Development of a Monoclonal Antibody-Based Immunochemical

Mar 13, 2014 - ABSTRACT: Liquiritin was reacted with a keyhole limpet hemocyanin (KLH) to synthesize a liquiritin−KLH conjugate as an immunogen for ...
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Development of a Monoclonal Antibody-Based Immunochemical Assay for Liquiritin and Its Application to the Quality Control of Licorice Products Shunsuke Fujii,† Osamu Morinaga,‡ Takuhiro Uto,‡ Shuichi Nomura,† and Yukihiro Shoyama*,‡ †

Department of Health and Nutrition, Faculty of Health Management, and ‡Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Nagasaki International University, 2825-7 Huis Ten Bosch, Sasebo 859-3298 Japan ABSTRACT: Liquiritin was reacted with a keyhole limpet hemocyanin (KLH) to synthesize a liquiritin−KLH conjugate as an immunogen for mice. A hybridoma cell line named 2F8 secreted a monoclonal antibody (mAb) against liquiritin, which was applied to an enzyme-linked immunosorbent assay (ELISA) for liquiritin. ELISA showed a good linear range from 0.39 to 25 μg/ mL of liquiritin. The maximum relative standard deviation (RSD) values for the intra-assay and interassay were approximately 5%. The recovery rates of liquiritin were in the range of 100.9−103.7%, and the concentrations of liquiritin in various licorice roots, as determined by ELISA, showed a good correlation with those analyzed by high-performance liquid chromatography (HPLC; R2 = 0.948). These results suggested that ELISA with anti-liquiritin mAb could be a simple, rapid, convenient, and accurate method for the high-throughput analysis of liquiritin in various licorice products including liqueurs, sweets, and food supplements. KEYWORDS: licorice, licorice products, liquiritin, glycyrrhizin, monoclonal antibody, enzyme-linked immunosorbent assay

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INTRODUCTION Licorice, the root of Glycyrrhiza species, is an herbal plant that is typically used as a taste-modifying food additive and is added to various foods such as snacks, candies, seasoning sauce, soy sauce, and drinks as a natural sweetener throughout the world.1 Licorice contains a large number of flavonoid glycosides and their aglycones, which are currently gaining much attention because of multiple biological activities such as antioxidant 2 and anti-inflammatory 3activities, prevention of inflammatory bone loss,4 antitumor activities,5 and inhibition of eotaxin-1 secretion.6 Liquiritin (Figure 1) is a major licorice flavonoid classified as a phloroglucinol, which is a simple phenolic compound exhibiting neuroprotective effects by preventing oxidation and apoptosis.7 Neurodegenerative conditions such as Alzheimer’s and Parkinson’s diseases could be treated with liquiritin by potentiating neurite outgrowth in PC 12 cells.8 Glycyrrhizin (Figure 1) is the main triterpenoid saponin in licorice root, and it is a quality control marker that also has various pharmacological activities.9−13 Depending on their concentration and pharmacological activities, liquiritin and glycyrrhizin have been considered as important constituents in the licorice food industry. Several techniques have been developed for analyzing liquiritin in licorice roots and its products: high-performance liquid chromatography (HPLC),14,15 HPLC coupled with photodiode array detection,16,17 ultraperformance liquid chromatography−mass spectrometry,18 and capillary electrochromatography.19 These analytical chromatography techniques were most frequently used for quantitative and/or qualitative liquiritin analysis; however, complicated pretreatment of sample extracts and use of several organic solvents in the mobile phase were needed to analyze liquiritin. Monoclonal antibody (mAb)-based immunochemical assays are inexpensive, fast, highly specific, sensitive, and promising © 2014 American Chemical Society

