Investigation for phytochemical composition, antioxidant activities and antibacterial properties of Achyranthes aspera L.

11 Haque et al. Int. J. Biosci. 2025 RESEARCH PAPER RESEARCH PAPER RESEARCH PAPER RESEARCH PAPER OPEN ACCESS OPEN ACCESSOPEN ACCESS OPEN ACCESS Investigation for phytochemical composition, antioxidant activities and antibacterial properties of Achyranthes aspera L. Mst. A khi Khatun, Md. Sohanur Rahman, Nusrat Rahman, Md. Omar Faruq, Md. Sarwar Parvez * Department of Botany, U niversity of Rajshahi, Rajshahi - 6205, Bangladesh Key words: Achyranthes aspera, Phytochemical , Antioxidant , Antibacterial activities http://dx.doi.org/10.12692/ijb/26.6.11 - 23 Article published on June 04 , 20 2 5 Abstract Plants and plant - based medicines form the foundation of many modern pharmaceuticals used to treat various ailments. Thus, this study aimed to investigate the phytochemical, antioxidant, and antibacterial properties of A. aspera leaves. Phytochemical screening revealed the presence of alkaloids, flavonoids, terpenoids, glycosides, carbohydrates, coumarins, and steroids in both ethanol and methanol extracts. However, cardiac glycosides and phenols were exclusively found in the ethanol extract. Methanol leaf extracts exhibited the highest levels of flavonoids (41.16 mg QE/gm) and phenols (9.99 mg GAE/gm), respectively. Significant antioxidant activity was observed with IC50 values of 237.83 μg/ml and 279.14 μg/ml for methanol and ethanol leaf extracts, respectively. The antibacterial efficacy of A. aspera leaf extracts was tested against Bacillus sp., E. coli, and Enterobacter sp. Methanolic extracts showed stronger antibacterial effects compared to ethanolic extracts, with the highest zone of inhibition (20.67 mm) against Bacillus sp. and the lowest MIC (75 mg/ml) and MBC (150 mg/ml) values. These findings suggest that A. aspera leaf extract could serve as a source of novel antioxidant and antibacterial compounds. * Corresponding Author: Md. Sarwar Parvez  [email protected]c.bd International Journal of Biosciences | IJB | ISSN: 2220-6655 (Print), 2222-5234 (Online) http://www.innspub.net Vol. 26, No. 6, p. 11-23, 2025 12 Haque et al. Int. J. Biosci. 2025 Introduction Therapeutic chemicals derived from natural sources have been used for thousands of years. Across the globe, people have traditionally employed a wide variety of medicinal plants to treat a range of ailments. Many of the chemicals found in these plants can be used to develop new drugs. These include secondary metabolites or phytochemicals such as tannins, terpenoids, alkaloids and flavonoids, which are abundant in plants. These compounds exhibit diverse bioactivities including antimutagenic, anticarcinogenic, antioxidant, antibacterial and antiinflammatory properties (Yen et al., 1993). The three main categories of plant secondary metabolites are terpenoids, alkaloids and polyphenols (phenolics). The antimicrobial activity of plants is largely attributed to these secondary metabolites (Keita et al., 2022). The use of medicinal plants particularly their phenolic constituents such as anthocyanins, flavonoids, tannins, stilbenes and phenolic acids as safe, effective and sustainable sources of natural antioxidants and free radical scavengers has long been recognized. The primary natural dietary antioxidants include carotenoids, phenolic compounds (especially flavonoids) and vitamins C and E. According to (Aguoru et al., 2015), nearly 80% of the global population now relies on plants for primary medical care. Medicinal plants provide a valuable source of lead compounds for the development of new drugs with fewer side effects. However, in the latter part of the 19th century, the pharmaceutical industry began to grow rapidly, prompting researchers to begin developing and characterizing various drugs derived from plant sources (Ovais et al., 2021). Achyranthes aspera L., a member of the Amaranthaceae family, is currently being evaluated for its medicinal properties. In northern Bangladesh this widespread plant is considered both a weed and an invasive alien species (Akter and Zuberi, 2009). According to (Talreja and Tiwari, 2023), A. aspera commonly known as "Prickly Chaff Flower," holds significant ethno-pharmacological value. It is commonly found as a roadside weed. A. aspera is a well-known