CHAPTER ONE

1.0 INTRODUCTION

1.1 Background of the study

Hemp (cannabis sativa) is an angiosperm belonging to the cannabaceae family and cannabis

genus. Hemp plant itself is easy to grow in temperate climates and requires good soil, fertilizer and

water but no pesticides nor herbicides. A hemp crop is usually harvested in 120 days after reaching a

height of 10-15 feet. The whole seed contains roughly 25 % protein, 30 % carbohydrates,

15%insolublefibre, carotene, phosphrous, potassium, magnesium, sulphur, calcium, iron and zinc as

well as vitamin E,C,B1,B2, B3, B6. Hemp seed is one of the best source of essential fatty acids with

perfect 3:1 ratio of omega-3-linolenic acid and omega-6-linoleic acid, good for strengthening the

immune system. It is also a good source of gamma linoleic acid. The high content of omega-6 &

omega-3 fatty acids and the relatively high phytosterol content of hemp make them beneficial to

cardiovascular health polyunsaturated for saturated fats can reduce the risk of sudden cardiac arrest

and fatal cardiac arrhythmia as well as reducing blood cholesterol levels and decreasing the cellular

proliferation associated with atheroscierosis. It is also a good source of gamma linoleic acid ( GLA ).

The GLA and vitamin D of hemp beneficial in preventing and treating Osteoporosis. Essential fatty

acids has been found capable of reversing scaly skin disorder, rheumatism, inflammation, diabetes,

excessive epidermal water loss, itch and poor wound beneficial for atopic eczema and psoriasis.

Traditional hemp formulas were applied topically to treat abscesses, boils, pimples and swellings.

The seed folk remedy for tumour and cancerous ulcers. The seed oil is also used in paint, shampoos

and [Link] is also used in cosmetics and body care product is antimicrobial, anti-inflammatory and

antiageing balances, skin pH and moisture levels.

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1.4 Aims and objectives

1.4.1 Aims

Furthermore, we revised the scientific literature regarding the potential use of hempseeds and their

derivatives as a dietary supplement for the prevention and treatment of inflammatory and chronic-

degenerative diseases on animal models and humans too.

1.4.2 Objectives

The goal of this review is to examine the scientific literature concerning the nutritional and

functional properties of hempseeds.

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 CHAPTER TWO

2.0 LITERATURE REVIEW

Cannabis sativa plant, known as hemp, is one of the oldest domesticated crops. Hemp has

been cultivated for years to produce paper, textiles, and cordage. When utilized as a food source,

hemp seeds are processed by cold pressing, resulting in oil and pressed cakes (flour), with alleged

excellent nutraceutical properties (Callaway, 2004). Hemp oil is rich in nutrients and health-

promoting components, including vitamins, mineral salts, amino acids, and phytosterols, etc. In this

sense, the most interesting property is the relatively high content of linolenic essential fatty acid,

belonging to the !-3 series. In general, hemp seed oil is high in polyunsaturated fatty acids (PUFAs),

especially linoleic (18:2, omega-6) and _-linolenic (18:3, omega-3) essential fatty acids (Kriese et

al., 2004). The omega-6 to omega-3 ratio omega!-6/omega-3) approaches 3:1 which is considered

desirable to reduce the risk of dyslipidaemia-associated diseases (Hamazaki, et al., 2001).

Hemp health benefits are also associated with phenolic compounds (Andre et al., 2012),

which occur in variable amounts in almost all classes of plant-derived foods and agro-industrial

residues. Epidemiological, clinical, and nutritional studies strongly support the evidence that dietary

phenolic compounds are effective in the prevention of common diseases, including cancer,

neurodegenerative diseases, gastrointestinal disorders, and others (Zhang and Tsao, 2016; Bilotto, et

al., 2013). A large number of polyphenols has been identified in hemp, especially flavonoids such as

flavanones, flavanols, flavonols, and isoflavones (Smeriglio, et al., 2016). Hemp flavones and

flavonols exert a wide range of biological effects, such as anti-inflammatory, anti-cancer, and neuro-

protective properties (Andre et al., 2010). In addition, apigenin has been shown to play anxiolytic

and estrogenic roles (Wang and Kurzer, 1998; Murthy et al., 2012). Several simple and complex

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lignanamides, also endowed with uncommon and varying bioactive properties, are among the most

abundant (poly-) phenolics of hemp seeds and cake flour (Yan et al., 2015).

Hemp seeds also supply a range of macro- and micro-elements to humans. The deficiency of mineral

nutrients, such as Fe, I, and Zn, is a growing nutritional problem in human populations, while uptake

of other elements, such as Ca, K, Mg, and Se, can be poor in the diets of some specific populations.

Although C. sativa contains only trace amounts of tetrahydrocannabinols (THCs), the psychotropic

components from the congener C. indica, its growing has been long forbidden in Western Countries

for the precautionary purpose (Callway, 2004). However, in the last decades a renewed interest in the

utilization of this crop with low-THC (0–0.2%), for non-drug purposes, and as a raw material to

produce gluten-free foods, fuels, textiles, paper or even biodegradable plastics, took place (Struik et

al., 2000). The versatility of hemp seeds may lead to the sustainable development of numerous

products to be employed in the food, cosmetic, therapeutic and nutraceutical industries (Rannali and

Venturi, 2004). Nowadays, cold pressed hemp seed oils, flour, and seeds are indeed commercially

available. This study reports on the physico-chemical, chemical, and biochemical characterization of

hemp seeds, oil, and flour (Cannabis sativa L.) from the monoecious genotype Fedora, which is

among the most popular hemp variety and it is listed within the EU database of admitted agricultural

species. The main aim of the research was to monitor the distribution of hemp seed components in

the flour or in the oil, after fractionation by cold-pressing. In particular, both the saponificable and

un-saponificable lipid fraction, accounting for 25–35% by weight of hemp seed, and the macro- and

micro-elements, as well as the total polyphenols content and the antioxidant activity (DPPH method),

have been determined. Finally, possible cultivar characteristic traits to be monitored for traceability

were assessed using ATR-FTIR spectroscopy.

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Figure: Cannabis sativa (a) plant and (b) see

2.1 Chemical Phenotype and Taxonomy of C. sativa L.

Elucidation of the cannabinoids’ biosynthetic pathway has been essential to demonstrate that the

concentration of each cannabinoids in the plant is genetically determined, so that various genotypes

related to different chemical phenotypes, diverging in types and concentration of cannabinoids (i.e.,

cannabinoids profile), exist. These phenotypes are known as “chemotypes” or “biotypes”, and three

different principal chemotypes were commonly identified on the basis of the two main cannabinoids

(i.e., THC and CBD) content and ratio:

 I is characterized by a low CBD/THC ratio (0.00–0.05), due to high THC content (>0.3% of

dry weight of the reproductive part of the female plant at flowering). This chemical

phenotype is also known as “drug type”, “THC-predominant”, or C. sativa L. subsp. Indica

and the varieties belonging to this chemotype are those commonly grown for

narcotic/recreational purposes.

 Chemotype II has both the two main cannabinoids, CBD and THC, in a content ratio

(CBD/THC) close to the unity (0.5–3.0), usually with a slight prevalence of CBD. This

chemical phenotype is also named “intermediate type” or “THC-intermediate”, and the

varieties belonging to this chemotype are mainly grown for medicinal use.

 Chemotype III is characterized by high CBD/THC ratio (15–25) due to high CBD amount

and low THC content, not over than 0.3% of dry weight of the reproductive part of the

5
 female plant at flowering. This chemical phenotype is also known as “non-drug type”, “fibre-

type”, “THC predominant”, or C. sativa L. subsp. Sativa, and the varieties belonging to this

chemotype are cultivated for industrial purposes, namely, for fibre, seeds, and their

derivatives.

2.2 Form of Hemp and Change in Composition

2.2.1 Whole seed.

The seed contains 33-35% oil (House et al., 2010). It typically contains 250-230 g/Kg oil and 200-

250 g/Kg crude protein (House et al., 2010). Most of the neutral detergent fiber is found in the seed

hull, hence shelled hemp seeds tend to have more fat and protein comparatively to whole seeds and

the protein is more readily digestible without fiber interference (House et al., 2010). The seed

contains about 25-35% fatty acid, 20-25% protein, 20-30% carbohydrates and 10-15% fiber (Leizer

et al, 2000). The whole seed contains 35.5% oil, 24.8% protein, 20-30% carbohydrates, 27.6% total

fiber (22.2% non-digestible and 5.4% digestible fiber) and 5.6% ash content (Aiello et al., 2016).

Much of the fiber is in the hemp seeds reside in the hulls (House et al., 2010). Fatty acids are

distributed amongst various parts of the seed in varying amounts, as discussed in Lipid section above

(Molleken et al., 1997).

Figure 6. Longitudinal section of hemp seed and its components. (Molleken et al., 1997).

The pericarp is the fruit-membrane, while the test is the seed-membrane and they both together

compose the shell (Molleken et al., 1997). The shell can be separated from the seed (i.e.

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dehulling/deshelling a seed). Oil. Cold-pressed oil does not utilize an organic solvent nor heat to

extract oil and therefore retains its beneficial bioactive compounds including natural antioxidants and

being free of chemical contamination as what would occur with solvent extraction (Teh et al., 2013;

Yu et al., 2005; Deferne et al., 1996). The dark greenish color of hemp seed oil is from the high

chlorophyll content present (Teh et al., 2013). The presence of antioxidants and other molecules that

stabilize cold-pressed oils gives them a long shelf life stability (Prescha et al., 2014). “Virgin” oil is

the oil recovered from the first fractions, or first press, and is the best quality (Deferne et al., 1996).

