1. Introduction
Natural products and health foods have recently received a lot of attention both by health professionals and the common population for improving overall well-being, as well as in the prevention of diseases. Epidemiological evidence has associated the frequent consumption of vegetables with decreased disease risk [1] -[3] . In line with this, different types of vegetables have been re-evaluated for recognition for possible candidature as functional foods, and as valuable sources of nutraceuticals. Functional foods refer to those whole, fortified, enriched or enhanced foods that provide health benefits beyond the provision of essential nutrients (e.g., vitamins and minerals), when they are consumed at efficacious levels as part of a varied diet on a regular basis [1] [4] . The term nutraceutical, which combines the word nutrition and pharmaceutical, is defined as a food that contains a medical health benefit beyond that of basic nutrition [1] [2] [4] .
One such vegetables with a potential for use as functional food is Tridax procumbens Linn (family Asteraceae), which is commonly called coat buttons. Its leaves are cooked and eaten as vegetable, and are also used as fodders [5] -[8] . Consequent upon this, Ikewuchi and colleagues investigated the nutritional potential of the leaves [9] -[11] . The leaves are also commonly used for medicinal purposes, because of their myriad of pharmacological properties. These include analgesic [12] , anti-anemic [13] , anti-arthritic [14] , anti-diabetic [15] -[17] , antihypertensive [18] [19] , anti-inflammatory, antioxidant [20] , antimicrobial [21] , antipyretic [6] , hepatoprotective [22] , hypocholesterolemic and weight reducing [23] properties. The present study reports the phytochemical composition of the leaves of Tridax procumbens, and in addition discusses the bioactivities of the detected compounds, with a view to highlighting the possibilities of the use of the leaves as a functional food, or as a source of nutraceuticals.
2. Materials and Methods
2.1. Collection of Plant Samples
Samples of fresh Tridax procumbens plants were collected from within the Choba and Abuja Campuses of University of Port Harcourt, Nigeria. Their identity was confirmed by Dr. M.C. Dike of Taxonomy Unit, Department of Forestry and Environmental Management, Michael Okpara University of Agriculture, Umudike, Abia State, Nigeria; and Mr. John Ibe, the Herbarium Manager of the Forestry Department, National Root Crops Research Institute, Umuahia, Nigeria. They were cleaned and their leaves were removed, oven dried at 55˚C and grounded into powder, which was stored in an air tight container, for subsequent use.
2.2. Determination of the Phytochemical Content of the Leaves
2.2.1. General Experimental Procedures
Analyses were carried out at Multi Environmental Management Consultants Limited, Igbe Road, Ikorodu, Lagos, Nigeria, with a Hewlett Packard HP 6890, GC apparatus, fitted with flame ionization detector, powered with HP Chemstation Rev. A 09.01 (1206) software, to identify and quantify compounds. Standard solutions were prepared in methanol for alkaloids, flavonoids, tannins and simple benzoic acid derivatives; acetone for carotenoids and lignans, methylene chloride for phytosterols; and ethanol for hydroxycinnamates. The linearity of the dependence of response on concentration was verified by regression analysis. Identification was based on comparison of retention times and spectral data with standards. Quantification was performed by establishing calibration curves for each compound determined, using the standards.
2.2.2. Determination of Alkaloid Composition
Extraction was carried out according to the method of Tram et al. [24] . The extract was subjected to gas chromatography on a capillary DB-5MS column (30 m × 0.25 mm × 0.25 μm film thickness). The inlet and detection temperatures were 250˚C and 320˚C. Split injection (split ratio of 20:1) was adopted. The carrier gas was nitrogen. The hydrogen and compressed air pressures were 1.97 and 2.67 kg/cm2. The oven was programmed initially at 60˚C for 5 min, ramped at 10˚C/min for 20 min, before ramping again at 15˚C/min for 4 min.
2.2.3. Determination of Carotenoid Composition
The extraction was carried out by a modification of the method of Rodriguez-Amaya and Kimura [25] . The carotenoids were successively extracted with acetone and a (1:1) mixture of diethyl ether and petroleum ether, were concentrated and re-dissolved in acetone before saponifying and re-extracting with a (1:1) mixture of diethyl ether and petroleum ether. The resultant extracts were dried and re-dissolved in petroleum ether and subjected to gas chromatography on a capillary ZP-5 column (30 m × 0.32 mm × 0.25 μm film thickness). The oven was programmed initially at 45˚C, held for 6 min, and ramped at 38˚C/min to 250˚C. Initial column head pressure at a constant flow mode was 0.243 kg/cm2.
