Evaluation Of Anti-Inflammatory and Anti-Bacterial Activity of Onion Peel (Allium Cepa. L) And Analysis of Its Bioactive Compounds

For millennia, natural products have played a crucial role in healthcare and disease prevention. Ancient civilizations, including those in India, China, and North Africa, have documented the use of natural resources to treat various ailments. The global transition towards processed food consumption, insufficient physical activity, sedentary lifestyles, and exposure to polluted environments has resulted in an increase in disorders such as obesity, cardiovascular diseases, diabetes, neurodegenerative conditions, and cancers. Oxidative stress is considered a primary factor in these health issues. Studies have demonstrated that plant-based bioactive phytonutrients can protect against damage caused by excessive free radical formation and oxidative stress. Onion peel has emerged as an excellent source of fructo-oligosaccharides, dietary fibers, polyphenols, and antioxidants[1]. The chemical composition of onions is influenced by factors such as environment, cultivar type, agricultural practices, ripening stage, and preservation duration. Notably, onion peel contains significantly higher levels of flavonoids than the edible portion. The yellow and purple colours of onions are attributed to two main subgroups of flavonoids, anthocyanins, and quercetin, along with their derivatives[2]. The outer layer of onions is particularly rich in major flavonoids, including quercetin di-glucoside, quercetin, quercetin aglycone, kaempferol, and quercetin 4-O-glucoside[3]. Extensive research on onion peel/waste and its extracts has revealed that they are a rich source of phytoconstituents with diverse therapeutic potential and pharmacological activities[4, 5]. A deeper understanding of these biological properties could facilitate further research and potential applications of onion waste in biomedical and pharmaceutical industries. This study aimed to evaluate the anti-inflammatory and antibacterial activities of onion peel extract and analyse its bioactive compounds.

MATERIALS AND METHODS

Dried onion peels were collected from different onion vendors at Perundurai, Erode, Tamil N?du.

Method Of Extraction

The scales were pulverized in laboratory mill and sieved. Particles smaller than 0.2 mm was used to experiments for extraction. Extraction was done by 2 hours heating under reflux condenser with 96 percent ethanol. (Proportion of solvent to material was:50 ml of solvent per 1 g of powder). Extracts were filtered under vacuum, and 20 microliters of clear supernatants were taken to analysis. Solvent was evaporated in rotary evaporator under vacuum in 40°C. The residue was collected[6, 7].

Fractionitation Of Extract by Solvent-Solvent Extraction Method

The ethanolic extract was dissolved in water and the aqueous suspension of ethanolic extract was sequentially extracted with petroleum ether, diethyl ether and ethyl acetate solvents (solvents are chosen based on their order of polarity i.e., non-polar to polar) using separatory funnel. 2g of the ethanolic extract was dissolved in 20 ml of water. Then the aqueous suspension of ethanolic extract was extracted with 40 ml of petroleum ether for 30 minutes which was repeated for at least three times. The petroleum ether phase(FS1) was collected and evaporated to dryness under vacuum. As in the similar way, the aqueous suspension of ethanolic extract was sequentially extracted with diethyl ether and ethyl acetate and evaporated to dryness to give the corresponding diethyl ether(FS2) and ethyl acetate(FS3) components respectively. The samples of respective solvents are taken for further analysis[8, 9].

Fractionation Scheme for Solvent-Solvent Extraction

Table:1 Solvent system for identification of flavonoids in TLC.

After performing TLC, the respective fractionated samples were undergone IR, MASS and NMR spectral analysis to confirm the major compound present in samples.

Evaluation Of Anti-Inflammatory Activity of Whole (Allium Cepa. L) Onion Peel Extract.

The anti-inflammatory property  of onion peel (Allium Cepa. L) extract was studied against carrageenan induced acute paw edema in rats.

Animals

Experiments were performed on Wister albino rat (300-350 gm) housed at 22 ± 2^o C under a 12-h light/12-dark cycle with free access to food and water. Experimental animals were obtained from the animal house of NCP, Erode(Institutional Animal ethical committee no: NCP/IAEC/UG-2022-23-06). Throughout the experiment period, the animals were housed in large spacious propylene cage boxes. The animals were with standard laboratory diet and water.

