Applied Food Research 2 (2022) 100051

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Applied Food Research

journal homepage: www.elsevier.com/locate/afres

Valorization of hemp hearts oils by advanced extraction techniques and

their comparative physicochemical characterization
Abhiroop Mookerjee, Venu Babu Borugadda, Ajay K. Dalai, Venkatesh Meda∗
Department of Chemical and Biological Engineering, College of Engineering, University of Saskatchewan, 57 Campus Drive, Saskatoon, SK S7N 5A9, SK, Canada

a r t i c l e i n f o a b s t r a c t

Keywords: Industrial hemp hearts were subjected to three different extraction techniques-advanced extraction techniques
Industrial hemp hearts like microwave and ultrasound extraction and primitive extraction technique like mechanical cold pressing in
Hemp oil order to extract oils. The extraction yield varied between 41.0 ± 2.1% (w/w) and 54.0 ± 2.7% (w/w). Linoleic acid
Advanced oil extraction techniques
(LA) was reported to be the major polyunsaturated fatty acid (PUFA) followed by 𝛼−Linolenic acid (ALA). The
Antioxidants
ratio of LA to ALA was found to be close to 3:1 which is considered optimal for nutrition. The ratio of unsaturated
Pharmaceuticals
Nutraceuticals fatty acids (UFA) to saturated fatty acids (SFA) was in the range of 6.7 to 9.1. Several minor compounds like ɣ-
Sitosterol, stigmasterol, rhodoxanthin, carotene, and methyl cholate as well as antioxidants like 𝛽-Carotene and
ɣ-Tocopherol have been identified in the oils. The physicochemical properties of the oils like the acid value,
iodine value, and oxidative stability indicated that they have higher quality and that the level of unsaturation
was very high. Furthermore, free radical scavenging activity of the oils revealed that the oils exhibited 94% of
activity in the first 2 h. A perfect combination of unsaturated fatty acids and saturated fatty acids, an appropriate
medley of different antioxidants not only qualifies hemp seed oil as an excellent source of nutrition but also as
an important plant-based (vegetable) oil for the higher value nutraceutical and pharmaceutical industries.

1. Introduction tionally, it is known that the presence of 𝛾-linolenic acid in hemp seed
oil makes it nutritionally superior to most comparable seed oils. Apart
Cannabis sativa is a widespread species in nature, which grows in a from THC and CBD, 30 different cannabinoids have also been reported
variety of climates and altitudes of the Asiatic subcontinent. Hemp is in trace amounts in hemp seed oils from certain cultivars (Citti et al.,
a variety of Cannabis sativa with a lower composition of the psychoac- 2019). The fatty acids are essentially important in pathogenesis, pre-
tive compound tetrahydrocannabinol (THC) > 0.3% (w/w). On the other vention of coronary heart diseases (Gobbo et al., 2016), hypertension,
hand, it is rich in another bioactive compound called cannabidiol (CBD). during pregnancy (Lee et al., 2018), breastfeeding, etc. The essential FA
Currently, different parts of hemp are being used industrially in the man- and 𝛼-linolenic acid present in the oils are ideal for the cosmetics and
ufacture of rope, textiles, paper, bioplastics, body care products, essen- wellness industries (Ahmad & Ahsan, 2020).
tial oils, medicine, nutritional supplements, insulation, biofuel, and even Antioxidants are recognized for their potential in health promotion
in construction (Small & Marcus, 2019; Cerino et al., 2020) (Figure S1). and prevention of aging-related diseases (Conti et al., 2016) as well as
One of the most important parts of the hemp plant for its use in the phar- in the cure of cancer (Arsova-Sarafinovska & Dimovski, 2013) and heart
maceutical and food industry is the seed, and oil extracted from hemp diseases. The antioxidant property of cold-pressed hemp seed oil was
hearts is believed to contain the ideal ratio of polyunsaturated fatty acids proven to be more effective in free radical elimination than extra-virgin
(PUFA) i.e., omega-6 and omega-3 fatty acids (Callaway, 2004). olive oils (Tubaro et al., 1998). Apart from the nutrient value of the
The level of PUFA in hemp is considered to be the highest for any FA content in hemp seeds, it contains other important compounds like
vegetable oils (80–81%). Recent reports have stated that the ideal ratio 𝛽−sitosterol, 𝛽−carotene, and salicylic acid (Oomah et al., 2002), which
for the consumption of omega-6 and omega-3 fatty acids by human be- complements the nutritional makeup of the oil and makes it functionally
ings must be between 2:1 and 3:1. A study comparing the fatty acids of more effective.
two different varieties of hemp has shown that the ratio of omega-6 and Extraction of seed oils and determination of the physicochemical and
omega-3 fatty acids is 2.5 and 2.7 respectively (Callaway, 2004). Addi- functional properties using different techniques has always been of key

Abbreviations: ALA, 𝛼−Linoleic acid; LA, Linoleic acid; AV, Acid Value; MW, Microwave extraction; UAE, Ultrasound-assisted extraction; SFA, Saturated Fatty
acid; cP, Centipoise; IV, Iodine Value; CP, Cold pressing; UFA, Unsaturated fatty acid; EPR, Electro Paramagnetic Resonance; GLA, 𝛾−Linolenic acid.
∗
Corresponding author.
E-mail address: Venkatesh.meda@usask.ca (V. Meda).

https://doi.org/10.1016/j.afres.2022.100051
Received 12 November 2021; Received in revised form 13 January 2022; Accepted 27 January 2022
Available online 30 January 2022
2772-5022/© 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
A. Mookerjee, V.B. Borugadda, A.K. Dalai et al. Applied Food Research 2 (2022) 100051

