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INDEX GJRMI - Volume 3, Issue 5, May 2014

MEDICINAL PLANTS RESEARCH

Pharmacology

ANTIBACTERIAL, ANTI-SWARMING POTENTIAL OF ETHANOL EXTRACTS OF PHYSALIS

MINIMA L. WHOLE PLANT AND URENA LOBATA L. ROOT ON CEPHALOSPORIN RESISTANT

PROTEUS SPECIES

Mamunur Roshid, Aktar Uzzaman Chouduri 184195

Bio-Technology

CATECHIN DETECTION IN CALLUS AND IN VITRO CULTURES OF THE EASTERN

STRAWBERRY TREE, ARBUTUS ANDRACHNE L., AN ENDANGERED MEDICINAL TREE IN

PALESTINE

Zahra Aljabari, Jawad Alzeer, Rami Arafeh

196205

Review Article

ANDROGRAPHIS PANICULATA A TRADITIONAL HERB WITH PHARMACOLOGICAL

PROPERTIES: A REVIEW

Nishan Chatterjee, Sunipa Biswas, Nimai Chandra Saha, Surjyo Jyoti Biswas 206214

INDIGENOUS MEDICINE

Ayurveda Dravya Guna

PHARMACOGNOSTICAL EVALUATION ON TANNIN CONTENT IN HARITAKI LEAVES

(TERMINALIA CHEBULA RETZ. - COMBRETACEAE) BEFORE AND AFTER FLOWERING-

FRUITING

Patil Sunny C, Harisha C R, Baghel A S, Dwivedi R R 215224

Ayurveda Dravya Guna

ANTIDYSLIPIDAEMIC EFFECT OF THE STEM BARK OF CHIRABILWA (Holoptelea integrifolia

Planch.) - A CLINICAL TRIAL

Sinimol T P, Shahul Hameed A 225231

COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R, PLANT ID TENDER LEAVES OF TERMINALIA BELLIRICA (GAERTN.)

ROXB., OF THE FAMILY COMBRETACEAE PLACE KOPPA, CHIKKAMAGALUR DISTRICT,

KARNATAKA, INDIA

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 184195

Global Journal of Research on Medicinal Plants & Indigenous Medicine || GJRMI ||

ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

ANTIBACTERIAL, ANTI-SWARMING POTENTIAL OF ETHANOL

EXTRACTS OF PHYSALIS MINIMA L. WHOLE PLANT AND URENA

LOBATA L. ROOT ON CEPHALOSPORIN RESISTANT PROTEUS SPECIES

Mamunur Roshid1, Aktar Uzzaman Chouduri

2*

1,2Department of Pharmacy, University of Rajshahi, Rajshahi-6205, Bangladesh

*Corresponding author: Email: [email protected], [email protected];

Phone: +88-0721-711110 (Office), +88-01712792350 (Cell); Fax: +88-0721750064

Received: 01/04/2014; Revised: 25/04/2014; Accepted: 02/05/2014

ABSTRACT

Swarming of Proteus bacteria has been implicated in pathogenesis. In previous study, eleven

Proteus strains isolated from municipal water were found to be resistant to cephalosporins and four

isolates, 11(Pv), 661(Pp), 911(Pm), and 912(Pm), were resistant to normal human serum. The

increasing evidence of antibiotic resistance necessitates medicinal plants to develop alternative

strategies of treatment. This study aimed to search medicinal plants with high antibacterial potentials

in order to manage antibiotic resistant uropathogens. Twelve specimens of nine medicinal plants

which are available locally were analyzed for their anti-infective properties against resistant

uropathogens using disc diffusion method. Remarkable antibacterial activities of ethanol extract of

Physalis minima whole plant followed by Azadirachta indica leaf, Asparagus racemosus root,

Phyllanthus emblica fruit, Urena lobata root and Tamarindus indica bark were found against eleven

test bacteria and eleven resistant Proteus isolates. Physalis minima extract showed the highest zone

of inhibition but it had no anti-swarming effect. Interestingly complete inhibition of swarming was

found by Urena lobata root extract at 500 g/ml concentration although its antibacterial activity was

very low or nil. Thus, the mixture of two extracts would be a powerful anti-infective agent to combat

UTI and/or wound infection caused by resistant Proteus bacteria. The extracts could be further

analyzed for the drug development.

KEY WORDS: Urena lobata L., Physalis minima L., antibacterial and anti-swarming activities,

cephalosporin resistant Proteus bacteria.

ABBREVIATIONS: UTI-Urinary tract infection, CAUTI-Catheter associated urinary tract

infection, ESBL- Extended spectrum -lactamase, NHS- Normal human serum.

Research Article

Cite this article:

Mamunur Roshid, Aktar Uzzaman Chouduri (2014), Antibacterial, anti-swarming potential of

ethanol extracts of Physalis minima L. whole plant and Urena lobata L. root on cephalosporin

resistant Proteus species, Global J Res. Med. Plants & Indigen. Med., Volume 3(5): 184195



INTRODUCTION

Thirty-one species of medicinal plants were reported by traditional healers as being used for UTIs, including leucorrhea, frequent or infrequent urination, cloudy urination, and burning sensations during urination (Hossan et al., 2010). The major parts (flower, bark, root, leaves) of one of these medicinal plants, Urena lobata Linn are used as folk medicine for UTIs (Nandwani et al., 2008; Hossan et al., 2010). U. lobata Linn (common name Ceasar weed) is native to China but it is available in many tropical countries including Bangladesh, India, South America, Africa, Australia, and the United States. Especially U. lobata roots have been shown to bear a broad-spectrum antibacterial activity against Gram-positive and Gram-negative microorganisms (Mazumder et al., 2001).

The Proteus pathogens are thought to be the principal cause of UTI, CAUTI and wound infections. We isolated pathogenic Proteus bacteria from municipal tap water (Wadud and Chouduri, 2013) that were multi-antibiotic resistant especially to cephalosporins (Chouduri and Wadud, 2013) and several pathogenic features of those isolates have already been reported (Chouduri et al., 2014; Chouduri and Wadud, 2014). Although the pharmacological industries have produced a number of new antibiotics in the last four decades, resistance to these drugs by microorganisms has increased. In general, bacteria have the genetic ability to transmit and acquire resistance to drugs, which are utilized as therapeutic agents (Cohen, 1992). The increasing evidence of antibiotic resistance among bacterial pathogens necessitates medicinal plants as an alternate therapy in restricting the resistant infectious organisms. Previously it had been reported that recently the extensive use of cephalosporins for the treatment of infectious diseases allows pathogens to be resistant to the antibiotics of cephalosporin group. Therefore, an urgent need is to search new antibiotic or an alternate therapy of infectious diseases. This study aimed to manage the emergence of antibiotic resistance by phytochemicals of selective medicinal plants. To serve the purpose here

nine medicinal plants (Table 1) having potential antimicrobial properties have been selected that are traditionally used as folk medicine for urological disorders.

Nwodo et al. (2011) found the significant antimicrobial activities in aqueous and alcoholic extract of Tamarindus indica bark. Fruit of Phyllanthus emblica Gaertn is commonly known as Indian gooseberry or amla. The alcoholic extract of Phyllanthus emblica exhibited strong and broad spectrum antibacterial activity against various pathogenic bacteria and numerous biological activities has also been reported (Ahmad et al., 1998; Khan, 2009; Khosla and Sharma, 2012). The root extract of Asparagus racemosus showed antibacterial activity against resistant uropathogens isolated from patients having UTI (Narayanan et al., 2011). The alcoholic extract of Azadirachta indica leaf showed potential antimicrobial activities including Proteus mirabilis (Yasmeen et al., 2012). Leaves of Abroma augusta Linn has been widely investigated and its antibacterial potentials have been reported by researchers (Saikot et al., 2012; Zulfiker et al., 2013). The extract of Mimosa pudica Linn root is an alternative wound healing agent widely used as folk medicine in Indian subcontinent for the treatment of vaginal and uterine complications. It is very useful in diarrhea, amoebic dysentery, bleeding piles and urinary infections (Joseph et al., 2013). The ethanol extract of Coccinia grandis leaves exhibited antimicrobial activity against biofilm and ESBL producing uropathogenic Escherichia coli strains UPEC-17 and -82 (Poovendran et al., 2011).

The general acceptance of traditional medicine for health care and the development of microbial resistance to several available antibiotics have led researchers to investigate the activity of medicinal plants against infectious diseases (Low et al., 2002; Yarnell, 2002). Therefore, the aim of this study was to evaluate the role of ethanolic fractions of the medicinal plants to interfere with the growth and virulence of multi-antibiotic, especially cephalosporin resistant uropathogenic Proteus bacteria isolated in our previous study.



Table 1: List of medicinal plants tested

Sl Scientific name Family Local name Plant part Abbreviation

1 Tamarindus indica Leguminosae Tetul Bark Ti-b

2 Phyllanthus emblica Phyllanthaceae Amloki/Amla Fruit Pe-f

3 Physalis minima Solanaceae Bontepari/Potka Whole plant Pm-w

4 Asparagus racemosus Asparagaceae Shotomuli Root Ar-r

5 Urena lobata Malvaceae Bonokra Root Ul-r

6 Urena lobata Malvaceae Bonokra Leaf Ul-l

7 Urena lobata Malvaceae Bonokra Fruit Ul-f

8 Urena lobata Malvaceae Bonokra Bark Ul-b

9 Azadirachta indica Meliaceae Neem Leaf Ai-l

10 Coccinia grandis Cucurbitaceae Telakucha Whole plant Cg-w

11 Abroma augusta Malvaceae Ulotcombol Leaf Aa-l

12 Mimosa pudica Leguminosae Lojjaboti Root Mp-r

MATERIALS AND METHODS

Plant material

Plant parts were collected from the

medicinal plant garden, Department of

Pharmacy, University of Rajshahi and around

Rajshahi City area, Bangladesh on Nov 2013,

and duly identified by a plant taxonomist Mr.

