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Page 1 of 36 Analytical Methods
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DOI: 10.1039/C8AY00661J
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4 1 Evaluation of colorimetric methods for quantification of
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6 2 citrus flavonoids to avoid misuse
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3 Rui Huang, Wenyan Wu, Shuyu Shen, Jiawen Fan, Yue Chang, Shiguo Chen* and
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11 4 Xingqian Ye*
Published on 11 May 2018. Downloaded by University of Saskatchewan on 11/05/2018 15:18:49.
Analytical Methods Accepted Manuscript

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13 5 Zhejiang University, Department of Food Science and Nutrition, Zhejiang Key
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16 6 Laboratory for Agro-Food Processing, Fuli Institute of Food Science, Hangzhou
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18 7 310058, China
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21 8 * Corresponding author: Shiguo Chen and Xingqian Ye, College of Biosystem
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23 9 Engineering and Food Science, Zhejiang University, Hangzhou 310058, China.
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26 10 E-mail: chenshiguo210@163.com, Tel: 86-571-88982151.; psu@zju.edu.cn.
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Analytical Methods Page 2 of 36
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4 23 Abstract: The content of flavonoids is important for evaluating the quality of
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6 24 citrus-related products. However, due to the different characteristics of the structures
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25 of flavonoids and the lack of a universal method, many methods for quantification
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11 26 have been misused. Here, we used twenty-one citrus flavonoid standards to evaluate

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13 27 four widely used colorimetric methods, and seven species of citrus as natural samples
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16 28 were used to verify the evaluation. The results indicated that NaNO2-Al(NO3)3-NaOH
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18 29 and AlCl3 methods could only detect some of the flavonoids and were inadequate to
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21 30 estimate the total flavonoid contents, and the 2,4-dinitrophenylhydrazine (2,4-DNPH)
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23 31 method might be affected by other carbonyl compounds in extracts. The Davis
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26 32 method was modified with naringin as the standard at the measuring wavelength of
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28 33 420 nm for grapefruit and pomelo, and with hesperidin as the standard at the
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34 measuring wavelength of 360 nm for other species. It was proved to be the most
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33 35 reliable method for the quantification of citrus flavonoids and was also recommended
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35 36 for laboratories and factories where needed.
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40 38 Keywords: Citrus flavonoids; quantification; colorimetric methods; misuse; HPLC;
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43 39 modified Davis method
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4 45 1. Introduction
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6 46 Citrus is a group of fruit crops belonging to the genus Citrus L of the Rutaceae
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47 family 1, mainly containing sweet orange, mandarin, grapefruit and pomelo, lemon,
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11 48 hybrid citrus, and kumquat, etc. The flavonoids in citrus fruits contribute to

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13 49 multi-health-promoting benefits, including the prevention of atherosclerosis 2, 3
,
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15 4, 5
16 50 neurodegenerative disease , cancer 6, and anti-inflammatory diseases 7as well as
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18 51 antimicrobial activity 8. Thus, the quantification of flavonoids is very important for
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21 52 the quality control of citrus and its derived products.
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23 53 To date, the analytical methods for the quantification of citrus flavonoids have
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26 54 mainly included spectrometric methods 9, 10, high performance liquid chromatography
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28 55 (HPLC) 11and thin layer chromatography 12
. Although HPLC coupled with a diode
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56 array detector (DAD) and mass spectrometer could provide specific information for
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33 57 the identification and quantification of each individual flavonoid in citrus, this method
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35 58 generally requires expensive equipment, various individual analytical standards and
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38 59 large amounts of time. UV/VIS spectrophotometric methods, due to their convenience,
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40 60 rapidity, repeatability, and reproducibility, are more appropriate for routine analyses
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43 61 and are important for quality control in citrus products 13. However, there are no
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45 62 universal methods suggested for estimating flavonoids 14
, which leads to severe
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48 63 problems for quality evaluation in real products. The methods that have been widely
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50 64 used in the quantification of citrus flavonoids include NaNO2-Al(NO3)3-NaOH,
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65 aluminum chloride and Davis methods (Table 1).
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55 66 Actually, these methods, based on different colorimetric mechanisms, are not
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4 67 always suitable for detecting citrus flavonoids. In addition, a number of different
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6 68 standards that are not the main flavonoids in citrus have been misused in the testing,
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69 which leads to the results of detection far from the actual content. For example, Lou et
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11 70 al. showed that the total flavonoid contents of immature and mature kumquat

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13 71 extracted in hot water were 326 QE mg/100g DW and 153 QE mg/100g DW,
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16 72 respectively, as determined by the aluminum chloride method. While the results
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18 73 detected by HPLC were 2990 mg/100g DW and 1768 mg/100g DW, which is ten
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21 74 times greater than that of the aluminum chloride method.
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23 75 Flavonoids are the major phenolic compounds belonging to secondary
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26 76 metabolites in citrus. They commonly share the same basic skeleton, the flavan
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28 77 nucleus, consisting of two aromatic rings (A and B) bound together by three carbon
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78 atoms and an oxygen atom that form a pyran ring (C) 1. Various flavonoids derived
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33 79 from the skeleton with different molecular structures can be divided into six classes:
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35 80 flavones, flavanones, flavonols, isoflavones, anthocyanidins, and flavanols 31. The
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38 81 differences in structures led to a different performance in colorimetry, which may
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40 82 affect the identification and quantification of total flavonoid contents. For example,
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43 83 the NaNO2-Al(NO3)3-NaOH method with rutin or catechin as standard and 510 nm as
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45 84 detection wavelength to determine total flavonoid content has been the most widely
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48 85 used method. Based on the principle of this method that only components with
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50 86 catechol hydroxyl groups can be detected, many flavonoids abundant in citrus without
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87 o-diphenols like hesperidin and naringin may not be detected, while some phenolic
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55 88 acids with catechol hydroxyl groups like caffeic acid and chlorogenic acid may
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4 89 exhibit considerable absorbance at 510 nm ; therefore, this method has low
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6 90 specificity. The main flavonoids identified in citrus fruits are flavanones, flavones,
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91 and flavonols, which vary in content among citrus species. Thus, choosing suitable
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11 92 methods and standards appears highly important for the colorimetric quantification of

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13 93 citrus flavonoids.
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16 94 Recently, the 2,4-DNPH method has been used to determine the content of
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18 95 flavanone in propolis 33; here, we used this method to detect citrus flavonoids, which
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21 96 consist mostly of flavanones. In this paper, twenty-one citrus flavonoid standards
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23 97 would be selected to evaluate and discuss the specificity of these four methods. Seven
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26 98 species of citrus as natural samples and HPLC as the reference method were chosen to
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28 99 verify the evaluation. The most reliable method would be recommended to avoid
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100 misuse in publications and industrial production.
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33 101 2. Experimental
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35 102 2.1. Samples
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38 103 Flavonoid analytical standards including hesperidin, narirutin, naringin,
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40 104 neohesperidin, poncirin, hesperetin, (±)-naringenin, eriodictyol, luteolin, apigenin,
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43 105 quercetin, kaempferol, quercitrin, and diosmetin were obtained from Sigma-Aldrich
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45 106 (St. Louis, MO, USA). Eriocitrin, nobiletin, tangeretin, rhoifolin, and sinensetin were
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48 107 acquired from Aladdin Chemical Reagent Co., Ltd. (Shanghai, China). Didymin was
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50 108 gained from J&K Scientific Ltd. (Beijing, China). Rutin was bought from Shanghai
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109 Chemical Reagent Co., Ltd. The specific information of twenty-one flavonoid
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55 110 standards is listed in Table 2. Seven species of citrus were purchased from Walmart in
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4 111 Hangzhou, Zhejiang; the detailed information is shown in Table S1.
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6 112 2.2. Chemicals and reagents
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113 HPLC-grade methyl alcohol was purchased from Sigma-Aldrich (St. Louis, MO,
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11 114 USA). The other reagents used were of analytical grade, and were purchased from the

