Antioxidant activity and total phenolic contents of dried and germinated legumes

Aisha Umar (  ash.dr88@gmail.com ) Institute of Botany, University of the Punjab Lahore, Pakistan Muhammad Tajammal Khan Department of Botany Division of Science and Technology University of Education Lahore, Pakistan Shanila Bukhari Institute of Botany, University of the Punjab Lahore, Pakistan Rehana Sardar Institute of Botany, University of the Punjab Lahore, Pakistan Kishwar Naheed Institute of Botany, University of the Punjab Lahore, Pakistan Hajira Younas Institute of Botany, University of the Punjab Lahore, Pakistan Saber Hussain Institute of Botany, University of the Punjab Lahore, Pakistan Raazia Alam Gillani Institute of Botany, University of the Punjab Lahore, Pakistan Rushaan Kauser Kiani Institute of Botany, University of the Punjab Lahore, Pakistan


Introduction
Legumes are cultivated and an essential part of human meals throughout the world 1 . The consumption of phenolic rich legumes is maximum in Asia than other continents due to therapeutic effects of numerous bioactive compounds that in uence the human metabolic machinery 2 . The legumes also contain protein and minerals with low glycemic index) and also maintain the bone health 3 .
The polyphenols of legumes play a vital role in metabolic and physiological processes 6 , which hinder the production of free radicals formed by the breakage of biological molecules (lipids, protein, DNA) in our body 7 . The water insoluble polyphenols act as anti-artherogenic, anti-in ammatory, antimutagenic and antimicrobial activities 8 .
Germination stage of legumes causes change in biological activities due to these compounds during respiration and new cell formation. This is an e cient stage to enhance the nutrient activities (amino acid, dietary bers, and soluble sugar) and various other compounds of legume and pulses 9 .
The aim of this study was to explore the total phenolic compounds (TPCs) and antioxidant activity of dried and germinated beans and lentils.

Sample preparation
Five types of beans including white chickpeas (Cicer arietinum), black chickpeas (Cicer arietinum), red beans (Phaseolus vulgaris), cowpea (Vigna unguiculata), mung beans (Vigna radiata) and two types of lentils masoor (Lens culinaris) and white lentils (Vigna mungo) were selected for analysis and purchased randomly from Gulberg, Lahore, Pakistan in 2019. Each sample was divided into six equal portions. Three portions of each sample were analysed for antioxidant activity of total phenolic contents, while the other three were subjected to germination.

Germination of samples and measurement
An aliquot of 2-3 g of each bean/lentil sample was cleaned with running water, and then soaked in water for 1 h at 28°C.
After removing excess water, seeds were placed into a sprouter (60 × 150 mm) and kept in the dark at 28°C for germination. The sprouts germination with radicle period was 120 h. The seeds were moistened with water after every 24 h. Incubate at 28 ºC till 90% beans were germinated. Elongation of the germinated bean/lentils radicles were taken by measuring scale (cm). Percentage Elongation of radicles were determined by %EL = L f -L o /L o × 100.

Extraction of samples
Each sample (2-3 g) was grinded and extracted in a capped centrifuge tube with 50 mL solvent of ethanol/water (50% v/v). At temperature (25ºC), the mixture was well shaken (300 rpm) for 3 h. After centrifugation, the mixture was placed under darkness for 12-24 h. Supernatant was taken from extracts into a new tube after centrifugation at 4000 rpm for 10 min. Residues left behind in the tube were also extracted with 10 mL of respective solvent. Both extracts were combined and stored at 4 º C in the dark until further analysis.

