2020 SCIENCELINE

O nline J ournal of A nimal and F eed R esearch

Volume 10, Issue 4: 167-171; July 27, 2020

STUDY OF THE FLAVONOIDS AND SECONDARY METABOLITES

OF THE ARGAN TREE (Argania spinosa L.)

Miloudi HILALI^, Hanae El MONFALOUTI and Badr Eddine KARTAH

Laboratory of Plant Chemistry and Organic and Bioorganic Synthesis, Faculty of Science, University Mohamed-V, Av. Ibn Battouta, BP 1014 Agdal-

Rabat, Morocco



Email: hilali400@yahoo.com ;

: 0000-0003-2062-2969

^Supporting Information

ABSTRACT: The separation and identification of the main phenolic compounds present in the co-product of the

Argan tree (Argania spinosa) were carried out using high performance liquid chromatography techniques

coupled with mass spectrometry (LC-ESI-MS/MS).The study was based on the retention times of the peaks of the

phenolic compounds in samples and was compared to those of the controls (reference compounds) and

supplemented by an analysis of the fragmentations of the molecules by mass spectrometry. Phenolic

compounds in the pulp of the Argan namely such as catechin (2.8%), epicatechin (14.7%), procyanidin (2.7%),

quercetin (1.6%), luteolin (0.2%) and naringenin (0.07%) were found. Phenolic acid is consisted of gallic acid

(5%) and protocatechuic acid (21.1%). These compounds are more dominant than flavonoids. The flavonoids-O-

rhamnoglucosides the most dominant compounds is isorhoifoline (7.2%) and hesperidin (4.5%) against rutin

(0.1%) and rhamnetin-O-rutinoside (0.5%) are less dominant. The main compounds are the hyperoside (13.4%)

and isoquercetin (10%). On the other hand, naringenin-7-O-glucoside constituted the most minority compound of

this type of flavonoid in the pulp of the fruit of the Argan tree (the percentage of naringenin-7-O-glucoside and

quercetin-3-O-arabinose is 15.3%). There are other phenolic compounds in the pulp of the Argan namely such as

catechin (2.8%), epicatechin (14.7%), procyanidin (2.7%), quercetin (1.6%), luteolin (0.2%) and naringenin

(0.07%). The main flavonoids found in the leaves of the Argan tree are Quercetin (21.73%), Myricetin (54.34%),

Hyperoside (8.69%), and also Myricetin-3-Ogalactoside (9.78%). Argan cake is rich in flavonoids. Among these,

epicatechin (110 mg/kg), catechin (11 mg/kg), protocatechic acid (15.2 mg/kg), vanillic acid (16.3 mg/kg) and

4-hydroxybenzyl alcohol (8.6 mg/kg) are higher in Argan cake. Argan oil is richer in tocopherol (597 to 775

mg/kg), Argan oil is rich in gamma tocopherol (631 mg/kg), and that make Argan valuable nutraceutical. The

study of the secondary metabolites and especially the flavonoids of the Argan tree was undertaken with the aim

of identifying new metabolites making it possible to increase the industrial than commercial value of the Argan

tree.

Keywords: Argan tree, Biological activity, Flavonoids, Metabolism, Phenolic acids.

INTRODUCTION

The Argan tree (Argania spinose L. Skeels) is used by the local populations : the wood and the woody shell of the fruit for

heating. The almond of the fruit is used in the production of Argan oil. The foliage and the pulp of the fruit and also the

oil cake (residue from the production of Argan oil) are designed for animal nutrition (Pumareda et al., 2006 ). The study of

the chemical composition of Argan derivatives was undertaken with the aim of identifying new metabolites allowing

increasing the industrial than commercial value of the Argan tree. Secondary metabolites are compounds naturally

biosynthesized by plants but which do not directly participate in plant metabolism. Many secondary metabolites have

therapeutic properties and are (or have been) used in human medicine (Gitton-Ripoll, 2009; Khallouki et al., 2017 ). A

systematic study of the secondary metabolites of the Argan tree has been developed since the 1990s in order to see to

what extent it is possible to increase the economic value of the Argan grove and hence promote its extension in the long

term. The results of this study have revealed a wide variety of secondary metabolites within the different parts of the

