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Rao et al. World Journal of Pharmaceutical Research
“PROTECTIVE EFFECT OF ULTRA SONIC BATH ASSISTED
METHANOL EXTRACT OF LEPIDIUM SATIVUM LINN. SEEDS BY
USING CHEMOTHERAPY INDUCED NEUROPATHY IN RATS.”
*A.T. Chenna Kesava Rao, B. Priyanka, K. Maheswari, G. Rambabu, J. Vidya Sagar
Department of Pharmacy, Vision College of Pharmacy, Boduppal, Hyderabad. Telangana.
INTRODUCTION
Medicinal Plants & Their Importance
A medicinal plant is any plant which, in one or more of its organs,
contains substances that can be used for therapeutic purposes, or which
are precursors for chemo-pharmaceutical semi-synthesis. When a plant
is designated as „medicinal‟, it is implied that the said plant is useful as
a drug or therapeutic agent or an active ingredient of a medicinal
preparation. Medicinal plants may therefore be defined as a group of
plants that possess some special properties or virtues that qualify them
as articles of drugs and therapeutic agents, and are used for medicinal
purposes. [Bailey, C.J. and Day, C et al. (1989)].
History of Plant Based Traditional Medicine
Plants have formed the basis of sophisticated traditional medicine (TM) practices that have
been used for thousands of years by people in China, India and many other countries. Some
of the earliest records of the usage of plants as drugs are found in the Artharvaveda, which is
the basis for Ayurvedic medicine in India (dating back to 2000 BCE), the clay tablets in
Mesopotamia (1700 BCE) and the Eber Papyrus in Egypt (1550 BCE). Other famous
literature sources on medicinal plant include “De Materia Medica,” written by Dioscorides
between CE 60 and 78 and “Pen Ts‟ao Ching Classic of Materia Medica” (written around
200CE).
Nowadays plants are still important sources of medicines, especially in developing countries
that still use plant-based TM for their healthcare. In 1985, it was estimated in the Bulletin of
the World Health Organization (WHO) that around 80% of the world‟s population relied on
medicinal plants as their primary healthcare source. Even though a more recent figure is not
World Journal of Pharmaceutical Research SJIF Impact Factor 6.805
Volume 5, Issue 12, 205-233. Review Article ISSN 2277– 7105
*Corresponding Author
A.T. Chenna Kesava Rao
Department of Pharmacy,
Vision College of Pharmacy,
Boduppal, Hyderabad.
Telangana.
Article Received on
23 Sept. 2016,
Revised on 13 Oct. 2016,
Accepted on 03 Nov. 2016
DOI: 10.20959/wjpr201612-7318
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available, the WHO has estimated that up to 80% of the population in Africa and the majority
of the populations in Asia and Latin America still use TM for their primary healthcare needs.
In industrialized countries, plant-based traditional medicines or phyto therapeutically are
often termed complementary or alternative medicine (CAM) and their use has increased
steadily over the last 10 years. In the USA alone, the total estimated “herbal” sales for 2005
was $4.4 billion, a significant increase from $2.5 billion in 1995. (Butlet MS.et al 2004)
However, such “botanical dietary supplements” are regulated as foods rather than drugs by
the United States Food and Drug administration(U.S FDA).
Role of Plants in Human History
Plants have also been used in the production of stimulant beverages (e.g. tea, coffee, cocoa,
and cola) and inebriants or intoxicants (e.g., wine, beer, kava) in many cultures since ancient
times and this trend continues till today. Tea (Camellia sinensis Kuntze) was first consumed
in ancient China (the earliest reference is around CE 350), while coffee (Coffea arabica L.)
was initially cultivated in Yemen for commercial purposes in the 9th century. The Aztec
nobility used to consume bitter beverages containing raw cocoa beans (Theobroma cacao L.),
red peppers and various herbs. Nowadays, tea, coffee and cocoa are important commodities
and their consumption has spread worldwide. The active components of these stimulants are
methylated xanthine derivatives, namely caffeine, theophylline and theobromine, which are
the main constituents of coffee, tea and cocoa, respectively. (K.G. Ramawat and J.M.
Mérillon et al, 2008).
Extraction methods from solid sample
Sample pretreatment
method Principles of the technique
Accelerated solvent Sample is placed in a sealed container and heated to a temperature
higher than its boiling point, causing pressure in the vessel to rise.
Automated Soxhlet A combination of hot solvent leaching and Soxhlet extraction; sample in thimble is first immersed in boiling solvent and then the
thimble is raised for Soxhlet extraction with solvent refluxing.
