totalphenoliccontentandantioxidantandantibacterial activitiesof...

Research ArticleTotal Phenolic Content and Antioxidant and AntibacterialActivities of Pereskia bleo

Mas Athira Johari and Heng Yen Khong

Faculty of Applied Sciences Universiti Teknologi MARA 94300 Kota Samarahan Sarawak Malaysia

Correspondence should be addressed to Heng Yen Khong khonghysarawakuitmedumy

Received 30 August 2018 Revised 27 November 2018 Accepted 12 December 2018 Published 2 January 2019

Guest Editor Ghulam Hussain

Copyright copy 2019Mas Athira Johari andHeng YenKhongis is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in anymedium provided the original work isproperly cited

Different solvent extracts of Pereskia bleo leaves were evaluated for total phenolic content (TPC) and antioxidant activitiesbased on the FolinndashCiocalteu test and DPPH scavenging activities e antibacterial activities against four bacteria namelyGram-positive bacteria Streptococcus pyogenes ATCC 19615 (SP) and Staphylococcus aureus ATCC 29737 (SA) and Gram-negative bacteria Escherichia coli ATCC 10536 (EC) and Pseudomonas aeruginosa ATCC 9027 (PA) were also performedbased on the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays e findingsdemonstrated that both the methanolic and chloroform extracts displayed strong activities against SA SP EC and PA while thehexane extract demonstrated the weakest activities towards all the four bacteria e methanolic extract also exhibited higherTPC and possessed higher antioxidant activity with the IC50 value 3383 microgmL compared to the chloroform and hexaneextracts As such the methanolic extract has a higher ability to scavenge free radical compared to other extracts Due to theinteresting result activities are shown by the methanolic and chloroform crude extracts of P bleo hence the study has beenextended to the isolation of bioactive compounds to uncover its great potential as a natural source for antibacterial andantioxidant agents

1 Introduction

Medicinal plants have been used to promote human healthand it has been documented since ancient times Eventhough accessibility to modern healthcare nowadays becomefaster and easier still there are people who prefer to promotehealth by using fresh medicinal plants which are locallygrown For example in the way to compliment the allopathicmedicine Malays prefer Jamu medicine and Chinese preferTraditional Chinese medicine (TCM) while Indians preferAyurvedic medicine erefore even though there is anabundance of modern medicine in the market traditionalmedicine still maintained its popularity in the developingworld

P bleo is well known as the medicinal plant of theCactaceae family which consists of 100 genera and about2000 species [1] e origin of the genus Pereskia is fromSouth America and they have been cultivated in tropicalcountries [2] such as Malaysia Indonesia Singapore and

India Some Pereskia species are used as traditional medi-cines to treat hypertension diabetes cancer high bloodpressure skin inflammation and skin injuries Previousreports revealed that the preparation methods of this plantvaried subject to the uses For example a decoction from theleaves is prepared and then used as a warm bath to alleviatemuscle ache while the leaves are boiled for making tea andthen drinking it warm or cold for prevention of cancer anddetoxification of the body

Previous phytochemical studies of Pereskia genus haverevealed the occurrence of a variety of compounds includingcarotenoids alkaloids flavonoid lactone sterols terpenoidsfatty acids phytosterol glycoside and phenolic compounds[1] Besides studies of Pereskia species on its biologicalactivities have been reported on antioxidant anticancerantinociceptive and antibacterial Moreover previousphytochemical studies of Pereskia genus have revealed theoccurrence of a variety of compounds Although there aremany reviews on the traditional uses for a variety of

HindawiAdvances in Pharmacological SciencesVolume 2019 Article ID 7428593 4 pageshttpsdoiorg10115520197428593

prophylactic and therapeutic purposes only few Pereskiaspecies have been investigated on its phytochemical andbiological activities erefore P bleo is selected to furtherstudy on its biological activities

e intention of this study is to explore the biologicalactivities of the P bleo crude extract in exploring the po-tential of natural sources for the drug discovery which can beused for further innovation development of pharmaceuticalmedicinal health and household products in order to in-crease its commercial values Studies onmedicinal plants notonly discover new therapeutics but also assist in settingappropriate guidelines and policy in the usage of the tra-ditional herbal medicine since there are scientific evidenceand proper understanding of traditional usage of the plantsavailable

