Uric Acid Inhibition Activity of Annona muricata L Leave Extract in Hyperuricemia induced Wistar Rat.

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Advances in Pure and Applied Chemistry, 2012,

2

(

1

): 81 - 105

Table of Content

Vol.

2

, No.

1

, 2012

Articles

Table of Contents

Articles

Uric Acid Inhibition Activity of

Annona muricata

L Leave

Extract in Hyperuricemia induced Wistar Rat

Sri-Wahjuni, Putra-Manuaba, I. B., Rahayu-Artini, N. P., and Wahyu-Dwijani, S.

Chemistry Department, Faculty of Math and Science, Udayana University,

Bali-Indonesia

81 - 90

Kinetics of Organic Dyes Degradation in Water Using

Vacuum Ultra Violet Radiation

Khaled M. Elsousy

Alaqsa University, Gaza, Gaza strip, Palestinian Territories

91 - 97

Occurrence and prevalence of four viruses infecting tomatoes

in Northern districts of West Bank, Palestinian Territories

Hazem Sawalha

Department of Biology and Biotechnology, Faculty of Arts and Sciences,

Arab American University of Jenin, Palestine

98-101

Study of Liquefied Petroleum Gas Heating Value

A Thermodynamics Approach

Niaz Bahar Chowdhury,

1

Dr. Md. Iqbal Hossain

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Advances in Pure and Applied Chemistry (APAC) 86 Vol. 2, No. 1, 2012, ISSN 2167-0854

www.worldsciencepublisher.org

Uric Acid Inhibition Activity of

Annona muricata

L Leave

Extract in Hyperuricemia induced Wistar Rat

Sri-Wahjuni, Putra-Manuaba, I. B., Rahayu-Artini, N. P., and Wahyu-Dwijani, S.

Chemistry Department, Faculty of Math and Science, Udayana University,

Bali-Indonesia

Abstract-This research aims to find a cure of gout, base on the utilization of Annona muricata. The research was

started with descriptive study to explore active components of Annona muricata leaf and followed by an experimental

study to investigate uric acid inhibition activity of the leaf extract in hyperuricemia induced wistar rat. We observed three dominant components, i.e. 2,3-dihidrobenzofuran; 3-ethoxy-1,4,4a,5,6,7,8,8a-octahydroisoquinoline; 2-cyclohexen-1-one, 4-hydroxy-3,5,6-trimethyl-4-(3-oxo-1-butenyl) which were probably active to inhibit uric acid formation in hyperuricemic induced wistar rat. In this study, n-buthnol was applied for partitioning the relatively pure compound. The n-buthanol extract obtained was then applied to cure hyperuricemic rat induced by a mixture of chicken livers and Gnetum gnemon a high purine diet. It was obtained, that the highest extract dose of 400 mg/kgBW was able to inhibit the formation of uric acid in hyperuricemic rat. It can be concluded that Annona muricata leaf extracted with n-buthanol in a dose of 400 mg/kg BW has an ability to inhibit further formation uric acid in hyperuricemic rat. Therefore, this natural plant is potent to develop for hyperuricemic medicine.

Keywords: gout, Annona muricata, hyperuricemic rat, active components

Introduction

Uric acid is a metabolic product of exogenous (brought in with food) or endogenous purine bases. This acid in most physiologic fluids is an end product of purine degradation. The serum urate level in a given patient is determined by the amount of purines synthesized and ingested, the amount of urate produced from purines, and the amount of uric acid excreted by the kidney (and, to a lesser degree, from the gastrointestinal tract).1,2Gout is an inflammatory arthritis caused by the deposition of monosodium urate crystals in tissues.1 This condition typically occurs after years of sustained hyperuricemia. It is estimated to affect 5.1 million people in the United States according to the most recent National Health and Nutrition Examination Survey (NHANES III).2 Gout affects approximately 2% of men older than

30 years and2% of women older than 50 years, and is the

most common form ofinflammatory joint disease in men

older than 40 years. Serum uric levels are, on average,

0.5 to 1.0 mg/dL higher in men than women, making

male sex a risk factor for hyperurisemia and gout. Lower serum uric levels in women are associated with the

presence of estrogen, which is thought to act as an

antihyperuricemic.3 In Indonesia, based on Health Survey

in the year of 2005, there were around 10-20% men and postmonopause women have a higher levels of uric acids than normal person.4 It was proven that, celery seed is

often used in treating this condition, as it possesses many anti-inflammatory compounds. Other helpful herbs include turmeric, boswellia, cayenne, colchicum and hyssop were also potent to treat hyperurisemia.

Clearly, uric acid is produced by purine nucleoside metabolism through hipoxanthin, xanthin, and guanin basic purine. Distortion of this metabolism leads to elevate level of uric acid and known as hyperuricemia.5

Annona muricata L is a traditional plant in Bali known as sirsak, empirically in Balinese traditional medicine was proven as a cure of hyperuricemia. This study was carried in order to investigate the component active of the plant that have an ability to inhibit further uric acid formation in the hyperuricemia wistar rat.

Methods

This study employs two research methods, i.e.

descriptive explorative to determine the active

components of Annona muricata L leaf extracted with

n-buthanol and followed by experimental study to observed their hyperuricemia activity.

Leaf extract was obtained through maseration process using methanol and followed by partition using n-buthanol. Crude extract obtained was the identified by applying GS-MS instrument.

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Sri-Wahjuni et al, APAC, Vol. 2, No. 1, pp. 86-90, 2012 87

study and devided into 5 groups. First group is a negative control group in which rat fed with a mixture of 4 g/kg

BW of Gnetum gnemon and 50 mL/kg BW of

chicken-liver juice in ad libitum manner. The second group is simillar to first group instead of delivering an anti-hyperuricemia medicine, allopurinol in a dose of 10 mg/kg BW oraly. The third group is simillar to the first group instead of delivering Annona muricata L extract in a dose of 100 mg/kg BW oraly. The fourth and five groups have similar treatment to the first group, instead of have extract of Annona muricata L in dose of 200 mg/kgBW and 400 mg/kgBW, respectively.

