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Physico-chemical investigation and antioxidant activity studies on extracts of Eruca sativa seed

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Along with the other medicinal plants Eruca sativa has remarkable potential against various diseases. Present work is based on the estimation of antioxidant potential of dist. Water, ethanolic and methanolic extracts of E. sativa seed oil. On phytochemical determination it was exposed that different plant extracts/fractions contains alkaloids, flavonoids, saponins, tannins, phenols, carbohydrates, steroids and proteins. Its physico-chemical screening was concluded the saponification value and fat value of the E. sativa seed oil. Antioxidant activity was evaluated by using DPPH free radical scavenging scheme. The maximum (30.60±1.1) anti-oxidative potential was exhibited by aqueous extract of E. sativa seed oil. IC50 was calculated maximum 126.2 in eq. extract of E. sativa seed oil. From current study, it is concluded that this plant seed oil could be used as a source of natural antioxidant and medicinal purposes.
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International Journal of Pharmaceutical Chemistry
ISSN: 2249-734X (Online)
Journal DOI:10.7439/ijpc
CODEN:IJPCI4 ( American Chemical Society)
* Correspondence Info
Asif Hanif Chaudhry
Department of Chemistry,
Quaid I Azam, University, Islamabad.
Geosciences Labs, Islamabad. Pakistan
E-mail: asif_hanif101@yahoo.com
Research Article
Physico- chemical investigation and antioxidant activity studies on
extracts of Eruca sativa seed
Saima Hamid1,3, Adila Sahar2, Farnaz Malik3, Shahzad Hussain3, Rashid Mahmood3, Kazi
Muhammad Ashfaq3, Tanveer Akhtar Malik3, Abbas Hassan4 and Asif Hanif Chaudhry4,5
1Department of Biochemistry, Pir Mehr Ali Shah Arid Agriculture University, Murree Road, Rawalpindi, Pakistan,
2Department of Chemistry, GC University, Faisalabad.
3National Institute of Health, Islamabad. Pakistan.
4Department of Chemistry, Quaid i Azam University, Islamabad.
5Geoscience Labs, Islamabad. Pakistan
1. Introduction
In recent years, because of increasing local, national and international interest, the demand for medicinal and aromatic plants increases
many folds in both developing and developed countries1. According to World Health Organization (WHO), more than 80% of the world‟s
population still relies on traditional medicines for their basic health needs. Medicinal plants contribution in disease prevention and control has
been attributed to antioxidant properties of their constituents, broadly termed as polyphenolic compounds. In addition to their role as antioxidants,
these compounds possesses broad spectrum of medicinal properties, such as anti-inflammatory, ant-microbial, anti-thrombic, cardio-protective
and vasodilatory2.
Scientists are taking keen interest in correlating phytochemical constituents with their pharmacological activities. Phytochemicals such
as flavonoids, terpenoids, lignans, glycosides and various alkaloids are well distributed in plant kingdom and analyzed scientifically for its
antiallergic activity3-4.
Eruca sativa locally known as Taramira is grown in different parts of Indo-Pak subcontinent. It has been cultivated since ancient times
in southern Europe and Central Asia. Eruca sativa Mill commonly referred to as “rocket salad, argula, roquette, or white pepper in English is a
member of brassicaceae family. It is minor oil crop and used in traditional medicines as remedies for different diseases. Its oil is mainly used in
industry in soap making, as an illuminating and lubricating agent, in massage and in medicines as well5-6. The extracted oil from the seed of E.
sativa is prohibited for eating purposes because of its pungent and obnoxious odour. The cake is used as manure for improving the soil physical
condition fertility and can also be used as nutritional feed for animals7.
E. sativa seed extract contains important secondary metabolite such as flavonoids, alkaloids, tannins, phenols, saponins, ascorbic acid
and those are used as remedies of many diseases and frequently required in traditional medicines. Essential oil especially erucic acids was present
in high concentration those are responsible for antibacterial activity, which could be used for the preparation of drugs required for human and
animal health8.
In traditional medicines, rocket species are recognized for their therapeutic properties such as astringent, digestive, diuretics, tonic,
laxatives, rubefacient and stimulants etc. Rocket salad species are rich in antioxidant compounds, as a source of vitamins like ascorbic acid,
carotenoids as well as polyphenols9.
