Analgesic property of Corchorus olitorius L

8/10/2019 Analgesic property of Corchorus olitorius L

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ABSTRACT


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ABSTRACT

BACKGROUND

Corchorus olitorius L. one of the abundant species throughout Bangladesh was tested

to evaluate its effect on pain relief on Swiss Albino mice model and for a preliminary

phytochemical screening.

OBJECTIVE

The methanol extract of aerial parts of Corchorus olitorius L. was examined for

peripheral analgesia by acetic acid induced writhing method and phytochemical

screening.

DESIGN

For the experiment a total of 35 adult mice (male: female = 3:2) were divided into 7

groups and 5 mice in each group. The Control group (1) was treated with only 1%

acetic acid by intra-peritoneal administration, Standard group (I) and Standard group

(II) were treated with Aspirin 200 mg/kg & 400 mg/kg and 1% acetic acid by intra-

peritoneal administration. Group 4, 5, 6 and 7 were treated with aerial parts (MeOH)

extract of Corchorus olitorius L. at 50 mg/kg, 100 mg/kg, 200 mg/kg and 400 mg/kg

body weight respectively, followed by 1% acetic acid was administered by intra-

peritoneal injection after 60 minutes. Acetic acid induced mice were subjected to

writhing for 15 minutes of observation. Meanwhile, the crude extract was screenedfor the presence of some phytochemicals such as, Saponins, Flavonoids, Alkaloids

and Tannins.

RESULT

Here, the results are expressed as Means. Here we found that the Control mean was

5.2, the Standard I mean was 3.2 and the standard II mean was 1.8. On the other hand,

the Corchorus olitorius L. (aerial parts) MeOH extract low dose (50 mg/kg) treated

mean was 4.2, moderate dose (100 mg/kg) treated mean was 3, medium dose (200

mg/kg) treated mean was 2.4 and high dose (400 mg/kg) treated mean was 2.2

Comparing with Control mean all the given doses significantly decreased the pain. So,

we can conclude that, methanol extract of aerial parts of Corchorus olitorius L.

possesses significant analgesic activities. In case of phytochemical screening wefound the presence of saponins, tannins, alkaloids and flavonoids in the extract.


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Chapter One

Plant profile of Cor chorus oli tor ius L.


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1.1 GENUS Corchorus :

Corchorus is a genus of about 40 100 species of flowering plants in the familyTiliaceae, native to tropical and subtropical regions throughout the world. Different

common names are used in different contexts, with jute applying to the fiber produced

from the plant (Wikipedia, Corchorus olitorius ). Jute was once known as the golden

fibre of Bangladesh, since it was the most important cash crop for the country. Two

species of jute (Corchorus capsularis L. and Corchorus olitorius L.) are being

cultivated in Bangladesh. Capsularis (deshi) has maximum use as vegetable than

Oloitorius (Tossa) due to its bitter taste. Jute leaves are being used as vegetables in

Africa, Middle East, Southeast Asia, including Bangladesh for a long time (hepzibah,

2008).

1.2 BOTANY OF Corchor us oli toriu s (L.):

Corchorus olitorius (jute) is a native plant of tropical Africa and Asia, and has since

spread to Australia, South America and some parts of Europe. Cultivation is

dependent on the climate, season, and soil. Almost 85% of the world's jute cultivation

is concentrated in the Ganges Delta (Indianjute.blogspot, Corchorus olitorius ).

This fertile geographic region is shared by both Bangladesh and India (West Bengal).

China also has a dominating place in jute cultivation. On a smaller scale, Thailand,

Myanmar (Burma), Pakistan, Nepal, and Bhutan also cultivate jute (Wikipedia,

Corchorus olitorius ).

Table 1: Scientific Classification of Corchoru s oli toriu s L. (USDA)

Kingdom Plantae

Division Magnoliophyta

Class Magnoliopsida

Order Malvales

Family Tiliaceae

Genus Corchorus L.

Species Corchorus olitorius L.


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1.3 LOCAL NAME:

Patshak.

1.4 GENERAL DESCRIPTION:

The plants are tall, usually annual herbs, reaching a height of 90-120 cm tall; stems

glabrous. Leaves 6-10 cm long, 3.5-5 cm broad, elliptic-lanceolate, apically acute or

acuminate, glabrous, serrate, the lower serratures on each side prolonged into a

filiform appendage over 6 mm long, rounded at the base, 3-5 nerved; petioles 2-2.5

cm long, slightly pubescent, especially towards the apex; atipules subulate, 6-10 mm

long. Flowers are pale yellow; bracts lanceolate; peduncle shorter than the petiole;

pedicles 1-3 and very short, Petals 5 mm long. Style short; stigma microscopically

papillose. The fruit is a many-seeded capsule 3-6.5 cm long, linear, cylindric erect,

beaked, glabrous, 10-ribbed, 5-valved; valves with stransverse partitions between the

seeds. Seeds trigonous, black (Kirtikar and Basu, 1975).

1.5 USEFUL PARTS:

Leaf and stem.

1.6 CHEMICAL CONSTITUENTS:

The leaves are reported to contain calories, H 2O, protein, fat, carbohydrate, fiber, ash,

Ca, P, Fe, Na, K, beta-carotene, thiamine, riboflavin, niacin, and ascorbic acid. Leaves

contain oxydase and chlorogenic acid (Chen and Saad, 1981).It also contain

phytochemicals including alkaloids, flavonoids, tannins, saponins, steroids and

phenols (Harborne, 1998). The seeds contain oil (Watt and Breyer-Brandwijk, 1962),

reportedly estrogenic (Sharaf et al, 1979), which contains palmitic, stearic, behenic,

lignoceiic, oleic,linoleic, and linolenic acids as well as large portions of B, Mn, Mo,

and Zn.

1.7 MEDICINAL USES:

The leaves are demulcent, diuretic, febrifuge and tonic. They are used in the treatment

of chronic cystitis, gonorrhoea and dysuria (Chopra. R. N et al, 1986). A cold infusion

is said to restore the appetite and strength (Duke,1983). It can also be used in other

medicinal purposes, such as:-It is used for anti-inflammatory treatment.


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- Its vitamin content is good for eyesight, as the vegetable contains beta-carotene.

-It is also been connected with curing the chronic inflammation of the urinary bladder

(Rullanamador.blogspot, Corchorus olitorius )

- It being rich in vitamins and minerals generally promotes good health and well-

being.

