w w w .ijir d.com Januar y, 2019 Vol 8 Issue 1 ISSN 2278 – 0211 (Online) Pr oximate Composition and Phytochemical Scr eening of Combr etum Her eroense Seed Powder Ndlovu Na Lectur er , Depar tment of Food Science and Nutr ition, Midlands State Univer sity, Zimbabw e Nkosinomsa Ndebele, Br ew ing Technician, Depar tment of Br ew ing, Delta Bever ages, Zimbabw e Siwela Nc Inter n Lab Technician, Depar tment of Higher Lear ning, Pear aon Institute, Midr and Abstr act: The objective of the study was to investigate the nutr itional and phyt ochemical pr oper ties of Combr etum her er oense t ea so as to deter mine its safety and quality. Two solvents; water and methanol: ethanol (50:50, v/ v) wer e used to obtain extr acts fr om the powder ed seed. The extr acts wer e subjected to qualitat ive phyt ochemical scr eening using standar d pr ocedur es. St andar d methods wer e used to measur e pr oximate composition (moistur e, cr ude fibr e, cr ude pr otein, fat and car bohydr ate). The tea was analysed for flavonoids, phenols, alkaloids, saponins, tannins, ter penoids, st er oids, glycoside and anthr aquinones. The pr oximate analyses showed that both extr acts exhibited a low-fat content , high fibr e and the highest value of car bohydr ate. Phytochemical scr eening r evealed the pr esence of six (alkaloids, glycosides, saponins, flavonoids, phenols and tannins) out of nine of the phytochemicals that wer e scr eened. The met hanol: ethanol extr act r evealedone mor e phytochemical pr oper ty (phytoster ols) that was not found in the aqueous ext r act. Both extr acts did not possess ter penoids, quinones and anthr aquinones. C her er oense tea has the potential to cont r ibute posit ively to t he health of consumer s as a bever age. Keywords: Combr etum her er oense, extr action, phytochemicals, pr oximate, tea 1. I ntroduction Plants used as food var y fr om r egion to r egion depending on availability and acceptability by people in each r egion. Some plants ar e impor tant sources of traditional bever ages including her bal indigenous tea plants for example tea br ew ed fr om Combr etum her er oense seeds (Motlhanka et al, 2008). Tea is consumed for its attractive ar oma and taste as w ell as the unique place it holds in the cultur e of many societies.In r ecent year s, tea and par ticular ly her bal tea gained incr easing consumer attention due to a gr ow ing aw ar eness of health benefits der ived fr om their consumption (Aoshima et al, 2007;McKay and Blumber g, 2002). Ther efor e, tea belongs to a r apidly expanding mar ket of ‘w ellness bever ages’ (Byun and Han, 2004). Combr etum her er oense also know n as the mouse ear ed Combr etum or Russet bush willow , belongs to the family Combr etaceae w hich consists of 18 gener a w ith the lar gest genus, Combr etum composed of about 370 species (Law r ence, 1951). This tr ee is most often seen ar ound pans, in r ocky ar eas and sometimes on-str eam banks. Russet-Bush w illow is a member of the Combr etum family and has the char acter istic four w inged pods with br ow n seeds found in the centr e. It is common in the low veld and usually seen in closely associated gr oups. It has an over all copper y appear ance often fr om Januar y to July owing to the pr esence of lar ge number s of r ed br ow n pods (Dr ummond, 1981). The seeds of Combr etum her er oense ar e r oasted and gr ound to a fine composite pow der w hich is br ew ed in water to make a pleasant tea. Tea fr om seeds of Combr etum her er oense is consumed in many r ur al ar eas in Zimbabw e w hile scarce scientific data on the composition of the tea exists. A decoction of the r oot bar k of C her er oense has been used as a r emedy for hear t diseases (Mabogo, 2012). Many studies have r evealed that plants possess antioxidant compounds w hich have anti-inflammator y, antimutagenic, antiather oscler otic, antitumor , anticarcinogenic, antibacter ial, and antiviral activities (Ivanisova et al., 2013). Ther e is a gr ow ing inter est in the use of the natur al phyt ochemicals pr esent in ber r y cr ops, teas, her bs, oilseeds, beans, fr uits and vegetables because of their health benefits (Kittset al, 2000). WHO, (2001) r epor ted that 50-80% of the population of developed and developing countr ies ar e mostly r elying on her bal plants for disease pr evention and pr omotion of health. Her