Antioxidant potential and quality benchmarking of commercial fennel seeds

Introduction

Fennel (Foeniculum vulgare Mill.) is a species belonging to the Apiaceae family, which is the largest and widely distributed plant family mainly in Asia, the Mediterranean region, and parts of Europe (Brahmi et al., 2013; Badgujar et al., 2014) [2,1] . Historically, fennel seeds have been used not only as spices but also as digestive stimulants and folk medicine (Noreen et al., 2023) [6] . Their efficacy in the treatment of diseases has been mainly attributed to the presence of volatile oils, phenolic acids, flavonoids, and other secondary metabolites which have been chemically and biologically characterized, therefore, demonstrate antioxidant, antimicrobial, and anti-inflammatory activities (Fadavi et al., 2022; Noreen et al., 2023) [3,6] . In fact, oxidative deterioration caused due to food exposure to the environment is a major toil to food systems that results in loss of food quality and safety in terms of sensory, nutritional, and health properties. Phenolics and flavonoids are the most important components of the plant defense mechanism involved in the neutralization of free radicals, chelation of metal ions, and electron donation (Noreen et al., 2023) [6] . Various authors have studied the antioxidant effects of fennel seed extracts by means of through in vitro model systems like DPPH and FRAP (Goswami & Chatterjee, 2014; Noreen et al., 2023) [5,6] . The different varieties and characteristics of the plant, its geographical origin, the time of harvest, the solvents used for extraction, and the analytical techniques employed, among other factors, contribute to the wide range of antioxidant values reported in the literature (Noreen et al., 2023) [6] . Hence the lack of consistency between research findings makes it difficult to compare one another and at the same time, it indicates the necessity of a standardized method for the evaluation of fennel seed quality (Brahmi et al., 2013) [2] .

International Journal of Agriculture and Food Science https://www.agriculturaljournals.com

Besides the antioxidant capacity, the physicochemical properties such as the moisture content, ash value, volatile oil yield, mineral impurities, and color features are very important for the quality, shelf life, and powder product.ion of spices (FSSAI, 2011; Noreen et al., 2023) [6,21] . Instrumental colour evaluation of commercially available fennel seed powder is scarcely reported in the literature. The present study uniquely documents quantitative L*, a*, and b* colour parameters, thereby establishing objective visual quality benchmarks for fennel seed powder. These colour attributes provide valuable indicators of raw material freshness, degree of processing, and market quality, complementing chemical and antioxidant assessments. To date, only a handful of works have investigated the association between the antioxidant potential and quality characteristics of fennel seeds in the Indian market even though India is the largest producer and consumer of spices.

Fulfillment of this research gap would be of benefit to both academic and industrial sectors in the efficient utilization of fennel as a natural functional ingredient.

Objectives of the Study

 To quantify the total phenolic and flavonoid content of commercially available fennel seed powder from Indian markets;  To evaluate its antioxidant capacity using DPPH radical scavenging and ferric reducing antioxidant power assays;  iii) To assess key physicochemical and colour attributes relevant to quality, purity, and storage stability; and

Materials and Methods

Materials

Fennel (Foeniculum vulgare Mill.) seed of a commercial brand under the provisions of FSSAI was bought off the local market of Anand (Gujarat, India). Seeds were decontaminated and then ground to powder by electric grinder. To get a uniform particle size, the powdered material was passed through a standard 35-mesh sieve. The sieved powder was stored in airtight containers at refrigeration temperature until further analysis.

Preparation of Fennel Seed Extract

For extract preparation, 2.5 g fennel seed powder was added in 50 ml of methanol-water (4:1, v/v) solvent and kept in a water bath at 37°C for 24 h as described by Murtaza et al.

(2021) and Anwar et al. (2009) [9,. 10] . After 24 h, the content was filtered using Whatman No. 1 filter paper to separate the solid particles. The filtrate (methanolic extract) obtained was ten times diluted and used for the determination of the total phenolic and total flavonoids content as well as antioxidant activity by DPPH assay and FRAP assay.

