Phytochemical Analysis, Antibacterial And Antioxidant .
Pharmacy & Pharmacology International JournalsResearch ArticleOpen AccessPhytochemical analysis, antibacterial and antioxidantactivity determination of Ocimum sanctumAbstractVolume 6 Issue 6 - 2018The objective was to analyse phytochemical constituents from the leaves of Ocimumsanctum using suitable solvents and extraction technique and to evaluate the in-vitroantibacterial and in-vitro antioxidant activities of leaf extract of Ocimum sanctum.In present work, Soxhlet extraction and maceration extractions were applied tothe fresh leaves of the Ocimum sanctum by using absolute ethanol. Phytochemicalanalysis for the important chemical constituents from ethanolic extract was carriedout. Antimicrobial activity of Ocimum sanctum extract was carried out using WellDiffusion method by comparing the clear inhibition zone of standard antibiotic andthe extracts on the Mueller Hinton agar. Antioxidant activity of Ocimum sanctum wascarried out performing total phenolic content test and DPPH to identify the percentageof scavenging by the chemical constituents. For phytochemical analysis, only test foralkaloids, test for terpenoids and test for carbohydrates showed positive results forOcimum sanctum extract. For antibacterial screening, all the concentrations of OSESEshowed negative results due to low concentration of extract being used. For antioxidantanalysis, total phenolic contents and DPPH radical scavenging showed antioxidantresult for OSESE. It is concluded that Ocimum sanctum is a very essential plantmedicinally. A long term research project is a must to evaluate the pharmacologicaluses of extracts with different solvents that can be used to isolate the pure and highyield of chemical constituents from the plants.Kang Zhi Xia, Nabila Perveen, Naeem HasanKhanDepartment of Pharmaceutical Chemistry, AIMST University,MalaysiaCorrespondence: Naeem Hasan Khan, Department ofPharmaceutical Chemistry, Faculty of Pharmacy, AIMSTUniversity, Bedong 08100, Kedah Darul Aman, Malaysia, Tel 006016 9372470, EmailReceived: November 18, 2018 Published: December 18,2018Keywords: soxhelt, maceration, ethanol, phytochemical, antibacterial, anti-oxidantIntroductionThe plant’s height is up to 1 m, with branches, carrying with apungent aromatic odour smell, the branchlets and new growthpubescent with soft white hairs. The Ocimum sanctum’s leaves withblades elliptic to elliptic-oblong approximately 3 to 6cm long, widthis1–2.5cm, cuneate to attenuate at base, obtuse to acute at apex,entire to remotely serrate at margins, pubescent on both surfaces butespecially on the nerves beneath. For the flowers terminal, the slenderracemes or panicles are 4 to 12cm long with the width of 1 to 1.5cm,the bracteoles are 2 to 3mm long, ovate, acuminate, ciliate; flowers inverticils, on the pedicels are 2 to 4.5mm long; at anthesis, the calyx cis 2.5mm long, in fruit up to 5mm long, glabrous within, the upper lipsuborbicular, reflexed, short-apiculate, the lower lip longer than theupper lip, the teeth 4, lanceolate; corolla pale pink, pale lavender orwhite, to 4mm long; filaments of stamens exerted, slender, the upperpair of each with a small, bearded basal appendage. The appearanceof fruit is purple-green to brown, broadly ellipsoid, approximately0.8–1.2mm long, smooth to minutely pitted, swelling in water.1,2 Theleaves and flowers of Ocimum sanctum are shown in Figure 1.Figure 1 The leaves and flowers of Ocimum sanctum.Other names of Ocimum sanctum are Tulsi, Tulasi, Gouri,Bhuteshta, Bhutaghini, Nagamata, Surasah, Mal-Tulasi, Krsiatulasi,Indian Basil, Holy Basil, Sacred Basil, Nalla Thulasi, Raihan, LoLe, Basil Icum, Basilic, Basilienkraut, Selasih, Kemangi, Basilico,Meboki, Selaseh, Belanoi, Sulasi, Man Jericao, Bazilik, Albahaca,Suwenda-Tala, Maduru-Tala, Basilkort, Horopa, Manghk, Krapow,Bai Horapa, Rau Que.3,4 It is available in India, Sri Lanka, Himalaya,Bangladesh, South West Asia, Burma, China, Thailand, Malaysia. Inaddition, it is also available at dry sandy areas in Hainan, Sichuan,Taiwan Cambodia, Indonesia, Laos, Myanmar, Philippines, Vietnam;Africa, South West Asia, Australia.2 Ocimum sanctum, the Queenof medicinal herbs is the holiest and the most valuable of the manySubmit Manuscript http://medcraveonline.comPharm Pharmacol Int J. 2018;6(6):490‒497.healing and ill-health giving herbs of the suitable way.5,6 The blessedBasil or Tulsi is significant in the traditional Ayurvedic and Unanisystem.5 In India, Ocimum sanctum are believed that it can be giventhe treatment of bronchitis, bronchial asthma, malaria, diarrhoea,dysentery, skin diseases, arthritis, painful eye diseases, increasein body temperature and also insect bite. More importantly it hasanticancer, antifungal, antihypergycaemic, antibacterial, in treatmentof nausea and vomiting, protection against liver and heart, analgesic,adaptogenic and diaphoretic actions. It improves the body immunesystem, reproductive system, CNS, CVS, gastrointestinal tractsystem, urinary system and also blood circulation.4 Table 1 show thenutritional facts about Ocimum sanctum.490 2018 Xia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, whichpermits unrestricted use, distribution, and build upon your work non-commercially.
