Chemical and Biological Studies on Oregano by MOSTAFA ABDEL LATIF ABDEL SALAM THESIS MASTER of SCIENCE in Agricultural Science (

Chemical and Biological Studies on Oregano

MOSTAFA ABDEL LATIF ABDEL SALAM

B.Sc. Agric. Sci. (Agric. Biochemistry), Fac. Agric., Ain Shams Univ., 2007

THESIS

Submitted in Partial Fulfillment of the Requirements for The Degree of

MASTER OF SCIENCE IN Agricultural Science (Agric. Biochemistry)

Department of Agricultural Biochemistry Faculty of Agriculture Cairo University Egypt

2012

SUPERVISION SHEET

Chemical and Biological studies on oregano

M.Sc. Thesis In Agric. Sci. (Agric. Biochemistry)

MOSTAFA ABDEL LATIF ABDEL SALAM B.Sc. Agric. Sci. (Agric. Biochemistry), Fac. Agric., Ain Shams Univ., 2007

SUPERVISION COMMITTEE

Dr. ABD El-MONEIM MOHAMED RADWAN AFIFY Professor of Agric. Biochemistry, Fac. Agric., Cairo University

Dr. SHERIF HELMY ESAWY Professor of Agric. Biochemistry, Fac. Agric., Cairo University

Dr. ESHAK MORAD ELHADIDY Researcher of Biochemistry, Food Technology Research Institute, ARC

ACKNOWLEDGEMENT

First of all thanks to GOD, by grace whom this work was accomplished. I would like to express my great appreciation and sincere gratitude to Dr. Abd-El Moniem Mohamed Radwan Afify, Professor of Biochemistry, Faculty of Agriculture, Cairo University, Dr. Sherif Esawy, Professor of Biochemistry, Faculty of Agriculture, Cairo University and Dr. Eshak Elhadidy, Researcher in Food Technology Research Institute, Agricultural Research Center for their supervision, continuous help during the course of the present study and in writing the thesis.

Deep thanks are also expressed to Dr. Hany Elshemy Professor of Biochemistry, Faculty of Agriculture, Cairo University for his valuable cooperation in the anticancer experiments.

DEDICATION

I dedicate this work to my parents and brothers for all the support they lovely offered during my post- graduate studies.

INTRODUCTION

A variety of herbs and herbal extracts are available on the market: they contain different phytochemicals with biological activity that can provide therapeutic effects. Most herbal products are used for health maintenance or for benign, self-limited conditions such as lower blood cholesterol concentrations, prevent some types of cancer, and stimulate the immune system. Besides herbal products for medicinal uses there are many culinary herbs (i.e., basil, caraway, cilantro, coriander, cumin, dill, oregano, rosemary, sage and thyme) that, if used generously to flavor food, provide a variety of active phytochemicals (Afify et al., 2012) that promote health and protect against chronic diseases (Craig et al.,1999). The use of medicinal plants with pharmaceutical properties has received increased interest and plays an important role in public health as antioxidant as well as anti-cancer (Afify et al., 2011a and b). Oregano is an aromatic plant of the Mediterranean ﬂora that has been commonly used for medical purposes (Bukovska et al., 2007 and Juhás et al., 2008). Oregano (Origanum syriacum L.) is a major non-wood forest product traditionally collected from the wild in Lebanon. To prevent overexploitation of the plant, the Ministry of Agriculture has regulated its collection. Origanum syriacum is used as a main ingredient of Zaatar, a local food prepared by mixing dried and crushed oregano leaves, sesame seeds, crushed sumac and olive oil. The plant is also processed as a spice and a herbal tea and constitutes a source material for the production of an essential oil that has been found to have antimycotic, insecticidal, nematicidal, allelochemical, antioxidant and

1 ovicidal properties (Atallah et al., 2011). Some previous studies have reported antioxidant and antimicrobial activities of oregano extracts in the inhibition of Helicobacter pylori growth (Chun et al., 2005 and Wojdylo et al., 2007). Moreover, it has been reported that the use of essential oils from oregano provide an interesting perspective in the prevention of neurodegenerative disorders (Loizzo et al., 2009).

Origanum syriacum L. is represented by three varieties, among which subsp. sinaicum Greuter and Burdet is native to Sinai, Egypt. It is commonly used by Bedouins (Boulos, 2002). A previous study of the chemical composition of the oil of these varieties revealed the existence of thymol, thymol-carvacrol and carvacrol- thymol chemotypes (Figuérédo et al., 2005).

