INTRODUCTION:

Fenugreek (Trigonella foenum-graecum L.) is an annual herbaceous plant belonging to the family Fabaceae and is extensively cultivated for its nutritional value and medicinal properties¹. The seeds and leaves are particularly rich in bioactive constituents, including saponins, flavonoids, alkaloids, and steroidal compounds, which contribute to its wide range of therapeutic applications^2,3.

Among its notable phytochemicals, trigonelline has attracted considerable scientific interest due to its antioxidant, antidiabetic, and potential anticancer properties⁴. Regular consumption of fenugreek has been associated with hypoglycemic, hypocholesterolemic, anti-inflammatory, and cardioprotective effects, leading to its increasing incorporation into functional foods and pharmaceutical formulations⁵. Similarly, antioxidant activity and phytochemical richness have been reported in several plant species analyzed using spectroscopic and chromatographic methods⁶.

Plant tissue culture offers a powerful biotechnological approach for the controlled production of plant-derived metabolites. Callus culture, which involves the growth of undifferentiated plant cells under defined conditions, allows manipulation of metabolic pathways through the use of specific growth regulators and culture environments⁷. Compared with conventional cultivation, in vitro callus systems enable continuous, year-round biomass production with reduced environmental variability and improved sustainability⁸. Efficient callus induction protocols have been reported in various plant species, including garlic stem discs, demonstrating the importance of optimizing hormonal combinations⁹.

Recent phytochemical studies in medicinal plants have demonstrated the presence of diverse bioactive compounds detectable through GC–MS and related analytical tools^10,11.

While the biochemical composition of fenugreek seeds has been extensively studied, limited information is available regarding the mineral, carbohydrate, and lipid composition of fenugreek callus tissue grown in vitro^12,13. Advanced analytical techniques such as GC–MS, FTIR, and phytochemical screening have been widely applied to characterize plant metabolites and bioactive compounds^14,15.

Therefore, the present study was designed to characterize the biochemical profile of T. foenum-graecum callus tissue using established analytical and chromatographic techniques.

Callus Induction:

Callus induction was successfully achieved under both hormonal treatments tested. The highest induction frequency (100%) was observed on MS medium supplemented with 2mg/L NAA alone, while a slightly lower frequency (97.33%) was recorded on MS medium containing 2mg/L NAA in combination with 1mg/L BAP. These results indicate that NAA alone was sufficient to promote efficient callus formation from fenugreek leaf explants. These results are consistent with previous findings demonstrating efficient callus formation in medicinal plants when auxin‑based media are used, as reported in garlic callus cultures⁹.

Cultures were maintained under controlled laboratory conditions until visible callus formation occurred. Induction frequency was calculated as the percentage of explants producing callus tissue in each treatment. The consistently high induction rates suggest that the resulting callus tissue was physiologically active and suitable for subsequent biochemical analyses.

Assay of Mineral Elements:

Fenugreek callus samples were oven-dried and ashed at 550°C until a constant weight was obtained. The ash was dissolved in 5mL of 1N hydrochloric acid (HCl) with gentle heating and continuous stirring to ensure complete dissolution. The solution was filtered, and the clear filtrate was used for mineral analysis.

Sodium (Na), potassium (K), and calcium (Ca) contents were determined using flame atomic emission photometry. Calibration curves were prepared using standard solutions ranging from 0.5 to 5ppm for each element, and concentrations were calculated according to AOAC methods¹⁶.

Phosphorus (P) was quantified spectrophotometrically using Barton’s reagent, with absorbance measured at 436 nm. Iron (Fe) and zinc (Zn) concentrations were determined by atomic absorption spectrophotometry using an air–acetylene flame at wavelengths of 248.3nm and 213.9nm, respectively. Elemental concentrations were calculated by comparison with standard calibration curves. Comparable mineral and phytochemical analyses have been performed in other medicinal plants using standardized extraction and quantification procedures¹⁴.

