Chemicals and Bioactivity Discrimination of Syconia of Seven Varieties of Ficus deltoidea Jack via ATR-IR Spectroscopic-Based Metabolomics

Introduction: Ficus deltoidea is one of the common Malaysian medicinal plants and currently commercialized as raw ingredients in some local food products. However, those products do not discriminate the varieties of Ficus deltoidea used. Methods: FTIR-based metabolomics coupled with chemometric technique was applied to discriminate chemical components in ethanolic extracts of syconia of seven varieties of Ficus deltoidea namely; var. deltoidea, var. trengganuensis, var. kunstleri, var. angustifolia, var. bilobata, var. intermedia and var. motleyana. Unsupervised multivariate data analysis (MVDA) including principal component analysis (PCA) was used as to evaluate chemical variability among the seven varieties. For discrimination, orthogonal partial least square discriminant analysis (OPLS-DA) was applied, while partial least square (PLS) was used to evaluate the relationship between the alpha-glucosidase inhibition, antioxidant activity and Ficus deltoidea varieties. Results: As a result, OPLS-DA successfully discriminated the seven varieties. The FTIR fingerprints which were responsible for the discrimination includes 1729, 1705, 1448, 1095, 453, 443 cm-1. In addition, PPLS model demonstrated the correlation between var. kunstleri, var. deltoidea and var. intermedia respective chemicals fingerprints and their bioactivity (DPPH, FRAP and α-glucosidase inhibition). Conclusion: The findings revealed that FTIR spectroscopy, in combination with MVDA, can be used for structural functional discrimination in relation to the sample bioactivity.


Sample collection and preparation
Syconia of seven varieties of Ficus deltoidea (3 accessions from each variety) were collected from the germplasm of Faculty of Bioresources and Food Industry, Universiti Sultan Zainal Abidin.All syconia were carefully selected to be identical in terms of colour and ripening stage.The syconia of the seven varieties were deposited as voucher specimens at the Faculty's herbarium.The syconia samples were dried Pharmacognosy Journal, Vol 10, Issue 6 (Suppl), Nov-Dec, 2018 and then ground into powder form and stored at -20ºC before extraction.

Extraction
Dried and powdered syconia were extracted individually with ethanol in a soxhlet apparatus.The extraction was repeated twice after decanting off prior extracts.Each combined ethanol were filtered, pooled and concentrated under reduced pressure at 40°C to yield crude ethanolic extracts (EE).The dried extracts were stored at -20 °C before used.

Biological activities assays
DPPH radical scavenging ability of samples were measured following, 8 and modified by. 9 FRAP was measured according to, 10 and modified by. 11he alpha glucosidase assay, was performed according to, 12 using alpha glucosidase enzymes from Saccharomyces cerevisiae at different concentrations (1.56 to 100 μg/mL) as substrate and quercetin as standard.

ATR-FTIR analysis
ATR-FTIR spectra of extracts were collected by using a Bruker Analitik IFS 66 FTIR spectrometer (Ettlingen, Germany) equipped with a DTGS KBr detector and a Golden Gate Single Reflection Diamond ATR accessory (incident angle of 45°).Spectra were recorded in the absorbance mode at mid infrared region (4000-400 cm -1 ) by using 16 scans and 4 cm -1 resolution.Three spectra were obtained for each sample.

Statistical analysis
Results are presented as the mean values of three different experiments and are presented as the mean ± standard deviation (SD).Analysis of variance (ANOVA) was used to determine the differences on antioxidant activities and alpha glucosidase inhibition activity.The alpha level of all analysis was at 0.05.

Data pre-processing and multivariate analysis
Each FTIR spectrum was baseline corrected and smoothed to seven data points, 13 by using a software to minimize the differences between spectra due to the baseline shifts.The spectra were then exported to ASCII file and excel files were prepared for chemometric analysis.The multivariate analyses ((PCA, OPLS-DA, OPLS-DA-HCA and PLS models) of FTIR data were produced using SIMCA-P software version 13 (Umetrics, Umea Sweden).

