الفهرس 
الغلافOnly 14 pages are availabe for public view
 |  | from | 232 |  |  |
| | | from | 232 | | |
Abstract
Introduction
Immunity is the ability of each body to defend itself against biological and chemical challenges and the immune system possesses cascade of potent mechanisms for defending and protecting the body against these challenges and maintaining the immune homeostatic balance during normal physiological circumstances (Ganeshpurkar & Saluja, 2018 and Chen et al., 2021). Once there is an external immune-stimulus that is defined by the body, an immune response begins to occur represented by activation of a series of immune cells which may give rise to cellular infiltration, swelling, redness, inflammation and allergy (Vesely et al., 2011). Various sever clinical and pathophysiological conditions comprising radiation therapy, chemotherapy and organ transplant immunosuppressive therapies in addition to antibiotics and cortisone therapy can aggravate immunosuppression (Kumar & Venkatesh, 2016). Cyclophosphamide which is among the most frequently used chemotherapy protocols for many malignancies is a strong immunosuppressive agent (Huang et al., 2013).
Cyclophosphamide (CP) is a well known broad-spectrum chemotherapeutic drug of a wide application in the era of cancer treatment due to its high efficacy in curing many types of solid and hematologic neoplasms (Ou et al., 2021) like breast cancer, prostate cancer, ovarian adenocarcinoma, leukemias and lymphomas (Reddy et al., 2016 and Harahap et al., 2021). Cyclophosphamide is one of the oldest and most widely prescribed synthetic nitrogen mustard alkylating cytostatic medicine. Alkylating drugs are one of the oldest well-established classes of anticancer drugs (Van de Perre et al., 2022). In general, the alkyl carbon groups of alkylating drugs act directly on the DNA double strand which alters the DNA helical structure by affecting the binding of chromatin protein and inducing DNA strand breaks, followed by genotoxic effects and tumor cell death (Peterson, 1980 and Lien & Ou, 1985). However, normal growing cells could also be affected through the same mode of action by its cytotoxic and genotoxic effects (Boisseaux et al., 2017; Grzesiuk et al., 2018 and Van de Perre et al., 2022). Thus, as well as killing tumor cells, CP damages a series of quickly proliferating normal cells, for instance, hepatocytes, bone marrow cells and gastrointestinal mucosal cells resulting in many side effects such as hepatotoxicity, decreasing immune function and inducing damage of the gastrointestinal mucosal barrier, respectively (Ou et al., 2021). Also, other severe side effects were detected such as nausea, vomiting, hair loss, anemia, leukocytopenia, thrombocytopenia and gonadal toxicity which lead to infertility (Harahap et al., 2021).
In an attempt to reduce the toxicity and deterioration of immune system accompanied to cyclophosphamide, several herbal remedies derived from traditional plants were trialed (Babich et al., 2020). Today, light was thrown on the role of herbal medications as they become of increasing importance in several medical applications since they are of natural sources, relatively lower cost, very low toxicity and high efficiency. They may be used as alternative or even adjuvant therapies for several metabolic disturbances like diabetes, hepatotoxicity and nepherotoxicity (El-Shobaki et al., 2015; Mahmoud et al., 2017 and El-Desouky et al., 2019). Also, it was mentioned previously that the use of antioxidants may reduce the toxicity of cyclophosphamide (Sieja &Talerczyk, 2004). Increased oxidative stress is nowadays highlighted as it is the causative etiology of many chronic diseases like diabetes mellitus, cardiovascular diseases, different malignancies, some neurodegenerative diseases like Alzheimer’s disease, dementia and Parkinson’s disease. Presence of free radicals normally occurs in the body cells as a part of their function but the imbalance between the production of these free radicals and the cellular antioxidant defense mechanisms lead to the state of increased oxidative stress (Mahmoud et al., 2018). Antioxidants are the defense system of the body against the damage of reactive oxygen species. Among the natural antioxidants are olive, guava and bitter orange which are plants of dual function serving as both edible and medicinal plants which are proved to be potent antioxidants and are expected to enhance the immune system. Also, Panax ginseng is a plant of potent antioxidant activity having many beifits and consumed in the South East Asian countries, especially Korea.
Olive tree (Olea europaea L.) which belongs to Oleaceae family is known to be native to the mediterranean region (Gilani & Khan, 2010 and Mannina & Segre, 2010) in which, olive tree is considered as the most important and precious fruit trees because of its prominent products which are; the table olives and the olive oil that representing main components of the mediterranean diet that are also consumed worldwide with large amounts (Nunes et al., 2022). Olive leaves (OLs) have strong antioxidant properties as they include a variety of bioactive components like polyphenols, triterpenoids, tocopherols and polysaccharides (Safarzadeh et al., 2020). OLs have been used for many purposes, since their polyphenolic content is greater than that of either whole fruit or even the extra virgin olive oil (Rocchetti et al., 2022). They are mainly used in folk medicine since they possess antidiabetic and cardioprotective properties (Acar-Tek & Ağagündüz, 2020).
Psidium guajava L which is known as guava is a fruitful evergreen shrub that is a member of the Myrtaceae family (Rajput & Kumar, 2021). Guava is planted in subtropical and tropical regions such as Africa, South Asia and South America. It is known to exert some therapeutic effects like; antioxidant, antibacterial, antivirus, hypoglycemic, anti-inflammatory and antitumor (Jiang et al., 2020). Guava is generally rich in bioactive compounds, particularly antioxidants and it is worth mentioning that the guava leaves (GLs) have higher antioxidant content than the fruit itself (Tran et al., 2020).
Citrus aurantium L. which belongs to the Rutaceae family and known as bitter orange or sour orange, is a popular and famous fruit that is mostly originating from the world tropical areas and subtropical areas (Karabıyıkl et al., 2014). Although the Citrus aurantium L. fruit possesses a bitter taste that renders it to be not attractive and not the fruit of choice for the human consumption, yet, the peel which represents almost one half of the fruit mass, contains the highest concentration of flavonoids in the citrus fruits and is used for the production of jam (Anagnostopoulou et al., 2005). The bitter orange peel was reported to have many health benefits based on its antioxidant activity such as anti-inflammatory, anticancer and antiviral effects (Sawalha et al., 2009).
