Phenotypic and Chemotypic Relations among Local Andrographis paniculata (Burm. f.) Wall Landrace Collection

Abstract

The relationship between the phenotypic and chemical composition of local Andrographis paniculata was evaluated in this study. Five seed collections were sourced from different regions of Thailand, namely Kamphaeng Saen (KS), Udon Thani (UT), Chiang Rai (CR), Chiang Mai (CM), and Ratchaburi (RB). They were cultivated in the same conditions, potted, and partially shaded (60%) in an open conventional greenhouse. The phenology and chemical composition of these plants were assessed at the commercial harvesting stage (ca. 90 days after planting). The results indicated that UT was morphologically distinctive, illustrating the highest edible biomass yield (aerial and mature leaf size). The above-ground parts (viz., leaves and stem) were then analyzed for bioactive compounds after maceration with 80% (w/w) ethanol. It was found that the highest lactone content (~14 mg/g extract) was obtained from leaf and stem extracts of all samples except KS. Nonetheless, total phenolics and flavonoids in the stem extract of KS were found to be the highest at 3.22 and 2.42 mg/g, respectively. Phytochemicals from both leaf and stem extracts were capable of high anti-oxidant activity (~70%) as determined by DPPH and ABTS assays. Chemically, RB contained the highest 14-deoxy-11,12-didehydroandrographolide (156.98 mg/g extract), while UT and CM contained up to 0.68 mg/g extract of neoandrographolide. Classification of the samples indicated a clear relationship between the morphological traits and chemical compositions. In conclusion, our findings suggest the variations in phenotypic and chemotypic relations across the different landraces of A. paniculata. In essence, the quantity of the consumable parts was essentially the marker to describe the quality of the phytochemical constituents. The overall outcome of this study was to select the physiological characteristics that could be used for further breeding programs of the ideal variety with high productivity and higher bioactive(s) content.

1. Introduction

Andrographis paniculata (Burm. f.) Wall, commonly known as “the king of bitter”, is an ethnomedicinal plant used across the Asian continent as an anti-pyretic, a detoxicant, and an analgesic. Reports also suggest that A. paniculata was traditionally utilized to alleviate dermatological, respiratory, and gastrointestinal complications caused by pathogenic bacteria and viruses [1,2,3,4,5]. The biomedical significance of A. paniculata is mainly attributed to the presence of andrographolides, a group of diterpenoid lactones widely recognized for their immunostimulant, anti-urolithiatic, anti-diabetic, anti-obesity, and anticancer activities [4,6,7,8,9,10,11,12,13]. In addition, diterpenoid derivatives, such as dehydroandrographolide, neoandrographolide, and andrographiside, are believed to contribute directly or synergistically to the observed medicinal properties of A. paniculata [14,15]. The phytochemicals from this plant have been one of the key active ingredients in several pharmaceutical formulations for the treatment of colds, HIV infection, hepatitis, diabetes, cancer, nephro-urological disorders, and even COVID-19 [3,16,17]. In Thailand, personal administration of dried aerial parts of A. paniculata has been suggested for use in primary health care to ease the symptoms associated with the common cold, including sore throat and non-infectious diarrhea [18,19] resulting in an ever-growing demand for pharmaceutical grade raw-material(s), and subsequent large-scale cultivation of A. paniculata in recent years [3,20,21,22]. Currently, the quality of A. paniculata is determined merely based on the active constituents with only one or two diterpenoid lactone constituents as marker(s). There have been no studies carried out on the biogenetic route between andrographolide and neoandrographolide or other related bioactive(s) present in this plant [23,24]. Dong et al. [25] demonstrated that the cumulative chemical profiling of A. paniculata was influenced by growth conditions, notably the habitat and geographical region of cultivation. Interestingly, these two factors also influenced biomass and secondary metabolite accumulation in the plants [23,26]. The variations in A. paniculata production characteristics and diterpene lactone concentration were also confirmed amongst different plant accessions obtained from Thailand, Indonesia, Vietnam, and India [18,27]. Notwithstanding, little is known about the relationships between their morphological and phytochemical variations. Therefore, it is deemed essential to establish a fingerprint that can help differentiate and distinguish the A. paniculata samples based on their geographical origins for a plant breeding program. The aim of the current study was to evaluate the phenotypic trait(s) that could wholly describe the plant’s yield and chemical constituents. There have not been many reports on the A. paniculata landraces grown in Thailand. Therefore, for future benefit, this research was conducted to explain the relationship and differences. The findings of this study will aid both growers and the pharmaceutical industry in selecting suitable varieties for processing and developing effective plant breeding programs that promote the commercial use of local crops as sustainable crops.

