EFFECT OF GERMINATION ON POLYPHENOL CONTENTS AND ANTIOXIDANT ACTIVITY IN MUSTARD SEEDS (BRASSICA NIGRA) GROWN IN ETHIOPIA

EFFECT OF GERMINATION ON POLYPHENOL CONTENTS AND ANTIOXIDANT ACTIVITY IN MUSTARD SEEDS (BRASSICA NIGRA) GROWN IN ETHIOPIA

Efrem, R. Negussie, B. Sekwati-Monang, R. I. Kobue-Lekalake, T. Selebatso, M. R. Setlalekgomo and G. D. Haki *

Botswana University of Agriculture and Natural Resources, Gaborone, Botswana.

ABSTRACT: Mustard seeds, in some countries, are used as a traditional medicine for the treatment of snakebite, toothache, and epilepsy, as well as to treat skin diseases because of their high sulphur content. The aim of this study was to investigate the effect of three germination periods on the polyphenol contents and antioxidant activity of mustard seeds. Phenolic compounds were determined by the Folin-Ciocalteau method, while antioxidant activity was determined using standard methods. The results indicated that the total phenol and flavonoid contents of the one, two, and three days germinated mustard seed were increased by 16%, 17%, 24% and 500%, 647%,747%, respectively. Antioxidant activity determined by reducing power assay (for one, two and three days of germinated mustard seed) also increased by 10%, 34%, 51%, respectively. Germination is thus a better technique to enhance the nutritional and pharmaceutical potential of mustard seed and can be used in the development of functional diets.

Keywords: Germination, Brassica nigra, Polyphenol contents, Antioxidant activity

INTRODUCTION: The mustard groups are formed by three species of the genus Brassica of the Cruciferae family: Brassica nigra, Brassica juncea and Brassica carinata. The use of the seeds in the food and beverage industry is immensely growing due to their nutritional and functional properties as they serve as a source for isothiocyanates which are responsible for the specific flavor of mustard ¹.

Ethanolic extract of B. nigra exhibited anti-proliferative activity in A549 and H1299 human lung cancer cells, probably through the induction of apoptosis and control of cell cycle through replication stress, resulting in fork-collapse DNA lesions ². Reports indicated the antimetastatic potential of B. nigra extract as it showed anti-migratory and anti-invasive activities, perhaps through down regulation of MMP2, MMP9, Snail, and E-cadherin ².

It contains unsaturated fatty acids (oleic, linoleic, and erucic acid), and its consumption may prevent memory loss caused by β-amyloid ³. The viscous liquid present in the seeds of B. nigra contains 25%–30% fat, sinapine, and singrin named glycoside and myrosin ⁴.

The Methanolic crude Extract (MCE) of B. nigra revealed the occurrence of alkaloids, flavonoids, tannin, diterpenes, glycosides, carbohydrates, phenols, fixed oils and fat. The fractions confirmed its anthelminthic and anxiolytic activity comparable to the standard drug diazepam 5 and its antidiarrheal potential was associated with flavonoid and tannin contents ⁶. Mustard seed is a good source of protein, fiber, minerals, vitamins, antioxidants, and phytonutrients, thus exhibiting several health benefits, including antimicrobial, antibacterial, anti-diabetic, and antimalarial ⁷.

In Ethiopia, the mustard seed brassica nigra grows in many parts of the country. The paste of grounded seeds of brassica nigra is commonly used as a traditional pungent spice for consuming raw and roasted beef. Most people in the country favour this spice for its sharp pungent taste. Mustard seeds do not have the pungent flavour naturally but is developed when the seeds are ground and steeped in water. Mustard leaf extracts possess antioxidant properties as they scavenge free radicals. This was demonstrated by a DPPH assay and can be attributed to the bioactive isothiocyanates present in mustard ⁸.

Leaves, fruits, and roots of Ethiopian Brassica nigra are used orally to treat wounds ⁹. Seeds collected from the northern part of Ethiopia showed good chemosuppressive and moderate chemo-prophylactic antimalarial activities against Plasmodium berghei infection in mice suggesting its potential for preventing malaria ¹⁰.

Germination of seeds is a process that starts with dry seeds imbibition of water and completes when the seeds embryonic axis gets longer. During this process, the seeds have manipulated the reserves within the storage tissues of the seed to support seedling growth and develop protective responses through the synthesis of phenolic and other compounds ¹¹. Germination enhanced several bioactive compounds such as γ-amino butyric acid, polyphenols, and vitamins, leading to greater bioactivity such as anti-diabetic, anti-bacteria, and anti-cancer effects while consuming the seeds ¹². It is also used for the reduction of antinutritive compounds such as tannins and phytates ¹¹. Germination also affects various legumes, cereals, and some vegetables ^13-18.

