1.1 Phytotherapy and medicinal plants
Phytotherapy is the study of the use of bioactive compounds and extracts from natural origin as medicines or health-promoting agents. Even though phytotherapy is usually regarded as "alternative medicine" in the Western countries when critically carried out, is considered an essential component of modern pharmacognosy. All living organism contains antioxidant metabolites and enzymes which prevent oxidative damage to cellular compounds such as lipids, proteins and DNA. Researches have found a correlation between oxidative damage and the occurrence of diseases. A large number of plants and their products and several plants derived dietary components have been evaluated as potential chemoprotective agents in the living cells. Medicinal plants have played a significant role in maintaining human health and improving the quality of human life for thousands of years. Much enthusiasm have been generated to explore the potential use of naturally occurring anti-inflammatory and antioxidant compounds to attenuate the xenobiotic compounds induced inflammatory reactions.
Curcumin (CCM) is a dietary antioxidant derived from turmeric (Curcuma longa) and has been in use since ancient times as therapeutic agents. It has been reported to be a potent anti-inflammatory, antioxidant, and anti-carcinogenic (Kawamori et al., 1999; Antunes et al., 2001; Khar et al., 2001). Curcumin also suppress bleomycin induced pulmonary fibrosis and toxicity induced by PQ (Punithavathi et al., 2000; Venkatesan et al., 2000). Ginkgo bilbola extract is considered to be a potent hepatoprotective agent against the carbon tetrachloride induced liver injury. Histopathological studies showed that CCl4 caused steatosis and hydropic degeneration of the liver tissue. Ginkgo bilbola pre-treatment exhibited protection, which confirmed the results of biochemical studies. (Ashoke et al., 2001)
The seed of Nigella sativa has been traditionally used for centuries in the Middle East, northern Africa, Far-East and Asia for the treatment of asthama. Recent research indicates that the extracted oil of Nigella sativa have the medicinal effects and repair the liver injury induced by carbon tetrachloride. (Kanter et al., 2004). The bark of Acacia arabica is traditionally used in disorders implicated with free radical damage to the tissues. Polyphenolic compounds of Acacia arabica possesed antioxidant property and metal chelating properties. ( Sundaram and Mitra, 2007). The roots of Hygrophila spinosa and Cassia Occidentalis are found to be hepatoprotective and also have the strong antioxidant activity against the carbon tetrachloride induced liver injury. The treatment of the aquous extract of plants roots increase the enzymes near to the normal enzyme level in the CCl4 induced liver damage. (Usha et al., 2007). Melilotis officinalis, Equistem maximum, Adiantum sp. have the high antioxidant property due to the presence of phenolic compounds. (Gymafi et al., 1999).
1.2 Sonchus asper
Sonchus asper (Sharp-fringed Sow Thistle, Spiny Sow Thistle, or Spiny-leaved Sow Thistle) is an annual plant with spiny leaves and yellow flowers resembling those of the dandelion. The leaves are bluish-green, simple, lanceolate, with wavy and sometimes lobed margins, covered in spines on both the margins and beneath. The base of the leaf surrounds the stem. The plant can reach 180cm (6 ft) in height. The leaves and stems emit a milky sap when cut. The flowers grow in clusters and the end of the stems. This plant is native to Pakistan, but is also a common weed in North American roadsides, landscapes, and pastures. Its edible leaves make a palatable and nutritious leaf vegetable. Sonchus asper is generly classified as,
Sonchus asper (L.) Hill, (Asteraceae) locally named as Mahtari used in food as a salad, in treatment of wound healing and anti-burning properties (Rehman, 2006). It is used in the treatment of cough, bronchitis and asthma (Koche et al., 2008). Likewise it is a powerful galactagogues when used orally and used for treatment of gastrointestinal infection and inflammation (Pieroni et al., 2002; Pieroni and Quave,2005). Sonchus asper possessed some bioactive compounds which control the fungal attack on human; therefore people used the dried plant powder against fungus (Rauf et al., 2007).
