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

Authors: O-Chul Kwon; Wan-Taek Ju; Hyun-Bok Kim; Gyoo-Byung Sung; Yong-Soon Kim
Journal: Journal of Food Quality
 (2019)

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## Abstract

Mulberry (Morus alba L.) has been used in East Asia (Korea, China, and Japan) as a medicine because of its various pharmacological effects including the excellent antioxidant properties of its fruit. This study analyzed extracts from 12 varieties of Korean mulberry fruit for flavonoids using ultrahigh-performance liquid chromatography coupled with diode array detection and quadrupole time-of-flight mass spectrometry (UPLC-DAD-QTOF/MS). Six quercetin derivatives were identified by mass spectrometry (MS) based on the [quercetin + H]+ ion (m/z 303), while four kaempferol derivatives were identified based on the [kaempferol + H]+ ion (m/z 287). Two new compounds (morkotin A and morkotin C, quercetin derivatives) were identified for the first time in mulberry fruit. The total flavonoid contents of the mulberry fruits 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. This study has, for the first time, evaluated the flavonoid chromatographic profiles of 12 varieties of Korean mulberry fruits in a following quali-quantitative approach, which will contribute to improved utilization of these fruits as health foods.


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## Body

## 1. 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 [2–4]. Mulberries are reported to exhibit numerous pharmacological activities, such as antidiabetic [5], antioxidant [6], anticancer [7], antistress [8], immunomodulatory [9], and hepatoprotective [10] activities. Therefore, mulberries should be considered as important potential sources of bioactive compounds that can be used for the prevention or treatment of many different 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 identified apigenin, caffeic acid, chlorogenic acid, gallic acid, luteolin, morin, rutin, kaempferol, and quercetin in mulberry fruit. These components and compounds have the potential to benefit human health because of their significant biological activities.Flavonoids are a large group of polyphenolic compounds composed of six subclasses: anthocyanins, flavan-3-ols, flavanones, flavones, flavonols, and isoflavones [16]. Flavonoids are present in mulberry fruit [11] and have biological and pharmacological activities that include antioxidative, anti-inflammatory, antiviral, antidiabetic, antihyperlipidemic, and antiobesity effects [17–19]. Lee et al. [20] and Chen et al. [21] identified and quantified anthocyanins and flavonoids in several mulberry varieties. Pawlowska et al. [11] used high-performance liquid chromatography (HPLC) to analyze the profile of flavonoids in Morus nigra and Morus alba fruits and identified the compounds using nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry (ESI-MS). They 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.This 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.


## 2. Materials and Methods

### 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). The fruit samples were cleaned, dried in a lyophilizer, and pulverized before storage at −18°C until required for analysis. HPLC-grade 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.


### 2.2. Sample Preparation and Extraction

Extracts were taken prepared from the samples as described by Kim et al. [22] with few modifications. The sample of powdered fruit sample (1 g) was mixed with 10 mL of methanol:water:formic acid (50 : 45 : 5, v/v/v) solution, containing 100 ppm of galangin (internal standard). The mixture was vortexed, stirred with shaking for 5 min at 200 rpm, and then centrifuged at 2000 g at 10°C for 15 min. The 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. The flavonoid-containing crude extract was further extracted and purified by solid-phase extraction using a Sep-Pak C18 cartridge (Waters, Milford, MA, USA). The cartridge was activated by washing with 2 mL of methanol and then conditioned with 2 mL of water. The diluted flavonoid extract was loaded onto the Sep-Pak C18 cartridge, and the impurities were removed by washing with 2 mL of water. The total flavonoid mixture was eluted from the Sep-Pak cartridge using 3 mL of methanol. The 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.


### 2.3. 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. The ultraviolet-vis spectra were collected in the range from 210 to 600 nm. The chromatographic conditions and equipment were as follows: column: Luna Omega 1.6μm C18, 150 × 2.1 mm (Phenomenex, Torrance, CA, USA); precolumn: SecurityGuard ULTRA Cartridges for UHPLC C18 for 2.1 mm i.d. column (Phenomenex); mobile phase: solvent A (0.5% formic acid in water) and solvent B (0.5% formic acid in acetonitrile); flow rate 0.3 mL/min; column temperature 30°C; total running time 60 min; injection volume 5 μL; and gradient condition: 0–2 min 7% (B), 24 min 15% (B), 40 min 30% (B), 48–50 min 60% (B), 53–54 min 90% (B), and 55–60 min 7% (B). The mass analysis conditions were as follows: sampling cone voltage 40 V; ion source temperature 120°C; desolvation temperature 400°C; desolvation gas flow 1000 L/h; cone gas 30 L/h; capillary voltage 3500 V; and mass range m/z 50–800 using positive ion electrospray.


