CHAPTER -4

DIURETIC ACTIVITY STUDIESON THREE MEDICINAL

PLANTS

133

4.1 INTRODUCTION

Abnormalities in fluid volume and electrolyte composition are common

and important clinical problems that can become life—threatening if untreated.

Drugs that block the transport functions of the renal tubules are important

clinical tools in the treatment of these disorders. Diuretics increase the rate of

urine flow and sodium excretion and are used to adjust the volume and I or

composition of body fluids in a variety of clinical situations, including

hypertension, heart failure, renal failure, nephrotic syndrome and cirrhosis.

Technically, the term "diuresis" signifies an increase in urine volume, while

"natriuresis" denotes an increase in renal sodium excretion.

By definition, diuretics are drugs that increase the rate of urine flow;

however, clinically useful diuretics also increase the rate of excretion of Na

(Natriuresis) and of an accompanying anion, usually Cl--Sodium chloride in

the body is the major determinant of extra cellular fluid volume and most

clinical applications of diuretics are directed towards reducing extra cellular

fluid volume by decreasing total body NaCl content.

Diuretics not only alter the excretion of Na but also may modify renal

handling of other cations such as K, Ca 2 and Mg2 and anions such as Cr,

HCO3 and H2 PO4 and uric acid'.

(1)

N )

NH

U')

CH2-

I1

H2NH2N

134

4.2 HISTORY OF DIURETICS

Calomel (mercurous chloride) had been used as a diuretic from the

time of Paracelsus and it was one of the constituents of the famous 'Guy's

Hospital Pill'2 . Organomercurials given by injection were introduced in the

1920s and dominated for nearly 40 years. The carbonic anhydrase (CAse),

inhibitors were developed in the1950s from the observation that early

sulfonamides caused acidosis and mild diuresis. The first modern orally

active diuretic, chlorothiazide (1) was discovered in 1957, and by early 1960s

Its congeners, (thiazide diuretics) were already in common use. Availability of

furosemide (2) and ethacrynic acid (3) by the mid 1960s revolutionized the

pattern of diuretic use. The K sparing diuretics spironolactone (4) and

triamterene (5) were developed in parallel to these2 . Most modern diuretics

were developed when side effects of antibacterial drugs were noted, which

included changes in urine composition and out put. Except for spironolactone

(4), diuretics were developed empirically, without knowledge of specific

transport pathways in the nephron.

(3)

- - Ijfl3

(4)

[I]

135

Cl Cl

0'I

H2C=C -c O—CH2—COOH

C2H5

NH2 N N^^f NH2

N :QNH2

(5)

4.3 RENAL ANATOMY AND PHYSIOLOGY

The basic urine forming unit of the kidney is the nephron, which

consists of a filtering apparatus, the glomerulus, connected to a long tubular

portion consisting of proximal and distal convoluted tubules, the loop of Henle

and a collecting duct, that reabsorbs and conditions the glomerular

136

ultrafiltrate. Each human kidney is composed of approximately one million

neph rons3.

Urine is first formed at the glomerulus, where by ultra filtration the

urinary fluid is freed from its nonfilterable cellular components, such as red

and white blood cells and the plasma proteins. During its passage along the

lumen of the nephron, this practically protein free fluid undergoes many

alterations in its composition. Some of its components are reabsorbed almost

completely into the renal blood vessels; for instance, 98 to 99% of the water

together with various electrolytes (Na', K, Cl- and HCO 3 ), glucose, and urea

filtered at the glomerulus is reabsorbed in the tubule. The remainder, about

1 .5 liters /day, is excreted as urine.

Table 4.1. Quantities of water, glucose and electrolytes filtered, excreted and

reabsorbed by kidneys of man (Mean Normal Values)4.

Plasma meq / 24hour PercentageConcn. meq /l Filtered Excreted [Reabsorbed Reabsorbed

Na 140 23,900 171 23,729 99.3

Cl 103 19,500 171 19,329 99.1

HCO327 5,100 2 5,098 99.9

K 4 684 51 633 80.6

ml/hour

H20 94.0% 169,000 1,500 167,570 99.1%

Glucose --- --- --- 100%

137

Many diuretic agents such as loop diuretics, thiazides, amiloride, and

triamterene exert their effects on specific membrane transport protein at the

luminal surface of renal tubular epithelial cells. Others exert osmotic effects

that prevent water reabsorption in the water—permeable segments of the

nephron e.g.(mannitol), inhibit enzymes e.g.(acetazolamide) or interfere with

hormone receptors in renal epithelial cells e.g.(spironolactone)5'6.

4.4 CLASSIFICATION OF DIURETICS

Historically, the classification of diuretics was based on a mosaic of

ideas such as site of action (loop diuretics), efficacy (high—ceiling diuretics),

chemical structure (thiazide diuretics), similarity of action with other diuretics

(thiazidelike diuretics), effects on potassium excretion (potassium—sparing

diuretics)6 , etc.

4.4.1 Carbonic anhydrase inhibitors

Carbonic anhydrase is present in many nephron sites, including the

luminal and basolateral membranes and cytoplasm of the epithelial cells and

the red blood cells in the renal circulation. However, the predominant location

of this enzyme is the luminal membranes of the proximal tubule cells, where it

catalyzes the dehydration of H2CO3, a critical step in the proximal

reabsorption of bicarbonate. Inhibitors of carbonic anhydrase thus block

sodium bicarbonate reabsorption, causing sodium bicarbonate diuresis and a

reduction in total body bicarbonate stores 1 .The prototypical carbonic

anhydrase inhibitor is acetazolamide (6). Carbonic anhydrase inhibitors also

138

increase phosphate excretion by unknown mechanism but have little or no

effect on the excretion of Ca 2 or Mg2

0

NH CC H3/L=N

si

802

NH2

(6)

The carbonic anhydrase inhibitors are unsubstituted sulfonamide

derivatives. The sulfonamide group is essential for activity. Alkyl

substitutions at this site completely block the effects on carbonic anhydrase

activity.

Carbonic anhydrase inhibition causes significant bicarbonate losses,

resulting in hyperchloremic metabolic acidosis. Because of the toxicity of this

acidosis and the fact that HCO3 depletion leads to enhanced NaCl

reabsorption by the remaining tubule segments in the nephron, the diuretic

effectiveness of acetazolamide decreases significantly with use over several

days. The combination of acetazolamide (6) with diuretics that block Na

reabsorption at more distal sites in the nephron causes a marked natriuretic

139

response in patients with low basal fractional excretion of Na (< 0.2 %) who

are resistant to diuretic monotherapy8.

Inhibition of carbonic anhydrase decreases the rate of aqueous humor

formation, causing a decrease in intraocular pressure. This effect is of value

in the management of several forms of glaucoma. Carbonic anhydrase

inhibitors can also be used to prevent altitude sickness 9. By decreasing

cerebrospinal fluid formation and by decreasing the pH of the cerebrospinal

fluid and brain, acetazolamide can enhance performance status and diminish

symptoms of mountain sickness 10 . Carbonic anhydrase inhibitors can be

useful for correcting a metabolic alkalosis, especially an alkalosis caused by

diuretic induced increase in H excretion'.

4.4.2 Inhibitors of Na - K - 2C1 symport ( Loop diuretics)

Loop diuretics inhibit the activity of the Na - K -2Cr symporter in the

thick ascending limb of the loop of Henle and thus loop diuretics selectively

inhibit NaCl reabsorption in the thick ascending limb of the loop of Henle11.

Inhibitors of Na + - K - 2C symport in the thick ascending limb are highly

efficacious, and for this reason, they sometimes are called high—ceiling

diuretics. The efficacy of loop diuretics is due to a combination of two factors;

(i) Approximately 25% of the filtered Na load normally is reabsorbed by the

thick ascending limb, and (ii) nephron segments past the thick ascending limb

do not possess the reabsorptive capacity to rescue the flood of rejectate

exiting the thick ascending limb. Inhibitors of Na + - K - 2C1 symport are a

140

chemically diverse groups. Only furosemide (2), bumetanide (7), ethacrynic

acid (3) and torsemide (8) are available currently in the United states'.

Furosemide (2) and bumetanide (7) contain a sulfonamide moiety. Ethacrynic

acid (3) is a phenoxyacetic acid derivative and torsemide (8) is a sulfonylurea.