Missiry and Gindy (2000) proposed that Eruca sativa seeds exhibits antidiabetic effect by reducing oxidative stress experimented in
rats. In addition ethanolic extract of Eruca sativa possesses significant anti-secretary, cytoprotective, and antiulcer activities against gastric
lesions experimentally induced in rats by elevating mucus synthesis and endogenous prostaglandins through its effective antioxidant activity10.
Present study focus on the phytochemistry of ethanlic and methanolic extract of E.
sativa seeds along with their antioxidant power.
2. Experimental
2.1. Collection and preparation of seed samples
Eruca sativa seed samples were purchased from local herbal store of Rawalpindi. Seeds samples were dried in shade and followed by
drying and finally converted into powered form (40 mash). This powder was again dried in the pre-set incubator at 50 oC for the removal of its
Abstract
Along with the other medicinal plants Eruca sativa has remarkable potential against various diseases. Present work is based on the
estimation of antioxidant potential of dist. Water, ethanolic and methanolic extracts of E. sativa seed oil. On phytochemical determination it
was exposed that different plant extracts/fractions contains alkaloids, flavonoids, saponins, tannins, phenols, carbohydrates , steroids and
proteins. Its physico-chemical screening was concluded the saponification value and fat value of the E. sativa seed oil. Antioxidant activity
was evaluated by using DPPH free radical scavenging scheme. The maximum (30.60±1.1) anti-oxidative potential was exhibited by aqueous
extract of E. sativa seed oil. IC50 was calculated maximum 126.2 in eq. extract of E. sativa seed oil. From current study, it is concluded that
this plant seed oil could be used as a source of natural antioxidant and medicinal purposes.
Keywords: Medicinal plants, Eruca sativa, Antioxidant, Alkaloids, Quercetin, DPPH free radical assay
Saima Hamid et al 161
IJPC (2014) 04 (04) www.ssjournals.com
moisture content. The samples was preserved in the sterilized clear bag, labeled properly and stored carefully in the refrigerator at 4oC for further
processing.
2.2 Proximate analysis
The seed samples of E. sativa were analyzed for protein, moisture, fiber, carbohydrates and ash contents by using AOAC, 1990.
2.2.5 Fat Content
The powered plant sample was analyzed for fat content according to standard method of AOAC (1990) and the results were expressed
in percentage11.
2.2.6 Estimation of Carbohydrate
The method used in this study is the modification of the protocol suggested by Khalifa12.
2.3 Preparation of plant extracts
2.3.1 Hot Water Extraction
About 5 gm of finely powdered seed sample was placed in a beaker and added in it 200 mL of D.H2O. Mixture was well shaken and
heated at about 30º- 40ºC for about 20 minutes with constant stirring. Aqueous extract was filtered and was additionally used for phytochemical
screening. The water extract was stored in air tight container and kept in refrigerator for further studies.
2.3.2 Solvent Extraction
About 25 gm of powdered plant material was evenly packed into a thimble and subjected to Soxhlet extraction method by using 250
mL of organic solvents (methanol and ethanol) separately. Extraction was done under normal conditions at room temperature. The extraction
process continues since the solvents develop into colorless. Extract recovered was filtered and heated at 30-40ºC on hot plate till evaporation.
Dried extract was stored at 4ºC for future use and analysis of phytochemical.
Extracts prepared were analyzed for the screening of bioactive compounds by standard methods13-15.
2.4 Qualitative estimations of phytochemicals
The identification or presence of Protein, Alkaloids, Reducing Sugars, Flavonoids, Phenols and Tannins, Steroids, Glycosides,
Carbohydrates, Terpenoids and Saponins by the standard identification methods and reagents.
2.5 Quantitative phytochemical screening
2.5.1 Alkaloids Determination
Alkaloids were quantified by using the protocol provided by Harborne (1973). Total 5 g sample was mixed thoroughly in (10%)
200mL acetic acid formulated in ethanol. Covered the solution and leave it for 4 hours at room temperature. After wards, extract was filtered and
left the filtrate to reduce it to one third to its actual volume on water bath. The concentrated solution of ammonium hydroxide was added till the
precipitation. The precipitates were washed with 0.2M ammonium hydroxide. A piece of filter paper was weighed and was set in the funnel for
filtration. During filtration the entire residue was left behind on filter paper. Then this residue was dried in oven at 50oC for absolute dryness and
weighed again. Alkaloid was quantified from the difference of paper with residue and empty filter paper. Furthermore experiment was repeated
thrice and the mean value was calculated.
