EVALUATION OF SELECTED PHARMACOLOGICAL ACITIVITES

OF CUSCUTA REFLEXA ROXB

BY
SAMIA GUL NIAZI
ROLL NO: PPFS13E017
THESIS SUBMITTED IN
PARTIAL FULFILLMENT OF
REQUIREMENTS FOR THE DEGREE OF
MASTER OF PHILOSOPHY IN PHARMACY
(PHARMACOLOGY)
SESSION 2013-2015

FACULTY OF PHARMACY,
UNIVERSTIY OF SARGODHA,
SARGODHA, PAKISTAN
 DEDICATED TO

My Affectionate PARENTS

So much of what I have become

Is just because of you,

And I want you to know that I

appreciate you

Thank you and love you.

 APPROVAL CERTIFICATE

It is certified that this thesis entitled “Evaluation of selected pharmacological activities of

Cuscuta reflexa Roxb.” has been prepared by Samia gul niazi under my supervision.
Furthermore, this thesis has not been previously presented for a higher degree. She has fulfilled
all the requirements and qualifies to submit this thesis for the degree of M.Phil
(Pharmacology).

SUPERVISOR

______________________
Dr. Alamgeer
Assistant Professor
Department of Pharmacology
Faculty of Pharmacy,
University of Sargodha, Sargodha
 i

CONTENTS
Sr. No Particulars Page No

List of tables v

List of figures vi

List of abbreviations viii

Acknowledgments x

Abstract xii

1 INTRODUCTION 1

1.1 Nephrotoxicity 1

1.1.1 Acute renal failure 2

1.1.2 Chronic renal failure 3

1.1.3 Nephrotic syndrome 3

1.1.4 Prevalence of nephrotoxicity 4

1.1.5 Role of immunosuppressive agents and other classes in nephrotoxicity 4

1.1.6 Nephrotoxicity management 5

1.2 Arthritis 7

1.2.1 Types of arthritis 7

1.2.2 Pathophysiology of rheumatoid arthritis 8

1.2.3 Medical treatment of rheumatoid arthritis 8

1.3 Ethnomedicine-a new focus 11

1.4 Introduction of medicinal plant selected for research work 11

 ii

1.4.1 Morphology of plant 12

1.4.2 Description of plant

14

1.4.3 Traditional names

14

1.4.4 Importance of plant in unani medicinal system

15

1.4.5 Reported Phytochemistry of Cuscuta reflexa Roxb.

15

1.5. Justification of current research work 19

1.6 Aims and objectives of present study 20

2 MATERIALS AND METHODS 21

2.1 Chemicals and materials used 21
2.1.1 Chemicals used in extraction of plant material 21
2.1.2 Chemicals used for phytochemical screening 21
2.1.3 Chemicals used for making buffer solution 21
2.1.4 Chemicals and drugs used in in-vivo experiments 22
2.1.5 Chemicals and drugs used in in-vivo experiments 22
2.2 Equipment used 22
2.3 Apparatus used 23
2.4 Experimental animals used 24
2.5 Methodology 24
2.5.1 Selection of plant material 24
2.5.2 Collection and authentication of plant 24
2.5.3 Preparation of plant extract 24
2.5.4 Preparation of stock solution of drug for in-vitro studies 25
2.6 Analysis on Plant extract 25
2.6.1 Solubility analysis for Extract 25
 iii

2.6.2 Preliminary Phytochemical analysis 26

2.6.3 HPLC Analysis of the extract 26
2.7 Pharmacological studies made on plant extract 29
2.7.1 Evaluation of Nephroprotective effect of Cuscuta reflexa Roxb. on 30
gentamicin induced nephrotoxicity in rats
2.7.1.1 Drug administration protocol 30
2.7.1.2 Sampling of Blood for Further Analysis 31
2.7.1.3 Histopathological examination 31
2.7.2 Evaluation of anti-arthritic effect of Cuscuta reflexa Roxb. 31
2.7.2.1 In-vivo anti-arthritic activity 31

2.7.2.1.1 Evaluation of anti-arthritic effect of Cuscuta reflexa on 32

formaldehyde induced arthritis in rats

2.7.2.1.2 Evaluation of anti-arthritic effect of Cuscuta reflexa on turpentine 32

oil induced arthritis in rats

2.7.2.2 In-vitro Anti-arthritic activity 33

2.7.2.2.1 Evaluation of anti-arthritic effect of Cucuta reflexa on inhibition of 33
protein denaturation (Fresh hens’s egg albumin)
2.7.2.2.2 Evaluation of anti-arthritic effect of Cuscuta reflexa on inhibition of 34
protein denaturation (bovine serum albumin)
2.8 Statistical analysis 35
3 RESULTS 36
3.1 Solubility analysis of Aqueous Methanolic extract of Cuscuta reflexa 36

3.2 Phytochemical Analysis of Aqueous Methanolic extract of Cuscuta 36

reflexa
3.3 HPLC analysis of Aqueous Methanolic extract of Cuscuta reflexa 37
3.4 Results of Nephroprotective activity 40
 iv

3.4.1 Effect of aqueous methanolic extract of Cuscuta reflexa on 40

Biochemical Parameters in gentamicin induced nephrotoxic rats.
3.4.2 Effect of aqueous methanolic extract of Cuscuta reflexa on physical 43
parameters in gentamicin induced nephrotoxic rats.
3.4.3 Effect of aqueous methanolic extract of Cuscuta reflexa on 43
histopathogical view in gentamicin induced nephrotoxic rats.
3.5 Results of In-vivo Anti-arthritic activity 49
3.5.1 Effect of aqueous-methanolic extract of Cuscuta reflexa against 49
formaldehyde induced arthritis in rats
3.5.2 Effect of aqueous-methanolic extract of Cuscuta reflexa against 49
turpentine oil induced arthritis in rats.
3.6 Results of In-vitro Anti-arthritic Activity 54
3.6.1 Effect of aqueous-methanolic extract of Cuscuta reflexa on protein 54
denaturation using fresh hen’s egg albumin
3.6.2 Effect of aqueous-methanolic extract of Cuscuta reflexa on of protein 54
denaturation using bovine serum albumin
4 DISCUSSION 57
4.1 Conclusion 65
5 REFRENCES 66
 v

LIST OF TABLES

Table No Particulars Page No.

Table 01 Important Phytochemicals of Cuscuta reflexa and their action 17

Table 02 Methods for phytochemical screening of different classes of 27

phytoconstituents in Cuscuta reflexa.
Table 03 Solubility Analysis of Aqueous Methanolic Extract of Cuscuta 38
reflexa using different solvents
Table 04 Preliminary phytochemical analysis of Cuscuta reflexa 38

Table 05 Preliminary HPLC Analysis of Cuscuta reflexa 39

Table 06 Biochemical Parameters Serum Urea, Serum Creatinine, Blood Urea 41

Nitrogen And Serum Uric Acid
Table 07 Weight of Kidney of Rats on treatment with extract of Cuscuta reflexa 44
(Aqueous methanolic extract)

Table 08 Histopathological analysis of effect of aqueous methanolic extract of 45

Cuscuta reflexa on animal model
Table 09 Effect of AMECR on formaldehyde induced arthritis in Sprague 50
dawley rats

Table 10 Effect of AMECR on turpentine oil induced arthritis in Sprague 52

dawley rats
Table 11 Effect of Cuscuta reflexa on protein (egg albumin) denaturation 55

Table 12 Effect of Cuscuta reflexa on protein (bovine serum albumin) 56

denaturation
 vi

LIST OF FIGURES
Figure No. Particulars Page No.
Figure 01 Role of immunosuppressive agents in nephrotoxicity (Adopted from Rick G. 6
Schnellmann and Katrina J. Kelly)

Figure 02 Mechanism of Nephrotoxicity caused by other classes of drugs 6

Figure 03 Pathophysiology of Rheumatoid Arthritis 9
Figure 04 Established RA is characterized by the presence of large bone erosions filled with 9
inflamed, synovially derived pannus tissues

Figure 05 Yellowish stem of Cuscuta reflexa on host plant 13

Figure 06 Cuscuta reflexa whole plant on host plant 13
Figure 07 Structural picture of phytochemicals found in Cuscuta reflexa 18
Figure 08 Research protocol of current studies 29
Figure 09 Peaks of Phytoconstituents in HPLC analysis. 39
Figure 10 Effect of AMECR on serum creatinine, urea, Blood urea nitrogen (BUN) and 42
Serum uric acid level of normal and gentamicin induced nephrotoxic rats.
Figure 11 Effect of AMECR on Kidney weight of animals from normal to Gentamicin 44
treated group
Figure 12 Histopathological view of kidney of normal group shows normal renal 46
parenchyma along with normal tubules and normal glomeruli
Figure 13 Histopathological view of Toxic control group that is dense pinkish 46
granulation. It is showing renal parenchyma with focal necrosis and
lymphatics infilterate.
Figure 14 Histopathological view of section of kidney given aqueous methanolic 47
extract of Cuscuta reflexa (200 mg/kg/ body weight) plus Gentamicin (100
mg/kg/body weight). Renal parenchyma showing moderate to sever
lymphatic infilterate.
Figure 15 Histopathological view of the section of kidney given aqueous methanolic 47
extract CR (400mg/kg/day) plus gentamicin (100 mg/kg/body weight)
 vii

Figure 16 Histopathological view of section of kidney given aqueous-methanolic 48

extract of Cuscuta reflexa (600 mg/kg/body weight) plus gentamicin (100
mg/kg/ body weight) . Renal parenchyma shows mild lymphatic infilterate
Figure 17 Effect of AMECR on formaldehyde induced arthritis in Sprague dawley rats. 51
Figure 18 Effect of AMECR on turpentine oil induced arthritis in Sprague dawley rats. 53
Figure 19 Effect of AMECR on percentage inhibition of protein denaturation 55
Figure 20 Effect of AMECR on percentage inhibition of protein denaturation 56
 viii

LIST OF ABBREVIATION

GFR Glomerular Filtration Rate

ARF Acute Renal Failure

CRF Chronic Renal Failure

ROS Reactive oxygen species

BUN Blood Urea Nitrogen

SE Selenium

NC Normal Control

TC Toxic Control

AMECR Aqueous Methanolic Extract of Cuscuta reflexa

SOD Super dioxide mutases

RA Rheumatoid arthritis

TNF Tumor necrosis factor

GS Gentamicin Sulphate

TS Test solution

BSA Bovine serum albumin

PBS Phosphate Buffer saline

GM-CSF Granulomonocyte-Colony stimulating factor

 ix

IL Interleukin

Th Helper T cells

NF-κB Nuclear factor κB

ACPA Anti-citrullinated protein antibodies

TGF Transforming growth factor

PDGF Platelet-derived growth factor

EGF Epidermal growth factor

COX Cyclooxygenase

PG Prostaglandin

DMARDs Disease-modifying antirheumatic drugs

NSAIDs Nonsteroidal anti-inflammatory drugs

ug Microgram

ml Milligram

mm Millimeter

p.o Per oral (orally)

RF Rheumatoid factor

SEM Standard error of mean

 x

ACKNOWLEDGEMENTS
First of all I am grateful to ALMIGHTY ALLAH who in every moment of my life always
with me and blessed me beyond my imagination, who helped me in every difficult time, who
loved me, who gave me strength and perseverance, whatever I am today is just because of
Allah Subhana-Tala. Countless salutation upon our beloved HOLY PROPHET HAZRAT
MUHAMMAD (P.B.U.H), the beacon of knowledge, who has guided his “Ummah” to seek
knowledge from cradle to grave and enabled me to win honor of life.
It is quite delectable to become and to avail this most propitious opportunity to articulate with
utmost gratification, my profound and intense sense of indebtedness to my ever affectionate
worthy supervisor Dr. Alamgeer, Assistant Professor Pharmacology, Faculty of Pharmacy,
University of Sargodha. His proficient counseling, valuable suggestions, boundless
forbearance, indefatigable help with anything, anywhere, anytime, consummate advice,
thought provoking instructions and giving me enough independence to decide the things
throughout the study. He helped me to grow in both my competence and in confidence as a
researcher.
I am unfathomable indebted to Dr. Sajid Bashir, Dean/Chairman, Faculty of Pharmacy, and
University of Sargodha for his keen interest, encouragement, full support and cooperation
during research work. I feel my words so shallow; they do not seem to be the same as felt k8to
be thankful to my worthy teacher who helped me a lot specially Prof. Dr. Riaz Hussain Dab
that without his help it was not possible for me to complete my research and to my class fellows
Ms. Umbreen Malik Uttra and Qurat-ul-Ain for their beneficial suggestions during my
research work. I am also very thankful to my uncle Dr. Imran Naseem who motivated me
throughout the duration of my research work. Sincere thanks to all laboratory staff members
of the Faculty of Pharmacy, University of Sargodha for their cooperation, especially Mr.
Ishfaq (material supplier), Mr. Nasir, Mr. Waqas (research lab attendants),
I have no words at command in acknowledging that all credit goes to my loving parents Sahib
Dad Khan Niazi and Farhat Naseem for their endless patience, sacrifices, prayers, continuous
encouragement and for instilling in me the love of reading and the pursuit of knowledge from
my early sapling years and to them I dedicate this thesis. Without their faithful love, care and
understanding, it would have been difficult for me to find the passion to succeed.
 xi

