**3.1 Antioxidant activity of fenugreek**

The fenugreek seeds contain polyphenolic compounds, which have been correlated to the beneficial health effects of fenugreek [55]. These polyphenolic compounds are known for several beneficial actions, such as antioxidant effect [56], cancer preventive activity [27], anti-diabetic effects [57] and hypocholesterolemic effect [12, 56].

In earlier studies Bors et al. [58] reported that the scavenging activities of phenolic substances are attributed to the active hydrogen donating ability of hydroxyl substituents. As an overall assessment, the presence of various phytochemicals, particularly naringenin and quercetin, may be responsible for the OH radical scavenging activity. Similarly, trigonellin isolated from ethanol extract of fenugreek seeds has been reported to reduce blood sugar and lipid profile in alloxane-diabetic rabbits [59]. This effect can be partially explained by the antioxidant properties of trigonellin due to its structural similarity to nicotinamide, which has an antioxidant effect [60].

Bukhari et al. [61] reported that fenugreek seed extract with methanol, ethanol, dichloromethane, acetone, hexane and ethyl acetate has a radical scavenging activity. In addition, Bhatia et al. [62] reported protective effect of fenugreek, on lipid peroxidation and on enzymatic antioxidants. Naidu et al. [63] reported that extracts of husk, fenugreek seed, and endosperm exhibited 72%, 64%, and 56% antioxidant activity respectively by free-radical scavenging method. Also, it was indicated that separation of fenugreek seeds into husk and endosperm could have advantage of process viability with respect to prior selective fractionation of bioactive components for their effective isolation.

In a similarly study, it was determined that fenugreek has a high phenolic content. Furthermore, antioxidant property was checked by reducing power, nitro blue tetrazolium (NBT) assay and H2O2 scavenging reported to show high superoxide and free radical scavenging [64].

Furthermore, Kaviarasan et al. [65] reported that 2,2′-Azinobis-(3 ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals are more reactive than 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and unlike the reactions with DPPH radical which involve H atom transfer, the reactions with ABTS radicals involve electron transfer process. In addition, Shang et al. [66] identified five different flavonoids in fenugreek seeds, namely vitexin, tricin, naringenin, quercetin, tricin-7-O-beta-D-glucopyranoside, and fenugreek seed extract was found to have significant antiradical and antioxidant properties depending on the concentration.

In line with above researchers [65], they reported that an aqueous methanolic extract of fenugreek seeds was investigated for antiradical and antioxidant activity in different model systems, and antiradical activity was associated with the polyphenolic contained in the extract. As a result, it was determined that fenugreek seeds provide some important factors responsible for the antioxidant potential and provide evidence for numerous in vivo beneficial effects of seeds reported in the literature.

Similarly, Belguith-Hadriche et al. [67] investigated the hypocholesterolemic and antioxidant activities of various extracts of fenugreek seeds (water, methanol, ethyl acetate, hexane, dichloromethane) in rats fed cholesterol, and ethyl acetate only for rats fed a cholesterol-rich diet (HCD). It has been found that fenugreek extracts reduce triglycerides and low density lipoprotein cholesterol (LDL-C) and increase high density lipoprotein cholesterol (HDL-C). Based on these results, it was reported that ethyl acetate extract of fenugreek seeds had a significant hypocholesterolemic effect and antioxidant activity in cholesterol fed rats.

Furthermore, Liu et al. [68] determined the lipid peroxidation (LPO) and cyclooxygenase enzyme (COX) inhibitory activities of hexane, ethyl acetate, methanolic and water extracts to investigate the functional food use quality of fenugreek. They found that the extracts inhibited LPO by 55–95%, COX-1 by 6–87% and COX-2 by 36–70% at 250 lg/ml, respectively. Also, the isolates, excluding the saccharides, inhibited LPO and COX-1 and COX-2 enzymes between the ranges of 8–89%, 4–51% and 15–70%, respectively, at 25 lg/ml. The fenugreek seeds that were studied afforded 3.9 g of triglycerides and fatty acids, 6 g of polysaccharides and 233 mg of flavone C-glycosides per 100 g of seeds. The strong antioxidant activity in the LPO assays of the aqueous extract of fenugreek seed might be attributed to the flavones C-glycosides [69].

