Antioxidant And Antiaging Activities Of Tannic Acid Biology Essay

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Reactive molecules and free radicals derived from molecular oxygen are called Reactive Oxygen Species (Paul Held, 2010). Through the number of different mechanisms ROS are formed which play a crucial role in normal cell transduction as well as cell cycle. ROS mainly generated through mitochondrial electron transport as byproducts. Two unpaired electrons make oxygen ready for radical formation. This leads to the creation of plenty of ROS such as: superoxide; hydrogen peroxide; hydroxyl radicals; hydroxyl ion; and nitric acid. Generally in all aerobic cells, ROS are in balance with biochemical antioxidants. Excess of ROS, reduction of antioxidants or both of them together in cells can cause an oxidative stress. ROS are key regulators in aging process. According to cellular and biological data which were obtained from various model organisms and systems, an increase of intracellular ROS will cause aging (E. Verdin et al., 2010). As a consequence of a failure in antioxidant defense mechanisms which protects an organism from oxidative damages, life span reduces (S. E. Schriner et al., 2005; V. I. Perez et al., 2008) and cardiomyophaty, neurodegeneration (R. M. Lebovitz et al., 1996) causes as well as cancer (D. C. Wallace, 2005) The genes which increase life span in some extent contain those concerned in oxidative stress response, while they were partly clustered in the IGF-1/insulin-like signaling pathway (M. Tatar et al., 2003; M. Barbieri et al., 2003)

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It's important to know that, ROS are not always harmful metabolic byproducts; when tightly regulated, ROS can act as intracellular signaling molecules (Scandalios JG, 2002; Klein JA and Ackerman SL, 2003) In living cells, the major source of endogenous ROS are hydrogen peroxide and superoxide anion, which are generated as byproducts of cellular metabolism such as mitochondrial respiration (Nohl H et al., 2003).

There is a growing awareness that oxidative stress plays a role in various clinical conditions such as aging, malignant diseases, diabetes, atherosclerosis, chronic inflammation, viral infection, and ischemia-reperfusion injury (Behrend L, et al., 2003; Apel K and Hirt H, 2004; Bergamini CM et al., 2004; Reddy MB and Clark L, 2004; Shah AM and Channon KM, 2004; Willner C neurodegenerative disorders, 2004).

1.2 Mechanism of Aging, longevity and stress resistance

Gradual change in an organism that leads to increased risk of weakness, disease, and death called aging (R). Over the whole adult life span of any living thing, it would be possible to observe this changing process in a cell, an organ, or the total organism. Biological function will reduce as well as the ability to adjust to any of the environmental stress. It is not obvious how to determine the rate of aging. However, there are some findings which agree that this process is actively regulated. For example, germ cells and transformed cells do not age, which indicates that aging is not a necessary feature of eukaryotic cells (R).In addition, the rate of aging is strikingly different in different species; e.g., the life spans of mice, canaries, and bats (all small warm-blooded animals) are 2, 13, and 30 years or more, respectively (for an extensive review of the phylogenetic variation of aging rates, see Finch 1990). In addition, control of the rate of aging by the endocrine system has been implicated in many different types of organisms; e.g., marsupial mice and certain species of salmon and insects undergo a rapid process of senescence following reproduction, and the endocrine system has been shown to delay senescence in the queen bee. Life span can also be extended in many species that enter a state of diapause or dormancy under unfavorable environmental conditions, an extreme example of which is the Caenorhabditis elegans dauer larva (R). The rate of aging is actively regulated. As with other biological processes, it may be possible to dissect the aging process using genetics, and in fact, a number of intriguing aging mutants have been isolated in several species while the most striking of these mutants have been isolated in C. elegans (for review, see Jazwinski 1996). Aging process in C.elegans is easily observable. Even as an untrained observer, the difference between old and young adult worms is so obvious. During the final stages of life, they become pale, lose turgor pressure, and appear flaccid and decrepit (see Johnson et al. 1984). There are popular theories of aging which have been discussed and influence the sudy of C. elegans aging.

I. Oxidative Damage

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There is a theory which brings up oxidative damages as a cause of aging. Conforming to this theory, it's possible to say that long-lived organisms are relatively more resistant to oxidative damages; e.g., through effective ways of scavenging reactive oxygen species (ROS).

