Composizione chimica e proprietà nutraceutiche

dei semi di canapa: un alimento antico con valori funzionali attuali

Dipartimento di Scienze e TecnologieAmbientali Biologiche e Farmaceutiche

15 febbraio 2019 Prof.ssa Severina Pacificoseverina.pacifico@unicampania.it

Fiera Internazionale della Canapa, Canapa Mundi, Roma F C LUniCampania

FoodChemLab

Cu

rren

t p

op

ula

tio

n

health trends

nutrition ⇢improvement of overall well-being

Well-being coincides with the search for a benefit, for which food is a means not only

to pursue sensory and emotional pleasure, but also to preserve physical health

The interest in health promotion and safeguard has reached a greater degree of scientific and institutional attention

When this connotation has a very marked character, it can be claimed as functional food.

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FoodChemLab

functional foodsfoods or dietary components that may provide a health benefit beyond basic nutrition

foods that by virtue of physiologically active food components provide health benefits beyond basic nutrition

similar in appearance to a conventional food, consumed as part of the usual diet, with demonstrated physiological benefits, and/or to reduce the risk of chronic disease beyond basic nutritional functions

Food fortified with added or concentrated ingredients to functional levels, which improve health and performance

functional

FOOD HEALTH BENEFITS

reduced risk of disease

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FoodChemLab

“any substance that may considered a food or part of food and provides medical or health benefits, including the prevention and treatment of disease. Such products may range from isolated nutrients, dietary, supplements and diets to genetically engineered ‘designer’ foods, herbal products, and processed foods such as cereals, soups, and beverages”

Nutraceutical

Nutrition +

PharmaCeutical

defence strategyplant’s defence against external aggressions UV irradiation,

microbial attacks, insect feeding deterrent, etc

visual ecological markers in flowers and fruits e.g. attract pollinators and animals for seed dispersal

bioactives for humans

health effects

sensory properties

antioxidant

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FoodChemLab

dietary seeds represent one of the richest source of nutraceuticals of the modern diet

Salvia hispanica L.

chia seeds high content in essential fatty acidsappreciable amount of phenol antioxidantsalternative to improve consumer’s health

Linum usitatissimum L.

flaxseeds

sc= seedcoatem= embryo secoisolariciresinol

diglucoside

herbacetin in its diglucoside form

Avena sativa L.

oat seeds

avenanthramides (AVAs)antioxidant

anti-inflammatoryantiproliferative

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FoodChemLab

Class - AngiospermaeSubclass - DicotyledoneaeOrder - UrticalesFamily - CannabaceaeGenus - CannabisSpecies - Cannabis sativa L.

Humulus lupulusCannabis sativa

Fiber hemp plants

Oilseed plants

Intoxicant plants

Phytochem Rev (2008) 7:615-639

Cannabis sativa L. is one of the earliest cultivated plant species, with a long and troublesome history, which strictly intertwines that of humans

use denial

survival

multi-purpose crop

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FoodChemLab

Scientific name and primary traits

Genome group Origin and diffusion (→) Common uses

C. sativa ssp. sativa

raramente psicoattivaResina bassaCBD=THC (1:1)

NLHNarrow leaflet hemp

Europe→ New World

Fiberand seed

THC ≤ 0.3%

C. indica ssp. chinensis

mediamente psicoattivaResina moderataCBD=THC (1:1)

BLHBroad Leaflet hemp

East Asia→ Europe→ New World

Fiberand seed

THC ≤ 0.5%

C. indica ssp. indica

Molto psicoattivaResina moderataCBD<THC (1:20)

NLDNarrow leaflet drug

South Asia→ Middle East→ Africa→ Europe→ New World

DrugFiber and seed

THC ≤ 20%

C. indica ssp. afghanica

moderatamente psicoattivaResina altaCBD=THC (1:1)

BLDBroad Leaflet drug

Afghanistan→ Europe→ New World

DrugTHC ≤ 10%

Hemp cvsCBD>THC (3:1)

