{"id":2029,"date":"2015-03-29T06:55:24","date_gmt":"2015-03-29T06:55:24","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=2029"},"modified":"2017-01-04T05:26:00","modified_gmt":"2017-01-04T05:26:00","slug":"an-experimental-evaluation-of-ageratum-conyzoides-on-membrane-stabilization-and-protein-denaturation-during-acute-inflammation-and-arthritis","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol4no2\/an-experimental-evaluation-of-ageratum-conyzoides-on-membrane-stabilization-and-protein-denaturation-during-acute-inflammation-and-arthritis\/","title":{"rendered":"An Experimental Evaluation of Ageratum conyzoides on Membrane Stabilization and Protein Denaturation during Acute Inflammation and Arthritis"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

Inflammation is the complex process which is frequently associated with pain and involved occurrences such as increased vascular permeability, protein denaturation and membrane alteration. Inflammatory diseases including different types of rheumatic diseases are a major cause of morbidity of the working force throughout world. This has been called the \u2018King of Human Miseries\u20191. Although rheumatism is one of the oldest known diseases of the mankind and affects a large percentage of population of the world, no substantial progress was seen till the synthesis of aspirin in 1899 by the German Company Bayer, the hint of which also was obtained from a plant, the Willow bark (Salix alba) used worldwide in folk medicine for the relief of aches, fever and rheumatic pain. Since then many compounds were introduced as a result of laboratory search for drugs with anti-inflammatory activity. Though many of them produced a dramatic symptomatic improvement in rheumatic processes, did not arrest the progress of the diseases process and all of them shared the common side effect i.e., gastro-intestinal irritations2.<\/p>\n

In India, many Ayurvedic practitioners are using various indigenous plants for the treatment of different types of arthritic conditions. Although the application of these medicaments has a sound tradition and a rational background according to the Indian system of medicine, perhaps it is essential to investigate the rationality of their use in modern scientific terms. The scientific studies to work out the actual efficacy and other limitations to these drugs would definitely widen their scope for future use if they come out to be really effective. This is particularly important, firstly due to the gravity of the problem of rheumatism and arthritis and secondly due to the absence of the right type of drug of synthetic origin for its treatment. The presently available drugs provide only symptomatic relief and are not free from side effects. The target should be to discover newer drugs from plant kingdom which may provide therapeutic cure and would be free from undesirable effects as well as economical, which would be accepted by the developing nations like India. [3]\n

Material and Methods<\/strong><\/p>\n

Plant material<\/strong><\/p>\n

The leaves of Ageratum conyzoides <\/em>Linn was collected from local area of muzaffarnagar, Uttar Pradesh, India in the month of February, 2011 and authenticated by Botanical Survey of India. (Vide no. CNH\/I-I\/ (292)\/2011\/Tech.II\/333).<\/p>\n

Preparation of extracts<\/strong><\/p>\n

Leaf was shade dried at room temperature and powdered to #40 mesh particle size. The powder (250 g) of crude plant was defatted with petroleum-ether and subjected to extraction with methanol by using Soxhlet apparatus. The extract was filtered and evaporated at 40\u00b0C under vacuum and the residue was freeze-dried.<\/p>\n

Phytochemical tests4<\/strong><\/p>\n

Azaretum conyzoides <\/em>leaf extract was separately tested for the presence of tannins, alkaloids, carbohydrates, steroids, flavonoids.<\/p>\n

Test for alkaloids<\/strong><\/p>\n

Mayer\u2019s test \u2013 2-3 ml. of solution of extract was added with few drops of Mayer\u2019s reagent.<\/p>\n

Dragendroff\u2019s test – 2-3 ml. of solution of extract was added with few drops of Dragendroff\u2019s reagent.<\/p>\n

Hagger\u2019s test – 2-3 ml. of solution of extract was added with few drops of Hagger\u2019s reagent.<\/p>\n

Test for tannins<\/strong><\/p>\n

2-3 ml. of solution of extract was added with few drops of 5% FeCl3\u00a0 solution.<\/p>\n

Test for flavonoids<\/strong><\/p>\n

2-3 ml. of solution of extract was added with 95% ethanol and neutral FeCl3 solution.<\/p>\n

