**The Effect of Hydro-Alcoholic Extract of Fenugreek Seeds on Female Reproductive Hormones in Mice**

Mehrdad Modaresi<sup>1</sup> , Behnaz Mahdian<sup>2</sup> , Alireza Jalalizand<sup>3</sup>

1 Department of Agriculture, Khorasgan Branch, Islamic Azad University, Isfahan, IRAN

2 Payam e Noor University, Isfahan Center, Isfahan, IRAN

3 Department of Plant protection, Khorasgan Branch, Islamic Azad University, Isfahan, IRAN

#### **Abstract**

Fenugreek is a plant from Fabeaceae family which is used for many medical plans. This study was conducted to study the effect of fenugreek seed's extract on reproductive system of female *Balb/C* mice .To this, mice's were divided in five groups: control, placebo, and three experimental groups. Each group had ten mice which their estrous cycles were synchronized for starting the study. Control group did not receive any drug. Placebo group received normal saline, and experimental groups were injected in peritoneum by 50,100, and 200 mg/kg extract every day until 20 days. After finishing injection, blood samples were taken. Hormone measuring (including FSH, LH, estradiol, and progesterone) was done using one way variance analysis of SPSS program at 95% probability level. Ovary slides were prepared and were studied using optical microscope. Obtained results, showed significant reduction in FSH, and LH levels and also significant increase in stradiol level in all experimental groups, but progesterone level was increased only in second experimental group. Histology results of ovary showed significant reduction in folliculogenesis of all three experimental groups. Also, increase in number of corpus luteum was highly significant. Furthermore, destruction of ovary tissues was observed in second experimental group. According to results, the extract of fenugreek seed stopped folliculogenesis trend and destroyed ovary tissue which shows its anti fertility effect in female mice.

**Keywords:** Fenugreek, reproductive hormones, mice

#### **1. Introduction**

Traditional medicine is a nature based science which is inherited from ancestors and includes plants, minerals and animal matters which are used as drugs [1] . In traditional medicine, pharmaceutical plants have a special place. Medicinal plant is a plant which has specific effective matters, is used in prevention or cure of illness, and has been mentioned in one of national valid Pharmacopoeia [2]. The importance of pharmaceutical plants is more obvious today and scientists of various countries are trying to identify medicinal plants, their properties and effective matters. Fenugreek is one of the old plants which have a wide range of medicinal properties: reducer of blood sugar and fat, anti diabetes, pain reliever, and anti cancer, increase in sex abil-

© 2012 Modaresi 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.

ity, and increase in milk production and worm killer [3] . Scientific name of fenugreek is *Trigonella foenum -graceum* L. which *trigonou* is derived from Latin and means triangle (because of triangle leaves), and *foenum-graceum* which means Greek hay is because of its different uses in ancient Greek. Also, because two seed pods are produced from main stem's nodes, these plants has been called "bull's horn" or "Goat horn " [4] . This plant is an annual, bush plant with 10 to 50 cm length which is sown in various regions of world like small Asia, Iran, Egypt, Algeria, India, Morocco, Italy, and Spain. The region of this plant has been known west of Asia [5] . Economical products of fenugreek are seeds and leaves, and reproduction of this plant is done by seeds which are sown in clay, calcareous lands in September. Seeds are sown together with clover seeds. Harvesting time is from June to July after harvesting, stems are being cut from the bottom and being dried [6] . This plant is one of the oldest pharmaceutical plants.

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cool place. After 24 hours, erlen contents were mixed completely using a shaker for five minutes. Then, after filtering the solution by filtrative paper and calculating extract residual in solution,

Female mice (*Balb/C*) in weight range of 25-40 g were taken from animal division of Isfahan Medical University. Samples were kept in similar conditions of water, food, light, temperature and moisture to environment adaption. These similar conditions were continued in injection time

Samples were divided to five groups with 10 mice in each group: one control group, one placebo group, and three treatment groups. Mean weight of all groups were 30 ±5 g and each group was kept in a separate cage. Prepared extract was injected in three concentrations according to body

Ten Injections were done between 8-10am in a twenty days period (every other day) . One day after progesterone injection, extract injection was started and one day after the last injection, blood sampling and autopsy were done. For studying drug effect on samples, all mice must be in similar estrous cycle. To this, 0.5 micro gram of cloprostenol was injected in peritoneum and three microgram of progesterone was injected under skin of all samples. One day after that, extract injection was started and one day after last injection, blood samples of heart were prepared to study variation of sexual hormones, and also autopsy was done to ovary histology of samples.

Obtained data were analyzed using one way variance analysis of *SPSS 11.5* program, and mean

Counting graafian follicles of prepared tissue slides and mean comparison of groups using Duncan test (p≤0.05) showed that all experimental groups had significant reduction in proportion to

concentration of extract in base solution was determined and doses were prepared.

**2.2. Animals**

**2.3. Study treatments**

**2.4. Statistical analysis**

**3. Results**

comparison was done using Duncan test.

**3.1. Number of graafian follicles**

control group and third group had the least.

Control group: non injected

Placebo group: 9% normal saline

group1: 50 mg/kg extract of body weight group2: 100 mg/kg extract of body weight group3: 200 mg/kg extract of body weight

too.

weight:

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It was used in old Egypt as incense, and for mummifying corpses and also for easy confinement and increase in milk production. Even nowadays Egyptian woman use this plant for curing menstrual pain, as a tea for stomach problems of tourists, and also as a complement matter for wheat and corn flours for baking breads and confectionaries [7] . In ancient Chinese drugs, seeds of fenugreek were used as strengthen drug [8] . The nature of plant and its seeds is dry and warm. Seeds are used as sterilizer, mild laxative, diuretic, in bronchial inflammation treatment, leprosy treatment, the treatment of hemorrhoids and mouth deodorant [9] . Also, it is laxative, anti inflammatory, joint pain reliever, pulmonary and bone tuberculosis. Meanwhile, it is used for increase in weight [6] . Seeds of fenugreek have constant oils, essence, alkaloids, flavonoids, saponin, sapogenin, mucilage, free amino acids, carbohydrate, fiber, phosphorus, sulfur, lecithin, iron, calcium, magnesium, potassium, sodium, coumarin, tannin, resin, pectin, niacin, and carotenodic compounds.

