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  • 1 Jiangsu University, 212013 Zhenjiang, China
  • 2 Nanyang Technological University, 637551, Singapore
  • 3 Jiangsu Provincial Academy of Chinese Medicine, 210028 Nanjing, China
Open access

Red Toon is a popular vegetable of favorable health benefits over Asia and Russia regions. In this study, isolation and identification of chemical constituents were performed to assess the quality of this functional food cultivated in various origins or harvested in different months. As a result, eight flavonol glycosides including rutin (I), myricitrin (II), quercetin-3-O-β-d-galactoranoside (III), quercetin-3-O-β-d-glucopyranose (IV), quercetin-3-O-α-l-arabinopyranoside (V), astragalin (VI), quercetin-3-O-α-l-rhamopyranoside (VII), and kaempferol-3-O-α-l-rhamopyranoside (VIII) were obtained. Among these, compounds III and V were isolated from Toona genus for the first time. Importantly, a rapid and convenient ultra-performance liquid chromatography (UPLC) method was developed to quantify the flavonol glycosides in Red Toon and validated for linearity, precision, stability, repeatability, and accuracy successively. In addition, it was found that total flavonoid glycosides (about 2.6%) in the food were kept at a higher level from April to June than other months of the year. Furthermore, their content in the Red Toon collected from ten different origins was also determined and compared, and the results suggested that the total flavonoid glycosides from Shandong Yantai were the highest, followed by Shandong Ximou, supporting a well-recognized viewpoint that Red Toon cultivated in Shandong Province, China, is considered genuine due to the best health benefits and flavor.

Abstract

Red Toon is a popular vegetable of favorable health benefits over Asia and Russia regions. In this study, isolation and identification of chemical constituents were performed to assess the quality of this functional food cultivated in various origins or harvested in different months. As a result, eight flavonol glycosides including rutin (I), myricitrin (II), quercetin-3-O-β-d-galactoranoside (III), quercetin-3-O-β-d-glucopyranose (IV), quercetin-3-O-α-l-arabinopyranoside (V), astragalin (VI), quercetin-3-O-α-l-rhamopyranoside (VII), and kaempferol-3-O-α-l-rhamopyranoside (VIII) were obtained. Among these, compounds III and V were isolated from Toona genus for the first time. Importantly, a rapid and convenient ultra-performance liquid chromatography (UPLC) method was developed to quantify the flavonol glycosides in Red Toon and validated for linearity, precision, stability, repeatability, and accuracy successively. In addition, it was found that total flavonoid glycosides (about 2.6%) in the food were kept at a higher level from April to June than other months of the year. Furthermore, their content in the Red Toon collected from ten different origins was also determined and compared, and the results suggested that the total flavonoid glycosides from Shandong Yantai were the highest, followed by Shandong Ximou, supporting a well-recognized viewpoint that Red Toon cultivated in Shandong Province, China, is considered genuine due to the best health benefits and flavor.

Introduction

Toona sinensis Roemer (T. sinensis), a widely grown deciduous arbor native to east and southeast Asia, has a cultivation history of longer than 2000 years in China [1]. Its fresh young leaves and shoots, well-known as Red Toon (Figure 1), are popularly consumed as a dietary vegetable or culinary ingredient in the Asia–Europe region due to their favorable health benefits and unique flavors [2]. In addition, as an easily accessible herbal material, the dried leaves of T. sinensis are also used medicinally to treat enteritis, dysentery, diabetes, infection, halitosis, vomiting, and itch, without any significant irreversible side effects after long-term conventional therapy in clinics [3]. Also, other organs including root, cortex, branch, flower, fruit, and seed have been broadly used for medicinal purposes for a long time by the folks nearby Asia.

Figure 1.
Figure 1.

Red Toon (left) and Toona sinensis Roemer (right)

Citation: Acta Chromatographica Acta Chromatographica 30, 1; 10.1556/1326.2017.00189

On the one hand, extensive researches have been accomplished to investigate various bioactivities of Red Toon and possible regulatory mechanisms in recent decades. For instance, it has shown exceptionally strong effect against both 1,1-diphenyl-2-picryl-hydrazil (DPPH) radical scavenging and lipid peroxidation assays in a comparative assessment of anti-oxidant capacity among 127 common and underutilized vegetables [4]. In other studies, it was also found that Red Toon extract possessed effective anti-oxidant activity against multiple oxidative systems in vitro, including the scavenging of free and superoxide anion radicals, reducing power, and chelating metals [5, 6]. Moreover, it was well demonstrated that Red Toon had anti-oxidative effects in vivo, and the researchers proposed that it could be a promising natural additive to improve serum superoxide dismutase content [7, 8]. Moreover, previous studies showed that its crude extract had a strong anti-proliferative effect on non-small cell lung cancer by regulating the expression of several important factors, such as B-cell lymphoma-2 (Bcl2), Bcl2 Associated X (Bax), cyclin D1, and cyclin dependent kinase 4 (CDK4) [9].

