STUDIES ON DISSOLUTION AND PREVENTION EFFECTS OF HAJRUL YAHOOD, SANG

SARMAHI AND PHYLLANTHUS NIRURI ON CALCIUM CONTAINING KIDNEY STONES IN

RATS

A Thesis Submitted In Partial Fulfillment for the Requirements for the Degree of Doctor of Philosophy

in Biochemistry

By

Dr. Ali Akbar Shah

MBBS, M.PHIL

Department of Biochemistry Faculty of Medicine & Allied Medical Sciences

Isra University, Hyderabad, Sindh 2017

STUDIES ON DISSOLUTION AND PREVENTION

EFFECTS OF HAJRUL YAHOOD, SANG SARMAHI AND PHYLLANTHUS NIRURI ON

CALCIUM CONTAINING KIDNEY STONES IN RATS

By

Dr. Ali Akbar Shah

NAME OF SUPERVISOR / CO SUPERVISORS Prof. Dr. Fatehuddin Khand ( Supervisor)

Prof. Dr. Rashid Ahmed Memon (Co-supervisor)

Prof. Dr. A. G. Arijo (Co-supervisor)

DEDICATION

This Research Work is dedicated

To

My Beloved Parents & Brother

CERTIFICATE

This is to certify that Dr. Ali Akbar Shah S/O Syed Ali Gohar Shah has

carried out research on the topic “STUDIES ON DISSOLUTION AND

PREVENTION EFFECTS OF HAJRUL YAHOOD, SANG SARMAHI AND

PHYLLANTHUS NIRURI ON CALCIUM CONTAINING KIDNEY STONES IN

RATS’’ under our supervision. The work embodied in this thesis is original

and distinct . His thesis is worthy of presentation to Isra University for the

award of the degree of Doctor of Philosophy in Biochemistry.

Signature of Supervisor

Prof. Dr. Fatehuddin Khand

Department of Biochemistry

Isra University, Hyderabad

Signature of Co-Supervisor Signature of Co-Supervisor Prof. Dr. Rashid Ahmed Memon Prof. Dr. Abdullah G.Arijo Department of Pathology Department of Parasitology Isra University, Hyderabad Sindh Agriculture University Tandojam

iv

ACKNOWLEDGEMENT

With the deep and profound sense of gratitude and thanks to the

almighty ALLAH for giving me the chance for completing this thesis. I am

greatly indebted to my respected Supervisor, Prof Dr. Fatehuddin Khand

and Co-supervisors Professor Dr. Rashid Ahmed Memon and Dr. A. G.

Arijo for their cooperation, guidance and constructive criticism in the

successful completion of this thesis and without their help, this manuscript

was not possible to complete. I am grateful to Prof. Dr. Ghulam Qadir Kazi,

the Vice Chancellor Isra University for his whole heartedly valuable co-

operation and support.

I am also very much grateful to Higher Education Commission of

Pakistan for awarding one year NRPU project.

My thanks are also to all my colleagues, friends and well wishers for giving

me the support and help during the course of conduct of this study.

v

ABSTRACT

Nephrolithiasis has severe ramifications with respect to health and

management cost. Current modalities of treatment though very effective in

getting provisional relief from stones are not devoid of side effects and also

fail to avert the recurrence, which is in fact the main concern of patients with

kidney stones. Hajrul yahood, Phyllanthus niruri and Cystone have been

used in folk medicine since ages and are well known for their lithotriptic and

anti- urolithic properties. The present study was carried out at the animal

house of Sindh Agriculture University Tando Jam to evaluate the litholytic

and anti -urolithic effects of Hajrul yahood, Sang sarmahi, Phyllanthus niruri

and cystone on glyoxylate induced nephrolithiatic rats.

For this purpose, seventy eight male wistar rats were equally divided

into thirteen groups of six rats each. Hajrul yahood, Sang sarmahi and

Phyllanthus niruri were administered either alone or in combination of equal

quantity of each by weight. Cystone given alone was also utilized as a

standard drug to compare the effects of Hajrul Yahood, Sang Sarmahi and

Phyllanthus Niruri on calcium oxalate kidney stones. Calcium oxalate

crystallization was induced by intraperitoneal injections of Glyoxalate prior to

start of the treatment in the litholytic groups and was administered along with

the test drugs in the groups. At the completion of treatment period, serum

samples from 42 rats in groups; and both the kidneys from all the

experimental rats were recovered. Serum was analyzed for the activity of

anti-oxidant enzymes superoxide dismutase (SOD), glutathione peroxidase

(GPX), catalase (CAT); and levels of calcium, magnesium, oxalate and

vi

creatinine. Right kidney was homogenized with phosphate buffer saline

(PBS), and centrifuged. Supernatant thus obtained was analyzed for the

levels of SOD, GPX, CAT, reduced glutathione (GSH) and malondialdehyde

(MDA). The left kidney was fixed in Bouin liquid, embedded in paraffin,

sectioned and stained with hematoxylin and eosin H&E for histological

examination under polarized light microscope.

Serum analysis results showed that the activity of antioxidant enzymes

and magnesium level in rat groups treated with cystone, combination, HY

and PN were lower in the order given as against the negative controls, but

were significantly higher than that of the positive controls (p<0.05). Serum

calcium level was found to be in normal range in cystone, combination, HY

and PN treated groups of rats, but it was lower than normal in positive

controls. Both serum oxalate and creatinine levels were detected to be

significantly lower in cystone, combination, HY and PN treated groups of rats

as compared with positive controls.

A similar comparison of the parameters measured in tissue samples

revealed that antioxidant enzyme activities and reduced glutathione level

were significantly enhanced in cystone, combination, HY, and PN treated

groups of rats as against the positive controls. In contrast, tissue MDA levels

were seen to be significantly lower in cystone, combination, HY and PN

treated groups of rats than the positive controls.

Histological findings of renal tissue sections were also consistent with the

serum and tissue chemistries showing lesser damage to the kidney tissue

vii

and calcification in cystone, combination and HY treated groups of rats as

against the positive controls.

Cystone in comparison to combined treatment exhibited better nephro-

protection against hyperoxaluria induced oxidative stress because of its

increased antioxidant enzyme activities and increased glutathione and

magnesium levels.

In conclusion, present study has demonstrated the litholytic, and

nephroprotective effects of cystone, HY and PN due to their high antioxidant

capacity to inhibit lipid peroxidation in glyoxylate induced hyperoxaluric rats

and also due to their ability to reduce oxalate synthesis. Sang sarmahi

however, in contrast to the general notion has failed to exhibit any significant

litholytic and anti urolithic effects at the dose used in present study.

viii

LIST OF ABBREVIATIONS

ABBREVIATION

TERM

BKN Bikunin

CAI Crystal adhesion inhibitor

CAT Catalase

CG Calgranulin

COD Calcium oxalate- dihydrate

COM Calcium oxalate- monohydrate and

EDTA Ethylene diamine tetraacetic acid

ESWL Extracorporeal shockwave lithotripsy

GAGs Glycosaminoglycans

GPX Glutathione peroxidase

GR. Glutathione reductase

GSH Reduced glutathione

GSSG Oxidized glutathione

H&E. Hematoxylin and eosin

HY HajrulYahood

IVU Intravenous urography

LoH Loop of Henle

MDA Malondialdehyde

NADP+ Nicotinamide adenine dinucleotide phosphate

NC Nephrocalcin

OSP Osteopontin

PBS, Phosphate buffer saline

PN Phyllanthus niruri

PPi Inorganic pyrophosphates

RTA Renal tubular acidosis

SOD Superoxide dismutase

SS Sang sarmahi

THP Tamm – Horsfall Protein

UPF-1 Urinary prothrombin fragment 1

UTF-1 Urinary trefoilfactor

ix

TABLE OF CONTENTS

Page # ACKNOWLEDGEMENT--------------------------------------------------------------- IV ABSTRACT------------------------------------------------------------------------------- V LIST OF ABBREVIATIONS----------------------------------------------------------- VIII TABLE OF CONTENTS-------------------------------------------------------------- IX LIST OF FIGURES--------------------------------------------------------------------- Xi LIST OF TABLES----------------------------------------------------------------------- Xii

CHPATER I ------------------------------------------------------------------------------

01 Introduction ------------------------------------------------------------------------------ 01 Objectives of the Study --------------------------------------------------------------- 06 Rationale of the Study ----------------------------------------------------------------- 07

CHPATER II ----------------------------------------------------------------------------- 08 LITERATURE REVIEW --------------------------------------------------------------- 08 1 Historical Background -------------------------------------------------------------- 08 2. Kidney Stones ----------------------------------------------------------------------- 09 2.1 Types of Kidney Stones-------------------------------------------------------- 09 3. Pathogenesis of Calcium Oxalate Stones ------------------------------------ 11 4. Pathophysiology of Calcium Oxalate Stones ----------------------------------- 12 5. Calcium Oxalate Crystal Development And Growth ----------------------- 17

5.1. Citrate --------------------------------------------------------------------------------- 18 5.2. Tamm – Horsfall Protein (THP) ---------------------------------------------- 20 5.3. Osteopontin (OSP) --------------------------------------------------------------- 21 5.4. Glycosaminoglycans (GAGs) ------------------------------------------------- 22 5.5. Magnesium (Mg++) --------------------------------------------------------------- 22 5.6. Nephrocalcin (NC) ---------------------------------------------------------------- 23 5.7. Calgranulin (CG) ------------------------------------------------------------------- 23 5.8. Urinary Prothrombin Fragment 1 (UPF-1) -------------------------------- 24 5.9. Bikunin (Bkn) ----------------------------------------------------------------------- 24 5.10. Phytate ------------------------------------------------------------------------------- 24

6. Kidney Stones- Diagnosis and Treatment --------------------------------------- 25 7. Other Remedies For Kidney Stones: ---------------------------------------------- 29

7.1 Phyllanthus Niruri (PN) --------------------------------------------------------- 29 7.2 Hajrul Yahood (HY) -------------------------------------------------------------- 34 7.3 Sang Sarmahi ---------------------------------------------------------------------- 36 7.4 Cystone ------------------------------------------------------------------------------- 37

CHAPTER III ---------------------------------------------------------------------------- 39 MATERIALS AND METHODS ------------------------------------------------------ 39 1. Animal Grouping And Housing ------------------------------------------------- 39 2. Induction Of Kidney Stones ----------------------------------------------------- 41 3. Assessment Of Antiurolithic Activity ------------------------------------------- 41 4. Assessment of Disolotion of Calcium Oxalte Crystals ------------------- 5. Measurement of Glutathione Peroxidase (GPX) Activity ---------------

42 43

6. Measurement of Superoxide Dismutase (SOD) Activity ---------------- 45

x

7. Measurement of Catalase (CAT) Activity ---------------------------------- 48 8. Determination of Reduced Glutathione (GSH) ----------------------------- 51 9. Determination of Malondialdehyde (MDA) ---------------------------------- 54 10. Determination of Serum Oxalate:---------------------------------------------- 11. Determination of Serum Calcium: --------------------------------------------- 12. Determination of serum Magnesium:------------------------------------------ 13. Determination of Serum Creatinine: ------------------------------------------

57 58 60 61

14. Statistical Analysis ---------------------------------------------------------------- 62

CHAPTER IV ---------------------------------------------------------------------------- 63 RESULTS -------------------------------------------------------------------------------- 63 CHAPTER V -----------------------------------------------------------------------------

115

DISCUSSION --------------------------------------------------------------------------- 115 CHPATER VI ----------------------------------------------------------------------------

122

CONCLUSION -------------------------------------------------------------------------- 122 CHPATER VII ---------------------------------------------------------------------------

123

RECOMMENDATIONS/ SUGGESTIONS --------------------------------------- 123 REFERENCES --------------------------------------------------------------------------

125

xi

IV-1

Graph showing SOD levels (U/ml) in tissue samples of 78 rats distributed equally into 13 groups. Columns and bars in the graph represent mean and standard deviation respectively ----------------------------------------------------------------

83

IV-2

Graph showing GPX levels (nM/min/mg) in renal tissue samples of 78 rats distributed equally into 13 groups. Columns and bars in the graph represent mean and standard deviation respectively --------------------------------------------------

84

IV-3

Graph showing CAT levels (nM/min/mg) in renal tissue samples of 78 rats distributed equally into 13 groups. Columns and bars in the graph represent mean and standard deviation respectively.--------------------------------------------------

85

IV-4

Graph showing GSH levels (µM/mg) in renal tissue samples of 78 rats distributed equally into 13 groups. Columns and bars in the graph represent mean and standard deviation respectively----------------------------------------------------------------

86

IV-5

Graph showing MDA levels (µmol/gWTW) in renal tissue samples of 78 rats distributed equally into 13 groups.Columns and bars in the graph represent mean and standard deviation respectively.------------------------------------

87

IV-6 Graph shwoing SOD levels (U/ml) in serum samples of 42 rats distributed equally into 13 groups. Columns and bars in

88

LIST OF FIGURES

Chapter Description Page#

II-1 Molecular mechanisms of kidney stone formation. 16

II-2 Chemical structure of citrate molecule.

19

II-3 Diagram of citrate absorption and metabolism in proximal convoluted tubule.

20

II-4 Natural look of Phyllanthus niruri plant.

32

II-5 Another natural look of Phyllanthus niruri, leaves are lush green, smooth and velvety.--------------------------------

32

II-6 Hajrul yahood -------------------------------------------------------

35

II-7 Sang sarmahi ------------------------------------------------------- 37

xii

the graph represent mean and standard deviation respectively---------------------------------------------------------------

IV-7

Graph showing GPX levels (nM/min/mL)in serum samples of 42 rats distributed equally into 13 groups.Columns and bars in the graph represent mean and standard deviation respectively-----------------------------------------------------------------

89

IV-8

Graph shwoing CAT levels (nM/min/mL)in serum samples of 42 rats distributed equally into 13 groups.Columns and bars in the graph represent mean and standard deviation respectively---------------------------------------------------------------

90

IV-9

Graph showing calcium levels (mg/dl) in serum samples of 42 rats distributed equally into 13 groups.Columns and bars in the graph represent mean and standard deviation respectively-------------------------------------------------------------

91

IV-10

Graph showing magnesium levels (mg/dl) in serum samples of 42 rats distributed equally into 13 groups.Columns and bars in the graph represent mean and standard deviation respectively------------------------------------------------------------

92

IV-11

Graph showing oxalate levels (µM/L) in serum samples of 42 rats distributed equally into 13 groups.Columns and bars in the graph represent mean and standard deviation respectively-------------------------------------------------------------

93

IV-12 Graph showing creatinine levels (mg/dl) in serum samples of 42 rats distributed equally into 13 groups.Columns and bars in the graph represent mean and standard deviation respectively-----------------------------------------------------------

94

IV-1 Group A (Negative Controls) ------------------------------

101 IV-2 GROUP B1(Positive controls) ---------------------------- 102

IV-3 GROUP B2(Hajrul yahood treated group )------------- 103

IV-4 Group B3 (Sang Sarmahi treated group) --------------

104 IV-5 Group B4 (Phyllanthus Niruri treated group) ---------- 105

IV-6 Group B5 (HY+SS+PN treated group) ----------------- 106

IV-7 Group B6 (Cystone treated group) ---------------------- 107

IV-8 Group C1 (Glyoxylate + Placebo treated group) ----- 108

IV-9 Group C2 (Glyoxylate + HY treated group) ----------- 109

IV-10 Group C3 (Glyoxylate + Sang sarmahi treated group)

110

xiii

IV-11 Group C4 (Glyoxylate + PN treated group) ----------- 111

IV-12 Group C5 (Glyoxylate + HY+SS+PN treated group) 112

IV-13 GROUP C6 (Glyoxylate + Cystone treated group) -- 113

xiv

LIST OF TABLES

Chapter Description Page

II-1 Different types of kidney stones, their incidence and major chemical composition -------------------------------------------------

09

IV –1 Renal tissue chemistry of 78 rats distributed equally into 13 different groups ---------------------------------------------------------

64

IV– 2 Serum chemistry of 42 rats distributed equally into 7 different groups---------------------------------------------------------

67

IV–3 SOD levels (U/ml) in renal tissue samples of 78 rats distributed equally into 13 groups ---------------------------------

69

IV–4 GPX levels (nM/min/mg) in renal tissue samples of 78 rats distributed equally into 13 groups ---------------------------------

70

IV–5 CAT levels (nM/min/mg) in renal tissue samples of 78 rats distributed equally into 13 groups ---------------------------------

71

IV–6 GSH levels (µM/mg) in renal tissue samples of 78 rats distributed equally into 13 groups ---------------------------------

72

IV–7 MDA levels (µmol/gWTW) in renal tissue samples of 78 rats distributed equally into 13 groups ----------------------------

73

IV–8 Analysis of variance of study variables measured in renal tissue samples of 78 rats distributed equally into 13 groups --------------------------------------------------------------------

74

IV–9 SOD levels (U/ml) in serum samples of 42 rats distributed equally into 7 groups -------------------------------------------------

75

IV–10 GPX levels (nM/min/mL)in serum samples of 42 rats distributed equally into 7 groups -----------------------------------

76

IV–11 CAT levels (nM/min/mL)in serum samples of 42 rats distributed equally into 7 groups -----------------------------------

77

IV–12 Calcium levels (mg/dl) in serum samples of 42 rats distributed equally into 7 groups-----------------------------------

78

IV–13 Magnesium levels (mg/dl) in serum samples of 42 rats distributed equally into 7 groups-----------------------------------

79

IV–14 Oxalate levels (µM/L) in serum samples of 42 rats distributed equally into 7 groups-----------------------------------

80

IV–15 Creatinine levels (mg/dl) in serum samples of 42 rats distributed equally into 7 groups-----------------------------------

81

IV–16 Analysis of variance of study variables measured in serum samples of 42 rats distributed equally into 7 groups-----------

82

1

CHPATER I

INTRODUCTION

A kidney stone, also known as a renal calculus, is a hard mass of

crystal aggregates formed in the kidney of sufferer from organic and

inorganic constituents present in the urine. Crystal aggregation and stone

formation are facilitated by matrix which comprises 2-5% of these stones.

