Effect of Preservation on Two Different Varieties of Vernonia amygdalina Del . ( Bitter ) Leaves

Vernonia amygdalina Del. is one of the leafy vegetables that can be used in an attempt to alleviate the problem of micronutrient malnutrition, prominent in tropical Africa. In order to ensure availability in non-growing areas or seasons, the vegetable needs to be preserved. Processing and preservation methods influence the nutrient content of vegetables. The present study was aimed at determining the effects of preservation on two different varieties of V. amygdalina (bitter) leaves (broad and small leaves). To this effect, evaluations were made on the chlorophyll content, phytochemicals, and antioxidant capacity of the two varieties of bitter leaf (V. amygdalina Del.) stored at 4 ̊C and −20 ̊C over a period of two weeks. Results showed a significant decrease in all parameters studied for both varieties at −20 ̊C except for the free radical reducing power (FRAP), DPPH radical scavenging activity (%RSA) and nitric oxide radical scavenging activity (%RSA) of V. amygdalina broad leaves where increase in scavenging activity was observed. Thus, it was concluded that to preserve the chlorophyll, phenol, total soluble proteins and reducing sugar levels, preservation at 4 ̊C is recommended. The present study finding would be useful during short-term preservation of bitter leaves for soup preparation and/or its aqueous extract for ethnomedicinal purposes, especially the small leaf variety.


Introduction
Vernonia amygdalina Del. (bitter leaf), a shrub or small tree that can reach 23 feet in height when fully grown with petiolate leaves of about 6 mm diameter and elliptic shape, belongs to the family Asteraceae.It is known as "Ewuro" in Yoruba, "Etidot" in Ibibio, "Onugbu" in Igbo, "Chusa-diki" in Hausa tribes and Origbo among the Urhobo and Itsekiri tribes in Nigeria [1].It is cultivated in Nigeria mainly for its nutritional value [2] [3].
V. amygdalina is a medicinal plant and fresh bitter leaf is of great importance in human diet because of the presence of vitamins and mineral salts [4].It is a very important protective food and useful for the maintenance of health and prevention and treatment of various diseases.The plant (especially the leaf) has been found useful in the ethnotherapy of diabetes [5]- [8], asthma, headache [9], skin infections such as ringworm, rashes and eczema, schistosormiasis, malaria [10], measles, diarrhea, tuberculosis, abdominal pain and intestine complaints as well as fevers, cough [11], induction of fertility in barren women [12]- [14] and hyperlipidemia [15].Bitter leaf also helps to cleanse vital organs of the body like the liver and the kidney.
Despite the wide application of bitter leaf and its high nutritional content, very little attempt has been made on preserving this vegetable.The vegetable is usually consumed either in the very fresh state or as dried leaves.
Hence the current study focuses on the effect of preservation on two different varieties of V. amygdalina (bitter) leaves.

Experimental Design
V. amygdalina Del. leaves' varieties of broad and small leaves were respectively collected from Harmony Path Farms in Otorho-Agbon, Delta State, Nigeria and put in well labeled polythene containers (two for each variety).The samples were then preserved in a refrigerator (4˚C) and freezer (−20˚C) for 2 weeks before leaf extraction.Fresh leaves (broad and slender) were also collected from the same pumpkin plants on the day of extraction to serve as control for the preserved ones.Thereafter, several biochemical analyses were carried out on the leaf extracts to ascertain the effect of preservation on the nutritional, phytochemical and antioxidant potentials of the preserved bitter leaves relative to the fresh control leaves.

Aqueous Leaf Extraction
1 g of broad and slender bitter leaf varieties were weighed, respectively, with an electronic balance (SPEC 21, England) and thereafter homogenized in a cold medium using 0.1 M phosphate buffer (pH 7.2) and the final volumes made to 50 ml with same buffer respectively, making a 2% w/v of the leaf extracts.The homogenates were filtered with a sieve cloth and the filtrate further centrifuged at 3000 g for 10 min.The later filtrates were used for subsequent analyses as the aqueous leaf extracts of both bitter leaf varieties (carefully labeled).

Estimation of Chlorophyll Content
The chlorophyll content of the leaves was determined according to the method described by [18] [19] with some little modifications.About 500 mg of leaf samples were weighed and ground with 50 ml of distilled water in a pestle and mortar.It was then centrifuged at 4000 rpm for 10 min till a clear supernatant was obtained.Absorption was measured, using a spectrophotometer at 645 and 663 nm and chlorophyll content (mg/g fw) was calculated using the equation: Three replicates were made of each treatment.

