Biogenesis of C-Glycosyl Flavones and Profiling of Flavonoid Glycosides in Lotus (Nelumbo nucifera)

Flavonoids in nine tissues of Nelumbo nucifera Gaertner were identified and quantified by high-performance liquid chromatography with diode array detector (HPLC-DAD) and HPLC-electrospray ionization-mass spectrometry (HPLC-ESI-MSn). Thirty-eight flavonoids were identified; eleven C-glycosides and five O-glycosides were discovered for the first time in N. nucifera. Most importantly, the C-glycosyl apigenin or luteolin detected in lotus plumules proved valuable for deep elucidation of flavonoid composition in lotus tissues and for further utilization as functional tea and medicine materials. Lotus leaves possessed the significantly highest amount of flavonoids (2.06E3±0.08 mg 100 g−1 FW) and separating and purifying the bioactive compound, quercetin 3-O-glucuronide, from leaves showed great potential. In contrast, flavonoids in flower stalks, seed coats and kernels were extremely low. Simultaneously, the optimal picking time was confirmed by comparing the compound contents in five developmental phases. Finally, we proposed the putative flavonoid biosynthesis pathway in N. nucifera.


Introduction
Component analysis of bioactive compounds including flavonoids, proanthocyanins and essential oils, has been the target of much research [1][2][3]. Among these, flavonoids are the prevalent phytochemicals highlighted in recent research because of their diverse biological activities, including antioxidant, antiinflammatory, antiallergic, antimutagenic, antibiotic and anticarcinogenic properties [4]. Additionally, flavonoids have also gained the attention of many researchers as chemotaxonomic markers [5].
Flavonoids, with a C6-C3-C6 skeleton, are biosynthesized by a series of condensation reactions between hydroxycinnamic acid (B ring) and malonyl residues (A ring). As a result, these compounds exhibit a two-absorption band structure: a cinnamoyl system at 300-400 nm (Band I) and a benzoyl system at 240-280 nm (Band II). In plants, flavonoids are found in various modified forms, generated by hydroxylation, methylation, acylation and glycosylation, among which glycosylated flavonoids are by far the most common natural compounds. In addition, glycosylation may occur as C-glycosyl flavonoids with formation of a C-C bond by direct linkage of the sugar to the basic skeleton of the flavonoid. So far, C-glycosylation has been found only at the C6 and/or C8 position.
Nelumbo nucifera Gaertner, considered to be a traditional ornamental and medicinal plant, has been cultivated all over China. It is usually used as a vegetable or in Chinese traditional herbal medicines, and each part of the lotus is valuable. For example, lotus seed kernels, rhizomes, young leaves and flower petals are usually used as raw food materials. Lotus leaves and plumules have been highly valued in traditional Chinese medicine. Moreover, petals and stamens, rich in bioactive components such as flavonoids and alkaloids, are used in the treatment of tissue inflammation, cancer, skin diseases and for use as antidotes [6].
Weishan Lake, the fifth biggest fresh water lake in China, possesses more than thirty thousand hectares of N. nucifera cultivation. According to preliminary statistics, it can produce approximately two thousand kilograms of dried seed kernels and ten thousand kilograms of dessicated lotus leaves each year [7]. The remaining tissues of lotus are mostly wasted every year. Therefore, the comprehensive development of uses for various lotus tissues is believed to be a promising avenue of inquiry. This study profiled the flavonoids in various tissues in lotus, and plentiful C-glycosyl flavones were detected in lotus plumules for the first time. Therefore, the application of lotus plumules in medicine is intriguing and likely to be the next frontier.

