Online Version ISSN: 1314-412X

June 2012Volume 4, Number 2

2012

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Genetics and Breading

Atanas Atanasov (Bulgaria)Ihsan Soysal (Turkey)Max Rothschild (USA)Stoicho Metodiev (Bulgaria)

Nutrition and Physiology

Nikolai Todorov (Bulgaria)Peter Surai (UK)Zervas Georgios (Greece)Ivan Varlyakov (Bulgaria)

Production Systems

Dimitar Pavlov (Bulgaria)Dimitar Panaiotov (Bulgaria)Banko Banev (Bulgaria)Georgy Zhelyazkov (Bulgaria)

Agriculture and Environment

Georgi Petkov (Bulgaria)Ramesh Kanwar (USA)

Product Quality and Safety

Marin Kabakchiev (Bulgaria)Stefan Denev (Bulgaria)Vasil Atanasov (Bulgaria)

English Editor

Yanka Ivanova (Bulgaria)

2012

Volume 4, Number 2June 2012

Online Version ISSN: 1314-412X

Influence of different factors on tannins and flavonoids extraction of some thyme varieties representatives of thymol, geraniol and citral chemotype

1 2G. Zhekova * , D. Pavlov

1 Institute of Roses, Essential and Medical Cultures, 49 Osvobozhdenie, 6100 Kazanlak, Bulgaria2 Department of Plant Production, Faculty of Agriculture, Trakia University, 6000 Stara Zagora, Bulgaria

Abstract. Three chemotype thyme – thymol “Nemska zima”, citral – “Slava” and geraniol – “Pagane” with purpose obtaining of extract for cosmetics and pharmaceutics application were investigated in the present work. The extraction was carried out as stationary periodical process with ratio between dry herb and solvent 1:10, temperature from 20ºC to 60ºC, duration from 1 to 5 hours with solvents – 60% ethanol and 85% glycerin. The obtained extracts were determined the content of tannins and flavonoids. The data were presented as four factors analysis – type of solvent, thyme variety, temperature and duration of process. The results showed that the thyme variety has the strongest influence on extracted tannins and flavonoids, followed by solvent type, temperature and duration of the process. The extract obtained from variety “Nemska zima” has the highest content of derived substances followed by “Slava” and “Pagane”. The ethanol derives tannins and flavonoids in bigger extent than the glycerin. The differences between the two solvents are most significant at temperature 20ºC.

Keywords: thyme, tannins, flavonoids, extraction

AGRICULTURAL SCIENCE AND TECHNOLOGY, VOL. 4, No 2, pp 148 - 153, 2012

aren't subject to standardization. (Kamedulski, 1980). For Introduction pharmaceuticals preparation (cough syrups – Tusirol and Hustagil) thyme extracts obtained by solvents are used: ammonia 10%, Thyme is an aromatic perennial herb of the Lamiaceae family glycerin 85% and ethanol 90% in ratio 1:20:70 (Bulgarian Drag characterized with wide species diversity. The thyme contains 0,5 – Agency, 2007; Bulgarian Drag Agency, 2008). The data indicated in 2,0% essential oil with main constituent depending on species and literature about obtaining of extraction are related mostly to thyme, variety. (Jia et al., 2010; Cases et al.,2009; Georgiev and Stoyanova, thymol chemotype. What are the possibilities for extraction of other 2006). In the different parts of the plants phenols, vitamins and other hemotype established in Bulgaria varieties "Slava" (Thymus substances, obtained mainly by extraction are in great interest in vulgaris, citral chemopype) and "Pagane." (Thymus marschalianus, food industry and cosmetics (Guilen and Manzanos, 1998; Zheng geraniol chemotype) are not completely established yet. and Wang, 2001; Stoilova et al.,2008).

