Phytochemical characterization, minerals and antioxidant properties of leaf extract of Piper guineenses schum thonn (piperaceae) obtained from Owerri environs

 Corresponding author: Irenus Chinonye IWU Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. Phytochemical characterization, minerals and antioxidant properties of leaf extract of Piper guineenses schum thonn (piperaceae) obtained from Owerri environs Irenus Chinonye IWU 1, *, Veronica Ifeoma Ukachukwu 1, Leonard Chukwuemeka ANYIKA 2, Ifeoma Chinwendu OBIAGWU 1, Maryann Chioma IGBOMEZIE 1 and Miracle Oluchukwu EZEKOYE 1 1 Department of Chemistry, Federal University of Technology, Owerri. 2 Department of Chemistry, Madonna University Elele River State. World Journal of Advanced Research and Reviews, 2025, 27(01), 1907-1920 Publication history: Received on 03 February 2025; revised on 18 July 2025; accepted on 21 July 2025 Article DOI: https://doi.org/10.30574/wjarr.2025.27.1.0736 Abstract Piper guineense Schum Thonn (Piperaceae) is a common medicinal plant employed for its numerous values which has drawn a lot of attention and a lot of research is still ongoing about the usefulness of the plant. The phytochemical screening of the leaf extract revealed the presence of tannins. saponins, alkaloids, flavonoids, cardiac glycosides, steroids and phenols, determination of some the phytochemicals indicated that the extract contains tannins 12.50 mg/100 g, saponins, 30.60 mg/100 g ,alkaloids, 0.30 mg/100 g and flavonoids, 36.90 mg/100 g. Mineral determination revealed the presence of important minerals such as Phosphorus 0. 35%, Calcium 1.07%, Magnesium 0. 46%, Potassium, 2.60%, Sodium 82.52 ppm, Manganese 300.74 ppm, Iron 315. 99 ppm , Zinc 25. 20 ppm, and Copper 7. 72 ppm. GC/MS analysis gave 34 compounds with their molecular formulas, weights and structures. The antioxidant activities were measured by the ferric reducing antioxidant power (FRAP) and 2, 2-diphenyl-1-picrylhydrazl hydrate (DPPH) assay methods. The extract had 84.303% scavenging activity at concentrations of 100 mg/ml as against that of butylated hydroxytoulene 84.303% at concentration of 80 mg/ml. Similarly, it showed a 91. 495% inhibition of free radical at concentrations of 80 mg/ml as against 99.640% by gallic acid at same concentration. Quantification of antioxidant activity of the extracts showed a dose dependent antioxidant activity comparing favorably with those of standard antioxidant. The extract is a strong antioxidant and free radical scavenger and could be used as a natural antioxidant in processes requiring natural antioxidant. Keyword; Antioxidants; Phytochemicals; Minerals; Free radicals 1. Introduction Piper guineense (black pepper) belongs to the Piperaceae family. The fruit (pepper) is used as spices having pungent aromatic smell which is responsible for its use as a flavoring and seasoning agent [1]. Piper guineense leaves has been reported to be useful in treatment of pseudofolliculitis Barbae (after shave rash). [2] The presence of flavonoids, alkaloids, tannins, and saponins, has also been indicated in the plant. It contains essential vitamins, phytochemicals, and other nutrients [3][4]). Earlier Studies have shown Piper guineense as a potential candidate for bio-insecticide and could serve as an alternative to synthetic insecticides. [5]. In another reports, the constituents of the seeds extracts of Piper guineense has been shown to contains secondary metabolites such as carotenoids anthraquinones, steroids, phenols, flavonoids and terpenes. [6]. The plant is a promising therapeutic agent that could have potential for the treatment of various pathogenic diseases. The use of Piper guineense as a possible agent in managing brain aging has been discussed because of its implications for practical brain function. [7] The plant contains an alkaloid (piperine) reported