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13 115 Sinopharm Chemical Reagent Co., Ltd.
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16 116 2.3. Sample pretreatment
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18 117 Citrus was divided into peel, pulp, and seed. The peel was dried using a drying
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21 118 oven at 60°C for about 48 h until the moisture levels became constant. The pulp was
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23 119 homogenized with a squeezer and then freeze-dried. The moisture content of each
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26 120 sample was recorded in Table S1. All of the dried peel and pulp was ground and
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28 121 sieved through a 40-mesh sieve. The powders were stored at -20°C until analysis.
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122 2.4. Extraction of flavonoids from citrus
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33 123 The extraction procedure was conducted according to the method described by
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35 124 Xi et al. 34 and some modifications were made. 2 g of peel or 4 g of pulp was extracted
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38 125 using 30 mL of methanol in a cylindrical glass reactor (inner diameter about 2.90 cm)
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40 126 with the help of ultrasound treatment (150 W, 50°C) for 30 min and then kept at room
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43 127 temperature for 12 h. After being centrifuged at 4500 g for 15 min at 4°C, the extract
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45 128 was decanted. The same procedure was repeated three times and the extracted
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48 129 supernatant was mixed and diluted with methanol to 100 mL and then stored at -20°C.
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50 130 2.5. The determination of total flavonoid content
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131 The total flavonoid content was determined by four widely applied colorimetric
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55 132 methods based on different principles and then compared with that of HPLC. All
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4 133 analyses were carried out in triplicate.
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6 134 2.5.1. The NaNO2-Al(NO3)3-NaOH colorimetric method
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135 The NaNO2-Al(NO3)3-NaOH colorimetric method was modified from the
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11 136 procedure reported by Navarro et al. 20. Briefly, 1 mL of extract was mixed with 4 mL

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13 137 of 30% ethanol and 0.3 mL of NaNO2 (5%, w/v). After 5 min of reaction, the mixture
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16 138 was reacted with 0.3 mL of Al(NO3)3 (10%, w/v) for 6 min. Then, 4mL of 1M NaOH
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18 139 was added and the mixture was adjusted to 10 mL with 0.4 mL of 30% ethanol. After
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21 140 incubation at room temperature for 10 min, the absorbance was read at 510 nm. The
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23 141 amounts of Al(NO3)3 and NaOH solutions were substituted by the same amount of 30%
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26 142 ethanol in blank. Similarly, 1 mL of twenty-one flavonoid standard solutions (100
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28 143 ppm) were reacted in the same way. The total flavonoid content of samples was
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144 expressed in rutin equivalent, and the calibration curve ranged from 0–500 µg mL-1.
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33 145 2.5.2. The aluminum chloride method
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35 146 The aluminum chloride method used was derived from the procedure described
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38 147 by Assefa et al. 9 with some modifications. Here, 1.5 mL of methanol was added to
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40 148 0.5 mL of the extract, followed by mixing with 0.1 mL of 10% AlCl3, 0.1 mL of 1 M
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43 149 potassium acetate and 2.8 mL of methanol. The mixture was vigorously shaken and
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45 150 left at ambient temperature for 30 min, then the absorbance was measured at 415 nm.
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48 151 The same procedure was conducted for 21 flavonoid standard solutions (100 ppm).
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50 152 The blank was prepared by replacing aluminum chloride with the same volume of
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153 methanol, and quercetin (concentration range from 0 to 100 µg/mL) was chosen as the
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55 154 reference standard to express the result.
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4 155 2.5.3. The modified Davis spectrophotometric method
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6 156 The Davis method described by Kim et al. was modified as the current
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157 procedure. 1 mL of diluted extract was mixed separately with 4 mL of distilled water,
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11 158 5 mL of diethylene glycol (90%) and 0.1 mL of 4 M NaOH. After 10 min water bath

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13 159 at 40°C and cooling to room temperature, the absorbance was detected at 360 nm and
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16 160 420 nm, respectively. The blank was comprised of the mixture without the addition of
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18 161 NaOH solution. This method was also used for analyzing 21 flavonoid standard
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21 162 solutions (200 ppm), and both hesperidin (0–400 µg/mL) and naringin (0–250 µg/mL)
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23 163 were used as the standard compounds at the wavelength of 360 nm and 420 nm,
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26 164 respectively.
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28 165 2.5.4. The 2,4-Dinitrophenylhydrazine colorimetric method
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166 The currently applied method was modified from the process used by Chang et al.
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33 167 . Here, 1 mL of extract was reacted with 2 mL of 2,4-DNPH reagent (1 g of
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35 168 2,4-DNPH in 2 ml of 96% sulfuric acid, diluted to 100 mL with methanol) and 2 mL
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38 169 of methanol at 50°C for 50 min. After cooling to ambient temperature, 5 mL of 10%
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40 170 potassium hydroxide in 70% methanol was added and incubated for 2 min. After that,
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43 171 1 mL of the mixture was combined with 4 mL of methanol and centrifuged at 1000 g
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45 172 for 10 min to remove the sediment. Then, 1 mL of supernatant was collected and
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48 173 diluted to 5 ml with methanol. The absorbance was measured at 476 nm. The blank
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50 174 was prepared by replacing the extract with methanol. Twenty-one flavonoid standard
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175 solutions (1000 ppm) were analyzed in the same way. Hesperidin and naringin were
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55 176 used to make the calibration curves (0–1500 µg/mL).
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4 177 2.5.5. HPLC method
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6 178 This procedure was similar to the method previously reported by Zhang et al. 19.
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179 Briefly, seventeen selected flavonoid standards were mixed in 80% methanol and
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11 180 dimethyl sulfoxide (1:1, v/v). After filtration with φ=13 mm PTFE membrane (0.45

