Antioxidant and anti inflammatory activity tectona grandis ijrpp

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International Journal of Research in Pharmacology and Pharmacotherapeutics (Research article)

PHYTOCHEMICAL SCREENING, ANTIOXIDANT AND ANTIINFLAMMATORY ACTIVITY OF DIFFERENT EXTRACTS FROM LEAF, STEM AND BARK OF TECTONA GRANDIS *1

Shruthi D.P, 2Sunith K.E, 3Haritha Kumari E, 4Govindappa M, 5Siddalingeshwara K.G Department of Biotechnology, Shridevi Institute of Engineering & Technology, Sira Road, Tumkur-572 106, Karnataka, India.

5 Department of Biochemistry, Tumkur University, Tumkur, Karnataka, India.

1

_________________________________________________________________________ ABSTRACT Interest in natural products as a source for innovation in drug discovery and agrochemicals is still growing worldwide. Natural products, whose immense diversity has been appreciated for many years, may become in a rich source of novel chemical structures. Our country is a rich source of both biological and chemical diversity, which may be useful as a source of novel chemical structures. The timber value of Tectona grandis has been well known from decades. Teak is the major exotic species found in tropical regions. The present study was meant to characterize pharmacological potential of different extracts from leaf, bark and stem of teak. The aim of present study to investigate the preliminary phytochemical screening of Tectona grandis. This work highlights on qualitative phytochemical investigation on leaf, bark and stem. Of the Tectona grandis. A comparative phytochemical analysis was carried to prove that the amount of phyto constituents varied with the fresh and dry stages of plants contributing to the activity of the extract. Total phenolic and saponnins content of both the extracts was estimated and was found to be more in the leaf, bark and stem giving positive results and the presence of steroids and Terpenoids also showed in leaf and stem. Antioxidant activity of extracts was carried out using ferric ion reducing antioxidant power (FRAP) and 2, 2-diphenyl-1- picrylhydrazyl (DPPH) assay and anti-inflammatory activity of the Tectona grandis extract were also made.

KEY WORDS: Antioxidant, anti-inflammatory activity, Tectona grandis, phytochemical screening.

INTRODUCTION Plants are the basis for traditional medicine systems and used for thousands of year in countries such as India and China. Numbers of medicinal plants and its derived extracts are used in the treatment of various disorders by Ayurveda, Unani and Siddha systems in India. Scientifically, few of pharmacological properties have been studied to support its traditional use 1. Throughout the world, many plant species are used for the treatment of inflammation and other diseases. Despite the availability of anti-inflammatory and analgesic _________________________________ * Corresponding author: *1 Shruthi D.P Department of Biotechnology, Shridevi Institute of Engineering & Technology, Sira Road, Tumkur-572 106, Karnataka, INDIA. E-mail: shruthidavasam@gmail.com

agents searching of newer therapeutic agent in this segment from the natural plants is still progressing due to presence of diverse chemical substances that have a better alternative and safer effect on inflammation without or lesser side effects. The World Health Organisation (WHO) estimates that 80% of the world’s inhabitants continue to rely on traditional medicines systems and its products 2.

Tectona grandis belonging to the family Verbinaceae is commonly called as teak. The various parts of the plants are reported to posses

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various activities. Some of the activities include its action as a cooling agent, laxative and sedative, bronchitis, as diuretic and in the treatment of urinary discharge, in the treatment of the common cold and headache, hair problems and in scabies 3, 4.

The various phyto constituents isolated from Tectona grandis are Juglone, which has been reported to posses anti-microbial activity 5, Betulin aldehyde is reported to posses anti tumor activity 6, Lapchol shows anti ulcerogenic activity 7. The present work was designed to give a detailed picture of antioxidant and anti inflammatory potentiality of extracts from different parts of Tectona grandis and also qualitative studies of photochemistry of the plants were studied.

MATERIALS AND METHODS Plant materials collection and identification Plant material was collected from the campus of Shridevi Institute of Engineering and Technology, Tumkur, Karnataka, India. The collected plant was authenticated from the Department of Botany, Manasa Gangotri, University of Mysore, Mysore, Karnataka, India and Government Ayurvedic College, Mysore, and herbarium was prepared.