Argan tree. Besides molecules frequently encountered in higher plants (triterpenes, sterols, flavonoids, etc.), molecules of

original structure and belonging to the group of flavonoids have been isolated. Multiple flavonoids, also extracted from

plant species other than Argan (El Kabouss et al., 2001; Zhar et al., 2016; Falode et al., 2019 ), have therapeutic

properties which are sufficiently encouraging to warrant further investigation (Guillaume et al., 2005). Some other

flavonoids seem to be involved in phytoprotective phenomena (Jiang et al., 2020 ). Applications in the food or cosmetology

fields are also being studied. All this clearly indicates that this chemical family of secondary metabolites has interesting

potential in many fields. As a result, the analysis of several parts of the Argan tree (wood, oil cake, shell and fruit pulp)

was carried out and many flavonoids of different and often original structures could be isolated and then identified, and

the properties of certain d 'have been evaluated (Safer, 2018; Hilali et al., 2020 ). This study briefly describes the

flavonoids of the Argan tree.

167

Citation: Hilali M, El MonfaloutiH and Kartah BE (2020). Study of the flavonoids and secondary metabolites of the Argan tree (Argania spinose L.). Online J. Anim. Feed

Res., 10(4): 167-171.

MATERIALS AND METHODS

Plant material

The fruits of the Argan tree and their leaves are harvested in the village of Tidzi province of Essaouira, The collected

samples are dried in the open air then pulped, crushed and finally reduced, separately, in fine powder (Bellefontaine et al.,

2011).

Figure 1 - Different parts of the Argan fruit

Identification of phenolic compounds

The separation and characterization of the main phenolic compounds present in the Argan fruit pulp were carried

out using high performance liquid chromatography techniques coupled with mass spectrometry (LC-ESI-MS/MS). This

method already applied on other plants (cocoa, lepechinia graveolens) (Sánchez ‐Rabaneda et al., 2003 ) is important for

the study of polyphenols. It makes it possible to determine the molecular weight and to give certain structural information

of the molecules (Adlouni, 2010 ). In our study, we based ourselves on the retention time of the peaks of the phenolic

compounds in our sample and compared to those of the control peaks of the reference compounds.

RESULTS AND DISCUSSION

Phenolic compounds of the pulp of the Argan fruit

The study of the phenolic composition of the pulp of Argan fruit has identified 16 phenolic compounds (Table 1). This

study was approached as a biochemical approach to establish a polyphenolic identity card and allows highlighting a fairly

significant structural diversity encompassing four main groups of phenolic compounds (El Kabouss et al., 2001; Charrouf

et al., 2007 ). Phenolic acids consisting of gallic acid (5%) and protocatechuic acid (21.1%). These compounds are more

dominant than flavonoids. In our study we did not find p-hydroxybenzoic acid among the phenolic acids in Argan pulp. The

flavonoids-O-rhamnoglucosides the most dominant compounds is isorhoifoline (7.2%) and hesperidin (4.5%) against rutin

(0.1%) and rhamnetin-O-rutinoside (0.5%) are less dominant. Flavonoids-O-glycosides: The main compounds are the

hyperoside (13.4%) and isoquercetin (10%). On the other hand, naringenin-7-O-glucoside constituted the most minority

compound of this type of flavonoid in the pulp of the fruit of the Argan tree (the percentage of naringenin-7-O-glucoside

and quercetin-3-O-arabinose is 15.3%), (compounds 11 and 12). There are other phenolic compounds in the pulp of the

Argan namely such as catechin (2.8%), epicatechin (14.7%), procyanidin (2.7%), quercetin (1.6%), luteolin (0.2%) and

naringenin (0.07%). It is noted that epicatechin was the most affordable compound in the pulp of the Argan fruit after

Protocatechuic acid (21.1%). The pulp of Argan fruit has been found to be rich in epicatechin and other catechic

derivatives whose natural antioxidant power is important according to numerous studies (Ba et al., 2010 ). However, such

combinations of natural phenolic compounds could be used for better preservation of Argan oil.