Special love and thanks to my loving and caring sisters Umbreen Liakat, Mehreen Sajjad,
Hamdia Ahsan, Amna Khurram and Aniqa Niazi and my brothers Khurram Shahzad and
Naveed Shahzad for their amicable attitude, love, immense orison, mellifluous affections,
inspiration, well-wishing, keen interest and for putting colors in my life which hearten me to
achieve success in every sphere of life. I offer my zealous thanks and deep appreciation to my
fellow colleague Imtiaz Mahmood Tahir who helped me in making photographs of
histopathology slides and my friends Anam adil, Ayesha Ahmad, Samra Sadiq, Fatima
Javaid, Sidra Anwar and Assra Umar for her invaluable support, favor, encouragement,
prayers and for wishing to see me glittering high in the skies of success. I just pray that ALLAH
bless her with everything.
SAMIA GUL NIAZI
 xii

ABSTRACT
Nephrotoxicity is renal dysfunction emerges out as a direct or indirect exposure of body to
environmental and industrial chemicals or any drug. The kidney is an organ which is mainly
targeted for the toxicity of drugs, pollutants, oxidative stress, and xenobiotics, in order to its
distinctive metabolism and major source of excretion. Acute renal failure is one of the renal
dysfunction which can be characterized by decrease in GFR, azotemia, changes in urine
volume and changes in the biochemical homeostatic processes. However obstructive renal
failure is characterized by obstruction in flow of urine due to any drug or its toxic metabolite
while chronic renal failure (CRF) is a major adverse effect of some drugs that normally present
as tubulointerstitial disease. Rheumatoid arthritis (RA) is a chronic inflammatory, autoimmune
and multisystem disorder. Drugs like DMARDS, NSAIDS, and corticosteroids have been
available for various type of arthritic diseases: nevertheless it is need of the hour to expose
such natural products which may be cost effective and causes less adverse reactions. Many
ethnic medicinal plants have been found having marked medicinal effects however no
scientific work has been done on them so these hypothesis opens the new horizons for the
treatment of different ailments.
The present study was conducted to evaluate the pharmacological activities of Cuscuta reflexa
Roxb. by performing in-vitro and in-vivo experiments for determining nephroprotective and

anti-arthritic potentials of aqueous-methanolic extract (AME). Pharmacological activates of

Cuscuta reflexa was evaluated by performing experiments on in-vivo models like Gentamicin
induced nephrotoxicity, Formaldehyde induced arthritis and turpentine oil induced arthritis and
two different in-vitro models to explore anti-arthritic potential such as inhibition of protein
(bovine serum albumin) denaturation and inhibition of protein (egg albumin) denaturation. The
plant extract were given at a dose level of 200, 400 and 600 mg/kg/p.o in Gentamicin induced
nephrotoxicity, turpentine oil and formaldehyde induced arthritis models. Cuscuta reflexa
displayed extremely significant results (p<0.001) and marked effect on biochemical
parameters like BUN, serum urea, creatinine and uric acid as well as arthritis and it also showed
dose dependent inhibitory effect against different diseases in rats. There was a marked
(p<0.001) reduction in BUN, serum urea , creatinine and uric acid level on eighth day in case
acute renal failure which has been shown through histopathological studies which is 18.370,
41.400, 3.267 and 0.740 respectively when compared with toxic control group that is 61.800,
 xiii

34.593, 5.714 and 1.878 and it also showed prominent effect on joint thickness and paw edema
of plant extract animals and at 6th hr. in case of turpentine oil and on 10th day in case of
formaldehyde induced arthritis, when compared with intoxicant treated animals. Aqueous
methanolic ek6xtract of dose of (70-30 ratio) 600 mg/kg of plant extract had an effect of 71.22
% on controlling turpentine oil induced joint edema which is comparable to aspirin (standard),
71.00 %. Similarly plant extract of dose of 600 mg/kg exhibited superior anti-arthritic activity
of 76.74 % as compared to 77.26 % at 100 mg/kg of aspirin (standard) against formaldehyde
induced arthritis.
The results indicated a significant concentration dependent increase in percentage protection
for all the two models after treatment with plant extract of different concentrations 25, 50, 100,
200, 400 and 800 ug/ml. Maximum inhibition of 89.307 %, 99.123 % at 800 μg/ml was
detected in case of bovine serum albumin denaturation method by plant extract and aspirin
(standard) respectively. In case of egg albumin denaturation method significant (P<0.001)
protection against protein denaturation viz., 93.510 % and 99.163 % was observed at 800 ug/ml
by plant extract and diclofenac sodium (standard respectively. Results of in-vitro tests
indicated that inhibition of protein denaturation (bovine serum and egg albumin) by AMECR
was comparable to standard drugs i.e. Aspirin and Diclofenac sodium. The results of effect of
AMECR showed that it exhibits almost equivalent or better protection against renal diseases
and RA.The biochemical parameters of kidney and serum marker of arthritis i.e. rheumatoid
factor was also reduced in treated arthritic rats. The overall severity of renal diseases is
determined by histopathological examination indicated that Cuscuta reflexa exerts a potent
protective effect gentamicin induced nephrotoxicity in rats. Moreover, phytochemical
investigation of plant extract using HPLC technique revealed the presence of polyphenols i.e.,
quercitin, gallic acid, Caffeic acid, p-coumeric acid, vanillic acid and trans-4-hydroxy-3-
methoxy cinnamic acid. It is conceivable, therefore; that Cuscuta reflexa possess possible
nephroprotective and anti-arthritic effect which could be due to the presence of above
mentioned phytoconstituents. However, further studies would be necessarily needed to
elucidate the exact mechanism(s) of anti-arthritic activity of selected plant and to establish the
real efficacy and safety in patients by following the FDA approved protocols
 1

SECTION I
1. INTRODUCTION
1.1 Nephrotoxicity

Most kidney problems occur when body is exposed to foreign agents such as certain drugs or
exogenous toxins. Nephrotoxicity is one of the kidney problems which can be defined as the
development of structural and functional kidney damages most probably due to the drugs or
medicines (Pani et al., 2011). Nephrotoxicity becomes one of the most important medical and
public health issue from last few years and its prevalence has been growing exponentially
throughout the world. Renal dysfunction arises as a result of exposure to external environment
or substances i.e. any drug or environmental chemicals (El-Tantawy et al., 2013). The renal
system especially kidney is a main target for the toxicity of drugs, environmental pollutants,
oxidative stress, and xenobiotics in order to its distinctive metabolism and source of excretion
(Uehara et al., 2007). Nephrotoxicity can be accessed by urine analysis, histopathology and
blood chemistry. In renal diseases, kidney damage occurs due to which the kidney becomes
unable to get rid of the waste products and urine due to which the electrolyte balance elevated
as well as the creatinine and urea level increases in the blood stream (Harlalka et al., 2006).
Drugs may affect one or more discrete regions of the kidney or it may target only one type of
cells (Zager et al., 1997). Pathophysiological responses of nephrotoxicity include:

1. Acute Renal Failure

2. Chronic Renal Failure
3. Nephrotic syndrome

1.1.1 Acute Renal Failure:

Acute renal failure (ARF) can be characterized by decrease in glomerular filtration rate (GFR),
azotemia, changes in urine volume and changes in the biochemical homeostatic processes.
Creatinine level usually increased in acute renal failure over a recognized baseline more than
1.5mg/dl. The mortality rate of acute renal failure is ranged from 15% to more than 60% (Hock
et al., 1995). Acute renal failure results mostly due to the direct kidney damage, impaired blood
 2

flow to the kidney and urine blockage in the kidney. Direct kidney damage mostly occurs due
to the hemolytic uremic syndrome, glomerulonephritis, cholesterol deposits and blood clots,
infection, thrombotic thrombocytopenic purpura (TTP), scleroderma, toxins, medications such
as antibiotics, certain chemotherapeutic agents, dyes used for imaging tests etc. (Liano et al.,
1996). Impaired blood flow to the kidney mostly occurs due to the blood or fluid loss, blood
pressure medications, heart disease, infections, medications such as aspirin, ibuprofen,
naproxen related drugs and severe allergic reactions (anaphylaxis) etc. Urine blockage in the
kidney may occurs due to the bladder cancer, blood clots in the urinary tract, cervical cancer,
colon cancer, kidney stones and nerve damage involving the nerves that control the bladder.
Drugs or the medications are leading cause of acute renal failure. Medications usually by three
mechanisms are reported to cause acute renal failure such as pre-renal ARF (60-70%), intrinsic
ARF (25-40 %) and obstructive ARF (10%). When the renal failure occurs due to the less
blood supply to the kidney then it is termed as the prerenal acute renal failure (Asangansi et
al., 2005). Drugs causing the prerenal acute renal failure are diuretics (with high osmolarity),
immunosuppressive agents (steroids) like cyclosporine and tacrolimus and non-steroidal anti-
inflammatory drugs (NSAIDS) like interleukins.

Reasons for the intrinsic acute renal failure are acute tubular necrosis, thrombotic
microangiopathy, acute interstitial nephritis (renal tubular and interstitial inflammation) (Zager
et al., 1997). Causes for acute tubular necrosis are drugs like amphotericin B, cisplatin ,
radiocontrast agents, pentamidine cocaine and combination therapies like statins plus
immunosuppressive. Clinical findings of acute tubular necrosis shows that urine creatinine /
serum creatinine is increased 2-3%. Thrombotic microangiopathy caused by
immunosuppressive drugs (cyclosporine, quinie.clopidogrel tacrolimus), agents used for
chemotherapy (bleomycin, cisplatin) and oral contraceptives with estrogen (Asangansi et al.,
2005). Clinical findings shows eosinophilia and leukocytes and casts are commonly present in
urine (proteinuria). Tubulointerstitial nephritis is caused by anti-epileptic drugs, penicillins,
cephalosporins and NSAIDS. However their clinical findings shows back pain and depression
and proteinuria (Zager et al., 1997).

Obstructive renal failure is determined by obstruction in flow of urine due to any medicine or
its poisonous metabolite. Obstruction, sometimes occurred within the tubules or in the uterus
due to drug induced crystal formation. Severe dehydration, metabolic disorders, bolus drug
 3

administration, underlying renal insufficiency is the major risk factors for underlying renal
failure. Obstruction outside the ureters can be due to drug induced retroperitoneal fibrosis
(Guo et al., 2002). Patients with human immunodeficiency virus (HIV) infection are at more
risk of developing obstructive acute renal failure because of compromised immunity level and
multiple drugs administration (Asangansi et al., 2005). Drugs causing obstructive acute renal
failure are acyclovir, indinavir and quinine.

1.1.2 Chronic Renal Failure:

Renal tubular syndrome and increase in the level of creatinine are the clinical manifestations
of chronic renal failure (CRF). Renal tubular syndrome can be characterized as proteinuria,
papillary necrosis, renal acidosis, renal loss of potassium and other electrolytes (Kleinknecht
et al., 1995). Pathophysiology of CRF is that such medications causes the chronic interstitial
fibrosis with or without papillary necrosis. However clinical findings of CRF shows flank pain,
hematuria and papillary sloughing. Medications used for CRF are NSAIDs (fenoprofen,
mefnamic acid, ibuprofen, and phenylbutazone), cidofovir, acyclovir, Indinavir, cyclosporine,
tacrolimus and phenacetin. Chronic renal failure may be reversible or irreversible (Agharazii
et al., 1993). 5-aminosalicylic acid, mesalamine and ifosfamide cause reversible injury while
lithium and cyclosporine are the cause of irreversible injury (Ponticelli et al., 2000).

1.1.3 Nephrotic Syndrome

Intense proteinuria and glomerular dysfunctions are the main causes of nephrotic syndrome.
The causes of nephrotic syndrome are the heavy metals like gold, lead etc., steroidal anti-
inflammatory drugs, penicillamine, interferons, and captopril.
If the offending agents used will be stopped the nephrotic syndrome can be reversed but if not
then they may cause irreversible injury (Decloedt et al., 2011).
 4

1.1.4 Prevalence of Nephrotoxicity

Renal tubular and interstitial inflammation like hypersensitivity reactions are the leading cause
of 15% of cases of acute renal failure. (Guo et al., 2002; Decloedt et al., 2011). Nephrotoxicity
is more prevalent in those patients who are already suffering from renal impairment. Incidence
of ARF due to the drugs is as high as 18.3% (Kleinknecht et al. 1987). Antibiotics such as
aminoglycosides induced nephrotoxicity cases are 36% of the total cases (Kaloyandes et al.,
2001). There are a lot of antibiotics which play an important role in the development of
nephrotoxicity because they are mainly excreted by the kidney so their proper dose adjustment
is necessary. Antibiotics causes nephrotoxicity are tetracycline, gentamicin, amikacin,
kanamycin, neomycin, streptomycin, sulphadiazine, trimethoprim, rifampicin and
amphotericin B. The mechanism of action of aminoglycosides is that they causes epithelial cell
damage which causes reduction in GFR that leads to tubular lumen obstruction. Glomerular
filterate accumulate across damaged epithelium and aminoglycosides collects in proximal
convulated tubules in cells which causes kidney cells damage and hence induced
nephrotoxicity. The nephrotoxic potential can be graded in such manner neomycin >
gentamicin > tobramycin (Hu et al., 1996).

