INTRODUCTION:

The use of plants in the treatment of ailments in traditional medicine began with the formation of social orders. Restorative spices are an important source of natural remedies for a variety of ailments in the traditional health paradigm. The antioxidant capabilities of plant-derived compounds may help to avoid oxidative stress in humans and animals.^1-2

Oxidative pressure is a major factor in the development of chronic illnesses such as cancer, cardiovascular disease, and diabetes mellitus.^3-5 Free radicals are produced as a consequence of normal metabolic processes. Responsive oxygen species (ROS) include free extremists such as hydroxyl radical (OH) and superoxide anion (O2), as well as non-free extreme species such as hydrogen peroxide (H2O2) and nitric oxide (NO), which are different types of initiated oxygen and are harmful to various physiologically significant atoms such as protein, lipids, cell membrane, DNA, and other cell components.^6-8 Natural antioxidants protect cells from oxidative damage, which aids in the prevention of different illnesses and maturation processes.⁹ The most basic action for managing the terrible situation is to administer a powerful anthelminthic remedy; nevertheless, there are few anthelminthic medications accessible, and those that are available cause opposition to cure in the majority of helminthes.^10-12 It is necessary to evaluate the regular remedies for the anthelmintic movement of natural drugs. Wild onion has received a lot of attention in recent years, and research has shown that it possesses a variety of healing benefits and pharmacological actions. The therapeutic use of Wild Onionpieces in many traditional homemade frameworks is known for several diseases, from which the current study proposes to focus on the in-vitro testing for its anti-diabetic, antioxidant, and anthelminthic qualities.^13-14

MATERIALS AND METHODS:

Materials:

The Wild Onion pieces were purchased from a local market. Albendazole was acquired as a gift sample from Leben Life science Pvt. Ltd. in Akola. Chloroform, ethyl acetate, methanol, and ethanol were obtained from S.D. Fine Labs in Mumbai. Loba Chem in Mumbai provided the potato starch and Di-nitro color reagent, while the local market supplied the commercial baker's yeast. All other chemicals and reagents used were analytical grade.

Methods:

Extraction Procedure:

Wild Onion parts, which were at that stage in the dried structure and had a strong cause for needing to be dried, were used as acquired with no more drying. The particles were coarsely pulverized and subjected to progressive dissolvable extraction using the soxhlet mechanical assembly. Each piece of material was extracted using a variety of solvents with increasing polarity, including chloroform, ethyl acetate, methanol, ethanol, and pure water. Each time, the material was dried and then extracted with the next highly polar solvent.^15-16 All concentrations were transferred to a Lab India rotating evaporator, and solvent remnants were removed using a desiccator containing silica gel.

Phytochemical Screening:

A phytochemical screening was done to assess the subjective substance organization of unrefined concentrates of wild onions. established screening tests employing usual conventions, methodologies, and reagents were undertaken using established approaches to identify the contents.^17-18

Evaluation of Antioxidant Activity by using in-vitro Assays:

DPPH Scavenging Effect:

A 40μl DPPH solution was vortexed with several concentrations and hatched in darkness. The absorbance was then estimated at 520nm. For clear insights to measure (IC50) mean quality and related standard error for each case, all computations were done using the Softmax PRO 4.3.2.LS software.^19-22 Butylated hydroxyl toluene was used as a source of perspective at concentrations of 0.1, 0.3, and 1mg/mL.

DPPH Scavenging Effect (%) =

Absorbance _(Control)- Absorbance _(Sample)

------------------------------------------------------ × 100

Absorbance _(Control)

Ferric-Reducing Antioxidant Power:

The concentrate was hatched in 0.2mol/l phosphate buffer (pH 6.6) containing K3Fe (CN)6. Trichloroacetic acid was added to the mixture, vortexed, and centrifuged. Water and ferric chloride were added to the supernatant, and the absorbance was measured at 700 nm. Ascorbic acid was used as a reference.^23-24

Metal-Chelating Capacity:

Extricates were hatched with FeCl2 (2mM). The reaction began upon the addition of 0.2ml of ferrozine (5mM). Eventually, absorbance was measured at 562nm. Ethylenediaminetetraacetic acid was employed as a prospective source, with FeCl2 and ferrozine serving as controls.^25-26 The restraint fraction of ferrozine-Fe⁺² complex formation was determined using the following formula:

Metal-Chelating Capacity (%) =

Absorbance _(Control)-Absorbance _(Sample)

------------------------------------------------------ × 100

Absorbance _(Control)

Phytochemical Content:

Estimation of Total Phenol Content:

The extracts were combined and treated with the Folin-Ciocalteu reagent. The sodium carbonate solution was then immediately vortexed into the mix. After 30 minutes of darkness, absorbance was estimated at 735nm.²⁷ The total phenol content of concentrates in mg of gallic acid equivalent (GAE) was calculated using the equation obtained from the linearity diagram. Absorbance = 0.0029 × (conc.) + 0.2496, with a correlation coefficient of 0.9929.

