Evaluation of Saponin Extract from Vitex doniana and Pentaclethra macrophylla for Antibacterial Activity

Saponins are pharmacologically active compounds that have been shown to ameliorate abnormal physiological processes and be aptly applied in folklore for the treatment of maladies occasioned by infectious agents. Consequently, saponins from Vitex doniana and Pentaclethra macrophylla were evaluated for antibacterial properties, as these herbs are used in folk medicine. Dried pulverized plant materials were defatted, and solvents with varying polarity were applied at varying ratios for the extraction of saponins. Phyto-chemistry was in accordance with standard methods, while an antibacterial assay was made through the agar well diffusion and micro broth dilution techniques. Phytochemical quantitation showed high concentrations of tannins, 231  ̆ 0.6 CE/g, and saponins, 58% from V. doniana. Similarly, P. macrophylla stem bark extract also showed high concentrations of tannins, 309  ̆ 2.42 CE/g, alkaloids, 71%  ̆ 0.5%, and saponins, 87%  ̆ 3.4%. The ethanol extracts of V. doniana inhibited the growth of Staphylococcus aureus (ATCC 11775) and a clinical strain with inhibition zone ranges of 15.5  ̆ 2.12 to 7.0  ̆ 0.0 (mm) against leaf extracts and 20.0  ̆ 1.41 to 7.0  ̆ 0.0 (mm) against stem bark extracts. Conversely, saponin extract from V. doniana showed a broad spectrum of activity, as it inhibited both Gram-negative and -positive test strains, E. coli clinical strain (20.0  ̆ 1.41 mm), P. aeruginosa clinical strain (18.5  ̆ 0.71 mm), E. coli ATCC 11775 (17.0  ̆ 0 mm), and S. aureus clinical strain (13.0  ̆ 1.41 mm). However, a broad spectrum was similarly achieved with P. macrophylla extracts, as all test bacteria genus was susceptible. Saponin fractions showed a high potency and broad spectrum antibacterial activity and thus a validation of the folklore applications and the potential for use as a drug or drug scaffold.


Introduction
The therapeutic properties of ethnobotanicals are adduced to the functional roles of their pharmacologically active phyto-constituents [1,2].Consequently, ethnobotanicals have been reported to possess ameliorative potentials towards some physiological anomalies including hyper/hypo-tension, atherosclerosis, hypo/hyper-glycaemia, erectile dysfunction, antiphlogistic, immunomodulatory effects, anti-allergic activities, anti-neoplastic properties, and the corrective effects/management of several metabolic syndromes in humans and livestock [3][4][5].In the same vein, functional properties of ethnobotanicals unconnected with the amelioration of abnormal physiological processes in humans and livestock include fungicidal, antibacterial, and antiviral activities [2,6].The plethora of desirable functions displayed by ethnobotanicals, as elicited by the myriad phyto-constituents, has been chronicled in several studies.
Vitex doniana and Pentaclethra macrophylla are folklore herbs common with herbal healers of Southeastern Nigeria; tribal communities in this geographical region use these herbs in folk medicine for the treatment of various ailments [7][8][9][10].V. doniana and P. macrophylla are respectively known as black plum and African oil bean commonly.Further, V. doniana and P. macrophylla belong to the Verbenaceae and Leguminosae families, respectively [11,12].These plants are widely distributed in the Eastern and Western parts of Nigeria [13][14][15].Decoctions of V. doniana are administered orally in the treatment of gastrenteritis, diarrhea, and dysentry [8,16,17].Similarly, the roots and leaves decoctions of P. macrophylla are used as a laxative and in the treatment of dysentery, while the ripe fruits and stem bark powder are applied externally to heal wounds and burns [10,18].Additionally, P. macrophylla have been used as analgesics, anthelmintics, and for the treatment of gonorrhea, diarrhea, convulsion, and inflammatory diseases [7,12,18,19].
Saponins constitute a major phytochemical grouping, and over 100 vascular plant species have been documented to possess various kinds of saponin [20].Saponins are structurally diverse and consist of polycyclic aglycones attached to one or more sugar side chains [21].Traditionally, they are subdivided into triterpenoid and steroid glycosides [21][22][23].Saponins exert a wide range of pharmacological activities including antibacterial, antifungal, antiviral, anti-parasitic, anti-inflammatory, hypocholesterolemic, and hypoglycemic [20,22,24].In cognizance of the folk practices of cold maceration in water or admixtures of locally brewed alcohol of V. doniana and P. macrophylla before administration, the reported work aimed at extracting saponins from V. doniana and P. macrophylla and respectively evaluating their antibacterial activity.

