INTRODUCTION:

People from different parts of the world depend on the natural products to treat diseases^1-2. Amaranthus caudatus grows well in different countries of Asia-Pacific regions. In India it grows with minimum irrigation during June to November³. It usually germinates well in winter and grows gradually up to a height of 5 meters⁴. The Amaranthus caudatus leaves are used in the preparation of spicy soup with onion, garlic, turmeric powder and salt, it can be used in seasoning the salads⁵. In South Africa, it is used as dry fine powder, so that even after season the nutrient property of this underutilized crop is readily availed⁶. Studies have demonstrated that the use of seeds in poultry results in an improvement in egg quality due to the presence of high-quality proteins⁷.

The different parts of these plants are used for various therapeutic purposes by indigenous tribes. The roots of this plant are used as antibacterial agents for sexually transmitted diseases like gonorrhoea, the leaves are used for the treatment of haemorrhoids, renal pathologies, antipyretic agent, celiac disease, antidiabetic, antimicrobial agent while the seeds are used to reduce extreme internal bleeding^8-9. The agricultural production is decreasing day by day due to urbanization, as the consequence there will be global demand for the natural products by 2050¹⁰. Hence there is a need to use the underutilized herb like Amaranthus caudatus to improve the growing needs of pharmaceutical industries.

The herbal extracts are prepared by various techniques like maceration, hydroalcoholic extraction after fermentation, continuous hot extraction with soxhlet apparatus, herbal aqueous extraction, etc¹¹. The soxhlet continuous hot methanolic extraction was proven to work against antimicrobial agents¹². The leaves were chosen because of their higher production rate when compared with the stem and root. GC-MS technique helps to reveal the different secondary metabolic constituents derived from carbohydrate, protein and fat metabolism in this extract during boiling^13-19.Antidiabetic and anticholesteremic activities were already proven in wistar albino rat experiment²⁵but the exact compound responsible for these activities is unknown. Therefore, this study will help to know the list of compounds present in methanolic leaf extract of Amaranthus caudatus (MEAC). Besides the phytoconstituents, in vitro antioxidant analysis was done with DPPH assay using UV vis spectrophotometer. This analysis will help by quantification before performing in vivo pre-clinical evaluation.

MATERIAL AND METHODS:

Plant seed procurement:

The specific indigenous collection of Amaranthus caudatus (IC-274455; Receipt No:DS-740/2023/44) was collected from Indian Council of Agricultural Research, National Bureau of Plant Genetic Resource, Shimla branch, Himachal Pradesh, India.

Plant cultivation and sample collection:

Land permission for plant cultivation was obtained from Dhanalakshmi Srinivasan University, Samayapuram, Trichy. Then plants were self-cultivated naturally without use of pesticides and fertilizers during winter season in December month. The protocol prescribed by the ICAR was followed during plant production. The fresh leaves were collected from plant at fortieth day, washed with running tap water, dried in sun shaded area and preserved in storage container for further analysis.

Chemicals utilized:

Methanol, aluminium chloride, quercetin, sodium hydroxide, sodium nitrite, Folin-Ciocalteu reagent, gallic acid, sodium carbonate were purchased from Merk, USA. Ascorbic acid and DPPH solution were procured from Himedia Laboratories Pvt. Ltd., India.

Materials utilized:

Soxhlet apparatus, number one Whatman filter paper, test tubes, electronic weighing machine, micropipette of different size, glass beaker 250ml, heating mantle and Labman double beam UV spectrophotometer advanced version, 2ml glass bottle.

Soxhlet extraction:

Thirty-five grams of dried leaves of Amaranths caudatus were crushed using a pestle and mortar. Then the fine powder was filled in porous thimble with thickness 25mm diameter and 80mm length. The mouth of the thimble was lubricated and tightened with the condenser in the upper portion and round bottomed flask of 250ml below in the lower portion. The inlet and outlet water tubes were ensured for their position. After completing the fixation of soxhlet apparatus, the round bottomed flask was placed in the heating mantle. Then the condenser was filled with 200ml of methanol through the funnel and heated using an isomantle. The temperature was kept at 65⁰C which is the standard boiling point for methanol. The solvent mixture evaporates well and the contents get recycled through the condenser. The condensate was collected in the reservoir until the presence of clear colourless fluid seen in the siphon arm. Then the filtered methanolic extract was transferred to the 250ml beaker which was placed in heating mantle and concentrated. The final product was collected in 2ml glass bottles and preserved in refrigerator at 4⁰C for future use²⁵.

