INTRODUCTION:

Plants have played a significant role in maintaining human health and improving quality of life for thousands of years. In particular, herbs have been used as food and for medicinal purposes for centuries. In herbal medicine, the term herb refers not only to seed producing plants but also the barks, root, leaves, seeds, flowers, and the fruit of trees. According to the World Health Organization, about three quarters of the world’s population relies on traditional medicine for primary health care needs and most of this treatment involves use of plant extracts or their active components¹ However, the mechanism of action of most herbal medicines has not been fully understood, and experience obtained from their traditional use over the years should not be ignored². Therefore it is prudent to look for options in herbal medicine for major chronic diseases. Ethiopia (East Africa) is very rich in natural resources and knowledge of traditional medicine and use of plants as a source of medicine are an innate and very important component of the health care system.

Ethiopian Abish (Trigonella foenumgraecum) is an annual herb that commonly called as fenugreek, belongs to the family Leguminosae widely grown in East Africa, Pakistan, India, Egypt, and Middle Eastern countries³. Due to its strong flavor and aroma, fenugreek in one of such plants whose leaves and seeds are widely consumed in African continent as well as in other oriental countries as a spice in food preparations, and as an ingredient in traditional medicine. It is rich source of calcium, iron, α-carotene and other vitamins [4]. Both leaves and seeds should be included in normal diet of family, especially diet of growing kids, pregnant ladies, puberty reaching girls and elder members of family because they have haematinic (i.e. blood formation) value⁵. Fenugreek seed is widely used as a galactagogue (milk producing agent) by nursing mothers to increase inadequate breastmilk supply⁶. The seeds of fenugreek contain L-lysine and L-tryptophan rich proteins, mucilaginous fiber and other rare phytochemical constituents such as saponins, coumarin, fenugreekine, nicotinic acid, sapogenins, phytic acid, scopoletin and trigonelline, which are thought to account for many of its presumed therapeutic effects, may inhibit cholesterol absorption and thought to help lower sugar levels^7-9. Therefore, fenugreek seeds are used as a traditional remedy for the treatment of free radical related chronic life style diseases such as diabetes and hypercholesterolemia^{10, 11}. It is reported to have restorative and nutritive properties and to stimulate digestive processes, useful in healing of different ulcers in digestive tract¹². Fenugreek has also been reported to exhibit pharmacological properties such as antitumor, antiviral, antimicrobial, anti-inflammatory, hypotensive and antioxidant activities.

The present studies were carried out to evaluate the effect of soaking and blanching on the yield, proximate composition and free radical scavenging activity (FRSA) of Ethiopian Abish seed flour.

MATERIALS AND METHODS:

Plant Material:

The seeds of Ethiopian Abish was purchased from local super market (Selam baltna), Addis Ababa, Ethiopia, and the same was authenticated in the Jimma Agricultural research center. The seeds were then cleaned in preparation for the study.

Chemicals and reagents:

2,2-Diphenyl-1-picryhdrazyl radical (DPPH), ethanol, methanol, tetra-oxo-sulphate VI acid, hydrogen peroxide, petroleum ether (boiling point 40–60^oC) were obtained from Sigma Chemical Co. (South Africa). All Chemicals and reagents were of analytical grade.

Methods:

Pre-treatment of Ethiopian Abish seeds by soaking:

Soaked Ethiopian Abish seeds were prepared by the method of Adeyemi¹³. Equal (100 g) quantities of the Ethiopian Abish seeds were soaked in 300 ml of distilled water for 6, 12, 18, 24, 36 and 48 h, respectively. The Ethiopian Abish seeds were then washed and dried in a cabinet dryer at 45 ± 2 ^oC for 24 h to moisture content of 10–13% before preparing into flour.

Pre-treatment of Ethiopian Abish seed by blanching:

Blanched Ethiopian Abish was prepared according to the method Xu and Chang¹⁴. Ethiopian Abish was divided into portions (100 g) and steeped in 300 ml of distilled water for 24 h. The steeped seeds were drained and placed on a tray in the steam cooker covered with lid and steamed over 2 L of boiling water under the atmospheric pressure for 10, 20, 30, 40 or 60 min, respectively and dried in a cabinet drier at 45 ± 2 ^oC for 24 h to moisture content of 10–11% before preparing into flour.

Preparation of Ethiopian Abish seed flour:

The untreated and treated Ethiopian Abish seed were milled using kenwood mixer (Model BL350, PK100/AD England). The milled flour was sieved to obtain a flour fraction of less than 250 ml, which yielded the full-fat Ethiopian Abish seed flour. Flour yield was calculated for all Ethiopian Abish seed products based on 100 g whole seeds (on d.b). The pretreated Ethiopian Abish seed flour samples were designated as soaked or blanched flour.

