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Article history: The present work reports the extraction of Moringa oil from the Moringao leifera Lam. (Moringaceae) seeds
Received 1 September 2015 using Soxhlet extraction. The effects of the various operating parameters such as temperature, solvent
Received in revised form ratio and solvent type on the extraction yield of the oil have been investigated. The extraction yield of
19 November 2015
the oil was varied with change in the operating temperatures (ranging from 80 to 110 C) as well as with
Accepted 4 December 2015
change in the solvents ratio (such as, chloroform:methanol (C:M) from 1:1, 2:1, 3:1, 4:1). The maximum
Available online 17 December 2015
extraction yield was obtained at C:M (3:1) ratio. The increase in the yield was also found with the increase
in the temperature. The fatty acid compositions of the Moringa oils extracted by the solvent extraction
Keywords:
Moringa oleifera Lam. seed
method have been analyzed using gas chromatography (GC). The change in the composition of the fatty
Moringa oil acid was seen with the change in the solvent media. Also, this extracted oil was analyzed by nuclear
Soxhlet extraction method magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR), thermo-gravimetric analysis
(TGA) analysis. From TGA analysis it was found that, the oil degrade at temperature about 425450 C.
The oil was also analyzed with the physicochemical properties and the phytochemical tests to conrm
the presence of saponins, avonoids, steroids, terpenoids, phenols, and triterpenoids. It was found that
the solvent extraction is an effective method for the Moringa oil extraction.
2015 Elsevier B.V. All rights reserved.
1. Introduction oleic acid (70%) and smells a pleasant peanut like fragrance. Ben
oil is more stable than canola oil, soybean oil, palm oil when
Moringa oleifera, known as Moringa, Drumstick, Malunggay is used in frying (Nguyen et al., 2011). Ben oil has been used in
an Indian tree grows in Asia, South America, Africa, the Caribbean the manufacturing of perfume, hair care products, as lubricants,
(Palafox et al., 2012). Moringa species is one of the most useful trees for edible purpose. Ben oil is considered equivalent to olive oil
in tropics and subtropics of Asia and Africa. Moringa belongs to the in terms of fatty acid composition (Zhao and Zhang, 2013). The
Moringaceae family. Almost all parts of Moringa such as ower, oil has fantastic cosmetic value; it is used in body and hair care
fruits, leaves, roots are edible and have been consumed as vegetable products as a moisturizer and as skin conditioner (Mahmood
(Abd El Baky Hanaa and El-Baroty Gamal, 2013). The root extract et al., 2010). The oil consists of various sterols such as campes-
possesses antimicrobial activity (Busani et al., 2012). The extracts terol, stigmasterol, clerosterol, -sitosterol, 5 -avenasterol and less
from the owers are found to have the hepatoprotective effect quantity of 24-methylenecholesterol, campestanol, stigmastanol
(Upadhyay et al., 2015). The seeds have the edible oil known as and 28-isoavenasterol (Anwar et al., 2007). Another potential use
Ben oil. The seed cake is one of the best natural coagulants and can of Moringa oil is as a biodiesel feedstock, as an alternative source
be effectively utilized for treatment and purication of the highly of oil, Moringa seeds have been proposed as a potential source to
turbid water (Zhao and Zhang, 2013). complement the mentioned feedstock (Palafox et al., 2012).
M. oleifera seeds contain 1947 % oil commercially known as Moringa seeds and oil are used in the treatment of arthri-
Ben oil and are rich in palmitic, stearic, behenic and oleic acids tis, rheumatism, and hypertension (Aviara et al., 2015). Moringa
(Ojiako and Okeke, 2013). Ben oil is reported to have very high oil is very stable due to the presence of the high oleic acid
and so can be used for the frying application (Zhao and
Zhang, 2013). Moringa seed oil is non-drying oil and can be
used for lighting as it burns without dense smoke. Because of
Corresponding author. Fax: +91 22 33611020. the capacity of the edible oil to absorb and retain oral fra-
E-mail addresses: dv.pinjari@ictmumbai.edu.in, dpinjari@gmail.com grances, it is valuable in perfume manufacturing. It is also used
(D.V. Pinjari).
http://dx.doi.org/10.1016/j.indcrop.2015.12.004
0926-6690/ 2015 Elsevier B.V. All rights reserved.
