Antimicrobial and Antioxidant Assessment of Trigonella foenum-graecum

Trigonella foenum-graecum is an imperative medicinal herb of Leguminosae family (Meena et al., 2021). Trigonella is the largest genera in the family Fabaceae (Ahlawat et al., 2023). It is one of the renowned Indian spices used in different cuisines and is commonly known as fenugreek (Qadir et al., 2022). The plant has a global distribution. India is the largest producer of fenugreek, followed by China, Pakistan, Iran, Afghanistan, Russia, Australia, Canada, USA and Europe (Singh et al., 2022).Fenugreek is the third largest seed spice after coriander and cumin (Shekhawat et al., 2023).T. foenum-graecum is a well-known anthelmintic, expectorant, hepatoprotective and immunity booster herb. Traditionally, it is used to cure gastric problems, joint pain, anxiety, pneumonia, bronchitis, fever, cold and cough. The plant is highly recommended for curing digestive problems, hair loss and reproductive health. The folkloric use of this medicinal herb is either inherited from elders or acquired through trials. These traditional uses also endorse multiple pharmacological efficacies of fenugreek (Sulieman et al., 2008).
       
Many studies have also documented the anticancer, anti-diabetic and anti-inflammatory role of fenugreek.The plant is effectively used in treating digestive problems, reproductive health problems and hypocholesterolemic issues (Syed et al., 2020). These pharmacological effects result from their diverse biologically active phytoconstituents, such as alkaloids, flavonoids, phenols, vitamins, minerals, amino acids, fibers and bioactive metabolites. These phytocompounds are mainly concentrated in their essential oil and bestow them numerous pharmacological effects. As a result, the essential oil help in curing many maladies in the case of human beings and animals (Salman and Qadeer, 2021). Moreover, EOs and their derivatives are increasingly used in the pharmaceutical and food industries. Numerous studies have highlighted the high efficacy of EOs against pathogenic microbes.
       
The EOs derived from spices are frequently used as flavouring, antimicrobial and antioxidant agents in food processing and pharmaceutical industries. The demand for herbal food preservatives and therapeutic agents is increasing daily. Hence, the present study was planned to evaluate the antimicrobial and antioxidant potential of T. foenum-graecum.

The work was carried out at Maharshi Dayanand University, Rohtak, Haryana (124001) from 2021-2023.The analytical-grade chemicals were procured from Sigma-Aldrich and Himedia. In the present study, we worked on the trigonella seeds. The seeds were purchased from the local market of Rohtak (Haryana), India-124001. The different plant parts of T. foenum-graecum are presented in Fig 1.
 

 
Extraction of trigonella seeds
 
The extraction was done using the Soxhlet apparatus (Fig 2). The thimble was prepared with 30 gm of seed powder. The extraction was performed in petroleum ether at 40-60°C for 4-5 hours. The extract was left to evaporate the solvent and to obtain the pure oil. The water droplets were removed using anhydrous sodium sulfate (Gungor et al., 2022).
 

 
Quantification of trigonella oil
 
The extracted oil yield was calculated using the standard equation given by Akbari et al., (2019). The Eq. was as follows:  
GC-MS analysis
 
GC-MS was carried out to identify the phytocompound composition of Trigonella seed oil. The analysis was done through394143602-GC Single Quadruple Mass Spectrometer SCION 436 GC; Fill Scan SIM, with CP-739651-CP8410 Liquid Auto Sample (Caputo et al., 2022).
 
Antimicrobial assessment
 
Microbial strains
The antimicrobial activity was carried out against four bacterial and two fungal strains as shown in Fig 3.
 

 
Disc-diffusion assay
 
The primary culture was obtained by inoculation and incubation (37°C overnight) of freshly prepared autoclaved nutrient broth media. The secondary culture was prepared by inoculating the freshly prepared autoclaved nutrient broth media from the primary culture and incubating for 3-4 hours to achieve the microbial concentration of 8 ×10⁸ cells/mL at 600 nm. The freshly prepared agar plates were inoculated with 100-150 µL of microbial suspension, followed by infusion of sterile discs with 30 µL of different concentrations of serially diluted Trigonella seed oil (stock solution of 10 mg/mL prepared in DMSO). The standard used for antibacterial and antifungal was Ciprofloxacin and fluconazole discs, respectively. In the case of fungal strains, the media was potato dextrose broth and incubation were done at 30±5°C for 48 hours (Errouane et al., 2020).
 
