1. INTRODUCTION:

Andrographolide (AP), a C₂₀ labdane diterpenoid is the bitter bioactive principle found in the aerial parts of the plant Andrographis paniculata (Burm f.) Nees (Family: Acanthaceae). It was found to exhibit various pharmacological actions including anti-inflammatory, anti-pulmonary, anti-viral, anti-cancer, anti-bacterial, anti-atheroscleron sis, neuroprotection and liver protection properties by Chakravarti and Chakravarti ¹.

Fig.1: Chemical Structure of Andrographolide

In literature study, HPLC and spectrophotometric methods for the determination of AP in A. paniculata and their report ² revealed that the HPLC method was found to be more accurate and sensitive as compared to the other methods. Some other methods including UV-Spectrophotometry³, Thin layer chromatography-image analysis method⁴, microemulsion electrokinetic capillary chromatography⁵, reverse phase high performance liquid chromatography (RP-HPLC), high speed counter current chromatography (Flow-injection chemiluminescence method⁶, electroanalytical method, miscellar electrokinetic capillary chromatography, HPLC⁷, rapid solution liquid chromatography (RRLC), high performance thin layer chromatography (HPTLC)⁸. fourier transform infrared spectroscopy (FTIR), ultra-performance liquid chromatography (UPLC) and chromatographic green method⁹ were also reported for the determination of AP in herbal formulations. Based on the literature survey, HPLC estimation method was found to be more sensitive and accurate for the determination of AP.¹⁰ reported the simplicity, quick standardisation, low operating cost and stability indicating properties of AP using TLC-image analysis method.¹¹ described the trace analysis of multi-pesticides present in the plant Morinda officinalis using GC-ECD. These days, the majority of research efforts are focused on combating dengue, viruses and other infectious diseases. Therefore, TLC, HPLC and GC-ECD methods to determine and quantify AP and to trace analyse the pesticides present in the four different samples obtained from the market compared with the control and standard samples in order to work against the above mentioned pharmacological conditions.

2. MATERIALS AND METHODS:

2.1 Chemicals and Reagents:

Four different Andrographis paniculata powder samples were purchased from the local market, one control sample and standard used were obtained as a gift sample from Natural Remedies Pvt Ltd. All chemicals and reagents used were of analytical grade.

2.2 TLC Parameters:

The TLC system used was pre-coated silica gel on aluminium plate. The solvent system used for the chamber saturation was chloroform: acetone: benzene (20:20:10 ratio). The developed plate were detected by observing at wavelengths 254 and 366nm and also by spraying vanillin- sulphuric acid reagent (1.0% Vanillin in ethanol + 10% Sulphuric acid in ethanol). The TLC plate were also run simultaneously with reference solution of andrographolide (100 μg/mL concentration).

2.3 HPLC Parameters:

The HPLC system consisted of Shimadzu LC-2030C Liquid Chromatograph consisting quaternary pump, UV detector, auto injector, column oven and supported by LC- Solution software.The standard and sample were injected into HPLC (20 μL each). The mobile phase used as solvent A was buffer solution (0.136 g potassium dihydrogen orthophosphate + 0,5 ml Orthophosphoric acid – 0.45 µ filter membrane; degassed in sonicator for 3 min) and acetonitrile as solvent B.

2.4 GC-ECD Parameters:

The GC-ECD system consisted of Agilent Gas Chromatograph with Electron Capture and Nitrogen phosphorus detector 7890A-ECD/NPD (EZ Chrome), Electrical stirrer (Remi Motors), Buchi Rotovapor R-200, Buchi Heating Bath B-490, Vacuum pump, Sartorius analytical weighing balance and Agilent Vacuum Manifold. The flow rate of the carrier gas (nitrogen) was 0.979 mL/min. The make-up gas (nitrogen) included was 29.629mL/min and 1.0 µL of the samples and standard were injected into GC.

2.5 Sample Preparation for AP Analysis using TLC:

200 mg of Andrographis paniculata extract were weighed, 50ml of methanol was added and sonicated for 3 minutes. After sonication, the volume was made up to 100ml with methanol. 0.5mg/ml Andrographolide (standard) in methanol were prepared simultaneously. 10 μl of sample, standard and control was spotted using TLC applicator.

