AROMATIC
AND MEDICINAL PLANTS
AMP01 ECBALLIUM ELATERIUM
L.: A SOURCE OF CUCURBITACIN E, A CYTOTOXIC TETRACYCLIC
TRITERPENOID
Ecballium elaterium L. Is a local medicinal plant which
stores several compounds, falling under the name of
cucurbitacins. Cucurbitacin E is obtained from the dried fruit
juice and has been found to possess cytotoxic properties. In this
project, a comparative study, regarding the cytotoxicity of
cucurbitacin E and busulphan on two human cell lines (ovarian and
stomach cell lines) was carried out.
The investigation was divided into two parts:
Part 1: To determine the inhibition of ovarian and stomach cancer
cell growth when these were exposed to different concentrations
of the two compounds. The cell viability was followed up from day
0 to day 11 and using the Exclusion Dye Technique (Trypan blue as
dye).
Part II: To observe any morphological changes provoked by
cytotoxic agents on the ovarian cancer cell line. The cells were
examined after one hour and twenty-four hours. The Papanicolau
staining procedure was followed.
From the first part of the investigation, it was observed that
cucurbitacin E had a marked effect on the ovarian cancer cell
line while busulphan showed a similar effect when exposed to
stomach cancer cell line.
From the second part of the investigation, it was found that
cucurbitacin E not only caused irreversible damage in the cell
membrane after one hour, but the damage was more pronounced after
twenty-four hours. These changes were apparent in untreated cells
or cells treated with busulphan.
For cucurbitacin E-treated ovarian cancer cells, it can be
concluded that since there was a pronounced cell kill exponential
curve (from part one) and it caused irreversible destruction of
the cytoplasm (from part II), then cucurbitacin E exerts its
action via the process of pinocytosis on the cell membranes.
Busulphan has no effect on the pinocytic process and so it exerts
its action on stomach cancer cells, by alkylation reactions at
the nucleus of cells.
Research Worker: Everaldo Attard
Project Supervisor: Prof. Anthony Scicluna-Spiteri
AMP02 CRATAEGUS MONOGYNA
JACQ.: A SOURCE OF OLEANOLIC ACID, A POTENTIAL
ANGIOTENSIN-CONVERTING ENZYME INHIBITOR
Crataegus monogyna Jacq., a local wild medicinal plant,
contains several compounds that possess various pharmacological
properties, including effects on the cardiovascular system. The
compound of interest in this dissertation was oleanolic acid, a
pentacyclic triterpenoid present in the plant in the free state
or salt form.
Phytochemical Investigation: This investigation was aimed at the
extraction of oleanolic acid and determination of its quality and
quantity by several analytical assays.
Oleanolic acid (0.0496 %) was extracted from air-dried fruit.
This involved Soxhlet extraction, partitioning of the compound in
diethyl ether, dissolution in acetone and preparative layer
chromatography, successively. The purity of oleanolic acid was
determined by TLC, IR and UV spectroscopy. The ethanolic extract
was tested for other compounds by spectrophotometric and
colorimetric techniques. It resulted that this extract contained
flavonoids and coumarins, but no catecholamines or organic acids.
The quantity of triterpenoids obtained from the plant material
was 0.8976 %, of which oleanolic acid constituted only 5.53 %.
Therefore the yield of oleanolic acid by the ripe fruit was low.
Pharmacological Investigation: This was aimed at assessing the
angiotensin-converting enzyme inhibitory activity of oleanolic
acid and the ethanolic extract when compared to a known ACE
inhibitory, captopril.
The technique was adapted from a diagnostic test kit (Sigma) and
employing a microtiter plate for the scanning of a large number
of samples simultaneously at a wavelength of 340 nm. It resulted
that captopril possessed the highest ACE blocking activity,
followed by oleanolic acid and the ethanolic extract (3.15 x 10-6
M, 1 x 10-4 M and 92.3 %), respectively.
It was concluded that oleanolic acid was present in the ethanolic
extract, together with other constituents. The yield of oleanolic
acid was low and therefore other environmental parameters should
be considered. The preparative layer chromatography was an
efficient purification process for this compound.