Animal ethical clearance was obtained from a local authority body at Veterinary Faculty Udayana University, Bali-Indonesia. Around 1 mL of blood was taken from rat heart aorta which was anesthesia before proceeding. The blood was then centrifuged for 15 minutes at the rate

of 3.000-3.500 rpm. Uric acid reagent, FS TBHBA

(2,4,6-tribromo-3-hydroxybenzoic acid) was then added to the serum obtained. The mixture was then incubated for 10 minutes at a temperature of 370C. Then, optical density of the mixture was determined using sphectrophotometer at 546 nm of wave number.

ANOVA was performed to determine the different effect amongst treatment with p<0.05 was consider significant.

Results

Descriptive study

Around 1,200 g of Annona muricata L. leaf powder

was macerated with methanol for overnight. From this, a number of 158 g crude extract was obtained. This crude extract was then tested for its antioxidant activity using DPPH test. The tes results was presented in Table 1.

Table 1. Antiozidant Activity Test of Annona muricata L Crude Extract

Sampl e

Time

(minutes) Test

Absorbance A 517 nm % inhibition 497 nm 517 nm 537 nm Crude extract

5 DPPH 0.714 0.785 0.698 0.0790 77.22

% Sampl

e

0.635 0.593 0.515 0.0180

60 DPPH 0.651 0.704 0.613 0.0720 85.42

% Sampl

e

0.527 0.508 0.468 0.0105

The crude extract was the purified by applying partition using petroleum ether, chlroform, n-buthanol, and water. Amongst them, in this research, it was obtained that partiton with n-buhanol produce the highest

anti-oxidant ativity indicates by their DPPH test. Therefore, the n-buthanol extract was then identified Phythochemically using a number of reagent as indicated in Table 2.

Table 2. Phythochemical Test of n-Buthanol Extract

N o.

Compounds Reagent Coloue Changes Resul

ts 1

.

Alkaloid Meyer

Wagner

Yellow - orange (without white precipitate)

Yellow - chocolate (without chocolate precipitate) - - 2 .

Flavonoid Wilstatter

NaOH 10 % H2SO4 concentrated

Bate Smith-Metacalf

Yellow - crimson Yellow - chocolate Yellow - crimson Yellow - red

+ + + + 3 .

Triterpenoid Lieberman-Burchard

H2SO4 10 %

Yellow - chocolate Yellow - chocolate

+ + 4

.

Saponin Hot water + HCl No foam formation -

5 .

Phenolate (tannin)

Hot water + FeCl3 Yellow – greenish black +

6 .

Steroid Lieberman-Burchard

H2SO4 10 %

Yellow - chocolate Yellow - chocolate

-

Remarks:

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Sri-Wahjuni et al, APAC, Vol. 2, No. 1, pp. 86-90, 2012 88

The most active extract was then identified by applying GC-MS, the chromatogram obtained was presented in Figure 1.

Figure 1. GC-MS Chromatogram of The Most Active Extract of Leaf Annona muricata L

Based on library data base of the GC-MS instrument, there were three compound detected as indicated in Table 3.

Table 3. Compound Identified Based on GC-MS Chromatogram

Peaks

Retentiom Time

(tR)

%

Area Compounds identified

Peak 1 11.29minutes 31.4

8

benzofuran,2,3-dihidro

Peak 2 18.07 minutes 11.7

1

3-ethoxy-1,4,4a,5,6,7,8,8a-octahydroisoquinoline

Peak 3 18.70 minutes 30.8

9

2-cyclohexen-1-one, 4-hydroxy-3,5,6-trimethyl-4-(3-oxo-1-butenyl

Experimental study

In this study, increase uric acid in wistar rat was achieved by intake of high purine diet. Rat were fed with a mixture of 4 g/kg BW of Gnetum gnemon with 50

mL/kg BW of chicken liver ad libitum. After

hyperuricemia condition was achieved, the rat then was fed with varies dose of leaf Annona muricata L extract, i.e 100 me/kgBW, 200 mg/kgBW, and 400 mg/kgBW. Other treatments are positive control using allopurinol and negative control. The uric acid concentration of hyperurisemia rat were presented in Table 4.

Table 4. Uric Acid Levels of Hyperurisemia Wistar Rat

Tratrment group Uric acid concentration (mg/dL)

Day-6 Day-9 Day-14 Day-18

Hyperurisemia Control (H)1

Control (H)2

Control (H)3

Control (H)4

Average 3.48 5.54 4.48 4.25 4.44 4.05 5.65 4.62 4.65 4.74 4.35 5.87 5.97 5.03 5.31 4.98 6.07 6.23 5.67 5.74 Allopurinol dose 10 mg/kg BW Control positive 1 Control positive 2 Control positive 3 Control positive 4

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Average 3.91 6.42 4.52 3.93

Extract dose of 100 mg/kg BW Treatment I1

Treatment I2

Treatment I3

Treatment I4

Average 4.81 4.19 4.50 4.92 4.61 7.18 6.11 5.73 6.04 6.27 3.35 3.00 4.19 3.77 3.58 3.08 2.65 3.96 3.50 3.30

Extract dose of 200 mg/kg BW Treatment II1

Treatment II2

Treatment II3

Treatment II4

Average 3.00 3.27 4.11 3.73 3.53 3,38 3,57 4,35 4,11 3,85 2,31 3,00 2,61 3,04 2,74 1,61 1,69 1,69 2,73 1,93

Extract dose of 400 mg/kg BW Treatment III1

Treatment III2

Treatment III3

Treatment III4

Average 3.69 3.81 3.77 4.04 3.83 4.69 5.15 4.19 7.69 5.43 3.00 3.77 2.73 5.19 3.67 3.00 3.00 2.31 3.96 3.09

Anova test indicates there was a significant different between treatment and control groups, indicate by p < 0.05.

