ةــــعماـــج لـــجـيج ىــــــــيــحي نب قيدصلا دمحم
Master's thesis
Branch: Biological Sciences Specialization: Biochemistry
Theme
Academic Year 2018-2019
Order number (library):……….…..….
يلك ـ ع ة ـــــ طلا مول ـــ عيب ـ حلا و ة ــــــ ةاي
سق ــــــ م : لا يب ولو جلا ايج ةيولخلا و ةيئيز
Faculty of Nature and Life Sciences Department of Molecular and Cellular
Biology
Phytochemical screening and evaluation of in vitro anti- inflammatory effect of hydro-methanolic extract of
foeniculum vulgare seeds
DEMOCRATIC AND POPULAR REPUBLIC OF ALGERIA MINISTRY OF HIGHER EDUCATION AND SCIENTIFIC RESEARCH
UNIVERSITY OF JIJIEL
Examiner’s committee:
President: Dr. LAHOUAL Asma Examiner: Mme. BENSAM Moufida Supervisor: Dr. CHERBAL Asma
Presented by:
BOUABDALLAH Mouna
BENHALLA Mouna
ةــــعماـــج لـــجـيج ىــــــــيــحي نب قيدصلا دمحم
Master's thesis
Branch: Biological Sciences Specialization: Biochemistry
Theme
Academic Year 2018-2019
Order number (library):……….…..….
يلك ـ ع ة ـــــ طلا مول ـــ عيب ـ حلا و ة ــــــ ةاي
سق ــــــ م : ةيولخلا و ةيئيزجلا ايجولويبلا
Faculty of Nature and Life Sciences Department of Molecular and Cellular
Biology
Phytochemical screening and evaluation of in vitro anti- inflammatory effect of hydro-methanolic extract of
foeniculum vulgare seeds
DEMOCRATIC AND POPULAR REPUBLIC OF ALGERIA MINISTRY OF HIGHER EDUCATION AND SCIENTIFIC RESEARCH
UNIVERSITY OF JIJIEL
Examiner’s committee:
President: Dr. LAHOUAL Asma Examiner: Mme. BENSAM Moufida Supervisor: Dr. CHERBAL Asma
Presented by:
BOUABDALLAH Mouna
BENHALLA Mouna
Acknowledgements
First and foremost we would like to extend a profuse gratitude and thanks to ALLAH for giving us the power and desire to complete this modest work.
It’s a genuine pleasure to express our profound appreciation and thanks for our supervisor Dr. Cherbal Asma for her wise guidance and significant support throughout the different stages that made this project stronger and for giving us all the freedom and advices to keep us right on track. Without her help and patience, this work would not have been carried out.
We acknowledge, with great respect, members of jury the president Dr.
Lahoual Asma, and the examiner Mme. Bensam Moufida, for accepting the evaluation of our graduation project and for their constructive comments and valuable opinions in improving this modest research.
We would like also to thank our families for their constant support, encouragement and their huge sacrifices.
Last but not least, we would like to express our warmest thanks to the
laboratory team for their help and patience. Similar thanks are due to all our
colleagues for their help, support and constructive comments. Finally we
thank everyone that gives us a moral or material support to accomplish our
project.
I dedicate this modest work to those whom I have loved so much with great affection, the poeple who made it possible,the successful completion of the project would be incomplete without them
All the words of the world can not express the love and respect that I door to my mother Naima and my father Mouhammed who tried always to make me happy ,for their support and their huge sacrifices.
To my sisters and brothers (Massika in the foremost, Moufida , especially my dear brother Azzouz and his wife Feyrouz , Nabila , Naziha , Abdou ,Ikram ) for their support and encouragement
To my small lovely angels Aridje , Souhayl , Assil and Louay …..my source of happiness
To my dear friend and colleague Mouna for her patience and support, I am very happy, lucky and proud for my work with her because I learned a lot of things from her (You were the best choice for me )
To all my friends, I love you all for your support, encouragement, motivation and continuous advices specially for Wahiba ,Chaima ,Nour elhouda, Assma,Zahra ,Rania,Souad,Samira,Sabah…and the list is long
To my partner Outekhdidjet who supported me materially and morally in all stages of this project, I will never forget that for him
To my Colleagues for their patience in the laboratory every time
To every professor teached me in good faith without racism and injustice through all my stages of study from primary to university
I would like also to dedicate this work for anyone who told me that I can’t do this thesis in English , you really excited me to learn a new useful language and present this project in new and innovative way
This project is dedicated also to sensitive and Intellectuals Algerians, hopefully may be create a sensitive
and intellectual Algeria.
Dedication
I dedicate this humble work to my dear parents, Karima and Zahir, for all their sacrifices, their love, their tenderness, their support through thick and thin and their prayers throughout my studies, to my dear brother and dear sister, for their encouragement, and their moral support, and to all my family, my grandmothers and grandfathers, aunts and uncles, for their support throughout my university career.
I’d like to thank all my friends and colleagues, for their support and assistance: Sabah, Samira and Souaad, Rania, Chaima and Zahra, and Also Salma.
And finally, I dedicate my deepest gratitude to my friend and my partner in this thesis, Mouna for her constant support and patience with me, and for her open-minded way of treating every person and information that comes her way.
May this work be the accomplishment of your vows so alleged, and flees from your infallible support.
Thank you for always being here for me.