## 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). The fruit samples were cleaned, dried in a lyophilizer, and pulverized before storage at −18°C until required for analysis. HPLC-grade 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.


## 2.2. Sample Preparation and Extraction

Extracts were taken prepared from the samples as described by Kim et al. [22] with few modifications. The sample of powdered fruit sample (1 g) was mixed with 10 mL of methanol:water:formic acid (50 : 45 : 5, v/v/v) solution, containing 100 ppm of galangin (internal standard). The mixture was vortexed, stirred with shaking for 5 min at 200 rpm, and then centrifuged at 2000 g at 10°C for 15 min. The 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. The flavonoid-containing crude extract was further extracted and purified by solid-phase extraction using a Sep-Pak C18 cartridge (Waters, Milford, MA, USA). The cartridge was activated by washing with 2 mL of methanol and then conditioned with 2 mL of water. The diluted flavonoid extract was loaded onto the Sep-Pak C18 cartridge, and the impurities were removed by washing with 2 mL of water. The total flavonoid mixture was eluted from the Sep-Pak cartridge using 3 mL of methanol. The 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.


## 2.3. 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. The ultraviolet-vis spectra were collected in the range from 210 to 600 nm. The chromatographic conditions and equipment were as follows: column: Luna Omega 1.6μm C18, 150 × 2.1 mm (Phenomenex, Torrance, CA, USA); precolumn: SecurityGuard ULTRA Cartridges for UHPLC C18 for 2.1 mm i.d. column (Phenomenex); mobile phase: solvent A (0.5% formic acid in water) and solvent B (0.5% formic acid in acetonitrile); flow rate 0.3 mL/min; column temperature 30°C; total running time 60 min; injection volume 5 μL; and gradient condition: 0–2 min 7% (B), 24 min 15% (B), 40 min 30% (B), 48–50 min 60% (B), 53–54 min 90% (B), and 55–60 min 7% (B). The mass analysis conditions were as follows: sampling cone voltage 40 V; ion source temperature 120°C; desolvation temperature 400°C; desolvation gas flow 1000 L/h; cone gas 30 L/h; capillary voltage 3500 V; and mass range m/z 50–800 using positive ion electrospray.