2. Materials and Methods

2.1. Plant Materials

The cultivating trial was conducted for a 3-month period, from July 2021 to September 2021. Dried seeds of A. paniculata were collected from different locations as shown in Supplementary Table S1. They were soaked in deionized water (DI) water for 48 hrs and thereafter placed on moist paper and maintained at room temperature (29 °C, 57.8% RH). After 15 days, the number of germinations was counted to calculate the percentage of seed germination [28]. The seedling(s) were transferred into 15 cm wells filled with a mixture of soil:sand media (2:1) and maintained at 27.4 °C, 65% RH for 15 days. The seedlings (~15 cm tall) were transplanted into 10-inch pots with the same growing media, each containing 2 plants, with n = 15 pots for each plant line. Plants were watered daily (at 100% evapotranspiration crops; etc.) and in the second week of cultivation, each pot was fertilized with 100 mL of liquid fertilizer (Chia Tai, Thailand, 15-15-15, 10 g/L) while maintained under 60% shade throughout the growth period [10]. The study was conducted in the nursery at the research farm of the Chiang Mai University, Faculty of Agriculture’s Mae Hia Agricultural Demonstration Research and Training Center located at 18°45′31.1″ N 98°55′47.1″ E with an elevation of 310 m above mean sea level. After collection, the plant specimens were separated and their morphological appearances were recorded with the voucher specimen numbers as shown in Supplementary Table S1 prior to sending them to the Department of Biology, Faculty of Agriculture, Chiang Mai University for taxonomic confirmation. The specimens were stored at the Plant Bioactive Compound Laboratory, Faculty of Agriculture, Chiang Mai University.

2.2. Growth and Morphology Parameters

To evaluate the various morphological parameters, we measured growth-attributing characteristics such as shoot length and the number of branches/plants. The fresh/dry weight of the separated leaves and stems of the whole plant was determined. When samples attained a constant weight while drying in the shade, their dry weights were measured. Additionally, the total fresh and dry herbage yield per plant was measured. The growth and yield attributes, viz., plant height (cm), stem diameter (mm), number of shoots, number of leaves, number of branches, number, and length (cm) of internodes of second-pair leaves, and canopy width (cm) of the main shoot were recorded before harvesting (ca. 90 days after planting). The leaf area was determined using a leaf area meter (LI-3100C, LI-COR, Nebraska, USA). Plants were removed from the pots, cleaned, and the biomass yield was recorded. They were then shade-dried at room temperature for 5 days until constant moisture content was attained (~6 ± 0.40% MC) [29]. The dried material was then ground to powder using a spice grinder (Spring Green Evolution, PG2500).

2.3. Moisture Content

The moisture content of the A. paniculata samples was obtained by measuring the percent weight after 12 h at 105 °C in a hot-air oven, as determined by the method of the Association of Official Analytical Chemists [30].

2.4. Chemical Analyses

The sample powder (20 g) was macerated with 500 mL of 80% (w/w) ethanol thrice at room temperature, and the supernatant was combined. The solvent was removed by high centrifugation, resulting in a concentrated extract. In the last step, the extract (1 g) was redissolved with 1.5 mL of 80% ethanol and used for further chemical analysis [30].

2.5. Total Phenolic Content

The total phenolic content was determined using the method described by Sunanta, Chung [30]. The ethanolic extract (30 µL) was mixed with 150 µL of Folin–Ciocalteu reagent and 120 µL of 7.5% (w/v) NaCO₃ solution. After incubating for 60 min, a UV-Vis spectrophotometer (SPECTROstar Nano, BMG LABTECH, Ortenberg, Germany) with a single UV-visible beam was used to determine the absorbance at 765 nm, and calibration standards using gallic acid were prepared at the concentration range of 30–300 mg/mL. The total phenolic content was given as the equivalent amount of gallic acid in milligrams per gram of the extract.

2.6. Total Flavonoid Content

Total flavonoid content was determined according to Sunanta, Chung [30] by adding 25 µL of the extract to 125 µL of distilled water. Following this, 7.5 µL of a 5% NaNO₂ solution was added to the mixture. After allowing the mixture to react for 5 min, 15 µL of 10% AlCl₃·6H₂O solution was added. Thereafter, 1 M NaOH solution (50 µL) and distilled water (27.5 µL) were added. The absorbance of the test solution was determined using the spectrophotometer at a wavelength of 510 nm. The calibration standards were prepared using catechin at a concentration of 15 mg/mL. The total amount of flavonoids was given in milligrams of catechin equivalent per gram of the extract.