Forced-flow leaching Sample is placed in a flow-through tube, and solvent is pumped or pushed through high pressure nitrogen gas, while the tube is heated
near the boiling point of solvent
Gas phase
After equilibrium, analytes partition themselves between a gas phase and the solid phase at a constant ratio with static headspace extraction, volatiles are sampled above the solid; with dynamic
headspace extraction, volatiles are sampled by continuously purging the headspace above a sample with inert gas, trapping them
on a solid medium, and then thermally desorbing them into a gas
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Ultrasonic bath
Introduction
Extraction techniques are widely accepted as a prerequisite for analytical determination of
both organic and inorganic analytes in a large variety of samples. As a part of an analytical
process, sample preparation is considered to be an essential step so that the entire process can
be simplified. In this case, the ability of many analytical systems to handle liquid samples has
brought about the development of separation methods which fulfill a main objective, i.e. to
obtain quantitative analyte leaching from the solid matrix using a suitable solvent, with little
or no matrix release, so that matrix effects can be kept to a minimum during the measurement
steps.
Fig 1.6 Ultrasonic Bath Sonicator
chromatograph.
Homogenization Sample is placed in a blender, solvent is added, and sample is homogenized to a finely divided state; solvent is removed for
further work-up.
Pervaporation
Volatile substances present in a heated donor phase placed inside a pervaporation module evaporate through a porous membrane and the vapour condenses on the surface of a cool acceptor stream on
the other side of the membrane.
Solid}liquid extraction Sample is shaken together with the appropriate solvent in a container and the liquid separated by filtration
Sonication
Finely divided sample in a container is immersed in ultrasonic bath
with solvent and subjected to ultrasonic irradiation; an ultrasonic probe or cell disrupter can also be used.
Soxhlet extraction
Sample is placed in a disposable, porous container (thimble);
constantly refluxing solvent flows through the thimble and leaches out analytes that are collected continuously.
Supercritical fluid
Sample is placed in flow-through container and a supercritical fluid (e.g. CO2) is passed through sample, after depressurization,
extracted analyte is collected in solvent or trapped on adsorbent and desorbed by rinsing with solvent.
Thermal A form of dynamic headspace analysis, but the sample is heated
(controlled) to much higher temperatures (as high as 3503oC).
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Sonication is usually recommended for pretreatment of solid environmental samples for the
extraction of nonvolatile and semi volatile organic compounds from solid, such as soils,
sludges and wastes. When comparing the different methods available for analyte extraction
from solid samples, sonication is considered as an effective method since unsophisticated
instrumentation is required and solid-liquid separations can usually be performed in a short
time using diluted reagents and low temperatures. To date, most of applications of ultrasonic
extraction have been carried out for organic compounds, but the usefulness of ultrasound for
element extraction is still to be explored. In general, the presence of an acidic liquid is an
important prerequisite for quantitative extraction to be achieved; nitric acid at low
concentration (e.g. 3-5% v/v) is usually chosen for extraction of elements from solid samples.
Quantitative extraction can be achieved for some analytes such as As, Cu, Pb, Cd, etc., from
plant and animal tissues. Nevertheless, incomplete extraction has been observed from
samples containing a typical inorganic matrix (e.g. sediment). It is believed that this finding
is related to the ability of ultrasound to penetrate the solid material. The extraction efficiency
obtained with ultrasound could be increased by addition of glass beads which promote
particle disruption by focusing the energy released by cavitation and by physical crushing.
Particle disruption could also be enhanced by increasing hydrostatic pressure and viscosity.
The use of a bubbling gas during sonication gives rise to an enhanced formation of H2O2 and
hydroxyl radicals (OH) thus aiding analyte extraction from oxidizable materials.
1.6.2 Principle
The high-frequency is generated electronically and the mechanical energy is transmitted to
the sample via a metal probe that oscillates with high frequency. The probe is placed into the
cell-containing sample and the high-frequency oscillation causes a localized low pressure
region resulting in cavitation and impaction, ultimately breaking open the cells. It uses sound
waves at frequencies above the range audible to humans to disrupt the plant cell wall, thereby
enhancing solvent penetration into the plant material and facilitating the release of extracts.
Fig1.7 Longitudinal sound waves
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1.6.3 Frequency
At high sonic frequencies, on the order of the MHz, the production of cavitation bubbles
becomes more difficult than at low sonic frequencies, of the order of the kHz. To achieve
cavitation, as the sonic frequency increases, so the intensity of the applied sound must be
increased, to ensure that the cohesive forces of the liquid media are over come and voids are
created. The broad classification of ultrasound as sound above 20 kHz and up to 100MHz can
be subdivided into two distinct regions Power and Diagnostic. The former is generally at
lower frequency end where greater acoustic energy can be generated to induce cavitation in
liquids, the origin of chemical effects. Sonochemistry normally uses frequencies between 20
and 40 kHz simply because this is the range employed in common laboratory equipment.
However since acoustic cavitation in liquids can be generated well above these frequencies,
recent researches into sonochemistry use a much broader range. High frequency ultrasound
from around 5MHz and above does not produce cavitation and this is the range used in
medical imaging.
Table 1.3 Frequency ranges of sound
Review of literature
Different genus with sativum species
Coriandrum sativum
Coriandrum sativum is a annual herb belonging to the family apiaceae. Its commonly known
as dhaniyalu in telugu, Dhania in oriya, kothmiri in gujarati. It is commonly used as a
digestant. Its fruits are considered carminative, diuretic, tonic, stomachic. and as a spice.