## 3. Results and Discussion

### 3.1. Isolation and Identification of Flavonoid Compounds in Mulberry Fruits

Mulberries contain bioactive components, such as alkaloids and flavonoids [23–25] and when dried possess antioxidant, antimicrobial, antidiabetic, antiobesity, and anti-inflammatory properties [26–34]. Qualitative analysis by the HPLC-DAD-ESI-MS/MS method has previously been used to identify six nonanthocyanin phenolics from two mulberry varieties: 4-caffeoylquinic acid, chlorogenic acid, protocatechuic acid, quercetin, rutin, and taxifolin. Three others were also tentatively identified [35]: 3,5-dicaffeoylquinic acid, taxifolin-hexoside, and kaempferol-hexoside.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). The chemical structures of the individual flavonoids were determined by analysis of fragment ion patterns. Six compounds (Peaks 1, 2, 4, 5, 6, and 9) were identified as quercetin derivatives by MS based on the presence of m/z 303 [quercetin + H]+. Four other compounds (Peaks 3, 7, 8, and 10) were assigned as kaempferol derivatives based on the presence of m/z 287 [kaempferol + H]+. The MS spectra of the flavonoids isolated from the mulberry fruit are shown in Figure 2. The following flavonoid compounds were isolated and identified from the mulberry fruits: Peak 1, quercetin 3-O-rutinoside-7-O-glucoside (morkotin A); Peak 2, quercetin 3,7-di-O-glucoside; Peak 3, kaempferol 3,7-di-O-glucoside; Peak 4, quercetin 3-O-rutinoside (rutin); Peak 5, quercetin 3-O-glucoside (isoquercitrin); Peak 6, quercetin 3-O-(6″-O-malonyl)glucoside; Peak 7, kaempferol 3-O-rutinoside (nicotiflorin); Peak 8, kaempferol 3-O-glucoside (astragalin); Peak 9, quercetin 3-O-(2″-O-malonyl)glucoside (morkotin C); and Peak 10, kaempferol 3-O-(6″-O-malonyl)glucoside (Table 1).Figure 1
LC chromatograms of flavonoids in samples of Korean mulberry fruits (Morus alba L.).Figure 2
Mass spectra of flavonoids detected in extracts of Korean mulberry fruits. (a) Glycoside derivative of quercetin; (b) glycoside derivative of kaempferol; (c) MS spectra of two new flavonoids detected from extracting Korean mulberry fruits.
(a)
(b)
(c)Table 1
Ten flavonoids isolated from Korean mulberry fruits (Morus alba L.) and their mass spectrometric data.
Aglycones
Glycosides
Acylations
Peak number
Individual flavonols
MW
Fragment ions (m/z)
Kaempferol (m/z 287)
Mono
Mal
8
Kaempferol 3-O-glucoside (astragalin)
448
471, 449, 287
10
Kaempferol 3-O-(6″-O-malonyl)glucoside
534
557, 535, 287
Di
7
Kaempferol 3-O-rutinoside (nicotiflorin)
594
617, 595, 449, 287
3
Kaempferol 3,7-di-O-glucoside
610
633, 611, 449, 287
Quercetin (m/z 303)
Mono
Mal
5
Quercetin 3-O-glucoside (isoquercitrin)
464
487, 465, 303
6
Quercetin 3-O-(6″-O-malonyl)glucoside
464
573, 551, 465, 303
9
Quercetin 3-O-(2″-O-malonyl)glucoside (morkotin C)a
550
573, 551, 303
Di
4
Quercetin 3-O-rutinoside (rutin)
610
633, 611, 465, 449, 303
2
Quercetin 3,7-di-O-glucoside
626
649, 627, 465, 303
Tri
1
Quercetin 3-O-rutinoside-7-O-glucoside (morkotin A)a
772
795, 773, 627, 611, 465, 303
All samples were analyzed in positive ion mode ([M + H]+) using UPLC-DAD-QTOF/MS. aNew flavonoid identified in mulberry fruit.The major peak of the mulberry fruit (Peak 4) generated the ion fragments ofm/z 633 [M + Na]+, 611 [M + H]+, 465 [M + H−Rham]+, 449 [M + H−Glu]+, and 303 [quercetin + H]+ and was identified as quercetin 3-O-rutinoside (rutin) (Figure 2(a)). Peak 7 generated the ion fragments of m/z 617 [M + Na]+, 595 [M + H]+, 449 [M + H−Rham]+, 433 [M + H−Glu]+, and 287 [kaempferol + H]+ and was identified as kaempferol 3-O-rutinoside (nicotiflorin) (Figure 2(b)). In analysis of flavonoid glycosides by UPLC-DAD-QTOF/MS, Jang et al. [36] reported that rutin and nicotiflorin were detected for the first time in Prunus tomentosa (Korean cherry, sweet cherry, and cherry). In the current study, morkotin A (MS fragments of m/z 795 [M + Na]+, 773 [M + H]+, 627 [M + H−Rham]+, 611 [M + H−Glu]+, 465 [M + H−Rut]+, and 303 [quercetin + H]+) and morkotin C (MS fragments of m/z 573 [M + Na]+, 551 [M + H]+, and 303 [quercetin + H]+) were newly identified in extracts of Korean mulberry fruit (Figure 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 [38–40], 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.