NLH × BLH Europe→ New World

Fiberand seed

THC < 0.5%

Sinsemilla cvsvariabilmente psicoattivaResina copiosa

NLD × BLD New World→ Europe→ Worldwide

DrugTHC < 0.1% to > 20%CBD < 0.1% to > 20%

Indica – erroneamente chiamata «sativa»

Afghan – erroneamente chiamata «indica»

THC cvs THC > 20%; CBD < 1.0%CBD cvs THC < 5.0%; CBD ≥ 4.0%

21 st century Cannabis taxonomy

hybrids genomes

geno

me lan

draces

C. in

dic

a s

ub

spec

ies

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FoodChemLab

seed

branch

leaf

root

flower

fruit

Cannabinoids content

high concentration

low concentration

null concentration

very low concentration

guilty of the Hemp Middle Age… F C LUniCampania

FoodChemLab

seed-membrane

fruit-membrane

shell

light-grey/green2.0-3.5 mm

True seed is enclosed within the pericarp, which forms

the protective hull or shell.

Most of the seed is filled by an embryo, principally the two cotiledons, which are rich in oils, proteins and carbohydrates.

A rudimentary nutritive tissue (endosperm, rich in aleuron bodies) is also present

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FoodChemLab

In Europe hemp seed was a famine foodfoxtail millet

broomcorn millet

rice

barley

until the 10th century

hempseed

Chinese tombs

employed

for at least 3000 years

on

e o

f th

e fi

ve g

rain

s o

f an

cien

t C

hin

a

food for both humans and livestock

British medieval hemp eeds

▪ The whole seed, including the hull, was eaten.

▪ Crushed peanut-butter type preparations have been produced from hempseed for centuries.

▪ The interest in producing commercial hulled hempseed for culinary purposes dates back only to about 1990.

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FoodChemLab

analgesic

hempseed pill

vs pulmonary diseases, anemia, gastric catarrh, scrofula, neurasthenia, asthenia, emaciation

in some areas of Southeast Asia

anci

ent

emp

loym

ent

as f

oo

dgrinding the seed in water

hull removal hempseed milk heating*

*until the proteins solidified into curds

butter type solid masspressing

uses for medicinal purposes

35.7% fructus cannabispurgative and hypotensive effect

for sores and skin diseases

coughs jaudicecolic

blood problems

for treating…

constipation F C LUniCampania

FoodChemLab

▪ 19th-20th centuries – in Russia the seed oil was employed for making soap, paints, varnishes

▪ Lighting oils (replaced by kerosene)

▪ 20th century usually employed as wild bird and poultry feed

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FoodChemLab

hemp seeds

cold pressing

oil seeds

hulling

hulled hemp

mixing

hemp butter

hemp cake

grinding

hemp meal

bake

hemp crispbread

Hempseed oil is commonly removed

mechanically by cold-pressing, which

provides hempseed cake as

by-product. This latter contains various

amounts of remaining fat. Oil

extraction could be achieved also using

heat or solvents. An expeller or a meal

with lower fat content is obtained.

hempseed cake

Hulled hemp seed, which is the whole seed with the crunchy outer shell removed, comes by quite a few names like hemp hearts, shelled hemp seed and hemp nut. Hulled seed was created essentially to make eating hemp seed more appealing and easier to eat.

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FoodChemLab

Whole seed Seed meal

Oil 35.5 11.1

Protein 24.8 33.5

Carbohydrates 27.6 42.6

Moisture 6.5 5.6

Ash 5.6 7.2

Energy (kJ/100g) 2200 1700

Total dietary fiber 27.6 42.6

Digestible fiber 5.4 16.4

Non-digestible fiber 22.2 26.2

Typical nutritional content (%) of hempseeda

acv Finola

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FoodChemLab

Callaway, J.C. Euphytica (2004) 140: 65.

