horticulturae Article Phytochemical Screening and Biological Activities of Diospyros villosa (L.) De Winter Leaf and Stem-Bark Extracts Oluwatosin Temilade Adu 1 , Yougasphree Naidoo 1 , Johnson Lin 2 , Temitope Samson Adu 3 , Venkataramegowda Sivaram 4,5 , Yaser Hassan Dewir 6, * and Antar Nasr El-Banna 7,8 1 2 3 4 5 6 7 8 * Citation: Adu, O.T.; Naidoo, Y.; Lin, J.; Adu, T.S.; Sivaram, V.; Dewir, Y.H.; El-Banna, A.N. Phytochemical Screening and Biological Activities of Diospyros villosa (L.) De Winter Leaf and Stem-Bark Extracts. Horticulturae 2022, 8, 945. https://doi.org/ 10.3390/horticulturae8100945 Academic Editors: Guillermo Cásedas, Cristina Moliner and Francisco Les Received: 30 August 2022 Accepted: 6 October 2022 Published: 14 October 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- School of Life Sciences, University of KwaZulu-Natal, Westville Campus, Private Bag X54001, Durban 4000, South Africa Department of Microbiology, School of Life Sciences, College of Agriculture, Engineering and Science, University of KwaZulu-Natal, Private Bag X54001, Durban 4000, South Africa Department of Physiological Sciences, Obafemi Awolowo University, Ile Ife 220005, Nigeria Laboratory of Biodiversity and Apiculture, Department of Botany, Bangalore University, Bangalore 560056, India V Sivaram Research Foundation, Vijayanagar, Bangalore 560056, India Plant Production Department, College of Food & Agriculture Sciences, King Saud University, Riyadh 11451, Saudi Arabia Federal Research Centre for Cultivated Plants, Julius Kühn-Institut (JKI), 38104 Braunschweig, Germany Genetics Department, Faculty of Agriculture, Kafrelsheikh University, Kafr El-Sheikh 33516, Egypt Correspondence: ydewir@ksu.edu.sa Abstract: This study aimed to evaluate the phytochemical components, antioxidant capacity, and antimicrobial effects of Diospyros villosa (L.) De Winter leaves and stem bark. The extracts were obtained using different media (methanol, chloroform, and hexane). The DPPH and FRAP methods were used to assess the antioxidant activity and the Folin–Ciocalteu method was used to determine the total phenolic contents of the crude extracts. The antimicrobial effects of the extracts against five pathogenic bacteria were determined using the MIC, MBC, and agar-well diffusion methods. Flavonoids, alkaloids, and phenols were identified in the D. villosa extracts. The mean concentrations of the methanolic leaf and stem-bark extracts against DPPH providing 50% inhibition were 9.53 ± 0.25 µg·mL−1 and 9.52 ± 0.30 µg·mL−1 , respectively. In addition, the total phenolic content within the test range of concentrations was found to be 28.45 ± 0.50 mg of gallic acid equivalent per g of sample extract [mg·g−1 (GAE)] (methanolic leaf extract) and 4.88 ± 0.36 mg·g−1 (GAE) (methanolic stem-bark extract). The methanolic leaf extracts further showed promising antimicrobial activity against Pseudomonas aeruginosa, Klebsiella pneumonia, Staphylococcus aureus, and methicillinresistant Staphylococcus aureus with inhibition zones of 18.0 ± 0.58, 23.5 ± 0.58, 20.0 ± 0.88, and 17.0 ± 2.0 mm, respectively which were comparable to the control (gentamicin and streptomycin). The results suggest that bioactive compounds are abundant in D. villosa leaves and stem bark and could serve as a potential source of natural antioxidants as well as an antibacterial agent for the treatment of pathogenic bacterial infections. Keywords: DPPH; free radicals; minimum inhibitory concentration; phenol content iations. 1. Introduction Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). The accumulation of free radicals at high levels is attributed to many pathological conditions and chronic diseases in humans [1]. In such situations, humans rely on plant sources of antioxidants for life maintenance and therapeutic measures. Certain research studies have further focused on various plant species that can circumvent the effects of oxidative damage incurred by the generation of free radicals [2–4]. High concentrations of antioxidants in some plant species serve to scavenge free radicals [5], whereas the identified antioxidants, flavonoids, and phenolic compounds have a wide range of structural Horticulturae 2022, 8, 945. https://doi.org/10.3390/horticulturae8100945 https://www.mdpi.com/journal/horticulturae Horticulturae 2022, 8, 945 2 of 21 features and descriptions as well as characteristics and biological consequences [6]. Bioactive compounds are also recognized for other multiple biological effects including their antimicrobial/bactericidal properties [7,8]. Rasheed et al. [9] indicated that Diospyros lotus contains both gallic acid (C7 H6 O5 ) and quercetin (C15 H10 O7 ). Diospyros montana has also been reported to contain 8-hydroxydiospyrin (C22 H14 O6 ) [10]. These active compounds have diverse defensive responses against different microbial strains through the generation of hydrogen peroxide [11] and alteration of the permeability of the microbial membrane [12,13]. Following these reports, the idea of investigating a novel South African plant for its antimicrobial activity and generation of free radicals comes to mind. Such plants may stand a better chance of alleviating the generation of free radicals and microbes in the disease state. Diospyros villosa (L.) De Winter is an African plant that occurs naturally in southern parts of the continent. D. villosa is a perennial, bushy evergreen plant with a height range of 1–4 m. The leaves are chartaceous, drying dull brown above and much paler beneath. The dimensional length and breadth of the leaf lamina are on average 3 cm and 1.5–6.5 cm, respectively. The shape of the leaves is always obovate but sometimes appears oblong. The leaf apex is usually broadly rounded and slightly emarginated and sometimes obtuse. The leaf base often has a cordate or round shape. The roots of the plant are used locally as toothbrushes and to treat oral infections [14]. This report gives an insight into the concept of this study in such a way as to provide scientific evidence for the medicinal use of the D. villosa plant in the pathogenesis of infection in the oral cavity. Escherichia coli was reported to be among the top pathogens that manifest in infections, which further result in cases of diarrhoeal illness [15]. Other aerobic Gram-negative bacteria such as Pseudomonas aeruginosa and Klebsiella pneumonia have been reported as opportunistic bacteria that further contribute to dental caries and sinusitis in orthodontic patients [16,17]. Gram-positive bacteria such as Staphylococcus aureus have been reported to co-exist with other microbial strains in the inflamed cavities of immunocompromised individuals and further induce bacterial reactivation in the infected cells [18]. Vellappally et al. [19] established that streptococci were not the only Gram-positive bacteria responsible for bloodstream infections and that S. aureus and methicillin-resistant S. aureus (MRSA) were often isolated in the hollow cavities of the body. In fact, the scourge MRSA accounted for almost 20–30% of all cases of oral infections [19]. At present, no studies are using D. villosa plant extracts against these bacteria strains. Hence, this study was geared toward making a significant contribution to the current search for ways to combat microbial strains involved in microbial infections and perhaps a contribution to existing knowledge of the antioxidant and antimicrobial properties of D. villosa extracts. To this end, the study intended to investigate the probable effects of D. villosa leaf and stem-bark extracts on identified Gram-negative and Gram-positive bacteria strains in close association with human body infections. 2. Materials and Methods 2.1. Chemicals 1,1-Diphenyl-2-picrylhydrazyl (DPPH), gallic acid, ethanoic acid, Folin–Ciocalteu, gentamicin, and streptomycin were procured from Sigma-Aldrich (St. Louis, MO, USA). Mueller Hilton agar media, hexane, methanol, and chloroform were purchased from Merck Chemical Co. (Durban, South Africa). 