QUALITATIVE ANALYSIS:

Methods of preliminary phytochemical analysis:

Experiments already proven the presence of phenol, flavonoid, carbohydrates, proteins, tannin, alkaloid, saponin, amino acids²⁰. Current research also revealed the presence of some more additional compounds as follows.

Effervescence test for confirmation of carboxylic acid:

1ml plant of MEAC was compounded with 2ml of sodium bicarbonate solution. Brisk effervescence confirms the carboxylic acid in MEAC.

Salkowski test for confirmation of steroids:

500μl of MEAC is added to 5ml of chloroform and equal amount of conc. sulfuric acid. Observation of red colour in the upper portion and yellowish green in the lower portion confirms the presence of steroids.

Testing method for confirmation of resin:

500μl of leaf concentrate is added with 3ml of copper sulphate, shaken for couple of minutes, until the emerald green colouration observed.

Total phenol content (TPC):

TPC was analysed by Folin Ciocalteu method. One ml of concentrated methanolic extract and one ml Gallic acid solution (0.007mg/ml to 1mg/ml) were taken separate tubes. Then the tubes were filled with 5ml of distilled water and 0.5ml of Folin Ciocalteau reagent. After mixing the contents are left for five minutes with addition of 1.5ml of twenty percentile sodium carbonate solution till the quantity reaches ten millilitres distilled water was added. This mixture was kept for couple of hours at lab temperature. After completing waiting period, dark blue colour was obviously visible. Rate of absorbance was detected at the range of 750nm with UV visible spectrophotometer. The aliquot was triplicated in 8 different serial dilutions as shown in the table 2. The blank value was obtained using standard reagent with adequate solvent. The graphical curvature was recorded using Gallic acid as reference for standardisation. The quantitative reports for total phenolic concentrate of the sample were noted in terms of mg of gallic acid reference (GAE)/100gram of dried mass³¹.

Total Flavonoid content (TFC):

TFC present in MEAC was quantified with the aluminium chloride colorimetric method. 1ml of required extract and 1ml standard quercetin solution (50 mg/ml) was taken in test tubes along with 4ml of distilled water, 0.3ml of 5% sodium nitrite solution in each tube. After completing waiting period of 5 minutes, 0.3ml of 10% aluminum chloride was added. After that 2ml of 1 M sodium hydroxide solution is taken. Finally, the volume was made up to 10ml with distilled water before final mix. Amber colour was observed. The rate of absorbance was detected at the range of 510nm with UV-visible spectrophotometer. The blank was performed with distilled water simultaneously the standard was performed with Quercetin in triplicates as shown in table-3. The graphical curve was plotted with standard quercetin. The data of total flavonoids in MEAC were noted in terms of mg of Quercetin equivalents/100g of dried mass^32-34_.

DPPH ASSAY:

0.1mM DPPH is prepared by mixing 3.94mg of DPPH powder was mixed with 100ml of methanol and stored in -20⁰C until needed.From this,0.1mM of DPPH is added to MEAC in various concentrations as depicted in table3. The mixture was left in room temperature for 30minutes. Then rate of absorbance is detected at 517nm with UV Spectrophotometer. Ascorbic acid is considered as the standard. Percentage of radical scavenging activity was calculated using the formula.

Percentage of Inhibition = (A_C – A_S)/A_C ×100, Where A_C indicates absorbance of the control and A_s was the absorbance in the presence of the leaf sample and IC₅₀ value was recorded^33-3⁷

STATISTICAL ANALYSIS:

The Statistical interpretation was done with SPPS software-20^th version. One-way ANOVA confirms the values were statistically important with P value. p<0.001 where the univariate analysis was adjusted to the R square at 0.990.

RESULTS:

Preliminary phytochemical analysis revealed the presence of resin, carboxylic acid and steroid in MEAC.

Estimation of Total Phenol Content:

The test sample was detected in the mean value: 0.58 mg/ml in the MEAC after triplication. The concentration of TPC was calculated with graph pad prism software with calibration plot, y=0.5343x+0. 0985.R²=0.9481. m value=0.5343, c value= 0. 0985.