i
Table of contents ………. i
List of abbreviations……… iv
List of figures……….. v
List of tables ……… vi
Introduction………. 1
I. Bibliographic section………... 2
I.1. Generalities on inflammation……….. 2
I.1.1. Definition………. 2
I.1.2. Inflammation stimuli……… 2
I.1.3. Pathophysiology of inflammation……… 2
I.1.3.1. Types of inflammation………. 2
I.1.3.1.1. Acute inflammation……….. 2
I.1.3.1.2. Chronic inflammation……… 3
I.1.3.2 Inflammatory cells………. 3
I.1.3.2.1. Monocytes and macrophages………. 3
I.1.3.2.2. Neutrophils………. 3
I.1.3.2.3. Mast cells………... 3
I.1.3.2.4. Dendritic cells (DCs)……… 3
I.1.3.2.5. Natural killer cells (NK cells)……….. 4
I.1.3.2.6. Innate lymphoid cells……… 4
I.1.3.2.7. Platelets………. 4
I.1.3.2.8. Epithelial cells……….. 4
I.1.3.3. Mediators of inflammation……….. 4
I.1.3.3.1.Vasoactive amines and peptides………. 5
I.1.3.3.2. Eicosanoids……… 5
I.1.3.3.3. Pro-inflammatory cytokines………. 5
I.1.3.3.4. Acute-phase proteins………... 5
I.1.3.4. The Inflammatory reaction process ……… 6
I.1.3.4.1. The silent phase ………..……….. 6
I.1.3.4.2. Vascular phase……….. 6
I.1.3.4.3. Cellular phase………. 7
I.1.3.4.4. Repair phase ………. 7
ii
I.1.4.Regulation of the inflammatory response………. 8
I.1.5.Biomarkers of inflammation ……… 9
I.1.6.Treatment of inflammation ………. 10
I.1.6.1. Steroidal anti-inflammatory drugs……….. 10
I.1.6.2. Non-steroidal anti-inflammatory drugs………. 11
I.1.6.3. Medicinal plants with anti-inflammatory activity……….. 11
I.2. Foeniculum vulgare……….. 13
I.2.1. Generalities………... 13
I.2.2. Botanic description……….. 13
I.2.3. Taxonomy……… 14
I.2.4. Phytochemical composition and nutritional value ……….. 14
I.2.4.1. Nutrients……… 15
I.2.4.2. Volatile compounds………..……… 15
I.2.4.3. Phenolic compounds ………... 16
I.2.5. Pharmacological activities……….. 17
I.2.6. Anti-inflammatory activity………. 18
II. Materials and Methods………. 19
II.1. Materials……… 19
II.1.1. Plant material………. …… 19
II.1.2. Animal material……….. 19
II.2. Methods………... 19
II.2.1. The preparation of hydro-methanolic extract of F. vulgare and extraction of phenolic compounds……… 19
II.2.2. Determination of total phenolic compounds………... 20
II.2.3. Determination of total flavonoids………... 20
II.2.4. Dosage of flavonols……… 21
II.2.5. GC-MS analysis………. 21
II.2.6. Evaluation of anti-inflammatory effect of Foeniculum vulgare seed……….. 21
II.2.6.1. The effect on protein denaturation………... 21
II.2.6.2. Stabilization of the red blood cell membrane………. 22
II.2.6.2.1. Preparation of the suspension of red blood cells……….. 22
II.2.6.2.2. Heat-induced hemolysis……….. 22
iii
II.2.6.3. The effect on proteases……….. 23
II.2.7. Statistical analysis………. 23
III. Results……… 24 III.1. F.vulgare extraction yield………... 24
III.2. Phenolic content of the hydro-methanolic extract………... 24
III.3. GCMS analysis………..…….. 25
III.4. Anti-inflammatory activity of F. vulgare..………... 26
III.4.1. Effect of hydro-methanolic extract of F. vulgare seeds on protein denaturation ………….. 26
III.4.2. Effect of hydro-methanolic extract of F. vulgare seeds on red blood cell membrane stabilization……….. 27
III.4.3. Effect of hydro-methanolic extract of F. vulgare seeds on protease activity ………. 28
IV. Discussion………..……… 30
V. Conclusion……….……….. 34
VI. References………. 35
Annexes
iv
List of abbreviations
A: Absorbance AA: Arachidonic acid
ALT: Alanine aminotransferase AST: Aspartate aminotransferase BCR: B Cell Receptor
BSA: Bovine serum albumin COX: Cyclooxygenase CRP: C-reactive protein
DAMPs: Damage-associated molecular patterns DNA: Deoxyribonucleic Acid
DPPH: 2,2-diphenyl-1-picrylhydrazyl EGA: Equivalent gallic acid
EQ: Equivalent of quercetin
GC-MS: Gas chromatography mass spectrometry G-CSF: Granulocyte Colony-Stimulating Factor
GM-CSF: Granulocyte Macrophage Colony- Stimulating Factor HCl: Hyperchlorite
IFNγ: Interferon gamma IL: Interleukin
LBP: Lipopolysaccharide-Binding Protein LOX: Lipooxygenase
LPS: LipopolySaccharide
MAPK: Mitogen activated protein kinases MBP: Mannose-binding protein
MCP-1: Monocyte chemoattractant protein 1 MHC II: Major histocompatibility complex class II MHC: Major Histocompatibility Complex
MIP: Macrophage Inflammatory Protein NF-B: Nuclear factor-kappa B
NO: Nitric Oxide
NOS: Nitric oxide synthase
NSAID: Non-Steroidal Anti-Inflammatory Drug OA: Oleic acid
PAMPs: Pathogen associated molecular patterns
v
PCT: Pro-calcitonin
PLA: Phospholipase A
PRRs: Pattern recognition receptors PUFA: Polyunsaturated fatty acids RBC: Red blood cell
RNA: RiboNucleic Acid
ROS: Reactive Oxygen Species Rpm: Tours per minute
TCR: T Cell Receptor TF: Transcription factors
TGF: Transforming Growth Factor Th: T helper
TLR: Toll-like receptor
TNF-α:Tumour Necrosis Factor alpha
UV: Ultraviolet
vi
List of figures
Figure 1 Overview of the cells and mediators involved in a local acute inflammatory response…. 7 Figure 2 Biomarkers reflecting the host response to harmful stimuli………... 10 Figure 3 Mechanism of action of conventional and COX-2 specific NSAID………... 11 Figure 4 Foeniculum vulgare (a) in its natural habitat (inflorescences and flowers); (b) population
of F. vulgare; (c) stem ; (d) fruits; (e) Seeds ; and (f) Leaves………… 14 Figure 5 The most abundant chemical components isolated from Foeniculum vulgare…………... 16 Figure 6 Chemical structure of caffeoyl isolated from Foeniculum vulgare………... 17 Figure 7 Chemical structure of quercetin isolated from Foeniculum vulgare………... 17 Figure 8 GC/MS profile result of F. vulgare, every peak determines a major component’s m/z
ratio………. 25
Figure 9 The percentage of the inhibition of proteins denaturation by the hydro-methanolic extract of F. vulgare and indomethacin at different concentrations………... 27 Figure 10 Percentage of stabilization of heat induced hemolysis by the hydro-methanolic extract of
F. vulgare and indomethacin at different concentrations……… 28 Figure 11 The percentage of the inhibition of trypsin by the hydro-methanolic extract of F. vulgare
and indomethacin at different concentrations………. 29
vii
List of tables
Table 1 Biomarkers of inflammation……… 9
Table 2 Natural compounds with anti-inflammatory activity ………... 12 Table 3 Nutrients found in dried fennel.………... 15 Table 4 Polyphenol, flavonoids and flavonols contents of the hydromethanolic extract of seeds of
F. vulgare………..………..
24
Table 5 Qualitative and quantitative compositions of F. vulgare methanolic extract………... 26
1
Introduction
Inflammation is the means by which the body deals with insult and injury that may be caused mechanically, chemically, physically or by microorganisms. Inflammation is a complicated and not fully understood communication between cellular and humoral elements. (Wu and al., 2006) However, its purpose is to rid the body of the foreign matter and disposes of damaged cells and initiates wound healing.
The increase in prevalence of multiple drug resistance and the negative side effects that Non- steroidal anti-inflammatory drugs and steroidal anti-inflammatory drugs represent has issued the necessity of the development of new anti-inflammatory drugs from alternative sources.
