Chemometric Analysis of Arbutin Derivatives from Paederia foetida and Vitis vinifera with Fourier Transform Infrared (FTIR)

Arbutin (β-arbutin) is a natural whitening compound of glycosylated derivatives from hydroquinone as tyrosinase inhibitors. This compound can be obtained from Ericaceae (berberry, strawberry, huckleberry), Saxifragaceae, Asteraceae, Rosaceae, Lamiaceae, Apiaceae and is found in pears (Pyrus communis L.). The percentage of arbutin in plants varies greatly according to species (17% in Arctostaphyllos uva ursi leaves, 5% in majorama leaves).1


INTRODUCTION
Arbutin (β-arbutin) is a natural whitening compound of glycosylated derivatives from hydroquinone as tyrosinase inhibitors. This compound can be obtained from Ericaceae (berberry, strawberry, huckleberry), Saxifragaceae, Asteraceae, Rosaceae, Lamiaceae, Apiaceae and is found in pears (Pyrus communis L.). The percentage of arbutin in plants varies greatly according to species (17% in Arctostaphyllos uva ursi leaves, 5% in majorama leaves). 1 Several attempts have been made to investigate safe and effective tyrosinase inhibitors from both natural and synthetic compounds. [2][3][4] Although there have been many studies and information about tyrosinase inhibitors, only few inhibitors can be applied due to their limitations in terms of cytotoxicity, selectivity and stability. 5,6 Skunk vine plant (Paederia foetida) and Grape (Vitis vinifera) are included.
Paederia foetida are one of the plants mentioned as having arbutin and have never isolated arbutin from these plants. This plant contains chemical compounds in the stem and leaves, namely asperuloside, deacetylasperuloside, scandoside, paederosid, paederosidic acid, gammasiitosterol, arbutin, oleanolic acid, and yawning oil. Grape plants (Vitis vinifera) have polyphenolic phytochemical content in the form of anthocyanins, tannins, flavonoids, resveratrol and phenolic acids. Polyphenols from grapes have a beneficial effect that can inhibit diseases such as heart disease, cancer, reduce plasma oxidation and slow aging.
In addition wine also has antioxidant, anticancer, anti-inflammatory, antiaging and antimicrobial effects. Another content contained in grape leaves is arbutin, mentioned that arbutin accumulates in grape leaves. 7,8 It is necessary, therefore, to identify and characterize arbutin compounds contained in Paederia foetida and Vitis vinifera using FTIR spectrophotometers and chemometric analysis.

Materials
Ethanol, ethyl acetate, chloroform, hexane, demineralized water and dimethyl sulfoxide (DMSO) were purchased from Merck. Indonesia. All chemicals and solvents were of analytical or pharmaceutical grade. Rotary Evaporatory (Buchi ® ) for vaporization of the solvents. IR data were collected on a IRPrestige-21 using a KBr pellet.

Extraction and partition
Paederia foetida L. and Vitis vinifera were weighed and extracted separately by maceration method using ethanol. The dried samples were weighed and then put into the maceration container, then 96% ethanol was added to be completely submerged. The maceration container was closed and stored for 3 x 24 hours in a place that is protected from direct sunlight while stirring occasionally. Then extracts were filtered and separated from their pulps and filtrates. The ethanol filtrate obtained was then collected and evaporated using the rotary evaporator. The liquidliquid partition was then carried out with n-hexane and water solvents. The n-hexan filtrate was collected and evaporated to obtain the dried extract. Then the ABSTRACT Introduction: Paederia foetida and Vitis vinifera were reported to have antioxidant activities due to their polyphenolic phytochemical contents. Arbutin may be included as active compounds in the plants. The chemometric analysis is used to identify the similarity of functional groups found in arbutin and that are in Paederia foetida and Vitis vinifera. Materials and Method: The ethanol extracts of Paderia foetida and Vitis vinifera were subjected to compound partition and characterization. Then a liquid-liquid partition is carried out using n-hexan and water solvents, followed by ethyl acetate and water partitions. This procedure is carried out continuously until the top layer is cleared. Partitioned compounds were analyzed using FTIR spectroscopy to identify functional groups. Furthermore, it was analyzed using the chemometric method (Principal Component Analysis and Cluster Analysis water phase is placed back into the separating funnel and repartitioned with ethyl acetate and water. This procedure was repeated 2 to 3 times.

Identification of phenolic compounds
To identify arbutin compounds includes polyphenol test, the extracts were added with 1% FeCl 3 solution. Results are shown in the form of green, red, purple, dark blue, blue, blackish blue, or blackish green. 9

Chemometric analysis
The Minitab software (Release 18; Minitab, State College, PA) was used for chemometric analysis including principal component analysis (PCA) and cluster analysis (CA) for the absorbance of FTIR spectra.

FTIR Spectroscopy
FTIR spectroscopy was used to determine the functional groups of a compound. From the IR spectra the arbutin compounds were    identified as hydroxyl groups, OH stretching (3380 cm -1 ), CH stretching (2989, 2929 and 2881 cm -1 ), and C-O stretching (1218 and 1291 cm -1 ).

Principal component analysis
Principal component analysis (PCA) is an unsupervised pattern recognition (Figures 1-4) method used in multivariate analysis. 9 In this study, PCA was accomplished using FTIR spectra absorbances of 4 evaluated arbutin compounds at 4 frequencies as shown in Tables 1 and  No Compounds Frequency     2. PCA provided score and loading plot, showing the distribution of the samples and variables employed on the principal components (PCs), respectively. The score plot ( Figure 5) exhibited patterns that may be correlated to sample characteristics. An eigenvalue of about 99.3% was achieved using two PCs. PC1 and PC2 described 95.3% and 4% of the variation, respectively. The study of the plote of loadings revealed distribution of variables and their correlations. From Figure 6, it is known that frequency regions at 1638.56 cm -1 and 3407.79 cm -1 make a larger contribution to the PCA model.
Cluster analysis (CA) is an algorithmic to construct hierarchy of clusters to assign observations to groups who are in the same class. In CA, clusters are visualized in dendogram graphs. Figure 7 shows a dendogram which is divided into two clusters: arbutin and Paederia foetida leaf with Euclidean distance 0.537, followed by Paederia foetida stem and Vitis vinifera.with Euclidean distance 1.157. In this study, CA results are in accordance with PCA results. They are in the same classes.

CONCLUSIONS
In this work, we partitioned the extract of Paederia foetida and Vitis vinifera extracts. Furthermore, we combined FTIR spectroscopy with chemometric analysis to exhibit the similarity in their physic and chemical properties. CA analysis showed that arbutin derivatives may be included in Paederia foetida (Euclidean distance 0.537) and Vitis Vinifera (Euclidean distance 1.157).