doi.org/10.36721/PJPS.2024.37.5.REG.1177-1187.1 Pak. J. Pharm. Sci., Vol.37, No.5, September-October 2024, pp.1177-1187 1177 Qualitative and quantitative phytochemical screening and antioxidant potential of Bulbine inflata (Asphodelaceae) Rebecca Opeyemi Oyerinde* and Ida Masana Risenga School of Animal, Plant and Environmental Sciences, University of the Witwatersrand, Johannesburg, South Africa Abstract: Bulbine inflata is one of the species in the genus Bulbine that are yet to be documented for potential medicinal uses. Hence, we carried out its preliminary phytochemical profiling and investigated its antioxidant potential. The leaves were dried using air- and freeze-drying techniques and were extracted by water, methanol, ethyl acetate and hexane. Various common colour tests were used for the presence of phytochemicals. Some of the phytochemicals were further quantified. Phosphomolybdate, 2, 2 diphenyl-1-picryhydrazyl, hydrogen peroxide and metal chelating assays were used to assess the antioxidant potential of B. inflata. Tannin, flavonoids, phenols, glycosides, steroids, coumarins, quinones, saponins and terpenoids were detected phytochemicals in B. inflata leaves. The highest total phenolic, flavonoid and tannin contents, as well as total antioxidant capacity, were recorded for water extract. B. inflata showed moderate to high antioxidant activities against DPPH, H2O2 and metal chelating. Freeze-dried samples presented with higher results than air-dried samples in most assays. The results showed the potential of B. inflata for medicinal uses and could expand the ethnomedicinal resources in the communities where it is prevalent and beyond. Keywords: Antioxidant activities, Bulbine species, drying methods, medicinal plants, secondary metabolites, solvents. Submitted on 12-12-2023 – Revised on 26-02-2024 – Accepted on 20-03-2024 INTRODUCTION Medicinal plant species have been shown to have curative and preventive effects against human ailments (Sofowora et al., 2013; Prasad, 2016) because of their antioxidant, anticancer, anti-inflammatory, antiseptic and antimicrobial, properties (Aye et al., 2019). Their associated positive effects on humans are attributed to the presence of phytochemicals/plant metabolites, which the plants produce primarily in response to biotic and abiotic stresses (Kumar et al., 2023). The health benefits associated with the presence of biologically active phytochemicals in plants offer great possibilities for their medicinal use and drug discovery (Sasidharan et al., 2011; Koche et al., 2016). Broadly, these phytochemicals are categorised as alkaloids, phenols, terpenoids and steroids and other chemicals (Koche et al., 2016). Each of these categories further comprises subgroups of phytochemicals. South Africa is one of the countries in the world that are richly endowed with a wide range of plant biodiversity (le Roux et al., 2017), and it is listed in the top five countries of the world with the highest percentage and number of medicinal plants (Chen et al., 2016). South Africa and other Southern African countries are home to a substantial number of plant families that have been used for medicinal purposes (Cock and Van Vuuren, 2020); one of which is Asphodelaceae (Fayisa and Mirete, 2022). Asphodelaceae comprises two subfamilies which are Alooideae and Asphodeloideae and 16 genera (Fayisa and Mirete, 2022). Some of the genera, such as Aloe, Kniphofia and Bulbine, are well known for medicinal and pharmacological uses. The genus Bulbine Wolf is so named because most of the species in the genus have ‘bulb-like’ tuberous roots. They are also succulent and perennials. The flowers are usually yellow with hairy filaments. The species in the genus are adapted to a wide range of habitats for efficient water use (Williamson and Baijnath, 1995; Naidoo et al., 2011; Bodede and Prinsloo, 2020). There are 78 approved species in the genus (Bodede and Prinsloo, 2020), most of which are indigenous and distributed across Southern Africa while about six are found in Australia (Naidoo et al., 2011; Bodede and Prinsloo, 2020; Gilbert, 2023). However, only 12, out of the 78 approved species, have been documented for medicinal properties and uses. Some of which are B. frutescens, B. abyssinica, B. natalensis, B. latifolia,and B.bulbosa (Bodede and Prinsloo, 2020). Species in this genus are useful for treating a wide range of health issues, which