Kenaf (Hibiscus cannabinus L.) Leaves and Seed as a Potential Source of the Bioactive Compounds: Effects of Various Extraction Solvents on Biological Properties

Hibiscus cannabinus (Kenaf) is a potential source of bioactive constituents and natural antioxidant. The current study determined the impact of various solvents on extraction yield, recovery of polyphenol and flavonoid, antioxidant, anticancer, and antibacterial properties of Kenaf leaves and seed. The powder of leaves and seed was separately extracted with n-hexane, ethyl acetate, ethanol, and water solvent. Among them, the ethanol extract of leaves and seed showed the highest extraction yield, and their GC-MS analysis revealed a total of 55 and 14 bioactive compounds, respectively. The total polyphenols (TP) and flavonoids (TF) content were quantified by a spectrophotometric technique where water extracts displayed a noteworthy amount of TP and TF content compared to other extracts. A similar demonstration was noticed in antioxidant activity, evaluated by DPPH (2,2-diphenyl-1-picrylhydrazyl) and hydrogen peroxide scavenging capacity. In addition, cytotoxicity and anti-lung cancer activity were identified against mouse embryonic fibroblast (NIH3T3) and human lung cancer (A549) cells. All extracts of leaves and seed were observed as non-toxic to the NIH3T3 cells, but slight toxicity was expressed by n-hexane extracts at the optimum dose (1000 µg/mL) of treatment. In parallel, n-hexane and ethanol extracts (leaves and seed) exposed promising anti-lung cancer activity at the same concentration. Furthermore, antibacterial activity was assessed using disc diffusion assay, and seed extracts exhibited a significant inhibition zone against Gram-positive and Gram-negative microorganisms. Overall, Kenaf seed extracted with polar solvents was found very potent in terms of important bioactive compounds and pharmacological aspects, which can be an excellent biological matrix of natural antioxidants.


Introduction
One of the common manifestations of living cells is the generation of harmful pro-oxidants and reactive oxygen species (ROS), produced either due to the biological dysfunctions or as a result of cellular metabolisms (byproducts) [1]. Such free radicals may induce oxidative damage to proteins, lipids, and nucleic acids and lead to several life-threatening conditions, including cancer, neurodegenerative

Gas Chromatography-Mass Spectroscopy (GC-MS) Analysis of Kenaf (Leaves and Seed) Extracts
The bioactive compounds of Kenaf leaves and seed (ethanol extract) were detected by GC-MS analysis using an Agilent Technologies 7890A capillary gas chromatograph, along with a mass spectrometer system. GC was equipped with a 30 m × 0.25 mm × 0.25 µm DB-5 capillary column. Initially, the instrument was maintained at a temperature of 100 • C for 2 min and 6 s. The temperature was risen to 300 • C at the rate of 25 • C/min and maintained for 20 min at the end of this period. The injection port temperature and the helium flow rate were ensured to be 250 • C and 1.5 mL/min, respectively. The ionization voltage was 70 eV. The sample was injected in split mode at 5:1. The MS scan range was set at 35-550 (m/z). The fragmentation patterns of mass spectra were compared with those stored in the using W8N05ST Library MS database. The percentage of each compound was calculated from the relative peak area of each compound in the chromatogram. The concept of integration used the Chem Station integrated algorithms. The total content of phenol in Kenaf leaves and seed extracts was assessed following the method of Folin-Ciocalteau [16]. The Folin-Ciocalteau reagent (200 µL, 1 N) was added in the test tube containing 1 mL of the sample (10 mg/mL). The volume of the mixture was increased by the addition of deionized water (1.8 mL) and kept (3 min at room temperature) for the reaction after the vortex. Afterwards, 400 µL of sodium carbonate (10% v/v) was added to the reaction mixture. Finally, the volume was adjusted up to 4 mL by adding deionized water (600 µL). The mixture was placed in dark ambience for incubation (1 h at room temperature), and the test was done in triplicates. The absorbance was measured against the blank (water) at 725 nm by the spectrophotometer (UV-1800 240 V, Shimadzu corporation, Kyoto, Japan). The TPC was calculated from a calibration curve (plotting the value of absorbance vs. concentration) using gallic acid and expressed as mg of GAE (gallic acid equivalent) per 100 g of extract.