2.2. Plant Collection Fresh samples of D. villosa (mature leaves and stem bark) were collected in April and August of 2019 in KwaZulu-Natal, Durban, South Africa (29◦ 84′ 33.6′′ S, 31◦ 4′ 12′′ E). The plant was identified and a voucher specimen was deposited in the Herbarium (01/18257) at the School of Life Sciences, University of KwaZulu-Natal. The collected plant parts (leaves and stem bark) were air-dried at room temperature for 45 days and crushed into a fine powder. The powdered samples were kept in a cool, dry place for extraction purposes. Horticulturae 2022, 8, 945 3 of 21 2.3. Plant Extraction Powdered samples of the plant weighing 8 g were heated to a temperature of 40 ◦ C for 15 min with 100 mL of 95% methanol in a round-bottom flask attached to a Soxhlet apparatus. The crude extract was retained and the process was repeated thrice. Successive extractions using chloroform and hexane, respectively, were carried out at 30 min intervals. The condensate was further evaporated to dryness under reduced pressure at 40 ◦ C in a rotary evaporator. The crude extract was stored at 4 ◦ C and used within 48 h for further Weight o f the dry extract ( g) tests. The extraction yield (%) = Weight o f dry sample used f or the extracton ( g) 2.4. Qualitative Phytochemical Tests The qualitative phytochemical constituent screening of the different extracts of the leaves and stem bark obtained from D. villosa was conducted using standard qualitative protocols [20–23]. 2.5. Gas Chromatography–Mass Spectrometry (GC–MS) Analysis The extracts were filtered using a Whatman No. 4 and subsequently via a 0.22 µL membrane filter. A GCMS-QP2010 Plus (Shimadzu, Tokyo, Japan) with a capillary chromatography column (30 cm × 0.25 mm ID × 0.25 µm film thickness of 5% phenylmethylsiloxane) was used for the GC–MS analysis of the extracts. At first, the instrument was fixed at a temperature of 50 ◦ C, sustained for 1.5 min, then increased to a temperature of 200 ◦ C using a pace of 4 ◦ C min−1 . The temperature was further increased to a temperature of 300 ◦ C using a pace of 10 ◦ C min−1 for 7 min. The injector and interface temperatures were 240 and 220 ◦ C, respectively. The helium flow rate was maintained at a rate of 1.2 mL min−1 , and 2 µL of each of the crude extracts was dissolved using different solvents (≥99%, GC grade, Sigma-Aldrich) and filtered through a 0.22 µm filter and subsequently injected into the ‘splitless’ mode system. The range and the total running time for the spectral scan were 40–500 m/z and 30 min, respectively. A comparison of each mass spectrum with the data published in Adams [24] and the Mass Spectral Search Program database of the National Institute of Standards and Technology, Washington, DC, USA, aided the identification of each component. 2.6. Fourier Transform Infrared (FT–IR) Analysis The FT–IR analysis was conducted to characterize the functional groups in the D. villosa leaf and stem-bark extracts that are responsible for the biochemical and molecular potential of the plants using a spectrophotometer (Perkin Elmer 100 FT–IR, Waltham, MA, USA). The spectra were examined and imaged at a range of 4000–400 cm−1 with a resolution of 4 cm−1 . KBr was used as a standard to analyze the samples by dispersing them uniformly in a matrix of dry KBr. 2.7. Sensitivity Test of Leaves and Stem-Bark Extracts of D. villosa on Test Microorganisms The antimicrobial activities of the methanolic, chloroformic, and hexanolic leaf and stem-bark extracts of D. villosa were investigated using the agar-well diffusion method. The discs were prepared using a Whatman No. 1 and obtained by punching and placing them in vials, which were further sterilized in an oven at 150 ◦ C for 15 min. The test microorganisms were reactivated on the nutrient agar broth and further incubated at 37 ◦ C overnight. The test microorganisms were also standardized at an optical density of 0.1 at 625 nm using a UV-vis spectrophotometer (Agilent Cary 60 Spectr., Santa Clara, CA, USA). Following this, 0.2 mL of the standardized test culture was added to 20 mL of molten Muller–Hilton agar and homogenized. This was then poured into sterile plates and allowed to solidify. Thereafter, the wells were aseptically bored into the inoculated Muller–Hilton agar plates using a 6mm sterile cork borer. The test solutions of extracts (100 µL) at graded concentrations of 0.625, 1.25, 2.5, 5, and 10 mg mL−1 already dissolved in 10% DMSO were then placed into each designated well on the plate, ensuring that there was no spillage. A standardized amount (10 µg·mL−1 ) of gentamicin and streptomycin was introduced into the residual wells on Horticulturae 2022, 8, 945 4 of 21 the plate, which was used as a control for the Gram-negative and Gram-positive bacteria strains, respectively. The plates were then allowed to diffuse at room temperature for 1 h in the medium and eventually incubated at 37 ◦ C for 16–18 h in an incubator. The clear zones of inhibition were noted, measured, and recorded in millimeters. Each extract’s activity was tested in triplicate. 2.8. Antioxidant Activity 2.8.1. DPPH Scavenging Activity The free radical scavenging activity of the extracts was determined by DPPH (1, 1-diphenyl-2-picrylhydrazyl) radicals [25]. An aliquot of 3 mL of 0.004% DPPH solution in 95% ethanol and 0.1 mL of each plant extract at concentrations of 15, 30, 60, 120, and 240 µg·mL−1 were mixed. The mixture was thoroughly mixed and allowed to sit for 30 min at room temperature. The procedure was repeated for the ascorbic acid (control). The decolorization of DPPH was ascertained by quantifying the absorbance at 517 nm. The control was prepared using 0.1 mL of each constituent and double-distilled water as a replacement for the plant extract or ascorbic acid. The percentage expression of DPPH radical scavenging activity by the plant extracts was calculated as thus: Absorbance o f Control − Absorbance o f Test samples × 100. Absorbance o f Control 2.8.2. Ferric-Reducing Antioxidant Potential (FRAP) Assay The FRAP assay was conducted as previously described by Juntachote and Berghofer [26]. Multiple graded concentrations of the extract (15, 30, 60, 120, and 240 µg·mL−1 ) of the extract (1 mL each) were added to 2.5 mL of phosphate buffer (0.2M, pH 6) and 2.5 mL of potassium ferricyanide (1% w/v). The resulting admixture was incubated for 20 min at a temperature of 50 ◦ C. Then, 2.5 mL of 10% trichloroacetic acid was added to the mixture. A quantity of 2.5 mL from each mixture was further diluted twice with deionized water, and 0.5 mL of 0.1% (w/v) FeCl3 was added. The absorbance was later determined at 700 nm after 30 min. The positive control used was ascorbic acid. The half-maximal inhibitory concentration (IC50 ) was calculated from the graph of absorbance against the concentrations of the extracts. The results were generated as thus:   Absorbance o f Control − Absorbance o f sample Scavenging Effect (%) = × 100. Absorbance o f control 2.8.3. Total Phenolic Content (TPC) TPC was investigated using the Folin–Ciocalteu colorimetric method with slight adjustments [27]. A volume of 0.1 mL of each extract was mixed thoroughly with 3 mL of distilled water and 0.5 mL of Folin–Ciocalteu reagent was also added to each sample extract. The mixture was allowed to sit at room temperature for 3 min and 2 mL of 20% sodium carbonate was added. The mixture was further incubated for 30 min at room temperature. The total phenolic content was measured at 725 nm using a spectrophotometer. Gallic acid was used as the positive control. The total phenol values were expressed as mg of gallic acid equivalents (GAE)/g of dry sample extracts. 