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People's Democratic Republic of Algeria Ministry of Higher

Education and Scientific Research

Dissertation for the award of the Master's Degree Field:

Natural and Life Sciences

Department: Biological Sciences

Speciality/ Fundamental and applied biology

Natural Sciences Department Theme:

Contribution to the phytochemical study and evaluation of

some biological activities of the raw extract of Ephedra
alata.
Presented by: Amina Mokhtari
Before a jury composed of:

Chairman: Dr. Krimate

Soumia Examiner: Dr. Bouti

karima Supervisor: Dr. Zebiri

Saliha

Co-supervisor: Dr. Tigrine Chafia

Academic year 2022/2023

 THANKS

First of all, I thank God, without whose power nothing can be done and nobody can

do anything. I thank Almighty God for having given me the courage, the patience, the

will and the strength to complete this modest work.

Our Prophet ‫ ﻣﺤﻤﺪ ﺻﻠﻰ ﻫﻠﻼ ﻋﻠﻴﻪ ﻭﺳﻠﻢ‬said, "Whoever does not thank people

does not thank Allah".

I would like to express my sincere thanks to the two professors who helped me

complete this humble work, zebiri Saliha and TIGRINE Chafia.

I would also like to thank the correction committee, Professor Bouti Karima and
Krimate Soumia.
 Dedication
I dedicate this modest work to myself and

To my dear mother and father

For Their love, Their kindness, Their sacrifice, Their

encouragement

To my sisters, my brother and my friends.

To all those who have sacrificed their time for science

and to all those who use science for good

and the prosperity of mankind.

Abstaract
summary

List of abbreviations

List of figures List

of tables

Table of contents
Introduction...............................................................................................................................1
Chapter I: Bibliographical research

I. Bibliographic search.................................................................Error! Bookmark not defined.

1. General information on Ephedra alata.......................Error! Bookmark not defined.
1.1. Presentation of the species Ephedra alata...........Error! Bookmark not defined.
1.2. Botanical description of Ephedra alata................Error! Bookmark not defined.
1.3 Systematic classificationError..............................! Bookmark not defined.
1.3. Geographical breakdown.......................................................................................6
1.4. Use of E. alata in traditional medicineError.....................! Bookmark not
defined.
2. Secondary metabolites....................................................................................................7
2.2. Alkaloids.....................................................................................................................8
2.3. Flavonoids................................................................................................................8
2.4. Tannins....................................................................................................................8
2.5. Saponosides..............................................................................................................9
3. Biological activities.........................................................................................................9
3.1. Antioxidant activity.....................................................................................................9
3.2. Anti-inflammatory activity......................................................................................10
3.3. Antifungal activity.....................................................................................................11
4. Main fungal strains studiedError...............................! Bookmark not defined.
4.1. Aspergillus flavus...................................................................................................11
4.2. Aspergillus niger........................................................................................................13
4.3. Aspergillus carbonarius.........................................................................................13
4.4. Aspergillus parasiticus...........................................................................................14
4.5. Aspergillus brasiliensis...........................................................................................15
4.6. Mucor.....................................................................................................................16
4.7. Candida albicans....................................................................................................17
 Chapter II: Materials and methods

II. Materials and methods.......................................................................................................22

1. Equipment............................................................................................................................22
1.1. Equipment for culture media and reagents...........................................................22
1.2. Biological materials...................................................................................................22
1.2.1. Plant material....................................................................................................22
1.2.2 Fungal strains..........................................................................................................23
2.................................................................................................................................................Methods 23
2.1. Preparation of the crude ethanolic extract............................................................23
2.2. Photochemical study.................................................................................................24
2.2.1. Determination of polyphenols..........................................................................24
2.2.2. Flavonoid assay..................................................................................................26
2.2.3. Phytochemical screening......................................................................................26
2.3. Antioxidant activity.....................................................................................................28
2.3.1 Bleaching test for β-carotene.................................................................................28
2.4. Anti-inflammatory activity in vitro.........................................................................29
2.5. Antifungal activity....................................................................................................30
Chapter III: Results and discussion

III. Results and discussion......................................................................................................35

1. Extraction yield................................................................................................................35
2. Phytochemical studies......................................................................................................35
2.1. Phytochemical screening...........................................................................................35
2.2. Total polyphenol content..........................................................................................37
2.3. Flavonoid assay.........................................................................................................38
3. Antioxidant activity.........................................................................................................39
3.1. Beta-carotene bleach test..........................................................................................39
4. Anti-inflammatory activity in vitro................................................................................42
5. Antifungal activity...........................................................................................................43
Conclusion................................................................................................................................46
Appendices...............................................................................................................................47
References................................................................................................................................50
Summary
Ephedra alata, or Alenda as it is known, is a plant belonging to the Ephedraceae
family that is widespread in Mediterranean countries. It is resistant to harsh climatic
conditions, such as those found in the desert and semi-desert areas of Algeria. It has been
used since ancient times in the medical field as a "medicinal plant" to treat a range of
illnesses including asthma and cancer.
This laboratory study was carried out to evaluate the antioxidant, anti-inflammatory
and antifungal activity of the ethanolic extract of Alenda stems harvested in Ghardaïa,
Algeria.
Phytochemical examination of this plant shows the presence of primary compounds
such as carbohydrates, and secondary compounds such as flavonoids, steroids and saponins
in the extract. Antioxidant activity tests using the betacarotene- leonic acid method also
revealed the presence of strong antioxidant activity. Add to this a laboratory study of anti-
inflammatory activity using the protein inhibition method, where we found that the
ethanolic extract of E. alata strongly inhibited the destruction of bovine serum albumin.
There was no activity of the extract against the mycotoxigenic fungal strains tested and
against Candida albicans. So, in general, Ephedra can be considered one of the useful
medicinal herbs that can be used for treatment or to extract medicines and supplements.
Key words:
Ephedra alata, ethanolic extract, antioxidant activity, anti-inflammatory activity,
antifungal activity, Algeria.
 ‫‪:‬ﻣﻠﺨﺺ‬
‫‪Ephedra alata‬ﺃﻭ ﻣﺎ ﻳﻌﺮﻑ ﺑﺎﻟﻌﻠﻨﺪﻯ ﻧﺒﺎﺕ ﻳﻨﺘﻤﻲ ﺇﻟﻰ ﻋﺎﺋﻠﺔ ﺍﺇﻟﻴﻔﻴﺪﺭﺍﺳﻲ ﻳﻨﺘﺸﺮ ﺑﻜﺜﻴﺮ‬
‫ﻓﻲ ﺩﻭﻝ ﺍﻟﺒﺤﺮ ﺍﺃﻟﺒﻴﺾ‬
‫ﺍﻟﻤﺘﻮﺳﻂ ﻭﻫﻮ ﻣﻘﺎﻭﻡ ﻟﻠﻈﺮﻭﻑ ﺍﻟﻤﻨﺎﺧﻴﺔ ﺍﻟﻘﺎﺳﻴﺔ ﺑﺤﻴﺚ ﻧﺠﺪﻩ ﻓﻲ ﻣﻨﺎﻃﻖ ﺻﺤﺮﺍﻭﻳﺔ‬
‫ﻭﺷﺒﻪ ‪.‬ﺑﺎﻟﺠﺰﺍﺋﺮﺍﺳﺘﻌﻤﻞ ﻣﻨﺬ ﺍﻟﻘﺪﻡ ﻓﻲ ﺍﻟﻤﺠﺎﻝ ﺍﻟﻄﺒﻲ "ﻋﺸﺒﺔ ﻃﺒﻴﺔ "ﻓﻲ‬
‫ﺍﻟﺘﺪﺍﻭﻱ ﻣﻦ ﻣﺠﻤﻮﻋﺔ ﻣﻦ ﺍﺃﻟﻤﺮﺍﺽ ﻛﺎﻟﺮﺑﻮ ‪.‬ﻭﺍﻟﺴﺮﻃﺎﻥ‬

‫ﺗﻢ ﺍﻟﻘﻴﺎﻡ ﺑﻬﺬﺍ ﺍﻟﻌﻤﻞ ﺍﻟﻤﺨﺒﺮﻱ ﻟﺘﻘﻴﻴﻢ ﺍﻟﻨﺸﺎﻁ ﺍﻟﻤﻀﺎﺩ ﻷﻟﻜﺴﺪﺓ‪ ،‬ﻻﻟﻠﺘﻬﺎﺏ ﻭ ﺍﻟﻤﻀﺎﺩ ﻟﻠﻔﻄﺮﻳﺎﺕ ﻟﻠﻤﺴﺘﺨﻠﺺ‬
‫ﺍﺇﻟﻴﺜﺎﻧﻮﻟﻲ ﻟﺴﻴﻘﺎﻥ ﺍﻟﻌﻠﻨﺪﻯ ﺍﻟﺘﻲ ﺗﻢ ﺟﻨﻴﻬﺎ ﻣﻦ ﻏﺮﺩﺍﻳﺔ ‪.‬ﺍﻟﺠﺰﺍﺋﺮ‬

‫ﻳﻈﻬﺮ ﺍﻟﻔﺤﺺ ﺍﻟﻨﺒﺎﺗﻲ ﺍﻟﻜﻴﻤﻴﺎﺋﻲ ﻟﻬﺬﻩ ﺍﻟﻨﺒﺘﺔ ﻭﺟﻮﺩ ﻣﺮﻛﺒﺎﺕ ﺍﻳﺾ ﺃﻭﻟﻴﺔ ﻣﺜﻞ ﻏﻠﻮﺳﻴﺪﺍﺕ‪ ،‬ﻭﻣﺮﻛﺒﺎﺕ ﺍﻳﺾ ﺛﺎﻧﻮﻳﺔ ﺍﻟﻘﺎﻟﻮﺑﺪﺍﺕ‬
‫ﻣﺜﻞ ﺍﻟﻔﺎﻟﻔﻮﻧﻮﻳﺪﺍﺕ‪ ،‬ﺳﺘﺮﻭﻳﺪﺍﺕ ﻭﺍﻟﺼﺎﺑﻮﻧﻴﻦ ﻓﻲ ﻣﺴﺘﺨﻠﺺ ‪ ،Ephedraalata‬ﻛﻤﺎ ﺑﻴﻦ ﺍﺧﺘﺒﺎﺭ ﺍﻟﻨﺸﺎﻁ ﺍﻟﻤﻀﺎﺩ ﻷﻟﻜﺴﺪﺓ‬
‫ﺑﺎﺳﺘﻌﻤﺎﻝ ﻃﺮﻳﻘﺔ ﺑﻴﺘﺎ ‪-‬ﻛﺎﺭﻭﺗﻴﻦ‪-‬ﺣﻤﺾ ﺍﻟﻠﻴﻮﻧﻴﻚ ﻭﺟﻮﺩ ﻧﺸﺎﻁ ﻗﻮﻱ ﻟﻤﻀﺎﺩﺍﺕ ﺍﺃﻟﻜﺴﺪﺓ‪ ،‬ﺿﻒ ﻟﺬﻟﻚ ﺩﺭﺍﺳﺔ ﺍﻟﻨﺸﺎﻁ‬
‫ﺍﻟﻤﻀﺎﺩ ﻻﻟﻠﺘﻬﺎﺑﺎﺕ ﻣﺨﺒﺮﻳﺎ ﺣﺴﺐ ﻃﺮﻳﻘﺔ ﺗﺜﺒﻴﻂ ﺍﻟﺒﺮﻭﺗﻴﻨﺎﺕ ﻭﻣﻦ ﺧﺎﻟﻞ ﺍﻟﻨﺘﺎﺋﺞ ﻧﺠﺪ ﺃﻥ ﺍﻟﻤﺴﺘﺨﻠﺺ ﺍﻟﻴﺜﺎﻧﻮﻟﻲ‬

