Phytochemical Profile of Trigonella caerulea (Blue Fenugreek) Herb and Quantification of Aroma-Determining Constituents

The herb of Trigonella caerulea (Fabaceae), commonly known as blue fenugreek, is used for the production of traditional cheese and bread varieties in the Alpine region. Despite its frequent consumption, only one study so far has focused on the constituent pattern of blue fenugreek, revealing qualitative information on some flavor-determining constituents. However, with regard to the volatile constituents present in the herb, the applied methods were insufficient and did not take relevant terpenoids into account. In the present study, we analyzed the phytochemical composition of T. caerulea herb applying a set of analytical methods, such as headspace-GC, GC-MS, LC-MS, and NMR spectroscopy. We thus determined the most dominant primary and specialized metabolites and assessed the fatty acid profile as well as the amounts of taste-relevant α-keto acids. In addition, eleven volatiles were quantified, of which tiglic aldehyde, phenylacetaldehyde, methyl benzoate, n-hexanal, and trans-menthone were identified as most significantly contributing to the aroma of blue fenugreek. Moreover, pinitol was found accumulated in the herb, whereas preparative works led to the isolation of six flavonol glycosides. Hence, our study shows a detailed analysis of the phytochemical profile of blue fenugreek and provides an explanation for its characteristic aroma and its health-beneficial effects.


Introduction
Trigonella caerulea (L.) Ser. (Fabaceae subfam. Papilionoideae), commonly known as blue fenugreek, is a flowering annual cultivated in the Alps and mountains of eastern and southeastern Europe [1,2]. Unlike the closely related fenugreek (T. foenum-graecum), which is a major component in most curry mixtures and therefore well-known and consumed around the world, T. caerulea is of regional importance [3][4][5]. Blue fenugreek seeds are used as a spice in Georgian cuisine and the Caucasus region, whereas the young seedlings are eaten with oil and salt [4][5][6][7]. In Switzerland, T. caerulea herb is added to the traditional Schabziger cheese in amounts of 2.0 to 2.5%, while it is mixed with flour for the flavoring of bread in South Tyrol, the German speaking part of Northern Italy [4,8]. For bread production, 2 g of blue fenugreek herb are added to 500 g of flour [9]. In South Tyrol, blue fenugreek is stored for a period of up to six months by many traditional farmers before being made commercially available. This measure should increase the aromatic flavor of the herb and the bread made thereof, respectively. The traditional usage of blue fenugreek in the Alps is reflected in its German name, where it is referred to as "Schabzigerklee" or "Brotklee", but also called by the ethically questionable term "Zigainerkraut", which means herb of the gypsies.
The few phytochemical studies on T. caerulea mostly focused on the seeds, which were analyzed together with the seeds of T. foenum-graecum and other Trigonella species [3,10,11].

Polar Metabolites
Polar primary metabolites were analyzed using a metabolomics approach [14]. The polar fraction of a chloroform-methanol-water extract was investigated by means of GC-MS and untargeted profiling. An example chromatogram and compound list of the commercial samples T. caerulea sample 1 to T. caerulea sample 3 (TC1-TC3) are provided in the supporting information ( Figure S1, Table S1). Two peaks were found dominating in the chromatogram, namely those of sucrose and the methylated sugar alcohol pinitol ( Figure 1). In addition, noteworthy signals were detected for fructose and glucose, the sugar alcohols mannitol, myo-inositol, and glycerol, as well as for malic acid, malonic acid, phosphate, and succinic acid.

Fatty Acid Profile
Fatty acid analysis was accomplished by esterification with methanol and subsequent gas chromatography coupled to flame ionization detection and mass spectrometry (GC-FID/MS) analysis. Palmitic acid and α-linolenic acid were found to be the most abundant fatty acids ( Figure 2). Moreover, significant amounts of linoleic acid and stearic acid were found in the herb of blue fenugreek, as well as small amounts of arachidic acid, myristic acid, and margaric acid. A sample chromatogram is depicted in Figure S2 and results are given in Table 1.

Fatty Acid Profile
Fatty acid analysis was accomplished by esterification with methanol and subsequent gas chromatography coupled to flame ionization detection and mass spectrometry (GC-FID/MS) analysis. Palmitic acid and α-linolenic acid were found to be the most abundant fatty acids ( Figure 2). Moreover, significant amounts of linoleic acid and stearic acid were found in the herb of blue fenugreek, as well as small amounts of arachidic acid, myristic acid, and margaric acid. A sample chromatogram is depicted in Figure S2 and results are given in Table 1.