-It contains vitamin E and other antioxidants. It is said to prevent wrinkles and

promote youthful looking skin.

-It is used to treat inflammation and pain such as arthritis , headache, stomach ache

and others.

-It being rich in fiber helps to control blood pressure, cholesterol build-up, diabetes

and prevents heart disease.

-Its leaves are rich in fiber and its slimy consistency when cooked is used to treat

various digestive problems such as diarrhea, stomach ache, dysentery, constipation

and ulcers.

-It is also claimed that together with other herbs it can cure cancer.

- In India infusion of leaves used as tonic and febrifuge.

-Cold infusion of leaves used as bitter tonic, used by patients recovering from

dysentery, to restore the appetite and improve strength.

- Powdered seeds with honey and ginger for diarrhea.

- Grains of the powder mixed with equal amounts of Curcuma longa used for acute

dysentery.

- Infusion of seeds for fever and liver congestion.

- Hindus reduce the plant to ashes, mix it with honey, and use it for obstruction of the

abdominal viscera.

- In South India, the dried plant is used as demulcent. Powder of leaves, 5 - 10 grains,

mixed with powdered tumeric in equal parts, used for dysentery (stuartxchange,

Corchorus olitorius )

1.8 THERAPEUTIC PROPERTIES:

Antioxidant activity:

Researchers studied to evaluate the protective effect of aqueous extract of Corchorus

olitorius L. leaves (AECO) against CdCl intoxication. In vivo assay, CdCl treated

rats exhibited a significantly increased intracellular Cd accumulation, oxidative stressand DNA fragmentation in the organs. In addition, the haematological parameters


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were significantly altered in the CdCl treated rats. Simultaneous administration of

AECO could significantly restore the biochemical, antioxidant and haematological

parameters near to the normal status. Presence of substantial quantity of phenolic

compounds and flavonoids in extract may be responsible for overall protective effect

(Dewanjee S et al, 2013)

Antibacterial and Antifungal activity:

A group of scientists described the antimicrobial activity of 3 extracts of Corchorus

olitorius L. Successive petroleum ether, methanol and ethyl acetate+water extracts of

C. olitorius leaves were tested (in vitro) for their antibacterial and antifungal activities

by agar-well diffusion assay. All extracts displayed varied levels of antibacterial or

antifungal activity. The petroleum ether extract of C. olitorius leaves presented a good

activity against Escherichia coli, Staphylococcus aureus and Yersinia enterocolitica.

The ethyl acetate+water extract presented a good activity against Geotrichum

candidum and Botrytis cinerea . The results obtained in this study appear to confirm

the antibacterial and antifungal potential of C. olitorius leaves (LHAN et al, 2007).

Arsenic Reduction activity:

A study was undertaken by scientists to evaluate the protective effect of an aqueous

extract of Corchorus olitorius L. leaves (AECO) against NaAsO 2 induced brain

toxicity in experimental rats. The animals exposed to NaAsO 2 for 10 days. Treatment

with AECO for 15 days prior to arsenic intoxication significantly improved

antioxidant markers in a dose dependant manner. Histological studies on the

ultrastructural changes of brain tissue supported the protective activity of the AECO.

The results suggest that treatment with AECO prior to arsenic intoxication has a

significant role in protecting animals from arsenic-induced toxicity (Das AK et al,

2010)

Gastroprotective activity:

According to previous studies of Corchorus olitorius L. evaluates the gastroprotective

effect of an ethanolic extract of C. olitorius against ethanol-induced gastric ulcers in

adult Sprague Dawley rats. Compared with the extensive mucosal damage in the ulcer

control group, gross evaluation revealed a marked protection of the gastric mucosa inthe experimental groups, with significantly preserved gastric wall mucus. The study


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reports the gastroprotective property of an ethanolic extract of C. olitorius against

ethanol-induced gastric mucosal hemorrhagic lesions in rats (Al Batran R et al, 2013).

Antihyperglycemic activity:

Researchers explored the suppressive effect of Corchorus olitorius L. leaves on

postprandial blood glucose levels in rats and humans. A soluble dietary fiber (SDF)

was extracted from the freeze-dried Corchorus olitorius L. leaves powder. An

elevation of the postprandial blood glucose level in rats given 1% or 2% SDF solution

orally together with 20% glucose solution was significantly suppressed as compared

with that observed in the control rats given only glucose solution. These results

indicate that the effective substance in Corchorus olitorius L. leaves for suppressing

blood glucose elevation is a kind of mucilaginous SDF (Innami S et al, 2005).


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Chapter Two

Analgesic profile


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2.1 ANALGESICS

Simply put, analgesics are a class of drugs used to relieve pain. The pain relief

induced by analgesics occurs either by blocking pain signals going to the brain or by

interfering with the brain's interpretation of the signals, without producing anesthesia

or loss of consciousness.

The word analgesic derives from Greek an- ("without") and -algia ("pain").

Analgesic drugs act in various ways on the peripheral and central nervous systems;

they include paracetamol (acetaminophen), the non-steroidal anti-inflammatory drugs

(NSAIDs) such as the salicylates, narcotic drugs such as morphine, synthetic drugs

with narcotic properties such as tramadol, and various others.

It should be noted that some references include aspirin and other non-steroidal anti-

inflammatory drugs (NSAIDS) in the class of analgesics, because they have some

analgesic properties. Aspirin and NSAIDS primarily have an anti-inflammatory

effect, as opposed to being solely analgesic.

In choosing analgesia, the severity and response to other medication determines the

choice of agent; the WHO pain ladder, originally developed in cancer-related pain, is

widely applied to find suitable drugs in a stepwise manner. The choice of analgesia is

also determined by the type of pain: for neuropathic pain, traditional analgesia is less

effective, and there is often benefit from classes of drugs that are not normally

considered analgesics, such as tricyclic antidepressants and anticonvulsants (Dworkin

RH, et al. 2003).

2.2 TYPES OF ANALGESICS

There are basically two kinds of analgesics:

Narcotics

Non-narcotics

2.2.A Narcotic analgesics

Narcotic analgesics are psychoactive compounds that relief pain by acting on CNS

system. Can cause numbness and induce a state of unconsciousness. They have the


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tendency to cause tolerance and dependence and have no anti - inflammatory effect.

