IRJCCS International Research Journal of Chemistry and Chemical Sciences Vol. 3(2), pp. 037-042, November, 2016. © www.premierpublishers.org. ISSN: XXXX-XXXX

Research Article

Free radical scavenging activity, phytochemistry and antimicrobial properties of tetraptera Seeds

O. U. Igwe*1 and H. C. Akabuike2

1*,2 Department of Chemistry, Michael Okpara University of Agriculture, Umudike, P.M.B. 7267 Umuahia, Abia State, Nigeria

This study is aimed at investigating the phytochemistry, free radical scavenging activity and antimicrobial properties of seeds. The results of the analysis of Tetrapleura tetraptera seeds showed the presence of phenols (0.34 %), flavonoids (0.91 %), alkaloids (0.52 %), tannins (0.23 %) and saponins (0.51 %). Vitamins include ascorbic acid (5.23 %), riboflavin (0.11 %), niacin (1.25 %), 휷- carotenoid (3.40 %) and thiamine (0.06 %). Proximate compositions were moisture (14.73 %), ash (7.30 %), crude protein (17.36 %), crude fibre (6.68 %) and lipids (35.19 %). Mineral elements were calcium (1.41 %), magnesium (0.48 %), potassium (0.56 %), sodium (0.21 %), phosphorus (0.32 %), iron (1.20 %), copper (0.13 %) and zinc (0.77 %). The seed extract showed potent free radical scavenging activity (6.72 – 22.8 %) using ascorbic acid as a standard (12.10 - 42.00 %). The seed extract exhibited significant antimicrobial activity (7.00 – 20.00 mm) and the zones of inhibition were compared with that of ciprofloxacin as standard (15.00 – 28.00 mm). From these investigations, seed extract of Tetrapleura tetraptera has proven to be considered an excellent source of nutriceuticals, a potential antioxidant and antimicrobial agents owing to its high level of phytochemical and vitamin constituents.

Keywords: Tetrapleura tetraptera, Free radical, Antimicrobial, Phytochemicals, Seed extract

INTRODUCTION

Primary and secondary metabolites of have especially in the lowlands and is single-stemmed. together played an invaluable role to ensure that man The tree is robust and perennial possessing dark remains healthy. Natural products obtained from green leaves, thick, woody base and spreading plants are very important in disease control and branches (Adetunji, 2007). The fruits are green when treatment which dates back several many years ago. tender and dark red-brown when fully ripe and are Till today people still patronize herbal preparations for about 22-27 cm long and 4-5 cm wide (Adetunji, certain ailments especially when modern medicine is 2007; Enwereuzoh et al. 2015). The fruit consists of a considered to have failed to provide a solution. fleshy pulp with some small, brownish-black seeds However, substances obtained from plants which are and possesses a fragrance, which has been secreted by these plants in response to stress or attributed to its insect repellent property (Okwu, 2003; other factors have been harnessed and mostly used Adetunji, 2007; Enwereuzoh et al. 2015; Akintola et as drugs and have proven to be efficacious. Most of al. 2015). these plants are still understudied and underutilized. Tetrapleura tetraptera is considered one of them. *Corresponding author: Dr. Okenwa U. Igwe, T. tetraptera belongs to the family Department of Chemistry, Michael Okpara University (Gbadamosi and Obogo, 2013). It is commonly found of Agriculture, Umudike, P.M.B. 7267 Umuahia, Abia naturally in the rainforest belt of tropical Africa State, Nigeria Email: [email protected]

Free radical scavenging activity, phytochemistry and antimicrobial properties of Tetrapleura tetraptera Seeds Igwe and Akabuike 038

The mature dry pods contain sweet smelling aromatic seeds was obtained by sieving with 1 mm aperture pulp and the brownish black seeds are found in them and the sample was stored in an airtight container for oil (Okwu, 2003; Adetunji, 2007; Akintola et al. 2015). extraction and laboratory analysis.