The purpose of the present study is to examine the influence of A dry material is used for the obtaining of cosmetic extracts in basic factors of extraction at different hemotype thyme and the which the cells are dead and their walls have the properties of simple possibility for obtaining cosmetic and pharmaceutical extracts rich of screens with better permeability (Topalov et al., 1983; Yankulov, biological activity substances.2000; Zhekova et al., 2011). According to Kamedulski (1980) the

main factors influencing on eliciting and speed of extraction are the selecting a suitable solvent and the creation of optimal conditions for

Material and methods the diffusion process. Important to the carry out of diffusion are the existence of concentration gradient in the system solvent – raw materials and ensuring the intensive molecules movement by The investigation was conducted with thyme varieties “Slava”, increasing the temperature. In this aspect Damyanova et al. (2008) “Nemska zima” and “Pagane”, cultivated in experimental filed in have been investigated temperature and duration of process Institute of Roses, Essential and Medical Cultures. The plants were influence on obtaining Thymus vulgaris extract with different ethanol gathered in stage full flowering, in sunny and dry weather, free of concentration. They constituted a full two levels, two-factor model of impurities and dried in ventilated room, in shady place up to 11-12% experiment and proved that the highest amount of biological activity moisture.substances has derived at 60ºC temperature and duration 5 hours Ethanol 60% and glycerin 85% were used to preparing of the independent of used solvent. The factor temperature has the bigger extracts. The extraction was carried out as stationary periodical influence on extraction, which was confirmed by the coefficients of process. The dry thyme grind in laboratory mill to 0,5 – 1,0 cm was the resulting equations. (Damyanova et al., 2008; Damyanova et al., thrown in flask and soaked with solvent at ratio 1:10. The 2004). Chizzola et al. (2008) has been pointed that the extracts temperature and duration of process were examined as two factors obtained with 60% ethanol reveal the strongest antioxidant activity, three levels experiment. which was related to better recovery of phenols compounds. The content of flavonoids in the extract was determined using

Water infusions are the simplest extractive forms of the thyme. modified method by Russian pharmacopeias (State Pharmacopoeia They are prepared easily, through material soaking at ratio water: of USSR) using rutin as a reference compound. Five ml of 0,2% material 1:20 to 1:50 with cold, warm and boiling water. These extract solution in 60% ethanol was mixed with 1ml aluminum extracts have the blemishes that are very dilute, perishable and trichloride in etanol (5%) and diluted with 60% ethanol to 25ml. Blank * e-mail: gzhekova@abv.bg

148

Solvent Ethanol 60%

Solvent Glycerin 85%

1

2

3

4

5

6

7

8

9

1

2

3

4

5

6

7

8

9

Table 1. Variants of the experiment and content of the extracted bioactive substances of thyme "Nemska zima"

No Duration, h -2Flavonoids mg/ml*10Тemp., ºC Tannins, %

0.82 ± 0.03

0.82 ± 0.02

0.70 ± 0.08

0.93 ± 0.02

0.97 ± 0.04

1.04 ± 0.04

1.07 ± 0.04

1.28 ± 0.03

1.16 ± 0.04

0.31 ± 0.02

0.43 ± 0.03

0.55 ± 0.02

0.37 ± 0.06

0.61 ±0.00

0.66 ± 0.06

0.64 ± 0.04

1.01 ± 0.00

1.04 ± 0.03

6.47 ± 0.05

6.56 ± 0.09

4.85 ± 0.29

6.24 ± 0.08

6.52 ± 0.10

7.13 ± 0.05

7.18 ± 0.37

8.26 ± 0.28

7.23 ± 0.28

2.77 ± 0.06

3.54 ± 0.09

4.33 ± 0.19

3,54 ± 0.25

5.18 ± 0.46

5.34 ± 0.01

5.22 ± 0.16

6.90 ± 0.01

7.12 ± 0.01

20

20

20

40

40

40

60

60

60

20

20

20

40

40

40

60

60

60

1

3

5

1

3

5

1

3

5

1

3

5

1

3

5

1

3

5

149

sample was prepared without aluminum trichloride and diluted to Results and discussion25ml. The absorption at 416nm was measured after 40 min. at room temperature against 60% ethanol. Solutions of rutin in ethanol The content of tannins and flavonoids in thyme varied (0,016, 0,024, 0,032, 0,04, 0,048 mg/ml) were used for calibration. considerably depending on the type of solvent, the thyme variety, the The amount of flavonoids in plant extract in rutin equivalent (RE) was temperature and the duration of the process. Into the variety calculated by the following formula: “Nemska zima” the best results of tannins and flavonoids with