Determination of Total Phenolic Content

The analysis of the total phenolic content in fennel seed extract was conducted using the Folin-Ciocalteu reagent, as described by Singleton and Rossi (1965) [11] . In a test tube, 0.1 ml of fennel seed extract was taken out, and the volume was then made to 1 ml with distilled water. Subsequently, the addition of 0.5 ml of each of the diluted Folin Ciocalteau reagent (1:1) and 10 ml of 7.5 per cent sodium carbonate was carried out. The content was mixed using a vortex mixer and incubated in the dark at room temperature for 30 min. For blank preparation, distilled water was used instead of the extract. The absorbance of the samples was measured against a blank at 750 nm using a spectrophotometer. The gallic acid standard curve was prepared using a known concentration of gallic acid (10-120 μg/ml) in a test tube and volume was made up to 1 ml with distilled water. Total phenolic content of sample was calculated based on the absorbance using equation of standard curve (Figure 1):

Where, y is absorbance at 750 nm, x is concentration in μg of gallic acid The result was expressed in terms of mg of gallic acid equivalent (GAE)/g of fennel seed powder.

Determination of Total Flavonoid Content

For the determination of total flavonoids content aluminum chloride method was used as described by Hayat et al.,

(2019) [13] with some modifications. To prepare the sample, 1 ml of the diluted fennel seed extract was measured and transferred into a 10 ml volumetric flask. Subsequently, 4 ml of distilled water was added to the flask. After that, 0.3 ml of a 5 per cent sodium nitrite solution was added to the flask. After allowing the mixture to react for 5 minutes, 0.3 ml of a 10 per cent aluminum trichloride solution was added to the flask. After that, 2 ml of a 1 M sodium hydroxide solution was added to the mixture. The volume of the flask was then made up to 10 ml using distilled water. The absorbance of the resulting solution was measured at a wavelength of 510 nm using a spectrophotometer.

In this method, a standard curve (Figure: 2) was prepared using quercetin as the standard. The flavonoid contents were then expressed as quercetin equivalents. The standard curve was prepared by using various concentrations of standard quercetin ranging from 0 to 500 µg using the same method instead of fennel seed extract in determining the total flavonoids content. Based on absorbance, the total flavonoids content of samples was calculated using equation of standard curve: y = 0.0005x + 0.0403 Where, y is absorbance at 510 nm, x is concentration in μg of quercetin. The results were expressed in terms of mg of Quercetin equivalent/g of fennel seed powder.

DPPH Radical Scavenging Activity

The radical-scavenging activity of spice extracts was evaluated by assessing their capacity to scavenge 2,2diphenyl-1-picrylhydrazyl (DPPH) radicals, according to the procedure of Brand Williams et al. (1995) [12] with some modification as given by Saeed et al. (2009) [14] . 1 ml of a diluted fennel seed extract was transferred to a test tube. Then, 3 ml of a 0.15 mM methanolic solution of 2,2diphenyl-1-picrylhydrazyl (DPPH) was added to the test tube, and the mixture was shaken using a vortex mixer. Subsequently, the test tubes were placed in a water bath at 37°C for 30 minutes. For the control, 1 ml of methanol was used instead of the fennel seed extract. The absorbance of the reaction mixture was then measured at a wavelength of 517 nm using a spectrophotometer, with the blank serving as the reference. Radical-scavenging activity was calculated as the percentage inhibition using the following formula: Radical-scavenging activity

Where, Ac = absorbance of control,

A = absorbance of sample

A standard Trolox curve was prepared using various concentrations of standard antioxidant (Trolox) in the range of 0 to 40 µg and using the same instead of fennel seed extract in the method for determining the radical scavenging activity by DPPH assay. Antioxidant activity of samples was calculated based on absorbance using the equation of standard curve: y = 2.5882x -2.9861 Where, y is per cent inhibition, x is concentration in μg of Trolox

The results were expressed in terms of mM Trolox equivalent/g of fennel seed powder.

