Antioxidant, antimicrobial, and preliminary phytochemical constituents of fermented seeds of Trigonella foenum-graecum

Trigonella foenum-graecum (Fenugreek) is among the earliest plants used as spices and medicine worldwide. This study determined the antioxidant activities of the fermented seeds of T. foenum-graecum by methods, ABTS 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) and DPPH (2,2-diphenyl-1-picrylhydrazyl). The phytochemical compounds, pH, and in vitro agar well (antimicrobial) assay of the sample was tested at three concentrations (100%, 50%, and 25%) against the selected pathogens (Staphylococcus aureus, Salmonella typhi, and Escherichia coli) by agar well diffusion method. There was a pH decline from 5.6 (on day 1) to 3.53 (on day 7) and finally 3.38 (on day 14). The antioxidant property of the 14th-day fermented T. foenum-graecum revealed a free radical scavenging potential in correspondence with the tested concentrations. Flavonoids, phenol, phytosterols, terpenoids, alkaloids, and tannins were detected in the samples fermented for 7 and 14 days, respectively. The zones of inhibition (in diameter) were in tune with the tested concentrations, and this ranged between 6mm – 15mm on day 7 and 6mm-17mm on day 14 against the test pathogens. Hence, this study validates the antimicrobial, and antioxidant activity of fermented seeds of T. foenum-graecum and supports its use as an herbal therapeutic agent against diseases associated with the test pathogen


Introduction
The effectiveness of many commonly used antibiotics is being compromised globally due to the emergence of multi-drug-resistant bacteria (Falcone & Paterson, 2016;Van Duin & Doi, 2017). Thus, a surge in the need to search for new antimicrobial agents to combat bacterial resistance. Plants with medicinal values constitute reliable sources of potent medicines. Going by worldwide records, around 258,650 species of plants, and more than 10% of such plants are used for the development of drugs against different ailments (Uniyal et al., 2006;Shinwari, 2010;Walter et al., 2011).
It is widely grown and frequently exported from India, China, Africa, Algeria, Saudi Arabia, Pakistan, Egypt, Turkey, Ukraine, Spain, and Italy (Salehi, 2008). In major Nigerian dialects, it is called Eru among the Yorubas, Kimba/Hulba among the Hausas, Mkpuru oka oyibo among the Igbos.
Owing to its numerous medicinal values and dietary fiber content, it is one of the oldest medicinal plants used in Rome and Egypt as "Hilba" tea to ease childbirth and increase milk flow, alleviate menstrual pains, and stomachrelated problems (Mortel and Mehta, 2013). Its seeds have been reported to possess some medicinal properties such as lactation aid, antidiabetic, hepatoprotective effects, and anticancer potentials (Srinivasan, 2006). There are extensive scientific reports on the hepatoprotective potentials of leaves and seeds of T. foenum-graecum against cancer (breast and colon) and their curative potentials for diabetes and hypercholesterolemia (Al-Oqail et al., 2013)

Plant Material
Dried T. foenum-graecum seeds were obtained from vendors in Kano, Northern region of Nigeria. The seeds were stored after identification and authentication (Assigned voucher number: UILH/004/1497/2022) at the Herbarium unit, Department of Plant Biology, University of Ilorin, Ilorin, Nigeria.

Test Microorganisms
Three bacterial pathogens used in the study, Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli and Salmonella typhi) were gotten from the Microbiology Laboratory of Al-Hikmah University, Ilorin, Nigeria, and maintained on Nutrient agar. They were further tested for viability at 37°C for 24 and maintained on agar slant at 4 o C till needed for further use. Turbidity of the test bacterial cultures was set at 0.5 McFarland turbidity standard; a few colonies of each organism from the subcultured agar plate were standardized using a UV spectrophotometer (Lemfield Medical England, MODEL 752G) to get an absorbance reading of (0.063-0.1) at a wavelength of 600 nm (equivalent to 0.5 McFarland turbidity standard) (Ochei & Kolhatkar, 2008).

Preparation of Fermented Seeds of T. foenumgraecum
The seeds were carefully sorted for healthy ones and stones were removed, they were rinsed using flowing tap water and later distilled water. The sample was drained for 15 mins. In a sterile Mason jar, 800 mL of distilled water and 200g of the cleaned seeds were combined (8:2 v/w). The jar was covered tightly and agitated daily to combine the content and facilitate the process of fermentation (Guarner & Schaafsma, 1998). The fermentation process was carried out for 14 days at room temperature after which the sample was centrifuged for 15 min at 14,000×g. The resulting supernatant was sifted using Whatman (no. 1) filter paper and kept for further use.