Chemicals

Carrageenan, Standard drug- Indomethacin.

Procedure

24 Male Wister rats were weighed and taken for the study. Rats were divided into four groups (n = 6), each receiving normal (control), Indomethacin (20 mg/kg p.o.) (reference standard), and 200 and 400 mg/kg of ethanolic extract of onion peel respectively, which are administered before 30 mins eliciting paw edema.  Carrageenan (0.1 ml of 1%) was injected into the sub plantar tissue of the right hind-paw of each rat to provide acute inflammation. The volume of the carrageenan injected into the foot was measured at 0, 30, 60, 120, and 180 minutes using a vernier[10, 11].

Evaluation Of Anti-Bacterial Activity of Whole Onion Peel (Allium cepa. L) Extract

Minimum Inhibitory Concentration

The onion peel extract was serially diluted to concentrations ranging from 800 to 25 µg/100 µL (800, 400, 200, 100, 50, and 25). These dilutions were individually added to the appropriate microbial growth medium in separate test tubes. Subsequently, each test tube was inoculated with the bacterial strains. The tubes were incubated overnight, after which the turbidity of the medium was assessed to evaluate bacterial growth.

Anti-Bacterial Activity

Antibacterial activity screening was performed for the ethanolic extract of onion peel using around  two-gram positive (Staphylococcus aureus, Bacillus subtillis) and two-gram negative bacterial strains(Proteus vulgaris,Escherichia coli). Various concentration of extract was used  (100µg/µl, 200µg/µl), 400µg/µl, 800 µg/µl) to test the antibacterial activity against  ciprofloxacin(10 µg/ml ) as the standard drug and ethanol as a control.

Procedure

The medium was prepared by heating a specified quantity of dehydrated medium in purified water using a water bath and subsequently distributing it into 100 ml conical flasks. These flasks were sealed with cotton plugs and sterilized in an autoclave at 121°C (15 lb.) for 45 min. Sterile universals were administered to 25 ml of the agar medium. Flat-bottomed Petri dishes were positioned on a level surface to ensure uniform thickness of the medium layer. Each universal was combined with 0.2 ml of various bacterial strains and nutrient agar medium in sterile petri dishes and then allowed to solidify. Using a cork-borer (6 mm), four wells were created in each agar plate containing the bacterial culture. Test solutions (100, 200, 400 and 800 µg/µl)) were prepared using ethanol as the solvent. Under aseptic conditions, 0.2 ml of each solution was introduced into the wells using a dropping pipette and labelled. The plates were maintained at room temperature for 3-5 hours to facilitate diffusion of the solution. For antibacterial activity testing, the Petri dishes were incubated at 37°C for 24 h. The diameter of the inhibition zone (mm) surrounding each well was then measured. The results were compared to those of ciprofloxacin (10 µg/ml) for antibacterial activity assessment[12, 13].

RESULTS AND DISCUSSION

Confirmatory Test

The confirmatory tests such  lead acetate and sodium hydroxide test shows positive response for the fractionated samples which indicates the presence of flavonoids[14].

Thin Layer Chromatography

From the TLC, R_f value of the fragmented samples were calculated and found to be 0.87,0.89,0.84 respectively.

Spectroscopic Analysis Of Fractionated Samples (Fs1-3) From Dried Onion Peel Extract

The proposed fragmentation pattern of  fractionated sample (FS1) is as follows: 

FT-IR analysis revealed peaks at 3504 cm^-1and 1078 cm ^-1,1166 cm ^-1,1205 cm ^-1,1267 cm^-1 which were consistent with the presence of hydroxyl groups. The peak at 1650 cm^-1 was attributed to the stretching vibration of a carbonyl group. Several others peaks were observed at 1620 cm ^-1, 1510 cm ^-1, and 1458 cm^-1 which were consistent with the presence of a phenyl ring skeleton. The peaks observed at 1701 cm^-1 also sug gested the presence of oxygen containing heterocycle. Mass M/Z=302.23 (C₁₅H₁₀O₇), 275.23(C₁₄H₁₁O₆), 261.20 (C₁₃H₉O₆), 231.22(C₁₃H₁₁O₄)

