ArticlePDF Available

A Comparison of the Cytotoxic potential of standardized aqueous and ethanolic extracts of polyherbal mixture comprised of Nigella sativa (seeds), Hemidesmus indicus (root) and Similax glabra (rhizome).

Authors:

Abstract and Figures

Background: A decoction (hot-water extract) comprised of Nigella sativa (seeds), Hemidesmus indicus (roots), and Smilax glabra (rhizome) has been reported to prevent chemically-induced hepatocarcinogenic changes in rats and to exert significant cytotoxic effects on human hepatoma (HepG2) cells. However, the decoction used in previous studies to determine cytotoxicity was not standardized. Further, during preparation of pharmaceuticals for clinical use, it is more convenient to use an ethanolic extract. Therefore this study was carried out to (a) develop standardized aqueous and ethanolic extracts of the plant mixture (N. sativa, H. indicus, and S. glabra) used in the preparation of the original decoction, and (b) compare the cytotoxic effects of these two extracts by evaluating cytotoxicity to the human hepatoma (HepG2) cell line. Methods: Aqueous and ethanolic extracts have been standardized by evaluating organoleptic characters, physicochemical properties, qualitative and quantitative analysis of chemical constituents, and analysis of High Performance Liquid Chromatography (HPLC) and Thin Layer Chromatography (TLC) profiles. Cytotoxic potentials of the above standardized extracts were compared by evaluating their effects on the survival and overall cell activity of HepG2 cells by use of the 3-(4, 5-dimethylthiazol-2yl) -2, 5 - biphenyl tetrazolium bromide (MTT) and Sulphorhodamine B (SRB) assays. Results: Results from MTT and SRB assays demonstrated that both extracts exerted strong dose-dependent in vitro cytotoxicity to HepG2 cells. The standardized aqueous extract showed a marginally (though significantly, P<0.05) higher cyotoxic potential than the ethanolic extract. Thymoquinone, an already known cytotoxic compound isolated from N. sativa seeds was only observed in the standardized ethanolic extract. Thus, compounds other than thymoquinone appear to mediate the cytotoxicity of the standardized aqueous extract of this poly-herbal preparation. Conclusion: It may be concluded that results obtained in the present study could be used as a diagnostic tool for the correct identification of these aqueous or ethanolic extracts and would be useful for the preparation of a standardized pharmaceutical product that may be used in the future for clinical therapy of hepatocellular carcinoma.
Content may be subject to copyright.
Pharmacognosy Research | November 2010 | Vol 2 | Issue 6 335
Address for correspondence:
Sameera R. Samarakoon, Institute of Biochemistry, Molecular
Biology & Biotechnology, University of Colombo, Cumarathunga
Munidasa Mawatha, 90, Colombo 3, Sri Lanka.
E-mail: ranganath148@yahoo.com
A comparison of the cytotoxic potential of standardized
aqueous and ethanolic extracts of a polyherbal mixture
comprised of Nigella sativa (seeds), Hemidesmus
indicus (roots) and Smilax glabra (rhizome)
Sameera R. Samarakoon, Ira Thabrew, Prasanna B. Galhena
1
, Dilip De Silva
2
, Kamani H. Tennekoon
Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo, Cumarathunga Munidasa Mawatha, 90, Colombo 3, Sri
Lanka,
1
Department of Biochemistry and Clinical Chemistry, Faculty of Medicine, University of Kelaniya, Talagoole Road, Ragama, Sri Lanka,
2
Department of Chemistry, Faculty of Science, University of Colombo, Sri Lanka
Submitted: 22-08-2010 Revised: 23-08-2010 Published: 12-01-2011
O R I G I N A L A R T I C L E
INTRODUCTION
Cancer is an important public health concern and is
considered to be the second largest common disease
in the world.
[1]
Hepatocellular carcinoma is the most
frequent primary liver cancer, fth commonest neoplasm
Background: A decoction (hot-water extract) comprised of Nigella sativa (seeds), Hemidesmus
indicus (roots), and Smilax glabra (rhizome) has been reported to prevent chemically-induced
hepatocarcinogenic changes in rats and to exert signicant cytotoxic effects on human hepatoma
(HepG2) cells. However, the decoction used in previous studies to determine cytotoxicity was not
standardized. Further, during preparation of pharmaceuticals for clinical use, it is more convenient
to use an ethanolic extract. Therefore this study was carried out to (a) develop standardized
aqueous and ethanolic extracts of the plant mixture (N. sativa, H. indicus, and S. glabra) used
in the preparation of the original decoction, and (b) compare the cytotoxic effects of these
two extracts by evaluating cytotoxicity to the human hepatoma (HepG2) cell line. Methods:
Aqueous and ethanolic extracts have been standardized by evaluating organoleptic characters,
physicochemical properties, qualitative and quantitative analysis of chemical constituents, and
analysis of High Performance Liquid Chromatography (HPLC) and Thin Layer Chromatography
(TLC) proles. Cytotoxic potentials of the above standardized extracts were compared by
evaluating their effects on the survival and overall cell activity of HepG2 cells by use of the 3-(4,
5-dimethylthiazol-2yl) -2, 5 – biphenyl tetrazolium bromide (MTT) and Sulphorhodamine B (SRB)
assays. Results: Results from MTT and SRB assays demonstrated that both extracts exerted strong
dose-dependent in vitro cytotoxicity to HepG2 cells. The standardized aqueous extract showed
a marginally (though signicantly, P<0.05) higher cyotoxic potential than the ethanolic extract.
Thymoquinone, an already known cytotoxic compound isolated from N. sativa seeds was only
observed in the standardized ethanolic extract. Thus, compounds other than thymoquinone appear
to mediate the cytotoxicity of the standardized aqueous extract of this poly-herbal preparation.
Conclusion: It may be concluded that results obtained in the present study could be used as a
diagnostic tool for the correct identication of these aqueous or ethanolic extracts and would
be useful for the preparation of a standardized pharmaceutical product that may be used in the
future for clinical therapy of hepatocellular carcinoma.
KEYWORDS: Cytotoxicity, Hemidesmus indicus, MTT and SRB assays, Nigella sativa, Smilax
glabra, Standardization
A B S T R A C T
P H C O G R E S .
Access this article online
Website:
www.phcogres.com
DOI:
10.4103/0974-8490.75451
Quick Response Code:
336 Pharmacognosy Research | November 2010 | Vol 2 | Issue 6
in the world, and third commonest cause of cancer-
related death. More than 500,000 new cases are diagnosed
annually worldwide and are most common in men in many
developing countries.
[2-4]
Hepatocellular carcinoma is very
common in the Asian region and it is strongly associated
with chronic liver infection or hepatitis, especially Hepatitis
B and C viruses. Other important risk factors include liver
cirrhosis from excessive alcohol consumption as well as
ingestion of aatoxin, a substance which is found in moldy
nuts and grain.
[4,5]
Chemoprevention, the prevention of cancer by ingestion
of chemical agents that reduce the risk of carcinogenesis,
is one of the most direct ways to reduce the morbidity and
mortality of cancer.
