Quantification of Total Flavonoid Content and antioxidant activity in comparison to a reference...

JPR:BioMedRx: An International Journal Vol.1 Issue 8.August 2013

Pal Tapas Kumar et al. /JPR:BioMedRx: An International Journal 2013,1(8),757-766

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Research Article Available online throughwww.jpronline.info

*Corresponding author.Tapas Kumar Pal, Professor,Department of Pharmaceutics,NSHM College of Pharmaceutical Technology,124, B L Saha Road, Kolkata, India.

Quantification of Total Flavonoid Content and antioxidant activity in comparison to areference flavonoid as in vitro quality evaluation parameter for assessing bioactivity of

biomarkers in herbal extracts or formulations

Pal Tapas Kumar*1, Kalita P1, Barman T K1, Chatterjee T K2, Maity S1

1NSHM College of Pharmaceutical Technology under NSHM Knowledge Campus,Kolkata- Group of Institutions, 124, B L Saha Road, Kolkata, India

2Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India

Received on:17-05-2013; Revised on:18-06-2013; Accepted on:23-07-2013

ABSTRACTThe present study involves in vitro quantification of total flavonoid content (TFC) and antioxidant activity of methanolic extract of twosample plants, Clerodendrum infortunatum and Biophytum sensitivum and comparison of their quality assessment of bioactivity withrespect to standard flavonoid, like Narigenin, Rutin & Quercetin. Clerodendrum infortunatum Linn. (Lamiaceae), commonly known as Bhantin Hindi, had been used in Indian folk medicine in the treatment of bronchitis, asthma, fever, burning sensation, diabetes, diseases of theblood, inflammation, and epilepsy. Biophytum sensitivum Linn. (Oxalidaceae) had also been found as traditional medicine in treating ailmentssuch as inflammation, arthritis, wounds, tumors and burns, gonorrhea, stomach ache, asthma, cough, degenerative joint disease, urinarycalculi, diabetes, snake bite, amenorrhea and dysmenorrhea. The total flavonoid content (TFC) of methanolic extract of C. infortunatum roots(MECI) and methanolic extract of B. sensitivum whole plant (MEBS) were determined by using aluminum chloride colorimetric method. Sincethese phenolic compounds and flavonoids are of pharmacological interest in traditional medicine, coupled with the use of these plant extractsand their formulations, prompted to evaluate C. infortunatum and B. sensitivum for their antioxidant potential by DPPH scavenging activityand reducing power ability. The percentage inhibition by DPPH method was compared in terms of IC50 value, which was found to be about34.46 mcg/ml and 12.30 mcg/ml of MECI and MEBS, when compared with IC50 value Quercetin (48.79 mcg/ml). The reducing capabilities byFRAP assay were also found to be in dose dependent manner. The present findings provided information about total flavonoid activity (TFC)which could further trigger partial or full isolation and standardization of the biomarker compounds like polyphenolics and flavonoids for invitro assessment and correlating their bioactivity of diabetes in comparison to a standard reference antidiabetic drug.

Key-words:Antioxidant; Biomarker; Flavonoid; Free radical scavenging; Polyphenolics; Soxhlet apparatus.

ISSN: 2321-4988

INTRODUCTION:The main characteristic of an antioxidant is its ability to trap freeradicals. Highly reactive free radicals and oxygen species are presentin biological systems from a wide variety of sources. These free radi-cals may oxidize nucleic acids, proteins, lipids or DNA and can initiatedegenerative disease. Antioxidant compounds like phenolic acids,polyphenols and flavonoids scavenge free radicals and thus inhibitthe oxidative mechanisms that lead to degenerative diseases. Freeradicals have one or more unpaired electrons and are produced innormal or pathological cell metabolism. Recently much attention hasbeen focused on reactive oxygen species (ROS) and free radicalswhich play an important role in the genesis of various diseases asantioxidant, antimicrobial, antiviral, antifungal, anti-inflammatory and

