Authentication of the Bilberry Extracts by an HPLC Fingerprint Method Combining Reference Standard Extracts

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Article Authentication of the Bilberry Extracts by an HPLC Fingerprint Method Combining Reference Standard Extracts

Bingbing Liu 1,2 , Tiantian Hu 1 and Weidong Yan 1,3,* 1 Department of Chemistry, Zhejiang University, Hangzhou 310027, China; [email protected] (B.L.); [email protected] (T.H.) 2 State Key Laboratory of Environmental and Biological Analysis, Hong Kong Baptist University, Kowloon Tong, Kowloon, Hong Kong 999077, China 3 Zhejiang Skyherb Biotechnologies Co., Ltd., Anji 313300, China * Correspondence: [email protected]; Tel.: +86-571-8795-1430; Fax: +86-571-8795-1895

Received: 18 April 2020; Accepted: 21 May 2020; Published: 28 May 2020

Abstract: A simple and fast high-performance liquid chromatography (HPLC) fingerprint method combining reference standard extract for the identification of bilberry extract was developed and validated. Six batches of bilberry extract collected from different manufactures were used to establish the HPLC fingerprint. Other berry extracts—such as blueberry extracts, mulberry extracts, cranberry extracts, and black rice extracts—were also analyzed for their HPLC chromatograms. The fingerprints of five batches of bilberry extract showed high similarities, while one batch was distinguished from others. Additionally, the content of anthocyanin Cyanidin-3-O-glucoside (Cy-3-glc) in each berry extract was analyzed and compared. The results indicate that this HPLC fingerprint method, combining reference standard extracts, could be used for the authentication and quality control of bilberry extracts.

Keywords: bilberry extract; anthocyanins; HPLC; ﬁngerprint; authentication

1. Introduction Vaccinium myrtillus L., also known as the bilberry, is a small shrub belonging to the Ericaceae family. It is a very diverse species which distributes in the northern hemisphere [1]. Bilberries are rich in anthocyanins, which are proved to be free radical scavengers and antioxidants [2–5], thus making them a good source for natural health-care foods. Anthocyanins are found with anti-inflammation, anti-tumor, and vision-improving activities [6–8]. Besides, it can aid the regulation of blood lipid levels and improve insulin resistance [3,6,7]. More importantly, bilberries contain a higher proportion of anthocyanins than other berries, such as the blueberry, mulberry, strawberry and cranberry [9–12]. Under these circumstances, the bilberry extract industry is starting to show strong momentum [13]. Bilberry extract has been recognized as a non-certified food additive by the US Food and Drug Administration (FDA), with US sales reaching USD 3.75 million in 2012 [14]. The price of bilberry extracts in the market has been soaring, but quality is uneven. The adulteration of cheaper anthocyanin-rich extracts such as blueberry extracts, mulberry extracts, and black rice extracts has been found. Thus, a feasible and reliable method for the authentication of bilberry extracts is highly required. Currently, research on anthocyanin in bilberries is still in the preliminary stage. The quantification of anthocyanin in bilberries and other plant extracts usually uses the ultraviolet spectrophotometry (UV) method and the HPLC (high-performance liquid chromatography) method [15]. The UV method has poor reproducibility and is susceptible to interference. Besides, the anthocyanin species cannot

Molecules 2020, 25, 2514; doi:10.3390/molecules25112514 www.mdpi.com/journal/molecules Molecules 2020, 25, 2514 2 of 12 be identified by the UV method. Thus, new technologies for the identification and quantification of anthocyanins in plants and plant extracts have come into sight. For example, the analytical characterization of anthocyanins in bilberry fruit and food products was achieved by HPLC, coupled with mass spectrometry (MS), in 2018 [16]. Besides, it was found that the content of the anthocyanins of bilberry was influenced by several conditions, such as plant species, place of origin, climate, and freshness of the fruit. For example, the anthocyanin content of wild bilberries is generally higher than that of cultivated bilberries. The HPLC fingerprint, combining standards, is a comprehensive method for the assessment of the quality and reliability of food and plant extracts [17–20]. The US FDA has suggested that herbal products should be evaluated by this strategy [21]. However, this requires a dozen of standards, since some standards are expensive and sometimes not available in the market, due to the difficulty of their preparation. On the other hand, the HPLC-mass spectrometry (MS) approach can identify components, but its accuracy and precision are inferior to the HPLC method; furthermore, the HPLC-MS instrument is expensive and more complicated than that of HPLC, requiring a professional background of its users [22]. Recently, our lab reported an HPLC method combining reference extracts, instead of individual standards [23], which makes up the deficiency mentioned above. The reference standard extract is used as a reference substance to identify and quantify the components of plant extract samples. Compared to the external standard method, it only requires standardization of the multiple markers of the reference standard extract in advance, which makes it economical and easy for the identification and quantification of plant components. The convenience and consistency of this method was validated in-house and its results showed no difference with that of the conventional external standards method [23]. Thus, this study aimed to develop an HPLC fingerprint method, combining reference standard extracts, for the authentication of bilberry extracts from other berry extracts, such as blueberry extracts, mulberry extracts, cranberry extracts, and black rice extracts. In addition, with only one standard obtained, we further quantified and compared the characteristic component Cyanidin-3-O-glucoside (Cy-3-glc) in each bilberry extract.

