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*Corresponding author: Farah T. O. Al-Jumaili
Copyright © 2023 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0.
Antioxidant and immunological evaluation of Ceratonia siliqua on Methotrexate
induced Albino mice male
Noor Khairullah Ibrahim, Farah T. O. Al-Jumaili * and Widad j. Atia
College of Biotechnology, AL-Nahrain University, Iraq,
International Journal of Science and Research Archive, 2023, 09(02), 325–331
Publication history: Received on 14 June 2023; revised on 21 July 2023; accepted on 24 July 2023
Article DOI: https://doi.org/10.30574/ijsra.2023.9.2.0581
Abstract
Carob (Ceratonia siliqua) plant was frequently used to cure a variety of health issues. This study aimed to evaluate the
total flavonoid, phytochemical compounds, antioxidant activity In vitro, as well as immunological potential of Ceratonia
siliqua methanolic extract in vivo through determination of total and absolute count of white blood cell on albino male
mice. The result showed that Ceratonia siliqua contain (224.5±37.86) of flavonoid compound, in addition of many
bioactive phytochemical compounds like (tannin, glycoside, alkaloid, polyphenol, and glycoside). Antioxidant activity
indicated that the ability of methanolic extract of Ceratonia siliqua plant to scavenging free radical In vitro in all
concentration uses (0.04, 0.08, 0.16, 0.32 and 0.64 mg/ml) when compared with trolox (vit E). Immunological
parameters indicated the ability of plant to enhance the immunity by increase WBCs count (7166 cell/cu.mm.blood) in
comparison with positive (methotrexate) and negative control, as well as increase in lymphocytes (3700
cell/cu.mm.blood) , neutrophil (2985 cell/cu.mm.blood) and monocyte (481 cell/cu.mm.blood) All of these effect
attributed to active phytochemical constituents in plant extract.
Keywords: Ceratonia siliqua; Flavonoid; Phytochemical; Methotrexate
1. Introduction
History of medicine and plants dates back to remote past when herbal treatment was the only answer to all kind of
ailments (1). Nowadays, greater emphasis was again being laid to phytotherapy all over the world (2). Medicinal plants,
also called medicinal herbs, have been discovered and used traditional medicine practices since prehistoric times. Plants
synthesis hundreds of chemical compounds for various functions, including defense and protection against insects,
fungi, diseases, and herbivorous mammals (3). The researches and utilization of herbal medicine in the treatment of
diseases increases every day. In the past, medicinal plants were believed traditionally to be a therapeutic agent for the
treatment of diseases such as typhoid, cholera, measles, etc (4). .
Carob (Ceratonia siliqua) was one of Asia and Africa’s popular nutritional and medicinal crops, This unique plant has an
outstanding functional properties and nutritional profile, carob has high sugar content, drought resistance and was very
economical, carob fruit consists of pulp and seed that are rich sources of different bioactive components, carob has wide
applications in various industries (food, pharmaceuticals and cosmetics) as an anti-oxidant, thickener, stabilizer, lactic
acid production and emulsions, the trend of moving toward natural products further highlights the use of carob in
different fields due to its excellent nutritional and therapeutic profile ,carob bean gum was widely used in the food
industry.(5)
In view of the present study focuses on the knowledge on medicinal uses of Ceratonia siliqua (carob) plant and the
scientific investigation to confirm their medicinal value that act as antioxidant, immunomodulator, antibacterial,
antifungal, anticancer, antidiabetic, analgesic and can prevent anemia in addition of cough and flu treatment.
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326
2. Material and methods
2.1. Ceratonia siliqua
The aerial part of plant (leaves) from Ceratonia siliqua was supplied from the local market of Baghdad during November
/2022 and recognized by Dr.Ibrahim S. Al-Jubouri, College of Pharmacy, AL-Mustansiriyah University, Iraq
2.2. Preparation of alcoholic Extract
Methanolic extract of Ceratonia siliqua was prepared according to (6), fifty grams of plant aerial part powdered and
extracted with 80% methanol (250 ml) at 65 ºC for 3 hours using the Soxhlet apparatus. The extract solution was
concentrated to dryness under reduced pressure in a rotary evaporator to yield dried crude extract, which was frozen
at -20 C until use to prepare the required doses
2.3. Dose of plant
In albino male mice, a dose of 300 mg/kg was tested depending on LD50 of Ceratonia siliqua to 50gm/kg.
2.4. In vitro analysis
2.4.1. Total flavonoid
The plant extract in weight (3.2 mg) was dissolved in 5 ml of 50% methanol solution 1 ml of a 5% (NaNo3) solution.
