**Effects of Heavy Metals on the Snails** *Helix aspersa* **Bioindicators of the Environment Pollution for Human Health**

Nedjoud Grara<sup>1</sup> ,Amira Atailia<sup>2</sup> , Mounir Boucenna<sup>2</sup> , Fadila Khaldi<sup>1</sup> ,Houria Berrebbah2 , Mohamed Reda Djebar<sup>2</sup>

1 Laboratory of Sciences and Technology of water and the environment, University of Mohammed cherifMessaadia, Souk-Ahras, Algeria 2 Laboratory of Cellular Toxicology. University of Annaba, Algeria

#### **Abstract**

In this study we were interested in the evaluation of toxicity sub-chronicle of the metal dust collected on the level of the iron and steel complex of EL-Hadjar**(Eastern Algeria) on** accumulating organizations bio and bio indicator of pollution *Helix aspersa.* The first results on the metabolic level show that metal dust causes a significant increase in proteins with a significant reduction in the Carbohydrates and lipids on the level of the two studied bodies (digestive Gland and the kidney). With regard to the bio markers we highlighted a reduction in the acetyl cholinesterase (AChE) activity at the level of the head. In addition, the exposure of *Helix aspersa*to metal dust induced a lipidic peroxidation with release of malondialdehyde (MDA) to the level of the studied bodies.

**Keywords:** *Helix aspersa,* dust metal, biomarkers, pollution, MDA, AChE, bioaccumulation, digestive gland, kidney.

## **1. Introduction**

The transfer of pollutants in the trophic networks is not limited to the organic compounds. The increase in the concentrations in elements traces metal (ETM) in the grounds – mainly due to the human activities[1].The central model of this study is the snail *Helix aspersa* for its capacities to accumulate the ETM with significant concentrations in its fabrics[2].The objective of this work is to study the effects of the stress oxidizing induced by metal dust of the iron and steel complex of EL-Hadjar (Annaba) on an accumulating metal bio, the gastropod terrestrial *Helix aspersa***.**

## **2. Materiel and Methods**

#### **2.1. Biological Material**

The biological material used is a terrestrial gastéropode: the snail Helix*aspersa*collected area of Guelma**(Eastern Algeria)**. The snails (of average Weight of 8,5 ± 0,15g) are high under the following optimal environmental conditions[2].

## **2.2. Chemical material (The Metallic Releases)**

The iron and steel complex of EL-Hadjar (Annaba) is at 15 km of the town of Annaba on the trunk road N44 (**(Eastern Algeria)** Metal dust in the study was collected manually .One analyzes chemical by atomic absorption was used to determine the composition of this dust. This analysis determined the presence of 07 heavy metals (Cu, Zn, Pb, Cr, Ni, Mn, Fe) [3].

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Figure (4) illustrates the variations of the rate of MDA on the level of digestive gland and of the kidney in the presence of metal dust, we note that in the presence of xenobiotic, the rate of MDA tends to increase in a manner proportions – dependent and very highly signifi cant between the rate on MDA for the treaties by the various concentrations on the level of the two bodies and this

**3.5. Effect of the metal rejections on the activity Acetylcholine Esterase (AChE)**

**Fig 1.** Evolution of the total proteins rate according to the increasing concentrations in metal dust.

**Fig 2.** Evolution of the Carbohydrates rate according to the increasing concentrations in metal dust.

Figure (5) illustrates the variations of the rate of AChE on the level of the head of snails; we note that in the presence of xenobiotic, the acetylcholine esterase rate tends to decrease in a manner proportions – dependent. The statistical analysis reveals a diff erence very highly signifi cant

**3.4. Effect of the metal rejections on the malondialdehyde (MDA)**

compared to the control for the treaties by the various concentrations tested.

always compared to the control.

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#### **2.3. Mode of treatment**

Treatment of the animals at summer carried out by addition of the increasing concentrations of metal dust in the food. We retained 4 concentrations and a pilot medium (100, 500, 1000, 1500 food µg/g).[2].After (28days) of treatment, the snails are dissected and digestive Gland (DG),the kidney (K) and Headare taken.

## **2.4. Measured parameters**

The proteins are quantified to the method of [4], the proportioning of the carbohydrates is carried by the method of [5] and lipid level's is given according to the method of [6] .The MDA is proportioned according to the method of [7] , the proportioning of the (AChE) is carried out according to the method of[8].

## **2.5. Statistical analysis of the results**

The results are compared by the nonparametric test of Kruskal-Wallis, This test is carried out using software of analysis of the data: Minitab (Version 14.0) [9].

## **3. Results**

## **3.1. Effect of the metal rejections on the total proteins**

Figure (1) illustrates the variations of the total protein on the level of the digestive gland and the kidney in the presence of metal dust. We note that at the treaties, the rate of total proteins tends to increase in manner proportions dependent in the digestive gland and kidney.

## **3.2. Effect of the metal rejections on the total of Carbohydrates**

Figure (2) illustrates the variations of the total of Carbohydrates on the level of the digestive gland and the kidney in the presence of metal dust. It is noticed that the rate of the carbohydrates at the level of digestive gland and kidney to decrease it in snails treated with the concentrations compared to the control.

## **3.3. Effect of the metal rejections on the Total lipids**

Figure (3) illustrates the variations of the total lipid levels on the level of the digestive gland and the kidney in the presence of metal dust. We note that in the presence of xenobiotic the lipid level in the digestive gland and kidney decreases in a significant way for the treaties by concentrations compared to the control.

#### **3.4. Effect of the metal rejections on the malondialdehyde (MDA)**

Figure (4) illustrates the variations of the rate of MDA on the level of digestive gland and of the kidney in the presence of metal dust, we note that in the presence of xenobiotic, the rate of MDA tends to increase in a manner proportions – dependent and very highly signifi cant between the rate on MDA for the treaties by the various concentrations on the level of the two bodies and this always compared to the control.

#### **3.5. Effect of the metal rejections on the activity Acetylcholine Esterase (AChE)**

Figure (5) illustrates the variations of the rate of AChE on the level of the head of snails; we note that in the presence of xenobiotic, the acetylcholine esterase rate tends to decrease in a manner proportions – dependent. The statistical analysis reveals a diff erence very highly signifi cant compared to the control for the treaties by the various concentrations tested.

**Fig 1.** Evolution of the total proteins rate according to the increasing concentrations in metal dust.

**Fig 2.** Evolution of the Carbohydrates rate according to the increasing concentrations in metal dust.

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In our work, we highlighted that the rate of proteins in the two bodies increases in a manner proportions –dependent in the presence of metal dust and of cadmium, these results go in the same direction as those of Masaya and *al*.,(2002)which highlighted a significant increase in the

The results concerning the evolution of the lipid level in the two bodies highlight a significant reduction in the lipids in treated snails and this in manner proportions – dependent by the various concentrations , Aurousseau(2002) suggest the free oxygenated radicals are toxic via the degradation of the lipids of which the β-oxidation [11].concerning the evolution of the Carbohydrates , we noted that this rate decreases in a manner proportions –dependent in the presence of metal dust in the two bodies chosen, this reduction would be due to the oxidation of the carbohydrates in the presence of the metal ions leading to the release of aldehyds and hydrogen peroxide, under the condition of stress, the reserves of carbohydrates are exhausted to satisfy the energy demands, these results are in conformity with those of EL-Wakil and Radwan (1991), which suggested that exposed to Endosulfane, the methyl parathion, of the quinalphos and to Nuvan (pesticides) would be the consequence of the direct use of glycogen for the generation of energy[12]. In the present study, toxicity in metal dust is at the origin of an increase in the rate of MDA which

The MDA is also under product of the biosynthesis of the prostaglandin [13] . Our results are in agreement with those of Viarengoand *al*.,(1990) [14] which studied the toxic effects of heavy metals on the peroxidation of the lipids at *Mytillusgalloprovinciallis*. Our results concerning the evolution of the rate of AChE during the various experiments, highlighted a reduction proportions dependent on the activity of this enzyme dice the weakest metal concentrations. Our results are in agreement with the study of Antonio and *al*., (2003)[15] The studied snails are edible species for the man and can cause significant metal concentrations within the organization of the human[1].The metals transferred at the humandy the consummation of gastropoda can be at the origin of oxidative stress which represents one of the factors potentiating the genesis ofplurifactorielles diseases such as the cardiovascular diseases, the diabetes, rheumatisms, asthma,

Our experiments show that the snails answer the criteria of the bioindicator to take part in the bio monitoring of the environment.De, more, these are edible species for the man, therefore it is appropriate attentive on the origin of snails to be collected in nature because, being likely to

[1] ScheiflerR.Evaluation de la biodisponibilité et des transferts de polluants métalliques et organiques dans les réseaux trophiques "sol-plante-invertébrés". Thèse de doctorat, Université

total protein rate under the effect of a chemical stress at different biological models[10].

is the principal active aldehyde of theperoxidation of acid membranes.

cancer,the neurodegenerative diseases and disease of Alzheimer[16].

de Franche-Comté, Besançon, France, 2002,196 p.

**4. Discussion**

**5. Conclusion**

**6. References**

contaminate the human.

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**Fig 3.** Evolution of the lipids rate according to the increasing concentrations in metal dust.

**Fig 4.** Evo lution of the MDA according to the increasing concentrations in metal dust.

**Fig 5.** Evolution of AChE activity according to the increasing concentrations in metal dust.

## **4. Discussion**

In our work, we highlighted that the rate of proteins in the two bodies increases in a manner proportions –dependent in the presence of metal dust and of cadmium, these results go in the same direction as those of Masaya and *al*.,(2002)which highlighted a significant increase in the total protein rate under the effect of a chemical stress at different biological models[10].

The results concerning the evolution of the lipid level in the two bodies highlight a significant reduction in the lipids in treated snails and this in manner proportions – dependent by the various concentrations , Aurousseau(2002) suggest the free oxygenated radicals are toxic via the degradation of the lipids of which the β-oxidation [11].concerning the evolution of the Carbohydrates , we noted that this rate decreases in a manner proportions –dependent in the presence of metal dust in the two bodies chosen, this reduction would be due to the oxidation of the carbohydrates in the presence of the metal ions leading to the release of aldehyds and hydrogen peroxide, under the condition of stress, the reserves of carbohydrates are exhausted to satisfy the energy demands, these results are in conformity with those of EL-Wakil and Radwan (1991), which suggested that exposed to Endosulfane, the methyl parathion, of the quinalphos and to Nuvan (pesticides) would be the consequence of the direct use of glycogen for the generation of energy[12].

In the present study, toxicity in metal dust is at the origin of an increase in the rate of MDA which is the principal active aldehyde of theperoxidation of acid membranes.

The MDA is also under product of the biosynthesis of the prostaglandin [13] . Our results are in agreement with those of Viarengoand *al*.,(1990) [14] which studied the toxic effects of heavy metals on the peroxidation of the lipids at *Mytillusgalloprovinciallis*. Our results concerning the evolution of the rate of AChE during the various experiments, highlighted a reduction proportions dependent on the activity of this enzyme dice the weakest metal concentrations. Our results are in agreement with the study of Antonio and *al*., (2003)[15] The studied snails are edible species for the man and can cause significant metal concentrations within the organization of the human[1].The metals transferred at the humandy the consummation of gastropoda can be at the origin of oxidative stress which represents one of the factors potentiating the genesis ofplurifactorielles diseases such as the cardiovascular diseases, the diabetes, rheumatisms, asthma, cancer,the neurodegenerative diseases and disease of Alzheimer[16].

## **5. Conclusion**

Our experiments show that the snails answer the criteria of the bioindicator to take part in the bio monitoring of the environment.De, more, these are edible species for the man, therefore it is appropriate attentive on the origin of snails to be collected in nature because, being likely to contaminate the human.

## **6. References**

[1] ScheiflerR.Evaluation de la biodisponibilité et des transferts de polluants métalliques et organiques dans les réseaux trophiques "sol-plante-invertébrés". Thèse de doctorat, Université de Franche-Comté, Besançon, France, 2002,196 p.