ANH(CH2)3CH3L0

COOH

H2N-02S

(7)

HN - Q\/N /CH3H3C 02S-NH-C-NH-CHUo CH3

(8)

Inhibitors of Na + - K - 2CL symport 12 ' 13 bind to the Na + - K - 2CL

symporter in the thick ascending limb and block its function, bringing salt

transport in this segment of the nephron to a virtual standstill. The molecular

mechanism by which this class of drugs blocks the Na + - K - 2C symporter

is unknown, but evidence suggests that these drugs attach to the Cl- binding

site located in the symporter's transmembrane domain 14 . They also inhibit

Ca 2+ and Mg 2+ reabsorption in the thick ascending limb by abolishing the

141

transepithelial potential difference that is the dominant driving force for

reabsorption of these cations.

Owing to blockade of the Na - K - 2C symporter, loop diuretics

increase the urinary excretion of Na and Cl - profoundly. Abolition of the

transepithelial potential difference also results in marked increase in the

excretion of Ca 2' and Mg 2 . All inhibitors of Na + - K - 2Cl symport increase

the urinary excretion of K and titratable acid15.

In addition to their diuretic activity, loop agents appear to have direct

effects on blood flow through several vascular beds 16 . The mechanisms for

this action are not well defined. Furosemide increases renal blood flow and

causes redistribution of blood flow within the renal cortex. Furosemide and

ethacrynic acid have also been shown to relieve pulmonary congestion and

reduce left ventricular filling pressure in congestive heart failure 17 before a

measurable increase in urinary output occurs, and in anephric patients.

A major use of loop diuretics is in the treatment of acute pulmonary

edema. A rapid increase in venous capacitance in conjunction with a brisk

natriuresis reduces left ventricular filling pressures and thereby rapidly

relieves pulmonary edema. Loop diuretics also are used for the treatment of

chronic congestive heart failure 18,19 when diminution of extra cellular fluid

volume is desirable to minimize venous and pulmonary congestion. Loop

diuretics are also employed in the treatment of edema and ascites of . liver

cirrhosis20 . In patients with a drug overdose, loop diuretics can be used to

142

induce a forced diuresis to facilitate more rapid renal elimination of the

offending drug. Loop diuretics, combined with isotonic saline administration to

prevent volume depletion, are used to treat hypercalcemia2

4.4.3 Inhibitors of Na-CI symport (Thiazide and Thiazidelike

diuretics)

The thiazide diuretics emerged during efforts to synthesize more potent

carbonic anhydrase inhibitors. However, unlike carbonic anhydrase inhibitors,

which primarily increase NaHCO3 excretion, thiazides were found

predominantly to increases NaCl excretion, an effect shown to be

independent of carbonic anhydrase inhibition.

Because the original inhibitors of Na t-CL symport were

benzothiadiazine derivatives, this class of diuretics are also known as thiazide

diuretics'. Subsequently, drugs that are pharmacologically similar to thiazide

diuretics but are not thiazides were developed and are called thiazidelike

diuretics'. The nature of the heterocyclic rings and the substitutions on these

rings may vary among the congeners, but all of them retain, in common with

the carbonic anhydrase inhibitors, an unsubstituted sulfonamide group.

Chlorothiazide (1), hydrochlorothiazide (9), chlorthalidone (10), indapamide

(11), metolazone (12), quinethazone (13), etc are some of the diuretics

belonging to this class.

143

HCl;jN

H2N - 02S02

(9)

:-

(10)

CH3

CI CO - NH

H2N-02S

( 11 ) t^,/

CI NCH

H2N-02S

0

CH3

(12)

HN

*'^f CA

H2N-O2S

0

(13)

Micropuncture and in situ microperfusion studies clearly indicate that

thiazide diuretics inhibit NaCl transport in the distal convoluted tubule (DCI).

Thiazides inhibit NaCl reabsorption from the luminal side of epithelial cells in

the DCT22'23

Inhibitors of Na + - Cl symport increase Na and Cl- excretion only

moderately and they have milder diuretic action than do the loop diuretics

because approximately 90% of the filtered Na load is reabsorbed before

reaching the distal convoluted tubule 24 ' 25 . Like inhibitors of Na + - K - 2Cl

144

symport, inhibitors of Na - Cl - symport also increase the excretion of K and

titratable acid. In contrast to the situation in the loop of Henle, where loop

diuretics inhibit Ca 2+ reabsorption, thiazides actually enhance Ca2

reabsorption in the distal convoluted tubule. Inhibition of Na t-Cr symporter in

the luminal membrane decreases intracellular Na levels, thereby increasing

the basolateral exit of Ca 2 via enhanced Na - Ca2 exchange26

Thiazide diuretics are used for the treatment of the edema associated

with heart (congestive heart failure), liver (hepatic cirrhosis) and renal

(nephrotic syndrome, chronic renal failure and acute glomerulonephritis)

disease.

Thiazide diuretics decrease blood pressure in hypertensive patients27

and they are used widely for the treatment of hypertension either alone or in

combination with other anti hypertensive drugs. They also reduce

cardiovascular morbidity and mortality in hypertensive patients 28. Thiazides

have proved useful in the treatment of kidney stones caused by

hypercalciuria. Approximately two—thirds of all renal stones contain calcium

phosphate or calcium oxalate. Many patients with such stones exhibit a renal

"leak" of calcium that causes hypercalciuria. This can be treated with thiazide

diuretics, which enhance calcium reabsorption in the distal convoluted tubule

and thus reduce the urinary calcium concentration 26. Since other halides are

excreted by renal processes similar to those for Cr, thiazide diuretics may be

useful for the management of Br intoxication.

145

4.4.4 Potassium—sparing diuretics

These are either aldosterone antagonist or directly inhibit Na channels

in the distal tubule and collecting duct cells to indirectly conserve K ions.

This type of diuretics are further classified into two types.

4.4.4.1 Inhibitors of renal epithelial Na channels

Triamterene (5) and amiloride (14) are two nonsteroidal organic bases

with identical action. Their most important action is to decrease K excretion,

particularly when it is high due to large K intake or use of a diuretic that

enhances K loss, along with a small increase in Na excretion'. The luminal

membrane of late distal tubule and collecting duct cells expresses a distinct

'amiloride sensitive' Na channel through which Na enters the cell down its

electro—chemical gradient which is generated by Na K ATPase operating at

the basolateral membrane. The higher permeability of the luminal membrane

for Na depolarizes the luminal membrane but not the basolateral membrane,

creating a lumen - negative transepithelial potential difference. This Na entry

partially depolarizes the luminal membrane creating a - 15 mV transepithelial

potential difference which promotes secretion of K into the lumen through K

channels. Though there is no direct coupling between Na and K channels,

more the delivery of the Na to the distal nephron, greater is its entry through

the Na channel. Because of this the luminal membrane is more depolarized

as a result the driving force for K secretion is augmented. Carbonic

anhydrase inhibitors, loop diuretics and thiazide diuretics increase the delivery

146

of Na to the late distal tubule and collecting duct, a situation that often is

associated with increased K and H excretion.

Considerable evidence indicates that amiloride (14)2930 and

triamterene (5) block the epithelial Na channels in the luminal membrane of

principal cells in the late distal tubule and collecting duct perhaps by

competing with Na for negatively charged areas within the pore of the Na

channel.

ci N CO—NH—C—NH2

NH

H2NNNH2

II

(14)

4.4.4.2 Antagonists of mineralocorticoid receptors (Aldosterone

antagonists)

Epithelial cells in the late distal tubule and collecting duct contain

cytosolic mineralocorticoid receptors that have a high affinity for aldosterone.

Aldosterone acts on the late distal tubule and collecting duct cells by

combining with an intracellular mineralocorticoid receptor which induces the

formation of aldosterone induced proteins (AlPs) which promote Na

reabsorption and K secretion.

13c ZNpO

CH3j'O

I THIt CH,0

(15)

Drugs such as spironolactone 3 ' (4) and eplerenone 32 (15) competitively

inhibit the binding of aldosterone to the minera loco rticoid receptors.

Spironolactone acts from the interstitial side of the tubular cell, combines with

the mineralocorticoid receptor and inhibits the formation of AlPs in a

competitive manner. Unlike the mineralocorticoid receptors - aldosterone

complex, the mineralocorticoid receptor - Spironolactone complex is not able

to induce the synthesis of AlPs. Since spironolactone and eplerenone block

the biological effects of aldosterone 33 , these agents also are referred to as

aldosterone antagonists'.