Biochemically the matrix of kidney stones consist of organic material mainly

comprised of proteins. (1)

Kidney stones on the basis of their crystalline composition are broadly

classified into calcium containing stones, uric acid / urate stones and struvite

stones. Well recognized risk factors involved in the causation of calcium

containing kidney stones are hypercalciuria, hyperoxaluria, hyperuricosuria,

low urine volume and decreased inhibitory activity against nucleation and

crystal aggregation of calcium salts, while for uric acid / urate stones are

hyperuricosuria, unduly low urinary pH and volume; and for struvite stones

are highly alkaline urine due to infection caused by urease producing

bacteria.

From the three types of kidney stones, calcium-containing stones

(which make up around 80-85% of all cases in the United States and in many

other countries of the world) are the most common type seen worldwide.

Calcium oxalate alone or combined with apatite or brushite is the most

frequent constituent of calcium-containing kidney stones. (2, 3) Primary

hyperoxaluria is mainly implicated in the formation of pure calcium oxalate

2

stones (4), while genesis of pure calcium phosphate stones are linked with

conditions such as primary hyperparathyroidism (5), distal tubular acidosis

(6) and use of carbonic anhydrase inhibitors.

Calcium is the predominant constituent of both pure and mixed type of

calcium- containing kidney stones. Some studies have revealed that those

people who take extra calcium had a higher tendency of developing calcium-

containing kidney stones as against those who do not take supplemental

calcium.(7) Similarly, in a study on postmenopausal women, it was observed

that those women who consumed 1000 mg of surplus calcium and 400 IU of

vitamin D daily for seven years were 17% more prone to develop kidney

stones as against those who were on a placebo.(8) A similar link between

surplus intake of calcium and kidney stone formation has been seen in The

Nurses' Health Study. (9)

Contrary to supplemental calcium, excessive intake of dietary calcium

is not reported to cause kidney stones, and seems to have protective effect

as calcium chelates ingested oxalate in the gastrointestinal tract. (8, 9)

However, owing to faulty dietary habits, the gut absorption of oxalate may

increase due to decline in the amount of calcium intake; this absorbed

oxalate then filters through the kidneys, as there is no known metabolic role

of oxalate in human body. Since oxalate compared to calcium is about 15

times more potent to cause calcium oxalate precipitation, hence

supersaturation of urine owing to hyperoxaluria can lead to precipitation of

calcium oxalate at physiological pH of urine. (10) Therefore, consumption of

diets low in calcium and high in oxalate such as green seedy and leafy

3

vegetables, tea, coffee and cola drinks (rich sources of oxalate), seem to

contribute a lot in the genesis of calcium containing kidney stones. (9) For

most individuals, however, other risk factors such as dehydration (owing to

any reason), increased consumption of animal protein, sodium, refined

sugars, fructose and fructose rich corn syrup (4), may be the main

perpetrators involved in the causation of calcium-containing kidney stones.

(11)

The mechanism of calcium nephrolithiasis is a complex phenomenon

not yet fully understood. However, supersaturation, crystal nucleation and

attachment to the surface of renal epithelial cells, are now generally agreed

upon as initiating events involved in the genesis of calcium containing kidney

stones. (12)

Supersaturation of urine concomitant to decrease in the level of

inhibitors of crystallization leads to the nucleation of a seed crystal. (13)

Heterogeneous nucleation is faster than the homogeneous nucleation as it

requires less energy. On adhering to cell surface, a seed crystal may

aggregate into a mineralized mass, the chemical composition of which largely

depends on the pH of urine. (14)

However, supersaturation of urine alone is not a sufficient condition for

the development of calcium-containing kidney stones; especially calcium

oxalate stones may have a more complex etiology. (15)

Normal urine contains chelating agents, such as citrate that chelates

free calcium thus inhibiting the crystal nucleation, growth, and aggregation of

4

calcium salts. Other endogenous inhibitors reported in literature include the

Tamm-Horsfall protein, Calgranulin, glycosaminoglycans, uropontin,

nephrocalcin, prothrombin F1 peptide, and bikunin . The biochemical

mechanisms involved in the inhibition of nephrolithiasis by these substances

are not yet fully understood and remain to be elucidated. However, when the

amounts of inhibitors of crystallization and aggregation are far less than their

normal concentrations, stones can develop in the kidneys of patients. (3, 16)

Calcium-containing kidney stones mostly result from a simultaneous

presence of multiple factors rather than a single one. Calcium stones may

develop in patients with metabolic conditions, such as distal renal tubular

acidosis (17), Dent's disease (15), primary hyperparathyroidism (18), primary

or enteric hyperoxaluria, Crohn's disease. (4) Recurrent calcium containing

kidney stone-formers are most often screened for above mentioned

metabolic disorders. The screening process involves chemical analysis of 24

hour urine samples for the amounts of promoters and inhibitors of

crystallization to find out the underlying cause.

Nephrolithiasis is a very common urological finding in Pakistan (19,

20) and costs the society a huge amount of money each year, both on

medical treatment and due to loss of work days. The economic

consequences of this disorder are enormous as it has a high recurrence rate.

Moreover, majority of the patients are males of 20-40 years age (an

important socio-economic group of any population). Calcium containing

kidney stones that cannot be passed out in urine usually obstruct the normal

flow of urine causing severe pain, haematuria and possibly infection or

5

kidney damage. Such patients are most often treated surgically either by

open surgery, percutaneous nephrolithotomy or extracorporeal shockwave

lithotripsy (ESWL), as no effective pharmacological treatment capable of

getting safe removal of these stones in situ is available as yet. The major

drawbacks of above mentioned medical treatments are that these are costly,

and have failed to decrease the rate of stone recurrence. As the kidney may

be surgically assaulted only a limited number of times, it is highly desirable to

develop such an efficient pharmacological / herbal treatment that could

successfully get rid of stones and also prevent the chances of their

recurrence. In this way the cost and morbidity associated with surgical and

ESWL treatments could be avoided saving billions to economy of the country.

Moreover, the pharmaceutical industrial exploitation of the treatment thus

developed can generate a substantial amount in foreign exchange as well.

6

1. OBJECTIVES OF THE STUDY

The major objective of this study was to develop an effective herbal

treatment for:

Dissolution of calcium-containing kidney stones in situ, and

Prevention of the recurrence of calcium-containing kidney stones.

The specific objectives of present study were to:

Evaluate litholytic and antiurolithic effects of Hajrul Yahood, Sang

Sarmahi, Phyllanthus Niruri and Cystone on calcium oxalate

crystallization in hyperoxaluric rats.

Compare litholytic and antiurolithic effects of combination (in equal

quantity by weight) of Hajrul Yahood, Sang Sarmahi, and Phyllanthus

Niruri with Cystone (taken as standard drug in present study) on calcium

oxalate crystallization in hyperoxaluric rats.

7

2. RATIONALE OF THE STUDY

To develop is it herbal treatment that can get safe removal of pre-

existing calcium-containing kidney crystal and also be able to prevent their

recurrence.

8

CHPATER II

LITERATURE REVIEW

1. HISTORICAL BACKGROUND

Kidney stones are known to mankind since antiquity. Hippocrates

(470-400 B.C) is accredited to be the first to describe signs, symptoms and

treatment for kidney stones. Avicenna (980 -1037) described differences

between kidney and ureteric stones and plainly stated that relaxation of

upper urinary tract allows stones to pass from kidneys into ureter. (21)

Ambroise Paré in1564 wrote about severe urinary pain that occurs if renal

stone becomes impacted anywhere in the urinary tract below kidney. De

Marchetti (1680) is credited as first who successfully removed renal stone by

giving cut in the lumbar region and gave subsequent description of surgical

removal of renal stone. Morris (1880) is recognized as the first surgeon of

earlier days who successfully performed nephrolithotomy. (21, 22)

Kelly (1900) is documented as the first who made successful removal

of stone with endoscope developed by Bozzini in1806. (21-23) Since then,

Intravenous urography and Extracorporeal shock wave lithotripsy (ESWL)

introduced in 1923 and in 1980 respectively, had revolutionized the

management of kidney stones.

9

2. KIDNEY STONES

Kidney stones are recognized to be more common in males than in

females, had affected about 12% population of the world with recurrence rate

of approximately 70-80% in males and 47-60% in females. (24, 25).

2.1 Types of Kidney Stones

Various types of kidney stones identified on the basis of their

crystalline composition are shown in Table II-1.

Table II-1. Different types of kidney stones, their incidence and major chemical composition

Type

Incidence

Major chemical composition

Calcium oxalate

70%

Calcium, Oxalate

Calcium phosphate

10%

Calcium, Phosphate

Struvite

10%

Calcium, Magnesium, Ammonium,

Phosphate

Uric acid

5-10%

Uric acid

Cystine

<1%

Cystine

Adapted from: Al-Atar et al., and Vijaya et al., (24, 25).

10

As can be seen from the table, calcium oxalate stones are most

common, followed by calcium phosphate, struvite, urate, cystine and other

rare stone types. (24, 25)

Calcium Oxalate Stones

Calcium oxalate stones which usually form in sterile acidic urine are

the most common type of kidney stones worldwide. The main crystalline

forms of calcium oxalate stones include calcium oxalate- monohydrate

(COM) and calcium oxalate- dihydrate (COD). COM is more stable and

prevalent than the COD. The solubility of calcium oxalate stones is largely

independent of pH. (24, 25)

Calcium Phosphate Stones

Calcium phosphate stones, the second most common type of kidney

stones are also formed in sterile urine. They exist in two crystalline forms

either as hydroxyapatite or brushite. (25, 26) Hydroxyapatite stones are

recognized to form in neutral or alkaline urine, while brushite stones in acidic

urine.

Struvite Stones

Struvite stones, also termed as magnesium ammonium phosphate

stones or triple phosphate stones or infection stones are caused by urease

producing bacteria such as Proteus species, Morganella morganii,

Ureaplasma urealyticum, Klebsiella, Serratia, Enterobacter, etc . (24, 25)

Urease produced by these bacteria splits urea into ammonia and carbon

dioxide resulting in highly alkaline urine (pH >7.5), conducive for precipitation

and hence formation of struvite stones.

11

Uric acid stones

Uric acid stones, which make up approximately 5-10% of kidney

stones, are formed in consistently acidic urine. Predisposing risk factors

characterized by hyperuricosuria in genetically predisposed persons include

gout, leukemia, lymphomas and inherited enzyme defects. (24, 25)

Cystine stones

Cystine stones account for less than 1% of kidney stones. These are

more prevalent in children and adolescents than in adults. The most common

cause of cystine stones is cystinuria- a genetic condition caused by inherited

defect in reabsorption of cystine from renal tubules. (24, 25)

Uncommon and rare stones

These include silicate, indigo, xanthine, indinavir, triamterene, galfenine and

antrafenine. (24 - 26)

3. PATHOGENESIS OF CALCIUM OXALATE STONES

Calcium oxalate stones are formed secondary to a condition called

hyperoxaluria (urinary oxalate concentration> 40mg/24 hours). It is an

inherited genetic disorder of oxalate metabolism (Endogenous over

production) which occurs in:

Inflammatory bowel disease

Chronic diarrhea

Ethylene glycol poisoning

12

Bowel luminal calcium normally chelates oxalate and prevents its gut

absorption. However, in the absence of calcium, free oxalate in gut lumen in

the presence of bile salts is rapidly absorbed into capillaries and excreted

into urine.

But, hyperoxaluria alone is not sufficient to cause calcium oxalate

stone formation. Other risk factors such as reduced urine volume,

hypocitraturia, hypomagnesuria and protein mal-absorption syndrome do

also contribute in the pathogenesis of calcium oxalate kidney stones. (24 -

27) In addition to this, several dietary and environmental risk factors have

also been documented in literature to play contributing role in the formation

of calcium oxalate kidney stones. (28, 29)

Genetic factors also seem to be involved in kidney stone formation,

since an increased incidence of kidney stones in twins has been reported;

32% in monozygotic versus 17% in dizygotic twins. Family clustering studies

had also demonstrated 20-40% increased tendency of kidney stone

formation in families with positive history. (30).

4. PATHOPHYSIOLOGY OF CALCIUM OXALATE

STONES

Pathophysiological mechanisms for the genesis of calcium oxalate

kidney stones involve following successive steps: depicted in Fig. II-1

Nucleation, in situ retention of crystals in urinary epithelium, crystal’s

growth, and crystal’s aggregation. (31, 32) Nucleation is the state of changing

of salts dissolved in urine into solid phase. High activity of free ions in

13

supersaturated urine converts solid phase (crystal formation) into stone

formation by epitaxial growth of crystals. (31-33).

Nucleation may be either homogenous or heterogeneous.

Homogenous nucleation is defined as spontaneous precipitation of solutes

into crystals in supersaturated urine, whereas heterogeneous nucleation is

precipitation of solutes into crystals occurring at lower degrees of saturation

of urine in the presence of nucleating facilitators such as epithelial cells,

urinary proteins, cells, crystals, etc., (31, 32).

Heterogeneous nucleation is faster than the homogenous nucleation,

because in heterogeneous nucleation a crystal surface (nidus) is already

available, while in homogeneous nucleation, a crystal is to be formed in liquid

with no surface. (4,25) Heterogeneous nucleation results in stone formation

when crystals become embedded in the urothelium of kidney tubular

epithelia. Here the growth and aggregation of crystals is critical to

nephrolithiasis. Calcium oxalate crystals grow faster at 37°C, and the rate of

growth of these increase with an increase in the level of supersaturation of

urine at acidic pH .(33-35).

Many theories of crystal growth and aggregation have been proposed

for explaining pathophysiology of nephrolithiasis. The three most widely

accepted are Randall’s plaque theory, free particle theory and fixed particle

theory. (31, 32, 36).

o Randall’s Plaque Theory

Randall`s plaques are the calcium phosphate deposits, which provide

a nidus for calculogenesis of calcium oxalate stone. A nidus of calcium

phosphate almost always develops in renal medulla and adheres to cell

14

membrane. Evidence supporting Randall`s plaque theory is presence of

calcium phosphate (apatite, brushite) crystals in the core of calcium oxalate

calculi, and also frequent presence of Randall`s plaque in kidney stone

patients compared to control subjects. (37).

However, Randall`s plaque has never been found in all patients of

kidney stones. (38) In patients of intestinal bypass with renal stones, several

nodular masses are visibly noted close to the openings of ducts of Bellini.

(39, 40) In a recent study, carried out on knockout mice, it has been

demonstrated that the nidus of calcium phosphate is not necessary for

calcium oxalate nucleation and growth. (40) Randall`s plaque theory has not

been supported in different studies of calcium oxalate calculogenesis.

o Free Particle Theory

The ‘Free Particle’ theory of calculogenesis describes that the salt

crystals grow in urine as free particles (homogeneous nucleation) till become

large enough to block the openings of collecting ducts and duct of Bellini,

resulting in severe renal colic. (41,42) However, as the urine is always

flowing, hence crystal conglomeration may not be possible in “urine flow”, this

is the only drawback due to which ‘Free Particle’ theory is not fully supported.