[2] Coeurdassier M., Saint-Denis M., Gomot-de Vaufleury A., Ribera D., BadotP.M.The garden snail *(Helix aspersa)* as bioindicator of organophosphorus exposure: effects of dimethoate on survival, growth and acetylcholinesterases activity. *EnvironmentalToxicology and Chemistry*2001; 20, 1951- 1957.

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**Honey Bee Venom Will Differentiate** 

, Zahra Nazari<sup>1</sup>

2 Department of Biology, Faculty of Science, Islamic Azad University, Damghan, Iran

Mohammad Nabiuni1,\*, Elham Azimi<sup>2</sup>

\*Corresponding author, Email: Nabiuni@tmu.ac.ir

under BV treatment and it may be useful in cell therapy.

**Keywords**: mesenchymal stem cells, Bee venom, osteogenic differentiation.

Tel: +98 9126609337, Fax: +98 26145100536

Abdolhosein Shiravi<sup>2</sup>

Tehran, Iran

**Abstract**

**1. Introduction** 

**Mesenchymal Stem Cells in to the Osteocyte**

,

1 Department of Cell and Molecular Biology, Faculty of Biological Sciences, Kharazmi University,

Umbilical cord (UC) is an important source of multipotential mesenchymal stem cells (MSCs). It has observed that bee venom (BV) is effective in survival and differentiation of the cells. We hypotheses that BV can cause differentiation of MSCs to osteocytes. The cells obtained from mouse UC tissues were digested and suspended in DMEM medium. After 24 hours, to induce osteogenic differentiation, cells were cultured for 14 and 21days respectively in DMEM medium contain different concentrations of BV (1,2,3,4,5,6 µgr/ml). Following the treatment, calcium's level in the cells was determined by Alizarin red staining. Cytotoxic effects of BV on MSCs were tested by MTT assay which are shown that BV inhibits MSCs growth. Furthermore, by Alizarin red test, we found that BV increases calcium level in MSCs on dose and time dependent manner. In conclusion we suggest that the MSCs from UC have differentiation potential to osteocyte

Umbilical Cord (UC) is a rich source of multipotential mesenchymal stem cells (MSCs). MSC's are a type of multipotent adult stem cell that was originally described as early as the 1960's in animal experiments [1]. Many studies have demonstrated that MSCs have an enormous therapeutic potential for cell therapy [2]. These cells are also considered to have regenerating potential for certain degenerative conditions. MSCs can be differentiate into bone, adipose, cartilage, muscle, and endothelium if these cells are cultured under specific conditions MSC's is obtainable from placental blood, bone marrow, fatty tissue and amniotic fluid. It is also easily obtainable from the tissue of the cord itself [3,4]. In this study MSC's present in the UC were selected. UC is a low cost source of MSCs with multiple differentiation capacities and so it is a much better source than other sources, such as bone marrow or fatty tissue [5,6]. Bee venom (BV) has been used for the treatment of chronic inflammatory diseases such as rheumatoid arthritis and relief of pain in oriental medicine. More recently, studies indicate that BV has potential role in cancer therapy [7,8]. BV contains a variety of biologically active components like melittin and phospholipase A2

> © 2012 Nabiuni et al.; licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

provided the original work is properly cited.

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## **Honey Bee Venom Will Differentiate Mesenchymal Stem Cells in to the Osteocyte**

Mohammad Nabiuni1,\*, Elham Azimi<sup>2</sup> ,

Abdolhosein Shiravi<sup>2</sup> , Zahra Nazari<sup>1</sup>

1 Department of Cell and Molecular Biology, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran

2 Department of Biology, Faculty of Science, Islamic Azad University, Damghan, Iran

\*Corresponding author, Email: Nabiuni@tmu.ac.ir

Tel: +98 9126609337, Fax: +98 26145100536

#### **Abstract**

Umbilical cord (UC) is an important source of multipotential mesenchymal stem cells (MSCs). It has observed that bee venom (BV) is effective in survival and differentiation of the cells. We hypotheses that BV can cause differentiation of MSCs to osteocytes. The cells obtained from mouse UC tissues were digested and suspended in DMEM medium. After 24 hours, to induce osteogenic differentiation, cells were cultured for 14 and 21days respectively in DMEM medium contain different concentrations of BV (1,2,3,4,5,6 µgr/ml). Following the treatment, calcium's level in the cells was determined by Alizarin red staining. Cytotoxic effects of BV on MSCs were tested by MTT assay which are shown that BV inhibits MSCs growth. Furthermore, by Alizarin red test, we found that BV increases calcium level in MSCs on dose and time dependent manner. In conclusion we suggest that the MSCs from UC have differentiation potential to osteocyte under BV treatment and it may be useful in cell therapy.

**Keywords**: mesenchymal stem cells, Bee venom, osteogenic differentiation.

## **1. Introduction**

Umbilical Cord (UC) is a rich source of multipotential mesenchymal stem cells (MSCs). MSC's are a type of multipotent adult stem cell that was originally described as early as the 1960's in animal experiments [1]. Many studies have demonstrated that MSCs have an enormous therapeutic potential for cell therapy [2]. These cells are also considered to have regenerating potential for certain degenerative conditions. MSCs can be differentiate into bone, adipose, cartilage, muscle, and endothelium if these cells are cultured under specific conditions MSC's is obtainable from placental blood, bone marrow, fatty tissue and amniotic fluid. It is also easily obtainable from the tissue of the cord itself [3,4]. In this study MSC's present in the UC were selected. UC is a low cost source of MSCs with multiple differentiation capacities and so it is a much better source than other sources, such as bone marrow or fatty tissue [5,6]. Bee venom (BV) has been used for the treatment of chronic inflammatory diseases such as rheumatoid arthritis and relief of pain in oriental medicine. More recently, studies indicate that BV has potential role in cancer therapy [7,8]. BV contains a variety of biologically active components like melittin and phospholipase A2

© 2012 Nabiuni et al.; licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

(PLA2) [9]. Previous observations have shown that bee venom or its components are eff ective in proliferation, survival and diff erentiation of the cells [10]. In this view the aim of this study was the examination of diff erentiating potential of BV on MSCs. Our hypothesis is that BV could cause diff erentiation of MSCs to osteocytes.

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**Figure 2.** Light microscope micrograph of the alizarin red staining of MSCs following exposure to diff erent concentrations of BV for 14 days. A: control group, B: 4µgr/ml BV treated cells, C: 5µgr/ml BV treated cells. D:

**Figure 3**. Light microscope micrograph of the alizarin red staining of MSCs following exposure to diff erent concentrations of BV for 21 days. A: control group, B: 4µgr/ml BV treated cells, C: 5µgr/ml BV treated cells. D:

6µgr/ml BV treated cells (200x magnification)

6µgr/ml BV treated cells (200x magnification)

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#### **2. Materials and methods**

UC from 1-2 pregnant mice were obtained from animal lab unit in Kharazmi university which were killed by ether and the harvested pieces of tissue were washed several times in sterile phosphate-buff ered saline (PBS) and then mechanically minced and enzymatically digested with 0.25% trypsin-EDTA (Gibco-Invitrogen) for approximately 10 min at 37 ˚C. Aft er centrifugation (1500rpm for 10 min), cells were collected and cultured in Dulbecco's modified Eagle's medium (Gibco-Invitrogen) supplemented by 10% fetal bovine serum (FBS), 100 U/ml penicillin-streptomycin (Gibco-Invitrogen). Cell cultures were maintained at 37 ˚C with a water-saturated atmosphere and 5% CO2 . Medium was replaced one to two times every week. Three to 5 days aft er initiating incubation, to calculate the proper doses of BV that cause diff erentiation of MSCs and also have low cytotoxicity, the cells were cultured with diff erent concentrations of BV (1–12 µgr/ ml) for 24h. Cell viability then measured by MTT assay. In continue, to induce osteogenic diff erentiation, the cells were treated by diff erent concentration of BV (4,5,6 µgr/ml) respectively for 14 and 21 days. Aft er these times, osteogenic diff erentiation was analyzed by alizarin red staining.

#### **3. Results**

Based on MTT assay, results the IC50 values of BV for MSCs were 7.5µgr/ml aft er 24h (fi g.1). Also the results showed that BV induces cell death in MSCs in high doses while, at low doses it could inhibit cell growth and induce diff erentiation. Maximum BV-induced cytotoxicity was evident aft er 24 h exposure to 15µgr/ml concentration. Our results from alizarin red staining illustrated that undiff erentiated MSCs (control group) showed almost no specific staining. Osteocyte-like cells could be seen aft er 14 days of osteogenic induction (Fig.2). But aft er 21 days of cultivation the overall color intensity of the diff erentiated MSCs was markedly enhanced (Fig.3).

**Figure 1.** Eff ect of BV on cell viability. MSCs were treated with diff erent concentration of BV for 24h.The cell viability was then determined by MTT assay, as described under Section 2. Data shown are the mean of three independent experiments ± SEM and are statistically significant in comparison to the control by one-way ANOVA.

**Figure 2.** Light microscope micrograph of the alizarin red staining of MSCs following exposure to diff erent concentrations of BV for 14 days. A: control group, B: 4µgr/ml BV treated cells, C: 5µgr/ml BV treated cells. D: 6µgr/ml BV treated cells (200x magnification)

**Figure 3**. Light microscope micrograph of the alizarin red staining of MSCs following exposure to diff erent concentrations of BV for 21 days. A: control group, B: 4µgr/ml BV treated cells, C: 5µgr/ml BV treated cells. D: 6µgr/ml BV treated cells (200x magnification)

#### **4. Summary and conclusion**

UC cells have many advantages because of the immaturity of newborn cells compared with other sources of stem cells. The UC appeared to be a source of fetal cells that could be easily used as multipotent stem cells [3]. Umblical cord derived mesenchymal stem cells can replicate stably in culture, possess the capability to differentiate into a wide variety of tissues after culture [5]. In this study we reported the osteogenic differentiation of MSCs, as characterised by alizarin red staining. By use of key markers of osteogenic differentiation (calcium deposition), we demonstrated that presence of BV can differentiate to osteocyte cells in the absence of osteogenic media. Osteogenic differentiation was greater when cultured-MSCs were treated with BV for 21 days rather than 14 days treatment. In conclusion, we suggest that BV can cause differentiation in the MSCs from UC and it may be useful in cell therapy.

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**Homocystein and Trace Elements Levels in** 

Department of Medical Physiology, College Of Medicine, University Of Babylon, Iraq

Saad Merza Al-Araji, Ala H. Abbase, Zainab F. Hassan,

**Associated Diseases**

patients groups in comparison with control group.

absorption spectrography. Ischemic heart disease .

**Abstract**

**1. Introduction** 

**Patient with Ischemic Heart Disease and some** 

This paper includes the determination homocysteine level and trace elements magnesium (Mg), zinc (Zn)and iron (Fe) in ppm (part per million); lead (Pb), cadmium (Cd ), selenium (Se ), chromium (Cr ) and germanium (Ge ) in ppb (part per billion)) in random serum of patients with pure ischemia, ischemia with hypertension and ischemia with diabetes. Homocysteine level was significantly increased ( P< 0.01) in pure ischemic patients, ischemia with hypertension and ischemia with diabetes in comparison with control group. A comparison had also been done between male & female groups in patients and control groups and no significant changes (P >0.05) were observed. The result of this study showed that concentration of the trace elements (Pb& Cd ) were significantly increased (P< 0.01) in patients groups in comparison with control group and the concentration of (Mg ,Zn, Se, Cr and Ge) were significantly decreased (P< 0.01) among

**Keywords:** Homocysteine, High performance liquid chromrtography, Trace elements ,Atomic

Homocysteine (Hcy) is an intermediate of methionine metabolism [1]. Elevated blood homocysteine concentration was an independent risk factor for cardiovascular disease [1, 2]. High-normal serum homocysteine concentrations are associated with an increased prevalence of carotid artery wall thickening [3] . The significance of the contribution of homocysteine to the variation of carotid intima-media thickness suggests a role for homocysteine as an independent risk factor for early carotid artery atherosclerosis in the asymptomatic subjects. Different studies indicated that the elevated level of total homocysteine (tHcy) had increased the risk of cardiovascular diseases and stroke [4]. Homocysteine is elevated in the case of inborn errors of methionine metabolism and excessive amount of homocysteine and its derivatives are found in blood and tissues of cardiovascular patients [5]. Moderate hyperhomocysteinemia up to (30 µmol/l ) is a major independent risk factor of a number of diseases characteristic of old ages , primarily occlusive vascular disease [6]. Non metallic elements (hydrogen, oxygen, carbon and nitrogen) make 99% of all the elements in human body, while major elements, which are calcium, magnesium, phosphorus, sulfur and chlorine, make 0.9% of the total. However, essential trace elements provide approximately less than 0.1% of human body [7]. Trace metals are metals in extremely small quantities,

> © 2012 Al-Araji et al.; licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

provided the original work is properly cited.