Since oleanolic acid was an effective ACE inhibitor, it was
evident that some constituents in the extract inhibited its
activity, therefore the anti-hypertensive activity of the extract
cannot be attributed to the inhibitory effect of oleanolic acid
on the angiotensin-converting enzyme.
Research Worker: Henrietta Borg
Project Supervisor: Prof. Anthony Scicluna-Spiteri
AMP03 STUDIES ON THE MICROPROPAGATION
OF THE LOCAL SQUILL: DRIMIA MARITIMA (L.)
STEARN
Drimia maritima is the only local medicinal plant which
was harvested and exported and thus, besides being important for
its medical properties it also contributed to the national
economy in the past. The development of rural areas into
agricultural or urban areas is endangering the plant. Thus the
need of conservation of this plant is being felt more than ever
before.
Micropropagation methods were thus investigated for their
potential to increase the stock of local squill. Explants were
taken from several parts of the plant for the studies. A surface
sterilisation technique had to be developed first since squill
was never studied before. After the explants, taken from various
plant parts, were surface sterilised successfully, experiments
were conducted to regenerate new plantlets from the explants.
Direct and indirect organogenesis techniques were investigated by
exposing the different explants to several types of nutrient
media.
Moreover, the production of secondary metabolites by in vitro
plant cell cultures was to be investigated. Nowadays, active
ingredients which used to be extracted from intact plants are
produced from fermenters in which plant cells are being cultured
in a liquid nutrient medium. Callus tissue had thus to be
initiated so that it could be then analysed for the presence of
the active glycosides.
Drimia maritima (L.) Stearn was regenerated successfully
using direct organogenesis of 4-5 mm explants consisting of bulb
scales joined at the base by a small piece of base plate. This is
the first record for Drimia species to obtain bulblet
formation on explants in vitro. The base plate explants
took an extremely long time and several subcultures on various
hormone concentrations were needed for the induction of bulblet
formation. Callus tissue was also successfully initiated on
explants retrieved from seedlings, but only after using unusually
high concentrations of auxins (4 mg/l 2,4-D + 2 mg/l NAA).
Indirect organogenesis, that is the formation of roots from the
callus tissue, was obtained when the callus tissue was removed
from high auxin medium to a hormone balanced medium. The
production of secondary metabolites by the callus tissue was not
conducted because the amount of callus tissue obtained from the
explants by the end of this study was not sufficient.
Research Worker: Etienne Cassar
Project Supervisor: Prof Radmilla Vujicic
AMP04 AN EVALUATION OF THE LOCAL
PUMPKIN SEED (CUCURBITA MAXIMA) AS AN
ANTHELMINTIC AGENT
A literature review was carried out on the use of the pumpkin
seed as an anthelmintic agent. Investigations were later made to
evaluate such claims.
For this purpose, faecal specimen from 135 dogs in Malta were
examined for cestodes. Three coprological methods were used
to aid the indemnification process: direct method, concentration
technique and flotation method. Twenty-four dogs tested were
positive. Out of this number twenty-one dogs naturally infected
with cestodes, were assigned to three groups: non-medicated,
control group (n=7), medicated dogs given a pumpkin seed Cucurbita
maxima mixture (n=7) and medicated dogs given a standarised
anthelminthic preparation, Vermox(R) (n=7). Medication was given
orally in a suspension formulation. Faeces were examined prior to
and after treatment for passed segments. In the nonmedicated
control group, segments were still detected after day 14. In the
second group of dogs, the segment count first increased reaching
a peak on the second day after treatment and then markedly
decreases until no segments were found by the sixth day after
treatment. As the pumpkin seed preparation was not accepted by
all dogs in this group (n=3), these had to be excluded from the
study. When the standardised preparation was administered
no segments were seen after the fourth day after treatment
(n=6). The latter preparation was well accepted by all dogs
without side-effects. Future studies should be carried out to
determine the anthelminthic constituents of the plant Cucurbita
maxima, thus enabling dose titration studies to be performed.
Moreover, the possible mode/s of action of the plant still need
to be elucidated.
Research Worker: Lucienne Farrugia
Project Supervisors: Prof. Anthony Scicluna-Spiteri & Dr.