2.5.2 Determination of Saponin
The protocol used in this study for the quantification of saponins was modified by Obadoni and Ochuko16. Total 20 g of powdered
seed sample was dispersed in 200 mL of ethanol (20%) in a glass beaker. This mixture was left in the water bath and heated at 550C for 4-5 hours
with constant shaking. Filtered the suspension and re-extracted the residue with 200 mL of 20% ethanol. Evaporate the collected extract up to
40mL by heating at 900C on water bath. Transfer the concentrated extract in a separating funnel and slowly added 20 mL of diethyl ether
followed by shaking. As a result ether and aqueous layer were obtained, and the aqueous layer was shifted to another flask whereas the layer
containing ether was thrown out. The purification process was repeated twice and added about 60 mL of n-butanol in the resultant concentrate.
Two times washing of purified butanol extracts was done with 10 mL sodium chloride solution (5%). Layer having sodium chloride was
discarded and the final extract was warmed slowly till evaporation. Afterwards the residue was desiccated in the oven to constant weight and
percentage of saponin was calculated.
2.5.3 Flavonoids Quantification
Total 10 g of sample was suspended in methanol having (80%) at normal temperature. After filtration of extract, weight of crucible
with solution was measured. Evaporate the solvent in the water bath at 60oC. After complete evaporation the extract material left were weighed
and flavonoids content was determined and percentage was calculated17.
2.5.4 Quantification of Phenolic Contents
In sample of aqueous extract, total phenolic content was assessed by Folin-Ciocalteu reagent18. Five gram of sample was weighed by
electric balance and dissolved in distilled water (Final volume 200mL). Then this mixture was heated along with continuous shaking at 450C for
15 minutes on hot plate and resultant solution was filtered. To 1 mL of plant extract, added in it 2 mL of 10% Folin-Ciocalteu reagent and 3mL of
sodium carbonate solution (2%) and left it for 20 min at room temperature. Spectrophotometric analysis of the sample was done at 765nm
whereas 1mg/mL of gallic acid was served as standard. Total content of polyphenol in plant extract was calculated using standard curve analysis
with gallic acid as standard expressed as equivalent to mg/g of extracted plant material. And procedure was repeated for triplicate analysis.
2.5.5 Quantification of Tannins
Quantification of tannins was carried out by Folin Ciocalteu method. 0.125g of dried plant sample was extracted with 25mL of 70%
acetone in mechanical shaker for 12 hrs at 60oC. The mixture was filtered by Whatman filter paper no.42 and added 0.5 mL of Folin Ciocalteu
reagent as well as 2.5 mL of Na2CO3 in filtrate. Spectrophotometric analysis was done at the wavelength of 725nm as compared to blank. Tannic
acid in plant extract was calculated using standard curve results prepared with tannic acid which was used as standard expressed as mg/g of
extracted compound. Experiment was repeated three times19.
2.6 Quercetin estimation by spectrophotometer
Quercetin was quantified by the modified protocol given by Patil20. 100 mg of grounded sample was placed in a volumetric flask and
80% ethanol was added in it, placed on shaker for complete extraction. Then this flask was placed in sonicator for about 10 minutes, followed by
filtration of the sample with the help of Whatman filter paper (No. 42), filtrate was poured in small falcon tubes and then stored at -20 °C for
further process.
Ethanolic extracts were thawed and vortexed for few minutes and absorbance was measured at 362nm via spectrophotometer. The
standard used in the analysis was quercetin with different concentrations i.e. 0.2-0.8 mg/mL. The calibration curve was drawn and expressed as
mg/g of extracted compound and experiment was repeated three times.
2.7 Assessment of antioxidant activity
2.7.1 DPPH Radical Scavenging Assay
The plant seed extract for free radical scavenging activity was determined by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) method by
applying the modified protocol of Lim et al 21. The reaction involved has
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DPPH + (H-A) → DPPH-H + (A)
(Violet color) (Light yellow color)
The antioxidant component in our plant extract undergo a reaction with DPPH by donating free electron, and the stable free radical
(DPPH) undergone the process of reduction and forms DPPH-H. This in turn, reduces the absorption from the DPPH radical to the reduced form.