[5]
Plants have been a prime source of
highly effective conventional drugs for the treatment of
many forms of cancer. It is estimated that approximately
60% of most effective antitumor/ anti-infectious drugs
already on the market or under clinical investigations
are of products or compounds derived from natural
products.
[6-9]
In Asian countries poly-herbal preparations are often used
by traditional medical practitioners for the treatment of
cancer.
[10]
One such herbal remedy prescribed to cancer
patients by a family of indigenous medical practitioners
in Sri Lanka, is a decoction (hot-water extract) prepared
from Nigella sativa (seeds), Hemidesmus indicus (roots)
and Smilax glabra (rhizome) (personal communication,
Ayurvedic physician, Dr, Nimal Jayathilaka). In previous
investigations, Iddamaldeniya et al.,
[11,12]
have demonstrated
that protection against diethylnitrosoamine (DEN)-
mediated carcinogenic changers in rat liver can be achieved
by long-term treatment with this decoction. Recent
investigations by Thabrew et al.,
[10]
have also shown that the
decoction can exert a signicant dose-dependent cytotoxic
effect on human hepatoma (HepG2) cells. However, the
decoction used in these investigations was not properly
standardized. Standardization is an essential measurement
for ensuring the quality control of herbal drugs.
[13]
To
develop a herbal extract with a pharmaceutical product
that can be used in modern clinical practice, pharmaceutical
companies prefer to use an alchoholic extract as the starting
material than an aqueous extract. Therefore the present
investigation was carried out to (a) develop standardized
aqueous (hot-water extract) and ethanolic extracts of
the herbal mixture previously utilized in the evaluation
of antihepatocarcinogenic
[11,12]
and cytotoxic activities,
[10]
and (b) compare the cytotoxic potential of the above
standardized extracts by evaluating their effects on the
survival and overall cell activity of HepG2 cells by use of
MTT and SRB assays.
MATERIALS AND METHODS
Collection of plant materials
Plant material for the preparation of extracts was
purchased from a reputed vendor of herbal material
used by traditional medical practitioners in Sri Lanka
(D. J. Fenando Pvt Ltd, Gabose lane, Colombo 13). The
identities were conrmed by the Botanist at Bandaranayaka,
Memorial Ayurvedic Research Institute (BMARI), Navinna,
Maharagam Sri Lanka. Voucher specimens of N. sativa
(seeds), H. indicus (roots), and S. glabra (rhizome) have
been deposited in the Institute of Biochemistry, Molecular
biology and Biotechnology, University of Colombo, Sri
Lanka. (Voucher specimen nos.UOC/IBMBB/2009/01,
UOC/IBMBB/2009/02 and UOC/IBMBB/2009/03).
Standardization of the aqueous and ethanolic extracts
Aqueous (hot-water) and ethanolic extracts of 15 different
samples of plant materials purchased at different times of
the year from the same vendor were standardized according
to the methods recommended by the World Health
Organization (WHO).
[14]
Estimation of organoleptic
characters, physicochemical properties, qualitative and
quantitative analysis of chemical constituents, and analysis
of HPLC, and TLC proles, were carried out.
Preparation of aqueous extract (hot-water extract)
Sixty grams (60 g) of plant material (composed of a mixture
of 20 g each of N. sativa seeds, H. indicus roots and S.
glabra rhizomes) was ground and boiled gently with 1.6 L
distilled water for approximately 3 h to reduce the volume
to 200 ml. The extract was then ltered through a layer of
muslin, ltrate centrifuged at 3000 g for 15 min to remove
any debris, and the supernatant freeze dried and stored at
-20
0
C until required.
Preparation of ethanolic extract
The dried, powdered plant material mixture (60g) was
subjected to soxhlet extraction with 80% ethanol (500 ml),
ltered, and evaporated to dryness under reduced pressure,
stored at -20
0
C until required.
Determination of Physicochemical parameters
Determination of pH
The pHs of the aqueous and ethanolic extracts were
determined using a pH meter (Fisher brand Hydrus 300,
USA), at room temperature.
Determination of water and ethanol-extractable
matter in the mixture of air-dried N. sativa seeds, H.
indicus roots and S. glabra rhizomes
Air-dried N. sativa seeds, H. indicus roots and S. glabra
Samarakoon, et al.: Cytotoxic role of a standardized poly-herbal mixture
Pharmacognosy Research | November 2010 | Vol 2 | Issue 6 337
rhizomes were mixed in equal proportions (3 g each) and
coarsely powdered; 4g of the above mixture was placed
in an accurately weighed glass-stoppered conical ask.
For estimation of water-extractable matter, distilled water
(100 ml) was added to the ask and weighed to obtain the
total weight including the ask. The contents were shaken
well and allowed to stand for 1 h. A reux condenser was
attached to the ask and boiled gently for 1 h, cooled and
weighed. The ask was readjusted to the original weight
with distilled water. The mixture was shaken well and
ltered rapidly through a dry lter. Then 25 ml of the
ltrate was transferred to a round-bottomed ask and
evaporated to dryness on a water bath. Finally, it was dried
at 105
º
C for 6 h, cooled in a desiccator for 30 min, and
immediately weighed. Same procedure was followed using
ethanol instead of distilled water to determine extractable
matter in ethanol. The extractable matter was calculated
as mg/g of air-dried material.
[14]
Determination of total ash content of extracts
Two grams of dried extract was placed in a crucible and
weighed. Dried material was spread in an even layer in the
crucible, and the material ignited by gradually increasing
the heat to 500-600
0
C until free from carbon, cooled in a
desiccator, and weighed. Total ash content was calculated
in mg/g of dried extract.
[14]
Determination of acid insoluble ash
Twenty-ve ml of 2M HCl was added to the crucible
containing the total ash, covered with a watch glass and
boiled gently for 5 min. The watch glass was rinsed with 5
ml of hot water and this liquid added to the crucible. The
insoluble matter was collected on an ashless lter paper and
washed with hot water until the ltrate was neutral. The
lter paper containing the insoluble matter was transferred
to the original crucible, dried on a hot plate and ignited to
constant weight. Acid-insoluble ash content was calculated
as mg/g of dried extract.
[14]
Determination of water-soluble ash
Twenty-five ml of water was added to the crucible
containing the total ash and boiled for 5 min. Insoluble
matter was collected on an ashless lter paper, washed
with hot water and ignited for 15 min at a temperature not
exceeding 450
0
C. The weight of the residue was subtracted
from the total ash and the water-soluble ash content
calculated as mg/g of dried extract.
[14]
Determination of foaming index
The dried water extract (1 g) was dissolved in 100 ml water,
the solution cooled and ltered. The ethanolic extract (1
g) was dissolved in 2.5 ml 95% ethanol, the nal volume
made up to 100 ml with distilled water, the solution cooled
and ltered. Each of the above ltrates were placed in test
tubes in a series of successive portions of 1,2,3, up to 10
ml and the volumes in each tube adjusted with distilled
water to 10 ml. The tubes were stoppered and then shaken
for 15 sec in a lengthwise motion (2 frequencies / sec).