Ferric Reducing Antioxidant Power (FRAP) Assay

The FRAP assay was measured as per the method given by Benzie and Strain (1999) [15] . Ninety microliters of diluted methanolic extract were mixed with 2.7 ml of FRAP reagent solution in 10 ml test tubes. The absorbance of the solution was measured at 595 nm using a spectrophotometer. Based on the equation determined from the standard curve, the results were expressed as mM Fe2+ per g of fennel seed powder. The standard curve was plotted by taking absorbance on Y-axis and varying from 200 to 1000 µM concentration of Fe2+ on X-axis. The reduction of absorbance was measured at 595 nm against a blank (methanol instead of sample) using a spectrophotometer.

Physicochemical Analysis

The gravimetric technique was used to determine the moisture content of fennel seed powder as described by Bukhari. Moisture content of fennel seed powder was determined by the gravimetric method using a hot air oven maintained at 102 ± 2 °C. Drying was continued until a constant weight was obtained, and moisture content was expressed on a percentage basis.

Ash and Ash Insoluble in Dilute HCl:

The ash and Ash Insoluble in Dilute HCl content of fennel seed powder was measured using the method prescribed in FSSAI (2021) [17] .

Volatile Oil Content

The volatile oil from fennel seed powder was extracted using the method described in FSSAI (2021) [17] . To obtain the total volatile oil insoluble in water, steam distillation was performed using Clevenger's apparatus. The apparatus consists of a round bottom flask, a separation tube, a wet condenser, and a heating mantle. First, 50 g of fennel seed powder was accurately weighed and transferred to a flask. Approximately 500 ml of distilled water was then added to the flask. For the extraction of fennel seed, the Clevenger asS.Em.bly was employed, which separates the volatile oil (which is lighter than water). The apparatus was cooled to room temperature by immersing it in a water bath. The content of volatile oil was calculated using equation as follows:

Volatile oil (% v/w) = Volume of volatile oil (ml) Weight of sample (g)

The volatile oil content is expressed as aper centage (v/w).

Colour Measurement

The colour value of fennel seed powder was measured by chroma meter. First calibration of chroma meter was done on white calibration plate provided by the manufacturer along with the instrument. For the analysis of sample, the appropriate amount of powder was taken in glass petri dish and colour measurement was performed according to the three-colour coordinates (L*, a*, b*) with chromameter with in the place with minimum interference of other light sources. The a* value indicates colour ranging from red (+) to green (-), the b* value represents colour ranging from yellow (+) to blue (-), and the L* value denotes sample luminance, which ranges from black (0) to white (100).

Statistical Analysis:

The obtained results were subjected to statistical analysis using Analysis of Variance (ANOVA) at a significance level of 5 per cent. For analyzing the data of each attribute under study, a statistical analysis was performed using the completely randomized design.

Results and Discussion:

In the present study the authors have worked out a detailed investigation of antioxidant potential, physicochemical quality and colour attributes of fennel seed powder, which may be very useful for the development of functional foods and natural antioxidant sources. The present work is a great demonstration of how different functional; compositional and technological parameters of the material can be correlated and evaluated simultaneously.