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13 181 µm pore size), 10 µL of both standard solutions and diluted extracts were injected into
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16 182 the HPLC system. A reverse phase column (ZORABX SB-C18, 250×4.6 mm internal
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18 183 diameter) was used to perform the chromatographic separation. A two-solvent
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21 184 gradient mobile phase system consisting of water with 0.1% formic acid (A) and
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23 185 methanol (B) was used. The gradient program was performed as follows: 0–20 min,
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26 186 37–50% B; 20–35 min, 50–80% B; 35–40 min, 80–100% B; 40–50 min, 100% B; and
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28 187 50–60 min, 100–37% B. The flow rate was 0.7 mL/min and the column temperature
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188 was maintained at 25°C. The detection wavelengths were set at 283 nm, 330 nm and
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33 189 367 nm for flavanones, flavones, and flavonols respectively. The flavonoids were
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35 190 identified and quantified by comparing the retention time and peak area with that of
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38 191 the corresponding standards. The total flavonoids were expressed in the summary of
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40 192 individual flavonoid content.
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43 193 3. Results and discussion
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45 194 Although there are a number of methods for the quantification of citrus
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48 195 flavonoids, they are either not specific for citrus or using wrong flavonoid standards
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50 196 for detecting. Thus, we evaluated four widely used colorimetric methods for the
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197 quantification of total flavonoids in citrus, using twenty-one commercial standards of
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55 198 citrus flavonoids. The results determined by these four colorimetric methods and
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4 199 HPLC method were compared and analyzed to recommend the most reliable method
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6 200 and avoid the misuse. Hesperidin, naringin, and rutin were used as examples to show
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201 the color change of these four colorimetric methods (Fig. S1).
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11 3.1. NaNO2-Al(NO3)3-NaOH colorimetric method
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13 203 This method has been applied to detect o-diphenols in the past 35
, but is now
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16 204 widely used to determine the total flavonoids in citrus . The principle of this
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18 205 method is that NaNO2 was first added to produce oxidation and form nitrous acid, Al3+
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21 206 was subsequently used to form red chelates with flavonoids at alkaline medium (Table
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23 207 S2) 13, 32, 36
. The absorbance of twenty-one citrus flavonoid standards at 510 nm
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26 208 determined by this method are shown in Table 3. The results showed that among the
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28 209 twenty-one flavonoid standards investigated, only eriocitrin, eriodictyol, luteolin,
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210 quercitrin, and rutin, which have an ortho-dihydroxyl group in the B-ring, showed a
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33 211 relatively strong absorbance at 510 nm. In contrast, flavanones like hesperidin,
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35 212 naringin, narirutin, and neohesperidin etc., which are abundant in citrus (Table 4),
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38 213 showed only a very weak absorbance at 510 nm. From this perspective, we could
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40 214 assume that the results of quantifying the total flavonoid content in citrus using this
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43 215 method would be considerably lower than actual values.
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45 216 3.2. Aluminum chloride method
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48 217 The principle of this method is that aluminum chloride can not only form acid
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50 218 stable complexes with flavones and flavonols, which have a C-4 carbonyl group and a
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219 C-3 or C-5 hydroxyl group, but also form acid unstable complexes with catechol
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55 220 hydroxyl groups in the A-ring or B-ring of flavonoids (Fig. 1-a) . In most cases,
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4 221 acetate buffer as a weak acid reaction environment (the optimum pH is around 5.0–
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6 222 5.5), is beneficial to the complexation . Any blockage of the hydroxyl groups by
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223 glycosylation or even methoxylation in the positions of 3, 5, 3’ or 4’ prevents
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11 224 chelation with aluminum chloride, reducing the bathochromic shifts to 415–420 nm 14.

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13 225 From the absorbance results of 21 flavonoid standards shown in Table 3, it was clear
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16 226 that with the exception of polymethoxylated flavones, other flavones and flavonols
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18 227 could be determined, while all the flavanones were found to have too low absorbance
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21 228 compared to quercetin to make meaningful contribution to total absorption. Therefore,
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23 229 it was predicted that the results of total flavonoid content in citrus determined by this
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26 230 method would be lower than the real values.
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28 231 3.3. Davis spectrophotometric method
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232 The principle of this method is that flavanones can react with alkali to generate
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33 233 2,6-dihydroxy-4-epoxy phenylacetone and p-hydroxybenzaldehyde. In the
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35 234 environment of diethylene glycol, the yellow compound of chalcone would be
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38 235 produced (Fig. 1-b) 37. The absorbance results in Table 3 showed that except for three
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40 236 selected polymethoxylated flavones with a very low absorbance at 420 nm, all the
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43 237 other flavonoids had meaningful individual absorbance contributing to the total
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45 238 absorbance. Based on the different maximum absorption wavelength and absorbance
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48 239 between hesperidin and naringin (Shown in Fig. 1-c), the reference compound and
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50 240 absorption wavelength should be chosen in terms of the citrus sample analyzed.
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241 According to this result and the flavonoid composition of various citrus in Table 4, we
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55 242 speculated that the measurement might be suitable for grapefruit and pomelo when
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4 243 naringin was used as the standard at the measuring wavelength of 420 nm and the
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6 244 estimation might be approximate for others when hesperidin was used as the standard
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245 at the measuring wavelength of 360 nm.
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11 3.4. 2,4-Dinitrophenylhydrazine colorimetric method
246

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13 247 Ketones and aldehydes which possess carbonyl groups can be detected by
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16 248 reacting with 2,4-dinitrophenylhydrazine and forming 2,4-dinitrophenylhydrazones.
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18 249 Chang et al. 33
have found that flavanones can interact with
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21 250 2,4-dinitrophenylhydrazine in acidic media (sulfuric acid) to form colored
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23 251 phenylhydrazones, while flavones and flavonols which have C2-C3 double bonds can
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26 252 not react with 2,4-dinitrophenylhydrazine. In our study, we found that the hydrazones
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28 253 of most of citrus flavanones showed maximum absorption at 476 nm (Fig. 1-d). As a
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254 consequence, 476 nm was chosen as the wavelength for all determinations. The
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33 255 results in Table 3 showed that flavanones and polymethoxylated flavones could be
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35 256 determined while other flavonoids could not be detected. Therefore, we consider that
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38 257 this method may be available for determining the total content of flavanones and
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40 258 polymethoxylated flavones if there no other disturbances.
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43 259 3.5. Comprehensive discussion of different methods based on each flavonoid
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45 260 subclass
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48 261 In the NaNO2-Al(NO3)3-NaOH method, all flavonoids have multi-vibration
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50 262 peaks at around 300–400 nm (Fig. 1-a), causing difficulties with choosing an optimal
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263 peak for detection, as reported by Pękal et al. 32. In the peak at around 400–600 nm,
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55 264 only one maximum absorbance was exhibited at 510 nm in flavonoids, including
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4 265 flavanones (eriocitrin and eriodictyol), flavone (luteolin), and flavonols (quercitrin
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6 266 and rutin). According to the results from Table 3 and the principle described in
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267 Section 3.1, this method is not selective for any flavonoid subclass but is selective for
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11 268 flavonoids with dihydroxyl groups in the B-ring. Interestingly, we found that

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13 269 quercetin with 3’,4’-dihydroxy groups showed much lower absorbance values than
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16 270 quercitrin (quercetin-3-L-rhamnopyranoside) and rutin (quercetin-3-rutinoside) at 510
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18 271 nm, which can be attributed to the appearance of a 3-hydroxy group in quercetin. The
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21 272 structure of luteolin, which shows even higher absorbance value than quercitrin and
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23 273 rutin, could be seen as the absence of 3-hydroxyl group in quercetin. The absence of
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26 274 the C2-C3 double bond (eriodictyol compared to luteolin) has a very limited effect on
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28 275 absorbance, while the substitution of the 7-hydroxyl group with a rutinoside
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276 (eriocitrin compared to eriodictyol) shows a relatively higher effect. The absorbance
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33 277 value of eriocitrin is similar to that of quercitrin. In terms of Al3+, which could chelate
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35 278 with C-4 carbonyl group and C-3 or C-5 hydroxyl group (as described in Section 3.2),
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38 279 we hypothesized that the C-3 hydroxyl group could chelate with it and affect its
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40 280 reaction with catecholic moieties in the B-ring (like quercetin compared to luteolin).
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43 281 Pękal et al. 32 made a similar speculation that the presence of 5-hydroxyl-4-keto in
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45 282 flavonoids could lessen complexation via dihydroxyl groups in the B-ring when
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48 283 comparing rutin and luteolin with catechins. Once the C-3 hydroxyl group was
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50 284 glycosylated, the chelation effect would be weakened (like quercitrin and rutin
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285 compared to quercetin). However, the glycosyl would still have some blockage effects
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55 286 on the reaction of Al3+ and ortho-dihydroxyl groups in the B-ring, regardless of the
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4 287 glycosylation in C-3 or C-7 (like quercitrin, rutin and eriocitrin compared to
5
6 288 eriodictyol and luteolin). The C2-C3 double bond has little effect on the chelation of
7
8
289 Al3+ and dihydroxyl groups in the B-ring (like eriodictyol compared to luteolin).
9
10
11 290 Overall, the structural differences that affect the absorption of flavonoids in this