‫ﻳﺜﺒﻂ‪E.alata‬ﺑﺸﻜﻞ ﻗﻮﻱ ﺗﺨﺮﻳﺐ ﺃﻟﺒﻮﻣﻴﻦ ﻣﺼﻞ ﺍﻟﺒﻘﺮﻟﻢ ‪ .‬ﻳﻈﻬﺮ ﻫﺬﺍ ﺍﻟﻤﺴﺘﺨﻠﺺ ﺍﻱ ﻧﺸﺎﻁ ﺿﺪ ﻣﺠﻤﻮﻋﺔ ﻣﻦ ﺍﻟﺴﺎﻻﻟﺖ ﺍﻟﻔﻄﺮﻳﺔ ﺍﻟﻤﻨﺘﺠﺔ‬
‫ﻟﻠﺴﻤﻮﻡ ‪.‬ﺍﻟﻔﻄﺮﻳﺔ‬

‫ﻋﻠﻰ ﺍﻟﻌﻤﻮﻡ ﻳﻤﻜﻦ ﺍﻋﺘﺒﺎﺭ ‪Ephedra‬ﻣﻦ ﺍﺃﻟﻌﺸﺎﺏ ﺍﻟﻄﺒﻴﺔ ﺍﻟﻤﻔﻴﺪﺓ ﺍﻟﺘﻲ ﻳﻤﻜﻦ ﺍﺳﺘﻌﻤﺎﻟﻬﺎ‬
‫ﻓﻲ ﺍﻟﺘﺪﺍﻭﻱ ﺃﻭ ﺍﺳﺘﺨﺮﺍﺝ ﻣﻨﻬﺎ ﺃﺩﻭﻳﺔ ﻭ ﻣﻜﻤﺎﻟﺖ‪.‬‬

‫‪:‬ﺍﻟﻤﻔﺘﺎﺡ ﺍﻟﻤﺴﺘﺨﻠﺺ‪،E alata‬ﺍﺇﻟﻴﺜﺎﻧﻮﻟﻲ ‪ ،.‬ﻣﻀﺎﺩ ﻷﻟﻜﺴﺪﺓ‪ ،‬ﻻﻟﻠﺘﻬﺎﺏ‪ ،‬ﻣﻀﺎﺩ ﻟﻠﻔﻄﺮﻳﺎﺕ‪. ،‬ﺍﻟﺠﺰﺍﺋﺮ‬

 Abstarct

Ephedra alata, or what is known as Ephedraceae, is a plant belonging to the Ephedra

family. It is widely spread in the Mediterranean countries and is resistant to harsh climatic
conditions, such that we find it in desert and semi-desert areas in Algeria. It has been used
since ancient times in the medical field as a "medicinal herb" in the treatment of a group of
diseases such as asthma and cancer....
This laboratory work was carried out to evaluate the anti-oxidant, anti-inflammatory
and anti-fungal activity of the ethanolic extract of Alanda stems harvested from Ghardaia,
Algeria.
Phytochemical examination of this plant shows the presence of primary compounds
such as glucides, and secondary compounds such as flavonoids, steroids and saponins in
the Ephedra alata extract. Testing of antioxidant activity using the beta-carotene-lionic

.
acid method also revealed the presence of strong antioxidant activity In addition to this, we
studied the anti-inflammatory activity in the laboratory according to the method of
inhibiting proteins. Through the results, we find that the ethanolic extract of Ephedra alata
strongly inhibits the destruction of bovine serum albumin. Despite these results, we did not
find any activity of the extract against a group of mycotoxinogene fungal strains. So, in
general, ephedra can be considered a useful medicinal herb that can be used for treatment
or extracted from it as medicines and supplements.
Key words: ethanolic extract, E. alata, antioxidant, anti-inflammatory, antifungal, Algeria.
 List of abbreviations

DNA : Deoxyribonucleic acid.

AlCl3 : Aluminium trichloride.

NSAIDS : non-steroidal anti-inflammatory

drugs.
AFB : Aflatoxins B.

AFG : Aflatoxins G.

BHT : Butyl hydroxy toluene.

BSA : Bovine serum albumin.

°C : Degrees Celsius.

CMI : Minimum Inhibitory Concentration.

DMSO : Dimethyl sulphoxide.

E. : Ephedra.

EAG : Gallic acid equivalent.

EQ : Extraction

ERO : Reactive oxygen species.

Fig : Figure.

HCl : Hydrochloric acid.

H2SO4 : Sulphuric acid.

m: metres.

mm : Millimetre.

MA : Malt Agar.

mg : Milligram.

ml : millilitre.

MS : secondary metabolites.

n: number.
HO- : Hydroxyl radical.

WHO : World Health Organization.

PDA : Potatoes dextrose agar.

PH : Hydrogen potential.

SD : Standard deviation.

μl: Microlitre.

VIT C : Vitamin C.

μg : Microgram.
 List of figures

Figure I. 1: Ephedra alata, A: General view. B: Flowering branches....................1

Figure I. 2: Morphology of male and female branches of Ephedra alata showing

male and female cones, A. Female branch, B. Female cone showing
membranous, winged bract, C. Male branch, D. Male cones showing
microsporangia)
..............................................................................................................
5

Figure I. 3: Geographical distribution of Ephedra alata in the world.....................6

Figure I. 4: the general chemical structure of polyphenols.....................................8

Figure I. 5: Microscopic appearance of the head of A. flavus...............................14

Figure I. 6 : of A. flavus under a photonic microscope (160x)..............................14

Figure I.7: Microscopic aspects of A. niger.........................................................15

Figure I. 8: A. carbonarius under a photonic microscope....................................16

Figure I. 9: A. parasiticus on light microscopy.....................................................17

Figure I. 10: A. brasiliensis, C. colonies ; f. conidiophores....................................18

Figure I. 11 : Mucor under a photonic microscope (400x)......................................19

Figure I. 12 : C. albicans on light microscopy.........................................................20

Figure II.1: E. alata stems A (fresh); B (dry); C (powder)...................................22

Figure II.2: Summary diagram of extraction stages..............................................24

Figure II.3 : Determination of total polyphenols in E. alata stems (n=4)

............25

Figure II.4 : The β-carotene bleach test prior to incubation....................................29

Figure II.5 : Principle of the disc method or aromatogram.....................................30

Figure II. 6: Petri dishes before incubation (contains whatman paper discs with
crude extract)......................................................................................31
Figure II. 7: Dilutions of E. alata extract................................................................32

Figure II. 8 : Image showing how to place dilutions..............................................33

Figure II. 9 : Petri dishes before incubation............................................................33

Figure III. 1 : Transformation of the colour from yellow to blue is due to the
presence of polyphenols in the ethanolic extract of E.alata: [1]
50µg/ml and [2] 100µg/ml)................................................................37

Figure III. 2: Gallic acid calibration line for the quantitative determination of total
polyphenols in E. alata crude extract.................................................38

Figure III. 3 : The colour change from transparent to yellow is due to the presence
of flavonoids in the ethanolic extract of E alata (100µg/ml)..............38

Figure III. 4 : Quercetin calibration line for the quantitative determination of total
flavonoids in E alata crude extract....................................................39

Figure III. 5: Prevention of β-carotene bleaching in the presence of ethanolic

extract of E alata and standard antioxidants: BHT and Vitamin C
............................................................................40

Figure III.6 : Bleaching kinetics of β-carotene in the presence and absence of

ethanolic extract of E. alata, BHT and vitamin C. Values
represent averages of 4 trials ±.........................................SD41

Figure III.7
:
The percentage of antioxidant activity of ethanolic extract of E alata,
BHT and vitamin C after 120min. E alata extract protected β-carotene
from bleaching. Values represent the means of 4 trials ±..............SD41

Figure III.8 : Inhibitory effect of BSA denaturation by ethanolic extract of E. alata

(500µg/ml) and diclofenac sodium (500µg/ml).................................42

Figure III.9 : Results of inhibition of the antifungal activity of the crude extract of
E.alata, after three days of...................................................ncubation43
Figure III.10 : Results of inhibition of antifungal activity, using dilutions of
E.alata extract (50mg/ml ; 25mg/ml ; 12.5mg/ml ;
6.25mg/ml), After three days of incubation........................................44
 List of tables

Table I.1: Systematic position of E. alata..................................................5

Table I.2: Systematic position of E. alata: The systematic position of A.

Flavus.......................................................................................13

Table I.3: The systematic position of A. niger........................................15

Table I.4: The systematic position of A. carbonarius.............................16

Table I.5: The systematic position of A. carbonarius.............................17

Table I.6: The systematic position of A. praslians..................................18

Table I.7: Mucor's systematic position....................................................19

Table I.8: The systematic position of Candida albicans.........................20

TableII.1 : Shaikh and Patil's method for carrying out this applied work
.......................................................................27

Table III.1: Results of phytochemical screening of ethanolic extract of

E. alat36
Introduction
 Introduction

Introduction
Throughout history, the plant kingdom has been an important source for the discovery
of new agents with enormous therapeutic effects in various fields of medicine. Different
types of plant extracts contain a wide range of phytochemicals that can benefit from
different therapeutic activities individually or through synergistic mechanisms
(Jerbi and al., 2016).
The use of plants for healing dates back to prehistoric times and is an ancient tradition
shared by all people on all continents; despite the efforts of chemists to synthesise new
molecules, more than 25% of drugs prescribed in developed countries are directly or
indirectly derived from plants (Benaissa and Boukhari, 2018). According to the World
Health Organization (WHO, 2008), more than 80% of the world's population uses so-
called traditional medicine to deal with their health problems (Salhi and al., 2010).
According to an estimate, more than 300,000 species are described throughout the world,
and 15% of them have been studied phytochemically, including 6% for their biological
activities (Negri and Tabach, 2013).
Oxidative stress corresponds to an imbalance between the generation of activated oxygen
species (EOA, reactive oxygen species (ROS)) and the body's antioxidant defences, in
favour of the former. Our lifestyle (smoking, obesity, intense physical exercise), but also
our poor eating habits and the consumption of foodstuffs contaminated by mycotoxins
abnormally increase the production of EOA in our body.
N the long term, this can contribute to the appearance of various pathologies linked to
aging such as cancer or cardiovascular diseases. For the sake of prevention, it will
therefore be necessary to have effective tools to correctly assess the oxidative stress status
in an individual in order to make the necessary corrections to optimise our antioxidant
defences and reduce the oxidative damage induced by EOA in the body. level of DNA,
proteins and lipids (Bourgou and al 2020). In addition, these diseases are accompanied by
microbial infections due to the weakening of the immune system.
o strengthen the spontaneous antioxidant system the consumption of natural antioxidants
from various food supplements and traditional medicines is necessary. In this context,
medicinal plants could have considerable antioxidant potential for protection against
oxidative stress.

