WO2021021050A1

WO2021021050A1 - Carob tree (ceratonia siliqua l.) leaves in the treatment of airway inflammation occurring in allergic asthma

Info

Publication number: WO2021021050A1
Application number: PCT/TR2020/050646
Authority: WO
Inventors: Nefise Nalan İMAMOĞLU ŞİRVANLI
Original assignee: T.C. Erci̇yes Üni̇versi̇tesi̇
Priority date: 2019-07-26
Filing date: 2020-07-23
Publication date: 2021-02-04
Also published as: TR201911349A2

Abstract

The invention is related to the potential of C. siliqua leaf extract and its phenolic compounds (gallic acid, epicatechin-3-gallate,epigallocatechin-3-gallate and chlorogenic acid) to be used as an anti-inflammatory and anti-asthmatic agent which have a therapeutic effect in the treatment of inflammatory diseases related to inflammation, in particular airway inflammation occurring in allergic asthma in medicine and health field.

Description

CAROB TREE ( Ceratonia siliqua L.) LEAVES IN THE TREATMENT OF AIRWAY INFLAMMATION OCCURRING IN ALLERGIC ASTHMA

Technical Field

The invention is related to the potential of carob tree (Ceratonia siliqua L.) leaf extract and its phenolic compounds [gallic acid (GAc), (-)-epicatechin-3-gallate (ECG), (-)-epigallocatechin- 3-gallate (EGCG) and chlorogenic acid (Cl Ac)] to be used as an anti-inflammatory and anti asthmatic agent which have a therapeutic effect in the treatment of inflammatory diseases related to inflammation, in particular airway inflammation occurring in allergic asthma in medicine and health field.

Known State of the Art (Prior Art)

Asthma is an allergic and inflammatory disease occurring in airways and lungs which is characterized by the excessive infiltration of leukocytes, especially eosinophils, into airways, airway hyperresponsiveness and mucus hypersecretion.

In the state of art; there is a great variety of steroid and non-steroid drugs used in the treatment of asthma which is inflammatory airway disease. These asthma medications are divided into two groups as controller and reliever (rescue) medications.

Controller Medications; these medications are used daily and for a long time to ensure that asthma is kept under control in the long term mainly by its anti-inflammatory effects. These medication groups; include inhaler and systemic glucocorticosteroids, leukotriene antagonists, long-acting inhaler beta2-agonists used in combination with inhaler steroids, long-acting oral beta2-agonists, sustained-released theophylline, chromones, anti-IgE, and other therapies allowing to reduce systemic steroid doses.

Reliever Medications; (Rescue or bronchodilator medications) these medications correct bronchoconstriction, relieve symptoms by acting rapidly and are used as needed. These medication groups; include rapid-acting inhaler beta2-agonists, inhaler anticholinergic drugs, short-acting theophylline and short-acting oral beta2-agonists.

The applications and possible side effects of asthma medication groups in the state of the said art are stated in detail below. CONTROLLER MEDICATIONS

Inhaler glucocorticosteroids

Inhaler steroids are the most effective anti-inflammatory drugs used today in the treatment of persistent asthma and therefore are preferred as drugs that provide the best control in the treatment of asthma. In the studies the efficiency of these drugs in reducing asthma symptoms, airway hypersensitivity, airway inflammation, frequency and severity of attacks, reducing asthma-related mortality, improving life quality, lung functions and consequently controlling asthma. However, these drugs require continuous use in asthma and if the treatment is discontinued, deterioration in clinical control occurs.

Local side effects of inhaler steroids are oropharyngeal candidiasis, hoarseness (dysphonia) and cough caused by upper respiratory irritation. Systemic side effects of inhaler steroids used in high doses for a long time are short stature in children, suppression of the suprarenal glands (adrenal suppression), skin thinning and easy bruising, decreased bone mineral density and osteoporosis.

Leukotriene Antagonists

Among leukotriene antagonists, only leukotriene receptor antagonists (montelukast and zafirlukast) are available in Turkey. Clinical studies have shown that leukotriene antagonists have a small and variable bronchodilator effect, reduce symptoms, including cough, provide improvement in lung function and reduce airway inflammation and asthma exacerbations.

Long-acting inhaler beta2-agonists

Long-acting inhaler beta2-agonists such as formoterol and salmeterol are not recommended for use alone since they don’t affect airway inflammation. When used in combination with inhaler steroids, they show the highest effect.

Inhaler beta2-agonists cause less systemic side effects (cardiovascular stimulation, skeletal muscle tremor and hypopotasemia) compared to the sustained-released oral form. Regular use of beta2-agonists can lead to tachyphylaxis (against brocoprotective, bronchodilator and side effects). Since salmeterol causes a possible increase in the risk of asthma-related death in a small group of patients, the use of long-acting beta2-agonists along with the recommendation of a physician and with steroids is recommended by the American Food and Drug Administration (FDA). Long-acting oral beta2-agonists

Long-acting oral beta2-agonists include sustained-release forms of salbutamol and terbutaline. In rare cases, these drugs are used when additional bronchodilation is required.

Its side effects are cardiovascular stimulation (tachycardia), anxiety and tremor in skeletal muscle and are more common than inhaler beta2-agonists. When used in combination with theophylline, undesired cardiovascular reactions can occur. Use as regular monotherapy is harmful and it should always be used in combination with inhaler steroids.

Anti-IgE

Anti-IgE (omalizumab) is indicated in patients susceptible to a perennial allergen (mite, mold, pet), with severe allergic asthma that cannot be controlled with inhaler steroids. It has been shown that it has a role in asthma control by reducing symptoms, the need for using relaxant drugs and exacerbations. However, these effects have not been confirmed in all studies. It is also a very costly treatment option.

In the studies, although anti-IgE treatment is considered to be quite safe, it is recommended that injections are applied in centers where appropriate conditions are provided since the risk of anaphylaxis (1/1000) is reported.

Long-acting Oral Systemic glucocorticosteroids

In severe and uncontrolled asthma, oral steroid therapy may be required for more than 2 weeks; however, the risk of side effects limits use.

Systemic side effects are osteoporosis, hypertension, diabetes, hypothalamic-hypophyseal- adrenal axis suppression, obesity, cataract, glaucoma, stria formation in skin and skin thinning leading to easy bruising and muscle weakness. Asthma patients using long-term systemic steroid should receive preventive treatment for osteoporosis. Discontinuation of oral steroids can rarely cause adrenal insufficiency or reveal an underlying disease such as Churg-Strauss syndrome. At the same time, close and careful medical surveillance is recommended in the treatment of systemic steroids in patients with asthma which have tuberculosis, parasitic infection, osteoporosis, glaucoma, diabetes, major depression or peptic ulcer. Herpes virus infections showing fatal courses have been reported even during short-term steroid therapy.

Other controller treatments

Various treatments have been proposed in order to reduce the dose of oral steroids required in steroid-dependent severe asthmatic patients. These drugs should be used in certain patients and under expert supervision. It has been stated that low-dose methotrexate provides a small general benefit in reducing steroid dose; however, its side effects are relatively frequent, the benefit which can be obtained from the drug is not worth the side effects.

Cyclosporine, gold and troleandromycin (macrolide group) are also used for the same purpose. Macrolides cause nausea, vomiting, abdominal pain and liver toxicity. Methotrexate can cause gastrointestinal symptoms, hepatic and diffuse pulmonary parenchymal disease, also show hematological and teratogenic effects.

Allergen immunotherapy

Today environmental protection and drug therapy form the basis of allergic asthma treatment. Allergen immunotherapy; can be considered only in mild asthma accompanied by allergic rhinitis and moderate cases in adult asthma if symptoms continue despite allergen avoidance and drug therapy.

Local and systemic side effects may occur while allergen immunotherapy is applied. Swelling, redness, and wide and painful reactions may occur locally at the injection site. Systemic reactions are also a life-threatening anaphylactic reaction and severe asthma exacerbations. Among patients with severe asthma, immunotherapy-related deaths have been reported.

RELIEVER MEDICATIONS

These medications that provide acute healing (bronchodilator; rescue) act rapidly to improve bronchoconstriction and are used to treat acute attack symptoms such as cough, wheezing and shortness of breath.

Rapid-acting inhaler beta2-agonists

They are used to eliminate bronchospasm in asthma exacerbations and to prevent bronchospasm to occur during exercise. Rapid-acting inhaler beta2-agonists should be used only when needed and at the lowest dose and frequency required. In our country, among the drugs belonging to this group, salbutamol, terbutaline and also formoterol which is a long- acting beta2-agonist are available. Formoterol can be used only as an anti-symptomatic in a patient under regular treatment with inhaled steroids.

Side effects similar to oral beta2-agonists can also be seen in rapid-acting inhaler beta2- agonists. However, oral beta2-agonists cause more side effects such as tremor and tachycardia compared to rapid-acting inhaler beta2-agonists. Systemic glucocorticosteroids

Systemic steroids are useful in the treatment of severe asthma attacks. This prevents the progression of exacerbation, reduces the need for appeal to emergency service and hospitalization, prevents early attack recurrence and reduces morbidity. Its effects in acute asthma become apparent 4-6 hours after use. Oral treatment is preferred. Typically, 40-60 mg/day prednisolone is given for 5-10 days in short-term oral steroid therapy. When symptoms decrease and lung function approaches the patient's best personal value, oral steroids are decreasingly discontinued and treatment is continued with inhaler steroids. Intramuscular injections are not superior to short-term oral steroid therapy.