bal medicines include her bs, her bal mater ials, her bal pr epar ations, and finished her bal pr oducts that contain par ts of plants or other plant mater ials as active ingr edient s (Faw zi, 2013;Scalber t et al, 2005). Her bal teas can easily be pr epar ed fr om any par t of a plant mainly its r oots, seeds, flow er , bar k and ber r ies. Often the pr epar ation of a herbal infusion may consist exclusively of one or mor e her bs and is per for med by means of decoction, infusion or macer ation (Visser , 2015). Plants typically contain mixtur es of differ ent INTERNATIONAL JOURNAL OF INNOVATIVE RESEARCH & DEVELOPMENT DOI No. : 10.24940/ ijir d/ 2019/ v8/ i1/ JAN19031 Page 93 w w w .ijir d.com Januar y, 2019 Vol 8 Issue 1 phytochemicals, also know n as secondar y metabolites that may act individually, additively, or in syner gy to impr ove health (Sr inivasahan and Dur air aj 2014). Phytochemical analysis is ver y impor tant as a key step to investigate the chemical composition of her bal tea and ensur e its quality and safety (Zhao et al, 2013). Thus, the pr esent w or k w as designed to car r y out pr oximate analysis and to scr een for phytochemical constituents of Combr etum her er oenseseed pow der . Know ledge of the phytochemicals contained in the seed pow der is key t o the safety of the consumer s. 2. Materials and Methods 2.1. St udy Set ting Extr action and chemical assays w er e done in the Food Chemistr y lab, in the depar tment of Food Science and Nutr ition at the Midlands State Univer sity. 2.2. Sour ce and Pr ocessing of Combr etum Her er oense Combr etum her er oense fr uits w er e har vested fr om 5r andomly selected t r ees in Gw anda Distr ict of Matebeleland South Pr ovince in Zimbabw e (29 o E, 21 o S). The fr uits wer e sor ted to r emove dir t and leaves after w hich the pods w er e manually r emoved to get the seeds. The seeds w er e roast ed at 650C for an hour and the seeds w er e cr ushed using a pestle and mor tar to pr oduce a pow der w hich w as then r oasted at 650C for 30 minutes. The pow der w as sieved to pr oduce a fine composite pow der and stor ed in glass vials at 4OC awaiting extr action and the r espective assays. 2.3. Deter minat ion of Pr oximate Composition of Combr etum Her er oense Seed Powder 2.3.1. Extr action One hundr ed gr ams of pow der ed Combr etum Her er oense w er e extr acted by macer ation (Chandr an et al., 2012) using either distilled w ater or methanol: ethanol (50:50; v/ v) solvent in a conical flask for 24 hour s, filter ed and evapor ated by a r otar y evapor ator at 40oC. The r esulting solution w as dr ied in an oven for 24 hour s and the extr acts kept at 4 oc until use. 2.3.2. Deter mination of Pr oximate Composition Fat fr om the Combr etum her er oense pow der w as extr acted in diethyl ether as descr ibed by the Association of Analytical Chemists (AOAC, 2000: method number 920.39). The moistur e, ash, cr ude pr otein and car bohydr ate content of the C her er oense pow der w er e deter mined as descr ibed by the Association of Analytical Chemists (AOAC, 2000: Methods 934.01 and 930.15, 942.05 and 954.01, r espectively). Each assay was car r ied out in duplicate. 2.3.3. Deter mination of Phytochemical Composition 2.3.3.1. Deter mination of Alkaloids Mayer ’s test was used to deter mine the pr esence of alkaloids (Vijayalakshmi et al., 2012). 50mg of solvent fr ee extr act w as stir r ed in 3ml of dilute hydr ochlor ic acid then filter ed thor oughly using Whatmann’s filter paper no. 1. Thr ee dr ops of Mayer ’s r eagent w er e added to 1ml of the filtr ate and a w hite cr eamy pr ecipitate indicated the pr esence of alkaloids. 2.33.2. Deter mination of Phytoster ols Phytoster ols w er e deter mined by the Liber dmann-Bur chad’s test as accor ding to Vijayalakshmi et al., (2012) w ith slight modifications. Briefly 50mg of extr act was dissolved in 2ml acetic anhydr ide then 1 dr op of concentr ated sulphur ic acid added along the side of the test tube. For mation of a blue-gr een colour indicated the pr esence of phytost er ols. 2.3.3.3. Deter mination of Ter penoids Ter penoids w er e deter mined as accor ding to the method used byVijayalakshmi et al., (2012). Briefly 2ml of Chlor ofor m and concentr ated sulphur ic acid w er e car efully added to 0.5ml of extr act. A r ed br ow n colour at i nter face indicated pr esence of ter penoids. 