Chemical characterization of Sonchus asper shows that it contains Flavonoids (Giner et al., 1993; Guirong et al., 1995; Guirong et al., 1996) and Sesquiterpene lactones glycosides (Hela et al., 2000). Nutritional analysis of Sonchus asper showed the presence of ascorbic acid and carotenoids (Guil-Guerreroet al., 1998). Flavinoids, Ascorbic acid and carotenoids possess antioxidant, anticancer; anti-inflammatory properties while Sesquiterpene lactones glycosides having antioxidant, anti-inflammatory and play important role in cardiovascular dysfunction.
1.3 Launea procumbens
Launea procumbens, (Asteraceae) found in waste places, vaccent lots and in cultivated fields through out the Pakistan. Launea procumbens was used as cattle's foods and as washing agent (Wazir et al., 2007). Chemical characterization showed that Launea procumbens composed of salicylic acid, vanllic acid, synergic acid, 2-methyl-resercinol and gallic acid (Shaukat et al., 2003). It has been used against plant pathogenic fungi, nematocides and as allelopathic for inhibition of plants (Shaukat et al., 2003). It is also used in the treatment of kidney disorders like painful urination and sexual diseases like gonorrhoea (Ahmad et al., 2006).
1.4 Antimicrobial screening and medicinal plants
For thousand years, mankind has learnt about the benefits of plant use to alleviate or cure diseases. Plant posses a number of new bio active compounds which are used in medicine. In ancient civilization plants extracts were used in curing and treatment of various diseases of various diseases. Now a day 30% of worldwide drugs are based on natural products isolated from medicinal plants (Grabley and Thiericke 1999). However, for some decades, there has been increasing interest in plant uses and the detection of their constituents with antimicrobial. Many efforts have been made to discover new antimicrobial compounds from various kinds of sources such as micro-organisms, animals, and plants. One of such resources is folk medicines. Systematic screening of them may result in the discovery of novel effective compounds. According to WHO reports most infectious disease has been controlled but still more than 40% deaths has been occurred due to infectious microbes in developing countries. In addition to diseases preservation of food is becoming a more complex problem, due to new products introduction in market which require more protection against pathogenic microbes (Marino, Bersani & Comi, 2001). There is therefore a great interest for new methods of making food safe which have a natural or green image. One such possibility is the use of essential oils as antibacterial additives. Some of the antimicrobial compounds produced by plants are active against plant and human pathogenic microorganisms (Mitscher et al., 1987). Antimicrobial agents, including food preservatives, have been used to inhibit food borne bacteria and extend the shelf life of processed food. Many naturally occurring extracts from plants, herbs and spices have been shown to possess antimicrobial functions and could serve as a source for antimicrobial agents against food spoilage and pathogens (Bagamboula, Uyttendaele & Debevere, 2003).
1.5 Cytotoxicity and Natural products
Cytotoxicity means toxic to cells, toxic agents are a chemical substance, an immune cell or some types of venom (e.g. from the puff adder or brown recluse spider). Cytotoxicity assays are widely used in pharmaceutical industry for screening of natural bioactive compounds and cancer therapy through MTT, SRB, WST and clonogenic assay. Cancer is an ailment that affects more or less 200 types of cells. The major characteristic is the lack of control of the cell proliferation, differentiation and death, invading organs and tissues. There are many difficulties in the treatment but the more frequently are the drug resistance, toxicity, and low specificity. Plant molecules, their semi-synthetic and synthetic derivatives are important sources of antitumour drugs. Currently, over 50% of drugs used in clinical trials for anticancer activity were isolated from natural sources or are related to them (Newman and Cragg, 2007). Hence, the search for natural products to be used in cancer therapy represents an area of great interest in which the plant kingdom has been the most important source, providing many anti-tumor agents with novel structures and unique mechanism of action (Chang et al., 1999). There are two main strategies for the selection of plant species in drug discovery, including anticancer drugs: random screening and ethnomedical knowledge. The second approach includes plants used in organized traditional medical systems like herbalism, folklore and shamanism (Pieters and Vlietnick, 2005).
1.5 Carbon tetrachloride (CCl4)
Carbon tetrachloride has chemical formula CCl4, and its molecular weight is 153.8 g/mol, has been in use as solvent in varnishes resins and as starting material in formation of many industrial organic compounds, it is estimated that the average daily intake of CCl4 for a general population is 0.1µg. (Abraham et al.,1999; ASDR, 2003).Exposure to toxic chemical through inhalation, ingestion or skin absorption; it is distributed through out the body with high concentration in liver, brain, kidney, muscles, fat tissue and blood (Ogeturk et al., 2004) damages various tissues especially liver (Szymonik-lesiuk et al., 2003).