Proximate analysis of Ethiopian Abish seed flour:

Moisture, ash, crude fat, crude fiber, crude protein (microKjeldahl N X 6.25) were determined by AOAC¹⁵ standard method while, carbohydrate was determined by difference. The values of analyses were means of three determinations.

Determination of the antioxidant activity by DPPH Assay:

The FRSA was determined using the DPPH assay as reported by Ansari et al.,¹⁶ with a little modification: 1 g of flour sample was mixed with 80% methanol (4:1 of methanol and water). This was incubated for 1 h at 37 ^oC in a shaking water bath and centrifuged (3500g at 4 ^oC) in an Eppendorf centrifuge (Model 5804 R, Germany) for 35 min. Ten times dilution of extracts was made with 80% methanol. About 100 µl of diluted extract was mixed with 100 µl of DPPH solution (0.1 mM) in a 96 well microplates. The mixture was kept in the dark at ambient temperature while the absorbance recorded at 517 nm after 20 min. Blank was made from 100 µl of DPPH and 100 µl methanol. The FRSA was calculated as

[1-(X-Y)] x 100

Where, X, is the absorbance of 100 µl of the diluted extract solution mixed with an equal volume of the DPPH solution; Y, is the absorbance of 10 µl of the diluted extract solution mixed with an equal volume of methanol and Z, is the absorbance of a blank prepared by mixing100 µl of the DPPH with an equal volume of methanol: water (4:1). Analysis was done in triplicates.

Statistical analysis:

All the grouped data were statistically evaluated with SPSS 16.00 soft ware. Hypothesis testing methods included one-way analysis of the variance followed by least significant difference (LSD) test. P<0.05 was considered to indicate statistical significance. All results are expressed as mean + standard deviation (SD).

RESULTS AND DISCUSSION:

Effect of pre-treatment on the yield of Ethiopian Abish seed flour:

Soaking of Ethiopian Abish seed resulted in higher yield of the flour with sample that was soaked for 48 h having the highest value of 90.92% (Table 1). Longer the soaking period, higher will be the yield. Moreover, progressive increase exists among all samples with the untreated having the least value of 73.47%. Soaking of cowpea prior to processing has been reported to soften the testa and cotyledons, which ensures easy removal of testa, milling of cotyledons and ease of further processing such as cooking¹⁷. This would probably explain the trend obtained for Ethiopian Abish seeds. Furthermore, conditioning with water to increase the moisture content of cereal grains has been shown to improve flour yield¹⁸. Blanching of the seeds also resulted in higher yields of the Ethiopian Abish seed flour with seeds blanched for 30 min having the highest yield of 92.21 (Table 1), which was gradually reduced to 81.22% as blanching time was increased to 60 min. Blanching had been reported to soften the texture of food material, thereby allowing easy milling which later resulted into higher yield¹⁹. Significant difference (P < 0.05) was noticed in most of the samples except for samples that were blanched for 30 and 40 min and samples that were soaked for 36 and 48 h which showed no significant difference from each other. Gradual reduction in yield as blanching time was increased from 40 to 60 min might be due to shrinkage and leaching of some components into the blanched water²⁰. Therefore, further blanching after 30 min may not be appropriate if higher yield is required.

Table 1. Effect of pre-treatment and time on the yield of Ethiopian Abish seed flour.

Sample	Processing time	Yield
N	0	73.47 ± 0.07d
S1	6h	76.26 ± 0.13d
S2	12h	80.32 ± 0.26c
S3	18h	81.68 ± 0.10c
S4	24h	87.64 ± 0.12ab
S5	36h	89.32 ± 0.08a
S6	48h	90.92 ± 0.05a
B1	10min	77.44 ± 0.12d
B2	20min	85.45 ± 0.04b
B3	30 min	92.21 ± 0.07a
B4	40min	89.19 ± 0.04a
B5	60min	81.22 ± 0.08c

Values are means of three determinations. Values with the same letter in the same column are not significantly different (P < 0.05).