Fl(mg/ml)=(As – Ao).k.5/m, solvent 60% ethanol (respectively 1,28% and 8,26mg in 100g where As is the absorption of sample, Ao – absorption of blank extract) were obtained when the temperature was 60ºC and the

sample, k is tgα of the calibration right, m – weight of the extraction. duration was 3 hours (Table 1). With solvent 85% glycerin, The tannins content in the extracts was determined according respectively 1,04% and 0,071 mg/ml were obtained at temperature

to Atanasova and Hristova (2009) after some modification. Five ml of 60ºC and extraction time 5 hours. 0,1% water solution of extract was transferred in conical flask, then There was a tendency in the glycerin extracts when duration of 5ml of indigo sulfuric acid solution and 150ml distilled water were process increases the amount of extracted substances increases added. 0,1N aqueous solution of KMnO4 was used for titration until also and respectively their values were the lowest in 1 hour and the the blue colored solution becomes golden yellow. The blank sample highest in the 5 hour. The same statement was not the same for was prepared with mixture of 5ml indigo carmine solution and 150ml ethanol extract, where the reduction of the extracted components to distilled water. Standard solution of indigo carmine was prepared as the 5-th hour of extraction is visible. The extraction time has different follows: 6g Indigo carmine was dissolved in 500ml distilled water by influence on the obtaining of active substance depending on solvent heating, after cooling 50ml of 95-97% H2SO4 was added, the used and the significant of the duration in different extraction will be solution is diluted to 1L and then filtered. too different.

The percentage of tannins was calculated using following The temperature had substantial effect on both type of equation: examined solvents. The extraction of bioactive substances was

Tannins (%) = (Vs – Vbl). 0,004157.F.10.100/m, improved by increasing it, which was explained by the intensification where: Vs is the volume of 0,1N aqueous solution of KMnO4 for of diffusion processes in the system and reducing the viscosity of the

the titration of the sample, ml; Vbl is the volume of 0,1N aqueous solvent. The values had to be conformable to thermal stability of the solution of KMnO4 for the titration of the blank sample, ml; 0,004157 solvent and active ingredients this factor reached and then to be is tannins equivalent in 1ml of 0,1N KMnO4; F – the factor of KMnO4; choosed an appropriate worked maximum. The best extraction of m – mass of the sample taken for the analysis; 100 – percent. bioactive substance was observed at 60 ºC (respectively 0.77%

All samples were measured in triple repetition and processed tannins and 0,063mg/ml flavonoids) in ethanol extracts of thyme by the methods of statistical and regression analysis. variety “Slava” as in a variety “Nemska zima” and the duration of the

150

process after the first hour is not substantial (Table 2). An increase of “Pagane” were similar to varieties "Slava" and "Nemska zima". the extracted components with increasing temperature and duration There were the best derived results at temperature 60ºC where the of the process was observed in the glycerin extracts and there was duration after 1 hour did not affect to yield (Table 3).the highest values at 5 hours extraction at 60 º C (respectively 0.55% The influence of tested factors (the type of solvent, thyme and 0,049mg/ml). variety, temperature and duration of the process) on extracting of

Changes in the extraction of active substances in thyme variety biologically active substances in the thyme extraction and their

Solvent Ethanol 60%

Solvent Glycerin 85%

Table 2. Variants of the experiment and content of the extracted bioactive substances of thyme "Slava."

No Duration, h -2Flavonoids mg/ml*10Тemp., ºC Tannins, %

Solvent Ethanol 60%

Solvent Glycerin 85%

Table 3. Variants of the experiment and content of the extracted bioactive substances of thyme "Pagane."