Total Flavonoid Content (TFC)
The total flavonoid content in Kenaf leaves and seed extracts was determined according to the previously described method [17] with some modifications [18]. In brief, 0.5 mL aliquot of extract (10 mg/mL) was mixed with 100 µL of aluminum nitrate (10% w/v), 100 µL of potassium acetate (1M), and 3.3 mL of ethanol. The mixture was vortexed and placed for incubation (40 min at room temperature). TF content of the extracts was measured at 415 nm by the spectrophotometer (UV-1800 240 V, Shimadzu corporation, Kyoto, Japan) and expressed in mg/100 g QE (quercetin equivalent).

DPPH Free Radical and Hydrogen Peroxide (H 2 O 2 ) Scavenging Activity
The antioxidant activity of Kenaf leaves and seed extracts was evaluated using DPPH free radical and H 2 O 2 , following the method of Braca et al. [19] and Adnan et al. (2020) [5]. For the DPPH, 3 mL of freshly prepared DPPH (0.004% w/v in methanol) was added to the 0.5 mL of stock solution. The reaction mixture was vortexed and placed in the dark ambience for incubation (30 min at room temperature). For H 2 O 2 scavenging, 0.6 mL of H 2 O 2 solution (4 mM prepared with 0.1 M phosphate buffer pH 7.4) was mixed with 0.4 mL of stock solution and then incubated for 10 min. The scavenging of DPPH and H 2 O 2 were measured at 517 nm and 230 nm, respectively by the spectrophotometer (UV-1800 240 V, Shimadzu Corporation, Kyoto, Japan). The percentage of scavenging capacity was calculated against negative control (methanol + DPPH) and (stock solution without H 2 O 2 ) expressed by the following equation: Scavenging effect (%) = [(Abs c − Abs s )/Abs c ] × 100, where Abs c is the absorbance of control; Abs s is the absorbance of DPPH radical/H 2 O 2 + sample (extract/standard).

Cell Culture, Cytotoxicity, and Anti-Lung Cancer Assay
The cytotoxicity and anti-lung cancer potentiality of various solvent extracts (leaves and seed) were evaluated against NIH3T3 and A549 cells, employing the WST assay kit. The NIH3T3 and A549 cells were cultured in the penicillin and streptomycin (PS)-incorporated DMEM and RPMI medium, respectively. The cells were incubated (CO 2 incubator at 37 • C for 24 h) and the quality of cells (confluences and morphology) were observed under light microscopy. The prepared cells were further used in cytotoxicity and anti-lung cancer analysis. Briefly, the NIH3T3 (5 × 10 4 ) and A549 (1 × 10 5 ) cells were seeded in the 96-well plates (containing DMEM and RPMI medium, respectively) and incubated (CO 2 incubator at 37 • C for 24 h) until they reached the desirable confluence (80-90%). The incubated cells were treated with the different concentrations (62.5, 125, 250, 500, and 1000 µg/mL) of extracts and incubated with a similar condition. Finally, Ex-CyTox reagent (10 µL) was added to each well and absorbance was recorded at 450 nm. As a negative control, 5% DMSO was used. From the obtained Life 2020, 10, 223 5 of 16 absorbance, the cytotoxicity and cell viability of the extracts were calculated according to the formula described elsewhere [15].

Antibacterial Activity
Antibacterial activity was analyzed using the disc diffusion method [15]. In brief, Mueller-Hinton agar was prepared and placed into Petri dishes for solidification (under laminar airflow). Gram-positive Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633), and Bacillus cereus (ATCC 14579) and Gram-negative Salmonella Typhi (ATCC 29629), Pseudomonas aeruginosa (ATCC 9027), and Escherichia coli (ATCC 8739) microorganisms were cultured overnight. Of each prepared bacteria, 100 µL (bacterial inocula were adjusted to 10 7 CFU/mL) was spread smoothly on the agar surface, and then a sterile disc (8 mm diameter) was laid upon an agar plate (seeded). Each extract with desired concentration (50 mg/mL) was loaded on these discs and incubated (at 37 • C for 24 h). The zone of inhibition was recorded and measured in mm. The 5% DMSO was used as a negative control, and as a positive control, ampicillin (25 µg/mL) was used.

