1GJRMI - VOlume 3, Issue 5, May 2014

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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
MI NI MA 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 I N VI TRO CULTURES OF THE EASTERN
STRAWBERRY TREE, ARBUTUS ANDRACHNE L., AN ENDANGERED MEDICINAL TREE IN
PALESTINE
Zahra Aljabari, Jawad Alzeer, Rami Arafeh

196205
Review Article
ANDROGRAPHI S PANI CULATA 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 HARI TAKI LEAVES
(TERMI NALI A 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 CHI RABI LWA (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 & I ndigen. Med. | Volume 3, Issue 5 | May 2014 | 184195

Global J ournal of Research on Medicinal Plants & I ndigenous Medicine || GJRMI ||

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

ANTIBACTERIAL, ANTI-SWARMING POTENTIAL OF ETHANOL
EXTRACTS OF PHYSALI S MI NI MA L. WHOLE PLANT AND URENA
LOBATA L. ROOT ON CEPHALOSPORIN RESISTANT PROTEUS SPECIES

Mamunur Roshid
1
, Aktar Uzzaman Chouduri
2
*

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

*Corresponding author: Email: auchow5@yahoo.com, auchow5.pharm@ru.ac.bd;
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), 66
1
(Pp), 91
1
(Pm), and 91
2
(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 & I ndigen. 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), 66
1
(Pp), 66
2
(Ph), 66
3
(Pp),
66
4
(Pp), 66
5
(Pp), 66
6
(Pp), 66
7
(Pp), 66
8
(Pp),
91
1
(Pm) and 91
2
(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 66
2
(Ph): 13 mm (Ul-r), 21 mm (Pm-w); in 66
6
(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: 91
1
(Pm), B: 91
2
(Pm), C: 66
2
(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), 66
1
(Pp), 91
1
(Pm), and 91
2
(Pm)
were found to be resistant to NHS (unpublished
data). Isolates 91
2
(Pm), 91
1
(Pm) and 66
2
(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
66
1
(Pp) 150.3 140.4 220.5 170.1 210.3
66
2
(Ph) 80.2 130.3 210.3 140.9 130.3 150.9
66
3
(Pp) 90.4 100.5 120.3 120.2 140.5
66
4
(Pp) 130.3 170.5 180.6 150.4 190.3
66
5
(Pp) 90.5 110.7 130.5 130.4 150.7
66
6
(Pp) 120.6 121.1 290.4 150.5 70.3 230.1 100.4 130.6
66
7
(Pp) 110.4 80.2 151.1 150.3 80.4 180.6 70.4
66
8
(Pp) 120.6 140.6 180.4 140.5 270.5
91
1
(Pm) 80.4 111.0 150.2 150.8 140.6
91
2
(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
91
1
(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
91
2
(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
66
2
(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.

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Source of Support: National Science and
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Conflict of Interest: None Declared



CATECHIN DETECTION IN CALLUS AND I N VI TRO 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.
2
Department of Applied Chemistry, Faculty of Applied Sciences. Palestine Polytechnic University, P.O.Box
198 Hebron, Palestine.
*Corresponding author: E-mail: arafeh@ppu.edu; 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 I N VI TRO CULTURES OF THE EASTERN STRAWBERRY TREE, ARBUTUS
ANDRACHNE L., AN ENDANGERED MEDICINAL TREE IN PALESTINE, Global J Res.
Med. Plants & I ndigen. 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.
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 GA
3
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 F
254
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
FeCl
3
solution (1.0 g of FeCl
3
dissolved in
100.0 ml water:methanol at 50:50% v/v for
visualization of total phenolic compounds.
Retention factor (R
f
) value for catechin was
measured by using the formula:
moves solvent distance
moves spot distance
=
Rf

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% H
3
PO
4
,

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 GA
3
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
I n vitro leaves 43.3
a
17.7
a
2.5
a

Wild plant leaves 28.3
b
13.4
b
0.5
b

Seeds derived callus 20.0
c
10.0
c
0.063
c

Figures with different letters are statistically different at p<0.05.


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
FeCl
3
solution. FeCl
3
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. andrachneafter being sprayed with FeCl
3
.

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.
andrachneplant.

Figure 5. HPLC chromatogram for catechin detection in A. andrachnecallus 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 GA
3
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.

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Source of Support: Deanship of Graduate
Studies and Scientific Research, Palestine
Conflict of Interest: None Declared



ANDROGRAPHI S PANI CULATA A TRADITIONAL HERB WITH
PHARMACOLOGICAL PROPERTIES: A REVIEW
Nishan Chatterjee
1
, Sunipa Biswas
2
, Nimai Chandra Saha
3
, Surjyo Jyoti Biswas
4*