2.2.4. Determination of Flavonoid Composition
The extraction was carried out according to the method of Millogo-Kone et al. [26] . The extract was subjected to gas chromatography on a capillary HP INNOWax column (30 m × 0.25 mm × 0.25 μm film thickness). The inlet and detection temperatures were 250˚C and 320˚C. Split injection (split ratio of 20:1) was adopted. The carrier gas was nitrogen. The hydrogen and compressed air pressures were 1.55 and 2.46 kg/cm2. The oven was programmed initially at 50˚C, ramped at 8˚C/min for 20 min, maintained for 4 min, ramped again at 12˚C/min for 4 min, and maintained for 4 min.
2.2.5. Determination of Hydroxycinnamates Composition
The extraction was carried according to method of Ortan et al. [27] . The extract was subjected to gas chromatography on HP-5 column (30 m × 0.32 mm × 0.25 μm film thickness). It was introduced via an all-glass injector working in the split mode, with nitrogen as the carrier gas, at a flow rate of 1 mL/min. The injection and detector temperatures were 260˚C and 300˚C. The oven was programmed at the start of the run from 170˚C to 250˚C at 5˚C/min.
2.2.6. Determination of the Lignans Composition
The extraction was carried out according to the method of Chapman et al. [28] . The extract was subjected to gas chromatography on a ZP-5 column (30 m × 0.32 mm × 0.25 μm film thickness). One microliter of sample was injected. The initial oven temperature was 40˚C, while the injector and transfer line temperatures were 250˚C and 280˚C. A solvent delay of 2.00 min was followed by ramping at 10˚C/min to a final temperature of 230˚C and held for 1.00 min.
2.2.7. Determination of Benzoic Acid Derivatives Composition
The extraction was carried out according to the method of Ndoumou et al. [29] . The extract was subjected to gas chromatography on a capillary HP 1 column (30 m × 0.25 mm × 0.25 μm film thickness). The inlet and detection temperatures were 250˚C and 320˚C. Split injection (split ratio of 20:1) was adopted. The carrier gas was nitrogen, at a pressure of 2.11 kg/cm2. The hydrogen and compressed air pressures were 1.97 and 2.25 kg/cm2. The oven was programmed initially at 60˚C for 5 min, ramped at 15˚C/min for 15 min, maintained for 1 min, and then ramped again at 10˚C/min for 4 min.
2.2.8. Determination of Phytosterols Composition
Extraction of oil was carried out according to AOAC method 999.02 [30] , while the analysis of sterols was carried out according to AOAC method 994.10 [30] . The resultant sterol derivatives were subjected to gas chromatography on a capillary HP INNOWax column (30 m × 0.25 mm × 0.25 μm film thickness). The inlet and detection temperatures were 250˚C and 320˚C. Split injection (split ratio of 20:1) was adopted. The carrier gas was nitrogen. The hydrogen and compressed air pressures were 1.55 and 2.46 kg/cm2. The oven was programmed initially at 60˚C, ramped at 10˚C/min for 20 min, maintained for 4 min, ramped again 15˚C/min for 4 min, and maintained for 10 min.
2.2.9. Determination of Tannins Composition
Extraction was carried out according to the method of Luthar [31] . The extract was subjected to gas chromatography on HP 5 column (30 m × 0.25 mm × 0.25 μm film thickness). The inlet and detection temperatures were 250˚C and 320˚C. Split injection (split ratio of 20:1) was adopted. The carrier gas was nitrogen. The hydrogen and compressed air pressures were 1.97 and 2.81 kg/cm2. The oven was programmed initially at 120˚C, before ramping at 10˚C/min for 20 min.
2.3. Derivation of Composition per Wet Weight from the Composition per Dry Weight
The compositions per wet weight of the determined parameters were derived from the compositions per dry weight and vice versa, using the following formula, adapted from Health Canada Official Methods [32] .
2.4. Data Analysis
Comparisons were based on simple percentages.
3. Results
The leaves had high flavonoids content (6477.706 g/kg) (Table 1). Twenty three known flavonoids were detected in the leaves. This consisted of 17.593% kaempferol, 12.538% (−)-epicatechin, 8.202% biochanin, 7.968% (+)-catechin, 7.875% apigenin, 6.254% naringenin, 6.124% daidzein, 4.418% quercetin, 4.376% butein, 3.895% robinetin, 3.047% baicalein, 2.986% nobiletin, 2.806% (−)-epigallocatechin, 2.444% genistein, 2.338% (+)- gallocatechin, 1.818% ellagic acid, 1.707% luteolin, 1.406% myricetin, 0.735% baicalin, 0.498% isorhamnetin, 0.404% (−)-epigallocatechin-3-gallate, 0.359% silymarin and 0.207% (−)-epicatechin-3-gallate.