They do not produce gastric irritation. There are two types of narcotic analgesics:

1. The opiates (a white liquid extract of unripe seeds of the poppy plant) belong

to the large biosynthetic group of benzylisoquinoline alkaloids.

2. The opioids (derivatives of opiates) are Opioids are any compound which

binds to opioid receptors in the central nervous system or gastointestinal tract.

There are four broad classes of opioids:

a. Endogenous opioid peptides (produced in the body): Endorphins,

dynorphins, enkephalins.

b. Opium alkaloids: Morphine, codeine, thebaine.

c. Semi-synthetic opioids: Heroin, oxycodone, hydrocodone, dihydrocodeine,

hydromorphine, oxymorphine, nicomorphine.

d. Fully synthetic opioids: Pethidine or demerol, methadone, fentanyl,

propoxyphane, pentazocine,buprenorphine, butrophanol, tramadol.

(Doyle D., et. al. , 2005)

Opiod analgesics exert their effects by binding with opioid receptors. Opioids specific

receptors are G - protein coupled seven transmembrane receptor families. These

receptors are divided into four broad classes named:

o delta () or DOP or OP1 o kappa () or KOP or OP2 o mu () or MOP or OP3 ando Nociceptin receptor or NOP or OP 4

According to their action on different types of opioid receptors, narcotic analgesics

are classified as: Full agonists: They stimulate all types of opioid receptors. Among these are

morphine, trimeperidine, fentanyl.

Partial agonist: They have limited intrinsic activity at the receptor, a measure

of the activation of the receptor by the drug. For instance Buprenorphine acts as a

partial agonist at mu opioid receptors, an antagonist at kappa opioid receptors, an

agonist at delta opioid receptors, and a partial agonist at ORL - 1 (or nociceptin)

receptors.


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Agonists - antagonists: They stimulate some types of opioid receptors and

block others. Example includes Pentazocine, Nalbuphine, and Nalorphine.

Pure antagonists: They bind to the opioid receptors with higher affinity than

agonists but do not activate the receptors. This effectively blocks the receptor, preventing the body from responding to opiates. Example includes Naloxone, and

Naltrexone.

2.2.B Non - narcotic analgesic

Non - narcotic analgesics also called non-opioid analgesics are compounds that relief

pain locally without causing any addiction or CNS depression. They exert anti -

inflamatory effect however cause gastric irritation. There are three types of non -

opioid analgesics:

1. Salicylates:

The nonacetylated salicylates have fewer gastrointestinal side effects and might be

considered in cases where GI distress is an issue. There are three broad types of

salicylates:

a. Salicylic acid: Methyl salicylate (topical use).

b. Na-salicylates: Benorylate.

c. Acetyl salicylic acid (Aspirin): Deflonisol.

2. Non-steroidal anti-inflammatory drugs:

Nonsteroidal anti-inflammatory drugs, usually abbreviated to NSAIDs but alsoreferred to as nonsteroidal anti - inflammatory agents/analgesics (NSAIAs) or

nonsteroidal anti-inflammatory medicines (NSAIMs) are a class of drugs that

provide analgesic and antipyretic (fever - reducing) effects, and, in higher doses, anti-

inflammatory effects. The most prominent members of this group of drugs are aspirin,

ibuprofen, and naproxen. Most NSAIDs act as nonselective inhibitors of the enzyme

cyclooxygenase (COX), inhibiting both the cyclooxygenase - 1 (COX - 1) and

cyclooxygenase - 2 (COX - 2) isoenzymes. NSAIDs are divided into several chemical

derivatives. Some of them are:

Propionic acids: Ibuprofen, Flurbiprofen, Fenoprofen, Naproxen, Naproxen

sodium, Ketoprofen, Ketoprofen, Oxaprozin.


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Acetic acids: Indomethacin, Indomethacin SR, Slindac, Tolmetin, Diclofenac,

Diclofenac, Etodolac, Etodolac, Ketorolac.

Fenamates: Meclofenamate, Mefenamic acid.

Non-acidic agent: Nabumetone To some patients NSAIDs can have significant adverse effects including gastropathy,

renal failure, and inhibition of platelet aggregation, irrespective of the route of

administration, with any of the nonselective medications. Trials may be needed to find

one that is efficacious for a given patient. To minimize the risk of renal failure,

including papillary necrosis, ensure adequate hydration and good urine output in all

patients on NSAIDs (sOxford Textbook of Palliative Medicine, 3rd ed.). The

nonselective medications are relatively contraindicated in the setting of significant

preexisting renal insufficiency. If bleeding is a problem, or coagulation or platelet

function is impaired, NSAIDs may be contraindicated. The new COX - 2 selective

inhibitors have less of these toxicities and may be indicated in high risk patients.

Acetaminophen:

Acetaminophen is the most commonly used over - the counter, non - narcotic

analgesic. Acetaminophen is a popular pain reliever because it is both effective for

mild to moderate pain relief and relatively inexpensive. If acetaminophen is not used

according to the directions on the label, serious side effects and possible fatal

consequences can occur. For example, taking more than 4000 mg/day or using it long

term can increase the risk of liver damage. The risk of liver damage with

acetaminophen use is also increased by ingesting alcohol.

2.3 Side effects of analgesics

Although analgesic compounds are meant to relieve pain, unfortunately that often

comes with different side effects. Some of them are temporary and go away as soon as

the drug is out of the system. Few analgesics on the other hand might cause

permanent damage in certain persons and are banned by various authorities.

Irrespective of their uses and mode of metabolism, common side effects of different

classes of analgesics are tabulated below (Driessen B., Reimann W., 1992).


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Table 2: Types of analgesics and their side effects.

Analgesic

GroupSide effects Remarks

N a r c o

t i c

O p

i a t e

Constipation, fatigue, dizziness,

anxiety, constricted pupils, itching,

tremors, spasms, sweating,

hallucinations, delirium and allergic

reaction

Raw opiate contains different

opioid and non-opioid alkaloids

which besides the analgesia

may trigger unexpected

physiological and psychological

condition.

O p i o i

d

E n

d o g e n o u s o p

i o i d

p e p

t i d e s

Increased pain

Only when peripheral

administration exceeds

hyperalgesia mediated by

immune cells (Hermanussen S.,

2004; Stein C., 1993).

O p

i u m

a l k a l o i

d s

Nausea, vomiting, and dry mouth.Particularly at the beginning of

treatment.

Impaired vision, respiratory

depression, euphoria.May occur at higher doses.

