Phytochemical screening and in-vitro biological properties of unprocessed and household processed fenugreek (Trigonella foenum-graecum Linn.) seeds and leaves

The impact of household processes on fenugreek leaves and seeds has been analyzed for total phenolic (TP) and total flavonoid content (TF), and in-vitro biological activities such as antioxidant, antimicrobial, and anti-inflammatory properties. Processes included air-drying for leaves and germinating, soaking, and boiling for seeds. Air-dried fenugreek leaves (ADFL) had high TP (15.27 mg GAE g−1 D.W.) and TF (7.71 mg QE g−1 D.W.) (milligram quercetin equivalents per gram dry weight). The TP contents of unprocessed, germinated, soaked, and boiled seeds were 6.54, 5.60, 4.59, and 3.84 mg gallic acid equivalents per gram of dry weight (mg GAE g−1 D.W.), respectively. The TF contents in unprocessed fenugreek seeds, germinated fenugreek seeds, soaked fenugreek seeds, and boiled fenugreek seeds (BFS) were 4.23, 2.11, 2.10, and 2.33 mg QE g−1 D.W., respectively. Sixteen phenolic and nineteen flavonoid compounds has been identified using high-performance liquid chromatography. Antioxidant activity using 2,2-diphenyl-1-picrylhydrazil (DPPH·), 2,2-azinobis (3-ethylbenothiazoline-6-sulfonic acid (ABTS+·), and ferric reducing antioxidant power (FRAP·) assays indicated that ADFL had the highest activity. Antimicrobial activity has been evaluated against each of the eight pathogenic bacterial and fungal strains. ADFL showed the strongest activity with minimum inhibitory concentrations values ranging from 0.03 to 1.06 and 0.04 to 1.18 mg ml·1 against bacterial and fungal strains, respectively. Anti-inflammatory activity was evaluated in-vitro against RAW 264.7 macrophage cells using the nitric oxide (NO) assay. Results revealed that ADFL had the highest cytotoxicity and anti-inflammatory activity according to the NO assay. Household processes significantly reduced the in-vitro biological properties of processed seeds.

Phytochemical screening is a method of investigating the existence or absence of potential phytochemical components in the plant. This assay can help scientists to diagnose the bioactive components in plants. It advises not only on the existence of therapeutic agents but also gives information about the presence of economically active ingredients, such as vitamins, phenolics, alkaloids, saponins, tannins, oils, gums, and precursors for the synthesis of complex compounds, etc. Phenolic constituents in plants have potential health activities principally due to their antioxidant activities, such as metal chelation, electrophile scavenging, and reactive oxygen species (ROS) scavenging and inhibition 1 .
Fenugreek (Trigonella foenum-graecum) is an annual plant belonging to the family Fabaceae. The green leaves and seeds of fenugreek are widely used in food and medicinal applications dating back to ancient times. However, the seeds are sour in taste due to the presence of bitter saponins, which limit their acceptability in foods 2 . It has been possible to decrease the bitter taste of fenugreek seeds by using diverse household processes, such as soaking, germination, boiling, fermentation, etc. 2,3 . Many extracts of each seed or leaf and its active components have been studied for their pharmacological effects and have been reported to have hypocholestrolaemic, antidiabetic, anti-inflammatory, antiulcer, analgesic, antipyretic, CNS-stimulant, antioxidant, wound healing and immune modulatory activity as well as gastro-protective and chemo-preventive activities 4 .
Newly, various actions and behaviours of macrophage cells play an important role in surviving homeostasis and normal physiological conditions by manufacturing a diverse range of biological impacts 5 . Macrophages (RAW 264.7) have been chosen because they are accepted as targeted single cells for assessing immune reactivity 6 . Macrophages can be induced by lipopolysaccharide (LPS) to generate pro-inflammation molecules (e.g., NO and PGE2) through enhancing intracellular signaling pathways including NF-κB and MAPK 7 .