The strength of discoloration from violet to light yellow color describes the scavenging ability of entire antioxidant compounds in the form of its
hydrogen donating capacity22.
Different concentrations of the E.sativa seed extract (0.2, 0.4, 0.6 and 0.8 mg/mL) were formulated in triplicates. In this method,
0.135mM of DPPH was made ready in absolute methanol (98%), thereafter 1mL of this DPPH solution was taken in a test tube and 1mL of plant
extract was mixed in it. Mixture was shaken or vortexed, allowed standing in the dark for half an hour in order to complete the reaction. The
spectrophotometer was set on 517nm and the absorbance was measured. Decrease in absorbance indicated the increase in anti oxidant ability. The
DPPH radical scavenging strength of plant seed extract was calculated by the equation given below:
DPPH Scavenging effect (%) = (Absorbance of control- Absorbance of sample) ×100
Absorbance of control
Where
Absorbance of sample = DPPH + aqueous seed extract
Absorbance of control = DPPH + methanol (absolute)
The 50% inhibition (IC50) was considered as the concentrations of samples that has the ability to inhibit 50% of scavenging activity
of DPPH radicals and was calculated via dose inhibition curve23.
2.8 Extraction of oil through soxhlet
About 10g of moisture free powder for sample was placed in thimble and thimble was then placed in the soxhlet‟s loading chamber.
The round bottom flask was filled with 300 mL of n-hexane. The temperature was set at medium mode of temperature. The solvent placed in the
flask were boiled and vaporized. The whole process was repeated many times to ensure the complete oil extraction. The solvent turned into
colorless, indicating the end point of the process. The oil was purified by placing it in rotary evaporator as a result the oil got free from the
solvent. The purified oil was stored in sterilized and labeled falcon tubes24.
2.9 Physiochemical properties of seed oil
Peroxide Value, acid Value, saponification Number, specific Gravity, pH of Oil and wax content were measured as per standard
methods.
3. Results and Discussion
The present study was carried out to assess bioactivity of Eruca sativa seeds along with physiochemical analysis of seed
extracts. Anti inflammatory and skin antiallergic activity was studied and the results are given in the following section.
3.1 Proximate analysis of plant sample
Proximate analysis of Eruca sativa seeds indicates the moisture content, crude protein, carbohydrate, crude fat and ash content Table
3.1. Based on these results Eruca sativa seeds provides good source of protein (30±1.2) and oil (29±1.6) as compared to other components.
Previous study had shown that seeds of Eruca sativa possess moisture content 4.1%, ash 6.6%, crude protein level 27.4% and oil content was
found to be 27.8%, however, oil contents in seeds depend on many factors including maturity of the seed as well as degree of plant irrigation5.
Similar results were proposed by Asmma (2013) which revealed that proximate composition of Eruca sativa seeds contain moisture 6.6%, crude
proteins 25.17%, and crude fiber 2.4% and total minerals ash 9.1%. Therefore results about proximate analysis of seeds are in closed agreement
with the results reported by different authors who indicate results studied were valuable and indicate standard values of E. sativa varieties.
3.2Qualitative and quantitative estimation of phyto- chemicals from Eruca sativa seeds
Qualitative analysis of Eruca sativa seed indicated the presence of diverse plants metabolites such as alkaloids, phenolic acid,
flavonoids, saponins and tannins (Table 3.2), whereas the quantification of phyto-constituent was mentioned in table 3.3.
Results depicts that E. sativa seed extracts possess significant amount of flavonoids (8.35±0.64), Phenols (14.13±1.40), alkaloids
(5.38±0.004), saponins (0.66±0.26) and are highly rich in tannins (52.98±4.29). Phytochemical study of seed of E. sativa showed presence of all
essential phyto constituents required for potential traditional medicine25-26. Eruca seed extract contains important secondary metabolite such as
flavonoids, alkaloids, tannins, phenols, saponins, ascorbic acid and those are used as remedies of many diseases and frequently required in
traditional medicines8. Inspite of that, it is mentioned in traditional pharmacopoeia and ancient literature that it does hold a numerous health
promoting chemical agents comprising carotenoids, vitamin C, fibers, and glucosinolates (GLs) used for several therapeutic properties. The
presence of large amount of these phytochemicals bestows high medicinal activities including antioxidant and anti allergic27.
