Chromatography and spectroscopic techniques plays a major role in phytochemicals profiling and are routinely utilized for qualitative and quantitative analysis of pharmaceutically and biologically active materials. The Rf value can be used to indicate the chemical nature (polar or non-polar) of the compounds present in the sample. Based on the comparison of the observed Rf values with literature data, compounds in the extract were identified as diterpenes (4-deoxy-11, 12-didehydroandrographolide (0.84), andrographolide (0.72), or flavones and glycosides (0.72) [16, 17]. Results confirmed the presence of phenolics, flavonoids, and their derivatives indicating the medicinal importance of A. paniculata. UV spectra of andrographolide are usually observed between 230 and 235 nm range; we also observed a sharp peak within this range confirming the presence of andrographolide in the methanol extract [16, 17]. Similarly, spectra for the phenolic compounds and flavones typically lie in the range of 230–290 nm or 300–350 nm [18, 19]. UV-Vis spectrum confirmed the presence of phenolic, flavones and their derivatives in the methanol extract.
The characteristic absorption band at the 1026.87 cm−1 and 894.75 cm−1 (C-N- and -C-H- out of plane stretch) suggested that the functional groups are amines (RNH2) and aromatic (C-C ring) in nature. The absorption band at 1377.80 cm−1 (-CH3-) indicated the presence of alkanes with weak intensity. Results confirmed that ethers, hydroxyl, alkanes, amines, aromatic, esters, aromatic nitro, and phosphine compounds were presented in the methanol extract of A. paniculata. The –OH group indicates the presence of flavonoids, alcoholic, and phenolic compounds. A. paniculata is an aromatic plant, which is evident by the presence of aromatic and oxidized nitrogen functional groups. Occurrence of alkaloids in the methanol extract also confirms the presence of alkanes, primary and secondary amines, and aromatic compounds. Previously, there have been several reports on the presence of alkanes, amines, ethers, hydroxyl, aromatic, esters group aromatic nitro, and phenolic groups [2, 20], but not the presence of phosphine groups in A. paniculata plant extracts, while in our study we report the presence of phosphine group based on FTIR spectrum.
Previously, Sharma and Sharma (2018) reported andrographolide (methanol leaf extract) compared to the standard and with an Rf value of 5.674 at 223 nm . Andrographolide peaks were identified by comparing the obtained chromatogram with those already reported with comparison of standard and obtained result at RT 4.338 at 229 nm. A retention time of 4.8 at 223 nm has also been reported for Andrographolide [22, 23]. Comparing with available standard data, the peaks for neoandrographolide were identified at RT 14.10 at 220 nm . The biological activities of plant extracts largely depends upon the employed extraction technique. Selection of the extraction method and the solvent used is of prime importance and should be optimized before final use. The process of separation, identification, and characterization of phytoconstituents from a plant extracts have always been a challenging task, but the technological advances, availability of several separation techniques, and detectors with wide range are tremendously speeding the separation and characterization process.
One of the most important techniques is the combination of liquid chromatography and mass spectrometry that symbolizes a robust, sensitive, quick, and reproducible analytical method for the identification and characterization of secondary metabolites present in bioactive extracts. Several studies have reported the presence of diverse secondary metabolites from leaves, root, stem, or from whole plants of A. paniculata extracts, such as andrographolide (major compound), neoandrographolide, 14-deoxy-11, 12-didehydroandrographide and isoandrographolide [8, 25, 26], 14-deoxy-17-hydroxyandrographolide, 12S-hydroxyandrographolide, andrographatoside, 14-deoxy-11, 12-didehydroandrographolide, andrographolide, andrographic acid, 14-deoxyandrographolide, andrographolide derivatives, andrograpanin, bisandrographolide, skullcapflavone I, 5-Hydroxy-7,8,2′,5′-tetra-methoxyflavone 5-O-glucoside, and 7-O-methylwogon [27, 28]. It might be possible that the anti-malarial potency of the methanol extract observed in our earlier studies  might be due to the synergistic activity among the diterpenes and flavones present in majority in the methanol extract. Diastereomers are stereoisomers and second group of isomers that do not display mirror image and also non-super imposable on one another; however, their chemical structures are qualitatively and quantitatively similar. They possess more than one stereogenic center and differ from each other in physical and chemical properties such as solubility or melting point. Due to the presence of diastereomers, we observed two bands in TLC in the Vis region, while five bands were visible in the UV region. This could be related to the achiral environment of the TLC plates that do not yield two different solute zones.