2.9. Test Micro-Organisms The bacteria strains used were identified as American-type collection culture strains obtained from the School of Pharmacy and Pharmacological Sciences, University of KwaZuluNatal. Three Gram-negative bacteria, namely E. coli (ATCC 35218), P. aeruginosa (ATCC 27853), and K. pneumoniae (ATCC 700603), and 2 Gram-positive bacteria, S. aureus (ATCC 33591) and MRSA (ATCC 43300), were used in this study. Horticulturae 2022, 8, 945 5 of 21 2.9.1. Determination of Minimum Inhibitory Concentration (MIC) of the D. villosa Extracts on Microorganisms The MIC of the extracts was determined using the method described by Akinpelu and Onakoya [28]. The extract was diluted in two folds and a portion of 2 mL of the extracts at different concentrations was mixed with 18 mL of sterilized molten Mueller–Hilton agar in order to achieve a final concentration regime of 0.313 mg·mL−1 to 0.01 mg·mL−1 . The resulting medium was transferred into sterile Petri dishes and allowed to set. The dry surface of the medium was achieved before streaking with 18 h-old standardized bacterial cultures. The plates were then incubated at 37 ◦ C for 48 h and later scrutinized for either the absence or presence of growth. The MIC was taken as the lowest concentration that prevents bacterial growth. 2.9.2. Determination of Minimum Bactericidal Concentration (MBC) of the Extracts on Test Microorganisms The minimum bactericidal concentration of the extract was determined according to Ferrazzano et al. [29]. The inoculum was taken on the line of streaks without visible growth in the MIC assay and subcultured on freshly prepared nutrient agar and incubated at 37 ◦ C for 48 h. The plates were later scrutinized for either the absence or presence of growth. The lowest concentration of the extracts with no indication of any growth on a new set of plates was considered as the MBC of the extracts. 2.10. Statistical Analysis The results were expressed as means ± SE. Statistical analysis was performed using Graph Pad Prism 5 (Graph Pad Software Inc., San Diego, CA, USA). All outcomes were compared with the control using both one-way and two-way analysis of variance (ANOVA) followed by a Bonferroni post hoc analysis. Effects were considered statistically significant at p value ≤ 0.05. 3. Results 3.1. Phytochemical Analysis The percentage extract yield of D. villosa leaves and stem bark are represented in Table 1. It was found that the methanol extract in the leaves produced the highest yield of phytochemicals, at about 10.8%, whereas the chloroform and hexane extracts in the leaves yielded 8.4% and 7.1%, respectively. Similarly, the methanol extract in the stem bark produced a yield of 9.2%, whereas the chloroform and hexane extracts yielded 7.9% and 10.3%, respectively. Table 1. Yield of extracts of D. villosa leaves and stem bark. Methanol Chloroform Hexane Leaf Extract Yield (%) Stem-Bark Extract Yield (%) 10.8 8.4 7.1 9.2 7.9 10.3 The results of the qualitative phytochemical assessment of the D. villosa leaves and stem-bark extracts are shown in Table 2. Phytochemical screening showed the presence of alkaloids, terpenoids, and phenols in the methanol extracts of D. villosa leaves. In addition, the presence of flavonoids was further observed in the methanol extracts of D. villosa stem bark. In addition, terpenoids, flavonoids, and phenols were shown to be present in both the chloroformic and hexanolic stem extracts of D. villosa. All the extracts from the plant’s leaves and stem cumulatively contained steroids, alkaloids, carbohydrates, and saponins. Horticulturae 2022, 8, 945 6 of 21 Table 2. Phytochemical screening of the methanol, chloroform, and hexane extracts of both the leaves and stem bark of D. villosa. Name of Test Alkaloids Leaves Mayer’s test Wagner’s test Protein Dragendorff Carbohydrates Benedict’s Steroids Lieberman-Burchard test Coumarins NaOH test Saponin Foam test Flavonoids Lead acetate Terpenoids Salowski’s Phenols FeCl3 test Stem Bark Methanol Chloroform Hexane Methanol Chloroform Hexane +++ + ++ ++ ++ +++ +++ +++ + + + + - + + + + - +++ + + ++ +++ +++ ++ ++ + + + + + + + + + + ++ ++ - + denotes presence; ++ denotes moderate presence; +++ denotes abundant presence; - denotes absence. 3.2. Gas Chromatography–Mass Spectrometry (GC–MS) Analysis The GC–MS analysis of both the leaves and stem of D. villosa showed the presence of certain bio-active compounds such as n-hexadecanoic acid, phytol, palmitoleic acid, eicosanoic acid and its derivatives, ascorbic acid and its derivatives, etc. (Tables 3–8). The retention time (RT), compound name, molecular formula, and peak height (%) are presented in Tables 3–8. Table 3. Compounds identified in the chloroform extracts of D. villosa leaves by GC–MS spectral analysis. S/N RT Compound Name 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 3.801 4.254 4.296 5.252 10.454 10.557 10.656 11.830 12.397 12.545 12.680 12.869 12.943 13.015 13.125 13.222 13.585 13.697 14.193 14.340 14.429 14.512 14.677 24. 14.749 25. 26. 27. 28. 14.981 15.304 15.440 15.550 1-Butene, 3-chloro-2-methyl1, 1-Dimethyl-3-chloropropanol Pentanoic acid, 2-hydroxy-4-methyl-(S)1-Butene,2,3,3-trimethyl 11-Methyldodecanol 11-Methyldodecanol 11-Methyldodecanol Vanillin 10-Methylnonadecane Eicosane Heptadecanoic acid, heptadecyl ester Phenol, 2, 4-bis(1,1-dimethylethyl)1-Dodecanol, 2-hexylEicosane 1-Dodecanol, 2-hexyl2(4H)-Benzofuranone, 5,6,7,7a-tetrahydro-4,4 Fumaric acid, ethyl 2-methyl allyl ester Decane, 2,3,7-trimethylTrans-3(10)-Caren-2-ol Decane, 2,3,7-trimethyl2,4a,8,8-Tetramethyldecahydrocyclopropa[d]n 2-Butanone, 4-(2,6,6-trimethyl-1-cyclohexen-1 Oxalic acid, 6-ethyloct-3-yl heptyl ester 1-{2-[3-(2-Acetyloxiran-2-yl)-1,1-dimethyl propyl]cycloprop-2-enyl} ethanone 11-Methyldodecanol 11-Methyldodecanol Tetradecanoic acid 11-Methyldodecanol Molecular Formula Height (%) C5 H9 cl C5 H11 clO C6 H12 O3 C7 H14 C13 H28 O C13 H28 O C13 H28 O C8 H8 O3 C20 H42 C20 H42 C34 H68 O2 C14 H22 O C18 H38 O C20 H42 C18 H38 O C11 H16 O2 C10 H14 O4 C13 H28 C10 H16 O C13 H28 C15 H26 C13 H22 O C19 H36 O4 1.08 0.87 7.45 0.93 1.14 1.21 1.02 0.28 0.67 0.53 0.25 5.54 0.87 1.12 0.98 2.52 1.19 0.33 1.04 0.33 1.23 1.02 0.39 C14 H20 O3 1.27 C13 H28 O C13 H28 O C14 H28 O2 C13 H28 O 0.68 0.67 3.13 0.80 Horticulturae 2022, 8, 945 7 of 21 Table 3. Cont. S/N RT Compound Name Molecular Formula Height (%) 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 15.685 15.879 16.320 16.410 16.679 16.761 16.970 17.538 17.741 18.168 18.753 21.024 21.795 21.991 22.537 22.935 24.190 1-Dodecanol, 2-Octyl 6-Methyl-cyclodec-5-enol Phytol, acetate 2-Pentadecanone,6,10,14-trimethyl3,7,11,15-Tetramethyl-2-hexadecen-1-ol Pentadecanoic acid 3,7,11,15-Tetramethyl-2-hexadecen-1-ol 5,9,13-Pentadecatrien-2-one,6,10,14-trimethy Hexadecanoic acid,methyl ester cis-13-Eicosenoic acid n-Hexadecanoic acid Eicosanoic acid Methyl 10-trans,12-cis-octadecadienoate Methyl 8,11,14-heptadecatrienoate Phytol cis,cis,cis-7,10,13-Hexadecatrienal Methyl 5,13-docosadienoate C20 H42 O C11 H20 O C22 H42 O2 C18 H36 O C20 H40 O C15 H30 O2 C20 H40 O C18 H30 O C17 H34 O2 C20 H38 O2 C16 H32 O2 C20 H40 O2 C19 H34 O2 C18 H30 O2 C20 H40 O C16 H26 O C23 H42 O2 0.52 3.88 4.14 3.77 0.91 0.48 1.41 0.64 1.74 0.63 21.20 0.40 0.45 1.22 5.21 14.36 0.41 Table 4. Compounds found in the chloroform extracts of D. villosa stem by GC–MS analysis. S/N RT Compound Name Molecular Formula Height (%) 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 10.830 10.925 10.991 11.050 13.890 14.524 16.326 16.682 16.976 18.586 19.546 20.233 22.405 22.435 22.620 Octadecanoic acid Octadecanoic acid Undecanoic acid n-Decanoic acid Disulfide,di-tert-dodecyl Phytol,acetate Phytol,acetate 3,7,11,15-Tetramethyl-2-hexadecen-1-ol 3,7,11,15-Tetramethyl-2-hexadecen-1-ol 1-(+)-Ascorbic acid 2,6-dihexadecanoate Eicosane 6,8a-Epidoxy-4a-methyl-2-oxo-3,4,4a,5,6,7,8 E,E,Z-1,3,12-Nonadecatriene-5,14-diol 2-(2-vinyloxy-ethoxy)-cyclohexanol Phytol C18 H36 O2 C18 H36 O2 C11 H22 O2 C10 H20 O2 C24 H50 S2 C22 H42 O2 C22 H42 O2 C20 H40 O C20 H40 O C38 H68 O8 C20 H42 C10 H14 O4 C19 H34 O2 C10 H18 O3 C20 H40 O 1.48 3.05 3.04 2.71 1.63 0.85 48.61 10.00 15.54 2.79 3.17 0.87 1.92 1.60 2.76 Table 5. Compounds identified in the methanol extracts of D. villosa leaves by GC–MS spectral analysis. S/N RT Compound Name Molecular Formula Height (%) 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 3.971 4.349 13.240 13.931 14.956 16.131 16.185 16.325 16.430 16.683 16.785 16.972 17.295 17.449 Toluene Tetrachloroethylene Nonanedioic acid, dimethyl ester Ethyl-1-thio-beta-d-glucopyranoside Methyl-21-methyldocosanoate Carda-4,20(22)-dienolide,3-(6-deoxy-3-O-methyl) Carda-4,20(22)-dienolide,3-(6-deoxy-3-O-methyl) Phytol acetate Cyclohexadecanone 3,7,11,15-Tetramethyl-2-hexadecen-1-ol Pseudosmilagenin bis(3,5-dinitrobenzoate) 3,7,11,15-Tetramethyl-2-hexadecen-1-ol Acetamide,N-methyl-N-[4-(3-hydroxypyrrolidinyl)-2-butynyl 1-oxaspiro(2,5)octan-4-one,2,2,6-trimethyl-trans C7 H8 C2 cl4 C11 H20 O4 C8 H16 O5 S C24 H48 O2 C30 H44 O9 C30 H44 O9 C22 H42 O2 C16 H30 O C20 H40 O C41 H48 N4 O13 C20 H40 O C11 H18 N2 O2 C10 H16 O2 2.05 2.33 1.24 0.66 1.12 0.68 0.67 9.80 1.10 4.98 0.52 7.28 0.20 0.65 Horticulturae 2022, 8, 945 8 of 21 Table 5. Cont. S/N RT Compound Name Molecular Formula Height (%) 15. 