Fenugreek has many pharmacological effects: results of researches show that diabetic mice (induces by stereptozotocine) which were cured by seed extract of fenugreek seeds, had an increase in weight and a reduction in ratio of kidney weight to body weight [10] .Also, reduction in blood fats was ascribed low carbohydrate absorption and fat absorption was ascribed to active presence of fibers [11]. Results of studies on wild mice showed that fenugreek increased excreting of acids and neutral stroles, and then saved cholesterol of body was decreased [12]. The effect of fenugreek on fat index of diabetic patients with high cholesterol showed that this plant reduced fats significantly [13]. Significant changes were observed in total cholesterol, LDL and triglyceride but no in HDL level. Flavonoides of fenugreek seeds have anti oxidant activities which play their roles via hydrogen reduction and deletion single oxygen. A lot of fenugreek seed poly phenoles prevent oxidative hemolysis and peroxidation of induced fats (by hydrogen peroxide in laboratory) of human's red cells. This study was conducted to evaluate the effect of hydroalcoholic extract of fenugreek on reproduction physiology of female mice.

#### **2. Materials and Methods**

#### **2.1. Extraction**

To prepare the extract of seeds, they were grinded completely and 30 g of obtained powder was poured in a sterilized erlen, 40 cc of physiological serum was added to it, and was located in a cool place. After 24 hours, erlen contents were mixed completely using a shaker for five minutes. Then, after filtering the solution by filtrative paper and calculating extract residual in solution, concentration of extract in base solution was determined and doses were prepared.

## **2.2. Animals**

Female mice (*Balb/C*) in weight range of 25-40 g were taken from animal division of Isfahan Medical University. Samples were kept in similar conditions of water, food, light, temperature and moisture to environment adaption. These similar conditions were continued in injection time too.

#### **2.3. Study treatments**

Samples were divided to five groups with 10 mice in each group: one control group, one placebo group, and three treatment groups. Mean weight of all groups were 30 ±5 g and each group was kept in a separate cage. Prepared extract was injected in three concentrations according to body weight:

Control group: non injected Placebo group: 9% normal saline group1: 50 mg/kg extract of body weight group2: 100 mg/kg extract of body weight group3: 200 mg/kg extract of body weight

Ten Injections were done between 8-10am in a twenty days period (every other day) . One day after progesterone injection, extract injection was started and one day after the last injection, blood sampling and autopsy were done. For studying drug effect on samples, all mice must be in similar estrous cycle. To this, 0.5 micro gram of cloprostenol was injected in peritoneum and three microgram of progesterone was injected under skin of all samples. One day after that, extract injection was started and one day after last injection, blood samples of heart were prepared to study variation of sexual hormones, and also autopsy was done to ovary histology of samples.

## **2.4. Statistical analysis**

Obtained data were analyzed using one way variance analysis of *SPSS 11.5* program, and mean comparison was done using Duncan test.

## **3. Results**

## **3.1. Number of graafian follicles**

Counting graafian follicles of prepared tissue slides and mean comparison of groups using Duncan test (p≤0.05) showed that all experimental groups had significant reduction in proportion to control group and third group had the least.

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There was significant reduction in LH level (mIu/ml) of all experimental groups according to

Mean comparison of estrogen level in blood serum of experimental and control groups using Duncan test (p≤0.05) showed significant increase in hormone level of all three experimental

Mean comparison results of progesterone level in blood serum of experimental and control groups showed increases in first group (50 mg/kg) and second group (100 mg/kg) which this increase was significant only for second group. Also, third group (200 mg/kg) showed significant

groups and second group (100mg/kg) had the highest hormone level.

**Graph 5.** Amount of Estrogen hormone in various groups

**3.5. Progesterone amount**

reduction in proportion to control group.

**3.3. LH amount**

mean comparison results.

**Graph 4.** LH amount of various groups

**3.4. Estrogen amount**

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**Graph 1.** The number of graafian follicles in various groups

Results of counting corpus leuteum of tissue slides showed significant increase in second experimental group (100 mg/kg) and significant reduction in third group (200 mg/kg) in proportion to control but there was no significant difference between first group (50 mg/kg) and control.

**Graph 2.** The number of corpus leuteum in various groups

#### **3.2. FSH amount**

Comparing FSH level (mIu/ml) in blood serum of experimental and control groups using Duncan test (p≤0.05) showed significant reduction all three experimental groups in proportion to control.

**Graph 3.** FSH amount of various groups

#### **3.3. LH amount**

There was significant reduction in LH level (mIu/ml) of all experimental groups according to mean comparison results.

**Graph 4.** LH amount of various groups

#### **3.4. Estrogen amount**

Mean comparison of estrogen level in blood serum of experimental and control groups using Duncan test (p≤0.05) showed significant increase in hormone level of all three experimental groups and second group (100mg/kg) had the highest hormone level.

**Graph 5.** Amount of Estrogen hormone in various groups

#### **3.5. Progesterone amount**

Mean comparison results of progesterone level in blood serum of experimental and control groups showed increases in first group (50 mg/kg) and second group (100 mg/kg) which this increase was significant only for second group. Also, third group (200 mg/kg) showed significant reduction in proportion to control group.

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decrease in corpus leuteum of second and third groups. On the other hand, progesterone increase can be ascribed to diosgenin compounds of fenugreek which are progesterone precursor.

