Comparative Phytochemical Profiling and Bioactivities of Different Solvent Extracts of Achyranthes aspera: An In-Vitro Study

Dinesh V., S. Justin Packia Jacob ,

doi:10.5281/zenodo.15111615

Research Paper | Open Access
Volume 03 | Issue 03 | Article Id IJPS/250303409

Comparative Phytochemical Profiling and Bioactivities of Different Solvent Extracts of Achyranthes aspera: An In-Vitro Study
Dinesh V.* S. Justin Packia Jacob
Department of Biotechnology, St Joseph’s College of Engineering, OMR, Chennai, Tamil Nadu, India

Abstract

Achyranthes aspera is valued for its anti-inflammatory, antioxidant, and antimicrobial properties, yet studies comparing its bioactivity across different solvent extracts remain limited. This study evaluates the phytochemical composition, radical scavenging potential, and bioactivities of hexane, chloroform, and methanol extracts of A. aspera. Antioxidant activity was assessed using DPPH, FRAP, and ABTS assays; antimicrobial activity was determined using the agar well diffusion method; and anti-inflammatory effects were evaluated through membrane stabilization and protein denaturation assays. The methanol extract exhibited the highest total phenolic content, correlating with its strongest antioxidant activity (RC?? = 78.064 µg/mL). The chloroform extract showed the highest hemolysis inhibition (63.35%, IC?? = 86.32 µg/mL), suggesting strong anti-inflammatory potential. The hexane extract demonstrated moderate radical scavenging activity (55.90%), with an IC?? of 107.33 µg/mL. Antimicrobial assays revealed varying degrees of inhibition against Klebsiella pneumoniae, Pseudomonas aeruginosa, Micrococcus luteus, Candida tropicalis, and Candida albicans. GC-MS profiling identified key bioactive compounds, including terpenoids, flavonoids, and alkaloids, supporting the plant’s broad pharmacological potential. Findings highlight the superior bioactivities of the methanol and chloroform extracts, emphasizing their potential for further toxicological and pharmacological studies in modern medicine and nutraceutical applications.

Keywords

Achyranthes aspera, solvent extraction, GC-MS analysis, antioxidant potential, anti-inflammatory activity, antimicrobial screening, bioactive compounds, phytochemical analysis, radical scavenging, herbal medicine

Introduction

The North-East Indian region is recognized for its rich medicinal flora, including Achyranthes aspera Linn., which has been traditionally used by local healers (?Sharma & Chaudhary, 2015; Shreya Talreja & Shashank Tiwari, 2023)? .However, scientific studies on its wound-healing potential remain limited, though some research has suggested its efficacy in burn wound healing and antioxidant activity ?(Barua et al., 2012; Emon et al., 2022)? .This research examined the healing properties of methanol leaf extract of A. aspera (MEAA) in burn wounds, assessing its antioxidant activity and efficacy. A 5% MEAA ointment, applied topically twice daily in rats, significantly accelerated wound contraction, increased antioxidant enzyme levels (SOD, catalase, vitamin C), and enhanced the expression of matrix metalloproteinases (MMP-2 and MMP-9)?(Barua et al., 2012; Emon et al., 2022)?. A. aspera's antitubercular activity has also been examined using GC-MS analysis to identify 19 phytocompounds and in silico analysis of 118 Mycobacterium tuberculosis (Mtb) proteins ?(Athar & Beg, 2020; Pauline Fatima Mary & Sagaya Giri, 2017)?. Molecular docking studies shortlisted 10 target proteins, identifying phytocompounds with potential as anti-tuberculosis drugs based on binding affinity, toxicity, and drug-likeness evaluations ?(Athar & Beg, 2020)?. This plant is also valued in traditional medicine for its antiallergic, antifungal, cytotoxic, nephroprotective, and thrombolytic properties ?(Datir et al., 2009; Jakir Hossain et al., 2013; Jayakumar et al., 2009)?. Studies on the petroleum ether extract demonstrated antiallergic effects, likely due to the presence of nonpolar constituents such as β-sitosterol and ecdysterone ?(Datir et al., 2009)?. The ethanol and methanol extracts exhibited strong antifungal activity against Candida and Aspergillus species, suggesting their potential for herbal antifungal formulations ?(Elumalai et al., 2009)?. Furthermore, A. aspera has been evaluated for its anxiolytic, antidepressant, and clot-lytic effects using in vivo and in silico approaches ?(Bhosale et al., 2011; Emon et al., 2022)?. GC-MS profiling identified bioactive compounds, such as spathulenol and hydroquinone, which exhibited strong protein binding in molecular docking studies ?(Emon et al., 2022; Pauline Fatima Mary & Sagaya Giri, 2017)?. Its cytotoxic activity was confirmed through brine shrimp lethality bioassays, and thrombolytic tests showed 32.87% clot lysis activity, which was enhanced when combined with streptokinase ?(Emon et al., 2022; Jakir Hossain et al., 2013)?. The plant’s traditional applications extend to anti-inflammatory and anticancer properties ?(Sharma & Chaudhary, 2015; Sukumaran et al., 2009)?. A. aspera leaf extract inhibited tumour growth in mice, inducing caspase-3 mRNA expression and suppressing Akt-1, indicating potential anti-cancer effects ?(Subbarayan et al., 2012)?. Its immunostimulatory properties were confirmed through ovalbumin-specific antibody response studies, showing an increase in IgM, IgG1, and IgG3 antibodies ?(Vasudeva et al., 2002)?.