### 3.2. Flavonoid Contents of Mulberry Fruit

The contents of the ten flavonoids isolated from the 12 varieties of mulberry fruit are shown in Table2. The 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. The 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 Thailand (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. These 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. Thus, the total flavonoid contents of mulberry can vary according to the plant variety, the parts of the plant, and the stages of fruit maturity.Table 2
Contents of ten flavonoids isolated from 12 varieties of Korean mulberry fruit (Morus alba L.).
Peak number
Data given in units of mg/100 g dry weight
Cheong-Il
Hwan Ship Jo Saeng
Su Hyang
Dae Shim
Cheong-Il 4X
180-11
Shim Heung
Cheong Su
180-12
Dae Dang Sang
Baek Ok Wang
181-18
1a
1.3 ± 0.2
1.8 ± 0.1
1.6 ± 0.1
1.6 ± 0.2
1.2 ± 0.1
0.8 ± 0.0
1.5 ± 0.0
0.6 ± 0.1
0.7 ± 0.1
2.1 ± 0.0
0.3 ± 0.0
0.7 ± 0.0
2
0.6 ± 0.0
1.0 ± 0.1
1.0 ± 0.1
0.6 ± 0.1
0.5 ± 0.0
0.4 ± 0.0
0.5 ± 0.1
0.1 ± 0.0
2.8 ± 0.3
0.8 ± 0.0
0.4 ± 0.0
0.3 ± 0.0
3
ND
ND
ND
ND
ND
ND
ND
ND
0.5 ± 0.0
0.3 ± 0.0
ND
ND
4
24.0 ± 1.5
37.9 ± 2.7
38.1 ± 1.7
66.1 ± 4.1
22.1 ± 1.2
39.0 ± 1.7
60.5 ± 5.8
24.8 ± 4.2
20.6 ± 1.9
67.8 ± 5.4
7.8 ± 0.5
24.0 ± 1.1
5
5.5 ± 0.2
7.6 ± 0.4
7.5 ± 0.2
11.0 ± 0.5
4.8 ± 0.2
5.2 ± 0.3
6.2 ± 0.7
3.0 ± 0.4
17.1 ± 1.6
8.9 ± 0.8
5.7 ± 0.4
3.4 ± 0.1
6
19.6 ± 1.2
21.2 ± 1.5
20.8 ± 0.7
26.7 ± 1.4
18.0 ± 0.9
12.9 ± 0.4
ND
5.2 ± 0.9
15.5 ± 1.3
ND
13.0 ± 0.9
6.3 ± 0.2
7
2.2 ± 0.1
2.9 ± 0.2
2.4 ± 0.1
5.3 ± 0.3
1.9 ± 0.1
1.6 ± 0.1
5.0 ± 0.5
0.5 ± 0.1
3.1 ± 0.3
4.4 ± 0.3
1.6 ± 0.1
1.3 ± 0.0
8
2.0 ± 0.1
4.1 ± 0.3
4.1 ± 0.1
3.1 ± 0.4
1.6 ± 0.0
1.9 ± 0.1
ND
1.3 ± 0.2
3.8 ± 0.2
2.2 ± 0.3
2.5 ± 0.1
4.7 ± 0.1
9a
0.9 ± 0.1
1.1 ± 0.1
1.0 ± 0.1
1.4 ± 0.1
0.8 ± 0.1
0.7 ± 0.1
ND
0.3 ± 0.1
0.7 ± 0.0
ND
0.7 ± 0.0
0.3 ± 0.0
10
2.4 ± 0.1
3.5 ± 0.3
3.1 ± 0.1
4.1 ± 0.5
2.4 ± 0.2
1.3 ± 0.0
ND
0.5 ± 0.1
3.0 ± 0.3
ND
3.0 ± 0.2
1.3 ± 0.0
Total
58.6 ± 3.1
81.1 ± 5.6
79.6 ± 2.9
119.9 ± 7.0
53.3 ± 2.7
63.8 ± 2.4
73.8 ± 6.9
36.3 ± 6.1
67.7 ± 5.6
86.5 ± 6.3
35.0 ± 2.3
42.4 ± 1.5
All samples were analyzed in positive ion mode ([M + H]+) using UPLC-DAD-QTOF/MS. Each value calculated as mean ± SD of three replicates using galangin as internal standard. Peak 1: quercetin 3-O-rutinoside-7-O-glucoside (morkotin A); Peak 2: quercetin 3,7-di-O-glucoside; Peak 3: kaempferol 3,7-di-O-glucoside; Peak 4: quercetin 3-O-rutinoside (rutin); Peak 5: quercetin 3-O-glucoside (isoquercitrin); Peak 6: quercetin 3-O-(6″-O-malonyl)glucoside; Peak 7: kaempferol 3-O-rutinoside (nicotiflorin); Peak 8: kaempferol 3-O-glucoside (astragalin); Peak 9: quercetin 3-O-(2″-O-malonyl)glucoside (morkotin C); Peak 10: kaempferol 3-O-(6″-O-malonyl)glucoside; ND: not detected. aNew flavonoid identified in mulberry fruit.Of the 10 flavonoids isolated from mulberry fruit (Table2), rutin showed the highest content (range 7.8 ± 0.5 to 67.8 ± 5.4 mg/100 g DW), being detected at a retention time of 13.56 min (Peak 4). The highest contents of rutin were found in the Dae Shim and Dae Dang Sang varieties (66.1 ± 4.1 and 67.8 ± 5.4 mg/100 g DW, respectively), while the lowest was found in