The pod is therefore used as a seasoning spice in Extraction of Oil Southern and Eastern Nigeria. The possesses a hard wood that is reddish in colour used for building The method reported by Igwe and Onuoha (2016) houses and other construction work (Okwu, 2003; was employed. In a typical extraction of oil from the Adetunji, 2007; Enwereuzoh et al. 2015; Akintola et seeds of T. tetraptera, 200 g of the powdered sample al. 2015). was introduced into a conical flask and 500 ml of 100 % methanol was added followed by continuous T. tetraptera has numerous traditional herbal stirring which lasted for about 5 h. The flask was applications in the treatment of diseases and covered with filter paper and cotton fastened with a infections. A decoction of T. tetraptera pod is used in paper tape to make it airtight. Stirring continued the the treatment of jaundice (Gbadamosi and Obogo, next day for another 5 h. The mixture was separated 2013). In West Africa, the fruit is widely used in using a Whatman No. 1 filter paper. The process was traditional remedies for the treatment of several repeated on the residue to ensure exhaustive conditions including convulsion, gastric ulcer, extraction. The filtrate was pooled together and the rheumatism, fevers, whitlow, skin rashes, smallpox, solvent was eventually evaporated in a rotary malaria and dysentery (Dalziel, 1955; Fleischer et al., evaporator to get the oil. 2006). An infusion of the whole fruit is taken as a recuperative tonic (Ojewole and Adesina, 1983). The Determination of Plant Chemicals plant is used in the management of leprosy and inflammation (Adetunji, 2007). It has been reported Alkaloids and phenols were determined according to that an infusion of the whole fruit is usually taken by the method of Harborne (1973) while tannin was convalescents to bathe in order to get relief from determined using the method of Van-Burden and feverish conditions, for constipation and as an emetic Robinson (1981). Saponin was determined using the (Adetunji, 2007). The methanolic extract of the leaf method of Obadoni and Ochuko (2002). Flavonoids has been reported to possess antioxidant effect on were determined according to the method of Boham carbon tetrachloride-induced hepatoxicity (Atawodi et and Kocipia (1994). Ascorbic acid was determined al., 2014) while the aqueous extract of the fruit using the method of the Association of Vitamin possesses hypoglycaemic properties (Lekana-Douki Chemists described by Kirk and Sawyer (1991). The et al., 2011). The plant is used in the treatment of B-complex vitamins (thiamine, riboflavin, and niacin) hypertension, asthma, epilepsy, schistosomiasis, and were determined according to SKALAR Analyzers for prevention of post-partum contraction (Ojewole method of Barakat et al. (1993) while carotenoid was and Adewunmi, 2004). determined according to the method described by James (1995). The macro and micro elements More emphasis and concern had been given to T. comprising potassium, sodium, magnesium, calcium, tetraptera fruits/pods, pulp and leaf by researchers phosphorus, nitrogen, iron, copper and zinc were and it appears there is a paucity of documented determined according to the method of Shahidi et al., information on the seeds. Therefore, this research is (1999). Protein, crude fibre, lipids, ash and moisture aimed at investigating the free radical scavenging were determined by the method described by James activity, phytochemistry and antimicrobial properties (1995). of T. tetraptera seeds. Antioxidant Activity Determination

MATERIALS AND METHODS The free radical scavenging activity of the oil fraction of the sample extract was determined using the 1,1- Sample Collection and Preparation diphenyl-2-picrylhydrazyl (훼, 훼-diphenyl-훽- picrylhydrazyl; DPPH) method described by Mature T. tetraptera fruits were bought from Ariaria Manzocco et al., (1998). 1.0 g of DPPH, a stable International Market, Aba, Abia State, Nigeria. The fruits radical was dissolved in 100 mL of methanol. 3.0 mL were identified and authenticated at the of different concentrations of the test sample were Unit of Forestry Department, Michael Okpara added to 3.0 mL of a 0.004 % methanol solution of University of Agriculture, Umudike, Abia State, DPPH and incubated for 30 min. at room Nigeria. The fruit brown pulp was separated from the temperature. The decrease in absorbance of the seeds by scraping it off with the aid of a sharp knife. solution brought about by the test samples was Removal of the seed coats was done with the aid measured at 517 nm using a spectrophotometer. of mortar and pestle to separate the endosperms Ascorbic acid, which is a known antioxidant, was from the seed coat. The endosperms were ground used as a reference standard. The radical into powder using a blender. Fine powder of the scavenging activity was calculated as the percentage

Free radical scavenging activity, phytochemistry and antimicrobial properties of Tetrapleura tetraptera Seeds Int. Res. J. Chem. Chem. Sci. 039

