Extraction Process and Content Determination of Total Flavonoids in Ginkgo (Ginkgo biloba L.) Leaves

Dengfeng ZOU Hua ZHU Binghua QIN Xiaohua WANG

Abstract［Objectives］ This study was conducted to determine capsaicin and dihydrocapsaicin in capsicum products.

［Methods］ The sample was ultrasonically extracted with anhydrous ethanol as the extraction solvent for capsaicin and dihydrocapsaicin, followed by centrifugation. The extract was subjected to HPLC separation with methanolwater (65∶35) as the mobile phase, and a fluorescence detector (Ex= 229 nm, Em = 320 nm) was used to detect capsaicinoids in the sample.

［Results］ Capsaicin and dihydrocapsaicin had a good linear relationship in the range of 1-200 mg/L (R21=0.999 8, R22=0.999 6). The detection limits were both 0.007 mg/kg; the quantification limits were both 0.02 mg/kg; the precision was 0.235% and 0.754%, respectively; and the average recoveries were 95.94% and 95.39%, respectively.

［Conclusions］ The method is simple, rapid, with good sensitivity and precision, and is suitable for detecting capsaicin substances in capsicum products.

Key wordsHPLCFLD; Capsaicin; Dihydrocapsaicin



Received: October 9, 2018Accepted: December 3, 2018

Wenjuan ZHENG (1986-), female, P. R. China, intermediate engineer, devoted to research about food detection.

*Corresponding author. Email: zhengwh218@163.com.

Capsaicin is a type of components in capsicum (Capsicum annuum), which is found in capsicum of Solanaceae and obtained by extraction of plant materials. Capsaicin is composed of 14 capsaicinoids, mainly capsaicin (C) and dihydrocapsaicin (DHC), the pungency degrees of which are almost two times of nordihydrocapsaicin, homodihydrocapsaicin and homocapsaicin. With the development and advancement of science and technology, capsaicin is used more and more widely. Capsaicin is used as a food additive in food processing. The detection of capsaicin is beneficial to the control of pungency degree. Capsaicin can be used as an analgesic and tranquilizer, and can also be used as a slimming product and a health care product［1-2］. Therefore, a highprecision method for detecting capsaicin can be established, which can better serve production and application.

At present, the detection methods of capsaicinoids include colorimetric method, thin layer chromatography, ultraviolet spectrophotometry, high performance liquid chromatography (HPLC), HPLCMS/MS, etc.［3-7］, among which HPLC is the most widely used method at the present stage. At present, the national standard GB/T 21266-2007 "Determination of total capsaicinoid content and representation of pungency degree in capsicum and its product" also uses HPLC for the detection of capsaicinoids. In the method of the national standard, the extracting solvent used for the extraction of capsaicinoids is a methanoltetrahydrofuran solution, while tetrahydrofuran is highly volatile, which poses a safety hazard to the health of the laboratory staff. In this study, the HPLC method for determination of capsaicin and dihydrocapsaicin in capsicum products was explored and optimized from extraction method and chromatographic conditions, and an HPLCFLD method for detection of capsaicin and dihydrocapsaicin was determined. The linearity, recovery rate and precision of the method were investigated. The method has the advantages of simple operation, high sensitivity and wide application range.

Materials and Methods

Instruments and Equipment

Waters e2695 high performance liquid chromatography (equipped with fluorescence detector, Waters, USA); ultrasonic cleaner (Kun Shan Ultrasonic Instruments Co., Ltd); pure water machine (Beijing Purkinje General Instrument Co., Ltd.); electronic balance (Sartorius, Germany); Centrifuge (Sigma, Germany); rotary evaporator (IKA, Germany).

Reagents and samples

Methanol (HPLC grade, Merck, Germany); ethanol (HPLC grade, Merck, Germany); tetrahydrofuran (analytically pure, Sinopharm Group); acetonitrile (HPLC grade, Merck, Germany); capsaicin and dihydrocapsaicin standard substances (Shanghai ANPEL); commercially available highlyhot chilli powder, chilli powder, chilli sauce and dried chilli.

