Quantification of total phenolics and flavonoids and evaluation of in vitro antioxidant properties of
methanolic leaf extr...
INTRODUCTION
Phytochemicals are naturally occurring
compounds of plant kingdom, such as medicinal plants,
vegetables, frui...
Turner, 1975).
Tannins
2mL of extracts were diluted with distilled water
and added with 2-3 drops of 5% Fecl3. Appearance ...
or a radical species. For the photometric assay, different
volumes of the extracts were taken in different test tubes.
The...
Chandran et al.,2013
Journal of Research in Plant Sciences (2013) 2(2): 196-204 200
(about 1:89 v/v) and equilibrated at 3...
Chandran et al.,2013
201 Journal of Research in Plant Sciences (2013) 2(2): 196-204
content of methanolic leaf extract wer...
Chandran et al.,2013
Journal of Research in Plant Sciences (2013) 2(2): 196-204 202
extract of T. asiatica when compared t...
Chandran et al.,2013
203 Journal of Research in Plant Sciences (2013) 2(2): 196-204
Cai YZ, Sun M, Xing J, Luo Q and Corke...
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Quantification of total phenolics and flavonoids and evaluation of in vitro antioxidant properties of methanolic leaf extract of tarenna asiatica – an endemic medicinal plant species

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Quantification of total phenolics and flavonoids and evaluation of in vitro antioxidant properties of methanolic leaf extract of tarenna asiatica – an endemic medicinal plant species

  1. 1. Quantification of total phenolics and flavonoids and evaluation of in vitro antioxidant properties of methanolic leaf extract of Tarenna asiatica – an endemic medicinal plant species of Maruthamali hills, Western Ghats, Tami Nadu. Keywords: Tarenna asiatica, total phenolics and flavonoids, antioxidant activities. ABSTRACT: The present study was aimed at to determine the preliminary phytochemical screening, quantification of total phenolics and flavonoids and to evaluate the antioxidant activities of an endemic medicinal plant species, Tarenna asiatica. The antioxidant activity of methanolic leaf extract of this species was assessed by DPPH• , ferrous ion chelating, reducing power and ABTS•+ radical scavenging activities. The total phenolics and flavonoids content of the extract were found to be 16.95µg of GAE/100mg extract and 3.72µg of QE/100mg extract respectively. The present study revealed that the methanolic leaf extract of this speices showed potent in vitro antioxidant activities and so it may be useful for their nutritional and medicinal properties. 196-204 | JRPS | 2013 | Vol 2 | No 2 This article is governed by the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited. www.plantsciences.info Journal of Research in Plant Sciences An International Scientific Research Journal Authors: Vishnu Chandran R1 , Nisha Raj1 , Jamuna S2 and Paulsamy S2 *. Institution: 1.Department of Biotechnology, SAS SNDP Yogam College, Konni, Kerala. 2. Department of Botany, Kongunadu Arts and Science College, Coimbatore, Tamil Nadu. Corresponding author: Paulsamy S. Email: paulsami@yahoo.com Web Address: http://plantsciences.info documents/PS0059.pdf. Dates: Received: 25 Apr 2013 Accepted: 07 May 2013 Published: 10 May 2013 Article Citation: Vishnu Chandran R, Nisha Raj, Jamuna S and Paulsamy S. Quantification of total phenolics and flavonoids and evaluation of in vitro antioxidant properties of methanolic leaf extract of Tarenna asiatica - an endemic medicinal plant species of Maruthamali hills, Western Ghats, Tami Nadu. Journal of Research in Plant Sciences (2013) 2(2): 196-204 Original Research Journal of Research in Plant Sciences JournalofResearchinPlantSciences An International Scientific Research Journal