medicinal plant traditionally used to treat a variety of ailments. The roots are employed as an astringent for treating stomachaches and wounds while leaf extracts have demonstrated antiperoxidative and thyroidstimulating properties. The plant exhibits a wide range of pharmacological effects including hepatoprotective, anti-allergic, cardiovascular, hypoglycemic, antidiabetic, analgesic, antiperiodic, antipyretic, purgative and laxative activities, making it effective in managing gastrointestinal issues and general body pain. According to (Hai-Anh et al., 2024), it contains high concentrations of essential phytochemicals such as phenolics, saponins, tannins, alkaloids, flavonoids, cardiac glycosides, steroids and phlobatannins. Achyranthes aspera is traditionally used to treat a variety of conditions including fever, wound healing, toothache, arthritis, gynecological disorders, urinary issues, insect bites, snake bites, abdominal tumors and stomach pain (Ribeiro, 2021). Given its rich phytochemical composition and wide range of medicinal uses, the present study aimed to investigate the phytochemical profile, antioxidant capacity, and antibacterial activity of A. aspera leaves. Medicinal plants with such potent phytoconstituents serve as valuable sources for the development of effective therapeutic agents. Materials and methods Samples Fresh plant material was gathered from the Rajshahi University region to guarantee that the plants were disease-free and healthy. The University of Rajshahi, Rajshahi, Bangladesh's Plant Taxonomy Laboratory identified the plant. 13 Haque et al. Int. J. Biosci. 2025 Chemicals Various tests were conducted using the following analytical-grade chemicals and reagents such as nitric acid (HNO 3 ), potassium nitrate, picric acid (2.4.6trinitro phenol), mercuric chloride, potassium iodide (KI), iodine, tannic acid, ferric chloride, lead acetate, copper sulfate (CuSO 4 ), sodium hydroxide (NaOH), hydrochloric acid (HCl), sulphuric acid (H 2 S0 4 ), alcoholic ferric chloride, glacial acetic acid, ninhydrin reagent, Molisch's reagent, gallic acid, Folin-ciocalteu reagent (FCR), sodium carbonate (Na 2 CO 3 ), aluminium chloride (AlCl 3 ), methanol, ethanol, quercetin, potassium acetate (C 2 H 3 KO 2 ), DPPH (2,2diphenyl-1picrylhydrazyl) reagent. Preparation of crude extracts After collection of the leaves were thoroughly washed 3-4 times through running tap water and dried under shade with room temperature (26 ± 2 °C). The dried leaves were made into fine powder by using grinder machine and stored in airtight glass container. Then, leaf powder was mixed with methanol and ethanol solvents in a ratio of 1:10 (w/v) and shaken for 48 h by a shaker machine. After that the solution was filtered via cotton cloth and Whatman No. 1 filter paper. The solvent was evaporated at 35 ± 2 °C in a water bath. Finally, semisolid crude extract was produced within 5 days and transferred into airtight screw cap vials and stored at 4°C for further analysis. Determination of phytochemicals With few modifications, the conventional methods of (Balamurugan et al., 2019) were used to perform a qualitative phytochemical analysis of A. aspera extracts. Qualitative phytochemical analysis Test for alkaloids Each extract was separately dissolved in diluted hydrochloric acid and then filtered. Wagner's test Take 1 ml of extract and add 3-5 drops of Wagner's reagent and observe for the formation of reddishbrown precipitate indicate positive result. Meyer's test 2 ml of extract and added 1 ml Mayer reagent, alkaloids give cream color precipitate with this reagent. Dragendroff's test 2 ml of extract and added 1 ml Dragendroff reagent, alkaloids give reddish brown precipitate with this reagent. Hager’s test 2 ml of extract and added 1 ml Hager reagent, alkaloids give yellow color precipitate with this reagent. Tannic acid test 2 ml extract and added 1 ml tannic acid, alkaloids give buff color precipitate with tannic acid. Test for Flavonoids (alkaline reagent test) 2 ml of extract was treated with 3-5 drops of 1 ml 10% NaOH solution. Observe for the formation of intense yellow color, which becomes colorless on addition of 0.5 ml diluted HCl indicates the presence of flavonoids. Test for Saponins (Foam test) 2 ml of extract was added 2 ml distilled water and shake 1 minute vigorously and