P.R. Bhutada et al. / Industrial Crops and Products 82 (2016) 7480 75
3. Characterization of extracted oil shows the details experimental process for determination of sec-
ondary metabolites (Ajibade et al., 2013).
3.1. Physico-chemical characteristics of extracted oil
3.3. Thermo-gravimetric analysis of Moringa oil
3.1.1. Determination of specic gravity and density
Specic gravity bottle (100 ml) was cleaned and dried in oven. The thermo gram of extracted Moringa oil was recorded using
The weight of the empty gravity bottle was obtained as W1 , the aluminum pan between temperature range 30500 C and under
weight of the gravity bottle with the water was taken as W2 , and inert atmosphere of N2 at a ow rate of 50 ml/min (Shimadzu,
the weight of the bottle with the oil was taken W3 . The specic Japan).
gravity and the relative density of the oil are calculated using the
formula below (Olushola, 2013): 3.4. Gas chromatographic analysis of Moringa oil
Weight of oil (W3 W1 )
Specic gravity = Fatty acid composition of the Moringa seed oil extracted by
Weight of equal volume of water (W2 W1 )
Soxhlet was determined using GC after derivatization to fatty acid
(6)
methyl esters (FAME). The preparation of the FAME was done by
standard IUPAC method. About 1 g of extracted oil was mixed with
g
Weight of oil 0.1 g sodium methoxide and 10 ml of methanol and then the mix-
Density( )= (7) ture was reuxed in water bath at 8090 C for about 1 h. The
ml Volume of oil
mixture was cooled and taken in separating funnel with the addi-
tion of hexane followed by water washing. Finally, some sodium
3.1.2. Determination of saponication value
sulphate was added to remove any traces of water in the FAME.
2 g of extracted oil weighted and taken in conical ask and
FAME separation and identication were carried out on a Shimadzu
approximately 25 ml 0.5 N alcoholic solution of potassium hydrox-
gas chromatograph model GC-1000, equipped with BPX70 (70%
ide (KOH) was added. The mixture was then gently boiled under
cyanopropyl polysilphenylene-siloxane) highly polar capillary col-
reux on the water bath for 1 h. After this the titration of the hot
umn and ame ionized detector. The amount of the sample injected
mixture was done by 0.5 N HCl, using phenolphthalein indicator.
was 1.0 l. Nitrogen ow rate of 4.0 ml/min was used as the car-
The end point of the titration was faint pink (Adejumo et al., 2013).
rier gas and a split injector was used with a split ratio of 50:1. The
injector and detector temperatures were set at 270 C and 280 C
28.05 respectively. The oven temperature was programmed as follow:
Saponication value = (Blank-sample)
Weight of oil sample the initial temperature was 180 C which was maintained for 4 min
(8) from 180 C to 190 C at 2 C/min, from 190 C to 270 C at 3 C/min
hold for 6 min, and then to a nal temperature 320 C hold for
another 6 min. Fatty acid methyl esters were quantied as per-
centages of the total methyl ester peak areas (Bezerra and Filho,
3.1.3. Determination of free fatty acid 2014).
Acid value is expressed as % free fatty acid calculated as oleic
acid. 1 g of oil was weighted accurately and taken in the conical 3.5. FTIR analysis of Moringa oil
ask. About 20 ml of neutral alcohol was added with few drops of
phenolphthalein and shaken vigorously. The solution was titrated FTIR analysis was used to reveal the functional groups present
with 0.5 M alkali solution with constant shaking until pink color in the extracted oil. The FTIR spectra of extracted oil samples was
remains constant. The acid value and the free fatty acid content recorded using FTIR spectrophotometer (Shimadzu 8400s, Japan)
were calculated as follows: (Adejumo et al., 2013) using ATR sampling technique by recording 45 scan in % transmit-
0.5 56.1 Burette reading tance mode in the range of 4000500 cm1 .