2. Results and Discussions

2.1. Optimization of the Elution Program The separation resolution of each peak, as well as the total HPLC analysis time, was used to evaluate the ﬁtness of the elution program. The separation resolution of each peak was obtained using Wufeng HPLC analysis software. Though elution program 2 (P2) had the shortest analysis time, the separation resolution of each peak was not proper, with some peaks overlapped and diﬃcult to identify. It was found that the separation resolution of all the anthocyanins was best under elution program 6 (P6) (Table1, chromatograms are shown in supporting information Figures S1–S6), so this was set as the optimum elution program for further use.

Table 1. Separation resolutions of the compounds under diﬀerent elution programs.

Compound P1 P2 P3 P4 P5 P6 Delphinidin-3-O-galactoside (Del-3-gal) 6.44 17.93 6.94 6.84 7.53 8.35 Delphinidin-3-O-glucoside (Del-3-glc) 4.45 3.11 1.55 2.85 2.84 2.93 Cyanidin-3-O-galactoside (Cy-3-gal) 2.11 2.38 2.66 2.64 2.65 2.63 Delphinidin-3-O-arabinoside (Del-3-ara) 2.72 1.63 1.61 1.72 1.76 1.84 Cyanidin-3-O-glucoside (Cy-3-glc) 2.43 1.57 1.59 1.74 1.77 1.83 Petunidin-3-O-galactoside (Pet-3-gal) 2.55 1.61 1.79 1.97 2.11 2.20 Cyanidin-3-O-arabinoside (Cy-3-ara) 1.11 - 2.20 1.98 1.94 1.88 Petunidin-3-O-glucoside (Pet-3-glc) 2.03 - 0.68 1.07 1.33 1.54 Malvidin-3-O-glucoside (Mal-3-glc) 2.13 1.78 1.40 2.55 2.75 2.86 Malvidin-3-O-arabinoside (Mal-3-ara) 1.41 2.94 1.58 2.34 4.33 7.23 Molecules 2020, 25, 2514 3 of 12

2.2. Similarity Analysis of HPLC Fingerprints of Bilberry Extract Samples As seen in Figures S1–S6, five batches of bilberry extracts from different cultivating regions share the same anthocyanin profiles according to the HPLC chromatogram, and one batch has the unique chromatogram. The similarities between the entire chromatographic profiles of the six batches of bilberry extract samples and the reference standard were calculated by the professional software Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version 2004A) [24]. The correlation coefficients of fingerprints from six batches were shown as 0.998, 0.993, 0.994, 0.997, 0.983 and 0.163. The results indicate that the five batches of bilberry extract samples from different plantations shared nearly the same correlation coefficients of similarities and one was different from others.

2.3. Establishment of the Bilberry Extract HPLC Fingerprint HPLC fingerprints of six batches of bilberry extracts from different manufactures were obtained (representative chromatogram is shown in Figure1B and detailed chromatograms are listed in Figures S7–S12). Since elution program 1 in this study was the same as the elution program of international commercial standard for bilberry extract SW/T 2-2013 [25], 10 characteristic peaks of the bilberry reference extract were identified according to international commercial standard directly, without preliminary identification of the reference standard extract. The HPLC fingerprints of six batches of bilberry extract from different regions were obtained, selected, and matched by the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version 2004A). Among these, five batches were in good accordance with that of the reference extract (Figure1A), but one batch (A6) only showed one characteristic peak (Figure1C), indicating that it might not be the genuine bilberry extract and needs further inspection by other technologies, such as HPLC-MS. The retention time and peak area of the bilberry reference extracts are shown in Table2, in addition to the relative retention time (RRT) and relative peak area (RPA) of the five similar bilberry extract samples, with respect to the reference peak (peak 5), which were compared by calculating the relative standard deviation (RSD). Good RSDs of the RRT and RPA (Table2) revealed that these samples had similar HPLC profiles, indicating that the characteristic HPLC fingerprint of bilberry extracts was applicable. This HPLC fingerprint method, combining reference standard extracts, is fast and easy and unusual samples can be subject to further inspection. In terms of identification, compared with methods such as HPLC combining external standards, it does not require the purchase of external standards, nor the laborious standards preparation procedure. For example, Muller used 15 anthocyanin standards to study the anthocyanin content of bilberry juices by HPLC-UV [26], the performance of which requires time to collect and prepare the standards. In terms of quantification, the HPLC fingerprint method combining reference standard extracts can provide accurate quantification of the components in samples. Since, in general, components of the reference standard extract are standardized before using as the reference standard for quantification [27–30]. The use of reference standard extract relieves the pressure of increasing demands for pure marker compounds [23]. Molecules 2020, 25, 2514 4 of 12 Molecules 2020, 25, x FOR PEER REVIEW 4 of 12