4.4.4.3 Effect of K - sparing diuretics on urinary excretion

Since the late distal tubule and collecting duct have a limited capacity

to reabsorb solutes, blockade of Na channels in this part of the nephron only

mildly increases the excretion rates of Na and Cl- (approximately 2% of

filtered load). Blockade of Na channels hyperpolarizes the luminal

147

membrane, reducing the lumen —negative transepithelial voltage and thus

148

indirectly inhibit K excretion. The effects of mineralocorticoid receptor

antagonists on urinary excretion are very similar to those induced by renal

epithelial Na - channel inhibitors.

Because of the mild natriuresis induced by Na -channel inhibitors,

these drugs seldom are used as sole agents in the treatment of edema or

hypertension. Rather, their major utility is in combination with other diuretics.

The ability of Na - channel inhibitors to reduce K excretion tends to off set

the kaliuretic effects of thiazide and loop diuretics. Consequently, the

combination of a Na - channel inhibitor with a thiazide or loop diuretic tends

to result in normal values 34 of plasma K. As with other K - sparing diuretics,

spironolactone (4) often is coadministered with thiazide or loop diuretics in the

treatment of edema and hypertension. Such combinations result in increased

mobilization of edema fluid while causing lesser perturbations of K

homeostasis. Spironolactone(4) is particularly useful in the treatment of

primary hyperaldosteronism and of refractory edema associated with

secondary aldosteronism. Spironolactone is considered the diuretic of choice

in patients with hepatic cirrhosis. Spironolactone, added to standard therapy,

substantially reduces morbidity and mortality35 and ventricular arrhythmias 36 in

patients with heart failure.

4.4.5 Osmotic diuretics

Osmotic diuretics are agents that are freely filtered at the glomerulus,

undergo limited reabsorption by the renal tubule, and are relatively inert

pharmacologically. Osmotic diuretics are administered in large enough doses

149

to increase significantly the osmolality of plasma and tubular fluid. The four

currently available osmotic diuretics' are glycerin (16), Isosorbide (17),

mannitol (18) and urea (19).

HOH

HO

OH

OH(16)

(17)

OH OHOHOH

LT

I I I IOH 011

(18)

0A

H2N NH2

(19)

An osmotic agent that is not transported causes water to be retained in

these segments and promotes a water diuresis. Osmotic diuretics limit water

reabsorption primarily in those segments of the nephron that are freely

permeable to water viz, the proximal tubule and the descending limb of the

loop of Henle. The presence of a non reabsorbable solute such as mannitol

(18) prevents the normal absorption of water by interposing a countervailing

osmotic force. As a result, urine volume increases in conjunction with mannitol

excretion 37 . The concomitant increase in urine flow rates decreases the

contact time between fluid and the tubular epithelium, thus reducing Na

150

reabsorption. However, the resulting natriuresis is of lesser magnitude than

the water diuresis, leading eventually to hypernatremia.

Osmotic diuretics increase the urinary excretion of nearly all

electrolytes, including Na', K, Ca 2+ Mg 2 , C, HCO 3 and PO43

A rapid decrease in glomerular filtration rate (GFR) i.e., acute renal

failure (ARF), is a serious medical condition that occurs in 5% of hospitalized

patients and is associated with a significant mortality rate. Acute tubular

necrosis i.e., damage to tubular epithelial cells, accounts for most cases of

acute renal failure. The renal protection afforded by mannitol (osmotic

diuretic) may be due to removal of obstructing tubular casts, dilution of

nephrotoxic substances in the tubular fluid, and I or reduction of swelling of

tubular elements via osmotic extraction of water38'39.

Another use for mannitol and urea is in the treatment of dialysis

disequilibrium syndrome. Too rapid a removal of solutes from the

extracellular fluid by hemodialysis or peritoneal dialysis results in a reduction

in the osmolality of the extracellular fluid. Consequently, water moves from

the extracellular compartment into the intracellular compartment, causing

hypotension and central nervous system symptoms. Osmotic diuretics

increase the osmolality of the extracellular fluid compartment and thereby shift

water back into the extracellular compartment.

By increasing the osmotic pressure of the plasma, osmotic diuretics

extract water from the eye and brain. All four osmotic diuretics are used to

151

control intraocular pressure during acute attacks of glaucoma and for short

term reductions in intraocular pressure both preoperatively and

postoperatively in patients. Mannitol and urea are also used to reduce

cerebral edema and brain mass before and after neurosurgery'.

4.4.6 Changes in urinary electrolyte patterns in response to

diuretic drugs.

Changes in urinary electrolyte patterns and the primary site of actions

are presented in Table (4.2.)°

Table 4.2 Changes in urinary electrolyte patterns in response to diuretic

drugs.

Urinary Electrolyte PatternsAgent

Na K CI

Carbonic anhydrase inhibitors + ++ (+)

Inhibitors of Na- K-2CrSymport (Loop ++ ++ ++diuretics)

Inhibitors of Na-CISymport (Thiazide + ++ +diuretics)

K Sparing agent + - +

Osmotic diuretics ++ + +

++ marked increase; + mild to moderate increase; (+) slight increase;

- decrease

152

4.5 PAST WORK ON DIURETIC ACTIVITY OF MEDICINAL PLANTS

Diuretic agents are most widely prescribed categories of drugs in the

world. But synthetic diuretics have serious side effects such as diabetonic

effect, electrolyte imbalance, impotence and hyperuricemia. Hence the

search for herbal drugs to act as diuretics are inevitable and can alter

therapeutic efficacy without any adverse effects 41 . Several indigenous drugs

have already been screened by various workers for their diuretic activity.

Four indigenous drugs viz. Boerhaavia repens, Boerhaavia rependa,

Pedalium murex and Tribulus terrestris have been tested for diuretic

activity by Krishnan Haravey 42. The effects of administration of Stevia

rebaudiana extracts on renal function and mean arterial pressure in normal

Wister rats have been evaluated by Melis 43 . The extract was found to induce

hypotension, diuresis and natriuresis. Traditional medicines reported to be

used as anti—hypertensives or diuretics from different regions in the world

have been investigated for inhibition of the angiotensin converting enzyme44.

Alcoholic extract of Aerva lanata was tested for diuretic activity in rats.

The parameters measured for diuretic activity were total urine volume,

sodium, potassium and chloride content. The results clearly indicated that the

alcoholic extract at a dose of 800 mg I kg acted as a diuretic, with respect to

control 45 . Natriuretic and diuretic responses induced by garlic powder in

anaesthetized dogs were evaluated by Pantoja 46 et al. Garlic extract also

inhibited sodium transporting epithelia and decreased ATPase activity47'48.

153

Diuretic and natriuretic effects of chromatographically purified fraction of garlic

(Allium sativum) have also been performed by Pantoja et a!49

The diuretic activity of the stem—bark extracts of Steganotaenia

araliacea and effects on urine electrolytes in rats was studied by

Agunu et a!50. Eighty species of vascular plants were tested for their ability to

inhibit the angiotensin converting enzyme which plays an important role in the

regulation of blood pressure and diuresis 51 . Diuretic effect of aqueous extracts

of Rosmarinus officinalis and Centaurium erythraea on Wistar rats have

been evaluated by Haloui et a!52. Hnatyszyn and co—workers have assessed

the diuretic activity of aqueous extract of Phyllanthus sellowianus

Euphorbia hirta is locally used in Africa and Australia to treat

numerous diseases, including hypertension and edema. The diuretic effect of

the E. hit-ta leaf extracts were assessed in rats using acetazolamide and

furosemide as standard diuretic drugs 54. In the Sri Lankan Traditional

Medicine, Spilanthes acmella flowers are claimed to possess powerful

diuretic activity. The diuretic potential of Spilanthes acmella flowers was

evaluated in rats using a cold—water extract. The extract caused marked

increase in urinary Na and K levels and a reduction in the osmolarity of

urine55. Maydis stigma (Corn silk) is a herbal drug reputed for the treatment

of urinary ailments in various traditional systems of medicine. Diuretic activity