Effect of soaking on the proximate composition of Ethiopian Abish seed flour:

Data on the effect of soaking and soaking time on the proximate composition of Ethiopian Abish seed flour is as presented in Table 2. Protein content of soaked Ethiopian Abish seed flour ranged from 53.99% and 59.03% while that from untreated seeds was 51.11%. Soaking of the seed was found to increase the protein content of the corresponding flour with the sample that was soaked for 36 h having the highest protein content of 59.03%. Moreover, the protein contents of all the soaked samples and the untreated one were significantly different (P < 0.05) from each other. About 5% decrease in protein was observed in the first 12 h of soaking while subsequent increase of 5–7% was observed between 18 and 48 h of soaking. During soaking, fermentation takes place. Proteolytic activity of the bacterial present in the soaking water probably increases the biological availability of essential amino acids²¹. This result is similar to the work of Yousif and El Tinay²² who observed a protein content decrease in the period 16–24 h of fermentation, however, after 24 h, the protein content started to increase in fermented rice. Yousif and El Tinay²³ had reported that the crude protein and non-protein nitrogen increased during the first stages of fermentation of maize. Also, Giami²⁴ reported that protein fractions (albumin and globulin) increased during fermentation, reaching their maximum levels on the 5th day, but declined thereafter. Protein content of oil bean had been reported to increase by fermentation^25,
26.

The fat content decreased from 16.04 in the untreated Ethiopian Abish seed flour to 13.08% after 48 h of soaking. The decrease trend was more significant (P < 0.05) between the 6th and 18th h of soaking. Thereafter, no significant decrease (P < 0.05) was noticed during the 36th and 48th h of steeping. The slight decrease in fat could be due to the action of lipase produced by micro-organisms as soaking period progressed²⁵. Similar trend was observed by Onimawo et al²⁷ in the fat content of fermented pumpkin seed. It was also reported by Achi²⁸ that fermentation led to a significant decrease in fat content of soy and yam flours.

The ash content increased from 4.42% in the untreated Ethiopian Abish seed flour to 4.95% after 48 h of soaking. Significant increase (P < 0.05) was also found to exist with increase in soaking time. Ash content increment is an indication of mineral increase.

Significant difference (P < 0.05) was noticed in the fiber content of un-soaked samples and soaked samples, however, no significant difference were observed to exist between the 12 and 24 h of soaking. Fiber is reported to help in the lowering of serum cholesterol, control blood sugar, increase bulk stool which may prevent colon cancer and several digestive disorders²⁹.

Significant differences (P < 0.05) were observed in the carbohydrate content of all the samples. Reduction in carbohydrate content especially within the 12–36 h of soaking period could be attributed to the possible bioconversion by the micro-organisms mainly yeast in the soaking water into other substances especially protein, in addition to the portion used as carbon and energy source by the organism²¹.

Effect of blanching time on the proximate composition of Ethiopian Abish seed flour:

Variations were observed in the protein content of Ethiopian Abish seeds subjected to different blanching time (Table 3). However, the sample which was blanched for 40 min had the highest protein content of 61.10%, while the protein content followed an increasing trend from 0 to 40 min of blanching but slight decease was observed as blanching time reached 60 min. The result obtained showed that blanching enhanced protein content, but longer period might result in protein reduction. Lower crude protein as blanching period increase to 60 min could be as a result of leaching of soluble components of the protein into the blanching water since the broth was discarded, some of such protein could have been lost³⁰.

The fat contents of all the blanched samples were lower than the un-blanched ones. This could be due to the loss of fat by the moist heat as this was observed to decrease as blanching time increased. Significant difference (P < 0.05) was noticed to exist among all the samples except for samples blanched for 10 and 20 min, which showed no significant difference between each other. The decrease might be due to the fact that direct heat helps to separate out oil from the cells of nuts and oil seed and subsequent removal during milling²⁰. Similar lower fat content was observed by Adeparusi³⁰ in blanched lima bean flour.

Table 2. Effect of soaking time (h) on the proximate composition of Ethiopian Abish seed flour (d.b).

Sample	Moisture content (%)	Protein (%)	Fat(%)	Ash(%)	Fiber (%)	Carbohydrate by difference (%)
N	10.38d	56.11e	16.04a	4.42e	2.45d	9.60d
S1	10.18f	53.99g	16.06a	4.75d	2.10e	11.92a
S2	10.33e	56.06f	15.11b	4.85b	2.59c	10.06b
S3	10.43c	58.18d	14.06c	4.82cb	2.64c	8.87d
S4	10.56b	58.51b	14.03c	4.85b	2.67bc	8.38e
S5	10.88a	59.03a	13.09d	4.75a	2.75ab	8.33f
S6	10.22f	58.36c	13.08d	4.95a	2.76a	9.63c

Values are means of three determinations. Values with the same letter in the same column are not significantly different (P < 0.05)

Table 3. Effect of blanching time (min) on the proximate composition of Ethiopian Abish seed flour (d.b).