inhibition of DPPH discoloration using the equation Flavonoids were the highest constituents followed by below: alkaloids and then saponins, phenols and lastly, tannins. According to scientific studies these detected phytochemicals possess medicinal properties. Also,  AAblanksample  % inhibition of DPPH radical 100 according to Mallikharjuna et al. (2007) plants have the limitless ability to synthesize phenols or their Ablank derivatives and their presence in all types of tissues is a characteristic feature of plants. Phenolic compounds are reported to have antioxidant, anti- Where, Ab l a n k is the absorbance of the control reaction solution (containing all reagents except the inflammatory and antimicrobial properties (Yadav and Agarwala, 2011). Flavonoids are polyphenolic test sample) and As a m p l e is the absorbance of the test compounds that have been reported to have sample. antiallergic, antioxidant, anticancer, vasodilatory, anti- inflammatory, antimicrobial and immune-stimulating Antimicrobial Screening activities (Igwe and Okwu, 2013a). Alkaloids are plant bases with pharmacological properties such as The in vitro antimicrobial activity of the sample extract hypotensive, anticonvulsant, antiprotozoal, was carried out for 24 h culture of three selected antimicrobial and antimalarial activities (Aiyelaagbe each of bacteria and fungi. The test organisms were and Osamudiamen, 2009). Tannins are reported to obtained from stock cultures at the Central possess anti-tumor, antiviral and antibacterial Laboratory services Unit of National Root Crops activities (Chung et al., 1998). The presence of Research Institute, Umudike, Abia State, Nigeria. tannins in a plant has been associated with ulcer With the aid of a single hole punch office paper management, wound healing, control of bleeding and perforator, circular discs of 5 mm diameter were cut burns in herbal medicine (Igwe and Okwu, 2013a). from Whatman No 1 filter paper. The paper discs Saponins possess antioxidant, anticancer, anti- were boiled in distilled water for an hour to remove inflammatory, hyperglycaemic and antifungal any residual preservatives. The boiled paper discs properties (Rausch et al., 2006). Phytochemicals were allowed to drain dry and they were wrapped in possess a lot of physiological properties which are aluminium foil and sterilized in an autoclave at 121 ℃ beneficial to human. Phytochemicals in plant-based for 15 min. They were however used within 48 h of foods can improve glucose metabolism as well as production. The sensitivity of each test enhance the overall health of diabetic patients by microorganism to the extract was determined using improving lipid metabolism, antioxidant status, the Disc Diffusion Technique (Pelczar and Chan, improving capillary function, and lowering blood 1977; Cheesbrough, 2000). A loopful of each test pressure and cholesterol (Kelble, 2006; Kasolo et al., sample organism was aseptically transferred into the 2010). surface of a sterile solid medium, appropriate for the test organism. Using a flamed glass hockey, the The vitamins constituents of T. tetraptera seeds is inoculum was spread evenly over the surface of the shown in Table 2. The seeds are sources of ascorbic medium, and then with the aid of a flamed pair of acid, riboflavin, niacin, 훽-carotenoid and thiamine. forceps, the extract bearing paper discs were Ascorbic acid and 훽-carotenoid were the most carefully placed on the surface of the inoculated abundant analytes in Table 2. Ascorbic acid is a good medium at some distance from one another. The antioxidant compound and has been involved in inoculated plates were incubated for 24 h in an wound healing and prevention of scurvy (Igwe and incubator at 37 ℃. They were examined daily for Okwu, 2013b). 훽-Carotenoid has also been touted as growth and for the presence of inhibition zones having the ability to scavenge free radicals (Igwe and around the paper discs. The level of sensitivity was Okwu, 2013b). The detection of ascorbic acid and 훽- determined by the diameter of the inhibition zone as carotenoid in the seeds of T. tetraptera suggests that measured with a transparent millimeter rule. the seeds may have high nutritional and medicinal values. Riboflavin, niacin, and thiamine are present in Statistical Analysis lower amounts. The proximate composition of the seed is displayed in Table 3. Lipid content was so Data were replicated three times and expressed as high meaning the seed is a good source of fat and oil. mean ± standard deviation (SD) in tables. Lipids play a significant role in the body metabolism. Apart from their energy-yielding function, they also constitute a component of the membrane structure. RESULTS AND DISCUSSION They protect the body from mechanical injury (Ononogbu, 2002). High protein content was also The phytochemical constituents of T. tetraptera observed in the seeds of T. teraptera. It means that seeds are shown in Table 1. The seeds contain these seeds are indeed nutritionally rich. The fibre phenols, flavonoids, alkaloids, tannins and saponins. content will provide bulky stools and this helps to

Free radical scavenging activity, phytochemistry and antimicrobial properties of Tetrapleura tetraptera Seeds improve gastrointestinal functions, preventing constipation and may thus reduce the incidence of Igwe and Akabuike 040

Table 1. Phytochemical constituents of T. tetraptera seeds

Phytochemicals Composition (%) Phenols 0.34 ± 0.10 Flavonoids 0.91 ± 0.12 Alkaloids 0.52 ± 0.08 Tannins 0.23 ± 0.02 Saponins 0.51 ± 0.01

Values are means ± standard deviation of three determination

Table 2. Vitamin Composition of T. tetraptera seeds

Vitamin Composition (%) Ascorbic acid 5.23 ± 0.21 Riboflavin 0.11 ± 0.02 Niacin 1.25 ± 0.25 훽-Carotenoid 3.40 ± 0.64 Thiamine 0.06 ± 0.02

Values are means ± standard deviation of three determinations

Table 3. Proximate composition of T. tetraptera seeds

Constituents Composition (%) Moisture content 14.73 ± 0.34 Ash content 7.30 ± 0.75 Crude protein 17.36 ± 1.10 Crude fibre 6.68 ± 0.71 Lipids 35.19 ± 0.18 Values are means ± standard deviation of three determinations.