Statistical Analysis
All data were expressed as mean ± standard deviation (SD) of several measurements. The obtained results (total phenol, flavonoid, and antioxidant activity) were compared among the composition using a paired t-test in order to observe the significant differences at the level of 5%. The paired t-test between the mean values was analyzed by MINITAB (version 17.0, Minitab Inc., State College, PA, United States). Data of cytotoxicity was analyzed using GraphPad Prism 6.0 statistical software. Values are expressed as mean ± SD (n = 3) and two-way ANOVA, followed by the Bonferroni test. There was a significant difference when comparing each column to all other columns at * p < 0.05, ** p < 0.01, and *** p < 0.001.

Extraction Yields
Various parts of a plant occupy a pool of bioactive compounds containing potential chemical groups which consistently protect both plants and humans from cellular oxidative damage [20]. However, to explore all these major classes of chemicals, efficient extraction techniques, such as maceration, subcritical water extraction, soxhlet extraction, supercritical fluid extraction, and ultrasonic-assisted extraction with suitable solvents are a prerequisite [21]. Importantly, plant-derived phytoconstituents compose numerous chemical characteristics and polarities that are not readily soluble in a particular solvent [22]. Therefore, various solvents with differing polarities must be studied to extract potential bioactive compounds from the plant [23]. Our study used different solvent types to evaluate the impact of extraction yield, and here, the seed extracts exhibited a higher yield than the leaves' extracts ( Figure 1). In both cases, the highest and lowest extraction yields were recorded for the ethanol (ETH) and n-hexane (NHX) extracts, respectively. After the ethanol extract, the yield differences were observed in another solvent extraction, such as ethyl acetate (EA), followed by the water (WT) extract for the Kenaf seed, and water followed by an ethyl acetate extract for kenaf leaves. Sim et al. [24] reported that pulsed ultrasonic-assisted Kenaf leaves extraction with water showed the highest extraction yield compared to methanol, ethanol, and acetone solvent. Yusri et al. [25] also followed a similar extraction method for Kenaf seed, and the highest extraction yield was obtained for the hexane extract. However, in our study, the conventional extraction method was followed, and the observed variation of yield extraction might have been due to the influence of a heterogeneous solvent used with different polarities, and the results are in line with the previously reported extraction yields of rice bran [26] and many medicinal plants [27].