In the past decade, great efforts have been made through phytochemical investigations and instrumental analyses to reveal the intrinsic chemical constituents responsible for these health benefits of this food [10, 11]. Twenty-three polyphenols have also been isolated and identified from the leaves, including 10 derivatives of gallic acid, 5 flavonoid aglycones, and 8 flavonol glycosides [12, 13]. Many of these compounds have shown favorable and powerful pharmacological effects on in vitro and in vivo models [14]. From the health point of view, it is an ideal dietary vegetable with natural antioxidants as well as a promising health-promoting food.

As a common knowledge, the geographic origins and harvest time can greatly influence the content and quality of bioactive chemical constituents in natural products [15]. Accordingly, in our previous studies, gallic acid, methyl gallate, and a few non-specific flavonoids such as rutin, kaempferol 3-O-β-d-glucopyranoside, and quercitrin, in Red Toon have been quantified by high-performance liquid chromatography (HPLC) [16, 17]. However, the existing analysis methods cannot well meet the high requirements for quality assessment of natural products. In this study, eight flavonol glycosides were isolated by column chromatography and subsequently identified by mass spectrometry (MS), proton nuclear magnetic resonance (1H-NMR), and carbon nuclear magnetic resonance (13C-NMR). Importantly, a rapid and convenient analysis method for determining the components in food materials, that is, ultra-performance liquid chromatography (UPLC) method was established to quantify the flavonol glycosides. Furthermore, linearity, precision, accuracy, stability, and repeatability of the established UPLC method were also validated and then used for quality assessment of Red Toon from various sources.

Experimental

Chemicals and Reagents

Silica gel (200–300 mesh, CP) and silica gel GF254 TLC plate were from Qingdao Haiyang Chemical Co. Ltd., China. D101 macroporous resin was from the Shanghai Mosutech Science Equipment Co. Ltd., China. C18 silica gel and C8 silica gel were the products from the SiliCycle Inc., Canada. Acetonitrile (ACN) of HPLC grade was purchased from Fisher Scientific, USA. Trifluoroacetic acid (TFA) was provided by Sigma-Aldrich Inc., USA. Methanol (MeOH) was from Merck KGaA, Germany. All the other chemicals used in the study were of analytical grade and obtained from Sinopharm Chemical Reagent Co., Ltd., China. Ultrapure water (>18.2 MΩ cm) was made by a Milli-Q Biocel system in our laboratory.

Instrumentation

A Shimadzu LC-20AP system equipped with a Thermo ODS column (10 mm × 250 mm, 10 μm) was used for semi-preparative HPLC separation of flavonol glycosides. Mobile phases consisting of solvent A (0.05% TFA in H2O) and solvent B (0.05% TFA in ACN) were pumped at a fixed flow rate of 5.0 mL min−1 through the column maintained at 35 °C. Time programs for gradient elution were as follows: 0–5 min, 5% B → 10% B; 5–15 min, 10% B; 15–75 min, 10% B → 30% B; 75–85 min, 30% B. The separation was monitored at 254 nm, and injection volume was 250 μL.

All mass spectra were taken under electrospray ionization (ESI) mode using a Thermo LXQ HPLC–ion trap–MS instrument via direct injection. 1H-NMR and 13C-NMR spectra were obtained by a Bruker AV-400 spectrometer using DMSO-d6 as solvent.

A Waters Acquity UPLC system was used for quantification, and data were acquired and processed using Empower software. The separation of samples was performed on an Acquity UPLC BEH Shield RP18 column (2.1 mm × 150 mm, 1.7 μm, Waters, Ireland) maintained at 35 °C, and mobile phases consisting of solvent A (0.05% TFA in ACN) and solvent B (0.05% TFA in H2O) were constantly pumped at a flow speed of 0.35 mL min−1. Time programs were as follows: 0–10 min, 18% B → 30% B; 10–17 min, 30% B. Ultraviolet (UV) wavelength was set at 350 nm, and 5 μL of solution was injected.

Sample Pretreatment

Ten Red Toon samples from various sources were harvested (Table 1), while a sample (code: G) was collected from March to October 2013, and their voucher specimens had been authenticated by Prof. Haile Ma and were deposited at School of Food and Biological Engineering, Jiangsu University, China. Additionally, the collected leaves were immediately dried under shade in a well-ventilated place to remove most of the moisture prior to further lyophilization for another 24 h. The raw material was then grounded into powder form and passed through a 40-mesh sieve. The fine powder obtained was stored in an electronic dry cabinet (relative humidity [RH], ≤40%) at room temperature.