1
 Introduction

Among the medicinal plants of the Algerian desert, the plant Ephedra alata (E. alata).
This plant is known as Alanda ‫ ))ﻋﻠﻨﺪﺍ‬in Arabic, is a species of the Ephedraceae
family. t is answered in several Arab countries including Algeria, Morocco, Egypt,
Palestine,
Saudi Arabia. E. alata is widely used in traditional medicine in the treatment of kidney
problems, bronchial asthma, circulatory system disorders, digestive system disorders and
bacterial and fungal infections as well as in the treatment of cancer (Bouafia et al., 2021).
The objective of the present study is to evaluate the antioxidant activities of an alcoholic
extract of the stems of E. alata, harvested from the wilaya of Ghardaïa. Based on this
hypothesis, the practical part of this study is organized as follows:
⮚ Phytochemical investigation which begins with preparation of the
alcoholic extract and calculation of the yield.
⮚ Evaluation of the total content of polyphenols and flavonoids.
⮚ Phytochemical screening to detect the different classes of families of primary
and secondary metabolites.
⮚ Evaluation of the antioxidant potential by the β-carotene bleaching test.
⮚ Evaluation of anti-inflammatory activity in vitro.
⮚ Evaluation of in vitro anti-fungal activity on 07 fungal strains involved in
the production of mycotoxins.
Our work is structured around three chapters:
The first chapter is a bibliographic review which includes general notions on the
studied species E. alata and basic notions on secondary metabolites, their biosynthesis and
the biological activities of bioactive molecules of this genus.
The second chapter presents the materials and methods used in this study. The third
chapter presents the results and their discussion and our manuscript is closed with a
conclusion.

2
 Chapter 01:
Bibliographic research
 Bibliographic research

I. Bibliographic research
1. General information about Ephedra alata
1.1. Presentation of the species Ephedra alata
E. alata (Fig.I.1) belongs to the Ephedraceae family. It is represented by several
vernacular names including Alenda in Arabic ‫ ))ﻋﻠﻨﺪﺍ‬ephedra in French and ephedra in
English (Al-Snafi, 2017).

Figure I.1: E. alata, A: general view. B: Flowering branches

(Boubekri and al.,2020)https://almerja.com/more.php?

idm=145054

The Ephedraceae family includes a single genus called Ephedra composed of nearly 40
species worldwide that grows wildly on rocky, sandy and clayey soils of arid environments
(Danciu et al., 2019).
1.2. Botanical description of Ephedra alata
Species belonging to the Ephedraceae family are herbaceous perennial plants that can
exceed 1m in height with a strong pine odor and an astringent taste (Abourashed et al.,
2003).

This species is known for its great capacity to survive in drought conditions, particularly
in the Saharan regions. It is a shrub reaching a height of 1-3 metres which has articulated
and strongly branched branches of a yellowish-green color with opposite leaves of small
size that follow one another from one node to another. The flowers appear in the form of
small whitish cones and are dioecious (Fig.I.2) (male and female flowers on different
plants). The fruits are surrounded by largely membranous bracts. This species has an
extremely powerful lateral root system (Ozenda, 2004; Palici, 2016).

4
 Bibliographic research

Figure I.2: morphology of male and female branches of E. alata showing the male and
female cones, A. Female branch, B. Female cone showing the membranous, winged bract
C. male branch, D. Male cones showing microsporangia (Samah and Usama, 2008).

1.3. Classification of Ephedra alata

Table I.1 Systematic position of E. alata (Ozenda, 1991).

Reign vegetal

Branch Spermaphytes

Sub-branch Gnetophyta

Class Genetopsida

Order Ephedrales

Family Ephedraceae

Type Ephedra

Species Ephedra alata

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1.4. Geographical breakdown

The genus is indigenous to temperate and subtropical latitudes of Europe, Asia and
America: particularly in northern and western China, northern India, Spain and the United
States. Ephedra plants grow along the Rocky Mountains Ephedra plants grow along the
Rocky Mountains (Al-Snafi, 2017).
In Africa it is found in Algeria, Egypt, Libya, Morocco, Tunisia Mauritania, Chad and
Mali. In Asia in Saudi Arabia, Iraq, Iran, Palestine Lebanon, Jordan and Syria, (Benarba
and al., 2021).
In Algeria E. alata is found in the northern and western Sahara in the sandy soils of the
regs and the sandy beds of the wadis; it is even found in the sand of the tropical level and
the Hamada of Tinghert wilaya of Illizi (Hadjadj and al., 2020).

Figure I.3: Geographic distribution of E. alata in the world (Caveney et al., 2001).
1.5. The use of E. alata in traditional medicine
The Chinese dispensary written in 1569 mentions that Ephedra species were valuable
as antipyretic diaphoretic circulatory stimulant and sedative for cough. However. Ephedra
has been used in traditional Chinese medicine to treat allergies asthma and lung congestion
chills colds hay fever coughs edema fever flu headaches and nasal congestion. The plant
was also traditionally used in Russia for respiratory disorders and rheumatism for many
centuries. Native Americans and Spaniards in the southwestern United States used Ephedra
for various medicinal purposes, including against venereal diseases (Al-Snafi, 2017).
An active ingredient was first isolated by Yamanashi 1885. In 1887 Nagai obtained the

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alkaloid in pure form and named it ephedrine. Pharmacological investigation indicated that
the drug was mydriatic toxic and sympathomimetic (Ebadi, 2007).
In Algeria E. alata is used for the treatment of cancer (Bouafia and al., 2021; Miara et
al., 2019), against flu, whooping cough and general weakness in herbal tea and by
inhalation as well as in the form of nasal drops against colds (Chehma and Djebar, 2008).
1. Secondary metabolites

Secondary metabolites (SM) are complex organic molecules synthesised by

autotrophic plants. They are generally characterised by low concentration in plant tissues
(Newman and Cragg, 2012).

Species of the genus Ephedra are known to be rich in various phytoconstituents such
as alkaloids, tannins, saponins, proanthocyanidins, phenolic acids and flavonoids (Hegazi
and El-lamey, 2011).
2.1. Polyphenols

Polyphenols, also called phenolic compounds, are very widespread substances in the
plant kingdom and constitute one of the most important groups. There are currently more
than 8,000 different phenolic structures known. Polyphenols are products resulting from
the secondary metabolism of plants. The term "phenolic compounds" covers a wide range
of possess an aromatic ring with one or more substituting hydroxyl groups (Urquiaga and
al., 2000) (Fig.I.4).

This family of secondary metabolites contains. We limit our study to flavonoids,

tannins and alkaloids.

Figure I.4: general chemical structure of polyphenols (Houba and Himeur, 2019).

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2.2. Alkaloids

These are basic nitrogen products of natural origin whose nitrogen atom is included in
a heterocyclic system and whose pharmacological activity is significant. Pseudo-alkaloids
are not derivatives of amino acids. They are then called terpene alkaloids and proto-
alkaloids are simple amines whose nitrogen is not included in a heterocyclic system.
Alkaloids also have the property of reacting with heavy metal salts, which allows
their easy characterisation (Mayer, Dragendorf, Wasicky, Bouchardat reagents).
Ephedra species are mainly appreciated in the medical field due to the presence
of phenylalanine-derived alkaloids, such as ephedrine and other related compounds
like pseudoephedrine, norpseudoephedrine, norephedrine, methylephedrine and
methylpseudoephedrine (Danciu and al., 2019).
2.3. Flavonoids
Flavonoids are plant pigments, simple or glycosylated, responsible for the colouring of
flowers, fruits and sometimes leaves. Flavones (for example, apigeno l13) and flavonols
(such as quercetol 14), colorless, have a co-pigment and protective role while yellow
flavonoids (chalcones such as isoliquiritigenin 16, aurones including hispidol 17 , and
yellow flavonols) and blue and red anthocyanosides are directly visible. Some are only
visible to insects, providing signaling for pollinators. Flavonoids, dissolved in the vacuoles
in the state of glycosides or in particular plastids, the chromosplasts, are present in the
cuticle and epidermal cells, ensuring the protection of tissues against, Harmful solar
radiation. Flavonoids (more than 3000) have a common biogenetic origin and flavonoids
sensu stricto can be distinguished from flavonic derivatives, anthocyanosides and
isoflavonoids. Flavones and flavonols represent 80% of flavonoids sensu stricto (Sabrina,
2003, p.21).
2.3. Tannins

In 1962, tannins were defined as water-soluble phenolic compounds, with a molecular

mass between 500 and 3000, having the property of precipitating gelatin and other proteins
and of being coloured by ferric salts (Sabrina, 2003, p.23).

