Method for determining squalene content of pandanus amaryllifolius Roxb. volatile compoistions

Technical Field

The invention relates to a technical field of chemical analysis, in particular to a volatile component from fragrant dew pocket in the method of determining the squalene content.

Background Art

fragrant dew pocketfragrant dew pocket leaf series pandanaceae dew pocket the (  Roxb   amaryllifolius Pandanus) fresh blade (alias: fragrant blue leaf , plate fragrance of her art remains with US, such as spot blue leaf). Herbaceous evergreen the pocket is dew, leaf about 30 cm, width is about 1.5 cm, the technical some drill, end apex thorn , leaf back tip with microspikes, vagina with narrow white film, no pharmaceutical preparations. ma Gulu islands to Indonesia of origin. It was found, in our country fragrant dew pocket acid red red soil, however, the temperature limit, is only suitable for in my semitropics southern to tropical region growing.

Are natural angle turtle alkene chainly triterpene compound, the natural active substance is important, is still main source of deep-sea fish, in the distribution of the plant is also very widely, but the content is not high. If the olive fruit, vegetable oil palm fruit a relatively high content in the, in which the other vegetable oil may also be detected, but content are generally very low.

Ren Zhujun the method of water vapor distillation (SD), a simple equation yin Guihao using supercritical CO₂ extraction (SFE-CO₂) method to extract, by gas phase chromatography mass spectrum (GC-MS) analysis method to study chemical composition of the volatile oil pocket leaf dew, the result of the relative content of higher squalene, are respectively a 21.03%, 4 . 229%.

 M.A Tan. Plant the branch isdew pocket in the same such as tree leaves (  Soland.var.laevis   tectorius Pandanus) extracting separation, the squalene compound, and through the MABA test indicates that the role having the biological activity of the anti-tuberculosis.

To date, the volatile ingredient fragrant dew pocket systematic analysis of reports also little. It is very necessary to carry out an in-depth to the volatile ingredient fragrant dew pocket extraction and separation, systematic quantitative and qualitative analysis, in order to further the development of dew wider application pocket leaf value provides a certain experiment according to the.

Content of the invention

The present invention provides a volatile component from fragrant dew pocket in the method of determining the squalene content.

The invention adopts the following technical scheme:

From of the present invention the volatile ingredient fragrant dew pocket in the method of determining the squalene content measured by high performance liquid chromatography, the mobile phase: methanol, flow rate 0.8-1.2 ml/min; detection wavelength: 204 nm; column temperature: the 25-35 [...] ; testing time: 30 min; sample size: the 10  l.

The velocity of flow of the mobile phase is preferably 1.0 ml/min.

Column temperature is preferably: the 30 [...].

The positive effect of this invention are as follows:

From of the present invention the volatile ingredient fragrant dew pocket in the method of determining in squalene content, whereas the peak area with the integral value of the linear relationship of well, sample stability, instrument precision, reproducibility and method for method specificity are consistent with requirements, for subsequent spinacene fast, accurate quantitative lay the foundation.

Description of drawings

Fig. 1 is a spectral diagram of squalene reference solution.

Figure 2 is a squalene for sample solution spectrum diagram.

Figure 3 is the supercritical extract fragrant dew pocket chromatogram for the sample.

Figure 4 is the chromatogram dogfishes a vertebrate.

Figure 5 is a blank control chromatogram.

Figure 6 is a curve chart of the squalene standards.

Mode of execution

The following embodiment of the present invention is described in further detail.

Embodiment 1

1 instrument and material

1.1 instrument

DIONEX3000-type high performance liquid chromatography (HPLC) instrument, DAD detector, Chromeleon chromatography workstation (the United States Company dai An);

AL104 type electronic balance (Mettler Toledo Shanghai instrument Company limited);

XS205DualRange type electronic balance (Mettler Toledo Shanghai instrument Company limited);

HN-1006-type ultrasonic cleaning machine (Guangzhou southern China Company limited of ultrasonic equipment);

HK-08B-shaped flowing water type Chinese medicine crusher (Guangzhou the rising sun is bright mechanical equipment Company limited);

National standard inspection sieve (zhejiang fada metal sand sifter factory)

1.2 material

Methanol (HPLC grade, Merck ltd.);

Squalene reference substance (140721-200601, Chinese food and drug testing Institute for identification and content determination, content 99.5%);

CO₂ (food grade, purity > 99.95%, bought from foshan shunde buddha roentgen gas Company limited); fragrant dew pocket leaf (batch number: 20130114B, 2013 years 1 month in the wanning xinglong oac farm, is identified to be (  Roxb   amaryllfoliu Pandanus) fragrant dew pocket the fresh leaf.

