**Toxic Effect of Fertilizers on Inferior Plants Resed as Biological Models**

Fadila Khaldi1, 2, Houria Berrebbah<sup>2</sup> and Med.Réda. Djebar<sup>3</sup>

1 Department of Biology,University of Mohammed Cherif Messaadia, Souk-Ahras, Algeria 2 Laboratory of Cell Toxicology, Department of Biology, Faculty of Sciences, Algeria

3 University of Badji Mokhtar, Annaba, Algeria

#### **Abstract**

The inopportune throws out of diverse substances in the atmosphere, constitutes without any doubt the obvious of environmental pollution by man. Among these substances, we are interested in the NPK (nitrate –phosphate-potassique).Nitrate fertilizers widely used in farming in our region - Annaba located in the eastern part of Algeria – and manufactured in the same region. In fact, the excessive fertilization, the intensive spreading of animal faeces and the industrial pollution are the accumulation sources of nitrate in vegetables, drilling and the underground waters.

The treatment by NPK affect the respiratory metabolism of mosses as well as the measure of the consumption of the oxygen shous the abviousness contrasted with a dampening of respiration but also of the photosynthesis.The perturbation of the respiration and photosynthesis of mosses can explain the degradation of the plant material and the disappearance of certain species from our ecosystem.

The effect of NPK indicate also the perturbation of enzymes antioxidants fonctions : GSH and GST.

**Keywords:** NPK, mosses, Cytotoxicity tests, respiratory and photosynthetic metabolism, Biomarkers; Antioxidant enzymes ,GSH,GST.

## **1. Introduction**

Bryophytes are particularly suitable organisms for the study of metal and organic pollutants. They owe this to their anatomical efficiency (high ratio surface / volume or surface area / mass, no waxy cuticle, of conducting vessels and real root system, easy to identify the annual growth) and physiological (photosynthetic activity continues at year round). They are therefore subject to the impact of pollutants in both dry depositions. Bioaccumulation of pollutants in plant species is an indicator of exposure. Indicators of effects of these pollutants can also be measured; they may be more defined, especially in the form of various biochemicals or physiological parameters (biomarkers) [1].

© 2012 Khaldi 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. Methods**

### **2.1. Sampling procedure of the lower plants**

The samples of Mousses (species *Leucodon sciuroides)* were taken in the area of Séraïdi, located at 850m above the sea (Annaba,Algeria). Our choice was made on this area because it is a zone considered as not polluted.

International Conference on Applied Life Sciences (ICALS2012)

**Figure 1.** Evolution of GST activity according to the

fertilizer concentrations (P ≤ 0,001).

0.001) compared with controls always.

Turkey, September 10-12, 2012

**Figure 2.** Evolution of GSH based on fertilizer con-

centrations (P ≤ 0,001).

**Figure 3.** Changes in chlorophyll (a, b, a + b) in Leucodon sciuroides treated by diff erent concentrations of NPK.

Fig. (3), highlights the changes in rates of chlorophyll *a, b* and (*a + b*) as a function of increasing concentrations NPK.Statistical analysis revealed a signifi cant diff erence between control and treated with the concentration (30 mM) for (Chl *b*) (P ≤ 0.05), while very highly signifi cant differences for all treated and all concentrations (10,20, 30.40 mM) (Chlorophyll *a, b* and *a + b*) (P ≤

**Figure 4.** Eff ects of NPK on photosynthetic metabolism of Mousses (Leucodon sciuroides) (P ≤ 0,001).

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#### **2.2. Tests of cytotoxicity for the moss**

NPK fertilizer was tested with four concentrations:10 ,20 ,30 and 40 mM. The solutions prepared with the various concentrations of NPKs are used for the imbibition of the samples of mousses. Approximately 1g of thallus was soaked in 100 ml of solution during 3 days [2].

#### **2.3. Determination of Glutathione (GSH) and activity Glutathione S-transferase (GST)**

The glutathione was assayed by the method of [3], based on measuring the absorbance of the 2-nitro-5 mercapturic resulting from the reduction of the acid 5-5 'thiol-bis-2-nitrobenzoic acid (DTNB) by the thiol groups (-SH) glutathione. The glutathione S-transferase activity is performed by the method of [4]. It is based on the conjugation reaction between GST and a substrate, CDNB (1-chloro 2, 4 dinitrobenzene).The GSH and GST biomarkers are expressed in µm/mg of protein. The protein level was measured according the method of [5].

## **2.4. Proportioning of chlorophyls**

The extraction of chlorophyls at summer was carried out according to the method of [6]. The formula related to solvent, enables us to calculate the values of chlorophylls [7].

## **2.5. Polarographic study**

The apparatus used is an electrode with oxygen (HANSATECH) which allows the measurement of the production of the oxygen uptake during a reaction. Its sensitivity makes it possible to detect concentrations of about 10µM [8].

## **2.6. Statistical study**

The statistical analysis was performed by Student t test used to compare between two samples (control and treated).This test is performed using the analysis software statistical processing of data: Minitab version 16.1.0. , n = 5 [9].

## **3. Results**

After 3 days of treatment, we found that glutathione-S-transferase tends to increase a dose-dependent manner. This increase was highest in the treaties with 40 mM concentration where the rate is: (0.103 (± 0.003)) µmole / min / mg of protein)(Fig.1).According to Fig.2, we find that glutathione levels decreased dose-dependent manner. Thus at 40 mM concentration, the GSH level is low (16.09 (± 0.49)) µmole / mg of protein) compared to the control of which is: (31.27 (± 0.21)) µmole / mg of protein).

**Figure 1.** Evolution of GST activity according to the fertilizer concentrations (P ≤ 0,001).

**Figure 2.** Evolution of GSH based on fertilizer concentrations (P ≤ 0,001).

**Figure 3.** Changes in chlorophyll (a, b, a + b) in Leucodon sciuroides treated by diff erent concentrations of NPK.

Fig. (3), highlights the changes in rates of chlorophyll *a, b* and (*a + b*) as a function of increasing concentrations NPK.Statistical analysis revealed a signifi cant diff erence between control and treated with the concentration (30 mM) for (Chl *b*) (P ≤ 0.05), while very highly signifi cant differences for all treated and all concentrations (10,20, 30.40 mM) (Chlorophyll *a, b* and *a + b*) (P ≤ 0.001) compared with controls always.

**Figure 4.** Eff ects of NPK on photosynthetic metabolism of Mousses (Leucodon sciuroides) (P ≤ 0,001).

This figure (4), illustrates the effect of different concentrations on the photosynthetic metabolism NPK foams where there has been a release of O2 in the medium for control samples and for samples treated with the four selected concentrations. 0n a marked increase in the amount of O2 produced in the middle as the fourth minute of recording for control samples. The minimum of this amount was recorded at the time (10min) for the sample treated with 30 mM concentration, which reached 10 nmol O2 / ml (Lower than the control: 11 nmol O2 / ml). While the sample treated with 20 mM concentration shows the highest amount produced until the time (10 min) or 15 nmol O2 /ml.On samples treated with other concentrations (10 and 40), have produced higher amounts of oxygen to those of the control resulting in stimulation of photosynthesis.

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NPK with glutathione. Our hypothesis is that induction of GST enzyme system can be explained by the entry of Xenobiotics (NPK) in plant cells (foam) and induction of detoxification system. The other aspect of our work was to measure the mean levels of chlorophylls *a, b* and (*a + b*), parameters that can tell us a possible state of stress due to the presence of a pollutant in mosses. In general, chlorophyll appears to be affected by the xenobiotic (NPK). This perturbation in the mean levels of chlorophyll *a, b* and (*a + b),* in these plants, explains the attenuation of photosynthetic activity. Our results agree with our previous work [2], which have demonstrated a disruptive effect of nitrate of ammonium on the biosynthesis of chlorophylls. Our results are quite revealing, and the NPK causes a stimulation of photosynthesis in mosses, is excessive production

of oxygen in the culture medium with a clear stimulation of respiratory metabolism.

impaired balance with other nutrients.

**5. Summary and conclusion**

Springer, Berlin,207-236.

[2] Khaldi F, 2003. Toxicité du nitrate d'ammonium NH4

Academie press, New York : pp.133.

tosynthesis.

**6. References**

Air pollution exposes plants to various forms of nitrogen that can be highly toxic (nitrogen dioxide, ammonia and ammonium). Among the reactions to the toxic effects of these compounds include: defoliation, training of larger cells thin-walled, yellowing, the lesions on some organelles of the plant and the reduction of drought resistance [14].The most important direct effect on vegetation results from the interaction of these various forms of nitrogen with other pollutants and

We can conclude that the NPK disrupts the photosynthetic metabolism and respiratory mousses. Our results are in perfect agreement with our previous work [2] and [15] which have demonstrated a stimulation of photosynthesis and respiration in these plants. But in higher plants one of the mechanisms of defense against air pollution is indeed a decrease in respiration and pho-

[1] Bargali R, 1998a. Mosses as passive and active monitors of trace elements.In Bargali R. Trace elements in terrestrial plants. An ecophysiological approach to biomonitoring and biorecovery.

[3] Weckberker, G., and Cory, G, 1988. Ribonucléotide reductase activity abd growth of glutathione

[4] Habig, W.H., M.J. Pabst and W.B. Jakoby, 1974.Gluthation-S-transferases: the first enzymatic step

[5] Bradford M , 1976 . A rapid and sensitive method for the quantification of microgram quantities

[6] Holden M,1975 .Chlorophylls in chemistry and biochemistry of plant pigment.2=nd Edition

Magister en Biochimie Appliquée. Université Badji Mokhtar, Annaba : 86pages.

inmercurapturic acid formation. Journal of Biologica lChemistry, 249: 7130-7139.

of protein utilizing the principle of protein-dye binding. Anal.Biochem.72:248-254.

depleted mouse leukemial 1210 cells in vitro. *Cacer letters* 40: 257-264.

paramécies, les mousses et les lichens. Effet sur leur métabolisme respiratoire. Mémoire de

NO3

sur trois modèles biologiques : les

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**Table 1.** Oxygen consumption (nmole/ml) in moss response to the NPK treatment (mM) (P ≤ 0,001).

The observation of the table (1) shows that the foams have a witness who starts breathing 340nmole O2 and reached 320 nmol O2 after 10min, the oxidation rate is an average of 2 nmol O2 / min.Ce treatment causes an acceleration observed from the 2nd minute especially in samples treated with 40 mM concentration where the rate of oxidation is 10 nmol of O 2 / min). Indeed, this speed is about 4, 6 and 8 nmol of O2 / min, respectively, in samples treated with 10, 20 and 30 mM of NPK.

#### **4. Discussion**

We propose in this work to proceed with the demonstration of the effect of NPK on foams, where we found a decrease in dose-dependent manner in the presence of GSH NPK. This condition can be explained by the direct connection of glutathione to the atoms of xenobiotic (NPK) as glutathione has a carboxylic acid group, an amine group, a group sulfhydryl (-SH) and two bypass likely peptide to be involved in reactions with other atoms. Its functional group-SH would then play an important role in binding to the xenobiotic [10].Our results agree with those of [11] and [12] in which the GSH level is decreased with increasing tolerance the accumulation of the pollutant for low concentrations .Our results show a significant increase of GST, in mosses in the presence of NPK; this increase is a response to oxidative stress caused by the presence of xenobiotics in the plant cell. The biotransformation enzymes are among the first to respond to the presence of a pollutant in a living organism [13]. This increase indicates a high rate of conjugation of atoms NPK with glutathione. Our hypothesis is that induction of GST enzyme system can be explained by the entry of Xenobiotics (NPK) in plant cells (foam) and induction of detoxification system.

The other aspect of our work was to measure the mean levels of chlorophylls *a, b* and (*a + b*), parameters that can tell us a possible state of stress due to the presence of a pollutant in mosses. In general, chlorophyll appears to be affected by the xenobiotic (NPK). This perturbation in the mean levels of chlorophyll *a, b* and (*a + b),* in these plants, explains the attenuation of photosynthetic activity. Our results agree with our previous work [2], which have demonstrated a disruptive effect of nitrate of ammonium on the biosynthesis of chlorophylls. Our results are quite revealing, and the NPK causes a stimulation of photosynthesis in mosses, is excessive production of oxygen in the culture medium with a clear stimulation of respiratory metabolism.

Air pollution exposes plants to various forms of nitrogen that can be highly toxic (nitrogen dioxide, ammonia and ammonium). Among the reactions to the toxic effects of these compounds include: defoliation, training of larger cells thin-walled, yellowing, the lesions on some organelles of the plant and the reduction of drought resistance [14].The most important direct effect on vegetation results from the interaction of these various forms of nitrogen with other pollutants and impaired balance with other nutrients.

## **5. Summary and conclusion**

We can conclude that the NPK disrupts the photosynthetic metabolism and respiratory mousses. Our results are in perfect agreement with our previous work [2] and [15] which have demonstrated a stimulation of photosynthesis and respiration in these plants. But in higher plants one of the mechanisms of defense against air pollution is indeed a decrease in respiration and photosynthesis.

#### **6. References**


[7] Arnon DI , 1949. Copper enzymes in isolated chloroplasts polyphenoloxidase in Beta vulgaris. Plant physiology 24 :1-15.