1,2,4
Post Graduate Department of Zoology, Midnapore College, Midnapore, West Bengal, India-721101.
3
Education Directorate, Government of West Bengal, Bikash Bhavan, Salt Lake, Kolkata, West Bengal India-
700091.
*Corresponding author: E-mail: surjyo@rediffmail.com
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),
ANDROGRAPHI S PANI CULATA A TRADITIONAL HERB WITH PHARMACOLOGICAL
PROPERTIES: A REVIEW, Global J Res. Med. Plants & I ndigen. 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 lowers the
systolic blood pressure of spontaneously
hypertensive rats possibly by reducing
circulating angiotensin converting enzyme in
the plasma as well as by reducing free radical
levels in the kidneys (Zhang and Tan 1996). A
hypotensive activity of AP in rats was also
reported by Yu et al., (2003). Further, studies
by Zhang et al., (1998) and Zhang and Tan
(1997) on cardiovascular activity of 14-
deoxyandrographolide and 14-deoxy-11, 12-
didehydroandrographolide revealed that AP
induced relaxation of the isolated rat thoracic
aortae. They also reported that 14-
deoxyandrographolide and 14-deoxy-11,12-
didehydroandrographolide significantly
decreased the mean arterial pressure and heart
rate of anaesthetized rats.
Antivenomic activities
It has been reported by Chang and But
(1987) that 10 cases of viper bites were cured
within 3-5 days by a formula which has AP as
primary constituent. It has been demonstrated
by Premendran et al., (2011) that AP has cobra
venom neutralizing activity at dose of 2 g/kg.
Further, methanolic extracts of AP has potent
Daboia russelli venom neutralizing activity and
could be used as snakebite evenomation
(Meenatchisundaram et al., 2009). Kale et al.,
2013 reported anti-scorpion venom activity of
AP when treated with 1g/Kg dose.
Effect on respiratory infections
Upon literature survey it was revealed that
there were contradictory reports of AP
administration against respiratory infections.
Thamlikitkul et al., (1991) administered AP
extract to cure cough and sore throat and
compared it with paracetamol administration;
they found that 6g AP powder administration
for 3 days reduced sore throat and fever
considerably. In a double blind study conducted
by Caceres et al., (1997) revealed that common
cold was prevented by 4% andrographolide.
Effect on hepatic enzymes
A crude extract of A. paniculata was
recently found to induce hepatic CYP1A1 and
CYP2B expression, based on observations of
significant increases in ethoxyresorufin O-
dealkylase (EROD) activity and
pentoxyresorufinO-dealkylase activities
(Jarukamjorn et al.,2006). Interestingly,
andrographolide plus typical CYP1A inducers,
including-naphthoflavone, TCDD, and
benz[a]anthracene, synergistically induced
CYP1A1 expression in mouse hepatocytes in
primary culture, and the synergism was blocked
by an AhR antagonist, resveratrol
(Jaruchotikamol et al.,2007).


Antidiabetic activity
It has been reported by Umamaheswari et
al., (2009) that oral administration of an
ayurvedic formulation of AP considerably
reduces streptozotocin induced diabetes
mellitus. Further ethanolic leaf extract of this
plant reduces oxidative stress and also have
antihyperglycaemic properties. The
antihyperglycaemic properties have been
attributed to andrographolide which is present
in leaves of AP in high amounts (Yu et al.,
2003). Antidiabetic property of aqueous extract
of AP was further confirmed by Borhanuddin et
al., (1994) and Husen et al., (2004).
Insecticidal activity
The ethanolic extracts of AP have been
reported to have insecticidal property which
was mainly attributed to andrographolides,
homoandrographolide and andrographin. A
study conducted by Elango et al., (2010)
revealed that hexane fractions of AP have
potent larvicidal, ovicidal and insect repellent
properties.
Anticancerous property
Zhou et al., (2006) reported that
andrographolide induced cell death which is
proapoptotic via Bcl2. Andrographolide
induced DNA fragmentation and enhanced
percentage of apoptotic cells has been reported
by Harjotaruno et al., (2007), in TD-47 human
breast cancer cell line by increasing expression
of p53, Bax and caspase 3 in a concentration
dependent manner and also lowering the
expression of Bcl2. Satyanarayana et al.,
(2004) reported cell cycle inhibition in MCF-7
cells which are human breast cancer cell line by
decreasing the expression of p27 and also
cyclin dependent kinase. Zhou et al., (2010)
demonstrated that Andrographolide is able to
significantly suppress both constitutively
activated and IL-6-induced STAT3
phosphorylation and subsequent nuclear
translocation in cancer cells. Such inhibition is
found to be achieved through suppression of
Janus activated kinase (JAK)1/2 and interaction
between STAT3 and gp130. They also
investigated the effect of andrographolide on
doxorubicin-induced apoptosis in human
cancer cells. Their observation revealed that
andrographolide could be a potential candidate
for treatment of cancer with combination with
other chemotherapeutic agents.
Adverse health effects
It has been reported that high doses of AP
extract causes gastric discomfort, vomiting,
head reeling and anaphylactic effects. When
being administered with anticoagulants, there
may be increased risk of bruising and bleeding
since Andrographis itself inhibits the platelet
aggregation and prevents blood clots
(Calabrese et al., 2000).
CONCLUSION
Kalmegh was used in Indian systems of
medicines for centuries. Literature survey
showed that extensive works has already been
done worldwide with regard to its efficacy
against various types of ailments. However it
would be prudent to investigate its constituents
singly and in combination against various other
diseased states, how they modulate
pathological changes and which form is more
potent or effective in treating disease. AP
showed consistent hepatoprotective effects in
animal models against various types of induced
hepatotoxicity however inconsistency was
found against bacterial investigations. This
might be due to collection of plant materials at
different time intervals, place of collection,
seasonal variation, different extraction
procedures, and its storage which might affect
its active compounds both quantitatively and
qualitatively. Antihyperglycemic activity of AP
extracts (both water and alcohol) was found to
be even better than commonly available
antidiabetic drugs in animal models. Further, it
shows a profound impact on blood pressure
thereby making it a good candidate for
understanding of its constituents on blood
pressure and its regulation. Effect of AP
extracts on reproductive system has been
carried out in animal model by some
investigators however till date its efficacy on
human reproductive system or on nervous


system has not been carried out, so focus can
be generated on these aspects for better drug
development. The information summarizes here
concerning Andrographis paniculata is
intended to serve as a reference and update
researchers with adequate information involved
in ethno-pharmacological research.
ACKNOWLEDGEMENT

Grateful acknowledgements are made to
Professor A. R Khuda-Bukhsh, Department of
Zoology, University of Kalyani and Dr. Prabir
De, Scientist, CCMB, Hyderabad for
inspiration and advice.