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Table 1. Flavonoids composition of Tridax procumbens leaves.
The leaves had high hydroxycinnamates (0.580 g/kg), tannins (0.813 g/kg) and phytosterols (0.250 g/kg), moderate benzoic acid derivatives (0.032 g/kg) and lignans (0.077 g/kg), and low carotenoids (0.001 g/kg) contents (Table 2). Five known carotenoids were detected in the leaves. This consisted of 62.608% lutein, 13.989% carotene, 11.328% antheraxanthin, 8.532% neoxanthin and 3.542% violaxanthin. Four known benzoic acid derivatives were detected in the leaves. This consisted of 46.091% ferulic acid, 22.624% 4-hydroxybenzaldehyde, 16.441% vanillic acid and 14.843% 4-hydroxybenzoic acid. The hydroxycinnamates fraction consisted 100% of caffeic acid, the tannins fraction consisted 100% of tannic acid, while the phytosterols fraction consisted of stigmasterol (80.853%) and sitosterol (19.146%). The leaves had moderate lignans (0.077 g/kg) contents (Table 3). Six known lignans were detected in the leaves. This consisted of 77.326% galgravin, 12.221% dehydroabietic acid, 7.837% retusin, 2.612% epieudesmin, 0.003% apigenin-4’, 7-dimethyl ether and 0.00002% (9E,12E, 15E)-9,12,15-octadecatrien-1-ol.
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Table 2. Benzoic acid derivatives, carotenoids, phytosterols, hydroxycinnamates and tannins compositions of Tridax procumbens leaves.
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Table 3. Lignans composition of Tridax procumbens leaves.
The leaves had very high alkaloids content (10.191 g/kg) (Table 4). Thirty nine known alkaloids were detected in the leaves. This consisted of 68.756% akuammidine, 16.508% voacangine, 4.944% echitamine, 2.3167% lupanine, 2.253% angustifoline, 1.902% augustamine, 0.549% crinamidine, 0.477% echitamidine, 0.327% cinchonidine, 0.324% oxoassoanine, 0.317% trigonelline, 0.178% crinane-3α-ol, 0.177% choline, 0.141% cinchonine, 0.117% indicine-N-oxide, 0.093% monocrotaline, 0.079% nitidine, 0.064% 6-hydroxypowelline, 0.050% 13-α-hydrorhombifoline, 0.043% acronycine, 0.041% powelline, 0.041% ambelline, 0.040% 6-hydroxyundula- tine, 0.040% dihydro-oxo-demethoxyhaemanthamine, 0.039% 9-octadecenamide, 0.037% ellipcine, 0.030% 1β,2β-epoxyambelline, 0.028% buphanidrine, 0.026% undulatine, 0.020% 6-hydroxybuphanidrine, 0.016% sparteine, etc.
4. Discussion
The leaves had higher catechin, gallocatechin, epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate, naringenin, apigenin, luteolin, kaempferol, myricetin and quercetin contents than lettuce and onions [33] . Amongst the biological activities of flavonoids are actions against free radicals, free radical mediated cellular signaling, inflammation, allergies, platelet aggregation, microbes, ulcers, viruses, tumors and hepatotoxins [34] . Luteolin has antibacterial, anti-inflammatory, anti-mutagenic, antioxidant [34] , anti-allergic, anti-andro- genic, anticancer, anti-diabetic, anti-estrogenic, antimicrobial, hypocholesterolemic, hypotensive, neuroprotective and radio-protective [35] activities. Kaempferol is known for its antibacterial, anti-inflammatory, antioxidant [34] , analgesic, anti-allergic, anticancer, anti-diabetic, antifungal, antiprotozoal, anti-osteoporotic, anti- thrombogenic, antiviral, cardioprotective, hepatoprotective, hypocholesterolemic, hypotriglyceridemic and neuroprotective [36] activities. Kaempferol-3-O-rutinoside has hypotensive activity [37] .
Apigenin has antibacterial, anti-inflammatory, diuretic, hypotensive, smooth muscle relaxation enhancing [34] , antioxidant, anti-carcinogenic, antidepressant, cardioprotective, hepatoprotective, anti-peroxidative [38] , anti-tumor, anti-proliferative, oxygenase inhibitory and apoptosis inducing [39] activities. Naringenin has anti- atherogenic, anticancer, anti-fibrogenic, anti-inflammatory, anti-mutagenic, antioxidant, hepatoprotective, hypocholesterolemic [40] , anti-ulcer and cardioprotective [41] properties.
