TABLE OF CONTENTS

1.1 Classification of Pain ............................................................................................... 1

1.2. Types of Pain .......................................................................................................... 2
1.3. Some other Types of Pain are Followings .............................................................. 3
1.3.1. Nociceptive ................................................................................................... 3
1.3.2. Neuropathic .................................................................................................. 4
1.3.3. Phantom pain ................................................................................................ 4
1.3.4. Psychogenic pain .......................................................................................... 4
1.3.5. Breakthrough pain ........................................................................................ 5
1.3.6. Incident pain ................................................................................................. 5
1.4. Process of Pain ........................................................................................................ 5
1.5. Diagnosis to a Pain.................................................................................................. 8
Management of pain ............................................................................................. 8
1.6. Medication for Pain................................................................................................. 8
1.6.1. Medication for Mild pain.............................................................................. 8
1.6.2. Medication for mild to moderate pain .......................................................... 8
1.6.3. Medication for moderate to severe ............................................................... 8
1.7. Assessment of Pain ............................................................................................... 10
1.8. Fever ..................................................................................................................... 12
1.9. Factor Affecting on Fever ..................................................................................... 12
1.10. Temperature Classification ................................................................................. 13
1.10.2. Normal temperature .................................................................................. 13
1.10.3. Fever ......................................................................................................... 13
1.10.4. Hyperthermia ............................................................................................ 13
1.10.5. Hyperpyrexia ............................................................................................ 13
1.11. Causes and Diagnosis of Fever ........................................................................... 14
1.12. Types of Fever .................................................................................................... 15
1.12.1. Continuous fever ...................................................................................... 15
1.12.2. Intermittent fever ...................................................................................... 15
1.12.3. Quotidian fever ......................................................................................... 15
1.12.4. Tertian fever ............................................................................................. 15
1.12.5. Quartan fever ............................................................................................ 15
1.12.7. Febrile neutropenia ................................................................................... 16

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 1.12.8. Persistent pyrexia of unknown origin (PUO) ........................................... 16
1.12.8.1. Diagnosis of PUO .................................................................................. 16
1.13.9. Drug which causing fever ......................................................................... 17
1.13. Mechanism of Drug’s Induced Fever ................................................................. 17
1.15. Hyperpyrexia....................................................................................................... 18
1.15.1 Causes of hyperpyrexia ............................................................................. 18
1.16. Difference between Hyperpyrexia and Hyperthermia ........................................ 18
1.17. Pathophysiology of Fever ................................................................................... 19
Http//www.google.com/ inhibition of prostaglandin synthesis researchgate.net/
download images.jpeg/files/C disk....................................................................... 20
1.18. Measurement of fever ......................................................................................... 23
1.18.1. Axilla ........................................................................................................ 23
1.18.2. Skin ........................................................................................................... 23
1.18.4. Rectal temperature .................................................................................... 23
1.19. Antipyretics ......................................................................................................... 24
1.19.1. Mechanism of Action of Antipyretics ...................................................... 24
1.19.2. Antipyretic therapy ................................................................................... 24
1.19.2.1. Aspirin ............................................................................................... 24
1.19.2.2. Paracetamol ........................................................................................ 25
1.19.2.3. Neproxin ............................................................................................ 25
1.19.2.4. Indomethacin ..................................................................................... 25
1.19.2.5. Anti-pyrine ......................................................................................... 25
1.19.2.6. Salicylamide ....................................................................................... 25
1.19.2.7. Steriods .............................................................................................. 25
1.19.3 Physical treatment ..................................................................................... 26
1.19.3.1. Alcohol sponging ............................................................................... 26
1.19.3.2. Terpid sponging ................................................................................. 27
1.19.3.3. Cold sponging .................................................................................... 27
1.19.3.4. Bed rest .............................................................................................. 27
1.19.3.5. Total body cooling ............................................................................. 27
1.20. Introduction of plants................................................................................ 28
1.20.1. Cyperus pertenuis ................................................................................... 28
1.20.2. Delphinium zelil........................................................................................ 29
1.21. Literature Review................................................................................................ 30
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1.22. Aims and Objectives ........................................................................................... 36
2.1. Experimental Animals .......................................................................................... 38
2.2. Drugs and Chemicals ............................................................................................ 38
2.3. Equipment’s .......................................................................................................... 38
2.4. Collection of Plants ............................................................................................... 38
2.5. Extracts Preparation .............................................................................................. 38
2.6. Phytochemical Screening ...................................................................................... 39
2.6.1. Solubility test .............................................................................................. 39
2.6.2. Test for Tannin ........................................................................................... 39
2.6.3. Tests for Alkaloids ..................................................................................... 39
2.6.3.1. Hager’s test .......................................................................................... 39
2.6.3.2. Wagnar’s test ....................................................................................... 39
2.6.3.3. Dragondroff test ................................................................................... 40
2.6.3.4. Mayer’s test .......................................................................................... 40
2.6.4. Test for saponin .......................................................................................... 40
2.6.5. Steroids test ................................................................................................ 40
2.6.6. Test for Cardiac glycosides ........................................................................ 40
2.6.7. Test for carbohydrates ................................................................................ 41
2.6.7.1. Molish test............................................................................................ 41
2.6.8. Test for amino acid ..................................................................................... 41
2.6.8.1. Ninhydrin test ...................................................................................... 41
2.6.9. Test for proteins .......................................................................................... 41
2.6.9.1. Biuretic test .......................................................................................... 41
2.6.10. Test for terpenes and sterols ..................................................................... 41
2.6.10.1. Libermaan burchard test .................................................................... 41
2.6.11. Total phenolic contents............................................................................. 41
2.6.12. Total flavonoid contents ........................................................................... 42
2.6.13. Assay of free redical scavenging activity by DPPH method .................... 42
2.7. Antipyretic Activity .............................................................................................. 43
2.7.1. Brewer’s yeast induced method.................................................................. 43
2.8. Analgesic Activity ................................................................................................ 43
2.8.1. Hot plate Assay........................................................................................... 44
2.8.2. Acetic induced writhing method ................................................................ 45
2.9. Acute Toxicity Study (LD50%) ............................................................................ 46
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2.10. Statistical Assay .................................................................................................. 46
3.1. Percentage Yield of Plant Extract ......................................................................... 47
3.2. Qualitative phyto-chemical Assay ........................................................................ 48
3.3. Total flavonoids Content....................................................................................... 50
3.4. DPPH free Redical Scavenging Activity .............................................................. 52
3.5 Antipyretic Activity Determination ....................................................................... 54
3.4. Results of effect of Cyperus pertenius ethanolic extract (C.p) on Brewer’s
yeast induced hyperpyrexia in rats ................................................................... 54
3.6. Analgesic Activity Determination ........................................................................ 57
3.6.1. Hot Plate method ........................................................................................ 57
3.6.2. Acetic induced writhing method ................................................................ 61
3.7. Acute Toxicity Test............................................................................................... 63
4.1 Percentage of Yield of plant extract Delphiniun zelil………………………………………….65

4.2. Qualitative phyto-chemical Assay ........................................................................ 65

4.3. Total Phenolic Content ......................................................................................... 66
4.3. Total Flavonoids Content ...................................................................................... 68
4.4. DPPH free Redical Scavenging Activity .............................................................. 70
4.5. Results and Effects of Delphinium zelil ethanolic extract (D.z) on
Brewer’s yeast induced hyperpyrexia in rats. ................................................. 72
4.6. Analgesic Activity Determination ........................................................................ 75
4.6.1. Hot Plate method ........................................................................................ 75
4.6.2. Acetic induced writhing method ................................................................ 79
4.7. Acute Toxicity Test............................................................................................... 81
5.1. Discussion ............................................................................................................. 83
5.2. Conclusions and Recommendations ..................................................................... 86
References .................................................................................................................... 87

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 List of Tables

Table 3.1 .Solubility results of C.pertenius extract ..................................................... 45

Table 3.2. Phytochemical results of C.pertenius extract .............................................. 46
Table 3 3. Estimation of total phenolic contents of C.pertenius ................................... 47
Table 3. 4. Estimation of total Flavonoids contents of C.pertenius .............................. 49
Table 3.5. Results of assay of antioxident activity by DPPH method at 517nm
percentage inhibition ..................................................................................................... 51
Table 3.6. Effect of different doses of Cyperus pertenius ethanolic extract on brewer’s
Yeast Induced Hyperpyrexia......................................................................................... 54
Table 3.7. Effect of different doses of Ethanolic extract of Cyperus pertenius and
pa.racetamol on Latency time on Hotplate method in Rats .......................................... 57
Table 3. 8. Decrease in latency time of C.pertenius E .................................................. 59
Table 3.9. Effect of different doses of Ethanolic extract of Cyperus pertenius and
diclofenac sodium on acetic induced writhing in rats ................................................... 60
Table 3.10. Acute toxicity test of Cyperus pertenius on behavioral and neurological
changes in rats ............................................................................................................... 62
Table 4.1 .Solubility results of D.zelil extract .............................................................. 63
Table 4. 2. Phytochemical results of D. zelil extract .................................................... 64
Table 4.3. Estimation of total phenolic contents of D.zelil ........................................... 65
Table 4.4. Estimation of total Flavonoids contents of D. zelil ...................................... 67
Table 4.5. Results of assay of antioxident activity by DPPH method at 517nm
percentage inhibition ..................................................................................................... 69
Table 4.6.Effect of different doses of Delphinium zelil ethanolic extract on brewer’s
Yeast Induced Hyperpyrexia......................................................................................... 72
Table 4. 7.Effect of different doses of Ethanolic extract of Delphinium zelil and
pa.racetamol on Latency time on Hotplate method in Rats .......................................... 75
Table 4. 8. Decrease in latency time of Dz.E................................................................ 77
Table 4. 9. Effect of different doses of Ethanolic extract of Delphinium zelil and
diclofenac sodium on acetic induced writhing in rats ................................................... 78
4. 10. Acute toxicity test of Delphinium zelil on behavioral and neurological changes
in rats ............................................................................................................................. 80

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 List of Figures

Figure 1. 1 transmission of pain ...................................................................................... 6

Figure 1. 2 transmission process ..................................................................................... 6
Figure 1. 3 Mode of action od NSAIDs .......................................................................... 8
Figure 1. 4 mode of action of NSAIDs ........................................................................... 9
Figure 1. 5 Pain asessment in childrens ........................................................................ 10
Figure 1. 6 Pain scale .................................................................................................... 11
Figure 1. 7 Inhibition of prostaglandins ........................................................................ 20
Figure 1. 8 Fresh and preserved rhizomes of Cyperus pertinius .................................. 27
Figure 1. 9 Delphinium zelil.......................................................................................... 29
Figure 3. 1 Graphical representation of standard curve of total phenolic contents of C.
pertenius ........................................................................................................................ 48
Figure 3. 2. Graphical representation of standard curve of total flavonoid contents of
C. pertenius .......................................................................................................................
Figure 3. 3. Graphical representation of anti-oxident of C.pertenius by DPPH method ..
Figure 3.4. Graphical representation of Effect of different doses of Ethanolic extract
of C.pertenius on rectal temperature by Brewer’s yeast method .................................. 55
Figure 3.5. Graphical representation of Effect Ethanolic extract of Cyperus pertenius
and paracetamol on Latency time on Hot plate method in Rats ................................... 58
Figure 3. 6 Graphical representation of effect of different doses of ethanolic extract of
Cyperus pertenius and diclofenac sodium on acetic induced writhing in rats .............. 61
Figure 4.1Graphical representation of standard curve of total phenol contents of
D.zelil ........ …………………………………………………………………………………..66
Figure 4. 2 Graphical representation of total flavonoid contents of D.zelil ................. .68
Figure 4. 3 Graphical representation of anti-oxident of D.zelil by DPPH method ....... 70
Figure 4. 4 Graphical representation of Effect of different doses of Ethanolic extract
of D.zelil on rectal temperature by Brewer’s yeast method .......................................... 73
Figure 4. 5 Graphical representation of effect of different doses of ethanolic extract of
Delphinium zelil and paracetamol on Latency time on Hot plate method in Rats ........ 76
Figure 4. 6 Graphical representation of Effect of different doses of Ethanolic extract
of Delphinium zelil and diclofenac sodium on acetic induced writhing in rats ............ 79

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 LIST OF ABBREVATIONS

ATP Adenosine triphosphate

COX-1 Cyclooxygenase-1
C0X-2 Cyclooxygenase-2
CBC Complete blood count
CRP C-reactive protein
CMC Corboxy methyl cellulose
CSF Cerebrospinal fluid
DF Drug fever
DDT Dichloro diphenyl trichloroethane
DMSO Di-methyl-sulfoxide
ESR Erythrocytes sedimentation rate
IL-1 Interlukin-1
IP Interaperitoneal
INF Interferon
JIA Juvenile idiopathic arthritis
NSAIDs Non-steroidal anti-inflammatory drugs
PUO Pyrexia of unknown origin
PGE2 Prostaglandin E2
TB Tuberculosis
TNF Tumor necrosis factor
URTI Upper respiratory tract infection
UTI Urinary tract infection
UC Ulcerative colitis
WBC white blood cells

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 ABSTRACT

Cyperus pertinius and Delphinium zelil both plants were frequently used in folk
medicine as a part of many medicinal preparations, including those which were used
to relieve pain and antipyretic, which grows abundantly at northern areas. Aim of the
present study was to validate the traditionally claims about both plants and also
effectiveness in pain and antipyretic by using hot plate analgesia, Acetic acid induced
writhing and Breawer‖s yeast induced method. In this study there is also the
comparison of two indigenous plants and their role in diseases. Special significant has
been put on treatment of disease with herbal products. Special emphasis has been put
on the pharmacological part and their activities in animals with underlying
mechanism. The experiment part of the study included extraction, phytochemical test
and evaluations, pharmacological evaluation encompasses antioxidant, analgesic,
antipyretic activities and acute toxicity study.

Antipyretic activity was determined by using the Brewer´s yeast induced method.
After oral administration there was 60.6% of decrease in rectal temperature after the
4th hour by the Cyperus pertenius at dose of 400mg/kg and 55.8% at the dose of
400mg/kg of Delphinium zelil when compared with control group

Analgesic activity was determined by using hot plate and acetic acid induced writhing
methods. Hot plate method produced maximum latency rate of 65 seconds at 150
minutes with the dose of 400mg/kg of Cyperus pertenius and 67.8 seconds at 150
minutes at the dose of 400mg/kg dose of Delphinium zelil. Acetic acid induced
writhing method produced 68.1% pain protection within 15-20 minutes at dose of
400mg/kg of Cyperus pertenius and 64.8% pain protection within 15-20 minutes at
dose of 400mg/kg of Delphinium zelil.

Acute toxicity (LD 50%) study was also done to checked the animal behavior changes
with maximum dose of 5000mg/kg and there was not seen any behavioral changes
with both plants extract at the maximum doses which showed that it is safe up to the
maximum dose of 5G This study indicates the presence of antipyretic activity possibly
mediated through the inhibition of prostaglandin pathway and analgesic activity
possibly regulated through central and peripheral pathways. Antioxident activity assay

viii
were performed and results produced were most likely due to presence of flavonoids
in the crude extract. This strengthen the results of the study and may be one of the
probable reason behind the effect of Cyperus pertinius and Delphinium zelil. The
presence of flavonoids, like baicalin and quencetin and alkaloids like boldine led to
the analgesic and antipyretic effect of cyperus pertenius and Delphinium zelil as
indicated by phytochemical analysis. Since the study was aimed to validate the
traditional uses and evaluation for the presence of antipyretic and analgesic activities.
So assumption were made about the proposed mechanisms on the basis of
resemblance of the results with the control

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1. Pain

The pain is the physical feeling which is caused by any disease, injury or
something that hurts the body. It may cause sadness mentally or emotionally.
Something that may cause trouble or make you feel irritate or angry.