interest to the oil industries. Cold pressing (CP) is a common and fre- derstand the nature of the oils in order to suggest probable areas of
quently used mechanical method for extraction (Çakaloğlu et al., 2018). application.
CP is advantageous since it operates at low temperatures without the use
of any organic solvent that might cause environmental or health haz- 2. Materials and experimental methods
ards. Moreover, the low temperature preserves the thermolabile com-
pounds present in the hemp seeds. The only disadvantage is that a huge 2.1. Cultivation, seeds collection, and processing
quantity of oil is left behind in the cake resulting in a low extraction ef-
ficiency (Aladić et al., 2015). The yield of oil obtained from grape seeds Industrial hemp hearts were procured from Northern Neutraceu-
ranged between 48.9% −73% (w/w) with the maximum yield of 73% ticals, Saskatchewan, Canada. The plants were grown in central
(w/w) being achieved for a preheating temperature of 90 °C, a dye di- Saskatchewan, near Saskatoon and had a harvest time of 4 months. Post-
ameter of 15 mm, and screw rotation speed of 40 RPM (Rombaut et al., harvest, the seeds were removed from the plants and dehulled and split
2015). using guarded dehullers.
Some novel methods have also been reported- microwave-assisted, 500 g of the hemp hearts were sieved using a 1.4 mm sieve (The
and ultrasound-assisted which are being recently used as an alternative W.S Tyler Company of Canada Limited). Seeds of particle size >1.4 mm
extraction technique to obtain oil from seeds including hemp (Jiao et al., were named as “whole seeds”. A part of the whole seeds was also ground
2014). The superiority of these methods lies in the fact that they of- using a kitchen coffee grinder which was measured to be 0.85 mm and
fer high extraction efficiency in considerably lesser amounts of time. less. These fine seeds were termed as “ground seeds”.
They promote extraction by breaking down the cell walls of the struc- Certified ACS grade hexane (98.5%), acetone, methanol (99.8%),
tures containing the oils and hence can also lead to the extraction of USP/FCC standard potassium iodide, and certified ACS grade potassium
essential nutraceutical and antioxidant compounds with lesser degener- hydroxide (85.8%), ACS grade starch for iodometry were purchased
ative effects on the cells. Ultrasound assisted extraction of hemp seed from Fisher Scientific, Edmonton, Canada. Laboratory grade distilled
oils was performed by Lin and his group as well as and Esmaeilzadeh water was used throughout the experiment. ACS grade phenolphthalein
Kenari and associates on hemp seeds and increased extraction yields and reagent grade carbon tetrachloride (99.9%), ACS certified sodium
were reported as compared to traditional extraction methods (Lin et al., thiosulphate pentahydrate (99.5–101%) crystals, Wij’s solution were
2012; Esmaeilzadeh & Dehghan, 2020). A slightly modified ultrasound purchased from Millipore Sigma, Oakville, Canada.
extraction method was also proposed in which solvent extraction using
a Soxhlet apparatus was supplemented using ultrasound applied using 2.2. Extraction of oils from hemp hearts
an ultrasound probe (Luque-García & Castro, 2004). This coupled ef-
fect of conventional Soxhlet extraction along with ultrasound extrac- 2.2.1. Ultrasound-assisted extraction
tion led to an increased oil yield. Rezvankhah and his team studied A GmbH UP400S Ultrasonic processor (Hielscher Ultrasonics, Tel-
the effects of Microwave assisted extraction on hemp seed oils and re- tow, Germany) with an H7 probe was used for the extraction. 25 g of
ported that the method was more time efficient as compared to tradi- both the whole and ground hemp hearts were mixed with 250 ml n-
tional Soxhlet extraction. The oils produced were also of higher qual- hexane and introduced in the ultrasonic processor with seed to solvent
ity (Rezvankhah et al., 2019). Similarly, it was also reported that Mi- ratio of 1:10. The working frequency of the device was maintained at
crowave assisted extraction of oils from pongamia pinnata seeds was 24 kHz with a 0.5 s pulse for all the experiments. The experimental
quicker and had no adverse effect on the quality of oils as compared to conditions used are tabulated in Table S1.
other conventional methods (Kumar et al., 2018). The solvent-oil mixture was separated from the extracted hemp
Previously, hemp did not receive significant attention because of le- hearts via vacuum filtration using a Buchner funnel and the oil was
gal issues concerned with hemp cultivation. According to the 2018 re- purified using a rotatory evaporator (Rotavapor®, Buchii, Farmingdale,
ports by Health Canada, 77,800 acres of industrial hemp were planted NY) and stored in the refrigerator at 4 °C for further analysis.
in Canada out of which 33,000 acres (38.5%) were planted in Alberta,
27,100 acres (35%) in Saskatchewan, and 11,500 acres (about 15%) in 2.1.2. Microwave-assisted extraction
Manitoba. It is estimated that roughly 90% of the total seed for hemp An Explorer Hybrid12 Discover microwave extractor (CEM, Toronto,
cultivation is produced in Canada which contains mostly Canadian de- ON) was used for the extraction of oils from the hemp hearts by varying
veloped varieties (Lepescu, 2019). Thus, it is evident that with increas- the temperature (65 °C, 70 °C) and time (10, 15, 20, 30 min). For each
ing awareness there is a significant increase in hemp cultivation as well experiment, 9 g (3 × 3 g) of the sample was added to 90 ml (3 × 30 ml)
as the number of hemp processing plants leading to more research fo- of n-hexane (sample: solvent= 1:10) and introduced into the microwave
cusing on hemp seeds, especially oils extracted from hemp hearts. extractor. After extraction, vacuum filtration was performed to separate
Although there are previous research reports concerning the extrac- the oil-solvent mixture from the extracted seeds. Finally, the mixture
tion of hemp oils, the literature is scanty and the scientific community was purified, and pure oil was collected using a rotary evaporator. The
lacks a thorough comparison between the effect of different extraction extracted oil was stored in glass vials in the refrigerator at 4 °C for further
techniques on the quality of oils produced from hemp seeds. Previous analysis.
research has mostly focused on the extraction and analysis of hemp seed
oil using a single technique (e.g., only cold pressing or microwave ex- 2.1.3. Cold pressing
traction) or a comparison between two techniques. However, a compre- Cold pressing was performed using an Oekotec screw-press (MON-
hensive comparative analysis of different extraction techniques (new FORTIS IBG, Nordrhein-Westfalen, Germany) using 2 kg of the dehulled
vs old) for hemp heart oils operating at different temperature ranges hemp hearts (Figure S2) with a moisture content of 9.5% (w/w). For
have not been performed previously. Thus, the major objectives of the each experiment, 600 g of the hemp hearts were mixed with 49.5 g
study were to (a) Compare the extraction efficiencies of three different (8.25% w/w) hulls and pressed using a 0.6 cm dye. A frequency of 24 Hz
extraction techniques-microwave, ultrasound (operating at higher tem- was maintained in the motor using an INVEOR transmitter (KOSTAL,
perature and more advanced) and cold pressing (operating at room tem- Dortmund, Germany). The pressed cake was collected and weighed for
perature and conventional extraction technique) for the extraction of further analysis. The obtained oil was separated from any debris using
hemp seed oils by varying particle size and extraction conditions to ob- a J6-M1 centrifuge (BECKMAN COULTER, Brea, CA) operating at 4200
tain maximum yield. (b) Analyze the physicochemical properties, fatty R.P.M for 10 min at a temperature of 18 °C.
acid composition, free radical scavenging activity, and inherent chemi- The Swedish tube method of oil extraction was performed on 2.22 g
cal composition of the extracted oils to determine the quality and, un- of hemp hulls to find the total quantity of oil present in the hemp