II. Metabolic Rate

This theory states that the rate of metabolism controls the duration of life span. This means the faster one species burns calories, the more rapidly it ages (R) In specific groups of animals, such as birds and mammals (excluding primates) a correlation between metabolic rate and life span certainly exist. Likewise, long-lived mutants exist in C. elegans that do not have lowered metabolic rates (Vanfleteren and DeVreese 1995, 1996).

III. Caloric Intake

In many ways the influence of environment on life span is well known, for instance, it can cause an improvement concerning individual's health or in contrast it also can cause illnesses. The effects of food as one of well-documented and very interesting environmental subjects on the rate of aging have been investigated. Vertebrates such as rats can live up to 60% longer than normal if they are fed a diet low in calories (R). In principle, caloric restriction could act to reset a normal control mechanism that governs the rate of aging. If an aging timer runs at different rates in different species, perhaps it can be reset within a single individual. Alternatively, caloric restriction could induce a type of aging process (or anti-aging process) that is fundamentally different from normal aging.

1.3. Polyphenols

Plenty of chemical compounds are synthesized by plants, which are not involved in their primary metabolism. These 'secondary compounds' with low molecular weight perform a numerous of ecological activities with the purpose of increasing the plants survival under stress (Winkel-Shirley, 2002). They also play an important role in the defense mechanism of plant against pathogenic stresses with antifungal, anti bacterial as well as antiviral activity (Bennett & Wallsgrove, 1994; Kuc, 1995).

Moreover, beneficial effects of fruits and vegetables on a wide spectrum of health related factors could be linked to secondary metabolites (Liu, 2003). Not only plants profit from secondary metabolites, meanwhile they have been subjected to different pharmaceutical, medicinal and nutritional researches. Their antioxidative effects (Duthie et al., 2003; Frei & Higdon, 2003; Ariga, 2004), their preventive effect on age- related diseases such as cancer (Lee & Lee, 2006; Duthie, 2007; Bonfili et al., 2008), cardiovascular diseases (Knekt et al., 2002; Manach et al., 2005) and neurodegenerative disorders (Ramassamy, 2006; Singh et al., 2008) in addition with, life span and stress resistance increasing capacities in various model organisms (Howitz et al., Wood et al., 2004; Baur et al., 2006; Wilson et al.,2006; Kampkötter et al., 2008; Saul et al., 2008; Pietsch et al., 2009) resulted in a large commercial marketing of polyphenolic food supplements. In the past, the antioxidant capacity of polyphenols considered to play a key role for their health benefits. Recent studies, however, leads to mention their antioxidant-independent or even pro-oxidant mechanisms (Akagawa et al, 2003; Nakagawa et al, 2004;. Galati et al, 2006;. Maeta et al, 2007.). Additionally the influence of polyphenols on gene expression by using different signaling cascades has been shown (Williams et al, 2004;.Narayanan 2006, Wilson et al, 2006;. Pietsch et al, 2009). Moreover, in literature diversity of polyphenol- mediated phenotypes both in quantity and quality are too high; therefore an explanation for positive effect on health through their antioxidant activity is not sufficient (Stevenson & Hurst, 2007). This has been supported through the studies of Doonan et al. (2008b), Gems & Doonan (2009) and Pun et al. (2010) supported. They showed that antioxidant effects and longevity in C.elegans are not correletaed

In reality, polyphenols seem to act through hormetic level. Their toxic potential uses for making plants pest repellents. In higher organisms this toxicity is only a mild stress, which can trigger hormesis (Lindsay, 2005, Mattson et al, 2007;. Son et al, 2008.).

Tannins are secondary metabolites with the molecular weight between 500 and 3000 Da, rarely up to 30.000 Da. They have polyphenolic structure which differs from other polyphenols especially in their ability to bind to proteins and precipitate them (Nadine saul et al, 2010). Through Hydrogen bonding, covalent bonding or hydrophobic interaction they can bind to proteins. Numerous free hydroxyl groups in tannins structures are crucial for these bonds and for the protein precipitation.There are two main groups of tannins: Hydrolysable tannins and condensed tannins. Tannic acid belongs to the first group of tannins (Hydrolysable tannins).Its chemical structure consists of five digallic acid residues which are linked covalently to a central glucose molecule. Because of its astringent feature

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Molecular structure of Tannic Acid

MW: 1701,22 g/ml

TA was used in various remedies for burns, diarrhea, poisonings .This gallotannin is found in various beverages (eg, red wine and tea) and food sources (eg, grapes and nuts). Several health benefits of TA have been reported, including anti-amyloidogenic (Ono et al., 2004), neuroprotective (Yazawa et al., 2006), antimicrobial (Chung et al., 1998a; Taguri et al., 2004; Kim et al., 2009), anticancer and antimutagenic (Huang et al., 1985; Kamei et al., 1999; Taffetani et al., 2005; Chen et al., 2009), and antioxidative (Lopes et al., 1999; Andrade et al., 2005, 2006) properties.