CNS depression as well as

headache, gastro - intestinal

disturbance, flush, skin rash,

tachycardia, sweating, hypotonia.

May occur after oral

administration of some

alkaloids.

S e m

i - s y n

t h e t

i c o p

i o i d s

Nausea, vomiting, urinary

retention, miosis, constipation.

Rare in medical doses, common

in pleasure doses.

Allergic reactions. Vary with individuals.

Physical dependence and develop

tolerance.Prolonged use.

Respiratory arrest.If taken in combination with

alcohol.

Liver failure.Reaction with other drugs or

history of hepatic disorders.

Loss of appetite, anxiety, muscle

weakness, diarrhea, fever or

Associated with withdrawal

(CESAR). Withdrawal effects


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Analgesic

GroupSide effects Remarks

flue like symptom. have been reported in newborn

patients whose mother was on

drug during pregnancy (Rao R.,

2002).

F u

l l y s y n

t h e t

i c o p

i o i d s

Diarrhea, nausea, constipation, dry

mouth, somnolence, confusion,

asthenia (weakness), sweating.

At more than 10 % of patient.

Abdominal pain, headache, fatigue,

dizziness, anorexia and weight loss,

nervousness, hallucinations,

anxiety, depression, flu - like

symptoms, dyspepsia (indigestion),

dyspnea (shortness of breath),

hypoventilation, apnea, urinary

retention.

At 3 to 10% of patients.

Aphasia. Associated with frequently use.

Suicidal ideation.Associated with post-acute

withdrawal effects.

Seizure. Only caused by Methadone.

Skin rash. Occurs in allergic persons.

Liver toxicity. Associated with an overdose.

Analgesic Group Side effects Remarks

N o n - n a r c o

t i c

S a l i c y

l a t e s

S a l i c y

l i c a c

i d

Stomach irritation.

Person can experience stomach

bleeding, pain, and ulcers if took

the drug with alcohol.

Skin peeling and blistering. When applied topically.

Allergic reaction.Individuals have allergies to

salicylic acid.

Reye's syndrome.

Severe illness occurs in children

characterized by acute


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encephalopathy and fatty liver.

N a - s a l

i c y l a t e s

Hepatotoxicity, gastric irritation,

mucosal lesions, worsening o pre-existing heart failure,

hyperventilation, fever, ketosis,

respiratory alkalosis, metabolic

acidosis.

Associated with overdose. Mayleads to total C. N. S. failure and

cause coma, cardiovascular

collapse and respiratory failure.

Hypersensitivity reactions. Individuals have allergies.

Reye's syndrome.

Severe illness occurs in children

characterized by acute

encephalopathy and fatty liver.

A c e

t y l s a

l i c y

l i c a c

i d ( A s p i r i n

)

Gastrointestinal system.Can occur in individuals on high

doses of aspirin.Gastric ulcers and gastric

bleeding.

Reye's syndrome.

Occurs in children with viral

illnesses such as influenza or

chicken pox who have been given

aspirin.

Liver and kidney failure. Continuous or high dose.

Loss of hearing.May also appears as pain or

bleeding in the ears.

Bleeding.Only in patient with thrombotic

problems.

o n - s e r o

a a n

- n a m m a o r y

d r u g

s ( N S A I D s )

P r o p

i o n

i c a c

i d s

Gastrointestinal lesions.

Both in oral and parental

administration can be avoided by

co administration of exogenous

prostaglandins (Elliott G. A.,

1988).

Nausea, headache, dizziness,

epistaxis, priapism.

Generally occurs when the

analgesia wear off.

Dyspepsia, diarrhea,

constipation. Associated with long term use.


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Raised liver enzymes and Salt

and fluid retention.

Associated with clinical

overdose.

Rash. Occurs if the person is allergic tothe drug compounds.

Photo toxicity.

Phototoxic reactions with UVA

as well as UVB in vitro

(Ljunggren B., 1985).

Loss of hearing ability.

Associated with long term use or

above clinical dosage (Curhan S.

G., 2010).

Hypertension and cardiac failure.

Associated with long term use or

occurs in patient with cardiac

dysfunction history (Hippisley-

Cox J., 2005).


N o n

- n a r c o

t i c

N o n

- s t e r o i

d a l a n

t i - i n

f l a m m a t o r y

d r u g s

( N S A I D s )

A c e

t i c a c i

d s

Lithium toxicity.

Some derivatives reduce lithium

its excretion by the kidneys

hence increases the risk of

lithium toxicity in patients

taking medication for depression

or bipolar disorder.

Ulceration along stomach and

GI tract, dyspepsia, heartburn

and mild diarrhea.

As a result of inhibition of the

production of prostaglandins in

the stomach and intestines.

Acute renal failure, chronic

nephritis and nephrotic

syndrome.

Because of elevations of serum

creatinine caused by some drugs

of this group.

Edema, hyperkalemia

(Akbarpour F., 1985),

hypernatremia, hypertension.

Due to reduced plasma renin

activity and aldosterone levels,

and increases sodium, potassium

retention and the effects of


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vasopressin.

Headache, sometimes with

vertigo and dizziness, hearingloss, tinnitus, blurred vision.


overdose and occurs in 10 - 20% of the patients.

Psychosis. Associated with prolonged use.

Hepatitis and bone marrow

damage..Less than 10 % of the patients.

Heart attacks and strokes.Occurs in patients with heart

disease.

F e n a m a t e s

Convulsions, nausea, vomiting,

vomiting blood, shallow

breathing, coma.


overdose.

Cardiovascular disorders, CNS

depression, dermatological

reactions, GI problems,

hematological diseases, hepatic

abnormalities renal

dysfunction.

Occurs less frequently in long-

term use.

Skin rash, itching and swelling,

sore throat and fever.

Associated with allergic

reactions.

N o n

-

a c i d i c

a g e n

t GI disorders.Extremely low risk o

happening.

A c e t a m

i n o p

h e n

Stomach bleeding (Rodrguez

L. A. G., 2000).Associated with prolonged daily

use.Infertility (Leffers H., 2010).

Kidney and liver damage

(Michael S., 2007).

Associated with long-term use as

well as patient with alcoholism

or liver disease history.

Blood cancer (Walter R. B.,

2011).Associated with chronic use.

Liver failure due to

hepatotoxicity. Caused by clinical overdose.


