The leaves of four plant foods commonly consumed in Nigeria namely Gnetum africanum (Igbo name: Ukazi), Gongronema latifolium (Igbo name: Utazi), Telfairia occidentalis (Igbo name: Ugu), Ocimum gratissimum (Igbo name: Nchoanwu), were each screened for the presence of known potential chemopreventive agents using paper chromatography, thin layer chromatography and various chemical tests. All four vegetables showed the presence of phenolic compounds, flavonoids, phytosterols, tannins, saponins, chlorophyll and glycosides. Only Telfairia occidentalis and Gnetum africanum showed traces of alkaloids

Iweala, E. E. J.

English

Covenant University Repository

Journal of Medicinal Food Plants Vol. 1(2), pp. 58 - 63, July – December 2009 Available online at htt://www.jmedfoodplants.com ISSN 1947-3710 © Iweala 2009    Correspondence to:  Emeka E.J. Iweala, Depart-ment of Biological Sciences, Biochemistry and Mo-lecular Biology Unit, Covenant University, P.M.B 1023, Ota, Ogun State, Nigeria. Email: emekaiweala2004@yahoo.co.uk  Full Paper Preliminary qualitative screening for cancer chemopreven-tive agents in Telfairia occidentalis Hook.f., Gnetum afri-canum Welw., Gongronema latifolium Benth. and Ocimum gratissimum L. from Nigeria   Emeka E.J. Iweala   Department of Biological Sciences, Biochemistry and Molecular Biology Unit, Covenant Uni-versity, Nigeria  Accepted 9 September 2009   The leaves of four plant foods commonly consumed in Nigeria namely Gnetum afri-canum (Igbo name: Ukazi), Gongronema latifolium (Igbo name: Utazi), Telfairia oc-cidentalis (Igbo name: Ugu), Ocimum gratissimum (Igbo name: Nchoanwu), were each screened for the presence of known potential chemopreventive agents using paper chromatography, thin layer chromatography and various chemical tests. All four vegetables showed the presence of phenolic compounds, flavonoids, phytoste-rols, tannins, saponins, chlorophyll and glycosides. Only Telfairia occidentalis and Gnetum africanum showed traces of alkaloids.   Keywords: Gnetum africanum, Gongronema latifolium, Telfairia occidentalis, Ocimum gratis-simum, chemopreventive agents    Introduction  Cancer is one of the leading causes of death worldwide [1, 2]. Cancer control involves concerted actions aimed at re-ducing the incidence and death that is as-sociated with it. Chemoprevention is one of the best strategies employed in cancer control and has evolved into one of the most exciting and promising area in can-cer research [3]. Chemoprevention in-volves the use of natural and synthetic compounds to inhibit, reduce or reverse carcinogenenesis [4]. A large number of potential chemopreventive agents have been identified from epidemiological sur-veys, experimental, preclinical and clinical observation and structural homology with known chemopreventive agents [5, 6].  In recent years, dietary constituents especially from plant foods have been found to protect against the occurrence of cancer [7, 8]. Some of these naturally oc-curring compounds include phenolic com-pounds, flavonoids, terpenoids, caroteno-ids,  phytosterols, isothiocyanates and other phytochemicals [9, 10].The me-chanisms underlying chemoprevention in-volves anti-oxidant, anti-inflammatory, immune-enhancing, and anti-hormone ef-fects. Other possible mechanisms include modification of drug-metabolizing en-zymes, and influences on cell cycling and differentiation, induction of apoptosis, and suppression of proliferation and angioge-nesis that play a role in the initiation and secondary modification of neoplastic de-velopment [11]. Plant foods including   Iweala      59  vegetables and fruits are largely con-sumed by tropical subpopulations [12]. Tropical plant foods elaborate an array of nutrient and non-nutrient substances with structural and chemical diversity and bio-logical activity capable of conferring pre-vention against cancer. This preliminary study is intended to identify possible po-tential chemopreventive agents in some commonly consumed Nigerian vegetables namely Gnetum africanum, Gongronema latifolium, Telfairia occidentalis, Ocimum gratissimum as a prelude to conducting more extensive studies on them.    Materials & Methods  Plant materials The leaves of four locally consumed plant foods namely Gnetum africanum, Gongronema latifolium, Telfairia occiden-talis and Ocimum gratissimum were bought from the local market at Nsukka town in Enugu State of Nigeria.    Preparation of plant materials  The leaves of the four plant food sam-ples were picked to remove infected ones and air dried at room temperature. The dried leaves were then ground to a coarse powder and stored in amber bags at room temperature.  Phytochemical screening Phytochemical screening for potential chemopreventive agents was done accord-ing to various screening methods [13, 14].  Test for phenolic substances  Plant material (5 g) was stirred with 50 mL of 2M HCl and heated in a water bath at 100 0C for 30 min, cooled and fil-tered. The filtrate was extracted into 30 mL of ether and the extract was concen-trated to dryness. Residue was dissolved in few drops of chloroform and chromato-graphed one dimensionally on silica gel plate (20 cm x 20 cm) in acetone–chloroform (1:9). Phenolic substances show a variety of colors including blue, gray, red and pink.   