16. 17.669 17.756 C17 H32 O2 C17 H34 O2 2.60 29.89 17. 17.925 C16 H26 O10 0.26 18. 19. 20. 21. 22. 23. 24. 25. 18.635 18.825 21.820 22.023 22.964 23.450 23.620 23.641 Methylhexadec-9-enoate Pentadecanoic acid,14-methyl-,methyl ester Methyl 2,3,4-tri-O-acetyl-6,7-di-O-methyl-beta-Dglucoheptopyranoside n-Hexadecanoic acid 1,1,1,4,7,7,7-Heptamethyl-4-vinyltrisilethylene 9,12-Octadecadienoic acid, methyl ester 11,14,17-Eicosatrienoic acid, methyl ester Heptadecanoic acid, 16-methyl-, methyl ester Acetamide, N-(4-piperidinylmehyl)3-[3-[1-Aziridinyl]propoxy]-2,5-dimethylpyraz 3-Tridecen-1-yne,(Z)- C16 H32 O2 C13 H32 Si3 C19 H34 O2 C21 H36 O2 C19 H38 O2 C8 H16 N2 O C11 H17 N3 O C13 H22 7.65 0.56 6.48 15.19 1.86 0.32 1.01 1.01 Table 6. Compounds identified in the methanol extracts of D. villosa stem by GC–MS spectral analysis. S/N RT Compound Name 1. 2. 3.965 4.343 3. 13.425 4. 5. 6. 7. 8. 14.339 14.462 15.858 16.325 17.766 9. 17.905 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 18.030 18.608 19.040 19.146 21.825 22.037 22.223 22.957 23.469 23.640 23.710 Toluene Tetrachloroethylene 2,2,6,7-Tetramethyl-10-oxatricyclo [4,3,1,0 (1,6)-decan-5-ol tau-Muurolol A-Cardinol 2-Methyltetracosane Phytol, acetate Hexadecanoic acid, methyl ester Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-methyl ester Borane, diethyl(1-ethyl-2-methyl-1-butenyl)-(Z) n-Hexadecanoic acid 2,7-dithiatricyclo [4,3,1,0(3,8)] decane,10-bromo Ethyl 15-methyl-hexadecanoate Methyl 10-trans, 12-cis-octadecadienoate 6-Octadecenoic acid, methyl ester, (Z)6-Octadecenoic acid, methyl ester, (Z)Heptadecanoic acid, 16-methyl-, methyl ester 1-[3-Aminopropyl]-2[1H]-pyridone Bicyclo [3,3,2] decan-9-one 1H-Inden-1-one,octahydro-7a-methyl-,trans Molecular Formula Height (%) C7 H8 C2 cl4 2.71 3.17 C13 H22 O2 0.34 C15 H26 O C15 H26 O C25 H52 C22 H42 O2 C17 H34 O2 2.41 3.28 1.53 0.99 26.89 C18 H28 O3 3.35 C11 H23 B C16 H32 O2 C8 H11 BrS2 C19 H38 O2 C19 H34 O2 C19 H36 O2 C19 H36 O2 C19 H38 O2 C8 H12 N2 O C10 H16 O C10 H16 O 0.73 25.93 0.40 1.51 10.79 8.20 1.47 2.01 0.91 2.59 0.81 Table 7. Compounds found in the hexane extracts of D. villosa leaves by GC–MS analysis. S/N RT Compound Name Molecular Formula Height (%) 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 3.836 3.885 3.985 4.639 5.425 5.678 5.924 6.061 6.308 6.611 6.650 6.740 3-Hexanone 2-Hexanone Cyclopentanol,1-methylCyclopentanone,3-methyl2-Pentanethiol,2-methylValeric acid,2-ethoxyethyl ester Pentanoic acid,2-propenyl ester 2-Furanmethanol, tetrahydro-5-methylOxalic acid, cyclohexyl ethyl ester 2-Pentene,4,4-dimethyl-,(E)2-Pentene,4,4-dimethyl-,(E)Phosphorus dibromide, cyclohexyl- C6 H12 O C6 H12 O C6 H12 O C6 H12 O C5 H14 S C9 H18 O3 C8 H14 O2 C6 H12 O2 C10 H16 O2 C7 H14 C7 H14 C6 H11 Br2 P 0.86 1.43 1.03 1.42 6.21 10.80 4.33 3.55 22.48 2.55 2.36 0.86 Horticulturae 2022, 8, 945 9 of 21 Table 7. Cont. S/N RT Compound Name Molecular Formula Height (%) 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 10.458 10.659 11.765 11.845 11.948 12.159 12.874 13.022 13.155 13.222 15.473 16.336 16.432 16.690 16.798 16.987 17.554 17.675 17.764 18.204 18.871 19.235 21.093 21.846 22.052 22.579 23.187 24.158 2-Isopropyl-5-methyl-1-heptanol 2-Isopropyl-5-methyl-1-heptanol Octadecanoic acid Tetradecanoic acid Octadecanoic acid 5,9-Undecadien-2-one,6,10-dimethyl-,(E) Phenol,2,4-bis(1,1-dimethylethyl)Hexadecanoic acid, butyl ester Dodecane,4-methyl2(4H)-Benzofuranone,5,6,7,7a-tetrahydro-4,4 Tetradecanoic acid Phytol,acetate 2-Pentadecanone,6,10,14-trimethyl3,7,11,15-Tetramethyl-2-hexadecen-1-ol Pentadecanoic acid 3,7,11,15-Tetramethyl-2-hexadecen-1-ol 5,9,13-Pentadecatrien-2-one,6,10,14-trimethyl Oxirane,hexadecylPentadecanoic acid,14-methyl-,methyl ester Palmitoleic acid n-Hexadecanoic acid 2-Hexadecene,3,7,11,15-tetramethyl-,[R-[R Eicosanoic acid 9,12-Octadecadienoic acid, methyl ester Methyl 8,11,14-heptadecatrienoate Phytol Butyl 9,12-Octadecadienoate cis,cis,cis-7,10,13-Hexadecatrienal C11 H24 O C11 H24 O C18 H36 O2 C14 H28 O2 C18 H36 O2 C13 H22 O2 C14 H22 O C20 H40 O2 C13 H28 C11 H16 O2 C14 H28 O2 C22 H42 O C18 H36 O C20 H40 O C15 H30 O2 C20 H40 O C18 H30 O C18 H36 O C17 H34 O2 C16 H30 O2 C16 H32 O2 C20 H40 C20 H40 O2 C19 H34 O2 C18 H30 O2 C20 H40 O C22 H40 O2 C16 H26 O 0.41 0.26 0.29 0.41 1.00 0.45 1.39 1.87 0.40 0.99 1.85 1.57 4.43 0.27 0.23 0.42 0.41 0.14 1.99 0.45 11.77 0.17 0.26 0.70 1.40 2.52 0.41 5.65 Table 8. Compounds found in the hexane extracts of D. villosa stem by GC–MS spectral analysis. S/N RT Compound Name 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 3.881 3.979 4.636 5.418 5.669 5.916 6.054 6.299 6.603 6.644 6.733 12.679 12.868 13.365 13.737 16. 14.014 17. 14.122 18. 19. 20. 14.156 14.313 14.429 2-Hexanone Cyclopentanol,1-methylCyclopentanone,3-methyl2-Pentanethiol,2-methylValeric acid, 2-ethoxy ethyl ester Pentanoic acid, 2-propenyl ester 2-Furanmethanol, tetrahydro-5-methylOxalic acid, cyclohexyl propyl ester 2-Pentene,4,4-dimethyl-,(Z)2-Pentene,4,4-dimethyl-,(Z)Cyclohexane,nitro Heptadecane,2,6,10,15-tetramethylPhenol,2,4-bis(1,1-dimethyl ethyl)1,6,10-Dodecatrien-3-ol,3,7,11-trimethyl,(E) Carophyllene oxide 12-Oxabicyclo [9,1,0]dodeca-3-7-diene,1,5, 5,8-tetramethyl-[1R,3E,7E,11R] 6-Isopropenyl-4,8a-dimethyl-1,2,3,5,6,7,8, 8a-octahydronaphthalene-2-ol Cubenol α-cadinol α-cadinol Molecular Formula Height (%) C6 H12 O C6 H12 O C6 H10 O C6 H14 S C9 H18 O3 C8 H14 O2 C6 H12 O2 C11 H18 O4 C7 H14 C7 H14 C6 H11 NO2 C21 H44 C14 H22 O C15 H26 O C15 H24 O 1.08 0.69 1.07 5.96 10.71 3.88 3.59 21.93 2.26 2.04 0.77 0.54 1.30 0.68 1.96 C15 H24 O 0.69 C15 H24 O 0.50 C15 H26 O C15 H26 O C15 H26 O 0.39 2.44 3.86 Horticulturae 2022, 8, 945 10 of 21 Table 8. Cont. S/N RT Compound Name 21. 14.684 22. 14.871 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 15.450 16.327 16.419 16.545 16.683 16.780 16.979 17.753 18.921 19.155 19.315 21.079 21.828 22.056 22.600 22.971 23.840 24.125 Octadecane,1-chloro 6-Isopropenyl-4,8a-dimethyl-1,2,3,5,6,7,8, 8a-octahydronaphthalene-2-ol Tetradecanoic acid Phytol, acetate 2-Pentadecanone,6,1014-trimethylCyclopentadecanone,2-hydroxy 3,7,11,15-Tetramethyl-2-hexadecen-1-ol Pentadecanoic acid 3,7,11,15-Tetramethyl-2-hexadecen-1-ol Pentadecanoic acid, 14-methyl-,methyl ester n-Hexadecanoic acid, ethyl ester Hexadecanoic acid, ethyl ester 2-Bromotetradecane Heptadecanoic acid 9,12-Octadecadienoic acid, methyl ester 8-Octadecenoic acid, methyl ester Phytol Methylstearate 1,E-11,Z-13-Octadecatriene 6-Octadecenoic acid,(Z)- Molecular Formula Height (%) C18 H37 cl 0.62 C15 H24 O 0.38 C14 H28 O2 C22 H42 O2 C18 H36 O C15 H28 O2 C20 H40 O C15 H30 O2 C20 H40 O C17 H34 O2 C16 H32 O2 C18 H36 O2 C14 H29 Br C17 H34 O2 C19 H34 O2 C19 H36 O2 C20 H40 O C19 H38 O2 C18 H32 C18 H34 O2 1.32 1.73 2.36 0.31 0.36 0.53 0.55 2.41 13.98 0.32 0.20 0.44 0.65 0.72 0.91 0.17 2.69 3.04 3.3. Antimicrobial Effects The zone of inhibition of D. villosa leaf and stem-bark extracts at 10 mg·mL−1 was significantly higher compared to the control (p < 0.05), except for the activity of the methanol stem-bark extract against E. coli, where the zone of inhibition at 10 mg·mL−1 was lower compared to the control (Table 9). At a concentration of 5 mg·mL−1 , the zones of inhibition of the