of M. stigma has been evaluated in adult male Wistar rats for eighty days by

Maksimovic et a!56

154

The effects of Clematis montevidensis on urinary excretion of water,

sodium and potassium were investigated in rats loaded with isotonic saline

solution. The infusions of the root and aerial part of C. montevidensis

showed a moderate diuretic activity. This effect could be due to the presence

of oleanolic acid (20) isolated from this plant57. The aerial parts of Bidens

odorata are used in Mexican folk medicine to treat renal diseases. The

diuretic response to aqueous extract of this plant was compared with that

induced by furosemide by Camargo et a! 58• Jawarish Zarooni Sada (JZS) is a

polyherbal preparation containing fifteen ingredients, mainly described to be

diuretic and nephroprotective and used widely in the Unani system of

medicine in the management of renal diseases. Diuretic and nephroprotective

effects of Jawarish Zarooni Sada have been evaluated by Afzal et a!59. Nedi

et al. have investigated the diuretic activity of different extracts of Carissa

edulis in rats using hydrochloro thiazide as a standard drug and the findings

support the traditional use of Carissa edulis as a diuretic agent60

Sripanidkulchai et al. have investigated the diuretic activity five Thai

indigenous medicinal plants in rats and have come to the conclusion that only

two plants viz. Ananas comosus and Carica papaya demonstrated

significant diuretic activity61

(20)

ro

OOH

155

Through ethnobotanical surveys in Guatemala, about 250 plants were

identified for use in the treatment of urinary ailments 62 . From 67 of these,

aqueous extracts were prepared to investigate their oral diuretic activity in

albino rats. The trials demonstrated that in 33 cases urinary excretion was not

significantly increased, in 20 cases intermediate activity was seen and in 14

cases high diuretic activity was noted. Oral administration of the water extract

of Spergularia purpurea at different doses produced a significant and dose

dependent diuresis and increase in electrolyte excretion. Chronic treatment

with S. purpurea decreased significantly urine osmolality, while a slight

increase in glomerular filtration rate was also observed 63

Erva Tostao is called "Punarnava" in India. It is botanically called

Boerhaavia diffusa. This plant has a long history of use by indigenous and

tribal people, and in Ayurvedic medicine in India where the roots are

employed for many purposes, including liver, gallbladder, kidney, renal and

urinary disorders64

156

The diuretic action of Erva Tostao (Boerhaavia diffusa) has been

studied and validated by scientists in several studies which help to explain its

long history of use in various kidney and urinary conditions 65 ' 66 . Researchers

showed in the mid 1950's that low dosages (10 mg/kg to 300 mg/kg)

produced strong diuretic effects while higher dosages (>300 mg /kg) produced

the opposite effects, reducing urine out put67. Other researchers who

followed, verified these diuretic and anti—diuretic properties as well as the

beneficial kidney and renal effects of Erva Tostao roots in animals and

humans 65,66

Extracts of 32 medicinal plants used popularly for their presumed

diuretic and / or anti hypertensive properties were tested for diuretic effects in

conscious unrestrained rats 68 . The most significant diuretic effect was

observed with Hedychium coronarium sheath and leaf—blade extracts.

Methanol extract of Strychnos potatorum seeds was evaluated for its

diuretic activity in Wister albino rats. Total urine volume, excretion of Na and

K ions and Cl - of drug treated groups increased significantly with respect to

control group and the diuretic effect was comparable with that of the standard

drug furosemide69. The aqueous extract of the bark of Raphanus sativus

has been tested for its antiuro lithiatic and diuretic activity by Vargas et a170.

Chloroform extracts of Equisetum fluviatile, E. hiemale Var. affine,

E. giganteum and E. myriochaetum were studied to determine diuretic

activity in mice using hydrochlorothiazide, spironolactone and furosemide as

standard drugs for comparison 71 . It was found that the most active plant was

157

E. hiemale Var. affine, followed by E. fluviatile, E. giganteum and

E. myriochaetum, producing an effect similar to that of hydrochlorothiazide in

relation to the excretion of sodium, potassium and chloride. An alcoholic

extract of Cucumis trigonus was studied for its diuretic activity in albino rats

using hydrochlorothiazide as a standard drug for comparison. The extract

exhibited a dose—dependent saluretic effect reaching a peak at 4 hours.

Unlike hydrochlorothiazide, the extract does not affect potassium excretion72.

Crude aqueous extract of Cleome rutidosperma was investigated for diuretic

and antibacterial activity by Bose et a!73. The diuretic activity was tested in

rats orally and compared with furosemide as the standard and the extract was

found to possess signifcant dose dependent diuretic activity.

Dandelion, Taraxacum officinale leaves have traditionally been used

to increase urine production and excretion. It is one of the most effective

diuretic herbs; its effect being comparable to that of the drug fruosemide. In

addition to its efficacy as a diuretic, Dandelion leaf has added benefits as a

rich source of potassium, replacing all potassium that is flushed from the body

via diuresis 74,75 . Diuretic action of an aqueous extract of Lepidium

latifolium has been studied by Eduardo et a!76 . Aqueous and alcoholic

extracts of Vitis vinifera leaves were tested for diuretic activity in rats by

Shastry et a177. using furosemide as a reference diuretic. The infusion and

the essential oil of Juniper berries, Juniperus communis as well as

terpinen-4—ol were tested for diuresis response in rats by Gordana et a178

and they came to the conclusion that the diuretic activity of Juniper berries

158

couldn't be attributed only to the essential oil but also to hydrophilic drug

constituents.

Intravenous administration of the aqueous extract of Retama raetam

produced a significant increment on diuresis from the second hour to the

fourth hour in normal rats 79 . The increase in diuresis was associated with an

elevation of glomerular filtration rate and a significant decrease of urinary

osmolarity. Diuretic activity of infusions of Artemisia thuscula was evaluated

in saline loaded rats by Benjumea et a! 80 . Urinary excretion of water, ph,

density, conductivity and Na , K and Cl content were investigated and the

drug showed potassium sparing effect.

In the Moroccan traditional medicine, the ripe fruits of Carum can'i and

the leaves of Tanacetum vulgare, two widelavailable plant materials, are

used as diuretics81 . The diuretic potential of aqueous extracts of Carum carvi

fruit (Caraway) and the leaves of Tanacetum vulgare (Tansy) in normal rats

after acute and sub—chronic oral administration was evaluated. Both extracts

increased urinary levels of Na and K, to about the same extent, while

furosemide (reference diuretic) increased urinary levels of only Na and

decreased urinary K. The diuretic activity of an infusion and the methanol

extract of Withania aristata was evaluated in laboratory rats by Martin

Herrera et al. Both the infusion and the methanol extract showed a significant

diuretic effect compared with non—treated controls, with notable increase in

the rate of water and sodium excretion. There was also a potassium retention

effect observed 82

159

4.6 SCOPE OF THE PRESENT STUDY

Phytochemicals have played a vital role in the treatment of diseases in

the past and will continue to do so in the future. Although synthetic drugs can

produce dramatic results in most cases, the side—effects associated with them

are a major concern. The source of many compounds used in modern

medicine today can be traced down to plant origin. Whether or not scientific

justification is available for the use of most plant products, the continued use

of these compounds is due to their safety profile, ease of availability and also

economic reasons. Each medicinal plant that has been used in the traditional

system of medicine must be scientifically tested in order to bring forth its

active principle that might be effectively used as a phytomedicine. However,

there should be adequate data from in-vivo and in-vitro studies to validate

the therapeutic potential claimed. There is a need to establish the

pharmacological activities for identifying and comparing the various

preparations for potency. Quite a good number of indigenous drugs have

been claimed to have diuretic effect in the Ayurvedic system of medicine and

several drugs have already been screened by various workers for their

diuretic activity. In the present study the diuretic activity of the methanolic

extracts of the roots of Dichrostachys cinerea, Hemidesmus indicus and

the thallus of a lichen, Parmelia perlata have been tested in male albino rats.

The advantage of traditional systems of medicine with respect to their safety

and efficacy could result in a better utilization of our herbal resources with

application of the scientific methods.

160

4.7 RESULTS

4.7.1 Dichrostachys cinerea

The root of Dichrostachys cinerea is extracted successively with

different organic solvents such as, petroleum ether (40 0-60°C), benzene,

chloroform, methanol and water. The methanolic extract was condensed and

evaporatd to dryness under vacuum (yield 23 g, 4.6%) and used for the

present study. The diuretic activity was evaluated on male albino rats using

Lipschitz's method 83 . The parameters measured for diuretic activity are total

urine volume, sodium, potassium and chloride contents of urine samples. The

results are presented in Tables 4.3 and 4.4. Urea was used as a reference

diuretic. All the results are expressed as mean ±standard error. Test of

significance is statistically analysed using Student's t' test84

The present study reveals that the methanolic extract of the root of

D. cinerea at the 250 mg/kg and 500mg/kg doses possess good diuretic

activity. The urine volume was found to be the maximum at 250 mg/kg dose.