[1] Modaresi M. Gholchobian H. (2012), Effect of hydro alcoholic extract of nettle on Immune

[2] Davazdah Emami, S.2003. Application of medicinal plants. Isfahan Agricultural Researches

[3] Mokhtari, M. Shariati,M, Ghahramani,R.2007. The effect of fenugreek (Triqonella foenumgreeum L .) on hormone variation of testpterone and spermatogenesis of rat . Medicinal plants

[6] Samsamshariat, H. Moattar, F.2008 .Medicinal plants and natural plants (medical materia).

[7] Ashish Toppo F, Akhand R, Pathak A. K. 2009.Pharmacological actions and potential uses of *Trigonella foenum-graecum*: A review, Asian Journal of pharmaceutical and clinical Research, vol.

[8] Yoshikawa M, Murakami T, Komatsu H, et al. . 2001 .Medicinal foodstuffs. IV, Fenugreek seed. (1): structures of trigoneosides Ia, Ib, IIb, IIIa, and IIIb, new furosranol saponins from the seeds of Indian *Trigonella foenum-graecum* (Fenugreek) seed extract as antineoplastic Agent, phytother.

[9] Bhatia K, Kaur M., Atif F., Ali M., Rchman H., Rahman S., Raisuddin S., 2005; Aqueous extract of T. fenum-graecum l. Ameliorates additives urotoxicity of buthionine sulfoximine and

[10] Wan- Li xue, Xuan- she Li, Jian Zhang, Young- Hui Liu, Zhi- lun wang and Ruijuan Zhang. 2007 Effect of Trigonella foenum- graecum (Fenugreek) extract on blood glucose blood lipid and hemorheological properties in streptozotocin induced diabetic mice. Asia PC J clin Nutr; 16

[11] Hannana JMA, Rokeya B, Faruque O, 2003. Effect of soluble dietary fibre fraction of Trigonella foenum-graecum on glycemic, Insulinemic, lipidemic and platelet aggregation status of Type2

[12] Sharma R.D. 1986 . An evaluation of hypocholesterolemic. Factor of fenugreek seeds (T. foenum-

[13] Awal MA, Rashid MU, Ahmad KW, Asadi ZS, Islam K. 1999. Effect of karela and fenugreek on lipid profile in hypocholesterolemic diabetic patients . Bangladesh J physiol pharmacol.; 15: 6-8.

[14] Tammanini C, Basini G, Grasselli F, Tirlli M. 2003 Nitric oxide and the ovary. J Anim SCi.; 81(E.

[15] Zamiri, M. 2006. Reporoduction physiology. Haghshenas press. Pages 112-114,127.

[4] Zargari.A. 2007 .Medicinal plants. Tehran university press.2nd volume. page 608. 637-642.

[5] Samsamshariat. H.2003 medicinal plant production. Maani press. Page 307-308

cyclophosphamide in mice. Food and Chemical Toxicology 44 ;1477-1750

diabetic model mice. J Ethnopharmacol; 88: 73-77.

graecum) in mice. Nutr. Rep. int.; 33: 669-77.

system in mice, *Asian J. Chemistry* 24(5) ; 2339-2341

magazine. 7th year. no: 25(winter).pages 12-20.

organization press. Page 10.12-14 .

Mashaal press. Page 79,333,336-337.

**5. References**

2, Issue 4,

Res; 15: 257-9.

(suppi): 422-426 .

Suppl.2): E1-E7.

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**Graph 6.** Amount of progesterone hormone in various groups

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

Obtained results show significant reduction in number of graafian follicles in all three experimental groups (50,100, and 200 mg/kg) . This reduction is more obvious in third experimental group which is in agreement with previous results [3] . In their study, because of some compounds like sapogenin and diosgenin extant in fenugreek seeds, which are precursor of progesterone and testosterone reducer, sperm production was decreased [3].

By decreasing FSH level in follicle liquid (one of the follicle growth affecting factors) IGFBP4.5 will be increased and action of proteases will be prevented, then, FSH antagonists will be increased and follicle will be evolved with atresia. GnRH stimulates also IGFBP4.5 production in granulosa cells of follicle and reduces IGFBP4.5 proteases, then, causes follicle atresia. Probably, secreting Aromatase preventing protein from dominant follicle is effective on the other follicles and cause growth stop and atresia. Furthermore, low concentration of leptin in follicle liquid may have negative effect on growth and ripening of ovules. Results of studies show that Nitric oxide (NO) prevents growth of follicles and repining of ovocytes via inducing apoptosis [14] . According to results, amount of FSH hormone decreased significantly in all experimental groups. High concentration of progesterone and low concentration of estrogen prevent follicle stimulating hormone. Meanwhile, it seems that opioid peptides of brain are mediums of this negative feedback [15] . On the other hand, inhibin hormone of dominant follicle prevents FSH secreting from pituitary, in very specific way. considering the results, LH hormone was reduced significantly in all experimental groups which can be because of :

Linoleic acid (CLA) has reducing effect on LH amount via decreasing leptine. Due to quite definite and significant relationship between leptine and nitric oxide in LH releasing from pituitary, Leptine reduction will be led to reduction in nitric oxide and then GNRH releasing [14] .

Fenugreek increases prolactin via affecting serotonergic system which will be led to prevention in GnRH releasing and then reduction in LH[14] .according to results, estrogen hormone was increased in all experimental groups. Seed extract of this plant has Gitogenin, Trigonelline, and Quercitin which have estrogen making activity. It seems that these three compounds play important roles in increasing estrogen by their biological activities. Considering the results, amount of progesterone hormone was increased significantly in second experimental group and was decreased significantly in third group. These increase and decreases are similar to increase and decrease in corpus leuteum of second and third groups. On the other hand, progesterone increase can be ascribed to diosgenin compounds of fenugreek which are progesterone precursor.