Determination of Total Flavonoid Contents of Extract
This was determined by colorimetry using a method described by [20] with some modifications.250 µl of the extract was added to 1.25 ml of distilled water and 75 µl of 5% NaNO 2 .After 5 min, 150 µl of 10% AlCl 3 .H 2 O was added, followed by 500 µl of 1 M NaOH and 275 µl of distilled water after 6 min.The solution was properly mixed and the colour intensity of the mixture read at 510 nm.Catechin was used as the standard.

Determination of Total Phenol Content of Extract
This was carried out according to the method described by [21]. 1 ml of Folin C reagent was added to 1 ml of the sample.After 3 min, 1 ml of saturated Na 2 CO 3 solution was added and the solution was made up to 10 ml with distilled water.The reaction mixture was kept in the dark for 90 min.The absorbance was read at 725 nml.Catechin was used as the standard.

Determination of Reducing Power of Extract
This was estimated using the method of [22].2.5 ml of 200 mM of phosphate buffer (pH 6.6) and 2.5 ml of 1% K 3 FeCN were added to various concentrations of the extract.The mixture was incubated for 2 min at 50˚C and then centrifuged at 1000 g for 8 min.5 ml of the supernatant was then mixed with 5 ml of distilled water and 1 ml of 0.1% FeCl 3 .The absorbance of the mixture was measured at a wavelength of 700 nm.Catechin was used as the standard.

2, 2-Diphenyl-1-Picrylhydrazyl Radical (DPPH • ) Scavenging Activity of Extract
This was estimated according to the method described by [23].To 0.3 ml of the extract, 2.7 ml methanolic solution of DPPH radical (6 × 10 −5 mol/l) was added.The mixture was shaken vigorously and left to stand for 60 min in the dark until stable absorption values could be obtained.The reduction of the DPPH radical was determined by measuring the absorption at 517 nm.
where, %RSA = Percentage of DPPH decolouration; A DPPH = absorbance of DPPH solution; A S = absorbance of the solution when the sample was added at a particular level.

Nitric Oxide Radical Scavenging Assay
The scavenging effect on nitric oxide (NO • ) radical was measured according to the method of [24].0.5 ml of the leaf extract was added in the test tubes to 1 ml of sodium nitroprusside solution (25 mM) and the tubes incubated at 37˚C for 2 h.An aliquot (0.5 ml) of the incubation solution was removed and diluted with 0.3 ml of Griess reagent (1% sulphanilamide in 5% H 3 PO 4 and 0.1% naphthylethylenediamine dihydrochloride).The absorbance of the chromophore formed was immediately read at 570 nm against distilled water as blank with catechin (50 µg) used as standard.Results were expressed as percentage radical scavenging activity (RSA): ΔAbs of sample % RSA=1 100.ΔAbs of control − ×

Reducing Sugar Determination Assay
This was carried out using the 3, 5-dinitrosalicylic acid (DNS) colorimetric method [25].1.5 ml of DNS reagent was added to 1.5 ml of leaf extract in a lightly capped test tube.(To avoid the loss of liquid due to evaporation, the test tube was covered with a piece of paraffin film since a plain test tube was used).Heat was applied to the mixture at 90˚C for 5 -15 min to develop the red-brown color.Then, 0.5 ml of a 40% potassium sodium tartrate (Rochelle salt) solution was added to stabilize the color.After cooling to room temperature in a cold water bath, the absorbance was recorded with a spectrophotometer at 575 nm.

Total Protein Determination by Biuret Method
Protein concentration of the various samples was determined by means of the Biuret method as described by [26] with some modifications: Potassium iodide was added to the reagent to prevent precipitation of Cu 2+ ions as cuprous oxide. 1 ml of the diluted sample was taken and added to 3 ml of Biuret reagent.The mixture was incubated at room temperature for 30 min after which the absorbance was read at 540 nm using distilled water as blank.Bovine serum albumin (BSA) was used as standard protein and the protein content of the samples was extrapolated from the BSA standard curve.