Extraction of plant material The fresh and dry T. grandis plant material were powdered and extracted with distilled water at room temperature for 24 h. and filtered to the help of Whatman No. 1 and fresh and dry plant materials were also used for extraction by 90% methanol using soxhlet apparatus for 72 h. All extracts were concentrated under reduced pressure using rotary evaporator and stored at 2 - 8 0C until the completion of qualitative phytochemical studies, antioxidant and ant-inflammatory activity.

Antioxidant activity Antioxidant activity of extracts was carried out using ferric ion reducing antioxidant power (FRAP) and 2, 2-diphenyl-1- picrylhydrazyl (DPPH) assay.

FRAP assay: FRAP reagents was freshly prepared by mixing 25 ml acetate buffer (300 mM, pH 3.6), 0.5 mL 2,4,6tris (2-pyridyl)-S-triazine (TPTZ) solution (10 mM TPTZ in 40 mM/l HCl) and 2.5mL FeCl3 (20 mM) water solution. Each sample (150 μl) (0.5 mg/ml) dissolved in methanol was added to 4.5 mL of freshly prepared FRAP reagent and stirred. After 5 min, absorbance was measured at 593 nm, using

FRAP working solution as blank 8,9 . A calibration curve of ferrous sulfate (100 to1000 μmol/l) was used and results were expressed in μmol Fe2+/mg dry weight extract. The relative activity of the samples was compared to L-ascorbic acid.

DPPH radical assay: The effect of plant extracts on DPPH radical was estimated using the method of Liyana-Pathirana and Shahidi 10. DPPH solution was freshly prepared by dissolving 24 mg DPPH in 100 ml methanol, stored at -20°C before use. 10 μl of the sample was added to 140 μl distilled water and allowed to react with 2850 μl of DPPH reagent (190 μl reagent + 2660 μl distilled water) for 24 h in the dark condition. Absorbance was measured at 515 nm. A linear standard curve between, 25 to 800 μM ascorbic acid, was obtained and expressed in μm AA/g fresh mass. Additional dilution will be needed if the DPPH value measured is over the linear range of the standard curve; mix 10 ml of stock solution in a solution of 45 ml of methanol, to obtain an absorbance of 1.1 ± 0.02 units at 517 nm using spectrophotometer 11. All determinations were performed in triplicate. The percentage inhibition of DPPH radical by the samples was calculated according to formula of Yen and Duh 12, % inhibition= [{Abs control - Abs sample}/Abs control] x 100, Where Abs control is the absorbance of the DPPH radical + ethyl acetate, Abs sample is the absorbance of DPPH radical+ sample extract/standard.

Anti-inflammatory activity Inhibition of albumin denaturation Methods of Mizushima and Kobayashi 13 and Sakat et al. 14 followed with minor modifications. The reaction mixture was consisting of test extracts and 1% aqueous solution of bovine albumin fraction, pH of the reaction mixture was adjusted using small amount at 37 0C. The extracts incubated (20min) and then heated to 51 0C the samples the turbidity was measured spectrophotometrically at 660nm. The experiment was performed in triplicate. Percent inhibition of protein denaturation was calculated as fallows. % inhibition= [{Abs control- Abs sample}/Abs control] x 100, Where Abs control is the absorbance without sample, Abs sample is the absorbance of sample extract/standard.

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(W/V) casein was added. The mixture was inhibited for an additional 20 min, 2ml of 70% perchloric acid was added to terminate the reaction.

Cloudy suspension was centrifuged, and the absorbance of the supernatant was read at 210nm against buffer as blank. The experiment was performed in triplicate. The percentage of inhibition of proteinase inhibitory activity was calculated.

Membrane stabilization test Preparation of red blood cells (RBCs) suspension Fresh whole human blood (10ml) was collected and transferred to the centrifuge tubes 14. The tubes were centrifuged at 3000 rpm for 10min and were washed three times with equal volume of normal saline. The volume of blood was measured and re constituted as 10% v/v suspension with normal saline.