  2. 2. INTRODUCTION Phytochemicals are naturally occurring compounds of plant kingdom, such as medicinal plants, vegetables, fruits, that work with nutrients and fibers to act against diseases or more specifically, provides protection against diseases (Chopra et al., 1984). Secondary metabolites of plants are the responsible chemical compounds for the medicinal properties of the respective plant species. Free radicals formed in human body are playing important role in different pathological conditions like tissue injury, inflammation, and neurodegenerative diseases. Antioxidants are very important chemicals which can protect the human body from these damages caused by free radicals. (Halliwell and Gutteridge, 1989). However, synthetic antioxidants are not much used for this carcinogenic effect (Buxiang and Fukuhara, 1997; Hirose, 1998). Therefore, investigation of natural antioxidants of plant origin is getting more attention (Kaur and Kapoor, 2001; Vinson, 2001). Tarenna asiatica L. (Rubiaceae) is a large shrub to small tree and is endemic to the Southern Western Ghats of Agasthyamalai, Anaimalai, Palani hills, Niligirs and Bababudangiri hills and distributed in Peninsular India, Malaysia and Sri Lanka. Fruit juice is applied on the eye lids to arrest infection. Malayali tribal in Javvadhu hills use T. asiatica for marriages, festivals and worship due to its aesthetic value (Ravikumar and Vijaya Sankar, 2003). The fruits are eaten by crows. The wood is used as fuel wood. It is a strong and used as a crobar also in Coimbatore district. Due to its medicinal properties the current study was aimed at to screen the phytochemicals, quantification of total phenolics and flavonoids and in vitro antioxidant activities such as DPPH• , ferrous ion chelating, reducing power and ABTS•+ radical scavenging activities. MATERIALS AND METHODS Collection of plant material The fresh leaves of Tarenna asiatica were collected from the Maruthamalai hills, Coimbatore district, Tamil Nadu, India. Collected plant materials were washed thoroughly in tap water, shade dried and then homogenized to fine powder and stored in air tight bottles. Preparation of plant extracts About 50g of powdered plant materials were extracted (50g/250mL) in a soxhlet extractor for 8 to 10 hours, by using methanol solvent. Then the extract was evaporated to dryness. Phytochemical Screening A portion of the concentrated extracts were used for the screening tests, both qualitative analysis and quantitative analysis using standard methods. Qualitative analysis of phytochemical constituents Alkaloids 1mL of extract was added with 2-3 drops of Dragendorff’s reagents. An orange red precipitate was produced with Dragendorff’s reagent would indicate the presence of alkaloids (Ciulci, 1994). Preparation of Dragendorff’s reagent: Hundred gram tartaric acid was dissolved in 400 mL water. To this, 8.5 g basic bismuth nitrate was added and the solution was shaken for 2 hours. 200 mL of 40 % potassium iodide was then added, and the solution was shaken vigorously. After 24 hours, the solution was filtered. Flavonoids To 4mL of the extract, a piece of magnesium ribbon was added followed by concentrated HCl drop wise. A colour ranging from crimson to magenta indicated the presence of flavonoids (Sofowora, 1993). Saponins 1mL of extract was 5mL of distilled water and shaken well. A persistent froth that lasted for at least 15 minutes indicated the presence of saponin (Brain and Chandran et al.,2013 197 Journal of Research in Plant Sciences (2013) 2(2): 196-204
  3. 3. Turner, 1975). Tannins 2mL of extracts were diluted with distilled water and added with 2-3 drops of 5% Fecl3. Appearance of blue-back or green black color indicates the presence of tannins (Mace and Gorbach, 1963; Ciulci 1994). Steroids 2mL of methanolic leaf extract were poured in test tube and evaporated to dryness. The residues were dissolved in acetic anhydride followed by the addition of chloroform. Conc.H2SO4 was added to the sides of the test tube. Formation of brown ring at the interface of the two liquids and a violet colour in the supernatant layer indicated the presence of steroids (Ciulci, 1994). Phenols Five mL of the concentrated extract was taken and 2mL of neutral ferric chloride solution was added. Appearance of violet colour indicates the presence of phenols (Krishnamoorthy, 1988). Glycosides Small amount of methanolic leaf extract was dissolved in 1mL of water followed by the addition