Microwave drying at one minute intervals to allow for cooling for twelve minutes improved oil yield

from seeds by 15% (Oomah et al., 2002).

2.2.2 Hemp seed meal/defatted flour.

Even after oil processing there is still 10% of residual oil, and has about 30-50% protein; meal is

used as source of vegetable protein and dietary fiber (House et al., 2010). As per FAO/WHO

recommendations, hemp seed meal contains nutritionally sufficient amounts of all the essential

amino acids necessary for infants and children (Malomo et al., 2014). In the hemp seed meal, there is

about 44.32% protein (Malomo et al., 2014). Others provide the range of 30-50% protein present

(House et al., 2010). The meal is a rich source of energy and protein (Russo et al., 2013). Hemp seed

meal is suitable as a human staple as well as animal feed (Deferne et al., 1996).

2.2.3 Hemp seed protein isolate.

Hemp protein isolate was found to consist of 84.15% protein and the amino acid content was mostly

unaffected by the isolation process, however there was a slight increase in arginine amino acid and

the arginine amino acid to lysine amino acid ratio increased with it (Malomo et al., 2014).

2.2.4 Digestibility and Susceptibility

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Digestibility. Malomo et al. in 2014 found that hemp seed protein, both as meal and isolate, were

fairly insoluble, not going higher than 30% across various pH. The low protein solubility obtained by

Malomo et al., 2014, was believed to be caused by protein aggregation of the major protein edestin.

The increased protein solubility of hemp seed meal may be due to the more native state of the

proteins, as compared to the hemp seed protein isolate, or the increased levels of nonprotein

components in hemp seed meal keep the protein in a more soluble structure (Malomo et al., 2014).

Solubility assessed is more for a food science base and Malomo et al., 2014, were studying hemp

seed to use as a food ingredient for product development for Food Industry, which may not compare

directly to digestibility of hemp seed protein in the gastrointestinal system of a human.

2.3 Biosynthetic Pathway Leading to Phytocannabinoids

The biosynthesis of cannabinoids from C. sativa has only been recently elucidated. The

precursors of cannabinoids actually originate from two distinct biosynthetic pathways: the polyketide

pathway, giving rise to olivetolic acid (OLA) and the plastidal 2-C-methyl-D-erythritol4-phosphate

(MEP) pathway, leading to the synthesis of geranyldiphosphate (GPP) (Sirikantaramas et al., 2007)

(Figure2). OLA is formed from hexanoyl-CoA, derived from the short-chain fatty acid hexanoate

(Stoutetal.,2012), by aldol condensation with three molecule of malonyl-CoA. This reaction is

catalyzed by are recently discovered polyketide synthase (PKS)enzyme and an olivetolic acid cyclase

(OAC) (Gagne etal.,2012). The geranylpyrophosphate: olivetolate geranyltransferase catalyzes the

alkylation of OLA with GPP leading to the formation of CBGA, the central precursor of various

cannabinoids (Fellermeier andZenk,1998). Three oxidocyclases will then be responsible for the

diversity of cannabinoids: the THCA synthase (THCAS) converts CBGA to THCA, while CBDA

synthase (CBDAS) forms CBDA and CBCA synthase (CBCAS) produces CBCA

(Sirikantaramasetal.,2004, 2005; Tauraetal.,2007b). Propyl cannabinoids (cannabinoids withaC3side-

chain, instead of a C5side-chain), such as tetrahydrocannabivarinic acid (THCVA), synthetized

8
froma divarinolic acid precursor, have also been reported in Cannabis (Flores-

SanchezandVerpoorte,2008).

Figure 2. The cannabinoid synthetic pathway: cannabigerolic acid (CBGA) is the common precursor

of all main cannabinoids. It is synthesized through an alkylation of the phenolic moiety of olivetolic

acid with the terpenoid component of geranyl pyrophosphate (GPP). The reaction is catalysed by a

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geranylpyrophosphate: olivetolate geranyltransferase (GOT). Olivetolic acid is originated in the

cytosolic polyketide pathway through an aldol condensation of hexanoyl-Coenzyme A (CoA) with

three molecules of malonyl-CoA, that is catalysed by the polyketide synthase (PKS) enzyme in the

presence of olivetolic acid cyclase (OAC). The GPP is synthesized by the plastidial methylerythritol

phosphate (MEP) pathway. In the cytosol, CBGA is converted into the acidic form of the three main

cannabinoids, tetrahydrocannabinol acid (THCA) that in the acidic form has no psychoactive

activity, cannabidiolic acid (CBDA) and cannabichromenic acid (CBCA). GPS: geranyl

pyrophosphate synthase; IPP: isopentenyl diphosphate; OAC: olivetolic acid cyclase.

2.4 Food Composition of Hempseed

Hemp seed has been recognized and valued as food, due to its nutritional properties, for both

animals and humans for a long time (Callaway, 2004). It is a rich source of proteins and PUFA, and

comes with considerable amount of fiber. It is a complete nutritional source with all essential amino

and fatty acids, as well as exhibiting pharmacological activity (Leizer et al, 2000). The moisture

content of the seeds is about 7.6-8.0% (Da Porto et al, 2011), and 6.5% for others (Callawy, 2004);

as long as it is below 10% moisture content it prevents the seeds from sprouting (Deferne et al.,

1996). Hemp seed naturally contains appreciable amounts of constituents such as omega-3 fatty

acids, antioxidants and others that provide nutritional and therapeutic benefits making it considered

as functional food, without the need to add any additional biochemical (Carvalho, et al., 2006).

2.4.1 Fatty Acid Composition

Fat represents the most important component of hempseeds, particularly from an industrial

point of view. Indeed, since hempseeds are oilseeds, the oil is the main food product of industrial

interest that it is possible to obtain from them. For this reason, hempseed’s fat is commonly called

oil. Several studies (Vvapartis et al., 2015; Galasso et al., 2016; Lan et al., 2019) demonstrated that

the oil content of hempseeds belonging to di_erent cvs ranges from 25 to 35% of the whole seed.

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Galasso and colleagues (Galasso et al., 2019) and Irakly and co-workers (Irakli et al., 2019),

analysing the seeds belonging to di_erent industrial hemp cvs, highlighted that this variation is

mainly due to the genotype. In addition, Irakli and colleagues (Irakli et al., 2019) also observed that

even environmental conditions such as geography, climatic conditions, and local agronomic factors

have an e_ect on the total oil content, though slighter than genotype. This evidence is in accordance

with the findings of Kriese and colleagues (Kiese et al., 2004) and Mihoc and co-workers (Mihoc et

al., 2012). In this latter study, the authors, analyzing the oil content of five Romanian industrial

hemp cvs grown in two consecutive years in the same site, found that environmental conditions such

as high monthly average temperature of 25–26 _C and a low rainfall level (223 mm during the

flourishing of plants) resulted in an incomplete maturation of hempseeds and in a decrease in oil

content.

As regards the chemical composition of the hempseed’s fat, it is needed to consider that most

publications on this argument are referred to the oil extracted from hempseed through specific

industrial methods, rather than to the whole seed, because of the high industrial relevance attributable

to hempseed oil. However, in this review, we have been focused primarily on the whole hempseed,

so, except in the case of a lack of literature data about whole hempseed, we have considered only the

publications about this matrix, excluding those specifically concerning the oil. The literature data

about the FA composition of the fat component of whole hempseed are reported in Table

2.4.2 Hemp Seed Protein

The protein content of whole hempseed can vary from 20 to 25% according to variety and

environmental factors. This amount can further increase in some hempseed-processed products such

as dehulled seed and hempseed meal or cake (also called hempseed flour), that is, the remaining

fraction of hempseed obtained after expelling its oil fraction (Mattila et al., 2018; House et al.,

2010,Oseyko et al., 2019; Siano et al., 2018). Mattila and colleagues (Mattila et al., 2018)

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demonstrated that in hempseed the proteins are mostly located in the inner layer of the seed, in fact

they found only a low quantity of total proteins in the hull. Therefore, the increase in the protein

content of processed products can be explained as a consequence of protein concentration after

removing some component of the whole seed that totally or almost lacks in protein, such as the hull,

where most of the fibre is located and the removal of which leads to a 1.5 times increase in both

protein and oil amount.

More proteins are also present when both hull and oil (the major component of whole hempseed) are

removed. Indeed, their removal leads to obtaining the hempseed-processed product with the highest

protein (up to over 50%) and the lowest fat content (even less than 10%, based on the type of

extraction methods used) (House et al., 2010).

Research about the hempseed proteins originated from the early 20th century and highlighted that the

two main proteins of hempseed are the storage protein albumin, a globular protein, and edestin, a

legumin. This latter is the most abundant component, constituting about 82% of total hemp protein

content. It is a hexamer of about 300 KDa composed by six identical subunits, each of which is in

turn made up of an acidic (AS) and a basic (BS) subunit with molecular weights of about 33.0 and

20.0 KDa, respectively, that are linked by one disulphide bond (Tang et al., 2006; Wang et al.,

2008). In addition to edestin, the other components that have been found were albumin components

(13% of total protein) and_-conglycinin (a vicilin-like protein) (up to 5% of total protein), the

presence of which has been recently confirmed also by Pavlovic and co-workers (Palvlovic et al.,

2018) and in a recent genome-wide study (Ponzoni et al., 2018) in which two albumin and one

vicilin-like encoding genes in addition to three edestin isoforms where identify, each encoded by a

different gene, discovering for the amount of sulphurated amino acids that are particularly abundant

in the isoform-type 3. Nevertheless, in the report of Pavlovic and co-workers (Palvlovic et al., 2018),

the authors found that edestin amounted to about 65% of total hempseed proteins. This result is in

line with what was found from the HPLC and MS/MS analysis performed by Mamone and

12
colleagues (Mamone et al., 2019), but it is less than what was obtained in the previous studies by

Tang and colleagues (Tang et al., 2006) and by Wang and co-workers (Wang et al., 2008).