Oryza sativa
Rice is the seed of the monocot plants Oryza sativa (Asian rice) or Oryza glaberrima
(African rice). As a cereal grain, it is the most important staple food for a large part of the
world's human population, especially in Asia and the West Indies. Rice is an ingredient of
many soups and dishes. When combined with milk, sugar and honey, it is used to make
desserts. In some regions, bread is made using rice flour.
Human hearing 16Hz – 18 kHz
Conventional power sound 20 kHz – 100 kHz
Extended range of sonochemistry 20 MHz – 2 MHz
Diagnostic ultrasound 5 MHz – 10 MHz
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Pisum sativum
It is a annual herb belonging to the family fabaceae. The mature seeds are rich in protein and
can be cooked as a vegetable or added to soups etc The sprouted seeds are added to salads,
soups bread etc The roasted seed is a coffee substitute.
Cannabis sativa
Cannabis sativa is an annual herbaceous plant in the Cannabaceae family. It has many uses
and has been used for thousands of years for paper, fabric, food and medicinally it is used in
treating chronic Pain, Headache, Gastrointestinal (Nausea, Anorexia, Abdominal pain,
Irritable bowel syndrome, Crohns disease, Ulcerative colitis, Chemotherapy)Chronic
anxiety., etc.
Different species with Lepidium genus
Lepidium meyenii
It is known commonly as maca, is an herbaceous. The prominent product for export is maca
flour, which is a baking flour ground from the hard, dried roots, "harina de maca." Maca flour
(powder) is a relatively inexpensive bulk commodity, much like wheat flour or potato flour.
In Peru, maca flour is used in baking as a flour base and a flavoring. These are biennial plant
or annual plant.
Lepidium latifolium
It is known by several common names including Pepperweed, Pepperwort, Dittander.
Lepidium perfoliatum
It is a species of flowering plant in the mustard family known by the common name clasping
pepperweed. It is native to Europe and Asia and it can be found in other parts of the world as
an introduced species. This is an annual or biennial herb belongs to family Brassicaceae. The
plant is used as an antiscorbutic.
Lepidium fremontii
It is commonly called as desert pepper weed. Family: Brassicaceae .Parts used are seeds and
is distributed in U.S.A. It is used as flavouring agent.
Ethanomedical information of Lepidium sativum
Garden cress has been considered as an important medicinal plant since vedic era. In
ayurveda it is described as hot, bitter, galactogogue and aphrodisiac and claimed to destroy
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vata (air) and kapha (phlegm). In unani system of medicine, seeds and leaves of this plant
have been reported to possess diuretics, aperients and aphrodisiac properties and are
recommended in inflammation, bronchitis, rheumatism and muscular pain. Lepidium sativum
is one of the most common plant species of genus Lepidium which is grown in Egypt for its
important uses in folk medicine for diabetes and antibacterial properties. A recent survey of
different regions of Saudi Arabia showed that the seeds are commonly used as febrifuge,
antirheumatic and he to enhance sexual desire (chopra rn). In china and other far eastern
countries, the seeds are dropsy. It is considered as the one of the better medicinal plants in
various African countries, where the seeds are chewed to cure throat disease, asthma and
headache and are useful for dieresis and rapid bone fracture healing. A tea spoon full of
garden cress seeds boiled in six ounces of water for ½ hour and the decoction with a table
spoon full of honey is given as an effective medicine to increase breast milk and sexual
disorders. Likewise, juice of the seed is also valued as medicine; one teaspoon obtained by
grinding with one ounce of water mixed with a glass full of tender coconut water given as a
folk medicine to cure diarrhea, dysentery, bleeding piles, scanty urination due to liver
disorders and irritation of the intestinal mucous membrane. Seeds roasted in ghee and mixed
with sugar are given as a tonic for a general weakness in young girls and after child birth to
increase breast milk and the oil extracted from seeds roasted in till oil is used as an analgesic
medicated oil in gout, rheumatism, glandular swelling, etc. It should be noted that this oil is
counter irritant and therefore, care must be taken to use a little at a time. (Pullaiah T,2006).
Pharmacological activities proved from Lepidium sativum
Lepidium sativum is reported to exhibit antihypertensive (Maghrani M, Zeggwagh NA,2005),
diuretic (Patel U, Kulkarni,2009), anti-inflammatory, analgesic, anticoagulant (Al-Yahya
MA, Mossa JS,1994), antirheumatic (Ahsan SK, Tariq M, Ageel M,1989), hypoglycemic
(Patole AP,1998), laxative, prokinetic (Rehman N, Journal of Ethnopharmacology. 2011),
antidiarrheal and antispasmodic (Rehman N, Mehmood MH et.al.,) properties. It has been
shown to possess antiasthmatic (Paranjape AN, Mehta AA, 2006) and bronchodilatory (Mali
RG, Mahajan SG, 2008) activity.