Phytochemicals from medicinal plants showing anti-inflammatory activities have the potential of filling this need because of the variety of their chemical structures and effects (Fabricant and Fanworth, 2001).Screening of various bioactive compounds from medicinal plants has led to the discovery of new medicinal drugs which have efficient protection and treatment roles in against various diseases (Kumar et al., 2004; Mukherjee et al., 2007).
Foeniculum vulgare commonly called fennel has been used in traditional medicine for a wide range of illnesses related to digestive, endocrine, reproductive, and respiratory systems. For this raison, in recent years, an increased interest in improvement of agricultural yield of fennel has encouraged cultivation of the plant on a large scale (Badgujar et al., 2014).
Our work aims to gather the fragmented informations available about anti-inflammatory effect of Foeniculum vulgare local to Algeria by:
The preparation of the hydro-methanolic extract from seed of Foeniculum vulgare previously dried and crushed.
Running a phytochemical screening in order to characterize the main bioactive chemical groups by colorimetric reactions and spectrophotometric assay of different polyphenolic groups and releasing a GC-MS analysis.
Evaluation of the anti-inflammatory activity of hydro-methanolic extracts of Foeniculum
vulgare seeds experimentally in vitro by studying the inhibition of protein denaturation,
stabilization of the red blood cell membrane and protease inhibition activity.
2
I. Bibliographic section
I.1. Generalities on inflammation I.1.1. Definition
Inflammation is the protective reaction of the body in response to stimuli such as injury, pathogens or damaged cells (Li et al., 2007). Local signals at the sites of inflammation mediate rapid cells mobilization, recruitment and dictate differentiation programs whereby these cells drive clearance of “inflammatory inducers” and promote resolution and restoration of tissue homeostasis (Brancaleone et al., 2014).This process consists of many other smaller processes such as phagocytosis and apoptosis, in order for the tissue to repair itself (Li et al., 2007).
I.1.2. Inflammation stimuli
Effective immunity is provided through natural pleiotropy or duality (polarity) of immune cells via acute inflammation to facilitate the organ ability to return to normal physiological function after encountering internal or external foreign elements, these elements could be:
- Microorganisms and allergens,(e.g., viruses, bacteria, parasites), - Biological, chemical or environmental hazards, carcinogens, - Useless or non-functional proteins or enzymes,
- Genetic and epigenetic defects (e.g., mutated DNA or RNA, hypo or hyper methylated genetic components),
- Useless cells (e.g., polyclonal B cell complexes, senescent and cancerous cells), - Oxidized metabolites (e.g., crystalline uric acid), (Khatami, 2011).
I.1.3. Pathophysiology of inflammation I.1.3.1. Types of inflammation
I.1.3.1.1. Acute inflammation
In general, an acute inflammatory response has a rapid onset and lasts a short while. Acute inflammatory process was recently defined as the balance between two highly regulated and biologically opposing arms termed apoptosis, growth-arresting, pro-inflammatory or tumoricidal and wound healing, growth-promoting, anti-inflammatory, tumorigenic responses of immune cells with intimate participation of vasculature (Khatami, 2008)
The principal mission of acute inflammation (immune surveillance) is to:
Encounter , process, digest, destroy and eliminate intrinsic or extrinsic foreign elements and infected or injured host tissue,
Resolve and terminate inflammation and repair and construct or remodel the injured host
tissue (Khatami, 2011).
3 I.1.3.1.2. Chronic inflammation
Chronic inflammation results when the inflammation persists. The beginning of chronic inflammation is often characterized by the replacement of neutrophils with macrophages and other immune cells, such as T cells (Medzhitov et al., 2008). During a chronic inflammatory state, granulomas are typically formed in a final attempt to wall off the host from pathogens. Persistent inflammation leads to increased cellular turnover and provides selection pressure that result in the emergence of cells that are at high risk for cancer. Chronic inflammation is also associated with a variety of cardiovascular, metabolic, and neurodegenerative diseases, as well as stroke and myocardial infarction (Hotamisligil et al., 2006).
I.1.3.2 Inflammatory cells
I.1.3.2.1. Monocytes and macrophages
The primary functions of monocytes and macrophages include cytokine production, antigen presentation, phagocytosis, migration, vascular functions, and immune-regulation. These cells can
“sense” pathogens or their products through the expression of pattern recognition receptors (PRRs) on their surface (Bonizzi et al., 2004).
I.1.3.2.2. Neutrophils
Neutrophils synthetize cytokines de novo and also express cytokines at a basal level from preformed stores (Pellmeet al., 2006). Neutrophils are also the favourite targets of pro-inflammatory cytokines (IL-1 and TNF-α) chemokines such as IL-8, and growth factors such as granulocyte/monocyte colony stimulating factor (G-CSF and GM-CSF) (Romagnani et al., 1994).
I.1.3.2.3. Mast cells
Mast cells are a pivotal modulator of the innate immune system. Other than directly contributing to degradation of bacteria or other noxious substances, they can also direct the activation and recruitment of appropriate immune cells to the site of infection to facilitate early clearance of the pathogen (Cavaillon, & Singer, 2018).
I.1.3.2.4. Dendritic cells (DCs)
DCs are major drivers of autoimmune and allergic inflammation, but it is very likely that they are
at the basis of basically every form of chronic and persistent inflammation (Cavaillon, & Singer,
2018).
4 I.1.3.2.5. Natural killer cells (NK cells)
NK cells are involved in the immune response to many microbial pathogens. They are recruited and/or activated via cytokines and chemokines secreted by sentinel cells (epithelial cells, macrophages, and dendritic cells) that initiate immune responses following pathogen detection (Cavaillon, & Singer, 2018).
I.1.3.2.6. Innate lymphoid cells
Recently, a new form of effector lymphocytes, called innate lymphoid cells (ILCs) has been described to be thoroughly involved in the inflammatory process (Spits et al., 2010; Spits et al., 2013). ILCs have morphological characteristics of lymphoid cells but lack rearranged antigen receptors (TCR and BCR), and lack markers associated with myeloid cells and granulocytes but express transcription factors (TF), surface markers, and effector molecules and can respond to environment signals with massive production of pro-inflammatory and immunoregulatory factors (Spits et al., 2013).
I.1.3.2.7. Platelets
Platelets are involved not only in the response to inflammation but also in a wide range of immune responses (Yeaman et al., 2010; Elzey et al., 2011). Platelet micro-particles (thrombin and cytokines) may be an important mechanism by which platelets affect other circulating blood cells (Elzeyet al., 2003).
I.1.3.2.8. Epithelial cells
The etiology of chronic inflammatory conditions involving the intestinal epithelium such as inflammatory bowel disease (IBD) are thought to involve an overactive immune response to commensal bacteria mediated by a defective epithelium (Podolsky et al., 2002). These responses are associated with subsequent activation of the innate and acquired immune systems, resulting in pro- inflammatory cytokine release and altered barrier function.
