Qualitative and quantitative analysis of flavonoids from 12 species of Korean mulberry leaves

The total flavonoids in leaves of 12 varieties of Korean mulberry (Morus alba L.) were determined. Seventeen flavonoids were isolated and analyzed using ultra-performance liquid chromatography coupled with diode array detection and quadrupole time-of-flight mass spectrometry (UPLC–DAD–QTOF/MS). To determine the flavonoid contents, HPLC analysis was performed on these 17 flavonoids. The total flavonoid contents of the 12 varieties of mulberry leaves ranged from 748.5 to 1297.9 mg, with the highest obtained from the Cheong Su variety (1297.9 ± 112.0 mg). Among the 17 flavonoids analyzed, quercetin 3-O-rutinoside (rutin) and quercetin 3-O-glucoside (isoquercitrin) had highest contents in the Cheong Su variety. Furthermore, the Dae Dang Sang variety gave the highest quercetin 3-O-rutinoside (rutin) content among the mulberry leaves investigated, at 425.5 ± 45.9 mg. Major flavonols from Dae Dang Sang were detected by UPLC–DAD–QTOF/MS. A total of 17 flavonoid compound peaks were identified in the analysis time range of 5–40 min, all of which were kaempferol and quercetin glycosides. Seven of the 17 compounds identified in mulberry leaves were unknown.


Introduction
Mulberry (Morus spp.) is a deciduous tree that belongs to the genus Morus from the Moraceae family that consists of 10-16 species and is widely distributed in tropical, subtropical, and temperate regions globally (Jeong et al. 2014). Mulberry has been used in East Asia (Korea, China, and Japan) as an herbal medicine due to its various pharmacological effects, including antihyperglycemic (Singab et al. 2005), antiallergic (Chai et al. 2005), and immunomodulatory activities (Bharani et al. 2010).
Flavonoids are a large group of polyphenolic compounds found in fruits, vegetables, and herbs (Enkhmaa et al. 2005). Plants of the genus Morus are known to be rich in flavonoids, including quercetin 3-(6-malonylglucoside), rutin, isoquercitin (Katsube et al. 2006), cyanidin 3-rutinoside, and cyanidin 3-glucoside (Chen et al. 2006). These compounds are known to have potential antioxidant properties and probable roles in preventing oxidative stressassociated diseases (Haminiuk et al. 2012). Several researchers have studied the isolation, identification, and contents of flavonoid components in various mulberry species. Lee et al. (2004) reported that the five flavonoid contents for 20 cultivars of mulberry fruits varied from 9.80 to 69.69 mg/100 g (dry weight) through quantitative analysis with high performance liquid chromatography (HPLC). Furthermore, Katsube et al. (2006) identified that quercetin 3-(6-malonylglucoside) was the most abundant active component in dried mulberry leaves. Recently, Thabti et al. (2012) reported the identification and quantification of phenolic acids and flavonol glycosides in leaves of three mulberry species (M. alba var. alba, M. alba var. rosa, and M. rubra) using HPLC-DAD and HPLC-MS. They also reported the first identification of kaempferol-7-O-glucoside, quercetin-3-O-b-glucoside-7-O-a-rhamnoside, and quercetin-3-O-rhamnoside-7-O-glucoside in mulberry leaves. Furthermore, Thabti et al. (2014) reported that the highest total flavonoid content among leaves of three mulberry species were detected in M. rubra. In Korea, flavonoids in various species of mulberry leaves have not been investigated and their compositions are unknown. Furthermore, the utilization of different Morus species has been attempted, with interspecific hybridization conducted to incorporate desirable characteristics for crop improvement. Improving the foliage characteristics of mulberry (Morus spp.), both quantitatively and qualitatively, is the long-term goal for mulberry breeders. Therefore, in this work, we have determined the flavonoid contents of leaves from 12 varieties of mulberry using ultra-performance liquid chromatography coupled with diode array detection and quadrupole time-of-flight mass spectrometry (UPLC-DAD-QTOF/MS). This study aimed to provide basic mulberry breeding information for commercial purposes and functional utilization.