Quantification of α-keto acids
Using ultra-high performance chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) and multiple reaction monitoring, we determined the contents of ten α-keto acids ( Figure 3) after conversion into their O-(2,3,4,5,6-pentafluorobenzyl)oxime derivatives by the method described by Noguchi et al. [15]. Among the thus quantified compounds were also the proposed key components pyruvic acid, α-ketoglutaric

Quantification of α-Keto Acids
Using ultra-high performance chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) and multiple reaction monitoring, we determined the contents of ten α-keto acids ( Figure 3) after conversion into their O-(2,3,4,5,6-pentafluorobenzyl)oxime derivatives by the method described by Noguchi et al. [15]. Among the thus quantified compounds were also the proposed key components pyruvic acid, α-ketoglutaric acid, α-ketoisovaleric acid, and α-ketoisocapronic acid ( Figure S3). Additional relevant α-keto acids were retrieved by means of GC-MS and the method of Lee et al. [16]. A total of eleven α-keto acids were found in considerable amounts and commercial standards were therefore obtained. Of the eleven standards, however, oxaloacetic acid did not show useful results and had to be excluded.

Quantification of α-keto acids
Using ultra-high performance chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) and multiple reaction monitoring, we determined the contents of ten α-keto acids ( Figure 3) after conversion into their O-(2,3,4,5,6-pentafluorobenzyl)oxime derivatives by the method described by Noguchi et al. [15]. Among the thus quantified compounds were also the proposed key components pyruvic acid, α-ketoglutaric acid, α-ketoisovaleric acid, and α-ketoisocapronic acid ( Figure S3). Additional relevant αketo acids were retrieved by means of GC-MS and the method of Lee et al. [16]. A total of eleven α-keto acids were found in considerable amounts and commercial standards were therefore obtained. Of the eleven standards, however, oxaloacetic acid did not show useful results and had to be excluded. The results of the three commercial samples TC1-TC3 are shown in Table 2. In all three samples, glyoxylic acid and α-ketoglutaric acid were the two dominating α-keto acids with amounts of 40 to 86 mg/kg dry plant material. Moreover, high amounts of pyruvic acid (8.1 to 14 mg/kg) were determined, whereas the remaining seven α-keto acids quan- The results of the three commercial samples TC1-TC3 are shown in Table 2. In all three samples, glyoxylic acid and α-ketoglutaric acid were the two dominating α-keto acids with amounts of 40 to 86 mg/kg dry plant material. Moreover, high amounts of pyruvic acid (8.1 to 14 mg/kg) were determined, whereas the remaining seven α-keto acids quantified in this study showed concentrations of 0.4 to 4.3 mg/kg. The total content of α-keto acids was found between 115 (TC3) and 185 (TC2) mg/kg, thus showing distinct variations. However, the three samples also differed with regard to their α-keto acid pattern, i.e., α-ketobutyric acid, showing 3 to 5 times higher amounts in sample TC2, or the ratio of the two major components ranging from 1.8 (TC1) to 0.8 (TC3). acids was found between 115 (TC3) and 185 (TC2) mg/kg, thus showing distinct variations. However, the three samples also differed with regard to their α-keto acid pattern, i.e., α-ketobutyric acid, showing 3 to 5 times higher amounts in sample TC2, or the ratio of the two major components ranging from 1.8 (TC1) to 0.8 (TC3).

