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In vitro anti-inflammatory activities of Loranthus micranthus

(Linn.) parasitic on Azadirachta indica

Channabasava and Govindappa M*

Abstract - The present work was aimed to evaluate the in vitro anti-inflammatory activity of crude extract of Loranthus micranthus and their phytochemical analysis. We have used four different solvents for extraction; the methanol extract was yielded all the potent phytochemicals at high percentage except saponins and athraquinones. The methanol extract possesses enough potential to reduce inflammation by in vitro and directs the importance of further research and development of novel anti-inflammatory activity.

Index Terms- Loranthus micranthus, anti-inflammatory, membrane stabilization, proteinase

1. Introduction

IJSElysosomal cRonstituents, activated neutrophil,

inhibitory, albumin denaturation

—————————— ——————————

Inflammation is a recurring state of cells and tissues as a self defence mechanism in response to external stimuli like pathogens, heat, injury, chemicals, and toxic substances or due to improper metabolic activities. Inflammatory response and damage upon extra cellular release is initiated by the release of
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*Corresponding author

Department of Biotechnology,
Shridevi Institute of Engineering & Technology, Sira Road, Tumkur-572 106, INDIA Email: dravid ateja07@gmail.com,
Phone: +91-9686114847, Fax: +91-816-2212628
bactericidal enzymes and proteases (Murugasan,
1981). Even inflammation is self defence mechanism; it is necessory to control to prevent further cellular damages because of free radicals, denatured proteins and DNA, pus cells and other toxins released during inflammation. Many herbals were recognized as important sources of medicinal copmounds from past centuries in treating inflammation. Despite of evolution in synthetic drugs, there has been more prominance was given to herbal medicines due to their fewer side effects and multiple therapeutic activities.

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Loranthus micranthus Linn (Loranthaceae) is a hemiparasitic plant growing on a variety of host trees and shrubs (Ali et al., 2005). It depends on its respective host for water and mineral nutrition, even though it produces its own carbohydrates through photosynthesis. It is used in folkloric medicine in the treatment of epilepsy, hypertension, headache, infertility, cancer and rheumatism (Griggs, 1991), and has been reported to have antidiabetic, antimicrobial

(Osadebe and Ukwueze, 2004), immunomodulatory

Loranthus micranthus (L.f) Ettingsh (Loranthaceae) growing on the host plant Azadirachta indica were collected in the month of April, 2009 during the flowering period at DC Bunglow, Tumkur, Karnataka, India and identified using authenticated herbarium from the Department of Studies in Botany, University of Mysore, Mysore and Government Ayurvedic College, Mysore. The plant material was washed with

distilled water, shade-air dried (26+20C) and
pulverized to a coarse powder in a mechanical grinder,

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and antimotility activities (Osadebe and Uzochukwu,
2006) and antimotility assay (Osadebe et al., 2012). The present work was aimed to carry out anti- inflammatory activities of Loranthus micranthus in four different solvent extracts viz., hexane, ethyl acetate, mathanol and distilled water by hypotonicity induced Human Red Blood Cell (HRBC) membrane stabilization, heat induced HRBC membrane stabilization, proteinase inhibitory activity and albumin denaturation methods.

2. MATERIALS AND METHODS

2.1.Collection and processing of plant

On the basis of present literature review and slight experimental modifications, the fresh leaves of
passed through a 40 mesh sieve and stored in air tight
container for further work.

2.2. Preparation of crude extracts

25 g/100ml of powdered leaf of Loranthus micranthus
was kept for solvent extraction in rotary shaker at
370C, 72 rpm for 48 h. The solvents hexane, ethyl acetate, methanol and distilled water were used with increasing order of their polarity. The solvent extract was cetrifuged at 6000 rpm for 10 min and then filtered with Whatman No. 1 filter paper and evaporated at a constant temperature of 620C in hot air oven until a very concentrated extract was obtained.

2.3. Phytochemical analysis

Phytochemical screening was performed according to

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the standard procedures and detected different phytoconstituents like carbohydrates, alkaloids, glycosides, saponins, phytosterols tannins, flavonoids, resins and proteins present in the plant extracts (Sofowora, 1993; Trease and Evans, 1989; Siddiqui and Ali, 1997).