Since the urine excretion is found to be the maximum at 250 mg/kg dose, Na',

K and Cl- concentrations in this particular dose have been determined. Urea

increased the urine volume 2.3 fold with respect to the control (Table 4.3.)

whereas methanolic extract of D. cinerea at 250 mg/kg increased the urine

volume around 2 fold. Urea increased the Na excretion level by 1.8 fold, K

excretion level by 1.2 fold and Cl - excretion level by 1.3 fold when compared

to that of the control. The methanolic extract of D. cinerea at 250 mg/kg dose

Table 4.3

Diuretic activity of methanolic extract ofDichrostachys cinerea root on rats

TreatmentDose Urine Volume

(mg/kg bw) (ml/rat)

Control Normal Saline, 25 ml 2.0 ± 0.08

100 2.1 ± 0.08

Dichrostachys cinerca 250 3.9 ± 0.16**

500 3.3±0.16**

Urea 750 4.6 ± 0.33**

Student's t-test was performed (Fisher, 1950).Each value is the mean ± SE of six rats weighing 250 - 300 g.Statistically significant from vehicle control ** P < 0.001

Table 4.4

Effect of methanolic extract of Dichrostachys cinerea

on Na', K+ and Cl concentration in the urine of albino rats

Electrolyte excretion

Treatment

Dose Na K Cl— Na/K(mg/kg bw) (meq / I) (meq I I) (meq / I) ratio

ControlNormal 52.60±2.4 98.08±2.8 107.36±6.1 0.54

Saline 25 ml

D.cinerea 250 73.37 ± 2 . 0** 165.92 ± 2 .4** 208.89 ± 5•7** 0.44

Urea 750 93.48±2.9** 114 . 70±2 . 0** 138.36±6.5 0.81

Student's t-test was performed (Fisher, 1950).Each value is the mean ± SE of six rats weighing 250 - 300 g.Statistically significant from vehicle control : ** P < 0.001

161

increased the Na excretion level by 1.4 fold, K excretion level by 1.7 fold

and Cl- excretion level by 2 fold. K and Cl- excretion levels are significantly

increased to nearly 1.5 fold when compared to that of urea (Table 4.4).

The sodium and potassium ratio is decreased to half fold when compared to

that of urea.

4.7.2 Hemidesmus indicus

The root of Hemidesmus indicus has also been extracted with various

solvents as mentiond above. Among the various extracts only the methanolic

extract has been subjected to the present analysis. The methanolic extract of

H. indicus was condensed and evaporated to dryness under vacuum (yield

21 g, 4.2%) and used. Male albino rats weighing between 250 —300 g have

been used for the study. The parameters taken for individual rat are body

weight before and after test period, total urine volume, concentration of Na,

K and Cl in urine, according to the method of Lipschitz et a!83. Urea is used

as a reference diuretic and the results are presented in Tables 4.5 and 4.6.

All the results are expressed as mean ± standard error. The data is analysed

using Student's 't' test for statistical significance 84

The present study reveals that the methanolic extract of the root of

H. indicus at the dose of 250 mg/kg shows significant diuretic activity and the

urine volume is the highest at this dose (Table 4.5.) beyond which it was

reduced. Therefore in the present study, the Na, K and Cl- concentrations at

this particular dose are determined (Table 4.6.) and there is no significant

Table 4.5

Diuretic activity of methanolic extract of Hemidesmus indicus root on rats

TreatmentDose Urine Volume

(mg/kg bw) (ml/rat)

Control Normal Saline, 25 ml 2.0 ± 0.08

100 2.0±0.08

Hemidesmus indicus 250 2.6 ± 0.08*

500 1.9 ± 0.12

Urea 750 4.6±0.33**

Student's t-test was performed (Fisher, 1950).Each value is the mean ± SE of six rats weighing 250 - 300 g.Statistically significant from vehicle control :*P < 0.05; ** P < 0.001

Table 4.6

Effect of methanolic extract of Hemidesmus indicuson Na+ , K+ and Clconcentration in the urine of albino rats

Electrolyte excretion

TreatmentDose Na Cl Na/K

'(mg/kg bw) (meq / I) (meq / I) (meq / I) ratio

ControlNormal

7.39 ± 2.2 33.82 ± 2.8 18.02 ± 4.1 0.22Saline 25 ml

H. indicus 250 7.39 ± 2.0 33.49 ± 2.6 15.17 ± 4.6 0.22

Urea 750 26.19 ± 2 . 8** 54.92 ± 4* 48.8 ± 4** 0.47

Student's t-test was performed (Fisher, 1950).Each value is the mean ± SE of six rats weighing 250 - 300 g.Statistically significant from vehicle control : < 0.05; ** P < 0.001.

162

change in these values compared to that of the control. Urea increased the

urine volume by about 2:3 times when compared with control whereas the

methanolic extract of H. indicus at 250 mgjkg dose increases the urine

volume by about 1.3 times, compared to that of the control. The Na value is

significantly increased, whereas K level is also slightly increased in the

urea treated group, when compared to that of the control.

4.7.3 Parmelia per!ata

The thallus of Parmelia perlata has been successively extracted with

petroleum ether (40 0-60°C), benzene ,chloroform , methanol and water and

the methanolic extract is used for the present study. This indigenous drug

has been tested for its diuretic activity and the relative diuretic potency has

been estimated and compared with urea. The method adopted during the

present investigation is that described by Lipschitz et a183. For diuretic activity,

healthy male albino rats weighing between 250-300 g are used The results

are statistically analysed using Student's 't' test84 and are presented in Tables

4.7. and 4.8.

The methanolic extract of Parmelia perlata produces significant dose

dependent decrease in urinary excretion with respect to the control. The

maximum decrease in urine output is observed for the higher dose ie.

500 mg /kg and the lower dose 100 mg/kg do not produce any change in

urinary excretion. There is no significant change in the concentration of Na,

K and Cl- in the drug treated group with respect to the control.

Table 4.7

Diuretic activity of methanolic extract of Parmelia perlata plant body on rats.

TreatmentDose Urine Volume

(mg/kg bw) (ml/rat)

Control Normal Saline 25 ml 2.0 ± 0.08

100 2.0±0.80

Parmelia perlata 250 1.3 ± 0.08**

500 0.9 ± 0.70*

Urea 750 4.6 ± 0.33**

Student's t-test was performed (Fisher, 1950).Each value is the mean ± SE of six rats weighing 250 - 300 g.Statistically significant from vehicle control: * P < 0.05, ** P < 0.001

Table 4.8

Effect of methanolic extract of Parmelia perlata

on Na, K and Clconcentration in the urine of albino rats.

Treatment

Electrolyte excretion

Dose Na meq/lK ci Na /K

mg/kg bw meq/l meq/l

ControlNormal 9.89 ± 2.2 51.79 ± 2.8 31.38 ± 4.1 0.19

Saline 25ml

Parmelia250 11.95±2.6 45.65± 4 23.58±3.1 0.26periata

Urea 750 26.74 ± 3 . 1* 56.52 ± 6 81.80 ± 4 . 6** 0.47

Student's t-test was performed (Fisher, 1950).Each value is the mean ± SE of six rats weighing 250 - 300 g.Statistically significant from vehicle control : * P < 0.05, ** P < 0.001

163

4.8 DISCUSSION

Herbs that stimulate the kidneys are traditionally used to reduce

edema. Herbal diuretics do not work the same way that drugs do and thus it

is unclear whether such herbs would be effective for this purpose. Solidago

cnadensis (Goldenrod) is considered one of the strongest herbal diuretics85

Animal studies show, at very high amounts, that dandelion, Taraxacum

officinale leaves possess diuretic effects that may be comparabale to the

prescription diuretic, furosemide Corn silk (Zea mays) has also long

been used as a diuretic, though a human study do not find that it increases

urine output86 . Aescin (21) isolated from horse chestnut seed, has been

shown to effectively reduce post surgical edema in preliminary trials87.

Horsetail 71 (Equisetum spp.) has a diuretic action that accounts for its

traditional use in reducing mild edema. The volatile oils in Juniper cause an

increase in urine volume and in this way can theoretically lessen edema 78,85

Cleavers, Galium aparine is one of numerous plants considered in ancient

times to act as a diuretic88. It is therefore used to relieve edema and to

promote urine formation during bladder infections. Medicinal plants are the

oldest known health—care products. Medicinal plants are also important for

pharmacological research and drug development, not only when plant

constituents are used directly as therapeutic agents, but also when they are

used as basic materials for the synthesis of drugs or as models for

pharmacologically active compounds.

