International Journal of Physical Sciences

The effects of various quantities of water were assessed on their potential to influence the content of flavonoid and anthocyanin contents in different tissues of cucumber plants grown hydroponically in the greenhouse. data from our study showed that flavonoid content was not significantly affected by different water quantities supplied to cucumber plants. However, the anthocyanin content in roots, leaves, and stem were significantly influenced by water levels. Lowest water quantity 2 to 6 L/h significantly increased the levels of anthocyanins in all tissues tested. Increasing water quantities to higher quantities significantly decreased the anthocynanin content in all tissues.


INTRODUCTION
Flavonoid and anthocyanins are known around the world as a wide group of natural products that consist of a carbon framework (Marais et al., 2006).They are polyphenolic plant secondary metabolites.Flavonoids have a great influence on colour pigmentation originating in plants and are also suggested to be beneficial to human health (Winkel, 2006).For example, the colour blue found in petals of flowers are due to anthocyanin (delphinidin-based).In some plant species, anthocyanins play a vital role in the autumn colours and photoprotection of leaf`s cells against stress (Ndakidemi and Dakora, 2003).It is probably a natural UV filter because of its ability to absorb light in the 280 to 315 nm regions.A wide range of plant flavonoids acts as a defence mechanism from microbes, insects and mammalian herbivory, namely; isoflavones, flavons and flavanones are recognized as constitutive antifungal plant agents (Ndakidemi and Dakora, 2003;Makoi et al., 2010).Flavonoids are also able to transform enzymatic and chemical reactions, which can impact human health negatively or positively (Beecher, 2003).They are also recognized for their antioxidant activity, antioxidants are compounds that protect cells from the harm reactive oxygen species can cause (Kukić et al., 2006).Due to anthocyanins ability to decrease inflammation and oxidative stress it also has useful effects on neurodegenerative processes in Parkinson's or Alzheimer's disease (Joseph et al., 2003; *Corresponding author.E-mail: laubscherc@cput.ac.za.Tel: +27 214603198.Lau et al., 2007).It was proven that berries increases dopamine discharge in the brain leading to a better communication between brain cells (Joseph et al., 2003).
Research on flavonoids revealed that they reduce chronic diseases, including cancer (Ramos, 2007;Chung et al., 2005).Cyanidin is an anthocyanin pigment originating in numerous berries (grapes, blackberry, blueberry, cherry, cranberry, raspberry etc.), apples, plums and red cabbage.It contains antioxidant, antiinflammatory and anticancer properties and has an important role in future cancer treatment (Fimognari et al., 2005).Anthocyanins of fruit extracts (blueberry, bilberry, cranberry, strawberry, raspberry) indicated to inhibit either the introduction of carcinogenesis or the development of tumors (esophageal, colon, liver).More specifically cyanidin and delphinidin inhibited the enlargement of particular tumor cells by inhibition of the epidermal growth-factor receptor (Liu et al., 2005) The synthesis and release of phenolic compounds such as flavonoid and anthocyanins are induced by various biotic and abiotic factors.Phosphorous deficiency can, for example, induce the accumulation of phenolics in plants (Ndakidemi, 2006).Studies conducted by Mattivi et al. (2006) showed that under limited water conditions anthocyanin build up increased through the stimulation of anthocyanin hydroxylation by up regulating the gene encoding the enzyme.In another case Castellarin et al. (2007b) stated that when berries of grapes were exposed to limited water conditions early sugars build up increased which accelerated anthocyanin synthesis.
Phenolic compounds such as anthocyanin and flavonoids have demonstrated a wide variety of biological activities mainly from their antioxidant background with the potential of health benefits to human beings (Hodek et al., 2002).Research evidence suggests that plants that are tolerant to limited water conditions build up three to four times more anthocyanins during dehydration in comparison to when they are in their fully hydrated state (Sherwin and Farrant, 1998).At altered water supply, phenolic compounds in cucumber may vary in quantity and can affect the quality, taste and possibly the digestibility.Additionally excessive phenolic compounds can cause browning and structural alterations that may adversely changes the functional properties of the proteins and their activities (Synge, 1975) in crops such as cucumber.The aim of this study was to assess the effects of various quantities of drip irrigated water on the flavonoid and anthocyanin content in Cucumis sativa L. grown in the hydroponic culture.

Site location and description
The research took place at the greenhouse of the Agronomy and Vegetables Department of the Cape Institute for Agricultural Training in Elsenburg South Africa during 2009-winter season and 2009 to 2010 summer seasons.The greenhouse had a fully automated fertigation system.The cucumber plants were placed in 20 black bags consisting of sawdust as a growth medium.The plants were irrigated via drip irrigation and plants were trained by polyethylene twines.

Experimental design and treatments
The experiment was set out in a randomised complete block design.The treatments included eight various water regimes.These different water regimes were 2 L/h (control), 4, 6, 8, 10, 12, 14 and 16 L/h.The plants received water five times a day, making it (10, 20, 30, 40, 50, 60, 70, and  ).This was a new high yield cucumber variety in South Africa.The trial was in a drain to waste system and ran over 3 months.There were 8 replicates.All the treatments received the same amount of nutrients.The electric conductivity of the nutrient solution was set at 1.65 mS cm -1 (Combrink, 2005).