of NaOH. Appearance of yellow colour indicates the presence of glycosides (Harborne, 1984). Terpenoids Methanolic leaf extracts of the study species taken in a test tube. To this, some pieces of tin and 3 drops of thionyl chloride were added. Formation of violet or purple colour indicates the presence of terpenoids (Trease and Evans, 1996). Triterpenoids 10mg of the extract was dissolved in 1mL of chloroform and 1mL of acetic anhydride was added following the addition of 2mL of conc.H2SO4. Formation of reddish violet colour indicates the presence of triterpenoids (Harborne, 1984). Quantitative analysis of phytochemical constituents Total phenolic content The total phenolic content of plant extracts was determined using Folin-Ciocalteu reagent according to the procedure described by Siddhuraju and Becker (2003). In this method, 20µg of the extract (dissolved in the respective solvent) was taken in a test tube and made up to the volume of 1.0mL with distilled water. Then 0.5mL of freshly prepared Folin-ciocalteu phenol reagent (1:1 with water) and 2.5mL of 20% sodium carbonate solution were added sequentially in each tube. The mixtures were agitated and left in the dark at laboratory temperature for 40min for the development of colour. The absorbance was recorded at 725 nm against the reagent blank using a spectrophotometer. A calibration curve of gallic acid was constructed, and linearity was obtained in the range of 10-50µg/mL. Using the standard curve, the total phenol content of the extract was calculated and expressed as gallic acid equivalent (GAE) mg/g extract. Total flavonoids content The total flavonoids content was estimated as per the modified method adopted by Zhshen et al. (1999). 1mL of leaf extract after proper dilution added with 2 mL of distilled water and then 0.15mL of sodium nitrite solution and incubated for 6 minutes. Then, 0.15mL of 10% aluminium chloride solution was added and allowed to stand for 6 min. Finally 2mL of 4% sodium hydroxide was made upto 5mL with distilled water and mixed well. At room temperature, after 15 minutes the absorbance was measured at 510nm. By using standard curve with quercetin, the total flavonoids content was estimated. Mean of the triplicates was expressed as (QE) mg/g extract on a dry weight basis. In vitro antioxidant activity DPPH▪ radical scavenging activity The antiradical scavenging efficiency was assessed using the DPPH▪ method as described by Blois (1958). In this method commercially available methanol soluble stable free radical DPPH▪ was used. In its radical form, DPPH▪ has an absorption band at 515nm, which disappears upon reduction by an antioxidant compound Chandran et al.,2013 Journal of Research in Plant Sciences (2013) 2(2): 196-204 198
  4. 4. or a radical species. For the photometric assay, different volumes of the extracts were taken in different test tubes. The volume was adjusted to 100μL with methanol. 5.0mL of 0.1mM methanolic solution of DPPH▪ was added to these tubes and shaken vigorously. The tubes were allowed to stand for 20 min at 27°C. The control was prepared as above but without the test extract and methanol was used for the baseline correction. Changes in the absorbance of the samples were monitored at 517nm. Results were compared with the activity of the standard, BHT. The per cent DPPH▪ discolouration of the samples was calculated using the following formula: DPPH radical scavenging activity (%) = [(Control OD – Sample OD/Control OD] X 100 Antioxidant activity of the methanolic leaf extract of the study species was expressed in IC50 [the microgram of extract to scavenge 50% of the DPPH▪ radicals] which was determined by linear regression. Lower the IC50 value, greater antioxidant activity. Ferrous ion chelating assay Ferrous ion chelating activity was determined according to the method of Sing and Rajini (2004). 