Table 1.

Red Toon samples from various geographic origins

CodeGeographic originsHarvest time
CityProvince
AXipingHenanJul, 2013
BMianyangSichuanJul, 2013
CYantaiShandongAug, 2013
DYunchengShanxiSep, 2013
EZhoukouHenanAug, 2013
FXimouShandongAug, 2013
GZhenjiangJiangsuAug, 2013
HDanyangJiangsuAug, 2013
IWenxianHenanAug, 2013
JYunchengShandongAug, 2013

Extraction, Isolation, and Identification

Red Toon powders (1.80 kg) were macerated in 9.0 L of CH2Cl2 for 2 h at room temperature, followed by Soxhlet extraction overnight to remove non-polar and less polar constituents. The residual plant material was dried and then refluxed in 50% EtOH for 2 h (9.0 L × 3). The solutions collected were pooled after suction filtration, and EtOH was completely removed under reduced pressure by rotary evaporator below 50 °C. The concentrated aqueous phase was subsequently partitioned using EtOAc for five times. The pooled EtOAc extraction solution was then subject to removal of EtOAc by rotorvap and concentration to be dryness by lyophilization.

The resulting EtOAc extract (120 g) was then dissolved in boiling water, and the aqueous solution was loaded into D101 macroporous resin (3.0 kg) column which was flushed with H2O and 20%–80% EtOH (v/v) sequentially. Both 40% EtOH and 60% EtOH elution were combined, concentrated, and dried to give out 25.2 g crude flavonoids, and 10.0 g of which was then separated on silica gel (1.50 kg) column by gradient CH2Cl2–MeOH–H2O (10:1:0.1–5:1:0.1, v/v/v; approximately 5 L for each gradient), C18 or C8 (200 g) column by gradient MeOH–H2O (20:80–100:0, v/v), and the aforementioned semi-preparative HPLC successively. The corresponding eluent was collected to yield eight amorphous yellow powders, I (20.3 mg), II (25.6 mg), III (33.3 mg), IV (105.0 mg), V (36.5 mg), VI (47.4 mg), VII (163.4 mg), and VIII (104.0 mg). Spectra of compounds IVIII were recorded by MS and NMR.

Optimization of Extraction Conditions

To optimize the extraction conditions of flavonol glycosides, L9(3)4 orthogonal experiment was designed and performed, in which four major affecting factors including extraction duration, solid–liquid ratio, MeOH concentration, and extraction method were investigated. The peak area of total flavonoid glycosides was used as the index of extraction efficiency. After optimization, the best condition was applied to prepare sample solution used for subsequent UPLC analysis. Kn (n = 1, 2 or 3) and R values are calculated according to the following equations:

Kn=thesumof peak area fromafactoratleveln/the number of experimentsatthe level
R=the maximumKnvaluethe minimumKnvalue

Preparation of Mixed Standard Solutions

Eight flavonol glycosides (14.50 mg for I, 1.30 mg for II, 1.30 mg for III, 16.85 mg for IV, 1.80 mg for V, 4.20 mg for VI, 39.55 mg for VII, and 23.15 mg for VIII) were precisely weighed and then dissolved in 60% MeOH and scaled to 250 mL as stock solution, and a series of dilutions was performed to prepare standard solutions at 1/2, 1/4, 1/8, 1/16, 1/32, and 1/64 of the original concentration of stock solution, respectively. Prior to any injection, all of the solutions were filtered through a 0.20 μm polytetrafluoroethylene (PTFE) membrane syringe.

UPLC Method Development

As shown in Figure 2, these flavonol glycosides have high similarity in their chemical structures, leading to analogous chromatographic behavior. To achieve an efficient separation of the analytes, UPLC conditions including column (Acquity BEH Shield RP18, 2.1 mm × 150 mm, 1.7 μm; Cortecs C18, 2.1 mm × 100 mm, 1.6 μm; and Acquity BEH C18, 2.1 mm × 150 mm, 1.7 μm), mobile phases (MeOH–H2O, ACN–H2O, and ACN–H2O containing 0.05%, 0.10%, or 0.30% TFA), column temperature (25 °C, 30 °C, and 35 °C), and flow rate (0.30 mL min−1, 0.35 mL min−1, and 0.40 mL min−1) were optimized for the purpose of high resolution, good peak shape, and reasonable analytical time.

Figure 2.
Figure 2.

Chemical structures of compounds IVIII isolated from Red Toon

Citation: Acta Chromatographica Acta Chromatographica 30, 1; 10.1556/1326.2017.00189

UPLC Method Validation

Linearity

The UPLC method to detect these flavonol glycosides was verified using the external standard method. To determine linearity, a series of prepared standard solutions was analyzed and the peak area (Y) versus the concentration of standard (X) was then plotted for the calibration curves by ordinary linear regression using Microsoft Excel.