methanol leaf extract as observed against S. aureus, methicillin-resistant S. aureus, and K. pneumoniae were found to be higher compared to the control (streptomycin and gentamicin) (Table 9). The zone of inhibition of the methanol stem-bark extract at 5 mg·mL−1 as observed against S. aureus was significantly higher compared to the control (p < 0.05). There was an effect of the chloroform extract of D. villosa leaves on S. aureus. The zone of inhibition of the chloroform leaf extract against K. pneumoniae was found to be higher compared to the control (gentamicin) at a concentration of 10 mg·mL−1 (Table 9). At all concentrations, the zone of inhibition of the hexane leaf extract against K. pneumoniae was lower compared to the control (gentamicin). Meanwhile, the zone of inhibition against K. pneumoniae at a concentration of 10 mg·mL−1 was higher compared to the control (Table 9). The zones of inhibition at all other doses were lower compared to the control. Following the serial dilution of 0.625 mg·mL−1 in the lowest concentration of the extract, all the bacterial strains were observed to be resistant to the activities of the methanolic leaf extract of D. villosa at a concentration of 0.01 mg·mL−1 (Table 10). In addition, the same trend was observed with the methanol stem-bark extract of the plant at 0.01 mg·mL−1 . It was also observed that the bacterial strains were sparred as there was no reactivity at a concentration of 0.01 mg·mL−1 . Horticulturae 2022, 8, 945 11 of 21 Table 9. Zone of inhibition (mm) of the graded doses of D. villosa leaf and stem-bark extracts against bacteria strains. Meth. Lv. Extr. Conc.(mg·mL−1 ) Meth. Stem Extr. Conc (mg·mL−1 ) Bacteria Strain 10 5 2.5 1.25 0.625 Control 10 5 2.5 1.25 0.625 Control E. coli P. aeruginosa S. aureus MRSA K. pneumonia 18.3 ± 0.67 18.0 ± 0.58 20.3 ± 0.88 17 ± 2 23.50 ± 0.50 19 ± 0 13.0 ± 0.58 15.3 ± 0.33 15.5 ± 1.50 21.5 ± 0.50 19.5 ± 0.5 12.0 ± 1.53 10 ± 1.15 12.67 ± 0.67 16.67 ± 3.33 19.67 ± 1.28 13.33 ± 0.88 12.33 ± 0.67 11.67 ± 0.88 13.67 ± 1.20 0.90 ± 0.10 0.90 ± 0 0.83 ± 0.10 0.2 ± 0 10.67 ± 5.46 21 ± 3.08 11.67 ± 0.77 15 ± 1.15 17.67 ± 1.45 19.3 ± 2.91 16.3 ± 0.88 14.3 ± 0.67 14.0 ± 0.58 16 ± 0 20.33 ± 1.33 15 ± 0.88 11.0 ± 0.58 11.3 ± 0.88 15.33 ± 0.88 17.33 ± 1.86 13.3 ± 1.05 0.9 ± 0 12.50 ± 0.50 13.33 ± 0.33 17.0 ± 2.08 12.3 ± 1.05 10.67 ± 0.33 10.33 ± 0.33 11.33 ± 0.67 11.67 ± 0.88 0.8 ± 0 0.9 ± 0.1 0.90 ± 0 0.63 ± 0.09 0.73 ± 0.12 23.3 ± 0.77 10.33 ± 0.29 5.45 ± 4.55 19 ± 1.15 21.67 ± 1.20 K. pneumonia Chl. Lv. Extr. Conc (mg·mL−1 ) 10 5 24.50 ± 0.5 22 ± 1.0 2.5 21.50 ± 1.50 1.25 20 ± 1.0 0.625 15.0 ± 2.0 Control 20 ± 0 Chl. Lv. Extr. Conc (mg·mL−1 ) 10 5 20.33 ± 1.33 20 ± 0 2.5 18.0 ± 0.58 1.25 16.33 ± 1.33 0.625 14.33 ± 2.33 Control 19 ± 0.88 K. pneumonia Hex. Lv. Extr. Conc (mg·mL−1 ) 10 5 17.50 ± 0.50 15 ± 2.0 2.5 14 ± 0 1.25 13 ± 1.0 0.625 0.85 ± 0.05 Control 19.5 ± 0.50 Hex. Stem-bark Extr. Conc (mg·mL−1 ) 10 5 2.5 24.0 ± 1.0 21.0 ± 2.0 20.5 ± 3.50 1.25 15.50 ± 3.50 0.625 13.50 ± 3.50 Control 21.50 ± 1.50 E. coli = Escherichia coli; P. aeruginosa = Pseudomonas aeruginosa; S. aureus = Staphylococcus aureus; MRSA = methicillin-resistant Staphylococcus aureus; K. pneumonia = Klebsiella pneumonia; Chl.= Chloroform; Hex = Hexane, Meth = Methanol; Lv = leaves; Extr. = Extract; Conc. = Concentration. Horticulturae 2022, 8, 945 12 of 21 Table 10. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of D. villosa leaf and stem-bark extracts against different bacterial strains. Meth. Lv. Extr. Conc. Bacteria strain K. pneumonia P. aeruginosa S. aureus MRSA E. coli MIC 0.01 0.01 0.01 0.01 0.01 (mg·mL−1 ) MBC 0.01 0.01 0.01 0.01 0.01 Meth. Stem Extr. Conc. MIC 0.01 0.01 0.01 0.01 0.01 (mg·mL−1 ) MBC 0.01 0.01 0.01 0.01 0.01 Chl. Lv. Extr. Conc. MIC 0.01 (mg·mL−1 ) MBC 0.01 Chl. Stem Extr. Conc. (mg·mL−1 ) MIC 0.01 MBC 0.01 P. aeruginosa = Pseudomonas aeruginosa; S. aureus = Staphylococcus aureus; MRSA = methicillin-resistant Staphylococcus aureus; K. pneumonia = Klebsiella pneumonia; Chl. = Chloroform; Hex = Hexane, Meth. = Methanol; Lv. = leaves; Extr. = Extract; Conc. = Concentration. 3.4. DPPH Radical Scavenging Activity The free radical scavenging activity and IC50 values of the leaf and stem-bark extracts are summarized in Figures 1 and 2, respectively. The simultaneous occurrence of a greater percentage of radical scavenging activity and lower IC50 values indicated excellent antioxidant activity. Out of all the different leaf extracts, the methanol leaf extract showed higher scavenging activity (85.29%) compared to the other extracts, whereas the ascorbic acid at the same concentration showed 92.82%, which are somewhat close to each other (Figure 1a). In addition, the methanol leaf extract showed excellent DPPH radical scavenging activity with an IC50 value of 9.53 µg·mL−1 compared to that of the ascorbic acid (10.3 µg·mL−1 ) (Figure 1b). In addition, both the chloroform and hexane leaf extracts showed weak antioxidant – behavior with higher IC50 values of 10.7 µg·mL−1 and 11.8 µg·mL−1 , respectively, compared to that of the ascorbic acid. The methanol and hexane stem-bark extracts also showed high scavenging activity at 85.76% and 87.22%, respectively, but these were not as high as the ascorbic acid (92.82%) (Figure 2a). The IC50 of both the methanol (9.53 µg·mL−1 ) and hexane (9.53 µg·mL−1 ) stem-bark extracts were lower compared to the ascorbic acid −1 ) of −1 −1 value 9.53 μg·mL ascorbic acid (10.3 μg·mL (10.0 µg·mL (Figure 2b). Meanwhile, the chloroform stem-bark extract indicated a higher IC50 compared to the ascorbic acid. Figure 1. Comparison of DPPH radical scavenging (a) IC50 values of D. villosa (%) of D. villosa leaf extracts in DPPH scavenging assay (b). Chl = Chloroform; Hex = Hexane, Meth = Methanol; Lv. = leaves; Extr. = Extract. Horticulturae 2022, 8, 945 13 of 21 Figure 2. Comparison of DPPH radical (a) IC50 values of D. villosa stem scavenging (%) of D. villosa stem-bark extracts in DPPH radical scavenging assay (b). Chl. = Chloroform; Hex = Hexane, Meth. = Methanol; Lv. = leaves; Extr. = Extract; Asc. Acid = Ascorbic acid. 3.5. Ferric-Reducing Antioxidant Potential The ferric-reducing potential and IC50 values of the leaf and stem-bark extracts of D. villosa are summarized in Figures 3 and 4, respectively. Among the different leaf extracts, the hexanic leaf extract showed high scavenging activity (85.2%), whereas the ascorbic acid indicated 79.3% at the same concentration (Figure 3a). The chloroform and methanol leaf extracts showed a higher reducing power compared to the ascorbic acid. Similarly, the methanol leaf extract showed ferric-reducing power with an IC50 value of 112 µg·mL−1 compared to that of the ascorbic acid (143 µg·mL−1 ) (Figure 3b). On the other hand, both the chloroform and hexane leaf extracts showed excellent antioxidant behavior with IC50 values of 11.0 µg·mL−1 and 13.3 µg·mL−1 , respectively, compared to that of the ascorbic acid (Figure 3b). The methanol and hexane stem-bark extracts also showed high scavenging activity at 85.8% and 87.22%, respectively, but these were not as high as the ascorbic acid (92.8%) (Figure 4a). The IC50 of both methanol and chloroform stem extracts was lower compared to that of the ascorbic acid (141.0 µg·mL−1 ) (Figure 4b). Similarly, the hexane stem-bark extract further indicated a higher IC50 compared to the ascorbic acid. Horticulturae 2022, 8, 945 14 of 21 Figure 3. Comparison of ferric-reducing power (a) IC50 values for D. villosa extracts of D. villosa leaf extract in ferric-reducing antioxidant potential (b). Chl. = Chloroform; Hex = Hexane, Meth. = Methanol; Lv. = leaves; Extr. = Extract; Asc. Acid= Ascorbic acid. Figure 4. Comparison of ferric-reducing power in (%) of D. villosa stem-bark extract (a), IC50 values for D. villosa stem ferric-reducing antioxidant potential (b). Chl. = Chloroform; Hex = Hexane, Meth. = Methanol; Extr. = Extract; Asc. Acid = Ascorbic acid. 3.6. Total Phenolic Content The TPC in the leaves and stem bark of D. villosa was estimated and analyzed. Oneway analysis of variance showed that there was a significant difference in the total phenol contents in D. villosa leaves and stem bark F(2, 6) = 225.8, p ≤ 0.001. The highest TPC in D. villosa leaves was found in the methanol extract (28.45 ± 0.50) mg gallic acid equivalent per gram of dry weight. Meanwhile, the highest TPC in the stem bark was found in the hexane extract (14.40 ± 0.58) mg gallic acid equivalent per gram of dry weight (Figure 5). ≤ 0.001. The highest TPC in Horticulturae 2022, 8, 945 15 of 21 Figure 5. Total phenol content of the solvent extracts of both D. villosa leaves and stem bark. F(2, 6) = 225.8, p ≤ 0.0001. * (methanol vs. chloroform), p < 0.05. * (hexane vs. methanol), ≤ 0.0001. * p < 0.05. Values are expressed as means ± SEM; n = 3/group. Chl. = Chloroform; Hex = Hexane, Meth. = Methanol; Lv. = leaves; Extr. = Extract. 