Evaluation of the Phenolic, Flavonoid, Antioxidant and Active ingredients in Lemon peel extract

Manvi Sharma¹, Aakansha¹, Madhulika Esther Prasad², Ajam Shaikh²,

Chinmoyee Maharana¹, Amit Gupta¹*

¹Department of Zoology, University of Jammu, Jammu and Kashmir, India.

²Department of Biochemistry and Biotechnology, School of Life Sciences,

Sardar Bhagwan Singh University, Dehradun, Uttarakhand, India.

³Department of Science, Faculty of Humanities Liberal Arts and Social Sciences,

Alliance University, Bengaluru, Karnataka, India.

*Corresponding Author E-mail: amit.gupta@jammuuniversity.ac.in

ABSTRACT:

Plants are rich in bioactive compounds and play an important role in the development of novel medications. In this study, lemon samples had been collected from different regions in the Jammu area, and their antioxidant and antibacterial qualities, and additionally their phenolic and flavonoid content, were explored. The purpose of this study was to determine the phenolic, flavonoid, antioxidant and antimicrobial content of a lemon peel extract. In order to achieve this objective, the Folin-Ciocalteu and aluminium chloride approaches was employed for calculating the overall amount of phenolic and flavonoid compounds of the lemon peel. The DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging technique was used to measure the antioxidant activity. Furthermore, antibacterial activity of lemon peel was assessed using the disc diffusion technique. From these studies, the results showed that the content of phenols and flavonoid ranged from 218.12 to 678.22 mg GAE/g and 8.06 to 171.37 mg quercetin/g, respectively. Its antioxidant activity of lemon peel was found to be greater, ranging from 24.49 to 65.71 mg AAE/g sample. Additionally, they demonstrated equal efficacy to the reference antibiotic i.e. Gentamicin and lemon peel of variable concentrations which showed maximum inhibition zones for Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli.

KEYWORDS: Lemon peel, Phenolic, Flavonoid, Antioxidant, Antimicrobial.

INTRODUCTION:

Lemon is the most prominent member of the Rutaceae family. Although the precise origin of the lemon is uncertain, historical evidence points to China, Northern Myanmar, or Assam as possible earliest cultivation locations. Lemons are ellipsoidal fruits that become yellow as they ripen from green.¹

A lemon is made up of three parts: the thickest outer layer, called the exocarp, which turns yellow as it ages and consists of oil glands that give lemons their aromatic qualities; the middle section, or mesocarp, typically white and spongy and rich in pectin and used to make jams and jellies; and the innermost part, called the endocarp, which typically separates into different segments and contains elongated cells where water, carbohydrates, and citric acid are accumulated. Endocarp is a light yellow in hue and has an acidic flavour.^2,3 Lemon seeds are tiny, oval-shaped, pale, and have a yellowish-white hue. Due to their bitter flavour and rough texture, seeds are often thrown away. On the other hand, lemon seeds include a lot of dietary fibre, which aids in preventing constipation. Limonoids, a kind of antioxidant found in lemon seeds, also help decrease cholesterol and stop the accumulation of fat in the liver, in addition to preventing the growth and spread of certain cancer cells.^4,5 Lemon seeds are also a great source of vitamin C, which is necessary for the manufacture of collagen, a protein that maintains the structure and flexibility of connective tissue, bones, and skin. The compounds citral, limonene, terpineol, geranyl acetate, and linalyl that occur in lemon are thought to have medicinal uses for treating kidney stones, lowering fevers, and adjusting pH. Lemon peel has more antioxidant and antibacterial action, as well as a high concentration of phytochemicals. Lemon peel contains potent antimicrobial and stringent in nature properties.^6,7

The literature describes a lemon tree with a robust spine that may reach a height of 6m. Unlike numerous other citrus species, the petioles of this species have narrow or no wings. The leaves are up to 14cm (4 in) long, dark green, leathery, evergreen, and oblong, elliptical, or oval. Purple flower buds unfold to reveal five white petals that can measure up to five centimetres in diameter.^8,9 The 7.5–12.5cm long, globose to rectangular fruits have smooth or bumpy rinds that are speckled with oil glands as they develop to yellow. According to FAO estimates, global citrus fruit production in 2020 was estimated to exceed 158 million metric tonnes. From this sum, 27million tonnes of citrus fruit were processed into juice. In this investigation, our team concentrated on phytochemical analysis, denaturation assay, phenolic and flavonoid content, including antioxidant and antimicrobial activity.