Likewise, the different solvent extracts of fenugreek seeds were used to examine the effects of extraction solvent on total phenolic content (TPC), DPPH and iron reducing antioxidant power (FRAP). It was observed that the extracts obtained using higher polar solvents were more effective than less ones, and the addition of 50% water to methanol, acetone or ethanol can enhance the extracting power and antioxidant activity estimation especially acetone and methanol. As a result, it was determined that the total phenolic content showed a good correlation with antioxidant activity FRAP and DPPH [70].

In similarly, Deshmukh et al. [71] reported that silver and iron oxide nanoparticles were successfully synthesized in a simple way at room temperature using an aqueous extract of fenugreek seeds. Then, all nanoparticles were characterized by various techniques to elucidate the stability and functionality of the nanoparticle. It has been determined that the nanoparticles synthesized with the assistance of ultrasound show higher stability and antibacterial and antioxidant activity due to the combined effect of ultrasound and biomolecules adhering to the surface of the nanoparticles.

Naidu et al. [6] also observed that the husk of fenugreek seeds contained higher total polyphenols (103.8 mg gallic acid equivalent/g and the total dietary fiber

#### *Assessment of Secondary Metabolites with Different Uses of Fenugreek DOI: http://dx.doi.org/10.5772/intechopen.99479*

(77.1 g/100 g), insoluble dietary fibers (31.9 g/100 g) and soluble dietary fibers (45.2 g/100 g). The bark, fenugreek seeds and endosperm extracts were reported to exhibit 72%, 64%, and 56% antioxidant activity, respectively, by the free radical scavenging method. As a result, separation of fenugreek seeds into husk and endosperms showed that the process viability advantage.

The antioxidant properties of germinated fenugreek seeds were examined in a study conducted by Dixit et al. [72]. Different fractions of germinated seeds were used at different levels to determine their antioxidant potential. Tests used are ferric reducing antioxidant power, DPPH, feriylmyoglobin / 2,2-azobis-3-ethylbenzthiazoline-6-sulfonic acid, pulse radiolysis, oxygen radical absorbance capacity and lipid peroxidation in rat liver mitochondrial preparations. An aqueous fenugreek fraction showed the highest antioxidant activity. Since the amount of phenolic and flavonoid compounds can be correlated with antioxidant activity, the contents of these extracts were measured and their polyphenols, flavonoids and other components were determined by HPLC analysis. This study reveals significant antioxidant activity in germinated fenugreek seeds, which may be due in part to the presence of flavonoids and polyphenols.

As seen from previous studies, the obtained different results may be attributed to different extraction methods and solvents used, different cultivars, growing conditions, maturity stage at harvest, or the storage conditions and time elapsed before the seeds were analyzed. Synthetic drugs used for the treatment of the diseases like cancer, diabetic and the antioxidants used for some treatment have side effects such as mutagenic and carcinogenic effects [73]. Some patients also have resistance to the synthetic drugs. To overcome this problem there is need to find effective natural drugs from traditional medicine. Therefore, fenugreek, which possesses phenolic compounds and antioxidant activity should have the ability to counteract these situations and might be a good candidate for a herbal drug.

#### **3.2 Antibacterial and antifungal effect of fenugreek**

The antibacterial activity of the plant extraction has been extensively investigated in many studies. Microbiological analyses revealed that fenugreek extracts exhibit antimicrobial activity against numerous bacteria [74]. Haouala et al. [75] determined the aqueous extracts obtained from various plant parts of fenugreek, different solvents such as methanol, petroleum ether and ethyl acetate fractions and their effects against fungal strains such as *Fusarium graminearum*, *Botrytis cinerea*, *Alternaria sp*., *Rhizoctonia solani* and *Pythium aphanidermatum*. It was found that all parts of the fenugreek exhibited antifungal potential and the magnitude of the effect varied according to plant parts and fungal species. So, they suggested that fenugreek is an important source of biologically active compounds that are useful for developing better and new antifungal drugs.

