Evaluation Of Antioxidant, Thrombolytic And Cytotoxic .
Journal of Applied Pharmaceutical Science Vol. 8(07), pp 051-056, July, 2018Available online at http://www.japsonline.comDOI: 10.7324/JAPS.2018.8709ISSN 2231-3354Evaluation of Antioxidant, Thrombolytic and Cytotoxic Potentialsof Methanolic Extract of Aporosa wallichii Hook.f. Leaves: AnUnexplored PhytomedicineShahana Sharmin1, Md. Tanvir Kabir1*, Md. Nasiful Islam1, Mohd. Raeed Jamiruddin1, Imon Rahman1, Arifur Rahman2,Mahboob Hossain3Department of Pharmacy, BRAC University, Dhaka, Bangladesh.Department of Pharmacy, Jagannath University, Dhaka, Bangladesh.3Microbiology Program, Department of Mathematics and Natural Sciences, BRAC University, Dhaka, Bangladesh.12ARTICLE INFOABSTRACTArticle history:Received on: 11/04/2018Accepted on: 08/06/2018Available online: 30/07/2018Aporosa wallichii Hook.f. is a medicinal plant, which belongs to Phyllanthaceae family. This plant was used inthis study for the determination of several pharmacological activities. This study involved experiments includingdetermination of antioxidant property by performing 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavengingassay and total phenolic content (TPC) test, determination of cytotoxic activity by brine shrimp lethality assay andthrombolytic property analysis. In this study, it was observed that for DPPH free radical scavenging assay; standard(ascorbic acid) provided the IC50 value of 75.6 μg/mL, whereas the extract provided the IC50 value of 58.7 μg/mL.In case of TPC test, plant extract showed 308.97 mg of gallic acid equivalents/g of extract. Brine shrimp lethalitybioassay revealed the cytotoxic property of the plant and the LC50 value of the plant extract was 26.7 μg/mL. Thenagain, the standard vincristine sulfate provided the LC50 value of 2.0 μg/mL. Moreover, the thrombolytic analysisshowed for standard (clopidogrel) 59.3% clot lysis and for extract 24.5 % clot lysis. This study revealed that this planthas significant antioxidant activity, moderate level of cytotoxic activity and thrombolytic activity.Key words:DPPH free radicalscavenging assay, Totalphenolic content, Brineshrimp lethality assay,Thrombolytic property,Cytotoxicity.INTRODUCTIONThe medicinal plants serve as a vital source of newpharmacologically active compounds (Atanasov et al., 2015).These agents are derived from different parts of plants and useddirectly as drug or as semi-synthesized or synthesized drug (Ghani,2003). Origin of Aporosa wallichii Hook.f is dry evergreen forestsof Meghalaya and Tripura in India, Bangladesh, Myanmar andThailand (Schot, 2004). The local name of this plant is Kokra inBangladesh and commonly known as Castoma (Hossain et al.,2015). The height of the tree can be up to 15 m and the diameteris about 15 cm. It has thick, rough, grooved and brownish colorbark. Plant’s stipules are narrowly ovate and the size is about 5-7Corresponding AuthorMd. Tanvir Kabir, Department of Pharmacy, BRAC University, Dhaka,Bangladesh. E-mail: tanvir kbr @ yahoo.com*mm 1.5-2.8 mm. Leafstalk is cylindrical, smooth and size up to6-19 mm 0.8-1.2 mm. Leaves narrowly egg-shaped to narrowlyelliptic and the size is up to 9-17.5 cm 3.5-6.5 cm, the base is thickand small basal glands is black in color. The color of the upper partof the leaves is bright to yellowish green and color of lower partof the leaves is reddish green or brownish. Cuspidate of Aporosawallichii Hook.f. is sharp, smooth, a little shiny and not fragile. Onthe surface of the plant’s leaf, irregular dense dots are present. Fruitsare smooth and ovoid. Young fruits are striped and the size is 9-11mm 6-9 mm and the color is brown to black. Each fruit containseither 1 or 2 seeds (Schot, 2004). Aporosa wallichii Hook.f. isone of the plants of a Phyllanthaceae family. Different medicinalplants contain many therapeutic activities to treat cancer, skindiseases, inflammation, diarrhea, dysentery, jaundice and headacheetc. (Rahman and Akter, 2013). A literature review of Aporosawallichii Hook.f. plant showed that no significant previous study 2018 Shahana Sharmin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial-ShareAlikeUnported License ).