Arshed Alom, Department of Botany,

University of Rajshahi, Bangladesh where a

specimen voucher (75/05.07.2008) was

recorded in the department herbarium for future

reference. Twelve specimens of nine medicinal

plants enlisted in table 1 were air-dried under

shade. A representative image of two plants

and plant parts has been shown in figure 1.

Once dried, the plant material was ground,

extracted by maceration for more than 72 hrs

with ethanol, filtered (Paper Whatman No. 3)

and the solvent was vacuum evaporated in a

Soxhlet apparatus (Rotary Evaporator, RE 300,

Bibby Sterilin Ltd, UK). Then solutions were

evaporated to dryness and further dilutions

were made in the same solvent to obtain the

required extract concentrations for the different

assays.

Bacterial strains

From our laboratory stock five Gram

positive bacteria, Staphylococcus aureus,

Streptococcus agalactiae, Bacillus cereus,

Bacillus megaterium, Bacillus subtilis, and six

Gram negative bacteria, Pseudomonas

aeruginosa, Shigella flexneri, Shigella

dysenteriae, Escherichia coli, Shigella sonnei,

Agrobacterium species, were used for

antibacterial activity assay of the plant extracts.

Eleven Proteus strains of four species: P.

vulgaris (hereafter termed as Pv), P. mirabilis

(Pm), P. hauseri (Ph), and P. penneri (Pp)

named as 11(Pv), 661(Pp), 662(Ph), 663(Pp),

664(Pp), 665(Pp), 666(Pp), 667(Pp), 668(Pp),

911(Pm) and 912(Pm) isolated from municipal

tap water (Rajshahi City, Bangladesh) in our

previous study (Wadud and Chouduri, 2013)

have been used. Those strains were multidrug

resistant to broad spectrum antibiotics and

possessed several pathogenic features including

swarming motility, urease production,

extracellular proteases, biofilm formation as

reported earlier (Chouduri and Wadud, 2013;

Chouduri et al., 2013; Chouduri and Wadud,

2014). Strains stored at 40C in Luria-Bertani

(LB) broth supplemented with 12% (v/v)

glycerol were freshly grown at 37C to carry

out this study.

Growth media and culture conditions

Nutrient agar media purchased from Difco,

USA was used for antibacterial activity assay



of the plant extracts. The bacterial strains were

incubated at 37C for overnight as described

elsewhere (Nesa et al., 2013; Chouduri and

Wadud, 2013). Fresh cell culture in nutrient

broth media prepared on water bath (Advantec

Lab-Thermo Shaker, TS-20, Toyo Kaisha Ltd)

with mild shaking at 37C was used to test the

swarming motility of Proteus strain.

Test for antibacterial activity

Plant extracts were tested for antibacterial

and specifically anti-Proteus activity using disk

diffusion method on nutrient agar media as

reported elsewhere (Dash et al., 2005; Parvin et

al., 2014). The extracts were separately

dissolved in 1 ml of ethanol and the filter paper

discs (6 mm diameter) were impregnated with

known amounts of test substances and prepared

disc with various potencies, 25 g to 1 mg/disc.

Discs were placed on pre-seeded bacterial

culture plates and then kept at low temperature

(4C) overnight to allow maximum diffusion of

the components. The plates were then allowed

to incubate at 37C for 18 hrs. Then the

diameter (in millimeter) of zone of inhibition

for each extract against tested microorganisms

was noted. Reference standard discs of

cefixime (5 g), ceftazidime (30 g),

kanamycin (30 g) (Hi-media, India) were used

as positive control and blank disc as negative

control.

Swarming motility test

Proteus strains were grown overnight in

10 ml of LB broth medium (1% Tryptone,

0.5% Yeast extract, and 0.5% NaCl) at 37C

with shaking (200 rpm). Then 5 l of fresh cell

culture was spotted at the center of LB agar

plates (LB medium containing 1.5% agar)

previously dried to remove water drops from

the surface of the agar medium as described in

other reports (Kwil et al., 2013) and incubated

at 37C for 24 hrs unless it is mentioned

otherwise. Then the mean diameters of

swarming zones measured in millimeter at

three different directions were used for

analysis.

Inhibition of swarming motility

The effects of plant extracts on swarming

motility of Proteus strains were assessed as

described in other report (Liaw et al., 2000;

Roshid et al., 2014). Briefly, an overnight

bacterial culture (5 l) was inoculated centrally

onto the surface of dry LB agar plates prepared

with extracts at various concentrations which

were then incubated at 37C for 24 hrs. The

perimetric distance of swarming motility was

assayed by measuring the fronts of swarming

areas in three different directions.

Data analysis

For data processing, the software Microsoft

Excel 2007 was used. Results of triplicate

experiments were averaged, and means

standard deviations were calculated.

RESULTS

Antibacterial activities of plant extracts

The ethanol extracts of the plant specimens

were tested for their antibacterial activities on

five Gram-positive and six Gram-negative

bacteria from our laboratory stock. The extracts

named Ti-b, Pe-f, Pm-w, Ar-r, Ul-r, and Ai-l

showed remarkable antibacterial activities with

a wide zone of inhibition whereas Ul-l, Ul-f,

Ul-b, Cg-w, and Mp-r were inactive in

antibacterial activities (Table 2). However, the

antibacterial potentials of the test extracts based

on their zone of inhibition were evaluated

where Pm-w was the best one showing 1522

mm clear zone on culture plate followed by Ai-l

(1820 mm), Ar-r (1019 mm), Pe-f (10

16 mm), Ul-r (814 mm) and Ti-b (913 mm).

The antibacterial potentials of the extracts Pm-

w and Ai-l against three Gram-positive bacteria,

S. agalactiae, B. megaterium, B. subtilis, and

two Gram-negative bacteria, P. aeruginosa, S.

flexneri, were very close and comparable to

that of reference antibiotic kanamycin (Table

2).



Figure 1: Effective medicinal plant species to cephalosporin resistant Proteus.

A: whole plant of Urena lobata L. (image courtesy- www.google.com.bd), B: leaf specimen of U. lobata L. washed with

water, C: fruits specimen of U. lobata L. under shade drying, D: whole plant of Physalis minima L.

(image courtesy- www.google.com.bd).

Table 2: Antibacterial activities of plant extracts on bacterial pathogens.

Test strains Diameter of zone of inhibition (mm) of test extracts (1 mg/disc) Kan

Ti-b Pe-f Pm-w Ar-r Ul-r Ul-l Ul-f Ul-b Cg-w Ai-l Aa-l Mp-r

Gram-positive

S. aureus 120.3 120.6 220.3 140.4 130.2 180.1 341.2

S. agalactiae 100.4 120.3 190.2 120.2 120.9 190.1 231.3

B. cereus 120.9 100.7 200.3 140.6 120.4 200.3 80.6 340.6

B. megaterium 90.4 120.7 220.4 110.9 110.2 180.5 230.5

B. subtilis 110.4 110.5 210.5 100.3 130.5 200.4 220.8

Gram-negative

P. aeruginosa 120.4 141.0 190.6 110.4 140.5 180.3 220.9

S. flexneri 130.2 160.8 200.2 130.6 130.7 190.8 251.5

S. dysenteriae 130.3 111.1 180.9 140.7 120.7 70.2 180.1 80.8 311.4

E. coli 121.1 140.8 210.3 190.8 80.6 80.1 190.1 400.8

S. sonnei 120.4 100.5 170.7 140.9 80.5 200.6 70.4 311.1

A. species 110.7 100.3 150.6 140.4 80.4 190.4 70.6 290.9 () sign indicates no activity. Values were expressed as mean SD (n=3). Ti-b: Tamarindus indica bark, Pe-f:

Phyllanthus emblica fruit, Pm-w: Physalis minima whole plant, Ar-r: Asparagus racemosus root, Ul-r: Urena lobata root,

Ul-l: Urena lobata leaf, Ul-f: Urena lobata fruit, Ul-b: Urena lobata bark, Cg-w: Coccinia grandis whole plant, Ai-l:

Azadirachta indica leaf, Aa-l: Abroma augusta leaf, Mp-r: Mimosa pudica root, Kan: Kanamycin (30 g/disc).

Screening of plant extracts for their abilities

to inhibit Proteus

Next our efforts aimed to search medicinal

plants to combat these strong cephalosporin

resistant Proteus isolates to control and manage

UTI caused by these bacteria. To do so, twelve

specimens of nine medicinal plants as enlisted

in table-1 were selected based on their reported

information. The extracts exhibiting high

antibacterial activities on several Gram-positive

and Gram-negative bacteria were used to test

whether they have any inhibitory effect on

multi-antibiotic resistant Proteus strains

isolated in our previous study (Wadud and

Chouduri, 2013). The extract Pm-w showed

remarkable zone of inhibition of Proteus strains

(22 mm) whereas no clear zone of inhibition

was observed for reference antibiotic cefixime

(Figure 2). A representative image has been

shown in figure 2. The extract Ul-r showed

clear zone of inhibition of Proteus strains but

relatively higher inhibition was found for the

strain 11(Pv). Then the extracts were screened

for their effects on swarming motility of the

test strains since swarming is one of the crucial

pathogenic factors of Proteus bacteria.