are majorly skin problems, rheumatism, fertility issues, gastrointestinal problems, sexually transmitted diseases and urinary tract infections (Coopoosamy, 2011; Bodede and Prinsloo, 2020). B. inflata is one of the Bulbine species that has no documented information for phytochemical screening and medicinal properties. In addition to the general characteristics of the species in the genus, B. inflata has inflated fruits (fig. 1) as its distinguishing trait, which suggests the species name ‘inflata’. Considering that about 80% of South African and other developing countries of the world directly or indirectly *Corresponding author: e-mail: rebecca.oyerinde@wits.ac.za Qualitative and quantitative phytochemical screening and antioxidant potential of Bulbine inflata (Asphodelaceae) Pak. J. Pharm. Sci., Vol.37, No.5, September-October 2024, pp.1177-1187 1178 use medicinal plants and their products (Chen et al., 2016; Volenzo and Odiyo, 2020) and that the increase in the world’s population has a proportional increase in the demand for medicinal plants (Chen et al., 2016), it is important to have knowledge of a wide range of plants that have medicinal properties rather than depending solely on those that are known and established. B. inflata may be another species with the potential for medicinal use just like other species in the genus. The aim of this work, therefore, was to carry out preliminary screening on some phytochemical constituents and antioxidant activities of B. inflata. MATERIALS AND METHODS All the reagents and chemicals that were used in this study were of analytical grades and they were bought from Sigma-Aldrich, USA. All absorbance readings were taken with a Thermo-Fisher Scientific Genesys (GEN10S UV-VIS) spectrophotometer. Plant material collection, preparation, extraction and yield The leaves of B. inflata (fig. 1) were harvested from the University of the Witwatersrand (26.1907°S, 28.0314°E), Johannesburg, South Africa in 2023. The species was identified by Dr Ida Risenga and the voucher specimen (IR01) was deposited at the herbarium of the same University. The leaves were cleaned and one half was air- dried in the oven (Binder oven) at 33±2C while the other half was freeze-dried (Zirbus technology freeze-dryer) at - 83C, both for 3 days. The dried leaves were subsequently ground to fine powder. Water and methanol as the polar solvents and ethyl acetate and hexane as the non-polar solvents, were used for the crude extraction of phytochemicals from the powdered leaves. Three grams of leaf powder was added to 25 ml of each solvent, agitated for 72 hrs in an orbital shaker at 150 rpm and subsequently centrifuged for 5 minutes at 3500 rpm. The supernatants, which served as the crude extracts, were decanted and kept in the refrigerator for this research while the residues were discarded. The percentage yield of extracted sample was calculated as: % yield  (dry extract (without solvent) ÷ weight of ground sample) × 100 Qualitative phytochemical screening Various colour tests for alkaloids, tannins, saponins, flavonoids, glycosides, steroids, volatile oils, coumarins, phlobatannins, quinones, terpenoids, cardiac glycosides and phenolics were carried out using standard published procedures (Gul et al., 2017; Tyagi, 2017; Roghini and Vijayalakshmi, 2018; Akinyemi and Oyelere, 2019; Shaikh and Patil, 2020; Teffo et al., 2022). Quantitative phytochemical screening Total flavonoid content (TFC) Four millilitre (4 ml) of distilled water was added to 0.3 ml of crude extract. Then, 0.3 ml of 5% sodium nitrate (NaNO3) was added, mixed thoroughly and allowed to rest for 5 minutes. Thereafter, 3 ml of 10% aluminium chloride (AlCl3) solution was added and left for 6 minutes, after which 2 ml of 1M NaOH solution was added. The mixture was made up to 10 ml by the addition of 0.4 ml of distilled water and then incubated for 1 hr in the dark at room temperature. The absorbance of the solution was measured at 510 nm wavelength and 80% methanol was used as the blank. A linear equation generated from quercetin standard calibration curve was used to calculate the TFC: y  0.2388x  0.0019 (r2  0.9997) Total phenolic content (TPC) TPC was determined by the Folin-Ciocalteu (FC) reagent assay. The FC reagent was diluted to 10% strength before its use. For each crude extract, 2 ml of 7.5% sodium carbonate (Na2CO3) was added to 0.2 ml of extract. This was followed by the