methods for the quantitative and/or qualitative analysis of natural bioactive compounds.20 Thus, immunoassays are a highthroughput screening method for a large number of samples. In a previous study, mAbs were prepared against glycyrrhizin with a fixed analytical system using an antiglycyrrhizin mAb.21,22 The glycyrrhizin contents in liqueurs, food supplements, and confectionery products were examined in this fixed analytical system using HPLC23,24 and an online coupled capillary isotachophoresis with capillary zone electrophoresis.25 mAb- or polyclonal antibody-based immunoassays for liquiritin have not been investigated and reported in the field of food products containing licorice. Furthermore, the analytical method for liquiritin in sweets, drinks, and food supplements has not been investigated. Here, we describe the production of anti-liquiritin mAb and its characterization for use in ELISA. Furthermore, the validation of ELISA was performed for the analysis of liquiritin and glycyrrhizin as quality control markers in licorice products including candies, soy sauce, beverages, beer, and food supplements as a natural sweetener or functional component.

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MATERIALS AND METHODS

Chemicals and Immunochemicals. Liquiritin (99% for crude drug test grade), human serum albumin (HSA), keyhole limpet hemocyanin (KLH), and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were purchased from Wako Pure Chemical Inustries, Ltd. (Osaka, Japan). Crude licorice roots were provided by Laboratory for Medicinal Plant Cultivation (Saga, Japan). Licorice products including candies, soy sauce, beverages, beer, and food

Received: Revised: Accepted: Published: 3377

August 4, 2013 March 12, 2014 March 13, 2014 March 13, 2014 dx.doi.org/10.1021/jf404731z | J. Agric. Food Chem. 2014, 62, 3377−3383

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Figure 1. Structures of liquiritin, glycyrrhizin, and structurally related compounds. supplements were purchased from a commercial market in Japan. Freund’s complete and incomplete adjuvants, polyethylene glycol (PEG), and horseradish peroxidase (HRP)-labeled anti-mouse IgG (Fc specific) were obtained from Sigma (Steinheim, Germany). All other chemicals were standard commercial products of analytical grade. Preparation of the Sample and Standard Solutions. Dried licorice root powder (100 mg) was extracted six times with 1.2 mL of methanol (MeOH) containing 0.1% NH4OH with sonication for 20 min and centrifugation at 9000g for 2 min. The combined supernatant was evaporated with N2 gas, and the residue was dissolved in 1.0 mL of MeOH containing 0.1% NH4OH. The prepared extract solution was filtered using a 0.45 μm Cosmonice W filter unit (Nacalai Tesque, Kyoto, Japan) and was used as the sample solution. Solid licorice products such as candies and food supplements were dissolved in 10 mL of H2O and then lyophilized. Freeze-dried licorice powder (200 mg) was extracted six times with 2.0 mL of MeOH containing 0.1% NH4OH. The rest of the freeze-dried licorice powder procedure was the same as that followed for the dried licorice root powder. ELISA was used to analyze liquid samples such as beverages, beer, and soy sauce without any pretreatments. Liquiritin was accurately weighed and dissolved in MeOH to prepare a 5 mg/mL stock solution. Several concentrations of liquiritin standard solutions were prepared by the serial dilution method (0.10, 0.20, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 μg/mL as liquiritin standard) using 20% MeOH. These samples and standard solutions were used for ELISA analysis. Synthesis of Antigen−Carrier Protein Conjugates. Liquiritin− carrier protein conjugates were synthesized using a model based on the periodate oxidation method.21 Liquiritin (5.0 mg) was dissolved in 0.4 mL of MeOH, and the solution was added dropwise to a sodium periodate (5.0 mg)/H2O (0.4 mL) solution. Next, the mixture was