100μL of 2mM FeSo4 and 300μL of 5mM ferrozine were mixed with different concentration of samples (50-250μg/mL3). The mixture was allowed to equilibrate for 10 min before measuring the absorbance. The ability of the sample to chelate ferrous ion was calculated by the formula of inhibition percentage as employed for DPPH• radical scavenging activity. EDTA was used as positive controls for comparison. Reducing power assay Total reducing power was determined as per the method of Yildirim et al. (2001). Different concentrations of methanolic leaf extract of T. asiatica (50-250μg/mL) were mixed with 1mL of 0.2M sodium phosphate buffer of 6.6pH and 1mL of 1% potassium ferric cyanide. Then it was incubated at 50° C for 20minutes. 1ml of 10% TCA was added to the mixture, which was then centrifuged at 3000rpm for 1min. Finally 2mL of the supernatant solution were mixed with equal volume of distilled water. Absorbance was measured at 700nm after the addition of 0.5mL of 1% FeCl3. Ascorbic acid was used as positive control for comparison. Total antioxidant activity It was performed according to the modified method of Siddhuraju and Manian, (2007). The ABTS▪+ radical cation (ABTS▪+ ) was generated by a reaction of 7 mmol/L ABTS▪+ and 2.45 mmol/L potassium persulfate after incubation for 16 h at laboratory temperature in dark. Blue – green ABTS▪+ was formed at the end of this period. Prior to assay, the solution was diluted in ethanol Chandran et al.,2013 199 Journal of Research in Plant Sciences (2013) 2(2): 196-204 Plant extract Phytochemical compounds Alkaloids Flavanoids Glycosides Steroids Phenols Tannins Saponins Terpenoids Triterpenoids Leaf ++ ++ +++ - + + - - - Table 1. Preliminary qualitative phytochemical analysis of methanolic leaf extract of Tarenna asiatica. ‘+’ – Presence of compounds; ‘-’ – Absence of compounds. Plant extract Extraction yield (%) Total phenolic content [GAE (µg/100mg)] Total flavanoid content [QE (µg/100mg)] Leaf 29.8 16.95±0.18 3.72±0.03 Table 2. Extraction yield, total phenolics and flavonoids content of methanolic leaf extract of Tarenna asiatica. Values were performed in triplicates and represented as mean ± SD. GAE - Gallic Acid Equivalent, QE - Quercetin Equivalent.
  5. 5. Chandran et al.,2013 Journal of Research in Plant Sciences (2013) 2(2): 196-204 200 (about 1:89 v/v) and equilibrated at 30°C to obtain an absorbance of 0.700±0.02 at 734 nm, the wavelength of maximum absorbance in the visible region. The stock solution of the sample extracts in ethanol was diluted such that, after introduction of a 10 μL aliquot of each dilution into the assay, they produced between 20 - 80% inhibition of the blank absorbance. After the addition of 1.0mL of diluted ABTS•+ solution to 10μL of sample extracts or Trolox standards (final concentration 0-15 μM) in ethanol, absorbance was recorded at 30°C, exactly 30 min after the initial mixing. Appropriate solvent blanks were also run in each assay. Triplicates were maintained for the experiments and the per cent inhibition of the blank absorbance at 734 nm was plotted as a function of Trolox concentration (Re et al., 1999). The unit of total antioxidant activity (TAA) was defined as the concentration of Trolox having the equivalent antioxidant activity expressed as μmol/g sample extracts on dry weight basis. STATISTICAL ANALYSIS The triplicates were maintained for all tests and the data were modified to ANOVA followed by DMRT. Significance at 5% level was calculated. RESULTS AND DISCUSSION Phytochemical analysis Preliminary qualitative phytochemical analysis This profile of phytochemicals in the leaf part of T. asiatica can provide ideas about the synthesis of certain bioactive compounds for the manufacturing of some useful drugs as reported by Yakubu et al. (2005) for Fadogia agrestis. Quantitative phytochemical analysis The percentage yield of crude methanolic leaf extract is 29.8. The total phenolics and flavonoids S. No. Sample concentration (µg/ml) % of inhibition IC50 value of leaf extract IC50 value of BHT 1. 50 33.20±0.32a 231.48±0.54 34.74±00.26 2. 100 42.05±0.09b 3. 150 52.96±0.31c 4. 200 53.65±0.23cd 5. 