No Duration, h -2Flavonoids mg/ml*10Тemp., ºC Tannins, %

1

2

3

4

5

6

7

8

9

1

2

3

4

5

6

7

8

9

0.29 ± 0.01

0.28 ± 0.01

0.31 ± 0.01

0.44 ± 0.01

0.45 ± 0.01

0.48 ± 0.01

0.55 ± 0.01

0.55 ± 0.01

0.55 ± 0.01

0.11 ± 0.01

0.17 ± 0.01

0.24 ± 0.02

0.19 ± 0.01

0.26 ± 0.02

0.31 ± 0.01

0.33 ± 0.01

0.40 ± 0.01

0.48 ± 0.02

2.70 ± 0.01

3.00 ± 0.09

3.13 ± 0.02

3.47 ± 0.01

3.54 ± 0.14

3.90 ± 0.01

4.14 ± 0.05

3.91 ± 0.18

4.11 ± 0.10

1.50 ± 0.06

1.54 ± 0.08

2.04 ± 0.07

1.68 ± 0.03

2.36 ± 0.08

2.67 ± 0.07

2.65 ± 0.01

2.99 ± 0.04

3.75 ± 0.01

20

20

20

40

40

40

60

60

60

20

20

20

40

40

40

60

60

60

1

3

5

1

3

5

1

3

5

1

3

5

1

3

5

1

3

5

1

2

3

4

5

6

7

8

9

1

2

3

4

5

6

7

8

9

0.48 ± 0.02

0.45 ± 0.02

0.44 ± 0.01

0.61 ± 0.01

0.62 ± 0.02

0.63 ± 0.03

0.72 ± 0.02

0.76 ± 0.02

0.77 ± 0.01

0.18 ± 0.01

0.28 ± 0.00

0.34 ± 0.02

0.28 ± 0.02

0.38 ± 0.02

0.42 ± 0.01

0.43 ± 0.01

0.52 ± 0.02

0.55 ± 0.01

4.95 ± 0.23

4.58 ± 0.25

4.89± 0.33

5.65 ± 0.08

5.75 ± 0.23

5.73 ± 0.21

6.19 ± 0.42

6.33 ± 0.37

6.20 ± 0.47

1.25 ± 0.10

2.23 ± 0.13

2.56 ± 0.15

2.32 ± 0.07

3.25 ± 0.16

3.53 ± 0.17

3.50 ± 0.01

4.31 ± 0.06

4.49 ± 0.04

20

20

20

40

40

40

60

60

60

20

20

20

40

40

40

60

60

60

1

3

5

1

3

5

1

3

5

1

3

5

1

3

5

1

3

5

151

interaction was different depending on the type of the extracted solvent as a factor was much more strongly exhibited in comparison compounds. The variety had the strongest influence on the degree with the tannins. The temperature as a factor in the flavonoids had of tannins extraction – 46,54% (Table 4). The temperature was slightly lower influence also. The influence of all factors was arranged on the second rank and the type of extraction on the third statistically significant at P<0,00001.please. The difference between the type of solvent and temperature The regression coefficient R² obtained from the design of was a small. The duration of the process had the insignificant present investigation for tannins was 0,9361 and for flavonoid was influence than the discussed factors (2,68%). The interactions 0,9332 that mean in 93% of research variants the change in solvent with variety, variety with temperature and solvent with extracted tannins and flavonoids amount was due to changes in the duration had some more important. The influence of all factors was value of the investigated factors. The type of row material in case the statistically significant at very high confidence level P <0,00001. variety of thyme had the strongest influence which was connected

Almost the same trend of influence of the tested factors in with amount of tannins and flavonoids in the herb. The next factor extraction of flavonoids was remained. The degree of influence of about significance influencing on flavonoids extraction was the type the variety was slightly lower – 43,82% (Table 5) in flavonoids, in of solvent followed of temperature and extraction time which showed comparison with the tannins. In flavonoids the influence of the type of that to be selected appropriate solvent was more important rather

Table 4. Influence of the factors on the extraction of tannins.