#### **5. References**


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, Mehrdad Modaresi<sup>2</sup>

**Determining Morphological Traits and Genetic** 

**Diversity of Rose Aphids Using RAPD and RFLP-**

1 Department of Plant Protection, Khorasgan (Isfahan) Branch, Islamic Azad University, Isfahan, IRAN 2 Department of Animal Science, Khorasgan (Isfahan) Branch, Islamic Azad University, Isfahan, IRAN

Rose is one of the most beautiful attractive flowers of world which is important in landscaping because of its unique botanical specifics. One of the most important pests of this plant is aphid. In this study, 135 aphid samples were collected from various regions of Isfahan landscapes. Morphological traits and mitochondrial gene sequencing were used for identifying them. According to morphological traits, these samples were belonging to *Macrosiphum rosae*,*aphis gossipy,* and *metopolophium dirhodum* species which diversity in their morphological traits was obvious. Studying the genetic diversity of 16 selected samples was done by RAPD-PCR molecular marker using three primers and RFLP-PCR using restriction enzyme *Rsa*I. Results showed high genetic diversity in studying population. Samples grouping were done better by RFLP marker than RAPD marker. So, all samples which were located in three groups by this method had also high relations in morphological traits. On the other hand, genetic differences were shown better by RAPD for insects of a group which didn't have similar morphological traits. Then, this method can be

Aphids, as an important group of insects which are belonged to Hemiptera, are very successful creatures with the most species diversity in temperate regions and worldwide distribution. There is few plant species in this area without any specific aphid [1] . They may cause loss of plants directly or indirectly. Plus the direct loss which is made by heavy feeding from sap and includes weakness of plant and finally reduction in yield, they cause indirect loss by honeydew secreting on leaves and branches which absorb dusts and also mold will start to grow and finally photosynthesis and yield will be reduced. Furthermore, aphids are very important economically because of transferring plant viruses and their related diseases [2 , 3 ] There have been identified more than 4000 insects and this number is increasing daily. Aphids are a little group in proportion to other insects groups but their diversity is very high because of polymorphism existence and creating new biological types [1]. Classification of aphids is according to their morphological traits like other insects. It means that main differences or main similarities of samples are being

, Azadeh Karimi<sup>1</sup>

**PCR Molecular Markers**

used to observe the highest diversity level of population.

**Keywords:** Rose , RAPD , RFLP-PCR

**1. Introduction**

Ali Reza Jalalizand<sup>1</sup>

Esmaeil Mahmoodi<sup>1</sup>

**Abstract**

© 2012 Jalalizand 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

,

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## **Determining Morphological Traits and Genetic Diversity of Rose Aphids Using RAPD and RFLP-PCR Molecular Markers**

Ali Reza Jalalizand<sup>1</sup> , Azadeh Karimi<sup>1</sup> , Mehrdad Modaresi<sup>2</sup> , Esmaeil Mahmoodi<sup>1</sup>

1 Department of Plant Protection, Khorasgan (Isfahan) Branch, Islamic Azad University, Isfahan, IRAN 2 Department of Animal Science, Khorasgan (Isfahan) Branch, Islamic Azad University, Isfahan, IRAN

#### **Abstract**

Rose is one of the most beautiful attractive flowers of world which is important in landscaping because of its unique botanical specifics. One of the most important pests of this plant is aphid. In this study, 135 aphid samples were collected from various regions of Isfahan landscapes. Morphological traits and mitochondrial gene sequencing were used for identifying them. According to morphological traits, these samples were belonging to *Macrosiphum rosae*,*aphis gossipy,* and *metopolophium dirhodum* species which diversity in their morphological traits was obvious. Studying the genetic diversity of 16 selected samples was done by RAPD-PCR molecular marker using three primers and RFLP-PCR using restriction enzyme *Rsa*I. Results showed high genetic diversity in studying population. Samples grouping were done better by RFLP marker than RAPD marker. So, all samples which were located in three groups by this method had also high relations in morphological traits. On the other hand, genetic differences were shown better by RAPD for insects of a group which didn't have similar morphological traits. Then, this method can be used to observe the highest diversity level of population.

**Keywords:** Rose , RAPD , RFLP-PCR

#### **1. Introduction**

Aphids, as an important group of insects which are belonged to Hemiptera, are very successful creatures with the most species diversity in temperate regions and worldwide distribution. There is few plant species in this area without any specific aphid [1] . They may cause loss of plants directly or indirectly. Plus the direct loss which is made by heavy feeding from sap and includes weakness of plant and finally reduction in yield, they cause indirect loss by honeydew secreting on leaves and branches which absorb dusts and also mold will start to grow and finally photosynthesis and yield will be reduced. Furthermore, aphids are very important economically because of transferring plant viruses and their related diseases [2 , 3 ] There have been identified more than 4000 insects and this number is increasing daily. Aphids are a little group in proportion to other insects groups but their diversity is very high because of polymorphism existence and creating new biological types [1]. Classification of aphids is according to their morphological traits like other insects. It means that main differences or main similarities of samples are being

© 2012 Jalalizand 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.