In contrast, some detrimental effects have also been highlighted, such as a potential pro-oxidant action (Khan et al., 2000; Varanka et al., 2001; Labieniec et al., 2003) and antinutritional effects, characterized by the decrease of ingestion, growth, net metabolizable energy, and protein digestibility (Mosha et al., 1995; Carbonaro et al., 2001; Samantha et al., 2004). These observations provoke the hypothesis that TA is a potent life-extending substance due to effects related to calorie restriction (CR) and/or the "Disposable Soma Theory."

1.4. The model organism Caenrhabditis elegans

To study aging and longevity, different model organisms were used by scientists, which possess shorter life span comparing to human. Ethical safety and the ability to repeat experiments on model organisms are other crucial factors. This includes especially the fruit fly Drosophila melanogaster (Helfand and Rogina, 2003), the yeast Saccharomyces cerevisiae (Gershon & Gershon, 2000), various rodents (Hazzard, 1991; Masoro, 1991) and Caenorhabditis elegans (Gershon & Gershon, 2002, Johnson, 2006, 2008). The existence of a variety of pollutant-induced genes in C.elegans (Custodia et al, 2001; Menzel et al, 2002) and its high sensitivity to environmental pollutants (Traunspurger et al., 1995), which can be absorbed as food through its thin cuticle is one of the particular interests of ecologists and ecotoxicologists. Nematode is an ecological wonder, as it has been found only in human habitats such as compost, which makes its original habitat still unknown (Kiontke & Brewhouse, 2006). Over the past decades Caenrhabditis elegans has been used as a model organism in researches in developmental biology, neurology as well as pharmacology. Sydney Brenner has established C.elegans as a model organism in the mid 1960s. With his works, he has tried to find an appropriate model which could be used to clarify the molecular mechanism of development and function of the nervous system (Strange 2006). C.elegans belongs to the phylum Nematoda (roundworm). C.elegans is an anatomically simple organism comprising under a thousand somatic cells. Its small size (1 mm long), short generation time and experimental flexibility, short life span (2-3 weeks) and ease of maintenance in the laboratory (tens of thousands can be grown on a 90-mm agar plate coated with bacteria) allow for inexpensive and rapid production of animals for experimental analysis. It contains the most important features of higher organisms, like a nervous system, an epidermis, a gastrointestinal tract and gonads (Jorgensen and Mango, 2002). Being transparent is also one of the advantages of the worms for research. It allows us to use a simple microscope, to view C. elegans internal organs/cells without any sort of invasive techniques. Moreover, visualization of cells and developmental processes such as cell migration or apoptosis in a living animal is possible. The worm can also easily be grown in liquid medium NGM (Nematode Growth Medium). In general adults C.elegans are hermaphroditic, about 0.2% of wild type populations consist of male (Hodgkin et al., 1979). At the same time that 300 offspring generated by Self-fertilized hermaphrodites, more than 1000 progeny can be produced by male-fertilized hermaphrodites. C.elegans is used for studying complicated diseases such as Alzheimer (Link, 2006; Morcos & Hutter, 2009), oncological disorders (Saito & van den Heuvel, 2002; Poulin et al., 2004; Jannot & Simard, 2006) and diabetes (Morcos and Hutter, 2009). Within associated diseases around 60% of human genes have a homologous gene in C.elegans (Wildner & Tovar, 1999, Saito & van den Heuvel, 2002), which makes it possible to apply the results directly in medical research. The metabolic rate and the rate of life cycle in C.elegans become greater by increasing the temperature in its surrounding.

Two days after hatching (at 20Ëš C), the worms begin to lay down eggs up to 300. Mainly on 4-5th day of adulthood the reproductive phase is completed which animal begins to age noticeably. This is mostly obvious in the reduction of pharynx pumping frequency as well as the deceleration of their movements. Generally after 30 days most of the animals are died.