2.7. Anti-Oxidant Activities

2.7.1. Free Radical Scavenging Activity

The method for analyzing free radical scavenging activity was followed according to Sunanta, Chung [30]. Experimentally, 25 µL of the crude extract was mixed with 250 µL of 0.20 mM DPPH (2,2-diphenyl-1-picrylhydrazyl) and incubated at room temperature, in the dark, for 30 min. The absorbance at 517 nm was read, and DPPH radical scavenging performance was calculated using the following formula:

DPPH radical scavenging activity (%) = [(Abs_control − Abs_sample)]/(Abs_control)] × 100

(1)

where Abs_control is the absorbance of DPPH radical mixed with methanol and Abs_sample is the absorbance of DPPH radical reacted with sample extract/standard.

2.7.2. Radical Cation Decolorization Assay

The method of Sangta, Wongkaew [31] was used and modified accordingly. The ABTS [2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] (1 mL) was diluted in 60 mL of 80% methanol, resulting in an absorbance of 0.7 ± 0.02 units at 734 nm. Equal volumes of 7.00 mM ABTS and 2.45 mM K₂S₂O₈ solution were made into the working solution. The extract (200 µL) was pipetted into microtiter wells filled with 500 µL ABTS and 500 µL of the working solution, agitated, and left to stand at room temperature for 30 min (the mixture was incubated in the dark, at room temperature, for 12–16 h). The ABTS scavenging potential was estimated using the equation:

ABTS radical scavenging activity (%) = [(Abs_control − Abs_sample)]/(Abs_control)] × 100

(2)

where Abs_control is the absorbance of ABTS radical mixed with 80% methanol and Abs_sample is the absorbance of ABTS radical reacted with sample extract.

2.8. Total Lactone

The total lactone content was analyzed using andrographolide as a standard according to the methodology described by Gajbhiye and Khristi [32]. For the spectrophotometric determination of total lactones, a 3,5-dinitro benzoic acid reagent was prepared by dissolving 1.0 g of the compound in 100 mL of ethanol. A potassium hydroxide reagent was prepared by dissolving 0.5 g in 100 mL of ethanol. Both reagents were prepared fresh during the analysis and stored in amber-colored bottles. For the spectrophotometric determination of the total lactones, UV-visible spectrophotometry was employed according to Aromdee [33]. Aliquots of the standard A. paniculata supplement (20 mg, Yanhee Fa Thalai Chon, Bangkok, Thailand) ranging from 0.10 to 1.00 mL were transferred to a test tube and made up to 2 mL with ethanol. Afterward, 0.5 mL of 3,5-dinitro benzoic acid and potassium hydroxide reagents were added to each aliquot and thoroughly mixed using a vortex. After 10 min, the absorbance was measured against a blank at 536 nm and used for sample analysis. Plant extracts were analyzed using the same procedure, and the andrographolide content of plant samples was calculated using the standard curve regression equation.

2.9. High Performance Liquid Chromatography (HPLC)

The HPLC conditions were as described by Jirakiattikul, Rithichai [34]. The sample extract was sonicated for 10 min and then passed through a 0.22 µm filter membrane. Ultra-high performance liquid chromatography was performed using a Nova-Pak C18 column (150 mm × 3.9 mm × 4 µm) (UHPLC, Nexera LC-30 A, Shimadzu) with a guard column. A mobile phase of phosphoric acid (A)-to-acetonitrile (B) at a volume ratio of 5 min, % A, % B; 4.5 min, % A, % B; 5.0 min, % A, % B; and 4.0 min, % B was delivered at a flow rate of 1.5 mL/min, over a total run time of 14 min. A sample (20 µL) was injected into the system. The concentrations of a standard (750 ppm and 1000 ppm) mixture containing 14-deoxyandrographolide and neoandrographolide in the range of 1.0 g were freshly prepared and injected. As a function of concentration, the peak area for each compound was plotted against calibration curves. To figure out how much of each compound was recovered, known amounts of reference standards (1 mg/mL) were added to the crude extracts.