Precision

The intra-day precision experiment was carried out by continuous six measurements of mixed standard solutions at highest, middle, and lowest concentrations, and a sample solution was freshly prepared within a single day. The inter-day precision was also determined by analyzing both the standard solutions and sample solution on three consecutive days as well. The relative standard deviation percentage (RSD, %) of their peak areas was then calculated.

Accuracy

The standards dissolved in 60% MeOH were added into known amounts of the samples with proper volume of 60% MeOH, making up to the desired volume. The sample solutions were prepared and analyzed using the developed method, and experiments were performed at each level in triplicate. The percentage recoveries were calculated based on the following formula: (detected amount − original amount)/spiked amount × 100.

Stability

The stability was studied based on the determination within 2 days. In details, a freshly prepared sample solution was injected immediately and then loaded after being stored at room temperature under shade for 2 h, 4 h, 6 h, 8 h, 24 h, 36 h, and 48 h. Then, the RSDs of their peak areas of were calculated.

Repeatability

Six aliquots of Red Toon sample solution were concurrently prepared and consecutively analyzed using the proposed method to evaluate the repeatability of the proposed method. Then, the RSDs of their contents were calculated.

Quality Assessment of Red Toon from Various Sources

The developed UPLC method was employed for the quality assessment of Red Toon from various sources based on the quantification of these eight flavonol glycosides in the leaves. In details, the samples harvested in continuous months (March 2013 to October 2013, 1 month interval, Jiangsu University campus) and cultivated in ten geographic origins were analyzed.

Results and Discussion

Isolation and Identification

After isolation and purification of Red Toon extract using open tubular column chromatography and semi-preparative HPLC, followed by chemical reactions, and 1H-NMR, 13C-NMR, and ESI–MS tests, eight compounds were identified as follows:

Rutin (I): pale yellow powder, positive HCl–Mg and Molisch reaction; 1H-NMR (DMSO-d6, 400 MHz) δ: 12.58 (1H, 5-OH), 7.54 (2H, brs, H-2′, 6′), 6.82 (1H, s, H-5′), 6.35 (1H, d, J = 2.0 Hz, H-8), 6.17 (1H, brs, H-6), 5.31 (2H, brs, H-l″), 5.07 (2H, brs, H-1″), 0.97 (3H, s, H-6″′).

Myricitrin (II): yellow powder, positive HCl–Mg and Molisch reaction; 1H-NMR (DMSO-d6, 400 MHz) δ: 12.67 (1H, s, 5-OH), 6.90 (2H, s, H-2′, 6′), 6.39 (1H, d, J = 2.0 Hz, H-6), 6.15 (1H, J = 2.0 Hz, H-8), 5.24 (1H, brs, Rha-H-1″), 3.77 (1H, dd, J = 9.4, 2.9 Hz, Rha-H-3″), 3.31 (1H, t, J = 9.5 Hz, Rha-H-4″).

Quercetin-3-O-β-d-galactoranoside (III): pale yellow powder; ESI–MS m/z: 927 [2M-H], 463 [M-H], 301 [M-Gal-H]; 1H-NMR (DMSO-d6, 400 MHz) δ: 7.68 (1H, dd, J = 8.4, 1.8 Hz, H-6′), 7.54 (1H, d, J = 1.8 Hz, H-2′), 6.80 (1H, d, J = 8.4 Hz, H-5′), 6.37 (1H, d, J = 1.8 Hz, H-8), 6.17 (1H, d, J = 1.8 Hz, H-6), 5.36 (1H, d, J = 1.8 Hz, H-1″), 3.17–3.66 (6H, m, H-2″, 6″).

Quercetin-3-O-β-d-glucopyranose (IV): yellow powder; UV (MeOH) nm: 255, 353; ESI–MS m/z: 927 [2M-H], 463 [M-H], 300 [M-H-Glc]; 1H-NMR (DMSO-d6, 400 MHz) δ: 12.65 (1H, s, 5-OH), 10.88 (1H, s, 4′-OH), 9.73 (1H, s, 7-OH), 9.24 (1H, s, 3′-OH), 6.19 (1H, d, J = 2.0 Hz, H-6), 6.41 (1H, d, J = 2.0 Hz, H-8), 6.84 (1H, d, J = 6.9 Hz, H-5′), 7.57 (1H, d, J = 2.0 Hz, H-2′), 7.58 (1H, dd, J = 2.0, 6.9 Hz, H-6′), 5.43 (1H, d, J = 7.3 Hz, H-1″).