Table No: 1. Estimation of Total phenolic contents with reference to Gallic acid Equivalent.

Gallic acid (mg/ml)	OD Values at 750nm in triplication			Mean Value
5	2.987	2.984	2.997	2.989
2.5	0.945	0.952	0.963	0.953
1.25	0.765	0.741	0.751	0.752
0.625	0.537	0.503	0.565	0.535
0.312	0.415	0.433	0.441	0..429
0.156	0.238	0..274	0.269	0.260
0.078	0.131	0.111	0.158	0.133
0.039	0.065	0.068	0.042	0.058

Estimation of Total Flavonoid Content:

The mean value of total flavonoid quantified was 0.44mg/ml in the MEAC after triplication equivalent to quercetin reference. The total flavonoid content was calculated with graph pad prism software with calibration plot, y=0.5177x+0. 1816.R²=0.9448. m value=0.5177, c value= 0. 1816.

Table No:2. Estimation of total flavonoid contents in mg/ml quercetin reference.

Quercetin (mg/ml)	OD Values at 510 nm in triplication			Mean Values
5	2.99	2.963	2.906	2.987
2.5	1.074	1.059	1.044	1.061
1.25	0.789	0.751	0.745	0.745
0.625	0.677	0.657	0.611	0.611
0.312	0.567	0.577	0.586	0.586
0.156	0.327	0.373	0.399	0.399
0.078	0.138	0.156	0.171	0.171
0.039	0.099	0.073	0.051	0.051

DPPH ASSAY:

IC50 value of MEAC in terms of ascorbic acid was 48.93mg/ml. Percentage of inhibition in DPPH Assay for ascorbic acid was depicted in the table-3.

GC-MS ANALYSIS:

The phytochemical analysis of methanolic aliquot was done with GC-MS apparatus- in the same method and machine set up as described in previous studies^21-24. GCMS analysis was done as prescribed in previous methods^26-32.

Fig.: 2. Total ion composition of different components seen in methanolic leaf extract of Amaranthus caudatus – GC-MS.

DISCUSSION:

Literature review for more than a decade shows the methanolic hot leaf extract of Amaranthus caudatus has multiple application in different clinical conditions, to improve the quality further there is a need to acquire a knowledge about the number of phytochemicals seen in the current investigation. The cold methanolic extract reveals the presence of Octanoic acid, 2-Pyrrolidinone, 3-Cyclopentylpropionic acid, 2-dimethylaminoethyl ester, Octadecanoic acid, 2-Ethylbutyric acid, Hexadecenoic acid, Dinoctyl phthalate, Cyclohexane acetic acid, Pentadecafluorooctanoic acid, Linalool oxide, alpha-Tocospiro B, Chondrillasterol in highest peak²⁹_.GC-MS analysis of present study was showed different phytochemicals in 28 high peaks and their medical uses were listed in the table 4. Among these phytochemicals, 13-docosenamide(Z)- reached with highest RT of 28.9844.

Table No: 3- Shows the comparative evaluation of antioxidant activities in MEAC with reference to ascorbic acid in DPPH assay.

S. No	Test sample concentrate (μg/ml)	Percentage of inhibition of sample in triplication			Mean value %
1.	Ascorbic acid(1 μg/ml)	91.46211	88.26041	87.19317	88.9719
2.	500μg/ml	68.83671	68..18036	66.38207	67.8762
3.	250μg/ml	62.9662	63.18036	62.11313	62.75347
4.	100μg/ml	59.55176	58..27108	55.92316	57.91533
5.	50μg/ml	55.17609	51.65422	39.48773	48.77268
6.	10μg/ml	26..254	25.93383	25.93383	26.04055

Table No.: 4. List of major phytocomponents revealed in high peaks during GC-MS analysis with their medical applications.

