Introduction

Naturally occurring substances have undergone a great deal of research and have shown fascinating biological and pharmacological properties, which indicates that they could be used as chemotherapeutic agents or as the building blocks for contemporary medications. But in order to use these resources effectively for the sake of human health, more research is required.¹ Because plant-derived pharmaceuticals have significantly enhanced human health and well-being, Herbs can serve two purposes in the creation of new medications: (a) they can serve as the model for the creation of natural medications, or (b) they can be used as a phytomedicine to treat diseases. Even while modern medicine has come a long way, there are still numerous illnesses and conditions for which there is no known treatment. Herbal plants are essential to human life.^2-3 As analgesics, antioxidants, antibacterials, antimalarials, and other therapeutic remedies, people have used the bark, berries, leaves, stems, flowers, and roots of plants. Flavonoids, phenolic acids, tannins, stilbenes, lignans, lignins, and coumarins are among the bioactive compounds found in medicinal plants; these compounds have a range of biological effects, such as antibacterial and antioxidant properties.⁴ Herbal remedies are frequently used in medical settings. The traditional medical system has long recognized the therapeutic potential of medicinal plants for treating a broad range of ailments. The development of safer drugs to treat inflammatory diseases, skin issues, fungal and microbial infections, diabetes, liver disorders, and gastrointestinal disorders has thus become essential. To this aim, different healthcare programs throughout nations have supplemented each other by separating active ingredients for use in contemporary medications and herbal remedies. secondary metabolites or phytochemicals that are present in medicinal plants and are in charge of their antibacterial, antifungal, and analgesic effects. Nonetheless, certain bacteria and fungi that are harmful to humans have become resistant to bioactive substances like flavonoids’ antibacterial and antifungal properties.⁵ The increasing prevalence of multiple drug-resistant microorganisms to conventional drugs has led to a growing interest in seeking natural sources of antimicrobial agents. The potential for plant-derived antimicrobial compounds to revolutionize modern medicine is vast and largely untapped, warranting further investigation in this field.

In Indian traditional medicine, Achyranthes aspera (A. aspera) Linn, commonly referred as chirchira in Hindi, is a source of high therapeutic properties. This native herb has long been used in Ayurvedic, Unani, and Siddha medicine, among other traditional medical systems. A. aspera leaves have been shown to have antibacterial qualities,⁶ which makes it beneficial for treating conditions involving the skin and teeth. This document lists the traditional uses, folk cures, ethnopharmacologic properties, and ethnopharmacologic properties of this herb. A. aspera Linn recently reported many potential activities such as nephroprotective,⁷ anticonvulsant,⁸ anticancer, ^9-10 wound healing activity,¹¹ anti-inflammatory^12-13 respectively. The annual, stiff, upright herb A. aspera Linn is frequently found as a weed all over India. The scientific community has recently become interested in A. aspera because of its possible pharmacological effects. Numerous features have been the subject of research, including its anti-inflammatory, anti-oxidative, anti-microbial, and cancer-fighting properties.¹⁴ Studies have also looked into its possible uses in the management of diseases like diabetes, high blood pressure, and gastrointestinal issues. Therefore, we selected A. aspera Linn for phytochemical and pharmacological screening to explore its possibilities and therapeutic potential. [Please include previous work published on this plant.

Hypothesis: A. aspera Linn has significant medicinal value due to its wide range of pharmacological properties, which can be further confirmed by pharmacological and phytochemical screening.

The goal is to identify the bioactive components of A. aspera Linn by doing a thorough phytochemical investigation, to assess its pharmacological characteristics, such as its antioxidant, antibacterial, and anti-inflammatory effects and to investigate its possible therapeutic uses in the management of long-term illnesses, to examine and gather data from earlier studies on A. aspera Linn in order to provide a scientific foundation for its therapeutic application. This study intends to aid in the creation of innovative herbal-based remedies for a range of illnesses by examining the bioactive components and therapeutic qualities of A. aspera Linn. 

