BIOACTIVE COMPOUND IDENTIFICATION, PHYTOCHEMICAL ESTIMATION, IN-VITRO ANTI-INFLAMMATORY AND ANTIOXIDANT ACTIVITY OF PUPALIA LAPPACEA
HTML Full TextBIOACTIVE COMPOUND IDENTIFICATION, PHYTOCHEMICAL ESTIMATION, IN-VITRO ANTI-INFLAMMATORY AND ANTIOXIDANT ACTIVITY OF PUPALIA LAPPACEA
A. Tamil Selvan * 1, N. Siva Subramanian 2, M. Ramadevi 2, B. Sree Giri Prasad 2 and Santhosh Kumar Muthu 2
Department of Pharmacology 1, Teegala Krishna Reddy College of Pharmacy, Meerpet, Saroor Nagar, Hyderabad - 500097, Andhra Pradesh, India.
Department of Biochemistry 2, PSG College of Arts and Science, Coimbatore - 641014, Tamil Nadu, India.
ABSTRACT: Pupalia lappacea Juss belongs to the family Amaranthaceae useful in the bone fracture treatment, antimalarial, pyrexia, wounds, cough, toothache and boils. The present investigation was aimed to identify the phytocompounds by GC-MS, investigate phytochemically and to screen anti-inflammatory and antioxidant property by in-vitro methods. Bioactive compounds were identified using GC- MS; functional groups were identified using FT-IR, phytochemical estimation was done for the phenolics, flavonoids, tannins, and alkaloids, in-vitro anti-inflammatory activity by heat-induced hemolytic and albumin denaturation method, in-vitro antioxidant by DPPH, SOD, NO, PPO and CAT methods. GC-MS analysis of the methanolic extract confirmed the presence of bioactive principles like phenols, tetradecanoic acid, pentadecanoic acid, Eicosatetraenoic acid, Hexatriacontane, eicosapentaenoic acid, and chloroundecane. Methanolic extract of aerial parts of Pupalia lappacea was estimated for the presence of secondary metabolites and screened for in-vitro anti-inflammatory and antioxidant effects. Aerial parts were extracted and estimated for total phenolics 248.19 ± 4.84, flavonoids 123.47 ± 0.19, tannins 57.12 ± 2.45 and alkaloids 69.29 ± 1.84 quantitatively reaction equivalent per gram of the extract. The FT-IR report showed the presence of functional groups like amines, ester, alkyl and other carbonyl groups. Significant RBC membrane stabilisation effect in heat-induced haemolysis method and egg albumin protein inhibition was studied for in-vitro anti-inflammatory action. The phytochemicals like alkaloids, phenolics, flavonoids and tannins present in the extract might be responsible for the desired anti-inflammatory effect of the methanolic extract, which was confirmed by the preliminary phytochemical tests and by estimation. Also, the extract showed anti-oxidant properties in DPPH, Superoxide dismutase and nitrogen oxide, polyphenol oxidase, and catalase methods compared with the standard ascorbic acid.
Keywords: |
Pupalia lappacea, Methanolic extract, Aerial parts, Diclofenac sodium, Egg albumin, Heat-induced hemolytic, Protein inhibition, Antioxidant
INTRODUCTION: Inflammation is a bodily response to injury, infection or destruction characterized by heat, pain, redness, swelling and disturbed physiological functions. It is a protective response to tissue injury caused by physical trauma, noxious chemical or microbial agents.
It is the body response to inactivate or destroy the invading organisms to remove the irritants and set the stage of tissue repair. It is released by the chemical mediators from injured tissues and migrating cells 1. Natural sources like plants, herbs have been used as an alternative therapy for the ailment of various diseases since from ancient times.
Present years, a lot of research has been explored in medicinal plants in the treatment of various stress-related disorders caused by metabolism of oxygen leads to generate free radicals.
Pupalialappacea belongs to the family Amaranthaceae is commonly known as Forest Burr or Creeping cock’s comb. Itis an erect or straggling under shrub found in the edges of fields, fruit orchards, dry scrub forests and waste places of Kashmir to Kauman at an altitude of 300-1050 m and in all states of India 2.