After allowing the tubes to stand for 15 min, the height
of foam was measured by means of a graduated tape with
millimeter scale.
[14]
Qualitative and Quantitative Analysis of chemical
constituents
Preliminary phytochemical screening of aqueous and
ethanolic extracts
The preliminary phytochemical screening of the aqueous
and ethanolic extracts was carried out using standard
laboratory procedures, to detect the presence of
different secondary metabolites such as alkaloids,
avonoids, saponins, tannins, steroids, reducing sugars, and
phenols.
[15-17]
Determination of polyphenolic content
The total polyphenolic content was estimated according
to the Folin-Ciacalteau method described by Spanos and
Worlstad.
[18]
The freeze-dried aqueous extracts were re-
dissolved in distilled water, ltered, and the concentrations
of each ltrate adjusted to 10 mg/ml with distilled water.
Each ethanolic extract was re-dissolved in 95% methanol
to a concentration of 10 mg/ml. Each aqueous extract and
ethanolic extract (0.05 ml) was diluted with distilled water
or 80% methanol (0.95 ml) respectively and mixed with
5 ml of 10-fold diluted solution of 2N Folin-Ciocalteau
reagent (Sigma-UK). Four milliliters of saturated sodium
carbonate solution were then added to the mixture and
shaken. After incubation at room temperature for 2 h the
absorbance of the reaction mixture was measured at 760
nm against a methanol blank. Gallic acid (Sigma-Aldrich
Chemie, Steinheim Germany) (0-100 mg/l) was used
as a standard to prepare a calibration curve. The total
phenolic content was expressed in mg of gallic equivalents
(GAE)/100 g of extract.
Determination of total avonoid content
Dried aqueous and ethanolic extracts were re-dissolved in
95% methanol to a nal concentration of 10 mg / ml. The
total avonoid content was determined using the Dowd
method as adapted by Meda et al.
[19]
Briey, 5 ml of 2%
aluminium trichloride (AlCl
3
) in methanol was mixed with 1
ml of extract (10 mg/ml). Absorbance readings at 415 nm
were taken after 10 min against a blank sample consisting
of 5-ml extract plus 5 ml methanol without AlCl
3
. The
total avonoid content was determined using a standard
curve prepared with quercetin (Sigma-Aldrich Chemie,
Steinheim Germany) (0-30 mg/l) as the standard. The
total avanoid contents are expressed as mg of quercetin
equivalent (QE)/100 g of extract.
Samarakoon, et al.: Cytotoxic role of a standardized poly-herbal mixture
338 Pharmacognosy Research | November 2010 | Vol 2 | Issue 6
Determination of heavy metal composition
Samples of (0.5 g) dried aqueous extracts and ethanolic
extracts of the mixture of plants comprised of N. sativa,
H. indicus, and S. glabra, were placed in clean silica crucibles,
digested with a mixture of acids having a ratio of conc.
HNO
3
to HClO
4
acid 1:1 and heated up to 135
o
C until a white
residue was obtained. The resulting dry inorganic residue in
each crucible was dissolved in 20 ml of distilled water and
used for the estimation of the heavy metal concentration.
The digested samples were analyzed for Cd and Pb, using
a graphite furnace atomic absorption spectrophotometer
(AAS; GBC 932 plus, Australia). A single-beam hollow
cathode lamp (GBC) was used for estimation of Cd and
Pb. Concentration of Hg was determined through AAS
using Cold Vapor technique and air-acetylene ame was
used for determination of As concentration. The metal
quantification was based on calibration curves, which
were determined through a series of concentrations
prepared by using the chemical standard with 1000 mg/l
concentration.
[20]
The concentration of the respective metals
in samples were expressed as mg of metal per kg (ppm). The
lowest detectable levels of Cd, Pb, As and Hg according to
methods used were 0.004 mg /L, 0.1 mg /L, 2 mg /L and
1.5 mg /L respectively.
Analysis of High-performance liquid chromatography
(HPLC) and Thin layer chromatography (TLC) proles
HPLC proles
For determination of the HPLC proles, the aqueous
extracts and ethanolic extract were re-dissolved in
distilled water, 80% methanol, respectively (20 mg/ml
concentration). After ltration through 0.45 µm, 13-mm
Millipore lters, 20 µl of each sample were injected into an
Intertsil ODS-3 C
18
, (250 x 4.0 mm, 5 µm) reversed phase
column of a High-Performance Liquid Chromatography
System (Schimadzu, Kyoto, Japan) connected to a UV-
Vis detector (Model SPD-10AVP). The HPLC analysis
was performed using a linear gradient of 80% water in
methanol to 100% methanol for 30 min, followed by100%
methanol for 50 min with a ow rate of 0.5 ml / min,
detection 254 nm. The peak areas and peak heights were
analyzed by the software package (CLASS-VP) provided
with the HPLC system. For determination of HPLC
prole of thymoquinone, the chemical purchased from
Sigma Aldrich, USA was re-dissolved in DMSO (1 mg/
ml concentration) and 10 µl of the sample was injected
into the HPLC system. HPLC analysis was performed as
described above for the aqueous and ethanolic extracts.
All solvents were HPLC grade and purchased from Fisher
Scientic International Company, UK.
TLC proles of aqueous and ethanolic extracts
Twenty microliters of each extract was spotted onto
TLC plates coated with silica gel (pre-coated, GF
254
)
and
separated using a variety of solvent systems. The best
separation of the aqueous extracts occurred in the solvent
system comprised of Butanol: Acetic acid: water (60: 15: 25
v/ v) while the best solvent system for separation of the
ethanolic extracts was found to be a mixture of Methanol:
Cyclohexane: Dichloromethane (6: 20: 74 v/v). Vanillin
sulphate spray reagent was used to visualize spots. All
solvents were analytical grade and purchased from Fisher
Scientic International Company, UK.
Cytotoxicity assays
Cell culture
HepG2 (human hepatoma) cells were harvested by
trypsinization, plated (5 x 10
3
cells/well) in 96-well cell
culture plate and maintained in Dulbecco's Modified
Eagle Medium (DMEM) for 24 h at 37
0
C in 95% air
/ 5% CO
2
atmosphere, with 95% humidity. Cultures
were exposed only to medium (1% DMSO, controls) or
medium containing different concentrations of aqueous
and ethanolic extracts dissolved in 1% DMSO (300 µg/ml
-4800 µg/ml), and incubated for 24 h. At the end of this
incubation period, cells were briey washed with Phosphate-
buffered saline (PBS). Fresh medium (200 µl) was then
placed in each well and Sulphorhodamine (SRB) and MTT
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assays performed as described below.
Sulphorhodamine (SRB) cytotoxicity assay
The SRB cytotoxicity assay was performed according to
the method of Mitry et al.