Side effects of short-term high-dose systemic therapy are rare. These side effects; can be considered as reversible glucose metabolism disorders, increased appetite, fluid retention, increased body weight, moon face, psychological state changes, hypertension, peptic ulcer and aseptic femur necrosis.

Anticholinergic drugs

Anti-symptomatic effect of the sole anticholinergic inhaler ipratropium bromide which can be used as short-acting is not as strong as inhaler beta2-agonists in patients with asthma. The use of inhaler ipratropium bromide with inhaler beta2-agonist in acute asthma attacks results in a significant additional improvement in lung functions and a decrease in hospitalization. In our country, it is available in the form of MDI in combination with salbutamol or as a nebul solution as a single drug.

Ipratropium inhalation may cause dryness or bitter taste in the mouth and prostatism complaints.

Theophylline

Short-acting theophylline or aminophylline can be used as an anti-symptomatic in asthma. The role of theophylline in the treatment of exacerbation is controversial. While short-acting theophylline does not provide bronchodilator benefit in addition to the rapid-acting beta2- agonist, it may be beneficial in stimulating respiratory drive and relieving diaphragm muscle fatigue.

Since this may cause undesirable effects, plasma levels should be measured if short-acting theophylline will be given to patients receiving sustained-release theophylline therapy. Short-acting oral beta2-agonists

It may be suitable to be used in a small number of patients who cannot use inhaler drugs; however, the prevalence of undesirable effects is higher than inhaler beta2-agonists.

Around the world, many people suffer from allergic inflammatory diseases such as asthma. Since all the anti-inflammatory or anti-asthma drugs mentioned above in details, which are currently used in the treatment of asthma today, can cause serious undesirable side effects, it has emerged the need to develop new and more effective therapeutic (curative) drugs which have no or fewer side effects in the treatment of asthma.

In the state of the art, it is stated that most medicinal plants can develop relief symptoms equally compared to conventional (common) agents in allergic inflammation.lt has been revealed with the studies which were performed in recent years that natural products such as medicinal plants have the potential in producing new drugs which do not have side effects and treat inflammation, and it has been shown that consumption of various vegetables and fruits in the daily diet reduces the prevalence of airway inflammation and childhood asthma. Today, since side effects occur very much in classical drug treatments and the cost of synthetic products is high, this situation created a need to research herbal drugs and to work on them in order to contribute to the treatment.

In the state of the art, it has been found that various polyphenols are highly effective in relieving allergic inflammation which is alleviated by the use of allopathic drugs and that flavonoids which are polyphenolic plant secondary metabolites have therapeutic effects on asthma-related symptoms.

The fruits and leaves of Ceratonia siliqua L. (C. siliqua) tree which is also known as carob in our country and belongs to the Fabaceae family are important sources of phenolic compounds consisting of various bioactive chemical components and are very rich in polyphenols and flavonoids. For this reason, extracts obtained from C. siliqua fruits and leaves have a high antioxidant capacity. In the prior art, it is stated that the high antioxidant properties of these extracts are associated with the high content of gallic acid, epigallocatechin gallate and epicatechingallate polyphenols, while C. siliqua leaves are better source in terms of phenolic compounds. The fact that C. siliqua leaf extract has high antioxidant activity indicates that it should be developed as a potential nutraceutical or pharmaceutical product. In the state of the art, it has been shown that carob (C. siliqua) plant has biological activities that are beneficial for health. Patent document EP1438058A1 is related to the antioxidant and antitumor activities of C. siliqua leaves and seed envelope extracts containing polyphenols. While patent document W02011089012A2 is related to the use of seed/seed flour (powder) obtained from the seeds of C. siliqua fruits in the treatment of diarrhea; Patent document US20150306023A1 is related to the use of the combination of C. siliqua seed extract and caffeine as an active thinning (weight loss) agent in cosmetics. However; there is no study about anti-inflammatory and/or anti-asthmatic effects of C. siliqua leaf extracts on asthma in the state of the art .

In the state of the art, human bronchial airway epithelial cells (BEAS-2B cells) are known as the most frequently used human bronchial epithelial cell line in the in vitro studies about asthma and airway inflammation since they resemble phenotypically to the primary bronchial epithelium and mimic the clinical conditions of asthma. Also in the state of the art, bacterial Lipopolysaccharide (LPS) which forms a large part of the outer membrane of gram-negative bacteria and is a glycolipid promotes various cells to stimulate signal pathway leading to the expression of immune-regulating or inflammatory cytokines. As a result of the activation of this signaling pathway which causes cellular activation, systemically and locally active pro- inflammatory molecules are released. The human airway epithelium is constantly exposed to gram-negative bacteria and endotoxins such as LPS due to inhalation of particles that are airborne and include bacteria and LPS. As a result of exposure to LPS, it is reported that the severity of asthma is increase.

In the state of the art, it is known that in allergic airway inflammation induced by pathogens or allergens, proinflammatory cytokines and chemokines such as IL-6, IL-8 and MCP-1, and proallergic cytokines such as IL-4, IL-5, IL-13 and GM-CSF are produced by airway epithelial cells. It has been stated that mRNA expression and protein levels of proallergic (IL- 4, IL-5, IL-13 and GM-CSF) and proinflammatory (IL-6, IL-8 and MCP-1) cytokines and chemokines are increased as a result of stimulation with allergen in BEAS-2B cells stimulated with an allergen obtained from house dust mite and that targeting these proinflammatory and proallergic chemokines and cytokines produced by airway epithelial cells during airway inflammation may provide an important treatment strategy in allergic airway inflammation in asthma.

In the state of the art, it is known that TLR4 receptor exists in mammalian cells is a signal- transmitter epithelial receptor that can be activated by bacterial LPS and it serves as an epithelial receptor for LPS in the airway inflammatory process in asthma. Also in the state of the art, it is known that LPS-induced TLR4 activation promotes inflammatory mechanisms such as NF-KB and JAK/STAT which are very important in the progress of asthma. Therefore, it is stated that blocking LPS-induced TLR4 activation can provide an important treatment strategy in asthmatic airway inflammation. Additionally, in the state of the art, it is stated that the cellular secretion and expression of IL-8 increase as a result of stimulation of the TLR4 signal by LPS. Over- secretion and -expression of IL-8 is one of the most important factors responsible for asthmatic airway inflammation. Inducing IL-8 via TLR4 signal pathway stimulates eotaxin-1 expression associated with asthmatic inflammation. For this reason, it is stated that inflammatory reactions in asthma can be prevented by regulating the IL-8 response via TLR4 signal pathway in airway epithelial cells.

In the state of the art, it is also stated that CCR3 receptor expressed on the surface of various types of immune cells and airway epithelial cells and eotaxin-1 from ligands that bind to this receptor are closely associated with asthma. Therefore, CCR3 receptor and eotaxin-1 have become an interesting possibility in the treatment and therapy of asthma. Eotaxin is shown as a possible target in drug development and it is stated that a useful therapeutic strategy can be developed in the treatment of asthma disease by regulating eotaxin production. Again similarly, in the state of the art, it is also stated that blocking of CCR3 receptor which is closely associated with asthma and allergy can show useful effects in the treatment and therapy of asthma.

In the state of the art, it is stated that through LPS-induced TLR4 signal pathway, the cellular secretion and expression of IL-8 increase and the increased IL-8 stimulates the expression of eotaxin-1 which is responsible for asthmatic airway inflammation.

In the prior art, STAT proteins are cytokine-inducible transcription factors and these proteins activate the STAT pathway by phosphorylation from tyrosine residues in consequence of cytokine stimulation. Also in the airway inflammation, the STAT pathway is activated in consequence of that STAT1 and STAT3 proteins are phosphorylated from tyrosine (tyr) residues (STAT1, from Tyr701; STAT3, from Tyr705) by cytokine stimulation. Still, in the state of the art, it is stated that STAT proteins mediate allergic responses in asthma, however, the roles of STAT1 and STAT3 proteins in these responses are not well defined. In addition, it has been shown that in the endotoxin-induced airway epithelium, transcription factors STAT1 and STAT3 play a role in the activation of IL-8 signal and subsequent eotaxin-1 and it has been stated that targeting STAT1 and STAT3 molecules may be beneficial for a novel asthmatic inflammation therapy. Similarly, it is reported that STAT3 is responsible for allergic inflammation in the airway epithelium of mice with chronic asthma and targeting this molecule may form the basis for a new asthma treatment.

Also in the prior art, the SOCS (supressors of cytokine signalling) family proteins have emerged as negative regulators of cytokine responses in inflammatory reactions and they negatively regulate the Cytokine-STAT pathway. It is stated that STAT3, SOCS3 and cytokines play a complementary role in the continuity and coordination of inflammation. STAT3 is the most important transcription factor promoted by IL-6 cytokine signal, the signal transduction of the IL-6 cytokine occur through the JAK/STAT3 pathway. The most important negative regulator of JAK/STAT3 activated by IL-6 is SOCS3. It is stated that the expression of SOCS3 proteins reduce in the airway cells which developed inflammation. In BEAS-2B cells exposed to LPS, it was shown that the expression of SOCS3 proteins is reduced, however, when kaempferol applied to the cells, kaempferol disrupts STAT3 signal pathway positively regulated by LPS by repairing in an opposite fashion. Therefore, the SOCS family proteins are shown as a target in the treatment strategy of allergic asthma.