RESULTS AND DISCUSSION
Most of the accessions showed DPPH free radical scavenging activity comparable to quercetin (standard).The IC 50 values of extracts (100 μg/mL) ranged from 13.58 ± 3.56 μg/mL to 79.3 ± 3.2 μg/mL, though they were less than that of quercetin (Table 1).In regards to the syconia, only few studies on some varieties of F. deltoidea have been found compared to the leaves part such as DPPH radical scavenging activity of syconia of var.deltoidea and var.intermedia (both with IC 50 ; 7.8 μg/mL). 14n FRAP assay, the ability of the plant/food extracts to reduce Fe 3+ to Fe 2+ were evaluated.The results showed that almost all extracts have strong reducing power.FRAP values of the extracts ranged from 1.1 (M234B) to 9.72 (K003) mmol/g which in comparison indicated significant differences (p<0.05) between all varieties (Table 1).FRAP of var.angusti- folia and var.kunstleri were found to be significantly lower (1.8 and 1.3 mmol/g) 15 than the similar varieties used in our study.The current study indicated strong α-glucosidase inhibition activity in most of the syconia extracts of the seven F. deltoidea varieties.Potential activity of all accessions was detected (IC 50 ) except from var. motleyana.
The representatives from the seven varieties with highest inhibition are shown in Figure 1.Among all accessions tested, the extracts of var.trengganuensis (T002) and var.kunstleri (K003) at different concentrations (1.56 to 100 μg/mL) exhibited the strongest activity with IC 50 of 36.7 and 37.8 μg/mL, respectively (Table 2).Our findings are consistently agreed with 16 who reported ethanol extract of Ipomoea aquatic had stronger α-glucosidase inhibition, in concentration dependent manner.The mean spectra of three different measurements were given in Figure 2.
The spectra showed broad peaks at wavenumbers in range 3600-3000 cm -1 and sharp peaks at 1604.77 and 1038 cm -1 .They suggested the presence of O-H bonds of either alcohols, phenols or water fraction at 3292.2 cm -1 , the sp 3 and sp 2 stretching of C-H bonds at bands ranging from 3000 cm -1 to 2850 cm -1 and the saturated fatty acids fraction at 2923 cm −1 . 17The C=O stretching of aldehyde group presence at 1734.3 cm -1 , 1 C=C stretching of alkenes and N-H bending of amines and amides at 1612.4 cm -1 , 1 and the vibration of N=O and stretching of C=C in aromatics at 1514.2 cm -1 , 1 were also assigned.Besides, other peaks at 1416 cm -1 were assigned as the alkane C-H bending, at 1368.6 cm -1 represent S=O, at 1162 cm -1 for carboxylic acids and 1043.5 cm -1 C-N for vibration of amines.In addition, the fingerprints of 1400 to 900 cm −1 may also be the carbohydrates signals. 17  FTIR data sets were subjected to principal component analysis (PCA) for unsupervised classification of syconia of the seven varieties of F. deltoidea.The first two principal components explained 80.2% of the total variance.The model revealed goodness of fit R 2 X (cum) and prediction of the model Q 2 (cum) with values of 0.986 and 0.973, respectively.The loading line plots showed that 2918, 2849, 1730 and 1708 cm -1 were the fingerprints contributed to the variation along PC1 Meanwhile, the loading plot along PC2 revealed that 1602 and 1440 cm-1 contributed the most to the variation (data not shown).In the present study, clustering of groups of varieties in PCA model of FTIR spectra were observed, though some of the varieties accessions were overlapped (Figure 3A).The overlapping of spectra was suggested to be due to similarity in their phytochemicals content.Meanwhile, the clusters differences were possibly due to other distinctive metabolites variation such exhibited by Sri Lanka green tea that clustered away from teas of other origins. 18thogonal partial least square-discriminant analysis (OPLS-DA) model was established as the supervised discrimination of syconia of seven varieties of F. deltoidea.The model was confirmed by the Q 2 Y and R 2 Y cumulatives, in which R 2 Y (cum) was 0.964 and Q 2 (cum) was 0.914.The first two PCs explained 39.2% of the variance.Six clusters were identified on PC1.The score plot showed that var.intermedia and var.bilobata were clustered together, revealing that the two varieties have phytochemicals similarity (Figure 3B).Var.trengganuensis, var.angustifolia and var.motleyana, discriminated in the positive quadrant of PC1, while, var.intermedia, var.bilobata, var.kunstleri and var.deltoidea were located into the negative quadrant of PC1, due to fingerprints at 1600, 1515, 1448, 1095 and 762 cm -1 .In addition, 1705, 443, 453, 484 cm -1 were the fingerprints that contributed to the discrimination of var.intermedia and var.angustifolia into the positive quadrant of PC2 (data not shown).Interestingly, var.bilobata and var.intermedia formed one cluster and discriminated themselves from var. kunstleri and var.deltoidea along PC2.The loading plot reveals that 1729, 1448, 1095, 1705, 443 and 453 cm -1 were the fingerprints with the highest contribution to the discrimination ((loading plot data not shown)).OPLS-DA-HCA was performed and similarities among the varieties were calculated using Euclidean distance and the clusters were established using ward hierarchical agglomerative method.the seven varieties of F. deltoidea showed discrimination which corresponded to bioactivity results (Figure 3D).