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A. Mookerjee, V.B. Borugadda, A.K. Dalai et al. Applied Food Research 2 (2022) 100051

hearts. The residual oil in the pressed cake was also determined and seed oils was determined by change in the intensity of the DPPH when
reported by the Analytical Service Laboratory at the KeyLeaf Life Sci- hemp seed oils were mixed (1 mg/ml) with a standard DPPH stock so-
ences laboratory (Saskatoon, SK) using the Swedish Tube method of lution prepared in methanol (0.11 g DPPH in 500 ml methanol). The
analysis (Troëng, 1955). Approximately 3.15 g of the initial hemp hearts test specimens were prepared by dissolving 0.2 g of hemp seed oils in
were also analyzed for their initial oil content using the aforementioned 1 ml of DPPH stock solution. Each test specimen was filled in melt-
method. ing point tubes and sealed with Teflon tape for analysis in triplicates at
room temperature. The antioxidant activity of the hemp seed oils was
2.2. Physicochemical characterization of the extracted hemp seed oils evaluated after every 6 h throughout 24 h, thereby slow reaction an-
tioxidants could quench the free radicals with time. After each analysis,
The initial moisture content of the seeds was measured by the tra- spectra were baseline corrected and double integrated using Win-EPR
ditional oven-dry method. In brief, 5 g of the whole and ground seeds software to determine the intensities to compare with the control spec-
were separately transferred to an aluminum tray and left overnight in imen (DPPH stock solution) without hemp seed oils. Free radical scav-
a SUPREMATIC laboratory oven (Hotpack, Canada) at a temperature of enging activity was calculated based on the following equation.
100 ± 3 °C for 24 h. The moisture content was calculated as follows. [ ]
Act ivit y = Ac − As ∕Ac ∗ 100 (3)
𝑤 − 𝑤𝑜
Moisture content = 𝑖 × 100 (1)
𝑤𝑖 Where, Ac : Double integrated average value of the control and As :
A Trace 1310 Gas Chromatograph equipped with a TSQ Duo Mass Double integrated average value of the specimen
Spectrometer (Thermo Fisher Scientific, Waltham, MA) was used for
identification of the chemical compounds present in the hemp heart 2.3. Statistical analysis
oils. The analysis was performed by maintaining the following condi-
tions: source temperature: 250 °C; helium flow rate: 1.2 mL/min; injec- All experimental data were analyzed using IBM SPSS Statistics soft-
tion temperature: 250 °C; split ratio: 50:1; split flow: 60 mL/min. Initial ware package (Version 28). The experimental data generated have been
oven temperature: 40 °C with a hold time of 1 min; increased to 150 °C reported as mean ± Standard Deviation (SD) of replicates unless other-
at 5 °C/min and finally, increased to 320 °C at 10 °C/min and held con- wise mentioned. The analysis of variance (ANOVA) was performed using
stant for 5 min. The hemp heart oils were dissolved in acetone for the the general linear model followed by Tukey’s Multiple Mean Compari-
preparation of test samples. The mass spectral data for the hemp oil sam- son test. The differences between means were considered significant at
ples were acquired from 50 to 650 m/z. The peaks were identified after P<0.05.
comparison against the standard NIST (National Institute of Standards
and Technology) library using ChromeleonTM 7.2 Chromatography Data 3. Results and discussion
System (CDS) software. Furthermore, tocopherols were measured and
identified from the GC–MS data after comparison against the standard The MC of the whole and ground seeds were reported to be
NIST library. 5.66 ± 0.06 and 5.93 ± 0.14 respectively which was similar to previ-
1 H NMR spectral analysis was used to determine the fatty acid com-
ous studies (Rezvankhah et al., 2019).
position and iodine value of the hemp heart oils at 500 MHz using an
Avance NMR spectrometer (Bruker, Milton, ON). In brief, 0.2 ml of the
3.1. Effects of particle size and ultrasound-assisted extraction (UAE)
extracted hemp seed oils and deuterated chloroform were pipetted into
process parameters on oil yield
NMR tubes (5 mm; Norell Standard Series, Sigma-Aldrich, USA) for the
analysis. Manual baseline correction and integration were applied using
Particle size is a major important to be considered during oil extrac-
the Topspin 4.0.7 software package (Bruker, Bremen, Germany) before
tion from seeds. In the present study, UAE was carried out using whole
processing the spectra for fatty acid composition and iodine value ac-
and ground seeds. It was noticed that the yield significantly increased
cording to the method of Popescu et al. (2015) and Vigli et al. (2003).
with a decrease in the particle size with yield% of 44.9 ± 0.76 (w/w)
The quantity of 𝛽-carotene was measured by the UV–VIS spectropho-
and 33.6 ± 0.66 (w/w) for the ground seeds as compared to yields of
tometer at 450 nm by the British standard method of analysis (BS 684,
43.3 ± 0.71% (w/w) and 28.3 ± 0.82% (w/w) for whole seeds for ultra-
1977) (Standard, 1977) The amount of 𝛽-carotene was measured using
sound times of 60 and 10 min respectively. It can be hypothesized that a
the following formula:
smaller seed size enhanced the contact surface area between the solvent
V × 383 × (As − Ab) and the seed surface, (Ntalikwa, 2021) causing better penetration of the
β − carotene = (2)
100 × W solvent inside the seeds leading to increased extraction of oil. These
where: V= Volume of the solution used for analysis; As-Ab = Absorbance results also lie in accordance with the study of Yunus who reported a
of the sample – Absorbance of the blank at 450 nm; W= Weight of the better oil yield from seeds having a lesser particle size (Yunus et al.,
sample in g and 383= Extinction co-efficient for carotenoids 2013).
The viscosity of the oils was measured using a DV-1+ Digital Vis- To study the effects of the different extraction parameters on UAE,
cometer (BROOKFIELD, MA, USA) at 25 °C and 40 °C. The acid value the solvent to seed ratio and the pulse-pulse ratio of the ultrasound de-
was measured according to AOCS official method Cd 3d-63 and the ox- vice were maintained at 10:1 and 0.5 (the acting on-off ratio was op-
idative stability of the extracted oils was determined by an accelerated timized at 1:1) respectively. The effect of time and ultrasonic power
oxidation test (Rancimat method) carried out at 110 °C according to EN on UAE were studied by varying the extraction time for 10, 30, and
14,112 standard procedure. A Metrohm 743 Rancimat (Metrohm AG, 60 min and power for 135 W and 200 W (Lin et al., 2012). From Fig. 1,
Switzerland) was used to conduct the stability tests. it is evident that the yield is affected by the ultrasound power. For a
Free radical scavenging activity of the extracted hemp seed oils was power of 200 W, the yield increases from 33.6 ± 2.4% (w/w) in 10 min
analyzed by measuring DPPH radical scavenging potential through Elec- to 46.8 ± 1.98% (w/w) in 30 min and then decreases to 44.9 ± 0.14%
tro Paramagnetic Resonance (EPR) spectrophotometer (BRUKER EMX (w/w) when exposed for 60 min. The same trend can be observed for ul-
EPR; Bremen, Germany). The following operating parameters were used trasound power of 130 W, where the yield increases from 32.5 ± 0.99%
for the DPPH radical quenching assay: microwave power 5.27 mW, cen- (w/w) to 38 ± 2.69% (w/w) and then decreases to 37.43 ± 0.49% (w/w)
ter field 3516.85 G, sweep width 100 G, operating frequency 9.8 GHz, for exposure times of 10, 30 and 60 min, respectively. However, it was
modulation amplitude 2 dB, receiver gain 30 dB, attenuation 16 dB, g- noted that the yields obtained under the latter condition were not sig-
factor 2, number of scans 2. Free radical quenching activity of the hemp nificantly different (Fig. 1).

