Biotechnology and Nanotechnology Research Centre, MARDI Headquarters, Serdang, P.O. Box 12301, 50774-Kuala Lumpur, Malaysia
Received date: September 21, 2015 Accepted date: October 09, 2015 Published date: October 16, 2015
Citation: Lazim MIM, Badruzaman NA, Peng KS, Long K (2015) Quantification of Cytokinins in Coconut Water from Different Maturation Stages of Malaysia’s Coconut (Cocos nucifera L.) Varieties. J Food Process Technol 6:515. doi:10.4172/2157-7110.1000515
Copyright: © 2015 Lazim MIM, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Coconut water (Cocos nucifera L.) is one of the world’s most versatile natural products because of its various applications. Multiple beneficial biochemicals such as vitamins, minerals, proteins, sugars and enzymes have been identified in coconut water. Phytohormones, particularly cytokinins, one of the most interesting components reported present in coconut water. Cytokinins had shown some significant anti-aging, anti-carcinogenic and anti-thrombotic effects that contributed to the various health benefits. In this study, two local coconut varieties: Malayan Green Dwarf (MGD) and Malayan Yellow Dwarf (MYD) were chosen to quantify the cytokinin compounds present in different maturation stage of coconut water using ultra-performance liquid chromatography. The maturation stage of coconut water was categorized into three groups: a) immature, 120-200 days, b) mature, 220-300 days and c) overly-mature, 320-380 days. Among two local coconut varieties, MGD was shown to have the highest content of cytokinins, which was 3.2841 μM. Different maturation levels of coconut water was found to have an effect on the cytokinins concentration in both MYD and MGD varieties, which was reported to be the highest at the immature and mature stage, respectively. Briefly, the trans-zeatin riboside was noted to be the major cytokinin compounds present in coconut water for both MGD and MYD varieties.
Coconut water; Cytokinins; Solid phase extraction (SPE); Ultra-performance liquid chromatography (UPLC); Malayan green dwarf (MGD); Malayan yellow dwarf (MYD)
Coconut water (coconut liquid endosperm) is widely consumed around the world since it is refreshing, high in nutritional values and contains many potential therapeutic properties . It was believed that coconut water is useful in the treatment of many health problems, including dehydration, digestive problems, fatigue, diarrhoea, kidney stones and constipation . It also uses as a growth promoting supplement in plant tissue culture industry . Phytohormones, particularly cytokinins, one of the most interesting components reported present in coconut water. Previous findings had shown some significant anti-aging, anti-carcinogenic and anti-thrombotic effects that contributed to the various health benefits . N6-isopentenyladenine, dihydrozeatin, trans-zeatin, kinetin, ortho-topolin, dihydrozeatin O-glucoside, trans-zeatin O-glucoside, trans-zeatin riboside, kinetin riboside and trans-zeatin riboside-5’-monophosphate are among the cytokinin compounds identified in coconut water [5-7].
The activity of cytokinins is essentially dependent on their chemical structures. All native cytokinins are derivatives of adenine with at least one substituent at the N6 position as summarized in Table 1. The most active cytokinin present in coconut water is trans-zeatin . Recent study has reported that trans-zeatin prevents the formation of amyloid β-protein, helpful in the treatment of neural diseases  and possesses an inhibitory effect on acetylcholinesterase. It can be used to treat Alzheimer’s disease or related neural dysfunctions [10,11]. The most abundant cytokinin present in coconut water is trans-zeatin riboside  and this compound was reported capable to impair the viability of neoplastic and colon carcinoma cell lines . DL-dihydrozeatin, another type of cytokinin compounds was reported involved in the regulation of cell division during cereal grain development. It also plays a role as an intermediate in the zeatin biosynthesis . Zeatin was known to have anti-ageing property or serves as youth-preserving agent. This type of cytokinin has no carcinogenic effect because it does not induce cell proliferation. Kinetin (N6-furfuryladenine)  was first identified in 1955 and exerts anti-ageing effects in plants, human skin cells and fruitflies [16,17].
Table 1: Cytokinins structure.