Phenolic compounds of Argan leaves

The study of the extracts of the leaves of the Argan tree by chromatography shows a higher content of flavonoids in

the leaves of the Argan tree (Table 2) which are flavonic molecules including (Myricetin 3-O-galactoside, the hyperoside,

quercitrine and myricitrine). The main flavonoids found in the leaves of the Argan are Quercetin, Myricetin and their

glycosides (Quercetin, Myricetrine, Hyperoside, and Myricetin 3-O-galactoside) (Tahrouch et al., 1998 ). The derivatives of

these aglycones represent 16.5% of the total flavonoids. The flavonoid extract of the leaves has a very interesting anti-

radical and antioxidant activity. It is currently marketed as a protective cosmetic active ingredient for extracellular

macromolecules in the skin such as collagen, glycoproteins, etc. (Charrouf et al., 2009 ).

Phenolic compounds in Argan oil

The proportion of phenols in Argan oil is low but their impact on its biological properties is very important. These are

caffeic acids, 4-hydroxybenzoic, vanillic, syringic, ferrulic 4-o-glycosylated, oleuropein, 3-hydroxypyridine (3-Pyridinol), 6-

168

Citation: Hilali M, El MonfaloutiH and Kartah BE (2020). Study of the flavonoids and secondary metabolites of the Argan tree (Argania spinose L.). Online J. Anim. Feed

Res., 10(4): 167-171.

methyl-3-hydroxypyridine and catechol, resorcinol, vanillyl alcohol, tyrosol, 4 hydroxy-3-methoxyphenethyl, epicatechin and

catechin (Guillaume et al., 2005 ).

Table 1 - Phenolic compounds of the pulp of the Argan fruit

MS/MS

(Experimental

method) Neutral

loss scan

MS/MS

(Experimental

method)

Production

MS/MS (Experimental

method) Precursor

scan

Retention

time (min)

N°

Compounds (%)

[M-H]

Fragments

Gallic acid (5.0)

Protocatechuic acid (21.8)

Catechine (2.8)

Isorhoifoline (7.2)

Epicatechin (14.7)

Procyanidin (2.7)

Rutin (0.1)

0.82

1.44

4.06

7.13

7.65

169

153

289

577

289

579

609

463

433

623

301

285

271

125

109

289

245

289,245

301

271

289

579

609

463

433

623

301

7.67

10.87

11.19

11.46

11.70

12.33

12.69

13.37

17.83

17.94

18.51

308

162

132

162

308

Hesperidin (4.5)

Hyperosid (13.4)

463.301

Isoquercitrine (10)

Quercetine-o-pentose

Naringenine-7-o-glucoside

Rhamnetine-o-rutinoside (0.5)

Quercetin (1.6)

315

151, 121, 107

119,109

Luteolin (0.2)

Naringenin (0.07)

271

Table 2 - Phenolic compounds of the Argan leaves

N°

Compounds (%)

Retention time (min)

Myricetine 3-Ogalactoside (9,78%)

Myricitrine (54,34%)

Hyperoside (8,69%)

3.4

3.9

Quercitrine (21,73%)

Compounds of tocopherols from Argan oil

The tocopherols were analyzed by HPLC on a column in the normal phase, directly from vegetable oil without

saponification. They were identified by comparison of their chromatogram with controls injected under the same

conditions. Their dosage was possible by the use of α-tocopherol. The results obtained are grouped in Table 3. Tocopherols

are natural antioxidants, they are molecules with carbon chains linked to a quinone function (Bouhadjra, 2011 ). In

vegetable oils there are four groups of tocopherols (a, b, g and d). Tocopherols have both a vitamin power (vitamin E, in

particular a tocopherol) and antioxidant properties (Landrier, 2011 ). In addition, these compounds can constitute an

analytical criterion for controlling the purity of oil. Argan oil is richer in tocopherol (597 to 775 mg/kg) than olive oil (50 to

150 mg/kg) and also than hazelnut oil (300 to 550 mg/kg) (Hilali et al., 2005, 2020 ). Tocopherols are natural

antioxidants, gamma tocopherol has the highest antioxidant power (Bourre and Clement , 1996). Rich in gamma

tocopherol, Argan oil is a valuable nutraceutical. Tocopherols (vitamin E) and polyphenols are natural antioxidants. These

play an essential role in the prevention of several diseases, because they are anti-free radicals (Carrouf, 2002 ).

