Bio-efficacy and Radical scavenging activity of Terminalia paniculata

 Corresponding author: Thippeswamy Basaiah. Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. Bio-efficacy and Radical scavenging activity of Terminalia paniculata Akarsh Subhakar, Brundha Kumar, Nandish Gurubasajar, Dhanyakumari Yomakesh and Thippeswamy Basaiah * Department of P. G. Studies and Research in Microbiology, Bioscience Complex, Jnanasahyadri, Kuvempu University, Shankaraghatta – 577 451, Karnataka, INDIA. World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 035–048 Publication history: Received on 12 December 2024; revised on 01 February 2025; accepted on 04 February 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.21.2.0068 Abstract The aim of this study is to qualitatively screen the phytochemicals, to assess antimicrobial and radical scavenging activity and to analyse the fingerprints of bioactive compounds by GCMS (Gas Chromatography and Mass Spectroscopy) in methanolic extract of Terminalia paniculata. Secondary metabolites including phenols, tannins, saponins and glycosides were present. The antimicrobial potential of methanolic extract of Terminalia paniculata by agar well diffusion assay showed maximum activity. The MIC (Minimum inhibitory concentration) of Terminalia paniculata by 96 well plate method ranged from 1.255×103μg/mL to 5×103μg/mL. The extract was screened for anti-oxidant activities by free radical scavenging activity DPPH assay (IC50value=3.499μg/mL), ABTS assay T. paniculata (IC50value=3.406μg/mL). In Ferric reducing antioxidant power (FRAP) assay Terminalia paniculata exhibited higher reducing ability. The total antioxidant capacity of the fractions was determined by phosphomolybdate method using ascorbic acid as a standard. Total phenolic contents in the extracts were determined spectrophotometrically according to Folin-Ciocalteu method. Gallic acid was used to set up the standard curve. Extract Terminalia paniculata demonstrated the highest phenolic content of 88.043 mg GAE/g and the GC-MS analysis was performed to evaluate the chemical constituents in the plant methanolic extract of Terminalia paniculata. With regard to the results the methanolic extract of Terminalia paniculata leaves could be an important source for the discovery of new plant-based drugs. Further research is needed for the identification and purification of chemical compounds and proving the efficacy of medicinal plants used by local people. Keywords: Terminalia paniculata; Gas Chromatography and Mass Spectroscopy (GC-MS); Anti-oxidant activity; Free radical and Anti-microbial activity 1. Introduction Medicinal plants have been used for centuries as a remedy for various human diseases. These plants owe their therapeutic power to substances, which they contain. For the evaluation of the biological activity of these plants, it is imperative to use appropriate biological tests and chemical screening methods [1]. The knowledge and the progress of the medical benefits of herbs have grown in both, developing and developed countries. Medicinal herbs have constituted the basis of alternative medicine and lead to be the main pathway for conceptualizing new drugs [2]. Natural products have been traditionally accepted as remedies for many diseases. The beneficial medicinal effects of plant products typically result from the combinations of secondary metabolites present in the plants. Plant extracts have been known since antiquity to possess notable biological activities, including antibacterial, antioxidant, and anticancer properties [3]. Therefore, there is a lot of on-going research on such substances for their potential usefulness as dietary supplements and as adjuvants for use in the therapeutic management of free radicals related disorders. And the importance of searching for and exploiting natural antioxidants, especially of plant origin, has increased greatly in World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 035–048 36 recent years [4]. During the search of plants as a source of natural antioxidants having anti-mutagenic and anticancer potentials, some