Method validation
To verify the reliability of the optimized separation method, the analytical parameters were determined for anthocyanins with cyanidin 3-O-b-D-glucopyranoside (Cy-3-Glc) and other flavonoids with quercetin 3-O-a-L-rhamnopyranosyl-(1R6)-b-D-glucopyranoside (rutin). Table S.1 shows the results of method validation for two external standards (Cy-3-Glc, rutin). The calibration curves were established with five concentrations of each standard in triplicate. Results showed good linearity in relatively wide concentration ranges for both standards at 525 and 350 nm (r 2 $0.9993). The limits of detection (LODs) and the limits of quantitation (LOQs) were separately defined as a signal-to-noise ratio of 3:1 and 10:1. The LOD and LOQ were obtained for Cy-3-Glc (0.51 and 1.68 mg mL 21 ) and rutin (S I, 0.28 and 0.94 mg mL 21 ; S II, 0.68 and 2.27 mg mL 21 ) (Table S.1).
The precision of the optimized method was studied by examining the repeatability (intra-day analysis, n = 6) and intermediate precision (inter-day analysis, n = 3) for all the compounds separated from petals, seed coats and plumules because these three tissues contained all the anthocyanins and flavonoid components. Six samples of these tissues were extracted and analyzed on the same day to determine the intra-day precision. Three samples per day were also extracted and evaluated on three consecutive days to determine the inter-day precision. The results showed that the relative standard deviations (RSDs) of the 38 compounds were less than 2.51% in inter-day test and less than 2.35% for intra-day analysis (Table S.
The fragment ions of f30 and f33 were similar with f8 and f15, suggesting that both possessed a rutinoside substitution.  2 and m/z 314 [Y 0 ] 2 of f32, f28 and f32 were assigned as quercetin and isorhamnetin derivatives, respectively. The interglycosidic linkage in f28 and f32 was C1RC2 due to the relatively higher abundance of the radical aglycone at m/z 300 and m/z 314 [Y 0 2H]2 to the aglycone product ion at m/z 301 and m/z 315 [Y 0 ] 2 [15]. As expected, the characteristic of UV absorbance wavelength differed notably with different glycosylation positions. For example, the Band I of flavonoid 7-O-glycoside caused bathochromic shifts compared with 3-O-glycoside [19]. By comparing the UV absorbance wavelength of Band I with earlier study in chrysanthemum flower, soybean and the pollen of Typha angustifolia, compounds f28, f30, f32 and f33 were assigned as   [20][21][22]. They were all reported in N. nucifera for the first time.
2.3 Identification of C-flavones. It was more challenging to identify the C-glycosides in lotus plumules due to a lack of previous reports ( Fig. 2, 3). From UV spectra, resistance to acidic hydrolysis and a series of fragments [(M2H)260] 2 , [(M2H)290] 2 and [(M2H)2120] 2 , it was deduced that a number of C-glycosyl flavones were present in lotus plumules. Abad-Garcia et al.

Flavonoid contents
3.1 Flavonoids among different tissues. Flavonoids in each sample were quantified semi-quantitatively by linear regression of rutin at 350 nm and Cy-3-Glc at 525 nm (Table S4, S5). The composition and content of flavonoids varied widely among tissues (Fig. 1, 3, 4). The total flavonoid content (TF), which was defined as the sum total of all the flavonoids measured in this study, was significantly higher in leaves [(2.0660.08)E3 mg 100 g 21 , FW] than other tissues (Table S4). Subsequently, flower petals, stamens and lotus plumules exhibited relatively high TF, varying from 402.960.30 to 496.060.31 mg 100 g 21 , while the lowest concentrations, 0.67 mg 100 g 21 , appeared in the seed kernels (Fig. 4). In addition, flavonoids in the seed coats and flower stalks were relatively low as well. The results were in agreement with reported data in various lotus tissues [11].
Corresponding to previous studies, quercetin derivatives were the dominant compounds ($65.74%) in almost all the tissues, while flower petals and stamens possessed the highest amounts of kaempferol glycosides [11,14,28]. Specifically, Qu-3-GlcA (f11) and Ka-3-GlcA (f17) were the prevailing compounds within two derivative groups (Fig. 1, S2). Additionally f14 (Ka-3-Rob) and f16 (Ka-3-Glc) were the predominant compounds in petals. It was reported that Qu-3-GlcA was partly responsible for the elevation of antioxidant activity and that quercetin metabolites could also contribute to antioxidant activity [29]. Lotus leaves produced up to 1.83E360.07 mg 100 g 21 of Qu-3-GlcA. In addition, Weishan Lake produced appropriately ten thousand kilograms of dessicated lotus leaves each year demonstrating it as a potential source of lotus for the separation and purification of Qu-3-GlcA.
3.2 Flavonoids among developing stages. Depending on the specific tissue, the TF content increased (leaves, petals, stamens, pistils and tori, and plumules), decreased (flower stalks, seed coats and kernels) or remained constant (lotus seedpods) as the tissues developed (Fig. 4). Qu-3-GlcA was the major flavonoid component contributing to the notably higher TF value observed in leaves, pistils and tori than in other tissues. TF content of leaves reached a maximum at stage 4, while the level was continuously increasing in pistils and tori. In contrast, Qu-3-GlcA led to the decrease of TF within flower stalks, seed coats and kernels. It was noteworthy that TF in seed kernels, although related to plumules, had a slightly increase due to the presence of isovitexin in later stages. The content changes in petals were relatively more complicated than in the tissues mentioned above. The content of Qu-3-GlcA reduced, while levels of Ka-3-Glc and Ka-3-GlcA increased as the petals matured. A relatively greater amount of both Ka-3-Glc and Ka-3-GlcA ultimately increased TF, achieving the highest amount at stage 3 (Fig. 4). Similarly, kaempferol glycosides, especially the compound Ka-3-GlcA, kept pace with the sustaining increase in TF content in flower stamens. The TF value showed that flavonoids in lotus seedpods remained constant during all five stages (Fig. 4). The composition of flavonoids in plumules, which were rich in C-flavonoids, was extremely different from other tissues. And TF content increased continuously in accordance with the C-glycosyl apigenin derivatives. It was no surprise that flavonoid C-glycosides have been receiving considerable attention due to their wide range of biological activities, including anti-inflammatory, antimicrobial, antioxidant, antinociceptive, sedative, antihepatotoxic, antidiabetic, antihypertensive and radioprotective properties [30,31]. The medical and nutritive applications of lotus warrant further exploration.