Acid value = (9)
Weight of oil sample
1H
Acid value 3.6. NMR analysis of Moringa oil
Free fatty acid = (10)
2 In addition to the GC analysis, 1 H NMR was employed to study
and compare the molecular structure of the Moringa seed oil
3.1.4. Determination of iodine value extracted using the solvent extraction technique. 1 H NMR of the
About 0.3 g of oil was taken in dry ask. Then 10 ml of car- extracted Moringa oil was analyzed using Agilent 400 MHz. The oil
bon tetrachloride solution was added followed by 25 ml of WiJs sample was dissolved in the deuterated chloroform and the spectra
solution and was kept in the dark place for 30 min. Then 10% KI were recorded (Almoselhy et al., 2014).
solution was added in 20 ml to the mixture and was shaken. After
that, 100 ml of distilled water was and was shaken vigorously. Then 4. Results and discussion
starch indicator was added to it and dark blue color was obtained.
The mixture was titrated with 0.1 N sodium thiosulphate till blue 4.1. Effect of temperature
to colorless point was obtained (Olushola, 2013). The iodine value
was calculated as follows: As shown in the Table 2, the yield of the Moringa seed oil changes
12.69
with the increasing temperature in the solvent extraction. When
Iodine value = (Blank-sample) N (11)
Weight of sample temperature increases (gradually) from 70 C to 100 C, the extrac-
tion yield was increases by about 10% for C:M (2:1) solvent ratio
3.2. Phytochemical screening and by about 10% for C:M (3:1) solvent ratio. Fig. 2(A) shows the
effect of temperature on the extraction yield of Moringa oil for
The phytochemical screenings were carried out to conrm the the solvent ratio of C:M (2:1). As the temperature increases from
presence or the absence of the secondary metabolitestannins, 70 C to 100 C extraction of oil was increased due to the increase
saponins, avonoids, phenols, terpenoids, and steroids. Table 1 of solubility and diffusivity which improves the mass transfer.
P.R. Bhutada et al. / Industrial Crops and Products 82 (2016) 7480 77
Table 1
Detailed description of experimental procedure for conrmation of presence of secondary metabolites.
Tannins 0.5 g of the moringa oil was mixed with 20 ml water. The mixture was boiled and ltered. Then few drops of 0.1% ferric
chloride were added. The development of the brownish green or blueblack color conrms the presence of tannins.
Saponins 2 g of moringa oil was mixed and boiled with 20 ml of the water and then ltered. 10 ml of this ltrate was further
mixed with 5 ml of distilled water and was shaken vigorously for stable persistent froth. The formation of the froth
conrms the presence of the saponins.
Flavonoids The known quantity of the oil was taken with the ammonia solution and the concentrated sulfuric acid was added and
was allowed to develop the color. Development of yellow color indicated the presence of the avonoids.
Steroids 0.5 g of the moringa oil was mixed with the 2 ml of acetic anhydride with further addition of concentrated sulfuric
acid and was allowed to develop color. The color change from violet to blue or green color conrms the presence of
the steroids.
Phenols 1 g of the oil was mixed with 20 ml water with the further addition of the ferric chloride. The appearance of the blue
color indicated the presence of the phenol.
Terpenoids 5 g of the oil was mixed with 2 ml of the chloroform. Further 3 ml of concentrated sulphuric acid was added to form
the layer. Development of the reddish brown color at interface indicates presence of the terpenoids.
Table 2
Comparison of extraction results with % yield and energy supplied.
Exp. Run Solvent Temp. ( C) Time (min.) Avg.% yield Std. Dev. Net energy supplied (kJ/g)
Fig. 2. (A) Effect of temperature on extraction yield for C:M (2:1); (B) effect of solvent typeon the extraction yield of moringa oil; (C) effect of solvent ratio on the extraction
yield of the Moringa oil.
78 P.R. Bhutada et al. / Industrial Crops and Products 82 (2016) 7480
Table 3 nicantly higher than that of the reported values and it will vary as
Physicochemical characteristics of extracted moringa oil.
per the geographical locations where Moringa plants are grown.