Root extract of Korean red Panax ginseng (C.A. Meyer) has been well known for being a potent antioxidant (Choi, 2008). It belongs to the family Araliaceae. The Korean Red Ginseng was recognized by the Korean ministry of food and drug safety for its 6 functions: potent antioxidant, improving fatigue, improving immunity, enhancing memory, improving blood circulation as it prevents aggregation of blood platelet and improving the health of post-menopausal women (Hyun et al., 2021).
The present study was designed to evaluate the possible immunomodulatory impact of some plants, namely; olive leaves, guava leaves and bitter orange peels in immunosuppressed rats induced by cyclophosphamide and comparing them to the well known immune boosting plant; Panax ginseng. This study also aims to evaluate to what extent these plants can combat the toxicity accompanied to cyclophosphamide injection.
AIM OF THE STUDY
2- Aim of the study
This study aims to evaluate the possible immunomodulatory effect of some plants namely: olive leaves, guava leaves and orange bitter peels and comparing them to the ginseng root extract as a plant with well-established immune boosting effect either each plant individualy or in a mixture of equal ratios in immunocompromised rat model induced by cyclophosphamide injection. These plants were selected from a group of plants on the basis of their antioxidant potency and their availability.
To achieve this objective the following investigations were done;
1. Growth parameters including; food intake, body weight gain, food efficiency ratio and different organ weight percents.
2. Complete blood count.
3. Immune parameters including IgA, IL6, CD4 & CD8.
4. Oxidative stress parameter; malondialdehyde (MDA).
5. Antioxidant parameters; reduced glutathione (GSH) & total antioxidant capacity (TAC).
6. Liver function and kidney function.
7. Histopathological examination of the spleen, thymus gland and liver tissues.
REVIEW OF LITERATURE
3-Review of literature
3.1- Immune system
Immune system is a composite inserted network of organs (thymus, bone marrow, spleen, lymph nodes and Peyer’s patches), and soluble mediators, developed to protect the organism from any foreign injury that menace completely the organism (Yatim & Lakkis, 2015). Immune system can do the immune response with many methods such as neutralizing foreign elements, removal of pathogenic microbes and the tumor cells instantly by phagocytosis (Lee et al., 2015; Puttur et al., 2019 and Tang et al., 2019). It is so elaborate protective strategy with microbes, pathogens, inflammatory diseases, autoimmune diseases and cancer as a result disorders of the immune system (Huang et al., 2020). The immune responses are traditionally categorized into innate and adaptive immune responses (Vesely et al., 2011; Hu et al., 2020 and Sette & Crotty, 2021).The effective immunity may either be acquired or inherent (Spellberg & Edwards, 2001). More than 1600 genes are involved in innate and adaptive immune reactions (Abbas et al., 2005).
3.1.1- Innate immunity
Innate immunity is called natural or inborn immunity, it is the first line of protection and indicates that defensive mechanisms that are existent before contagion. Its principal constituents are the epithelial membranes (which block pathogen admittance), phagocytic cells (macrophages), dendritic cells, natural killer cells and numerous plasma proteins and the complement system (Cruz et al., 2017).The innate immunity involved the natural biophysical and chemohumoral barriers (Iqbal & Rhee, 2020).
3.1.2- Adaptive immunity
The adaptive immune response is acquired by creating antigen through especially B and T (CD4+ T cells and CD8+T) lymphocytes out of a gene rearrangement development (Pancer & Cooper, 2006 and Roberts, 2015). The cellular immune responses are the main to the success of all vaccines against viral infection (Hu et al., 2020 and Sette & Crotty, 2021).
3.2- Factors participate to improve immune system functionality
Healthy balanced diet with increasing of vitamins and trace components supplements in case of need (Stephensen, 2001; Kańtoch et al., 2002; Combs, 2008; Yamshchikov et al., 2009; Gunville et al., 2013; Hemilä, 2017 and Martineau et al., 2017), especially zinc is an important micronutrient that is elaborated in the system of the innate and adaptive immune responses (Gammoh & Rink, 2017), moderate physical exercise improves defense action (Nieman & Wentz, 2019). Polyphenols are considered as source of flavonoids and phenolic acids which are all over parts in plants (Ma et al., 2014). Polyphenols are used to control intestinal mucosal immune reactions, allergic ailments, and antitumour immunity (Ding et al., 2018), natural antivirals such as coronaviruses (Cinatl et al., 2003 and Chen et al., 2004), protection of nasal and oropharyngeal mucosa where smoking influences both innate and adaptive immunity (Qiu et al., 2017).
3.3-Cellular and humoral immunity
Humoral immunity constituents as complement system and cellular constituents as neutrophils, monocytes, dendritic cells and innate lymphoid cells (Shishido et al., 2012). The cellular immune response is mainly mediated by lymphocytes and NK cells against infection (Russell & Ley, 2002 and Behrens et al., 2004). The lymphocytes and NK cells releases several cytokines in response to pathogenic antigenic (Behrens et al., 2004). Humoral immunity is an operation of acquired immunity revealed by the production of antibodies by B lymphocytes. It matures in bone marrow. B cells activated to proliferate into plasma cells to create antibodies. Antibodies are generated when the antigen bonds the B cell receptor (BCR) (Pal et al., 2016 a, b).
3.4- Immunomodulation
Immunomodulation could be defined as immune-stimulation or suppression of the immune response (Sharma et al., 2015 and Rasheed et al., 2016).
3.5- Immunomodulators
Immunomodulators is able to regulate the immune response. Recently, the bioactive compounds that are derived from the therapeutic plants have the immunoregulatory effect to regulate immune response (Brindha et al., 2016). Immunomodulators are divided into the immune stimulants that are able to stimulate the immune reaction against infection (Maggini et al., 2018). Other immunomodulators are able to suppress the immune reactions as in autoimmune diseases (Ilyas et al., 2016).