incubated 5 minutes. Observe the formation of persistence foam that confirms the presence of saponins. Test for Terpenoids (Salkowski's test) 2 ml of extract was mixed in 1 ml of chloroform and then 1.5 ml concentrated H 2 S0 4 carefully added to form layer. The reddish-brown ring of the mixture was formed show positive result for the presence of terpenoids. Test for Tannins (Braymen's test) 1 ml of extract was treated it with 1 ml of 10% alcoholic ferric chloride solution and observed for the formation of dark green or blue-black color. Test for cardiac glycosides (keller-killani test) 1 ml extract was treated it with 1 ml of glacial acetic acid and 2-3 drops of 5% ferric chloride solution. 14 Haque et al. Int. J. Biosci. 2025 After that, add 1 ml of conc. H₂SO 4 . Observe for a brown ring at the interface shows the presence of glycosides. Test for Phenol (Lead acetate test) 2 ml extract was added 1 ml 10% lead acetate solution and observe for the bulky white precipitate. Test for Coumarins 2 ml extract was added 3ml 10% NaOH and observed for the formation of yellow color for positive result. Test for Xanthoprotein 2 ml of extract was added 2 ml nitric acid. This mixture needed Sometimes heat for formation of light-yellow color. Then added 1 ml 40% NaOH and observed for the formation of Dark yellow or orange color. Test for Steroids 2 ml of extract was mixed in 1 ml of chloroform and concentrated 1ml H 2 SO 4 was carefully added to form red and yellowish green color for confirming positive result. Test for Proteins 1 ml extract was added 1 ml 40% NaOH. Then added few drops of 1% CuSO 4 for formation of violet or pink color that showed positive result for this test. Test for Amino acids (Ninhydrin reagent) 2 ml of extract was mixed with 2 ml ninhydrin reagent and boil for few minutes. Then observed for the formation of purple/blue color for ensuring positive result. Test for carbohydrates (Molisch's test) 1 ml extract was taken and added 3-5 drops of Molisch's reagent. In addition, added 1 ml H 2 SO 4 in this mixture and to strand for 2-3 min for red or dull violet color that ensuring the positive result. Test for Glycosides At first taken 2 ml extract and carefully added 1 ml conc. H 2 SO 4 . Then observed red color for ensuring the positive result. Quantitative phytochemical analysis Estimation of Total Phenolic Content in the Plant Extracts The total phenolic content (TPC) of different extracts was determined by UV-vis spectrophotometry using Folin-Ciocalteu (FC) assay (Alhakmani et al., 2013) with few modifications. The calibration curve was constructed with different concentrations (25 μg/ml, 50 μg/ml, 75 μg/ml, 100 μg/ml and 150 μg/ml) of gallic acid as the standard. For this test 1 ml (1000μg/ml) of plant extracts (Methanolic and ethanolic) were taken into test tubes separately. Then 3 ml of distilled water was added in each test tubes and after that mixed thoroughly with 1 ml of FolinCiocalteu reagent (1:10 v/v diluted with distilled water). The solutions were left to rest for 5 min before being added with 2 ml of 7.5% sodium carbonate (Na 2 CO 3 ) and stand 30 min of incubation at room temperature in the dark place. The absorbance of the samples was measured at 765 nm UV-vis spectrophotometer. Estimation of Total Flavonoid Content in the Plant Extracts The total flavonoid content (TFC) was estimated by aluminum chloride colorimetric method (Aruna et al., 2015) with slight modification. Different concentrations of quercetin (12.5-200 μg/ml) were used as a standard to created calibration curve. For this test 1 ml (1000 μg/ml) of plant extracts (ethanolic and methanolic) were taken into test tubes separately. Then 100µl of 10% aluminum chloride (AlCl 3 ) solution, 100µl of 1M potassium acetate (C 2 H 3 KO 2 ) solution and 2.8 ml of double distilled water added in each test tubes and mixed thoroughly. After 30 min of incubation at room temperature, the absorbance of the sample was measured at 415 nm with a UV-vis spectrophotometer. Determination of antioxidant activity DPPH (2,2-diphenyl-1-picrylhydrazyl) Radical Scavenging Assay Using a modified approach, the diphenylpicrylhydrazyl (DPPH) radical degradation was used to assess the antioxidant activity of methanolic, 15 Haque et al. Int. J. Biosci. 