Copyright: 2018 Xia et al.Phytochemical analysis, antibacterial and antioxidant activity determination of Ocimum sanctumTable 1 Nutritional highlight of Ocimum sanctumIn fresh Tulsi, five leaves were tested. (2.5g)Table ceration-Ethanolic SoxhletSample-8Flavonoids---9Carbohydrate Calories0.675Protein0.064gCarbohydrate0.108gTotal Fat0.015g presence, - absenceFiber0.098gMethodology for determination of antibacterialactivity Preparation of Luria Bertani broth mediaMethodsCollection and preparation of plant materialsGreen and fresh 557g of Ocimum sanctum was collected from theplants. The leaves were cleaned by distilled water and then leaveswere separated from the branches manually. The separated leaveswere weighed again and net weight was 349.64g and allowed forair drying under the room temperature to avoid destruction of activegroup in the leaves. The dried leaves were crushed by using hand intovery small pieces.MacerationThe 25.0g of crushed raw material was subjected to macerationwith 200ml of absolute ethanol in round bottom flask and sealed withthe aluminium foil and kept in the dark for seven days. The roundbottom flask was shaken throughout to ensure uniform and completeextraction. The mixture was filtered by using clean Muslin cloth andthe filtrate was collected in a cleaned beaker. The residue of macerationextract and filtrate of maceration were separated and being kept insidethe cabinet for further screening.Soxhlet extraction26.0g of the crushed powder form was placed inside a thimblealready fixed with the chromatographic paper. Ethanol added was350ml for the extraction and poured into the round bottom flask ofSoxhlet apparatus. The temperature was kept at 70 C and maintainedthroughout the process. The whole process took about 30 hours tocomplete till the clearance of colour extract. The residue of macerationextract and filtrate of maceration were separated and being kept insidethe cabinet for further screening.EvaporationThe evaporation was carried out from the extract (Soxhlet andMaceration) in a rotary evaporator. The temperature was set at 70 Cthroughout the evaporation and concentration. The extracts of Soxhletand maceration, after evaporation, were 88ml and 25ml respectively.Phytochemical screening7–12The results of phytochemical analysis is recorded and tabulated inTable 2.7–12Table 2 Results for qualitative phytochemical rateResidueEthanolic SoxhletSample1Alkaloids 2Reducing Sugar---3Saponins---4Terpenoids 5Antraquinones---6Glycosides---No.4912.0g of Luria Bertani broth was dissolved in 100ml distilled water.Then, 10ml of Luria Bertani broth was poured into each 4 universalbottles and subjected to the autoclave at high pressure saturated steam121 C for around 1 hour in the laboratory.13–17Preparation of Muller Hinton Broth38.0g Muller Hinton Broth was dissolved into 1000ml of distilledwater and poured into two 500ml of Scott bottles. After that, the twoScott bottles were taken to autoclave at high pressure saturated steam121 C for around 1 hour in the Biotechnology laboratory. After theautoclaving have done, the sufficient sterilized quantity of MullerHinton agar was poured into the sterilized petri plates and was allowedto solidify. Agar plates were stored in incubator at about 37 C.13–17Bacteria strains culturesBacteria strains of Bacillus subtilis, Pseudomonas aeruginosa,Escherichia coli and Streptococcus pyogenes were cultured usingLuria Bertani broth in 4 different universal bottles. The bacterialstrains stored in the universal bottles were left shaking incubator for24 hours at 37 C at 180rpm. The next days, the bacteria strains whichhas grown in Luria Bertani broth is then cultured into Muller Hintonagar.16Dilution of extracts16(dissolved in sterilized distilled water)a. To prepare 1.0mg/ml of the extract, 10mg of the extract