Active constituents isolated from Lepidium sativum
O
OH
O
sinapin
N
phenyl acetonitrile
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N
lepidine
N C S
benzyl isothiocyanate
O
OH
OO
HO
sinapic acid
2.6: Aim & Plan Of Work
• The plan of work involved collection and authentication of plant material, size reduction
after shade dried, extraction of powdered plant material using sonicator in methanol
(95%), phytochemical investigation of the extract and estimation of protective effect on
neuropathic pain.
AIM OF THE INVESTIGATION
Neuropathic pain is defined as the pain condition that results from damage affecting
peripheral nerves, posterior roots and spinal cord or certain regions of brain. Increased
neuronal excitability is thought to be the underlying mechanism involved in all forms of
neuropathic pain. Tricyclic antidepressants, often the first choice causes sedation and
cardiovascular issues and are only partially effective. Opioids prescribed for moderate to
severe pain sometimes avoided because of the potential dependence and tolerance as their
side effects. To overcome the side effects we made an attempt to develop newer drug.
Literature supports the involvement of adenosine receptors in pain. We framed the work to
study the adenosine based treatment for chemotherapy induced neuropathic pain. Adenosine
was found to be significant as it is involved in nociception.
Bearing the above points in mind Lepidium sativum is selected which is used for various
ailments like oxidative stress (anti oxidant), diabetics, cancer, aging, fatigue, pain. Hence to
identify a new drug an attempt is made to investigating the activity of Lepidium sativum to
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treat neuropathic pain by carrying out pharmacological studies. The present study was aimed
to carry out the in vivo chemotherapy induced neuropathic pain screening models.
Lepidium sativum Description
Taxonomical Classification (George H.M Lawerence,1959.)
Kingdom = Plantae
Division = Angiospermae
Class = Dicotyledonae
Sub Class = Polypetalae
Series = Thalamiflorae
Order = Parietales
Family = Cruciferae ( Brassicaceae)
Genus = Lepidium Linn
Species = Lepidium sativum Linn sp.
Description
It is an erect branched, glabrous herb with 60 cm height. Leaves are entire or pinnately
dissected, variously lobed often with linear segments; upto 6 -5 cm long and lobes are 0.7 –
1.2 to 0.3 – 0.6 cm size, upper leaves usually entire and 2 to 3 cm long, oblanceolate, sessile.
Recemes are 7 to 15 cm long axillary and terminal; flowers are pale pink; pedicels are 3 to
5mm long. Pods are obovate are broadly elliptical, roundate, emarginated slightly but thickly
winged above.
Fig: 3.1 Lepidium sativum plants
Fig: 3.2 Lepidium sativum Flowers Fig: 3.3 Lepidium sativum Leaf
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Fig: 3.4 Lepidium sativum Germinated seeds Fig: 3.5 Lepidium sativum Flex seeds
Seeds are small oval shaped pointed and triangular at one end, smooth about 2 to 3 mm long
1 to 1.5 mm wide, reddish brown and arrow present on both surfaces, extending upto two
thirds down wards, a slight wing like extension present on both the edges of seed when
soaked in water seedcoat swell and gets covered with a transparent, colourlessmucilage.
Distribution
Plant is a native of Mediterranean region. Now, it is being cultivated through out India very
likely indigenous in West Asia. In India it is mainly cultivated in U.P., Rajasthan, Gujarath,
Maharastra and Madhya Pradesh. (Indian medicinal plants Kirtikar et al.).
Cultivation & Harvesting
It is a cool season annual plant, cultivated throughout India. It has long leaves at the bottom
of the stem and small bright green feather like once arrange on opposite side of its stalk at the
top. There are plain broad leaf and curly leaf that differ in texture but not taste. Garden cress
can be harvested throughout the year whether in door or out door and is cut when the sprouts
are 5 to 10cm tall. Cress grows in well worked soil with good drainage. It flourishes in shade
or semi shade and can tolerate a wide range of temperature. while preparing the soil, it should
be dug and mixed with a well balanced fertilizer. The seed sowed thickly a 5 – 6 cm deep in
wide rows, 45 – 60 cm apart to have a continuous crop. The leaves should not remain wet
more than the requirement since the soil that lodges there when water splashes is impossible
to washout damaging the leaf. Cress has no serious pest problem. Its growth is rapid and
harvesting can begin in the same month as sowing with yields reaching as high as 6 tonnes
per hectare. The plant prefers light (sandy), medium (loamy) and heavy (clay) soils. It can
grow in semi shade (light woodland) or even without any shade. It requires moist soil and
also some shade during the summer to prevent heat from running straight to seed. It can be
grown at all elevations, the whole around. However, the best crop is obtained in the winter
season. Seeds are sown in the plains from September to February and on the hills, from
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March to September. They are sown thick and covered until germination begins. In few days
sowing the plants are ready for cutting. To get a continuous supply of leaves, sowing is done
in succession at intervals of 8 days. Garden cress leaves are consumed raw in salads; they are
also cooked with vegetable curries and used as fodder for horses, camels, etc. firm and evenly
colored rich green specimen. Its stem should be stuck in a water filled glass, then should be
selected for storage. Cress with any signs of slime, wilting or discolouration should be
avoided. The cress can be stored under refrigeration in plastic for up to five days. Its stem
should be stuck in a water filled glass, the glass bagged and refrigerated to prolong life. Until
they are needed for use, the leaves should be left on the stem. (The wealth of india new delhi
vol 6, 1962).