Quercetin-3-O-α-l-arabinopyranoside (V): yellow powder; UV (MeOH) nm: 255, 354; ESI–MS m/z: 433 [M-H], 301 [M-Ara-H]; 1H-NMR (DMSO-d6, 400 MHz) δ: 7.57 (1H, d, J = 2.1 Hz, H-2′), 7.62 (1H, dd, J = 2.1, 8.0 Hz, H-6′), 6.85 (1H, d, J = 8.0 Hz, H-5′), 6.42 (1H, d, J = 2.4 Hz, H-8), 6.21 (1H, d, J = 2.4 Hz, H-6), 5.39 (1H, d, J = 5.2 Hz, H-1″), 3.72 (1H, m, H-2″), 3.65 (1H, m, H-3″), 3.53 (1H, m, H-5″).

Astragalin (VI): yellow powder; positive HCl–Mg reaction; 1H-NMR (DMSO-d6, 400 MHz) δ: 12.55 (1H, s, 5-OH), 8.03 (2H, d, J = 8.7 Hz, H-2′, 6′), 6.86 (2H, d, J = 8.7 Hz, H-3′, 5′), 6.36 (1H, d, J = 2.0 Hz, H-8), 6.18 (1H, d, J = 2.0 Hz, H-6), 5.42 (1H, d, J = 7.5 Hz, H-1″).

Quercetin-3-O-α-l-rhamopyranoside (VII): yellow powder; UV (MeOH) nm: 254, 348; ESI–MS m/z: 895 [2M-H], 447 [M-H], 301 [M-Rha-H]; 1H-NMR (DMSO-d6, 400 MHz) δ: 7.27 (1H, brs, J = 2.1 Hz, H-2′), 7.23 (1H, d, J = 8.0 Hz, H-6′), 6.86 (1H, d, J = 8.0 Hz, H-5′), 3.97 (1H, brs, H-2″), 6.38 (1H, brs, H-8), 6.18 (1H, brs, H-6), 5.22 (1H, brs, H-1″), 3.49–3.51 (1H, m, H-3″), 3.12~3.16 (2H, m, H-4″, 5″), 0.79 (3H, d, J = 6.0 Hz, H-6″); 13C-NMR (DMSO-d6, 100 MHz) δ: 178.2 (CO), 164.9 (C-7), 161.8 (C-5), 157.7 (C-9), 156.9 (C-2), 148.9 (C-4′), 145.7 (C-3′), 134.7 (C-3), 121.6 (C-1′), 121.2 (C-6′), 116.1 (C-5′), 115.9 (C-2′), 104.5 (C-10), 102.3 (C-1″), 99.2 (C-6), 94.1 (C-8), 71.7 (C-4″), 71.0 (C-3″), 70.8 (C-2″), 70.5 (C-5″), 17.9 (C-6″).

Kaempferol-3-O-α-l-rhamopyranoside (VIII): UV (MeOH) nm: 263, 342; ESI–MS m/z: 862 [2M-H], 431 [M-H], 285 [M-Rha-H]; 1H-NMR (DMSO-d6, 400 MHz) δ: 7.66 (2H, d, J = 8.0 Hz, H-2′, 6′), 6.86 (2H, d, J = 8.0 Hz, H-3′, 5′), 5.92 (1H, brs, H-8), 5.78 (1H, brs, H-6), 5.28 (1H, brs, H-1″), 3.97 (1H, brs, H-2″), 3.46 (1H, d, J = 8.0 Hz, H-3″), 3.06–3.14 (2H, m, H-4″, 5″), 0.77 (3H, d, J = 6.0 Hz, H-6″); 13C-NMR (DMSO-d6, 100 MHz) δ: 176.3 (CO), 161.3 (C-7), 160.4 (C-5, 4′), 157.7 (C-9), 155.5 (C-2), 133.8 (C-3), 130.7 (C-2′, 6′), 121.3 (C-1′), 115.8 (C-3′, 5′), 101.8 (C-10), 101.8 (C-1″), 71.6 (C-4″), 70.8 (C-2″, 3″), 70.6 (C-5″), 17.9 (C-6″).

Extraction Condition

The design and results of L9(3)4 orthogonal experiments were presented in Table 2, showing the extraction efficiency obtained under nine different conditions. K and R values were calculated and listed, which indicated that solid–liquid ratio was the most important factor in comparison with other three factors. According to the R values, the effect of each factor on the extraction efficiency was decreased in the order: B > C > D > A. In addition, it was suggested that the highest extraction yield would be achieved under the optimum conditions A2B3C3D1, namely, 250 mg of Red Toon powder was sonicated in 10.0 mL of 60% MeOH for 30 min.

Table 2.