Xanthine oxidase assay Xanthine oxidase activity was assayed spectrophotometrically at 300 nm as described by Yamamoto et al. 16. Briefly, the reaction mixture consisting of 500 μl of solution A (0.1Mphosphate buffer containing 0.4mM xanthine and 0.24 mM NBT), 500 μl of solution B (0.1 M phosphate buffer containing 0.0449 units/ml xanthine oxidase) and 50 μl of a 10% of each solvent extracts were incubated in a cuvette at 37 0C for 20 min. The enzyme activity was expressed as the increment in absorption at 300 nm per unit time.

Heat induced hemolytic The reaction mixture (2ml) consisted of 1 ml of test sample solution and 1 ml of 10% RBCs suspension, instead of test sample only saline was added to the control test tube. Aspirin was used as a standard drug. All the centrifuge tubes containing reaction mixture were incubated in water bath at 56⁰C for 30min. At the end of the incubation the tubes were cooled under running tap water. The reaction mixture was centrifuged at 2500 rpm for 5 min and the absorbance of the supernatants was taken at 560 nm 14. The experiment was performed in triplicates for all the test samples. Percent membrane stabilization activity was calculated by the formula mentioned below.

RESULTS AND DISCUSSION Phytochemical screening Phytochemical analysis of methanol and water solvent extract for saponins, phenolic compounds, anthraquinones, steroids, terpenoids, tannins and alkaloids are done. Plant extracts have all the phytochemical except anthraquinones and alkaloids. The presence of saponnins and phenolic compounds was observed in higher amount than compared to steroids, tannins and terpenoids. The phytochemical studies were presented in Table 1 and 2 with solvent extract.

Protein inhibitory action The test was performed according to the modified method of Oyedepo et al.15and Sakat et al.14. The reaction mixture (2ml) was containing 0.06mg trypsin, 1ml of 20mM Tris HCl buffer (pH7.4) and 1ml test sample of different concentrations of different solvents. The reaction mixture was incubated at 37⁰C for 5min and then 1ml of 0.8%

Table 1: Phytochemical analysis of different plant parts (20ml distilled water) Leaf Tests

Stem

Bark

Fresh

dry

Fresh

dry

Fresh

dry

Saponins

+ve

+ve

+ve

+ve

+ve

-ve

Phenols

+ve

+ve

+ve

+ve

+ve

-ve

Anthaquinones

-ve

-ve

-ve

-ve

-ve

-ve

Steroids

+ve

-ve

+ve

-ve

-ve

-ve

Tannins

+ve

+ve

-ve

+ve

-ve

-ve

Terpenoids

+ve

+ve

-ve

+ve

-ve

-ve

Alkaloids

-ve

-ve

-ve

-ve

-ve

-ve

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Table 2: Phytochemical analysis of different plant parts (99% methanol

Leaf Tests

Stem

Bark

Fresh

dry

Fresh

dry

Fresh

dry

Saponins

+ve

+ve

+ve

+ve

+ve

-ve

Phenols

+ve

+ve

+ve

+ve

+ve

+ve

Anthaquinones

-ve

-ve

-ve

-ve

-ve

-ve

Steroids

+ve

+ve

+ve

-ve

-ve

-ve

Tannins

+ve

+ve

-ve

+ve

-ve

-ve

Terpenoids

+ve

-ve

-ve

+ve

-ve

-ve

Alkaloids

-ve

-ve

-ve

-ve

-ve

-ve

Antioxidant activity of Tectona grandis extract Antioxidant status of all extracts was checked by DPPH and FRAP free radical. Its ability to scavenge those free radicals at different concentrations was analysed. Plant at a concentration of 0.1mg/ml, the scavenging activity of the endophytes reached at high concentration,

shows the dose response curve of DPPH radical scavenging activity of ethyl acetate of plant extract (leaf, stem and flower). The study on antioxidant activity of Tectona grandis with its crude ethanol extracts by H2O2 scavenging activity, DPPH and FRAP proved its potential. The fig 1 and Table 3 will show the DPPH and FRAP antioxidant activity.