2.10. Metabolite Profiling

The LC-MS/MS analysis was performed on an Agilent LC-QTOF 6500 system with an Agilent ZORBAX Eclipse XDB column-C18 (2.1 mm × 50 mm × 1.8 μm). Water containing 0.1% formic acid was used as the mobile phase, while acetonitrile containing 0.1% formic acid was used as the eluent in a gradient mode. The injection volume was 20 μL, and the temperature of the column was maintained at 30 °C. The flow rate remained constant across the gradient. The UPLC system was coupled to a QTOF mass spectrometer (6500 series: Model-G6545B) with an AP-ESI Dual Spray source. The parameters for the analysis were set using the positive ion mode, and spectra were acquired over the mass range of 120–1000 m/z. The MS data were processed with MassHunter v B.08.00, Rapid Control version 2.9 (Agilent Technologies). This software is integrated with libraries to give a list of possible elemental formulas. The MassHunter software (Agilent Technologies, Santa Clara, CA, USA) was used to refine the data. An MS/MS fragment matching and an error of less than 5 ppm were adjusted to determine the precision of the compounds’ confirmation [35,36].

2.11. Statistical Analysis

The experiment utilized a completely randomized design (CRD) with ten replicates. All experimental data were expressed as the mean plus the standard deviation (SD). A one-way analysis of variance (ANOVA) and Duncan’s multiple range tests at a significance level of 0.05 were conducted to determine the significance of the difference between samples of each type of A. paniculata (SPSS Institute, Armonk, NY, USA). Principal component analysis (PCA) was utilized to summarize the visual differences between the chemical components of the extracts, utilizing XLSTAT version 2018.5 (Suite NY, New York, NY, USA). To display the metabolite profile clustering, the m/z data were subjected to an online web server program, Heatmapper (heatmapper.ca) [37].

3. Results and Discussion

3.1. Taxonomical and Physiological Characteristics

The taxonomical characteristics of the A. paniculata collection are shown in Figure 1. Overall, they were branched, herbaceous plants growing to a height of 30–110 cm. All samples had stems that were acutely quadrangular, slender, highly branched, easily broken, dark green in color, square in cross section with longitudinal furrows and wing-like projections along the angles. The leaves were simple, opposite, lanceolate, glabrous, and lance-shaped. The leaf margins were found to be acute, entirely or slightly undulating, and the upper leaves were typically bract-like with a short petiole. The flowers were small and borne in spreading racemes. The plant’s inflorescence was present, terminal and axillary in the panicle, usually 10–30 mm long; the bracts were small; and the pedicel short. The results of morpho-physiological evaluation(s) are also mentioned in

Table 1. Physiological characteristics of Andrographis paniculata collected from various locations.

Specimen Location	Plant Height (cm)	Stem Diameter (mm)	Numbers of Shoots (n)	Numbers of Leaves (n)	Numbers of Branches (n)	Internode Length (cm)	Canopy Width (cm)	Leaf Area Index		Aerial Fresh Weight Yield (g/Plant)	Dried Leaf Biomass (g/ Plant)
								Young Leaf Area	Mature Leaf Area
KS	21.85 ± 3.00 a	12.82 ±0.88 b	11.82 ± 1.63 bc	28.24 ± 5.02 c	5.61 ± 0.72 b	6.98 ± 2.31 ab	20.73 ± 4.19 a	14.96 ± 0.40 b	23.88 ± 0.49 b	21.16 ± 5.78 c	7.97 ± 2.25 c
UT	14.89 ± 3.54 b	11.81 ±0.95 c	10.68 ± 2.77 c	23.02 ± 4.30 d	6.87 ± 0.65 b	6.37 ± 2.31 b	14.66 ± 4.40 c	17.43 ± 0.43 a	30.30 ± 0.61 a	37.83 ± 5.78 a	14.61 ± 2.25 a
CR	22.06 ± 3.72 a	14.53 ±0.98 a	13.11± 2.86 b	31.14± 4.46 b	9.88 ± 0.89 a	7.52 ± 3.18 ab	22.84 ± 7.31 a	12.01 ± 0.43 c	21.35 ± 0.55 c	33.61 ± 5.78 ab	13.01 ± 2.25 a
CM	22.27 ± 3.40 a	15.06 ±3.64 a	15.15± 3.56 a	29.91 ± 4.44 c	9.37 ± 0.84 a	7.70 ± 1.72 a	20.12 ± 3.38 a	9.28 ±0.53 d	16.70 ± 0.49 d	27.75 ± 5.78 b	11.01 ± 2.25 b
RB	20.91± 2.77 a	13.92 ±0.93 b	13.77 ± 4.17 b	35.59± 24.34 a	9.63 ± 0.61 a	7.85 ± 2.09 a	19.93 ± 2.26 b	8.91 ±0.24 d	24.31 ± 0.56 b	26.6 ± 5.78 b	10.65 ± 2.25 b

KS = Kamphaeng Saen, UT = Udon Thani, CR = Chiang Rai, CM = Chiang Mai, RB = Ratchaburi. Values are mean ± SD. Values followed by a different letter in the same column are significantly different (p < 0.05) by Tukey’s honesty significant difference test.

