**Total Growth of Tomato Hybrids Under Greenhouse Conditions**

Humberto Rodriguez-Fuentes1, Juan Antonio Vidales-Contreras1, Alejandro Isabel Luna-Maldonado1 and Juan Carlos Rodriguez-Ortiz2 *1Department of Agricultural and Food Engineering, Faculty of Agriculture, Autonomous University of Nuevo Leon, Escobedo, Nuevo Leon, 2Faculty of Agriculture, Autonomous University of San Luis Potosi, San Luis Potosi, Mexico* 

#### **1. Introduction**

Often in intensive production of tomato, the fertilization is applied by the farmers without consider the suitable doses in order to cover nutritional requirements according to crop physiological stages. Thus, appropriate crop management is a strategic demand to maintain or increase tomato production. In spite of many researchers conducted experiments in this subject and data is available about physiological stage requirements for plant nutrition, only few studies have been focused to nutritional parameters. The crop growth curves and nutrient uptake for tomato may determine uptake rate for a particular nutrient eluding possible deficiencies and superfluous fertilizer consumption. The daily rates of nutrient uptake are depending on crop and wheater (Scaife and Bar-Yosef, 1995; Honorato *et al*., 1993; Magnificent *et al*., 1979; Miller *et al*, 1979); however, crop requirements and opportune fertilizer applications, are little known in many of fresh consumption crops. In Mexico, vegetable production is located at desert areas in the north and middle of the country where water shortages have constraints with impact on of water demands the crops of tomato, pepper and cucumber. Thus, the surface for crop production in greenhouses has increased from 350 ha in 1997 (Steta, 1999) to about 5000 ha in 2006 (Fonseca, 2006), because the increasing demand for quality products and the risk of losses on field for crop production.

Imas (1999) found that nutrient uptake and fertilization recommendations are conditions depending of crops. For example, tomato crops under hydroponic greenhouse environments have averaged 200 t ha-1 which is significantly higher than the 60-80 t ha-1 yield, typically observed in an open field. In contrast to tomato crops grown in an open field, nutrient uptake in greenhouse environment can be duplicated or triplicated. In practical terms, crop growth cycle is divided according to physiological stages thence different concentrations or amounts of nutrients have to be applied according to recommendations given by Department of Agriculture. In the tomato production are considered four physiological stages: establishment-flowering, flowering-fruit set, ripening of tomato fruit (the first-crop harvest and last harvest on the crop). In each stage concentrations of nitrogen (N) and phosphorus (K) are increasing while nitrogen-phosphorous are decreasing because potassium is uptaken in large quantities during the reproductive stage of the crop (Zaidan

Total Growth of Tomato Hybrids Under Greenhouse Conditions 65

Fig. 1. Tunnel-type greenhouse used for experimentation located in Marin, Nuevo Leon, Mexico.

Fig. 2. Vinyl bag used for growing tomato in a hydroponics system.

and Avidan, 1997). In order to determine nutrient uptake more accurately, crop growth can be scheduled by chronological periods of sampling and analysis nearest one another.

The nutrient demand is the maximum amount of nutrient that a crop needs in order to meet their metabolic growth demand and development and that is calculated to maximize production goals and domestic demand price (nutritional optimal concentration of total biomass (air and / or root) at harvest time); however this criteria is not yet thought by the farmers. An appropriate method for such calculation is to use mass balance concept in a hydroponic system. In this way, nutrimental control is more efficient in the nutritive solution and its effect can be seen rapidly in the plants (Steiner, 1961). The method before mentioned is based on the composition of plant dry matter which consists of 16 essential nutrients; however, only thirteen are directly uptaken by plants from soil. Therefore, if the amount of total dry matter production (root + aerial part) during the cycle of growth and development as well as nutrimental concentration in each physiological stage is determined, the amount of nutrients that the plant absorbs can be estimated. With this analysis it is possible to establish a program of daily/weekly fertilization for crop production. This would result in a substantial economic savings on fertilizer costs and also in a decrease of negative environmental impact for its inappropriate applications.

#### **2. Tomato hybrids analysis**

The field study was conducted on the farm "El Cuento" located in the town of Marin, Nuevo León, Mexico (latitude N 25° 53' and longitude W 100° 02' and 400 m above sea level), where two greenhouses were used (Figure.1), first one of these greenhouses was a tunnel type of 900 m2 (75 m x 12 m and 7 m high) and second one was multi-Korean tunnel type of 1300 m2 (45 m x 30 m). It was used an open hydroponics system during the tests of two indeterminate hybrid tomatoes beef type.