The leaf paste of Pupalialappacea with edible oil (Sesamum or Carthamus) is an effective and inexpensive treatment of bone fracture for human beings as well as cattle. Stems used as a toothbrush, for treating toothache. A poultice of the fresh leaves is used in the treatment of boils, new and chronic wounds. A decoction of the black powder of the plant is drunk to cure piles and for enema, fever, and malaria 3.
MATERIALS AND METHODS:
Collection and Extraction: The aerial parts were purchased from Dr. Madhava Chetty, Sri Venkateswara University, Tirupathi and was air dried until free from moisture. Then they were subjected to size reduction to get coarse powder of desired particle size. The powdered drug was subjected to extraction with petroleum ether and methanol in a Soxhlet extractor, the temperature was maintained on an electric heating mantel with thermostat control.
The extracts were then concentrated to ¾ th of their original mass using rotary vapor apparatus. The concentrated extract was then transferred to a china dish and evaporated on a thermostat controlled water bath till it formed a thick paste. The thick mass was vacuum dried in a desiccator till it is free from moisture.
Phytochemical Test: Phytochemical tests on the extract and fractions were performed using standard procedures 4.
Estimation of Secondary Metabolites:
Determination of Total Phenolic contents: The amount of total phenolics in the extract was determined with the Folin-Ciocalteu reagent. Gallic acid was used as a standard, and the total phenolics were expressed as mg/g gallic acid equivalents (GAE). Concentration of 0.01, 0.02, 0.03, 0.04 and 0.05 mg/ml of gallic acid were prepared in methanol. Concentration of 0.1 and 1mg/ml of plant extract were also prepared in methanol and 0.5 ml of each sample were introduced into test tubes and mixed with 2.5 ml of a 10 fold dilute Folin- Ciocalteu reagent and 2 ml of 7.5% sodium carbonate. The tubes were covered with parafilm and allowed to stand for 30 min at room temperature before the absorbance was read at 760 nm spectrometrically.
All determinations were performed in triplicate. The Folin-Ciocalteu reagent being sensitive to reducing compounds including polyphenol is producing a blue color upon a reaction which is measured spectrophotometrically 5.
Determination of Total Flavonoids Content: The total flavonoids content of each plant extract was estimated by the method described by Zhishen et al. 6 Based on this method, each sample (1.0 ml) was mixed with 4 ml of distilled water and subsequently with 0.30 ml of a NaNO2 solution (10%). After 5 min, 0.30 ml AlCl3 solution (10%) was added followed by 2.0 ml of NaOH solution (1%) to the mixture. Immediately, the mixture was thoroughly mixed, and absorbance was then determined at 510 nm versus the blank. A standard curve of Rutin was prepared (0-12 mg/ml), and the results were expressed as Rutin equivalent (mg Rutin/gm dried extract).
Determination of Tannin Contents: Tannin content in each sample was determined using insoluble polyvinylpolypyrrolidone (PVPP), which binds tannins as described by Makkar et al. 7 Briefly, 1 ml of extract dissolved in methanol (1 mg/ml), in which the total phenolics were determined, was mixed with 100 mg PVPP, vortexed, kept for 15 min at 4 °C and then centrifuged for 10 min at 3,000 rpm. In the clear supernatant, the non-tannin phenolics were determined the same way as the total phenolics 10. Tannin content was calculated as a difference between total and non-tannin phenolic content.
Determination of Alkaloids Content: 5 g of the sample was weighed into a 250 ml beaker, and 200 ml of 10% acetic acid in ethanol was added and covered and allowed to stand for 4 h. This was filtered, and the extract was concentrated on a water bath to one-quarter of the original volume.
Concentrated ammonium hydroxide was added dropwise to the extract until the precipitation was complete. The whole solution was allowed to settle, and the precipitated was collected and washed with dilute ammonium hydroxide and then filtered. The residue is the alkaloid, which was dried and weighed. Atropine Equivalent was used as a standard 8.
FT-IR Analysis: The FT-IR analysis of the plant extract in KBr pellets by using FT-IR spectroscopy Shimadzu, IR affinity 1, Japan) at a moderate scanning speed between 4000-400 cm-1. The peak values (wave numbers) and the possibility of the functional group were shown.