. Among all accessions, UT was found to be the smallest in plant height (~15 cm), stem diameter (~12 cm), and number of shoots, leaves, and branches, with the smallest canopy size of ~14.6 cm. CR and CM were found to be the tallest plants (~22 cm) with the largest stem diameter (~15 mm). KS and UT had the same stem diameter, number of shoots, number of leaves, number of branches, and length of internodes of the second-pair leaves (~12 mm, ~11, ~23, ~6, and ~6 cm, respectively), but less than those of the CR, CM, and RB. In CM, the leaf area index in young leaves and old leaves was the smallest (~9 and ~16 cm, respectively). In KS, the diameter of the stem, the number of shoots, the number of branches, and the length of the internodes of the second-pair leaves were found to be the smallest, approx. 12 mm, 11 cm, and 6 cm, respectively (Table 1).

In concurrence with the findings mentioned in Table 1, the A. paniculata leaf and stem per plant had a significant direct effect on its phytochemical, particularly total andrographolide, yield. Aerial fresh weight yield (per plant) and dried leaf biomass (per plant) of the A. paniculata varieties 90 days after planting were found to be significantly different (Table 1). The highest values were 37.83 and 14.61 g/plant, respectively, found in CM and RB, which had fresh leaf weight yields of 27.75 and 26.6 g/plant and dry leaf weight yields of 11.01 and 10.65 g/plant, respectively. KS had the lowest fresh and dry leaf weight yields of 21.16 and 7.97 g/plant for the A. paniculata planted at different planting distances. It was found that the yield of fresh and dry leaf weights also differed statistically. A. paniculata yield per plant was determined by the number of primary branches and plant spread. The plant height and number of secondary branches had a negligible indirect negative effect on the total yield per plant [38]. Optimizing plant growth and the biosynthesis of desirable secondary metabolites has been a primary objective in commercial plant production. However, environmental factors and agricultural practices can have a substantial effect on plant growth and crop quality [39,40,41,42]. Plant growth optimization is also deemed essential to improve the secondary metabolite quality [43,44].

3.2. Chemical Properties

The presence of multiple pharmacological properties makes andrographolide a potential therapeutic agent [45]. Andrographolide contains an α-alkylidene-butyrolactone moiety and three hydroxyls at C-3, C-19, and C-14 that are responsible for its cytotoxic effects against numerous cancer cell lines [45]. Andrographolide is abundant in leaves and can be readily isolated as a crystalline solid from crude plant extracts [6,46,47,48,49,50]. In CM for both edible parts shown in

Table 2. Chemical properties of Andrographis paniculata.

