UPLC-DAD-QTOF / MS Analysis of Flavonoids from 12 Varieties of Korean Mulberry Fruit

Mulberry (Morus alba L.) has been used in East Asia (Korea, China, and Japan) as a medicine because of its various pharmacological eects including the excellent antioxidant properties of its fruit. is study analyzed extracts from 12 varieties of Koreanmulberry fruit for avonoids using ultrahigh-performance liquid chromatography coupled with diode array detection and quadrupole time-of-ight mass spectrometry (UPLC-DAD-QTOF/MS). Six quercetin derivatives were identied by mass spectrometry (MS) based on the [quercetin +H] ion (m/z 303), while four kaempferol derivatives were identied based on the [kaempferol +H] ion (m/z 287). Two new compounds (morkotin A and morkotin C, quercetin derivatives) were identied for the rst time inmulberry fruit.e total avonoid contents of themulberry fruits ranged from 35.0± 2.3mg/100 g DW in the Baek OkWang variety (white mulberry) to 119.9± 7.0mg/100 g DW in the Dae Shim variety.is study has, for the rst time, evaluated the avonoid chromatographic proles of 12 varieties of Koreanmulberry fruits in a following quali-quantitative approach, which will contribute to improved utilization of these fruits as health foods.


Introduction
Mulberry belongs to the Moraceae family, which includes 37 genera and over 1100 species [1] and has long been used as a traditional medicine and food in Korea, China, and Japan [2][3][4].Mulberries are reported to exhibit numerous pharmacological activities, such as antidiabetic [5], antioxidant [6], anticancer [7], antistress [8], immunomodulatory [9], and hepatoprotective [10] activities.erefore, mulberries should be considered as important potential sources of bioactive compounds that can be used for the prevention or treatment of many di erent medical conditions.
In Korea and other countries, mulberry fruits are used in many commercial products, such as frozen desserts, ice cream, jam, juice, marmalade, paste, pulp, tea, and wine [11,12].In particular, mulberry fruit juice has been used as a folk remedy for treating aphtha, asthma, colds, coughs, diarrhea, dyspepsia, edema, fevers, headache, hypertension, and wounds [13].Mulberry fruits contain a variety of chemical components, including anthocyanins, sugars, organic acids, free amino acids, vitamins, and micronutrients [14].Chu et al. [15] have previously identi ed apigenin, ca eic acid, chlorogenic acid, gallic acid, luteolin, morin, rutin, kaempferol, and quercetin in mulberry fruit.ese components and compounds have the potential to bene t human health because of their signi cant biological activities.
ey also found that the red pigment of M. nigra fruit contained four anthocyanins, which were identified as cyanidin 3-O-glucoside, cyanidin 3-O-rutinoside, pelargonidin 3-O-glucoside, and pelargonidin 3-O-rutinoside using HPLC-photodiode array detection-ESI-MS analysis.is study investigated the flavonoids from 12 varieties of Korean mulberry fruits using a quali-quantitative approach to establish their nutrient profiles and to promote the further development of Korean mulberry resources for use in food and medicine.e fruit samples were cleaned, dried in a lyophilizer, and pulverized before storage at −18 °C until required for analysis.HPLCgrade solvents (acetonitrile, methanol, and water) and formic acid were purchased from Fisher Scientific (Fair Lawn, NJ, USA) and Junsei Chemical (Tokyo, Japan), respectively.Galangin (Sigma Aldrich, St. Louis, MO, USA) was used as internal standard for the flavonoids.