1.1.5 Role of Immunosuppressive agents and other classes of drugs in Nephrotoxicity:

Cyclosporine A and tacrolimus (FK 506) are the common immunosuppressive agents
predominantly produce the nephrotoxic effects. Cyclosporine A produce the nephrotoxic
effects by acting at the several sites in the kidney and also causes damage to endothelial and
tubular cells (Liano et al., 1996). Endothelial cell injury results in to the increased vascular
permeability and hypovolemia which ultimately activates the sympathetic nervous system and
increase the endothelin and thromboxane A2 and causes decrease in concentration of nitric
oxide and vasodilatory prostaglandins (PG) (Mohana et al., 2012). Cyclosporine sensitize the
vasculature, activates the renin-angiotensin system which produce the vasoconstriction and
ultimately leads towards the hypertension and due to vasoconstriction, decrease in GFR occur
(Figure 01). Other classes of the drugs causes nephrotoxicity by reducing the GFR by more
than one mechanism either alone or in combination. The toxicants biotransformed in to the
reactive moiety and produces the reactive oxygen species (ROS) in the susceptible cells and
 5

ultimately results into the oxidative damage and cell injury (Figure 02). Other drugs include
Anticancer drugs (Adriamycin, cisplatin, methotrexate), antiviral agents (acyclovir, cidovir),
vasoactive agents (nonsteroidal anti-inflammatory drugs (NSAIDS) angiotensin-converting
enzyme inhibitors (captopril, enalopril) heavy metals (cadmium, gold, mercury) other drugs are
acetaminophen, halothane, methoxyflurane, cimetidine and hydralazine (Sarumathy et al.,
2011). Advancement of nephrotoxicity includes the antioxidant defense system against injuries
created by reactive oxygen species (Okokon et al., 2011). Antioxidants can be categorized in
3 groups vitamins antioxidants, enzymatic antioxidants and miscellaneous antioxidants.

1.1.6 Nephrotoxicity management:

Nephrotoxicity can be managed by taking such steps like dialysis. Fluid volume should be
replaced by adjusting the doses of nephrotoxic drugs. In the case of acute interstitial nephritis,
steroids should be administered. If a drug shows some symptoms of nephrotoxicity try to avoid
its next exposure. If the cause of nephrotoxicity is not confirm, seize the use of all the drugs
(Sarumathy et al., 2011). Therefore medicinal plant was selected for evaluation of its effect in
present study. For the evaluation of nephroprotective effect, in the present study the Cuscuta
reflexa plant has been selected on the basis of its traditional use.
 6

Figure 01: Role of immunosuppressive agents in Nephrotoxicity (Adopted from Rick G. Schnellmann
and Katrina J. Kelly)

Figure 02: Mechanism of nephrotoxicity caused by other classes of drugs

 7

1.2 Arthritis

It is a combination of two Greek words “Arthro” mean joints and “Itis” mean inflammation,
Inflammation of one or more joints in body is usually termed as Arthritis.
According to the US national library of medicine, it is stated that if you are facing some trouble
in moving around or feeling some stiffness or pain in the body, you could have arthritis.
Arthritis in most cases causes swelling and pain in joints. There are more than 100 different
types of arthritis (Macon et al., 2012) among them osteoarthritis and rheumatoid arthritis (RA)
are most common. Other forms are the psoriatic arthritis, Lupus, Gout and related autoimmune
diseases. Persistent joint synovial fluid inflammation is termed as the rheumatoid arthritis
(Kalla et al., 2003).

1.2.1 Types of Arthritis

Osteoarthritis is the most common form of the arthritis and it occurs in any joint of the body
(Bridges et al., 1992) but most often affects the hands and weight-bearing joints such as the
knee, hip and facet joints in the spine (Brandt et al., 2009). Pain is the principal symptom of
osteoarthritis which aggravates during activity but relieves during rest, other are stiffness in
the joints after long period of inactivity and swelling in the joints. Rheumatoid arthritis (RA)
is a long lasting disease as compared to that of osteoarthritis and it effects all joints of the body
except lower back and it is systemic inflammatory disorder (Efthimiou et al., 2010). Hallmark
of RA is persistent symmetric polyarthritis that affects the hands and feet, muscle aches and
pain. It especially effects the joints of hands, wrist, feet, knees etc. Lupus arthritis presented
with collagen vascular disorder is usually termed as lupus. Clinical presentation includes
constant joint pain, skin rash, kidney disorder, lung fibrosis etc. Gouty arthritis is a painful
condition characterized by the deposition of uric acid crystals in the joints that ultimately
causes the inflammation. It most often effects the wrist, knee and big toe joints (Chen et al.,
2008).

.
 8

1.2.2 Pathophysiology of Rheumatoid Arthritis

Rheumatoid arthritis is a long lasting disease as compared to that of osteoarthritis and joint
destruction occurs in a cascade manner that starts by an "Autoimmune reaction" and ends by joint
destruction. Anti-citrullinated protein antibodies (ACPA) are produced early by plasma cells
during the preclinical phase of rheumatoid arthritis. ACPA can stimulate osteoclast
differentiation and lead to initial bone loss (Boldt et al., 2012). These early changes may initiate
in the bone marrow adjacent to the joint. Synovitis at the onset of clinical disease leads to
production of cytokines, which stimulate osteoclast genesis by inducing expression of
Receptor activator of nuclear factor κB ligand, and synergize with Receptor activator of
nuclear factor κB ligand to enhance bone erosion (Figure 03 & Figure 04).
Complication of RA are osteoporosis, carpal tunnel syndrome, heart problems and lung
diseases. However other complication are shortness of breath, enlarged spleen reduce the
number of red blood cells (anemia), Sjögren's syndrome and sclerotic (Hurkmans et al., 2009).

1.2.3 Medical treatment of Rheumatoid Arthritis

Treatment of rheumatoid arthritis is most successful when there is close cooperation between
the doctor, patient, and family members. Regular exercise and the daily physical activities are
usually recommended to the patients of RA (Fiona, 2013).
Two classes of medications are used in treating rheumatoid arthritis:
 Fast-acting "first-line drugs”: The first-line drugs are used to reduce pain and
inflammation (Scott et al., 2010)
 Slow-acting "second-line drugs": The slow-acting second-line drugs promote disease
remission and prevent progressive joint destruction.
 9

Figure 03: Pathophysiology of Rheumatoid arthritis

Figure 04: Established RA is characterized by the presence of large bone erosions filled with inflamed,
synovial derived pannus tissues.
 10

 Fast-acting "first-line drugs’’:

Drugs included in the first line drugs include; NSAID’s (Tarp et al., 2012) acetylsalicylate
(aspirin), naproxen (Naprosyn), ibuprofen (Advil, Medipren, Motrin), etodolac (Lodine) ) and
Corticosteroid medications. Most common side effects of NSAID’s include the abdominal
pain, stomach pain, stomach ulcer etc. to prevent or cure these side effects usually following
drugs are used (Radner et al., 2012, McCormack, et al., 2011) sucralfate (Carafate), proton-
pump inhibitors (Prevacid and others) and misoprostol (Cytotec). Newer NSAIDs include
selective Cox-2 inhibitors, such as celecoxib (Celebrex), which offer anti-inflammatory effects
with less risk of stomach irritation and bleeding risk (Chen et al., 2008) Corticosteroids are
more potent then the NSAID,s.

 Slow-acting "second-line drugs”:

Slow-acting "second-line drugs" are known as the disease-modifying anti-rheumatic drugs or

DMARDs (Deighton et al., 2009). First line drugs only reduces the pain or inflammation, they
do not necessarily prevent joint destruction or deformity.
These "second-line" or "slow-acting" medicines may take weeks to months to become
effective. They are in use from long period of time even years at varying doses. If maximally
effective, DMARDs can promote remission, thereby retarding the progression of joint
destruction and deformity (Scott et al., 2010). Sometimes a number of DMARD second-line
medications are used together as combination therapy. As with the first-line medications, the
doctor may need to try different second-line medications before treatment is optimal. Drugs
categorized under the second line drugs are; Hydroxychloroquine (Plaquenil), Sulfasalazine
(Azulfidine), Methotrexate (Wasserman et al., 2011), Azulfidine is used to treat rheumatoid
arthritis in combination with anti-inflammatory medications, Gold salts, D-penicillamine
(Depen, Cuprimine), cyclophosphamide (Cytoxan), chlorambucil (Leukeran), cyclosporine
(Sandimmune) Newer "second-line" drugs (DMARDs) for the treatment of rheumatoid
arthritis include; leflunomide (Arava) and "biologic" medications etanercept (Enbrel),
infliximab (Remicade), anakinra (Kineret), adalimumab (Humira), rituximab (Rituxan),
 11

abatacept (Orencia), golimumab (Simponi), certolizumab pegol (Cimzia), tocilizumab

(Actemra), and tofacitinib (Xeljanz) (Edwards et al., 2004).

1.3 Ethnomedicine- A new focus

Based on the theories and beliefs of different cultures, we have collection of knowledge, skills
and practices, helps in the maintenance of health as well as in prevention, diagnosis,
improvement or treatment of physical and mental illness (Mahmood et al., 2011).During the
last five years, more than 13000 plants have been studied (Dahanukar et al., 2000), and all over
the world the focus on plant research has increased.
Due to the easy and continuous availability (Bhattarai et al., 1989), better compatibility
(Kamboj et al., 2000) and cost effectiveness of the herbal medicines, they are more preferred
over the conventional drugs. In many social settings the herbal medicines processes more
significance over the conventional drugs because the herbal medicines have less potential of
toxicity and side effects (Khanna et al., 1986), higher safety and improved efficacy. Pakistan
is also gifted with rich source of herbal medicinal plants. The variety of climate, multiple type
of soil conditions, variety in ecological regions and close contact of people with the natural
environment have brought the region of the people close to the medicinal impact of the herbs
of this area. A study shows that Pakistan has about 6 thousands wild plant species among which
only 400-600 plants are in practice of the physicians. Many new discoveries have been made
with the passage of time in the best interest of humanity to provide them relief from various
diseases and sufferings (Shinwari et al., 2011). This study is in series of this great line of
identification of the plants and application of their medicinal effect on human beings. It focuses
on the evaluation of the nephroprotective activity and anti-arthritic activity of selected
medicinal plant Cuscuta reflexa.

1.4 Plant Used for pharmacological activities

Cuscuta reflexa (also known as Amar bail) is commonly found in the plains of India and
Pakistan. In different languages it have different names.
 12

1.4.1 Morphology of plant:

Cuscuta reflexa Roxb. is obligate twining climber holoparasite (Bleischwitz et al., 2010). The
seeds of Cuscuta reflexa are also very important and used for different diseases traditionally
in china where they are called tu si zi (Vijikumar et al., 2011). The seeds of Cuscuta reflexa
are without chlorophyll and that’s the reason the plant of Cuscuta reflexa is unable to prepare
its own food so they depend on the host plant for its food (Khan et al., 2010). However in
studies of some areas shows that Cuscuta reflexa has little amount of chlorophyll so some
photosynthesis may occur as shown in the figure (Figure 05 & Figure 06). Filamentous stem
of Cuscuta reflexa grow near the host plant so that it can prepare the food because of the
absence of chlorophyll in the plant. The transfer of inorganic substance and liquid material
between parasitic plant and host occur through xylem connections however the organic
substances are moved through phloem connections (Chatterjee et al., 2010).
 13

Figure 05: Yellowish stem of Cuscuta reflexa on host plant (Gupta et al., 2010)

Figure 06: Cuscuta reflexa whole plant on host plant (Katiyar et al., 2012)
 14

1.4.2 Description of plant:

Kingdom----------------------Plantae.
Sub-kingdom--------------------Tracheobionta
Super Division-------------------spermatophyta
Division---------------------------Angiosperma
Class ------------------------------Eudicots
Subclass--------------------------Asterids
Order----------------------------Solanales
Family--------------------------Cuscutaceae
Genus ---------------------------Cuscuta
Species---------------------------reflexa (Vijikumar et al., 2011)

1.4.3 Traditional Names:

Hindi---------------------------------Amrabela
Bengali-------------------------------Akashbel
Urdu----------------------------------Akasbel
Sanskrit ------------------------------Ammravalli
Tamil----------------------------------Verillakothan
Punjabi--------------------------------Zarbut
English--------------------------------Dodder Plant
Malayalam--------------------------Moodillathali
(Vijikumar et al., 2011)
 15

1.4.4 Importance of Cuscuta reflexa Roxb. in Unani medicinal system:

Cuscuta sp. consists of almost 100 to 170 species all over the world (Nikam et al., 2014). In
unani medicinal system it is named as Aftimmoon and kasoos while in Ayurveda it is known
as ammara villi (Aslam et al., 2013). Unani scholars used Cuscuta reflexa in several
neurological, psychological and skin disorders (Rampratap et al., 2010). Therapeutic actions
which are observed from this plant are that it have antimicrobial (Inamdar et al., 2011),
anticonvulsant (Borole et al., 2011), diaphoretic, cardio tonic (Shikha et al., 2013), antiseptic,
hepatoprotective (Balakrishnan et al., 2010) purgative, antioxidant (Sakshy et al., 2010)
diuretic and anti-bacterial and induced alopecia (Pandit et al., 2008) properties. These actions
observed during the trials of unani medicines so this plant is of great value n unani medicinal
system. Cuscuta reflexa is also used as medicine of choice for black bile (which occur by the
derangement of sauda). Moreover in unani medicinal system Cuscuta reflexa is not only used
as single medicine for variety of diseases but also used as important compound for several
unani formulations so it increases its action spectrum. Its important medicinal actions are that
it act as demulcent, carminative, anthelmintic, resolving and deosorbent. However
therapeutically it is used in the treatment of worm in festation, epilepsy, jaundice, facial palsy
and inflammation (Aslam et al., 2013).
Above mentions uses and actions are purely worked done in unani medicinal system where
different formulations of this plant are available. Some of these formulation are made of from
single plant while in some unani formulation the plant is compounded with several other
medicinal plants.