2.4. Membrane stabilization test

2.4.1. Preparation of Red Blood cells (RBCs)

suspension

Fresh whole human blood (5 mL) was collected and transferred to the centrifuged tubes containing Heparin
rpm for 20 min. and the hemoglobin content of the supernatant solution was estimated spectrophotometrically at 560 nm. Diclofenac was used as standard and a control was prepared by distilled water instead of hypo saline to produce 100
% hemolysis without plant extracts. The percentage of HRBC hemolysis and membrane stabilization or protection was calculated by using the following Formula:
% of Hemolysis = (Optical density of test sample /

IJSEOptical densityRof control) X 100

or EDTA or Sodium citrate to prevent clotting. The
tubes were centrifuged at 3000 rpm for 10 min and were washed three times with equal volume of normal saline. The volume of the blood was measured and reconstituted as 10% v/v suspension with normal saline (Sadique et al., 1989).
2.4.2. Hypotonicity induced human red blood cell (HRBC) membrane stabilization method (Gandhisan et al., 1991)
The reaction mixture consists of 1.0 mL of test sample of different concentrations (40µg – 200 µg) in 1 ml of
0.2 M phosphate buffer and 0.5 mL of 10% HRBC
suspension, 0.5 ml of 0.25 % hyposaline were incubated at 370C for 30 min and centrifuged at 3,000
% Protection = 100 – [(Optical density of test sample /
Optical density of control) X 100]

2.4.3. Heat induced human red blood cell (HRBC)

membrane stabilization method (Shinde et al., 1999)
The reaction mixture in heat induced hemolysis consists of 1.0 mL of test sample of different concentrations (40µg – 200 µg) in normal saline and
1.0 mL of 10% RBC suspension. Diclofenac sodium was taken as a standard drug. Control was prepared by distilled water instead of normal saline to produce 100
% hemolysis without plant extracts. All the tubes containing reaction mixture were incubated in a water bath at 560 C for 30 min. After incubation, the tubes
were cooled under running tap water. The reaction

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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. The percentage of HRBC hemolysis and membrane stabilization or protection was calculated by using the following Formula:
% of Hemolysis = (Optical density of test sample / Optical density of control) X 100
% Protection = 100 – [(Optical density of test sample /
Optical density of control) X 100]

2.5. Proteinase inhibitory activity

inhibitory activity was determined.
Percentage inhibition = 100 - [(optical density of sample) / (optical density of control) × 100]
2.6. Albumin denaturation method (Dey et al., 2011; Chandra et al., 2012)
Antiinflammatory activity involves inhibition of albumin denaturation. The reaction mixture consists of
1.0 mL of distilled water containing varied concentrations of plant extracts or standard (80µg –
400 µg), 0.2 mL of .05 % BSA and 1.8 mL of 0.2 M
phosphate buffered saline (pH 6.4). The mixtures were
Anti-inflammatory activIity waJs performeSd according ER
to the modified method of Oyedepo et al. (1995). The reaction mixture contains 1.0 mL of test sample of different concentrations (40µg – 200 µg), 0.5 mL of
20 mM Tris HCl buffer (pH 7.4) containing 0.06 mg trypsin, and the mixture was incubated at 37oC for 5 min and then 0.5 mL of 0.8% (w/v) casein was added. The mixture was incubated for an additional 20 min.
1.0 mL of 70% perchloric acid was added to terminate the reaction. Cloudy suspension was centrifuged and the absorbance of the supernatant was read at 280 nm against buffer as blank. Diclofenac sodium was used as standard drug. The experiment was performed in
triplicate. The percentage inhibition of proteinase
incubated at 37ºC for 15 minutes and then heated at
70ºC for 5 minutes. After cooling, the absorbance was measured spectrophotometrically at 660 nm against a blank. Diclofenac sodium was used as standard drug and the percentage inhibition of protein denaturation was calculated by using the following formula:
Relative % of inhibitory activity = (A- Amin) / Amax - Amin ) x 100
Where,
A – Abs of Sample
Amin – Abs of control
Amax – Highest Abs of Standard
The experiment was performed in triplicate

3. Results

3.1.Phytochemical analysis

Phytochemical analysis of different solvents yielded

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different phytochemicls (Table. 1). Compared to other solvents, the methanol extracts yielded well distinguishable phytochemical compounds and revealed the presence of carbohydrates, cardiac glycosides, proteins, amino acids, phytosterols, alkaloids, phenols, tannins and reducing sugars at higher rate. Whereas, the hexane and ethyl acetate extracts yielded less quantity of phytochemicals. More phenolic substances observed in methanol extract
followed by aqueous extract.

stabilization method

In hyposaline condition, red blood cells undergo exosmosis leads to either cell shrinkage or membrane rupture causing haemolysis. The extent of haemolysis indicates the state of memrane stabilization. As the decrease in haemolysis, percentage of memrane stabilization increases and the absornance which can be read at 560 nm.
The percentage of hemolysis was found more in
hexane extract (65.74 %) and it is further increased as

extracts from L. micranIthus lJeaves SER

Phytochemicals

Hexane

Extracts

Ethyl Methanol

Acetate

Distilled water

(37.44 %) and distilled water extract (45.52 %) were
shown significant inhibition of hemolysis which in

Carbohydrate + +++ +++ +

Cardiac

Glycosides + ++ +++ +

Fixed oils and

-- - - -

turn expressess the active membrane stabilization when compared to Diclofenac Sodium (39.28 %), the

Fats

Proteins and

Amino acids

- - ++ ++

reference standard (Table. 2).