Experimental methods

Chromatographic conditions

Column: Symmetry C18 column (4.6 mm * 150 mm, 5 μm); mobile phase: methanol∶water (V/V)=65∶35; excitation wavelength: 229 nm; emission wavelength: 320 nm; column temperature: 30 ℃; injection volume: 10 μl; flow rate: 1 ml/min.

Preparation of standards

A certain amount of each of capsaicin standard and dihydrocapsaicin standard (0.010 0 g) was weighed accurately, dissolved in anhydrous ethanol and diluted to 10 ml, as a standard stock solution (the mass concentrations of capsaicin and dihydrocapsaicin were both 1 mg/ml). Each standard stock solution was diluted with anhydrous ethanol to 1, 5, 10, 40, 80, 100 and 200 mg/L standard working solutions. The solutions were freshly prepared for use.

Pretreatment of samples

A certain amount of capsicum product (2.500 g) was accurately weighed into a 50 ml centrifuge tube, added with 25 ml of anhydrous ethanol, and vortexmixed for 1 min. The mixture was ultrasonically extracted in a water bath at 50 ℃ for 30 min, cooled to room temperature, and centrifuged at 5 000 r/min for 5 min. The extract was transferred to a 100 ml pearshaped bottle, and continuously extracted for 3 times. The extracts were combined and rotaryevaporated to 10-20 ml at 50 ℃. The extract was then diluted with anhydrous ethanol to 25 ml, and filtered with 0.22 μm microporous organic filter membrane for later detection. If the contents of capsaicin and dihydrocapsaicin in the sample are high, the extract should be diluted to the range of the standard curve and then detected.

Results and Discussion

Selection of detector

In this study, 10 mg/L capsaicin and dihydrocapsaicin mixed standard and sample were detected with a UV detector (280 nm) and a fluorescence detector (Ex=229 nm, Em=320 nm) at the corresponding detection wavelength. The sensitivity under the two detection conditions was compared according to the response values.



Fig. 1UV chromatogram of capsaicin and dihydrocapsaicin standards

It could be seen intuitively from Fig. 1 and Fig. 2 that at the same standard concentration, the response value of the fluorescence detector was much larger than the response value of the UV detector. Therefore, fluorescence detection is more sensitive for the analysis of capsaicin and dihydrocapsaicin, and is more suitable for the detection of capsaicinoids in samples.



Fig. 2Fluorescence chromatogram of capsaicin and dihydrocapsaicin standards

Optimization of pretreatment method

In GB/T 21266-2007, the pretreatment method is to extract a sample with methanol and tetrahydrofuran (V∶V=1∶1) as the extraction solvent in a water bath condition at 60 ℃. During the extraction, the sample is sealed with plastic wrap on which several small holes are made with a needle, and ultrasonically shaken for 30 min, followed by filtration; and the residue was extracted twice using the mixed solvent together with the filer. The filtrates were collected and concentrated at 70 ℃ to 10-20 ml and diluted with the mixed solvent to 50 ml. We found that the extraction steps of capsaicinoids in this method were cumbersome, and filter was used for multiple times during the extraction process, which greatly increased the detection time, which is not conducive to simultaneous extraction of large batches of samples. In this study, centrifugation was used instead of filter paper to save cost, simplify operation, shorten time, reduce loss, and improve sample extraction rate and recovery.

