FLAVONOID DISTRIBUTION IN FOUR VARIETIES OF Ficus deltoidea (JACK)

We have previously reported the antioxidant and neuroprotective activities of the aqueous extract of four varieties of Ficus deltoidea ( Fd ) (Moraceae) namely var kunstleri ( Fdvk ), var angustifolia ( Fdva ), var deltoidea ( Fdvd ) and var intermedia ( Fdvi ). In this study, flavonoid constituents in aqueous leaf and fig extracts of the four varieties were analyzed and characterized using liquid chromatography mass spectrometer quadrupole-time of flight (LCMS-Q-TOF) via hierarchical cluster analysis (HCA) technique. The HCA dendrogram revealed that the abundant flavonoids among the eight samples are epi catechin, quercetin-3-rutinoside, quercetin 5,4'-di- O -beta-D-glucopyranoside, myricetin and naringenin. The study found that the distribution of the flavonoids differed between the four varieties and varied within the plant parts. To date, the flavonoid distribution of the different plant parts of the four varieties has not been documented. A positive correlation was observed between flavonoid constituents present and radical scavenging activities of the aqueous extracts. new biomarkers for disease and drugs. Analysed data from LCMS-Q-TOF has been used to examine the untargeted metabolites in plant samples and to find the ones with statistically significant variations in abundance within a set of experimental versus internal and external database [29]. In analysing LCMS-Q-TOF data, hierarchical clustering analysis (HCA) is an accurate technique in which results are represented as a two-dimensional diagram known as a dendrogram. HCA demonstrate a clear clusterisation of plant specimens selecting the highest discriminating ions given by the complete data analysis and this lead to the specific identification of metabolites [12]. Continuing our interest in the biological activities of the plant, this study was aimed at identifying flavonoids in the aqueous leaf and fig extracts of four varieties of Fd namely var angustifolia ( Fdva ), var deltoidea ( Fdvd ), var intermedia ( Fdvi ), and var kunstleri ( Fdvk ) via LCMS-Q-TOF method. Distribution of the flavonoids was determined by HCA technique and correlation of flavonoid constituent and radical scavenging activities was made.


INTRODUCTION
Ficus deltoidea (Mas Cotek) has long been used as traditional medicine in Malaysia and exists in seven varieties [39]. Malay traditional practitioners extensively consume Mas Cotek as tea. It is also used both externally and internally to treat an extensive list of conditions; including wounds, headache, fever [20], diabetes [31], toothache and as herbal drink for women to strengthen the uterus after childbirth [27] as well as other diseases related to oxidative stress. The reported biological activities of this plant include antioxidant, antihyperglycemic, anti-inflammatory, antiulcerogenic and antinociceptive ( [36]; [15]; [26]; [27]; [2]). [2] reported that almost all of the parts of Fd plant including the root, bark, leaf and fig have their own medicinal properties. Previous studies on the aqueous extracts showed that it contained antioxidant flavonoids including flavan-3-ol monomers namely catechin, epicatechin, gallocatechin and epigallocatechin [22]. It was earlier reported to contain polyphenols, flavonoids and tannins but was absent of alkaloids, triterpenes and saponins ( [38]; [9]). [33] have reported flavonoids as the antioxidant scavengers of a wide range of ROS and inhibitors of lipid peroxidation. This class of natural products represents the most abundant antioxidants in the diet and they have gained tremendous interest as potential therapeutic agents against a wide variety of diseases, most of which involve oxidant damage [25]. Flavonoids may interfere in several of the steps that lead to the development of malignant tumors, including protecting DNA from oxidative damage, inhibiting carcinogen activation, and activating carcinogen-detoxifying systems [7,13].
Today, liquid chromatography mass spectrometer quadrupole-time of flight (LCMS-Q-TOF) has been shown to be a powerful tool in the search of new biomarkers for disease and drugs. Analysed data from LCMS-Q-TOF has been used to examine the untargeted metabolites in plant samples and to find the ones with statistically significant variations in abundance within a set of experimental versus internal and external database [29]. In analysing LCMS-Q-TOF data, hierarchical clustering analysis (HCA) is an accurate technique in which results are represented as a two-dimensional diagram known as a dendrogram. HCA demonstrate a clear clusterisation of plant specimens selecting the highest discriminating ions given by the complete data analysis and this lead to the specific identification of metabolites [12].
Continuing our interest in the biological activities of the plant, this study was aimed at identifying flavonoids in the aqueous leaf and fig extracts of four varieties of Fd namely var angustifolia (Fdva), var deltoidea (Fdvd), var intermedia (Fdvi), and var kunstleri (Fdvk) via LCMS-Q-TOF method. Distribution of the flavonoids was determined by HCA technique and correlation of flavonoid constituent and radical scavenging activities was made.