However, the biological activity of these plants strongly depends on their composition. Thymol and carvacrol are two compounds present in oregano with proved antioxidant and antimicrobial properties. Carvacrol has also shown an anti- proliferative activity in tumor cells of Hela (Masteli et al., 2008). Numerous studies have demonstrated that spices have potent antioxidant properties, mostly due to the quantity and quality of polyphenolic compounds present in them (Cervato et al., 2000).

Oregano is used as a medicinal plant with health imparting properties such as powerful anti-bacterial and anti-fungicidal properties (Elgayyar et al., 2001). The compounds for these biological properties are polyphenolic secondary metabolites such as rosmarinic acid, carvacrol, caffeic acid, thymol (Zheng and Wang, 2006). These

2 compounds have been shown to have anti-carcinogenic, antimicrobial, antiviral, hypolipidemic, anti-mutagenic, anti-inﬂammatory and anti- cardiovascular disease properties (Lampe, 2003 and Srinivasan, 2005). Thymol and carvacrol belong to the most active antioxidants found in essential oils (Monteiro et al., 2000). Ground oregano, which is rich in carvacrol, thymol and various phenolic acids including caffeic acid, p -coumaric acid and rosmarinic acid (Shan et al.,2005).

Oregano phenolics have signiﬁcant antioxidant activity and are effective in the inhibition of all phases of the peroxidative process by neutralizing free radicals, blocking the peroxidation catalysis by iron and interrupting the lipid radical chain reactions (Dornan et al., 2003). Traditional medicinal practitioners have claimed for centuries that extracts from plant can be effectively used for the evaluation of different type of ulcers. Except for the use of appropriate vaccine for the treatment ulcers caused by infection, some treatments are available today to cure ulceration. It is not surprise, that a considerable interest has been taken by researcher to examine these number of traditional plant remedies, used for treating ulcers (Lawande, 2012).

The cause of ulceration in patients is mainly due to hyper secretion of gastric juice and also due to hyper secretion of pepsin. An ulcer is basically an inflamed break in the skin or the mucus membrane lining the alimentary tract. Ulceration occurs when there is a disturbance of the normal equilibrium caused by either enhanced aggression or diminished mucosal resistance. About 19 out

3 of 20 peptic ulcers are duodenal. Gastric ulcers, found in the stomach wall, are less common. The gastric mucosa is continuously exposed to potentially injurious agents such as acid, pepsin, bile acids, food ingredients, bacterial products (Helicobacter pylori) and drugs. These agents have been implicated in the pathogenesis of gastric ulcer, including enhanced gastric acid and pepsin secretion, inhibition of prostaglandin synthesis and cell proliferation growth, diminished gastric blood flow and gastric motility. (Grossman, 2009). Some previous studies have reported antioxidant and antimicrobial activities of oregano extracts in the inhibition of Helicobacter pylori growth (Wojdylo et al., 2007).

The main of the study was to determine the antioxidant constitutes (polyphenols, flavonoids, volatile oil and carotenoids) and evaluate the potential anticancer activity of volatile oil and ethanol extract of O. syricum on HepG2 and EAC cell lines. Also, estimate the effectiveness of leaves extract in preventing the formation of gastric ulcer by indomethacin gastric damage in albino rats and antibacterial activity of volatile oil and ethanol extract on pathogenic positive and negative gram bacteria.

REVIERW OF LITETRATURE

1. Oregano plant

Oregano is the common name for a general aroma and flavor primarily derived from aplethora of plant genera and species used all over the world as a spice, but usually refers to the genus Origanum, the European oregano, the name of which is derived from the Greek words oros, mountain and hill, and ganos, ornament. At least 61 species of 17 genera belonging to six families are mentioned under the name oregano. The family Lamiaceae (Labiatae) is considered to be the most important group containing the genus Origanum that provides the source of well-known oregano spices – Turkish and Greek types. Two genera of the Verbenaceae family (Lanata and Lippia) are used for production of oregano herbs (Bernath, 1996). Oregano is an annual, perennial and shrubby herb that is native of the Mediterranean, Euro-Siberian and Irano-Siberian regions (Bayramoglu et al., 2008) .

The genus Origanum L., belonging to the family Labiatae (Lamiaceae), contains aromatic and medicinal plants. They are small shrubs or perennial herbs with several stems, ascending or erect, subsessile or petiolate leaves and flowers in verticillasters aggregated in dense or loose spikes which are arranged in a paniculate or corymb form inflorescence. Most of the Origanum species are native to the Mediterranean Basin, and they have a very local distribution around it (Baser, 2002). Origanum syriacum L. is an aromatic, herbaceous and

5 perennial plant growing wild in the Sinai Desert of Egypt (Kamela et al., 2001).