International Conference on Applied Life Sciences (ICALS2012)

**Rare Plant Species of the Protected Area of** 

In this study, we collected and determined rare plant species from protected areas of kalmand-Bahadoran located in 30-105 km SE of Yazd City in the Yazd Province. Analyses of the flora showed that, there is 148 vascular plant species in this area. Threatened species of this region analyzed, according to the IUCN criteria. On the basis of this study, four categories of rare species so called Endangered, vulnerable, lower risk and data deficient are determined and the list of these species has been presented. Result showed that, there is34 threatened plant species in this protected area. Finally, floristic composition, and species richness of this area is discussed.

Nowadays the procedure of investigation, recognition, and maintenance of herbal species, especially useful and rare ones has gained vital importance in the world. It constructs a foundation for sustainable development and presents principle and logical utilization of nature and natural resources and is defined as a basis for protecting and maintaining herbal species and genetic treasure. Therefore, aiming at recognition and introduction of rare and useful species of plants and animals all over the world and adopting necessary approaches to prevent from their extinction, International Union for Conservation of Nature and Natural Resources (IUCN) has been

Among studies conducted in Iran by Iranian and foreign botanists about collection and identification of plants we may refer to Iran's flora[12], Iranica flora [14], Orientalis flora [3], Iran's herbs [9], Iran's flora [2], Iran'Asastragalus bisulcatus(astragalus spp) [8], study of Iran's desert flora and herbs [7]and etc.; however, although some floras are argent for research and educational purposes, there not onlydo existed documented floras published regarding various parts of Iran but also there are less studies about rare species according to patterns and criteria of IUCN or-

To achieve this goal, one protected zone of Yazd province i.e.Kalmand-Bahadoran protected zone is selected. The reason for selecting this zone stem from this fact those plants of this area is to some extent being preserved from livestock foraging and human destruction and therefore study of this area flora is applicable and beneficial. Up to now flora of some protected zones like Turan and Kavir[14, 15], Arasbanan[1], are being investigated. In Yazd province there are some investigations conducted on province total-floras [10] and various regions including Kalmand-

**Kalmand-Bahadoran, Yazd Province, Iran**

Faculty of Natural Resources, Yazd University, Yazd, Iran

**Keywords:** Flora, Biologic form, rare species, Kalmand-Bahadoran.

Ali Akbar Karimian

**Abstract**

**1. Introduction**

established.

ganization.

© 2012 Karimian.; 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.

Turkey, September 10-12, 2012

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## **Rare Plant Species of the Protected Area of Kalmand-Bahadoran, Yazd Province, Iran**

Ali Akbar Karimian

Faculty of Natural Resources, Yazd University, Yazd, Iran

#### **Abstract**

In this study, we collected and determined rare plant species from protected areas of kalmand-Bahadoran located in 30-105 km SE of Yazd City in the Yazd Province. Analyses of the flora showed that, there is 148 vascular plant species in this area. Threatened species of this region analyzed, according to the IUCN criteria. On the basis of this study, four categories of rare species so called Endangered, vulnerable, lower risk and data deficient are determined and the list of these species has been presented. Result showed that, there is34 threatened plant species in this protected area. Finally, floristic composition, and species richness of this area is discussed.

**Keywords:** Flora, Biologic form, rare species, Kalmand-Bahadoran.

## **1. Introduction**

Nowadays the procedure of investigation, recognition, and maintenance of herbal species, especially useful and rare ones has gained vital importance in the world. It constructs a foundation for sustainable development and presents principle and logical utilization of nature and natural resources and is defined as a basis for protecting and maintaining herbal species and genetic treasure. Therefore, aiming at recognition and introduction of rare and useful species of plants and animals all over the world and adopting necessary approaches to prevent from their extinction, International Union for Conservation of Nature and Natural Resources (IUCN) has been established.

Among studies conducted in Iran by Iranian and foreign botanists about collection and identification of plants we may refer to Iran's flora[12], Iranica flora [14], Orientalis flora [3], Iran's herbs [9], Iran's flora [2], Iran'Asastragalus bisulcatus(astragalus spp) [8], study of Iran's desert flora and herbs [7]and etc.; however, although some floras are argent for research and educational purposes, there not onlydo existed documented floras published regarding various parts of Iran but also there are less studies about rare species according to patterns and criteria of IUCN organization.

To achieve this goal, one protected zone of Yazd province i.e.Kalmand-Bahadoran protected zone is selected. The reason for selecting this zone stem from this fact those plants of this area is to some extent being preserved from livestock foraging and human destruction and therefore study of this area flora is applicable and beneficial. Up to now flora of some protected zones like Turan and Kavir[14, 15], Arasbanan[1], are being investigated. In Yazd province there are some investigations conducted on province total-floras [10] and various regions including Kalmand-

© 2012 Karimian.; 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.

Bahadoran and KuheBafgh protected zones [6]as well as other locations. However, there is not any documented report about Yazd's protected zones plants.

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**Scientific Name Rare class Biologic form**

With respect to the fact that there is not complete information available on the regions' flora in the past years and yet no exact investigation is being carrying out regarding rareness of species in Iran, about 34herbalspecies were identified as endangered class in this region that mostly classified as lower risk category. Table 1 shows rare and vulnerablespecies of the above mentioned

PrangoscheilanthifoliaBoiss. LR He.

Centaureagaubae (Bornm.) wagenitz LR He. CentaureaispahanicaBoiss. LR He. Cirsiumspactabilis DC. LR He. Cousiniapiptocephala Bunge. LR He. EchinopsceratophorusBoiss. LR He. JurineabungeiBoiss. DD He. Jurinea radians Boiss. Subsp radians DD He.

OnosmastenosiphonBoiss. LR He.

 Alyssum bracteatumBoiss. , Buhse LR Th. IsatisrugulosaBge.exBoiss. LR Th. SamerariaelegansBoiss. LR Th. Sterigmostemumlongistylum(Boiss).Bornm. LR Th.

AcanthophyllumchloroleucumRech.f ., Aell DD Ch.

Nepetasaccarata Bunge. LR Th. NepetaSatureioidesBoiss. LR Th. ZatariamultifloraBoiss. LR Ch.

Allium chloroneurumBoiss. LR Cr.

Astragalus (Choronopuse) jesdianusBoiss. , Buhse LR He. Astragalus (choronopus) vanilla Boiss. LR He.

region.

**Apiaceae**

**Asteraceae**

**Boraginaceae**

**Brassicaceae**

**Caryophyllaceae**

**Lamiaceae**

**Liliaceae**

**Papilionaceae**

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## **2. Study area**

Kalmand-Bahadoran protected zone with approximately 255 thousands hectare situated 30 to 105 kilometers far in southeast of Yazd alongside Yazd-Kerman road at 31° and 20' of north latitude and 54° and 30' of east longitude. The zone mean attitude is 1616 above sea level and it is claimed as Kalmand-Bahadoran protected zone in 1994. The highest point is Medvar Mountain with altitude of approximately 3290 meters and the lowest part is positioned in Mahdiabad plain with an altitude of 1400 meters. Based on meteorological statistics annual precipitation average is 100 millimeters. The highest amount of rainfall occurs in January–February (22/21 millimeter). As it is observed in ambro-thermic diagram of the region drought period is started from the second half of March (beginning of Farvardin) and continues till second part of November (beginning of Azar). Maximum temperature average is 29/44 and coldest average is 5/220 C; moreover annual humidity average is 30%.

## **3. Methods of investigating rare and endangered species**

Researchers use various criteria for identification, investigation, and classification of rare species like limited geographical propagation and low population; in 1985, Grime, beside above criteria, considered hard bio-environmental conditions and heavy environmental changes as important factors in determining rare species. Rabinowitz(1981) identified some rare species based on geographical range of propagation, habitat features, and population size. Fielder and Ahouse(1992) described rare classes according to species, spatial dispersion and their chronological resistance. In order to determine and classify rare species, some researchers use various routinesliketaxonomy, chronology, endemism, the quality of settlement and natural proliferation, the manner of plants utilization by human beings, livestock, wildlife, and finally illness and diseases, lack of bio-reactions that causes population reduction and therefore species extinction.

In this study, some criteria such as limited geographical propagation, human utilization of plants, livestock, wildlife, population amount, biologic form, how to settle and natural reproduction, have been used in determining rare species classes of investigated zone. Among 8 classes ofrare plants, based on IUCN classification principles, we have identified 4 classes as follows:


## **4. Results**

Preliminary Results achieved from KalmandBahadoranprotected zone show that there are 148 herbal species in this zone. Among them rare species were extracted and their biologic form featureswere determined as in table 1.

With respect to the fact that there is not complete information available on the regions' flora in the past years and yet no exact investigation is being carrying out regarding rareness of species in Iran, about 34herbalspecies were identified as endangered class in this region that mostly classified as lower risk category. Table 1 shows rare and vulnerablespecies of the above mentioned region.



International Conference on Applied Life Sciences (ICALS2012)

recent droughts caused their population and frequency to be declined meaningfully.According to Zohri view point important Iran's locations regarding floristic enrichment, percentage of exclusive and rare species, are Alborz and Zagrosmountain ranges and some single mountains like Karkas, Shirkooh in Yazd, and south Kerman mountains. The investigated region is relatively near Shirkooh and its endangered species is not so much as indicated in table 1. The endangered species are mostly among lower Risk class and their biologic form character is described as hemicryptophyte47%, Therophyte23.5%, Chameophyte12%, and rarely Phanerophyteand

[1] Assadi, M., 1988. Plants of Arasbaran Protected area, N.W. of Iran (Part II). Jour. Bot.4 (1): 1.59. [2] Assadi, M., 1989-1996. Flora of Iran, volume, 1-14, Ministry of forest and rangelands publication,

[3] Fielder, P.L. and Ahouse, J.J. 1992, Hierarshiesof cause: toward an understanding of rarity in vascular plant species. In: Fieldr, P.L. and Jain, S.K. (Eds.). Conservation Biology, the Theory, and Practice of Nature Conservation, Preservation and Management. Chapman and Hall, New-York.

[4] Grime, P.P., 1985. The C-S-R model of primary plant strategies- Origins, implications and tests. In Gottlieb, L.D. and Jain, S.K. (Eds.). Plant Evolutionary Biology. Chapman and Hall, London. [5] IUCN. 1981 How to use the IUCN Red Data Book Categories. Threatened Plants Committee

[6] Karimian, A.A. 2001. Investigation of plant species in KalmandBahadoran Protected Area,

[7] Leonard, J., 1981 – 1992. Contribution an' I' etude de la Flore et de la Vegetation des deserts de I'

[8] Masumi. A.A. 1986-1995. Astragaluses of Iran, Volume 1-3, Ministry of forest and rangelands

[9] Mobayen, S. 1975-1994. Vegetation's of Iran, (Vascular Plants) volume 1-4, Tehran University

[12] Parsa, Ahmad, 1978-80. Flora of Iran, Vol. 1-2, Ministry of Culture and Higher Education of

[13] Rabinowitz, D., 1981. Seven forms of rarity, In: Synge, H. (Ed.). The Biological aspects of rare

[14] Rechinger, K.H. (Ed.), 1963-1993. Flora Iranica. Vol. 1-172. AkademischeDruck-U Verlagsanstalt,

[15] Rechinger, K. H., 1977. Plants of the Touran protected Area (Iran). Iran. Jour. Bot. 1(2): 155-180.

[11] Parsa, Ahmad, 1948-60. Flore de I' Iran. Vol. 1-8, Ministered de I 'Education. Teheran.

Ministry of Environmental protection Publication, Iran, Pp. 44.

[10] Mozafarian, V., 2001. Flora of Yazd, Yazd publication, Iran, Pp.473.

plant conservation. John Wiley and Sons, New-York, pp.: 205-217.

Iran. Fasc. 1-10. Jard, Botanique National de Belgique.

NanoPhanerophyte.

**6. References**

Iran.

Secretariat. IUCN, Kew.

publication, Iran.

Publication, Iran.

Graz, Austria.

Islamic Republic of Iran., Tehran.

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**Tab 1.** List of Rare plant species of Kalmand Bahadoran Yazd Province

Rare categoriesinclude:*Endangered species facing extinction (En),vulnerable species (Vu), and Lower Risk species (LR), Data Deficient species (DD)*

Biologic form include: *Throphytes(Th),Criptophytes(Cr),Hemicryptophytes(He), Phanerophytes(Ph.), Nanophanerophytes (Nph)*

#### **5. Summary and Conclusions**

The investigated region is situated in the heart of Iran's central plateau and is assumed as IranoT ouranian'svegetativeregion. Totally, regarding plants, this regionis less enriched than the whole country. The existence of limited number of tree and shrub species even in sparse and sporadicform shows that this area is poor concerningvariety and amount of woody species. Based on information achieved from this research most of the herbal species included at firstperennialshrub species that may tolerate drying conditions and regarding low precipitation they can continue their survival and finely reproduce in rainy years and secondlyannual species that are drought escape and whendrought is prevailed they biologically turn to dormant.Herbal speciesof the region are mainly belonging to *Astraceae*, *Paplionaceae*,and*Brassicaceae*families. Herbal species were to some extent immune from destruction in recent years since they are protected by Environmental Protection Agency and because livestock manager were removed from the region; however, recent droughts caused their population and frequency to be declined meaningfully.According to Zohri view point important Iran's locations regarding floristic enrichment, percentage of exclusive and rare species, are Alborz and Zagrosmountain ranges and some single mountains like Karkas, Shirkooh in Yazd, and south Kerman mountains. The investigated region is relatively near Shirkooh and its endangered species is not so much as indicated in table 1. The endangered species are mostly among lower Risk class and their biologic form character is described as hemicryptophyte47%, Therophyte23.5%, Chameophyte12%, and rarely Phanerophyteand

#### **6. References**

NanoPhanerophyte.