Brief Description and Purposes of The Invention

In the invention, it is basically purposed that C. siliqua leaf extract and/or its phytochemicals can be used as a therapeutic potential anti-inflammatory and anti-asthmatic agent in the treatment of airway inflammation occurring in allergic asthma.

In the invention, the anti-inflammatory/anti-asthmatic effects of the C. siliqua leaf extract and the phenolic compounds contained within it and the possible molecular mechanisms underlying these effects are examined in vitro for the first time in BEAS-2B cells in which inflammation is developed with bacterial LPS.

With the invention, first leading data were obtained for developing new pharmaceutical compositions, nutraceuticals, herbal drugs or natural dietary preparations of C. siliqua leaf extract and/or pure compounds which don’t have side effects as a new and alternative therapeutic agent for anti-inflammatory or anti-asthma drugs used today in the treatment of airway inflammation occurring in allergic asthma and can cause serious side effects.

In the invention, carob tree leaf extract can be used as dry or liquid in the treatment of airway inflammation occurring in allergic asthma.

In the invention, carob tree leaf extract can be used as a drug and/or dietary supplement in the treatment of airway inflammation occurring in allergic asthma. With the invention, it was aimed to investigate the effects of C. siliqua leaf extracts and the phenolic compounds which are determined to be found in the extracts on possible signal pathways contributing to asthma progress in the human bronchial airway epithelial cells (in BEAS-2B cells) which developed inflammation with LPS and mimic the clinical condition of asthma.

As a result of qRT-PCR analyses performed in the invention; it has been determined that C. siliqua leaf extracts and/or its pure compounds effectively inhibit mRNA expressions of proinflammatory cytokines (IL-6) and chemokines (IL-8 and MCP-1) which are produced by airway epithelial cells in the allergic airway inflammatory process in asthma and showing the increase with LPS.

As a result of qRT-PCR analyses performed in the invention; it has been determined that C. siliqua leaf extracts and/or its pure compounds also exhibit strong inhibitory properties on the TLR4 receptor, which plays an important role in the airway inflammatory process in asthma.

With the invention, it has been demonstrated for the first time that C. siliqua leaf extracts and its pure compounds can provide an important treatment strategy as a therapeutic agent in asthmatic airway inflammation by regulating IL-8 response via the TLR4 signal pathway.

With the invention, it has been determined that C. siliqua leaf extracts and its pure compounds strongly inhibit mRNA and protein expressions of the CCR3 receptor which is expressed on the surface of airway epithelial cells and showing the increase with LPS.

Similarly, with the invention, C. siliqua leaf extracts and its pure compounds have been seen to strongly inhibit mRNA and protein expressions of the eotaxin-1 ligand that binds to the CCR3 receptor and showing the increase with LPS.

With the invention, it has been revealed for the first time that C. siliqua leaf extracts and its pure compounds can be developed as a therapeutic agent in the treatment of asthma and can provide a useful treatment strategy by blocking CCR3 activation which is closely associated with asthma and allergy and by regulating the production of eotaxin-1 associated with asthmatic inflammation shown as a possible target in drug development.

With the invention, it has been detected that C. siliqua leaf extracts and pure compounds contained within the extracts effectively inhibit phosphorylations of STAT1 and STAT3 proteins which mediate allergic responses in asthma and showing the increase with LPS, in a dose-dependent manner. With the invention, it has been determined that C. siliqua leaf extracts and/or its pure compounds increase the expression of the SOCS3 protein belonging to the SOCS family proteins shown as a target in the treatment strategy of allergic asthma by negatively regulating the Cytokine-STAT pathway and thereby they disrupt the STAT3 signal pathway positively regulated by the LPS.

The results obtained with the invention were compared with the results obtained from Dexamethasone (Dex), which is used as an anti-asthmatic drug in the treatment of asthma in the clinic and is determined as positive control in the study. As a result of comparisons, it was found that extract and/or pure compounds whose anti-inflammatory/anti-asthmatic effects were investigated had inhibitory effects on signaling pathways causing asthma pathogenesis similar to positive control Dex or more effectively than Dex.

With the invention, it was revealed for the first time that in airway inflammation induced by endotoxines, such as LPS, C. siliqua leaf extracts and/or its pure compounds can prevent destructive asthmatic reactions by disrupting TLR4-STAT1/3 signal paths and consequently by reducing IL-8 response and subsequent eotaxin-1 activation.

As a result, with the discovered invention, C. siliqua leaf extracts and/or its pure compounds whose anti-inflammatory/anti-asthmatic effects were investigated, were determined to have strong anti-inflammatory and anti-asthmatic potential for the first time.

Definitions of Drawings Illustrating the Invention

Figure 1: Cell viability (%) graph obtained as a result of incubation of BEAS-2B cells with increasing concentrations of YLE extract for 6, 12 and 24 hours. *p<0.001, **p<0.05 comparison with control group. (n=3).

Figure 2: Cell viability (%) graph obtained as a result of incubation of BEAS-2B cells with increasing concentrations of MLE extract for 6, 12 and 24 hours. *p<0.001,**p<0.05, ***p<0.01 comparison with control group. (n=3).

Figure 3: Cell viability (%) graph obtained as a result of incubation of BEAS-2B cells with serially increasing concentrations of Gallic acid (GAc) for 6, 12 and 24 hours. *p<0.001 comparison with control group. (n=3).

Figure 4: Cell viability (%) graph obtained as a result of incubation of BEAS-2B cells with serially increasing concentrations of Epigallocatechin-3-gallate (EGCG) for 6, 12 and 24 hours. *p<0.001, **p<0.05 comparison with control group. (n=3). Figure 5: Cell viability (%) graph obtained as a result of incubation of BEAS-2B cells with serially increasing concentrations of Epicatechin-3-gallate (ECG) for 6, 12 and 24 hours. *p<0.05, **p<0.001 comparison with control group. (n=3).

Figure 6: Cell viability (%) graph obtained as a result of incubation of BEAS-2B cells with serially increasing concentrations of Chlorogenic acid (CIAc) for 6, 12 and 24 hours. *p<0.05, **p<0.01 comparison with control group. (n=3).

Figure 7: The expression levels of IL-6, IL-8 and eotaxin-1 genes in the BEAS-2B cells incubated with LPS (2 pg/mL) for 6, 12 and 24 hours.

Figure 8: The expression levels of IL-6, IL-8 and eotaxin-1 genes in the BEAS-2B cells incubated for 2, 4, 6 and 12 hours with 25 and 50 pg/mL concentrations of YLE and MLE extracts after being developed inflammation with LPS.

Figure 9: The expression levels of IL-6, IL-8 and eotaxin-1 genes in BEAS-2B cell groups incubated with Dex (1 pM) for varying times before and after inflammation development.

Figure 10: The effects of YLE and MLE extracts on mRNA expression of proinflammatory IL-6 produced in response to LPS in BEAS-2B cells. *p<0.001, **p<0.05 comparison with control group, ^#p<0.001 comparison with LPS group. (n=2).

Figure 11: The effects of YLE and MLE extracts on mRNA expression of proinflammatory IL-8 produced in response to LPS in BEAS-2B cells. *p<0.001 comparison with control group, ^#p<0.001 comparison with LPS group. (n=2).

Figure 12: The effects of YLE and MLE extracts on mRNA expression of proinflammatory MCP-1 produced in response to LPS in BEAS-2B cells. *p<0.001, **p<0.01, ***p<0.05 comparison with control group, ^#p<0.001, ^##p<0.05 comparison with LPS group. (n=2).

Figure 13: The effects of pure compounds contained within extracts on mRNA expression of proinflammatory IL-6 produced in response to LPS in BEAS-2B cells. *p<0.001, **p<0.01, ***p<0.05 comparison with control group, ^#p<0.001 comparison with LPS group. (n=2).

Figure 14: The effects of pure compounds contained within extracts on mRNA expression of proinflammatory IL-8 produced in response to LPS in BEAS-2B cells. *p<0.001 comparison with control group, ^#p<0.001 comparison with LPS group. (n=2).

Figure 15: The effects of pure compounds contained within extracts on mRNA expression of proinflammatory MCP-1 produced in response to LPS in BEAS-2B cells. *p<0.001, **p<0.01, ***p<0.05 comparison with control group, #p<0.001, ##p<0.05 comparison with LPS group. (n=2).

Figure 16: The effects of pure compounds and YLE and MLE extracts on the level of IL-6 protein released from LPS-induced BEAS-2B cells into the culture medium. *p<0.001, **p<0.01 comparison with control group, ^#p<0.001 comparison with LPS group. (n=3).

Figure 17: The effects of pure compounds and YLE and MLE extracts on the level of IL-8 released from LPS-induced BEAS-2B cells into the culture medium. *p<0.001, **p<0.05 comparison with control group, ^#p<0.01 comparison with LPS group. (n=3).

Figure 18: The effects of pure compounds and YLE and MLE extracts on the level of MCP-1 released from LPS-induced BEAS-2B cells into the culture medium. *p<0.01, **p<0.05, ***p<0.001 comparison with control group. (n=3).

Figure 19: The effects of YLE and MLE extracts on TLR4 mRNA expression in LPS- induced BEAS-2B cells. *p<0.001 comparison with control group, #p<0.001 comparison with LPS group. (n=2).