Material and Methods

Selection procurement of plant materials

A. aspera whole plant was collected from our herbal garden in Bhopal, Madhya Pradesh. The collection was conducted during the flowering stage to ensure maximum phytochemical constituents. A total of 2kg samples were collected to maintain uniformity in the study. The plant materials were authenticated by Dr. Awadesh Kumar Shukla, Department of Botany, Dr. Ram Manohar Lohia Avadh University, Ayodhya, India, and a voucher specimen was deposited in the herbarium for future reference. The whole plant was selected for preliminary phytochemical screening and antioxidant activity evaluation. 

Preparation of extract

The plant materials were gathered, cleaned, dried (or freeze-dried), and powdered to produce a coarse powder for extraction. The entire A. aspera plant was roughly pulverized, sieved twenty times, and allowed to dry for two weeks in the shade. The dry powder was first treated for 24 hours at 60 to 80 °C with petroleum ether to eliminate fatty components. The plant extraction was performed using a Soxhlet apparatus with methanol as the solvent. The extracts were collected using Whatman filter paper and then dried or concentrated using a rotary vacuum evaporator. The yield percentage was determined after storing the extracts in desiccators.^15-16 

Phytochemical Evaluation Parameters

Qualitative Chemical Tests

Qualitative chemical tests were conducted on the plant extract, confirming the presence of alkaloids, glycosides, volatile oils, flavonoids, tannins, etc.¹⁷ 

Identification Test (TLC study)

Thin Layer Chromatography (TLC) was performed using conventional procedures. AAME (2 mg/ml) was dissolved in suitable solvents in small quantities. The mobile phase concentrations were adjusted to create solvent systems. The Rf values were determined using a UV-TLC chamber and compared to reference values. The TLC study is shown in Figure 3.^18-20 The Rf values interpreted and were compared to the reference values. 

FTIR study

The FTIR study of AAME was performed and identified the occurrence of bioactive compounds and their active groups using Agilent FTIR Cary 630 instruments.^21-23 

Determination of total phenolic content (TPC)

0.5 ml of AAME extract and 2.5 ml of the Folin-Ciocalteu reagent were used to assess the phenolic content of AAME. Then after were added 50 ml of distilled water and 2 ml of a 7.5% sodium carbonate solution. The phenolic content of AAME samples was calculated in milligrams of gallic acid equivalents per gram of AAME using the absorbance at 765 nm. ^24-25

Figure 1: The standard curve of Gallic Acid Click here to view Figure

Total flavonoid content (TFC)

TFC was determined using rutin as a standard. A 2% methanolic solution of aluminum chloride (1 ml) was added to serially prepared test samples of AAME. Then after UV-visible spectrophotometer was used to measure the absorbance at 420 nm following 15 minutes at room temperature. The TFC results were expressed in quercetin equivalents (µg/ml).^26-28

Figure 2: Quercetin’s standard curve Click here to view Figure

In-vitro antioxidant activity assessment:

Antioxidant activity was determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method. A 0.1 mM DPPH solution was prepared in methanol, and the antioxidant determination was performed using the DPPH standard curve. The ascorbic acid and methanol extract test sample stock solution (10 mg/ml) was serially diluted to concentrations of 2, 4, 6, and 8 mg/ml. One milliliter of DPPH solution (0.1 mM) was added to 50 µl of each test sample, followed by the addition of methanol to achieve a final volume of 3 ml. After thorough shaking, the mixture was left to stand at room temperature for 30 minutes. The absorbance was measured at 517 nm using methanol as the reference. Ascorbic acid was used as the standard antioxidant. The percentage of DPPH free radical scavenging activity (FRSA) was calculated using the formula^29-30:

% (𝐹𝑅𝑆𝐴) = (𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 – 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑡𝑒𝑠𝑡 𝑠𝑎𝑚𝑝𝑙𝑒) 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑋100 

Separation of chemical constituent

A 9.3 g methanolic extract was subjected to liquid chromatography on a silica gel column and eluted using petroleum ether, ethyl acetate, and methanol in increasing polarity. Bioactive compounds, including PAL-1, were obtained from methanol extracts. The structures of these compounds were determined using IR and 1H NMR spectral data. The separation and purification of each component were thoroughly explained in the experimental section. A schematic representation of the isolation procedure is provided in Figure 3. 