International Conference on Applied Life Sciences (ICALS2012)

**The Alkaline Phosphatase Levels in the Seminal** 

**Plasma and Sperms of Sub-Fertile Patients and** 

Faris N. A. Alhady Alibawi 1, Sahib Y. Al-Morshidy 2, Ali G. Alhuweizi <sup>1</sup>

This study examined 110 semen specimens collected from sub-fertile and Normospermic men after a period of abstinence from 3-5 days. The samples were collected in Fertility Center Laboratories/ Al- Saader Hospital/ Najaf province in the period from November 2009 to May 2010. This study aimed to comparison between the concentrations of the enzyme alkaline phosphatase (ALP) in seminal plasma of different in sub-fertility groups include: Asthenospermia (AS), Oligoasthenospermia (OAS), Azoospermia and Normospermia (NS) to study the effect of ALP levels

The results revealed that a significant increase (p <0.01) in the concentration of ALP in the seminal plasma compared to the levels of this enzyme in sperm per each group of this study: NS, AS and OAS. The comparison study of the seminal plasma ALP enzyme level indifferent groups of this study, revealed a significant decrease in the levels of ALP enzyme in the seminal plasma of Oligoasthenospermic(p <0.05) and Azoospermic (p <0.01) patients compared to seminal plasma of Normospermic men, while this decrease was insignificant in the seminal plasma of Asthenospermic patients . The results showed non significant decrease (p>0.05) in ALP enzyme activity of the asthenospermic patient`s sperm, while a significant decrease (p <0.05) of this enzyme in Oligoasthenospermic sperms compared to the levels of this enzyme in Normospermic sperm. It was concluded that the level of ALP in the seminal plasma and sperms correlated with the

The alkaline phosphatase secret in seminal fluid by Epididymis of rabbet and dogs (1;2), while it was secreted by prostate and testis in human (3). All Isoenzymes of this enzyme is encoded by three genes in the body, which includes the placental alkaline phosphatase , intestinal and Osteohepatic, this differences of the isoenzymes refer to the addition of gluco groups to the peptide chains to form glucoproteinsand thus these isoenzymes differ in their different characteristics such as behavior which is expressed during the Electrophoresis (4). According to the differences of isoenzymes of alkaline phosphatase can diagnosis of seminal fluid quality (5). The alkaline phosphatase

**Normospermic Men**

University of Kufa, Najaf, Iraq

concentration of the sperms.

**1. Introduction** 

College of Science, University of Babylon, Babylon, Iraq

in the quality of sperm in sub-fertile patients.

1

2

**Abstract**

© 2012 Alibawi 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.

Turkey, September 10-12, 2012

<sup>217</sup> ISALS

## **The Alkaline Phosphatase Levels in the Seminal Plasma and Sperms of Sub-Fertile Patients and Normospermic Men**

Faris N. A. Alhady Alibawi 1, Sahib Y. Al-Morshidy 2, Ali G. Alhuweizi <sup>1</sup>

1 College of Science, University of Babylon, Babylon, Iraq

2 University of Kufa, Najaf, Iraq

#### **Abstract**

This study examined 110 semen specimens collected from sub-fertile and Normospermic men after a period of abstinence from 3-5 days. The samples were collected in Fertility Center Laboratories/ Al- Saader Hospital/ Najaf province in the period from November 2009 to May 2010.

This study aimed to comparison between the concentrations of the enzyme alkaline phosphatase (ALP) in seminal plasma of different in sub-fertility groups include: Asthenospermia (AS), Oligoasthenospermia (OAS), Azoospermia and Normospermia (NS) to study the effect of ALP levels in the quality of sperm in sub-fertile patients.

The results revealed that a significant increase (p <0.01) in the concentration of ALP in the seminal plasma compared to the levels of this enzyme in sperm per each group of this study: NS, AS and OAS. The comparison study of the seminal plasma ALP enzyme level indifferent groups of this study, revealed a significant decrease in the levels of ALP enzyme in the seminal plasma of Oligoasthenospermic(p <0.05) and Azoospermic (p <0.01) patients compared to seminal plasma of Normospermic men, while this decrease was insignificant in the seminal plasma of Asthenospermic patients . The results showed non significant decrease (p>0.05) in ALP enzyme activity of the asthenospermic patient`s sperm, while a significant decrease (p <0.05) of this enzyme in Oligoasthenospermic sperms compared to the levels of this enzyme in Normospermic sperm.

It was concluded that the level of ALP in the seminal plasma and sperms correlated with the concentration of the sperms.

## **1. Introduction**

The alkaline phosphatase secret in seminal fluid by Epididymis of rabbet and dogs (1;2), while it was secreted by prostate and testis in human (3). All Isoenzymes of this enzyme is encoded by three genes in the body, which includes the placental alkaline phosphatase , intestinal and Osteohepatic, this differences of the isoenzymes refer to the addition of gluco groups to the peptide chains to form glucoproteinsand thus these isoenzymes differ in their different characteristics such as behavior which is expressed during the Electrophoresis (4). According to the differences of isoenzymes of alkaline phosphatase can diagnosis of seminal fluid quality (5). The alkaline phosphatase

© 2012 Alibawi 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.

is intended to be more effective in the Leyding cell , transitional and Fibroblast in seminiferous tubules (6), but on the other hand not see any effectiveness of this enzyme in Sertoli cell.

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The ALP enzyme level in the seminal plasma and sperms was estimated by colorimetric method

Analysis of data was performed by usingStatistical Package for (SPSS) (Version 17). Results are expressed as mean ± S.E. Statistical differences were determined by Least Significance Differences

The results showed significant decrease in sperm concentration in (OAS) group (p<0.01), and (p<0.05) in (AS) group compared to (NS). The sperm motility percent and grade activity in (AS) and (OAS) groups were significant decrease (p<0.01) compared to (NS), also there was a significant increase (p<0.01) in abnormal sperm morphology percent in all infertile groups compared

The comparison between the ALP concentration in seminal plasma and sperms showed significant increase (p<0.01) of ALP level in the seminal plasma compared to sperm in (AS), (OAS) and (NS) specimens. Also the results revealed a significant decrease (p<0.01) in seminal ALP concentration in (OAS) and azoospermia compared to (NS), while this decreasment is non significant (p>0.05) in (AS). Also there was a significant decrease (p<0.01) of sperm ALP level in (OAS) com-

> **Asthenospermia Mean ± SE**

Grade activity 3.714 ± 0.106 1.913 ± 0.168\*\* 1.500 ± 0.177\*\* ---

59.761 ± 3.423 76.956 ± 2.82\*\* 87.391 ± 2.492

68.6 ± 6.2 54.4 ±3.1 45.1 ±26.8

**Table 1.** Seminal Fluid Parameters and Alkaline Phosphatase (ALP) Level in Infertile groups and Normosper-

74.285 ± 4.257 57.826 ± 5.258 \* 7.700 ± 1.955 \*\* ---

65.00 ± 2.182 28.913 ± 2.981\*\* 25.227 ± 2.662\*\* ---

273.6 ± 29.9 218.5± 26.8 179.2± 13.0 \* \* 156.7± 15.0 \*

by using a kit manufactured by Biolabo: France Company.

(LSD) test for multiple comparisons between different groups.

**Normospermia Mean ± SE**

**Statistical analysis:** 

**3. Results**

to normospermia.

pared to (NS) Table-1-.

**Seminal fluid parameters Mean ± SE**

Sperm concentration (million/ml)

> Sperm motility percent

Abnormal sperm morphology percent

ALP Level in seminal plasma (IU/L)

ALP Level in sperms (IU/L)

\* Significant difference (p<0.05) \*\* Significant difference (p<0.01)

mia.

Turkey, September 10-12, 2012

**OligoAsthenospermia Mean ± SE**

\*\*

\* \*

**Azoospermia Mean ± SE**



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The alkaline phosphatise from the enzymes that cause loss of the phosphorus Group, which is effective in several tissues, including bone, liver, kidney, bowel, lung, and placenta in addition to the reproductive system. The amount of most alkaline phosphatise in the bulls excreted from the seminal vesicles in addition to the testis and epididymis, which constitutes a small percentage of concentration in semen (7).Another study indicated the existence of significant difference in the concentration of the alkaline phosphatise in dogs that underwent a process of Vasectomization for those that did not take place in this process (8). Also observed significant decrease in the levels of one isoenzyme of alkaline phosphatase secreted from germ cells, which called Placentallike alkaline phosphatase, in men who have undergone the process of Vasectomy So that it is possible to use the measuring of this enzyme concentration in the semen of these patients, to see the success of vasectomy (3;5). The strains of rabbits that have high fertility, have higher levels of seminal phosphatase enzymes compared to those that have low fertility (9;10).

The seminal alkaline phosphatase in boar inhibited by Theophylline, Caffeine and Pentoxifylline which lead to the improvement of sperm parameters (11) but the same research added that this result may be different when studied on human sperm.

This study aimed to study the correlation between the level of Alkaline Phosphatase and male infertility

## **2. Materials and Methods**

#### **Semen specimens**

Semen specimens were collected from Normospermic (NS), Asthenospermic (AS), Oligoasthenospermic (OAS) and Azoospermic patients by masturbation after three days of sexual abstinence. The specimens were allowed to liquefy at 37C°, and then seminal fluid analysis was performed to determine the sperm parameters include: sperm concentration, sperm motility percent, grade activity and abnormal sperm morphology percent.

#### **Preparation of Seminal Fluid specimens for estimation of Alkaline Phosphatase**

One ml of each specimen was centrifuged (3000 rpm for10 minutes) after the Seminal Fluid Analysis was performed to obtain the seminal plasma as supernatant and pellet. The supernatant (Seminal Plasma) was transferred to the other tube and then used for estimation the level of ALP enzyme. The pellet was washing by using one ml of Normal Saline and mixed well , then recentrifuged again (3000 rpm for 5 minutes), then the supernatant was removed completely. The ALP enzyme connected with the sperm plasma membrane, so that the sperm should be crushed by wood stick, then 0.5 ml of distilled water was added to the specimens contain the sperms for disruption the cell membrane due to the different of osmotic pressure . The seminal plasma and sperm suspension was froze untile the test.

#### **Alkaline phosphatase concentration:**

The ALP enzyme level in the seminal plasma and sperms was estimated by colorimetric method by using a kit manufactured by Biolabo: France Company.

#### **Statistical analysis:**

Analysis of data was performed by usingStatistical Package for (SPSS) (Version 17). Results are expressed as mean ± S.E. Statistical differences were determined by Least Significance Differences (LSD) test for multiple comparisons between different groups.

## **3. Results**

The results showed significant decrease in sperm concentration in (OAS) group (p<0.01), and (p<0.05) in (AS) group compared to (NS). The sperm motility percent and grade activity in (AS) and (OAS) groups were significant decrease (p<0.01) compared to (NS), also there was a significant increase (p<0.01) in abnormal sperm morphology percent in all infertile groups compared to normospermia.

The comparison between the ALP concentration in seminal plasma and sperms showed significant increase (p<0.01) of ALP level in the seminal plasma compared to sperm in (AS), (OAS) and (NS) specimens. Also the results revealed a significant decrease (p<0.01) in seminal ALP concentration in (OAS) and azoospermia compared to (NS), while this decreasment is non significant (p>0.05) in (AS). Also there was a significant decrease (p<0.01) of sperm ALP level in (OAS) compared to (NS) Table-1-.


\* Significant difference (p<0.05)

\*\* Significant difference (p<0.01)

**Table 1.** Seminal Fluid Parameters and Alkaline Phosphatase (ALP) Level in Infertile groups and Normospermia.

### **4. Discussion**

The results of the current study showed, the parameters of semen and sperm for patients with infertility significant decrease (p <0.05) in the sperm concentration for (AS) and (OAS) patients compared to (NS) men, this result is agree with other studies (12; 13). The concentration of sperm in the (NS) is higher than the other infertility groups due to several factors, including: a defect in the action of hormones or bacterial infections that affect the male reproductive system, or varicocele.

International Conference on Applied Life Sciences (ICALS2012)

[1] Frenette, G.; Dube, J.Y. and Tremblay, R.R. (1986) . Origin of alkaline phosphatase of canine

[2] Muller, B. (1983) Genital tract proteins in the male rabbit: Alkaline phosphatase enzyme action

[3] Lewis-Jones, D. I., Johnson, P. M. Desmond, A. D. and McLaughlin, P. J. (1992) Germ cell alkaline phosphatase in human seminal plasma following vasectomy. Br. J. Urol. *69*: 418-420.

