Proximate composition, and total phenols, tannins, flavonoids and saponins, and in vitro ruminal fermentation activity of fenugreek cut at three maturity stages

H Abdouli, M Hadj Ayed, M Elham, B Nabila* and M Remedios Alvir Morencos**

Département de Production Animale, Ecole Supérieure d’Agriculture de Mateur, 7030 Mateur, Tunisia
* Corresponding author: Tel : +216 23 386 314 ; Fax: (00 216) 72 485 088
Abdouli@topnet.tn
* Département de Production Animale, Institut Supérieur Agronomique de Chatt Mariem, Université de Sousse
** Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica de Madrid, Ciudad Universitaria, 28040 , Madrid, Spain

Abstract

To evaluate the effect of fenugreek growth stage on its  total phenols(TP), tannins (TT), flavonoids (TF) and saponins (SAP) contents,  and the  impact of TT and SAP on in vitro ruminal fermentation activity,  fenugreek was cut at vegetative (V), full flowering (FF) and well-developped pods (WDP) and served for proximate composition, TP, TT, TF, SAP determinations, and for in vitro gas production with (+PEG) or without (-PEG) polyethylene glycol, and with (+SAP) or without (–SAP) its water extract containing saponins.

DM was low (10.03%) at V and increased (P<0.05) at FF (13.04%) and WDP (28.73%). There were a consistent decrease (P<0.05) in CP and an increase (P<0.05) in NDF and ADF as fenugreek matured. TP decreased (P<0.05) by 0.005 g/100 g DM d-1 from 1.26 to 0.88 g/100g DM. TF  decreased (P<0.05)  by 0.006 g/100 g DM d-1 from 0.77 to 0.26 g/100g DM. TT (0.29-0.33 g/100g DM) and SAP (5.79-7.02 g/100 g DM) tended to decrease (P>0.05) from V to WDP. Potential gas production (As) and rate constant were not different (P>0.05) between the three maturity stages while there was a difference (P<0.05) in As between +PEG (35.79 ml/200 mg DM) and –PEG (31.88 ml/200 mg DM). The effect of PEG addition was independent (P>0.05) of the maturity stage. Gas production profiles for +SAP and -SAP were not adequately described by the exponential models with or without lag. Cumulative gas production was not different (P>0.05) between fenugreek cut at V and FF and was the lowest (P<0.05) at WDP. The removal of saponins (-SAP) decreased (P<0.05) gas production. SAP effect was significant (P<0.05) at V and FF not at WDP. Results from this study indicated that the vegetative stage is adequate for cutting fenugreek for food or medicinal uses and at well-developed pods growth stage for use as a forage. The use of fenugreek in ruminant nutrition may profit from in-depth studies on impact of fenugreek genotypes and their cultivation conditions on their antinutritional factors, feed utilization efficiency and products quality 

Key words: Fenugreek, flavonoids, gas production, tannins, phenols, saponins

Introduction

Fenugreek (Trigonellajoenum graecum L.) is an annual legume grown as a spice, a medicinal plant and a forage. This geographically-widespread multiple purpose use is due to fenugreek low  water requirement and dry land adaptation (Moyer et al 2003), high nutritive value (Mir et al 1998, Upadhyay et al 1977))  and because of the release of high yielding  and high quality genotypes (Mir et al 1993, Thomas et al 2006). Fenugreek foliage, like most medicinal plants, contains secondary metabolites like saponins, flavonoids, alkaloids and tannins (Sauvaire et al 1996,Taylor et al 1997) some of which may, at high levels, have detrimental effects on its nutritive value.  Although, Thomas et al (2006) reported that leaves and seed of 75 different Trigonella accessions did not inhibit the growth of enteric bacteria and yeast and, therefore, were suitable for forage development and animal consumption, Acharya et al (2010), Taylor et al (2002) and Fazli and Hardman (1968) showed that phytochemicals in fenugreek can vary depending not only on the genotype of the plant but also on the environment under which is grown and their interaction. Plants use these compounds to defend themselves against various threats and the nature and levels of such resistance chemicals were found to change as plants become more mature (Varma et al 2007; Kale 2010). To authors knowledge, none of the existing study investigated the evolution of bioactive compounds during fenugreek plant growth yet there is a need to evaluate the bioactive properties and chemical composition at different maturity stages of the plant either for therapeutic or forage use. Therefore, the objective of this study was to evaluate the total phenols, tannins, flavonoids and saponins,  and their  effects on in vitro ruminal fermentation activity of the unique local  cultivated fenugreek genotype cut at three maturity stages.