Trevor Zammit
AMP05 A FEASIBILITY STUDY OF THE
CULTIVATION OF NERIUM OLEANDER L. IN MALTA
A previous investigation carried out on Nerium oleander
Linn. gave positive results in the quantity of cardiac glycoside
Oleandrin. When this quantity was compared with that of
other countries, it was found that the local shrub may supply
more glycoside. However it was noticed that there was a
difference in glycosidal content according to the season when the
cuttings were taken. This thus left many areas where one
could specialise and a study was carried out to investigate the
effect of the seasonal variations on the content of oleandrin.
The aim of this study was to find out the content of oleandrin in
the cuttings collected all throughout the year and to estimate
the feasibility of producing the glycoside locally. The
method used was extraction with ethanol and readings were taken
using the UV spectrophotometer. It was observed that the
content of oleandrin remained high during most of the months and
is estimated to be quite feasible to produce when compared with
the cost of oleandrin on the local market.
Research Worker: Corinne Briffa
Project Supervisor: Prof. Anthony Scicluna-Spiteri
AMP06 STUDIES ON IN VITRO
PROPAGATION AND ROOTING OF DWARF ALMOND (P. TENELLA)
The effects of various propagation media on in vitro
development of P. tenella cultures obtained from the
Institute for Biological Research - Belgrade were studied.
Plantlets were grown on two-phase Murashige and Skoog (MS)(1962)
multiplication media which proved to be excellent for growth
purposes of P. tenella, and then subcultured onto solid
phase media containing variable amounts of inorganic salts and
plant growth substances to induce elongation and rooting.
Chilling the plantlets at 4°C for 30 days was a highly effective
mode to induce elongation of growing shoots.
Supplementation of multiplication media with GA3
(1mg/l) promoted shoot elongation and reduced plantlet necrosis.
The level of auxin necessary for rooting, contrary to other
members of Prunus seems to be relatively low.
In fact rooting was obtained at a very low concentration
(0.5mg/l) or in the absence of IBA. These studies
confirm that although at times, the complete absence of cytokinin
may be implicated in lack of rooting, it is not absolutely
necessary for root induction.
The salts of Lepoivre (LP) have proved to be a superior
ingredient in root inducing protocols than MS salts and this
could probably be due to the decreased amount of ammonium salt
and increased concentration of calcium in LP salts.
Research Worker: Kenneth Mifsud
Project Supervisor: Prof Radmilla Vujicic
AMP07 THE ISOLATION AND POTENTIAL USE
OF GLYCOLIC ACID PRESENT IN THE LOCAL PLANT ERICA
MULTIFLORA L.
Erica multiflora L. 1753 is a perennial, evergreen
shrub widely distributed in the lowlands of the Mediterranean
Basin.
Literature was reviewed for the medicinal properties and
constituents of the plant. Not much information was gathered but
it was found out that Erica multiflora L. has been used in
traditional medicine as an astringent, urinary antiseptic and
diuretic.
Glycolic acid (alpha-hydroxyacetic acid) was the active component
responsible for this diuretic potential (Balansard, 1951). Other
constituents of the shrub are tannins, concrete oil and
cyanogenic glycosides. The botanical aspect and habitat of the
plant were also reviewed.
Glycolic acid was extracted by the following procedure adapted
from that described originally by Balansard (1951). Decoctions
were prepared from the plant, which were then precipitated by
lead II acetate to remove the tannins. 96% ethanol was added to
the filtrates and any excess lead was decomposed by sodium
phosphate. The solutions were filtered and then bleached by
activated carbon. After evaporation, the residues were extracted
with a mixture of diethyl ether and petroleum ether. The solvent
was evaporated and the residues were collected.
Titrations were performed for quantitative determination of the
acid. The residues were dissolved in distilled water and to each
of the above solution, potassium iodate solution was added and
after half an hour, the solutions were titrated with 80% 10 Molar
hydrochloric acid.
The percentage content of glycolic acid in the decoctions and the
percentage recovery of the acid from the residues were
calculated. A cream containing 2% glycolic acid was formulated,
using the residues as a source of the acid and Aqueous Cream
(also prepared by myself) as the base.
The cream was tested for its irritancy/intolerability on five
volunteers. In only one case, a slight prickling sensation was
felt. No visible hypersensitivity reactions were observed.