Various investigations have established that carbon tetrachloride (CCl4) is a potent environmental hepatotoxin (Guven et al., 2003) causes many dysfunctions of kidneys, lungs, testis and brain as well as in blood by generating free radicals (Charbonneau et al., 1986; Ozturk et al., 2003). It has been reported that CCl4 causes renal injuries in rats and humans (Ogeturk et al., 2005).
CCl4 is known to produce reactive oxygen species (ROS), decreases GSH of phase II enzyme, and reduces antioxidant enzyme and antioxidant substrates to induce oxidative stress that is an important factor in acute and chronic liver injury. The injuries induced by CCl4 are resulted from free radicals and lipid per oxidation causes hepatic cell damage. CCl4 requires bioactivation by phase I cytochrome P450 system in liver to form reactive metabolic trichloromethyl radical (CCl3*) and proxy trichloromethyl radical (*OOCCl3). These free radicals can bind with polyunsaturated fatty acid (PUFA) to produce alkoxy (R*) and peroxy radicals (ROO*), that, in turn, generate lipid peroxide, cause damage in cell membrane, change enzyme activity and finally induce injury or necrosis (Weber et al., 2003).
Carbon tetrachloride reduce the total protein content in blood as well as induce the depletion of hepatic and renal antioxidant enzymes such as catalase (CAT), peroxidase (POD) and super oxide dismutase (SOD). The depletion of these antioxidant enzymes are due to the controlling action against peroxy radicals produce by the CCl4 (Adewole et al., 2007) Carbon tetrachloride induces reactive oxygen species (ROS) and oxidative DNA damages, with the formation of DNA adducts, genetic mutation, strand breakage and chromosomal alterations. DNA strand breaks are especially important in inducing mutations, such as deletions and translocations, in affected cells undergoing replication with error-prone repair or without proper repair. Moreover, extensive DNA strand breaks without prompt repair may cause cell death and compensatory cell regeneration (Jia et al., 2002).
1.6 Potassium bromate (KBrO3)
Potassium bromate has chemical formula KBrO3 and its molecular weight 166g/mol, is an oxidizing agent that has been used as a food additive in the treatment of flour and barley. KBrO3 has been used as a main constituent in cold-wave hair solutions and in cosmetic industries. Potassium bromate is also found as by product formed by ozanization of water causes disinfections and has been classified as 2B group toxic chemical a possible human carcinogen (IARC, 1986).
KBrO3 causes renal cell and thyroid carcinomas in rats, hamsters and mice when exposed chronically (Kurokawa et al., 1990). It has been investigated that potassium bromate produced free oxygen radicals causes oxidative stress and DNA damages (Umemura et al., 1998; Murata et al., 2001). KBrO3 causes nephrotoxicity and hepatotoxicity decreases the tissue soluble proteins, antioxidant enzymes like CAT, SOD and Peroxidase. The decreases of antioxidant enzymes levels are due to active oxygen species produced by potassium bromate. Potassium bromate depleted GSH content in liver and renal tissue which causes decreases in phase II metabolizing enzymes like GSHpx and GSR. KBrO3 also increases MDA causes lipid peroxidation and liver profile including ?-gt, alkaline phasphatases (ALP) (Farombi et al., 2002).
1.7 Scavenging effects of antioxidant enzymes and oxidative stress
Oxygen is necessary for living organism for the production of energy to biological processes. However, oxygen-centred free radicals and other reactive oxygen species (ROS), like superoxide (O2¯), hydroxyl (OH¯), peroxyl (ROO¯), peroxinitrite (ONOO¯), and nitric oxide (NO¯) radicals; which are produced in body by metabolism of lipids, oil and other food ingredients or through exposure of industrial pollutions, various types of dangerous rays and chemical toxins like CCl4 , Potassium bromate (Atta-ur-Rahman & Choudhary, 2001), bind with polyunsaturated fatty acid which generate lipid peroxidation, cause damage in cell membrane, tissue damage, oxidative stress change enzyme activity and finally induce injury or necrosis (Weber et al., 2003) protein degradation, DNA damages, histopathalogical abnormalities cancer, hormonal disturbance diabetes, cardiovascular diseases (Khan and Ahmed, 2009).