The medical applications of tannin plants arise from their affinity for proteins: they
have an anti-diarrheal effect, and externally, they waterproof the superficial layers of the
skin, are vasoconstrictors and limit fluid loss. These properties, added to their antiseptic
effect,

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make them interesting molecules for tissue regeneration in the event of superficial injury or
burn, and make them usable in the treatment of infectious diarrhea. The officinal tannin
used as an astringent in dermatoses and burns, and as a hemostatic is also extracted
(Sabrina, 2003, p.24).
These compounds are synthesised in large quantities in the stems of the Ephedra species
belonging to both continents, Eurasian and American. These molecules contribute to the
astringent taste of Ephedra (soni, 2004).
2.4. Saponosides

Saponosides constitute a large group of glycosides very common in plants. They are
characterized by surfactant effects giving them the property of forming foaming solutions
when dissolved in water. They can be classified into two groups according to the nature of
their genin which can be steroidal or triterpene. Steroidal genins have a C27 backbone and
six rings. Steroidal saponosides are found in many plants, but they are also characteristic of
starfish. Some were used for a time in the synthesis of steroids (diosgenins of Dioscorea).
Thus sarsapogenin, coming from the hydrolysis of sarsaparilloside 35, was used as a raw
material for the synthesis of steroids (Sabrina, 2003, p.29).
Triterpene saponosides often have a pentacyclic backbone, oleananes or ursanes. The
saccharide chains of saponosides are most often formed from 2 to 10 osebanals, linked to
genin by an ester or ether type bond (Sabrina, 2003, p.29).
3. Biological activities

The plant has shown a wide range of pharmacological activities, including

antimicrobial (Hibi et al., 2022), anti-inflammatories Benarba et al. (2021), and
antidiabetic, cardiovascular, nervous, respiratory, immunological, antipyretic, analgesic
and many other pharmacological effects. Among which we cite the most important.
3.1. Antioxidant activity
Oxidation represents an essential process in the metabolism of the body's aerobic cells. It
involves the oxygen molecule whose production by uncontrolled metabolic pathways leads
to the formation of reactive oxygen species (ROS) such as the free radicals superoxide O2,
hydroxyl HO, alkoxyl RO and peroxyl RO2 .These radicals are involved in oxidative
stress which is characterized by an imbalance between the production of ROS
(prooxidants) and the elimination of these species by the antioxidant defence mechanism.
Oxidative stress can cause damage to molecules such as lipids, DNA, carbohydrates and
proteins. Free radicals are also involved in the development of many pathologies such as

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obesity, cancer,

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diabetes, inflammation, atherosclerosis and degenerative diseases (Al-Laith,

2010; Codoñer-Franch et al., 2011 ; Rashid et al., 2013).
Antioxidants constitute a family of substances capable of neutralising free
radicals and thus preventing the occurrence of diseases associated with oxidative
stress. Among the best-known natural antioxidants, we can cite αtocopherol (vitamin
E), ascorbic acid (vitamin C) and phenolic compounds (Kulawik and al., 2013).
Ephedra is a natural source of phenolic compounds, giving it high antioxidant
capacity. Research has reported the presence of various phenolic compounds in Ephedra,
which contribute significantly to its antioxidant activity (Al-Rimawi and al., 2017).
The antioxidant effect of the extracts of this plant was evaluated by the β-carotene
bleaching test.
3.2. Anti-inflammatory activity

Inflammation is the response of living vascularized tissues to an attack. The role of this
inflammatory response is to eliminate the attacking agent and allow tissue repair. It
therefore makes it possible to maintain the integrity of the "self" (Boudjida and Halit-
Sahnoun, 2017).
E. alata has been used in traditional herbal medicine to treat inflammatory diseases
(Kmail and al., 2017).
The aqueous extract of E.sinica exhibits complement inhibitory property in both
animal and human serum. This could explain the use of the plant in traditional Chinese
medicine in the case of acute nephritis (Ling, 1995). Furthermore, Hikino and colleagues
(1980) suggested that pseudoephedrine is the active principle responsible for the anti-
inflammatory activity shown by E. intermedia. Konno and his colleagues (1979) reported
that the aerial part of Ephedra species contains ephedroxane which was also found to
possess anti-inflammatory activity.
Anti-inflammatories are defined as substances that act on the pain and swelling that
appear following an attack by a pathogen. They block the secretion or action of certain
chemical mediators of inflammation (such as prostaglandins) and therefore reduce the
sensation of pain but also inflammation (Hajjaj, 2017; Orliaguet and al., 2013). They are
used when the inflammatory reaction continues abnormally (chronic inflammation) and
causes tissue damage. These molecules are classified into steroidal anti-inflammatories
(cortisone and derivatives), non-steroidal anti-inflammatories and natural anti-
inflammatories such as photochemical compounds from the plant kingdom.

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3.3. Antifungal activity

Antifungals (or antifungals) take their name from the Latin fungus which means
mushrooms. These are therefore medications capable of treating mycoses, that is to say
infections caused by microscopic fungi.
An antifungal will act either by directly attacking the fungal wall, thus causing the
death of the cell (fungicidal action). Or by blocking cell division, thus stopping the
reproduction of fungi (fungistatic action) (Nabti and Boulberhane, 2017). Among the
researchers who used the plant E. alata to detect antifungal activity (Hibi and al., 2022).
The aqueous extract of E. alata exhibits significant inhibitory potential in vitro and in vivo
against the growth, as well as the production of aflatoxins, by the aflatoxigenic mould
Aspergillus flavus (Al-Qarawi et al., 2011; Parsaeimehr et al.,2010). According to Ghanem
and El-Magly (2008), the acetonitrile extract of E.alata shows antifungal activity
especially on Aspergillus fumigatus and the ethanolic extract of the aerial part of E. alata.
alata acts as a fungal agent and against C. albicans (Danciu et al., 2019).
4. Main fungal strains studied
Mycotoxins are secondary metabolites produced by moulds belonging mainly to the
genera Aspergillus, Penicillium and Fusarium.
Aspergillus are microscopic fungi that contaminate crops in the fields or during storage in
silos or granaries (Barros et al. 2005). When climatic conditions are favourable, certain
strains of the Aspergillus genus produce aflatoxins which are secondary metabolites known
to be carcinogenic, immunosuppressive and teratogenic (WHO, 2006).

4.1. Aspergillus flavus

A. flavus is widespread in warm climates and occupies very diverse ecological

niches (Wilson et al., 2002). This species lives in the soil in a saprophytic state but is
capable of causing grain rot (Horn and Dorner, 1998). The taxonomy of the genus
Aspergillus was first established by Micheli (1729). Aspergillus belong to the phylum
Ascomycetes
The colonies on potato agar, PDA, incubated at 25°C develop rapidly, they are olive
to lime green in colour with a cream underside; their texture is woolly to cottony and
somewhat granular. A clear or brown exudate may be present in some isolates (Tabuc,
2007). A. flavus produces aflatoxin B which is the most powerful natural carcinogen
known to date.

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Table I.2: The systematic position of A. Flavus is summarized as follows:

Reign Fungi

Division Ascomycota

Class Eurotiomycetes

Subclass Eurotiomycetidae

Order Eurotiales

Family Trichocomaceae

Gender Aspergillus

Species Aspergillus Flavus

Figure I.5: Microscopic appearance of the head of A. flavus (Cahagnier, 1997).

Figure I.6: A. flavus under Photonic microscope

(160x) (https://www.gettyimages.fr/photos/aspergillus-
flavus).

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4.2. Aspergillus niger

Table I.3: The systematic position of A. niger (Alexopoulos, 1979).
Reign Fungi

Branch Amastigomycota

Sub-branch Deuteromycotina

Class Deutoromycetes

Order Moniliales

Family Moniliaceae

Gender Aspergillus

Species Aspergillus niger

THERE. niger or black Aspergillus, is a filamentous ascomycete fungus of the order

Eurotiales. It is one of the most common species of the genus Aspergillus which appears as
a black mould on fruits and vegetables (Rapper and Fennell, 1977).

Figure I.7: Microscopic aspects of A. Niger

(https://www.inspq.qc.ca/moisissures/fiches/aspergillus-niger).

4.3. Aspergillus carbonarius

Table I.4: The systematic position of Aspergillus carbonarius is summarized as follows

(Gams and al., 1985).

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Reign Fungi

Branch Amastigomycota

Sob-branch Deuteromycotina

Class Deuteromycetes

Order Hyphomycetes

Family Moniliaceae

Gender Aspergillus

Species Aspergillus carbonarius

A. carbonarius has a thallus with septate mycelium carrying numerous erect,

unbranched conidiophores ending in a vesicle. The species is biseriate (Phialides carried
on metules or sterigmata). The conidia are black in colour and have a warty shape with a
diameter of 7-9 μm. A. carbonarius has grayish yellow sclerotia (Gams and al., 1985). The
species A. carbonarius of the Nigri section constitutes the main producer of ochratoxin A
(OTA).

Figure I.8: A. carbonarius under Photonic microscope (100x)

(https://library.bustmold.com/fr/aspergillus/aspergillus-carbonarius
/)

4.4. Aspergillus parasiticus

This species is characterized by a dark yellow green, flaky thallus and granular and
dense colonies on Malt Agar (MA), with a colorless to light beige reverse. The
conidiophore, 250-500 μm in size, pale brown in colour with an echinulate wall.

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The aspergillus heads are mainly uniseriate, characterized by spherical vesicles of 30-35
μm in diameter, three-quarters covered, giving greenish phialides of 7-11 μm carrying
spherical conidia of 4-6 μm in diameter in a radial arrangement, which sometimes differs
from 'HAS. flavus characterized by smaller conidia. The sclerotia occasionally produced
in young isolates, spherical 400-800μm in diameter, are often white at first and turn black
over time. The mycotoxins produced by this species are AFB1, AFB2, AFG1 and AFG2
(Pitt and Hocking, 1997).
Table I.5: The systematic position of A.parasiticus is summarized as follows:
Reign Fungi

Division Ascomycota

Class Eurotiomycetes

Order Eurotiales

Family Aspergillaceae

Gender Aspergillus

Species Aspergillus parasiticus

Figure I. 9: A. parasiticus on light microscopy (600x) ;

(http://bioimagen.bioucm.es/foto/6524).
4.5. Aspergillus brasiliensis

The name brasiliensis refers to the locality where the crop was isolated. Since its

discovery, the species has also been found in other localities, but we propose to use

the name "brasiliensis".

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 Bibliographic research
keep the name to avoid confusion, as the epithet was used and quoted in

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various publications. Colony initially white then dark brown to black. Exudates absent,

underside cream to light brown. Cnidial heads globose at first and later radiating,

occasionally developing into several columnar conidia, thick, smooth walls. The size of a

condial (3. 5-4.5 mm), it produces tensidol A and B, and pyrophen (Varga et al., 2007).

Figure I.10: A. brasiliensis, C. colonies ; f. conidiophores (Varga et al., 2007).

brasiliensis is a fungus and one of the most common species of the genus Aspergillus. This
common food contaminant is ubiquitous in soil and is also regularly reported in enclosed
spaces such as industrial sites. Unlike other Aspergillus species, A brasiliensis rarely
causes illness in humans. However, in those rare cases, it can cause a serious lung disease
called aspergillosis (Patterson et a l., 2016).

4.6. Muco

Table I.7: The systematic position of Mucor is summarised as follows (Walther et al.,
2013)

Kingdom Fungi

Branching: Mucoromycota

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Sub-branch Mucoromycotina

Order Mucorales

Family Mucoraceae

Type Mucor

The work of Schipper (1967, 1969, 1970, 1976) contributed to the morphological
description of Mucor. Mucor are characterised by their rudimentary organisation. They
have a vegetative apparatus, the thallus, which corresponds to a bundle of more or less
branched filaments or hyphae that form the mycelium (Zycha et al., 1969).

The mycelium, which is the vegetative part of the fungus, grows rapidly and can
branch out to colonise the environment (these by Morin-Sardin, 2016).