GC-MS Analysis: GC-MS analysis of these extracts was performed using a Perkin-Elmer GC Clarus 500 system and Gas chromatograph interfaced to a Mass spectrometer (GC-MS) equipped with an Elite-I, fused silica capillary column (30 mm × 0.25 mm 1D × 1µMdf, composed of 100% Dimethylpolysiloxane). For GC-MS detection, an electron ionization system with ionizing energy of 70eV was used.
Helium gas (99.999%) was used as the carrier gas at constant flow rate 1 ml/min, and an injection volume of 2 µl was employed (split ratio of 10:1); Injector temperature 250 °C; Ion source temperature 280 °C. The oven temperature was programmed from 110 °C (isothermal for 2 min.), with an increase of 10 °C/min, to 200 °C, then 5 °C/min to 280 °C, ending with a 9 min isothermal at 280 °C. Mass spectra were taken at 70 eV; a scan interval of 0.5seconds and fragments from 45 to 450 Da.
Total GC running time was 36 min. The relative % amount of each component was calculated by comparing its average peak area to the total areas; software adapted to handle mass spectra, and chromatograms was a Turbomass 9.
Interpretation on mass spectrum GC-MS was conducted using the database of national Institute Standard and Technology (NIST) having more than 62,000 patterns. The spectrum of the unknown component was compared with the spectrum of the known components stored in the NIST library. The Name, Molecular weight, and structure of the components of the test materials were ascertained. The results were shown in Table 2.
In-vitro Pharmacological Screening heat Induced Haemolytic Method:
Preparation of Red Blood Cells (RBC’s Suspension): Fresh whole human blood 10 ml was collected and transferred to the centrifuge tubes. The tubes were centrifuged at 3000 rpm for 10 min and were washed three times with equal volume of normal saline. The volume of blood was measured and reconstituted as 10% v/v suspension with normal saline.
The reaction mixture 2 ml consisted of 1ml of the test sample solution and 1ml of 10% RBC’s suspension, instead of test sample the only saline was added to the control test tube. Aspirin was taken as a standard drug. All the centrifuge tubes containing reaction mixture were incubated in water bath at 56 °C for 30 min 10.
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. The experiment was performed in triplicates for all the test samples. Percentage membrane stabilization activity was calculated using the formula:
Percentage of inhibition = 100 × {Vt – Vc – 1}
Where, Vt = Optical density of test, Vc = Optical density of control
Inhibition of Albumin Denaturation: The reaction mixture (5 ml) consisted of 0.2 ml egg albumin from fresh hen’s egg, 2.8 ml of phosphate buffered saline (PBS, pH 6.4) and 2 ml of varying concentrations of extract so that so that final concentrations become 50, 100, 200, 400 and 800 µgm/ml. A similar volume of double distilled water served as control. Then the mixtures were incubated at 37 ± 2°C in a BOD incubator for 15 mins and then heated at 70 °C for 5 min. After cooling the absorbance was measured at 660 nm (SHIMADZU, UV 1800) by using the vehicle as blank.
Diclofenac sodium at the final concentration of 50, 100, 200, 400 and 800 µgm/ml was used as a reference as reference drug and treated similarly for absorbance 11. The percentage inhibition of protein denaturation was calculated by using the following formula:
Percentage inhibition = 100 × [Vt – Vc/1]
Where, Vt = absorbance of the test sample, Vc = absorbance of control
Anti-oxidant Screening:
DPPH Radical Scavenging Assay: The free radical scavenging activity was measured in-vitro by DPPH assay. About 0.3mM solution of DPPH in methanol was prepared, and 1ml of this solution was added to 1 ml of both the extract at different concentrations (100, 200, 300, 400 and 500 µg/ml). The mixture was shaken and allowed to stand at room temperature for 30 min, and the absorbance was measured at 517 nm using a spectrophotometer. Lower absorbance of the reaction mixture indicates higher free radical scavenging activity. The percent DPPH scavenging effect was calculated using the following equation.