antitumoral1. A great number of aromatic and other medicinal plantscontain natural phytoconstituents that exhibit antioxidant properties.An antioxidant is a molecule capable of slowing or preventing theoxidation of other molecules. Oxidation is a chemical reaction thattransfers electrons from a substance to an oxidizing agent. Oxidationreactions can produce free radicals, which start chain reactions thatdamage cells. Antioxidants terminate these chain reactions by remov-ing free radical intermediates, and inhibit other oxidation reactions bybeing oxidized themselves. As oxidative stress might be an importantpart of many human diseases, the use of antioxidants in pharmacol-ogy is intensively studied, particularly as treatments for cardiovascu-lar and neurodegenerative diseases. Antioxidants are also widely usedas ingredients in dietary supplements in the hope of maintaining healthand preventing diseases such as diabetes, cancer and coronary heartdisease. A wide range of antioxidants from both natural and syntheticorigin had been proposed for use in the treatment of various degen-erative human diseases.2 Flavonoids and Phenolic compounds arethe major bioactive phytoconstituents having such reactive oxygen



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species, which play an important role in control & prevention oftissue damage by their inherent antioxidant activity3. Hence herbaldrugs / herbal preparations containing such phytoconstituents aregaining importance in the prevention and treatment of various organtoxicities due to xenobiotic / environmental challenges4.

Herbal medicines have been regarded as popular remedies for dis-eases widely used by a vast majority of the world’s population5.Plants in all facets of life have served a valuable starting material fordrug development. Herbal plants contain many pharmacologicallyactive compounds like flavonoids, alkaloids, tannin, steroids, glyco-sides, phenols, fixed oils, which are stored in their specific parts ofleaves, bark, flowers, seed, fruits, root etc.6. Biophytum sensitivum(family- Oxalidaceae) had been well explored to ascertain its bioactiv-ity in different pharmacological and pathological situations as anti-malarial, anti-dengue, anticancer, anti-inflammatory, chemo-protec-tive, antidiabetic and wound healing activities of their different parts7-

11. The biochemical properties12 of the plant showed the presence ofamentoflavone, 3', 8?- biapigenin, proanthocyanidins and other phe-nolic compounds.

Phyto-medicinal importance of various species of Clerodendrumgenus had also been reported in various indigenous systems of medi-cines and in folk medicines, specifically in Indian, Chinese, Thai,Korean, Japanese systems of medicine for the treatment of variousdiseases such as syphilis, typhoid, cancer, jaundice and hyperten-sion. The most important constituents of Clerodendrum infortunatumare phenolics, flavonoids, steroids, terpenoids, fixed oils and sugars.The Clerodendrum infortunatum roots are externally used for tu-mors and skin diseases as paste. Thus Clerodendrum infortunatumroot extracts may have potential pharmacological activities like woundhealing, anti-venom and anti-fertility properties. The powder / pasteform of the extracts of root, stem and leaves of Clerodendrum genusare reported to be used as medicine for the treatment of asthma,diabetes, cataract, malaria, and diseases of blood, skin and lung13.Preliminary phytochemical studies showed the presence of flavonoids,tannins, and saponins in MECI. Flavonoids and tannins are well-known polyphenolic natural antioxidants, which may be responsiblefor the antioxidant role.

Flavonoids are potent antioxidants and have aroused considerableinterest recently because of their potential beneficial effects on hu-man health in fighting degenerative diseases. The capacity of fla-vonoids to act as antioxidants depends upon their molecular struc-ture. The position of hydroxyl groups and other features in the chemi-cal structure of flavonoids are important for their antioxidant and freeradical scavenging activities. Quercetin, the most abundant dietaryflavonol, is a potent antioxidant because it has all the right structuralfeatures for free radical scavenging activity14. Based on recent ad-vances and involvement of oxidative stress in complicating DiabetesMellitus, efforts are on to find suitable herbal antidiabetic medicineswith antioxidant properties. Flavonoids like Naringenin, Rutin havealso been reported to possess antidiabetic activity15-17.