Figure 20: The effects of pure compounds contained within extracts on TLR4 mRNA expression in LPS-induced BEAS-2B cells. *p<0.001 comparison with control group, #p<0.001 comparison with LPS group (n=2).

Figure 21: Western blot images showing the effects of YLE and MLE extracts on TLR4 protein expression in LPS-induced BEAS-2B cells.

Figure 22: Western blot images showing the effects of GAc, EGCG and ECG compounds on TLR4 protein expression in LPS-induced BEAS-2B cells.

Figure 23: Western blot images showing the effects of CIAc on TLR4 protein expression in LPS-induced BEAS-2B cells.

Figure 24: The effects of YLE and MLE extracts on CCR3 mRNA expression in LPS- induced BEAS-2B cells. *p<0.001 comparison with control group, ^#p<0.001 comparison with LPS group. (n=2).

Figure 25: The effects of pure compounds contained within extracts on CCR3 mRNA expression in LPS-induced BEAS-2B cells. *p<0.001 comparison with control group, ^#p<0.001 comparison with LPS group. (n=2). Figure 26: The effects of YLE and MLE extracts on the mRNA expression of Eotaxin-1 in LPS-induced BEAS-2B cells. *p<0.001 comparison with control group, ^#p<0.001 comparison with LPS group (n=2).

Figure 27: The effects of pure compounds contained within extracts on Eotaxin-1 mRNA expression in LPS-induced BEAS-2B cells. *p<0.001 comparison with control group, ^#p<0.001 comparison withEPS group. (n=2).

Figure 28: Western blot images showing the effects of YLE and MLE extracts on CCR3 protein expression in LPS-induced BEAS-2B cells.

Figure 29: Western blot images showing the effects of GAc, EGCG and ECG compounds on CCR3 protein expression in LPS-induced BEAS-2B cells.

Figure 30: Western blot images showing the effects of CIAc on CCR3 protein expression in LPS-induced BEAS-2B cells.

Figure 31: Western blot images showing the effects of YLE and MLE extracts on Eotaxin-1 protein expression in LPS-induced BEAS-2B cells.

Figure 32: Western blot images showing the effects of GAc, EGCG and ECG compounds on Eotaxin-1 protein expression in LPS-induced BEAS-2B cells.

Figure 33: Western blot images showing the effects of YLE and MLE extracts on p-STATl (Tyr701) protein level in LPS-induced BEAS-2B cells.

Figure 34: Western blot images showing the effects of GAc, EGCG and ECG compounds on p-STATl (Tyr701) protein level in LPS-induced BEAS-2B cells.

Figure 35: Western blot images showing the effects of CIAc on p-STATl (Tyr701) protein level in LPS-induced BEAS-2B cells.

Figure 36: Western blot images showing the effects of YLE and MLE extracts on p-STAT3 (Tyr705) protein level in LPS-induced BEAS-2B cells.

Figure 37: Western blot images showing the effects of GAc, EGCG and ECG compounds on p-STAT3 (Tyr705) protein level in LPS-induced BEAS-2B cells.

Figure 38: Western blot images showing the effects of CIAc on p-STAT3 (Tyr705) protein level in LPS-induced BEAS-2B cells.

Figure 39: Western blot images showing the effects of YLE and MLE extracts on SOCS3 protein expression in LPS-induced BEAS-2B cells. Figure 40: Western blot images showing the effects of GAc, EGCG and ECG compounds on SOCS3 protein expression in LPS-induced BEAS-2B cells.

Figure 41: Western blot images showing the effects of CIAc on SOCS3 protein expression in LPS-induced BEAS-2B cells.

Detailed Description of The Invention

The invention is related to the use of Carob tree (Ceratonia siliqua) leaf extracts and/or gallic acid (GAc), (-) epigallocatechin gallate (EGCG), (-)-epicatechin gallate (ECG) and chlorogenic acid (CIAc) phenolic compounds contained within extracts as an anti inflammatory and anti-asthmatic agent which have therapeutic effect in the treatment of inflammatory diseases associated with inflammation, in particular airway inflammation occurring in allergic asthma.

Carob tree (Ceratonia siliqua) leaf extracts and/or gallic acid (GAc), (-) epigallocatechin gallate (EGCG), (-)-epicatechin gallate (ECG) and chlorogenic acid (CIAc) compounds contained within extracts of the invention are used in the treatment of inflammatory diseases related to inflammation, including gastrointestinal diseases such as rheumatoid arthritis, tendonitis (tendon inflammation), polymyalgia rheumatic, rheumatic gout disease, inflammation-related cardiovascular diseases, cancer, diabetes, obesity, inflammation-related gastritis, diarrhea and gastroenteritis and inflammatory intestinal diseases such as Crohn's and ulcerative colitis as well as asthma.

In addition to carob tree leaves, due to being sources of phenolic compounds and their high antioxidative properties, its fruits, seed envelopes, seeds and/or barks can also be used as an anti-inflammatory and anti-asthmatic agent in the treatment of inflammatory diseases related to inflammation, such as asthma, in particular in the treatment of airway inflammation occurring in allergic asthma. However, since the leaves of the carob tree have more polyphenolic content and higher antioxidant capacity than other parts of the carob tree (fruit, seed envelope, seed and bark), the leaves of the carob tree are richer in polyphenols and are a better source of antioxidant compared to other parts of the plant.

In the invention, 70% methanol and 70% ethanol extracts were prepared from the leaves belonging to mature (bearing fruits for years) and young (not bearing fruits yet) Ceratonia siliqua trees and phenolic contents of extracts were compared with each other. Thus, it was aimed to determine whether there is a difference between the leaves of mature and young C. siliqua trees belonging to the same species in terms of phenolic compound amount and content. For this purpose, Mature tree Leaf Methanol (MLM), Young tree Leaf Methanol (YLM), Mature tree Leaf Ethanol (MLE) and Young tree Leaf Ethanol (YLE) extracts were prepared from the leaves belonging to mature and young C. siliqua trees. Then, total phenol and total flavanoid amounts in the prepared extracts were determined. As a result of the analysis, it was detected that it is as MLM <YLM < MLE < YLE when sorted in terms of total phenol amount C. siliqua leaf extracts contain from the lowest to the highest and also as MLM < YLM < MLE <-YLE terms of total flavonoid amount. Thus, it was determined that 70% ethanol extracts have more phenol and flavonoid amount than 70% methanol extracts and YLE extract has maximum total phenol and flavonoid amount (Table 1). Phenolic compound contents of the extracts were determined by LC/MS/MS analysis. As a result of content analyses performed with LC/MS/MS, it was determined that gallic acid (GAc), chlorogenic acid (CIAc), epigallocatechin-3-gallate (EGCG) and epicatechin-3-gallate (ECG) compounds are found in YLE extract; GAc, EGCG and ECG compounds are found in YLM, MLE and MLM extracts (Table 2). In consequence of the analysis, it was detected that YLE extract differs from other extracts both in terms of CIAc content and having the highest amount of phenol and flavonoids. Thus, YLE extract was determined as C. siliqua leaf extract whose effect will be investigated within the scope of the invention. In addition to this, it was decided to investigate the OYE extract, which does not contain CIAc and has the second highest amount of phenol and flavonoid, within the scope of the study. Thus, it was aimed to determine whether two C. siliqua leaf extracts, which differ in their CIAc content, will also differ in terms of their effects on allergic airway inflammation.

The effects of C. siliqua leaf extracts (YLE and MLE extracts) and GAc, EGCG, ECG and CIAc compounds detected to be found within the extracts, the effect of which was investigated within the scope of the invention, on the viability of BEAS-2B cells were analyzed by using MTT method in a dose and time dependent manner. As a result of MTT analysis, 12-hours incubation of BEAS-2B cells with the concentrations of 25 and 50 pg/mL YLE extract (Figure 1), 25 and 50 pg/mL MLE extract (Figure 2), 5 and 10 pM GAc (Figure 3), 10 and 20 pM EGCG (Figure 4), 40 and 80 pM ECG (Figure 5) and 40 and 80 pM CIAc (Figure 6) was determined as the optimal time and concentrations which do not reduce the viability of BEAS-2B cells.