Chapter 4: Extraction and qualitative analysis of extracts
MATERIALS AND METHODS
Plant material
The Plant material was collected from the distributor SHRI-SHAIL MEDI-FARM, Nagpur,
Maharashtra, India. It was identified by Prof. Dr. B.Pratibha Devi Department of Botany,
Osmania University and Hyderabad. A voucher no.032 of the plant was deposited in the
Department of Botany, Osmania University, and Hyderabad. Air-dried under the shed at
room temperature. Dried seed material was pulverized and the powder kept in polyethylene
bags.
Preparation of extract
Accurately weighed plant material was soaked in the conical flask by using methanol solvent.
Extraction was done by using ultra sonic bath sonicator. Solvent recovery done by using
simple distillation method. Extract was collected and stored in refrigerator.
Preliminary phytochemical screening
Tests for Alkaloids (Kokate, 2006b)
A small portion of the solvent free petroleum ether, alcohol extracts were stirred separately
with few drops of dilute hydrochloric acid and filtered. The filtrate was tested with various
reagents for the presence of alkaloids.
Mayer‟s Reagent – Cream colored precipitate.
Dragendroff‟s Reagent – Orange - brown colored precipitate.
Hager‟s Reagent – Yellow Colored precipitate.
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Wagner‟s Reagent – Reddish brown precipitate
Test for Saponins
The extract was diluted with 20ml of distilled water and it was agitated in a graduated
cylinder for 15mins. The formation of 1cm. layer of foam shows the presence of saponins.
Test for Phenolic Compounds and Tannins
Small quantities of the extracts were taken separately in water and test for the presence of
phenolic compounds and tannins was carried out with the following reagents.
Dilute ferric chloride solution (5%) – Violet color.
1% solution of gelatin containing 10% sodium chloride – White precipitate.
10% lead acetate solution – White precipitate.
Test for Flavonoids
Extracts were taken with aqueous sodium hydroxide solution – blue to violet color
(anthocyanins) yellow color (flavones), yellow to orange (flavonones).
With concentrated sulfuric acid – yellow to orange color (anthocyanins) yellow to orange
(flavones), orange to crimson (flavonones).
Shinoda‟s Test: Small quantities of the extracts were individually dissolved in alcohol, to
them piece of magnesium followed by concentrated hydrochloric acid drop – wise added
and heated. Appearance of magenta color shows the presence of flavonoids.
Test for Proteins (Khandelwal, 2006c)
Small quantities of the extract was dissolved in water and treated with the following
reagents.
Biuret test: An equal volume of 5% sodium hydroxide and 1% copper sulfate solution
was added – appearance of pink or purple color shows the presence of free amino acids or
proteins.
Millon‟s Reagent: Appearance of red color shows the presence of protein and free amino
acid.
Test For Terpenoids
Noller’s test
A pinch of powder was taken in a dried test tube. A bit of tin foil and 0.5ml of thinly chloride
was added and heated gently. Formation of pink colour indicates the presence of terpenoids.
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Test For Carbohydrates (Kokate et al.,2006a)
Molisch’s test
The substance was treated with α-napthal and concentrated sulphuric acid. Formation of
violet colour indicates the presence of carbohydrates.
Chemicals
Chemicals were obtained from the sources mentioned as follows. Pregabalin, Ketamine was
purchased from NIMS medicals Hyderabad. Vincristine was purchased from NIMS medicals
Hyderabad The Plant material was collected from the distributor SHRI-SHAIL MEDI-
FARM, Nagpur, Maharashtra, India.
All chemicals and reagents used were of analytical grade.
Experimental animals
Adult male Wistar rats weighing 200-250gm were used in the pharmacological studies. The
inbred animals were taken from the animal house in Peerzadiguda, uppal, Hyderabad 500008.
The animals were housed in groups of 6 per cage. They were maintained in well-ventilated
room at a temperature of 22-24ºc with relative humidity at 45-55% and natural 12h: 12h day-
night cycle in propylene cages. All the experiments were carried out between 10:00 am to
5:00 pm. The animals were housed one week prior to experiments to acclimatize laboratory
temperature. Food was withdrawn 3hrs before and during experiment.
Preparation of drug solution
Drug was dissolved in saline and administered to animals through intravenous and intra
peritoneal routes.