Flavonoids are low molecular weight bioactive polyphenols consist-ing of 5 major subgroups: flavones, flavonols, flavanones, flavanolsand anthocyanidins. Dietary polyphenols are chemicals of plant ori-gin that are abundant in fruit, vegetables, chocolate, and nuts, as wellas in beverages such as tea, coffee, wine, and soy milk18-19. There arethousands of natural polyphenols in the plant kingdom (and in de-rived foods), all of which share the basic structure of an aromatic ringwith attached hydroxyl groups. Despite promising experimental re-sults from in vitro and animal studies establishing the antioxidanteffect reported with a variety of medicinal herbs, standardization ofthese herbs, the extracts prepared there from and the formulationshave been scarcely studied for in vitro quantification of total fla-vonoids and total phenolics as standard quality parameter againststandard flavonoids with an eye to compare their bioactivity. Modernformulators prefer using whole plants rather than extracting and iso-lating single components from them with an objective to producesynergistic therapeutic effects and also lessen the chances of sideeffects from any one isolated component. Several herbs are oftenformulated together to enhance effectiveness and synergistic ac-tions and to reduce toxicity [20].

MATERIALS AND METHODS:

Plant materialThe whole plant of Biophytum sensitivum was collected locally fromthe forest of Midnapore, West Bengal and Clerodendruminfortunatum was collected from Ischadagharia village of Kamrup,Assam. The plant materials were identified and authenticated taxo-nomically by an expert taxonomist at the Central National Herbarium,Botanical Survey of India, Botanical Garden, Howrah-711103, WestBengal, India. A voucher specimen of each of the collected sampleswas deposited in the institutional herbarium for future reference.

Preparation of extracts by hot extractionThe collected whole plants were thoroughly washed, cleaned, driedunder shade and powdered by a mechanical grinder to obtain a coarsepowder and then passed through 20# mesh sieve. A quantity of 500 gof the dried pulverized whole plant of Biophytum sensitivum wasextracted with methanol successively in a soxhlet apparatus at a tem-perature not exceeding 60 deg C. Petroleum ether was used in initialstep of extraction for defatting the plant materials. The successivemethanolic extracts were mixed & concentrated at reduced tempera-ture on a rotary vacuum evaporator and stored at 4 deg C until furtheruse. The yield was found to be around 8.26 % (w/w) with respect todried whole plant of Biophytum sensitivum. The biologically potentdry extract (MEBS) had been used for further study.

Similarly, 500 g of the dried pulverized roots of Clerodendruminfortunatum were taken in another soxhlet apparatus separately.After initial defatting with Petroleum ether, successive hot extractionin a soxhlet apparatus at a temperature not exceeding 70 deg C werecarried out. Then the extract obtained had been filtered and concen-



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trated at reduced temperature on a rotary vacuum evaporator and standard flavonoid for comparison or Naringenin equivalent whenNaringenin used as standard flavonoid for comparison.

Antioxidant activity (DPPH free radical scavenging)Antioxidants through their scavenging power are useful for the man-agement of these diseases. DPPH stable free radical scavengingmethod is an easy, rapid and sensitive way to survey the antioxidantactivity of a specific compound or plant extracts22, 23. The free radicalscavenging activity of the Extracts, MEBS & MECI were measured interms of hydrogen donating or free radical scavenging ability byusing the stable 1,1-diphenyl-2-picryl hydrazyl radical (DPPH). Dif-ferent concentration of methanolic plant extract (1.0 ml, MEBS &MECI each separately) and test sample tablet powder prepared withMEBS were mixed in separate test tubes, each with a mixture of 2.5 mlof methanol and 5 ml (100 µM) DPPH-methanol solution and thereaction mixture was set aside in the dark at room temperature for 30min, thereafter the absorbance was recorded at 517 nm. The antioxi-dants react with DPPH and get reduced to DPPH-H form, therebydecreasing in absorbance value. IC50 value is the concentration ofsample, required to scavenge 50% of DPPH free radicals. The per-centage inhibition of DPPH radical was calculated by comparing theresults of the test samples with those of the control (not treated withextract) using the following equation.

Percentage inhibition = (1– absorbance of test/absorbance of con-trol) × 100

IC50 values have been calculated with comparison to standard refer-ence flavonoid (like Quercetin) from the graph of concentration ver-sus % Inhibition.