I.1.3.3. Mediators of inflammation
A variety of chemical mediators from circulation system, inflammatory cells, and injured tissue
actively modulate the inflammatory response (Halliwell et al., 2015). They include vasoactive
amines and peptides (e.g., bradykinin), eicosanoids, pro-inflammatory cytokines and acute phase
proteins.
5 I.1.3.3.1.Vasoactive amines and peptides
Histamine is released in a quantity of few pictograms from basophils to maintain acute-phase response during inflammation events (Gilfillan et al., 2011).
Serotonin is produced via decarboxylation of tryptophan, and it is stored in the granule of platelets (Platko et al., 2015).
Bradykinin, Similar to histamine and serotonin, it can increase the synthesis of prostaglandins and produces pain locally (Hsieh et al., 2014).
I.1.3.3.2. Eicosanoids
Arachidonic acid is one of the most important substrates in the synthesis of important mediators of the inflammation called eicosanoids (Mak et al., 2013), which include the products of 5- lipoxygenase (leukotriene and5-hydroxyeicosatetraenoic acid), cyclooxygenases (prostaglandins and thromboxanes), and 12-1ipoxygenase (12-hydroxyeicosatetraenoic acid) (Piomelli et al., 2013;
Lieberman et al., 2013).
Prostanoids, formed by cyclooxygenase-l, are important in many physiological functions including regulation of platelet aggregation (Honn et al., 2013).
Prostaglandins (prostaglandin E2 and prostaglandin b) are essentially involved in conserving the inflammatory process by increasing the vascular permeability and strengthening the effect of other inflammatory mediators (Johnson et al., 2012).
I.1.3.3.3. Pro-inflammatory cytokines
In addition to many stromal cells, fibroblasts, and endothelial cells, every cytokine can be released from many cell types (Seta et al., 2012).Cytokines have important effects in the activity of many cells. However, their most significant importance is in regulating the immune system (Guo et al., 2013).Interleukin (IL)-1β, IL-8, tumor necrosis factor alpha (TNF-α), IL-6, and IL-12 are the most remarkable cytokines (Rubin et al., 2009).
I.1.3.3.4. Acute-phase proteins
The interleukins have a strong effect on liver cells and stimulate them to create a class of proteins named acute-phase proteins (Jiang et al., 2011). These laters include:
Fibrinogen A soluble protein present in blood plasma, from which fibrin is produced by the action of the enzyme thrombin; it has a vital role in fibrin peptides generation and clotting.
Haptoglobin Combines to iron-containing haemoglobin and decrease the levels of iron needed by
bacteria for their metabolism, thus decreasing their growth.
6 Complement component C3 C3a excites the basophilic cells, and C3b aid phagocytes to identify pathogens.
Mannose-binding protein (MBP) it binds to mannose-containing sugars, lying on the surface of a microorganism, and it makes it easier for phagocytes to identify pathogens (Gebhardt et al., 2009).
Serum amyloid A this protein reduces platelet activation and fever, but its physiological role is not well understood yet (Mackiewicz et al., 1993).
C-reactive protein (CRP) This protein can combine to phosphoryl choline, which is available on the surface of microorganism and injured cells. CRP assists phagocytes to identify pathogens or damaged cells (Pepys et al., 2012).
I.1.3.4. The Inflammatory reaction process
The hallmarks of a localized acute inflammatory response, first described almost 2000 years ago, are swelling (tumor), redness (rubor), heat (calor), pain (dolor), and loss of function. Within minutes after tissue injury, there is an increase in vascular diameter (vasodilation), (Judith et al., 2009).
I.1.3.4.1. The silent phase
The very first event of the inflammatory reaction is based upon the reaction of resident cells of the damaged tissue. Among these resident cells, mast cells and macrophages are playing a determinant role in alerting the body to tissue injury, by releasing mediators, such as nitric oxide (NO), histamine, kinins, cytokines, or prostaglandins. The activation of mast cell leads to subsequent release of mast cell granule contents (D’Andrea et al.,2000). The activation of monocytes and macrophages leads to the release of inflammatory cytokines such as interleukin (IL)-1, -6, and -8 (Naldiniet al., 1998). Epithelial cells can also release cytokines (IL-6, IL-8) (Asokananthan et al., 2002; Kong et al., 1997).
I.1.3.4.2. Vascular phase
Many of the vascular changes that occur early in a local response are due to the direct effects of
plasma enzyme mediators such as bradykinin and fibrinopeptides, which induce vasodilation and
increased vascular permeability. Some of the vascular changes are due to the indirect effects of the
complement anaphylatoxins (C3a, C4a, and C5a), which induce local mast cell degranulation with
release of histamine causing vasodilation and smooth muscle contraction. The prostaglandins can
also contribute to the vasodilation and increased vascular permeability (Judith et al.,2009 ).
7 I.1.3.4.3. Cellular phase
Within a few hours of the onset of vascular changes, neutrophils adhere to the endothelial cells, and migrate out of the blood into the tissue spaces (Figure1). These neutrophils phagocytize pathogens and release mediators. Among the mediators are the macrophage inflammatory proteins (MIP-1 and MIP-2), chemokines that attract macrophages to the site of inflammation. These macrophages are activated cells that exhibit increased phagocytosis and release of mediators and cytokines(IL-1, IL-6, and TNF) that induce coagulation, an increase in vascular permeability and increase expression of adhesion molecules on vascular endothelial cells (Judith et al., 2009).
I.1.3.4.4. Repair phase
The duration and intensity of the local acute inflammatory response must be carefully regulated to control tissue damage and facilitate the tissue repair mechanisms that are necessary for healing.
TGF has been shown to play an important role in limiting the inflammatory response by promoting accumulation and proliferation of fibroblasts and the deposition of an extracellular matrix that is required for proper tissue repair (Judith et al., 2009).
Figure 1: Overview of the cells and mediators involved in local acute inflammatory response
(Judith et al., 2009) .Tissue
damage leads to the formation of complement products that act as opsonins, anaphylatoxins, and chemotactic agents. Bradykinin and fibrinopeptides induced by endothelial damage mediate vascular changes. Neutrophils generally are the first leukocytes to migrate into the tissue, followed by monocytes and lymphocytes. Only some of the interactions involved in the extravasation of leukocytes are depicted.8 I.1.4.Regulation of the inflammatory response
Since an uncontrolled or prolonged release of inflammatory cytokines can cause serious damage to the surrounding tissues, a tight regulation of this mechanism is necessary. Thus, cells express anti- inflammatory cytokines such as IL-10 and IL-1. (Lawrence et al., 2007)
Macrophages during resolution engulf apoptotic cells (e.g., apoptotic neutrophils) as part of clearance of debris and switch to a phenotype characterized by impaired pro-inflammatory cytokine expression but heightened expression of TGFβ, IL-10, and lipoxins, which relates to pro-resolving, antifibrotic, and wound repair functions (Serhan et al., 2007, 2009, 2010).