FigureFigure 1. 1. RepresentativeRepresentative HPLC HPLC (high-performance (high-performance liquid liquid chromatography) chromatography) chromatograms chromatograms of the of referencethe reference extract extract (A), the (A), bilberry the bilberry extract extract samples samples (B), and (B), bilberry and bilberry extract extract sample sample A6 (C). A6 Peak (C). identification:Peak identiﬁcation: 1, Delphinidin-3- 1, Delphinidin-3-O-galactosideO-galactoside (Del-3-gal); (Del-3-gal);2, Delphinidin-3- 2, Delphinidin-3-O-glucoside (Del-3-glc);O-glucoside 3, Cyanidin-3-(Del-3-glc);O-galactoside 3, Cyanidin-3- (Cy-3-gal);O-galactoside 4, Delphinidin-3- (Cy-3-gal);O-arabinoside 4, (Del-3-ara); Delphinidin-3- 5, Cyanidin-3-O-arabinosideO- glucoside(Del-3-ara); (Cy-3-glc); 5, Cyanidin-3- 6, Petunidin-3-O-glucosideO-galactoside (Cy-3-glc); (Pet-3-gal); 6, Petunidin-3- 7, Cyanidin-3-O-galactosideO-arabinoside (Pet-3-gal); (Cy-3- 7, O O O ara);Cyanidin-3- 8, Petunidin-3--arabinosideO-glucoside (Cy-3-ara); (Pet-3-glc 8, Petunidin-3-); 9, Malvidin-3--glucosideO-glucoside (Pet-3-glc); (Mal-3-glc); 9, Malvidin-3- 10, Malvidin-3--glucosideO- (Mal-3-glc); 10, Malvidin-3-O-arabinoside (Mal-3-ara). arabinoside (Mal-3-ara).

Table 2. The retention time (tR), peak area (PA), relative peak relative retention time (RRT), and relative peak area (RPA) of 10 characteristic peaks in the bilberry reference extract and bilberry extract samples (n = 3).

Componenta tR (min)b RRT Peak area (mAU)b RPA Average RSD (%) Average RSD (%) 1 14.68 0.75 0.58 2065.633 1.15 0.53 2 15.94 0.82 0.17 2514.939 1.34 5.69 3 17.14 0.89 0.30 1686.999 0.89 6.40

Molecules 2020, 25, 2514 5 of 12

a b b MoleculesComponent 2020, 25, x FORtR (min) PEER REVIEW RRT Peak area (mAU) RPA 5 of 12 Average RSD (%) Average RSD (%) 41 17.95 14.68 0.94 0.75 0.28 0.581616.961 2065.633 1.03 1.15 4.66 0.53 5 (S)2 18.80 15.94 1.00 0.82 0 0.17 1864.444 2514.939 1.00 1.34 4.36 5.69 3 17.14 0.89 0.30 1686.999 0.89 6.40 64 19.80 17.95 1.07 0.94 0.10 0.28813.383 1616.961 0.43 1.03 7.25 4.66 57 (S)20.84 18.80 1.13 1.00 0.22 01224.583 1864.444 0.71 1.00 6.03 4.36 86 21.43 19.80 1.19 1.07 0.09 0.101528.597 813.383 0.87 0.43 3.96 7.25 7 20.84 1.13 0.22 1224.583 0.71 6.03 98 27.09 21.43 1.63 1.19 0.93 0.091599.29 1528.597 0.88 0.87 0.11 3.96 109 29.88 27.09 1.84 1.63 1.19 0.93 261.977 1599.29 0.20 0.88 40.03 0.11 10 29.88 1.84 1.19 261.977 0.20 40.03 a Component: 1, Del-3-gal; 2, Del-3-glc; 3, Cy-3-gal; 4, Del-3-ara; 5, Cy-3-glc; 6, Pet-3-gal; 7, Cy-3-ara; a Component: 1, Del-3-gal; 2, Del-3-glc; 3, Cy-3-gal; 4, Del-3-ara; 5, Cy-3-glc; 6, Pet-3-gal; 7, Cy-3-ara; 8, Pet-3-glc; 9, 8,Mal-3-glc; Pet-3-glc; 10, 9, Mal-3-ara. Mal-3-glc;b Retention 10, Mal-3-ara. times and b Retention peak areas times of the and ten peaks peak inareas bilberry of the reference ten peaks extract. in bilberry reference extract. 2.4. HPLC Chromatograms of the Other Extract Samples 2.4. HPLC Chromatograms of the Other Extract Samples Then, HPLC chromatograms of the other extract samples are obtained. Seven batches of blueberry extractsThen, were HPLC divided chromatograms into two groups, of wherethe other two batchesextract showedsamples chromatogram are obtained. type Seven as inbatches Figure 2ofA blueberryand the other extracts five batcheswere divided showed into chromatogram two groups, typewhere as two in Figure batches2B; showed however, chromatogram these chromatograms type as inare Figure quite 2A diff anderent the from other that five of batches the bilberry showed extract chromatogram fingerprint. type The as in di ffFigureerent 2B; chromatograms however, these of chromatogramsblueberry extracts are obtained quite different may be because from that of several of the factors, bilberry such extract as genotypes, fingerprint. cultivation The different regions, chromatogramssoil quality, ages, of treeblueberry age, and extracts harvest obtained periods, may all be of because which couldof several affect factors, the anthocyanin such as genotypes, content cultivationof blueberries. regions, soil quality, ages, tree age, and harvest periods, all of which could affect the anthocyanin content of blueberries.