Results and analysis of L9(3)4 orthogonal experiments

No.Extraction duration (A, min)Solid–liquid ratio (B, g/mL)MeOH concentration (C, %)Extraction method (D)Peak area
1151:1020Ultrasonic770048
2151:2040Vortex1968515
3151:4060Reflux2521656
4301:1040Reflux1121311
5301:2060Ultrasonic2465624
6301:4020Vortex2012406
7451:1060Vortex1071583
8451:2020Reflux1688284
9451:4040Ultrasonic2395909
K1175340698764714902461877194
K21866447204080718285781684168
K31718592230999020196211777083
R1478551322343529375193026

Method Validation

Linearity

As shown in Table 3, high correlation coefficients (r2 > 0.9990) of the calibration curves were obtained, which demonstrated that there were good linear relationships between the peak areas and the concentrations of these eight flavonol glycosides.

Table 3.

Calibration curves of eight flavonol glycosides

AnalytesRegression equationsr2Linearity range (μg mL−1)
IY = 17,846,994.7975X − 2855.93681.00000.906–58.0
IIY = 18,181,045.8308X − 15.66671.00000.0813–5.20
IIIY = 17,510,327.8403X + 510.61490.99990.0813–5.20
IVY = 19,544,405.7695X − 5127.88511.00001.05–67.4
VY = 16,102,734.1350X1.00000.113–7.20
VIY = 23,225,706.4419X − 1793.71260.99920.263–16.8
VIIY = 21,648,729.3620X − 7446.43100.99922.47–158
VIIIY = 19,831,426.2776X − 6003.79311.00001.45–92.6

Precision

The results of the intra-day and inter-day precision test were shown in Table 4. The RSDs of the peak areas were less than 2%, indicating that the analysis of these eight flavonol glycosides was precise.

Table 4.

Precision, stability, and repeatability of eight flavonol glycosides

AnalytesIntra-day precision (n = 6, RSD, %)Inter-day precision (n = 3, RSD, %)Repeatability (n = 6, RSD, %)Stability (n = 8, RSD, %)
Standard solutionSample solutionStandard solutionSample solution
LMHLMH
I1.171.641.060.581.971.670.470.900.640.52
II0.832.030.281.081.361.650.321.671.271.64
III1.271.150.200.591.591.770.290.820.760.49
IV0.830.781.390.191.841.000.400.290.540.85
V1.451.191.680.711.391.820.231.391.850.84
VI1.850.500.970.170.931.350.260.470.140.46
VII0.750.150.140.170.351.920.280.350.360.29
VIII1.250.220.140.100.680.910.100.560.210.57

Accuracy

The results obtained from recovery test have been summarized in Table 5. It can be seen that the percentage recoveries of these eight flavonol glycosides were both within the range of 96.3%–105.0%, with the RSD values ranging from 1.68% to 3.97%, verifying the good accuracy of the proposed UPLC method.

Table 5.

Results of recovery test of eight flavonol glycosides (n = 3)

AnalytesLowMiddleHigh
Added (μg)Recovery (%)RSD (%)Added (μg)Recovery (%)RSD (%)Added (μg)Recovery (%)RSD (%)
I19.4096.303.0738.8098.902.9458.20102.12.45
II9.35105.02.6218.70103.53.6628.0599.802.78
III14.50101.22.8829.0097.902.4043.5098.602.71
IV75.0098.601.78150.0101.31.87225.0101.51.86
V10.66103.93.9721.32102.42.9631.9897.602.80
VI22.48100.52.5544.96102.21.8467.4498.903.06
VII650.097.703.03130097.301.961950103.21.68
VIII270.0103.42.42540.0100.62.81810.096.403.74

Stability

As shown in Table 4, the RSD was ranged from 0.29% to 1.64%, suggesting that the sample solution was stable at room temperature within 48 h.

Repeatability

The results of repeatability were shown in Table 4. The RSD of the contents of eight flavonol glycosides in the vegetable was from 0.14% to 1.85%, demonstrating a good repeatability of the developed analytical method.

UPLC Profile of Eight Flavonoid Glycosides

After optimization of UPLC chromatographic conditions, both mixed standard solution and sample solution were analyzed. The chromatograms were presented in Figure 3. It can be seen that eight flavonol glycosides have been well separated within 18 min by the newly proposed UPLC method, showing that it was much more efficient than the HPLC method developed in our previous studies for individual qualification of rutin, kaempferol 3-O-β-d-glucopyranoside, or quercitrin in Red Toon.

Figure 3.
Figure 3.

UPLC chromatograms of mixed standard solution (A) and sample solution (B)

Citation: Acta Chromatographica Acta Chromatographica 30, 1; 10.1556/1326.2017.00189

Quality Assessment of Red Toon from Different Sources

As shown in Table 6 and Figure 4, the contents of eight beneficial flavonol glycosides differed largely in Red Toon harvested from continuous 8 months in a same production site. The total amount of glycosides in the food began to accumulate quickly from March in the early half of the year. Afterwards, the content was maintained at a much higher level from April to June, followed by a sharp decrease to about 1.0% until October. In addition, it was noticed that the change in the content of compound VI (astragalin) presented almost the same trend as the total flavonol glycosides over 8 months, and the content of compounds IV was kept at a lower level within the same period.

