Abstract
The focus of this study was to evaluate the antioxidants and antimycobacterial activitiesof extracts of Schkuhria pinnata. Serial exhaustive extraction procedurewas employed using solvents of varying polarity to obtain the desired extracts. Thin layerchromatography and standard chemical tests were used to analyze phytochemicalsconstituents. Free radical scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) methods wereused to detect the presence of antioxidant compounds. Antimycobacterial activity wasevaluated using microdilution and bioautography assays. A variety of secondary metabolitessuch as flavonoids, tannins, and alkaloids were detected in the extract. Ethyl acetate andacetone extracts had high antioxidant activity on chromatograms eluted in ethylacetate/methanol/water while methanol extract at various concentrations had the bestscavenging activity. The minimum inhibitory concentration (MIC) values ranged from 0.02 to2.50 mg/mL. Total phenol content was 55.33 ± 3.51 mg of gallic acid equivalent (GAE)/g andhigher when compared with flavonoids (4.00 ± 0.35 mg of quercetin equivalent [QE]/mg) andtannin content (28.00 ± 1.73 mg of GAE/g). The most effective antimycobacterial activityagainst Mycobacterium smegmatis was observed with the lowest inhibitoryconcentrations of acetone (0.27 mg/mL), dichloromethane (0.32 mg/mL), and ethyl acetate(0.32 mg/mL) in that order. In massive extraction, hexane and dichloromethane had thegreatest inhibitory bands on benzene/ethanol/ammonium hydroxide bioautograms.Antimmycobacterial activity gives promising potential leads of S pinnataextracts to be used in the development of antimycobacterial drugs. The presence ofantioxidant and antimycobacterial compounds requires further isolation andpurification.
Keywords: medicinal plants, phytochemicals, antioxidants, antimycobacterial activity
Mycobacterium smegmatis is a Gram positive and acid fast bacterium that fallsunder the Mycobacteriaceae family, which includes Mycobacterium tuberculosis,Mycobacterium fortuitum, Mycobacterium abscessus, andMycobacterium chelonae, that have shown resistance against many antibioticsdue to their protective outer layer.1M smegmatis lives in aggregated layers of community called biofilm and arecommonly found on plants, soil, and in water.2
M smegmatis have been reported to cause diseases such as skin and soft tissueinfections, and bone diseases.3 The organism can be transmitted as a results of contaminated materials during invasiveprocedures, which can result in infection.4 Best and Best3 reported that M smegmatis is resistant to isoniazid and rifampicin, 2widely used antibiotics for treatment of tuberculosis.
Other debilitating effects resulting from the treatment of M smegmatisinfection include prolonged antibiotic therapy, which is toxic to the patients, and surgicaldebridement of infected tissues, which is expensive to poor rural people.3 The rise in bacterial resistance against a broad spectrum of antibiotics as well ashigh cost of therapies is a major concern, especially to the rural poor. There is, thus aspike in the surge in trying to identify plant derived drugs that are nontoxic,cost-effective, and possess improved biological efficacy.
Plants have been used by humans for thousands of years for various purposes, including foodtransport and as medicine.5–7 Sofowora8 reported that about 80% of the population in developing countries use medicinal plantsfor their primary health care needs. Plants produce primary and secondary metabolites, whichthey use in their metabolisms and defense against invading pathogens. Various studies haveindicated that theses metabolites possess healing power that can be used in treatment ofchronic as well as infectious diseases.9,10 Bioactive metabolites have consistently provided a platform for new drug leads againsta host of diseases.