Ferric reducing antioxidant power (FRAP) assayReducing power of the methanolic plant extracts, MEBS & MECI,was determined on the basis of their antioxidant principles to formcolored complex with potassium ferricyanide, Trichloroacetic acid(TCA) and Ferric chloride (Fecl3). 1.0 ml each of different concentra-tions (40 - 200 µg/ml) of the extract fractions had been mixed withpotassium ferricyanide (2.5 ml, 1%) and 2.5 ml of 0.2 M phosphatebuffer (pH 6.6). The mixture was incubated at 50° C for 30 min. Further,2.5 ml TCA (10% w/v) was added to it and centrifuged at 3000 rpm for10 min. 2.5 ml of supernatant was taken, diluted with 2.5 ml distilledwater and shaken with 0.5 ml Ferric chloride solution (0.1% w/v. freshlyprepared). The absorbance was measured at 700 nm24. Quercetin atdifferent concentrations (40 – 200 µg/ml) had been used as a refer-ence standard to compare antioxidant activity and the control solu-tion was prepared as above, but contained distilled water instead ofextract samples or quercetin. Higher absorbance of the reaction mix-ture indicated higher reducing power25, 26.

Experimental animalsHealthy young Albino rats weighing between 120 g to 200 g wereprocured from Indian Institute of Cultivation of science, Kolkata. Theanimals were individually housed in polypropylene cage and the room

Total Flavonoid content had been evaluated as Quercetin equivalent(mg/g of dry extract or % per 100 g of dried herbs) when quercetin wasused as standard flavonoid for comparison or Rutin equivalent (mg/g of dry extract or % per 100 g of dried herbs) when Rutin used as

Estimation of total flavonoid content (TFC)Formation of acid stable complexes with the C-4 keto group and eitherthe C-3 or C-5 hydroxyl group of flavones and flavonols in additionwith aluminium chloride is the basic principle. Aluminium chloridealso forms acid labile complexes with the ortho - dihydroxyl groups inthe A- or B-ring of flavonoids. The total flavonoid content was deter-mined according to the aluminium chloride colorimetric method [21].Both the methanolic extracts, MEBS & MECI that have been dilutedwith methanol to come under the linearity range (2 mL, 0.3 mg/mL)were individually mixed with 0.1 mL of 10% w/v aluminium chloridehexahydrate, 0.1 mL of 1 M potassium acetate and 2.8 mL of distilledwater. After 40 minutes incubation period, at room temperature, theabsorbance of the reaction mixture was determined spectrophoto-metrically at 415 nm. Sample blank was prepared in similar way byreplacing aluminium chloride with distilled water. Sample and sampleblank of all extracts were prepared and their absorbance was mea-sured at 415 nm with a double beam UV/Visible spectrophotometer,SHIMADZU UV 1800 (Japan). All prepared solutions were filteredthrough whatmann filter paper (no 41) before measuring. To performthe calculations of total flavonoid content in the plant extract, a stan-dard calibration curve is needed which is obtained from a series ofdifferent concentrations of a standard reference flavonoid (like Quer-cetin, Rutin, Naringenin).

Preparation of herbal tablets from Biophytum sensitivum whole plantdry extractThree batches of tablet formulations, each containing 200 mg of MEBSper tablet were prepared separately by non aqueous wet granulationmethod using different proportions of excipients as BS 1, BS 2, andBS 3. All the ingredients were passed through mesh no. 60# andmixed with 1% aerosil (Aerosil-200) and 1% of magnesium stearate.The micromeritic properties were evaluated for all the granules ofthree batches of Tablets to ensure that pre compression blends pos-sess good flow properties and good compressibility. Tablets werecompressed each of 675 mg average weight on a 10-station MiniPress-I rotary tablet compression machine fitted with a set of 16 mm X8 mm Caplet shaped die-punches. No manufacturing defects wereobserved in tablets like capping, lamination and chipping. The physi-cal properties of tablet were determined and the results of the unifor-mity of weight, hardness, drug content and friability of the tablets aregiven in Tables 2. The drug content was evaluated based on per centTFC of the extract with respect to a standard Flavonoid.

stored at 4 deg C until further use. The yield was found to be around7.00% (w/w) with respect to dried roots of Clerodendruminfortunatum. Dried extracts (MECI) were kept in refrigerator andused for further study.



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condition was maintained at temperature of 25±5 deg C and humidity45±5 per cent with 12 hr day and night cycle. The animals were fedwith Pellet chew feed standard diet and water ad libitum. All thestudies were conducted in accordance with the Institutional AnimalEthical committee of NSHM College of Pharmaceutical Technology.