Discusions

Discriptive study

As can be seen in Table 2, n-buthanol fraction based on phytochemical test was positively containing flavonoid, triterpenoid, and phenolate indicates by the colour changes for all compounds type tested. This is because of n-buthanol is a polar solvent with 3.9 of polarity index.6 Generally, the present of glucose bind to flavonoid group results in the compound easier to solve on water and polar solvent.7 Tannin group is a phenolate compound, that has a tendency to solve in water and polar solvent. On the other hand, triterpenoid group of compound is a pentacyclic compound tend to solve in nonpolar solvent. GC-MS analysis confirms three important compounds observed as indicates on Table 3. All of these compounds are benzofuran,2,dihidro, 3-ethoxy-1,4,4a,5,6,7,8,8a-octahydroisoquinoline, and 2-cyclohexen-1-one, 4-hydroxy-3,5,6-trimethyl-4-(3-oxo-1-butenyl. The present of triterpenoid is probably due to the present of hydroxide group on the structure. The extract that contain all these thee compound was then tested for their anti-hyperurisemia activity.

Experimental study

A number of 20 wistar rat were adapted in a laboratory condition. Then, all of these rats were fed with high purine diet, i.e. a mixture of 4 g/kg BW of Gnetum gnemon and 50 mL/kg BW of chicken liver and mix with 100 g pelete a standard diet for rat. On day-6 and day-9, about 1 mL of blood were taken from the heart aorta of the rat to determine the increase of uric acid. Before treatment uric acid serum level of the rats were

determined. In this study, uric acid levels of normal rats were in the range of 1.7 – 3.0. After induction with high purine diet the uric acid levels of the rat was increase roughly, in which all rats have uric acid levels above 3 mg/dL, on average of 4.74

r

0.665 mg/dL. It can be said, that all experimental rats are in hyperuricemia condition.

Rats induced hyperuricemia were achieved during 9 days after feeding with high purine diet. Then, on day-10 all experimental rats receive treatment for decreasing uric acid levels. Five groups of experiment were carried out as mentioned on the method. The treatment was stopped on the day-18 and uric acid leves were detrmined for all experimental rats.

In this study we obtain that for positive control group treated with allopurinol, there is a 51.93% decrease of uric acid levels, their uric acid levels become 3.93

r

0.995 mg/dL. For the varies extract treatment, i.e. dose of 100, 200, and 400 mg/kg BW, the uric acid decrease levels obtained are 63.98%, 86.29%, and 61.50%, respectively. Therefore, the optimum dose of 200 mg/kg BW produces the highest decrease.

Allopurinol was applied in this study as a positive control, since this medicine is a cure for hyperuricemia case. In low dose this compound has an ability to inhibit the formation of xanthine oxidase enzyme.8 Allopurinol dose of 10 mg/kg BW applied is on the basis of Zhao et al, (2005), they obtain this dose was effective to decrease uric acid levels until 125.59

r

1.49 on their mice experimental study.9

Conclusion

This study investigates the application of natural plant,

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compounds, i.e. benzofuran,2,3-dihidro,

3-ethoxy-1,4,4a,5,6,7,8,8a-octahydroisoquinoline, and

2-cyclohexen-1-one, 4-hydroxy-3,5,6-trimethyl-4-(3-oxo-1-butenyl) were identified. However, this still need to be further investigated. Our study also gained that the extract leaf of this plant is potent to develop as a cure for hyperuricemia, since we obtain that the dose of 200 mg/kg BW of rat is effective to decrease uric acid levels. This also need to be investigaed further, whether that will give simmilar effect on human.

Akcnowledgment

The authors would like to thank Staff of UPT Lab. Analitik Udayana University for access and aid of their fasilities for managing the research. Thanks also to Mr. Priono from Kristallindo for providing reagents for uric analysis, Mr. Rudy at Forensic Laboratory Police Department for help in assessing the GC-MS data. And special thank to Vetinery Board for providing rat for this experiment.

References

1. Stankov, M., Predrag, D., and Stankov, D. 2003. Determination of uric acid in human serum by an enzymatic method using N-methyl-N -(4-aminophenyl)-3-methoxyaniline reagent. J.Serb.Chem.Soc. 68(8–9)691– 698.

2. Mandell, B. F. 2008. Clinical manifestations of

hyperuricemia and gout. Cleveland Clinic Journal of

Medicine Volume 75, Supplements 5 July 2008.

3. Luk AJ, Simkin PA. Epidemiology of

hyperuricemia and gout. Am J Manag Care. 2005;11 (15

Suppl):S435-S442; quiz S465-S468 [Review].

4. Saraswati-Sylvia. 2009. Diet Sehat Untuk

penyakit Asam Urat, Diabetes, Hipertensi, dan Stroke, Yogyakarta, A-Plus Books.

5. Harn-Shen, C. 2011. Clinical implications of the

metabolic syndrome and hyperuricemia. Journal of the Chinese Medical Association 74 (2011) 527-528.

6. Snyder, C. R., et al, 1997. Practical HPLC

Method Development, Second Edition. New York:John Wiley and Sons, Lnc, Hal 722-723.

7. Markham, K. R., 1988, Cara Mengidentifikasi

Flavonoid, ITB, Bandung, Hal 15-17.

8. Tjay, T.H., dan Raharja., 2002, Obat-Obat

penting, Khasiat, Penggunaan dan Efek-Efek sampingnya, Edisi V, Cetakan ke-2, Penerbit PT. Eleks Media Komputindo Kelompok Gramedia, Jakarta.