medicinal plants and fruits have been extensively investigated [5]. The present study aims to determine the bio efficacy and radical scavenging activity of Terminalia paniculata, an ethnomedicinal plant. It involves collecting taxonomic characters, extracting crude compounds, screening phytochemicals, assessing antimicrobial and radical scavenging activity, and analyzing bioactive compounds fingerprints using GCMS. The extracts from medicinal plants exhibit antimicrobial, anti-inflammatory, and antioxidant activities, potentially inhibiting bacteria, fungi, viruses, and protozoa growth and potentially treating resistant microbial strains. The study also aims to analyze the fingerprints of bioactive compounds in the plants extract [6]. 2. Materials and Methods 2.1. Collection and identification of plant samples Healthy plant samples were collected aseptically, photographed, documented, and placed in paper bags. Taxonomic features were identified using standard floras and manuals, and documented in the laboratory [7]. 2.2. Preparation of plant materials Plant samples were thoroughly washed, dried, and pulverized in a mechanical grinder to obtain a fine powder. The pulverized materials were stored in zip lock polythene covers until used for extraction [8]. 2.3. Extraction of Phyto-compounds from plants The crude compound from plants was extracted using cold maceration. The pulverized plant material was immersed in methanol and agitated for 48-72 hours. The solvent-containing phyto-compounds were separated and filtered. The solvent was evaporated, and the crude extract was dried to obtain a persistent weight. The physical properties of the extract were examined, and the percentage yield was calculated. The dried extract was stored in a refrigerator for further use [8]. Yield of crude extract (%) = Weight of the plant crude extract Weight of the pulverized plant material taken ×100 2.4. Qualitative screening of secondary metabolites in plants extract The groups of secondary metabolites in plants extract were determined by implementing the standard protocol of qualitative tests of Phyto-chemicals [9]. 2.4.1. Test for Phenols (Ferric Chloride Test) The Ferric Chloride Test indicates the presence of phenols when 2 mL of plant extract is treated with 5% FeCl3 solution, resulting in a deep blue or black color. 2.4.2. Test for Tannins (Gelatin Test) A mixture of plant extract, gelatin, and 10% NaCl was mixed, resulting in a white precipitation indicating the presence of tannins. 2.4.3. Test for Alkaloids (Mayer’s Teat) To the 2 mL of extract, 2 mL of Mayer’s reagent was added. An organic dull white or cream color precipitate indicates the presence of alkaloids. 2.4.4. Test for Flavonoids (Alkaline Reagent Test) About 2 mL of plant extract was treated with 20% NaOH solution. A formation of intense yellow colouration confirms the presence of flavonoids. World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 035–048 37 2.4.5. Test for Terpenoids (Salkowski’s Test) The presence of terpenoids was confirmed by adding 2 mL of extract, 2 mL of chloroform, and 2 mL of concentrated H2SO4 to the mixture, resulting in a reddish-brown monolayer colouration. 2.4.6. Test for Steroids (Liebermann-Burchard Test) The presence of steroids was detected by adding 2 mL of acetic anhydride to 0.5gm of plant extract and 2 mL of concentrated H2SO4, resulting in a color change from violet to blue or green. 2.4.7. Test for Saponins (Foam Test) The plant extract was agitated with 20 mL of distilled in a graduated cylinder for 15minutes. The formation of foam layer about 1cm indicates the presence of saponins. 2.4.8. Test for Glycosides (Keller-Killiani Test) The presence of glycosides was detected when 2 mL of plant extract was treated with glacial acetic acid, FeCl3 solution, and concentrated H2SO4, resulting in a dark brown color ring. 2.4.9. Test for Protein and Amino acids (Ninhydrin Test) To 2 mL of plant extract, 3-5 drops of freshly prepared 2% ninhydrin reagent was added and heated on water bath. The reaction mixture turns to blue color confirms the presence of proteins and amino acids. 