Figure 2: Antibacterial activities of extracts on cephalosporin resistant Proteus isolates.

Extracts at 1 mg/disc concentration were used. The diameters of zone of inhibition in isolate 11(Pv): 8 mm (Ul-b),

15 mm (Ul-r), 22 mm (Pm-w), 11 mm (Cg-w); in 662(Ph): 13 mm (Ul-r), 21 mm (Pm-w); in 666(Pp): 23 mm (Ai-l),

15 mm (Ar-r), 12 mm (Ti-b), 12 mm (Pe-f), 29 mm (Pm-w). Reference standard discs Cfx: cefixime (5 g)

and Cfd: Ceftazidime (30 g).

Figure 3: Effects of extracts on Proteus swarming.

Three top swarmer strains, A: 911(Pm), B: 912(Pm), C: 662(Ph) were subjected to a test for swarming motility on LB agar

plate in the presence of U. lobata bark (), leaf (), fruit (), and root () extract at 500 g/ml concentration and the absence of extract (). The U. lobata root extract strongly inhibited the swarming of all test strains.

Effects of plant extracts on swarming

motility of Proteus strain

Eleven Proteus isolates found to be

strongly resistant to cephalosporin by disc

diffusion method as reported earlier (Chouduri

and Wadud, 2014) were subjected to a

bactericidal activity assay by NHS where four

isolates 11(Pv), 661(Pp), 911(Pm), and 912(Pm)

were found to be resistant to NHS (unpublished

data). Isolates 912(Pm), 911(Pm) and 662(Ph)

were strong swarmer on LB agar media

(Chouduri et al., 2014), therefore, these isolates

were undertaken to a test of swarming in the

presence of various concentrations of plant

extracts especially U. lobata extracts (Figure 3)

since major parts of this plant are used as folk

medicine for UTI (Nandwani et al., 2008;

Hossan et al., 2010). No noticeable effects of

the extracts except Ul-r were found on the

swarming motilities of the test strains (Figure

3). The extract Ai-l accelerated the swarming of

Proteus isolates about 2 fold. However,

interestingly complete inhibition of swarming

was found by the extract Ul-r at 500 g/ml

concentration (Figure 3) although its

antibacterial activity was nil or very low by

disc diffusion method (Figure 2, Table 3). The

lag phase of swarming continued up to 4 hrs of

incubation and the basal swarming starts after 4

hrs of incubation in the presence of the extracts.

The zigzag pattern of swarming curves was a



consequence of swarming-plus-consolidation

cycle of the strains. However, anti-swarming

effect of U. lobata root extract can be of

interest to develop phytomedicine for the

management and control of UTI and/or wound

infection caused by antibiotic resistant Proteus

bacteria. Moreover, the extract of Physalis

minima had no anti-swarming effect although

its antibacterial activity was stronger than that

of others.

Table 3: Antibacterial activities of extracts on cephalosporin resistant Proteus isolates.

Proteus

strains

Diameter of zone of inhibition (mm) of test extracts (1 mg/disc) Cfx

Ti-b Pe-f Pm-w Ar-r Ul-r Ul-l Ul-f Ul-b Cg-w Ai-l Aa-l Mp-r

11(Pv) 70.6 160.8 220.2 160.4 150.2 80.2 110.1 180.3

661(Pp) 150.3 140.4 220.5 170.1 210.3

662(Ph) 80.2 130.3 210.3 140.9 130.3 150.9

663(Pp) 90.4 100.5 120.3 120.2 140.5

664(Pp) 130.3 170.5 180.6 150.4 190.3

665(Pp) 90.5 110.7 130.5 130.4 150.7

666(Pp) 120.6 121.1 290.4 150.5 70.3 230.1 100.4 130.6

667(Pp) 110.4 80.2 151.1 150.3 80.4 180.6 70.4

668(Pp) 120.6 140.6 180.4 140.5 270.5

911(Pm) 80.4 111.0 150.2 150.8 140.6

912(Pm) 80.3 110.7 140.9 171.2 280.7

() sign indicates no activity. Values were expressed as mean SD (n=3). Ti-b: Tamarindus indica bark, Pe-f: Phyllanthus emblica fruit, Pm-w: Physalis minima whole plant, Ar-r: Asparagus racemosus root, Ul-r: Urena lobata root,

Ul-l: Urena lobata leaf, Ul-f: Urena lobata fruit, Ul-b: Urena lobata bark, Cg-w: Coccinia grandis whole plant, Ai-l:

Azadirachta indica leaf, Aa-l: Abroma augusta leaf, Mp-r: Mimosa pudica root, Cfx: Cefixime (5 g/disc).

Table 4: Kinetics of swarming motility

Proteus

strain

Extracts Rate of swarming (mm/h)

03 h 34 h 45 h 56 h 67 h 78 h 89 h

911(Pm) Control 1.03 1.62 11.50 4.00 3.50 3.50 5.50

Ul-b 0.98 1.25 10.00 2.00 2.04 8.00 7.00

Ul-l 1.02 1.50 17.50 2.50 2.35 9.00 9.50

Ul-f 0.95 1.62 20.50 1.50 1.45 10.00 9.50

Ul-r 0.89 1.25

912(Pm) Control 0.97 1.50 13.50 5.00 7.00 9.00 6.50

Ul-b 1.10 1.62 21.50 2.00 1.50 10.00 12.00

Ul-l 1.09 1.12 14.50 3.00 2.96 14.00 6.50

Ul-f 0.94 1.37 16.00 5.50 6.00 9.50 2.50

Ul-r 0.95 1.12

662(Ph) Control 1.03 1.25 2.75 3.00 2.00 4.00 5.50

Ul-b 0.92 1.37 9.50 3.50 6.50 13.00 4.89

Ul-l 1.17 1.87 13.50 3.00 2.88 10.50 5.50

Ul-f 0.88 1.13 7.50 7.00 6.95 10.50 9.50

Ul-r 1.11 2.00



Kinetics of U. lobata extract-induced

swarming motility

Three Proteus isolates exhibiting enhanced

swarming motility were assessed for their

abilities to swarm onto the LB agar plate in the

presence of U. lobata extracts. The velocities of

swarming (mm/h) of the test strains in the

presence of U. lobata root extract were found

to be zero after the lag phase (4 hrs) (Table 4)

and the swarming velocities for other extracts

were instantaneously accelerated just after the

lag phase (4 hrs) that were the maximal

velocities up to 7 hrs of incubation period. The

similar duration of lag phase (4 hrs) of clinical

isolates of P. mirabilis was reported by

Rauprich et al. (Rauprich et al., 1996). The

rapid onset of swarming after lag phase is

possibly due to the formation of elevated

number of flagella on bacteria or multi-

nucleation in the first generation of swarmer

cells. The cycle time of swarming-plus-

consolidation phase found by Rauprich et al.,

(1996) is about 3.5 hrs at 37C on 1.5% agar

plate. In this study, the high swarming

velocities after lag phase at 45 hrs and the subsequent gradual decline of the velocity up to

7 hrs indicated the first cycle of swarming-plus-

consolidation phase and the following high

velocities at 78 hrs indicated the second cycle of swarming-plus-consolidation phase

resembling the findings of Rauprich et al.

(1996). However, only the U. lobata root

extract showed significant inhibition of

swarming of the test strains and no noticeable

effects of other test extracts on swarming were

observed.

DISCUSSION

Among the most common infections UTI is

affecting humans and represent a serious health

problem for millions of people each year.

Proteus is an important opportunistic

uropathogen, frequently isolated from

catheterized patients or individuals with

structural abnormalities of the urinary tract

(Khalid et al., 2013; Hoban et al., 2012; Alves

et al., 2014) although it does not commonly

cause UTI in the normal host. UTI is

commonly managed with antibiotic therapy but

the increasing evidence of antibiotic resistance

is restricting the therapeutic option. Thus the

acceptance of traditional medicine as an

alternative form of health care and the

development of microbial resistance to the

available antibiotics have led researchers to

investigate the antimicrobial activity of herbal

extracts.

The World Health Organization reported

that about 80% of the worlds population depends primarily on traditional medicine that

mainly involves the use of plant extracts (Low

et al., 2002). The screening of plant extracts

and plant products has shown that medicinal

plants represent a potential source of new anti-

infective agents. For instance, cranberry has

long been of interest for its beneficial effects in

preventing UTI (Ahuja et al., 1998; Howell et

al., 1998; Howell and Foxman 2002; McCall et

al., 2013). Plants containing flavonoids,

terpenoids, steroids, phenolic compounds and

alkaloids have been reported to have

antimicrobial activity. Three compounds

(kaempferol, quercetin, tiliroside) isolated from

ethyl acetate fraction of U. lobata leaf showed

strong antimicrobial activities against

Escherichia coli, Bacillus subtilis, Klebsiella

pneumoniae, Bacillus polyxyma and Candida

albicans (Adewale et al., 2007). But in this

study, the ethanol extract of U. lobata leaf

showed no antibacterial activities against test

bacterial pathogens including Proteus. In

contrast, U. lobata root had a significant anti-

swarming effect on Proteus isolates although

its antibacterial activity was very low. It has

been reported that U. lobata root has no

significant toxic effects on serum total proteins,

albumin and globulins (Omonkhua and

Onoagbe, 2011). Therefore, U. lobata root can

be used as a source of alternative anti-infective

agent for the treatment of UTI and wound

infection caused by antibiotic resistant bacteria.