addition of 0.75 ml of diluted FC reagent and 7 ml of distilled water. The solution was incubated for 2 hrs in the dark at room temperature. The absorbance of the solution was measured at 765 nm wavelength and 80% methanol was used as the blank. A linear equation generated from gallic acid standard calibration curve was used to calculate the TPC: y  0.0495x  0.0259 (r2  0.9994) Total tannin content (TTC) The TTC from the crude extract of B. inflata leaves was determined as follows: 0.1 ml of crude extract was added to 7.5 ml of distilled water. Thereafter, 0.5 ml of undiluted FC reagent was added, which was followed by the addition of 1 ml of 35% sodium carbonate (Na2CO3). The volume was made up to 10 ml by the addition of 0.9 ml of distilled water. The solution was incubated at room temperature in the dark for 30 minutes. The absorbance of the solution was measured at a wavelength of 725 nm (Lahare et al., 2021; Teffo et al., 2022). A linear equation generated from gallic acid standard calibration curve was used to calculate the TTC: y  0.046x  0.0264 (r2  0.9833) Total proanthocyanidin content (TPAC) The vanillin-methanol method was adopted for the determination of TPAC (Oyedemi et al., 2010). Crude extract (0.5 ml) was added to 3 ml of 4% vanillin- methanol solution, which was followed by the addition of 1.5 ml of HCl. The mixture was mixed vigorously and incubated for 15 minutes in the dark at room temperature. The absorbance of the solution was measured at a Rebecca Opeyemi Oyerinde and Ida Masana Risenga Pak. J. Pharm. Sci., Vol.37, No.5, September-October 2024, pp.1177-1187 1179 wavelength of 500 nm. A linear equation generated from catechin calibration curve was used to calculate the TPAC: y  0.9554x  0.0003 (r2  0.9927) In vitro antioxidant activity assays Total antioxidant capacity by phosphomolybdate method and three antioxidant assays, i.e. 2, 2 diphenyl-1- picryhydrazyl (DPPH) scavenging activity assay, hydrogen peroxide (H2O2) assay and metal chelating assay, were investigated in this study. All assays were carried out using various volumes (10, 20, 30, 40 and 50 l) of crude extracts in capped test tubes. Each assay was done in triplicate. After the required incubation period for each assay, the solutions were dispensed into cuvettes and the spectrophotometer earlier described was used to measure the absorbances at required wavelengths. The antioxidant activity percentage of B. inflata leaf extract, against DPPH and H2O2 radicals, was calculated as: % activity  A0  A1 A0  100 A0 and A1 represented the absorbance of the control and the extracted sample, respectively. A linear graph equation generated from the various volumes against the percentage activity was used to calculate the IC50 for DPPH and H2O2. IC50 is the half-maximal inhibitory concentration of the extract required to inhibit the oxidative activity of the tested radicals by 50%. Total antioxidant capacity (TAC) by phosphomolybdate method The TAC of B. inflata leaves was measured using phosphomolybdate reagent and ascorbic acid as the standard. A volume of 0.1 ml of the crude extract for each solvent and each drying method was added to 1 ml of the reagent (made by combining equal volumes of 28 mM sodium phosphate, 0.6 M H2SO4 and 4 mM ammonium molybdate) in capped tubes. The solutions were heated in a water bath at 95˚C for 90 minutes. The solutions were left to cool completely after which the absorbance was measured at 695 nm wavelength against 80% methanol as blank (Prieto et al., 1999). A linear equation generated from ascorbic acid calibration curve was used to calculate the TAC: y  0.0036x  0.1161 (r2  0.9985) Diphenyl-1-picryhydrazyl (DPPH) scavenging activity assay A stock solution of DPPH was made by dissolving 50 mg of DPPH in 100 ml of 80% methanol. DPPH work solution was prepared by mixing one part of the stock solution with four parts of 80% methanol. For the scavenging of DPPH by the crude extract of B. inflata leaves, 700 l of the work solution was added to various volumes (as above) of extract. The final volume was made up to 1 ml by the addition of respective solvents. The reaction was allowed to take place in the dark for 45 minutes at room temperature. The absorbance of the mixture was measured at a wavelength of 517 nm. The control reaction was made up of 700 l of DPPH work solution mixed with 300 l of solvent (without the extract) while 80% methanol was