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2.4 THE MAJOR CLASSES

1) PARACETAMOL AND NSAIDS

The exact mechanism of action of paracetamol/acetaminophen is uncertain, but it

appears to be acting centrally. Aspirin and the other non-steroidal anti-inflammatory

drugs (NSAIDs) inhibit cyclooxygenases, leading to a decrease in prostaglandin

production. This reduces pain and also inflammation (in contrast to paracetamol and

the opioids).

Paracetamol has few side effects and is regarded as very safe, although excessive

doses can lead to kidney and liver damage in the form of analgesic nephropathy and

paracetamol hepatotoxicity, respectively. NSAIDs predispose to peptic ulcers, renal

failure, allergic reactions, and occasionally hearing loss, and they can increase the risk

of hemorrhage by affecting platelet function.The use of aspirin in children under 16

suffering from viral illness may contribute to Reye syndrome.

2) COX-2 INHIBITORS

These drugs have been derived from NSAIDs. The cyclooxygenase enzyme inhibited

by NSAIDs was discovered to have at least 2 different versions: COX1 and COX2.

Research suggested that most of the adverse effects of NSAIDs were mediated by

blocking the COX1 (constitutive) enzyme, with the analgesic effects being mediated

by the COX2 (inducible) enzyme. The COX2 inhibitors were thus developed to

inhibit only the COX2 enzyme (traditional NSAIDs block both versions in general).

These drugs (such as rofecoxib and celecoxib) are equally effective analgesics when

compared with NSAIDs, but cause less gastrointestinal hemorrhage in particular.

However, post-launch data indicated increased risk of cardiac and cerebrovascular

events with these drugs due to an increased likelihood of clotting in the blood due to a

decrease in the production of protoglandin around the platelets causing less clotting

factor to be released, and rofecoxib was subsequently withdrawn from the market.

The role for this class of drug is debated.


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3) OPIATES AND MORPHINOMIMETICS

Morphine, the archetypal opioid, and various other substances (e.g. codeine,

oxycodone, hydrocodone, diamorphine, pethidine) all exert a similar influence on the

cerebral opioid receptor system. Tramadol and buprenorphine are thought to be partial

agonists of the opioid receptors. Dosing of all opioids may be limited by opioid

toxicity (confusion, respiratory depression, myoclonic jerks and pinpoint pupils), but

there is no dose ceiling in patients who tolerate this. Opioids, while very effective

analgesics, may have some unpleasant side-effects. Up to 1 in 3 patients starting

morphine may experience nausea and vomiting (generally relieved by a short course

of antiemetics). Pruritus (itching) may require switching to a different opioid.

Constipation occurs in almost all patients on opioids, and laxatives (lactulose,

macrogol-containing or co-danthramer) are typically co-prescribed.

When used appropriately, opioids and similar narcotic analgesics are otherwise safe

and effective; however risks such as addiction and the body becoming used to the

drug (tolerance) can occur. The effect of tolerance means that drug dosing may have

to be increased if it is for a chronic disease this is where the no ceiling limit of the

drug comes into play. However what must be remembered is although there is no

upper limit there is a still a toxic dose even if the body has become used to lower

doses.

4) SPECIFIC AGENTS

In patients with chronic or neuropathic pain, various other substances may have

analgesic properties. Tricyclic antidepressants, especially amitriptyline, have been

shown to improve pain in what appears to be a central manner. The exact mechanism

of carbamazepine, gabapentin and pregabalin is similarly unclear, but these

anticonvulsants are used to treat neuropathic pain with modest success (Oxford

Textbook of Palliative Medicine, 3rd ed.).

2.5 MECHANISMS OF PAIN INDUCTION IN ACETIC ACID INDUCED

WRITHING METHOD OF ANALGESIC ACTIVITY SCREENING

Intraperitoneal administration of acetic acid (1%) causes localized inflammation in

mice. Following inflammation, there is biogenesis of prostaglandins (fromcyclooxygenase pathway) and leukotrienes (lipooxygenase pathway). The released


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prostaglandins, mainly prostacyclin (PGI 2) and prostacyclin-E have been reported

responsible for pain sensation. The exact mechanism by which prostaglandin produce

pain are not still clear but there are a number of proposed mechanisms of action:

All kinds of pain or noxious stimuli (nociception) are conveyed by specific nerves

called un-myelinated C fiber and myelinated A- fibers, the former being slow.

Conducting and the later being fast conducting. It has been investigated that un-

myelinated C fiber are the most usual conveyer of two. The prostaglandin and other

liberated products of inflammation serve as noxious stimuli. They are supposed to

sensitize C fibers and subsequently reduce pain threshold. The C fibers get stimulated

and cause enhanced release of tachykinins, mainly substance P and neurokinins. It is

the substance P released in excessive amount following the stimulation of C fibers

that has been held responsible for sensation of pain in animal.

The precise mechanism by which substance P arouses pain sensation is not well

documented. But upon release, substance P and others tachykinins bind to specific

receptors (NK 1, NK 2, and NK3) that are G-protein coupled. Among these receptors,

substance P is specifically bound to NK 1. After binding of substance P to the

respective receptor, there is stimulation of phospholipase C resulting in the formation

of two second messengers-inositol triphosphate (IP 3) and diacylglycerol (DAG). IP 3

causes the exocytotic release of Ca ++ stored intra-cellular and DAG activates protein

kinase C which then causes the influx of Ca ++ through the voltage gated Ca ++ channel.

These happenings may have role in the neural processing of the pain sensation and its

subsequent conveyance to higher centers of the brain. Prostaglandin also potentate the

pain producing activity of bradykinins and other autacoids (Bamigbade TA, et al.

September 1997).


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22

2.6 SCHEMATIC PRESENTATION OF PAIN INDUCTION

Acetic acid induced

(1%)

Inflammation in-situ

Localized biogenesis of prostaglandin

Prostacyclin

(PGI2) & (PGE2)

Stimulation un-myelinated C fiber and myelinated A- fibers

Release of substance P and other nociceptive neurokinins

Nociception or pain

Biological response

Writhing

Flow chart 1: Schematic presentation of pain induction.


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23

Chapter Three

Rational for the current project


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Since Corchorus olitorius L. has been described by folk practitioners to possess

Antinoceciptive property and there was no extensive pharmacological studies

regarding analgesic effect with this species in Bangladesh, so we have selected

Corchorus olitorius L. for my present study. The objective of the present work was to

explore the possibility of developing new and effective as well as natural drugs for the

treatment of peripheral pain.