The chloroform extract of P. minima

exhibited remarkable cytotoxic activities on

NCI-H23 (human lung adenocarcinoma) cell

line at dose- and time-dependent manners

(Leong et al., 2011). The strong antibacterial



activity of ethanol extract of P. minima leaf has

been reported (Gavimath et al. 2012). In this

study, we found strong inhibition of antibiotic

resistant Proteus isolates by the treatment of

ethanol extract of P. minima whole plant. Thus,

P. minima can also be the plant of interest for

the treatment and control of antibiotic resistant

uropathogens.

Howell et al., (1998) determined that

proanthocyanidins isolated from the cranberry

fruit inhibit P-fimbrial adhesion in vitro, and

thus may be the compounds responsible for the

beneficial effect on UTI prevention (Howell et

al., 1998). The urine of humans who consumed

cranberry juice cocktail also exhibited anti-

adhesion activity (Howell and Foxman, 2002),

which suggests that a certain level of

absorption occurred and that bioactive

proanthocyanidins and/or their metabolites

have been excreted in the urine to inhibit

adhesion. The bactericidal activities of

anacardic acid and totarol (a diterpene

extracted from the totara tree) on methicillin

resistant strains of S. aureus and the synergistic

effect of these compounds associated with

methicillin have been reported (Muroi and

Kubo, 1996). Therefore, more studies

pertaining to the use of plants as therapeutic

agents should be emphasized, especially those

related to the control of antibiotic resistant

microbes.

CONCLUSION

The ethanol extract of Physalis minima

whole plant showed strong antibacterial

activities against cephalosporin resistant

uropathogen Proteus and the extract of Urena

lobata root showed strong anti-swarming effect

on Proteus. Therefore, a mixture of two

extracts would be a powerful anti-infective

agent to combat UTI caused by antibiotic

resistant Proteus. This study could offer

scientific basis for the in-depth evaluation of

ethanol extract of P. minima whole plant and

U. lobata root. The phytochemical(s) in P.

minima and U. lobata extracts having the

potential antibacterial activities and anti-

swarming effect are remain to be identified and

are required to go through the toxicity analyses

before they can be safely applied.

ACKNOWLEDGEMENTS

Authors wish to thank the Department of

Pharmacy, University of Rajshahi, Bangladesh

for providing laboratory facilities to carry out

the entire experiments. We thank the Ministry

of Science and Technology, Government of the

People's Republic of Bangladesh for the NST

fellowship provided to author MR to carry out

the research.

REFERENCES

Adewale AO, David AA, Abiodun OO, Craig

OA (2007). Studies on antimicrobial,

antioxidant and phytochemical analysis

of Urena lobata leave extract. J. Phys.

Nat Sci. 1(2): 1220.

Ahmad I, Mehmood Z, Mohammad F (1998).

Screening of some Indian medicinal

plants for their antimicrobial properties.

J. Ethnopharmacol. 62: 183193.

Ahuja S, Kaak B, Roberts J (1998). Loss of

fimbrial adhesion with the addition of

Vaccinium macrocarpon to the growth

medium of P-fimbriated Escherichia

coli. J. Urol. 159: 559562.

Alves MJ, Barreira JC, Carvalho I, Trinta L,

Pereira L, Ferreira IC, Pintado M

(2014). Propensity for biofilm

formation by clinical isolates from

urinary tract infections: developing a

multifactorial predictive model to

improve the antibiotherapy. J. Med.

Microbiol., 63: 471477.



Chouduri AU, Wadud A (2013). Strong

cephalosporin resistant

uropathogen, Proteus mirabilis, in

urban tap water harbors a risk to public

health, Bangladesh. Glob. Adv. Res. J.

Microbiol. 2: 164171.

Chouduri AU, Wadud A, Islam AU (2014).

Extended spectrum multi-drug

resistance versus pathogenic factors-

swarming, proteases, and urease- of

Proteus species. Int. Res. J. Microbiol.

5: 815.

Chouduri AU, Wadud A (2014). Twitching

motility, biofilm communities in

cephalosporin resistant Proteus spp and

the best in vitro amoxicillin

susceptibility to isolates. Am. J.

Microbiol. Res. 2: 815.

Cohen ML (1992). Epidemiology of drug

resistance: implications for a

postantimicrobial era. Science 257:

10501055.

Dash S, Nath LK, Bhise S, Bhuyan N (2005).

Antioxidant and antimicrobial activities

of Heracleum nepalense D Don root.

Trop. J. Pharm. Res. 4: 341347.

Hoban DJ, Lascols C, Nicolle LE, Badal R,

Bouchillon S, Hackel M, Hawser S

(2012). Antimicrobial susceptibility of

Enterobacteriaceae, including molecular

characterization of extended-spectrum

beta-lactamaseproducing species, in urinary tract isolates from hospitalized

patients in North America and Europe:

results from the SMART study 20092010. Diagnostic Microbiol. Infec.

Dis., 74: 6267.

Hossan MS, Hanif A, Agarwala B, Sarwar MS,

Karim M, Rahman MTU, Jahan R,

Rahmatullah M (2010). Traditional use

of medicinal plants in Bangladesh to

treat urinary tract infections and

sexually transmitted diseases. Ethnobot.

Res. Appl. 8: 6174.

Howell AB, Vorsa N, der Marderosian A

(1998). Inhibition of the adherence of P-

fimbriated Escherichia coli to

uroepithelial-cell surfaces by

proanthocyanidin extracts from

cranberries. New England J. Med. 339:

10851086.

Howell AB, Foxman B (2002). Cranberry juice

and adhesion of antibiotic-resistant

uropathogens. JAMA 287: 30823083.

Joseph B, George J, Mohan J (2013).

Pharmacology and traditional uses of

Mimosa pudica. Int. J. Pharm. Sci.

Drug Res. 5: 4144.

Khalid MIH, Teh LK, Lee LS, Zakaria ZA,

Salleh MZ (2013). Genome sequence of

Proteus mirabilis strain PR03, isolated

from a local hospital in

Malaysia. Genome Announ., 1: e003

2713.

Khan MA, Khan H, Khan S, Mahmood T,

Khan PM, Jabar A (2009). Anti-

inflammatory, analgesic and antipyretic

activities of Physalis minima Linn. J.

Enz. Inhib. Med. Chem. 24: 632637.

Khosla S, Sharma S (2012). A short description

on pharmacogenetic properties of

Phyllanthus emblica. Spatula DD. 2:

187193.

Kwil I, Kazmierczak D, Rozalski A (2013).

Swarming growth and resistance of

Proteus penneri and Proteus vulgaris

strains to normal human serum. Adv.

Clin. Exp. Med. 22: 165175.

Leong OK, Muhammad TST, Sulaiman SF

(2011). Cytotoxic activities of Physalis

minima L. chloroform extract on human

lung adenocarcinoma NCI-H23 cell

lines by induction of apoptosis.

Evidence-Based Compl. Alter. Med.

2011: 110.



Liaw SJ, Lai HC, Ho SW, Luh KT, Wang WB

(2000). Inhibition of virulence factor

expression and swarming differentiation

in Proteus mirabilis by p-

nitrophenylglycerol. J. Med. Microbiol.

49: 725731.

Low CAM, Regnier TJC, Korsten L (2002).

Medicinal bulbous plants of South

Africa and their traditional relevance in

the control of infectious diseases. J.

Ethnopharmacol. 82: 147154.

Mazumder UK, Gupta M, Manikandan L,

Bhattacharya S (2001). Antibacterial

activity of Urena lobata roots.

Fitoterapia 72: 927929.

McCall J, Hidalgo G, Asadishad B, Tufenkji N

(2013). Cranberry impairs selected

behaviors essential for virulence in

Proteus mirabilis HI4320. Can. J.

Microbiol. 59: 430436.

Muroi H, Kubo I (1996). Antibacterial activity

of anacardic acids and totarol, alone and

in combination with methicillin, against

methicillin-resistant Staphylococcus

aureus. J. Appl. Bacteriol. 80: 387394.

Nandwani D, Calvo JA, Tenorio J, Calvo F,

Manglona L (2008). Medicinal plants

and traditional knowledge in the

Northern Mariana Islands. J. Appl.

Biosci. 8: 323330.

Narayanan AS, Raja SSS, Ponmurugan K,

Kandekar SC, Natarajaseenivasan K,

Maripandi A, Mandeel QA (2011).

Antibacterial activity of selected

medicinal plants against multiple

antibiotic resistant uropathogens: a

study from Kolli hills, Tamil Nadu,

India. Beneficial Microbes 2: 235243.

Nesa L, Salam A, Islam AU, Chouduri AU

(2013). Multi-drug resistant Neisseria

gonorrhea among hotel-based sex

workers in Rajshahi, Bangladesh. Int. J.

Microbiol. Res. 4: 167176.

Nwodo UU, Obiiyeke GE, Chigor VN, Okoh

AI (2011). Assessment of Tamarindus

indica extracts for antibacterial activity.