Amino acid Wheat (14%) Soy bean (32%) Hemp seed (25%) Egg white (13%)

alanine 0.50 1.39 1.28 0.83

arginine 0.61 2.14 3.10 0.68

aspartic acid 0.69 3.62 2.78 1.23

cystine 0.28 0.54 0.41 0.29

glutamic acid 4.00 5.89 4.57 1.67

glycine 0.71 1.29 1.14 0.50

histidine* 0.27 0.76 0.71 0.28

isoleucine* 0.53 1.62 0.98 0.74

leucine* 0.90 2.58 1.72 1.08

lysine* 0.37 1.73 1.03 0.74

methionine* 0.22 0.53 0.58 0.47

phenylalanine* 0.63 1.78 1.17 0.76

proline 1.53 1.65 1.15 0.50

serine 0.70 1.54 1.27 0.92

threonine* 0.42 1.35 0.88 0.58

tryptophan* 0.51 0.41 0.20 0.20

tyrosine 0.40 1.14 0.86 0.46

valine 0.61 1.60 1.28 0.98* essential

Typical protein content (%) of each food is reported in brackets

Individual amino acid valuefor each food is given in g per 100 g

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FoodChemLab

albuminedestin

opened flexible structure closed non-flexible structure

Seven edestin forms have been identified from the seed of the most known Italian hemp variety Carmagnola. They belong to a multigene family with two distinct types of edestin.

aids in DNA repair at a cellular level.

high digestibility (88%–91%)

compatible with the human digestive system

closely resembles the globulin in blood plasma

Hemp seeds can contain up to 36% protein

hemp seed protein is free of the tryspin inhibitors which block protein absorption that is found in abundance in soy. Hemp protein is also free of oligosaccharides found in soy, which cause stomach upset and gas.

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FoodChemLab

Docimo et al., 2014. Plant Physiol Biochem. 2014;84:142-148.

Wabs (water absorption) decreased, observed as the level of hemp flour increased, could be attributed to the dilution of gluten–protein fractions of wheat flour, responsible for water binding during dough mixing at 30 °C.

Increasing hemp flour level, bread volume was also impaired in comparison with the control

Formulation with the increasing amount of hemp flour yielded bread crumb, characterized by decreasing cohesiveness and increasing chewiness.

Appearance of cross-section of bread a. Control breadb. Bread with 5% of hemp flourc. Bread with 10% of hemp flourd. Bread with 20% of hemp flour

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FoodChemLab

Pojić et al., Journal of Food Quality38(2015) 431–440

Typical fatty acid profiles (%) of hemp and other seed oils

5 8 7 6 5 4 10 15

2 3 1 3 11 < 1 4 0

9 11 11 15 22 60 23 76

56 55 48 15 63 23 55 8

22 21 13 61 < 1 13 8 < 1

4 1 17 0 0 0 0 0

2 < 1 3 0 0 0 0 0

2.5 2.7 4.1 0.2 > 100 1.8 6.9 > 100n6/n3 ratio

black currant linseed sunflower rapeseed soy olive

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FoodChemLab

Callaway, J.C. Euphytica (2004) 140: 65.

80-90 g/100 g total FA

Δ6-desaturase

EPA

DMGLA PG-1

52-62%

12-23%

processing storage age of the samples

variability of the fatty acid profile

LNA18:4ω3Stearidonic

acid

20:4ω3Eicosatetraenoic

acid

20:5ω3EPA

22:5ω3Clupanodonic acid

22:6ω3docosahecaenoic

acid (DHA)

LA18:3ω6γ-linolenic

acid

20:3ω6Dihomo-

γ-linolenic acid

20:4ω6arachidonic

acid (AA)

22:6ω6adrenic acid

22:5ω6docosapentaenoic

acid

PG1

PG2

PG3

promotes the AA retention within the cell membrane

out of the cell membrane

+ platelet aggregation+ inflammation

Proper ratios of fatty acids provide proper amounts of prostanoids and leukotrienes which have anti-thrombotic, anti-vasoconstrictive and anti-inflammatory properties

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blood cholesterol levels

blood pressure

insulin dependence in diabetics

tumor growth

cancer

fat metabolism

membrane fluidity

metabolic rate

anti-inflammation status

The benefits of ω-3 PUFA are not only

present when taken in large quantities but the

regular intake of recommended levels (2-2.5%

of caloric intake/day) can be sufficient to

provide many of its nutritional qualities.