2 Materials and Methods

21 Plant Material e studied plant sample which is theleaves of P bleo was collected from Perlis Malaysia esample was air-dried at a room with room temperature andgood air ventilation A dried sample was cut into smallerpieces to make it easier for it to be grounded using an electriccutting mill to produce fine powder sample

22 Extraction e air-dried leaves (926 g) of P bleo wereextracted at 24 hours interval by the cold extraction methodand the process was repeated twice using methanol as asolvent Solvents removal under diminished pressure using aBUCHI model R-200 rotavapor produces 391371 g dark-green methanol crude extracts Chlorophyll was removedfrom the methanol extract before being performed thefractionation e fractionation process which was per-formed using liquid-liquid partition have yielded 0334 ggreen hexane fraction 58916 g dark-yellow chloroformfraction and 253989 g dark-brown methanol fraction

23 Total Phenolic Content e FolinndashCiocalteu test waschosen to measure TPC of P bleo extracts is test wasperformed by referring to the method developed by Veliogluet al [3] with some modifications e crude sample wasprepared by liquefying 10mg of the extract in 10mL of thesolvent to yield a concentration of 1mgmL About 100 microL ofthe extract (1mgmL) was combined and mixed with075mL of the FolinndashCiocalteu reagent (diluted 10-fold withdeionized water previously) in the test tube e liquidmixture was allowed to stand for 5 minutes at a roomtemperature e mixture was then added about 075mL ofsodium carbonate (Na2CO3) and the test tube was shakengently to mix them After 90minutes the absorbance of themixture was measured using the UV-Vis spectrophotometerat 725 nm

A calibration curve of standard reference was establishedusing gallic acid (range of concentration from 001 to005mgmL) as standard references plotted TPC wasrevealed as gallic acid equivalents in milligrams per 100g ofthe extract

24 Antioxidant Activity e DPPH was used to determinefree radical scavenging activity as previously described byShimada et al [4] About 394mg ofDPPHwas first dissolved in100mL of ethanol to a concentration of 01mL About 1mL ofDPPH solution was added to 3mL of the samples with differentconcentrations (250 125 625 3125 and 1562μgmL)

For the control test the same amount of ethanol wasadded All the mixture was mixed well by shaking vigorouslyand left to stand for 30 minutes at a room temperature Afterthat the UV-Vis spectrophotometer was used to measurethe value of absorbance of each mixture at 517 nm ecalculation for the percentage of inhibition (I) of theDPPH radical is as follows

I Ao minus As

Ao

1113888 11138891113890 1113891 times 100 (1)

where AS represents the absorbance value of the sample whileA0 represents the absorbance value of the control reaction(contain all reagents except the sample) A graph of inhibitionpercentages (I) against concentrations of the sample wasplotted From the graph 50 inhibition (IC50 value) providesthe value of concentrations for each sample All experimentswere carried out in triplicate to minimize the precision errorMean plusmn SD of triplicates was reported as IC50 values

25 Antimicrobial Activity Antimicrobial activities wereconducted using MIC and MBC assays e MIC method asdescribed by Gulluce et al [5] involves the broth micro-dilution technique using 96-well microplates In this studyfour types of bacteria are used which are SP SA EC and PA

26 Minimum Inhibitory Concentration e MIC repre-sents the lowest concentration that had absence of micro-scopically noticeable growth e interpretation of in vitrodata is based on attainable sample concentrations Nutrientbroth (NB) was prepared as the medium About 36mg crudeextract samples were weighted and dissolved in 2mL DMSO(stock solution 1800 μgmL) Inoculations of the microbialstrains were produced from 24 hours broth cultures andsuspensions regulated to 05 McFarland standard turbidityAbout 100 microgmL sterile NB was added into 96-well plate inrows B to H After that about 100 microgmL of the stock solutionwas added into rows A and B Well-mixed mixture of sampleandNB at row Bwere shifted to each well in order to achieve atwo-fold serial dilution of samples (1800 900 450 225 11255625 2813 and 1407 μgmL) Lastly about 100 microgmL in-cubated bacteria were added to all wells (from A to H) e96-well plate was capped with a lid airtight and incubated for24 hours at 37degC e microbial growth was recognized byturbidity and the presence of pallet at the base of the wells