9. Zhao, X., Zhu, X. dan Pan, Y., 2005, Effects Of

Cassia Oil On Serum and Hepatic Uric Acid Levels In

Oksonate-Induced Mice and Xantine Dehiydrogenase and

Xantin Oksidase Aktivities In Mouse Liver, Journal Of

Ethnopharmacology, (http:/

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Kinetics of Organic Dyes Degradation in Water Using

Vacuum Ultra Violet Radiation

Khaled M.

Elsousy

Alaqsa University, Gaza, Gaza strip, Palestinian Territories

Corresponding: Dr. Khaled M. Elsousy, khasousi@yahoo.com

Abstaract: In this study vacuum ultraviolet (VUV) radiation (185 nm wave length) was used in the presence of

atmospheric oxygen as an advanced oxidation technique. Six organic dyes were examined as model pollutants (methyl violet, methyl blue, brilliant green, malachite-green, Remazole blue B and picric acid). Picric acid as the most persistent one was selected for more detailed kinetic investigations. The influence of each of the related main parameters was studied, Radiation time, salinity, pH, temperature and radiation intensity. Kinetics of the oxidation reaction was studied. COD was also followed up. It follows from the results that vacuum-UV radiation of 185 nm in the presence of atmospheric oxygen is an efficient method for the oxidation process. Four of sex examined dyes were degraded in different rates according to persistency of each pollutant dye. The reaction rates were in the order of:

(methyl violet > methyl blue >brilliant green > remazole > malachite green> picric acid). In the case of picric acid; the reaction was promoted by rising the temperature, raising or lowering pH above and below pH 7.0, increasing radiation dosage. The rate was inhibited by increasing salinity and buffering. The present technique was found promising in the elimination of the persistent organic pollutants out of the treated water.

Keywords: Advanced oxidation process - Vacuum ultraviolet - Wastewater treatment, Radiochemistry, Organic pollutants.

1. Introduction

During recent years the Advanced Oxidation

Processes (AOPs) have become an important group of

techniques to the treatment of hazardous water contaminants, with an increasing number of feasible applications. (Ahmed et al. 2011) Normally, the main reason for the use of AOPs is the resistivity and/or the toxicity of pollutants that makes unfeasible the biological treatment. Although AOPs use different reacting systems, all are characterized by the same chemical feature: production of OH free radicals. These radicals are extraordinarily reactive species. (Ahmed et al. 2010) The oxidation reactions involving hydroxyl radical and organic substrates in aqueous solution may be classified with respect to their character to: (Bossmann et. al. 1998) a. Abstraction of hydrogen: .OH +RH R. + H2O

b) Addition reactions: .OH + PhX HOPhX.

c) Electron transfer: .OH +RH [+R-H] + HO-

AOPs are characterized by a free radical mechanism initiated by the interactions of photons of a proper energy level with the molecules of chemical species present in

the solution such as H2O2 or with a catalyst surface such

as TiO2. (US.EPA 1998; Bakardjieva et al 2005;

Bakardjieva et al 2004).

Peroxone is another technique including the oxidation of organic pollutants using ozone with H2O2 was also

reported.(Ben Abdelmelek et al. 2011; Ben Abdelmelek et al. 2010; Chu et al. 2022) Many studies were conducted concerning the elimination of the hardly-oxidized water pollutants by different catalytic methods using hydrogen peroxide as the main source of OH free radicals. Persisting pollutants such as phenol derivatives, (Ben Abdelmelek et al. 2011; Ben Abdelmelek et al. 2010) aromatic pesticides, (Chu et al. 2011; Chu et al. 2010) fuel additives, dyes, and some other pharmaceutical drugs has been treated. (Radjenovic et al. 2009; Gaya et al. 2008)

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Khaled M. Elsousy, APAC, Vol. 2, No. 1, pp. 91-97, 2012 92

reaction rate of compounds of interest with hydroxyl radicals. Aqueous electronsare powerful reducing agents and act as nucleophiles. They react with substrates with one-electron transfer process. One of the most important reactions of aqueous electrons is with halogenated organic compounds resulting elimination of halide anions.

H2O (L) + hȞ (< 190 nm) ĺ H. + .OH ļ H+ + e- + .OH

Carbon centered radicals, formed by their reaction with hydroxyl radicals, may result in the formation of higher molecular weight compounds hindering mineralization and TOC (Total Organic Carbon) reduction. Thisproblem may be surmounted by bubbling oxygen gas. (Oppenländer 2003b)

It was reported that (VUV) radiation produced by an immersed Xe-excimer light source (172 nm) was investigated for the oxidative degradation of organic pollutants in aqueous systems. ( Hashem et al. 1997; Oppenländer and Gliese 1997; Chong et al. 2010) It was shown that the rates of degradation of the substrate decrease in the order of the applied processes, VUV/O3 >

O3 > VUV.

Influence of the oxygen concentration on the rate and reaction pathway of the degradation of organic compounds in aqueous solution by VUV-irradiation (Xe-excimer: 172 nm) was reported. (Heering 2004) The reaction rate was found to be strongly influenced by the concentration of dissolved molecular oxygen in the volume of primary reactions.

In the present research; 185 nm, VUV, radiation from a low pressure mercury lamp in the presence of atmospheric oxygen will be studied as advanced oxidation technique, the process is thought to be simple, cheap and clean where no chemicals are to be used. Six organic dyes will be tested as model pollutants, the method efficiency and the oxidation kinetic parameters will be investigated on the most persisting one of them.

2. Experimental

2.1Materials.

Picric acid; (2,4,6-trinitrophenol) was locally prepared by the nitration of phenol, (Brewster et al. 1977) Remazole blue B commercial grade was purchased from (Brilliant Blue Daystar-LP, India), methyl blue, methyl violet and malachite green, Solid sodium hydroxide and Phosphoric acid 85% ~15M were analytical grade of (Merck-Germany). Distilled water of (TDS 60 ppm) was used in all the experimental activities.