used for the blank (Teffo et al., 2022). The percentage scavenging of DPPH and IC50 were calculated as earlier described. Hydrogen peroxide (H2O2) assay A 40 mM solution of H2O2 was prepared by mixing phosphate buffer and 30% H2O2. Thereafter, 600 l of 40 mM H2O2 solution was added to various volumes (as stated earlier) of crude extract. The volume was made up to 1 ml by the solvent used for the extraction. The solution was incubated for 10 minutes in the dark and at room temperature, after which the absorbance was measured at a wavelength of 230 nm. The phosphate buffer, without the H2O2, was used as the blank, while 600 l of 40 mM H2O2 solution combined with 400 l of solvent (without the extract) was used as the control (Teffo et al., 2022). The percentage scavenging of H2O2 by B. inflata and IC50 was calculated as earlier described. Metal chelating assay Various volumes (specified earlier) of B. inflata leaf crude extracts were made up to 1 ml with the various solvents used for extraction. Thereafter, 50 l of 2 mM FeCl3 was added to the 1 ml mixture (of extract and solvent) and shaken vigorously with a vortex. Subsequently, 200 l of 5 mM ferrozine was added and the mixture was shaken again. The mixture was then incubated in the dark and at room temperature for 10 minutes and the absorbance was taken at a wavelength of 562 nm. 1 ml of solvent mixed with 50 l of 2 mM FeCl3 and 200 l of 5 mM Ferrozine served as control while 80% methanol was used as the blank (Pavithra and Vadivukkarasi, 2015; Rakesh et al., 2021). The difference between the absorbance of samples and the control was taken as the actual sample absorbance. STATISTICAL ANALYSIS Values obtained from quantitative phytochemical screening assays and in vitro antioxidant assays were carried out in triplicates. Pearson Correlation Coefficient (r) was used to assess the relationship between Total Antioxidant Capacity (TAC) and quantified phytochemicals. R Studio (R 4.2.3) was used to analyse all collected data. The obtained values were expressed as mean ± standard deviation (SD). One-way Analysis of variance (ANOVA) was used to determine significant differences at P ≤ 0.05. Thereafter, Tukey HSD posthoc test was used to determine where the significant differences lie. Qualitative and quantitative phytochemical screening and antioxidant potential of Bulbine inflata (Asphodelaceae) Pak. J. Pharm. Sci., Vol.37, No.5, September-October 2024, pp.1177-1187 1180 RESULTS Yield The percentage of extraction yields, based on solvents and drying methods of B. inflata leaves, are shown in fig. 2. Methanol and water, which are the polar solvents, gave more extraction yield in comparison with the non-polar solvents, which are hexane and ethyl acetate. Except for methanolic extract, the yields of freeze-dried samples were slightly higher than those obtained from air-dried samples. The highest yield was in the water extract of freeze-dried sample at 37.11% while the lowest yield was in the hexane extract of air-dried sample at 7.77%. The order of yield percentage according to solvents was (water, methanol, ethyl acetate and hexane (fig. 2). Fig. 1: B. inflata showing the leaves, fruits and flowers. Fig. 2: The yield percentage of B. inflata leaf extracts using various solvents and two drying methods. Qualitative phytochemical screening The detection of some secondary metabolites such as phenolics, tannins, terpenoids, glycosides, flavonoids among others, in the powdered dried leaves of B. inflata was presented in table 1. Most tested phytochemicals were detected in the extracts of polar solvents, especially in water, while extracts of ethyl acetate gave the least indication of the presence of the phytochemicals. Tannin was detected in all extracts of used solvents and in both drying methods. Fig. 3: Total phenolic content (TPC) of B. inflata, dried under two conditions and extracted with four different solvents. Bars represented mean values with standard deviation (SD) and different alphabets implied significant difference (P ≤ 0.05). Fig. 4: Total flavonoid content (TFC) of B. inflata, dried under two conditions and extracted with four different solvents. Bars represented mean values with SD and different alphabets implied significant difference (P ≤ 0.05). Phenolics were detected in both drying methods and all tested solvents, except for hexane. Saponin and phlobatannin were found only in the methanolic