FigureFigure 2. HPLCHPLC chromatograms chromatograms of of the the blueberry blueberry extracts, extracts, ( (AA),), chromatogram chromatogram type type of of B2 B2 and and B3 B3 samples;samples; ( (BB)) chromatogram chromatogram type type of B1, B4, B5, B6 and B7 samples.

Likewise,Likewise, four four batches batches of of mulberry mulberry extracts extracts were were analyzed; they they all all showed showed the the same chromatogramschromatograms (Figure 33),), whichwhich werewere didifferentﬀerent from thatthat ofof thethe bilberrybilberry extractextract ﬁngerprint.fingerprint.

400.00 [mAU]

319.00

238.00

157.00

76.00

-5.00 0.00 6.10 12.20 18.30 24.40 30.50 36.60 42.70 48.80 54.90 61.00 [min]

Figure 3. HPLC chromatograms of the mulberry extracts.

Chromatograms of the two batches of cranberry extracts were divided into two groups (Figure 4A,B), which were also different from bilberry extracts.

Molecules 2020, 25, x FOR PEER REVIEW 5 of 12

4 17.95 0.94 0.28 1616.961 1.03 4.66 5 (S) 18.80 1.00 0 1864.444 1.00 4.36 6 19.80 1.07 0.10 813.383 0.43 7.25 7 20.84 1.13 0.22 1224.583 0.71 6.03 8 21.43 1.19 0.09 1528.597 0.87 3.96 9 27.09 1.63 0.93 1599.29 0.88 0.11 10 29.88 1.84 1.19 261.977 0.20 40.03 a Component: 1, Del-3-gal; 2, Del-3-glc; 3, Cy-3-gal; 4, Del-3-ara; 5, Cy-3-glc; 6, Pet-3-gal; 7, Cy-3-ara; 8, Pet-3-glc; 9, Mal-3-glc; 10, Mal-3-ara. b Retention times and peak areas of the ten peaks in bilberry reference extract.

2.4. HPLC Chromatograms of the Other Extract Samples Then, HPLC chromatograms of the other extract samples are obtained. Seven batches of blueberry extracts were divided into two groups, where two batches showed chromatogram type as in Figure 2A and the other five batches showed chromatogram type as in Figure 2B; however, these chromatograms are quite different from that of the bilberry extract fingerprint. The different chromatograms of blueberry extracts obtained may be because of several factors, such as genotypes, cultivation regions, soil quality, ages, tree age, and harvest periods, all of which could affect the anthocyanin content of blueberries.

Figure 2. HPLC chromatograms of the blueberry extracts, (A), chromatogram type of B2 and B3 samples; (B) chromatogram type of B1, B4, B5, B6 and B7 samples.

MoleculesLikewise,2020, 25, 2514four batches of mulberry extracts were analyzed; they all showed the same6 of 12 chromatograms (Figure 3), which were different from that of the bilberry extract fingerprint.

400.00 [mAU]

319.00

238.00

157.00

76.00

Molecules 2020, 25, x FOR PEER REVIEW 6 of 12 -5.00 0.00 6.10 12.20 18.30 24.40 30.50 36.60 42.70 48.80 54.90 61.00 [min] Figure 3. HPLC chromatograms of the mulberry extracts. Figure 3. HPLC chromatograms of the mulberry extracts. Chromatograms of the two batches of cranberry extracts were divided into two groups Chromatograms of the two batches of cranberry extracts were divided into two groups (Figure (FigureMolecules4A,B), 2020 which, 25, x FOR were PEER also REVIEW di ﬀerent from bilberry extracts. 6 of 12 4A,B), which were also different from bilberry extracts.

Figure 4. HPLC chromatograms of the cranberry extracts, (A), chromatogram type of E1 sample; (B) chromatogram type of E2 sample.