12
13 291 method could be put in the following order: catecholic moiety in the B-ring > C-3
14
15
16 292 hydroxyl group > glycosylation in C-3 or C-7 > C2-C3 double bond. This method
17
18 293 suits the flavonoid determination of samples, in which the main flavonoids have a
19
20
21 294 catecholic moiety in the B-ring and no C-3 hydroxyl group.
22
23 295 In the aluminum chloride method, the maximum absorption wavelength showed
24
25
26 296 a strong correlation with the subclasses of flavonoids (Fig. 1-b). All of the flavanones
27
28 297 exhibited a maximum absorbance at 305–309 nm and 374–383 nm owing to the
29
30
298 shared presence of the C-5 hydroxyl group. The maximum absorbance of flavones
31
32
33 299 depends on whether C-5 has a methoxy group or a hydroxyl group. Also, 415 nm is
34
35 300 widely chosen as the measuring wavelength, with the quercetin containing C-3 and
36
37
38 301 C-5 hydroxyl groups and an ortho-dihydroxyl group in the B-ring giving the highest
39
40 302 absorbance among all of the flavonoids selected at this wavelength. The absorbance
41
42
43 303 of kaempferol (a structure without an ortho-dihydroxyl group in the B-ring compared
44
45 304 with quercetin) was higher than that reported for quercitrin and rutin (structure with
46
47
48 305 glycosylated C-3 hydroxyl group compared with quercetin), which means that the C-3
49
50 306 hydroxyl group has a greater effect on the chelation with Al3+ than the catecholic
51
52
307 moiety in the B-ring. Comparing the absorbance and structure of eriodictyol with that
53
54
55 308 of luteolin, we can see the importance of the C2-C3 double bond on the reaction.
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4 309 Flavanones without a C2-C3 double bond all had a very low absorbance at 415 nm.
5
6 310 Flavones like nobiletin, tangeretin, and sinensetin without a C-5 hydroxyl group had
7
8
311 no absorbance at 415 nm. Instead, whether the presence of an ortho-dihydroxyl group
9
10
11 312 in the B-ring or not seems to have a limited impact on the absorption at 415 nm,

12
13 313 although lacking a catecholic moiety may affect the chelation with Al3+ and reduce the
14
15
16 314 bathochromic shifts (comparing the structure and absorbance of luteolin with
17
18 315 diosmetin and apigenin at 100 ppm); this discovery disagreed with the data put forth
19
20 33
21 316 by Chang et al. . Therefore, this method is suitable for the determination of
22
23 317 flavonols and flavones with a C-5 hydroxyl group.
24
25
26 318 The Davis method was created for the determination of citrus flavonoids and 420
27
28 319 nm was generally chosen as the detection wavelength. However, not all flavonoids
29
30
320 had a maximum absorbance at this point. Also, there was a significant difference in
31
32
33 321 the absorption value between naringin or narirutin and other flavonoids., which is
34
35 322 related to the B-ring and glycosylation in flavonoids. Thus, the Davis method with
36
37
38 323 naringin as a standard at the measuring wavelength of 420 nm was only suitable for
39
40 324 samples mainly containing naringin or narirutin. Occasionally, we found that some
41
42
43 325 flavanones like hesperidin and neohesperidin have a maximum absorbance at 360 nm
44
45 326 under the reaction conditions of the Davis method. What surprised us was that the
46
47
48 327 absorption difference of almost all flavanones we selected at 360 nm was not huge.
49
50 328 Considering that hesperidin was the most common flavonoid in citrus except naringin,
51
52
329 we chose it as the other standard at the measuring wavelength of 360 nm to make a
53
54
55 330 measurement which can supplement the deficiency of the Davis method.
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4 331 2,4-Dinitrophenylhydrazine was thought to only react with flavanones in all
5
6 332 subclasses of flavonoids 33. It is also shown in our study (except for polymethoxylated
7
8
333 flavones). The nucleophilic addition of the -NH2 group to the C=O carbonyl group
9
10
11 334 with the loss of an H2O molecule to form 2,4-dinitrophenylhydrazone is the

12
13 335 mechanism of this method (Table S2) 38. We speculate that the presence of the C2-C3
14
15
16 336 double bond may have a conjugative effect with the C=O carbonyl group, resulting in
17
18 337 diminishing the electronic need of the reactive site. Additionally, polymethoxylated
19
20
21 338 flavones (like nobiletin, tangeretin, and sinensetin) showed high absorption using this
22
23 339 method, which can be attributed to the methoxyl groups. Therefore, this method is
24
25
26 340 suitable for the determination of flavanones and polymethoxylated flavones.
27
28 341 3.6. Flavonoid contents of various citrus fruits detected by different analytical
29
30
342 methods
31
32
33 343 To evaluate the specificity of four colorimetric methods in real samples, we
34
35 344 tested the flavonoid contents in the peel and pulp of seven species of citrus; the results
36
37
38 345 were compared with those determined by HPLC.
39
40 346 Information on regression equations in various methods using different standards
41
42
43 347 and absorption wavelengths are listed in Table S3. Fig. S2 and Table S4 showed some
44
45 348 information about HPLC chromatograms and standard curves of the seventeen
46
47
48 349 flavonoid standards as well as flavonoids in some samples. Table 5 summarized that
49
50 350 total flavonoid contents of seven different citrus fruits, including the peel and pulp
51
52
351 obtained by different methods. The values gained from four different colorimetric
53
54
55 352 methods all showed a significant correlation (p < 0.05) with that from HPLC (shown
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4 353 in Table 6); their simple linear regression plots are shown in Fig. 2. The
5
6 354 NaNO2-Al(NO3)3-NaOH method showed the lowest correlation coefficient (R = 0.539)
7
8
355 with the HPLC method compared to the other three spectrometric methods (R = 0.9,
9
10
11 356 0.971, 0.96 respectively), which verified the low specificity of this method. As for the

12
13 357 values for the aluminum chloride method, in spite of the high correlation coefficient
14
15
16 358 shown with the HPLC method values (R = 0.9), the low content of flavonols and
17
18 359 flavones with the C-5 hydroxyl group in citrus (Table 4) made the slope of the
19
20
21 360 regression line much higher than that seen for other methods (Fig. 2). Based on this,
22
23 361 these two methods were considered inappropriate for the determination of total
24
25
26 362 flavonoid contents in citrus. This was similar to that which was hypothesized in
27
14, 32
28 363 Section 3.1 and 3.2, and was consistent with the studies of Mammen & Daniel .
29
30
364 The indexes of the correlation coefficient (R = 0.96) and the slope (0.9384) in the
31
32
33 365 regression line between the 2,4-DNPH and HPLC method seemed perfect. However,
34
35 366 the intercept of the regression line (-20.145) was much higher than that in other
36
37
38 367 methods, perhaps due to other components containing carbonyl groups like phenolic
39
40 368 acids in extracts. This may be the reason why 2,4-DNPH method values were much
41
42
43 369 higher. The results determined by the Davis method showed huge differences when
44
45 370 different standard compounds and absorption wavelengths were chosen. On the basis
46
47
48 371 of the speculation and analysis in Section 3.3 and 3.5, the modified Davis method
49
50 372 with naringin as the standard at the measuring wavelength of 420 nm for grapefruit as
51
52
373 well as pomelo, and hesperidin as the standard at the measuring wavelength of the
53
54
55 374 measuring wavelength of the measuring wavelength of the measuring wavelength of
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4 375 the measuring wavelength of the measuring wavelength of the measuring wavelength
5
6 376 of 360 nm for other species was considered better. This method not only had the
7
8
377 highest correlation coefficient (R = 0.971) but its slope (1.0254) and intercept (-0.144)
9
10
11 378 of regression were also the closest to one and zero, respectively, indicating that the