As a general consensus, free particle theory is not considered as a

sufficient theory to explain the process of calculogenesis, hence another

theory termed as “fixed particle theory” was proposed. (24).

o Fixed Particle Theory

The ‘Fixed Particle’ theory says that the crystal formation and

adhesion to epithelial cells of tubule of renal medulla is essential for

calculogenesis. (41,42)) Since, multiple layers of calcium oxalate crystals

15

become deposited over attached nidus, eventually terminating into kidney

stone formation.

The ‘Fixed Particle’ theory is supported by evidence that dead cells

provide surface for nidus attachment, and crystal growth to stone formation.

(43) Calcium oxalate crystals initiate oxidative stress by generating reactive

oxygen species (ROS) (44) which are responsible for more cell injury,

dystrophic changes, dystrophic calcification, nidus formation, crystallization

and stone growth. ROS also disturbs mitochondrial membrane permeability

and ATP production and aggravates cell injury. (44) Previous studies had

reported that the calcium oxalate crystals bind irreversibly to tubular epithelial

cells and nucleate cell surface directly, followed by endocytosis. (45)This

evidence indicates following underlying pathogenic mechanisms:

Calcium oxalate crystals may directly act as nidus for calculogenesis

even in the absence of Randall’s plaque, and

Tubular epithelial cell injury is an important event in calcium oxalate

calculogenesis. (41 - 45)

16

Figure II-1. Molecular mechanisms of kidney stone formation. Adapted from; Aggarwal et al., 2013 (34).

o Nanobacteria or Nanoparticle

Earlier studies had reported presence of nanosized particles in kidney

stones, hence it was hypothesized that the nanosized particles do play a role

in calculogenesis by promoting nucleation of apatite on cell surfaces. (46,

47).

Several authors are of the view that the nanosized particles are

actually “nanobacteria”. Nanobacteria are gram negative cytotoxic bacteria

17

found in the blood of human and bovine origin, which do play a role not only

in calculogenesis but also in various other diseases. (48).

Colonization of epithelial cells of kidney induces cell damages,

dystrophic calcification and hence calculogenesis. Deposition of calcium

minerals over dead cells is termed as bio-mineralization which is induced by

nanobacteria. However, viability of nanobacteria is questionable, as they

don’t show any bacterial growth in culture media. (49) Hence majority of the

authors are of the opinion that the nanobacteria should be re-termed as

“nanoparticles”. Nanoparticles are assembled into subunits of crystal

particles which are assembled as hybrid inorganic material i.e. a calcium

oxalate calculus. (47) The identity of nanosized particles remains to be

elucidated in future for better understanding of calculogenesis. (24).

5. CALCIUM OXALATE CRYSTAL DEVELOPMENT

AND GROWTH

A lot of pathogenic mechanisms such as Randall’s lesion, ‘free’ or

‘fixed’ particle, or nanoparticles growth had been proposed long ago, but the

exact mechanism of calcium oxalate nephrolithiasis remains an enigma. (50)

Some studies suggest that calcium oxalate crystals aggregate in urine

and deposit as nidus or on nidus or a template for stone growth. (51, 52)

Other studies suggest a role of epitaxial process whereby calcium oxalate

crystals attach to a surface for nephrolithiasis. Nonetheless, it is not clear

from available literature that how calcium oxalate crystals which also form in

the urine of normal subjects grow to form stone in stone-formers. (24) It has

been documented by many investigators that the urine of recurrent stone-

18

formers is deficient in certain chemicals (called inhibitors of kidney stone

formation) that hinder the processes of nucleation, growth, conglomeration

and renal epithelial cell attachment of crystals. The well characterized

inhibitors of calcium nephrolithiasis are:

5.1 Citrate

Citrate is one of the most active calcium chelating agents and chelates

calcium in 1:1concentration. This means if citrate concentration is more than

calcium then free calcium ions will be zero and there will be no nucleation

and crystal growth of calcium oxalate. This is the reason as to why low citrate

carries a tendency of calculogenesis. Citrate excretion in urine maintains low

phosphate and prevents precipitation of calcium phosphate salts. (33, 53)

Citrate performs two important functions in urine;

Chelates urinary calcium

Acts as urinary base.

19

5.2 Tamm – Hors fall Protein (THP)

Tamm-Horsfall protein also called as uromodulin is most abundant

urinary protein. It is expressed by epithelial cells of thick ascending limb of

Loop of Henle (LoH). Renal tubules produce approximately 100 mg of THP

daily. A significant correlation was found between THP and Citrate in stone

forming subjects. (33, 53) THP shows a double role in calcium oxalate

calculogenesis such as: it inhibits precipitation and conglomeration of calcium

oxalate and hydroxyapatite crystals.

In an experimental study on mice deficient in THP, it was observed

that those mice who received calcium overload developed calcium crystals in

76%, whereas controls did not show any change. THP is supposed to act

synergistically with osteopontin as its expression is increased in calcium

overload. THP reduction is observed in human beings with kidney stones.

Some patients showed THP molecular abnormality. THP deficiency has been

positively correlated with degree of calculi formation. (33, 53).

5.3 Osteopontin (OSP)

It is also termed as nephropontin or uropontin. Normal secretion of

OSP is approximately 4mg/24 hours. OSP is found in large concentration in

kidney stones. In vitro studies had shown that OSP inhibits nucleation, crystal

growth, and crystal aggregation of calcium oxalate crystals. It increases

adhesion force between crystal and carboxylate. Immunogold labeling has

shown that OSP is located mostly on surfaces of apatite crystals in particular

at junction of crystal-organic layers. It is demonstrated in in-vitro studies that

16-28nM of OSP produces a 50% reduction in calcium oxalate crystals

20

growth and aggregation. An in-vivo study demonstrated that 131 nM

concentration of OSP is sufficient to inhibit crystal formation and growth.

Experimental studies had shown that OSP deficient mice rapidly developed

calcium oxalate kidney stones. (33, 53)

Role of OSP is not yet clear in human kidney stone formation. Some

studies report that the concentration of OSP is reduced in stone formers

while other studies could not find any difference in OSP levels. (33, 53)

5.4 Glycosaminoglycans (GAGs)

Glycosaminoglycans include hyaluronic acid, dermatan-SO4, heparan-

SO4, and chondoitin-SO4. Several studies had reported role of GAGs in

prevention of kidney stones either by inhibiting or delaying the process of

nucleation. For example;

Chondroitin sulphate–delays nucleation

Dermatan sulphate – inhibits nucleation

Hyaluronic acid is expressed by damaged distal renal tubule

epithelium and inhibits crystal binding and retention.

It is suggested that GAGs protect against cytotoxic insults of oxalate

ions and calcium oxalate crystal induced injury. A few studies had reported

reduced urinary GAGs concentration in stone formers. However, other

studies had failed to see any relationship between urinary GAGs levels and

calcium oxalate calculogenesis. (33, 53).

21

5.5 Magnesium (Mg++)

Approximately 70-80% of serum magnesium is filtered through

glomerular capillaries and is reabsorbed through whole length of renal tubule.

Proximal tubule reabsorbs approximately 5-15% of Mg++, 60-70% is

reabsorbed by thick ascending limb of LoH, 5-10% by distal collecting tubule

and the remaining 3% of total filtered Mg++load is excreted in urine.

Magnesium is an important inhibitor of calcium oxalate nucleation, crystal

growth and aggregation. (33, 54)

In in vitro experiments It has been demonstrate that Mg++inhibits

calcium-oxalate and calcium-phosphate crystal formation and aggregation,

and urinary pH determines inhibitory activity of magnesium. However,

therapeutic benefits of magnesium oxide have not been observed in

recurrent calcium stone formers. (33, 54).

5.6 Nephrocalcin (NC)

Nephrocalcin belongs to osteo-calcin group of bony proteins.

Approximately 1-2 % of bony protein comprises of NC. Nephrocalcin is an

important chelator of calcium and apatite crystals. It is expressed by renal

tubular epithelial cells. Carboxylation of NC depends upon vitamin K

availability and is essential for its biological activity. Nucleation of COM

crystals is inhibited by NC. Synthetic alteration in carboxylation of NC leads

to nucleation, crystal growth and aggregation of calcium oxalate crystals. (33,

54).

5.7 Calgranulin (CG)

22

Calgranulin is also termed as calprotectin and it belongs to S 100

family of calcium binding proteins. Calgranulin is a powerful inhibitor of

nucleation, crystal growth and aggregation of calcium oxalate crystals. (33)

5.8 Urinary Prothrombin Fragment 1 (UPF-1)

Urinary prothrombin fragment 1 is present in calcium containing kidney

stones. UPF-1 is a potent urinary inhibitor of calcium oxalate crystal growth

and aggregation. (33).

5.9 Bikunin (BKN)

Bikunin is a light chain peptide of inter-a-inhibitor. It is suggested to

prevent adhesion of calcium oxalate crystals to tubular and urinary epithelial

cells in normal persons. BKN activity is mostly observed in proximal

convoluted tubules. In hyperoxaluric rats, BKN expression is increased.

Stone formers as compared to non-stone formers had a 50% reduction in

BKN concentration. (33)

5.10 Phytate

Phytate is a storage compound for phosphorus in plants. It is a most

powerful inhibitor of nucleation, crystal growth and aggregation of calcium

oxalate. In the Nurses’ Health Study II (NHS-II), females with highest quintile

of phytate intake were found to have a reduced risk of kidney stone formation

as compared to lowest quintile. (33)

23

a. Pyrophosphates (PPi)

Pyrophosphates are an important component of hydroxyl-apatite and

calcium oxalate kidney stones. These are natural inhibitors of nucleation,

crystal growth and aggregation of calcium salts. Hydroxyapatite precipitation

is inhibited by PPi in in vitro studies. Humans with kidney stones in

comparison to normal controls had very low urinary PPi concentrations. (33,

54).

b. Urinary Trefoilfactor-1 (UTF-1)

Urinary trefoilfactor-1is normally found in overlying gastric mucosa in

normal persons. UTF-1 is a novel calcium oxalate crystal growth inhibitor as

it has been detected by mass spectrometry. Crystal inhibitory activity

detected has been similar to NC. (33, 53)

c. Crystal Adhesion Inhibitor (CAI)

Crystal adhesion inhibitor, a natural stone formation inhibitor, is a 39

kDa peptide expressed by tubular epithelial cells. CAI blocks adhesion of

calcium oxalate crystals to epithelial cell surface, thus prevents nucleation,

crystal growth and crystal conglomeration. (33, 53)

6. KIDNEY STONES- DIAGNOSIS AND TREATMENT

Diagnosis of kidney stones is essential for proper management.

Significant advancement has been made in clinical investigations. Presently

many invasive and non- invasive techniques are available to investigate the

kidney stones in clinical practice. (55)

Laboratory Investigations

Following is a list of laboratory and radiological investigations:

24

Urinalysis

Urine culture and sensitivity

Kidney function tests

Chemical composition of stones – by different methods like FTIR, X

ray Diffraction.

X- rays

X rays are performed to detect radio opaque kidney stones.

However, radiolucent stones are not visible on X rays.

Intravenous Urography (IVU)

IVU is x-raying of urinary tract after injecting a dye in the venous

blood. The dye is filtered through glomeruli and fills the urinary tract. X rays

are taken at different intervals of 30 minutes, 1 hour, 2 hour and so on until

whole dye is washed out. X rays are taken to check stones as filling defects

anywhere in urinary tract. (55)

Sonography

Ultrasonography is a non-invasive testing, easily available and gives

better results for diagnosis of kidney stones. Nowadays it is combined with X

rays. (55)

Renal Endoscopy

Nephroscopy is carried out for diagnostic and therapeutic purpose.

25

Computer Tomography

Helical Imaging

Computerized tomography is sophisticated investigation having various

modifications according to visualization. CT scanning provides more detailed

information of kidney and urinary tract. (55)

Treatment of Kidney Stones

Kidney stones may be managed by conservative or surgical treatment.

Conservative treatment is either specific (stone etiology based) or

nonspecific. Conservative clinical management may help in safe removal of

small stone and concretions.

Nonspecific Conservative Treatments:

These are recommended to every patient suffering from kidney stones

and include:

Fluid and flush therapy

Fluid therapy is mainstay of conservative management of kidney

stones. Sufficient water intake in short intervals of time and after every

voiding helps to wash out crystals and small concretions. A daily urinary

output of over 2 liters must be ensured as hydration prevents urine

stagnation and super-saturation of crystal particles. (22, 56, 57) Lemon and

orange juices increase urinary citrate, hence are good natural home

remedies which dilute urine, alkalinize urine and prevent crystallization of

particles in urinary tract. Flush therapy is recommended with excessive

26

parenteral fluids and diuretics concomitantly to wash out urinary tract of

crystals. (22, 56, 57)

Dietary Restrictions:

Animal Protein

Increased animal protein intake has been linked to increased

incidence of kidney stones in several epidemiological studies. Since dietary

proteins increase uric acid, oxalate and calcium load, hence stone patients

are advised to reduce their intake. (22, 56, 57)

Sodium

Sodium and salt restriction has been advised as primary recommendation

for prevention of kidney stones (58), as high dietary sodium intake alters

urinary pH, increases urinary calcium excretion and reduces urinary citrate

concentration. (22, 59).

Oxalate containing foods

Oxalate is ubiquitous in seedy and green leafy vegetables; hence

stone patients are advised to consume milk or milk products while eating

them. Tea, coffee and nuts also contain abundant oxalate (22, 60); hence

their consumption is also restricted.

Physical parameters

Physical parameters such as increased BMI and Waist size should be

controlled as these are positively correlated with frequency of kidney stones.

(60).

27

Specific conservative treatments

Specific conservative treatments are stone etiology based and are

considerably helpful to reduce the rate of stone recurrence. These are mostly

prescribed to correct the underlying metabolic derangement responsible for

the recurrence. For example, thiazides are given to treat idiopathic

hypercalciuria; allopurinol to prevent hyperuricosuria; cholestiramin,

pyridoxine and oral calcium to control hyperoxaluria; and K-Mg citrate to

avoid hypocitraturia owing to distal RTA. (61)

Surgical treatment

Nowadays, surgical treatment is freely available for both complicated

and uncomplicated kidney stones. (22, 60)

Lithotripsy

Shock wave lithotripsy is least invasive option for getting rid of small stones.

(60, 62).

7. OTHER REMEDIES FOR KIDNEY STONES:

Currently, a lot of interest has developed in alternative remedies of

kidney stones, in particular the herbs many of which have been reported in

literature as remedy for kidney stones. (63) In present study, Phyllanthus

niruri, Hajrul yahood, Sang sarmahi and Cystone have been evaluated for

their antiurolithic and dissolution effects on calcium oxalate crystallization in

hyperoxaluric rats in order to rationalize their use.

28

7.1 Phyllanthus Niruri (PN)

Phyllanthus Niruri (PN), commonly known as Seed-under-leaf and/or

stone breaker, is a tropical plant found along coastal areas. This plant

belongs to the genus “Phyllanthus” of “Phyllanthaceae” family. (63, 64)

Synonym terms used for PN in literature are:

P. carolinianus

P. fraternus,

P. lathyroides

P, sellowianus

P. kirganella

P.lonphali

Nymphanthus niruri

Hundreds of names are in use for Phyllanthus niruri in local languages

from different geographical areas of the world. Some of the local names for

Phyllanthus niruri are enlisted below (63- 65):

29

Chancapiedra,

quebrapedra,

stone-breaker,

arranca-pedras,

punarnava, amli,

bhonya,

bhoomiamalaki,

bhui-amla,

bhuiamla,

bhuianvalah,

bhuimy-amali,

bhuin-amla,

bhumyamalaki,

cane peas senna,

carry-me-seed,

creole senna,

daunmarisan,

derriere-dos,

deye do,

erva-pombinha,

elrageig,

elrigeg,

evatbimi,

gale-wind grass,

graine en bas fievre,

hurricane weed,

jar-amla,

jar amla,

kizhanelli,

malva-pedra,

mapatan,

para-parai mi,

paraparai mi,

pei,

phyllanto,

pombinha,

quinine weed,

sachafoster,

cane senna,

creole senna,

shka-nin-du,

viernessanto,

ya-taibai,

yaa tai bai,

yah-tai-bai,

yerba desanpablo

Phyllanthus niruri plant:

Phyllanthus niruri plant (Fiqs II-4 & II-5) grows approximately 50-70 cm

tall. It bears herbaceous branches which are arranged in ascending order.