2.4.10. Test for Carbohydrates (Fehling’s Test) To 2 mL of extract, equal volume Fehling’s solution A and Fehling’s solution of B was added and then heated on boiling water bath. The formation of brick red precipitate indicates the presence of carbohydrates. 2.5. Antimicrobial activity of methanol extract of plants 2.5.1. Agar well diffusion assay The study investigated the effectiveness of a methanolic extract of plants in inhibiting pathogenic microorganisms using an Agar well diffusion assay. The extract was inoculated onto Mueller Hinton Agar and Sabouraud’s Dextrose Agar plates and a concentration of different plants extracts was added to the plates. The zone of inhibition around the wells was measured, indicating the compound's antimicrobial activity. The larger the zone, the more effective the extract was in inhibiting the pathogens [10]. 2.5.2. Minimum Inhibitory Concentration (MIC) of plants extract The study tested the effectiveness of plants extracts in inhibiting pathogenic microorganisms using a modified resazurin 96-well micro titre plate broth dilution assay. The extract was diluted in a concentration range of 5000 to 9.76≈10µg/mL for bacterial pathogens and 10,000 to 19.53≈20µg/mL for fungal pathogens. The MIC value of the pigment extract was determined by incubating the extract with resazurin dye for 1-2 hours. The lowest concentration without a color change was considered the MIC value [10]. 2.6. Antioxidant activity of plants extract 2.6.1. DPPH+ assay The 1,1-Diphenyl-2-picrylhydracyl (DPPH+) radical scavenging activity of plants extract was performed as described by Baliyan et al. [11]. The assay involved dissolved plant extract and ascorbic acid in methanol, with DPPH radical solution added to each tube. The reaction mixture was incubated at 37℃ for 30 minutes, with methanol used as a control. The effectiveness of plant extract to scavenge the DPPH+ radicals was determined by using the bellow equation. DPPH+ radical scavenging activity (%) = (Ac−As) Ac × 100 Where, Ac is absorbance of the control and As is the absorbance of the tested sample. World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 035–048 38 2.6.2. ABTS•+ assay The 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS•+) radical scavenging activity of the plants extract was performed [12]. The assay involved mixing 0.2 mL of methanol-prepared plant extract with 1.8 mL of ABTS•+ radical solution, using ascorbic acid as a reference standard, and measuring optical density at 734nm. The percentage of ABTS•+ free radicals scavenged was calculated by using fallowing formula ABTS•+ radical scavenging activity (%) = (A0−A1) A0 × 100 Where, A0 is absorbance of the control and A1 is the absorbance of the tested sample. 2.6.3. FRAP assay Ferric reducing power Assay (FRAP) of plants extract was determined [13], A mixture of plant extract, phosphate buffer, and potassium ferricyanide was mixed and incubated at 50°C for 20 minutes. After cooling, trichloroacetic acid and ferric chloride were added, and absorbance was measured at 700 nm against a blank, with ascorbic acid as the standard. An increase in the absorbance with increase in concentration of plant extract/standard indicates the increasing capacity of ferric ion reducing power of plant extract. 2.6.4. Total antioxidant activity The total antioxidant activity of plants extract was determined by phospho-molybdenum assay as described by Chaves et al. [12]. Plant extract was mixed with phospho-molybdenum reagent, incubated at 95°C for 90 minutes, and absorbance measured against methanol, using ascorbic acid as a standard. 2.6.5. Total phenolic content in the plants extract The total phenolic content of the plants extract was determined by using Folin-Ciocalteu (FC) method. The study involved mixing plant extract with FC reagent, adding sodium carbonate solution, and incubating tubes for 30 minutes. Gallic acid concentration was used as a standard. The total phenolic content of the plants extract will be express as gallic acid equivalent in mg/g (GAE mg/g extract) [14]. 