wasdissolved using 10ml of sterilized distilled water.b. To prepare 5.0mg/ml of the extract, 50mg of the extract wasdissolved using 10ml of sterilized distilled water.c. To prepare 10.0mg/ml of the extract, 100mg of the extract wasdissolved using 10ml of sterilized distilled water.Well Diffusion test preparation in laminar air flowcabinet16,17i.ii.iii.iv.v.vi.vii.viii.1mg/ml of ciprofloxacin was used as a positive control andsterilized distilled water was used as a negative control.Marker pen was used to label the bottom of the prepared petridish.100µl of bacterial strain (Bacillus subtilis) was spread onto thesurface of agar by using spreader.Five different spot corresponding to the label around 6 to 8mmwere punched aseptically by using cork-borer.Each holes was filled with ciprofloxacin, sterilized distilledwater, 1mg/ml of extract, 5mg/ml of extract and 10mg/ml ofextract respectively by using 100µl micropipette.The procedure was repeated for another three bacterial strains(Pseudomonas aeruginosa, Escherichia coli and Streptococcuspyogenes).After that, the agar plates were covered and subjected to incubateat 37 C for 24 hours.Antimicrobial activity is determined by measuring with theinhibition zone.Citation: Xia KZ, Perveen N, Khan NH. Phytochemical analysis, antibacterial and antioxidant activity determination of Ocimum sanctum. Pharm Pharmacol Int J.2018;6(6):490‒497. DOI: 10.15406/ppij.2018.06.00223
Copyright: 2018 Xia et al.Phytochemical analysis, antibacterial and antioxidant activity determination of Ocimum sanctumResults of antibacterial activityResult for the Well Diffusion Test. Table 3 indicates the zone ofinhibition in mm. In present antibacterial study, two different extractswith three different concentrations were investigated to detect thezone of inhibition against Bacillus subtilis, Pseudomonas aeruginosa,Streptococcus pyogenes and Escherichia coli. Ciprofloxacin was usedas a positive control and sterilized distilled water was used as a negativecontrol. As a result, there is no antibacterial inhibition zone occurredin the most of the agar plates. This may be due to low concentration492of the extract being used. Low concentration of the Ocimum sanctumwas unable to produce any antibacterial effect against the bacteriastrains in the agar plate. Figure 2, Figure 4, Figure 6 and Figure 8shows the ethanolic maceration extract and Figure 3, Figure 5, Figure7 & Figure 9 indicates the ethanolic Soxhlet extract on the agar plate.All Figures shown at lower region, from 2–9, showed that the agarplates do not have any antibacterial activity by well diffusion method.There was an absence of zone of inhibition for each of the differentconcentration used by the extract solution.16,17Table 3 Zone of inhibition in mmMicroorganismsBacillus subtilisPseudomonas aeruginosaStreptococcus pyogenesEscherichia coliConcentrated extract1 mg/ml5 mg/ml10 mg/mlCiprofloxacinSterilized distilled waterEthanolic Maceration---24-Ethanolic Soxhlet---24-Ethanolic Maceration---26-Ethanolic Soxhlet---24-Ethanolic Maceration---26-Ethanolic Soxhlet---26-Ethanolic Maceration---24-Ethanolic Soxhlet---24-Figure 2 Ethanolic maceration extract on the agar plate.Figure 5 Ethanolic Soxhlet extract on the agar plate.Figure 3 Ethanolic Soxhlet extract on the agar plate.Figure 6 Ethanolic maceration extract on the agar plate.Figure 4 Ethanolic maceration on the agar plate.Figure 7 Ethanolic Soxhlet extract on the agar plate.Citation: Xia KZ, Perveen N, Khan NH. Phytochemical analysis, antibacterial and antioxidant activity determination of Ocimum sanctum. Pharm Pharmacol Int J.2018;6(6):490‒497. DOI: 10.15406/ppij.2018.06.00223