SS MS F p %d. f.

2.49483

5.66089

2.63694

0.32581

0.26288

0.05139

0.20330

0.20208

0.10026

0.05324

0.03707

0.03368

0.01588

0.06618

0.01959

{1}Extract

{2}Variety

{3}Temperature

{4}Duration

Extract*Variety

Extract*Temperature

Variety*Temperature

Extract*Duration

Variety*Duration

Temperature*Duration

Extract*Variety*Temperature

Extract*Variety*Duration

Extract*Temperature*Duration

Variety*Temperatura*Duration

1*2*3*4

4064.03

4610.73

2147.76

265.37

214.11

41.86

82.79

164.59

40.83

21.68

15.09

13.72

6.47

13.48

3.99

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

0.0001

0.0000

0.0003

20.51

46.54

21.68

2.68

2.16

0.42

1.67

1.66

0.82

0.44

0.30

0.28

0.13

0.54

0.16

1

2

2

2

2

2

4

2

4

4

4

4

4

8

8

2.49483

2.83044

1.31847

0.16290

0.13144

0.02570

0.05082

0.10104

0.02507

0.01331

0.00927

0.00842

0.00397

0.00827

0.00245

Table 5 Influence of the factors on the extraction of flavonoids

SS MS F p %d. f.

139.208

219.530

83.111

12.312

12.279

4.260

4.625

10.255

3.047

1.706

1.030

2.173

1.244

2.932

3.228

{1}Extract

{2}Variety

{3}Temperature

{4}Duration

Extract*Variety

Extract*Temperature

Variety*Temperature

Extract*Duration

Variety*Duration

Temperature*Duration

Extract*Variety*Temperature

Extract*Variety*Duration

Extract*Temperature*Duration

Variety*Temperatura*Duration

1*2*3*4

4354.01

3433.13

1299.74

192.55

192.03

66.62

36.17

160.38

23.82

13.34

8.05

16.99

9.72

11.46

12.62

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

0.0000

27.79

43.82

16.59

2.46

2.45

0.85

0.92

2.05

0.61

0.34

0.21

0.43

0.25

0.59

0.64

1

2

2

2

2

2

4

2

4

4

4

4

4

8

8

139.208

109.765

41.556

6.156

6.140

2.130

1.156

5.128

0.762

0.426

0.257

0.543

0.311

0.367

0.403

152

than to be seek in other factors changes for this type of active for their extraction was necessary also.ingredients. The type of solvent and temperature had almost the The interpolated values of examined factor for the different same effect for tannins (20,51% and 21,68%), which showed with a biological active ingredients showed different influence at the properly selected solvent and increasing the temperature of process relevant levels.

0,

,5

Profiles for Predicted Values and Desirability

Desirability

Fla

vono

ids

7,89

404,

3541

8140

8

Temperature DurationVarietyExtraction

1 3 5EthanolGlicerine

N zima PaganeSlava

20 40 60

Figure 1. A profile of predictable influence on flavonoids extraction of detected factors.

10,000

3,2483

-1,000

,34382

0,

,5

1,

Profiles for Predicted Values and Desirability

1,10

01,5

4881

-,00

24

Tann

ins

DesirabilityTemperature DurationVarietyExtraction

1 3 5EthanolGlicerine

N zima PaganeSlava

20 40 60

Figure 2. A profile of predictable influence on tannins extraction of detected factors