Materials and Methods

Cultivation of Fenugreek

The cultivation was conducted under rain-fed conditions at the teaching farm of ESA–Mateur, 60km northeast of Tunis, Tunisia. The soil was clayey–loam. Fenugreek (of a local genotype) was sown November 18, 2010 at the rate of 70 kg of seed per hectare in rows with a 0.15 m spacing. No fertilizer nor herbicide were used. The mean annual rainfall and temperature were 488.3 mm and 16.8 ºC, respectively.

Fenugreek was cut between February and May, 2011 at three successive maturity stages: vegetative (V; 14 February, 2011), full flowering (FF; 22 march, 2011), and well-developed pods (WDP; 09 may, 2011).  At each sampling time, approximately 10 Kg of fresh forage was collected immediately after cutting approximately 0,05 m on the soil level from different areas. Collected forage was put in black plastic bags and immediately transported to the laboratory where 2 to 4 subsamples were taken for dry matter determination, 2 for proximate composition, secondary metabolites and fermentation determinations.

Proximate composition Analysis

Dry matter (DM) was determined at 104 °C for 24 h while all other analyses were done on samples dried at 60 °C and ground in a mill to pass through a 0.5 mm screen.  Ash content was determined by igniting the ground sample at 550 ° C in a muffle furnace for at least 6 h (overnight).  The Association of Official Analytical Chemists (AOAC 1984, methods 7.033-7.037) was used for crude proteins (CP) determination. Acid detergent fiber (ADF) and neutral detergent fiber (NDF) were determined as described by Van Soest et al(1991) but sodium sulphite and alpha amylase were omitted from the NDF procedure.  Lignin content was determined by acid detergent lignin analysis of ADF residues (AOAC 1984, methods 7.074-7.077).  

Secondary metabolites analysis

Total phenols and tannins

Samples were analyzed for extractable total phenols (TP) and tannins (TT). Dried ground sample (100 mg) was mixed with 2x5 ml of ethyl ether containing 1% acetic acid and stirred for 2x 15 minutes. The contents were centrifuged (10 min, 2000 rpm) and the supernatants were discarded.  The residue was stirred with 2x5 ml of 70 % (v/v) acetone for 2x1h, centrifuged (20minutes, 2000 rpm), filtered on whatman 54 and adjusted to 50 ml with distilled water. Total phenols were determined with the Folin-Ciocalteau reagent (Makkar et al 1993). For each extract, 2x1ml  aliquots were taken and each was mixed with 0.5ml of 1 N Folin-Denis reagent and,  5 minutes later,  2.5ml of sodium carbonate  (20% , w/v) were added. The absorbance was measured at 750nm after 40 minute of reaction against a reagent blank. A standard calibration plot was generated using known concentrations of Gallic acid. The concentrations of phenols in the test samples were calculated from the calibration plot and expressed as mg gallic acid equivalent of phenol/100g of dry sample. Extractable tannins were determined as the difference in total phenols measured by Folin-Ciocalteau reagent as above before and after treatment of 6 ml of the extract with 300 mg of insoluble polyvinyl polypyrrolidone (PVPP) and centrifugation (20 min, 2000 rpm).

Total flavonoids determination

Dried ground sample (100 mg) was mixed with 2x5 ml of ethyl ether and stirred for 2x 1h. The contents were centrifuged (10 min, 2000 rpm) and the supernatants were discarded.  The ether was evaporated overnight and the residue was stirred with 2x5 ml of 80 % (v/v) methanol for 2x5h, centrifuged (20minutes, 2000 rpm), filtered on wathman 54 and adjusted to 50 ml with distilled water. Total flavonoids content was measured with the aluminum chloride colorimetric method as described by Patel et al (2010) with some modifications. For each extract, 2x2.5 ml aliquots were taken and each was mixed with 0.15 ml of 5% NaNO2  and, 5 minutes later,  0.15 ml of 10% ALCL3 in 80% methanol were added. After 6 min, 1ml of 1 M NaOH was added and the total volume was made up to 5 ml with distill water. Then the solution was mixed well and the absorbance was measured against a freshly prepared reagent blank at 510 nm. A standard calibration plot was generated using known concentrations of catechin (+catechin, cat №:88191-48-4, Sigma).Total flavonoids (TF) content of the extracts was expressed as percentage of catechin equivalent per 100 g dry weight of sample.

Saponins determination:

Crude Saponins was extracted as described by Nwosu (2011) with some modifications. One gram of the sample was extracted with 40 ml of acetone for 3 h in a Soxhlet extractor fitted with a reflux condenser and a flask,  and then  with 40 ml of methanol in another flask for another 3 h to remove crude saponins.