Sample Preparation and Extraction.
Extracts were taken prepared from the samples as described by Kim et al. [22] with few modifications.
e mixture was vortexed, stirred with shaking for 5 min at 200 rpm, and then centrifuged at 2000 g at 10 °C for 15 min.e supernatant was filtered through a syringe filter (0.45 μm, PTFE; Whatman, Maidstone, UK), and 0.5 mL of flavonoid extract was diluted with water to a final volume of 5 mL.e flavonoid-containing crude extract was further extracted and purified by solid-phase extraction using a Sep-Pak C 18 cartridge (Waters, Milford, MA, USA).e cartridge was activated by washing with 2 mL of methanol and then conditioned with 2 mL of water.e diluted flavonoid extract was loaded onto the Sep-Pak C 18 cartridge, and the impurities were removed by washing with 2 mL of water.e total flavonoid mixture was eluted from the Sep-Pak cartridge using 3 mL of methanol.
e purified extract was concentrated by evaporation under a stream of nitrogen gas and then redissolved in 0.5 mL of methanol:water:formic acid (50 : 45 : 5, v/v/v) solution without internal standard (galangin) before instrumental analysis.All experimental samples were analyzed in triplicate.

Analysis Conditions for UPLC-DAD-QTOF/MS.
An ultra-high-performance liquid chromatography (UPLC) system with a diode array detector (DAD) set at 280 and 320 nm coupled to a quadrupole time-of-flight mass spectrometer (QTOF/MS, Waters) was used for analysis.e ultraviolet-vis spectra were collected in the range from 210 to 600 nm.
In the present study, flavonoids from the fruits of 12 mulberry varieties (Morus alba L.) were analyzed by UPLC-DAD-QTOF/MS.Ten mulberry flavonoids were identified on the chromatogram between 5 and 20 min (Figure 1).e chemical structures of the individual flavonoids were determined by analysis of fragment ion patterns.2(c)).Ju et al. [37] reported seven new quercetin (morkotin A, B, and C) and kaempferol (moragrol A, B, C, and D) compounds by UPLC-DAD-QTOF/MS analysis of flavonoids from mulberry leaves.However, the quercetin derivatives morkotin A and morkotin C have not previously been reported in mulberry fruit.Although quercetin and kaempferol are reported to exhibit a range of biological activities, including anti-inflammatory, anticancer, antiobesity, antioxidant, antihypercholesterolemic, and antiatherosclerotic activity [38][39][40], the biological activities of morkotin A and morkotin B are unclear at this time.As newly identified components of mulberry fruit, these compounds need further investigation to determine their beneficial effects to human health.