Therefore from the results obtained, it was concluded that the
cream can be used safely for topical application. Erica
multiflora L. qualifies as a good source of glycolic acid and can
be incorporated in topical formulations. The plant can substitute
the synthetically produced acid or glycolic acid-containing
plants which do not grow in the Maltese Islands, that are
currently being incorporated in commercially available skin care
products.
Research Worker: Maureen Delia
Project Supervisor: Prof Anthony Scicluna-Spiteri
AMP08 PHYTOCHEMICAL AND MICROSCOPICAL
ANALYSIS OF HYOSCYAMUS ALBUS
Man has found either by accident or method of trial and error,
since early times, during his tireless quest for food, that
certain wild plants help to alleviate some of his sufferings from
diseases and injuries. One of such plant is Hyoscyamus albus
Common Name: Henbane, White Henbane, Russian Henbane, Jusquiame
(France), Mammazejzy, (Maltese), Henbel, (Anglo-Saxon).
Parts Used: Fresh Leaves, Flowering Tops, Branches and Seeds.
Habitat: Native of the Mediterranean region; found also in Russia
and the south of Europe. (1) The plant grows abundantly in arid
places, Malta, Gozo and Comino (2) and in disturbed habitats,
especially rubble, old walls and fortifications (3). It is
therefore a plant that occurs frequently in waste, sandy places,
by roadsides, on rubbish heaps, near old buildings, on chalky
grounds and particularly near the sea.
The plant can easily be distinguished from the other species of
hyoscyamus by the bracts, as well as the leaves being all stalked
and by the pale yellow colour of the flowers (1).
The plant is a biennial herb, sometimes annual or perennial,
erect, branched densely and flowers all year round, with flowers
generally pale yellow with brown centre. The leaves are greyish
with soft hairs and all petiolate, corolla without purple veins
but sometimes with purplish throat (3).
Aim: To investigate: the microscopic characteristics, and the
chemical composition of Hyoscyamus albus as identification and
authentication of the crude drug.
Study 1: Preparation of crude drug.
Various parts of the plant, roots, flowering tops, leaves and
branches were separated and dried in an oven at 100 C to 105 C
for 24 hours.
Study 2: Loss of weight on drying:
This was determined by drying separately exactly 10g each of
fresh: flowering tops, braches, roots and leaves, in an oven at
100 C to 105 C for 24 hours.
Study 3: Microscopic analysis. Small quantity of dried powered
leaves was taken into a clean slide. Three drops of chloral
hydrate solution was added. The cover slide was carefully placed
from one edge to prevent the formation of air bubbles. The slide
was then flamed twice to remove any trapped air bubbles; heating
was stopped as soon as bubbles started forming, and two drops of
chloral hydrate solution ran under the cover slide. The slide was
then viewed from low to high magnification. An upper epidermis
leaf fragment of about 5mm by 5mm was heated for 15 minutes in
chloral hydrate solution on a water bath. It was then transferred
on to a slide and covered with a cover slid. The specimen was
observed with the micrometer ocular and a low power objective.
The same procedure was repeated for a lower epidermis leaf
fragment
Study 4: Subjective drug testing:
Odour, taste, and Touch, were determined. The plants parts used
were; leaves, flowering tops and branches, mixed together, in the
ratio 3:2:1.
Study 5: Determination of total alkaloid content:
The total alkaloid content (calculated as Hyoscyamine) of the
leaves, flowering tops, roots and branches were determined by
quantitative analysis (extraction followed by titration)
Study 6: Qualitative analysis by chromatography:
Qualitative analysis of the leaves, roots, branches and flowering
tops extracts were carried out by thin layer chromatography.
High total alkaloid content (calculated as hyoscyamine) was found
in the flowering tops, 0.26. The least total alkaloid content was
observed with the branches. The roots should also be included as
part of the plant part used, based on it’s high total
alkaloid content.
The zones in the chromatograms obtained with the extract
solutions were similar to those in the chromatograms obtained
with the reference solutions, with respect to their position
(hyoscyamine in the lower quarter, hyoscine in the upper quarter)
and their colour (orange-red). The zones in the chromatograms
obtained with the extract solutions were almost equal in size to
that obtained, with the corresponding reference solution, using
the same spotting technique and volume. The chromatograms can
thus be use as quick identification for the crude drug.