Induction of diabetesRats were allowed to fast for overnight and a single dose ofStreptozotocin (STZ) (dissolved in ice cold phosphate buffer of pH6.8) at a dose of 60 mg/ kg B. Wt. (i.p.) has been administered toindividual fasted rats. The blood glucose level was checked beforeand 72 h after STZ injection. The Glucometer was initially calibratedwith maximum 660 mg/dL glucose concentration according to specifi-cation. The Blood is taken out of Rat’s tail vein and immediatelyspread on the marked end of the Gluco-strip, which is the inserted inthe Glucometer through the electrodes. After few seconds theglucometer displays the blood glucose level. Hyperglycemia wasconfirmed by elevated glucose levels, determined at 72 h. The ani-mals with blood glucose concentration more than 250 mg/dL wereconsidered to be diabetics and used for the experiment.

Experimental protocol for anti-diabetic activity [27]

The prepared dried herbal extracts (MECI & MEBS), optimized formu-lation of BS tablets (BS 3) and standard antidiabetic drug,Glibenclamide samples were finely powdered and suspended in 0.1%w/v aqueous suspension of sodium carboxy methyl cellulose (SCMC).A suitable dose of each of MECI, MEBS, BS 3 tablet (eqv. to 200 mg/kg of B. Wt.) and a standard dose of Glibenclamide (eqv to 10 mg/kgof B. Wt.) were daily administered orally by intragastric (i.g) route byusing an intragastric tube to the STZ induced hyperglycemic rats.The stock suspensions were prepared in such a way that that dailydosage to be fed to the rats using the intragastric tube should notexceed beyond 1.0 /1.5 ml at a time.

Thirty six rats were divided into six groups, (n=6).Group I: Normal control; Received only 0.1% SCMC 2ml/kg per oral.Group II: Disease control; Received STZ 60 mg/kg (i.p) + 0.1% SCMC2ml/kg per oral.Group III: Standard. STZ 60 mg/kg (i.p) + Glibenclamide (10 mg/kg peroral)Group IV: Test 1. STZ 60 mg/kg (i.p) + MECI oral suspension (eqv 200

mg/kg of B. Wt.).Group V: Test 2. STZ 60 mg/kg (i.p) + MEBS oral suspension (eqv 200mg/kg of B. Wt.).Group VI: Test 3. STZ 60 mg/kg (ip) + BS 3 Tablet powder oral suspen-sion (eqv 200 mg of MEBS)

As expected, administration of STZ led to the elevation of Bloodsugar level, which was maintained in the Disease (Diabetes) controlGroup II during the entire period of study and Group III animals aredaily fed with standard Glibenclamide (suspended in 1% w/v SCMCsuspension) at a dose of 10 mg/kg of B. Wt. in each. Similarly STZinduced diabetic rats in Group IV & V received a daily dose of MECI& MEBS (suspended in 1% w/v SCMC suspension) in dose of 200mg / kg of B. Wt. in each rat. Group VI received a daily dose ofpowdered BS 3 tablets (suspended in 1% w/v SCMC suspension) indose of 200 mg / kg of B. Wt. in each rat. The blood samples from thetail vein were collected and analyzed for blood glucose content at 0th,1st, 3rd, & 22nd hr on the First day (single dose, short term study) andsubsequently on 7th, 14th & 21st day for comparative anti-hyperglyce-mic activity in multi-dose long term study.

RESULTS AND DISCUSSION:Flavonoids play vital role in scavenging the free radicals to preventor reduce the oxidative damage encountered in human degenerativediseases. Hence, the methanolic extract of root bark of C.I. (MECI)and methanolic whole plant extract of B.S. (MEBS) were prepared andtheir total flavonoid content were quantified with respect to standardflavonoids. In the study, Quercetin, Rutin, and Naringenin were takenas standard flavonoids. Both these extracts (MECI and MEBS) con-tain flavonoids as essential phytoconstituents and sample batch ofTablets with MEBS (200mg per tablet) had also been evaluated. Inthis experiment it was found that MEBS having much more TFC activ-ity in comparison to MECI. Equivalents Quercetin and Rutin activi-ties of both methanolic extracts had low TFC as compared to that ofNaringenin activity. In our study we have also assessed thatmethanolic root extract of Clerodendrum infortunatum (MECI) andmethanolic whole plant extract of Biophytum sensitivum(MEBS) haspotential antidiabetic and antioxidant activities; which are standard-ized in vitro with reference to standard Flavonoid (like Quercetin)and in vivo against standard antidiabetic drug like Glibenclamide.