Fig 2 Mechanism of Oxidative stress
The human body composed of many defense mechanisms to ameliorate this oxidative stress, including a system of antioxidant enzymes like SOD, POD and GPx in addition to non-enzymatic compounds such as a-tocopherol, -carotene and ascorbic acid, and some micronutrient elements such as zinc and selenium etc and protect the body from degenerative diseases. Super oxide dismutase converts these free super oxides radical into HOOH which was further ameliorated by POD, CAT and GPx into HOH to detoxify the radicals (Halliwell and Gutteridge, 1999).
Fig 3 Mechanism of enzyme action in mammalian cells
The superoxide dismutases which are the product of highly conserved gene convert the free oxygen radical to H2O2. This molecule which is itself toxic for the cells is broken down to release hydroxyl radical (*OH), a reactive species which is more toxic either *O2 and H2O2. The enzymes responsible for converting H2O2 to other harmless substances are catalase and Glutathione peroxidase. Thus this enzyme family may act in a sequential fashion to dismutate the one toxic oxygen species to another which then can be rapidly broken down to non toxic byproducts (Schinina et al., 1987). Catalase is also an antioxidant enzyme and primarily located in the cytosolic peroxisomes. Its function is to detoxify H2O2 to oxygen and water (Freeman et al., 1986). Glutathione peroxidase is a cytosolic enzyme and also eliminates H2O2, but in comparison to catalase, GPX has a wider range of substrate including lipid peroxides. The kinetics of this enzyme is very complex, but it though to have a greater affinity for H2O2 as compared to catalase. Glutathione peroxidase (GPX) primarily functions to detoxify low level of H2O2 in the cells (Heffner, 1989).
1.8 Silver staining Technique (AgNORs assay) and oxidative stress
Tumor cells identification is a well known diagnostic problem. It is very difficult to differentiate neoplastic cells from macrophages and particularly, reactive mesothelial cells on morphological grounds. Therefore number of tests including DNA flow cytometry, Restriction Enzyme Fragment Length Polymorphism (RFLP), PCR Sequencing, monoclonal and polyclonal antibodies has been employed to distinguish the benign from the neoplastic cells (Cheah et al., 1996). A comparatively simpler technique used for this purpose is the silver staining of nucleolar organizer regions (NORs). Interphase AgNORs are the structural and functional units of the nucleolus which contain all the essential components for the synthesis of ribosomal RNA. The two argyrophilic proteins which are associated with rRNA transcription and processing are nucleolin and nucleophosmin (Drenzini, 2000). These proteins are argyrophilic and are easily stained by silver stains. After silver-staining, the NORs can be identified as black dots present throughout the nucleolar area. The number and size of NORs reflect cell activity, proliferation and transformation and help to distinguish benign from malignant cells (Rocher, 2000). A number of studies carried out in different tumour types demonstrated that malignant cells frequently present a greater AgNOR count than corresponding non-malignant cells (Bocking et al., 2001).
1.9 Telomerase activity and TRAP assay
Telomerase is a ribonucleo protein that shortens the ends of eukaryotic chromosomes occurring during the cell cycle. This RNA-dependent DNA polymerase provides the basis for an unlimited proliferation. Its activity is absent in most normal somatic cells, but is present in over 90% of cancerous cells and in vitro-immortalized cells (Shay and Bacchetti, 1997). Telomerase consists of two fundamental components, an RNA component (in humans, hTR or hTERc) and a reverse transcriptase component (hTERT) (Bettendorf, 2003).The RNA component of telomerase acts as the template for telomeric repeat synthesis. In man, hTR is transcribed by RNA polymerase II and its mature transcript consists of 451 nucleotides (Feng et al., 1995). The hTR gene was cloned and localized to chromosome 3q26.3 (Zhao et al., 1998, Soder et al., 1997). The template for hTERT activity lies in nucleotides 46 to 53. Although there is a variation of hTR RNA sequences among telomerase RNAs, there is a remarkably conserved secondary structure from ciliates to vertebrates. This indicates an essential role for the structure in enzyme function (Chen et al., 2000).