The Total Phenolic Content and Total Flavonoid Content

The total phenolic content (TPC) and total flavonoid content (TFC) of fennel seed powder were determined as 9.55 ± 0.23 mg GAE g⁻¹ and 7.76 ± 0.28 mg QE g⁻¹, respectively (Table 1). These data attest that fennel seeds are a potentially good source of phenolic antioxidants which are known for their activity in radical scavenging and inhibition of oxidative stress. The TPC value obtained has well matched with the phenolic content of fennel extracts previously reported. Liu et al., (2008) [18] stated a content of 7.54 mg g⁻¹ phenolics in ethanolic fennel extract and Murtaza et al., (2021) [9] registered TPC being in the range of 1.02 -1.26 mg GAE g⁻¹ depending on the extraction solvent used. On the other hand, the TFC figures determined here are within the wide range reported by different authors, although direct comparisons at the absolute level are impeded by the fact that different factors such as cultivar, agroclimatic condition, extract solvent polarity and analytical procedures used might have been variable. With the phenolic and flavonoid contents quantified, it can be inferred that the sample of fennel seed powder analyzed in this study had a phytochemical composition matching those of reference high-grade quality fennel materials thus confirming its nutraceutical potential. The Antioxidant Activity The antioxidant capacity of fennel seed powder was determined by DPPH radical scavenging and ferric reducing antioxidant power (FRAP) assays with the results summarized in Table 2. The DPPH radical scavenging activity was found to be 6.79 ± 0.24 mM Trolox equivalents g⁻¹, whereas the FRAP value was 16.88 ± 0.72 mM Fe²⁺ g⁻¹. These findings highlight the potent antioxidant nature of fennel seed powder and align with previous studies that have shown a high level of radical scavenging and reducing abilities in fennel extracts and essential oils. Bilawar et al., (2022) [19] observed a DPPH inhibition of about 56.9% and high FRAP values for fennel oil, attributing the antioxidant properties mainly to trans-anethole, fenchone, and estragole.

In the same vein, Noreen et al., (2023) [6] illustrated strong antioxidant activity in fennel extracts from both aqueous and organic solvents, with considerable changes depending on the polarity of the solvent used. The simultaneous use of DPPH and FRAP tests gives a comprehensive view of how antioxidants work. Saxena et al., (2016) [20] investigated the essential oil of ninety-one fennel samples gathered from different regions in India. They found that the essential oil content ranged between 1.0 and 3.3 per cent. The main compounds of the essential oil were anethole, α-pinene, βpinene, myrcene, camphene, cymene, 4-allyl anisole, estragol, geranyl acetate, γ-terpinene, and p-anisaldehyde. The corroboration of the assay results in the present work indicates that both hydrogen atom transfer and single electron transfer pathways, most likely phenolic acids and flavonoids, contribute to the antioxidant activity in fennel seeds. [6,21] were close to ours, thus indicating the present sample's stability. The total ash content determined on dry matter basis was 8.74 ± 0.24% and that talks about the mineral content of the fennel seeds and is also consistent with the literature data. The ash insoluble in dilute HCl was very low (0.074 ± 0.005%), which means that the presence of extraneous inorganic matter such as sand, soil, and that the whole process from harvest to packaging was well done and of good quality. The volatile oil, which is a main quality factor for fennel since it determines the aroma and also has a bioactive property, concentration was 1.56 ± 0.05% (v/w) and is within the range of values reported in the literature for the fennel seeds of the right maturity stage. According to the Food Safety and Standards Authority of India (FSSAI), 2023 specifications for fennel (Saunf) powder, the permissible limits include a moisture content not exceeding 12.0% (w/w), total ash (on a dry basis) not more than 9.0% (w/w), acid-insoluble ash (on a dry basis) not exceeding 2.0% (w/w), and a volatile oil content (on a dry basis) of not less than 1.0% (v/w). The results obtained in the present study were within the prescribed FSSAI limits, indicating good physicochemical quality and compliance with food safety standards. El-Gamal and Ahmed (2017) [22] studied the effect of different maturity stages on the essential oil content of fennel. They found that depending on the maturity level the essential oil content of the fennel varied between a low of 1.21 per cent and a high of 2.85 per cent.

The essential oil content differences in the past research were explained by the variation in genotype, environmental factors, and extraction methods while the oil content we determined depicts the good quality raw material and favorable processing conditions.

Colour Characteristics

The instrumental colour analysis of the fennel seed powder (Table 4) revealed an L* value of 62.23 ± 0.62, indicating comparatively high lightness, while the negative a* value (-5.91 ± 0.16) reflects a greenish hue and the elevated b* value (33.10 ± 0.42) denotes a strong yellow component.