Saponins content was measured with the vanillin –perchloric acid colorimetric method as described by Wong et al (2007) with some modifications. A volume of 0.05 ml of the crude extract was dried at 70 °C in a water-bath for 2 h. Following this, 0.1 ml 5% vanillin-glacial acetic acid solution and 0.4 ml perchloric acid were added. The tube containing the mixture was vortex stirred  to ensure complete distribution, plaggued with glass marble,  transferred to a water-bath at 70 °C for 15 min, then removed and placed in ice-water to cool. Following this, 2.5 ml glacial acetic acid was added to each tube. Then the solution was mixed well and the absorbance was measured against a freshly prepared reagent blank at 540 nm. A standard calibration plot was generated using known concentrations of saponin (cat №: 8047-15-2, Riedel de Haen). Saponins (SAP) content of the extracts was expressed as percentage of quillaja equivalent per 100 g dry weight of sample.  

In Vitro fermentation 

In vitro gas production with or without PEG 

Parameters of in vitro gas production were determined according to the Menke and Steingass method (1988) in 60 ml plastic syringes. Rumen fluid was brought in a pre-warmed thermos flask from the slaughter house within 20 minutes before inoculation, filtered under CO2 through eight layers of cheese cloth and mixed with two volumes of McDougall‘s synthetic saliva warmed to 39°C. Using an automatic volume dispenser, 30 ml of the buffered rumen fluid solution was introduced under CO2 in each syringe containing 200 mg of ground sample (-PEG) or 200 mg of ground sample and 200 mg PEG (+PEG). All syringes were shaken and placed in the incubator at 39 °C. Samples were incubated in duplicate together with two syringes containing only buffered rumen fluid solution and two containing, each, buffered rumen fluid solution+ 100 mg PEG to serve as blanks. Cumulative gas volume measurements were read after 2, 4, 6, 8, 10, 12, 14, 16, 20 and 24 h of incubation. After each reading, the content in the syringe was shaken properly to ensure proper mixing of the substrate. Cumulative gas production data were fitted to the exponential model as: 

y = A × (1 − exp^−ct)

where ‘y’ is the cumulative volume of gas produced (ml/200 mg DM) at time ‘t’ (h), ‘A’ the asymptotic gas volume from the fermentable fraction  (ml/200 mg DM)  and ‘c’ the fractional rate of gas production (h^-1).

In vitro gas production with or without saponins extract

Parameters of in vitro gas production were determined as above but after saponins extraction (+SAP) or saponins extraction and removal (-SAP). In the latter case, dried ground sample (200 mg) was mixed with 5 ml of boiling distilled water, stirred for 1h and centrifuged (20 min, 2000 rpm) and the supernatant  was discarded. The extraction was repeated 4 times.  In the case of +SAP, dried ground sample (200 mg) was mixed with 5 ml of boiling distilled water and stirred for 4 h. In both cases, after the last extraction process, water was allowed to evaporate overnight at 60°C and the residue was suspended in 10 ml of McDougall‘s synthetic saliva and transferred to the syringe.

In vitro true degradability

Duplicate ground samples (0.5 g) were weighed into 80 ml glass tubes and mixed with 50 ml of pre-warmed buffered rumen fluid solution (1v rumen fluid + 4 v McDougall‘s synthetic saliva) prepared as described above. The mixture was gassed with CO2 and the tube was then sealed with a rubber cork fitted with a Bunsen gas release valve as described by Tilley and Terry (1963).Tubes were incubated at 39 °C in a water bath covered with a black plastic sheet to ensure darkness. At the end of the incubation period (24 h), the fermentation was stopped by the addition of 1 ml of 5% HgCl2. The tube content was filtered through a 42 μm nylon cloth (used for In Situ bag confection), washed with distilled water and quantitatively transferred into previously weighed P2 sintered crucibles (40- 90 μm pore size). The residue was refluxed with neutral detergent solution (without sodium sulfite) for 1 h, washed and ignited at 550 °C. The in vitro true dry matter digestibility (IVDMD) was derived as the difference between the weight of DM incubated and NDF residue.