2-3 ml. of solution of extract was added with 95% ethanol and lead acetate solution.<\/p>\n

Test for carbohydrate<\/strong><\/p>\n

Molish test<\/strong><\/p>\n

2-3 ml. of solution of extract was added with few drops of alpha napthol solution in alcohole, shaked well and add conc.H2SO4\u00a0from sides of the test tube.<\/p>\n

Test for steroid<\/strong><\/p>\n

Salkowski reaction<\/strong><\/p>\n

2-3 ml. of solution of extract was added with 2 ml. of chloroform and 2 ml. of\u00a0 conc. H2SO4.<\/p>\n

Isolation of flavonoides<\/strong><\/p>\n

Methanol part of extract (5 gm) was successively extracted with Ethyl acetate, and then the Ethyl acetate extract was concentrated to dryness. The\u00a0 Ethyl acetate extract was subjected to a column of silica gel (60-120 mesh; 3 cm dia, X 60 cm length) being eluted a gradient of Pet ether Ethyl acetate with increasing polarity. 10 main fraction were collected and individual fraction were tested for presence of the active bi-flavonoid compounds. Thus from fraction of ether\/ Ethyl acetate (10:90), compound I was separated by using preparative TLC using Hexane\/ Ethyl acetate in ratio of 8:2 system as eluent.<\/p>\n

There were two different spots on the TLC plate, when illuminated with UV light with Rf\u00a0value of compound 0.28 respectively from point of origin of sample. The compound with Rf\u00a0value of 0.28, showed relatively higher concentration of flavonoid tests, (Shinoda and NaOH). Compound labeled as AZ-1 (Rf\u00a00.28). The isolated compound\u00a0 was identified after analyzing spectra obtained from IR Spectra.<\/p>\n

Preparation of test sample<\/strong><\/p>\n

Sample solutions for in- vitro <\/em>studies were prepared by dissolving 10 mg of dried extract of Azaretum conyzoides <\/em>leaf in 0.5 ml water and with phosphate buffer salt solution. According to concentration range from 50 \u00b5g\/ml, 100 \u00b5g\/ml and 250 \u00b5g\/ml and also by dissolving 5 mg of isolated compound of leaf extract in 0.25 ml water and with phosphate buffer salt solution according to concentration 100 \u00b5g\/ml.<\/p>\n

In-vitro<\/strong> anti-arthritic study<\/strong><\/p>\n

Membrane stabilizing activity<\/strong><\/p>\n

The test was followed by the method described by shinde et al<\/em> (1999) [5] with some modifications. Whole human blood obtains from a healthy human volunteer and transferred to heparinised centrifuge tube. The blood was washed three times with isotonic buffer solution (154 mM NaCl) in 10 mM sodium phosphate buffer solution (pH-7.4) for 10 mins at 3000g. The test sample considered to stock erythrocyte (RBC) suspension(0.5 ml) mixed with 5 ml of hypotonic solution (50 mM NaCl) in 10 mM sodium phosphate buffer solution (pH-7.4) containing the leaf extract and isolated compound solution or indomethacine (0.1 mg\/ml). The control sample considered of 0.5 ml of RBC suspension mixed with hypotonic buffer saline solution alone. The mixture was incubated for 10 mins at room temperature and centrifuge for 10 mins at 3000g and the absorbance of supernatant was measured at 540 nm. Each experiment was carried out triplicate and the average was taken. The percentage inhibition of heamolysis or membrane stabilization was calculated by following equation.<\/p>\n

% inhibition of heamolysis = 100 X (A1- A2\/A1)<\/p>\n

Where<\/p>\n

A1 = Absorption of hypotonic buffer solution alone.<\/p>\n

A2\u00a0= Absorption of test sample in hypotonic solution.<\/p>\n

Effect on protein denaturation<\/strong><\/p>\n

Test solution (extract and isolated compound) containing different concentration of plant extract or indomethacine (100\u00b5g\/ml) was mixed with 1 ml of egg albumin solution (1mM) and incubated at 27\u00b11\u00b0C for 15 mins. Denaturation was induced by keeping the reaction mixture at 70\u00b0C in a water bath for 10 mins. After cooling the turbidity was measured spectrophotometrically at 660nm6,7. Percentage inhibition of denaturation was calculated from control where no drug was added. Each experiment was carried out in triplicate and the average was taken. Percentage inhibition = (Abs control\u00a0 \u2013 Abs sample) \u00a0X \u00a0100\/\u00a0 Abs control<\/p>\n