2.2 Instruments

The main kinetic investigation system was a semi-continuous system as shown in (fig. 1, a low pressure mercury, VUV, 185 nm lamp (S415 ROL-Rcan- USA) is contained inside of a sealed stainless steel cell (S300- S2R,OZAP-USA) which is protecting the electrical components of the system. The system also includes air pumpforthe continuous aeration of the reaction media, a thermo-statated water bath, and a circulation system for the treated water.

Spectrophotometer (Spectrum-900-USA) has been used for the spectroscopic measurements. A Hanna pH meter was used to adjust the pH of different solutions.

2.3 Methodology

The reaction kinetic study was conducted to compare similar samples of different pollutants; the samples have been treated identically under the same conditions of pollutant concentration (mg/l), circulating rate (0.65 dm3/ min), aeration rate (6.0 watt pump), working temperature (25.0 Co) and reaction volume (2500 ml).

Substrates depletion rates were followed spectrophotometrically. For each pollutant separately substrate concentration through the reaction term was determined. Each dye was followed at the proper Ȝmax

(picric acid at 355 nm, remazole at 570 nm, methyl blue at 665 nm, methyl violet at 590 nm and malachite green at 625 nm). Absorption bands Ȝmax were obtained

practically, Calibration curves of concentration against absorbance were obtained for the six dyes.

Kinetic investigation was performed such that: in each experimental run, reaction vessels were prepared with the proper volume (500 to 2500ml) of the reaction solution. The (observed reaction half-life) t1/2 was obtained for

each run. The half-life was approved because of the ignorance of the exact reaction order since many reactions are expected to proceed on the same time. Samples of picric acid were read spectrophotometrically with 0.02 mg/L detection limit at 355 nm Ȝmax which was

determined experimentally. Reaction parameters such as salinity (ionic strength), temperature, light intensity and reaction pH) were investigated and the resultant half-lives were plotted against readings (or values) of each parameter to clarify the relation graphically. Phosphoric acid (0.1M; H3PO4), and (0.1M; NaOH) were used to

adjust the reaction pH at the values of (5, 6, 7, 8 and 9).

3. Results and Discussion

3.1 Influence of the organic pollutant resistivity.

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Table 1: Organic pollutants degradation Half-lives (t1/2), (2500 ml total volume, T= 25±0.5 0C, circulating rate, 6.5

dm3/min).

Pollutant die Methyl violet Methyl blue Brilliant green Malachite green Remazole blue B Picric acid

Ȝmax , nm 590 665 523 625 570 355

t1/2 , hrs 1.5 1.83 1.7 3.11 Colloid 4.73

3.2 Effect of salinity

The influence of the treated water salinity on the observed reaction half-life was studied. Salinity of course is mainly proportional to ionic strength. The reaction was conducted under fixed conditions of temperature, circulating power and reaction total volume. In the studied range of [NaCl] the reaction half-life was increasing by increasing [NaCl], direct relation was observed up to 6000 mg/l sodium chloride concentration as shown in (fig. 2), the optimum oxidation rate can be achieved in desalinized treated water. In practical water treatment processes; water salinity is expected to have a negative influence on the rate of degradation. The influence of aqueous NaCl is not clear, wither it is up to free radical scavenging or simply the loss of UV photons by scattering with ions.

3.3 Effect of the volume of the treated water

Degradation rate was lowered by increasing the treated water volume. Reaction half-life was followed. It was increasing directly by increasing the volume of the treated water in the studied range, the relation is plotted in (fig. 3). The conversion rate is decreasing by decreasing the radiation intensity per unit volume of the treated water in the studied range (3.0 to 30.0 Watt per liter).

3.4 Influence of temperature

Increasing temperature has a promotional influence on the oxidation reaction in the range of (25-60 0C). It is clear in (Fig. 4) that an inverse relationship was obtained between ln 1/(t1/2) and 1/T, where a straight-line was

obtained. The result means that the process has an overall Arrhenius type behavior. It was expected according to post studies (Oppenländer et al 2005) that: the radiation intensity is affected by changing the environment temperature. In that case it may affect the UV light intensity and so the reaction kinetics, Arrhenius type relation has been observed in this case, which means a very weak influence of temperature on the radiation intensity under the present reaction conditions.

3.5 Effect of pH

Effect of pH on the oxidation reaction was examined in the pH range (4 to 10). (Fig. 5) shows the values of T1/2 corresponding to each pH value.The oxidation rate is

increasing by increasing each of OH- and H+ concentrations. It means that there are two accelerating roles for each of H+ and OH- according to various

mechanisms. Comparison of t1/2 in buffered and

non-buffered pH 7 reactions which were respectively (14.73 and 4.71 hours) implies that the presence of ions in the treated water again has a retarding influence on the degradation rate.

The last results show that the treatment of basic polluted water is easier and faster than the treatment of neutral and acidic polluted water under the same conditions. It is not recommended of course to increase the basicity of the treated water to get a faster process but at least it should be known that if the treated water was basic by chance the process is expected to be faster. 3.6 COD follow up.

Degradation was followed by measuring chemical oxygen demand COD at different time intervals. One model run was performed. Picric acid oxidation derivatives are thought to go further oxidation by time. After the breakage of aromaticity, ketones, aldehydes and carboxylic acids are thought to precede oxidation (Qian-Rong L. 2006). The final expected products are mostly CO2 and H2O. Degradation conditions were (250mg

picric acid in 2500 ml, distilled water, 25oC, 6.5 dm3/min circulating rate). (Fig. 6) insures the degradation process, t1/2 of the COD depletion was 6.5 where the oxidation

process is continuous for the derivatives as well as the starting compound picric acid.

