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In mining, geology, and quarry production, it is well known that the properties of aggregates,
such as size and shape, are very important information for particle characterization and the
optimization of production. For the last fifteen years, image analysis techniques have been
used for aggregate particle measurement, which increases speed and accuracy of analysis.
There are a number of methods for measuring aggregate particle size and shape in image
analysis, the stability of measurement methods is very important.
Palynology is the study of plant pollen, spores and certain microscopic plankton organisms
(collectively termed palynomorphs) in both living and fossil form. Botanists use living pollen
and spores (actuopalynology) in the study of plant relationships and evolution, while
geologists (palynologists) may use fossil pollen and spores (paleopalynology) to study past
environments, stratigraphy (the analysis of strata or layered rock) historical geology and
paleontology.
Melissopalynology is the study of pollen in honey, with the purpose of identifying the source
plants used by bees in the production of honey. This is important to honey producers because
honey produced by pollen and nectar from certain plants as mesquite, buckwheat, tupelo or
citrus trees demand a higher price on the market than that produced by other plant sources.
Some plants may produce nectar and pollen that is harmful to human health. A careful
monitoring of the pollen types found in honey may identify these toxic sources and the honey
produced may be kept out of the commercial market.
Pollen grains are showing two nuclei. The smaller (generative) nucleus forms two sperm
cells. Endoplasmic reticulum threads present throughout the pollen grain and several
mitochondria and plastids containing starch.
2
PLATE 1
Fig: 1. Mother Plants
A.
Yellow variety B. Pink variety C. Orange variety
Fig: 2. Mother Plants with Scale Reading
3
OBJECTIVES
Mirabilis jalapa Linn. belongs to the family Nyctaginaceae and is a large herbaceous plant
grown in gardens throughout India. The present project work entitled
"Studies on
antimicrobial, biochemical and image analysis in Mirabilis jalapa
varities" contains
following objectives:
1. Staining of pollen grain and observation of meiotic stages.
2. Phytochemical studies of 3 different colored plant varieties of M.jalapa by TLC
3. Comparative Evaluation of Antimicrobial Activities of plant leaf Extract (3 different
colored varieties) of Mirabilis jalapa.
4. Image analysis of data got by experimentation using software.
4
REVIEW OF LITERATURE
Nature has been a source of medicinal agents for thousands of years and an impressive
number of modern drugs have been isolated from natural sources, many based on their use in
traditional medicine. Various medicinal plants have been used for years in daily life to treat
disease all over the world. They have been used as a source of medicine. The widespread use
of herbal remedies and healthcare preparations, such as those described in ancient texts like
the Vedas and the Bible, has been traced to the occurrence of natural products with medicinal
properties. In fact, plants produce a diverse range of bioactive molecules, making them a rich
source of different types of medicines. Higher plants, as sources of medicinal compounds,
have continued to play a dominant role in the maintenance of human health since ancient
times
1
. Over 50% of all modern clinical drugs are of natural product origin
2
and natural
products play an important role in drug development programs in the pharmaceutical
industry
3
.
There has been a revival of interest in herbal medicines. This is due to increased awareness of
the limited ability of synthetic pharmaceutical products to control major diseases and the
need to discover new molecular structures as lead compounds from the plant kingdom. Plants
are the basic source of knowledge of modern medicine. The basic molecular and active
structures for synthetic fields are provided by rich natural sources. This burgeoning
worldwide interest in medicinal plants reflects recognition of the validity of many traditional
claims regarding the value of natural products in health care.
The relatively lower incidence of adverse reactions to plant preparations compared to modern
conventional pharmaceuticals, coupled with their reduced cost, is encouraging both the
consuming public and national health care institutions to consider plant medicines as
alternatives to synthetic drugs. Plants with possible antimicrobial activity should be tested
against an appropriate microbial model to confirm the activity and to ascertain the parameters
associated with it. The effects of plant extracts on bacteria have been studied by a very large
number of researchers in different parts of the world
4-6
. Much work has been done on
ethnomedicinal plants in India
7-9
. Interest in a large number of traditional natural products
has increased
10
. It has been suggested that aqueous and ethanolic extracts from plants used in
allopathic medicine are potential sources of antiviral, antitumoral and antimicrobial
agents
11, 12
. The selection of crude plant extracts for screening programs has the potential of
5
being more successful in initial steps than the screening of pure compounds isolated from
natural products
13
.
This plant is 50-100 cm high. It has antifungal, antimicrobial, antiviral, antispasmodic,
antibacterial, diuretic, carminative, cathartic, hydragogues, purgative, stomachic, tonic and
vermifuge properties.
14
This plant contains alanine, alphaamyrins, arabinose, beta-amyrins,
campesterol, daucosterol and dopamine
15
, and is used to treat conjunctivitis, edema, fungal
infections, inflammation, pains and swellings.