Definition

The international Association for study of pain (IASP) defined as “pain is an

unpleasant sensory and emotionally experience which is associated with tissues
damage temporary or permanently.‖

Pain is the distressing felling which is often caused by damaging stimuli such
as burning of finger, putting alcohol on a cut etc (Naro, Leo, et al., 2015).

Medically it is diagnosis as just a symptom.

Pain may alarm the person to withdraw from damaging condition, to protect a
damaged body part while it is healed and to avoid the same condition in future. (A. V.
Holden & W. Winlow, 1984) Most of the pain resolves once the harmful stimuli is
removed and body is healed up, but it may contineous to exit without being affected
by removal of stimulus and appearent healing of body. some time it may arise in
absence of any detectable disease or stimuli (Kreitler & Beltrutti, 2007).

Simple medication of pain is useful in 20-70% cases (Moore et al., 2013).

Pain is the most common physician consultation in most countries and it is major
symptom in medical conditions which can interfere the quality of life and internal
functioning of body (Milton, 2013).

1.1 Classification of Pain

In 1994 the international Association for study of pain (IASP) classify the pain
on specific characteristics such as Region of body involved (Abdomen, upper limbs,
lower limbs) Dysfuntion of any system such as ( Nervous system, Gestro-intestinal
tract), Duration and pattern of occurance, Intensity and onset time, Causes (Treede et
al., 2008).

However clifford and J.woolf classify pain into three other classes

1
  Norciceptive
 Inflammatory pain
 Pathological pain which is caused by any disease (Woolf, 2010).

Pain is usually temporary and last for only until the harmful agent is removed
or underlying damaging, pathological condition is recovered. But in some painful
condition such as Rheumatoid arthritis, cancer and iodiopathic pain may be prolong
for years. The pain which recovered quickly is called acute pain and pain which is
long lasting is called as chronic pain. The variation between acute and chronic
depends upon duration of time period. The two commonly used markers are

The pain less than 30 days may be acute pain

The pain which remain for more than 3 month is said to be chronic pain (Lindenbaum
& Milia, 2012).

1.2. Types of Pain

Acute pain

Chronic pain

(A) Acute pain

Acute pain is uncomfortable feeling due to tissue damage, injury, or

inflammation that goes away with healing of injury. It is caused by trauma, injury,
cancer, infection and surgerical process. It can produce change in autonomic nervous
system by which increase in heart rate, sweating, and blood pressure. It can also
disturb the normal daily activity and sleep. It is last for less than 30 days. It may be
managed by simple analgesic, NSAIDs (non-steroidal anti-inflammatory drugs) like
diclofenac sodium, mafenemic acid etc (Pasero & McCaffery, 1999).

(B) Chronic pain

Chronic pain can be a major problem for some people and it effect the daily
life and quality of life. The chronic pain may be caused by change in nociception,
injury, or disease and may result from current or past damage to peripheral nervous
system. The duration of chronic pain is long which is more than 3 months to years. It

2
is pain like cancer pain or rheumatoid pain. It is managed by strong pain relief e.g
opioids, morphine like drugs (Calvino & Grilo, 2006).

1.3. Some other Types of Pain are Followings

1.3.1. Nociceptive

Norciceptive pain is the pain which is caused by stimulation of sensory nerve

fibers that may reply to stimuli approaching. It may be classified according to the
mode of harmful stimuli. The most commonly classes are

 Thermal stimuli
 Mechanical stimuli
 Chemicals
Some nociceptive response to more than one of them are said to be poly-
modal.Nociceptive pain may also be divided into

(a) Visceral pain

Visceral structure is very sensitive to stretching, inflammation or ischemia’s

but insensitive to other stimuli that may produce pain in other structure like cutting or
burning. It is diffused, difficult to locate and often referred to a distant usually super-
facial structure. It may accompanied as nausea and vomiting and may be said that dull
or deep squeezing.

(b) Deep somatic pain

It is initiated by stimulation of norciceptor in bones, ligands, tendons, blood

vessel and muscles. It is dull irritating localised pain e.g broken of ulna bone produce
pain in arm.

(c) Super-facial somatic pain

Super-facial somatic pain is initiated by activation of norciceptor in the skin and other
super-facial tissues. It is defined and clearly located pain like minor wound or minor
burn (Main & Spanswick, 2000).

3
Http//google.com./basic types of pain pain science.com/download images.jpeg/files/C
disk
Fig 1.1: Types of pain

1.3.2. Neuropathic

The pain which is feels due to damaging of nervous system by any disease or
pathological change (Majumder & Farquhar-Smith, 2007).

1.3.3. Phantom pain

Phantom pain is pain which is felt when loss of any body part or no more
signals is received from brain. Some time it is said to be phantom pain. Pain felt by
chopped legs and arm (Kooijman et al., 2000).

1.3.4. Psychogenic pain

It is sometime call as psych logia. The pain due to emotional or mental

disturbance rather than physiological origin e.g headache, back-pain. Sometime
stomach pain is also diagnosis as psychogenic pain by some physicians .In the view of
general public psychogenic pain is not real pain (Engel, 1959).

4
1.3.5. Breakthrough pain

Breakthrough pain is short term acute that comes on instantly and is not
reduced by regular patient pain management. It is common in cancer patients who
often have background of pain which is treated by medication. The features of
breakthrough pain in the cancer pain may differ from person to person according to
causes. Management of this pain is by opioids or fentanyl (Haugen et al., 2010).

1.3.6. Incident pain

Pain that comes as the result of an activity such as movement of arthritis joint.

Effects of pain on functioning

The pain may affect the person physiologically and psychologically. The
patient who have acute or chronic pain may experience impairment in attention,
working memory, mental flexibity, problem solving and information processing -
speed. Acute or chronic pain increase depression or anxiety, fear and anger in the
person (Hart et al., 2003).

Duration of pain

Duration of pain depend on type of pain present.

 Acute pain is for less than 30 days

 Duration of Chronic pain is last for more 3 month to several years (Pasero et
al., 1999).
Pain can be good
Sometime pain can produce positive results like
 It can serve as a protection process and warning the person when belonging to
feel the pain related to presence of painful stimuli.
 It can drive the avoidance process.
 It can have legislative effect
 It can proceed weak effect like decrease appetite, increase heart rate, decrease
sleep and increase respiratory rate.

1.4. Process of Pain

There are 4 steps which are involved how our body process pain

5
 (a) Transduction
(b) Transmission
(c) Perception
(d) Modulation
(A) Transduction

Norciceptor received pain signal message from skin, bones, deep tissue, and
viscera. These message signals are actually chemicals that become in an action
potential.

(B) Transmission

This message is than transmitted via peripheral nervous system to spinal cord
and then to the dorsal horn where they are transfer to the thalamus and store in and
sent to cerebral cortex. From here message actually hurts.

(C) Perception

The cerebral cortex recognized where the pain is actually from or rather is
going to be felt. It can create measureable references for pain so that it has an
intensity, quality and location. Because of the pain which can be feel by many
dimensions which can be associated with emotional, feeling of anxiety, fear or
pleasure.

(D) Modulation

Pain can also include facilitation and inhibition. Body action against the
message is to prevent, decrease, minimize the pain message (Gatchel, 1996).

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 Http//www.google.com./pain pathway pain genes journal.plos.org/download
images.jpeg/files/C disk

Fig 1.2: Pain pathway

Http//www.google.com./pain pathway journals.plos.org/download images.jpeg/files/C

disk

Fig 1.3. Transmission of pain

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 In the figure there is the matrix of pain which showed how the pain is start and feel by the
nerves

1.5. Diagnosis to a Pain

Pain is the symptom of many medical conditions. Diagnosis of pain depend

upon time of onset, location, intensity, pattern of occurance and quality of pain e.g
chest pain with extreme heaviness may indicate as Myocardial Infarction and chest
pain as tearing as Aortic Dissection (McNally, 1990).

Management of pain

Poor quality of pain treatment is throughout the surgical wards, intensive care
unit, accident, emergency department and general-wards have been seen in world-
wide. This neglection is extended to all ages from neonate to adult. The international
Association for the pain study (IASP) support that relief from pain is human right and
it should be treated as a disease and should not forget due to which may produce
many problems in future.

1.6. Medication for Pain

The world health organization (WHO) has mentions a pain ladder for treating
the pain. There are 3steps which are involved for selection of drugs in management of
pain.

1.6.1. Medication for Mild pain

For the treatment of mild pain paracetamol (acetaminophen) or non-steriodal

anti-inflammatory drugs (NSAIDs) like ibuprofen, diclofenac sodium etc are used.

1.6.2. Medication for mild to moderate pain

In this NSAIDs or paracetamol NSAIDs combination are used with weak

opioids like tramadol which may relief the pain immediately as compared when given
single.

1.6.3. Medication for moderate to severe

When we treated moderate to severe pain it depend on type of pain which may
be acute or chronic. On the basis of type of pain can result in different medication

8
being prescribed. Some medication may work good in acute pain and other in chronic.
Acute pain medication is rapid in onset like post-operative pain or trauma injury
immediate relief of pain medicine is used but in chronic long lasting continue
medication is used in cancer therapy (Fishman, 2012).

Mode of action analgesic

Http//www.google.com./nonsteroidal anti-inflammatorydrugs socratic.org/download

images.jpeg/files/C disk

Fig 1.4: Mode of action of NSAIDs

The figure showed the mechanism of action of pain relievers which block the COX-
1and COX-2 by acting on the cyclooxygenase enzyme

9
 Http//www.google.com/ pharmacologic management of chronic pain
synape.koreamed.org/ download images.jpeg/files/C disk

Figure 1.5: Mode of action of NSAIDs

This figure show that how the medication cover the pain from mild to severe pain

1.7. Assessment of Pain

Self -reporting is most reliable way to measure pain. But it is very difficult to
assess the severity pain in children and for this different type of methods are used like
Oucher Scale, pointing to schematics of faces showing different pain levels or locate
the pain on body parts.

10
 Http//www.google.com/ horizontal pain measurement scale pain assessment
shutterstock.com/ download images.jpeg/files/C disk

Fig 1.6: Pain Asessement in childrens

Http//www.google.com/ pediatric pain assessment nhpco.org/ download

images.jpeg/files/C disk

Fig 1.7: Pain scale

This figure show that the pain scale in the children which assess the severity of pain in
children

11
1.8. Fever

Definition

’Fever is defined as having the temperature above the normal range due to an
increase in body’s temperature set-point or less than ≥38.1°C temperature. It is also
called pyrexia or febrile response’’ (Herzog & Coyne, 1993).

There is no single upper limit for normal temperature with source using the
range from 37.5-38.3°C (Axelrod & Diringer, 2008).This increase in body
temperature produce muscle contraction and causes the feeling of cold. The result in
more heat production and effort to converse heat. When the body temperature return
to normal, person feel hot and sweating may produce (Diringer MN,. May 2008).

Fever do not commonly higher than 41-42°C (Mahadevan & Garmel, 2012). It
may be cause of many medical conditions which may not be serious or serious
problems, which included viral, bacterial, fungal infections which causes common
cold, urinary tract infection, malaria and meningitis. Non-infectious causes of causing
fever are deep vein thrombosis, vacuities, or side effect of many medication
(Mahadevan & Garmel, 2012).

About 75%of adult may have fever who are seriously ill and about 30% of
children developing fever when treating other medical problems (Sullivan & Farrar,
2011).

It is some time useful mechanism when treating diseases fever may not
increase (Mohammed & Ahmed, 2012).

1.9. Factor Affecting on Fever

There are different types of factors on which temperature of body is depending

which including

 Age
 Sex
 time of day
 surrounding temperature
 activity of person.

12
 Increase in temperature is not always fever e.g rise of body temperature during
exercise is not fever as set-point is normal. On other hand a normal temperature may
be fever, if it is unusually higher for person e.g weak elderly people have decrease
ability to produce enough body heat so normal temperature may represent clinically as
fever (Rolo, 2009).

1.10. Temperature Classification

Temperature can be classified in to different classes depending upon the rise in

temperature. It can be classify into following

1.10.1. Hypothermia

Hypothermia in which body temperature is less than 35°C or (Marx et

al., 2013).

1.10.2. Normal temperature

The range of normal body temperature is 36.5-37.5°C or 97.7-99.5°F

(Hutchison et al., 2008).

1.10.3. Fever

In the fever the body temperature start from 37.5-38.3°C or 99.5-100.9°F

(Axelrod & Diringer, 2008).

1.10.4. Hyperthermia

Hyperthermia is when body temperature is 37.5-38.3°C or 99.5-100.9°F

(Laupland, 2009).

1.10.5. Hyperpyrexia

When the body temperature is above the 40°C or greater than 104°F is said to
be hyper-pyrexia (Trautner et al., 2006).

13
Table 1.1: Difference between fever and hyperthermia

Fever Hyperthermia
Temperature above the normal range due to Increase in temperature over the
raise in body’s set-point set-point
Contration in muscle may cause in production Too much heat is produce
of heat
It may be central regulation No
Heat is lost by sweating. No loss of heat
Different antipyretic drugs are used for Only physical measure
treatment

1.11. Causes and Diagnosis of Fever

There are two types of main causes of fever and on which diagnosis of fever is
dependent

1. Infections which is caused by different bacteria, viruses, fungus and T.B

Diagnosis of infectious causes of fever are

 Different conditions effect to infection e.g neonate, young infants, sickle cell
anemia, IV catheters
 Alteration of fever chills and fever high up-to 40°C
 Viral fever may be short duration
 Infection specific diseases are pneumonia, tonsilitis. If fever is without any
causes then laboratory test may conforms the diagnosis.
2. Noninfectious causes of fever are drugs, allergy, malignancies, immunization and
periodic fever

Diagnosis of noninfectious causes of fever are

 Always presence of Low grade fever

 Chill and fluctuation of fever are absent
 History of patient
 Negative culture of bacteria in stool and urine
 Not responding to antibiotics but fever is down when taking of steroids (El-
Radhi et al., 2009).

14
1.12. Types of Fever

The method of changing of temperature from time to time may hint for good
diagnosis

1.12.1. Continuous fever

In the continuous fever temperature of body remain above the normal range
throughout day and does not alter more than 1°C within 24 hours e.g lobar
pneumonia, typhoid, urinary tract infection. Typhoid fever may show a specific
changes in body temperature with slow stepwise increase and a high level.

1.12.2. Intermittent fever

The increase in temperature is for some time and return back to normal after
certain period of time e.g. malaria, pyrexia etc.

It may be divided into following

1.12.3. Quotidian fever

In this there is a period of 24 hour in rise of temperature e.g malaria like

plasmodium falciperum.

1.12.4. Tertian fever

Alteration of temperature is for 48 hours typically plasmodium vivex or

plasmodium ovale malaria.

1.12.5. Quartan fever

Re-occurrence of fever after 72 hours typically of plasmodium malaria malaria

(Chouhan, 2011; Deyashi et al., 2011).