Selection of extracting solvent

The extraction system selected in the national standard is a methanoltetrahydrofuran mixed solution (V/V=1∶1), but the tetrahydrofuran solvent is highly toxic and volatile, posing a potential threat to the health of the experimenter. This study tested five kinds of extracting solvents: methanol, acetonitrile, ethanol, methanoltetrahydrofuran mixed solution (V/V=1∶1), and ethanoltetrahydrofuran mixed solution (V/V=1∶1) for extracting dried chilli, highlyhot powder and chilli sauce, and it was found that methanoltetrahydrofuran and anhydrous ethanol had a better extracting effects on capsaicinoids. Because anhydrous ethanol is humanfriendly and environmentally friendly, and is more advantageous compared with tetrahydrofuran in price, so anhydrous ethanol was selected as the extracting solvent.

Selection of ultrasonic time

One part of each of commercially available dried chilli, highlyhot chilli powder and chilli sauce was selected, respectively, for the exploration and study of the time during the three times of ultrasonic extraction. The results showed that with ethanol as the extracting solvent, when the extraction time was 30+30+30 and 30+20+10 min, the contents of capsaicin and dihydrocapsaicin in the sample were significantly higher than those in the samples extracted for 30+10+10, 20+10+10 and 10+10+10 min. When the extraction time was 30+30+30 and 30+20+10 min, the contents of capsaicin and dihydrocapsaicin in the samples had no big difference, and the extraction effects were basically the same, indicating that capsaicin and dihydrocapsaicin in the samples were almost extracted completely, so the ultrasonic time was selected as 30+20+10 min.

Table 1Effect of extracting solvent on extraction of capsaicinoids

Extracting solvent

Dried chilli∥mg/kg

CDHC

Highlyhot chilli powder∥mg/kg

CDHC

Chilli sauce∥mg/kg

CDHC

Methanol 2 340.7221 738.000262.190171.923 88.223 53.318

Acetonitrile 2 425.7161 791.967268.082176.853 89.584 56.058

Ethanol 2 485.0011 840.429292.839195.535 99.126 60.065

Methanoltetrahydrofuran (V/V=1∶1)2 450.1621 797.783282.776183.459 92.179 56.103

Ethanoltetrahydrofuran (V/V=1∶1)2 412.4981 772.499271.560176.612 88.451 53.149

Table 2Effect of extracting time on extraction of capsaicinoids

Ultrasonic time∥min

Dried chilli∥mg/kg

CDHC

Highlyhot chilli powder 2#∥mg/kg

CDHC

Chilli sauce∥mg/kg

CDHC

30+30+302 485.4711 831.076292.256196.36498.73160.652

30+20+102 486.4471 832.660291.194196.55298.09159.946

30+10+102 309.3531 782.219288.897195.88894.85856.702

20+10+102 278.0521 721.303283.958194.14391.35554.792

10+10+102 223.5861 682.474280.128187.06485.81553.935

Recovery test

One sample of each of commercially available dried chilli, chilli powder, and chilli sauce was selected and added with standards at such three levels as 50, 100, and 150 mg/kg, and capsaicin contents were determined according to the method in "Pretreatment of samples". The results are shown in Table 3. The recovery values of capsaicin and dihydrocapsaicin were in the range of 90.21%-104.7%, with an average recovery of capsaicin 95.94% and an average recovery of dihydrocapsaicin of 95.39%.

Wenjuan ZHENG et al. Determination of Capsaicin and Dihydrocapsaicin in Capsicum (Capsicum annuum) Products by High Performance Liquid ChromatographyFluorescence Detection (HPLCFLD)

Table 3Results of recovery of capsaicin and dihydrocapsaicin in capsicum samples

Sample

Backgroundvalue∥mg/kg

CDHC

Amount of addedstandard∥mg/kg

CDHC

Determinedvalue∥mg/kg

CDHC

Recovery∥%

CDHC

Averagerecovery∥%

CDHC

Dried chilli 2 481.1281 835.75050502 526.2331 883.00590.2194.5195.9495.39

1001002 576.6981 932.99095.5797.24

1501502 619.5781 972.49092.3091.16

Chilli powder 353.245219.3085050399.665266.64392.8494.67

100100457.945320.008104.70100.70

150150500.789362.88898.3695.72

Chilli sauce 95.33260.1055050143.997107.11597.3394.02

100100196.832158.255101.5098.15

150150231.367198.66090.6992.37

Precision

A chilli powder sample was selected, and added with capsaicin and dihydrocapsaicin mixed standard solution at a concentration of 100 mg/kg. The sample solution was detected for 9 times repeatedly with an injection volume of 10 μl. The RSD of capsaicin was determined to be 0.235% (n=9), and the RSD of dihydrocapsaicin was 0.754% (n=9).