Table 4. Mineral elements composition of T. tetraptera seeds

Mineral elements Composition (%) Calcium 1.41 ± 0.21 Magnesium 0.48 ± 0.02 Potassium 0.56 ± 0.03 Sodium 0.21 ± 0.03 phosphorus 0.32 ± 0.05 Iron 1.20 ± 0.05 Copper 0.13 ± 0.03 Zinc 0.77 ± 0.04

Values are means ± standard deviation of three determinations metabolic diseases. The mineral elements analyzed The free radical scavenging activity of the seed and found in the seeds of T. tetraptera were calcium, extract of T. tetraptera is shown in Table 5. The magnesium, potassium, sodium, phosphorus, iron, extract showed comparable free radical scavenging copper and zinc (Table 4). Of all these elements, activity with the ascorbic acid used as a standard. calcium, iron and zinc were found in high amounts. The presence of phenols, flavonoids, saponins, Calcium is important for strong bone and teeth ascorbic acid and 훽–carotenoid in the extract was formation. Iron is needed in building red blood cells responsible for the free radical scavenging activity while zinc is important for wound healing and in exhibited by this extract (Rausch et al., 2006; Yadav carbohydrate metabolism (Igwe and Okwu, 2013b). and Agarwala, 2011; Igwe and Okwu, 2013a; Igwe and Okwu, 2013b). Table 6 shows the antimicrobial activity of T. tetraptera seed extract. The extract

Free radical scavenging activity, phytochemistry and antimicrobial properties of Tetrapleura tetraptera Seeds showed significant inhibitions against the comparable to that obtained using ciprofloxacin as a microorganisms and zones of inhibition were Int. Res. J. Chem. Chem. Sci. 041

Table 5. Free radical scavenging activity of extract of T. tetraptera seeds

Concentration (mg/ml) Radical Scavenging (%) Ascorbic acid Extract

4.0 12.10 ± 0.92 6.72 ± 0.54 8.0 22.00 ± 0.45 10.0 ± 0.3 12.0 25.00 ± 1.75 15.5 ± 1.55 16.0 38.00 ± 0.01 18.9 ± 0.08 20.0 42.00 ± 0.06 22.8 ± 0.18

Values are means ± standard deviation of triplicate results.

Table 6. Antimicrobial activity (zone of inhibition) of T. tetraptera seed extract

Microorganism T. tetraptera (mm) Ciprofloxacin (mm) S. aureus 7.00 ± 1.41 18.00 ± 0.70 E. coli 9.00 ± 0.14 24.00 ± 0.28 P. aeruginosa 15.00 ± 0.80 15.00 ± 0.80 C. albicans 14.00 ± 0.70 21.00 ± 0.90 A. niger 20.00 ± 0.71 23.00 ± 0.70 P. notatum 16.00 ± 0.90 28.00 ± 0.20

Values are means ± standard deviation of triplicate results.

standard antimicrobial agent. The order of inhibition flavour industries where they could be used as spices against the tested organisms was A. niger > P. cum antimicrobial and antioxidant agents. notatum > P. aeruginosa > C. albican > E. coli > S. ACKNOWLEDGEMENTS aureus. An important characteristic of plant extracts and their components is their hydrophobicity, which The authors are grateful to Mr. I. K. Ndukwe of Plant enables them to partition the lipids of the bacterial Taxonomy Section, Forestry Department, Michael cell membrane and mitochondria, disturbing the cell Okpara University of Agriculture, Umudike, for structures and rendering them more permeable identifying and authenticating the plant sample. (Sikkema et al., 1994). The presence of phytochemicals in the extract, such as phenols, flavonoids, alkaloids, tannins and saponins could be COMPETING INTERESTS the reason behind the strong antimicrobial activity exhibited by the extract (Chung et al., 1998; Rausch Authors have declared that no competing interests et al., 2006; Aiyelaagbe and Osamudiamen, 2009; exist. Yadav and Agarwala, 2011; Igwe and Okwu, 2013a).

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Free radical scavenging activity, phytochemistry and antimicrobial properties of Tetrapleura tetraptera Seeds