in-vitro anti-inflammatory, anti-oxidant...

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ASIO Journal of Medical & Health Sciences Research (ASIO-JMHSR)

Volume 1, Issue 1, 2016: 05-11

Dids: 02.2016-14482937; dids Link: http://dids.info/didslink/10.2016-29157168/

IN-VITRO ANTI-INFLAMMATORY, ANTI-OXIDANT AND THROMBOLYTIC ACTIVITIES OF METHANOLIC EXTRACT OF TRADITIONALLY USED MEDICINAL

PLANT IXORA VILLOSA

Md. Abul Kalam Azad1, Md. Qamrul Ahsanϯ 2, Abdullah Al Faruq3, Syed Mohammed Tareq4,

M. Mohi Uddin Chowdhury5

1Department of pharmacy, Southern University Bangladesh, Chittagong-PIN Code 4000, Bangladesh, 2Department of pharmacy, Southern University Bangladesh, Chittagong-PIN Code 4000, Bangladesh, 3Department of pharmacy, Southern University Bangladesh, Chittagong-PIN Code 4000, Bangladesh, 4Department of pharmacy, Southern University Bangladesh, Chittagong-PIN Code 4000, Bangladesh, 5Department of pharmacy, Southern University Bangladesh, Chittagong-PIN Code 4000, Bangladesh.

ARTICLE INFO ABSTRACT

Research Article

Corresponding Author:

Md. Qamrul Ahsan

Department of Pharmacy,

Southern University of

Bangladesh, Chittagong-PIN

Code 4000, Bangladesh

Email: [email protected]

The aim of this work was to investigate the phytochemical screening, in-vitro

anti-inflammatory activity, antioxidant and thrombolytic activities of crude

methanolic extract of dry and fresh leaves of Ixora villosa (IxV). Chemical test

revealed the presence of glycosides, steroids, tannins, flavonoids, reducing

sugars, and amides in the plant extract. Anti-inflammatory and Antioxidant

activities of the crude extract were determined by egg albumin denaturation and

DPPH free radical scavenging method respectively with minor modification. The

methanolic extract showed moderate anti-inflammatory potential at highest

dose (500 µg/ml) used in the study. The test results were compared with the

activity of positive control, Acetyl salicylic acid. The antioxidant activity test

showed a gradual decrease in absorbance with increase in sample concentration.

Significant IC50 value was obtained by the test extract (83.91µg/ml) in

comparison with standard drug, Ascorbic acid (52.23 µg/ml). During

thrombolytic activity test, the test results of clot lysis were negligible in

comparison with Streptokinase and distilled water used as positive control and

negative control respectively. In conclusion, the methanolic extract from dry

leaves of IxV could be used as potential source of new antioxidant and anti-

inflammatory agents.

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Keywords: Ixora villosa, anti-inflammatory, antioxidant, and thrombolytic.

1. INTRODUCTION

Medicinal plants have been of age long remedies for human diseases because they contain components of therapeutic value [1]. Ixora is a genus of flowering plants in the Rubiaceae family. It consists of tropical evergreen trees and shrubs and holds around 500 species with its centre of diversity in Tropical Asia i.e. Malaysia. It also grows commonly in subtropical climates in the United States, such as Florida. Members of Ixora prefer acidic soil, and are suitable choices for bonsai [2]. The leaves of this genus are used to treat diarrhea; roots in hiccough, fever, sore, chronic ulcers and skin diseases; meanwhile flowers are used in catarrhal bronchitis and dysentery [3].

In Bangladesh, IxV is largely located in forests of Chittagong, Chittagong Hill Tracts, Sylhet and cox’s Bazar districts. It is called ‘Bjantjhara phul’ by the tribal people. A paste of the leave fruits of IxV is taken traditionally for the treatment of abdominal pain.