052Sharmin et al. / Journal of Applied Pharmaceutical Science 8 (07); 2018: 051-056has been conducted on this plant (Fabricant and Farnsworth, 2001).However, previous studies on various species of Phyllanthaceaefamily showed powerful antioxidant, anthelmintic, antimicrobial,anti-diarrheal, anti-tumor, anti-inflammation and insecticidalproperties (Li, 2000). Aporosa lindleyana is also from the samegenus and has antioxidant, anti-amylase and lipid-loweringproperties (Kathirgamanathar et al., 2018). This plant also hasantimicrobial, analgesic (Srikrishna et al., 2008) and antidiureticactivity (Ganegamage et al., 2014). Other species like Baccaureaparviflora, Antidesma tomentosum, Aporosa aurea, and Mallotuspaniculatus have various pharmacological properties includingcytotoxicity and antitrypanosomal activity (Mohmod et al., 2015).Croton gratissimus also has an acceptable rate of antiplasmodialactivity. On the other hand, Croton argyratus has excellentantiprotozoal activity (Abdullah et al., 2007). So, there is a highpossibility of the presence of different types of pharmacologicalproperties in Aporosa wallichii Hook.f. plant. Free radicals areresponsible not only for aging but also for many age-relateddiseases (Harman, 2009). Evidence from various sources suggeststhat free radicals trigger cell death mechanisms in the body such asapoptosis and necrosis (Chatterjee et al., 2011). Blockage of veinsis a thrombosis which affects different organs and this thrombosiscan cause various pathological conditions (Bekker et al., 2009).Antithrombotic and thrombolytic therapies have a great effecton thrombosis and they play crucial roles in the management ofthromboembolic disorders (Hirsh et al., 2008). Cytotoxic propertyis very important to destroy cancer cells. Therefore, this plantAporosa wallichii Hook.f. can be a potential source of medicinalproperties and for this purpose, this study was focused on to findout the antioxidant, cytotoxicity and thrombolytic activity of theplant.MATERIALS AND METHODSCollection of plant materialsThe leaf part of Aporosa wallichii Hook.f. plant wascollected in May 2017 from Moulovibazar district of Sylhetdivision; after that, it was sent to the National HerbariumBangladesh (NHB), Mirpur, Dhaka for verification. It wasauthenticated by NHB and the plant accession number was alsoprovided (DACB-44996).Preparation of the extractThe leaves were washed with clean water to removethe plant scrap and dust particles. Then the cleaned leaves wereallowed to dry under the sun for a day after which the leaveswere dried for 1 hour at 30-40 C in a hot air oven. After that, thedry and crusty leaves were ground with coarse dust using a highcapacity grinding machine. Approximately 900 g of powder wassoaked in 2.5 L of methanol for a period of 2 days at a normalambient temperature (22-25 C) with occasional stirring. Afterthat filtration was done by using a cotton filter (pore size: 110mm), then the maximum amount of solvent was evaporated byusing rotary evaporator at 100 rpm at 30 C. Then the extract ofthe leaves was placed under laminar airflow cabinet to vaporizethe solvent completely from the extract and it was used to avoidany possibility of microbial growth in the extract while drying.Finally, 22.4 g of plant leaf extract was obtained and it was keptin a dry and cool place with proper labeling. Antioxidant, brineshrimp lethality assay and thrombolytic studies were conductedwith this extract.ChemicalsThe chemicals were gallic acid [Sigma-Aldrich, USA],sodium chloride [Sigma-Aldrich, USA], Folin-Ciocalteu reagent[Sigma-Aldrich, USA], vincristine sulphate [Sigma-Aldrich,USA], clopidogrel [Sigma-Aldrich, USA], 2,2-Diphenyl-1Picrylhydrazyl (DPPH) [Sigma-Aldrich, USA]., sodium carbonate[Merck, India] and ascorbic acid (ASA) [Merck, India], dimethylsulfoxide (DMSO) [Fisher Scientific, UK]. All the chemicals usedin this study were of analytical grade.Total phenolic content (TPC)Folin-Ciocalteu chemical easily oxidizes the phenolswhen these chemicals added in this ionic phenolic solution, thephenols easily oxidizes by reagents (Singleton et al., 1999). Whenthe oxidation procedure completed in the solution yellow colorof Folin-Ciocalteu chemical turned into dark blue. This colorchanged mixture measured in a 760 nm in UV spectrophotometer.Value of the absorbance plotted in the gallic acid calibration curveand data was evaluated as gallic acid equivalents (GAE) (Wolfeet al., 2003).DPPH free radical scavenging assayDPPH free radical scavenging