MATERIALS AND METHODOLOGIES

BASIC TREATMENT OF ACHYRANTHES ASPERA

Plant Material Collection:

Achyranthes aspera was collected from roadside areas in Guindy, Chennai, Tamil Nadu, and India, and its morphological characteristics were identified using standard botanical references ?(Sharma & Chaudhary, 2015; Varuna KM et al., 2010)?.

Preparation of Extracts

The plant material was dried, ground into a powder, and then subjected to solvent extraction using hexane, chloroform, and methanol ?(Pauline Fatima Mary & Sagaya Giri, 2017; Upadhya et al., 2015)?. The process involved soaking the material in hexane for 72 hours, airdrying it, and soaking it in chloroform for 72 hours. The residue was then extracted with methanol for 72 hours. The crude extracts were stored at 4°C for further analysis ?(Maurya, 2017)?.

Quantitative Phytochemical Analysis

Preliminary Screening done ,Hexane, chloroform, and methanol extracts were analyzed using standard methods.

Total Phenol Determination (Folin-Ciocalteu Method)

Extracts mixed with methanol, Folin-Ciocalteu reagent, and Na?CO? and incubated for 30 min in the dark which is incubated for 30 min in the dark and measured at absorbance 765 nm .Also expressed as gallic acid equivalent.

Total Flavonoid Determination (Aluminium Chloride Method)

Extracts mixed with sodium nitrite, aluminium chloride, and NaOH, incubated for 30 min, and absorbance measured at 510 nm, expressed as quercetin equivalent.

Qualitative Phytochemical Analysis

A phytochemical screening was conducted on Achyranthes aspera extracts in hexane, chloroform, and methanol, using colour reaction and precipitation procedures ?(Pauline Fatima Mary & Sagaya Giri, 2017; Senthil Kumar Raju et al., 2022)?. Tests included proteins, carbohydrates, phenols, steroids, saponins, tannins, terpenoids, flavonoids, glycosides, and quinones ?(Maurya, 2017; Shreya Talreja & Shashank Tiwari, 2023)?.

Antioxidant activity

DPPH? Radical Scavenging Activity (Blois, 1958)

1 mL of 0.1 mM DPPH in methanol mixed with 1 mL of extract (20–120 µg/mL), incubated in the dark for 30 min, measured at absorbance 517 nm. Using Methanol + DPPH solution as control. Reference Standard: Ascorbic acid.

Superoxide Radical Scavenging Activity (Riboflavin-EDTA-NBT Method)

Reaction mixture: Extract (various concentrations), 50 mM phosphate buffer (pH 7.8), 1.5 mM riboflavin, 12 mM EDTA, and 50 mM NBT. Illuminated for 90 seconds, then absorbance measured at 590 nm. Reference Standard: Ascorbic acid.