MATERIALS AND METHODS:

Collection of samples:

The study performed an experiment from August to September of 2024 at the University of Jammu's Department of Zoology. We purchased the lemons for this investigation from the local market in Jammu. First, the lemons were rinsed with fresh water to eliminate the dust; next, the lemon peels were separated and crushed using a mortar and pestle to make the fine powder. One gram of freeze-dried sample and five millilitres of normal saline were combined in a magnetic stirrer that ran at rapid speed for about five minutes. The mixture was then centrifuged for ten minutes at 4500rpm. Following that, filter paper had been employed to hold and filter the supernatant. The obtained supernatants had been incorporated together with the leftover residue, which was extracted once more with the same protocols. After that, a rotatory evaporator operating at 40°C was used to dry the mixed supernatant. After being precisely weighed and kept at -20°C in a glass vial with a screw top, the dried extract was utilised for the assay by dissolving it in PBS at the necessary concentration.

Phytochemical analysis:

The confirmatory tests were carried out in keeping with standard methods pertaining to the qualitative phytochemical assessment of lemon peel extract with the objective to recognise the metabolites that are secondary (tannins, saponins, flavonoids, alkaloids, phenols, glycosides, steroids, and terpenoids). In addition, various compounds were reported in the lemon peel, as already mentioned in the literature (Fig. 1).

Estimation of total phenolic and flavonoid content:

Spectrophotometric analysis of the lemon peel extract's total phenolic content was conducted employing the Folin-Ciocalteu test, with gallic acid serving as a reference.¹⁰ We combined 0.5mL of lemon peel extract, distilled water (3000µl), and Folin-Ciocalteu reagent (250µl), then agitated the reaction mixture to create it. After 5 minutes in the dark, 1 mL of 7.5% Na2CO3 was added and incubated at room temperature for 90 minutes in the dark. In addition, a reagent blank was created using distilled water. The absorbance was measured around 760nm employing a double beam UV/Vis spectrophotometer compared to the manufactured reagent blank. The extract's total phenolic component content has been calculated in milligrams of gallic acid equivalent (GAE) per 100grams of sample (mg GAE/100g).

The flavonoid content was measured using the aluminium chloride assay method. In brief, an aliquot (0.5mL) of lemon peel extract had been added to an 8 mL test tube holding two millilitres of distilled water. 0.15mL of 5% NaNO2 was introduced to each test tube. After 5 minutes of incubation, 0.15mL of 10% AlCl3 was added. Following a minute, 1millilitre of 1 M NaOH was added, and the volume was then adjusted using distilled water (5ml). The resultant solution's absorbance had been determined at 510nm for approximately 10minutes. The total flavonoid content of the collected samples has been determined as mg of quercetin equivalent per 100g of sample (mg CE/100g sample) using catechin as the reference. Each sample underwent three separate analyses.¹¹

Antioxidant assay:

The antioxidant activity of the lemon peel extract was measured using the DPPH technique with slight alterations. A tiny quantity of methanol was employed to disperse 20mg of DPPH in a 500mL volumetric flask. The flask had been filled entirely with methanol once the DPPH had completely dissolved. Methanol (3ml) and DPPH (2ml) solutions were combined to create the control. A stock solution of ascorbic acid was also made by dissolving 50mg of the acid in 100millilitres of methanol. By creating several concentrations from the stock solution, the calibration curve may be generated.¹²

Three millilitres of normal ascorbic acid and two millilitres of DPPH solutions were added to each test tube. The test tubes were subsequently sealed with aluminium foil and left in the dark for thirty minutes. The lemon peel extracts were divided into fractions of 0.1, 0.2, 0.4, 0.8, 1.6, and 2.4mL for the samples. A final amount of eighty percent aqueous methanol was incorporated into each solution to increase the total volume to 4mL. The combination spent thirty minutes in the dark. Finally, the absorbance was determined to be 517nm.¹²

Denaturation assay:

For this experiment, 0.10ml of lemon peel extract was used at a concentration of 31.25–1000μg/ml. Mix with 2.40ml of Ovalbumin (OVA, 4000µg/ml) solution. After being produced, 1N hydrochloric acid at pH 6.3 was added to the extract solution. The solution was then incubated for 20minutes at 37°C. After that, the reaction mixture was heated for one minute to 71°C. Each sample received 2.5mL of PBS, which has a pH of 6.3, after the freezing solution. Using a UV spectrophotometer, the absorbance of the final reaction mixture was determined at 660nm.¹³ The proportion of protein denaturation inhibition has been calculated using the following formula, and the product control was made with OVA.

where test is mean absorbance of lemon peel extract and reagents and control is mean absorbance of solvents and reagents without extract.