Atropine appeared on the same spot with hyoscyamine on the
chromatograms. This indicates that, atropine is likely not a
constituent of the plant, but a product of hyoscyamine changes
during extraction.
The distinctive microscopic characteristics of the stomata and
trichome, as described in the results:
“Epidermal cell: Wavy anticlinal walls with smooth cuticle
Stomata: Anisocytic and anomocytic: more abundant on the lower
epidermis.
Trichome: 1 Glandular; uniseriate stalk composed of two to six
cells with, thin smooth walls and multi-cellular head: more
abundant.
2.Covering, less abundant, unseriate and conical, composed of,
from two to four thin-walled smooth cells.
Both trichomes were found scattered or attached to fragments of
the epidermis and were frequently broken. They were both
un-branched and non collapsible.
Calcium Oxalate Crystals: Less abundant, occurs in the spongy
mesophyll and are of varied shapes and sizes”, can serve as
diagnostic characteristics for the crude drug.
Research Worker: Atabongnkeng Michael Fuangunyi
Project Supervisor: Prof. Anthony Scicluna-Spiteri
AMP09 PHYTOCHEMICAL AND PHARMACOLOGICAL
ANALYSIS OF LOCALLY CULTIVATED MOMORDICA
CHARANTIA
Momordica charantia is a member of the Cucurbitaceae
family. Gourd family, is widely used in traditional
medicine in the Orient and Carribean. Some common names
used for Momordica charantia are Balsam Pear, Boston Pear, Bitter
Gourd, Bitterweed, Bitter melon, Carillon Kho Qua and K’u
Kua.
The general description of the plant is as follows- ‘Vines
with square sided stems, slender, weak, creeping or climbing
stems, musky odour’ (T Johnson 1990). Leaves are dark
green, alternate with 5 to 7 toothed leaflets. the fruit is
1 to 8 inches long or more, bright orange when ripe, oval,
pointed, fleshy, splits into 3 parts which curl back. These
enclose irregularly shaped elliptic brown seeds.
The major aim of the project was:
To investigate the insulin content of various parts of the plant
i.e. fruit, leaves and seeds. This was then to be compared
with standard human insulin.
Study 1 – Different parts of the plant were separated,
dried, and after appropriate treatment were boiled in distilled
water for 3 hours.
Study 2 – In this study fresh fruit and seeds were used
. Finely divided fruit was placed in a conical flask
and 20ml of distilled water, 90ml of 95% ethanol and 7.2ml
of sulphuric acid were added to the fresh fruit. This
mixture was mixed for 20 minutes. The mixture was then
divided into two portions i.e. solution A and solution B.
To solution A, 60ml of distilled water and 250ml of 95% ethanol
were added simultaneously. Solution B was further
divided into two i.e. solution C and solution D. To
solution C, 60ml of distilled water were added. To solution
D, 250ml of ethanol were added. Acetone was then added
until a white precipitate was formed. Samples from the
three mixtures were taken and zinc solution and EDTA added to
increase the purity of the solution.
Study 3 - Qualitative analysis of extracts obtained from
study 1 and study 2 were carried out by performing thin layer
chromatography.
Study 4 – Quantitative determination of insulin content
within different plant parts was carried out using UV
spectrophotometry. The results obtained were compared with
absorbance of standard insulin.
Study 1 - After processing the dried plant parts the
following solutions were obtained: Leaves (Dark brown colour
solution), Fruit (yellowish brown solution) and Seeds (Pale
yellowish solution)
Study 2 – On precipitation of the hypoglycaemic agent by
using acetone, a cloudy precipitate was obtained. The
precipitate was left for several hours in a refridgerator.
After this time a white precipitate in all containers was
obtained, however the quality varied in each container.
Study 3 – Plates were sprayed with ninhydrin and after
placing plates within the oven at 120C for about 20 minutes, a
yellow colour (spots) was developed.
Study 4 – Two peaks were obtained at 226nm and a shoulder
peak at 273nm. Seeds gave the greatest absorbance value,
followed by fruit.