2.7. GC-MS (Gas Chromatography and Mass Spectroscopy) analysis of plants extract The array of Phyto-compounds present in the plants extract was determined by GC-MS analytical technique. GC-MS analysis on plants extract was performed using a Shizuzu QP 2010 Plus instrument. The RtxR-5 column was used and the sample was filtered using a Whatman filter paper No 1. The ion sources were maintained at 250℃ and the mass spectrum of compounds was obtained by ionization of electrons at 70eV. The procedure took 22.5 minutes, including a three-minute solvent delay, and involved 30 and 500 Da atomic units [15]. 3. Results 3.1. Area of sampling The plant samples were collected from Kuvempu University at Shankaragatta (13°35'48.3"N 75°49'21.3"E) of Shivamogga district (Figure 1). Figure 1 Location and geographical area of sampling plots World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 035–048 39 3.2. Identification of plant samples by morphological characters Terminalia paniculata Roth. (TP), which belongs to Combretaceae family, is a tropical tree with a large natural distribution in Western Ghats, India [16] and taxonomic classification was summarized in Table 1. The tree is commonly known as Kindal Golden coloured flowers and deep red colored fruits of T. paniculata give splendid colouration to the entire forest area due to the formation of a large number of flowers and fruits in a single individual and population dominance (Figure 2) [17]. Figure 2 Terminalia paniculata 3.3. Physical properties and yield of the extract The crude methanolic extract of Terminalia paniculata was yellowish-brown in color, yield and percentage of the obtained crude extract was 1.7749g and 7.0996% for the dried plant powder. 3.4. Qualitative screening of secondary metabolites in plant extract This work investigates the phytochemical characteristics of medicinal plants, encompassing bioactive components such as proteins, amino acids, alkaloids, flavonoids, terpenoids, phenols, and tannins. Qualitative phytochemical screening was used to confirm the presence of phenols, tannins, saponins and glycosides in the methanolic extract of T. paniculata (Table 1). Table 1 Phytochemicals in Terminalia paniculata extract Sl. No. Phytochemicals Plants Sample 01. Phenols Present 02. Tannins Present 03. Alkaloids Absent 04. Flavonoids Absent 05. Terpenoids Absent 06. Steroids Absent 07. Saponins Present 08. Glycosides Present 09. Proteins/Amino acids Absent 10. Carbohydrates Absent 3.5. Antimicrobial activity of methanol extract of plant The study evaluates the antimicrobial potential of Terminalia paniculata leaves' methanolic extract against pathogenic bacterial and fungal strains was tabulated in table 2 and table 3. Results show Terminalia paniculata's leaves exhibit antibacterial activity against both gram-positive and gram-negative bacteria (Figure 3 & 4). World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 035–048 40 Table 2 Antimicrobial activity of standard antibiotics and antifungal agents Test Pathogenic Bacteria Antibacterial Activity of Standard Antibiotics(1mg/mL) Negative control Streptomycin Chloramphenicol Ciprofloxacin DMSO (10%) Zone of inhibition in diameter (mm) E. coli 21 24 25 00 E. faecalis 23 22 28 00 K. pneumoniae 22 25 28 00 P. aeruginosa 19 23 30 00 S. aureus 19 17 29 00 S. typhi 22 24 30 00 Test Pathogenic Fungi Antifungal Activity of Standard Antifungal Agents(10mg/mL) Negative control Fluconazole Clotrimazole DMSO (10%) Zone of inhibition in diameter (mm) A. brasiliensis 20 17 00 C. albicans 13 11 00 Table 3 Antimicrobial activity of methanolic extract of Terminalia paniculata Test Pathogenic Bacteria Plant Extract (mg/mL) Antibacterial Activity of Plant Extract Terminalia paniculata 20 10 05 2.5 Zone of inhibition in diameter (mm) E. coli 20 18 17 16 E. faecalis 19 19 16 15 K. pneumoniae 23 18 16 15 P. aeruginosa 24 19 18 16 S. aureus 23 19 16 16 S. typhi 24 20 19 16 Test Pathogenic Fungi Plant Extract (mg/mL) Antifungal Activity of Plant Extract Terminalia paniculata 20 10 05 2.5 Zone of inhibition in diameter (mm) A. brasiliensis 23 21 19 16 C. albicans 19 17 12 10 