Fig. WormAtlas. Cold Spring Harbor Laboratory Press. doi:10.3908/wormatlas.1.1

Fig2. WormAtlas. Cold SpringHarbor Laboratory Press. doi:10.3908/wormatlas.1.1

Studying dauer larvae formation by biologists is also one of the interesting aspects Of C.elegans. In presence of unfavorable environment, L2 larvae have the opportunity to change their progress instead of reproduction to dauer formation. However after few months with the improvement in their conditions they become to adult worms (Vanfleteren & Braeckman, 1999; Hu, 2007; Wang et al, 2009). Several genes and pathways related to aging and stress physiology were discovered by exploring the amazing life extension in dauer larvae (Kenyon et al, 1993; Riddle & Albert, 1997).

C. elegans is also a traditional model in aging research and provides a powerful model to simulate neurodegenerative diseases and aging processes which could be used to dissect the molecular and cellular pathways in these important areas. There are lots of important factors which introduce C.elegans as a perfect model organism in aging research concluding; easy cultivation, short life span, short generation time, existence of long and short mutants as well as the complete genome sequencing with a relatively high compliance to human genome.

With the help of genome sequencing the similarities between different species among eukaryotes was verified which successfully concluded that 50% of the C.elegans genes have homolog in the human genome. In conclusion, these similarities give us the ability of finding a significant relation between knowledge which we obtain by the molecular pathways and mechanisms involved in worm's neurophysiology to human physiology and diseases (Conn M, 2008). Therefore C. elegans has been used to explain the complicated mechanisms involved in human aging processes and neurodegenerative diseases. Locomotion, feeding, defecation, egg laying, dauer larva formation, sensory responses to touch, smell, taste, and temperature are some of the worm's complicated behavioral features which C.elegans in a spite of its simple anatomy; shows as well as other complex behaviors like male mating, social behavior, and learning and memory ( Rankin, 2002; de Bono, 2003).

Its body has an unsegmented, cylindrical structure like other nematodes which posses an outer tube as well as an inner tube while pseudocoelomic space separates these both tubes. Under the cuticle, hypoderm is located which is an epithelium. Whole surface of worm covers with cuticle. The secretion of cuticle will be done with hypodermal cells. Hypodermal cells are mainly multinucleate and act as a storage site for lipids as well as other molecules (Hedgecock et al. 1987; White 1988) , they are also incorporate in phagocytosis of apoptotic cells (Sulston et al. 1983; Lints and Hall 2009a).

To gain mutants various methods have been used such as using chemical mutagenesis, genetic transformation as well as exposure to ionizing radiation (Anderson 1995; Jorgensen and Mango 2002). There is a possibility to keep different strains of C.elegans as frozen stocks for a long time, in this condition as well as other unpleasant conditions; they will switch to a stage called dauer larva which has a longer life span than the normal one (Casasad and Russel 1975). To prolong life span as well as causing a delay in aging process in C.elegans at least four methods are already known. These include i) Hormesis, ii) CR effect, iii) different genetic manipulation and iv) Treatments with synthetic or natural substances. C.elegans has numerous of convenient characteristics which are desirable for laboratory studies in aging, synaptic transmission, cell division and sensory physiology. Under low-dose of various chemicals and physical stressors such as applying heat Hormetic induced longevity has been increased (Yashin et al. 2001; Hartwig et al. 2009; le Bourg, 2009). Cyper et al (2006) have proved relevance between involved Genes in dauer formation consisting of daf-16, daf-18 and daf-12 and mediating the hormetic durability. In nematodes CR effect can be triggered in various ways, using an axenic medium in which either E. coli or another species are available can cause life extension with 50- 80% (Walker et al., 2005). Meanwhile over 270 genes have been introduced which can change the shutting down of C.elegans life span drastically (Partridge, 2008). Collins et al. (2006) offered an overview in which substances are able to prolong C.elegans life span without causing any genetic manipulation. These include antiepileptic, antioxidants such as Vitamin E and two plant extracts from blueberries (rich in polyphenols) as well as Gingko Biloba. One of the particular relevant studies is the life span increasing properties of polyphenols resveratrol, in which a controversial discussion still exists. Although a life span increasing effect was detected by Wood et al. (2004), Viswanathan et al. (2005) and Gruber et al. (2007), Bass et al. (2007) only observed a variable and weak effect. Other natural substances with the life prolonging effect on C.elegans are Quercetin which icludes polyphenols (Kampkötter et al, 2007b, 2008; Saul et al, 2008; Pietsch et al, 2009), α-Lipoic acid (Brown et al., 2006) and Huminfeed ® (Steinberg et al., 2007).

