Exploring the Antioxidant and Anxiolytic Biological Activities of Spinacea Oleracea Seed Extract

Please cite this article as: Keerthana V et al., Exploring the Antioxidant and Anxiolytic Biological Activities of Spinacea Oleracea Seed Extract. American Journal of Pharmacy & Health Research 2025. Research Article www.ajphr.com 2025, Volume 13, Issue 09 ISSN: 2321–3647(online) Exploring the Antioxidant and Anxiolytic Biological Activities of Spinacea Oleracea Seed Extract K. Ravi Shankar1, V. Keerthana1*, S. Amala1 1.Department of pharmacology, Aditya College of pharmacy, Surampalem, Andhra Pradesh, India ABSTRACT Spinacia oleracea is a green leafy vegetable widely consumed for its nutritional value. The present research work is focused on spinacia oleracea seed extract for evaluation of in vitro antioxidant and in vivo anxiolytic activities. Spinach leaves antioxidant properties are well documented but the bioactive potential of seeds remains unexplored. Hence this research work aims to explore the antioxidant and anxiolytic effects of Spinacia oleracea seed extract using standard in vitro and in vivo methods. The in vitro antioxidant potential of the spinacia oleracea seed extract at dose of 50,100,300 and 500µg/ml were assayed using hydroxyl radical scavenging, nitric oxide, and DPPH radical scavenging assay and results are compared with standard reference drug Gallic acid at concentration 2.5µg/ml. In behavioral studies, spinacia oleracea seed extract at dose of 75mg/kg and 150mg/kg was tested on albino mice subjected to standard behavioral paradigms such as elevated plus maze, open field test, hole board test, and ymaze. The results of these effects were compared to standard anxiolytic drug diazepam. The results obtained were encouraging and indicated significant antioxidant and anxiolytic effect. The FTIR studies revealed that the seed extract was rich in phenolic compounds known for its antioxidant property. Keywords: Spinacia oleracea; antioxidant; DPPH; anxiolytic; elevated plus maze; FTIR *Corresponding Author Email: [email protected] Received 01 August 2025, Accepted 18 August 2025 Keerthana et al., Am. J. Pharm Health Res 2025;13(09) ISSN: 2321-3647 www.ajphr.com 23 INTRODUCTION Anxiety is a complex emotional and physiological response characterized by feelings of tension, worry, and physical changes such as increased heart rate and restlesness29,32. While mild anxiety can serve as a normal and adaptive response to everyday challenges, excessive or persistent anxiety may interfere with an individual’s daily functioning and quality of life30,31. Anxiety disorders represent one of the most prevalent mental health conditions globally, affecting over 264 million people across all the age groups, according to the world health organization. Spinacia oleracea39,41,47 is a green leafy vegetable known for its nutritional benefits belongs to the family amaranthacea commonly known as spinach and Hindi palak6,7,35. This is abundantly grown in all parts of Andhra pradesh8,34,35. The natives of surampalem and naykampalli are using the seeds of Spinacea oleracea plant for nervousness related problems, based on the folkloric usage of this plant, a study was taken up to evaluate the possible biological activities like in vitro antioxidant and anxiolytic activity. MATERIALS AND METHOD Collection of plant material: The seeds of Spinacia oleracea42,47 were collected from different parts of East Godavari district, Andhra Pradesh. Extraction The obtained seeds were dried under shade and powdered. The coarsely powdered seeds of Spinacia oleracea43,44,45 were subjected to successive extraction using ethanol in a Soxhlet apparatus. The resulting extract was concentrated by removing the solvent through distillation, followed by evaporation on a water bath to obtain a dried extract and stored in a dessicator for further analysis. Experimental animals Albino mice of either sex, weighing 20-25g were used. Three mice in each group were housed in a cage under standard laboratory conditions and acclimatized for 7 days before starting the experiment. The animal protocol was approved by the institutional animal ethics committee (IAEC) and the animals were taken care as per CPCSEA guidelines. FTIR studies FTIR spectroscopy was performed to