DPPH scavenging effect (%) = [(A0 – A1)/ AO] × 100
Whereas, A0 is the absorbance of the control reaction mixture and A1 is the absorbance of the plant extract or standard at different concentrations 12.
Super Oxide Dismutase: The assay of superoxide dismutase was done according to the method of Das. In this method, 1.4 ml aliquots of the reaction mixture (comprising 1.11 ml of 50 mM phosphate buffer of pH 7.4, 0.075 ml of 20 mM L-Methionine, 0.04 ml of 1% (v/v) Triton X-100, 0.075 ml of 10 mM Hydroxylamine hydrochloride and 0.1ml of 50 mM EDTA) was added to 100 μl of the sample extract and incubated at 30 ºC for 5 min. 80 μl of 50 μM riboflavin was added, and the tubes were exposed for 10 min to 200 W-Philips fluorescent lamps. After the exposure time, 1ml of Greiss reagent (a mixture of an equal volume of 1% sulphanilamide in 5% phosphoric acid) was added, and the absorbance of the color formed was measured at 543 nm. One unit of enzyme activity was measured as the amount of SOD capable of inhibiting 50% of nitrite formation under assay conditions 13.
Nitric Oxide Radical Scavenging Activity: 3 ml of 10 mM sodium nitroprusside in 0.2 M phosphate buffered saline (pH 7.4) was mixed with different concentrations (250- 1250 µg/ml). After the incubation time, 0.5 ml of Griess reagent (1% sulfanilamide, 0.1% naphthyl ethylenediamine dihydrochloride in 2 % H3PO4) was added. The absorbance of the chromophore formed was measured at 546 nm. Percentage of radical scavenging activity was calculated 14.
% NO radical scavenging activity = (Control OD-Sample OD / Control OD) × 100
Polyphenol Oxidase Activity: Assay of Polyphenol oxidase activity was carried out according to the procedure of Sadasivam and Manickam 15. To 2.0 ml of plant extract and 3.0 ml of distilled water added and mixed. 1.0 ml of catechol solution (0.4 mg/ml) added to the above solution, and the reactants were quickly mixed. The enzyme activity was measured as the change in absorbance/min at 490 nm.
Catalase Activity: Catalase activity was assayed by the method of Sinha 16. The enzyme extract (0.5 ml) was added to the reaction mixture containing 1ml of 0.01 M phosphate buffer (pH 7.0), 0.5 ml of 0.2 M H2O2, 0.4 ml H2O and incubated for the different period.
The reaction was terminated by the addition of 2 ml of acid reagent (dichromate/acetic acid mixture) which was prepared by mixing 5% potassium dichromate with glacial acetic acid (1:3 by volume). To the control, the enzyme was added after the addition of acid reagent. All the tubes were heated for 10 min, and the absorbance was read at 610 nm. Catalase activity was expressed in terms of μmoles of H2O2 consumed/min/mg protein.
Statistical Analysis: Data analyzed using One Way Analysis of Variance (ANOVA, SPSS Version 16) and expressed as mean ± SEM and comparisons were done using Tukey’s test as post-hoc. Difference between means was regarded as significant at P<0.01.
RESULTS: The percentage yield of the methanolic extract was 20% w/v of methanol. The phytochemical evaluation showed the presence of alkaloids, terpenoids, flavonoids, carbohydrates, tannins, phenolic compounds, saponins, and phytosterols.
Phytochemical estimation showed the presence of total phenolics 248.19 ± 4.84 mg of tannic acid equivalent per gram of the extract, flavonoids 123.47/0.19 mg of rutin equivalent per gram of the extract, tannin 57.12 ± 2.45 mg of gallic acid equivalent per gram of the extract and alkaloids 69.29 ± 1.84 mg atropine sulphate equivalent per gm of the extract. The results were shown in Table 1. Also, The FT-IR showed the presence of functional groups like amines, ester, alkyl and other carbonyl groups. The results were shown in Table 2. GC-MS result was shown in Table 3.