1.12.6. Remittent fever

Temperature always above the normal range for whole day. Changes in
temperature is more than 1°C in a day e.g infective endocarditis.

15
1.12.7. Febrile neutropenia

In this fever the immune system is not properly functioning due to lack of
neutrophil and by this bacterial infection may spread more rapidly. This is usually
medically urgent attention e.g fever in chemotherapy (Fauci, 2008).

1.12.8. Persistent pyrexia of unknown origin (PUO)

In this fever there is no localized signs and symptom with minimum duration
of 3 weeks. It can be evaluated and attack quickly in children other than in an elder.
Laboratory investigation is done on the basis of children health and level of
investigation is dependent on history like

 Traveling
 Exposed to infection
 Exposed to animals
 Ingestion of raw milk

1.12.8.1. Diagnosis of PUO

For the diagnosis of PUO early test like chest x-rays, C.B.C, urine test is
performed. If the examination failed for diagnosis then additional investigation and
infrequently invasive techniques will be done for proper diagnosis. The PUO is better
observed in children due to quickly occurrence of infection other than in adult (El-
Radhi et al., 2009).

1.12.8.2. Causes of PUO

Table 1.2: Causes of PUO

Causes Reasons
Localized Sinusitis Standard sinus radiogragh is not performed
Endocarditis May have cardiac defect previously
Occult abscess No clinical signs
Systemic Viral Fever is major sign of is disease
T.B Tuberlin test is negative
Kawasaki Unknown reason fever present
disease

16
1.13.9. Drug which causing fever

Fever may be due to presence of some drug side effects. It is common

condition in which administration of drug there is increase in body temperature which
is normalized after the withdrawal of drug therapy.

There are no specific tests to diagnosis for drug fever.

Drug fever may occur after start of drugs therapy and patient do not have any
serious side effects. Body temperature may return normal at adequate level like
cytotoxic or other drugs that cause hyperpyrexia may cause to increase in body
temperature.

1.13. Mechanism of Drug’s Induced Fever

 Some drugs when taken act as antigen and produce IL-1 which make antigen
anti-bodies complex with leukocytes and cause fever. Penicillin is good
example.

 Cytotoxic drugs and some antibiotic which are derived from microorganism
and contaminated with endotoxin may causes to increase body temperature.

 Some drugs have direct pyrogenic effect on hypothalamic center like cocaine

 Some drugs can also increase in body temperature by increasing the heat
production such as atropine

 Interferon are used for treatment of hepatitis B and C which are endogenous
pyrogen and causes fever (Treede et al., 2008).

Hyperthermia

Hyperthermia is unable to waste heat at enough time due to failure of

thermoregulatory mechanism, or maximum production of heat at normal rate of loss
of heat e.g malignant hyperthermia.

Causes of hyperthermia

Main cause is dehydration which causes

 Decrease in sweating

17
  Increase in body temperature
 Infections
 Vasoconstriction
Hyperthermia is also related with fever

Table 1.3: Difference between fever and hyperpyrexia

Fever Hyperthermia
Common Relatively rare
Feeling cold Feeling hot
Temperature is usually 38-41°C Fever may exceed up-to 42°C
Antipyretic drugs is used for treatment Only physically measurement

1.15. Hyperpyrexia

Hyperpyrexia in which extreme increase in body temperature greater than or

equal to 41.5°C or 106.7°F which is considered as medical emergency as it may leads
to significant side effects.

1.15.1 Causes of hyperpyrexia

Main causes of hyperpyrexia are

 Intracranial hemorrhage
 Sepsis
 Kawasaki syndrome
 Neuroleptic malignant syndrome
 Serotonin syndrome
 Infections

Infections are most common cause of fever however as the body temperature
increases other cause become more common. Infections which are associated with
hyperpyrexia are measles, retroviral infections (McGugan, 2001).

1.16. Difference between Hyperpyrexia and Hyperthermia

In the hyperpyrexia thermoregulatory mechanism set the body’s temperature

above the normal and then generate heat to achieve this temperature but in

18
hyperthermia failure of thermoregulatory mechanism and body temperature rises
above the set point due to external sources (Fauci, 2008).

1.17. Pathophysiology of Fever

Hypothalamus regulate the body temperature. It is some time called as

thermostat (Fauci & Langford, 2010). The fever is activated by pyrogen which causes
to release the prostaglandin E2 (PGE2).This prostaglandin act on hypothalamus which
generate a systemic response back to body which causing heat- creating effects to
body to match new temperature level. When the set-point is increased body also
increase its temperature through both active generation of heat and keeping of heat.
Through the peripheral veso-constriction both reduce heat loss by skin and cause to
feel cold. If blood temperature is not match then brain sets a new set-point in
hypothalamus and bring to cause shivering in order to use muscle movement to
produce more heat. When hypothalamus set-point move back either naturally or with
medication, then this process is reversed and sweating is produced used to cool body
by veso-dilation and no sweating.

Pyrogens that causing fever

The pyrogen is a substance that cause fever in body. These may be either
internal (Endogenous) or external (Exogenous).

(A) Endogenous

In all endogenous pyrogenicity is produced by cytokines, which are the parts

of immune system. When immune system is activated, there is production of
cytokines which causes to increase in thermoregulatory set-point. The major
cytokines are

 Interleukin 1 (IL 1)
 Interleukin 6 (IL 6)

1. Interkeukin1 (IL1)

These cytokines are present inactive form and converted into active form by
enzymatically before release in circulation. These are stored in secreting cells of

19
cytoplasm and major site of removal is kidney. These are in three forms in which two
act as agonist like ILα and Ilβ and one as antagonist IL ra acts on hypothalamus to
increase in body temperature. It also plays an important role in activation of T and B
cells.

2. Tumor necrosis factor (TNF)

The cytokines which are released from macrophages and monocytes are tumor
necrosis factor. These are also endogenous pyrogen which directly act on
hypothalamus and initiate fever. TNF has significant role in different biological
function with sharing of IL1 like increase in cytotoxic effects and phagocytic effect,
increase in defense mechanism against infections.

3. Interleukin6 (IL 6)

It is secreted by macrophages and T lymphocytes. It also activates immune

system. It initiate fever with combination of IL1 and TNFα.It is also responsible for
stimulation of acute phase protein and production of neutrophils. The level of IL6 is
increased in many diseases like sepsis, kawasaki disease, autoimmune disease and
infections.

Http//www.google.com/ inhibition of prostaglandin synthesis researchgate.net/

download images.jpeg/files/C disk
Fig 1.8: Inhibition of prostaglandins

20
(B) Exogenous

Most of the mechanism which are involved in exogenous pyrogen are through
the initiation of IL-1 which is released due to macrophages and monocytes which
leads to initiate fever. Other mechanisms which are involved are following

 Bacteria produce some endotoxin which are acting on hypothalamus and

disturbing the set point
 Some bacteria produce exotoxin that may activate macrophages and
monocytes to release IL-1
 Exogenous pyrogen may cause the lymphocytes to release of lymphokines
specially INF-Y which stimulate macrophages and monocytes to generate IL-
1
 Microbial pyrogens
 Gram positive bacteria

The cell wall is made up of peptidoglycan and this peptidoglycan may act as
pyrogen in body

 Gram negative bacteria

Gram negative produce some type of endotoxin which disturb the set-point in
hypothalamus .Endotoxin is heat stable factor.

 Viruses

Viruses may cause fever by directly acting on macrophages and monocytes

which produce interleukins

 Fungi

Fungi increase body temperature and disturb set-point when these are present
in blood circulation. Fungi is exogenous pyrogen that may be live are dead fungus
product.

 Non microbial pyrogens

Lymphocytes and monocytes are mononuclear cells that are present in

peripheral blood and spread in different organs like lymph nodes, placenta, lungs and

21
subcutaneous tissues. These are responsible for IL-1 production and ultimate initiation
of fever. These cells play a major role in defense system, phagocytosis, T
lymphocytes, activation and destruction of tumor cells. The main products of this
system are IL-1 and TNF.

i) Thermoregulation

Hypothalamus acts as thermostat in body which regulated body temperature.

The balance between heat loss and heat production required thermoregulation.
Hypothalamus regulate this mechanism by

i. Heat production

Different types of mechanism are involved in heat production like,

 At rest, different organ like heart, liver, brain, viscera, muscles, pancreas and
adrenal gland supply heat to cellular level in the form of adenosine
triphosphate (ATPs)

 Brown fat in scapula and neck area produces heat in newborn and infants

 Adults and older children save heat by veso-constriction and produce heat by
shivering in response to cold .The flow of heat is through blood circulation
which is regulated by CNS. In cold environment, when outside temperature is
decreased the flow of blood supply to skin is low which cause in preservation
of heat. And in hot environment core temperature is increased blood flow
toward skin is fast which cause s to loss of heat through skin by sweating (El-
Radhi et al., 2009).

ii. Heat loss

Heat is lost from body by different physical methods which are radiation,
evaporation, convection and conduction.

 60% of heat is lost through skin by radiations.

 ¼ of heat is lost by evaporation through skin. Lungs play an important role in
it, evaporation occurs as water is transformed from liquid to gas.
 12% of heat is lost by convection which is transferred with movement of air
that surrounding the skin surface.

22
  3% of heat is lost by conduction. It is more common when a person is lying on
hot surface than on standing position due to increase in surface area.

1.18. Measurement of fever

Measurement of body temperature is very important to observed increase in

body temperature and to identify the disease. Different type of methods and
instruments are used to observed body temperature depend on different sites.

Major sites of temperature measurement

Measurement of body temperature depend on site at which body temperature

is observed are.

1.18.1. Axilla

For axillary measurement of temperature mercury thermometer is used which

have mercury level in it, and on mercury level temperature should be confirmed.
Thermometer bulb is placed in halfway between anterior and posterior of axilla. Arm
should be extended and kept against chest wall. Body temperature should be noted
within 5 minutes. This technique is safe, easy to use and comfortable.

1.18.2. Skin

The thermometer used for this method is simple, plastic, disposible encased
with liquid crystal and placed on forehead. Change in color of substance occur if
increase in temperature. This method is most convenient, safe, easy to use, give rapid
result and home usage.

1.18.3. Sublingual

Like axillary method mercury thermometer is used for sublingual.

Thermometer bulb is placed under tongue and level of mercury shows the result.
Duration of measurement is three minutes and used mostly for children older than 5
years.

1.18.4. Rectal temperature

Mostly glass and digital thermometer is used. Thermometer should be inserted

in proper position of children by separating the buttock at the distance of 5 cm into

23
rectum. Lubricant jelly should be applied on tip of thermometer before insertion.
Time of notification not more than three minutes (El-Radhi et al., 2009).

1.19. Antipyretics

Antipyretics are drugs which lower fever. Today fever is considered as

discomfort for patients. In earlier time’s physician had applied different physical
methods to lower body temperature. Cinchona bark was used as antipyretic in
eighteen century. Salicylic acid was prepared from glucoside salicin in 1838 which
was active component of willow bark. Acetyl salicylate was 1st synthetic antipyretic
which was prepared in 1853 and supplied in market 1899.By this different antipyretic
have been introduced but effect of acetaminophin is latest. Today commonly used
antipyretics are aspirin, paracetamol, NSAIDs etc.

1.19.1. Mechanism of Action of Antipyretics

Antipyretics decrease the thermoregulatory hypothalamus set-point by acting

on centrally. This is due to inhibition of cyclooxygenase (COX).This enzyme convert
arachdionic acid to prostaglandin and leukotriens. Fever is induced by different
prostaglandins and most essential is PGE2.Some physiological changes may come
when decrease in set-point of hypothalamus center which included decrease in heat
production, increase in blood supply toward skin and increase in loss of heat by
radiation, evaporation, convection result in decrease in body temperature.
prostaglandin may also causes broncho-dilation and have effect on kidney and gestro-
intestinal tract. Antipyretics do not directly show effect on pyrogen formation, or loss
of heat by sweating. Their efficacy is depends on dose, height and ratio of absorption
of drug.

1.19.2. Antipyretic therapy

1.19.2.1. Aspirin

Aspirin decrease the temperature by inhibition of prostaglandin E2 enzyme. It

may cause GI disturbance, Asthma, skin rashes, bleeding and peptic ulceration. Its
half-life is 20 minutes.

24
1.19.2.2. Paracetamol

It Inhibit the prostaglandin synthesis with half-life of 2 hours. Over dose may
cause liver damage, liver cirrhosis and liver necrosis. Some common side effects are
GI disturbance, Gastritis and hepatitis etc.

1.19.2.3. Neproxin

It is NSAIDs with long half-life of 14 hours. Twice is taken for best effect. It
is suitable in cancer patients.

1.19.2.4. Indomethacin

It may cause GI disturbance and CNS symptom like headache, convulsion,

dizziness, confusion and vertigo. It is not suitable in children due to severe side
effects.

1.19.2.5. Anti-pyrine

It is drug which was used as antipyretic in all over the world but now it is
restricted due to toxicity specially a-granulocytosis.

1.19.2.6. Salicylamide

It is less effect than paracetamol but rarely used as antipyretic drug.

1.19.2.7. Steriods

Steriods are also used as antipyretic. In patient who receiving long term
steriods treatment do not reduce fever in responsive to infections. This is due to
decrease in interleukin1production by macrophages and inhibition of prostaglandin
release.

25
 Table 1.4: Drugs which decrease body temperature

Drug Mechanism Half Dose Common side Frequency

life effects
Paracetamol Inhibit 2 hours 15mg/kg Skin rashes, Every 6 hours
prostaglandin hepatitis ,liver
synthesis damage
Aspirin Prostaglandin 20 min Not clear in Peptic Not clear
inhibition fever ulceraion,
asthama
Ibuprofen Cyclooxygenase 2 hours 6-10mg/kg Prolong 4-6 hours
inhibition bleeding
,gastitis,
dizziness
Mefenamic Inhibit 2 hours 6.5/kg/day GI disturbance, 6-8 hours
acid cyclooxygenase rashes
Neproxin Inhibition of 12-17 5mg/kg/day Heartburn, Every 8 hour
sod complex hours indigetion,GIdi
enzyme of sturbance
prostaglandin
Diclofenac Inhibition of 1-2 12.5mg/day Stomach pain, 10-12 hours
sod prostaglandin hours GI disturbance,
synthesis due to sweating
inhibition of
cyclooxygenase

1.19.3 Physical treatment

Different physical methods are applied to lower the body temperature which
are following.

1.19.3.1. Alcohol sponging

This method reduces the fever very quickly. Use spirit or ethyl alcohol in
water for it with carefully and is superior to torpid sponging. Avoid direct inhalation

26
which may cause asthma or hypo-glycemia.(Chandra & Bhatnagar, 2002; El-Radhi
et al., 2009)

1.19.3.2. Terpid sponging

Used only for short term treatment which may cause the production heat to
achieve hypothalamic set-point and not lower the body temperature. This method
causes un comfort and shivering (Chandra & Bhatnagar, 2002).

1.19.3.3. Cold sponging

It is restricted due to cause shivering and veso-constriction which leads to

increase in temperature but effective in summer or core temperature is increased (El-
Radhi et al., 2009).

1.19.3.4. Bed rest

Extreme physical activity causes to increase in body temperature in some

person. Bed rest does not have notable role in lowering body temperature (El-Radhi et
al., 2009).

