Nutrition is an important aspect of public health because it is linked to many significant diseases and health problems. Multiple epidemiologic studies have shown that the consumption of vegetable foods could be a public health issue. Polyphenols are the most abundant antioxidants in the diet, and consumption of foods and beverages containing polyphenols may have significant public health implications. Based on existing research, polyphenols may be useful in primary and secondary prevention, particularly concerning diseases associated with oxidative damage such as obesity, diabetes, cardiovascular disease, and cancers.1 Finding alternative and complementary ways to reduce the redox processes might have a beneficial interest in the context of developing countries.
The Flora of Kwilu Kwango in Bandundu area (Democratic Republic of Congo) is rich in traditional foods that are mostly unexploited. For this reason, Mbemba et al assessed the nutritional value of some traditional foods by determining their relative amino acid composition, in order to contribute to the equilibration of the diet in the population of this area severely affected by malnutrition 2 and diseases such as konzo, a toxico-nutritional neurological disease in which oxidative damage plays a decisive role.3 Among traditional foods studied by Mbemba et al, we focus our research on four species of plants commonly used as traditional vegetables to Kenge city in Bandundu area: Entada gigas (L.) Fawc. & Rendle, Psophocarpus scandens (Endl.) Verdc, Salacia pynaertii De Wild, and Tetrorchidium congolense J.Léonard.
Entada gigas, native from Central America, Caribbean, and Africa, is commonly known as sea heart. It is a vegetable flowering plant of the pea family, Fabaceae. Seeds of E. gigas have not been used by people as food but it is a very popular seed used in the preparation of traditional concoctions for curing diseases in Nigeria.4 Young leaves are consumed as a vegetable in DRC where it was called Futi futi or Nzembo futi.2 Psophocarpus scandens (Fabaceae) is a vigorous, perennial vine with mauve, blue, lilac or white flowers. The primary use of P. scandens is as food in DRC where it was commonly called kikalakasa.2 Salacia pynaertii (Celastraceae) is a liana native of Africa. The young leaves are eaten as a vegetable in DRC and it is called Mbondi in the local language. They are usually pounded and cooked with peanuts, caterpillars or mushrooms and local tomatoes.2,5 Tetrorchirdium congolense (Euphorbiaceae) is native to tropical Africa. This plant is used medicinally in Central Africa, where the bark latex is taken with palm wine to treat diarrhea.6 It is called in Bandundu local language “Nkelekete. Young leaves are consumed as a traditional vegetable in DRC.2
To our knowledge, few investigations have been performed on the phytochemical composition and the biological properties such as antioxidant capacities of these food plants from Bandundu. The present work aimed to determine the microscopic features of these vegetables and to investigate the antioxidant activities of their methanolic extracts using ABTS and DPPH assays. Thin Layer Chromatography (TLC) and High-Performance Liquid Chromatography (HPLC-DAD) were used to achieve phytochemical analysis of phenolic compounds.
MATERIALS AND METHODS
Plants have been harvested in Kenge (S04° S1’ E16° S8’), a commune in Pelende-Nord sector, Kwango district, Bandundu Province in the Democratic Republic of the Congo.
The leaves of Psophocarpus scandes, Entada gigas, Salacia pynaerti and Tetrorchidium congolense were collected from the territory of Kenge, Bandundu (Democratic Republic of Congo, DRC). The collected plants were identified by experts of the herbarium of Kinshasa ( National Institute of Studies and Research in Agronomy, Department of Biology, Faculty of Science, University of Kinshasa, DR Congo ) and Professor Lukoki. They were dried at the room temperature. Once in the laboratory, the leaves were dried at 37 ° C in an oven while calculating the rate of moisture and were finely ground in a high-speed mill (Retsch ZM 100 Model) sieved at 180 μm particle size. The powdered leaves were stored in the dark at room temperature and used for solvent extraction.
Chemicals and reagents
All solvents were of analytical grade and purchased from Merck (Germany) and VWR Chemicals Prolabo (Leuven, Belgium). Caffeic acid, gallic acid, rutin, hyperoside and isoquercitrin (HPLC grade); 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), 2-aminoethyl-diphenylborat, dimethyl sulfoxide (DMSO), Folin-Ciocalteu reagent and potassium persulfate were purchased from Sigma (Bornem, Belgium). 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) was purchased from Eastman Kodak (Rochester, NY, USA). Water was treated in a Milli-Q water ultra-purification system (Easy Pure Purification System) before use.
Preparation of extracts
Methanolic extracts were prepared by extracting successively 10 g of leaves powder with methanol HiPerSolv Chromanorm from VWR Chemicals Prolabo (Leuven, Belgium) to obtain 200 mL of percolate. Evaporation of the solvent was performed under reduced pressure (40°C) followed by a 48-72 h vacuum drying to provide the dry extracts which were weighed and kept in hermetic and dark flasks at 4 °C.