to be an antioxidant, anti-depressant, and central nervous system stimulant. This alkaloid and other related compounds are neuroprotective agents that reduce lipid oxidation and inhibit tangles in the brain tissues. Further studies has revealed World Journal of Advanced Research and Reviews, 2025, 27(01), 1907-1920 1908 that consumption of aqueous leaf extract of Piper guineense causes dyslipidemia and elevation of liver enzymes but no significant effect on hematological indices. [8] The leafy vegetable contains varying amount of the proximate compounds, vitamins and phytochemicals [9]. Reports has documented the pharmaceutical evidence of Piper guineense on reproduction; which suggested that it is an essential therapeutic input on reproductive health. Piper guineense is an important ingredient for pharmaceuticals and could be used to ameliorate reproductive dysfunction. [10]. Researchers have reported that Piper guineense leaves are aseptic in nature, with the ability to relieve flatulence. It is also useful for treating intestinal diseases, cough, bronchitis and rheumatism. [11]. Studies have shown that the plant possesses antioxidant, anticonvulsant, anti-inflammatory, and neuropharmacological activities ([12]. All these reports are fascinating and made the study of the plant interesting. It is a known fact that the full constituents of this plant have not been fully elucidated. Our work is focused on determining the mineral, phytochemicals, and antioxidant properties of the leaves of our locally grown Piper guineense 2. Material and methods 2.1. Plant materials The sample, which is the Leaves of Piper guineense (uziza) was collected from the farm within Owerri environs in Imo State of Nigeria. They were identified by Prof. Mbagwu of Department of Plant Science and Biotech, School of Agriculture And Agricultural Technology, Federal University of Science and Technology, Owerri. The leaves were washed and air dried at room temperature and pulverized. The powdered sample was stored in air tight container 2.2. Phytochemical screening The phytochemical analyses of the sample extracts were carried out using A. O. A. C official methods of analysis [13] The presence of tannins, alkaloids, flavonoids, saponins (steroids and terpenoids), phenols, and cardiac glycoside were tested. The procedure were as follow, 5g of the plant sample was soaked in 100 cm3 of water and another 5 g of the sample was soaked in 20% acetic acid in ethanol and a third percolated in 80% ethanol, they were kept for 4 hours and extracted 2.2.1. Test for tannins 5 cm3 of the water extract was measured into a 250 cm3 beaker and a 1% gelatin solution containing sodium chloride was added. Formation of white precipitate indicates the presence of tannins 2.2.2. Test for alkaloids 5 cm3 of the acetic acid in ethanol extract was treated with Meyers’ reagent. Formation of yellow precipitate indicated presence of alkaloids. 2.2.3. Test for flavonoids 5 cm3 of the water extracts was treated with few drops of sodium hydroxide solution. Formation of intense yellow colour, which becomes colourless on addition of dilute acid, indicates the presence of flavonoids. [14] 2.2.4. Test for saponins 2 cm3 of distilled water was added to 1ml of the extract and shaken vigorously (in the presence or absence of olive oil). Formation of persistent frothing foaming indicated presence of saponin. 2.2.5. Test for steroids 5 cm3 of the water extract was treated with chloroform and filtered. The filtrates were treated with few drops of acetic anhydride, boiled and cooled. Conc. Sulphuric acid was added. Formation of brown ring at the junction indicates the presence of sterols [14] 2.2.6. Test for Glycosides 5 cm3 of the water extract was treated with equal amount of Fehling solutions of A and B and boiled. The appearance of a reddish brown precipitate indicates the presence of cardiac glycoside. World Journal of Advanced Research and Reviews, 2025, 27(01), 1907-1920 1909 2.2.7. Test for phenols - ferric chloride test 5 cm3of water extract was treated with 3 drops of FeCl3. Formation of dark-brown precipitate indicated the presence of phenols. 