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FoodChemLab

Extraction methods employed otherwise affect oil content and its recovery and quality

Cold-pressing CO2 Extraction Soxhlet Extraction

preserve antioxidant substances…

triacylglycerols (TAG)

Hemp seed oil

tocopherolspolyphenolsfree fatty acidspigments

dietary minerals

phytosterolsvitamins

Non-TAG or unsaponifiable fraction

β-sitosterol

campesterol

Δ5-avenasterol15.1

8% o

f H

SO u

n-s

apo

nif

iab

le m

atte

r (1

.84-

1.92

%)

cholesterol absorption via crystallization and co-precipitationPLASMA

Intestinal lumen cholesterol solubility by excluding it from micelles

cholesterol absorption

effective in cholesterol uptake inhibition

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FoodChemLab

Vit

amin

E v

itam

ers

chain-breaking antioxidant

HS vitamins and minerals mg/100 g

Vitamin E 90.0

Thiamine (B1) 0.4

Riboflavin (B2) 0.1

Phosphorous 1160

Potassium 859

Magnesium 483

Calcium 145

Iron 14

Sodium 12

Manganese 7

Zinc 7

Copper 2

The ratio between the tocopherols in hemp seed oil was found as 5:2:90:3 for α/β/γ/δ-tocopherol, respectively.

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FoodChemLab

Carotenoids and chlorophyllsThe intensive dark green color feature of hempseed oil is associated with the high content of total chlorophylls, which can have many adverse effects…

1. chlorophylls are susceptible to photo-oxidation under the exposure of light, which causes the color change of the oil from dark green to yellow.

2. the high content of chlorophyll could give rise to the instability of free fatty acids in the oil, accelerating the rancidity and result in a rancid aroma.

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FoodChemLab

JUSO-31

Carmaleonte*

Finola

Finola dehulled

UAM

n-hexane

ethanol

+

UAM

UAM = Ultrasound Assisted Maceration

Exploiting the nutraceutical value of hempseed oils…

*hemp variety for fiber production of monoecious type, derived from C. sativa cv. Carmono[Carmagnola x Ermes] x Chamaelon

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FoodChemLab

UV-Vis spectra in the wavelength range 250-750 nm of oil samples: (a) CsC; (b) CsJ; (c) CsF; (d) CsFd. In the grey box the UV-Vis spectrum of the reference cold-pressed commercial oil is reported. Carotenoid region is underlined in orange, whereas chlorophyll in green.

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FoodChemLab

A) 1H-NMR and B) 13C-NMR spectra in CDCl3 of hempseed oil

through UAM in n-hexane of commercial hemp seeds previously grounded. ALA = α-linolenic acid; LA = linoleic acid; OA = oleic acid.

TIC chromatogram recorded in negative ion mode of hempseed oil through UAM in n-hexane of commercial hemp seeds. ALA = α-linolenic acid; GA = gadoleic acid; LA = linoleic acid; OA = oleic acid; PA = palmitic acid; SA = stearic acid; SDA = stearidonic acid. Peaks not assigned are not fatty acid compounds.

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FoodChemLab

ω6/ω3 ratio of UAM oils and commercial cold-pressed oil

JUSO-31

Carmaleonte

Finola

Finola dehulled

Finola

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FoodChemLab

Peak n. Rt

(min)Tentative assignment

Formula

[M-H]- found

(m/z)

[M-H]- calc.