27MinimumBactericidal Concentration MBC was used toreconfirm the results of MIC by determining the number ofthe surviving organism through observing the growth of thebacteria MBC represents the concentration at which 99 ofthe bacteria were killed About 10 μgmL of the solution inthe 96-well plate (from MIC analysis) at the first clear stage

2 Advances in Pharmacological Sciences

was transferred on the Petri dish containing nutrient agar(NA) using micropipette and spread using sterilized cottonbude Petri dish was incubated for 24 hours at 37degC If theNA inside the Petri dish is clear during observation meaningno bacteria growth so theMBC result is the same as theMICresult Meanwhile if the NA turns cloudy or not clear theMBC result showed that concentration is one step higherthan MIC e agar was prepared by dissolving NA powder(20 gL) in distilled water and then mixed e mixture wasautoclave at 121degC for 15 minutes When the NA solution iswarm enough the media is poured aseptically into Petridishes

3 Results and Discussion

31 Total Phenolic Content TPC activity is the process tofigure out the amount of phenolic content in the samplesPhenolic compounds that contained in the plants have redoxproperties and the properties allow them acting as antioxi-dants [6 7] e results (Table 1) showed that the methanolicextract exhibited higher TPC as compared to the chloroformand hexane extracts which are approximately about 4082mgGAEg for methanolic extract 3191mg GAEg for chloro-form extract and 252mg GAEg for hexane extract Higherphenolic content in the methanolic extract is responsible forbioactivity therefore this extract is expected to exhibit goodresult in antioxidant and antibacterial activities

32 Antioxidant Activity Scavenging activity of DPPH isbased on one-electron reduction which represents the freeradical reducing activity of antioxidants Ascorbic acid (AA)and quercetin (Qc) were used as positive control All thesamples were run in triplicates e results (Figure 1)showed percentage inhibition of DPPH radical of thePereskia bleo crude extract and standard at different con-centrations e lowest IC50 was detected in the methanolicextract (PbM) followed by hexane extract (PbH) andchloroform extract (PbC) with IC50 values of 3383 microgmL14355 microgmL and 37941 microgmL respectively As the lowerIC50 value possesses a higher antioxidant activity themethanol extract has a higher ability to scavenge free radicalcompared to hexane and chloroform extracts High anti-oxidant activity showed by the methanolic extract has apositive relationship with TPC activity where high TPCgives a high antioxidant capacity due to the linear correlationbetween the two parameters Previous studies have shownthat the capacity of the antioxidant is highly associated withthe total flavonoid content and total phenolic compounds ofthe plant leavesrsquo crude extract [8ndash10] Finding from thisstudy was supported by the findings reported by Sharif et al[11] where the leaves of the P bleomethanol extract showedthe lowest IC50 with a value of 6875 microgmL e study alsoreported the presence of high amounts of phytol fatty acidand sterols in the methanolic extract

33 Antimicrobial Activity Antimicrobial activities ofP bleo extracts against all four tested bacteria namelyStaphylococcus aureus (SA) Streptococcus pyogenes (SP)

Pseudomonas aeruginosa (PA) and Escherichia coli (EC)were qualitatively and quantitatively appraised by thepresence or absence of bacteria based on minimum in-hibitory concentration (MIC) and minimum bactericidalconcentration (MBC) assays Based on the MIC values themethanolic extract displayed strong activities towardsGram-positive bacteria SA and SP with the MIC value225 microgmL as well as that of against Gram-negativebacteria PA and EC with the MIC value 450 microgmL Inaddition the chloroform extract also exhibited strongactivities against SA SP and EC with the MIC value225 microgmL as well as that of against PA with the MICvalue 450 microgmL However the hexane extract demon-strated weak activities towards all four bacteria with theMIC value 1800 microgmL (Table 2)