Putative flavonoid biosynthesis pathway of lotus
Taking into consideration previous studies and these compounds newly detected in lotus tissues, we propose the putative flavonoid biosynthesis pathway in N. nucifera (Fig. 5) [32,33]. It begins from coumaroyl-CoA and malonyl-CoA. Centered on naringenin, the pathway is divided into nine sub-pathways for synthesis of anthocyanins and flavonoids, with the enzymes of F39H, FNS, F3H, FLS and F3959H. Thereinto, seven subpathways synthesize different aglycones of flavonols and flavones (diosmetin, luteolin, quercetin/isorhamnetin, kaempferol, apigenin, myricetin and syringetin), while the remaining two subpathways synthesize anthocyanins (delphinidin/petunidin/malvidin, and cyanidin/peonidin). This completes the first important modification of hydroxylation of flavonoids. Subsequently, the resulting secondary metabolites are glycosylated by glycosyltransferase at different positions, resulting in various flavonoid glycosides. Among them, O-glycosyl flavonoids are most prevalent in almost all the tissues, whereas C-glycosyl flavonoids appear in lotus plumules in large amounts. C-flavonoid glycosyltransferase (CGT) is firstly observed in lotus. Considering that CGT is significantly active only in lotus plumules, it may indicate the specific medicinal value of lotus embryos distinctly from its other tissues. However, the exact flavonoid biosynthesis pathway in lotus tissues still needs to be confirmed by further molecular and biochemical evidence.

Conclusions
In this study, we proposed two sensitive, reliable and reproducible solvent systems that could separate C-glycosyl flavonoids in lotus plumules, plus anthocyanins and other flavonoids from the remaining tissues. Flavonoids in nine tissues were studied, and we found that lotus leaves possessed the highest amount of flavonoids, followed by flower petals, lotus plumules and stamens. Moreover, we determined the optimum harvest time for vegetable, tea or medicinal purposes. Overall, thirty-three flavonoids were identified, in which eleven C-glycosides and five O-glycosides were detected for the first time in lotus tissues. The detection of plentiful C-glycosyl flavonoids has enhanced our understanding of flavonoid biosynthesis in lotus. These findings demonstrate the importance of further study of flavonoid Cglycosides because of their wide range of biological activities that could prove vital in the use of lotus plumules for medical and nutritional applications.

Plant materials
Nine different tissues including leaves, flower petals, flower stamens, flower pistils and tori, flower stalks, lotus seedpods, seed coats, seed kernels and lotus plumules of N. nucifera were collected at Weishan Lake (35u109N, 116u679E) in mid-July, 2010 (the authority responsible for the Weishan Lake Wetland Park). No specific permissions were required for these locations/activities, and this study did not involve endangered or protected species. Each tissue was divided into five developmental stages except for lotus plumules since the lotus seed of stage 1 was too unripe to be separated from the seed kernels. There were three divisions of developmental phases: flowers (petals/stamens/pistils and tori/ stalks), fruit (seed pods/seed coats/kernels/plumules), and leaves. Each phase was replicated three times using three individual plants (Fig. S3). These materials were first kept overnight in water, then were powdered in liquid nitrogen with mortars and pestles and subsequently stored at 240uC for latter analysis. All concentrations used in this study were calculated from fresh weight.