12
13 379 values obtained by modified Davis method were similar to those of the HPLC method
14
15
16 380 in the quantification of citrus flavonoids. Therefore, we regarded the modified Davis
17
18 381 method as the most reliable method for determining the content of citrus flavonoids
19
20
21 382 among the four widely used colorimetric methods.
22
23 383 3.7. Discussion on the anti-interference of each colorimetric method
24
25
26 384 As we have discussed above, the low content of citrus flavonoids determined by
27
28 385 the NaNO2-Al(NO3)3-NaOH method and the AlCl3 method was due to their principles.
29
30
386 The first method could only detect compounds with catechol hydroxyl groups, and the
31
32
33 387 latter one suits for the determination of flavonols and flavones with a C-5 hydroxyl
34
35 388 group. However, many flavonoids abundant in citrus like hesperidin and naringin
36
37
38 389 have no o-diphenols and belong to flavanones. Therefore, the inaccuracy of these two
39
40 390 methods was mainly owing to their unsuitability, but not other interferences.
41
42
43 391 The flavonoid content values detected by 2,4-DNPH method in real sample
44
45 392 extracts were much higher than that of HPLC method, perhaps due to other
46
47
48 393 components containing carbonyl groups like phenolic acids in extracts. So the
49
50 394 anti-interference measure of this method was to determine the content of other
51
52
395 components with carbonyl groups in extracts, or to separate flavonoids from the
53
54
55 396 extracts firstly and then to detect. No matter which measure was taken, the procedure
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4 397 would become much complicated and not be suitable for quick detection.
5
6 398 The Davis method was thought to be the most specific for the determination of
7
8
399 flavanones and the interference was limited. Based on the different maximum
9
10
11 400 absorption wavelengths and absorbance between hesperidin and naringin, the

12
13 401 anti-interference measure we have taken was to choose the appropriate reference
14
15
16 402 compound and absorption wavelength in terms of the citrus sample analyzed. This is
17
18 403 what we called “the modified Davis method”, and this method has been proved to be
19
20
21 404 quick, easy to operate and reliable.
22
23 405 4. Conclusion
24
25
26 406 Here, we evaluated four colorimetric methods for the quantification of total
27
28 407 flavonoids in citrus, and found that method misuse indeed existed. Two widely used
29
30
408 colorimetric methods (NaNO2-Al(NO3)3-NaOH and AlCl3) could only detect some of
31
32
33 409 the flavonoids and were inadequate for the estimation of total flavonoid contents in
34
35 410 citrus. The NaNO2-Al(NO3)3-NaOH method was suitable for the determination of
36
37
38 411 flavonoids with catecholic moieties in the B-ring and no C-3 hydroxyl group, while
39
40 412 the AlCl3 method could be used for the determination of flavonols and flavones with a
41
42
43 413 C-5 hydroxyl group. The 2,4-DNPH method was suitable for the determination of
44
45 414 flavanones and polymethoxylated flavones. However, other unknown components
46
47
48 415 containing carbonyl groups in extracts strongly affected this method, which suggests
49
50 416 that the 2,4-DNPH method was not suitable for use with citrus. The modified Davis
51
52
417 method (by choosing naringin as the standard at the wavelength of 420 nm for
53
54
55 418 grapefruit and pomelo, and hesperidin as the standard at the wavelength of 360 nm for
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4 419 other species) which could be interchangeable with the HPLC method to some extent
5
6 420 was considered the most reliable method for the determination of citrus flavonoids.
7
8
421 Therefore, we recommended the use of the modified Davis method in laboratories and
9
10
11 422 factories with limited access to HPLC to avoid the current misuse of colorimetric

12
13 423 methods for the quantification of citrus flavonoids as well as for other plant
14
15
16 424 flavonoids.
17
18 425 5. Conflicts of interest
19
20
21 426 The authors declare that they have no conflicts of interest.
22
23 427 Supporting information
24
25
26 428 Specific information of citrus species and HPLC chromatograms were shown in
27
28 429 supplementary materials.
29
30
430 Acknowledgements
31
32
33 431 This research project was supported by the Key Research and Development
34
35 432 Project of Zhejiang Province, China (2015C02036) and National Natural Science
36
37
38 433 Foundation of China (C200501).
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4 507 Figure Captions
5
6 508 Fig. 1. Spectral analysis of 21 flavonoid standards determined by four colorimetric
7
8
509 methods in the wavelength range of 300-600 nm
9
10
11 510 2-a NaNO2-Al(NO3)3-NaOH method

12
13 511 2-b AlCl3 method
14
15
16 512 2-c Davis method
17
18 513 2-d 2,4-DNPH method
19
20
21 514 Fig. 2. Simple linear regression plots between four spectrophotometric methods and
22
23 515 HPLC method for flavonoids determination of 14 citrus samples
24
25
26 516
27
28 517
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518
31
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33 519
34
35 520
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38 521
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40 522
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43 523
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3 529 Table 1 Examples of colorimetric methods for quantifying total flavonoid contents in
4 530 citrus
5
6 Methods Standard compounds Wavelength (nm) References
7
Rutin 15-19
8 NaNO2-Al(NO3)3-NaOH 510
9 Catechin 20-24
10 415 9, 25
AlCl3 Quercetin
11
430 26, 27

12 Hesperidin 420 28, 29
13 Davis
Naringin 420 30
14
15
531
16
17
18 532
19
20 533
21
22
23 534
24
25 535
26
27
28 536
29
30 537
31
32
33 538
34
35 539
36
37
540
38
39
40 541
41
42 542
43
44
45 543
46
47 544
48
49
50 545
51
52 546
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55 547
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3 549 Table 2 The specific information of 21 flavonoid standards used in this study
4
5
Structural Molecular Molecular
6 Flavonoids Systematic name
7 formula formula weight
8
9
10 Rutinoside;
11

12 Neohesperidoside
13
14 Ru= Nh=
15
16
17
Flavanones
18
19
20
21 3´,5,7-trihydroxy-4´- R1=OH; R2=OH;
22 Hesperetin C16H14O6 302.28
methoxyflavanone R3=OCH3
23
4´,5,7- R1=OH; R2=H;
24 (±)- Naringenin C15H12O5 272.25
25 trihydroxyflavanone R3=OH
26 3´,4´,5,7- R1=OH;
Eriodictyol C15H12O6 288.25
27 tetrahydroxyflavanone R2=R3=OH
28 3´,5,7-trihydroxy-4´-
29 R1=Ru; R2=OH;
Hesperidin methoxyflavanone-7- C28H34O15 610.56
30 R3=OCH3
31 rutinoside
32 4´,5,7-
33 R1= Ru; R2=H;
Narirutin trihydroxyflavanone- C27H32O14 580.53
34 R3= OH
7-rutinoside
35
36 5,7-dihydroxy-4´-
R1= Ru; R2=H;
37 Didymin methoxyflavanone-7- C28H34O14 594.56
R3=OCH3
38 rutinoside
39 3´,4´,5,7-
40 R1= Ru;
Eriocitrin Tetrahydroxyflavanon C27H32O15 596.53
41 R2=R3=OH
42 -7-rutinoside
43 4´,5,7-
44 R1= Nh; R2=H;
Naringin trihydroxyflavanone- C27H32O14 580.541
45 R3=OH
7-neohesperidoside
46
47 3´,5,7-trihydroxy-4´-
R1= Nh; R2=OH;
48 Neohesperidin methoxyflavanone-7- C28H34O15 610.56
R3=OCH3
49 neohesperidoside
50 5,7 -dihydroxy-4´-
51 R1= Nh; R2=H;
Poncirin methoxyflavanone-7- C28H34O14 594.56
52 R3=OCH3
53 neohesperidoside
54
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3
4
5
Flavones
6
7
8
9 3´,4´,5,6,7,8- R1= R2=R3= R4=
10 Nobiletin C21H22O8 402.39
hexamethoxyflavone R5=R6=OCH3
11