(m/z)

error

(ppm)

RDB

MS/MS fragment ions

(m/z)

1 0.304 Tetrahexoside C24

H42

O21

665.2148

701.1915

[M+Cl]-

665.2146

0.3 4 665.2185(100); 485.1509(8.8); 443.1393(5.7); 383.1194(53.0); 341.1070(10.9); 221.0669(8.6); 179.0569(24.7); 119.0358(5.7); 89.0228(7.7)

2 0.320 Trihexoside C18

H32

O16

503.1621

539.1389

[M+Cl]-

503.1618 0.7 3 503.1640(100); 341.1097(11.9); 323.0974(7.8); 281.0851(8.1); 251.0750 (2.0); 221.0666 (54.2); 179.0555(46.5); 161.0455(13.3); 131.0357

(1.4); 119.0347(9.8); 113.0245(14.8); 101.0238(10.9); 89.0244(29.3)

3 2.505 Rutin C27

H30

O16

609.1466 609.1461 0.8 13 609.1498(100); 301.0357(28.6); 300.0276(63.9); 271.0241(41.3); 255.0290(22.5); 243.0294(7.98); 227.0363(2.9); 178.9981(3.2); 151.0031(4)

4 2.783 Hesperidin C28

H34

O15

609.1823 609.1825 1.0 12 609.1858(27.9); 325.0718(6.8); 301.0714(100); 286.0486(11.6); 257.0817(6.2); 242.0568(7.9); 241.0488(4.8); 227.0328(2.1); 214.0638(4.9);

199.0385(5.1); 164.0115(6.7); 151.0028(1.9); 125.0245(2.4)

5 3.088 quercetin-3-O-(6”-O-acetyl)hexoside C23

H22

O13

505.0989 505.0988 0.3 13 505.1008(49.8); 301.0353(27.9); 300.0273(100); 271.0245(60.9); 255.0288 (25.6); 243.0297(17.8); 227.0339(7.9); 211.0380(3.7);

199.0378(5.9); 151.0027(7.8)

6 4.011 trihydroxyoctadecenoic acid C18

H34

O5

329.2330 329.2333 -0.1 2 329.2333(100); 229.1439; 211.1329; 171.1030; 139.1167; 127.1128

7 5.023 dihydroxyoctadecenoic acid C18

H32

O4

311.2227 311.2228 -0.3 3 311.2239(96); 293.2119(16.2); 275.2020(11.7); 235.1714(17.1); 223.1708(100); 87.0462(9.2)

8 5.171 6,7-epoxycannabidiolic acid C22

H30

O5

373.2018 373.2020 -0.7 8 373.2013(95.4); 329.2122(100); 311.2011(67.4); 271.1676(17.7); 259.1675(11.6); 205.1211(28.1); 173.0971(33.4)

9 5.344 dihydroxylinoleic acid C18

H34

O4

313.2383 313.2384 -2 2 313.2375(100); 295.2260(11.2); 277.2152(10.2); 183.1381(66.5); 129.0918(16.1); 99.0807(16.5)

10 5.945 6,7-epoxytetrahydrocannabinolic

acid

C22

H30

O5

373.2020 373.2020 -0.1 8 373.2030(100); 329.2127(46); 311.2022(31.2); 287.2007(5.5); 271.1711(17.5); 259.1701(6.4); 205.1231(23.5); 191.1078(15.8); 179.1076(8.5);

173.0969(28.8); 158.0728(5.6); 123.0815(2.2)

11 6.301 hydroxy linoleic acid C18

H32

O3

295.2279 295.2279 -0.6 3 295.2275(100); 277.2170(74.5); 195.1384(18.8); 171.1024(21.8)

12 6.683 cannabielsoic acid A C22

H30

O5

373.2020 373.2020 0.1 8 373.2040 (43.2); 355.1924(100); 337.1811(11.7); 329.2142(7.6); 311.2017(57.6); 309.1842(17.8); 293.1907(8.9); 291.1767(12.2)

287.1282(40.1); 173.0971(58.5)

13 6.880 6,7-epoxycannabichromenic acid

C22

H30

O5

373.2016 373.2020 -1.2 8 373.2027(100); 329.2116(21.3); 311.2019(41.4); 271.1700(17.3); 205.1235(23.7); 173.0971(21.5); 158.0745(7.1); 137.0956(5.9)