2025 ethanolic and aqueous extracts (Alvarez-Jubete et al., 2010) as a standard ascorbic acid was utilized. For this assay 2 ml of methanol and ethanol solution of plant extract or standard at different concentration (25 µg/ml, 50 µg/ml, 100 µg/ml, 200 µg/ml, 400 µg/ml) was taken in test tubes separately. 3 ml of methanol and ethanol solution of DPPH was added in to the test tubes. The test tubes were incubated at room temperature for 30 minutes in dark place to complete the reaction. Then the absorbance of the solution was measured at 519 nm using a spectrophotometer against control. A typical control solution contains all reagents except plant extract or standard solution. The percentage (%) scavenging activity was calculated from the following equation: % SCV= {(A 0 -A 1 )/A 0 }*100 Where, A 0 is the absorbance of the blank, and A 1 is the absorbance of the extract/standard. Determination of antibacterial activity Plant materials and bacterial species In the present investigation, ethanolic and methanolic crude extract of A. aspera leaves were used for antibacterial activity by disc diffusion assay. Three bacterial species were used to study to determine the antibacterial activity. The bacterial species were collected from Microbiology Laboratory, Department of Botany, University of Rajshahi. The concentrations (200 mg/ml, 400 mg/ml, 600 mg/ml, 800 mg/ml) of A. aspera were made from the mother extracts by using standard formula for respective solvents. Culture preparation of bacterium The studied species maintained in laboratory by 33% glycerol stock culture in eppendorf tube. During this investigation, all the bacterial species were cultured on nutrient agar medium. This step was repeated to go a single colony. Single colony was then transferred into nutrient broth medium and incubated at 30 ± 2 °C for 24 hours. After 24 hours, broth culture of bacteria was used as inoculums. Inoculation, discs placing and incubation In vitro antibacterial activities of the extracts were measured by employing standard agar disc diffusion method (Bauer et al., 1966; Barry, 1980). In the disc diffusion method, the disc was placed aseptically over the bacterial culture on Mueller Hinton Agar Medium plates and incubated at 30 ± 2 °C for about 24 hours. After incubation for about 24 hours the antibacterial activities were determined by measuring the diameter of the inhibition zone in mm. The experiment was replicated three times to confirm the reproducible results. Sterile, blank paper discs were impregnated with sterile solvents (Methanol and Ethanol leaf extract) were used and tetracycline contained paper disc were used as positive control each time. Determination of the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) The minimum inhibitory concentrations (MIC) of the extracts were determined for each of the test organisms in triplicate in test tubes. According to (Doughari et al., 2007) with some modification that 0.5 ml of varying concentrations of the extracts in test tubes nutrient broth was added and then a loopful of the test organism, previously diluted to 0.5 Mc Farland turbidity standard was introduced. The procedure was repeated on the test organisms using the standard antibiotics tetracycline. A tube containing nutrient broth only was seeded with the test organisms as described above to serve as controls. The culture tubes were then incubated at 30 ± 2 °C for 24 h. After incubation the tubes were then examined for microbial growth by observing for turbidity. To determinate the MBC for each set of test tubes in the MIC determination, a loopful of broth was collected from those that did not show any growth and inoculated onto sterile Nutrient agar by streaking. Nutrient agar plates only were also streaked with the respective test organisms to serve as controls. All the plates were then incubated at 37°C for 24 h. After incubation the concentration at which no visible growth was seen was noted as the Minimum Bactericidal Concentration (MBC). 16 Haque et al. Int. J. Biosci. 