3.3 Quercetin determination in Eruca Sativa seeds
The concentration of Quercetin in Eruca sativa seeds was found to be 0.03326±0.002 (y=32.58x, R2=0.971) using UV/Vis
spectrophotometric analysis as shown in fig 3.2. Quercetin which is a typical example of the flavonol subclass is used as a nutritional supplement
found in fruits and vegetables. It has the capacity to stop the oxidation of LDL by scavenging free radicals and chelating the transition metal ions.
The quercetin may assist to prevent several diseases, such as atherosclerosis, cancer and chronic inflammation by a mechanism which retards
oxidative degradation28.
3.4 Determination of antioxidant activity of Eruca sativa seeds
For the assessment of plant extracts for antioxidant activity, the DPPH scavenging protocol is quite easy and sensitive. Numerous
methods are employed for the evaluation of free radical scavenging assay but the stable 2, 2-diphenyl-1-picryl-hydrazyl radical (DPPH) is
considered more important due to its availability and ease.
The antioxidant assay of the aqueous extract of Eruca sativa seed was investigated using DPPH scavenging assay. The ability of
DPPH to decolorize a free radical depends upon the presence of antioxidant. The DPPH is a stable radical, having an odd electron which shows
maximum absorbance at 517 nm. By accepting an electron by DPPH, provided by antioxidant compound, it decolorizes the DPPH which is
calculated quantitatively by the change in absorbance of solution. The plant extract exhibited a significant dose dependent inhibition of DPPH
activity with a 50% inhibition at a concentration of as compared with reference standard gallic acid which was also reported by Goyal29.
Scavenging activity of plant extract is a function of its concentration, as the concentration of compounds to be tested raises; there is an
increase in total radical scavenging where as lower IC50 value of any extract reflects improved protective action30. IC50 and anti radical power
(ARP) for DPPH radical- scavenging activity was calculated graphically by using standard curve (y=0. 079, R² = 0.959) for Eruca seeds and (y =
0.095x, R² = 0.974) for gallic acid. IC50 value of Eruca sativa was 126.2 µg/mL, with reference to (y=0.079x, R2=0.959 (Fig 3.1).
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The IC50 was calculated for Eruca seeds and that of gallic acid (standard) are enlisted in table 3.5. By changing the concentrations of
test compounds scavenging effect was increased. The IC50 value for seed extract was comparatively higher than the IC50 of gallic acid
(126.2μg). DPPH radical scavenging activity of the extracts expressed as IC50 garden rocket was previously studied which showed that ethanolic
extract showed high antioxidant activity (1.0 mg/mL) where as aqueous extract exhibits moderate DPPH radical scavenging activity of (3.1
mg/mL) as reported by Ismail et al31. Table 3.1: Proximate analysis (%) of Eruca sativa seeds
E. sativa seeds
Dry Matter
Moisture
content
Ash
content
Crude
Protein
Crude Fat
content
Carbohydrate
content
Sample I
94.5±2.5
5.5±0.5
2.9±0.6
30±1.2
29±1.6
3.55±0.6
Sample II
92.5±2.2
5.1±0.5
2.5±0.5
29±1.1
28±1.5
3.45±0.2
Sample III
93.5±1.8
5.9±0.5
2.3±0.4
27±1.0
26±1.2
3.33±0.4
Values are expressed in terms of Mean ±SD after triplicate analysis
Table 3.2: Qualitative analysis of phytochemicals from Eruca sativa seeds
S. No
Phytochemicals
E. Sativa
I II III
1
Alkaloids
+
+
+
2
Flavonoids
+
+
-
3
Phenols and tannins
+
+
+
4
Saponin
+
+
+
5
Terpenoids
+
+
+
6
Carbohydrates
+
+
-
7
Protein and amino acids
+
+
+
8
Reducing sugars
+
+
+
9
Cardiac glycoside
+
+
+
10
Steroids
+
-
-
Triplicate analysis (n=3)
Distilled water =I, ethanol=II. Methanol=III. Present +, Absent -, (n=3)
Table 3.3: Quantitative analysis of phytochemicals from plant extracts
Eruca sativa seeds
Flavonoids
%
Alkaloids
%
Saponin
%
Tannins
%
Phenols
%
Sample I
8.35±0.64
5.38±0.004
0.66±0.26
52.98±4.33
14.13±1.40
Sample II
8.05±0.54
5.18±0.05