Plant Materials
The fresh leaf and fig of four varieties of Fd namely Fdvk, Fdva, Fdvd, and Fdvi, were collected from Kuala Pilah, Negeri Sembilan, Malaysia and were identified by Mr. Kamarudin Saleh (botanist) from Forest Research Institute Malaysia (FRIM). The samples were cleaned, dried under room temperature and finely ground. 100 g of each ground sample was heated separately in 500 ml of distilled water by continuous stirring at 60°C for 1 hour. The aqueous extracts were then filtered, concentrated under reduced pressure and stored at -20°C before being subjected to further analysis.

Total Flavonoids Content (TFC) Assay
Total flavonoids content were measured by a colorimetric assay developed by [37]. A 1 ml aliquot of appropriately diluted sample or standard solutions of catechin (20,40,60,80, and 100 mg/ml) was added to a 10 ml volumetric flask containing 4 ml distilled water. At zero time, 0.3 ml 5% NaNO2 was added to the flask. After 5 minutes, 0.3 ml 10% AlCl3 was added. At 6 minutes, 2 ml 1 M NaOH was added to the mixture. Immediately, the reaction flask was diluted to volume with the addition of 2.4 ml of distilled water and thoroughly mixed. Absorbance of the pink coloured mixture was determined at 510 nm against water blank. Total flavonoid of extract was expressed in milligram catechin equivalents (mg CE/g extract). All analyses were run in three replicates and mean values were recorded.

1,1-diphenyl-2-picrylhydrazyl (DPPH) Radical Scavenging Assay
This assay was carried out as described by [28] with some modification [4]. Stock solutions of crude extracts were prepared as 1 mg/ml in methanol. The solutions were diluted to different concentrations (250, 125, 62.5 and 31.3 μg/ml in methanol) in a 96-well microtiter plate. Then, 5 μl of DPPH solution (prepared as 10 mg/ml in methanol) were added to each well. The plate was shaken gently and placed in the dark for 30 minutes at room temperature. The absorbance was then measured at 517 nm. Percentage of inhibition was calculated using the following formula: % Inhibition = x 100% D e c e m b e r 1 8 , 2 0 1 4

Liquid Chromatography Conditions
Chromatography was performed on Liquid Chromatography of 1200 Rapid Resolution Series (Agilent Technologies, Santa Clara, CA, USA) consisting of a binary pump, degasser, 96-well plate autosampler with thermostat, thermostat column compartment and 6520 Q-TOF mass spectrometer equipped with a dual-ESI source. The column used was a Zorbax Eclipse Plus C18 with column ID of 1.8 μm particle size and 2.1 x 100 mm column dimensions [3]. The temperature was maintained at 40°C during the run.

Solvent System Conditions
The mobile phase (A) consisted of 0.1% formic acid (Supelco, Inc) in water and (B) 0.1% formic acid in acetonitrile. The flow rate was 0.25 mL/min and the injection volume was 2 μL. A linear gradient was developed over 36 minutes from 5% to 95% of mobile phase (B). Total run time was 48 minutes for each analysis. ESI source settings were as follows: V Cap 4000 V, skimmer 65 V and fragmentor 125 V.

Mass Spectral Conditions
Mass spectral acquisition range selected was from 50 to 1700 m/z. The nebulizer was set at 45 psig and the drying gas nitrogen was set at a flow rate of 12 L/min. Drying gas temperature was maintained at 350°C. Data was acquired at a rate of 2.5 spectra/second with a stored mass range of 50 to 1000 m/z. Internal reference ions were used to correct mass accuracy. Autocalibration parameters were chosen to average five scans and reference mass correction was enabled throughout the run.

Determination of Total Flavonoids Content (TFC) and Radical Scavenging Activities of the Aqueous Extracts
The total flavonoids content in the aqueous extracts of each of the four variety was determined through a linear catechin standard curve (y = 0.002x + 0.014; R 2 = 0.999) where x is the catechin concentration in mg/L and y is the absorbance reading at 510 nm. The total flavonoids content of each extract of the four varieties of Fd were expressed in mg catechin/g extract or (mg CE/g extract). Table 1  [38] studied the content of flavonoids in the aqueous leaf extract of Fdva and Fdvd using colorimetric assay. The authors found that the TFC of the aqueous leaf extract of Fdvd was higher than the value for Fdva with 42.63 and 27.35 mg quercetin/g extract, respectively. Their results showed the same trend as found in our study (89.75 and 61.10 mg GAE/g extract for Fdvd and Fdva, respectively) with TFC value of Fdvd about 1.5 times higher than Fdva. The TFC values of the aqueous extracts of previous studies may be different with our study due to the employment of quercetin as a standard instead of catechin.  To date, no report has been found on the radical scavenging activity of the aqueous extract on any of the varieties of Fd. However, a study on methanolic extracts of F.microcarpa found DPPH radical scavenging activity of 7.3 and 21.4 μg/ml of fruits and leaf, respectively [6]. These values are comparable to the values found in our study.