4´,5,6,7,8- R5=H; R1= R2=
12 Tangeretin C20H20O7 372.37
13 pentamethoxyflavone R3= R4=R6=OCH3
14 3´,4´,5,6,7- R1=H; R2=R3=
15 Sinensetin C20H20O7 372.37
Pentamethoxyflavone R4=R5=R6=OCH3
16
17 4´,5,7- R1= R3=R5=H;
18 Rhoifolin trihydroxyflavone-7- R4= R6= OH; C27H30O14 578.52
19 neohesperidoside R2=Nh
20
3´,4´,5,7 – R1= R3=H; R2=
21 Luteolin C15H10O6 286.24
22 tetrahydroxyflavone R4= R5= R6=OH
23 R1= R3=H;
24 3´,5,7 -trihydroxy-4´-
Diosmetin R2= R4=R5=OH; C16H12O6 300.26
25 Methoxyflavone
R6=OCH3
26
27 4´,5,7 – R1= R3=R5=H;
Apigenin C15H10O5 270.24
28 trihydroxyflavone R2=R4=R6=OH
29
30
31
Flavonols
32
33
34 Rh=
35 3,3´,4´,5,7-
36 Quercetin R1= R2=R3=OH C15H10O7 302.24
pentahydroxyflavone
37
38 3,4´,5,7-
Kaempferol R1=H; R2=R3=OH C15H10O6 286.24
39 tetrahydroxyflavone
40 3,3´,4´,5,7-
41 pentahydroxyflavone- R1= R2=OH;
42 Quercitrin C21H20O11 448.38
3-L- R3=Rh
43
44 rhamnopyranoside
45 3,3´,4´,5,7-
R1= R2=OH;
46 Rutin pentahydroxyflavone- C27H30O16 610.52
47 R3=Ru
3-rutinoside
48
49 550
50
51
52 551
53
54 552
55
56 553
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DOI: 10.1039/C8AY00661J
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3 554 Table 3 The absorbance of twenty-one flavonoid standards determined by four
4 555 colorimetric methods
5
6 NaNO2-Al(NO3)3
7 Flavonoids AlCl3 Davis 2,4-DNPH
-NaOH
8
9 Wavelength 510 nm 415 nm 360 nm 420 nm 476 nm
10
Absorbance Flavanones
11

12 Hesperidin 0.0074 ± 0.0040 0.0205 ± 0.0016 0.3215 ± 0.0068 0.1689 ± 0.0040 0.1387 ± 0.0068
13
14 Narirutin 0.0097 ± 0.0009 0.0195 ± 0.0015 0.2106 ± 0.0086 0.5735 ± 0.0089 0.1492 ± 0.0038
15 Didymin 0.0006 ± 0.0006 0.0311 ± 0.0009 0.4158 ± 0.0048 0.0732 ± 0.0021 0.1768 ± 0.0044
16
17 Eriocitrin 0.0938 ± 0.0016 0.0138 ± 0.0009 0.1474 ± 0.0046 0.0938 ± 0.0021 0.0153 ± 0.0035
18 Naringin 0.0094 ± 0.0011 0.0197 ± 0.0001 0.2092 ± 0.0015 0.5646 ± 0.0021 0.1666 ± 0.0009
19
20 Neohesperidin 0.0034 ± 0.0015 0.0146 ± 0.0012 0.2325 ± 0.0026 0.1332 ± 0.0031 0.1704 ± 0.0042
21
22 Poncirin 0.0001 ± 0.0002 0.0187 ± 0.0018 0.2846 ± 0.0088 0.0544 ± 0.0054 0.1896 ± 0.0009
23 Hesperetin 0.0166 ± 0.0005 0.0243 ± 0.0015 0.1612 ± 0.0012 0.0662 ± 0.0013 0.3028 ± 0.0088
24
25 (±)- Naringenin 0.0252 ± 0.0015 0.0174 ± 0.0003 0.2184 ± 0.0022 0.3141 ± 0.0029 0.3195 ± 0.0038
26
27 Eriodictyol 0.1503 ± 0.0035 0.0240 ± 0.0004 0.3508 ± 0.0055 0.1875 ± 0.0047 0.0447 ± 0.0031
28 Flavones
29
Nobiletin 0.0028 ± 0.0015 0.0003 ± 0.0006 0.6994 ± 0.0036 0.0015 ± 0.0008 0.1550 ± 0.0046
30
31 Tangeretin 0.0034 ± 0.0019 0.0011 ± 0.0007 0.3718 ± 0.0121 0.0041 ± 0.0001 0.1895 ± 0.0107
32
33 Rhoifolin 0.0035 ± 0.0013 0.0976 ± 0.0008 0.7428 ± 0.0055 0.2484 ± 0.0024 0.0021 ± 0.0024
34 Sinensetin 0.0042 ± 0.0015 0.0040 ± 0.0012 0.5333 ± 0.0029 0.0132 ± 0.0014 0.0963 ± 0.0017
35
36 Luteolin 0.1687 ± 0.0005 0.2874 ± 0.0058 0.7083 ± 0.0107 1.6182 ± 0.0074 0.0289 ± 0.0025
37
Diosmetin 0.0000 ± 0.0000 0.2100 ± 0.0080 0.8482 ± 0.0267 0.6647 ± 0.0225 0.0000 ± 0.0000
38
39 Apigenin 0.0000 ± 0.0000 0.2005 ± 0.0039 1.0229 ± 0.0056 1.6443 ± 0.0076 0.0016 ± 0.0028
40
41 Flavonols
42
Quercetin 0.0279 ± 0.0037 0.5592 ± 0.0075 0.8290 ± 0.0057 0.0909 ± 0.0042 0.0057 ± 0.0040
43
44 Kaempferol 0.0000 ± 0.0000 0.3846 ± 0.0111 0.2986 ± 0.0070 0.9249 ± 0.0091 0.0000 ± 0.0000
45
46 Quercitrin 0.0993 ± 0.0008 0.2061 ± 0.0003 0.3754 ± 0.0023 0.5163 ± 0.0086 0.0050 ± 0.0023
47
Rutin 0.1185 ± 0.0015 0.1689 ± 0.0100 0.2327 ± 0.0012 0.4297 ± 0.0183 0.0070 ± 0.0034
48
49 556 Notes: 1. The concentration of each authentic standard are 100 ppm in NaNO2 method and AlCl3
50 557 method, 200 ppm in Davis method and 1000 ppm in 2,4-DNPH method.
51
558 2. Results were shown as average ± SD (n=3).
52
53 559
54
55
56
57
28
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560 Table 4 Flavonoid composition of citrus peels and pulps determined by HPLC
8