250 68.26±0.43e Table 3. DPPH• radical scavenging activity of methanolic leaf extract of Tarenna asiatica. Values were performed in triplicates and represented as mean ± SD. Mean values followed by different superscript in a column are significantly different (p<0.05). Butylated Hydroxyl Toluene (BHT). S. No. Sample concentration (µg/ml) % of inhibition IC50 value of leaf extract IC50 value of EDTA 1. 50 48.68±0.12a 152.90±0.04 120.77±0.05 2. 100 49.43±0.34ab 3. 150 58.80±0.13b 4. 200 61.04±0.45c 5. 250 66.66±0.15d Table 4. Ferrous ion chelating activity of methanolic leaf extract of Tarenna asiatica. Values were performed in triplicates and represented as mean ± SD. Mean values followed by different superscript in a column are significantly different (p<0.05). Ethylene Diamine Tetra Acetic acid (EDTA).
  6. 6. Chandran et al.,2013 201 Journal of Research in Plant Sciences (2013) 2(2): 196-204 content of methanolic leaf extract were 16.95µg of GAE/100mg and 3.7295µg of QE/100mg (Table 2). Phenolic compounds are known as powerful chain breaking antioxidants and they may contribute directly to antioxidative action (Lu et al., 2011). Flavanoids possess a broad spectrum of chemical and biological activities including radical scavenging properties (Prasad et al, 2009). In vitro antioxidant activity DPPH• radical scavenging assay DPPH• is a relatively stable free radical. The assay is based on the measurement of the scavenging ability of antioxidants towards the stable free radicals DPPH• (Halliwell and Gutteridge, 2007). Free radical scavenging capacities of methanolic leaf extract was measured by their ability to trap free radical DDPH• . The percentage of inhibition was increased with the increasing concentration of the sample from 50-250µg/ mL (Table 3). The IC50 value of the extract was 231.48 which is comparable to that of the standard, BHT. DPPH• test provides information on the reactivity of test compounds with a stable free radical. Because of its, addition of electron to 2, 2-Diphenyl-Picryl Hyrazyl radical (DPPH• ) gives a strong absorption band at 517nm in visible spectroscopy. The efficacies of antioxidants are often associated with their ability to scavenge stable free radicals (Krishnaraju et al., 2009). It has been shown that the scavenging effects on the DPPH• radical increased sharply with the increasing concentration of the samples and stands to a certain extent (Jamuna et al., 2012; Karthika et al, 2012) and hence are said to be strongly dependent on the extract concentration. Ferrous ion metal chelating assay Metal ion chelating activity may be due to the chelating agents, which form sigma bonds with the metal and effective as secondary antioxidants because they reduce the redox potential, there by the oxidized form of the metal ion (Gulcin et al., 2004). The results of antioxidant activity of the methanolic leaf extract of T. asiatica based on metal chelating activity are given in Table 4. The percentage of metal chelating activity was determined to be sample concentration dependent and it was increased with the increase of concentration of extract from 50-250µg/mL. The percentage of inhibition of the metal chelating was varying from 48.68% at 50µg/ mL to 66.66% at 250µg/mL. The IC50 values of the methanolic leaf extract of the study species was 152.90µg/mL and it was comparable to that of the standard drug, EDTA. Similar trend of metal ion scavenging activity was observed in the species, Cyperus rotundus (Shajiselvin and Kottai Muthu, 2011; Thambiraj and Paulsamy, 2012). Reducing power assay It is based on the principle that the substances of reduction potential react with potassium ferricyanide (Fe+++ ) to form potassium ferrocyanide (Fe++ ) which reacts with ferric chloride and form ferrous complex that has an absorption maximum at 700nm. Generally, the level of reducing power of a compound is one of the indicators to show the degree of efficiency of its antioxidant property. Change in yellow colour in to various shades of green and blue colour in the present test for the phenolic leaf extract T. asiatica indicates the presence of antioxidant property for this species. Fig. 1 shows the reductive capabilities of methanolic leaf Figure 1. Reducing power activity of methanolic leaf extract of Tarenna asiatica.