Identification of Compounds Present in the Aqueous Leaf and Fig Extracts of Four Varieties of Fd
The   Fig. 1(a) and confirmed by comparison with standard. Epicatechin is a flavan-3-ol belonging to catechin family and can be found in natural products or medicinal herbs. It commonly exists in monomeric and oligomeric forms [21]. Employing the DPPH method, [22] had identified 18% antioxidant activity of epicatechin (tR, 19.4

min; m/z, 289 [M-H]
-) in the aqueous infusion of leaf of Fd. It showed a strong fluorescent peak using HPLC-PDA-MS 2 , which gave rise to MS 2 ions at m/z 245, 205, and 179. Their results were confirmed by co-chromatography with an authentic standard and it indicates that epicatechin found in Fd may be utilized as a potential antioxidant. Similarly, [6] also used DPPH method and found that epicatechin from the ethyl acetate fraction of aerial root and bark of Ficus microcarpa exhibited excellent antioxidant activity.
As shown in Fig. 1(b), peak 2 (tR, 9.361 min) indicated the presence of rutin (quercetin-3-rutinoside) [M+H] + at m/z 611.1611. Rutin is a flavonol and comprises quercetin and the disaccharide rutinose [35]. The first documented report on flavonoids profiling in different varieties of the hot and cold aqueous extracts of Fd leaf namely Fdvk and Fdva by [14] using reversed-phase HPLC reported a higher rutin content in Fdvk compared to Fdva. In contrast, the results obtained in our study indicated that the content of rutin in the aqueous extracts of Fdvk is similar to Fdva based on the dendrogram shown in Fig. 3. A study by [24] found that rutin have beneficial effect on spatial memory along with the concentration of brain neurotransmitters in aged rats. Rutin also showed antidiabetic potential in streptozotocin-induced diabetic istar rats as reported by [18].

Peak 3 (tR, 9.377 min) had a [M+H]
+ at m/z 627.1587 and was identified as quercetin 5,4'-di-O-beta-D-glucopyranoside as shown in Fig. 1(c). [19] had earlier reported a relative molecular mass of 626 for the compound from its FD-MS spectrum. Its aglycone, quercetin has often been proven by in vitro tests to act as a powerful antioxidant. The compound being a major constituent of the flavonoid intake, has been suggested to be a key in fighting several chronic degenerative diseases. It also exhibited a marked neuroprotective effect in in vitro experiments and this is mainly explained by its antioxidant capacity and ability to scavenge free radicals [11]. To date, there has been no comparative study reported on the quercetin or quercetin derivatives from the four varieties of Fd.
Peak 4 (tR, 9.553 min) showed an m/z 319.0435 corresponding to [M+H] + and was identified as myricetin as shown in Fig.  1(d). Myricetin (3,3',4',5,5',7-hexahydroxylflavone) is a natural flavonol with hydroxyl substitutions at the 3,3',4',5,5' and 7 positions [34]. A study by [8] had reported the myricetin capacity in protecting PC12 cells from oxidative insult (H2O2) and at the same time, it increases the cell survival. A study by [32] found myricetin as the major flavonoid in the ethanolic leaf extract of Ficus carica using reversed-phase HPLC and analyzed by UV/Vis array and electrospray ionization (ESI)-mass spectrometry (MS) detectors. Interestingly, [22] who studied Fd did not find myricetin in the aqueous extract. There has also been no other report on its detection from any variety of Fd species.
Peak 5 (tR, 16.013; m/z, 273.0770) was identified as naringenin as shown by Fig. 1(e). Naringenin (4′,5,7trihydroxyflavanone) is also known as aglycone of naringin and classified as a flavanone [23]. Naringenin extracted from the methanol leaf extract of F.benjamina was also reported to possess cytotoxicity against T-lymphoblastic leukemia (CEM-SS) cell line [5]. To date, there has been no report on the isolation or detection of this compound from

Hierarchical Cluster Analysis (HCA) of Aqueous Leaf and Fig Extracts of Four Varieties of Fd
HCA is the most popular clustering technique to evaluate the quality of the medicinal plants [17] and it is a simple method of grouping a set of available data based on their similarities [30]. The presentation of HCA results as a dendrogram makes it such a way the relationships can be more readily visualized. In this study, HCA using Euclidean distances and average linkage method was used for clustering its ability to separate the data into four clusters. In the dendrogram shown in Table 3 To the best of our knowledge, no comparative study regarding this analysis of plant parts of the aqueous extracts of the four varieties of Fd has been documented. In addition, the distribution and intensity of flavonoid constituents in the aqueous leaf and fig extracts of the four varieties were seen to be consistent with their radical scavenging activities as measured by the DPPH assay. Both Fdvd and Fdvi (leaf and fig extracts) exhibited strong antioxidant potential which may be related to the high amount of epicatechin, in this plant variety. A study by [16] had reported the scavenging activity of hydroxyl groups present on ring B of flavonoids, specifically the structure 3'-4' catechol (catechol moiety). The presence of a hydroxyl group in position 3 added to the catechol structure of flavonoid (as shown in Fig. 3) was found to increase tenfold scavenger activity towards free radicals. extract. This is also supported by our previous study which found strong antioxidant and neuroprotective effects for the same two plant varieties [10]. D e c e m b e r 1 8 , 2 0 1 4