HE - ANTIBACTERIAL PROPERTIES CERATONIA Artémisia

ACADEMIA Letters
ANTIBACTERIAL PROPERTIES OF CERATONIA

SILIQUA L ETHANOLIC EXTRACT AND ARTEMISIA
HERBA ALBA ESSENTIAL OIL
adjal fatima
guitone asma
almi sana
Abstract
Algeria, by its geographical location, offers rich and diverse vegetation including aromatic
and medicinal plants. Among these plants, our interest has focused on the Ceratonia siliqua L.
and artemisia herba alba for the medicinal and aromatic properties of its compounds (terpene,
flavonoid, and essential oil..).
The main objective of this work consisted to evaluate the antibacterial activity of the
ethanolic extracts and the essential oil of these plants, which are widely used in traditional
medicine, in the state of Biskra (Southeast Algeria), for their medicinal and therapeutic prop-
erties. Both Artemisia Herba Alba essential oil and Ceratonia Siliqua ethanolic extract were
used for determination of their minimal inhibitory concentration (MIC) and minimal bacteri-
cidal concentration (MBC).
The extraction of the essential oil of artemisia herba alba was performed by hydro-distillation
(Clevenger type) and had yielded 1.34% (m/m). The ethanolic extract was performed by a
soxhlet extractor with a yield value of 16.92%.
The results of the antibacterial activity against seven pathogenic bacteria (Escherichia
coli, Staphylococcus aureus, Pseudomonas aeruginosa, klebsiella oxytoca, Bacillus cereus,
listeria monocytogenes, Salmonella typhi) by the agar disk diffusion method; showed that the
Academia Letters, July 2021 ©2021 by the authors — Open Access — Distributed under CC BY 4.0
Corresponding Author: adjal fatima, f.adjal@univ-biskra.dz

Citation: Fatima, A., Asma, G., Sana, A. (2021). ANTIBACTERIAL PROPERTIES OF CERATONIA
SILIQUA L ETHANOLIC EXTRACT AND ARTEMISIA HERBA ALBA ESSENTIAL OIL. Academia
Letters, Article 1943. https://doi.org/10.20935/AL1943.
1
essential oil of artemisia herba alba has a very important antibacterial activity compared to
ceratonia siliqua ethanolic extract
Keywords: Artemisia Herba Alba; Ceratonia Siliqua; Essential Oil; Ethanolic Extract;
Antibacterial Activity
1. INTRODUCTION
In recent years there has been a growing interest in researching and developing new antimicro-
bial agents from various sources to combat microbial resistance. Therefore, greater attention
has been paid to the screening of antimicrobial activity and its evaluation methods.
In traditional medicines, medicinal plants have been used since ancient times in the treat-
ment of various diseases and health disorders. Among these plants we mention, Artemisia
Herba Alba (wormwood or chih in Arabic) and ceratonia siliqua (kharoub in arabic).
The wormwood plant treats digestive diseases, as the parts of the plant are used without
the roots to solve stomach problems, including stomach discomfort, diarrhea, constipation,
gastrointestinal cramps, poor digestion, abdominal worms problems, and persistent vomiting
[1]. Also, this plant helps in treating bronchitis even children by applying olive oil to the
patient from the side of the chest and adding plant powder.
Several scientific studies have also proven the efficacy of Artemisia herba alba as an anti-
diabetic [2], anti-parasitic, antiviral, antipyretic, antispasmodic [3-5], and antioxidant agent
[6].
Carob plant helps absorb fluids in the intestine in particular, and the digestive system in
general, and it also helps to get rid of diarrhea in children, and it is recommended to feed it
to young children; Where carob powder is placed with any other fruit, such as apples, this
method is very useful for diarrhea, whether in young or old. Helps get rid of stomach acidity;
Because it is alkaline; Where the carob fruits are crushed after raising the seeds from them,
then they are soaked in water, boiled for a period of not less than ten minutes, and three spoons
are taken before eating each meal or when feeling sour. Carob is a de-worming and diuretic.
Several studies have shown that ceratonia has an activity Antimicrobial and Antioxidant
[7, 8], Anti-diabetic, Anti-inflammatory [9].