Cytokine expression by neutrophils can be easily modulated by the T-cell-derived regulatory cytokines: positively by Th1-type cytokines such as IFNγ and negatively by Th2-type cytokines such as IL-10, IL-4, and IL-13 (Cassatella et al., 1999).
Mast cells secrete IL-10 that can suppress the production of pro-inflammatory cytokines and
chemokines and enhance the ability of DCs to decrease T cell proliferation and cytokine
secretion. (Grimbaldeston et al., 2007).
9 I.1.5.Biomarkers of inflammation
A biomarker is defined as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention Biomarkers (Table 1)(Atkinson et al, 2001).
Table1: Biomarkers of inflammation.
The
biomarker Secretion Role Normal
value References
C - reactive protein (CRP)
Primarily released by the liver in response to Interleukin-6 (IL- 6).
Binds to pathogens and damaged cells, and to phagocytic cells.
Activates the complement system when bound to one of its ligands.
<2 mg/L
(Pepys, 2003) (Poon, 2010)
Pentraxin 3
Produced in response to TNF-α, IL-1β, and TLR signaling in several cell types (macrophage, endothelial and epithelial cells, and fibroblasts)
Acts as a fluid-phase pattern recognition molecule with a protective role in resistance against fungal, bacterial, and viral pathogens.
(Moalli,2011) (Foo, 2015) (Garlanda, 2016) ( Bottazzi,2016)
Procalcitonin
Precursor of calcitonin. In
inflammation and in sepsis, PAMPs as well as DAMPs can stimulate cells in various tissues to produce and secrete PCT
Augments the
inflammatory response triggered by LPS, TNFα, or IFNγ and modulates gene
expression of inducible nitric oxide synthase.
<0.05 ng/mL
(Hoffmann et al, 2001)
(Sridharan et al, 2013)
Interleukin 6
IL6 release is directly induced by the
primary cytokines TNFα and IL1
Exhibits pro- and anti- inflammatory
properties and has numerous biological functions in
inflammatory processes, immune reactivity, oncogenesis, and haematopoiesis.
(Song et al, 2005)
Lipopolysacch aride-Binding Protein (LBP)
Acute inflammation- phase protein synthesized in the liver
Binds to LPS, a cell membrane component of Gram (-) bacteria, mediating its binding to receptors on monocytes and macrophages.
secretion of IL-1 and TNFα.
between 1 and 20 μg/mL
(Park et al, 2013)
10 Acute-phase reactants
A variety of diseases and tissue damages, such as infections, trauma, burns, surgeries, or autoimmune diseases, induce inflammatory reactions mediated, for example, by interleukin-6 leading to a reprogramming most notably in liver parenchymal cells with profound changes in the plasma proteome (Figure 2). Changes on transcriptional and on posttranscriptional levels lead either to reduced protein levels (negative acute-phase reactants, e.g., albumin) or higher levels of so-called positive acute-phase reactants, such as C-reactive protein (CRP), pro-calcitonin (PCT), serum amyloid A, or fibrinogen.(Cavaillon, & Singeret al, 2018).
Figure 2: Biomarkers reflecting the host response to harmful stimuli (Cavaillon, & Singer et al., 2018).
Local or systemic activation of cells of the immune system will release cytokines that might contribute to remote organ injury and to induction of downstream proteins, such as CRP, PCT, or LBP, that indicate with different kinetics the underlying host response to the pathogen. Changes in expression of cytokines or downstream mediators may inform about severity and prognosis but also potentially reflect eradication or propagation of pathogens and response to treatment.I.1.6.Treatment of inflammation
I.1.6.1. Steroidal anti-inflammatory drugs
The corticosteroids, which are cholesterol derivatives are potent anti-inflammatory agents, they exert various effects that result in a reduction in the numbers and activity of immune-system cells (Judith et al., 2009).
Corticosteroid treatment causes a decrease in the number of circulating lymphocytes as the result
either of lysis of lymphocytes (lympholysis) or of alterations in lymphocyte-circulation patterns,
Corticosteroids also reduce both the phagocytic and killing ability of macrophages and neutrophils,
they reduce the expression of class II MHC molecules and IL-1 production by macrophages and
stabilize the lysosomal membranes of participating leukocytes (Judith et al., 2009).
11 Corticosteroids increase transcription of the NF-B inhibitor (I-B) which prevents NF-B activation of a number of genes (stops T-cell activation and cytokine production) (Judith et al., 2009).
I.1.6.2. Non-steroidal anti-inflammatory drugs
NSAIDs reduce pain and inflammation by blocking cyclooxygenases (cox), enzymes that are needed to produce prostaglandins. Most NSAIDs block two different cyclooxygenases called Cox-l and Cox-2 (figure 4). Cox-2, found in joint and muscle, contributes to pain and inflammation but can cause bleeding because they block the Cox-l enzyme that protects the lining of the stomach from acid. (Saraf et al., 2008). So a selective NSAID that blocks Cox-2 but not Cox-1 might reduce pain and inflammation in joints but leave the stomach lining intact (Vane et al., 1998).
Figure 3: Mechanism of action of conventional and COX-2 specific NSAIDs (Vane et al., 1998).
I.1.6.3. Medicinal plants with anti-inflammatory activity
Natural compounds contributed significantly to drug improvement and has paved the way for
new pharmacological treatments of various inflammatory diseases (Maione et al., 2009; Bonito et
al., 2011; Maione et al., 2013; Muhammad et al., 2015) by studying models of inflammation in vitro
and in vivo that have led to the identification of a variety of natural extracts with proven anti-
inflammatory activities (Table 2)(Sultana and Saify, 2012; Orlikova et al., 2014). Although the anti-
inflammatory functions of these natural extracts were initially described, it was the key role of
12 follow-up phytochemical and pharmacological studies that led to the identification and characterisation of a variety of natural active compounds (Mózsik et al., 2009;Patrignani&Patrono, 2015).
Table 2: Natural compounds with anti-inflammatory activity (Maione et al., 2016).
Botanical name Part used
Chemical Constituent
Mode of action
Type of inflammation
Reference Terminalia chebula
(chebulic)
Fruits 1,2,3,4,6- pentao- galloyl-betaD-
glucose
In vitro hyaluronidase
inhibitory activity
joint inflammation
Kim et al.
(2010)
Withaniasomnifera (l.)
Dunal (indien Ginseng)
Roots Withaferin A In vivo modulation
of coX-2 expression
joint inflammation
Kim &
Kim (2014) Citrus unshiu
(mandarin)
Fruits citrusina XI In vitro decrease in
No production in
lPs- stimulated
raw cell
skin inflammation
Noh et al.
(2015)
Curcuma longa L.
(Turmeric)
Rhizomes Curcumin In vitro/in vivo modulation
of skin disruption
skin inflammation
Fang et al.
(2003)
Andrographispanicu lataNees (green
chireta)
Whole Plant
Andrograpanin In vitro inhibition of iNos, TNF-α
and Il-6 production
cardiovascular inflammation
liu et al.
(2008)
Psoraleacorylifolia l.