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Source of Support: NIL Conflict of Interest: None Declared


PHARMACOGNOSTICAL EVALUATION ON TANNIN CONTENT IN
HARI TAKI LEAVES (TERMI NALI A CHEBULA RETZ. -COMBRETACEAE)
BEFORE AND AFTER FLOWERING-FRUITING
Patil Sunny C
1
*, Harisha C R
2
, Baghel A S
3
, Dwivedi R R
4

1
PG Scholar, Department of Basic Principles, IPGT & RA, Gujarat Ayurved University, Jamnagar, Gujarat,
India. 361 008

2
Head, Pharmacognosy Lab, IPGT & RA, Gujarat Ayurved University, Jamnagar, Gujarat, India. 361 008

3
Associate Professor, Department of Basic Principles, IPGT & RA, Gujarat Ayurved University, Jamnagar,
Gujarat, India. 361 008

4
Professor and Head, Department of Basic Principles, IPGT & RA, Gujarat Ayurved University, Jamnagar,
Gujarat, India. 361 008
*Corresponding Author: Email: sunnypatil125@gmail.com Mob: +919067965954
ABSTRACT
Haritaki (Terminalia chebula Retz., Fam. Combretaceae) is one of the most abundantly used
plant for various ailments in Ayurvedic classics. In folklore practice, it is used in the treatment of
asthma, hiccough, sore throat, vomiting, bleeding piles, diarrhea, dysentery, heart and bladder
diseases etc. Though whole plant of Haritaki possesses high medicinal value, fruit is the part which
is used most in therapeutics. The medicinal efficacy of Haritaki can be attributed to various chemical
constituents present in it. Tannin is one of these, which is responsible for various therapeutic
properties of Haritaki. The present work is carried out to study the tannin content and its localization
in Haritaki leaves before and after floweringfruiting in which it was observed that leaves of
Haritaki before flowering-fruiting are having more concentration of tannin than after flowering-
fruiting leaves. It was also noted that fruit of Haritaki which is a totally new entity formed in the
plant shows recognizable amount of tannin content in it. This work may provide incentive for proper
evaluation of the chemical constituents which exist there in the leaves and later get transported to the
fruits.
KEY WORDS: Haritaki, Medicinal value, Pharmacognosy, Tannin, Terminalia chebula Retz.

Research Article
Cite this article:
Patil Sunny C, Harisha C R, Baghel A S, Dwivedi R R (2014),
PHARMACOGNOSTICAL EVALUATION ON TANNIN CONTENT IN HARI TAKI
LEAVES (TERMI NALI A CHEBULA RETZ. -COMBRETACEAE) BEFORE AND AFTER
FLOWERING-FRUITING, Global J Res. Med. Plants & I ndigen. Med., Volume 3(5): 215224


INTRODUCTION
Medicinal plants are being used for
combating various human ailments since the
dawn of civilization. Ayurveda is an applied
science related to medicine in which foremost
emphasis has been given to the treatment of
diseases using various medicinal herbs.
Haritaki (Terminalia chebula Retz., Fam.
Combretaceae) is one of the most extensively
used plants described in most of the ancient
classics of Ayurveda. Owing to their natural
origin, cost effectiveness and lesser side
effects, there has been growing interest in
exploiting the biological activities of different
Ayurvedic medicinal herbs since last one or two
decades.
T. chebula has been extensively used not
only in Ayurveda but also in Siddha (S. Aruna
et al., 2014), Unani, Homoeopathic medicine
and has become a cynosure of modern
medicine (Anwesa Bag et al., 2013). Because
of its extraordinary power of healing, it is
known as King of Medicines (Kshirod K.
Gupta et al., 2013) and is always listed at the
top of the list of Ayurvedic Materia Medica.
Therapeutic efficacy of this plant can be
attributed to the large number of different types
of phyto-constituents present in it. It has been
demonstrated to possess multiple
pharmacological and medicinal activities, such
as antioxidant, antimicrobial, antidiabetic,
antiarthritic, anti-inflammatory, antimutagenic,
antiproliferative, hepato-protective, radio-
protective, cardio-protective, gastrointestinal
motility and wound healing activity (Anwesa
Bag et al., 2013). Fruit of T. chebula,
commonly known as Harad in Hindi, is the
part used for medicinal purpose which
possesses diverse health benefits. It has been
used as traditional medicine for household
remedy against various human ailments since
antiquity. The observed health benefits of this
fruit may be credited to the presence of various
phytochemicals like tannin, polyphenols,
terpenes, anthocyanins, flavonoids, alkaloids
and glycosides (Anonymous, 2010).
Tannin is one of the important constituents
responsible for medicinal efficacy of the fruit.
Thus in this study a detailed pharmacognostical
examination of T. chebula leaves before and
after floweringfruiting has been carried out
with the hypothesis that the leaves of Haritaki
before flowering would have more
concentration of tannin than after flowering-
fruiting leaves. This study may offer immense
opportunity for researchers engaged in
validation of the active ingredients present in
the medicinal plant and also contribute to know
the actual plant physiology about the
transportation of secondary metabolites.
Collection and preservation of the sample
Fresh leaves of Terminalia chebula Retz.
were collected from the botanical garden of
Institute for Post Graduate Teaching and
Research in Ayurveda, Gujarat Ayurveda
University (GAU), Jamnagar in the month of
April 2013 and November 2013 before and
after flowering-fruiting respectively as per
collection standards (API, 2001). Fruits were
also collected from the same location. The
plant specimen was authenticated by the
Pharmacognosist, GAU, Jamnagar, India. A
sample specimen was deposited to
Pharmacognosy Lab. (Specimen No.- Phm.
6123/2013) for future references. Leaves and
fruits were separated, shade dried, pulverized,
sieved through 80 no. mesh and preserved in an
air tight glass bottle.
Macroscopic study
Macroscopic characters of leaves before
and after floweringfruiting and those of fruit
were analyzed systematically and their
morphological characters like size, shape etc.
were noted down.