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#### **5. Acknowledgements**

This project was done in laboratory of Cell and Developmental Biology at Kaharazmi University and the authors wish to thanks Prof. Shahrbanoo Oryan for her support.

#### **6. References**


## **Homocystein and Trace Elements Levels in Patient with Ischemic Heart Disease and some Associated Diseases**

Saad Merza Al-Araji, Ala H. Abbase, Zainab F. Hassan,

Department of Medical Physiology, College Of Medicine, University Of Babylon, Iraq

#### **Abstract**

This paper includes the determination homocysteine level and trace elements magnesium (Mg), zinc (Zn)and iron (Fe) in ppm (part per million); lead (Pb), cadmium (Cd ), selenium (Se ), chromium (Cr ) and germanium (Ge ) in ppb (part per billion)) in random serum of patients with pure ischemia, ischemia with hypertension and ischemia with diabetes. Homocysteine level was significantly increased ( P< 0.01) in pure ischemic patients, ischemia with hypertension and ischemia with diabetes in comparison with control group. A comparison had also been done between male & female groups in patients and control groups and no significant changes (P >0.05) were observed. The result of this study showed that concentration of the trace elements (Pb& Cd ) were significantly increased (P< 0.01) in patients groups in comparison with control group and the concentration of (Mg ,Zn, Se, Cr and Ge) were significantly decreased (P< 0.01) among patients groups in comparison with control group.

**Keywords:** Homocysteine, High performance liquid chromrtography, Trace elements ,Atomic absorption spectrography. Ischemic heart disease .

## **1. Introduction**

Homocysteine (Hcy) is an intermediate of methionine metabolism [1]. Elevated blood homocysteine concentration was an independent risk factor for cardiovascular disease [1, 2]. High-normal serum homocysteine concentrations are associated with an increased prevalence of carotid artery wall thickening [3] . The significance of the contribution of homocysteine to the variation of carotid intima-media thickness suggests a role for homocysteine as an independent risk factor for early carotid artery atherosclerosis in the asymptomatic subjects. Different studies indicated that the elevated level of total homocysteine (tHcy) had increased the risk of cardiovascular diseases and stroke [4]. Homocysteine is elevated in the case of inborn errors of methionine metabolism and excessive amount of homocysteine and its derivatives are found in blood and tissues of cardiovascular patients [5]. Moderate hyperhomocysteinemia up to (30 µmol/l ) is a major independent risk factor of a number of diseases characteristic of old ages , primarily occlusive vascular disease [6]. Non metallic elements (hydrogen, oxygen, carbon and nitrogen) make 99% of all the elements in human body, while major elements, which are calcium, magnesium, phosphorus, sulfur and chlorine, make 0.9% of the total. However, essential trace elements provide approximately less than 0.1% of human body [7]. Trace metals are metals in extremely small quantities,

© 2012 Al-Araji et al.; licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

almost at the molecular level, that reside in or are present in animal and plant cells and tissue. Trace metals are a group of metals that include both heavy and transitional elements present in micrograms quantities in the blood. There are divided into two groups, essential for health and have no known biological function.

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**22 39%**

Pure ischemic patients 17 31 6 14

Control 20 36 20 48

Ischemic patients with hypertension 10 18 6 14 Ischemic patients with diabetes 8 15 10 24

Total 55 42

**Male**

Control 9.58 ±0.93 9.11±0.65

Ischemic patients 24.11\*\*±5.45 25.4\*\*±2.5

Ischemia with diabetic 22.16\*\*±3.59 21.26\*\*±1.44±

Ischemia with hypertension 20.84\*\*±3.38 23.9\*\*±4.85

**Table 3.** The mean serum level of homocysteine in male and female patients groups and control group

Comparison between ischemic male or female and similar sex healthy controls results indicate an increase in level of homocysteine among patients groups. This elevation in homocysteine concentration value was highly significant (P < 0.01). When similar comparisons were done between ischemia with hypertension and ischemia with diabetes and control group a similar behavior were observed (P < 0.01).Table 3 shows the mean values of serum level of homocysteine in male and female compared with the mean values in control group. No significant changes was ob-

served when comparison was done between two age groups as shown in table 4.

**Mean ± SD Groups**

**35 61%**

Table 2 show the sex distribution for all patients in this study. Number of female patients in ischemia with diabetes was more than male patients while in ischemia and ischemia with hyperten-

**Male Female**

**Figure 1.** Sex distribution of total ischemic patients.

sion male patients were more than female.

**Table 2.** Sex distribution in the study groups

Patients

\*\* P < 0.01

**Groups**

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**Number Male % Female %**

> **Female Mean ± SD**

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 In human body, there are some metals which naturally exist and are essential to human health. These essential metals at trace levels play vital role when present in human body and may cause some diseases when present beyond specific concentrations [8, 9].

## **2. Designing**

## MATERIALS AND METHODES

**Patients:** The study was conducted during the period from Augest, 2008 to May , 2009 in Merjan hospital in Hilla city , Babylon province, Iraq. A total 57 patients were included from urban and rural area(35 males and 22 females) (Table 3-1). Twenty three patients had pure ischemic heart disease (17 males and 6 females), 18 had ischemic heart disease with hypertension (10 males and 6 females),18 had ischemia heart disease with diabetes (8 males and 10 females ). The age of the patients varying between 40-85 years old (mean± SD 57.82±10.25).

**Control Group:** The control group consisted of 40 healthy person who were chosen as healthy, non smokers , didn't have any history of chronic disease and didn't take any treatment for chronic diseases

## **3. Results**

The patients and control were divided according to ages into two groups group 1 in which the age range between 40-60 year and age group two in which the age is > 60 year as shows in table 1.


**Table 1.** Patients and control number according to age group

The sex distribution of total ischemic patients, was clearly obvious . The highest percentage in male was 61% while female was 39% ( figure 1).

**Figure 1.** Sex distribution of total ischemic patients.

Table 2 show the sex distribution for all patients in this study. Number of female patients in ischemia with diabetes was more than male patients while in ischemia and ischemia with hypertension male patients were more than female.


#### **Table 2.** Sex distribution in the study groups

Comparison between ischemic male or female and similar sex healthy controls results indicate an increase in level of homocysteine among patients groups. This elevation in homocysteine concentration value was highly significant (P < 0.01). When similar comparisons were done between ischemia with hypertension and ischemia with diabetes and control group a similar behavior were observed (P < 0.01).Table 3 shows the mean values of serum level of homocysteine in male and female compared with the mean values in control group. No significant changes was observed when comparison was done between two age groups as shown in table 4.


\*\* P < 0.01

**Table 3.** The mean serum level of homocysteine in male and female patients groups and control group


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**Pure ischemia Ischemia with hypertension Ischemia with diabetes Mg Zn Fe Mg Zn Fe Mg Zn Fe**

> 1.01 ±0.13

> 1.41 ±1.23

> 1.3 ±1.06

0.73 ±0.11

> 0.68 ±0.2

0.96 ±0.18

Measurements of trace elements in two age groups (40-60) and > 60 years in all patients groups

shows no significant changes in their levels (p > 0.05 ) as shown in table 7 .

14.25 ±1.08

14.5 ±1.36

14.43 ± 1.27

**Table 7.** Comparison of levels of (Mg , Zn and Fe ) between two age groups { age group 1 (40-60 year) and

There is a significant decrease in the level of magnesium, zinc ,selenium , chromium & germanium in all patients groups in comparism with control group (P < 0.01) & significant increase level of both lead & cadmium in all patients group than that of control group (P < 0.01) while there is

**hypertension Control Pure ischemia Trace** 

Mg 19.59±1.58 14.38±2.64\*\* 14.44±1.27\*\* 15.25±3.66\*\*

Zn 1.16±0.22 0.66±0.15\*\* 0.68±0.18\*\* 0.8±0.26\*\* Fe 1.06±0.18 1.33±1.36 1.30±1.06 0.95±0.19 Pd 0.11±0.17 6.39\*\*±2.89 5.63\*\*±1.73 3.58\*\*±1.06

Cd 0.00±0.00 5.07\*\*±2.69 4.78\*\*±1.83 4.89\*\*±1.75 Se 103.64±11.95 53.37\*\*±14.85 61.53\*\*±11.17 61.81\*\*±7.19 Cr 55.21±6.93 32.42\*\*±6.95 32.05\*\*±11.77 31.02\*\*±3.21 Ge 42.37± 4.76 18.51\*\*±6.14 16.9\*\*±5.19 14.61\*\*± 6

The relationship between increased homocysteine and heart disease is well established in the medical community. Unlike the other three predictors of heart disease which are cholesterol, triglycerides and C Reactive Protein, homocysteine levels are influenced by what the person does not eat rather than what he does eat. This is due to the fact that homocysteine is a sulphydrylcontaining amino acid derived from demethylation of methionine. Nutritional deficiencies in the

1.5 ±1.77

1.15 ±0.76

1.33 ±1.36

no significant changes in the level of iron (p< 0.5) as shown in table 8.

**Table 8.** Trace elements level in control & patients groups. \*\* (P < 0.01)

**Age groups**

40-60 14.43

>60 14.32

All ages 14.38

age group 2 (>60 year)}. **Trace elements level:**

±2.33

±3.03

±2.46

**elements**

g/mlμ

ng/ ml

**4. Discssion**

0.73 ±0.17

0.6 ±0.12

0.66 ±0.16 Turkey, September 10-12, 2012

17.9 ±3.73

13.14 ±1.95

15.26 ±3.66

**Ischemia with** 

0.95 ±0.23

0.68 ±0.24

0.8 ±0.26 <sup>255</sup> ISALS

0.94 ±0.19

0.96 ±0.19

0.95 ±0.18

**Ischemia with diabetes**

**Table 4.** Comparison of levels of homocysteine (µmol/l) between two age groups ( age group 1 (40-60) and age group 2 (>60))

There is a highly significant increase in homocysteine level in ischemic patients compared with control values (P < 0.01).Also there is significant increase in homocysteine level in patients with ischemia with hypertension & ischemia with diabetes compared with control patients as shown in table 5.


\*\* P < 0.01

**Table 5.** The mean serum level of homocysteine( µmol/l) in different patients groups and control groups

There is no significant changes in serum homocysteine level between pure ischemic patients , ischemia with hypertension and ischemia with diabetes ( P >0.05).

Measurements of trace elements in serum of two age groups (40-60) and > 60 years shows no significant changes in their levels (p > 0.05).Table 6 shows comparison of trace metal concentration in control group between two age groups


**Table 6.** Comparison of trace metal concentration in control group between two age groups


Measurements of trace elements in two age groups (40-60) and > 60 years in all patients groups shows no significant changes in their levels (p > 0.05 ) as shown in table 7 .

**Table 7.** Comparison of levels of (Mg , Zn and Fe ) between two age groups { age group 1 (40-60 year) and age group 2 (>60 year)}.

#### **Trace elements level:**

There is a significant decrease in the level of magnesium, zinc ,selenium , chromium & germanium in all patients groups in comparism with control group (P < 0.01) & significant increase level of both lead & cadmium in all patients group than that of control group (P < 0.01) while there is no significant changes in the level of iron (p< 0.5) as shown in table 8.