Catechins are antimicrobial [34] , anti-allergic, anti-carcinogenic, anti-diabetic, antihypertensive, anti-in- flammatory, anti-mutagenic, anti-obesity, antioxidant, anti-platelet, anti-proliferative, anti-tumorigenic, anti- ulcer, chemo-preventive, hypocholesterolemic and neuroprotective [42] agents. (+)-Catechin has been reported to have cardioprotective [34] , antispasmodic, bronchodilator and vasodilator [43] activities. Epicatechin, epicatechin gallate and epigallocatechin gallate provide protective benefits by their free radical scavenging ability and their inhibition of eicosanoid synthesis and platelet aggregation [34] . In addition, epicatechin has antioxidant [34] and hypoglycemic activities [44] .
Diadzein and genistein have antithrombotic, hypocholesterolemic, hypotensive [45] , anticancer and estrogen- like properties [34] . Genistein also has anti-diabetic, anti-inflammatory, anti-obesity, antioxidant and cardio- protective [46] activities. Nobiletin has anti-atherosclerotic, anti-dyslipidemic, antihypertensive, anti-inflam- matory, antioxidant, anti-resorptive, antithrombotic [47] , anti-angiogenic, anti-carcinogenic, anti-dementia, cognitive impairment improving [48] and antitumor properties, and enhances adiponectin secretion [49] .
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Table 4. Alkaloids composition of Tridax procumbens leaves.
Studies have shown that ellagic acid has anti-allergic, anticancer, anti-depressant, anti-diabetic, anti-inflam- matory, antimalarial, antioxidant, anti-wrinkle, neuroprotective [50] , anti-apoptotic, anti-atherogenic, anti-pro- liferative and chemo-preventive [51] activities. Silymarin is an anti-arthritic, anti-carcinogenic, anti-diabetic, anti-hypercholesterolemic, anti-inflammatory, anti-obesity, antioxidant, cardioprotective, gastroprotective, hepatoprotective, immunomodulatory, neuroprotective and neurotropic [52] agent.
Myricetin exhibits antibacterial, anti-gonadotropic, antioxidant [34] , anti-carcinogenic, anti-diabetic, anti- hyperlipidemic, anti-inflammatory, antimicrobial, antioxidant, anti-platelet, antiviral and cytoprotective [53] activities. Biochanin A has been reported to have anticancer, anti-inflammatory, antimicrobial, antioxidant, immunomodulatory and hepatoprotective [54] activities. Robinetin is a chemopreventive [55] agent; while butein is an antioxidant, pro-apoptotic and hepatoprotective [56] agent.
Baicalein is a potent free radical scavenger and xanthine oxidase inhibitor, thus improving endothelial function and conferring cardiovascular protective actions against oxidative stress-induced cell injury. Baicalein has anti-arteriosclerotic, antibacterial, antihypertensive, anti-inflammatory, anti-mitogenic, antioxidant, anti-proli- ferative, anti-thrombotic, antitumor, antiviral and cardio-protective [57] activities. Quercetin has been reported to have anti-allergic, anti-arthritic, antibacterial, anti-carcinogenic, anti-cancer, anti-cataractogenic, anti-diabetic, antihypertensive, anti-inflammatory, antioxidant, antiviral, cardio-protective, gastro-protective, hepatoprotective and hypocholesterolemic [34] [58] activities.
The carotene and lutein contents of the leaves were higher than those of cabbage, but lower than those of amaranth [59] . Carotenoids have been widely accepted as safe chemicals for food supplementation and neutraceutical purposes due to their intense coloring abilities, their role as precursors of vitamin A, and their antioxidant activity in animals [34] [60] . They have been shown to be beneficial in preventing major health problems in developing and developed countries, including cancers, cardiovascular and coronary heart diseases, photosensitivity disorders, age-related macular degeneration and cataract [60] . Carotenes and lutein protect against uterine, prostate, breast, colorectal and lung cancers [34] . Carotenes, in addition, have pro-vitamin A and antioxidant [34] activities.