stirred at room temperature for 30 min. After the oxidative reaction, KLH (5.0 mg) was added to 50 mM carbonate buffer (0.8 mL; pH 9.6) and was stirred at room temperature for 6 h. The oxidation reaction mixture was dialyzed against H2O at 4 °C for 2 days and then lyophilized. Mice were then immunized with the liquiritin−KLH conjugate (3.6 mg), whereas the liquiritin−HSA conjugate (7.5 mg), which was prepared using the same method as for the liquiritin−KLH conjugate, was used for ELISA. Analysis of the Liquiritin−Carrier Protein Conjugate by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). A previous study described how the hapten liquiritin−HSA conjugate was determined by MALDITOF.26 Liquiritin−HSA conjugate (1−10 pmol) was mixed with a 103fold molar excess of sinapinic acid in an aqueous solution containing 0.15% (v/v) trifluoroacetic acid. The mixture was subjected to a JEOL mass spectrometer (JMS) time-of-flight (TOF) mass monitor and irradiated with an N2 laser (337 nm, 150 ns plus). The ion formed by each pulse was accelerated by a 20 kV potential into a 2.0 m evacuated tube. Immunization, Hybridization, and Purification of mAb. All procedures and animal care were approved (approval no. 50) by the Committee on Ethics of Animal Experiments, Faculty of Pharmaceutical Sciences, Nagasaki International University; the procedures were conducted according to the Guidelines for Animal Experiments of the Faculty of Pharmaceutical Sciences, Nagasaki International University. BALB/c male mice (6 weeks old) were intraperitoneally injected with the liquiritin−KLH solution four times. The first immunization was a 1:1 emulsion in Freund’s complete adjuvant (0.5 mL, 50 μg of liquiritin−KLH). Two weeks later, a second immunization was repeated with the same amount of antigen, but the liquiritin−KLH solution was emulsified in Freund’s incomplete adjuvant. After 2 weeks, a third immunization was administered, which consisted of 3378

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liquiritin−KLH (100 μg) dissolved in 0.5 mL of phosphate-buffered saline (PBS). The fourth and final immunization was repeated according to the method of the third immunization. Three days after the fourth immunization, immune splenocytes were exenterated and fused to a hypoxanthine−aminopterin−thymidine (HAT)-sensitive mouse myeloma cell line (SP2/0) using the PEG method. After HAT selection, the 2F8 hybridoma cell line secreting anti-liquiritin mAbs was cloned using a limited dilution method. The 2F8 hybridoma cell line was cultured at 37 °C under 5% CO2 atmosphere in enriched RPMI1640−Dulbecco’s−Ham’s F12 medium containing 10% fetal bovine serum. The mAbs that were prepared from the 2F8 hybridoma cell line were purified using a Protein G FF column (GE Healthcare UK Ltd., Amersham, UK). The cultured medium containing IgG (400 mL) was loaded into the column and was eluted with 100 mM citrate buffer (pH 2.7). The eluted IgG solution was adjusted to pH 7.0 with 1 M Tris-HCl buffer (pH 9.0) and then dialyzed against H2O at 4 °C for 2 days. After lyophilization, anti-liquiritin mAb (7.52 mg) was obtained. Direct ELISA. Liquiritin−HSA conjugate and other proteins (100 μL, 1 μg/mL) were dissolved in a 50 mM carbonate buffer (pH 9.6), loaded into a 96-well immunoplate, and incubated for 1 h. The plate was washed three times with PBS containing 0.05% Tween 20 (PBST). The plate was treated with 300 μL of PBS containing 5% skimmed milk (SPBS) for 1 h to reduce nonspecific reactions. After blocking, the plate was washed with PBST three times, and it was then reacted with 100 μL of anti-liquiritin mAb for 1 h. The plate was washed with PBST three times and incubated with 100 μL of a 1000fold diluted HRP-labeled anti-mouse IgG solution for 1 h. After three washings of the plate with PBST, 100 μL of substrate solution of 0.1 M citrate buffer (pH 4.0) containing 0.003% (v/v) H2O2 and 0.3 mg/mL ABTS was added to each well and incubated for 20 min. The absorbance was measured with an Immuno Mini NJ-2300 microplate reader (Nalge Nunc, Roskilde, Denmark) at 405 nm. All reactions were conducted at 37 °C. Competitive ELISA. Liquiritin−HSA (100 μL, 1 μg/mL) was dissolved in a 50 mM carbonate buffer (pH 9.6) and was loaded onto the 96-well immunoplate, which was then treated with 300 μL of SPBS to reduce nonspecific reactions. Fifty microliters of various liquiritin concentrations or samples dissolved in 20% MeOH solution were incubated with 50 μL of anti-liquiritin mAb solution (105.5 ng/mL) for 1 h. After three washings with PBST, the plate was then reacted with 100 μL of HRP-labeled anti-mouse IgG solution for 1 h. The plate was washed three times with PBST, and 100 μL of ABTS solution was added to each well and incubated for 20 min. Absorbance was recorded at 405 nm by a microplate reader. The glycyrrhizin contents in various foods containing licorice were determined by following a previously described method.22 Recovery Experiments. Various amounts of liquiritin standards (25, 50, and 100 μg) were spiked into dried licorice root powder (10 mg). Spiked samples were extracted six times with MeOH containing 0.1% NH4OH under sonication, and the combined supernatant was evaporated with N2 gas. The residue was dissolved in 1.0 mL of MeOH containing 0.1% NH4OH and was filtered using a Cosmonice Filter W. The recovery experiment was performed by using a competitive ELISA. The recovery of spiked liquiritin was calculated as follows:

recovery (%) =

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RESULTS AND DISCUSSION Direct Determination of the Hapten−Carrier Protein Conjugate by MALDI-TOF MS Analysis. We evaluated the hapten number of liquiritin−HSA conjugate by MALDI-TOF MS analysis. A broad peak coinciding with the liquiritin−HSA conjugate was detected around m/z 68500 (data not shown). This result and the molecular weight of approximately 66400 for HSA revealed that the calculated value of the liquiritin component (MW = 418.4) was 4.83, indicating that at least four molecules of liquiritin were coupled with one molecule of HSA. Thus, the prepared liquiritin−HSA conjugate was suitable for use in ELISA by comparing these results to those of a previous study.27 The mAb was tested against liquiritin using a liquiritin− bovine serum albumin (BSA) conjugate as an immunogen. However, a suitable and specific mAb for liquiritin could not be obtained. Because the maximum molecular weight that can be detected by MALDI-TOF MS analysis is 260,000, determining the hapten liquiritin−KLH conjugate number using the MALDI-TOF MS system was impossible because the molecular weight of KLH is about 800,000. Thus, the liquiritin−KLH conjugate was prepared using the same procedure as for the liquiritin−HSA conjugate and was used as an immunogen for the anti-liquiritin mAb production without determination of the hapten liquiritin−KLH conjugate number. Production and Characterization of Anti-liquiritin mAb. The immune splenocytes were fused with SP2/0 myeloma cells by using an established procedure in this laboratory. The 2F8 hybridoma cell line that produced mAb reactive to liquiritin was obtained and classified as IgG2a, which has a κ light chain. The reactivity of anti-liquiritin mAbs was tested against the liquiritin−HSA conjugate and various proteins such as HSA, BSA, ovalbumin (OVA), and KLH with various anti-liquiritin mAb concentrations (Figure 2). Anti-liquiritin mAb did not recognize the HSA, BSA, OVA, and KLH carrier proteins; however, it specifically recognized the liquiritin portions in the liquiritin−HSA conjugate molecule. Furthermore, the absorbance, which was approximately 1.0, was obtained under the

measured amount of liquiritin − 298.07 × 100 spiked amount

The amount of liquiritin in the unspiked sample was determined to be 298.07 (μg) in dried licorice root powder (10 mg) by HPLC. For each level, three samples were analyzed. Determination of Liquiritin Concentration by HPLC. Analytical HPLC was performed using the LC-8020 model III (Tosoh Co. Ltd., Tokyo, Japan). The column used was a 150 mm × 4.6 mm i.d., 3 μm, TSK gel ODS-100Z (Tosoh Co. Ltd.) at room temperature. The mobile phase was water/acetonitrile/acetic acid (82:18:1, by volume), and the flow rate was 0.7 mL/min. UV absorbance detection at 280 nm was used; in this case, the retention time of liquiritin was 14.6 min.