e evaluation of antibacterial activity was extended toMBC From the result the MBC values were confirmed bythe absence of bacterial growth on the nutrients agarstreaked from the lowest clear MIC values Both methanolicand chloroform extracts of P bleo showed strong inhibitionactivity towards the tested bacteria SA SP PA and ECMostof the bacterial strains used in this study are related to thefood spoilage For example SA considered as one of themostcommon sources of food-borne disease while EC and PAproduce toxins that induce human gastroenteritis diseasesMoreover antibacterial activities studied by Abbdewahab[12] against two bacteria which are Salmonella choleraesuisand Pseudomonas aeruginosa has reported that methanoland hexane extracts of P bleo show positive result ininhibiting both bacteria e dichloromethane extract of Pbleo also demonstrated a good antibacterial effect againstStaphylococcus aureus [2 12] Besides antibacterial meth-anol leavesrsquo extract of P bleo also show great antifungalactivity against a plant pathogenic fungus Cladosporiumcucumerinum [13] As the methanolic and chloroform ex-tracts of P bleo showed strong inhibition activity towards thetested bacteria the present study suggested that this plantextract could have a great potential to be used as foodpoisoning control and natural preservatives in preservingfood for replacing chemical preservative

4 Conclusions

e methanolic and chloroform extracts of P bleo showedstrong inhibition activity towards the tested bacteria SA SPPA and EC e methanolic extract exhibited highest TPC(4082 mg GAEg) and possess highest antioxidant activity(IC50 value 3383 microgmL) compared to chloroform andhexane extracts Linear correlation among two parameters ofthe high TPC value which will give great antioxidant ca-pacity are proved in this study where high antioxidant

Table 1 Total phenolic content of P bleo hexane chloroform andmethanolic extracts

Sample extract Total phenolic content (mg GAEg extract)Hexane 2520 plusmn 001Chloroform 3191 plusmn 001Methanolic 4082 plusmn 001

Advances in Pharmacological Sciences 3

activity demonstrated by the methanolic extract is backed bythe high TPC value Due to the interesting results exhibitedby the methanolic and chloroform extracts of P bleo thestudy has extended to the isolation of bioactive compoundsto uncover its great potential natural source for antibacterialand antioxidant agents

Data Availability

No data were used to support this study

Conflicts of Interest

e authors declare that there are no conicts of interestregarding the publication of this paper

References

[1] S Zareisedehizadeh C H Tan and H L Koh ldquoA review ofbotanical characteristics traditional usage chemical com-ponents pharmacological activities and safety of Pereskia bleo(Knuth) DCrdquo Evidance-Based Complementary and Alterna-tive Medicine vol 2014 Article ID 326107 2014

[2] S I A Wahab A B Abdul S M Mohan A S Al-ZubairiM M Elhassan and M Y Ibrahim ldquoBiological activities ofPereskia bleo extractsrdquo International Journal of Pharmacologyvol 5 no 1 pp 71ndash75 2009

[3] Y S Velioglu G Mazza L Gao and B D Oomah ldquoAnti-oxidant activity and total phenolics in selected fruits vege-tables and grain productsrdquo Journal of Agricultural and FoodChemistry vol 46 no 10 pp 4113ndash4117 1998

[4] K Shimada K Fujikawa K Yahara and T NakamuraldquoAntioxidative properties of xanthan on the autoxidation ofsoybean oil in cyclodextrin emulsionrdquo Journal of Agriculturaland Food Chemistry vol 40 no 6 pp 945ndash948 1992

[5] M Gulluce H Ozer O Baris D Daferera F Sahin andM Polissiou ldquoChemical composition of the essential oils of Salviaaethiopis Lrdquo Turkish Journal of Biology vol 30 pp 231ndash233 2004

[6] A B Shoib and A M Shahid ldquoDetermination of totalphenolic and avonoid content antimicrobial and antioxi-dant activity of a root extract of Arisaema jacquemontiiBlumerdquo Journal of Taibah University for Science vol 9 no 4pp 449ndash454 2015

[7] M A Soobrattee V S Neergheen A Luximon-RammaO I Aruoma and T Bahorun ldquoPhenolics as potential an-tioxidant therapeutic agents mechanism and actionsrdquo Mu-tation Research-Fundamental and Molecular Mechanisms ofMutagenesis vol 579 no 1-2 pp 200ndash213 2005