Moreover, these latter authors also found that the solubility of hempseed proteins at acidic Ph was

lower than that of soy proteins, and this may be due to the formation of covalent disulphide bonds

among individual molecules of edestin, resulting in insoluble protein aggregates. Another physio-

chemical property investigated was the thermal one. It was found that the denaturation temperature

of hempseed proteins was 920C. This evidence is in agreement with what was observed by Raikos

and colleagues (Raikos et al., 2015) about the effect of heat treatment (800C or over) on the structural

characteristics of hempseed proteins and consequently, on their digestibility.

Indeed, high temperatures can lead the proteins to unfold and to expose their hydrophobic groups,

favoring protein-protein interactions instead of the protein-water ones, and, hence, the formation of

insoluble protein aggregates to which digestive enzymes cannot access. In this context, Wang and

co-workers (Wang et al., 2008), investigating on the in vitro digestibility of hempseed protein

isolates, had previously shown that untreated hempseed proteins were much more digestible

compared to soy proteins. The high digestibility of hempseed proteins has been confirmed also by

Mamone and colleagues (Mamone et al., 2019), who observed that only a handful of peptides

survived to an in vitro digestion process. On the contrary, Lin and colleagues (Lin et al., 2020)

observed that heat pre-treatment on hempseed proteins at 100 0C for 15 or 30 min, improved their

digestibility. The authors explained this evidence as a consequence of the improvement in the

digestive enzymes’ bio-accessibility on target proteins through increased exposure of susceptible

peptide bonds after protein unfolding.

Regarding the nutritional value of hempseed proteins, it is important to consider that the nutritional

quality of a protein is defined by its amino acid composition and by its digestibility and

bioavailability. The protein’s amino acid composition along with the individual’s amino acid

requirement, are important to establish the amino acid score, which is the relative contribution that

13
the amino acids contained in the protein meet the individual’s amino acid requirement, whereas, the

protein digestibility is closely related to the bioavailability of its amino acids, since it measures the

degree to which the protein is digested and their components the amino acids are absorbed by the

gastrointestinal tract and, hence, introduced into the human body. Several authors investigated the

hempseed proteins’ amino acid composition (Russo et al., 2015).

2.5 Hempseed Anti-nutritional Compounds

The anti-nutritional factors are biological compounds present in human or animal foods that reduce

nutrients’ bioavailability or food intake, or the metabolism of which may lead to the release of toxic

products, thereby contributing to impaired gastrointestinal and metabolic performance. Generally,

compounds such as saponins, phytic acid, alkaloids, certain oligosaccharides, protease inhibitors,

cyanogenic glycosides, glucosinolates, and tannins are traditionally incorporated into this group.

Referring to hempseed, only a few reports investigated about the antinutritional compounds in this

seed, and the antinutritional factors that have been considered were phytic acid, trypsin inhibitors,

condensed tannins, cyanogenic glycosides, and saponins.

2.6 Other Anti-nutrients.

Antinutritional factors like condensed tannins, phytic acid and trypsin inhibitors are in low

concentrations in hemp seed (Aiello et al., 2016). These antinutritional components (such as

condensed tannins, trypsin inhibitors, phytic acid and saponins) present in hemp seed can reduce the

availability of proteins by precipitation or inhibiting digestive enzymes (Russo et al., 2013). These

components can also limit absorption of vitamins and minerals by chelation or complex formations

(Russo et al., 2013). Metabolism of certain substances can lead to release of toxic products (Russo et

al., 2013).

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2.6.1 Phytic acid. Phytic acid presence in hemp defatted flour ranged from 61.2 -74.1 g/Kg (6-7%)

(Russo et al., 2013).

1.6.2 Tannins. Tannins can be found in the plant kingdom, they are one of their chemical defenses

by facilitating protein aggregation (McGee, 2004). The astringency of the tannins interferes with

digestive enzymes and meant for self-preservation to deter planting-eating animals (McGee, 2004).

Condensed tannins presence in hemp defatted flour ranged from 1.36 – 2.14 g/Kg which is

considered non-toxic (Russo et al., 2013).

2.6.3 Cyanogenic glycoside. Cyanogenic glycoside presence in hemp defatted flour ranged from

0.09 –0.23 g/Kg (Russo et al., 2013).

2.6.4 Trypsin inhibitor activity. Trypsin inhibitor activity in hemp defatted flour ranged from 10.8-

28.4 unit/mg (Russo et al., 2013).

2.6.5 Saponins. Saponins are bitter defensive compounds that are soap-like emulsifying defenses

(McGee, 2004). Saponin presence in hemp defatted flour was around 69.0 mg/Kg (Russo et al.,

2013).

2.7 Micronutrients.

There are various micronutrient components present in hemp seed, both vitamins and minerals as

well as much more. Hemp seed oil has a rich array of minerals like phosphorous, magnesium,

calcium, sulfur and potassium, as well as modest quantities of zinc and iron (Deferne et al., 1996).

2.7.1 Phenols. Phenolics in the ten Canadian cultivars ranged from 1368-5160 mg/100g gallic acid

equivalent (GAE) (average of 2224 mg/100 GAE) (Vonapartis et al., 2015). Table 11 below provides

details of the ten Canadian cultivars regarding phenolics and tocopherol contents.

15
2.7.2 Carotene. Hemp seed is also a fair source of a precursor for Vitamin A, carotene (Deferne et

al., 1996). Hemp seed oil contains between 2-5.3 mg/100g oil of carotenoid content (Oomah et al.,

2002).

2.7.3 Chlorophyll. The chlorophyll within mature hempseeds is what gives hempseed oil its natural

dark green color, but it also hastens the auto-oxidation of oil when it is exposed to light (Callaway,

2004; Teh et al., 2013).

2.7.4 Cannabinoids. Cannabidiol (CBD) has been found in oil, but generally isn’t supposed to be

there and is considered a contaminant from poorly washed seeds that still have flower residue or

adherent resin (Leizer et al., 2000; Deferne et al., 1996). Regarding hemp seeds, THC and other

cannabinoids are at their highest concentration on the surface of the seed coat, and, generally,

administration via gastrointestinal system (i.e. food) has much less bioavailability of THC than with

smoking tending to have less as psychoactive effect (Petrovic et al., 2015). In the Croatian market,

out of the eleven different hemp seeds analyzed, the THC (the psychoactive compound) content

ranged from 3.04 to 69.50 mg/Kg, while CBD ranged from 4.18 to 243.68 mg/Kg, and CBN

(degradation product of THC) ranged from 1.85 to 8.44 mg/Kg (Petrovic et al., 2015). Gross

chemotype of hemp plants are determined by the ratio of specific cannabinoids (Leizer et al., 2000).

2.8 Brief Processing, Uses and Cultivation

2.8.1 Processing and Uses.

Hemp is a versatile crop that has uses for nearly every component of the plant. Figure 1 and Figure 2

below depicts the versatility and processes; Figure 1 is regarding the whole plant, while Figure 2 is

regarding the whole seed. The bast fiber, for example, is taken from the stalk and is used, even in

present day, for paper, durable fabrics and rope (Vonapartis, 2015; Callaway, 2004). Hemp fibers are

durable and used to make clothing, sails and paper, most famously, the first copies of the Bible and

16
the U.S. Constitution were written on hemp paper (Ranalli et al., 2004). Hemp seeds are used for

nutrition, and the oil can be extracted by various methods, some being cold-pressed, solvent or

supercritical CO2, among others. The oil has been used for ink for printers, soaps, detergents,

lubricants, and wood preservatives (Oomah et al., 2002; Russo et al., 2013), as well as emollient

body care products and fuel from lighting oil (Deferne et al., 1996; Herer, 2000).

Figure 3: Hemp seed processing and products (Crawford, 2012)

As Figure 2 depicts, the whole seed, after harvesting and cleaning, can be used for snacks, baking,

cooking, as Nut-butter, energy bars, nondairy milk and cheese as well as spreads and dips; typically

eaten raw to maintain nutritional value, as the PUFA oil doesn’t do well with high heat and the

unsaturated bonds in fatty acids deteriorate (House et al., 2010; Carvalho et al., 2006), but has also

be eaten cooked or roasted by various cultures worldwide throughout history (Callaway, 2004). The

whole seed can be hulled, i.e. the outer hard shell is removed; the hulls can be used as dietary fiber,

mulch and animal feed, and the hulled hemp seeds (also known as hemp hearts) can be used for

similarly as whole hemp seeds but they will be more susceptible to oxidation since the protective

outer hard shell has been removed, but without the dietary fiber that is in the hull so fats and proteins

17
within the hemp hearts are more easily digested (House et al., 2010). Both the whole hempseed and

the hulled hempseed can be used to make oil begetting an unrefined oil and defatted seed meal/meat

(even with the oil extracted from process, there is still beneficial residual oil in the meal): the

unrefined oil can be used for cooking, salad dressings and as a dietary supplementation; the defatted

seed meal can be used as supplementary protein powder, baking flour and animal feed (House et al.,

2010). The hemp seed oil can be refined and that oil can be used in cosmetic products, massage and

bath oils, in industry and as detergents and soaps (for instance Dr. Bronner’s soap products contain

hemp seed oil).