4. Conclusion

The primary goal of this project was to investigate the VUV degradative oxidation of persistent industrial organic pollutants. The method was found efficient for four per six examined pollutants. As expected, the present method is more efficient at higher temperatures and higher intensity of VUV radiation. The present technique can be preferable over other techniques in two ways: it is simple and clean method where no chemicals are needed; only atmospheric oxygen. It can be suggested for practical applications after case study is conducted concerning the real pollutant/s specifically beside the other treated water conditions.

References:

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S. Ahmed, M.G. Rasul, W.N. Martens, R. Brown, M.A. Hashib, Advances in heterogeneous photocatalytic degradation of phenols and dyes in wastewaters: A Review. Water, Air and Soil pollution 215 (2011) 3-29. S. Bakardjieva, J. Subrt, V. Stengl, M. J. Dianez, M. J. Sayagues, Photoactivity of anatase-rutile TiO2nanocrystalline mixtures obtained by heat treatment

of homogeneously precipitated anatase. Applied Catalysis B: Environmental 58 (2005) 193-202.

S. Bekkouche, M. Bouhelassa, N.H. Salah, F.Z.Meghlaoui, Study of adsorption of phenol on titanium oxide (TiO2). Desalination 166 (2004) 355-362.

F. J. Benitez, J. L. Acero, F. J. Real, G. Roldan, Ozonation of pharmaceutical compounds: Rate constants and elimination in various water matrices. Chemosphere 77.1 (2009) 53-59.

F. L. Ben Abdelmelek, E. C. Wert, S. A. Snyder, Evaluation of UV/H2O2 treatment for the oxidation of

pharmaceuticals in wastewater. Water Research 44.5 (2010) 1440-1448.

S. Ben Abdelmelek, J. Greaves, K. P. Ishida, W. J. Cooper, W. Song, Removal of pharmaceutical and personal care products from reverse osmosis retentate using advanced oxidation processes. Environmental Science & Technology 45.8 (2011) 3665-3671.

S. H. Bossmann, E. Oliveros, S. Gob, S. Siegwart, E. Dahlen, L. Payawan, M. Straub, M. Worner, A. Braun, New evidence against hydroxyl radicals as reactive intermediates in the thermal and photochemically enhanced Fenton reactions. J. Phys. Chem 102 (1998) 5542-5550.

R.Q. Brewster, C.A. Vanderwarvf , W.E. Mcewen, Unitized experiments in organic chemistry, 4th edition, 1977, PP 394.

M.N. Chong, B. Jin, C.W.K. Chow, C. Saint, Recent developments in photocatalytic water treatment technology: A review. Water Research 44 (2010) 2997-3027

T.Chu, S.K. Umamaheshwar, A. Mumper, Removal of sulfadiazine, sulfamethizole, sulfamethoxazole, and sulfathiazole from aqueous solution by ozonation. Chemosphere 79.8 (2010) 814-820.

W. Chu, Y.R. Wang, H.F. Leung, Synergy of sulfate and hydroxyl radicals in UV/S2O8-/H2O2 oxidation of

iodinated X-ray contrast medium iopromide. Chemical Engineering Journal 178 (2011) 154-160.

M.C. Dodd, H.P.E. Kohler, U. Von Gunten, Oxidation of Antibacterial compounds by ozone and hydroxyl radical: Elimination of biological activity during aqueous ozonation processes. Environmental Science & Technology 43.7: 2498-2504.

Gaya UI, Abdullaha AH. 2008. Heterogeneous photocatalytic degradation of organic contaminants over titanium dioxide: A review of fundamentals, progress and problems. Journal of Photochemistry and Photobiology C: Photochemistry Reviews.9 (2009) 1-12.

T.M. Hashem, M. Zirlewagen, A.M. Braun Simultaneous photochemical generation of ozone in the gas phase and

photolysis of aqueous reaction systems using one VUV light source, Water Science and Technology, 35. 4 (1997) 25–30.

W. Heering, UV bources-basics, properties and applications, International ultra violet association, 6. 4 (2004) 7-13.

D. F. Ollis, E. Pelizzetti, N. Serpone, Destruction of water contaminants. Environ. Sci. Technol. 25. 9 (1991) 1523-1529.

T. Oppenländer, Photochemical Purification of water and air, advanced oxidation processes (AOPs): Principles, reaction mechanisms, and reactor concepts. Wiley-VCH, Germany (2003b) 73-91.

T. Oppenländer., S. Gliese, Mineralization of organic micropollutants (homologous alcohols and phenols) in water by vacuum-UV-oxidation (H2O-VUV) with an

incoherent xenon-excimer lamp at 172 nm.

Chemosphere, 40. 1(2000) 15-21

T. Oppenländer, J. Burgbacher, M. Kiermeier, K. Lachner, H.Weinschrottin, Improved vacuum-UV (VUV)-initiated photomineralization of organic compounds in water with a xenon excimer flow-through photoreactor (Xe2* lamp, 172 nm) containing an axially centered ceramic oxygenator. Chemosphere 60.3 (2005) 302-9.

A. B. Prevot, M. Vincenti, A. Bianciotto, E. Pramauro, Photocatalytic and photolytic transformation in aqueous solutions. App. Catal. B: Environ; 22 (1999) 149-158. L. Qian-Rong, G. Cheng-Zhi, D. Yan, y. Hao, Z. Jun-Ying, Photo-degradation of nitrobenzene using 172 nm excimer UV lamp.Materials, 133, Issue: 1. 3 (2006) 68-74

J. Radjenovic, M. Godehardt, M. Petrovic, A. Hein, M. Farre, M. Jekel, D. Barcelo, Evidencing Generation of Persistent Ozonation Products of Antibiotics Roxithromycin and Trimethoprim. Environmental Science & Technology 43.17 (2009) 6808-6815.

Vitae

Dr. Khaled M. Elsousy, was born in Gaza/ Palestinian territories in 1962. He obtained a Bs.C. in chemistry/ physics in 1985, Msc. in physical chemistry from METU-Ankara-Turkey 1988 and Ph.D. degree from ainshams Uni. Cairo Egypt in 2001.