Determination of TPCs
Folin-Ciocalteu reagent was used to determine the total phenolic contents accordingly Xu and Chang 10 . Extract (50 µl) was homogenized with 250 µl of Folin-Ciocalteu reagent, 750 µl of 7% Na 2 CO 3 (W/V) and 3 mL of distilled water and left for 8 min. Add 950 µl of distilled water again and allow to settle for 2 h in room T. Absorbance (765 nm) was measured by using distilled water as a blank in UV/Visible spectrophotometer (Schimadzu UV 1700). All determinations were done in triplicate (n = 3). The quantity of total phenolic contents was determined from the standard curve of gallic acid and stated as gallic acid equivalent (mg GAE/g) of beans and lentils. The linearity range of the calibration curve of Gallic acid was 2 µg-40 µg mL −1 and r 2 = 0.9991 ( Figure 1).

Determination of antioxidant capacity (AC)
(A) DPPH (2, 2-diphenyle-1-picrylhydrazyl radical). The method modi ed by Xu and Chang 10 was used to determine antioxidant activity. Ethanolic DPPH solution of 3.8 mL (0.1 mM) was mixed in 0.2 mL of legume extract. Vortexing this mixture for 1 min and then placed in darkness (30 min). Absorbance was measured at 517 nm of the resulting solution (A sample ) by a spectrophotometer (Schimadzu UV 1700) using ethanol as a blank. For the control set, respective extraction (0.2 mL) was added into 3.8 mL of DPPH solution and absorption (A control ) was measured at 517 nm. The DPPH discoloration of the sample was calculated in percent according to the equation = [1-(A sample /A cont )] × 100. The antioxidant content was determined using BHA as an external standard. A calibration curve of BHA was used to calculate the AOs with a unit of micrograms of BHA equivalents per 100 g of dried beans or germinated bean with sprouts (µg BHA/100g) under the same experimental conditions. (B) ABTS (2,2'-azinobis-(3-ethylbenzothiazoline-6-sulphonate). Add 3.0 mL of diluted ABTS (0.1 mM) solution (A734 nm) to 0.2 mL of the legumes extracts, the absorbance was taken exactly 10 min after the initial mixing. The ABTS scavenging activity of each seed extract was calculated as a difference between the initial absorbance and that after reacting for 10 min. Methanol was used as the blank solution. All determinations were performed in triplicate. The antioxidant content was determined using BHA as an external standard. A calibration curve of BHA was used to calculate the AOs with a unit of micro grams of BHA equivalents per 100 g of dried beans or germinated bean with sprouts (µg BHA/100g) under the same experimental conditions.

Statistical analysis
Results were expressed as mean ± standard deviation. Data were statistically analysed using software SPSS. Signi cance difference in total phenolic contents and antioxidant activities between germinated and non-germinated beans and lentils was determined at p < 0.05 by applying ANOVA of means. The p < 0.05 was considered statistically signi cant.

Effects of germination time on radicles elongation
Elongation of the germinated bean/lentils radicles exhibited a quick tendency during the germination period of 120 h. The elongation of radicles during germination time of 120 h in cowpea, red beans and lentils masoor was 23.3, 21.5 and 14.5 cm than other beans/lentils. The elongation percentage of radicles during germination time of 72 h was decreased in Phaseolus vulgaris, Lens culinaris, and Vigna unguiculata than other beans and lentils. It suggested that the elongation e cacy of radicles were decreased with the extension of germination time. Hence, the most effective elongation period of radicles was the early 1-3 days of germination ( Figure 2).
The percentage (%) elongation of radicles in the germinated beans/lentils were 87.32%, 77.33%, and 72.59% in Lens culinaris, Phaseolus vulgaris, and Vigna radiata sprouts, respectively in a time-dependent manner and reached the peak on 72 h, but the values reduced in percentage elongation from 87.32-56% and 25% on day 4 and 5 respectively in Lens culinaris. The elongation percentage of radicles was 48-77.33% and 36% in Phaseolus vulgaris and 41-72.59% and 28% in Vigna radiata on day 4 and 5, respectively. The least percentage was observed in Vigna mungo (25.86%) on day 5 ( Figure 3).