Numerous techniques had been utilized to quantify the cytokinins, including gas or liquid chromatography coupled with mass spectrometry (e.g. GC-MS, LC-MS), capillary chromatography such as capillary electrophoresis (CE) and micellar electrokinetic capillary chromatography (MECC) as well as enzyme-linked immunosorbent assays (ELISA) or radioimmunoassays (RIA) [18,19]. Recent advances in instrumentation like ultra-performance liquid chromatography (UPLC) and the range of detectors available nowadays enable analytical scientists to identify and quantify several cytokinins [20-22]. It was known that cytokinins presents in coconut water at a very low concentration, therefore, pre-concentration of the cytokinins was achieved using solid-phase extraction (SPE) with Strata C18-E cartridges . In our study, we have developed an UPLC-PDA method to quantify few cytokinin compounds (trans-zeatin, trans-zeatin riboside, trans-zeatin glucoside, DL-dihydrozeatin and kinetin) in a single run. This analytical approach is more sensitive and able to provide higher efficiency with better separation within a short period of time. The aim of this study is to quantify the cytokinin compounds from different maturation stages of coconut water. Two local coconut varieties were chosen, namely Malayan Green Dwarf (MGD) and Malayan Yellow Dwarf (MYD).
Cytokinin standards: trans-zeatin riboside (ZR), kinetin (K) and DL-dihydrozeatin (DZ) were obtained from Sigma-Aldrich (St. Louis, MO, USA); trans-zeatin (Z) was purchased from Acros (Geel, Belgium) and trans-zeatin glucoside (ZG) was obtained from Santa Cruz, (California, USA). All the standards were dissolved in 0.01N NaOH, and the stock standards solution were stored at the temperature of -20°C. Chromatographic grade chemicals: methanol (Merck, Darmstadt, Germany), acetic acid and ethanol were purchased from Fischer Scientific (Hanover Park, IL, USA) and sodium hydroxide was obtained from Fluka (Steinheim, Swirtzeland). Strata C18-E cartridge was purchased from Phenomenex (USA). Ultrapure Mili-Q water (Waters, Milford, MA, USA) was used throughout the study.
Coconut water sample preparation
A total of two local coconut fruits: Malayan Green Dwarf (MGD) and Malayan Yellow Dwarf (MYD) were collected from Mardi Hilir Perak station. Both coconut varieties were harvested at different maturation stages, starting from days 120 until days 380. We have divided the maturation level of coconut water into three categories: a) immature coconut, IMC (120-200 days); b) mature coconut, MC (220-300 days); c) overly-mature coconut, OMC (320-380 days). The maturation stages of coconut fruits were authenticated by local coconut planter whom has wide experiences. Coconut water was freshly extracted from the coconut fruit and filtered through muslin cloth. The filtrated coconut water was pooled in plastic bag and stored in -20°C until further use.
Pre-concentrate of coconut water extract via solid phase extraction
Coconut water (75 mL) was undergoing freeze dried (Martin Christ Alpha, Germany) process before subjected to solid phase extraction (SPE). The freeze dried extract was dissolved with 5 mL of 0.01N NaOH solution and filtered using 0.45 μm pore size before applied into Strata C18-E cartridge. The procedure of pre-concentrate of cytokinins was followed from Mooi et al. method . Briefly, Strata C18-E cartridge was conditioned subsequently with 2 mL of each methanol-acetic acid (100:1, v/v), methanol-water-acetic acid (50:50:1, v/v/v), methanolwater- acetic acid (30:70:1, v/v/v) and lastly with 2 mL of ultrapure water. Then, the filtered samples were transferred into Strata C18-E cartridge, followed by flushing with 10 mL of acidic distilled water (pH 3). Finally, the component of interest was eluted out with 3 mL ethanol-water-acetic acid (80:20:1, v/v/v). A pressurized stream of nitrogen gas was directed perpendicular to the surface of the liquid to remove the solvent. Prior to UPLC analysis, samples were dissolved in 1 mL of 0.01N NaOH and filtered with 0.2 μm nylon syringe filter. All analyses were done in triplicate.
UPLC conditions and procedure
The UPLC analysis was performed through Acquity UPLC system linked concurrently with a photodiode array (PDA) system and the data was analyzed using Waters Empower 2 software (Water Corp, USA). At the column temperature of 50°C, cytokinin standards were separated using isocratic elution program of methanol and water (10:90) on Acquity UPLC®BEH C18 (2.1 × 100 mm, 1.7 μm Waters, USA) column with the flow rate of 0.45 mL/min. A total of 1 μL sample was injected and monitored at the wavelength of 270 nm. Quantification was made using calibration curves obtained by injecting known amounts of cytokinin standards (trans-zeatin glucoside, transzeatin, DL-dihydrozeatin, kinetin and trans-zeatin riboside) as external standards with known retention times.