Many studies have indicated the effectiveness of fenugreek extracts against *Helicobacter pylori* [76–80]. In a study conducted with honey produced with different plant pollens, the highest antibacterial activity against *Staphylococcus aureus*, *Pseudomonas aeruginosa*, and *Escherichia coli* was found in honey produced with fenugreek pollens [79].

Since cysteine-rich peptides, defensins have strong antifungal activity, the methanol-soluble fraction of fenugreek extract has been studied against nematodes and has been found to show nematicidal activity. It has also been reported to significantly cause the death of *Meloidogyne javanica* larvae [80]. Laroubi et al. [81] studied the prophylaxis effect of fenugreek seeds on renal stone formation in rats. They reported that, the fenugreek can be used in the treatment of patients with calcic urolithiasis.

In addition, Shaheed et al. [82] recorded that the inhibition results for each *Proteus mirabilis* and *E. coli* reached 10.5 and 10.0 mm, 9.0 and 9.5 mm, respectively at 50 mg/ml, and 13.0 and 11.5 mm, 10.5 and 7.5 mm, respectively at 100 mg/ml. Similarly, Hamadii [83] showed the highest percentage of inhibition against *Proteus mirabilis* and *E. coli* at 50 mg/ml from alcoholic fenugreek seed extract.

In another study, the antibacterial effects of fenugreek oil against *Escherichia coli, Samonella, typhimurium, Taphylococcus aureu, Aspergillus niger* microorganisms were investigated. These microorganisms have been chosen as common causes of some human and animal diseases and are contaminants that damage certain foods and are resistant to antibiotics. These results showed that fenugreek oil was stated to be suitable for human consumption and eating [84].

Similarly, it was determined that fenugreek essential oil showed the highest activity against *E. coli*, the inhibition zone reached 21 mm at 100% concentration, and against *Staphylococcus aureus*, it was found to reach 17 mm at 80% concentration [85]. Also, Sulieman et al. [86] found that the inhibition results for *Salmonella* reached 16 mm at 100% concentration. Thereby, these results are promising and may contribute to the future development of natural bio pesticides for the control of fenugreek for the microorganisms. Likewise, antibacterial effect was determined against *Escherichia coli* with an inhibition value of 20 mm at 100% concentration of fenugreek seed oil, while the antimicrobial activity was not detected at 50% and 90% concentrations [86]. As a result, fenugreek extracts show antibacterial activity against many gram positive and gram negative bacterial isolates. In addition, fenugreek seeds are potential sources of new antibacterial compounds as emphasized by the antibacterial activity of their different extracts. So, identification of different effective bacteria from crude plant extracts will assist in the development of drugs against pathogenic microorganism.

#### **3.3 Anticancer effect of fenugreek**

Cancer remains one of the leading causes of death worldwide. Flavonoids could also significantly contribute to fenugreek's anticarcinogenic properties. Fenugreek constitutes valuable raw material for the pharmaceutical industry that has long searched for effective cures for cancer.

Previous studies reported that fenugreek seeds have a preventive effect on cancer as in experimental models of cancer using cell lines or experimental animals.

Earlier studies revealed that seed extract of fenugreek importantly inhibits 7,12-dimethyl benz(a)anthracene-induced mammary hyperplasia and decreases its ratio in rats. It was also advised that anti-breast cancer preventive effect of fenugreek could be depending on the increasing apoptosis [87]. Furthermore, alcoholic whole plant extracts of fenugreek effected in vitro cytotoxicity against different human cancer cell lines such as IMR-32, a neuroblastoma cell line, and HT29, a cancer cell line [88].

A selective cytotoxic effect of fenugreek extract in vitro to a panel of cancer cell lines has been observed, including T-cell lymphoma by Alsemari et al. [89]. In addition, Sebastian and Thampan [90] and Prabhu and Krishnamoorthy [91] examined the growth of MCF-7 cells, which is an estrogen receptor positive breast cancer cell line, with ethanol extracts of fenugreek, and reported that the ethanol extract of fenugreek decreased cell viability and induced early apoptotic changes such as inversion of phosphatidyl serine and decreased mitochondrial membrane potential.