Sample	Moisture content (%)	Protein (%)	Fat (%)	Ash (%)	Fiber (%)	Carbohydrate by difference (%)
N	10.38c	56.11e	16.04a	4.42e	2.45e	9.60d
B1	10.12e	56.40d	12.13b	4.58c	2.57d	13.20b
B2	10.80b	57.09c	12.10b	4.66c	2.61cd	11.74c
B3	10.26d	58.53b	11.15c	4.75c	2.63bc	11.68d
B4	9.99f	61.10a	10.22d	4.76b	2.66ba	10.27e
B5	10.95a	54.48f	10.08e	4.88a	2.69a	15.92a

Values are means of three determinations. Values with the same letter in the same column are not significantly different (P < 0.05).

Significant increase (P < 0.05) was found in the ash content of the blanched and the untreated samples, however, no significant difference were found at 10–30 min of blanching. Higher ash content was reported by Adeparusi³⁰ in blanched lima bean flour.

Fiber contents also increased from 2.45% in the untreated to 2.69% after blanching the sample for 60 min. The hull of matured Ethiopian Abish seed sample consists mainly of fiber. The release of fat during blanching makes the cotyledon sticky and the separation of cotyledon from the hull more difficult thereby contributing to the increase in ash and fiber. Fiber content had been reported to be enhanced by blanching in sorghum flour³¹.

Effect of pre-treatment on FRSA of Ethiopian Abish seed flour

The DPPH radical scavenging activities of the soaked samples is as shown in Fig. 1. After 18th h of soaking, the FRSA increases from 58.34% in the raw sample to 61.28% which is about 6% increase (Table 4). No significant difference was observed during 0–12 h of soaking however significant increase (P < 0.05) was observed at 18th h after which a significant decrease occurred (18–36 h). During soaking, fermentation occurs. Fermenting conditions had been reported to increase the FRSA level of cabbage³²

Fig. 1 Effect of Soaking on the FRSA of Ethiopian Abish seed flour.

Fig. 2. Effect of Blanching on the FRSA of Ethiopian Abish seed Flour

Table 4. Effect of Pre-treatment and time on the antioxidant level of Ethiopian Abish seed flour

Sample	Antioxidant level (% inhibition)	% Increase in antioxidant level
N	58.34bc	0
S1	59.11b	1.59
S2	58.05c	-0.60
S3	61.28a	6.08
S4	56.85d	-3.09
S5	54.73f	-7.47
S6	55.45e	-5.97
N	58.33a	0
B1	41.67b	-34.48
B2	42.54b	-32.68
B3	40.25c	-37.42
B4	37.36d	-43.38
B5	37.78d	-42.53

Values are means of three determinations. Values with the same letter in the same column are not significantly different (P < 0.05). N: Raw Ethiopian Abish seed (untreated); S1: Seed soaked for 6 h; S2: Seed soaked for 12 h; S3: Seed soaked for 18 h; S4: Seed soaked for 24 h; S5: Seed soaked for 36 h; S6: Seed soaked for 48 h; B1: Seed blanched for 10 min; B2: Seed blanched for 20 min; B3: Seed blanched for 30 min; B4: Seed blanched for 40 min; and B5: Seed blanched for 60 min.

Blanching resulted in lower FRSA, the longer the blanching time the lower the activity (see Fig. 2). About 33–43% loss was observed in the blanched samples (Table 4). In general, the loss of the DPPH free radical scavenging capacities could be partly due to soluble antioxidants in leached water and heat effect¹⁴. This work is in consonance with other researchers’ work who found out that thermal treatment decreases the FRSA content in all vegetables and fruits^{33- 36,14}.

CONCLUSION:

From this study, it could be suggested that Ethiopian Abish seed is a promising source of protein and FRSA as earlier reported by other researchers. Pretreatment by soaking and blanching resulted in enhancement of the yield and the protein content. The FRSA was moderately increased by soaking while blanching reduced it. Therefore, soaking could be advised as a pretreatment for Ethiopian Abish seed in order to preserve and enhance the yield, protein content and the antioxidant activity. The process of extraction and identification of active principles responsible for the observed pharmacological properties of Trigonella foenum graecum seed through bioactivity guided fraction is under progress to understand the possible mechanism of action of T.foenum seeds. Utilization of this seed will be of advantage to mankind and increased in its consumption will help in prevention of chronic life style diseases.

ACKNOWLEDGEMENTS:

Our gratitude goes to SRM Management, for their encouragement and Mr. G. Manoharan (Lecturer), MTU for his valuable statistical and computational assistance.

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Received on 25.03.2011 Modified on 03.04.2011

Research J. Pharm. and Tech. 4(7): July 2011; Page 1154-1158