Figure 1: plant Ixora villosa 5

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Md. Qamrul Ahsan et al. / ASIO Journal of Medical & Health Sciences Research (ASIO-JMHSR), 1(1), 2016:05-11

Doi: 10.2016-74831794; doi link: http://doi-ds.org/doilink/10.2016-95119637/

It is also used in high blood pressure [4-5]. The genus

Ixora has been reported to possess different classes of

compounds mainly triterpenoids (lupeol, urosilic acid,

oleanolic acid betunolic acid, amyrins, etc.), aromatic

acrid oils, tannins, saponins, carbohydrate, fatty acids,

flavanoids (rutin, formononetin, β-sitosterol, quercetin

and kaempferol) and sterols [6-11]. Out of many species

of Ixora much research was done on I. coccinea (IC) and

some part of the work on I. chinensis, I. javanica, I.

finlaysoniana, I. parviflora and I. macrothyrsa [12]. The IC

flower contained ursolic acid chemotype; Geranyl

acetate (8.74%), Linalyl acetate (6.79%), Neryl acetate

(6.49%) is the major monoterpenes and terpineol

acetate [13]. In addition, different extracts IC50 have

been to anti-oxidant, anti-inflammatory, anthelmentic,

anti-asthmatic, anti-diarrhoeal, hepatoprotective, wound

healing, anti-ulcer, cyto-toxic, antitumor, hypoglycaemic

and Hypolipidaemic activity [14]. So far, no work has

been reported on IxV extracts by using different

solvents. Altogether we performed in-vitro anti-

inflammatory, antioxidant and thrombolytic activity to

provide a scientific evidence for its traditional claim.

2. MATERIALS AND METHODS Collection of plant The traditional practitioners called locally as “Kabiraj”

were the main reliable informer about the traditional

uses of this plant. The fresh leaves of IxV were collected

during the month of July to August from the hilly areas of

Bandarban district of Chittagong division in Bangladesh.

Taxonomical identification of this plant was made by the

experts of Bangladesh Forest Research Institute (BFRI)

Herbarium, Chittagong. The herbarium sheet was

prepared following the standard procedure and

specification suggested by the expert of the herbarium

and preserved.

Extraction of plant materials After removal of extraneous materials, the leaves were dried in shade for a week. Then they were ground into coarse powder with the help of a grinder. The powder were stored in an airtight container and kept in a cool, dark and dry place until extraction was commenced. For hot extraction, about 120 gm of plant powder was subjected with 800 ml of methanol (97.7%) continuously in a Soxhlet Apparatus for 48 hrs and the obtained extract was collected, filtered and made to evaporate the solvent below 50°C temperature and reduced pressure. After evaporation of the solvent, a gummy concentrate was obtained which was designated as hot methanolic crude extract [15]. Phytochemical evaluation The phytochemical group tests were carried out by the

earlier reported methods [16]. The preliminary

phytochemical investigation was performed to find out

the presence of glycosides, steroids, tannins, flavonoids,

saponins, reducing sugars, gums and amides in the plant

extract.

Anti-inflammatory activity Heat induced protein denaturation method was applied

to evaluate the anti-inflammatory activity of the plant

extract [17]. Acute toxicity study was performed and the

LD50 was found to be 2000 µg/ml. So a dose of 500 µg/ml

was chosen as higher dose for this test. Other doses were

selected by reducing it by half two times (250 µg/ml &

125 µg/ml). Egg albumin collected from fresh eggs of

hen was the source of protein. In brief, there were three

groups of each containing three test tubes and

designated as standard, methanolic extract and control

group. 3 ml of 5% egg albumin solution was poured in

each test tube followed by 1ml Acetyl salicylic acid, 1 ml

test extract and 1ml methanol for standard, test extract

and control groups respectively. Then the mixtures were

incubated at 37±2 ºC in an incubator for 15 minutes and

then heated at 57 ºC for 20 min. After cooling, their

absorbance was measured at 660 nm by using vehicle as

blank. The data were reported in the chart below their

absorbance was measured at 660 nm by using vehicle as

blank. At this wavelength maximum absorbance 660nm

was observed. The Percent inhibition of protein

denaturation was calculated as follows.