Nitric oxide (NO) is a signaling molecule that plays a crucial role in the way inflammation develops. It has anti-inflammatory activity in normal physiological situations, but it is regarded as a pro-inflammatory mediator in unusual conditions due to overproduction. NO has been generated and released to the endothelial cells. It is an active neurotransmitter at the neuronal synapses, and it also helps to regulate programmed cell death regulation. NO participates in the way the inflammatory disorder in joints, intestines, and in the upper and lower respiratory systems. Hence, NO inhibitors represent significant therapeutic advances in the control of inflammatory diseases 8 . It also has an important property as a bio-regulation molecule in the nervous, immune, and cardiovascular systems. The permanent release of NO is associated with several diseases, including; inflammation, cancers, and arthritis 9 .
RAW 264.7 macrophages induced by LPS are used to assess the in-vitro potential inhibitory effects of antiinflammatory compounds. LPS is considered one of the most important stimuli used to upgrade pro-inflammatory protein arranging, such as inducible nitric oxide synthase (i-NOS) and cyclooxygenase-2 (COX-2), which are in charge of the high standards of prostaglandin spotted in various inflammatory confuses. Furthermore, i-NOS develop huge quantities of NO, which is considered to be important for showing a necessary role in the inflammation 10 . Beside, the highly reactive nitric oxide becomes more sensitive when it is linked with oxygen and thus produces highly reactive compounds which can cause some provisions cellular damage such as; cellular DNA fragmentation and lipid peroxidation 11 .
This work aimed to investigate the influences of household treatments, including air-drying for fresh fenugreek leaves and each soaking, germination, and boiling for edible fenugreek seeds, on both phenolic, flavonoid and isoflavonoid components, as well as on their in-vitro antioxidant, antimicrobial and anti-inflammatory activities of the selected samples.
The profile of phenolic, flavonoid and isoflavone components. Vegetables and cereals (including seed and sprouts) are excellent sources of phenolic components. Legumes contain a high concentration of isoflavonoids 18 .
The phytochemical screening of each phenolic, flavonoid and isoflavonoid component evaluated using of HPLC has shown in the Table 1.The highest concentrations have obtained by the alcoholic crude extract of ADFL, UFS and GFS.
The contents (mg 100 g −1 D.W.) of phenolic compounds in ADFL were declined in the following order; oleoropin > chlorogenic acid > pyrogallol > ellagic acid > ferulic acid > salicylic acid > p-hydroxy benzoic acid > coumarin > benzoic > caffeine > 4-amino benzoic > 3-OH tyrosol > vanillic > catechol > caffeic. This finding is compatible with Hussain et al. 12 who found that, among the hydroxyl benzoic acid derivatives, the most abundant acids present in fenugreek leaves were m and p-hydroxy benzoic acid, β-resorcylic acid, gentisic acid, and gallic acid. In the case of hydroxyl cinnamic acid derivatives, the major acids present included; o and m-coumaric acid and sinapic acid. HPLC analysis showed the absence of α-resorcylic acid, vanillic acid, and p-coumaric acid in air-dried fenugreek leaves.
Antioxidant activity of the selected samples. "The most common antioxidant methods are ABTS ·+ and DPPH • . DPPH free radical (DPPH · ) does not require any special preparation; in contrast, the ABTS radical cation (ABTS ·+ ) has generated by enzymes or chemical reactions 20 ". "Another important difference is that ABTS ·+ Table 1. Phenolic, flavonoids and isoflavonoids screening of crude alcoholic extracts of air-dried fenugreek leaves (ADFL), unprocessed fenugreek seeds (UFS), soaked fenugreek seeds (SFS), germinated fenugreek seeds (GFS) and boiled fenugreek seeds (BFS) by HPLC (mg 100 g −1 D.W.). Rt retention time, ND not detected. www.nature.com/scientificreports/ can dissolve in aqueous and organic media, due to their hydrophilic and lipophilic nature of the compounds in samples. On the other side, DPPH can only dissolve in organic media, especially in ethanol, this being an important limitation when interpreting the role of hydrophilic antioxidants. Both radicals show similar bi-phase kinetic reactions with many antioxidants. Although, the ferric reducing antioxidant power (FRAP) method is based on the reduction of a ferroin analogue, the Fe 3+ complex of tripyridyltriazine Fe (TPTZ) 3+ to the intensely blue-colored Fe 2+ complex Fe (TPTZ) 2+ by antioxidants in acidic medium. However, the reducing capacity does not necessarily reflect antioxidant activity, as has been suggested by Katalinic et al. 21 and Wong et al. 22 ". The results of the antioxidant activity of crude extracts of each studied samples have shown in Figs. 2, 3 and 4. These results indicated that at 1000 μg ml −1 ADFL had the maximum antioxidant activity (81.11% and 75.01%) with IC 50 = 330 μg ml −1 , as shown in Table 2, according to both DPPH and ABTS methods, respectively. ADFL has the maximum potential (57.88 μM) for the reduction of ferric ions into ferrous ions at 800 μg ml −1 according to FRAP assay. Furthermore, UFS at 1000 μg ml −1 exhibited a higher radical scavenging activity against DPPH and ABTS free radicals(70.04% and 71.40%) than SFS (61.00% and 52.50%), GFS (62.91% and 57.00%) and BFS (56.00% and 52.41%), respectively. According to the FRAP assay, at 800 μg ml −1 , UFS showed more potential (57.60 μM) for ferric ions reduction than SFS (19.00 μM), GFS (57.52 μM) and BFS (18.29 μM).