compared and they are being located in their own location [2] . Phylogenic classification is based on evaluation history of species, genera, and families and classifies them according to common ancestors and hosts. In this type of classification, apomorphic traits are used [4]. Primary classifications were not based on phylogenic specifics and were according to personal tastes because the importance of phylogenic specifics was not known. Aphid classification is being discussed very much today especially in one main category: their family numbers[4] . .There have been many morphological anatomical studies conducted on aphids which have prepared background for systematic studies about them [5] . Aphids have high ability for adaption and changing and their morphology is being affected by environmental factors. Many ecological physiological factors affect morphological form of aphids [6] . Considering rich vegetation coverage in Iran and high diversity in roses, many aphids' species are not collected yet and probably this diversity in vegetation coverage and weather conditions have been led to high genetic diversity in aphid population of Iran. Molecular methods are appropriate way for responding main basic questions about genetic diversity and more systematic relationships about live organisms. because of high inter species and intra species diversity in populations of some insects like tripses, white flies, and aphids, morphological tool is not efficient for dividing them, then using molecular markers can be very effective [7, 8] . In high taxonomic levels, to study phylogenic relations and also creating and determining classification systems, molecular markers are efficient resources. In molecular level, they help highly in expanding the concept of species. In population level, they explain direct relationship between heredity patterns, distribution, and colonization with temporal geographical distribution. Molecular markers have been used in last years to basic and applied studies of various organisms. So that discovering various molecular markers have caused high progresses in genetic studies [9] . DNA molecule is the base of genetic differences between two live organism and DNA fingerprint is one of the current methods for determining biological identity of live organisms. By comparing electrophoresis profile of DNA we can realize their differences. DNA polymorphism is the base of many genetic studies [10] . Some of PCR based DNA markers are: RAPD, PCR-RFLP, and AFLP [11] . This markers have been used for various purposes like: creating genetic maps, map of traits in diversing populations, saturating genome places with marker , people fingerprints, germplasm analysis, measuring the genetic distance of people, and evaluating parents portions in back cross. This method is a valuable tool in molecular genetics science which is used easily in map creating and fingerprint application [12] . There has not reported any research about morphological and genetic differences of colorful biotypes of rose aphids and then many researches can be done in this category. These studies can be a good introduction for next studies about resistance of rose varieties to these biotypes, their biological differences and their geographical distribution [13] . These types of researches cause more knowledge about pests and then we can find better ways for fighting against pests.

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C for one hour. Then 300 micro liter phenol - chloroform was added to each

C in 2000g. Finally 300 micro liter of 70% ethylic alcohol was added to depsit and

are interested mainly to this type of branches. Samples were transferred to laboratory and their

At first, microscopic slides were prepared from healthy samples using boiling with Canadabalsam method. Samples identification was done using different identification keys extant for Iran

DNA extraction was conducted using Kawasaki method (2005) with a little change: aphids were washed by distilled water and located in 1.5 ml micro tubes plus 100 micro liter extraction buffer. Then samples were grinded for one minute to obtain a homogenized suspension. After that another 200 micro liter extraction buffer and 30 micro liter of protease K were added to each tube and

micro tube and tubes were centrifuged for five minutes in 1200g. Upper part was collected, was transferred to new micro tube with 300 micro liter of phenol – chloroform and then centrifuged again. Then, upper part was mixed with 300 micro liter of Isopropanol and 30 micro liter of so-

and 25 micro liter of twice sterilized distilled water was added to them and were kept at -20°

Polymerase chain reaction was conducted using prepared mixture made by Amplicon Company (Japan). Substances of reaction were Perimx: 12.5 micro liters, primer: 0.5 ml, 7 micro liters of twice distilled water, pattern DNA: 1 micro liter. Three primers were used in this study: UBC90 (5 -GGGGGTTAGG), R108 (5 -GTATTGCCCT), and R157 (5 -GCTGTAGTGT). Thermal plan of RAPD reaction was: six minutes at95°c, 40 cycles with 45 seconds at 94°c, 90 seconds at 32°c, 90

Evaluating of PCR product was done on Agarose gel (1.2%). Nine micro liters of each reactions product plus one micro litter loading buffer (6X from 0.25%Boromo phenol blue and 40% w/v of sucrose) were poured in gel wells and electrophoresis was done at 2.5 volts/cm. After electrophoresis, gel was colored in methyl bromide solution (1µg/ml) for 10-15 minutes and then was decolorized in distilled water for 5-7 minutes. RAPD bands were observed under UV lamps and were shot using Uvtech machine. Evaluating of PCR product was done on Agarose gel (1.2%). Execu-

To data analysis of electrophoresis results, existence or none existence of each band was recorded in Excel as one and zero, respectively. Cluster analysis of isolates was conducted using UPGMA

tion buffer of electrophoresis machine was TAE (0.04M Tris-acetate and 0.001 M Na2

**2.4. RAPD-PCR reaction for evaluating genetic diversity of aphids**

insects were swept by brush and were stored in 70% alcohol.

dium – acetate (3M) and was incubated for ten minutes at -20°

seconds at 72°c, and final amplification with 8 minutes at 72°c.

were centrifuged in 2000g for ten minutes at 4°

**2.5. Data analysis of RAPD-PCR**

method and *NTSYS V 2.2* program.

**2.2. Morphological characterization of aphids**

aphid fauna.

were incubated at 60°

ten minutes at 4°

**2.3. DNA extraction of roses**

Turkey, September 10-12, 2012

C. Then samples were centrifuged for

C.

EDTA).

C. After eliminating alcohol, micro tubes were dried

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#### **2. Materials and Methods**

#### **2.1. Sampling and counting aphids**

In order to determine the number of rose aphids, sampling was done from Isfahan province. Because that aphid colonies take place mostly in 10 -15 cm end of branches, about 15 cm of twigs were cut and locate in plastic bags. Sampling was done from young branches because aphids are interested mainly to this type of branches. Samples were transferred to laboratory and their insects were swept by brush and were stored in 70% alcohol.

#### **2.2. Morphological characterization of aphids**

At first, microscopic slides were prepared from healthy samples using boiling with Canadabalsam method. Samples identification was done using different identification keys extant for Iran aphid fauna.