2.6 Sample Preparation for AP Analysis using HPLC:

2g of Andrographis paniculata roots/stems/leaves powder was weighed, 75ml of methanol was added and boiled on water bath for 20 min at 70-80 ºC. Then, the sample was cooled and filtered in a separate 250ml beaker. The extraction procedure was repeated 3 more times with 50ml methanol or until extract turns colorless. The filtrate was collected and concentrated to less than 100ml and transferred to 100ml flask, beaker was rinsed with methanol and transferred to same volumetric flask, the volume was made up to 100ml, mixed well and filtered the solution through 0.2 µ membrane filter paper. After filtration, samples were transferred to injector vial for loading HPLC instrument filter with syringe filter then transferred to injector vials. Before being used, all of the solutions were cooled to room temperature and kept at 4 °C.

2.7 Sample Preparation for AP Analysis using GC-ECD:

10g of sample was weighed and transferred to 500 ml beaker containing 350ml of mixture of acetonitrile and water was added. After that, the beaker was blended at high speed for 5mins using electrical stirrer and filtered. After filtration, filtrate was transferred to separating funnel and 100ml of hexane was added and shaken for 5 minutes. Then the hexane layer was transferred to the round bottom flask through a glass funnel plugged with cotton and anhydrous sodium sulphate and the process was repeated twice. After collection of hexane layer in round bottom flask, it was kept for rotary evaporation, dried and evaporated the hexane layer and made up to 10 ml in volumetric flask. From that, 1ml of sample was pipetted out and transferred into the quenchers containing mixture of salt (MgSO₄ + Psa + C18 + Gcb) in each quenchers, mixed well for 2 min and kept for centrifugation for 6min. After centrifugation, the supernatant layer was transferred to injector vial and loaded to GC-ECD system.

2.8 Standard Pesticides Preparation for GC-ECD Analysis:

Standard concentration was prepared as mentioned in the USP by suitably dissolving an appropriate quantity of the individual reference standard in n-hexane and diluting with the same solvent. Working Standard was prepared by diluting the stock solution to 50ml as mentioned in the USP. 2.5 ml of the working standard concentration I solution is further diluted to 25 ml [Working standard solution (II)]. The pesticide residues present in the sample were identified by comparing the retention times of the peaks present in the chromatogram of the extract with that of standard. The standard was stored at 2-5^oC.

2.9 Calculation:

calculations was carried out using excel sheet. For TLC, the spotted bands of the samples, standard and control were detected at 264 nm using TLC visualizer. Then, the plates were dried at the plate heater and spots were detected at 366 nm. For HPLC, the percentage of andrographolide, neoandrographolide, 14-deoxy-11-12 didehydroandrographolide and andrograponin contents present in the samples and standards from the peak areas were calculated using the formula:

Area of the sample

Weight of the standard (mg)

Sample dilution (ml)

Purity of the standard (%)

Area of the standard

Standard dilution (ml)

Weight of the sample (mg)

The percentage of total andrographolide was calculated as a sum of andrographolide, neoandrographolide, 14-deoxy-11-12-didehydroandrographolide and andrograponin. For GC-ECD analysis, the pesticide residues present in the samples, standard and control were calculated using the formula:

Residual pesticides (ppm)	=	Sample area	x	Standard weight
		Standard area		Sample weight

X	Standard dilution	x	Standard purity	x	1000
	Sample dilution		100

3. RESULTS AND DISCUSSION:

In this research work, the presence of andrographolide was analysed for the four marketted samples, one control and one standard sample gifted from the Natural Remedies Pvt. Ltd. using HPLC, TLC and GC-ECD methods. The TLC system analysis revealed the presence of andrographolide, neoandrographolide, 14-deoxy-11-12-didehydroandrographolide and andrograponin in all four samples, control and standard spots as given in Fig. 2 after the TLC plate was dried and detected on TLC visualizer at 264 and 366 nm in the solvent system (chloroform, acetone and benzene). For HPLC, the percentage of total andrographolide was calculated as a sum of andrographolide, neoandrographolide, 14-deoxy-11-12-didehydroandrographolide and andrograponin contents present in the standards and samples were calculated from the peak areas and given in Table 1 and 2 and Fig. 3-9. The results revealed the presence of all the above mentioned chemical constituents in both the samples and standards. As compared to four samples, sample C was foun to be the highly quantified with andrographolide.