9 Flavonoids ）
（mg/g DW）
Content（
10
11 Peels Mandarin,
Grapefruit Pomelo Ponkan Tangerine Lemon Sweet orange
12 Pulps Owari unshǔ
13 Flavanones 283nm
14
1.5492 ± 0.2145 0.2626 ± 0.0830 84.8236 ± 3.0925 64.0564 ± 2.3540 50.6882 ± 1.0707 13.8471 ± 0.1826 34.7838 ± 0.3581
15 Hesperidin
16 0.4148 ± 0.0280 0.1534 ± 0.0177 9.0871 ± 0.0950 15.4646 ± 0.2872 11.9907 ± 0.1795 12.6640 ± 0.1601 7.7711 ± 0.2048
17 1.7341 ± 0.3333 0.1957 ± 0.0877 11.7622 ± 0.4140 1.2110 ± 0.0399 1.5372 ± 0.0919 1.4538 ± 0.0374 2.2585 ± 0.0577
18 Narirutin
2.2081 ± 0.2210 ND 4.1273 ± 0.0355 1.5559 ± 0.0241 1.5614 ± 0.0178 0.6716 ± 0.0134 1.9208 ± 0.0912
19
ND 0.0850 ± 0.0299 2.7832 ± 0.1094 1.0890 ± 0.0373 0.6706 ± 0.0943 0.0744 ± 0.0537 1.3141 ± 0.0210
20 Didymin
ND ND 0.8518 ± 0.0083 0.8606 ± 0.0131 0.4623 ± 0.0083 ND 0.8083 ± 0.0426
21
22 1.789 5 ± 0.4364 ND 0.4420 ± 0.0271 0.1576 ± 0.0882 0.2279 ± 0.0631 7.9547 ± 0.0654 ND
Eriocitrin
23 0.7971 ± 0.0554 ND 0.0846 ± 0.0259 0.0788 ± 0.0198 ND 6.3422 ± 0.0681 ND
24 23.0505 ± 3.6412 44.5892 ± 3.1509 0.1807 ± 0.0527 0.1692 ± 0.0446 0.1620 ± 0.0419 0.8601 ± 0.0243 ND
25 Naringin
6.5388 ± 0.6223 1.3414 ± 0.1396 ND ND ND 0.2517 ± 0.0061 ND
26
27 Neohesperidin
21.0729 ± 3.3946 0.3553 ± 0.0970 ND ND ND 0.9069 ± 0.02362 ND
28 2.6986 ± 0.2644 0.0675 ± 0.0079 ND ND ND ND ND
29 0.1222 ± 0.0116 0.1262 ± 0.0096 0.1058 ± 0.0170 0.4931 ± 0.0211 0.6303 ± 0.0147 0.1189 ± 0.0085 ND
30 Poncirin
0.0462 ± 0.0030 0.0302 ± 0.0021 ND 0.0525 ± 0.0036 0.0487 ± 0.0015 ND ND
31
ND ND 0.0537 ± 0.0126 0.2001 ± 0.0037 0.2856 ± 0.0056 ND ND
32 Hesperetin
33 ND ND ND 0.0298 ± 0.0010 ND ND ND
34 ND 0.0628 ± 0.0038 ND 0.0450 ± 0.0102 0.0521 ± 0.0025 0.0567 ± 0.0043 ND
35 (±)-Naringenin
ND ND ND ND ND ND ND
36
0.1755 ± 0.0200 1.6521 ± 0.3856 ND 0.2916 ± 0.0815 0.1209 ± 0.0042 0.8190 ± 0.0306 ND
37 Eriodictyol
38 ND 0.1982 ± 0.0063 ND ND ND ND ND
39
40 29
41
42
43
44
45
46
47
Published on 11 May 2018. Downloaded by University of Saskatchewan on 11/05/2018 15:1
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6
Flavones 330nm
7
8 Nobiletin
0.4812 ± 0.0694 0.0502 ± 0.0041 0.6596 ± 0.0200 7.5112 ± 0.2150 4.7388 ± 0.0906 0.0693 ± 0.0107 0.8274 ± 0.0072

9 ND ND ND 0.0389 ± 0.0020 0.0256 ± 0.0058 ND ND
10 0.3067 ± 0.0383 0.0284 ± 0.0026 0.2815 ± 0.0090 4.5876 ± 0.1206 3.4638 ± 0.0615 0.0533 ± 0.0023 0.1815 ± 0.0027
11 Tangeretin
ND ND ND 0.0283 ± 0.0020 0.0219 ± 0.0049 ND ND
12
0.7169 ± 0.1296 2.9548 ± 0.1583 0.5989 ± 0.3024 0.5348 ± 0.0090 0.1904 ± 0.0049 2.8673 ± 0.0390 0.2042 ± 0.0030
13 Rhoifolin
14 ND ND ND ND ND 0.8431 ± 0.0167 ND
15 0.0525 ± 0.0029 ND 0.1367 ± 0.0079 0.6961 ± 0.0330 0.5028 ± 0.0077 ND 0.6824 ± 0.0084
16 Sinensetin
17
0.0867 ± 0.0088 0.0733 ± 0.0021 0.2795 ± 0.0169 0.0902 ± 0.0294 0.0799 ± 0.0070 0.1963 ± 0.0036 ND
18 Diosmetin
19 ND ND ND ND ND ND ND
20 Flavonols 367nm
21 ND 0.1104 ± 0.0020 0.1619 ± 0.0068 0.1208 ± 0.0026 0.1189 ± 0.0055 ND ND
22 Kaempferol
23
0.2179 ± 0.0172 0.1765 ± 0.0154 0.0890 ± 0.0157 ND ND 0.1712 ± 0.0104 ND
24 Quercitrin
25 ND ND ND ND ND ND ND
26 51.3558 ± 8.2615 50.7225 ± 2.9541 102.3584 ± 3.7935 81.2537 ± 2.7721 63.4696 ± 1.3910 29.4487 ± 0.2069 40.2519 ± 0.3986
27 SUM
12.7037 ± 1.1937 1.7908 ± 0.1708 14.1507 ± 0.1261 18.1095 ± 0.3318 14.1107 ± 0.2057 20.7727 ± 0.2465 10.5002 ± 0.3381
28
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DOI: 10.1039/C8AY00661J
1
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3 562 Table 5 Total flavonoid contents of citrus peels and pulps determined by different
4 563 methods
5
6 NaNO2 AlCl3 Davis 2,4-DNPH
7 Methods
510 nm 415 nm 360 nm 420 nm 476 nm
8 HPLC
9 Standard
Rutin Quercetin Hesperidin Naringin Hesperidin Naringin
10 compound
11