Within the scope of the invention, the optimal incubation times and application methods of LPS, Dexamethasone (Dex), YLE and MLE extracts and pure compounds determined to be found within the extracts to be used during the studies were determined according to expression levels of IL-6, IL-8 and eotaxin-1 genes related to inflammation as a result of mRNA expression studies made by using qRT-PCR. Accordingly, it has been observed that 12-hours incubation period with 2 pg/mL LPS increased at the highest rate expression levels of IL-6, IL-8 and eotaxin-1 genes related to inflammation in BEAS-2B cells and thus, it was considered as optimal incubation period in which inflammation was induced (Figure 7). With the invention, the optimal incubation period to reveal therapeutic effects of YLE and MLE extracts in inflammation was determined to be 12-hours period in which mRNA expressions of IL-6, IL-8 and eotaxin-1 genes were inhibited at the highest rate (Figure 8). Accordingly, as the optimal mode of administration and incubation time to reveal therapeutic effects of YLE and MLE in LPS-induced inflammation; it was determined that firstly inflammation was developed with LPS (2 pg/mL, 12 hours) in BEAS-2B cells and then the cells were incubated for another 12 hours with 25 and 50 pg/mL concentrations of YLE and MLE extracts (Figure 8). The optimal incubation time and mode of administration determined in the extracts are also regarded as the optimal incubation time and mode of administration for pure compounds (GAc, EGCG, ECG and ClAc) contained in extracts. The concentrations of extracts and pure compounds to be used in the studies conducted within the scope of the invention were specified according to the results of MTT test obtained in consequence of 12-hours incubation of YLE and MLE and pure compounds contained in the extracts with BEA-2B cells. Accordingly, the two highest concentrations of the extracts and its pure compounds indicated above, which were determined by MTT analysis and did not reduce the viability of BEAS-2B cells in 12-hours incubation, were determined as the most appropriate extract and pure compound concentrations to be used within the scope of the invention (Figures 1-6). With the invention, as the most appropriate mode and time of administration of Dex to be used as a positive control in the inhibition of LPS-induced inflammation in BEAS-2B cells; it was determined that firstly inflammation was developed with LPS (2 pg/mL, 12 hours) in BEAS- 2B cells, and then the cells were incubated for another 4 hours with Dex (lpM) in which mRNA expressions of IL-6, IL-8 and eotaxin-1 genes were most inhibited (Figure 9). Thus, ELISA, qRT-PCR and western blot analyses were performed by determining the most appropriate incubation times and modes of administration of LPS, Dex, YLE and MLE extracts and pure compounds.

With the invention, it has been identified that C. siliqua leaf extract and/or its pure compounds have anti-inflammatory and anti-asthmatic effects in BEAS-2B cells that develop inflammation with LPS and mimic the clinical conditions of asthma , and thus, the possibilities of C. siliqua leaf extract and its pure compounds to be used as a potential anti inflammatory and anti-asthmatic agent in the treatment of asthma disease have been revealed for the first time. In the invention, the effects of C. siliqua leaf extract and its pure compounds on possible signal pathways contributing to progress of asthma were compared with the results obtained from Dexamethasone (Dex) used as an anti-asthmatic drug in asthma treatment in clinic and as positive control in the study and it was identified that C. siliqua leaf extract and its pure compounds have inhibitory effects on signal pathways which cause asthma pathogenesis similarly to Dex or more effectively than Dex and are indicated below. Within the scope of the invention, in the BEAS-2B cells which develop inflammation with LPS, the effects of MLE and YLE extracts prepared from the leaves of mature and young C. siliqua trees belonging to the same species and pure compounds found in the extracts on mRNA transcriptions and protein levels released into the culture medium of proallergic (IL-4, IL-5, IL-13 and GM-CSF) and proinflammatory (IL-6, IL-8 and MCP-1) cytokines and chemokines were analyzed by qRT-PCR and ELISA analyses, respectively.

As a result of qRT-PCR analyses conducted in respect of the invention, it has been shown that LPS effectivelt induced mRNA transcriptions of proinflammatory cytokines (IL-6) and chemokines (IL-8 and MCP-1) in BEAS-2B cells of LPS, and that the extract and/or pure compounds used in the study reduced mRNA levels of proinflammatory cytokines and chemokines which increased with LPS, similarly to positive control Dex (Figure 10-12). It has been detected that YLE and MLE extracts inhibited mRNA expressions of IL-6, IL-8 and MCP-1 which increased with LPS similarly and there was no difference between them in terms of effect. It was observed that both extracts decreased IL-6 mRNA level, which increased with LPS, in a dose dependent manner, however, other extract concentrations other than the 50 pg/mL concentration of the YLE extract could not bring the increased IL-6 mRNA level to the level in the control group (Figure 10). Nevertheless, the concentrations of 25 and 50 pg/mL of both extracts were found to effectively reduce proinflammatory IL-8 mRNA transcription, which increased with LPS, similar to the positive control Dex (Figure 11). Likewise, it was found that both extracts reduced proinflammatory MCP-1 mRNA transcription, which increased with LPS, in a dose dependent manner and that 50 pg/mL of concentrations effectively inhibited MCP-1 mRNA expression similarly to positive control Dex (Figure 12). It has been shown that among the pure compounds contained in the extracts, the level of IL-6 mRNA, which increased with LPS, was reduced by 80 pM concentration of ECG (Figure 13); the level of IL-8 mRNA was reduced by all pure compound concentrations (Figure 14); MCP-1 mRNA level was effectively reduced by all concentrations of ECG and Cl Ac compounds (Figure 15). On the other hand, it has been shown that all pure compound concentrations contained in the extracts effectively inhibited proinflammatory IL-8 mRNA expression, which increased with LPS, similarly to positive control Dex (Figure 14).

As a result of the qRT-PCR studies carried out within the scope of the invention, it was determined that LPS did not induce mRNA expressions of proallergic (IL-4, IL-5, IL-13 and GM-CSF) cytokines in BEAS-2B cells.

As a result of the ELISA studies carried out within the scope of the invention, it was shown that LPS effectively increased the amount of all proinflammatory cytokines (IL-6) and chemokines (IL-8 and MCP-1) released into the culture medium from BEAS-2B cells compared to the control group (Figure 16-18). Additionally, it was determined that YLE and MLE extracts could not reduce the level of IL-6, which increased with LPS in the culture medium with LPS, similar to the positive control Dex (Figure 16), whereas other extract concentrations other than 25 pg/mL concentration of MLE extract effectively reduced the level of IL-8 released into culture medium (Figure 17) and also 25 pg/mL concentrations of both extracts effectively reduced the level of MCP-1 (Figure 18) released into the culture medium similar to the positive control Dex. It was observed that YLE and MLE extracts had a similar effect on IL-6, IL-8 and MCP-1 levels, which increased with LPS in culture medium. Nearly all of the pure compounds contained in the extracts were observed to effectively reduce the level of proinflammatory cytokines and chemokines, which exhibit an increase with LPS in culture medium (Figure 16-18). In particular, ClAc reduced the levels of IL-6 (Figure 16) and IL-8 (Figure 17), and GAc reduced the level of MCP-1 (Figure 18) more effectively than positive control Dex.

Additionally, as a result of ELISA studies, it was determined that LPS did not induce the release of proallergic (IL-4, IL-5, IL-13) cytokines from BEAS-2B cells into the culture medium, similar to the results obtained from the qRT-PCR analysis. Thus, the results obtained from ELISA and qRT-PCR studies are consistent and the findings obtained showed that extract and/or pure compounds whose anti-inflammatory/anti-allergic effects were investigated have a strong anti-inflammatory potential.

In the invention, the effects of C. siliqua leaf extracts and its pure compounds on TLR4 actavation stimulated by LPS in BEAS-2B cells were determined with qRT-PCR and western blot analyses. As a result of qRT-PCR analysis conducted, it was observed that TLR4 mRNA expression effectively increased in BEAS-2B cells stimulated by LPS; YLE and MLE extracts potently inhibited TLR4 mRNA expression, which increased with LPS, in a dose dependent manner (Figure 19). In particular, it was determined that concentrations of 50 pg/mL of both extracts inhibited mRNA expression of TLR4 similar to the positive control Dex. It has been shown that all pure compounds contained in the extracts potently inhibited the expression of TLR4 mRNA induced by LPS similarly to positive control Dex (Figure 20). Similar to the results obtained from qRT-PCR analysis, in western blot analysis it was also observed that TLR4 protein expression in BEAS-2B cells stimulated with LPS increased effectively (Figures 21-23). It was determined that YLE and MLE extracts inhibited TLR4 protein expression, which increased with LPS, more effectively than the positive control Dex in a dose dependent manner (Figure 21). It was observed that GAc (Figure 22) and ClAc (Figure 23) compounds contained in the extracts inhibited LPS-induced TLR4 protein expression more than other pure compounds in a dose-dependent manner. Thus, the results obtained from western blot analysis were consistent the results obtained from qRT-PCR analysis and it was revealed that the extract and/or its pure compounds, whose effects have been investigated, have a strong inhibitory effect on TLR4 receptor playing an important role in the airway inflammatory process in asthma.

As a result of the qRT-PCR and western blot studies carried out with the invention, it was revealed that both mRNA and protein expression of TLR4 effectively increased in BEAS-2B cells stimulated with LPS, however, C. siliqua leaf extracts and pure compounds effectively inhibited TLR4 activation stimulated with LPS. Also, as a result of ELISA and qRT-PCR studies conducted within the scope of the invention, it was disclosed that both the protein level released into culture medium and mRNA expression of IL-8 effectively increased in BEAS-2B cells stimulated with LPS and that C. siliqua leaf extracts and pure compounds potently inhibited both cellular secretion and mRNA expression of IL-8, which increased with LPS. With these obtained results, it was revealed that the cellular secretion and expression of IL-8 increased by stimulating TLR4 signal by LPS and C. siliqua leaf extracts and pure compounds inhibited the cellular secretion and mRNA expression of IL-8 by blocking LPS- induced TLR4 activation. Thus, it has been demonstrated for the first time that C. siliqua leaf extracts and/or its pure compounds can provide an important treatment strategy as a therapeutic agent in asthmatic airway inflammation by regulating IL-8 response via the TLR4 signal pathway. In the invention, the effects of C. siliqua leaf extracts and pure compounds on the CCR3 receptor and eotaxin-1 binding to this receptor in LPS-induced BEAS-2B cells were determined by qRT-PCR and western blot analyses.