Figure 3: Chromatographic separation of Ethyl acetate extract of the Achyranthas aspera plant. Click here to view Figure

Most of flavonoid biocompounds are dissolved is ethyl acetate solvent. Therefore mostly select this solvent. It is semi polar organic solvent due to this reason, easily to evaporate and TLC separation fast. Bioactive compound PAL-2 20mg successfully was isolated (80ethylacetate:10 methanol) and fraction-3 PAL-3 respectively. These isolated bioactive compounds stored properly for the further investigation.^28-30

Assessment of In-vitro antimicrobial activity of AAME

The antimicrobial activity of AAME was evaluated using the agar well diffusion method against Gram-negative (Escherichia coli, Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus, Bacillus subtilis) bacteria.

Using agar-agar and 8% nutritious broth in double-distilled water, nutrient broth medium (NBM) was created.  It was autoclaved for 25 to 30 minutes to sterilize it at 15 Ibs pressure. After growing the bacterial strains in an agar gel petri plate and aseptically adding 15 milliliters of NBM, the plates were left to stabilize at room temperature. Bacterial cell cultures were maintained in peptone saline solution by routine sub-culturing. The cultures were then kept at 37°C for a whole day. ^31-34 

Agar plate preparation

Bacterial strains were streaked across the sterile surface of agar plates to spread the inoculum evenly. After curing at room temperature, wells of 9 mm diameter were drilled into the agar plates. AAME at concentrations of 50, 100, 150, and 200 mg/ml was prepared using dimethyl sulfoxide (DMSO) as the solvent. Ofloxacin (10 mg/ml) was used as a standard antibiotic. The plates were incubated at 37°C for 24 hours. The zone of inhibition was measured using a calibrated digital Vernier caliper. Each test was performed in triplicate.^33-34 

Results

It was found that the extract from the AAME stem segment had a 9.31% yield. The methanolic extract of the entire AAME component was semisolid, and all of the extracts had a dark brown color. In phytochemical screening tests, the methanolic extract of AAME was shown to include proteins, amino acids, carbohydrates, glycosides, tannins, saponins, alkaloids, phenolic compounds, and flavonoids.^35–38 Semi-qualitative information on the extract’s active elements is provided by the bioactive compounds. A phytochemical analysis revealed a variety of phytoconstituents in the methanolic extract of AAME. Consequently, more in-vivo studies were conducted on AAME.

The TLC study of AAME was studied, calculated Rf vale and interrelated the Rf value. The Rf value represented and found to be Ferulic acid, Caffic acid, Oleanolic acid, Ursolic acid, Flavonoid-VII, Phenolic acid-VI, Flavonoid-III, and Flavonoid-I were present. The TLC values showed that AAME test sample having potential sources of phenol and flavonoid components which revealed that AAME having potential activities.^19-20 The computed Rf values for the various sites were then compared to the reference values from studies. Results are shown in table 1.

Table 1: SolventS, spots and Rf values of AAME

 The Quercetin equivalent of phenols was used to calculate the TPC of the AAME. For the concentration of TPC, there are strong linear correlations between the standard and sample extracts. It was found that the TFC was 35.9±0.13µg/ml quercetin equivalent using the aluminum chloride colorimetric method.

The gallic acid equivalent of phenols was used to calculate the TPC of AAME. For the concentration of total phenol, there are strong linear correlations between the standard and sample extracts. It was calculated using the Folin-Ciocalteu method and was found to be 9.9±70.15 µg/ml of equivalent to gallic acid, the results demonstrated that AAME had the greatest potential. 