Preparation of working concentration of the sample
The filtrate was diluted serially to have different concentrations (100%, 50%, and 25%). From a tube containing 100% sample, 10 mL was transferred into a clean tube containing 10% sterile distilled water to generate 50% concentration. Another 10 mL was transferred from the tube containing 50% concentrated sample into a new clean tube of 10 mL of sterile distilled water to generate 25% concentration (Al-Bakri and Afifi, 2007).
Antimicrobial activity of fermented seeds of T. foenum-graecum The activity of the sample against each strain was tested by the Agar well diffusion method (Valgas et al., 2007). Into approximately 20mL of sterile molten nutrient agar medium, a sterile syringe was used to introduce (by aseptic means) 0.5mL of standardized inoculum of the isolates. The impregnated dishes were allowed to solidify at room temperature on the lab flat surface, then, a sterile cork borer measuring 8mm in diameter was used to bore a well in the impregnated dish. A quantity (0.1mL) of the fermented sample was introduced into the well and the plates were incubated for 24 hours at 37 °C. The clear zone without growth was recorded after 18-24 hours. Plates were prepared in triplicates for each tested concentration of the sample.

ABTS Radical Scavenging Activity
The antioxidant activity of the sample was assayed by the ABTS cation scavenging activity approach described by Gulcin et al. (2011). By this method, a 7 mM solution of ABTS was reacted with 2.45 mM of potassium persulfate solution and left in the dark overnight to give a dark-colored solution of ABTS radical cations. Before use, the resulting ABTS radical cation was thinned with methanol (50%) for the first absorbance (at 745 nm) of 0.70±0.02 at a temperature control of 30°C. Subsequently, free radical scavenging activity was estimated by mixing the test studied sample (300 μl) with 3.0 mL of working standard of ABTS in a microcuvette. The reduction in absorbance was determined every minute up to 6 min after mixing the solution. Then, the inhibition (%) was determined using the formula: ABTS scavenging activity (%) = (A0-A1)/ A0 x 100 while A0 represents the absorbance of the control, A1 represents the absorbance of the sample.

Results
The value of ABTS antioxidant activity of T. foenum-graecum during the fermentation period increased along with the concentration ( Figure  1).

Figure 1: ABTS Radicals Scavenging Activity of Fermented Seeds of T. foenum-graecum
The antioxidant activity of the sample by the DPPH method increased correspondingly with the concentration tested ( Figure 2).  (Table 1).

Discussion
The concentration-dependent manner of antioxidant activity reported in this study indicated the free radicals scavenging property of the T. foenum-graecum Previous studies have shown that plants harbor some compounds with anti-inflammation and antioxidant activities (Arora & Pandey-rai, 2014;Benzidia et al., 2018).
Similar antioxidant ability of fenugreek has also been reported by Naidu et al. (2011) Phytochemical compounds are natural biologically active compounds occurring in plants, which confer enormous medicinal and nutritional benefits on humans (Hasler & Blumberg, 1999).
Fenugreek harbors secondary metabolites such as flavonoids, alkaloids, and tannins (Taylor et al., 2006) which in abundance, may predominantly have deleterious effects on its nutrients. However, microorganisms associated with fermentation can alter the food sample by causing the structural breakdown of the walls of the sample, leading to the synthesis and release of new bioactive compounds (Liu et al., 2017;Ricci et al., 2019). Hence, in this study, the phytochemical screening of T. foenum-graecum conveyed the presence of flavonoids, alkaloids, phenol, phytosterols, terpenoids, and tannin which may individually or collectively, attributed to the antibacterial properties of fenugreek seeds as previously reported (Khursheed et al., 2012).
Flavonoids are ever-present polyphenolic compounds occurring in numerous derivatives such as glucosides, aglycones, and methylated (Narayana et al., 2001;Mahmomoodally et al., 2005). They have numerous biochemical properties with the best, being their antioxidant potential (Kelly et al., 2002). Terpenoid shows immunobiologically properties and may have therapeutic importance on various infections. Tannins have well-known antimicrobial and astringent activity (Chung et al., 1998).
The decline in the pH value of the fermented sample means an increase in the acidity of the sample which may be due to the production of organic acid-producing organisms e.g., Lactic acid bacteria (Broadbent et al., 2010). which creates stressful conditions for spoilage-causing microorganisms and outcompete them in foods by reducing the pH (Meyer et al., 2009).
The concentration-related zone of inhibition obtained by different concentrations of the sample against the test organisms (S. aureus, E. coli, and S. typhi) agrees with the work of Ritu et al. (2010) which reported the activity of fenugreek seed extracts against E. coli, L. acidophilus, B. cereus, and Pneumococcus. The wider zone of inhibition recorded against S. typhi than E. coli authenticates the work of other researchers where a broad spectrum of antagonistic activity was reported against Gramnegative pathogens (Liasi et al., 2009;Grosu-Tudor et al., 2014;Setyawardani et al., 2014;Jose et al., 2015). However, the work of Kloos (1998) is in disagreement with previous reports that Staphylococci (a Gram-positive organism) develops resistance swiftly to antibiotics. Anas et al. (2008) contradict this as a higher degree of antagonistic activity was reported against Grampositive organisms than Gram-negative organisms.

Conclusion
From this study, fermented seeds of T. foenumgraecum harbor phytochemical constituents with several health importance. It has also shown antioxidant and antimicrobial activity against selected microorganisms and thus, can be exploited for the development of safe antimicrobial agents.