S.No	Name of the compound	Retension time	Area	Height	Medical uses
1.	DL-Proline	12.164	878651	202691	Proper joint and muscle functioning .
2.	Niacinamide	12.499	177164	35824	Form of vitamin B3 which maintain healthy cells.
3.	Succinic acid1	12.858	185640	31032	Standard agent in acid base anaysis.
4.	1,6-Anhydro-2	13.168	830159	120210	Used as modified sugars.
5.	2,4-Di-tert-butylphenol	14.266	169535	40178	Intermediate agent in production of antioxidants.
6.	Benzoic acid	14.266	936948	140006	Food preserevative agent.
7.	Uracil	14.844	552091	51671	Anticancer agent
8.	Isosaxole 3 carboxylic acid	15.160	165684	35463	Antimicrobial agent
9.	1-Naphthalenemethanamine	15.325	250573	36872	Antifungal agent
10.	Diethyl phthalate	15.519	547179	115457	Analgesics used as coating aspirin tablets
11.	1,2,3,5-Cyclohexanetetrol	15.673	956265	110655	Plant metabolite used as glycosidase inhibitor.
12.	1,6 octadiene	16.098	63345	20470	Fragrance ingredient in cosmetic industry
13.	Cyclohexylamine	16.538	135167	17721	Intermediate agent used in pharmaceutical industry as bronchodilator
14.	Oxanonanoic acid.	17.738	359651	68540	Cardiac diseases and pulmonary hypertension.
15.	6 Hyroxy 4	17.955	108102	33386	Coumarin derivative,anticancer agent
16.	n pentadecanol	18.152	62850	22916	Lubricative agent in lotion and creams.
17.	3 Pentadecanol	18.300	52333	17710	Pulmonary surfactant and mycobactericidal agent
18.	Isopropyl myristate	18.595	186173	56637	Skin moisturizer.
19.	Hexyl octyl ester	19.821	55953	13180	Plant metabolite used as flavouring agent.
20.	Octadecanoic acid	20.258	54369	12933	Anticancer,Hypocholesterolemic agent.
21.	Benzene acetic acid	20.342	77789	17930	Manufacture substitued amphitamine
22.	9H -Pyridol	20.607	178524	17930	Treat neurological disorders
23.	Cis-Sinapyl alcohol	20.741	89343	23993	Disinfectant.
24.	6 Methylheptanoic acid	21.010	89226	22108	Lubricative agent.
25	Chondrillasterol	25.610	989148	108115	Antibacterial agent
26.	Hexadecenoic acid	26.220	91502	25232	Antimicrobial and antioxidant agent.
27.	1Heptanol	28.241	42277	11751	Treat prolonged arrythmias
28.	13-Docosenamide,(Z)-	28.984	1111296	193484	Antimicrobial agent and surfactant.

CONCLUSION:

Many compounds with antiseptic properties were revealed in GC-MS analysis of MEAC which can be isolated to decrease inflammatory reactions. 13-docosenamide(Z), 1 heptanol, hexadecenoic acid, chondrosterol were some of the major compounds which reached highest peaks. Our research study reveals MEAC inhibited free radicals and has shown decreasing antioxidant capacity in a phytoconstituent based approach. The compounds like Phenol, Flavonoid and various listed compounds may be responsible for these properties. Free radical scavenging activity of MEAC will helps to protect us from various inflammatory diseases. The selection of compounds from the list help to characterize their activity in various diseases. Further compound isolation from the higher peak compounds can be done based on individual needs.

ACKNOWLEDGEMENTS:

We are thankful to Dhanalakshmi Srinivasan University, Samayapuram, Trichy, Tamil Nadu for funds provided for the conduct of this study and Dr. Mohar Singh in charge of National Bureau of Plant Genetic Resource, Shimla branch for seed arrangement.

ABBREVATIONS:

MEAC- Methanolic Extract of Amaranthus caudatus.

NBPGR-National Bureau of Plant Genetic Resources.

RT- Retention Time.

GC-MS-Gas Chromatography Mass spectrometry.

ANOVA-Analysis of Variations.

IC-50-Half Maximal Inhibitory Concentration.

DPPH-2,2, diphenyl-1-picrylhydrazyl.

ABTS- 2,2'-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid.

ICAR-Indian Council of Agricultural Research

SPSS-Statistical package of Social Sciences

GAE-Gallic acid equivalent.

GA/g-Gallic acid per gram.

mg-Milli gram.

OD-Optical density.

CONFLICTS OF INTEREST:

No conflicts of interest.

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Received on 05.10.2024 Revised on 19.02.2025

Accepted on 24.04.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):141-146.

DOI: 10.52711/0974-360X.2026.00022

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