2025 Results Qualitative phytochemical analysis The findings of the qualitative analysis of the phytochemical screening of A. aspera leaf extracts in methanol and ethanol were shown in Table 1. and included 14 components. Out of the two solvents the ethanolic leaf extract included nine different compounds: (alkaloids, flavonoid, terpenoids, glycosides, cardiac glycosides, phenol, coumarins, xanthoproteins and steroids). In contrast, methanolic leaf extracts had a maximum of seven different compounds: (alkaloids, flavonoids, terpenoids, glycosides, coumarins, steroids and carbohydrates). In contrast, methanol leaf extracts did not contain cardiac glycosides, xanthoprotein and phenol, while ethanol leaf extracts did not contain carbohydrates. Methanolic and ethanolic leaf extracts did not include saponins, tannins, proteins and amino acids. Due to its ability to identify the top nine phytochemicals, ethanol solvents outperformed methanol solvents. Table 1. Phytochemical screening of methanol leaf extract and ethanol leaf extract of A. aspera. Name of the phytochemicals Name of specific test Methanol Ethanol Alkaloids Dragendroff's test - - Hager's test - - Wagner's tes t + + Mayer's test - - Tannic acid test + + Flavonoids Alkaline test + + Saponins Foam test - - Terpenoids Salkowski's test + + Tannins Braymen's test - - Glycosides NaOH test + + Cardiac Glycosides Killer - killiani test - + Phenol Lead acetate t est - + Coumarins 10%NaOH test + + Xanthoprotein HNO3 test - + Steroids Salkowski test + + Proteins FeCl3 test - - Amino acids Ninhydrin reagent - - Carbohydrates Molich's test + + Total phenol content The findings of the total phenol content of A. aspera in methanol and ethanol leaf extracts are shown in Table 2. The maximum concentration of phenolic components was found in the methanolic leaf extract of A. aspera, which measured 9.99 ± 0.43 mg GAE/g, which was statistically significant when compared to ethanolic leaf extracts (2.44 ± 0.46 mg GAE/g) at the p<0.05% level. Total flavonoid content A. aspera leaf extracts in methanol and ethanol are tested for total flavonoid content (TFC) and the findings are shown in Table 2. Quercetin equivalents per gram of dry weight were used to express the combined flavonoid concentration of the two extracts. Comparative analysis showed that the methanolic leaf extract from A. aspera had the highest flavonoid content (41.16 ± 0.93 mg QE/g), while the ethanol leaf extract had the lowest amounts (16.07 ± 0.28 mg QE/g), which is not statistically significant. Antioxidant activity by DPPH method The IC 50 values of the methanolic and ethanolic leaf extracts of A. aspera as well as the percentage of scavenging activity of the leaf extracts are shown in Figure 1. and Figure 2. 17 Haque et al. Int. J. Biosci. 2025 Table 2. Total phenol and flavonoid content of methanol and ethanol leaf extracts of A. aspera. Plant Name Solvents Equivalents per gm dry weight of extract Total Phenol Content (mg GAE/gm) Total Flavonoid Content (mg QE/gm) A. aspera Methanol 9.99 ± 0.43 a 41.16 ± 0.93 a Ethanol 2.44 ± 0.46 b 16.07 ± 0.275 b Note: Values are mean ± Standard error (n = 3). Catalog ‘a, b’ reveals statistically significant differences between the column of TPC and TFC calculated by t-test (p < 0.05). The identical designation (e.g., ’a’) do not differ significantly. They calculated their decreasing power using the 50% inhibition (IC 50 ) values. Ascorbic acid has an IC 50 of 46.24 ± 1.52 μg/ml. As evidenced by their lower IC 50 value, plants have superior antioxidant activity. Strong antioxidant activity was indicated by the scavenging activity range of A. aspera methanolic extracts (39.44 ± 0.45% to 54.96 ± 0.09%), as well as the lowest IC 50 values (237.83 ± 1.70 μg/ml) and ethanolic extracts (37.64 ± 0.55% to 56.77 ± 0.16%) and the highest IC 50 values (279.14 ± 2.51 μg/ml). The methanolic extracts were non-significant with methanolic extracts at the p < 0.05 level. Fig. 1. Scavenging activity of standard, methanolic and ethanolic leaf extract of A. aspera. The antibacterial activity of methanol leaf extract of A. aspera The antibacterial activity of A. aspera methanol leaf extract against three bacterial strains is shown Figure 3. Against Bacillus sp., the methanolic leaf extract showed the highest inhibitions zone 20.67 ± 0.32 mm in 800 mg/ml concentration and Enterobacter sp. was showed lowest inhibition zone 7.25± 0.27 mm in 600 mg/ml concentration respectively. Positive control exhibited zone of inhibition against all the organisms tested. The range of inhibition zone was 31.20 ± 0.13 - 34.09 ± 0.19 mm. Antibacterial activity of ethanol leaf extract of A. aspera The antibacterial activity of A. aspera methanol leaf extract against three bacterial strains is shown Figure 4. In ethanol leaf extract, against Bacillus sp showed the highest inhibition zone (17.76 ± 0.47mm) in 800 mg/ml concentration and lowest inhibition zone 7.98 ± 0.22 mm was found in 800 mg/ml concentration against Enterobacter sp. respectively. Positive control exhibited zone of inhibition against all the organisms tested. The range of inhibition zone was 30.21 ± 0.30 - 33.30 ± 0.16 mm. 18 Haque et al. Int. J. Biosci. 