TABLE 1: ESTIMATION OF SECONDARY METABOLITES
Total Phenolics
mg Tannic acid/g extract |
Flavonoids mg Rutin/g extract | Tannins mg of Gallic acid/gm of extract | Alkaloids mg Atropine RE/g extract |
248.19 ± 4.84 | 123.47 ± 0.19 | 57.12 ± 2.45 | 69.29 ± 1.84 |
Values are means of three independent analysis of the extract ± standard deviation (n=3)
TABLE 2: FT-IR ANALYSIS OF THE METHANOLIC EXTRACT PUPALIA LAPPACEA
S. no. | Peak value nm-1 | Functional group |
1
2 3 4 5 6 7 8 9 10 |
3404.87
2969 1753 1571 1480 1226 1262 1119 829 764 |
Amine group or amide
Alkyl C-H stretch Ester C=O Stretch Amines bending Amines bending Amines stretching Amines stretching Amines stretching ortho substitution ortho substitution |
TABLE 3: COMPONENTS DETECTED IN AERIAL PARTS OF PUPALIA LAPPACEA
S. no. | R.T | Compound name | Formula | Peak area % | M.W |
1 | 6.908 | Phenol | C6H6O | 4.69 | 94 |
2 | 8.964 | 1-Chloroundecane | C11H23Cl | 0.09 | 190 |
3 | 10.440 | Napthalene | C10H8 | 0.02 | 128 |
4 | 12.601 | p-tert-Butyl cyclohexyl-acetamide | C12H22O2 | 0.05 | 198 |
5 | 13.101 | 4-tert-butylcyclohexyl acetamide | C12H22O2 | 0.11 | 227 |
6 | 14.757 | 1-Dodecanamine, N,Nidimethyl | C15H33N | 1.26 | - |
7 | 14.909 | Phenol, 2,4-BIS(1,1Dimethane, N,N-Dimethyl-1-Pentadecane | C14H22O | 1.57 | 206 |
8 | 17.128 | Undecanal,2-methyl | C17H37N | 0.78 | 255 |
9 | 17.208 | Ethanol,2-(dodecycloxy)- | C12H24O | 0.93 | 184 |
10 | 17.294 | Tetradecanoic acid | C14H30O2 | 0.57 | 230 |
11 | 17.446 | Hexadecanoic acid, methyl ester | C9H9NO3SSi | 0.58 | 249 |
12 | 17.742 | Cis-10-Heptadecinoic acid | C14H28O2 | 0.72 | 228 |
13 | 19.469 | Ascorbic acid | C16H30O2 | 1.09 | 254 |
14 | 20.145 | Hexadecanoic acid | C18H36O2 | 1.66 | 284 |
15 | 20.200 | Diethylene glycol monodocecyl ether | C16H34O3 | 0.68 | 274 |
16 | 21.399 | Octadecanoic acid | C19H38O2 | 0.86 | 298 |
17 | 21.597 | Octadec-9-enoic acid | C18H34O | 8.21 | 282 |
18 | 21.633 | 14-Petadecenoic acid | C15H28O2 | 5.04 | 240 |
19 | 21.805 | Stearic acid, Octadenoic acid | C18H36O2 | 9.96 | 284 |
20 | 22.011 | Methyl 17-methyl octadecanoate | C20H40O2 | 2.35 | 312 |
21 | 22.054 | Hexatriacontane | C36H74 | 0.58 | 506 |
22 | 22.781 | 5,8,11,14-Eicosatetraenoic acid | C21H34O2 | 1.01 | 318 |
23 | 23.307 | 11,14,17-Eicosapentanoic acid | C20H30O2 | 8.62 | |
24 | 23.450 | Propyl 5,8,11,14,17 eicosapentaenoate | C23H36O2 | 1.11 | 344 |
25 | 24.161 | Dotriacontane | C32H66 | 2.20 | 450 |
26 | 24.999 | Methyl 4,7,10,13,16,19 docosahexaenoate | C23H34O2 | 2.51 | 342 |
27 | 25.538 | Hexatriacontane | C36H74 | 3.61 | 506 |
28 | 25.846 | 5,8,11,14,17-Eicosapentanoic acid | C21H32O2 | 4.61 | 316 |
29 | 25.930 | Butyl 4,7,10,13,16,19-docosahexaenoate | C26H40O2 | 2.15 | 384 |
30 | 27.253 | Dotriacontane | C32H66 | 2.89 | 450 |
31 | 31.667 | Phenol | C6H6O | 1.11 | 450 |
32 | 32.729 | 1-chloroundecane | C11H23Cl | 0.08 | 190 |
33 | 34.080 | Napthalene | C10H8 | 0.24 | 128 |
34 | 34.886 | Tritiobutylcyclohexyl acetate | C12H22O2 | 0.08 | 198 |
The extract was tested for its anti-inflammatory action in in-vitro by a heat-induced hemolytic method in RBC and protein inhibition by egg albumin denaturation method. The methanolic extract inhibited membrane stabilization effect by inhibiting hypotonic induced lysis of erythrocyte membrane. Stabilization of lysosomal membrane was important in limiting the inflammatory response by preventing the release of lysosomal constituents of activated neutrophil such as bacterial enzyme and proteases which cause further tissue inflammatory and damage 17.