Table 3 - Composition of tocopherols in Argan oil (mg / kg).

Tocopherols

Argan oil

γ-tocopherol

δ-tocopherol

α-tocopherol

β-tocopherol

Total

631,3 mg/kg

59,5mg/kg

26,6 mg/kg

717.4 mg/ kg

R₁

R₂

CH₃

Figure 2 - Structure of tocopherols in Argan oil (R1 = R2 = CH3: α- tocopherol; R1 = CH3, R2 = H: β- tocopherol; R1 = H; R2

= CH3: γ- tocopherol; R1 = R2 = H: δ- tocopherol)

169

Citation: Hilali M, El MonfaloutiH and Kartah BE (2020). Study of the flavonoids and secondary metabolites of the Argan tree (Argania spinose L.). Online J. Anim. Feed

Res., 10(4): 167-171.

Phenolic compounds of Argan cake

The cake from the extraction or meal is currently used as feed for fattening cattle. It is rich in carbohydrates and

proteins (46.6% to 49%) and contains an important pharmacodynamic group consisting of saponins (Guillaume et al,

2005) and also contains an important group of flavonoids (Table 4). The Argan cake is rich in flavonoids, sixteen

flavonoids were found. Among these, epicatechin, catechin, protocatechic acid, vanillic acid and 4-hydroxybenzylic alcohol

are higher in Argan cake. On the other hand, epicatechin is a powerful antioxidant from the flavonoid family. A recent

American study shows that epicatechin has positive health effects and shows that it can significantly reduce the risks of

coronary heart disease and stroke during regular consumption (Rees et al., 2018 ).

Table 4 - Polyphenols from Argan cake

N°

Phenolic compound

Catechol

Resorcinol

Concentration (mg / kg)

1.4

1.3

4-hydroxybenzyl alcohol

Vanillin

8.6

1.1

Tyrosol

6.2

P-hydroxybenzoic acid

(4-hydroxyphenylacetic) acid Alcohol

vanillic alcohol

14.1

1.0

3.6

3.4-dihydroxybenzyl alcohol

Methyl 3.4-dihydroxybenzoate

vanillic acid

Hydroxytyrosol

Protocatéchic acid

Syringic acid

0.9

1.6

16.3

0.9

15.2

6.6

Epicatechine

Catechin

110.1

CONCLUSION

Argan cake is rich in flavonoids. Among these, epicatechin (110 mg/kg), catechin (11 mg/kg), protocatechic acid (15.2

mg/kg), vanillic acid (16.3 mg/kg) and 4-hydroxybenzyl alcohol (8.6 mg/kg) are higher in Argan cake. Argan oil is richer in

tocopherol (597 to 775 mg/kg), Argan oil is rich in gamma tocopherol (631 mg/kg), and this metabolite making Argan oil

is a valuable nutraceutical. The flavonoid extract of the co-products of the Argan tree has a very interesting anti-radical

and antioxidant activity. It is currently marketed as a cosmetic active protector of the skin's extracellular macromolecules

such as collagen, glycoproteins for this reason the co-product of Argan has become very important in the cosmetic field

and its most widespread marketed.

DECLARATIONS

Corresponding author: hilali400@yahoo.com

Authors' contribution

The author reviewed the document and contributed to the development of the content.

Availability of data

The data can be availed to the journal upon request.

A conflict of Interest

The author declare that there is no conflict of interests regarding the publication of this paper

Acknowledgments

The author would like to thank my fellow plant chemistry lab colleagues for their invaluable help and especially Mr.

Mohamed Greih. To all the people who contributed directly or indirectly to the realization of this work, I send them my

warmest thanks. The author has stated that he is not taking any funding to do this work or to publish this article.

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Citation: Hilali M, El MonfaloutiH and Kartah BE (2020). Study of the flavonoids and secondary metabolites of the Argan tree (Argania spinose L.). Online J. Anim. Feed

Res., 10(4): 167-171.