Because of the observed life-modulating properties in some polyphenols or polyphenol-rich extracts we believe that this group needs a closer look. Therefore, this work is devoted to Tannic Acid and plant polyphenolic extracts from Pelargonium sidoides.

1.5. Pelargonium sidoides

Pelargonium sidoides belongs to family Geraniaceae. Plant with tuberous roots, ovate-cordate shaped, slightly silky and fairly aromatic leaves, small tubular flowers that are dark purplish red to nearly black (van Wyk and Wink, 2004). When they are cultivated, P. sidoides plants are evergreen when they are cultivated (Van der Walt and Vorster, 1988) however, in nature at some district they die back in winter time (White, 2004-5, pers. obs.). This species is mostly distributed across central to eastern parts of South Africa, but nowadays they also grown in other regions (van Wyk and Wink, 2004). Being different from the rest of its genus in morphological features, and its progress into a highly successful herbal medicine make this species interesting.

Medicinal use of Pelargonium sidoides (Umckaloabo)

Pelargonium sidoides tuberous and woody roots were used in South Africa for its medicinal effects (Gerardy, 2002; andVan der Walt and Vorster, 1988), especially in the form of infusion to treat diarrhea, cold and respiratory infections including tuberculosis (Bladt, 1977; and Van Wyk and Gericke, 2000). P. sidoides have been targeted in different countries to produce commercial products such as (Medicerb UK, Pelargonium Syrup, Bioharmony Africa and Umckaloabo®, Spitzner) which are used to treat upper respiratory tract infections.

Clinical trials

Matthys et al (2003) has conducted a randomized, double-blind, placebo controlled trail with 468 adults, in which the effect of plants root extract (EPs7630®) to treat acute bronchitis was obvious following only mild or unserious adverse effects. Chuchalin et al. (2005) also has investigated the effect of (EPs7630®) in a same clinical trial was based at six outpatients clinics in Germany including 124 patients Treatment was successful and there wasn't any report about serious side effects. Chemical, immunological and antibacterial assays were conducted to present a reasonable basis for the successful treatment of acute bronchitis with P.sidoides extract (Kayser andKolodziej, 1997; Kolodziej et al., 2003; Kolodziej et al., 2005; Seidel and Taylor, 2004 and Trun et al., 2006). The main ingredients in extracts which derive from roots of P.sidoides are the coumarins mainly umckalin and 6, 8-dihydroxy-5, 7-dimethoxycoumarin (Kayser and Kolodziej, 1997), gallic acid and its methyl ester (Kayser and Kolodziej, 1997), (+)-catechin (Kolodziej et al., 2003), certain fatty acids (Seidel and Taylor, 2004) and tannins (Kolodziej et al., 2005).

Antibacterial and Antifungal effect

The antibacterial effect of P.sidoide root and leaf extracts was examined against a range of pathogenic bacteria causing respiratory tract infections (Kayser and Kolodziej, 1997). Umckalin and 6, 8-dihydroxy-5, 7-dimethoxycoumarin are the most active compounds in P. sidoides roots extracts by showing respectively Minimum inhibitory concentrations (MIC) between 200-500 µg/ml and 220-500 µg/ml (Kayser and Kolodziej 1997). Afterwards, an impressive effect of P. sidoides against multi resistant Staphylococcus aureus strains was announced by Kolodziej et al. (2003). In addition, the growth of several fungal species causing respiratory tract infections such as Aspergillus niger, Fusarium oxysporum and Rhizopus stoloniferIts were inhibited by P. sidoides ethanolic root extracts (Mativandlela et al, 2006).

Immunomodulatory effect

Functional bioassays make the stimulation of the immune system's measurement possible. Such assays usually use a step in the non-specific immune system and consider a parameter, which normally relates the changes of concentration in vivo with activation of the immune defense. Different functional bioassays were used to measure the stimulation of the immune defense [extracellular Leishmania growth assay (Kayser et al, 1997), INF activity (Marcucci et al, 1992), TNF-release (Wagner and Juric, 1991) and biomedical assay for inorganic nitric oxidie (iNO) - release (Ding et al, 1998)]. None of the test substances was significantly effective against extracellular, promastigote forms of Leishmani. However, all the pelargonium extracts, gallic acid and its methyl ester were effective against amastigotes in murine macrophages (Kayser et al, 2001). This information suggests an indirect effect of the tested substances on Leishmania by stimulating the cytotoxic macrophage functions.