Fig. 2: A TLC Chromatogram of market available samples, control and standard

Table 1: Standard Values of Andrographolide and other three constituents using HPLC

Standard	Andrographalide	Neoandrographolide	14-deoxy-11-12-didehydro andrographolide	Andrograponin
Standard weight	26.59	5.16	4.77	1.51
Standard Dilution	100	100	100	100
Purity	99	99	99	95
Standard Area	3916348	200229	488273	84837
	4164813	214841	517662	89053
	4032931	206368	500825	86943
Average	4038031	207146	502253	86944
	3.08	3.54	2.94	2.42

Table 2: Estimation of Total Andrographolide Content Present in the Samples Using HPLC

Sample Name	Sample Weight	Sample Dilution	Andrographolide		Neoandrographolide		14-deoxy-11-12-didehydro Andrographolide		Andrograponin		Total (%)
Sample Name	Sample Weight	Sample Dilution	Area	Result	Area	Result	Area	Result	Area	Result
Sample A	2029.2	100.0	191819.0	0.062	7006.0	0.009	50220.0	0.0	9542.0	0.008	0.10
Sample B	2003.1	100.0	2355727.0	0.767	40627.0	0.050	382859.0	0.2	28285.0	0.023	1.02
Sample C	2044.5	100.0	5035490.0	1.606	127457.0	0.154	686959.0	0.3	32513.0	0.026	2.10
Sample D	2031.8	100.0	920392.0	0.295	41564.0	0.050	438020.0	0.2	29449.0	0.024	0.57

Fig. 3: HPLC Chromatograms of Standard A

Fig. 4: HPLC Chromatograms of Standard B

Fig. 5: HPLC Chromatograms of Standard C

Fig. 6: HPLC Chromatograms of Samples A

Fig. 7: HPLC Chromatograms of Samples B

Fig. 8: HPLC Chromatograms of Samples C

Fig. 9: HPLC Chromatograms of Samples D

The pesticide residues present in the sample were identified by comparing the retention times of the peaks present in the chromatogram of the extract with that of the standards. The results showed the presence of mixture of pesticides including delta-HCH, cyclorpyrifos-methyl, parathion methyl, vinchlozoline, alachlor, heptachlor, paraxon ethyl, chlorpyrifos-ethyl, dichloflunid, bromofos-methyl, heptachlorepoxide, procymidon, beta-endosulphon etc in the standards which was in the range of 7.600 ppm and the pesticide residues present in the samples was around 0.032 – 0.091 ppm which is lesser than the standards. The GC-ECD chromatogram of the standards and the samples were given in the Tables 3-6. and in Fig. 10 to 14

Table 3: List of Pesticides Present in the Sample A

Name	RT (min)	Area (Hz-s)	Area %	Amount (ppm)
Alpha-HCH	13.013	289.072	5.359	0
HCB	13.451	418.442	7.757	0.001
Delta-HCH	15.835	1764.966	32.719	0.003
Chlorpyrifos-ethyl	21.076	198.607	3.682	0.001
2,4 DDE	25.118	128.797	2.388	0
2,4 DDD	27.571	275.378	5.105	0.001
4,4 DDD	29.872	45.326	0.84	0
2,4 DDT	30.072	209.216	3.878	0.001
4,4 DDT	32.194	188.896	3.502	0
Cypermethrin-II	43.412	44.217	0.82	0.001
Cypermethrin-III	43.919	1491.232	27.645	0.015
Fenvalerate-I	47.585	340.159	6.306	0.008
			Sum	0.032

Table 4: List of Pesticides Present in the Sample B

Name	RT (min)	Area (Hz-s)	Area %	Amount (ppm)
Alpha-HCH	12.971	147.036	0.944	0
Beta-HCH	14.304	2354.307	15.107	0.009
Gamma-HCH	14.596	7134.315	45.78	0.012
Delta-HCH	15.841	1816.741	11.658	0.003
Chlorpyrifos-ethyl	21.073	384.857	2.47	0.002
2,4 DDD	27.637	52.844	0.339	0
4,4 DDD	29.877	18.931	0.121	0
2,4 DDT	30.054	222.33	1.427	0.001
4,4 DDT	32.175	84.947	0.545	0
Cypermethrin-II	43.457	503.996	3.234	0.017
Cypermethrin-III	43.925	1354.256	8.69	0.014
Fenvalerate-I	47.511	1509.289	9.685	0.033
			Sum	0.091