extracts. Quinones were detected only in the polar solvent extracts of freeze-dried samples. As indicated in the percentage yield, most of the phytochemicals were detected in polar solvents and freeze-dried samples in comparison with non-polar solvents and air-dried samples (table 1). Quantitative phytochemical screening Total phenolic content (TPC) Ground dried leaves of B. inflata showed high total phenolic content (TPC) as shown in fig. 3. Three out of Rebecca Opeyemi Oyerinde and Ida Masana Risenga Pak. J. Pharm. Sci., Vol.37, No.5, September-October 2024, pp.1177-1187 1181 the four solvents were able to extract between 218-446 mg gallic acid equivalent per g dry weight (mg GAE/g DW) of B. inflata leaf sample. Hexane was the only solvent that did not extract a relatively high amount of TPC (<60 mg GAE/g DW). Air-dried water extract significantly (p ≤ 0.05) had the highest TPC value (446.56 mg GAE/g DW), while air-dried hexane extract had the least TPC content (25.40 mg GAE/g DW). Except in water extract, all freeze dried samples had higher TPC than the air-dried samples. Fig. 5: Total tannin content (TTC) of B. inflata, dried under two conditions and extracted with four different solvents. Bars represented mean values with SD and different alphabets implied significant difference (P ≤ 0.05). Fig. 6: Total proanthocyanidin content (TPAC) of B. inflata, dried under two conditions and extracted with four different solvents. Bars represented mean values with SD and different alphabets implied significant difference (P ≤ 0.05). Total flavonoid content (TFC) Total flavonoid content (TFC) in B. inflata leaves was significantly (p ≤ 0.05) the highest in freeze-dried water extracted sample (99.75±10.29 mg quercetin equivalent (QE)/g DW) while the least was recorded in air-dried hexane extract (22.95±1.41 mg QE/g DW). For each solvent, TFC in freeze-dried samples were significantly higher than in air-dried samples (fig. 4). Fig. 7: Total antioxidant capacity/reducing power of B. inflata, dried under two conditions and extracted with four different solvents. Bars represented mean values with SD and different alphabets implied significant difference (P ≤ 0.05). Fig. 8: Scavenging of DPPH by B. inflata, dried under two conditions and extracted with four different solvents. Total tannin content (TTC) In B. inflata, quantitative analysis showed the highest presence of tannins in freeze-dried water and methanol as well as air-dried water extracted samples (138.47±0.72; 135.10±0.84 and 133.89±11.71 mg GAE/g DW, respectively). The least amount of tannin was recorded in the non-polar hexane extracted samples (29.66±2.29 and 28.39±1.52 mg GAE/g DW for air-dried and freeze-dried samples, respectively) (fig. 5). Total proanthocyanidin content (TPAC) Total proanthocyanidin content was highest in air-dried methanolic extract of B. inflata (18.12±0.91 catechin equivalent (CE)/g DW) and freeze-dried ethyl acetate extract (18.02±0.65 mg CE/g DW) (fig. 6). Surprisingly, the TPAC extracted by water was at its lowest level (fig. 6) in contrast with other tested phytochemicals (figs. 2-4). Qualitative and quantitative phytochemical screening and antioxidant potential of Bulbine inflata (Asphodelaceae) Pak. J. Pharm. Sci., Vol.37, No.5, September-October 2024, pp.1177-1187 1182 In vitro antioxidant activities Phosphomolybdenum assay for total antioxidant capacity (TAC) In B. inflata, TAC was higher in freeze-dried samples than in the air-dried samples (fig. 7). In addition, samples extracted with polar solvents had higher TAC in comparison with non-polar solvents. Freeze-dried water extract had the highest TAC (1289.79±2.74 mg ascorbic acid equivalent (AAE)/g DW), while air-dried hexane extract had the lowest TAC (87.75±0.28 mg AAE/g DW) (fig. 7). In most cases, The TAC has very strong correlation with quantified phytochemicals, i.e. TPC, TFC, TTC and TPAC (table 2). Fig. 9: Inhibition of H2O2 by B. inflata, dried under two conditions and extracted with four different solvents. Fig. 10: Metal chelation of iron III ions by B. inflata, dried under two conditions and extracted with four different solvents. Diphenyl-1-picryhydrazil (DPPH) assay Water extract of B. inflata leaves had the highest DPPH inhibition capacity for both freeze-dried (84.6±0.23%) and air-dried (79.18±2.04%) samples (fig. 8). Small concentrations of water extracts of freeze- and air-dried B. inflata leaves (0.81±0.22 