Schkuhria pinnata is a herbaceous and exotic plant that belong to the familyAsteraceae. The species within this family have distinctive phytochemicals that differentiatethem. S pinnata is recorded to grow in some regions in South America and insome African countries namely Zimbabwe and South Africa. It grows in cultivated lands, alongroadsides and fields.11 It has been employed as a herbal remedy for kidney, liver, renal problems, malaria,diabetes, allergies, yeast infections, prostate inflammation, digestive disorders, andintestinal gas.12,13 The plant has been reported to have therapeutic effects in the treatment of eyeinfections, pneumonia, heart water, diarrhea, wound infections, and retained placenta in livestock.14,15 Extracts of S pinnata have also been reported to be effective againstthe pathogens that cause mastitis in dairy cattle Mupfure et al.16
Medicinal plants are considered to have less or no side effects, affordable, and readilyavailable to the community. However, clinical trials for the biological activities frommedicinal plants are necessary to give a clear understanding of the safety and efficacy ofmedicinal plants by traditional healers and other herbalist.17 The focus of this study is to assess the phytochemical, antioxidant, andantimycobacterial activities of S pinnata extracts.
Methods
Plant Collection
Schkuhria pinnata (Lam.) Kuntze ex Thell was collected at the Universityof Limpopo, South Africa. Voucher specimen was identified at Larry Leach herbarium (UNIN12298). Plant materials were dried at ambient temperature at the Microbiology Department,University of Limpopo. The roots of the plants were separated and discarded. The remainingplant materials were milled to fine powder using a grinding machine (Trf400) (animalration shredder hammer mill foliage machine) at the school of Agricultural andEnvironmental Sciences (University of Limpopo). The powdered material was stored in thedark at room temperature in an air-tight container until further use.
Extraction Procedure
Serial Exhaustive Extraction
Finely ground plant material (5 g) was exhaustively extracted with 50 mL ofn-hexane. The bottle was shaken for 1 hour at 200 rpm on a series 25shaking machine (New Brunswick Scientific Co, Inc). The supernatant was filtered usingthe Whatman No. 1 filter paper into preweighed bottles and the process was repeated 3times. The same procedure was followed, on the same plant residues with 50 mL ofchloroform, dichloromethane, ethyl acetate, acetone, ethanol, and methanol toexhaustively extract compounds of varying polarities. The supernatants collected weredried under a stream of air, after which the mass was determined, and extractsreconstituted in acetone at a concentration of 10 mg/mL.
Qualitative Phytochemical Constituent Analysis
Phytochemical constituents were analyzed using thin layer chromatography (TLC).Briefly, 10 µL of extracts were loaded on aluminum-backed TLC plates. Three solventsystems of varying polarity, benzene/ethanol/ammonium hydroxide (BEA) (90:10:1)(nonpolar/basic); chloroform/ethyl acetate/formic acid (CEF) (5:4:1) (intermediatepolarity/acidic); ethyl acetate/methanol/water (EMW) (40:5.4:5) (polar/neutral) wereused to elute TLC plates in saturated tanks.18 Developed plates were observed under ultraviolet light at 254 and 365 nm for thepresence of quenching and fluorescing compounds, respectively, and thereafter sprayedwith vanillin sulfuric acid reagent (0.1 g vanillin [Sigma]:28 methanol:1 mL sulfuricacid). Plates were heated at 110° C for optimal color development.
Preliminary Biochemical Analysis of Phytochemicals
Acetone plants extracts were tested for the presence of saponin, phlobatannin, tannins,terpenes/terpenoids, steroids, cardiac glycosides, and flavonoids using the standardprocedures as described by Borokini and Omotayo.19
Quantitative Analysis of Total Phenolic, Flavonoids, and Tannins Content
Total Phenol Content
Total phenolic contents (TPC) in the S pinnata extracts wereestimated, following the method of Singleton et al.20 Aliquots of 1.0 mL of water or acetone extracts were mixed with 5 mL of 10-folddiluted Folin-Ciocalteu reagent and 4 mL of 7% sodium carbonate(Na2CO3) solution. The mixture was allowed to stand for 90minutes at room temperature and the absorbance was measured at 550 nm. Results wereexpressed as milligrams of gallic acid equivalents per gram of extract (mg GAE/g).