Bark is light green, smooth and velvety. Plant bears numerous flowers which

are pale green in color and later flush red. Its fruit is tiny smooth capsulated

with contained seeds.

Plants Parts Used: All parts of plant are of equal medicinal importance. (63-

65)

30

Figure II-4. Natural look of Phyllanthus niruri plant

Figure II-5. Another natural look of Phyllanthus niruri, leaves are lush green, smooth and velvety.

31

Phytochemical isolated from PN

Following Phytochemicals have been isolated from PN:

Alkaloids

Astragalin

Corilagin

Ellagitannins

Hypophyllanthin

Lintetralins

Methyl-salicylate

Niruretin

Niranthin

Repandusinic acids

Niruriside

Saponins

Nirurine

Norsecurinines

Rutin

Tricontanol

Quercetol

Brevifolin

Carboxylic acid

Cymene

Ellagic acid

Gallotechins

Geraniin

Lignans

Nirtetralin

Nirurin

Phyllochrysine

Phyllanthin

Phyllanthine

Phyltetralin

Phyllanthenol

Triacontanal

Quercitrin

Phyllanthus Niruri as Traditional Medicine

Phyllanthus niruri is a famous plant of Ayurvedic system of medicine

used in Southeast Asia for problems of digestive system, liver and kidney

diseases, urinary and genital problems and spleen disorders. In Peru and

Brazil, it is used as herbal remedy for kidney stones.

Some established therapeutic activities of PN reported in literature (63 - 67) are following:

32

Analgesic

Antimalarial

Antinociceptive

Anti-inflammatory

Antiviral

Antispasmodic

Antibacterial

Antihelminthic

Antimutagenic

Liver tonic

Hepato-protective

Appetizer

Digestive

Choleretic

Carminative

Diuretic

Stomachic

Tonic

Laxative

Stone breaker

7.2 Hajrul Yahood (HY)

Hajrul yahood, also called as Jews stone and in English as Lapis

judaicus (Fig. II-6), is a fossilized stone found in Europe, North Africa and

Middle East. (68) It is easily available in the market places of Iran, Iraq,

Afghanistan, Jordan, India, and Pakistan. (69-71) It has been used since

ages to treat urinary diseases in both east and west (72). Dioscorides

Pedanius was the first to use Hajrul yahood to treat urinary stones. According

to Faridi et al (73) Ibn Sina, a Persian scientist of medieval period regarded

Hajrul yahood as the most effective drug to treat urinary stones. Hajrul

yahood is also one of the ingredients of Cystone-a herbal preparation of

Himalaya drug company used to treat kidney stones. At present very little is

known about its precise composition and mechanism of action although a few

studies have clearly shown its anti-urolithic and lithotriptic effects. (74) The

results of these studies demonstrated that it has the potential to decrease the

33

concentration of kidney stone promoters and increase the concentration of

kidney stone inhibitors. (74)

Figure II-6 Hajrul yahood

Composition of Hajrul yahood

Main components of Hajrul yahood identified in an Iranian study by Faridia et

al., (75) are:

CaO 49.77%

MgO 4.28%

SiO2 1.07%

Fe2O3 0.50%

Al2O3 0.33%

Sr 0.08%

Trace components (less than 0.001%) of Hajrul yahood include:

34

Phosphorus

Chlorine Nickel Palladium Sodium Sulfur Titanium Chromium Copper Potassium Manganese Palladium Gadolinium Bismuth Ruthenium Indium Cerium

7.3 Sang Sarmahi

Sang means stone, Sar means head & Mahi means Fish, so Sang

sarmahi ( Fig. II-7) is a stone obtained from the head of the fish. This stone is

endowed with the power to break urinary stones and has been used since

antiquity to treat urinary stone disease (Tibb-e-Nabwi). Up to now very little is

known about the composition and pharmacological activity of Sang sarmahi

as no scientific study has been carried out on it as yet. However, its

widespread use in unani medicine to treat kidney stones as well as

unpublished manuscripts about its benefits inspired us to use this stone in

the present study.

35

Figure II-7 Sang sarmahi

7.4 Cystone

Cystone is a polyherbal as well as polymineral formulation of Himalaya drug

company of India that follows the Ayurvedic system of medicine . Cystone is

by far the best herbal drug used to treat urolithiasis as it possesses both

antiurolithic and lithotriptic properties. (76, 77)

36

The ingredients / Tablet of cystone includes:

Ingredients Quantity in mg

Didymocarpus pedicellata 130

Saxifraga ligulata 98

Rubia cordifolia 32

Cyperus scariosus 32

Achyranthes aspera 32

Onosma bracteatum 32

Vernonia cenerea 32

Shilajeet 26

Hajrul yahood bhasma 32

Mujumdar et al., (78)

The results of more than 50 clinical trials have shown that cystone is highly

effective in the treatment of urinary tract stones as it causes their

fragmentation and easy expulsion, hence provides symptomatic relief. It is

also claimed to prevent recurrence of kidney stones by inducing urinary

output along with reduction in the levels of promoters and increase in the

levels of inhibitors of stone formation. (76, 79, 80).

37

CHAPTER III

MATERIALS AND METHODS

This study employed rat urolithiasis model to investigate the

antiurolithic activity and kidney stone crystal dissolution power of Hajrul

Yahood, Sang Sarmahi, Phyllanthus Niruri and Cystone. Rat urolithiasis

model was chosen because of many resemblances between experimental

nephrolithiasis induced in rats and nephrolithiasis caused in human beings.

(81, 82) In this study Cystone was also utilized as a standard drug to

compare the effects of Hajrul Yahood, Sang Sarmahi and Phyllanthus Niruri

on calcium oxalate kidney stones.

1. ANIMAL GROUPING AND HOUSING:

Seventy eight male Wistar rats of eight weeks age (weight range 250-

350 g), purchased from Pioneer company were kept in polypropylene rat

cages type1(SRP01) under a controlled 12 h light/dark cycle at 23 to 24 ,

and 50 to 60 % humidity. All animals had ad libitum access to standard chow

and tap water. After 3 days in cages, the rats were randomly divided into 3

groups namely A, B and C. Group A was comprised of six rats, B and C of 36

rats each.

Group A rats were untreated and served as negative controls.

Group B rats were given intraperitoneal injection of 60 mg/kg of glyoxylate

five times a week to induce calcium oxalate crystal deposition in the kidney.

After one week, these rats were randomly divided into 6 equal sub-groups (6

rats in each sub-group) i.e. B1, B2, B3, B4, B5, and B6.

38

Group B1 rats were given double distilled deionized water as placebo only

and served as positive controls.

Group B2 rats were given orally through gavage 30mg/kg of Hajrul Yahood

aqueous suspension.

Group B3 rats were given orally through gavage30mg/kg of Sang Sarmahi

aqueous suspension.

Group B4 rats were given orally through gavage30mg/kg of Phyllanthus

Niruri crude aqueous suspension .

Group B5 rats were given orally through gavage30mg/kg of combination of

equal quantities by weight of Hajrul Yahood + Sang Sarmahi+ Phyllanthus

Niruri aqueous suspension.

Group B6 rats were given 30mg/kg cystone (a polyherbal ayurvedic

preparation)

Group C rats were randomly divided into six equal sub-groups (6 rats in

each sub-group) viz. C1, C2, C3, C4, C5, and C6.

Rats in group C1 simultaneously received intraperitoneal injection of 60

mg/kg of glyoxylate + placebo and served as control group.

Rats in group C2 simultaneously received intraperitoneal injection of 60

mg/kg of glyoxylate + 30 mg/kg of Hajrul Yahood

Rats in group C3 simultaneously received intraperitoneal injection of 60

mg/kg of glyoxylate + 30 mg/kg of Sang Sarmahi

Rats in group C4 simultaneously received intraperitoneal injection of 60

mg/kg of glyoxylate + 30 mg/kg of Phyllanthus Niruri

39

Rats in group C5 simultaneously received intraperitoneal injection of 60

mg/kg of glyoxylate + 30 mg/kg of a combination of equal weight of Hajrul

Yahood+ Sang Sarmahi + Phyllanthus Niruri

Rats in group C6 simultaneously received intraperitoneal injection of 60

mg/kg of glyoxylate + 30 mg/kg of cystone

2. INDUCTION OF KIDNEY STONES:

Calcium oxalate crystal deposition in the kidney of rats was induced by

intraperitoneal injection of glyoxylate (purchased from Sigma-Aldrich).

Glyoxylate was selected for this purpose because this compound has been

shown to be a faster and more reliable method for induction of calcium

oxalate crystals than previous experimental rat models of calcium oxalate

stone formation. (83)

3. ASSESSMENT OF ANTIUROLITHIC ACTIVITY:

On day eight of experiment, rats in groups A and C were anaesthetized

(by administering a combination of Ketamine 80mg/kg and diazepam

10mg/kg intra peritoneally) as reported by Green et al., (84) and blood

samples were collected from the retro-orbital region. These samples after

clotting at room temperature (for 15-30 minutes) were centrifuged at 1,000 x

g for 15 minutes in a refrigerated centrifuge (Labnet Z326 Hi-

Speed Centrifuge). The resulting supernatant (serum) was aspirated with a

clean medicinal dropper and stored at -80 and was analyzed within a

month for the activity of antioxidant enzymes superoxide dismutase (SOD),

glutathione peroxidase (GPX) and catalase by kit methods (85, 86) using a

40

SpectraMax Plus 384 micro-plate reader; and calcium, magnesium, oxalate

and creatinine by commercial kit methods (87,88), employing a Microlab 300

Semi-automatic biochemistry analyzer .

After collection of blood samples, these rats were sacrificed by

cervical dislocation and their abdomen was opened to excise both the

kidneys. The left kidney was weighed and rinsed with phosphate buffer saline

to remove any RBCs. Thereafter, a 10% homogenate was prepared by

homogenizing the tissue in 5-10ml of ice cold buffer, pH 7.4 i.e. 50mM

phosphate buffer saline containing 1 mM of EDTA per gram tissue. The

contents were centrifuged at 10,000X g for 15 min at 4ºc, and the

supernatant thus obtained was analyzed for the levels of reduced glutathione

(GSH), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione

peroxidase (GPX) and catalase by standard methods. (89, 90)

o Tissue Fixation, Sectioning and Staining

The right kidney was fixed in bouin liquid for 24-48 hours, dehydrated

in ascending grades of alcohol, cleared in xylene , embedded in paraffin and

were cut at 3–4 μm intervals on a rotary microtome floated in hot water bath

at 42°C and mounted on thoroughly cleaned gelatinized glass slides

appropriately numbered with a diamond pencil. The slides were placed on

hot plate at 37°C for 24 hours for fixation of sections and thereafter stained

with hematoxylin and eosin (H&E). Tissue slices were photographed using

optical microscopy under polarized light. (91-93)

41

4. ASSESSMENT OF DISSOLUTION OF CALCIUM

OXALATE CRYSTALS

At the end of 14 days treatment, all group B rats were sacrificed under

anesthesia, and both the kidneys were excised. These kidneys were

processed in a similar manner as reported for rats in groups A, C1, C2, C3,

C4, C5, and C6.

5. MEASUREMENT OF GLUTATHIONE PEROXIDASE (GPX) ACTIVITY:

Cayman GPX assay kit purchased from Al-Madina traders was used

to measure GPX activity indirectly by a coupled reaction with glutathione

reductase (GR). Oxidized glutathione (GSSG) produced by reduction of

hydroperoxide by GPX was recycled to its reduced state (GSH) by GR and

NADPH.

ROOH+2GSH ROH + GSSG + H2O

GSSG + NADPH + H+ 2 GSH + NADP +

The oxidation of NADPH to NADP+ was accompanied by a reduction

in the absorbance at 340 nm. The rate of decrease in the absorbance at

340nm was directly proportional to GPX activity.

o Reagent preparation

1. GPX Assay Buffer (10X)

Assay Buffer (3 ml) of each vial was diluted with 27 ml of HPLC-grade

water. The final Assay Buffer (pH 7.6) contained 50 mM Tris-HCl + 5 mM

EDTA. This was stable for 6 months.

42

2. GPX Sample Buffer (10X)

Diluted 2 ml of GPX Sample Buffer concentrate with 18 ml of HPLC-grade

water. This final Sample Buffer (pH 7.6) containing 50 mM Tris-HCl + 5 mM

EDTA + 1 mg/ml BSA) was used to dilute the GPX control and the GPX

samples prior to assaying. When stored at 4°C, it was stable for one month.

3. Glutathione Peroxidase (Control)

To avoid repeated freezing and thawing, the vial containing 50 μl of

bovine erythrocyte GPX was aliquoted into several small vials and stored at -

20°C. Before use, 10 μl of the enzyme transferred to another vial was diluted

with 490 μl of diluted Sample Buffer while kept on ice. The diluted enzyme

was stable for four hours on ice. A 20 μl aliquot of this diluted enzyme per

well caused a decrease of approximately 0.051 absorbance unit/minute

under the standard assay conditions.

4. GPX Co-Substrate Mixture

Each vial contained a lyophilized powder of NADPH, glutathione, and

glutathione reductase. To each reconstituted vial, 6.0 ml of HPLC-grade

water was added and mixed well. This reconstituted reagent was kept at

25°C while assaying and then stored at 4°C, at that temperature the

reconstituted reagent remained stable for 2 days.

GPX Cumene Hydroperoxide

A 2.5 ml vial of Cumene Hydroperoxide, supplied with the 96-well kit,

was stored at -20°C when not in use. The reagent was ready to use as

supplied.

43

o Assay Protocol

1. Added 120 μl of Assay Buffer and 50 μl of co-substrate mixture to 3 non-

enzymatic wells.

2. Added 100 μl of Assay Buffer, 50 μl of co-substrate mixture, and 20 μl of

diluted GPx (control) to 3 enzymatic wells.

3. Added 100 μl of Assay Buffer, 50 μl of co-substrate mixture, and 20 μl of

sample to 3 sample wells

4. The reaction was initiated by adding 20 μl of Cumene Hydroperoxide to

all these wells.

5. The plate after careful shaking for a few seconds was measured at 340

nm.

6. MEASUREMENT OF SUPEROXIDE DISMUTASE (SOD) ACTIVITY:

Cayman Superoxide Dismutase Assay Kit that is able to measures all

three types of SOD (Cu/Zn-, Mn-, and Fe-SOD) was employed for the

recording SOD activity.

Biochemical reaction scheme of SOD assay

44

o Reagent preparation

1. Assay Buffer

Assay buffer concentrate (3.0 ml) was diluted with 27.0 ml of HPLC-

grade water. The final assay buffer (pH 8.0), comprised of 50 mM Tris-HCl +

0.1 mM diethylenetriaminepentaacetic acid (DTPA) and 0.1 mM

hypoxanthine was used to dilute the radical detector.

2. Sample Buffer

3.0 ml of assay buffer concentrate was diluted with 18 ml of HPLC-

grade water. This final sample buffer (50 mM Tris-HCl, pH 8.0) was used to

prepare the SOD standards and diluting the XA and SOD samples prior to

assaying.

3. Radical Detector

50 μl of 250 μl tetrazolium salt solution supplied in a vial was

transferred to another vial containing 19.95 ml of diluted Assay Buffer and

covered with tin foil. The prepared Radical Detector, stable for 2 hours was

sufficient for 96 wells.

4. SOD Standard

100 μl of bovine erythrocyte SOD (Cu/Zn) supplied in vials were

thawed and stored on ice for preparing the standard curve. The unused

enzyme in vials was stored at -20°C. The enzyme was stable for at least 2

freeze/thaw cycles.

45

5. Xanthine Oxidase

Prior to use, 1 vial containing 150 μl of xanthine oxidase was thawed

and 50 μl of that was transferred to another vial containing 1.95 ml of dilute

sample buffer. This thawed and diluted xanthine oxidase, stored on ice, was

used within 1 hour as it remained stable for that time duration only. Additional

xanthine oxidase was prepared if needed. Any unused enzyme was

discarded.

o Standard preparation

20 μl SOD standard was diluted with 1.98 ml of dilute sample buffer to

obtain the stock SOD solution. In 7 clean glass test tubes, marked as A to G,

SOD stock and diluted Sample Buffer were added in amounts as shown in

table below:

Superoxide dismutase (SOD) standards

Tube

SOD stock

(µl)

Sample buffer

(µl)

Final SOD activity

(U/ml)

A 0 1000 0

B 20 980 0.005

C 40 960 0.010

D 80 920 0.020

E 120 880 0.030

F 160 840 0.040

G 200 800 0.050

o Assay Protocol

200 μl of the diluted Radical Detector and 10 μl of Standard (contained in

tubes A-G) were added per well in SOD standardwells on the plate.