Finally, four batches of black rice extract were analyzed; they all showed the same chromatograms (Figure 5), which were also different from bilberry extracts. By comparing the HPLC chromatograms of the bilberry reference standard extract with those of the blueberry,Figure 4. HPLCHPLC mulberry, chromatograms chromatograms cranberry, of of and the the cranberryblack cranberry rice extracts,extracts, extracts, ( (AA it),), canchromatogram chromatogram be concluded type type that of of E1 E1 the sample; sample; anthocyanin ( (BB)) profilechromatogram of bilberry extracttype of E2 is quitesample. different from that of the other berry extracts. The bilberry extract is rich in anthocyanins, containing more than 10 kinds of anthocyanins, while other berry extracts Finally, four batches of black rice extract were analyzed; they all showed the same chromatograms only Finally,contain fourone tobatches four kinds of black of anthocyanins rice extrac.t Therefore,were analyzed; this HPLC they fingerprintall showed method the same can (Figure5), which were also di ﬀerent from bilberry extracts. effectivelychromatograms distinguish (Figure the 5), bilberry which were reference also different standard from extract bilberry from otherextracts. plant extracts. By comparing the HPLC chromatograms of the bilberry reference standard extract with those of 400.00 the blueberry, mulberry, cranberry,[mAU] and black rice extracts, it can be concluded that the anthocyanin

profile of bilberry extract319.00 is quite different from that of the other berry extracts. The bilberry extract is rich in anthocyanins, containing more than 10 kinds of anthocyanins, while other berry extracts

only contain one to four238.00 kinds of anthocyanins. Therefore, this HPLC fingerprint method can effectively distinguish the bilberry reference standard extract from other plant extracts.

157.00400.00 [mAU]

319.0076.00

238.00-5.00 0.00 5.90 11.80 17.70 23.60 29.50 35.40 41.30 47.20 53.10 59.00 [min] Figure157.00 5. HPLC chromatograms of the black rice extracts. Figure 5. HPLC chromatograms of the black rice extracts.

By comparing the HPLC76.00 chromatograms of the bilberry reference standard extract with those of the blueberry,2.5. Validation mulberry, of the HPLC cranberry, Quantification and black Method rice extracts, it can be concluded that the anthocyanin proﬁle of bilberry extract is-5.00 quite diﬀerent from that of the other berry extracts. The bilberry2 extract is The linearity regression0.00 curve5.90 for11.80 Cy-3-glc17.70 23.60 was29.50 35.40y = 29430.1414341.30 47.20 53.10x 59.00− 0.37173, r = 0.9988. The [min] linearityrich in anthocyanins, range was 0.02 containing mg/mL–0.12 more mg/mL. than 10 The kinds LOD of (limit anthocyanins, of detection) while was other 0.007 berry mg/mL, extracts and only the LOQ (limit of quantification) was 0.02 mg/mL. Figure 5. HPLC chromatograms of the black rice extracts. Good precision, as revealed in the relative standard deviations (RSDs) for peak areas, was found (Table2.5. 3). Validation of the HPLC Quantification Method For the stability test, the RSDs of the peak areas of analytes were < 0.34%, indicating that the standardThe solutionlinearity wasregression stable for curve 24 h forat ambient Cy-3-glc temperature. was y = 29430.14143 x − 0.37173, r2 = 0.9988. The linearityThe rangerepeatability was 0.02 of mg/mL–0.12 the method wasmg/mL. tested The by LOD the determination(limit of detection) of a standardwas 0.007 solution mg/mL, of and Cy-3- the glc.LOQ The (limit RSDs of ofquantification) peak areas were was calculated, 0.02 mg/mL. which fell within the ranges specified, as shown in Table 4. Good precision, as revealed in the relative standard deviations (RSDs) for peak areas, was found (Table 3). For theTable stability 3. Stability, test, the precision, RSDs of and the repeatability peak areas of of the analytes Cy-3-glc were quantification < 0.34%, method. indicating that the standard solution was stable for 24 h at ambient temperature. Stability Inter-day precision Intra-day precision Repeatability The repeatabilityItem of the method was tested by the determination of a standard solution of Cy-3- (n = 1) (n = 1) (n = 1) (n = 6) glc. The RSDs of peak areas were calculated, which fell within the ranges specified, as shown in Table 4. RSD (%) 0.34 0.30 0.90 0.98

Table 3. Stability, precision, and repeatability of the Cy-3-glc quantification method.

Stability Inter-day precision Intra-day precision Repeatability Item (n = 1) (n = 1) (n = 1) (n = 6) RSD (%) 0.34 0.30 0.90 0.98

Molecules 2020, 25, 2514 7 of 12 contain one to four kinds of anthocyanins. Therefore, this HPLC ﬁngerprint method can eﬀectively distinguish the bilberry reference standard extract from other plant extracts.

2.5. Validation of the HPLC Quantiﬁcation Method The linearity regression curve for Cy-3-glc was y = 29430.14143x 0.37173, r2 = 0.9988. The − linearity range was 0.02 mg/mL–0.12 mg/mL. The LOD (limit of detection) was 0.007 mg/mL, and the LOQ (limit of quantiﬁcation) was 0.02 mg/mL. Good precision, as revealed in the relative standard deviations (RSDs) for peak areas, was found (Table3).

Table 3. Stability, precision, and repeatability of the Cy-3-glc quantiﬁcation method.