Quantity of total phenolic in dried and germinated seeds
The result of our study indicated that TPCs in all beans and lentils have been signi cantly increased (p < 0.05) after germination (Table 1). Literature cited that legume fractions and germination time were affected (p < 0.05) on TPC. Total phenolic contents were signi cantly (p < 0.05) increased in a time-dependent manner after the germination than the original concentration of leguminous seeds. In this study, the germinated sample of red beans (Phaseolus vulgaris) exhibited the maximum amount of TPCs (15.40 mg GAE/g), which was low in white chickpeas (Cicer arietinum) (2.52 mg of GAE/g).
In white lentils of this work, TPCs value was 8.93 mg GAE/g) and 6.62 mg GAE/g in germinated and dried seeds, respectively. The TPCs of Lens culinaris (1.49 mg GAE/g) were lower amongst the remaining samples of this study (Table  1).
In this research work, the TPCs of germinated legumes were observed in the following order:    Dried beans/lentils (DB/L), Germinated beans/lentils (GB/L).
Estimation of radical scavenging activity. DPPH •+ and ABTS •+ are a stable, simple and quick organic radical used to estimate the antioxidant capacity useful to evaluate the free radical scavenger activity in legumes. The antioxidant action of beans is interlinked with the quantity of polyphenols. The antioxidant capacities signi cantly differ amongst the samples of this work. The observed DPPH and ABTS scavenging capacity of the ethanol-based extracts of beans and lentils were given, which signi cantly higher in germinated red beans ( Table 2). The scavenging activity in percentage inhibition (ABTS) of germinated beans and lentils extract decreased in the following order: Phaseolus vulgaris > Lens culinaris > Vigna radiata > Vigna mungo > Vigna unguiculata > Cicer arietinum (black) > Cicer arietinum (white). Whereas in DPPH the order of inhibition in germinated beans/lentils was Phaseolus vulgaris > Vigna mungo > Vigna radiata > Vigna unguiculata > Cicer arietinum (black) > Lens culinaris > Cicer arietinum (white) ( Table 2).
Signi cant positive correlation was observed between phenolics and antioxidant activity by DPPH and ABTS scavenging activity in the various legume extracts. The data revealed that germinated beans possessed highest total phenolic contents with antioxidant activity than lentils. The free radical scavenging activity of beans and lentils increased in both germinated beans and lentils along total phenolic contents. It means total phenolic contents are the major contributor in analysis of antioxidant capacity of beans and lentils. In this study, red beans (Phaseolus vulgaris) were stronger free radical scavengers, because of high level of total phenolic contents and antioxidant capacity.

Conclusion
The cultivars of beans and lentils processed different amount of total phenolic contents as well as antioxidant activity. The germinated beans and lentils contained more phenolic contents that decrease signi cantly in dried beans. The germinated beans were valuable source of natural antioxidants and sprouting was the best stage to increase few nutritional ingredients and antioxidants in beans and lentils.

Discussion
Elongation e cacy of radicles was effective in rst three days 11 , similarly to results of this study, contrary to 3 to 4-daygermination in work of Zhou and Zhang 12 . Legumes are famous for total phenolic compounds rather than rice, corn, millet and wheat. Major classes of phenolic contents in beans and lentils have been gaining popularity due to antioxidant activity. The levels of natural endogenous antioxidants (e.g., phenolics, tocopherols; vitamin C) vary during seed germination of legumes. Federica et al. 6 analysed 14 polyphenolic compounds ranging from 3 mg kg −1 for dehulled red lentils to 1630.5 mg kg −1 for ruviotto beans.
Legume fractions and germination time were affected on TPC. The total phenolic contents were increased after the germination. Similarly, the TPC contents were signi cantly (p < 0.05) increased with the germination time in seed hulls, radicles, cotyledons of legume 13 and lentil seeds. Contrary results were shown by lentils and beans of Aguilera et al. 14