Figure I.11: Mucor under a light microscope (400x)

https://www.istockphoto.com/fr/photo/mucor-mold-champignon-noir-champignons-de-
moule-%C3%A0-pain-illustration-3d-gm1328467090-412509211

4.7. Candida albicans

Candida albicans, an opportunistic polymorphic yeast, is the most common fungal

pathogen in humans. It is responsible for a variety of oral candidiasis and vaginitis, two
widespread infections. It varies in shape from round to elongated. Commensal in the
digestive tract of humans, mammals and birds, it is opportunistic and becomes pathogenic
in favourable conditions (Segal, 2005).

Table I.7: The systematic position of C. albicans.

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Kingdom Fungi

Branch Ascomycota

Class Sassharomycetes

Order Saccharomycetales

Family Saccharomycetaceae

Type Candida

Species Candida albicans

Figure I.12: C. albicans under light microscopy (600x)

(https://www.istockphoto.com/fr/search/2/image-film?phrase=candida+albicans).

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 Chapter 02:
Materials and methods
 Materials and methods

I. Materials and methods

Our experimental work was carried out in two laboratories. We carried out the in
vitro antioxidant, antifungal and anti-inflammatory activity of the E. alata species at the
Laboratory of Ethnobotany and Natural Substances (LESN) at ENS Kouba from March to
July 2023. The antifungal activity w a s carried out at the Laboratoire de Biologie des
Systèmes Microbiens at the Ecole Normale Supérieure d'Alger (LBSM-ENS-Kouba) in
July 2023.

1. Hardware

1.1. Equipment for culture media and reagents:

The equipment, culture media, glassware and solvents used are included in
Appendix 1.

1.2. Biological materials

1.2.1. Plant material

The aerial parts of E. alata were collected in February 2023 at Oued El Sebseb in the
Ghardaïa region. The plant was identified by Mr Toumi Mouhamed (Professor at the
University of Algiers 1 Ben Yousef Benkhedda). The plant was dried in the shade at room
temperature until it was completely dry. The dried stems were ground to powder using a
grinder (Fig.II.1).

Figure II.1: E. alata Alenda stems, A (fresh); B (dry); C (powder).

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 Materials and methods

1.2.2 Fungal strains

In our study, we used six (06) fungal strains: Aspergillus flavus NRRL 1829; A.
niger; A. carbonarius M333; A. parasiticus; A. brazilians ATCC 16404, Mucor,
Candida albicans belonging to the LBSM collection.

2. Methods
2.1. Preparation of the crude ethanolic extract

After grinding the dried stems and obtaining a powder, a crude extract was extracted.
A quantity of the powder was extracted by maceration in ethanol in three stages as
follows (Fig. II.2):

⮚ Mix 50g of powder with 500ml of ethanol and place in a water bath for 20min
at 40°C with occasional stirring.
⮚ Perform filtration using a 4μm porosity sintered glass and a vacuum pump.
⮚ Evaporate the solvent to dryness in a rotary evaporator at 45°C u n t i l a
brown paste is obtained.
⮚ Store the extract in a dark place in the refrigerator at +4°C.

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 Materials and methods

Figure II.2: Summary diagram of the extraction stages.

1.2. Photochemical study

1.2.1. Determination of polyphenols

The total polyphenol content in the ethanolic extract of E. alata stems was
measured using the Follin-Ciocalteu reagent method.

a. Principle

Polyphenols are assayed using the method described by (Dewanto et al., 2002). The
principle of the method is based on the oxidation of phenolic compounds by the "Folin-
Ciocalteu" reagent, which is a mixture of yellow phosphotungstic acid and
phosphomolybdic acid complexes. This oxidation leads to the formation of
24
 Materials and methods

of a new blue molybdenum-tungsten complex that absorbs at 750 nm (Haddouchi et al.,

2016).

b. How it works
Polyphenols were assayed using a colorimetric method cited in (Tigrine and Kameli,
2021) according to the following protocol:

⮚ A total of 250 μl of extract (50 μg/ml final concentrations) was mixed with 3500
μl of distilled water and 250 μl of Folin-Ciocalteu reagent.
⮚ After incubating for 3 minutes in the dark, 1 ml Na2CO3 (20%) was added.
⮚ The mixture was incubated at 100°C for 1 minute.
⮚ Absorbance was measured at 685 nm against a blank containing all the
solutions except the extract, which was replaced by ethanol.
⮚ The total amount of polyphenols was estimated using a calibration curve with
gallic acid. The results are expressed in milligrams.
gallic acid equivalents (mg GAE) per gram of extract (mg GAE/g extract).

Figure II.3: Determination of total polyphenols in E. alata stems (n=4).

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 Materials and methods

1.2.2. Flavonoid assay

a. Principle

Flavonoids are quantified using a colorimetric method described by (Dewanto et al.,

2002). The principle of the method is based on the oxidation of flavonoids by two
colourless reagents, sodium nitrite and aluminium chloride. This leads to the formation of a
brownish complex that absorbs at 510 nm.

b. How it works
The method cited in (Tigrine and Kameli, 2021) was used to quantify the total
flavonoids present in the ethanolic extract of E. alata stems:
⮚ In this experiment, 1000 μL of t h e extract solution (100 μ g / m L ) was
mixed with 1000 μL of 2% aluminium trichloride (AlCl3) solution prepared
in
methanol, in a test tube.
⮚ A blank was prepared containing a mixture of AlCl3 and ethanol.
⮚ The experiment was repeated three times. The tubes were incubated at
room temperature for 10 minutes.
⮚ Optical density was measured at a wavelength of 430 nm.
Flavonoid concentration was calculated from the standard curve of
quercetin, and the results were expressed as the number of mg quercetin equivalent
for each gram of extract.

2.2.3. Phytochemical screening

In order to highlight the presence or absence of certain compounds belonging to the

chemical families of secondary metabolites, we carried o u t specific phytochemical tests
based on colour, turbidity or precipitation reactions, using the methods described in the
literature (Chaouche et al., 2011). Phytochemical tests carried out on the variety are used to
search for bioactive substances (primary or secondary metabolites). They are carried out
either on the powder or on the 10% infusion. The characterisation methods used are based
on those described by Harborne and Raaman. The metabolites sought are total tannins, gall
tannins, mucilage, leuco-anthocyanins, saponosides, anthocyanins, carotenoids, starch,
glucosides, proteins, reducing sugars, flavonoids, senosides, free quinones, alkaloids and
lipids (Harborne et al., 1998; Raaman, 2006).

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 Materials and methods

We chose the method of Shaikh and Patil in order to carry out this applied work, and it
is presented in a table so that all spies are carried out with an extract at the concentration
of

1 μg/mL (Table Ⅱ.1).

Table Ⅱ.1: Phytochemical screening experiments

Phytochemical compounds Protocols Observation

sought
Proteins Biuret reaction 1 ml Na OH + 1ml extract (1 Colour change to violet.
μg/mL) + 5 drops of CU SO4
Amino Ninhydrin 1 ml of extract + 3 drops of reagent Appearance of
acids reaction Put Ninhydrin in a bain-marie for 1 purple colouring.
min.
Molish reaction 2 ml extract + 3 drops α naphthol + Appearance of a purple ring
Primary metabolites

1 ml sulphuric acid H2SO4.

Benedict's 1 ml extract + 1 ml Benedict's reagent Appearance of a brick-
reaction (Na OH + CU SO4) Heat in a water red precipitate.
bath for 1
min.
Reaction 1 ml extract + 1 ml Barfoed reagent, 1 ml extract + 1 ml Barfoed
from heated for 2 min. reagent, heated for 2
Barfoed min.
Iodine reaction 1 ml of extract + 1 drop of iodine Appearance of a blue
diluted in distilled water. discolouration.
Test of 1 ml extract + 1 to 2 ml Dragendroff A reddish-brown precipitate
Dragendroff reagent.
Mayer test 1 ml of extract + 1 to 2 drops of the A creamy white precipitate
Secondary metabolites

Mayer reagent. yellow.

Bouchardat test 6 ml of extract + a few drops of A reddish-brown colour.
Bouchardat reagent (dilute iodine
solution).
Saponins Saponin test 1 ml extract + 5 ml distilled water, Appearance of foam.
well shaken.
Flavonoids Shinoda test 100 μl of extract + 1 ml of ethanol + Pink solution.
Magnesium ribbon fragment + a few
drops of HCL.

27
 Materials and methods
Phytosterols Salkowski test 1ml extract + a few drops of A red precipitate.
H2SO4

28
 Materials and methods

2.3. Antioxidant activity

2.3.1 β-carotene bleach test

In this test, antioxidant capacity is determined by measuring the inhibition of volatile

organic compounds and conjugated peroxide radicals resulting from the oxidation of
linoleic acid (Tepe et al., 2005).
In another way, this test is based on the extract's ability to inhibit the formation of
conjugated diene hydroperoxide during the oxidation of linoleic acid.

⮚ Briefly, 0.5 mg of β-carotene was dissolved in 1 ml of chloroform, 25 μ l

of linoleic acid and 200 mg of Tween 40 were added.
⮚ The chloroform was completely evaporated using a steam extractor at 45°C.
Next, 100 ml of distilled hydrogen peroxide was added with stirring.
⮚ In test tubes, 2.5 ml of this reaction mixture called "β-carotene/linoleic
acid emulsion" was added to 0.35 ml of ethanolic extract (2
mg/ml).
⮚ The same procedure with the positive controls: BHT and vitamin C, the blank
(emulsion without β-carotene and without extract) and the positive control
which
contains the emulsion and the solvent.
⮚ The experiment was repeated four times. The tubes were shaken
and incubated in a water bath at 50°C.
⮚ Readings were taken at 490 nm every 30 min for 2 hours, starting before incubation
(t0, t30, t60, t90, t120).
⮚ The oxidation of the previous emulsion is monitored by measuring the absorbance of
each sample immediately after preparation (t=0 min) and at intervals of 30 min
until the end of the experiment (t=120 min) at 490 nm. Antioxidant activity is
expressed as a percentage of inhibition compared with the control according to the
following equation:

AA(%) = (1-[A (t=0)- A(t=120)/ A0(t=0) - A0(t=120)]) ×100

AA: Antioxidant activity

A(t=0): absorbance of extract at 0 min, A(t=120): absorbance of extract at 120 min
A0(t=0): absorbance of control at 0 min, A0(t=120): absorbance of control at 120 min

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 Materials and methods

Figure II.4: The β-carotene bleach test before incubation.