Figure 2. Reactivity of anti-liquiritin mAb against liquiritin−HSA conjugate and various proteins. 3379

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antibody concentration (105.5 ng/mL) selected for competitive ELISA. Assay Sensitivity and Specificity. The assay sensitivity was validated by using anti-liquiritin mAb with competitive ELISA. After incubation of anti-liquiritin mAb with competing liquiritin antigens, any mAb binding free liquiritin was washed out from the ELISA plate. The mAb binding to the liquiritin− HSA conjugate was bound to an enzyme-labeled secondary antibody and reacted to the substrate solution. Under these conditions, the full measuring range of the assay extended from 0.39 to 25 μg/mL (0.93−59.75 μM), as indicated in Figure 3.

Table 1. Cross-Reactivities of Anti-liquiritin mAb against Various Compounds compound

classification

IC50 (μM)

liquiritin liquiritigenin hesperetin hesperidin (±)-naringenin (±)-eriodictyol apigenin luteolin myricetin quercetin fisetin kaempferol isorhamnetin (−)-epigallocatechin (−)-epicatechin daidzein rutin glycyrrhizin 3-monoglucuronyl-glycyrrhetic acid glycyrrhetic acid saikosaponin a ginsenoside Rb1 peaoniflorin

flavanone glycoside flavanone flavonoid glycoside flavanone flavanone flavanone flavone flavone flavonol flavonol flavonol flavonol flavonol flavanol flavanol isoflavonoid flavonoid glycoside triterpenoid triterpenoid triterpenoid triterpenoid triterpenoid monoterpenoid

8.60 33.09 17.83 >100 79.41 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100

ELISA can now be developed for aglycone liquiritigenin using the anti-liquiritin mAb. A previous study developed an ELISA system for the aglycone of baicalin, baicalein, using anti-baicalin mAb, which had lower CRs against baicalein.28 Assay Variation. The intra-assay (well-to-well) and interassay (plate-to-plate) variations were measured with a competitive ELISA because reproducibility and precision are important criteria for an immunoassay. The maximum relative standard deviations (RSDs) of intra-assay and interassay were 4.08 and 5.46%, respectively (Table 2). These results showed that competitive ELISA was accurate and precise for liquiritin.

Figure 3. Calibration curve of liquiritin using anti-liquiritin mAb by competitive ELISA.

The cross-reactivities (CRs) of the anti-liquiritin mAb against the structurally related compounds were checked for the evaluation of assay specificity using competitive ELISA. The CRs were calculated and indicated as IC50 (μM).

Table 2. Validations among ELISA Runs for the Analysis of Liquiritin

IC50 (μ M) = concn (μ M) of liquiritin or various structurally related

CVa (%)

compds yielding A /A 0 = 50%

A is the absorbance in the presence of the test compound, and A0 is the absorbance in the absence of the test compound. As indicated in Table 1, the mAb showed that the CRs against hesperetin (IC50 = 17.83 μM), liquiritigenin (IC50 = 33.09 μM), and naringenin (IC50 = 79.41 μM) have a flavanone molecular skeleton (Figure 1). On the other hand, flavones such as flavonols, flavanone, flavanols, isoflavonoids, flavonoid glycosides, and other tested compounds were not recognized by the anti-liquiritin mAb (Table 1). Thus, the hypothesis that the mAb simultaneously recognized the hydroxyl group at the C7 position and the hydrogen at C2 position as essential functions (Figure 1) was formulated. The anti-liquiritin mAb that was obtained was comparatively recognized by liquiritigenin, and the ELISA that used the antiliquiritin mAb performed in a linear range from 1.56 to 50 μg/ mL (6.09−195.12 μM) for liquiritigenin (date not shown). Thus, the CR against liquiritigenin may have been an unexpected advantage from this experiment because an

liquiritin (μg/mL)

intra-assay

interassay

0 0.39 0.78 1.56 3.13 6.25 12.50 25.00

0.60 1.81 4.08 2.71 1.73 0.95 1.12 0.62

3.49 1.25 1.23 3.29 4.29 3.20 5.22 5.46

Data are mean ± SD for three replicate wells and three plates for each concentration within one plate from three consecutive days.