14 7.169 γ-linolenic acid C18

H30

O2

277.2169 277.2173 -1.5 4

15 7.471 cannabielsoic acid B C22

H30

O5

373.2020 373.2020 -0.1 8 373.2042(23.5); 355.1924(100); 329.2120(45.6); 311.2015(35.1); 287.1287(36.8); 271.1704(19.6); 259.1347(14.2); 243.1414(26.4);

205.1238(9.9); 191.1070(22.7); 173.0965(11.0)

16 7.486 cannabidiolic acid C22

H30

O4

357.2076 357.2071 1.3 8 357.2067(100); 339.1958(67.7); 313.2160(34.4); 311.2007(45.8); 295.2073(11); 289.1433(16.9); 271.1329(32.8); 245.1537(82.2);

227.1431(28.2); 203.1071(5.2); 179.1063(25.5); 170.0730(25.8); 137.0968(5); 107.0494(18.7)

17 7.718 stearidonic acid C18

H28

O2

275.2014 275.2017 -0.9 5 275.2013(100); 231.2114(40.3); 203.1772(1.3)

18 8.448 α-linolenic acid C18

H30

O2

277.2176 277.2173 1.4 4 277.2177(100); 259.2072(3.1); 233.2275(2.3); 205.1952(1.3); 127.0758(1.2)

19 9.388 linoleic acid C18

H32

O2

279.2331 279.2330 0.5 3 279.2330(100); 261.2225(4.3)

20 9.68 tetrahydrocannabinolic acid C22

H30

O4

357.2070 357.2071 -0.4 8 357.2082(100); 339.1972(1.8); 313.2186(68.2); 311.2019(11.0); 245.1547(27.1); 229.1243(6.5); 203.1090(11.3); 191.1069(19.1);

179.1070(7.1)

21 10.030 cannabichromenic acid C22

H30

O4

357.2070 357.2071 -0.4 8 357.2082(100); 339.1981(34.5); 313.2164(37.3); 203.1065(17.6); 191.1068(33.8)

22 10.176 oxohexadecanoic acid

(oxopalmitoleic acid)

C16

H30

O3

269.2117 269.2122 -1.9 2 269.2120(100); 225.2235(15)

23 10.206 palmitoleic acid C16

H32

O2

255.2332 255.2330 1.0 1 255.2335(100); 219.8542(7.1); 201.8346(12); 166.8656(13.9)

24 10.520 oleic acid C18

H34

O2

281.2491 281.2486 1.8 2 281.2490(100)

25 11.411 stearic acid C18

H36

O2

283.2643 283.2643 0.2 1 283.2646(100)

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FoodChemLab

ω-6:ω-3 =2.23

α-linoleic acid

linolenic acid

4.152 mg eq CBD per g oil 11.53x

IA = 2.55 ± 0.23

Cannabidiolic Acid as a Selective Cyclooxygenase-2 Inhibitory Component in Cannabis

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XIC m/z 373.2

TIC

XIC m/z 357.2

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CBD-A

CBG-A

…altro metodo estrattivo…altra cv

ALALA

9831,13

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UAM

n-hexane

defatted hempseed

hempseed oil +

EtOH extract

UAM

ethanol

SiO2

CClignanamide fraction

UAM = Ultrasound assisted

maceration CC = Column

Chromatography

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FoodChemLab

Trolox®

Trolox®

Cs-EtOAcL/L Cs-EtOAc

L/L

RSC = Radical Scavenging Capacity

DPPH and ABTS methods data…

ID50

= 10.27 μg/mL

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FoodChemLab

a

a

ccb

dd

e2D NMR hemp seed ethanolic extract

eb

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FoodChemLab

fen

ola

mm

idi

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FoodChemLab

lignanammidi1% dei metaboliti del seme

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lignanamide fraction

LC HRMS data of compounds in lignanamide fraction (LF)

UPLC-QTOF- MS total ion chromatogram (TIC) acquired in negative mode

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SH-SY5Y ctrl SH-SY5Y Cs-EtOAcL/L

-treated SH-SY5Y vinblastine-treated

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…clear morphological changes were observed

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Experimental factors tested: i) pre-seeding soil fertilization: Fertilized (F) vs non-fertilized control (NoF); ii) foliar fertilization: foliar fertilized (Fol) vs non-fertilized control (NoFol); iii) crop density: 60 plant m2 (D1) vs 30 plants m2 (D2). The abovementioned factors were fully combined obtaining 8

treatments (F-Fol-D1, F-Fol-D2, F-NoFol-D1, F-NoFol-D2, NoF-Fol-D1, NoF-Fol-D2, NoF-NoFol-D1, NoF-NoFol-D2), each one arranged in a 1200 m2 plot.