Total Flavonoid Content
Total flavonoid contents were quantified using a modified colorimetric method asdescribed by Tambe and Bhambar.21 Briefly, 5 mL of water or acetone extract was mixed with 0.3 mL of 5% sodiumnitrite for 5 minutes in a test tube. Then 0.3 mL of 10% aluminum chloride was added.After 6 minutes, 2 mL sodium hydroxide was added to stop the reaction and the mixturewas further diluted with distilled water up to 10 mL. The absorbance was immediatelymeasured at 510 nm and results were expressed as milligrams of quercetin equivalents pergram of extract (mg of QE/g).
Total Tannin Content
Total tannin contents were measured following the procedure of Tambe and Bhambar.21 Briefly, 0.5 mL of Folin-Ciocalteu reagent and 1 mL of 35%Na2CO3 solutions were added in 10 mL of sample extract. Theabsorption was measured at 725 nm after 45 minutes of incubation at room temperature.Results were expressed as milligrams of gallic acid equivalents per gram of extract (mgof GAE/g).
Antioxidant Activity
Qualitative DPPH Assay
Aluminum-baked TLC plates coated with silica were used to detect the presence ofantioxidant compounds from the plant extracts. Ten microliters of plant extracts wereloaded on the plates and developed in 3 solvent systems of varying polarity, BEA(90:10:1) (nonpolar/basic); CEF (5:4:1) (intermediate polarity/acidic); and EMW(40:5.4:5) (polar/neutral). Thereafter the plates were dried under a stream of air atroom temperature for about 1 minute and sprayed with 0.2%2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH; Sigma) in methanol. A positive result wasindicated by the presence of yellow bands against the purple background.22
Quantitative Total Antioxidant Activity Assay
DPPH scavenging activity was designed following the method described formerly by Brand-Williams.23 Briefly, 2.0 mL of the extracts or standards was added to 5 mL of DPPH solution(0.1 mM in methanol), vortexed vigorously and incubated in dark for 30 minutes at roomtemperature. The decolorization of DPPH was measured against a blank at 517 nm.Percentage scavenging was calculated as
Ferric Reducing Power
Ferric ion reducing power from S pinnata extracts was determined.Various concentrations of plant extracts (1 to 0.0625 mg/mL) were prepared in testtubes. Ascorbic acid was used as a standard control and a blank solution was preparedwithout adding extracts. Two milliliters of 0.2 M sodium phosphate buffer and 2 mL of 1%potassium ferricyanide were added to the test tubes containing extracts of differentconcentrations. The solution was mixed well and incubated in a water bath at 50°C for 20minutes. After incubation, 2.5 mL of 10% trichloroacetic acid was added into the testtubes and centrifuged at 650 rpm for 10 minutes. The supernatant was mixed with 10 mL ofdistilled water and 1 mL of freshly prepared ferric chloride solution (0.1%) and thesolution was mixed. The absorbance of the solution was recorded at 700 nm against theblank solution.24
Bacterial Species
The test organism M smegmatis ATCC 1441 was obtained from the Schoolof Molecular and Cell Biology, University of Witwatersrand. The bacterial specie wasgrown and maintained in Middlebrook 7H9 (Fluka M0178) broth with glycerol (Fluka 49769)or Tween 80 (Fluka 93780) and Middlebrook Oleic Albumin Dextrose Catalase (OADC) growthsupplement (Fluka M0553).
Minimum Inhibitory Concentration Determination
The minimum inhibitory concentration (MIC) values were determined using the serialmicroplate method developed by Eloff.25 Minimum inhibitory concentration is described as the lowest concentration of thecompounds inhibiting the growth of microorganisms. Dried extracts were redissolved inacetone to a concentration of 10 mg/mL of crude extracts. The plant extracts wereserially diluted 50% with water in 96-well microtiter plates. Bacterial cultures weresubcultured and transferred into fresh Middlebrook 7H9 broth and 100 μL of the culturewas transferred into each well and appropriate acetone blanks were included. Themicrotitre plate was incubated at 37°C for 24 hours. After incubation, 20 μL ofp-iodonitrotetrazolium violet (Sigma) (INT) dissolved in water wasadded to each of the microplate wells as an indicator of growth. The covered microplateswere incubated for 30 minutes at 35°C and 100% relative humidity for color development.All determinations were carried out in triplicate. Microorganism growth led to theemergence of a purple-red color resulting from the reduction of INT to formazan. Clearwells indicate the presence of compound in the extracts that inhibited the growth of themicroorganisms tested.