2.3. Alkaloid determination 20g of the plant sample was weighed into a beaker and soaked with 400 cm3 of 20% acetic acid in ethanol and covered to stand for six hours. It was filtered using watman no1 filter paper. The filtrates were concentrated using a water bath to one quarter of the original volume. The alkaloid in the extract was precipitated out using concentrated ammonium hydroxide which was added drop by drop until precipitation was completed. The solution was allowed to settle and cool. The precipitate was filtered using whatman filter paper, dried and weighed. [15] 2.4. Saponin determination 20g of the plant sample was weighed into a beaker and socked with 400 cm3 of 20% ethanol and stirred using a glass rod. The mixture was allowed to stand for four hours and filtered using watman no 1 filter paper. This procedure was repeated thrice and the combined filtrate was heated over water bath to concentrate it with continuous stirring while the temperature was maintained at 55 °C. The mixture was extracted with 200 cm3 of 98% diethyl ether and separated with separating funnel. The aqueous layer was collected while the ether layer was discarded. The procedure was repeated thrice and the combine aqueous phase was concentrated. It was then treated with 60 cm3 of n-butanol and washed with 10 cm3 of 5% sodium chloride. The solution was heated to dryness and weighed. The saponin content was calculated in percentages. [15] 2.5. Flavonoid determination 20 g of plant sample was weighed into a beaker and soaked with 400 cm3 of 80% methanol with continuous stirring. The mixture was allowed to stand for four hours and filtered with whatman filter paper. The filtrates were heated over water bath to dryness and weighed [16] 2.6. Tannin determination 20 g of plant sample was weighed into a beaker and soaked with 400 cm3 of distilled water. The mixture was stirred vigorously with a glass rod and allowed to stand for four hour. The mixture was filtered into a volumetric flask and heated over a water bath and concentrated to one quarter the original volume. The concentrated solution was allowed to cool and acidified with aqueous drops of hydrochloric acid and treated with ethyl acetate. The mixture was separated in a separating funnel. The procedures was repeated thrice, the aqueous layer were collected and heated to dryness and weighed. [17] 2.7. Mineral determination by ashing method . The macro elements such as calcium, sodium, potassium, phosphorus and magnesium were determined according to the method described by previous. Researchers. [18]. 5.0 g of dried plant sample was weighed into a petri-dish. The sample was ashed for six hours at a temperature of 500 oC in a muffle furnace. The ash was cooled and extracted with 2-3 cm3 of hydrochloric acid (SG 1-4) and evaporated to dryness. The residue was re-extracted with 20 cm3 of 25% V/V HCl and transferred into a volumetric flask and diluted to the mark with water. The elementary determinations were achieved by direct aspiration of the ash solution into the flame of the AAS (Atomic Absorption Spectrophotometer) by the spray chamber through the capillary tube and nebulizer. Each metal was determined by using the metal’s hollow cathode lamp. The readings were taken in triplicate and the mean value was obtained. 2.8. Preparation of samples for GC/MS analysis Two hundred grams of sample was soaked in 400 cm3 ethanol for 48 hours and then extracted. The extract was reextracted using chloroform to obtain chloroform soluble extract. This was centrifuged at 10, 000 rpm for 20 minutes and the clear supernatant oil was subjected to GC-MS analysis. 