Determination of Total Flavonoids Content: Concentrates of 1mg/mL were placed in suspension in 80% ethanol. Sodium acetic acid, aluminum chloride solution, ethanol, and water were combined and incubated with the varied extracts for 30 minutes. The absorbance was computed at 415 nm.²⁸ The monotone diagram was applied to derive the equation used to plot the data in mg QE/g extracts. Absorbance = 0.0161 × (conc.) + 0.025, with a r2 value of 0.9965.

Anthelminthic Property:

Collection of Test Organisms (Earthworms) and Authentication:

The earthworms of the species Phertima Posthuma were used in the investigation. They were collected from the wet soil of the nursery at Shri Shivaji College of Horticulture in Amravati and authenticated at Shri Shivaji College of Agriculture in Amravati using the confirmation ID (825 (AB)/2023-24). The worms were used to study anthelminthic motility because they are physically and physiologically similar to roundworms found in the digestive tract. The Phertima Posthuma were housed in a compartment with a large aperture for the air section, and the container was filled with new soil and water on a regular basis to provide the worms with a familiar environment until the study was completed.

Evaluation of Anthelminthic Property:

Worms Motility Assay (WMA):

Before beginning the experiment, the earthworms were taken from the container and rinsed with distilled water. HPMC was utilized to thicken the concentrate extract solution in distilled water, using albendazole as a reference. A dose of 200mg/5mL was employed. Seven distinct groups were tested in vitro for antihelminthic activity. A single earthworm was put in three distinct Petri dishes for each of the three groups. Phertima Posthuma worms have been employed in antihelminthic research due to their physical and physiological similarities to gastrointestinal roundworms. The study materials show how long it took the earthworms to become paralyzed and die after being put to the suspensions on the Petri plates.^29-31 As long as the individual worms in each petri dish stay paralyzed and die, they are examined in detail, along with their corresponding time of death.

Statistical Analysis:

The experiments, conducted in triplicate to ensure accuracy, yielded mean values which were then calculated. All numerical results have been meticulously presented in the form of mean values accompanied by their respective standard deviations, providing a comprehensive overview of the data obtained.

RESULTS AND DISCUSSION:

Phytochemical Screening: During the phytochemical screening, alkaloids and tannins were identified in the concentrates. Notably, flavonoids and phenols were also present in the concentrate of Wild onion. This comprehensive analysis revealed the diverse chemical composition of the concentrate, providing valuable insights into its phytochemical properties.

Evaluation of Antioxidant Activity by using in-vitro Assays:

DPPH Scavenging Effect:

The standard calibration for gallic acid and quercetin was investigated, along with the DPPH scavenging capacity of several wild onion extracts and BHT. The aqueous extract demonstrated the maximum scavenging activity (65.80±7.45) at 3 mg/ml, followed by ethanolic, ethyl acetate, methanolic, and chloroform extracts. At a dosage of 1 mg/ml, the aqueous extract had the maximum scavenging activity (58.30±1.43), followed by ethanolic, ethyl acetate, methanolic, and chloroform extracts. The aqueous extract demonstrated the maximum scavenging activity (38.67±4.01) at a concentration of 0.3 mg/ml, followed by ethanolic, ethyl acetate, methanolic, and chloroform extracts.

Table 1: DPPH radical scavenging activity inhibition (% ± SD)

Samples	3 mg/ml	1 mg/ml	0.3 mg/ml
Chloroform	30.91±7.04	24.17±3.25	08.07±9.87
Ethyl acetate	47.78±0.64	41.58±1.47	12.37±1.44
Methanol	34.56±0.46	27.00±3.77	10.97±4.47
Ethanol	59.63±6.16	45.03±3.15	14.63±1.28
Aqueous	65.80±7.45	58.30±1.43	38.67±4.01
Standard (BHT)	70.67±3.40	67.17±4.31	40.83±7.69

Figure 1: (A) Gallic acid standard curve for total phenolic content determination and (B) Quercetin standard curve for flavonoid content determination.