Statistical analysis<\/strong><\/p>\n

The experiment data was expressed as mean \u00b1 SEM, the significance of difference among the various treated groups and control group were analyzed by the means of one \u2013way ANNOVA followed by dunnett\u00a0 t-test.<\/p>\n

Results<\/strong><\/p>\n

Phytochemical screening<\/strong><\/p>\n

The results of the test for chemical constituents are as follows<\/p>\n

Preliminary phytochemical investigation reveals the presence of flavonoides. Methanolic extract have showed significant activity in the some of the parameters at higher concentrations (Table 1).<\/p>\n

Table 1: Preliminary\u00a0 phytochemical\u00a0 investigation<\/strong><\/p>\n

 <\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n
Test<\/strong><\/td>\nExtract<\/strong><\/td>\nResult<\/strong><\/td>\n<\/tr>\n
(+)<\/strong><\/td>\n(-)<\/strong><\/td>\n\u00a0<\/strong><\/td>\n<\/tr>\n
Test for alkaloid<\/td>\n<\/td>\n<\/td>\n<\/tr>\n
1. Mayer\u2019s test<\/td>\nMethanolic extract of leaf<\/td>\n(+) (+) (+)<\/td>\n<\/tr>\n
2. Dragendroff\u2019s test<\/td>\nMethanolic extract of leaf<\/td>\n(+) (+) (+)<\/td>\n<\/tr>\n
3. Hagger\u2019s test<\/td>\nMethanolic extract of leaf<\/td>\n(+) (+)<\/td>\n<\/tr>\n
Test for tannin<\/p>\n

(Extract with 5% FeCl3\u00a0solution)<\/td>\n

Methanolic extract of leaf<\/td>\n(+) (+)(+)<\/td>\n<\/tr>\n
Test for favonoid Extract of alcohol + neutral FeCl3\u00a0solution)<\/td>\nMethanolic extract of leaf<\/td>\n(+) (+) (+)<\/td>\n<\/tr>\n
<\/td>\n<\/td>\n<\/td>\n<\/tr>\n
Test for carbohydrate (Molish test)<\/td>\nMethanolic extract of leaf<\/td>\n(+)<\/td>\n<\/tr>\n
Test for steroid (Salkowaski test)<\/td>\nMethanolic extract of leaf<\/td>\n(+) (+) (+)<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Isolation of flavonoides<\/strong><\/p>\n

Compound AZ-1<\/strong><\/p>\n

Brownish yellow crystal, mp. 2250C, Rf value\u00a0 0.28, FT-IR (Jasco-5300) (KBR) V max\/cm: 3414 (-OH), 1651 (Unsaturated \u2013C=O), 2928 (C-Me), 1072 cm-1 glycosidic (C-O) groups that are found in flavonoids8-9.<\/p>\n

Membrane stabilizing activity<\/strong><\/p>\n

Different concentration (100, 250 \u00b5g\/ml) of extract showed significant membrane stabilizing activity and isolated compound and indomethacine have significant inhibitory activity (63.69% and 69.31% respectively) which is shown in table 2.<\/p>\n

Table 2: Effect of methanol extract and isolated compound (flavonoides)\u00a0o<\/strong>f Azaretumconyzoides<\/em> leaves on membrane stabilizing activity<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n
Samples<\/strong><\/td>\nConcentration<\/strong><\/p>\n