Int. J. Mol. Sci. 12: 63856396.

Omonkhua AA, Onoagbe IO (2011).

Evaluation of the long-term effects of

Urena lobata root extracts on blood

glucose and hepatic function of normal

rabbits. J. Toxicol. Env. Health Sci. 3:

204213.

Parvin S, Kader MA, Chouduri AU,

Rafshanjani MAS, Haque ME (2014).

Antibacterial, antifungal and

insecticidal activities of the n-hexane

and ethyl-acetate fractions of

methanolic extract of the leaves of

Calotropis gigantea Linn. J. Pharma.

Phytochem. 2: 4751.

Poovendran P, Vidhya N, Murugan S (2011).

Antimicrobial activity of Coccinia

grandis against biofilm and ESBL

producing uropathogenic E. coli. Glob.

J. Pharmacol. 5: 2326.

Rauprich O, Matsushita M, Weijer CJ, Siegert

F, Esipov SE, Shapiro JA (1996).

Periodic phenomena in Proteus

mirabilis swarm colony development. J.

Bacteriol. 178: 65256538.

Roshid M, Wadud A, Chouduri AU (2014).

Potent inhibition of swarming in

uropathogenic Proteus bacteria by

ethanol extracts of Phyllanthus emblica

fruit and Tamarindus indica bark. Int.

Res. J. Biol. Sci. 3: 17.

Saikot FK, Khan A, Hasan MF (2012).

Antimicrobial and cytotoxic activities

of Abroma augusta Lnn. leaves extract.

Asian Pac. J. Trop. Biomed. 2: S1418

S1422.



Wadud A, Chouduri AU (2013). Microbial

safety assessment of municipal water

and incidence of multi-drug resistant

Proteus isolates in Rajshahi,

Bangladesh. Curr. Res. Microbiol.

Biotechnol. 1: 189195.

Yarnell E (2002). Botanical medicines for the

urinary tract. World J. Urol. 20: 285293.

Yasmeen R, Hashmi AS, Anjum AA, Saeed S,

Muhammad K (2012). Antibacterial

activity of indigenous herbal extracts

against urease producing bacteria. J.

Anim. Plant Sci. 22: 416419.

Zulfiker AHM, Roy PP, Momin MAM, Khan

MS, Bulbul IJ, Ahmed T, Rana MS

(2013). Investigation of antioxidant and

antimicrobial potential of chloroform

and petroleum ether extracts of selected

medicinal plants of Bangladesh. British

J. Med. Medical Res. 3: 4.

Source of Support: National Science and

Technology (NST) Fellowship, Government

of Bangladesh

Conflict of Interest: None Declared




CATECHIN DETECTION IN CALLUS AND IN VITRO CULTURES OF THE

EASTERN STRAWBERRY TREE, ARBUTUS ANDRACHNE L., AN

ENDANGERED MEDICINAL TREE IN PALESTINE

Zahra Aljabari

1, Jawad Alzeer

2, Rami Arafeh

1*

1, 3

Biotechnology Research Center. Palestine Polytechnic University. P.O. Box 198, Hebron, Palestine. 2Department of Applied Chemistry, Faculty of Applied Sciences. Palestine Polytechnic University, P.O.Box

198 Hebron, Palestine.

*Corresponding author: E-mail: [email protected]; Tel: +970-22231921 ext. 137; Fax: +970-2231921 ext. 119


ABSTRACT

The Eastern Strawberry tree, Arbutus andrachne L., is a medicinal evergreen small tree naturally

distributed from Eastern Mediterranean to the Northern Black Sea region. In Palestine, the tree is

known for its high medicinal value and recently has been included within the endangered species.

For conservation and utilization of A. andrachne we investigated the presence of catechin, an

antioxidant and active flavonoid in the ethylacetate fraction in leaves of wild plant material and also

in the extract of callus and the in vitro grown vegetative tissues. HPLC analysis of catechin revealed

0.063% in callus extract, 2.5% in the in vitro growing tissues and 0.5% in wild growing plants. In

vitro propagation and callus culture are promising approaches for the secondary metabolites

production in the case of A. andrachne.

KEY WORDS: Arbutus andrachne L., callus culture, catechin, medicinal plant, secondary

metabolites.

Research Article

Cite this article:

Zahra Aljabari, Jawad Alzeer, Rami Arafeh (2014), CATECHIN DETECTION IN CALLUS

AND IN VITRO CULTURES OF THE EASTERN STRAWBERRY TREE, ARBUTUS

ANDRACHNE L., AN ENDANGERED MEDICINAL TREE IN PALESTINE, Global J Res.

Med. Plants & Indigen. Med., Volume 3(5): 196205



INTRODUCTION

The Eastern Strawberry Tree, Arbutus

andrachne L. (Ericaceae), is one of the

important medicinal trees in the Eastern

Mediterranean region. It is a medium evergreen

tree grows in mountainous rocky habitats with

alkaline soil. The plant parts are known to have

valuable medicinal values due to its content in

antioxidants and natural pigments that makes it

a multiple uses plant. In the traditional folk

medicine, it is used as astringent and for

urinary antiseptic treatments, treatment for

aching joints and wounds and against some

cancer types (Said et al., 2002; Serce et al.,

2010). Furthermore, a cosmetic value of the

dried leaves' powder as skin whitening agent in

face masks has been described by Issa et al.

(2008). Recently, the plant gained higher

attention after being listed in databases of the

endangered species in Palestine (Eshtayeh &

Jamous, 2002) and Israel (ROTEM, 2002). The

species is progressively encountering genetic

erosion after being collected for its medicinal

and cosmetic uses. The unplanned expansion in

agricultural activities, overgrazing and

collection for fire contribute in the genetic

erosion of A. andrachne. Following to the

continuous increase in demand for the plant

during the last few years several attempts have

addressed the propagation and conservation of

A. andrachne in which some successes have

been achieved, examples are discussed in

(Bertsouklis & Papafotiou, 2009; Karam & Al-

Salem, 2001; Kose, 1998; Mostafa, et al., 2010;

Tilki & Guner, 2007 ).

Catechins are polyphenolic flavonoids that

can be found in wide range of natural sources

including leaves of herbs like green tea

Camellia sinensis, fruits like apples Malus

pumila, fruit skin, juice and oil seed of grapes

Vitis vinifera, wood and bark of trees in the

genus Acacia reviewed in (Ruidavets, et al.,

2000) and (Iacopini, Baldi, Storchi, &

Sebastiani, 2008). Catechin exists in different

chemical forms like epicatechin, epicatechin-3-gallate, epigallocatechin, epigallocatechin-3-gallate, +catechin and +gallocatechin. They exhibit many biological

activities accountable for their medicinal

values. They play an antioxidant, anti-cancer,

anti-angiogenic, anti-mutagenic, hypo-

cholesterolemic, anti-ageing, anti-diabetic, anti-

bacterial, anti-HIV and anti-inflammatory

effects (Al-Hanbali, et al., 2009; Ivanov, et al.,

2011; Sakar, et al., 1991; Suzuki, et al., 2005;

Zaveri, 2006). Saker et al. (1991) have

described some chemical constituents present

in A. andrachne, namely +catechin,

epicatechin and arbutin in addition to other constituents in the tree bark such as

monotropein and unidoside.

Recently, A. andrachne is being

overexploited and attempts are focused to

conserve it and supply sufficient material for

propagation and utilization. In this study, we

present the use of A. andrachne material

obtained by in vitro culture for the production

of secondary metabolites, particularly catechin,

as a representative of flavonoids in comparison

to its presence in the leaves of wild trees.

MATERIALS AND METHODS

Plant material:

Seeds of A. andrachne were collected in

November 2007 from wild growing trees West

of Hebron city [N:313200, E:350542]. Plant characterization was carried out by Dr.

Rami Arafeh and a voucher specimen of the

sampled plant was deposited in the

Biotechnology Research Center at Palestine

Polytechnic University. Ripe fruits were soaked

in tap water for 72 h before the seeds were

separated manually and washed from the fruit

pulp. Seeds were stored at room temperature at

212C to be used for experimental work.

Callus induction and maintenance:

Since A. andrachne seeds exhibit

physiological dormancy (Karam & Al-Salem,

2001; Mostafa, et al., 2010), a pretreatment for

the seeds was carried out by soaking for 24 h in

a solution of 5.0 mg/l GA3 at room temperature.

Seeds then were surface sterilized in 5% v/v

solution of commercial bleach for 20 min then

washed 3X with autoclaved deionized water.



Callus tissue was initiated from germinating

seeds that cultured on the surface of solid

Gamborgs B5 medium with vitamins (Gamborg et al., 1968) and supplemented with

1.0 mg/l 2,4D. The medium was also supplemented with 3.0% w/v sucrose in

addition to 0.1% w/v polyvinylpyrrolidone

(PVP) as antioxidant to prevent tissue

browning. Seeds were inoculated on 6.0 cm

Petri dishes in the growth room for six weeks in

full darkness. Induced callus clumps were

transferred to WP media supplemented with 2.0

mg/l TDZ, 0.5 mg/l NAA, and 1.0 g/l PVP.

Five calli pieces (~ 5.0 mm diameter) were

placed on the surface of the media under cold

fluorescent light at 4550 mol.m-2.sec-1. Culture growth conditions were adjusted as

described in Shatnawi et al., (2010).