2.8.2 Cultivation

Hemp seed plants are ideally highly branched and can produce a high yield of seed, about half to a

full ton of seed per hectare; seed which has high amounts of good quality oil as well as protein

(Deferne et al., 1996). For producing seed, the males can be removed after pollination to leave more

space for the females and their seeds thrive (Deferne et al., 1996). Using a monoecious variety can

double the yield of seeds produced but the sexual type (hermaphrodite) can be afflicted with

inbreeding depression (Deferne et al., 1996). By “topping” (or “crowning”), which is cutting the

apical meristem of the plant, when they are 30-50 cm high can increase the number of flowers per

plant, thereby increasing the number of seeds made (Deferne et al., 1996). To increase seed yields,

the plants must be sown in lower densities as compared to growing hemp fiber, although if grown too

sparse, it leaves space for weeds to grow (Deferne et al., 1996).

Harvesting is difficult to find the optimal time to harvest, being that maturation of seeds do not

happen simultaneously; the moisture content of the seed crop increases, oil yield is lowered and taste

is modified with presence of unripe seeds (Deferne et al., 1996). Once harvested, the seeds go

through a drying process to decrease moisture content to limit sprouting during storage (Deferne et

al., 1996).

18
The varieties of hemp that were encountered in the present research: Finola is a variety of hemp that

has been accepted by Canada in 1998, and European Union in 2004 and is a main Finnish variety

that is well acclimated to northern climates for seed production; it produces more seed than any other

strains to date (Callaway, 2004). It is relatively short compared to other varieties, being only 1.5 m at

maturity, it is well suited for modern agricultural machines. It is frost tolerant to about -5 C at all

stages of life, and during the early maturation it is drought tolerant. It does not require herbicides or

pesticides (Callaway, 2004). Aiello et al., 2016 studied the French cultivar Futura. The ten Canadian

cultivars assessed were Alyssa, Anka, CanMa, CFX1, CFX2, CRS1, Dolores, Finola, Jutta and

Yvonne (Vonapartis et al., 2015).

Two groups were studied, the first were Italian dioecious varieties (Carmagnola, Carmagnola

selezionata, Fibranova) and the second were French monoecious varieties (Fedora 17, Felina 32, and

Ferimon) (Russo et al., 2013). Four hemp cultivars were analyzed in four different states which

were, whole hemp seed, de-hulled hemp seed, hemp seed meal, and hemp hulls (House et al., 2010).

The varieties were Finola, USO-14, USO-31 and Crag; the two USO’s are dual-purpose (fiber and

grain) crops that are early maturing with significant stalk yield and seed yield potentials of 400Kg

per acre (House et al., 2010). Crag and Finola are Finnish variety that have a shorter stature and are

very early maturing with high seed yield potential (House et al., 2010). Uniko-B is a unisex female

variety that can obtain high seed yields (Deferne et al., 1996). The variety of the seed used is one of

the factors that could lead to varying compositions found.

Fatty Acids An aggregate of 33 distinctive unsaturated fats, primarily unsaturated fats, have been

recognized in the oil of Cannabis seeds. Linoleic corrosive (53–60% of aggregate unsaturated fats),

linolenic corrosive (15–25%), and oleic corrosive (8.5–16%) are most basic. Other unsaturated fats

are γ-linolenic corrosive (1–4%), stearidonic corrosive (0.4– 2%), eicosanoic corrosive (<0.5%), cis-

vaccenic corrosive, and isolinolenic corrosive. The immersed unsaturated fats are palmitic corrosive

(6–9%), stearic corrosive (2–3.5%), arachidic corrosive (1–3%), behenic corrosive (<0.3%), myristic

19
corrosive, lignoceric corrosive, caproic corrosive, heptanoic corrosive, caprylic corrosive, pelargonic

corrosive, capric corrosive, lauric corrosive, margaric corrosive, and isoarachidic corrosive. The

unsaturated fat range of Cannabis seeds does not altogether shift in oil created from medication

(THC) or low-THC (hemp, fiber) sort Cannabis for the THC substance of Cannabis seeds and seed

oil

2.9 Implications on human health.

Used topically, hemp seed oil strongly absorbs UVC (100-290nm) and UVB (290-320), has

transmittance with UVB and UVA (320-400 nm), and has relatively high shielding power (SPF) and

protection factor (PFA) scores, therefore can be used topically as a UV protectant to protect our skin

and limit incidence of skin cancer (Oomah et al., 2002). Hemp seed oil soaps can be used for

cleanliness. Hemp seeds can be ingested in a variety of forms. Diet is essential; it has been

recognized for its vital role in controlling of noncommunicable chronic diseases (de Oliveira Barbosa

Rosa et al., 2015).

In an animal study, consumption of vegetable protein instead of animal protein has shown better

results in lowering cholesterol concentrations (de Oliveira Barbosa Rosa et al, 2015). Higher levels

of vegetable protein have been associated with greater satiety, greater reduction in weight, lowering

blood triglycerol levels, reduction in blood pressure, all with no negative consequences in bone

mineral density and renal function (de Oliveira Barbosa Rosa et al, 2015). Phytosterols which are

present solely in plants help against dyslipidemia by lowering absorption of cholesterol in the

intestine; they compete with cholesterol to be absorbed and the loser of the two (either cholesterol or

sterols (but with higher concentrations of sterols, cholesterols tend to lose)) are unabsorbed and lost

in stool (de Oliveira Barbosa Rosa et al, 2015).

The recommended daily amount of fat is 15% minimum for adult men and 20% minimum of child

bearing aged women and people with an underweight BMI, and a maximum of 30-35%; PUFA is 6

20
to 11% of total energy intake; omega 6 is 2.5-9% of total energy intake; omega 3 is 0.5-2% total

energy intake; the source stated no rationale for the n-6:n-3 ratio but to follow recommendations

which at the lows begets 5:1 ratio and at the highs 4.5:1 ratio (Food and Agriculture Organization,

2010).

From another source, the recommended daily amount of fat is 20-35% total energy intake for adults;

omega 6 is 5-10% total energy intake; omega 3 is 0.6-1.2% total energy intake; no mention of n-6: n-

3 ratio but going by lower and the higher recommendations we get the same ratio of 8.3:1 (Otten et

al., 2006). The recommendation for adults for carbohydrates is to consume 45-65% total energy

intake (Otten et al., 2006). The recommendation for adults for protein is to consume 10-35% total

energy intake (Otten et al., 2006). Table 13 shows these figures for adults as well as for children in

different age ranges. MUFA are fatty acids with only one double bond and some examples are: oleic

acid (18:1 n-9), palmitoleic acid (16:1 n-7) and elaidic acid (18:1 n-9). MUFA are generally found in

vegetable oils and have been found to positively modulate serum lipids, blood pressure and insulin

sensitivity in individuals demonstrating cardiovascular disease (CVD) and metabolic syndrome (de

Oliveira Barbosa Rosa et al., 2015). Intake of MUFA should be around 15-20% of total energy (de

Oliveira Barbosa Rosa et al, 2015).

Hemp seed oil has a high P/S ratio of 10.5 which has been shown to favorably reduce serum

cholesterol, production of arteriosclerotic plaque and a reduced risk for heart disease (Da Porto et al,

2011). Two essential PUFAs are LA and ALA, and they are considered essential fatty acids because

they cannot be produced endogenously (de Oliveira Barbosa Rosa et al, 2015). LA is a precursor to

arachidonic acid (20:4n-6), and ALA is a precursor to both eicosapentaenoic acid (EPA) (20:5n-3)

and docosahexaenoic acid (DHA) (22:6n-3) (de Oliveira Barbosa Rosa et al, 2015). Essential fatty

acids are not utilized by the body as energy sources, rather the serve the purpose of raw materials to

make the structure of the cell (i.e. phospholipid bilayer cell membrane) and as precursors for

21
biosynthesis for the regulatory biochemical in our bodies (Callaway et al., 1996). Omega-6/omega-3

ratio is thought to be ideal between 5:1 to 10:1 by some (de Oliveira Barbosa Rosa et al, 2015).

Earlier ratio considerations given by FAO and WHO in 1995 were in between 10:1-5:1 but more

recent evidence suggests that a more optimal range is 3:1-2:1, which is similar to the ratios of the

Mediterranean and Japanese diets which tend to have a lower prevalence of coronary heart disease

(Callaway, 2004; Teh et al., 2013).

22
 Chapter three

Chapter Three

3.1 SAMPLE COLLECTION AND PREPARATION

3.1.1 SAMPLE COLLECTION

Fresh samples of hemp seed were collected from national drug law enforcement agency Ado-Ekiti,

Ekiti State around January 2020

3.1 3.2

Fig 3.1 and 3.2- Dried hemp seeds

3.1.2 SAMPLE PROCESSING

The hemp seeds were carefully separated and sorted, air-dried for seven days. The dried seeds

were milled into fine powder, stored in sample bottles, and refrigerated pending chemical analysis.