Mirabilis jalapa L. (Nyctaginaceae) is a tropical American herb
that is commonly cultivated
in North America where it is perennial
in the south and warm west and annual in the north. In
Mirabilis jalapa pollen performance was influenced by the
number of competing pollen
grains or pollen tubes, but was not
influenced by potential genetic differences with load
diversity
16
.
B P Cammue et al., has isolated from seeds of Mirabilis jalapa L. two antimicrobial peptides,
designated Mj-AMP1 and Mj-AMP2, respectively. These peptides were also active on two
tested Gram-positive bacteria but were apparently nontoxic for Gram-negative bacteria and
cultured human cells
17
.
J Kataoka et al., 1991, cloned a cDNA for Mirabilis antiviral protein (MAP), a ribosome-
inactivating protein (RIP), which inhibits the mechanical transmission of plant virus and the
in vitro protein synthesis of both prokaryotes and eukaryotes
18
. A potent antiviral activity
was found in extracts from a yellow flower cultivar of Mirabilis jalapa L. (Nyctaginaceae) in
root, leaf, and stem tissues and in in vitro cultured cells
19,20
.
Extracts of Mirabilis jalapa (Nyctaginaceae), containing a ribosome inactivating protein
(RIP) called Mirabilis antiviral protein (MAP), were tested against infection by potato virus
X, potato virus Y, potato leaf roll virus, and potato spindle tuber viroid. Several plants, such
as Pelargonium hortorum, Chenopodium album, C. amaranticolor, Capsicum frutescens,
Azadirachta indica, Vitis vinifera, and Rosa banktia, possess antiviral factors. Plant-derived
antiviral compounds are active against plant, animal, and human viruses. Plant antiviral
compounds are grouped as furocoumarins, alkaloids, terpenoids, lignins, and specific
proteins. Among plant-derived antiviral proteins, a group called ribosome-inactivating
6
proteins (RIPs), which are widely distributed in higher plants, hold promise for agricultural
and pharmaceutical applications
21
.
7
MATERIALS AND METHODS
Study species
Mirabilis jalapa has tubular
flowers are fragrant and vary in color among plants. The self-
compatible,
perfect flowers each have 56 stamens and a single-ovulate
ovary. An individual
flower opens for one night in the early
evening, the exact time depending on temperature and
relative
humidity, and closes early the next morning. An individual plant
produces between
25 and 75 flowers in one flowering season.
Growth form: Annual or perennial herb.
Size: 0.5 - 2 m tall.
Root : a swollen and somewhat tuberous taproot.
Stems: usually several, erect to slightly decumbent, branching, light or bright green,
sometimes with a yellow or pink hue, mostly hairless, but sometimes hairy near the base or
even glandular-hairy further up; if hairy, often in two lines.
Leaves: about midstem and above, opposite, on 1 - 7 cm long stalks, somewhat elongate
triangular to egg-shaped or lance-shaped, 4 - 14 cm long, 2 - 9 cm wide, with blunt or
indented bases, and non-toothed edges.
Inflorescence: of several, terminal or axillary, compact clusters with one to fifteen flowers
on short, 0.5 - 5 mm long stalks, and each cluster subtended by a pair of 2 - 17 mm long leaf-
like bracts. Each flower sits atop a green, 0.5 - 1.5 cm tall, bell-shaped cup (involucre)
formed by five fused bracts with triangular tips.
Flowers: pink or yellow or orang, sometimes white or striped, usually hairless, 3 - 5 cm long,
radially symmetric, funnel-shaped with a long, narrow tube, and five, abruptly flared lobes.
Sepals: showy, brightly colored, not green, and mimicking petals. The five sepals are fused
for most of their length, constricted above the ovary into a long, narrow tube, then separated
into five, abruptly flared lobes.
Petals: none.
Stamens: five, long, and extending beyond the sepal tube.
Pistil: with one, single-chambered, superior, somewhat globular ovary; one, long, threadlike
style, which extends beyond the stamens; and a rounded, head-like stigma.
Fruit: a hard, dark brown or nearly black, 0.7 - 1.1 cm long, broadly ellipsoid to inversely
egg-shaped, one-seeded, nut-like achene, which is tightly enclosed by the remnant bract cup
8
(involucre). The achene is round or slightly five-angled in cross-section, smooth or
inconspicuously bumpy or warty, and either hairless or hairy.
Plant Materials
M. jalapa (yellow, orange and pink flower cultivars) were collected from plants grown in the
garden of the GITAM Institute of sciences, GITAM University, Visakhapatnam during
winter season.
Meiosis in flower buds of Mirabilis jalapa
Materials: M.jalapa flower bud, Tween 80, acetocarmine stain, glass slides, cover slips,
blotting paper.
Procedure:
1. Select appropriate flower buds of different size from the inflorescence.
2. Fix them in Tween 80 fluid, which is used as fixative.
3. Take a preserved flower bud and place it on a glass slide.
4. Separate the anthers and discard the other parts of the bud.
5. Put one or two drops of acetocarmine and squash the anthers.
6. Leave the material in the stain for five minutes.
7. Place a cover slip over them and tap it gently with a needle or pencil.
8. Warm it slightly over the flame of a spirit lamp.
9. Put a piece of blotting paper on the cover slip and apply uniform pressure with the
thumb.