He worked as a chemistry instructor in Alaqsa University/Gaza. His research interest includes Physical chemistry fields such as kinetics, heterogeneous catalysis, advanced oxidation technology, adsorption methods of purification and water treatment methods in general.

He is the peer reviewer of XX. He is a member of Arabian journal of chemistry and Tenside detergents and surfactants.

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Fig. 1. kinetic installation system.

0 5 10 15 20 25 30 35

0 1 2 3 4 5 6 7

[NaCl] x 1000, mg/ L

t

1/

2

,

h

rs

Fig. 2. Effect of [NaCl] on the degradation half-life of picric acid using vacuum UV. (25±0.50C, [picric acid]0=100

mg/l, reaction volume 2500ml, 6.5 dm3/min circulating rate).

0 1 2 3 4 5 6

0 1 2 3 4 5

Reaction volume, L t1/2, hrs.

Fig. 3. The relation between degradation half-life of picric acid and the amount of the treated water, (25±0.5 0C,

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-2 -1.8 -1.6 -1.4 -1.2 -1

2.8 2.9 3 3.1 3.2 3.3 3.4

1/T x 10-3, K-1

ln

1

/(

t1/2

), s

-1

Fig. 4. Effect of temperature change on the VUV oxidative degradation of picric acid, (reaction volume = 2500ml,

[picric acid]0=100 mg/l, 0.65 dm3/min circulating rate).

Fig. 5.

Influence of changing pH on picric acid degradation Half-live (t1/2), (2500ml total volume, T= 25±0.5 0C, 6.5

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Fig. 6. COD decay with process time, (2500mL total volume, T= 25±0.50C, circulating rate, 6.5 L/min). [picric

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www.worldsciencepublisher.org

Occurrence and prevalence of four viruses infecting tomatoes

in Northern districts of West Bank, Palestinian Territories

Hazem Sawalha

Department of Biology and Biotechnology, Faculty of Arts and Sciences, Arab American University of Jenin, Palestine

Email: hsawalha@aauj.edu

Abstract

–

Studying tomato viruses during 2003/2004 using immunosorbent assays revealed that tomato yellow leaf

curl virus (TYLCV) was the most dominant disease infecting tomato plants in the northern districts of the West Bank particularly Jenin and Tobas as its incidence ranged from 28-93%. The maximum disease incidence of 90-93% was recorded in Al-Far'a region of Tobas district. Cucumber mosaic virus (CMV) was the second most prevalent virus of tomato in the studied regions as its incidence ranged from 15-51%. Tomato infection with tomato mosaic virus (TMV) and potato virus Y (PVY) was considered insignificant as these viruses were detected in very few tomato samples.

Keywords – Tomato; Viruses; Palestine.

Introduction

Like many other developing countries, Palestine has an important agricultural sector, which has been considered the backbone of its economy. This was particularly true in the years after instituting its autonomy with the establishment of the National Palestinian Authority in 1994. Since then the agriculture became the largest contributor to the country’s gross product and provided employment to the economically active population (Palestinian Central Bureau of Statistics (PCBS) 2008).

Vegetables are the most important crops grown in the country, occupying 18734.4 hectares, with an annual production of 645157 metric tons. More than 31% of those vegetables are tomatoes, and the remainder include thirty-five other vegetable crops, of which cucumbers are the most important. Tomato is the main greenhouse crop but it is also cultivated in low tunnels and in open fields, comprising a total annual production of 203999 metric tons (PCBS 2008). Several viruses are known worldwide to attack tomatoes causing serious damage and yield reduction. TMV, TYLCV, CMV and PVY are of the ten most viruses affecting the crop elsewhere (Oetting and Yunis, 2004, Agrios, 1997, Pico et. al. 1996, Sawalha. (2009a, 2009b, 2010).

Viral diseases of tomatoes occasionally cause serious damages and large economic losses. The amount of loss depends on the viral disease involved, the strain of the virus, the variety of tomato, the age of the plant at infection time, the presence of other diseases, and the

extent that viruses have spread during the planting of the crop (Walkey 1985).

Tomato production in Palestine, however, has not yet reached its full potential due to many factors related to climate and poor management, and above all, due to the high level of pests and diseases (PCBS 2008). In regard to diseases, viral diseases may be considered an important factor responsible for the recurrent crop failure in the country forcing many farmers to switch their choice of crop production from tomato to other vegetables (Sawalha 2010, Ministry of Agriculture, personal communication).

As tomato is the most important vegetable crop in the country, this research aims to study and identify the viruses affecting this crop and to shed light on their occurrence and prevalence. Northern districts including Jenin and Tobas were selected for this study because tomato production in those regions is 48250 metric tons and they occupy about 47% of the total tomato production in the West Bank part of Palestine (PCBS 2008).

Materials and Methods

Regions of study

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Hazem Sawalha., APAC, Vol. 2, No. 1, pp. 98-101, 2012 99

fields located in Jenin were selected in Zababdeih, Al-Jededeih, and Qabatyya regions. Two fields were selected in Tobas; one of them was in Kashda village and the other was selected in Al-Far’a region a few hundred meters away from the town’s midpoint (Fig 1).

Growing season

The experiments were carried out during the summer growing season because this season is considered the main planting one of tomato in the regions of study throughout the year. In this season, seeds are sown during April and May and the seedlings are transplanted from the middle of May until the middle of June.

The pattern of tomato cultivation in the regions of study

Tomato is planted in the studied area in rows with a width of about 1 to 1.2m. The lands are first prepared by hoeing twice and then adding manure. Chemical fertilizers are added in three equal doses; the first when preparing the land, the second after transplanting and the third at fruit set.