2.4. Anti-inflammatory activity in vitro

Inhibition of protein denaturation
a. Principle
Denaturation affects almost all the physico-chemical properties of molecules, varies
considerably with the various physical and chemical agents that cause it and also with the
character and concentration of protein solutions (Mizushima and Kobayashi, 1968). This
denaturation is often associated with inflammation, so inhibition of protein denaturation
has been widely used as an in vitro screening model for assessing anti-inflammatory
activity (Chaiyana et al., 2016).

b. How it works
The in-vitro inhibitory effect of E. alata gum extracts was determined using the
method described by (Habibur et al., 2012).

c. Carrying out the test

Solutions of 0.5 ml were prepared, consisting of 0.45 ml of 2% BSA solution and 0.05
ml with a concentration of 500µg/ml of the aqueous extract and standard diclofenac, which
is an anti-inflammatory drug. The samples were incubated at 37°C in a water bath for 20
min, then at 57°C for 30 min. After the samples had cooled, 2.5 ml of phosphate buffer (pH
=6.3) was added to each tube. The control was prepared without extract. The turbidity of
the albumin solution was monitored by reading the absorbance at 660 nm. The percentage
inhibition was calculated using the following formula:

𝑝𝑜𝑢𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒𝑑′𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 % = (𝐴𝑏𝑠𝐶- 𝐴𝑏𝑠𝑇 / 𝐴𝑏𝑠𝐶) × 100

30
 Materials and methods

Abs C: Control absorbance.

Abs T: Test absorbance.

2.5. Antifungal activity

This study was carried out at the LBSM laboratory at ENSK. The antifungal activity
of the extract was determined using the diffusion method (disc method) in an agar medium
(Rasooli et al., 2008).
a. Principle
The aromatogram is a method inspired by the antibiogram which can be used to
determine the growth inhibiting activity of plant extracts (Chapuis., 2007). The principle of
this method is based on the power of migration of the essential oil or aqueous extract by
diffusion in the agar (Bauer et al., 1966). This method enables the antibacterial effect of an
extract to be demonstrated by measuring the inhibition diameter in millimetres around a
disc impregnated with an extract (Khebichat et al., 2013). Depending on the inhibition
diameter, the strains studied can be classified as sensitive or resistant (Fig II.5).

Figure 1.5: Principle of the disc method or aromatogram.

⮚ Preparation of fungal suspensions

The fungal strains to be tested are inoculated into Petri dishes containing nutrient agar,
then incubated at 28°C for 4 to 5 days. From these young cultures, using a sterile platinum
loop, 3 to 5 well-isolated colonies, perfectly

31
 Materials and methods

The identical samples were taken and the platinum loop was then discharged into 10 ml of
sterile physiological water containing 80% NaCl. The fungal suspensions are then
thoroughly homogenised using a vortex.

⮚ Preparing the discs

Using a cookie cutter, 6mm diameter discs of Wattman N⁼°40 paper are made. These discs
are then autoclaved in a sterile test tube.
b. How it works
• Seeding
The semi-solid PDA medium is poured into Petri dishes at a rate of 15 to 20 ml in each
dish 5 of a physiological solution containing fungal species are added using micropipettes.
After solidification of the culture medium,
• Disc deposits
Under aseptic conditions, sterile Wattman paper discs 6mm in diameter are placed on the
previously seeded agar surface using sterile forceps. Each disc was impregnated with 5μl
o f crude E. alata extract. All the tests, including the controls, were repeated 02 times
under the same experimental conditions (Fig II.6).

Figure II.6: Image showing how to place the dilutions.

• Incubation:
Petri dishes were kept at 4°C for 1 hour to allow the
extrac
t to diffuse, then incubated at 28°C for 4 days and Candida albicans at 37°C for 4 days.

32
 Materials and methods

• Expressing results
The results are read by measuring the inhibition diameter formed around the disc in
millimetres where the microorganisms tested did not grow as an average (El Hanbali.,
2009).

FigureII.7: Petri dishes before incubation.

⮚ Preparation of dilutions
F r o m a stock solution of the extract at a concentration of 100 mg/ml, we have
prepared a range of dilutions as follows (Fig II.8):
⮚ We take 1ml of crude extract.
⮚ To this is added 1 ml of sterile distilled water, giving the first solution with a
concentration of 50 mg/ml (a).
⮚ Take 1ml of solution a and add 1ml of sterile distilled water to obtain the
second concentration b (b=25mg/ml).
⮚ 1ml of sterile distilled water is added to 1ml of extract with a concentration of
25mg/ml, to obtain a new concentration of extract C(c=12.5mg/ml).
⮚ In the same way as above, we obtain an extract with a new
concentration (d=6.25mg/ml) (Fig II.8).

Figure II. 8: Dilutions of E. alata extract.

33
 Materials and methods

⮚ We tested these dilutions with the antibiogram (Fig II. 9).

Figure II. 9: Petri dishes before incubation.

34
 Results and discussion

22
 Results and discussion

Chapter 03:
Results and discussion
 Results and discussion

III. Results and discussion

1. Extraction yield

The yield of the ethanolic extract of E. alata represents the ratio between the weight of
the crude extract (18-18.5 g) and the weight of the dry plant matter (50 g). It is expressed as a
percentage according to the following formula:

Knowing that:
Pext : Weight of crude extract in grams (g).
Psèche : weight of dry plant matter in grams (g).

The yield of the ethanolic extract of E. alata was estimated at 36.5 ± 0.5 %. This value is
considered a good yield in comparison with other small studies on the same plant. For
example, Benarba et al (2021) obtained a yield of 15% from a methanolic extract. This
difference can be attributed to several factors, such as the nature of the solvent used in the
extraction, the plant species, the organ used, the drying conditions and the metabolic
composition specific to each plant species. Other factors also play an important role in
explaining this difference, such as pH, temperature, the ratio of the quantity of material to the
volume of solvent, time intervals, etc.

2. Phytochemical studies

2.1. Phytochemical screening

Phytochemical screening showed that the ethanolic extract of E. alata is fairly rich in
secondary metabolites, namely alkaloids, flavonoids, saponins and phytosterols. As far as
primary metabolites are concerned, it contains only reducing sugars (Table III.1).

35
 Results and
discussion

TableIII.1: Results of phytochemical screening of ethanolic extract of E. alat

 Results and
discussion
2.2. Total polyphenol content
The polyphenol content of the ethanolic extract of E. alata was determined by the
addition of Foline Ciocalteu reagent and sodium carbonate. The appearance of a blue
colour proves the richness of this species in phenolic components (Fig.III.1).

Figure III.1: Transformation of the colour from yellow to blue is due to the presence of
polyphenols in the ethanolic extract of E. alata:
[1] 50µg/ml and [2] 100µg/ml).

The quantitative analysis of total polyphenols was determined from the linear
regression equation of the calibration curve, plotted using gallic acid as the standard. The
values obtained are expressed as mg gallic acid equivalent per g of extract (mg GAE/g
extract). The quantities of polyphenols are determined by the equation: y = 0.109 x - 0.055
(Fig.III.2).

The assay results show that the extract is rich in polyphenols with a concentration of
216.5mg EAG/g crude extract. This value is higher than that found by Jaradat et al. (2015)
whose value was 54.66 mg EAG/g dry extract as well as that found by Danciu et al. (2019)
and Ibragic and Sofic (2015) whose values were 156.22 mg EAG/g and 53.3 mg EAG
respectively. The use of different extraction solvents has an impact on the total amount of
polyphenols (TPC) present in Ephedra extracts, leading to significant variations (Al-
Rimawi et al., 2017).
 Results and
discussion

FigureIII.2: Gallic acid calibration line for the quantitative determination of

total polyphenols contained in E. alata crude extract.

The assay results show that the extract is rich in polyphenols with a concentration of 141.9 ± 7.2
mg EAG/g crude extract.

2.3. Flavonoid assay

Total flavonoids were determined by the aluminium trichloride method (AlCl3 ) and
flavonoid content was expressed as milligram quercetin equivalent per gram extract (mg
EQ/g extract).
After the addition of aluminium trichloride (AlCl3 ), a yellow colour appeared,
confirming the presence of flavonoids in the extract (fig.III.3).

Figure III.3: Colour change from transparent to yellow due to the presence of flavonoids in
the ethanolic extract of E alata (100µg/ml).

The quantity of total flavonoids i n E alata extract was assessed using the aluminium
trichloride method.
 Results and
discussion
The content of total flavonoids in the two concentrations of extract was calculated using
the linear regression equation of the calibration curve established with quercetin, expressed
in mg quercetin equivalents per g extract mg EQ/g extract (fig.III.4).

FigureIII.4: Quercetin calibration line for the quantitative determination of total

flavonoids in E alata crude extract.

The assay results show that the extract is rich in flavonoids with a concentration of 18
mg EAG/g crude extract. According to the previous results, the E. alata plant is rich in
flavonoids compared to other species of the genus E. phedra. According to Jaradat et al.
(2015) the total flavonoid content was around 54.66 mg REU/g. Another recent study on
the same plant (Al-Rimawi et al., 2017) revealed that E. alatacon contains flavonoids with
a concentration of 19.5 mg catechin/g. Despite the large amount of flavonoids found in this
study, it remains a lower value than previous studies and this is probably due to ethanol
which has a low extraction capacity compared to methanol (Danciu et al., 2019).

3. Antioxidant activity

3.1. Beta-carotene bleach test

The β-carotene bleaching test was carried out to determine the protective effect of E.
alata extract against heat-induced linoleic acid degradation and oxidation, which causes β-
carotene oxidation and discolouration (Fig.III.5).
 Results and
discussion

FigureIII.5: Prevention of β-carotene bleaching in the presence of ethanolic extract of E

alata and standard antioxidants: BHT and V.C.

Polyunsaturated fatty acids, such as linoleic acid, are easily oxidised by atmospheric
oxygen. This autooxidation leads to chain reactions with the formation of coupled double
bonds, and later to secondary products such as aldehydes, ketones and alcohols (Berk et al,
2011). In the β- carotene-linoleic acid system, β-carotene undergoes rapid discolouration in
the absence of antioxidant. The presence of antioxidants (such as phenolics) can 'neutralise'
the 'linoleate' free radical and any other free radicals formed in the system, and negatively
affect the extent of β-carotene damage (Tepe et al, 2011).

The kinetics of β-carotene bleaching and its inhibition by ethanolic extract of E alata,
BHT and vitamin C, show that they significantly (p ˂ 0.05) inhibit the coupled oxidation of
linoleic acid/β-carotene compared with the positive control (Fig.III.6). Indeed, the results
show that the antioxidant activity of BHT was measured at 72.6% close to the ethanolic
extract estimated at 67.4%. However, the effect of vitamin C seems to be weak with a
percentage of 8.1% (Fig.III.7).
 Results and
discussion

FigureIII.6: Bleaching kinetics of β-carotene in the presence and absence of

ethanolic extract of E. alata, BHT and vitamin C. Values represent the means of
4 trials ± SD.