2.3.3.4. Deter mination of Glycosides Legal’s test was per for med accor ding toVijayalakshmi et al., (2012) to deter mine the pr esence of glycosides. Br iefly 2 ml chlor ofor m and 10% Ammonia solution w er e added to 2ml of the C her er oense seed ext r act. The formation of a pink colour confir med the pr esence of glycosides. 2.3.3.5. Deter mination of Saponins A fr oth test w as done to deter mine the pr esence of saponins w her e 2ml of distilled w ater w er e added to 5g of the extr act and the r esulting mixtur e was agitated vigor ously for 7 minutes. Foam pr oduction confir med the pr esence of saponins (Liu et al, 2009). 2.3.3.6. Deter mination of Flavonoids 3 ml of 2% sodium hydr oxide w er e added to 3ml of the extr act. The colour changes w er e r ecor ded and a yellow colour signifies flavonoids pr esence (Liu et al., 2009). INTERNATIONAL JOURNAL OF INNOVATIVE RESEARCH & DEVELOPMENT DOI No. : 10.24940/ ijir d/ 2019/ v8/ i1/ JAN19031 Page 94 w w w .ijir d.com Januar y, 2019 Vol 8 Issue 1 2.3.3.7. Deter mination of Phenols The pr esence of phenols w as deter mined by the Lead acetate method as accor dingto Vijayalakshmi et al., (2012). Br iefly, 5mg of the seed extr act w as dissolved in distilled w ater and 3ml of 10% lead acetate solution w as added. A bulky w hite pr ecipitate indicates the pr esence of phenols. 2.3.3.8. Deter mination of Quinones Concentr ated sulphur ic acid (1ml) w as added to 1ml of the seed extr act. A r ed colour indicated the pr esence of quinones (Vijayalakshmi et al., 2012). 2.3.3.9. Deter mination of Anthr aquinones The pr esence of anthr aquinones w as deter mined by adding 3 dr ops of 2% hydr ochlor ic acid to 0.5ml of the seed extr act. A r ed colour ed pr ecipitate indicated the pr esence of anthr aquinones (Vijayalakshmi et al., 2012). 2.3.3.10. Deter mination of Tannins The pr esence of tannins w as deter mined using the fer r ic chlor ide test w her e 5mg of extr act was dissolved in 5ml of distilled water and 3 dr ops of neutr al 5% fer r ic chlor ide solution added. A blue gr een colour indicated the pr esence of tannins (Vijayalakshmi et al., 2012). 2.4. Statistical Analysis Data is expr essed as mean ±SD. Gr aph Pad Pr ism Ver sion 5 (Gr aph-pad Softwar e Inc., San Diego, USA) statistical package was used to analyse data. An unpair ed t- test w as used to analyse the data. The level of significance w as set at P<0.05. 3. Results and Discussion 3.1. Pr oximate Composition of Combr etum Her er oense Seed Powder The r esults of pr oximate composition of Combr etum her er oense seed pow der ar e show n in table 1.Both the aqueous and methanol: ethanol (50:50v/ v) extr acts w er e obser ved to have a low -fat content (2.53% and 2.64% r espectively), high cr ude fibr e (34.81% and 35.02% r espectively) and the highest value for car bohydr ate content (43.21% and 43.17% r espectively).The methanol: ethanol extr act r evealed a significantly higher cr ude fibr e content compar ed to the aqueous extr act. Cr ude fibr e ser ves to enhance the efficiency of digestion by stimulating per istaltic action and enhancing the movement of food thr ough the alimentar y canal. It is also know n to pr event colon cancer . Food fibr e has been show n to aid absor ption of dietar y miner als as w ell as r educe absor ption of cholester ol (Aliyu et al., 2009). In tea how ever , cr ude fibr e impr oves the sensor y appeal of the bever age by pr oviding a filter system to pr event the leaching of plant mater ial fr om the tea bag into the infusion (Waldr on et al, 2003). Pr evious r esear cher s also indicated positive association betw een fibr e content and keeping quality of the tea. Parameter (% ) Aqueous Methanol: Ethanol P- value Moistur e 10.05±0.02 10.02±0.01 0.0955 Cr ude Fibr e 34.81±0.01 b 35.02±0.02 a 0.0478 Cr ude pr otein 4.01±0.03 3.83±0.02 0.0896 Fat 2.53±0.01 2.64±0.10 0.3652 Ash 5.03±0.14 5.25±0.01 0.2698 Car bohydr ate 43.21±0.01 43.17±0.02 0.2396 Table 1: Pr oximate Composition of Combr etum Her er oense Seed Powder a, b within Row Means with Differ ent Super scr ipts Differ Significant ly at P ˂ 0.05 Data Is Expr essed as Mean ± SD 3.2. Qualitative Phyt ochemical Analysis of Combr etum Her er oense Seed Powder The r esults of the phytochemical content of Combr etum her er oense seed pow der ar e show n in table 