the Baek Ok Wang variety (white mulberry; 7.8 ± 0.5 mg/100 g DW). Butkhup et al. [42] reported that the major flavonoid compounds in eight varieties of mulberry fruit were (+)-catechin (309.26–750.01 mg/100 g DW), procyanidin B1 (62.59–224.41 mg/100 g DW), quercetin (5.36–58.42 mg/100 g DW), rutin (18.73–26.90 mg/100 g DW), and (–)-epicatechin (8.47–29.21 mg/100 g DW). Our study found that the major flavonoid in mulberry fruit was rutin (Peak 4), at levels higher than those found by Butkhup et al. [42]. Radojkovic et al. [44] reported that rutin was predominant among the six compounds (gallic acid, chlorogenic acid, ferulic acid, sinapic acid, rutin, and quercetin) identified in the dried extracts of mulberry fruit from two species (43.5 mg/100 g for M. alba fruit and 72.6 mg/100 g for M. nigra fruit). Katsube et al. [45] have also reported that rutin was the main flavonol glycoside of M. alba. Similarly, the present study also found rutin to be the most abundant of the ten flavonoids isolated from each of the 12 varieties of mulberry fruit except for Baek Ok Wang (white mulberry).The content of quercetin 3-O-glucoside (isoquercitrin) (Peak 5) ranged from 3.0 ± 0.4 to 17.1 ± 1.6 mg/100 g DW with the highest content found in the 180-12 variety (17.1 ± 1.6 mg/100 g DW) and the lowest in the Dae Shim variety (3.0 ± 0.4 mg/100 g DW). Pawlowska et al. [11] reported that the contents of quercetin 3-O-glucoside in M. alba and M. nigra fruits were 29 and 34 mg/100 g DW, respectively, which are higher concentrations than the levels observed in the present study. Overall, of the ten flavonoids assayed, the two new compounds (morkotin C and A), as well as quercetin 3,7-di-O-glucoside, kaempferol 3,7-di-O-glucoside, nicotiflorin, kaempferol 3-O-glucoside (astragalin), and kaempferol 3-O-(6″-O-malonyl)glucoside, were found at low levels. Kaempferol 3,7-di-O-glucoside (Peak 3) was detected in only two varieties: 180-12 (0.5 ± 0.0 mg/100 g DW) and Dae Dang Sang (0.3 ± 0.0 mg/100 g DW). Quercetin 3-O-(6″-O-malonyl)glucoside (Peak 6) and morkotin C (Peak 9) were not detected in the Shim Hueng and Dae Dang Sang varieties, while astragalin (Peak 8) was not detected in the Shim Hueng variety. Flavonols such as kaempferol and quercetin are bioactive compounds, possessing antioxidant and anti-inflammatory properties [46] and have been used for the prevention and treatment of cancers, cardiovascular disease, neurological and metabolic disorders, and bone diseases [39, 47]. In mulberries, kaempferol and quercetin are present mainly as glycosides such as rutin, isoquercitrin, astragalin, kaempferol-3-(6-O-malonyl)-β-D-glucoside, and quercetin-3-O-(6-O-malonyl)-β-D-glucoside, with contents varying according to variety, cultivation, harvest period, maturity, and heat-processing method [48].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). The 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. These 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. The 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. The 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. Therefore, 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.