3.6- Immunomodulatory response
Phytochemicals such as, polysaccharides, flavonoids, alkaloids, lactones, terpenoids and glycosides are presented in various plants (Gupta et al., 2014). Immunomodulators are categorized into immunostimulants immunoadjuvants and immunosuppressants (Vetvicka & Vetvickova, 2012) and Patil et al., 2012). Two major compounds are able to boost immune reactions containing adjuvants and immunostimulants. An adjuvant is a constituent combined with an antigen for cumulative its immune reaction, but an immunostimulant can prompt the immune reaction without injection with an antigen (Carter & Reed, 2010). Plants which controlled anticancer and antioxidant properties have immunomodulatory characteristic (Boudjeko et al., 2015 and Zhang et al., 2018).
3.7- Immunoadjuvants
Immunoadjuvants are considered as immune stimulants to enhance the efficiency of chemotherapy or vaccine. They are influenced by the cellular and humoral immune levels (Gertsch et al., 2011). Immunoadjuvant also belongs to the department of immunomodulatory agents which have more ability to boost the usefulness of the vaccines (Khajuria et al., 2007and Gupta et al., 2011).
3.8- Immunostimulants
Immunostimulants or immunopotentiators are functionary by innate and acquired immune reaction (Giorgi, 2019).
Immunostimulants are desirable constituents that motivate the immune system of humans and animals for inhibition of diseases and improvement of the body’s natural reluctance to various bacterial and viral infections. These biologically diligent substances are the products resulting from natural sources or synthetically made with dissimilar chemical properties and mechanisms of action. Immunostimulants prompt synthesis of definite cytokines and antibodies for the treatment of contagious diseases. Two main groups of immunostimulants encompass are the specific immunostimulants acting as antigen for an incentive of immune reactions (e.g., vaccines), and the non-specific immunostimulants without antigenic possessions enhancing immune reactions to other antigens (e.g., adjuvants and non-specific immunostimulators) (Barman et al., 2013 and Pujol et al., 2015).
Moreover, immunostimulants were categorized based on their origin and method of action (Labh & Shakya, 2014). Natural crops can act as immune stimulating agents and numerous phytochemicals that can boost immune function, such as flavonoids, terpenoids, lignans, polyphenolics, sulfides, and saponins, have been well-known and some effective and natural antioxidant composites are considered promising cures for chronic illnesses (Chen et al., 2018).
3.9- Immunosuppressants
Immunosuppressive agents are a class of drugs reducing the strength of the body’s immune system. Steroids, anti-proliferative agents, calcineurin inhibitors and target of rapamy¬cin inhibitors are different types of immuno-suppressive agents (Narayanaswamy, 1981 and Taylor et al., 2005). Medicines like azathioprine, cyclophosphamide, cyclosporine A, pencillamine etc. Which are used as immunosuppressors have a main disadvantage connected to their cytotoxicity and side effects (Vigila & Baskaran, 2008 and Gupta et al., 2010). Immunotherapy is the cure of a disease by making, improving or controlling the immune reaction. Immunotherapies, produced to get or augment an immune reaction, are categorized as immunostimulants. Furthermore, immunotherapies are able to diminish or suppress the immune functions are called immunosuppressants (Koido et al., 2013).
Immunosuppressive medications comprising glucocorticoids are widely used for the administration of inflammatory illnesses. These drugs suppress several immune functions by affecting gene transcription actions (De Bosscher et al., 2008 and Perretti & D’acquisto, 2009).
3.10- Cyclophosphamide (CP)
Cyclophosphamide (CP) is an immunosuppressive chemotherapeutic medicine, which affects mostly the B cells. It is presently used to treat ailments in which autoantibodies play a pathogenic role, such as rheumatoid diseases (Agarwal et al., 1999; Dooley & Nair, 2008 and Li et al., 2018). Depending on the dosage and controlling of administration, cyclophosphamide can also affect T cells (Brode & Cooke, 2008).
Cyclophosphamide is a widely used as an alkylating medication for the treatment of various kinds of cancers such as lymphoma, myeloma, and chronic lymphocytic leukemia, it is having actual immunosuppressive action which cross links the DNA of actively dividing cells in that way inhibiting the both cellular and humoral immune responses (Shruthi et al., 2018). It can be used as immunosuppressive agent to research the immunomodulatory effects of plant extracts (Raj & Gothandam, 2015 and Yadav et al., 2015).
3.11- Immunostimulants and plants
Some nutritional and herbal supplements are motivated as immune stimulants. The concern with these products is that they can precipitate an immune reaction and interpose with immunosuppression therapy. Echinacea, ginseng, astragalus, and vitamin c are examples of herbs and supplements that have an immune stimulating effects. Vitamin C may also be used to stimulate wound healing (Mecadon, 2021).
There are several kinds of stimulants with diverse mechanisms and functions for example microbial products, intricate carbohydrates (e.g., schizophyllan, glucans, lentinan, scleroglucan, bestatin, statolon, acemannan), vaccines, immunoenhancing medicines (e.g., isoprinosine, levamisole, fluoro-quindone, avridine, polyribonucleotides), dietary factors (e.g., lipids, carotenoids, vitamins, selenium), cytokines (e.g., macrophage stimulating factor, interleukin-2, tumor necrosis factor and interferon) and plant extracts (e.g., lectins, mitogens) (Galeotti, 1998). Pharmaceutical plants have been known as immunostimulants, growth developers, appetite motivation and antimicrobial due to the presence of bioactive materials as flavanoids, pigments, alkaloids, terpenoids, steroids, phenolics, and essential oils (Van Hai, 2015).