The seeds were sown in a mixture of peat moss and perlite (1:1 v/v) in containers of 200 cavities. The transplanting was realized at 40 days after sowing. Plants were transplanted inside of 2 gallons polythene bags (white outer and black inner, Fig.2) with a mixture of perlite and peat moss (1:1, v/v). In the Korean type greenhouse it was settled 50% of plants of the hybrids Cayman (1700) and Charleston (1700). In the tunnel type greenhouse it was established 2220 plants of the hybrid Charleston, in both cases it was used a density of 2.5 plants m-2. The hydroponic irrigation system and nutrition of the plants were conducted with emitters calibrated to 4 min L-1. It was drained of 10 to 15% of the hydroponic solution applied. The nutrimental solution used was suggested by Rodríguez *et al.* (2006).

Plant sampling and were conducted according to tomato crops and greenhouse type (Fig 3). On plant sampling, three plants, visually uniform, were removed from the hydroponic system (substrate) every 15 days after plant plantation total dry matter was determined considering root + leaves + stems + flowers and fruits (if they were in the plant) To obtain the biomass model in both production and the nutrient uptake, roots, stems, leaves, and fruits were included. The samples were analyzed in the Laboratory of Soil, Water and Plant Analysis at the Agronomy School of Autonomous University of Nuevo Leon, where plant specimens were washed with deionizer water to obtain their dried weights. In addition to this parameter, dry matter, nitrogen, phosphorus, potassium, calcium and magnesium were determined per plant sample.

and Avidan, 1997). In order to determine nutrient uptake more accurately, crop growth can

The nutrient demand is the maximum amount of nutrient that a crop needs in order to meet their metabolic growth demand and development and that is calculated to maximize production goals and domestic demand price (nutritional optimal concentration of total biomass (air and / or root) at harvest time); however this criteria is not yet thought by the farmers. An appropriate method for such calculation is to use mass balance concept in a hydroponic system. In this way, nutrimental control is more efficient in the nutritive solution and its effect can be seen rapidly in the plants (Steiner, 1961). The method before mentioned is based on the composition of plant dry matter which consists of 16 essential nutrients; however, only thirteen are directly uptaken by plants from soil. Therefore, if the amount of total dry matter production (root + aerial part) during the cycle of growth and development as well as nutrimental concentration in each physiological stage is determined, the amount of nutrients that the plant absorbs can be estimated. With this analysis it is possible to establish a program of daily/weekly fertilization for crop production. This would result in a substantial economic savings on fertilizer costs and also in a decrease of

The field study was conducted on the farm "El Cuento" located in the town of Marin, Nuevo León, Mexico (latitude N 25° 53' and longitude W 100° 02' and 400 m above sea level), where two greenhouses were used (Figure.1), first one of these greenhouses was a tunnel type of 900 m2 (75 m x 12 m and 7 m high) and second one was multi-Korean tunnel type of 1300 m2 (45 m x 30 m). It was used an open hydroponics system during the tests of

The seeds were sown in a mixture of peat moss and perlite (1:1 v/v) in containers of 200 cavities. The transplanting was realized at 40 days after sowing. Plants were transplanted inside of 2 gallons polythene bags (white outer and black inner, Fig.2) with a mixture of perlite and peat moss (1:1, v/v). In the Korean type greenhouse it was settled 50% of plants of the hybrids Cayman (1700) and Charleston (1700). In the tunnel type greenhouse it was established 2220 plants of the hybrid Charleston, in both cases it was used a density of 2.5 plants m-2. The hydroponic irrigation system and nutrition of the plants were conducted with emitters calibrated to 4 min L-1. It was drained of 10 to 15% of the hydroponic solution

Plant sampling and were conducted according to tomato crops and greenhouse type (Fig 3). On plant sampling, three plants, visually uniform, were removed from the hydroponic system (substrate) every 15 days after plant plantation total dry matter was determined considering root + leaves + stems + flowers and fruits (if they were in the plant) To obtain the biomass model in both production and the nutrient uptake, roots, stems, leaves, and fruits were included. The samples were analyzed in the Laboratory of Soil, Water and Plant Analysis at the Agronomy School of Autonomous University of Nuevo Leon, where plant specimens were washed with deionizer water to obtain their dried weights. In addition to this parameter, dry matter, nitrogen, phosphorus, potassium, calcium and magnesium were

applied. The nutrimental solution used was suggested by Rodríguez *et al.* (2006).

be scheduled by chronological periods of sampling and analysis nearest one another.

negative environmental impact for its inappropriate applications.