Table 4Linear regression equations and correlation coefficients of standard curves and minimum detection limits and quantification limits of the determination method

AnalyteLinear equation Correlation coefficient Linear range∥mg/kgDetection limit∥mg/kg Quantitation limit∥mg/kg

CapsaicinY=1.92e+0.006X+2.19e+0.0060.999 61-2000.0070.02

Dihydrocapsaicin Y=1.92e+0.006X-1.28e+0.0060.999 81-2000.0070.02

Linear range, detection limit and quantitation limit

At first, 1, 5, 10, 40, 80 and 200 mg/L of capsaicin and dihydrocapsaicin standard working solutions were prepared, respectively, and determined according to conditions in "Chromatographic conditions". Under the analysis, standard curves were drawn with mass concentrations and peak areas, and the peak areas of capsaicin and dihydrocapsaicin showed a good linear relationship with the mass concentrations of capsaicin and dihydrocapsaicin, respectively. The corresponding detection limits of capsaicin and

(Continued on page 198)

Agricultural Biotechnology2019, 8（1）: 188-189, 193

Inspection and Testing

Zhaoqin WANG et al. Study on Colloidal Gold Immunochromatography Assay for Rapid Detection of Spectinomycin

Study on Colloidal Gold Immunochromatography Assay for Rapid Detection of Spectinomycin

Zhaoqin WANG1,2, Yuping WAN1,2, Xiaosheng WU1,2, Yu ZHANG1,2, Fangfang JIA1,2, Guangyao HAN1,2, Zhengxue PENG3, Fangyang HE1,2*

1. Beijing Kwinbon Biotechnology Company, Beijing 102206, China; 2. Beijing Engineering Research Centre of Food Safety Immunodetection, Beijing 102206, China; 3. Beijing Wanger Biotechnology Company, Beijing 102206, China

Abstract［Objectives］ This study was conducted to establish a rapid detection method for spectinomycin in pork, chicken, fish, shrimp flesh and water.

［Methods］ A test strip for rapid detection of spectinomycin in milk was developed by colloidal gold immunochromatography assay.

［Results］ The test strip had a detection limit of 50 μg/kg to milk with a detection time of 15 min, and the false positive rate and false negative rate were both 0.

［Conclusions］ The method is accurate, simple, reliable and convenient, and is suitable for rapid onsite spectinomycin detection.

Key wordsSpectinomycin; Gold immunochromatography assay rapid test strip; Rapid detection



Received: June 30, 2018Accepted: October 9, 2018

Supported by Beijing Training Project for the Leading Talents in Science and Technology (Z171100001117158).

Zhaoqin WANG (1969-), male, P. R. China, researcher, PhD, devoted to research about food safety inspection.

*Corresponding author. Email: 524198094@qq.com.

Spectinomycin is an aminocyclitol antibiotic produced by Streptomyces cerevisiae, which has a strong effect on Gramnegative bacteria, a weak effect on Grampositive bacteria and a certain effect on mycoplasma. It is a new type of antibiotic that specially used for treating gonorrhea and has strong antibacterial activity against Neisseria gonorrhoeae［1-2］.