Thin Layer Chromatography (TLC) analysis
The phytochemical screening was performed following some standard lab techniques.9 Aqueous extracts at the concentration of 10 mg/mL were analyzed by TLC on normal phase Silica gel 60 F254 plates (Merck), using a 10 μL deposit and as eluent either:
Ethyl acetate, formic acid, glacial acetic acid and water (100:11:11:26; v/v/v/v)
Toluene/ethyl acetate (9:1; v/v)
Flavonoids and phenolic acids were revealed using Natural Products-PEG reagent and observed under UV-365 nm light. Flavonoids were detected as yellow-orange fluorescent spots and phenolic acid as blue fluorescent spots.10 Chlorogenic acid, Kampferol 3-O-glucoside (astragalin), quercetin 3-O-galactoside (hyperoside), quercetin 3-O-rhamnoside (quercitrin) and quercetin 3-O-rutinoside (rutin) were used as standards.
High-Performance Liquid Chromatography (HPLC-DAD) analysis
Analytical HPLC- DAD separations were carried out on a Hypersil ODS® RP18 column as described previously.11 Chlorogenic acid, rutin, and isoquercitrin were used as standards.
Determination of total phenolic content
Total phenolic content of methanolic extracts (Methanol 80%) was determined according to the Folin-Ciocalteu method as described previously.12 A calibration curve of gallic acid (0.025-0.4 mg mL-1) was prepared, and phenolic contents were determined in triplicate from the linear regression equation of this curve. The results were expressed as milligrams of gallic acid (GA) equivalent per gram of dried matter.
The flavonoid content of the extracts was determined by UV-Vis spectrophotometry13 Results are expressed in mg equivalent of quercetin per g (mg QE/g) of dry vegetal material using the following equation y= 0.0232x + 0.1535 (R2= 0.945).
The extraction of tannins was carried out according to the adapted method used by Zhang et al, 2008.14 Tannin extract contents were quantified by vanillin method using the procedure reported by Sun et al. 1998.15 This method is based on the ability of vanillin to react with tannins units in the presence of acid to produce a colored complex measured at 500 nm.
Determination of anthocyanins
The determination of the anthocyanin content of the extracts was performed by the procedure reported by Adedapo et al.16
ABTS radical scavenging capacity
ABTS assay was based on the method described by Kapepula et al.17
DPPH radical scavenging capacity
DPPH assay was performed according to the method described previously by Floegel et al (2011) with slight modifications.18 A solution of 0.004% of DPPH in 80% (v/v) methanol was prepared one hour before use. The absorbance of the solution was adjusted to 0.75±0.03 at 517 nm using fresh 80% (v/v) methanol. Then 0.02 mL of standard or sample were mixed with 1.98 mL of DPPH solution and incubated for 30 min in the dark covered with aluminum foil. The decrease of absorbance was monitored at 517 nm with Spectrophotometer Hewlett-Packard 8453. The antiradical capacity analysis was performed on dichloromethane and methanolic dry extracts. Quercetin was used as positive control and ABTS•+, DPPH• scavenging activities of extracts were expressed as IC50 (IC50 is the amount of antioxidant necessary to decrease the initial concentration of radical by 50%).
Results were expressed as mean values ± standard deviation (SD). IC50 were calculated with GraphPad Prism 6.0 under application of the function “log (inhibitor) vs. normalized response-variable slope” after converting the concentrations into their decimal logarithm. One-way analysis (ANOVA) and Student’s paired t-test were used to compare scavenging capacities determined by ABTS and DPPH assays and the level of statistical significance was set at p < 0.05, for two-sided testing.
Botanical microscopic characters
Powders of the leaves of the different vegetables selected were treated with Steimetz reagent and showed the following specific botanical microscopic characters. Entada gigas revealed fragments of parenchyma with acicular raphides of calcium oxalate, rectangular epidermal cells containing crystals, paracytic stomata. Also reticulate vessels, smooth no glandular trichrome and few starch grains were observed (Figure 1). Psophocarpus scandens leaves powder revealed abundant prisms of calcium oxalate, starch grains, fragments of lignified fibers, diacytic stomata, circular epidermal cells, spiral vessels and unicellular non grandular trichomes. Salacia pynaertii showed the presence of fibers containing crystals, epidermis fragments containing numerous paracytic stomata, cluster crystals (druse), spiral vessels, few starch grains and sclerotic cells (Figure 1). Leaf powder of Tetrochirdium congolense exhibited palisade parenchyma, anticlinal beaded cell walls, numerous anisocytic stomata, lipid droplets, unicellular nongrandular trichoma, punctuated vessels and fibers with crystals.
The results of phytochemical screening showed that flavonoids, terpenes, steroids and quinones are present in all the species studied. However, E. gigas and P. scandes contain also alkaloids, S. pynaertii, and T. congolenses contain also anthocyanins. Iridoids were present in P.scandens and S. pynaertii.