Inhibition<\/strong><\/td>\n

Absorption at 540 nm<\/strong><\/p>\n

 <\/td>\n

% of <\/strong><\/p>\n

 <\/td>\n<\/tr>\n

<\/td>\n<\/td>\n<\/td>\n<\/td>\n<\/tr>\n
Hypotonic solution<\/td>\n50mM<\/td>\n0.730 \u00b1 0.06\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 –<\/td>\n<\/td>\n<\/tr>\n
Methanolic extracts<\/td>\n50 \u00b5g\/ml<\/td>\n0.480 \u00b1 0.05<\/td>\n34.24<\/td>\n<\/tr>\n
100 \u00b5g\/ml<\/td>\n0.384 \u00b1 0.37*<\/td>\n47.39<\/td>\n<\/td>\n<\/tr>\n
250 \u00b5g\/ml<\/td>\n0.330 \u00b1 0.24**<\/td>\n54.79<\/td>\n<\/td>\n<\/tr>\n
Isolated compound<\/td>\n100 \u00b5g\/ml<\/td>\n0.265 \u00b1 0.16**<\/td>\n63.69<\/td>\n<\/tr>\n
Indomethacine<\/td>\n100 \u00b5g\/ml<\/td>\n0.224 \u00b1 0.29 **<\/td>\n69.31<\/td>\n<\/tr>\n
Each value represents the mean\u00b1 SEM; n=3, *p<0.05 and **p<0.01 when compared with blank<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Protein denaturation activity<\/strong><\/p>\n

Different concentrations (100, 250 \u00b5g\/ml) of extracts and isolated compound have remarkable inhibitory activity on protein denaturation when compared with the control, which is shown in table 3.<\/p>\n

Table 3: Effect of methanol extract and isolated compound\u00a0<\/strong>(flavonoides) of Azaretumconyzoides<\/em>\u00a0 leaves on protein denaturation<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n
Samples<\/strong><\/td>\nConcentration\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 % of Inhibition of protein<\/strong><\/td>\n<\/tr>\n
denauration<\/strong><\/td>\n\u00a0<\/strong><\/td>\n<\/tr>\n
<\/td>\n<\/td>\n<\/tr>\n
Hypotonic solution<\/td>\n50mM<\/td>\n–<\/td>\n<\/tr>\n
Methanolic extracts<\/td>\n50 \u00b5g\/ml<\/td>\n24.68<\/td>\n<\/tr>\n
100 \u00b5g\/ml<\/td>\n45.65<\/td>\n<\/td>\n<\/tr>\n
250 \u00b5g\/ml<\/td>\n56.42<\/td>\n<\/td>\n<\/tr>\n
Isolated compound<\/td>\n100 \u00b5g\/ml<\/td>\n76.46<\/td>\n<\/tr>\n
Indomethacine<\/td>\n100 \u00b5g\/ml<\/td>\n82.67<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Discussion<\/strong><\/p>\n

Inflammation is the response of living tissue injury. It involves a complex array of enzyme activation, mediator release, extravastation of fluid, cell migration, tissue breakdown and repair10.<\/p>\n

The vitality of cells depends on the integrity of their membrane, exposure of RBC to injurious substance such as hypotonic medium results in lysis of its membrane accompanied by haemolysis and oxidation of haemoglobin11,12. The haemolytic effect of hypotonic solution is related to exessesive accumulation of fluid within the cell resulting in the rupturing of its membrane. Such injury to RBC membrane will further render the cell more susceptible to secondary damage through free radical induces lipid peroxidation. It is therefore expected that compound with membrane stabilizing properties, should offer significant protection of cell membrane against injurious substance13,14,15. Compounds\u00a0 with\u00a0 membrane stabilizing properties are well known for their ability to interfere with the release of phospholipases that trigger the formation of inflammatory mediators.[16]\u00a0The extract and isolated compound has shown significant membrane stabilizing activity, which suggests that\u00a0 its\u00a0 anti arthritic activity observed in this study, may be related to the inhibition of release of phospholipases that trigger the formation of inflammatory mediators.<\/p>\n

Denaturation of proteins is well documentated cause of inflammation and rheumatoid arthritis. Several anti-inflammatory drugs have shown dose dependent ability to inhibit thermally induce protein denaturation17<\/strong>. Ability of Azaretum conyzoides <\/em>extracts and isolated compound to bring down thermal denaturation of protein is possible contribution factor for its anti-inflammatory activity.<\/p>\n

