High Performance Liquid Chromatography (HPLC) Profile Bioactive Phytochemicals in Selected Medicinal Plants

standards of selected phytochemical compounds, including gallic acid, quercetin, rutin, caffeic acid and catechin, were prepared for qualitative and quantitative analysis. Accurately weighed amounts of each standard were dissolved in HPLC-grade methanol to obtain stock solutions at a concentration of 1 mg/mL. Serial dilutions of the stock solutions were prepared to generate calibration standards of varying concentrations. These standard solutions were used to construct calibration curves and to facilitate the identification and quantification of corresponding phytochemicals in the plant extract (Dai and Mumper, 2010). 2.5 HPLC Instrumentation and Chromatographic Conditions HPLC analysis was carried out using a reversed-phase HPLC system equipped with a quaternary pump, autosampler, column oven and UV/Diode Array Detector . Chromatographic separation was achieved on a C18 reversed-phase column (250 × 4.6 mm, 5 μm particle size). The mobile phase consisted of solvent A, which was water containing 0.1% formic acid and solvent B, which was acetonitrile or methanol. Elution was performed using a gradient program to allow effective separation of compounds with varying polarities. The flow rate was maintained at 1.0 mL/min, and the column temperature was set at 30 °C. An injection volume of 20 μL was used for both sample and standard solutions. Detection was carried out at wavelengths ranging from 254 to 360 nm depending on the phytochemical class under investigation and the total run time was between 30 and 45 minutes. These chromatographic conditions enabled efficient resolution of a wide range of phytochemical constituents present in the plant extract (Snyder et al., 2010). 2.6 Identification of Phytochemical Compounds The identification of phytochemical constituents in the plant extract was achieved by comparing the retention times and UV absorption spectra of detected peaks with those of authentic reference standards analyzed under identical chromatographic conditions. Further confirmation of compound identity was obtained by comparing the chromatographic profiles and spectral characteristics with published literature data. This approach ensured reliable identification of the bioactive compounds present in the extract (Dai and Mumper, 2010). 2.7 Quantification of Phytochemicals Quantitative analysis of the identified phytochemicals was performed using the external standard calibration method. Calibration curves were constructed by plotting peak areas against corresponding concentrations of the reference standards. The concentrations of phytochemicals in the plant extract were calculated using the regression equations obtained from the calibration curves. Results were expressed as milligrams of compound per gram of dry extract (mg/g), providing a quantitative estimate of the bioactive constituents present in the plant material (Khoddami et al., 2013). 3. Results In Table 1, the bioactive compounds of Chromolaena odorata HPLC analysis revealed a diverse phytochemical profile dominated by phenolic compounds and alkaloids. Ellagic acid showed a high concentration (72.95 μg/ml), indicating strong antioxidant potential. Naringenin, naringin, rutin and kaempferol further confirm the abundance of flavonoids. The avocado seed extract in Table 2 contained various flavonoids and phenolic compounds, with flavones and kaempferol occurring prominently. Catechin and resveratrol were detected, reflecting antioxidant