#### **2.3. DNA extraction of roses**

DNA extraction was conducted using Kawasaki method (2005) with a little change: aphids were washed by distilled water and located in 1.5 ml micro tubes plus 100 micro liter extraction buffer. Then samples were grinded for one minute to obtain a homogenized suspension. After that another 200 micro liter extraction buffer and 30 micro liter of protease K were added to each tube and were incubated at 60° C for one hour. Then 300 micro liter phenol - chloroform was added to each micro tube and tubes were centrifuged for five minutes in 1200g. Upper part was collected, was transferred to new micro tube with 300 micro liter of phenol – chloroform and then centrifuged again. Then, upper part was mixed with 300 micro liter of Isopropanol and 30 micro liter of sodium – acetate (3M) and was incubated for ten minutes at -20° C. Then samples were centrifuged for ten minutes at 4° C in 2000g. Finally 300 micro liter of 70% ethylic alcohol was added to depsit and were centrifuged in 2000g for ten minutes at 4° C. After eliminating alcohol, micro tubes were dried and 25 micro liter of twice sterilized distilled water was added to them and were kept at -20° C.

## **2.4. RAPD-PCR reaction for evaluating genetic diversity of aphids**

Polymerase chain reaction was conducted using prepared mixture made by Amplicon Company (Japan). Substances of reaction were Perimx: 12.5 micro liters, primer: 0.5 ml, 7 micro liters of twice distilled water, pattern DNA: 1 micro liter. Three primers were used in this study: UBC90 (5 -GGGGGTTAGG), R108 (5 -GTATTGCCCT), and R157 (5 -GCTGTAGTGT). Thermal plan of RAPD reaction was: six minutes at95°c, 40 cycles with 45 seconds at 94°c, 90 seconds at 32°c, 90 seconds at 72°c, and final amplification with 8 minutes at 72°c.

Evaluating of PCR product was done on Agarose gel (1.2%). Nine micro liters of each reactions product plus one micro litter loading buffer (6X from 0.25%Boromo phenol blue and 40% w/v of sucrose) were poured in gel wells and electrophoresis was done at 2.5 volts/cm. After electrophoresis, gel was colored in methyl bromide solution (1µg/ml) for 10-15 minutes and then was decolorized in distilled water for 5-7 minutes. RAPD bands were observed under UV lamps and were shot using Uvtech machine. Evaluating of PCR product was done on Agarose gel (1.2%). Execution buffer of electrophoresis machine was TAE (0.04M Tris-acetate and 0.001 M Na2 EDTA).

## **2.5. Data analysis of RAPD-PCR**

To data analysis of electrophoresis results, existence or none existence of each band was recorded in Excel as one and zero, respectively. Cluster analysis of isolates was conducted using UPGMA method and *NTSYS V 2.2* program.

#### **2.6. RFLP-PCR reaction**

One of the molecular markers which are used for diversity studying is pattern of amplified genome segments or RFLP. To this, lepF: 5 -ATTCAACCAATCATAAAGATATGGG-3 and lepR: 5 - TAAACTTCTGGATGTCCAAAAAATCA-3 primer pairs were used in polymerase chain reaction.

International Conference on Applied Life Sciences (ICALS2012)

rose aphids. The size of obtained bands was estimated about 100-2000 bp. Among used primers, UBC 90 and R 108 showed the highest polymorphism with 25 and 24 amplified segments respectively (figure1). According to cluster analysis rose aphids divided into three groups: the first group (A) consisted of 5 aphid samples from various regions of Isfahan. Second group (B) consisted of nine samples which were different in color. Members of this group were black and green and were belonging to *Aphis gossypii* and *Macrosiphum rosae*. Two samples of rose aphids (C2 and V1) were located in C group which C2 was green to grey colored and V7 was brown colored and both were located in *Macrosiphum rosae* species. Results of this study showed that dividing rose aphids by RAPD-PCR has a close relation with their morphological traits but not

UBC - R108

**Figure 1.** Band figures of rose aphids amplified segments using UBC-90, and R-108 primers in Agarose

**Figure 2.** Cluster analysis of rose aphids using data of RAPD-PCR. Group A:, group B:, and group C:

To this, PCR product of mitochondrial gene was digested by *Rsa*I restriction enzyme. The cutting distribution pattern of aphid mitochondrial gene is presented in figure 3. The enzyme could show seven band pattern of polymorphism on mitochondrial genome of aphid samples. Cluster analysis of this polymorphism was done using *NTSYS V 2.2* program (Figure4). As it was shown in cladogram, aphids are dividing in three groups according to RFLP-PCR. Group A were red aphids and were very similar morphologically. In this grouping many of black and green aphids (7 samples) was located in group B and the rest (4 samples) were located in group C. This group-

**3.3. Genetic diversity of rose aphids using RFLP-PCR**

with their color (figure2).

gel 1.2%

1 2 3 4 5 6 7 8 9 10 11 12 13 14

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1 2 3 4 5 6 7 8 9 10 11 12 13 14

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PCR substances were: PCR buffer, dNTP (200MM), Mgcl2 (2MM), primers (25 pichomole of each), DNA polymerase Taq (1.25) and pattern DNA (100 nanog.). Primary denaturing of PCR was done for 6 minutes at 95° C, amplification in 35 cycles as 0.75 minute at 94° C, 1.5 minutes at 55° C, 1.5 minutes at 72° C and final amplification 5 minutes at 72° C.then,amplified segments were cut in enzymatic digestion reaction by *Rsa*I enzyme at 37° C for three hours and cutting pattern of genomic segments was observed by electrophoresis on Agarose gel (1.5%).To data analysis of polymorphism results, existence or none existence of each band was recorded as one and zero, respectively. Cluster analysis was conducted using UPGMA and Jacquard coefficients and *NT-SYS V 2.2* program and then its cladogram was drawn.