As a result of qRT-PCR analyses carried out regarding the invention, it was found that mRNA expression of CCR3 was effectively increased in BEAS-2B cells stimulated by LPS (Figure 24-25); C. siliqua leaf extracts (Figure 24) and pure compounds (Figure 25) strongly inhibited CCR3 mRNA expression, which increased with LPS, similar to the positive control Dex. YLE and MLE extracts of C. siliqua was observed to effect CCR3 expressions which increased with LPS. Also, as a result of qRT-PCR analyses carried out regarding the invention, it was found that mRNA expression of eotaxin-1 was effectively increased in BEAS-2B cells stimulated by LPS (Figure 26-27); C. siliqua leaf extracts (Figure 26) and pure compounds (Figure 27) strongly inhibited eotaxin-1 mRNA expression, which increased with LPS, similar to the positive control Dex. It was observed that YLE and MLE extracts of C. siliqua had an effect on eotaxin-1 mRNA expression, which increased with LPS, in a dose dependent manner (Figure 26).

As a result of the western blot analyses carried out regarding the invention, it was found that CCR3 protein expression in LPS-stimulated BEAS-2B cells increased (Figure 28-30); other extract concentrations of YLE extract except for the concentration of 25 pg/rnL inhibited CCR3 protein expression, which increased with LPS, more effectively than the positive control Dex (Figure 28). Among the pure compounds contained in the extracts; concentrations of EGCG of 20 mM (Figure 29), ECG of 80 pM (Figure 29), and ClAc of 40 and 80 pM (Figure 30) decreased CCR3 protein levels, which increased with LPS, more effectively than Dex. Likewise, as a result of western blot analyses carried out regarding the invention, it was found that eotaxin-1 protein expression was increased in BEAS-2B cells stimulated by LPS (Figure 31-32); all concentrations of C. siliqua leaf extracts (Figure 31) and pure compounds (Figure 32) inhibited eotaxin-1 protein expression, which increased with LPS, more effectively than positive control Dex.

The results obtained from qRT-PCR and western blot analyzes performed within the scope of the invention showed that C. siliqua leaf extracts and pure compounds have a potent inhibitory effect on the CCR3 receptor closely associated with allergy and the Eotaxin-1 ligand binding to this receptor. Hence, with the invention, it has been revealed for the first time that C. siliqua leaf extracts and/or its pure compounds can be developed as a therapeutic agent in the treatment of asthma and can provide a useful treatment strategy by blocking CCR3 activation which is induced by LPS and by regulating the production of eotaxin-1 associated with asthmatic inflammation shown as a possible target in drug development.

With the invention, it was observed that both cellular secretion and mRNA expression of IL-8 were increased by stimulating the TLR4 signal by LPS, and in parallel with the increase in IL- 8, both mRNA and protein expressions of eotaxin-1 increased effectively. It was revealed that C. siliqua leaf extracts and pure compounds inhibited cellular secretion and mRNA expression of IL-8 by blocking LPS-induced TLR4 activation, and in parallel with IL-8 inhibition, they strongly inhibited mRNA and protein expressions of eotaxin-1.

In the invention, the effects of C. siliqua leaf extracts and pure compounds on the phosphorylations of STAT1 and STAT3 proteins in LPS-induced BEAS-2B cells were determined by western blot analysis. As a result of the western blot analysis, it was observed that the phosphorylation levels of STAT1 and STAT3 proteins in BEAS-2B cells stimulated with LPS increased (Figure 33-38) and STAT pathway was activated.lt was observed that YLE and MLE extracts decreased the phosphorylation of STAT1 protein, which increased with LPS, more effectively than the positive control Dex in a dose dependent manner while 50 pg/mL concentration of the YLE extract had the strongest inhibition (Figure 33). Among the pure compounds contained in the extracts, ECG (Figure 34) and Cl Ac (Figure 35) were determined to have a strong inhibitory effect on the phosphorylation of STAT1 protein. It was observed that YLE and MLE extracts decreased the phosphorylation of STAT3 protein, which increased with LPS, similarly to the positive control Dex in a dose dependent manner while 50 pg/mL concentration of the MLE extract reduced the phosphorylation of STAT3 protein more effectively than other extract concentrations (Figure 36). Among the pure compounds contained in the extracts, EGCG (Figure 37), ECG (Figure 37) and ClAc (Figure 38) were determined to inhibit the phosphorylation of STAT3 protein more effectively than positive control Dex. Additionally, YLE and MLE extracts were found to be more effective in inhibiting the phosphorylation of the STAT1 protein. The results obtained from Western blot analyses showed that C. siliqua leaf extracts and/or pure compounds have an inhibitory effect on the phosphorylations of STAT1 and STAT3 proteins that mediate allergic responses in asthma. Thus, it has been demonstrated with the invention that C. siliqua leaf extracts and/or pure compounds diminished STAT activation by blocking the signaling pathway dependent on LPS-induced TLR4 activation. With the obtained results, it was revealed for the first time that in airway inflammation induced by endotoxines, such as LPS, C. siliqua leaf extracts and/or its pure compounds can prevent destructive asthmatic reactions by disrupting TLR4- STAT1/3 signal paths and consequently by reducing IL-8 response and subsequent eotaxin-1 activation.

In the invention, the effects of C. siliqua leaf extracts and pure compounds on the expression of SOCS3 protein in LPS-induced BEAS-2B cells were determined by western blot analysis. As a result of the western blot analysis, it was observed that SOCS3 protein expression decreased in BEAS-2B cells stimulated by LPS, while YLE and MLE extracts increased the expression of the SOCS3 protein, which decreased with LPS, by repairing oppositely (Figure 39). It was observed that 50 pg/mL concentration of MLE extract increased the expression of SOCS3 protein, which decreased with LPS, more effectively than other extract concentrations. Among the pure compounds contained in the extracts, it was determined that concentrations of EGCG of 10 mM (Figure 40) and Cl Ac of 40 pM (Figure 41) increased the expression of SOCS3 protein compared to the cell group treated with LPS. The effects of C. siliqua leaf extracts and pure compounds on the expression of SOCS3 protein in LPS-stimulated BEAS-2B cells were found to be consistent with their effects on STAT3 phosphorylation. The results obtained from the western blot analysis performed within the scope of the invention showed that by increasing the expression of SOCS3 protein, C. siliqua leaf extracts and pure compounds disrupted the STAT3 signal pathway positively regulated by LPS.

Consequently, with the invention, Ceratonia siliqua leaf extracts and pure compounds were revealed in vitro for the first time to have a therapeutic potential in airway inflammation associated with endotoxin. As a result of the studies carried out within the scope of the invention, it was determined that C. siliqua leaf extracts and/or pure compounds reduced effectively the mRNA and protein expressions of proinflammatory cytokines and chemokines, which increased with LPS, by inhibiting TLR4, CCR3 and STAT1/3 activations in LPS- stimulated airway epithelial cells and increasing the expression of SOCS3 protein. Thus, it was revealed for the first time that concentrations of C. siliqua leaf extracts and/or pure compounds which are non-toxic for cellular model of allergic asthma can suppress the production of IL-8 and subsequent increase of Eotaxin-1 by blocking TLR4 signal pathway causing asthma pathogenesis, and thus they can provide an useful treatment strategy by being developed as an anti-inflammatory and anti-asthmatic agent in the treatment of asthma disease.

All of the methods used in creating the invention are disclosed below:

Preparation of Ceratonia siliqua (C. siliquaiheaf Extracts Within the scope of the invention, 70% methanol and 70% ethanol extracts from the leaves belonging to two different Ceratonia siliqua trees, mature (bearing fruits for years) and young (not bearing fruits yet) trees, were prepared and the extracts were compared in terms of phenolic compound contents. Thus, it was aimed to determine whether there is a difference between the leaves belonging to mature and young C. siliqua trees in terms of phenolic compound content.

Preparation of 70% Methanol Extracts

The leaves belonging to two differentC. siliqua trees, young and mature, were powdered by grinding separately on a mechanical mill. 100 grams were taken from the obtained C. siliqua leaf powders and extracted in the 70% methanol (3x0.5 L) at 37°C for 3 days. Consequently, mixture of 10.5 ml of methanol and 4.5 ml of distilled water was used for 1 g of C. siliqua leaf powder. On the other hand, the range of 3.5 ml-14mL of methanol and 1.5 ml-6ml of distilled water for 1 g of C. siliqua leaf powder can be used for preparing 70% methanol leaf extract from C. siliqua tree. Then, methanol was removed from the extracts under vacuum by combining obtained liquid extracts and then the extracts were lyophilised. Thus, extract samples of dry Young tree Leaf Methanol (YLM) and Mature tree Leaf Methanol (MLM) were obtained. Lyophilized samples were stored at-20°C until analysis.

Preparation of 70% Ethanol Extracts

The leaves belonging to two differentC. siliqua trees, young and mature, were powdered by grinding separately on a mechanical mill. 100 grams were taken from the obtained C. siliqua leaf powders and extracted in the 70% ethanol (3x0.5 L) at 37°C for 3 days. Consequently, mixture of 10.5 mL of ethanol+4.5 mL of distilled water was used for 1 g of C. siliqua leaf powder. On the other hand, the range of 3.5 ml-14mL of ethanol and 1.5-6 mL of distilled water for 1 g of C. siliqua leaf powder can be used for preparing 70% ethanol leaf extract from C. siliqua tree. Then, ethanol was removed from the obtained liquid extracts under vacuum and then the extracts were lyophilised. Thus, extract samples of dry Young tree Leaf Ethanol (YLE) and Mature tree Leaf Ethanol (MLE) were obtained. Lyophilized samples were stored at -20°C in order to be used in the study.