2 methods and results

2.1 preparation of reference solution

Angle squalene contrast approximately 0.01g, precision stability, adding methanol to dissolve, and constant volume to 5 ml brown bottle in, as squalene reference substance storage liquid.

Precision move spinacene control reserve liquid 3 ml, with 25 ml in brown measuring flask, add methanol dilution to scale, with capillary membrane (0.45 the  m) cvvhdf, as squalene reference solution.

2.2 preparation of sample solution

Supercritical takes the fragrant dew pocket CO₂ extract about 0.02g, precision stability, adding methanol ultrasonic the dissolved, and constant volume to 25 ml in brown measuring flask, with capillary membrane (0.45 the  m) cvvhdf, as for sample solution.

2.3 the choice of the detection wavelength is

Squalene reference substance ultraviolet wavelength spectrum display spinacene maximum absorption wavelength as 203.9 nm, the volatile ingredient fragrant dew pocket in the ultraviolet wavelength spectrum of squalene basically in accordance with the contrast product, a wavelength of maximum absorbance of 204.6 nm. In the chromatographic conditions that the lower corner squalene the maximum absorption wavelength is 204 nm. As shown in Figure 1, 2 is shown.

2.4 the choice of mobile phase

Squalene is unsaturated olefin compound, a reversed phase chromatographic detection supercritical CO₂fragrant dew pocket of extraction of squalene content in the composition, according to literature reports, squalene component separation using methanol: acetonitrile (60:40),

Methanol: acetonitrile (40:60), methanol: water (99:1) mobile phase, and the like; adjustment of the proportion of the mobile phase, in methanol to obtain a better separation effect, reference substance retention time of 18.040 min, sample-retaining time is 18.047 min, retention time of the two basically the same. Chroatogram as shown in Figure 3.

2.5 column temperature selection

Experiment on column temperature is inspected, in order to select the appropriate column temperature. Inspection with the temperature of the 25 [...] , the 30 [...] , the 35 [...]. The experiment shows that, high temperature rise of the column, retention time is shortened, the retention time is 20.07 min, 18 . 12 min, 16 . 47 min. In the 25-35 the [...][...] range, good degree of separation of the chromatographic peaks, column temperature is diagonal squalene of no significant influence on the analysis, as shown in table 1. Comprehensive consideration, choose the 30 [...] to a proper temperature.

Table 1 peak area of   column temperature the impact of the diagonal squalene

2.6 flow rate selection

Inspection experiment on velocity, according to the 2.2 methods for preparing the sample solution, the detection of the selected wavelength, composition and proportion of the fluidity, the detection time, the injection volume is a certain chromatographic conditions, in order to respectively 0.8 ml/min, 1.0 ml/min, 1.2 ml/min flow rate to be measured.

Result display, retention time is shortened with the pace of the velocity of flow, the retention time is 22.68 min, 18 . 14 min, 15 . 12 min, flow rate is 0.8 ml/min, , chromatographic the peak compares obviously spreading, flow rate is 1.2 min/ml time, affect the degree of separation of the chromatographic peak, taking into consideration, the experiment using 1.0 min/ml flow rate.

2.7 analysis conditions

Chromatographic column: Agilent   Eclipse   XDB-C₁₈ (4.6 mm × 150 mm, 5 µm),

Column temperature is: the 30 [...]. Mobile phase: methanol, flow rate 1.0 ml/min; detection wavelength: 204 nm; testing time: 30 min; sample size: the 10  l.

2.8 the methodology study

2.8.1 specificity test

Respectively absorbing control solution, the test sample solution fragrant dew pocket and a negative control solution (methanol) the the 10  l, into the liquid chromatography, by 2.7 the chromatographic conditions under determination. As a result, the negative control is the same as in the squalene contrast peak is not see chromatographic peak retention time, note the diagonal squalene measuring the content of solvent without interference. As shown in Figure 3-5.

2.8.2 standard curve plotting

Precision pipetting spinacene reference solution 1, 4, 6, 10, 12 the  l, into the liquid chromatography, by 2.7 chromatographic condition under measurement, the peak area of the record. Integral value in the peak area (y) as ordinate, contrast specimen handling quantity (x) as abscissa, by linear regression, from the regression equation of squalene y=88.897x + 1.4269 (r=0.9999, n=5). As shown in Figure 6.