The observed diterpenes were classified into monomers and polymers of diterpene lactones and can be classified as penta-cyclic or macrocyclic diterpenoids depending on their skeletal core . The monomers possess multiple hydroxyls group that displays a characteristic fragmentation behavior by successive loss of one or more than one H2O molecules . The positive ion mode was much more efficient for the analysis of these kinds of compounds. Kumar et al. (2018) also reported fragmentation of Andrographolide from the methanol extract of aerial parts of A. paniculata, but the fragmentation pattern differed at m/z 303 and 275 due to loss of CO in reverse Diels-Alder (RDA) . The biological function of a specific diterpenoid cannot be representative for the whole class of molecules due to their diverse nature and often restricted distribution . Andrographolide, a labdane diterpenoid, found to be present in highest concentrations is known for wide range of therapeutic properties. It has been found to be effective in in vitro and in vivo anti-plasmodial assays demonstrating its importance in anti-malarial drug discovery programs. It is known to target protein and nucleic acid synthesis acting as “transcription blocker” . Andrographolide has been gaining attention of the scientific community due to its broad biological activities such as anti-angiogenic, immune-stimulating properties, anti-cancer, anti-inflammatory, cardio-protective properties and anti-HIV , anti-feedant, anti-viral, anti-bacterial, and anti-diabetic activities . Neoandrographolide is anti-inflammatory, anti-oxidant, lipid lowering, and cardio protective molecule. It is known to be a chemo-sensitizer and is known to inhibit iNOS and COX-2 expression [33, 34]. Owing to these biological properties and its presence in the A. paniculata methanol extract, it should be evaluated for its anti-malarial potential. Andrographolactone has not been reported with any biological activity till date and has been recently identified and characterized . 14-Deoxy-11,12-didehydroandrographolide has been reported to possess promising anti steatohepatitis, anti-liver fibrosis, anti-oxidant, and anti-inflammatory activities . 14-Dehydroxy-11,12-didehydroandrographolide is also known to inhibit biofilm formation when used in combination with azithromycin and gentamicin  preventing the development of multiple drug resistance . Experimental results suggested the use of 14-dehydroxy-11,12-didehydroandrographolide in combination therapies to combat biofilm associated infections. To our knowledge, till date andrographic acid has not been reported for any biological activity.
Flavones are the most common naturally occurring extensively dispersed group of low molecular weight phenolic compounds having a benzo-γ-pyrone structure. They are known to be synthesized by plant against microbial infections and their activity depends on their structural category, hydroxylation, conjugations and degree of polymerization [38,39,40]. The presence of functional hydroxyl group is responsible for the antioxidant activities of these polyphenolic compounds making them molecules of interest for nutraceutical, pharmaceutical and medicine industries for treating an array of human diseases such as cardiovascular, cancers, and other age-related diseases [41, 42]. Flavones have the capacity to induce protective enzymatic pathways and also plays major role in inflammation suppression by inhibiting xanthine oxidase, cyclo-oxygenase, lipoxygenase, and phosphoinositide 3-kinase . There are several reports that suggest flavones to be protective against many infectious and degenerative diseases.
Skullcapflavone I, an important flavonoid, was found in high concentrations in the methanolic extract of A. paniculata that previously displayed in vitro anti-malarial activity . Flavonoids are known to possess several biological activities [44, 45]; however, there are no reports on the anti-parasitic activity of Skullcapflavone I and thus, it should be evaluated for its anti-malarial potential. 5-Hydroxy-2',7,8-trimethoxyflavone and tetramethoxyflavone are able to inhibit bacterial activity against sensitive and multidrug-resistant strains of Mycobacterium tuberculosis . We hypothesize that neoandrographolide, andrographolactone, 14-dehydroxy-11,12-didehydroandrographolide, andrographic acid, skullcapflavone I, 5-Hydroxy-2′,7,8-trimethoxyflavone, and tetramethoxyflavone might be lead molecules for infectious disease drug development programs and could be an alternative to counter the emerging anti-malarial drug resistance phenomenon.