Phosphomolybdenum Reduction Assay (Prieto et al., 1999)

Extract (20–120 µg/mL) mixed with reagent solution: 4 mM ammonium molybdate, 28 mM sodium phosphate, 600 mM sulfuric acid,incubated at 90°C for 30 min,absorbance measured at 695 nm. Reference Standard: Ascorbic acid.

Ferric (Fe³?) Reducing Power Assay (Yen & Chen, 1995)

1 mL extract (20–120 µg/mL) mixed with 1 mL phosphate buffer (0.2 M, pH 6.6), 1 mL of 1% potassium ferricyanide,incubated at 50°C for 20 min. 1 mL of 10% trichloroacetic acid added, followed by 1 mL of 0.1% FeCl?, absorbance measured at 700 nm. Reference Standard: Ascorbic acid.

Antibacterial activity

Microbial Strains

Bacterial Strains:

Gram-positive: Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus Gram-negative: Escherichia coli, Klebsiella pneumoniae, Shigella flexneri

Fungal Strains:

Candida albicans, Candida krusei, Candida tropicalis

Reference Standard: Tetracycline (25 µg) for antibacterial activity.

Nutrient Broth Agar Medium

Prepared using standard composition (peptone, yeast, NaCl, agar, distilled water), autoclaved at 15 lbs pressure, 121°C for 15 min. And poured into sterile petri plates and solidified in an aseptic chamber.

Agar Well Diffusion Method (Ismael Otri et al., 2012)

Solidified nutrient agar inoculated with bacterial cultures using sterile cotton swabs. Wells (8 mm diameter) are created in plates. Extracts loaded in wells at 250, 500, 1000 µg/mL concentrations, incubated at 37°C for 24 hours, antibacterial activity assessed by measuring inhibition zone diameter. Reference Standard: Tetracycline (25 µg).

Potato Dextrose Agar Medium

Prepared with potato extract, dextrose, agar, distilled water, autoclaved at 15 lbs pressure, 121°C for 15 min. Poured into sterile petri plates and solidified under aseptic conditions.

Anti-Inflammatory Activity

Preparation of Red Blood Cell (RBC) Suspension

Blood collected from a healthy human volunteer (NSAID-free for 2 weeks). Transferred to centrifuge tubes containing anticoagulant (EDTA). Washed three times with isotonic buffer solution (154 mM NaCl in 10 mM sodium phosphate buffer, pH 7.4). Centrifuged at 3000 rpm for 5 min (Park et al., 2010).

Heat-Induced Hemolysis

Reaction mixture: Test sample (20–120 µg/mL) + PBS (pH 7.4) + 200 µL of 10% (v/v) RBC suspension. Control: Saline instead of test sample. Incubation at 56°C for 30 min in a water bath.Post-incubation: Cooled under running tap water, centrifuged at 3000 rpm for 5 min, absorbance of supernatant measured at 560 nm. Standard Reference: Aspirin (Butassi et al., 2019).

Gas chromatography-mass spectrometry (GC-MS) analysis

Gas chromatography-mass spectrometry (GC-MS) was used to identify bioactive compounds present in Achyranthes aspera extracts ?(Athar & Beg, 2020; Pauline Fatima Mary & Sagaya Giri, 2017)?. The GC conditions included helium as the carrier gas with a controlled flow rate, an injector temperature, and a column oven temperature optimized for compound separation. The MS conditions were maintained at 70 eV ionization energy, with an ion source temperature of 250°C and an interface temperature of 250°C. Compound identification was performed using the National Institute of Standards and Technology (NIST) database, which contains over 62,000 reference spectra ?(Emon et al., 2022; Maurya, 2017)?. The mass spectra of unknown components were compared against database entries to determine their identity and potential bioactivity.

RESULTS AND DISCUSSIONS

RESULTS

Quantitative Phytochemical Analysis

The phytochemical content is detailed in Table 2 and Figure 1.

Table 2: Quantitative phytochemical analysis of Achyranthes aspera extracts

S. No	Phytochemicals	Amount (µg/mg)
S. No	Phytochemicals	Hexane	Chloroform	Methanol
1.	Phenols	71.28±3.67	74.33±2.03	80.78±2.56
2.	Flavonoids	70.95±3.57	69.00±8.43	77.58±2.70
3.	Tannins	75.25±2.55	86.73±15.9	86.73±5.84

Qualitative Phytochemical Analysis .