Plant material and reagents
Leaves from 12 mulberry species were collected from the Sericulture and Apiculture Division of the Department of Agricultural Biology, RDA (Jeon-Ju, South Korea). All mulberry leaf samples were cleaned and dried in a lyophilizer. All dried samples were pulverized and stored below -18°C prior to analysis. HPLC-grade acetonitrile, methanol, and water were obtained from Fisher Scientific (Fair Lawn, NJ, USA). Formic acid was purchased from Junsei Chemical (Tokyo, Japan). Galangin (Sigma Aldrich Co., St. Louis, MO, USA) was used as the internal standard solution.

Preparation of samples for instrument analysis
Sample extraction was conducted according to the method described by Kim et al. (2012) with minor modifications. The powdered leave (1 g) was mixed with 10 mL of acidified hydroalcoholic solvent (methanol/water/formic acid = 50:45:5, v/v/v) containing galangin (100 ppm) as internal standard. To extract flavonoids, the mixture was vortexed, stirred with a shaker (5 min, 200 rpm, and room temperature), and then centrifuged for 15 min at 3000 rpm and 10°C. The supernatant was filtered using a syringe filter (0.45 lm, PTFE, Whatman, Kent, England). A 0.5-mL aliquot of the filtrate was then diluted with water to a final volume of 5 mL. The flavonoid extract was then purified and isolated by solid-phase extraction using Sep-Pak C-18 cartridge (Waters Co., Milford, MA, USA). Sep-Pak activation was performed by washing the cartridge with methanol (2 mL), followed by water (2 mL) for conditioning. The diluted extract was then loaded onto the cartridge and impurities were removed by washing with water (2 mL). Finally, the flavonoid mixture was eluted using methanol (3 mL). The purified extract was then concentrated by blowing with N 2 gas, and then redissolved in the extraction solvent (0.5 mL) without internal standard prior to instrument analysis. All experimental analyses were performed in triplicate.