It showed the maximum inhibitory of 68.02 at 800 µg/ml. Diclofenac sodium showed the maximum inhibitory of 79.93 at 100 µg/ml. Proteinases have been implicated in arthritis and inflammatory reactions. Neutrophils are known to be a rich source of proteinases. The results were shown in Table 3.
Also, in the egg albumin inhibitory method the extract produced dose-dependent inhibitory of protein denaturation. Denaturation of tissue protein was one of the well-documented causes of the inflammatory and arthritic diseases. The extract produced 110.00 of a maximum of protein denaturation at 800 µg/ml compared with the diclofenac sodium 190.00 at 800 µg/ml of concentration. The results were shown in Table 4. All the enzymes studied shown significant antioxidant activity in-vitro method as they were compared with the standard drug ascorbic acid. The results were shown in Table 5-9.
TABLE 4: IN-VITRO ANTI-INFLAMMATORY ACTIVITY IN HEAT INDUCED HAEMOLYTIC METHOD OF METHANOLIC EXTRACT OF PUPALIA LAPPACEA
S. no. | Extract | Concentration (μg/ml) | Absorbance | % Inhibition of denaturation |
1
2 3 4 5 |
Control
Pupalia lappacea Pupalia lappacea Pupalia lappacea Diclofenac sodium |
--
100 200 500 20 |
0.080 ± 0.00
0.123 ± 0.00 0.136 ± 0.00 0.156 ± 0.00 0.183 ± 0.00 |
--
26.38 ± 0.00 38.67 ± 0.01 66.63 ± 0.11 89.49 ± 0.12 |
TABLE 5: IN-VITRO ANTI-INFLAMMATORY ACTIVITY BY INHIBITION OF ALBUMIN DENATURATION OF METHANOLIC EXTRACT OF PUPALIA LAPPACEA
S. no. | Extract concentration (μg/ml) | Absorbance (nm) | % Inhibition of denaturation |
1
2 3 4 5 6 7 8 9 10 |
Pupalia lappacea 50
Pupalia lappacea100 Pupalia lappacea 200 Pupalia lappacea 400 Pupalia lappacea 800 Diclofenac sodium 50 Diclofenac sodium 100 Diclofenac sodium 200 Diclofenac sodium 400 Diclofenac sodium 800 |
0.113 ± 0.003
0.203 ± 0.003 0.467 ± 0.003 0.373 ± 0.086 0.597 ± 0.117 0.183 ± 0.003 0.313 ± 0.003 0.533 ± 0.066 0.587 ± 0.074 0.620 ± 0.030 |
47.7 ± 0.003
52.7 ± 0.333 75.7 ± 0.333 85.3 ± 0.333 110 ± 0.500 105 ± 0.333 110 ± 0.333 142 ± 0.333 119 ± 0.333 190 ± 40.2 |
TABLE 6: NITRIC OXIDE RADICAL SCAVENGING ASSAY
Sample | Concentration (µg) | Percentage activity (%) | IC50 (µg/TRV) |
Pupalia lappacea extract |
100
200 300 400 500 |
13.12 ± 0.14
25.22 ± 0.55 39.22 ± 0.52 42.21 ± 1.22 63.85 ± 1.20 |
391.5 ± 0.04 |
Ascorbic acid |
5
10 15 |
22.72 ± 1.01
43.78 ± 1.06 36.67 ± 0.52 |
11.42 ± 3.17 |
Note: TRV – Total reaction volume
TABLE 7: DPPH RADICAL SCAVENGING ACTIVITY OF PUPALIA LAPPACEA EXTRACT
Sample | Concentration (µg) | Percentage activity (%) | IC50 (µg/TRV) |
Pupalia lappacea extract |
100
200 300 400 500 |
17.28 ± 1.23
26.55 ± 1.05 37.85 ± 0.52 44.65 ± 1.01 60.08 ± 0.89 |
416.01 ± 0.11 |
Ascorbic acid |
5
10 15 |
22.72 ± 1.01