Phytochemical analysis, antibacterial and antioxidant activity determination of Ocimum sanctumCopyright: 2018 Xia et al.493was Gallic acid and the measurement was done at λ max 765nm.Gallic acid standard calibration curve was generated as shown inGraph 1 and the regression value was 0.9884. From the absorbanceobtained, the amount of the phenolic content measured as Gallicacid equivalent (GAE) using FC method. The total phenolic contentobtained at the concentration of 1.0mg/ml was found to be the highestwhen compared to other lower concentration extract solution. As aresult, the higher the concentration of the stock solution used for thisanalysis, the higher the Gallic acid equivalent (GAE).19–25Figure 8 Ethanolic maceration extract on the agar plate.Graph 1 Standard graph of the Gallic acid plotted from the series ofabsorbance obtained from UV- Visible Spectrophotometer.Extract preparationi.Figure 9 Ethanolic Soxhlet extract on the agar plate.Pseudomonas aeruginosaii.iii.Figure 2 & Figure 3iv.Escherichia coliv.Figure 4 & Figure 5Bacillus subtilisvi.Figure 6 & Figure 7To prepare 1mg/ml of the extract solution, 10mg of the extractwas dissolved using 10ml of ethanol.To prepare 0.8mg/ml of the extract solution, 8ml of the 1mg/mlof the extract solution was dissolved using 10ml of the ethanol.To prepare 0.6mg/ml of the extract solution, 7.5ml of the 0.8mg/ml of the extract solution was dissolved using 10ml of the ethanol.To prepare 0.4mg/ml of the extract solution, 6.67ml of the 0.6mg/ml of the extract solution was dissolved using 10ml of the ethanol.To prepare 0.2mg/ml of the extract solution, 5ml of the 0.4mg/mlof the extract solution was dissolved using 10ml of the ethanol.To prepare 0.1mg/ml of the extract solution, 5ml of the 0.2mg/mlof the extract solution was dissolved using 10ml of the ethanol.Preparation of sample extract, blank and standard16,17Streptococcus pyogenesi.Figure 8 & Figure 9All Figures from 2–9 showed that the agar plates do not haveany antibacterial activity determination by well diffusion method.There was an absence of zone of inhibition for each of the differentconcentration used by the extract solution.ii.iii.Antioxidant activity determinationMethodology for determination of antioxidant activityTotal phenolic contents analysis (TPC) : For the total phenoliccontents analysis, Folin-Ciocalteau (FC) assay was applied toOcimum sanctum ethanolic Soxhlet extract to find out the totalphenolic content of this extract. In this analysis, the standard used18iv.v.vi.0.5ml of the extract solution (1.0mg/ml) was added with the2.5ml of the 0.75% sodium bicarbonate and 2.5ml of the 1%Folin–Ciocalteu’s reagent.The sample mixtures were incubated at 45 C for 15 minutes. TheUV absorbance was detected at λ max 765nm.The steps were repeated by using other concentration of extractsolution (0.8mg/ml, 0.6mg/ml, 0.4mg/ml, 0.2mg/ml and 0.1mg/ml).Blank was prepared by using ethanol instead of extract solution.The same steps were prepared for the Gallic acid (standard) andthe calibration line was plotted.The total phenolic content was calculated in term of Gallic acidequivalent (mg of GAE/g of extrtact) by using formula of:Citation: Xia KZ, Perveen N, Khan NH. Phytochemical analysis, antibacterial and antioxidant activity determination of Ocimum sanctum. Pharm Pharmacol Int J.2018;6(6):490‒497. DOI: 10.15406/ppij.2018.06.00223
Copyright: 2018 Xia et al.Phytochemical analysis, antibacterial and antioxidant activity determination of Ocimum sanctumC (A/B) x dilution factorC, Total phenolic content; A, x value; x, regression line; B,concentration of extractDPPH free radical scavenging assay16i.ii.iii.iv.v.vi.measured at 517nm against a blank by using UV-Visiblespectrophotometer.vii. The percentage of DPPH scavenging activity was calculatedusing the following equation: % Scavenging of test sampleDifferent concentration of extract solution (0.03125, 0.0625,0.125, 0.25, 0.5 and 1.0mg/ml) were prepared as well as control(methanol