Organoleptic study of dried powders
Dried powders of both the sample leaves
and of fruit were evaluated for organoleptic
characters like texture, taste, odour and colour
etc.
Microscopic study
Free hand transverse sections of leaves
through midrib including its petiole and of fruit
were taken and washed with chloral hydrate
solution. Sections were first observed in
distilled water then stained with phloroglucinol
and concentrated HCl (Khandelwal K.R et al.,
1996).
Histochemical analysis
Histochemical analysis was carried out to
determine the tannin content and its
localization in both the samples of leaves as
well as in fruit (Krishnamurty KV, 1988).
Powder microscopy
Dried powders of leaves and fruit were
studied following standard procedures (Trease
GE et al., 2002). The micro photographs were
taken by Carl zeiss trinocular microscope.
RESULTS
Macroscopical study
Leaves
Macroscopic investigation of both the leaf
samples showed similar characteristics. Leaves
were 1020 cm long, sub-opposite, simple;
exstipulate; petiolate; laminae broadly elliptic
to elliptic-oblong, rarely ovate, the bases
obtuse, the margins entire, the tips acute,
glabrescent, with two large glands at the top of
the petiole. (Plate A, ab)
Fruit
Single seeded drupe of Haritaki was
glabrous, sub globose to ellipsoid in shape,
smooth and golden yellow in colour. It became
ridged, wrinkled and brown when dried. (Plate
A, cd)

Organoleptic study of dried powders of
leaves and fruit
Organoleptic study of powders of both the
leaf samples was carried out to see any
difference in the organoleptic characters of leaf
before and after flowering-fruiting. Characters
of fruit powder were also observed. Results are
tabulated in Table-1.

TABLE 1: Organoleptic characters of dried powders of T. chebula fruit and leaves before and
after flowering-fruiting

Sr.
No.
Parameter Leaf: before
flowering-fruiting
Leaf: after
flowering-fruiting
Fruit
1 Colour Light greenish Light yellowish Yellowish brown
2 Odour Characteristic Characteristic Aromatic, slightly
astringent
3 Taste Astringent Less astringent Astringent, sweet
4 Texture Rough Rough Rough



Microscopical study
Histological and histochemical evaluation of
petiole before flowering-fruiting
T.S of the petiole was more or less circular
in shape, showed epidermis, cortex, vascular
bundles and central pith. (Plate C)
Epidermis was single layered, made up of
barrel shaped cells arranged compactly and
covered with thick cuticle. Hypodermis was
present just beneath the epidermis, made up of
compactly arranged single layered collenchyma
cells. Hypodermal cells were rich in tannin
content filled with brownish material. Cortex
consisted of parenchymatous cortical cells
enriched by chlorophyll pigments and rosette
crystals of calcium oxalate and some of the
cortical cells were filled with brown content

(tannin). Endodermis was single layered,
consisted of some rosette crystals. Continuous
bands of pericyclic fibers were present beneath
the endodermis followed by distinct vascular
bundles. Adjacent cells of the vascular bundles
were found to be greatly filled with tannin
content. Vascular bundles were open and
collateral in which phloem was present beneath
the pericyclic fibers consisting of sieve
elements and phloem fibers while xylem was
radially arranged beneath the phloem along
with xylem parenchyma and xylem fibers.
Centrally located large pith showed presence of
parenchymatous cells showing some starch
grains and also filled with some tannin content.
leaf before flowering-fruiting
T.S of the leaf through mid rib showed
distinct upper and lower epidermis and
centrally located vascular bundles (Plate C).
Upper and lower epidermis was single
layered, covered with the cuticle; lower
epidermis was interrupted by stomata. Beneath
the upper epidermis, 23 layers of pallisade
parenchyma and beneath the lower epidermis
67 layers of spongy parenchyma were present.
Upper epidermis i.e. Pallisade cells were
strongly filled with the tannin content as
compared to the lower spongy parenchyma.
The mesophyll tissue consisted of large
quantity of oil globules and prismatic crystals.
Mesophyll, upper pallisade parenchyma and
lower spongy parenchyma were separated by
vascular trends. Vascular bundles were situated
at the centre of the midrib, made up of phloem
situated towards lower epidermis, xylem
situated towards upper epidermis along with
few elements of xylem parenchyma cells.
Ground tissue of the midrib portion i.e.
parenchyma cells were strongly filled with
tannin content, heavily at adjacent cells of the
vascular bundles. Some of the central pith
parenchyma cells were enriched by tannin
content.

petiole after flowering-fruiting
T.S. of the petiole was more or less circular
in shape, showed epidermis, cortex, vascular
bundles and central pith. (Plate C)
Epidermis was single layered, made up of
barrel shaped cells arranged compactly and
covered with thick cuticle. Hypodermis was
present just beneath the epidermis, made up of
compactly arranged single layered collenchyma
cells. Hypodermal cells were showing less
localization of tannin. Cortex consisted of
parenchymatous cortical cells enriched by
chlorophyll pigments and rosette crystals of
calcium oxalate. Very poor amount of brown
content (tannin) was noted in the cortical cells.
Endodermis was single layered consisting of
some rosette crystals. Continuous bands of
pericyclic fibers were present beneath the
endodermis followed by distinct vascular
bundles. Adjacent cells of the vascular bundles
showed little concentration of tannin content.
Vascular bundles were open and collateral in
which phloem was present beneath the
pericyclic fibers consisting of sieve elements
and phloem fibers, while xylem was radially
arranged beneath the phloem along with xylem
parenchyma and xylem fibers. Centrally
located large pith was having parenchymatous
cells showing some starch grains and also filled
with some tannin content which was not so
prominent.
leaf after flowering-fruiting
T.S of the leaf through mid rib showed
distinct upper and lower epidermis and
centrally located vascular bundles (Plate C).
Upper and lower epidermis was single
layered, covered with the cuticle in which
lower epidermis was interrupted by stomata.
Beneath the upper epidermis 23 layers of
pallisade parenchyma and beneath the lower
epidermis 67 layers of spongy parenchyma
were present. Upper epidermis i.e. pallisade
cells were showing slightly more tannin content
as compared to the lower spongy parenchyma.