Ferric-Reducing Antioxidant Power: The study demonstrates the ferric-reducing antioxidant activity of several wild onion extracts and ascorbic acid. The aqueous extract demonstrated the maximum scavenging activity (3.2090±0.0210) at a concentration of 3 mg/ml, followed by methanolic, ethanolic, chloroform, and ethyl acetate extracts. At 1mg/ml, the aqueous extract had the maximum scavenging activity (2.8980±0.0936), followed by methanolic, ethanolic, chloroform, and ethyl acetate extracts. The aqueous extract demonstrated the maximum scavenging activity (1.2257±0.0528) at 0.3 mg/ml, followed by methanolic, ethanolic, chloroform, and ethyl acetate extracts.

Table 2: Ferric-Reducing Antioxidant Power (% ± SD)

Samples	3 mg/ml	1 mg/ml	0.3 mg/ml
Chloroform	0.7040±0.0867	0.2143±0.0084	0.0477±0.0042
Ethyl acetate	0.6154±0.0285	0.0847±0.0110	0.0190±0.0036
Methanol	1.7140±0.0875	1.4720±0.0339	1.0283±0.1198
Ethanol	1.2424±0.0101	0.9977±0.1124	0.3130±0.0300
Aqueous	3.2090±0.0210	2.8980±0.0936	1.2257±0.0528
Standard (Ascorbic acid)	3.5870±0.0874	3.4547±0.0852	3.0787±0.0587

*Total antioxidant capacity is expressed as ascorbic acid equivalent/g extract ±SD.

Metal-Chelating Capacity:

The study examines the metal-chelating properties of several Wild Onion extracts with EDTA. The aqueous extract demonstrated the maximum scavenging activity at a concentration of 3mg/ml (96.33±15.66), followed by ethanolic, chloroform, methanolic, and ethyl acetate extracts. For the 1mg/ml sample, the aqueous extract showed the maximum scavenging activity (91.11±5.66), followed by ethanolic, chloroform, methanolic, and ethyl acetate extracts. At 0.3mg/ml, the aqueous extract had the maximum scavenging activity (25.24±6.17), followed by chloroform, ethanolic, methanolic, and ethyl acetate extracts.

Table 3: Metal-Chelating Capacity (% ± SD)

Samples	3 mg/ml	1 mg/ml	0.3 mg/ml
Chloroform	87.78±0.64	41.58±1.47	24.37±1.44
Ethyl acetate	51.74±1.82	27.78±3.32	14.51±1.64
Methanol	59.63±6.16	45.03±3.15	16.07±4.47
Ethanol	91.48±9.19	90.33±4.19	16.98±5.72
Aqueous	96.33±15.66	91.11±5.66	25.24±6.17
Standard (EDTA)	98.87±0.14	96.60±3.39	95.75±0.08

*Total antioxidant capacity is expressed as EDTA equivalent/g extract ± SD.

Phytochemical Content: According to this study, the pieces have a significant concentration of flavonoids and phenolic chemicals. The aqueous extract had a greater total phenolic content (118.04±1.94) than other Wild Onion extracts, including ethanolic, chloroform, methanolic, and ethyl acetate. The extracts of ethyl acetate showed the lowest flavonoid content (51.032±0.043). The aqueous extract contained the greatest phenolic content (53.35±2.02), followed by ethanol, methanol, chloroform, and ethyl acetate. The extracts of ethyl acetate exhibited the lowest flavonoid content at 26.11±10.005. The total flavonoids were represented as mg quercetin equivalent/g extract, and the total phenols as mg gallic acid equivalent/g extract.

Figure 2: Total Phenolic and Flavonoids Contents of Wild OnionExtract

Anthelminthic Property:

Worms Motility Assay (WMA):

According to the data, the aqueous extract exhibited paralytic effects more earlier, and all worms died sooner than the other extract trials.