Cytokinin standards profiling using UPLC-PDA
A reversed phase UPLC-PDA method was used in the simultaneous determination of different cytokinin compounds present in coconut water. Briefly, a total of 1 μL of cytokinin standards or samples was injected into UPLC system and each of the cytokinin compounds was separated through Acquity [email protected] C18 column (2.1 × 100 mm; 1.7 μm) using isocratic elution program of methanol-water (10:90, v/v), with the flow rate set at 0.45 mL/min and column temperature of 50°C. The separation of different cytokinin compounds was detected at the wavelength of 270 nm. Quantification was made using calibration curves obtained by injecting known amounts of cytokinins standards; trans-zeatin glucoside (ZG), trans-zeatin (Z), DL-dihydrozeatin (DZ), kinetin (K) and trans-zeatin riboside (ZR) as external standards with known retention times. The analyses indicated that the retention times for the analytes increased in the following order: ZG<Z<DZ<K<ZR throughout the isocratic mobile phase as shown in Figure 1.
Quantification of cytokinins at the different maturation stages of MGD and MYD coconut water
The presence of cytokinins in coconut water was pre-concentrate first by subjecting coconut water to SPE columns (Strata C18-E, Phenomenex; 500 mg, 3 mL) cartridge as a pre-treatment to achieve high extraction efficiency and at the same time to minimize the sample matrix effects. Two local coconut varieties; Malayan Green Dwarf (MGD) and Malayan Yellow Dwarf (MYD) were chosen for this study. Each coconut variety at different maturation level was selected to determine the presence of each cytokinin compounds (Figure 2). Briefly, it was divided into 3 categories: a) immature coconut, IMC (120-200 days); b) mature coconut, MC (220-300 days); c) overlymature coconut, OMC (320-380 days).
In general, it was found that the cytokinins concentration of each type of coconut water was varied with the different maturation period. The amount of trans-zeatin was noted decreased at IMC stage of MGD coconut water (Figure 3a). However, the concentration of trans-zeatin of this coconut variety was increased gradually with the maturation period and reached maximum concentration of 0.1365 μM at the maturation days of 300. When reached OMC stage, the amount of trans-zeatin was found decreased and remained no much changes for another 2 months before start decreased at the maturation days of 380. However, coconut water in MYD variety showed a reversed trend. The amount of trans-zeatin was increased, start from days 120 until days 160 (IMC), which is the highest concentration of cytokinin (0.0694 μM). Then, it decreased dramatically when reached at the MC and OMC stages with the cytokinins content range in between 0.0048 and 0.0180 μM. During the MC and OMC stages (days 220 to 380), the amount of trans-zeatin was found higher in MGD variety compared to MYD variety (Figure 3a).
trans-zeatin riboside was not detected at early stage of maturities for MGD variety. But, the amount of trans-zeatin riboside showed a gradually increment, start from days 140 (0.0204 μM) until reached maximum concentration of 0.6171 μM at the MC stage (days 300) before it decreased and remained constant at the OMC stages (days 320 to 380). The amount of trans-zeatin riboside in MYD variety was also noted to be high at early maturation stages (days 120 to 160) and then decreased throughout the maturation periods. In the MC stage, the amount trans-zeatin riboside was found increased, starting from days 240 until days 300 and remained constant in the OMC stages (Figure 3b).
DL-dihydrozeatin was not detected at the early stage of maturation for both coconut water samples. Amount of DL-dihydrozeatin was firstly detected at the days 140 in MGD variety (0.0144 μM) and days 160 (0.0046 μM) in MYD variety. The amount of DL-dihydrozeatin present in MGD variety was decreased from the early stages, but, it showed an increase in coconut water with the maturation days of 220 to 300. Whereas, the amount of DL-dihydrozeatin present in MYD variety was decreased from days 160 and only showed an increase after 300 maturation days (Figure 3c). Kinetin only detected in IMC stages (days 120 to 160) of MGD variety and the amount was fall in the range of 0.0121-0.0355 μM. However, the lowest concentration of kinetin was detected in both coconut varieties, start from days 180 to 380 (Figure 3d). On the other hand, trans-zeatin glucoside was not detected at all maturation stages, either in MGD or MYD variety.
trans-zeatin riboside was showed to be the highest cytokinin concentration found in both MGD and MYD varieties. This finding was supported by the presence of trans-zeatin riboside as the most abundant types of cytokinins in coconut water as reported by Van and Drewes . Both trans-zeatin and trans-zeatin riboside was confirmed present in MGD variety at different stages of maturation, with the maximum amounts detected in mature green coconuts (days 300). The presence of these two cytokinins in coconut water showed its important role in the developing of coconut. They may also play a significant role in the nutrition of plant and tissue cultures. The transzeatin and trans-zeatin riboside content of coconut water was found to be consistently high at mature and overly-mature levels of coconut water.