Table 5: List of Pesticides Present in the Sample C

Name	RT (min)	Area (Hz-s)	Area %	Amount (ppm)
Delta-HCH	15.838	1884.964	25.761	0.003
Chlorpyrifos-ethyl	21.085	118.806	1.624	0
Alpha-ENDOSULPHON	25.38	344.707	4.711	0.001
4,4 DDE	27.07	185.998	2.542	0
2,4 DDD	27.578	145.106	1.983	0.001
4,4 DDD	30.005	13.801	0.189	0
2,4 DDT	30.022	147.527	2.016	0
Endosulphon	31.938	1327.488	18.142	0.004
Lamda-cyhalothrin	38.467	210.964	2.883	0.001
Cypermethrin-ii	43.385	139.652	1.909	0.005
			Sum	0.043

Table 6: List of Pesticides Present in the Sample D

Name	RT (min)	Area (Hz-s)	Area %	Amount (ppm)
Alpha-HCH	12.98	769.521	6.706	0.001
Beta-HCH	14.304	1126.623	9.818	0.004
Gamma-HCH	14.595	2672.486	23.289	0.005
Delta-HCH	15.84	1182.768	10.307	0.002
Chlorpyrifos-ethyl	21.091	577.625	5.034	0.002
Alpha-endosulphon	25.374	552.294	4.813	0.001
4,4 DDE	27.076	288.946	2.518	0.001
2,4 DDD	27.645	61.079	0.532	0
4,4 DDD	29.877	46.846	0.408	0
2,4 DDT	30.038	405.254	3.532	0.001
4,4 DDT	31.938	1605.54	13.991	0.004
Cypermethrin-I	43.174	85.511	0.745	0.002
Cypermethrin-III	43.908	2100.768	18.307	0.021
			Sum	0.044

Fig. 10: GC-ECD Chromatogram of the Standard

Fig. 11: GC-ECD Chromatogram of the Sample A

Fig. 12: GC-ECD Chromatogram of the Sample B

Fig. 13: GC-ECD Chromatogram of the Sample C

Fig. 14: GC-ECD Chromatogram of the Sample D

4. CONCLUSION:

Our research strategically addresses global health challenges, particularly focusing on combating dengue and viral infections. The exploration of A. paniculata has uncovered a myriad of pharmacological activities, notably its pronounced antimicrobial and antiviral properties. In light of the contemporary surge in the adoption of Ayurveda and natural herbal products, our study aims to contribute valuable insights into the safety and potency of human consumption. The meticulous evaluation of samples, employing advanced analytical techniques, not only confirms the therapeutic potential of A. paniculata but also underscores its safety profile. This provides a solid foundation for its integration into modern healthcare practices. The TLC results revealed the well-developed spots of andrographolide along with different constituents present in all four samples of A. paniculata. The HPLC method was applied for the determination of andrographolide in the market available samples and the results showed that the sample C was highly quantified with andrographolide which is summed up with other constituents including neoandrographolide, 14-deoxy-11-12-didehydroandrographolide and andrograponin. GC-ECD analysis identified and quantified various pesticide residues present in the samples of A. paniculata as compared with the standards.This research serves as a testament to the synergy between traditional knowledge and cutting-edge scientific methodologies, offering substantial contributions to addressing current health concerns.

5. ACKNOWLEDGMENT:

We thank the Management of Natural Remedies Pvt. Ltd. and Principal of KLE College of Pharmacy for giving constant support and encouragement for writing this research article.The authors received no financial support for the research, authorship and publication of this article.

6. CONFLICT OF INTEREST:

The authors declare no potential conflicts of interest with respect to research, authorship and/or publication of this article.

7. ABBREVATIONS:

TLC - Thin layer chromatography, HPLC - High Performance Liquid Chromatography and GC-ECD Gas Chromatography-electron capture detector, HCH- Hexachlorocyclohexane, DDE –dichlorodiphenyldichloroethane, DDT-Dichlorodiphenyltrichloroethane,DDD-dichlorodiphenyldichloroethane.

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Received on 17.05.2024 Revised on 10.11.2024

Accepted on 07.04.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):69-75.

DOI: 10.52711/0974-360X.2026.00011

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.