3.2 PROXIMATE COMPOSITION

3.2.1 CRUDE FAT

A soxhlet flask (250ml) was dried in an oven at 100 oC transferred to the desicator to cool

with the laboratory temperature and weight of the flask was measured. 0.25g of the sample was

weighed in to the labeled thimble. 200ml of the petroleum ether was measured and then added to the

dry 250ml flask. The covered porous thimble with the sample was placed in the condenser of the

soxhlet arrangement that was assembled. The sample was extracted for 5hrs and the porous thimble

was removed with care and the petroleum ether in the top container (tube) and the extracting flask

were collected for the recycling or re-use through distillation. The extraction flask with the oil was

23
oven dried at 105oC for 1hr. the flask containing the dried oil was cooled in the desicator and the

weight of the cooled flask with the dried oil was measured. .

CALCULATIONS:

W0 = weight of the empty porous thimble

W1 = weight of the thimble + sample

W2 = weight of extraction flask

W3 = weight of the extraction flask + ether

Percentage of total crude fat = W3 – W2 X 100

W1 – W0

3.2.2 MOISTURE CONTENT

1.0g of the sample weighed into the silica dish. The silica dish and the content placed in the

oven at 105oC for 24hrs and were cooled in the desicator. The content with container was weighed

and later placed back into the oven for another 24hrs to ensure complete drying. The cooling process

in the desicator was repeated before taking the final weight.

3.2.3 CRUDE PROTEIN

The total Nitrogen was determined by the Kjeldahl procedure. 1.0g of the sample was

weighed into the conical flask and 20ml of the 1.25% of the H 2SO4 added and allowed to boil gently

for 30minutes and maintained the volume by gradually balancing the content with the pipette filler

filtered with the buckner funnel and rinsed well with hot de-ionized water. The materials were

scrapped back into the flask with spatula. 200 ml of the boiling 1.25% NaOH was added and allowed

to boil gently for 30minutes using cooling pipette filler to maintain a constant volume, it was filtered

through the buckner funnel. The residue was washed through with hot distilled water. It was rinsed

with 10.0% HCL and later twice with industrial methylated spirit. After proper rinsing, it was

allowed to dry in the oven. It was later ashed in the furnace at 500oC.

24
3.2.4 CRUDE FIBRE

Apparatus

Muffle Furnace, Conical Flask, Weighing Balance, Poplin Cloth, Oven, Heater, Buckner

Filtration system, Crucible and Spatula.

Procedure

2.0g of the pulverized sample was weighed into the 1L. Conical flask capacity 200ml of

1.25% sulphuric acid was added and allowed to boiled gently for 30minutes and maintained volume

by gradually balancing the contents with pipette filler. It was filtered through the Buckner funnel and

rinsed well with hot de-ionized water. The material was scrapped back into the flask with spatula.

200ml of boiling 1.25% of NaOH was added, allowed to boil gently for 30minutes using the pipette

filler to maintain the volume during reaction. It was later filtered through the Buckner funnel. The

residue was rinsed with 10% HCL, and then followed by acetone. The residue was then dried in oven

over night at 100oC. it was transferred into the desicator to cool before weighing. After ashing, it

was transferred into the desicator to cool and the weighed.

CALCULATION

%Crude Fibre = W1 – W2

W0

W0 = weight of the sample

W1= weight of the sample out of desicator after oven dried at 110oC

W2= weight of the sample after ashing in the furnace at 550oC

3.2.5 ASH CONTENT

1.0g of the pulverized sample was weighed an placed in the petri dish on the laboratory bench

before the analysis. The sample in the petri dish was emptied into the crucible. The crucible and the

25
sample was place into the furnace at 550oC for 4h. The sample in the crucible was then placed into

the desicator for cooling. It was repeated until the final weight was obtained.

3.2.6 CARBOHYDRATE

The carbohydrate analysis was got by getting the difference between the summation of values of

crude protein, crude fat, ash, crude fibre and moisture level of the sample.

Amino Acids

3.5.1 Extraction

Extraction and the instrumentation analyses were carried out as reported by following the

modified method of AOAC (2006) and Danka et al. (2012) in the “Simultaneous Identification and

Determination of Total Content of Amino Acid in Food Supplements-Tablets by Gas

Chromatography”.

3.5.2 Procedure

The dried and pulverized sample was made to be free of water by ensuring constant weight

for a period of time in the laboratory. The sample of 0.5g was weighed into 250ml conical flask

capacity. The sample was defatted by extracting the fat content of the sample with 30ml of petroleum

spirit three times with Soxhlet extractor that was equipped with [Link] sample was hydrolyzed

three times for complete hydrolysis to be achieved for the totality of amino acid recovery. The

pulverized and defatted sample was soaked with 30ml of 1M potassium hydroxide solution and was

incubated for 48 hours at 1100C in hermetically closed borosilicate glass container. After the alkaline

hydrolysis, the hydrolysate was neutralized to get pH in the range of 2.5-5.0. The solution was

purified by cation exchange solid-phase extraction. The amino acids in purified solution were

derivatised with ethylchloroformate by the established mechanism.

[Link] Derivatisation Mechanism

26
 The derivatisation of the extracted amino acids for the volatility sake in the gas chromatography

with ethylchloroformate is as described with the reaction below:

Figure

[Link] Mechanism of Amino Acids

The derivatising reagent was removed by scavenging with nitrogen gas for proper mop up of the

excess reagent. The derivatised amino acid that was free of derivatising reagent was made up to 1ml

in a vial for gas chromatography analysis.

[Link] GC Conditions for Amino Acids

GC: HP 6890 powered with HP ChemStation Rev. A 09.01 (1206) software

Injection temperature: Split injection

Split ratio: 20:1

Carrier gas: Hydrogen

Flow rate: 1.0ml/min

Inlet temperature: 2500C

Column type: EZ

Column dimensions: 10m x 0.2mm x 0.25µm

Oven programme: Initial@ 1100C

First: Ramp @ 270C/min for 4mins to 3200C

27
 Second: Constant for 5 mins at 3200C

Detector: PFPD

Detector temperature: 3200C

Hydrogen pressure: 20psi

Compressed air: 35psi

[Link] Determination of Amino Acids Quality Parameters

Predicted Protein Efficiency Ratio (P-PER): The predicted protein efficiency ratio (P-PER) was

calculated using the equation derived by Alsmeyeret al. (1974):

P-PER= -0.468+0.454 (Leu)- 0.105 (Try).

Estimation of Isoelectric Point (pI): The isoelectric point (pI) was calculated using the equation

form:

n
I Pm=∑ I Pi Xi❑
I=1

whereIPm is the isoelectric point of the mixture of amino acids, IPi is the isoelectric point of the ith

amino acid in the mixture and Xi is the mass or mole fraction of the i th amino acid in the mixture

(Finar, 1975; Olaofe and Akintayo, 2000).

Amino Acids Scores: The determination of amino acid scores was first based on whole egg hen’s

egg (Paul et al., 1976). In this method both essential and non essential amino acids were scored. The

amino acid score was also calculated using the following formular:

Amino Acids Score= (amount of amino acid per test protein (mg/g)) / (amount of amino acid
per protein in reference pattern (mg/g)) (FAO/WHO, 1973).

28
In the calculation based on amino acid scoring pattern, Met +Cys and Phe + Tyr were each taken as

a unit. Also, only essential amino acids determination was scored. Amino acid score was also

calculated based on the composition of amino acid obtained in the sample compared with the

suggested pattern of requirement for pre- school children (2-5). Here, Met+Cys and Phe+ Tyr were

each taken as a unit. Also, only essential amino acids including His were scored (FAO/WHO/UNU,

1985).

Essential Amino Acids and Biological Value: The essential amino acid index (EAAI) and

biological value (BV) were calculated using the methods and equation of Oser (1959).

Biological value = 1.09 (EAAI) - 11.73 (Oser, 1959).

Leucine/Isoleucine Ratio: Leucine/Isoleucine ratio, their differences and their percentage

differences were calculated. The various amino acid groups into class and their percentage values

were also calculated (Niemanet al., 1992).

Computation of Lys/Trp and Met/Trp: The ratios of Lys/Trp (L/T) and Met/Trp (M/T) were

computed.

The calculations of other determinations such as total amino acid (TAA), total essential

amino acid (TEAA), total acidic amino acid(TAAA), total basic amino acid (TBAA), total essential

aliphatic amino acid(TEAIAA), total aromatic amino acid (TArAA), total sulphur amino acid

(TSAA) and their percentages were calculated. Percentage of cystine in TSAA (%Cys in TSAA) and

Leu/Ile were also calculated.

3.6 Fatty Acids Methyl Ester Analysis

50mg of the extracted fat content of the sample was saponified (esterified) for five (5)

minutes at 950C with 3.4ml of 0.5M KOH in dry methanol. The mixture was neutralized by using

0.7M HCl. Three ml (3ml) of 14% boron triflouride in methanol was added. The mixture was heated

29
for 5 minutes at the temperature of 90 0C to achieve complete methylation process. The fatty Acid

Methyl Esters were thrice extracted from the mixture with redistilled n-hexane. The content was

concentrated to 1ml for gas chromatography analysis and 1µl was injected into the injection port of

GC.