According to the previous data on TP and TF, there is a linear correlation between the TP and TF and each of the free radicals (DPPH and ABTS) scavenging activities and the reduction of ferric ions into ferrous ions.
Household treatments of the fenugreek seeds reduced the in-vitro antioxidant property. These results are compatible with the earlier findings of Hooda and Jood 13 and Shakuntala et al. 14 . In contrast, Pandey and Awasthi 15 approved that soaking and germination enhanced the total phenolic content and the antioxidant activity of fenugreek seed flour compared to raw seeds flour.   Table 3. The data illustrated that crude extract of the ADFL exhibited the highest antibacterial activity against B. cereus, Staph. aureus, Staph. scuiri and S. typhi with inhibition zones 11.87, 11.61, 11.60 and 11.30 mm, respectively, compared to the positive control (tetracycline). On the other side, UFS had the maximum activity against both S. enterica (11.11 mm) and S. typhi (10.35 mm). Soaked and germinated fenugreek seeds (SFS and GFS) had the highest activity against   www.nature.com/scientificreports/ S. typhi (9.52 and 9.71 mm), S. enterica (9.34 and 9.52 mm) and P. aeruginosa (9.00 and 9.15 mm), respectively. Finally, BFS had the greatest activity on E. coli O157 H7, S. enteric and P. aeruginosa with inhibition zones were 8.50, 8.18 and 8.12 mm, respectively. As illustrated in Fig. 5, ADFL had the highest MIC values (0.04 mg ml −1 ) against each of Staph. Scuiri and P. aeruginosa, but the lowest MIC value (0.10 mg ml −1 ) was observed against E. coli O157 H7.On the other side, UFS had the highest MIC value (0.03 mg ml −1 ) against P. aeruginosa and the lowest MIC value (0.19 mg ml −1 ) against Staph. aureus. The maximum MIC values for each SFS, GFS, and BFS were against P. aeruginosa with 0.17, 0.06, and 0.27 mg ml −1 , respectively.
Antifungal activity and MIC of the studied samples. Antifungal activities of the crude extracts of selected samples against eight micotoxigenic fungal strains are shown in Table 4. Data showed that ADFL had the highest activity against A. ochraceus and F. proleferatum with zones of inhibition found to be 11.01, and 10.74 mm, respectively. Also, UFS and SFS had the highest activity against A. ochraceus (10.75, and 8.38 mm, respectively). Germinated seeds exhibited the greater property against both of A. ochraceus and A. purasiticus with inhibition zones found to be 9.18 and 9.02 mm, respectively. Finally, BFS showed the highest antifungal activity against A. carbonarius, A. flavus and P. verrucosum with inhibition zones 8.11, 8.00, and 8.00 mm, respectively. Figure 6 illustrates the MIC values of the tested samples against each studied fungal strain. The most effective levels of MIC values were recorded at 0.04 and 0.21 mg ml −1 with ADFL and GFS, respectively, against A. wasterdijikia. Whereas, UFS, SFS, and BFS crude extracts recorded the most significant levels of MIC values, which were 0.11, 0.41, and 0.80, respectively, against A. purasiticus.