potential. Ribalinidine and lunamarin indicate alkaloid and glycoside presence. In Table 3, the stem extract of Dialium guineense showed high levels of ribalinidine, aglycones and flavonoids, with ribalinidine exhibiting a notably high concentration. Catechin, resveratrol and kaempferol further contributed to the phenolic richness. The presence of lunamarin and gallocatechin indicates glycosidic and antioxidant activity, suggesting the stem as a potent source of bioactive compounds. In Table 4, the HPLC profiling of the bark revealed flavones, flavon3ol and catechin as major constituents, alongside ribalinidine and lunamarin. Kaempferol and resveratrol were also detected, supporting antioxidant activity. The bark extract demonstrated a balanced composition of phenolics, flavonoids and alkaloids, which may account for its traditional use in managing infections and inflammatory conditions. The leaf extract in Table 5 exhibited a rich and complex phytochemical profile, dominated by ellagic acid, ferulic acid and vanillic acid, indicating strong antioxidant capacity. Multiple flavonoids including rutin, naringenin and kaempferol were detected. In Table 6, the stem of Napoleonaea imperialis contained appreciable levels of phenolic acids such as vanillic, ferulic and coumaric acids. Alkaloids including ephedrine and sparteine were prominent. Flavonoids such as rutin and kaempferol were also identified. The root extract in Table 7 showed a predominance of proanthocyanins and flavonoids, including flavon3ols and naringenin. Alkaloids such as ribalinidine and sparteine were present alongside phenols and steroids. The detection of humulone derivatives suggests additional bioactivity. The seed extract of Napoleonaea imperialis in Table 8 demonstrated high levels of tannins, ribalinidine, cardiac glycosides and cyanogenic glycosides. Flavonoids such as rutin, kaempferol, catechin and naringenin were also abundant. The leaf extract of Napoleonaea imperialis in Table 9 contained substantial amounts of tannins, flavonones and anthocyanins, reflecting strong antioxidant capacity. Alkaloids such as ephedrine, sparteine and ribalinidine were detected alongside cardiac glycosides. The diversity of phenolics, flavonoids and alkaloids suggests broad therapeutic potential, particularly in antimicrobial and anti-inflammatory applications. Fig 1: HPLC Analysis of Dialium guineense leaf Fig 2: HPLC Analysis of Napoleonaea imperialis Stem Fig 3: HPLC Analysis of Napoleonaea imperialis Seed Table 1: Bioactive Compounds of Chromolaena odorata Compound Retention Area Height External Units Epihedrine 1.006 6391.6332 500.813 17.4924 μg/ml Ribalinidine 4.100 5724.7406 448.262 13.2075 μg/ml Ellagic acid 9.146 13615.5420 1054.478 72.9528 μg/ml Spartein 12.016 4414.7548 346.275 8.2461 Ppm Naringin 14.310 4133.8336 324.646 39.1548 l μg/ml Lunamarin 20.166 13869.6608 1053.816 1.1526 ppm Quinine 25.570 12106.8242 937.585 144.3547 ppm Kaempferol 29.456 10548.5846 780.061 4.3066 μg/ml Naringenin 32.263 17268.1446 1314.844 40.9997 μg/ml Rutin 35.140 4462.9954 350.224 12.2142 μg/ml Resveratrol 40.080 3869.9483 303.931 10.9246 μg/ml Quinine 45.233 8713.6257 680.538 69.5336 μg/ml Table 2: Bioactive Compounds of Avocado (Persea americana) Seed Component Retention Area Height External Unit Resveratrol 0.426 6108.0233 55.456 6.7431 μg/ml Catechin 2.423 3584.7027 68.471 10.1194 μg/ml Ribalinidine 5.506 3438.8984 68.471 12.3962 μg/ml Flavonones 16.900 4273.9892 85.054 9.1471 μg/ml Flavone -3-ol 18.013 3668.8441 73.766 10.3883 μg/ml Flavone 23.483 