In a study conducted by Shabbeer et al. [92] treatment with fenugreek extract showed growth inhibitory effects on breast, pancreatic and prostate cancer cell lines but primary prostate or immortalized prostate cells remained unaffected.

#### *Assessment of Secondary Metabolites with Different Uses of Fenugreek DOI: http://dx.doi.org/10.5772/intechopen.99479*

In addition, in a dietary study involving fenugreek seed powder, it reduced colon tumor incidence and hepatic lipid peroxidation in rats treated with 1,2-dimethylhydrazine and also increased catalase, superoxide dismutase, glutathione S-transferase and glutathione peroxidase activities in the liver [93].

Li et al. [94] recorded that diosgenin modulates the STAT3 signaling pathway in hepatocellular carcinoma by suppressing the activation of c-Src, JAK1 and JAK2. They also noted that diosgenin reduced the expression of various STAT3-regulated genes, inhibited proliferation, and potentiated the apoptotic effects of paclitaxel and doxorubicin, which could be a new and potential treatment option for hepatocellular carcinoma and other cancers. Also many researchers reported that diosgenin exhibited anticancer and antiaging activities, as well as cardioprotective and contraceptive properties [44–49].

In addition, in different studies with diosgenin, it has antiproliferative activity such as prostate cancer (PC-3 and DU-145 cells) [95], colon cancer (HCT-116 and HT-29 cells) [96], erythroleukemia (HEL cells) [97], carcinoma (A431, Hep2 and RPMI 2650 cells) [98], stomach cancer (BGC-823 cells) [99], lung cancer (A549 [100], breast cancer (MCF-7) [101], hepatocellulacarcinoma (HepG2 and HCC cells) [102] and human chronic myeloid leukemia (CML) (K562 cells) [103].

As a result of the studies mentioned above, the role of fenugreek seeds and its main active ingredients as new supplements in diet-based preventive / therapeutic strategies to potentially alleviate human diseases remains an important area of study for future research [104].

## **3.4 Hypoglycemic effect, anti-inflammatory and immunological activity of fenugreek**

Immunological changes include altered levels of cytokines and chemokines, changes in the numbers and activation states of various leukocyte populations, apoptosis, and fibrosis during diabetes. Therefore, treatment of diabetes and its complications may include pharmacological strategies to reduce inflammation [105]. Laroubi et al. [81] studied the prophylaxis effect of fenugreek seeds on renal stone formation in rats. And they said that the fenugreek can be used in the treatment of patients with calcic urolithiasis. Chauhan et al. [106] reported an antiinflammatory potential of fenugreek. Jung et al. [107] observed a reduction in the production of several inflammatory mediators, including NO and interleukins 1 and 6, in murine macrophages which had been pretreated with diosgenin and stimulated with lipopolysaccharide/interferon-.

In addition, Roberts [108] said that the gum, composed of galactose and mannose, is associated with reduced glycemic effect. Also, the hypoglycemic effect of fenugreek has been especially documented in humans and animals with type 1 and type 2 diabetes mellitus.

Xue et al. [109] reported that the fenugreek extract can lower kidney/body weight ratio and blood glucose and also improves hemorheological properties in experimental diabetic rats following repeated treatment for 6 weeks. A study on animals evaluated the hypoglycemic effects of the fenugreek seeds on dogs. The seeds lowered blood glucose levels, plasma glucagons and somatostatin levels; carbohydrate-induced hyperglycemia also was found to be reduced [110]. Most of the studies with polar fractions of fenugreek seeds point toward a strong anti-inflammatory and anti-arthritic activities mediated through anti-oxidant mechanisms [68, 111, 112].

In addition, Sharma et al. [113] recorded that guar gum of fenugreek prevents the rapid uptake of glucose in the small intestine, aids in blood sugar retention in diabetic patients and may also be effective in the treatment of hypercholesterolemia.
