Analysis of Flavonoid in Medicinal Plant Extract Using Infrared Spectroscopy and Chemometrics

Infrared (IR) spectroscopy combined with chemometrics has been developed for simple analysis of flavonoid in the medicinal plant extract. Flavonoid was extracted from medicinal plant leaves by ultrasonication and maceration. IR spectra of selected medicinal plant extract were correlated with flavonoid content using chemometrics. The chemometric method used for calibration analysis was Partial Last Square (PLS) and the methods used for classification analysis were Linear Discriminant Analysis (LDA), Soft Independent Modelling of Class Analogies (SIMCA), and Support Vector Machines (SVM). In this study, the calibration of NIR model that showed best calibration with R 2 and RMSEC value was 0.9916499 and 2.1521897, respectively, while the accuracy of all classification models (LDA, SIMCA, and SVM) was 100%. R 2 and RMSEC of calibration of FTIR model were 0.8653689 and 8.8958149, respectively, while the accuracy of LDA, SIMCA, and SVM was 86.0%, 91.2%, and 77.3%, respectively. PLS and LDA of NIR models were further used to predict unknown flavonoid content in commercial samples. Using these models, the significance of flavonoid content that has been measured by NIR and UV-Vis spectrophotometry was evaluated with paired samples t-test. The flavonoid content that has been measured with both methods gave no significant difference.


Introduction
Indonesia shows an amazing diversity of plants species that have been associated with the human health from time immemorial. Many of them were reported to have various desirable activities; however, only 20-22% were cultivated [1]. Research of Indonesian medicinal plants using modern laboratory facilities has been started since 1970, but only about 200 plants were studied. This figure shows a very small portion of the overall number of medicinal plant species that were reported [2]. Therefore, the analysis of chemical constituents would help in determining various biological activities of plants.
Studies have shown that many plants have chemical components and biological activities. The most important of these bioactive constituents of plant are alkaloids, flavonoids, terpenoids, steroids, tannins, and saponins [3]. Flavonoids are the most common and widely distributed group of plant phenolic compounds, occurring virtually in all plant parts, particularly the photosynthesising plant cells [4]. Flavonoids have been reported to exert multiple biological effects, including antioxidant, free radical scavenging abilities, antiinflammatory, and anticarcinogen [5].
Infrared spectroscopy is a technique based on the vibrations of the atoms of a molecule. The advantage of the infrared technique is that it can be nondestructive, requires a relatively small amount of sample, is fast, and is accurate [11,12]. Infrared technique does not require a reagent, so this method is more ecofriendly. It has been proved to be a powerful analytical tool used in many fields [12]. In recent years, NIR combined with chemometrics has attracted considerable attention in chemical content analysis [13]. NIR 2 Journal of Analytical Methods in Chemistry spectroscopy also shows promising ability for discrimination of similar biological materials, such as pea [14], fruits [15], and wine [16]. Some papers have been published regarding NIR quantitative analysis of active compound concentration in herbal products [17]. Multivariate statistical methods are very useful for processing of IR spectra. The big advantage of multivariate statistical methods is their capability to extract the information of IR spectra and explore this spectral information for qualitative or quantitative applications. The most frequently used of multivariate statistical methods (often called chemometric methods) are Linear Discriminant Analysis (LDA) and Partial Least Squares (PLS) regression [18].
The objective of this research is to develop a simple, rapid, and validated model of IR spectra for the determination of the flavonoid content. Furthermore, IR spectroscopy and chemometric methods were applied for determining flavonoid content in commercial samples.

Material and Reagents.
In this study, samples used were leaves samples collected from Materia Medica Botanical Garden, Malang, Indonesia (Table 1). Methanol, ethanol, Folin-Ciocalteu, potassium acetate (E. Merck, Darmstadt, Germany), and quercetin (Sigma-Aldrich) were of analytical grade reagent. Aquadest and Aerosil were of pharmaceutical grade. The solvents were used without further purification. Commercial extract capsules, Stimuno® and Daun Salam®, were purchased from a local pharmacy in Jember, East Java, Indonesia (October 2015).

Extraction Methods.
Dry leaves samples were mixed and finely powdered. 80.0 g of powdered sample was extracted with 800 mL of methanol in an ultrasonicator for an hour and continued being extracted by maceration for 24 hours. The extract was filtered through Whatman filter paper and then the solvent was evaporated using a rotavapour at 60 ∘ C. Extract was dried using Aerosil to yield dry extract.