**2. Tomato hybrids analysis** 

determined per plant sample.

two indeterminate hybrid tomatoes beef type.

Fig. 1. Tunnel-type greenhouse used for experimentation located in Marin, Nuevo Leon, Mexico.

Fig. 2. Vinyl bag used for growing tomato in a hydroponics system.

Total Growth of Tomato Hybrids Under Greenhouse Conditions 67

accumulation of intense biomass was observed (Figures. 4 and 5) until the establishment of the harvest. In last stage of crop growth the dry matter is decreasing as well as the nutrient uptake. Bugarin *et al*. (2002), coincided in their research due in same crop growth stage any

Accumulation of dry matter production had a linear behavior up to 96-day initial crop period. Thereafter, an erratic behavior was observed probably as a consequence of the conditions of high temperatures and low relative humidity occurred in those greenhouse designs. This condition caused a severe reduction in dry matter accumulation, low pollination of fruit and fruit set. Sampling finished after 170 days since transplanting stage; however, plant health recovery did not occurred, reducing drastically fruit production to about one third of the best period, from 2.5 to 0.8 kg m2 after 125 days since transplanting. The same reduction is normal but it occurs gradually as the harvest progresses and the

Observed data sets were analyzed for dry matter production during 96 days after transplanting. Three models of linear fitting (Table 1 and Figures 6, 7, 8) with R2 values of 0.96, 0.99 and 0.91 for hybrids: Cayman (CAI), Charleston greenhouse Korean (CHK) and Charleston in greenhouse Tunnel (CHT), respectively, were obtained. The R2 value for the average of both hybrids was 0.96 (Table 1, Figs. 6 and 7) to predict possible accumulation of DM with the models obtained for each hybrid, is to believe the 170 days scheduled time to

nutritional deficit decreases production.

Fig. 4. Tomato plants (variety Charleston) in breaking stage.

**4. Accumulation of dry matter production** 

remove crop residues and begin the next crop season.

number of clusters**.** 

Fig. 3. Tomato plants inside of the experimental greenhouse.

To determine the dry weight at constant weight, samples were milled and then placed in a forced convection oven (Riossa model F-62, Mexico) at 70o to 80oC. Samples were screened through a 50 μm mesh. Determination of total nitrogen was done by the Kjeldhal method (Rodríguez and Rodríguez, 2011). A wet digestion microwave (MARSX, EMC Corporation, North Carolina, USA) was used in order to analyze P, K, Ca, and Mg. Three dried samples of 0.5 ± 0.001g were placed in a digestion flask (Teflon PFA vessels of a capacity of 120 mL) and then 5 mL of HNO3 were added. The instrument was programmed with a ramp of 15 min to reach a temperature of 180 degrees Celsius and a pressure of 300 PSI and keeping these conditions for 5 minutes. Finally, to the samples were left to cool for 15 minutes.

The dry matter accumulated values in the hybrid tomato were analyzed with the DM Sigma Plot 10.0 Program and Microsoft Excel Office 2003 software and it was found that the best fitted linear regression model. To calculate the equations were considered the average values of both hybrids.

#### **3. Flowering period**

The flowering period occurred after 20 days after plant plantation, fruits set showed up 10 days later and until 55 days after transplanting. During the beginning of flowering there was a low accumulation of dry matter. On the fruit set and harvest time of fruit an

To determine the dry weight at constant weight, samples were milled and then placed in a forced convection oven (Riossa model F-62, Mexico) at 70o to 80oC. Samples were screened through a 50 μm mesh. Determination of total nitrogen was done by the Kjeldhal method (Rodríguez and Rodríguez, 2011). A wet digestion microwave (MARSX, EMC Corporation, North Carolina, USA) was used in order to analyze P, K, Ca, and Mg. Three dried samples of 0.5 ± 0.001g were placed in a digestion flask (Teflon PFA vessels of a capacity of 120 mL) and then 5 mL of HNO3 were added. The instrument was programmed with a ramp of 15 min to reach a temperature of 180 degrees Celsius and a pressure of 300 PSI and keeping these conditions for 5 minutes. Finally, to the samples

The dry matter accumulated values in the hybrid tomato were analyzed with the DM Sigma Plot 10.0 Program and Microsoft Excel Office 2003 software and it was found that the best fitted linear regression model. To calculate the equations were considered the average

The flowering period occurred after 20 days after plant plantation, fruits set showed up 10 days later and until 55 days after transplanting. During the beginning of flowering there was a low accumulation of dry matter. On the fruit set and harvest time of fruit an

Fig. 3. Tomato plants inside of the experimental greenhouse.

were left to cool for 15 minutes.

values of both hybrids.