#### **3. Results**

#### **3.1. Morphological traits of rose aphids**

Isfahan has its own aphid's fauna for roses like every other place. Wide sampling of all Isfahan places prepared 135 samples totally. According to morphological traits, three aphid species were collected from Isfahan roses. The most abundant species was *Macrosiphum rosae* which? samples were belonged to this species. This species which is known as rose aphid too has been sawn from average to big sizes, with long or spindle shaped bodies in green, yellow, pink and red – brown colors. According to previous reports, this species is distributed all over Iran and can be collected all seasons except summer. The most important morphological characteristics of this species are ?. The second species which was collected less in this study was *Metropolophium dirhodum*. Wingless members of this species are all long spindle – shaped with green color or green – yellow and an obvious bright green back stripe. Antennas are bright and the end of third and fifth part and appendix of final part were dark to black. Antenna length is about 0.6 of total length, and third part has 1 to 3 secondary \* in its bottom. The others have a green abdomen without sclerotium spots. Hair formula of tarsus first part was 3-3-3 and tail had 9-12 hairs too. The length of wingless members bodies were from 1.6 to 3.3 mm. This characteristic is completely in accordance to Black man and Eastop (2000) reports. The main host of this insect is rose too and has been reported from many places of Iran. The third species was *aphis gossypii*. This egg shaped insect is about 1.8 mm and is sawn in many colors. Same are green or yellowish green and the other are grey to green. It has been reported from all places of Iran and various hosts. We must announce that some samples were not completely in accordance to identification Key and were ascribed to these species because of showing the most important characteristics. One of important specifics of aphids is similarity of their morphological traits which make their identification difficult [14] .

#### **3.2. RAPD-PCR reaction**

In this study 16 aphid samples which had high morphological differences were used to study their genetic diversity. All used primers of this study showed good polymorphism in studied rose aphids. The size of obtained bands was estimated about 100-2000 bp. Among used primers, UBC 90 and R 108 showed the highest polymorphism with 25 and 24 amplified segments respectively (figure1). According to cluster analysis rose aphids divided into three groups: the first group (A) consisted of 5 aphid samples from various regions of Isfahan. Second group (B) consisted of nine samples which were different in color. Members of this group were black and green and were belonging to *Aphis gossypii* and *Macrosiphum rosae*. Two samples of rose aphids (C2 and V1) were located in C group which C2 was green to grey colored and V7 was brown colored and both were located in *Macrosiphum rosae* species. Results of this study showed that dividing rose aphids by RAPD-PCR has a close relation with their morphological traits but not with their color (figure2).

**Figure 1.** Band figures of rose aphids amplified segments using UBC-90, and R-108 primers in Agarose gel 1.2%

**Figure 2.** Cluster analysis of rose aphids using data of RAPD-PCR. Group A:, group B:, and group C:

#### **3.3. Genetic diversity of rose aphids using RFLP-PCR**

To this, PCR product of mitochondrial gene was digested by *Rsa*I restriction enzyme. The cutting distribution pattern of aphid mitochondrial gene is presented in figure 3. The enzyme could show seven band pattern of polymorphism on mitochondrial genome of aphid samples. Cluster analysis of this polymorphism was done using *NTSYS V 2.2* program (Figure4). As it was shown in cladogram, aphids are dividing in three groups according to RFLP-PCR. Group A were red aphids and were very similar morphologically. In this grouping many of black and green aphids (7 samples) was located in group B and the rest (4 samples) were located in group C. This grouping has a high accordance to morphological results of aphid population. For instance, C2, C6, and C9 which were related to numbers 109,105 and 108 (studied samples) respectively were located in one group. In morphological traits all these aphids are red. These results show efficiency of this marker for for classifying and identifying the aphid population, along with their phenotypic characteristics. In second group also samples were located which were very similar morphologically and were different only in color and some other characteristics.

International Conference on Applied Life Sciences (ICALS2012)

completing effective controlling strategies [15] . Then, one of the other goals of studying genetic diversity and population structure is identifying the factor which plays more important role in evolution of that population and also how that factor determines genetic structure of population and its evolution potential[5] . Developing molecular technics and using genetic markers in last decades have led to developing tools and fast, cheap, and accurate methods for identifying creatures. So, polymerase chain reaction (PCR) and molecular methods based on PCR have had an important role in developing biological sciences. In recent years, many efforts have been done for using molecular methods in entomology. For example Wagou et al. (1996) used molecular markers for identifying parasite insects and studying their biology. Genome sequencing and molecular markers is an appropriate replacing method for morphological methods in studying aphids' epidemiology [13] RAPD and AFLP molecular markers have high application as population markers in studies about population diversity of living things especially insects [7] Chen et al (2008) studied genetic diversity of cabbage aphids with molecular methods and reported that when it was not possible to divide aphid populations by morphological traits, RAPD and AFLP markers divided them well. In current study, rose aphids were studied for morphological and molecular properties. According to morphological findings, all collected samples were belonging to three species: *macrosiphum rosae, aphis gossypii,* and *metopolophium dirhodum*. Previous studies confirm it too. These three aphid species have been identified before on roses of Isfahan [16] this study was done to clarify structure of rose aphid's populations from morphological and genetic aspects and also for evaluating use of RAPD and RFLP markers for grouping these populations. UBC-90 primers showed the highest polymorphism in this study. Also, primers could present significant relationship with morphologic traits of rose aphids. Polymorphism analysis of these markers with NTSYS program showed that RFLP markers acted better than RAPD in locating close populations with similar morphological traits in similar clads. Considering high distribution of this pest on various plants, for better realizing the population structure of this pest, it is better to study its genetic diversity on other hosts in various regions. On the whole, results of this study showed that morphological and genetic diversity of this aphid is relatively high,

and then effective factors in its creation and expanding must be studied.

[1] Dixon A F G. 1998. Aphid ecology. Second edition, UK, Chapman and Hall.

natural enemies and control. Elsevier science publishers B V, PP:51- 77.

Guide, 2nd edn. England, John Wiley & Sons Ltd.