3.7. Fourier Transform Infrared (FT–IR) Spectral Analysis – The FT–IR spectra of the D. villosa leaf and stem-bark extracts using methanol, chloro– form, and hexane as the media for extraction are presented in Figure 6. The spectra analysis revealed the vibrational frequencies of the various functional groups observed to be present in the crude extracts. Absorption peaks with a broad range of 2500–3300 cm−1 were char−1 – 6a,b). The acteristic of the hydroxyl (O-H) group, particularly from carboxylic acid (Figure − 1 peaks around 1650–1750 cm were assigned to the carbonyl (C=O) group. The stretching −1 peaks at 1599 and 1605 cm−–1 occurred due to the presence of (C≡N) nitrile. The C-H −1 of (C≡N) nitrile. (hydrocarbon) stretches appeared at 2927 cm−1 (Figure 6c,d). Thecepeak at 2849 cm−1The was −1 meant to further characterize the (C-H) stretching vibration. The strong peaks absorbed−1at 1735 and 1617 cm−1 (Figure 6c,d) were the (C=O) stretches in the aldehydes and ketones. The peaks at 3369 and 3309−1 cm−1 (Figure 6c,d) occurred due to the presence of the (O-H) −1 functional group. In addition, the strong peaks at 2916 and 2848 cm−1 , as well as at 2917 − 1 −1 and 2849 cm (Figure 6e,f), were due to the presence of the (C-H) functional group. −1 Horticulturae 2022, 8, 945 16 of 21 – spectra of crude plant extracts of D. villosa (a) crude leaf methanol extract; (b) crude Figure 6. FT–IR stem-bark methanol extract; (c) crude leaf chloroform extract; (d) crude stem-bark chloroform extract; (e) crude leaf hexane extract; (f) crude stem-bark hexane extract at room temperature (24 ◦ C). 4. Discussion – Several studies have reported that herbal antioxidants act against free radicals [30–32]. The presence of secondary metabolites such as terpenoids, alkaloids, flavonoids, and phenolic compounds have been implicated as antioxidant factors in different plant materials [33]. It is interesting to note that the methanol extract of D. villosa (both leaves and stem bark) showed the presence of flavonoids and alkaloids. Following the qualitative phytochemical analysis, the methanol leaf extract presented with a color intensity to indicate the presence of terpenoids, flavonoids, and even phenolic compounds. It is without a doubt that the Horticulturae 2022, 8, 945 17 of 21 confirmed compounds (terpenoids, flavonoids, and phenols) could provide the justifiable underlying factors for the antioxidant activity of D. villosa. This confirms the findings of Echeverría et al. [34] that showed that the antioxidant activity of natural flavonoids of Chilean flora (Flora of Chile) is a result of the embedded hydroxyl group for the oxygenationsubstitution pattern. The presence of a hydroxyl group in the D. villosa leaves and stem bark supports hydroxylation as the plant’s mechanism for exhibiting its antioxidant function and thereby conferring stability to free radicals. In addition, this study revealed the presence of alkaloids in the methanol leaf and stem-bark extracts only. Many alkaloids are potent antioxidants and are used for the treatment and/or management of skin cancer [35]. The detection of alkaloids in the plant extracts is consistent with Dangoggo et al. [36], who revealed that the aqueous extract of Diospyros mespiliformis (ebony diospiros) leaves was quite rich in alkaloids and may further be responsible for the effective inhibitory effect on DPPH, thereby helping in the detoxification of the generated radical oxygen species. The GC–MS analysis indicted the existence of a number of phytocompounds such as n-hexadecanoic acid; phytol; palmitoleic acid; oxalic acid; 3,7,11,15-Tetramethyl-2hexadecen-1-ol; acetate; alpha-cadinol; tau-Muurolol; eisosane; and vitamin E. The major compounds found in the methanol leaf extracts were 11, 14, 17-eicosatrienoic acid, methyl ester (15.19%), pentadecanoic acid, and methyl ester (29.89%), whereas in the methanol stem extracts, n-hexadecanoic acid (25.93%), hexadecanoic methyl ester (25.93%), and methyl 10-trans, 12-cis octadecadienoate (10.79%) were present. In the chloroform leaf extract, the major compounds were n-hexadecanoic acid (20.12%) and cis, cis, cis-7, 10, 13-Hexadecatrienal (14.36%), whereas phytol acetate (48.61%) and 3,7,11,15-tetramethyl-2hexadecen-1-ol (15.54%) were found in the chloroform stem extract of D. villosa. Similarly, oxalic acid, cyclohexyl ethylester (22.48%), and n-hexadecanoic acid (11.77%) were found in the hexane leaf extract, whereas in the hexane stem extract, oxalic acid, cyclohexylpropyl ester (21.93%), n-hexadecanoic acid, and ethyl ester (13.98%) were found in high proportions. The high concentrations of valeric acid in the hexane extracts of the leaves and stem bark may be associated with the antioxidant activity of the extracts. These results support the findings of Vishwakarma et al. [37], where valeric acid isolated from Valeriana wallichii was scientifically proven to have anti-inflammatory properties by reducing lipid peroxidation and restoring glutathione levels in intracerebrovascular streptozotocin-induced neurodegeneration, and further suggests that it could be used in the management of inflammatory diseases. Phytol is not just a diterpene compound but can also act as an anti-inflammatory, anticancer, antimicrobial, and diuretic. Phytol acetate as found in the D. villosa extracts was revealed to be in high concentrations and could be used as a novel class of pharmaceuticals as a therapeutic measure for rheumatoid arthritis and especially for chronic inflammatory diseases. This is further corroborated by Ogunlesi et al. [38], where phytol increased oxidative burst in vivo and further corrected the effect of the genetic polymorphism in the translational model of arthritis. Phytol may further be considered a novel class of drugs for treating chronic inflammatory diseases. In fact, phytol as an acyclic diterpene alcohol could be considered a precursor for the industrial synthesis of vitamin E [38]. Among the found compounds, n-hexadecanoic acid, hexadecanoic acid, and palmitoleic acid have antioxidant, hypocholesterolemic, nematicide, pesticide, and lubricating properties [39]. In addition, n-hexadecanoic acid and ethyl ester have antitumor, antifungal, and antibacterial properties. Hexadecanoic acid as found in D. villosa possesses antioxidant and haemolytic properties and is also an effective pesticide. Similarly, Rathee et al. [40] reported that the methanolic extract of Mentha longifolia showed remarkable antioxidant activity via the reactive oxygen species scavenging efficacy and lipid peroxidation inhibition. In this study, the methanol extracts of D. villosa (both leaves and stem bark) showed notable antioxidant activity in comparison with the reference drug (ascorbic acid). This activity could further be associated with the presence of alkaloids and flavonoids as well as the phenolic content. The observed lower IC50 values of these extracts support the relevance of D. villosa leaves and stem bark as a potential organic source of antioxidants and hence they can be used for the prevention of free-radical-mediated Horticulturae 2022, 8, 945 18 of 21 diseases. Furthermore, the results of the DPPH radical