Quantification of flavonoids in mulberry leaves
To determine the flavonoid contents in 12 varieties of mulberry leaves, HPLC was performed using the 17 flavonoids isolated from the mulberry leaves. As shown in Table 2, the total flavonoid contents ranged from 748.5 to 1297.9 mg for the 12 varieties of mulberry leaves. The variety with the highest total flavonoid content was Cheong Su (1297.9 ± 112.0 mg). For comparison, Thabti et al. (2012) reported that the total flavonoid content of Morus rubra ranged from 193.87 to 398.33 mg RE/100 g DW, and was quantified as 450 mg (aqueous extracts) for the  Su Hyang (3) Dae Shim (4) Cheong-Il 4X (5) 180-11 (6) Shim Heung (7) Cheong Su (8) 180-12 (9) Dae Dang Sang (10) Baek Ok Wang (11) 181-18 (12  In Cheong Su, quercetin 3-O-rutinoside (rutin) and quercetin 3-O-glucoside (isoquercitrin) had the highest contents among the 17 flavonoids analyzed. Furthermore, in Dae Dang Sang, the quercetin 3-O-glucoside (isoquercitrin) content was lower than that of Cheong Su, while the quercetin 3-O-rutinoside (rutin) content was the highest among the mulberry leaves investigated, at 425.5 ± 45.9 mg. Major compounds (quercetin 3-O-rutinoside (rutin) and quercetin 3-O-glucoside (isoquercitrin)) were detected at retention times of 13.5 and 14.01 min in the LC chromatogram (peaks 8 and 10) from Dae Dang Sang (Fig. 1). Both Cheong Su and Dae Dang Sang had similar flavonoid contents. Buckwheat and some plant leaves have been shown to have rutin contents of 4-9% dry weight depending upon the stage of plant development (Kalinova et al. 2006). For dry fruits and vegetables, the rutin content was found to show little variation, ranging from 0.15 to 0.18% dry weight (Kalinova et al. 2006). Furthermore, isoquercitrin is a natural flavonoid glucoside found in medicinal and dietary plants, such as vegetables, herbs, and flowers, and, together with rutin, is a major glycosidic form of natural flavonol quercetin. Another study reported the isolation of isoquercitrin from Annona squamosa and demonstrated its protective action on diabetes mellitus, which is possibly mediated by enhanced insulin synthesis/secretion and/or decreased glucose-6-phosphatase activity (Panda and Kar 2007). From these results, we concluded that Cheong Su and Dae Dang Sang were good varieties for breeding and functional food development.
In contrast, from Cheong-Il 4X, polyphenol compound quercetin 3-O-(6 00 -O-malonyl) glucoside was detected at 361.4 ± 29.1 mg. In a previous study, ethanol extraction was performed on Cheong-Il and quercetin 3-O-(6 00 -Omalonyl) glucoside, a main bioactive substance with antidiabetic and antiarteriosclerosis properties, was determined to have a content of 143.25 mg/100 g, while quantitative changes in the six polyphenols in Cheong-Il, which is a mulberry cultivar widely used as a material in mulberry leaf tea, were investigated for three different heat pretreatments: steaming, roasting, and microwaving (Choi et al. 2015). Our data shows that the quercetin 3-O-(6 00 -Omalonyl) glucoside content of Cheong-Il was 283.9 ± 28.8 mg. The each other reason was due to other cultivation region and climate, even though same variety phenol compound. In contrast, quercetin 3-O-(6 00 -O-malonyl) glucoside was not detected on Cheong Su and Dae Dang Sang. Peaks 1, 2, 3, 5, 8, 10, 11, and 15 corresponded to quercetin derivatives, as confirmed by [quercetin?H] ? ion peak by MS,while others (4,6,7,9,12,13,14,16,and 17) (Fig. 3a). Quercetin 3-O-rutinoside-7-O-rhamnoside had the highest content at 11.31 min. Quercetin O-glycosides are quercetin derivatives with at least one O-glycosidic bond, and are widely distributed in plants. Quercetin 3-O-glycosides can occur as monosaccharides with glucose, galactose, rhamnose, or xylose. These compounds are found in various fruits, vegetables, and other anatomical parts of plants (Wiczkowski and Piskuła 2004). Quercetin 3-O-glucoside has been found, among others, in mango fruit (Berardini et al. 2005), whereas quercetin 3-O-rhamnoside has been detected in spinach (Kuti and Konuru 2004) and peppers (Materska et al. 2003). Quercetin 3-O-b-glucoside-7-O-arhamnoside has been isolated from leaves of Cotoneaster species (Kicel et al. 2016) and some Italian Aconitum species (Braca et al. 2003). In contrast, compounds 4, 7, 13, and 17 all showed an intense MS2 ion at m/z 287, suggesting that they were glycoside derivatives of kaempferol. Quercetin have been reported many beneficial effects, such as anti-inflammatory, antihypertensive, vasodilator effects, antiobesity, antihypercholesterolemic and antiatherosclerotic activities (Sultana and Anwar 2008;Salvamani et al. 2014). In addition, kaempferol have been shown to augment the body's antioxidant defense and reducing the risk of cancer (Chen and Chen 2013). However, the studies for biologically active of derivatives of quercetin and kaempferol in mulberry leaves are scarce, and it still remains to be seen whether these derivatives can really help augment biological effects in in-vivo of human. Thus, the 17 compounds including seven new compounds identified in mulberry leaves need that further investigation conducted to verify beneficial health effects for biologically active of human.

Conclusion
Flavonoid components extracted from leaves of 12 mulberry varieties from Korean cultivars were quantified. UPLC-DAD-QTOF/MS analysis was used, and the mulberry leaves constituents were analyzed using complementary information obtained from LC spectra, MS ions, and MS/MS fragments. The seven of the 17 compounds identified were observed in mulberry leaves, and further research will be devoted for evaluating their biological activities. This information on the concentration of functional materials in mulberry leaves could contribute to the development and promotion of processed functional products and facilitate the possible industrial use of mulberry, which could enhance the overall profitability of sericulture.