1.19.3.5. Total body cooling

Different methods are used to cool the total body surface by cool blankets, ice
packs, air conditioner and taking shaver which decrease in body temperature
(Chandra & Bhatnagar, 2002).

27
1.20. Introduction of plants

1.20.1. Cyperus pertenuis

Fig 1.9: Fresh and preserved rhizomes of Cyperus pertinius

This plant belonging to family cypereceae and genus have more than 5500
species. Cyperus pertenuis is traditionaly used with others as an antipyretic and
hepato-protective.

Common name Naghar motha

Habitat banghal
Parts used roots/stems
Kingdom plantae
Family Cyperaceae
Order Poales
Genus Cyperus
Specie pertenuis

It is good remedy for indigesion because of constituents present in it as most

of the constituents that were present in cyperus roduntus (Panda, 1999).

Previously Cyperus scariosus was said to be Cyperus pertenuis but in 2012

Cyperus scariosus was redefined and Cyperus pertenuis name was given. It is 45cm
to 80cm long with 3.5mm thick stems.

28
Followings are the major constituents in it. Fats, Sugar, Gum, Carbohydrates,
Essential oil, Starch, Fiber and alkoloids.

The contituents of Cyperus pertenuis give protection against many diseases

that are involved in production of free redicals. Alkalois, phenol, and flavonoids are
responsible for anti-oxident activity. Other constituents are cyperene, cypriol,
sugeonol etc.

Cyperus pertenuis is traditionally used in following symptoms

For chronic fever, torpid liver, in ascites, Dyspenea and in epilepsy.

Oils of Cyperus pertenuis was used in perfumes (Panda, 1999).

1.20.2. Delphinium zelil

Old name of delphinium zelil was delphinium samibarbatum and commonly

used name is Asbarg (asbar) larkspur. Delphinium genus has more than 300 species
and have yellowing flowers which is attractive for industerial and commercial plant. It
is native in Iran and Afghanistan at high mountains. It is 20 inch long with long
flowering season. It is cultivated by seeds and is suitable for hybridization.

Fig 1.10: Delphinium zelil

29
Common name Asbarg(a sbar)larkspur.
Habitat northern Iran high attitude mountain and Afghanistan
Parts used roots/stems/flowers
Kingdom Plantae
Family Ranunculaceae
Order Ranunculales
Genus Delphinium
Specie zelil

Traditionally it was used as:

Anti-inflammatory, Anti-pyretic, Demulcent, Detersive, Resolvent.

Extract of flower is used as diuretics and anodyne. The major constituents

present are following.

Alkoloids, Fats, Tannins, Saponin, Flavonoids, Phenols, Proteins, Steroids ,

Essentional oils

The constituents that give the protection against free redical are flavonoids
alkolods and phenol etc. The name delphinium came from greek word ―Delpi‖ which
mean dolphin due to resembling of their flowers shape to dolphin. Delphinium genera
are divided into Annual dephinium species and Perennial delphinium species
(Ramezani & Zarif Ketabi, 2003).

1.21. Literature Review

Euruca sativa plant of family Brassicaceae had been studied for its analgesic
and antipyretic activity. The leaves of this plant were used and methanolic extract
were made in 1:1.The ME extract of doses 125mg/kg and 250mg/kg were
administered. Diclofenac sodium (9mg/kg PO) was administered as reference. MEES
reduced the acetic induced writhing by 32% and 46%at 125mg/kg and 250mg/kg
when compared with diclofenac sodium (9mg/kg p/o) which induced 57%.HOT plate
and tail flicking test showed more positive response at (250mg/kg p/o) when
compared with standard (Al-Enazi et al., 2014).

30
 The ethanolic root extract of C. zambesicus was evaluated for analgesic and
Antipyretic effect in mice. The pain was induced by the 1% acetic acid and
2.5%formalin induced paw licking. The pyrexia was induced by the brewer’s yeast.
The animals were grouped into six groups with six animals in each. First group
received only (0.9% Nacl).Second received normal standard ASA (100mg/kg). The
third, fourth, fifth, and six received doses of (21-81 mg/kg) extracts. There was
decreased in abdominal writhing and paw licking which showed decreased in central
pain. The extract doses showed the decreased in rectal temperature when compared
with standard due to prostaglandin synthesis (Okokon,Nwafor 2009).

The methanolic extract of Diclipter averticillata stems with the leaves was
investigated for Analgesic and Antipyretic effect on the animal models. The extract
shown significantly decrease in acetic induced numbers of writhing at all the doses as
the dose dependent pattern when it was compared with standard (paracetamol
150mg/kg). The extract was also efficient on yeast induced hyperpyrexia in rats
(Saleem, Ahmad, Ahmad, Hussain, Bukhari, et al., 2015).

The aqueous extract Adasoniya digitala was evaluated for analgesic and
antipyretic effect on the animal models. The extract showed markly decreased in the
number of paw licking and number of abdominal writhing in mice at the doses of
(400-800mg/kg). The extract showed significantly decreased in rectal temperature
which was induced by the brewer’s yeast at the dose of (800mg/kg) (Ramadan et al.,
1994).

Antipyretic effect of aqueous extract of Alstoniascholaris L.leaf were studied

by using the brewer’s yeast induced hyperpyrexia in rats. Paracetamol was used as
positive control drug. When compared the temperature changes between two
different groups which received plant extract and paracetamol. The result showed
there was no significantly difference in it (Ramadan et al., 1994).

Antipyretic and analgesic effect of Nelumbonucifera rhizome was studied. The

methanolic extract was analyzed by using brewer’s yeast induced pyrexia and hotplate
and acetic acid induced writhing. Different doses of ME of 200,300 and 400mg/kg. It
was observed that the reduction in temperature is dose dependent manner. There was
also increased in hotplate cutoff time and decreased in abdominal writhing when
compared with paracetamol (150mg/kg) as standard (P. K. Mukherjee et al., 1997).

31
Analgesic effect of plant terphosia purpurea was studied .The ethanolic extract of
plant was made which was used for investigation the activity. Four groups of mice 6
in each were used by using hotplate and acetic acid induced writhing method. Group
one normal control received (0.9%Nacl p/o) group two positive control received
diclofenac sodium (25mg/kg p/o) group three ,fourth received (200mg/kg and
400mg/kg p/o) .The TPEE at doses (400mg/kg p/o)significantly reduced the thermal
and chemical induced in mice when compared with standard (Hajare et al., 2000).

Antipyretic effect of leaves and latex of Euphorbia helioscopia was observed.

Mathanolic extract was made and observed on brewer’s yeast induced rats. The
extract was administered orally at doses of (100,200,300mg/kg).The maximum effect
was seen at (300mg/kg p/o) 45.3% when compared with positive control. The extract
may inhibit the prostaglandin and other mediators which was responsible for pyrexia
(Ahmad et al., 2013; Saleem, Ahmad, Ahmad, Hussain, & Bukhari, 2015).

Antipyretic effect of diospyros lotus root extract was investigated on mice.

Different types of plant extracts with chloroform, hexane, ethylene acetate were used
.Paracetamol was used as standard (100mg/kg).50mg/kg and 100mg/kg were
administered orally. Chloroform and ethylene acetate fraction reduced 69.2% and
72.2% respectively while hexane extract did not show any effect.100mg/kg showed
the significant effect by inhibiting the prostaglandin synthesis (Uddin et al., 2014).

(Garg & Khosa, 2008) investigate the antipyretic effect of Aqueous extract of
Cynodon dactylon (poaceae) on rats by brewer’s yeast induced pyrexia. Five groups
were made with six rats in each group. The first group served as normal, second
served as standard received paracetamol (100mg/kg). Third, fourth and fifth received
plant extract with different doses of (200,400,600mg/kg).The maximum effect was
seen in 600mg/kg dose when compared with positive control by inhibiting the pyrexia
induced mediators.

The ethanolic extract of stems of plant Enantia chlorantha olive of family

annonaceae was studied on Albino rat for analgesic and antipyretic effect. Five groups
were made in which there six rats in each. First group was served as control which
was received (.9%Nacl) second group was positive control which received
(indomethacin 10mg/kg) Third ,fourth and fifth groups received ethanolic extracts of
doses 50, 100,and 200mg/kg. The extract reduced the writhing induced by acetic acid

32
and increased the response time of hotplate. The extract also exhibited as antipyretic
at the doses of 100-200mg/kg when compared with standard (indomethacin).The
extract may had some flavonoids and alkaloids which showed the analgesic and
antipyretic effects (Hajare et al., 2000).

The effect of different Anti-depressent and anxiolytic drugs like specific

serotanin reuptake inhibitor (SSRI), specific non epinephrine reuptake inhibitor
(SNRI) tricyclic antidepressent and anti convulsant drugs lamotrigine on the Albino
rats. All the animals were divided into five groups. First group served as normal
(0.9%Nacl). Second served as positive control which received paracetamol
(150mg/kg) or ibuprofen (5mg/kg).Third, fourth and fifth groups received different
types of antidepressent drugs. The pain was induced by intra-peritoneal injection of
2%acetic acid or by 5% formalin injection (I/p) and by thermal stimulation hotplate
method to observed the analgesic effect. The result showed that different types of
antidepressents like (citalopram 3mg/kg, duloxamine 25mg/kg fluroxamine 40mg/kg
and lamotrigine ) were effective in reduction of pain when compared with standard
(Magni, 1991)

The antipyretic effect was investigated on ethanolic extract of Aerva lanata of

family Amaranthaceae on the Albino rat by using the Breawer’s yeast induced
pyrexia. Five groups were made in each there were six animals. First group was
normal (.9%NaCL).Second group was positive control received paracetamol
(150mg/kg).Groups three, fourth ,and fifth received ethanolic extracts of ( 50, 100
,400mg/kg), Extract of 400mg/kg possessed significant effect when compared with
standard due to inhibition of prostaglandin mechanism (K.S.Kumar and
kuppast,2014).

The ethanolic extract of seeds of Daucus carota was investigated for analgesic
activity on the Albino mice by using acetic acid induced writhing and hotplate
methods. Different doses of 100,200 and 400mg/kg were used. Maximum effect was
seen at the dose of 400mg/kg by increasing the response time and decreasing the
writhing in mice (Vasudevan et al., 2006).

Antipyretic effect was carried out to determine the effect of methanolic extract
of Abutilon mauritianum of family Malvaceae by using the brewer’s yeast and
lipopolysaccharide (.3microgram/kg) induce pyrexia methods in Albino rats.

33
Ethanolic extract at the doses of 150mg/kg and 300mg/kg were used. Maximum effect
was seen at the dose of 600mg/kg which reduced the rectal temperature induced by
brewer’s yeast. The temperature may be down due to presence of tannin, flavonoids
and alkaloids in the extract which inhibition of prostaglandin (Akapa et al., 2014).

Analgesic effect of plant Angellica pubescens (Ap) was studied. Different

types of extract were used like methanolic, choloform and ethylene acetate. Analgesia
was induced by 1% acetic acid and hotplate methods. There was decreased in writhing
and increased in response time when compared with standard drug ASA (150mg/kg)
(Chiu et al., 2009).

The aqeoues extracts of Nigella sative was studied on mice and rats for
analgesic and antipyretic activity. Different groups were made to check the analgesic
activity on Albino mice with different doses. It was seen that there was significant
increase in hot plate reaction time. The suspension of N sativa decreased the pyrexia
and rectal temperature in rats which was induced by brewer’s yeast when compared
with standard (Al-Ghamdi, 2001).

The roots extract of plant Clitoria ternatea shows the analgesic and antipyretic
effect. The methanolic extract reduced the vascular per-ability induced by the acetic
acid in rats. There was markely reduced the number of writhing at 400mg/kg dose.
The extract also exhibited a significant decreased of pyrexia when induced by the
Brewer’s yeast in rats which showed the prostaglandin inhibition in rats (Devi et al.,
2003; M Gupta et al., 2005).

This study was carryout on Ocimuna sanctumto investigate the analgesic and
antipyretic effect. The methanolic extract and aqueous suspension was used. Both the
methanolic extract and aqueous suspension showed the analgesic effect in mice by
using the hotplate and tail flicking methods. The result showed the increased in tail
flicking reaction time. The suspension reduced the typhoid paratyphoid A/B vaccine
induced pyrexia which was weaker than standard (300mg/kg sodium salicylaye)
(Godhwani et al., 1987).

The methanolic extract of seeds of Hyocyamus nigar was used to investigate

the analgesic and antipyretic effect in the experimental animals at the different doses.
Different groups of mice were used to evaluated the analgesic effect and there was

34
significantly increased in hotplate reaction time and decreased in writhing response
which was dose dependent. The pyrexia was induced in rats by brewer’s yeast method
and there was decreased in rectal temperature when methanolic extract was given to
rats and when it was compared with standard (Begum et al., 2010).

The plant Garcinia hanburyi was assessed for antipyretic and analgesic effect.
The ethylene acetate extract was used on the experimental animal models. There was
decreased in acetic acid induced writhing response and increased in the hotplate time
response. Moreover the extract showed the excellent antipyretic effect when tested in
yeast induced hyperthermia in rats, when it was compared with standard. The
analgesic and antipyretic was caused due to prostaglandin synthesis (Panthong et al.,
2007).

The methanolic extract of Caessalpinia bonducella leaves were evaluated for

analgesic and antipyretic effects on mice and rats models. Different doses of
50,100,200mg/kg were used. Acetic acid induced writhing and hotplate methods
were used to assessed the analgesic effects .The dose of 200mg/kg showed the
maximum effect in the reduction of numbers of writhing and increased in response
time of hotplate, when compared with morphine and aspirin standard. The extract also
showed the significant reduction of pyrexia in rats (Malaya Gupta et al., 2003).

The ethanolic root extract of C. Zambesicus was evaluated for analgesic and
antipyretic effect in mice. The pain was induced by the 1% acetic acid and 2.5%
formalin induced paw licking. The pyrexia was induced by the brewer’s yeast. The
animals were grouped into six groups with six animals in each. First group received
only (0.9% Nacl).Second received normal standard ASA (100mg/kg). The third,
fourth, fifth, and six received doses of (21-81 mg/kg) extract. There was decreased in
abdominal writhing and paw licking which showed decreased in central pain. The
extract doses showed the decreased in rectal temperature when compared with
standard due to prostaglandin synthesis (Okokon & Nwafor, 2010).

The methanolic extract of Dicliptera verticillata stems with the leaves was
investigated for Analgesic and Antipyretic effect on the animal models. The extract
shown significantly decreased in acetic induced numbers of writhing at all the doses
as the dose dependent pattern when it was compared with standard (paracetamol

35
150mg/kg). The extract was also efficient on yeast induced hyperpyrexia in rats
(Lamien et al., 2006).

The aqueous extract Adasoniyadigitala was evaluated for analgesic and

antipyretic effect on the animal models. The extract showed markely decreased in the
number of paw licking and number of abdominal writhing in mice at the doses of
(400-800mg/kg). The extract showed significantly decreased in rectal temperature
which was induced by the brewer’s yeast at the dose of (800mg/kg) (Ramadan et al.,
1994).

Antipyretic effect of aqueous extract of AlstoniascholarisL. Leave were

studied by using the brewer’s yeast induced hyperpyrexia in rats. Paracetamol was
used as positive control drug. When compared the temperature changes between two
different groups which received plant extract and paracetamol. The result showed
there was no significantly difference in It (Ruiz et al., 2015).

Antipyretic and analgesic effect of Nelumbo nucifera rhizome was studied.