characterize the functional groups present in the Spinacia oleracea seeds extract. The spectra were recorded in the range of 4000-400 cm-1at a resolution of 4 cm-1 using an FTIR spectrometer. The resulting spectra were analyzed for the identification of Keerthana et al., Am. J. Pharm Health Res 2025;13(09) ISSN: 2321-3647 www.ajphr.com 24 characteristic peaks corresponding to specific functional groups, provided insights into chemical constituents of the extract. Determination of in vitro antioxidant activity using DPPH scavenging method DPPH (1,1 diphenyl, 2-picryl hydrazyl) was used to study the free radical scavenging effect of Spinacia oleracea seed extract1,2,3. Different concentrations of extract (50,100,300,500 µg/ml) were prepared in methanol. 1 ml of extract at various concentrations (50,100,300,500 µg/ml) is added to 0.1 ml of prepared DPPH solution and kept in dark for 30 minutes. Incubated samples were checked for absorbance at 517nm using the following equation and expressed as percent inhibition and the obtained results were compared with that of the standard Gallic acid of concentration (2.5µg/ml)1,4,5. % RSA = [(A0-A1/A0)] *100 Where A0 indicates control absorbance A1 indicates sample absorbance Hydroxyl radical scavenging assay method: For each individual concentration of extract, 1ml of the reagent solution was added11, followed by the sequential addition of 1 ml of 1.5Mm Feso4, 0.7 ml of 6mM H2O2, and 0.3ml of 20mM sodium salicylate12. The resulting mixture was incubated for approximately 1 hour13, after which the absorbance was measured at 562nm and % inhibition is calculated by using the formula and compared with that of standard Gallic acid(2.5µg/ml)14. % RSA = [ (A0-A1/A0)] *100 Where, A0 indicates control absorbance A1 indicates sample absorbance Nitric oxide scavenging assay For each individual concentration of the extract, 0.5ml of phosphate-buffered saline and dissolved sodium nitroprusside were added and thoroughly mixed17,18. The mixture was then incubated at 250C for 2 hours. Following incubation, 0.5ml of the reaction mixture was withdrawn from each sample and mixed with 0.5ml of Griess reagent. The resulting solution was measured at 546nm19. The percentage of nitric oxide scavenged by the test extract was then calculated using the formula and compared with standard Gallic acid(2.5µg/ml)20,21. % RSA = [ (A0-A1/A0)] *10 Where, Keerthana et al., Am. J. Pharm Health Res 2025;13(09) ISSN: 2321-3647 www.ajphr.com 25 A0 indicates control absorbance A1 indicates sample absorbance Determination of in vivo anxiolytic activity by behavioral tests Experimental design: The animals were divided into four groups, each group containing 3 mice. Group 1: control Group 2: animals treated with low dose of extract (75mg/kg) Group 3: animals treated with high dose extract (150mg/kg) Group 4: animals treated with standard drug diazepam (1 mg/kg) Elevated plus maze test The elevated plus maze (EPM) is a widely utilized behavioral model for assessing anxiety-like behavior in rodents. In this study group 2 and group 3 were administered a low and high dose of the test extract, respectively for 7 consecutive days. Group 4 received diazepam (1mg/kg) one hour prior to the experiment as a standard anxiolytic reference. On 7th day, the behavioral activity of each group was assessed and compared based on parameters such as the number of entries and time spent in open arms, as well as the number of entries and time spent in the closed arms of the maze. Open field test The apparatus consists of a contained area with surrounding walls to prevent the animal from escaping58, typically marked with a grid to distinguish center and peripheral squares. Animals were divided into four groups. Group 2 and Group 3 received low and high dose of test extract, respectively for 7 consecutive days. On the 7th day, one hour after extract administration, the animals were placed in a corner of the open field apparatus, and their behavior was recorded for 5 minutes. Parameters observed included the number of center square crossings, peripheral square crossings, and readings. As a standard, diazepam was administered to group 4 and the same behavioral parameters were recorded. Hole board apparatus The apparatus consists of an open field arena equipped