[4] Seargeant, L. E. and Stinson, R. A. (1979) Evidence that three structural genes code for human

[5] Kutzler, M. A.; Solter P.F.; Hoffman, W. E. and Volkmann, D. H.(2003) Characterization and localization of alkaline phosphatase in canine seminal plasma and gonadal tissues.

[6] Gunawardana, VK. (1990). Ultrastructural localization of alkaline phosphatase in intertubular

[7] Zakrzewska, H.; Udala, J. and Blaszczyk, B. (2002). In vitro influence of sodium fluoride on ram

[8] Stornelli, A.; Arauz, M.; Baschard, H. and R-L de la sota.(2003) Unilateral and bilateral vasectomy

[9] Elkomy, A.E.; El-SeibyM.E. and K.I. Kamel,(2008)Comparative study of semen quality and free aminoacid content in seminal plasma between high andlow motile sperm rabbit bucks. Egypt.

[10] El-Seiby, M.E.; Elkomy A.E. and K.I. Kamel,(2008)Evaluation of spermatological parameters and free amino acids composition of Black Baladi, New Zeland White andV-Line rabbits buck under

[11] Glogowski, J.; Danforth, D.F. and Ciereszko, A.(2002) Inhibition of alkaline phosphatase activity

[12] Chia, S-H.; Lim, S-T.A.; Tay, S-k. and Lim, S-T.(2000).Factors associated with male infertility: a

[13] Okonofua, F.; Menakaya, U.; Onemu, S.O.; Omo-Aghoja, L.O. and Bergstorm, S.(2005). A casecontrol study of risk factors for male infertility in Nigerria Asian J. Andrology, *7*(4): 351-361. [14] Pal, P.C.; RajalakShmi, M.; Manocha, M.; Sharma, R.S.; Mittal, S., and Rao, O.N.(2006). Semen quality and sperm functional parameters in fertile India men. Andrologia, *38*:20-25.

[15] Bell, D.J. and Lake, P.E.(1962).Comparison of phosphomonoesteraseactivities in the seminal plasmas of domestic cock, tom turkey, boar, bull, buck, rabbit and man. J ReprodFertil , *3*: 262-268.

[16] Singer,R.;Barnet, M.;Allalouf, D.;Schwartzman, S.; Sagiv, M.; Landau, B.; Segenreich,E.; Servadio,C.(1980) Some properties of acid and alkaline phosphatase in seminal fluid and isolated

seminal plasma. Archives of Andrology.*;16*:235-241.

and site of synthesis. Andrologia.*15*: 676-681.

alkaline phosphatase. Nature *281*: 152-154.

tissue of the testis in domestic fowl. Tissue cell.; *22*:113-121.

semen quality and enzyme activities. Fluoride. *35*: 153-160.

winter egyptian condition. Egypt. Poult. Sci., *28*(2): 617-631.

sperm. Archives of andrology, *5*:195-199.

in the dog. Blackwell Synergy. Reproduction in domestic animals. *38*(1):1.

of boar semen by petoxifylline,caffeine and theophylline. J. Andrology, *23*(6).

case- control study of 218 infertile and 240 fertile men. BJ. *101* (1): 55-61.

Theriogenology, *60*:299-306.

Poult. Sci.,*28*(2): 633-649.

**5. References**

Turkey, September 10-12, 2012

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Also the results revealed significant increase in abnormal sperm morphology percent in (AS) and (OAS) patients compared to (NS), this result agree with the study of (14), that the normospermic men have 75.67% normal sperm morphology as compared to other groups of infertility.

The results showed significant increase (p <0.01) in the concentration of the alkaline phosphates (ALP) enzyme in the seminal plasma of AS and OAS patients compared to the concentration of ALP enzyme in sperm. The place and the amount of secretion of the ALP enzyme different according to the type of organism varies from one species to another (1). The studies conducted with human have indicated that the secretion of ALP enzyme from the prostate and testis (3) , while the another study revealed that this enzyme in rabbits and dogs is secreted by the epididymis (2). There is another study conducted on one of thecattle breeds showed an inverse relationship between the ALP concentration and sperm concentration (15), so this variation may be due to the presence more than one source of the ALP enzyme secretion (16;17).

The comparison study of the concentration of seminal plasma ALP between the different infertility groups and normospermia showed non significant decrease of ALP in (AS) and significant decrease in (OAS) and Azoospermia. The significant differences of ALP concentration refer to sperm number only not for the sperm motility percent or grade activity, this result agree with the other study (3), which are revealed to the existence of a positive relationship between sperm counts and concentration of this enzyme in the semen samples. Another study found that the concentration of spermatozoa increase with the reduction of ALP, while the sperm concentration increase with the increasing of ALP , and the same study showed that the lowest activity of ALP enzyme in azoospermic patients compared to another groups of infertility (16). The levels of ALP enzyme increased in the first split ejaculate compared to the second split is because the largest amount of the first split of ejaculate secreted from prostate (18).

The ALP enzyme in the sperm showed a significant decrease in sperm of AS and (OAS) patients compared to (NS) , this significant decrease in (OAS) may be refer to the decline sperm count in this group of patients. Low and Saltiel; 1988 (19) revealed, that the ALP enzyme linked to the sperm cell membrane by Phosphatidylinositolglycan located on the outer surface of the sperm. The site of ALP enzyme is in the plasma membrane in addition to cytoplasmic droplet and acrosome body of the sperm (20; 21). The ALP enzyme act through hydrolysis of phosphate ester of the nucleotide , sugars and ATP and has a potential role in removing phosphorus from Adenosine Monophosphate (AMP), also works to prevent the addition carbohydrates groups to glycoproteins in the surface of sperm (22). The another study indicated that the ALP enzyme present in the chloride channels in the sperm (23), and that have a role in the acrosome reaction (24), and thus there is a positive linear relationship between the level of this enzyme and sperm concentration in the semen samples of NS, AS and OAS.

#### **5. References**


[17] McLaughlin, P. J.; Lewis-Jones, I.; Hutchinson, G. E. and Johnson, P.M. (1986) Placental-Type alkaline phosphatase in human seminal plasma from fertile and infertile men. Fertil. Steril. *46*:934-937.

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, Miguel A Alvarez<sup>2</sup>

**Influence of Lactose and Sucrose on Growth** 

Department of Biotechnology, University of Mostaganem, Mostaganem, Algeria

2 Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Villaviciosa, Spain

**of** *Streptococcus thermophilus*

María Fernández <sup>2</sup> , Ahmed Bensoltane <sup>3</sup>

<sup>3</sup> Department of Biology, University of Oran, Algeria

compared to that quantified with strains BN2 and BN3.

Rabha Bennama 1, Victor Ladero <sup>2</sup>

1

**Abstract**

**1. Introduction**

**and Acetaldehyde Production by Three Strains** 

This investigation describes three strains of *Streptococcus thermophilus* on the basis of production of acetaldehyde as aroma compounds. The strains under study (BN1, BN2 and BN3) were isolated from Algerian raw milk and were identified according to microbiological, biochemical, and genetic criteria. The growth of the strains and the determination of acetaldehyde were performed on M17 medium added to 0.5 and 3% (w/v) of lactose and sucrose. It was observed that the produced biomass (log cfu/ml), reached high values in the presence of 3% (w/v) of lactose and sucrose compared to that posted with 0.5% (w/v). The strains appeared to produce acetaldehyde. This production was more powerful in the case of concentration 3% (w/v) of lactose and sucrose. Strain BN1 produced approximately 205±75µmol and 218±90µmol of acetaldehyde respectively in the presence of 3% (w/v) of lactose and sucrose. This ratio was significantly higher (P<0.01)

**Keywords**: *Streptococcus thermophilus*, growth, lactose, sucrose, acetaldehyde production.

Fermented dairy products have become commonly consumed food in many countries around the world. These products were industrially developed using lactic acid bacteria, which were at the origin of an individual transformation process that affected the texture, flavour, quality, and the conservation of fermented dairy products [1]. *Streptococcus thermophilus* is one of the species that plays a great, interesting role for its contribution to the rapid transformation of lactose milk in lactate, the secretion of exopolysaccharides, synthesis of vitamins like folic acid, and production of some flavour compounds such as acetaldehyde [2]. Acetaldehyde is the major component responsible for the typical flavour of yogurt and a number of cheeses [3-4]**.** It is produced by the two yoghurt bacteria: *S. thermophilus* and *Lactobacillus bulgaricus*, but *S. thermophilus* species is deemed to be a good acetaldehyde producer [3]. The exact mechanism for the production of acetaldehyde from *S. thermophilus* is not well understood. In general, it is formed directly from the pyruvate decarboxylation through the action of the pyruvate decarboxylase or indirectly from the acetyl – CoA, through the pyruvate dehydrogenase and aldehyde dehydrogenase [5- 6]**.** Moreover, acetaldehyde can be produced through the serine hydroxyl-methyl transferase

> © 2012 Bennama 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.

Turkey, September 10-12, 2012

,

<sup>223</sup> ISALS


## **Influence of Lactose and Sucrose on Growth and Acetaldehyde Production by Three Strains of** *Streptococcus thermophilus*

Rabha Bennama 1, Victor Ladero <sup>2</sup> , Miguel A Alvarez<sup>2</sup> , María Fernández <sup>2</sup> , Ahmed Bensoltane <sup>3</sup>

1 Department of Biotechnology, University of Mostaganem, Mostaganem, Algeria

2 Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Villaviciosa, Spain

<sup>3</sup> Department of Biology, University of Oran, Algeria

#### **Abstract**

This investigation describes three strains of *Streptococcus thermophilus* on the basis of production of acetaldehyde as aroma compounds. The strains under study (BN1, BN2 and BN3) were isolated from Algerian raw milk and were identified according to microbiological, biochemical, and genetic criteria. The growth of the strains and the determination of acetaldehyde were performed on M17 medium added to 0.5 and 3% (w/v) of lactose and sucrose. It was observed that the produced biomass (log cfu/ml), reached high values in the presence of 3% (w/v) of lactose and sucrose compared to that posted with 0.5% (w/v). The strains appeared to produce acetaldehyde. This production was more powerful in the case of concentration 3% (w/v) of lactose and sucrose. Strain BN1 produced approximately 205±75µmol and 218±90µmol of acetaldehyde respectively in the presence of 3% (w/v) of lactose and sucrose. This ratio was significantly higher (P<0.01) compared to that quantified with strains BN2 and BN3.

**Keywords**: *Streptococcus thermophilus*, growth, lactose, sucrose, acetaldehyde production.

## **1. Introduction**

Fermented dairy products have become commonly consumed food in many countries around the world. These products were industrially developed using lactic acid bacteria, which were at the origin of an individual transformation process that affected the texture, flavour, quality, and the conservation of fermented dairy products [1]. *Streptococcus thermophilus* is one of the species that plays a great, interesting role for its contribution to the rapid transformation of lactose milk in lactate, the secretion of exopolysaccharides, synthesis of vitamins like folic acid, and production of some flavour compounds such as acetaldehyde [2]. Acetaldehyde is the major component responsible for the typical flavour of yogurt and a number of cheeses [3-4]**.** It is produced by the two yoghurt bacteria: *S. thermophilus* and *Lactobacillus bulgaricus*, but *S. thermophilus* species is deemed to be a good acetaldehyde producer [3]. The exact mechanism for the production of acetaldehyde from *S. thermophilus* is not well understood. In general, it is formed directly from the pyruvate decarboxylation through the action of the pyruvate decarboxylase or indirectly from the acetyl – CoA, through the pyruvate dehydrogenase and aldehyde dehydrogenase [5- 6]**.** Moreover, acetaldehyde can be produced through the serine hydroxyl-methyl transferase

© 2012 Bennama 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.

(SHMT), which catabolizes threonine into acetaldehyde and glycine [3]. SHMT is not the only shunt involved in the formation of acetaldehyde in yogurt, but also in the formation of glycine, serine, and significant amounts of folic acid [7].

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Bacteria obtain energy by multiple ways. Most of this energy is used in the biosynthesis of many metabolites or anabolism. Performance of diff erent metabolic reactions depends essentially on the nature of the carbon source and its concentration**.** Thus in this work, the importance of the carbon source was checked with strains BN1, BN2, and BN3 of *S. thermophilus* by studying their growth in the presence of lactose and sucrose. These have been incorporated to M17 medium at fi nal concentration of 0.5 and 3% (w/v). It is well known that *S. thermophilus* exhibits a highly affi nity to grow on lactose**;** sucrose can also be used nevertheless with lower effi ciency than lactose [10-11]. This affi nity towards both carbon sources is related to the presence of a large variety of important genes and enzymatic equipment of sugar metabolism and central carbon pathways [11-12].