Saponins - - - ++ Phytosterol + ++ +++ + Alkaloids - ++ +++ ++ Flavonoids - + +++ +++ Phenol + + ++ ++ Tannins + + ++ ++

Gums and

mucilages - - + ++ Anthraquinones ++ - - -

Reducing sugars - ++ + +

Note: (-): not detectable. (+): Low quantities. (++): average quantities. (+++): high quantities. Repeated the each experiment thrice

Table 2: Hypotonicity induced HRBC membrane

Concen tration of Test / Std

(µg)

% of hemolysis

Concen tration of Test / Std

(µg)

Hexan e extrac t

Ethyl acetate extract

Methan ol extract

Distille d water

extract

Diclofen ac Sodium (Std)

80

77.202

56.461

72.209

56.387

58.480

160

73.568

52.679

71.365

55.066

56.350

240

72.026

48.825

68.612

54.552

52.606

320

67.254

45.888

67.033

51.395

47.356

400

65.748

37.444

48.091

45.521

39.280

*Repeated the experiment thrice

3.2.Hypotonicity induced HRBC membrane

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At final concentrations the percentage of membrane stabilization was found more in ethyl acetate extract (62.55 %) even efficient than the reference standard Diclofenac Sodium (60.71%). Hexane extract (34.25

%) was shown least stabilization activity (Fig. 1).

Fig 1: Hypotonicity induced HRBC

70 stabilization of L. micranthus extracts

60

50

40

30 Hexane extract

20 Ethyl acetate extract

Methanol extract

10 Distilled water extract

Diclofenac sodium (Std)

(58.46 %). hexane extract (36.26 %) was shown least stabilization activity (Fig. 2).

Table 3: Heat induced HRBC membrane stabilization method (% of hemolysis)

*Repeated the experiment thrice

Fig 2: Heat induced HRBC stabilization of L.

0 70

0 100 200 300 400 500

60

Concentration (μg/ml)

50

micranthus extracts

3.3.Heat induced HRBC membrane stabilization 40

method 30

The percentage of hemolysis was found more in 20

10

hexane extract (63.73 %) and it is further increased as 0

Hexane extract

Methanol extract

)

concentration decreases. But methanol extract (44.28
%) and ethyl acetate extract (45.27 %) were shown significant inhibition of hemolysis when compared to Diclofenac sodium (41.53 %), the reference standard (Table. 3).
At final concentrations the percentage of membrane stabilization was found more in methanol extract (55.71 %) and ethyl acetate extract (54.72 %) when
compared to reference standard Diclofenac sodium

0 100 200 300 400 500

Concentration (μg/ml)

3.4.Proteinase inhibitory activity

Trypsin is an endoprotease enzyme found in the pancreatic juice which catalyzes the hydrolysis of peptide bonds formed from the carboxyl groups of basic amino acids. The inhibition of trypsin activity was found to be dose dependent and shown more inhibitory activity in distilled water extract (67.43 %) and methanol extract (64.31 %). The inhibition shown

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by reference standard Diclofenac sodium (71.37 %)

Fig 4: Albumin denaturation test for different


was just higher than that of plant extracts. At final concentrations hexane extract (30.38 %) was shown least proteinase inhibitory activity compared to all extracts (Fig. 3).

Fig 3: Proteinase inhibitory activity of L.

120

100

80

60

40

20

0

solvent extracts of L. micranthus

Hexane extract

Ethyl acetate extract

Methanol extract

Distilled water extract

Diclofenac sodium (Std)

80 micranthus extracts

70

60

50

40

30 Hexane extract

Ethyl acetate extract

20 Methanol extract

10 Distilled water extract

0 Diclofenac sodium (Std)

0 100 200 300 400 500

Concentration (μg/ml)

3.6.IC50 values of L. micranthus extracts in response to different anti-inflammatory methods

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0 100 200 300 400 500

Concentration (μg/ml)

3.5.Albumin denaturation test

Anti-inflammatory activity involves inhibition of albumin denaturation by noncovalent change in the structure and loss of its solubility. The increased

Fig 5: IC50 values of L. micranthus

extracts in response to different anti-

inflammatory methods

50

45

40

35

30

25

20

15

10 IC50 NA*

5

0

percentage of inhibition of albumin denaturation
expressess the anti-inflammatory efficacy of plant

Hexane extract

Ethyl acetate extract

Methanol extract

Distilled water extract

Diclofenac Sodium (Std)

extracts. Here the percentage of inhibition was carried out in realation with reference standard Diclofenac sodium. The realtive percentage of inhibition was significantly more in methanol extract (84.92 %) comapared to all other extracts. But hexane extract (13.56 %) was shown very least denaturation
inhibitory activity (and Fig. 4).