Al-Abdeen 23 studied the antibacterial activity of aqueous and some organic compound extracts of stems, leaves, seeds, and roots of fenugreek against three Gram-negative and one Gram-positive bacteria by welldiffusion and colony-assay methods. The microorganisms were Staph.aureus, E.coli, P.aeruginosa and K. spp. All plant extracts did not exhibit any inhibitory activity against any of the microorganisms tested by the welldiffusion and colony-assay techniques. Abdalah 24 found that the extract of fenugreek seeds at concentrations of 1000, 500, and 250 mg ml −1 inhibited the growth of Streptococcus pyogenes. The methanolic extract of fenugreek   In-vitro anti-inflammatory activity of the selected samples. Industrial steroidal and non-steroidal anti-inflammatory drugs have a wide range of side effects. So, diverse works are interested in finding anti-inflammatory agents from natural sources 28 .
Our work is the first study reports the impact of household techniques on the cytotoxic activity against RAW 264.7 macrophage cell line and the anti-inflammatory property by NO assay of both fenugreek leaves and seeds crude extracts.
Cell cytotoxicity assay. Results presented in Table 5 illustrated that ADFL crude extract exhibited the highest cytotoxic activity (89.03%) on the RAW 264.7 cell line at a concentration of 100 µg g −1 . Furthermore, UFS at a concentration of 100 µg g −1 had higher activity (73.21%) than the processed samples, SFS, GFS and BFS, which  Table 5. Cytotoxicity of crude extracts of the studied samples against RAW 264.7 macrophage cell line. *Each value represents the mean ± standard deviation *The same letter of denoting over values proves that they are not at significantly different at (p ≤ 0.05), and comparison is done according to treatments. *(-) means unspecified IC 50 (µg g −1 ). www.nature.com/scientificreports/ showed 38.66%, 63.00%, and 18.19% cytotoxic activity, respectively, at the same concentration. So, the household treatments declined the cytotoxic property of the fenugreek seeds against the macrophage cell line but were not affected by the fenugreek leaves. These may be due to a decrease in TP, TF, and AOX activity after each soaking, germinating and boiling process. Table 6 demonstrated that ADFL at 100 µg g −1 exhibited the maximum inhibition (76.11%) of NO molecules. On the other side, at 100 µg g −1 , UFS showed higher inhibition (62.11%) than each of SFS (39.91%), GFS (56.12%), and finally, BFS (33.11%).

Plant materials.
Our used of plant material and all methods in our research complies with all applicable local, regional, national, and international regulations.
Air-dried fenugreek leaves (ADFL). Fresh and healthy leaves of fenugreek (Trigonella foenum-graecum Linn.) were purchased from the local market of Egypt in December 2019 and identified by the Faculty of Science, Cairo University. The leaves have washed thoroughly with tap water, and the surface water has removed by air-drying under shade for 15 days. The leaves have subsequently dried in a hot air-oven at 50 °C for 4 h, homogenized to a fine powder and then stored at 4 °C.
Fenugreek seeds. Two kilograms from fenugreek seeds were also purchased from the local market of Egypt, identified by the Faculty of Science, Cairo University, and divided into four groups, as the followings; 1st group (Untreated fenugreek seeds, UFS), 500 g of seeds were manually cleaned to remove dust and foreign particles, crushed into a fine powdered flour with the help of Moulinex blender LM 241 and were sieved in a 0.5 mm mesh size. The powdered flour has been kept at 4 °C to prevent changes till further analyses.
2nd group (Soaked fenugreek seeds, SFS), 500 g of raw seeds were soaked in tap water at the ratio of 1:5 (w/v) for 12 h at room temperature. After pouring off the soaking water, seeds were air-dried in shade for 5 days followed by hot air-oven drying at 50 °C for 4 h in a conventional oven, and stored at 4 °C.
3rd group (Germinated fenugreek seeds, GFS), 500 g of raw seeds were soaked in tap water at the ratio of 1:5 (w/v) for 12 h at room temperature. After pouring off the soaking water, seeds were kept in the dark for germination (tied in cotton cloth) at 20 °C for 60 h in darkness. After harvesting the sprouts, they were air-dried for 5 days followed by hot air-oven drying at 50 °C for 4 h in a conventional oven, and stored at 4 °C.