43.78 ± 1.06 36.67 ± 0.52 |
11.42 ± 3.17 |
Note: TRV – Total reaction volume
TABLE 8: SUPEROXIDE OXIDE RADICAL SCAVENGING ACTIVITY OF PUPALIA LAPPACEA EXTRACT
Sample | Concentration (µg) | Percentage activity (%) | IC50 (µg/TRV) |
Pupalia lappacea extract |
100
200 300 400 500 |
18.03 ± 1.42
32.90 ± 1.53 41.23 ± 0.52 53.13 ± 1.25 62.25 ± 1.52 |
401.6 ± 0.16 |
Ascorbic acid |
5
10 15 |
22.72 ± 1.01
43.78 ± 1.06 36.67 ± 0.52 |
11.42 ± 3.17 |
Note: TRV – Total reaction volume
TABLE 9: POLYPHENOL OXIDASE ACTIVITY OF PUPALIA LAPPACEA EXTRACT
Sample | Concentration (µg) | Percentage activity (%) | IC50 (µg/TRV) |
Pupalia lappacea extract |
100
200 300 400 500 |
16.87 ± 1.04
27.67 ± 1.53 38.93 ± 1.52 54.18 ± 1.51 68.85 ± 1.64 |
363.2 ± 0.23 |
Ascorbic acid |
5
10 15 |
22.72 ± 1.01
43.78 ± 1.06 36.67 ± 0.52 |
11.42 ± 3.17 |
Note: TRV – Total reaction volume
TABLE 10: CATALASE ACTIVITY OF PUPALIA LAPPACEA EXTRA
Sample | Concentration (µg) | Percentage activity (%) | IC50 (µg/TRV) |
Pupalia lappacea extract |
100
200 300 400 500 |
15.43 ± 1.34
26.70 ± 0.93 37.35 ± 1.42 50.33 ± 1.11 62.75 ± 1.84 |
398 ± 0.40 |
Ascorbic acid |
5
10 15 |
22.72 ± 1.01
43.78 ± 1.06 36.67 ± 0.52 |
11.42 ± 3.17 |
Note: TRV – Total reaction volume
DISCUSSIONS: The enzyme phospholipase A2 Is known to be responsible for the formation of mediators of inflammatory such as prostaglandins and leukotrienes which by attracting polymorphonuclear leucocytes to the site of inflammation would lead to tissue damage probably by the release of free radicals.
Phospholipase A2 comments phospholipids in the cell membrane into arachidonic acid, which is highly reactive and is rapidly metabolized by cyclooxygenase to prostaglandins, which are major components that induce pain inflammation. Erythrocytes have been used as a model system by some scientists to investigate the interaction of drugs in the membranes.
The erythrocyte membrane resembles lysosomal membrane and as seen the erythrocyte could be extrapolated to the stabilization of lysosomal membrane 18. The vitality of cells depends on the integrity of their membrane, exposure of RBC’S to a major substance such as hypotonic medium results in lysis of its membrane accompanied by hemolysis and oxidation of hemoglobin. An injury to RBC membrane will further render the cell more susceptible to secondary damage through free radical-induced lipid peroxidation 19. It was therefore that phytochemicals present in the extract stabilize the RBC membrane should offer significant protection of the cell membrane against injurious substances. Compounds with the stabilizing properties as well known for their ability to interfere with the release of phospholipases that trigger the formation of inflammatory mediators.