Plant Materials
The leaves and stem bark of V. doniana and P. macrophylla were collected from the woodlands of Obollo-afor (6.92 ˝N, 7.52 ˝E) and Orba (6.51 ˝N, 7.27 ˝E), respectively, in Enugu North Senatorial Zone, Enugu State, Nigeria.The plants were identified taxonomically by Mr JO Ozioko as Vitex doniana and Pentaclethra macrophylla, and voucher specimens (ViD/a03 and PeM/05) were deposited at the herbarium of the Department of Botany, University of Nigeria, Nsukka.The plant materials were collected in line with the ethical guide of the University of Nigeria, Nsukka, on herbal research.

Maceration and Extraction of Plant Materials
Fresh leaves and fleshy stem bark of V. doniana and P. macrophylla were harvested, thoroughly rinsed with distilled water, and air-dried at room temperature (28 ˘2 ˝C).Dried plant portions were mechanically pulverized using a hammer mill (Henan Always Machinery, Zhengzhou, China).Ethanol extraction was carried out in accordance with the method described by [3].About 50 g of the pulverized leaves and stem bark was macerated in 200 mL of absolute ethanol (BDH Chemicals, Birmingham, England) for 4 h.Each portion was filtered using Whatman No. 1 filter paper (Sigma-Aldrich, Johannesburg, South Africa) and filtrates concentrated to dryness at steady air current.Extracts were stored in sterile containers until further use.

Phytochemical Assay
Ethanol extracts of V. doniana and P. macrophylla leaves and stem bark were screened for the presence of saponins, alkaloids, tannins, anthraquinones, glycosides, flavonoids, and sterols following the methods of Trease and Evans [25] and Harbone [26].However, quantitation of these phytochemicals was in accordance with standard protocols [27,28].

Saponin Extraction
Extraction of saponin from the stem bark of V. doniana and P. macrophylla was carried out in accordance with the method Ajibade and Famurewa [29] with some modifications.Briefly, 100 g of the pulverized stem bark was defatted with 300 mL of petroleum ether (BDH Chemicals, Birmingham, England).The resulting marc was air-dried and depigmented in 300 mL of chloroform (BDH Chemicals, Birmingham, England).Afterwards, 300 mL of methanol (BDH Chemicals, Birmingham, England) was subsequently used to extract saponin form the defatted samples.The mixture was allowed to stand overnight, and the resulting aqueous residue was filtered and the filtrate evaporated to dryness.The methanol extract was further fractionated with 200 mL of distilled water-butanol (1:1 v/v) to get butanol extracts, which were precipitated with 50 mL of acetone (BDH Chemicals, Birmingham, England) to obtain crude saponins.

Test Bacteria
Clinical isolates of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were obtained from the clinical diagnostic laboratory of Microbiology Department, University of Nigeria Nsukka, while type cultures (Escherichia coli ATCC 11775, Pseudomonas aeruginosa ATCC 10145, and Staphylococcus aureus ATCC 12600) were obtained from Bioresource Development and Conservation Project (BDCP), Nsukka.The inoculum size of each test bacterial strain was standardized as described by the Clinical and Laboratory Standards Institute [30].

Antibacterial Assay
Ethanol extracts (leaves and stem bark) and crude saponin extracts of V. doniana and P. macrophylla were assayed for antibacterial activity using the agar well diffusion technique.A stock solution of 100 mg/mL concentration of each extract was prepared, from which varying concentrations (50, 25, 12.5, and 6.25 (mg/Ml)) were achieved.About 100 µL of standardized test bacterial suspension (1.5 ˆ108 CFU/mL) was seeded onto sterile Mueller-Hinton agar plates (Conda, Madrid, Spain) and spread evenly to achieve a confluent lawn of microbial growth [3].The plates were left to dry, and a sterile cork borer 6 mm in diameter was used to bore wells.Afterwards, about 100 µL of each plant extract was introduced into the well.Plates were incubated at 37 ˝C for 24 h, and the bacterial inhibition zone diameter was measured on the plates to the nearest millimeter (mm).Ciprofloxacin (Oxoid, Hampshire, UK) served as a positive control, and experimentation was performed in triplicate assays.