**Table 8.** Trace elements level in control & patients groups. \*\* (P < 0.01)

#### **4. Discssion**

The relationship between increased homocysteine and heart disease is well established in the medical community. Unlike the other three predictors of heart disease which are cholesterol, triglycerides and C Reactive Protein, homocysteine levels are influenced by what the person does not eat rather than what he does eat. This is due to the fact that homocysteine is a sulphydrylcontaining amino acid derived from demethylation of methionine. Nutritional deficiencies in the

vitamin cofactors (folate, vitamin B12, and vitamin B6) required for homocysteine metabolism may promote hyperhomocysteinaemia [10]. This means that increased homocysteine levels are associated with increased risk of cardiovascular disease and then tHcy measurement will become another useful marker of vascular risk, multivitamin therapy will be another therapeutic option for people at risk of atherothrombotic vascular disease, and fortification of food with folic acid will rise high on the political and public health agenda. Homocysteine level in all patients with heart disease in this study ( pure ischemia, ischemia with hypertension and ischemia with diabetes ) were found to be elevated significantly(P < 0.01) compared with control groups. Only about two-thirds of all episodes of symptomatic atherothrombotic vascular disease in developed countries can be attributed to established genetic and environmental vascular risk factors [11]. An additional causal vascular risk factor may be raised plasma levels of homocysteine (hyperhomocysteinaemia).

International Conference on Applied Life Sciences (ICALS2012)

The results of this study show that there was a reduction in iron serum level of ischemia with diabetes patient and an elevation in patients with ischemia and ischemia with hypertension compared with control group. However, these changes were not significant (P>0.05). This was explained due to the fact that ischemic heart diseases, diabetes and hypertension as diseases occurs with variant risk factors mostly not affect the levels of iron if these diseases occur from risk factors other than those in the previous points. The results reveal a significant increase p<0.01 in serum lead of ischemic heart disease, hypertension and diabetes patients where when compared with the corresponding control values. Till (1997), believed that there was no known biological requirement for germanium (Ge) which was regarded as not an essential element [21, 22]? However, recent study observed that different symptoms in the deficiency of germanium such as cardiovascular disease, atherosclerosis, higher risk for several cancers, osteoporosis, and arthritis

[1] Allon ,N.; Friedman G.; Bostom, Jacob Selhub; Andrew, S. Levey; and Irwin, H. Rosenberg .(2001) The Kidney and Homocysteine Metabolism , J Am Soc Nephrol 12: 2181–2189.

[2] Ford, E. S., Smith, S. J., Stroup, D. F., Steinberg, K. K., Mueller, P. W. & Thacker, S. B. (2002) Homocysteine and cardiovascular disease: a systematic review of the evidence with special emphasis on case-control studies and nested case-control studies. Int. J. Epidemiol. 31:59-70. [3] Willinek, Winfried, A.; Ludwig, Malte; Lennarz, Martina; Höller, Tobias; Stumpe, Klaus O.(2000). High-normal serum homocysteine concentrations are associated with an increased risk of early atherosclerotic carotid artery wall lesions in healthy subjects Journal of Hypertension: , 18 ( 4) :

[4] Bostom, AG.; Silbershatz, H.& Rosenberg,IH. (1999). Nonfasting plasma total homocysteine levels and ll-cause and cardiovascular disease mortality in elderly Framingham men and women.

[5] Mudd, SH.;Skovby, F.; Levy ,HL.; Pettigrew, K.D.;Wilcken, B.; Pyeritz, RE.; Andria, G.; Boers ,G.H.; Bromberg, IL.& Cerone, R. (1985) The natural history of homocystinuria due to cystathionine

[6] Boushey ,CJ.; Beresford ,SA.; Omenn ,G.S. and Motulsky ,A.G. (1995) A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. Probable benefits of increasing folic

[7] Conor R. (2007), Food Science & Technology and Nutrition & Dietetics,Company/Brand:

[9] Khurshid, S. J.and Qureshi, I. H.(1984). The role of inorganic elements in the human body, The

[10] Auer, J.;Berent , R. and Eber , B.(2001) . Homocysteine and Risk of Cardiovascular Disease. J Clin

weakened immune system, decreased oxygen [23].

**5. References**

425-430.

*Arch Intern Med* . **159**:1077–80

Blackwell Publishing

Nucleus, 21: 3-23.

Basic Cardiol . 4: 261-264.

beta-synthase deficiency. Am J Hum Genet 37: 1-31.

[8] WHO, World Health Organization, Cadmium, Geneva, 1992

acid intakes. *J Am Med* Assoc 274**:** 1049-1057.

Turkey, September 10-12, 2012

<sup>257</sup> ISALS

Mild hyperhomocysteinaemia occurs in approximately 6% of the general population [12,13 ]. Patients with mild hyperhomocysteinaemia are typically asymptomatic until the third or fourth decade of life when premature coronary artery disease develops, as well as recurrent arterial and venous thrombosis.The elevation of serum level of homocysteine (hyperhomocysteinemia) in this study could be considered as a risk factor for cardiovascular disease. Research has shown that increased homocysteine level is associated with both the hyperinsulinemia seen with insulin resistance and increased urinary albumin excretion. It is also associated with low serum levels of vitamin B12 [14]. However, supplementation with vitamin B12 has resulted in reduction of homocysteine levels, but as failed to show subsequent reductions in incidence of cardiovascular disease [15]. Trace elements are micro-nutrients, present in blood and tissues. They are essential for enzymatic activities and metabolic processes and even for vital functions [16 , 17] . Some trace metals have antiviral activity, while others may alter the genome of the viruses enhancing their virulence [18]. Several trace elements are of great importance in a number of biological processes, mostly through the following terms:


It is therefore reasonable to assume that these minerals would also exert an action, either directly, or indirectly, on the cardiac cell, on the blood vessel walls, on the blood-pressure-regulating centers, or on other systems related to cardiovascular function. Astudy showed that the mean values of serum magnesium were lower in patients with acute myocardial infarction than in non cardiac patients. The authors suggested that a lowering or elevation of serum trace elements could be useful in the diagnosis of recent infarction and could possibly have other implications [19]. Table 6 shows that serum magnesium level was decreased in all patients in comparison with control & this is in agreement with most studies [20]. The disturbances of magnesium metabolism may have profound effect on the contractile state of vascular smooth muscle and as a result on blood pressure. Accordingly, magnesium is particularly important for assessment of hypertension. [20] Reported that magnesium deficiency has a role in the pathogeneses of hypertension.

The results of this study show that there was a reduction in iron serum level of ischemia with diabetes patient and an elevation in patients with ischemia and ischemia with hypertension compared with control group. However, these changes were not significant (P>0.05). This was explained due to the fact that ischemic heart diseases, diabetes and hypertension as diseases occurs with variant risk factors mostly not affect the levels of iron if these diseases occur from risk factors other than those in the previous points. The results reveal a significant increase p<0.01 in serum lead of ischemic heart disease, hypertension and diabetes patients where when compared with the corresponding control values. Till (1997), believed that there was no known biological requirement for germanium (Ge) which was regarded as not an essential element [21, 22]? However, recent study observed that different symptoms in the deficiency of germanium such as cardiovascular disease, atherosclerosis, higher risk for several cancers, osteoporosis, and arthritis weakened immune system, decreased oxygen [23].

#### **5. References**


[11] Stampfer ,M.J.; Malinow, M.R.;Willett ,W.C.; Newcomer, L.M.; Upson ,B.;Stephen, N.;Adler, Dianne, B. Gasbarra.(1999). A Pocket Manual of Differential Diagnosis, Lippincott Williams & Wilkins, ISBN: 0-78171-943-7.

International Conference on Applied Life Sciences (ICALS2012)

**Total Phenolic Content, Antioxidant,** 

1 Department of Plant Sciences, Quaid-i-Azam University Islamabad

2 College of Pharmacy, University of Hawaii at Hilo, USA

\*Corresponding author: sami\_jan69@yahoo.com

candidates for natural antioxidants and anticancer.

**1. Introduction**

Samiullah1, 2, \*, Asghari Bano<sup>1</sup>

**Abstrat**

**Antimicobial and Anticancer Activities of** 

, Sisay Girmay<sup>2</sup>

Anticancer activity against Human lung carcinoma (LU-1) and Human prostrate carcinoma (LnCap) along with antimicrobial and antioxidant activity on DPPH ((1,1)-diphenyl-2-picrylhydrazyl) and Hydrogen peroxide radicals scavenging activity and the contents of total phenolic and flavonoids were assessed in methanol extract of *Lespedeza bicolor*. The highest content of total phenolic content was detected in the arial part of *Lespedeza bicolor* (0.5-1.7 mg gallic acid equiv./g), while the highest content of total flavonoids was found in the aerial part of *Lespedeza bicolor* (0.102- 0.148 mg/g D/W). *Lespedeza bicolor* arial parts and root extract showed IC50 value of 12.5µg/ml and 50µg/ml against human lung carcinoma (LU-1) whereas, ≤ 12.5 µg/ml and 12µg/ml were calculated against Human prostrate carcinoma (LnCap) cell line. MIC value of 20-35 µg ml−1 has been observed gainst *Aspergillus fumigates, Aspergillus niger, Fusarium solani* and *Mucor sp* in comparision with 1-2.5µg/ml of Terbinafine used as a standard fungicide. MIC value of 20 µg/ml and 35 µg ml−1 of *Lespedeza bicolor* arial parts and root extract against bacterial pathogen *Klebsiella pneumonia*  and 20-50 µg ml−1 against *Enterococcus* has been measured. DPPH radical scavenging activity of *Lespedeza bicolor* with IC50 values of ≤ 50 µg/ml and ≤ 200 µg ml−1 was observed whereas, hydrogen peroxide scavenging activity with IC50 values of ≤ 25 µg/ml for arial parts and ≤ 50 µg ml−1 for the root extract of *Lespedeza bicolor* has been shown with galllic acid (R2= 0.819) and ascorbic acid (R2= 0.728). These data suggested that the methanolic extract of *Lespedeza bicolor* could be potential

**Lespedeza Bicolor Turcz (Papilionaceae)**

© 2012 Samiullah et al.; licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

provided the original work is properly cited.

**Keywords:** Lespedeza bicolor, anticancer activity, antioxidant, Antimicrobial

*Lespedeza bicolor* Turcz (Papilionaceae) commonly called; bush clover has been collected from natural high saline and arid habitat of District Mardan, Pakistan (34° 05' to 34° 32' north latitudes and 71" 48' to 72° 25' east longitudes. According to [1] Six pterocarpans isolated from the root bark of *Lespedeza bicolor* has exposed significant levels of bacterial neuraminidase inhibitory activity with IC50 = 0.09-3.25µM. *Lespedeza bicolor* constituents including flavonoids, alkaloids, terpenes, organic acids, and stigmasterols have been screened for anti-inflammation, reducing blood sugar, antioxidation anti-radiation, anticancer, and anti-tumor by [2]. The work of [3] showed that the total amount of hydrolyzed amino acid was 148.95 mg/100g, free amino acids

Turkey, September 10-12, 2012

and Ghee Tan<sup>2</sup>

<sup>259</sup> ISALS


## **Total Phenolic Content, Antioxidant, Antimicobial and Anticancer Activities of Lespedeza Bicolor Turcz (Papilionaceae)**

Samiullah1, 2, \*, Asghari Bano<sup>1</sup> , Sisay Girmay<sup>2</sup> and Ghee Tan<sup>2</sup>

1 Department of Plant Sciences, Quaid-i-Azam University Islamabad

2 College of Pharmacy, University of Hawaii at Hilo, USA

\*Corresponding author: sami\_jan69@yahoo.com

#### **Abstrat**

Anticancer activity against Human lung carcinoma (LU-1) and Human prostrate carcinoma (LnCap) along with antimicrobial and antioxidant activity on DPPH ((1,1)-diphenyl-2-picrylhydrazyl) and Hydrogen peroxide radicals scavenging activity and the contents of total phenolic and flavonoids were assessed in methanol extract of *Lespedeza bicolor*. The highest content of total phenolic content was detected in the arial part of *Lespedeza bicolor* (0.5-1.7 mg gallic acid equiv./g), while the highest content of total flavonoids was found in the aerial part of *Lespedeza bicolor* (0.102- 0.148 mg/g D/W). *Lespedeza bicolor* arial parts and root extract showed IC50 value of 12.5µg/ml and 50µg/ml against human lung carcinoma (LU-1) whereas, ≤ 12.5 µg/ml and 12µg/ml were calculated against Human prostrate carcinoma (LnCap) cell line. MIC value of 20-35 µg ml−1 has been observed gainst *Aspergillus fumigates, Aspergillus niger, Fusarium solani* and *Mucor sp* in comparision with 1-2.5µg/ml of Terbinafine used as a standard fungicide. MIC value of 20 µg/ml and 35 µg ml−1 of *Lespedeza bicolor* arial parts and root extract against bacterial pathogen *Klebsiella pneumonia*  and 20-50 µg ml−1 against *Enterococcus* has been measured. DPPH radical scavenging activity of *Lespedeza bicolor* with IC50 values of ≤ 50 µg/ml and ≤ 200 µg ml−1 was observed whereas, hydrogen peroxide scavenging activity with IC50 values of ≤ 25 µg/ml for arial parts and ≤ 50 µg ml−1 for the root extract of *Lespedeza bicolor* has been shown with galllic acid (R2= 0.819) and ascorbic acid (R2= 0.728). These data suggested that the methanolic extract of *Lespedeza bicolor* could be potential candidates for natural antioxidants and anticancer.