[21]
Cell survival was determined
after exposure to different concentrations of the aqueous
extracts or ethanolic extracts for 24 h. Cells were xed with
50 µl of ice-cold 50% trichloroacetic acid solution by gently
adding on top of the medium overlaying the cells. The
plates were then incubated for 60 min at 4ºC. Wells were
rinsed ve times with tap water and then cells were stained
with 0.4% SRB solution (100 µl stain/well) for 15 min at
room temperature. After staining, SRB solution was poured
off, unbound dye was removed by washing ve times with
1% acetic acid solution and left to air dry. The bound SRB
dye was then solubilized by adding unbuffered Tris-base
solution (200 µl/well), and plates were placed on a plate
shaker for 1 h at room temperature. Plates were then read
at OD 540 nm, using a microplate reader (EL
x
800 Universal
Micro Plate Reader, BIO-TEK INSTRUMENTS, USA)
and the results expressed as a percentage of control values.
Overall cell activity – MTT assay
Effect on overall cell activity was determined by
performing the MTT assay based on the method of
Oka et al.
[22]
The MTT assay measures the metabolism of
3-(4, 5-dimethylthiazol-2yl) -2, 5 biphenyl tetrazolium
bromide to form an insoluble formazan precipitate by
Samarakoon, et al.: Cytotoxic role of a standardized poly-herbal mixture
Pharmacognosy Research | November 2010 | Vol 2 | Issue 6 339
mitochondrial dehydrogenases only present in viable cells.
After exposure of cells to different concentrations of the
aqueous extract or ethanolic extract for 24 h, twenty (20
μl) microlitres of MTT solution was added to the 200 μl
medium in each well of the 96-well plate, and the plate was
incubated at 37
o
C for 4 h. The medium was then removed
by aspiration. Finally, 100 μl isopropanol / HCl was added
per well, the plate was shaken for a further 30 min and the
absorbance at 620 nm was measured using a microplate
reader EL
x
800 Universal Microplate Reader, BIO-TEK
INSTRUMENTS,USA) and the results expressed as a
percentage of control values.
Statistical analysis
Statistical analysis of the results obtained in each
experiment was carried out by use of the MINITAB 14
statistical software package.
RESULTS
Physicochemical parameters
Physicochemical parameters of the aqueous and ethanolic
extracts were determined according to the methods
recommended by World Health Organization (WHO). As
apparent from Table 1, the percentage yield of ethanolic
extract and ethanol-extractable matter were greater than the
percentage yield of aqueous extract and water-extractable
matter. A higher content of total ash, acid-insoluble ash
and water-soluble ash were found in the aqueous extract
in comparison to the ethanolic extract. Analysis of heavy
metals (Cd, Hg, Pb and As) showed the absence of
detectable levels of these metals in both aqueous and
ethanolic extracts.
The organoleptic properties of aqueous and ethanolic
extracts
As seen in Table 2, both the aqueous and ethanolic extracts
had similar organoleptic properties except for the darker
color of the aqueous extract.
Samarakoon, et al.: Cytotoxic role of a standardized poly-herbal mixture
Table 1: Physicochemical parameters
Parameter Aqueous
extract
Ethanolic
extract
Yield (%) on dry wt. basis
(Mean±SD)
15.242 ± 0.319 21.892 ± 0.231
Extractable matter (mg/
g,Mean%±SD)
132.5 ± 2.260 178.5 ± 3.340
Total ash (Mean%±SD) 5.9068 ± 0.449 2.2131 ± 0.129
Acid-insoluble ash (Mean%±SD) 0.3791 ± 0.065 0.2174 ± 0.007
Water-soluble ash (Mean%±SD) 3.8832 ± 0.345 0.6155 ± 0.027
Foaming index <100 <100
pH value of (Mean±SD) 4.86 ± 0.130 4.72 ± 0.070
Heavy metal content (ppm)
Pb ND ND
Cd ND ND
As ND ND
Hg ND ND
Values are expressed as mean ± S.D., n = 15. ND = Not detected
Table 2: Organoleptic properties of aqueous and
ethanolic extracts
Name of the
extract
Appearance Color Taste Smell
Aqueous extract Liquid Dark Brown Astringent Pungent
Ethanolic extract Liquid Brown Astringent Pungent
Table 3: Phytochemical analysis of water extract
and ethanolic extracts
Components Water extract Ethanolic extract
Alkaloids + +
Flavonoids +++ +++
Saponins +++ +
Tannins +++ +++
Steroids +++ +++
Reducing sugars +++ +++
Phenols +++ +++
+++ = appreciable amount; + = Trace amount
Table 4: Total polyphenol content and avonoid
content in aqueous and ethanolic extracts
Name of the extract Total polyphenols
(mg GAE/100g ±SD)
Total Flavonoids
(mg QE/100g ±SD)
Aqueous extract 23.80 ± 4.563 4.566 ± 1.004
Ethanolic extract 69.40 ± 5.029 5.518 ± 1.022
Values are expressed as mean ±S.D., n = 15. Polyphenolic content is expressed
as gallic acid equivalents (GAE) while total avanoid contents are expressed as
Quercetin equivalents (QE).
Table 5: TLC analysis of aqueous and ethanolic
extracts
Name of the extract Solvent system Rf values of
the spots
Hot-water extract Butanol: Acetic acid: Water,
(60: 15: 25 v/ v)
0.055*
0.417*
0.484
#
0.747*
0.857
$
0.923*
Ethanolic extract
Methanol: Cyclohexane: 0.033*
Dichloromethane 0.080*
(6: 20: 74 v/v) 0.127
#
0.273
#
0.320
#
0.360
$
0.380
$
0.460*
0.633*
0.753
$
0.990
$
*- intense,
$
- Moderately intense,
#
- Faint
340 Pharmacognosy Research | November 2010 | Vol 2 | Issue 6
Figure 2: Effect of aqueous and ethanolic extracts on overall cell
viability-MTT assay. Mean±S.D.
Figure 1: (a) HPLC prole of aqueous extract, (b): HPLC prole of ethanolic extract (c): HPLC prole of Thymoquinone
Figure 3: Relative cell survival-SRB assay. Mean± S.D.
Phytochemical analysis and total polyphenol and
avonoid contents of aqueous and ethanolic extracts
Phytochemical screening carried out using standard
laboratory procedures demonstrated the presence of
alkaloids, avanoids, saponins, tannins, steroids, reducing
sugars and phenolic compounds in both the aqueous and
Samarakoon, et al.: Cytotoxic role of a standardized poly-herbal mixture
Pharmacognosy Research | November 2010 | Vol 2 | Issue 6 341
ethanolic extracts. Higher saponin content was found in the
aqueous extract than in the ethanolic extract [Table 3]. As
seen in Table 4, a greater content of total polyphenolic and
avanoid compounds were found in the ethanolic extract
when compared to the aqueous extract.
TLC analysis of water extract and ethanolic extracts.Table 5
summarizes the R
f
values of spots visible in the TLC
proles of the aqueous and ethanolic extracts.
HPLC analysis of the aqueous and ethanolic extracts
Each extract was subjected to reverse phase chromatography
as described in the ‘Materials and Methods’ section. As
apparent in Figures 1a and 1b, most of the major peaks in
the aqueous extract appeared at retention times < 20 min
while in the ethanolic extract, major peaks with retention
times up to 40 min were also observed. This prole was
observed in all 15 samples of each extract that was analyzed.