4thgroup (Boiled fenugreek seeds, BFS), 500 g of raw seeds were put in 2 L beaker containing 1250 ml tap H 2 O (1:5 w/v). The sample was boiled on a hot plate for 10 min and then, after water was discarded, the boiled seeds were air-dried in shade for 5 days followed by hot air-oven drying at 50 °C for 4 h in a conventional oven, and stored at 4 °C.
Extraction procedure of samples. One hundred grams of each sample were extracted with 1 L (1:10 w/v) 80% ethanol in distilled H 2 O by sonication for 60 min. Extraction had repeated three times. After filtration, each extract condensed to dryness (resulting in crude extracts) using a rotary evaporator at 40 °C. The obtained Table 6. Anti-inflammatory activity of crude extracts of selected samples against nitric oxide (NO). *Each value represents the mean ± Standard deviation **The same letter of denoting over values proves that they are not at significantly different at (p ≤ 0.05), and comparison is done according to treatments. *(-) means unspecified IC 50 (µg g −1 ). Inhibition of NO (%) (Mean ± STDEV*) www.nature.com/scientificreports/ residue has collected to calculate the yield and finally stored in the freezer for further biochemical and in-vitro biological analyses. A known weight of each crude extract obtained from rotary evaporation has dissolved in each ethanol 80% for determination of TP, TF, DPPH · , ABTS +· , and FRAB · , and in dimethyl sulphoxide (DMSO) for both in-vitro antimicrobial and anti-inflammatory activities.
Recovery of the extract was calculated as yield (%) using the following equation: where W f is the final weight of the crude extract and W i is the initial weight of the tested sample.
Determination of total phenolic content (TP). Total phenolic content of ethanol extracts was evaluated according to Singleton and Rossi 29 , using of Folin-Ciocalteau reagent. Total phenolic content was expressed as mg of gallic acid equivalents (GAE) per g dr weight (D.W.) of sample. All determinations were performed in triplicates.
Determination of total flavonoid content (TF). Total flavonoid content was analyzed by a spectrophotometric method described by Boateng et al. 30 . Total flavonoid content was expressed as mg of quercetin equivalents (QE) per g D.W. of sample. All determinations were performed in triplicates.

Fractionation of phenolic and flavonoid components by HPLC.
A high-performance liquid chromatography system equipped with a variable wave length detector (Agilant technologies, Germany) 1200 series. Also the HPLC was equipped with auto-sampler, Quaternary pump degasser and column compartment set at 35 °C. Analyses were performed on a C18 reverse phase (BDS 5 μm, Labio, Czech Republic) packed stainless-steel column (4 × 250 mm, i.d.).To determine phenolic and flavonoids compounds, samples were prepared according to the method described by 31 . All chromatograms were plotted at 280 nm to estimated phenolic compounds and at 330 nm for flavonoids and isoflavonoids. All components were identified and quantified by comparison of peak areas with external standards.

Antioxidant activity of alcoholic extracts of fenugreek samples. Determination of DPPH • scaveng-
ing activity. In order to determine DPPH radical-scavenging activity, a method described by Moure et al. 32 was used with minor modification. Various concentrations of each sample (200, 400, 600, 800 and 1000 µg g −1 ) were prepared from the stock solution (10 mg ml −1 ). The DPPH radical-scavenging activity in the extracts was expressed as percentage inhibition activity. The percentage inhibition activity was calculated from [(A C − A S )/A C ] × 100. A C is the absorbance of the control, and A S is the absorbance of the sample. The analyses were carried out in triplicate.
Determination of ABTS •+ scavenging activity. The free radical-scavenging activity has been determined by the ABTS radical cation decolorization assay described in 33  Agar disc-diffusion assay. Agar disc-diffusion assay was used for evaluation the antimicrobial activity of alcoholic extracts against some foodborne pathogenic bacteria and fungi according to Kavanagh 35 method. The plates were incubated for 24 h at 37 °C and after the incubation period, the diameters of the cleared zones of inhibition (millimeter) were measured. Dimethyl sulphoxide (DMSO) was used as the negative control, tetracycline used as positive control. Mean and standard deviation (STDEV) values were tabulated.