Total Phenol and Flavonoid Content
Four different extracts (NHX, EA, ETH, and WT) of Kenaf leaves and seed were analyzed to quantify phenolic and flavonoid content. The phenolic content was assessed by gallic acid and expressed as GAE per 100 g of dry extract ( Figure 3A), whereas flavonoid content was estimated by quercetin and expressed as QE per 100 g of dry extract ( Figure 3B). The highest amount of phenolic and flavonoid content was recorded in seed compared to leaves. Particularly, WT extract demonstrated the highest total phenol content for both Kenaf seed (754.6 ± 3.14 mg/100 g dry extract) and leaves (418.7 ± 3.47 mg/100 g dry extract). Similarly, the maximum flavonoid content was also detected from the WT (425.33 ± 4.39 and 299.17 ± 3.43 mg/100 g dry extract) extract of both Kenaf seed and leaves. However, the other solvent seed extracts of EA followed by ETH and NHX, as well as leaf extracts of ETH, followed by EA and NHX were noted as significant for both TPC and TFC. The highest TPC and TFC observed in the water extract might have resulted from its high polarity index [31]. The important phytoconstituents of a plant can be polar or nonpolar in nature-mainly, phenolic compounds possess abundant hydroxyl groups, which are responsible for dissolving of the polar solvent [32]. In addition, the ethyl acetate extract displayed better extraction of TPC and TFC, which confirmed that this solvent is also efficient for extracting secondary metabolites. It is stated that the ethyl acetate solvent has potential for the extraction of phenolic compounds, and this statement is in agreement with the result found from the EA extract of Kenaf seed [33]. Besides, noteworthy TPC and TFC manifested by the ethanol extract might be owing to the presence of methyl radicles, which can easily conjugate with phenolic or flavonoid compounds and allow efficient solvation [34]. In contrast, hexane showed poor extraction of TPC and TFC, which could be attributed to the lower polarity or strong non-polar nature of the solvent [35].
Four different extracts (NHX, EA, ETH, and WT) of Kenaf leaves and seed were analyzed to quantify phenolic and flavonoid content. The phenolic content was assessed by gallic acid and expressed as GAE per 100 g of dry extract ( Figure 3A), whereas flavonoid content was estimated by quercetin and expressed as QE per 100 g of dry extract ( Figure 3B). The highest amount of phenolic and flavonoid content was recorded in seed compared to leaves. Particularly, WT extract demonstrated the highest total phenol content for both Kenaf seed (754.6 ± 3.14 mg/100 g dry extract) and leaves (418.7 ± 3.47 mg/100 g dry extract). Similarly, the maximum flavonoid content was also detected from the WT (425.33 ± 4.39 and 299.17 ± 3.43 mg/100 g dry extract) extract of both Kenaf seed and leaves. However, the other solvent seed extracts of EA followed by ETH and NHX, as well as leaf extracts of ETH, followed by EA and NHX were noted as significant for both TPC and TFC.
The highest TPC and TFC observed in the water extract might have resulted from its high polarity index [31]. The important phytoconstituents of a plant can be polar or nonpolar in naturemainly, phenolic compounds possess abundant hydroxyl groups, which are responsible for dissolving of the polar solvent [32]. In addition, the ethyl acetate extract displayed better extraction of TPC and TFC, which confirmed that this solvent is also efficient for extracting secondary metabolites. It is stated that the ethyl acetate solvent has potential for the extraction of phenolic compounds, and this statement is in agreement with the result found from the EA extract of Kenaf seed [33]. Besides, noteworthy TPC and TFC manifested by the ethanol extract might be owing to the presence of methyl radicles, which can easily conjugate with phenolic or flavonoid compounds and allow efficient solvation [34]. In contrast, hexane showed poor extraction of TPC and TFC, which could be attributed to the lower polarity or strong non-polar nature of the solvent [35].   Figure 4A,B displays the percentage inhibition of the DPPH and OH free radical scavenging capacity of Kenaf leaves and seed extracts. The activity notably varied among the various extracts, and the overall higher percentage of scavenging capacity was observed in the seed extracts compared to leaves. In both cases, the antioxidant capacity of ethanol and ethyl acetate extract was almost identical; even no noticeable changes in regard to activity were noted for the DPPH and H2O2 free radical scavenging. However, in the case of DPPH, the highest antioxidant activity of Kenaf seed was recorded for WT (73.12 ± 2.06%), followed by ethyl acetate (54.45 ± 1.38%), ETH (52.49 ± 1.12%), and hexane (28.09 ± 2.61%) extract. However, Kenaf leaves also exhibited 65.35 ± 1.86% DPPH scavenging for the WT, 46.27 ± 1.71% (EHT), 43.19 ± 2.15% (EA), and 21.07 ± 1.65% (NHX) extracts. These findings were analogous with many previous studies, where authors concluded that relatively high polar  Figure 4A,B displays the percentage inhibition of the DPPH and OH free radical scavenging capacity of Kenaf leaves and seed extracts. The activity notably varied among the various extracts, Life 2020, 10, 223 10 of 16 and the overall higher percentage of scavenging capacity was observed in the seed extracts compared to leaves. In both cases, the antioxidant capacity of ethanol and ethyl acetate extract was almost identical; even no noticeable changes in regard to activity were noted for the DPPH and H 2 O 2 free radical scavenging. However, in the case of DPPH, the highest antioxidant activity of Kenaf seed was recorded for WT (73.12 ± 2.06%), followed by ethyl acetate (54.45 ± 1.38%), ETH (52.49 ± 1.12%), and hexane (28.09 ± 2.61%) extract. However, Kenaf leaves also exhibited 65.35 ± 1.86% DPPH scavenging for the WT, 46.27 ± 1.71% (EHT), 43.19 ± 2.15% (EA), and 21.07 ± 1.65% (NHX) extracts. These findings were analogous with many previous studies, where authors concluded that relatively high polar solvent extracts, such as water, methanol, and ethanol exposed higher DPPH free radical potential than the non-polar solvent extracts. Likewise, DPPH, a similar pattern of antioxidant activity, was observed in the case of the H 2 O 2 scavenging test. Here, Kenaf seed extracts demonstrated strong hydroxyl radical scavenging activity, ranging from 67.69 ± 1.46 to 26.41 ± 2.58%, whereas leaf extracts manifested between 53.86 ± 0.43 and 19.52 ± 1.83%.