0.58±0.23
50.98±2.29
13.99±1.29
Sample III
8.01±0.74
5.08±0.19
0.61±0.56
49.98±1.89
14.93±1.31
Values are shown in form of Mean±SD after triplicate analysis
Fig 3.1: Quercetin determination in Eruca sativa seeds
y = 32.58x
R² = 0.971
0
0.5
1
1.5
2
2.5
3
0 0.02 0.04 0.06 0.08 0.1
Absorbance
Concentration (mg/ml)
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Table 3.4: DPPH Radical Scavenging assay of aqueous extract of Eruca sativa seeds
TEST
DPPH Free Radical Scavenging Activity
(%age)
Seed Extracts
(µg/mL)
Eruca Sativa Seeds
Gallic acid
(Standard)
20µg/mL
10.37±0.04
11.0±0.1
40 µg/mL
14.72±0.07
16.26±0.3
60 µg/mL
25.22±0.3
30.0±0.6
80 µg/mL
30.60±1.1
38.54±1.0
Values are shown in the form of mean±SD after triplicate analysis
Fig 3.2: DPPH Free Radical Scavenging Activity of aqueous extract of Eruca sativa seed
Table 3.5: IC50 and anti radical power (ARP) of aqueous seed extract and gallic acid
Sample
IC50(µg)
ARP
Gallic acid
104.38
9.58×10-3
Eruca sativa Seed
126.2
7.92×10-3
Table 3.6: Physiochemical Properties of Eruca sativa seed oil
Oil Parameters
Values
Acid value
0.338±0.0062 mg KOH/g oil
Saponification value
174.43±3.03 g of NaOH/100 g oil
Peroxide value
6.66±1.527 meq 2/kg oil
Wax contents
17.073±0.050%
Specific gravity(30 ºC)
0.8005±0.000361
pH
7.103±0.015
Color
Dark Yellow
State at room temperature
Liquid
Values expressed in term of mean ±standard deviation after triplicate analysis
3.5 Physiochemical properties of Eruca sativa oil
Physiochemical parameters of oil were determined which are given in table 4.6. The Eruca seed oil was greenish brown in color,
liquid at room temperature and even in a refrigerator. It was found that the specific gravity of Eruca oil was lower than values reported by
Flanders and Abdulkarim5. According to Rudan and Klofutar. (1999), specific gravity of oils (vegetable) at any specific temperature when
compared to water rises as the average molecular mass decreases32.
Saponification number is an indicator of the average molecular weight and chain length which is inversely proportional to the
molecular weight of the lipid33. The saponification number of the Eruca oil was found to be 174.43g NaOH/100g oil which is closed to
saponification value of 168.1% reported by Flanders and Abdulkarim5. Lower values of saponification prove that they have greater molecular
mass comparing to that of common oils. This parameter is dependent upon the extent of unsaturation which describes their iodine number along
with the free fatty acid value34.
0
10
20
30
40
50
60
70
80
20
40
60
80
%age inhibition
Conc (µg/ml)
Gallic acid
E.Sativa
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The Eruca seed oil had a wax content of 2.14±0.023% as shown in table 4.6. Waxes are high melting- point 25esters of long chain
carboxylic acids and long-chain alcohols. During the refining process, wax was removed from crude oils to clarify the oil. They have intense
applications in cosmetic and pharmaceutical industry, lubricants, food products and polymers as well.
The peroxide value of Eruca sativa seed oil was found to be 6.66±1.527 meq 2/kg oil as given in table 3.6. In order to determine
quality of fats and oils, this parameter is highly significant because it suggests the oxidative constancy of the oil for the period of storage. Fat or
oil which is processed from premium quality oil seed kernels produces the new peroxide values to visualize like the oil35.
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... The existence of saponins and alkaloids in rocket extract produce an enhancement in sperm viability and motility. This increase may be due to that, rocket extract have the ability to improve testes growth and enhance the spermatozoa maturation, proliferation and differentiation [27].Furthermore, rocket have aphrodisiac effect. In seeds, the aphrodisiac effect may be duo to isothiocyanates and desulfoglucosinolates agent [28]. ...
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