The leaves had higher caffeic, vanilic and 4-hydroxybenzoic acids, but lower ferulic acid contents than red cabbage [61] . Phenolic acids are powerful antioxidants and have been reported to demonstrate antibacterial, antiviral, anti-carcinogenic, anti-inflammatory and vasodilatory actions [61] . 4-Hydroxybenzaldehyde is used as a flavor and fragrance agent. Caffeic acid has anti-diabetic [50] , antioxidant [62] , anti-inflammatory and photo-protective [63] activities. 4-Hydroxybenzoic acid esters (also called parabens), are widely used as antimicrobial agents in a large variety of food, pharmaceutical, and cosmetic products due to their excellent antimicrobial activities, low toxicity and stability over a wide pH range [64] . 4-Hydroxybenzoic acid has antifungal, anti-mutagenic, anti-sickling, estrogenic and antimicrobial [62] activities. Vanillic acid (4-hydroxy-3-methox- ybenzoic acid) is used as a flavoring agent. It has anti-sickling, anthelmintic, hepatoprotective, immune-mod- ulating, anti-inflammatory, antifungal, anti-mutagenic, estrogenic and antimicrobial [62] [65] activities. It also inhibits snake venom 5’-nucleotidase [62] [65] . Ferulic acid has antioxidant, hypocholesterolemic, anti-inflam- matory, photo-protective, cardio-protective and sperm viability boosting activities [62] .
The leaves had higher total phytosterols, stigmasterol and sitosterol contents than those of white cabbage and onions [66] . Phytosterols have anti-inflammatory [34] , hypocholesterolemic, anti-cancer [34] [66] , anti-ulcer, anti-fungal, anti-atherogenic, antioxidant [67] and estrogenic [68] activities. Stigmasterol has anti-osteoarthritic, anti-hypercholestrolemic, cytotoxicity, antitumor, thyroid inhibiting, hypoglycemic, anti-mutagenic, antioxidant, anti-inflammatory, anticonvulsant and sedative [69] , chemopreventive, anticancer, estrogenic [70] activities. β-Sitosterol has been reported to have anti-inflammatory, anti-neoplastic, immune-modulating, antipyretic [34] , cholesterol-lowering, antipyretic, antineoplastic, anti-diabetic, cardio-protective, estrogenic [68] , antimicrobial, antioxidant, chemopreventive, anticancer and antipyretic [71] activities. It is used in the treatment of benign prostate hyperplasia [71] , and alters sex steroid levels [68] . Sitosterylglucoside has been identified as an “adaptogens” [67] . According to Berger and colleagues [67] , adaptogens are a group of natural plant products which promote overall health without the rapid response normally elicited by a drug and without the side effects associated with any drug used.
The leaves had higher total tannin content than red pepper [72] , but lower tannic acid content than cabbage [73] . Tannins have antioxidant, antimicrobial [34] , anticancer [74] activities. Tannic acid has antihypertensive, anti-diarrheal, anti-cancer, anti-asthmatic, cardioprotective, anti-diabetic, anti-cataractogenic, tumor inhibition, anti-inflammatory, anti-adipogenic [74] and hepatoprotective [75] activities.
The leaves had higher total lignans contents than garlic and lettuce [76] . Lignans have antioxidant, anticancer, anti-diabetic, antiviral, bactericidal, anti-inflammatory, anti-atherosclerotic, cardio-protective [77] , anti-hepato- toxic, antitumor, antiplatelet aggregation [78] and estrogen-like [76] activities. Galgravin has been reported to exhibit anti-inflammatory, anti-malarial, anti-platelet, anti-resorptive [79] , anti-rheumatoid, neurotrophic and neuroprotective [28] activities.
Akuammidine has antibacterial, antifungal [80] , antimalarial, anti-inflammatory, hypotensive, skeletal muscle relaxant, local analgesic and anti-depressant [81] activities. Voacangine has been reported to have cardiovascular toning, central nervous system depressant, anti-convulsive, anti-pyretic, analgesic, local anesthetic, acetyl cholinesterase inhibitory [82] , anti-angiogenic [83] and anti-mycobacterial [84] activities. Studies have shown that echitamine has diuretic, hypotensive [85] , central nervous system depressant and antiepileptic [86] properties. Augustifoline is a bacteriostatic agent [87] while lupanine is an anti-arrhythmic, hypotensive, hypoglycemic, β-glucosidase inhibitory, bacteriostatic [87] , oxytocic and central nervous system depressant [88] agent.
Finally, the present study showed that the leaves of Tridax procumbens contain a variety of biologically active phytochemicals. The beneficial roles of these bioactive phytochemical constituents can be harnessed in the diet, making them veritable tools for nutritional therapy. This, therefore, emphasizes the potential of these leaves as a functional food.
NOTES
*Corresponding author.