Broth dilution assay
 
For minimum inhibitory concentration, 20 µL aliquot (prepared in 0.85% of NaCl) was added to test tubes containing different concentrations of test samples, followed by a standard incubation period. The test tube without turbidity (MIC) was further tested for minimum bactericidal concentration. For MBC, 10 µL of non-turbid culture was again incubated at optimum temperature and duration. The microbial growth was observed visually (Errouane et al., 2020).
 
Antioxidant activity
 
DPPH assay
 
The assay was performed according to Akhlaghi and Najafpour-Darzi (2021). The 0.1 mM DPPH was prepared in methanol. One mL of methanolic DPPH was mixed with 1 mL of different concentrations of essential oils. The solution was left in the dark for 30 minutes. The solution was stirred vigorously and then poured into quartz cuvettes. The fluctuations in the absorbance were monitored at 517 nm. Pure methanol was used to zero down the spectrophotometer. Ascorbic acid was used as standard. The assay was done in triplicates. The radical inhibition by DPPH assay was calculated as follows Eq 2:
                                    
Where,
A_control = Absorbance of the control reagents excluding test sample.
A_sample = Absorbance of the test sample.
 
ABTS Assay
 
ABTS radical cation was prepared by mixing an aqueous ABTS solution (7 mM) with an aqueous solution of K₂S₂O₈ (140 mM). The solution was kept for 16 hours in the dark followed by dilution in methanol up to an absorbance of 0.8 at 734 nm. The one mL ABTS solution was dissolved with one mL concentrations of different essential oils. After vigorous stirring, the solution was poured into quartz cuvettes. The spectrophotometer was zeroed down with pure methanol. Ascorbic acid was used as the positive control. The decreased absorbance for each sample was registered at 734 nm. The readings were taken thrice to deduce the mean value (Kaviarasan et al., 2007).

Yield of oil
 
The oil extracted from Trigonella seed was yellow in colour with a viscous texture. The oil had a characteristic aroma of fenugreek seeds. The yield of trigonella seed oil was calculated using Eq. 1.
Trigonella oil specific density = 0.98 g/mL.
The volume of the obtained oil = 20 mL.
Mass = Density × volume; 0.98 g/mL × 19.6 mL= 19.6 gm
   
The yield of oil obtained from Trigonella seeds was 4.35%.
 
GC-MS results
 
GC-MS analysis revealed the presence of numerous phytocompounds in Trigonella seed oil. The different retention times represented the presence of different phytocompounds. Dibutyl.pthalate was recognized as the major phytocompound of Trigonella seed oil. Other compounds namely Benzaldehyde 4-(1-methylethyl), Cinnamaldehyde, n-Pentadecanol, Phenol, 2,4-bis (1,1-dimethyl ethyl), Aspidospermidin-17-ol, 1-acethyl-19,21-epoxy-15,16-dimethoxy, Murolan-3,9 (11) diene-10-peroxy, Pthalic acid butyl hexyl ester and i-Proxy 7,10,13,16,19-docosapent have also been identified. The physicochemical properties of different phytocompounds of Trigonella seed oil are shown in Fig 4.
 

 
Antimicrobial activity
 
The maximum antibacterial activity was observed against E. coli. In fungal strains, the oil had shown a zone of inhibition against F. oxysporum only. In the case of MIC, the oil had shown good MIC except forS. aureus. Though the oil had no bactericidal potential but it has shown fungicidal potential against A. oryzae. The antimicrobial results are shown in Table 1 and 2.
 

 

 
Antioxidant activity
 
The compounds with lesser absorbance are found to have higher free radical scavenging activity and antioxidant potential. In the present study, Trigonella oil was also reported to have significant antioxidant potential (Fig 5). ABTS assay has given more pronounced antioxidant results than the DPPH assay.