6312.0334 125.373 17.2746 μg/ml Aglycone 24.916 3275.3754 65.634 7.7131 ppm Lunamarin 32.563 3545.0768 70.367 33.6262 ppm Gallocatechin 35.806 3739.7866 74.523 13.4230 ppm Isoflavonoids 38.563 3726.4203 74.465 5.0253 ppm Kaempferol 39.776 2050.3854 57.237 10.9861 μg/ml Kaempferol 40.076 66.5142 35.450 0.3564 μg/ml Table 3: Bioactive Compounds of Dialium guineense Stem Component Retention Area Height External Unit Resveratrol 1.883 10310.4636 79.009 11.3824 μg/ml Catechin 4.770 7981.3190 148.708 22.5308 μg/ml Ribalinidine 8.750 17777.7308 374.284 64.0834 μg/ml Flavonones 15.696 15628.3172 330.432 33.4475 μg/ml Flavan-3-ol 18.260 10767.5819 226.431 30.4883 μg/ml Flavone 24.106 4624.6554 97.990 12.6566 μg/ml Aglycone 29.116 17704.3660 374.940 41.6918 μg/ml Lunamarin 31.433 8046.0438 183.516 76.3193 μg/ml Gallocatechin 32.346 13533.5810 279.188 48.5752 μg/ml Isoflavonoids 39.246 5364.3732 113.520 7.2342 μg/ml Kaempferol 45.270 5218.4190 149.341 27.9606 μg/ml Kaempferol 45.570 137.3300 75.440 0.7358 μg/ml Table 4: Bioactive Compounds of Dialium guineense Bark Component Retention Area Height External Unit Rasveratol 0.306 7747.0627 285.288 10.6774 Catechin 2.420 3648.4088 285.401 19.4950 Ribalinidine 5.506 3437.7298 269.451 6.7335 Flavonones 16.896 4255.4497 334.689 1.1533 Flavon-3-ol 18.016 3691.5902 289.353 13.6479 Flavone 23.483 6295.0994 493.007 17.2283 Aglycone 24.916 3293.1976 258.448 7.7551 Lunamarin 32.563 3529.8671 277.175 33.4819 Gallocatechin 35.806 3738.6794 293.649 5.4091 Isoflavonoids 38.563 3750.8060 294.034 5.0582 Kaempferol 39.900 3964.3128 312.169 7.8819 Table 5: Bioactive Compounds of Dialium guineense Leaf Component Retention Area Height External Units Proanthocyanin 0.286 2674.4902 83.255 2.8995 Ribalinidine 2.390 12130.7858 228.502 5.8944 Ellagic acid 4.120 6336.6840 119.930 6.8944 Ellagic acid 6.016 18229.8623 337.706 22.7844 Spartein 7.470 8324.8153 157.319 2.5994 Ferullic acid 10.366 19541.1716 363.724 25.8994 Epicatechin 12.970 6168.0984 115.724 8.9944 Sapogenin 15.460 4894.9617 92.004 7.3899 Vanillic acid 17.966 11156.5757 209.679 14.6773 Anthocyanin 22.730 9463.5916 177.253 4.3899 Tannin 25.650 10016.2504 187.036 4.8993 Tyrosol 27.536 11395.4124 213.413 13.8993 Kaempferol 29.860 5371.1982 101.221 2.3788 Naringenin 32.996 14311.5574 265.886 18.7884 Rutin 34.600 5899.8182 111.776 2.8995 Naringenin 36.876 6791.8962 128.573 3.7882 Resveratol 39.200 10114.9720 189.466 2.8994 Resveratrol 42.276 3497.2340 65.306 1.0883 Protocatechuic 44.170 10556.7706 196.213 1.0884 Table 6: Bioactive Compounds of Napoleonaea imperialis Stem Component Retention Area Height External Units Proanthocyanin 0.330 5743.6906 191.344 5.4893 Epihedrine 1.370 12308.3341 252.395 6.8944 Ribalinidine 3.196 7287.1844 181.144 3.0893 Ellagic acid 4.093 7507.2951 183.663 9.0433 Coumaric acid 5.486 12528.8822 245.191 8.4783 Spartein 7.093 17818.5769 432.684 9.4933 Ferullic acid 9.026 10027.9036 243.817 16.7884 Sapogenin 17.033 10810.2516 261.989 16.2793 Vanillic acid 19.113 15829.8099 383.021 20.0892 Anthocyanin 23.306 3631.7268 89.200 2.8994 Epicatechin 25.080 9936.8383 241.806 13.7882 Tyrosol 28.593 3561.4480 86.290 5.9022 Kaempferol 30.986 6168.6561 149.389 2.8993 Rutin 34.116 7294.7142 176.567 8.9543 Hydroxytyrosol 39.026 4541.0092 109.798 6.0893 Resveratrol 43.983 9610.1972 141.130 6.9888 Table 7: Bioactive Compounds of Napoleonaea imperialis Root Component Retention Area Height External Units Proanthocyanin 0.123 3359.9492 360.197 15.8993 naringin 2.700 2436.2256 191.759 4.2833 Flavon-3-ol 5.793 5819.4134 456.746 