Sample	Leaf
	Lactones (%)	Phenolic (mg/g)	Flavonoid (mg/g)	Antioxidant (%)		HPLC Lactones (mg/g)
				DPPH•	ABTS•+	14-Deoxy-11,12-didehydroandrographolide	Neo Andrographolide
KS	9.35 ± 1.92 b	3.22 ± 0.22 a	2.42 ± 0.09 a	72.48 ± 2.03 b	76.55 ± 14.65 a	0.01 ± 0.00 a	0.11 ± 0.07 a
UT	13.41 ± 1.68 ab	1.96 ± 0.17 b	1.17 ± 0.09 bc	74.77 ± 2.05 a	73.71 ± 8.98 bc	0.02 ± 0.00 a	0.06 ± 0.036 b
CR	13.91 ± 0.03 ab	1.69 ± 0.01 c	1.36 ± 0.50 bc	74.37 ± 3.39 a	75.08 ± 11.49 b	n/d	0.09 ± 0.04 b
CM	14.43 ± 0.58 ab	2.29 ± 0.06 a	1.57 ± 0.18 b	72.36 ± 1.15 b	69.86 ± 10.43 c	0.01 ± 0.00 a	0.04 ± 0.03 c
RB	13.79 ± 2.19 ab	1.45 ± 0.02 d	1.06 ± 0.05 d	73.66 ± 1.53 b	70.78 ± 10.78 c	n/d	0.09 ± 0.02 b
Sample	Stem
	Lactones (%)	Phenolic (mg/g)	Flavonoid (mg/g)	Antioxidant (%)		HPLC Lactones (mg/g)
				DPPH•	ABTS•+	14-Deoxy-11,12-didehydroandrographolide	Neo Andrographolide
KS	9.63 ± 0.68 c	4.79 ± 0.48 a	3.40 ± 0.15 a	74.74 ± 2.40 a	76.16 ± 12.13 ab	148.65 ± 1.58 c	0.58 ± 0.17 b
UT	11.66 ± 0.51 ab	1.88 ± 0.01 c	1.50 ± 0.03 c	73.89 ± 1.73 ab	81.54 ± 8.24 a	104.57 ± 2.66 d	0.68 ± 0.01 a
CR	12.82 ± 0.94 ab	3.27 ± 0.40 b	2.31 ± 0.32 b	72.36 ± 0.75 ab	75.01 ± 12.12 ab	148.51 ± 1.26 b	0.57 ± 0.15 b
CM	13.15 ± 0.88 a	3.68 ± 0.42 b	2.88 ± 0.32 ab	72.70 ± 1.87 ab	69.48 ± 17.37 c	153.98 ± 3.67 ab	0.68 ± 0.01 a
RB	14.11 ± 0.27 a	3.27 ± 0.34 b	2.51 ± 0.12 ab	71.45 ± 3.20c	72.03 ± 18.39 ab	156.98 ± 4.55 a	0.09 ± 0.02 c

The sample accessions were taken from KS = Kamphaeng Saen, UT = Udon Thani, CR = Chiang Rai, CM = Chiang Mai, RB = Ratchaburi. Values are mean ± SD. Values followed by a different letter in the same row are significantly different (p < 0.05) by Tukey’s honesty significant difference test. Abbreviation: DPPH, 2,2-diphenyl-1-picrylhydrazyl.

, the total lactone was ~9.00–14.00 mg/g, while KS had the lowest. The amount of total lactone did not differ much between these utilizable parts. The phenolic and flavonoid contents were significantly higher in the KS, CM, and RB (3.00–5.00 mg/g and 2.50–3.00 mg/g, respectively), and the stem part contained slightly higher amounts. Flavonoids are phenolic group constituents, and flavones were found to be more prevalent in fresh A. paniculata. The total flavonoid content of this plant collection ranged between 3.40 and 1.06 mg/g. The antioxidant activities as determined by DPPH^• and ABTS^•+ were around 73% of crude extract, while the activities were significantly lower in the CM for both edible parts. It is also worth highlighting that ethanol was chosen in the extraction process due to its higher solubility, as reported previously [51].

The results of the andrographolide contents from different parts are mentioned in Table 2. The HPLC chromatograms are displayed in Supplementary Figure S1. The number of diterpene lactones, namely 14-deoxy-11,12-didehydroandrographolide (>100 mg/g) and neo-andrographolide (>0.50 mg/g), was much higher in the stem than contained in the leaf tissue. The leaf ethanolic fraction from landraces KS, UT, and CM contained 14-deoxy-11,12-didehydroandrographolide at ~0.01 mg/g, while neo-andrographolide was the highest in the KS (~0.11 mg/g), then in the UT, CR, and RB (0.06–0.09 mg/g) and the lowest in the CM (~0.04 mg/g). In stem, the UT sample illustrated the lowest content of 14-deoxy-11,12-didehydroandrographolide 105 mg/g), whereas neo-andrographolide was the highest (~0.68 mg/g). The neo-andrographolide was found in the highest amount in UT and CM (~0.7 mg/g), followed by the KS and CR (~0.6 mg/g), and the RB was the lowest (0.09 mg/g). Chromatography and spectroscopic techniques play a major role in phytochemical profiling and are routinely utilized for qualitative and quantitative analysis of pharmaceutically and biologically active materials [52,53]. Changes in the amount of andrographolide could be caused by the way samples are prepared for HPLC measurement, but the overall trend is the same. The variation in the proportion of andrographolides may also be attributable to differences in sample origin, their respective genotypes, and variable expression of genes such as the WRKY (“Worky”) transcription factors [32,54,55].