FigureIII.7: The percentage of antioxidant activity of ethanolic extract of E alata, BHT

and vitamin C after 120min. E alata extract protected β-carotene from bleaching. Values
represent means of 4 trials ± SD.
 Results and
discussion
4. Anti-inflammatory activity in vitro

Protein denaturation has been correlated with the development of inflammatory

disorders. Therefore, the ability of a substance to prevent protein denaturation is an
important step in the development of potential anti-inflammatory drugs (Rjeibi et al.,
2020). In the present study, the ability of the ethanolic extract of E. alata to block the
thermal denaturation of albumin (BSA) was explored. As shown in Figure x, ethanolic
extract of E. alata and diclofenac sodium inhibited heat-induced albumin denaturation.
However, ethanolic extract of E. alata exerted a moderate inhibition which was estimated
at (30.09±1.52)% compared to diclofanac which exerted an almost complete inhibition
with a percentage of 96.99 ± 0.26 (Fig.III.8).

FigureIII.8: Inhibitory effect of BSA denaturation by ethanolic extract of E. alata

(500µg/ml) and diclofenac sodium (500µg/ml).

A recent study on the same species showed that the methanolic extract of the pulp
had an inhibitory effect of 82.2%, which was similar to the effect of the standard anti-
inflammatory agent indomethacin, with a percentage inhibition of 82.7% (Mufti et al.,
2023). Another study showed that Ephedra nebrodensis extracts could protect proteins
against denaturation (Hamoudi, 2021). In the same context, various medicinal plant
extracts have been evaluated for their ability t o inhibit protein denaturation (Osman et al.,
2016; Dharmadeva et al., 2018; Gunathilake et al., 2018). In addition, inhibition of BSA
denaturation has been suggested to be responsible for the anti-inflammatory effects of
various NSAIDs, such as diclofenac
 Results and
discussion
sodium, salicylic acid, indomethacin and flufenamic acid (Alamgeer et al., 2017).

5. Antifungal activity

In this study we used the agar diffusion technique to test the antifungal potential of E.
alata extract on a group of fungi: Aspergillus niger. A. carbonarius M333, A. parasiticus,
A. brazilian ATCC 16404, A. flavus and candida albicans.
The results obtained were negative, with no zone of inhibition in any of the E. alata
concentrations tested (from 100 mg/ml (crude ethanolic extract of E. alata) - 0.5 mg/ml
(crude ethanolic extract of E. alata) to 0.5 mg/ml (crude ethanolic extract of E. alata).
6.5mg/ml) (Fig.III.9- Figure III.10). On the other hand, it was found that there was
activity for this plant with the solvent acetonitrile, (Ghanem and El-Magly 2008).
Ghanem and El-magly (2008) explain the high sensitivity of A. fumigatus to one of the
chemical groups present in the acetonitrile fraction, even at low concentrations compared
to the other fractions. In addition, the ethanolic extract and the aqueous extract showed
remarkable antifungal activity against A. fumigatus.
The results of Hibi et al (2022) show that the aqueous extract of E. alata exerts an
inhibitory activity on all the strains tested with a variable percentage of inhibition: 64.44%
on A. ochraisus. 60% on A. flavus and 55.55% on A. niger. On the other hand, methanoic
and ethyl acetate extracts had no activity against these species.

FigureIII.9: Results of inhibition of the antifungal activity of the crude extract of

E.alata, after five days of incubation.
In other studies on the antifungal activity of E. alata dissolved in methanol, it was
found that there was anti-Candida albicans activity and that the inhibition diameter was 10
mm (Danciu et al., 2019).
 Results and
discussion

Figure III.10: Antifungal activity inhibition results, using dilutions of E. alata

extract (50mg/ml; 25mg/ml; 12.5mg/ml and 6.25mg/ml), after three days
incubation.
Alqarawi et al., 2011 highlighted the inhibitory effect of E. alata plant extract on
nucleic acid synthesis in A. flavus. A comparison of the growth inhibition of crude extracts
and their respective dilutions shows a stronger dependent effect of extract concentrations.
In general, the antifungal activity of the extract dilutions was weaker than that of the crude
extracts. These results revealed that the antifungal activity of the crude extracts was indeed
enhanced by increasing the extract concentration. The inhibitory activity of the extracts
was concentration dependent. This finding is in agreement with the report by Banso et al
(1999), who also observed that higher concentrations of antimicrobial substances resulted
in greater growth inhibition.
Research was carried out to evaluate the effect of the E. alata range plant as a biological
strategy for inhibiting the growth traits and aflatoxin production (in vitro and in vivo) of A.
flavus (Alqarawi et al., 2011). The results showed that E. alata aqueous extract had
significant inhibitory potential against growth as well as aflatoxin p r o d u c t i o n by the
seed-borne aflatoxigenic mould (A. flavus). In addition, it was found that the addition of 1
and 2% (w/w) E. alata plant powder to maize and soya beans respectively reduced
aflatoxin contamination and improved their nutritional value (total nitrogen content, fibre
content), total lipid content and ash content under storage conditions. The results observed
here suggest the use of E. alata as an alternative non-chemical means of controlling
aflatoxin contamination of fodder in Saudi Arabia. The results of t h e antifungal
activity of the various E. alata extracts revealed that the intensity of this activity varied
according
t o several factors, including the solvent used, the extraction method and the strain studied.
 Results and
discussion

Conclusion
 Conclusion

Conclusion

In recent years, there has been growing interest in the use of natural antioxidants and
antimicrobials. Many researchers have been interested in biologically active compounds
isolated from plant extracts.

The primary objective of this study is in the same context. We are interested in
evaluating the antioxidant, anti-inflammatory and antifungal activity of ethanolic extracts
of E. alata. Antioxidant activity w a s carried out using the β-carotene bleach test and the
chelation test, anti-inflammatory activity by inhibiting protein denaturation, and antifungal
activity by the antibiograme technique. The present study gave satisfactory results on the
extraction and phytochemical screening of ethanolic extracts of E. alata. The results
showed that the phytochemical screening of the aqueous extracts of E. alata revealed a
richness in secondary ethanolic molecules such as saponosides, tannins, flavonoids,
glycosides, proteins, triterpenoids and alkaloids; these indicators varied greatly depending
on the technique applied and whether or not the ethanolic extracts were rich in
phytochemical molecules.

Evaluation of the anti-inflammatory activity showed that ethanolic extracts of E. alata

have a strong anti-inflammatory potential. As for the antifungal activity, there was no
activity against the species strains tested.

As far as the antifungal results are concerned, our results are in line with several studies on
the plant, which have shown that organic extracts (methanoic, ethanolic, etc.) have no
activity against the champion species tested. This preliminary study has led us to change
the extraction method in future studies.

In general, according to the results obtained, ephedra can be considered as a treatment

for cancer and other traditional uses, thanks to its great capacity as an antioxidant and anti-
inflammatory.

46
 Appendices
Appendix 1: Table of biochemical and microbiological equipment and materials

Refrigerator, freezer, hot plate, bain-marie, Bunsen

burner, vortex, spectrophotometer, autoclave, precision
Equipment
balance, digital balance, testing device, etc.
pump filtration, steam-rotation, proofer.
PDA
Culture media
Physiological water, Distilled water, Gentian violet,
Lugol's, Alcohol, Fuchsin, Immersion oil, Glycerol,
DMSO, Antibiotic discs, 2% aluminium trichloride
(AlCl3), Iodine, Ninhydrin reagent, reagent, Benedict's
reagent, Barfoed's reagent, Dragendroff's reagent,
Reagents and solvents
Mayer's reagent, Bouchardat's reagent, magnesium,
Sulphuric acid (H2SO4), HCL, ethanol and methanol,
Sodium carbonate (Na2CO3) 20%, Benedict's reagent
Folin-Ciocalteu, linoleic acid, BHT, β-carotene,
Tween 40, Whatman paper, Agar.
. Swabs, beakers, test tubes, pasteur pipettes, Petri dishes,
micropipettes (10 to 100 μl and 1000μl), forceps,
graduated cylinder, rack, brown flasks, Erlenmeyer flasks,
Glassware and blue and yellow and transparent tips, Eppendorf, sterile
small
equipment dry tubes, cuvettes, funnel, pissette, test tube rack,
graduated cylinder, flask.
volumetric flask, round-bottomed flask.

Appendix 1: Preparation of the PDA.

The potato infusion was prepared by boiling 200g of potato slices (washed but
unpeeled) in a litre of water for 30 minutes at 1

47
hour. The resulting broth filtrate is then diluted with distilled water to a final volume of
one litre. To this is added 12 g of dextrose and agar powder before autoclaving at 100 kPa
for 15 minutes.

48
References
 1. References

2. Abourashed E.A., Al-Alfy T., Khan A., Walker L. (2003). Ephedra in perspective-
acurrentre view. Phytotherapy Research, 17(7),703-712.
https://doi.org/10.1002/ptr.1337.

3. Alexopoulos J., Mims W. (1979). Introductory Mycology: 1-613. Cited by Briki

Khaoula ,2013.

4. Al-laith A. (2010). Antioxidant components and antioxidant/antiradicall activities of

deserttruffle (Tirmanianivea) fromvarious Middle Easternorigins. J. Food Compos.
Anal. 23(1): 15-22.

5. Al-qarawi A., Abd Allah F., Abeer H. (2011). Ephedra alata as biologically-based
strategy inhibit aflatoxigenic seedborne mold. African J. Microbiol. Res. 5:2297-
2303.

6. Al-rimawi F., Abu-lafi S., Abadi J., Alamarneh A., Sawahreh R., Odeh I. (2017).
Analysis of phenolic and flavonoids of wild ephedra alata plant extracts by lc/pda and
lc/ms and their antioxidant activity. African journal of traditional, complementary and
alternative medicines, 14(2), Art. 2.
https://doi.org/10.21010/ajtcam.v14i2.14

7. Al-snafi, A. (2017). Therapeutic importance of Ephedra alataand ephedra e-a

review. Indo Am J P Sci, 4, 399-406.
https ://doi.org/10.5281/zenodo.376634

8. Barros D., Torres A., Chulze S. (2005). Aspergillus flavus population isolated
fromsoil of Argentina speanut-growing region. Sclerotia production and
toxigenicprofile. J. Sci. Food. Agric. 85 :2349-2353.

9. Bauer A W., Kirby M M., Sherris J C., Turck M. (1966). Antibiotic susceptibility
testing by a standardized single disk method. American journal of clinicalpathology,
45 (4_ts), 493.

50
10. Benarba B., Douad O., Gadoum C., Belhouala K., Mahdjour S. (2021).
Phytochemical Profile, Antioxidant and Anti-Inflammatory Activities of Ephedra
alata Decne Growing in South Algeria.
https ://doi.org/10.20944/preprints202108.0296.v1

11. Bouafia M., Amamou F., Gherib M., Benaissa M., Azzi R., Nemmiche S. (2021).
Ethnobotanical and ethno medicinal analysis of wild medicinal plants traditionally
used in Naâma, southwest Algeria. Vegetos, 34(3), 654-662. https
://doi.org/10.1007/s42535-021-00229-7.

12. Boubekri A., Ababou M., Kartit N., Doghmi N., Bakkali H. (2020) Ephedra alata
intoxication ( about a case). PAMJ Clinical Medicine, 3. https
://doi.org/10.11604/pamj-cm.2020.3.120.23391.