10,000

3,2483

-1,000

,34382

1,

153

The ethanol extracted more completely than glycerine tannins Bulgarian Drug Agency, Patient leaflet, 1/25.02.2008.as well as flavonoids. That was shown from the interpolated levels of Bulgarian Drug Agency, Patient leaflet, 5/17.12.2007.factors influence and the profile of predictable and optimization of Cases A, Perez B, Navarrede P, Mora E, Peria B, Pelluzo A, Calvo process. The extracts obtained from variety “Nemska zima” were R, Ron D and Varela F, 2009. Variability in the chemical composition more abundant with extracted biological active substances followed of wild Thymus vulgaris L., Acta Horticulturae, 826, 159-166of variety “Slava” and variety “Pagane” that came from thyme Chizzola R, Michitsch H and Franz C, 2008. Antioxidative varietals special feature and the existed diversity in this plant genus. Properties of Thymus vulgaris Leaves: Comparison of Different The thyme representative of indicated chemotype showed Extracts and Essential Oil Chemotypes. Journal of Agricultural and differences in the amount of tannins and flavonoids (Figures 1 and Food Chemistry, 56, 16, 6897–6904.2). An increasing of the temperature from 20ºС to 60ºС had the Damyanova S, Stoyanova A and Damyanov D, 2004. Technology expected positive impact on the extraction process of the studied for plant extracts. Thyme, Scientific Session University of Rousse components. Regarding to the duration of the process, it is clear that "Angel Kanchev", 41, 1.2, 94-97. the extraction process to 3 hours ran intensive, after that the change Damyanova S, Tasheva S, Stoyanova A and Damyanov D, 2008. in the values of the extracted substances was low and the process Investigation of Extracts from Thyme (Thymus vulgaris L.) for can be regarded as unprofitable. Application in Cosmetics, Jeobp 11, 5, 443-450.

Georgiev E and Stoyanova A, 2006. Guide to the specialist of the flavoring industry, BNAEOPC, Plovdiv, 212-214. Guilen M and Manzanos M, 1998. Study of the composition of the Conclusiondifferent parts of a Spanish Thymus vulgaris L. Plant Food Chemistry, 63, 3, 373-383.

The variety “Nemska zima” was the most appropriate about Jia HL, Ji QL, Xing SL, Zhang PH, Zhu GL and Wang XH, 2010.

extraction in the aspect of source of tannins and flavonoids for the Chemical composition and antioxidant, antimicrobial activities of the

cosmetic. The extracts obtained from the “Nemska zima” exceed the essential oils of Thymus marschallianus Will. and Thymus proximus

other two varieties by 30% to 60% more tannins and flavonoids in the Serg, Journal of Food Science,75, 1,59-65.

ethanol extract and 30% to 60%more tannins and 30% to 40% more Kamedulski V, 1980, Technology of plant extraction for

flavonoids in glycerin extract.phytochemistry, New in natural and synthetic aromatics products

The ethanol is better for extracts and derives in a bigger extent manufacture, UFT, Plovdiv, 99-116.

tannins as well as flavonoids in comparison to glycerin. The State Pharmacopeia of USSR, 1989. Moscow, Medicina, 2, 324-

differences between the two solvents are most significant at 20ºC. 333.

The increasing of temperature decreases the differences, which is Stoilova I, Bail S, Buchbauer G, Krastanov A, Stoyanova A,

more strongly expressed in flavonoids. The temperature effects in a Schmidt E and Jirovetz L, 2008. Chemical composition, olfactory

stronger degree on the extracting ability of glycerine and when evaluation and antioxidant effects of an essential oil of Thymus

cosmetic procedure requires the use of natural solvent (other than vulgaris L. from Germany. Natural Product Communications, 3, 7,

ethanol) the improving of the quality of the extracts can be found in 1047-1050.

this direction.Topalov V, Gavrailov M and Yankulov Y, 1983. Herbals and herbal gathering, Hr. G. Danov, Plovdiv, 226-227.Yankulov Y, 2000. A main aromatic plants, Sofia, 114-120Zhekova G, Dzhurmanski A and Nikolova M, 2011. Essential oil Referencescontent and composition of Thyme “German winter”. Agricultural Science and Technology, 3, 2, 123-125.Atanasova M and Hristova V, 2009. Determination of tannins Zheng W and Wang S, 2001. Antioxidant activity and phenolic content by titrimetric method for comparison of diferent plant compounds in selected herbs. Journal of Agricultural and Food species, Journal of the University of Chemical Technology and Chemistry, 49, 11, 5165-5170. Matallurgi, 44, 4, 413-415.