Cytotoxicity and Anti-Lung Cancer Activity
The cytotoxicity of the Kenaf leaf and seed extracts was determined against NIH3T3 cells. Figure  5A displays the Kenaf seed extracts which expressed slight toxicity with increasing concentrations in a dose-dependent fashion. Notably, the hexane and ethyl acetate extracts showed higher cytotoxicity of23.64 ± 0.12% and 18.77 ± 0.13%, respectively, and cell growth inhibition was detected at a higher Interestingly, an interplay relationship exists between antioxidant activity and the quantity of secondary metabolites [36]. Usually, phenolics are regarded as the predominant antioxidant components, and scavenging activities of these components are directly proportional to the total content of phenolics [18]. Hence, the observed higher antioxidant activity in our study might be attributed to secondary metabolites, such as higher content of polyphenol and flavonoids of the extracts. Nevertheless, these outcomes indicate that this plant has remarkable scavenging capacity.

Cytotoxicity and Anti-Lung Cancer Activity
The cytotoxicity of the Kenaf leaf and seed extracts was determined against NIH3T3 cells. Figure 5A displays the Kenaf seed extracts which expressed slight toxicity with increasing concentrations in a dose-dependent fashion. Notably, the hexane and ethyl acetate extracts showed higher cytotoxicity of23.64 ± 0.12% and 18.77 ± 0.13%, respectively, and cell growth inhibition was detected at a higher concentration (1000 µg/mL). In the case of Kenaf leaves ( Figure 5B), a similar cytotoxicity pattern was also exposed by the hexane and ethyl acetate extracts. Here, cell growth inhibition was recorded as 16.37 ± 0.20% and 12.7 ± 0.37%, respectively, at the same concentration. However, treatment of water and ethanol extracts (both seed and leaves) did not reveal any obvious toxicity toward the NIH3T3 cells, even at the higher concentration treatment, which indicates that these extracts are comparatively safe than the others. The observed healthy cells after the treatment of water and ethanol extract might be caused by their higher content of polyphenol, flavonoid, and promising antioxidant activity, which acted as nutrients for the cell growth [37]. In contrast, the toxicity demonstrated by hexane extract might be attributed to its poor antioxidant capacity, which affected the cells by producing reactive oxygen species [38]. Figure 6A,B displays the anti-lung cancer activity of Kenaf seed and leaves against A549 cells. Here, NHX extract exposed the highest anti-lung cancer activity in a concentration-dependent response, where treatment of the optimal concentration (1000 µg/mL) revealed 37.4% cell death by Kenaf seed and 29.6% by Kenaf leaves. The comparable accord was also observed for ETH and EA extract. In Kenaf seed, cell viability was reduced to 33.8% by ETH, while EA decreased 29.7% cell viability at the highest concentration. In contrast, ETH (26.4%) and EA (23.6%) of Kenaf leaves also manifested moderate anticancer activity. Interestingly, NHX demonstrated toxicity against NIH3T3 cells, and ETH was found to be safe for NIH3T3 cells; however, during an anti-lung cancer test, both extracts exhibited remarkable inhibition of A549 cell growth, which concludes that the ETH extract is relatively safer than the other extracts. Previously, a study conducted by Wong et al. revealed that the optimum concentration of Kenaf seed extract strongly inhibited the cell growth of HeLa (CCL-2), breast cancer (MCF-7), colon cancer (HCT-116), and lung cancer (SK-LU1) [39], which also support our findings. Importantly, the treatment of 5% DMSO in the NIH3T3 and A549 cells did not expose any notable toxicity, wherein 90% of cell confluence was observed (visual observation), which suggests that the solvent (5% DMSO) did not have a toxicological effect. 