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FoodChemLab

Peak

n.

Rt

(min)

Tentative assignment Formula [M-H]- found (m/z) [M-H]-

calc.(m/z)

error

(ppm)

RDB MS/MS fragment ions (m/z)

1 0.305 Trihexoside C18

H32

O16

503.1629 503.1631 2.3 3 221.0660; 179.0557; 161.0434; 119.0337; 113.0229

2 0.313 Dihexoside C12

H22

O11

341.1092

377.0861 [M+Cl]-

387.1115 [M+HCOO]-

341.1089 0.8 2 179.0566; 161.0466; 149.0446; 143.0321; 131.0386; 119.0345; 113.0244;

101.0345; 89.0244

3 0.327 dihexosyl propanetriol C15

H28

O14

431.1417 431.1406 2.5 2 341.1085; 179.0547; 161.0467; 131.00339; 119.0356

4 0.348 citric acid C6H

8O

7191.0196 191.0197 -0.7 3 111.0093; 87.0082

5 0.331 malic acid C4H

6O

5133.0145 133.0142 1.9 2 115.0039

6 0.460 isocitric acid C6H

8O

7191.0203 191.0197 3.0 3 111.0089

7 1.347 dihydroxybenzoic acid

hexoside

C13

H16

O9

315.0720 315.0722 -0.5 6 153.0202; 152.0128; 109.0287; 108.0228

8 1.456 hydroxytyrosol hexoside C14

H20

O8

315.1079

351.0851[M+Cl]-

315.1085 -2.0 5 153.0562; 123.0451

9 1.874 caffeoyl-tartaric acid

(isomer 1)

C13

H12

O9

311.0409 311.0403 0.1 8 179.0347; 149.0092; 135.0457

10 2.088 caffeoyl-tartaric acid

(isomer 2)

C13

H12

O9

311.0406

623.0896 [2M-H]-

311.0403 -0.8 8 179.0348; 149.0093; 135.0453; 134.0375

11 2.449 hydroxybenzoic acid

hexoside

C13

H16

O8

299.0072 299.0072 -0.1 6 137.0239

12 2.524 caffeic acid hexoside C15

H18

O9

341.0874 341.0878 -1.2 7 179.0347; 135.0453

13 2.566 p-coumaroyl-tartaric

acid (isomer 1)

C13

H12

O8

295.0458 295.0459 -0.5 8 163.0407; 149.0097; 119.0507

14 2.807 dihydroxycoumarin

hexoside

C15

H16

O9

339.0716 339.0722 -1.6 8 177.0189; 133.0291; 105.0337

15 2.984 p-coumaroyl-tartaric

acid (isomer 2)

C13

H12

O8

295.0462 295.0459 2.9 8 163.0404; 119.0506

16 3.102 p-coumaric acid

hexoside

C15

H18

O8

325.0928 325.0929 -0.3 7 163.0393; 119.0499

LC-MS/MS data of polyphenolic and not polyphenolic constituents from hemp oil hydroalcoholic extract (A)

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Peak

n.