Table 1: Standard calibration curve of Reference flavonoid

Sl No Quercetin[R2 = 0.998] Rutin[R2 = 0.9993] Naringenin[R2 = 0.9991]Concentration Absorbance Concentration Absorbance Concentration Absorbance(mcg/ml) at 415 nm (mcg/ml) at 415 nm (mcg/ml) at 415 nm

1 0 0 0 0 0 02 11.04 0.022 20.6 0.092 46.2 0.0123 22.08 0.058 41.2 0.174 92.4 0.0254 33.12 0.077 61.8 0.253 138.6 0.0365 55.2 0.135 103 0.417 184.8 0.0476 77.28 0.187 144.2 0.579 231 0.0597 110.4 0.279 - - - -



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Fig 1: Standard Calibration curve of Quercetin

Fig 2: Standard calibration curve of Rutin

Fig 3: Standard calibration curve of Naringenin

Calibration curve of Quercetin

Fig.4: Estimation of Total Flavonoid activity & their comparison:

Batch Avg Wt Hardness Friability Thickness Disintegration Avg. Drug Content/tabCode [Mean+SD] [Mean+SD] [Mean+SD] [Mean+SD] Time (TFC activity

(n = 10) (mg) (n = 10) (n = 10) (%) (n = 10)(mm) [Mean+SD] eqv to mg of(Kg/cm2) (n = 10) (min) Quercetin)

BS 1 0.672+0.12 3.72+0.07 0.34+0.02 3.7+0.05 27+0.51 1.816BS 2 0.674+0.08 3.42+0.22 0.39+0.02 3.8+0.45 28+0.27 1.844BS 3 0.675+0.05 3.31+0.43 0.41+0.02 3.9+0.25 20+0.34 1.858

Table 2: Evaluation data of the Compressed Tablets from Biophytum sensitivum whole plant dry extract

Average weight of BS Tablets = 675.7 mg (Optimized Formulation BS 3) (Each Tablet contains 200 mg of Dry extract of whole plant of Biophytum sensitivum)TFC of each Tablet equivalent to (9.27*200/1000) = 1.854 mcg of Quercetin ActivityTFC of each Tablet equivalent to (5.79*200/1000) = 1.158 mcg of Rutin ActivityTFC of each Tablet equivalent to (77.22*200/1000) = 15.444 mcg of Naringenin Activity

Table 3: Total flavonoid activity of Plant Extract and a herbal tablet formulation

Sample of PlantExtract/ Tablet Concn of # Eqv. TFC Activity (mcg/ml) of sample Eqv. TFC Activity (mcg/mg) of Dry Extractprepared Sample inwith Plant Ext. Stock solution

(mg/ml) Quercetin Rutin Naringenin Quercetin Rutin Naringenin

Methanolic dry Extract ofBiophytum sensitivum 2.014 19.17 ± 0.33 11.98 ± 0.21 159.78 ± 2.78 9.52 5.95 79.33MEBS Tablet (200 mg MEBS per tab) 2.123 19.67 ± 0.99 12.30 ± 0.62 163.94 ± 8.33 9.27 5.79 77.22Methanolic dry Extract of

Clerodendrum infortunatum 2.436 14.10 ± 0.52 8.81 ± 0.32 117.52 ± 4.31 5.79 3.62 48.24

[# n = 3, value includes Mean & Std Dev.]