The aqueous extract showed a paralysis time of 3.52±0.158 minutes and a death time of 2.74±0.247. The ethanolic extract took the longest time for paralysis and death, at 6.34±0.46min and 5.11±1.54min, respectively. The periods necessary for paralysis and death in chloroform, ethyl acetate, and methanolic extracts were 6.98±1.52, 5.48±1.30, and 7.08±1.40 min, respectively. The timeframes required for death are 5.81±1.56, 11.31±1.48, and 4.55±1.10min. The anthelmintic activity of extracts was examined and compared to the reference standard albendazole, which revealed 3.36±0.189 for paralysis and 1.51±0.157 minutes for death.

Figure 3: Graphical Representation of time required for the Paralysis and Death of the Earthworms

CONCLUSION:

The trial proof acquired from laboratory experiments not only sheds light on the rationale behind the traditional use of these components as antioxidants and anthelmintics but also has the potential to revolutionize the field of natural medicine. Our research findings strongly support the belief that the healing properties attributed to this plant will likely replace synthetic drugs in the foreseeable future. This shift could be particularly beneficial given the rising concerns surrounding drug interactions and the development of drug resistance, making the cultivation and utilization of these natural remedies a promising alternative in the realm of healthcare.

REFERENCES:

1. Aggarwal H, Kotwal N. Foods used as ethno-medicine in Jammu. Studies on Ethno-Medicine. 2009 Jan 1;3(1):65-8.

2. Thippeswamy NB, Naidu KA. Antioxidant potency of cumin varieties—cumin, black cumin and bitter cumin—on antioxidant systems. European Food Research and Technology. 2005 May; 220:472-6.

3. Freeman GG, Mossadeghi N. Influence of sulphate nutrition on the flavour components of garlic (Allium sativum) and wild onion (A. vineale). Journal of the Science of Food and Agriculture. 1971 Jul; 22(7): 330-4.

4. Nguyen NH, Driscoll HE, Specht CD. A molecular phylogeny of the wild onions (Allium; Alliaceae) with a focus on the western North American center of diversity. Molecular Phylogenetics and Evolution. 2008 Jun 1; 47(3):1157-72.

5. Simin N, Mitić-Ćulafić D, Pavić A, Orcic D, Nemes I, Cetojevic-Simin D, Mimica-Dukić N. An overview of the biological activities of less known wild onions (genus Allium sect. Codonoprasum). Biologia Serbica. 2020 Feb 27;41(2).

6. Kousalya M, Geetha P, Jesuraja A, Kumar MV. In-Vitro study of anthelmintic activity of Eclipta prostrata (L) y various Extracts. Research Journal of Pharmacy and Technology. 2017; 10(1):58-60.

7. Babu ND, Kodithala S, Murali R, Srinivasan N. Anthelmintic activity of methanolic bark extract of Buchanania axillaris (Desr.). Research Journal of Pharmacy and Technology. 2018; 11(4): 1298-300.

8. Shagana S, Navenaa S, Vijay J, Vishali S, Rajendran V. Investigation of in vitro Anthelmintic activity of Ocimum basilicum Linn. (Lamiaceae). Research Journal of Pharmacy and Technology. 2021; 14(1):52-4.

9. Koleva II, Van Beek TA, Linssen JP, Groot AD, Evstatieva LN. Screening of plant extracts for antioxidant activity: a comparative study on three testing methods. Phytochemical Analysis: An International Journal of Plant Chemical and Biochemical Techniques. 2002 Jan; 13(1):8-17.

10. Lim YY, Lim TT, Tee JJ. Antioxidant properties of several tropical fruits: A comparative study. Food chemistry. 2007 Jan 1;103(3):1003-8.

11. Tachakittirungrod S, Okonogi S, Chowwanapoonpohn S. Study on antioxidant activity of certain plants in Thailand: Mechanism of antioxidant action of guava leaf extract. Food Chemistry. 2007 Jan 1; 103(2):381-8.

12. Seifried HE, McDonald SS, Anderson DE, Greenwald P, Milner JA. The antioxidant conundrum in cancer. Cancer Research. 2003 Aug 1; 63(15): 4295-8.

13. Eswaraiah MC, Reddy DP, Illendula S. In-vitro anthelmintic activity of Tamilnadia uliginosa fruits. Research Journal of Pharmacy and Technology. 2019; 12(1): 321-3.

14. Sutar NG, Giri MA, Kendre PN, Syed NL, Singh J. Phytochemical Investigation and Pharmaological Screening of Alienthus excelsa Linn. Leaves as Anthelmentic activity in Earthworm. Research Journal of Pharmacy and Technology. 2010; 3(1): 244-6.

