Effects of Tannin Extract from Gongronema latifolium Leaves on Lipoxygenase Cucumeropsis manii Seeds

Lipoxygenase (EC 1.13.11.12) was partially puri�ed from germinated seeds of Cucumeropsis manii to a puri�cation fold of 47.14, enzyme activity recovery of 72.18%, and speci�c activity of 326.25 units/mg protein, using a three-step process of centrifugation, ammonium sulphate precipitation and gel �ltration. �inetic properties show maximum activity at pH 6.0 and at optimum temperature of 40C. Inhibitory effects of the extract from Gongronema latifolium and two other known antioxidants: ascorbic acid and propyl gallate on lipoxygenase from Cucumeropsis manii were studied. Result shows presence of inhibition with IC50 of 4.2 × 10 ± 0.09 × 10 g/L, 4.3 × 10 ± 0.11 × 10 g/L and 7.9 × 10 ± 0.11 × 10 g/L for the extract from ascorbic acid and propyl gallate, respectively. e extract when compared to the other antioxidants exhibits a competitive mechanism of inhibition. is tannin extract could be included during food processing as preservative against food deterioration that might be caused by oxidative enzymes such as lipoxygenase.


Introduction
Lipoxygenase (EC 1.13.11.12) (LOX) catalyzes the addition of molecular oxygen to the cis, cis-1, 4-pentadiene system in polyunsaturated fatty acids forming hydroperoxides and is widely distributed in plants [1], animals, and microorganisms [2].e products of the degradation of polyunsaturated fatty acids and their derivatives can react with proteins, peptides, and aminoacids, which can result in off-�avour production and rancidity of oils thereby lowering nutritional values of oil-based foods [1].
Oleaginous seeds are known to contain LOX [3].e seeds of Cucumeropsis manii are oil seeds [4], having mostly polyunsaturated fatty acids and are of high nutritive value and thus potential for human and livestock feed [5][6][7].Cucumeropsis manii seed kernels are major soup ingredients and they are used as a thickner and �avour component of soups [4,8].Many fats-and oil-based foods including these soup thickeners are susceptible to deterioration as a result of enzymatic activities (of which lipoxygenase is one of them), thereby posing a storage problem [9].Very few reports are available on lipoxygenases belonging to this family of Cucurbitaceae [10].
Owing to the uncertain future for chemical preservatives and public concern over residues in prepared products, there has been an increasing interest in the use of natural enzyme inhibitors as a nonchemical method for food preservation.
Gongronema latifolium is an herbaceous nonwoody plant from the family of Asclepiadaceae.It has milky or clear latex, widespread in the tropical, and subtropical regions especially in Africa and South America, with a moderate representation in Northern and South Eastern Asia [11].In South Eastern and South Western Nigeria, Gongronema latifolium (whose leaves are bitter) is commonly called "utazi" and "arokeke", respectively, and is primarily used as spice and vegetables in traditional folk medicine [12].Earlier reports on extract from this plant have focused mainly on their medicinal properties [13,14], with little attempts at investigating their food preservative potentials.Sueeri [15] reported the use of bittering agents in brewing to produce the characteristic �avour, foam stability, and preservative properties in beer.Adenuga et al. [16] have used Nigerian bitter vegetables of Gongronema latifolium, Vernonia amygdalina, and Garcia kola as substitutes for commercial hops in lager beer production.Apart from this, there is a dearth of information on the preservative potential of extracts from this plant with particular reference to its effect on some food-qualityrelated enzymes.It has been observed that the extracts of Gongronema latifolium contain phytochemical compounds including alkaloids, saponins, tannins (�avonoids), and glycosides [17].Studies have shown that these phytochemicals found in Gongronema latifolium may in�uence cellular proteins with enzymic activities.Tannins have been shown to be strong inhibitors of oxidative enzymes present in foodstuffs [18].Makkar and Singh [19] noted that extracts of tannin rich leaves of Oak (Quercus incana) inhibited various microbial enzyme activities of bovine rumen.Min et al. [20], while investigating the antibacterial activity of tannin extracts from perennial plants on mastitis pathogens, observed that the source and concentration are important factors that in�uence antimicrobial activity of tannins.erefore, this report focuses on studies on the inhibitory effects of tannin extract from the leaves of Gongronema latifolium on lipoxygenase from Cucumeropsis manii seeds with a view to contribute to the knowledge that will assist in the processing of the seeds of Cucumeropsis and the leaves of Gongronema latifolium into value-added products.