The results related to the increase in biomass (log cfu/ml) and pH variations aft er 8h of growth on LM17 and SM17 media are illustrated in fi gures 1 and 2. From these results, it appears that strains displayed a high level of growth with lactose and sucrose used at 3% (w/v). Comparatively to the initial rate of inoculation, the strains showed an average increase in the biomass of 3.20±0.09 and 3.10±0.14 log cfu/ml respectively in the presence of 3% (w/v) of lactose and sucrose. A signifi cant diff erence (P<0.05) in the biomass was only observed between the sucrose used at

During the growth, a signifi cant decrease in the pH of the cultures was observed. The pH values decreased, in average, from 7.0 to 4.5 in the presence of 3% (w/v) of both carbon sources (fi gure 2). The pH decreasing over time raises the existence of a specifi c metabolic activity in the diff erent fermentative pathways used by *S. thermophilus*, which acidifi es the medium by the production of lactate or other acids [11]. Furthermore, it is important to point that the level of growth of the strains was not infl uenced by the decrease in pH, even aft er 24h of incubation (data not shown).

**3. Results and Discussion**

0.5 and 3% (w/v).

media.

**Fig 1.** Mean values of increase in biomass (log cfu/ml) of the strains BN1, BN2 and BN3 after 8h of fermentation at 42°C in LM17 and SM17

**3.1. Growth of Strains in the Presence of Lactose and Sucrose**

Turkey, September 10-12, 2012

**Fig 2.** Mean values of pH measured after 8h of fermentation at 42°C in LM17 and SM17 media inoculated with strains BN1, BN2 and BN3.

<sup>225</sup> ISALS

Concerning this compound, the focus was on improving among other things of biosynthesis conditions, the composition of the culture media. In this context, this work is interested to the screening of three strains of *S. thermophilus* by studying the effect of carbon source (lactose and sucrose) on the growth and production of this aromatic compound.

## **2. Materiel and Methods**

## **2.1. Strains and Culture Conditions**

Three strains of *S. thermophilus* BN1, BN2, and BN3 isolated from Algerian raw milk were used in this study. Strains were identified by phenotypic and biochemical criteria and confirmed by molecular methods as described by Bennama et al. [8]. They were routinely grown on M17 medium [9] containing 0.5% (w/v) of lactose (LM17) and incubated anaerobically at 42°C.

## **2.2. Fermentation and Growth Parameters**

Experimental cultures for growth were established using M17 medium containing as sole carbon sources 0.5 and 3% (w/v) of lactose or sucrose (LM17 or SM17) (Biochemika); adjusted to pH 7.0. Fermentation was initiated by inoculating the media with 1% (1x108 cfu/ml) overnight cultures of the studied strains. After 8h of fermentation at 42°C, viable bacterial counts were performed by serial dilution in peptone-saline water [[(1 g.l-1) and NaCl (8.5 g.l-1)]. Selected dilutions were then plated onto LM17 agar. Plates were incubated at 42°C for 48h; growth is expressed as log cfu/ml and analyzed by comparison to the initial rate of inoculation. Acidification that developed in the cultures was measured with a pH meter (Hanna Instruments, pH210 microprocesor pH meter). All experiments were triplicated.

## **2.3. Acetaldehyde Estimation**

Estimation of acetaldehyde was carried out under the same conditions outlined above with LM17 or SM17. In order to avoid evaporation, it should be noted that cultures destined for these experiments were prepared in centrifuge tubes hermetically sealed. However, acetaldehyde was determined after 8h of fermentation at 42°C. It was measured by spectrophotometer (Jenway J7305) using an assay kit (R-Biopharm: enzymatic bioanalysis, Germany), based on the reduction of the NAD to NADH in the presence of aldehyde dehydrogenase. All assays were repeated three times.

#### **2.4. Data Analysis**

In all experiments mentioned above, the values of results are the mean ±standard error. Statistical analysis was done with student's test.

## **3. Results and Discussion**

#### **3.1. Growth of Strains in the Presence of Lactose and Sucrose**

Bacteria obtain energy by multiple ways. Most of this energy is used in the biosynthesis of many metabolites or anabolism. Performance of diff erent metabolic reactions depends essentially on the nature of the carbon source and its concentration**.** Thus in this work, the importance of the carbon source was checked with strains BN1, BN2, and BN3 of *S. thermophilus* by studying their growth in the presence of lactose and sucrose. These have been incorporated to M17 medium at fi nal concentration of 0.5 and 3% (w/v). It is well known that *S. thermophilus* exhibits a highly affi nity to grow on lactose**;** sucrose can also be used nevertheless with lower effi ciency than lactose [10-11]. This affi nity towards both carbon sources is related to the presence of a large variety of important genes and enzymatic equipment of sugar metabolism and central carbon pathways [11-12].

The results related to the increase in biomass (log cfu/ml) and pH variations aft er 8h of growth on LM17 and SM17 media are illustrated in fi gures 1 and 2. From these results, it appears that strains displayed a high level of growth with lactose and sucrose used at 3% (w/v). Comparatively to the initial rate of inoculation, the strains showed an average increase in the biomass of 3.20±0.09 and 3.10±0.14 log cfu/ml respectively in the presence of 3% (w/v) of lactose and sucrose. A signifi cant diff erence (P<0.05) in the biomass was only observed between the sucrose used at 0.5 and 3% (w/v).

During the growth, a signifi cant decrease in the pH of the cultures was observed. The pH values decreased, in average, from 7.0 to 4.5 in the presence of 3% (w/v) of both carbon sources (fi gure 2). The pH decreasing over time raises the existence of a specifi c metabolic activity in the diff erent fermentative pathways used by *S. thermophilus*, which acidifi es the medium by the production of lactate or other acids [11]. Furthermore, it is important to point that the level of growth of the strains was not infl uenced by the decrease in pH, even aft er 24h of incubation (data not shown).

**Fig 1.** Mean values of increase in biomass (log cfu/ml) of the strains BN1, BN2 and BN3 after 8h of fermentation at 42°C in LM17 and SM17 media.

**Fig 2.** Mean values of pH measured after 8h of fermentation at 42°C in LM17 and SM17 media inoculated with strains BN1, BN2 and BN3.

#### **3.2. Acetaldehyde Production**

The ability to produce acetaldehyde from a carbon source was determined in strains BN1, BN2, and BN3 in the presence of 0.5 and 3% (w/v) lactose and sucrose. The acetaldehyde amounts were quantifi ed aft er 8h of incubation at 42°C (fi gures 3, 4). The results indicated clearly that the three strains produced acetaldehyde, but the production appeared closely related to the concentration of carbon source and strain-dependent. The amounts of acetaldehyde formed by the strains were proportional to the concentration of carbon source. In the presence of 3% (w/v) of either lactose or sucrose, the strains formed a high amount of acetaldehyde compared to the 0.5% (fi gures 3, 4**),** with the exception of the strain BN2, which formed about 83.50±4.90 and 85.20±4.00 µ mol respectively with 0.5% and 3% (w/v) of sucrose (P> 0.05). However, the most potent acetaldehyde producer was BN1 strain. It was capable of producing up to 205±75µmol and 218±90µmol of acetaldehyde with 3% (w/v) of lactose and sucrose respectively. This ratio was signifi cantly higher (P <0.01) than the one produced by the BN2 and BN3 strains, which produced 70.50±3.70, 85.20±4.00, 54±12 and 80±7µmol, respectively. These results indicate that there is variability in the amounts of acetaldehyde formed by the strains of *S. thermophilus* under study. This variability has been widely reported by many authors [3-13]**.** Moreover, Ayhan et al. [13] noticed a large variability in the amounts of acetaldehyde produced by 30 strains of *S. thermophilus*. Chaves et al. [3] signaled that the production of acetaldehyde appeared to be strain specifi c and variable. The same authors reported that the high levels of acetaldehyde were obtained when L-threonine was added to the culture medium. This observation was also reported by Bennama et al**.** [4].

**Fig 3.** Acetaldehyde amounts (μmol) formed by the strains BN1, BN2 and BN3 after 8h of fermentation at 42°C in LM17 medium.

\*\*: Highly signifi cant diff erence (P<0.01) compared to mean values of acetaldehyde obtained with BN2 and BN3 strains.

\*: No signifi cant diff erence.

**Fig 4.** Acetaldehyde amounts (μmol) formed by the strains BN1, BN2 and BN3 after 8h of fermentation at 42°C in SM17 medium.

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of acetaldehyde in milk fermented with *S. thermophilus.* They confirmed that the major production of acetaldehyde was related to the glycolitic pathway. These observations explain the origin of acetaldehyde amounts formed by strains under study especially by BN1 strain. Furthermore, Oïzer and Atasoy [14] reported that lactose hydrolysis induced by the β-galactosidase caused a significant increase in the level of acetaldehyde in yoghurt samples prepared using viscous starter cultures. The results of this study show that production of acetaldehyde by BN1 strain seemed efficient with lactose and sucrose, whereas for BN3 strain, the production was more

In this study, it was found that strains synthesized significant amounts of acetaldehyde in the presence of 3% (w/v) lactose and sucrose. However, with 0.5% (w/v) small quantities were formed. The results reveal that acetaldehyde production was strain-specific and influenced by the concentration of carbon source added to the medium. Following these promising findings, it appears that depending on the strains of *S. thermophilus*, a determined concentration of carbon source constitutes one of the optimal conditions for the synthesis of acetaldehyde in this thermophilic species. This property, mainly for sucrose, could be useful in dairy technology to enhance

[1] Tamime, A. Y., 2002. Fermented milks: a historical food with modern applications-a review.

[2] Delorme, C., 2008. Safety assessment of dairy microorganisms: *Streptococcus thermophilus*,

[3] Chaves, A.C.S.D., M. Fernandez, A.L.S. Lerayer, I. Mierau, M. Kleerebezem, and J. Hugenholtz, 2002. Metabolic Engineering of Acetaldehyde production by *Streptococcus thermophilus.* Applied

[4] Bennama, R., Rechidi-Sidhoum, N. and A. Bensoltane, 2011. Effect of threonine on growth and acetaldehyde production by *Streptococcus thermophilus*. World Applied Sciences Journal, 15 (2):

[5] Ott, A., J. E. Germond, and A. Chaintreau, 2000. Origin of acetaldehyde during milk fermentation using 13C-labeled precursors. Journal of Agriculture and Food Chemistry, 48: p. 1512-1517. [6] Bongers, S. R., M.H.N. Hoefnagel, and M. Kleerebezem, 2005. High-Level acetaldehyde Production in *Lactococcus lactis* by metabolic engineering. Applied and Environmental

[7] Ott, A., L.B. Fay, and A. Chaintreau, 1997. Determination and origin of the aroma impact compounds of yogurt flavour. Journal of Agricultural and Food Chemistry, 45: p. 850-858. [8] Bennama, R., M. Fernández, V. Ladero, M. A. Alvarez, N. Rechidi-Sidhoum, and A. Bensoltane, 2012. Isolation of an exopolysaccharide-producing *Streptococcus thermophilus* from Algerian raw

important with 3% (w/v) of sucrose.

natural production of this aroma compound by *S. thermophilus*.

European Journal of Clinical Nutrition, 56 (4): p. S2-S15.

and Environmental Microbiology, 68 (11): p. 5656-5662.

International Journal of Food Microbiology, 126: p. 274- 277.

cow milk. European Food Research Technology, 234: p. 119-125.

**4. Conclusion**

**5. References**

p. 160-163.

Microbiology, 71 (2): p. 1109-1113.

Turkey, September 10-12, 2012

<sup>227</sup> ISALS

\*\*: Highly signifi cant diff erence (P<0.01) compared to mean values of acetaldehyde obtained with BN2 and BN3 strains.

\*: No signifi cant diff erence.

According to Ott et al. [5]**,** the production of acetaldehyde in milk by lactic acid bacteria seems to be strain-dependent too. These authors also showed that glucose appeared as the main precursor

## **4. Conclusion**

In this study, it was found that strains synthesized significant amounts of acetaldehyde in the presence of 3% (w/v) lactose and sucrose. However, with 0.5% (w/v) small quantities were formed. The results reveal that acetaldehyde production was strain-specific and influenced by the concentration of carbon source added to the medium. Following these promising findings, it appears that depending on the strains of *S. thermophilus*, a determined concentration of carbon source constitutes one of the optimal conditions for the synthesis of acetaldehyde in this thermophilic species. This property, mainly for sucrose, could be useful in dairy technology to enhance natural production of this aroma compound by *S. thermophilus*.

#### **5. References**


[9] Terzaghi, B. E., and W. E. Sandine, 1975. Improved medium for lactic streptococci and their bacteriophages. Applied and Environmental Microbiology, 29: p. 807-813.

International Conference on Applied Life Sciences (ICALS2012)

**Characterization of** *Quercus* **Species Distributed** 

, Mohammad H. Brake<sup>2</sup>

**in Jordan Using Molecular Markers**

, Jamil N. Lahham<sup>1</sup>

3 Plant Production Department, Collage of Food and Agricultural Sciences, King Saud

Genetic diversity among 25 natural populations of three different species of *Quercus* in Jordan at molecular levels using Random Amplified Polymorphic DNA (RAPD) primers was assessed. Significant molecular variations among and within 25 *Quercus* populations were estimated. Twenty-seven polymorphic markers and 5917 scored bands were generated using six RAPD primers. Based on RAPD data, the populations were grouped together in the same cluster according to species regardless to local of collections. This study has emphasized the ability of the molecular markers in the determining the genetic diversity among and within the populations of *Quercus* and the resulted high genetic variability could be utilized in implications of improving

1 Department of Biological Sciences, Faculty of Science, Yarmouk University

conservation, restoration, and reforestation strategies of *Quercus* in Jordan.