Plant harvest, sample preparation and metabolite extraction
After 91 days each plant for each treatment was harvested.The stems, leaves, roots were cut up for each treatment and placed in separate paper bags.The plant organs were oven-dried at 60°C for 48 h, weighed, ground into fine powder (0.85 mm) and stored prior to bioassay for flavonoids and anthocyanins.About 0.1 g of ground powder (root, leaves and stem) was weighed and mixed with 50 ml of acidified methanol prepared from a ratio of 79:20:1 MeOH H2O HCl.The mixture was incubated for 72 h in darkness for autoextraction, filtered through Whatman paper No. 2 and absorbance of the clear supernatant measured spectrometrically at 300, 530, and 657 nm using acidified methanol as standard.Concentrations of flavonoids were measured at 300 nm and expressed as Abs gDM −1 (Mirecki and Teramura, 1984), while anthocyanin concentration was measured as Abs530 −1/3Abs657 (Lindoo and Caldwell, 1978) and expressed as Abs gDM −1 .

Statistical analysis
The analysis was performed using STATISTICA software (StatSoft Inc., 2007 Tulsa, OK, USA).One-way ANOVA analysis was used to compare treatment means of the metabolites in plant organs.

RESULTS
In this study, the flavonoids and anthocyanins (Abs gDM −1 ) in tissues (roots, leaves, and stem) of cucumber were extracted in aqueous methanol (10 g of seed in 50 ml of acidified MeOH), and their concentrations measured spectrophotometrically.The data from our study showed that flavonoid content was not significantly affected by different water quantities supplied to cucumber plants (Table 1).However, the anthocyanin content in roots, leaves, and stem were significantly influenced by water levels (Figures 1 to 3) and (Table 1).For example, lowest water quantity 2 to 6 L/h significantly (P≤0.001)increased the levels of anthocyanins in roots.
As water quantities increased, the anthocynanin content in roots also decreased (Figure 1 and Table 1).The lowest anthocyanin content in roots was recorded in the treatment supplied with 16 L/h of water.
Figure 2 and Table 1 show the effect of different water quantities on the anthocyanin content in leaves.The data showed that the levels of anthocyanin content in leaves were significantly (P≤0.001)affected by water quantities.Relative to higher water levels, 12 to 16 L/h, the lower water levels 2 to 6 L/h exhibited greater concentrations of anthocyanins in leaves (Figure 2 and Table 1).The concentration of anthocyanins in stems of cucumber plants differed significantly (P≤0.001) with the various water quantities supplied.Lowest water quantities 2 to 4 L/h produced stems which had significantly higher anthocyanin content than those supplies with 10 to 16 L/h.The lowest concentration of anthocyanin in stems was recorded in a treatment supplied with water at a rate of 16 L/h.

DISCUSSION
In this study, roots, leaves and stems of cucumber were assayed for flavonoids and anthocyanins with the objective of quantifying the levels of these phenolic  compounds at different exposure to water quantities.Our findings suggest that the C. sativa L. exposed to different water regimes resulted in significantly increased anthocyanin content.Plants that received the least amount of water 2 L/h (control treatment) had the highest anthocyanin content compared with those supplied with the greater quantities such as 16 L/h.The higher anthocyanins present in different cucumber organs supplied with low water quantities could serve as defence molecules against abiotic stresses such as water stress.This report is consistent with the finding that the presence of anthocyanins in plant tissues provided protection of plant tissues against abiotic stress such as water (Chalker-Scott, 1999).In a similar study, Mattivi et al. (2006) and Castellarin et al. (2007b) reported anthocyanin increases in plant tissues exposed to limited water conditions.This increase in anthocyanin is due to the stimulation of anthocyanin hydroxylation by up regulating the gene encoding its enzyme (Mattivi et al., 2006;Castellarin et al., 2007b).It can also be as a result of increased early sugar accumulation which as a result accelerates anthocyanin production (Castellarin et al., 2007b).Plants that contain a high amount of anthocyanin can survive droughts for an extended periods of time.During this procedure, resurrection plants restore their great central vacuole with smaller vacuoles and increase their anthocyanin content three to fourfold over normal levels which assist the plant in withstanding droughts (Farrant, 2000;Sherwin and Farrant, 1998).
In conclusion, we have shown that the concentration of anthocyanins in tissues of cucumber differed markedly between different water treatments tested, with plants supplied with lowest quantities (2 L/h) showing higher levels of anthocyanins relative to highest water quantities (16 L/h).The higher concentration of these compounds at lower water level is as a result of a mechanism adopted by plants to protect them from abiotic constraints such as water stress to reduce evapo-transpiration (Chalker-Scott, 1999).

Figure 1 .
Figure 1.Effect of different amounts of water quantities on anthocyanins in the root of Cucumus sativa as measured during 2010.Mean values within each bar followed by different letter differ significantly at P ≤ 0.005 according to Fishers least significance difference.

Figure 2 .
Figure 2. Effect of different amounts of water quantities on anthocyanins in the leaves of Cucumus sativa as measured during 2010.Mean values within each bar followed by different letter differ significantly at P ≤ 0.05 according to Fishers least significance difference.

Figure 3 .
Figure 3.Effect of different amounts of water quantities on anthocyanins in the stems of Cucumus sativa as measured during 2010.Mean values within each bar followed by different letter differ significantly at P ≤ 0.05 according to Fishers least significance difference.

Table 1 .
Effect of different amount of water quantities on flavanoids and anthocyanins of Cucumus sativa stems as measured in Abs g.DM −1 during 2010.