After 6 min ago( 1 ml ) of a 10% (AlCl3) solution was additional ,then the mixture was leave for a more 5 minutes before
10 ml of a 10% NaOH solution was added. The mixture had been completed to 50 ml with distilled water (DW) and
mixed very well. Finally absorbance was measured at 450 nm with a spectrometer after 15 min. A similar procedure
had been applied to six concentrations (2.5, 5, 10, 20, 40 and 80 µg) of rutin slandered, a standard curve was drawn .
The total flavonoid content was determined sing a curve-fitting equation of the standard curve (7).
2.5. Determination of the active phytochemicals in of Ceratonia siliqua plant
According to (8), the qualitative phytochemicals investigations of Ceratonia siliqua were carried out as follows as.
2.5.1. Detection of Tannins tests
A few drops of the 1% Lead acetate solution have been added to the alcoholic plant extract. A gelatinous or white
precipitate have been made that the presence of tannin.
2.5.2. Detection of glycosides
A liquate of 1 ml of the alcoholic plant extract was mixed with 2 ml of the Benedict reagent, then place the mixture in a
boiling water bath for 5 minutes and left to cool. The red deposit indicated that a presence of polysaccharides.
2.5.3. Detection of alkaloids (Dragangroff test)
A solution of 60 mg of Bismuth sub-nitrate have been dissolved in 0.2 ml HCl (solution A) and Solution B contains 600
mg potassium iodide in 1 ml D.W The solution A + B were mixed and added to the plant alcoholic extract, an orange to
brown color will detected expression the presence of alkaloids.
2.5.4. Detection of the Saponins
These detection process will be happening by shaking the solution of the plant alcoholic extract well. Formation of foam
at the top of the extract will be show presence of saponins.
2.5.5. Detection of Flavonoids
The test of Alkaline reagent: was prepared by using Sodium hydroxide solution had been mixed with restricted amount
of plant extract solutions and left, a bright yellow color was showed that presence of flavonoids.
2.5.6. Detection of Polyphenolic Compound
A little few drops of 3% ferric chloride (FeCl3) solution have been added to the plant alcoholic extract solution a brown
deposition will be showen.
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2.6. In vivo analysis
2.6.1. Laboratory Animals
Albino male mice (Mus musculus) were the laboratory animals.They were supplied by the Biotechnology Research
Center (Al-Nahrain University). Their ages at the start of experiments were 8-10 weeks , and their weight was 23-27
grams. They were distributed into groups, and each group was kept in a separate plastic cage (details of these groups
are given in the section of experimental design). The animals were maintained at room temperature , and had free excess
to food (standard pellets) and water (ad libitum).
2.7. Experimental Design
The experimental designs were divided in four groups each group contain six animles as the following:
Group I: Mice were administrated with D.W (negative controls).
Group II: Mice were administrated with Ceratonia siliqua methanolic extract at dose of (300mg/kg).
Group III: Mice were administrated with the MTX (1st and 2ed days) + Ceratonia siliqua methanolic extract (from
3rd to 7th day).
Group IV: Mice were administrated with the MTX (7th days) at dose of (200mg/kg).
2.8. Total and absolute count of leucocytes
Blood samples were collected by heart puncture using a disposable insulin syringe (1ml). The method of (9) was
followed.
A drop of blood was smeared on a clean slide and air-dried. The smear was stained with Leishman stain for 5 minutes
and buffered for 10 minutes, and then washed with tap water. The slide was air-dried, and then examined under oil
immersion lens (100X). At least 100 leucocytes were examined, and percentage of each cell type was recorded, while
absolute count of each type of leucocytes was obtained by using (9).
3. Results and discussion
3.1. In vitro Analyses
3.1.1. Total flavonoid
The total flavonoid of Ceratonia silique methanolic extract was (224.5±37.86) table (1).
Table 1 Concentration of total flavonoid of Ceratonia silique
Mean ± Std
Total Flavonoid
224.5±37.86
Ceratonia siliqua
3.1.2. Phytochemical contents
Ceratonia silique methanolic extract had many chemical composition (phytochemical ) as showed in table (2)
Table 2 Investigation of main bioactive phytochemical compounds of Ceratonia silique
Test name
Alcoholic extract
Tannins
++ve
Glycoside
-ve
Alkaloids
+ve
Saponins
-ve
Flavonoids
+ve
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Polyphenols
+ve
3.1.3. Reductive Ability (FRAP assay)
In all tested concentrations (0.04, 0.08, 0.16, 0.32 and 0.64 mg/ml), the absorbance of Ceratonia siliqua methanolic
extract was significantly higher than trolox (vitamin E), and such findings suggest that the plant extract was more
effective than trolox in the reductive ability, which was concentration-dependent, as showed in table (3).