and 1.22±0.64 mg/ml, respectively) were required to scavenge 50% of DPPH (IC50 values, table 3). Samples extracted with non-polar solvents, hexane and ethyl acetate, were not as potent as those extracted with polar solvents i.e., water and methanol. The least inhibition capacities were recorded for hexane extracts at 27.21±5.79% and 28.76±5.08%, for freeze-dried and air-dried samples, respectively (fig. 8), with corresponding IC50 values of 13.75±7.21 mg/ml and 9.23±2.59 mg/ml for freeze-dried and air-dried samples, respectively (table 3). Hydrogen peroxide (H2O2) assay Ethyl acetate extract from air-dried leaves of B. inflata had the highest antioxidant activity against H2O2, scavenging 95.9% of H2O2 (fig. 9) with IC50 of 1.98±0.04 mg/ml (table 3). Similarly, methanolic extracts had IC50 of 2.50±0.14 and 2.32±0.18 mg/ml, for both air- and freeze-dried samples, respectively (table 3). However, hexane extracts had the least antioxidant capacity just as seen in the other tested parameters (table 3 and fig. 9). Metal chelating assay The colour of the Fe-ferrozine complex deepened when extracts of B. inflata leaf were added as shown by the absorbance values (fig. 10). Methanolic extracts for both air- and freeze-dried samples as well as ethyl acetate extracts of freeze-dried samples had the highest colour intensity and absorbance values at 50 µl. On the other hand, hexane extract of freeze-dried showed the least colour intensity and absorbance (fig. 10). DISCUSSION The concentration of bioactive compounds in plants is usually low, hence, suitable solvents, as well as extraction methods with minimal or no damage to the compounds, are requisites for optimal yield (Dhanani et al., 2017). Higher polarity of solvents have been linked with higher extraction yield (El Mannoubi, 2023). In addition, different drying methods have been shown to affect the yield of bioactive compound extraction (Poorgharib et al. 2023). Among the four solvents that were used in this study, water has the highest polarity while hexane has the least (Adeleye and Risenga, 2022). Thus, the percentage yield of extracted phytochemicals from B. inflata was directly proportional to the polarity of solvents (fig. 2). The reduced yield and lack of detection of some phytochemicals could be attributed to (i) The solubility of the bioactive compounds in the solvents as well as the nature of the plant material (Teffo et al., 2022). (ii) The inability of a drying method to effectively disrupt cellular components for optimal bioactive compound release and extraction (Kolawole et al., 2018). In B. inflata, most phytochemicals were detected in the freeze-dried, polar solvent extracted samples. B. inflata presented with substantial amount of phenolics and flavonoids (fig. 3 and 4). Rebecca Opeyemi Oyerinde and Ida Masana Risenga Pak. J. Pharm. Sci., Vol.37, No.5, September-October 2024, pp.1177-1187 1183 Table 1: Colour test for phytochemical screening of B. inflata samples. Phytochemicals Air-dried Freeze-dried Hexane Ethyl acetate Methanol water Hexane Ethyl acetate Methanol water Saponins - - + - - - + - Terpenoids - - +++ + + - +++ ++ Glycosides - - - +++ - - - +++ Steroids - - +++ + - - ++ +++ Volatile oils +++ - - - ++ + - - Coumarins + - - - ++ - - +++ Phlobatannins - - - - - + - Alkaloids - - - - - - - - Phenolics - ++ +++ +++ - ++ +++ ++ Tannins  Bromine water test  FeCl3 test +++ - ++ - ++ - + - +++ - ++ - ++ + ++ - Quinones - - - - - - + +++ Cardiac glycosides - - - ++ - - ++ - Flavonoid  NaOH test  Conc. H2SO4  Conc. HCl +++ - - - - - - - - - - - ++ - - + - - - - - ++ + - -, +, ++ and +++ represent absence, low, moderate and high detection of phytochemicals, respectively. Table 2: The Pearson’s Correlation Coeffiecient (r) between total antioxidant capacity (TAC) and quantified phytochemicals (TPC, TFC, TTC and TPAC) of B. inflata leaves. TAC TPC TFC TTC TPAC AD-Hex 0.984324* 0.804906* 0.631798** 0.372141 FD-Hex 0.654654** 0.066983 0.715554** 0.953821* AD-EA 0.978883* 0.978450* 0.992336* 0.632381** FD-EA 0.618590** 0.641053** 0.999424* 0.927247* AD-Meth 0.081211 0.775506** 0.739186** 0.393159 FD-Meth 0.991799* 0.365263 0.995981* 0.862886* AD-Water 0.785714** 0.841698* 0.778434** 0.716934** FD-Water 0.912245* 0.586712*** 0.810885* 0.214809 *, ** and *** depict a ‘very strong (0.80-1.00), strong (0.60-0.79) and moderate 0.4-0.59)’ correlation. Table 3: Half-maximal inhibitory concentration (IC50) of B. inflata on various oxidative molecules. Drying method/solvent IC50 + SD (mg/ml) DPPH H2O2 AD-Hex 9.23 ± 2.59ac 7.93 ± 0.56a FD-Hex 13.75 ± 7.21c 14.16 ± 0.97b AD-EA 2.84 ± 0.174ab 1.98 ± 0.04c FD-EA 4.55 ± 0.04ab 5.81 ± 0.63d AD-Meth 2.36 ± 0.07ab 2.50 ± 0.14c FD-Meth 1.51 ± 0.11b 2.32 ± 0.18c AD-Water 1.23 ± 0.64b 4.05 ± 0.38e FD-Water 0.81 ± 0.22b 8.62 ± 0.57a Different superscripts for each molecule implied significant differences AD: air-dried, FD: freeze-dried, Hex: hexane, EA: ethyl acetate, Meth: methanol. Qualitative and quantitative phytochemical screening and antioxidant potential of Bulbine inflata (Asphodelaceae) Pak. J. Pharm. Sci., Vol.37, No.5, September-October 2024, pp.1177-1187 1184 The phenolic class of phytochemical comprises the largest group of bioactive compounds in plants; while the flavonoids are one of the four groups of phenolics constituting about 66% of phenolic content in dietary plant products (Ozcan et al., 2014; Kumar and Goel, 2019). The presence of the phenol group confers antibacterial, antioxidant, vasodilating and anticancer properties on plants; and makes the plants serve protective roles against mutagenic and degenerative health issues (Ozcan et al., 2014; Koche et al., 2016; Kumar and Goel, 2019). flavonoids have also been reported to have a reducing effect on Type 2 diabetes and cardiovascular diseases (Di Lorenzo et al., 2021). Thus, the consumption of phenolic-rich plant products offer a wide range of benefits to humans (Lin et al., 2016; Rahman et al., 2021). Tannins, which were detected in B. inflata (fig. 5), have antioxidant properties thus, are effective as free radical scavengers. They inhibit the growth of antibiotic-resistant bacteria species as well as the ulcer-causing Helicobacter pylori. They have also been shown to have antiviral, antidiabetic and anti-inflammatory properties. Thus, they are able to prevent chronic diseases and inflammation- associated ailments (Kibiti and Afolayan, 2015; Sieniawska, 2015). Proanthocyanidins, also known as condensed tannin, were present in B. inflata (fig. 6). They are one of the major group of tannin with complex structures, which make them resistant to hydrolysis and are associated with bitter taste of plant products (Sieniawska, 2015; Koche et al., 2016; Singh and Kumar, 2019). They also have the health benefits associated with tannins and polyphenols. These compounds have been detected in other Bulbine species such as B. natalensis (Yakubu and Afolayan, 2009) and B. abyssinica (Kibiti and Afolayan, 2015; Teffo et al., 2022). However, the content of proanthocyanidin was lowest in the water extract of B. inflata. This could be attributed to the resistance of proanthocyanidin to hydrolysis unlike the hydrolysable tannin group. Importantly, all the phytochemicals that were detected in other Bulbine species of medicinal importance such as B. abyssinica leaves (Teffo et al., 2022), B. natalensis stems with the exception of anthraquinones (Yakubu and Afolayan, 2009) and B. frutescens (Shikalepo et al., 2018) plants except alkaloids were found in B. inflata (table 1). Hence, B. inflata may serve medicinal purposes like other members of the family. Free radicals, such as hydrogen peroxide, are molecules that have unbound/unpaired electrons in their outer layer, which make them unstable and highly reactive. They are formed naturally in human body as byproducts of various physiological and biological activities and a moderate amount of free radicals is essential for human physiological functions. However, when they are in excess, their activities result in oxidative stress, which have degenerative effects that are implicated in various ailments, some of which are lethal (Pham-Huy et al., 2008; Jakubczyk et al., 2020; Munteanu and Apetrei, 2021). Antioxidants are naturally occurring or synthetic compounds that can scavenge and take up the unpaired electrons from free radicals, making them stable and less reactive (Pham-Huy et al., 2008; Gulcin and Alwasel, 2022). While synthetic antioxidants may be more effective, available and stable than their natural counterpart, natural antioxidants are safer (Gulcin and Alwasel, 2022). The total antioxidant capacity by the phosphomolybdenum assay is based on the ability of antioxidants, such as those that are present in plant extracts, to reduce molybdenum VI (MoVI) to molybdenum V (MoV). The reduced MoV forms a green