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Fig.5: Estimation of Antioxidant Activity by DPPH method & their comparison

Fig.6: Estimation of DPPH scavenging activity w.r.t Standard Flavonoid



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Fig.7. Comparison of % Inhibition of DPPH activity w.r.t Standard Flavonoid

Fig.8: Estimation of Reductive ability by FRAP assay w.r.t. Standard Flavonoid



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Fig.9: Comparison of Reductive Ability w.r.t. Standard Flavonoid

Table 4: Comparison of DPPH radical scavenging activity of Plant Extract and a herbal tablet formulation

Test Samples Concentration (mcg/ml) Absorbance at 517 nm % Inhibition IC50 (mcg/ml)

Control (DPPH) 0.24 mM 3.81 Quercetin 22.08 2.968 22.10 48.79

44.16 2.392 37.2255.20 1.911 49.8477.28 1.252 67.14110.40 0.681 82.13

MEBS 20.14 3.638 4.51 12.30 40.28 3.537 7.1760.56 3.294 13.54100.70 2.883 24.33201.40 2.135 43.96

Tablets With MEBS 20.21 3.686 3.25 13.5240.41 3.511 7.8560.62 3.171 16.77101.04 2.791 26.75202.07 2.219 41.76

MECI 22.60 3.285 13.78 34.4644.20 2.871 24.6567.80 2.382 37.48113.00 1.214 68.14226.00 0.267 92.99



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Table 5: Comparison of reductive ability of Plant Extract and a herbaltablet formulation

Test Samples Concentration Absorbance(mcg/ml) at 700 nm

Quercetin 40.00 2.42160.00 2.548100.00 2.855200.00 2.917

MEBS 42.56 0.31763.78 0.458106.3 0.795212.6 1.171

Tablets With MEBS 40.41 0.26860.62 0.386101.04 0.738202.07 1.255

MECI 46.52 0.83369.78 1.059116.3 1.662

232.6 2.089

Table 6: Effect of Single dose administration of Herbal Extracts & Formulated BS Tablets on Blood Glucose level (mg/dL) in STZinduced Diabetic Rats

Group Treatment Blood Glucose Level (mg/dL)(Dose, mg/ Kg of B.W., PO) Hour 0 Hour 1 Hour 3 Hour 15 Hour 22

Group I 1% w/v SCMC (Oral) 105.3 + 6.43 104.7 + 6.66 103.7 + 7.57 103.3 + 8.14 1033 + 8.72(Normal Control) (0.63) (1.58) (1.90) (2.22)Group II STZ (IP) + 1% w/v SCMC (Oral) 304.0 + 7.94 305.3 + 8.14 306.3 + 8.14 309.7 + 6.66 311.3 + 3.00(Diabetic Control) (-0.44) (-0.77) (-1.86) (-2.41)Group III STZ (IP) + Glib in 1% w/v SCMC (Oral) 289.3 + 4.51 282.7 + 3.06 278.7 + 3.06 276 + 3.00 274.7 + 2.52(Standard) (2.30) (3.69) (4.61) (5.07)Group IV STZ (IP) + MECI in 1% w/v SCMC (Oral) 286.0 + 4.00 285.0 + 4.00 283.0 + 3.06 280.3 + 3.00 279.0 + 2.52(Test 1) (0.35) (1.05) (1.98) (2.45)Group V STZ (IP) + MEBS in 1% w/v SCMC (Oral) 309.3 + 3.06 307.0 + 3.21 304.0 + 2.65 300.7 + 3.21 299.3 + 3.79(Test 2) (0.75) (1.72) (2.80) (3.23)Group VI STZ (IP) + Powdered BS Tab in 301.3 + 3.51 300.7 + 1.53 298.0 + 2.00 297.3 + 1.53 295.7 + 1.15

1% w/v SCMC (Oral Dose 200 mg/kg BW)(Test 3) (0.22) (1.11) (1.33) (1.88)

(Short Term Single dose study till 22 hours) (Values are mean + SE from 6 rats in each group, Figures in parenthesis indicates % fall in BGL as comparedto Hour 0)

Table 7: Effect of Multi dose administration of Herbal Extracts & Formulated BS Tablets on Blood Glucose level (mg/dL) in STZ inducedDiabetic Rats

Group Treatment Blood Glucose Level (mg/dL)(Dose, mg/ Kg of B.W., PO) Day 3 Day 5 Day 10 Day 15 Day 21