1.4.5 Reported Phytochemistry of Cuscuta reflexa Roxb. :

Various climbing parasitic plants contains different phytochemical. Out of these

phytochemicals flavonoids and triterpinoid are very useful and have great medicinal value.
Flavonoids, which are largest naturally occurring phenols, found in Free State as well as in
bound form as glycoside. Its contains three acetate and one phenyl propane unit (Fawzy et al.
2008)
 16

However different researches shows that Cuscuta reflexa have chemical like cuscutin (Shikha
et al., 2013), quercitin, kaempferol, sigma sterol, beta sterol, berginin (Patel et al., 2012),
amabelin, duicitol, myrcitin, kaempferol-3-o glycoside, Astragalin, benzopyrones,
glucopyranoside, myrcitin, procainamide, beta sitosterol, flavonols, qurcetin-3-o glycoside.
There are nine such phytochemical which are firstly analyzed from genus Cuscuta namely
coumarin, alpha amyrin, beta amyrin, alpha amyrin acetate, beta amyrin acetate, oleanolic acid,
oleanolic acetate lupeol and sigma sterol (Sakshy et al., 2010).
Following four chemical have great value however due to structural difference they have
different polarities (Figure 07). The structures of these phytoconstituents have been made by
their spectral data analysis (Shailajan et al., 2011).
Lupeol, phytochemical found in Cuscuta reflexa, have complicated pharmacology in human
model as it also have anti-protozoal, antimicrobial, anti-inflammatory, anti-tumor and
chemoproptective properties (Gallo et al., 2010).
 17

Table 01: Important phytochemicals of Cuscuta reflexa and their action

Important Phytochemical found in Actions

Cuscuta reflexa
Quercitin Anti-allergic (Olszanecki et al., 2008)
Anti-Inflammatory (Katiyar et al., 2012)
Antitumor (Kim et al., 2008)
Immunomodulatoy (Bischoff et al., 2008 ,
Abdel-Raheem et al., 2009)

Kaempferol Anti-diabetic (Fawzy et al., 2008)

Anticancer (Huang et al., 2010)
Beta sito sterol Anticarcingenic (Choi et al., 2002)
Anticancer (Imanka et al., 2008)

Lupeol Anti-malarial (Fotie et al., 2006)

Hepatoprotective (Prasad et al., 2008)
 18

Figure 07: Structural picture of phytochemical found in Cuscuta reflexa (Shailajan et al.,
2011)
 19

1.5 Justification of Research work:

Nephrotoxicity is worldwide problem we are facing these days and no specific medicine is
available in the market for the treatment and prevention of acute renal failure so the precious
life of millions of renal patients are under threat. The present research aim and focus to work
on this issue from medicinal perspective to find out an effective drug to treat drug induced
nephrotoxicity. Nephrotoxicity is actually renal disorder or dysfunction that occur as direct or indirect
result of exposure to environmental and industrial chemicals or any drug which is the most common
problem in Pakistan. (Asangansi et al., 2005). Another very common disease in Pakistan is
rheumatoid arthritis which is systemic inflammatory disease of joints causes functional disability and
hence reducing the life quality. Current therapies includes the use of DMARD shows marked effect on
the arthritic patient as compared to NSAIDs but they are not cost effective for the patient. However on
the other hand non-selective NSAIDs cause several side effects which limits their use in rheumatoid
arthritis. Another class of drugs introduces is “biologics” which have prominent effect in reducing the
joint inflammation and swelling and but it is also very expensive and causes life threatening infection.
So all current therapies have certain shortcomings due to which use of herbal medicines and medicinal
plants is increasing day by day. Traditional users used medicinal plants for nephrotoxicity and RA.
Cuscuta reflexa has been used commonly in the Pakistan, India and Bangladesh and known as
miracle plant due to their effect in different conditions. Literature of Scientific research shows
that it may be used as hepatoprotective agent, diuretic, antitumor agent, anti-inflammatory and
antioxidant. However from centuries traditional users are using this plant in inflammation,
arthritis and some other conditions. Phytochemical analysis shows the presence of flavonoids
and phenolic phytochemical analysis of Cuscuta reflexa shows the presence of flavonoids and
phenolic which play very important role in reducing the nephrotoxicity. Cuscuta reflexa has
also been used in the rheumatoid arthritis among the traditional user however no scientific
work was done in the past to check the effect of plant in this disease. Therefore Cuscuta reflexa
was selected for scientific evaluation of its traditional uses for the treatment of RA and
nephrotoxicity.
 20

1.6 Aims and objectives of present study

Since centuries various parts of the Cuscuta reflexa have been used empirically used by
traditional herbal practitioners for treatment of various diseases like alopecia, liver diseases,
and hemiplegia and as diuretic. The present investigation has been conducted to evaluate the
nephroprotective and anti-arthritic activities of Cuscuta reflexa in various in-vitro and in-vivo
experiments using animal models and phytochemical analysis of plant was also performed. All
these experiments were conducted with the following aims and objectives:

1. In-vivo studies were conducted to scientifically validate the alleged traditional use
of selected plant in the treatment of renal diseases and in arthritis .
2. In-vitro studies to explore the effect of Cuscuta reflexa in inhibition of protein
denaturation.
3. Phytochemical studies to assess and characterize active phytoconstituents of selected
plant in aqueous-methanolic extract.
 21

SECTION II
2. MATERIALS AND METHOD

2.1 Chemicals and material used:

For performing pharmacological activities different chemical and materials have been used.

2.1.1 Chemicals used for the extraction of plants

 Methanol (RCI Labscan Limited, Bangkok, Thailand)

 Distilled water

2.1.2 Chemicals used for phytochemical screening

 Xylene (Sigma Aldrich Laborchemikalien GmbH, Seelze)

 Paraffin ( Merck, Germany)
 Sodium hydroxide (Riedel-de-haen,USA)
 Chloroform (Sigma Aldrich Laborchemikalien GmbH, Seelze)
 Sulfuric acid (Merck, Germany)
 Ammonia (Riedel-de-haen,USA)
 Ferrous chloride (Sigma Aldrich Laborchemikalien GmbH, Seelze)
 Mayer’s reagent (Sigma Aldrich Laborchemikalien GmbH, Seelze)
 Degendroff solution (Sigma Aldrich Laborchemikalien GmbH, Seelze)

2.1.3 Chemicals used for making buffer solution

 Disodium hydrogen phosphate (Merck, Germany)

 Sodium chloride (Sigma-Aldrich, USA)
 Potassium dihydrogen phosphate (Riedel-de-haen,USA)
 Hydrochloric acid (Riedel-de-haen,USA)
 Potassium chloride (Sigma Aldrich Laborchemikalien GmbH, Seelze
 22

2.1.4 Chemicals and drugs used in in-vivo experiments

 Formalin (VWR, International Ltd, Poole, England)

 Turpentine oil (UNI-CHEM, Germany)
 Gentamicin sulphate 80mg/2ml ( Novartis Pharmaceutical Ltd)
 Gentamicin sulphate 40mg/ml (Novartis Pharmaceutical Ltd)
 Methanol (Riedel-de-haen, USA)
 DMSO (Sigma Aldrich Laborchemikalien GmbH, Seelze)

2.1.5 Chemicals and drugs used in in-vitro experiments

 Diclofenac sodium (Sigma-Aldrich, USA)

 Aspirin (Sigma Aldrich Laborchemikalien GmbH, Seelze)
 Bovine serum albumin (Sigma-Aldrich, USA)
 Egg albumin from fresh hen’s egg

2.2 Equipment used:

 Incubator
 Light microscope (Olympus optical Co. Tokyo, Japan)
 UV-Visible Spectrophotometer
 Blood collection tubes
 Electrical grinder
 Vortex mixer (PCSIR)
 Rotary evaporator (Stuart re300b)
 Electrical weighing balance (Ar2140)
 Sami-automatic chemistry analyser BA-88 (Mindry)
 Centrifuge machine (Sigma)
 Oven (Memert)
 HPLC with UV-visible detector
 Digital Vernier caliper
 23

 pH meter
 Microscope
 Chemical kits for estimation of BUN, Serum urea, Uric acid and creatinine by Human
diagnostics

2.3 Apparatus used:

 Beaker (100ml, 1000ml)
 Measuring cylinder (100ml, 1000ml)
 Weighing balance
 Stirrer
 Round bottom flask
 Funnel
 Whatmann filter paper (44mm)
 Syringe filter (0.22μm)
 Eppendorf tubes
 Test tubes
 Test tubes stand
 Pipette (1ml, 5ml,10ml)
 Iron stand
 Burner
 Petri dishes
 Disposable Syringe (1cc)
 Nasogastric tube
 Gloves
 24

2.4 Experimental Animals used:

The animals used for in-vivo analysis were Sprague dawley rats of either sex male and
female. All the experimental animals were provided recommended national and housing
conditions as guided by National Research Council USA. Animals were placed in ideal
conditions of animal house by maintaining the temperature 25 ℃ ± 5 ℃ for seven days
(Katiyar et al., 2012).

2.5 Methodology:

2.5.1 Selection of plant material:

Plant selection was made on available literature review regarding traditional scientific work.
Therefore such plant was selected that has been traditionally used in inflammatory disorders
and other disorders like liver and renal diseases.

2.5.2 Collection and authentication of plant:

Plant Collection and Identifications was the first step. Cuscuta reflexa is twining parasitic herb
whose stems were growing on the plant of zizphus sp. were collected from the local region of
District Faisalabad. The plant was identified by Dr. Mansoor Ahmed, Department of
taxonomy, University of Agriculture, Faisalabad. Voucher specimens of this plant was
deposited under the voucher number 225-1 respectively in Faculty of Pharmacy, University of
Sargodha for future reference.

2.5.3 Preparation of Plant Extract:

Second step was washing and shade drying of plant. The plant were collected from the aerial
part of other plant so it was separated and properly washed with water. After washing the plant
was kept under shade for few days until it was completely dried and ready for use. Grinding
 25

and storage was third step in which plant which was dried under shade were firstly chopped
and grinded in motor and pestle and then in the electrical grinder to get fine powder (Gilani et
al., 2005). After grinding, powdered material was stored in amber color glass container. Then
grounded plant materials were soaked in aqueous-methanolic mixture (70:30) for 7 days at
room temperature (Katiyar et al., 2012). After seven days of occasional shaking, the soaked
material was filtered through muslin cloth to separate the solvent. The filtrate was filtered again
through Whatmann filter paper no. 1 to separate the tiny particles of macerated material. After
filtration, the solvent removed by using the rotary evaporator at the temperature below 50
Celsius, into a solid extract which was then stored in glass container to avoid leaching.

2.5.4 Preparation of stock solution of drug for in-vitro studies:

In-vitro studies were performed to determine the anti-arthritic effect of aqueous methanolic
extract of Cuscuta reflexa (AMECR) by using the method of protein denaturation. For this
purpose stock solution of different concentrations 25, 50, 100, 200, 400, 800 microgram/ml
were prepared. The highest concentration that is 800 microgram/ml was prepared by taking the
20 mg of the AMECR in 25 ml of the methanol (solution in the extract is 100 % dissolved) and
then 12.5 ml from above solution was taken and 12.5 ml of the distilled water was added to
make 400 µg/ml. after this 12.5 ml from the solution was taken to prepare the 200 µg/ml by
adding the 12.5 ml of water and so on.

2.6 Analysis on Plant extract

Aqueous methanolic extract of Cuscuta reflexa (AMECR) was checked by performing

different analysis.

2.6.1 Solubility analysis for extract:

In order to check the solubility of the extract, solubility analysis was conducted in distilled
water, methanol, olive oil, n-hexane and 5 % DMSO. An equal amount of extract (which was
up to 0.2 gm) was weighed on digital weighing balance and taken in different eppendorf tubes.
 26

Now one ml of each solvent was poured into eppendorf tubes and shaken vigorously and then
these tubes was also shaken on vortex mixture for 2-4 mins. After that solution was allowed to
settle down and solubility was determined by observing for the presence of any sediment
material. Solubility study shows that extract was sparingly soluble in distilled water however
it was completely soluble in methanol and also sparingly soluble in 5 % DMSO solution.
However extract was completely insoluble in other solvents.