OH

HO/IOH

164

H

HC

H at,,,

HOH

(21)

Animal techniques for evaluating diuretics have been critically reviewed

by Baer89 , Ginsburg 90 and Lazarow91 . Rats are convenient animals for

screening of diuretic agents because of the cost and ease in maintaining large

number of animals. In the present study diuretic activites of the methanolic

extracts of three plants viz. Dichrostachys cinerea, Hemidesmus indicus

and Parmelia perlata have been evaluated in male albino rats. Out of three

drugs tested for diuretic activity only Dichrostachys cinerea gave promising

results. Urea increases the urine volume 2.3 fold whereas D. cinerea

methanolic extract at 250 mg/kg dose increases the urine volume around 2

165

fold. Potassium and chloride excretion levels, for D. cinerea are even greater

than that of the reference diuretic. Since the methanolic extract of D. cinerea

acts as a potent kaliuretic, the sodium and potassium ratio is decreased to

half fold when compared to urea. Previous study on the diuretic activity of

Aerva lanata also indicated similar results45

Methanolic extract of H. indicus root at 250 mg/kg dose shows

significant diuretic activity, but there is no appreciable change in electrolyte

excretion. The data reveals that the methanoilc extract of H. indicus acts as a

good aquaretic rather than a kaliuretic or saluretic. The diuretic effect of

H. indicus is not comparable to that of urea, which is a known osmotic

diuretic. A valuable diuretic should cause marked natriuresis also. This is not

observed in the case of H. indicus, which is more aquaretic. Therefore

H. indicus can possibly be used as a diuretic in combination with other herbs

only, which are natriuretic and kaliuretic.

The methanolic extract of Parmelia perlata was found to exhibit

antidiuretic effect where there is dose dependent decrease in urinary

excretion. The maximum decrease in urinary excretion is observed for

500 mg/kg dose. The decease in urinary excretion is unaccompanied by

appreciable change in electrolyte excretion. The sustained oliguric action

(reduction in passage of urine) is the action which is clinically useful in

combating the polyuria of diabetes insipidus. In the absence of antidiuretic

hormone (ADH), large volumes of diluted urine are excreted (diabetes

insipidus). Under the influence of antidiuretic hormone, the distal tubule and

166

the collecting duct of the nephron become permeable to water, leading to a

reduction in the total urine volume. The electrolyte pattern of the urine,

however, is not altered. Absence of antidiuretic hormone causes diabetes

insipidus. Drugs like morphine, nicotine (22) and barbiturates can stimulate

the release of antidiuretic hormone. Alcohol and chlorpromazine (23), on the

other hand, depress antidiuretic hormone release 92

C1 N

Me

N"

NkH Me

(22)

(23)

The antidiuretic activity may also be assayed on animals. Lindquist and

Rowe have used the rabbit to assay the antidiuretic activity of vasopressin

(ADH)93 . Larson carried out studies 94 which indicate that enzyme processes

in the liver and kidney attack vasopressin and terminate its activity. However

mechanism behind the antidiuretic activity of P. perlata is not known, the

antidiuretic activity of this plant drug could be due to the phytoconstituents of

the plant species.

167

In the case of D. cinerea and H. indicus, lower dosage (250 mg/kg)

produces strong diuretic effect where as higher dosage (500 mg/kg)

decreases the urine flow.

The diuretic action of Erva Tostao64 (Boerhaavia diffusa) has been

studied and validated by Scientists in several studies, which help to explain its

long history of use in various kidney and urinary conditions. Researchers

showed in the mid 1950's that low dosages (10 mg/kg to 300 mg/kg) of

B. diffusa produced strong diuretic effects while higher dosages (>300

mg/kg) produced the opposite effect, reducing urine output". Other

researchers who followed, verified these diuretic and anti-diuretic properties

as well as the beneficial kidney and renal effects of Erva Tostao roots in

animals and humans65'66

In the case of D. cinerea maximum diuretic effect is observed in 250

mg/kg dose and it is found to decrease beyond that. In the case of

H. indicus also maximum diuretic effect is observed in 250 mg/kg dose and

an opposite effect is observed at higher dose 500 mg/kg, reducing urine out

put. Perhaps the higher dose used in the study may be inhibitory to the

mechanism of diuresis. In H. indicus at 250 mg/ kg dose the increase in

urine volume is unaccompanied by a corresponding increase in electrolyte

excretion, thus the extract behaves like a water diuretic rather than a kaliuretic

or saluretic.

Aminoacids and flavonoids are reported to possess calculi dissolving

ability95 and diuretic activity96 . In the present study, the diuretic activity of

168

D. cinerea and H. indicus may be due to the presence of these polyphenolic

compounds. In similar studies, the fungus Polyporus umbellatus is reported

to possess strong diuretic action 97 . The major components isolated form it are

proteins (which are composed of aminoacids) and ergosterol. Similary a

flavonoid isolated from Helichrysum bracteatum exhibited significant diuretic

activity in rats98 . Lepidium latifolium containing phenols and sterols was

found to exhibit significant diuretic activity76

Flavonoids are a group of polyphenolic compounds, which are widely

distributed throughout the plant kingdom. Several double—blind trials have

found that 400 mg per day of coumarin, a flavonoid found in a variety of

herbs, can improve many types of edema, including lymphedema after

surgery9902. A group of semisynthetic flavonoids known as

hydroxyethylrutosides are also beneficial for some types of edema' 03-105

A combination of the flavonoids diosmin and hesperidin has been investigated

for the treatment of a variety of venous circulation disorders' 06 . Because

coumarin, hydroxyethylrutosides and diosmin are not widely available in the

United States, other flavonoids, such as quercetin, rutin, or anthocyanosides

(from bilberry) have been substituted by doctors in an attempt to obtain similar

benefits. In one study, quercetin in amounts of 30-50 mg per day corrected

abnormal capillary permeability (leakiness), 107 an effect that might improve

edema. A similar effect has been reported with rutin at 20 mg three times per

day108

169

Diuretics remain the cornerstone' for the treatment of edema or volume

overload, particularly that owing to congestive heart failure, ascites, chronic

renal failure and nephrotic syndrome.

The medicinal plants of present investigation contain several types of

phytochemicals including flavonoids that are believed to be responsible for

their diuretic action.

The exact site of action of these herbal diuretics is difficult to delineate

in this preliminary studies. As urea was the only drug taken as the standard

during the present investigation, the diuretic activity of all the drugs was tested

for a maximum period of five hours only.

4.9 CONCLUSIONS

Traditional medicines are used by about 60 per cent of the world's

population. These are not only used for primary health care not just in rural

areas in developing countries, but also in developed countries as well where

modern medicines are predominantly used. While the traditional medicines

are derived from medicial plants, minerals and organic matter, the herbal

drugs are prepared from medicinal plants only. Use of plants as a source of

medicine has been inherited and is an important component of the health care

system in India. In the Indian systems of medicine, most practitioners

formulate and dispense their own recipes, hence this requires proper

documentation and research. The major hindrance in the amalgamation of

herbal medicines into modern medical practices is the lack of scientific and

170

clinical data and better understanding of efficacy and safety of the herbal

products. Herbs that stimulate the kidneys were traditionally used to reduce

edema. In the present investigation three medicinal plants viz.

Dichrostachys cinerea, Hemidesmus indicus and Parmelia perlata which

are used by traditional medical practioners for the treatment of urinary

disorders have been screened experimentally on saline—loaded albino rats for

diuretic activity. The diuretic activity has been determined with reference to

urea as standard. All the three drugs exhibited varied degrees of diuretic

activity. Out of the three drugs tested, D. cinerea gave promising results

where significant increase in urine volume and electroyte excretion were

observed. H. indicus showed moderate increase in urinary excretion which

was unaccompanied by any significant change in electrolyte excretion.

Parmelia perlata showed anti-diuretic effect with decrease in urinary

excretion was produced. However a detailed long perspective study must be

performed to explore and establish these drugs into routine therapeutic

practices.