16.37 ± 0.20% and 12.7 ± 0.37%, respectively, at the same concentration. However, treatment of water and ethanol extracts (both seed and leaves) did not reveal any obvious toxicity toward the NIH3T3 cells, even at the higher concentration treatment, which indicates that these extracts are comparatively safe than the others. The observed healthy cells after the treatment of water and ethanol extract might be caused by their higher content of polyphenol, flavonoid, and promising antioxidant activity, which acted as nutrients for the cell growth [37]. In contrast, the toxicity demonstrated by hexane extract might be attributed to its poor antioxidant capacity, which affected the cells by producing reactive oxygen species [38]. Here, NHX = n-Hexane extract, EA = Ethyl acetate extract, ETH = Ethanol extract, and WT = Water extract. Data was analyzed using GraphPad Prism 6.0 statistical software. Values are expressed as mean ± SD (n = 3), and a two-way ANOVA andBonferroni test were applied. They were significantly different when comparing each column to all other columns at * p < 0.05, ** p < 0.01, and *** p < 0.001. Figure 5. Cytotoxicity of various solvent extracts (NHX, EA, ETH, and WT) of Kenaf seed (A) and leaves (B). Here, NHX = n-Hexane extract, EA = Ethyl acetate extract, ETH = Ethanol extract, and WT = Water extract. Data was analyzed using GraphPad Prism 6.0 statistical software. Values are expressed as mean ± SD (n = 3), and a two-way ANOVA andBonferroni test were applied. They were significantly different when comparing each column to all other columns at * p < 0.05, ** p < 0.01, and *** p < 0.001.
Life 2020, 10, x FOR PEER REVIEW 13 of 17 Figure 6A,B displays the anti-lung cancer activity of Kenaf seed and leaves against A549 cells. Here, NHX extract exposed the highest anti-lung cancer activity in a concentration-dependent response, where treatment of the optimal concentration (1000 µ g/mL) revealed 37.4% cell death by Kenaf seed and 29.6% by Kenaf leaves. The comparable accord was also observed for ETH and EA extract. In Kenaf seed, cell viability was reduced to 33.8% by ETH, while EA decreased 29.7% cell viability at the highest concentration. In contrast, ETH (26.4%) and EA (23.6%) of Kenaf leaves also manifested moderate anticancer activity. Interestingly, NHX demonstrated toxicity against NIH3T3 cells, and ETH was found to be safe for NIH3T3 cells; however, during an anti-lung cancer test, both extracts exhibited remarkable inhibition of A549 cell growth, which concludes that the ETH extract is relatively safer than the other extracts. Previously, a study conducted by Wong et al. revealed that the optimum concentration of Kenaf seed extract strongly inhibited the cell growth of HeLa (CCL-2), breast cancer (MCF-7), colon cancer (HCT-116), and lung cancer (SK-LU1) [39], which also support our findings. Importantly, the treatment of 5% DMSO in the NIH3T3 and A549 cells did not expose any notable toxicity, wherein 90% of cell confluence was observed (visual observation), which suggests that the solvent (5% DMSO) did not have a toxicological effect. Previously, Kenaf seed-mediated silver nanoparticles showed promising anti-lung cancer activity [15]. It was reported that the plant possesses bioactive compounds which induce mitochondrial damage by elevating the superoxide level that suppresses cancer cell growth through reduction of ATP synthesis [40]. Some secondary metabolites may also attack the DNA, resulting in abundant ROS production and apoptosis-inducing enzyme activation, subsequently leading to cell Previously, Kenaf seed-mediated silver nanoparticles showed promising anti-lung cancer activity [15]. It was reported that the plant possesses bioactive compounds which induce mitochondrial damage by elevating the superoxide level that suppresses cancer cell growth through reduction of ATP synthesis [40]. Some secondary metabolites may also attack the DNA, resulting in abundant ROS production and apoptosis-inducing enzyme activation, subsequently leading to cell death [41].