without extract sample).Different concentration of extract solution was subjected to theuniformly mixing.5.0ml of methanolic solution of DPPH reagent was addedindividually to each of the different concentration of the extractsolution.The mixture samples were then subjected to vortex for fewminutes.The mixture samples were incubated at room temperature in thedark for around 30 minutes.The absorbance for each concentration mixture samples was494control absorbance test absorbancecontrol absorbance 100viii. All the procedure was repeated for BHT solution (standard) ofdifferent concentration (similar to the previous concentration).ix. The 50% inhibitory concentration value (IC50) for both extractswere calculated and it is indicated as the effective concentrationof the sample that is required to scavenge 50% of the DPPH freeradicals.x. The data for both extract sample and standard were obtained andtabulated.Results of antioxidant activityThe results are shown in Table 4, Table 5, Table 6.OSESETable 4ConcentrationyxABDilution factorC0.3008 x 0.073mg/ml0.073mg/ml0.1mg/ml 0.0001g/ml1/10000(A/B) x dilution factor (0.073/0.0001) x1/10000 0.073 mg GAE/g0.084mg/ml0.084mg/ml0.2mg/ml 0.0002g/ml1/5000(A/B) x dilution factor (0.084/0.0002) x1/5000 0.084 mg GAE/g0.297mg/ml0.297mg/ml0.4mg/ml 0.0004g/ml1/2500(A/B) x dilution factor (0.297/0.0004) x1/2500 0.297 mg GAE/g0.513mg/ml0.513mg/ml0.6mg/ml 0.0006g/ml3/5000(A/B) x dilution factor (0.513/0.0006) x3/5000 0.513 mg GAE/g0.06480.1mg/ml0.067 0.3008x 0.06480.3008 x 0.00220.3008x 0.06480.090 0.2mg/ml0.3008x 0.06480.3008x 0.02520.3008x 0.06480.154 0.4mg/ml0.3008x 0.06480.3008x 0.08920.3008x 0.06480.219 0.6mg/ml0.3008x 0.06480.3008x 0.1542Citation: Xia KZ, Perveen N, Khan NH. Phytochemical analysis, antibacterial and antioxidant activity determination of Ocimum sanctum. Pharm Pharmacol Int J.2018;6(6):490‒497. DOI: 10.15406/ppij.2018.06.00223
Copyright: 2018 Xia et al.Phytochemical analysis, antibacterial and antioxidant activity determination of Ocimum sanctum495Table Continued.ConcentrationyxABDilution factorC0.3008x 0.679mg/ml0.679mg/ml0.8mg/ml 0.0008g/ml1/1250(A/B) x dilution factor (0.679/0.0008) x1/1250 0.679 mg GAE/g0.961mg/ml0.961mg/ml1.0mg/ml 0.001g/ml1/1250(A/B) x dilution factor (0.961/0.001) x1/1000 0.961 mg GAE/g0.06480.2690.8mg/ml 0.3008x 0.06480.3008x 0.20420.3008x 0.06480.354 1.0mg/ml0.3008x 0.06480.3008x 0.2892Table 5 UV absorbance of Gallic acid in various concentrationS. NoConcentration of Gallic acid / 50.80.294610.377Table 6 Dilution factors for Gallic acid in total phenolic analysisConc. Of Sample (mg/mL)1001010.10.20.30.40.60.8Stock solution (ml)100010010111111Vol. of stock solution (ml)111123468Vol. of 95% methanol (ml)999987642Total Volume (ml)101010101010101010Dilution 50001/1250DPPH free radical scavenging analysis18Table 8 Percentage scavenging of BHT and OSESE in DPPH assayTable 7, Table 8Table 7 Absorbance value of standard (BHT) and extract of Ocimumsanctum at 517
antibacterial and in-vitro antioxidant activities of leaf extract of Ocimum sanctum. In present work, Soxhlet extraction and maceration extractions were applied to the fresh leaves of the Ocimum sanctum by using absolute ethanol. Phytochemical analysis for the important chemical constituents from ethanolic extract was carried out. Antimicrobial activity of Ocimum sanctum extract was carried .
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Designed by Cardiff Archaeological Illustration and Design Software: Adobe Creative Suite 4 Design Premium ARCHAEOLOGICAL EVALUATION OF THE EXTRAMURAL MONUMENTAL COMPLEX (‘THE SOUTHERN CANABAE’) AT CAERLEON, 2011. Contents Introduction 1 Background 3 Project Aims & Objectives 9 Methodology 11 Results of the excavations 13 Trench 1 15 Trench 2 25 Trench 3 29 Trench 4 33 Trench 5 39 Trench 6 .