assay was performed todetermine the antioxidant activity of Aporosa wallichii. Methanolplant extracts showed free radical scavenging activities on stable2,2-diphenyl-1-picrylhydrazyl radicals were measured for this test(Braca et al., 2001). At different concentrations, plant extractswere mixed with 2,2-diphenyl-1-picrylhydrazyl (DPPH) solution.In methanol or aqueous solution, it generated stable free radicalsby the delocalization of the free electrons; which in turn produceda deep purple colored solution. Absorbance values of theseconcentrations were calculated at 517 nm in UV spectrometer andthe decreasing value of DPPH at 517 nm is directly proportionalto the radical scavenging activity (Brand-Williams et al., 1995).Percentage of inhibition of DPPH free radical (I%) wascalculated by using the following equation:(I%) ((Absorbance of blank Absorbacne of sample)/(Absorbance of blank)) 10050% of inhibition (IC50) of extract concentration wascalculated from the graph; where the percentage of inhibition (I%)was plotted against extract concentration.Brine shrimp lethality assayArtemia salina shrimp used in this assay. Its offspringwas hatched in replicated seawater to grow nauplii (Meyer et al.,1982). By adding the calculated amount of dimethylsulfoxide(DMSO), the sample was prepared in desired concentration bydilution. The nauplii were counted with visual examination andwere placed in vials which contained around 5 mL simulatedseawater. Subsequently, various concentrations of samples wereadded to the tubes by micropipette and vincristine sulfate wasused as positive control. These tubes were then left in a dry placefor 24 hours at room temperature. Finally, survivors were countedafter 24 hours (Hossain et al., 2012). Percentage (%) of mortality
Sharmin et al. / Journal of Applied Pharmaceutical Science 8 (07); 2018: 051-056was calculated by using following equation:053(%) of clot lysis ((released clot weight)/(clot weightafter clot disruption)) 100.Percentage (%) of mortality ((Number of nauplii taken Number of nauplii alive)/(Number of nauplii taken)) 10050% of lethal concentration (LC50) of extractconcentration was calculated from the graph plotted percentage ofmortality against concentration.Thrombolytic activityThrombus hampers the normal flow of blood to cells andtissues by blocking the blood vessel which can lead to lack of bloodand oxygen. So, thrombolytic medications such as urokinase,clopidogrel, and streptokinase remove this thrombus and help tokeep cells and tissues in normal condition (Prasad et al., 2006).In this study, fresh human blood was collected and blood sampleswere taken in three different pre-weighed sterile microbes andallowed to incubate at 37 C for 45 min. When the clot was formed,the upper fluid was entirely discharged from all micro-tube lines.Clopidogrel was used as positive control and water (distilled) wasused as negative control. Each test tube was filled with 100 μlof plant extract and then microtubes were incubated for 90 minat 37 C. Afterward, the liquid was removed which was releasedfrom the clot and again the tubes were weighed to see the weightdifference when the clot disruption occurred (Ali et al., 2014).Percentage of clot lysis was calculated by the followingequation:RESULTS AND DISCUSSIONAntioxidant propertyIn this experiment, methanol extract of Aporosa wallichiiHook.f. leaves were tested properly through DPPH assay and TPCto determine the antioxidant property of this plant (Pavithra et al.,2009). Antioxidant activity is very important in preventing freeradical reactions because they can neutralize free radical by theirreducing ability.Determination of DPPH radical scavenging activityHere, the percentage of inhibition of ascorbic acid andmethanol extract in different concentrations were obtained (Table1). It was found that DPPH free radical scavenging activity wasincreasing along with increasing concentration of the methanolextract (Figure 1). As the reference standard, ascorbic acid wasused in this experiment for which IC50 value was 75.6 µg/mL. Onthe other hand, the IC50 value of the methanol extract of Aporosawallichii Hook.f. leaves were 58.7 μg/mL (Table 1). This resultindicates the presence of DPPH free radical scavenging activity;which specifies the presence of antioxidant activity in Aporosawallichii Hook.f.Table 1: Evaluation of DPPH free radical scavenging activity of methanol extract of Aporosa wallichii Hook.f. leaves.Concentration(µg/mL)Absorbance of ascorbicacidAbsorbance of