While the entire plant has been reported to be of therapeutic value, the leaves were found to contain the highest concentration of useful terpenoids, including andrographolide, followed by the stems, roots, and lastly the seeds [56,57]. However, it is known that all plant parts contain extractable bioactive active compounds [47,58,59]. The major bioactive classes of A. paniculata are terpenoid lactones and flavonoids, which are responsible for pharmacological activities such as analgesic, anti-cancer, anti-diabetic, anti-fertility, anti-inflammatory, anti-malarial, anti-microbial, anti-oxidant, anti-pyretic, anti-viral, anti-retroviral, anti-venom, cardioprotective, hepatoprotective, immunomodulatory, and neuroprotective properties [60,61]. Reportedly, A. paniculata contains over 20 diterpenoids and over 10 flavonoids [62,63], while andrographolide is the most common diterpenoid found in A. paniculata, making up about 1–6% of the dried whole plant, stem and leaf extracts, respectively [42,58,59]. Deoxyandrographolide, neoandrographolide, 14-deoxy-11,12-didehydroandrographide, and isoandrographolide are the other principal diterpenoids along with flavonoids and polyphenols [42,58,64,65,66]. In vitro study advised that 14-deoxy-11,12-didehydroandrographolide and neoandrographolide possess immunostimulatory, anti-infectious, anti-atherosclerotic, anti-inflammatory, anti-microbial, and anti-hepatotoxic properties [6,67]. In addition, 5-hydroxy-7,8-dimethoxyflavone, 5-hydroxy-7,8,2′,5′-tetramethoxyflavone, 5-hydroxy-7,8,2′,3′-tetramethoxyflavone, and 5-hydroxy-7,8,2′-trimethoxyflavone are structurally related flavonoids. The principal flavonoids isolated were 7-O-methylwogonin and 2′-methyl ether [48,64,68,69].

Flavones are the most prevalent naturally occurring, widely dispersed group of low molecular weight, benzo-γ-pyrone-structured phenolic compounds. Several studies suggest that flavones are protective against numerous infectious and degenerative diseases. Their activity against microbial infections is dependent on their structural category, hydroxylation, conjugations, and degree of polymerization, and they are known to be produced by plants [70,71,72]. The antioxidant activities of these polyphenolic compounds are due to the presence of a functional hydroxyl group, making them molecules of interest to the nutraceutical, pharmaceutical, and medical industries as therapeutic agents for a variety of human diseases, including cardiovascular, cancerous, and age-related diseases [73,74]. Flavones can induce protective enzymatic pathways and play a significant role in inflammation suppression by inhibiting xanthine oxidase, cyclo-oxygenase, lipoxygenase, and phosphoinositide 3-kinase [75,76]. It has been reported that 14-deoxy-11,12-didehydroandrographolide has promising anti-steatohepatitis, anti-liver fibrosis, antioxidant, and anti-inflammatory properties [77]. The 14-dehydroxy-11,12-didehydroandrographolide is also known to prevent the development of multiple drug resistance (MDR) when combined with azithromycin and gentamicin [76,78]. Andrographolactone was recently identified and characterized [79]; no biological activity has been reported to date.

3.3. Chemometric Relationship

Several chemical variables were used to understand the relationship between the morphological data of the five A. paniculata varieties used in this study and their chemical properties. These variables are shown in Table 1 and Table 2. The morphological scale is represented in the graph in Figure 2A. As shown, representing the bioactive compound relationship as a score curve, all patterns were evenly distributed in the scatter plot, with 95.47% in PC1 and 7.98% in PC2. As shown in Figure 2C, all patterns were evenly distributed across the plot, with 99.98% in PC1 and 1.34% in PC2.

As mentioned in Table 1, the physiological data were computed using principal component analysis (Figure 2). The figures A and B variables of morphology were divided into two categories: UT observed in the mature leaf area (14.61 g) and aerial fresh weight (37.7 g) was the greatest and thus distinguished from the other variants by the data. The biplot analysis in Figure 2B revealed that UT was distinguished by variables such as aerial fresh weight and mature leaf area. The other four species were associated with young leaf area, dried leaf biomass, No. of branches, stem diameter, No. of shoots, internode length, and young leaf area to a lesser extent.

