. The methodological investigation shows that the linear relationship, precision, stability, repeatability, recovery rate, etc. all meet the analysis requirements. The method is accurate and can be used as an effective method for quantitative determination of squalene in licorice.

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