Antimicrobial activity:

Utilising the disc diffusion method (also known as the Kirby-Bauer method) (Bauer et al. 1966), the antimicrobial capacity of a crude extract of lemon peel was evaluated. Multiple dilutions of the lemon peel extract, that ranged from 4 mg/disc to 0.008 mg/disc, were inserted onto sterile commercial blank discs with a 6.0 mm diameter. The discs were kept at -5°C before being used. A turbidometer was used to modify the overnight broth cultures so that they produced about 10⁸ CFU/ml. Discs impregnated with lemon peel extract (20μl) were arranged on agar plates and incubated for a full day at 37°C. Vancomycin discs (30μl) served as the positive control, while pure DMSO (20μl) served as the negative control. The clear zone of inhibition was then measured to the closest millimetre (mm) in order to assess the antibacterial activity.¹⁴

RESULTS:

Total phenolic and flavonoid content calculation:

In this study, we measured the total phenolic and flavonoid content, which varied widely among the samples and ranged from 218.12 to 678.22mg GAE/g and 8.06 to 171.37 mg quercetin/g of fresh weight. The studies revealed the comparative amount of phenolic and flavonoid content from the standard sample (ascorbic acid) with lemon peel extract. It shows a decrease in absorbance of flavonoid content in the presence of different concentrations of lemon peel extract, and this may indicate that lemon peel extract possessed antioxidant capacity.

Antioxidant activity:

In Fig. 2, lemon peel extract effectively scavenges DPPH radicals at a concentration of 2000µg/ml (65.71%), compared to ascorbic acid (2000µg/ml, standard). While ascorbic acid's (P<0.001) antioxidant potential was found to be higher than that of lemon peel extract (P<0.01), the study did find that lemon peel extract exhibited significant antioxidant activity; this could be explained by the extract's higher observed antiradical properties, which are primarily caused by phenolic compounds, including phenolic hydroxyls.

Table 1: Total phenolic and flavonoid contents of aqueous extract of lemon peel extract

Lemon peel, aqueous extract (Doses)	Total phenolic mg GAE/g extract	Total flavonoids mg quercetin/g extract
62.5 µg/ml	218.12 ± 4.94	8.06 ± 1.78
125 µg/ml	292.45 ± 6.92	35.07 ± 3.04
250 µg/ml	381.32 ± 8.28*	61.6 ± 4.82
500 µg/ml	491.37 ± 6.18**	117.38 ± 8.16*
1000 µg/ml	571.38 ± 9.26**	148.81 ± 7.94**
2000 µg/ml	678.22 ± 10.84**	161.37 ± 8.06**
Ascorbic acid, 2000 µg/ml	797.28 ± 9.34***	208.74 ± 7.44***

One-way ANOVA test performed, *P<0.05; **P<0.01 and ***P<0.001

Protein denaturation assay:

Lemon peel extract suppressed protein denaturation (ovalbumin) in a concentration-dependent way throughout 31.25–1000μg/ml (Fig. 3). Diclofenac sodium (in the dosage range of 50-2000μg/ml) was utilised as the reference medication, which likewise displayed dose-dependent suppression of protein denaturation. The higher absorbance of lemon peel extract compared to the control suggests that an increased dosage has a stabilising effect on proteins (denaturation is slowed). It follows that purification of these crude extracts will undoubtedly result in a large rise in pharmacological activity, which may even approach that of the reference medication.

Fig. 3: Protein denaturation assay of lemon peel extract. Values were expressed in the form of Mean±S.E using one-way ANOVA test (*P<0.05; **P<0.01 and ***P<0.001).

