Method for simultaneously detecting four isomers of resveratrol in peanut

Abstract

The present application relates to a method for simultaneously detecting four isomers of resveratrol in peanut by using ultra performance liquid chromatography during the operation procedure and accurately controlling the detection conditions. The method of the invention can achieve continuous sample injection, and perform sample analysis in batch. The detection time for each sample is only about 10 min, which greatly improves the detection efficiency. Further, the ultra performance liquid chromatography has high sensitivity and a low detection limit. The method of the invention adopts ethanol extraction under heating in the earlier stage of extraction, and controls the temperature of the extraction, so as to achieve effective extraction of four isomers of resveratrol, time saving, and efficient extraction and separation.

Claims

1. A method for simultaneously detecting four isomers of resveratrol in a sample to be tested, the method comprising the steps of: 1) pretreating the sample to be tested to obtain a pretreated sample to be tested, 2) detecting the four isomers of resveratrol from the pretreated sample to be tested using an ultra performance liquid chromatography method with a C.sub.18 column having a column temperature controlled at 33 to 38 C., a photodiode array (PDA) detector having simultaneous detection wavelengths of 285 nm for 2-dimensional operation and 210 to 400 nm for 3-dimensional operation, and a mobile phase containing methanol (B) and 0.1% aqueous solution of formic acid (D), wherein the elution conditions for the method are as follows: 0 min: 10% B, 90% D; 0.5 min: 10% B, 90% D; 2 min: 25% B, 75% D; 3.5 min: 30% B, 70% D; 4 min: 35% B, 65% D; 5 min: 50% B; 50% D; 7 min: 90% B, 10% D; 7.2 min: 10% B, 90% D; and 10 min: 10% B, 90% D; and the flow rate of the mobile phase is 0.4 to 0.5 mL/min; wherein the sample to be tested is selected from the group consisting of a fresh sample, a dry sample and a third sample; and wherein the pretreatment of the fresh sample to be tested or the dry sample to be tested comprises the steps of: 1) drying the fresh sample to be tested or the dry sample to be tested to a moisture content of less than 5%, to obtain a dried sample; 2) crushing and sieving the dried sample with a 60 mesh sieve, to obtain a sieved and powdered sample; 3) extracting the sieved and powdered sample with 80% ethanol extracting solution under vibration at 70 to 80 C. in a constant-temperature water bath until all of the resveratrol and its glycosides are dissolved in the ethanol extracting solution, to obtain a mixture; and 4) centrifuging the mixture obtained from the extraction step to obtain a supernatant, and filtering the supernatant with a 0.2 to 0.25 m filter membrane, to obtain a pretreated sample to be tested; and/or the pretreatment of the third sample to be tested comprises the steps of: 1) extracting the third sample to be tested with 80% ethanol extracting solution under vibration at 70 to 80 C. in a constant-temperature water bath until all of the resveratrol and its glycosides are dissolved in the ethanol extracting solution, to obtain a mixture; and 2) centrifuging the mixture obtained from the extraction step to obtain a supernatant, and filtering the supernatant with a 0.2 to 0.25 m filter membrane, to obtain a pretreated sample to be tested.

2. The method according to claim 1, wherein the fresh sample to be tested comprises peanut sprout or peanut skin; the dry sample to be tested comprises peanut root or peanut kernel; and the third sample to be tested comprises peanut butter.

3. The method according to claim 1, wherein the drying method for the fresh sample to be tested is lyophilization by lyophilizer; and the drying method for the dry sample to be tested is oven-drying with hot air at a temperature of 50 to 60 C.

4. The method according to claim 1, wherein the column temperature is 35 C.

5. The method according to claim 1, wherein the flow rate of the mobile phase is 0.45 mL/min.

6. The method according to claim 1, wherein: a). in steps 3) and 4) of the pretreatment of the fresh sample to be tested or the dry sample to be tested: 3) the sieved and powdered sample is placed in 80% ethanol extracting solution and extracted under vibration at 130 to 150 r/min for 45 to 50 min in a constant-temperature water bath at 80 C., wherein the mass-volume ratio of the sample to the ethanol extracting solution is 1-1.5:50, to obtain a mixture; and 4) the mixture obtained from the extraction step is subjected to centrifugation, and the supernatant is filtered with a 0.22 m filter membrane, to obtain a pretreated sample to be tested; b). in steps 1) and 2) of the pretreatment of the third sample to be tested: 1) the third sample is placed in 80% ethanol extracting solution and extracted under vibration at 130 to 150 r/min for 45 to 50 min in a constant-temperature water bath at 80 C., wherein the mass-volume ratio of the sample to the ethanol extracting solution is 1-1.5:50, to obtain a mixture; and 2) the mixture obtained from the extraction step is subjected to centrifugation, and the supernatant is filtered with a 0.22 um filter membrane, to obtain a pretreated sample to be tested; c). in the detection step of the one or more samples to be tested: the flow rate of the mobile phase is 0.45 mL/min.