IVDMD= (DM Incubated-NDF residue)x 100/ DM Incubated  

Statistical analysis

The rate and extent of in vitro gas production were calculated by non-linear regression of SAS. Proximate composition and secondary metabolites contents data were subjected to one-way analysis of variance (ANOVA). The used model was: Yi= μ+ MSi + ei, where μ is overall mean, MSi is the maturity stage  effect, and  ei is error term.  The significant differences between maturity stage means were identified using Tukey test. Fermentation parameters data were subjected to two-way analysis of variance using the GLM. The used model was: Yijk= αi +βj+λij+ eijk , where αi is the maturity stage effect, βj is the lack or presence of PEG  or saponins extract effect,  λij in the interaction of the two factors effect, and  eijk is error term. The significant differences between maturity stage means were identified using Tukey test, and the effect of the second factor (PEG or SAP)  within each maturity stage was tested using ‘Lsmeans fact1*fact2/slice= fact1’ statement.

Results

Proximate composition and In vitro true dry matter digestibility (IVDMD)

The result of the detailed proximate composition of fenugreek as affected by maturity stage is presented in Table 1. DM was low (10.0%) at V and increased at FF (13.0%) and WDP (28.7%). Generally there was a consistent decrease in CP and an increase in NDF and ADF as fenugreek matured. IVDMD decreased from 83.5% at V to 74.8 and 71.5% at FF and WDP, respectively.

Secondary metabolites

Quantitative estimation of fenugreek secondary metabolites contents are shown in table 2. Total phenols and flavonoids contents were higher at V than at FF or WDP. Phenols content decreased by 0.005 g/100 g DM d-1 from 1.26 to 0.88 g/100g DM. Flavonoids content decreased by 0.006 g/100 g DM d-1 from 0.77 to 0.26 g/100g DM. Total tannins and saponins contents tended to decrease from V to WDP maturity stages.

Effect of PEG on In vitro gas production parameters 

Cumulative volume (G), asymptote (A) and the fractional rate (c ) of in vitro gas production  after 24 h of incubation of fenugreek with (+PEG) or without (-PEG) polyethylene glycol  are summarized in Table 3. All parameters were not different between the three maturity stages while there were differences in G and A between +PEG and –PEG. The effect of PEG addition was independent of the maturity stage. The cumulative gas produced profiles are shown in fig.1.

Figure 1. In vitro gas production profiles of fenugreek cut at three maturity stages incubated with (+PEG) or without (-PEG) polyethylene glycol. V: vegetative, FF: full flowering, and WDP: well-developed pods

Effect of saponins extraction on In vitro gas production.

Gas production profiles for fenugreek incubated with (+ SAP) or without (-SAP) its aqueous extract are shown in Figure 2. They were not adequately described by the exponential models with or without lag. Therefore, only the cumulative volume of gas produced at 12 h (G12) or 24 h (G24) are given (Table 4). Both G12 and G24 were not different between fenugreek cut at V and FF and both were the lowest at WDP maturity stage. The removal of saponins  in water extract before fenugreek incubation decreased gas production. Saponins extraction effect was significant at V and FF not at WDP maturity stages. 

Figure 2. In vitro gas production profiles of fenugreek cut at three maturity stages incubated  with (+sap) or without its water extract (-sap) containing saponins. V: vegetative, FF: full flowering, and WDP: well-developed-pods

Discussion

In the present study, fenugreek was first cut 11 weeks post sowing (WAS) at the vegetative maturity stage. Its DM content was yet 10 % and was made mostly of soluble material since its fiber content was less than 22%. CP accounted for 28.2 % of the DM which was much higher than the values of 17.3% and 21.7% reported for 9-wk-old greenhouse grown fenugreek by Mir et al (1995) and Mir et al (1997, respectively. The difference between our value and the reported ones may be due to differences in plant genotypes and growing conditions more than to maturity stage. The greenhouse conditions might have been more favorable for fenugreek growth than the ones in our study (field, 80kg seeds/ha, no fertilize). This was likely plausible in view of the higher NDF (32.6 %DM) and ADF (29.4%DM) contents and lower IVDMD (59.3) reported by Mir et al (1997). In the present study, the ADF level in fenugreek cut at V maturity stage was, unexpectedly, slightly higher than NDF (21.9 vs 21.1 % DM) which indicated the lack of hemicelluloses at this growth stage and that fenugreek contained some components less soluble in acid than in neutral detergent. With regard to this, Van Soest (1982) reported that neutral-detergent dissolves pectins and tannins while acid-detergent recovers tannin-protein complexes and some of the pectins. Again, fenugreek cut at V maturity stage, had higher ADL content (10.2% DM) than 9-wk-old greenhouse grown fenugreek reported by Mir et al (1997). At the subsequent maturity stages, DM content increased to reach levels of 13.0% at FF (i.e. 16 WAS) and 28.7% at WDP (i.e. 22 WAS).