Carbohydrate Analysis by HPLC:

The carbohydrate composition of T. foenum-graecum callus tissue was analyzed using high-performance liquid chromatography (HPLC). Samples were prepared following standard extraction procedures and injected into the HPLC system equipped with an amino (R-NH₂) column and a refractive index (RI) detector. HPLC and related chromatographic techniques have been widely used for profiling sugars and other metabolites in plant tissues, supporting the reliability of the present analytical approach¹⁵.

The mobile phase consisted of acetonitrile and distilled water (80:20, v/v) at a flow rate of 1 mL/min, with an injection volume of 100μL. Individual sugars were identified and quantified by comparing their retention times with those of authentic standards analyzed under identical chromatographic conditions.

Fatty Acid Analysis by GC–MS:

Fatty acids were extracted from callus tissue using the Soxhlet extraction method in accordance with AOAC guidelines¹⁷. The extracted lipids were analyzed using gas chromatography–mass spectrometry (GC–MS) for both qualitative and quantitative assessment. GC–MS is one of the most powerful tools for identifying lipid‑derived and volatile compounds in plant extracts, and its effectiveness has been demonstrated in several RJPT studies^10,11.

GC–MS analysis was performed using a DB-Wax-5ms capillary column (30m × 0.25mm, 0.25μm film thickness) with helium as the carrier gas. The oven temperature was programmed from 55°C to 265°C to achieve optimal separation. Mass spectra were recorded in full-scan mode over a range of 35–500m/z. Fatty acids were identified by comparison with spectra from the Wiley and NIST mass spectral libraries.

Experimental Design and Statistical Analysis:

The experiment was conducted using a completely randomized design (CRD) with three replicates per treatment, each replicate consisting of 15 culture tubes. All experimental data were analyzed using XLSTAT software.

A two-way analysis of variance (ANOVA) was applied to assess the effects of the experimental treatments. Mean comparisons were performed using Fisher’s least significant difference (LSD) test at a significance level of p≤0.01.

RESULTS AND DISCUSSION:

Mineral Content:

The mineral composition of Trigonella foenum-graecum callus tissue is presented in Table 1.

Table 1. Mineral composition of Trigonella foenum-graecum callus tissue cultivated in vitro (mg/100 g dry weight)

Mineral	Content
Potassium (K)	2725
Sodium (Na)	365
Calcium (Ca)	92
Phosphorus (P)	0.70
Iron (Fe)	2.00
Zinc (Zn)	0.80

The distribution of mineral elements in fenugreek callus tissue is illustrated in Figure 1.

Figure 1. Mineral composition of Trigonella foenum-graecum callus tissue cultivated in vitro (mg/100g dry weight; log scale).

Potassium (K) was the predominant mineral detected (2725mg/100g dry weight), followed by sodium (365 mg/100g) and calcium (92mg/100g), whereas phosphorus, iron, and zinc were present at comparatively lower levels.

The accumulation of mineral elements in callus tissue may be related to the nutrient composition of the Murashige and Skoog (MS) medium and the high metabolic activity of actively proliferating undifferentiated cells. Similar mineral distribution patterns have been reported for callus cultures of other medicinal plants grown under in vitro conditions.

Potassium and calcium play essential roles in enzymatic activity, energy metabolism, and cellular division, which may explain their higher accumulation in rapidly growing callus tissue. The presence of essential micronutrients such as iron and zinc, even at lower concentrations, indicates the nutritional relevance of fenugreek callus tissue and supports its potential as a controlled source of mineral elements.

RESULTS AND DISCUSSION:

Carbohydrate Composition:

The carbohydrate composition of Trigonella foenum-graecum callus tissue determined by HPLC is presented in Table 2.

Table 2. Carbohydrate composition of Trigonella foenum-graecum callus tissue cultivated in vitro (g/100 g dry weight)

Sugar	Content
Fructose	15.8
Glucose	9.3
Sucrose	6.7
Raffinose	9.5

The carbohydrate profile of fenugreek callus tissue determined by HPLC is illustrated in Figure 2.