According to Blanco et al. (2003) Telomerase enzyme contains a catalytic subunit (TERT), a RNA subunit (TR), and several protein components including the TEP1 associated protein. The RNA subunit serves as a template for reverse transcription in telomere DNA synthesis. The protein subunit TERT constitutes, with the TR, the minimum structure of telomerase; both are sufficient to reverse transcribed telomere DNA from the RNA subunit to extend telomeres.
Telomeric repeat amplification protocol (TRAP) assay was used to measure the telomerase activity in different type of malignancies. This assay has much application to measure the telomerase activity. TRAP assay was introduced by Kim et al. (1994) by using the radiolabeled phosporus and the activity of enzymes was estimated from the radioactivity in a specific photographic film.
1.11 Mechanism of Telomeric Repeats Amplification Protocol (TRAP) assay
The basic mechanism under which telomerase acts is the extension of substrate (TS19) primer by the addition of hexameric (TTAGGG)n repeats. The extended primer sequence is then amplified by using thermocycler and the amplified product will shows about the telomerase activity. Using the knowledge of the low substrate specificity of telomerase, the Fujkus, (2006) replaced the oligonucleotide of a natural telomeric sequence by the substrate primer (TS) of a non-telomeric sequence. This made it possible to specifically amplify (using PCR with a reverse primer, CX) the products of telomerase-mediated extension of the substrate oligonucleotide, while suppressing false-positive amplification products, for example those due to amplification of genomic telomeres or catenation of primer-dimmers (Fujkus, 2006).
Fig. 1: Scheme of TRAP assay for telomerase activity. Telomerase first extends substrate primer (TS) of non-telomeric sequence with telomeric repeats (in italics).These primary telomerase products are specifically amplified by PCR with reverse primer (CX) and TS as a forward primer. Asterisks indicate designed mismatches in CX that reduce formation of primer-dimers. Taq polymerase and CX primers may be separated from the rest of the reaction mix by wax barrier, or added during heat inactivation of telomerase. All reaction components may also be present in the initial reaction mix when using hot-start DNA polymerase for the PCR step (polymerase is then activated simultaneously with inactivation of telomerase) (Fujkus , 2006).
The present project was designed for phytochemical and pharmacological characterization of Sonchus asper and Launea procumbens both in vitro and in vivo against CCl4 and KBrO3 induced oxidative stress in rats. The main objectives of this project are
1) Phytochemical characterization through
- Preliminary Phytochemical qualitative and quantitative screening
- Trace metal and elemental analysis
- Nutritional characterization
- Determination of total flavinoids and phenolic compounds via Uv-spectrophotometre
- Detection of bioactive compounds through thin layer chromatography
- High performance chromatographic analysis for identification of bioactive compounds
2) In vitro screening of Sonchus asper and Launaea procumbens
- Antibacterial Screening
- Antifungal activities of Sonchus asper and Launaea procumbens
- Evaluation of cytotoxicity through brine shrimp assay
- Phytotoxic characterization through radish root inhibition assay
- Antitumer characterization through potato disc assay
- Antioxidant scavenging evaluation through
- 1. DPPH free radical scavenging assay
- 2. Phosphomolybdenum assay
- 3. ABTS radical cation assay
- 4. Antioxidant activity determined by beta carotene bleaching method
- 5. Superoxide radical scavenging activity
- 6. Hydroxyl radical scavenging activity
- 7. Hydrogen peroxide-scavenging activity
- 8. Reducing power
- 9. Chelating activity on Fe2+
3) In vivo evaluation of Sonchus asper and Launaea procumbens against oxidative stress caused by CCl4 and KBrO3 in various tissues of rats through
- * Serum marker liver function and kidney function test
- * Urine profile
- * Evaluation of antioxidant and phase II metabolizing enzymes
- * RIA characterization of T3, T4, TSH, FSH, LH, E2, prolactin, cortisol, and testosterone hormone
- * Insulin analysis via ELISA
- * DNA damages through DNA fragmentation and ladder assay
- * Telomerase cancer marker enzymes activity through TRAP assay
- * Microscopic histopathalogical evaluations of H.E staining
- * To determine histological changes.
- * To count nucleolus by AgNORs technique