(Babchi)
Seeds Psoralidin In vitro/in vivo inhibition of
coX- 2 and 5-loX
activity
Lung, neuro and gastrointestinal
inflammation.
Yang et al.
(2011)
Cratoxylumformosu m (Jack) Benth. &hook.f.
(mampat)
Leaves Formosumone A
In vitro inhibition of
nitric oxide (No) production in
microglial cells
Lung, neuro and gastrointestinal
inflammation.
Xiong et al.
(2014)
13 I.2. Foeniculum vulgare
I.2.1. Generalities
Fennel (Foeniculum vulgare) is one of the oldest herbs native to the Mediterranean region and belongs to the Apiaceae family (Hornok, 1992), a widespread family that includes 300 genus and 3000 aromatic herbaceous species (Hay et al., 1993). Fennel is a well-known aromatic medicinal plant which is used in traditional medicine as spice and for various purposes in the food, cosmetic, and medical industries (Telci et al., 2009). It is known by the name of Fenouil in French and Shmr in Arabic.
It is cultivated and also widespread in many parts of Mediterranean and Middle East countries such as Italy, Turkey and Iran (Marino et al., 2007; Altameme et al., 2015b). The increasing commercial value of fennel necessitates the need to identification, recognizing and conservation of the existing diversity.
I.2.2. Botanic description
Foeniculum vulgare is a periodic herb with potent medicinal importance. It is an aromatic plant,
with a height of 1 to 2 m, grows in many parts of Europe, the Mediterranean, and Asia. Fennel is a
perennial herb with grooved stems, intermittent leaves, fluffy with a blade divided into thin pieces,
petiole with sheath; flowers are usually bisexual, regular or irregular, with yellow umbrella in the
form of oval beads (Pourabbas et al., 2011; Bernath et al., 1996). Fennel has small seed with a
length of about 8 mm and a width of 3 mm with an aromatic odor and sweet taste. Fennel seeds
have narrow, long, cylindrical appearance (figure 4) and dimensions vary depending on plant
growth (Ahmadi et al., 2007 ; Moura et al., 2005).
14 Figure 4: Photography of Foeniculum vulgare. (a) in its natural habitat (inflorescences and flowers); (b) population of F. vulgare ; (c) stem ; (d) fruits; (e) Seeds ; and (f) Leaves (Badgujar et al., 2014).
I.2.3. Taxonomy (Badgujar, 2014) Fennel belongs to:
Kingdom: Plantae Division: Tracheophyta Subdivision: Spermatophytina
Class: Magnoliopsida
Order: Apiales
Family: Apiaceae
Genus: Foeniculum
Species: Vulgare
Botanical name: Foeniculum vulgare.
I.2.4. Phytochemical composition and nutritional value
Fennel is used in various traditional Forms of medicine, its stem, fruit, leaves and seeds, are
medicinally used for the treatment of a variety of illness. Moreover, as a typical seasonal fresh fruit,
fennels are an important constituent of the regional diet of Africa and other regions. Different
15 varieties of fennel parts are widely used in many of the cooking dishes all over world (Badgujar et al., 2014).
I.2.4.1. Nutrients
Carbohydrates are the most abundant macronutrients in all the parts of the plant. Proteins and fats are the less abundant macronutrients. The inflorescences and stems revealed the highest fat content and reducing sugar content amongst all the parts of fennel. On the basis of the proximate analysis, it can be calculated that a fresh portion of 100 g of these parts yields, on average, 94 Kcal of energy (Badgujar et al., 2014). Fennel contains about 21 different fatty acids, some of them include caproic acid, caprylic acid, capric acid, oleic acid, linoleic acid, 𝛼-linolenic acid, arachidic acid, eicosanoic acid, cis-11,14-eicosadienoic acid and cis-11,14,17-eicosatrienoic acid . Thus, polyunsaturated fatty acids (PUFA) are the main group of fatty acids present in all the fennel parts (Badgujar et al., 2014).
Fennel nutrients are listed in table 3.
Table 3: Nutrients found in dried fennel (Badgujar et al., 2014)
macronutrients Protein, Total lipid (fat), Carbohydrate, Sugars Minerals Calcium Ca ; Iron Fe ; Magnesium Mg; Phosphorus, P
Potassium K ; Sodium Na ; Zinc Zn
Vitamins Vitamin C ; Thiamin B-1 ; Riboflavin B-2 ; Niacin B-3 Vitamin B-6 ; Folate ; Vitamin A ; Vitamin E ; Vitamin K Lipids Saturated Fatty acids ; Monounsaturated Fatty acids ;
Polyunsaturated Fatty acids Essential
amino acids Leucine ; Isoleucine ; Phenylalanine ; Tryptophane Nonessential
amino acid Glycine ; Proline
I.2.4.2. Volatile compounds
The anise odor of F. vulgare is due to its essential oil, which has been reported to contain more than 87 volatile compounds (Badgujar et al., 2014; Zoubiri et al., 2010; Telci et al., 2009). The accumulation of these volatile compounds inside the plant is variable in any of its parts, namely, roots, stem, shoots, flowers, and fruits (Badgujar et al., 2014 ; D´ıaz-Maroto et al., 2006 ; Gross et al., 2009).
The fruits of sweet fennel contain essential oil which is rich source of anethole, limonene,
fenchone, estragole and camphene among them the anethole is the most important constituent with
16 determinant role in quality of the essential oil of seeds (Gross et al., 2002; Hameed et al., 2015a).
This depends upon internal and external factors affecting the plant such as genetic structures and ecological conditions (Telci et al., 2009).
The chemical composition of the Algerian F. vulgare seed oil was different as compared with Turkish (Badgujar et al., 2014; Zoubiri et al., 2010). The hexane extracts of fennel contains 78 compounds; the major compounds (figure 5) were identified as 1,3-benzenediol, 1- methoxycyclohexene, o-cymene, sorbic acid, 2-hydroxy-3-methyl-2 cyclopenten-1-one, estragole, limonene-10-ol, fenchone, and 3-methyl-2-cyclopenten-1-one (Badgujar et al., 2014 ; Esquivel- Ferriño et al.,2012; Diao et al.,2014). Trans-anethole , a phenylpropanoid, was found to be the main component.
Figure 5: the most abundant chemical components isolated from Foeniculum vulgare (Badgujar et al., 2014).
I.2.4.3. Phenolic compounds
The phenolic compounds present in F.vulgare are considered to be associated with the prevention of diseases possibly induced by oxidative stress such as cardiovascular diseases, cancer and inflammation (Badgujar et al., 2014).