Table 6.

Content of eight flavonol glycosides in Red Toon harvested in different months (n = 3)

AnalytesMean (mg g−1, n = 3)
MarchAprilMayJuneJulyAugustSeptemberOctober
I0.1310.1820.0620.1050.0270.0470.0450.040
II0.0080.0110.0060.0090.0020.0160.0130.023
III0.0390.0580.0280.0600.0150.0220.0180.023
IV0.2010.3500.1480.3050.0810.1210.1000.115
V0.0170.3370.0110.0190.0070.0070.0080.010
VI0.0860.6550.8820.9320.0600.0240.0370.050
VII0.6630.7200.7180.7470.3670.5470.4290.382
VIII0.6210.2520.7460.4870.4650.2440.3370.307
Total1.7662.5652.6012.6641.0241.0270.9870.940
Figure 4.
Figure 4.

Total content of eight flavonol glycosides of Red Toon harvested in different months (A) and collected from various geographic origins (B)

Citation: Acta Chromatographica Acta Chromatographica 30, 1; 10.1556/1326.2017.00189

The contents of eight flavonol glycosides in Red Toon collected from ten geographic origins were also determined. It can be seen that the total amount of them in the vegetable produced from Shandong Yantai (C) was the highest (2.815%), with Shandong Ximou (F) the second highest at 2.074% (Table 7), supporting a well-recognized viewpoint that Red Toon cultivated in Shandong Province is considered genuine due to the best health benefits and the flavor of its leaves steadily exhibited in the consumption over a very long period. Furthermore, the change on the content of quercetin-3-O-α-l-rhamopyranoside in Red Toon harvested in different areas has exhibited almost the same trend as the total flavonol glycosides, which suggested that it could represent the other compounds to be used to distinguish the geographic origins of Red Toon.

Table 7.

Content of eight flavonol glycosides in Red Toon from various geographic origins (n = 3)

AnalytesMean (mg g−1, n = 3)
ABCDEFGHIJ
I0.6400.0310.1600.0290.0420.0780.0470.0900.0410.023
II0.0120.0150.0180.0040.0040.0300.016N.D.0.0140.003
III0.1110.0230.0540.0200.0320.0210.0220.0370.0350.018
IV0.3580.1200.4590.1070.1500.1350.1210.1930.1500.071
V0.0670.0170.0550.0110.0230.0170.0070.0210.0220.012
VI0.1040.0360.0570.0100.1800.0160.0240.1030.0270.011
VII0.4151.0401.5201.2700.7351.4420.5470.7700.7260.514
VIII0.1530.4320.3300.1540.1800.3350.2440.7950.1790.059
Total1.2801.7142.8151.6401.3462.0741.0281.9281.1940.711

As we all know, intrinsic enzymes play an important role in the synthesis and hydrolysis of chemical constituents in plant during its lifespan. Therefore, exploration on the enzymes in Red Toon has attracted attention and is ongoing in our research facility by using prevailing proteomics research technologies such as MALDI–TOF/TOF–MS, NanoLC-Orbitrap MS, peptides mass fingerprinting, and de novo sequencing [18, 19]. In addition to chemical hydrolysis of these glycosides to their aglycones, the potential relationship between the bioactive flavonol glycosides and relevant enzymes and possible conversion mechanisms in biosynthesis and hydrolysis will be investigated in further research [20, 21].

Conclusion

Isolation, identification, and quantification of chemical constituents from Red Toon were conducted. Eventually, eight flavonol glycosides were obtained. Afterwards, a UPLC–UV method was developed to determine these compounds in the leaves and a validation was further fulfilled, which was well demonstrated to be a reliable and convenient analytical method in this application. Then, based on the difference in the content of eight flavonol glycosides, the quality of Red Toon harvested from ten geographic origins or eight months was assessed and compared, suggesting that harvest time is preferred to be from April to June and Shandong Province in China is the best production site for this vegetable.

Acknowledgments

The authors gratefully acknowledge the financial supports provided by the National Natural Science Foundation of China (81303313 and 81303174), the China Postdoctoral Science Foundation (2016M590425), the Program for Graduate’s Innovative Research of Jiangsu (CXLX13_691), and the Program for Undergraduate's Scientific Research of Jiangsu University (15A156).

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If the inline PDF is not rendering correctly, you can download the PDF file here.

  • 1.

    Yang, H.; Gu, Q.; Gao, T.; Wang, X.; Chue, P.; Wu, Q.; Jia, X. Pharmacogn. Mag. 2014 , 10 , 185 190 .

  • 2.