For the chemical analysis (Figure 2C), the UT had the highest antioxidant potential while illustrating low phytochemical content. However, the other samples corresponded with higher concentrations of the observed chemical compounds (Figure 2D). Obviously, the PCA patterns of the morphological and phytochemical data were in-line, indicating that the plants with smaller physiological characteristics illustrated higher amounts of active ingredients. However, the antioxidant activities were conversely lower. Chlorophyll, which is the primary pigment used in photosynthesis, is frequently used as a marker to determine plant biomass [12]. Multiple studies have demonstrated a significant correlation between chlorophyll content and secondary metabolites such as total phenolic compounds, flavonoid content, and anthocyanin content [9,10]. The research conducted by Stratil, Klejdus [80] revealed a highly significant correlation between total phenolic content and antioxidant activity. In the common case, the DPPH- and ABTS-scavenging activity were related to the total amount of phenolic and flavonoid compounds [81]. Antioxidant compounds are key elements for the prevention of diseases caused by free radicals and for protecting the nervous system and memory [82]. The main antioxidants in A. paniculata are phenolic acids, flavonoids, and lactones that possess anti-cancer, neuroprotective, and neurotrophic properties [30,51,83,84]. More work regarding the metabolomic profiling of the biosynthesis of active ingredients in this plant needs to be further investigated.

3.4. Metabolite Profiling of A. paniculata Leaf Constituents

In the present study, A. paniculata was analyzed for metabolite profiling using LC-ESI-MS/MS. A total of 110 metabolites were identified in Supplementary Table S2. The heatmap displays 2 major distinctive clusters with the UT, projected separately from the others (Figure 3A). The CM and RB were closely related in the metabolite profile. Similarly, in the bi-plot analysis, only 44.25% across PC1 (23.05%) and PC2 (21.20%) were considered (Figure 3B), commercial (Figure 3C) mass spectral data of supplementary Table S2 mass spectral libraries and from literature. The samples of CM and RB were cast away from the others by compounds of positive fragment dodemorph (282.28 m/z) (3), phytosphingosine (318.30 m/z), C16 Sphinganine (274.27 m/z), Xylostasin (212.21 m/z) (30,31,39), Triethyl phosphate (205.06 m/z) (63) and(S)-2-Methylbutanal 121.09 m/z) B (105). The CR, KS, and RB were projected with the compounds Oleamide, 6,10,14-Trimethyl-5,9,13-pentadecatrien-2-one, and Inulobiose with a 282.28 m/z of the positive ion of 280.26 m/z and 166.06 m/z, respectively. The rest of the metabolites were shared across the samples, which were general chemotypes of A. paniculata. In other words, terpenoid lactones and flavonoids were commonly identified using LC–MS/MS analysis [61], as previously reported. Terpenoid lactones such as neoandrographolide (510.29 m/z), 14-deoxyandrographolide (340.21 m/z), andrograpanin (453.13 m/z), andrographin (382.42 m/z), and andrographolide (476.26 m/z) were identified from the ethanolic extract. Andrographidine F (527.65 m/z), andrographiside (542.46 m/z), andrographidine B (484.43 m/z), andrographidine A (472.53 m/z), 5-hydroxy-7,8,2′-trimethoxyflavone-5-glucoside (489.16 m/z), and andrographidine D (460.38 m/z) were identified as new flavone glycosides.

The observed diterpenes were categorized as monomers and polymers of diterpene lactones and as pentacyclic or macrocyclic diterpenoids based on their skeletal core [85]. The monomers contain multiple hydroxyl groups that exhibit a unique fragmentation behavior characterized by the successive loss of one or more H₂O molecules [85]. The positive ion mode was significantly more effective for analyzing these types of compounds. Kumar et al. [61] also reported fragmentation of andrographolide from the methanol extract of A. paniculata aerial parts, but the fragmentation pattern differed at m/z 303 and 275 due to the loss of CO in reverse Diels-Alder (RDA). Due to their diverse nature and often restricted distribution, the biological function of a particular diterpenoid cannot be generalized to the entire class of molecules. A. paniculata is an active ingredient in the numerous formulations listed in the Thai national herbal medicine list. Because of this, A. paniculata has been chosen as a marker for standardizing raw and commercialized herbal products using HPLC and LC-MS methods for quality control and quality assurance [86].

4. Conclusions

The present study evaluated physiological traits that could be used for further propagation of high-yielding ideal varieties and found high bioactive content differences between A. paniculata cultivars. To establish this, the relationship between the phenotypic and chemical composition of local A. paniculata varieties was evaluated. The results indicated that the species contained a substantial amount of total lactone, while phenolic and flavonoid content varied among the tested samples. The amounts of 14-deoxy-11,12-didehydroandrographolide and neo-andrographolide had a better influence on the antioxidant potential than other phytochemical ingredients. Overall, morphological traits (i.e., higher aerial fresh weight and larger mature leaf size) are indicative of higher phytochemical yield for its utilization for consumption and pharmaceutical purposes.