and
Duenas et al. 15 . No change in TPCs was shown by kidney beans, but the values were highly decreased in lentils 16 . Our results regarding TPCs were signi cantly higher than common bean seed coats (0.69-3.32 mg GAE/g) reported by Chávez-Mendoza et al. 13 .
The TPCs of Lens culinaris (1.49 mg GAE/g) were lower amongst the remaining samples of this study (Table 1) Contrarily, maximum values were reported in red and green lentils 7,30 .
There are two schools of thought behind low level of TPCs during germination: 1) sprouting causes the reduction in avan-3-ols and anthocyanin 15 ; 2) reactive oxygen species (ROS) released from metabolically active cells of seed, which in uenced the biological process of seed germination. ROS as a messenger transmits environmental signals during seed germination and responsible for lowering the antioxidant activity of bean and lentils after germination 31 . TPCs were decreased in peanuts, soybeans and lentils after germination 32 contrary to this study (Table 1), while Khang et al. 33 values were signi cantly increased during germination in all legumes. Zhaohui et al. 34 investigated TPCs and antioxidant activity of germinated Mung beans, soybeans and black beans sprouts were highest initially (44.87-90.31%) then decreased.
In white lentils of this work, the TPCs were comparable to faba beans (7.11 mg gallic acid/g) 35 and lupine seeds (8.56 mg gallic acid/g) 36 . Emily et al. 37 worked on 14 Canadian pulses included beans, peas, lentils and analyzed antioxidant activity (1.16 to 7.45 mg GAE/g DW) and revealed that samples with dark testa (black lentils and diavoli beans) possessed higher antioxidant activity than pale testa.
Antioxidant capacity (AC) of legumes is due to active micro and macro elements (polysaccharides, vitamins, amino acids proteins) 38 . In this study, germinated lentils (Masoor) contained maximum ACs than dried lentils (Masoor) similarly the work of Dalaram et al. 39 but contrary to lentils of Gubanenko et al. 40 and Zhao et al. 29 . Researchers supported that AOA (Antioxidant activity) increased as the germination time increased 25 , e.g., in mung beans, soybeans, black beans 33 , lentil seeds 41 , kidney beans 14,16 , chickpea seeds, fenugreek seeds, lentil seeds 26 and lupine seeds 36 . Though, black beans did not change AOA during the germination process 42 .
Gubanenko et al. 40 revealed that seedlings of lentils show a slightly higher antioxidant activity than chickpea sprouts, whereas seeds of green pea, chickpea 43 and faba beans 39 dramatically contained maximum TAC. The seed coat of common bean exhibited greater antioxidant capacity (23.86-84.10%) than the cotyledon (0.66-29.77%) of all bean varieties 13 .
The most famous legumes with maximum antioxidant compounds in dietary bres were found in chickpeas (Cicer arietinum), pulses 44 , white and red beans (Phaseolus vulgaris), which promote the health and prevent chronic diseases 45 .
The result of this study regarding scavenging activity was similar to Saleh et al. 25 . They explained common beans (84.52%) contained highest radical scavenging activity than lupine seeds (78.29%). The values of lentils scavenging capacity of this study were signi cantly higher than 38.5% 29 .
The antioxidant activity of beans increased after germination and decreased in lentils depends upon type and conditions of germination 46 , similarly to this study, where free radical scavenging activity increased in germinated seeds depend on the time taken for germination. Wang et al. 47 worked on Chinese beans, spring bay beans, black beans, pearl beans and determined the strong positive relationship between total phenolic contents and antioxidant activities. Black, red, green beans, red kidney beans and soybeans possessed higher total phenolic contents and antioxidant capacity, whereas red and yellow lentils (dhal) and chickpea possessed lower capacity 48 .

Conclusion
This study concluded that germinate legumes exhibited the highest total phenolic contents (TPCs) and antioxidant capacity which decreased signi cantly in dried samples. This study also suggested that germinated beans are a valuable source of commercial natural antioxidants.

Declarations
Con ict of interest Authors declare no con ict of interest. Aisha Umar contributed to complete this work in all aspects.

Competing interests
There is no any con ict of interest.  Kinetic changes of radicle length (cm) of beans/lentils with germination time. Percentage elongation of radicles (beans/lentils) germination with time.