MATERIALS AND METHODS:

Materials: The mustard seeds were purchased from Merkato market in Addis Ababa city, Ethiopia. The plant species used were selected according to documented traditional use acquired through personal interviews with the indigenous communities. Chemicals and Reagents used were ethanol, methanol, sodium hypochlorite, potassium ferricyanide, ascorbic acid, trichloroacetic acid, ferric chloride, Trolox (6-hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid), DPPH (2,2-diphenyl-1-icrylhydrazyl), ABTS (2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) and Folin–Ciocalteu reagent and all were of analytical grade.

Germination and Powder Preparation of the Mustard Seeds: The mustard seeds were first disinfected based on the procedures used by Cevallos-Casals et al. ¹¹. Briefly, 500g mustard seeds were sterilized with 70% ethanol for 2.5m, followed by the addition of 2.5% sodium hypochlorite for 15m. Ethanol and sodium hypochlorite were removed by rinsing with sterile water. After disinfection, mustard seeds were allowed to imbibe water for 24h at room temperature.

Then water was removed, and seeds were dark-germinated in sterile plates with humidified cotton at 28^oC. The cotton was kept moist by spraying with sterile water as needed. After one, two and three days of germination periods the germinated mustard seeds were dried at 40^oC in an oven for 48 h. The dried germinated mustard seeds were milled and the flour were passed through a sieve of 0.5mm. The powder of germinated mustard seeds was kept in a dark brown glass bottle and stored in a refrigerator until extraction.

Extraction of the Powder of Mustard Seeds: The powder of raw and germinated mustard seeds was extracted following the procedure used by Zakia et al. ¹⁹. Briefly, 5g of mustard seeds powder was extracted by stirring with 50 mL of methanol/water (4:1) at 25°C for 24 h using a temperature shaker incubator (ZHWY-103B) and then filtered through Whatman No. 4 paper. The residue was then extracted with two additional 50 mL portions of methanol/water. The combined methanol/water extracts were evaporated at 40°C to dryness using a rotary evaporator (Stuart R3300) and re-dissolved in methanol/water at the 10 mg/mL concentration and stored in a dark brown glass bottle at 4°C for further use.

Analysis of the Antioxidant Activity:

Determination of DPPH Free Radical Scavenging Activity: The effect of methanol/water (4:1) extracts on DPPH radical was estimated according to Kirby and Schmidt ²⁰. A 0.004% solution of DPPH radical solution in methanol was prepared and then 4 mL of this solution was mixed with 1mL of the various concentrations (12 – 416 µg/mL) of the extracts in methanol/water. Finally, the samples were incubated for 30m in the dark at room temperature. Scavenging capacity was read in a spectrophotometer (Perkin Elmer Lamda 950 UV/Vis/NIR) by monitoring the decrease in absorbance at 517nm. A standard curve was prepared with ascorbic acids. Results were given as mg ascorbic acids equivalents per g dry weight (mg AE/g). All tests were performed in triplicate.

Determination of ABTS Free Radical Scavenging Activity: The ABTS free radical scavenging activity was determined based on the method developed by Re et al ²¹. A stable stock solution of ABTS^•+ was produced by reacting a 7 mM/L aqueous solution of ABTS with 2.45 mM/L potassium per sulphate (final concentration) and allowing the mixture to stand in the dark at room temperature for 12–16 h before use. ABTS^•+ solution (3.9 mL) was added to 0.1 ml of the test samples and mixed thoroughly. The blank was 3.9 mL of ABTS^•+ solution with 0.1mL of 80% ethanol. The reactive mixture was allowed to stand at room temperature for 6m and the absorbance was immediately read with a spectrophotometer (Perkin Elmer Lamda 950 UV/Vis/NIR) at 734 nm. A standard curve was prepared with Trolox. Final results were given as mM Trolox equivalents per g dry weight (mM TE/g). All tests were performed in triplicate.