Preparation of standard solutions and flavonoid extractions
Standards of Cy-3-Glc and rutin were accurately weighted, separately dissolved in 0.1% HCl-MeOH and MeOH and finally diluted to appropriate concentrations to establish calibration curves at 525 and 350 nm. The others were prepared with MeOH for co-elution with samples and acid hydrolysed solutions.
The extraction method of flavonoids was modified from that of Chen et al [28]. Appropriate amounts of most materials were extracted with methanol-water (70:30, v/v), while petals were extracted with 70% methanol aqueous solutions containing 0.1%

Acid hydrolysis of flavonoids extraction
The filtered extract solutions of petals, seed coats and plumules including all the flavonoid components were dried in a rotary evaporator (35uC), re-dissolved in 4 mL 1.5 M HCl in a methanolwater solution (50:50, v/v) and then heated in a capped tube at 90uC for 2 h. The hydrolysate obtained was partially purified on the Oasis HLB Cartridge (Milford, MA, USA) before HPLC-DAD and HPLC-MS n analysis.

HPLC-DAD Systems and Conditions
The chromatographic separation was performed on a Dionex (Sunnyvale, CA, USA) system including a P680 HPLC pump, an UltiMate 3000 autosampler, a TCC-100 thermostatted column compartment and a PDA100 photodiode array detector. An aliquot of 10 mL solution was injected and analyzed on an ODS-80Ts QA C18 column (250 mm64.6 mm, Tosoh, Tokyo, Japan), which was protected with a C18 guard cartridge (Shanghai ANPEL Scientific Instrument, Shanghai, China). Chromatograms were acquired at 525 and 350 nm for anthocyanins and the other flavonoids, respectively, and photodiode array spectra were recorded from 200 to 800 nm. The mobile phase system for all tissues except for plumules was established based on the solution system in blueberry with little modification [34]. Finally, eluent A was 0.1% TFA aqueous solution; eluent B was 15% methanol in acetonitrile (solution system I, S I). A gradient elution as follows was used: 16% B at 0 min, 23% B at 10 min, 26% B at 30 min. The flow rate was 0.8 mL min 21 .
The separation of flavonoids from lotus plumules was accomplished using the following solvent and gradient: A, 10% formic acid in water; B, formic acid-acetonitrile-water (10:40:50, v/v/v); constant gradient from 15 to 45% B within 75 min, at a flow rate of 1.0 mL min 21 (solution system II, S II). Unfortunately, two peaks were difficult to achieve in simultaneous separation with the others. Another gradient elution was obtained with a linear elution gradient protocol of 0 min, 15% B; 55 min, 45% B, at a flow rate of 0.8 mL min 21 . Column temperature was maintained at 35uC for all analyses.

HPLC-MS n System and Conditions
HPLC-ESI-MS n analysis for anthocyanins and other flavonoids were carried out in an Agilent-1100 HPLC system coupled with a DAD system and a LC-MSD Trap VL electrospray ion mass spectrometer (Agilent Technologies, Palo Alto, CA, USA). The HPLC separation conditions were the same as mentioned above. Anthocyanins were adopted in PI mode and other flavonoids were employed both in PI and in NI mode. The MS conditions were as follows: capillary voltage, 4.0 kV; a nebulization pressure, 241.3 kPa; and a gas (N 2 ) temperature, 350uC; flow rate, 8.0 L min 21 . Capillary offset and exit voltage were separately 74.7 V and 113.0 V for both PI and NI mode. MS spectrum was recorded over the range from m/z 50 to 1000.
Since flavonoid C-glycosides in lotus plumules needed higher collision energies to fragment than O-glycosides, the MS condition was almost the same as the method above with a little modification. Capillary offset and exit voltage were improved to 89.2 V and 151.8 V in PI and NI mode, respectively.

Statistical Analysis
One-way analysis of variance test (ANOVA) was performed by SPSS 18.0 (SPSS Inc., Chicago, IL). Post hoc comparisons were accomplished with Duncan's test using the same statistical package. The differences were considered to be significant when p,0.05.