Table 3 Reductive Ability (FRAP assay) of Ceratonia siliqua methanolic extract
Reductive Ability Absorbance (Mean ± SD)
Con. (mg/ml)
Ceratonia siliqua
Trolox (Vitamin E)
0.64
0.375±0.11
0.211 ± 0.015
0.32
0.281±0.010
0.132 ± 0.007
0.16
0.252±0.009
0.114 ± 0.004
0.08
0.248±0.008
0.108 ± 0.001
0.04
0.245±0.008
0.101 ± 0.001
3.2. Evaluation of methanolic extract of Ceratonia siliqua on immunological parameters
3.2.1. Total white blood cell (WBC) counts
The typical WBC count in mice the range is 2000 to 10,000 per microliter. This value had been shown in the control
negative (group no.1), in which the total count was 4500 cell/cu.mm.blood ,as showed in table (4)An increase in the
WBC count was observed in mice administered Ceratonia siliqua (group no.2), the measurement of WBC was 7166
cell/cu.mm.blood .When interact Ceratonia siliqua with MTX (group no.3), the count increased to 5971
cell/cu.mm.blood, but MTX decrease the WBC count, which this appearance at mice group no.4), in which the total count
was 3000 cell/cu.mm.blood , as showed in table (4).
Table 4 The mean of total WBC count in albino male mice
No
Groups
Dose
Mean ± SD (cells/cu.mm.blood)
I
Control negative
---------
6001±0.152
II
Ceratonia siliqua
300mg/kg
7166±0.264
III
Ceratonia siliqua + Methotrexate
300+200mg/kg
5971±0.300
IV
Methotrexate
200 mg/kg
3000±0.404
3.3. Differential count of WBCs
3.3.1. Total lymphocyte count.
In the control negative group (group no.1), the total lymphocyte count was 3161 cell/cu.mm.blood ,as showed
in table (5).
In mice (group no.2) treated with Ceratonia siliqua lymphocytes increased to 3700 cell/cu.mm.blood ,when it
compared with control negative.
In mice treated with Ceratonia siliqua and MTX (group no.3), the count of lymphocyte also increased to 3151
cell/cu.mm.blood, but when treated with MTX (group no.4), the lymphocyte count decreased to 1800
cell/cu.mm.blood.
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3.3.2. Total neutrophils count
Neutrophils are a kind of white platelet (WBC or granulocyte) that shield us from diseases. In mice, Neutrophils are the
first of all cells reach on the scene when we experience microorganisms.
In the control negative (group no.1), the total neutrophils was 1430 cell/cu.mm.blood, as showed in table (5).
In mice treated with Ceratonia siliqua (group no.2), neutrophils count increased to 2985 cell/cu.mm.blood,
when it compared with control negative.
In mice treaded with Ceratonia siliqua and MTX (group no.3), the result showed also increased in neutrophils
count (2400), but when treated with MTX (group no.4), the neutrophils decreased to 1080 cell/cu.mm.blood .
3.3.3. Total monocytes count
Monocytes are a type of WBC. They help fight bacteria, viruses, and other infections in the body. Along with other types
of WBC, monocytes are a key element of your immune response. Monocytes in mice equal or less than 3%.
In the control negative (group no.1), the total count of monocytes was 170cell/cu.mm.blood, as showed in table
(5).
In mice treated with Ceratonia siliqua (group no.2), monocytes count increased to 481 cell/cu.mm.blood.
In mice treaded with Ceratonia siliqua and MTX (group no.3), monocytes count also increased to 420
cell/cu.mm.blood. But when treated with MTX (group no.4), the count of monocytes was decreased to 120
cell/cu.mm.blood.
Table 5 The mean of differential Lymphocyte, Neutrophil and Monocyte in albino male mice
NO.
Groups
Dose
Mean ± SD (cells/cu.mm.blood)
Lympho
Neutro
Mono
I
Control negative
---------
3161
2415
425
II
Ceratonia siliqua
300 mg/kg
3700
2985
481
III
Ceratonia siliqua + Methotrexate
300+200mg/kg
3151
2400
420
IV
Methotrexate
200 mg/kg
1800
1080
120
Plants are of great interest in drug discovery and are the main source of our modern medicine. About 25% of modern
medicines were derived from a plant origin and only 5–15% of plants are being investigated for their medicinal use (10)
Today, medicinal herbs and functional foods were being widely studied resulting inlucrative therapeutic potentials (11)
Where the secondary metabolites, which possessed antioxidant properties can fight several pathological disorders such
as cancer, heart disease, hypertension, dementia, and stroke (12). .