The mesophyll tissue consisted large quantity
of oil globules and prismatic crystals.
Mesophyll, upper pallisade parenchyma and
lower spongy parenchyma were separated by
vascular trends. Vascular bundles were situated
at the centre of the midrib, made up of phloem
situated towards lower epidermis, xylem
situated towards upper epidermis along with
few elements of xylem parenchyma cells.
Ground tissue of the midrib portion i.e.
parenchyma cells were with tannin content,
which was not so prominent. Some of the
central pith parenchyma cells were showing
little tannin content.


fruit
Transverse section of the fruit showed the
outer epicarp, middle mesocarp and inner
endocarp. (Plate A, e-f) Outer epicarp was
made up of single layer of rectangular to barrel
shaped epidermal cells compactly arranged
without intercellular spaces with cuticle. On
histochemical evaluation, epidermal cells were
seen filled with the tannin content. Largely
occupied mesocarp cells were made up of
parenchyma cells interrupted by groups of
sclereids, stone cells, fibers and vascular
bundles. Histochemical evaluation also
revealed sclereids and adjacent vascular tissues
i.e. xylem parenchyma and fibers which were
thickly enriched with the tannin content.
Endocarp attached with the seed was showing
cells having somewhat little isolated tannin
content.
Powder microscopy of leaf before flowering-
fruiting
The diagnostic characters of the powder of
leaf before flowering-fruiting revealed
epidermal cells with rich tannin content, more
quantity of tannin in the fragments of pallisade
parenchyma, trichomes heavily filled with
tannin content and lower fragments of spongy
parenchyma cells rich in tannin content. Oil
globules, stomata, prismatic and rosette
crystals, were the other characters noted in the
powder microscopy. (Plates D1 and D2)
Powder microscopy of leaf after flowering-
fruiting
The diagnostic characters of the powder of
leaf after flowering-fruiting revealed epidermal
cells with less tannin content, less quantity of
tannin in the fragments of pallisade
parenchyma, trichomes poorly filled with
tannin content and lower fragments of spongy
parenchyma cells showing somewhat absence
of the tannin content. Oil globules, stomata,
prismatic and rosette crystals, were the other
characters noted in the powder microscopy.
(Plates D1 and D2)
Powder microscopy of fruit
On organoleptic study, fruit powder of
Haritaki showed yellow brown colour,
aromatic and slightly astringent odour,
astringent taste which later ends in sweet and
rough texture.


Diagnostic characters of the powder
revealed presence of sclereids out of which
some were pitted with wide lumen, mesocarp
cells, pitted stone cells with wide lumen, fibers,
tannin contents, simple and compound starch
grains and vascular strands (Plate B).
DISCUSSION
Macroscopic examination of leaves before
and after flowering-fruiting did not show any
prominent difference in their characteristics
while their organoleptic evaluation showed
recognizable difference in the colour and taste
parameters. Histological and histochemical
evaluation of petiole before flowering-fruiting
revealed hypodermal cells, parenchymatous
cortical cells and adjacent cells of vascular
bundles filled with rich tannin content whereas
in the evaluation of petiole after flowering-
fruiting, these areas showed poor tannin content
or somewhat absence of tannin content in the
cells. Histological and histochemical evaluation
of leaf before flowering-fruiting revealed upper
epidermis i.e. pallisade cells, ground tissue of
the midrib portion i.e. parenchyma cells and
adjacent cells of vascular bundles having rich
tannin content whereas the evaluation of leaf
after flowering-fruiting showed very poor
localization of tannin content in the cells of
these areas. On histological and histochemical
evaluation of fruit, it was observed that
sclereids and adjacent vascular tissues i.e.
xylem parenchyma and fibers were thickly
enriched with the tannin content. Cells of
endocarp attached with the seed and epidermal
cells were also seen filled with the tannin
content. In powder microscopy, leaf before
flowering-fruiting revealed trichomes,
epidermal cells, spongy and palisade
parenchyma cells which were filled with rich
amount of tannin content whereas powder
microscopy of leaf after flowering-fruiting
showed very poor tannin content in these parts
of the leaf. Powder microscopy of fruit showed
the characters like sclereids, stone cells, fibers,
simple and compound starch grains, mesocarp
cells with tannin, vascular strands and tannin
contents. Thus histological, histochemical and
powder microscopical examinations suggest
presence of abundant tannin content in the leaf
of Haritaki before flowering-fruiting and its
recognizable absence or less localization in the
leaf after flowering-fruiting. Histochemical and
powder microscopical examinations of fruit
reveals noticeable presence of tannin content in
it. Thus there is scope to the possibility that
tannin content in the leaves before fruiting got
transported to fruit and thus leaves after fruiting
are showing comparatively less tannin content.
CONCLUSION
Detailed pharmacognostical evaluation of
T. chebula leaves before and after flowering-
fruiting suggests that the leaves of Haritaki
before flowering-fruiting are having more
concentration of tannin than after flowering-
fruiting leaves. Fruit which is a totally new
entity formed in the plant shows recognizable
amount of tannin content in it. This work may
provide incentive for the future research works
carried out for the evaluation of variations in
the chemical constituents of the plant.

REFERENCES

Anonymous, (2010) Quality Standards of
Indian medicinal Plants, Vol. 1, ICMR,
New Delhi, p. 205
Anwesa Bag, Subir Kumar Bhattacharyya, Rabi
Ranjan Chattopadhyay, Asian Pac J
Trop Biomed. 2013 March; 3(3): p.
244252

Govt. of India, (2001) The Ayurvedic
pharmacopoeia of India. New Delhi:
Government of India Ministry of Health
and Family Welfare Department of
Indian System of Medicine &
Homoeopathy; p. 47


Khandelwal K.R., Kokate C.K., Gokhale S.B.
(1996). Practical Pharmacognosy
Techniques and Experiments. Nirali
Prakashan, Pune, p. 1039
Krishnamurty K.V., (1988) Methods in the
Plant Histochemistry. Madras:
Vishwanandan Pvt, Limited; p. 174