3.6.1 GC Conditions for Fatty Acids Analysis

GD: HP 5890 Powered with HP ChemStation Rev. A 09.01 (1206)

Software
Injection temperature : Split injection
Split ratio: 20:1
Carried gas: Nitrogen
Inlet temperature: 2500C
Column type: HP 5
Column dimensions: 30m x 0.25mm x 0.25µm
Oven programme: Initial temperature @ 500C
First ramping @ 100C/min for 20min, maintained for 4min
Second ramping @ 150C/min for 4min, maintained for 5min
Detector: PFPD
Detector temperature: 3200C
Hydrogen pressure: 20psi
Compressed air: 30psi

3.7 Phospholipids Analysis

Modified method of Raheja et al. (1973) was employed in the analysis of the extracted oil

phospholipids content determination.

0.01g of extracted fat was added to the test tubes. To ensure complete dryness of the oil for

phospholipids analysis, the solvent was completely removed by passing stream of nitrogen gas on the

oil. 0.40ml of chloroform was added to the content of the tube and it was followed by the addition of

0.10ml chromo-genic solution. The content of the tube was heated at the temperature of 100 0C in a

water bath for about 1 minute 20 seconds. The content was allowed to cool to the laboratory

30
temperature and 5ml of hexane was added and the tube with its content shaken gently several times.

The solvent and the aqueous layers were allowed to be separated. The hexane layer recovered and

allowed to be concentrated to 1.0ml for gas chromatography analysis using pulse flame photometric

detector (Rahejaet al., 1973).

3.7.1 GC Conditions for Phospholipids Analysis

GD: HP 5890 Powered with HP ChemStation Rev. A 09.01 (1206)

Software
Injection temperature : Split injection
Split ratio: 20:1
Carried gas: Nitrogen
Inlet temperature: 2500C
Column type: HP 5
Column dimensions: 30m x 0.25mm x 0.25µm
Oven programme: Initial temperature @ 500C
First ramping @ 100C/min for 20min, maintained for 4min
Second ramping @ 150C/min for 4min, maintained for 5min
Detector: PFPD
Detector temperature: 3200C
Hydrogen pressure: 20psi
Compressed air: 30psi

31
 Chapter four

4.0 Result and discussion

4.1 LIPIDS ANALYSIS

NAME AMOUNT (MG/100G)

PHOSPHATIDYLETHANOLAMINE 36.55473
PHOSPHATIDYLCHOLINE 109.48657
PHOSPHATIDYLSERINE 2.77480
LYSOPHOSPHATIDYLLCHOLINE 27.20290
PHOSPHATIDYLLINSITOL 16.25332

Table 4.1 shows the level of various phospholipids in the samples. Phospholipids are not essential

nutrients, they are just another lipid acids. They can be broken down in the body and utilized for

energy. Phospholipids intervene in prostaglandin signal pathways as a raw material used by lipase

enzymes to produce the prostaglanding precursors. In plants, they serve as raw material to produce

jasmonic acid, a plant hormone similar in structure to prostaglandins that mediates defensive

response against pathogens. Phosphatidylcholine was the highest concentrated entity in the samples

having values of 109.49mg/100g. Phosphatidylethanolamine was the second largest concentrated

entity in the samples having values of 36.55mg/100g. Phosphatidylethanolamine has been shown to

be able to propagate infectious prions without the assistance of any protein ornucleic acid. This is a

unique character of phosphatidylethanolamine (Deleault et. al ., 2012). Phosphatidylethanolamine is

thought to play a role in blood clotting as it works with phosphatidylserine to increase the rate of

thrombin formation (Majumber et. al ., 2011).

32
4.2 STEROL ANALYSIS

NAME AMOUNT (MG/100G)

CHOLESTEROL 1.76098 X 10-6
CHOLESTANOL 6.84982 X 10-4
ERGOSTEROL 6.22140 X 10-6
CAMPESTEROL 106.07191
STIG-MASTEROL 24.21528
SAVANASTEROL 9.65588
SITOSTEROL 292.90989

The sterol levels are shown in table 4.2; the values of cholesterol in the three samples were

apparently higher than all other sterols identified in the samples with value 1.76098 X 10-6mg/100g.

Cholesterol is needed by the body in small amount as the body uses it to build cell membrane

structure; make hormones; produce vitamin D, produce bile acids which help the body to digest fat

and absorb important nutrients. Some seafood, including shrimp, lobster, and certain fish, contain

moderately high amount of cholesterol (60-100g/half cup serving). Products that contain milk (e.g.

cheese and ice cream) are moderately sources of cholesterol, whereas margarine does not contain

cholesterol.

Metabolic studies in human have indicated that a high cholesterol diet induce both increased LDL

synthesis and reduce receptor dependent fractional removal rate of LDL particles (Packard et. al.,

1983). Stigmasterol which can be used in the manufacture synthetic progesterone, a hormone that

plays an important physiological role in the regulatory and tissue rebuilding mechanism related to

estrogen has an extremely low concentration in the three samples.

4.3 FATTY ACID METHYL ESTHERS ANALYSIS (PS)

NAME AMOUNT (MG/100G)

Caproic acid C6:0 0.000000
Caprylic acid C8:0 0.000000
Capric acid C10:0 0.000000
Dodecanoic acid C12.0 0.269604
Myristic acid C14:0 0.521985
C14 : 1 (CIS-9) 0.013660
Palmitic acid C16:0 14.881540
C16 : 1 (CIS-9) 2.649521

33
Stearic acid C18:0 3.201341
C18 : 1 (TRANS-6) 0.038854
C18 : 1 (CIS-6) 7.579090
C18 : 1 (TRANS-9) 0.003516
C18 : 1 (CIS-9) 21.623612
C18 : 1 (TRANS-11) 0.000000
C18 : 2 (CIS-9,13) 43.490906
C18 : 2 (TRANS-9,12) 0.253909
Behenic acid C20:0 0.112522
C18 : 3 (CIS-6,9,12) 2.150774
C20 : 1 (CIS-11) 0.190666
C18 : 3 (CIS-9,12,15) 1.462272
C20 : 2 (CIS-11,14) 0.170841
Behenic acid C22:0 0.099672
C20 : 3 (CIS-8,11,14) 0.881541
C22 : 1 (CIS-13) 0.146912
C20 : (CIS-11,14,17) 0.066084
C20 : 4 (CIS-5,8,11,14) 0.000000
C22 : 2 (CIS-11,13) 0.069883
Lignoceric acid C24:0 0.012307
C20 : 5 (CIS-5,8,11,14,17) 0.052831
C24 : 1 (CIS-15) 0.012307
C22 : 6 (CIS-4,7,10,13,16,19) 0.043851

4.4 FATTY ACID METHYL ESTHERS ANALYSIS (GS)

NAME AMOUNT (MG/100G)

C6 : 0 0.000000
C8 : 0 0.000000
C10 : 0 0.000000
C12 : 0 0.547078
C14 : 0 0.365188
C14 : 1 (CIS-9) 0.009402
C16 : 0 15.009985
C16 : 1 (CIS-9) 2.075985
C18 : 0 2.607357
C18 : 1 (TRANS-6) 0.026739
C18 : 1 (CIS-6) 11.421962
C18 : 1 (TRANS-9) 0.002420
C18 : 1 (CIS-9) 20.853361
C18 : 1 (TRANS-11) 0.000000
C18 : 2 (CIS-9,13) 43.354974
C18 : 2 (TRANS-9,12) 0.274758
C20 : 0 0.080066
C18 : 3 (CIS-6,9,12) 1.338093
C20 : 1 (CIS-11) 0.136669
C18 : 3 (CIS-9,12,15) 1.330500
C20 : 2 (CIS-11,14) 0.129573
C22 : 0 0.068594

34
 C20 : 3 (CIS-8,11,14) 0.087642
C22 : 1 (CIS-13) 0.101878
C20 : (CIS-11,14,17) 0.045479
C20 : 4 (CIS-5,8,11,14) 0.000000
C22 : 2 (CIS-11,13) 0.048768
C24 : 0 0.008470
C20 : 5 (CIS-5,8,11,14,17) 0.036354
C24 : 1 (CIS-15) 0.008470
C22 : 6 (CIS-4,7,10,13,16,19) 0.030236

It is found that the ratio of essential fatty acids in hemp oil is close to perfect: Omega-6 and Omega-3

as 3.0:1 – 3.7:1, while in linseed oil is 1:3.6. Hemp oil also contains biologically valuable gamma-

linolenic acid, which is quite rare in vegetable raw materials. Nowadays, it is widely recognized the

exceptional importance of Omega-3 polyunsaturated fatty acids for maintaining physical and mental

health and preventing a number of diseases.

4.5 Discussion

A brief discussion regarding the processing done to hemp upon harvest showed some versatility of

the plant. The chunk of this paper was on the macro- and micro-nutrients found in hemp and its

various forms. There are many nutriments when it comes to hemp nutriture; from the plethora of

EFA present in an optimal ratio found in the oil, to the mostly complete (very few in the plant

kingdom are complete proteins), easily digestible protein, to the carbohydrates and fiber supporting

macronutrient consumption and digestion, to the vitamins, minerals and bioactive compounds. There

are many components in hemp seed that are beneficial to human health; the majority of the oil is

PUFA, with a good amount of omega-3 fatty acids (ALA and even the products that occur

endogenously via elongation and desaturation), the bioactive components like the tocopherols,

phenolic acids, chlorophyll, terpenes, Beta-sitosterol, methyl salicylate and even THC and CBD that

further contribute to health. Hemp compares favorably to other plants with regards to ingestion,

human health and effect on environment, and should be more prevalent in our food system.