Different extraction procedures did not effect amount of active
ingredient obtained but had a great influence on the amount of
impurities present within the sample. From the TLC study it
was found out that active ingredient present within the plant
corresponds quite well to the human insulin preparation. It
was concluded that 0.1137mg of p-insulin are present in 1ml (when
considering crude fruit extract). Maximal percentage
insulin was found to be 0.2965. This was found in the seed
extract.
Research Worker: Darleen Zerafa
Project Supervisor: Prof. Anthony Scicluna-Spiteri
AMP10 ECBALLIUM ELATERIUM (L.)
A.RICHARD IN MALTA: THE GROWTH, QUALITY AND POTENTIAL OF THE MALTESE SQUIRTING
CUCUMBER AS A SOURCE OF THE ANTI-CANCER TETRACYCLIC TRITERPENOID,
CUCURBITACIN E
Ecballium elaterium L. is a local medicinal plant,
containing elaterium, an extract rich in cucurbitacins (Cu),
particularly cucurbitacin E (CuE).
The aims of this dissertation were: to determine the potential
production of CuE in tissue culture. to investigate further the
effects of the compound on normal and malignant cell lines.
A series of experiments correlating biomass accumulation and
secondary metabolite production were carried out. Two experiments
were carried out simultaneous to study the quality of calluses,
the quantitative accumulation of cucurbitacins in tissue culture,
and the effects of different PGRs on a particular callus strain.
The best callus strain was strain 14 obtained from a seed excised
from an immature fruit collected from M'Scala. The Cu and CuE
contents correlated with the chlorophyll content of the calluses,
indicating that a photosynthetic callus is capable of producing
secondary metabolites. The PGR treatments of choice were
NAA/BAP for maximum biomass accumulation and 2,4-D/Ki for the
optimum cucurbitacin accumulation. The initial mixed PGR grid was
carried out on strain 3 that was abundant at that time. Further
experiments were carried out with strain 14. It was observed that
when performing the above-mentioned 2-PGR grids, a growth-linked
accumulation of secondary metabolites was observed. The lower
metabolite yields with the 2,4-D/Ki combination prompted further
investigations on the behaviour of callus in suspension with the
above-mentioned treatments. It was observed that 2,4-D/Ki-treated
calluses showed the greatest degree of metabolite leakage
To regenerate plants from tissue culture, surface sterilised
fruit were aseptically excised, the immature seeds taken and
germinated on MS medium. The sterile plantlets obtained were
excised and the cotyledons were cultured on MS medium containing
0.1 mg/L NAA and 1 mg/L BAP for four weeks, to enhance bud
multiplication. Explants were then treated with various plant
growth regulators (PGRs) or additives (i.e. IAA, IBA, NAA, 2,4-D,
Ki, BAP, GA3 and charcoal). Shoots developed on most
media but elongation was best observed with GA3. Some
of the GA3-treated plantlets were treated for one week
with IAA (auxin shock) that did not actually produce any effects
on shoot, root or callus proliferation. With unsuccessful
rooting, half of the plantlets were then treated with rooting
hormone powder (NAA), and all planted in Jiffy® pots
and allowed to grow in a hot room. The plants that were given the
auxin shock and then treated with rooting hormone showed the
greatest mean apical height over a period of 75 days. The GA3-treated
plantlets developed with greater difficulty especially the
non-rooting hormone treated ones.
From the pharmacological point of view, CuE was tested against
five cell types; breast carcinoma (ZR-75-1), melanoma (COLO 679),
prostate (PC-3), fibroblasts (L929) and peripheral lymphocytes,
all obtained from the ECACC except for the peripheral lymphocytes
that were obtained from healthy human male volunteers.
The cultured cells were treated with different CuE
concentrations. Specific control drugs were used for different
cell lines. The total counts, viability, cytotoxicity,
proliferation, cell morphology and apoptosis, were studied over a
period of 72 h, in most cases. Control and untreated cells were
assayed likewise.
CuE exhibited a marked effect on PC-3 cells at a median
inhibitory concentration (IC50) of 9.35 nM and
moderate effects on COLO 679 and ZR-75-1 cells (IC50 =
0.87 and 1.95 mM, respectively).
Parameters that showed a reduction in cell viability were
prominent with the drug, as compared to the controls.