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A. Mookerjee, V.B. Borugadda, A.K. Dalai et al. Applied Food Research 2 (2022) 100051

Table 1
Effects of MW Time and Temperature on the Ex-
traction Yield∗ .

Time (min) Temperature ( °C) Yield (%)

10 70 40.31 ± 1.14a
15 65 47.43 ± 1.6b
20 70 45.00 ± 1.46b
30 65 53.77 ± 2.05c
∗
Values as reported as mean ± SD (n = 2).
Means in the same column followed by different
superscripts are significantly different (P < 0.05).

the yields except for the minimum and maximum values obtained (Table
S2).
The effect of different ultrasound conditions upon the solvent re-
covery percentage is illustrated in the bar chart in Figure S4. It is noted
that the maximum recovery percentages are noted for UAE conditions of
P200, T30 (70 ± 1.48% v/v); P200, T10 (76.8 ± 3.1% v/v) and P80, T30
Fig. 1. Change in yield percentages concerning the time for ultrasound power (68 ± 3.14% v/v), respectively which were not significantly different.
of 130 W and 200 W respectively. Values are represented as mean ±SD (n = 2).
However, there were significant differences between the recovery% of
Comparisons have been performed separately for US power 130 W and 200 W.
some of the groups (P200, T60; P130, T60; and P200, T30) as indicated
Data represented by different letters under each comparison set are significantly
different (P < 0.05).
in Figure S4.

3.2. Impact of microwave-assisted extraction on oil yield

These results indicated that the ultrasound is more effective in the The effects of two independent properties- temperature and time
first 30 min and with an increase in time, a decreasing trend in the yield have been studied to understand the impact of microwaves on the ex-
was noticed. This may be attributed to the fact that due to increased time traction yield (Table 1). Microwaves work by rupturing and damaging
periods; ultrasound waves can disrupt more hemp heart cell walls lead- cell walls as well as the lipoprotein membrane surrounding the individ-
ing to increased penetration of the solvent inside the cells resulting in ual lipid bodies, thus causing the release of oil from the cell (Hu et al.,
more extraction. Again, the slight decrease in the extraction yield can 2017). A 10:1 solvent to solid ratio was maintained as it was previously
be hypothesized to have been caused by prolonged exposure time as observed that a higher solvent to sample ratio led to a higher extraction
a result of which ultrasound oxidation of the edible oils take place ei- yield (Rezvankhah et al., 2019).
ther by thermal degradation or sonolysis, which can be attributed to the It was noticed that the extraction yield gradually increased with a
phenomenon of “cavitation”. Cavitations are micro-mechanical shocks subsequent increase in the time and temperature of the MW extractor.
affecting the structural and functional components of the cells and such From Table 1, it is noticeable that the extraction yields obtained by dif-
an increased phenomenon in the cells lead to lipid oxidation and de- ferent MW conditions are significantly different (P < 0.05). The yield
terioration (Jahouach-Rabai et al., 2008). This phenomenon may have increased drastically from 40.31 ± 1.15% (w/w) for MW time of 10 min
resulted in a slight decrease in the oil yield upon increasing the ultra- to about 47.43 ± 1.6% (w/w) for 15 min and finally the maximum yield
sound time. Further, an increase in the rate of cavitation leads to ex- of 53.77 ± 2.05% (w/w) was obtained at 30 min extraction time. The
cessive breakdown of the cell membranes and cell walls, thus exposing slight decrease in the extraction yield for the temperature and time com-
the hydrophobic fatty acid tail of the phospholipids. This might cause bination of 70 °C and 20 min can be hypothesized as follows. Due to the
binding of the oil to the phospholipid and lower its extractability by the elevated temperature of the solvent for a longer period, the phospholipid
solvent. Therefore, excessive ultrasound can have a delirious effect on cell wall and cell membrane disintegrates exposing the fatty acid tail
extraction (Gonzalez & Barrett, 2010). which in turn binds with the free oil and thus decreases its extractabil-
Again, the ultrasound time was optimized at 30 min and 60 min ity by the solvent (Gonzalez & Barrett, 2010). The slight decrease in the
respectively to understand the effect of UAE power on the yield (%). extraction yield can also be attributed to the partial thermal decompo-
Ultrasound powers of 20 W, 130 W, and 200 W were used for UAE time sition of the extracted oils as a result of exposure to microwaves at an
of 60 min which is represented in Figure S3. On the other hand, 130 W elevated temperature for a prolonged period (Rezvankhah et al., 2019).
and 200 W were used for 30 min. The yields obtained by varying the This was however not observed when the MW extraction time was
ultrasound power at 60 min was not found to be significantly different 10 min. Also, contrary to the research conclusions by Rezvankah and his
(Figure S3). group, it was observed that time plays a significant role in the extraction
With an increase in power, the yield gradually increased and the yield using microwaves at a constant temperature.
maximum yields of 46.8 ± 3.25% (w/w) and 44.9 ± 3.11% (w/w) were The effects of different process parameters like MW time and tem-
produced at 200 W for 30 min and 60 min respectively (Table S2). It perature on the solvent recovery coefficient are represented by a bar
can be hypothesized that when the ultrasound power is low, the energy chart in Figure S5.
produced by the ultrasound probe is not enough to cause the propaga- The maximum and minimum solvent recoveries of 36.67 ± 1.8%
tion of ultrasound pressure waves to cause cavitation (for the release of (v/v) and 17.78 ± 0.9% (v/v) were achieved for an MW temperature of
oils). The results obtained are on par with previous research led by Lin 65 °C for 30 min and 70 °C for 10 min respectively.
et al., who also reported that the maximum extraction yield is achieved
for 200 W. The slight decrease in the yield% for 130 W and 60 min run 3.3. Extraction of hemp hearts oil via cold pressing
time may be attributed to sonolysis and the cavitation phenomena. The
maximum extraction yield (46.8 ± 3.25% w/w) was obtained for ul- The extraction yield obtained by cold pressing was calculated to be
trasound power of 200 W and 30 min which was considered for further 41.07 ± 0.5% (w/w). The% yield from the hemp hulls was reported to
experiments. However, no significant differences were reported between be 2.87 ± 0.1% (w/w) which indicated that the oil contribution by the