scavenging ability showed that the methanol leaf and stem-bark extracts can prevent radical-induced oxidative damage. This is reflected in the phenol content in the methanol leaf and stem-bark extracts. Accordingly, Saeed et al. [41] established a correlation between the health benefits of polyphenolic-rich plants and their antioxidant properties, and the possible mechanism responsible for the phenolic activity could be the redox properties of its hydroxyl group. The functional groups in any bio-organic compound influence the biological activities of the compound. The influence is a result of the contribution of the embedded functional groups to the inherent properties of the compounds such as solubility, stereochemistry, partition coefficient, acid–base properties, etc. All these proficiencies are believed to induce the metabolic extraction, absorption, distribution, and toxicity of bioactive molecules [42]. Therefore, the analysis of the functional group showed that it performs a dynamic function in identifying the physicochemical properties of the extracts. The detection of the functional groups, therefore, helped to assess the structure–function relationship of the bio-organic compound. In this study, the FT–IR spectral analysis of the leaf and stem-bark extracts of D. villosa showed the presence of phytochemicals carrying a hydrogen-bonded OH functional group. The functionality of most phenolic compounds such as tannins and flavonoids owes to the presence of a hydroxyl functional group [43]. Although the mechanism of antibacterial activity could not be ascertained in this study, it was however noted that higher zones of inhibition were produced by the graded doses of the methanol leaf extract compared to those of the stem bark. It is not a mere coincidence that the chloroform and hexane extracts showed antibacterial activity against K. pneumonia only. The presence of alkaloids in the methanol extracts may be identified as an additional key factor for the antibacterial activity of the D. villosa plant. This is supported by Bai et al. [39]’s evidence and confirms the antibacterial activity of the alkaloids as well as the mechanism of action through intercalation with bacterial DNA. There has been motivation and justification for the production of new antimicrobial agents to treat infections [44]. The newest trend shows that plant-based antimicrobial agents have high medicinal efficacy since they pose no hazardous threats to human life [45]. The fact that plant extracts produce zones of inhibition against different bacteria strains indicates their antimicrobial activity and further confirms their use as anti-infection agents. In addition, the production of zones of inhibition against both Gram-negative and Gram-positive bacteria shows their applicability for a wide spectrum of activity. The methanol extract of D. villosa leaves further indicates a higher zone of inhibition against S. aureus, P. aeruginosa, and K. pneumoniae compared to conventional antibiotics of high concentrations. Although the mechanisms of action of D. villosa are yet to be ascertained, there is no doubt that the chemical contents of the plants, such as phenols, flavonoids, and alkaloids, are much more likely to be responsible for antimicrobial activities. This is similar to Linuma et al. [46], where the flavonoid contents of the extract were revealed to be a typical phytochemical responsible for microbial inhibition. In addition, the methanol stem-bark extract of D. villosa further showed a reaction against these strains but it was not as high as the control drug. The observed reduction in the degree of inhibitory activity could be ascribed to the lower concentration of phenolic content in the stem-bark extract of D. villosa. Vaquero et al. [47] indicated that phenolic compounds possess high antibacterial effects. Similarly, Majhenič et al. [48] found that methanol extracts of Paullinia cupana (guarana) seed showed high antibacterial activity due to their high phenolic contents [48]. Hence, it can be said that D. villosa possesses exceptional phytochemicals that account for bacterial metabolism inhibition. 5. Conclusions The present study indicated that the leaf and stem-bark extracts of D. villosa displayed the occurrence of flavonoids, alkaloids, phenols, and even terpenoids. The leaves of the plant further revealed strong antioxidant activity owing to the high concentration of total phenolic content as well as the absorption peaks with a wide range for the hydroxyl group. In addition, the plant showed strong antimicrobial activities against Gram-negative and Horticulturae 2022, 8, 945 19 of 21 Gram-positive bacteria strains with a minimum inhibitory concentration of 0.01 mg·mL−1 . Therefore, this study suggests that the leaf and stem-bark extracts of D. villosa are a notable source of natural antioxidant and antimicrobial agents. It is expected that this study could lead to the design of bio-compounds that could be used to investigate novel and efficacious antioxidants as well as the antimicrobial agents of plant origin. Further research is needed to isolate the active molecules from the crude extract and also to evaluate in detail the in vivo biological activities of these isolated compounds. Author Contributions: Conceptualization and methodology, O.T.A. and Y.N.; investigation, O.T.A., Y.N. and J.L.; formal analysis and data curation, O.T.A., Y.N. and J.L.; writing—original draft preparation, O.T.A., Y.N., J.L. and V.S.; writing—review and editing, T.S.A., V.S., Y.H.D. and A.N.E.-B.; validation and visualization, T.S.A., V.S., Y.H.D. and A.N.E.-B.; Supervision, Y.N. All authors have read and agreed to the published version of the manuscript. Funding: The authors acknowledge the Researchers Supporting Project number (RSP-2021/375), King Saud University, Riyadh, Saudi Arabia. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All data are presented within the article. Acknowledgments: The authors acknowledge the Researchers Supporting Project number (RSP2021/375), King Saud University, Riyadh, Saudi Arabia. The authors are thankful to the TWAS/ National Research Foundation (NRF) for their financial support and the University of Kwa-ZuluNatal, South Africa, for providing the research facilities for this work. Conflicts of Interest: The authors declare no conflict of interest. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Sugamura, K.; Keaney, J.F., Jr. Reactive oxygen species in cardiovascular disease. Free Radic. Biol. Med. 2011, 51, 978–992. [CrossRef] [PubMed] White, B.L.; Howard, L.R.; Prior, R.L. Polyphenolic composition and antioxidant capacity of extruded cranberry pomace. J. Agric. Food Chem. 2009, 58, 4037–4042. [CrossRef] [PubMed] Ali, M.; Cheng, Z.; Ahmad, H.; Hayat, S. Reactive oxygen species (ROS) as defenses against a broad range of plant fungal infections and case study on ROS employed by crops against Verticillium dahliae wilts. J. Plant Interact. 2018, 13, 353–363. [CrossRef] Das, S.K.; Prusty, A.; Samantaray, D.; Hasan, M.; Jena, S.; Patra, J.K.; Samanta, L.; Thatoi, H. Effect of Xylocarpus granatum Bark Extract on amelioration of Hyperglycaemia and oxidative stress associated complications in STZ-induced diabetic mice. Evid.-Based Complement. Altern. Med. 2019, 2019, 8493190. [CrossRef] [PubMed] Pandey, K.B.; Rizvi, S.I. Plant polyphenols as dietary antioxidants in human health and disease. Oxidative Med. Cell. Longev. 2009, 2, 270–278. [CrossRef] [PubMed] Balasundram, N.; Sundram, K.; Samman, S. Phenolic compounds in plants and agri-industrial by-products: Antioxidant activity, occurrence, and potential uses. Food Chem. 2006, 99, 191–203. [CrossRef] Jagani, S.; Chelikani, R.; Kim, D.S. Effects of phenol and natural phenolic compounds on biofilm formation by Pseudomonas aeruginosa. Biofouling 2009, 25, 321–324. [CrossRef] [PubMed] Borras-Linares, I.; Stojanovic, Z.; Quirantes-Pine, R.; Arraez-Roman, D.; Švarc-Gajic, J.; Fernadez-Gutierrez, A.; Segura-Carretero, A. Rosmarinus officinalis leaves as a natural source of bioactive compounds. Int. J. Mol. Sci. 2014, 15, 20585–20606. [CrossRef] [PubMed] Rasheed, K.; Zhang, X.J.; Luo, M.T.; Zheng, Y.T. Anti-HIV-1 activity of phenolic compounds isolated from Diospyros lotus fruits. Phytopharmacology 2012, 3, 199–207. Rauf, A.; Uddin, G.; Patel, S.; Khan, A.; Halim, S.A.; Bawazeer, S.; Ahmad, K.; Muhammad, N.; Mubarak, M.S. Diospyros, an under-utilized, multi-purpose plant genus: A review. Biomed. Pharmacother. 