Quantification of Total Phenol Amount in C. siliqua Leaf Extracts

The total amount of phenol contained in the extracts was calculated by using Folin-Ciocalteu method as equivalent to gallic acid. To a 10 mL vessel containing 6 mL of distilled water, were added 100 pL of sample solution and 500 pL of Folin-Ciocalteu reagent. After 1 minute, it was completed with water to 10 mL by adding 1.5 mL of 20% aqueous NaiCC _. Reagent mixture which does not contain extract was used as control. After incubated for 2 hours at 25°C, its absorbance was measured at 760 nm and total phenolic substance amounts were calculated by using the gallic acid calibration curve and the results were given in mg GAE/g_extract as equivalent to gallic acid (GAE). The experiments were performed in three parallel and the result were given as mean values.

Quantification of Total Flavonoid Amount in C. siligua Leaf Extracts

1 ml of extract was mixed with 0.3 mL of 5% NaNCE solution at t=0 minute, after addition of 0.3 mL of 10% AICI_3.6H₂O solution at t=5 minutes, at t=6 minutes, 2 ml of 1 M NaOH solution was added and mixed by adding 2.4 ml of water. Absorbance against blank was measured at 510 nm and calculated by using the calibration curve of Catechin. Total flavonoid amounts of the extracts were given in (CE) mg CE/g_extract as equivalent to catechin. All measurements were made in 3 parallels and mean values were taken.

Determination of Phenolic Compound Contents of C. siligua Leaf Extracts by LC/MS/MS Analysis

The contents of gallic acid (GAc), epicatechin-3-gallate (ECG), epigallocatechin-3-gallate (EGCG), catechin (C), epicatechin (EC), epigallocatechin (EGC), ellagic acid (EAc), theophylline (T) and chlorogenic acid (CIAc) in the extracted prepared from C. siliqua leaves were analyzed by using LC/MS/MS device. In LC/MS/MS analysis, the contents of compounds in the extracts were detected exactly by comparing mass/weight (m/w) ratios and amounts were detected againts standards in ppb level. Mean values were given by analyzing all standard and exemplary solutions for three times.

LC/MS/MS Analysis Conditions;

Device: Shimadzu LC/MS 8040

Ionization Mode: Electrospray Ionization (ESI), negative ionization

Column: Restek Cl 8 (3.0 x 70 mm, 3 pm particle diameter)

Column Temperature: 40°C

Flow rate: 0.4 mL/min

Mobile Phase: A) water with 1% acedic acid; B) methanol with 1% acedic acid Flow Plan: Starting with 30%B, then increasing to 60% B in the first 3 min, increasing to 60% B next 10 min

BEAS-2B Cell Culture

Human bronchial airway epithelial cell line used in the invention, BEAS-2B cells were cultured in a 75 cm² flask in the RPMI-1640 medium containing 10% FBS, 2 mM L- glutamine, 100 U/ml penicillin, 100 pg/ml streptomycin at 37°C and in the medium with 5% C0₂ until reaching 70-75% confluence. Medium of the cells was changed every two days and their viabilities, proliferation rates and morphological structures were monitored with inverted microscope. Sufficiently proliferated cells were removed from bottom of culture plate by using 0.25% Trypsin/EDTA. Some of the cells collected from the culture plates were frozen at -80 degrees by taking their cryo, and the culture was continued by passaging a portion to another flask.

MTT dimethyl thiazolyl-2)2,5-diphenyl tetrazolium bromide) Viability Test

MTT experiment is based on the principle of converting yellow colored tetrazolium salt (3- (4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) into purple colored formazan crystalline by being reduced by succinate dehydrogenase enzyme found in mitochondria of living cells. The effects of test substances [C. siliqua leaf extracts (YLE and MLE extracts) and pure compounds (GAc, ECG, EGCG and ClAc) contained in the extracts], the effect of which was investigated within the scope of the invention, on the viability of BEAS-2B cells were analyzed by using MTT test in a dose and time dependent manner. The YLE and MLE extracts whose effects were investigated within the scope of the invention were dissolved in DMSO, while pure compounds were dissolved in deionized water or PBS buffer according to their solubility.

In the MTT analysis, BEAS-2B cells were planted in a 96-well culture plate in 100 mΐ RPMI- 1640 medium at 20.000 cells in each well. Cells were incubated in a sterile oven with C0₂ for 24 hours in order to hold on the bottom of culture plate. At the end of the incubation time, the media in the wells were removed and the cells were incubated for 6, 12 and 24 hours with different concentrations of test substances whose effects were investigated. Concentrations between 12.5-150 pg/mL (12.5, 25, 50, 75, 100, 125 and 150 pg/mL) for the extracts whose effects on cell viability were investigated and serially increasing concentrations between 1.25- 80 mM (1.25, 2.5, 5, 10, 20, 40 and 80 mM) for pure compounds were used. 3 wells were used for each concentration of each test substance and 100 pL of each concentration was added to the cells. Only medium was added to 3 wells containing cells but not test substance, these wells were used as positive control in the study. Only medium was added to 3 wells not containing cells and test substance and these wells were used as negative control in the study. The examined extracts dissolve in DMSO, whereas the final concentration of DMSO in the culture medium is lower than 1%. Additionally, a medium containing DMSO amount in the highest concentration of the extracts was added to 3 wells containing cells but not test substance, and these wells were used as the DMSO control group in the study. At the end of the specified incubation times, all media containing and not containing test substances were removed from the medium and 10 mΐ of MTT reagent in 100 mΐ of medium was added to the cells. Then, cells were allowed to incubate for 2 hours at 37°C. Thereafter, purple colored formazan product formed was allowed to dissolve by adding 100 mΐ of DMSO as a solvent solution to each well. The absorbance value of the formazan in each well was measured in a microplate reader at a wavelength of 570 nm and cell viability % was calculated from the measured absorbance values.

Enzyme Linked Immunosorbent Assav-ELISA

In BEAS-2B cells that develop inflammation with LPS, the effects of the test substances, whose effects were investigated, on cellular secretion levels of pro-allergic (IL-4, IL-5, IL-13 and granulocyte macrophage-colony stimulating factor; GM-CSF) and pro-inflammatory (IL- 6, IL-8 and monocyte chemotactic protein; MCP-1) cytokines and chemokines in culture medium were determined by using ELISA method.

For this purpose, BEAS-2B cells were plated in a 6 cm² culture plate in 2 mL of RPMI-1640 medium with 5xl0⁵ cells in each well and inoculated in a sterile oven with C0₂ for 24 hours to hold on the bottom of the culture plate. After 24 hours, media in wells were removed from the medium and cells were incubated with LPS (2 pg/mL) to trigger inflammation for 12 hours. Cells in which inflammation was induced with LPS were incubated again with test substances at doses determined by MTT analysis for 12 hours while the cell group used as positive control in the study was incubated with Dexametazon for 4 hours. At the end of the incubation period, the media in the wells were collected and cell-free culture media were stored at -20°C until the time to be experimented. Each of the proinflammatory and proallergic cytokines and chemokines released into the culture medium was measured according to the kit procedure by using the materials in the commercial ELISA kits and analyzed in the Microplate reader. All analyzes were repeated 3 times and the concentration of each protein was calculated from the standard curve.

RNA Isolation, cDNA Synthesis and Quantitative Real-Time-Polymerase Chain Reaction (qRT-PCR) Analysis

The mRNA transcript levels of TLR4, CCR3, Eotaxin-1, IL-4, IL-5, IL-6, IL-8, IL-13, GM- CSF, MCP-1 and control genes which play a role in the pathogenesis of asthma were determined quantitatively by using real-time PCR. For this, firstly, total RNAs were isolated from BEAS-2B cells. For total RNA isolation, commercially available Trizol kit (TriPure Isolation Reagent, Roche) was used and the protocol provided by the kit manufacturer was applied.For total RNA isolation, BEAS-2B cells were plated in a 6 cm² culture plate in 2 mL of RPMI-1640 medium with 5xl0⁵ cells in each well and inoculated in a sterile oven with CO2 at 37°C for 24 hours to hold on the bottom of the culture plate. After 24 hours, media in wells were removed from the medium and then, cells were incubated with LPS (2 pg/mL) to trigger inflammation for 12 hours. Then, cells in which inflammation was induced with LPS were incubated again with test substances at doses determined by MTT analysis for 12 hours while the cell group used as positive control in the study was incubated with Dexametazon (1 pM) for 4 hours. At the end of the incubation time, media containing LPS and test substances or LPS and Dexametasone in the wells were removed and the cells were washed once with cold PBS. After this step, all works were performed over ice. Then, 1 mL of cold PBS was added to the cells and the cells were collected in the sterile tube by scraping the cells from the bottom of culture plate with cell scraper. This process was repeated 3 times. Then, the cell suspension in the tube was centrifuged at 4°C at 4000 rpm for 10 min. At the end of the centrifuge, the supernatant was removed, 500 pL of trizol was added to the cell pellet and total RNA isolation was carried out by following the isolation protocol. The purity and amount of RNAs obtained were calculated spectrophotometrically by optical density measurement at 260 and 280 nm wavelengths.