a

Recovery of Liquiritin by Competitive ELISA in Spiked Samples. The recovery experiment confirmed the accuracy of this assay system. Various amounts of liquiritin (25−100 μg) were added to the dried powder licorice root, and the amount of liquiritin was measured by competitive ELISA. The recovery was calculated, and the rates were between 100.9 and 103.7% with low RSD values (0.10−4.02%) as indicated in Table 3. 3380

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These results show that an ELISA using the anti-liquiritin mAb can be routinely used for phytochemical investigations involving crude plant extracts.

Table 4. Liquiritin and Glycyrrhizin Contents in Various Licorice Products Determined by ELISAa contents (g/kg)

Table 3. Recovery of Liquiritin Determined by ELISA in Spiked Samples spiked level (μg)

measured amounta (μg)

RSDb (%)

recoveryc (%)

25 50 100

323.71 ± 0.31 348.50 ± 14.00 401.75 ± 14.12

0.10 4.02 3.51

102.6 100.9 103.7

liquiritin

glycyrrhizin

glycyrrhizin/ liquiritinb

27.15 ± 2.22

46.03 ± 3.93

1.70

sample licorice root (JP grade)

contents (g/kg) solid sample food suppl 1 food suppl 2 candy

Data are mean ± SD from triplicate analyses for each sample. b Relative standard deviation. cRecovery (%) = (measured amount − 298.07)/spiked amount × 100. a

liquid sample

Correlation of Liquiritin Contents in Licorice Roots Determined by ELISA and HPLC. To confirm the correlation results of liquiritin determination between ELISA and HPLC, the contents of liquiritin in various licorice roots using ELISA and HPLC were also analyzed. The concentrations of liquiritin, as determined by ELISA, were 0.95 ± 0.03, 3.11 ± 0.08, 1.34 ± 0.01, 0.17 ± 0.01, 0.78 ± 0.04, and 2.12 ± 0.08 g/kg dry weight root for samples 1, 2, 3, 4, 5, and 6, respectively. These values and HPLC liquiritin concentrations (concentrations were 0.92 ± 0.01, 3.02 ± 0.10, 1.39 ± 0.06, 0.20 ± 0.01, 0.94 ± 0.02, and 2.73 ± 0.01 g/kg dry weight root for samples 1, 2, 3, 4, 5, and 6, respectively) were similar. The correlation coefficient (R2) between the analytical value of liquiritin from the ELISA and that from HPLC was 0.948 (Figure 4). This high correlation

soy sauce 1 soy sauce 2 soy sauce 3 soy sauce 4 soy sauce 5 soy sauce 6 beverage 1 beverage 2 beer

liquiritin

glycyrrhizin

1.18 ± 0.07 6.24 ± 0.56 1.67 ± 0.09 0.02 ± 0.00 trc 0.07 ± 0.00 contents (mg/L) liquiritin 214.45 249.41 235.46 272.99 226.70 124.82 tr 0.30 tr

± ± ± ± ± ±

14.60 7.96 4.46 9.43 3.13 8.02

± 0.01

glycyrrhizin 93.55 24.94 166.22 71.37 128.00 96.12 1.31 8.78 1.06

± ± ± ± ± ± ± ± ±

6.22 0.58 1.73 2.47 2.16 2.98 0.07 0.09 0.09

glycyrrhizin/liquiritinb 5.29 0.01

glycyrrhizin/liquiritinb 0.44 0.10 0.71 0.26 0.56 0.77 29.27

Data are mean ± SD from triplicate analyses for each sample. Glycyrrhizin/liquiritin = glycyrrhizin contents/liquiritin contents. ctr, trace (LOD ≤ tr ≤ LOQ). a b