Kshirod Kumar Gupta, Girish Chandra Joshi,
AYU, 2013; Jul-Sep; 34(3): p. 331334
S. Aruna, L.V. Nandakishore, Scholars
Academic Journal of Biosciences, 2014;
2(2): p. 132136
Trease G.E., Evans WC (2002), Trease and
Evans Pharmacognosy, Harcourt brace
& Co. 1999, p. 512, 547

Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 5 | May 2014 | 225231

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


ANTIDYSLIPIDAEMIC EFFECT OF THE STEM BARK OF
CHIRABILWA (Holoptelea integrifolia Planch.) - A CLINICAL TRIAL
Sinimol T P
1
*, Shahul Hameed A
2

1
Lecturer, Department of Dravyagunavijnan, Ahalia Ayurveda Medical College, Palakkad, Kerala, India
2
Associate Professor, Department of Dravyagunavijnan,Govt. Ayurveda College, Kannur, Kerala, India
*Corresponding author: Email: drsinitp@gmail.com; Mob: 9446519427

ABSTRACT
The powder of stem bark of Chirabilwa (Holoptelea integrifolia Planch.) was clinically
evaluated to find out its effect in altered serum lipid levels and signs & symptoms of dyslipidaemia.
The statistical analysis of the change before and after treatment in values of Lipid profile and signs &
symptoms of dyslipidaemia were the assessment criteria. It was found that the study drug Chirabilwa
(Holoptelea integrifolia Planch.) was effective in reducing Total Cholesterol, Low Density
Lipoprotein, Very Low Density Lipoprotein & Triglycerides and in increasing High Density
Lipoprotein. Chirabilwa (Holoptelea integrifolia Planch.) was also effective in reducing the
symptoms of dyslipidaemia such as difficulty in physical activity, breathlessness on slight exertion,
excess sweat, dizziness and lassitude.
KEYWORDS: Dyslipidaemia, Chirabilwa, Holoptelea integrifolia Planch., Cholesterol

Research Article
Cite this article:
Sinimol T P, Shahul Hameed A (2014), ANTIDYSLIPIDAEMIC EFFECT OF THE STEM
BARK OF CHIRABILWA (Holoptelea integrifolia Planch.) - A CLINICAL TRIAL,
Global J Res. Med. Plants & Indigen. Med., Volume 3(5): 225231



INTRODUCTION
Non communicable diseases (NCDs) are
the major killer diseases in the world today,
causing more deaths than all other diseases
combined. The first WHO (World Health
Organisation) global status report on non
communicable diseases of year 2010 confirms
that 36.1 million people died from non
communicable diseases in 2008 (Margaret
Chan, 2011). World Health Organisation
predicts that non communicable diseases will
cause over three quarters of all deaths in 2030
(Fuster V, Kelly BB, 2010). India is also
passing through an epidemiological and
demographic transition leading to the
emergence of non communicable diseases as
major health problem, estimated to account for
53% of all deaths and 44% of disability in 2005
(Samu.K, 2012).
Dyslipidaemia, a major non communicable
disease is a major health problem in both
developed and developing nations (Margaret
Chan, 2011). It is an extremely important
condition, principally because of its
contribution to atherogenesis and as it is an
independent and modifiable risk factor for
Cardio Vascular Disease (CVD), together with
Hypertension, smoking and sedentary habits
(Poss J et al., 2011). The ever increasing
epidemic nature of the disease prompts the
scientific researchers to understand the
dimension of the condition (Li YP et al, 2012).
Modern medications available to dyslipidaemia
are costly, giving only temporary relief and are
potentially hazardous (Robert. K. Murray et al.,
2000). Traditional Ayurvedic approaches to
health have become all the more relevant in the
present century in the context of universal rise
of NCDs (non communicable diseases).
Ayurvedic approach to these conditions is
holistic, which is a combination of diet,
exercise, awareness of environmental
influences and the use of medications.
From Ayurvedic classics, an exact
correlation of dyslipidaemia to any disease
cannot be made. Scientific treatment can be
adopted by analyzing dosha dooshya vikriti
(imbalance of functional and structural entities
of our body).

Unhealthy diet and activities that vitiate
doshas (functional entities of our body) leads to
vitiation of srotases (channels in the body). It is
the root cause for all diseases. The abnormality
in dhatus (tissues) brings about abnormality in
srotases (channels) and vice-versa. This leads
to impairment of agni (digestive fire) and
formation of ama (undigested food). This is
followed by faulty dhatuparinama (formation
of tissues) and vitiated medodhatu (lipids) is
formed (Yadavji Trikamji, 2006).

Chirabilwa (Holoptelea integrifolia
Planch.) is a large decidous tree distributed
throughout the greater parts of India upto an
altitude of 2000 feet. It is a drug which has
been mentioned by various Ayurvedic books as
an ingredient in many formulations indicated
for the management of derangement of meda
(body fat). It possesses tikta rasa (bitter taste),
kashaya rasa (astringent taste), laghu (easily
digestible property), rooksha guna (dry
property), ushna veerya (hot potency) and katu
vipaka (pungent post metabolic effect)
(Chunekar K.C, 1995). Chirabilwa is included
in many medohara (lipid curtailing) groups by
various Ayurvedic text books. But no research
has been conducted yet to clinically evaluate its
antidyslipidaemic effect as a single drug. Hence
a clinical study to evaluate the
antidyslipidaemic effect of the stem bark of
Chirabilwa was taken up.
For the clinical study, a total of 32 patients
fulfilling the diagnostic criteria of
dyslipidaemia approaching the outpatient
department of Government Ayurveda Medical
College and Hospital, Trivandrum were
selected for the study. The National Institutes
of Health's National Cholesterol Education
Program (NCEP) Adult Treatment Panel III
(ATPIII) guidelines were used as the diagnostic
criteria. The patients were assigned into a
single group taking into consideration the
inclusion and exclusion criteria for the