Results and Discussion
Results on the effects of preservation (refrigeration at 4˚C and freezing at −20˚C) on the chlorophyll content, phytochemicals, and antioxidant capacity of the two varieties of bitter leaf (V.amygdalina) studied over a period of two weeks are shown below.The results showed a significant decrease (p < 0.05) in the chlorophyll content of both species of V. amygdalina at 4˚C and −20˚C compared to the respective controls (Figure 1).From the results, at 4˚C, percentage decrease in chlorophyll a and b contents is 72.33% for V. amygdalina broad while the percentage decrease was 51.48% for V. amygdalina small when compared to the respective controls.Thus, V. amygdalina broad lost more of its chlorophyll content at 4˚C compared to V. amygdalina small.However, at −20˚C, the decrease in cholorophyll a and b for both varieties shows no statistical difference.Thus at freezing condition, the chlorophyll content of V. amygdalina is significantly lost irrespective of specie.Cătunescu et al. [27] showed a decrease in chlorophyll contents of minimally processed parsley (Petroselinum crispum), dill (Anethum graveolens) and lovage (Levisticum officinale) subjected to cold storage at 4˚C.Agüero et al. [28] also reported decrease in the chlorophyll content of external, middle, and internal zones of lettuce heads (Lactuca sativa L.) stored at 0˚C -2˚C and 97% to 99% relative humidity.Enzymatic degradation of chlorophyll a and b caused by chlorophyllase may occur during frozen storage [29].During storage, vegetables containing chlorophyll undergo changes or loss of color [30]- [32].Environmental factors such as light, temperature, humidity, oxygen and ethylene [33] [34] and inner plant factors such as chlorophyllase and magnesium dechelactase activity [35] [36] are responsible for the loss of chlorophyll pigments during green vegetable storage.
Chlorophyll content was determined along the storage period in order to assess both the visual quality of the samples (green index) and its nutritional quality.Chlorophyll is considered crucial to final product acceptance, because green color is associated with fresh vegetables quality [37].Also, chlorophyll may prove to have disease-protective effects attributed to diets rich in green vegetables because its content in these plants is much higher in concentrations than the widely studied phytochemicals [38].
Figure 2 shows that refrigeration at 4˚C and freezing at −20˚C for two weeks significantly (p < 0.05) affected the phenolic content extract of both varieties of bitter leaf.V. amygdalina small showed a more significant decrease at 4˚C compared to V. amygdalina broad while the changes showed no significant difference (p < 0.05) at −20˚C between both varieties.In cherries, the amount of total phenolics was evaluated throughout frozen storage for 6 months at −23˚C and −70˚C by [39] and they reported 25% degradation after 3 months and 50% after 6 months at the higher temperature, with minimal changes at the lower temperature.Thus, the decrease in phenolic compounds in vegetables could be both temperature and time dependent.The phenolic composition of fruit and  V. amygdalina broad (mg AA eq/g fw) V. amygdalina small (mg AA eq/g fw) Figure 2. Effect of preservation on total phenol content extract (mg AA eq/g fw) of leaves.4˚C, leaves preserved by refrigeration (4˚C) for two weeks; and −20˚C, leaves preserved by freezing (−20˚C) for two weeks.
vegetables is also dependent on commodity, cultivar, maturity stage and post-harvest storage handling and storage conditions.Since phenolic compounds are antioxidants, they are subject to oxidation during storage and processing.Chemical degradation of phenols can still occur during storage, depending on available oxygen and exposure to light [40].Phenolic compounds are also water-soluble, rendering them susceptible to leaching.In terms of flavonoid content, V. amygdalina broad showed a significant increase in flavonoid content after refrigeration at 4˚C (Figure 3).However, at −20˚C, there was a decrease in the flavonoid content which is highly significant (p < 0.05).V. amygdalina small however, showed significant (p < 0.05) decrease in flavonoid content at both 4˚C and −20˚C.Phenol and flavonoid are antioxidants that play major roles in the protection of cells from lethal effects of free radicals and their derivatives [41].A diet rich in antioxidant compounds (like phenols and flavonoids) therefore helps to strengthen the antioxidant-based defense system in the human body [42] [43].
Total soluble protein and reducing sugar of V. amygdalina broad showed decrease at 4˚C which became highly significant (p < 0.05) at −20˚C (Figure 4, Figure 5).V. amygdalina small on the other hand showed highly significant decrease at both temperatures.The decrease in total sugars may be attributed to conversion of glucose into starch during storage.Also, hydrolysis of sucrose could account for the decrease [44].At low temperature, the linear amylose molecules aggregate together to crystallize, and the water held between their structures is released which could lead to decrease in total soluble sugar [45].Singh et al. [45] also reported decrease in refrigerated (4˚C) and frozen (−18˚C) Indian water chestnut.
In this study, the ferric reducing antioxidant power (FRAP) of the fresh and preserved bitter leaves was determined and reported (Figures 6-8).Free radical reducing power (FRAP), DPPH radical scavenging activity (%RSA) and nitric oxide radical scavenging activity (%RSA) of V. amygdalina broad leaves were significantly (p < 0.05) increased at both 4˚C and −20˚C with a higher level at −20˚C.However, the nitric oxide radical scavenging activity (%RSA) showed a slight decrease at −20˚C compared to the level at 4˚C.In like manner, V. amygdalina small leaves showed significant increase in DPPH radical scavenging activity at temperatures 4˚C and −20˚C, with a higher scavenging activity at −20˚C.The FRAP was also significantly decreased at both storage conditions while for the nitric oxide radical scavenging activity (%RSA), a decrease was observed only at −20˚C while remaining constant at 4˚C when compared to the control.The results generally show that there was increase in reducing power of the stored vegetable at both storage conditions.The result of the scavenging activity assays indicates that the vegetable was potentially active at both refrigerating and freezing conditions.This suggests that the plant extract contains compounds that are capable of donating hydrogen to free radicals (chemical species with lone pair electrons) thereby reducing such radicals or reactive species to their inert states and this could help to prevent free radical-induced cellular damage in individuals that consume this vegetable as part of their regular diets [24] [41].