Total crude extract

Three different sources of plant tissues

were used for the total crude extraction and

later for catechin detection; a) leaves from a

wild tree harvested in mid September 2008, b)

in vitro vegetative parts cultured on WP

medium for four months. The medium was

supplemented with 6.0 mg/l zeatin; c) callus

that have been grown and maintained for 4

months on WP medium supplemented with 2.0

mg/l TDZ + 0.5 mg/l NAA. Plant material was

air dried at room temperature then ground to a

fine powder with mortar and pestle. One gram

of powder was soaked in 100.0 ml ethylacetate

(EtOAc) or 5% methanol. The mixture was

shaked for 48 h at 100 rpm then centrifuged for

15 min at 5000 rpm. The supernatant was

separated then air dried under fume hood. The

percentage yield of the dry extracts was

calculated for both solvents as described by

Alzeer et al., (2014).

Qualitative and quantitative analyses of

catechin

Analytical TLC

For the qualitative detection of phenolic

compounds particularly +catechin, TLC

analysis was performed from the procedure

described in Wagner & Bladt (2009) with the

following modifications; the TLC analysis was

run on a precoated TLC plates (Macherey-

Nagel, Dueren, Germany, Cat# 818133). The

plates were covered with silica gel layer of 0.20

mm, 60 F254 with UV indicator. The mobile

phase composed of 50 ml of chloroform:

acetone: acetic acid at 65:21.5:13.5% v/v/v.

TLC plates were run for three consecutive

times in the mobile phase and after each run the

plates were air dried. The detection of phenolic

compounds was visualized with a UV lamp at

254 nm. Finally, Plates were sprayed with

FeCl3 solution (1.0 g of FeCl3 dissolved in

100.0 ml water:methanol at 50:50% v/v for

visualization of total phenolic compounds.

Retention factor (Rf) value for catechin was

measured by using the formula:

moves solventdistance

moves spotdistance = R f

Analytical HPLC:

HPLC analysis was conducted in the Center

for Chemical and Biological Analyses at Al-

Quds University. The HPLC setting as

described in Bramati et al., (2002) was

followed; quantitative analysis of catechin was

performed on HPLC (Alliance, Waters 2692

separation module) using the analytical column

RP18 Waters Symmetry Shield TM

, (5.0 m,

4.6250 nm). Samples for the analysis were

prepared by dissolving 100.0 mg of the dried

ethanol extract obtained from in vitro, wild

material leaves, and callus in 0.7 ml HPLC-

grade 8% methanol. Total run time was

adjusted to 21 min using the following gradient

elution, 98% A, 2% B (019) min, 80%A, 20%B at 20 min, then back to 98% A, 2% B at

21 min, A: buffered water 1% H3PO4, B:

acetonitrile. Flow rate was adjusted at 1.0

ml/min, UV detection was adjusted at 280 nm

and the injection volume was 40 l. Serial

reference standard solutions of +catechin (0, 1,

20, 40, 60, 80, and 100 ppm) were prepared by

dissolving catechin in a HPLC-grade 99.9%

methanol to construct the calibration curve.

Percentage yield of catechin from different

sources was calculated in mg per 100 mg of

plant dried material against external catechin

standard using the following equation: W/W%



= (C FVD100%)/W, where C is catechin

concentration in the sample (mg/ml)

extrapolated from the calibration curve linear

regression, FV is the final volume of the

sample in milliliters, D is the dilution factor,

and W is the sample weight in milligrams.

RESULTS

In vitro seed germination

High germination percentage (70%)

followed by callus growth was observed after

the seeds have been pretreated with GA3 and

cultured on B5 medium supplemented with 1.0

mg/l 2,4-D. Callus from germinated seeds,

cotyledons and roots were successfully grown

on WP medium supplemented with 2.0 mg/l

TDZ, 0.5 mg/l NAA and 1.0 g/l PVP (Figure

1).

Crude extract yield

One gram of dried leaves powder from wild growing tree, leaves from in vitro plants

and dried callus was immersed in EtOAc or in

5% methanol in order to collect total crude

extracts. Results indicate higher yield of crude

extract in the in vitro grown tissues than the

wild material or from callus tissue (Table 1).

Furthermore, the EtOAc fraction was almost

double amount than the 5% methanol fraction

in the three tissues used (Table 1). For the TLC

and HPLC analysis the fraction derived from

EtOAc was used.

Figure 1. Callus induction after six weeks from (a) cotyledons,(b) seeds, and (c) roots on B5

media supplemented with 1.0 mg/l 2,4-D under full dark, (d) subcultured callus on WP media

with 2.0 mg/l TDZ and 0.5 mg/l NAA under light condition.

Table 1. Yield percentage of crude extract in 1.0 g of dried callus from different tissues.

Catechin yield in EtOA fraction calculated by HPLC analysis.

Type of explant EtOAc Methanol

5%

Catechin % in mg/100g

In vitro leaves 43.3 a 17.7

a 2.5

a

Wild plant leaves 28.3b 13.4

b 0.5

b

Seeds derived callus 20.0c 10.0

c 0.063

c

Figures with different letters are statistically different at p



Qualitative and quantitative detection of

catechin

TLC analysis of catechin:

Extracts derived from wild tree leaves, in

vitro growing vegetative parts and callus tissue

were investigated by TLC analysis for the

detection of catechin. Catechin in the 5%

methanol fraction was not detected whereas

significant amount was revealed in the EtoAC

extract particularly in both in vitro and wild

material. Catechin identification was first

visualized with a UV lamp then stained with

FeCl3 solution. FeCl3 provided an excellent

mean to selectively visualize phenolic

flavonoids (Yadav & Agarwala, 2011). The Rf

values of the major spot in wild and in vitro

leaves were dominantly observed in accordance

to the reference values of catechin at Rf = 0.4

(Figure 2). Slightly less polar compounds were

also observed at lower Rf values. According to

the results revealed by TLC analyses, extract

from callus tissue of A. andrachne did not show

any traces of catechin (Figure 2, lane 5). After

three consecutive runs of the TLC plate, mix

spot was not separated during the three runs.

This clearly indicates the presence catechin

from in vitro leaves match well with reference

catechin (Figure 2). Catechin from in vitro

material showed darker and higher

concentration spot than the wild material.

HPLC analysis of catechin

HPLC analysis was carried out on the

EtOAc extract of in vitro, wild plant leaves and

callus of A. andrachne. Under the

chromatographic conditions described in the

methodology, the retention time of catechin

was 12.563 to 12.615 min. The chromatograms

for catechin in the wild leaves, in vitro

vegetative tissues and callus extract of A.

andrachne are shown in (Figure 3, 4 and 5).

Percentage yield of catechin from different

sources was calculated in mg per 100 mg of

plant dried material against external catechin

standard (Table 1).

Figure 2. TLC plate spotted with 3.0 l of EtOAc extract from different sources of

A. andrachne after being sprayed with FeCl3.

1.0 l of (R) Catechin standard, (1) in vitro grown A. andrachne, (2) mix 1 and R, (3) in vivo grown A. andrachne, (4)

mix 3 and R, (5) callus extract of A. andrachne and (6) mix 5 and R.



Figure 3. HPLC chromatogram for catechin detection in A. andrachne in vitrogrown leaves.

Figure 4. HPLC chromatogram for catechin detection in the leaves of wild grown A.

andrachne plant.

Figure 5. HPLC chromatogram for catechin detection in A. andrachne callus extract.



Results indicate congruency between TLC

and HPLC analysis. The revealed TLC pattern

indicated catechin in both wild and in vitro

material with a higher concentration regarding

the in vitro grown material. TLC could not

reflect any traces of catechin in callus tissue,

however, the HPLC analysis of callus extract

showed catechin at a retention time of

12.563 min (Figure 5). Furthermore, HPLC

chromatograms showed increase in

concentrations of other chemical compounds

from in vitro grown material at the retention

time of 7.816, 8.295 and 15.507 min. The

presence of catechin in A. andrachne grown

under in vitro conditions indicates that the plant

tends to produces catechin under controlled

environment in higher concentration than the

wild material of September harvest.

DISCUSSION

Based on a recent literature review, this is

the first study that addressed the detection of a

flavonoid compounds in the in vitro grown

tissues of A. andrachne. Our results indicated

that this plant species, if grown under in vitro

conditions, produces higher concentration of

catechin compared to the wild growing plant.

In studies on other medicinal plants, higher

secondary metabolites content was detected in

the in vitro growing material compared to wild

ones (Arafeh et al., 2006); Narula et al. 2004).

Higher yield of volatile oils (camphor and

borneol) were detected in Salvia fruticosa in

vitro microshoots more than greenhouse

growing plants (Arikat et al., 2004). Karam et

al. (2003) also reported that the yield of

rosmarinic acid in the in vitro grown S.

fruticosa was higher (2.15.1 mg/100 mg dry weight) than in the leaves or roots (0.21 or 0.72

mg/100 mg dry weight, respectively) of

greenhouse-grown plants.

It is well documented in literature that

under controlled environment some plants tend

to produce higher yield of secondary

metabolites than their naturally growing

counterparts. Example is in the study of Arikat,

et al. (2004) and the review of Karuppusamy,

(2009) and Hussain et al. (2012). The likely

reason behind the difference was attributed to

the presence of plant growth regulators and

elicitors and precursors of the secondary

metabolites in the growth media.