CONCLUSION
In general, the results show potential α-glucosidase inhibitory and antioxidant activities of syconia extracts of almost all varieties.Among all Ficus deltoidea varieties, most promising antioxidant and anti hyperglycemic activities were found in var.kunstleri (K003, K217, K313), var.trengganuensis (T002) and var.angustifolia (A171, A321).OPLS-DA model of FTIR data successfully discriminated the varieties, while PLS model had identified the correlation between var.kunstleri, var.deltoidea and var.intermedia bioactivity and their respective chemicals fingerprints, those were also identified.Thus, their syconia part were determined to be a good source of natural antioxidants as well as α-glucosidase inhibitors.Instead of being underutilized, the syconia of these targeted varieties (elite accessions) could be developed as ingredient in food products and health supplement in local agro-industry besides the widely studied Ficus deltoidea leaves.

Figure 1 :
Figure 1: Bioactivities of ethanolic extracts of the seven varieties of Ficus deltoidea syconia.A) DPPH free radical scavenging activity B) α-glucosidase inhibition.Data represented by the most active accession of each variety.Values are the means ± standard error based on three different experiments.

Figure 2 :
Figure 2: ATR-FTIR spectra of ethanolic extracts of syconia of the seven varieties of Ficus deltoidea.

Figure 3 :
Figure 3: Unsupervised and supervised multivariate data analysis (MVDA) of ATR-IR spectra to discrimination of chemical variability and the relationship between the alpha glucosidse inhibition, antioxidant activity and Ficus deltoidea varieties.A) Principal component analysis (PCA), B) Orthogonal partial least square discriminant analysis (OPLS-DA), C) Orthogonal partial least square discriminant analysis hierarchical cluster analysis (OPLS-DA-HCA) and D) Partial least square (PLS) model indicating the relationship between chemicals and extracts bioactivities.

Table 2 : α-glucosidase inhibition of ethanolic extracts of syconia of seven varieties of Ficus deltoidea. Accession Code α-Glucosidase Inhibition (IC 50 ; μg/mL)
The resulting dendrogram of the OPLS-DA shows the clustering of the seven varieties of F. deltoidea extracts.Three clusters (C1-C3) were observed.Accessions of var.trengganuensis and var.motleyana clustered in C1; var.kunstleri and var.deltoidea in C2; and var.angustifolia, var.intermedia and var.var.bilobata in C3 (Figure3C).HCA model of OPLS-DA gave distinctive clusters involving all accessions of each variety.This indicated that supervised HCA of OPLS-DA model was a better representation than HCA of PCA model in discussing hierarchy of chemicals clusters of the syconia of seven varieties of F. deltoidea.