10. Observe the slide under the light microscope for different pollen grain meiotic stages at
10X, 45X and 100X.
Image Analyser v 1.31
The GSA Image Analyser v 1.21 (2009) is a program for the scientific evaluation of 2D
images (image analysis). The possibilities of the program are varied and can be divided in
three main groups:
9
x Object surface calculation
x Object length calculation
x Object counting
Surface calculation
The surfaces of all recognized objects are automatically calculated. In addition, the surface
calculation of single objects is possible. The program offers the possibility to calculate the
relations of the ascertained surfaces to each other and to the whole image or background.
Object length calculation
The GSA Image Analyser has a set of functions which have been developed especially for the
direct length calculation of very complex and much ramified objects. In addition to the direct
length calculation, an indirect calculation with a grid intersection (developed by Tennant) is
possible. For this function, the grid size can be freely defined.
Object counting
The automatic count-function determines the number of all recognised objects and, in
addition, offers the possibility to count overlapping objects apart. This separation can make
over the object form, the object surface and over the object intensity.
The grid-intersection-count-function serves for the indirect object number regulation. Such
procedures are described, e.g., by Buerker, Fuchs-Rosenthal, Thoma, shilling, Tuerk.
Beside the automatic count-functions, the program has two manual count-procedures
included. One method marks the objects inside of the image and the other cuts the image into
several smaller parts to make it easier for human eyes to follow many small objects.
Picture import
The GSA Image Analyser can read almost all known picture formats (JEPEG, GIF, TIFF,
BMP, PNG). In addition, the program provides interfaces to scanners, microscopes and
digital cameras as well as to video cameras (Video grabber, TV cams).
10
Picture creation and manipulation
A picture production by means of input device (graphics tray, mouse) is integrated in the
program and allows a cloning of objects. In addition, there exist picture manipulation tools
and different filter functions.
Calibration
This function allows calibrating the input tools as well as determining the picture resolution
(DPI) with picture formats who do not contain this information.
This program is designed to solve many problems. It has been used for counting cells and
calculating the size of plant.
Image Analyzer is a robust, proprietary, real time image analysis system. The engine is
supplied with an API, is external dependency independent and supports multiple platforms
(Windows/LINUX/Unix etc). It is small in footprint, fast and accurate when identifying
pornographic or inappropriate image content within all popular digital data transmission
protocols.
Anti microbial activity
Sample extraction
The determined Fresh plant leaves (200g) were ground, extracted with Diethyl ether, ethyl
acetate and methanol separately and filtered. The plant residue was re-extracted by adding
above solvents and filtered again after 48hs. Such procedure was repeated every 72hs,
completing three filtration processes. The filtrate was concentrated on a rotary evaporator at
45ºC for solvent elimination, and the extracts were kept in sterile bottles under refrigerated
conditions until use. The final volume is adjusted to a concentration of 1 mg/ml with the
above solvents separately.
11
Test microorganisms
The microbial strains are identified strains and were obtained from the MTCC, IMTECH,
Chandigarh, India. The bacterial strains studied are Eschericia coli MTCC 739,
Staphylococcus aureus MTCC 737, Klebsiella pneumoniae MTCC 109, Bacillus subtilis
MTCC 441 and Aspergillus niger MTCC 282.
Antimicrobial assay
The antibacterial assays were performed by the agar well diffusion method. Petri dishes (200
mm) were poured with nutrient agar (HI-Media) and allowed to solidify to make base layers.
The seed layers were prepared by inoculating 10mL of test organism suspension in 100 mL
Mueller-Hinton agar(for bacteria) and Sabouraud Dextrose agar (for fungi) and wells, 6 mm
in diameter, were made in the agar medium with the help of a sterile steel borer. About 100
L of each extract was added aseptically in wells. All the plates were incubated at 37 ± 1°C
for 24 hours in the upright position. At the end of the incubation times, the diameters of the
inhibition zones were measured in millimeters. Ampicillin (20 g/mL) and solvent (diethyl
ether, ethyl acetate, and methanol 20 g/mL) were used as antimicrobial compounds against
text microorganisms. The tests were conducted in triplicate.
Thin layer chromatography (TLC)
Thin layer chromatography (TLC) is a chromatography technique used to separate mixtures.
The present study is performed on a sheet of glass, which is coated with a thin layer of
adsorbent material, usually silica gel. This layer of adsorbent is known as the stationary
phase.
After the sample has been applied on the plate, a solvent or solvent mixture (known as the
mobile phase) is drawn up the plate via capillary action. Because different analytes ascend
the TLC plate at different rates, separation is achieved.
The TLC analysis was performed on glass slides pre-coated with silica gel G/GF
(E-Merck
grade). Before use, glass slides were pre-washed with methanol, and dried in an oven at
105°C for 1 hour.
12
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