including licorice. In a previous study, HPLC was most frequently used for liquiritin and/or glycyrrhizin analysis in numerous licorice roots that were used in traditional Chinese/ Korean medicine.14−17,29 Previously, glycyrrhizin and flavonoid concentrations including liquiritin in Glycyrrhiza species were determined by HPLC,30 and licorice roots were applied to the glycyrrhizin ELISA system for quality control.22 The quality control using both liquiritin and glycyrrhizin ELISA systems was not reported; thus, in this study the quality control of the licorice products was demonstrated by ELISA systems using the anti-liquiritin mAb and anti-glycyrrhizin mAb, respectively. The calculated glycyrrhizin/liquiritin rate varied from 0.01 to 29.27 in all licorice products except for candies, beverages, and beer, as indicated in Table 4. Japanese Pharmacopoeia (JP) grade licorice root had a glycyrrhizin/liquiritin value of 1.70 that was obtained by ELISA. The difference of the glycyrrhizin/ liquiritin value may be caused by the quality of the licorice plants because the liquiritin and glycyrrhizin components fluctuated with collection year and cultivation environment.31 Furthermore, different licorice processing techniques and various preparation methods of licorice products could have affected the amount of glycyrrhizin/liquiritin in the licorice. Therefore, determining the amount of liquiritin in the licorice plants and products was necessary for quality control just as glycyrrhizin analysis of the Japanese Pharmacopoeia was regulated and reported by previous studies.19,23−25,32 Because licorice is used worldwide as a natural sweetener for food, it is an important herbal plant.1 Liquiritin is one of the major flavonoid glycosides in licorice and is employed as a quality control marker for licorice in foods and phytochemical products because it has a variety of pharmacological activities.2−8 Various analytical methods that have used chromatography have been reported;14−17,29 however, an antibody-based immunochemical assay system has not been established for liquiritin in licorice plants and products.

Figure 4. Correlative relationship of liquiritin contents in various licorice roots between the ELISA and HPLC.

indicates that the developed ELISA system could be useful in determining liquiritin in licorice crude extracts without any complicated pretreatments and without the influence of liquiritigenin on this assay system. Furthermore, competitive ELISA was at least 20 times more sensitive for liquiritin than compared with HPLC under the study conditions. Determination of Liquiritin and Glycyrrhizin in Various Licorice Products for Quality Control Demonstrated by ELISA. Table 4 shows the quantitative analysis results of ELISA for liquiritin and glycyrrhizin in licorice root (Japanese Pharmacopoeia grade) and various foods and drinks 3381

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In this study, an established 2F8 hybridoma cell line secreted mAb reactive to liquiritin, and an ELISA system using antiliquiritin mAb was developed. Validation of ELISA confirmed its sensitivity, reproducibility, and accuracy from various validation experiments, and a correlation coefficient (R2) of 0.948 was obtained between the ELISA and HPLC methods. The newly established immunochemical approach compared with instrumental analysis such as HPLC uses less organic solvent and is a cleaner analytical system; thus, the new immunochemical approach is better for the environment. Furthermore, an advantage of the developed ELISA system is that it can be directly applied to impure samples such as crude extracts and licorice products because complicated pretreatments are not necessary for the new system. These results show that ELISA can be a simple, fast, and convenient highthroughput analysis for numerous licorice samples and may apply to a small amount of analytical target samples. In conclusion, the ELISA that was established in this study is a very useful technique for samples with many impurities such as crude extracts from the licorice root and its final products including functional foods that contain several hundred components. Moreover, the ELISA system can be useful for analyzing liquiritin in the manner that glycyrrhizin was analyzed in phytochemical products and medicine. There have been no previous papers indicating an immunoassay system for liquiritin; thus, this is the first time that an immunoassay system for liquiritin has been developed.

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AUTHOR INFORMATION

Corresponding Author

*(Y.S.) Phone: 81-956-20-5653. Fax: 81-956-20-5740. E-mail: [email protected]. Funding

This research was supported by faculties of Health Management and Pharmaceutical Sciences, Nagasaki International University. This work was also supported in part by the Laboratory for Medicinal Plant Cultivation (Saga, Japan). Notes

The authors declare no competing financial interest.

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