3.12- Plants
The immunostimulant plants contain many bioactive constituents, including alkaloids, flavonoids, coumarins, glycosides, gums, polysaccharides, phenols, tannins, terpenes, and terpenoids, which possess various biological activities that help health. These effects include antioxidant (Kumar & Pandey, 2013), antidiabetic (Gothai et al., 2016 and Hasrini et al., 2017), antiobesity (Yuliana et al., 2011), diuretic (Yuliana et al., 2013), anticancer (Ramos, 2007 ; Nobili et al., 2009; Park et al., 2014 and Zhang et al., 2017), and anti-inflammatory (Hossen et al., 2015) and (Vilar et al., 2016) activities, which improve the immune system (Zakaria-Rungkat & Prangdimurt, 2003 and Mukherjee et al., 2014).
3.13- Olive Leaves (Olea europaea L. folium)
Olive tree (Olea europaea, Oleaceae) leaves have an inclusive use in conventional plant medication against several diseases especially in mediterranean area (Hassen et al., 2015 and Boss et al., 2016). The plant is cultivated in Arabian Peninsula, the Indian subcontinent and Asia (Somova et al., 2003). Olive leaves are utilized as a source of bioactive composites as polyphenols, few quantities of phenols and acids (Khemakhem et al., 2017). Drying environments (Silva et al., 2006), agriculture zone (Bilgin & Şahin, 2013), extraction procedure (Rafiee et al., 2011 and Bilgin & Şahin, 2013), and cultivar (Japón-Lujan & de castro, 2006 and Rafiee et al., 2011) are the factors that may influence the qualitative and quantitative phenolic structure of olive leaves (Boudhrioua et al., 2009 and Brahmi et al., 2012). Leaves represent about 5% of the load of olives; have been recycled in folk medicine as cures for treating diabetes or cardiovascular ailments (Şahin & Bilgin, 2018). In ancient Egypt, olive leaves were first used to treat numerous illnesses such as fever, cough and cystitis (Şahin & Bilgin, 2018). Using of bioactive compounds from olive fruit and leaves as an active antioxidants (Cádiz-Gurrea et al., 2021), anti-inflammatory and antineoplastic (Alipieva et al., 2014).
3.13.1- Chemical characteristics of olive leaves (OLs)
3.13.1.1- Polyphenols in olive leaves
Polyphenols are mainly categorized by chemical structure. They are renowned with other chemical composites by the amalgamation of one or additional hydroxyl composites with aromatic rings (phenols). These molecules can be sub categorized into flavonoids, phenolic acids and tannins (Szliszka & Krol, 2011).
Polyphenols are displayed a beneficial impacts on our health comprising antioxidants antimicrobial, anti-inflammatory, antiviral, anticancer, and immunomodulatory actions (Scalbert et al., 2005; Chiurchiù & Maccarrone, 2011; Marzocchella et al., 2011; Izzi et al., 2012; Benvenuto et al., 2013; Bilgin & Şahin, 2013; Lall et al., 2015 and Vallianou et al., 2015). Polyphenol content of olive tree leaves (OLs) is greater than that detected in more virgin olive oil or the fruit (El & Karakaya, 2009).
OLs may be deemed as both, tailings from olive oil production and therapeutic and aromatic plants, enclose a large diversity of phenols including oleuropeosides (24.5% oleuropein and 1.1% verbascoside); flavonols (rutin); flavones (1.4% luteolin-7-glucoside, apigenin-7-glucoside, diosmetin-7-glucoside, luteolin and diosmetin); flavan-3-ols (catechin), exchanged phenols (0.7% tyrosol, 1.5% hydroxytyrosol, vanillin, vanilic acid and caffeic acid), oleoside and secoiridoid glycoside (oleuricine A and oleuricine B) (Ferreira et al., 2007; Omar, 2010; Wainstein et al., 2012 ; Rahmanian et al., 2015; Lockyer et al., 2016; Giacometti et al., 2018; Putnik et al., 2018; Jung et al., 2019 and Vizza et al., 2019).
Oleuropein, in OLs presents antiviral characteristics, protects enzymes and hypertensive cell death in cancer patients, restrains cardiac diseases and increases the lipid metabolism to limit obesity problems (Japón-Luján & de Castro, 2006).
3.13.2- The uses of olive leaves (OLs)
Olive leaves are used for the treatment against stomach ache, diarrhea, urinary tract contagions and mouth detergent. Boiling OLs extract is used for declination of hypertension through duresis and is taken also for bronchial asthma (Sabry, 2014).
3.13.3- Effects of OLs extracts
3.13.3.1- Antioxidant effect
Antioxidant action of OLs phenolic composites reactive oxygen (RO) and nitrogen species are important in normal physiological mechanisms of energy resource, purification, chemical signaling and immune response. They are constantly created in the human body and are organized by endogenous enzymes for instance glutathione peroxidase, superoxide dismutase and catalase. This destruction has been related to an increased risk of cardiovascular illness, cancer, and additional chronic diseases. Consumption of antioxidants from the food will decrease the risks of chronic illnesses (Dimitrios, 2006). Oleuropein has an effective antioxidant and anti-inflammatory properties due to its ability to remove Cu and Fe (Andrikopoulos et al., 2002). The oleuropein prevented lipoxygenases, without influence on the cyclo-oxygenase way (Visioli & Galli, 2002). Hydroxytyrosol and oleuropein are scavengers of the 1, 1- diphenyl-2- picrylhydrazyl (DPPH) radical (Gordon et al., 2001). Enhancement of repeated olive with OLs lead to an appreciable reluctance to oxidative decline because of the phenolic antioxidant content of the leaves and extract (Bouaziz et al., 2008 and Andreadou et al., 2006).
3.13.3.2- Anti-inflammatory effect
The anti-inflammatory mechanisms are activated by OLEs (Boss et al., 2016). The polyphenols of olive leaves prevent the development of proinflammatory cytokines, comprising IL-1β, TNF-α, IL-6, and prostaglandin E2 (Ahmed et al., 2004 and Murakami et al., 2007). Luteolin, another olive flavonoid, prevents IL-1-induced COX-2 expression via c-Jun N terminal kinase (JNK), ERK and NF-B involvement (Lamy et al., 2015). The capacity of olive polyphenols to down-regulate COX-2 in the cancer cells, thus resulting in tumor growth stopping (Camargo et al., 2010 and Llorente-Cortés et al., 2010).