Anti-oxidant activity

The free radical scavenging capacity of the extract was

determined using DPPH (1,1-diphenyl-2-picryl hydrazyl)

[18]. Freshly prepared DPPH solution (0.004% w/v) in

methanol was taken in test tubes and methanolic extract

of IxV was added in varying concentrations (10, 20, 40,

60, 80 and100 µg/ml) to every test tube so that the final

volume was 3 ml. For positive control, a series of same

concentration were made by ascorbic acid with

methanol and 0.004 % (w/v) DPPH solution was added.

Control sample was prepared containing the same

volume without any extract. The mixture was shaken

vigorously and left to stand for 30 min in the dark and

the absorbance was measured at 517nm. The DPPH was

showed maximum absorbance at this wavelength.

Quantitatively, the half maximal inhibitory concentration

(IC50) was determined graphically. The Percent

scavenging activity was calculated as Eq.1.

Thrombolytic Activity The thrombolytic activity of the plant extract was evaluated following a standard method [19] in which streptokinase (SK) was used as positive control and Doubled distilled water (DDW) as negative control. 5 ml of venous blood was drawn from healthy volunteers, was distributed in five different previously weighed alpine tubes (0.5 ml/tube) and the tubes were incubated for a period of 45 minutes at 37°C. After 45 minutes when clot formation occurred, the serum portion was removed

completely without hampering the clot and each tube

was weighed again to determine weight of the clot

formed in the alpine tubes (clot weight = weight of clot

6


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containing tube – weight of tube alone). 100 µl of

streptokinase (SK), methanolic extract and methanol

were added as positive control, test extract and negative

control to the marked tubes. All the tubes were then

incubated for a period of 90 minutes at 37°C and were

observed for clot lysis. The percentage of clot lysis was

estimated from the differences in weights measured

before and after clot lysis by following formula.

3. RESULTS & DISCUSSION The result of the phytochemical screening revealed the presence of important chemical components like glycosides, steroids, tannins, flavonoids, reducing sugars and amides in the plant in the Table 1.

Table 1: Presumed chemical groups for the plant

S.L No. Phytochemicals Methanolic extract

1 Alkaloids --

2 Glycosides ++

3 Steroids ++

4 Tannins ++

5 Flavonoids ++

6 Saponins --

7 Reducing sugars ++

8 Gums --

9 Amides ++

“++” Indicates presence of chemicals; “--” Indicates absence of chemicals

The methanolic extract of the plant significantly inhibited the heat induced protein (albumin) denaturation which suggests the evidence of anti-inflammatory activity of the plant. The plant extract exhibited dose depended increase of activity ranging from 36.52% to 53.76% inhibition of protein

denaturation by different doses. The positive control, Acetyl salicylic acid showed maximum inhibition of 95.36% by 500µg/ml dose. The activity of plant extract is considered as moderate in comparison with the positive control groups as mentioned in Table 2.

Table 2: Spectrophotometric determination of anti-inflammatory activity

Test group(s) Concentration (µg/mL)

Absorbance (at 660 nm)

Mean absorbance SEM % MIPD

Control (Methanol)

---

0.330 0.3303

0.0004

0 0.331

0.330 Positive control (ASA)

500 µg/ml

0.017 0.0153 0.0020

95.36* 0.017

0.012

250 µg/ml

0.046

0.0470

0.0012

85.77* 0.046 0.049

125 µg/ml

0.065

0.0683

0.0022

79.32* 0.069 0.071

MEIV 500 µg/ml

0.159 0.1527

0.0040

53.76* 0.148

0.151

250 µg/ml

0.199

0.1897

0.0061

42.56* 0.188 0.182

125 µg/ml

0.199 0.2097

0.0069

36.52*

0.218 0.212

ASA = Acetyl Salicylic acid; MEIV= Methanol extract of Ixora villosa; MIPD= Mean inhibition of protein denaturation; SEM = Standard Error of Mean.