FTIR study of AAME

Figure 4 reports the FTIR spectrum of AAME leaves. The OH group of phenolic compounds is represented by the absorption band at 3438.03 cm–1. It attests to the plant’s phenolic component content. The results of AAME are listed in Table 2. It

Figure 4: FTIR study of AAME  Click here to view Figure

Table 2: Analysis of AAME’s FTIR spectrum

 Antioxidant evaluation of AAME Extract

AAME composition was selected based on its distinct in-vitro efficacy, or % free radical scavenging activity. It was shown that AAME whole component extracts has potent antioxidant qualities.^23-24 Table 5 and Figure 3 show the findings of the DPPH method’s examination of the AAME capacity to scavenge free radicals. When compared to the conventional ascorbic acid, the test samples’ comparative antioxidant activity amply demonstrated the impact of AAME. 

Table 3: Antioxidant effects of AAME and Ascorbic acid

“(Mean ± SD, n = 3)”

Separation of chemical constituent

A silica gel column (100-200 mesh) was used to isolate bioactive compounds, were 9.3 gm AAME loaded in column chromatography. It was then eluted using a step-by-step gradient using petroleum ether: ethyl acetate, followed by methanol (90:10, 80:10, 70:20, 60:30, and 50:50 by volume) solvents, as indicated in Table 4. After collecting 25 fractions, the identical fractions check by Thin Layer Chromatography (TLC), and were identical fractions were combined. For the detection of flavonoid bioactive compounds spots were identified by applying a 10% methanol: sulfuric acid spray reagent.^25-27 

Fraction-1

Using the gradient elution technique, the residue (450 mg) from these fractions was collected on a silica gel (100–200 mesh) column using methanol: ethyl acetate (70:30). Methanol, glacial acetic acid, toluene, and ethyl acetate make up the residue on TLC. A single, noticeable pinkish red patch with an Rf value of 0.44 was visible in 3.5:1:0.1:0.1. The Rf value, FTIR interpretation data and reference sources revealed that isolated compounds was derivative of quercetin (Flavonoid-III). Addition ferulic acid, Caffic acid, Oleanolic acid, Ursolic acid, Flavonoid-VII, Phenolic acid-VI, Flavonoid-III, and Flavonoid-I were present in AAME. These results also indicated that AAME whole part having potential activities.^28-30 

Antimicrobial test

The agar well diffusion technique was used to determine the zone of inhibition for the entire AAME. The antimicrobial spectrum demonstrated that AAME has concentration-dependent efficacy against both gram-positive and gram-negative bacteria. When tested against S. aureus, S. bacillus, E. coli, and P. aureginosa at 50 to 200 mg/ml, the AAME sample was found to be effective (Maximum ZI among all test samples). Comparing AAME to the ofloxacin standard, the results demonstrated its antibacterial activity (Fig. 5). The antibacterial effectiveness of AAME against bacterial infections is shown in Table 8. 

Table 4: Antimicrobial efficacy assessment of AAME

All results are shown in form of mean ± SEM, n = 3. For every bacterial strain, data were evaluated using a two-way ANOVA, Dunnett’s multiple comparisons test, and ****p<0.0001.

Figure 5: Antimicrobial assessment of AAME Click here to view Figure

Discussion

The amount of alkaloids, tannins, polyphenols, and flavonoids in the AAME was determined using Thin Layer Chromatography (TLC) and validated against reference standards. These bioactive molecules, particularly flavonoids, play a crucial role in shielding biological systems from oxidative stress and damage caused by free radicals. Oxidative stress is a key factor in the pathogenesis of numerous diseases, including cardiovascular disorders, neurodegenerative conditions, and various cancers. The significant presence of bioactive compounds in AAME suggests its potential role in mitigating oxidative stress and associated disease progression.