Origanum syriacum subsp. sinaicum (Boiss.) Greuter & Burdet (Boulos, 2002).

Kingdom: Plantae

Phylum: Magnoliophyta

Class: Magnoliopsida

Order: Lamiales

Family: Lamiaceae

Genus: Origanum

Species: Origanum syriacum L.

2. Utilization of oregano

Oregano is an herb that has been cultivated for centuries in the Mediterranean area, although it now can be found on most continents. Actually, there is not simply one "oregano," but rather several species that may contribute to the oregano used for culinary purposes. Origanum vulgare, a member of the plant family Lamiaceae, is generally the spice variety sold as oregano in Europe and the United States. Medicinal uses for oregano date back to the ancient Greek and Roman empires where applications of the leaves were used to treat such maladies as skin sores and relieve aching muscles and as an

6 antiseptic. Oregano also has been used in traditional medicines for such ailments as asthma, cramping, diarrhea, and indigestion. In Greece, an oregano infusion is still used as a folk remedy against colds and upset stomach and to maintain general health. Based on the current scientific literature, oregano extracts and individual constituents consistently have demonstrated antimicrobial actions in vitro toward food-borne pathogens, although the capacity to counter human infections is not well studied. Oregano contains several potent antioxidants that may contribute to the findings in preliminary studies that oregano exhibits benefits toward the cardiovascular and nervous systems, relieves symptoms of inflammation, and modulates blood sugar and lipids. Well-controlled human studies substantiating these health effects are lacking (Singletary, 2010). The worldwide market of oregano has increased with exceptional examples such as USA where the procapita consumption has augmented over 3800% between the years 1940 to 1985 (Kintzios,2002). As well as European countries importation. In this market context, oregano is the most widespread species (Spade et al., 1996).

Oregano has several properties that deserve further characterization and confirmation. It is clear that oregano's chemical constituents can suppress the growth of a broad range of microorganisms in vitro. The conditions under which it can act effectively to counteract microbial contamination in food products need to be better examined. Particularly valuable will be characterization of the conditions under which its constituents may

7 combat human infection (Aligiannis et al., 2001).There is limited but suggestive evidence that oregano bioactive components may suppress inflammation and improve blood glucose and lipid regulation. Such properties warrant additional preclinical evaluation to determine the active agents, potential mechanisms, and consistency among experimental models. In particular, confirmation of the potential capacity of oregano to decrease hyperglycemia and enhance insulin sensitivity and the doses necessary to do so warrant further investigation. Lastly, despite numerous reports that oregano's phytochemicals have antioxidant actions in vitro, the in vivo evidence in animals is limited and in humans is unsubstantiated (Nurmi et al., 2006). This lack of effect clinically in part may be due to poor oral bioavailability that has been observed. Thus, it will be of value to determine the specific oregano extracts, active constituents, effective doses, and conditions for which oregano may have measurable impact on markers of antioxidant status in humans after supplementation (Nurmi et al., 2006). There are various reports on the traditional medicinal uses European oregano has as a carminative, diaphoretic, expectorant, emmenagogue, stimulant, stomachic and tonic. In addition, it has been used as a folk remedy against colic, coughs, headaches, nervousness, toothaches and irregular menstrual cycles. Turkish villagers have traditionally used kekik water, the aromatic water obtained after removing essential oil from the distillate of oregano herbs, which has in recent years become a commercial commodity (Baser, 2002 and Kintzios, 2002) .Nevertheless, many of the studies confirmed benefits of oregano for human health and its use

8 for the treatment of a vast list of ailments, including respiratory tract disorders such as cough or bronchial catarrh (as expectorant and spasmolytic agent), in gastrointestinal disorders (as choleretic, digestive, eupeptic and spasmolytic agent), as an oral antiseptic, in urinary tract disorders (as diuretic and antiseptic) and in dermatological affections (alleviation of itching, healing crusts, insect stings), viral infections and even cancer (Baricevic and Bartol, 2002).