Spectinomycin is a good animal feed additive that promotes animal growth and is widely used as a veterinary drug in animal husbandry. However, some unscrupulous traders are driven by economic interests and abuses antibiotics, leaving a large amount of antibiotics in animal products. After longterm consumption of animalderived foods containing antibiotic residues, human body will produce antibiotic resistance, which damages the balance of invivo environment, causing dysbacteriosis as well as a series of problems of drug residue in food and environment［3-4］. Therefore, governments and international organizations pay much attention to the pollution of antibiotic residues in animal foods, and it has become a hot public health issue involving human health.

At present, domestic analytical methods for spectinomycin mainly include GCMS, GC, HPLC and HPLCMS/MS［1-10］. Instrument methods have the advantages of high sensitivity and accurate results, but the capital and personnel input is high. Therefore, in view of the shortcomings existing in spectinomycin detection methods, a method for detecting spectinomycin in milk samples by colloidal gold immunochromatography was designed. The method has good specificity, high sensitivity, easy operation and low detection cost, and is suitable for screening and testing of batch samples. It is an ideal rapid screening means, and can better meet the requirements to detection of dairy products conducted by enterprises and government function supervision departments in China.

Materials and Methods

Materials and instruments

Standards such as spectinomycin and various crossreactive drugs (purity≥95%) were purchased from Beijing Research Center for Certified Reference Materials; chloroauric acid (HAuCl4?3H2O) and ovalbumin (OVA) were purchased from Sigma; trisodium citrate and potassium carbonate were purchased from Beijing Baixin Reagent Co., Ltd.; sample extractant and phosphate buffer were provided by Beijing Kwinbon Biotechnology Company; and vortex mixer and homogenizer were purchased from Hunan Xiangli Scientific Instrument Co., Ltd.

Methods

Preparation of spectinomycin hapten

The mixture of spectinomycin (0.66 g), carboxymethoxylamine (0.27 g) and pyridine (1 ml) in DMSO (20 ml) was stirred and distilled at room temperature for 20 h to remove solvent. The mixture was then purified by column chromatography and crystallized in ethanolwater system, obtaining the hapten product.



Fig. 1Syntheiss of spectinomycin hapten

Preparation of immunogen——Synthesis of spectinomycin haptenbovine serum albumin (BSA) conjugate

A certain amount of spectinomycin hapten (38 mg) was dissolved with 5 ml of water. Then, 100 mg of BSA was dissolved with 5 ml of water. The spectinomycin hapten water solution was added into the BSA water solution, obtaining a mixture which was stirred with a magnetic stirrer for 24 h. The mixture was subjected to dialysis with 0.01 mol/L PBS for 3 d, and the solution was replaced for 3 times every day, to remove unreacted macromolecules. Centrifugation was finally performed at 12 000 r/min for 30 min, obtaining a supernatant, which was subpackaged and stored at -20 ℃.

Preparation of coating antigen——Synthesis of spectinomycin haptenovalbvmin (OVA) conjugate

Spectinomycin hapten (22 mg) and N,N′carbonyldiiazole (CDI, 15mg) were dissolved with 1.5 ml of N,Ndimethylformamide (DMF) and stirred for 1 h of reaction at room temperature, obtaining reaction liquid A. Then, 36mg of ovalbvmin was completely dissolved in 3.5 ml of 50 mmol/L sodium carbonate solution. Reaction liquid A was added into the solution dropwise, to allow 24 h of reaction at room temperature. The reaction liquid was subjected to dialysis with 0.01 mol/L PBS for 3 d, and the solution was replaced for 3 times every day, to remove unreacted macromolecules. Centrifugation was finally performed at 12 000 r/min for 30 min, obtaining a supernatant, which was subpackaged and stored at -20 ℃.

Preparation of colloidal goldlabeled antispectinomycin monoclonal antibody

Under magnetic stirring, pH of colloidal gold was adjusted with 0.2 mol/L potassium carbonate to 7.2. Antispectinomycin monoclonal antibody was added into the colloidal gold solution at a rate of 60 μg/ml, followed by stirring uniformly and standing at room temperature for 10 min. 10% BSA was added to adjust the colloidal gold solution to a final concentration of 1% (volume percentage), followed by standing for 10 min. The solution was then subjected to centrifugation, washing and resuspension, and finally stored at 4 ℃.