Determination of Total Reducing Power: Total reducing power was carried out according to the method established by Oyaizu ²². One milliliter of different concentrations (31.25 – 500 μg/mL) of mustard seeds extract was mixed in a test tube with a 2.5 mL of 0.2M phosphate buffer (2.5 mL), pH 6.6 and 2.5 ml of 1% potassium ferric cyanide. The mixture was incubated at 50°C for 20 min to reduce ferric cyanide into Ferrocyanide. Then 2.5 mL of 10% trichloroacetic acid was added to the mixture and centrifuged at 6,000 rpm for 10 m. The upper layer (2.5 mL) was transferred into another tube, mixed with 2.5 mL deionized water and 0.5mL ferric chloride (0.1%), and left to react for 10 m. Finally, the absorbance of the reaction mixture was measured at 700 nm using a UV-VIS spectrophotometer (Perkin Elmer Lamda 950 UV/Vis/NIR). A standard curve was prepared with ascorbic acids. Final results were given as mg ascorbic acids equivalents per g dry weight (mg AE/g). All tests were performed in triplicate.

Analysis of the Polyphenol Contents:

Determination of Total Phenolic Content: The total phenolic content of the extract was determined by the modified Folin-Ciocalteu method used by Afolayan et al. ²³. The extract (1mL) was mixed with 2.5mLFolin-Ciocalteu reagent (previously diluted with distilled water 1:10 v/v) and 2mL (75 g/L) of sodium carbonate. The mixture was vortexed for 15s and allowed to stand for 30m at 40°C for colour development. The absorbance was measured at 765 nm using a UV-VIS spectrophotometer (Perkin Elmer Lamda 950 UV/Vis/NIR). Total phenolic content was expressed as mg Gallic acid equivalent per g dry weight (mg GAE/g) using the equation obtained from the calibration curve prepared by Gallic acid.

Determination of Total Flavonoid Content: The Total flavonoid content was determined using a spectrophotometric method based on a flavonoid–aluminium complex formation ²⁴. One milliliter of the extract was placed in a 10mL volumetric flask. Five milliliters of bi-distilled and 0.3mL of sodium nitrite were added and mixed. About 0.6 mL of 10% A1Cl₃. 6H₂O was added after 5m. Vigorously added two milliliters of NaOH (1M) 5m later in the solution mixture. The absorbance was immediately read with a spectrophotometer (Perkin Elmer Lamda 950 UV/Vis/NIR) at 510 nm. Total flavonoid content was expressed as mg catechin equivalent per g dry weight (mg CE/g) using the equation obtained from the standard calibration curve prepared from catechin.

Statistical Analysis: The results were given as means and SD of three measurements. The data were tested by one-way ANOVA using SPSS 20.0 (SPSS 2006) statistical software package, followed by the Tukey post hoc test. Differences of p<0.05 were considered significant.

RESULTS: 

Polyphenol Content and Antioxidant Activity: The total phenol, total flavonoid, and antioxidant activity of a three, two, and one day germinated mustard seeds were significantly higher (p<0.05) than the raw mustard Table 1 and 2. The total phenol content of the first, second, and third days of germinated mustard seed was increased by 16%, 17%, and 24%, and the total flavonoid contents were drastically increased by 500%, 647%, and 747%, respectively. Among the three antioxidant activity assays, the reducing power assay exhibited the highest increment, 10%, 34%, and 51% in 1, 2, and 3days of germination periods, respectively. In contrast, antioxidant activity increments by TEAC and DPPH assays were 8% and 5% after 3days of germination, respectively Table 2. In general, the accumulation of total phenol and flavonoid contents as well as the order of antioxidant activity of all assays on the germinated seeds revealed a consistent trend as shown in Tables 1 and 2, where 3 days germinated > 2days germinated > 1day germinated > raw mustard seeds.

Even though the effects of germination on different types of seeds have been investigated, there have been no or few reports, especially on the effect of germination on the polyphenol contents and antioxidant activity Brassica nigrafrom Ethiopia. Therefore, this work aimed to report the effect of germination on the polyphenol contents and antioxidant activity of mustard seeds (Brassica nigra) grown in Ethiopia.

TABLE 1:  THE EFFECT OF GERMINATION ON TOTAL PHENOL AND TOTAL FLAVONOID CONTENTS

Different superscripts in the same column mean significant difference (P ≤ 0.05).

TABLE 2:  THE EFFECT OF GERMINATION ON ANTIOXIDANT ACTIVITIES

Different superscripts in the same column mean significant difference (P ≤ 0.05).