Qualitative Antibacterial Activity (Bioautography)
For bioautographic analysis 20 μL of each extract was loaded on the TLC plates. Theplates were developed in mobile phases as previously mentioned. The chromatograms weredried at room temperature for about 4 days to remove the solvents used to developchromatograms. The chromatograms were sprayed with overnight culture of Msmegmatis until completely wet and were incubated at 37°C in a humidifiedchamber for 24 hours. The plates were sprayed with INT (Sigma) and incubated for afurther 24 hours. The presence of clear bands on the plates against a purple backgroundindicates growth inhibition.26
Results
Phytochemical Constituents
Phytochemical constituents from the crude extracts were analyzed using aluminum-backedTLC plates, which were developed in solvent systems of different polarity (BAE, CEF, andEMW) and sprayed with vanillin–sulfuric acid reagent for color development. Solvent systemCEF followed by BEA separated more bands of phytochemical constituents that react withvanillin–sulfuric reagent while EMW separated fewer bands (Figure 1).
Figure 1.
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Preliminary Biochemical Analysis of Phytochemicals
Various standard phytochemical tests were conducted to test for the presence of differentcompounds. S pinnata extracts tested positive for all testedphytoconstituents, namely: tannins, saponins, phlabotannins, terpenoids, alkaloids,steroids, and cardiac glycosides (Table 1). The presence of these secondary metabolites may be responsible infighting against diseases.
Table 1.
Phytochemical Constituents From Schkuhria pinnata Extracts.
| Phytochemical Constituents | Reactiona |
|---|---|
| Tannins | + |
| Saponins | + |
| Phlabotannis | + |
| Flavonoids | + |
| Terpernoids | + |
| Alkaloids | + |
| Cardiac glycoside | + |
| Steroids | + |
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a + indicates presence.
Quantitative Analysis of Total Phenolic, Flavonoids, and Tannins Content
The extracts had high concentrations of phenolic and tannin contents and low flavonoidcontents (Table 2).
Table 2.
Determined Total Phenol, Flavonoid, and Tannin Content From Schkuhriapinnata Extracts.
| Sample | Total Phenol (mg of GAE/g) | Total Tannin (mg of GAE/g) | Total Flavonoid (mg of QE/mg) |
|---|---|---|---|
| Schkuhria pinnata | 55.33 ± 3.51 | 28.00 ± 1.73 | 4.00 ± 0.35 |
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Abbreviations: GAE, gallic acid equivalent; QE, quercetin equivalent.
Qualitative DPPH Assay
S pinnata sample was extracted with solvents of varying polarities. Theethyl acetate and acetone extracts had strong antioxidant activity from all solventsystems. In BEA and CEF solvent systems, the compounds did not migrate; best separationwas observed in EMW solvent system (Figure 2). It can be concluded that the extracted compounds that showed activityin this assay were polar.
Figure 2.
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Quantitative Total Antioxidant Activity Assay
The quantitative antioxidant activity from S pinnata extracts wasperformed using the DPPH free radical scavenging activity assay. Methanol extracts had thegreatest antioxidant activity when compared with other extracts at all concentrations,followed by ethyl acetate and acetone extracts. The lowest percentage scavenging activitywas observed with the dichloromethane extracts (0.02 mg/mL) (Figure 3).
Figure 3.
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Ferric Reducing Power
The ferric reducing power of S pinnata extracts was evaluated atdifferent concentrations in comparison with ascorbic acid the positive control. Theabsorbance was observed to be increased as concentration increases; the same trend wasobserved with the positive control (Figure 4).