Also oregano plant has been used for the treatment of various diseases due to its antioxidant, anti-microbial and anti-parasitic properties. Considering these medicinal properties, studies are ongoing to use oregano for the treatment of severe diseases such as cancer, heart disease and muscular degeneration. Also, it is used for the treatment of flatulence, bloating and other indigestion problems. It enhances the secretion of saliva and serves in improving digestion. It is also effective in overcoming menstrual cramps and promoting men-striation. Some people use oregano as a natural remedy to treat cold and flu symptoms. Herbal tea prepared with oregano helps in alleviation of headache, urinary problems, lung disorders, diarrhea, nausea, vomiting and jaundice. Oregano boiled in plain water can be used as mouthwash. Gargling with this water can help to prevent tooth infection and sore throat. Oregano oil applied directly to the infected tooth helps in combating toothache. Ground oregano leaves soothe arthritis pain, insect bites and other skin problems. Its bitter taste and strong aroma help in controlling head lice. Creosote bush has been used with many medicinal

9 purposes: analgesic, diuretic, emetic, antipyretic antiviral, antimicrobial, gout, arthritis, sinusitis, anemia and fungus infections in skin (Raissouni, 2005). Oregano has been used in the traditional medicine for treatment of a variety of human diseases of bacterial and fungal origin (Lacoste et al., 1996) .Thymol and carvacrol are commonly used in the food industry and in cosmetics as preservatives and antioxidants (Lacoste et al.,1996 and Manou et al.,1998). Thymol is also used in mouth rinse for oral hygiene against caries-related pathogens (Kato et al., 1990). They possess multiple biological properties such as antiinﬂammatory, antibacterial ,antifungal, anti- carcinogenic, and antiplatelet (Baker, 2008).Thymol and carvacrol were reported to exhibit in vitro acetylcholinesterase inhibitory activity (Jukic et al., 2006) and to show antispasmodic activity in the isolated ileum, vas deferens and tracheal preparations (Astudillo et al., 2004).

Soliman et al. (2007) mentioned that the essential oil and 70% ethanol extract of O. Syriacum L. subsp sinaicum exhibited powerful tocolytic effects in the rat uterus possibly through antagonizing receptor-dependent mechanism (oxytocin-induced contraction) and independent mechanism (KCl-induced contraction).

Many studies in vitro revealed new potential therapeutic effects of Origanum, in the treatment of Alzheimer’s disease, as an anti-inflammatory agent in a cellular model of atherosclerosis, a suppressor of food bacterial growth and enterotoxin synthesis, and as a way of increasing the sensitivity of some antibiotics

10 against resistant bacteria (Aslim and Yucel, 2008 and De Souza et al., 2010).

Awaad et al. (2011) reported that the acute treatment of the total ethanol extract of Origanum syriacum L. plant was well tolerated. Moreover, indicates that the test plants may be useful in the protection against inflammatory diseases, especially if free radicals are a part of its pathophysiology. However the sufficient activity of Origanum syriacum L extract in hepatic protection against administration of paracetamol in an over dose as liver injury drug. All results shows the role of Origanum extract in liver amelioration to be in a healthy status and it is more effective than sylimarin as hepatoprotective or therapeutic drug but it is superior as therapeutic than hepatoprotective also it is sufficient to decrease the oxidative stress on liver as mentioned in magnification of glutathione- antioxidant system and detoxification in liver with glutathione path way (Abeer et al., 2010).

3. Active ingredients of oregano

Although abundant chemical compounds have been isolated from oregano, the most important group, from a commercial and application point of view, refers to its volatile oils, basically composed of terpenoids. However, composition may vary significantly among different genotypes. Oregano species are rich in phenolic monoterpenoids such as carvacrol and secondly thymol (Fig.1). It is quite easy to distinguish the difference between the pungent smell of

11 oregano and the sweet smell of marjoram. In the first group are a number of chemically related compounds such as γ-terpinene, p- cymene, thymol and carvacrol methyl ethers, thymol and carvacrol acetates; also compounds such as p-cymenene, p-cymen-8-ol, p- cymen-7-ol, thymoquinone and thymohydroquinone are also present. In the second group,α-thujene, cis-sabinene, and trans-sabinene hydrate acetates, cis and trans-sabinol and sabina ketone can also be found (Skoula and Harbone, 2002).

Fig. 1. Structure of (a) thymol and (b) carvacrol. Bin et al. (2005) carried out comprehensive analysis of polyphenols in the extracts of Lamiaceae spices. Furthermore, 26 compounds in oregano, such as phenolic acids (caffeic acid, p- coumaric acid, rosmarinic acid, kaempferol and caffeoyl derivatives), volatile compounds (carvacrol) and flavonoids.

A range of phenolic compounds has been identified in oregano including rosmarinic, caffeic, and p-coumaric acids and caffeoyl derivatives, the phenolic monterpenes, carvacrol and thymol, and flavonoids, luteolin, apigenin, myricetin and quercetin (Shan et al., 2005; Yanishlieva et al., 2006 and USDA 2007) (Fig.2). Although most research interest has centered upon oregano as an essential oil,

12 and its constituent compounds carvacrol and thymol, which are believed to cause its antioxidant activity, it has been shown that water extracts, which isolate a range of different compounds, are rich in phenolic acids and flavonoids also have strong antioxidant activity (Triantaphyllou et al., 2001).