The methanolic extract was analyzed by using brewer’s yeast induced pyrexia and
hotplate and acetic acid induced writhing. Different doses of ME of 200,300 and
400mg/kg. It was observed that the reduction in temperature is dose dependent
manner. There was also increased in hotplate cutoff time and decreased in abdominal
writhing when compared with paracetamol (150mg/kg) as standard (P. Mukherjee et
al., 1996).

1.22. Aims and Objectives

 The aims of this work is to evaluate the analgesic, Anti-pyretic activities of

these plants
 To prepare ethanolic extract of these plants
 To analyze the phytochemical constituents of these plants
 Quantitative analysis of the total phenolic and flavonoids contents
 To estimate the antioxidant potential of these plants by using the DPPH
method and reducing power assay
 To determine the analgesic activity of these plants in experimental animals by
using the following method

36
 To analyze the antipyretic activity of these plants by using the Brewer´s yeast
induced method

37
2.1. Experimental Animals

Albino rats of both sex weighing about 150-250g were used. The animals were
purchased from university of Lahore (defense road campus) Lahore. All the animals
were kept in animal house of Riphah international university Lahore with the standard
condition and standard diet and water. All the animals must be kept at 25°C± 2°C
with humidity of 45%-55% in the alternate of dark and light cycle. All the animals
must be free to access of food and water throughout the study (Saleem, Ahmad,
Ahmad, Hussain, & Bukhari, 2015).

2.2. Drugs and Chemicals

Diclofenac sodium and Paracetamol were gifted from Sami Pharmaceutical

Lahore. Brewer’s yeast was purchased from local market of Lahore. The Ethanol,
methanol, DMSO, C.M.C and acetic acid were used. All the chemicals of analytical
grade were used as provided by university.

2.3. Equipment’s

Digital electronic balance (FA2014, YOKE,Galvano), centrifuge machine

(Pro-economic),Vortex mixer (VM300), Grinder (Waves Pakistan), Microwave oven
(DHG9053A), Spectrophotometer (UV1900,Yoke Galva no), Selecta -pro-s (Merck),
Disposable syringes, Measuring cylinder.

2.4. Collection of Plants

The two plants named as Cyperus pertanius and Delphinium zelil were
purchased from local market of Lahore and these plants were identified from the
botany department of Punjab University Lahore, Lahore campus.

2.5. Extracts Preparation

The Cyperus pertenius and Delphinium zelil 250g each were grinded by using
the mechanical grinder in to coarse powder form. Then these powder were stored into
an air tight container separately and kept at cool and dry place. The powder sample
was taken into round flask beaker of (1L) and these were soaked into 70% aqueous
Ethanolic solvent for 15 days with occasionally shaking and stirring. Then filter the

38
whole mixture through muslin cloth and wattmann no 1filter paper. Filtrate were left
for 7 days for complete evaporation and dryness in the oven at the 37°C.After 7 days
a brownish solid and semi-solid were obtained and weighed to found the %age yield.

2.6. Phytochemical Screening

Chemical tests were performed on aqueous and Ethanolic extract by using the
standard procedures to see the constituents present.

2.6.1. Solubility test

Different solvents like Methanol, chloroform, water, normal saline, DMSO

were taken to find out the solubility with equal amount of both extracts (w/v).The
result were expressed in the term of soluble, Insoluble and partial soluble.

2.6.2. Test for Tannin

Dried powder samples of 0.5g was boiled in 20ml of water in each test tube.
Then a few drops of 0.1%ferric chloride were added in each, blackish blue or
brownish green color appears which showed the presence of tannin (Saleem et al.,
2014).

2.6.3. Tests for Alkaloids

Ethanolic extracts of both plants were added in 5% C.M.C to form

suspension.5ml of each were taken and treated with 4ml of 1% Hcl. Then warmed on
water bath for few minutes and filtered. Then these filtrate were divided into two test
tubes (A. Kumar et al., 2009).

2.6.3.1. Hager’s test

The aliquot of 5ml was mixed with few drops of hager’s reagent (standard
aqueous solution of picric acid). Appearance of yellow precipitates indicates the
presence of alkaloids.

2.6.3.2. Wagnar’s test

Aliquot of 5ml was mixed with few drops of wagnar’s reagents. Appearance
of reddish brown precipitate showed the presence of alkaloids.

39
2.6.3.3. Dragondroff test

Aliquot 0f 2ml was mixed with 1ml of dragondroff reagent. Formation of

reddish brown or blackish precipitate indicate the presence of alkaloids.

2.6.3.4. Mayer’s test

Filtrate of 1ml was mixed with mayer’s reagent. Appearance of precipitates at

the bottom showed the presence of alkaloids (Ayoola et al., 2008).

2.6.4. Test for saponin

About 1g of powder samples was mixed and boiled in 10ml of water in each
test tube at water bath and filtered.5ml of each filtrate were mixed with 5ml of D/W
and shaken very well for persistent froth. Then this frothing was mixed with 5 drops
of olive oil in each. Formation of emulsion showed the presence of saponin (Edeoga
et al., 2005).

2.6.5. Steroids test

Ethanolic of 5ml extract of each plant was mixed with 2ml of chloroform and
concentrated sulphuric acid was added in each extract along the wall of the test tubes
.A red color appear at the bottom of the test tubes which indicates the presence of
steroids. Another test was performed for conformation.5ml of extract of each plant
was taken in test tubes and added 2ml of chloroform in each test tube and added few
drops of H2so4 and few drops of acetic acid along the wall of test tubes. Formation of
greenish color conform the presence of steroids.

2.6.6. Test for Cardiac glycosides

(keller-killani test)

Each extract of 5ml was taken in test tubes and few drops of Fecl3 was added
and mixed very well. Then a few drops of glacial acetic acid and concentrated H 2so4
along the wall of the test tubes very carefully. Radish brown coloration at the junction
of the layer indicates the presence of glycosides (Parekh & Chanda, 2007).

40
2.6.7. Test for carbohydrates

2.6.7.1. Molish test.

Ethanolic extract of 2ml each was taken in test tube and 2ml of molish reagent
was added in each test tube. Then mixed on the vortex mixer for few minute and
added concentrated H2so4 along of the test tubes drop wise. Voilet ring was made at
the inter phase which showed the presence of carbohydrates.

2.6.8. Test for amino acid

2.6.8.1. Ninhydrin test

A mixture of 5ml suspension of each was boiled after adding 2ml of 0.2%
ninhydrin solution in each. Formation of violet appearance indicates the presence of
amino acid (Saleem et al., 2014).

2.6.9. Test for proteins

2.6.9.1. Biuretic test

5ml of ethanolic extract of each were treated with 1% strong base (sodium or
potassium hydroxide) and added a few drops of copper sulphates in each test tube
.Formation of purple color in solution indicates the presence of proteins.

2.6.10. Test for terpenes and sterols

2.6.10.1. Libermaan burchard test

A mixture of 5ml of each extract was taken in the test tube, and mixed with
3ml of chloroform and dried. Then 2ml of concentrated H2so4 was added and heated
for 2-3 minutes. A grayish solution was formed which showed the presence of
terpenes (Yadav & Agarwala, 2011).

2.6.11. Total phenolic contents

Folin-ciocalteu reagent method was used with some modification.1ml of

ethanolic extract (1mg/ml) was taken in DMSO and added 2.0ml of 10% folin-
ciocalteu reagent and 1ml of 15% sodium bicarbonate (Na2co3). This mixture was
incubated for 15 minutes and then absorbance was measured at 760nm.Gallic acid

41
was used as standard. The results was found and expressed as the gallic acid
equivalent (mg/g of extracted compound) by using the standard curve. Total phenolic
contents of both plants extract were determined by using the above procedure and
sample solution of ethanolic extracts were made separately (Saleem et al., 2014).

2.6.12. Total flavonoid contents

Aluminium chloride colorimetric method was used with little

modification.1ml of plant extract was taken (mg/ml in solution of DMSO) and mixed
with 2ml of methanol, 0.2ml of 10% aluminium chloride, 0.2ml of 1M potassium
acetate and 4.5ml of D/W was mixed and kept at room temperature for 2 hours. The
absorbance was measured at 420nm. The rutin was used as reference (1mg/ml). By
using the standard curve results was determined and were expressed as rutin
equivalent (mg/g of extracted compound) The above procedure was used for both
ethanolic extract of plants and determined the flavonoid contents (Saleem et al.,
2014).

2.6.13. Assay of free redical scavenging activity by DPPH method

The DPPH scavenging activity of ethanolic extract of both plants were

observed by using 1,1-diphenyl-2-picryl-hydrazyl (DPPH) assay.0.1mM solution of
DPPH was prepared in the methanol. Different concentrations of both plants extract
(0.2-6.4mg/ml) were prepared in DMSO. Rutin was used as reference.1ml of extract
and 1ml of rutin was mixed with 3ml of 0.1mM solution Of DPPH in test tube
separately and kept it at room temperature in the dark place for 30 minutes. Blank was
prepared similarly which contain 1ml of DMSO in the place of extract or standard and
3ml of DPPH was added in it. After the 30 minutes absorbance was measured at
517nm with specro-photometer. Each sample absorbance measured triplicate for
conformation (Tona et al., 1998).

Formula of scavenging activity =

%age scavenging effect = (A-blank-A sample)/A-blank x100
A blank = absorbance of blank
A sample = absorbance of sample

42
2.7. Antipyretic Activity

2.7.1. Brewer’s yeast induced method

Antipyretic activity was carried out according to method (Ramadan et al.,

1994) with little modification. The activity was performed on albino rats of either sex
weighing of (150-180g) five animals in each group. Pyrexia was induced in rats by
injecting 20% (w/v) aqueous suspension of Brewer's yeast subcutaneously. After 18
hours, the animals developed 0.5°C or more raise in the rectal temperature (about
60% of the total number of animals injected) was taken in the study. At different time
rectal temperature was noted. Similarly, over- night fasted normal animals were
divided into different groups of 5animals in each and the experiment was carried out
in the same manner as described above. Percentage reduction in rectal temperature
was calculated by considering the total fall in temperature to normal (Saleem, Ahmad,
Ahmad, Hussain, & Bukhari, 2015).

Group I was normal control (10 ml/kg 5% C.M.C)

Group II was standard control (100mg/kg Paracetamol)
Group III was treated with Ethanolic extract of D.zelil (100mg/kg)
Group IV was given Ethanolic extract of D.zelil (200mg /kg)
Group V was received Ethanolic extract of D.zelil (400mg/kg)
Group VI was received Ethanolic extract of C. Pertenius (100mg/kg)
Group VII was administered Ethanolic extract of C.pertenius (200mg/kg)
Group VIII was received Ethanolic extract of C.pertenius (400mg/kg)

After the 18 hours of injection of brewer’s yeast the rectal temperature was
noted carefully in each animal and different doses of treatment was given one by one
to all the groups. The rectal temperature was measured at regular intervals of 0, 30
minutes, 1 hour, 2 hours, 3 hours, and 4 hours. The temperature of standard control
was compared with all treated groups.

2.8. Analgesic Activity

The analgesic activity was measured in 12 hours fasted albino rat of either
sex weighing of (150–180 g) each group contain five animals by Acetic acid-induced

43
writhing (Koster et al., 1959) and hotplate method (Saleem, Ahmad, Ahmad, Hussain,
& Bukhari, 2015; Taranalli et al., 2008).

2.8.1. Hot plate Assay

Analgesic activity determined by the Hot plate method described by (Naveed

et al, 2012) with the little modifications. The experiment was performed on albino
rats of either sex weighing of (150-180g). All the animals were fasted 18hours before
the start of experiment. In the experiment, the hot plate was maintained at 55 ± 1°C
(Harward apparatus). Animals were placed one by one on the heated surface to obtain
the animal response to heat-induced pain. When the animals were jumping out from
the apparatus or licking of paws it indicated the pain response in the animals and
observe them in the transparent glass box of (25cm high and 30cm diameter).The
animal which have more than 40 seconds latency time was rejected from the study.
Latency time during which animal remain on the hot plate. The animal were divided
in to eight following groups with (N=5) with different doses were administered to all
the animals (Purnima et al., 2010; Saleem, Ahmad, Ahmad, Hussain, & Bukhari,
2015).

Group I was kept as control (10 ml/kg 5% C.M.C)

Group II was kept as standard received paracetamol (400mg/kg)
Group III was received Ethanolic extract of D.zelil (100mg/kg)
Group IV was received Ethanolic extract of D.zelil (200mg /kg)
Group V was received Ethanolic extract of D.zelil (400mg/kg)
Group VI was treated with Ethanolic extract of C. Pertenius (100mg/kg)
Group VII was administered Ethanolic extract of C.pertenius (200mg/kg)
Group VIII was treated with Ethanolic extract of C.pertenius (400mg/kg)

After the 30 minutes of treatment the animals were place on the hot plate
gently and latency time was measured with out of jumping and licking of hands and
paws of animal .A cut off time of 90 seconds applied on all the animals for prevention
of tissue damage. Latency time of each group was measured at regular intervals at 0
minutes, 30 minutes, 60 minutes, 90 minutes and 120 minutes. Then latency time of
control group was compared with treated groups

44
2.8.2. Acetic induced writhing method

Analgesic activity was also determined by acetic induced writhing method,

described by (nesa et al,2014) with the little modifications. Experiment was
performed on albino rats of either sex weighing of (150-180g) n=5. Food of all the
animals was withdrawn 5 hours before the start of experiment with free excess of
water. Animals were divided in following eight groups with animals (n=5) and
respected doses were administered to all the groups (Ramadan et al., 1994; Saleem,
Ahmad, Ahmad, Hussain, & Bukhari, 2015).

Group I was kept as control (10 ml/kg 5% C.M.C)

Group II was kept as standard received diclofenac sodium (10mg/kg)
Group III was received Ethanolic extract of D.zelil (100mg/kg)
Group IV was received Ethanolic extract of D.zelil (200mg /kg)
Group V was received Ethanolic extract of D.zelil (400mg/kg)
Group VI was treated with Ethanolic extract of C. Pertenius (100mg/kg)
Group VII was administered Ethanolic extract of C.pertenius (200mg/kg)
Group VIII was treated with Ethanolic extract of C.pertenius (400mg/kg)

Then after the 30 minutes of treatment, animals were administered 1ml of the
1%acetic acid intra-peritoneally, at the rate of 1ml/100g. After the five minutes no of
contraction which are actually writhes were notes at the regular interval of time 0-5,
5-10,10-15,15-20 minutes. The percentage of analgesic was calculated by the
following formula given

%Analgesic activity = (A-B)/A×100

Where A = Average no of writhing of control group.
Where B = Average no of writhing of treated group.

45
2.9. Acute Toxicity Study (LD50%)

Study of acute toxicity of plants extract were performed according to

guidelines of organization of Economic Cooperation and development (OECD423,
2001). This study was conducted on 20 rats of either sex weighing of more than 150g
approximately. All the animals were fasted and only excess of water to them. Animals
(n=5) were treated with the single oral dose of ethanolic extract at dose of 500mg ,
1000, 3000,5000mg/kg respectively. Control group received only 5% C.M.C
suspension. Experimental animals were observed for behavioral changes and toxicity
sign like jumping, convulsion, twitching, sedation, loss of light reflex and mortality
(Botham, 2004).