Materials. Cucumeropsis manii seeds and the leaves of
Gongronema latifolium used for this study were procured from Orba Modern Market in Udenu Local Government Area of Enugu State, Nigeria and identi�ed at the Botany Department of the University of Nigeria.
2.2.Methods.Linoleic acid (99% Sigma) used as a substrate was prepared in solubilized state as described [21].Other analytical grade quality chemicals were purchased from local commercial sources and were freshly prepared unless otherwise stated.

Preparation of Plant
Extract.is was carried out using the method of Kubicka and Troszunska [18].e leaves of Gongronema latifolium were collected fresh, sun-dried and powdered.is was then subjected to extraction with 70% acetone at the ratio of 1 : 7 (w/v) in a shaking incubator for 30 min.e mixture was then centrifuged and the supernatant was collected.e pellets were extracted twice with the same solution.e resultant extract was evaporated under vacuum and analyzed for total phenolics according to the methods of Julkunen-Tiitto [22].

2.�. Isolation and Puri�cation of Lipoxygenase.
Four days germinated (in the dark) white melon seeds were used for this experiment.One hundred grams of ground seeds were used for the extraction of LOX in 0.05 M phosphate buffer, pH 7.5 as in the method of Buranasompob et al. [23].e crude LOX obtained was partially puri�ed using a modi�cation of the method of Malekian et al. [9].Solid ammonium sulphate was added to each sample to obtain 70% saturation.e sample was allowed to stand for 24 hr and then centrifuged at 9000 ×g for 10 min at 4 ∘ C. e precipitate was redissolved in 0.01 M phosphate buffer pH 7.5.is solution was introduced on a sephadex G-50 column previously equilibrated with 0.01 M phosphate buffer pH 7.5.3 mL, fractions were collected aer every 5 min interval, and the active fractions were pooled and designated as the partially puri�ed enzyme.
2.5.Lipoxygenase Assay and Protein Determination.LOX activity was assayed according to the methods of Aurand et al. [21], with slight modi�cations.LOX activity was measured with a JENWAY 640 5 UV/VIS spectrophotometer (Beckman Instruments, Inc., Huston, TX, USA) at 234 nm at 30 ∘ C. e cuvette contained 2.9 mL of substrate solution and was placed in the sample compartment of the spectrophotometer.0.1 mL of the enzyme solution was rapidly added, mixed, and the increase in absorbance (A) versus the blank was recorded for every 5 sec.One unit of LOX activity was de�ned as the change in absorbance of 0.001 U/min in 3 mL volume and 1-cm light path using linoleic acid as substrate at 234 nm.Protein was determined by the method of Lowry et al. [24].
2.6.Effect of Substrate Concentration.e kinetics for lipoxygenase reaction (Km and Vmax) were determined in duplicate using linoleic acid stock solution diluted with 0.01 M borate buffer pH 7.5 to attain different concentrations (0-3 mM).e reaction was followed in the spectrophotometer at 30 ∘ C. LOX activity at each substrate concentration was determined as described under the assay section.

Optimum pH and
Temperature of LOX Activity.e following buffer systems were used at concentrations of 0.02 M: sodium acetate buffer (pH 5.0-6.0);sodium phosphate buffer (pH 7-8); Tris-Hcl, (pH 9.0-10.0).LOX activity at each pH was determined as described in assay method.Also, LOX activity was determined at 30, 40, 50, 60, and 70 ∘ C, respectively as described in the assay method.To minimize lag phase, the assay cocktail was preincubated in a circulating water bath to the required temperatures for 600 sec followed by the introduction of 0.1 mL of the enzyme solution.is was rapidly mixed and assayed as earlier described.