DISCUSSION:

Polyphenol Content and Antioxidant Activity: The polyphenol content and antioxidant activity of germinated mustard seeds in this study revealed that they were synthesized during the germination periods, and the polyphenols generated were responsible for th enhanced antioxidant activity. Reports elsewhere have indicated that structural properties of substances belonging to the subgroups of phenolic acids, flavonols, flavanones, dihydrochalcones and flavanols influence antioxidant activities ²⁵. Moreover, higher antioxidant activity was observed on germinated seeds during water imbibition. Other antioxidant compounds reported in mustard seeds include 3,4-dihydroxybenzoic acid, ferulic acid, sinapic acid, and rutin in black mustard seeds ²⁶. Extraction of phenolic compounds from mustard seeds is best achieved when a mixture of water and acetone in equal proportions is utilized ²⁶. The rise in polyphenols and antioxidant activity after germination revealed in this work agrees with previous findings. A strong correlation between antioxidant activity with total phenolic content was reported ^{27, 28}. Cevallos-Casals et al. ¹¹ testified a rise in total phenols by 435% and antioxidant activity by 566% in germinated Brassica juncea. In addition, Fernandez-Orozco et al. ²⁹ have stated an increment of total phenolic content by 55% and antioxidant capacity (TEAC) by 23% for germinated chickpeas. El-Mergawi and Taie ³⁰ studied germinated Faba bean cultivars. They reported an increase in total phenols by 24.3%, total flavonoid by 76.1%, and antioxidant activity (DPPH) by 65%, while the antioxidant activity of lentils by hexane extract increased by 480% and total phenolic content by 77.4% ³¹. Similarly, Suryanti et al. ³² showed that for 4 days of germinated lead tree seeds, the antioxidant activity increased by178% while total phenolic content by 630%. Furthermore, Tarzia et al ¹⁸ reported that total phenolic compounds of methanol extracts of germinated chickpeas were increased by 92.8% and antioxidant activity by 493% as compared to raw seeds. In the present study, a drastic rise in the total flavonoid contents of germinated mustard seeds was observed compared to polyphenols. A similar trend was shown in the work of Herchia et al. ¹⁵ who stated that the total flavonoids of germinated flaxseed were increased by 32.5%, though the total phenolic acid content and antioxidant activity decreased as compared to raw seeds. In germinated faba be cultivars, the four flavonoid constituents, myricetin, daidzein, apigenin and quercetin, were increased by 228%, 527%, 366%, and 538%, respectively ³⁰. These results indicated that the germination process is one of the effective methods for the accumulation of flavonoids in seeds and hence higher antioxidant activity. The flavonoid is one of the most widespread and diverse groups of polyphenols in Brassica species ³³. The antioxidant ability of flavonoids is related to the number and position of hydroxyl groups in the molecule. Compounds with three hydroxyl groups on the B ring of flavonoids have a high antioxidant activity ³³. The three-day germinated mustard seeds exhibited the strongest antioxidant activity, which is due to the higher polyphenols content. This conclusion is further validated by computing the correlation coefficient between polyphenol contents and antioxidant activity. A positive linear correlation was observed where the correlation coefficients were in the order of R² = 0.9 Table 3. Moreover, the correlation coefficients between total phenol and total flavonoid and between the three antioxidant analysis methods (DPPH, TEAC, and reducing power) were in R² = 0.9.

TABLE 3: THE CORRELATION COEFFICIENT BETWEEN POLYPHENOL CONTENT AND ANTIOXIDANT ACTIVITY

CONCLUSION: The phytochemicals of Ethiopian mustard, B. nigra seeds changed after germination, and its polyphenol profile and antioxidant activities increased with an increase in germination days. Germination can thus enhance the bioactive compounds in mustard seeds allowing the plant for efficient food and nutraceutical uses.

CONTRIBUTION OF AUTHORS: All authors contributed equally to this work.

ACKNOWLEDGEMENT: The laboratory of aroma research for the food industry in The Katholieke Universiteit Leuven (KU Leuven), technology campus, Ghent, Belgium, is acknowledged.

CONFLICTS OF INTEREST: All authors declare that there is no conflict of interest for the manuscript.

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    How to cite this article:

    Efrem M, Negussie R, Sekwati-Monang B, Kobue-Lekalake RI, Selebatso T, Setlalekgomo MR and Haki GD: Effect of germination on polyphenol contents and antioxidant activity in mustard seeds (Brassica nigra) grown in Ethiopia. Int J Pharm Sci & Res 2022; 13(10): 4087-92. doi: 10.13040/IJPSR.0975-8232.13(10).4087-92.

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