2.6.2 Preliminary Phytochemical analysis:

After the solubility analysis of the plant extract, preliminary phytochemical analysis was done
to determine the important phytochemical to determine that which phytochemical was
responsible for required pharmacological activity. For this purpose standard procedure was
performed which are given below in Table no. 02.
Note: An equal amount of extract was used for each procedure that is 0.2 gm.

2.6.3 HPLC Analysis of the extract:

First of all we have taken 50 mg of extract and then added 16 ml of deionized distilled water
and 24 ml of methanol (HPLC grade). Now the solution was shaken for five minutes and then
added 10 ml of 6 Molar HCl and kept in oven at 90 degree Celsius for 2 hours. Now it was
filtered through micron filter of 0.2-0.4 microns. Now sample is prepared to run in to HPLC.
Separation of desired phytochemicals from hydroalcholic extract of plant is now analyzed in
HPLC for determining flavonoid and phenolic compound by using ultraviolet visible detector.
Shim-pack CLC-ODS (C-18) columns of dimensions 25cm X 4.6mm was used. Mobile phase
consist of solution A and B and their composition was:
A: (H2O: AA (acetic acid) -94:6, of pH=2.27
B: CAN (Acetonitrile 100%)
The mobile phase was moved through column at the rate of 1 ml/min for ten minutes. The peak
was observed at 280 nm using UV visible detector. The peaks of standard samples were
compared afterwards. To check out the peaks and retention time for samples.
(Sheth et al., 2012).
 27

Table 02: Methods for phytochemical screening of different classes of phytoconstituents in

Cuscuta reflexa.

Name of
Method Appearance Result
Phytoconstituents

0.2 gm extract +5 ml Formation of yellow Shows presences of

Flavonoids NaOH + 5ml diluted HCl color that disappear Flavonoid.
Investigation after some time (Roopashree et al,
2008)
0.2 gm extract +5 ml Stable foam formed Shows presences of
distilled water → heated Saponins.
Saponins on boiling water (Roopashree et al,
2008)

0.2gm extract was Formation of layer Shows presences of

dissolved in 2ml was observed. Terpenes.
chloroform+3 ml conc. Reddish brown liquid (Tadesse et al, 2012)
Terpinoids
Sulfuric acid. The was formed.
mixture was added with
care.
0.2 gm extract was Rose pink color Shows presences of
filtered after boiling it appeared. Anthraquinones.
with hydrochloric acid (Tadesse et al, 2012)
(10 %) for 4-5 min. Now
allow the solution to cool
Anthraquinones
and then add equal
amount of chloroform.
The mixture was heated
after adding some drops
of 10 % NH3.
 28

0.2 gm of the extract was Solution in both Shows presences of

heated with 5 ml 2N portions becomes Alkaloidal contents.
hydrochloric acid. When turbid. (Tadesse et al, 2012)
it attains the room
temperature the filterate
Alkaloidal divide into two portions.
Few drops of Mayer’s
reagent was added to one
of them. Two drops of
Degendroff solution was
added in the next portion.
0.2 gm of extract Dark green color Shows presences of
dissolved in 10 ml of appears. Tanins.
distilled water and heated (Abbas et al, 2012)
Tanins on boiling water bath.
Investigation After boiling mixture
was filtered and 5 %
(w/V) FeCl3 was added
to filterate.
 29

2.7 Pharmacological studies made on plant extract:

Formaldehyde-
induced arthritis
In-vivo studies
Turpentine oil
induced arthritis
Anti-arthritic
activity Protein
denaturation by
bovine serum
Pharmacological albumin
In-vitro studies
activities of Protein
Cuscuta reflexa to denaturation by
be performed fresh hen's egg
albumin

Gentamicin
Nephroprotective
In-vivo studies induced
activity
nephrotoxicity

Figure 08: Research protocol of current studies.

 30

2.7.1 Evaluation of Nephroprotective effect of Cuscuta reflexa Roxb. on gentamicin

induced nephrotoxicity in rats:

Evaluation of Nephroprotective potential of AMECR against gentamicin induced

nephrotoxicity in rats. The experimental procedure was as follows that twenty five Sprague
dawley rats of either sex were arranged from University of Agriculture, Faisalabad in the
month of September. After seven days animals were divided into total five groups.
These groups were named as:
 Group one: Control Group (given routine diet)
 Group two: Toxic Control Group (receive Gentamicin 100mg/kg)
 Group three: Gentamicin plus Aqueous-methanolic Extract treated group (given dose
of 200 mg/kg)
 Group four: Extract treated group (given dose of 400 mg/kg)
 Group five: Extract treated group (given dose of 600 mg/kg)
After dose adjustment Injection gentamicin sulphate (40mg/ml) and 80mg/2ml manufactured
by Novartis Pharmaceuticals were given to the rats intraperitonealy for eight consecutive days.
Gentamicin was given at the rate of 100 mg /kg body weight for producing nephrotoxicity in
rats which is approved dose for this purpose (Lakshmi et al., 2010).

2.7.1.1 Drug administration protocol:

Drug was administered according to following protocol:

Group one: Normal control: routine diet plus normal saline for eight consecutive day.
Group two: Toxic Control: Routine diet plus gentamicin (100 mg/kg day)
Group three: Treated group: Routine diet plus gentamicin (100mg/kg/ day) then after
two hour aqueous-methanolic (70-30) extract of plant with the dose of 200 mg/kg/day
dissolve in methanol solution for eight consecutive days:
Group four: Treated group: Routine diet plus gentamicin (100mg/kg/ day) then after
two hour aqueous-methanolic (70-30) Extract of plant with the dose of 400 mg/kg/day
dissolve in methanol solution for eight consecutive days:
 31

Group five: Treated group: Routine diet Plus gentamicin (100mg/kg/ day) then after
two hour methanol: water Extract of plant with the dose of 600 mg/kg/day dissolve in
methanol solution for eight consecutive days.

2.7.1.2 Sampling of Blood for Further Analysis:

After the completion of drug administration protocol, at the eight day rats were sacrificed
and blood was collected by reteroorbital technique. After collecting the blood it was
centrifuged at 370 RPM for ten minutes and then refrigerated at -20 ℃ for biochemical analysis
of blood.

2.7.1.3 Histopathological examination:

The kidneys were weighed using digital electronic balance and to avoid any changes kidneys
were fixed in 10 percent neutral formalin immediately after sacrificing the animal. (Preethi et
al., 2009). After dehydrating kidneys in graded alcohol they were embedded in paraffin. The
thickness of the section of kidney prepared were 5 𝜇m.
Now these sections were placed on glass slides using Mayer’s egg albumin as binder. Section
of kidney were stain with H&E stain and drop of DPX poured on the slide and covered with
cover slip. Now slides were placed in the oven for fixation. Light microscope of Olympus
optical Co. Tokyo Japan were used to examine slides.

2.7.2 Evaluation of Anti-arthritic effect of Cuscuta reflexa Roxb. :

2.7.2.1 In-vivo Anti-arthritic activity

Anti-arthritic activity was performed to determine the effect of Cuscuta reflexa in arthritis.
Evaluation of anti-arthritic activity of AMECR against formaldehyde induced and turpentine-
oil induced arthritis in rats was done. The experimental procedure as follows that fifty Sprague
dawley rats of either sex were arranged from agriculture university Faisalabad. All the animal
 32

was kept under standard condition in the animal house in neat and clean cages for seven day.
For this time period animals was given routine standard diet. After seven day animals were
divide into five group for each experiment. Each group contain five animal models (Singh et
al., 2010). Following study design was performed for the study of the effect AMECR.

2.7.2.1.1 Evaluation of anti-arthritic effect of Cuscuta reflexa on formaldehyde induced

arthritis in rats

 Group One: Arthritic Control: Given Formaldehyde (2%) only at 1st and 3rd day.
 Group two: Extract treated group: Given Formaldehyde (2%) 0.1 ml for first and third
day plus aqueous methanolic extract for eight days consecutively (200 mg/kg/day)
 Group three: Extract treated group: Given Formaldehyde (2%) 0.1 ml for first and
third day plus aqueous methanolic extract for eight days consecutively (400 mg/kg/day)
 Group four: Extract treated group: Given Formaldehyde (2%) 0.1 ml for first and
third day plus aqueous methanolic extract for eight days consecutively (600
mg/kg/day).
 Group five: Given Formaldehyde (2%) 0.1 ml for first and third day plus Aspirin
(standard drug).
All the groups receive formaldehyde after half an hour of administration of extract/ drug or
vehicle into the sub plantar surface. (Kumar et al., 2013). Arthritis was determined by
measuring the mean increase in the diameter of joint of animals for a period of ten days using
Vernier calipers.

2.7.2.1.2 Evaluation of anti-arthritic effect of Cuscuta reflexa on turpentine oil induced

arthritis in rats:

Five groups of animals was used in study. Animals were fasted over night before the
experiment. Groups follows drug as following way:
 Group one: Arthritic Control: Receive drug only at the rate of 0.02ml/kg/day
 Group two: Extract treated group: Receive Extract for one day. (200
mg/kg/day)
 33

 Group three: Extract treated group: Receive Extract for one day. (400
mg/kg/day)
 Group four: Extract treated group: Receive extract for one day. (600
mg/kg/day).
 Group five: Receive Aspirin 100 mg/kg body weight.
Thirty minutes after receiving the extract turpentine oil was given at the rate of 0.02 ml on the
knee joint to induce arthritis (Kumar et al., 2013).Evaluation of anti-arthritic activity was done
after one, two, three, four, five and sixth hour of injecting the turpentine oil.
For determination of the percentage inhibition of joint edema was measure by following
formula:
% inhibition = (𝑉𝐶 − 𝑉𝑇) × 100
VC
Where,
VC: paw/joint edema of control group
VT: paw/joint edema of test group

2.7.2.2 In-vitro Anti-arthritic Activity

These methods are used to evaluate effect of aqueous methanolic extract of Cuscuta reflexa.

2.7.2.2.1 Evaluation of anti-arthritic effect of Cuscuta reflexa on inhibition of protein

denaturation (Fresh hen’s egg albumin)

Reaction mixture (5 ml) was prepared having 0.2ml of fresh eggs albumin + 2.8 ml of PBS
(pH 6.3) plus 2 ml of varying concentration of aqueous-methanolic extract. Using this reaction
mixture final concentration are prepared that is of 25, 50, 100, 200, 400 and 800 µg/ml .Similar
volume of double-distilled water served as control. Reaction mixtures were incubated at
(37±2) ºC in a BOD incubator for not more than 15 min and then heated at specific temperature
that is 70 ºC for almost 5 min. After cooling, their absorbance was measured at 660 nm.
Diclofenac sodium was used as reference drug (Chandra et al., 2012). The percentage
inhibition of protein denaturation was calculated by using the following formula
 34

Percentage inhibition = (Abs control – Abs sample) X 100/ Abs control

2.7.2.2.2 Evaluation of anti-arthritic effect of Cuscuta reflexa on inhibition of protein

denaturation (bovine serum albumin)

Following procedure was followed for this method (Sheelarani et al., 2014) 5 % aqueous
Bovine serum albumin (BSA) was prepared by adding 5gm of BSA in 95ml of water.
Phosphate Buffer Saline of pH 6.3 was prepared 8 gm of NaCl + 0.2 gm of KCL +1.44 gm of
disodium hydrogen phosphate (Na2HPO4) + 0.25 gm of potassium dihydrogen phosphate
(KH2PO4) was dissolved in 800 ml of water: Test solution (0.5 ml) was prepared which consist
of 0.05ml of TS of various concentrations (25-800 µg/ml) plus 0.45 ml of BSA (5 % Aqueous
solution). Test control Solution (0.5ml) was prepared which consist of 0.05 ml of distilled
water and 0.45 ml of bovine serum albumin (BSA) (5 % aqueous solution). Product control
solution was prepared which consist of 0.05 ml of TS of different concentrations (25 to 800
µg/ml) plus 0.45 ml of distilled water. Now at the end Standard Solution (0.5ml) was prepared
which consist of 0.05 ml Diclofenac sodium (100, 250, 500, 1000 µg/ml) Plus 0.45 ml of BSA
(5 % Aqueous concentration). pH of all the solution was adjusted to 6.3 by the use of 1N
hydroalcholic acid (Sheelarani et al., 2014). Now all of above solutions were incubated at
specific temperature i.e, 37 celsius for almost twenty minutes and temperature was increased
progressively up to 57 celsius for three minutes only. Solution were allowed to stand for twenty
five minutes for cooling and then add 2.5 ml of phosphate buffer to the solution mentioned
above. Now all the solutions were ready for determination of their absorbance in UV visible
spectrophotometer. After buffering the resultant solutions absorbance was measured in UV
visible spectrophotometer at 416 nm. (Sheelarani et al., 2014).
The % age inhibition of protein denaturation was determined by caclcution of following
formula:
Percentage inhibition:
= 100 - Absorbance (O.D) of TS – Absorbance (O.D) of Product control solution * 100
Absorbance (O.D) of Test Control solution
 35

2.8 Statistical Analysis:

After collecting the data, Statistical analysis was performed using 2 way ANOVA followed by
Boneferroni posttest where p<0.005 was considered statistically significant.
 36

SECTION III

3. RESULTS

3.1 Solubility analysis of Aqueous Methanolic Extract Cuscuta reflexa:

Plant extract is the semi solid mass which is given to Experimental animal in liquid form.
Different extract have solubility in different solvents. For this purpose a solubility analysis was
performed to determine that in which solvent extract is soluble. Hydro alcoholic extracts are
usually completely soluble in distilled water, methanol and ethanol. The extract of Cuscuta
reflexa have different solubility in this regard. The extract of Cuscuta reflexa was sparingly
soluble in distilled water and ethanol but have solubility in methanol solution and 5 % DMSO
(Table 03).