2. Added 200 μl of the diluted Radical Detector and 10 μl of sample per well.

46

3. The reaction was initiated by rapidly adding 20 μl of diluted xanthine

oxidase to all the wells in use and precise time of start of reaction was noted.

4. After shaking the 96-well plate covered with the plate cover for a few

seconds to mix, the plate was incubated on a shaker for 30 minutes at room

temperature. The absorbance was measured at 440nm using a plate reader.

7. MEASUREMENT OF CATALASE (CAT) ACTIVITY:

For measurement of catalase activity, a Cayman Catalase assay kit

was purchased.

2H2O2 Catalase O2 + 2H2O (Catalytic activity)

8. REAGENT PREPARATION:

1. Catalase Assay Buffer

2.0 ml of 5.0 ml catalase assay buffer contained per vial was diluted

with 18.0 ml of HPLC-grade water. The final assay buffer (comprised of 100

mM potassium phosphate, pH 7.0) was used in the assay.

2. Catalase Sample Buffer

5.0 ml of 10 ml Catalase Sample Buffer contained in a vial was diluted

with 45 ml of HPLC-grade water. The prepared Sample Buffer contained 25

mM potassium phosphate (pH 7.5), 1 mM EDTA and 0.1% BSA, and was

used to dilute the formaldehyde standards, CAT control and CAT samples

prior to assaying. The diluted Sample Buffer remained stable for at least two

months at 4°C.

47

3. Catalase Formaldehyde Standard

Formaldehyde standard (4.25 M) was used as supplied.

4. Catalase (Control)

Lyophilized powder of bovine liver CAT (positive control) supplied in a

vial was reconstituted by adding 2 ml of diluted Sample Buffer to the vial and

vortex mixed. 100 μl of the reconstituted enzyme was then diluted with 1.9

ml of diluted sample buffer. A 20 μl aliquot of this diluted enzyme per well

caused an absorbance of approximately 0.29 after subtracting the

background absorbance.

The diluted enzyme was stable for 30 minutes, while the reconstituted

Catalase control remained stable for 1 month at -20°C.

5. Catalase Potassium Hydroxide

Each vial contained 4 ml of 10 M potassium hydroxide (KOH). The

reagent was ready to use as supplied.

6. Catalase Hydrogen Peroxide

40 μl of 8.82 M solution of H2O2 supplied in a vial was diluted with 9.96

ml of HPLC-grade water. This diluted Hydrogen Peroxide solution remained

stable for 2 hours.

7. Catalase Purpald (Chromogen)

Each vial contained 4 ml of 4-amino-3-hydrazino-5-mercapto-1, 2, 4-

triazole (Purpald) in 0.5 M hydrochloric acid. The reagent was ready to use

as supplied.

48

8. Catalase Potassium Periodate

Each vial contained 1.5 ml of potassium periodate in 0.5 M potassium

hydroxide. The reagent was ready to use as supplied.

9. Formaldehyde Standard Preparation

10 μl of Catalase Formaldehyde Standard was diluted with 9.99 ml of

diluted Sample Buffer to obtain a 4.25 mM formaldehyde stock solution.

Seven clean test tubes were taken and labelled as A to G. Formaldehyde

stock and dilute sample buffer were added to each as shown in table below:

Catalase (CAT) standards

Tube

Formaldehyde

(µl)

Sample buffer

(µl)

Final concentration (µM formaldehyde)

A 0 1000 0

B 10 990 5

C 30 970 15

D 60 940 30

E 90 910 45

F 120 880 60

G 150 850 75

Assay Protocol

1. Added 100 μl of diluted Assay Buffer, 30 μl of methanol, and 20 μl of

standard (tubes A-G) to Formaldehyde Standard Wells.

2. Added 100 μl of diluted Assay Buffer, 30 μl of methanol, and 20 μl of

diluted Catalase into two positive control wells.

3. Added 100 μl of diluted Assay Buffer, 30 μl of methanol, and 20 μl of

sample into two sample wells.

49

4. The reaction was initiated by rapidly adding 20 μl of diluted H2O2 to the

wells. Plate was covered with plate cover and incubated on shaker for

twenty minutes at 25 0C.

5. 30 μl of diluted KOH was then added to each well to terminate reaction.

Thereafter, 30 μl of Catalase chromogen was added to each well and the

plate was covered with plate cover and incubated on shaker for 10

minutes at 25 0C.

6. After addition of 10 μl of Catalase potassium periodate to each well ,

plate was again covered with plate cover and incubated on shaker for 5

minutes at 25 0C.

7. The absorbance was measured at 540 nm.

9. DETERMINATION OF REDUCED GLUTATHIONE

(GSH)

Cayman glutathione assay kit was used to measure reduced

glutathione. Glutathione (GSSG) is a tripeptide (γ-glutamylcysteinylglycine)

distributed in cells compartements..Reduced glutathione (GSH) is a substrate

for glutathione transferase and donates electrons to glutathione peroxidase.

Glutathione recycling is shown below:

50

Reagent Preparation

1. GSH MES Buffer (2X)

60mL of the buffer comprised of 0.4 M (2-N-morpholino)

ethanesulphonic acid, 0.1 M phosphate, and 2 mM EDTA, pH 6.0, was

diluted with 60 ml of HPLC-grade water before use.

2. GSSG Standard

Each vial of the standard containing 2 ml of 25 μM GSSG in MES

Buffer was stored at 0-4°C and used as supplied.

3. GSH Co-Factor Mixture

Lyophilized powder of NADP+ and glucose-6-phosphate contained in

a vial was reconstituted with 0.5 ml of water and mixed well. This mixture was

sufficient for 96 wells.

4. GSH Enzyme Mixture

Vial containing glutathione reductase (GR) and glucose-6-phosphate

dehydrogenase (G6PD) in 0.2 ml buffer was mixed vigorously with 2 ml of

diluted MES buffer and stored at 0-4°C.

51

5. GSH DTNB

To Lyophilized powder of Ellman’s reagent i.e., DTNB (5, 5’-dithio-bis-

(2-nitrobenzoic acid) in a vialwas added 0.5 ml H2O and mixed well.

Reconstitution was done about 10 minutes prior to use. 4-6 vials were used

each time the Assay Cocktail was prepared.

Standard Preparation

In each of 8 test tubes marked as A-H, GSSG Standard and MES

Buffer were added as shown below:

Glutathione (GSH) standards

Tube

Standard GSSG (µl)

MES buffer

(µl)

Final conc. GSSG (µM)

Equivalent total GSH

(µM)

A 0 500 0 0

B 5 495 0.25 0.5

C 10 490 0.5 1.0

D 20 480 1.0 2.0

E 40 460 2.0 4.0

F 80 420 4.0 0.8

G 120 380 6.0 12.0

H 160 340 8.0 16.0

Assay protocol

1. 50 μl of standard were added (tubes A-H) per well on the plate

2. 50 μl of sample was added to each of the wells.

3. Plate was covered by plate cover

4. Following reagents were mixed in a 20 ml vial to prepare the Assay

Cocktail;

52

o MES Buffer (11.25 ml),

o Reconstituted Cofactor Mixture (0.45 ml),

o Reconstituted Enzyme Mixture (2.1 ml),

o water (2.3 ml), and

o Reconstituted DTNB (0.45 ml).

5. After removing the cover, 150 μl of the freshly prepared Assay Cocktail

was added to each of the wells containing standards and samples using a

multichannel pipette.

6. Absorbance was measured at 410 nm.

10. DETERMINATION OF MALONDIALDEHYDE

(MDA):

.Malondialdehyde (MDA), a naturally occurring lipid peroxidation

product of polyunsaturated fatty acids is used as an indicator of oxidative

stress. Thiobarbituric Acid Reactive Substances (TBARS) measurement is a

well-established method for screening and monitoring lipid peroxidation.

Reagent Preparation

1. Thiobarbituric Acid

Vial containing 2 g of thiobarbituric acid (TBA) was ready to use for

preparing the Color Reagent.

53

2. TBA Acetic Acid

To 40 ml of TBA acetic acid, 160 ml of HPLC-grade water was added

slowly. This solution was used for preparing the Color Reagent.

3. TBA Sodium Hydroxide (10X)

20 ml of NaOH was added to 180 ml of HPLC-grade water. This

diluted NaOH solution was used for the preparation of the Color Reagent.

4. TBA Malondialdehyde Standard

Each vial contained 500 μM MDA in H2O. It was ready to use for the

preparation of the standard curve.

5. TBA SDS Solution

Each vial contained a solution of sodium dodecyl sulfate (SDS). The

solution was ready for assay use.

6. Color Reagent Preparation:

o 530 mg TBA was transferred to 200mL beaker containing 50 mL of

diluted TBA Acetic Acid Solution.

o 50 ml of diluted TBA Sodium Hydroxide was added and mixed until the

TBA was completely dissolved.

o The solution remained stable for 24 hours.

Standard Preparation

o Taken 250 μl of the MDA Standard and diluted it with 750 μl of H2O to

obtain a stock solution of 125 μM.

o Taken 8 test tubes and labeled as A-H.

o 125 μM MDA stock solution and water was added to each tube as

depicted in Table below;

54

Malondialdehyde (MDA) colorimetric standards

Tube

MDA (µl)

Water (µl)

MDA conc. (µM)

A 0 1000 0

B 5 995 0.625

C 10 990 1.25

D 20 980 2.5

E 40 960 5.0

F 80 920 10.0

G 200 800 25.0

H 400 600 50.0

Assay Protocol

1. Vial caps were labeled with standard identification number

2. 100 μl of sample or standard was added to labeled 5 ml vial.

3. 100 μl of SDS solution was added to vial and vortex mixed

4. 4 ml of Color reagent was added into each vial forcefully

5. Vials were caped and placed in foam in upright for boiling in boiling water

for 1 hour

6. Removed vials were Incubated for 10 minutes on ice bath to stop reaction

and then

centrifuged at 1,600 x g at 4°C for 10 minutes (Vials may appear clear or

cloudy, cloudiness will clear upon warming to room temperature).

7. 150 μl was transformed from each vial into clear plate and the

absorbance was read at 540 nm.

55

11. DETERMINATION OF SERUM OXALATE:

Oxalate (C2O42-), in the form of Oxalic acid is present in many foods

and beverages (e.g. spinach, tea etc.). It accumulates in many plant tissues

and play role in regulating pH, osmosis and calcium storage. In animals,

oxalate is either absorbed from dietary intake or produced from glycolate

metabolism in liver. Under normal conditions, the daily oxalate load can be

excreted by kidney. However, hereditary defects can cause an increased

level of oxalate, which leads to hyperoxaluria, and results in the formation of

kidney stones. Therefore, measurement of oxalate level is useful for the

prevention, diagnosis and monitoring of kidney stones. In this assay, Oxalate

reacts with Oxalate Converter & Oxalate Enzyme Mix to form an

intermediate, which in turn reacts with a highly specific probe to generate

color at 450 nm. The assay kit can detect Oxalate levels lower than 20 µM.

Kit Contents:

Components K663-100

Cap Code

Part Number

Oxalate Assay Buffer 25 ml WM K663-100-

1

Oxalate Development Buffer 15 ml NM

K663-100-2

Oxalate Converter 0.2 ml Purple K663-100-

3

Oxalate Enzyme Mix (lyophilized) 1 vial Green

K663-100-4

Oxalate Probe (lyophilized) 1 vial Red K663-100-

5

Oxalate Standard (lyophilized) 1 vial Yellow

K663-100-6

56

Oxalate Assay Protocol

Standard Curve Preparation: Diluted Oxalate Standard to 1 mM (1 nmol/μl)

by adding 10 μl of 100 mM Oxalate Standard to 990 μl dH2O.After well

mixing, added well. 0, 2, 4, 6, 8, and 10 μl of the 1 mM Oxalate Standard into

a series of wells in a 96-well plate. Adjusted the volume to 50 μl/well with

Oxalate Assay Buffer to generate 0, 2, 4, 6, 8, and 10 nmol/well of Oxalate

Standards.

Oxalate Converter: Added 2 μl of Oxalate Converter to each Standard and

samples.After well mixing, contents were incubated at 37ºC for 1 hour.

Reaction Mix: For each well, prepared 50 µl of Reaction Mix containing:

Oxalate Development

Buffer 46 μl Oxalate Enzyme Mix 2 µl Oxalate Probe 2 µl

Added 50 µl of the Reaction Mix to each well containing the Standard and the

samples and mixed well.

Measurement: Incubated the contents at 37ºC for 60 min. and measured the

absorbance at 450nm.

12. DETERMINATION OF SERUM CALCIUM:

Principle

Arsenazo 3 [ 2, 7bis 2-arsonophenylazo) 1,8 dihydroxynapthalene 3,6

disulphonic acid ] in neutral medium forms a blue complex with calcium ,the

colour intensity of which is directly proportional to the serum total calcium

concentration.

57

Reagents composition :

Reagent =R

M E S , PH 6.50 100 mmol/l

Arsenazo 3 200mol/l

Calcium Standard 10 mg /dl

wavelength =620nm

Temperature= 37 ºC

Reagent ®

Distilled

urotec

standard

Sample

Blank

1000

20l

-

Calibration

(standard)

1000l

-

20l

-

Test

(sample)

1000l

-

-

20l

After well mixing, the contents were incubated at 25 ºC for 15 minutes and then the absorbance was measured at 620nm.

13. DETERMINATION OF SERUM MAGNESIUM:

Principle

Magnesium ions form a purple colored complex with xylidyl blue in

alkaline solution, in presence of GEDTA, which complexes calcium ions. The

58

reaction is specific for magnesium and the intensity of the purple color

developed is proportional to the serum magnesium concentration .

Reagents

Reagent solution ( R1 ) 12 x 25 ml

Components and Concentrations of R1

Reagent:

Ethanolamine pH 11.0 750 mmol/L

GEDTA (Glycoletherdiamine-tetraacetic acid 60mol/L

Xlydyl blue

Detergents 110 mol/L

Assay Procedure

Wavelength 520nm

Temperature 37 ºC

Measurments Against reagent blank

Blank sample or standard

Sample or standard - 10 L

Dist. Water 10 L -

Reagent 1000 L 1000 L

Mixed and read absorbance against blank after 5-6 min. at 37 ºC.

14. DETERMINATION OF SERUM CREATININE:

Principle

Creatinine forms a yellow_orange compound in alkaline solution with

picric acid. At the low picric acid concentration used in this method a

prescription of protein does not take place. The concentration of the dyestuff

59

formed is a measure of the creatinine level in serum .As a result of the rapid

reaction, later secondary reactions do not cause any interference.

Reagents

Reagent 1 :R1

Picric acid 8.73 mmol/L

Reagent 2:R2

Sodiun hydroxide 312.5 mmo/L

Disodium phosphate 12.5 mmo/L

Creatinine 2.0mg/dL

Assay Procedure

The reaction temperature of the standards and samples were

maintained at 37 ºC

Wavelenghath : 500nm.

Temperature 37 ºC

Sample standard

Reagent 1 500ul 500ul

Reagent 2 500ul 500ul

Serum or 1+99 diluted urine 100ul -

Standard 100ul

60

Mixed and measured the absorbance of the sample (A s1) and

standard (A st) after 1 min at 500nm.

15. STATISTICAL ANALYSIS:

Results are reported as mean ± standard deviation (S.D). One-way

ANOVA was utilized to find out the significance of differences in the mean

values of the parameters measured among different groups of rats.

Independent samples test was used to discover significant differences

in the mean values of the parameters measured in two rat groups. A p-value

of < 0.05 was considered statistically significant

61

CHAPTER IV

RESULTS

Renal tissue chemistry of 78 rats is presented in table IV-1, while

serum chemistry of 42 rats is depicted in table IV-2.

Renal tissue SOD, GPX, CAT, GSH and MAD levels determined in 13

groups of rats are summarized in tables IV.3 to IV.7 and graphs IV-1 to IV-5

respectively, whereas in table IV.8, one way analysis of variance statistics for

above mentioned renal tissue variables has been presented.