Assessment of effectiveness of Lepidium sativum seeds extract for neuropathic pain
using chemotherapy induced neuropathic pain model
Chemotherapy induced neuropathic pain was performed following the method described by
Joseph et al., 2003. Rats were first treated with vincristine sulphate (100 µg/kg, i.v). A single
intravenous dose of vincristine causes painful peripheral neuropathy. Baseline readings for
mechanical hyperalgesia and thermal hyperalgesia are noted. After 5days of administration of
vincristine test drug and standard drug are administered for 5days. The total period of study
lasts for 10 days. Mechanical hyperalgesia and thermal hyperalgesia of all groups were
measured during test drug administration in order to confirm the development of neuropathic
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pain. hyperalgesia of test drug treated group were compared with vincristine treated group in
order to confirm the effectiveness of drug in neuropathic pain.
Experimental groups
The rats were divided into four groups consisting of 6 per each group
Group I -Vincristine (100 µg/kg i.v.)
Group II - Vincristine (100 µg/kg i.v.) + test extract (50 mg/kg i.p.)
Group III- Vincristine (100 µg/kg i.v.) + test extract (100 mg/kg i.v.)
Group IV- Vincristine (100 µg/kg i.v.) + Pregabalin (80 mg/kg i.p.)
Behavioural tests
Mechanical hyperalgesia (Decosterd et al., 1998).
Mechanical hyperalgesia was tested by using the pin prick test. Animals were placed on the
elevated grid; a pin prick test was performed using a safety pin. The lateral plantar surface of
the right hind paw was briefly stimulated at intensity sufficient to indent but not penetrate the
skin (pin prick test). The duration of paw withdrawal was recorded, with an arbitrary minimal
time of 0.5 (sec) and a maximal cut off 15 (sec).
Thermal Hyperalgesia (Hot Plate Test) (Jain V et al., 2009).
The thermal nociceptive threshold, as an index of thermal hyperalgesia, was assessed by the
Eddy‟s hot plate, which is an instrument designed by Eddy and co-workers to assess thermal
sensitivity. The plate was preheated and maintained at a temperature of 40 ± 2.0°C. The rat
was placed on the hot plate and nociceptive threshold, with respect to licking of the hind paw,
was recorded in seconds. The cut-off time of 20 s was maintained.
Statistical analysis
The data are represented as mean±S.E.M and statistical significance between groups were
analyzed by means of student paired t-test, one way ANOVA followed by Dunnet‟s t-test, for
behavioral parameters as applicable and two way ANOVA for motor coordination. P<0.05
implies significance. All the statistical analysis was carried out using graph pad prism 5.0
version software.
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Table no. 5.1 Preliminary phytochemical screening of Lepidium sativum seed extract
Table5.2: Base line readings of Eddy’s hot plate for category 1:
Paw licking
Animal 1 ( head) 8
Animal 2 (body) 7
Animal 3 (tail) 3
Animal 4 (head +body) 2
Animal 5 (body + tail) 5
Animal 6(head + body +tail) 6
Fig 5.2 Base line readings of Eddy’s hot plate for category 1
Extract Alkaloids Carbo
hydrates Glycosides Tannins
Amino
acids Flavanoids Saponins Inulin
Tri
terpenoids
Methanol + + _ + + + _ + +
‘+’ indicates-presence of constituents ‘-’ indicates-absence of constituents
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Table 5.3: Base line readings of Eddy’s hot plate for category 2:
Paw licking
Animal 1 (head) 7
Animal 2 (body) 6
Animal 3 (tail) 5
Animal 4 (head +body) 7
Animal 5 (body + tail) 6
Animal 6 (head + body +tail) 5
Fig 5.3 Base line readings of Eddy’s hot plate for category 2:
Table 5.4: Base line readings of Eddy’s hot plate for category 3
Paw licking
Animal 1 ( head) 7
Animal 2 (body) 6
Animal 3 (tail) 7
Animal 4 (head +body) 8
Animal 5 (body + tail) 5
Animal 6 (head + body +tail) 7
Fig 5.4 Base line readings of Eddy’s hot plate for category 2:
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Table 5.5: Base line readings of Eddy’s hot plate for category 4
Paw licking
Animal 1 ( head) 6
Animal 2 (body) 4
Animal 3 (tail) 6
Animal 4 (head +body) 4
Animal 5 (body + tail) 6
Animal 6 (head + body +tail) 7
Fig 5.5: Base line readings of Eddy’s hot plate for category 4:
Table 5.6: Base line readings of actophotometer for category 1:
Readings \ count for 5 mins
Animal 1 ( head) 148
Animal 2 (body) 455
Animal 3 (tail) 343
Animal 4 (head +body) 341
Animal 5 (body + tail) 350