7. The method according to claim 1, wherein the method further comprises performing quantitative detection of the four isomers of resveratrol using a method, comprising the steps of: A. injecting standard substances of the four isomers of resveratrol selected from the group consisting of trans-resveratrol, trans-resveratrol glucoside, cis-resveratrol and cis-resveratrol glucoside in different concentrations into the C.sub.18 column respectively, and detecting concentrations and peak areas of the standard substances by the ultra performance liquid chromatography method of claim 4, and plotting standard curves according to the detected concentrations and peak areas of the standard substances; and B. substituting the peak areas of the four isomers of resveratrol from the sample to be tested measured in claim 4 into the standard curves respectively to obtain the concentrations of the corresponding substances in the sample to be tested, and calculating the contents of the four isomers of resveratrol in the sample to be tested.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the UPLC spectrum of four isomers of resveratrol;

(2) FIG. 2 shows the UPLC spectrum of four isomers of resveratrol in peanut sprout;

(3) FIG. 3 shows the UPLC spectrum of three isomers of resveratrol in peanut skin.

(4) FIG. 4 shows the UPLC spectrum of one isomer of resveratrol in peanut butter;

(5) FIG. 5 shows the HPLC spectrum of four isomers of resveratrol;

(6) FIG. 6 shows the UPLC spectrum of resveratrol extracted from peanut skin by the method described in the Comparative Example.

SPECIFIC MODES FOR CARRYING OUT THE EMBODIMENTS

(7) The following examples are intended to illustrate the invention, but not to limit the scope of the invention.

Example 1

(8) The present Example relates to a method for qualitative and quantitative detection of resveratrol in peanut sprout, comprising the steps of:

(9) 1. Pretreatment of the sample to be tested:

(10) 1) some fresh peanut sprouts were placed in a lyophilizer to be lyophilized for 48 h;

(11) 2) the lyophilized peanut sprouts were pulverized with a high-speed pulverizer, and sieved with a 60 mesh sieve, and then the powder is collected and stored at low temperature in dark;

(12) 3) 1 g of powder of peanut sprouts was weighed accurately, and added into a 150 ml triangular flask with stopper, 50 ml ethanol solution (80%, v/v) was added, and then extraction was carried out in a constant temperature water bath oscillator (80 C., 140 r/min) for 45 min; and

(13) 4) the extracting solution was centrifuged at 5000 rpm for 10 min, and the supernatant was passed though a 0.22 m microfiltration membrane to obtain a sample to be tested, wherein exposure to sunlight was avoided in the whole process;

(14) 2. The sample to be tested was analyzed and detected by ultra performance liquid chromatography, the instrument conditions were as follows:

(15) Liquid Phase Conditions:

(16) Column: Waters ACQUITY UPLC HSS C.sub.18 column (2.1 mm100 mm, 1.8 m); PDA detector, a column temperature of 35 C., a detection wavelength of 285 nm for 2D and of 210 to 400 nm for 3D; an injection volume of 1 an injection mode of autoinjection. The mobile phase was methanol (B) and 0.1% aqueous solution of formic acid (D), and the elution conditions were as follows: 0 min: 10% B, 90% D; 0.5 min: 10% B, 90% D; 2 min: 25% B, 75% D; 3.5 min: 30% B, 70% D; 4 min: 35% B, 65% D; 5 min: 50% B; 50% D; 7 min: 90% B, 10% D; 7.2 min: 10% B, 90% D; 10 min: 10% B, 90% D. The flow rate was 0.4 to 0.5 mL/min;

(17) 3. Quantitative detection

(18) the quantitative detection method comprised the following steps: standard curve method was used, and it comprised plotting a standard curve with the concentrations of the standard solutions as the X-axis (mg/L) and the peak areas as the Y-axis, and calculating the linear regression as follows:

(19) trans-resveratrol y=8957.3x2488 R.sup.2=0.9999

(20) trans-resveratrol glucoside y=4695.4x1687.8 R.sup.2=0.9997

(21) cis-resveratrol y=6894.1x+32769 R.sup.2=0.9989

(22) trans-resveratrol glucoside y=4047.5x+4740.6 R.sup.2=0.9979

(23) to ensure that the linear relationship is good (R.sup.2>0.997);

(24) the concentration of each isomer of resveratrol in the sample was calculated by the following formula (mg/kg):
C=(Ab)1/ac1/W1/1000

(25) wherein:

(26) C=concentration of the isomer of resveratrol in the sample to be tested;

(27) A=peak area of the isomer of resveratrol;

(28) W=sample weight (g);

(29) A=slope;

(30) B=intercept;

(31) C=dilution multiple.