F. vulgare has been reported to contain phenolic acids like caffeoyl (figure 6), 3-O-
caffeoylquinic acid, 4 O-caffeoylquinic acid, 5-Ocaffeoylquinic acid, 1,3-O-di-caffeoylquinic acid,
1,4-O dicaffeoylquinic acid, and 1,5-O-di-caffeoylquinic acid (Faudale et al., 2008)
17 Figure 6: Chemical structure of caffeoyl isolated from Foeniculum vulgare (Badgujar et al., 2014).
Flavonoids are abundant in the plants of Apiaceae family and are generally considered as an important category of antioxidants in the human diet. Amongst the flavonoids present in F. vulgare, the most prevalent are quercetin-3-O- glucoside (figure 7), quercetin-3-glucuronide, isoquercetin, quercetin-3-arabinoside, kaempferol-3-glucuronide, kaempferol-3-arabinoside, and isorhamnetin glucoside (Kunzemann et al., 1977).
Figure 7: Chemical structure of quercetin isolated from
Foeniculum vulgare (Badgujar et al.,2014).
I.2.5. Pharmacological activities
Foeniculum vulgare is officially noted in Ayurvedic Pharmacopoeia as an important part of polyherbal formulations in the treatment of different diseases and disorders. A number of biological-pharmacological studies have been undertaken to evaluate the indigenous uses of F.
vulgare. Few extracts of F. vulgare and isolated compounds have been evaluated for several
activities, namely, antiaging, antiallergic, anticolitic, antihirsutism, anti-inflammatory, antimicrobial
and antiviral, antimutagenic, antinociceptive, antipyretic, antispasmodic, antistress, antithrombotic,
anxiolytic, apoptotic, cardiovascular, chemomodulatory action, antitumor, diuretic, estrogenic
properties, expectorant, galactogenic, hypoglycemic, hypolipidemic and hepatoprotective effects.
18 Furthermore, It has gastrointestinal, memory-enhancing, nootropic, and oculohypotensive activities (Pradhan, 2008; Koppula and Kumar, 2013; Rahimi, 2013; Rasul, 2012; Tripathi, 2013).
Fennel essential oil has a valuable antioxidant, antibacterial, anticancer and antifungal activities (Moura et al., 2005; El-Awadi and Esmat, 2010; Altameme et al., 2015a).
I.2.6. Anti-inflammatory activity
Kataoka (2002) evaluated the anti-inflammatory activity of a methanolic extract of F. vulgare fruit in rats and mice using three screening protocols, namely, carrageenan-induced paw edema, arachidonic acid-induced ear edema, and formaldehyde-induced arthritis. These are widely used for testing non-steroidal anti-inflammatory drugs. Oral administration of methanol extract of F. vulgare fruit to rat and mice exhibited inhibitory effects against acute and subacute inflammatory diseases.
For acute inflammation, methanol extract (200 mg/kg) exhibits significant inhibition of paw edema
(69%) induced by carrageenan injection as compared to the control group of animals. Methanol
extract of F. vulgare also inhibits ear edema (70%) induced by arachidonic acid in mice. The level
of serum transaminase, aspartate aminotransferase (AST), and alanine aminotransferase (ALT)
significantly increases in the presence of methanolic extract of F. vulgare on inflammation induced
by formaldehyde as compared to control group. The assessment of AST and ALT levels provides a
good and simple tool to measure the anti-inflammatory activity of the target compounds. Choi and
Hwang (2004) suggest that F. vulgare may act on both cyclooxygenase and lipoxygenase pathways.
19
II. Materials and Methods Materials II.1. Materials
II.1.1. Plant material
Foeniculum vulgare seeds were obtained from a local market in Jijel in 2019 and then dried in the dark and in room temperature and crushed to give a powder from which the hydro-methanolic extract was made.
II.1.2. Animal material
Two rabbits, obtained from a farm in the region of Ouled rabah in Jijel, were used to collect blood that is required for the anti-inflammatory study. They were placed in a plastic cage with standard food and water. They have been placed in the laboratory animal house of the Faculty of Nature and Life Sciences of the University of Jijel for 4 days for adaptation.
II.2. Methods
II.2.1. The preparation of hydro-methanolic extract of F. vulgare and extraction of phenolic compounds
The extract was prepared with a sieve, 30 g of the plant powder was then stored in an opaque bottle to prevent oxidation of their components (Awika et al., 2005).
Extraction of phenolic compounds from the seed powder of the plant was carried out by a solid- liquid method according to the procedure of Owen and Johns (1999) slightly modified.
It is carried out in two stages:
Maceration: it consists in leaving the powder of the plant material in prolonged contact with a solvent at room temperature to extract the active principles. Thirty grams of the plant powder was macerated at room temperature for 72 h in 300 ml of 80/20 (v/v) water-methanol at a solid/liquid ratio of 1/10 (w/v) under continuous stirring by a magnetic stirrer.
Evaporation: the hydro-methanolic extract or macerate was filtered by Whattman paper (Chevoleau et al., 1992). Then, this solution was concentrated at 40°C in a Rotavapor® 300 rotary evaporator (Naczk and Shahidi, 2006). After evaporation, the extract is introduced into a Memmert-type oven at 40°C until its weight is stabilized to give the weight of the crude extract.
The extraction yield was calculated according to Stanojevic et al. (2009) by the ratio between the
weight of the extract after the evaporation of the solvent (g) and the weight of the test portion (g)
powder.
20 It is expressed as a percentage according to the following formula:
Where:
Pf: weight of the extract after evaporation of the solvent (g) P °: weight of the test portion (g)
The dried extract was stored in a dry sterile flask in the dark at 4°C for pharmacological tests.
II.2.2. Determination of total phenolic compounds
The Folin-Ciocalteu method is one of the most suitable methods in most studies concerning natural antioxidants and is considered to be the best method for the quantification of total polyphenols (Spignon et al., 2007). The principle of this method is based on the ability of a phenol to reduce yellow Folin-Ciocalteu reagent consisting of phosphor-tungstic and phosphomolybdic acids in an alkaline medium to a mixture of blue tungsten and Molybdenum. The blue color produced is proportional to the amount of polyphenols present in the analyzed extract (Ribéreau- Gayon et al., 1982)
The level of phenolic compounds of the extracts was estimated by the Folin-Ciocalteu reagent according to the procedure of Othman et al. (2007), with some modifications.
An aliquot of 0.2 ml of the extract at (125µg / ml, 250 μg / ml, 2mg / ml) or standard solution was mixed with 1.5 ml of Folin-Ciocalteu reagent (1/10). After 5 min at room temperature and in the dark, 1.5 ml of a 7.5% Na₂CO₃ solution was added to the mixture. Then, the latter was incubated in the dark at room temperature for 90 minutes. Absorbance against a blank was measured at 750 nm. The average content of total polyphenols is expressed as mg of gallic acid per g of crude extract (mg EGA/g). The results were carried out 3 times and the average value was calculated.
II.2.3. Determination of total flavonoids
Flavonoids are the most abundant class of polyphenols in nature (Kumaran and Karunakaran, 2007). The principle of their dosage is based on the property of these compounds to chelate aluminum ions. In the presence of aluminum chloride (AlCl₃), the flavonoid-Al complex can be formed between the oxygen atoms carried by carbons 4 and 5 of the flavonoids. The yellow color
Extraction yield (g / 100 g of powder) = 𝑷𝒇
𝑷°
×
10021 thus obtained is proportional to the amount of flavonoids in the extract (Ribérau-Gayon, 1968;
Berset, 2006).