2.10. Statistical Assay

Data were analyzed by excel sheet and graph pad prism. All the results were
expressed as a mean ± SEM. Statistical analysis was represented find out variance by
using TWO WAY ANOVA followed by mean P-value <0.05 and P <0.001 were
considered as statistical significant.

46
3.1. Percentage Yield of Plant Extract

The %age yield calculated was 3.16%. The extract was packed into air tight
container and was stored in the refrigerator at 8°C

The percentage yield of dried plant extract was calculated as follow:

Weight of finely divided plant powder (whole) = 250gms

Weight of concentrated Cyperus pertenius ethanolic extract = 7.9gms

%age yield of Ethanolic extract =7.9gms/1000gms× 100 = 0.7% (w/w)

7.9/250×100 = 3.16% in 250 grams

Table 3.1: Solubility results of C.pertenius extract

Ethanolic extract of Cyperus pertenius was soluble in ethanol, DMSO, C.M.C and
chloroform when dissolved in 1:1(w/v), and slightly soluble in ethyl acetate. It was
insoluble in water and normal saline

Solvent Ratio Presence

Water 1:1 -

Normal saline 1:1 -

Chloroform 1:1 +++

DMSO 1:1 +++

C.M.C 1:1 +++

Soluble = +++
Partial soluble = ++
Insoluble = -

47
3.2. Qualitative phyto-chemical Assay

The phytochemical constituents of ethanolic extract of C. pertenius ar present

in Table 3.2. The results showed that presence of tannin, steroids, alkaloids,
carbohydrates etc

Table 3.2: Phytochemical results of C. pertenius

Phytochemical Test performed Ethanolic extract

Tannin Ferric chloride test +

Alkaloids Hagar’ test +

Wagner test +
Mayer test +
Dragondorff test +
Saponin Foam test -

Steriods Ring test +

Cardiac glycosides Ring test +

Terpenes Salkowski test +

Carbohydrates Molish test +

Proteins and Biuret test -

amino acid Ninhydrin test +

Lipid and fixed oils Spot test +

Phenols FC method +

Flavonoids Aluminium chloride test +

Phytosterols Libermann-burchard test +

+ detected -- Not detected

48
3. 1 Total phenolic content

The amount of total phenolic contents in ethanolic extract of C. pertinius was

determined by the Folin-ciocalteu reagent method with little modifications. Gallic
acid was used as standard dissolved in DMSO. Cyperus pertenius has the total
phenolic content of 0.83 mg/ml.

Table 3.3: Total phenolic content of C. pertenius

Sr no Concentration of gallic Absorbance at 720nm SEM

acid (mg/ml)

1 0.02 0.127 ± 0.006

2 0.04 0.187 ± 0.011

3 0.08 0.254 ± 0.023

4 0.16 0.408 ± 0.032

5 0.32 0.508 ± 0.029

6 0.64 0.7743 ± 0.027

7 1.28 1.33 ± 0.1

8 C.p 0.834 ± 0.028

Gallic acid was used as standard. Data were expressed as mean ± SEM (1mg/ml
concentration was used to find out total phenolic content)

49
 Y = 0 .6 6 6 1  X + 0 .4 2 2 0
2
R = 0 .9 2 1

A b s o rb a n c e a t 7 6 0 n m 2 .0

1 .5

1 .0

0 .5

0 .0
0 .0 0 .5 1 .0 1 .5
C o n c e n tr a t io n (m g /m l)

Fig 3.1: Graph of standard curve of total phenolic contents of C.pertenus

Value of R square obtained from linear regression curve with 95% confidence limit
was 0.921
Equation
Y = 0.6661*X+0.4220
Percentage of total phenolic content = 42.1%

Result

Total phenolic content were measured by standard curve of regression line and
gallic acid equivalents (µg/ml) percentage was 42.1%

3.3. Total flavonoids Content

Aluminium chloride colorimetric method was used to determine the total

flavonoid contents in the ethanolic extract of C. pertenius with little modifications.
Rutin was taken as standard which was dissolved in DMSO.

50
Table 3.4: Total Flavonoids content of Cyperus pertenius

Sr no Concentration of rutin Absorbance at 420 nm

(mg/ml) SE M

1 0.02 0.026 ± 0.004

2 0.04 0.038 ± 0.003

3 0.08 0.049 ± 0.002

4 0.16 0.084 ± 0.002

5 0.32 0.168 ± 0.003

6 0.64 0.258 ± 0.021

7 1.28 0.471 ± 0.058

8 C.p.E 0.280 ± 0.025

Rutin was used as standard. Data were expressed as mean ± SEM (1mg/ml
concentration to find out total flavonoids content)

51
 Y = 0 .3 2 8 0 X + 0 .0 3 2 4 3
0 .6 R 2 = 0 .9 8 7 6

A b s o rb a n c e a t 4 2 0 n m

0 .4

0 .2

0 .0
0 .0 0 .5 1 .0 1 .5
r u tin c o n c e n tr a tio n (m g /m l)

Fig 3.2. Graph of standard curve of total flavonoid contents of C. pertenius

Value of R square obtained from linear regression curve with 95% confidence limit
was 0.9876.

Equation

Y = 0.3280X+0.03243

Percentage of total flavonoids content = 47.1%

Result

Total flavonoids content were measured by standard curve of regression line

and represented as rutin equivalents (µg/mL) Percentage was 47.1

3.4. DPPH free Redical Scavenging Activity

Percentage inhibition at 517nm was assayed by DPPH method to estimate

scavenging activity of Cyperus pertenius at different concentrations of 20, 40, 80,
160, 320 and 640 (µg/ml). It was calculated as Ethanolic extract 82.0 ± 1.05, 83.1 ±
0.757, 84.1 ± 0.589, 87.1 ± 0.810, 88.0 ± 0.479, 89.2 ± 0.18. Antioxidant activity was
increased as concentration of extract was increased and was comparable to standard

52
ascorbic acid that showed percentage inhibition 85.0 ± 0.169, 87.3 ± 1.02, 89.0 ±
0.541, 93.2 ± 1.16, 94.4 ± 0.06 and 95.2 ± 0.718

Table 3.5: Results of percentage of Redical scavenging action

Concentration Ascorbic acid Ethanolic extract of

(µg/ml) (%) C.pertenius (%)
20 85.0 ± 0.169 82.0 ± 1.05

40 87.3 ± 1.02** 83.1 ± 0.757*

80 89.0 ± 0.541** 84.1 ± 0.589ns
160 93.2 ± 1.16*** 87.1 ± 0.810**
320 94.4 ± 0.06** 88.0 ± 0.479***
640 95.2 ± 0.718** 89.2 ± 0.189ns

Data was expressed as mean ± SEM. *P<0.05 was considered as significant when
compared with standard to ethanolic extract.

150
p e rc e n t in h ib itio n o f ra d ic a l s c a v a n g in g a c tiv ity

a s c o r b ic a c id ( % )

E th a n o lic e x t r a c t o f C . p e r te n iu s ( % )

100

50

0
0

0
0

4
2

r u t i n c o n c e n t r a t i o n (  g /m l)

Fig 3.3: Graph of anti-oxident of C.pertenius by DPPH method

53
3.5 Antipyretic Activity Determination

3.4. Results of effect of Cyperus pertenius ethanolic extract (C.p) on Brewer’s

yeast induced hyperpyrexia in rats

Experimental studies showed that pyrexia level in the normal control was 38.63° ±
0.135, 38.66 ± 0.129, 38.6 ± 0.122, 38.55 ± 0.115, 38.55 ± 0.124 at 0, 1st, 2nd, 3rd and
4th hour. While in the positive control (standard paracetamol) pyrexia was
38.45±0.064, 38.06 ± 0.015, 37.83 ± 0.025, 37.60 ± 0.037,37.50 ± 0.05 at 0, 1st ,2nd
,3rd and 4th hour. The result of Cp.E at 100mg/kg dose was before pyrexia 36.81 ±
0.094 and at 0 hour 38.57 ± 0.055,1st hour 38.41 ± 0.046, 2nd hour 38.30 ± 0.053, 3rd
hour 38.20 ± 0.04, 4th hour 38.12 ± 0.043.The results of 200mg/kg dose were before
pyrexia 36.96 ± 0.08 and at 0 hour 38.40 ± 0.06, 1st hour 38.14 ± 0.07, 2nd hour 38.0 ±
0.68, 3rd hour 37.87 ± 0.073, 4th hour 37.68 ± 0.068.The antipyretic effect of Cp.E at
400mg/kg dose was before pyrexia 36.95 ± 0.18 and after pyrexia at 0 hour 38.46 ±
0.076,1st hour 38.14 ± 0.04,2nd hour 37.97 ± 0.06, 3rd hour 37.77 ± 0.056 and 4th hour
37.53 ± 0.043.Brewer’s induced pyrexia in rats. There was remarkable reduction in
pyrexia in standard control group. It also represented that Cp.E reduced the pyrexia at
different doses. The percentage decrease in pyrexia was calculated by the formula

% Decrease in fever = B-C/B-A*100

Where

B = temp at 0 hr

C = temp at n hr

A = temp before pyrexia

54
 Table 3.6: Effect of different doses of Cyperus pertenius ethanolic extract on
brewer’s Yeast Induced Hyperpyrexia in Rats

Groups Rectal temperature (C)

Before yeast After drug administration

administration
0hr 1hr 2hr 3hr 4hr

Normal control 37.06 ± 0.02 38.63 ± 0.135 38.66 ± 0.129 38.6 ± 0.12 38.56 ± 0.115 38.55 ± 0.0108

Positive 36.82 ± 0.143 38.45 ± 0.064 38.06 ± 0.015* 37.83 ± 0.025* 37.6 ± 0.037*** 37.48 ± 0.05***
28.2% 44.9% 61.6% 68.8%
control

Cp100mg/kg 36.8 ± 0.094 38.57 ± 0.055 38.41 ± 0.046ns 38.3 ± 0.053ns 38.2 ± 0.039* 38.12 ± 0.043*
10.2%

16.3% 22.7% 27.1%

Cp200mg/kg 36.96 ± 0.08 38.4 ± 0.06 38.14 ± 0.069* 38.0 ± 0.068* 37.87 ± 37.68 ±
** **
0.073 0.068
18.7% 30.3% 37.2%

52.5%

Cp400mg/kg 36.95 ± 0.184 38.45 ± 0.08 38.14 ± 0.04* 37.97 ± 37.77 ± 37.53 ±
** ** ***
0.058 0.056 0.042
21.1% 32.2% 45.0% 60.6%

Results are expressed as mean ± SEM (n=5), analysed by two ways ANOVA.
P values are considered non-significant (ns) as P significant (*),
P<0.01 more significant (**), P<0.001 highly significant (***) when compared with
normal control group when compared with standard group

55
 3 9 .0
C p 40 0m g/kg

3 8 .5 C p 20 0m g/kg
C p 10 0m g/kg
te m p e ra tu re ( C )

3 8 .0 sta nd ard co ntrol

n orm al co ntrol
3 7 .5

3 7 .0

3 6 .5

3 6 .0
hr

hr

hr
a
a

r
xi
xi

2h
1

4
e
re

yr
py

r p
re

te
fo

af
be

tim e (h r)

Fig 3.4: Graph of Effect of different doses of Ethanolic extract of C.pertenius on

rectal temperature by Brewer’s yeast method

56
3.6. Analgesic Activity Determination

3.6.1. Hot Plate method

Hot Plate method was used to determine the analgesic activity. Paracetamol
400mg/kg was used as standard or positive control and it give the maximum latency
time when it was compared with normal control and were statistically significant
result whereas different doses of100mg/kg, 200mg/kg and 400mg/kg ethanolic extract
of C.p showed the dose dependent effect when compared with standard. The results
were statistically highly significant (***) when different doses of plant extract were
compared with normal control group. Dose 100mg/kg produced the maximum latency
time of 45.7 seconds at 150 minutes.200mg/kg of dose showed the maximum latency
time of 59.6 second at 150 minutes. Dose of 400mg/kg produced the maximum
latency time of 65.4 seconds at 150 minutes. Whereas PCM 400mg/kg showed the
maximum latency time of 89.9 second at 150 minutes. The results of Cp.E were
compared with normal control group found to be highly significant.

57
 Table 3.7: Effect of different doses of Ethanolic extract of Cyperus pertenius and
paracetamol by using hot plate method in Rats

Groups Before drug After drug administration

administratio
n
30 min 60min 90 min 120 min 150 min

Normal control 13.7 ± 0.57 14.95 ± 0.74 16.1 ± 1.14 15.02 ± 0.68 15.45 ± 1.06 18.17 ± 1.62

Positive 14.5 ± 1.04* 31.9 ± 1.89* 45.5 ± 2.15** 58.07 ± 2.48** 74.9 ± 3.01*** 89.9 ± 1.28***
control(PCM
400mg/kg)
Cp100mg/kg 15.27± 1.43ns 22.7 ± 1.30ns 28.5 ± 0.54* 34.7 ± 2.52* 41.4 ± 2.4* 45.7 ± 2.01*

Cp 200mg/kg 15.5 ± 1.16 28.6 ± 1.18* 38.07 ± 1.7* 45.25 ± 1.5** 53.8 ± 1.62** 59.6 ± 1.63***

Cp400mg/kg 14.6 ± 0.992 28.7 ± 0.82* 37.6 ± 1.97** 45.02 ± 2.12*** 54.9 ± 1.93*** 65.4 ± 2.01***

Results are expressed as mean ±SEM (n=5), analysed by two ways ANOVA. P values
are considered non-significant (ns) as P significant (*), P<0.01
more significant (**), P<0.01 highly significant (***) when compared with normal
control group

58
 100

80 n o rm a l s ta n d a rd

c o n tro l s ta h d a rd

C p 1 0 0 m g /k g

c p 2 0 0 m g /k g
L a te n c y tim e

60
c p 4 0 0 m g /k g
(s )

40

20

0
in

in

in

in
s
se

te

m
o

u
d

60

90

0
in

12

15
re

m
o

30
ef
b

tim e (m in )

Fig 3. 5. Graph of Effect Ethanolic extract of Cyperus pertenius and paracetamol

on Latency time on Hot plate method in Rats

59
 Table 3.8: Decrease in latency time of Cp.E

Groups Respose time (Sec)

Before drug administration After drug administration
0 time
Normal control 13.7 ± 0.565 30 min 14.95 ± 0.742
60 min 16.1 ± 1.135
90 min 15.02 ± 0.682
120 min 15.45 ± 1.06
150 min 18.17 ± 1.62
Standard control 14.5 ± 1.04 30 min 31.9 ± 1.89
60 min 45.5 ± 2.15
90 min 58.07 ± 2.48
120 min 74.9 ± 3.01
150 min 89.9 ± 1.28
Cp 100mg/kg 15.27 ± 1.43 30 min 22.7 ± 1.3
60 min 28.5 ± 0.541
90 min 34.7 ± 2.52
120 min 41.4 ± 2.40
150 min 45.7 ± 2.01
Cp200mg/kg 15.5 ± 1.16 30 min 28.6 ± 1.18
60 min 38.07 ± 1.7
90 min 45.25 ± 1.5
120 min 53.8 ± 1.62
150 min 59.6 ± 1.63
Cp400mg/kg 14.6 ± 0.992 30 min 28.7 ± 0.82
60 min 37.6 ± 1.97
90 min 45.02 ± 2.12

120 min 54.9 ± 1.93

150 min 65.4 ± 2.00
Data were expressed in mean ± SEM

60
3.6.2. Acetic induced writhing method

Acetic induced writhing method was used to determine the analgesic activity.
It was used to measure the pain by peripheral mechanism. Diclofenac sodium was
used as standard control. When positive control group was compared with standard
control group the results were highly significant Diclofenac sodium showed the
maximum induction of pain at 15-20 minutes against chemically induced pain.
Different doses of Ethanolic extract of Cp i.e.100mg/kg, 200mg/kg, 400mg/kg
displayed 48.8%, 59% and 68% of pain protection within 15-20 minutes respectively.
There was significant reduction in writhing in the whole treated groups when these
were compared with the standard group which showed the highly significant.