Carotenoids have attracted the interest of researchers from diverse fields including chemistry, biochemistry, food science and technology, medicine, pharmacy and nutrition for more than a century and these fascinating compounds continue to be intensely investigated. In nature, carotenoids are mainly responsible for the red, yellow and orange colors. However, in green plant tissues, the color of carotenoids is masked by the more dominant pigment, chlorophyll and becomes evident only during the degradation of chlorophyll. This phenomenon can be seen during the ripening of fruits as well as in autumn leaves. In food, in addition to their function as the natural pigments and pro-vitamin A precursor role of certain carotenoids, these compounds can be used as food additives for coloring (European Parliament and Council Directive, 1994). The carotenoids are a group of yellow-orange-red pigments found in a variety of fruits and vegetables as well as in algae, fungi and bacteria. Carotenoids cannot be synthesized in the body and are present solely as a result of ingestion from other sources, either ingestion of the plant itself or a product from an animal that has consumed that plant source, e.g. egg yolks are yellow because of the carotenoids they contain. Often, the colors of the carotenoids

(a) (b)

(e) (f) Fig. 2. Structure of (a) p-coumaric acid, (b) caffeic acid, (c) Rosmarinic acid, (d) apigenin, (e) Luteolin and (f) Quercetin. present in plants are masked by chlorophyll, to the extent that some of the highest levels of carotenoids are found in dark green leafy vegetables such as kale and spinach (Sies and Stahl, 2003). Carotenoids in oregano according to National Nutrient Database for Standard Reference Release (USDA, 2007) contain 4112 mcg β- carotene, 60 mcg β-Cryptoxanthin and 826mcg Lutein and zeaxanthin.

Zein et al. (2011) studied minerals composition of wild and cultivated O. syriacum, and we identify 15 elements. It seems that the

14 cultivated population, before and after flowering, has a higher amount of minerals than the wild population and the cultivated leaves before flowering have a higher amount of minerals than cultivated leaves after flowering. Blooming peaks are the poorest in minerals. Cultivated Origanum may be a dietary source of minerals and trace elements. For example, 1 g of cultivated leaves before flowering may contain 1.25mg of Mn; 40mg of Ca; 5mg of Fe; 65mg of K (recommended daily dose: 5mg for Mn,100-150mg for Ca;10 to 18mg for Fe; 2 to 4g for K). Some others elements are detected as traces (Co<0.03; Cd<0.01; As<0.01and Hg<0.01).

Zein et al. (2011) studied fatty acids composition from leaves and bloom peaks from wild and cultivated O. syriacum. 16 fatty acids were identified. Chromatography analysis of fatty acids methyl esters showed that myristic acid was the predominant component in both wild and cultivated leaves after flowering (61.50 and 79.07%, respectively) and in blooming peaks (84.15 and 76.33%, respectively). There was no trace of myristic acid before flowering. Myristic acid is particularly effective at causing the liver to synthesize cholesterol. It is mainly used for soap manufactures, flavorings, cosmetics, perfumes, also as raw materials in spices and in the pharmaceutical domain. Myristoleic acid was found at a lesser amount after flowering, it may have a cytotoxic effect against prostatic cancer (Iguchi et al., 2001). The human body can produce all but those 2 fatty acids out of the ones it needs. They must be

15 supplied in diet. Hence, linoleic acid and linolenic acid are essential fatty acids for humans. In the body, essential fat y acids are primarily used to produce hormone-like substances that regulate a wide range of functions, including blood pressure, blood clotting, blood lipid levels, the immune response, and the inflammation response to injury infection. The predominant fatty acid in leaves before flowering was the linolenic acid (51.73% for wild and 49.34% for cultivated Origanum). Linoleic acid amount was less than 10%. These results allow the classification of the O. Syriacum leaves before flowering for both 2 populations as source of essential fatty acids. It is not the same for leaves after flowering.

4. Antioxidant activity of oregano

Natural antioxidants are able to protect from free radicals and retard the progress of many chronic diseases and lipid oxidative rancidity in foods (Gulcin et al., 2003).

Herbs and spices intake, especially in therapeutic dosages, may help decrease inflammation, removal of free radicals before damage occurs of cells DNA and RNA, elimination of damaged molecules, and the prevention of mutations. Many culinary herbs and spices, for example, rosemary, oregano, cinnamon, basil and cloves, are inherently high in antioxidant activity measured in vitro (Kim et al., 2011).

Considering the possibility of undesirable influences of oxidized lipids on the human organism, it is of essential