10.6733 Citrulline 8.436 4721.8424 371.188 14.2019 Ribalinidine 10.793 4775.5038 375.479 3.8992 Naringenin 12.450 4743,7844 372.795 13.8992 Epicatechin 14.040 4579.5053 360.005 2.7883 Spartein 15.076 1038.4858 81.526 3.2744 Phenol 19.756 3930.6483 309.146 10.2844 Flavonones 21.553 5025.1523 394.848 11.4933 Steriods 24.580 4953.9757 389.494 10.3844 Adhumulone 31.363 4199.9884 329.743 0.3922 Cohumulone 34.120 2174.5382 171.250 1.5944 Humulone 36.010 663.9956 52.301 5.7833 Catechin 39.900 2206.4649 173.478 4.3883 Resveratrol 41.836 3159.5558 248.559 2.4933 ppm Table 8: Bioactive Compounds of Napoleonaea imperialis Seed Component Retention Area Height External Units Proanthocyanin 0.080 247.0756 139.945 0.2780 Lunamarin 0.230 2673.5748 77.285 3.1316 Cardiac glycoside 2.390 12463.7692 207.166 8.1210 Flavan-3-ol 4.120 6453.7512 109.082 3.7489 Tannin 6.016 18473.9050 303.874 12.3374 Anthocyanin 7.466 8209.3358 141.562 10.5609 Ribalinidine 10.366 19406.2195 325.524 8.3217 Naringenin 12.970 6015.1932 102.717 5.4013 Spartein 15.460 4747.4720 81.456 6.3810 Rutin 17.966 10838.3774 185.757 10.0885 Cyanogenic glycoside 20.313 12126.0843 207.446 16.2985 Flavonones 22.730 9256.9310 157.532 5.1604 Steriods 25.650 9899.8924 166.836 12.7357 Kaempferol 27.536 11173.4073 189.669 7.7378 Epicatechin 29.860 5193.3534 89.413 7.7911 Phytate 32.996 14260.2702 237.682 14.3753 Flavone 34.600 5663.5741 98.627 5.2717 Catechin 36.876 6483.6329 112.946 1.7081 Resveratrol 39.200 9912.2862 168.507 3.7661 Sapogenin 42.276 3472.9824 58.519 2.8537 Epihedrine 44.170 10566.2594 175.989 15.4054 Table 9: Bioactive Compounds of Napoleonaea imperialis Leaf Component Retention Area Height External Units Sapogenin 0.140 4083.6304 389.518 1.7930 Narigenin 3.473 4093.0642 320.769 2.6958 Anthocyanin 4.613 6355.7564 494.097 7.6049 Epihedrine 9.480 4859.7778 380.588 1.7854 Dihydrocytisine 10.866 4680.9656 366.718 3.1593 Kaempferol 13.126 6488.6312 507.928 4.4925 Cyanogenic glycoside 14.886 5010.1896 392.627 1.4510 Aphyllidine 16.956 3363.0461 263.532 1.5098 Steroid 19.440 4580.4180 359.146 3.1713 Tannin 22.883 8235.4138 642.944 11.0566 Flavonones 25.713 7931.5974 618.930 5.3411 Catechin 28.606 2956.9610 231.988 3.5605 Flavone 30.280 5551.2211 435.394 3.6569 Proanthocyanidin 32.730 4433.1010 347.266 2.4434 Ribalinidine 34.633 4104.5175 321.457 5.0940 Spartein 36.896 7473.4783 583.325 9.2781 Oxalate 38.526 4454.0796 349.461 1.5898 Cardiac glycoside 39.326 8756.4876 683.810 7.8816 Phytate 40.326 6201.0704 486.517 4.2924 Ammodendrine 40.953 3567.9760 280.808 0.6006 Aphyllidine 41.816 5879.1171 494.615 1.2378 4. Discussion High Performance Liquid Chromatography profiling revealed a wide diversity of bioactive phytochemicals across Chromolaena odorata, Persea americana (avocado seed), Dialium guineense (stem, bark and leaf), and Napoleonaea imperialis (stem, root, seed and leaf). The detected compounds mainly belonged to phenolics, flavonoids, alkaloids, glycosides and related secondary metabolites, confirming the rich phytochemical nature of the studied plants. In Chromolaena odorata, ellagic acid and naringenin were among the most abundant compounds, alongside significant levels of naringin, rutin, kaempferol and resveratrol. Ellagic acid is a potent antioxidant and anti-inflammatory polyphenol, previously reported as a major constituent of C. odorata extracts (Akinmoladun et al., 2010). The presence of quinine, sparteine and ephedrine further highlights the alkaloid richness of the plant, which may explain its traditional antimicrobial and wound-healing