4
A. Mookerjee, V.B. Borugadda, A.K. Dalai et al. Applied Food Research 2 (2022) 100051

hemp hulls can be considered negligible and we can assume that the & Khanam, 2019). The main component present in the oils is found
major fraction of the oil was extracted from the hemp hearts only. The to be the triglyceride (trilinolein) with a relative area% 49.65 ± 2.5
amount of residual oil in the pressed cake for each of the three trials and 43.16 ± 2.2 for oils obtained by UAE and CP, respectively. Trili-
was found to be 6.47 ± 0.2% (w/w). nolein is a naturally occurring triglyceride that has anti-isthmic, anti-
arrhythmic, and antioxidant properties (Chan et al., 2005) and has
3.4. Comparative analysis of three different extraction techniques found profound uses in the pharmaceutical, cosmetics, and food indus-
try.
A comparative analysis of the extraction yields is presented in Figure Linoleic acid (LA/n-6 PUFA) was the second most significant com-
S6. Results reported that there existed a significant difference between pound present in the extracted oils with a relative area% of 8.94 ± 0.4
the yield obtained by CP and MW, but not between CP and UAE or and 12.08 ± 0.6 for UAE and CP, respectively. Here, we notice that the
UAE and MW (Figure S6). MW extraction produced the maximum oil relative percentage of trilinolein is more in the case of UAE as com-
yield of 54 ± 3.8% (w/w) followed by 47 ± 1.97% (w/w) for UAE, and pared to CP and vice-versa for LA. This can be attributed to the effects
41 ± 2.82% (w/w) for CP. This can be attributed to microwaves causing of microwaves which might have caused hydrolysis of the trilinolein
increased rupture of the cell walls resulting in lipolysis and proteoly- molecule into linoleic acid. UAE extracted oil showed a lower% of LA,
sis of the cell wall materials, and thus enhancing the penetration of the which is similar to the results obtained by Devi and Khanam (Devi &
solvent inside the cells to extract the oil. Moreover, a temperature of Khanam, 2019).
70 °C and MW exposure time of 15 min (optimum condition for MW The presence of PUFA in food is essentially important for cardio-
extraction) led to a movement in the media due to a reduction in sol- vascular health. Data from clinical studies and meta-analyses led by
vent viscosity which enhanced the extraction yield (Rezvankhah et al., Marangoni and his team report the existence of a positive correlation
2019). The mechanical effect of ultrasound promotes the release of sol- between high dietary intakes of LA and the improvement of cardiovascu-
uble compounds from the plant material disrupting the cell walls, en- lar health (Marangoni et al., 2020). It also influences long-term glycemic
hancing mass transfer, and facilitating the solvent to access the cellular control and insulin resistance. The abundance of Linoleic acid in hemp
contents (Cravotto et al., 2008). On the other hand, microwaves lead to seed oils was also reported by other researchers (Latif & Anwar, 2009;
an increased solvent penetration into the matrix due to the movement Leizer et al., 2015). The presence of palmitic acid and oleanolic acid
of dissolved ions (since microwaves promote the rotation of molecu- in very low amounts was noted for UAE and CP oils respectively. Oleyl
lar dipoles by disrupting the weak hydrogen bonds) and as a result, oleate (previously not identified) was also reported in UAE oil samples.
causes increased extraction of intracellular materials. It can further be Apart from the presence of fatty acids and triglycerides, numer-
hypothesized that ultrasound waves may have caused the lipoprotein ous other compounds like sterols, vitamins, and carotenoids were also
cell membrane to break and retain some of the oils, thus reducing its identified in the oils. Among them, the notable ones are ɣ-Sitosterol,
extractability which led to a lower yield (%) as compared to MW ex- ɣ-Tocopherol, stigmasterol, rhodoxanthin, carotene, methyl cholate,
traction. Cold pressing is a mechanical extraction technique and hence methyl glycocholate, and demecolcine. Their ranges of application
the yield is fairly less compared to the other two techniques. Overall, it are varied, ɣ-Sitosterol has anti-bacterial and anti-fungal properties
can be concluded that the yield obtained by each extraction technique (Hernández-Hernández et al., 2016). Stigmasterol, a phytosterol has
was more compared to the yields produced by hemp cultivars in Turkey been documented as an immunomodulator that possesses a great thera-
(Bouayoun et al., 2018) and Pakistan (Anwar et al., 2006). peutic potential (Antwi et al., 2017). Overall, the results showed a gen-
The extraction rates for each extraction method were compared to eralized GC–MS spectrum of the chemical compounds present in hemp
find the most time-effective extraction technique. The extraction rate heart oils. The results indicate that the components present in the oils
was calculated as follows: vary relatively and depending upon the different extraction techniques
adapted, may result differently due to the nature of heating mechanisms
Ext ract ion rate = Weight of the ext ract ed oil (g)∕Total time of reaction (min)
involved as well as other physical treatments followed.
Extraction rate studies showed a significant difference in the extrac-
tion rates between UAE, MW and CP (Figure S7). The rate for CP was the 3.5.2. Fatty acid composition and iodine value by 1 H NMR
least (0.18 ± 0.01 g/min) whereas, it was maximum 0.45 ± 0.01 g/min The relations between the chemical groups generating the NMR spec-
for MW extraction, followed by UAE at 0.33 ± 0.03 g/min. This can tral signals and their chemical shifts in CDCl3 solvent have been used as
be attributed to the rupture of cell walls due to microwaves and ultra- previously reported (Vigli et al., 2003; Popescu et al., 2015).
sound which caused an increased exposure of the intracellular materials The integrals of the 1 H NMR values are strictly proportional to the
to the solvent, resulting in enhanced extraction over a shorter time pe- number of protons present at each chemical shift. From the 1 H NMR
riod. The only limitation was the difference in extraction times for each spectra, the fatty acid composition was calculated by combining the
reaction. The extraction rates have been calculated based on the experi- empirical formulas listed in Table S3 and various signal integrals from
ments which gave the maximum oil yield for each extraction technique. Table S4.
Thus, calculation of the extraction yield based on a specific time for each From Table 3, it is evident that Linoleic acid (LA) was the major
technique can be considered as the future scope of the study. PUFA calculated by 1 H NMR. The maximum concentration of LA was
calculated for CP (65.4 ± 0.7 mol%) followed by MW (63.6 ± 0.1 mol%)
3.5. Characterization of the hemp heart oils and UAE (57.0 ± 0.13 mol%) which are comparable to the ones re-
ported from hemp seed cultivars of Pakistan (Anwar et al., 2006) and
3.5.1. Chemical composition by GC–MS analysis Italy (Porto et al., 2012) and slightly more than from the cultivars of
GC–MS analysis of the oil samples obtained from different extrac- Morocco (Bouayoun et al., 2018). Apart from LA, ALA was also found
tion techniques was performed to analyze the differences in the chem- to be present in the hemp oil samples in minor quantities ranging from
ical composition, tocopherols, and other minor compounds present. 18.6 ± 0.17 mol% for MW extracted oils to 23.9 ± 0.2 mol% for UAE
The GC–MS values for MW oils and UAE oils were identical, thus the oils which was comparable to the Italian cultivars (Porto et al., 2012)
MW data has not been reported in the study. The major compounds and slightly higher than Polish (Siudem et al., 2019) and Pakistani culti-
present in the oils are presented in Table 2. The present study com- vars (Anwar et al., 2006). Numerous studies have repeatedly shown that
pared the GC–MS results based on FA profiles for each process. The ma- ALA (𝜔−3 FA) has significantly helped in enhancing the heart health
jor components revealed from the GC–MS readings were linoleic acid, (Freeman, 2009; Balanzá-Martínez et al., 2011) and hence their health
trilinolein, palmitic acid, and oleyl oleate which was in accordance benefits cannot be ignored. It has also been reported that ALA has in-
with previous studies ( Porto et al., 2012; Aladić et al., 2015; Devi hibitory effects on cancer and tumor growth (Leizer et al., 2015). The