Figure 4.
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Minimum Inhibitory Concentration Determination
Antimycobacterial activity of S pinnata extracts was evaluated usingmicrodilution assay. The lowest MIC value was observed with acetone extracts (0.27 mg/mL)followed by ethyl acetate (0.32 mg/mL) and dichloromethane (0.32 mg/mL). The highest MICvalue was observed with hexane extracts (2.5 mg/mL) (Table 3). The total activity is the values in which1 g of dried plant material can be diluted and still inhibit the growth of microorganism.Acetone extracts indicated the highest total activity (Table 3).
Table 3.
The Minimum Inhibitory Concentration (MIC) of the Plant Extracts With Their TotalActivity Values.
| H | C | D | EA | A | E | M | Rifampicin |
|---|---|---|---|---|---|---|---|
| MIC Values (mg/mL) | |||||||
| 2.5 | 0.43 | 0.32 | 0.32 | 0.27 | 0.37 | 0.53 | 0.08 |
| Total Activity (mL) | |||||||
| 26 | 337 | 478 | 353 | 521 | 368 | 306 | |
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Abbreviations: H, hexane; C, chloroform; D, dichloromethane; EA, ethyl acetate; A,acetone; E, ethanol; M, methanol.
Qualitative Antibacterial Activity (Bioautography)
Bioautography assay was used to detect the presence of antimycobacterial compounds in theplant extracts on TLC plates. Figure5 shows the presence of antimycobacterial compounds from chloroform and ethylacetate extracts that were separated in CEF and EMW solvent systems.
Figure 5.
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Discussion
Drugs derived from medicinal plants are developed from plant phytochemical constituentssuch as alkaloids, flavonoids, tannins, terpenoids, and saponins, which are of greatimportance in human’s health, veterinary, and agriculture. Analysis of plant phytochemicalconstituents is necessary for the synthesis of drugs and other therapeutic agents. Afterextraction, the plant extracts were reconstituted to a certain concentration with acetonebased on reports by Eloff25 that it is nontoxic to microorganism and can dissolves compounds of varyingpolarities. TLC was employed in the screening of the phytochemical profiles of Spinnata, as it was considered to be highly efficient.27 CEF solvent system was observed to be the best mobile phase that separated most ofvanillin reactive compounds, followed by BEA. And the least compounds were separated in EMWsolvent system (Figure 1). Differentcolors observed on the chromatograms shows that S pinnata has differentcompounds of varying polarities. Since solvent system CEF separate compounds of intermediate polarity,15 this suggest that S pinnata has high amount of intermediatecompounds. The bands that turn blue on the chromatogram when sprayed with vanillin reagentdepict the presence of terpenes.28
Chemical quantitative tests were carried out on S pinnata extracts todetect the presence or absence of secondary metabolites. Metabolites present in Spinnata are known to have various pharmacological actions in human.29 The result of phytochemicals screening for S pinnata (Table 1) shows the presence oftannins, saponins, phlabotannins, flavonoids, terpernoids, alkaloids, cardiac glycoside, andsteroid compounds. Oryema et al30 also detected the presence of alkaloids, steroids, and terpenoids in Spinnata extracts. Ethanol extracts of S pinnata have beenreported to possess the sterol triterpenes and flavonoids compounds.31 Researches have reported the anticancer activity of S pinnataextracts of which triterpenes compounds are responsible for the activity.32–34 Sesquiterpene lactones and eucannabinolide are compounds that have been isolated fromS pinnata extracts and the family Asteraceae have beenreported to mostly possess these compounds.35
Phenolic compounds are the largest group of phytochemicals which have been recorded fromevery plant part36; the total phenol content was determined to be highest at 55.33 GAE/g. this may bedue to the presence of flavonoids, tannins, and alkaloids, which are part of phenolic group.It has been reported that phenol compounds are responsible for biological activities such asantioxidants, antibacterial, antimalarial, and antidairearhea.37,38 Tawaha et al39 reported on the high phenolic content from plants falling under the familyAsteraceae. This is the first study to report the total tannin and flavonoids content ofS pinnata extracts. The tannin content of 28 GAE/g was observed to behigher