The gradual decrease of absorbance with increasing doses suggests the potential anti-oxidant activity of the

plant extract. The methanolic extract of IxV showed 61.98% inhibition of DPPH activity at 100 μg/ml dose

7






where the standard drug, Ascorbic acid showed 94.29% inhibition by the same dose (Table 3). The value of IC50 of the plant extract is significant (Table 4). Graphical

data showed the linear anti-oxidant activity of the plant extract (Figure 2).

Table 3: Determination of radical scavenging activity by DPPH

Test groups Concentration (µg/ml) Absorbance (517 nm)

% Inhibition of DPPH

Control (Methanol) -- 0.805 0

Ascorbic Acid ( Standard )

10 0.715 11.18

20 0.641 20.37 40 0.508 36.89 60 0.323 59.87 80 0.195 75.78

100 0.046 94.29

MEIV (500 µg/100µl)

10 0.773 3.98 20 0.702 12.79 40 0.613 23.85 60 0.516 35.91 80 0.424 47.33

100 0.306 61.98

MEIV= Methanolic extract of Ixora villosa

y1: standard; y2: MEIV; MEIV= Methanolic extract of Ixora villosa

Figure 2: Determination of Anti-oxidant activity and IC50

Table 4: IC50 value for plant extract and Standard drug

Treatment groups IC50

Standard (ascorbic acid) 52.23 μg/ml Methanolic extract of Ixora villosa 83.91 μg/ml

Comparatively less thrombolytic activity was achieved by the methanolic plant extract. The extract showed 22.98% lysis of clotted blood at 500 µg/100µl doses but

the response of positive control, Streptokinase was 81.57% (Table 6 & Table 7).

8






Table 5: Thrombolytic activity of negative control (Methanol)

N

o. o

f b

loo

d

Sp

eci

me

n

Wt

of

Em

pty

a

lpin

e t

ub

e

(gm

) W

1

Wt

of

tub

e w

ith

clo

t (g

m)

W2

Wt

of

tub

e w

ith

clo

t a

fte

r ly

sis

(gm

) W

3

Wt

of

clo

t (g

m)

W2

-W1

Wt

of

lysi

s cl

ot

(gm

) W

2-W

3

% o

f ly

sis

Me

an

ly

sis

(%)

SD

SE

M

To

tal

lysi

s

1 0.924 1.322 1.312 0.398 0.010 2.513

2.11

0.84

0.34

2.1

1 ±

0.3

4

2 0.923 1.575 1.557 0.652 0.018 2.761 3 0.968 1.485 1.475 0.517 0.010 1.934 4 0.937 1.414 0.477 1.409 0.005 1.048 5 0.933 1.367 0.466 1.362 0.006 1.288 6 0.929 1.465 0.536 1.448 0.017 3.172

SD: Standard deviation; SEM: Standard error of mean; values are expressed as mean ± SEM

Table 6: Thrombolytic activity of methanol extract of Ixora villosa

E

xtr

act

No

. of

blo

od

Sp

ecim

en

Wt

of

Em

pty

alp

in t

ub

e

(gm

) W

1

Wt

of

tub

e w

ith

clo

t (g

m)

W2

Wt

of

tub

e w

ith

clo

t a

fte

r ly

sis

(gm

), W

3

Wt

of

clo

t (g

m)

W2

-W1

Wt

of

lysi

s cl

ot

(gm

) W

2-W

3

% o

f ly

sis

Me

an

ly

sis

(%)

SD

SE

M

To

tal

lysi

s

MEIV

1 0.741 1.289 1.146 0.548 0.143 26.09

22.98 3.92 1.59

22

.98

± 1

.59

2 0.811 1.335 1.218 0.524 0.117 22.33

3 0.827 1.343 1.198 0.516 0.145 28.11 4 0.792 1.310 1.187 0.518 0.123 23.75

5 0.828 1.418 1.316 0.590 0.102 17.29 6 0.806 1.378 1.262 0.572 0.116 20.28

SD: Standard deviation; SEM: Standard error of mean; values are expressed as mean ± SEM; MEIV= Methanol extract of Ixora villosa.