Mostafa et al., (2010) studied the arbutin (a

hydroquinone found in leaves of A. andrachne)

content and compared the yield between in

vitro-grown material and samples from wild

plant material collected in August, October and

December. They reported higher arbutin yield

in the three wild samples compared to the in

vitro-grown one. In our work as revealed by

HPLC analysis, the catechin content in the in

vitro growing material was nearly three times

more than the wild grown material. Some

plants tend to accumulate certain secondary

metabolites when planted under in vitro

conditions in higher quantities than the wild

material, examples were documented in

Hashimoto et al. (1993).

Seeds treated prior to the in vitro culture

showed comparable results (70% germination)

with the finding of Mostafa et al. (2010) (at

least 80%), however, in their work, they used

agar-gelled water supplemented with GA3 at

2.0 mg/l.

In the present study, the total catechin

content measured in the in vitro vegetative

parts was 2.5 mg/100 mg, which is five times

more than the material in the wild

0.5 mg/100 mg and 40 times in callus tissue.

This catechin concentration in callus was not

surprising since callus is undifferentiated mass

of cells and it is proved that secondary

metabolism in plant cells is tightly linked to its

differentiation state. When cells are completely

undifferentiated, secondary metabolite

pathways are partially shut off. A similar

conclusion is observed in the in vivo leaf

tissues of tobacco, when they are differentiated

cells they are able to synthesis enzymes like

chalcone synthase which is involved in

flavonoids biosynthesis pathway. However,

callus generated from the leaf tissues, which

contains partially or totally undifferentiated

cells are unable to produce as much of this



enzyme like their growing tissues in natural

conditions.

CONCLUSION

This study demonstrated that in vitro

growing tissues particularly callus,

accumulated the antioxidant flavonoid catechin.

This would eventually decrease the pressure on

a threatened wild plant in nature and provide

new alternative source for secondary

metabolites production via in vitro culture

techniques.

ACKNOWLEDGEMENTS

We would like to thank the Deanship of

Graduate Studies and Scientific Research for its

support in this project. We also would like to

thank Dr. Muhannad Quree from the Faculty of Pharmacy at Al-Quds University for helping

in the HPLC analysis and Mr. Zaid Al-Taradeh

for his kind technical help in the laboratory.

REFERENCES

Al-Hanbali, M., Ali, D., Bustami, M., Abdel-

Malek, S., Al-Hanbali, R., Alhussainy,

T., et al. (2009). Epicatechin suppresses

IL-6, IL-8 and enhances IL-10

production with NF-kappaB nuclear

translocation in whole blood stimulated

system. Neuro Endocrinol Lett, 30 (1),

131138.

Alzeer, J., Vummidi, B, R., Arafeh, R.,

Rimawi, W., Saleem, H. & Luedtke, N.

W. (2014). The influence of extraction

solvents on the anticancer activities of

Palestinian medicinal plants. Journal of

Medicinal Plant Research. 8, (8), 408415,

Arafeh R.M, Shibli R.A, Mahmoud M.A, &

M.A, S. (2006). Callusing, cell

suspension culture and secondary

metabolites production in Persian

oregano (Origanum vulgare L.) and

oregano (O. syriacum L.). Jordan J

Agric Sci, 2, 274282.

Arikat, N. A., Jawad, F. M., Karam, N. S., &

Shibli, R. A. (2004). Micropropagation

and accumulation of essential oils in

wild sage (Salvia fruticosa Mill.).

Sientia Hort, 100, 193202.

Bertsouklis, K. F., & Papafotiou, M. (2009). In

vitro propagation of Arbutus andrachne

L. . Acta Hortic. , 813, 477480

Bramati, L., Minoggio, M., Simonetti, P.,

Mauri, P., & Pietta, P. (2002).

Quantitative Characterization of

Flavonoid Compounds in Rooibos Tea

(Aspalathus linearis) by LCUV/DAD. Journal of Agricultural and Food

Chemistry, 50 (20), 55135519.

Eshtayeh M.S, & Jamous, R. M. (2002). The

BERC red list of threatened plants of

the West Bank and Gaza Strip. BERC

publications.

Gamborg O.L, Miller R.A, & K, O. (1968).

Nutrient requirements of suspension

culture of soybean root cells.

Experimental Cell Research, 50, 15158.

Hashimoto, T., Yun, D.J, & Yamada, Y.

(1993). Production of tropane alkaloids

in genetically engineered root cultures.

Pyhtochemistry, 32, 713718.

Hussain, M. S., Fareed, S., Ansari, S., Rahman,

M. A., Ahmad, I. Z., & Saeed, M.

(2012). Current approaches toward

production of secondary plant

metabolites. J Pharm Bioallied Sci,

4(1), 1020.

Iacopini, P., Baldi, M., Storchi, P., &

Sebastiani, L. (2008). Catechin,

epicatechin, quercetin, rutin and

resveratrol in red grape: Content, in

vitro antioxidant activity and



interactions. Journal of Food

Composition and Analysis, 21, 589598.

Issa, R., Afifi, F., & Amro, B. (2008). Studying

the anti-tyrosinase effect of Arbutus

andrachne L. extracts. Int. J. Cosmet.

Sci, (30), 271276.

Ivanov, S. A., Nomura, A., Malfanov, I. L.,

Sklyar, I. V., & Ptitsyn, L. R. (2011).

Isolation of a novel catechin from

Bergenia rhizomes that has pronounced

lipase-inhibiting and antioxidative

properties. Fitoterapia, (82), 212218.

Karam N.S., Fawzia M.J., Arikat, N. A., &

Shibli, R. A. (2003). Growth and

rosmarinic acid accumulation in callus,

cell suspension, and root cultures of

wild Salvia fruticosa. Plant Cell, Tissue

and Organ Culture, (73), 117121.

Karam, N. S., & Al-Salem, M. M. (2001).

Breaking dormancy in Arbutus

andrachne L. seeds by stratification and

gibberellic acid. Seed Science and

Technology, 29, 5156.

Karuppusamy, S. (2009). A review on trends in

production of secondary metabolites

from higher plants by in vitro tissue,

organ and cell cultures. Journal of

Medicinal Plants Research, 3(13),

12221239.

Kose, H. (1998). Studies on the germination of

some woody ornemental plants existing

in Turkish Flora. 1. Arbutus unedo L.

and Arbutus andrachne L. Anadolu. .

Journal of Aegean Agricultural

Research Istitute, 8, 5565.

Mostafa S.E., Karam N.S., Shibli R. A., &

Alali, F. Q. (2010). Micropropagation

and production of arbutin in oriental

strawberry tree (Arbutus andrachne L.).

Plant Cell Tissue and Organ Culture,

103(111121).

Narula, A., Kumar, S., Bansal, K. C., &

Srivastava, P. S. (2004).

Biotechnological approaches towards

improvement of medicinal plants. In:

Srivastava, P. S., Narula, A., Srivastava,

S. eds. Plant Biotechnology and

Molecular Markers. Anamaya

Publishers. New Delhi., 78116.

ROTEM. (2002). Constructing Red Number for

endangered plant species. Israel flora as

a test case.

Ruidavets, J. B., Teissedre, P. L., Ferrie`res, J.,

Carando, S., Bougard, G., & Cabanis, J.

C. (2000). Catechin in the

Mediterranean diet: vegetable, fruit or

wine?. Atherosclerosis, 153(107117).

Said, O., Khalil, K., Fulder S., & Azaizeh, H.

(2002). Ethnopharmacological survey

of medicinal herbs in Israel, the Golan

Heights and the West Bank region.

Journal of Ethnopharmacology, 83,

251265.

Sakar, M. K., Berkman, M. Z., Calis, I., &

Ruedi, P. (1991). Constituents of

Arbutus andrachne L. Fitoterapia,

62(2), 176177.

Serce, S., Ozgen M, Torun, A.A, & Ercis, L.S.

(2010). Chemical composition,

antioxidant activities and total phenolic

content of Arbutus andrachne L. (Fam.

Ericaceae) (the Greek strawberry tree)

fruits from Turkey. Journal of Food

Composition Analysis, 23(6), 619623.

Shatnawi, M., Al-Fauri, A., Megdadi, H., Al-

Shatnawi, M.K., Shibli, R., Abu-

Romman, S. & Al-Ghzawi, A. (2010).

In Vitro multiplication of

Chrysanthemum morifolium Ramat and

it is responses to NaCl induced salinity.

Jordan Journal of Biological Sciences,

3, (3), 101110.



Suzuki, T., Someya, S., Hu, F., & M., T.

(2005). Comparative study of catechin

compositions in five Japanese

persimmons (Diospyros kaki). . Food

Chemistry, 93, 149152.

Tilki, F., & Guner, S. (2007). Seed germination

of three provenances of Arbutus

andrachne L in response to different

pretreatments, temperature and light.

Propagation of Ornamental Plants 7(4),

175179.

Yadav, R. N. S., & Agarwala, M. (2011).

Phytochemical analysis of some

medicinal plants. Journal of Phytology,

3, 1014.

Wagner H, & Bladt S, (2009). Plant Drug

Analysis: A Thin Layer

Chromatography Atlas. 2nd

ed.,

Springer-Verlag, Berlin Heidelberg.

Zaveri, N. T. (2006). Green tea and its

polyphenolic catechins: Medicinal uses

in cancer and noncancer applications.

Life Sciences, 78, 20732080.