Constituents Obtained FAO/WHO standard
Density (gm/cm3 ) 0.24 0.009 4.5. Phytochemical analysis of extracted Moringa oil
Specic gravity 0.98 0.006 0.9
Acid value (mg KOH/gm) 26.22 1.210 4 The test for the presence of the avonoids, saponins, phenol,
Free fatty acid (mg KOH/gm) 13.11 0.615 5.77.28 tannins, and terpenoids were done and found to be positive. These
Saponication value (mg KOH/gm) 172.16 0.945 181.4
chemical constituents are known for many therapeutic values and
Iodine value 75.06 0.943 80106
various applications will be determined on the basis constituents
present in the oil. The avonoid test showed the presence of yellow
color, the froth formation was seen in the saponins test, reddish
However, when temperature exceed beyond the 100 C the yield
brown color indicated the presence of the terpenoids, and phenols
decreases. The possible reason behind this nature may be the reduc-
test gave the light red color. Grey color was seen in the tannins test
tion and breakdown of antioxidant molecule at high temperature.
and red color for the steroid test. The avonoids are reported to have
Another possibility may be the decomposition of active ingredi-
the antifungal, antibacterial and antioxidant properties (Emmanuel
ents presents in the oil (Hossain et al., 2009). Also, from Table 2 it
et al., 2014).
is clear that, as the temperature increase, the yield of the Moringa
oil increases and the energy supplied per unit of extracted oil was
4.6. Gas chromatography (GC) analysis of extracted Moringa oil
decreases. The highest extraction yield of oil (41%) was obtained
at the solvent ratio of 3:1 (C:M) and temperature of 100 C with
Fig. 3(A) shows the GC chromatogram of extracted oil which
expense of energy requirement of 5.211 kJ/g of oil.
conrms the presence of the various fatty acids such as palmitic,
stearic, arachidic and behenic acids and the main unsaturated
4.2. Effect of solvent type fatty acid is oleic acid (70.5%), with small amounts of palmitoleic,
linolenic, and eicosenoic acids (Table 4). Since, the extracted oil
The solvents used for the extraction in this work, were both polar contained a substantial amount of behenic acid (4.9%), it can be
(methanol) and non-polar (chloroform, petroleum ether). Polarity used as a natural source of behenic acid, which has been used as an
is dened as the relative ability of the molecule to engage in the oil structuring and solidifying agent in margarine, shortening, and
strong interactions with the other polar molecules. Polarity rep- foods containing semi-solid and solid fats, eliminating the need to
resents the ability of the molecule to enter into interactions of hydrogenate the oil (Abdulkarim et al., 2005). The high percentage
all kinds. Relative polarity is the sum of all possible interactions of oleic acid in the oil makes it desirable in terms of nutrition and
(Hossain et al., 2009). It was found that, as the polarity of the solvent high stability cooking and frying oil. High oleic-acid vegetable oils
decreases, the extraction yield oil increases as shown in Fig. 2(B). such as high-oleic corn, sunower and canola have been found to
The yield for the methanol is low as compared to the yield for the have enough oxidative stability to be used in demanding applica-
chloroform and petroleum ether as shown in Table 2. Polar solvents tions such as frying (Petukhov et al., 1999; Warner and Knowlton,
are highly capable of extracting the nonfat material such as antiox- 1997). In addition high-oleic oils have low saturated fatty acid lev-
idants, tocopherol, pigments, etc., non polar solvents along with els. Therefore high-oleic oils can be viewed as a healthy alternative
the non-fatty matter also extract the oil present in the seeds. The to partially hydrogenated vegetable oils.
oil extracted using only methanol was red in color whereas the oil
extracted using the chloroform and the petroleum ether was yel- 4.7. FTIR analysis of extracted Moringa oil
low in color. The red color of the oil may be due to the presence of
the pigments and the tocopherol present in the Moringa seed. FTIR analysis as shown in Fig. 3(B) clearly shows one peak
of ester functional group (C O stretch) at 1773 and peak at
1162 shows ether linkage and broad peak of 3414 (O H stretch)
4.3. Effect of solvent ratio
and 750 peaks of ortho-substituents. The peak in the region
35003200 cm1 indicates the presence of the alcohol or phenol
The extraction of the Moringa oil was performed using the sol-
(O H stretch). The elongated U shape around this region shows
vent mixture of chloroform and methanol in various ratios. The
the presence of the alcohol group. This functional group appears
comparison of the extraction of Moringa oil with respect to various
predominantly in the protein and fatty acid structures present
solvent ratios is shown in Fig. 2(C). The solvent mixture contains
in Moringa seeds. Due to the high content of protein present in
both the polar (methanol) and the nonpolar (chloroform) solvent.