**Keywords:** Lespedeza bicolor, anticancer activity, antioxidant, Antimicrobial

## **1. Introduction**

*Lespedeza bicolor* Turcz (Papilionaceae) commonly called; bush clover has been collected from natural high saline and arid habitat of District Mardan, Pakistan (34° 05' to 34° 32' north latitudes and 71" 48' to 72° 25' east longitudes. According to [1] Six pterocarpans isolated from the root bark of *Lespedeza bicolor* has exposed significant levels of bacterial neuraminidase inhibitory activity with IC50 = 0.09-3.25µM. *Lespedeza bicolor* constituents including flavonoids, alkaloids, terpenes, organic acids, and stigmasterols have been screened for anti-inflammation, reducing blood sugar, antioxidation anti-radiation, anticancer, and anti-tumor by [2]. The work of [3] showed that the total amount of hydrolyzed amino acid was 148.95 mg/100g, free amino acids

© 2012 Samiullah et al.; licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

were 106.39 mg/100g and that of γ-aminoisobutyric acid was recorded 12.57 mg/100g in *Lespedeza bicolor* stem extract. The contents of neutral lipids, glycolipids, and phospholipids in *Lespedeza bicolor* seed detected by [4] were 71.75%, 23.26% and 4.99% respectively. 12 flavonoids including Quercetin, kaempferol, trifolin, isoquercetin, homoorientin, and orientin has been isolated from *Lespedeza* bicolor by [5]. N, N-dimethyltryptamine isolated from *Lespedeza bicolor* **var. japonica** has uterus contracting action in 1-2 × 10-6 dilution. The [6] work revealed that the Leaves, shoots and inflorescences of *Lespedeza bicolor* have been used in the treatment of acute and chronic nephritis, azothemia and dieresis.

International Conference on Applied Life Sciences (ICALS2012)

Several flavonoids and tannins isolated from medicinal plants have been discovered for their significant role in antibacterial, antifungal and anti-inflammatory activities. It is, therefore, possible that the present activities observed with this extract in the study may be attributable to its total

In table 1 total phenolic content (TPC) was shown in the range of 1.23-1.70 mg/g of the *lespedeza bicolor* extract using a standard curve of gallic acid (R2= 0.783). The total flavonoids are in the range

**Metabolites** *Lespedeza* **Arial** *Lespedeza* **Root**

Tannins (mg/g. D/W) 0.193±0.014 0.064±0.326 Total Flavonoids (mg/g. D/W) 0.148±0.003 0.102±0.001 Alkaloids (mg/g. D/W) 1.8±0.150 1.4±0.255 Saponins (mg/g. D/W) 2.0±0.215 2.2±0.137

(mg/g. D/W) 1.669±0.06 1.23±0.121

**Table 1.** Tannins, total Flavonoids content (TFC), Alkaloid and Saponins content of methanolic extract of

The methanolic extracts of *Lespedeza bicolor* arial parts and root were significantly active against the fungal pathogens studied. The arial parts of *Lespedeza bicolor* showed the broadest spectrum of activity against *Aspergillus fumigates, Aspergillus niger, Fusarium solani* and *Mucor sp* with MIC value of 20-35 µg ml−1 than the root extract shown in table 2. MIC value of 20 and 35 µg ml−1 of *Lespedeza bicolor* arial parts and root extract against bacterial pathogen *Klebsiella pneumonia* and 20-50 µg ml−1 against *Enterococcus* has been shown in table 2. Penicillin and Chloramphenicol with MIC value of 1.5-2.5 µg ml−1 has been used as a positive control against *Klebsiella* and *Enterococcus* specie. To better understand the antioxidant potential of *Lespedeza bicolor* extracts of root and arial parts were evaluated for radical scavenging activity against DPPH. Fig.1 illustrated a significant decrease in the concentration of DPPH due to scavenging activity of the extract. DPPH radical scavenging activity of *Lespedeza bicolor* arial parts and root extract with IC50 values of ≤ 50 and ≤ 200 µg ml−1 respectively with galllic acid (R2= 0.871) and ascorbic acid (R2= 0.780) was shown in table. 3 whereas, hydrogen peroxide scavenging activity with IC50 values of ≤ 25 for arial parts and ≤ 50 µg ml−1 for the root extract of *Lespedeza bicolor* has been shown in the same table with galllic acid (R2=

Cytotoxicity results against LU-1 and LnCaP cell lines are summarized in table 3. *Lespedeza bicolor* arial parts and root extract showed IC50 value of 12.5 and 50 µg/ml against LU-1 whereas, ≤ 12.5 and 12 µg/ml were calculated against LnCaP cell line. Interestingly, *Lespedeza bicolor* possessed the highest inhibition potential against human lung carcinoma (LU-1) and human prostrate carci-

noma (LnCaP) cell lines indicating its ultimate potential for biopharmaceutical uses.

Data are expressed as mean±SEM (n = 3) of three independent experiments

phenolic, total flavonoids and tannins contents.

from 0.102-0.148 mg/g D/W shown in Table 1.

All data expressed as (mg/g. Dry Weight)

Total phenolic content

Lespedeza bicolor arial parts and root

0.819) and ascorbic acid (R2= 0.728).

Turkey, September 10-12, 2012

<sup>261</sup> ISALS

## **2. Materials and Methods**

#### **Extraction**

Fresh aril parts and root of *Lespedeza bicolor* (300g) were collected, rinsed with distilled water and air dried for 12 days. The leaves were ground into powder, then soaked in 80% methanol and incubated for two weeks at room temperature (25 °C). The mixture was filtered twice, using whatman-41 filter paper. The extracts were dried by removing the methanol using a rotary film evaporator.

#### **Preliminary phytochemical screening**

Phytochemical screening of the *Lespedeza bicolor* was performed to detect the presence of different classes of constituents, such as alkaloids, flavonoids, saponins, steroids, terpenes, Coumarins, Anthraquinone, phlobatannins, Cardiac glycosides and tannins [7]. Total phenolic contents of *Lespedeza bicolor* were determined by the Folin-Ciocalteu colorimetric method [8]. Tannin content was determined by using [9] method. Total Flavonoids content was determined according to the standard protocol [10]. The absorbance was measured immediately at 510 nm spectrophotometer. Alkaloid content was determined by [11] method using 10% acetic acid followed by concentrated ammonium hydroxid. Saponin contents were calculated as percentage of the dried fraction using [12] method.

#### **Antibacterial and antifungal assays**

Antibacterial activity of *Lespedeza bicolor* crude extracts was determined by the agar well diffusion method [13]. The agar tube dilution method was used for determination of antifungal activity of methanolic extracts of *Lespedeza bicolor*.

#### **Antioxidant potential of Lespedeza bicolor**

The antioxidant activity of *Lespedeza bicolor* crude extract was assessed in DPPH radical scavenging system using gallic acid and ascorbic acid as a positive control, and the decrease in absorbance was determined at 517 nm [14]. The ability of the extracts to scavenge hydrogen peroxide was determined according to the method of [15].

#### **Anticancer activities**

The cytotoxic potential of the total methanolic extract of *Lespedeza bicolor* was determined in the human lung carcinoma (LU-1) and human prostrate carcinoma (LnCaP) cell line at the highest concentration of 20 µg/mL with sulforhodamine B (SRB) method [16].

## **3. Results and discussion**

Several groups of polyphenols (anthocyanins, tannins, flavanones, isoflavones, resveratrol and ellagic acid) are currently used in nutraceuticals industries and functional foods [17]. MIC of *L.bicolor* crude extract against *E. coli* and *B. subtilus,* was found 0.5 mg/ml [18].

Several flavonoids and tannins isolated from medicinal plants have been discovered for their significant role in antibacterial, antifungal and anti-inflammatory activities. It is, therefore, possible that the present activities observed with this extract in the study may be attributable to its total phenolic, total flavonoids and tannins contents.

In table 1 total phenolic content (TPC) was shown in the range of 1.23-1.70 mg/g of the *lespedeza bicolor* extract using a standard curve of gallic acid (R2= 0.783). The total flavonoids are in the range from 0.102-0.148 mg/g D/W shown in Table 1.


Data are expressed as mean±SEM (n = 3) of three independent experiments All data expressed as (mg/g. Dry Weight)

**Table 1.** Tannins, total Flavonoids content (TFC), Alkaloid and Saponins content of methanolic extract of Lespedeza bicolor arial parts and root

The methanolic extracts of *Lespedeza bicolor* arial parts and root were significantly active against the fungal pathogens studied. The arial parts of *Lespedeza bicolor* showed the broadest spectrum of activity against *Aspergillus fumigates, Aspergillus niger, Fusarium solani* and *Mucor sp* with MIC value of 20-35 µg ml−1 than the root extract shown in table 2. MIC value of 20 and 35 µg ml−1 of *Lespedeza bicolor* arial parts and root extract against bacterial pathogen *Klebsiella pneumonia* and 20-50 µg ml−1 against *Enterococcus* has been shown in table 2. Penicillin and Chloramphenicol with MIC value of 1.5-2.5 µg ml−1 has been used as a positive control against *Klebsiella* and *Enterococcus* specie.

To better understand the antioxidant potential of *Lespedeza bicolor* extracts of root and arial parts were evaluated for radical scavenging activity against DPPH. Fig.1 illustrated a significant decrease in the concentration of DPPH due to scavenging activity of the extract. DPPH radical scavenging activity of *Lespedeza bicolor* arial parts and root extract with IC50 values of ≤ 50 and ≤ 200 µg ml−1 respectively with galllic acid (R2= 0.871) and ascorbic acid (R2= 0.780) was shown in table. 3 whereas, hydrogen peroxide scavenging activity with IC50 values of ≤ 25 for arial parts and ≤ 50 µg ml−1 for the root extract of *Lespedeza bicolor* has been shown in the same table with galllic acid (R2= 0.819) and ascorbic acid (R2= 0.728).

Cytotoxicity results against LU-1 and LnCaP cell lines are summarized in table 3. *Lespedeza bicolor* arial parts and root extract showed IC50 value of 12.5 and 50 µg/ml against LU-1 whereas, ≤ 12.5 and 12 µg/ml were calculated against LnCaP cell line. Interestingly, *Lespedeza bicolor* possessed the highest inhibition potential against human lung carcinoma (LU-1) and human prostrate carcinoma (LnCaP) cell lines indicating its ultimate potential for biopharmaceutical uses.