5
A. Mookerjee, V.B. Borugadda, A.K. Dalai et al. Applied Food Research 2 (2022) 100051

Table 2
Chemical composition of hemp heart oils∗ .

Name of compound Molecular Formula Retention time (min) Relative Area (%)

UAE CP

Palmitic acid C16 H32 O2 27.677 1.55±0.01 ND

Linoleic acid C18 H32 O2 30.118 8.94±0.4a 12.08±0.6b
Psi, psi Carotene C40 H66 37.716 ND ND
ɣ-Tocopherol C28 H48 O2 39.768 3.64±0.04a 1.83±0.01b
Stigmasterol C29 H48 O 41.512 ND ND
ɣ-Sitosterol C29 H50 O 41.995 7.96±0.1a 7.04±0.1a
Betulin/Lupeol C30 H50 O2 42.616 0.96±0.02a 0.82±0.01a
Rhodoxanthin C40 H50 O2 43.121 ND 2.99±0.02
Oleyl oleate C36 H68 O 45.395 1.85±0.01 ND
Oleanolic acid C30 H48 O3 45.488 ND 0.59±0.01

Trilinolein C57 H98 O6 49.646 49.65±2.5a 43.16±2.2b

Decanedioic acid C36 H66 O8 51.557 ND ND
Demecolcine C21 H25 NO5 53.294 1.09±0.03a 1.04±0.02a
Methyl glycocholate C36 H69NO 6Si13 54.468 ND 3.54±0.17
Methyl cholate C25 H42 O5 59.39 ND ND

ND: Not Detected;.

∗
Values are represented as mean ± SD; Means in the same row followed by different super-
scripts (a, b) are significantly different (P < 0.05).

Table 3
Fatty acid composition of the hemp hearts with different extraction techniques (% molar)∗ .

Name of the extraction technique Linoleic acid Linolenic acid Oleic acid SFA LA: ALA UFA: SFA

Microwave 63.6 ± 0.1a 18.6 ± 0.17a 8.3 ± 0.3a 9.5 ± 0.4a 3.4 9.6
Ultrasound 57.0 ± 0.13b 23.9 ± 0.2b 10.4 ± 0.1ab 8.9 ± 0.2a 2.4 10.3
Cold Pressing 65.4 ± 0.7a 18.1 ± 0.5a 12.4 ± 1.0b 4.1 ± 0.4b 3.6 23.2
∗
Values are reported as mean ± SD; Means in the same column followed by different superscripts (a,b) are found
to be significantly different (P < 0.05).

consumption of omega−6 and omega−3 Fatty acids in a ratio of 3:1 is cholesterol and preventing cardiovascular diseases (Bouayoun et al.,
considered optimal for nutrition (De La Paz et al., 2014; Leizer et al., 2018). A MUFA, oleic acid was also found in the hemp seed oil sam-
2015). The ratios of LA and ALA in our MW and CP samples are close to ples ranging between 8 and 11 w/w%. Total FA composition results
3:1 and hence they can be considered optimal for consumption (Table 3). show that Linoleic, Linolenic, and Oleic acid comprised around 90% of
The concentration of SFA was also found to be very less which indicates the total fat content which was similar to previous research findings
that hemp seed oil is suitable for dietary intake. The ratio of UFA to (Oomah et al., 2002).
SFA was also found to be significantly different between the extraction Vitamin E has been studied to function primarily as a chain-breaking
methods and they lie between 9.6 and 23.2 with the highest ratio of antioxidant and it prevents the propagation of lipid peroxidation. GC–
23.2 being observed for CP oils (Table 3). The difference in the ratios of MS studies of our oil samples have shown the presence of ɣ-Tocopherol
UFA and SFA between the oils obtained can be explained as follows. Mi- (UAE:3.64 ± 0.18% CP:1.83 ± 0.1%). ɣ-Tocopherol and Vitamin E pos-
crowaves are nonionized electromagnetic waves which causes a rotation sess anti-inflammatory properties that are beneficial for the treatment
of the molecular dipoles. Due to the presence of moisture in the seeds, of cardiovascular disease and prostate cancer (Jiang et al., 2001). Be-
the rotation of the water molecules leads to a disruption in the weak hy- sides, it also has antioxidant effects in very low quantities (Câmara et al.,
drogen bond which destabilizes the arrangement of water molecules and 2014). Although previous studies have shown the presence of 𝛼, ɣ, and
as a result, the individual water molecules break up into its constituent 𝛿 Tocopherols in hemp oils (Anwar et al., 2006; Latif & Anwar, 2009),
H+ and OH− ions. The free H+ in the medium causes hydrogenation of the quantities of 𝛼 and 𝛿 tocopherols have been significantly less com-
the unsaturated fatty acids into saturated fatty acids. Similarly, ultra- pared to ɣ-tocopherols. The major tocopherol reported in our oils was ɣ-
sound pressure waves also disrupt the hydrogen bonds present in the Tocopherol which was similar to previous results (Oomah et al., 2002).
water content of the cells and similarly causes hydrogenation of the The vitamin E profile of oil extracted from hemp seeds can also be com-
UFA to SFA. Cold pressing takes place at room temperature and hence pared to those of peanut oil, olive oil as well as soybean oils. From the
there is no disruption of hydrogen bonds of the water content of the spectrophotometric analysis, minute quantities of 𝛽−Carotene have also
cells. The mechanical pressure applied to extract the oils is insignificant been reported in our oils. The concentration of 𝛽−Carotene reported
as compared to the energy induced by microwaves or ultrasound. As in MW oils was more (1.66 ± 0.08 mg/100 g) compared to CP oils
a result, there is no hydrogenation leading to conversion of the UFA to (0.38 ± 0.02 mg/100 g).
SFA. Thus, there exists a considerable difference in the UFA:SFA ratio for Very small quantities of 𝛽−Carotene in hemp seed oils cultivated in
UAE and MW as compared to CP extraction. We also know that unsatu- Manitoba were also reported which confirms our results (Oomah et al.,
rated fatty acids are less stable than saturated fatty acids and hence oxi- 2002). Dietary carotenoids (especially 𝛽−Carotene) are thought to pro-
dation caused by heat generated due to microwaves/ ultrasound waves vide health benefits in combatting certain types of cancers and eye dis-
might also be a factor for conversion of the UFA (especially linoleic acid) eases. It is also believed to have added benefits since it has the potential
into hydroperoxides and hydroxy acids leading to a lower UFA:SFA ra- to be converted to Vitamin A (Kim et al., 2021).
tio for the MW and UAE oils. This high ratio, especially in the case Iodine value is an important indicator of the degree of unsaturation
of CP oils can also be considered favorable for the reduction of blood in the oil. Oils having a higher concentration of unsaturated FA have a