Instruction for authors

Preparation of papersPapers shall be submitted at the editorial office typed on standard typing pages (A4, 30 lines per page, 62 characters per line). The editors recommend up to 15 pages for full research paper ( including abstract references, tables, figures and other appendices)

The manuscript should be structured as follows: Title, Names of authors and affiliation address, Abstract, List of keywords, Introduction, Material and methods,Results, Discussion, Conclusion, Acknowledgements (if any), References, Tables, Figures.The title needs to be as concise and informative about the nature of research. It should be written with small letter /bold, 14/ without any abbreviations. Names and affiliation of authorsThe names of the authors should be presented from the initials of first names followed by the family names. The complete address and name of the institution should be stated next. The affiliation of authors are designated by different signs. For the author who is going to be corresponding by the editorial board and readers, an E-mail address and telephone number should be presented as footnote on the first page. Corresponding author is indicated with *. Abstract should be not more than 350 words. It should be clearly stated what new findings have been made in the course of research. Abbreviations and references to authors are inadmissible in the summary. It should be understandable without having read the paper and should be in one paragraph. Keywords: Up to maximum of 5 keywords should be selected not repeating the title but giving the essence of study. The introduction must answer the following questions: What is known and what is new on the studied issue? What necessitated the research problem, described in the paper? What is your hypothesis and goal ?Material and methods: The objects of research, organization of experiments, chemical analyses, statistical and other methods and conditions applied for the experiments should be described in detail. A criterion of sufficient information is to be

possible for others to repeat the experi-ment in order to verify results.Results are presented in understandable tables and figures, accompanied by the statistical parameters needed for the evaluation. Data from tables and figures should not be repeated in the text.Tables should be as simple and as few as possible. Each table should have its own explanatory title and to be typed on a separate page. They should be outside the main body of the text and an indication should be given where it should be inserted.Figures should be sharp with good contrast and rendition. Graphic materials should be preferred. Photographs to be appropriate for printing. Illustrations are supplied in colour as an exception after special agreement with the editorial board and possible payment of extra costs. The figures are to be each in a single file and their location should be given within the text. Discussion: The objective of this section is to indicate the scientific significance of the study. By comparing the results and conclusions of other scientists the contribution of the study for expanding or modifying existing knowledge is pointed out clearly and convincingly to the reader.Conclusion: The most important conse- quences for the science and practice resulting from the conducted research should be summarized in a few sentences. The conclusions shouldn't be numbered and no new paragraphs be used. Contributions are the core of conclusions. References:In the text, references should be cited as follows: single author: Sandberg (2002); two authors: Andersson and Georges (2004); more than two authors: Andersson et al.(2003). When several references are cited simultaneously, they should be ranked by chronological order e.g.: (Sandberg, 2002; Andersson et al., 2003; Andersson and Georges, 2004).References are arranged alphabetically by the name of the first author. If an author is cited more than once, first his individual publications are given ranked by year, then come publications with one co-author, two co-authors, etc. The names of authors, article and journal titles in the Cyrillic or alphabet different from Latin, should be transliterated into Latin and article titles should be translated into English. The original language of articles and books translated into English is indicated in