Morphologically, the cancer cells exhibited nuclear and
cytoplasmic changes such as condensation of chromatin, an
increase in the nuclear to cytoplasmic ratio, and rounding up of
the cytoplasm. Surface blebbing and morphological signs of
apoptosis occurred in all cancer cell types. In the agarose-gel
electrophoresis analysis, DNA ladder characteristic of apoptosis,
was exhibited by the CuE treatment on both PC-3 and ZR-75-1 cell
lines, as for tamoxifen and mesterolone. Negligible cytotoxic
effects were observed on the L929 cells and human T-lymphocytes
as compared to the control. The fact that this compound did not
damage human T lymphocytes but instead stimulated their
activation and proliferation is a positive sign demonstrating
that CuE is non- cytotoxic to normal cells and does not induce
apoptotic changes seen with several other drugs. It was also
observed that the compound is non-toxic even after the cells had
been stimulated with PHA.
The common parameter studied in the two different research
fields, was CuE. The plant has been found to be a potential in
vitro source for this compound and the latter has shown to be
effective against certain cancer cell lines, not investigated or
partly investigated previously. From the conclusions reached,
research work could be directed towards bioreactor studies to
optimise further, the yield of secondary metabolites in tissue
culture, especially Cu and CuE. Also, animal trials, especially
toxicological studies should be performed to determine the toxic
effects of CuE on various organs and the immune system in vivo.
Research Worker: Everaldo Attard
Project Supervisor: Prof. Anthony Scicluna-Spiteri & Prof.
Mark P. Brincat
AMP11 THE ANTHELMINTHIC PROPERTIES OF SOME MALTESE PLANTS
A bio-assay for in vitro screening of compounds
with potential anthelminitc activity is proposed using the free living nematode
Caenorhabditis elegans (Rhabditida) as a model.
The aqueous, methanolic, acetone, ethyl acetate, chloroform, and petroleum ether
extracts of Ruta chalepensis leaves (Rutaceae), Punica granatum (Punicaceae)
fruit pericarp and the seeds of two local Cucurbita maxima (Cucucbitaceae)
cultivars were screened for anthelmintic activity using the proposed bio-assay.
The essential oil of R. chalepensis has also been assessed for
anthelmintic activity. The activities of the extracts were compared to the drugs
ivermectin and piperazine citrate as gold standards.
Preliminary screening and subsequent concentration-response
analysis showed that the most potent anthelmintic extracts in decreasing rank of
potency were R. chalepensis essential oil (IC50 = 342.3 μg ml-1)
> P. granatum aqueous extract (IC50 = 852.8μg ml-1)
> C. maxima “Hamra” aqueous extract (IC50=1076 μg ml-1)
> C. maxima “Torka” aqueous extract (IC50 = 1230μg ml-1).
The calculated IC50 for ivermectin and piperazine citrate were equal
to 646 hg
ml-1 and 173 μg ml-1 respectively.
Analysis of co-variance showed that the anthelmintic
concentration-response profiles of all four extracts were not statistically
significant from that shown by piperazine citrate at p=0.05. Only the
concentration-response profile of P. granatum was significantly different
from that shown by ivermectin at p=0.05.
Other extracts showing appreciable anthelmintic activity
included C. maxima “Hamra” methanolic extract, C. maxima “Torka”
methanolic extract, and the P. granatum chloroform extract exhibiting 72,
61, and 49% inhibition of motility at a concentration of 10 mg ml-1.
The methanolic and acetone extracts of P. granatum fruit pericarp also
showed interesting partial inhibition of motility and lysing of nematode
cuticles though complete inhibition of motility was not observed.
The results obtained tend to confirm the local ethnomedical
use of these plants for de-worming practices. These results also indicate
potential anthelmintic activity of the constituents found in these plants
against nematodes of veterinary importance including the trichostrongyloids, to
which C. elegans is most closely related according to current
phylogenetic classifications.
Research Worker: Joseph Vella
Project Supervisor: Prof. Anthony Scicluna-Spiteri & Dr. Everaldo Attard
AMP12
The Chemistry and Pharmacology Of the Local Capparis
spinosa L.
Capparis spinosa
is a member of the genus Capparis, which has been established to posses
culinary and medicinal properties. The caper plant metabolises a large number
of secondary metabolites mainly flavonoids, alkaloids, terpene glycosides,
organic acids and glucosinolates.