Consumption of LA as compared to ALA within the past 100-150 years has increased exponentially

(Leizer, 2000), which has many unfavorable repercussions (Simopoulos, 2016), hemp seed, and
35
flaxseed, can help to increase our consumption of ALA to bring more balance of eicosanoid

production to reach homeostasis. There are some antinutritional components of hemp seed which

reduce absorption of nutrients, but more studies need to be done to test the extent of the disturbance

of absorption and analyze how much is absorbed; factoring in antinutritional components, how much

nutrients do we absorb. Other future research recommendations would be to study further the

composition of hemp seed on its own and in relation to others, study the digestibility, assimilation

and utilization of these components, as well as deciphering healthy doses.

4.6 Conclusions

On the basis of a systematic approach and analysis of chemical and technological lipidcontaining

systems, in particular on the oxidation and scorching of unsaturated and saturated acylglycerols in

oils, seeds, and products, the purpose and methodology of studies are considered, taking into account

the provisions of the systemic health concept. The content of fatty acids, phospholipids, vitamins A

and E, their degree of unsaturation the obtained pressed hemp oil has a high biological value. In

terms of tocopherols content, hemp pressed oil significantly outweighs sunflower, sesame and

amaranth oils. It is found that the ratio of essential fatty acids in hemp oil is close to perfect: Omega

6 and Omega-3 as 3.0:1 – 3.7:1, while in linseed oil – 1:3.6. Hemp oil also contains biologically

valuable gamma-linolenic acid. In chemical terms and less integral value, hemp oil is better stored at

8±2 °C without light access.

4.7 Recommendation

The results have shown that the samples have a good lipid profile. The high level of phospholipid

can reduce the cholesterol level of the blood. The good level phospholipids and sterols in the samples

make them beneficial for human health. It is therefore recommended for consumption.

4.8 REFERENCES
36
Aiello, G., Fasoli, E., Boschin, G., Lammi, C., Zanoni, C., Citterio, A., & Arnoldi, A. (2016).

Proteomic characterization of hempseed (Cannabis sativa L.). Journal of Proteomics, 147,

187-196.

Andre, C.M.; Hausman, J.-F.; Guerriero, G. Cannabis sativa: (2016). The Plant of the Thousand and

One Molecules. Frontier Plant Sciences., 7, 19.

Andre, C.M.; Larondelle, Y.; Evers, D. (2010). Dietary antioxidants and oxidative stress from a

human and plant perspective: A review. Current Nutritional of Food Sciences, 6, 2–12.

Angelova, V., Ivanova, R., Delibaltova, V., & Ivanov, K. (2004). Bio-accumulation and distribution

of heavy metals in fibre crops (flax, cotton and hemp). Industrial Crops and Products, 19,

197-205.

Bender, D. A. (2006). Bender's dictionary of nutrition and food science (8th ed.). Cambridge,

England: Woodhead Publishing Limited.

Bilotto, S.; Spagnuolo, C.; Russo, M.; Tedesco, I.; Laratta, B.; Russo, G. L. (2013). Dietary

phytochemicals in chemoprevention of cancer: an update. Immunology. Endocr. Metab.

Agents Med. Chem, 13, 2–24.

Calder, P. C., PhD. (2015). Functional roles of fatty acids and their effects on human health. Journal

of Parenteral and Enteral Nutrition, 39, 18S-32S.

Callaway, J. C. (2004). Hempseed as a nutritional resource: An overview. Euphytica, 140, 65-72.

Callaway, J. C., Tennila, T., & Pate, D. W. (1996). Occurrence of “omega-3” stearidonic acid (cis-

6,9,12,15-octadecatetraenoic acid) in hemp (Cannabis sativa L.) seed. Journal of Industrial

Hemp Association, 3 (2), 61-63. Retrieved November 14, 2016. I couldn’t find online, I got it

from Cal Poly SLO’s inter-loan library.

37
Callaway, J.C. Hempseed as a nutritional resource: (2004). An overview. Euphytica 140, 65–72.

Carvalho, I. S., Miranda, I., & Pereira, H. (2006). Evaluation of oil composition of some crops

suitable for human nutrition. Industrial Crops and Products, 24, 75-78.

Chaddha, A., & Eagle, K. A. (2015). Omega-3 fatty acids and heart health. American Heart

Association: Cardiology Patient Page.

Contento, I. & Koch, P. (2016). Contento, Powerpoints, Chapter 16. Lecture presented at Food

Science and Nutrition 415 Nutrition Education class in Building 10 Room 124, San Luis

Obispo, CA. Dr. Doris Derelian presented slides in class during Fall 2016 quarter.

Crawford, F., Deards, B., Moir, B., & Thompson, N. (2012). Human consumption of hemp seed:

Prospects for Australian production. Australian Bureau of Agriculture and Resource

Economics and Sciences, 1-18. Retrieved October 23, 2016,

Da Porto, C., Decorti, D., & Tubaro, F. (2011). Fatty acid composition and oxidation stability of

hemp (cannabis sativa L) seed oil extracted by supercritical carbon dioxide. Industrial Crops

and Products, 36, 401-404.

Da Porto, C., Decorti, D., & Tubaro, F. (2011). Fatty acid composition and oxidation stability of

hemp (cannabis sativa L) seed oil extracted by supercritical carbon dioxide. Industrial Crops

and Products, 36, 401-404. Eicosanoid. (n.d.). Retrieved December 26, 2016.

Encyclopaedia Britannica (Ed.). (1998). Iodine Value. Retrieved December 20, 2016.

Erasmus, U. (1986). Fats and Oils. Burnaby, BC: Alive Books. British Colombia, Canada.

Evans, R. (Director). (1942). Hemp for Victory (Video file). USA: United States Department of

[Link] November 10, 2016,

38
Fernandez, A. Z., Graces, M. F., Alvarado-Castillo, C. P., & Estrada, O. (2010). Eicosanoid

Biosynthesis Food and Agriculture Organization of the United Nations (FAO), 2010. Fats and

fatty acids in human nutrition: report of an expert consultation. Rome. FAO Food and

Nutrition paper 91. 10-14,

Galasso, I.; Russo, R.; Mapelli, S.; Ponzoni, E.; Brambilla, I.M.; Battelli, G.; Reggiani, R. Variability

in Seed Traits in a Collection of Cannabis sativa L. Genotypes. Front. Plant Sci. 2016,

Gibbson, G. H. (2013). What is Atherosclerosis? Retrieved November 10, 2016.

Hampson, A. J., Axelrod, J., & Grimaldi, M. (2013). Cannabinoids as antioxidants and

neuroprotectants. Retrieved October 13, 2016.

Hampson, A. J., Grimaldi, M., Lolic, M., Wink, D., Rosenthal, R., & Axelrod, J. (2000).

Neuroprotective antioxidants from marijuana (Abstract). Annals of the New York Acadamy

of Sciences. Hemp. (n.d.). Retrieved November 2, 2016

Herer. J. (2000). The Emperor Wears No Clothes (11th ed., 13th printing). Van Nuys, CA: AH HAn

Publishing. ISBN: 1-878125-02-8. Library of Congress No. 90-164252

Hillig, K. W. (2005). Genetic evidence for speciation in Cannabis (Cannabaceae). Genetic Resources

and Crop Evolution, 52, 161-180.

Hillig, K. W., & Mahlberg, P. G. (2004, June). A chemotaxonomic analysis of cannabinoid variation

in Cannabis (Cannabaceae). American Journal of Botany, 91, 966-975.

House, J. D., Neufeld, J., & Leson, G. (2010). Evaluating the quality of protein from hempseed

products through the use of digestibility-corrected amino acid score method. Journal of

Agricultural and Food Chemistry, 58.

39
House, J.D.; Neufeld, J.; Leson, G. (2010). Evaluating the Quality of Protein from Hemp Seed

(Cannabis sativa L.) Products Through the use of the Protein Digestibility-Corrected Amino

Acid Score Method. J. Agric. Food Chem., 58, 11801–11807.

Kaul, N., Kreml, R., Austria, A., Richard, M., Edel, A. L., Dibrov, E., Hirono, S., Zettler, M. E.,

Pierce, G. N. (2008). A comparison of fish oil, flaxseed oil and hempseed oil

supplementation on selected parameters of cardiovascular health in healthy volunteers.

Journal of the American College of Nutrition, 27(1), 51-58.

Irakli, M.; Tsaliki, E.; Kalivas, A.; Kleisiaris, F.; Sarrou, E.; Cook, C.M. E (2019). ect of Genotype

and Growing Year on the Nutritional, Phytochemical, and Antioxidant Properties of

Industrial Hemp (Cannabis sativa L.) Seeds. Antioxidants, 8, 491.

Kriese, U.; Schumann, E.; Weber, W.E.; Beyer, M.; Bruhl, L.; Matthaus, B. (2004, ). Oil content,

tocopherol composition and fatty acid patterns of the seeds of 51 Cannabis sativa L.

genotypes. Euphytica 137, 339–351.

Lan, Y.; Zha, F.; Peckrul, A.; Hanson, B.; Johnson, B.; Rao, J.; Chen, B. (2019). Genotype x

Environmental E_ects on Yielding Ability and Seed Chemical Composition of Industrial

Hemp (Cannabis sativa L.) Varieties Grown in North Dakota, USA. J. Am. Oil Chem. Soc.

96, 1417–1425.

Le Troedec, M., Rachini, A., Peyratout, C., Rossignol, S., Max, E., Kaftan, O., Fery, A., Smith, A.