Quantification of volatile constituents in commercial samples
Volatile constituents of blue fenugreek herb were quantified using headspace-GC-MS/MS and external calibration with eleven compounds, which were found in relevant concentrations (Figures 4 and S4). The results are given in Table 3 and show three dominating constituents in all three samples, namely tiglic aldehyde, trans-menthone, and camphor. The amounts on camphor (10 mg/kg) and trans-menthone (8.3 to 8.6 mg/kg) were comparable in all three samples, whereas the concentration on tiglic aldehyde was differing significantly with 8.4, 17, and 6.8 mg/kg, respectively. Other compounds found in considerable amounts were The results are given in Table 3 and show three dominating constituents in all three samples, namely tiglic aldehyde, trans-menthone, and camphor. The amounts on camphor (10 mg/kg) and trans-menthone (8.3 to 8.6 mg/kg) were comparable in all three samples, whereas the concentration on tiglic aldehyde was differing significantly with 8.4, 17, and 6.8 mg/kg, respectively. Other compounds found in considerable amounts were benzaldehyde, phenylacetaldeyhde (hyacinthin), safranal, and bornyl acetate, being present in amounts of 1 mg/kg or above. Moreover, all quantified compounds (except p-cymene in samples TC1 and TC2) showed concentrations above their respective olfactory threshold values (Table 3), and thus contribute to the aroma of blue fenugreek herb. Five compounds were found to predominantly affect the odor of blue fenugreek, namely the aldehydes tiglic aldehyde, phenylacetaldehyde, and n-hexanal, as well as methyl benzoate and trans-menthone (Table 4). Other compounds with values significantly above their olfactory thresholds were camphor, menthol, and benzaldehyde, as well as p-cymene in sample TC3.

Isolation and Identification of Flavonoids
In order to detect eventual non-volatile secondary metabolites, ultra-high performance liquid chromatography coupled to photodiode array detection (UHPLC-PDA) analysis of a crude methanol extract of blue fenugreek herb was performed ( Figure S5). Thereby, several flavonoids were detected, showing characteristic UV spectra with absorption maxima at 249 to 265 nm and at 347 to 354 nm, respectively. Additional UHPLC-MS/MS analysis revealed the two major flavonoids to be triglycosides, whereas the minor components showed two sugar moieties. The fragmentation pattern of the flavonoids revealed both hexoside as well as deoxyhexoside moieties, while the remaining aglycone fragments pointed at kaempferol and quercetin scaffolds. As no absolute determination of the structures could be performed without isolating the respective constituents, larger amounts of plant material were extracted and made subjects for a preparative phytochemical work-up.
Repeated chromatographic separation using liquid-liquid fractionation, vacuum liquid chromatography, size exclusion chromatography, and semi-preparative HPLC led to the isolation of the two major flavonoids (1 and 2) along with four minor constituents (3 to 6). After 1D and 2D nuclear magnetic resonance (NMR) spectroscopic experiments and comparison of the acquired data with the data obtained from the literature,