3.13.3.3- Anti-atherosclerotic effect of olive leaves
The phenolic composites of OLs and olive oils are considered as antioxidant-rich diets have been related to a reduced prevalence of heart disease, inhibiting oxidation and deferring atherosclerosis. It may comprise phospholipase C motivation and arachidonic acid metabolism, and decrease hydrogen peroxide (Dimitrios, 2006).
3.13.3.4- Anti-cancer effect
Olive leaves include many bioactive composites have antioxidant, antimicrobial, antihypertensive, antiviral, anti-inflammatory, hypoglycemic, neuroprotective and anticancer properties (Somova et al., 2003; Bouaziz & Sayadi, 2005; Micol et al., 2005; Pereira et al., 2007; Goulas et al., 2009; Fares et al., 2011; Seddik et al., 2011 and Wainstein et al., 2012).
3.13.4- Potential side effects and drug interactions
The OLs extract boosts an immune response to liberation of toxins from pathogens which have been damaged, in this case by the reaction continues as follows: OLs composites attack and destruction the cells cause release of toxins collapse and are absorbed by surrounding tissues, which were already display symptoms caused by the contagion of the pathogens. This increase in the concentration of toxins degenerates the original symptoms and stimulates a further immune reaction from the body producing histamine release, swelling and pain the body ramps up its detoxification and purging processes which may consequence in the other adverse symptoms are the following: dull headaches, muscle and joint pain, feverishness and sweating, nausea, sore throat and nasal passages, vaginal irritation especially in the case of yeast/fungal infections (Sabry, 2014).
3.14- Guava Leaves (Psidium guajava L.)
Guava (Psidium guajava L.) is originated in Asia, Africa, Europe, and South America. It is considered an inborn to Mexico (Heinrich et al., 1998 and Leonti et al., 2001). The guava tree, pertinence to the Myrtaceae family, is a very unparalleled and traditional plant due to its pharmaceutical and nutritive impacts. The parts of the guava tree have been implicated for curing stomachache, diabetes and diarrhea.
Guava leaves (GLs) are dim green, elliptical, oval and categorized by their obtuse-type apex. GLs are a rich source of great-value nutritious medicinal composites (Kumar et al., 2021). Guava leaves with the pulp and seeds used to treat respiratory, gastrointestinal diseases and increased the count of platelets in patients anguish from dengue fever antispasmodic, cough sedative, anti-inflammatory, antidiarrheic, antihypertension, antiobesity and antidiabetic characteristics (Chen & Yen, 2007 and Laily et al., 2015). GLs are as an antitumor and reduce effects of cytotoxic agents as cyclophosphamide, corticosteroids, azathioprine and cyclosporin (Ashraf et al., 2016 and Jiang et al., 2020).
3.14.1- Chemical characteristics of guava leaves (GLs)
Guava leaves (GLs) comprise 82.47% moisture, 3.64% ash, 0.62% fat, 18.53% protein, 12.74% carbohydrates, 103 mg ascorbic acid, and 1717 mg gallic acid equivalents (GAE)/g total phenolic composites (Shabbir et al., 2020). GLs comprise flavonoids, quercetin, saponins, alkaloids, pectin or water-soluble fibers, tannins comprising phe¬nols, and essential oils that have antibacterial cures (Vijayakumar et al., 2015). Tannins are famous as psiditannins and have also antibacterial, neutralizing, absorbent and astringent composites. Crude GL comprises 9% tannins and 0.4% essen¬tial oils that are greenish containing eugenol, 6% fat oil, 3% dammar, and 3% mineral salts (Harrizul et al., 2010).
3.14.1.1- Polysaccharides
Polysaccharides extracted from GLs have an antioxidant, anti-inflammatory, antidiabetic, immunomodulatory and antitumor activities (Luo et al., 2019).
3.14.1.2- Proteins
GLs comprises 9.73% protein on a dehydrated weight basis (Rahman et al., 2013).
3.14.1.3- Minerals and vitamins
GLs are a source of calcium, potassium, sulfur, sodium, iron, boron, magnesium, manganese, and vitamins C and B. The higher concentrations of Mg, Na, S, Mn, and B in GLs creates them an extremely suitable choice for our food (Adrian et al., 2015). The GLs have a high concentrations of Ca, P, Mg, Fe, and vitamin B than that in guava fruit. The greater vitamin C content in GLs are improving the immune system (Kumar et al., 2021).
3.14.1.3- Phenolic compounds
Several secondary metabolites existing in GLs contain phenolic acids, flavonoids (quercetin, hesperetin, kaempferol, quercitrin, rutin, catchin, and apigenin), triterpenoids, sesquiterpenes, glycosides, alkaloids, and saponins (Díaz-de-Cerio et al., 2016). As well as, the GLs contain kaempfertin, isoquinoline, and corilaginoline alkaloids (Taha et al., 2019).
3.14.2- Traditional uses of GLs
By tradition, decoction from guava (leaves and bark) is used to cure dysentery, sore throats, diarrhea, vomiting and for controlling of menstrual cycles. Leaf extract of guava has antimicrobial, anti-inflammatory analgesic, antioxidant and hepatoprotective activities. These properties are may due to the presence of phenolic composites in guava (Lozoya et al., 2002).
3.14.3- Bioeffects of GLs
Separately from the fruit, GLs have probable health benefits also, some of which are; protection from cancer, controlling blood pressure, curing diarrhea and resolving bowel problems. It also assistances in losing weight, increases tonicity of skins, cures cough and cold, constipation, dysentery and scurvy (Palozza & Krinsky, 1992). The public sorts of guava around the world contain apple guava, cherry guava, and strawberry guava. This common fruit is a factory of nutrients. Possible health benefits of guava and its leaves are regulation of breath due to folate content in the latter. Guava thus forms an excellent medication for treating problems related to oral cavity (Kafle et al., 2018).