171

4.10 EXPERIMENTAL

4.10.1 Collection of plant materials

Roots of Dichrostachys cinerea and Hemidesmus indicus were

collected respectively from Thirukurunkudi and Thenmalal of Tirunelveli

District of Tamil Nadu and Parmelia perlata was collected from Thantrikudi,

Dindigul District of Tamil Nadu, India in the month of September. Plants were

identified by Dr. V. Chelladurai, Research Officer (Botany), Survey of

Medicinal and Aromatic Plants Unit—Siddha, CCRAS, Palayamkottai,

Tirunelveli District, Tamil Nadu, India and voucher specimens have been

deposited at the Department of Chemistry, Manonmaniam Sundaranar

University, Tirunelveli District, Tamil Nadu, India [Dichrostachys cinerea

(MSU 051), Hemidesmus indicus (MSU 052) and Parmelia perlata (MSU

053)].

4.10.2 Preparation of plant extracts

.

The plant materials were thoroughly washed with water, cut into small

pieces, dried under shade for two weeks and powdered. The powdered plant

materials were individually and successively extracted with petroleum ether

(40°-60°C), benzene, chloroform, methanol and water. The last trace of the

solvent was removed under reduced pressure distillation and the crude

extract was dried in a vacuum desiccator and used for the experiments. Dried

methanolic extracts were weighed exactly and used for the evaluation of

diuretic activity.

172

4.10.3 Animals

Male Wister albino rats weighing between 250 - 300 g were used for

the study. They were given standard rodent diet (Lipton India Pvt. Ltd.

Mumbai) and tap water ad Jib/turn. They were housed under standard animal

husbandry conditions and had no access to water or food, during the test

period. For each experiment rats were randomly selected into groups

comprising of 6 rats.

4.10.4 Diuretic activity

4.10.4.1 Diuretic activity of D. cinerea

The modified method of Lipschitz83 et a! (1943) was employed for the

evaluation of diuretic activity. Rats were divided into five groups of six rats,

each weighing 250-300 g and were fasted and deprived of water for eighteen

hours prior to the experiment. On the day of experiment, the methanolic

extract of D. cinerea was suspended in normal saline at three different doses

namely 100, 250 and 500 mg/kg body weight and were administered orally to

three different groups. A group of rats receiving normal saline (25 mI/kg bw)/

alone was taken as the control and another group of rats received urea (750

mg in 25 ml of normal saline) as a reference drug. The fluid intake was the

same in all cases, i.e. 25 ml/kg of the body weight. Immediately after

administration, the rats were placed in metabolic cages (3 in each cage)

specially designed to separate urine and faeces. Animals were kept at room

temperature of 25 0 ± 0.5°C, throughout the experiment. The urine was

collected in measuring cylinders up to 5 h after dosing. During this period, no

food or water was made available to animals. The total volume of urine

collected was measured for both control and drug treated groups. Since in

173

the process of feeding, the animals lose all their urine, it was considered

important at the end of the experiment to expel the urine from the bladder by

pulling the base of the tail. The parameters taken for individual rat were body

weight before and after test period, total urine volume, concentration of Na,

K and Cl- in urine.

Analytical procedure

Na and K concentrations were determined by using a flame

photometer109 (Sistronics-128, Chennai) and Cl- concentration was estimated

by titration with silver nitrate solution (N150) using 3 drops of 5% potassium

chromate solution as an indicator.

Statistical analysis

All the results are expressed as mean ± standard error. The data was

analysed using Student's 't' test 84 for statistical significance and P < 0.05 was

considered significant.

4.10.4.2 Diuretic activity of H. indicus

Diuretic activity of methanolic extract of H. indicus was performed

using the modified method of Lipschitz 83 et a!) as mentioned under

D. cinerea.

4.10.4.3 Diuretic activity of P. perlata

The modified method of Lipschitz83 et al was used for the evaluation of

diuretic activity of methanolic extract of P. perlata as mentioned under

D. cinerea.

174

4.11 REFERENCES

1. Edwin K. Jackson, Diuretics. In: Goodman & Gilman's The

Pharmacological Basis of Therapeutics, eleventh Eds. Laurence

L. Brunton, John S. Lazo and Keith L. Parker, McGraw - Hill, New york

pp 737 - 769 (2006).

2. Tripathi, K.D.(5th Eds.) In : Essentials of Medical Pharmacology,

Jaypee Brothers Medical Publishers (P) LTD. New Delhi Pp 561 - 574

(2004).

3. Cannon, P.J. and Kilcoyne, M.K. "Ethacrynic Acid and Furosemide

Renal Pharmacology and Clinical Use." In : C.K. Friedberg (Eds.)

Current Status of Drugs in Cardiovascular Diseases, Grune and

Stratton, New York pp 243 -262 (1969).

4. Andrejus, K. and Joseph, H. B. (2nd Eds.) In: Essentials of Medicinal

Chemistry., John Wiley & Sons, New York pp 345 - 362 (1988).

5. Edwards, K.D.G. (Eds.) Drugs Affecting Kidney Function and

Metabolism. Progr. Biochem. Pharmacol., 71 -538 (1972).

6. Puschett, J.B. Cardiology., 84 (Supp 12), 4(1994).

7. Greene, M. K. etal. Br. Med. J., 283, 811 (1981).

8. Knauf, H. and Mutschler, E. Clin. Pharmacokinet., 34, 1-24 (1998).

9. Coote, J. H. Trends Pharmacol. Sc., 12, 450 - 455 (1991).

10. Zell, S.C. and Good man, P.H. West J. Med., 148, 541 (1988).

11. Hebert, S.C. Semin. Nephro!., 19, 504-523 (1999).

12. Xu, J.C., Lytle, C., Zhu, T.T., et al. proc. Nat!. Acad. Sd. USA., 91,

2201 —2205(1994).

175

13. Shilliday, I. R. etal. Nephrol. Dial. Transplant, 12, 2592 (1997).

14. Isenring, P. and Forbush, B. J. Biol. Chem., 272, 24556 - 24562

(1997).

15. Wilcox, C.S. Semin. Nephrol., 19, 557 - 568 (1999).

16. Dormans, T.P., Van Meyel, J. J., Gerlag, P.G., et al. J. Am. Coil.Cardiol., 28, 376-382 (1996).

17. Cotter, G. etal. Lancet., 351, 389 (1998).

18. Vargo, D.L. etal. Clin. Pharmacol. Ther., 57, 601(1995).

19. Fans, R., Flather, M., Purcell, H. et al. mt. J. Cardiol.., 82, 149 - 158

(2002).

20. Forns, X. etal. Semin. liver Dis., 14, 82(1994).

21. Rossi, S. (Eds.2004) Australian Medicines Handbook . Adelaide:

Australian Medicines Handbook. ISBN 0 -95 78521 - 4 - 2 (2004).

22. Monroy, A., Plata, C., Hebert, S.C. and Gamba, G. Am. J. Physiol.

Renal Physiol., 279, F 161 - F 169 (2000).

23. Bachmann, S., Velzquez, H., Obermutler, N., et al. J. Clin. Invest., 96,

2510 -2514(1995).

24. Stanton, B. A. J. Am. Soc. Nephrol., 1, 832 (1990).

25. Stokes, J. B. Kidney mt., 36, 427 (1989).

26. Friedman, P.A. and Bushinsky, D.A. Semin. Nephrol., 19, 551 - 556

(1999).

27. Saito, F. and Kimura, C. Hypertension., 27, 914-918 (1996).

176

28. Chobanian, A., Bakris, G., Black, R. et al. Hypertension., 42, 1206 -

1252 (2003).

29. Bush, A. E., Suessbrich, H., Kunzelmann, K. et al. Pflagers Arch., 432,760 - 766 (1996).

30. Kleyman, T.R., Sheng, S., Kosari, F. and Kieber Emmons, T.

Semin. Nephrol., 19, 524 - 532 (1999).

31. Quzan, J., Perault, C., Lincoff, A.M., Carre, E. and Mertes, M. Am. J.Hypertens., 15, 333 - 339 (2002).

32. White, W.B., Carr, A.A., Krause, S. et al. Am. J. Cardiol., 92, 38 - 42

(2003).

33. Weinberger, M.H., Roniker, B., Krause, S.L. and Weiss, R.J.Am. J. Hypertens.,15, 709-716 (2002).

34. Hollenberg, N.K. and Mickiewicz, C.W. Am. J. Cardiol., 63, 37 B-41 B

(1989).

35. Pitt, B., Zannad, F., Remme, W. et al. New Eng!. J. Med., 341,

709 —717 (1999).