Stability Inter-Day Precision Intra-Day Precision Repeatability Item (n = 1) (n = 1) (n = 1) (n = 6) RSD (%) 0.34 0.30 0.90 0.98

For the stability test, the RSDs of the peak areas of analytes were < 0.34%, indicating that the standard solution was stable for 24 h at ambient temperature. The repeatability of the method was tested by the determination of a standard solution of Cy-3-glc. The RSDs of peak areas were calculated, which fell within the ranges speciﬁed, as shown in Table4.

Table 4. The recovery rate of the Cy-3-glc in bilberry extract.

No. Original (µg) Added (µg) Found (µg) Recovery yield (%) RSD (%) 1 505.13 479.04 988.04 100.81 2 505.86 479.04 998.14 102.76 3 506.42 479.04 981.56 99.19 1.29 4 505.94 479.04 983.62 99.72 5 505.41 479.04 985.15 100.15 6 505.78 479.04 982.97 99.61

The recovery test was conducted to evaluate the accuracy of this method. The recovery test of the Cy-3-glc standard solution was obtained by adding a known amount (479.04 µg) of Cy-3-glc standard solution to the six bilberry extract sample solutions. As shown in Table4, the recovery rate for Cy-3-glc was within the range of 99.61% and 102.76% and the RSD for recovery rate was 1.29%.

2.6. Quantiﬁcation of the Cy-3-glc in Bilberry Extract Samples The proposed HPLC method was successfully applied to the quantiﬁcation of anthocyanin Cy-3-glc in bilberry extract samples, using the external standard calibration method. The contents of Cy-3-glc in bilberry extract samples were calculated as follows,

C η = x Cm where Cx is the concentration (mg/mL) of Cy-3-glc in bilberry extract sample solution, Cm is the concentration (µg/mL) of bilberry extract solution, and η is the content of Cy-3-glc in bilberry extract sample. As shown in Table5, the contents of Cy-3-glc in bilberry extract samples were within the range of 62–77 µg/mg, with a slight diﬀerence. Molecules 2020, 25, 2514 8 of 12

Table 5. Contents of Cy-3-glc in bilberry extract samples (n = 3).

Sample No. Cm (µg/mL) Cx (µg/mL) η (µg/mg) A1 1504 97.792 65.021 A2 1513 115.54 76.365 A3 1512 98.380 65.066 A4 1509 93.638 62.053 A5 1504 98.175 65.276

2.7. Comparison of the Cy-3-glc Content in the Other Extracts The Cy-3-glc contents in several batches of bilberry extracts, blueberry extracts, mulberry extracts, cranberry extracts, and black rice extracts were determined and presented in a whisker plot. A whisker plot is suitable to show the quantitative differences between the extracts. It can be clearly seen in Figure6 that extracts from di fferent origins had different Cy-3-glc contents for the blueberry extract, mulberry extract, cranberry extract and black rice extract samples, which were in the range of 8.3–594.5 µg/mg. Meanwhile, in bilberry extract samples, the Cy-3-glc contents were similar, which were in the range of 62–77 µg/mg. According to the HPLC analysis, these extracts contained fewer kinds of anthocyanins, where Cy-3-glc was the main anthocyanin component. As for cranberry extracts, theseMolecules contained 2020, 25 minimal, x FOR PEER Cy-3-glc REVIEW content: 18.7–33 µg/mg. 8 of 12

FigureFigure 6. 6. ComparisonComparison of of the the contents contents of of cyanidin-3- cyanidin-3-OO-glucoside-glucoside in in different diﬀerent plant extracts. 3. Materials and Methods 3. Materials and Methods 3.1. Chemicals and Reagents 3.1. Chemicals and Reagents HPLC-grade acetonitrile and methanol were purchased from Sigma-Aldrich (St. Louis, USA). All otherHPLC-grade chemicals acetonitrile and reagents and weremethanol of analytical were pu graderchased and from obtained Sigma-Aldrich from Sinopharm (St. Louis, Chemical USA). AllReagent other Co.chemicals Ltd. (Shanghai, and reagents China). were Bilberry of analytical reference grade standard and obtained extract from (European Sinopharm Pharmacopoeia Chemical Reagentreference Co. standard) Ltd. (Shanghai, and cyanidin-3- China).O -glucosideBilberry reference chloride standard (98%) was extract purchased (European from J&K Pharmacopoeia Scientiﬁc Ltd. reference(Beijing, China). standard) The and information cyanidin-3- onO berry-glucoside extracts chloride is listed (98%) in Table was6. purchased from J&K Scientific Ltd. (Beijing, China). The information on berry extracts is listed in Table 6.

Table 6. Information about the extract samples.