Solvent extracts

Hypotonicity induced HRBC Heat induced HRBC Proteinase inhibitory Albumin denaturation

*NA – 50 % inhibition not attained, repeated the experiment thrice

The half maximal (50 %) inhibitory concenatration has got more significance which shows the potency of drugs and helps in drug formulation and dose fixation.

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The IC50 values of L. micranthus extracts in response to different anti-inflammatory methods were shown comparatively near values with few exptions except hexane extract. Hexane extract was failed to exhibit 50
% of effectiveness in all assays (and Fig. 5).

4. Discussion

Concentrating on the aims of preseent studies; the human red blood cell membrane is analogous to the lysosomal membrane (Chou, 1997) and its stabilization indicates the stabilization of lysosomal membranes. Stabilization of the membranes of these
standard Diclofenac sodium (58.46 %).
Trypsin is well recognized as an activator of Proteinase-Activated Receptor-2 (PAR2). Studies have demonstrated that trypsin and PAR-2 induces inflammatory responses through p65-NF-kB pathway in many cell types (Liang et al., 2011) and via the production of cytokines, such as IL-6, IL-8, and prostaglandin (Asokananthan et al., 2002; Johansson et al., 2005). PAR-2 activation results in proinflammatory effects including vasodilatation, edema, reflux esophagitis, leukocyte-endothelial
damage and exacerbaItion

Jof the iSnflammatory

Eal., 2007). EveRn trypsin plays an important benificial

cells inhibits lysis and subsequent release of the
cytoplasmic contents which in turn limits the tissue
interactions and damage the lining of gastrointestinal tract (Busso et al., 2007; Saifeddine., 1996; Georgie et
response (Charles et al., 2008). Exposure of red blood cells to hypotonic medium, heat, injurious substances such as methyl salicylate or phenylhydrazine results in the lysis of membranes accompanied by haemolysis and oxidation of haemoglobin (Feirrali et al., 1992). At final concentrations the percentage of membrane stabilization by hypotonicity induced was found more in ethyl acetate extract (62.55 %) even efficient than the reference standard Diclofenac Sodium (60.71%). In heat induced, methanol extract (55.71 %) was
shown significant effect when compared to reference
role in gastro intetstinal tract system, it is necessory to control the trypsin activity in an unconditional events. In our studies trypsin inhibitory activity was dose dependent and shown comparable effect in distilled water extract (67.43 %) and methanol extract (64.31
%) when compared to reference standard Diclofenac sodium (71.37 %).
Maintenance of structural heirachy of proteains by cell system is necessory for proper functioning of metobolic activities. In humans, albumin is the most

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abundant plasma protein, accounting for 55–60% of the measured serum protein (Gosling, 1995). Modification of the amino, carboxyl or disulfide groups of the albumin may results in its deanturation (Norman et al., 1971). Denaturation of protein constituents and intercellular fluid is a well referable cause of inflammation in rheumatoid arthritis (Mizushima et al., 1964). Extracellular proteases from plasma transudate infiltrating leukocytes, chondrocytes and synovial cells may degrade albumin
more on in vivo anti-inflammatory studies with purification.

5. Conclusion

Phytochemical and in vitro anti-inflammatory studies of Loranthus micranthus were shown good results. Among hexane, ethyl acetate, methanol and distilled water, methanol was the good choice as a solvent in extraction of well distinguishable phytochemicals. Methanol extract has shown comparatively significant anti-inflammatory effects for all anti-inflammatory
(Barnnart et al., 1968)Iand Jit may beSpossible in ER
conditions like tissue injury, redox reactions due to free radicles, activation of trypsin induced proteolysis, heat, chemicals, antigens and by metabolic impairments. Chronic inflammation leads to cardiovascular disease, cancer, diabetes, degenerative joint diseases and neurodegenerative diseases (Lucas et al., 2011). Relatively methanol extract (84.92 %) has inhibited albumin denaturation and can be choosen for further optimal studies by purification.
IC50 values of L. micranthus extracts were very less and few crude extracts were shown even lesser IC50 values than the reference standard towords some
assays. The effective crude extracts can be concetrated
tests. It can be considered in future for optimal studies
and also for isolation and identification of the exact anti-inflammatory phytochemical in Loranthus micranthus.

6. Acknowledgement

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