3.2 Phytochemical Analysis of Aqueous Methanolic Extract Cuscuta reflexa:

The phytochemical analysis was performed to determine the phytoconstituents. Preliminary

experiments were performed to determine the classes of all the phytoconstituents. The results
of these investigation show the presence of Flavonoid, Glycosides and phenols (Table 04).
+: Minimum presence of chemical constituent
++: Normal presence of chemical constituent
+++: Maximum presence of chemical constituents
–: Absence of chemical constituent
 37

3.3 HPLC analysis of Aqueous Methanolic Extract Cuscuta reflexa:

The aqueous methanolic extract was run through high performance liquid chromatography.
HPLC analysis was done to determine the presences of Flavonoids and phenolic and
Triterpinoids. The peaks of all the phytoconstituents were compared with standards. All the
standard were purchased from sigma & co. UK. Retention time and quantity was also checked
and recorded for record. The HPLC analysis shows the presences of coumeric acid, quercitin,
gallic acid and other phenolic compounds (Table 05, Figure 09).
 38

Table 03: Solubility Analysis of Aqueous Methanolic Extract of Cuscuta reflexa using
different solvents

NAME OF List of solvents used

EXTRACT
Distilled Methanol Ethanol Ethanol: n-hexane DMSO
Water Water 5%
METHANOL: Sparingly Highly Sparingly Insoluble insoluble soluble
Water (70: 30) soluble soluble soluble

Table 04: Preliminary phytochemical analysis of Cuscuta reflexa

Sr. Name of Phytoconstituents Aqueous-

no. Methanolic Extract

1 Alkaloids ++

2 Flavonoids +++

3 Triterpinoids +++

4 Saponins +

5 Anthraquinones +

6 Tanins --

7 Glycosides ++
 39

Table 05: Preliminary HPLC Analysis of Cuscuta reflexa.

NAME of Retention Area Area Quantity

compound time
mV.s % Ppm
Quercitin 2.893 20.776 0.1 1.06

P-coumeric acid 17.280 73.255 2.8 0.94

Vanillic acid 13.007 128.1662 4.9 7.93

Gallic acid 4.160 23.380 0.9 2.82

Caffeic acid 11.887 37.435 1.4 1.78

Trans-4-hydrpxy- 24.787 302.780 11.6 1.05

3-methoxy
cinnamic acid

Figure 09: Peaks of Phytoconstituents in HPLC analysis

 40

3.4 Results of Nephroprotective Activity

3.4.1 Effect of aqueous methanolic extract of Cuscuta reflexa on biochemical parameters
of gentamicin induced nephrotoxic rats

Nephroprotective activity was determined by analysis the plant extract through different ways.
Biochemical, physical and histopathological parameters were used to determine the
nephroprotective effect of plant. Biochemical parameters are serum uric acid, serum urea, and
blood urea nitrogen and serum creatinine. All these parameters of five groups were determined
therefor serum urea, serum uric acid, blood urea nitrogen and serum creatinine level of normal
control, toxic control and extract treated groups (200mg/kg, 400mg/kg , 600mg/kg ) were
analyzed after sacrificing the sprague dawley on eighth day and also there blood sample was
taken. For analyzing the biochemical parameter specific kits were used under standard
conditions. Results of these parameters shows that extract of Cuscuta reflexa have prominent
Nephroprotective effect. However the dose of 400 mg/kg and 600 mg/kg have more prominent
effect then dose of 200mg/kg. Phytochemical analysis shows that extract of Cuscuta reflexa
contains such phytoconstituents which have nephroprotective potential (Table 06, Figure 10).
Normal values of these Parameters are as follows:
a. BUN 8-18 mg/dl
b. Serum Urea 10-50 mg/dl
c. Serum Uric Acid 0.6-1.1 mg/dl
d. Serum Creatinine M( 3-7) F(2.5-7.5)
 41

Table 06: Biochemical Parameters Serum Urea, Serum Creatinine, Blood Urea Nitrogen and
Serum Uric Acid

Biochemical Normal control Toxic control Gentamicin Gentamicin Gentamicin

Parameters plus AMECR Plus AMECR plus AMECR
200 mg/kg 400mg/kg 600mg/kg

Serum Urea

43.400±0.510*** 61.800±0.583*** 48.400±0.510*** 44.600±0.509*** 41.400±0.510***

Blood Urea
Nitrogen
18.397±0.088*** 34.593±0.547*** 24.946±0.659*** 22.896±0.257*** 18.370±0.328***

Serum
Creatinine 4.310±0.073*** 5.714±0.126*** 4.676±0.068*** 3.968±0.034*** 3.267±0.076***

Serum Uric
0.654±0.021*** 1.878±0.048** 0.909±0.013*** 0.865±0.017*** 0.740±0.032***
Acid

Values are expressed as ±SEM (n=5), by two way ANOVA and p˂0.05 was considered as
significant as compared to arthritic control where ***= p˂0.001.
 42

80
Serum urea
*** BUN
60
*** Serum Creatinin
***

mg/dl
*** Serum Uric acid
40 ***

***
***
***
20
***
*** *** ***
** *** *** ***
0

kg

kg

kg
NC

TC

g/

g/

g/
m

m
0

0
20

40

60
R

R
EC

EC

EC
M

M
+A

+A

+A
G

Groups

Figure 10. Effect of AMECR on serum creatinine, urea, Blood urea nitrogen (BUN) and serum
uric acid level of normal and gentamicin induced nephrotoxic rats.
NC: Normal control; TC: Toxic control (Gentamicin 100mg/kg/day); G+AMECR 200
mg/dl: Gentamicin 100mg/kg/day plus aqueous-methanolic extract of Cuscuta reflexa 200
mg/kg/day; G+AMECR 400 mg/dl: Gentamicin 100mg/kg/day plus aqueous-methanolic
extract of Cuscuta reflexa 400 mg/kg/day; G+AMECR 600 mg/dl: Gentamicin 100mg/kg/day
plus aqueous-methanolic extract of Cuscuta reflexa 600 mg/kg/day
 43

3.4.2 Effect of aqueous methanolic extract of Cuscuta reflexa on physical parameters in

gentamicin induced nephrotoxic rats.

All the animals used were weighed on day one and also after the intervals so that at the end
changes in their weight can be recorded. At the eighth day before scarifying the rats were
weighed again. The weight change was recorded and analyzed (Table 07, Figure 11).

3.4.3 Effect of aqueous methanolic extract of Cuscuta reflexa on histopathogical view in

gentamicin induced nephrotoxic rats:

Histopathological study was done to determine the Nephroprotective effect of aqueous

methanolic effect of plant. Histopathological findings showed the presence of tubular
degeneration, sever tubular interstitial infiltration and tubular necrosis providing the degree of
destruction of tubular cell damage. The picture of normal group shows normal epithelial cell
lining and zero degree of tubulointerstitial infiltration and normal parenchyma with normal
tubules and glomeruli. The picture of kidneys on treatment with Cuscuta reflexa show normal
tubules and glomeruli (Table 08, Figure 12 to Figure 16).
 44

Table 07: Weight of Kidney of Rats on treatment with extract of Cuscuta reflexa (Aqueous
methanolic extract)

Group of Animals Mean kidney weight

Normal control 0.544± 0.011

Toxic control 0.702± 0.009

G+AMECR 200 mg/kg 0.624± 0.009

G+AMECR 400 mg/kg 0.606± 0.009

G+AMECR 600 mg/kg 0.586± 0.005

0.8
Normal control
Toxic control
Kidney weight(g)

0.6
G+AMECR 200mg/kg
G+AMECR 400mg/kg
0.4 G+AMECR 600 mg/kg

0.2

0.0
ES
G
N
A
H
C
T
H
IG
E
W
EY
N
ID
K

Figure 11: Effect of AMECR on Kidney weight of animals from normal Gentamicin treated
groups
 45

Table 08: Histopathological analysis of effect of aqueous methanolic extract of Cuscuta

reflexa on animal model (Sprague dawley rats)

Groups Tubular Tubulo- Tubular necrosis

degeneration interstitial
infiltration TN

TD TIN

Normal Control NIL NIL NIL

Toxic control Severe Severe Moderate

Aqueous –
methanolic Moderate Mild Nil
extract 200mg/kg
Aqueous –
methanolic Mild Moderate Nil
extract 400mg/kg
Aqueous –
methanolic Mild Nil Nil
extract 600mg/kg
 46

Figure 12: Histopathological view of kidney of normal group shows normal renal parenchyma
along with normal tubules and normal glomeruli (NORMAL CONTROL)

Figure 13: The figure shows the histopathological view of Toxic control group that is dense
pinkish granulation. It is showing renal parenchyma with focal necrosis and lymphatics
infilterate
 47

Figure 14: The histopathological view of section of kidney given aqueous methanolic extract
of Cuscuta reflexa (200 mg/kg/ body weight) plus Gentamicin (100 mg/kg/body weight). Renal
parenchyma showing moderate to sever lymphatic infilterate.

Figure 15: The histopathological view of the section of kidney given aqueous methanolic
extract (400mg/kg/day) plus gentamicin (100 mg/kg/body weight)
 48

Figure 16: The histopathological view of section of kidney given aqueous-methanolic extract
of Cuscuta reflexa (600 mg/kg/body weight) plus gentamicin (100 mg/kg/ body weight) . Renal
parenchyma shows mild lymphatic infilterate.
 49

3.5 Results of In-vivo Anti-Arthritic activity:

3.5.1 Effect of aqueous methanolic extract of Cuscuta reflexa against formaldehyde
induced arthritis:
Arthritis was induced by formaldehyde (2%) in Sprague dawley rats and assessment was made
on tenth day after starting the experiment. Reading for determination of the extent of the
increase in joint size was started from day one. Reading was taken on after one hour of injection
of formaldehyde. Next dose of formaldehyde was given on the same time at third day. Reading
of this experiment shows effect of AMECR is very significant at maximum dose that is 600
mg/kg body weight as compared to minimum dose. The result of maximum dose (76.74 %)
was comparable to standard drug aspirin (77.32 %) which showed that results of this
experiment were very significant (p<0.001) (Table 09, Figure 17)

3.5.2 Effect of aqueous methanolic extract of Cuscuta reflexa against turpentine oil
induced arthritis:

Turpentine oil induced arthritis was one day study in which the arthritis was induced by
turpentine oil (0.02). Reading for determination of the extent of increase in joint diameter was
done at after one hour and then after every hour. After six hours the results showed that the
result of maximum dose of AMECR (71.22 %) was comparable to the standard drug aspirin
(71 %) as compared to result of minimum dose (66.57 %) which showed that results of this
experiment were very significant (p<0.001) (Table 10, Figure 18)
 50

Table 09: Effect of AMECR on formaldehyde induced arthritis in Sprague dawley rats

Treatment Increase in paw diameter (mm)

Group
On day 2 On day 4 On day 6 On day 8 On day 10

Athritic
Control 8.480±0.046 10.417±0.220 13.767±0.377 16.317±0.296 18.167±0.300
(3ml/kg)
Standard 5.050±0.050*** 5.667±0.060*** 4.800±0.153** 4.267±0.093*** 4.120±0.040***
Aspirin
(100ml/kg) (40.44 %) (45.59 %) (65.13 %) (73.84 %) (77.32%)
AMECR
7.167±0.101*** 6.767±0.101*** 5.800±0.760*** 5.400±0.870*** 5.250±0.290***
200mg/kg
(15.48 %) (35.03 %) (57.87 %) (66.90 %) (71.10 %)

AMECR
6.550±0.058*** 6.417±0.120*** 5.433±0.093*** 5.050±0.050*** 4.867±0.044***
400mg/kg
(22.75 %) (38.39 %) (60.53 %) (69.06 %) (73.20 %)

AMECR
6.250±0.058*** 6.050±0.029*** 5.067±0.44*** 4.690±0.097*** 4.225±0.025***
600mg/kg
(26.29 %) (41.92 %) (63.19 %) (71.25 %) (76.74 %)

Values are expressed as ±SEM (n=5), by two way ANOVA followed by Boneferroni test and
P˂0.05 was considered as significant as compared to arthritic control where ***= (very
significant) P˂0.001.
 51

Increase in joint diameter (mm)

20
AC
Standard Aspirin
15
AMECR 200 mg/kg
AMECR 400 mg/kg
10 AMECR 600mg/kg
***
*** *** **
*** *** ***
*** *** *** ***
*** ******
*** *** *** *** ***
5 ***

0
y
2
y
4
y
6
y
8 10
Da Da Da Da y
Da
Time

Figure 17: Effect of AMECR on formaldehyde induced arthritis in Sprague dawley rats.
 52

Table 10: Effect of AMECR on turpentine oil induced arthritis in Sprague dawley rats.