3.14.3.1- Anticancer/ Antitumor Activity of GLs
Biswas et al., (2019) demonstrated that terpenoids and flavonoids in GLs were displayed antitumor effects by suppressing the signal assignment, tumor cell adhesion, an inhibition to tumor angiogenesis and cell production. Previously, Kim, (2011) reported that kaempferol may have anticancerous characteristics.
3.14.3.2- Antioxidant activity of GLs
The GL is a immunostimulant component owing to the existence of a great level of antioxidant and phenolic composites as gallic acid, pyrocatechol, taxifolin, ellagic acid, ferulic acid, and few others, is responsible for the antioxidant roles of GLs. (Chen & Yen, 2007; Laily et al.,2015 and Farag et al., 2020). In addition to the seven flavonoids were mentioned above (Taha et al., 2019). These composites are the main composites responsible for the antioxidant properties of GLs (Kumar et al., 2021).
3.14.3.3- Guava leaves for Cold and Cough
GLs have been established to be active in curing cold and cough. Guava is rich source of ascorbic acid and iron by virtue of which it diminishes lungs congestion & excretion of mucous and simultaneously retains the respiratory tract restricted of any unfriendly pathogen. These constituents in guava act similar to miracle in curing influenza (Jaiarj et al., 1999 and Kafle et al., 2018). Fruit mainly the raw ones or decoction made from kind of immature leaves is quite useful in discharging cold and cough. It works by the dissolution of mucus polymers thus loosening cough and decreasing further mucus production, keep the respiratory tract, throat, and lungs free of microbes and inhibits present in microbial action due to its astringent properties. Vitamin c is existing in good concentration in guava which has been found to be very active in treating cold and cough related to bacteria or virus. Roasted ripe guava is used as a home remedy against great cases of cough and cold and crowding in many villages of India (Kafle et al., 2018).
3.14.4- Guava leaves, potential side effects and drug interactions
Carbohydrate and nutritional polyphenols in GLT which are related to digestive enzymes and have an important in health over poor absorption of eating sugar or lipids (Kaneko et al., 2013). Furthermore, the guava tea showed no interaction with medicine (Matsuda et al., 2007).
3.15- Bitter orange peels (Citrus aurantium L.)
Citrus aurantium (C. aurantium) or bitter orange or seville orange or sour orange is belonging to the family Rutaceae (Periyanayagam et al., 2013). C. aurantium is a small citrus tree, about five meters tall, with scented white flowers, creating in eastern Africa, Arabia, and Syria, and cultivated in Spain, Italy, and North America (Calabrese, 1992 and Nicolosi et al., 2000). C. aurantium is utilized for food, scent and medical application. Fruit, peel, leaves, flowers, seeds, and essential oil (EO) of C. aurantium are used in perfumes and cosmetics (Karoui et al., 2010). The peel encompasses flavones, the alkaloids synephrine, octopamine, N-methyltyramine, carotenoids (Suryawanshi, 2011), particularly hesperidin, naringin and alkaloids, mostly synephrine, with valuable medical impacts on human health (Pellati et al., 2002).
3.15.1- Chemical characteristics of C. aurantium
C. aurantium contains vitamins, minerals, phenolic composites, and terpenoids.C. aurantium contains flavonoids belonging to phenolics have a vital role due to their physiological and pharmacological role and their health advantages. C. aurantium flavonoids are comprising flavones, flavanones, flavonols and anthocyanins. Flavonoids are existing in Citrus fruits as glycosyl products. Aglycones are mostly existing in specific portions of the fruit as peel and seeds, due to their lipophilic nature and therefore their low solubility in water. For glycoside forms, O-glycosides, C-glycosides, rutinosides, glucosides, and neohesperidosides are public (Khan et al., 2018; Marya et al., 2018 and Nabavi et al., 2018). Bitter orange peel, seeds, and leaves comprise flavonoids with limonene, hesperidin, neohesperidine (L), naringin (K), tangaretin (F), which are flavonoids constituents of bitter orange is revealed to be more than the flower and leaves (Pellati et al., 2002). Dehydrated whole plant encompasses isoquinoline alkaloid- Synephrine, 5-methyl tyramine in China (Guo, 1983), and triterpene - limonin, nomilin are used in Sudan (Khalid et al., 1986).
3.15.2- Traditional uses of C. aurantium
The peel and dehydrated immature fruit of orange plants have been treated gastrointestinal troubles, respiratory syndromes as agent for cough (Chen et al., 2004; Fang et al., 2009 and Parle & Chaturvedi, 2012), insomnia, stress disorders, epilepsy, and anxiety (Parle & Chaturvedi, 2012 and Pimenta et al., 2016). In herbal medication the C. aurantium is used as a stimulant and appetite suppressant. In traditional Chinese medicine it has been cured nausea, indigestion, constipation, cancer, cardiovascular effect and sedative (Suryawanshi, 2011).
3.15.3- Bioeffects of bitter orange peels
3.15.3.1- Antioxidant activity of C. aurantium
The Citrus peel is a good source of great content natural bioactive composites and has good antioxidant action (Yi et al., 2008). C. aurantium peel is a source of natural antioxidants (Tundis et al., 2012 and Marzouk, 2013).
3.15.3.2- Anti-inflammatory activity of C. aurantium
Kim et al., (2012) explained the anti-inflammatory response of the C. aurantium flavonoid, namely, nobiletin, naringin, and hesperidin. The flavonoids have the ability to restrain the mRNA and protein expression of COX-2 and iNOS, by descriptive their anti-inflammatory activity (Kang et al., 2011).
3.15.3.3- Cytotoxic and anticancer effects of C. aurantium
The polysaccharides from C. aurantium have numerous pharmacological action such as cytotoxic and anticancer activity (Lee et al., 2015 and Suntar et al., 2018).