6
A. Mookerjee, V.B. Borugadda, A.K. Dalai et al. Applied Food Research 2 (2022) 100051

Fig. 2. Effect of different extraction techniques on the IV of the oil (n = 2).

Values are represented as mean ± SD (n = 2). Mean data represented by different Fig. 3. Effect of different extraction techniques on the AV of hemp oil (n = 2).
letters are significantly different (P < 0.05). Values are represented as mean ± SD (n = 2). Mean data represented by different
letters are significantly different (P < 0.05).

higher iodine value and vice versa (Dawodu et al., 2015). Iodine value
thus depends upon the number of double bonds present in the oil. Exper-
extracted oil (cruder) which makes it more viscous when compared to
imental results showed no significant differences between the mean IV
the other oils.
which ranged between 150 and 170 g I2 /100 g oil and is similar to the IV
The same trend is also observed at temperatures of 40 °C. More-
of hemp seed oils obtained by the research led by Anwar (Anwar et al.,
over, viscosity is one of the main factors governing oil absorption and
2006), Latif (Latif & Anwar, 2009) and Chen (Chen et al., 2010) (Fig. 2).
drainage (Ziaiifar et al., 2008) i.e., more the viscosity of the oil, the
The maximum IV (161.5 ± 4.6 g I2 /100 g oil) was recorded for CP oils
slower is the oil drainage. The viscosity reported in our samples vary
whereas the minimum IV of 152.96 ± 2.82 g I2 /100 g oil was recorded
between 20 cP and 50 cP at 25 °C and 15 cP and 35 cP at 40 °C which
for MW oils. The high IV is an indicator of the high degree of unsatu-
can be considered a bit low since vegetable oils like canola, olive, corn,
ration present in the oils. On the other hand, a higher temperature and
peanut, and soybean usually have a viscosity in the range of 55–75 cP
microwave irradiation especially in the case of MW extraction caused
at 20 °C and 30–45 cP at 40 °C (Ziaiifar et al., 2008).
some oxidative damage to the oil resulting in a lower IV (Pop, 2018).
Acid value (AV) is a measure of the amount of FFA present in the
Although MW resulted in higher extraction yield, the composition of the
oils and it gives an idea about the suitability of oil for its direct con-
oil was reported to be slightly different relative to the other extraction
sumption or industrial use (Al-Bachir, 2015). From the experimental
methods studied.
outcomes, it is observed that the AVs are not significantly different for
UAE and MW. However, there is significant difference between the AV
3.5.3. Other physicochemical properties obtained by UAE and CP as well as MW and CP (Fig. 3). A maximum AV
From Newton’s law of viscosity, it was concluded that our oil samples of 2.81 ± 0.11 mg KOH/g of oil was obtained for MW oils followed by AV
are Newtonian since the viscosity does not vary considerably by varying of 2.64 ± 0.04 mg KOH/g of oil for UAE oil. The AV, however, dropped
RPM at a particular temperature. abruptly to 0.81 ± 0.05 mg KOH/g of oil for the CP oils (Fig. 3). It can
The viscosities of the oils obtained are reported to be significantly be hypothesized that as a result of the heat generated due to the prop-
different among two different extraction techniques for a particular tem- agation of ultrasound pressure waves (for UAE) and elevated reaction
perature and RPM. The following viscosities are observed at different temperatures (for MW) extraction, the hydrolytic and lipolytic activities
temperatures and RPM ranges. At temperature of 25 °C and RPM 3: vis- become relatively high (Dawodu et al., 2015). This caused the triglyc-
cosity of MW, UAE and CP oils are found to be 32.0 ± 1.2 cP, 29.6 ± 1.5 erides present in the oils to break down into FFA, deteriorating the oil
cP and 47.4 ± 2.4 cP respectively; temperature of 25 °C and RPM 6: vis- (Dawodu et al., 2015). On the other hand, CP takes place at room tem-
cosity of MW, UAE and CP oils are found to be 30.2 ± 1.8 cP, 29.1 ± 1.5 peratures and hence the lipase activity was minimum compared to the
cP and 46.0 ± 2.3 cP respectively; temperature of 40 °C and RPM 3: Vis- other extraction techniques. However, all the reported values obtained
cosity of MW, UAE and CP oils are found to be 17.6 ± 2.0 cP, 21.5 ± 1.1 from the experiments lie within the range of 0.8 and 2.8 mg KOH/g for
and 33.1 ± 1.7 respectively; temperature of 40 °C and RPM 6: viscosity virgin and non-virgin edible oils (Standards., 1999, 2001) which is an
of MW, UAE and CP oils are found to be 14.8 ± 2.0 cP, 19.8 ± 1.0 cP, indication of their safety in consumption.
31.6 ± 1.6 cP respectively. The induction time is defined as the time required for the conductiv-
It was noticed that the viscosity for each of the oils decreases with ity of the water to be increased rapidly and was used as an indication
an increase in the temperature. This can be analyzed from the thermo- of the oxidative stability of our samples.
dynamic perspective. As the temperature of the system increases, the It is known that oxidation is an exponential chain reaction process
molecules gain energy, and the kinetic energy of the molecules increases leading to the generation of free radicals. The Rancimat results in our
leading to less intermolecular interactions. Thus, the viscosity decreases experiments indicate that CP oils have the maximum induction time of
with an increase in temperature. 2.56 ± 0.02 h followed by both UAE (0.65 ± 0.01 h) as well as MW
The CP oils reported the highest viscosity. Cold pressing is essen- (0.69 ± 0.02 h) extracted oils (Fig. 4). Statistical analysis on the exper-
tially a physical mechanism to separate the oil from the cells with the imental results reported that the means of the induction time obtained
help of shear and inter-molecular elasticity and pressure. Thus, even by UAE and MW are not significantly different. On the other hand, there
after centrifugation, some dispersed or slightly soluble phospholipids, exists a significant difference between UAE and CP as well as MW and
lipoproteins, minerals, and polysaccharides might still be present in the CP oils.