with 16 evenly spaced holes each measuring 3 cm in diameter. Animals were divided into four groups and treated with their respective test and standard substances. One hour following the administration of the extract, each animal was individually placed on a corner of the apparatus. The number of head dipping behaviors exhibited by each animal was recorded for 5 minutes. Y-maze test Keerthana et al., Am. J. Pharm Health Res 2025;13(09) ISSN: 2321-3647 www.ajphr.com 26 The apparatus consists of three identical arms arranged at 120-degree angles, forming a y-shaped structure. Animals were divided into four groups and treated with their respective substances. Group 2 and group 3 received low and high dose of the test extract for 7 consecutive days, while group 4 received diazepam as the standard drug. On the 7 th day, one hour after extract administration for group 2 and group 3 and 30 minutes post diazepam administration for group 4, each animal was placed at the center of the maze and allowed to explore freely for 5 minutes and recorded the number of visits into three arms. RESULTS AND DISCUSSION FTIR findings Figure: 1 FTIR spectral peaks of Spinacia oleracea FTIR analysis was carried out to identify the functional groups present in the Spinacia oleracea seed extract and to confirm its bioactive compound The FTIR spectrum of the ethanolic seed extract of Spinacia oleracea reveals a complex mixture of bioactive constituents: a broad absorption around 3300cm⁻¹ indicates extensive hydrogen bonded O–H and N–H stretching, characteristic of phenolic compounds and proteins; prominent bands at 2920 and 2850cm⁻¹ arise from asymmetric and symmetric C–H stretching of –CH₂– groups in long chain fatty acids, while a sharp ester carbonyl stretch at 1740cm⁻¹ further confirms lipid content. In the mid‑region, the AmideI (≈1650cm⁻¹) and AmideII (≈1540cm⁻¹) bands attest to peptide backbones, and bending vibrations near 1450cm⁻¹ reinforce the presence of aliphatic chains. Below 1300cm⁻¹, strong C– O–C and C–O stretching vibrations (≈1240 and 1075cm⁻¹) point to polysaccharides or glycosides linkages, while the fingerprint region (900–600cm⁻¹) displays aromatic C–H bends and C–C skeletal modes associated with phenolic rings as shown in the Figure 1. Collectively, Keerthana et al., Am. J. Pharm Health Res 2025;13(09) ISSN: 2321-3647 www.ajphr.com 27 these features demonstrate that the seed extracts rich in phenolics, proteins, lipids, and carbohydrates, consistent with its known antioxidant, nutritional, and structural properties. Antioxidant activity The antioxidant effect of ethanolic seed extract of Spinacia oleracea was evaluated using standard protocols hydroxyl radical scavenging, nitric oxide scavenging, and DPPH method 1,3 . The IC 50 values so obtained were compared with standard reference Gallic acid (2.5µg/ml) 2,4 . Figure: 2 Hydroxyl radical scavenging activity The above graph indicates the hydroxyl radical scavenging activity of Spinacia oleracea seed extract at different concentrations (50,100,300,500µg/ml) compared to a standard antioxidant, Gallic acid (2.5µg/ml). The results are represented in terms of % Radical scavenging activity (%RSA) and IC 50 values. The data indicate a concentration -dependent increase in %RSA for Spinacea oleracea. It is shown in Figure 2. 50 100 300 500 2.5 41.35 58.92 70.16 84.63 81.2 64.7 64.7 64.7 64.7 79.2 0 100 200 300 400 500 600 Spinacia oleracea (50μg/ml) Spinacia oleracea (100μg/ml) Spinacia oleracea (300μg/ml) Spinacia oleracea (500μg/ml) Gallic acid (Std) (2.5μg/ml) 1 2 Concentration (μg/ml) % RSA IC50 Keerthana et al., Am. J. Pharm Health Res 2025;13(09) ISSN: 2321-3647 www.ajphr.com 28 Figure: 3 DPPH radical scavenging activity The above graph presents the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity of Spinacia oleracea seed extract at different concentrations (50,100,300,500 µg/ml), compared to the standard antioxidant, Gallic acid(2.5µg/ml). The results displayed in terms of concentration, % radical scavenging activity (%RSA), IC 50 values. A concentration dependent increase in %RSA was observed with increasing concentrations of spinacia oleracea extract. At the highest concentration (500µg/ml), the extract showed the greatest %RSA, indicating enhanced antioxidant activity. In