As it is known, oxidative stress occurs when there is an imbalance as it is known, oxidative stress occurs when there is
an imbalance between antioxidation and oxidation, there was a mechanism in metabolism consisting of endogenous
and exogenous antioxidants to counteract the oxidative stress that occurs in normal physiological conditions (13).
However, oxidative stress that occurs when the antioxidant system of metabolism is not sufficient to respond to
different xenobiotics can cause various chronic and degenerative disorders such as cancer, arthritis, aging, autoimmune
disorders, cardiovascular and neurodegenerative diseases ,carob contains a wide range of biologically active
compounds, including polyphenols, sugars, cyclitols, amino acids, fibers, and minerals, whereas carob seeds contain
gum, polyphenols, and proteins (14). Due to its chemical composition, carob and its ethanolic extract have a strong
antioxidant activity and possess several valuable therapeutic functions, such as lipid-lowering , anti-proliferative, anti-
cardiovascular, and nephroprotective properties (15) . In addition, carob has exhibited significant pharmacological
activities in the digestive tract including antidiarrheal, antibacterial, anti-ulcer, and anti-inflammatory actions, and
possesses a laxative effect on gastrointestinal propulsion (16). Despite carob’s importance in functional food
development, most Arab countries use carob to make a popular drink consumed mainly in the month of Ramadan. Carob
is also used in the preparation of special traditional types of Arabic confectionery. Due to the nutritional value of carob
powder water extract which is known to contain dietary fiber, tannins, flavonoids, and gallic acid, which account for its
antioxidant properties, Phenolic compounds as depicted in carob pods are good electron donors and could terminate
the radical chain reaction by converting free radicals into more stable products (17), The antioxidant activity of
phenolics depends on the number and substitution of the hydroxyl group. As such, carob’s antioxidant activity can thus
International Journal of Science and Research Archive, 2023, 09(02), 325–331
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be attributed to the presence of gallic acid, protocatechuic, catechin, p-hydroxybenzoic, and vanillic acid, according to
the study of ,the methanolic extracts from a variety of Moroccan carob barks showed good antioxidant power In vitro.
It was previously reported C. siliqua leaves from Morocco exhibited high antioxidant activity In vitro, the DPPH
discoloration degree in a solution reveals the ability of extracts to release H+ protons and it is likely due to the presence
of products having the capacity to interact with free radicals as electron donors and, therefore, inhibiting the ROS such
the hydroxyl radical, superoxide anion (18).
Compound identification using High Performance Liquid Chromatography (HPLC) showed that carob contain flavonoids
of quercetin glycosides, catechin and epicatechin gallate. Flavonoids are considered as plant secondary metabolites that
numerous pharmacological functions are attributed to them including antioxidant, anti-mutagenic, antibacterial, anti-
angiogenic, anti-inflammatory, anti-allergic, enzyme modulation, and anti-cancer (19) .they are defined as
phytochemicals which exist either as free aglycones or glycosidic conjugates. Flavonoids are polyphenolic with a wide
range of structures Based on this diversity, they are categorized mainly into flavones, flavanols, isoflavones, flavonols,
flavanones, flavanonols, and chalcones. The diverse structures of flavonoids have resulted in many properties including
anti-cancer and anti-inflammatory effects (20). Recently, it has been shown that flavonoids can affect immune system
response and might have immune-modulator effects.
4. Conclusion
Ceratonia species have long been used as traditional herbal remedies for many diseases related to antioxidant ,
immunity and respiratory tract, or even in; digestive tract, malignancy, etc.
Compliance with ethical standards
Disclosure of conflict of interest
No conflict of interest to be disclosed.
Statement of ethical approval
As per international standard or university standard written ethical approval has been collected and preserved by the
authors.
References
[1] Welz, A. N., Emberger-Klein, A., and Menrad, K. (2018). Why people use herbal medicine: insights from a focus-
group study in Germany. Biomedcentral complementary and alternative medicine. 18(1): 92.
[2] Raman, R. P. (2017). Applicability, feasibility and efficacy of phytotherapy in aquatic animal health management.
American Journal of Plant Sciences.8(02): 257
[3] Gershenzon J., and Ullah C (January 2022). "Plants protect themselves from herbivores by optimizing the
distribution of chemical defenses". Proc Natl Acad Sci USA.119 (4).