Source of Support: Deanship of Graduate

Studies and Scientific Research, Palestine

Conflict of Interest: None Declared




ANDROGRAPHIS PANICULATA A TRADITIONAL HERB WITH

PHARMACOLOGICAL PROPERTIES: A REVIEW

Nishan Chatterjee1, Sunipa Biswas

2, Nimai Chandra Saha

3, Surjyo Jyoti Biswas

4*

1,2,4Post Graduate Department of Zoology, Midnapore College, Midnapore, West Bengal, India-721101.

3Education Directorate, Government of West Bengal, Bikash Bhavan, Salt Lake, Kolkata, West Bengal India-

700091.

*Corresponding author: E-mail: [email protected]


ABSTRACT

Andrographis paniculata (Kalmegh) is herb belonging to family Acanthaceae and cultivated

widely in India. This plant is known for its wide range of pharmacological properties and various

traditional uses. Considerable efforts are being made by various scientists to validate its utility

through scientific and pharmacological screening. The reported biological activities are antibacterial,

antifungal, antiviral, anthelmintic, anticancer, hyperglycemic, anti-inflammatory, antivenomic,

antiasthmatic, hepatoprotective and used to treat cold and fever. This review intends to integrate

traditional knowledge, summarizes modern scientific findings and suggests areas where further

research can be conducted.

KEYWORDS: Andrographis paniculata, Pharmacological properties, Kalmegh

Review Article

Cite this article:

Nishan Chatterjee, Sunipa Biswas, Nimai Chandra Saha, Surjyo Jyoti Biswas (2014),

ANDROGRAPHIS PANICULATA A TRADITIONAL HERB WITH PHARMACOLOGICAL

PROPERTIES: A REVIEW, Global J Res. Med. Plants & Indigen. Med., Volume 3(5): 206214



INTRODUCTION

Andrographis paniculata (AP) commonly

known as Kalmegha in Hindi, Kalamegha in

Sanskrit and Kalmegh in Bengali is an erect

herb belonging to family Acanthaceae which

grows in many South east Asian countries and

in India. Other vernacular names of AP have

been listed in table 1. The plant is highly

praised for its therapeutic potential in Indian

phytotherapy and traditional medicine. Both

crude and alcoholic extracts of AP have been

reported to have wide variety of

pharmacological activities viz. antibacterial,

antifungal, antiviral, anthelmintic, anticancer,

hyperglycemic, anti-inflammatory,

antivenomic, in alleviation of upper respiratory

tract infections, hepatoprotective, preventive

effects against cold (Dey et al., 2013, Datta et

al., 2012, Coon and Ernst, 2004, Akbar, 2011).

The herb grows upto 34 feet in height, the leaves are lanceolate and 23 inches long. The

flowers are small, solitary and flowering time is

from September to December. Traditionally the

leaves of this herb are used for bronchitis,

worm infestation, influenza and dyspepsia. The

expressed juice of the leaves is a domestic

remedy in flatulence and diarrhoea. This plant

is also known as King of bitters which was used for centuries both in Indian and Chinese

systems of medicines where both fresh and

dried leaves and roots were used as folkloric

medicines and traditional household remedies.

Traditionally this plant was used as powder,

raw juice, decoction either singly or in

combination with other plant extracts for

various types of ailments. There is need for

critical evaluation since few scientists have

reported side effects of AP (Akbar, 2011). This

review is intended to give a view mainly on the

biological activities of AP, the compounds

isolated, their pharmacological properties,

clinical and laboratory investigations and their

safety evaluation.

Table 1: Vernacular names of Andrographis paniculata

Phytochemical studies:

It has been reported by various

investigators that AP contains lactones,

diterpenes, alkanes, ketones and aldehydes and

flavonoids. Though flavonoids are mainly

found in roots but the aerial parts are

predominant in alkanes, aldehydes and ketones.

The intense bitter taste of the leaves is due to

presence of large amounts of kalmeghin and

andrographolide. Deoxyandrographolide, 19-

Sl.No. Vernacular Names

1. Sanskrit: Bhunimba

2. Hindi: Kirayat

3. Bengali: Kalmegh

4. Telugu: Naelavemu

5. Tamil: Nilavaembu, Siriyanangai

6. Oriya: Bhuinimba

7. Chinese ChuanxinLian

8. Kannada Nelabevu

9. Thai FaThalai Chon

10. Malay HempeduBumi

11. Punjabi Chooraita



D-glucoside has been isolated from leaves of

AP (Weiming and Xiaotian, 1982). 12 new

flavonoids and 14 new diterpenoids have been

isolated from the aerial parts of AP (Chen et

al., 2006a, Chen et al., 2006b). Li et al., (2007)

isolated andrographic acid which is a new

diterpenoid and two new ent-labdane

diterpenoid glycosides from the aerial parts of

the plant. On the other hand 1, 8-dihydroxy-3,

7-dimethoxyxanthone, 4,-8-dihydroxy-2, 7-

dimethoxyxanthone, 1, 2-dihydroxy-6,8

dimethoxyxanthone and 3-7- 8 trimethoxy-1-

hydroxyxanthone were isolated from roots (

Dua et al., 2004, Akbar, 2011).

Medical formulations

There are many formulations commercially

available in the market both nationally and

internationally, where the major constituent is

Andrographolide. They are Bhunimbadi khada,

kalansundarras, kalpataruras, Andrographis

200 and 400, Liv 52, andrographis 60V caps,

Ilogen excel etc (www.iherbs.com,

www.ayurvedicherbs.com,

www.paradiseherbs.com).

Pharmacological activities

Antimicrobial effect:

It has been experimentally proved that

crude powder from the aerial parts of the plant

shows no antimicrobial activity but its aqueous extract of leaves exhibit significant

antimicrobial activity against Gram positive S.

aureus, and Gram negative Pseudomonas

aeruginosa. Mishra et al., (2009) found that the

IC50 of methanol extract was 7.2microgram/ml

against Plasmodium falciparum.

Andrographolide, neoandrographolide, 14-

deoxy-11, 12-didehydro andrographolide

showed antiviral activity against herpes

simplex virus or HSV1 (Wiart et al., 2005).

Antiprotozoal and antihelmenthic property

Dua et al., (2009) reported antiprotozoal

activity of some xanthones isolated from roots

of AP, xanthones reduced the growth of T.

brucei, T. cruzi and L. infantum. Further, it has

been reported by other workers that water

extract of the leaves of AP showed significant

filaricidal properties in canines (Dutta and

Sukul 1982). Kaleysa 1975 reported significant

anti-helminthic action of AP against Ascaris

lumbricoides.

Immunomodulatory properties

Puri et al., (1993), reported several

immuno-stimulatory agents from AP.

According to Puri intragastric administration of

ethanolic extract of aerial parts of AP at a rate

of 1 mg/kg body weight to mice stimulated

antibody production, it was also responsible for

delayed hypersensitivity reaction in sheep red

blood cells. The whole extract of AP was more

effective than andrographolide or neo

andrographolide alone suggesting other

constituents may involve in the immuno-

stimulant response/process. Sheeja et al.,

(2006) showed that administration of

cyclophosphamide increased cytokine TNF which was considerably reduced with

administration of AP extract.

Anti-inflammatory activities

Chiou et al., (2000) reported suppression of

inducible NO synthase (iNOS) by

andrographolide. Other investigators reported

inhibitory effects of neoandrographolide on

prostaglandin E2 and NO production in LPS

stimulated murine macrophage (Liu et al.,

2007a, Liu et al., 2007b, Abu-Ghefreh et al.,

2009). Suppression of NO production in

activated macrophages in vitro and ex vivo by

neoandrographolide isolated from AP was also

reported by Batkhuu et al., (2002). Methanolic

extract of this plant inhibited formation of ROS

(reactive oxygen species) which completely

inhibited carrageenan induced inflammation as

reported by Sheeja et al., (2006).

Antioxidant properties

Since this plant contains higher flavonoid

and phenolic content, this might attribute to its

antioxidant capabilities. It has been reported

that suppression of rat neutrophil ROS

production by diterpenoid lactone

andrographolide (Shen et al., 2000, Shen et al.,

2002). Verma and Vinayak (2008) reported



antioxidant action of AP on lymphoma. It has

been reported that activities of mitochondrial

electron transport chain complexes were

decreased in different brain parts by

pretreatment with water and ethanolic extracts

of AP (Das et al., 2009). Ojha (2009) reported

antioxidant activity of AP in ischemic

myocardium of rats. It might be that the

neoandrographolide present in the plant extract

take up the free oxygen radical due to the

donating activity of allylic hydrogen of the

unsaturated lactone ring.

Effects on Reproductive system

The therapeutic efficacy of AP needs

critical evaluation because there are many

reports that the extract of this plant affects the

reproductive system. Some studies reported

that AP extract may be used as an antifertility

agent. When dry leaf powder were

administered to male albino rats there was a

decrease in spermatogenesis, increased

abnormal sperm head morphology, decreased

sperm motility, regression of cells of Leydig,

degeneration of epididymis and prostrate

(Akbarsha et al., 1990). It has also been

reported that intra-peritoneal injection of aerial

parts of AP to female albino mice caused

abortion and also it prevented implantation of

embryos. Panossian et al., (1999) reported that

treatment with extract of AP did not alter

progesterone levels in pregnant rats. Since

experimental investigations contradicted each

other there is ample scope of research in this

direction.

Cardiovascular activity

The aqueous extract of AP l

1GJRMI - VOlume 3, Issue 5, May 2014

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