NIR Spectra Acquisition.
Samples were scanned with a Brimrose, Luminar 3070 (Brimrose Corp, Baltimore, MD), with an integrating sphere. Before samples were measured, the instrument was warmed up for 30 minutes. The monochromator entrance slit was set on 500 pm, the amplifier was set on 200. the response time is smooth (1 ms), and light intensity was set on 14 volts. The wavelength range of spectra is from 8500-2000 nm and the data were measured in 5 nm intervals, which resulted in 120 points reflection.

FTIR Spectra
Acquisition. FTIR spectrometer (Alpha FTIR Spectrometer from Bruker optic), equipped with a deuterated triglycine sulphate (DTGS) as a detector and a germanium as beam splitter, interfaced to computer operating under Windows-based system, and connected to software of OPUS operating system (Version 7.0 Bruker optic), was used during FTIR spectra acquisition. A few drops of each sample were positioned in contact with attenuated total reflectance (ATR) plate.
FTIR spectra were collected at frequency regions of 4000-650 cm −1 by coadding 32 scans and at resolution of 4 cm −1 . All spectra were substracted against a background of air spectra. After every scan, a new reference of air background spectra was taken. The ATR plate was carefully cleaned by scrubbing with isopropyl 70% twice followed by drying with soft tissue before being filled in with the next sample, making it possible to dry the ATR plate. These spectra were recorded as absorbance values at each data point in replicate two times.

Determination of Total Flavonoids Content.
The flavonoids content was determined by aluminum chloride method using quercetin as a reference compound [19]. Sample was prepared by mixing 0.5 mL of 4 mg/mL sample extract in ethanol with 3 mL of ethanol, 0.2 mL of 10% aluminum chloride, and 0.2 mL of 1 M potassium acetate and then diluted to 25 mL with distilled water. After incubation at room temperature for 30 min, the absorbance of the mixture solution was measured at 432 nm using spectrophotometer (UV-Vis Hitachi U 1800). Various standard solutions of quercetin (2.0 up to 15.0 g/mL) were prepared from two stock solutions by dilution with ethanol.

Total Flavonoids Content.
The results for total flavonoids content in samples are presented in Table 3. The total flavonoids measurements were distributed around 4.03 up to 51.49 mg quercetin equivalence (QE)/g extract. Figure 1 showed NIR spectra of quercetin, dry extract, Aquadest, and Aerosil. Those spectra have a different intensity and typical characteristic of absorption bands. In the PLS calibration models, the evaluation of the linearity method was carried out in order to show a proportional relationship between the absorbance of NIR spectra versus the concentrations    used. LOOCV was performed as follows: one sample was left out from the calibration set, a model was built with the remaining samples in the calibration set, then the left-out sample was predicted by this model, and the procedure was repeated by leaving out each sample in the calibration set.

Calibration and Classification of NIR Models.
2 and the root mean square error of prediction (RMSEP) of LOOCV were 0.9986664 and 0.9136531, respectively (Figure 3).
Twofold cross-validation was used to validate the developed model using independent samples (test set). Five medicinal plant extracts were used as test set. 2 and RMSEP were 0.9823225 and 2.6224468, respectively (Figure 4).  The ability of NIR model (LDA, SIMCA, and SVM) to classify samples in flavonoid and matrix category can be seen through the accuracy of classification models. Table 4 shows 100% of accuracy, which means that the model could classify fifteen training set samples in a correct category. Figure 5 showed FTIR spectra of quercetin, dry extract, Aquadest, and Aerosil. Those spectra have similar intensity and characteristic of absorption bands in some segment of wavenumber. The results obtained from the PLS in terms of 2 , RMSEC for normal spectra, and segmentation were presented in Table 5. PLS calibrations in segment C (1650-1400 cm −1 ) revealed the highest of 2 and the lowest of RMSEC compared with other segments (Table 5). 2 and RMSEC were 0.8653689 and 8.8958149, respectively. However, this result was not good due to the fact that 2 was less than 0.99 and RMSEC was high.

Calibration and Classification of FTIR Models.
The ability of FTIR model (LDA, SIMCA, and SVM) was less than 100%, which means that the model could not classify fifteen training set samples in a correct category (Table 6).

Application of Commercial
Samples. PLS and LDA developed models of NIR spectra were further used to predict flavonoid in commercial samples. The results of flavonoids content in samples measured by NIR and UV-Vis spectrophotometry method are presented in Table 7. The paired samples t-test shows that flavonoid content that has been measured with both methods gave no significant difference ( > 0.05). Furthermore, all of the commercial samples were in the flavonoid category in LDA model.

Conclusion
The NIR spectroscopy combined with multivariate calibrations methods can be used to determine flavonoid in medicinal plant extract. The suggested method is simple, selective, validated, and ecofriendly.