plantextractAscorbic acid inhibition(%)Methanol extractinhibition 24.60.9770.4970.57419.47.0Blank% o f I n h i b it i o n100M e t h a n o l e x t r a c t I n h ib itio n ( % )y 0 .1 3 1 x 4 2 .3 0 7R ² 0 .5 0 4 68060A s c o r b ic a c id I n h ib itio n ( % )y 0 .1 5 1 4 x 3 8 .5 1 8R ² 0 .6 2 7 740200100IC50 value in µg/mL(Methanol extract)75.658.7Absorbance 0.617C o m p a r is o n B e t w e e n S t a n d a r d a n d S a m p le0IC50 value in µg/mL (Ascorbic acid)200300400500600C o n c e n t r a t io n ( µ g /m l)Fig. 1: Comparison of % inhibition between ascorbic acid and methanolextract.Total phenolic content (TPC)In total phenolic content test methanol extract showeda significant level of reducing power. In this test, the gallic acidused as standard and methanol extract’s absorbance plotted ingallic acid calibration curve (Figure 2). The absorbance of theplant extract was high for that reason the obtained TPC valuewas 308.97 mg of GAE/g of extract (Table 2). This TPC valueindicated that Aporosa wallichii Hook.f. has antioxidant activity.Cytotoxic propertyBrine shrimp lethality assayThe brine shrimp lethality assay was used to assess thecytotoxic property of methanol extract (Runa et al., 2013). At
054Sharmin et al. / Journal of Applied Pharmaceutical Science 8 (07); 2018: 051-056different concentrations; standard and extract provided differentpercentages of mortality (Table 3). Vincristine sulfate was usedin this experiment as a standard (positive control), in which2.0 μg/mL was obtained as the value of LC50, compared to thestandard methanol extract of the Aporosa wallichii Hook.f. leavesgave 26.7 μg/mL as the value of LC50 (Table 3). Percentage ofmortality was found to increase with increasing concentrations ofvincristine sulfate and methanol extract (Figures 3 and 4). Thisstudy indicated the methanol extract of Aporosa wallichii Hook.f.leaves have cytotoxic activity.Table 2: Total phenolic content (TPC) profile of the plant extract of Aporosawallichii Hook.f. leaves.Name ofextractPlant partThe absorbance ofmethanol plant extractTotal phenolic content(mg of GAE/g of extract)MethanolextractLeaves ofAporosa wallichii2.5308.97Table 3: Brine shrimp lethality profile of the plant extract of Aporosa wallichii Hook.f. leaves.Concentration(µg/mL)% of Mortality(Vincristine sulphate)0.039LC50 µg/mL(Vincristine sulphate)Concentration(µg/mL)% of Mortality(Methanol extract of 06.25500.6255012.5601.256025702.57050702.0Table 3: Brineshrimp lethality profile580 of the plant extract of Aporosa wallichii Hook.f.100 leaves.80902009020100400 LC50100% ofLC50 µg/mLMortalityS t a n d a r d c u r v e o f(VincristineG a llic a c id(Vincristinesulphate)sulphate)A b sorb an ce0 .80.0390.0780 .60.1560.3120 .40.6251.250 .22.55 0 .010 0202020303040502.060708090 4 06080C100o n c e n t r a t io n ( µ g / m l )Concentration(µg/mL)0.7811.5623.125y 0 .0 0 8 1 x 6.25- 0 .0 0 0 7R ² 0 .9 9 7 512.52550100200100400Fig. 2: Gallic acid’s standard curve for the total phenolic content test.Fig. 2: Gallic acid’s standard curve for the total phenolic content test.E f f e c t s o f V in c r is t in e S u lf a t e o n n a u p lii100Cytotoxicproperty% o f M o r t a lit y26.710Concentration(µg/mL)80Brine shrimp lethality assayy 3 .5 4 2 9 x 4 2 .8 4 260LC50 µg/mL(Methanol extract of leaves)% ofMortalityµg/mL(Methanolas a (Methanolresult, the clot becomes soluble and blood flow is restored.extract ofHere,extractof extract showed much lower level of thrombolyticmethanolleaves)leaves)activity than standard (Figure 5). In thrombolytic activity test,2020 clopidogrel was used as a positive control; because it is a blood30 thinning agent (Maegdefessel et al., 2010). Clopidogrel gave50 59.3% clot lysis, distilled water was used as a negative control,60 which provided26.72.5% clot lysis and methanol extract of Aporosa70 wallichii Hook.f. leaves showed 24.5% clot lysis (Table 4). After70 comparing the clots lysis value of plant extract with the positive80control value, it was observed that Aporosa wallichii Hook.f.90100 revealed thrombolytic activity.Table 4: Evaluation and results of the thrombolytic activity.Name of samplesW1W2W3W4W5% of clotlysisPlant ti-platelet agent)as 331.5150.7280.0182.5R ² 0 .6 8 8 7The brine shrimp lethality assay was used to assess the cytotoxic property of methanolextractHere, W1 Micro-tube weight, W2 Clot with micro-tube weight, W3 40Clot with micro-tube weight after clot disruption, W4 Clot weight after clot(Runa et al., 2013). At different concentrations; standard and extract provided differentdisruption, W5 Released clot weight.20percentages of mortality (Table 3). Vincristine sulfate was used in this experiment as a standard0CONCLUSION051052 0 obtained2 5 as the value of LC50, comparedto the(positivecontrol),in which2.0 1μg/mLwasC o n c e n t r a t io n ( µ g /m L )The methanol extract of the Aporosa wallichii Hook.f.standard methanol extract of the Aporosa wallichii Hook.f. leaves gave 26.7 μg/mL as the valueleaf was investigated to evaluate the therapeutic properties. In thisFig. 