81.54

2.11

22.98

0

10

20

30

40

50

60

70

80

90

% C

lot

Ly

sis

Treatment groups

Streptokinase (standard)

Methanol (control)

MEIV

Figure 3: Thrombolytic activity of the plant extract

9






Table 7: Thrombolytic activity of positive control, Streptokinase

4. CONCLUSION

The methanolic extract of IxV chosen for the present study have shown the presence of important chemical groups like glycosides, steroids, tannins, flavonoids, reducing sugars, and amides. In anti inflammatory activity test, the plant extract significantly inhibited the protein denaturation at maximum dose used for the

study purpose. The test result can be concluded as the extract having moderate anti inflammatory in comparison with the activity of standard drug. The plant extract contains potential anti-oxidant activity. During thrombolytic activity test, the methanolic extract exhibited mild clot lysis activity as compared with the activity of positive control, Streptokinase.

No

. of

Blo

od

sp

eci

me

n

Wt

of

Em

pty

alp

ine

tu

be

(gm

)

W1

W

t o

f tu

be

wit

h

clo

t (g

m)

W2

Wt

of

clo

t (g

m)

W2

-W1

Me

an

Wt

of

clo

t

(gm

)

Wt

of

tub

e w

ith

clo

t a

fte

r ly

sis

(gm

)

W3

W

t o

f ly

sis

clo

t

(gm

)

W2

-W3

Me

an

Wt

of

lyse

d

clo

t (g

m)

Me

an

ly

sis

(%)

Me

an

ly

sis

(%)

SD

SE

M

To

tal

lysi

s

01

0.936 1.368 0.432

0.445

0.965 0.403

0.408 91.69

81

.54

11

.11

3.7

05

81

.57

± 3

.70

5

0.912 1.364 0.452 0.947 0.417

0.938 1.389 0.451 0.985 0.404

02

0.945 1.457 0.512

0.487

0.994 0.463

0.421 86.45 0.965 1.424 0.459 1.034 0.390

0.956 1.446 0.490 1.036 0.410

03

0.921 1.408 0.487

0.417

0.933 0.475

0.401 96.16 0.957 1.32 0.363 0.973 0.347

0.945 1.346 0.401 0.965 0.381

04

0.946 1.444 0.498

0.471

1.035 0.409

0.386 81.95 0.924 1.436 0.512 1.031 0.405

0.914 1.317 0.403 0.973 0.344

05

0.921 1.393 0.472

0.482

1.092 0.301

0.318 65.98 0.965 1.511 0.546 1.186 0.325

0.921 1.349 0.428 1.021 0.328

06

0.926 1.412 0.486

0.495

1.063 0.349

0.367 74.14 0.925 1.451 0.526 1.094 0.357

0.931 1.404 0.473 1.009 0.395

07

0.951 1.276 0.325

0.418

0.998 0.278

0.379 90.67 0.962 1.423 0.461 1.012 0.411

0.943 1.411 0.468 0.963 0.448

08

0.921 1.365 0.444

0.425

0.986 0.379

0.366 86.12 0.942 1.325 0.383 1.014 0.311

0.926 1.374 0.448 0.966 0.408

09

0.938 1.411 0.473

0.441

1.096 0.315

0.352 79.82 0.956 1.384 0.428 1.028 0.356

0.946 1.368 0.422 0.983 0.385

10

0.948 1.440 0.492

0.476

1.214 0.226

0.297 62.39 0.953 1.389 0.436 1.008 0.381

0.942 1.442 0.500 1.158 0.284

SD: Standard deviation; SEM: Standard error of mean; values are expressed as mean ± SEM

10






ACKNOWLEDGEMENTS We are grateful to the Authority of Southern University

Bangladesh, providing laboratory and other relevant

facilities, and to the person who helped us in any way

during the tenure of our study.

DECLARATIONS Funding: This project was founded by CHASA (Centre

for Health Agriculture and Socio Advancement).

Conflict of interest: None of the author had conflict

interest.

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