[2] Rezvani, A. 2001. Identification key of Iran aphids. Iran organization of agricultural researches,

[3] Blackman R L, Eastop V F .2000. Aphids on the World's Crops: an Identification and Information

[4] Ilharco F A, Van Horten A. 1987. Systematics. In: Minks A K, Harrewijn P. Aphids Their biology,

**5. References**

training and advisory.

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**Figure 3.** Polymorphism (restriction pattern) of mitochondrial gene of rose aphid in enzyme digesting using RsaI restriction enzyme on Agarose gel 1.5% in TBE buffer

**Figure 4.** Cladogram of cluster analysis of aphids' polymorphism in RFLP-PCR using NT SYS V2.2 program

## **4. Summary and conclusion**

Population structure of each organism is dependent to amount and distribution of within and between populations genetic diversity. Genetic structure of a population shows evolution history and potential of it for evolution and adaption to environment [5] . Every agricultural ecosystem is facing to environment changes like cultivating resistant cultivars, fungicides, pesticides, fertilizers, irrigation and crop rotation, then pests and plant pathogens are constantly evolving and changing to adapt to these changes. So, having knowledge about power of pest populations in evolution and adaption to environment and host is very important for completing effective controlling strategies [15] . Then, one of the other goals of studying genetic diversity and population structure is identifying the factor which plays more important role in evolution of that population and also how that factor determines genetic structure of population and its evolution potential[5] . Developing molecular technics and using genetic markers in last decades have led to developing tools and fast, cheap, and accurate methods for identifying creatures. So, polymerase chain reaction (PCR) and molecular methods based on PCR have had an important role in developing biological sciences. In recent years, many efforts have been done for using molecular methods in entomology. For example Wagou et al. (1996) used molecular markers for identifying parasite insects and studying their biology. Genome sequencing and molecular markers is an appropriate replacing method for morphological methods in studying aphids' epidemiology [13] RAPD and AFLP molecular markers have high application as population markers in studies about population diversity of living things especially insects [7] Chen et al (2008) studied genetic diversity of cabbage aphids with molecular methods and reported that when it was not possible to divide aphid populations by morphological traits, RAPD and AFLP markers divided them well. In current study, rose aphids were studied for morphological and molecular properties. According to morphological findings, all collected samples were belonging to three species: *macrosiphum rosae, aphis gossypii,* and *metopolophium dirhodum*. Previous studies confirm it too. These three aphid species have been identified before on roses of Isfahan [16] this study was done to clarify structure of rose aphid's populations from morphological and genetic aspects and also for evaluating use of RAPD and RFLP markers for grouping these populations. UBC-90 primers showed the highest polymorphism in this study. Also, primers could present significant relationship with morphologic traits of rose aphids. Polymorphism analysis of these markers with NTSYS program showed that RFLP markers acted better than RAPD in locating close populations with similar morphological traits in similar clads. Considering high distribution of this pest on various plants, for better realizing the population structure of this pest, it is better to study its genetic diversity on other hosts in various regions. On the whole, results of this study showed that morphological and genetic diversity of this aphid is relatively high, and then effective factors in its creation and expanding must be studied.

#### **5. References**


[5] Mcdonald B. 1997. The population genetics of Fungi: tools and techniques. Phytopathology, 87: 448-453.

International Conference on Applied Life Sciences (ICALS2012)

, S.T.Tawfeek<sup>2</sup>

The purpose of this research is to synthesize several new pyrazolopyrimidine containing an arylazo function containing electron with drawing groups and benzothiazole moiety, the substituted 5-arylazopyrazolopyrimidine were prepared by reaction of aryl azopyrazole with airelidene of 2- cyanomethyl benzothiazole under basic condition in boiling ethanol. The structure of arylazopyrazolopyrimidine dyes were established by their element analysis and spectral data (MS,

H-NMR). The antibacterial properties of these dyes have been investigated.

Innovations in azo dye based on heterocyclic systems have been made as a result of intensive studies stimulated by the mounting need for bright dyes. Generally many of heterocyclic azodyes show dramatic bathochromic shifts combined with brilliance of shade and high tinctorial strength compared with conventional anthraquinone dyes and aminobenzene azodyes[1-4]. In spite of the large number of arylazopyrazol dyes reported in literature, only very few condensed pyrazole derivatives carrying arylazo functions on the pyrazole ring have been reported,

In continuation of the increasing interest in synthesis of condensed arylazopyrazole new dyestuffs [3], the present work deals with novel synthesis of condensed arylazopyrazolopyrimidines derivatives and studying their printing properties using silk screen and heat transfer printing techniques on polyester and polyamide fabrics. The antibacterial activity of these dyes was also studied, where recently in the textile industrial sector [5], there has been increasing interest in the manufacture of clothing and products with antibacterial properties. Clothing of textile can act as carrier for microorganisms such as pathogenic or odor –generating bacteria and moulds [6]. The textile material is known to be susceptible to microbial attack, in contact with the human body it offers an ideal environment for microbial growth providing oxygen, water and warmth, and nutrients from spillages and body exudates [7]. This often leads to objectionable odor, dermal infection product deterioration, allergic responses and other related diseases which necessitate

**Keywords:** pyrazolopyrimidine – benzothiazole – antibacterial activity

**Synthesis and Characterization of** 

**Arylazopyrazolopyrimidines Dyes and** 

**Studying their Antibacterial Activity**

, M. A. Elkashouti<sup>1</sup>

1 Textile division, National Research Center, Cairo, Egypt 2 Faculty of applied art, Helwan University, Cairo, Egypt

K.A.Ahmed<sup>1</sup>

**Abstract**

IR and 1

**1. Introduction**

© 2012 Ahmed 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.

the development of clothing products with antimicrobial properties [8].

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and Sh. S. Mohamed<sup>1</sup>

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.