2.8.1. System calibration OF AGILENT 7890A GC-MS Prior to calibration, tables are selected from the sequence menu, sequence table is created by filling the sample name, sample type, the external standard mode for calibration is clicked for sequence parameters through the edit /sequence parameter. The sample is acquired by injecting 3 cm3 of PAH standards of 0. 1, 0. 5. 1. 0, 5. 0, 10 mg/cm3 in CH2Cl2 for a five level calibration. The GCMS automatically integrates the chromatograms of each component of the standards and label each peak with the corresponding name given by the manufacturer. Subsequently other levels of calibration were World Journal of Advanced Research and Reviews, 2025, 27(01), 1907-1920 1910 added through the calibration /add level menu. The concentration of each calibration level is added in the calibration table or through the add level dialog box. The correlation factor is checked with the minimum set value at 0. 99. Finally the calibration is saved through the path file /save method 2.8.2. Analysis of sample extract 3 cm3 dichloromethane is injected and used as a blank, the 3 cm3 of extract after concentration into the injection port. If the peaks generated are above scale, the extract diluted with dichloromethane and re-analyzed. Surrogate quality control is run after every 20 samples of every batch , recovery should be between 80-120% ,if not, the system is checked 2.9. Data analysis Integration is done automatically for all PAH components 2.10. Antioxidant evaluation of the plant The antioxidant activity of the extract of the plant was carried out using the ferric reducing antioxidant power (FRAP) and 1, 1-Diphenyl-2-picrylhydrazl (DPPH) assay methods. 2.11. 2, 2-diphenyl-1-picrylhydrazl hydrate (DPPH) 2.11.1. DPPH spectrophotometric assay The scavenging ability of the natural antioxidants of the leaves towards the stable free radicals was determined as follows; The leaf extract (40 cm3) was added to 1 cm3 of 0. 1 mM methanolic solution of DPPH and 0. 9 6cm3 of methanol. The mixture was allowed to react at room temperature for 30 minutes. Methanol served as the blank and DPPH in methanol without the leaf sample served as the positive control, while butylated hydroxytoluene (BHT) served as reference. After 30 minutes of incubation, the discolouration of the purple colour was measured at 518nm in a spectrophotometer (Genesys 10-S, USA). The radical scavenging activity was calculated. 2.11.2. Ferric reducing antioxidant property The principle focus of this assay was the quantification of ferric degradation product, by its condensation with the extract. The reducing property of the extracts was determined by standard method. 0. 50 cm3 of the extract was mixed with 0. 50 cm3 of 200 mM sodium phosphate buffer, pH 6. 6 and 0. 50 cm3 of 1% potassium Ferro cyanide. The mixture was incubated at 50 oC for 20 minutes, thereafter 0. 50 cm3 of 10% trichloroacetic acid was added and centrifuge at 2000 rpm for 10 minutes, 1 cm3 of the supernatant was mixed with 1 cm3 of distilled water and 0. 2 cm3 of ferric chloride and absorbance was measured at 700nm. 3. Results and Discussion Table 1 Result of phytochemical screening of the crude extracts S/N Phytochemicals Inference 1 Saponin + 2 Flavoniod + 3 Alkaloid + 4 Tannins + 5 Cardiac Glycosides + 6 Steroids + 7 Phenols + Key; + = present - = negative The phytochemical screening of the leaf extract revealed the presence of tannins. saponins, alkaloids, flavonoids, cardiac glycosides, steroids and phenols World Journal of Advanced Research and Reviews, 2025, 27(01), 1907-1920 1911 Table 2 Result of phytochemical quantification Phtochemicals Quantity in Mg/100g Tannins 12. 50 Flavonoid 36. 90 Saponins 30. 60 Alkaloid 0. 