A comparison of the HPLC proles of the aqueous
and ethanolic extracts (Figures 1a and 1b) with that of
thymoquinone (a cytotoxic component present in N. sativa
seeds) showed (Figure1c) that thymoquinone (retention
time of 35.98 min) was only present in the ethanolic
extracts and not in the aqueous extracts.
Effect on overall cell viability-MTT assay
The effects of the aqueous and ethanolic extract on
overall activity of HepG2 cells were tested by MTT
assay [Figure 2]. Both the aqueous extract and ethanolic
extracts demonstrated a dose-dependent reduction in the
overall activity of HepG2 cells with the maximum effect
at concentrations > 4800 µg/ml. The dose causing 50%
inhibition, ED
50
`s of 24-h post incubation periods of
aqueous extract and ethanolic extract were 1600 µg/ml,
and 2400 µg/ml respectively. The inhibition by aqueous
extract was marginally (though signicantly) greater (P<
0.05) than that mediated by the ethanolic extract at all the
different concentrations tested.
Relative cell survival-SRB assay
Results of the SRB assay are summarized in Figure 3. As
evident from this gure, a dose-dependent inhibition of
cell survival as assessed by the SRB assay was observed
with both aqueous and ethanolic extracts. As was observed
in the MTT assay, the inhibition by aqueous extract was
signicantly (P < 0.05) greater than that mediated by the
ethanolic extract at all concentrations tested.
DISCUSSION
Aqueous and ethanolic extracts prepared from a mixture
containing equal proportions of N. sativa seeds, H. indicus
roots and S. glabra rhizomes were standardized by evaluating
different parameters such as organoleptic characters,
physicochemical properties, chemical constituents, HPLC,
and TLC proles. The percentage yield of the ethanolic
extract and ethanol-extractable matter were greater than the
percentage yield of aqueous extract and water-extractable
matter. A higher content of total ash, acid-insoluble ash and
water-soluble ash were found in the aqueous extract. Our
phytochemical screening revealed that both aqueous and
ethanolic extracts contained secondary metabolites such as
alkaloids, avanoids, saponins, tannins, steroids, reducing
sugars and phenolic compounds. However, higher saponin
content was found in the aqueous extract than in the
ethanolic extract. HPLC proles showed the presence of
both polar and non-polar compounds in the two extracts.
A higher aggregation of polar compounds was seen in the
aqueous extract. In all 15 samples (purchased at different
times during the year) analyzed by TLC, the same number
of spots with the same Rf values were observed. In the
HPLC analysis also similar peak proles was observed in
all the samples analyzed. These results show that there was
batch to batch consistency of the plant materials used in the
preparation of the extracts. The therapeutic value of any
drug depends not only on its clinical efcacy, but also in its
lack of toxic side-effects. In this investigation, heavy metal
analysis showed that the aqueous and ethanolic extracts
did not contain detectable levels of Cd, Pb, As and Hg. A
previous in vivo investigation by Iddamaldeniya et al.,
[23]
has
also shown that the aqueous extract (decoction) does not
produce any signicant toxic effects. These results indicate
that the extracts are relatively safe to use for therapeutic
purposes.
The results of this study also demonstrated that both
the standardized aqueous extract and ethanolic extract
prepared from N. sativa (seeds), H. indicus (roots), and
S. glabra (rhizome) could exert a strong dose-dependent
cytotoxicity to human hepatocellular carcinoma (HepG2)
cells in vitro as assessed by the inhibitory effects in the
MTT and SRB assays. Results with the aqueous extract
provide support for the dose-dependent in vitro cytotoxic
potential of the decoction reported recently by Thabrew et
al.
[10]
When comparing the cytotoxic potential of these two
extracts, the aqueous extract showed a marginally (though
signicantly, P<0.05) higher activity than the ethanolic
extract at all concentrations tested. Thymoquinone is an
active compound isolated from N. sativa seeds that has been
shown to be cytotoxic to several parental and multidrug-
resistant human cancer cells.
[24,25]
HPLC proles showed
that thymoquinone was present in the ethanolic extract
but not in the aqueous extract. Thus, compounds other
than thymoquinone appear to mediate the cytotoxicity
of the standardized aqueous extract of this poly-herbal
preparation.
Samarakoon, et al.: Cytotoxic role of a standardized poly-herbal mixture
342 Pharmacognosy Research | November 2010 | Vol 2 | Issue 6
CONCLUSIONS
Standardization of aqueous and ethanolic extracts of
a mixture of N. sativa (seeds), H. indicus (roots), and S.
glabra (rhizome) has been carried out according to WHO
guidelines. Data obtained may be used as a diagnostic
tool for the correct identication of these aqueous or
ethanolic extracts. Of the two extracts, the aqueous extract
demonstrated marginally greater cytotoxicity despite the
absence of thymoquinone in this extract and a higher
number of chemical components being extracted into
the ethanolic extract as evident from the TLC and HPLC
proles. Results obtained in the present study would be
useful for the preparation of a standardized pharmaceutical
product that may be used in the future for clinical therapy
of hepatocellular carcinoma.
ACKNOWLEDGMENTS
The financial support provided by the National Science
Foundation (NSF), Sri Lanka, and the assistance given by
Mrs. Menuka Arawwawala and Mr. Asitha Siriwardana, ITI
Colombo, to conduct the phytochemical analysis are gratefully
acknowledged.
REFERENCES
1. Coleman WB, Tsongals GJ. The molecular basis of human
cancer. New Jersey: Humana Press Inc; 2002. p. 3.
2. Irfan A, Dileep NL. Malignant tumours of liver. Surgery
2006;25:34-41.
3. Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma.
Lancet 2003;362:1907-17.
4. Natcher Conference Center National Institutes of Health
Bethesda. Maryland: Screening, Diagnosis, and Management;
Apr 1-3. 2004.
5. Jang M, Cai L, Udeani GO, Slowing KV, Thomas CF, Beecher
CW, et al. Cancer chemopreventive activity of resveratrol, a
natural product derived from grapes. Science 1997;275:218-20.
6. Shoeb M. Anticancer agents from medicinal plants. Bangladesh.
J Pharmacol 2006;1:35-41.
7. Reichert JM, Wenger JB. Development trends for new cancer
therapeutics and cancers. Drug Discov Today 2008;13:30-7.
8. Cragg GM, Newman DJ. Plants as a source of anti cancer
agents. Ethnopharmocology. Oxford UK: Eolss Publishers;
2006.
9. Wang MW, Hao X, Chen K. Biological screening of natural
products and drug innovation in China. Phil Trans R Soc B
2007;362:1093-105.
10. Thabrew MI, Mitry RR, Morsy MA, Hughes RD. Cytotoxic
effects of a decoction of Nigella Sativa, hemidesmus indicus,
and smilax glabra on human hepatoma Hep G2 cells. Life Sci
2005;77:1319-30.
11. Iddamaldeniya SS, Thabrew I, Wickramasinhe SMDN,
Ranathunga N, Thammitiyagoda MG. Protection against
diethylnitrosoamine-indused hepatocarcinogenesis by an
indigenous medicine comprised of Nigella sativa, Hemidesmus
indicus, and Smilax glabra. J Carcinog 2003;2:6-11.