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2. DETAILS EXPERIMENTAL
2.1. Materials and Procedures
Plant material
The aerial parts of A. herba-alba and Ceratonia siliqua L, were collected in the Biskra region,
South east Algeria, collected in mai 2017,
Carob tree
The carob tree (Figure 1) is an evergreen sclerophyllous tree or shrub, which can reach 7 to
20 m in height and a circumference at the base of the trunk of 2 to 3 m. It is honey, its honey
is good. It has a smooth, gray bark when the plant is young and brown, rough in adulthood.
Its reddish wood is very hard. The carob tree can live up to 200 years [10]
Family: Fabaceae, Genus: Ceratonia,
Algerian vernaculars: elkharoub,
Kingdom: Plantae
Class: Magnoliopsida
Order: Fabales
Family: Fabaceae (Leguminosae)
Genus: Ceratonia
Species: C. siliqua
Subspecies: C. siliqua
Scientific Name: Ceratonia siliqua L.
Algerian vernaculars: elkharoub

3
Fig. 1. Ceratonia siliqua L.

4
Artemisia herba alba
White mugwort “Artemisia herba alba” (Figure.2), is a herbaceous plant with woody, branched
and tomentose stems 30 to 50 cm long. The leaves are short, sessile, pubescent and silvery.
Fig.2. Artemisia Herba Alba
The flower heads are grouped in small, elongated, narrow 1.5–3 mm panniculus containing
3-6 yellowish flowers. The outer bracts of involucre are orbicular and pubescent [11,12]. The
composition and yield of an H.E varies depending on the part of the plant from which it is
extracted [13].
Kingdom: Plantae
Order: Asterales
Family: Asteraceae
Species:A. herba-alba
Genus: Artemisia
Algerian name: chih

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Essential oil Extraction:
The extraction of the essential oil of Artemisia herba alba was performed by hydrodistillation
using a Clevenger apparatus.
Extraction of phenolic compounds
The dried powder of bulbs parts of ceratonia silqua (100g) were extracted with absolute
ethanol for 6 hours using soxhlet apparatus. The solvent was removed under reduced pressure
using a Bushi rotavapor.
Antibacterial activity
The bacteria were cultivated nutrient agar (NA) at 37 ± 0.2°C.Susceptibility of the tested
organism to the extracts was determined by employing the disc-diffusion method [14]. To
study the antibacterial activity of Artemisia herba Alba essential oil and Ceratonia siliqua L
ethanolic extract, we use the method of diffusion assay, for seven bacterial strains (Escherichia
coli, Staphylococcus aureus, Pseudomonas aeruginosa, klebsiella oxytoca, Bacillus cereus,
listeria monocytogenes, Salmonella typhi )
The bacterial sample was suspenssed for 18 to 24 hour to each one of the bacteria and
prepared with physiologique water (NaCl).
The sterile medium (15ml) in petri dishes was uniformly smeared with Mueller Hinton,
where four discs (6mm diameter) were placed in the sides of each Petri dish, to which
the extracts (Essential oil and ethanolic extract) were added with a different concentration.
Then the sample was incubated to 24 hour at 37°C
3. RESULTS AND DISCUSSION

The calculation of the yield R in percent % is then carried out according to the formula:
R% = M0/M1 * 100
The result obtained in our case is:
R% = 7.5 /700*100 = 1.07% in the case of essential oil
And R % = (25.38 / 150) *100 = 16.92 % in the case of ethanolic extract
The physicochemical properties of the essential oil of Artemisia herba-alba are presented
in the table 1:

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Table 1: Physico-chemical characteristics of the Artemisia herba-alba essential oil

7
The zero value of optical rotation, indicate the presence of a racemic mixture. The deter-
mined acid and saponification numbers show few free acids in the essential oil of Artemisia
herba-alba.
3.1. Antibacterial activity