Test for flavonoids  Plant material (5 g) was hydrolyzed by heating in 2M HCl at 100 0C for 30 min, cooled and filtrate was extracted in 30 mL ethylacetate and the extract was concen-trated to dryness. Residue was dissolved in few drops in Forestal-butanol-acetic ac-id-water (BAW) and phenol-water (phOH-H2O) solvents. Flavones and glycosylfla-vones give varying yellow colours while biflavonyls give brown colours. Also, 1 mL of dilute ammonia solution was added to about 2 mL of the ethyl acetate extract and shaken. A yellow color at the lower ammonia layer is a positive indication for flavonoids.   Test for anthocyanadins and chalcones  Plant material (5 g) was hydrolyzed in HCl for 30 min at 100 0C. Hydrosylate was filtered and the filtrate was washed twice with ethyl acetate. The aqueous layer was heated at 80 0C to remove all the ethyl acetate and extracted into amyl alcohol. The extract was concentrated to dryness. Residue was dissolved in few drops of me-thanolic-HCl and chromatographed on sili-ca gel and paper in BAW and water. An-thocyanidins give red, purple, and magen-ta colors while chalcones give red and brown colors.  Test for phytosterols Plants tissue (5 g) was defatted three times with ether and extracted into hot methanol and concentrated to a small vo-lume. Sample was chromatographed on silica gel in chloroform and detected with 50% H2SO4. Brown and violet colors are positive indicators.   Test for chlorophyll  All procedures are carried out in the dark. 2 g of fresh plant tissue was ground and extracted with 80% acetone and Ca-CO3 (to prevent formation of pheophytin) until all the color was released from the tissue. The extract was made up to 100 J Med Food Plants 1(2):58-63.      60  mL with acetone and stored in an amber bottle in a refrigerator. The absorbance was read at 645 nm, 652 nm and 663 nm and the following calculation was made:   Total Chlorophyll = 100 X A652nm or 278 X A652nm                 36  Test for tannins  Plant tissue (1 g) was extracted in 50 mL of distilled water and filtered. The fil-trate was concentrated and chromato-graphed on silica gel in chloroform–acetone–formic acid (75:16.5:8.5). Also, 1 g of plant tissue was boiled in 10 mL of 45% ethanol for 3 min, cooled and fil-tered. Filtrate was divided into three por-tions. To one portion was added few drops of lead subacetate and observed for for-mation of gelatinous precipitate. To another portion was added 1 mL of bro-mine water and observed for pale brown portion precipitates. The last portion was diluted with distilled water and few drops of ferric chloride added and observed for a transient greenish to black color.   Test for saponins  Plant tissue (5 g) was extracted with 25 mL methanol for 5 min at 50 0C in a water bath. Filtrate was evaporated and mixed with 2.5 mL water and extracted with 15 mL of butanol and the butanol layer was then chromatographed on silica gel in chloroform–methanol–water. Also 1 g of plant tissue was boiled with 10 mL of distilled water for 5 min and decanted while still hot. 2 mL of the filtrate was di-luted with 8 mL water and vigorously sha-ken and observed for a stable froth. To another 2 mL of filtrate was added 4 drops of olive oil, shaken and observed for for-mation of emulsion.  Test for alkaloids Plant tissue (5 g) was mixed with 10 mL of 10% ammonia solution and ex-tracted with 25 mL ethanol at 60 0C for 15 min. The extract was concentrated and chromatographed on silica gel in ethyl acetate–methanol–water (100:13.5:10). Blue or yellow fluorescence and brown or orange spots with Dragendorff’s reagent are positive. Also 1 g of plant tissue was boiled with 5 mL of 2% HCl and filtered. The filtrate was divided into four portions each of which is added 2 drops of Mayer’s reagent, Dragendorff’s reagent, Wagner’s reagent and picric acid solution to observe cream coloured, orange coloured, red-dish–brown and yellow precipitate respec-tively.  Test for glycosides  Plant tissue (5 g) was mixed with 50 mL of water and the solution heated for 5 min in a water bath, filtered and the fil-trate was divided into two portions. Fehl-ing solutions (1 mL) were added to 10 mL of the filtrate until alkaline and heated for 3 min. A brick red precipitate is positive. For cyanogenic glycosides, 5 g of tissue was mixed with 20 mL of water and heated in water bath with sodium picrate paper. A change from yellow to orange is positive. For cardiac glycosides, 5 g was extracted with 29 mL of water and fil-tered. The filtrate was evaporated and re-sidue dissolved in 10 mL glacial acid with 3 drops of ferric chloride.  Results & Discussion  A summary of the screening results of the four local plant foods is presented in Table 1. The four plant foods showed the presence of phenoilc substances. G. latifo-luim had the highest amount followed by G. africanum, T. occidentalis and O. gra-tissimum had the least. All the samples indicated the presence of flavonoids. The level of flavonoids was highest in G. latifo-lium and G. africanum. All the plant food samples showed the presence of phytoste-rol and chlorophyll. G. latifolum contained a higher level of phytosterol and chloro-phyll than the other samples.  Tannins were found to be present in all the sam-ples, with O. gratissimum and T. occiden-talis having higher levels than G. afri-canum and G. latifolium.   