**3. Flowering period** 

accumulation of intense biomass was observed (Figures. 4 and 5) until the establishment of the harvest. In last stage of crop growth the dry matter is decreasing as well as the nutrient uptake. Bugarin *et al*. (2002), coincided in their research due in same crop growth stage any nutritional deficit decreases production.

Fig. 4. Tomato plants (variety Charleston) in breaking stage.

### **4. Accumulation of dry matter production**

Accumulation of dry matter production had a linear behavior up to 96-day initial crop period. Thereafter, an erratic behavior was observed probably as a consequence of the conditions of high temperatures and low relative humidity occurred in those greenhouse designs. This condition caused a severe reduction in dry matter accumulation, low pollination of fruit and fruit set. Sampling finished after 170 days since transplanting stage; however, plant health recovery did not occurred, reducing drastically fruit production to about one third of the best period, from 2.5 to 0.8 kg m2 after 125 days since transplanting. The same reduction is normal but it occurs gradually as the harvest progresses and the number of clusters**.** 

Observed data sets were analyzed for dry matter production during 96 days after transplanting. Three models of linear fitting (Table 1 and Figures 6, 7, 8) with R2 values of 0.96, 0.99 and 0.91 for hybrids: Cayman (CAI), Charleston greenhouse Korean (CHK) and Charleston in greenhouse Tunnel (CHT), respectively, were obtained. The R2 value for the average of both hybrids was 0.96 (Table 1, Figs. 6 and 7) to predict possible accumulation of DM with the models obtained for each hybrid, is to believe the 170 days scheduled time to remove crop residues and begin the next crop season.

Total Growth of Tomato Hybrids Under Greenhouse Conditions 69

Fig. 6. Dry matter and total accumulated average in CAI: Caiman; DM A: Dry Matter

Fig. 7. Dry matter and total accumulated average in CHK: Charleston Korean; DM A: Dry

average; ADT: After day trasplanting.

Matter average; ADT: After day trasplanting.

Fig. 5. Pruning of unnecessary tomato shoots.

Fig. 5. Pruning of unnecessary tomato shoots.

Fig. 6. Dry matter and total accumulated average in CAI: Caiman; DM A: Dry Matter average; ADT: After day trasplanting.

Fig. 7. Dry matter and total accumulated average in CHK: Charleston Korean; DM A: Dry Matter average; ADT: After day trasplanting.

Total Growth of Tomato Hybrids Under Greenhouse Conditions 71

Fig. 9. Linear model obtained from total dry matter considering both hybrids.

Although the stage of harvest of tomato is not completed successfully because of environmental factors, It was possible to estimate dry matter production, the rate of increase in dry matter was higher and similar for CAI and CHT, but was lower for CHK. The average yield of the two hybrids was considered adequate to estimate dry matter production in tomato crop in this geographical area. Once we determined the removal of nutrients such as nitrogen, phosphorus, potassium, among others, and generate models of extraction of these, it can be calculated the amount of each nutrient applied and thus may establish a program of fertilization to ensure a sustainable production system; such program may be adjusted by

The authors would like to thank to M Sc. Gerardo Jiménez García† his contribution to

Bugarin, M. R.; Galvis, A.; Sanchez, P. y García, D. (2002). *Acumulación diaria de materia seca y* 

De Koning, A.N.M. (1989). *Development and growth of a commercially grown tomato crop*. Acta

Galvis, S. A. (1998). *Diagnóstico y simulación del suministro edáfico para cultivos anuales.* Tesis Doctoral en Ciencias. Colegio de Postgraduados. Montecillo, Mexico.

*de potasio en la biomasa aérea total de tomate*. Terra Latinoamericana. Vol.20, No.4, pp.

**5. Conclusion** 

several cycles of this same culture.