complex with the phosphate component of the reagent, which shows the highest absorption at 695 nm in the spectrophotometer (Bhatti et al., 2015). The intensity of the green colour is a measure of the reduction from MoVI to MoV, which is directly proportional to higher absorbance and the total antioxidant capacity (TAC)/reducing power of the plant extract. With these parameters, B. inflata, showed antioxidant and reducing properties, with the freeze-dried water extract presenting the highest TAC. Similarly, aqueous extract of B. abyssinica showed the highest TAC in comparison with other solvents (Idamokoro and Afolayan, 2020). DPPH is a stable free radical molecule that is often used to test and measure the antioxidant capacity of materials such as plant extracts (Kedare and Singh, 2011). The deep purple colour of DPPH reduces when it is mixed with a reducing agent, such as antioxidants. The antioxidants donate electrons, which pair with the unpaired electrons of DPPH, t reducing it to DPPH.H (2,2-diphenyl-1- picrylhydrazine). The degree of discoloration of the DPPH purple colour is a measure of the antioxidant potency of the plant extracts (Kedare and Singh, 2011). Hydrogen peroxide (H2O2) is a member of the reactive oxygen species (ROS), which are molecules that are responsible for oxidative stress (Jakubczyk et al., 2020). It is important that there is a balance between their production and elimination by antioxidants, in order to prevent their potential degenerative damage. Medicinal plants, including those in the Bulbine genus, have been shown to be highly effective in scavenging these molecules. The IC50 for DPPH scavenging by B. inflata was within the range of other Bulbine species, e.g. 0.0962, 0.1380 and 4.79 mg/ml for the aqueous and methanolic leaf extract of B. abyssinica; 0.053 and 0.601 mg/ml for the aqueous methanolic leaf extract of B. frutescens and B. natalensis (Ghuman et al., 2019; Idamokoro and Afolayan, 2020; Teffo et al., 2022). Examples in the genus Bulbine include B. abyssinica (Kibiti and Afolayan, Rebecca Opeyemi Oyerinde and Ida Masana Risenga Pak. J. Pharm. Sci., Vol.37, No.5, September-October 2024, pp.1177-1187 1185 2015; Teffo et al., 2022); B. frutescens (Shikalepo et al., 2018; Ghuman et al., 2019) and B. natalensis (Ghuman et al., 2019). Similarly, B. inflata presented with high antioxidant capacity against these molecules, not only because of their high scavenging percentages, but also because of the IC50 values of the extracts. Essential heavy metals, such as iron, cobalt, copper and zinc, are necessary components for normal physiological and biological functions in humans. However, heavy metals can become toxic when they are accumulated beyond the required threshold in the human systems, causing metal poisoning, oxidative stress and the consequent associated health complications (Gulcin and Alwasel, 2022). Metal chelation is a therapeutic process in which antioxidants are used to inhibit metal toxicity by binding to the metals, thus preventing their harmful effects. Higher affinity for metal binding corresponds to higher antioxidant capacity. Ferrozine has a high binding affinity for iron (Fe) ions and when there is contact between these molecules, an Fe-ferrozine complex is formed immediately (Gulcin and Alwasel, 2022). In this research, ferric ion (Fe3+) was used as opposed to the commonly used ferrous ion (Fe2+) and the complex formed was a light purple/lilac solution. The observed result was opposed to what is reported in literature, that the addition of extracts with antioxidant/chelating properties should prevent or inhibit the formation of Fe- ferrozine complex, leading to a reduction in colour intensity (Adjimani and Asare, 2015; Gulcin and Alwasel, 2022). Instead, the addition of B. inflata extracts increased the colour intensity of the solution. The probable explanation was that since B. inflata leaf extract has been shown to be a good reducing agent as reported earlier, the Fe (III) ion used for this assay was reduced to Fe (II) ion by the extracts. The Fe (II) ions then bound with the ferrozine to give deeper colour intensity. The second probable explanation was that B. inflata leaf was rich in iron. This could be in line with the finding that Fe was one of the mineral components in one of the Bulbine species, B. abyssinica (Kibiti and Afolayan, 2018). 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