Treatment Increase in joint diameter (mm)

group

At first hour At 2nd hour At 3rd hour At 4th hour At 5th hour At 6th hour

Arthritic
control
8.893±0.087 13.013±0.373 16.433±0.262 17.917±0.203 19.000±0.126 21.083±0.484

Standard 6.843±0.047***
7.207±0.012*** 7.367±0.101*** 6.580±0.012*** 6.320±0.012*** 6.173±0.018***
Aspirin

(18.95 %) (59.95 %) (63.27 %) (66.73 %) (71 %)

(47.41 %)

AMECR
200mg/kg 7.770±0.067*** 7.747±0.039*** 7.543±0.015*** 7.367±0.018*** 7.230±0.026*** 7.047±0.032***
(12.62 %) (40.46 %) (54.09 %) (58.88 %) (61.94 %) (66.57 %)

AMECR
400mg/kg 7.430±0.015*** 7.327±0.09*** 7.243±0.022*** 7.090±0.017*** 6.913±0.019*** 6.743±0.043***
(16.45 %) (43.69 %) (55.92 %) (60.42 %) (63.61 %) (68.01 %)

AMECR
600mg/kg 6.993±0.060*** 6.667±0.056*** 6.453±0.019*** 6.243±0.022*** 6.120±0.015*** 6.067±0.01***
(21.36 %) (48.76 %) (60.73 %) (65.15 %) (67.70 %) (71.22 %)

Values are expressed as ±SEM (n=5), by two way ANOVA followed by Boneferroni test and
p˂0.05 was considered as significant as compared to arthritic control where ***= p˂0.001.
 53

Increase in joint diameter (mm)

25
AC
Standard Aspirin
20
AMECR 200 mg/kg
15 AMECR 400 mg/kg
***
AMECR 600 mg/kg
*** ***
10 *** ***
*** ***
*** *** *** *** *** ***
*** *** *** *** ***
*** *** *** ***
*** ***

0
r

ur

ur

ur
ur

ur
ou

ho

ho

ho

ho
ho
th

h
d
1s

3r

4t

5t

6t
2n

Time

Figure 18: Effect of AMECR on turpentine oil induced arthritis in Sprague dawley rats.
 54

3.6 Results of In-vitro Anti-arthritic studies

3.6.1 Effect of aqueous-methanolic extract of Cuscuta reflexa on protein denaturation

using Fresh hen’s egg albumin:

In-vitro studies was conducted to determine the effect of aqueous methanolic extract on protein
denaturation. Firstly solution of different concentrations (25, 50, 100, 200, 400, 800) of extract
and standard diclofenac sodium was prepared and then their absorbance was check at 416 nm
wave length in UV visible spectrophotometer. The result shows that the higher concentration
solution shows the maximum percentage inhibition in protein denaturation and its result is
comparable to that of standard solution.

3.6.2 Effect of aqueous-methanolic extract of Cuscuta reflexa on of protein denaturation

using bovine serum albumin:

In-vitro studies performed using the bovine serum albumin solution to check the effect of
AMECR on protein denaturation. For this purpose solution of different concentration of
AMECR and standard aspirin was prepared and their absorbance was checked at 416 nm
wavelength in UV visible spectrophotometer. Result shows that the higher concentration of the
AMECR shows the maximum percentage inhibition in protein denaturation which was
comparable to the standard aspirin as compared to lowest concentration of AMECR.
 55

Table 11: Effect of Cuscuta reflexa on protein (egg albumin) denaturation

Treatment Percentage inhibition of protein (egg albumin) denaturation

group 25 µg/ml 50 µg/ml 100 µg/ml 200 µg/ml 400 µg/ml 800
µg/ml
Diclofenac 77.420±0. 81.990±0. 85.390±0. 92.177±0. 96.413±0. 99.163±
Sodium 190 397 207 270 215 0.037

AMECR 61.633±0. 70.577±0. 78.417±0. 83.767±0. 87.357±0. 93.510±

109*** 183*** 132*** 113*** 204*** 0.140***

Values are expressed as ±SEM (n=3), by two way ANOVA followed by Boneferroni test and
p˂0.05 was considered as significant as compared to arthritic control where ***= p˂0.001.
protein (egg albumin) denaturation

100 ***
***
Percentage inhibition of

*** Standard Diclofenic sodium

***
80 AMECR
*** ***
60

40

20

0
25 50 10
0
20
0
40
0
80
0
Concentration (µg/ml)

Figure 19: Effect of AMECR on percentage inhibition of protein denaturation.

 56

Table 12: Effect of Cuscuta reflexa on protein (bovine serum albumin) denaturation.

Treatment Percentage inhibition of protein (BSA) denaturation

group
25 µg/ml 50 µg/ml 100 µg/ml 200 µg/ml 400 µg/ml 800
µg/ml
Standard 78.607±0. 84.480±0.26 87.590±0.031 91.353±0.095 94.580±0.142 99.123±0
Aspirin 680 3 .068

AMECR 67.417± 72.493± 78.470± 81.363± 85.417± 89.307±

0.083*** 0.209*** 0.133*** 0.110*** 0.090*** 0.553***

Values are expressed as ±SEM (n=3), by two way ANOVA followed by Boneferroni test and
p˂0.05 was considered as significant as compared to arthritic control where ***= p˂0.001.
protein (Bovine serum albumin) denaturation

100 ***
Percentage inhibition of

*** ***
Standard Aspirin
***
80 ***
AMECR
***

60

40

20

0
25

50

0
10

20

40

80

Concentration (µg/ml)

Figure 20: Effect of AMECR on percentage inhibition of protein denaturation

 57

SECTION IV
4. DISCUSSION
Since centuries medicinal plant have great importance among traditional healers. They used
plants for various ailments because plants have such constituents which have therapeutic effect
as well. The ethnobotanical studies revealed the medicinal effect of around 8000 plant’s
species (Murugeswaran et al., 2014). Therefore plants are essential element of human and
animal health. According to world health organization (WHO), the use of herbal medicine
gaining prominence among developing counties therefore 80 % of the world population is now
whirling towards herbal drugs to avoid the side effects of allopathic medicines (Alves, et al.,
2007) During the past few years, therapeutics benefits of more than 13,000 plants have been
inspected (Dahanukar et al., 2000). Herbal medicines depends upon the phytochemical
constituents of plants that uphold the health conditions and offer the cure from ailments with
lesser adverse effects (Alves, et al., 2007). The renal disorders are of rising health concern and
nephropathy is the most common disorder among these. Renal disorders are the tenth major
cause of mortality in the world (Pani et al., 2011) and ninth major cause of death in USA
(Javaid et al., 2012). Acute renal failure (ARF) is one of the renal disease which is life
threatening these days if not managed properly. ARF cause the sudden decrease in Glomerular
filtration rate (GFR) which causes increase in serum urea and creatinine level (Ibrahim et al.,
2006, Ghaisas at al., 2010). Arthritis is another disease which is very common these day. Most
of the researchers claimed that these days it is the leading cause of disability in the patients
with no proper cure. Presently, all over the world efforts are being made to discover the curative
agents with novel effects for the treatment of renal and joint diseases. It was hypothesized that
by exploring certain selected herbal medicines from medicinal plants on modern lines would
contribute to the management of renal diseases as well as arthritis and/or finding of some novel
compounds for its treatment. Due to these reasons, the present investigation was taken to study
in detail the nephroprotective and anti-arthritic effect of selected medicinal plant Cuscuta
reflexa Roxb. It has wide distribution in India, Afghanistan and Pakistan. As it is twining
parasitic herb which depends on the host plants for their existence, food and shelter. Cuscuta
reflexa grows on the host plant so it also contains certain phytoconstituents of that host (Nikam
et al, 2014). Genus Cuscuta ranges in severity based on its species and the species of host and
the time of attachment (Kumar et al,. 2012). The plant also have bitter and astringent taste and
 58

sticky and dry in nature (Sharma et al,. 2013). Different extracts of Cuscuta reflexa are used
for different disorders for example petroleum ether extract of full plant has been used for its
psychopharmacological activity (Pal et al,. 2003) and methanolic extract was used for anti-
steroidogenic activity (Gupta et al., 2003) and anti-fertility activities (Pal et al., 2003). Its
ethanolic extract has been used in hypotension and bradycardia (Gilani et al,. 1992) and also
in anti-microbial activities (Amaresh et al., 2014). The effects of Cuscuta reflexa were
evaluated pharmacologically in sprague dawley rats by inducing nephrotoxicity and arthritis.
The data obtained have clearly shown that the AMECR produced a dose dependent decrease
in both diseases that is case of nephrotoxicity and arthritis in animal model (Table 06 to Table
12). It was also noted that degree of nephroprotective and anti-arthritic effect produced by the
extract of this plant was much higher in higher dose as compared to low doses in sprague
dawley rats. This has supported an earlier finding that AMECR have better effect on
inflammation and in liver diseases when its dose has been increased (Katiyar et al., 2012,
Balakrishnan et al., 2010). Focusing on the renal diseases first, it was investigated that another
widespread disorder is nephrotoxicity which is caused by toxins (if body exposed to them for
long period) and continuous use of medicines. According to previous studies certain agents
which was used as curative agent may also cause renal disorders if used for long duration and
at the end these renal disorders leads to ARF, nephrotic syndrome and chronic interstitial
nephritis (Asangani et al., 2012). Details of these studies showed that compounds like carbon
tetra chloride (CCL4), ethylene glycol and heavy metals like cadmium lead and arsenic induces
nephrotoxicity when body exposed to them. Therefore if patients who already have some renal
problems, given medicines which have renal excretory route also accumulate in the body and
may worsen the condition (Mohana et al., 2012).
In present study we have used the aqueous methanolic extract of Cuscuta reflexa for evaluation
of selected pharmacological activities (Nephroprotective and anti-arthritic). Therefore its
preliminary phytochemical investigation was conducted initially to determine the
phytoconstituents so that we can analyze its proposed effect, presence of flavonoids like
quercitin and Kaempferol and tri terpinoids like lupeol showed that plant may have significant
result in nephroprotective and anti-arthritic activity. Previous literature shows presences of
several medicinal important phytochemicals in Cuscuta reflexa like cardiac glycosides,
Saponins, alkaloids, terpinoids and flavonoids however Tanins were absent in aqueous
methanolic extract (Sharma et al., 2012). However other studies shows that ethanolic extract
 59

contain Flavonoids, tannins, alkaloids and terpinoids and phytosterols, alkaloids and
Triterpinoids are present in chloroform extract. Phytochemical studies on Cuscuta reflexa by
researcher shows that its chloroform extract has also been used for anti-tumor activity
(Chatterjee et al., 2011). HPLC analysis was also done to check the presences of flavonoids.
Results of previous HPLC studies shows the presences of flavonoids like quercitin, kaempferol
(Shailajan et al., 2011), coumeric acid. However present HPLC analysis of the AMECR
showed the presences of flavonoid like quercitin, and phenolics like gallic acid, vanillic acid,
cinnamic acid and caffeic acid. By evaluating the results of HPLC analysis and the
results of present study that the kampherol and quercitin may be responsible for the
nephroprotective activity (Bischoff et al. 2008) of Cuscuta reflexa.
Studies have already been reported that Nephroprotective activity can be performed by
inducing the nephrotoxicity by gentamicin which is an approved medicine for inducing
nephrotoxicity (Hussain et al., 2012). After inducing nephrotoxicity by aminoglycoside
(gentamicin) and delivering the different doses of AMECR (200mg/kg, 400 mg/kg, 600 mg/kg
body weight) the animals were sacrificed. Gentamicin induced nephrotoxicity causes marked
increase in the serum level of specific markers like Creatinine, Urea, Uric acid and BUN
(Gaikwad et al, 2012, Bibu et al., 2010). Gentamicin is aminoglycoside which shows
concentration dependent renal toxicity in both man and animal (Whiting et al., 1981, Sharma
et al., 2011). Now a days use of antibiotics is very common. It is reported that aminoglycosides
are one of the commonly used antibiotic which are widely used for gram negative bacterial
infections and other diseases. Studies also shows that they are also important due to its potential
bactericidal effects, its cost effectiveness and also less vulnerability for antibiotic resistance
and past antibiotic activities (Balakumar et al., 2010) but according to previously reported
toxicological studies its adverse reactions like it have potential to cause nephrotoxicity and
ototoxicity limits its use in various disorders (Bibu et al., 2010). Neomycin have great
nephrotoxic potential then gentamicin then tobramycin (Mohana et al,. 2012). Gentamicin is
broad spectrum antibiotic belongs to aminoglycosides and much effective in septicemia but
due to its nephrotoxic and ototoxic potential its use had been reduced (Bhushan et al., 2012).
Therefore its use have been limited against bacterial strains resistant to other antibiotics where
it has been used as effective therapeutic tools (Salgado et al., 2007). Gentamicin shows the
increase in the no. of reactive oxygen species (ROS) like superoxide radicals (Okokon et al.,
2011, Bledsoe et al., 2006), -OH radicals (Balakumar et al., 2008) and Reactive nitrogen
 60