This was approved by treating macrophage cultures with samples and finding TNF as well as iNO in cell supernatants.

Alongside with the antibacterial and antifungal properties of the constituents of P. sidoides extracts there is some molecular evidence, which proposes their immunomodulating capacities. By means of these capabilities, immune system could be able to resist against the viruses which often cause respiratory tract infections. Moreover, stimulation of the non- specific immune system with three ingredients of P. sidoides extracts including umckalin, gallic acid and 6, 8-dihydroxy-5, 7-dimethoxycoumarin was proved (Kolodziej et al. 2003). The stimulation was caused through gene expression inducing of tumor necrosis factor (TNF).

There are three isolated compounds from P.sidoides extract which show the greatest interferon (INF)-like cytoprotective property among others. This includes Gallic acid, (+)-catechin and umckalin. (Kolodziej et al, 2003). In a study which was conducted by Trun et al. (2006), they have concluded that in a present of an infectious agent EPs7630 shows an immunological effect. This can explain the condition in which immune system is in conflict with infection and may demand for extra help. To specify the compound(s) in charge of immunological activity on the molecular level (Trun et al., 2006;Kolodziej et al., 2003), Kolodziej et al. (2005) used reverse transcription (RT)-PCR. Turn et al (2006) suggested that all of the tested compounds including P. sidoides and Phyllanthus amarus extracts containing polyphenols; and chosen standards of simple phenols, proanthrocyanidins, flavan-3-ols and hydrolysable tannins were able of increasing iNOS and cytokine mRNA levels in parasitized compared to non infected cells. Kolodziej et al. (2003) concluded that there is a likelihood that the P. sidoides extracts activity is synergically related to the overall action of the root extract, or unknown compounds.

to the powerful overall action of the root extracts, or then unknown compounds were

responsible for the activity.

Collectively, these research findings clearly show that constituents of P. sidoides extracts

are effective against bacteria and also stimulate the non-specific immune system, thus

helping the body combat infectious agents, specifically viruses, and their harmful effects

on cells. It can be concluded that umckalin, the compound quantified in this study, is an

antibacterial agent with INF-like cytoprotective activity that induces the expression of

TNF in vitro.

1.6. Our Aims

2, 2-Diphenyl-1-picrylhydrazyl (DPPHËš) free radical scavenging activity

One of the most important properties of antioxidants is their abilities to trap free radicals. The radical scavenging activity of antioxidant compounds was carried out following the modified method described by Blois [R]. 2, 2-Diphenyl-1-picrylhydrazyl (DPPH˚) after reacting with hydrogen donors will be reduced to hydrazine. DPPH˚ in the form of radical has purple color which reduces after adding antioxidant agents and changes into pale yellow color. This interaction can be used to measure the antioxidant activity of different samples. Briefly 0.2 mM of DPPH˚ in methanol was prepared and 500 µl of this solution was added to 500 µl of our samples (EPs7630®, pelargonium sidoides methanolic extract, Tannic acid and EGCG as positive control) at different concentration After Thirty minutes incubation in the dark at room temperature, the absorbance was measured at ƛ max = 517 nm using Biochrom WPA Biowave II UV-spectrophotometer. The ability to scavenge the DPPH˚ radicals was calculated using the following equation:

DPPHo Scavenging effect (%) = [(A0 - A1)/A0] x 100

Whereas A0 is the absorbance of the control reaction and A1 is the absorbance of samples with different concentrations.

Superoxide anion radical inhibition activity

Superoxide anion scavenging activity of antioxidant compounds was calculated using method of Robak and Gryglewski (Robak and Gryglewski 1988) with some modifications. Oxidation of NADH in PMS-NADH system caused superoxide radicals which were assayed by reduction of NBT. Different concentration of our samples (EPs7630®, pelargonium sidoides methanolic extract, Tannic acid and EGCG as positive control) as well as 150 μl of water (control) were added to the mixture of solutions containing 150 μl of NADH (156 μM), 150μl of NBT (630 μM) in 400 μl of 0.1 M phosphate buffer pH 7.4. The reference solution contained 150 μl of NBT (630- μM) in 700 μl of 0.1 M phosphate buffer pH 7.4. By adding 0.1 ml PMS (30 µM) at the final step to mixture the reaction was started. 5 minutes later, the absorbance was measured at ƛ max = 560 nm using Biochrom WPA Biowave II UV spectrophotometer. The percentage inhibition of superoxide anion scavenging anion activity was calculated using the following formula:

% inhibition= [(A0- A1) / A0] x 100

Whereas A0 is the absorbance of the control reaction and A1 is the absorbance of various concentrations of samples.