Flavonoid Contents of Mulberry Fruit.
e contents of the ten flavonoids isolated from the 12 varieties of mulberry fruit are shown in Table 2. e total flavonoid contents of the mulberry fruit ranged from 35.0 ± 2.3 mg/100 g DW in the Baek Ok Wang variety (white mulberry) to 119.9 ± 7.0 mg/ 100 g DW in the Dae Shim variety.e total flavonoid contents (0.06-6.54 mg CE (catechin equivalents)/100 g DW) determined in five other Korean mulberry varieties (Pachungsipyung, Whazosipmunja, Suwonnosang, Jasan, and Mocksang) by Bae and Suh [41] were lower than those found in the present study.Butkhup et al. [42] reported that the total flavonoid content of eight major mulberry varieties from ailand (Nakhonratchasima 60, Buriram 60, Chumphon, Wavee, Chiang Mai, Pikultong, Kamphaengsaen, and Kamnanchul) ranged from 69.58 to 211.01 mg CE/100 g DW.Mahmood et al. [43] reported that the total contents of flavonols (myricetin, quercetin, and kaempferol) of four mulberry fruits (Morus laevigata, M. macroura, M. nigra, and M. alba) at different stages of maturity (unripe, semiripe, and fully ripe) ranged from 28.3 to 221.8 mg/100 g DW.
ese results for total flavonoid contents from Butkhup et al. [42] and Mahmood et al. [43] are similar to those found in the present study.Another study found that the total flavonoid contents in the fruits, leaves, and roots of two mulberry varieties (M.alba and M. nigra) ranged between 0.8948 and 67.369 mg RE (rutin equivalents)/g of dried extract, with the contents in the leaves and roots being higher than those in the fruit [44].
Recently, Ju et al. [37] reported that the total contents of the 17 flavonoids isolated from the leaves of 12 mulberry varieties ranged from 748.5 to 1297.9 mg/100 g DW.In addition, various flavonoid derivatives (moragrols A-D, kaempferol 3-O-rhamnoside-7-O-glucoside, morkotin B, and quercetin 3-O-rhamnoside-7-O-glucoside) other than those found in mulberry fruits in this study were found in mulberry leaves.us, the total flavonoid contents of mulberry can vary according to the plant variety, the parts of the plant, and the stages of fruit maturity.
An analysis of anthocyanins in different types of berry showed that blackcurrants contain the highest levels (5521 nmol/g), followed by blueberries (4810 nmol/g), cranberries (725 nmol/g), and redcurrants (328 nmol/g).e anthocyanins in blackcurrants were responsible for 73% of the total antioxidant capacity, whereas vitamin C contributed only 18% [46].In comparison, the total amount of anthocyanins in M. nigra and M. alba, made up of flavonols such as kaempferol and quercetin, was reported as 27 mg/10 g of fresh berries with cyanidin 3-O-glucoside being the major anthocyanin (66.3%), followed by cyanidin 3-O-rutinoside (27.8%), pelargonidin 3-O-glucoside (4.4%), and pelargonidin 3-O-rutinoside (1.5%) [11].Overall, the quercetin and kaempferol contents of blueberries, cranberries, and redcurrants are lower than those of mulberries without anthocyanins as found in the present study.ese results show that mulberry fruit is a significant source of flavonoids, which have protective effects on human health, as suggested in recent epidemiological and experimental studies [12,41,42,44].
Mulberry can grow in various climatic and soil conditions.e variation of flavonoid contents in mulberry could be caused by various factors, such as growing conditions, regional differences, climatic conditions, and genetic differences.For example, phenylalanine ammonia-lyase is reported as the key enzyme in the formation of flavonoids in mulberry leaves.
e expression of the phenylalanine ammonia-lyase gene can be induced by environmental factors such as low temperatures, and content of flavonoids can be increased under such conditions [49].However, there is a general lack of studies on the variation of flavonoid compounds in mulberry fruit caused by environmental factors, such as growing conditions, regional differences, and climatic conditions.In addition, there is little information available regarding the occurrence of flavonoid compounds in fruit according to plant variety for Korean mulberries, and these compounds have received little attention regarding their biological health effects on humans.erefore, further studies that build upon the present work are required to highlight the bioactive properties of flavonoid compounds in mulberry fruit and to promote further development of valuable mulberry resources in Korea.

Conclusions
e contents and composition of 10 flavonoids, isolated and identified in 12 varieties of Korean mulberry fruits, varied considerably.An UPLC-DAD-QTOF/MS system was used with complementary information obtained from LC spectra, MS ions, and MS/MS fragments to identify the constituents of the mulberry fruits.To the best of our knowledge, two out of the 10 compounds have been identified in mulberry fruits for the first time, so further research will be needed to evaluate their biological activity.e quantitative analysis of the functional flavonoids available in mulberry fruits provided by this study will help to evaluate the standards and physiological aspects of Korean mulberry fruits and so will encourage their cultivation and use.

Figure 2 :
Figure 2: Mass spectra of avonoids detected in extracts of Korean mulberry fruits.(a) Glycoside derivative of quercetin; (b) glycoside derivative of kaempferol; (c) MS spectra of two new avonoids detected from extracting Korean mulberry fruits.
2.1.Materials and Reagents.Mulberry fruits of 12 varieties were collected from the Sericulture and Apiculture Division (Department of Agricultural Biology, Rural Development Administration, Jeon-Ju, Republic of Korea).

Table 1 :
Ten flavonoids isolated from Korean mulberry fruits (Morus alba L.) and their mass spectrometric data.All samples were analyzed in positive ion mode ([M + H] + ) using UPLC-DAD-QTOF/MS.a New flavonoid identified in mulberry fruit.