Table 3 .9. Effect of different doses of Ethanolic extract of Cyperus pertenius and
diclofenac sodium on acetic induced writhing in rats

Treated No. of writhing at various intervals

Groups 0-5 mins 5-10 mins 10-15 mins 15-20 mins
NormalControl 41± 0.479 35 ± 0.707 32 ± 0.645 29 ± 0.645
10ml/kg
Diclofenac 14 ± 0.854 11 ± 0.479 9 ± 0.289 8 ± 0.289
** ** *** ***
sodium 65% 68% 71% 72.8%
10mg/kg
C.p 28 ± 0.750 21 ± 0.479 17 ± 0.625 15 ± 0.479
ns * * **
100mg/kg 30.3% 40% 45.1% 48.8%
C.p 21 ± 0.479 18 ± 0.645 14 ± 0.250 12 ± 0.479
* * * **
200mg/kg 47.5% 48.5% 56% 59%
C.p 18 ± 0.645 14 ± 0.250 12 ± 0.408 9 ± 0.250
* ** ** ***
400mg/kg 55% 60% 62.3% 68%

Results are expressed as mean ± SEM (n=5), analysed by two ways ANOVA.
P values are considered non-significant (ns) as P significant (*),
P<0.01 more significant (**), P<0.001 highly significant (***) when compared with
normal control group

61
 50
n o rm a l c o n tro l c o n tro l s ta n d a rd

C p 1 0 0 m g /k g C p 2 0 0 m g /k g

40 C p 4 0 0 m g /k g
N o . o f w r it h n in g s

30

20

10

0
10

0
5
0-

-1

-2
5-

10

15

tim e (m in )

Fig 3.6: Graph of effect of different doses of ethanolic extract of Cyperus

pertenius and diclofenac sodium on acetic induced writhing in rats

62
3.7. Acute Toxicity Test

Acute oral toxicity was analysed in rat which showed that Cp extract up to
5000mg /kg body weight did not show any sign of behavioral and neurological
toxicity in the rats. There was no relative difference in the body weights of control
and treated rats. Aqueous ethanolic extract of plant was used in acute toxicity study
and there was no lethality up to the dose of 5000mg/kg which showed the safety of
the extract.

63
Table 3.10: Acute toxicity test of Cyperus pertenius on behavioral and
neurological changes in rats

Days Behavioral changes Presence

30 min -24 hours Skin changes No
Aggressivity No
Pain No
Sleep No
Salivation No
Sensitivity to sound No
Breathing No
7 day Skin changes No
Aggressivity No
Pain No
Sleep No
Salivation No
Sensitivity to sound No
Breathing No

14 day Skin changes No

Aggressivity No
Pain No
Sleep No
Salivation No
Sensitivity to sound No
Breathing No

There was no behavioral changes seen in the study start from dose of 300mg/kg up to
5000mg/kg from time start 30 minutes to 24hr and up to 7 days.
Results are expressed as mean ± SEM (n=5), analysed by two ways ANOVA. P
values are considered non-significant (ns) as P significant (*),
P<0.01 more significant (**), P<0.001 highly significant (***) when compared with
normal control group

64
4.1. Percentage yield of plant extract of Dilphnium zelil
The percentage yield of dried plant extract was calculated as follow:
Weight of finely divided plant powder (whole) = 500g
Weight of concentrated Cyperus pertenius ethanolic extract = 12.7g
%age yield of Ethanolic extract = 12.7g/1000gx100 = 1.27% (w/w)
12.7/500x100 = 2.54% in 250g

Table 4.1: Solubility results of D.zelil extract

Ethanolic extract of Delphinium zelil was soluble in ethanol, DMSO, C.M.C and
chloroform when dissolved in 1:1(w/v), and slightly soluble in ethyl acetate. It was
partial soluble in water

Solvent Ratio Presence

Water 1:1 ++

Normal saline 1:1 ++

Chloroform 1:1 +++

DMSO 1:1 +++

C.M.C 1:1 +++

Soluble = +++
Partial soluble = ++
Insoluble = -

4.2. Qualitative phyto-chemical Assay

The phytochemical constituents of ethanolic extract of D. zelil are present in

table 4.2. The results showed that presence of tannin, steroids, alkaloids,
carbohydrates, phenol, amino acid etc

65
Table 4. 2. Phytochemical results of D. zelil extract

Phytochemical Test performed Ethanolic extract

Tannin Ferric chloride test +

Alkaloids Hagar’ test +

Wagner test +
Mayer test +
Dragondorff test +

Saponin Foam test +

Steriods Ring test +

Cardiac glycosides Ring test +

Terpenes Salkowski test -

Carbohydrates Molish test +

Proteins and Biuret test -

amino acid Ninhydrin test +

Lipid and fixed oils Spot test +

Phenols FC method +

Flavonoids Aluminium chloride test +

Phytosterols Libermann-burchard test +

+ (ve) Detected
_ (-ve) Not detected

4.3. Total Phenolic Content

The amount of total phenolic contents in ethanolic extract of D.zelil was

determined by the Folin-ciocalteu reagent method with little modifications. Gallic
acid was used as standard dissolved in DMSO.

66
 Table 4.3: Estimation of total phenolic contents of D.zelil

Sr no Concentration(1mg/ml) Absorbance at 765nm SEM

1 0.02 0.286 ± 0.031

2 0.04 0.411 ± 0.054

3 0.08 0.450 ± 0.054

4 0.16 0.520 ± 0.069

5 0.32 0.628 ± 0.02

6 0.64 0.839 ± 0.038

7 1.28 1.279 ± 0.049

8 D.z.E 0.782 ± 0.024

Data were expressed as mean ± SEM (mg/ml) concentration was used to find the total
phenolic contents

67
 Y = 0 .6 6 6 1  X + 0 .4 2 2 0
2
R = 0 .9 1

1 .5

A b s o rb a n c e a t 7 6 0 n m

1 .0

0 .5

0 .0
0 .0 0 .5 1 .0 1 .5
C o n c e n tr a t io n (m g /m l)

Fig 4.1. Graph of standard curve of total phenol contents of D.zelil

Value of R square obtained from linear regression curve with 95% confidence limit
was 0.912

Equation

Y= 0.6661*X+0.4220

Percentage of total phenolic content = 45.25%

Result

Total phenolic content were measured by standard curve of regression line and
gallic acid equivalents (µg/ml).percentage was 45.2%

4.3. Total Flavonoids Content

Aluminium chloride colorimetric method was used to determine the total

flavonoid contents in the ethanolic extract of D. Zelil with little modifications. Rutin
was taken as standard which was dissolved in DMSO.

68
Table 4.4: Estimation of total Flavonoids contents of D. zelil

Sr no Concentration (mg/ml) Absorbance at 420nm

SE M

1 0.02 0.026 ± 0.002

2 0.04 0.038 ± 0.0018

3 0.08 0.049 ± 0.002

4 0.16 0.084 ± 0.0017

5 0.32 0.158 ± 0.0012

6 0.64 0.258 ± 0.021

7 1.28 0.473 ± 0.018

8 D.z.E 0.230 ± 0.004

Data were expressed as mean ± SEM (mg/ml) concentration was used to find the total
contents

69
 Y = 0 .3 2 8 0 X + 0 .0 3 2 4 3
0 .6 2
R = 0 .9 8

A b s o rb a n c e a t 7 6 0 n m

0 .4

0 .2

0 .0
0 .0 0 .5 1 .0 1 .5
C o n c e n tr a tio n (m g /m l)

Fig 4. 2. Graphical representation of total flavonoid contents of D.zelil

Value of R square obtained from linear regression curve with 95% confidence limit
was 0.9876.

Equation

Y = 0.3280X+0.03243

Percentage of total flavonoids content = 40.3%

Result

Total flavonoids content were measured by standard curve of regression line

and represented as rutin equivalents (µg/mL).Percentage was 40.3%

4.4. DPPH free Redical Scavenging Activity

Antioxident activity was measured by using the DPPH free redical

scavenging activity at 517nm.The antioxident activity of ethanolic extract showed
significant action on the free redical by DPPH. Different concentration of 0.2, 0.4,
0.8, 1.6, 3.2, 6.4 (mg/ml) of plant extract was used. It was calculated as 2.56 ± 0.176,
3.8 ± 0.058, 11.3 ± 0.306, 27.46 ± 0.120, 48.26 ± 0.120, 73.33 ± 0.145 of ethanolic

70
extract of D zelil. Antioxident activity of plant was increased as concentration of
extract was increased and was compared to standard rutin that showed percentage
inhibition 71.73 ± 0.327, 74.40 ± 0.252, 75.26 ± 0.088, 76.47 ± 0.176, 78.0 ± 0.576,
and 81.20 ± 0.058

Table 4. 5. Results of assay of antioxident activity by DPPH method at 517nm

percentage inhibition

Sr DPPH free redical scavenging Activity

no
Concentrations(mg/ml) Rutin Ethanolic extract
1 0.2 71.73 ± 0.327 2.56 ± 0.176
2 0.4 74.40 ± 0.252 3.80 ± 0.058
3 0.8 75.26 ± 0.088 11.30 ± 0.306*
4 1.6 76.47 ± 0.176 27.46 ± 0.120*
5 3.2 78.00 ± 0.576 48.26 ± 0.120*
6 6.4 81.20 ± 0.058 73.33 ± 0.145*
Data was expressed as mean ± SEM. *P<0.05 was considered as significant when
compared with standard to ethanolic extract.

100
R u tin ( % )

E th a n o lic e x tra c t o f D . z e lil e x tr a c t(% )

p e r c e n t in h ib it io n o f r a d ic a l s c a v a n g in g a c tiv ity

80

60

40

20

0
0

0
.2

.4

.8

.6

.2

.4
0

C o n c e n tr a t io n (m g /m l)

Fig 4.3: Graph of anti-oxident of D.zelil by DPPH method

71
4.5. Results and Effects of Delphinium zelil ethanolic extract (D.z) on Brewer’s
yeast induced hyperpyrexia in rats.

Experimental studies showed that pyrexia level in the normal control was 38.42 ±
0.05,38.38 ± 0.05,38.35 ± 0.042, 38.31 ± 0.06, 38.29 ± 0.05, at 0,1st, 2nd, 3rd and 4th
hour. While in the positive control (standard paracetamol) pyrexia was 38.42 ± 0.130,
37.87 ± 0.107, 37.71 ± 0.086, 37.5 ± 0.072, 37.40 ± 0.065, at 0 , 1st , 2nd , 3rd and 4th
hour. The result of Dz.E at 100mg/kg dose was before pyrexia 37.0 ± 0.057 and at 0
hr 38.40 ± 0.05, 1st hr 38.18 ± 0.056, 2nd hr 38.08 ± 0.064,3rd hr 37.93 ± 0.07, 4th hr
37.85 ± 0.065.The results of 200mg/kg were before pyrexia 37.1 ± 0.48 and at 0 hr
38.18 ± 0.01,1st hr 38.05 ± 0.09, 2nd hr 37.91 ± 0.093, 3rd hr 37.78 ± 0.102, 4th hr
37.65 ± 0.112.The antipyretic effect of Dz.E at 400mg/kg dose was before pyrexia
36.9 ± 0.20 and after pyrexia at 0 hr 38.45 ± 0.037, 1st hr 38.18 ± 0.054, 2nd hr 38.02 ±
0.03, 3rd hr 37.86 ± 0.04 and 4th hr 37.74 ± 0.07 Brewer’s induced pyrexia in rats.
There was remarkable reduction in pyrexia in standard control group. It also
represented that Dz.E reduced the pyrexia at different doses. The percentage decrease
in pyrexia was calculated by the formula

% decrease in fever = B-C/B-A×100

Where
B = temp at 0 hour
C = temp at n hour
A = temp before pyrexia

72
 Table 4.6. Effect of different doses of Delphinium zelil ethanolic extract on
brewer’s Yeast Induced Hyperpyrexia

Groups Rectal temperature (°C)

Before yeast After drug administration

administration
0hr 1hr 2hr 3hr 4hr

Normal control 36.93 ± 0.114 38.42 ± 0.05 38.38 ± 0.05 38.35 ± 0.042 38.31 ± 0.06 38.29 ± 0.05
** ** **
Positive 36.62 ± 0.224 38.17 ± 0.13 37.87 ± 0.101 37.71 ± 0.86 37.5 ± 0.07 37.4 ± 0.07***
19.4% 29.6% 43.2% 48.0%
control
Dz100mg/kg 37.0 ± 0.057 38.40 ± 0.04 38.18 ± 0.056ns 38.08 ± 0.064ns 37.93 ± 0.07* 37.85 ± 0.08*
15.7% 22.8% 33.5% 39.2%

Dz200mg/kg 37.1 ± 0.481 38.18 ± 0.01 38.08 ± 0.083ns 37.91 ± 0.093* 37.78 ± 0.093* 37.62 ± 0.115**
12% 25% 37.0% 42.3%

Dz400mg/kg 36.9 ± 0.202 38.45 ± 0.03 38.18 ± 0.054* 38.02 ± 0.028** 37.86 ± 37.67 ± 0.088***
17.4% 27.7%
0.034** 42.8%

38.0%

Results are expressed as mean ± SEM (n=5), analysed by two ways ANOVA. P
values are considered non-significant (ns) as P significant (*),
P<0.01 more significant (**), P<0.001 highly significant (***) when compared with
normal control group

73
 3 9 .0
D z 4 0 0 m g /k g
D z 2 0 0 m g /k g
D z 1 0 0 m g /k g
3 8 .5 s ta n d a rd c o n tro l
n o rm a l c o n tro l
te m p e ra tu re ( C )

3 8 .0

3 7 .5

3 7 .0

3 6 .5
hr

hr

hr
a
se

hr
xi

4
do

re

2
py
re
fo

r
te
be

af

tim e (h r)

Fig 4.4. Graph of Effect of different doses of Ethanolic extract of D.zelil on rectal
temperature by Brewer’s yeast method

74
4.6. Analgesic Activity Determination

4.6.1. Hot Plate method

Hot Plate method was used to determine the analgesic activity. Paracetamol
400mg/kg was used as standard or positive control and it give the maximum latency
time when it was compared with normal control and were statistically significant
result whereas different doses of100mg/kg, 200mg/kg and 400mg/kg ethanolic extract
of D.Z showed the dose dependent effect when compared with standard. The results
were statistically highly significant when different doses of plant extract were
compared with normal control group. Dose 100mg/kg produced the maximum latency
time of 53.5 ± 1.01 seconds at 150 minutes. 200mg/kg of dose showed the maximum
latency time of 63.02 ± 1.231 second at150 minutes. Dose of 400mg/kg produced the
maximum latency time of 67.8 ± 1.61 seconds at 150 minutes. Whereas PCM
400mg/kg showed the maximum latency time of 89.9 second at 150 minutes. The
results of Dz.E were compared with normal control group found to be highly
significant.