Data are expressed as mean±SEM (n = 3) of three independent experiments

\*Terbinafine 1-2.5μg/ml is used as a standred fungicide, \*Penicillin and Chloramphenicol 1-3.5μg/ml is used as a standred antibiotics.

**Table 2.** Antifungal activities (expressed in MIC) of methanolic extracts of Lespedeza bicolor arial parts and root

**Fig 1.** Analysis of DPPH Radical Scavenging activity of methanolic extract of arial parts and root of *Lespedeza bicolor*

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In conclusion, the high antimicrobial, antioxidant and cytotoxic potential of *Lespedeza bicolor* highlight the need of further investigations to isolate the active principle and their subsequent evaluation. The results also suggest the presence of biologically active principles which may be worth further investigation and elucidation. Further studies are in fact currently under way to

The authors would like to thank the Higher Education Commission of Pakistan for providing the

[1] Woo Hyun Sim; Kim Dae Wook; Curtis-Long Marcus J; Lee Byong Won; Lee Ji Hye; Kim Jun Young; Kang Jae Eun; Park Ki Hun. 2011. Potent inhibition of bacterial neuraminidase activity by pterocarpans isolated from the roots of Lespedeza bicolor. *Bio-Orgainc and Medicinal Chemistry* 

[2] Zhang, Fan; Qi, Xiaohua; Zou, Mingqiang; Xie, Ruili; Li, Jinfeng; Zhang, Honggui. 2008. Research progress of extraction and analysis of flavonoids and chemical constituents

[4] Kim, Hyang Ran; Koh, Moo Seok; Yang, Hee Cheon. 1987. Studies on the lipid composition of bush clover (Lespedeza bicolor) seed. *Hanguk Yongyang siklyong Hakhoechi*. 16(3): 75-84. [5] Glyzin, V. I.; Ban'kovskii, A. I.; Zhurba, O. V.; Sheichenko, V. I. 1970. Flavonoids

[8] Lee YR, Woo KS, Kim KJ, Son JR, Jeong HS (2007)*.* Antioxidant activities of ethanol extracts from

[9] Van-Burden T.P, Robinson WC (1981). Formation of complexes between protein and tannin acid.

[10] Sakanaka, S., Y. Tachibana and Y. Okada, 2005. Preparation and antioxidant properties of extracts

[11] A. Sofowara. "Medicinal plants and Traditional Medicine in Africa", Spectrum Books Ltd, Ibadan,

[12] Carron, E.A, J.M. Maran, L. Montero, A. Fernandozalgo and A.Dominguez. 1987. Antimicrobial properties of some extracts obtained from some Mediterranean plants of medicinal value. *Plantes* 

[13] Washington, J.A. and V.L. Sutter. 1980. "Dilution susceptibility test: agar and macro-broth dilution procedures, In: E.H. Lennette, A. Balows, WJ. Hausler Jr., and J.P. Truant (Ed,).

isolate and characterize the active principle(s) of the crude extract.

in Lespedeza bicolor Turcz. *Shizen Guovi Guoyao.* 19(12): 2884-2885. [3] Chen BH, Li SH, Lin JX, Zhuang HR. J Fujian Normal University 2003;19:86.

of Lespedeza bicolor. *Khimiya prirodnykh soedinenii.* 6(4): 473-474.

[7] Wagner H, Bladt S, Zgainski EM. Plant drug analysis. Berlin: Springer; 1984.

germinated specialty rough Rice. Food Sci. Biotechnol*.* 16(5): 765 – 770.

of Japanese persimmon leaf tea (kakinoha-cha). Food Chem., 89: 569-575.

[6] Trumpe TE, Gulyaev VG. Med Help 1994;2:47.

*Medicinales et Phytotherapie*, 21: 195-202.

J. Agric Food Chen. 1: 77-82.

Nigeria. 1993: p. 289.

**4. Conclusion**

**6. References**

**5. Acknowledgement**

necessary financial support for the study.

*Letter*. 21 (20): 6100-6103.

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**Fig 2.** Analysis of Hydrogen Peroxide Radical Scavenging activity of methanolic extracts of arial parts and root of *Lespedeza bicolor bicolor*


Data are expressed as mean±SEM (n = 3) of three independent experiments

\*1Human lung carcinoma \*2Human prostrate carcinoma

Colchicine with IC50 values 0.02±0.002 is used as Standard anticancer drug

**Table 3.** Cytotoxicity against (LU-1) and (LnCaP) and antioxidant activities of methanolic extracts of *Lespedeza bicolor* arial parts and root expressed as IC50 (μg ml−1)

## **4. Conclusion**

In conclusion, the high antimicrobial, antioxidant and cytotoxic potential of *Lespedeza bicolor* highlight the need of further investigations to isolate the active principle and their subsequent evaluation. The results also suggest the presence of biologically active principles which may be worth further investigation and elucidation. Further studies are in fact currently under way to isolate and characterize the active principle(s) of the crude extract.

## **5. Acknowledgement**

The authors would like to thank the Higher Education Commission of Pakistan for providing the necessary financial support for the study.

## **6. References**


[14] Ruch, R.J., Cheng, S.J., Klaunig, J.F., 1989. Prevention of cytotoxicity and inhibition of intracellular communication by antioxidant catechins isolated from Chinese green tea. Carcinogenesis 10, 1003–1008.

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, Hadjira Hannachi,

2 Laboratory of aquatic and terrestrial ecosystems (Ecostaq), Badji-Mokhtar University, Algeria.

This study was conducted at the National Park of El Kala (Algeria) which has a mosaic of habitats, and located in north-eastern Algeria. This rich landscape induces another one; it is the ecological biodiversity of the park. We are interested in a particular species of mammal that is the Black Rat (*Rattus rattus*) and specifically in identifying different species of parasites that her little body homes and vehicles. The Black Rat's skin is very noisy. We identified mites (*Dermanysus bacoti*), ticks (*Ixodes ricinus*), fleas (*Nosopsyllus fasciatus* and *Xenopsylla chiopis*), lice (*Poluplax sp*)

The Muridae family is the most diversified at globe level, including more than 700 species and 120 kinds amongst Rattus which accounts 50 species [8]. South – eastern Asian originally species has become cosmopolitan through times. In fact, from the Far East, the black Rat has conquered all the continents following Man everywhere [6]. At the National Park El Kala (North – east of Algeria) we identified *Rattus rattus* (relying on morphological, craniological and caryological approaches). The rodent *Rattus rattus* (Linnaeus, 1958) is a devastating species, reported as resistant

The black rat (*Rattus rattus*) which is a small unit of the biological system is a very rich and diverse synopsis. Indeed, this micro-ecosystem supports the installation and development of a mosaic parasite, which exists, co-evolves, multiplies and spreads even in the ecosystem. The parasite is bad for the host; it can only harm his life, and whatever its form is horizontal or vertical parasitism. The black rat is observed at the park in urban, sub-urban nature reserve, the forest ... etc. We used rat traps that were placed in different environments to capture it. After capture we identified the species according to body size of individuals recovered alive (performed by the method of Chap-

*Rattus rattus* **Parasites of El-Kala** 

1 SNV Institute, Department of Biology, El-Tarf University, Algeria.

3 Vectorial systems ecology service, Pasteur Institute of Algeria.

4 Laboratory of Animal Ecology, Faculty of Sciences, El-Manar University, Tunis.

**National Park (Algeria)** 

M'Barek Chetoui4 and Zihad Bouslama<sup>2</sup>

\*Corresponding author, Email: becir-f@hotmail.com

**Keywords**: Black Rat, ectoparasites, tick, flea, Algeria.

to plague *Yersinia pestis*, and also a vector of several pathogens.

Farida Becir1, 2, \*, Idir Bitam<sup>3</sup>

**Abstract**

and sandflies (*Flebotomus sp.).*

**1. Introduction**

© 2012 Becir et al.; licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the

original work is properly cited.

pelier), Then, we collected all suspected parasites kinds of the species.

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## *Rattus rattus* **Parasites of El-Kala National Park (Algeria)**

Farida Becir1, 2, \*, Idir Bitam<sup>3</sup> , Hadjira Hannachi,

M'Barek Chetoui4 and Zihad Bouslama<sup>2</sup>

1 SNV Institute, Department of Biology, El-Tarf University, Algeria.

2 Laboratory of aquatic and terrestrial ecosystems (Ecostaq), Badji-Mokhtar University, Algeria.

3 Vectorial systems ecology service, Pasteur Institute of Algeria.

4 Laboratory of Animal Ecology, Faculty of Sciences, El-Manar University, Tunis.

\*Corresponding author, Email: becir-f@hotmail.com

#### **Abstract**

This study was conducted at the National Park of El Kala (Algeria) which has a mosaic of habitats, and located in north-eastern Algeria. This rich landscape induces another one; it is the ecological biodiversity of the park. We are interested in a particular species of mammal that is the Black Rat (*Rattus rattus*) and specifically in identifying different species of parasites that her little body homes and vehicles. The Black Rat's skin is very noisy. We identified mites (*Dermanysus bacoti*), ticks (*Ixodes ricinus*), fleas (*Nosopsyllus fasciatus* and *Xenopsylla chiopis*), lice (*Poluplax sp*) and sandflies (*Flebotomus sp.).*

**Keywords**: Black Rat, ectoparasites, tick, flea, Algeria.

## **1. Introduction**

The Muridae family is the most diversified at globe level, including more than 700 species and 120 kinds amongst Rattus which accounts 50 species [8]. South – eastern Asian originally species has become cosmopolitan through times. In fact, from the Far East, the black Rat has conquered all the continents following Man everywhere [6]. At the National Park El Kala (North – east of Algeria) we identified *Rattus rattus* (relying on morphological, craniological and caryological approaches). The rodent *Rattus rattus* (Linnaeus, 1958) is a devastating species, reported as resistant to plague *Yersinia pestis*, and also a vector of several pathogens.

The black rat (*Rattus rattus*) which is a small unit of the biological system is a very rich and diverse synopsis. Indeed, this micro-ecosystem supports the installation and development of a mosaic parasite, which exists, co-evolves, multiplies and spreads even in the ecosystem. The parasite is bad for the host; it can only harm his life, and whatever its form is horizontal or vertical parasitism.

The black rat is observed at the park in urban, sub-urban nature reserve, the forest ... etc. We used rat traps that were placed in different environments to capture it. After capture we identified the species according to body size of individuals recovered alive (performed by the method of Chappelier), Then, we collected all suspected parasites kinds of the species.

© 2012 Becir et al.; licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **2. Material and Methods:**

Forty three black Rats (twenty fi ve males and eighteen females) captured during the year 2011 in diff erent biotopes of El Kala National Park, whose geographical coordinates are 36° 43' N. to 36° 57' N. and of 7° 43' E. to 8° 37' E. The park extends on a 78 400 hectars surface [5].

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**Fig 3.** Flea : D : Xenopsylla cheopis (female); E : Nosopsylla sp. (male)

**Fig 4.** C : Lice: Flebotomus sp.

**Fig 5.** G: lice (Poluplax sp)

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Ratt raps were put randomly in diff erent biotopes (cork forests, alder forest, scrubland…etc.) where daily visits where planned.

As soon as captured, samples were cleaned of parasites (direct collecting method). Collected parasites were conserved in alcohol 70 % and identifi ed at Pasteur Institute of Algeria (vectorial systems ecology service) according to identifi cation keys.

## **3. Results :**

Identifi ed parasites are either insects (fl eas, lices or phlebotomes) or acarides (ticks or mites), with the majority of fl eas (*Xenopsylla cheopis*).

**Fig 1.** majority of ectoparasites.

**Fig 2.** Aacarids : A et B Mite: Dermanyssus bacoti (A : dorsal side, B ventral side) ; F Tick : Ixodes ricinus (adult female)

**Fig 3.** Flea : D : Xenopsylla cheopis (female); E : Nosopsylla sp. (male)

**Fig 4.** C : Lice: Flebotomus sp.

**Fig 5.** G: lice (Poluplax sp)

#### **4. Discussion**

These parasites are hematophagous arthropods that live on several domestic and wild animals. These parasites are themselves carriers of very harmful dseases [7] . Ticks are considered as vectors of pathogens (Bacterian, viral, parasitarian) the most important all over the world after mosquitos. They can transmit several pathogen organisms like *Borrelia, Rickettsia, Bartonella, Coxiella*, *Ehrlichia*, *et Anaplasma.* Fleas and louses can also transmit some pathogens like *Bartonella*, *Rickettsia* and espcially *Yersinia* for fleas.