Table1. Representation of pipetting schedule

Blank control

Control

Blank sample

Sample

Buffer (0.1 M)

1000 µl

800 µl

900 µl

800 µl

Water

-

100 µl

-

-

Sample

-

-

100 µl

100 µl

NADH(156µM)

-

100 µl

-

100 µl

NBT (630 µM)

100 µl

100 µl

100 µl

100 µl

PMS (30 µM)

100 µl

100 µl

100 µl

100 µl

Worm's strains and culture conditions

Following C. elegans strains were obtained from Caenorhabditis Genetic Center (CGC): N2, BA17, fem-1 (hc17) which is fertile at 20oc and infertile at 25oc, TJ375, (hsp-16.2/GFP) and TJ356 (DAF-16:GFP). For maintenance and growth of C.elegans, Nematode growth medium (NGM) were used at 20oc (Brenner 1974). Age-synchronized worms were raised from eggs. Plates were seeded with live Escherichia coli OP50 which was also obtained from Caenorhabditis Genetic Center (CGC). E. coli OP50 is a uracil auxotroph whose growth is limited on NGM plates (wormbook.org). To obtain eggs hermaphrodites were treated with sodium hypochlorite and sodium hydroxide, killing adult worms but not their eggs (Apfeld and Kenyon 1999).

Freezing and recovery process in C. elegans

Caenorhabditis elegans can be frozen and stored in liquid nitrogen (−196 °C) (Brenner, 1974). To freeze the worms successfully we need to use animals at the correct stage of their life cycle. Adding Glycerol to the freezing media and gently operating the cooling process to -80˚C are the other important factors. Freshly starved young larvae (L1-L2 stage) are best choice which can survive freezing process best, however, well-fed animals, dauers, eggs and adults will not survive.The final volume of glycerol in the freezing solution should be 15 % and advisable decrease in temperature is 1°C per minute.

The CGC uses two solutions for freezing C. elegans: a Liquid Freezing Solution (Brenner, 1974) and Soft Agar Freezing Solution (Leon Avery, personal communication).

Preparing synchronous cultures of C.elegans

To start each experiment with the worms in the same age it is necessary to obtain eggs for each strain. First we need to wash gravid hermaphrodites on NGM plates (Nematode Growth Medium) several times. With the aid of pipetting, the worms which were floating in sterilized water will be collected in a sterile tube with the total volume of 3.5 ml. After adding fresh mixture of 0.5 ml 5 M NaOH and 1 ml 5% sodium hypochlorite, the tube was shaked very well every 2 minutes for approximately 10 minutes followed by 30 seconds centrifuge at 1300 g to release eggs. After removing the supernatant, 5 ml sterilized water was added for repeating the centrifuge process.

At the final step 1 ml sterilized water was added to the liquid containing eggs. To start each experiment the eggs were transferred to new NGM or S-medium plate which contains E.coli OP50.

Lifespan assay

3 days old adult hermaphrodite worms were placed in groups of 25 individuals and transferred onto small plates (35 mm-Diameter, CELLSTAR, Greiner bio-one GmbH, Germany) containing NGM with 100 µl spot of Escherichia coli OP50 (obtained from the Caenorhabditis Genetic Center) and treated with 50 µg/ml of EPs7630® along also with 10 µM of Tannic acid. Aqueous solutions of both EPs7630® and Tannic acid were used in all experiments. To avoid Progeny worms were transferred daily to new plastic Petri dishes containing E. coli OP50 together with 50µg/ml EPs7630® or 10 µM Tannic acid. Animals were checked and counted daily, considered as dead when they failed to respond to gentle touch with platinum wire. Nematodes suffering from internal hatching and those that escaped from the NGM agar were considered as censor.