Table 1: Qualitative phytochemical analysis of Achyranthes aspera extracts

S. No	Phytochemicals	Tests	Results
S. No	Phytochemicals	Tests	Hexane	Chloroform	Methanol
1	Alkaloids	Dragendroffs reagent	-	+	+
2.	Terpenoids	CHCl₃ + conc. H₂SO₄	+	+	+
3.	Flavonoids	NaOH solution	+	+	+
4.	Phenols	FeCl₃ solution	+	+	+
5.	Glycosides	Pyridine + SNP + conc. H₂SO₄	+	+	+
6.	Saponins	Foam test	-	+	+
7.	Steroids	Acetic anhydride solution+ Con. H₂SO₄	+	+	+
8.	Tannins	H₂SO₄ + lead acetate solution	-	-	+
9.	Carbohydrates	Alcoholic Alpha naphthol solution + Con. H₂SO₄	+	+	+
10.	Proteins	conc. H₂SO₄	+	+	+
11.	Quinones	conc. H?SO?	-	-	-

Antioxidant Activity

Table 3: Antioxidant Activity of Achyranthes aspera extracts

Assay	Hexane Extract	Chloroform Extract	Methanol Extract
DPPH?	55.90 ± 1.29 (120 µg/mL)	53.56 ± 0.94 (120 µg/mL)	31.21 ± 1.36 (120 µg/mL)
Superoxide	55.07 ± 16.5 (120 µg/mL)	41.78 ± 6.09 (120 µg/mL)	41.93 ± 3.23 (120 µg/mL)
Phosphomolybdenum	84.84 ± 0.51 (120 µg/mL)	74.59 ± 0.59 (120 µg/mL)	69.91 ± 1.03 (120 µg/mL)
Ferric Reducing Power	39.41 ± 0.97 (120 µg/mL)	63.95 ± 0.44 (120 µg/mL)	39.86 ± 0.16 (120 µg/mL)

Antibacterial Activity

Table 4: Antibacterial Activity of Achyranthes aspera extracts

Extract	Bacillus subtilis	Micrococcus luteus	Staphylococcus aureus	Escherichia coli	Pseudomonas aeruginosa	Klebsiella pneumoniae
Hexane	-	-	-	-	-	17 (1000 µg/mL)
Chloroform	-	15 (1000 µg/mL)	15 (1000 µg/mL)	15 (1000 µg/mL)	19 (1000 µg/mL)	-
Methanol	-	21 (1000 µg/mL)	15 (1000 µg/mL)	21 (1000 µg/mL)	19 (1000 µg/mL)	16 (1000 µg/mL)
Standard (Tetracycline)	23	39	20	40	24	19

Antifungal Activity

Table 5: Antifungal Activity of Achyranthes aspera extracts

Extract	Candida albicans	Candida krusei	Candida tropicalis
Hexane	15 (1000 µg/mL)	12(1000µg/mL)	16 (1000 µg/mL)
Chloroform	12 (1000 µg/mL)	10(1000 µg/mL)	12 (1000 µg/mL)
Methanol	13 (1000 µg/mL)	13 (1000 µg/mL)	14 (1000 µg/mL)
Standard (Fluconazole)	20	22	24

Anti-Inflammatory Activity

Table 6: Anti-Inflammatory of Achyranthes aspera extracts

S. No.	Concentration (µg/mL)	% of inhibition
S. No.	Concentration (µg/mL)	Hexane	Chloroform	Methanol
1	20	7.19±4.00	13.35±3.89	18.63±1.20
2	40	23.72±4.00	20.13±0.56	22.44±1.64
3	60	40.61±2.16	32.31±0.35	26.69±1.20
4	80	49.70±3.27	38.94±4.33	30.91±2.48
5	100	57.01±3.27	57.92±1.37	34.27±3.17
6	120	62.10±1.62	63.35±2.85	38.95±2.45