Rt

(min)

Tentative assignment Formula [M-H]- found

(m/z)

[M-H]-

calc.(m/z)

error

(ppm)

RDB MS/MS fragment ions (m/z)

17 4.207 Dihydrokaempferol hexoside C21

H22

O11

449.1093 449.1089 0.8 11 287.0556; 269.0447; 259.0612; 243.0641; 125.0246

18 6.237 Rutin C27

H30

O16

609.1477 609.1461 2.6 13 609.1501; 301.0356; 300.0278; 271.0252; 255.0307

19 6.264 quercetin-O-hexoside 1 C21

H20

O12

463.0891 463.0882 1.9 12 463.0909; 301.0365; 300.0288; 271.0257; 255.0308; 243.0310

20 6.342 quercetin-O-hexuronide C21

H18

O13

477.0686 477.0675 2.4 13 477.0718; 301.0370; 300.0295; 283.0266; 255.0308; 211.0408; 178.9996; 151.0048;

121.0302

21 6.397 quercetin-O-hexoside 2 C21

H20

O12

463.0895 463.0882 2.8 12 463.0942; 301.0382; 300.0304; 271.0273; 255.0323; 243.0331; 151.0022

22 6.521 kaempferol-O-hexoside1 C21

H20

O11

447.0937 447.0933 1.2 12 285.0406; 284.0340; 257.0474; 255.0307; 229.0508; 227.0357

23 6.918 kaempferol-O-rutinoside C27

H30

O15

593.1517 593.1512 0.9 13 285.0410; 284.0331; 257.0464; 255.0310; 229.0504; 227.0350

24 7.104 N-caffeoyltyramine C17

H17

NO4

298.1091 298.1085 2.0 10 178.0514; 135.0455

25 7.108 isorhamnetin-O-rutinoside C28

H32

O16

623.1839 623.1618 3.4 13 315.0571; 314.0489; 299.0266; 271.0272; 243.0317

26 7.111 kaempferol-O-hexuronide C21

H18

O12

461.0742 461.0725 3.6 13 285.0409; 257.0455; 229.0498

27 7.125 kaempferol-O-hexoside 2 C21

H20

O11

447.0945 447.0933 2.7 12 285.0411; 284.0331; 255.0306; 227.0350

28 7.217 hesperidin/neohesperidin C28

H34

O15

609.1810 609.1825 -2.5 12 301.0718; 286.0486

29 7.389 isorhamnetin-O-hexuronide C22

H20

O13

491.0832 491.0831 0.2 13 315.0524; 301.0370; 300.0289; 271.0261; 255.0310; 243.0307

30 7.684 N-feruloyl tyramine1 C18

H19

NO4

312.1240 312.1241 -0.4 10 190.0506; 178.0506; 148.0530; 147.0450; 135.0448; 134.0358

31 8.039 quercetin 3-O-(acetyl-hexoside) C23

H22

O13

505.0999 505.0988 2.2 13 301.0357; 300.0273; 271.0232; 255.0297

32 8.53 N-feruloyl tyramine 2 C18

H19

NO4

312.1242 312.1241 0.3 10 190.0508; 178.0508; 147.0452; 135.0448

33 8.881 kaempferol hexuronyl methyl

ester

C22

H20

O12

475.0890 475.0882 1.7 13 285.0413; 284.0332; 255.0303; 229.0520; 227.0355

LC-MS/MS data of polyphenolic and not polyphenolic constituents from hemp oil hydroalcoholic extract (B)

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Extracted ion chromatogram (XIC) of the ions related to main quercetin glycosides. TOF MS/MS of [M-H]- ions related to compounds 18 (B), 19 (C), 20 (D), 21 (E), and 31 (F).

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PCA Biplot representing the relationship between investigated hempseed oils (variables) and their percentage content (observations) in non-phenolic constituents (no-Ph), simple phenols derivatives (Ph) and flavonoids (Fla). Observations are also plotted as histograms. A) Data acquired for D1 hempseed oils

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PCA Biplot representing the relationship between investigated hempseed oils (variables) and their percentage content (observations) in non-phenolic constituents (no-Ph), simple phenols derivatives (Ph) and flavonoids (Fla). Observations are also plotted as histograms. B) data acquired for D2 hempseed oils.

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PCA Correlation circle and Observations map from data acquired for D1 hemp oils

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PCA Correlation circle and Observations map from data acquired for D2 hemp oils

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