## 3.1. Isolation and Identification of Flavonoid Compounds in Mulberry Fruits

Mulberries contain bioactive components, such as alkaloids and flavonoids [23–25] and when dried possess antioxidant, antimicrobial, antidiabetic, antiobesity, and anti-inflammatory properties [26–34]. Qualitative analysis by the HPLC-DAD-ESI-MS/MS method has previously been used to identify six nonanthocyanin phenolics from two mulberry varieties: 4-caffeoylquinic acid, chlorogenic acid, protocatechuic acid, quercetin, rutin, and taxifolin. Three others were also tentatively identified [35]: 3,5-dicaffeoylquinic acid, taxifolin-hexoside, and kaempferol-hexoside.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). The chemical structures of the individual flavonoids were determined by analysis of fragment ion patterns. Six compounds (Peaks 1, 2, 4, 5, 6, and 9) were identified as quercetin derivatives by MS based on the presence of m/z 303 [quercetin + H]+. Four other compounds (Peaks 3, 7, 8, and 10) were assigned as kaempferol derivatives based on the presence of m/z 287 [kaempferol + H]+. The MS spectra of the flavonoids isolated from the mulberry fruit are shown in Figure 2. The following flavonoid compounds were isolated and identified from the mulberry fruits: Peak 1, quercetin 3-O-rutinoside-7-O-glucoside (morkotin A); Peak 2, quercetin 3,7-di-O-glucoside; Peak 3, kaempferol 3,7-di-O-glucoside; Peak 4, quercetin 3-O-rutinoside (rutin); Peak 5, quercetin 3-O-glucoside (isoquercitrin); Peak 6, quercetin 3-O-(6″-O-malonyl)glucoside; Peak 7, kaempferol 3-O-rutinoside (nicotiflorin); Peak 8, kaempferol 3-O-glucoside (astragalin); Peak 9, quercetin 3-O-(2″-O-malonyl)glucoside (morkotin C); and Peak 10, kaempferol 3-O-(6″-O-malonyl)glucoside (Table 1).Figure 1
LC chromatograms of flavonoids in samples of Korean mulberry fruits (Morus alba L.).Figure 2
Mass spectra of flavonoids detected in extracts of Korean mulberry fruits. (a) Glycoside derivative of quercetin; (b) glycoside derivative of kaempferol; (c) MS spectra of two new flavonoids detected from extracting Korean mulberry fruits.
(a)
(b)
(c)Table 1
Ten flavonoids isolated from Korean mulberry fruits (Morus alba L.) and their mass spectrometric data.
Aglycones
Glycosides
Acylations
Peak number
Individual flavonols
MW
Fragment ions (m/z)
Kaempferol (m/z 287)
Mono
Mal
8
Kaempferol 3-O-glucoside (astragalin)
448
471, 449, 287
10
Kaempferol 3-O-(6″-O-malonyl)glucoside
534
557, 535, 287
Di
7
Kaempferol 3-O-rutinoside (nicotiflorin)
594
617, 595, 449, 287
3
Kaempferol 3,7-di-O-glucoside
610
633, 611, 449, 287
Quercetin (m/z 303)
Mono
Mal
5
Quercetin 3-O-glucoside (isoquercitrin)
464
487, 465, 303
6
Quercetin 3-O-(6″-O-malonyl)glucoside
464
573, 551, 465, 303
9
Quercetin 3-O-(2″-O-malonyl)glucoside (morkotin C)a
550
573, 551, 303
Di
4
Quercetin 3-O-rutinoside (rutin)
610
633, 611, 465, 449, 303
2
Quercetin 3,7-di-O-glucoside
626
649, 627, 465, 303
Tri
1
Quercetin 3-O-rutinoside-7-O-glucoside (morkotin A)a
772
795, 773, 627, 611, 465, 303
All samples were analyzed in positive ion mode ([M + H]+) using UPLC-DAD-QTOF/MS. aNew flavonoid identified in mulberry fruit.The major peak of the mulberry fruit (Peak 4) generated the ion fragments ofm/z 633 [M + Na]+, 611 [M + H]+, 465 [M + H−Rham]+, 449 [M + H−Glu]+, and 303 [quercetin + H]+ and was identified as quercetin 3-O-rutinoside (rutin) (Figure 2(a)). Peak 7 generated the ion fragments of m/z 617 [M + Na]+, 595 [M + H]+, 449 [M + H−Rham]+, 433 [M + H−Glu]+, and 287 [kaempferol + H]+ and was identified as kaempferol 3-O-rutinoside (nicotiflorin) (Figure 2(b)). In analysis of flavonoid glycosides by UPLC-DAD-QTOF/MS, Jang et al. [36] reported that rutin and nicotiflorin were detected for the first time in Prunus tomentosa (Korean cherry, sweet cherry, and cherry). In the current study, morkotin A (MS fragments of m/z 795 [M + Na]+, 773 [M + H]+, 627 [M + H−Rham]+, 611 [M + H−Glu]+, 465 [M + H−Rut]+, and 303 [quercetin + H]+) and morkotin C (MS fragments of m/z 573 [M + Na]+, 551 [M + H]+, and 303 [quercetin + H]+) were newly identified in extracts of Korean mulberry fruit (Figure 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 [38–40], 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.