Similarly, serum SOD, GPX, CAT, calcium, magnesium, oxalate and

creatinine levels estimated in seven groups of rats are summarized in tables

IV-9 to IV-15 and graphs IV-6 to IV-12 respectively, while table IV-16

presents one way analysis of variance statistics for above measured serum

variables.

62

Table IV-1. Renal tissue Chemistry of 78 rats distributed equally into 13 different groups.

Group Animals SOD

(U/mg) GPX

(nM/min/mg)

CAT (nM/min

/mg) GSSH

(µM/mg)

MDA (µmol/gWTW)

A (Negative controls)

1 198 291 899 4.11 1.89

2 199 286 878 4.32 1.78

3 190 295 910 5.23 2.1

4 203 279 978 5.99 1.98

5 189 289 896 5.58 2.9

6 191 301 867 4.89 3.21

B1 (Positive controls)

1 86 108 311 3.5 7.01

2 90 99 361 3.59 6.89

3 89 83 316 3.61 6.01

4 91 89 264 3.68 8.78

5 83 130 299 3.78 9.09

6 88 134 254 3.89 9.12

B2 (H.Y treated)

1 123 139 509 3.5 5.9

2 136 159 576 3.8 1.21

3 129 162 593 3.9 4.78

4 143 173 560 4.3 3.97

5 139 183 565 4.2 1.78

6 140 170 585 4.41 2.19

B3 (S.S treated)

1 91 110 298 3.31 7.97

2 98 107 269 3.34 4.2

3 93 93 307 3.56 0.73

4 89 97 309 3.49 6.4

5 96 131 289 3.61 5.98

6 107 139 399 3.7 7.87

63

B4 (P.N treated)

1 110 131 408 3.3 5.99

2 120 119 519 3.7 4.89

3 119 143 599 3.8 4.01

4 117 129 512 4.6 4.78

5 108 121 501 3.98 2.1

6 103 110 429 4.12 3.89

B5 (H.Y+S.S+P.N

treated)

1 101 168 575 3.9 3.12

2 145 160 599 4.1 2.13

3 150 172 698 4.89 3

4 149 133 593 5.3 4.79

5 147 141 561 3.94 2.1

6 155 139 497 3.99 2.98

B6 (Cystone

treated)

1 164 190 691 3.69 3.98

2 145 168 592 4.73 1.82

3 158 176 599 4.91 2.32

4 156 167 619 5.11 1.24

5 150 189 601 5.2 1.76

6 154 186 649 4.79 2.1

C1

(Glyoxylate+Plac

ebo treated)

1 87 70 251 3.0 9.01

2 83 69 269 3.2 7.89

3 71 81 223 3.3 11.9

4 79 77 287 3.45 9.78

5 76 48 237 3.49 10.99

6 73 66 298 3.7 7.1

C2

(Glyoxylate+H.Y

treated)

1 107 120 429 3.4 5.9

2 114 114 466 3.51 4.21

3 109 121 509 3.62 7.78

4 106 122 411 3.73 3.97

5 100 116 491 3.77 5.78

64

6 104 99 387 3.8 5.19

C3

(Gloxylate+S.S

treated )

1 79 57 307 2.9 9.99

2 77 77 317 2.99 7.91

3 85 69 323 3.12 3.01

4 81 81 331 3.3 5.78

5 83 107 285 3.25 6.41

6 80 79 321 3.31 7.87

C4

(Glyoxylate+P.N

treated)

1 89 89 402 3.27 6.99

2 103 93 481 3.2 4.2

3 95 99 389 3.31 4.5

4 99 119 413 3.93 5.4

5 92 111 409 3.91 5.98

6 87 115 428 3.76 5.95

C5

(Glyoxylate+H.Y

+S.S+P.N

treated)

1 95 139 473 3.87 4.12

2 105 108 498 3.70 5.13

3 121 121 467 3.4 4.15

4 118 131 489 3.84 6.73

5 123 136 304 4.1 3.1

6 127 143 494 3.97 8.98

C6

(Glyoxylate+Cys

tone treated )

1 126 145 534 3.7 3.98

2 129 139 567 4.7 4.79

3 130 149 578 4.8 2.98

4 132 152 499 3.9 4.24

5 144 158 551 4.6 3.79

6 137 165 512 3.98 9.01

65

Table IV-2. Serum chemistry of 42 rats distributed equally into 7 different groups

Group

Animals

SOD (U/ml)

GPX (nM/min/ml)

CAT (nM/min/ml)

Ca++ (mg/dl)

Mg++ (mg/dl)

Oxalate (µM/L

Creatinine (mg/dl)

A (Negative controls)

1 184 199 889 10.1 2.81 9.1 0.6

2 177 256 907 9.5 2.17 7.8 0.4

3 196 187 810 10.3 1.95 6.9 0.5

4 180 267 910 10.9 1.98 5.99 0.6

5 187 274 942 9.1 2.21 7.91 0.7

6 189 236 708 10.34 2.7 8.72 0.8

C1 (Glyoxylate +Placebo treated)

1 56 77 356 6.1 1.3 43 1.9

2 87 79 359 6.7 1.4 41 2.3

3 76 91 371 6.5 1.5 42 2.4

4 54 67 367 8.3 1.3 42.1 2.8

5 61 57 337 6.3 1.56 44 2.45

6 78 76 354 9.1 1.55 36 2.12

C2 (Glyoxylate

+H.Y treated)

1 106 118 598 8.1 1.79 24.76 1.12

2 119 121 481 7.96 1.99 22.7 1.34

3 109 129 567 8.4 1.96 23.89 1.42

4 122 111 471 7.90 2.04 24.9 1.29

5 129 127 513 8.3 1.97 25.1 1.49

6 128 117 488 7.98 1.89 24.6 1.5

C3 (Glyoxylate

+S.S treated)

1 76 64 396 7.1 1.43 34 2.15

2 98 87 369 7.8 1.4 40 1.98

3 91 79 401 7.9 1.5 45 1.91

4 63 91 402 8.1 1.52 39.9 1.79

5 89 117 387 8.5 1.6 38 1.99

6 74 89 399 10.2 1.58 39 2.39

C4 (Glyoxylate

+P.N treated)

1 100 98 451 8 1.69 25 0.91

2 105 101 481 8.7 1.75 27 0.97

3 124 119 489 8.9 1.89 29 1.56

4 110 129 456 9.1 1.71 39.1 1.79

5 115 119 498 8.74 1.81 37.5 1.93

6 108 123 467 10.6 1.77 35.45 2.01

C5 (Glyoxylate+H.Y+S.S+P.N

treated)

1 123 129 571 8.9 1.8 17 1.35

2 125 118 594 10.7 1.5 19.87 1.23

3 129 138 607 9.98 2.07 18.9 1.56

4 131 140 589 10.23 2.09 21.78 1.38

66

5 135 143 591 8.6 1.07 24.7 1.51

6 105 149 600 11.8 2.7 23.56 1.29

C6 (Glyoxylate +Cystone treated)

1 135 151 601 9.87 1.81 14.57 0.9

2 117 149 611 8.09 1.92 17.8 1.1

3 109 163 677 11.4 1.99 19.89 0.56

4 148 165 598 7.98 2.56 21.89 0.92

5 129 159 647 8.07 2.78 22.12 1.12

6 152 157 612 8.70 2.02 21.95 1.35

67

Table IV.3. SOD levels (U/ml) in renal tissue samples of 78 rats distributed equally into 13 groups

Group

N

Mean

SD

95%

Confidence

Interval for

Mean

Minimum

Maximum

p-

value

Lower

Bound

Upper

Bound

A (Negative control) 6 195.00 5.76 188.95 201.04 189.00 203.00

˂0.001

B1 (Positive control) 6 87.83 3.97 83.66 92.00 80.00 91.00

B2 (H.Y treated) 6 135.00 7.29 127.34 142.65 123.00 141.00

B3 (S.S treated) 6 95.66 6.43 88.90 102.42 89.00 107.00

B4 (P.N treated) 6 112.83 4.95 107.63 118.03 106.00 119.00

B5 (H.Y+S.S+P.N treated) 6 141.16 17.93 122.34 159.99 105.00 151.00

B6 (Cystone treated) 6 154.50 3.72 150.58 158.41 150.00 161.00

C1 (Glyoxylate +Placebo

treated)

6 78.16 5.23 72.67 83.65 71.00 85.00

C2 (Glyoxylate +H.Y

treated)

6 106.66 3.07 103.43 109.89 102.00 110.00

C3 (Glyoxylate +S.S

treated)

6 80.83 2.04 78.69 82.97 77.00 83.00

C4 (Glyoxylate +P.N

treated)

6 94.16 4.91 89.00 99.32 89.00 101.00

C5

(Glyoxylate+H.Y+S.S+P.N

treated)

6 114.83 12.27 101.95 127.71 95.00 127.00

C6 (Glyoxylate +Cystone

treated)

6 133.00 5.25 127.48 138.51 128.00 141.00

68

Table IV.4. GPX levels (nM/min/mg) in renal tissue samples of 78 rats distributed equally into 13 groups

Group

N

Mean

SD

95%

Confidence

Interval for

Mean

Minimum

Maximum

p-

value

Lower

Bound

Upper

Bound

A (Negative

control)

6 290.16 8.30 281.45 298.88 276.00 301.00

˂0.002

B1(Positive

control)

6 107.16 21.08 85.03 129.29 83.00 134.00

B2 (H.Y treated) 6 164.33 15.30 148.27 180.39 139.00 183.00

B3 (S.S treated) 6 112.83 18.44 93.47 132.18 93.00 139.00

B4 (P.N treated) 6 125.50 11.41 113.52 137.47 110.00 143.00

B5

(H.Y+S.S+P.N

treated)

6 152.16 16.37 134.98 169.35 133.00 172.00

B6 (Cystone

treated)

6 179.33 9.95 168.88 189.77 167.00 189.00

C1 (Glyoxylate

+Placebo

treated)

6 68.50 11.91 55.99 81.00 48.00 83.00

C2 (Glyoxylate

+H.Y treated)

6 115.33 8.47 106.43 124.22 99.00 122.00

C3 (Glyoxylate

+S.S treated)

6 78.33 16.57 60.94 95.72 57.00 107.00

C4(Glyoxylate

+P.N treated)

6 104.33 12.37 91.34 117.31 89.00 119.00

C5 (Glyoxylate

H.Y+S.S+P.N

treated)

6 129.66 13.04 115.97 143.36 108.00 143.00

C6 (Glyoxylate

+Cystone

treated)

6 151.33 9.09 141.79 160.87 139.00 165.00

69

Table IV.5. CAT levels (nM/min/mg) in renal tissue samples of 78 rats distributed equally into 13 groups

Group

N

Mean

SD

95%

Confidence

Interval for

Mean

Minimum

Maximum

p-

value

Lower

Bound

Upper

Bound

A (Negative

control)

6 904.66 39.09 863.63 945.69 867.00 978.00

˂0.001

B1 (Positive

control)

6 300.83 37.51 261.46 340.20 254.00 361.00

B2 (H.Y treated) 6 564.66 29.77 533.41 595.91 509.00 589.00

B3 (S.S treated) 6 311.83 45.11 264.48 359.17 269.00 399.00

B4 (P.N treated) 6 494.66 68.76 422.50 566.82 408.00 599.00

B5

(H.Y+S.S+P.N

treated)

6 587.16 65.45 518.47 655.85 497.00 698.00

B6 (Cystone

treated)

6 625.16 38.23 585.04 665.28 592.00 691.00

C1 (Glyoxylate

+Placebo

treated)

6 260.83 29.08 230.31 291.35 223.00 298.00

C2 (Glyoxylate

+H.Y treated)

6 448.83 47.62 398.85 498.81 387.00 509.00

C3 (Glyoxylate

+S.S treated)

6 314.00 15.27 297.97 330.02 287.00 331.00

C4 (Glyoxylate

+P.N treated)

6 420.33 32.37 386.36 454.30 389.00 481.00

C5 (Glyoxylate

+H.Y+S.S+P.N

treated)

6 454.16 74.67 375.79 532.53 304.00 498.00

C6 (Glyoxylate

+Cystone

treated)

6 540.16 30.96 507.66 572.66 499.00 578.00

70

Table IV.6. GSH levels (µM/mg) in renal tissue samples of 78 rats distributed equally into 13 groups

Group

N

Mean

SD

95%

Confidence

Interval for

Mean

Minimum

Maximum

p-

value

Lower

Bound

Upper

Bound

A (Negative control) 6 5.02 0.72 4.25 5.78 4.11 5.99

˂0.03

B1 (Positive control) 6 3.67 0.14 3.52 3.82 3.50 3.89

B2 (H.Y treated) 6 4.01 0.26 3.73 4.29 3.70 4.41

B3 (S.S treated) 6 3.50 0.15 3.34 3.66 3.31 3.70

B4 (P.N treated) 6 3.91 0.26 3.63 4.19 3.60 4.30

B5 (H.Y+S.S+P.N

treated)

6 4.36 0.51 3.81 4.90 3.90 5.10

B6 (Cystone treated) 6 4.73 0.53 4.17 5.30 3.71 5.20

C1 (Glyoxylate

+Placebo treated)

6 3.35 0.18 3.15 3.55 3.10 3.60

C2 (Glyoxylate +H.Y

treated)

6 3.63 0.15 3.47 3.80 3.40 3.80

C3 (Glyoxylate +S.S

treated)

6 3.14 0.17 2.96 3.32 2.90 3.31

C4 (Glyoxylate +P.N

treated

6 3.56 0.33 3.20 3.91 3.20 3.93

C5 (Glyoxylate

+H.Y+S.S+P.N

treated)

6 3.81 0.24 3.55 4.07 3.41 4.10

C6 (Glyoxylate

+Cystone treated)

6 4.28 0.55 3.69 4.86 3.50 4.80

71

Table IV.7. MDA levels (µmol/gWTW) in renal tissue samples of 78 rats distributed equally into 13 groups

Group

N

Mean

SD

95%

Confidence

Interval for

Mean

Minimum

Maximum

p-

value

Lower

Bound

Upper

Bound

A (Negative control) 6 2.31 0.59 1.68 2.93 1.78 3.21

˂0.03

B1 (Positive control) 6 7.81 1.34 6.40 9.22 6.01 9.12

B2 (H.Y treated) 6 3.30 1.86 1.35 5.25 1.21 5.90

B3 (S.S treated) 6 5.52 2.72 2.66 8.38 0.73 7.97

B4 (PN treated) 6 4.27 1.30 2.90 5.64 2.10 5.99

B5 (H.Y+S.S+P.N

treated)

6 3.02 0.97 1.99 4.04 2.10 4.79

B6 (Cystone

treated)

6 2.20 0.94 1.21 3.19 1.24 3.98

C1 (Glyoxylate

+Placebo treated)

6 9.44 1.82 7.53 11.35 7.10 11.90

C2 (Glyoxylate

+H.Y treated)

6 5.47 1.38 4.02 6.92 3.97 7.78

C3 (Glyoxylate

+S.S treated)

6 6.82 2.37 4.34 9.31 3.01 9.99

C4 (Glyoxylate

+P.N treated)

6 5.50 1.03 4.42 6.58 4.20 6.97

C5 (Glyoxylate

+H.Y+S.S+P.N

treated)

6 5.36 2.15 3.11 7.62 3.10 8.98

C6 (Glyoxylate

+Cystone treated)

6 4.79 2.14 2.54 7.05 2.98 9.01

72

Table IV.8. Analysis of variance of study variables measured in renal tissue samples of 78 rats distributed equally into 13

groups

Tissue

Sum of

Squares

df Mean

Square

F p-value

SOD (U/mg)

Between

groups

80523.33 12 6710.27

116.74

<0.0001

Within

groups

3736.00 65 57.47

Total 84259.33 77

GPX(nM/min/mg)

Between

groups

226590.48 12 18882.54

98.98

<0.0001

Within

groups

12399.66 65 190.76

Total 238990.15 77

CAT

(nM/min/mg)

Between

groups

2190363.94 12 182530.32

87.03

<0.0001

Within

groups

136314.00 65 2097.13

Total 2326677.94 77

GSH (µM/mg)

Between

groups

59.04 12 4.92

6.69

<0.0001

Within

groups

47.75 65 0.73

Total 106.80 77

MDA

(µmol/gWTW)

Between

groups

325.66 12 27.13

9.34

<0.0001

Within

groups

188.82 65 2.90

Total 514.48 77

73

Table IV.9. SOD levels (U/ml) in serum samples of 42 rats

distributed equally into 7 groups

Group

N

Mean

SD

95%

Confidence

Interval for

Mean

Minimu

m

Maximu

m

p-

value

Lower

Boun

d

Upper

Boun

d

A (Negative

control)

6 185.5

0

5.92 179.2

8

191.7

1

177.00 193.00

<0.00

1

C1

(Glyoxylate+

Placebo

treated)

6 68.66 13.5

0

54.49 82.83 54.00 87.00

C2

(Glyoxylate

+H.Y

treated)

6 118.8

3

8.72 109.6

7

127.9

9

109.00 129.00

C3

(Glyoxylate

+S.S

treated)

6 81.83 13.0

1

68.17 95.49 63.00 98.00

C4

(Glyoxylate

+P.N

treated)

6 110.3

3

5.60 104.4

4

116.2

2

105.00 119.00

C5

(Glyoxylate+

H.Y+S.S+P.