World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 035–048 41 a b Figure 3 Antibacterial activity of (a) standard antibiotics (b) Terminalia paniculata Note: A: Streptomycin; B: Chloramphenicol; C: Ciprofloxacin; D: DMSO (10%); E.C: E. coli; E.F: E. faecalis; K.P: K. pneumonia; P.S: P. syringae; S.A: S. aureus; S.T: S. thypi; a b Figure 4 Antifungal activity of (a) standard antibiotics (b) Terminalia paniculata Note: D: DMSO (10%); E: Clotrimazole; F: Fluconazole. C.A: C. albicans; A.B: A. brasiliensis 3.5.1. Minimum inhibitory concentration (MIC) of plant extract The MIC of Terminalia paniculata, a methanolic extract, was determined to be 1.255×103μg/mL, which effectively prevents visible growth of the test strain under controlled conditions. 3.6. Antioxidant activity of plants extract 3.6.1. DPPH+ assay The study tested the free radical scavenging and hydrogen atom donating properties of plant extracts using DPPH radical scavenging assay. T. paniculata showed the highest antioxidant potential, while standard ascorbic acid had the lowest yield. T. paniculata exhibited the highest antioxidant potential (IC50value=3.499μg/mL) and the antioxidant potential of standard ascorbic acid was found to be (IC50value = 1.353 μg/mL) shown in Figure 5a. 3.6.2. ABTS•+ assay The ABTS assay was used to determine the free radical scavenging activity of plant samples, revealing its sensitivity to antioxidant activity due to its faster reaction kinetics. In the present study T. paniculata exhibited the anti-oxidant potential of (IC50 value=3.406μg/mL) and standard (ascorbic acid) was found to be (IC50value = 1.353 μg/mL) (Figure 5b). World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 035–048 42 Figure 5 (a) DPPH+ radical scavenging activity of extracts and reference standard and (b) ABTS radical scavenging activity of extracts and reference standard 3.6.3. FRAP assay This method is based on the principle of reduction of ferric tripyridyl-s-triazine complex (colour less complex) to ferrous coloured (blue coloured complex). Increased absorbance of the reaction mixture indicates increased reducing capacity. Terminalia paniculata exhibited higher reducing ability (Figure 6). Figure 6 FRAP assay values of Terminalia paniculata with standard 3.6.4. Total antioxidant activity The total antioxidant capacity of the fractions was determined by phosphomolybdate method using ascorbic acid as a standard in this review the total antioxidant activity of methanolic extract of Terminalia paniculata were studied which exhibited minimum activity when compared to standard (ascorbic acid) (Figure 7). Similar results were recorded by Elghiet et al. [18] which demonstrated a bad correlation with flavonoids and phenolics content due to its ability to detect other compounds as ascorbic acid, carotenoids and α-tocopherol. World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 035–048 43 Figure 7 Comparison of Total antioxidant activity between Terminalia paniculata and standard 3.6.5. Total phenolic content in the plant extract Total phenolic contents in the extracts were determined spectrophotometrically according to Folin-Ciocalteu method. Gallic acid was used to set up the standard curve. The content of phenolic compounds of the samples was expressed as gallic acid equivalents (GAE) in mg per gram dry weight and the absorbance was measured at 765 nm using a spectrophotometer. Extract Terminalia paniculata demonstrated the highest phenolic content of 88.043 mg GAE/g summarized in Figure 8a & 8b. Figure 8 (a) Standard graph of Gallic acid (b) Total phenolic content of extract 3.7. GC-MS analysis of plants extract The GC-MS Analysis of Terminalia paniculata was carried out for the identification of bioactive phytochemicals. The GCMS chromatogram spectra obtained for the extract revealed that T. paniculata is plenteously rich in bioactive compounds in leaf (Figure 9). A total of 45 effective compounds were identified from the chromatogram. The bioactive compounds were predicted by their retention time (RT), peak area percentage (%) and molecular weight with the help of NIST Library summarized in Table 6.