No. Extract kind Batch No. Manufacture Bilberry dry extract Bilberry reference standard − J&K Scientific Ltd. CRS extract A1 Bilberry extract 161004 Longze Biotech. Co., Ltd. A2 Bilberry extract 20160709 Luzhijian Biotech. Co., Ltd. Anhui, Shangshan Biotech. A3 Bilberry extract − Co., Ltd. Xian, Haotian Biotech. Co., A4 Bilberry extract B1161101 Ltd. Zhejiang, Huisong Biotech. A5 Bilberry extract 161222 Co., Ltd. Shanxi, Baicaocui Biotech. Co., A6 Bilberry extract 160725 Ltd. Shanxi, Haolin Biotech. Co., B1 Blueberry extract 161208 Ltd. B2 Blueberry extract 20161126 Longze Biotech. Co., Ltd. Xian, Haotian Biotech. Co., B3 Blueberry extract Bl161202 Ltd. Baoji, Runmu Biotech. Co., B4 Blueberry extract Rm161025 Ltd. Baoji, Runmu Biotech. Co., B5 Blueberry extract Rm161025 Ltd. Shanxi, Baicaocui Biotech. Co., B6 Blueberry extract − Ltd.

Molecules 2020, 25, 2514 9 of 12

Table 6. Information about the extract samples.

No. Extract kind Batch No. Manufacture Bilberry reference Bilberry dry extract CRS J&K Scientiﬁc Ltd. standard extract − A1 Bilberry extract 161004 Longze Biotech. Co., Ltd. A2 Bilberry extract 20160709 Luzhijian Biotech. Co., Ltd. A3 Bilberry extract Anhui, Shangshan Biotech. Co., Ltd. − A4 Bilberry extract B1161101 Xian, Haotian Biotech. Co., Ltd. A5 Bilberry extract 161222 Zhejiang, Huisong Biotech. Co., Ltd. A6 Bilberry extract 160725 Shanxi, Baicaocui Biotech. Co., Ltd. B1 Blueberry extract 161208 Shanxi, Haolin Biotech. Co., Ltd. B2 Blueberry extract 20161126 Longze Biotech. Co., Ltd. B3 Blueberry extract Bl161202 Xian, Haotian Biotech. Co., Ltd. B4 Blueberry extract Rm161025 Baoji, Runmu Biotech. Co., Ltd. B5 Blueberry extract Rm161025 Baoji, Runmu Biotech. Co., Ltd. B6 Blueberry extract Shanxi, Baicaocui Biotech. Co., Ltd. − B7 Blueberry extract Shanxi, Longfu Biotech. Co., Ltd. − C1 Mulberry extract Rm161007 Baoji, Runmu Biotech. Co., Ltd. C2 Mulberry extract Rm161007 Baoji, Runmu Biotech. Co., Ltd. C3 Mulberry extract 150523 Huzhou, Liuyin Biotech. Co., Ltd. C4 Mulberry extract 160819 Shanxi, Baicaocui Biotech. Co., Ltd. D1 Cranberry extract Rm161025 Baoji, Runmu Biotech. Co., Ltd. D2 Cranberry extract Shanxi, Longfu Biotech. Co., Ltd. − E1 Black rice extract 160623 Shanxi, Haolin Biotech. Co., Ltd. E2 Black rice extract HM20160407/s Hubei, Zixin Biotech. Co., Ltd. E3 Black rice extract Anhui, Shangshan Biotech. Co., Ltd. − E4 Black rice extract BR161001 Xian, Haotian Biotech. Co., Ltd.

3.2. Sample Preparation

3.2.1. Preparation of Cy-3-glc Standard Solutions Stock standard solutions of Cy-3-glc were prepared by dissolving 10 mg of cyanidin-3-O-glucoside chloride into a 25 mL volumetric flask with HCl/methanol (2:98, v/v) to volume, mixed. Of the stock solution, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mL was transferred to a 10 mL volumetric flask and diluted with 10% aqueous phosphoric solution. Thus, a series of standard solutions, at concentrations of 0.01, 0.02, 0.04, 0.06, 0.08, 0.10, and 0.12 mg/mL, were obtained. Each standard solution was prepared every day and passed through a 0.45 µm PTFE filter before HPLC injection.

3.2.2. Preparation of Reference Extract Solutions Stock solutions were prepared by dissolving 125 mg of bilberry reference extract into a 25 mL volumetric flask with HCl/methanol (2:98, v/v) to volume, mixed. Of the above solution, 2 mL was accurately transferred into a 10 mL volumetric flask, diluted with 10% aqueous phosphoric solution prior to the HPLC analysis. Each sample solution was passed through a 0.45 µm PTFE filter prior to the HPLC injection.