All the samples showed the presence of saponins. Only T. occidentalis showed the presence of alka-  Iweala      61  loids, while others showed slight traces of alkaloids. Generally, glycosides were   Table 1.  Result of qualitative phytochemical screening for potential chemopreventive agents in Gnetum afri-canum, Gongronema latifolium, Telfairia occidentalis and Ocimum gratissimum. Potential chemopreven-tive agents  G. africanum  G. latifolium   T. occidentalis  O. gratissimum Phenolic compounds  ++ +++ + + Flavonoids  ++ ++ + + Phytosterols  + ++ + + Chlorophyll  +++ +++ ++ + Tannins  ++ + +++ +++ Saponins  ++ + +++ ++ Alkaloids  + ++ ++ - Glycosides  ++ ++ ++ + Anthocyanidins/chalcones  +++ + + + + indicates presence; - indicates absence   found to be present in all the samples with O. gratissimum showing very slight traces. All the samples were positive for cyano-genic glycoside while all except G. latifo-lium were seen to contain cardiac glyco-sides. Anthracene glycosides were absent in all the samples. All the samples showed presence of anthocyanidins/chalcones with G. africanum showing the highest level.    The concept of chemoprevention has assumed a global significance as a result of its acceptance in the management, prevention and treatment of a wide range of life threatening diseases such as can-cer, diabetes and coronary diseases and in the maintenance of good health [15].The enormous benefits to public health which the lowering of the incidence of cancer can generate are invaluable. A large number of chemopreventive agents identified in plants have considerably contributed to the concept of cancer prevention and con-trol [16]. The chemical diversity of these naturally occurring substances increases the likelihood of their existence in tropical plant foods. There is an abundance of plant foods in the tropics especially Nige-ria and their consumption by large tropical subpopulations can be correlated with the relatively and comparatively low incidence of cancers in the tropics.  The four plant foods investigated namely Gnetum africanum, Gongronema latifolium, Telfairia occidentalis and oci-mum gratissimum are common tropical plant foods whose leaves are consumed especially in South Eastern Nigeria. The preliminary screening of these plant foods using simple chemical tests, paper chro-matography showed that they contain some known potential cancer chemoper-ventive agents such as flavonoids, tan-nins,   chalcones, anthocyanidins, phytos-terols, chlorophyll, saponins, glycosides and alkaloids. The presence of these bio-active phytochemicals have been reported in several African plants [17]. Phytosterols which are terpenoid compounds have been reported to inhibit certain cancers in animals [18]. Flavoniods, anthocyanadins and chalcones are ubiquitous phytochemi-cals with pronounced bioactivity. Some of these compounds such as apigenin and licochalcone have showed chemopreven-tive properties against several cancers [19-21]. Saponins such as diosgenin have been found to have therapeutic and che-mopreventive effects [22]. Tannins con-sumed in large quantities as fruits and vegetables on a daily basis such as ellagi-tannins are also effective against some cancers [23]. The chemopreventive prop-J Med Food Plants 1(2):58-63.      62  erties of alkaloids and glycosides have al-so been reported [24, 25]. Chlorophylin found in chlorophyll produces an anti-promoting effect on skin cancer in mice [26]. All the four plants almost had similar constituents of these substances except for alkaloids which was only prominent in Telfairia occidentalis. These chemopreven-tive agents are able to enhance host pro-tective systems such as detoxification en-zymes against carcinogens. Combinatorial effects are even more effective due to the synergistic actions of phytochemicals [27]. Accordingly, it would be anticipated that consumers of these tropical plant foods would have greater protection against specific carcinogenic compounds than non-consumers. This expectation is in ac-cord with epidemiological studies that show inverse relationships between con-sumption of vegetables and fruits and oc-currence of cancer. [28, 29]. Further in-vestigations involving specific characteri-zation of these potential chemopreventive agents and their effects in animal models are being carried out in our laboratory.     References 1. Ilson DH. Esophageal cancer chemotherapy: recent advances. Gastrointest Cancer Res. 2(2):85-92. 2008. 2. Tran E, Rouillon F, Loze JY, Casadebaig F, Phi-lippe A, Vitry F, Limosin F. Cancer mortality in patients with schizophrenia: an 11-year pros-pective cohort study. Cancer.115(15):3555-62. 2009. 3. Greenwald P, Kelloff G.J. 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Preliminary qualitative screening for cancer chemopreventive

agents in Telfairia occidentalis Hook.f., Gnetum africanum

Welw., Gongronema latifolium Benth. and Ocimum gratissimum L. from Nigeria

http://eprints.covenantuniversity.edu.ng/175/1/JMedFoodPlants.pdf

Preliminary qualitative screening for cancer chemopreventive agents in Telfairia occidentalis Hook.f., Gnetum africanum Welw., Gongronema latifolium Benth. and Ocimum gratissimum L. from Nigeria

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