[8] B Hassanbaglou A A Hamid A M Roheeyati et alldquoAntioxidant activity of diyenerent extracts from leaves ofPereskia bleo (Cactaceae)rdquo Journal of Medicinal Plants Re-search vol 6 no 15 pp 2932ndash2937 2012

[9] K S Sim S Nurestri and A Norhanom ldquoPhenolic contentand antioxidant activity of crude and fractionated extracts ofPereskia bleo (Kunth) DC (Cactaceae)rdquo African Journal ofPharmacy and Pharmacology vol 4 pp 193ndash201 2010

[10] R A Mustafa A A Hamid S Mohamed and F A BakarldquoTotal Phenolic compounds avonoids and radical scav-enging activity of 21 selected tropical plantsrdquo Journal of FoodScience vol 75 no 1 pp C28ndashC35 2010

[11] KM Sharif MM Rahman J Azmir et al ldquoEthanol modisectedsupercritical carbon dioxide extraction of antioxidant richextract from Pereskia bleordquo Journal of Industrial and Engi-neering Chemistry vol 21 pp 1314ndash1322 2015

[12] S I Abbdewahab N M Ain A B Abdul M M E Taha andT A T Ibrahim ldquoEnergy-dispersive x-ray microanalysis ofelementsrsquo content and antimicrobial properties of Pereskiableo and Goniothalamus umbrosusrdquo African Journal of Bio-technology vol 8 no 10 pp 2375ndash2378 2009

[13] L Rahalison M Hamburger K Hostettmann et alldquoScreening for antifungal activity of Panamanian plantsrdquoInternational Journal of Pharmacognosy vol 31 no 1pp 68ndash76 2008

Table 2 Inhibitory concentration ofMIC activity for crude extractsof Pereskia bleo

Sample extract SA SP PA ECHexane 1800 1800 1800 1800Chloroform 225 225 450 225Methanolic 225 225 450 450Note SA Staphylococcus aureus SP Streptococcus pyogenes PA Pseudo-monas aeruginosa EC Escherichia coli (unit in μgmL)

QcAAPbH

PbMPbC

000

2000

4000

6000

8000

10000

12000

0 100 200 300 400 500 600

AA IC50 1563 (microgmL)Qc IC50 3125 (microgmL)

PbM IC50 3383 (microgmL)

PbC IC50 37941 (microgmL)

PbH IC50 14355 (microgmL)

Figure 1 Graph for percent inhibition of DPPH radical of P bleo crude extract and standard at diyenerent concentrations


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prophylactic and therapeutic purposes only few Pereskiaspecies have been investigated on its phytochemical andbiological activities erefore P bleo is selected to furtherstudy on its biological activities

e intention of this study is to explore the biologicalactivities of the P bleo crude extract in exploring the po-tential of natural sources for the drug discovery which can beused for further innovation development of pharmaceuticalmedicinal health and household products in order to in-crease its commercial values Studies onmedicinal plants notonly discover new therapeutics but also assist in settingappropriate guidelines and policy in the usage of the tra-ditional herbal medicine since there are scientific evidenceand proper understanding of traditional usage of the plantsavailable

2 Materials and Methods

21 Plant Material e studied plant sample which is theleaves of P bleo was collected from Perlis Malaysia esample was air-dried at a room with room temperature andgood air ventilation A dried sample was cut into smallerpieces to make it easier for it to be grounded using an electriccutting mill to produce fine powder sample

22 Extraction e air-dried leaves (926 g) of P bleo wereextracted at 24 hours interval by the cold extraction methodand the process was repeated twice using methanol as asolvent Solvents removal under diminished pressure using aBUCHI model R-200 rotavapor produces 391371 g dark-green methanol crude extracts Chlorophyll was removedfrom the methanol extract before being performed thefractionation e fractionation process which was per-formed using liquid-liquid partition have yielded 0334 ggreen hexane fraction 58916 g dark-yellow chloroformfraction and 253989 g dark-brown methanol fraction