3.2.3. Preparation of Sample Solutions The bilberry extract sample solution was prepared by preparing 125 mg of bilberry extract in HCl/methanol (2:98, v/v) in 25 mL flasks; 3 mL of this solution was transferred into a 10 mL volumetric flask, diluted with 10% aqueous phosphoric solution, prior to the HPLC analysis. Each sample solution was passed through a 0.45 µm PTFE filter, prior to the HPLC injection. Other berry extract samples were prepared by the same procedure indicated above. Molecules 2020, 25, 2514 10 of 12

3.3. Screen of the Chromatographic Elution Program The HPLC analysis was performed on a Wufeng HPLC unit (Wufeng Co., Shanghai, China), coupled with an ultra-violet detector (1290 VWD). Separation was achieved on a Hedera ODS-2 C18 column (4.6 mm 250 mm, 5 µm; Nacilai Tesque Inc., Japan). The detection wavelength was set at 535 × nm and the ﬂow rate was 1 mL/min. The injection volume was 20 µL. The injections were performed in triplicate for each sample. According to the European Pharmacopeia, eluent A was 8.5% aqueous formic acid solution, and eluent B was acetonitrile/methanol/formic acid/water = 22.5:22.5:8.5:41.5 (v/v/v/v). Six gradient elution programs were performed, and the one with the optimum resolution was set as the optimum elution program. Program 1 (P1): 0–35 min: 7% B–25% B, 35–45 min: 25% B–65% B, 45–46 min: 65% B–100% B, and 46–50 min: 100% B for 10 min. Program 2 (P2): 0–15 min: 7% B–25% B, 15–25 min: 25% B–65% B, 25–26 min: 65% B–100% B, and 26–30 min: 100% B for 5 min. Program 3 (P3): 0–5 min: 7% B–25% B, 5–25 min: 25% B–55% B, 26–35 min: 55% B–100% B, and 36–40 min: 100% B for 5 min. Program 4 (P4): 0–5 min: 7% B–25% B, 5–35 min: 25% B–55% B, 36–45 min: 55% B–100% B, and 46–50 min: 100% B for 5 min. Program 5 (P5): 0–5 min: 7% B–25% B, 5–35 min: 25% B–45% B, 36–45 min: 45% B–100% B, and 46–50 min: 100% B for 5 min. Program 6 (P6): 0–5 min: 7% B–25% B, 5–35 min: 25% B–40% B, 36–45 min: 40% B–100% B, and 46–50 min: 100% B for 5 min. The sample solutions were prepared with the authentic bilberry reference extract.

3.4. Establishment of the HPLC Fingerprint of Bilberry Extracts To establish the representative HPLC chromatogram, six batches of bilberry extracts, as well as the reference bilberry extract, were analyzed under the optimized HPLC condition. Then, seven batches of blueberry extracts, four batches of mulberry extracts, two batches of cranberry extracts, and four batches of black rice extract samples were analyzed, and their HPLC chromatograms were compared.

3.5. Validation of the HPLC Quantiﬁcation Method According to the guidelines for the validation of drug quality standards of the China Food and Drug Administration (CFDA) [31], the linearity regression curves for each component were obtained by plotting the peak areas (y) against the concentrations of the Cy-3-glc standard solution. The limit of detection (LOD) was determined by considering a signal to noise ratio (S/N) of 3 and limit of quantiﬁcation (LOQ) was determined by considering a signal to noise ratio (S/N) of 10. The precision of the HPLC method was evaluated by analyzing the replicated Cy-3-glc standard solution of the same sample (0.08 mg/mL) for three times within one day and on two consecutive days. The stability test was determined by sampling the same standard solution in 2, 4, 6, 8, 10, 12, and 24 h. The repeatability was determined by preparing six bilberry extracts sample solutions independently and calculating the contents of the Cy-3-glc and the RSD.

4. Conclusions In this study, an easy HPLC fingerprint method, combining reference standard extracts, was set up to authenticate the anthocyanin profile of bilberry extracts. By using this method, one batch of bilberry extract was distinguished from others. In addition, it has been found that bilberry extract is unique for its anthocyanin profile, which is more plentiful than other berry extracts. Besides, the content of anthocyanin Cy-3-glc was determined in each extract. Furthermore, if more standards are used to quantify the reference standard extract of the bilberry in advance, this easy and effective HPLC Molecules 2020, 25, 2514 11 of 12 method could be used for the quality control of bilberry extracts, offering a substitute for the external standard calibration method.

Supplementary Materials: The following are available online. Figures S1–S6: Chromatogram under elution program 1–6; Figures S7–S12: HPLC chromatogram of the bilberry extract sample A1–A6; Figure S13. HPLC chromatogram of the blueberry extract sample B1–B7; Figure S14. HPLC chromatogram of the mulberry extract sample C1–C4; Figure S15. HPLC chromatogram of the black rice berry extract sample E1–E4. Author Contributions: B.L. conceived and designed the experiment, analyzed the data, and wrote the manuscript. T.H. designed and performed the experiment. W.Y. conducted the funding acquisition, project administration, and supervision of this study. All authors have read and agreed to the published version of the manuscript Funding: The authors appreciate funding from the Center for Food Evaluation, State Administration for Market Regulation of China (Program: Study on the quality standards of raw materials for health foods). Acknowledgments: We thank Yong Cheng (from Zhejiang Skyherb Biotechnologies Co., Ltd.) for providing materials for the experiment and conducting the validation of this HPLC method. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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Sample Availability: Samples of the compounds are not available from the authors.

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