401-409, ISSN 0187-5779

Hort. Vol.260, pp. 267-273, ISSN 0567-7572

**6. Acknowledgment** 

accomplish this research.

**7. References** 

Fig. 8. Dry matter and total accumulated average in CHT: Charleston Tunnel; DM A: Dry Matter average; ADT: After day trasplanting.


Table 1. Dry matter and total accumulated average in two hybrid tomatoes. CAI: Caiman; CHK: Charleston grown in Korean greenhouse; CHT: Charleston in tunnel; DMA: Dry matter average (g/plant). ADT: after day transplanting.

Figure 9 shows the estimated model with the average values in the three systems evaluated, if there were some mathematical models of nutrient removal, we could estimate the dose for each nutrient to apply, once it has the most of the nutrients, it is possible calculate a fertilization program for growing tomatoes, a further advantage of this procedure is that it could adjust fertilization with irrigation, if available, a pressurized irrigation system.

Fig. 9. Linear model obtained from total dry matter considering both hybrids.

#### **5. Conclusion**

70 Horticulture

Fig. 8. Dry matter and total accumulated average in CHT: Charleston Tunnel; DM A: Dry

35 48.69 30.77 30.77 36.74 50 76.81 60.47 66.66 67.98 65 116.16 108.98 98.76 107.96 80 184.80 153.06 150.00 162.62 96 260.32 201.80 272.77 244.96

R2 0.9655 0.9949 0.9095 0.9672

Rate of Increase of MS g/day/plant 3.5017 2.8607 3.7098 3.3699

Table 1. Dry matter and total accumulated average in two hybrid tomatoes. CAI: Caiman; CHK: Charleston grown in Korean greenhouse; CHT: Charleston in tunnel; DMA: Dry

Figure 9 shows the estimated model with the average values in the three systems evaluated, if there were some mathematical models of nutrient removal, we could estimate the dose for each nutrient to apply, once it has the most of the nutrients, it is possible calculate a fertilization program for growing tomatoes, a further advantage of this procedure is that it

could adjust fertilization with irrigation, if available, a pressurized irrigation system.

DMA (g/plant) CAI CHK CHT Average

Matter average; ADT: After day trasplanting.

ADT

matter average (g/plant). ADT: after day transplanting.

Although the stage of harvest of tomato is not completed successfully because of environmental factors, It was possible to estimate dry matter production, the rate of increase in dry matter was higher and similar for CAI and CHT, but was lower for CHK. The average yield of the two hybrids was considered adequate to estimate dry matter production in tomato crop in this geographical area. Once we determined the removal of nutrients such as nitrogen, phosphorus, potassium, among others, and generate models of extraction of these, it can be calculated the amount of each nutrient applied and thus may establish a program of fertilization to ensure a sustainable production system; such program may be adjusted by several cycles of this same culture.

#### **6. Acknowledgment**

The authors would like to thank to M Sc. Gerardo Jiménez García† his contribution to accomplish this research.

#### **7. References**


**Part 4** 

**Postharvest Physiology** 


**Part 4** 

**Postharvest Physiology** 

72 Horticulture

Gary, C.; Jones, J.W. & Tchamitchian, M. (1998). *Crop modelling in horticulture: State of the art*.

Heuvelink, E. & Marcelis, L.F.M. (1989). *Dry matter distribution in tomato and cucumber*. Acta

Honorato, R.; Gurovich, L. y Piña R. (1993). *Ritmo de absorción de N, P y K en pepino de semilla*.

Imas, P. (1999). *Manejo de Nutrientes por Fertirriego en Sistemas Frutihortícolas*. XXII Congreso

Magnífico, V.; Lattancio, V. & Sarli, G. (1989). *Growth and nutrient removal by broccoli raab*. J.

Miller, C.H.; Mccollum, R.E. & Claimon, S. (1979). *Relationships between growth of bell peppers* 

Rodríguez, J. (1990). *La fertilización de los cultivos: Un método racional*. Facultad de Agronomía.

Rodríguez F., H. y Rodríguez Absi, J. (2011). *Métodos de Análisis de Suelos y Plantas. Criterios* 

Rodríguez F., H.; Muñoz, S. y Alcorta G., E. (2006). *El tomate rojo. Sistema hidropónico*. Editorial Trillas S.A. de C. V. (Primera edición). México. ISBN 968-24-7606-2 Scaife, A. & Bar-Yosef, B. (1995). *Nutrient and fertilizer management in field grown vegetables*. IPI

Steiner, A.A. (1961). *A universal method for preparing nutrient solutions of certain desired composition*. Plant and Soil. Vo.15, pp. 134-154, (print version) ISSN: 0032-079X Steta, M. (1999). *Status of the greenhouse industry in Mexico.* Acta Hort. Vol.481, pp. 735-738.