parenthesis after the bibliographic reference (Bulgarian = Bg, Russian = Ru, Serbian = Sr, if in the Cyrillic, Mongolian = Мо, Greek = Gr, Georgian = Geor., Japanese = Jа, Chinese = Ch, Arabic = Аr, etc.)The following order in the reference list is recommended:Journal articles: Author(s) surname and initials, year. Title. Full title of the journal, volume, pages. Example:Simm G, Lewis RM, Grundy B and Dingwall WS, 2002. Responses to selection for lean growth in sheep. Animal Science, 74, 39-50Books: Author(s) surname and initials, year. Title. Edition, name of publisher, place of publication. Example: Oldenbroek JK, 1999. Genebanks and the conservation of farm animal genetic resources, Second edition. DLO Institute for Animal Science and Heal th, Netherlands.Book chapter or conference proceedings: Author(s) surname and initials, year. Title. In: Title of the book or of the proceedings followed by the editor(s), volume, pages. Name of publisher, place of publication. Example: Mauff G, Pulverer G, Operkuch W, Hummel K and Hidden C, 1995. C3-variants and diverse phenotypes of unconverted and converted C3. In: Provides of the Biological Fluids (ed. H. Peters), vol. 22, 143-165, Pergamon Press. Oxford, UK.Todorov N and Mitev J, 1995. Effect of level of feeding during dry period, and body condition score on reproductive perfor-

thmance in dairy cows,IX International Conference on Production Diseases in Farm Animals, Sept.11 – 14, Berlin, Germany, p. 302 (Abstr.).Thesis:Penkov D, 2008. Estimation of metabolic energy and true digestibility of amino acids of some feeds in experiments with muscus duck (Carina moshata, L). Thesis for DSc. Agrarian University, Plovdiv, 314 pp.

The Editorial Board of the Journal is not responsible for incorrect quotes of reference sources and the relevant violations of copyrights.

Volume 4, Number 2June 2012

CONTENTS

Genetics and Breeding

Nutrition and Physiology

Production Systems

Agriculture and Environment

Product Quality and Safety

Polymorphism of the C3 component of the complement system and its impact on serum lysozyme concentrations and complement activityTs. Koynarski, L. SotirovUse of cluster analysis for selection of quality at the early stages of wheat breedingI. Stoeva, I. Belchev, K. Kostov, I. Ivanova, E. PenchevGenetic relationship among Nigerian indigenous sheep populations using blood protein polymorphism

M. Akinyemi, A. SalakoEffect of melatonin treatment on fertility, fecundity, litter size and sex ratio in ewesG. BonevThe effects of productive status and age on blood serum parameters before oestrous synchronisation in Awassi and Awassi crosses sheep breedG. Bonev, R. Slavov, S. Georgieva, P. Badarova, S. Omar

Chemical composition and nutritive value of fungal treated acha strawA. Akinfemi Carcass traits, intestinal morphology and cooking yield of broilers fed different fermented soyabean meal based dietsM. M. Ari, B.A. Ayanwale, T. Z. Adama, E.A. Olatunji

Possibilities for application of plant promotor Immunocitofit in integrated pepper productionS. Masheva, V. Todorova, G. ToskovStudy on vegetative and generative manifestations as well as yield from glasshouse tomatoes when foliar fertilizers are appliedN. Valchev, S. MashevaPlant species screening for biofumigant activity against soil-borne pathogens and root-knot nematodesS. Masheva, V. Yankova, G. Toskov

Water quality assessment from own source at poultry farm located in rural region in South BulgariaR. Stefanova, G. Kostadinova, N. GeorgievaInfluence of different factors on tannins and flavonoids extraction of some thyme varieties representatives of thymol, geraniol and citral chemotypeG. Zhekova, D. PavlovIdentification of pathogenic races of wheat common bunt using differential lines in Lorestan provinceZ. Noruzi, V. Mahinpoo, M. Mohamedi, F. Fayazi, F. Jalali, A. Sohilinejad, M. DarvishniaReview of species of the phylum Acanthocephala from the Middle-Bulgarian RegionZ. Dimitrova, N. DimitrovaEffect of experimentally polluted water on the morphological characteristics on the leaves of two varieties of Triticum aestivum L. grown on different soil types K. Velichkova, D. Pavlov, D. NinovaEvaluation the relative resistance of 14 corn cultivars to Fusarium maize rot infection L. Ashnagar, S. Rezaei, M. Darvishnia, M. Zamani

Modeling of spectral data characteristics of healthy and Fusarium diseased corn kernelsTs. Draganova

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