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 2022% were cultivated .
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 .
therefore , the analysis of chemical constituents would help in determining various biological activities of plants .
the most important of these bioactive constituents of plant are alkaloids , flavonoids , terpenoids , steroids , tannins , and saponins .
flavonoids are the most common and widely distributed group of plant phenolic compounds , occurring virtually in all plant parts , particularly the photosynthesising plant cells .
flavonoids have been reported to exert multiple biological effects , including antioxidant , free radical scavenging abilities , anti - inflammatory , and anticarcinogen .
several analytical techniques have been developed for determining total flavonoids concentration such as gas chromatographic ( gc ) , mass spectrometry , thin layer chromatography , uv spectrometry , and high performance liquid chromatography ( hplc ) .
these methods are precise but all time - consuming , requiring many reagents , and costly .
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 ] .
it has been proved to be a powerful analytical tool used in many fields . in recent years ,
nir spectroscopy also shows promising ability for discrimination of similar biological materials , such as pea , fruits , and wine .
some papers have been published regarding nir quantitative analysis of active compound concentration in herbal products .
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 .
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 .
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 .
commercial extract capsules , stimuno and daun salam , were purchased from a local pharmacy in jember , east java , indonesia ( october 2015 ) .
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 60c .
samples were scanned with a brimrose , luminar 3070 ( brimrose corp , baltimore , md ) , with an integrating sphere .
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 85002000  nm and the data were measured in 5  nm intervals , which resulted in 120 points reflection .
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 4000650  cm by coadding 32 scans and at resolution of 4  cm .
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 .
the flavonoids content was determined by aluminum chloride method using quercetin as a reference compound .
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 .
calibration and classification model for determination of flavonoids content were formed by training set samples that consists of fifteen medicinal plant extracts , quercetin , aquadest , and aerosil .
the training set samples of medicinal plant extracts have been traced having varied flavonoid content which is expected to represent variations of flavonoid content of all plants .
the linear discriminant analysis ( lda ) , soft independent modelling of class analogies ( simca ) , and support vector machines ( svm ) were used to develop classification model .
matrix category was sample without flavonoids content ( aquadest and aerosil ) and flavonoid category was sample with flavonoid content ( leaves extracts and quercetin ) .
partial least square ( pls ) was used to develop calibration model for total flavonoids content .
the pls model was then validated with leave - one - out cross - validation ( loocv ) and 2-fold cross - validation ( five test set samples ) . the training set and test set samples were shown in table 2 .
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 of flavonoid .
the correlation data of pls model in figure 2 showed good performance of pls model , indicated by coefficient of determination ( r ) higher than 0.99 and the low value of rmsec .
r and the root mean square error of calibration ( rmsec ) were 0.9916499 and 2.1521897 , respectively .
therefore the calibration model can be used as a tool to predict the concentration of flavonoid content in medicinal plant . in order to validate the developed model ,
leave - one - out cross - validation ( loocv ) and 2-fold cross - validation were 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 .
r 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 ) .
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 r , rmsec for normal spectra , and segmentation were presented in table 5 .
pls calibrations in segment c ( 16501400  cm ) revealed the highest of r and the lowest of rmsec compared with other segments ( table 5 ) .
r and rmsec were 0.8653689 and 8.8958149 , respectively . however , this result was not good due to the fact that r was less than 0.99 and rmsec was high .
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 ) .
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 ( p > 0.05 ) .
furthermore , all of the commercial samples were in the flavonoid category in lda model .
the nir spectroscopy combined with multivariate calibrations methods can be used to determine flavonoid in medicinal plant extract .
 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 r2 and rmsec value was 0.9916499 and 2.1521897 , respectively , while the accuracy of all classification models ( lda , simca , and svm ) was 100% . 
 r2 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 . 
1. Introduction
2. Materials and Methods
3. Result and Discussion
4. Conclusion