13. Boudjida L., Halit-Sahnoun R. (2017) Intérêt de l'électrophorèse capillaire dans le

diagnostic du syndrome inflammatoire (Doctoral dissertation, Université Mouloud
Mammeri.

14. Benaissa R., Boukhari L. (2018) Etude antibactérienne des extraits phénoliques de la
farine de la pulpe de caroube Cerratonia Siliqua ,.Master 2 :Science de la nature et de
la vie,. Université, Saad Dahleb Blida ,1p.

15. Cahagnier B. (1997) Moisissures des aliments peu hydraté; technique et

documentation; Lavoisier; Paris: 405p.

16. Carlile M. (1995) The Success of the Hypha and Mycelium. In The Growing Fungus
SE 1 pp. 3-19. 978-0-412-46600-7.

51
17. Danciu c., Muntean D., Alexa E., Farcas C., Camelia O., Istvan Z. ( 2019)
Phytochemical Characterization and Evaluation of the Antimicrobial,
Antiproliferative and Pro-Apoptotic Potential of Ephedra alata Decne.
Hydroalcoholic Extract against the MCF-7 Breast Cancer Cell Line, Molecules, 24,
13, 4p.

doi :10.3390/molecules24010013

18. Habibur R., Chinnaeswaraiah M., Vakati K., Madhavi P.(2012) In-vitro studies
suggest probable mechanism of Eucalyptus oil for anti-inflammatory and anti-
arthritic activity. International Journal of Phytopharmacy, 2(3) : 81-83.

19. Hadjadj K., Belkacem B., Guerine L. (2020) Importance thérapeutique de la plante
Ephedra alata subsp. Alenda dans la médecine traditionnelle pour la population de la
région de Guettara ( Djelfa, Algérie). Le jeunai, Revue de Botanique. https
://doi.org/10.25518/0457-4184.1956

20. Hajjaj G. (2017) Phytochemical screening, toxicological study and pharmacological

valorisation of Matricaria chamomilla L. and L'ormenis mixtaL. (asteraceae). PhD
thesis, Mohammed V University, Morocco. 216.

21. Hamoudi M., Amroum D., Baghiani A., Khennouf S., Dahamna S. Antioxidant,
Anti-Inflammatory, and Analgesic Activities of Alcoholic Extracts of Ephedra
Nebrodensis from Eastern Algeria. Turk. J. Pharm. Sci. 2021, 18, 574-580.

https://doi.org/10.4274/tjps.galenos.2021.24571.

22. Harborne A.J. (1998) Phytochemicalmethods a guide to modern techniques of plant

analysis. Springer science & business media.

23. Hegazi, G., El-lamey, T. (2011) In vitro production of somephenolic compounds

from Ephedra alata Decne. I. Applied Environmental and Biological Science, 1,
158- 163.

52
24. Hibi Z., Makhlouf A., Azzi R. (2022) Ethnobotanical, phytochemical characterization
and biological activities of Ephedra alata Decne extracts, growing wild in Bechar
region, south west of Algeria. South Asian Journal of Experimental Biology, 12(1), 35-
45

25. Horn B., Dorner J.(1998) Soil populations of Aspergillus species from section Flavia
along a transect through peanut growing regions of the United States.

States. Mycologia, 90, 767-776.

26. Houba Z., Himeur H. (2019) Contribution to the phytochemical and biochemical
study (In vitro and In vivo) of female cones of Ephedra alata DC. From the Oued
Souf region.
https ://www.semanticscholar.org/paper/Contribution-%C3%A0-l'%C3%A9tude-
phytochimique-et-biochimique-Houb

27. Jerbi A., Zehri S., Abdnnabi R., Gharsallah N., Kammoun M. (2016) Essential
Oil Composition, Free-Radical-Scavenging and Antibacterial Effect from Stems
of Ephedra alata alenda in Tunisia. Journal of Essential Oil Bearing Plants.19 (6),
1503-1509

28. Khebichat A. (2013) Evaluation de l'activité antibactérienne et antifongique des

cendres de bois du chene vert " Kourriche ou Ballot " (Quercus ilex).mémoire de
master 2 : BIOCHIMIE Appliquée.Université Abou Bekr Belkaid-Tlemcen, 32p.
29. Kmail A., YoussiI B., Zaib H., Imtara H., Saad B. (2017) In vitro evaluation of anti-
inflammatory and antioxidant effects of Asparagus aphyll us L., Crataegus azarolus
L., and Ephedra alata Decne.in monocultures and co-cultures of HepG2 and THP-1-
derived macrophages. Pharmacogn. Commn, 7(1):24-33p.

30. Kulawik P., Özogul F., Glew R., Özogul Y. (2013) Significance of antioxidants for
seafoodsafety and humanhealth. J. Agric. Food Chem. 61(3) :475-491.

53
31. Mizushima Y., Kobayashi M.(1968) Interaction of anti-inflammatory drugs with
serum proteins, especial ly with some biologically active proteins. J. Pharm
Pharmac, 20: 169-173.

32. Morin-Sardin S., Rigalma K., Coroller L., Jany L., Coton E. (2016) Effect of
temperature, pH, and water activity on Mucor spp. growth on synthetic medium,
cheese analog and cheese. Food Microbiology56, 69-79.

33. Nabti H., Boulberhane S., (2017) Phytochemical study and evaluation of antibacterial
activity and antifungal activity of the two plants Artemi siacompestris L. and
Ephedra alata alenda Staph: 92.

34. Orliaguet G, Gall O, Benabess-Lambert F. (2013) New developments in steroidal and

non-steroidal anti-inflammatory drugs. Le Praticien en Anesthésie Réanimation ;
17(5) : 228-23

35. Ozenda, P. (2004). Flora and vegetation of the Sahara. CNRS.

36. Ozenda P. (1991). Flora and vegetation of the Sahara. Centre National De La
Recherche Scientifique, Paris (3rd Ed.). 662 p

37. Palici F. (2016) Valorisation des activités biologiques de certaines espèces végétales
sahariennes nord-africaines [These de doctorat, Bordeaux]. https
://www.theses.fr/2016BORD0321.

54
38. Pilar P., Manuel M., Miguel A. Spectro photo metric quantitation of antioxidant
capacity through the formation of a phosphor molybdenum complex: specific
application to the determination of vitamin E. Analytical Biochemistry. 1999 ;
269(2) :337-41. http ://dx.doi.org/10.1006/abio.1999.4019 ; Pmid :10222007

39. Pitt J., Hocking A.(1997) (Eds) Fungi and foodspoilage. Blackie, London (1997)

40. Raman N. (2006) Phytochemical techniques. Ed. Publishing Agency, New Delhi,
306p.

41. Rashid K, Sinha K, Sil PC. (2013) An updateon oxidative stress-mediatedorgan

pathophysiology. Food Chem. Toxicol,62: 584-600.

42. Rasooli I., Fakoor M., Yadegarinia D., Gachkar L., Allameh A., Rezaei B. (2008)
Anti mycotoxigenic characteristics of Rosmarinus officinalis and Trachyspermum
copticum L. essential oils. International J of Food Microbiology.122 :135-139.

43. Rjeibi I., Ben Saad A., Ncib S. (2020) Brachychiton populneus as a novel source of
bioactive ingredients with therapeutic effects: antioxidant, enzyme inhibitory, anti-
inflammatory properties and LC-ESI-MS profile. Inflammopharmacol 28, 563-574
https://doi.org/10.1007/s10787-019-00672-8

44. Sabrina Krief. (2003) Métabolites secondaires des plants et comportement animal:
surveillance sanitaire et observations de alimentation des chimpanzees (Pan
troglodytes schweinfurthii) en Ouganda. Biological activities and chemical study of
consumed plants, MNHN PARIS, 238.

45. Samah G., Usama E. (2008) Antimicrobial Activity and Tentative Identification of
Active Compounds from the Medicinal Ephedra alata Male Plant,
EXPERIMENTAL STUDY, 3(1).

55
46. Segal E.(2005) Candida ,still number one -what dowe know and where are going
from there .Mycoses 48Suppl1,3-11

47. Soni M., Carabin I., Griffiths J., Burdock G. (2004) Safety of ephedra: lessons
learned. Toxicology Lettersn, Vol. 150, 97-110.

48. Stajich J., Berbee M., Blackwell M., Hibbett D., James T., Spatafora J., Taylor J.
(2009). The fungi. Current biology : CB 19, R840-5.

49. Tabuc C. (2007) Fungal flora of different substrates and optimal conditions for

production of mycotoxins. Thesis doct.Univ. Toulouse :190 pp.

50. Tepe B., Daferera D., Sokmen A., Sokmen M., Polissiou M. (2005) Antimicrobial and
antioxidant activities of the essential oil and various extracts of Salviato mentosa
Miller (Lamiaceae). Food chemistry, 90(3),333-340.

51. Urquiaga I., Leighton F. (2000) Plant Polyphenol Antioxidants and Oxidative Stress.
Biological Research, 33(2), 55-64.

https ://doi.org/10.4067/S0716-97602000000200004

52. Walther G., Pawlowska J., Alastruey-Izquierdo A., Wrzosek M., Rodriguez-Tudela,
J., Dolatabadi S., Chakrabarti, A., Hoog, G. (2013) DNA barcoding in Mucorales :
Aninventory of biodiversity, Persoonia: MolecularPhylogeny and Evolution of
Fungi30, 11-47.

53. World Health Organization (WHO) (2006). AFRO Food Saf. News, Issue No 2. July.
Food Safety (FOS).
56
54. Zycha H., Siepmann R., Linneman, G. (1969). Mucorales, Cramer. Keys (A revision
of Zycha). 355pp.

55. Patterson, Thomas F. (2016) Practice Guidelines for the Diagnosis and Management
of Aspergillosis, Clinical Infectious Diseases, 63, 15 August, 1-60.

56. Varga J., Kocsube S., Toth B., Frisvad J., Perrone G., Susca A., Meijer M., and
Samson. R. (2007) Aspergillus brasiliensis sp. Nov. a biseriate black Aspergillus
species with world-wide distribution, International Journal of Systematic and
Evolutionary Microbiology, 57, 1925-1932, p4.

2. Website
1. https://www.gettyimages.fr/photos/aspergillus-flavus

2. https://www.inspq.qc.ca/moisissures/fiches/aspergillus-niger

3. https://library.bustmold.com/fr/aspergillus/aspergillus-carbonarius

4. http://bioimagen.bioucm.es/foto/6524

5. https://www.istockphoto.com/fr/photo/mucor-mold-champignon-noir-champignons-
de-moule-%C3%A0-pain-illustration-3d-gm1328467090-412509211

6. https://www.istockphoto.com/fr/search/2/image-film?phrase=candida+albicans

57