Animal 6 (head + body +tail) 335
Fig 5.6: Base line readings of actophotometer for category 1
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Table 5.7: Base line readings of actophotometer for category 2:
Readings \ count for 5 mins
Animal 1 (head) 220
Animal 2 (body) 342
Animal 3 (tail) 227
Animal 4 (head +body) 256
Animal 5 (body + tail) 452
Animal 6 (head + body +tail) 204
Fig 5.7 Base line readings of actophotometer for category 2:
Table 5.8: Base line readings of actophotometer for category 3:
Readings \ count for 5 mins
Animal 1 (head) 341
Animal 2 (body) 421
Animal 3 (tail) 356
Animal 4 (head +body) 265
Animal 5 (body + tail) 227
Animal 6 (head + body +tail) 376
Fig 5.8 Base line readings of actophotometer for category 3:
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Table 5.9: Base line readings of actophotometer for category 4:
Readings \ count for 5 mins
Animal 1 (head) 456
Animal 2 (body) 236
Animal 3 (tail) 274
Animal 4 (head +body) 345
Animal 5 (body + tail) 271
Animal 6 (head + body +tail) 143
Fig 5.9 Base line readings of actophotometer for category 4:
Table 5.10: Actophotomer readings when vincristine induced (Category1):
Day1 Day2 Day3 Day4 Day5 Day6
Animal 1 (head) 145 138 128 118 98 85
Animal 2 (body) 425 389 357 298 276 232
Animal 3 (tail) 324 318 297 257 232 198
Animal 4 (head +body) 324 298 276 243 208 187
Animal 5 (body + tail) 328 317 298 253 231 187
Animal 6 (head + body +tail) 317 298 254 221 197 167
Fig 5.10 Actophotomer Readings When Vincristine Induced (Category1):
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Table 5.11 Actophotometer readings when vincristine is induced: (Category 2)
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
Animal 1 (Head) 162 158 144 136 128 119
Animal 2 (Body) 200 191 188 178 162 156
Animal 3 (Tail) 200 196 178 160 142 128
Animal 4 (Head + Body) 196 172 164 158 144 122
Animal 5 (Body + Tail) 245 218 185 172 165 148
Animal 6 (Head + Body + Tail) 233 218 189 171 155 132
Fig 5.11 Actophotomer Readings When Vincristine Induced:(Ct-2).
Table 5.12 Actophotometer readings when vincristine is induced: (Category 3)
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
Animal 1 (Head) 160 152 132 122 111 98
Animal 2 (Body) 222 198 188 172 152 123
Animal 3 (Tail) 218 192 179 162 151 132
Animal 4 (Head + Body) 216 201 189 165 148 125
Animal 5 (Body + Tail) 252 232 199 187 165 148
Animal 6 (Head + Body + Tail) 253 231 197 171 149 113
Fig 5.12 Actophotomer Readings When Vincristine Induced:(Ct-3)
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5.13 Actophotometer readings when vincristine is induced: (Category 4)
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
Animal 1 (Head) 158 132 111 96 82 72
Animal 2 (Body) 260 243 221 188 162 126
Animal 3 (Tail) 210 196 178 165 132 108
Animal 4 (Head + Body) 192 172 144 128 94 81
Animal 5 (Body + Tail) 236 222 190 172 152 128
Animal 6 (Head + Body + Tail) 253 232 203 171 142 98
Fig 5.13 Actophotomer Readings When Vincristine Induced:(Ct-4)
Table 5.14: Actophotomer readings when test extract(50µg\ml) induced
Day1 Day2 Day3 Day4 Day5 Day6
Animal 1 (head) 87 101 123 135 142 156
Animal 2 (body) 243 287 308 376 398 432
Animal 3 (tail) 198 243 265 294 308 334
Animal 4 (head +body) 194 221 254 294 308 334
Animal 5 (body + tail) 195 241 267 302 327 335
Animal 6(head + body +tail) 175 208 234 276 308 324
fig 5.14: Actophotomer readings when test extract(50µg\ml) induced
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Table 5.15: Actophotomer readings when test extract(100µg\ml) induced
Day1 Day2 Day3 Day4 Day5 Day6
Animal 1 ( head) 91 111 126 141 149 160
Animal 2 (body) 261 294 317 384 401 446
Animal 3 (tail) 200 249 272 301 318 346
Animal 4 (head +body) 201 234 267 303 321 354
Animal 5 ( body + tail ) 200 254 273 318 330 345
Animal 6(head + body +tail) 184 215 245 286 318 348
Fig 5.15: Actophotomer readings when test extract(100µg\ml) induced
Table 5.16: Eddy’s hot plate readings when vincristine induced
Day 1 Day 2 Day 3 Day 4 Day 5
Animal 1 (head) 7 7 4 3 4
Animal 2 (body) 5 5 4 2 1
Animal 3 (tail) 4 4 3 2 1
Animal 4 (head +body) 6 5 4 2 1
Animal 5 (body + tail) 4 4 3 2 1
Animal 6(head + body +tail) 5 4 3 2 1
fig 5.16: Eddy’s hot plate readings when vincristine induced
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Table: 5.17 Eddy’s hot plate readings when vincristine is induced: (category 2)
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