comparison, Gallic acid, used as a reference standard at 2.5µg/ml, exhibited superior %RSA and a significantly lower IC 50 value. It is shown in Figure 3. Figure: 4 Nitric oxide scavenging assay 0 100 200 300 400 500 600 spinacia oleracea( 50μg/ml) spinacia oleracea( 100μg/ml) spinacia oleracea( 300μg/ml) spinacia oleracea( 500μg/ml) Gallic acid (2.5μg/ml) 1 2 concentration(μg/ml) % RSA IC 50 0 100 200 300 400 500 600 spinacia oleracea( 50μg/ml) spinacia oleracea( 100μg/ml) spinacia oleracea (300μg/ml) spinacia oleracea(500(μg/ml) Gallic acid (2.5μg/ml) 1 2 concentration (μg/ml) % RSA IC50 Keerthana et al., Am. J. Pharm Health Res 2025;13(09) ISSN: 2321-3647 www.ajphr.com 29 The above graph presents the Nitric oxide scavenging activity of Spinacia oleracea seed extract at different concentrations (50,100,300,500 µg/ml), compared to the standard antioxidant, Gallic acid(2.5µg/ml). The results displayed in terms of concentration, % radical scavenging activity (%RSA), IC 50 values. A concentration dependent increase in %RSA was observed with increasing concentrations of Spinacia oleracea extract. The highest activity noted at 500µg/ml, indicating that the extract becomes more effective at higher doses. It is shown in Figure 4. Anxiolytic activity The anxiolytic activity was assessed using elevated plus maze, open field apparatus, hole board test, y-maze. All these methods are widely acceptable for the screening of behavioral studies. Hence we have taken up the experiment using these standard protocols. Elevated plus maze Figure: 5 Effect of ethanolic seed extract of Spinacia oleracea on mice using elevated Plus maze. The above graph demonstrates that Spinacia oleracea seed extract exhibits dose-dependent anxiolytic activity in mice. The higher dose (150 mg/kg) closely mimics the anxiolytic profile diazepam. These findings support the efficacy of Spinacia oleracea in modulating anxietyrelated behavior through elevated plus maze performance has shown in Figure 5. Open field test 0 50 100 150 200 250 control low dose of extract(75/kg) High dose of extract(150mg/kg) Diazepam(1mg/kg) Elevated plus maze Time spent in open arm Time spent in closed arm Number of entries into open arm Number of entries into closed arm Keerthana et al., Am. J. Pharm Health Res 2025;13(09) ISSN: 2321-3647 www.ajphr.com 30 Figure: 6 Ethanolic seed extract of Spinacia oleracea on albino mice in open field test. The above graph indicates that the ethanolic seed extract of Spinacia oleracea enhances exploratory behavior and reduces in a dose-dependent manner in albino mice. The high dose (150 mg/kg) produced effects similar to the standard anxiolytic, Diazepam, as evidenced by increased center crossings and rearing activity which is shown in Figure 6. Hole board test Figure: 7 Effect of ethanolic seed extract of Spinacia oleracea on albino mice in hole board apparatus. The results from the hole board test indicates that Spinacia oleracea seed extract promotes anxiolytic activity in a dose dependent manner. The high dose (150 mg/kg) substantially increased head-dipping behavior and it is indicated in Figure 7. Y-maze 0 10 20 30 40 50 60 70 80 90 1 2 3 4 5 Open field test Treatment Center square crossings Peripheral square crossings Number of rearings 0 2 4 6 8 10 12 14 16 18 Control Low dose of extract(75mg/kg) High dose of extract(150mg/kg) Diazepam(1 mg/kg) Number of head dippings Keerthana et al., Am. J. Pharm Health Res 2025;13(09) ISSN: 2321-3647 www.ajphr.com 37 CONFLICT OF INTEREST: The authors declare that they have no competing interests. ACKNOWLEDGMENT: The authors would like to express their gratitude to all those who contributed to the successful completion of this article. REFERENCES: 1. Ravishankar K, PVSCP S, Kiranmayi GV. 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K., & Bhattacharya, S. K. (1992). Effects of stress on exploratory behavior in the elevated plus-maze. Indian Journal of Experimental Biology, 30(8), 696–698. 58. Sathish Kumar, B. P., et al. (2011). Ethanol effects on anxiety-like behavior in rodents: A review. Pharmacology Biochemistry and Behavior, 99(3), 480– 486. https://doi.org/10.1016/j.pbb.2011.05.012 AJPHR is Peer-reviewed monthly Rapid publication Submit your next manuscript at [email protected] / [email protected]