216.18(C₁₂H₈O₄), 152.10 (C₇H₄O₄), 134.13 (C₈H₆O₂). NMR (ppm)=7.14(Aromatic CH), 3.85(-OH group), 7.10(C₄H₈O₂). Hence the sample FS1 is predicted to contain Quercetin.

       
           
       
IUPAC NAME: 2-(3,4-dihydroxy phenyl)-3,5,7-trihhydroxy chromen-4-one

       
           
       
 Molecular weight=316.26228

FT-IR analysis revealed peaks in range of 1250-1050 cm ^-1which were consistent with the presence of hydroxyl groups. The peak at 1649 cm^-1 was attributed to the stretching vibration of a carbonyl group. The strong peak at 2850 cm ^-1indicates the presence of methoxy group. Several other peaks were observed at 1620,1510 and 1458 cm ^-1, which were consistent with the presence of a phenyl ring skeleton. The peaks observed at 1733 cm ^-1 also suggested the presence of oxygen containing heterocycle. Mass m\z =316.26 (C₁₆H₁₂O₇), 288.25 (C₁₅H₁₂O₆), 274.26 (C₁₅H₁₄O₅), 244.24 (C₁₄H₁₂O₄), 228.24 (C₁₄H₁₂O₃), 172.13 (C₇H₈O₅), 148.15 (C₉H₈O₂). NMR (ppm)= 7.14 (Aromatic CH), 7.10 (C₄H₈O₂), 3.85 (S,CH-OH), 2.4-4.4 (S, OCH₃). Hence, the fractionated sample (FS2) was predicted to be contain Isorhamnetin.

       
           
       
Molecular weight=316.26228

IUPAC: 3,5,7-trihydroxy-2-(4 hydroxy-3-methoxy phenyl) chromen -4-one

 FT-IR analysis revealed peaks in range of 1250-1050 cm^-1 which were consistent with the presence of hydroxyl groups. The peak at 1650 cm^-1 was attributed to the stretching vibration of a carbonyl group. Several other peaks were observed at 1580 cm ^-1,1510 cm ^-1 and 1458 cm ^-1, which were consistent with the presence of a phenyl ring skeleton. The peaks observed at 1733 cm ^-1 also suggested the presence of oxygen containing heterocycle.Mass m\z =286.24 (C₁₅H₁₀O₆), 258.22 (C₁₄H₁₀O₅), 214.21 (C₁₃H₁₀O3), 198.21 (C₁₃H₁₀O₂), 152.10(C₇H₄O₄), 118.13 (C₈H₆O). NMR (ppm)=7.14 (C₆H₆), 7.10 (C₄H₈O₂), 3.85 (S, CH-OH). Hence, the fractionated sample(FS3) was predicted to be contain Kaempferol.

       
           
       
Iupac Name :3,5,7-trihydroxy-2-(4-hydroxyphenyl) chromen-4-one

Anti-Inflammatory Activity

The data represented as mean ± SEM. The standard drug indomethacin 20 mg/kg produce significant reduction in paw thickness and treatment with ethanolic extract of onion peel reduced the carrageenan induced paw edema in dose dependent manner.

Minimum Inhibitory Concentartion

The onion peel extract did not show any visible growth at the lowest concentration of  200µg/ 100 µl.

Anti-Bacterial Activity

 The  ethanolic extract of onion peel on gram positive and gram negative organisms at a  concentration range of 100, 200, 400, 800 µg/µl and standard (ciprofloxacin) 10 µg/ml showed significant inhibition in concentration dependent manner. In this study,ethanolic extract of onion peel showed maximum inhibition at 800 µg/µl against all organisms which is compared with standard ciprofloxacin drug.