Sigmaplot 8.0. (2003). *Users Manual.* Exact Graphs for Exact Science*.* Systat Software Inc,

Zaidan, O. & Avidan, A. (1997). *Greenhouses tomatoes in soilless culture*. Ministry of Agriculture*,* Extension Service, Vegetables and Field Service Departments, USA.

Pontificia Universidad Católica de Chile. Santiago de Chile. 406p.

Bulletin No. 13. International Potash Institute, Basel, Switzerland).

Jour. Amer. Soc. Hortic. Sci. Vol.104, pp. 852 – 857. Print ISSN: 0003-1062 Ortega B., R.; Correa-Benguria, M. y Olate M., E. (2005). *Determinación de las curvas de* 

*(Capsicum annuum L.) and nutrient accumulation during ontogeny in field environments*.

*acumulación de nutrientes en tres cultivares de lilium spp. para flor de corte*. Agrociencia

*de Interpretación.* Editorial Trillas S.A. de C. V. (Segunda edición). México. ISBN 978-

Argentino de Horticultura. Septiembre – Octubre, 1999. Argentina.

Amer. Soc. Hort. Sci. 104 (2): 201 – 203. ISSN (print): 0394-6169

Sci. Hort*.* Vol.74, pp. 3-20, ISSN 03044238

Hort. Vol.260, pp. 149-157. ISSN 0567-7572

Vol.40, No.1, pp. 77-88, ISSN 1405-3195

607-17-0593-8.

ISSN 0567-7572

USA.

Cien. Inv. Agr. Vol.20, pp. 169-172 ISSN 0718-3267

**5** 

*Lithuania* 

**Chemical Composition and** 

Audrius Sasnauskas and Juozas Lanauskas

**Antioxidant Activity of Small Fruits** 

Pranas Viskelis, Ramune Bobinaite, Marina Rubinskiene,

*Institute of Horticulture, Lithuanian Research Centre for Agriculture and Forestry* 

Small fruits contain significant levels of micronutrients and phytochemicals with important biological properties. Consumption of small fruits has been associated with diverse health benefits, such as prevention of heart disease, hypertension, certain forms of cancer and other degenerative or age-related diseases (Manach et al 2004, Santos-Buelga and Scalbert 2000, Hummer and Barney, 2002). These beneficial health effects of small berry fruits could mostly be due to their particularly high concentrations of natural antioxidants (Wang et al., 1996), including phenolic compounds, ascorbic acid and carotenoids. Because of the high contents and wide diversity of health-promoting substances in berries, these fruits are often referred to as natural functional products (Bravo, 1998; Joeph et al., 2000). The chemical composition of berry fruits has previously been shown to be affected by the environmental conditions under which these plants are grown. However, accumulating data suggest that the genotype has a profound impact on the concentration and qualitative composition of phytochemicals and other important constituents of berries. Studies have reported that standard cultivars of dark-fruited berries present a higher antioxidant content compared to vegetables or other foods (Wang et al., 1997). The influence of the genotype is of increasing interest, and several studies, particularly those addressing antioxidants, have been published on this topic

Therefore, the aim of this study was to evaluate the quality parameters (the total amounts of phenolic compounds, anthocyanins and ascorbic acid as well as radical scavenging capacity) related to the fruits of *Sambucus, Aronia, Ribes, Hippophae rhamnoides* and *Rubus* cultivars.

The amount of total phenolics in the fruit extracts was determined with the Folin-Ciocalteu reagent according to the method of Slinkard and Singleton (1977) using gallic acid as a standard. The reagent was prepared by diluting a stock solution (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) with distilled water (1:10, v/v). Samples (1 ml in duplicate) were aliquoted into test cuvettes, and 5 ml of Folin-Ciocalteu's phenol reagent and 4 ml of Na2CO3 (7.5%) were added. The absorbance of all samples was measured at 765 nm using a Genesys10 UV/VIS spectrophotometer (Thermo Spectronic, Rochester, USA) after

**1. Introduction** 

(Connor et al., 2002, Lister et al., 2002).

**2. Materials and methods** 