Figure 2. Carbohydrate composition of Trigonella foenum-graecum callus tissue cultivated in vitro (g/100 g dry weight).

Fructose and raffinose were the predominant sugars detected in the callus tissue, followed by glucose and sucrose at comparatively lower concentrations.

The predominance of fructose reflects active carbohydrate metabolism associated with rapid cell division in undifferentiated callus tissue. Raffinose, a member of the raffinose family oligosaccharides, is known to play an important role in carbon storage and stress tolerance in plant cells, and its accumulation may represent a metabolic adaptation of callus tissue to in vitro culture conditions.

Similar carbohydrate profiles have been reported in callus cultures of other medicinal plants, where sugar composition is influenced by the carbon source in the culture medium and the physiological state of actively growing cells.

RESULTS AND DISCUSSION:

Fatty Acid Composition:

The fatty acid composition of Trigonella foenum-graecum callus tissue analyzed by GC–MS is presented in Table 3.

Table 3. Fatty acid composition (%) of Trigonella foenum-graecum callus tissue cultivated in vitro

Fatty acid	Percentage (%)
α-Linolenic acid (C18:3)	19.4
Linoleic acid (C18:2)	14.7
Oleic acid (C18:1)	12.7
Palmitic acid (C16:0)	11.7
Stearic acid (C18:0)	7.9
Arachidic acid (C20:0)	7.6
Eicosenoic acid (C20:1)	9.7
Heneicosanoic acid (C20:2)	5.5
Others	10.8

The fatty acid profile of fenugreek callus tissue determined by GC–MS is illustrated in Figure 3.

The fatty acid profile of fenugreek callus tissue was characterized by a predominance of unsaturated fatty acids, with α-linolenic acid representing the major component, followed by linoleic and oleic acids. Saturated fatty acids such as palmitic and stearic acids were detected at moderate levels. This predominance of unsaturated fatty acids is consistent with previous findings reported for fenugreek and other medicinal plants cultivated under in vitro conditions^5,3.

Unsaturated fatty acids are essential constituents of cellular membranes and play a critical role in maintaining membrane fluidity and metabolic activity in actively dividing plant cells. Fatty acid biosynthesis in callus tissue is influenced by several factors, including nutrient availability, carbon source, temperature, and culture conditions. The lipid profile observed in this study indicates that fenugreek callus tissue is metabolically active and capable of synthesizing a diverse range of fatty acids under controlled in vitro conditions.

CONCLUSIONS:

The present study demonstrated that Trigonella foenum-graecum callus tissue cultivated in vitro possesses a diverse and valuable biochemical composition, including essential mineral elements, active carbohydrate constituents, and a lipid profile dominated by unsaturated fatty acids.

The high accumulation of potassium, together with the presence of essential macro- and micronutrients such as phosphorus, iron, and zinc, highlights the nutritional relevance of the callus tissue.

In addition, the predominance of fructose and raffinose reflects an active carbohydrate metabolism characteristic of rapidly proliferating undifferentiated cells. The fatty acid profile, characterized by a high proportion of unsaturated fatty acids, further indicates the metabolic competence of fenugreek callus tissue under controlled culture conditions.

Overall, the findings suggest that callus culture represents a viable and sustainable alternative to whole-plant cultivation for obtaining biochemically valuable compounds. Optimization of culture conditions and further evaluation of biological activities may enhance the potential application of fenugreek callus tissue in pharmaceutical and nutritional fields.

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Received on 10.02.2026 Revised on 07.03.2026

Accepted on 30.03.2026 Published on 08.07.2026

Available online from July 13, 2026

Res. J. Pharmacognosy and Phytochem. 2026; 18(3):233-237.

DOI: 10.52711/0975-4385.2026.00033

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