It have numerous pharmacological action such as cytotoxic and anticancer activity (Lee et al., 2015 and Suntar et al., 2018). Han et al., (2012) and Shen et al., (2017) explained how the C. aurantium polysaccharides are motivating the production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Furthermore; polysaccharides from C. aurantium have numerous pharmacological action such as cytotoxic and anticancer activity (Lee et al., 2015 and Suntar et al., 2018).
It is used to cure indigestion, diarrhea, dysentery, constipation and dry cough (Wu et al., 2014 and Shen et al., 2017). C. aurantium is also used as agent for insomnia, anxiety and epilepsy (Pimenta et al., 2016). C. aurantium is considered as an antioxidant, cardioprotective, antiproliferative, anticancer and hypolipidemic (Prouillet et al., 2004; Tanaka et al., 2013; Tenore et al., 2013 and Cirmi et al., 2017). The C. aurantium is comprising vitamins, minerals, phenolic compound and terpenoids.
3.15.4- Side effects and drug interactions of C. aurantium
The side effect of a great amounts of orange peel have produced intestinal colic, convulsions, and death in children. It can be safely eaten during pregnancy. C. aurantium suppressed the human cytochrome P450 3A (CYP3A) which aids the liver discard from several toxins so, C. aurantium interacted with medications that are metabolized by CYP3A (Suryawanshi, 2011).
3.15.5- Safety and toxicity of C. aurantium
Decoctions of C. aurantium substantially are improved blood levels of cyclosporine that causing toxicity (Bent et al., 2004). C. aurantium has a hypertension properties due to it comprises synephrine and octopamine (Nykamp et al., 2004) and arrhythmias (Bui et al., 2006), stroke and even death (Bouchard et al., 2005).
3.16- Ginseng roots (Panax ginseng)
Ginseng is a member of the Araliaceae family it includes 8 to 13 species of the Panax genus, comprising Panax ginseng (Yun, 2001). The genus name Panax is derivative from “Panacea” meaning treatment for all ailments. P. ginseng is used as herbal cure in east Asian countries since it is found in the mountains of Manchuria, China, more than 5,000 years ago (Nair et al., 2012 and Ratan et al., 2021). It has utilized its active roles in an extensive range of pharmacological consumptions (Buettner et al., 2006).
Ginseng, perennial plant, is cultivated in northern parts of Asia and used for diabetes, and male erectile dysfunction while American ginseng moreover recycled for diabetes and also applied for decreasing the risk of the public cold and flu (Zhu et al., 2020). Ginseng enhancement the immunity, memory, and blood circulation, fatigue relief, antioxidation and mitigation of menopausal signs. It also reportedly defends from cancer; cardiac diseases; inflammatory disorders; bacterial, viral or other microbial diseases; and neurological ailments (Ratan et al., 2020). The active constituents in Korean red ginseng (KRG) include ginsenosides, phenolic composites, alkaloids, peptides, polysaccharides, and polyacetylene (Baeg & So, 2013). Ginsenosidese is extracted from the roots of P. ginseng and is regarded as one of the main immunomodulatory constituents. The pharmacological characteristics of ginseng have been verified by modern science, and its proven medicinal properties comprise immune reaction boosting (Kang & Min, 2012), anti-inflammatory (Yun & Yi, 2020), hepatoprotective (Huu Tung et al., 2012), antiobesity (Li & Ji, 2018), antimicrobial (Iqbal & Rhee, 2020), cognition improvement (Park et al., 2019), and antioxidant activities (Chung et al., 2016). The root of ginseng is used for curing cardiovascular ailments (Kim, 2018), autoimmune illnesses (Lee et al., 2019), Alzheimer’s illness (Kim et al., 2018), stress-induced diseases (Lee & Rhee, 2017), ocular disease (Kim et al., 2020) and diabetes (Yuan et al., 2012).
3.16.1- Chemical characteristics of ginseng
P. ginseng 200 elements have been insulated (Roberts, 2015) and almost 100 substances have been identified. The P. ginseng substances contain ginsenosides, polysaccharides, amino acids, volatile oils, and polyacetylenes (Liu et al., 2020).
3.16.1.1- Saponins
The total ginsenosides (TGS) can be categorized as dammarane-type, ocotillol-type, and oleanane-type oligoglycosides (Kim, 2012). The ginseng saponins are dammarane-kind which includes protopanaxadiol-type and protopanaxatriol-type, ocotillol-type, and oleanane-type, though notoginseng comprises only the first two sorts (Zhang, 2004).
3.16.1.2- Polysaccharides
Ginseng polysaccharides are separated into: starch-like glucans that comprise dextran and arabinogalactan and ginseng pectin is mostly composed of galactose, galacturonic acid, arabinose, and rhamnose (Zhao & Lu, 2019).
3.16.1.3- Amino acids
Ginseng comprises at least 18 amino acids. Fourteen of these amino acids are comprising arginine, glutamic acid, aspartic acid, glycine, leucine, alanine, proline, lysine, serine, threonine, proline, phenylalanine, isoleucine, and tyrosine (Sun et al., 2016).
3.16.2- Traditional uses of ginseng
Ginseng root has been used for many goals since its overview comprising helping psychiatric and neurological disorders and increasing vitality. Ginseng moreover has a long history in mythology, herbalism, and remedy. Its medicinal properties comprise the therapy of aging, psychiatric complications, and interior organ dysfunction (Kim et al., 2018).
3.16.3- Effects of ginseng roots
3.16.3.1- Antibacterial effects of ginseng
P. ginseng extract or its constituents possess bactericidal characteristics by decreasing the cytotoxicity and DNA mutagenesis induced by Helicobacter pylori, and can diminish proinflammatory activity in gastric mucosal cells (Hong et al., 2021).
3.16.3.2- Antiviral effect of ginseng
Ginseng extract may be an active modulator for both natural and adaptive immunity regarding viral contagion as influenza virus (Hong et al., 2021).