2025 Fig. 2. IC 50 values of A. aspera methanolic and ethanolic leaf extract. Catalog a-c reveals statistically significant differences between the bars calculated by DMRT test (p < 0.05). Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) MIC is important in diagnostic laboratory to confirm resistance of microorganisms to an antimicrobial agent and also to monitor the activity of new antimicrobial agents. MBC is the lowest concentration of antibiotic required to kill an organism (Mathews, 2002). Results of Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of three different extract are shown in Figure 5. The result of methanol extract showed in the range from 75-500 mg/ml. The lowest MIC value 75 mg/ml was recorded against Bacillus sp. and the highest MIC value was 500 mg/ml was recorded against Enterobacter sp. The other tested bacteria E. coli had medium MIC value (100 mg/ml). Fig. 3. Antibacterial activity of methanol leaf extract of A. aspera against three human pathogenic bacteria. The lowest MBC value 150 mg/ml was recorded against Bacillus sp. and highest MBC value 650 mg/ml was recorded against Enterobacter sp. The MIC value of ethanol extract was range from 100 19 Haque et al. Int. J. Biosci. 2025 mg/ml to 700 mg/ml. The lowest MIC value 100 mg/ml was recorded against Bacillus sp. and highest MIC value 700 mg/ml was recorded against Enterobacter sp. The other tested bacteria E. coli had medium MIC value 125 mg/ml. The lowest MBC value 175 mg/ml was recorded against Bacillus sp. and highest MBC value 850 mg/ml was recorded against Enterobacter sp. Discussion Medicinal plants are an important source of basic healthcare services for rural populations. Originally herbal remedies were made up of crude pharmaceuticals including tinctures, teas, powders, poultices and other herbal preparations (Balick and Cox, 1997). The secondary metabolites of medicinal plants particularly terpenoids, phenolic compounds and alkaloids are primarily responsible for their therapeutic effects. It can be utilized to look for bioactive substances that might be synthesized and prepared into very beneficial medications (Harborne, 1998). Fig. 4. Antibacterial activity of ethanol leaf extract of A. aspera against three human pathogenic bacteria. In the present work A. aspera leaf extract showed the presence of alkaloids, flavonoids, terpenoids, glycosides, cardiac glycosides, phenol, coumarins, xanthoproteins and steroids. The earlier reports confirmed the presence of similar phytochemicals in A. aspera (Manandhar et al., 2021; Thapa et al., 2023). On the other hand, some research confirmed the presence of saponins, tannin, protein and amino acid (Thapa et al., 2023) while our studies giving negative results. The presence of phytochemical constituents such as flavonoids, triterpenoids, polyphenolic compounds and steroids that have antibacterial and antioxidant properties. Alkaloids are essential for the invention of strong analgesic, cytotoxic, antispasmodic and antibacterial effects in both conventional and current medicine (Faruq et al., 2024). The presence of significant amounts of phenol and flavonoids are potent antioxidants, free radical scavengers and metal chelators as well as inhibit lipid peroxidation and exhibit various physiological activities, such as anti-bacterial, anti-viral, anticancer, anti-inflammatory, anti-allergic etc. (Nayan et al., 2021) reported that A. aspera leaf contained flavonoids at 17.59 µg QE/mg and 297.20 mg QE/100g (Imtiaz et al., 2020), which is approximately closer to our result. Phenolic compounds are widely distributed in plants and have shown to possess antimicrobial properties. Some previous studies reported that phenolic content 22.282 gm/100 gm (Kodidhela, 2023), 209.007 μg GA/mg (Manandhar et al., 2021), 461.09 mg GA/100gm (Imtiaz et al., 2020) was found in leaf