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Chapter Four

Materials & Methods


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Local Name: Patshak

Scientific Name: Corchorus olitorius L.

Accession no: 38715

Place of collection: Rayer bazar

Date of collection: 10-9-2013

Part use for extraction: Aerial parts

Weight of powder used for extraction: 150gm

Methanol used for extraction: 750ml

Temperature used for evaporation: 40 0C

Date of extraction: 15-11-2013Weight of extraction: 5.16gm


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4.1 Place and Period of study

The study was carried out in the Pharmaceutical Biotechnology Lab of Department of

Biotechnology & Genetic Engineering, University of Development Alternative

(UODA), Dhanmondi, Dhaka during the period of September 2013 to December

2013.

Figure 2: Animal cage at animal laboratory of University of Development Alternative

4.2 Principle of methodologies

In the present study we choosed maceration approach for extraction using methanol as

menstruum (Handa S. S. et. al. , 2008). Acetic acid induced visceral pain model

(Koster R., et. al. , 1959 and Dambisya Y. M., et. al. , 1999) was used to evaluate

extract's analgesic efficacy. Finally, writhing data were compared using t-test at a

significance of


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1. Dried aerial parts powder of Corchorus olitorius L.

2. Methanol 750 ml

3. Distilled water

4. Beakers

5. Hot water bath

6. Drier

7. Weight machine

8. Glass rods

9. Tissue paper

10. Spatula

11. Titrating pipette

12. Foil papers

13. Vial.

14. Blender machine

15. Scissor.

4.5 Procedure of extraction

Extraction procedure of Corchorus olitorius L. (aerial parts) with non-polar

(methanol) solvent:

1. After blending of the Corchorus olitorius L. (aerial parts), it was powder like.

Further filter the powder was taken out for extraction.

2. Measured the dried powder of Corchorus olitorius L. by digital balance and

took 150 gm powder of aerial parts.

3. We have taken a beakers and poured out with methanol as per account as 5:1,

i.e. 750ml for 150 gm.

4. Stirred slowly with glass rod or any stainless steel rod to mix up the solventand dry sample for making soft liquid.

5. Continued stirring after a few minutes and maintained it for one hour and

covered up the beakers with aluminum foil.

6. Left the system for overnight for 48 hours.

7. Filtered the solvent with thin cloth (white color) and took the extract to the

water bath then wait till we got dried extract (crude drug).


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8. 5.16 gm extraction was found after 5 days of evaporation, where the

temperature was always maintained at 40C and finally it was collected by spatula in

a marked glass vial.

9. The extracted residues were kept in plastic jars and the vial containing extract

kept in refrigerator at temperature of 4-8C.

4.6 MATERIALS AND METHODS FOR THE EXPERIMENT OF

ANALGESIC EFFECT:

The methanol extract of Corchorus olitorius L. aerial parts was subjected to screening

for analgesic activity by well recognized method for peripheral analgesia: Acetic acid

induced writhing method (Koster et al, 1959; whittle, 1964; Vogel & Vogel, 1997)

4.7 PRINCIPLES

In the method, acetic acid is administered intra-peritoneal to the experimental animals

to create pain sensation. As a result, the animals squirms their body at regular interval

out of pain. This squirm or contraction of the body is term as writhing. As long as

the animals feel pain, they continue to give writhing. Each writhing is counted and

taken as an indication of pain sensation. Any substance that has got analgesic activity

is supposed to lessen the number of writhing of animals within in a given time frame

and with respect to the control group. The writhing inhibition of positive control was

taken as standard and compared with test samples and control. As positive control,

any standard NSAID drug can be used. In the present study, Aspirin was used to serve

the purpose.

4.8 EXPERIMENTAL ANIMALS

In the present studies, Swiss Albino mice of 4-5 weeks from both sexes, weighed

between 15 to 20 g were used. The animals were obtained from International Centre

for Diarrheal Disease Research (ICDDR, B), Bangladesh. Mice were housed in iron

cages over dried wooden chips. Tagging was done by drawing red or black stripes on

the mices tail to distinguish them within a specific cage.


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4.9 REAGENTS, CHEMICALS, AND EQUIPMENTS FOR SCREENING OF

ANALGESIC ACTIVITY

Disprin (300 mg) Acetic acid DMSO (as suspending agent) Sterile disposable syringe (1 ml, 100

divisions)

Electronic and digital balance Stop watch Micropipette Yellow tip Plastic gloves Corchorus olitorius L. (aerial parts) extract

4.10 EXPERIMENTAL DESIGN

For the experiment, a total of 35 adult mice (male: female = 3:2) were taken, divided

into 7 groups, 5 mice in each group. Then 0.4 gm of the Corchorus olitorius L. aerial

parts (methanol) extract was taken in a vial and dissolved in DMSO (net volume 1 ml)

and 300 mg Aspirin was dissolved in distilled water (net volume 1 ml) in another vial.

Meanwhile we also made the 1% acetic acid solution in a 100ml beaker. Then the

Control group was treated with only 1% acetic acid by intra-peritoneal administration,

Standard group-I and Standard group-II was treated with Aspirin 200 mg/kg & 400

mg/kg respectively; and 1% acetic acid by intra-peritoneal administration. Group M,

Figure 3: Modeled experimental mouse.

Figure 4: stop watch


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N, O and P were treated with aerial parts extract of Corchorus olitorius L. at 50

mg/kg, 100 mg/kg, 200 mg/kg and 400 mg/kg body weight respectively, followed by

1% acetic acid was administered by intra-peritoneal injection after 60 minutes. Acetic

acid induced mice are subjected for writhing for 15 minute observation.

Figure 7: Micropipette

Figure 6: Gavaging of mice.Figure 5: Preparation of extract


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The summary of experimental design is given below:

Extraction of Corchorus olitorius L. with non-polar (MeOH) solvent.