12. Iddamaldeniya SS, Thabrew I, Wickramasinhe SM, Ranathunga
N, Thammitiyagoda MG. A long term investigation of anti
hepatocarcinogenic potential of indigenous medicine comprised
of Nigella sativa, Hemidesmus indicus, and Smilax glabra. J
Carcinog 2006;5:11-4.
13. Sanjay J, Sweta S, Rakesh B, Praveen K. Standardization of
‘Dashamularishta’: A Polyherbal Formulation. Phramocog J
2009;1:215-20.
14. Organization Mondiale De La Sante. Quality control methods
for plant materials,559, Rev.1, Original English, World Health
Organization; 1992. p. 8-67.
15. Harbone JB. Phytochemical methods. 2
nd
ed. London:
Chapmann and Hall; 1984. p. 8-33.
16. Evans WC. Trease and Evans Pharmacognosy. Hartcourt Brace
and Company Asia Pvt. Ltd; Singapore: 1997.
17. Harborne JB. Phytochemical Methods. London: Chapman and
Halls; 1998.
18. Spanos GA, Worlstad RE. Inuence of processing and storage
on the phenolic composition of Thompson seedless grape juice.
J Agric Fd Chem 1990;38:1565-71.
19. Meda A, Lamien CE, Romito M, Millogo J, Nacoulma OG.
Determination of total phenolic, avonoid and proline content
in Burkina Fasan honey, as well as their radical scavenging
activity. Fd Chem 2005;91:571-7.
20. Csuros M, Csuros C. Environmental sampling analysis. London:
Lewis Publishers CRC Press Company; 2002. p. 372.
21. Mitry RR, Sarraf CE, Havlik R, Habib NA. Detection of adenovirus
and initiation of apoptosis in hepatocellular cells after Ad-p53
treatment. Hepatology 2000;31:885-9.
22. Oka M, Maeda S, Koga N, Kato K, Saito T. A modied colorimetric
MTT assay adapted for primary cultured hepatocytes: Application
to proliferation and cytotoxic assay. Biosci Biotechnol Biochem
1992;56:1472-3.
23. Iddamaldeniya SS, Wickramasinhe SM, Thabrew I,
Thammitiyagoda MG, Ranathunga N. An investigation of tocxic
effect of a herbal formulation with anti carcinogenic properties.
Cey J Med Sci 2005;48:12-23.
24. Worthen DR, Ghosheh O, Crooks PA. The in vitro anti-tumour
activity of some crude and puried compounds of black seed,
Nigella sativa. Anticancer Res 2001;18:1527-32.
25. Rooney S, Ryan MF. Effects of alpha- hederin and thymoquinone,
constituents of Nigella sativa, on human cancer cell lines.
Anticancer Res 2005;25:2119-204.
Source of Support: National Science Foundation (NSF), Sri
Lanka, Conict of Interest: None declared.
Samarakoon, et al.: Cytotoxic role of a standardized poly-herbal mixture
... HPLC analysis of these two extracts (aqueous and ethanolic) of the herbal formulation has shown the presence of both polar and non-polar compounds in their analysis. Furthermore, a higher aggregation of saponins has also been observed in the aqueous extract [5]. However, a concise bio-assay guided fractionation has not been carried out for the isolation of individual plant components of this standardized herbal formulation. ...
Article
Full-text available
A Sulforhodamine-B assay-guided fractionation on Nigella sativa seeds was conducted to determine the presence of cytotoxic compounds against human hepatoma (HepG2) cells. Initially, a freeze-dried sample of Nigella sativa seeds was sequentially extracted into solvents of increasing polarities. Crude extracts from the sequential extraction of Nigella sativa seeds in chloroform and ethyl acetate showed the highest cytotoxicity. The combined mixture of these two extracts was subjected to bioassay guided fractionation using a modified Kupchan method of partitioning, followed by Sephadex® LH-20 chromatography. This chromatographic separation process resulted in a column fraction with a convincing IC50 (half-maximal inhibitory concentration) value of 13.07µg/ml, which is considerable for developing therapeutic drug leads against human hepatoma. Reversed phase High-Performance Liquid Chromatography (HPLC) was finally conducted for the same column fraction, and the result indicates the presence of one or several main cytotoxic compounds against human HepG2 cells.
... Black cumin is also categorized as a plant with anti-inflammatory and antioxidant potential (Jerrine et al., 2017;Ramadan, 2007). Nutrients and antioxidant compounds such as phenolic compounds, flavonoids, polyunsaturated fatty acids, phospholipids, amino acids, proteins, carbohydrates, crude fibers, terpenoids and saponins are present in the essential oil isolated from black cumin (Samarakoon et al., 2010). The main bioactive compounds present in Nigella sativa L. seeds are thymoquinone, p-cymene and carvacrol (Palabıyık and Aytaç, 2018). ...
... Black Cumin have the potential to be developed into dietary supplements as food preservative and for the improvement of human nutrition and health. In addition, the seeds could be useful for therapeutical purposes and could be developed as anticancer agent and as foodborne preservative as well as for the treatment of chronic inflammatory pathologies associated with overproduction of nitric oxide (Samarakoon et al., 2010).Black cumin seeds have saponin and alpha hederin and in trace amount has carvone,limonene and citronellol, as well as prove relatively good amounts of different minerals such as a Fe,Ca,K,Zn,P,Cu (Forouzanfar et al., 2014). The major merits of herbal medicine seem to be their perceived efficacy, long term treatment with lesser efficacy. ...
Article
Full-text available
Background: Origanum vulgare (referred to as Spanish thyme and wild marjoram),is a member of the plant family Lamiaceae. Oregano contains potent components which contributes towards the cardiovascular and nervous systems, relieves symptoms of inflammation, and modulates blood sugar and lipids. Coffee which contains polyphenols attributes to a number of pharmacological activities that include antioxidant, antiinflammatory, immunomodulatory, anti-microbial, anti-cancer, cardioprotective and neuroprotective effects. Nigella sativa, black caraway is also called kalonji or nigella, and more common in the Far East, Mideast, Bangladesh, India and Africa. Nigella sativa contains active ingredients, in particular, thymoquinone, the main active constituent known for anti-inflammatory effect. Aim: To evaluate the antiinflammatory effect of oregano, coffee and black cumin formulation. Materials and methods: This study includes the usage of about 1g of oregano, coffee and black cumin extracts mixed with 100 mL of distilled water and boiled for 15 minutes, filtered and again concentrated till 10mL.The antiinflammatory activity of this formulation is assessed by using Bovine Serum Albumin assay. Results: The anti-inflammatory activity of oregano, coffee and black cumin aqueous formulation shows about 65% of inhibition when compared with standard.
... High-Performance Liquid Chromatography (HPLC) analysis of these two extracts (aqueous and ethanolic) of the herbal formulation has shown the presence of both polar and non-polar compounds in their analysis. Furthermore, a higher aggregation of saponins has also been observed in the aqueous extract [5]. However, a concise bio-assay guided fractionation has not been carried out for the isolation of individual plant components of this standardized herbal formulation. ...