  7. 7. Chandran et al.,2013 Journal of Research in Plant Sciences (2013) 2(2): 196-204 202 extract of T. asiatica when compared to the standard, ascorbic acid. As responded for DPPH• and ferrous ion chelating activities, the reducing power also increased with the increase of concentration of the samples. The extract of T. asiatica showed the highest reducing ability (absorbance 0.493) for 250µg/mL. However, the activity was lesser than the standard, ascorbic acid (absorbance 1.393). ABTS•+ cation radical scavenging activity The decolorization of the ABTS•+ , through measuring the reduction of the radical cation as the percentage inhibition of absorbance at 734nm (Re et al., 1999). ABTS•+ was generated by (Wolfenden et al., 1982) incubating ABTS•+ chromophore through the reaction. The reduction of the 2,2’-azinobis (3- ethylnenzo thiazoline sulphonate) radical cation (ABTS•+ ) has widely used to measure the antioxidant capacity of natural extract (Cai et al., 2004; Cai et al., 2006). The presence of chemical compounds in the tested extracts that inhibit the potassium persulfate activity may reduce the production of ABTS•+ . This study reports that the methanolic leaf extract of T. asiatica have highest antioxidant activity, 3186 ± 21.10μmolTE/g extract (Table 8). It shows that the methanolic leaf extract of T. asiatica possessed the highest ABTS•+ scavenging capacity. Quantitative phytochemical analysis indicated that the plant extract contains significant amounts of phenolics and flavonoids compounds such as total phenolic and flavanoids. These classes of secondary metabolites are playing major role for antioxidant and free radical scavenging effect (Sreeram et al., 2005; Gill et al., 1999; Hazra et al., 2008). The antioxidant activity of phenolic compounds is mainly due to their redox properties, which play an important role in neutralizing free radicals, quenching singlet and triplet oxygen and flavanoids are wide spread in all natural compounds and posses a broad spectrum of biological activities. Due to the presence of phytochemicals in general and phenolics and flavonoids in large amount in particular, this species, T. asiatica may has effective free radical scavenging activity. CONCLUSION On the basis of the results obtained in the present study, it was concluded that the methanolic leaf extract of T. asiatica possess the significant antioxidant activity. These findings suggest that this plant is a potential source of natural antioxidant. However, further studies are needed to understand the mechanism of action extract and to isolate the compounds responsible for such activities. ACKNOWLEDGMENT The authors are gratefully thankful to Dr. M. Aruchami, Secretary and Director, Kongunadu Arts and Science College, Coimbatore for permitting to do the work in the Botany Department of his institute under consultancy scheme. They sincerely acknowledge Dr. S. Paulsamy, Associate Professor in Botany, Kongunadu Arts and Science College for offering his expertise to carry out the work. REFERENCES Blois MS. 1958. Antioxidant determination by the use of a stable free radical nature. Nature. 26: 181(4617): 1199- 1200. Brain KR and Turner TD. 1975. The Practical Evaluation of Phytopharmaceuticals. Wright Scientechica, Bristol.; 57-58. Buxiang S and Fukuhara M. 1997. Effects of co- administration of butylated hydroxytoluene, butylated hydroxyanisole and flavonoids on the activation of mutagens and drug metabolizing enzymes in mice. Toxicology.; 122(1-2): 61–72. Cai Y, Luo Q, Sun M and Corke H. 2004. Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer. Life Sci.; 74(17): 2157-2184.
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