interventional study.
Inclusion criteria: Patients of both sexes in the
age group of 1670 years, diagnosed to have
dyslipidaemia.
Exclusion criteria: Pregnant and lactating
woman, patients undergoing other treatments
for dyslipideamia, patients taking any long term
medication, known cases of Hypothyroidism,
Coronary Artery Disease (CAD), liver disease,
renal disease.
Preparation of the drug
The stem bark of Chirabilwa (Holoptelea
integrifolia Planch.) was collected from the
herbal garden of Pharmacognosy Unit, Govt.
Ayurveda College, Trivandrum, India during
January 2011. The plant identification was
done by Mr. G.R. Jayakumar, Senior Research
officer, Pharmacognosy Unit, Govt. Ayurveda
College, Trivandrum. Voucher specimens were
preserved in the Herbarium at the Department
of Dravyaguna vijnana, Ahaliya Ayurveda
College, Kerala, India. The collected stem
barks of Chirabilwa (Holoptelea integrifolia
Planch.) were washed well to remove mud and
soil contaminants, dried in shade and powdered
using a micropulveriser to the mesh size 80.
60g of fine powder was weighed out and sealed
in polythene packets. 6 gm of powder was
given orally, twice daily before food with luke
warm water for 30 days.
Clinical Trial
The Clinical research design was approved
by the Institutional ethics committee,
Government Ayurveda Medical College,
Trivandrum, India.
Investigations: Routine Urine examination and
biochemical examination with Serum Total
Cholesterol (S. TC), Serum High Density
Lipoprotein (S. HDL), Serum Low Density
Lipoprotein (S. LDL), Serum Very Low
Density Lipoprotein (S. VLDL) and Serum
Triglycerides were performed before and after
the study.
Assessment Criteria: The patients were
assessed based on the clinical parameters
explained in Table 2 and laboratory findings.
The clinical parameters were graded as follows.
All the symptoms except Incidence of
hypersomnia & dizziness were graded as:
Grade 0- not noted /not present, Grade 1 mild,
Grade 2- moderate, Grade 3-severe. Incidences
of hypersomnia & dizziness were graded as:
Grade 0- never, Grade 1 less frequent, Grade
2- moderately frequent and Grade 3- Highly
frequent.
Statistical analysis: Values obtained were
statistically evaluated using pairedt test and
Wilcoxons signed rank test. In the entire
statistical test, the P-values less than 0.05 were
considered to be statistically significant.
RESULTS
46.88% of cases were distributed among the
age group of 5059 years, which showed this
age group is more prone to the development of
dyslipidaemia. 68.75% had vatakapha prakriti
(body constitution), vata and kapha are the
main doshas involved in the pathogenesis of
dyslipidaemia. 87.5% of cases belonged to
middle class. 71.88% were residing in urban
area indicating the high prevalence of the
disease in urban area. 90.63% were taking both
vegetarian & non vegetarian food showing the
role of non-vegetarian diet in causing the
disease. 81.25% patients had regular pattern of
eating, 78.13% patients used only coconut oil
for cooking, 84.38% had moderate appetite,
59.38% were stout, 59.38% had weight
between 6575 kg, 96.88% of patients never
smoked, 63% of patients never consumed
alcohol, 37.5% patients had less sleep. 59.38%
patients had no exercise, lack of exercise is
considered to be a main cause of the disease.


Table-01. Analysis of effect of treatment on Serum Lipid Profile
Mean SD KS P MD Z or t P
TC-BT 241.56 35.80 0.167 0.024* -30.50 4.581$ 0.001*
TC-AT 211.06 26.42 0.158 0.040*
HDL-BT 50.50 9.90 0.105 0.200 5.66 2.643# 0.013*
HDL-AT 56.16 11.33 0.127 0.200
LDL-BT 158.75 28.00 0.153 0.056 30.34 4.33 $ 0.0001*
LDL-AT 128.41 26.51 0.188 0.005*
VLDL-BT 32.25 20.53 0.233 0.000* 5.72 4.18 $ 0.0004*
VLDL-AT 26.53 15.78 0.216 0.001*
TG-BT 161.44 102.57 0.234 0.001* -27.19 4.339$ 0.001*
TG-AT 134.25 87.35 0.242 0.001*
*: Significant at 5% level (P<0.05), KS: Kolmogorov Smirnov test, $: Wilcoxons Signed Rank test, #: Paired t test

Table - 02. Analysis of signs & symptoms of dyslipidaemia
Signs & Symptoms
Before treatment After treatment Wilcoxons
test
Grade Grade
3 2 1 0 3 2 1 0 Z P
% % % % % % % %
Difficulty in physical
activity
12.5 12.5 12.5 62.5 0 3.125 12.5 84.375 3.145 0.002
*
Breathless-ness on
slight exertion
9.375 6.25 9.375 75 0 0 12.5 87.5 2.460 0.014
*
Breathlessness
without known
reason
3.125 0 0 96.875 0 3.125 0 96.875 1.000 0.317
Shallow breathing
3.125 0 3.125 93.75 0 3.125 0 96.875 1.414 0.157
Excess thirst
0 9.375 6.25 84.375 0 0 3.125 96.875 1.518 0.129
Excess hunger
0 3.125 6.25 90.625 0 0 3.125 96.875 1.000 0.317
Excess sweat
12.5 9.375 9.375 68.75 0 0 15.625 84.375 2.598 0.009
*

Dizziness
12.5
15.625
9.375 62.5 0 6.25 12.5 81.25 3.025 0.002
*
Lassitude
31.25 25 9.375 34.375 0 6.25 25 68.75 4.086 0.001
*
Incidence of
Hypersomnia
0 0 6.25 93.75 0 0 6.25 93.75 1.342 0.180
Bromhid-rosis
3.125 0 0 96.875 0 0 0 100 1.342 1.000
Decrease in sexual
vigour
3.125

0 0 96.875 3.125 0 0 96.875 1.342 1.000

* Significant at 5% level (P<0.05)