Lipoxygenase Inhibition Studies. e enzyme inhibitory
effect was tested by adding different volumes of the solution (0-9.1 mg/mL) of the extract to the incubation mixture.e lipoxygenase activity was monitored aer 5 min of incubation as an increase in the absorbance at 234 nm which re�ects the formation of hydroperoxylinoleic acid in the presence and absence of the extract as in the LOX assay section.e extinction coefficient of 25 mM −1 cm −1 was used for calculation of enzyme activity.is was also repeated with two known inhibitors of lipoxygenase: propyl gallate and ascorbic acid under the same condition.Each of the inhibitors was incubated in 50 mM phosphate buffer pH 7.5 with the enzyme before it was transferred to a cuvette containing the assay cocktail to start the reaction.e Lineweaver-Burk plot was applied to determine the kinetic mechanism of inhibition.F 2: Lineweaver-Burk plot for the determination of Km and Vmax.
2.9.Statistical Analysis.Each experiment was performed in triplicate.Results are expressed as the mean ± S.D. Statistical analysis was performed using SPSS package and statistical signi�cance is expressed as,   .For kinetic studies, each experiment was performed in duplicate and result expressed as the mean.

3.�. En�yme �uri�cation.
Lipoxygenase was puri�ed about 47.14-fold with protein recovery of about 72.18% and LOX-speci�c activity of 326.25 units/mg protein.e puri�cation pro�le is shown in Table 1.

Effect of Substrate
Concentration on LOX Activity.e linoleic acid hydroperoxidation rate followed Michaelis-Menten kinetic equation (Figure 1). Figure 2 shows the Lineweaver-Burk plot for the determination of Km and Vmax.A plot of the reciprocal of hydroperoxidation activity rates (1/V) versus the reciprocal of linoleic acid concentrations (1/[S]) ranging from 0.0 to 3 mM was constructed.
Vmax was calculated from the intercept to be 1.44 ± 0.02 units/mg/min, while Km value for linoleic acid for the partially puri�ed LOX extract was 1.61 ± 0.019 mM (by extrapolating the Lineweaver-Burk plot to the hypothetical point where 1/  ).