Conclusion
Investigations carried out in the current study showed that at both preservation conditions, chlorophyll content V. amygdalina broad (mg AA eq/g fw) V. amygdalina small (mg AA eq/g fw) Figure 3.Effect of preservation on total flavonoid content extract (mg AA eq/g fw) of leaves.4˚C, leaves preserved by refrigeration (4˚C) for two weeks; and −20˚C, leaves preserved by freezing (-20˚C) for two weeks.V. amygdalina broad (mg/g fw) V. amygdalina small (mg/g fw) V. amygdalina broad (mg/g fw) V. amygdalina small (mg/g fw)    was significantly decreased at p < 0.05 for both bitter leaf varieties.Chlorophyll content is used to assess both the visual quality of the samples (green index) and its nutritional quality.Since chlorophyll is considered crucial to the final product acceptance; the decrease in chlorophyll as observed in the current study could affect the final product acceptance of the vegetable.Phenol and flavonoid contents are antioxidants that can be more easily preserved at 4˚C compared with −20˚C.However, irrespective of temperature, there was a significant reduction in total soluble protein and reducing sugars.Antioxidants studied also showed a significant increase at a storage temperature of 4˚C.Thus, it could be concluded from the current study that V. amygdalina stored at 4˚C retains more of its nutritional and medicinal properties compared with when stored at −20˚C.This finding would be useful during short-term preservation of bitter leaves for soup preparation and/or its aqueous extract for ethnomedicinal purposes, especially the small leaf variety.

Figure 1 .
Figure 1.Effect of preservation on chlorophyll a and b contents (mg/g fw) of leaves.4˚C, leaves preserved by refrigeration (4˚C) for two weeks; and −20˚C, leaves preserved by freezing (−20˚C) for two weeks.

Figure 4 .
Figure 4. Effect of preservation on total soluble protein content (mg/g fw) of leaves.4˚C, leaves preserved by refrigeration (4˚C) for two weeks; and −20˚C, leaves preserved by freezing (−20˚C) for two weeks.

Figure 5 .
Figure 5.Effect of preservation on total reducing sugar content (mg/g fw) of leaves.4˚C, leaves preserved by refrigeration (4˚C) for two weeks; and −20˚C, leaves preserved by freezing (−20˚C) for two weeks.

Figure 6 .
Figure 6.Effect of preservation on free radical reducing power (FRAP) of leaves.4˚C, leaves preserved by refrigeration (4˚C) for two weeks; and −20˚C, leaves preserved by freezing (−20˚C) for two weeks.

Figure 7 .
Figure 7. Effect of preservation on DPPH radical scavenging activity (%RSA) of leaves.4˚C, leaves preserved by refrigeration (4˚C) for two weeks; and −20˚C, leaves preserved by freezing (−20˚C) for two weeks.

Figure 8 .
Figure 8.Effect of preservation on nitric oxide radical scavenging activity (%RSA) of leaves.4˚C, leaves preserved by refrigeration (4˚C) for two weeks; and −20˚C, leaves preserved by freezing (−20˚C) for two weeks.