Group I 1% w/v SCMC (Oral) 114.7 + 1.53 114.3 + 1.53 113.3 + 1.53 113.0 + 2.00 112.3 + 2.08(Normal Control) (0.35) (1.22) (1.48) (2.09)Group II STZ (IP) + 1% w/v SCMC (Oral) 313.3 + 2.08 305.0 + 4.36 301.0 + 3.46 272.0 + 4.58 268.3 + 4.51(Diabetic Control) (2.65) (3.93) (13.18) (18.51)Group III STZ (IP) + Glib in 1% w/v SCMC (Oral) 277.7 + 3.21 253.0 + 2.00 214.7 + 4.51 177.7 + 4.16 146.7 + 2.08(Standard) (8.89) (22.69) (36.01) (47.17)Group IV STZ (IP) + MECI in 1% w/v SCMC (Oral) 290.3 + 5.13 264.3 + 4.04 231.0 + 3.61 193.7 + 2.52 158.0 + 1.00(Test 1) (8.96) (20.43) (33.28) (45.57)Group V STZ (IP) + MEBS in 1% w/v SCMC (Oral) 279.7 + 4.04 253.3 + 4.73 214.3 + 3.06 171.3 + 6.51 138.0 + 5.86(Test 2) (9.44) (23.38) (38.76) (50.66)Group VI STZ (IP) + Powdered BS Tab in 1% w/v 280.3 + 4.51 260.3 + 3.79 237.7 + 4.51 195.7 + 2.52 145.0 + 3.00

SCMC (Oral Dose 200 mg/kg BW)(Test 3) (7.14) (15.20) (30.18) (48.27)

(Long Term Single dose study till 21 days with daily once single dose) (Values are mean + SE from 6 rats in each group, Figures in parenthesis indicates %fall in BGL as compared to Day 3)

Antioxidants through their free radical scavenging power are alsouseful for the management and prevention of human illness includingneurodegenerative diseases, diabetes, cancer, cataracts, cardiovas-cular disease and AIDS. Free radicals’ posses the ability to reduce theoxidative damage. DPPH stable free radical scavenging assay is aneasy, rapid and sensitive method to quantify the antioxidant activityof a specific flavonoid or plant extracts. This study provides informa-tion on the reactivity of test compound with a stable free radical sinceodd electron of DPPH gives strong absorption band at 517nm (violetcolour) and when it is quenched by the extract, there is a decrease inabsorbance. Methanolic extract of C.I. and B.S. (MECI & MEBS)showed a very promising activity in scavenging DPPH free radicalwith a level of inhibition of 68.14% at 113.00 µg/ml concentration and24.33% at 100.70 µg/ml concentration, respectively in case of MECIand MEBS; when correlated with a standard flavonoid, Quercetin(82.13% inhibition at 110.40 µg/ml concentration) and found to bedose dependent. The reductive ability of a compound may serve as asignificant indicator of its potential antioxidant activity. Reductive



757-766

abilities of MEBS & MECI had been assessed by the Fe3+ to Fe2+

transformation, where the change in the optical density is measuredat 700 nm. Increase in optical density indicates higher reductive abil-ity of the phytoconstitutent. Reductive abilities of MEBS & MECIwere also found to be in dose dependent manner when comparedwith Quercetin. (MECI showing higher reductive ability as comparedto MEBS). The present study showed that all extracts (MECI &MEBS) as well as the tablet formulation made with MEBS demon-strated different extent of TFC, different antioxidant activity and dif-ferent reductive abilities, which were correlated with the similar activ-ity of a standard flavonoid and ultimately established as routine invitro quality parameters in standardization and assessing bioactivityof the phytoconstituent or biomarker in herbal extract or any formula-tion containing similar polyphenolic, antioxidant phytoconstituemtslike flavonoids.

ACKNOWLEDGMENTThe authors acknowledge grateful and heartfelt thanks to Depart-ment of Pharmacology, for their valuable cooperation for this study.The authors would like to express gratitude to the authority of WestBengal University of Technology and NSHM college of Pharmaceu-tical Technology for providing necessary infrastructural and logisticsupport

CONFLICT OF INTEREST STATEMENTWe declare that we have no conflict of interest.

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Quantification of Total Flavonoid Content and antioxidant activity in comparison to a reference...

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