Measurement of minimum inhibitory concentration (MIC)
. MIC against fungi was studied by using the technique of Perrucci et al. 36 . The prepared plates were centrally inoculated with 3 μl of fungal suspension (10 8 CFU ml −1 ; In-vitro anti-inflammatory activity. Cell cultivation. RAW 264.7 macrophage cell line was acquired from the ATCC (American type culture collection). Cells were cultivated in RPMI, 1640 medium (Institute of Roswell Park Memorial), and subjoined with 1% pen/strep and 10% heat-inoperative fetal bovine serum. Cells were transferred in a moistened incubator, in an ambient of 5% CO 2 at 37 °C, and they were subculture two times before the assay.
Proceedings. The following proceedings were obtained in a sterile area using a laminar bio safety flow cabinet class II level (Baker, SG403INT, Sanford, ME, USA). RAW 264.7 cells were suspended in RPMI medium. After 24 h of seeding 1 × 10 5 cells per well (in 96-well plates) and incubated for one day for the assay. Cells were then processed with the specimens at different concentrations of 10, 20, 40, 80 and 100 µg g −1 and incubated for 60 min. Cells were then enhanced with 10 μg ml −1 of LPS, as a negative control, for another one day. The supernatant was transferred wisely to a new 96-well plate and processed for NO determination, while the cells stayed in the old plate were used for the MTT protocol, to determine the percentage of the viable cells. Specimens (stock) were dissolute in DMSO, and the working specimens were prepared in the media. Viable cells were determined by the reduction of mitochondrial dependence of yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to purple formazan 37 . The percent of change in the number of viable cells was measured according to the following equation: As R* is reading of the extract, and R 0 reading of the control.
Nitric oxide (NO) protocol. The production of nitric oxide was processed by determining nitrite in the supernatants of cultivated RAW 264.7 macrophages. The protocol was processed with slight modification as previously described 38 . After pre-incubation for one day of RAW 264.7 cells (1 μg ml −1 ) with LPS (10 μg ml −1 ), the quantity of nitrite, which is considered a stable NO-metabolite, and used as an indicator of NO-production in the culture medium, it was estimated using a reagent of (0.1% naphthyl ethylene diamine dihydrochloride + 1% sulfanil amide + 2.5% phosphoric acid), and this reagent is commonly known as Griess reagent. A 50 μl volume of the Griess reagent was mixed with 50 μl of the cell culture medium. Afterward, the mixture was incubated at ambient temperature for 15 min, and the absorbance was measured using a microplate multi-well reader (Model 3350, Hercules, California, USA, Bio-Rad Laboratories Inc.) at 540 nm. In every single experiment, a new culture medium was used as a blank. The amount of nitrite was estimated from a sodium nitrite standard curves phrased in the following equation: Statistical analysis. All assays used in this work were evaluated triple times, and the data obtained were represented by the mean ± standard deviation (STDEV). Statistical Analysis Software (SAS 9.1) was applied for the statistical analysis of data, and IC 50 was calculated by using of Graphed prisms. One-way analysis of variance (ANOVA) was used to analyze the difference between groups by applying the least significant difference (LSD) test with 1% and 5% levels of significance (p < 0.05).

Conclusions
Fenugreek seeds have a bitter taste due to saponins and specific smell due to alkaloids and volatile oils, which limit their useand acceptability in the food industry. It has been possible debittered by using various household treatments such as soaking, germinating, boiling, etc. Among all the studied samples, ADFL exhibited the highest in-vitro antioxidant, antibacterial, antifungal, and anti-inflammatory properties due to their high contents of phenolics, flavonoids, and isoflavonoids. Each soaking, germinating, and boiling treatments lowered the in-vitro biological activities because a decreasing occurred in TP, TF, and AOX activities. Finally, air-dried fenugreek leaves and unprocessed seeds must use in both industrial and pharmaceutical fields as an excellent natural antioxidant, antibacterial, antifungal and anti-inflammatory agents, as well as a natural source of potential phenolics, flavonoids, isoflavonoids, steroidal saponins, alkaloids, etc. in medicinal field.

Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or non-profit sectors.