15. Tehseen F, Ghori SS, Dawood M, Tazeen S, Sara K, Samad A. Phytochemical screening and invitro anthelmintic activity of a novel polyherbal formulation. Research Journal of Pharmacy and Technology. 2021; 14(5): 2742-4.

16. Rajesh R, Selvakumar K. Evaluation of in vitro Anthelmintic activity of Lannea coromandelica (Houtt.) Merr. against Pheretima posthuma and Ascardia galli. Research Journal of Pharmacy and Technology. 2022; 15(6): 2539-42.

17. Ivanova AV, Gerasimova EL, Brainina KZ. Potentiometric study of antioxidant activity: development and prospects. Critical Reviews in Analytical Chemistry. 2015 Oct 2; 45(4):311-22.

18. Kang MY, Tsuchiya M, Packer L, Manabe M. In vitro study on antioxidant potential of various drugs used in the perioperative period. Acta Anaesthesiologica Scandinavica. 1998 Jan; 42(1):4-12.

19. Stephens NG, Parsons A, Brown MJ, Schofield PM, Kelly F, Cheeseman K, Mitchinson MJ. Randomised controlled trial of vitamin E in patients with coronary disease: Cambridge Heart Antioxidant Study (CHAOS). The Lancet. 1996 Mar 23; 347(9004): 781-6.

20. Mishra A, Pradhan DK, Mishra MR, Behera R, Panda AK, Vaishnaw SK. In-vitro Anthelmintic Activity of Musa sapientum Linn. Flower. Research Journal of Pharmacy and Technology. 2011; 4(1): 89-91.

21. Chiranjeevi U, Kamarajan K, Manna PK. Investigation of in-vitro anthelmintic activity of Lindernia madayiparense. Research Journal of Pharmacy and Technology. 2018; 11(1):183-6.

22. Gayathiri NM, Sudhakar P, Manimekalai P, Sabarinath C. In Vitro Anti-helminthic Activity of hydroalcoholic extract of Martynia annua L. and Pentanema indicum. Research Journal of Pharmacy and Technology. 2019; 12(8):3847-50.

23. Wu X, Gu L, Holden J, Haytowitz DB, Gebhardt SE, Beecher G, Prior RL. Development of a database for total antioxidant capacity in foods: a preliminary study. Journal of Food Composition and Analysis. 2004 Jun 1; 17(3-4):407-22.

24. MR A. Antioxidant study of pulps and peels of dragon fruits: a comparative study. International Food Research Journal. 2010; 17: 367-75.

25. Potapovich AI, Kostyuk VA. Comparative study of antioxidant properties and cytoprotective activity of flavonoids. Biochemistry (Moscow). 2003 May; 68:514-9.

26. Korotkova EI, Karbainov YA, Shevchuk AV. Study of antioxidant properties by voltammetry. Journal of Electroanalytical Chemistry. 2002 Jan 11; 518(1): 56-60.

27. Deepa VS, Kumar PS, Latha S, Selvamani P, Srinivasan S. Antioxidant studies on the ethanolic extract of Commiphora spp. African Journal of Biotechnology. 2009; 8(8).

28. Moon JK, Shibamoto T. Antioxidant assays for plant and food components. Journal of Agricultural and Food Chemistry. 2009 Mar 11; 57(5): 1655-66.

29. Tachakittirungrod S, Okonogi S, Chowwanapoonpohn S. Study on antioxidant activity of certain plants in Thailand: Mechanism of antioxidant action of guava leaf extract. Food Chemistry. 2007 Jan 1; 103(2):381-8.

30. Chaves N, Santiago A, Alías JC. Quantification of the antioxidant activity of plant extracts: Analysis of sensitivity and hierarchization based on the method used. Antioxidants. 2020 Jan 15; 9(1):76.

31. Veeru P, Kishor MP, Meenakshi M. Screening of medicinal plant extracts for antioxidant activity. Journal of Medicinal Plants Research. 2009 Aug 1; 3(8):608-12.

Received on 03.10.2024 Revised on 05.12.2024

Accepted on 15.01.2025 Published on 05.03.2025

Available online from March 11, 2025

Res. J. Pharmacognosy and Phytochem. 2025; 17(1):9-13.

DOI: 10.52711/0975-4385.2025.00003

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