3: Percentage (%) of mortality and predicted regression line of vincristinestudy,it was clearly observed that Aporosa wallichii Hook.f. reasewithincreasingconcentrations50sulfate.Fig. 3: Percentage (%) of mortality and predicted regression line of vincristine sulfate.various therapeutic potentials. This study showed that this plant hasof vincristinesulfate and methanol extract (Figure 3 and 4). This study indicated the methanolThrombolyticactivityan acceptable level of antioxidant and thrombolytic property alongPlasminogenenzymeisusuallyactivatedbywith the moderate level of the cytotoxic property. Furthermore,extract of Aporosa wallichii Hook.f. leaves have cytotoxic activity.thrombolytic agents and it also removes fibrin bonds in blood,additional investigations on Aporosa wallichii Hook.f. to find out
Sharmin et al. / Journal of Applied Pharmaceutical Science 8 (07); 2018: 051-056unidentified biological properties; will help in the development ofnew and effective therapeutic agent in the field of medicine.055% o f M o r t a lit y2015; 33(8):1582-1614.Bekker J, Ploem S, Jong KP. Early Hepatic Artery Thrombosisafter Liver Transplantation: A Systematic Review of the Incidence,E
Antioxidant property In this experiment, methanol extract of Aporosa wallichii Hook.f. leaves were tested properly through DPPH assay and TPC to determine the antioxidant property of this plant ( Pavithra et al., 2009). Antioxidant activity is very important in preventing free radical reactions because they can neutralize free radical by their
The antioxidant parameters including Superoxide dismutase (SOD), glutathione peroxidase and glutathione levels were evaluated [16,17]. Then the animals were sacrificed under anaesthesia using diethyl ether and the tissue samples of liver, kidney and heart were collected for evaluation of antioxidant levels in tissues. Antioxidant Assays
antioxidant, which accounts for the scavenging of free radicals and protective effect on antioxidant enzymes (Ravi et al., 2004). Total phenolics of the seeds said have an important antioxidant activity (Bajpai et al., 2005). The fruit is rich in sugar, mineral salts, vitamins C. Fruit of Syzygium cumini contain malic acid and a small quantity of
Abdullah Sadik Girisgin4, Said Bodur5 ABSTRACT Objective: The eligibility for thrombolytic therapy for patients who present to the emergency department with Acute Ischaemic Stroke (AIS) has been researched in
Evaluation of antioxidant activities of flower extract (fresh and dried) . eliminate free radicals and other reactive oxygen and nitrogen species, and these . Table.1 Phytochemical and antioxidant activity analysis of water and ethanol extract of fresh flowers of Saraca indica
antioxidant methodologies such e) cationic radical (ABTS) scavenging, reducing power, 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging, total antioxidant and metal chelating activities. The search and evaluation of 1,2,4-triazole
evaluation of the free radical scavenging activity of the drug sample. All eight marketed hepatic formulation showed the capacity of scavenging of free radical. The lowest antioxidant activity is shown by Udiliv i.e. 8.97% and silybon shows the highest antioxidant activity i.e. 60.86%.
Theoretical Evaluation of Antioxidant Activity of Tea Catechins N.S. Labidi1*, L. Guerguer 1, A. Kacemi 1 1. Institut des Sciences, Département des Sciences de la Matière, Centre Universitaire de Tamanrasset, BP (10034) Sersouf- Tamanrasset (11000)-Algeria Abstract To quickly evaluate the antioxidant activity of tea catechins epicatechin (EC), -
perpustakaan berupa buku, literatur, jurnal maupun e-journal. Disediakan juga hot spot area maupun anjungan komputer. c. Heregistrasi Kuliah Setiap awal semester dan awal tahun akademik agar dapat mengikuti kegia-tan akademik mahasiswa harus melaksanakan heregistrasi. Ketentuan ten- tang heregistrasi dapat dilihat pada pedoman akademik universitas. Penen-tuan kelas paralel dilakukan pada .