## 3.2. Flavonoid Contents of Mulberry Fruit

The contents of the ten flavonoids isolated from the 12 varieties of mulberry fruit are shown in Table2. The 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. The 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 Thailand (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. These 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. Thus, the total flavonoid contents of mulberry can vary according to the plant variety, the parts of the plant, and the stages of fruit maturity.Table 2
Contents of ten flavonoids isolated from 12 varieties of Korean mulberry fruit (Morus alba L.).
Peak number
Data given in units of mg/100 g dry weight
Cheong-Il
Hwan Ship Jo Saeng
Su Hyang
Dae Shim
Cheong-Il 4X
180-11
Shim Heung
Cheong Su
180-12
Dae Dang Sang
Baek Ok Wang
181-18
1a
1.3 ± 0.2
1.8 ± 0.1
1.6 ± 0.1
1.6 ± 0.2
1.2 ± 0.1
0.8 ± 0.0
1.5 ± 0.0
0.6 ± 0.1
0.7 ± 0.1
2.1 ± 0.0
0.3 ± 0.0
0.7 ± 0.0
2
0.6 ± 0.0
1.0 ± 0.1
1.0 ± 0.1
0.6 ± 0.1
0.5 ± 0.0
0.4 ± 0.0
0.5 ± 0.1
0.1 ± 0.0
2.8 ± 0.3
0.8 ± 0.0
0.4 ± 0.0
0.3 ± 0.0
3
ND
ND
ND
ND
ND
ND
ND
ND
0.5 ± 0.0
0.3 ± 0.0
ND
ND
4
24.0 ± 1.5
37.9 ± 2.7
38.1 ± 1.7
66.1 ± 4.1
22.1 ± 1.2
39.0 ± 1.7
60.5 ± 5.8
24.8 ± 4.2
20.6 ± 1.9
67.8 ± 5.4
7.8 ± 0.5
24.0 ± 1.1
5
5.5 ± 0.2
7.6 ± 0.4
7.5 ± 0.2
11.0 ± 0.5
4.8 ± 0.2
5.2 ± 0.3
6.2 ± 0.7
3.0 ± 0.4
17.1 ± 1.6
8.9 ± 0.8
5.7 ± 0.4
3.4 ± 0.1
6
19.6 ± 1.2
21.2 ± 1.5
20.8 ± 0.7
26.7 ± 1.4
18.0 ± 0.9
12.9 ± 0.4
ND
5.2 ± 0.9
15.5 ± 1.3
ND
13.0 ± 0.9
6.3 ± 0.2
7
2.2 ± 0.1
2.9 ± 0.2
2.4 ± 0.1
5.3 ± 0.3
1.9 ± 0.1
1.6 ± 0.1
5.0 ± 0.5
0.5 ± 0.1
3.1 ± 0.3
4.4 ± 0.3
1.6 ± 0.1
1.3 ± 0.0
8
2.0 ± 0.1
4.1 ± 0.3
4.1 ± 0.1
3.1 ± 0.4
1.6 ± 0.0
1.9 ± 0.1
ND
1.3 ± 0.2
3.8 ± 0.2
2.2 ± 0.3
2.5 ± 0.1
4.7 ± 0.1
9a
0.9 ± 0.1
1.1 ± 0.1
1.0 ± 0.1
1.4 ± 0.1
0.8 ± 0.1
0.7 ± 0.1
ND
0.3 ± 0.1
0.7 ± 0.0
ND
0.7 ± 0.0
0.3 ± 0.0
10
2.4 ± 0.1
3.5 ± 0.3
3.1 ± 0.1
4.1 ± 0.5
2.4 ± 0.2
1.3 ± 0.0
ND
0.5 ± 0.1
3.0 ± 0.3
ND
3.0 ± 0.2
1.3 ± 0.0
Total
58.6 ± 3.1
81.1 ± 5.6
79.6 ± 2.9
119.9 ± 7.0
53.3 ± 2.7
63.8 ± 2.4
73.8 ± 6.9
36.3 ± 6.1
67.7 ± 5.6
86.5 ± 6.3
35.0 ± 2.3
42.4 ± 1.5
All samples were analyzed in positive ion mode ([M + H]+) using UPLC-DAD-QTOF/MS. Each value calculated as mean ± SD of three replicates using galangin as internal standard. Peak 1: quercetin 3-O-rutinoside-7-O-glucoside (morkotin A); Peak 2: quercetin 3,7-di-O-glucoside; Peak 3: kaempferol 3,7-di-O-glucoside; Peak 4: quercetin 3-O-rutinoside (rutin); Peak 5: quercetin 3-O-glucoside (isoquercitrin); Peak 6: quercetin 3-O-(6″-O-malonyl)glucoside; Peak 7: kaempferol 3-O-rutinoside (nicotiflorin); Peak 8: kaempferol 3-O-glucoside (astragalin); Peak 9: quercetin 3-O-(2″-O-malonyl)glucoside (morkotin C); Peak 10: kaempferol 3-O-(6″-O-malonyl)glucoside; ND: not detected. aNew flavonoid identified in mulberry fruit.Of the 10 flavonoids isolated from mulberry fruit (Table2), rutin showed the highest content (range 7.8 ± 0.5 to 67.8 ± 5.4 mg/100 g DW), being detected at a retention time of 13.56 min (Peak 4). The highest contents of rutin were found in the Dae Shim and Dae Dang Sang varieties (66.1 ± 4.1 and 67.8 ± 5.4 mg/100 g DW, respectively), while the lowest was found in the Baek Ok Wang variety (white mulberry; 7.8 ± 0.5 mg/100 g DW). Butkhup et al. [42] reported that the major