The antibacterial activity of the Ethanolic extract of the Ceratonia siliqua plant, and the EO of
artimissia is evaluated on seven pathogenic germs after 24 hours of incubation at an adequate
temperature of 37C °.
This activity is evaluated by the aromatogram method. The antibacterial power of ethano-
lic extract and EO is obtained by measuring the diameter of the zone of inhibition in (mm)
using
a caliper vernier.
The results of the sensitivity of bacteria to ATB (antibiotic), DMSO (dimethyl sulfoxide),
ethanolic extract and EO are summarized in (figure 3)
Fig. 3. Histogram shows the zones of inhibition for all bacterial strains of ethanolic extract
and pure EO
The results of the inhibition zones diameters (Figure .3), reveal that the stock solution
(EO) of artemissia herba plant exerts a considerable antibacterial on all bacterial strains (Es-
cherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, klebsiella oxytoca, Bacillus

8
cereus, listeria monocytogenes, Salmonella typhi), while the ethanolic extract of Ceratonia
siliqua has different antibacterial activity against Staphylococcus aureus, listeria monocyto-
genes, Bacillus cerichlebsi, against , Pseudomonas aeruginosa, Salmonella typhi are resistant.
3.2. Determination of MIC by the agar diffusion method:

The parameter most often used to evaluate the effect of an antibiotic is the minimal inhibitory
concentration (MIC). It corresponds to the minimum concentration of antibiotic which inhibits
the visible growth of the germ in 24 hours.
Fig.4: The Inhibition Zones of Diluted Essential Oil Tested
The results obtained from the MIC for the Artemisia harba alba essential oil are shown in
the following figure (fig. 5):

9
Fig .5: Histogram Showing The Inhibition Zones For All The Bacteria Strains OfDiluted Eo

10
The results of the antibacterial activity of the essential oil of mugwort showed that all bac-
teria are sensitive at the 1/2 dilution, except for Salmonella bacteria which showed resistance
to this dilution. On the other hand, the 1/4 dilution showed that all bacteria are sensitive for
this dilution except Salmonella typhimurium and Escherichia which are resistant.
We also note that the Bacillus cereus and Listeria monocytogenes strains are sensitive to
EO diluted to 1/8.
These results also shows that there is only one effect of the 1/16 dilution, that was with
the bacterium listeria monocytogenes, but this result, do not take it into consideration because
the diameter of the inhibition area is less than 8mm.
So:
The MIC (minimum inhibitory concentration) of EO for the strains of Pseudomonas aerug-
inosa, klebsiella oxytoca and of Staphylococcus aureus is 0.5g / ml (1/4), whereas the strains
of Escherichia coli have an MIC of 1g / ml.
In the case of the ethanolic extract of ceratonia plant, the results obtained showed that the
1/4 dilution was sufficient to inhibit the growth of the bacterium listeria monocytogenes (MIC
= 0.25 g / ml), while Staphylococcus aureus showed some resistance at this concentration
and sensitivity at a concentration of 0.5 g / ml, while the strain of Bacillus cereus showed
resistance at all concentrations. On the other hand, the dilution (1/2) exhibits antibacterial
activity against Staphylococcus aureus
Fig. 6: The Zones of Inhibition For Different Dilution of The Ceratonia Ethanolic Extract
(Three Bacterial Strains)

11
3.4. Determination of the Minimum Bactericidal Concentration (MBC):
There are two kinds of effects of EO against bacteria: lethal or bactericidal activity and inhibi-
tion of bacterial growth or bacteriostatic activity. The most common estimation of bactericidal
activity is the determination of minimum bactericidal concentration (MBC) which is defined
as the concentration killing 99.9% or more of the initial inoculums [15].
Most often the action of essential oils is likened to a bacteriostatic effect. However, some
of their chemical constituents seem to have bacterial properties. [16]
Figures 7 represent the reinjection of certain experimental tubes by streaks on agar for
EO and ethnolic extract. This reinjection enabled us to determine the different MCB of the
concerned strains.
The results of the bactericidal effect of the different concentrations of EO of Artemisia
herba-alba and the ethanolic extract of Ceratonia siliqua are respectively shown in the tables:
Fig. 7 : the reinjection of certain experimental tubes by streaks on agar for EO and ethnolic
extract
We note from these results that all types of bacteria reactivate at all concentrations of
essential oil and ethanolic extract, except the 1/2 dilution which has shown its effectiveness in
killing bacteria.
CONCLUSIONS
Medicinal plants are still the reliable source of active ingredients known for their therapeutic
properties.