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**Assessment of Phylogenetic Inter-Relationships** 

Mud crab of the genus *Scylla* possessed almost similar characteristics despite the established key identification which contributed to a slight confusion when inspecting them at a morphological level. Therefore, this phylogenetic study may give an insight to differentiate between species at a molecular level. This study examined 520 base pairs (bp) of the mitochondrial cytochrome *c* oxidase I (COI) gene from 60 individuals belonging to the genus *Scylla*, a group of mud crabs that inhabit over vast geographic areas ranging from south eastern and eastern Africa to Southeast Asia and Indo-Pacific regions. The samples were taken from various locations throughout peninsular Malaysia, Sabah and Sarawak. The nucleotide sequences were subjected to phylogenetic analyses by using the neighbour – joining (NJ) and maximum parsimony (MP) methods. All two methods revealed the reciprocally monophyletic relationship between *Scylla olivacea*, *S. paramamosain*, *S. tranquebarica* and *S. serrata*. The clustering of all four species of *Scylla* samples into four different clades suggested that their genetic identity belongs to their respective species, thus strongly supporting their status as different species available throughout Malaysia. Overall, the present study was able to be a reference on the phylogenetic relationships and assessment of

Taxonomy is the foundation of traditional conservation practices [1, 2] and understanding the taxonomic details of a species is central to the development of successful management strategies for sustainable fisheries resources. Mitochondrial DNA (mtDNA) has been one of the most widely used molecular markers for phylogenetic and taxonomic studies in animals [3] due to its maternal mode of inheritance and mainly non-recombining nature [4]. Though mtDNA sequence data have proved valuable in determining phylogenetic relationships, there are considerable differences in the characteristics of different types of gene and it is crucial that the choice of gene is appropriate to the problem being tackled [5]. Cytochrome oxidase subunit I (COI) is the largest of the three mitochondria-encoded Cytochrome Oxidase subunits, and is one of the largest protein-coding genes in the metazoan mitochondrial genome which has been used as a target

**in Mud Crab Genus Scylla (Portunidae) Based** 

**on Mitochondrial DNA Sequence**

Darlina Md. Naim1,\*, Hurul Adila-Aida Mohamad

School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia

<sup>2</sup>Centre for Marine and Coastal Studies, Universiti Sains Malaysia, Penang, Malaysia

and Siti Azizah Mohd. Nor1,2

genetic structure of *Scylla* sp. in Malaysia.

**1. Introduction** 

**Keywords:** mud crab, Scylla, phylogenetics, COI

Rosly<sup>2</sup>

**Abstract**

1

© 2012 Naim et al.; licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the

original work is properly cited.

gene for a number of molecular phylogenetic and identification studies [6].

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In Algeria, a small number published works were realized on the role of pathogen, the biodiversity and the dynamic of ticks, fleas, none on louses of different mammals. The study done by Bitam *et al.* on [1] and [2], allowed the detection of *Rickettsia* by PCR, on fleas, on differents ticks speaces (*Rhipicephalus sanguineus, Hyalomma marginatum, Rhipicephalus turanicus*) and on fleas (*Ctenocephalides canis*, *Xenopsylla cheopis)* from different mammals. In Bitam *et al.* [3] and [4] allowed the detection by PCR *Yersinia*, on fleas *Xenopsylla cheopis* captured on anthrophile mammals, and *Bartonella* on puces *Xenopsylla cheopis, Archeopsylla erinacei* and *Leptopsylla segnis*  collected on insectivores as well as rodents.

#### **5. References**


## **Assessment of Phylogenetic Inter-Relationships in Mud Crab Genus Scylla (Portunidae) Based on Mitochondrial DNA Sequence**

Darlina Md. Naim1,\*, Hurul Adila-Aida Mohamad Rosly<sup>2</sup> and Siti Azizah Mohd. Nor1,2

1 School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia

<sup>2</sup>Centre for Marine and Coastal Studies, Universiti Sains Malaysia, Penang, Malaysia

#### **Abstract**

Mud crab of the genus *Scylla* possessed almost similar characteristics despite the established key identification which contributed to a slight confusion when inspecting them at a morphological level. Therefore, this phylogenetic study may give an insight to differentiate between species at a molecular level. This study examined 520 base pairs (bp) of the mitochondrial cytochrome *c* oxidase I (COI) gene from 60 individuals belonging to the genus *Scylla*, a group of mud crabs that inhabit over vast geographic areas ranging from south eastern and eastern Africa to Southeast Asia and Indo-Pacific regions. The samples were taken from various locations throughout peninsular Malaysia, Sabah and Sarawak. The nucleotide sequences were subjected to phylogenetic analyses by using the neighbour – joining (NJ) and maximum parsimony (MP) methods. All two methods revealed the reciprocally monophyletic relationship between *Scylla olivacea*, *S. paramamosain*, *S. tranquebarica* and *S. serrata*. The clustering of all four species of *Scylla* samples into four different clades suggested that their genetic identity belongs to their respective species, thus strongly supporting their status as different species available throughout Malaysia. Overall, the present study was able to be a reference on the phylogenetic relationships and assessment of genetic structure of *Scylla* sp. in Malaysia.

**Keywords:** mud crab, Scylla, phylogenetics, COI

## **1. Introduction**

Taxonomy is the foundation of traditional conservation practices [1, 2] and understanding the taxonomic details of a species is central to the development of successful management strategies for sustainable fisheries resources. Mitochondrial DNA (mtDNA) has been one of the most widely used molecular markers for phylogenetic and taxonomic studies in animals [3] due to its maternal mode of inheritance and mainly non-recombining nature [4]. Though mtDNA sequence data have proved valuable in determining phylogenetic relationships, there are considerable differences in the characteristics of different types of gene and it is crucial that the choice of gene is appropriate to the problem being tackled [5]. Cytochrome oxidase subunit I (COI) is the largest of the three mitochondria-encoded Cytochrome Oxidase subunits, and is one of the largest protein-coding genes in the metazoan mitochondrial genome which has been used as a target gene for a number of molecular phylogenetic and identification studies [6].

© 2012 Naim et al.; licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Mud crab of the genus Scylla belongs to the Portunidae family. They are also known as swimming crabs which characterized by their broad paddles of the flattened fifth pair of legs. The discoverer recognized the existence of only one species, Scylla serrata (Forskål, 1775), until Estampador's [7] revision made clear that there are more than one species exists. Numerous convincing arguments for the recognition of the species involved were made to minimize the confusion of the taxonomic nomenclature [8] until genetic studies have come up to support and correct the findings [9].

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, 0.5 pmol of each forward and reverse primer and 1.6 µl (20 ng) of DNA from each

target region of the COI gene in the mtDNA genome of all sampled individuals. Heavy strand primer Mtd-10 5'- T TGA TTT TTT GGT CAT CCA GAA GT - 3' [11] and light strand primer C/N 2769 5'- TT AAG TCC TAG AAA ATG TTG RGG GA - 3' [16] were used to amplify a 542 bp region of the COI region. Each PCR amplification was performed in a total volume of 20 µl of PCR mixture consisting of 10x PCR buffer, 2.5 mM dNTP mixture, 5U of *i-Taq* DNA polymerase,

sample. Thermal cycling conditions (on a G-STORM; Gene Technologies Ltd.) were 35 x [94°C for 30 s, 50°C for 30 s, 72°C for 1 min, 94°C for 3 min] and a final incubation at 72°C for 5 min.PCR products were then purified and sent to First Base Laboratories Sdn Bhd (1st BASE) for sequencing.

The program MEGA ver. 4.0 [12] was used to visualize and align all sequences, including COI partial sequences of *S. serrata* obtained from Genbank (Genbank accession no: GU055497.1 – GU055506.1). The resulting sequences for each individual were then aligned using CLUSTAL W ver. 1.6 with default settings and were manually checked and trimmed in the BIOEDIT ver 7.0.9 sequence editing program; alignments were subsequently revised by eye in an effort to maximize positional homology. All aligned sequences were then imported into Basic Local Alignment

The sequence characteristics of the COI region were calculated using MEGA ver. 4.0 [12]. The phylogenetic relationships among haplotypes were reconstructed using maximum parsimony (MP) and neighbour joining (NJ; [13]). We conducted MP and NJ phylogenetic analysis in MEGA ver. 4.0 [12]. Neighbour joining analysis was executed using non-parametric re-sampling procedure with 10,000 replicates. We performed MP analysis under the HKY+I model using the heuristic search algorithm with tree bisection reconnection (TBR) branch swapping and 100 random sequence addition replicates. Both trees reconstructions included 5 sequences of indigenous portunid from Genbank; *Portunus pelagicus* (Genebank accession no: GQ272560.1 – GQ272564.1). The genetic distance, *Gst* between populations was calculated using the Tamura-Nei distance [14] based on unequal base frequencies and unequal ratios of transitions to transversions (Ti:Tv) implemented in mega ver. 4.0 [12]. The frequency of each haplotype, haplotype diversity (*h*) (i.e., the probability that two randomly selected haplotypes are present in the sample) and the nucleotide diversity (π) within populations and geographical regions was estimated using ARLEQUIN ver. 3.11 [15].

Overall 50 partial sequences (see Supplementary Material for Genbank accession number for each sequence analysed) of 520 base pairs (bp) each of the mtDNA COI gene correspond to 50 individuals representing three *Scylla* species (*S. olivacea, S. paramamosain* and *S. tranquebarica*) and 10 COI partial sequences of *S. serrata* obtained from Genbank (Genbank accession no: GU055497.1 – GU055506.1) were analyzed. All the sequences show high sequence similarity (99-100%) to their respective species sequence in Genbank, indicate that misidentification of species does not occur in this study. The 520 bp *Scylla* sequences comprised 109 variable sites, of which 102 are parsimony informative sites (Table 1). This high number of parsimony-informative sites indicates that COI mtDNA is capable to be an informative locus candidate for phylogenetic studies [16] as well

as for population differentiation and population genetic structure (Rosly et al., in press).

25mM MgCl2

**2.3. Alignment and sequence properties**

**2.4. Phylogenetic analysis**

**3. Results and Discussions**

Search Tool (BLAST) software to ensure the identity of the samples.

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Several genetic methods using allozyme electrophoresis, mitochondrial genes and nuclear genes [10] havebeen carried out in attempt to justify species in this genus and to date, there are four distinct species wererecognized namely Scylla serrata, S. paramamosain, S. tranquebarica and S. olivacea [10]. However, the relationships among these species are still not clear. Therefore, it is imperative that data collected from COI gene sequence be employed for defining the phylogenetic relationships among species within the Scylla genus. In this current study, the sequence of COI gene has been employed to reappraise the taxonomic status and unravel the phylogenetic relationships among species within genus Scylla.

## **2. Materials and Methods**

#### **2.1. Sample collection**

A total of 50 individuals were caught from eight different mangrove swamps across Malaysia, two within the east coast Peninsular (Terengganu, *n* = 10; Kelantan, *n* = 11), three within the west coast of Peninsular Malaysia (Kuala Perlis, *n* = 5; Perak, *n* = 5; Penang, *n* = 5), one within the south Peninsular (Johor, *n* = 4) and the other two within Sabah and Sarawak (Sarawak, *n* = 6; Sabah, *n* = 4) [Figure 1]. Mud crabs were caught using five to seven crab pots baited with fish laid at suitable mangrove area (spaced approximately 100 m apart) and lifted and re-baited every 24 hours for three successive days. More details on sampling method were described in Rosly et al. (in press). From each specimen, approximately 5-10 g muscle tissue was removed from a single claw and placed in 95% alcohol.