Discussion
The first interesting finding of our detailed phytochemical analyses was revealed using metabolic profiling, by which we discovered pinitol as one of two major carbohydrates ( Figure S1, Table S1). The relative concentration was more or less the same in all of the investigated samples and comparable to that of sucrose, the second highly abundant carbohydrate in blue fenugreek herb. A study on the occurrence and accumulation on pinitol in T. foenum-graecum found the compound to undergo seasonal variation, thereby showing an increase in content in the leaves during the generative period of plant vegetation [30]. In contrast, the content in the stems remained stable for the whole time of the investigations (56 to 126 days after sowing). Eighty-six days after sowing, which was comparable to the harvesting date of our study, T. foenum-graecum showed about half of the amount of sucrose and the same level as glucose in the leaves. Although no quantification of the polar metabolites was conducted in our study, at the same time point, the amount of pinitol in T. caerulea was significantly higher than that of glucose, and rather comparable to the level of sucrose. Apart from the role of pinitol in plant carbohydrate metabolism, the compound was also found to exhibit anti-hyperglycemic effects in vivo, which seem to derive from insulin-sensitizing or insulin-mediating properties, respectively [31][32][33].
Other compounds with health-beneficial effects present in blue fenugreek herb were linoleic acid and α-linolenic acid. In all of the investigated samples, the latter compound was found to be one of the two dominant fatty acids, which was only surpassed by palmitic acid ( Figure S2, Table 1). The fatty acid composition was also the topic of a comparison study of fenugreek and blue fenugreek seeds, which reported high amounts of linoleic acid and α-linolenic acid in the two species and much lower amounts of palmitic acid [11].
In the same study, the occurrence of flavonoids was also proposed, however, with no details on eventual flavonoid types or sugar moieties. A metabolite profiling study on the seeds of three Trigonella species, also showed the occurrence of flavonoids [3], namely C-and O-glycosides of apigenin and luteolin, respectively. However, no assignment of the sugar moieties or eventual linkages were given, indicating the limitations of the metabolomics approach for many secondary metabolites. Furthermore, even more interestingly, no mentions on the occurrence of flavonol derivatives were made, and thus on the flavonoid types detected in our study. Therefore, different scaffolds seem to be present in the seeds and the herb of T. caerulea, with the herb containing predominantly di-and triglycosides of kaempferol and quercetin ( Figure 5). This is of interest as a commercial dietary supplement containing blue fenugreek and kale leaves (4:1) is standardized on the content of flavonoids [13], even though no reports on the flavonoid composition of T. carulea herb have been made so far. This preparation, a hydroethanolic (36%) extract, is intended to prevent skin aging by the antioxidative properties of its ingredients, which was partly demonstrated in a recent study [13]. The identification of the major flavonoids in blue fenugreek by our work might lead to the compounds responsible for the skin-protecting effect and thus lay the basis for future compound-related investigations.
Referring to the study of Ney [8], which so far was the only detailed phytochemical investigation of blue fenugreek herb, the author reported pyruvic acid, α-ketoglutaric acid, α-ketoisovaleric acid, and α-ketoisocaproic acid as being key components of T. caerulea herb. Ney [8] determined the α-keto acids after a reduction to the respective amino acids and subsequent ion exchange chromatography. In our work, we chose the conversion of the α-keto acids to their O-(2,3,4,5,6-pentafluorobenzyl)oxime derivatives and quantification via LC-MS/MS ( Figure S3, Table 2). We thus quantified most of the α-keto acids described by Ney [8], including the supposed key components. Of those, pyruvic acid and even more α-ketoglutaric acid were indeed found in high amounts in all of the investigated samples. However, α-ketoisovaleric acid and α-ketoisocaproic acid were present in lower amounts and in the range of other keto acids, such as α-ketobutyric acid, α-ketovaleric acid, and α-ketoanteisocaproic acid. In addition, we found high concentrations of glyoxylic acid, being the major α-keto acid in two of three commercial samples (Table 2). Even more differing than the results of the α-keto acids, were our findings on the aldehyde composition. Using headspace GC-MS/MS, we determined a completely different profile of aldehydes than Ney [8], who was using thin layer chromatography after conversion to the respective dinitrophenylhydrazone derivatives. With tiglic aldehyde, n-hexanal, benzaldehyde, phenylacetaldehyde, and safranal, we identified five aldehydes with concentrations above 1 mg/kg in the dried herb (Table 3), which were not mentioned by Ney [8]. Out of these, tiglic aldehyde, phenylacetaldehyde, and n-hexanal were found to have a great impact on the aroma of blue fenugreek herb (Table 4). Other compounds clearly affecting the flavor of blue fenugreek were methyl benzoate and trans-menthone. With odor activity values above fifty, these five components should also play a role in the aroma of the traditional Schabziger cheese, for which blue fenugreek herb is added in concentrations of 2.0 to 2.5% [8]. For the flavoring of bread in South Tyrol, instead, only tiglic aldehyde and phenylacetaldehyde would reach the respective concentrations [9]. Apart from trans-menthone, we also determined high amounts of the monoterpene camphor, with a concentration of around 10 mg/kg in all measured samples (Table 3).
Certainly, the explanation of plant aromas by odor activity values is oversimplified as olfactory thresholds were determined in aqueous solutions and do not necessarily show the same values in other matrices. In addition, synergistic and masking effects can affect flavor perception [17]. Still, the findings of our study contribute to the knowledge on blue fenugreek herb and thereby provide an explanation for its characteristic aroma. As the aroma of the herb is said to be increasing over time, further studies will concentrate on eventual processes occurring during storage.
With regard to the initially mentioned high amounts on oxalic acid (1.25%) and the recommended maximum daily intake of no more than 100 mg oxalates for people affected by hyperoxaluria [5], 320 to 400 g of Schabziger cheese or bread made out of 2 kg blue fenugreek-containing flour would therefore have to be consumed. Although these amounts are rather high, the intake of oxalate for people at risk must not have to be underestimated, especially when additional oxalate-rich foods (spinach, rhubarb), teas (licorice), or dietary supplements are consumed.
To summarize, with the results of our study, new and significant knowledge on the constituent pattern of T. caerulea, a plant of growing popularity, is presented. Our findings give explanations for the smell and taste of blue fenugreek and thus for its culinary use. Moreover, with the identification of the major flavonoids, our study reveals the compounds responsible for the supposed beneficial antioxidant effects in nutraceutical preparations. LC-MS grade formic acid, Diaion HP-20, and Sephadex LH-20 were purchased from Sigma Aldrich. Silica gel (40-63 µm) for column chromatography, TLC plates (silica gel 60 F254), acetonitrile and water (both of LC-MS grade), gradient grade methanol, and other (analytical grade) solvents were obtained from VWR International GmbH (Darmstadt, Germany). Water used for isolation was doubly distilled in-house. Dimethyl sulfoxide-d 6 (99.80%, Lot S1051, Batch 0119E) for NMR spectroscopy was purchased from Euriso-top GmbH, Saarbrücken, Germany, and conventional 5 mm NMR sample tubes were obtained from Rototec-Spintec GmbH (Griesheim, Germany).
UHPLC-MS/MS analyses were carried out on a Shimadzu Nexera 2 liquid chromatograph connected to an LC-MS triple quadrupole mass spectrometer using electrospray ionization (Shimadzu, Kyoto, Japan). A Phenomenex Luna Omega C18 column (100 × 2.1 mm, 1.6 µm particle size, Phenomenex, Aschaffenburg, Germany) was employed for the analysis of extracts, fractions, and pure compounds during isolation. Quantification of α-keto acids was accomplished with a Phenomenex Kinetex Biphenyl column (100 × 2.1 mm, 1.7 µm particle size). Headspace GC-MS/MS analyses were performed on a Trace 1310 gas chromatograph equipped with split/splitless (SSL) and programmable temperature vaporizer (PTV) inlets and a TSQ Duo mass spectrometer and the GC-MS instrument used for fatty acid and polar metabolite analysis was a Focus GC gas chromatograph equipped with an SSL inlet, a flame ionization detector and an ISQ mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA). Column used for all GC analyses was a ThermoFisher TG-5SilMS (30 m × 0.25 mm × 0.25 µm). NMR spectra were recorded using a Bruker Avance III 400 NMR spectrometer (Bruker, Rheinstetten, Germany) operating at 400.33 MHz for the proton channel and at 100.66 MHz for the 13 C channel by means of a 5 mm PABBO broad-band probe with a z gradient unit.