Antibacterial Activity
The antibacterial activity of Kenaf leaves and seed extracts are presented in Table 2. Both Gram-positive (Staphylococcus aureus, Bacillus cereus, and Bacillus subtilis) and Gram-negative (Salmonella Typhi, Escherichia coli, and Pseudomonas aeruginosa) microorganisms were tested through the disc diffusion method. Results revealed that Kenaf seed extracts were very effective against both Gram-positive and Gram-negative bacteria. Ethyl acetate, ethanol, and water extract exposed antibacterial activity against B. cereus, E. coli, and B. subtilis microorganisms. Here, the most remarkable inhibitory effect was observed against E. coli bacteria, where the zone of inhibition was recorded for 15.2 ± 0.72 mm for ethyl acetate extract. In the case of leaf extracts, the zone of inhibition recorded for ETH and WT were the most potent among all other extracts. These results indicate that the Kenaf plant possesses a broad spectrum antipathogenic effect. Moreover, both leaves and seed extracts of NHX did not show any inhibitory effect against any bacteria. On the other hand, both leaf and seed extracts did not manifest any activity against Gram-positive (Staphylococcus aureus) and Gram-negative (Salmonella Typhi and Pseudomonas aeruginosa) microorganisms. In the case of the negative control (5% DMSO), no activity was recorded, which indicates that the 5% DMSO did not have an influence on the extract's activity. Values are presented as mean inhibition zone (mm) ± SD of three replicates, -: no activity, NHX = n-Hexane extract, EA = Ethyl acetate extract, and WT = Water extract.
The observed antibacterial efficiencies of the Kenaf plant might be due to the higher content of phenols and flavonoids [42]. It is stated that the secondary metabolites present in the plant are the key source of diverse pharmacological actions [43]. These metabolites also provide natural defensive pathways to inhibit various insects and pathogens, such as viruses and fungi [44]. In addition, GC-MS analysis of Kenaf leaves and seed exposed several potential bioactive constituents, which might play a crucial role either by interrupting normal cellular functions [45] or destabilizing the bacterial membrane [46].

Conclusions
This research explored the various solvent extractions of Kenaf leaves and seed and their pharmacological potentials. Among the solvents tested, ethanol was the efficient solvent, and demonstrated the highest extraction yield and potential bioactive compounds. In contrast, the water solvent remarkably influenced phytochemical content. In the case of leaves and seed, water extracts significantly impacted on the recovery of the highest total polyphenol, flavonoids, and manifestation of noteworthy antioxidant activity. In vitro cytotoxic activity revealed that extracts of Kenaf leaves and seed were non-toxic to the healthy (NIH3T3) cells, except n-hexane extracts, which expressed slight toxicity. Besides, n-hexane and ethanol extracts manifested promising anti-lung cancer activity against A549 cells, where ethanol extracts were comparatively safer than other extracts. During the antibacterial test, extracts of Kenaf seed exhibited significant activity against Gram-positive and Gram-negative microorganisms, as evidenced by the notable zone of inhibition. This work collectively concluded that Kenaf seed extracts was found very potent in terms of important bioactive compounds and pharmacological aspects, which can be an excellent biological matrix of natural antioxidants.