The content of flavonoids was determined spectrophotometrically according to the method of (Djeridane et al., 2006) with some modifications. An aliquot of 1.5 ml of the 2 mg / ml extract or standard solution was mixed with 1.5 ml of 2% AlCl₃. Thirty minutes later, the absorbance against blank was determined at 430 nm using the SPECORD® 50 PLUS Spectrophotometer.
The results were expressed in mg equivalent of quercetin per g of crude extract (mg EQ/g). Results were carried out 3 times and the average value was calculated.
II.2.4. Dosage of flavonols
The content of flavonols was determined by the modified colorimetric method described by (Shehata et al., 2009). An aliquot of 1 ml of 2 mg/ml extract was mixed with 1 ml of 2% AlCl3 and 3 ml of a 5% sodium acetate solution was added. The mixture was incubated for 30 min at room temperature and in the dark. The absorbance was read at 440 nm using the spectrophotometer. The results were expressed in mg equivalent of quercetin per gram of crude extract (mg EQ/g).
II.2.5. GC-MS analysis
The phytochemical investigation of methanolic extract was performed on a GC-MS equipment shimadzu QP2010 mass spectrometer, with electron impact ionization (70ev). An SE 30 capillary column (0,25х25mm) coated with 5% diphényle and 95% diméthylpolysiloxane was used. Oven temperature was programmed to rise from 50 to 250 at a rate of 50°C/min. Transfer line temperature was 2500C.The carrier gas used was helium with a flow of 1,5 ml /min and a split ratio of 20:0. Scan time and mass range were 0,50 s and 40-350 m/Z , respectively. The results were compared by using the NIST mass spectral library of the CG/MS data system. Determination of the percentage composition was based on peak area.
II.2.6. Evaluation of anti-inflammatory effect of Foeniculum vulgare seed
The anti-inflammatory activity in vitro has been studied by three methods: protein denaturation, stabilization of red blood cell membrane and the effect on protease activity.
II.2.6.1. The effect on protein denaturation
The inhibition of protein denaturation was carried out according to the method of kar et al.
(2012). Three solutions were prepared as follows:
- The negative control solution (0.5ml) contains 0.45 ml of BSA at 5% in distilled water and 0.05
ml of distilled water.
22 - Standard solution (0.5ml) contains 0.45 ml of 5% BSA in distilled water and 0.05 ml of
different concentrations of indomethacin (50, 100, 150, 200, 250 µg / ml).
- Test solution (0.5ml) contains 0.45 ml of 5% BSA in distilled water and 0.05 ml of different concentrations of the extract (50, 100, 150, 200, 250 µg / ml).
All the above solutions were adjusted to pH 6.3 using 1N HCl, the samples were incubated at 37°C for 20 min and the temperature was raised to maintain the sample at 57 °C for 3 min. After cooling, 2.5 ml of phosphate buffer solution (6.3 pH) was added. The absorbance was measured using a visible UV spectrophotometer at 416 nm. The percentage of inhibition of protein denaturation was calculated as follows:
The control represents 100% of the protein denaturation. The results were compared with the standard solution of indomethacin (250 μg/ml).
II.2.6.2. Stabilization of the red blood cell membrane
The principle of this method is based on membrane stabilization of red blood cells by the extract referring to a standard solution. This method is carried out according to Kar et al. (2012) with some modifications.
II.2.6.2.1. Preparation of the suspension of red blood cells
The blood was collected (2ml) from rabbits and mixed with an equal volume of a sterilized Alsevers solution (2% glucose, 0.8% soduim citrate, 0.5% citric acid and 0.42% in distilled water) and centrifuged at 3000 rpm. The packed cells (pellet) were washed with iso-saline water and put together with normal saline to prepare a 10% (v / v) suspension and conserved at 4°C before use.
II.2.6.2.2. Heat-induced hemolysis
Different concentration of Foeniculum vulgare extract and indomethacin were prepared (50,100, 200, 500 and 1000 μg / ml in normal saline, indomethacin was used as a standard and physiological saline was used as a negative control.
These concentrations were mixed separately with 1 ml of phosphate buffer, 2 ml of hypo-saline and 0.5 ml of the 10% red blood cell suspension.
All the test mixtures were heated at 56°C for 30 min and centrifuged at 2500 rpm for 10 minutes.
Then the absorbance was measured using a visible UV spectrophotometer at 560 nm.
The percentage of inhibition (%) =𝑨 𝒄𝒐𝒏𝒕𝒓𝒐𝒍−𝑨 𝒆𝒙𝒕𝒓𝒂𝒄𝒕
𝑨𝒄𝒐𝒏𝒕𝒓𝒐𝒍
× 100
23 The percentage of the stabilization of red blood cell membrane or red blood cells hemolysis inhibition was calculated using the following formula:
II.2.6.3. The effect on proteases
The test was performed using the slightly modified method of Oyedapo et al. (1995) and Sakat et al. (2010). The reaction mixture (2 ml) contained 0.06 mg of trypsin, 1 ml of 20 mM Tris-HCl buffer pH 7.4 and 1 ml of the extract dissolved in distilled water and F. vulgare extract at different concentrations (50, 100, 150, 200, 250 μg/ml). Indomethacin was used as a standard and distilled water was used as a negative control. The reaction mixture was incubated at 37°C for 5 minutes, and then 1 ml of the 0.8% (w / v) casein prepared in the buffer solution was added.
The mixture was inhibited for further 20 minutes then 2 ml of acetic acid was added to stop the reaction. The suspension was centrifuged and the absorbance of the supernatant was read at 210 nm against the buffer solution blank. The experiment was performed three times and the percentage inhibition of the protease inhibitory activity was calculated using the following formula:
II.2.7. Statistical analysis
The results of different evaluations are given as mean ± standard deviations. The statistical analysis is carried out using ANOVA test coupled with two ki test by GraphPad Prism 8 software.
The value found by calculating the p value may indicate that:
p> 0.05 = the difference is not significant (ns).
0.05> p> 0.01 = the difference is significant (*).
0.01> p> 0.001 = the difference is highly significant (**).
p <0.001 = the difference is very highly significant (***).
The percentage of stabilization (%) = 𝑨𝑪𝒐𝒏𝒕𝒓𝒐𝒍 −𝑨𝑻𝒆𝒔𝒕
𝑨𝑪𝒐𝒏𝒕𝒓𝒐𝒍
×100
The percentage of inhibition (%) = 𝑨 𝒄𝒐𝒏𝒕𝒓𝒐𝒍−𝑨 𝒆𝒙𝒕𝒓𝒂𝒄𝒕
𝑨𝒄𝒐𝒏𝒕𝒓𝒐𝒍