Survival assay

By adding an acute, lethal concentration of juglone at 300 μM Oxidative stress was induced. Juglone (5-hydroxy-1, 4-naphthalenedione) was solved in ethanol (abs.) to prepare its stock solution. Synchronized N2 worms were cultured in a suspension containing E. coli OP50 in S-medium at the concentration of 1x109 cell/ml in the presence of 50 µg/ml EPs7630®, 100 µg/ml pelargonium sidoides extract and 10 µM of Tannic acid on the day after hatching for 48 h. In this experiment control group consists of untreated worms. On the third day juglone was added to the medium and the survivors counted every two hours.

Measurement of intracellular H2O2 in C.elegans

Intracellular levels of hydrogen peroxide (H2O2)-related ROS were investigated in model organism C.elegans using 2, 7-dichlorofluorescein diacetate (DCF-DA). DCF-DA oxidizes by ROS (Reactive Oxygen Species) which converts the molecule to a highly fluorescent molecule (DCF) 2, 7-dichlorofluorescein (DCF). For time to time the increase of fluorescence signal will occur by producing of more ROS. Age synchronized worms (N2-wild type) were treated with 50 µg/ml of EPs7630®, 100 µg/ml pelargonium sidoides extract and 10 µM of Tannic acid on the day after hatching for 72 h.60 worms from control (untreated worms) and treatment were collected into 100 μL PBS with 0.1% Tween-20 (PBST). To break the outer cuticle, worms were homogenized, and the homogenates were pipetted into 96-well plates (Greiner 96 Flat Bottom Transparent Polystyrol ) which contained 50 μM DCF-DA. A microplate fluorescence reader (Safire2; XFLUOR4SAFIREII- AUTOSA) read samples at 37°C every 10 min for 2.5 h at excitation 485 nm and emission 530 nm.

DAF-16 localization via fluorescence microscopy

In TJ356 strain of C.elegnas, the gene coding for green fluorescent protein (GFP) is fused to the daf-16 gene (Henderson and Johnson 2001). To investigate the effect of EPs7630® and Tannic acid on the intracellular distribution of DAF-16, worms were incubated in S-medium with E.coli OP50 containing 50 mg/ml of EPs7630®, 100 µg/ml of Pelargonium sidoides extract and 10 µM of Tannic acid for 1 h. In this experiment, worms under heat shock stress (37 ËšC for 15 min), oxidative stress (20 µM juglone) together with treated worms with 220 µM of EGCG also used as positive controls. DAF-16 localization was examined in approximately 20 animals per treatment. Worms from each sample were mounted onto glass slide and paralyzed by 10 mM sodium azide in PBS. Fluorescence images were taken at constant exposure times (Nikon-eclipse 90i, Nikon digital sight DS-Qi1Mc; 40Ã- objective; Nikon Imaging Center, University of Heidelberg).

Quantitation of hsp-16.2:GFP expression

To quantify the expression of hsp-16-2/GFP under oxidative stress TJ375-(hsp-16.2/GFP) strain was used. The worms were first treated with 50 µg/ml of EPs7630®, 100 µg/ml pelargonium sidoides extract and 10 µM of Tannic acid from the L1 stage for 48 h, then after adding 20 µM juglone (a quinone known to induce superoxide radicals in C. elegans) for 24 h to the medium the expression of hsp-16-2 was measured by directly observing the fluorescence of the GFP reporter. Worms from treatment as well as control (untreated) were mounted onto glass slide and paralyzed By 10 mM sodium azide in PBS (Sultson and Hodgkin 1988) Fluorescence images were taken at constant exposure time 4 seconds using 20x objective of a microscope (Nikon-eclipse 90i, Nikon Imaging Center, Heidelberg University) with digital camera (Nikon digital sight DS-Qi1Mc). The images were analyzed by using Image J (NIS-Elements AR 3 software). For measuring the mean pixel density, the anterior part of the worms was outlined, following by inversion of images into black & white.

LC-MS /HPLC

Statistical analysis

To find out statistical analysis and calculate mean lifespan, lifespan data from the day of hatching were subjected to Kaplan-Meier survival analysis using StatView 5 (SAS) software. To compare all groups with each other log rank (Mantel-Cox) test was applied. Animals which were exploded or bagged considered as censor at the time of the event and were incorporated into the data set (Apfeld and Kenyon 1999).

All statistical comparison between controls and treatment were done using two-tailed unpaired Student's t-test, assuming equal variance. All figures indicate means and standard errors of the mean. P < 0.05 was remarked as statistically significant.