**Figure 1.** Sampling sites and species distribution in Malaysia. Each colour represents each species as indicated in the legend and the size of fractions corresponds to the frequency of samples obtained.

#### **2.2. Total DNA extraction, polymerase chain reaction (PCR) and sequencing**

Total genomic DNA from muscle tissue was extracted following the protocols from AquaGenomic Solution Kit (BioSyntech, USA). Polymerase chain reaction (PCR) was used to amplify the target region of the COI gene in the mtDNA genome of all sampled individuals. Heavy strand primer Mtd-10 5'- T TGA TTT TTT GGT CAT CCA GAA GT - 3' [11] and light strand primer C/N 2769 5'- TT AAG TCC TAG AAA ATG TTG RGG GA - 3' [16] were used to amplify a 542 bp region of the COI region. Each PCR amplification was performed in a total volume of 20 µl of PCR mixture consisting of 10x PCR buffer, 2.5 mM dNTP mixture, 5U of *i-Taq* DNA polymerase, 25mM MgCl2 , 0.5 pmol of each forward and reverse primer and 1.6 µl (20 ng) of DNA from each sample. Thermal cycling conditions (on a G-STORM; Gene Technologies Ltd.) were 35 x [94°C for 30 s, 50°C for 30 s, 72°C for 1 min, 94°C for 3 min] and a final incubation at 72°C for 5 min.PCR products were then purified and sent to First Base Laboratories Sdn Bhd (1st BASE) for sequencing.

#### **2.3. Alignment and sequence properties**

The program MEGA ver. 4.0 [12] was used to visualize and align all sequences, including COI partial sequences of *S. serrata* obtained from Genbank (Genbank accession no: GU055497.1 – GU055506.1). The resulting sequences for each individual were then aligned using CLUSTAL W ver. 1.6 with default settings and were manually checked and trimmed in the BIOEDIT ver 7.0.9 sequence editing program; alignments were subsequently revised by eye in an effort to maximize positional homology. All aligned sequences were then imported into Basic Local Alignment Search Tool (BLAST) software to ensure the identity of the samples.

#### **2.4. Phylogenetic analysis**

The sequence characteristics of the COI region were calculated using MEGA ver. 4.0 [12]. The phylogenetic relationships among haplotypes were reconstructed using maximum parsimony (MP) and neighbour joining (NJ; [13]). We conducted MP and NJ phylogenetic analysis in MEGA ver. 4.0 [12]. Neighbour joining analysis was executed using non-parametric re-sampling procedure with 10,000 replicates. We performed MP analysis under the HKY+I model using the heuristic search algorithm with tree bisection reconnection (TBR) branch swapping and 100 random sequence addition replicates. Both trees reconstructions included 5 sequences of indigenous portunid from Genbank; *Portunus pelagicus* (Genebank accession no: GQ272560.1 – GQ272564.1). The genetic distance, *Gst* between populations was calculated using the Tamura-Nei distance [14] based on unequal base frequencies and unequal ratios of transitions to transversions (Ti:Tv) implemented in mega ver. 4.0 [12]. The frequency of each haplotype, haplotype diversity (*h*) (i.e., the probability that two randomly selected haplotypes are present in the sample) and the nucleotide diversity (π) within populations and geographical regions was estimated using ARLEQUIN ver. 3.11 [15].

## **3. Results and Discussions**

Overall 50 partial sequences (see Supplementary Material for Genbank accession number for each sequence analysed) of 520 base pairs (bp) each of the mtDNA COI gene correspond to 50 individuals representing three *Scylla* species (*S. olivacea, S. paramamosain* and *S. tranquebarica*) and 10 COI partial sequences of *S. serrata* obtained from Genbank (Genbank accession no: GU055497.1 – GU055506.1) were analyzed. All the sequences show high sequence similarity (99-100%) to their respective species sequence in Genbank, indicate that misidentification of species does not occur in this study. The 520 bp *Scylla* sequences comprised 109 variable sites, of which 102 are parsimony informative sites (Table 1). This high number of parsimony-informative sites indicates that COI mtDNA is capable to be an informative locus candidate for phylogenetic studies [16] as well as for population differentiation and population genetic structure (Rosly et al., in press).


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**Figure 3.** Maximum Parsimony (MP) phylogram showing the relationships among cytochrome c oxidase I (COI) sequences of Scylla species and 5 outgroups of genus Portunus analyzed in the present study. The number at each node represents the bootstrap percentage value based on 1000 replicates and applies the close-neighbour interchange (CNI) using the initial tree by random addition of 100 replicates for the maxi-

This study has highlighted the monophyly pattern of mud crab genus *Scylla* as evident by the consistent of four major clades in both phylogenetic trees (Figures 2 and 3). All samples were clustered according to their respective species; *S. tranquebarica*, *S. paramamosain, S. serrata* and *S. olivacea* with both trees showed that *S. tranquebarica* branched the earliest from all the species analysed in this study (Figures 2 and 3).The generic relationships revealed by our neighbourjoining (NJ) and maximum parsimony (MP) tree are highly in agreement with the previous molecular phylogeny study of *Scylla* based on COI sequence data [10]. However, our current results provide new insights into the classifications of the genus *Scylla* and presents additional evident regarding their inter-relationships that were not included and discussed in previous studies.

mum parsimony (MP) analysis generated using MEGA ver. 4.0 [17] software.

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**Table 1.** Summary of number of haplotypes, nucleotide diversity (π) and haplotype diversity (h) with standard deviation (SD), nucleotide composition, transition, transversion and multiple substitution dataset for each species implemented in ARLEQUIN ver. 3.11 software [25]. The number of sites was generated by using MEGA ver. 4 [17]. n = sample size; Ti = transition; Tv = transversion.

**Figure 2.** Neighbour-joining (NJ) phylogram showing the relationships among cytochrome c oxidase I (COI) sequences of Scylla species and 5 outgroups of genus Portunus analyzed in the present study. The number at each node represents the bootstrap percentage value based on 10000 pseudoreplications for the neighborjoining (NJ) analysis generated using MEGA ver. 4.0 [17] software.

**Figure 3.** Maximum Parsimony (MP) phylogram showing the relationships among cytochrome c oxidase I (COI) sequences of Scylla species and 5 outgroups of genus Portunus analyzed in the present study. The number at each node represents the bootstrap percentage value based on 1000 replicates and applies the close-neighbour interchange (CNI) using the initial tree by random addition of 100 replicates for the maximum parsimony (MP) analysis generated using MEGA ver. 4.0 [17] software.

This study has highlighted the monophyly pattern of mud crab genus *Scylla* as evident by the consistent of four major clades in both phylogenetic trees (Figures 2 and 3). All samples were clustered according to their respective species; *S. tranquebarica*, *S. paramamosain, S. serrata* and *S. olivacea* with both trees showed that *S. tranquebarica* branched the earliest from all the species analysed in this study (Figures 2 and 3).The generic relationships revealed by our neighbourjoining (NJ) and maximum parsimony (MP) tree are highly in agreement with the previous molecular phylogeny study of *Scylla* based on COI sequence data [10]. However, our current results provide new insights into the classifications of the genus *Scylla* and presents additional evident regarding their inter-relationships that were not included and discussed in previous studies.

The construction of phylogenetic trees using NJ and MP analyses produced identical tree topologies with strong levels of support for all nodes (Figures 2 and 3). Additionally, clear relationships among mud crab species was observed in both trees and supports the placement of outgroup taxa, *Portunus pelagicus* at the base of both trees (Figures 2 and 3). The relative efficiencies of the NJ and MP in obtaining the correct topology for phylogenetic inference were studied by computer simulation. The NJ method gives a correct topology even when the distance measures used are not unbiased estimators of nucleotide substitutions, while for the MP method, both the weighted and unweighted parsimony are generally less efficient than the NJ method even in the case where the MP method gives a consistent tree. However, the NJ and MP analysis in our study returned a similar tree topology (see Figures 1 and 2), as demonstrated by several other studies at different taxa. Thus, the same tree topology (although different bootstrap values) that demonstrated by both of the phylogenetic trees has highly support the inter-taxa relationship among mud crabs genus *Scylla* sampled from Malaysia.

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The relatively deep gene trees clearly show a large divergence between mud crab genus *Scylla* analysed in this study (Figures 1 and 2). This was further corroborated by the pairwise genetic distance among the four species of *Scylla* which ranges from *Gst* = *0*.086-0.198 (mean±SD; 0.146±0.261) for COI gene (Table 2). Other studies, particularly in marine fishes also show a large genetic distance between species. The intraspecific and interspecific differentiation in various crustacean taxa has been reported to be ranges at *Gst* = 0.00-1.47 and *Gst* = 5.2-31.6 respectively. Accepting this threshold as valid, the genetic distances between four species of mud crab in our study falls within the range of interspecific differentiation of crustacean taxa, thus further supported the study by Fuseya and Watanabe [8] which reported that there exist at least three distinct species within the genus *Scylla* (see e.g. [10, 12]).


**Table 2.** Pairwise Tamura-Nei genetic distances (Gst) for COI gene sequence among Scylla.

In conclusion, in this study we were able to provide useful insights into phylogenetic relationships and the genetic identity of *Scylla* species from mangrove areas in Malaysia. However, further studies using larger samples from other areas of its geographical distribution, sequence data from other mtDNA regions, and information based on nuclear DNA markers are required to support our findings.

#### **4. Acknowledgements**

We thank the Fisheries Research Institute of Penang for their assistance in sample collection. This project was funded by Universiti Sains Malaysia through research grant 304/PBIOLOGI/6311003.

#### **5. References**


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**Comparison of the MAKLER & HINRICHS** 

**(1993) Technique Versus Application of Hepes** 

**Lysis Solvent in Determining the Activities of** 

*Plasmodium berghei***- Infected Erythrocytes**

, Hasidah M. Sidek, Salmijah Surif

Shafariatul A.I\*

**Abstract** 

pLDH assay

**1. Introduction**

Department of Biomedical Science, Faculty of Allied

and simple device with fast and precise respond.

Health Sciences, Universiti Kebangsaan, Kuala Lumpur, Malaysia

*Plasmodium Lactate Dehydrogenase* **(pLDH) in** 

Plasmodium lactate dehydrogenase ( pLDH) is a clinically valuable diagnostic indicator for malaria disease. In this preliminary report, there are some considerable interest in measurements of pLDH assay. All these years, pLDH was evaluated according to method which was established by Makler and Hinrichs in 1993. This method has became very popular among malaria researches . In this study , we tried to describe an alternative way or method for measuring pLDH assay. The method was used Hepes lysis to disrupt the parasitized erythrocyte membrane and thus released the pLDH in the solution. Measurement was done based on the intensity of chromatographic changes in color through ELISA reader at 650nm. The Hepes lysis technique was recently introduced in few years ago. Both Makler (1993) and Hepes lysis techniques have demonstrated their ability and efficiency for assessing the pLDh assay . Based on the results, Makler and Hinrich ( 1993) technique have shown that pLDH assay activities were higher than Hepes lysis technique significantly ( 2 way ANOVA). The results were also supported by the microscopic view on both techniques .The experiment were conducted at 10% parasitemia and 30% parasitemia. Further investigation are needed in order to create and have more robust lactate sensing format

**Keywords:** Plasmodium lactate dehydrogenase , Makler and Hinrich (1993), Hepes lysis ,

*Plasmodium lactate dehydrogenase* (pLDH) is an essential metabolic enzyme that responsible for energy production in the parasite and parasite development. It is present in all malarial parasites of man and other animal host. pLDH catalyzes dehydrogenation of lactate and generates pyruvate by using NAD† as a cofactor. For more efficient and reliable test for pLDH activity, it can easily be differentiated from host lactate dehydrogenase (LDH) with 3-acetylpyridine adenine dinucleotide (APAD), an analogue of nicotinamide adenine dinucleotide (NAD) (Makler et. al 1993). pLDH can utilize APAD at 200 fold more rapidly and effectively than host LDH isoforms.

> © 2012 Shafariatul et al.; licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

provided the original work is properly cited.

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