Figure 1. DPPH scavenging activities

% of inhibition

Concentration in Âľg/ml

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Table 3.Total antioxidant (FRAP) activities Extracts Leaf Stem Flower

FRAP 1623.21±0.06a 1432.64+0.08d 810.14±1.2f 773.32+1.4g 1611.26+0.06b 1368.57±0.09e 1648.52±0.06c 64.84±1.5h

Fresh Dry Fresh Dry Fresh Dry

Ascorbic acid BHT The total phenolic content of the metabolites of host plant extract are shown and they had the highest total phenolic content (TPC). The Phenolic compounds in the plant extract may have contributed significantly to their antioxidant activity.

Anti inflammatory properties Inhibition of albumin denaturation

followed by different subscripts are significantly different at P<0.05, SE-standard error of the mean.

Repeated the experiments three times for each replicates, According to Duncan’s Multiple Range.

Test (DMRT), values followed by different subscripts are significantly different at P<0.05, SEStandard error of the mean.

Statistical analysis

Denaturation of proteins is a well documented cause of inflammation. As part of the investigation on the mechanism of the anti-inflammation activity, ability of different solvent plant extract protein denaturation was studied. It was effective in inhibiting heat induced albumin denaturation.

Maximum inhibition 89.61% was observed from methanol extract followed by ethanol 86.81% and water 51.14%. All the solvent extracts inhibited the albumin denaturation, the methanol extract stood first compared to ethanol and water extracts.

Aspirin, a standard anti-inflammation drug showed the maximum inhibition 75.89% at the concentration of 200 μg/ml. Repeated the experiments three times for each replicates and results were presented in Table. 4. According to Duncan’s Multiple Range Test (DMRT), values

Analysis of variance (ANOVA) was used to determine the significance of difference between treatment groups (p < 0.05). Means between treatment groups were compared for significance using Duncan’s new Multiple Range post test.

Proteinase inhibitory activity The T.grandis different solvent extracts exhibited significant antiproteinase activity represented in Table 4. The maximum inhibition was observed from methanol extract (83.91%) in decreasing order was ethanol (81.17%) and water (61.73%).

The methanol and ethanol extract have showed highest proteinase inhibitory activity compared to water extract. The standard drug aspirin have showed the maximum proteinase inhibitor activity is 92.83%

Table 4. Effect of water extracts on albumin denaturation, membrane stabilization and proteinase inhibitory activity Xanthane oxidase percentage inhibition Test sample Fresh extract Leaf Bark Stem Dry extract Leaf Bark Stem Aspirin (200μg/ml)

Albumin denaturation

Membrane stabilization

Proteinase inhibition

Xanthane oxidase

89.61±0.06a 51.14±0.08e 86.81±0.06b

78.82±0.04b 52.31±0.06d 76.65±0.05b

83.91±0.03b 64.84±0.06d 81.17±0.03c

41.13±0.07f 36.92±0.08f 38.97±0.08f

86.26±0.06b 48.22±0.07f 82.11±0.06c 75.89±0.06d

74.63±0.06c 50.72±0.06d 72.46±0.04d 85.92±0.02a

79.33±0.06c 64.84±0.06d 77.55±0.05c 92.83±0.03a

40.16±0.07f 34.86±0.08f 36.75±0.08f 95.96±0.03f

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Xanthine oxidase assay

6.

The maximum inhibition of xanthine oxidase was observed from methanol extract (41.13%) followed by ethanol (38.97%) and water (36.72%).

Maximum inhibition was noticed in methanol and ethanol extracts compared to the water extract. The standard drug aspirin have showed the maximum proteinase inhibitor activity is 95.96% (Table 4).

7.

CONCLUSION In this study results indicate that the different solvent extracts of T.grandis possess antioxidant and anti-inflammatory properties. These activities may be due to the strong occurrence of polyphenolic compounds such as tannins, steroids, phenols, terpenoids and Saponins. The extract fractions serve as free radical inhibitors or scavenger or acting possibly as primary oxidants and inhibited the heat induced albumin denaturation and proteinase activity and stabilized the Red Blood Cells membrane. The solvent fractions exhibited a moderate xanthine oxidase (XO) inhibitory activity and therefore may due to presence of bioactive constituents and these can useful in the treatment of xanthine oxidase induced diseases. This paper proposing its potential application as a lead compounds for designing potent anti-inflammatory activity and they can be used for treatment of various diseases like bronchitis, biliousness, hyperacidity, dysentery, diabetes, leprocy, and inflammatory.

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