Generally, as fenugreek matured, there was a consistent decrease in CP which dropped to 13.4 % of DM at WDP and an increase in NDF and ADF which reached levels of 44.4 and 36.8 % DM, respectively. IVDMD decreased to 74.8 and 71.5% at FF and WDP, respectively. Similar changes, but of different magnitude, were reported by Mir et al (1995) and Mir et al (1997) for greenhouse grown fenugreek cut 9, 15 or 19 WAS. Their values for fenugreek at 19 WAS were 12.9-15.7 %DM for CP, 38.7-47.3 %DM for NDF, 33.7-36.7%DM for ADF and 53.9-66.2% for IVDMD. Such values, except the ones for IVDMD, were in agreement with the values obtained in the present study for fenugreek cut at WDP.

Among the secondary metabolites determined in the present study, only TP and TF contents decreased from V to WDP. Even at this late maturity stage at which usually fenugreek is cut for hay, the levels of TP and TF were very high indicating that their biological functions were preserved. The antioxidant and antimicrobial activities of extracts of fenugreek leaves have been demonstrated even when phenols and flavonoids contents varied with the extracting solvent from 1.5 to 4.9 and 0.16 to 0.47 mg /g, respectively (Ramya et al 2011). Furthermore, it is reported that polyphenols contents in fenugreek depend not only on the genotype but also on the degree of its environment threats. In this line, Avtar et al (2003) reported that the levels of TP and some of their activities increased in response to infection and decreased at higher disease severity levels. Gupta and Singh (2002) investigated the changes in anti-nutritional factors at growth stages of fenugreek leaves of four genotypes and found that TP and flavonols increased with growth of leaves in two genotypes while they increased upto the second cutting and decreased thereafter in the others genotypes. In this study, TP and flavonols ranged from 11.1 to 17.5 and 2.0 to 5.8 mg/g, respectively.

It is interesting to point out that in the present study, while TP contents decreased as fenugreek aged, TT contents changed very little and ranged from 0.29 to 0.33 g/100 g DM. At these levels, tannins reduced in vitro gas production as suggested from the results of fenugreek incubated with or without PEG. This adverse effect was independent of the maturity stage and was noted on the potential and cumulative gas produced after 24 h of incubation not on the rate constant. Mir et al (1997) reported inconsistent results concerning the fenugreek growth effect. In one experiment, cumulative in vitro gas production of 9-wk-old fenugreek was lower than that of 15-wk-old fenugreek which was lower than that of 19-wk-old fenugreek whereas in another experiment, there were no difference between gas production of 15-wk-old and 19-wk-old fenugreek. It is worth noting that even for similar CP, NDF and ADF contents of 19-wk-old fenugreek in this experiment and of fenugreek cut at WDP stage in the present study, potential gas production values were different (52.4 vs 35.8 ml/200 mg DM) and rate constants were similar (0.10 vs 0.11 h-1).

The SAP levels in fenugreek were not different between the maturity stages and were very high ranging from 5.79 to 7.02 g equivalent quillaja/100g DM. To our knowledge, there is no data in the literature to compare with. However, such levels may be specific to the genotype used and grown under the conditions described above. Unexpectedly, In vitro incubation of fenugreek without its aqueous extract which is assumed to remove saponins reduced gas production when fenugreek was cut at V or FF not at WDP. As such, the presence of saponins appeared to have positive effect on rumen microbial activity. However, hot water extract is not specific for saponins removal and other soluble organic materials could have been removed as well. This was most likely the case since the reduction in gas production occurred for fenugreek cut at V or FF not for fenugreek cut at WDP stage which was poorer in CP and richer in NDF. Wu et al (1994) reported that administration of up to 8 g/d of yucca extract (containing  sarsaponin as  main component) into the rumen of dairy cows fed diets containing 1.2% urea did not affect ruminal NH3-N concentrations,  pH,  or volatile fatty acids. In an in vitro ruminal fermentation experiment, Hu et al (2006) reported that 24 h gas production, NH3-N concentration and protozoal counts were significantly reduced, while microbial protein yield was increased by tea saponins addition

Conclusions

• The presented data for proximate composition and for secondary metabolites and their effects on ruminal fermentation assessed by in vitro gas production suggested cutting fenugreek at vegetative stage for food or medicinal uses and at well-developed pods growth stage for use as a forage.

• The use of fenugreek in ruminant nutrition may profit from in-depth studies on impact of fenugreek genotypes and their cultivation conditions on their antinutritional factors, feed utilization efficiency and products qualit

Acknowledgement

The authors thank  Mr Tayachi  Lassad  for his help  in laboratory analyses

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Received 18 November 2011; Accepted 16 December 2011; Published 4 January 2012

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