Effect of treatment
1. Effect of treatment on Serum Lipid
Profile
The drug was statistically effective
(P<0.05) in reducing Total Cholesterol, LDL,
VLDL & Triglycerides and in increasing HDL
(Table No. - 1).
2. Effect of treatment on Signs &
Symptoms of dyslipidaemia
The drug was statistically effective
(P<0.05) in reducing difficulty in physical
activity, breathlessness on slight exertion,
excess sweat, dizziness and lassitude. The drug
was statistically not (P>0.05) effective in
reducing breathlessness without known reason,
shallow breathing, excess thirst, excess hunger,
Incidence of hypersomnia, bromhidrosis,
decrease in sexual vigour (Table No. - 2).
DISCUSSION
Dyslipidaemia is a term used to denote
abnormal fractions of circulating lipids or
lipoproteins in blood. Dyslipidaemia as such is
not mentioned in Ayurvedic classical texts. In
Ayurvedic context, Dyslipidaemia can be
considered as a particular condition of
increased medodhatu (lipids) associated with
increased kapha dosha and deranged kleda
(unhealthy secretions) in the body. Agnisada
(decreased digestive fire) and increased kapha
dosha lead to the formation of ama (undigested
food). Due to obstruction of nourishment
pathways to other dhatus (tissues), only
medodhatu (lipids) gets nourished at the cost of
other dhatus (tissues). Meda (lipids) with its
high picchila (sticky) property causes lepana
(coating) of srotases (channels of body),
gradually blocking them. As a result of
srotorodha (obstruction in channels of our
body), the activities of vata dosha also get
increased resulting in the symptoms of
dyslipidaemia (Srikantha Murthy K.R, 2005).
Probable mode of action of the drug
One basic principle of Ayurveda is that
similar factors cause increase and dissimilar
factors cause decrease of our body constituents.
Based on this principle, mode of action can be
explained. As stated earlier, dyslipidaemia is
caused by the excessive indulgence of factors
causing increase of medodhatu (lipids), ie.
substances having madhura rasa (sweet taste),
seeta veerya (cold potency), guru guna
(difficult to digest property), snigdha gunas
(unctuous property) and sedentary life style.
(Acharya Yadunandana Upadhyaya, 2005).
Thus treatment should be with drugs having
tika katu kashaya rasa (bitter, pungent and
astringent taste), ushna veerya (hot potency)
and laghu rooksha guna (which are easy to
digest and dry). The study drug Chirabilwa is
having tika kashaya rasa (bitter and astringent
taste), laghu rooksha guna (which is easily
digestible and dry), ushna veerya (hot potency)
and katu vipaka (pungent post digestive effect),
thus possessing all the above properties
(Chunekar K.C, 1995).
Drugs having katu rasa (pungent taste),
ushna veerya (hot potency) and laghu guna
(easily digestible) can increase the strength of
agni (digestive fire) which may act on
improperly digested food. These properties can
pacify kapha and dessicate vitiated meda
(lipids) and kleda (unhealthy secretions), the
major contributing factors for the condition.
Thus it can remove the obstruction of channels.
Laghu rooksha guna (which is easy to
digest and dry) and katu rasa (pungent taste)
can act as lekhana (dessicate and scrape away
the morbid tissues especially meda and kapha).
Chirabilwa is included in lekhana
mahakashaya group (group of drugs that
dessicate and scrape away the morbid tissues
especially meda and kapha) of Caraka Samhita
(Yadavji Trikamji, 2006); Shleshma
samsamana (groups of drugs which decrease
kapha), several kaphamedohara groups (groups
of drugs which decrease meda and kapha) like
Saalasaradi, Varanadi, Aragwadadi, Asanadi
and Arkadi of Susruta Samhita (Yadavji
Trikamji, 2006); Asanadi, Varanadi,
Aragwadadi and Arkadi groups of Ashtanga
Hridaya (Hari Sadasiva Sastri Paradakara,
2005). Drugs having these properties and ushna


veerya can also act as pramathi (drugs that
expel cumulative mala from channels). Hence
the mode of action of Chirabilwa can be
attributed principally to its laghu rooksha guna
(which is easy to digest and dry), ushna veerya
(hot potency), tikta kashaya rasa (bitter and
astringent taste) and katu vipaka (pungent post
digestive effect), with which it decreases meda
(lipids) by means of lekhana (dessicate and
scrape away the morbid tissues especially meda
and kapha) and pramathi (drugs that expel
cumulative excretory products from channels)
modes.

-sitosterol is a chemical constituent of
Chirabilwa (Ram.P.Rastogi. & Mehrotra B.N,
1970). It is to be noted that -sitosterol is used
as a dietary supplement to lower blood
cholesterol. -Sitosterol inhibits cholesterol
absorption in the intestine. When the sterol is
absorbed in the intestine, it is transported by
lipoproteins and incorporated into the cellular
membrane. Phytosterols and phytostanols both
inhibit the uptake of dietary and biliary
cholesterol, decreasing the levels of LDL and
serum total cholesterol. Because the structure
of -sitosterol is very similar to that of
cholesterol, -sitosterol takes the place of
dietary and biliary cholesterol in micelles
produced in the intestinal lumen. This causes
less cholesterol absorption in the body (Fauci &
Longo, 2008).
CONCLUSION
Chirabilwa (Holoptelea integrifolia
Planch.) is a classical drug possessing lekhana
(dessicate and scrape away the morbid tissues
especially lipids and kapha dosha) action. The
study drug is effective in raising Serum High
Density Lipoprotein and decreasing Serum
Total Cholesterol, Serum Low Density
Lipoprotein, Serum Very Low Density
Lipoprotein and Triglycerides. The drug
reduces some signs and symptoms of
dyslipidaemia like difficulty in physical
activity, breathlessness on slight exertion,
excess sweat, Dizziness and lassitude.
Therefore it can be concluded that the study
drug, the powder of the stem bark of
Chirabilwa (Holoptelea integrifolia Planch.),
Family -Ulmaceae] is effective in
dyslipidaemia.

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