Peels
12 b a c
Grapefruit 16.598±0.109 4.224±0.034 50.628±0.940 45.515±0.839c 85.751±2.138e 73.697±1.859d 51.356±8.262c
13
14 Pomelo 9.416±0.041b 3.615±0.064a 46.598±2.179c 55.562±2.425e 84.092±0.885g 72.262±0.770f 50.723±2.954d
15 Mandarin,
16 7.592±0.257a 8.283±0.287a 85.018±1.304c 45.774±0.761b 127.471±2.643f 109.921±2.298e 102.358±3.794d
Owari unshǔ
17
Ponkan 20.363±0.754b 6.163±0.053a 80.058±1.861d 29.061±0.704c 113.960±2.240f 98.193±1.948e 81.254±2.772d
18 b a b f d
19 Tangerine 21.465±0.394 5.036±0.084 76.573±0.822 e
24.902±0.535 83.043±5.241 71.341±4.557 63.470±1.391c
20 Lemon 22.423±1.222b 6.325±0.109a 21.049±1.308b 26.839±0.168c 38.767±3.303e 32.843±2.872d 29.449±0.207c
21 Sweet orange 7.363±0.571a 3.721±0.227a 40.527±1.799d 16.228±0.213b 57.416±8.005e 23.229±3.480c 40.252±0.399d
22
Pulps
23 b a d
Grapefruit 6.109±0.043 0.948±0.024 16.589±0.251 12.156±0.184c 44.923±1.792f 38.625±1.558e 12.704±1.194c
24
25 Pomelo 2.772±0.012c 0.331±0.007a 5.301±0.087d 3.066± 0.040c 31.865±0.779f 27.272±0.677e 1.791±0.171b
26 Mandarin,
27 4.120±0.066a 1.402±0.014a 17.130±0.663d 9.003±0.236b 47.441±3.069f 40.797±2.669e 14.151±0.126c
Owari unshǔ
28
Ponkan 6.546±0.135b 0.938±0.028a 22.234±1.073d 7.822±0.052b 45.941±2.961f 39.508±2.575e 18.110±0.332c
29 b a b e d
30 Tangerine 5.415±0.062 0.985±0.014 15.052±0.339 c
6.036±0.127 40.916±3.231 35.145±2.809 14.111±0.206c
31 Lemon 8.231±0.044b 1.958±0.017a 13.853±0.172d 11.610±0.255c 28.816±0.180g 24.618±0.156f 20.773±0.247e
32 Sweet orange 2.304±0.063b 0.247±0.016a 12.520±0.265e 5.704±0.069c 49.446±1.025g 41.255±0.891f 10.500±0.338d
33
564 Note: 1. The results were shown as average ± SD (n=3) and expressed as mg/g DW.
34
35 565 2. Different letters represent statistically significant differences (p < 0.05).
36
37 566
38
39 567
40
41
568
42
43
44 569
45
46 570
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48
49 571
50
51 572
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54 573
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56 574
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DOI: 10.1039/C8AY00661J
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3 575 Table 6 Correlation coefficients (R) between the citrus flavonoid contents obtained
4 576 from five different methods
5
6 NaNO2- Modified
7 Methods AlCl3 2,4-DNPH HPLC
Al(NO3)3-NaOH Davis method
8
9 NaNO2-
– – – – –
10 Al(NO3)3-NaOH
11
AlCl3 0.706** – – – –

12 Modified
13 0.564* 0.829** – – –
Davis method
14
15 2,4-DNPH 0.412 0.789** 0.95** – –
16
17 HPLC 0.539* 0.9** 0.971** 0.96** –
18
577 * and ** denotes significantly (P<0.05) and highly significantly (P<0.01) correlated.
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DOI: 10.1039/C8AY00661J
1
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3 Hesperidin Sinensetin
Flavanones Naringin Flavones Rhoifolin
4
1.0 1.0
Neohesperidin Tangeretin
0.9 0.9
5 0.8
Eriocitrin
(±)-Naringenin 0.8
Nobiletin
Luteolin
6 0.7
Narirutin
0.7
Apigenin
Poncirin Diosmetin
7
Absorbance
Absorbance 0.6 Hesperetin 0.6
8 0.5
Eriodictyol
Didymin
0.5
9 0.4 0.4
10 0.3 0.3
11
0.2 0.2

12 0.1 0.1
0.0
13
0.0
300 350 400 450 500 550 600 300 350 400 450 500 550 600
14 λ (nm) λ (nm)
15 0.8
16 Flavonols
17
0.7 Rutin
Quercitrin
18 0.6 Kaempferol
Quercetin
19
Absorbance
0.5
20 0.4
21 0.3
22 0.2
23 0.1
24
25
0.0
300 350 400 450 500 550 600
26 λ (nm)
27
28 1-a
29
30
31
32 Hesperidin Tangeretin
33 0.8 Flavanones Hesperetin 0.8 Flavones Luteolin

Poncirin Apigenin
34 0.7 Eriocitrin
Eriodictyol
0.7 Diosmetin
Nobiletin
35 0.6 Neohesperidin
Naringin
0.6 Rhoifolin
Sinensetin
36
Absorbance
Absorbance
0.5 (±)-Naringenin 0.5

Narirutin
37 0.4
Didymin 0.4
38 0.3 0.3
39 0.2 0.2
40 0.1 0.1
41 0.0 0.0
300 350 400 450 500 550 600
300 350 400 450 500 550 600
42 λ (nm) λ (nm)
43 Quercitrin
44 0.8
Flavonols
Quercetin
45
Rutin
0.7 Kaempferol
46 0.6
47
Absorbance
0.5
48 0.4
49
0.3
50
51
0.2
52 0.1
53 0.0
300 350 400 450 500 550 600
54 λ (nm)
55
56 1-b
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DOI: 10.1039/C8AY00661J
1
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3 1.0 Flavanones
Hesperidin
Naringin 1.0 Flavones
Sinensetin
Rhoifolin
4 0.9
Narirutin
Neohesperidin 0.9
Nobiletin
Tangeretin
5 0.8 (±)-Naringenin 0.8 Diosmetin
Poncirin Apigenin
6 0.7
Hesperetin 0.7
Luteolin
Absorbance
Absorbance
7 0.6 Eriocitrin 0.6
Eriodictyol
0.5 0.5
8 0.4
Didymin
0.4
9 0.3 0.3
10 0.2 0.2
11
0.1 0.1

12 0.0
300 350 400 450 500 550 600
0.0
300 350 400 450 500 550 600
13 λ (nm) λ (nm)
14 1.0 Flavonols
Rutin
Quercetin
15 0.9
Quercitrin
Kaempferol
16 0.8
17 0.7
Absorbance
18 0.6
0.5
19 0.4
20 0.3
21 0.2
22 0.1
23 0.0
300 350 400 450 500 550 600
24 λ (nm)
25
26 1-c
27
28
29
Hesperidin Luteolin
30 0.5 Flavanones Hesperetin 0.5 Flavones Apigenin
Poncirin
31 Eriocitrin
Sinensetin
Tangeretin
32 0.4
Narirutin
(±)-Naringenin
0.4 Diosmetin
Nobiletin
33 Eriodictyol Rhoifolin
Absorbance
Absorbance
Neohesperidin
34
0.3 0.3
Naringin
Didymin
35 0.2 0.2
36
37 0.1 0.1
38
39
0.0 0.0
300 350 400 450 500 550 600 300 350 400 450 500 550 600
40 λ (nm) λ (nm)
Flavonols
41 0.5 Kaempferol
Rutin
42 Quercitrin
0.4
43 Quercetin
44
Absorbance
0.3
45
46 0.2
47
48 0.1
49
50
0.0
300 350 400 450 500 550 600
51 λ (nm)
52
1-d
53
54
55 Fig. 1
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DOI: 10.1039/C8AY00661J
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120 120
4
HPLC method value (mg/g DW)

5 100 100
6 80 80
7
60
8 60
9 40
40
10 20 y = 2.2806x + 13.577
11 20 y = 10.446x + 3.5381
R = 0.539

0 R = 0.9
12 0 5 10 15 20 25 0
13 NaNO2-Al(NO3)3-NaOH method value 0 2 4 6 8 10
14 (mg/g DW) AlCl3 method value (mg/g DW)
15
16
17 120 120

18 100 100
19
20 80 80
21 60 60
22
23 40 40
24 20 y = 1.0254x - 0.144 20 y = 0.9384x - 20.145
25 R = 0.971 R = 0.96
0 0
26
0 20 40 60 80 100 0 50 100 150
27
Modified Davis method value (mg/g DW) 2,4-DNPH method value (mg/g DW)
28
29
30
Fig. 2
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DOI: 10.1039/C8AY00661J
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13 Misused colorimetric methods for citrus flavonoids quantification were evaluated and
14
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16 the most reliable method was recommended.
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33 0.8 Flavanones Hesperetin 0.8 Flavones Luteolin

0.5 (±)-Naringenin 0.5

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