2017, 91, 714–730. [CrossRef] Kumar, S.; Abedin, M.M.; Singh, A.K.; Das, S. Role of Phenolic Compounds in Plant-Defensive Mechanisms. In Plant Phenolics in Sustainable Agriculture; Springer: Berlin/Heidelberg, Germany, 2020; pp. 517–532. Gorniak, I.; Bartoszewski, R.; Kroliczewski, J. Comprehensive review of antimicrobial activities of plant flavonoids. Phytochem. Rev. 2019, 18, 241–272. [CrossRef] Singh, A.K.; Singla, P. Root Phenolics Profile Modulates Microbial Ecology of Rhizosphere. In Plant Phenolics in Sustainable Agriculture; Springer: Singapore, 2020; pp. 555–578. Horticulturae 2022, 8, 945 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 20 of 21 Cirera, J.; Da Silva, G.; Serrano, R.; Gomes, E.; Duarte, A.; Silva, O. Antimicrobial activity of Diospyros villosa root. Planta Med. 2010, 76, P454. [CrossRef] Brando, R.; Miliwebsky, E.; Bentancor, L.; Deza, N.; Baschkier, A.; Ramos, M.; Fernandez, G.; Meiss, R.; Rivas, M.; Palermo, M. Renal damage and death in weaned mice after oral infection with Shiga toxin 2-producing Escherichia coli strains. Clin. Exp. Immunol. 2008, 153, 297–306. [CrossRef] Kitada, K.; De Toledo, A.; Oho, T. Increase in detectable opportunistic bacteria in the oral cavity of orthodontic patients. Int. J. Dent. Hyg. 2009, 7, 121–125. [CrossRef] [PubMed] Flynn, T.R. Evidence-Based Principles of Antibiotic Therapy. In Evidence-Based Oral Surgery; Springer International Publishing: Cham, Switzerland, 2019. [CrossRef] Vila, T.; Sultan, A.S.; Montelongo-Jauregui, D.; Jabra-Rizk, M.A. Oral candidiasis: A disease of opportunity. J. Fungi 2020, 6, 15. [CrossRef] [PubMed] Vellappally, S.; Divakar, D.D.; Al Kheraif, A.A.; Ramakrishnaiah, R.; Alqahtani, A.; Dalati, M.; Anil, S.; Khan, A.A.; Harikrishna Varma, P. Occurrence of vancomycin-resistant Staphylococcus aureus in the oral cavity of patients with dental caries. Acta Microbiol. Immunol. Hung. 2017, 64, 343–351. [CrossRef] Harborne, J.B. Phenolic compounds. In Phytochemical Methods; Springer International Publishing: Cham, Switzerland, 1973; pp. 33–88. Treare, G.; Evans, W. Pharmacognosy, 17th ed.; Bahive Tinal: London, UK, 1985; p. 149. Sofowora, A. Recent trends in research into African medicinal plants. J. Ethnopharmacol. 1993, 38, 197–208. [CrossRef] Evans, W. Trease and Evans’ Pharmacognosy, Hartcourt Brace and Company; Asia Pvt. Ltd.: Singapore, 1997; pp. 226–227. Adams, R.P. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry; Allured Publishing Corporation: Carol Stream, IL, USA, 2007. Barros, L.; Falcao, S.; Baptista, P.; Freire, C.; Vilas-Boas, M.; Ferreira, I.C. Antioxidant activity of Agaricus sp. mushrooms by chemical, biochemical and electrochemical assays. Food Chem. 2008, 111, 61–66. [CrossRef] Juntachote, T.; Berghofer, E. Antioxidative properties and stability of ethanolic extracts of Holy basil and Galangal. Food Chem. 2005, 92, 193–202. [CrossRef] Shao, Y.; Xu, F.; Sun, X.; Bao, J.; Beta, T. Identification and quantification of phenolic acids and anthocyanins as antioxidants in bran, embryo and endosperm of white, red and black rice kernels (Oryza sativa L.). J. Cereal Sci. 2014, 59, 211–218. [CrossRef] Akinpelu, D.; Onakoya, T. Antimicrobial activities of medicinal plants used in folklore remedies in south-western. Afr. J. Biotechnol. 2006, 5, 1078–1081. Ferrazzano, G.F.; Scioscia, E.; Sateriale, D.; Pastore, G.; Colicchio, R.; Pagliuca, C.; Cantile, T.; Alcidi, B.; Coda, M.; Ingenito, A. In vitro antibacterial activity of pomegranate juice and peel extracts on cariogenic bacteria. Biomed. Res. Int. 2017, 2017, 2152749. [CrossRef] Wangia, C.O.; Orwa, J.A.; Muregi, F.W.; Kareru, P.G.; Cheruiyot, K.; Kibet, J. Comparative anti-oxidant activity of aqueous and organic extracts from Kenyan Ruellia lineari-bracteolata and Ruellia bignoniiflora. Eur. J. Med. Plants 2016, 17, 1–7. [CrossRef] Dintcheva, N.T.; Arrigo, R.; Baiamonte, M.; Rizzarelli, P.; Curcurto, G. Concentration-dependent anti-/pro-oxidant activity of natural phenolic compounds in bio-polyesters. Polym. Degrad. Stab. 2017, 142, 21–28. [CrossRef] Tatipamula, V.B.; Killari, K.N.; Ketha, A.; Sastry, V.G. Taxithelium napalense acts against free radicals and diabetes mellitus. Bangladesh J. Pharmacol. 2017, 12, 197–203. [CrossRef] Muchirah, P.N.; Waihenya, R.; Muya, S.; Abubakar, L.; Ozwara, H.; Makhokha, A. Characterization and anti-oxidant activity of Cucurbita maxima Duchesne pulp and seed extracts. J. Phytopharm. 2018, 7, 134–140. [CrossRef] Echeverria, J.; Opazo, J.; Mendoza, L.; Urzua, A.; Wilkens, M. Structure-activity and lipophilicity relationships of selected antibacterial natural flavones and flavanones of Chilean flora. Molecules 2017, 22, 608. [CrossRef] [PubMed] Ahsan, H.; Reagan-Shaw, S.; Eggert, D.M.; Tan, T.C.; Afaq, F.; Mukhtar, H.; Ahmad, N. Protective effect of sanguinarine on ultraviolet B-mediated damages in SKH-1 hairless mouse skin: Implications for prevention of skin cancer. Photochem. Photobiol. 2007, 83, 986–993. [CrossRef] [PubMed] Dangoggo, S.; Hassan, L.; Sadiq, I.; Manga, S. Phytochemical analysis and antibacterial screening of leaves of Diospyros mespiliformis and Ziziphus spina-christi. J. Chem. Eng. 2012, 1, 31–37. Vishwakarma, S.; Goyal, R.; Gupta, V.; Dhar, K.L. GABAergic effect of valeric acid from Valeriana wallichii in amelioration of ICV STZ induced dementia in rats. Rev. Bras. Farmacogn. 2016, 26, 484–489. [CrossRef] Ogunlesi, M.; Okiei, W.; Ofor, E.; Osibote, A.E. Analysis of the essential oil from the dried leaves of Euphorbia hirta Linn (Euphorbiaceae), a potential medication for asthma. Afr. J. Biotechnol. 2009, 8, 7042–7050. Bai, L.P.; Zhao, Z.Z.; Cai, Z.; Jiang, Z.H. DNA-binding affinities and sequence selectivity of quaternary benzophenanthridine alkaloids sanguinarine, chelerythrine, and nitidine. Bioorg. Med. Chem. 2006, 14, 5439–5445. [CrossRef] Rathee, J.S.; Hassarajani, S.A.; Chattopadhyay, S. Antioxidant activity of Mammea longifolia bud extracts. Food Chem. 2006, 99, 436–443. [CrossRef] Saeed, N.; Khan, M.R.; Shabbir, M. Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L. BMC Complement. Altern. Med. 2012, 12, 221. [CrossRef] Horticulturae 2022, 8, 945 42. 43. 44. 45. 46. 47. 48. 21 of 21 Olivares-Vicente, M.; Barrajon-Catalan, E.; Herranz-Lopez, M.; Segura-Carretero, A.; Joven, J.; Encinar, J.A.; Micol, V. Plantderived polyphenols in human health: Biological activity, metabolites and putative molecular targets. Curr. Drug Metab. 2018, 19, 351–369. [CrossRef] Diaz, P.; Jeong, S.C.; Lee, S.; Khoo, C.; Koyyalamudi, S.R. Antioxidant and anti-inflammatory activities of selected medicinal plants and fungi containing phenolic and flavonoid compounds. Chin. Med. 2012, 7, 26. [CrossRef] Fair, R.J.; Tor, Y. Antibiotics and bacterial resistance in the 21st century. Perspect. Med. Chem. 2014, 6, 25–64. [CrossRef] Anand, U.; Jacobo-Herrera, N.; Altemimi, A.; Lakhssassi, N. A comprehensive review on medicinal plants as antimicrobial therapeutics: Potential avenues of biocompatible drug discovery. Metabolites 2019, 9, 258. [CrossRef] Iinuma, M.; Tsuchiya, H.; Sato, M.; Yokoyama, J.; Ohyama, M.; Ohkawa, Y.; Tanaka, T.; Fujiwara, S.; Fujii, T. Flavanones with potent antibacterial activity against methicillin-resistant Staphylococcus aureus. J. Pharm. Pharmacol. 1994, 46, 892–895. [CrossRef] Vaquero, M.R.; Alberto, M.R.; De Nadra, M.M. Antibacterial effect of phenolic compounds from different wines. Food Control 2007, 18, 93–101. [CrossRef] Majhenic, L.; Škerget, M.; Knez, Ž. Antioxidant and antimicrobial activity of guarana seed extracts. Food Chem. 2007, 104, 1258–1268. [CrossRef]