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Table 2: determination of the Bacteriostatic and bactericidal effect of Artemisia herba Alba
essential oil
Table 3: determination of the Bacteriostatic and bactericidal effect of ceratonia siliquaethano-

lic extract
The essential oil and polyphenol extraction and antibacterial properties of the extract of
two algerian medicinal plants, Mugwort and Ceratonia Siliqua respectively was studied and
major
conclusions are as follows:
1. These results clearly show that the essential oil of mugwort has a broad spectrum of
inhibitory activity on the bacterial strains tested.

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2. karob ethanolic extract gave a very important antibacterial effect on Bacillus cereus,
Staphylococcus aureus, listeria monocytogenes.
References
1. Z. Ghrabi, R.L. Sand, “Artemisia herba alba Asso”. A Guide to Medicinal Plants in
North Africa, 49 – 49, 2008
2. A. Boudjelal, L. Siracusa, C. Henchiri, M. Sarri, B. Abderrahim, F. Baali, G. Ruberto,

“Antidiabetic Effects of Aqueous Infusions of Artemisia herba-alba and Ajuga iva in
Alloxan-Induced Diabetic Rats”, Planta Med, Vol 81, pp 696-704. 2015
3. Y. Yin, F.Y. Gong, X. XinWu, Y. Sun, Y.Li, T. Chen and Q. Xu, “Antiinflammatory
and immunosuppressive effect of flavones isolated from Artemisia vestita”, J thnophar-
macol, Vol 120, 1–6, 2008.
4. J. Yashphe, I. Feuerstein, S. Barel, and R. Segal, “The antibacterial and antispasmodic

activity of Artemisia herba alba Asso. II. Examination of essential oils from various
chemotypes”, International journal of crude drug research, Vol 25, pp 89-96, 1987.
5. O.Sekiou, M. Boumendjel, F. Taibi, , A. Boumendjel and M. Messarah, “Mitigating

effects of antioxidant properties of Artemisia herba alba aqueous extract on hyperlipi-
demia and oxidative damage in alloxan-induced diabetic rats”, Archives of physiology
and biochemistry, Vol 125, pp 163-173, 2019.
6. K. B. Othmen, W. Elfalleh, J. M. G. Beltrán, M. Á. Esteban, and M. Haddad, “An in

vitro study of the effect of carob (Ceratonia siliqua L.) leaf extracts on gilthead seabream
(Sparus aurata L.) leucocyte activities. Antioxidant, cytotoxic and bactericidal proper-
ties”, Fish & Shellfish Immunology, Vol 99 , pp 35-43, 2020.
7. A.Azab, “Carob (Ceratonia siliqua): health, medicine and chemistry”, Eur Chem Bull,
Vol 10, pp 456-469, 2017.
8. M. N. Rejeb, D. Laffray, and P. Louguet, “Modification de la conductance stomatique

de diverses origines tunisiennes de caroubier (Ceratonia siliqua L.) soumises à une con-
trainte hydrique prolongée “ ..L’amélioration des plantes pour l’adaptation aux milieux
arides. France: John Libbey Eurotext, pp 149-158, (1991).