**Keywords:** Quercus spp, genetic diversity, RAPD markers, conservation, restoration

*Quercus* L. (Oak) is one of the exceptionally important woody genera worldwide. It is a large genus in the family Fagaceae with about 600 species growing in a wide range of habitats and distributed in temperate and subtropical regions of the northern hemisphere (Yilmaz et al. 2011). Members of the genus grow as shrubs and trees and form prominent deciduous forests or evergreen woodlands with a range of distribution extending from cold latitudes to tropical Asia and

In Jordan, the genus constitutes an important component of the forest ecosystems in the Mediterranean topographic zone. Three species of the genus are known to occur naturally in this region with a general range of distribution extending from Aum Qais in the North to Tafilah in the South. These species are *Quercus calliprinos L., Quercus ithaburensis, and Quercus infectoria* (Kasapligil 1956, Long 1957, Zohary 1961; 1962; 1973). *Q. calliprinos* is the most widespread and evergreen species; distributed throughout the Mediterranean region from Ajloun in the North through Salt and Fuhais to Tafilah and Shobak in the South. *Q. infectoria* is the least abundant and deciduous species; restricted largely to the Northern parts of the country. *Q. ithaburensis* is intermediate in terms of distribution and abundance; it forms deciduous forests in the Northern

2 Science Department, Faculty of Science, Jerash University

Mohammad S. Jawarneh<sup>1</sup>

Hussein M. Migdadi<sup>3</sup>

University, Saudi Arabia

**1. Introduction**

the Americas (Manos et al. 1999).

**Abstract**

© 2012 Jawarneh 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.

Turkey, September 10-12, 2012

, Ahmad Ali El-Oqlah<sup>1</sup>

, Riad Muhidat<sup>1</sup>

<sup>229</sup> ISALS

,


## **Characterization of** *Quercus* **Species Distributed in Jordan Using Molecular Markers**

Mohammad S. Jawarneh<sup>1</sup> , Mohammad H. Brake<sup>2</sup> , Riad Muhidat<sup>1</sup> , Hussein M. Migdadi<sup>3</sup> , Jamil N. Lahham<sup>1</sup> , Ahmad Ali El-Oqlah<sup>1</sup>

1 Department of Biological Sciences, Faculty of Science, Yarmouk University

2 Science Department, Faculty of Science, Jerash University

3 Plant Production Department, Collage of Food and Agricultural Sciences, King Saud University, Saudi Arabia

#### **Abstract**

Genetic diversity among 25 natural populations of three different species of *Quercus* in Jordan at molecular levels using Random Amplified Polymorphic DNA (RAPD) primers was assessed. Significant molecular variations among and within 25 *Quercus* populations were estimated. Twenty-seven polymorphic markers and 5917 scored bands were generated using six RAPD primers. Based on RAPD data, the populations were grouped together in the same cluster according to species regardless to local of collections. This study has emphasized the ability of the molecular markers in the determining the genetic diversity among and within the populations of *Quercus* and the resulted high genetic variability could be utilized in implications of improving conservation, restoration, and reforestation strategies of *Quercus* in Jordan.

**Keywords:** Quercus spp, genetic diversity, RAPD markers, conservation, restoration

## **1. Introduction**

*Quercus* L. (Oak) is one of the exceptionally important woody genera worldwide. It is a large genus in the family Fagaceae with about 600 species growing in a wide range of habitats and distributed in temperate and subtropical regions of the northern hemisphere (Yilmaz et al. 2011). Members of the genus grow as shrubs and trees and form prominent deciduous forests or evergreen woodlands with a range of distribution extending from cold latitudes to tropical Asia and the Americas (Manos et al. 1999).

In Jordan, the genus constitutes an important component of the forest ecosystems in the Mediterranean topographic zone. Three species of the genus are known to occur naturally in this region with a general range of distribution extending from Aum Qais in the North to Tafilah in the South. These species are *Quercus calliprinos L., Quercus ithaburensis, and Quercus infectoria* (Kasapligil 1956, Long 1957, Zohary 1961; 1962; 1973). *Q. calliprinos* is the most widespread and evergreen species; distributed throughout the Mediterranean region from Ajloun in the North through Salt and Fuhais to Tafilah and Shobak in the South. *Q. infectoria* is the least abundant and deciduous species; restricted largely to the Northern parts of the country. *Q. ithaburensis* is intermediate in terms of distribution and abundance; it forms deciduous forests in the Northern

© 2012 Jawarneh 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.

and middle parts of the country, mainly around cities of Irbid, Jarash, Salt, and Fuheis. Information on levels of genetic diversity within and among populations of Quercus species in Jordan per se is lacking. Knowledge of the genetic variation of this important genus provides a robust framework for follow-up systematic studies and facilitates its use in genetic conservation and rehabilitation. In addition, this information will help understand the dynamics of the population genetics of *Quercus*, its evolutionary trends, and its responses to changes in the environment.

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generated a total of 27 polymorphic markers (alleles). In total, 5917 data points (bands) could be scored with an average of 986.2 bands per primer pair across the genotypes, thereby confirming the high multiplex ratio expected for the RAPDs. The ability of different primer to generate RAPD markers varied from 4 to 5 markers with an average of 4.5 markers per primer pair across all genotypes. On a per-population basis, the number of markers generated by the primer pairs ranged from 32.4 for OPD 17 to 47.9 for OPA 20 with an average of 39.4 markers per primer. The percentage of polymorphic among the primers generated was100% polymorphic markers.

Polymorphic bands ranged in size from 250 to 790 bp. The size out of this range was not considered in the analysis). The densely stained markers were considered in scoring. The total bands for each primer ranged from 810 for primer OPD-17 to 1197 bands for OPA-20 using 389 plants

> **Polymorphic markersc**

OPA 12 4 43.4 4 100 280-700 1086 OPA 17 5 42.0 5 100 250-750 1050 OPA 19 4 35.5 4 100 300-790 887 OPA 20 5 47.9 5 100 250-680 1197 OPB 5 4 35.5 4 100 400-780 887 OPD 17 5 32.4 5 100 380-750 810 Total 27 27 5917 Average 4.5 39.4 4.5 100 986.2

a Total number of differently sized RAPD markers amplified across all 25 populations, b Average number of RAPD bands scored per population, c Total number of RAPD markers found to be polymorphic across the 25populations, d

Based on the Jaccard coefficients index (Jaccard, 1974), a genetic similarity matrix was constructed using the RAPD data to assess the genetic relatedness among the 25 *Quercus* populations. The within population means were used to construct the similarity matrix. The mean similarity indices ranged from 0.24 between population 4 and population 11 to 0.84 within the population

The results showed based on RAPD product data that populations from the different locality represent species tend to grouped together in the same cluster figure (1). The species of the 25 *Quercus* populations clustered into two main clusters; the first cluster consists of the populations belong to *Quercus ithaburensis* and the second cluster consists of the populations belong to the

**Polymorphic markers %**

**Size range (Bp)**

**Total no. of bandsd**

representing 25 *Quercus* populations.

**markersa**

**Average bandsb**

**Table 1.** The features of RAPD primers selected in *Quercus* genetic diversity

Total number of RAPD bands ( data points) scored for all populations

number 8 and 0.48 for over all populations.

*Quercus infectoria* and *Quescus Calliprinos* species.

**Primer Total** 

Turkey, September 10-12, 2012

<sup>231</sup> ISALS

In this study, the classical technique of random amplified polymorphic DNA (RAPD) is employed to investigate for the first time levels of genetic diversity of natural populations of the genus *Quercus* in Jordan. In Palestine the *Quercus boissieri* Reut. an associated species within the *Quercus calliprinos*–Pistacia palestine association of the Mediterranean sclerophyllous broad-leaf forests. The use of random amplified polymorphic DNA (RAPD) markers,within- and amongpopulations genetic diversity of *Quercus boissieri* in Palestine , as influenced by geo-climatic parameters (Schiller, et al., 2006).

## **2. Materials and methods**

Samples of the three different Jordanian *Quercus* species were collected from the field during October, November and December of 2008. Samples were randomly selected from different populations distributed over various geographical regions in Jordan. Samples were identified cautiously and taxa names were confirmed following Zohary, (1962). Fresh leaf samples were stored at -20 for DNA extraction.

Leaves collected from each individual tree were manually ground in liquid nitrogen with a mortar and pestle, to a fine powder and DNA was extracted according to the protocol of Genomic DNA Purification Kit form Fermentas and then the DNA samples were stored at -20ºC until use.

Six DNA samples were tested using 60 primers from Operon kits A,B and D. Primers that amplified consistently reproducible polymorphisms were selected and used to analyze all of the 25 *Quercus* populations. Only six RAPD primers were used to amplify the 25 populations.

RAPD reactions were performed in total volume of 15 µl according to standared protocol (Sambrook *et al.,*1989). The amplification products were loaded using 1.8% agarose gel electrophoresis at 100 volts for 2 hrs using horizontal gel electrophoresis apparatus. The amplified products were visualized and documented by gel documentation system. 100 bp ladders were used as a DNA marker to estimate the molecular weights of the amplified products.

Data generated from RAPD analysis were analyzed using Jaccard similarity coefficients (Jaccard, 1908). These similarity coefficients were used to construct dendrograms using the unweighted pair group method with arithmetic average (UPGMA) employing SAHN (sequential, agglomerative, hierarchical, and nested clustering) using the NTSYSpc (ver.2.10) program, (Rohlf, 2005).

#### **3. Results**

A total of 60 RAPD primers were evaluated for their ability to amplify polymorphic regions from six randomly selected populations. Of the 60 primers, 6 amplified consistently reproducible polymorphisms, and so these were used to analyze all of the 25 *Quercus* populations. The features of the primers across the tested populations are summarized in Table 1. The 6 primer generated a total of 27 polymorphic markers (alleles). In total, 5917 data points (bands) could be scored with an average of 986.2 bands per primer pair across the genotypes, thereby confirming the high multiplex ratio expected for the RAPDs. The ability of different primer to generate RAPD markers varied from 4 to 5 markers with an average of 4.5 markers per primer pair across all genotypes. On a per-population basis, the number of markers generated by the primer pairs ranged from 32.4 for OPD 17 to 47.9 for OPA 20 with an average of 39.4 markers per primer. The percentage of polymorphic among the primers generated was100% polymorphic markers.

Polymorphic bands ranged in size from 250 to 790 bp. The size out of this range was not considered in the analysis). The densely stained markers were considered in scoring. The total bands for each primer ranged from 810 for primer OPD-17 to 1197 bands for OPA-20 using 389 plants representing 25 *Quercus* populations.


**Table 1.** The features of RAPD primers selected in *Quercus* genetic diversity

a Total number of differently sized RAPD markers amplified across all 25 populations, b Average number of RAPD bands scored per population, c Total number of RAPD markers found to be polymorphic across the 25populations, d Total number of RAPD bands ( data points) scored for all populations

Based on the Jaccard coefficients index (Jaccard, 1974), a genetic similarity matrix was constructed using the RAPD data to assess the genetic relatedness among the 25 *Quercus* populations. The within population means were used to construct the similarity matrix. The mean similarity indices ranged from 0.24 between population 4 and population 11 to 0.84 within the population number 8 and 0.48 for over all populations.

The results showed based on RAPD product data that populations from the different locality represent species tend to grouped together in the same cluster figure (1). The species of the 25 *Quercus* populations clustered into two main clusters; the first cluster consists of the populations belong to *Quercus ithaburensis* and the second cluster consists of the populations belong to the *Quercus infectoria* and *Quescus Calliprinos* species.

International Conference on Applied Life Sciences (ICALS2012)

*Quercus ithaburensis* genetic material represented by the three main assemblages of its distribu-

In this study it was possible to show that the amplification products from six random primer RAPD assay were sufficient to discriminate among and within population of *Quercus* species for each location. Also, the assay was useful in discriminating among plants of the same location. The ability to distinguish between closely related individuals was simply a function of the observed number of RAPD bands.The results of RAPD markers were compared in a genetic diversity of *Quercus* species the differences in the level of polymorphism detected by the markers and evaluating the potential of these markers in assessing the genetic variation in 25 population of *Quercus* to three species *Quercus ithaburensis*, *Quercus infectora*, and *Quercus calliprinos*. that matching result come into view the morphological result. The classes of molecular markers adopted in this study deserve additional discussion. The key of the success of multilocus PCR-based markers has to found in their high multiplex ratio. In fact, owing to their own genetic nature, RAPD assays detect simultaneously many loci randomly distributed in the genome. Moreover, compared to SSRs, these marker systems allow a more precise estimate of marker allele frequencies at single

From the similarity matrix the highest values of similarity between populations was found between the populations Aqraba, Makhraba, Ashah and Umm Qiass these population are cluster together in the hierarchical cluster constructed on the base of the genetic similarity values. These population belong to the same species *Quercus ithaburensis* and the population are found in the same region and located at the same elevation. The results obtain confirm once again the great versatility, reliability and precision of the techniques based on molecular markers, which can be used to aid the classical evaluation of the differentiation between population based on the observation of morphological characteristics. Our molecular results also in agreement of those Cottrell et al. (2003) who used six microsatellite markers found high expected heterozygosity values in Quercus robur and Quercus petraea populations. ranging from 0.87 to 0.92 and from 0.76 to 0.82 in Q. crispula populations (Ohsawa et al., 2007d ). And with those of SCHILLER et al 2003 who found that Quercus aegilops L. ssp. ithaburensis populations were aggregated according

In conclusion, the variations among *Quercus* species studied at molecular levels indicated that there is a high variation among these populations and the RAPD technique was useful for studying genetic variability of *Quercus*. The wide geographical distribution of *Quercus* populations across different environments means that this species has good genetic resources to fill the gap between northern natural distribution sites with the southern natural distribution site. *In-situ* as well as *ex-situe* conservation, restoration, and reforestation should be done in the nearest popula-

[1] Awishi M (1967) Taxonomic–geographic studies in the Middle- East oaks. Dissertation, Hebrew

loci and faster estimate of population polymorphisms over several loci.

tion in this region.

to main geographic regions.