The in-vitro<\/em> anti-arthritic activity of Azaretum conyzoides <\/em>found may be due to the presence of flavonoides.<\/p>\n

References<\/strong><\/p>\n

    \n
  1. Chatterjee, G.K., and Pal, S.P. \u201cSearch for anti\u00adinflammatory agents from Indian Medicinal Plants\u201d – A review. Indian Drugs, <\/em>21: 413 (1984)<\/li>\n
  2. Rainsford, K.D. and Whitehouse, M.W. \u201cAnti\u00adinflammatory\/anti-pyretic salicylic acid esters with low gastric ulcerogenic activity.\u201d Agents Action, <\/em>10: 451-55 (1980).<\/li>\n
  3. Huang, K.C. The Pharmacology of Chinese Herbs. CRC Press, London, 199 (1999).<\/li>\n
  4. Khandelwal, K.R. Techniques and Experiments, Practical Pharmacognosy,\u00a0 9th\u00a0Ed. Nirali Prakashan, 149-59 (2002)<\/li>\n
  5. Shind, U.A., Phadke, A.S., Nair, A.M., Mungantiwar, A.A., Dikshit, V.J.and Saraf, V.O. \u201cMembrane stabilizing activity – a possible mechanism of action of anti-inflammatory activity of cedrus deodara wood oil.\u201d Fitoterapia<\/em>, 70: 251-57 (1999)<\/li>\n
  6. Mizushima Y. Screening test for anti – rheumatic drugs. Lancet <\/em>2: 443 (1966).<\/li>\n
  7. Elias, G. and Rao, M.N. \u201cInhibition of albumin denaturation and anti-inflammatory activity of dehydrozingerone and its analogs\u201d. Indian. J. Exp. Biol., <\/em>26: 540-42 (1988)<\/li>\n
  8. Kalsi PS. In: Spectroscopy of organic compounds, 5thEd. Newage publisher 88 (2002).<\/li>\n
  9. Masbry, T.J. and Markham, K.P. \u201cThe systematic Identification of flavonoids\u201d, Springer verlag, 41(1970).<\/li>\n
  10. Vane, J.R. and Bolting, R.M. \u201cNew inside into the mode of action of anti-inflammatory drugs\u201d. Inflame.Res.<\/em> 44: 1-10 (1995).<\/li>\n
  11. Augusto, O., Kunze, K.L. and Montellano, P.R. \u201cNphenylprotophorphyrin formation in the haemogolobinphenylhydrazine reaction\u201d. J. Biol. Chem. <\/em>257: 6231-41(1982)<\/li>\n
  12. Ferrali, M., Signorni, C., Ciccoli, L. and Comporti, M. \u201cIron release and membrane damage in erythrocytes exposed to oxidizing agent, phenylhydrazine, divicine and isouramil.\u201d Biochem. J <\/em>285: 295-301 (1992).<\/li>\n
  13. Maxwell, SRJ. \u201cProspect for the use of antioxidant therapies\u201d. Drugs <\/em>49: 345-61 (1995)<\/li>\n
  14. Liu, G.T., Zhang, T.M., Wang, B.E. and Wang, Y.W. \u201cProtective action of the seven natural phenolic compounds against peroxidase damage to biomembranes\u201d. Biochem. Pharmacol. <\/em>43: 147-152 (1992)<\/li>\n
  15. Prenez, R.M., Prenez, S., Zavala, M.A. and Salazar, M. \u201cAntiinflammatory activity of the bark of hippocratea excelsa\u201d. J. Ethanopharmacol.\u00a0 <\/em>47: 85-90 (1995)<\/li>\n
  16. Aitadafoun, M., Mounieri, C., Heyman, S.F., Binistic, C., Bon, C. and Godhold, J. \u201c4-Alkoxybenzamides as a new potent phospholipase A2 inhibitors.\u201d Biochem. Pharmacol<\/em>. 51: 737-42 (1996)<\/li>\n
  17. Grant, N.H., Alburn, H.E. and Kryzanauskas, C. \u201cStabilisation of serum albumin by anti-inflammatory drugs\u201d. Biochem. Pharmacol. <\/em>19: 715-22 (1970).<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"

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