N treated)

6 124.6

6

8.23 116.0

2

133.3

1

109.00 131.00

C6

(Glyoxylate

+Cystone

treated)

6 131.6

6

16.8

9

113.9

3

149.3

9

109.00 152.00

74

Table IV.10. GPX levels (nM/min/mL)in serum samples of 42 rats distributed equally into 7 groups

Group

N

Mean

SD

95%

Confidence

Interval for

Mean

Minimum

Maximum

p-

value

Lower

Bound

Upper

Bound

A (Negative

control)

6 236.50 36.25 198.44 274.55 187.00 274.00

<0.001

C1(Glyoxylate+

Placebo

treated)

6 74.50 11.51 62.41 86.58 57.00 91.00

C2 (Glyoxylate

+H.Y treated)

6 120.50 6.68 113.48 127.51 111.00 129.00

C3 (Glyoxylate

+S.S treated)

6 87.83 17.37 69.60 106.06 64.00 117.00

C4 (Glyoxylate

+P.N treated)

6 114.83 12.46 101.75 127.91 98.00 129.00

C5

(Glyoxylate+

H.Y+S.S+P.N

treated)

6 136.16 11.05 124.56 147.76 118.00 149.00

C6 (Glyoxylate

+Cystone

treated)

6 157.33 6.12 150.90 163.75 149.00 165.00

75

Table IV.11. CAT levels (nM/min/mL)in serum samples of 42 rats distributed equally into 7 groups

Group

N

Mean

SD

95%

Confidence

Interval for

Mean

Minimum

Maximum

p-value

Lower

Bound

Upper

Bound

A (Negative

control)

6 861.00 87.03 769.66 952.33 708.00 942.00

<0.0001

C1 (Glyoxylate+

Placebo treated)

6 357.33 11.91 344.83 369.83 337.00 371.00

C2 (Glyoxylate

+H.Y treated)

6 519.66 51.55 465.56 573.76 471.00 598.00

C3 (Glyoxylate

+S.S treated)

6 392.33 12.64 379.06 405.60 369.00 402.00

C4 (Glyoxylate

+P.N treated)

6 473.66 18.71 454.02 493.30 451.00 498.00

C5 (Glyoxylate+

H.Y+S.S+P.N

treated)

6 592.00 12.04 579.35 604.64 571.00 607.00

C6 (Glyoxylate

+Cystone

treated)

6 624.33 31.16 591.63 657.03 598.00 677.00

76

Table IV.12. Calcium levels (mg/dl) in serum samples of 42 rats distributed equally into 7 groups

Group

N

Mean

SD

95%

Confidence

Interval for

Mean

Minimum

Maximum

p-

value

Lower

Bound

Upper

Bound

A (Negative

control)

6 10.0 0.64 0.26 9.36 9.10 10.71

˂0.01

C1 (Glyoxylate+

Placebo treated)

6 6.80 1.170 0.47 5.93 6.10 8.39

C2 (Glyoxylate

+H.Y treated)

6 8.03 0.07 0.03 7.95 7.93 8.11

C3 (Glyoxylate

+S.S treated)

6 7.30 1.05 0.42 7.16 7.10 9.37

C4 (Glyoxylate

+P.N treated)

6 8.90 0.86 0.35 8.09 8.00 9.91

C5 (Glyoxylate+

H.Y+S.S+P.N

treated)

6 9.10 1.02 0.41 8.96 8.90 11.10

C6 (Glyoxylate

+Cystone treated)

6 9.70 1.36 0.55 7.58 7.98 10.45

77

Table IV.13. Magnesium levels (mg/dl) in serum samples of 42 rats distributed equally into 7 groups

Group

N

Mean

SD

95%

Confidence

Interval for

Mean

Minimum

Maximum

p-

value

Lower

Bound

Upper

Bound

A (Negative

control)

6 2.30 0.36 1.92 2.68 1.97 2.81

<0.03

C1 (Glyoxylate+

Placebo treated)

6 1.43 0.11 1.31 1.55 1.30 1.56

C2 (Glyoxylate

+H.Y treated)

6 1.94 0.08 1.85 2.02 1.79 2.01

C3 (Glyoxylate

+S.S treated)

6 1.50 0.07 1.42 1.58 1.40 1.60

C4 (Glyoxylate

+P.N treated)

6 1.77 0.07 1.69 1.84 1.69 1.89

C5 (Glyoxylate+

H.Y+S.S+P.N

treated)

6 1.87 0.48 1.36 2.37 1.07 2.50

C6 (Glyoxylate

+Cystone treated)

6 2.18 0.39 1.76 2.59 1.81 2.78

78

Table IV.14. Oxalate levels (µM/L) in serum samples of 42 rats distributed equally into 7 groups

Group

N

Mean

SD

95%

Confidence

Interval for

Mean

Minimum

Maximum

p-value

Lower

Bound

Upper

Bound

A (Negative

control)

6 7.73 1.14 6.53 8.94 5.99 9.10

<0.0001

C1

(Glyoxylate+

Placebo

treated)

6 41.35 1.37 39.90 42.79 39.00 43.00

C2 (Glyoxylate

+H.Y treated)

6 24.32 0.897 23.38 25.26 22.70 25.10

C3 (Glyoxylate

+S.S treated)

6 39.31 1.62 37.61 41.02 37.00 41.00

C4 (Glyoxylate

+P.N treated)

6 32.17 5.922 25.95 38.39 25.00 39.10

C5

(Glyoxylate+

H.Y+S.S+P.N

treated)

6 20.96 2.91 17.90 24.02 17.00 24.70

C6 (Glyoxylate

+Cystone

treated)

6 19.70 3.02 16.52 22.87 14.57 22.12

79

Table IV.15. Creatinine levels (mg/dl) in serum samples of 42 rats distributed equally into 7 groups

Group

N

Mean

SD

95%

Confidence

Interval for

Mean

Minimum

Maximum

p-value

Lower

Bound

Upper

Bound

A (Negative

control)

6 0.60 0.14 0.45 0.74 0.40 0.80

<0.0001

C1

(Glyoxylate+

Placebo

treated)

6 2.32 0.19 2.12 2.53 2.10 2.60

C2

(Glyoxylate

+H.Y treated)

6 1.36 0.14 1.20 1.51 1.12 1.50

C3

(Glyoxylate

+S.S treated)

6 2.03 0.20 1.81 2.25 1.79 2.39

C4

(Glyoxylate

+P.N treated)

6 1.52 0.48 1.02 2.03 0.91 2.01

C5

(Glyoxylate+

H.Y+S.S+P.N

treated)

6 1.38 0.12 1.25 1.51 1.23 1.56

C6

(Glyoxylate

+Cystone

treated)

6 0.99 0.26 0.71 1.27 0.56 1.35

80

Table IV-16. Analysis of variance of study variables measured in serum samples of 42 rats distributed equally into 7 groups

Serum

Sum of

Squares

df

Mean

Square

F

p-value

SOD (U/ml)

Between

groups

51515.14 6 8585.85

70.89

<0.0001

Within

groups

4238.50 35 121.10

Total 55753.64 41

GPX

(nM/min/mL)

Between

groups

103568.14 6 17261.35

57.29

<.0001

Within

groups

10544.33 35 301.26

Total 114112.47 41

CAT

(nM/min/mL)

Between

groups

1035664.95 6 172610.82

100.68

<0.0001

Within

groups

60000.66 35 1714.30

Total 1095665.61 41

calcium

(mg/dl)

Between

groups

165.72 6 27.62

24.80

<0.0001

Within

groups

38.97 35 1.11

Total 204.69 41

magnesium

(mg/dl)

Between

groups

5.47 6 0.91

45.81

<0.0001

Within

groups

0.69 35 0.020

Total 6.16 41

oxalate (µM/L)

Between

groups

5259.05 6 876.50

57.53

<0.0001

Within

groups

533.22 35 15.23

Total 5792.27 41

Serum

creatinine

(mg/dl)

Between

groups

105.98 6 17.66

53.54

<0.0001

Within

groups

11.54 35 0.33

Total 117.52 41

81

Figure IV-1.SOD levels (U/ml) in tissue samples of 78 rats distributed equally into 13 groups. Columns and bars in the graph represent mean and standard deviation respectively.

82

Figure IV-2. GPX levels (nM/min/mg) in renal tissue samples of 78 rats distributed equally into 13 groups. Columns and bars in the graph represent mean and standard deviation respectively.

83

Figue IV-3 CAT levels (nM/min/mg) in renal tissue samples of 78 rats distributed equally into 13 groups. Columns and bars in the graph represent mean and standard deviation respectively.

84

FigureIV-4. GSH levels (µM/mg) in renal tissue samples of 78 rats distributed equally into 13 groups. Columns and bars in the graph represent mean and standard deviation respectively.

85

Figure IV-5. MDA levels (µmol/gWTW) in renal tissue samples of 78 rats distributed equally into 13 groups. Columns and bars in the graph represent mean and standard deviation respectively.

86

Figure IV-6. SOD levels (U/ml) in serum samples of 42 rats distributed equally into 13 groups. Columns and bars in the graph represent mean and standard deviation respectively.

87

Figure IV-7. GPX levels (nM/min/mL)in serum samples of 42 rats distributed equally into 13 groups. Columns and bars in the graph represent mean and standard deviation respectively.

88

Figure IV-8 CAT levels (nM/min/mL)in serum samples of 42 rats distributed equally into 13 groups. Columns and bars in the graph represent mean and standard deviation respectively.

89

Figure IV-9. Calcium levels (mg/dl) in serum samples of 42 rats distributed equally into 13 groups. Columns and bars in the graph represent mean and standard deviation respectively.

90

Figure IV-10. Magnesium levels (mg/dl) in serum samples of 42 rats distributed equally into 13 groups. Columns and bars in the graph represent mean and standard deviation respectively.

91

Figure IV-11. Oxalate levels (µM/L) in serum samples of 42 rats distributed equally into 13 groups. Columns and bars in the graph represent mean and standard deviation respectively.

92

Figure IV-12. Creatinine levels (mg/dl) in serum samples of 42 rats distributed equally into 13 groups. Columns and bars in the graph represent mean and standard deviation respectively.

93

Tissue Chemistry

Glyoxylate treated rats (GroupB1) as against the normal control rats

(Group A) were found to have significantly decreased tissue mean levels for

SOD, GPX, CAT, and GHS; and increased for MDA (p<0.001).

Hajrul yahood treated group of rats (Group B2) compared to the

positive controls (Group B1) had significantly higher renal tissue levels for

SOD (p<0.001), GPX (p<0.001), CAT (p<0.001), and GSH (p<0.02); and

lower for MDA (p<0.001)

Similarly, renal tissue levels for SOD, GPX, CAT, and GSH were seen

to be significantly higher and of MDA lower (p<0.001) in both B5 & B6 groups

of rats than that of the rats in B1 group.

Likewise, renal tissue SOD and CAT levels were significantly higher

and of MDA lower (p<0,001) in Phyllanthus niruri treated group of rats (Group

B4) than that of the controls (Group B1).

With exception to renal tissue SOD activity (p<.03), no significant

change (p>0.05) in the mean levels of GPX, CAT, GSH, and MAD was

noticed between Sang sarmahi treated rats (Group B3) and controls (Group

B1).

o Tissue SOD

Highly significant differences (p<0.001) in tissue mean SOD activity

was seen among A (control), B (litholysis) and C (antiurolithic) groups of rats.

Lowest SOD activity was observed in C1 and B1groups of rats i.e. 78.1 ± 5.2

and 87.8 ± 3.9 U/mg respectively. Highly significant improvement in SOD

activity was observed in cystone treated rats, followed by rats treated with

94

combination of HY+SS+PN, HY, and PN. Sang Sarmahi showed relatively

less significant improvement in SOD activity (Table IV-3 and figure IV-1)

o Tissue GPX

Tissue GPX activity showed significant differences among control,

litholysis, and antiurolithic groups of rats (p<0.002). Rats of groups C1 and

B1were found to exhibit lowest mean GPX activity i.e. 68.5 ± 11.9 and 107.1±

21.0 nM/min/mg respectively. Significant improvement in GPX activity was

noted in cystonetreated rats, followed by rats treated with combination of

HY+SS+PN, HY, and PN. Sang Sarmahi treated rats showed non-significant

rise in GPX activity. (Table IV-4 and figure IV-2).

o Tissue CAT

Tissue CAT activity significantly varied among control, litholysis and

anti- urolithic groups (p<0.001). Rats in groups C1 and B1showed lowest

CAT activity i.e. 260.8 ± 29.08 and 300.8 ± 37.5 nM/min/mg respectively.

Significant improvement in CAT activity occurred in cystone treated rats

followed by rats treated with combination of HY+SS+PN,HY and PN. Non-

significant rise in catalase activity was seen in Sang Sarmahi treated rats

(Table IV-5 and figure IV-3).

o Tissue GSH

Tissue GSH levels showed significant differences among control,

litholysis and antiurolithic groups (p<0.03). Lowest mean GSH concentrations

i.e. 3.35 ± 0.18and 3.67 ± 0.26 µM/mg were respectively noted in C1 and

B1groups of rats. Highest increase in GSH concentration was observed in

cystone treated rats followed by rats treated with combination of

95

HY+SS+PN,HY and PN. Sang Sarmahi group of rats did not exhibit any

significant rise in GSH level (Table IV-6 and figure IV-4).

o Tissue MDA

Tissue MDA levels were significantly different among control, litholysis

and anti- urolithic groups (p≤0.03).Highest tissue MDA level was detected in

group C1 rats i .e. 9.44 ± 1.82µmol/gWTW. MDA levels were significantly

decreased in Cystone treated rats followed by rats treated with combination

of HY+SS+PN, HY and PN (TableIV-7 and figure IV-5).

Comparison of measured parameters between cystone treated B

group rats and rats treated with combination of HY+SS+PN showed that

there was no significant difference (p>0.05) in the mean tissue levels for

SOD, CAT, GSH and MDA. Only tissue GPX activity was found to be

significantly higher (p<0.006) in Cystone treated group than the group of rats

on combined treatment.

A similar comparison between C group rats treated with cystone and

with combination of HY+SS+PN showed that cystone treated group had

significantly elevated activity of antioxidant enzymes SOD (p<0.01), GPX

(p<0.008) and CAT (p<0.02).

Serum Chemistry

Sera of rats of control group A and antiurolithic groups (C1 to C6)

were analyzed for SOD, GPX, CAT, calcium, magnesium, oxalate and

creatinine levels.

In comparison to group C1 rats (controls) group C2 rats were found to

have significantly increased mean serum SOD, GPX, CAT and magnesium

levels; and decreased oxalate and creatinine levels (p<0.0001).

96

Comparison of serum parameters between C1 and C3 groups

revealed that group C3 rats had significantly raised CAT (p<0.001) activity

and decreased oxalate (p<0.04) and creatinine levels (p<0.03).

A similar comparison of serum parameters between C1 and C4 groups

of rats showed that group C4 rats had significantly increased SOD

(p<0.0001), GPX (p<0.0001) , CAT (p<0.0001),magnesium (p<0.0001) and

calcium (p<0.01) levels; and decreased oxalate and creatinine (p<0.004)

levels.

Likewise, group C5 rats as against group C1 had significantly higher

SOD (p<0.0001), GPX (p<0.0001), CAT (p<0.0001), calcium (p<0.001) and

magnesium (p<0.056) levels; and decreased oxalate and creatinine

(p<0.001) levels.