The phytochemical screening of AAME confirmed the presence of alkaloids, polyphenols, flavonoids, glycosides, triterpenoids, tannins, and other bioactive constituents, which exhibit strong antioxidant potential. Flavonoids and polyphenols, in particular, are well-recognized for their potent antioxidant properties and their ability to enhance the body’s natural defense mechanisms. Studies have shown that rutin and its metabolites, which contain vicinyl dihydroxyl groups, contribute to the destruction of harmful microorganisms and offer protective effects against a variety of infectious bacterial and viral diseases. Additionally, rutin has been linked to the prevention of glucose-induced radical formation, thereby playing a role in diabetes management.^28-30

Furthermore, gallic acid (GA), a well-known polyphenol (3,4,5-trihydroxybenzoic acid), has been identified as possessing both antioxidant and pro-oxidant activities.^31-33 This dual role of GA can be beneficial in modulating cellular responses to oxidative stress and promoting immune responses. The polyphenolic components in A. aspera extract have demonstrated immunomodulatory and anti-cancer properties, emphasizing its therapeutic potential. Moreover, the PCA extract contains known phenolic acids and flavonoid components such as kaempferol, quercetin, chrysin, quinic acid, and chlorogenic acid, all of which contribute to its bioactivity.^34-36

Despite these promising findings, further in-depth studies are required to validate the specific therapeutic activities of these bioactive compounds. Advanced analytical techniques, including High-Performance Liquid Chromatography (HPLC), Mass Spectrometry (MS), and Nuclear Magnetic Resonance (NMR) spectroscopy, should be employed to identify and quantify these compounds with greater accuracy.^37-38 Additionally, in vivo and clinical studies are essential to substantiate the pharmacological relevance of AAME and its bioactive constituents in disease prevention and management.

Conclusion

Natural antioxidants derived from food supplements and traditional medicines offer significant potential in preventing oxidative damage, in contrast to synthetic therapies, which often come with undesirable side effects. The findings of this study provide valuable insights into the potential application of natural antioxidants, either as standalone treatments or in combination with innovative chemical formulations, to counteract oxidative stress-related diseases. The strong antibacterial activity observed in the AAME whole plant extract can be attributed to its diverse array of physiologically active compounds. These bioactive constituents contribute to the extract’s broad-spectrum antimicrobial efficacy, making it a promising candidate for further research in the development of alternative therapeutic agents. However, to fully establish the therapeutic potential of AAME, additional studies are needed to validate the bioefficacy, safety, and mechanisms of action of its active compounds through rigorous preclinical and clinical evaluations.

Acknowledgement

The author sincerely expresses gratitude to the Institute of Pharmacy, Dr. Ram Manohar Lohia Avadh University, Ayodhya, Uttar Pradesh, India, for providing the necessary support for the research work. Special appreciation is extended to the Department of Pharmacy, Guru Ghasidas Vishwavidyalaya, Bilaspur, Chhattisgarh, India, for facilitating the sampling and testing process. The author is also deeply grateful to the Faculty of Pharmacy, Swami Vivekanand Subharti University, Meerut, Uttar Pradesh, India, for their valuable guidance in procuring satellite data. Additionally, heartfelt thanks are extended to the Pacific College of Pharmacy, Pacific Academy of Higher Education & Research University, Udaipur, for their invaluable guidance and support in the writing and drafting of this article.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of Interest

The authors do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval.

Informed Consent Statement

This study did not involve human participants, and therefore, informed consent was not required.

Clinical Trial Registration

This research does not involve any clinical trials.

Permission to reproduce material from other sources

Not Applicable

Author Contributions

Ajay Kumar Shukla: Conceptualization, Research work, Writing Original Draft

Aarti Tiwari: Checking article Draft

Vimal Kumar Yadav: Data Collection, Analysis

Vishnu Prasad Yadav and Kunal Agam Kanaujia: Writing – Review & Editing

Manoj Kumar Mishra: Visualization, Supervision, Project Administration.

Maya Sharma and Garima Verma: Funding Acquisition, Resources, Supervision.

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Abbreviations

AAME: Achyranthes aspera Linn methanol extract

TLC: Thin-layer chromatography

DPPH: 2,2-Diphenyl-1-picrylhydrazyl

FTIR: Fourier-transform infrared spectroscopy