30 Alkaloids are vast and vary a lot in their activity when ingested by man and livestock. Some alkaloids are useful and important in medicine and constitute most of the valuable drugs currently used by humans. Their amount in the sample is 0. 30 mg/100 g, . tables (1, 2). They are among the most efficient therapeutically significant plant substances. Pure isolated alkaloids and their synthetic derivatives are used by Etinomedicinal practitioners for their analgesic, antispasmodic and bactericidal effects [19]. They exhibit marked physiological activity when administered to animals; the high alkaloid content of these samples may be the reason for their use in the treatment of cough, wounds, and rheumatism and skin infections. Saponins was found to be available at 30.60 mg/100 g in the leaf of the plant tables (1 and 2). Some of the general characteristic of saponins includes; hemolytic activity and cholesterol binding properties. They boast several health benefits. They control immunological responses, protecting the body from bacteria, viruses, and fungi, having antibacterial, antiinflammatory, and immune-boosting properties. They are oxidative stress scavengers and antioxidants. Their hypoglycemic properties help to minimize insulin surges and maintain normal blood sugar levels. Saponins can reduce body fat and cholesterol, which helps to improve cardiovascular health, and are have molluscicidal, anthelmintic, insecticidal, analgesic, antiviral, anticancer, sedative, and anti-tumor activities. [20] The flavonoid content of was found to be 30.90 mg/100 g as shown in Table (1, and 2). Flavonoids are distributed group of polycyclic compounds characterized by a common Benzo pyrone ring structure that has been reported to act as antioxidants in many biological systems. . They possess free radical scavenging activities, having multiple biological activities including – vasodilatory, anti-carcinogenic, anti-allergic, antiviral, estrogenic effects as well as being inhibitors of phospholpase H-2, cycloxygenase, and glutathione reductase and xanthine oxidase. [21]. They possess antiinflammatory and analgesic effect as well as anti-cancer properties. [22] Tannins are a type of polyphenol that has several therapeutic and medical uses. 12. 5 mg/100 g was detected in the plant, tables 1 and 2. Their pharmacological qualities includes; the ability to heal wounds, prevent dysentery, fight bacteria, and reduce inflammation, been anti-toxic, anti-cancerous, antiallergic, and anti-inflammatory. [23]. Cardiac glycosides was detected in the extract, they are compounds that lower heart rate and increase heart muscle contraction force without increasing oxygen consumption. They are useful in the treatment of congestive heart failure and cardiac arrhythmia. . They do this by activating the heart muscle, which enhances cardiac output and efficiency. They also aid in the deceleration of irregular heartbeats. Apart from their beneficial effects on heart health, cardiac glycosides also exhibit anticancer capabilities. There is a growing interest in polyphenolic compounds as therapeutic agents against many diseases such as cardiac and cerebral ischemic, arteriosclerosis and rheumatic or pulmonary diseases. [24], [25] The activated phagocytic cells are known to produce potentially destructive oxygen species like super oxide anion (O2- ), hydrogen peroxide (H2O2) and Hypochloric acid (HOCl) during chronic inflammatory disorder. Many polyphenolics are known to exhibit antioxidant properties; they are free radical’s scavengers. Phenolic flavonoids are also excellent hydroxyl scavengers. These properties promote health, and prevent certain chronic disorders such as cancer, cardiovascular diseases, diabetics and arthritis. The presence of phenols means that the extract could act as antiinflammatory, anticlothing, antioxidants, immune enhancers and hormone modulators. Phenols have been the subject of extensive research as disease preventive agents. [24] [26]. The have the ability to block specific enzymes that causes inflammations. They modify the prostaglandin pathways and thereby protect platelets from clumping. Table 3 Result of mineral element determination Mineral elements Piper guineense LAB ID: 202001529 Nitrogen (N) 3. 