Freezedrying colloidal goldlabeled antispectinomycin monoclonal antibody onto microwell agent

Into a microwell plate, 100 μl of colloidal goldlabeled antispectinomycin monochonal antibody was added. The microwell plate was then added into a freeze drier, prefreezed at -50 ℃ for 3 h and vacuumdried for 15 h. It was finally taken out as the microwell agent with colloidal goldlabeled antispectinomycin monoclonal antibody which was sealed and preserved.

Detection method

100 μl of a tobedetected solution was transferred with a micropipettor to microwells, which was mixed uniformly with the microwell agent. After incubation at room temperature (20-25 ℃) for 3 min, 70 μl of the mixture was vertically dropped onto an assembled test strip. Timing was started when the liquid began to flow, and after 10 min of reaction, the detection result was judged according to the diagram and read through a colloidal gold analyzer. The judgment at other time was noneffective.



Fig. 2Schematic diagram of spectinomycin colloidal gold test strip

Detection limit of sample

Blank milk samples were added with spectinomycin standard to mass concentrations of 0, 30, 40, 50, 60 and 70 μg/L, respectively. The obtained liquids were determined with three batches of test stripes according to "Detection method", with five repetitions, followed by the determination of the detection limit.

Specificity of test strip

500 μg/L neomycin, streptomycin, gentamicin, apramycin, kanamycin and other drugs were prepared, respectively. The obtained liquids were tested with the test strip, with three repetitions, followed by the determination of the test strip’s specificity.

Accuracy

According to document from Ministry of Agriculture, the accuracy of colloidal gold immunochromatography is expressed as false positive rate and false negative rate［11］. 50 blank milk samples and 50 positive milk samples prepared by adding spectinomycin into blank milk sample to a concentration of 50 μg/L were tested with the test strip, followed by the calculation of the false positive rate and false negative rate.

Repeatability of test strip

Test strips were randomly drawn from three batches, and positive milk sample prepared by adding spectinomycin into blank milk sample to 50 μg/L were detected with the selected test strips with three repetitions. The repeatability of the test strip was judged according to the test results.

Stability of test strip

Enough test strips were preserved at 2-8℃, and a certain amount of test strips were taken out every other month for the determination of 10 blank milk samples and 10 positive milk samples added with spectinomycin to a mass concentration of 50 μg/L with two repetitions. The stability of the test strip was judged according to the test results.

Results and Analysis

Detection limit of sample

Milk samples, which had the spectinomycin concentrations of 0, 30, 40, 50, 60 and 70 μg/L, respectively, were detected according to "Detection method", so as to determine the detection limit. The lowest standard mass concentration only shown line C was selected as the detection limit of the test strip. It could thus be known that the detection limit of the test strip was 50 μg/kg, as shown in Table 1.

Table 1Detection limit of the test strip for rapid detection of spectinomycin

Spectinomycin concentration∥μg/L03040506070

Detection result ---+++

- indicates negative, and + indicates positive.

Specificity

The test strip was used to detect 500 μg/L neomycin, streptomycin, gentamicin, apramycin, kanamycin and other drugs, and the results were all negative, suggesting that no cross reaction happened with other drugs, and the test strop has high specificity.

Accuracy

The test strip was used to detect 50 blank milk samples, all of which produced a negative result, and the false positive rate was lower than 5%. 50 positive milk samples added with spectinomycin to a mass concentration of 50 μg/L were also detected and all showed a positive result, and the false negative rate was 0, which was satisfactory.

Repeatability and stability

The test strip was verified to have good intrabatch and interbatch repeatability.

The test strip could be preserved at 2-8 ℃ for 12 months, and from the 13th month, the failure and false negative phenomena were observed.