    Yang, H.; Gu, Q.; Xu, Y.; Cheng, B.; Chue, P.; Wu, Q.; Shen, Y. J. Liq. Chromatogr. Relat. Technol. 2015 , 38 , 687 691 .

  • 3.

    Chen, H. M.; Wu, Y. C.; Chia, Y. C.; Chang, F. R.; Hsu, H. K.; Hsieh, Y. C.; Chen, C. C.; Yuan, S. S. Cancer Lett. 2009 , 286 , 161 171 .

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4.

    Yang, R.; Tsou, S.; Lee, T.; Wu, W.; Hanson, P.; Kuo, G.; Eagle, L. M.; Lai, P. J. Sci. Food Agric. 2006 , 86 , 2395 2403 .

  • 5.

    Hseu, Y. C.; Chang, W. H.; Chen, C. S.; Liao, J. W.; Huang, C. J.; Lu, F. J.; Chia, Y. C.; Hsu, H. K.; Wu, J. J.; Yang, H. L. Food Chem. Toxicol. 2008 , 46 , 105 114 .

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6.

    Yang, H. L.; Chen, S. C.; Lin, K. Y.; Wang, M. T.; Chen, Y. C.; Huang, H. C.; Cho, H. J.; Wang, L.; Kumar, K. J.; Hseu, Y. C. J. Ethnopharmacol. 2011 , 137 , 669 680 .

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7.

    Chen, G. H.; Huang, F. S.; Lin, Y. C.; Hsu, C. K.; Chung, Y. C. J. Funct. Foods 2013 , 5 , 773 780 .

  • 8.

    Lin, M. J.; Chang, S. C.; Jea, Y. S.; Liao, J. W.; Fan, Y.K.; Lee, T. T. J. Appl. Anim. Res. 2016 , 44 , 395 402 .

  • 9.

    Hseu, Y. C.; Chen, S. C.; Lin, W. H.; Hung, D. Z.; Lin, M. K.; Kuo, Y. H.; Wang, M. T.; Cho, H. J.; Wang, L.; Yang, H. L. J. Ethnopharmcol. 2011 , 134 , 111 121 .

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    Cheng, K. W.; Yang, R. Y.; Tsou, S. C. S.; Lo, C. S. C.; Ho, C. T.; Lee, T. C.; Wang, M. J. Funct. Foods 2009 , 1 , 253 259 .

  • 11.

    Wu, J. G.; Peng, W.; Yi, J.; Wu, Y. B.; Chen, T. Q.; Wong, K. H.; Wu, J. Z. J. Ethnopharmacol. 2014 , 154 , 198 205 .

  • 12.

    Wang, K. J.; Yang, C. R.; Zhang, Y. J. Food Chem. 2007 , 101 , 365 371 .

  • 13.

    Zhang, W.; Li, C.; You, L. J.; Fu, X.; Chen, Y. S.; Luo, Y. Q. J. Funct. Foods 2014 , 10 , 427 435 .

  • 14.

    Shen, Y. P.; Yin, H. W.; Chen, B.; Xia, G. H.; Yang, H.; Jia, X. B. Pharmacogn. Mag. 2012 , 8 , 49 53 .

  • 15.

    Sun, J.; Li, Y. Z.; Ding, Y. H.; Wang, J.; Geng, J.; Yang, H.; Ren, J.; Tang, J. Y.; Gao, J. Brain Res. 2014 , 1589 , 126 139 .

  • 16.

    Chen, B.; Ma, L.; Wang, X.; Shen, Y.; Jia, X. Pharmacogn. Mag. 2013 , 9 , 103 108 .

  • 17.

    Shen, Y. P.; Yang, H.; Xia, G. H.; Wang, J. J.; Cai, B. C.; Jia, X. B. Acta. Chromatogr. 2013 , 25 , 687 701 .

  • 18.

    Sun, X. X.; Zhang, L. T.; Cao, Y. Q.; Gu, Q. Y.; Yang, H.; Tam, J. P. Pharmacogn. Mag. 2016 , 12 , 270 276 .

  • 19.

    Yang, H.; Shen, Y. P.; Xu, Y.; Maqueda, A. S.; Zheng, J.; Wu, Q. N.; Tam, J. P. Int. J. Nanomed. 2015 , 10 , 4947 4955 .

  • 20.

    Yang, H.; Yin, H. W.; Shen, Y. P.; Xia, G. H.; Zhang, B.; Wu, X. Y.; Cai, B. C.; Tam, J. P. J. Clean. Prod. 2016 , 131 , 10 19 .

  • 21.

    Yang, H.; Yin, H. W.; Wang, X. W.; Li, Z. H.; Shen, Y. P.; Jia, X. B. Pharmacogn. Mag. 2015 , 11 , 636 642 .

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