**5. Refrences**

tions within the same geographic region.

University of Jerusalem

Turkey, September 10-12, 2012

<sup>233</sup> ISALS

**Figure 1.** Dendrogram of 25 *Quercus* L. (1=Ber Adbagat, 2= Al-Rashadia, 3= Heisha, 4= Dana,5= Achtifina, 6=Ebein, 7= Enbeh,8= Anjara, 9= Rahaba,10= Fuheis,11= Gelad, 12= Kufour Houda, 13= Zobia, 14= Bargesh 15= Ebein, 16= Ashah, 17= Umm Qaiss, 18= Aosra, 19= Jeneen Safa, 20= Aqraba, 21= Kufour Kifya, 22= Makhraba, 23= Alouk, 24= Jobbah, 25= Gelad) generated by UPGMA cluster analysis of the genetic similarity values. Adbagat, 2=

#### **4. Discussion**

The 25 populations analyzed in this study represented *Quercus* species from a wide range of geographical areas in Jordan**.** In this work we followed the nomenclature used in the previous workers (Kasapligil 1956, Long 1957; Zohary 1962, 1973). Our results showed that Jordan has at least three *Quercus* species and each has its morphological characters.

The current study uses the RAPD-PCR based protocol to assess genetic variability of the *Quercus* species in Jordan. Genetic diversity determines the adaptive potential of a species and is an essential component of the stability of ecosystems. Analysis of within- and among-population genetic diversity is a fundamental step in the development of strategies for conservation of genetic resources and, consequently, of their adaptability. With its oak forests that comprise *Quercus ithaburensis*, *Quercus boissieri*, and *Quercus calliprinos*, is in a geographically peripheral position to the main area of distribution of these species in the Mediterranean basin (Awishi, 1967). According to (Safriel et al. 1994), unlike core populations, peripheral ones may be tolerant to environmental extremes and changes because of their higher genetic variability, which has resulted from fluctuating selection. It is also likely that peripheral populations evolve resistance to extreme conditions; therefore, they should be treated as a biogenetic resource, to be used for rehabilitation and restoration of damaged ecosystems. Owing to their long life cycle, forest trees are among the species that cannot migrate or adapt quickly enough to cope with the rapid changes imposed on the environment by human activity, and this could create ecological and forest management problems. Thus, attention should be given to in situ and ex situ conservation of the varieties of 1956, also Owing the

*Quercus ithaburensis* genetic material represented by the three main assemblages of its distribution in this region.

In this study it was possible to show that the amplification products from six random primer RAPD assay were sufficient to discriminate among and within population of *Quercus* species for each location. Also, the assay was useful in discriminating among plants of the same location. The ability to distinguish between closely related individuals was simply a function of the observed number of RAPD bands.The results of RAPD markers were compared in a genetic diversity of *Quercus* species the differences in the level of polymorphism detected by the markers and evaluating the potential of these markers in assessing the genetic variation in 25 population of *Quercus* to three species *Quercus ithaburensis*, *Quercus infectora*, and *Quercus calliprinos*. that matching result come into view the morphological result. The classes of molecular markers adopted in this study deserve additional discussion. The key of the success of multilocus PCR-based markers has to found in their high multiplex ratio. In fact, owing to their own genetic nature, RAPD assays detect simultaneously many loci randomly distributed in the genome. Moreover, compared to SSRs, these marker systems allow a more precise estimate of marker allele frequencies at single loci and faster estimate of population polymorphisms over several loci.

From the similarity matrix the highest values of similarity between populations was found between the populations Aqraba, Makhraba, Ashah and Umm Qiass these population are cluster together in the hierarchical cluster constructed on the base of the genetic similarity values. These population belong to the same species *Quercus ithaburensis* and the population are found in the same region and located at the same elevation. The results obtain confirm once again the great versatility, reliability and precision of the techniques based on molecular markers, which can be used to aid the classical evaluation of the differentiation between population based on the observation of morphological characteristics. Our molecular results also in agreement of those Cottrell et al. (2003) who used six microsatellite markers found high expected heterozygosity values in Quercus robur and Quercus petraea populations. ranging from 0.87 to 0.92 and from 0.76 to 0.82 in Q. crispula populations (Ohsawa et al., 2007d ). And with those of SCHILLER et al 2003 who found that Quercus aegilops L. ssp. ithaburensis populations were aggregated according to main geographic regions.

In conclusion, the variations among *Quercus* species studied at molecular levels indicated that there is a high variation among these populations and the RAPD technique was useful for studying genetic variability of *Quercus*. The wide geographical distribution of *Quercus* populations across different environments means that this species has good genetic resources to fill the gap between northern natural distribution sites with the southern natural distribution site. *In-situ* as well as *ex-situe* conservation, restoration, and reforestation should be done in the nearest populations within the same geographic region.

#### **5. Refrences**

**Figure 1.** Dendrogram of 25 *Quercus* L. (1=Ber Adbagat, 2= Al-Rashadia, 3= Heisha, 4= Dana,5= Achtifina, 6=Ebein, 7= Enbeh,8= Anjara, 9= Rahaba,10= Fuheis,11= Gelad, 12= Kufour Houda, 13= Zobia, 14= Bargesh 15= Ebein, 16= Ashah, 17= Umm Qaiss, 18= Aosra, 19= Jeneen Safa, 20= Aqraba, 21= Kufour Kifya, 22= Makhraba, 23= Alouk, 24= Jobbah, 25= Gelad) generated by UPGMA cluster analysis of the genetic similarity

The 25 populations analyzed in this study represented *Quercus* species from a wide range of geographical areas in Jordan**.** In this work we followed the nomenclature used in the previous workers (Kasapligil 1956, Long 1957; Zohary 1962, 1973). Our results showed that Jordan has at

The current study uses the RAPD-PCR based protocol to assess genetic variability of the *Quercus* species in Jordan. Genetic diversity determines the adaptive potential of a species and is an essential component of the stability of ecosystems. Analysis of within- and among-population genetic diversity is a fundamental step in the development of strategies for conservation of genetic resources and, consequently, of their adaptability. With its oak forests that comprise *Quercus ithaburensis*, *Quercus boissieri*, and *Quercus calliprinos*, is in a geographically peripheral position to the main area of distribution of these species in the Mediterranean basin (Awishi, 1967). According to (Safriel et al. 1994), unlike core populations, peripheral ones may be tolerant to environmental extremes and changes because of their higher genetic variability, which has resulted from fluctuating selection. It is also likely that peripheral populations evolve resistance to extreme conditions; therefore, they should be treated as a biogenetic resource, to be used for rehabilitation and restoration of damaged ecosystems. Owing to their long life cycle, forest trees are among the species that cannot migrate or adapt quickly enough to cope with the rapid changes imposed on the environment by human activity, and this could create ecological and forest management problems. Thus, attention should be given to in situ and ex situ conservation of the varieties of

least three *Quercus* species and each has its morphological characters.

values.

**4. Discussion** 

[1] Awishi M (1967) Taxonomic–geographic studies in the Middle- East oaks. Dissertation, Hebrew University of Jerusalem

[2] Aykut Y, Uslu E, Babac M (2011) Cytogenetic studies on Quercus L. (Fagaceae) species belonging toIlex and Cerris section in Turkey. CARYOLOGIA 64: 297-301

International Conference on Applied Life Sciences (ICALS2012)

, L Karimzadeh<sup>1</sup>

**Honey Bee Venom Modulates Hyperglycemia** 

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

2 Department of Biology, Faculty of Agriculture and Basic Science, Payamnoor University, Tehran, Iran.

Polycystic Ovarian Syndrome (PCOS) is inflammatory disease characterized by hyperandrogenemia, hyperthecosis and chronic anovulation. Honey bee venom (HBV) contains a variety of biologically active components having various pharmaceutical properties. This study was designed to detect the possibility of HBV application as an anti-inflammatory therapeutic agent. To induce PCOS, 2mg/100gr B.W Estradiol Valerate (EV) was subcutaneously injected to induce PCOS in mature Wistar rats then ovaries and serum from three groups of EV-induced PCOS, HBV-treatment and normal intact animals were collected for histological comparison and blood sugar test. As a result, a significant increase in ovarian weight was observed in experimental group rather than controls. Furthermore, in HBV-treated group a significant decrease was observed in ovary weight comparing with experimental group (P<0.01). The results obtained from Chemo Luminesance Immuno Assay (CLIA) declared that testosterone and Estradiol levels in experimental group significantly increased (P<0.001). These hormones were decreased in animals treated with HBV. Blood sugar level showed reduction in HBV-treated rats. Thickness of theca layer, number and diameter of cysts significantly decrease in HBV group comparing to PCOS group. Moreover, corpus luteum, as a sign of ovulation, was observed in HBV-treated ovaries. In conclusion our results suggest that beneficial effect of HBV against PCOS may be mediated by the inhibitory

**Ovarian Syndrome-Induced Wistar Rats**

, F Poyanmanesh<sup>2</sup>

M Nabiuni<sup>1</sup>

Tehran, Iran.

**Abstract**

effect of HBV on TNF-α level.

**1. Introduction**

, S Nasri<sup>2</sup>

**in Response to Hyperandrogenism in Polycystic** 

© 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.

**Keywords**: Polycystic ovarian syndrome, honey bee venom, blood sugar, theca layer

Polycystic Ovarian Syndrome (PCOS) is an inflammatory disease characterized by hyper androgenemia, hyperthecosis, hyperglycemia and chronic anovulation(1-6). Honey bee venom (HBV) contains a variety of biologically active components like peptides (Melittin and Apamin), enzymes and biologically active amines (histamine, epinephrine. It has shown that HBV has analgesic, anti-cancer and anti-inflammatory activity. Melittin, the major active ingredient of BV, has been reported to induce apoptosis and to possess anti-proliferation effects (7-9). TNF-α is a key inflammatory stimulus which plays a main role in regulating normal activity of ovary in follicular growth and luteal stages. It's over expression in adipose tissue leads to obesity and

Turkey, September 10-12, 2012

, Z Nazari<sup>1</sup>

<sup>235</sup> ISALS


## **Honey Bee Venom Modulates Hyperglycemia in Response to Hyperandrogenism in Polycystic Ovarian Syndrome-Induced Wistar Rats**

M Nabiuni<sup>1</sup> , S Nasri<sup>2</sup> , F Poyanmanesh<sup>2</sup> , L Karimzadeh<sup>1</sup> , Z 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 Agriculture and Basic Science, Payamnoor University, Tehran, Iran.

#### **Abstract**

Polycystic Ovarian Syndrome (PCOS) is inflammatory disease characterized by hyperandrogenemia, hyperthecosis and chronic anovulation. Honey bee venom (HBV) contains a variety of biologically active components having various pharmaceutical properties. This study was designed to detect the possibility of HBV application as an anti-inflammatory therapeutic agent. To induce PCOS, 2mg/100gr B.W Estradiol Valerate (EV) was subcutaneously injected to induce PCOS in mature Wistar rats then ovaries and serum from three groups of EV-induced PCOS, HBV-treatment and normal intact animals were collected for histological comparison and blood sugar test. As a result, a significant increase in ovarian weight was observed in experimental group rather than controls. Furthermore, in HBV-treated group a significant decrease was observed in ovary weight comparing with experimental group (P<0.01). The results obtained from Chemo Luminesance Immuno Assay (CLIA) declared that testosterone and Estradiol levels in experimental group significantly increased (P<0.001). These hormones were decreased in animals treated with HBV. Blood sugar level showed reduction in HBV-treated rats. Thickness of theca layer, number and diameter of cysts significantly decrease in HBV group comparing to PCOS group. Moreover, corpus luteum, as a sign of ovulation, was observed in HBV-treated ovaries. In conclusion our results suggest that beneficial effect of HBV against PCOS may be mediated by the inhibitory effect of HBV on TNF-α level.