Figure 4 summarizes the total cytokinin compounds presents at different coconut water maturation stages. Generally, the cytokinins concentration was found to be the highest at the immature stage (IMC) for MYD variety (0.9845 μM). Whereas, it showed the highest amount in coconut water of MGD at the MC (1.9021 μM) and OMC stages (1.0201 μM). Previous studies were mainly focusing on young coconut water (IMC). There is no research or finding reported on the study of coconut water at the MC or OMC maturation stages. Based on our findings, the concentration of cytokinins detected in our local coconut species was higher than the previous cytokinins concentration reported on young green coconut water by Ge . The cytokinins concentration was exhibited higher at MC and OMC stages in MGD variety.
Confirmation on the presence of cytokinins in local coconut water was performed by spiking the sample with the appropriate amount of mixture standards (Figure 5a-c). It was clearly shown that each of cytokinin peaks was increased after spiking the pre-concentrate coconut water with the cytokinins standards mixture. Our findings on the presence of significant amount of zeatin derivatives and kinetin at the MC and OMC stages of Malaysia’s coconut water indicated the potential usage of this maturation ranged of coconut water for daily consumption to maintain good health. Different composition of cytokinin compounds was detected at the different maturation stage of coconut water. Several factors, including plant development, cell cycle, different harvesting stage, soil and environmental conditions may contribute to the composition of cytokinins presents in coconut water. The changes in cytokinin levels associated with plant development have been reported earlier [25-27]. The cell cycle also reported to have an effect in cytokinin levels in coconut water, whereby the cytokinin concentration was noted to be the highest at the late S phase and during the M phase . Furthermore, environmental factors were reported have an influence on the cytokinins content. It was positively correlated with the levels of minerals nutrient [29,30] but showed a decreased by water stress . This factor was supported by Hirose et al. findings , whereby the environmental factors such as light, water and nutrition were affecting the chemical profile of coconut water. Inorganic nitrogen sources, such as nitrate, are also a major factor that regulating gene expression of adenosine phosphate-isopentenyltransferase (IPT), a key enzyme of cytokinin biosynthesis .
The levels of active cytokinins in plants are expected to be regulated by the rates of biosynthesis, inter-conversion, transport and degradation . During the maturation period, coconut water was used to form coconut flesh on the inner portion of the coconut fruit. The conversion of coconut water to coconut flesh was a general phenomenon in coconut fruits regardless of the coconut varieties [32,33]. However, the starting time for the formation of coconut flesh can be different among different coconut varieties and these variations were dependent on the different harvesting stages and coconut variety.
Hence, future studies should be carried out to determine the factors that contribute to the desirable chemical composition of cytokinins for a specific purpose. Although coconut water has been well studied in terms of its chemical content, there may be unknown solutes that play a contribution role to its special biological effects. To the best of our knowledge, there is no report on the quantification of cytokinin concentration of coconut water from green and yellow varieties of coconut fruits in Malaysia. With the development of more advanced detection techniques, screening can be intensified to detect novel compounds of medicinal values present in coconut water.
The amount of cytokinins presents in coconut water was found to be maturation level dependent. The highest concentration of cytokinins was detected in MGD variety. trans-zeatin riboside was reported to be the major cytokinin compounds presents in both MGD and MYD varieties. Total cytokinins concentration was noted to be the highest at the immature stage (IMC) of MYD variety. However, in MGD variety, the high content of cytokinins was shown at MC and OMC stages. With better understanding on the cytokinins composition of coconut water obtained from OMC fruits, it is possible to process and market OMC coconut water as a refreshing drink since it still contains a significant amount of cytokinin compounds.
This work was financially supported by the Development Fund, Malaysian Agricultural Research and Development Institute (MARDI) under 10th Malaysia Plan. We would like to express our gratitude to all staffs for the technical support in this research.
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