Discussion
e result recorded here suggests that the puri�cation procedure used here is suitable for puri�cation of LOX from Cucumeropsis manii to the extent of our puri�cation.Ammonium sulphate is the most frequently used method at the �rst stage of enzyme puri�cation and gave a good separation of the protein.e enzyme recovery is quiet low especially when compared with results obtained by a number of researchers [2,25], this might be as a result of differences in enzyme extraction method and the assay conditions [26].
A Km value of 1.61 ± 0.019 mM obtained in this experiment suggests low affinity of this LOX from Cucumeropsis manii for linoleic acid especially when compared with those observed in literature 0.131 ± 0.019 mM for barley lipoxygenase [27],   0.67 M for durum wheat lipoxygenase [28].Low affinity is an indication that lipoxygenase requires the use of high substrate concentration for its assay.e optimum pH of 6.0 obtained in this report is within the range of pH optimum for plant lipoxygenases (5.5-7.5)[25].An optimum temperature of 40 ∘ C suggests that LOX from C. manii might not be suitable for operation at very high temperatures, but it is in agreement with the result of Koksel et al. [27], who determined maximum linoleic acid hydroperoxidation activities at 40 ∘ C. ough temperatures of 25-30 ∘ C were used in the past for studying LOX activity by a number of researchers.
In this work, the LOX inhibitory properties were tested on partially puri�ed LOX from Cucumeropsis manii.Food spoilage caused by oxidative rancidity involves a reaction between the lipid and molecular oxygen.e reaction takes place at the double bonds of unsaturated fatty acids and can be accelerated by singlet oxygen, free radicals, metal ions (iron, cooper, and cobalt), light, radiation, and enzymes containing a transition metal prosthetic group such as lipoxygenases [29].LOX speci�cally oxygenates polyunsaturated fatty acids and/or their esters and acylglycerols containing the cis, cis-1, 4 pentadiene double-bond system located between carbons 6-10 counting from the methyl terminus [30].Consequently it causes off-�avour and off-odour in food.ere is little published information on the inhibition of LOX in Cucumeropsis manii, especially in regard to storage characteristics of its value-added products.Lipoxygenases are sensitive to antioxidants and the most common mechanism of action may consist in either inhibition of lipid hydroperoxide formation due to scavenging of lipidoxy or lipidperoxy radicals formed in the course of enzymic peroxidation or via chelation of its nonheme bound iron or by reduction of its ferric form, suggesting a competitive kind of inhibition [28].However, noncompetitive or mixed competitive/noncompetitive inhibition of LOX has been reported [26].e inhibitory properties of the extract from Gongronema latifolium, ascorbic acid, and npropyl gallate were compared using linoleic acid as substrate.e strongest lipoxygenase inhibition was obtained for the crude extract (IC 50 = 4.2 × 10 −3 ± 0.09 × 10 −3 g/L).Although ascorbic acid showed the stronger LOX inhibition effect (IC 50 = 4.3 × 10 −2 ± 0.11 × 10 −2 g/L), than propyl gallate (8.0 × 10 −2 ± 0.09 × 10 −2 g/L), the difference between the inhibitory effects of the crude extract and ascorbic acid is not signi�cant   0. Baraniak and Krzepilko [26] observed that phenolic compounds which are endogenous inhibitors in plant sources could act mostly as scavengers of free radicals rather than as inhibitors of lipoxygenase generating free radicals released from fatty acid oxidation.Kubo et al. [31] have observed that anacardic acid competitively inhibited various prooxidant enzymes involved in the production of the reactive oxygen species and acts by chelating divalent metal ions such as Fe 2+ or Cu 2+ from the prosthetic group of enzymes.Baysal and Demirdoven [29] have observed the presence of Fe 2+ in LOX, though Trop et al. [32] had earlier suggested that lipoxygenase is the only oxidative enzyme which is not inhibited by those substances which bind to the prosthetic group of enzymes.ese controversies are subject of further research.

Conclusion
Preservation technologies aimed at inactivating enzymes with deteriorative actions could include the use of enzyme inhibitors such as the tannin extract from the leaves of Gongronema latifolium.is work suggests that Cucumeropsis-manii-based food products could be protected against LOX using tannin extract from this plant instead of heating which could destroy other important nutrients inherent in them or using chemical preservatives which will raise acceptability questions by consumers.

Figure 3 (
a) shows the activity pro�le for white melon seed lipoxygenase across the pH range of 3-10, used in this study.e optimum pH was at 6.0.Maximum lipoxygenase activity was observed at 40 ∘ C (Figure 3(b)).

F 3 :F 4 :
(a) p� pro�le for the activity of lipoxygenase from Cucumeropsis manii seeds.(b) Optimum temperature for LOX activity from C. manii.Inhibition studies on Cucumeropsis manii seed LOX.(a) shows a competitive inhibition with the crude extract.(b) shows a competitive inhibition with ascorbic acid.(c) Noncompetitive inhibition with propyl gallate.
F 1: Michaelis-Menten plot showing the effect of substrate concentration on LOX.
2: Results of lipoxygenase effects of crude Gongronema latifolium and two other known antioxidants.Lipoxygenase activity was assayed as change in absorbance at 234 nm aer 5 min of incubation in the presence and absence of the extract, propyl gallate, and ascorbic acid, respectively.e equation           .was applied in the calculation of lipoxygenase activity where  is the value of absorbance increase,  is the vol of incubation mixture,  is the extinction coefficient for linoleic acid (25 × 10 −3 mol  L  cm −1 ),  is the length of the cuvette (1 cm), and  is the volume of enzyme (0.1 mL).Experiments were conducted in triplicates and   0.05.