than that of flavonoids (4 QE/mg). Natural antioxidants have been reported to protectagainst chronic diseases and oxidative stress. The presence of antioxidant compounds wasindicated by the yellow bands against the purple background on the TLC plates. The intensityof the yellow color depends on the quantity and nature of compounds present in extracts atthat area.40 The antioxidant activity observed might be due to phytochemical constituents whichhave been found to be present in S pinnata extracts. The qualitativeantioxidant assay indicated that methanol extracts had the highest scavenging activity whencompared with the positive control at all concentrations. Masevhe et al41 indicated that S pinnata had weak antioxidant activity. However, themethanol extracts have been reported to have high antioxidant activity.42,43 The results from qualitative analysis and quantitative analysis do not correlate asthe ethyl acetate and acetone extracts were observed to have high antioxidants activity withmethanol extract not showing activity. The lack of activity with the methanol extract mightbe due to synergistic mechanism of compounds and the evaporation of solvent systems. Allplant extracts had high antioxidant activity at high concentration (10 mg/mL), and at lowconcentration, only methanol and hexane had high activity (Figure 3). There are different mechanisms by whichantioxidants prevent oxidative stress and ferric reducing power falls under one of themechanisms. The results indicated that S pinnata had high ferric reducingpower when compared with the positive control (Figure 4). Tannins have been reported to have ionchelator activity, which might be responsible for the reducing power of the plant extracts.44
Medicinal plants are considered the greatest source of antimicrobial drugs.45 A white area against a pink color on bioautograms indicates that chloroform and ethylacetate extracts have antimycobacterial compounds (Figure 5). Alkaloids and flavonoids were also reportedfor their antibacterial activity.46,47 Antibacterial activity has been reported from other Asteraceae species.48 The MIC was observed from acetone extracts followed by ethyl acetate anddichloromethane extracts. The activity might be due to the presence of saponins, glycosides,steroids, and polyphenols compounds.49 Antibacterial activity of the same plant have also been reported by Masevhe et al.41 The lower the MIC value, the higher the total activity volume. This was observed withacetone extracts (Table 3).Total activity is referred to as; the amounts in which the active compounds in dried plantmaterial can be diluted and still inhibit the growth of the microorganism.50 The white area on bioautograms developed under solvent system CEF, could be explainedby evaporation of solvent system, which might have not evaporated properly or the lowconcentration of the active compounds from the extracts under the tested condition or bydisruption of synergistic mechanism between active compounds caused by TLC separation.51,52
Conclusion
The observed results indicated that S pinnata possess compounds ofintermediate polarity. The plant has the potential biologically active compounds which canbe used in the development of new drugs. The antioxidant and antimycobacterial compoundsdetected require isolation and characterization. Further studies should be conducted for thecytotoxic effects of the plant extracts to address the safety of the plant.
Acknowledgments
We would like to thank the National Research Foundation and University of Limpopo forfinancial support. We would like to thank Mr I. Njanje and Dr V. P. Bagla forproofreading.
Footnotes
Author Contributions: PM was involved with conception and design of the study. MVM carried out the experimentsand analyzed the data.
Declaration of Conflicting Interests: The authors declared no potential conflicts of interest with respect to the research,authorship, and/or publication of this article.
Funding: The authors disclosed receipt of the following financial support for the research,authorship, and/or publication of this article: Financial assistance was provided by theUniversity of Limpopo and National Research Foundation.
ORCID iD: Peter Masoko, PhD 
https://orcid.org/0000-0002-8076-083X
Ethical Approval: The study protocol was confirmed by University of Limpopo Ethics Committee(TREC/248/2017: IR).
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