Animal 1 (Head) 3 2 2 1 1 0
Animal 2 (Body) 2 2 1 1 1 0
Animal 3 (Tail) 2 2 1 1 0 0
Animal 4 (Head + Body) 2 1 1 1 0 0
Animal 5 (Body + Tail) 3 2 2 1 1 0
Animal 6 (Head + Body + Tail) 3 2 1 1 0 0
Fig: 5.17 Eddy’s hot plate readings when vincristine is induced: (category 2)
Table 5.18: Eddy’s hot plate readings when vincristine is induced: (category 3)
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
Animal 1 (Head) 2 1 1 0 0 0
Animal 2 (Body) 3 2 2 1 1 0
Animal 3 (Tail) 3 2 1 0 0 0
Animal 4 (Head + Body) 2 1 1 0 0 0
Animal 5 (Body + Tail) 3 2 2 1 1 0
Animal 6 (Head + Body + Tail) 3 2 2 1 1 0
Fig 5.18: Eddy’s hot plate readings when vincristine is induced: (category 3
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Table 5.19 Eddy’s hot plate readings when vincristine is induced: (category 4)
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
Animal 1 (Head) 3 2 2 1 0 0
Animal 2 (Body) 3 2 2 1 1 0
Animal 3 (Tail) 2 1 1 0 0 0
Animal 4 (Head + Body) 3 2 1 1 1 0
Animal 5 (Body + Tail) 2 1 1 1 0 0
Animal 6 (Head + Body + Tail) 3 2 2 1 1 0
Fig: 5.19 Eddy’s hot plate readings when vincristine is induced: (category 4)
Table 5.20: Eddy’s hot plate readings when test extract(50µg\ml) induced
Day 1 Day 2 Day 3 Day 4 Day 5
Animal 1 ( head) 5 6 6 7 8
Animal 2 (body) 2 3 4.5 6 7
Animal 3 (tail) 1.5 2 3.5 4 6
Animal 4 (head +body) 2 3.5 5 6 8
Animal 5 (body + tail) 1.5 2 3.5 5 6
Animal 6 (head + body +tail) 2 2.5 4 5.5 7
Fig: 5.20 Eddy’s hot plate readings when test extract(50µg\ml) induced
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Table 5.21: Eddy’s hot plate readings when test extract(100µg\ml) induced
Day 1 Day 2 Day 3 Day 4 Day 5
Animal 1( head) 6 6.5 7 8 9
Animal 2 (body) 3 3.5 5 7 8.5
Animal 3 (tail) 2 3 4 6 7
Animal 4 (head +body) 3 5 6 7 9
Animal 5 (body + tail) 2 3 4 6 7
Animal 6 (head + body +tail) 3 4 5 6 8
Fig 5.21 Eddy’s hot plate readings when test extract(100µg\ml) induced
Table 5.22: Eddy’s hot plate readings when standard(pregabalin) drug induced
Day 1 Day 2 Day 3 Day 4 Day 5
Animal 1 (head) 10 10 11 12 12
Animal 2 (body) 10 11 11 11 12
Animal 3 (tail) 10 10 12 12 12
Animal 4 (head +body) 10 10 11 11 12
Animal 5 (body + tail) 10 11 11 12 12
Animal 6 (head + body +tail) 10 10 11 11 12
fig: 5.22: eddy’s hot plate readings when standard(pregabalin) drug induced
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Table 5.23: Actophotometer readings when standard(pregabalin) drug induced
Day1 Day2 Day3 Day4 Day5
Animal 1 (head) 100 142 162 190 220
Animal 2 (body) 104 158 172 185 210
Animal 3 (tail) 110 154 184 216 255
Animal 4 (head +body) 118 162 194 216 250
Animal 5 (body + tail) 140 180 240 290 311
Animal 6 (head + body +tail) 123 162 212 276 320
Fig 5.23: Actophotometer readings when standard(pregabalin) drug induced
CONCLUSION
The Plant material was collected from the distributor Shri-Shall Medi-Farm, Nagpur and
Maharashtra, India. It was identified by prof. Dr. Mrs. B. Prathibha Devi. Department of
Botany, Osmania University and Hyderabad. A Voucher No. 032 of the plant was deposited
in the Department of Botany, osmania University and Hyderabad. Air-dried under the shed at
room temperature. Dried seed material was pulverized and the powder kept in polyethylene
bags.
Extraction was done by using ultra sonic bath sonicator. Solvent recovery done by using
simple distillation method. Extract was collected and stored in refrigerator. Preliminary
phytochemical screening results shown that, Alkaloids carbohydrates, tannins, aminoacids,
flavonoids are present so we concluded that the plant material contains the activity towards
neuropathic pain, thus we undergone animal studies. To identify pharmacological properties
against neuropathy of our seed extract we undergone Behavioral tests. In that we performed
thermal hyperalgesia tests and locomotor activity studies. By conducting Thermal
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Hyperalgesia test on animals by using Eddy‟s hot plate and by observing the results we
concluded that plant extract has shown some activity towards neuropathy by comparing with
standard. By conducting Locomotor Activity on animals by using Actophotometer and by
oberserving the results we concluded that plant extract has shown some activity towards
neuropathy by comparing with standard. Finally, we concluded that Lepidium sativum seeds
extract having anti-neuropathic activity.
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