Total RNA samples isolated from BEAS-2B cells were transformed into cDNA by reverse transcription method. To perform cDNA synthesis from total RNA, commercially available cDNA synthesis kit (Transcriptor High Fidelity cDNA Synthesis Kit) was used and cDNA synthesis was performed in accordance with the protocol provided by the kit manufacturer. For this, total RNA and kit mixtures belonging to each sample were separately incubated at appropriate temperatures for certain times, and thus reverse transcriptions of RNAs to cDNA were provided. Real-time-PCR was performed on the LightCycler 480 device in accordance with the protocol provided by the kit manufacturer by using primer-probes specific to the genes to be examined (LightCycler 480 Probes Master kit and RealTime ready Designer/Catalog Assays primer probes) with cDNA of each sample obtained. Thus, mRNA transcript levels of the examined genes were determined quantitatively. The results were normalized by the expression level of GAPDH control gene which is known to be expressed in each tissue. Thus, whether each examined gene was expressed in BEAS-2B cells or changes in expression level were determined. All analyses were performed in duplicate.

Western Blot Analysis

The effects of test substances whose effects were investigated on expression levels of TLR4, CCR3, Eotaxin-1, STAT1, p-STATl, STAT3, p-STAT3 and SOCS3 proteins, which play a role in the pathogenesis of asthma in BEAS-2B cells in which inflammation was induced by LPS, were determined by Western Blot analysis. The total cell lysate was used for TLR4, CCR3, Eotaxin-1, STAT1, p-STATl, STAT3,p-STAT3 and SOCS3 proteins. For this, firstly, BEAS-2B cells were plated in a 110 mm cell culture plate in 6 mL of RPMI-1640 medium with lxlO⁶ cells and inoculated in a sterile oven with CO2 at 37°C for 24 hours to hold on the bottom of the culture plate. After 24 hours, media in petri plates were removed from the medium and then, cells were incubated with LPS (2 pg/mL) to trigger inflammation for 12 hours. Then, cells in which inflammation was induced with LPS have incubated again with test substances at doses determined by MTT analysis for 12 hours while the cell group used as a positive control in the study was incubated with Dexametazon (1 mM) for 4 hours. At the end of the incubation times, protein isolation was made from the cells. For this, media containing LPS and test substances or LPS and Dexametasone in the petri plates were removed from medium and the cells were washed once with cold PBS. After this step, all works were performed over ice. Then, 1 mL of cold PBS was added to the cells and the cells were collected in the sterile tube by scraping the cells from the bottom of the culture plate with cell scraper. This process was repeated 3 times. The cell suspensions collected in the tube were centrifuged at 4000 rpm at 4°C for 10 minutes, and at the end of the centrifuge, cell pellets were obtained by removing supernatants. Then, total cell lysates were prepared from the obtained cell pellets.

Preparation of Total Cell Lysate

RIPA (radioimmunoprecipitation assay) lysis buffer containing protease and phosphatase inhibitor cocktail was used for the preparation of total cell lysates from BEAS-2B cells. According to the size of cell plate, 40-100 pL of RIP A (50mM Tris-HCl; pH 7.4, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 200 mM PMSF, 100 mM sodium orthovanadate (Na3V04) and protease inhibitor cocktail) solution was used.

Protein concentrations in total cell lysates were measured using Pierce™ BCA (Bicinchoninic acid) protein analysis kit (Thermo Scientific, Asheville, NC) in accordance with the protocol provided by the kit manufacturer.

40 pg of protein was loaded into gel wells from each protein sample, and proteins were electrophoresed in 8-15% SDS-PAGE according to the size of the examined protein. Then, the proteins were transferred to PVDF membrane by wet transfer method. Then, the membrane was subjected to blocking for 3 hours with TBS-T buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl and 0.1% Tween 20) containing %5 nonfat dry milk to prevent nonspecific bindings. After blocking, the membrane was removed from the milk powder medium by washing with TBS-T buffer 3 times for 10 minutes. The membrane was then incubated with a specific primer antibody (Anti -human; TLR4, CCR3, Eotaxin-1, STAT1, p- STAT1, STAT3, p-STAT3 or SOCS3) in a suitable dilution prepared in TBS-T at 4°C overnight. After incubation, non-specific bindings were removed from the medium by washing the membrane with TBS-T buffer 3 times. The membrane was then incubated with horseradish peroxidase (HRP) linked secondary antibody in appropriate dilution prepared in TBS-T for 1 hour at room temperature. At the end of the incubation time, the membrane was washed again with TBS-T buffer 3 times for 10 minutes, and after the washing process, the examined target protein was determined by using chemiluminescence detection reagents (ECL). The resulting chemiluminescence signals were imaged in a gel imaging system (Image Quant 350, GE Healthcare) connected to a computer with a suitable software. On the same membranes, appropriate sized control proteins (GAPDH, b-actin or b-tubulin proteins) selected according to the molecular weights of the examined target proteins were then imaged by using antibodies. Images of the bands obtained in the gel imaging system were analyzed using the Image J program, and the expression level of the examined target protein was normalized with the expression level of the control protein displayed on the same membrane.

Statistical Analysis

Data of each group in in vitro experiments were evaluated as mean ± Standard Deviation (SD). Statistical analyses were conducted using SPSS ver. 15.0 computer program. To determine the effect of C. siliqua leaf extract and pure compounds on airway inflammation and allergic responses in BEAS-2B cells, differences between the means of the treatment groups were analyzed by using One-way ANOVA post hoc Dunnett multiple comparison test. The differences were considered statistically significant at p<0.05, p<0.01 and p<0.001.

Table 1

*: mean ± SD

Table 2

Claims

1. The use of Carob tree (Ceratonia siliqua) leaf extracts and/or gallic acid (GAc), (-) epigallocatechin gallate (EGCG), (-)-epicatechin gallate (ECG) and chlorogenic acid (ClAc) compounds contained within the extracts as an anti-inflammatory and anti asthmatic agent which have a therapeutic effect in the treatment of inflammatory diseases related to inflammation, especially airway inflammation occurring in allergic asthma.

2. The use according to claim 1; wherein its fruits, seed envelopes, seeds and/or barks as well as the leaves of the Carob tree ( Ceratonia siliqua ) are also used as an anti inflammatory and anti-asthmatic agent with a therapeutic effect in the treatment of inflammatory diseases associated with inflammation, especially in the treatment of airway inflammation occurring in allergic asthma.

3. The use according to claim 1; wherein carob tree leaf extract is used in dry form.

4. The use according to claim 1; wherein carob tree leaf extract is used in liquid form.

5. The use according to claim 1; wherein inflammatory disease associated with inflammation is gastro-intestinal diseases such as rheumatoid arthritis, tendonitis (tendon inflammation), polymyalgia rheumatic, rheumatic gout disease, inflammation- related cardiovascular diseases, cancer, diabetes, obesity, inflammation-related gastritis, diarrhea and gastroenteritis and inflammatory intestinal diseases such as Crohn's and ulcerative colitis as well as asthma.

6. A drug and/or dietary supplement for use in the treatment of inflammatory disease associated with inflammation, wherein it includes Carob tree ( Ceratonia siliqua ) leaf extracts and/or gallic acid (GAc), (-) epigallocatechin gallate (EGCG), (-)-epicatechin gallate (ECG) and chlorogenic acid (ClAc) phenolic compounds contained within the Carob tree ( Ceratonia siliqua) leaf extracts.

7. A method of preparing Carob tree ( Ceratonia siliqua ) leaf extracts, the method comprises the process steps; • Powdering the leaves belonging to C. siliqua tree by grinding,

• Extracting the powdered leaves belonging to C. siliqua tree in alcohol,

• Obtaining liquid extracts,

• Removing alcohol from liquid extracts under vacuum,

• Lyophilising the extracts,

• Obtaining dry extracts

8. The method of preparing Carob tree (Ceratonia siliqua) leaf extracts according to claim 7; wherein powdered leaves belonging to C. siliqua tree are extracted in ethanol and/or methanol.

9. The method of preparing Carob tree (Ceratonia siliqua) leaf extracts according to claim 7; wherein it is prepared in dry or liquid form.

10. The method of preparing Carob tree (Ceratonia siliqua) leaf extracts according to claim 7; wherein the mixture of 3.5 ml-14 ml of alcohol and 1.5 ml- 6ml of distilled water is used for 1 g of carob tree (Ceratonia siliqua) leaf.

11. The method of preparing Carob tree (Ceratonia siliqua) leaf extracts according to claim 7; wherein the mixture of 3.5 ml-14 ml of ethanol or methanol and 1.5 ml- 6ml of distilled water is used for 1 g of carob tree (Ceratonia siliqua) leaf.

12. The method of preparing Carob tree (Ceratonia siliqua) leaf extracts according to claim 7; wherein the mixture of 10.5 ml of alcohol and 4.5 ml of distilled water is used for 1 g of carob tree (Ceratonia siliqua) leaf.

13. The method of preparing Carob tree (Ceratonia siliqua) leaf extracts according to claim 7; wherein the mixture of 10.5 ml of ethanol or methanol and 4.5 ml of distilled water is used for 1 g of carob tree (Ceratonia siliqua) leaf.