Moreover, it has also been shown that the deformability and cell volume of RBC’s are closely related to the intercellular content of the calcium stabilization of lysosomal membrane is important in limiting the inflammation response by preventing the release of lysosomal constituents of activated neutrophil such as a bactericidal enzymes and kills the proteases, which cause further tissue inflammation and damage upon extracellular release 20. The membrane stabilizing the activity of extract may be due to the presence of flavonoids, alkaloids, tannins and or saponins present in the extract. The extract prevented protein denaturation effect on egg albumin solution. Denaturation of proteins is a well-documented cause of inflammation in conditions like rheumatoid arthritis.
Thus, protection against protein denaturation which was the main mechanism of action NSAID’s. Production of auto-antigen in certain rheumatic diseases may be due to in-vitro denaturation of proteins. Mechanism of denaturation probably involves an alteration in electrostatic, hydrogen, hydrophobic and disulfide bonding. Neutrophils contain many neutral serine proteinases in their lysosomal granules. Leukocytes proteinases play an important role in the development of tissue damage during inflammatory reactions, and a significant level of protection was provided by proteinases inhibitors 21.
The components present in the methanol extract of aerial parts of Pupalia lappacea were identified GC-MS analysis. The active principles with their retention time (RT), molecular formula, molecular weight (MW) and concentration (%) in the methanol extract of leaves of Pupalia lappacea were presented in Table. Thirty-eight compounds were identified in the methanol extract of Pupalia lappacea aerial parts. Plants are a natural source of biologically active compounds known as phytoconstituents 22, 23, 24. Preliminary analysis of Pupalia lappacea for chemical constituents showed the presence of alkaloids, amino acids, glycosides, flavonoids, glycosides, saponins, tannins, starch, steroids, terpenoids, and coumarins. The prevailing compounds were ascorbic acid (23.36%), phenol (4.69%), 10-heptadecenoic acid (3.74%), octadec-9-enoic acid (8.21%) stearic acid (9.96), 11,14,17-Eicosa pentanoic acid (8.62) and 14-pentadecanoic acid respectively.
In the present study, the methanolic extract was allowed to run in the GC – MS for 34 minutes, nearly 38 compounds were detected in the extract within the specified time limit. Among the identified phytochemicals ascorbic acid 25, Hexadecanoic acid 26, phenol 27, 1-Chloroundecane, Hexatriacontane, and Stearic acid, octadecanoic acid compounds have antioxidant activity. Thus, this type of GC-MS analysis is the first step towards understanding the nature of active principles in this medicinal plant, and this type of study will be helpful for further detailed study.
CONCLUSION: Hence, the methanolic extract of Pupalia lappacea possesses membrane stabilization properties, limiting protein denaturation process and white blood cell anti-migration properties. Therefore, the extract leads to effective RBC membrane stabilization and protein inhibition, denaturation both contributing to invitro anti-inflammatory activity. Further, definitive studies were necessary to ascertain the mechanism and constituents behind its anti-inflammatory actions.
ACKNOWLEDGEMENT: Nil
CONFLICT OF INTEREST: Nil
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How to cite this article:
Selvan AT, Subramanian NS, Ramadevi M, Prasad BSG and Muthu SK: Bioactive compound identification, phytochemical estimation, in-vitro anti-inflammatory and antioxidant activity of Pupalia lappacea. Int J Pharmacognosy 2014; 1(9): 596-04. doi: 10.13040/IJPSR.0975-8232.1(9).596-04.
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Article Information
8
596-604
644
1617
English
IJP
A. T. Selvan *, N. S. Subramanian, M. Ramadevi , B. S. G. Prasad and S. K. Muthu
Department of Pharmacology , Teegala Krishna Reddy College of Pharmacy, Meerpet, Saroor Nagar, Hyderabad, Andhra Pradesh, India.
tamilselvanpharmacologist@gmail.com
15 July 2014
21 August 2014
28 August 2014
http://dx.doi.org/10.13040/IJPSR.0975-8232.IJP.1(9).596-04
01 September 2014