[4] Mintah, S. O., Asafo-Agyei, T., Archer, M. A., Junior, P. A. A., Boamah, D.,Kumadoh, D., and Agyare C. (2019).
Medicinal plants for treatment of prevalent diseases. In Pharmacognosy-Medicinal Plants. Intech Open.
[5] Zunaira Basharat, Muhammad Afzaal, Farhan Saeed, Fakhar Islam, Muzzamal Hussain, Ali Ikram, Muhammad
Usama Pervaiz and Chinaza Godswill Awuchi. (2023). Nutritional and functional profile of carob bean (Ceratonia
siliqua): a comprehensive review. International Journal of Food Properties. 26:1, 389-413.
[6] Ibraheem, R. M., Mhawesh, A. A., and Abood, K. W. (2018). Estimation of the whole flavonoid, antioxidant,
antibacterial challenge concerning Viola odorata (banafsha) methanolic extract. Iraqi Journal of Agricultural
Sciences. 49(4) 655–662.
[7] Sakanaka, S., Tachibana, Y., and Okada, Y. (2005). Preparation and antioxidant properties of extracts of Japanese
persimmon leaf tea (kakinoha-cha). Food Chemistry.89(4)569–575.
[8] Sticher, O. (2008). Natural product isolation. Natural Product Reports, 25(3).
[9] Sood, R. (1986). Hematology for Students and Practitioners. Jaypee Brothers, New Delhi, India
International Journal of Science and Research Archive, 2023, 09(02), 325–331
331
[10] Khan F., Sarker M.M.R., Ming L.C., Mohamed I.N., Zhao C., Sheikh B.Y., Tsong H.F., and Rashid
M.A.(2019).Comprehensive review on phytochemicals,pharmacological and clinical potentials of gymnema
sylvestre. Front. Pharmacol.10:1–19.
[11] Atanasov A.G., Zotchev S.B., Dirsch V.M., Orhan I.E., Banach M., Rollinger J.M., Barreca D., Weckwerth W., and Bauer
R., Bayer E.A.,.(2021). Natural products in drug discovery: Advances and opportunities. Nat. Rev. Drug Discov.
20:200–216.
[12] Fanzo J., Hunter D., Borelli T., and Mattei F.(2013). Diversifying Food and Diets: Using Agricultural Biodiversity
to Improve Nutrition and Health. Routledge;London, UK
[13] Pizzino G., Bitto A. (2014).Interdonato M., et al. Oxidative stress and DNA repair and detoxification gene
expression in adolescents exposed to heavy metals living in the Milazzo-Valle del Mela area (Sicily, Italy) Redox
Biology.2:686–693.
[14] Zhu B.-J., Zayed M.Z., Zhu H.-X., Zhao J., and Li S.-P.(2019). Functional polysaccharides of carob fruit: Areview.
Chin. Med.14:1–10.3.
[15] Rtibi K., Selmi S., Jabri M.-A., El-Benna J., Amri M., Marzouki L., and Sebai H. (2016).Protective effect of Ceratonia
siliqua L. against a dextran sulfate sodiuminduced alterations in liver and kidney in rat. J. Med. Food. 19:882–889.
[16] Rtibi K., Selmi S., Grami D., Amri M., Eto B., El-Benna J., Sebai H., and Marzouki L. (2017).Chemical constituents
and pharmacological actions of carob pods and leaves (Ceratonia siliqua L.) on the gastrointestinal tract: A
review. Biomed.Pharmacother.93:522–528.
[17] Ayache S.B., Saafi E.B., Emhemmed F., Flamini G., Achour L., and Muller C.D.(2020).Biological activities of aqueous
extracts from Carob. Molecules. 25:1–16.
[18] Tagne, R.S., Telefo, P., Nyemb, J.N., Yemele, D.M., Njina, S.N., Goka, S.M.C.,Lienou, L.L., Kamdje, A.H.N., Moundipa,
P.F., and Farooq, A.D.(2014). Anticancer and antioxidant activities of methanol extracts and fractions of some
Cameroonian medicinal plants. Asian Pac. J. Trop. Med. 7, S442–S447.
[19] Cardenas C, Quesada AR., and Medina MA.(2011). Anti-angiogenic and anti-inflammatory properties of kahweol,
a coffee diterpene. PLoS ONE 6:e23407.
[20] Ravishankar D., Rajora AK., Greco F., and Osborn HM.(2013). Flavonoids as prospective compounds for anti-
cancer therapy. Int J Biochem Cell Biol.45:2821–31.