Similarly, group C6 rats compared to group C1 rats had significantly

increased serum SOD (p<0.0001), GPX (p<0.0001), CAT (p<0.0001),

calcium (p<0.03) and magnesium (p<0.001) levels, while decreased oxalate

(p<0.0001) and creatinine (p<0.0001) levels.

Comparison of serum parameters investigated in rats of groups C5

and C6 showed that there was no significant difference (p<0.05) in the mean

values for SOD, calcium, magnesium and oxalate. However, group C6 rats

as against C5 showed significantly increased serum GPX (p<0.002) and CAT

(p<0.04) activity; and decreased creatinine level (p<0,008).

o Serum SOD

Serum SOD activity was significantly different among controls and

antiurolithic groups of rats (p<0.001). Group C1 rats showed the minimum

97

SOD activity i.e. 68± 0.6 U/ml. Significant improvement in SOD activity was

observed in C6 rats followed by rats in groups C5, C2 and C4. Non-

significant (p>0.05) increase in SOD activity was noticed in group C3 rats

(Table IV-9 and figure IV-6).

o Serum GPX

Serum GPX activity differed significantly among controls and

antiurolithic groups of rats (p<0.0001). Lowest GPX activity i.e., 74.5 ± 11.52

was noted in group C1 rats.Significant increase in GPX activity was seen in

C6 group rats followed by rats of groups C5, C4 and C2 (Table IV-10 and

figure IV-7)

o Serum CAT

Serum CAT activity showed significant differences among controls and

anti- urolithic groups of rats (p< 0.0001). Least CAT activity was found in C1

group of rats.CAT activity significantly improved in group C6 rats followed by

in rats of groups C5,C4 and C2 (Table IV-11 and figure IV-8).

o Serum Calcium and magnesium

Highest and lowest serum calcium and magnesium concentrations

were seen in rats of groups C6 and C1 respectively (Tables IV-12 and IV-13,

figure IV-9 and IV-10).

98

o Serum oxalate and creatinine

Highest and lowest serum oxalate and creatinine levels were detected

in rats of groups C1 and C6 respectively (Tables IV-14 and IV-15, figure IV-

11 and IV-12).

HISTOPATHOLOGICAL OBSERVATIONS

99

Figure IV-1. Group A (Negative controls) Kidney section showing essentially normal histological structure (H&E stain × 100)

100

FigureIV-2. Group B1 (Positive controls) Kidney sections showing calcium crystals , focal glomerulosclerosis and focal

tubular epithelial degeneration (H&E stain × 400)

Tubular epithelial degeneration

Calcium crystals

Focal Glomerulosclerosis

101

Figure IV-3. Group B2 (Hajrul yahood treated group) Kidney sections showing intact and essentially normal renal cellular architecture (H&E stain × 100)

102

Figure IV-4. Group B3 (Sang sarmahi treated group) Tissue section of kidney showing edematous interstices, vacoulation, and atrophy of renal tubules; tissue details are faintly visible. 2+ calcification is visible (H&E stain x 400). .

Calcification

Vacoulation

Edematous interstices

Atrophy of renal tubules

103

Figure IV-5. Group B4 (Phyllanthus niruri treated group) Renal tissue section shows mild to moderate interstitial edema and degeneration of tubular epithelial cells. 1+ calcification is visible (H&E stain x 400).

Calcification

Interstitial edema

Degeneration of tubular epithelial cells

104

Figure IV-6. Group B5 (HY+SS+PN treated group) Renal tissue section shows intact histological architecture. Normal glomeruli and tubules with intact basement membranes, mesangium and blood capillaries with red blood cells inside are seen. Interstitial spaces are normal (H&E stain x100)

105

Figure IV-7. Group B6 (Cystone treated group) Renal tissue section shows intact histological architecture. Normal glomeruli and tubules with intact basement membranes, mesangium and blood capillaries with red blood cells inside are seen. Interstitial spaces are normal (H&E stain x 100).

106

Figure IV-8. Group C1 (Glyoxylate + placebo treated group) Tissue section of kidney shows severe edematous interstices, large vacoulations, hydropic changes, and shrinkage of renal tubules. 4+ calcification and inflammatory cells are visible (H&E stain x 400).

Inflammatory cells

Calcification

Glomerular distortion

Interstitial edema

shrinkage of renal tubules

107

Figure IV-9. Group C2 (Glyoxylate + HY treated group) Renal tissue section shows intact histological architecture. Normal glomeruli and tubules with intact basement membranes, mesangium and blood capillaries with red blood cells inside are seen. Interstitial spaces are normal (H&E stain x 400).

108

Figure IV-10. Group C3 (Glyoxylate + Sang sarmahi treated group) Tissue section of kidney showing edematous interstices, vacoulation, and shrinkage of renal tubules. Glomeruli are collapsed; tissue details are not clearly visible. 3+ calcification and inflammatory cells are visible. (H&E stain x 400).

Shrinkage of renal tubules

Collapsed tubules

Vacoulation

Inflammatory cells

Interstitial edema

Calcification

109

Figure IV-11. Group C4 (Glyoxylate + PN treated group) Tissue section of kidney shows an edematous interstices, minimal vacoulation, hydropic changes and increased vascularity. 1+ calcification is present while inflammatory cells are not visible. (H&E stain x 400).

Edematous interstices

Calcification

110

Figure IV-12. Group C5 (Glyoxylate + HY+SS+PN treated group) Renal tissue section showing intact histological architecture . Tubules with intact basement membranes, mesangium and blood capillaries with red blood cells inside are seen. Interstitial spaces are normal (H&E stain x 400).

111

Figure IV-13. Group C6 (Glyoxylate + Cystone treated group) Kidney section showing essentially normal cellular structure and architecture (H&E stain × 400).

112

Physical Health of the Rats

General physical health of the rats in urolithic groups B1 and C1, and

in Sang Sarmahi treated group became deteriorated by the end of the

treatment period owing to reduced water and food intakes resulting in

decreased physical activity and decline in the weight. On the contrary, rats in

in both dissolution and antiurolithic groups treated with Hajrul yahood,

Phyllanthus niruri, Combination and Cystone were observed to maintain their

normal appetite, physical activity and weight throughout the study period.

113

CHAPTER V

DISCUSSION

Since nephrolithiasis is a multifactorial disorder, its treatment may also

involve multiple chemical substances with antispasmodic, antioxidant and

anti-inflammatory properties. In this connection medicinal plants or known

folkloric herbal medicines claimed to be effective in the treatment of kidney

stones, are worth to be exploited as these contain a variety of chemicals that

can target multiple steps involved in the pathogenesis of kidney stones.

In present study preventive and curative effects of Phyllanthus niruri,

Hajrul yahood, Sang sarmahi, and Cystone have been evaluated in

glyoxylate induced rat urolithiasis model.

Phyllanthus NirurI

Phyllanthus niruri is popularly called stone breaker as it causes

fragmentation and expulsion of kidney stones. The plant is reported to

contain more than 50 chemical compounds including alkaloids, flavanoids,

lignans and triterpenes which are attributed for its therapeutic potential. (66,

94)

Alkaloids are reported to exert antispasmodic activity necessary for

smooth muscle relaxation, mostly evidenced in the urinary tract, thereby

facilitating the expulsion of urinary calculi. (66)

Triterpenes are reported to prevent calcium oxalate induced cell

cytotoxicity (95), reduce urinary availability of stone forming substances, (96)

and reduce the levels of promoters of crystal formation in the urine. (97)

114

Lignins and phyllanthin present in the methanol extract of PN are

reported to increase the uric acid excretion through kidneys in induced

hyperuriceamic rats. (98)

Many investigators (99-102) have found inhibitory effects of PN on

calcium oxalate crystal formation, growth, aggregation and adhesion to renal

epithelial cells.

The finding of the present study that glyoxylate treated rats as against

the controls (untreated rats) had significantly decreased antioxidants levels

both in the renal tissue and serum samples, while increased MDA levels in

tissue samples and of oxalate and creatinine in serum samples confirms the

observation that glyoxylate increases oxalate excretion and calcium oxalate

crystal precipitation which in turn triggers oxidative stress by producing

intracellular oxygen derived free radicals that have the potential to damage

biological membranes. (103) Damage to kidney is also evident from the

renal tissue section of glyoxylate treated rats (Figs.IV-2 and IV-11) which

shows glomerulosclerosis, tubular epithelial degeneration and 4+ calcification.

In current research study PN was found to significantly increase

antioxidants levels and decrease lipid peroxidants load in glyoxylate treated

rats as indicated by tissue SOD, GPX, CAT, GSH and MDA levels.

Simultaneous treatment of rats with glyoxylate and PN exhibited more

effective antioxidant and anti-peroxidant role of PN. This improvement in

antioxidant and lipid peroxidant status of PN treated rats can be attributed to

triterpenes and quercetin content of PN that seems to have protected the

renal tubular epithelial cells against the cytotoxicity caused by hyperoxaluria

115

induced by glyoxalate. (96) In this respect the finding of present study is in

full agreement with the findings of other researchers. (104-109)

Serum calcium concentration was found to be near normal in PN

treated rats compared to placebo control group of rats whose calcium levels

were lower than the normal. This finding of present study is in contrast with

the reports of (110,111) but in agreement with the findings of (64, 87,112).

The anti- urolithic effect of PN can also be attributed to significantly higher

magnesium levels found in PN treated group of rats than in the rats of

placebo control group. This is because magnesium forms soluble salts with

oxalate thereby decreasing the amount of oxalate available for calcium

oxalate salt formation and precipitation. (113)These results are in line with

the reports of many other investigators. (87,110,114) Serum oxalate levels in

the PN treated rats as against the placebo control group of rats were

significantly decreased, possibly due to inhibition of the enzyme glycolate

oxidase that converts glyoxalate into oxalate. (74, 87, 115) The nephro-

protective effect of PN is very much evident from the normal serum creatinine

levels as compared with placebo control group rats whose serum creatinine

levels were significantly elevated due to outflow obstruction of the renal

tubules by renal stones. (106,111)The histological examination of PN treated

rat kidney tissue sections (Fig. IV-5) showed few calcium oxalate crystal

fragments and less tubular atrophy than that of placebo controls. This

indicates that PN contains chemicals capable of disintegrating calcium

oxalate kidney stone crystals. (100,106).

116

Hajrul Yahood

In present study Hajrul yahood has been shown to be a highly

effective drug for the dissolution and prevention of kidney stones. The

antioxidant enzymes activities were found to be near normal i.e slightly lower

than the negative controls but significantly higher than the positive controls.

The normal activity of SOD, GPX and CAT and decrease in MDA levels

detected in HY treated group of rats could be due to increased level of

magnesium and decreased level of oxalate in serum that have resulted in

decreased urinary supersaturation and hence calcium oxalate precipitation,

which is the main trigger for the production of oxygen derived free radicals.

(74) This observation is in agreement with (117-120). Hajrul yahood contains

silicon dioxide (75) that is known to produce soluble oxalate salts i.e., calcium

oxalate dihydrate and calcium oxalate trihydrate, both of which are highly

unstable and soluble than calcium oxalate monohydrate. (121,122) Serum

calcium and magnesium levels in HY treated group of rats were found to be

within normal limits, which could be due to high calcium and magnesium

content of Hajrul yahood. (123,124) Serum creatinine levels were also found

to be normal. These observations are in accord with the findings of (74,125-

127).The histological examination of the rat kidney sections treated with

Hajrul yahood showed intact and essentially normal histological architecture

in both preventive and dissolution groups of rats (Figs.IV-3 and IV-9). This

confirms the findings that Hajrul yahood has ability to dissolve and prevent

calcium oxalate kidney stones. (128-130).

117

Sang sarmahi

Sang sarmahi experimented first time in present study was found to be

less effective in the dissolution and prevention of kidney stones at the dose

used. As compared to controls, SS treated group of rats were found to have

significantly raised SOD activity in tissue samples (p<0.03); increased CAT

activity (p<0.001) and decreased oxalate (p<0.04) and creatinine (p<0.03)

levels in serum. The serum creatinine levels were however, quite high

signifying obstruction to the urinary outflow due to kidney stones in the renal

tubules .The histological examination of the kidney sections showed calcium

crystals, atrophic tubules and tubular epithelial degeneration in both preventive

and dissolution groups of rats (Figs.IV-4 and IV-10). This indicates that Sang

sarmahi had neither stone dissolving nor stone preventing effect at the

concentration used in present study.

Cystone

In present study cystone has exhibited the highest level of nephron-

protection against hyperoxaluria induced oxidative stress as is evident from the

serum creatinine levels of cystone treated rats. This is because of highest

antioxidant enzymatic activity observed in both serum and tissue samples of

cystone treated rats. These findings of present study are in accord with the

reports of many other investigators. (117-120,125-127) Cystone because of its

antioxidant properties has averted the lipid peroxidation normally caused by

hyperoxaluria as is obvious from significantly lower levels of MDA in cystone

treated group of rats compared to other rat groups. These results of present

study are in line with (117-120,125). Serum calcium levels were found to be

normal in cystone treated group of rats. This is in contrast with the findings of

118

many other researchers (110,111), but consistent with the findings of (85, 87).

Serum magnesium levels were also seen to be higher in the cystone treated

rats as compared to other rat groups. The normal concentration of both

calcium and magnesium in cystone treated group of rats can possibly be due

to Hajrul Yahood Bhasma (one of the ingredients of cystone) which contains

high amounts of calcium and magnesium. (75) This observation is in full

agreement with the reports of (87,110,114). Serum oxalate levels in cystone

treated rats were significantly lower than the rats of other groups possibly due

to inhibition of the enzyme glycolate oxidase that converts glyoxalate into

oxalate. (87,115, 116) The histological examination of the rat kidney sections

treated with cystone showed intact and essentially normal histological

architecture in both preventive and dissolution groups of rats (Figs.IV-7 and IV-

13).These observations are consistent with the findings of many other

investigators (104,128-130) and validates the claim that cystone has ability to

prevent calcium oxalate crystallization and dissolve in situ any preformed

calcium oxalate crystals.

Combination of HY+SS+ PN

This combination contained equal quantity by weight of Hajrul Yahood

+ Sang Sarmahi+ Phyllanthus Niruri. Treatment of rats with this combined

therapy for studying its stone dissolution and prevention effects showed that

it had almost similar effects as that of cystone.

Comparison of measured parameters in renal tissue and serum

samples between cystone treated rats and rats treated with combination of

HY+SS+PN for studying dissolution effects of these on calcium oxalate

crystals induced as a result of glyoxylate treatment showed that there was no

119

significant difference (p>0.05) in the mean tissue levels for SOD, CAT, GSH

and MDA. Only tissue GPX activity was found to be significantly higher

(p<0.006) in cystone treated group than the group of rats on combined

treatment.

A similar comparison between rats treated with cystone and with

combination of HY+SS+PN for evaluation of antiurolithic effects of both types

of treatments indicated that there was no significant difference (p>0.05) in the

mean MDA level in renal tissue samples and SOD, calcium, magnesium and

oxalate levels in serum samples. However, cystone in comparison to

combined treatment exhibited significantly increased antioxidant enzymes

activities and nephro-protective role.

The histological examination of the rat kidneys treated with HY+SS+PN

showed intact and essentially normal histological architecture in both

preventive and dissolution groups of rats (Fig.IV-6 and IV-12). This is clearly

because of stone dissolving and stone breaking effects of HY and PN

respectively.

120

CHPATER VI

CONCLUSION

Results of present study suggest tremendous lithotriptic, anti-urolithic

and nephro-protective potential of Cystone, Combination and Hajrul Yahood

due to their high antioxidant capacity to inhibit lipid peroxidation in glyoxylate

induced hyperoxaluric rats and also to their ability to reduce oxalate

synthesis.

121

CHPATER VII

RECOMMENDATIONS/ SUGGESTIONS

Cystone and combination of Hajrul yahood, Sang sarmahi and

Phyllanthus niruri are highly recommended as an alternative medicine for the

treatment and prevention of calcium oxalate kidney stones as these are

effective in prevention and dissolution/ disintegration of calcium oxalate

crystals. They are also claimed to be cheap, easily available and devoid of

any side effects . Further research studies are warranted to be carried out to

identify the active ingredients present in them and their mode of action in the

dissolution and prevention of calcium oxalate crystals. With regard to Sang

sarmahi further studies are necessary to be carried out with higher doses to

justify or reject the notion that Sang sarmahi has stone disintegrating effects.

125

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