Antimicrobial activity:

At concentrations ranging from 4mg/disc to 0.25 mg/disc, lemon peel extract inhibited the growth of Pseudomonas aeruginosa and Staphylococcus aureus, with diameters of 15.1, 14.2, 13.5, 10.4, and 9.4mm for Pseudomonas aeruginosa and 4 mm, 13.6, 11.4, and 10.2mm for Staphylococcus aureus. Since no inhibition zones were seen, the growth of E. coli was not suppressed by lemon peel extract. Vancomycin, the positive control, exhibited inhibition diameters ranging from 16.0 to 22.0mm, whereas the DMSO negative control likewise did not exhibit any inhibitory effect.

DISCUSSION:

The Rutaceae family includes the adaptable citrus plant. In the genus Citrus, there are about 140 genera and 1300 species. The word for lemons is derived from the ancient French word "limon". There are numerous distinct names for lemons available. The majority of the phenols and flavonoids in citrus fruits have been discovered to originate from the peel, which makes up about half of the fruit mass. Of the phenolic chemicals found in various plant components, flavonoids make up the biggest category and can exist in both free and glycoside forms.^3-5 In the literature, plant materials with an aromatic structure that has one or more hydroxyl groups on it are known as phenolic compounds. There are around 8000 naturally occurring plant phenolics, half of which are flavonoids. The majority of the antioxidant activity found in plants or plant-derived products is attributed to the biggest class of phytochemicals, known as phenols. It has been discovered that they possess a wide range of biological properties, such as anti-inflammatory, anti-ulcer, anti-arthritic, antiangiogenic, and protein kinase inhibition.^13,14 The flavonoids are composed of two benzene rings bridged by a propane unit. Flavones and flavonols are probably the most broadly dispersed of all the phenols. Due to their antioxidant properties, flavonoids are especially helpful in preventing cardiovascular disease, some types of cancer, and age-related cellular component deterioration. They can scavenge harmful free radicals such super oxide and hydroxyl radicals because of their polyphenolic content. In this study, we determined the total phenolic and flavonoid content and also analysed its anti-oxidant and antimicrobial activity.

Several techniques have been used to evaluate antioxidant activity in vitro for the purpose of enabling fast screening of compounds, as molecules that have low antioxidant activity in vitro will probably have minimal effect in vivo. It is well recognised that free radicals contribute significantly to a broad range of clinical symptoms. Free radicals are opposed by antioxidants, which also shield us from a number of illnesses. They work by scavenging reactive oxygen species or safeguarding our defence systems from antioxidants. The capacity of natural products to donate electrons may be tested using DPPH purple-coloured solution bleaching. The technique works by scavenging DPPH through decolorizing the DPPH solution following the addition of a proposed antioxidant, i.e., lemon peel extract. The strength and concentration of the antioxidants are correlated with the degree of colour change.¹² The substance being tested exhibits significant capacity to neutralise free radicals, assuming there is a big drop in the absorbance of the reaction mixture. The results of this investigation indicate that the extract from lemon peels had a much greater inhibitory percentage and a positive correlation with the total phenolic and flavonoid content.

More than 80% of people worldwide rely on traditional medicine for their basic healthcare requirements, according to the World Health Organisation (WHO). Plants possess chemical compounds that have specific physiological effects on humans, which give them their therapeutic worth. The antibacterial properties of naturally occurring antimicrobial compounds and plant extracts, particularly citrus species, are widely studied using disc diffusion techniques.^8-10 Utilising discs as reservoirs for the substance solution under investigation is the foundation of these assays. In summary, the extract from lemon peel showed encouraging antibacterial properties by preventing the development of bacterial infections.

CONCLUSION:

To extract the citrus plant (lemon) fractions that inhibit bacterial activity and isolate the antimicrobial component using fractionation guided by bioassay, further research has to be done. Subsequently, the structure of fractions that inhibit bacterial activity and boost antioxidant activity—which is directly connected with total phenolic and flavonoid content—should be examined using nuclear magnetic resonance (NMR) and C13 investigations.

ACKNOWLEDGEMENT:

We gratefully acknowledge the support of the Department of Biotechnology (DBT), Government of India, through the DBT Builder Program and seed grant (UoJRF No. DRS/24/4889-94). This support was instrumental in facilitating the research and development activities for this project.

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Received on 19.09.2024 Revised on 14.01.2025

Accepted on 23.04.2025 Published on 08.11.2025

Available online from November 13, 2025

Research J. Pharmacy and Technology. 2025;18(11):5245-5250.

DOI: 10.52711/0974-360X.2025.00756

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