Field visits and sample collection

Field visits in the studied regions began in July and continued until the end September. Samples were collected at least one month after the transplanting of tomato seedlings to ensure that tomato had become infected after being exposed for a sufficient period to the viral source. The selected fields were visited weekly in a regular manner and leaf samples were collected randomly from top parts of plants. The samples were labelled and kept frozen for laboratory testing.

Virus identification and incidence

Virus identification was done serologically using virus-specific antibodies purchased from international companies. Thus, indirect enzyme-linked immunosorbent assay (I-ELISA) adopted by Clark et. al (1986) was used to test the plant samples for CMV, PVY and TMV. With respect to the other case, the triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) was used to test the TYLCV infection. TAS-ELISA was used according to Macintosh et. al. (1992), Muniyappa et. al.

(1991), Sawalha et. al. (2000: 339) and Sawalha (2000: 24-89). The antibodies of CMV, PVY and TMV and the goat anti-rabbit conjugate were purchased from Bioreba, Inc. The polyclonal and monoclonal antibodies of TYLCV, along with the rabbit anti-mouse conjugate, were purchased from Adgen Ltd (Scotland, UK.). The

results of the ELISA tests were recorded one hour after the substrate incubation took place using the automated ELISA-Reader. The light absorbance was measured for ELISA wells at 405 nanometer (Sawalha (2009c)

Statistical analysis

Statistical analysis of the data was done using the Two-Sample Tests of Proportions (TSTP) to compare virus occurrence in the studied regions. The results were analysed using a level of significance when Ȑ = 0.05 (Lind et. al. 2005).

Results

Virus identification and incidence

ELISA tests showed that the TYLCV, CMV, TMV and PVY parasitize tomato fields in different percentages. ELISA readings recorded for the virus-infected samples were at least eight times greater than the readings recorded for the virus-free samples (Fig 1).

Fig 1: ELISA de te ction of tomato viruse s showing the diffe re nce be twe e n positive and ne gative control sample s

0 0.2 0.4 0.6 0.8 1 1.2 1.4

TYLCV CMV TMV PVY

Tom ato virus

Ab s o rb a n c e a t 4 0 5 n m Positive sample Negative sample

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Hazem Sawalha., APAC, Vol. 2, No. 1, pp. 98-101, 2012 100

Fi g 2: Ave rage i n ci de n ce of tom ato vi ruse s i n the stu dy re gi ons i n 2003/2004

0 10 20 30 40 50 60 70 80 90 100

Kashda Al-Far'a Qabatyya Al-Zababdeih Al-Jededeih

Re gion Pe rc e n ta g e o f in fe c ti o n TYLCV CMV TMV PVY

Statistical analysis revealed that the proportions of TYLCV in the studied regions were greater than CMV, TMV and PVY. The maximum computed Z values (Ȑ = 0.05) for the TYLCV compared with the other viruses were recorded in Al-Far'a region followed by Kashda, Qabatyya, Al-Zababdeih and Al-Jededeih. No significant difference was recorded between TYLCV and CMV infecting tomato fields in Qabatyya region as the computed Z value was much less than the critical one (Table 2)

Table 2: Statistical analyses and the Z value of the TSTP. The Z table = 1.65

Region Virus combination Computed Z value Decision

Kashda TYLCV/CMV 6.74 S

TYLCV/TMV 9.80 S

TYLCV/PVY 10.16 S

Al-Far'a TYLCV/CMV 8.66 S

TYLCV/TMV 11.81 S

TYLCV/PVY 12.54 S

Qabatyya TYLCV/CMV 0.14 NS

TYLCV/TMV 6.63 S

TYLCV/PVY 8.30 S

Al-Zababdeih TYLCV/CMV 2.10 S

TYLCV/TMV 5.15 S

TYLCV/PVY 6.17 S

Al-Jededeih TYLCV/CMV 2.20 S

TYLCV/TMV 5.55 S

TYLCV/PVY 5.83 S

S: Significant, NS: Non significant

Discussion

Tomato viruses were identified serologically using I-ELISA for CMV, and TMV, PVY, and TAS-I-ELISA for TYLCV. TYLCV is the most prevalent virus threatening tomato fields in the studied regions. The higher incidence of TYLCV (31-93%) is attributed to the abundance of whiteflies (Bemisia tabaci) and the overlapping that occurs between tomato growing seasons in the regions (Sawalha 2010). The 93% incidence of the virus in Al-Far’a may be attributed also to the warm climate which helps the whiteflies to appear earlier and to reproduce more compared with the other regions (PCBS 2005, Sawalha 2010). In this regards, Nava-Camberos et. al.

(2001) reported that the warm climate is the best condition for the whitefly development, fecundity and survival. Similar results were recorded in Jordan where Al-Musa and Mansour (1983) reported that TYLCV was the predominant virus affecting tomatoes in the Jordan Valley. CMV was detected to be the second-most prevalent virus affecting tomato fields in the studied regions. This infection with CMV may be attributed to allate aphid species which are usually ample in the region, especially the green peach aphid (Myzus persicae

Sulzer) and the melon aphid (Aphis gossypii Glover). The virus has a wide host range (800 species) from which it can be acquired by aphids and transmitted in a non-persistent manner and can also be mechanically transmitted (Oetting and Yunis, 2004, Trigiano et. al.

2004). Another reason for the wide spread occurrence of CMV in the studied regions may be attributed to the intensive and annual culturing of cucurbits including cucumber, squash and melon in the region. According to PCBS (2008), these crops are planted in a wide area with an annual production of 15376 and 9250 metric tons in Jenin and Tobas districts, respectively. These crops are the most suitable hosts for CMV, so their presence in the tomato growing sites makes them the viral source from which the allate aphids transmit the disease to tomato (Oetting and Yunis 2004, Sacristian et. al. 2004). The

Table 1: Number of samples collected from the studied region and the percentages of infection with TYLCV, CMV, TMV and PVY.