88% Phosphorous (P) 0. 35% Calcium (Ca) 1. 07% World Journal of Advanced Research and Reviews, 2025, 27(01), 1907-1920 1912 Magnesium (Mg) 0. 46% Potassium (K) 2. 60% Sodium (Na) 82. 52 ppm Manganese (Mn) 300. 74 ppm Iron (Fe) 315. 99 ppm Zinc (Zn) 25. 20 ppm Copper (Cu) 7. 73 ppm Results obtained from the mineral determination revealed the presence of important minerals such as Nitrogen, Phosphorus, Calcium, Magnesium, Potassium, Sodium, Manganese, Iron, Zinc, and Copper, table( 3). These minerals are vital to the body and are needed for its proper functions and development. Iron, magnesium and calcium contents of black pepper have been reported to be high [3]. Results also showed that the leaves were rich in calcium, magnesium and iron. 3.1. Result of GC/MC spectrum of n-haxane extract of Piper guineense leaf Figure 1 GCMS spectrum of sample The GC/MS spectrum of the n-haxane extract of Piper guineense showed 34 major peaks, their intensity and elution time of bioactive compounds present in the extract. These compounds according to studies have medicinal values. 3.2. Compounds present in GC/MS analysis of n-haxane extracts of Piper guineense The major peaks were analyzed, the name of the compound, their molecular weight, molecular formula and retention time are structure Table 4 Compounds obtained from GC-MS analysis of sample S/N Name of Compounds Molecular weight (mw) Molecular formula Retention time RT(min) Structure of the compounds 1 Cayophylene 204 C15 H24 7. 990 World Journal of Advanced Research and Reviews, 2025, 27(01), 1907-1920 1913 2 Cis-α-Bisebolene 204 C15 H24 8. 309 3 Naphthalene, 1, 2, 3, 5, 6, 8αhexahydro-4, 7dimethyl-1-[1 methylethyl], (1Scis) 204 C15 H24 8. 936 4 Benzene, 1, 2, 3-trimethoxy-5-[2propenyl] 208 C12 H16 O3 9. 128 5 Benzene, 1, 2, 3 trimethoxy-5- [2propenyl] 208 C12 H12 O3 9. 404 6 1-Hydroxy-1, 7-dimethyl-4isopropyl-2-7-dimethyl-4-isopropyl2-7-cyclodecadiene 222 C15 H26 O 9. 542 7 Apiol 222 C12 H14 O4 9. 967 8 3, 5-Dimethoxy cinnamic acid 208 C11 H12 O4 10. 095 9 3, 7, 11, 15-Tetramethyl-2hexadecen-1-ol 296 C20 H40 O 11. 264 10 n-Hexadecanoic acid 256 C16H32 O2 12. 529 11 1-Eicosanol 298 C20 H42 O 12. 954 12 Phytol 296 C20 H40 O 13. 166 World Journal of Advanced Research and Reviews, 2025, 27(01), 1907-1920 1914 13 9, 12, Octadecadienoic acid 280 C18 H32 O2 13. 592 14 Octadecadienoic acid 284 C18 H36 O2 13. 698 15 Butanoic acid, 2, {1-adamantyl} -3oxo, ethyl ester 264 C16 H24 O3 14. 995 16 Ethyl Iso-allocholate 436 C26 H44 O5 15. 600 17 Pyroidine-1-{1-oxo 10-octadecynyl} 333 C22 H39 NO 15. 664 18 Octanoic acid, 6-ethyl-3-octyl ester 284 C18 H36 O2 15. 845 19 2-4-methy-6-(2, 6, 6-trimethylcy clohex-1-enylhexa-1, 3, 5) trienylcyclohex-1-en-1 carboxaldehyde 324 C23 H32 O 15. 909 20 2H-Benzo[F]owireno[2, 3E]benzofuran-B {9H}one, 9{1, 3benzodiowol-5ylmethylamino}melhylocha 399 C23 H29 NO5 16. 079 21 2-4-methyl-6-{2, 6, 6bimethylcyclohex-1-enyihewa-1, 3, 5-trienyl}cyclohex-1-en-1carboxaldehyole 324 C23 H32 O 16. 484 World Journal of Advanced Research and Reviews, 2025, 27(01), 1907-1920 1915 22 Linoleic acid methyl ester 308 C20 H36 O2 16. 674 23 Bis-(3, 4-dimethoxyphenyl)- hydroxyacetic acid methyl ester 362 C19 H22 O7 17. 003 24 Androstane-3-one, 17-melhoxy-3methoxine, {5α, 17β} 333 C21 H35 NO2 17. 503 25 Cinamidine, 3-oxo 315 C17 H17 NO5 17. 790 26 Vitamin E 430 C29 H50 O2 18. 098 27 Cinamidine, 3-oxo 315 C17 H17 NO5 18. 491 28 Stigmasterol 412 C29 H48 O 18. 640 29 β Sitosterol 414 C29 H50 O 18. 863 30 EthylIso-allocholate 436 C26 H44 O5 19. 044