species (Walker et al,. 1999). Studies shows that ability of gentamicin to ROS like; superoxide
anions, hydrogen peroxides and hydroxyl radicals in power house of cell mitochondria causes
antioxidant deficiency leading to oxidative stresses in the renal proximal tubules
(Maldonado et al., 2003; Yanagida et al., 2004). ROS lead to cell injury as well as cell death
and necrosis within the cell which leads to nephrotoxicity. (Cuzzocrea et al., 2002). Different
enzymes and hydroxyl radical (-OH) scavengers like dimethylthiourea, superoxide
dismutase (SOD) and antioxidants may be available from diet like vitamin E, selenium
(SE) , Ascorbic acid and carotenoids can diminished the ROS and reduce the glomerular
filtration rate (GFR) (Poormoosavi et al., 2010). So in present studies gentamicin (Novartis
Pharmaceuticals) was used to induce nephrotoxicity by above described mechanism with the
approved dose of 100 mg/kg/body weight (Table 06, Figure 11). Results shows that effect of
the low dose (200 mg/kg body weight) was not up to mark (Figure 12) however by increasing
the dose the effect of AMECR was prominent which shows that Cuscuta reflexa have marked
effect on the kidney by reducing ROS (Table 07 & Figure 13, 14). Statistical analysis shows
significant results (P <0.001) of the plant extract. All the results were compared with the
normal control and toxic control groups of the animal model.
Arthritis is a disorder which is chronic in nature which causes inflammation in several joints
specifically tendon, synovium muscle and cartilages. Several classes of medicines like
NSAIDs, DMARDS and biologics are currently being used in the treatment of RA but due to
their side effects, less cost effectiveness and toxicities limiting their use and in return they are
leaving a space for new and effective natural agents which have fewer side effects and are
more cost effective. From thousands of years plants have been used traditionally for different
diseases. However from past few decades researchers focus on the medicinal plant for the
treatment of RA (Kumar et al., 2013, Gokhale et al., 2002, Kore et al., 2011). So keeping in
mind above properties of medicinal plant investigation was made on Cuscuta reflexa, by both
ways in-vivo as well as in-vitro (Suresh et al., 2011), which was used traditionally for the
treatment of arthritis and other inflammatory disorders (Udavant et al., 2012). The results of
such investigations shows dose dependent effect of AMECR in both in-vivo animal models
and also in in-vitro models.
The investigations was confirmed by performing in-vivo experiment on animal model.
Studying the previous literature arthritis was induced by formaldehyde and turpentine oil
which are the common methods for determining the effect of medicinal plant in rats (Rajendran
 61

et al., 2010). In this studies one of the main reason for choosing rats as animal in this anti-
arthritic activity is that there is strong similarities and correlation between rheumatic condition
which are seen in human model and efficacy in this in-vivo model (Kumar et al., 2013, Harris
et al., 1990). The determination of joint swelling is apparently simple, sensitive and
quick procedure for evaluating the degree of inflammation and the therapeutic effects of
drugs. Chronic inflammation involves the release of number of mediators like cytokines (IL-
1B and TNF-alpha), GM-CSF, interferon’s and PGDF. These mediators are responsible
for the pain, destruction of bone and cartilage that can lead to severe disability (Lam et
al., 2004). Rheumatoid arthritis (RA) is basically type of arthritis, a chronic inflammatory
disease characterized by joint swelling of joints and inflammation of synovial along with
cartilage and bone damage and lead to chronic disability (Kumar et al., 2013). About one
percent of the population of the world specially female is in greater ratio then male as their
bones and cartilages are weak then male. RA developed through number of pro-inflammatory
chemical molecules which are released by macrophages including ROS and mediators
such as prostaglandins, interleukins (IL), leukotrienes ( Arachidonic acid metabolites) and
cytokines. The regulation of these mediators through arachidonic acid metabolism by
targeting the enzymes like cyclooxygenase and lipoxygenase are the potential target for
chronic inflammatory conditions. (Tripathy et al, 2010 , Kore et al., 2011). There is an increase
in synthesis of 5- Hydroxy triptamine(5-HT) among whole central nervous system during
the mild phase of the disorder (14 to 21 days after inoculation) , further increase is restricted
to the spinal cord (Chitme et al., 2009). It has been reported that RA is characterized by
inflammatory process which have three significant step of development including first step of
increase in the vascular permeability due epithelial cells contraction followed by stasis of blood
and then the second step including the outside movement of leukocyte due to chemotaxis by
chemical mediators and then the third step was granuloma formation. (Raphael et al., 2003).
Literature shows that histamine is one of the important chemical mediator which is sole
responsible for inflammation through increasing the vascular permeability of vessels followed
by sudden slowing of blood (Okhawa et al., 1979). Anti-arthritic activity was investigated in
acute models of arthritis in rats as the arthritis was induced by sub-plantar injection of
formaldehyde and turpentine oil.
It was reported that formaldehyde and turpentine oil administration causes release of various
mediators like histamine, serotonin (initial phase), kinins (middle phase) and PG (final phase)
 62

that play an important role in the development of inflammation (Amresh et al., 2007).
AMECR have inhibited the all phases showing that the extracts can destroy the mediators like
histamine, interleukin, bradykinins and prostaglandins (PG). In case of formaldehyde induce
arthritis AMECR have specific inhibitory actions which shows that this extract have their anti-
arthritic activity by means of inhibiting the synthesis ,release or actions of the several chemical
mediators (Rao et al., 2003). Phytoconstituents responsible for the anti-arthriticf activity are
triterpinoid, cardiac glycosides, flavonoids, phenolic compounds , sterols and saponins present
in AMECR so that is the reason for suppressing edema in animal model (El-Desouky et al.,
2009).
Formaldehyde is well known irritant which may cause mild inflammation to ulceration. It also
causes accumulation of inflammatory cells to multiple joints of body .If inhaled it may cause
the upper respiratory tract infection and it may also induce irritation to fore-stomach depending
upon dose (WHO Report Vol. 88). Formaldehyde induced arthritis is the model used
commonly for the determining the anti-arthritic activity of the extract under examination. After
inducing the injection of formaldehyde in a concentration of 2 % W/V at the rate of 0.1 ml, it
causes the release of chemical mediators which are responsible for arthritis like histamine,
serotonin and PG (Bansod et al., 2010). After inducing arthritis by formaldehyde in the
arthritic control group and then comparison was made by giving the p.o doses of extract
(200mg.kg, 400 mg/kg and 600mg/kg). Results was determined by using Vernier caliper which
has shown a marked increase and then clear decrease in the size of ankle joint showing that the
extract had marked anti-arthritic activity (Table 09). In experiment, standard dose of 2%
formaldehyde was administered on first and third day in arthritic control group which
produced the swelling in the ankle and paw joint. However AMECR was used in the different
doses (200 mg/kg/body weight, 400 mg/kg/body weight and 600 mg/kg/body weight). The
maximum dose showed marked effect on reducing the formaldehyde induced arthritis as
compared to low dose so the effect of maximum dose of the extract was comparable to the
standard drug (Nair et al., 2011) (Figure 03).The standard drug used in this case was aspirin
which shows very less increase in the diameter of paw diameter as it was target specific and
have immediate effect (p<0.001) throughout experiment. The main reason behind inducing the
inflammation through formaldehyde was that it was biphasic in nature that is it showed
neurogenic elements and effects the brain activity firstly then leads its effect to tissue mediated
response (Owoyele et al., 2011). In case of formaldehyde neurogenic phase is considered the
 63

direct reason for stimulation of swelling in paw along with centrally mediated effects of pain
but releasing mediators of pain however in late phase other chemical mediators released which
produces other effects (Cheeke et al., 2006). Certain phytoconstituents like essential oils was
able to block both neurogenic and tissue mediated response but other phytoconstituents like
triterpinoids, flavonoid and phenolics compounds have more prominent effect on the second
phase (Kangralkar et al., 2010).
Further experiment in which arthritis was induced by turpentine oil. The reason for use of
turpentine oil was that it was triphasic in nature. It induces arthritis in three phases. In its first
phase it releases the chemical mediator histamine and serotonin and in second phase it releases
kinin like chemical substances and then in third phase it releases COX and lipoxygenase
products however same experimental protocol was as followed as in case of formaldehyde.
Turpentine was administered in the rate of 0.02 ml in subplantar injection. The result was noted
for one day at different hours which suggests that maximum dose (600 mg/kg body weight) of
AMECR have marked effect in reducing the arthritis in rats ankle joint which was comparable
to standard drug than the minimum dose of AMECR (200 mg/kg body weight) (Table 10).
However, maximum inhibition of ankle and paw edema was seen during the late
phase of inflammation, thus suggesting a prominent cyclooxygenase/lipoxygenase inhibitory
activity (Gupta et al., 1999).
Following the in-vivo evaluation of anti-arthritic activity of Cuscuta reflexa, it was further
conformed by employing different in-vitro models .Previous studies have been reported that
protein denaturation is one of the leading cause of inflammatory as well as arthritic diseases
(Chandra et al., 2012, Sharavan et al., 2011) and due to this protein denaturation production
of auto antigen occur which causes certain rheumatic diseases. However in-vitro test was also
performed to check the effect of extract in rheumatoid arthritis. Previous literature review
showed that the main mechanism involved in protein denaturation is characterized by change
or alteration in the hydrophobic, electrostatic hydrogen and disulphide bonding among the
protein molecule (Gupta et al., 1999). The most common in-vitro methods to check the anti-
arthritic activity through protein denaturation method is by using bovine serum albumin and
fresh hen’s egg albumin (Sharavan et al., 2011). BSA have ability to show the effects which
are correlated with type III hypersensitivity reactions mostly found in the disease like systemic
lupus erythematosus (SLE) , glomerulonephritis and RA and its can be reduced by using certain
NSAIDS like mefnamic acid, diclofenac sodium and salicylic acid which reduces the protein
 64

denaturation changing the pH (Lam et al., 2004). It has been reported that NSAID’s causes the
inhibition of synthesis of prostaglandins (PG) by inhibition of cyclooxygenase pathway which
is its important mechanism regarding its use in arthritis. It has also been investigated that
NSAID’s cause improvement in RA not only by inhibition of PG synthesis but also by reducing
the O2- production polymorpho nucleus and macrophages which shows the prevention of
tissue damage through oxygen free radicals (Biemond et al., 1986). So current study is focusing
not only the in-vivo effects of AMECR but also extract was investigated through in-vitro
models. For this purpose solution of various concentration was prepared that is 25,
50,100,200,400,800 (Table 11, 12) and solution of higher concentration showed prominent
effect on protein denaturation. The results was significant (P < 0.001). Thus the result of
present study shown that aqueous methanolic extract of Cuscuta reflexa is capable to reduce
the production of autoantigen which indirectly reduces the protein denaturation and hence
inhibits arthritis. The extract shows dose dependent response and it is also assumed that the
effect is mainly due to presence of phytochemicals like flavonoids, alkaloids and steroid.
 65

4.1 Conclusion
Based on the above discussed findings, it may be concluded that aqueous extract of Cuscuta
reflexa (whole plant) produce significant and consistent nephroprotective and anti-arthritic
effects in diseased sprague dawley rats. The site of action of test plant extracts were kidney
and knee joint. In the kidney, they have found to maintain the biochemical parameters like
BUN, Serum urea, creatinine and uric acid because it can neutralize the ROS and in arthritic
rats it reduces the inflammation. Further, its antioxidant activity was proved which was the
main reason for using this plant in acute renal failure and RA. Gentamicin induces
nephrotoxicity by the mechanism of direct tubular necrosis that primarily targets in proximal
tubules. While arthritis was induced by formaldehyde and turpentine oil which causes
production of certain inflammatory chemicals like kinins and COX. Thus, effect of the aqueous
methanolic extract of this plants may be suggested to have been produced through a number
of mechanisms including nephroprotective and anti-arthritic effect.
The active principles in the test plants appear to be more effective and helpful in effecting the
diseased rats. In addition flavonoids and phenolic compounds detected in Cuscuta reflexa
could also be held responsible for its activities. Since effects of plant in ARF showed that
presences of polyphenols in extract is also responsible for its antioxidant activity. Virtually,
test plants was observed to increase and maintain the level of biochemical parameters and
reduces the inflammation and produced no visible signs of toxicity or adverse effects in the
treated animals. Nevertheless, further phytochemical, pharmacological and toxicity studies
would be needed to further pinpoint and isolate the active principle(s) in this plant and to
confirm their exact mechanism(s) of action in ARF and Rheumatoid arthritis.
 66

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