0.4 gm extract was dissolved in DMSO whose net volume 1ml and 300 mg aspirin was dissolved in

distilled water whose net volume 1 ml

Taken 35 adult mice and divided into following groups

Control

5 mice

were

treated

with

only 1%

acetic

acid

injectio

n

Standard I

Aspirin

200 mg/kg bw

5 mice were

treated aspirin

and 1% acetic

acid injection

Standard II

Aspirin

400mg/kg bw

5 mice were

treated aspirin

and 1% acetic

acid injection

Dose-M

50 mg/kg

bw

5 mice were

treated with

low dose of

Corchorus

olitorius L.

extract and

1% acetic

acid

injection

Dose-N

100 mg/kg

bw

5 mice were

treated with

moderate

dose of

Corchorus

olitorius L.

extract and

1% acetic

acid

injection

Dose-O

200 mg/kg

bw

5 mice were

treated with

medium

dose of

Corchorus

olitorius L.

extract and

1% acetic

acid

injection

Dose-P

400 mg/kg

bw

5 mice

were

treated

with high

dose of

Corchorus

olitorius L.

extract and

1% acetic

acid

injection

Acetic acid induced mice were subjected for 15 minute

observation of writhing.

Flow chart 2: Summary of experimental design.


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4.11 Procedures of vehicle, extract and standard drug sample preparation

All reagents and extract solution needed were prepared prior to the experiment. In

current experiments we used 1% acetic acid aqueous solution as vehicle to induce

visceral pain. Solution of commercial aspirin tablet in distilled water was used as

standard. Plant extract was made into solution using Dimethyl Sulfoxide (DMSO).

4.11.A Preparing plant extracts

A freshly cleaned, sterilized, dry vial was taken. Using a glass pipette, 1 ml distilled

water was taken and the upper meniscus level marked using a marker on the outer

surface of the vial. The distilled water was discarded and 0.4 g of the extract was

taken in the vial. Then Dimethyl Sulfoxide (DMSO) was slowly pippetted into the

vials till the upper meniscus touches 1ml mark, while dissolving the extract with the

help of a spatula.

4.11.B Preparing Dispirin solution

Another freshly cleaned, sterilized, dry vial was taken and the 1ml measurement

marked on the outside surface. In this vial 1 Dispirin tablet (300 mg) was dissolved in

1ml of distilled water. Dispirin is made by Reckitt Benckiser Bangladesh Ltd. and is

readily found at any local pharmacy. This was used as a standard.

Figure 7: Preparing aspirin solution

4.11.C Preparing 1% acetic acid solution

1% acetic acid was made in a beaker adding 1 part acetic acid and 99 parts of distilled

water. This was used as control and to induce abdominal constriction in the rodents.


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4.11.D Routes of administration

A. Acetic acid solution was administered intraperitonealy into the left lower

quadrant of mice using a hypodermic needle.

B. Aspirin and plant extract solutions were gavaged orally using blunt end yellow

tip pipette.

Figure 8: Injection of 1% acetic acid in mice

4.12 PROCEDURES

After 60 minutes of dosing of all the test samples in each mouse, 1% acetic acid is

induced in intra-peritoneal and after the administration of acetic acid, number of

squirms or writhing were counted for 15 minutes for each mouse. Note that first 5

minutes is not counted for data analysis. Only 10 minutes is counted for data analysis.

Figure 9: Writhing of mice.


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35

Flow chart 3: Schematic diagram of the induction and counting of writhing.

4.13 Phytochemical analysis of Corchoru s olitori us L. (aerial parts) extract:

4.13.A Appliances (Necessary equipments and materials):

Apron Beakers Spatula Measuring pipette Digital balance

Plant extract Distilled water Spirit lamp Gloves Filter paper 5% FeCl 3 Funnel

1% Picric acid

0 min.

Time allowed for absorption Counting ofwrithing

60 min. 75 min.

Oral administration oftest samples

Intra-peritoneal administration ofacetic acid (1 )


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37% HCl Ethanol 10% w/v Ethanol solution

4.13.B Saponin analysis:

Preperation of stock solution for saponin analysis:

0.5gm plant extract was taken by using digital balance in a beaker. Then we added 10

ml water with it. After dissolving plant extract into water we heated the solution for 5

minutes. Then we kept it for cooling.

Methods for saponin analysis:

We took 1ml stock solution in a beaker. Then we added 1ml distilled water into it.

After that we vigorously shaked the beaker and kept it in the working bench for

observation.

Observation:

Bubble was formed in the beaker and persisted for some times.

Result:

Saponins are present in Corchorus olitorius L. (aerial parts) MeOH extract.

4.13.C Tannin analysis:

Preperation of 5% FeCl 3 solution:

0.5 gm FeCl 3 was taken by using digital balance in a beaker. Then we added 5ml

water into it and mixed it. Then we adjusted the volume upto 10 ml by adding water.

Methods for Tannin analysis:

At first we took 0.5gm plant extract using digital balance in a beaker. Then we added

10 ml water with it. After dissolving plant extract into water filtered it using filter

paper. Then we added a few drops (3-4) 5% FeCl 3 in the filtrate solution and

observed it.

Observation:

Green precipitation was formed into the beaker.

Result:

Tannins are present in Corchorus olitorius L. (aerial parts) MeOH extract.


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4.13.D Alkaloid analysis:

Preperation of 10% w/v ethanol stock solution:

2 gm plant extract was taken using digital balance in a beaker. Then we added ethanol

in the beaker and mixed it and adjusted the volume upto 20 ml.

Preperation of 1% picric acid solution:

2 ml water was taken in a beaker and 0.05 (50 mg) picric acid was added into it by

mesuring with digital balance and mixed it. Then we added water to adjust the volume

upto 5 ml.

Methods for alkaloid analysis:

2 ml of 10% w/v ethanol stock solution was taken into a beaker. Then we added 0.2ml

37% HCl. Then we shaked and mixed it. After that 1 ml of 1% picric acid solution

was added and observed it.

Observation:

Yellow color precipitation was observed.

Result:

Alkaloids are present in Corchorus olitorius L. (aerial parts) MeOH extract.

4.13.E Flavonoid analysis:

Methods for flavonoid analysis:

5 ml 10% w/v ethanol solution was taken into a beaker. Then we added 0.1 ml 37%

HCl and 4-5 drops of 5% FeCl 3 into it and observed it.

Observation:

Color was changed of the solution and green precipitation was observed.

Result:

Flavonoids are present in Corchorus olitorius L. (aerial parts) MeOH extract.


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Chapter FiveResults & Discussion


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5.1 Result of analgesic effect of Corchoru s oli toriu s L. (aerial parts)

The MeOH extract of Corchorus olitorius L. (aerial parts) was subjected to screening

for analgesic activity by acetic acid induced writhing inhibition method in mice. Intra-

peritoneal administration of acetic acid (1%) causes localized inflammation in mice.