Extraction and Isolation of Secondary Metabolites
A total of 855 g dried and ground plant material was extracted 5 times with a mixture of acetone-water (80:20) using ultra-sonication for 15 min and subsequent maceration for 24 h each. After combining the extracts, the acetone was evaporated and the remaining water was subsequently extracted with ethyl acetate followed by 1-butanol to give 20.5 g and 25.2 g of extract, respectively, after evaporation of the solvents. The remaining water layer yielded 71.2 g. The 1-butanol phase was dissolved in 400 mL water and chromatographed over Diaion HP-20 material, using subsequent elution with water, methanol 25% (400 mL), methanol 50% (600 mL), methanol 75% (600 mL) and methanol 100% (600 mL).
The methanol 50% fraction (2.40 g) was also chromatographed with Sephadex LH-20 giving 10 fractions, of which fractions 8 and 9 were further separated by semipreparative HPLC using water (A) and acetonitrile (B) as solvents with the following gradient 15% B to 30% B in 30 min, and to 50% B in 40 min. Thus, 10.1 mg of compound 3 and 10.5 mg of compound 5 were obtained.
The methanol 75% fraction (2.57 g) was as well-subjected to Sephadex LH-20 using methanol-water (50:50) as an eluent and obtaining 12 fractions. Fractions 9 and 10 were chromatographed with semipreparative HPLC in the same manner as before, yielding 15.6 mg of compound 4 and 12.8 mg of compound 6.

Analysis of Polar Metabolites
Analysis of polar metabolites was accomplished with GC-MS and the method adapted by Fiehn [14]. Briefly, 10 mg of plant material was suspended in 100 µL of water and vortexed for 5 min before adding 300 µL of methanol and 100 µL of chloroform. The extraction was incubated for 1 h over ice and 100 µL of the supernatant (water-methanol) was dried under a stream of nitrogen overnight. An amount of 100 µL of a solution of 20 mg/mL methoxylamine hydrochloride in pyridine was added to the dried extract and incubated at 30 • C for 60 min. Subsequently, 150 µL of MSTFA containing 1% TMCS was added and the reaction was incubated for another 60 min at 45 • C. An amount of 1 µL of the derivatized sample was analyzed by GC-MS with the following parameters: 100 • C hold for 5 min, 25 • C/min to 160 • C hold 1 min, 10 • C/min to 300 • C hold for 12 min. MS parameters: full-scan 50-500 m/z. Compounds were identified using the NIST database version 2020.