2.Non- polar solvents yield mor e lipophilic components, w hile alcoholic solvents give a larger spectr um of polar mater ial. Therefor e, as can be seen in table 2 it was easier to detect ster oids in the Methanol: Ethanol extr action unlike the aqueous one since methanol has the ability to dissolve both polar and non-polar mater ials. Natural pr oducts belonging to saponins, phenols, tannins, and flavonoids w er e all show n to be pr esent in both the aqueous and methanol: ethanol extr act. The aqueous extr act w as used in this study since the C her er oense seeds ar e usually used as a hot w ater decoction to make tea. The estimated amount of phytochemicals detected in the extr acts was based on the intensity of colour change obser ved dur ing scr eening. Saponins have been r epor ted to possess analgesic pr oper ties and centr al ner vous system activities (Mandal et al, 2001), w hile Podolak et al., (2010) have r epor ted cytotoxic effects of saponins. Table 2 show s the pr esence of tannins in C her er oense t ea. Tannins have tr aditionally been consider ed anti-nutr itional, how ever the effect of their anti-nutr itional pr oper ties depend lar gely on their chemical str uctur e and dosage. It has been r epor ted that if ingested in excessive quantities, tannins inhibit the absor ption of miner als, such as ir on w hich may, if pr olonged, lead to anaemia. Lalitha, (2012) added that tannins ar e know n to inhibit pathogenic fungi w hich thr eaten human health, ther efor e the consumption of teas containing tannins can contr ibute positively to the health of consumer s. Tannins ar e used as antiseptic and this activity is due to pr esence of the phenolic gr oup. Common examples of hydr olysable tannins include theaflavins (fr om tea), INTERNATIONAL JOURNAL OF INNOVATIVE RESEARCH & DEVELOPMENT DOI No. : 10.24940/ ijir d/ 2019/ v8/ i1/ JAN19031 Page 95 w w w .ijir d.com Januar y, 2019 Vol 8 Issue 1 daidezein and genistein. In Ayur veda, for mulations based on tannin-r ich plants have been used for the tr eatment of diseases like leucor r hoea, r hinor r hoea and diar r hoea(Doughar i et al ,2009). Ster ol is soluble in ethanol to a small extent because of some hydr oxyl gr oups pr esent in its str uctur e. The study show ed the pr esence of ster ols in the Methanol: Ethanol extr act (table 2). How ever , ster ols ar e not soluble in water because of their lar ge size; solvation in water is not possible.Ter penoids w er e found pr esent in both aqueous and Methanol: Ethanol extr acts. Ter penoids have been shown to decr ease blood sugar level in animal studies (Mandal et al, 2009). Table 2 indicates the pr esence of alkaloids in C. her er oense seed pow der .Resear ch show s that alkaloids exhibit mar ked physiological effects such as antibacter ial (Mabhiza et al., 2016) and analgesic effects. Flavonoids ar e phenolics compounds and act as pr imar y antioxidants (Abdel-moneim et al, 2012). Flavonoids w er e found to be pr esent in both extr acts (Table 2).Flavonoids play an impor tant r ole in bio- activities of tea (Anesini et al, 2008). The chemical composition of tea var ies and lar gely depends on climatic conditions, hor ticultur al pr actices, soil, gr ow th altitude (Pelillo et al, 2002).The phenolic hydr oxyl gr oup in flavonoids is a str ong antioxidant capable of effectively scavenging r eactive oxygen species (Pr ior et al, 1999). Parameter Aqueous Methanol: Ethanol Alkaloids + + Ster oids + Ter penoids Glycosides + + Saponins + + Flavonoids + + Phenols + + Quinones Anthr aquinones Tannins + + Table 2: Phytochemical Pr oper ties of Combr etum Her er oense Seed Powder +: Pr esent; -: Negative 3. Conclusion We conclude that C her er oenseseed pow der has the potential to benefit the health of consumer s if consumed as a tea. How ever , C her er oense tea must not be consumed excessively consider ing the fact that it has tannins w hich mayinter fer e w ith miner al absor ption in the body. 4. Acknowledgements The author s w ould like to acknow ledge the technical assistance pr ovided by the lab technicians in the depar tment of Food Science and Nutr ition, Midlands State Univer sity. 5. Conflict of I nterest The author s declar e that they have no conflict of inter est. 6. References i. Abdel Moneim, A.E. and K.M. El-Deib, 2012. 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