(2011). Influence of chemical treatments on adhesion properties of hemp fibres (Abstract).

Journal of Colloid and Interface Science, 356(1), 303-310.

Lehne, R. A. (2010). Pharmacology for Nursing Care (7th ed.). St Louis, MO: Saunders Elsevier.

40
Leizer, C., Ribnicky, D., Poulev, A., Dushenkov, S., & Raskin, I. (2000). The composition of Hemp

seed oil and its potential as an important source of nutrition. Journal of Nutraceuticals,

Functional and Medical Foods, 2(4), 35-53.

Lin,Y.; Pangloli, P.; Meng, X.; Dia,V.P. E_ect of heating on the digestibility of isolated hempseed

(Cannabis sativa L.) protein and bioactivity of its pepsin-pancreatin digests. Food Chem.

2020, 314, 126198.

Malomo, S. A., He, R., & Aluko, R. E. (2014). Structural and Functional Properties of Hemp Seed

Protein Poducts. Journal of Food Science, 79(8).

Mattila, P.; Mäkinen, S.; Eurola, M.; Jalava, T.; Pihlava, J.-M.; Hellström, J.; Pihlanto, A. (2018).

Nutritional Value of Commercial Protein-Rich Plant Products. Plant Foods Hum. Nutr.73,

108–115.

Mihoc, M.; Pop, G.; Alexa, E.; Radulov, I. (2012) Nutritive quality of romanian hemp varieties

(Cannabis sativa L.) with special focus on oil and metal contents of seeds. Chem. Cent. J. 6,

122.

Molleken, H., & Theimer, R. R. (1997). Survey of minor fatty acids in Cannabis sativa L. fruits of

various origins. Journal of International Hemp Association, 4(1), 13-17. Retrieved December

15, 2016.

Murthy, K.N.C.; Kim, J.; Vikram, A.; Patil, B.S. (2012). Differential inhibition of human colon

cancer cells by structurally similar flavonoids of citrus. Food Chem. 132, 27–34.

Oomah, B. D., Busson, M., Godfrey, D. V., & Drover, J. C. (2002). Characteristics of hemp

(Cannabis sativa L.) seed oil. Food Chemistry, 76, 33-43. Retrieved November 13, 2016.

41
Oseyko, M.; Sova, N.; Lutsenko, M.; Kalyna, V. (2019). Chemical aspects of the composition of

industrial hemp seed products. Ukr. Food J. 8, 544–559.

Otten, J. J., Hellwig, J. P., & Meyers, L. D. (Eds.). (2006). Dietary Refernce Intakes: The essential

guide to nutrient requirements. Washington DC: National Academy of Science.

Palmer, B. (2011). High on Environmentalism: Can hemp clothing save the planet? Retrieved

November 3, 2016.

Pavlovic, R.; Panseri, S.; Giupponi, L.; Leoni, V.; Citti, C.; Cattaneo, C.; Cavaletto, M.; Giorgi, A

(2019). Phytochemical and Ecological Analysis of Two Varieties of Hemp (Cannabis sativa

L.) Grown in a Mountain Environment of Italian Alps. Front. Plant Sci. 10, 1265.

Petrovic, M., Debeljak, Z., Kezic, N., & Dzidara, P. (2015). Relationship between cannabinoids

content and composition of fatty acids in hempseed oils. Food Chemistry, 170, 218-225.

Ponzoni, E.; Brambilla, I.M.; Galasso, I. (2018). Genome-wide identification and organization of

seed storage protein genes of Cannabis sativa. Biol. Plant, 62, 693–702.

Prescha, A., Grajzer, M., Dedyk, M., & Grajeta, H. (2014). The antioxidant acitivty and oxidative

stability of cold-pressed oils. Journal of American Oil Chemists Society, 91, 1291-1301.

Raikos, V.; Duthie, G.; Ranawana, V. (2015). Denaturation and Oxidative Stability of Hemp Seed

(Cannabis sativa L.) Protein Isolate as A_ected by Heat Treatment. Plant Foods Hum. Nutr.

70, 304–309.

Ramadan, M. F., & Moersel, J. (2006). Screening of the antiradical action of vegetable oils. Journal

of Food Composition and Analysis, 19, 838-842.

Ranalli, P., & Venturi, G. (2004). Hemp as as raw material for industrial applications. Euphytica,

140, 1-6.

42
Ranalli, P.; Venturi, G. (2004). Hemp as a raw material for industrial applications. Euphytica 140, 1–

6.

Russo, R. (2013). Variability in Antinutritional Compounds in Hempseed Meal of Italian and French

Varieties. Plant 1, 25.

Russo, R., & Reggiani, R. (2013). Variability in antinutritional compounds in hempseed meal of

Italian and French varieties. Plant, 1(2), 25-29. Sawler, J., Stout, J. M., Gardner, K. M.,

Hudson, D., Vidmar, J., Butler, L., Page, J. E., Myles, S. (2015, August 26). The Genetic

Structure of Marijuana and Hemp (N. A. Tinker, Ed.). PLoS ONE.

Russo, R. and Reggiani, R. (2015). Evaluation of Protein Concentration, Amino Acid Profile and

Antinutritional Compounds in Hempseed Meal from Dioecious and Monoecious Varieties.

AJPS 06, 14–22.

Siano, F.; Moccia, S.; Picariello, G.; Russo, G.; Sorrentino, G.; Di Stasio, M.; La Cara, F.; Volpe, M.

(2018). Comparative Study of Chemical, Biochemical Characteristic and ATR-FTIR

Analysis of Seeds, Oil and Flour of the Edible Fedora Cultivar Hemp (Cannabis sativa L.).

Molecules, 24, 83.

Simopoulos, A. P. (2004). Omega-6/Omega-3 Essential Fatty Acid Ratio and Chronic Diseases.

Food Reviews International, 20(1), 77-90.

Simopoulos, A. P. (2016). An increase in omega-6/omega-3 fatty acid ratio increases the risk for

obesity. Nutrients, 8 (1) 28).

Smeriglio, A.; Galati, E.M.; Monforte, M.T.; Lanuzza, F.; D’Angelo, V.; Circosta, C. (2016).

Polyphenolic Compounds and Antioxidant Activity of Cold-Pressed Seed Oil from Finola

Cultivar of Cannabis sativa L. Phytother. Res. 30, 1298–1307.

43
Struik, P.C.; Amaducci, S.; Bullard, M.J.; Stutterheim, N.C.; Venturi, G.; Cromack, H.T.H. (2000).

Agronomy of fibre hemp (Cannabis sativa L.) in Europe. Ind. Crop Prod. 11, 107–118.

Tang, C.-H.; Ten, Z.; Wang, X.-S.; Yang, X.-Q. (2006). Physicochemical and Functional Properties

of Hemp (Cannabis sativa L.) Protein Isolate. J. Agric. Food Chem. 54, 8945–8950.

Teh, S., & Birch, J. (2013). Physicochemical and quality characteristics of cold-pressed hemp, flax

and canola seed oils. Journal of Food Composition and Analysis, 30, 26-31.

Utiger, R. D. (2009, December 21). Prostaglandin. Retrieved December 10, 2016.

Vonapartis, E., Aubin, M., Seguin, P., Mustafa, A. F., & Charron, J. (2015, May). Seed composition

of ten industrial hemp cultivars approved for production in Canada. Journal of Food

Composition and Analysis, 39, 8-12.

Vonapartis, E.; Aubin, M.-P.; Seguin, P.; Mustafa, A.F.; Charron, J.-B. (2015). Seed composition of

ten industrial hemp cultivars approved for production in Canada. J. Food Compos. Anal. 39,

8–12.

Wang, C.; and Kurzer, M.S. (1998). Effects of phytoestrogens on DNA synthesis in MCF-7 cells in

the presence of estradiol or growth factors. Nutr. Cancer. 31, 90–100.

Wang, X.-S.; Tang, C.-H.; Yang, X.-Q.; and Gao, W.-R. (2008). Characterization, amino acid

composition and in vitro digestibility of hemp (Cannabis sativa L.) proteins. Food Chem,

107, 11–18.

Wysoczanski, T., Sokola-Wysoczanska, E., Pekala, J., Lochynski, S., Czyz, K., Bodkowski, R.,

Herbinger, G., Patkowska-Sokola, B., and Librowski, T. (2016, March). Omega-3 fatty acids

and their role in central nervous system-a review (Abstract). Current Medicinal Chemistry,

23(8), 816-831.

44
Yan, X.; Tang, J.; dos Santos Passos, C.; Nurisso, A.; Simões-Pires, C.A.; Ji, M.; Lou, H.; Fan, P.

(2015). Characterization of lignanamides from hemp (Cannabis sativa L.) seed and their

antioxidant and acetylcholinesterase inhibitory activities. J. Agr. Food Chem., 63, 10611–

10619.

Yu, L. L., Zhou, K. K., & Parry, J. (2005). Antioxidant properties of cold-pressed black caraway,

carrot, cranberry and hemp seed oils. Food Chemistry, 91, 723-729.

Zhang, H.; and Tsao, R. (2016). Dietary polyphenols, oxidative stress and antioxidant and anti-

inflammatory effects. Curr. Opin. Food Sci, 8, 33–42.

Zock, P. L., Blom, W. A., Nettleton, J. A., and Hornstra, G. (2016, September 20). Progressing

insights into the role of dietary fats in the prevention of cardiovascular disease. Current

Cardiology Reports, 18(11) 43

45