14
9. M. Ait Chitt, M. Belmir et A. Lazrak, “Production des plantes sélectionnées et gref-
fées du caroubier“, Transfert de technologie en Agriculture, N°153, IAV Rabat, pp.1-4.
2007.
10. I. Batlle, and J. Tous. “Carob tree.”Ceratonia siliqua 92 (1997).
11. R. Segal, L. Eden, A. Danin, M. Kaiser, and H. Duddeck, “Sesquiterpene lactones from
Artemisia herba alba”, Phytochemistry, Vol 24, pp 1381-1382, 1985.
12. P. Ozenda, “ Flore du sahara”, Edition CNRS, 2e édition, 416-442, 1983.
13. G. Roulier, “Les huiles essentielles pour votre santé”, Ed. Dangles, France, p.336,2000.
14. O. Yesil-Celiktas, P. Nartop, A. Gurel, E. Bedir and F. Vardar-Sukan, “Determination

of phenolic content and antioxidant activity of extracts obtained from Rosmarinus of-
ficinalis calli”. Journal of plant physiology, Vol 164, pp 1536-1542. 2007.
15. N. Canillac, A. Mourey “Antibacterial activity of the essential oil of Piceaexcelsa on

Listeria, Staphylococcus aureus and coliform bacteria”. Food Microbiol. Vol 18, pp
261– 268, 2001.
16. S. Ayadi, C. Jerribi et M. Abderrabba, “Extraction et Etude Des Huiles Essentielles

De Rosmarinus officinalis cueillie dans trois régions différentes de La Tunisie”. J. Soc.
Alger. Chim. Vol 21, pp 25-33, 2011.

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ACADEMIA Letters

ANTIBACTERIAL PROPERTIES OF CERATONIA

Corresponding Author: adjal fatima, f.adjal@univ-biskra.dz

Corresponding Author: adjal fatima, f.adjal@univ-biskra.dz

Corresponding Author: adjal fatima, f.adjal@univ-biskra.dz

Corresponding Author: adjal fatima, f.adjal@univ-biskra.dz

Fig.2. Artemisia Herba Alba

Corresponding Author: adjal fatima, f.adjal@univ-biskra.dz

Extraction of phenolic compounds

3. RESULTS AND DISCUSSION

Corresponding Author: adjal fatima, f.adjal@univ-biskra.dz

Corresponding Author: adjal fatima, f.adjal@univ-biskra.dz

3.1. Antibacterial activity

Corresponding Author: adjal fatima, f.adjal@univ-biskra.dz

3.2. Determination of MIC by the agar diffusion method:

Fig.4: The Inhibition Zones of Diluted Essential Oil Tested

Corresponding Author: adjal fatima, f.adjal@univ-biskra.dz

Corresponding Author: adjal fatima, f.adjal@univ-biskra.dz

Corresponding Author: adjal fatima, f.adjal@univ-biskra.dz

Corresponding Author: adjal fatima, f.adjal@univ-biskra.dz

Table 3: determination of the Bacteriostatic and bactericidal effect of ceratonia siliquaethano-

Corresponding Author: adjal fatima, f.adjal@univ-biskra.dz

2. A. Boudjelal, L. Siracusa, C. Henchiri, M. Sarri, B. Abderrahim, F. Baali, G. Ruberto,

4. J. Yashphe, I. Feuerstein, S. Barel, and R. Segal, “The antibacterial and antispasmodic

5. O.Sekiou, M. Boumendjel, F. Taibi, , A. Boumendjel and M. Messarah, “Mitigating

6. K. B. Othmen, W. Elfalleh, J. M. G. Beltrán, M. Á. Esteban, and M. Haddad, “An in

8. M. N. Rejeb, D. Laffray, and P. Louguet, “Modification de la conductance stomatique

Corresponding Author: adjal fatima, f.adjal@univ-biskra.dz

10. I. Batlle, and J. Tous. “Carob tree.”Ceratonia siliqua 92 (1997).

12. P. Ozenda, “ Flore du sahara”, Edition CNRS, 2e édition, 416-442, 1983.

14. O. Yesil-Celiktas, P. Nartop, A. Gurel, E. Bedir and F. Vardar-Sukan, “Determination

15. N. Canillac, A. Mourey “Antibacterial activity of the essential oil of Piceaexcelsa on

16. S. Ayadi, C. Jerribi et M. Abderrabba, “Extraction et Etude Des Huiles Essentielles

Corresponding Author: adjal fatima, f.adjal@univ-biskra.dz

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