In this present study, great
attention has been devoted to the secondary metabolites that are present in the
plant with the scope of correlating the pharmacological activity of extracts to
their content of secondary metabolites.
Aerial parts have been used for
this study, mainly consisting of leaves and branches. Five extractions were
carried out on five aliquots of plant material, with five solvents namely water,
water/methanol, methanol, chloroform and petroleum ether. The solvents have a
decrease in polarity when going from water to the petroleum ether.
The extracts obtained were dried
and the highest content of extract was observed for the aqueous solvent (3.015
%), followed by the aqueous/methanol solvent (2.401 %), methanol (1.398 %), then
the chloroform (0.196 %) and finally petroleum ether (0.020 %).
The first three extracts
contained important secondary metabolites in the order of terpenoids, flavonoids
and alkaloids, respectively. This was confirmed by the phytochemical
colorimetric tests carried out, using six tests. The three classes of
metabolites were determined by the triphenyltetrazolium test, the acidified
vanillin test and the Dragendorff’s test. Three other tests used were the
xanthoproteic test for proteins, the Fehling’s test for carbohydrates and the
Sudan IV test for fats, all of which are considered as primary metabolites.
The brine shrimp test proved a
high activity of the aqueous, followed by the methanol and finally the aqueous/methanolextract
(LC50 = 0.014 %, 0.0475 % and 0.08 %, respectively). Values below
0.1 % are expected to exhibit in vitro
cytotoxicity
to various cancer cell lines.
From the results, it was
concluded that the extraction was successful yielding important metabolites with
high activity against the brine shrimp. Considerations, for further
phytochemical analysis and further in vitro pharmacological testing, have
been suggested.
Research Worker: Marie Josette Parnis
Project Supervisor: Dr. Everaldo Attard
AMP13
Urtica dioica
Agglutinin: A
Lectin from Stinging Nettle
The stinging nettle (Urtica dioica L.) is a local medicinal plant that
contains the plant lectin, Urtica dioica Agglutinin (UDA). UDA which is
the first lectin to be isolated from a member of the Urticaceae family is a
small monomeric protein with high contents of glycine, cysteine and trytophan.
UDA is able to agglutinate erythrocytes, induces considerable amounts of
antiviral activity in the cell cultures, and stimulates proliferation of
lymphocytes.
In this dissertation a comparative study, regarding the agglutination capacity
of UDA and PHA (Phaseolus Vulgaris Agglutinin) was carried out.
The investigation was divided into two parts:
q
Part I: The isolate and purify UDA from the leaves, stem and root
of the stinging nettle.
q
Part II: To observe the agglutination properties of UDA isolated
from the different plant parts.
From the first part of the investigations it was observed that the leaves
contain the highest amount of UDA (0.6530 %). Then the roots (0.4899 %) and
lastly the stems (0.1634 %) follow. One may assume that it is reasonable that
the leaves and roots contain higher amounts because the rhizomes are the site of
synthesis of the protein and the leaves the storage organs of the protein. To
confirm the protein content of these three extracts, three colorimetric tests
were used, that is, xanthoproteic, ninhydrin and biuret tests. All three
extracts gave a positive result to these tests.
In the second part of the investigation, the haemagglutination activity of the
extracts were compared to those of PHA. A micro-titre plate method was used in
which case the content of haemoglobin, in agglutinates that form during the
agglutination assay, was assayed at a wavelength of 405 nm. It was found that
for all the extracts and PHA, agglutination was concentration dependant.
Moreover, after 60 minutes a decline of agglutination was observed for all the
extracts and PHA. The maximum agglutination activity took place at the
60-minute interval for all the extracts and PHA, however that agglutination
activity of the PHA was considerably less than that of the UDA.
From this study it can be concluded that the extraction of the UDA was
successful, and this being confirmed by the phytochemical tests. The
agglutination of UDA in the three extracts gave a positive response compared to
the PHA. This provides ground for further research on the characteristics of the
local Urtica dioica agglutinin and studies on lymphocyte activation and
co-activity with anticancer drugs.
Research Worker: Bernardette Rossi
Project Supervisor: Dr. Everaldo Attard
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