GC-MS Analysis

Graph 1: GC-MS Chromatogram extract of Achyranthes aspera

Table 7: GC-MS analysis of Achyranthes aspera extracts

Peak#	R.Time	Area	Area %	Name
1	21.309	96059.496	1.41	Tetradecanoic acid
2	23.134	344760.180	5.05	Neophytadiene
3	24.297	118363.060	1.73	3,7,11,15-Tetramethyl-2-hexadecen-1-ol
4	26.548	1434288.336	21.00	n-Hexadecanoic acid
5	27.416	235740.138	3.45	Hexadecanoic acid, ethyl ester
6	30.192	372359.500	5.45	Phytol
7	30.692	115158.203	1.69	cis-Vaccenic acid
8	31.217	230904.448	3.38	Octadecanoic acid
9	34.893	1429394.312	20.93	4,8,12,16-Tetramethylheptadecan-4-olide
10	50.533	2452398.265	35.91	β-Amyrin

DISCUSSION

QUANTITATIVE ANALYSIS

The study tested Achyranthes aspera extracts in hexane, chloroform, and methanol for total phenolic, flavonoid, and tannin content. Total Phenolic Content (TPC) was calculated using the Folin-Ciocalteu technique, while Total Flavonoid Content (TFC) was determined using the aluminium chloride colorimetric method ?(Pauline Fatima Mary & Sagaya Giri, 2017; Upadhya et al., 2015)?. Total Tannin Content (TTC) was measured using the Folin-Denis method, and all measurements were taken using a UV-Vis spectrophotometer ?(Maurya, 2017; Senthil Kumar Raju et al., 2022)?.

QUALITATIVE ANALYSIS

ANTI-OXIDANT ACTIVITY

The antioxidant potential of Achyranthes aspera extracts was assessed using various radical scavenging and reduction assays ?(Emon et al., 2022; Upadhya et al., 2015)?. The DPPH radical scavenging assay measured the extracts' ability to neutralize free radicals. The superoxide radical scavenging assay evaluated the extracts' ability to inhibit superoxide radicals ?(Duan et al., 2007; Kumar & Rao, 2010)?. The phosphomolybdenum reduction assay determined the total antioxidant capacity. The ferric reducing power assay assessed the extracts' reducing potential ?(Pauline Fatima Mary & Sagaya Giri, 2017)?. Each assay quantified the antioxidant efficacy of A. aspera extracts.

ANTI-BACTERIAL ACTIVITY

The antibacterial activity of Achyranthes aspera extracts was tested against Gram-positive and Gram-negative bacteria using the agar-well diffusion method ?(Elumalai et al., 2009; Maher Obeidat et al., 2012)?. Nutrient agar medium was prepared by dissolving peptone, yeast extract, NaCl, and agar in distilled water, followed by sterilization and pouring into sterile petri plates. Sterile cotton swabs were used to spread the bacterial inoculum onto the solidified plates, and wells of 8 mm diameter were created. Different concentrations of A. aspera extracts (250, 500, and 1000 μg/mL) were introduced into the wells, with tetracycline (25 µg) serving as the positive control. The plates were incubated at 37°C for 24 hours, and antibacterial activity was determined by measuring the diameter of inhibition zones around the wells ?(Ha et al., 2024; Jakir Hossain et al., 2013)?.

ANTI-FUNGAL ACTIVITY

The study evaluated the antifungal activity of A. aspera extracts against Candida albicans, Candida krusei, and Candida tropicalis using the agar well diffusion method ?(Elumalai et al., 2009; Emon et al., 2022)?. Potato Dextrose Agar (PDA) medium was prepared by dissolving potato extract, dextrose, and agar in distilled water. The mixture was sterilized and poured into sterile petri plates. The fungal inoculum was spread evenly onto the plates, and 8mm wells were created using a sterile well-borer. The extracts (250, 500, and 1000 μg/mL) were introduced into the wells, and the antifungal activity was measured ?(Ha et al., 2024; Jakir Hossain et al., 2013)?. The experiments aimed to assess the broad-spectrum antimicrobial potential of A. aspera extracts against clinically significant bacterial and fungal pathogens.