3.16.3.3- Antifungal effects of ginseng
The fungicidal consequence of ginseng, with ginsenosides viewing antifungal action by disturbing cell membranes (Chen et al., 2019). P. ginseng and its many constituents showed pharmacological effects versus viruses, bacteria or other microorganisms, demonstrating its possible adaptogenic behavior (Hong et al., 2021).
3.16.3.4- Anticancer effect of ginseng
Researches have showed that ginseng and its extracts as compound K and ginsenosides significantly reduced concentrations of some inflammatory cytokines (Cho et al., 2013 and Sun et al., 2016). The ginsenoside 20(R)-Rg3 suppress cancer extension in ovarian cancer cells by weakening the expression of hypoxiainducible factor-1a (Liu et al., 2014). In addition, ginsenoside Rd depress control expression of iNOS, COX-2, NF-kB , destroy the phosphorylation of extracellular signal-regulated kinase and ginsenosides Rd and Rh2 inhibit tumor passage and metastasis in liver cancer (Kang et al., 2019). The ginseng extract had an immunomodulatory effect on ailments (Guan et al., 2015). Anticancer effects of ginseng or its extracts are because of the stimulation of the immune regulatory pathways in the human body as result the growth in natural killer cells both action and numbers (Kim et al., 1990, Yun, 2001, Du et al., 2008, Byeon et al., 2012 and Riaz et al., 2019). Ginseng decreased toxicities as medication-induced immunosuppression, diaphragm muscle & renal toxicity, drug-induced weight loss, decline in cancer chemotherapy connected to nausea, fatigue and vomiting (Yun, 2001 ; Park et al., 2015; Takeda & Okumura, 2015 and Riaz et al., 2019).
3.16.3.5- Ginseng and immunity
Ginseng as a dietary addition was said to restrain the immune system (Beutler, 2004).
MATERIAL AND METHODS
4-Materials and methods
4.1- Materials
4.1.1- Natural plants
The plants were used in this study which were: guava leaves (Psidium guajava L.), olive leaves (Olea europaea L. folium) and bitter orange peels (Citrus aurantium L.) in addition to the Panax ginseng root extract as a reference immunomodulatory plant.
The three plants that were used in this study were selected from a group of seven plants. After screening for the antioxidant activity and the total polyphenolic content of all seven plants, the three plants were selected on the basis of the antioxidant activity and the polyphenolic content in addition to their availability.
The seven plants were obtained as follows; Guava leaves (Psidium guajava L.), olive leaves (Olea europaea L. folium), basil leaves (Ocimum canctum) and berry leaves (Rubus fruticosus) were collected from herbarium of faculty of agriculture, Cairo university, while bitter orange peels (Citrus aurantium L.) and prickly pears (Opuntia ficus indica) were obtained from the local market at their ripening season. Ginkgo biloba was obtained from Al-Orman garden at its ripening season. However, Panax ginseng root extract was obtained from Yanbian, Bitoshan Ltd., (South Korea).
4.1.2- Cyclophosphamide
Cyclophosphamide (CP) used for induction of immunosuppression was purchased from Sigma-Aldrich company, (USA) with a Cat. No. of “PHR1404-1G”.
4.1.3- Ingredients of the diet
Ingredients used for formulation of the diet (vitamin and salt mixtures) were obtained from Fluka (Germany) and BDH (England) Chemical Companies. Casein was obtained from alahram laboratory chemicals (Egypt) while, cellulose was purchased from the laboratory of Rasayan, fine Chemical limited, Mumbai, India.
The other ingredients of the diet were obtained from the local market. Solvents used for extraction of the agro- wastes and for polyphenolic determination by HPLC were purchased from Sigma-Aldrich (analytical grade for HPLC analysis).
4.1.4- Diagnostic kits
The kits used for the determination of the plasma malondialdehyde (MDA) with a Cat. No. of E-EL-R3034 was obtained from Elabscince, (China). Kits used for reduced glutathione (GSH), cluster differentiation 4 (CD4) and cluster differentiation 8 (CD8) by ELISA technique were obtained from Sunlong Biotech Company (China) with Cat. No. of E-EL-0060, SL1410Ra, SL1201Ra and SL0172Ra, respectively. ELISA kits for determination of interleukin 6 (IL-6) with a Cat No. K0331229 and immunoglobulin A (IgA) with a Cat No. K3231104 were purchased from Koma Biotech., Seoul (South Korea). Kit used for spectrophotometric determination of total antioxidant capacity (TAC) was obtained from biodiagnostic Co. Egypt. Total antioxidant capacity (TAC) measured by colorimetric method with a kit of Cat. No. of TA2513 and obtained from Biodiagnostic Co.31, Egypt. Kits used for spectrophotometric determination of liver and kidney functions were obtained from MG Co. Egypt.
4.1.5- Animals
Male Wistar albino rats used in this study were obtained from the central animal house, national research centre, Dokki, Egypt. The study protocol was submitted to the scientific committee at the national research centre (NRC, Dokki, Egypt) and was approved with an approval no. of 18- 106. The animal experiment was carried out in compliance with the rules and the guidelines of the institutional animal care and ethics committee of the NRC.
4.2- Methods
4.2.1- Preparation of agro-waste and some plants
Each of the fresh olive leaves (OL), guava leaves (GL), bitter orange peels (BOP), berry leaves and gingko leaves were washed with running tap water and then, they were all as well as peeled prickly pears allowed to dry in an air ventilated oven at a temperature of 40ºC until complete dryness. Then, the dried peels and the dried leaves of each of guava, olive, berry and gingko as well as the dried prickly pears fruits and pellets of the panax ginseng (PG) roots each of them was grinded alone into a fine powder using electric miller; Minta, Egypt. Then, a mixture from the powder of the selected items (OL, GL, BOP and ginseng) was formulated with equal ratio. A portion this formula was subjected to extraction procedures for further determination of polyphenolic content by HPLC, while the other portion was stored at -20º C in deep freezer until being used in the feeding experiment.