75
 Table 4. 7. Effect of different doses of Ethanolic extract of Delphinium zelil and
paracetamol on Latency time on Hotplate method in Rats
Groups Before drug After drug administration
administration

30 min 60min 90 min 120 min 150 min

Normal control 15.06 ± 1.41 15.43 ± 1.14 16.8 ± 1.2 13.9 ± 0.58 16.63 ± 17.05 ± 0.71
1.53

Positive control(PCM 14.57 ± 1.94* 23.8 ± 1.71* 37.95 ± 53.67 ± 68.2 ± 82.2 ±
400mg/kg) 1.50** 2.05*** 2.81*** 4.05***
Dz 100mg/kg 15.27 ± 1.63ns 23.0 ± 1.39ns 31.9 ± 2.74* 42.7 ± 49.4 ± 53.5 ± 1.01**
1.32* 0.722**
Dz 200mg/kg 16.7 ± 1.61* 25.6 ± 1.35* 37.22 ± 46.82 ± 55.02 ± 63.02 ±
** ** ** ***
1.07 1.5 0.98 1.231
* ** **
Dz 400mg/kg 16.9 ± 1.97 27.7 ± 1.98 36.3 ± 1.45 45.22 ± 54.2 ± 67.8 ±
** *** ***
1.18 0.98 1.61
Results are expressed as mean ± SEM (n=5), analysed by TWO WAYS ANOVA. P
values are considered non-significant (ns) as P significant (*),
P<0.01 more significant (**), P<0.01 highly significant (***) when compared with
normal control group

76
 100 n o rm a l s ta n d a rd
s ta n d a rd c o n tro l

D z 1 0 0 m g /k g

80 D z 2 0 0 m g /k g

D z 4 0 0 m g /k g
L a te n c y tim e

60
(s )

40

20

0
in

in

in

in
es
se

m
ut
do

60

90

0
in

12

15
re

m
fo

30
be

tim e (m in )

Fig 4.5. Graph of effect of different doses of ethanolic extract of Delphinium zelil
and paracetamol on Latency time on Hot plate method in Rats

77
 Table 4.8: Decrease in latency time of Dz.E
Groups Response time (Sec)
Before drug After drug administration
administration
0 time
Normal control 15.1 ± 1.41 30 min 15.43 ± 1.14
60 min 16.8 ± 1.27
90 min 13.9 ± 0.6
120 min 16.63 ± 1.53
150 min 17.1 ± 0.71
Standard control 14.6 ± 1.9 30 min 23.8 ± 1.71
60 min 37.95 ± 1.5
90 min 53.7 ± 2.05
120 min 68.02 ± 2.81
150 min 82.1 ± 4.05
Dz 100mg/kg 15.27 ± 1.65 30 min 23.0 ± 1.39
60 min 31.9 ± 2.79
90 min 42.8 ± 1.32
120 min 49.9 ± 0.722
150 min 53.5 ± 1.01
Dz200mg/kg 16.7 ± 1.61 30 min 25.7 ± 1.35
60 min 37.2 ± 1.05
90 min 46.82 ± 1.53
120 min 55 ± 0.983
150 min 63 ± 1.21
Dz400mg/kg 16.9 ± 1.97 30 min 27.7 ± 1.98
60 min 36.3 ± 1.45
90 min 45.02 ± 1.18

120 min 59. ± 0.98

150 min 67.8 ± 1.6

Results are expressed as mean ± SEM (n=5), analysed by two ways ANOVA. P
values are considered non-significant (ns) as P significant (*),
P<0.01 more significant (**), P<0.01 highly significant (***) when compared with
normal control group

78
4.6.2. Acetic induced writhing method

Acetic induced writhing method was used to determine the analgesic activity.
It was used to measure the pain by peripheral mechanism. Diclofenac sodium was
used as standard control. When positive control group was compared with standard
control group the results are maximum. Diclofenac sodium showed the maximum
induction of pain 74% at 10-15 minutes against chemically induced pain. Differents
doses of Ethanolic extract of Dz i.e.100mg/kg, 200mg/kg, 400mg/kg given the
maximum induction of pain 54.8%, 63.2% and 64.5% at 15-20 minutes respectively.
There was significant reduction in writhing in the whole treated groups when these
were compared with the standard group which showed the highly significant

Table 4.9: Effect of different doses of Ethanolic extract of Delphinium zelil and
diclofenac sodium on acetic induced writhing in rats

Treated No. of writhing at various intervals

Groups 0-5 mins 5-10 mins 10-15 mins 15-20 mins
Control 39 ± 1.58 36 ± 0.854 32 ± 0.479 21 ± 1.19
10ml/kg
Diclofenac 17 ± 0.854 13 ± 0.408 11 ± 0.479 8 ± 0.408
** ** *** ***
sodium 56.4% 63.8% 65.6% 74%
10mg/kg
Dz 26 ± 1.75 21 ± 0.975 18 ± 0.500 14 ± 0.707
ns * ** **
100mg/kg 33.3% 42% 50% 54.8%
Dz 21 ± 0.479 17 ± 0.479 13 ± 0.645 11 ± 0.500
* ** ** ***
200mg/kg 46% 51.1% 59.3% 63.2%
Dz 17 ± 0.408 15 ± 0.408 12 ± 0.629 11 ± 0.408
** ** *** ***
400mg/kg 56.4% 58.1% 62.5% 64.5%

Results are expressed as mean ± SEM (n=5), analysed by two ways ANOVA. P
values are considered non-significant (ns) as P significant (*),
P<0.01 more significant (**), p<0.01 highly significant (***) when compared with
normal control group

79
 n o rm a l sta n d a rd

50
c o n tro l sta n d a rd D z 2 0 0 m g /kg

D z 1 0 0 m g /kg D z 4 0 0 m g /kg

40
N o . o f w r it h n in g s

30

20

10

0
10

0
5
0-

-1

-2
5-

10

15

tim e (m in )

Fig 4.6. Graph of Effect of different doses of Ethanolic extract of Delphinium zelil
and diclofenac sodium on acetic induced writhing in rats

80
4.7. Acute Toxicity Test

Acute oral toxicity was analysed in rat which showed that Dz extract up to
5000mg /kg body weight did not show any sign of behavioral and neurological
toxicity in the rats. There was no relative difference in the body weights of control
and treated rats. Aqueous ethanolic extract of plant was used in acute toxicity study
and there was no lethality up to the dose of 5000mg/kg which showed the safety of
the extract.

81
Table 4. 10. Acute toxicity test of Delphinium zelil on behavioral and neurological
changes in rats

Days Behavioral changes Presence

30 min-24 hours Skin changes No
Aggressivity No
Pain No
Sleep No
Salivation No
Sensitivity to sound No
Breathing No

7 day Skin changes No

Aggressivity No
Pain No
Sleep No
Salivation No
Sensitivity to sound No
Breathing No

14 day Skin changes No

Aggressivity No
Pain No
Sleep No
Salivation No
Sensitivity to sound No
Breathing No

There was no behavioral changes seen in the study start from dose of 300mg/kg up to
5000mg/kg from time start 30 minutes to 24hr and up to 7 days.

Results are expressed as mean ± SEM (n=5), analysed by two ways ANOVA.
P values are considered non-significant (ns) as P significant (*),
P<0.01 more significant (**), P<0.01 highly significant (***) when compared with
normal control group

82
5.1. Discussion

The pain is the physical condition which may be due to any disease, injury and
trauma, which may cause irritation and sadness mentally or emotionally in body. It
may also be due to physical stress on body. (Naro, Calabrò, et al., 2015) Pain is just a
symptom which may arise from any destruction in body cells and alarming the person
to withdraw from damaging conditions, to protect a damaged part of body while
healed and to avoid the same condition in future (A. Holden & W. Winlow, 1984).

Most of the pain recovered with simple medication in 20-70%cases (Chaparro

et al., 2013).

Pain is usually for short time and is only until the harmful agent is removed or
pathological condition is recovered. But some time pain is for long time. Therefore
pain is divided into two type acute or chronic pain. The pain which is from simple
damaging of body cells and recovered within from few days to month is called acute
pain, and for it simple medication are used to treat it like NSAIDs . The pain which
remained for long time from months to years is called chronic pain. Chronic pain is
recovered with strong pain killer, opioids and morphine like substances. Pain in
cancer or rheumatoid arthiritis is good examples (Calvino & Grilo, 2006).

Most of the pain is recovered with NSAIDs, opioids or morphine like drugs
but these substances produced severe adverse effects like peptic ulceration,
hepatotoxicity, skin rashes etc. Therefore natural products have been used for
alternative to these drugs which may suppress the pain with less adverse effects.

In many clinical condition fever is occur due to presence of substances called

pyrogens, which may increase in body temperature. Pyrogens are exogenous or
endogenous. Most common pyrogens are microorganisms, fungi, or toxins. Many
toxins, infections and other mediators are act as endogenous pyrogen. Gram negative
bacteria produce some toxin which acts as endogenous pyrogen. Gram positive
bacterial cell wall has peptidoglycan which may cause fever in body. Exogenous
pyrogen acts as endogenous pyrogen by the formation of monocytes and macrophages
through the host cell stimulation. Endogenous pyrogens may be produced locally or
systematically, then enter in circulation and causes increase in body temperature and
produced fever. Many cytokines like IL-1, IL-6, TNF, interferon causes fever in body.

83
IL-I causes fever within 10 minutes when produced and on reaching to hypothalamus
(Chandra & Kumar Bhatnagar, 2002). PGE2 prostaglandin is produced when these
cytokines acts on brain blood vessels. Fever is autonomic, behavioral and endocrine
response which is control by hypothalamus in brain (Kunnaja et al., 2014).

Yeast induced pyrexia is called pathogenic fever which acts on

thermoregulatory mechanism and cause to production of prostaglandins, which may
cause increase in body temperature. Formation of these prostaglandins which act as
pyretic agents in body. The antipyretic effect of NSAIDs is due to inhibition of PGE2
synthesis and release in body from the hypothalamus in the brain.

A number of plants extract modifying the enzyme of cyclooxygenase pathway

which inhibit the leukotriene and prostaglandins synthesis by inhibition of COX-I and
COX-II pathway.(Ganguly et al., 2013) The increase in body temperature due to
factor like exercise or increase in ambient temperature paracetamol does not influence
on this body temperature and does not decrease the body temperature. A common
available drug such as ibuprofen, diclofenac, mefenamic acid and indomethacin may
have the therapeutic effects in reduction of pain, fever and inflammation in body, but
have severe side effects such as ulceration, GIT tracts infections, irritation,
hepatotoxicity and hypotension etc. Therefore natural products are alternative to these
for treatments and are consider to be used for inhibition of prostaglandins biosynthesis
by the plant extracts of Delphinium zelil and Cyperus pertenius which may cause in
blockage of COX-I and COX-II enzymes and acts as antipyretic and analgesic
substances (Somchit et al., 2014).

Phytochemical analysis of the Cyperus pertenius and Delphinium zelil showed

the presence of tannins, saponin, phenols, flavonoids, terpenes and sterols. It was
reported that phenols and flavonoids were responsible for antibacterial, anticancer and
anti-inflammatory activities.(S. Kumar & Pandey, 2013) Alkaloids like boldine have
ability to blockage the synthesis of prostaglandin PGE2 (Backhouse et al., 1994).
Similarly flavonoids like baicalin and quercetin have ability to suppress TNF and
exert antipyretic effect (Kim et al., 2004). Due to the importance of flavonoids,
phenols, tannins and other metabolites in ant- oxidant activity, these components were
estimated for anti-oxidant by DPPH method (Abo-Dola et al., 2015). DPPH method
was performed on both plant extracts with different standards like (ascorbic acid and

84
rutin) were used. Highest value of scavenging was seen at the dose of 640µglmg and
Cyperus pertenius showed 89% when compared with standard ascorbic acid and
Delphinium zelil shown 73% at the same dose when compared with standard rutin.

Present study was conducted to evaluate the analgesic and antipyretic

activities of both ethanolic extracts of Cyperus pertinius and Delphinium zelil plant.
Aqueous ethanolic extracts of Cyperus pertenius and Delphinium zelil produced
significant (P<0.05) antipyretic effect in the dose dependent manner. The plant
extracts inhibit the prostaglandin synthesis due to presence of alkaloids like boldine,
flavonoids like quercetin, baicalin and phenols. Analysis of extracts showed they
contain Alkaloid and flavonoids which possessed antipyretic activity. The plant
extracts at the dose of 100mg∕kg showed slightly decreased in rectal temperature.
There was more decrease in rectal temperature at the dose of 200mg∕kg, but at the
dose of 400mg∕kg extracts showed reduction in rectal temperature not efficient as
compared to standard (paracetamol) drug.

The analgesic activity was investigated against the thermally and chemically
induced pain stimuli to evaluate the central and peripheral pain inhibiting pathways by
the extracts of Cyperus pertenius and Delphinium zelil. Hot plate and acetic induced
writhing tests were used to be seen the analgesic activity of extracts. Hot plate method
was used to seen the central analgesic effect of extracts and acetic induced writhing
method was used to display peripheral analgesic effect of the extracts. Extracts
exhibited dose dependent analgesic activity in the hotplate and acetic induced
writhing methods. In the pain histamines, bradykinins and prostaglandins are
produced. The plant extracts inhibit the prostaglandins and histamine synthesis and
release due to presence of alkaloids, boldine, flavonoids like baicalin,quercetin etc
(Kim et al., 2004). The results of analgesic activity of Cyperus pertenius and
Delphinium zelil were remarkable same as that of diclofenac sodium which was used
as standard in the current experiment. The extracts produced analgesic effect in dose
dependent manner, as dose was increased there was more reduction of pain. At dose
of 400mhlkg there was more reduction in pain but not efficient like standard
diclofenac sodium drug. This indicated that the effect might be due to inhibition of
prostaglandins and histamines. It may inhibit the COX-I and COX-II enzymes.
Diclofenac sodium was used as standard due to its analgesic effect and causes
reduction in pain.

85
5.2. Conclusions and Recommendations

In this study phytochemicals and pharmacological evaluation of analgesic and

antipyretic activities of extracts of Cyperus pertenius and Delphinium zelil was
carried out. It is concluded from current study that ethanolic extracts of Cyperus
pertenius and Delphinium zelil produced antipyretic effects which may be due to
inhibition of prostaglandins similar to paracetamol which was used as standard.

Analgesic activity was also assessed by hotplate method and acetic induced
writhing method and result produced are near to the result of NSAIDs (non-steroidal
anti-inflammatory) drugs like diclofenac sodium in the dose dependent manner. The
experimental evidence suggests that extracts reduce the pain. This is due to inhibition
of cyclooxygenase enzyme and certain metabolites like bradykinin, histamine and
prostaglandin which cause pain in body. So it is concluded from the previous
experimental study that plants Cyperus pertunius and Delphinium zelil has significant
analgesic and antipyretic effects. Although this study was preliminary steps toward
screening of these plants extract, which proves the way for further attention and
research to identifying the active compounds which are responsible for
pharmacological activities. Further studies could be undertaken to elucidate the exact
mechanism by which these plants extract exert their analgesic and antipyretic effects.
Moreover the phytochemical and pharmacological exploration is required to see the
other activities of these important plants.

86
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