Presentation
Full-text available
A Sulforhodamine-B assay-guided fractionation on Nigella sativa seeds was conducted to determine the presence of cytotoxic compounds against human hepatoma (HepG2) cells. Initially, a freeze-dried sample of Nigella sativa seeds was sequentially extracted into solvents of increasing polarities. Crude extracts from the sequential extraction of Nigella sativa seeds in chloroform and ethyl acetate showed the highest cytotoxicity. The combined mixture of these two extracts was subjected to bioassay guided fractionation using a modified Kupchan method of partitioning, followed by Sephadex® LH-20 chromatography. This chromatographic separation process resulted in a column fraction with a convincing IC50 (half-maximal inhibitory concentration) value of 13.07µg/ml, which is considerable for developing therapeutic drug leads against human hepatoma. Reversed phase High-Performance Liquid Chromatography (HPLC) was finally conducted for the same column fraction, and the result indicates the presence of one or several main cytotoxic compounds against human HepG2 cells.
... Working solutions were prepared in RPMI immediately before use. We assessed the toxicity of the drugs using the sulforhodamine B assay as previously described [12] and found that all the test drugs were not toxic to the macrophages at the concentrations that were used in this study ( Table 1, and Figure S1 in Supplementary Materials). We used hydrocortisone (catalog no. ...
Article
Full-text available
Ion transport modulators are most commonly used to treat various noncommunicable diseases including diabetes and hypertension. They are also known to bind to receptors on various immune cells, but the immunomodulatory properties of most ion transport modulators have not been fully elucidated. We assessed the effects of thirteen FDA-approved ion transport modulators, namely, ambroxol HCl, amiloride HCl, diazoxide, digoxin, furosemide, hydrochlorothiazide, metformin, omeprazole, pantoprazole, phenytoin, verapamil, drug X, and drug Y on superoxide production, nitric oxide production, and cytokine expression by THP-1-derived macrophages that had been stimulated with ethanol-inactivated Mycobacterium bovis BCG. Ambroxol HCl, diazoxide, digoxin, furosemide, hydrochlorothiazide, metformin, pantoprazole, phenytoin, verapamil, and drug Y had an inhibitory effect on nitric oxide production, while all the test drugs had an inhibitory effect on superoxide production. Amiloride HCl, diazoxide, digoxin, furosemide, phenytoin, verapamil, drug X, and drug Y enhanced the expression of IL-1β and TNF-α. Unlike most immunomodulatory compounds currently in clinical use, most of the test drugs inhibited some inflammatory processes while promoting others. Ion pumps and ion channels could therefore serve as targets for more selective immunomodulatory agents which do not cause overt immunosuppression.
... Working solutions were prepared in RPMI immediately before use. We assessed toxicity of the drugs using the Sulforhodamine B assay as previously described [12], and found that all the test drugs were not toxic to the macrophages at the concentrations that were used in this study (Table 1 below, and Figure S1 in Supplementary Materials). We used hydrocortisone (catalog no. ...
Preprint
Full-text available
Ion transport modulators are most commonly used to treat various non-communicable diseases including diabetes and hypertension. They are also known to bind to receptors on various immune cells, but the immunomodulatory properties of most ion transport modulators have not been fully elucidated. We assessed the effects of thirteen FDA approved ion transport modulators namely ambroxol HCl, amiloride HCl, diazoxide, digoxin, furosemide, hydrochlorothiazide, metformin, omeprazole, pantoprazole, phenytoin, verapamil, drug X and drug Y on superoxide production, nitric oxide production and cytokine expression by THP-1 derived macrophages that had been stimulated with ethanol-inactivated Mycobacterium bovis BCG. Ambroxol HCl, diazoxide, digoxin, furosemide, hydrochlorothiazide, metformin, pantoprazole, phenytoin, verapamil and drug Y had an inhibitory effect on nitric oxide production, while all the test drugs had an inhibitory effect on superoxide production. Amiloride HCl, diazoxide, digoxin, furosemide, phenytoin, verapamil, drug X and drug Y enhanced the expression of IL-1β and TNF-α. Unlike most immunomodulatory compounds currently in clinical use, most of the test drugs inhibited some inflammatory processes while promoting others. Ion pumps and ion channels could therefore serve as targets for more selective immunomodulatory agents which do not cause overt immunosupression.
... All drugs were used at concentrations that were below their respective maximum free plasma concentrations when administered to humans at therapeutic doses (see Table 1). We also assessed the toxicity of the test drugs to THP-1 derived macrophages using the sulforhodamine B assay as described previously [5], and all the test drugs were only used in subsequent experiments at concentrations that were not toxic to the macrophages ( Figure S1 in Supplementary Materials). ...
Article
Full-text available
There is an urgent need for better and safer therapeutic interventions for tuberculosis (TB). We assessed the effects of FDA-approved ion transport modulators, namely, ambroxol HCl, amiloride HCl, diazoxide, digoxin, furosemide, hydrochlorothiazide (HCTZ), metformin, omeprazole, pantoprazole, phenytoin, verapamil, and drug X and Y on the growth of free and intracellular Mycobacterium bovis BCG. Free and intracellular M. bovis BCG were cultured in the presence or absence of the test drugs for 3 to 9 days and then quantified. For both free and intracellular bacteria, cultures that were exposed to furosemide, phenytoin, or drug Y yielded lower bacteria counts compared to drug-free controls (p < 0.05). The same was observed with diazoxide, HCTZ, verapamil, and drug X, but only for intracellular M. bovis BCG (p < 0.05). To assess the effects of the drugs on bactericidal activity of rifampicin, free and intracellular M. bovis BCG were treated with rifampicin alone or in combination with each of the thirteen test drugs for 3 to 9 days. For extracellular bacteria, higher bacteria clearance rates were observed in cultures exposed to rifampicin in combination with amiloride HCl, diazoxide, digoxin, furosemide, HCTZ, metformin, pantoprazole, phenytoin, drug X, or drug Y than those exposed to rifampicin alone, indicating that rifampicin had a synergistic effect with these test drugs. Rifampicin was also synergistic with ambroxol HCl, diazoxide, digoxin, furosemide, HCTZ, omeprazole, pantoprazole, phenytoin, verapamil, and drug X against intracellular M. bovis BCG. The antimycobacterial properties exhibited by the ion transport modulators in this study make them viable candidates as adjuncts to the current anti-TB regimens.
Chapter
Hemidesmus indicus (L.) R. Br. is commonly known as Indian Sarsaparilla or Anantmula. Traditionally, it has been utilised as a vital herb for the treatment of several disorders. Indian Sarsaparilla is rich in a wide range of phytoconstituents such as pregnane glycosides, steroids, terpenoids, aromatic aldehydes, lignans, saponins, flavonoids and aliphatic acids which may further contribute to its pharmacological properties. This chapter gathers and compiles the traditional ethnobotanical and ayurvedic aspects of H. indicus and recently updated knowledge regarding the pharmacology, phytochemistry, adulteration, and current trends of this medicinally important herb in the field of modern phytomedicine. It also presents the ayurvedic pharmacology of this herb and summarizes the biomedical researches in as much as it helps glean a better understanding of H. indicus safety and effectiveness in humans, and describes the various natural products and polyherbal medicines containing H. indicus.