Analysis of Fatty Acid Profile
A total of 50 mg of plant material was analyzed for the fatty acid composition by directly weighing into a 5 mL reaction tube. An amount of 1000 µL of 2.5% H 2 SO 4 in methanol was added and incubated at 80 • C for 1 h. After cooling at room temperature, 500 µL of n-hexane was added, followed by 1500 µL of saturated sodium chloride solution in water. The n-hexane phase was transferred into a 2 mL glass vial. An amount of 1 µL was directly injected into the GC-MS. The GC parameters were as follows: 50 • C for 5 min and heated with 5 • C/min to 160 • C hold 1 min, 5 • C/min to 300 • C and hold for 5 min. MS parameters: full-scan from 50-500 m/z, ion source temperature: 280 • C. Relative quantification was done under the same conditions using FID.

Analysis of Volatile Constituents
An amount of 100 mg of dried and ground plant material was directly weighed into a 20 mL headspace vial, 100 ng of internal standard (toluene-d 5 ) was added, and the vial was incubated for 30 min at a temperature of 90 • C. An amount of 1000 µL of the headspace was taken with a heated syringe and injected into the GC-MS. The GC program was as follows: 35 • C hold 1 min, 5 • C/min to 120 • C and hold 1 min, 30 • C/min to 300 • C and hold 1 min. The MS parameters were as follows: 43-300 m/z scan with MS Source at 280 • C. Quantification of compounds was achieved by external calibration using original references standards. Odor activity values for each compound were calculated by dividing the obtained concentration by the respective olfactory activity threshold [34].

Analysis of α-Keto Acids
An amount of 1000 mg of dried and ground plant material was extracted using pressurized solvent extraction (70 • C, 100 bar, 1 min heat up, 5 min hold, 2 min discharge) using one cycle of n-hexane and three cycles of methanol, in principle, following the extraction procedure reported by Hidalgo et al. [12]. The methanol extract was evaporated to dryness and reconstituted in 1000 µL of methanol. Following the procedure of Noguchi et al. [15], which was used for quantitation, 5 µL of the solution (or standard solution) was mixed with 45 µL of a mixture of acetonitrile and 0.1% (w/w) NaOH solution (1:1). An amount of 20 µL of this mixture was treated with a mixture of 10 mg/mL PFBHA and kept at room temperature for 30 min before adding 10 µL of acetone and 100 µL of acetonitrile-NaOH 0.1% (w/w) solution (1:1). For qualitative analysis, the PFBOximes were further derivatized using MSTFA (after evaporation of the solvent) and analyzed using GC-MS/MS, following the method described by Lee et al. [16], thus retrieving the most relevant α-keto acids for quantitative analysis. For quantitation of α-keto acids, LC-MS/MS was applied in the multiple reaction mode using external calibration with α-keto acid standard solutions over a range of 0.9 to 600 µmol/L. Transitions used for quantification and respective collision energies are given in the supporting information ( Figure S3).

Conclusions
Using a set of analytical techniques, we identified and quantified the key constituents responsible for the smell and taste of T. caerulea herb, which is used for flavoring bread and cheese in some regions of the Alps, and which has also become increasingly popular in other parts of Europe. After pyruvic acid and α-ketoglutaric acid were previously determined as important components for the taste of blue fenugreek, we proved glyoxylic acid as another relevant α-keto acid, showing the highest amount in two of the three measured samples. Apart from the importance of α-keto acids, we demonstrated the contribution of several volatiles to the aroma of T. caerulea herb, such as tiglic aldehyde, phenylacetaldehyde, methyl benzoate, n-hexanal, and trans-menthone. Thus, our study also provides an explanation for the characteristic smell of blue fenugreek herb. As a last outcome of our study, the major flavonoids present in T. caerulea herb were identified and, subsequently, the value-determining constituents of blue fenugreek nutraceutical preparations.