seeds there is also a contribution in this region from the N H
The ratio of the nonpolar solvent was changed and the difference
stretching of amide groups. The peaks at 2920 cm1 and 2852 cm1
in the extraction yield of the oil was noted. With the increase in
are assigned to symmetrical and asymmetrical stretching of the
the nonpolar solvent from 1:1 to 3:1 the yield in the extraction of
C H of CH2 group present in fatty acids. One peak in the region
the oil also increases. However, when the ratio of nonpolar solvent
17601665 cm1 shows the presence of the ester functional group.
in the mixture was increased further then the yield was depressed.
The peak in the region 30002700 cm1 shows the presence of the
This is because, nonpolar solvent may be unable to extract the toco-
carboxylic acid. The peak in the region of 15001450 cm1 shows
pherols, pigments, antioxidants and the other substances present
the presence of the benzene (C C) (Arajo et al., 2010).
in the oil.
4.8. NMR analysis of extracted Moringa oil
4.4. Physico-chemical analysis of extracted Moringa oil
It was observed from the 1 H NMR analysis Fig. 3(C), there were
The physicochemical properties such as density, specic grav- no signals for hydroperoxides (primary oxidation products) and
ity, acid value, saponication value, iodine value, were analyzed aldehydes (main secondary oxidation products), indicating that no
and are tabulated in Table 3. The specic gravity, iodine value and oxidative degradation was taking place. The presence of these oxi-
saponication value of the extracted oil matches with the reported dation products in the ranges 8.098.19 ppm for hydroperoxides
values (Table 3). The acid value and free fatty acid content was sig- proton, and 9.309.90 ppm for aldehydes, indicates oxidation of
P.R. Bhutada et al. / Industrial Crops and Products 82 (2016) 7480 79
Fig. 3. (A) Gas chromatic analysis of Moringa oil; (B) FTIR Spectra of Moringa oil; (C) 1 H NMR spectra of oil extracted by solvent extraction; (D) TGA analysis of Moringa oil.
Table 4
Relative percent composition of fatty acid in Moringa oleifera seed oil.
Abdulkarim et al. (2005) Sunga and Whitby (1995) Dahot and Memon (1985) Ferrao and Ferrao (1970)
these edible oils (Alonso-Salces et al., 2011). The presence of sterol (I). The CH2 O between tetrahydropyran ring ( O ) and ester group
(-sitosterol) in extracted oil could be deduced from the signal shows peak at 5.3 (J) (more downeld because of electronegative
0.84 ppm due to methylic protons in position C18 of the steroidal group on both sides). The solvent (deuterated chloroform) shows
skeleton (Almoselhy et al., 2014). The peak in the region 0.840.85 peak at 7.3 (K).
(A) is the methyl proton, splitted into triplets. The acyl group in
the region 1.121.16 (B) has been splitted into multiplets. Other 4.9. TGA analysis of extracted Moringa oil
aliphatic protons are splitted in the region of 1.572.29 (CE). The
alcoholic OH shows triplet at 3.433.44 (F) (downeld shifting of The thermo-gravimetric analysis of the extracted Moringa oil
protons of OH due to electro negativity of oxygen). Esteric proton was carried out to characterize the decomposition stages and
shows peak at 3.62 (G). Glyceryl group shows peak at 4.084.13 thermal stability of the Moringa oil as shown in Fig. 3(D). This
(H). The CH2 O of tetrahydropyran ring shows peak at 4.254.28 thermo gravimetric curve veries the sample heterogeneity, since
80 P.R. Bhutada et al. / Industrial Crops and Products 82 (2016) 7480
the intermediates formed are a mixture of several components. palm and coconut biodieseldiesel blending on their physico-chemical
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