Book
The book is in the field of theory and research in health sciences. TİP 2 DİYABETES MELLİTUS’TA İNKRETİN BAZLI TEDAVİLERDE KULLANILAN İLAÇLARIN ADMET SONUÇLARININ İN SİLİKO OLARAK KARŞILAŞTIRILMASI: SWISSADMET VE ADMETSAR are given in chapter 57.
Book
Temel Fetal Kalp Muayenesi
Article
Standardization of herbal formulations is essential in order to assess the quality of drugs, based on the concentration of their active principles. The present work is an attempt to standardize Dashamularishta, a traditional formulation, used in the normalization of physiological processes after child birth. Four marketed preparations and three in-house preparations were used for the study. The various parameters performed included organoleptic characteristics, physicochemical and toxicological parameters. HPTLC was carried out for quantitative analysis of piperine in all the formulations. The results obtained may be considered as tools for assistance to the regulatory authorities, scientific organizations and manufacturers for developing standards.
Article
Malignant tumours of the liver are classified as primary or secondary (metastatic). Some of the tumours present with deranged liver functions tests and symptoms, but most remain asymptomatic with normal liver function tests.Common primary tumours of the liver are hepatocellular carcinomas and cholangiocarcinomas. The former are confined to the liver and usually originate in cirrhotic livers. Cholangiocarcinomas arise from the intra-or extrahepatic biliary tree. Secondary tumours of the liver account for 95% of hepatic malignancies, are manifestations of systemic spread of the primary, and usually indicate end-stage disease. Metastases from colorectal cancer and neuroendocrine tumours are two types of cancer in which liver resection is potentially curative.More liver tumours are being discovered incidentally due to improved imaging techniques; CT, MRI and PET offer earlier diagnosis and staging. Tumour markers and liver biopsy help in diagnosing selected cases.Treatment of liver tumours requires a multidisciplinary approach. This should be offered in tertiary referral centres equipped with a full range of diagnostic and therapeutic facilities. Surgery offers a potential cure for liver tumours. New chemotherapeutic agents and ablation techniques are promising and are being evaluated in the clinical setting. Recent medical advances have made treatment of malignant liver tumours safer and potentially curative.
Article
Plant-derived compounds have played an important role in the development of several clinically useful anti-cancer agents. These include vinblastine, vincristine, the camptothecin derivatives, topotecan and irinotecan, etoposide, and paclitaxel (Taxol®). Several promising new agents are in clinical development based on selective activity against cancer-related molecular targets, including flavopiridol and Combretastatin A4 phosphate. Recently, plants have yielded several agents showing anti-AIDS activity, and one of these, (+)-calanolide A, is in clinical development.
Article
The science of molecular biology has been responsible for a dramatic change in our understanding of cancer. This understanding has led to improvements in the ability to diagnose and treat certain malignancies. Unfortunately, despite these advances in patient care, molecular biology remains an elusive and perplexing topic for many physicians. While a minority are able to stay abreast of advances in the field of molecular biology, this often requires a rather manic reading of the literature and a keen eye for new and significant information. Oncology was one of the first fields to benefit from advances in molecular biology and undoubtedly will benefit from additional advances in the future. For these reasons, it is important that each practitioner of oncology have at least a general understanding of the molecular mechanisms at work in the clinic. An alternative to an exhaustive reading of the literature is a well-written textbook. The textbook
Article
Methodology for measurement of phenolics in fruit juices using HPLC separation and diode array detection is presented. Quantitation of phenolic acids and flavonol glycosides was achieved with minimum sample preparation. Procyanidin quantitation, however, required removal of interfering compounds with Sephadex LH-20 minicolumn chromatography. Good reproducibility and high recoveries (up to 92.3%) were achieved in procyanidin isolation. The methods were used to study the effect of SO2, enzymatic clarification, fining, bottling, concentration, and storage on the phenolic composition of Thompson Seedless grape juice. SO2 addition during processing resulted in higher levels of phenolic acids and procyanidins, but it had no apparent effect on the quercetin glycoside composition. Oxidation of caftaric acid to 2-S-glutathionylcaftaric acid was evident in juices processed both with and without SO2. Enzymatic clarification caused hydrolysis of caftaric, coutaric, and quercetin derivatives, but it showed no effect on the 2-S-glutathionylcaftaric acid. Procyanidins demonstrated sensitivity to the heat applied during bottling and concentration. Storage of concentrates for 9 months at 25°C led to the formation of (hydroxymethyl)furfural (HMF) (up to 33.5 mg/L), extensive oxidation of cinnamics, and total loss of procyanidins and quercetin glycosides. Colorimetric measurement of phenolics showed no correlation with the HPLC quantitation.
Chapter
IntroductionProblems Associated with SamplingSynple Fractions and Sample PreservationSampling of Lakes. Rivers, and GroundwaterSampling on Ice, Snow, Rain, Dew, and FogReferences
Chapter
IntroductionWhy Sample Water?Elements of the Sampling PlanTypes of SamplesSampling ProgramsSampling EquipmentPumps and Representative SamplesAdvancements in SamplingPreserving Sample IntegrityConclusion
Article
Transcription of the p53 gene can regulate progression of apoptosis in a wide variety of tissues. Three categories of human hepatocyte culture have been used to show the initiation of apoptosis after treatment with p53-bearing adenovirus. Chang liver cells are derived from normal liver tissue and express native p53, whereas hepatocellular carcinoma (HCC)-derived cell lines were Hep3B (p53-deleted) and PLC/PRF/5 (p53-mutant). Cultures were infected with Ad-p53 (15 particles per cell; 36 hours), and after treatment, morphological changes in all cell categories were observed by electron microscopy. Infection was evident in the cytoplasm of all treated cell types: after entry across the plasma membrane viruses translocated and came to rest surrounding and adjacent to nuclei, cytoplasm proximal to nuclear membranes became dense with virus- and membrane-derived debris, but intact viruses did not enter nuclei. Apoptosis, recognized morphologically by characteristic chromatin and cytoplasmic condensation, occurred more frequently in HCC-derived cells, and the ultimate fate of apoptotic bodies was phagocytosis and degradation by neighboring cells.
Article
Several honey samples (27) from Burkina Faso were analyzed to determine their total phenolic, flavonoid and proline contents as well as their radical scavenging activity. These samples consisted of 18 multifloral, 2 honeydew and 7 unifloral honeys, derived in the latter cases from flowers of Combretaceae, Vitellaria, Acacia and Lannea plant species. The total phenolic contents varied considerably with the highest values obtained for honeydew honey. Similarly, much variation was seen in total flavonoid and proline content, with Vitellaria honey having the highest proline content. Vitellaria honey was also found to have the highest antioxidant activity and content. The correlation between radical scavenging activity and proline content was higher than that for total phenolic compounds. This suggests that the amino acid content of honey should be considered more frequently when determining its antioxidant activity.