7
A. Mookerjee, V.B. Borugadda, A.K. Dalai et al. Applied Food Research 2 (2022) 100051

Fig. 4. Effect of different extraction techniques on the Induction time of oils.

Values are represented as mean ± SD (n = 2). Mean data represented by different
letters are significantly different (P < 0.05).

Table 4
Free radical scavenging activity of the hemp seed
oils∗ .

Extraction Technique Antioxidant Activity (%)

2h 20 h

MW 95.0 ± 0.4 ab 98.0 ± 0.2 a

UAE 94.0 ± 0.7 a 97.0 ± 0.3 b
CP 98.0 ± 1.0 b 99.0 ± 0.1 c
∗
Values are represented as mean ± SD (n = 2).
Means in the same column followed by different su-
perscripts are found to be significantly different (P <
0.05).
Fig. 5. EPR spectra for blank and CP hemp seed oil (a) and their double inte-
grated spectra (b).
The presence of antioxidants like tocopherols in higher concentra-
tions in CP oils can be a reason for a higher induction time since we
know that these antioxidants act as free radical scavengers and singlet antioxidants that are present in the hemp seed oils. Upon analyzing the
oxygen quenchers (Kasote et al., 2015). It is also known that factors like spectra at 20 h, enhanced scavenging activity was observed by the an-
the processing and storage treatment as well as the fatty acid compo- tioxidant compounds present in the hemp seed oils. After 20 h, 1 – 3%
sition of the oil play a key role in determining the oxidative stability increment in the antioxidant activity was noticed relative to at 2 h, i.e.,
(De almeida et al., 2019). Since cold pressing took place at room tem- within 20 h of reaction with DPPH, hemp seed oils quenched or neu-
perature (22 °C), it was highly likely that the oxidation reaction did not tralized the synthetic-free radicals present in the DPPH, primary and
initiate during its storage leading to a higher induction time during the secondary oxidation products from the hemp seed oils. As discussed in
analysis. the previous section, GC–MS analysis of the hemp seed oils revealed the
Electro paramagnetic resonance spectroscopy has found a wide vari- presence of 𝛾-tocopherols, sitosterols, stigmasterol that may be hypoth-
ety of applications in detecting free radicals owing to its high sensitivity esized to be primarily responsible for the antioxidant properties shown
to the unpaired electrons. The close interaction of DPPH with the free by the oils (Câmara et al., 2014). The free radical scavenging activity
radical scavengers present in the hemp seed oils presented a reduced sig- of the hemp seed oils showed that they are capable of quenching the
nal intensity relative to the DPPH stock solution EPR spectrum. DPPH reactive oxygen species and their derivatives in short time (within 2 h)
scavenging activity of the hemp seed oils is reported in Table 4; the addi- owing to the fast reactivity of the antioxidant compounds in hemp seed
tion of 0.2 g of hemp seed oils to 1 ml of DPPH stock solution quenched oils. Therefore, it was anticipated that the presence of antioxidants in
more than 93% of free radicals that are present in DPPH for all the oil the hemp seed oils, could be a value-added feedstock for the produc-
samples in the first two hours. The declining trend in the spectral inten- tion of pharmaceuticals and nutraceuticals as reported by Polovka in
sity for the oil sample is observed from Fig. 5(a) and (b) relative to the his research (Polovka et al., 2007).
DPPH spectra (Blank). Among the three extraction techniques, UAE and
MW oils have lower antioxidant activity relative to CP oil. During the 4. Conclusion
extraction, cold-pressed seeds and oil were not exposed to the higher
temperature like MW and UAE techniques. Due to the high-temperature This study has made a comparative analysis of the extraction effi-
processing, thermally sensitive antioxidant compounds were destroyed ciencies of new and emerging extraction techniques with preexisting
in MW and UAE hemp seed oils (Olczyk et al., 2017). On the other hand, ones operating over a range of temperatures. The maximum extraction
CP hemp seed oil was extracted at room temperature, thereby, the an- yield of 54% (w/w) was produced by MW extraction at a solute: solvent
tioxidant activity of the CP is higher than MW and UAE. Further, the an- ratio of 1:10, run time of 30 min and temperature of 65 °C followed
tioxidant activity of the oils was evaluated at 20 h, therefore the DPPH by UAE oils (47% w/w at an ultrasound power of 200 W for 30 min).
stock solution allows the dissolution of both DPPH and lipid-associated Cold pressing produced the minimum yield of 41% (w/w) (run time

8
A. Mookerjee, V.B. Borugadda, A.K. Dalai et al. Applied Food Research 2 (2022) 100051

60 min). The maximum extraction rate of 0.45 g oil/min extraction was Bouayoun, T., Stambouli, H., Ez, Y., Bouri, A. E., Farah, A., & Tabyaoui, M. (2018). Hemp
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Declaration of Competing Interest cesses for hemp (Cannabis sativa) seed oil considering physical, chemical and
industrial-scale economic aspects. Journal of Cleaner Production, 207, 645–657.
The authors declare that there are no known competing financial 10.1016/j.jclepro.2018.10.036.
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interests or personal relationships that could have appeared to influence vent extraction of hemp (Cannabis sativa L.) seed oil using RSM: Evaluation of ox-
the work reported in this paper. idative stability and physicochemical properties of oil. Food Science Nutrition, 8(9),
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