**Keywords**: Polycystic ovarian syndrome, honey bee venom, blood sugar, theca layer

## **1. Introduction**

Polycystic Ovarian Syndrome (PCOS) is an inflammatory disease characterized by hyper androgenemia, hyperthecosis, hyperglycemia and chronic anovulation(1-6). Honey bee venom (HBV) contains a variety of biologically active components like peptides (Melittin and Apamin), enzymes and biologically active amines (histamine, epinephrine. It has shown that HBV has analgesic, anti-cancer and anti-inflammatory activity. Melittin, the major active ingredient of BV, has been reported to induce apoptosis and to possess anti-proliferation effects (7-9). TNF-α is a key inflammatory stimulus which plays a main role in regulating normal activity of ovary in follicular growth and luteal stages. It's over expression in adipose tissue leads to obesity and

© 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.

insulin-resistance in humans and rodents. This factor by stimulating mitotic activity in undifferentiated theca cells and increasing steroidogenic cells causes PCOS (10-13). Whereas the antiinflammatory and anticancer effects of HBV were proved, we examined sugar levels in rats with PCOS before and after treatment via HBV. We also measured plasma testosterone and theca layer thickness, the commonly used index of PCOS (1). We hypothesized that HBV can modulate hyperglycemia in bee venom-treated rat with PCOS in response to androgenemia, compared with age-matched controls.

International Conference on Applied Life Sciences (ICALS2012)

sugar level (P<0.01), and its reduction in rats treated with honey bee venom was outstanding

**Diagram 1.** Diff erent sugar level in control and polycystic rats (n=10). In polycystic group, a signifi cant in-

**Cont SD SEM PCOS SD SEM PCOS+BV SD SEM con vs pcos con vs** 

(pg/ml) 14.5 1.958 0.6191 54.778 8.599 2.866 21.889 4.485 1.495 \*\*\* P<0.001 \* P<0.05 \*\*\* P<0.001

(ng/dl) 62.5 9.925 3.138 345.9 112.99 35.73 115.9 39.145 12.379 \*\*\* P<0.001 ns P>0.05 \*\*\* P<0.001

weight (mg) <sup>13</sup> 3.232 1.022 20.5 4.577 1.447 <sup>15</sup> 4.714 1.491 \*\* P<0.01 ns P>0.05 \* P<0.05

**Table 1.** Bee venom treatment eff ects in polycystic ovarian syndrome (PCOS). Baseline parameters of polycys-

In order to determine follicular development, follicles were classifi ed based on morphology and diameter into 6 groups consisting of: primordial, primary and preantral (<600µm), antral (600- 1000µm), cystic follicles, and corpus luteum. Decrease in the number of primary follicles, antral follicles and corpus luteums was signifi cant with P<0.001, in primordial follicles with P<0.01, and in preantral follicles with P<0.05. In PCOS ovaries, some large cystic follicles with thick theca

In rats treated with honey bee venom, the number of primordial and preantral follicles and corpus luteums increased, whereas, the number of cysts and thickness of theca layer in antral follicles decreased, which these changes were signifi cant in comparison with sham group. Also, some corpus luteums were observed in this group which was considered as a sign of relative

99.8 11.144 3.524 157.2 44.183 13.972 110.7 23.281 7.362

tic ovarian syndrome (PCOS) rats (n=10) and control (n=10) and bee venom –treated rats (n=10).

layer were observed. In this group, no corpus luteum as a sign of ovulation was seen.

improvement in PCOS ovaries. (Diagram 2 and 3).

crease was observed in sugar level. \*\*\*P< 0.001, PCOS vs. control, HBV vs. PCOS group.

(P<0.05). (Diagram1).

Estradiol

Testosteron

Ovarian

Theca layer -late antral follicles (micrometer)

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**pc+bv**

ns P>0.05

\*\*\* P<0.001 **pcos vs pc+bv**

> \*\* P<0.01

## **2. Materials and Methods**

Experiments were performed on Female Wistar rats (170±20g). Before and during the experiment they were housed in special cages with a standard space and under controlled cycle of light and darkness (lights on from 06:00 to 20:00), humidity 55±15% and temperature range of 20-24˚c and free access to water and commercial food (Behparvar Com., Iran). Induction of PCOS was administered using 1mg/100gr B.W intramuscular injection of EV. After verifying the induction of PCOS, experimental group was divided into two groups: PCOS group and PCOS+BV group. PCOS+BV received 0.5 mg/kg BV sc for 14 days, continuously (7). PCOS group in this period of time received physiological saline solution. At around 09:00 am, trunk blood was collected and the serums were separated using 4000 RPM centrifuge for 10 min. The ovaries were separated from the twisted oviduct tubes and were placed in bouin fixative for histological analysis; fixed samples were kept in alcohol solutions of 20 to 100% for a period of 45 min for dehydration and afterwards in alcohol/xylen (50:50) and xylen (3 times) for clearing and blocked in paraffin. The samples were sliced in 7 micron thickness using a microtome and the sections were placed on slides previously coated with gelatin and then stained with hematoxylin-eosin for histological observation. Serological analysis was performed to measure sugar level and hormone alterations. Testosterone and estradiol detected by Chemo Luminesance Immuno Assay (CLIA). In order to detect suger, glucose kit (GOD\_PAP 90014) was used. The one-way ANOVA and INSTAT software were used to determine the statistical significance of differences between the values for the experimental and control groups. Data are expressed as means ± standard errors (S.E.M) and the results are taken from at least three independent experiments performed in triplicate. P-values of 0.05 or less were considered statistically significant.

## **3. Results**

The ovaries were also precisely weighted and a significant increase was observed in experimental group rather than controls. Furthermore, in bee venom-treated group a significant decrease was observed in ovary weight comparing with experimental group (P<0.01). The results obtained from CLIA declared that testosterone and Estradiol levels in experimental group significantly increased (P<0.001). These hormones were decreased in animals treated with bee venom, and comparing with animals in control group they were regulated. Reduction observed in testosterone and Estradiol levels was significant with P<0.05. These data including raise in androgens showed that induce of syndrome was absolutely successful. What more is bee venom managed to reduce Estradiol and testosterone.(Table1) sugar level fundamentally adjusts its production, which in this study, PCOS induction by means of Estradiol volerate led to a significant raise in sugar level (P<0.01), and its reduction in rats treated with honey bee venom was outstanding (P<0.05). (Diagram1).

**Diagram 1.** Diff erent sugar level in control and polycystic rats (n=10). In polycystic group, a signifi cant increase was observed in sugar level. \*\*\*P< 0.001, PCOS vs. control, HBV vs. PCOS group.


**Table 1.** Bee venom treatment eff ects in polycystic ovarian syndrome (PCOS). Baseline parameters of polycystic ovarian syndrome (PCOS) rats (n=10) and control (n=10) and bee venom –treated rats (n=10).

In order to determine follicular development, follicles were classifi ed based on morphology and diameter into 6 groups consisting of: primordial, primary and preantral (<600µm), antral (600- 1000µm), cystic follicles, and corpus luteum. Decrease in the number of primary follicles, antral follicles and corpus luteums was signifi cant with P<0.001, in primordial follicles with P<0.01, and in preantral follicles with P<0.05. In PCOS ovaries, some large cystic follicles with thick theca layer were observed. In this group, no corpus luteum as a sign of ovulation was seen.

In rats treated with honey bee venom, the number of primordial and preantral follicles and corpus luteums increased, whereas, the number of cysts and thickness of theca layer in antral follicles decreased, which these changes were signifi cant in comparison with sham group. Also, some corpus luteums were observed in this group which was considered as a sign of relative improvement in PCOS ovaries. (Diagram 2 and 3).

International Conference on Applied Life Sciences (ICALS2012)

observed in ovary after bee venom treatment, can also be considered as a confirmation for this syndrome progress. Our results confirm that Bee venom caused a decrease in follicular theca layer in PCOS rats, which is actually because of increased lipolysis and decreased hypertrophy of this layer. Due to this decrease, the androgens and steroids produced by this layer also decrease and consequently the total levels of serumic estrogen and androgens reduce by honey bee venom. In this regard we have demonstrated that bee venom injection produces a significant

[1] Repaci, A., Gambineri, A., Pasquali, R., 2011. The role of low-grade inflammation in the polycystic

[2] Sathyapalan, T., Atkin, L., 2010. Mediators of Inflammation in Polycystic Ovary Syndrome in

[3] Panidis D, Kita M, Katsikis I, Karkanaki A, Karayannis V, Rousso D., 2006. Μechanisms of infertility in polycystic ovary syndrome. Aristotle University Medical Journal. 33(2), 67-77. [4] Kelly, C., Lyall, H., Petrie, J., Gould, G., Connell, J., Sattar, N., 2001. Low Grade Chronic Inflammation in Women with Polycystic Ovarian Syndrome. J Clin Endocrinol Metab. 86(6),

[5] Benson, S., Janssen, O., Hahn, S., Tan, S., Dietz, T., Mann, K., Pleger, K., Schedlowski, M., Arck, P., Elsenbruch, S., 2008. Obesity, depression, and chronic low-grade inflammation in women with

[6] Baravalle, C., Salvetti, N., Mira, G., Pezzone, N., Ortega, H., 2006. Microscopic characterization of follicular structure in letrosole-induced poly cystic ovarian syndrome in the rat. Archives of

[7] Luo, H., Zuo, X., Li, T., Zhang, J., 2006. Effect of bee venom on adjuvant induced arthritis in rats.

[8] Park, H., Son, D., Lee, C., Choi, M., Lee, U., Song, H., Lee, J., Hong, J., 2007. Melittin inhibits inflammatory target gene expression and mediator generation via interaction with IκB kinase.

[9] Son, D., Lee, J., Lee, Y., Song, H., Lee, C., Hong, J., 2007. Therapeutic application of antiarthritis, pain-releasing, and anti-cancer effects of bee venom and its constituent compounds.

[10] Spaczynski, R., Arici, A., Duleba, A., 1999. Tumor Necrosis Factor-a Stimulates Proliferation of

[11] Norata, G., Tibolla, G., Seccomandi, P., Poletti, A., Catapano, A., 2006. Dihydrotestosterone Decreases Tumor Necrosis Factor- a and Lipopolysaccharide-Induced Inflammatory Response

[12] McGrath, K., McRobb, L., Heather, A., 2008. Androgen therapy and atherosclerotic cardiovascular

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anti-hyperglycemia effect in PCOS Wistar rats.

**5. References**

2453–2455.

medical research. 37, 830-839.

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**Diagram 2.** Diff erent follicular groups in control and polycystic ovaries (n=10). In ovaries of polycystic group, a signifi cant increase and decrease was observed in number of cysts and number of corpus luteums respectively. \*\*\*P< 0.001, \*\*P< 0.01, \*P< 0.05. (PMF, Pre Mordial Follicle; PF, Primary Follicle; PAF, Preantral Follicle, AF, Antral Follicle; CF, Cystic Follicle; CL, Corpus Luteum.)

**Diagram 3.** Diff erent follicular types in ovaries of polycystic and bee-venom treated polycystic group. In polycystic ovaries treated with honey bee venom (n=10), a signifi cant increase was observed in all follicular clusters (except primordial follicles) rather than polycystic group. The primordial follicles were not signifi cantly increased. Moreover, a signifi cant decrease was seen in the number of ovarian cysts. \*\*\*P<0.001, \*\*P< 0.01. (PMF, Pre Mordial Follicle; PF, Primary Follicle; PAF, Preantral Follicle, AF, Antral Follicle; CF, Cystic Follicle; CL, Corpus Luteum.)

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

Insulin sensitivity and hyperglycemia is directly related to androgen levels (14). These fi ndings suggest that hyperandrogenism play a role in the development of insulin resistance and hyperandrogenism in PCOS. It is considered metformin as a treatment for PCOS which has been shown to inhibit the NF-ҡB activation and also reduce sugar level in PCOS woman (15). Then we can also name honey bee venom as a similar factor decreasing sugar level. Histological changes observed in ovary after bee venom treatment, can also be considered as a confirmation for this syndrome progress. Our results confirm that Bee venom caused a decrease in follicular theca layer in PCOS rats, which is actually because of increased lipolysis and decreased hypertrophy of this layer. Due to this decrease, the androgens and steroids produced by this layer also decrease and consequently the total levels of serumic estrogen and androgens reduce by honey bee venom. In this regard we have demonstrated that bee venom injection produces a significant anti-hyperglycemia effect in PCOS Wistar rats.

#### **5. References**


[13] Demissie, M., Lazic, M., Foecking, E., Aird, F., Dunaif, A., Levine, J., 2008. Transient prenatal androgen exposure produces metabolic syndrome in adult female rats. Am J Physiol Endocrinol Metab. 295, 262–268.

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, Mounir Boucenna<sup>2</sup>

,Houria Berrebbah2 , Mohamed Reda Djebar<sup>2</sup>

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, di-

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***.**

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 follow-

**Effects of Heavy Metals on the Snails** *Helix* 

*aspersa* **Bioindicators of the Environment** 

**Pollution for Human Health**

,Amira Atailia<sup>2</sup>

University of Mohammed cherifMessaadia, Souk-Ahras, Algeria 2 Laboratory of Cellular Toxicology. University of Annaba, Algeria

1 Laboratory of Sciences and Technology of water and the environment,

Nedjoud Grara<sup>1</sup>

gestive gland, kidney.

**2. Materiel and Methods**

ing optimal environmental conditions[2].

**2.1. Biological Material** 

**1. Introduction**

Fadila Khaldi<sup>1</sup>

**Abstract**

© 2012 Grara 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.

Turkey, September 10-12, 2012

,

<sup>241</sup> ISALS