TLC and HPLC-DAD analysis have shown the presence of polyphenolic compounds in all methanolic extracts. TLC fingerprints of extracts showed the presence of glycosylated flavonoids (yellow, orange and green fluorescent spots) and phenolic acids (blue fluorescent spots) as major compounds (Figure 2). This was confirmed by HPLC-DAD analysis (Figure 3). By comparison with the retention factor, the coloration of spots, retention time and UV-spectrum of standards used, some phenolic compounds could be identified in these extracts. E. gigas contains, quercetin-3-O-rutinoside and kaempferol 3-O-glucoside; P. scandens contains quercetin 3-O-rhamnoside; S. pynaerti contains kaempferol 3-O-glucoside and T. congolense contains kaempferol 3-O-glucoside and quercetin 3-O-rhamnoside (quercitrin). Further chromatographic and spectroscopic studies are needed to characterize the others unknown compounds.
Results from the quantitative determination of anthocyanins, flavonoids, tannins, and total phenolic content are summarized in Table 1.
Anthocyanin and tannin contents were determined as catechin equivalents in milligrams per gram of dry weight (mg CE/g DW), while total polyphenol contents were calculated as gallic acid equivalents in milligrams per gram of dry weight (mg GAE/g DW). Flavonoid content was determined as quercetin equivalents in milligrams per gram of dry weight (QE/g DW). The total phenolic contents varied significantly (P <0.05) between the studied vegetables. Results showed that S. pynaertii is the vegetable with the highest amount of total phenolic compounds and anthocyanins. P. scandens had the highest amount of flavonoids. Furthermore, concentrations of tannins were also high in the studied vegetables, with higher values in P. scandens leaves, followed by the leaves of T. congolense.
Radical scavenging activity of tested traditional food items, determined by two biochemical in vitro methods namely ABTS and DPPH assays is presented in Table 2 and is expressed as IC50 values.
IC50 is the amount of antioxidant necessary to decrease the initial concentration of radical by 50%. Lower IC50 value indicates a higher antioxidant activity. Quercetin-equivalent (QE) is calculated as IC50 of quercetin used as positive control divided by IC50 of plant extract. All the extracts had significant scavenging effects with antiradical activities connected to their ability to scavenge free radicals according to their IC50 and quercetin equivalent (Q-E) values. IC50 and Q-E values for extracts showed that S. pynaertii is the most active followed by T. congolense, E. gigas and P. scandens in ABTS assay; while in DPPH assay, T. congolense is the most active.
Considering the microscopic examination of the four vegetables, the presence or shape of specific crystals of calcium oxalate, grain starch, epidermal cells, fibers, cuticle, stomata, and vessels allowed the identification of these four species.
There is few reports on the phytochemical screening of these Congolese traditional vegetables. However, Fankam et al.20 Reported the presence of alkaloids; phenols and tannins in E. gigas from Cameroon but flavonoids, quinones and terpenoids were not detected. Some studies showed that seeds from E. gigas used in the preparation of traditional concoction for curing diseases in Nigeria contained essential minerals and high amount of anti-nutrient factors such as oxalate, phytate, and tannins.4,21 These anti-nutrient factors are reduced through processing of the vegetables. Oxalate content of leaves of E. gigas is in accordance with microscopic analysis that showed abundant fragments of parenchyma with cells containing calcium oxalate crystals. Previous studies on P. scandens, S. pynaertii and T. congolense reported their highest protein and mineral contents.2,22,23 Interestingly, S. pynaertii constitute the richest vegetable in methionine and cysteine among all studied traditional vegetables in Bandundu.24 Methionine and cysteine are sulphur amino acids, essential for the detoxification of cyanogen glycosides implicated in the occurrence of konzo. S. pynaertii is an unconventional green leafy vegetable consumed by populations of Popokabaka, another area severely affected by konzo.5
Many studies were performed to assess the nutritional value of P. scandens. Leaves of P. scandens were showed to contain lipids and proteins with high nutritive quality. One of the most important traditional use of this plant is to increase the quantity and quality of milk produced by nursing mothers.22Our results obtained from ABTS and DPPH assays show that all the extracts possessed antioxidant properties. Our results were correlated between the two chemical methods used. The small differences observed between the two methods could be attributed to the fact that DPPH assay only detect hydrophilic antioxidants in the contrary of ABTS assay. The highest radical-scavenging capacity of S. pynaertii and T. congolense extracts is in adequacy with their higher total phenolic content.
Earlier studies have reported that the selected vegetables have a reasonable nutritive value. They are good source of essential amino acids and minerals especially for children who are exposed to many diseases.2,22,23 The high nutritive value of these traditional vegetables associated with their important antioxidant activities could contribute to a diversification of the diet in Bandundu’s population, and could then provide benefits leading to a protection against oxidative damage under different conditions including konzo.
The microscopic analysis of powders from leaves of selected species allowed the identification of specific botanical microscopic characters. TLC and HPLC-DAD analysis indicated that phenolic compounds are major secondary metabolites of leaves from E. gigas, P. scandens, S. pynaertii and T. congolense. All extracts exhibited good antioxidant activity with S. pynaertii and T. congolense as the most actives. However, these antioxidant in vitro activities should be complemented in the future by in vivo evaluation in healthy humans. Further studies are then needed to evaluate the cellular antioxidant and the in vivo antioxidant activities of these traditional vegetables and particularly in their cooked forms. This could lead to the valorization of these traditional vegetables, which could be promoted as food or as food supplements with high antioxidant capacity.