The result shows that, in the sample of squalene 0.2606 the the g-3.1276   g with its peak area integral value within the range of the linear relationship is good.

2.8.3 precision tests

In the concentration of 0.1563 mg/ml reference solution dogfishes continuous sampling 6 times, each time for the injection volume is the 10  l, in accordance with the 2.7 peak area of the chromatogram under measuring conditions, results as shown in table 2.

Table 2 experimental result   precision

As a result, RSD=0.68%, indicate that instrument precision are good.

2.8.4 repeatability test

Taking the same batch of supercritical CO₂ extraction of the volatile ingredient fragrant dew pocket 20 mg, parallel 6 parts, separately according to 2.2 method for preparing the sample solution. Precision absorbing sample solution the 10  l, into the liquid chromatography, by 2.7 chromatographic conditions under the peak area of the measuring, computing sample content.

Results as shown in table 3

Table 3 supercritical extract   fragrant dew pocket experimental repeatability of the results for the test solution

Results RSD=1.20% (n=6), good repeatability of the method.

2.8.5 sample application recovery ratio test

Sample recovery process is adopted, the known squalene content of the same supercritical CO₂fragrant dew pocket of extraction of the volatile ingredient, about 0.0100g, precision stability, parallel 9 parts, separately according to about the 1 [...] 0.8, 1:1, the 1 [...] 1.2 the mass ratio by adding corresponding amounts of squalene reference substance, as 2.2 method for preparing the sample solution, the 2.7 chromatographic conditions for measuring a sample, the peak area of the record, calculating sample application recovery ratio, results shown in table 4.

Table 4 sample selection   fragrant dew pocket supercritical extract of recovering experimental results

As a result, the recovery rate 95%-105% between, in accordance with the Chinese Pharmacopoeia 2010 year part 1st Edition, squalene low, in, the concentration for the average recovery rate respectively 101.63%, 98 . 53%, 100 . 52%, RSD are respectively 1.37%, 0 . 86%, 2 . 37%.

2.8.6 stability test

Taking the same batch of supercritical CO₂ extraction of the volatile ingredient fragrant dew pocket for sample solution, according to the above-mentioned chromatographic condition respectively for 0, 1, 2, 4, 8, 12, 24h into the liquid chromatography, by 2.7 peak area of the chromatogram under measuring conditions, the content of the calculation of the sample. Results as shown in table 5

Table 5   stability test results

Results RSD=0.47% (n=6), for the sample that the 24h the inner basic stability.

2.9 squalene content determination

Taking 3 batch of supercritical CO₂ extraction of the volatile ingredient fragrant dew pocket , 0.02g, precision stability, according to the 2.2 method for preparing the sample solution, precise absorbing sample solution the 10  l, according to 2.7 measured under conditions of liquid-phase chromatography, the peak area of the recorded value, calculating the squalene content.

Results as shown in table 6

Table 6   fragrant dew pocket squalene ingredient in the supercritical extract of assay results

3 conclusion

Adopts the high performance liquid chromatography to supercritical CO₂ extraction of the volatile ingredient fragrant dew pocket the squalene in qualitative and quantitative analysis, the squalene standard chromatographic behavior contrast can be known:

(1) in the corresponding chromatographic conditions, squalene standard and fragrant dew pocket volatile supercritical CO2 extraction of the methanol for the retention time in the test solution for 18 min corresponding chromatographic peaks appeared nearby, further evidence of the volatile ingredient fragrant dew pocket containing a certain amount of squalene.

(2) experiment confirms the squalene high performance liquid chromatographic analysis condition is: the mobile phase: methanol, flow rate 1.0 ml/min; detection wavelength: 204 nm; column temperature: 30 the [...] ; testing time: 30 min; sample size: the 10  l. This assay condition, the test sample solution with other components of squalene chromatographic peaks can reach the chromatographic peak baseline separation, squalene retention time of 18.047 min. And the methodology study, the result shows that, in the sample of squalene 0.2606 the the g-3.1276   g with its peak area integral value within the range of the linear relationship is good, squalene sample application recovery ratio (low, middle, high plurality of concentration sample application recovery ratio are 101.63%, 98 . 53%, 100 . 52%), sample stability, instrument precision, reproducibility and method for method specificity are consistent with requirements, for subsequent spinacene fast, accurate quantitative lay the foundation.

Although have already shown and described the embodiment of the present invention, for the ordinary technicians in this field are concerned, can be understood without departing from the principle of the invention and the spirit of the in these embodiment can carry out various changes, modification, replacement and modification, the scope of the present invention is that by the attached limit and its equivalent.