applications. Similar flavonoid dominance has been reported by Owoyele et al. (2008), who associated these compounds with anti-inflammatory activity. The avocado seed extract demonstrated a flavonoid-rich profile, characterized by catechin, flavones, flavan-3-ols, kaempferol and isoflavonoids. These findings agree with reports by Dabas et al. (2013), who identified avocado seeds as rich sources of antioxidant flavonoids and phenolic compounds. The detection of lunamarin and ribalinidine also suggests the presence of glycosidic and alkaloid compounds, contributing to the seed’s bioactivity. For Dialium guineense, clear variation in phytochemical composition was observed among stem, bark and leaf. The stem contained high levels of ribalinidine, catechin, flavonones and aglycones, indicating strong antioxidant and antimicrobial potential. The bark showed a similar flavonoid pattern but with relatively lower concentrations, while the leaf exhibited the highest diversity, including ellagic acid, ferulic acid, vanillic acid, tannins, anthocyanins and multiple flavonoids. This supports earlier findings by Ajiboye et al. (2016), who reported that D. guineense leaves possess higher phenolic content and antioxidant activity compared to other plant parts. In Napoleonaea imperialis, distinct phytochemical distribution was also evident across plant parts. The stem contained abundant phenolic acids (vanillic, ferulic and coumaric acids) and flavonoids such as rutin and kaempferol, alongside alkaloids like ephedrine and sparteine. The root extract showed the presence of proanthocyanins, flavon-3-ols, phenols and humulone derivatives, indicating possible antimicrobial and anti-inflammatory properties. The seed extract was particularly rich in tannins, cardiac glycosides, cyanogenic glycosides and flavonoids, while the leaf showed high levels of tannins, anthocyanins, flavonones and alkaloids. These observations are consistent with earlier phytochemical screenings of N. imperialis, which reported flavonoids, tannins and alkaloids as dominant constituents (Okoye et al., 2014). In essence, the HPLC results confirm that the studied plants are rich reservoirs of biologically active compounds, with flavonoids and phenolic acids consistently dominant. Variations among species and plant parts highlight the influence of anatomical origin on phytochemical composition. The detected compounds support the ethnomedicinal uses of these plants and provide a scientific basis for their antioxidant, antimicrobial and anti-inflammatory potentials. 5. Conclusion and Future Considerations This study demonstrated that Chromolaena odorata, Persea americana seed, Dialium guineense, and Napoleonaea imperialis possess rich and diverse phytochemical profiles as revealed by HPLC analysis. The dominance of flavonoids, phenolic acids, alkaloids and glycosides across different plant parts supports their wide application in traditional medicine and suggests strong antioxidant, antimicrobial and anti-inflammatory potentials. Variations in phytochemical composition among species and plant organs highlight the influence of botanical origin on bioactive content. Future studies should focus on bioactivity-guided fractionation, in vivo pharmacological evaluation, and toxicity profiling to establish safety and therapeutic efficacy. Additionally, advanced techniques such as LC–MS/MS and metabolomics are recommended for precise compound identification and to explore synergistic interactions among phytochemicals. 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