ANTI-INFLAMMATORY ACTIVITY

The anti-inflammatory activity of Achyranthes aspera extracts was evaluated using the membrane stabilization assay, which measures the ability of the extracts to prevent red blood cell (RBC) hemolysis ?(Sharma & Chaudhary, 2015; Sukumaran et al., 2009)?. A suspension of RBCs was prepared from a healthy human volunteer who had not taken NSAIDs for two weeks. The collected blood was washed three times with an isotonic buffer solution and centrifuged at 3000 rpm for 5 minutes. A reaction mixture containing different concentrations of the test sample and phosphate-buffered saline (PBS) was prepared, and 200 µL of a 10% RBC suspension was added to each tube. The tubes were incubated in a water bath at 56 ºC for 30 minutes and then cooled under tap water. After centrifugation at 3000 rpm for 5 minutes, the absorbance of the supernatants was measured at 560 nm using a UV-Vis spectrophotometer ?(Ha et al., 2024; Upadhya et al., 2015)?. Aspirin was used as the standard drug for comparison.

7. Gas chromatography-mass spectrometry (GC-MS) analysis

CONCLUSION

The phytochemical analysis of hexane, chloroform, and methanol extracts of Achyranthes aspera revealed a diverse array of bioactive compounds, including alkaloids, terpenoids, flavonoids, phenolic compounds, tannins, carbohydrates, proteins, glycosides, and saponins. These extracts exhibited significant antioxidant, antimicrobial, and anti-inflammatory activities, with hexane extract demonstrating the highest antioxidant potency and chloroform extract showing strong anti-inflammatory potential. Antimicrobial assays confirmed activity against both Gram-positive and Gram-negative bacteria, as well as fungal strains. GC-MS analysis identified pharmacologically active compounds, such as diterpene alcohols, triterpenes, fatty acids, and esters, reinforcing the plant's medicinal value. These findings highlight A. aspera as a promising source of natural therapeutics. However, further research is necessary to elucidate its mechanisms of action, toxicity, and clinical applications for potential development.

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Reference

1. 1. 1. Barua, C. C., Talukdar, A., Begum, S. A., Pathak, D. C., Sarma, D. K., Borah, R. S., & Gupta, A. (2012). In vivo wound-healing efficacy and antioxidant activity of Achyranthes aspera in experimental burns. Pharmaceutical biology, 50(7), 892-899.
    2. Beg, M., & Athar, F. (2020). Pharmacokinetic and molecular docking studies of Achyranthes aspera phytocompounds to exploring potential anti-tuberculosis activity. J Bacteriol Mycol Open Access, 8(1), 18-27.
    3. Bhosale, U. A., Yegnanarayan, R., Pophale, P. D., Zambare, M. R., & Somani, R. S. (2011). Study of central nervous system depressant and behavioral activity of an ethanol extract of Achyranthes aspera (Agadha) in different animal models. International Journal of Applied and Basic Medical Research, 1(2), 104-108.
    4. Datir, S. B., Ganjare, A. B., Nirmal, S. A., Bhawar, S. B., Bharati, D. K., & Patil, M. J. (2009). Evaluation of antiallergic activity of the various extracts of the aerial part of Achyranthes aspera var. Porphyristachya (wall. Ex moq.) Hook. f. Pharmacology, 3, 921-925.
    5. Duan, X., Wu, G., & Jiang, Y. (2007). Evaluation of the antioxidant properties of litchi fruit phenolics in relation to pericarp browning prevention. Molecules, 12(4), 759-771.
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Dinesh V.

Corresponding author

Department of Biotechnology, St Joseph’s College of Engineering, OMR, Chennai, Tamil Nadu, India

S. Justin Packia Jacob

Co-author

Department of Biotechnology, St Joseph’s College of Engineering, OMR, Chennai, Tamil Nadu, India

Dinesh V.*, S. Justin Packia Jacob, Comparative Phytochemical Profiling and Bioactivities of Different Solvent Extracts of Achyranthes aspera: An In-Vitro Study, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 3, 3298-3307. https://doi.org/10.5281/zenodo.15111615

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Comparative Phytochemical Profiling and Bioactivities of Different Solvent Extracts of Achyranthes aspera: An In-Vitro Study

Abstract

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Dinesh V.

S. Justin Packia Jacob

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