flavonoid compounds in eight varieties of mulberry fruit were (+)-catechin (309.26–750.01 mg/100 g DW), procyanidin B1 (62.59–224.41 mg/100 g DW), quercetin (5.36–58.42 mg/100 g DW), rutin (18.73–26.90 mg/100 g DW), and (–)-epicatechin (8.47–29.21 mg/100 g DW). Our study found that the major flavonoid in mulberry fruit was rutin (Peak 4), at levels higher than those found by Butkhup et al. [42]. Radojkovic et al. [44] reported that rutin was predominant among the six compounds (gallic acid, chlorogenic acid, ferulic acid, sinapic acid, rutin, and quercetin) identified in the dried extracts of mulberry fruit from two species (43.5 mg/100 g for M. alba fruit and 72.6 mg/100 g for M. nigra fruit). Katsube et al. [45] have also reported that rutin was the main flavonol glycoside of M. alba. Similarly, the present study also found rutin to be the most abundant of the ten flavonoids isolated from each of the 12 varieties of mulberry fruit except for Baek Ok Wang (white mulberry).The content of quercetin 3-O-glucoside (isoquercitrin) (Peak 5) ranged from 3.0 ± 0.4 to 17.1 ± 1.6 mg/100 g DW with the highest content found in the 180-12 variety (17.1 ± 1.6 mg/100 g DW) and the lowest in the Dae Shim variety (3.0 ± 0.4 mg/100 g DW). Pawlowska et al. [11] reported that the contents of quercetin 3-O-glucoside in M. alba and M. nigra fruits were 29 and 34 mg/100 g DW, respectively, which are higher concentrations than the levels observed in the present study. Overall, of the ten flavonoids assayed, the two new compounds (morkotin C and A), as well as quercetin 3,7-di-O-glucoside, kaempferol 3,7-di-O-glucoside, nicotiflorin, kaempferol 3-O-glucoside (astragalin), and kaempferol 3-O-(6″-O-malonyl)glucoside, were found at low levels. Kaempferol 3,7-di-O-glucoside (Peak 3) was detected in only two varieties: 180-12 (0.5 ± 0.0 mg/100 g DW) and Dae Dang Sang (0.3 ± 0.0 mg/100 g DW). Quercetin 3-O-(6″-O-malonyl)glucoside (Peak 6) and morkotin C (Peak 9) were not detected in the Shim Hueng and Dae Dang Sang varieties, while astragalin (Peak 8) was not detected in the Shim Hueng variety. Flavonols such as kaempferol and quercetin are bioactive compounds, possessing antioxidant and anti-inflammatory properties [46] and have been used for the prevention and treatment of cancers, cardiovascular disease, neurological and metabolic disorders, and bone diseases [39, 47]. In mulberries, kaempferol and quercetin are present mainly as glycosides such as rutin, isoquercitrin, astragalin, kaempferol-3-(6-O-malonyl)-β-D-glucoside, and quercetin-3-O-(6-O-malonyl)-β-D-glucoside, with contents varying according to variety, cultivation, harvest period, maturity, and heat-processing method [48].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). The 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. These 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. The 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. The 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. Therefore, 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.


## 4. Conclusions

The 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. The 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.



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*Source: 1528917-2019-01-08.xml*