       
Medicinal plants have always remained integral to treating routine maladies and maintaining healthcare. They are an affluent reservoir of various phytocompounds (Singh et al., 2022). These phytocompounds endow diverse therapeutic attributes to the plants. The phytocompounds are mainly concentrated in the plant essential oil. Essential oils are used as food additives. They not only enhance the food taste but also preserve the food items. In developing countries, the fenugreek plant can be used as a nutrient supplement to avoid fungal infections, oxidative stress and associated health issues (Gurjar et al., 2016). Considering the various traditional medicinal uses of Trigonella foenum-graecum, we extracted essential oil from Trigonella seeds in the present study. The yield of oil obtained in the current study (4.35 %) was compared to a previous where they reported a 5.55±0.05% yield (Akbari et al., 2019). The difference in yield may be due to the difference in solvent used. The seed oil was characterized by GC-MS. They also analyzed the chemical composition of Trigonella seeds oil and reported approximately 23 compounds (Akbari et al., 2019). The Trigonella essential oil composition was also evaluated by Qadir et al., (2022) and the findings were in concordance with the present study’s findings. Though we also analyzed the chemical composition of Trigonella oil but the phytochemical composition obtained in the present study was different from the previously documented results. In the present study, Dibutyl. phthalate was obtained as the major phytocompound. In other studies, linoleic acid was reported as the major phytocompound (Ali et al., 2012; Ciftci et al., 2011). The difference may be due to one or more of the following reasons: (a) different variety types, (b) growth stages, (c) climatic conditions, (d) harvesting time and others (Behbahani et al., 2019; Lal et al., 2017).
       
Spice essential oils are good antimicrobial agents and are effective against many bacterial and fungal strains. Therefore, in the present study, we screened Trigonella essential oil against a panel of bacterial and fungal strains. The oil had shown significant antifungal activity but almost negligible antibacterial activity. Sulieman et al., (2008) also studied the antimicrobial efficacy of Trigonella seed oil. They reported that the oil possesses higher antifungal activity than antibacterial activity (Sulieman et al., 2008). Yousefipour et al., also reported the antimicrobial activity of Trigonella aqueous extract (Yousefipour et al., 2022).
       
The antioxidant potential of essential oils improves the immune system of human beings. They act as good free radical scavengers, avoiding oxidative stress and associated disorders. In the present study, we carried out DPPH and ABTS assays to assess the antioxidant potential of Trigonella seed oil. Priya et al., (2011) also reported the antioxidant potential of hydroalcoholic extract of trigonella seeds (Priya et al., 2011). Though both the assays in the present study have shown significant results but the maximum activity was obtained with the ABTS assay. It may be primarily because ABTS radicals are more reactive than DPPH assay.

Trigonella foenum-graecum is an important dietary spice with plenty of phytocompounds. The presence of different phytocompounds was confirmed by GC-MS. GC-MS has shown Dibutyl.pthalate as the major phytocompound of trigonella seed oil along with Benzaldehyde 4-(1-methylethyl), Cinnamaldehyde, n-Pentadecanol, Phenol, 2,4-bis (1,1-dimethyl ethyl), Aspidospermidin-17-ol, 1-acethyl-19,21-epoxy-15,16-dimethoxy, Murolan-3,9 (11) diene-10-peroxy, Pthalic acid butyl hexyl ester and i-Proxy 7,10,13,16,19-docosapent. These phytocompounds are backed scientifically by various therapeutic potentials. Though the oil has shown less antibacterial activity but has shown significant antifungal activity. The antioxidant assays also confirmed the radical scavenging activity of Trigonella seed oil. The maximum antioxidant activity was obtained with ABTS assay. Further in-vivo and clinical trials are needed to explore the full efficacy of Trigonella essential oil.

Financial assistance from the Council for Scientific and Industrial Research (CSIR), New Delhi and Science and Engineering Research Board-Department of Science and Technology (SERB-DST), Government of India, New Delhi, Fund for Improvement of SandT Infrastructure in Universities andHigher Educational institutions (FIST), Department of Science and Technology, Govt. of India, New Delhi is thankfully acknowledged.
 
Author contribution
 
Neetu Singh designed the paper, wrote the paper draft and performed the experiments; Surender Singh Yadav and Balasubramanian Narasihman supervised the study, read the draft and gave valuable suggestions.
 
Ethical approval
 
Not required.

There is no conflict of interest.