Collectively, these values define the characteristic greenishyellow colour associated with high-quality fennel seed powder. Fennel seed powder typically exhibits a greenishyellow to light brown color. This hue arises primarily from natural pigments and compounds inherent in fennel seeds (Foeniculum vulgare), influenced by factors like ripeness and processing (Badgujar et al., 2014) [1] . Fresh fennel seeds are greenish-yellow due to residual chlorophyll, transitioning to yellowish-brown tones from carotenoids and other phytochemicals as they mature and dry into powder. Powdered form retains these traits, appearing greyish-brown to grayish-yellow, as confirmed in phytochemical screenings of seed powder (Alam et al., 2019) [24] . Notably, quantitative instrumental colour data for commercially available fennel seed powder remain scarcely reported in the literature, particularly in the Indian context. In this regard, the present findings provide one of the few objective colour benchmarks for commercial fennel seed powder, offering reproducible indicators of raw material freshness, processing quality, and market acceptability. The inclusion of instrumental colour evaluation alongside antioxidant and physicochemical parameters strengthens the comprehensive quality assessment and enhances the practical relevance of the study for functional food formulation and spice standardization.

References

1. Brand-Williams W, Cuvelier ME, Berset C. Use of a free radical method to evaluate antioxidant activity. LWT Food Sci Technol. 1995;28(1):25-30.
2. Hayat K, Abbas S, Hussain S, Shahzad SA, Tahir MU. Effect of microwave and conventional oven heating on phenolic constituents, fatty acids, minerals and antioxidant potential of fennel seed. Ind Crops Prod. 2019;140:111610. DOI:10.1016/j.indcrop.2019.111610.
3. Saeed MK, Ejaz N, Ahmed I, Ashraf M, Iqbal I, Khokhar ZU. Nutritional evaluation and DPPH assay of Pakistani variety of fennel (Foeniculum vulgare). In: Proc Int Conf Complex Med Eng. IEEE; 2009. p. 1-4.
4. Benzie IFF, Strain JJ. Ferric reducing/antioxidant power assay. In: Methods Enzymol. 1999;299:15-27. doi:10.1016/S0076-6879(99)99005-5.
5. Bukhari H, Shehzad A, Saeed K, Sadiq BM, Tanveer S, Iftikhar T. Compositional profiling of fennel seed. Pak J Food Sci. 2014;24(3):132-136.
6. Food Safety and Standards Authority of India (FSSAI). Manual of Methods of Analysis of Foods: Spices and Condiments. New Delhi: Ministry of Health and Family Welfare; 2021.
7. Liu H, Qiu N, Ding H, Yao R. Polyphenols content and antioxidant capacity of 68 Chinese herbals suitable for medical or food uses. Food Res Int. 2008;41(4):363370. DOI:10.1016/j.foodres.2007.12.012.
8. Bilawar SS, Sree KP, Thamburaj S, Palanivel R, Kalakandan SK. Chemical composition and antioxidant activity study of cumin and fennel oil. Pharma Innov J. 2022;11(8):207-213.
9. Saxena SN, Kakani RK, Rathore SS, Meena RS, Vishal MK, Sharma LK, Singh B. Genetic variation in essential oil constituents of fennel (Foeniculum vulgare Mill.) germplasm. J Essent Oil Bear Plants. 2016;19(4):989-999. DOI:10.1080/0972060X.2016.1191378.
10. Faten AE, Farag M, Hanan AK. Chemical analysis of fennel seeds, basil leaves and their essential oil contents. J Biol Chem Environ Sci. 2011;6(2):127-145.
11. El-Gamal S, Ahmed HMI. Influence of different maturity stages on fruit yield and essential oil content of fennel (Foeniculum vulgare Mill.). J Plant Prod. 2017;8(1):127-133. doi:10.21608/jpp.2017.37825.
12. Food Safety and Standards Authority of India (FSSAI). Food Safety and Standards (Food Products Standards and Food Additives) Regulations, 2011: Chapter 2.9. New Delhi: FSSAI; 2023.
13. Alam P, Abdel-Kader MS, Alqarni MH, Zaatout HH, Ahamad SR, Shakeel F. Chemical composition of fennel seed extract and determination of fenchone in commercial formulations by GC-MS method. J Food Sci Technol. 2019;56(5):2395-2403. DOI:10.1007/s13197-019-03695-9.