Minimum Inhibitory Concentration Determination
The minimum inhibitory concentrations (MICs) of ethanol extracts, of the leaves and stem bark, and of the crude saponin fractions of V. doniana and P. macrophylla were respectively determined using the microbroth dilution technique [31].The MICs were recorded as the lowest concentrations of the extract, which showed inhibition of bacterial growth.

Statistical Analysis
Assays were conducted in triplicate, and all data was subjected to analysis of variance (ANOVA) using the IBM Statistical Package for Social Scientists (IBM-SPSS) version 22 (IBM Coorp, Armonk, NY, USA, 2013).The results were presented as mean values with standard deviation.

Results
Quantitative assessment of V. doniana and P. macrophylla showed the presence of saponins, tannins, flavonoids, anthroquinones, and alkaloids, respectively.However, these phytochemicals were present at varying concentrations, and saponins were observed in higher measures than other constituents (Table 1).The quantities of secondary metabolites detected/quantified might have been predicated on the solvent of extraction, a position advanced on the premise that solvent polarity plays an integral role on the nature of phytochemical extracted.Nonetheless, ethanol served as the extraction solvent for the plant materials used for these studies due to the fact that locally brewed alcohol (ethanol) is used in folk practices for the preparations of the decoctions involving V. doniana and P. macrophylla administered in herbal remedies for disease conditions.Antibacterial susceptibility assay showed that V. doniana inhibited the growth of the Gram-positive bacteria but not the Gram-negatives tested; inhibition zone diameters recorded against the type culture of Staphylococcus aureus (ATCC 11775) and the clinical strain ranged from 15.5 ˘2.12 to 7.0 ˘0.0 (mm) against the leaf extracts and from 20.0 ˘1.41 to 7.0 ˘0.0 (mm) against the stem bark extracts (Table 2).Conversely, P. macrophylla extracts showed activity against both Gram-positive and -negative test bacteria strains; leaf extract concentration of 50 mg/mL showed inhibition zone diameters (mm) of 20.0 ˘1.41 (S. aureus clinical strain), 18.5 ˘0.71 (P.aeruginosa clinical strain), 17.0 ˘0 (P.aeruginosa ATCC 10145), and 14.5 ˘2.12 (E. coli ATCC 11775), respectively.A similar pattern of activity was achieved with the stem bark extract, and the inhibition zone diameters ranged from 23.0 ˘2.83 to 17.0 ˘0 (mm) at 50 mg/mL (Table 2).
Saponin extracts of both V. doniana and P. macrophylla, respectively, exhibited activity across the divide of Gram status (Table 3).At 50 mg/mL, saponin extracted from V. doniana yielded inhibition zone diameters (mm) of 20.0 ˘1.41 (E. coli clinical strain), 18.5 ˘0.71 (P.aeruginosa clinical strain), 17.0 ˘0 (E. coli ATCC 11775), and 13.0 ˘1.41 (S. aureus clinical strain), respectively, against the listed test bacteria.Similarly, the saponin extracts of P. macrophylla showed activity against over 80% (5/6) of the test isolates, with inhibition zone diameters ranging from 18.5 ˘0.71 against P. aeruginosa to 10.5 ˘0.71 against S. aureus (both test bacteria are clinical strains).Consequently, saponin extracts from both V. doniana and P. macrophylla, respectively, inhibited the growth of all bacterial strains tested, with the exception of S. aureus ATCC 12600 (Table 3).The minimum inhibitory concentrations obtained from the results of the micro-broth dilution assay showed varying threshold concentrations for the growth inhibition of test bacteria; however, the MICs ranged from 0.12 to 0.78 (mg/mL), as is shown in Table 4.