(32) In order to judge the appearance time of the four isomers, standard samples were introduced to obtain FIG. 1. The sample was detected to obtain the chromatogram as shown in FIG. 2, the content of trans-resveratrol (trans-Chun) in the peanut sprout sample was calculated to be 0.030 mg/kg, the content of trans-resveratrol glucoside (trans-Gan) was 0.099 mg/kg, the content of cis-resveratrol (cis-Chun) was 0.025 mg/kg, and the content of cis-resveratrol glucoside (cis-Gan) was 0.049 mg/kg.

Example 2

(33) The present Example relates to a method for qualitative and quantitative detection of resveratrol in peanut skin, comprising the steps of:

(34) 1. Pretreatment of the sample to be tested:

(35) 1) some peanut skin samples were oven-dried in a blast air oven at 55 C. for 9 h;

(36) 2) the treated peanut skins were pulverized by a high-speed pulverizer, and sieved with a 60 mesh sieve, and then the powder was collected and stored at low temperature in dark;

(37) 3) 1 g peanut skin powder was weighed accurately, and added into a 150 ml triangular flask with stopper, 50 ml ethanol solution (80%, v/v) was added, and then extraction was carried out in a constant temperature water bath oscillator (80 C., 140 r/min) for 45 min; and

(38) 4) the extracting solution was centrifuged at 5000 rpm for 10 min, the supernatant was passed though a 0.22 m microfiltration membrane to obtain a sample to be tested, wherein exposure to sunlight was avoided in the whole process.

(39) The steps of instrumental analysis and data processing were the same as those in Example 1.

(40) The obtained chromatogram was shown in FIG. 3. The content of trans-resveratrol (trans-Chun) in the peanut skin sample was calculated to be 1.492 mg/kg, the content of trans-resveratrol glucoside (trans-Gan) was 0.073 mg/kg, the content of cis-resveratrol (cis-Chun) was 0.489 mg/kg, and cis-resveratrol glucoside (cis-Gan) was not detected.

Example 3

(41) The present Example relates to a method for qualitative and quantitative detection of resveratrol in peanut butter, comprising the steps of:

(42) 1) 1 g peanut butter sample was weighed accurately, and added into a 150 ml triangular flask with stopper, 50 ml ethanol solution (80%, v/v) was added, and then extraction was carried out in a constant temperature water bath oscillator (80 C., 140 r/min) for 45 min; and

(43) 2) the extracting solution was centrifuged at 5000 rpm for 10 min, the supernatant was passed though a 0.22 m microfiltration membrane to obtain a sample to be tested, wherein exposure to sunlight was avoided in the whole process.

(44) The steps of instrumental analysis and data processing were the same as those in Example 1.

(45) The obtained chromatogram was shown in FIG. 4, the content of trans-resveratrol glucoside (trans-Gan) in the peanut butter sample was calculated to be 2.572 mg/kg, and trans-resveratrol (trans-Chun), cis-resveratrol (cis-Chun) and cis-resveratrol glucoside (cis-Gan) were not detected.

Comparative Example 1

(46) By using high performance liquid chromatography instead of ultra performance liquid chromatography for detection, the actually measured appearance time of standard samples of the four isomers of resveratrol were as follows: trans-resveratrol at 14.663 min, cis-resveratrol at 17.069 min, trans-resveratrol glucoside at 10.681 min, and cis-resveratrol glucoside at 12.326 min, as shown in FIG. 5. In addition, the whole process of sample detection needs 35 min, whereas the ultra performance liquid chromatography of the present invention only needs 10 min.

Comparative Example 2

(47) Compared with Example 2, the difference of the comparative example 2 lied in that peanut skin was detected, and the pretreatment method thereof was as follows: 3 g peanut skin was weighed accurately, placed in a 150 ml triangular flask with stopper, added with 45 ml of absolute ethanol with a volume fraction of 75%, ultrasonically extracted at 50 C. for 64 min, and then centrifuged at 5000 r/min for 10 min; and the supernatant was sieved with a 0.22 m filter membrane to give a sample to be tested.

(48) Only two active ingredients could be detected in the sample prepared by the above method, as shown in FIG. 6, and the ingredients were trans-resveratrol glucoside (trans-Gan) and cis-resveratrol (cis-Chun), respectively. It is further demonstrated that the pretreatment method described in the present invention can be used to extract the resveratrol sufficiently.

(49) In the drawings of the present invention, there is a portion where the characters and the spectrum overlap. Since the appearance time determines the type of the substance, clear disclosure is not affected even if the characters overlap the spectrum.

(50) While the present invention has been described in detail by way of general description, specific embodiments and tests, modifications and improvements that may be made without departing from the spirit of the invention are within the scope of the invention as claimed.