Following inflammation, there is biogenesis of prostaglandins (cyclooxygenase

pathway) and leukotrienes (lipooxygenase pathway). The release of prostaglandins,

mainly prostacyclin (PGI 2) and to lesser extent prostaglandin-E have been held

responsible for pain sensation.

Table 3: 1% Acetic acid induced analgesic effect of methanol extract of Corchorus

olitorius L. (aerial parts) on Swiss Albino mice model system

Data given as Number of writhing .

SerialControl

10ml/kg

Standard

I

200mg/kg

Standard

II

400mg/kg

Dose-M

50mg/kg

Dose-N

100mg/kg

Dose-O

200mg/kg

Dose-P

400mg/kg

1 5 3 1 6 3 3 3

2 5 2 2 3 2 2 2

3 5 4 1 4 2 2 2

4 5 2 1 4 5 3 3

5 6 5 4 4 3 2 1

Sum 26 16 9 21 15 12 11

Mean 5.2 3.2 1.8 4.2 3 2.4 2.2

SD 0.44721 1.3038 1.3038 1.0954 1.2247 0.5477 0.8367

SE 0.1996 0.5821 0.5821 0.4890 0.5468 0.2445 0.3735

T-value 3.2444 5.5155 1.889822 3.7730 8.8544 7.0711

P-value 0.01 0.0005 0.05 0.005 0.0005 0.0005Significance

level 1% 0.05% 5% 0.50% 0.05% 0.05%

5%

Significance

Level

Significant Significant Significant Significant Significant Significant


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Analgesic activity of Corchorus olitorius L. (aerial parts) methanol extract in Swiss

Albino mice

Different doses (mg/kg)

Bar diagram 1: Analgesic activity of Corchorus olitorius L. ( aerial parts) MeOH

extract by acetic acid induced writhing method.

5.2 DISCUSSION

From the analgesic effect analysis by the number of squirms or writhing counted for

10 minutes after 1% acetic acid injection, we found that the Control mean was 5.2, the

Standard I mean was 3.2 and the Standard II mean was 1.8. On the other hand the

Corchorus olitorius L. (aerial parts) MeOH extract low dose (50 mg/kg) treated mean

was 4.2, moderate dose (100 mg/kg) treated mean was 3, medium dose (200 mg/kg)

treated mean was 2.4 and high dose (400 mg/kg) treated mean was 2.2. Comparing

with Control mean all the given doses significantly decreased the pain.

0

1

2

3

4

5

6


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5.3 Result of phytochemical analysis of Corchorus olitorius L. (aerial parts)

MeOH extract:

Table 4: Result of phytochemical analysis of Corchoru s olitor iu s L. (aerial parts)

MeOH extract:

Serial Name of

phytochemicals

Changes observed in

solution

Result

1. Saponin Bubble forms and

persist for some time

Present

2. Tannins Green precipitation Present

3. Alkaloids Yellow precipitation Present

4. Flavonoids Green precipitation Present

From the phytochemical analysis of plant extract, we found that plant extract contains

saponins, tannins, alkaloids and flavonoids.


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Chapter Six

Conclusion


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From our scientific investigation we have observed that the methanolic extract of

Corchorus olitorius L. (aerial parts) has analgesic activity and has the presence of

some phytochemicals like saponins, tannins, alkaloids and flavonoids. As a result, we

can conclude that Corchorus olitorius L., one of the available species in Bangladesh,

has some desired chemical entities that confirm its analgesic activity. Planned and

systematic cultivation and proper scientific investigations, both pharmacological and

phytochemical, of this plant are sure to produce a large variety of new drugs and

pharmaceutical raw materials of natural origin. Thus the indigenous Corchorus

olitorius L. promises to be a very good source of new drugs and pharmaceutical raw

materials in the country.


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Chapter Seven

References & Bibliography


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45

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Hassandarvish P, Fouad M, Golbabapour S, Talaee S. Gastroprotective effects of

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Bamigbade TA, Davidson C, Langford RM, Stamford JA Actions of tramadol, its

enantiomers and principal metabolite, O-desmethyltramadol, on serotonin (5-HT)

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By hepzibah on July 24th, 2008, Saluyot (Jute Leaves): The Wow Vegetable. Chen, T.S. and Saad, S. 1981. Folic acid in Egyptian vegetables: The effect of

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www.stuartxchange.com/Pasau.html


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Appendix


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49

APPENDIX

Calculation of SD, SE and unpaired t-value used in different biological studies:

SD=

1

2

n

x x

Where,

SD = Standard deviation X = Value of individual observation

X = Mean value

n = Number of observation

SE = n

SD

Where,

SE = Standard error

SD = Standard deviation

n = Number of observation

t value, t =

nS

mS

y x2

2

2

1

0


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50

Abbreviation


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51

C : Degree Celsius

AECO : Aqueos extract of Corchorus olitorius

B : Boron

bw : body weight

Ca : Calcium

Cdcl 2 : Cadmium chloride

cm : Centimeter

CNS : Central nervous system

CO : Corchorus olitorius

COX : Cyclo Oxygnase

DAG : Diacylglycerol

DMSO : Ddimethyl sulfoxide

DNA : Deoxyribonucleic acid

et al : et alia = and other people

etc : et cet. era = and the others

Fe : Ferrus

Fecl 3 : Ferric chloride

GI : Gastrointestine

gm : gram

GST : Glutathaione S-transferase

HCl : Hydrogen chloride

ICDDRB, B : International Center for Diarrheal Diseases and Research,

Bangladesh

IP3 : inositol triphosphate

K : Potassium

L. : Linnaeus (Name of scientist)

MeOH : Methanol

mg/kg : milligram/kilogram

mg/ml : milligram/milliliter

ml : milliliter

mm : millimeter

Mn : ManganeseMo : Molybdenum


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Na : Sodium

NaASO 2 : Sodium arsenite

NK : Natural Killer Cell

NSAID : non-steroidal anti-inflammatory drugs

P : Phosphorus

PGI 2 : Prostacyclin

SD : Standard Deviation

SDF : Soluble dietary fiber

SE : Standard Error

UODA : University Of Development Alternative

USDA : United States Department of Agriculture

WHO : World Health Organization

Zn : Zinc

Analgesic property of Corchorus olitorius L

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