23 Total Phenolic Content e FolinndashCiocalteu test waschosen to measure TPC of P bleo extracts is test wasperformed by referring to the method developed by Veliogluet al [3] with some modifications e crude sample wasprepared by liquefying 10mg of the extract in 10mL of thesolvent to yield a concentration of 1mgmL About 100 microL ofthe extract (1mgmL) was combined and mixed with075mL of the FolinndashCiocalteu reagent (diluted 10-fold withdeionized water previously) in the test tube e liquidmixture was allowed to stand for 5 minutes at a roomtemperature e mixture was then added about 075mL ofsodium carbonate (Na2CO3) and the test tube was shakengently to mix them After 90minutes the absorbance of themixture was measured using the UV-Vis spectrophotometerat 725 nm

A calibration curve of standard reference was establishedusing gallic acid (range of concentration from 001 to005mgmL) as standard references plotted TPC wasrevealed as gallic acid equivalents in milligrams per 100g ofthe extract

24 Antioxidant Activity e DPPH was used to determinefree radical scavenging activity as previously described byShimada et al [4] About 394mg ofDPPHwas first dissolved in100mL of ethanol to a concentration of 01mL About 1mL ofDPPH solution was added to 3mL of the samples with differentconcentrations (250 125 625 3125 and 1562μgmL)

For the control test the same amount of ethanol wasadded All the mixture was mixed well by shaking vigorouslyand left to stand for 30 minutes at a room temperature Afterthat the UV-Vis spectrophotometer was used to measurethe value of absorbance of each mixture at 517 nm ecalculation for the percentage of inhibition (I) of theDPPH radical is as follows

I Ao minus As

Ao

1113888 11138891113890 1113891 times 100 (1)

where AS represents the absorbance value of the sample whileA0 represents the absorbance value of the control reaction(contain all reagents except the sample) A graph of inhibitionpercentages (I) against concentrations of the sample wasplotted From the graph 50 inhibition (IC50 value) providesthe value of concentrations for each sample All experimentswere carried out in triplicate to minimize the precision errorMean plusmn SD of triplicates was reported as IC50 values

25 Antimicrobial Activity Antimicrobial activities wereconducted using MIC and MBC assays e MIC method asdescribed by Gulluce et al [5] involves the broth micro-dilution technique using 96-well microplates In this studyfour types of bacteria are used which are SP SA EC and PA

26 Minimum Inhibitory Concentration e MIC repre-sents the lowest concentration that had absence of micro-scopically noticeable growth e interpretation of in vitrodata is based on attainable sample concentrations Nutrientbroth (NB) was prepared as the medium About 36mg crudeextract samples were weighted and dissolved in 2mL DMSO(stock solution 1800 μgmL) Inoculations of the microbialstrains were produced from 24 hours broth cultures andsuspensions regulated to 05 McFarland standard turbidityAbout 100 microgmL sterile NB was added into 96-well plate inrows B to H After that about 100 microgmL of the stock solutionwas added into rows A and B Well-mixed mixture of sampleandNB at row Bwere shifted to each well in order to achieve atwo-fold serial dilution of samples (1800 900 450 225 11255625 2813 and 1407 μgmL) Lastly about 100 microgmL in-cubated bacteria were added to all wells (from A to H) e96-well plate was capped with a lid airtight and incubated for24 hours at 37degC e microbial growth was recognized byturbidity and the presence of pallet at the base of the wells

27MinimumBactericidal Concentration MBC was used toreconfirm the results of MIC by determining the number ofthe surviving organism through observing the growth of thebacteria MBC represents the concentration at which 99 ofthe bacteria were killed About 10 μgmL of the solution inthe 96-well plate (from MIC analysis) at the first clear stage









4 Conclusions






Data Availability




References
















QcAAPbH

PbMPbC

000

2000

4000

6000

8000

10000

12000

0 100 200 300 400 500 600













Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of









4 Conclusions






Data Availability




References
















QcAAPbH

PbMPbC

000

2000

4000

6000

8000

10000

12000

0 100 200 300 400 500 600













Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of



Data Availability




References
















QcAAPbH

PbMPbC

000

2000

4000

6000

8000

10000

12000

0 100 200 300 400 500 600













Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of








Arthritis










AddictionJournal of







Journal of


Pharmaceutics



Volume 2018



Canadian Journal of






of


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