Extraction and inhibition of α-glucosidase activity
The compounds from A. paniculata were extracted by maceration at room temperature. The results showed that the extraction yield using different concentrations of ethanol and water solvents differed slightly (Table 1). The highest yield was obtained using 50% ethanol, and the lowest using water, indicating that different concentrations of solvent extraction affected the level of metabolite extracted.
The effect of different extraction solvent concentrations on α-glucosidase inhibitory activity was also determined. The assay was based on the principle that α-glucosidase will hydrolyze glucose in the substrate (p-nitrophenyl-α-D-glucopyranoside) to α-D-glucose and p-nitrophenol so that inhibitory activity can be measured based on the amount of p-nitrophenol produced. Table 1 shows that α-glucosidase inhibitory activity was highest in the 50% ethanol extract followed by 70%, 30% ethanol, water and pure ethanol. This result showed that a combination of water and ethanol could extract more polar and semi-polar compounds that are known to have α-glucosidase inhibitory activity.
HPLC fingerprint and andrographolide content
Each extract from A. paniculata was analyzed using HPLC to determine the effect of the extraction solvent concentration on the composition of the extracted metabolites. Fig. 1 shows the fingerprint chromatogram of the A. paniculata extracts. Overall, 23 peaks with a percentage area of more than 5% were detected in the extracts. Peak 15 (andrographolide) was the major peak in A. paniculata with the highest intensity and peak area in all extracts. The fingerprint chromatograms obtained of all samples had a similar pattern with peaks 2, 7, 8, 10, 11, 13, 15, and 21 appearing in every sample extract. The differences between the peaks were mostly in peak height and area because each solvent used for extraction exhibited a different polarity and ability for extracting the chemical compounds.
Another difference was that some peaks, such as peaks 12 and 22, appeared only in the ethanol extract thus indicating a typical peak for the fingerprint pattern of ethanol extracts. Peak 1 also appeared in the 30%, 50% ethanol, and water extracts. The extraction solvents with a greater polarity also led to a greater number of detected peaks, the water extracts exhibiting more detected peaks than the other extracts (Fig. 1). These results agreed with a previous study that adding more water to ethanol increased its polarity, thus increasing the yield of diterpenoid lactones .
Andrographolide is one of the main bioactive compounds present in A. paniculata. The present study determined the andrographolide levels in extracts from five different treatments (Table 1). The highest andrographolide levels were found in the 50% ethanol extract, followed by pure ethanol, 70% ethanol, 30% ethanol, with the lowest in the water extract. These results indicated that the amount of andrographolide extracted depended on the polarity of the extraction solvent. A previous study has shown that andrographolide has a lactone ring which is chemically very vulnerable, reactive and easily rearranged. Opening the lactone ring of andrographolide is the initial stage of the decomposition process. In water, this ring opening occurs through hydrolysis, whereas in ethanol it occurs through a trans-esterification mechanism, with hydrolysis being faster than trans-esterification. Therefore, the rate of andrographolide decomposition depends on the type of solvent. Kumoro et al.  reported that adding water leads to the conversion of andrographolide to deoxyandrographolide through the hydrolysis process, thus reducing the andrographolide levels in the extracts.
Classification of A. paniculata extracts
The HPLC fingerprint chromatograms for the A. paniculata extracts used in the present study exhibited a similar pattern, only differing in the peak height and area which corresponded with the level of compound extracted by the different solvent extraction treatments. Differentiating treatments based on HPLC fingerprint chromatograms alone is not easy, so chemometrics analysis is also necessary. Principal component analysis (PCA) can be used to classify or group the extracts according to their solvent extraction treatment. The peak area of the eight major peaks (Peaks 2, 7, 8, 10, 11, 13, 15, and 21) were used as a variable.
Before using PCA, the variable was pretreated by autoscaling. Pretreatment of data is an important step before chemometric analysis to obtain a meaningful result because the quality of the input data greatly affects the quality of the output of the analysis. A common autoscaling method uses the standard deviation as a scaling factor to produce a good analytical output from PCA chemometric analysis techniques .
Using PCA, the samples were grouped according to their solvent extraction treatment based on their chemical composition. This multivariate analysis works by simplifying the observed variables by reducing the number of dimensions to give an overview of sample groups using the principal component (PC) . Figure 2A shows the PCA score plot for the A. paniculata extracts where the extracts were grouped according to their solvent extraction treatment. Samples with a similar profile of the metabolite will be grouped together and those with a dissimilar profile will form a separate group. The two principal components, PC1 and PC2, explaining most of the variance are used in the analysis. In the present study, the cumulative percentage of the two PCs used was 89% of the total variance. According to Varmuza (2001) , if the cumulative percentage of PC1 and PC2 is greater than 70%, the score plot offers a good two-dimensional visualization.
The PCA biplot is a combination of the score and loading plots. The loading plot provides information on how strongly each variable affected the principal component. Figure 2B shows the PCA biplot of the A. paniculata extracts with the variables which contributed most to its grouping. We found that peaks 5 and 7 contributed strongly to the grouping of the 50% and 70% ethanol extracts of A. paniculata.