Discussion
The applications of saponins as pharmacologically active agents are described in various literary documentations on applications and potentials.Anticipated pharma activity includes anti-lipid peroxidation of plasma lipoprotein with the concomitant effect as a reduction in the risk of atherosclerosis [5], anti-diabetic activities from steroidal saponins of the Polygonatum kingianum in origin [32], and in the treatment of other ailments of human metabolic disorder and infectious agents [33].The phyto-constituent analyses of the extracts of V. doniana and P. macrophylla indicated varying concentrations of tannins, flavonoids, anthroquinones, and alkaloids.The qualitative assay showed trace to non-detectable levels of falvonoids, anthroquinones, and alkaloids; however, upon quantitation, using a more sensitive technique, lower concentrations of these phytochemicals were detected.Nonetheless, saponins were detected in high concentrations without recourse to the techniques applied, thus an indication that the herbs are rich sources of saponins.
Antibacterial activity assessment of the ethanol extracts of V. doniana and P. macrophylla against Gram-positive and -negative bacteria showed a broad spectrum of activity for P. macrophylla, as all the genus bacteria tested were susceptible following zones of inhibition.Conversely, only Gram-positives were susceptible to the ethanol extract of V. doniana.The precise component(s) that may have potentiated activity alone, or in combination, against test bacteria from the respective extracts of V. doniana and P. macrophylla is (are) unknown and are not within the purview of this study.However, a phyto-constituent guided bio-assay, with combinatorial of respective phyto-constituent in a factorial, may be ideal to give an insight into the role of respective constituents on the herbal extracts.
In the same vein, various folkloric herbs have been validated for activity against several bacterial etiologic agents associated with human and livestock disease [34].Similar activity has been shown to exist in Vitex species collected from the Southern African region [1].Although, geographical boundaries impacts the variance of the pharmacologically active constituent of plants following prevailing conditions, which includes the interaction with other plants, pathogens, and weather conditions, as well as other biotic and abiotic factors.
Furthermore, in the assessment of saponin extracts from both V. doniana and P. macrophylla for activity against the same test bacteria used with the ethanol extracts, activity was recorded against the entire test bacteria genus.Saponin extracts from V. doniana showed more activity against both Gram-positive and -negative bacteria, indicating a broader spectrum of activity as compared to the ethanol extract of same plant.The ethanol extraction of V. doniana stem bark and leaves might only be able to extract constituents with activity against the Gram-positives.On the other hand, the saponin extracted may have been void of any adjuncts with activity inhibitory properties, as it is peculiar with plant extracts.It is worth mentioning that, the antibacterial activity recorded against the saponin extracted from V. doniana showed larger inhibition zone diameters as compared to the saponin extract from P. macrophylla.In effect, this phenomenon may be understood as structural variance in the aglycone backbone of the saponins from both plants.Whether or not the saponin's aglycones are triternoid, steroidal, and/or a combination of both, the objectives of this study did not address whether the variation in activity is largely attributed to these differences.
Besides the obvious, namely, that saponins represent an important class of plant's secondary metabolites and has ameliorated physiological anomalies in humans and livestock, it has also contributed significantly to the bio-economy.The World Health Organisation's estimation that about 60% of the world's rural dwellers depend on herbal remedies [35] connotes the imperativeness of the exploration of phytochemicals for anti-infective properties.Hence, the efficacies of these herbs in folk medicine may be adduced to the antimicrobial activities of respective phyto-constituents, or a combination thereof, vaguely understood due to a paucity of information on the advanced concept.

Conclusions
The applications of V. doniana and P. macrophylla in folk medicine for the treatment of various ailments may be validated by the antibacterial activity recorded in this study.Additionally, the saponin extract from both plants showed high potency and a broad spectrum of activity against both Gram-positive and -negative bacteria tested.The saponins from V. doniana were active against bacterial species that were not susceptible to the ethanol extracts of the same plant.Saponins constituting important phytochemical grouping suggest the imperativeness of further studies on the saponins from V. doniana and P. macrophylla to elucidate the nature of the aglycone structures in these plants for novelty and the possibility for use as a scaffold in drug design.

Table 1 .
Quantitation of the phytochemical composition of ethanol extracts of Vitex doniana and Pentaclethra macrophylla.

Table 2 .
Bacteria growth inhibition zones of ethanol extract of V. doniana and P. macrophylla.

Table 3 .
Bacteria growth inhibition zones of saponin extract of V. doniana and P. macrophylla stem bark.
CPF: ciprofloxacin; values are mean zone of inhibition (mm) ˘standard deviation of three replicates; -: no activity.