36. Ramires, F.J., Mansur, A., Coelho, 0. et al. Am. J. Cardiol., 85,

1207-1211 (2000).

37. Warren, S.E. and Blantz, R.C. Arch. Intern. Med., 141, 493 (1981).

38. Kellum, J.A. Kidney mt .Suppl., 66, S 67 - S 70 (1998).

39. Solomon, R., Werner, C., Mann, D., Di Elia, J. and Silva, P. New Engl.

J. Med., 331, 1416 - 1420 (1994).

40. Puschett, J.B. and Winaver, J. Effects of diuretics on renal function In:

Handbook of Physiology. Sec. 8 . Vol.2. (Windhager, E.E. eds.).

Oxford University Press, New York, pp 2335 - 2404 (1992).

177

41

Vetrichelvan, T. and Jegadeesan, M. Hamdard., Vol. X LIV (4),125— 128 (2000).

WIA

Krishnan Haravey, S. Ind. Jour. Med. Res., 54, 774— 778 (1966).

43

Melis, M.S. Journal of Ethnopharmacology., 47,129 — 134 (1995).

44. Klaus, H., Ulf, N., Ulla WS. , Anne, A., Lene, G., Sreedharan, R. and

Palpu, P. Journal of Ethnopharmacology., 48, 43-51(1995).

45. Vetrichelvan, M., Jegadeesan, M., Senthil Palaniappan, M., Murali, N.

P. and Sasikumar, K. Indian Journal of Pharmaceutical Science July

-August, 300 - 302 (2000)

EEO

Pantoja, C.V., Chiang, L.Ch., Norris, B.C. and Concha, J.B. Journal of

Ethnopharmacology., 31, 325 -331 (1991).

47

Norris, B.C., Pantoja, C.V., Concha, J.B. and Chiang, L.Ch.

Pharmacology., 33, 157 - 166 (1986).

48. Norris, B.C., Pantoja, C.V., Chiang, L.Ch., Concha, J.B.,

Neumann, V.R. and Ferrada, O.V. Journal of Ethnopharmacology.,

31,309-318(1991).

49. Pantoja, C.V., Norris, B.C. and Contreras, C.M. Journal of

Ethnopharmacology., 52,101 - 105 (1996).

50. Agunu, A., Abdurahman, E.M., Andrew, G.O. and Muhammed

Z. J. Ethnopharmacol., 96 (3), 471 - 475 (2005).

51. Adsersen, A. and Adsersen, H. J. Ethnopharmacol., 58 (3),

189-206 (1997).

52. Haloui, M., Louedec, L., Michel, J. B. and Lyoussi, B.

J. Ethnopharmacol., 71 (3), 465 - 72 (2000).

178

53. Hnatyszyn, 0., Mino, J., Gorzalczany, S., Opezzo, J., Ferraro, G.,

Coussio, J. and Acevedo, C. Phytomedicine., 6(3), 177-179 (1999).

54. Johnson, P.B., Abdurahman, E.M., Tiam, E.A., Abdu Aguye, I. and

Hussaini ,l. M. J. Ethnopharmacol., 65(1), 63-69 (1999).

55. Ratnasooriya, W.D., Pieris, K. P., Samaratunga, U. and Jayakody, J.

R. J. Ethnopharmacol., 91 (2- 3), 317-320 (2004).

56. Maksimovic, Z., Dobric, S., Kovacevic, N. and Milovanovic, Z.

Pharmazie., 59 (12), 967 - 71(2004).

57. Alvarez, M.E., Maria, A.O., Villegas, 0. and Saad, J.R. Phytother.Res., 17 (8), 958 - 960 (2003).

58. Camargo, M.E., Berdeja, B. and Miranda G., J. Ethnopharmacol.,95 (2 -3), 363 - 366 (2004).

59. Afzal, M., Khan, N.A., Ghufran, A., lqbal, A. and Inamuddin, M.

J. Ethnopharmacol., 91(2 - 3), 219 - 23 (2004).

60. Nedi, T., Mekonnen, N. and Urga, K. J. Ethnopharmacol., 95 (1),57-61 (2004).

61. Sripanidkulchai, B., Wongpanich, V., Laupattarakasem, P.,

Suwansaksri, J. and Jirakulsomchok, D. J. Ethnopharmacol., 75(2 -3),185-90 (2001).

62. Caceres, A., Giron, L.M. and Martinez, A.M., J. Ethnopharmacol.,19(3), 233-245 (1987).

63. Jouad, H., Lacaille Dubois, M.A. and Eddouks, M.J. Ethnopharmacol.,75(2-3), 219-223 (2001).

64. Gaitonde, B.B., etal. Bull. Haffkine. Inst., 2,24(1974).

65. Singh, R. P., etal. J. Res. Edt,. Ind Med., 71,29-35 (1992).

179

66. Mishra, J. P., etal. Indian J. Pharmacy., 12, 59(1980).

67. Chowdhury, A.,et al. Ann. Biochem. Exp. Med., 15,119-126(1955).

68. Ribeiro Rde A, de Barros F., de Melo, M.M., Muniz, C., Chiela, S.,

Wanderley M. das G., Gomes, C., and Trot in, G. J. Ethnopharmacol.,

24(1), 19-29(1988).

69. Biswas, S., Murugesan, 1., Maiti, K., Ghosh, L., Pal, M. and Saha, B.P.

Phytomedicine., 8 (6), 469 - 71(2001).

70. Vargas, R., Perez, R.M., Perez, S., Zavala, M.A. and Perez, C.

J. Ethnopharmacol., 68 (1 -3), 335-8 (1999).

71. Perez Gutierrez, R.M., Laguna, G.Y. and Walkowski, A.

J. Ethnopharmacol., 14 (2 -3), 269 - 72 (1985).

72. Naik, V.R., Agshikar, N.V. and Abraham, G.J., J. Ethnopharmacol.,

3(1), 15-19(1981).

73. Bose, A., Gupta, J.K., Dash, G.K., Ghosh, T., Si, S., Panda, D.S.,

Indian J. Pharm. Sc., 69, 292 - 294 (2007).

74. Hook, I., McGee, A., Henman, M. et a!, mt. J. Pharmacog., 31 (1),

29-34(1993).

75. Racz, K.E., Racz, G. and Solomon, A. planta med., 26(3), 212-217

(1974).

76. Eduardo Navarro, Josefina Atonso, Rosario Rodriguez, Juan Trujillo

and Jose Boada, Journal of Ethnopharmacology., 41, 65 - 69

(1994).

77. Shastry, C.S., Ashok, K., Aravind, M.B. and Joshi, S.D. Indian Drugs.,

39 (9), 497 - 499 (2002).

180

78. Gordana Stanic, Ita Samarzija and Nikola Blazevic, Phytotherapy

Research., 12 (7), 494 - 497 (1998).

79. Maghrani, M., zeggwagh, N.A., Haloui, M. and Eddouks, M. Journal

of Ethno pharmacology., 99 (1), 31 - 35 (2005).

80. Benjumea, D., Abdala, S., Hernandez Luis, F., Perezpaz, P. and

Martin Herrera, D. Journal of Ethnopharmacology., 100 (1 - 2), 205

- 209 (2005).

81. Sanaa Lahlou, Adil Tahraoui, Zafar Israili and Badiaa Lyoussi, Journal

of Ethnopharmacology., 110 (3), 458 - 463 (2007).

82. Martin Herrera, D., Abdala, S., Benjumea, D. and Perez Paz, P.

Journal of Ethnopharmacology., 113 (13), 487 - 491 (2007).

83. Lipschitz, W.L., Haddidian, Z. and Kerpscar, A. Journal of

Pharmacology and Experimental Therapeutics., 79, 97 - 110

(1943).

84. Fisher, R.A., Statistical Methods for Research workers., Oliver and

Boyd, Edinburgh (1950).

85. Tyler V. Herbs of Choice. The Therapeutic Use of Phytomedicinals.,

Pharmaceutical Products Press, New York,pp 74-77 (1994).

86. Doan, D.D., Nguyen, N.H., Doan, H.K. et al. J. Ethnopharmacol., 36,

225-31 (1994).

87. Dm1, D., Bianchini, M., Massa, T. and Fassio, T. Minerva Med., 72,

2319-22 (1981).

88. Mills, S.Y. Out of the Earth: The Essential Book of Herbal Medicine.

London , Viking Arkana pp 493-4(1991).

181

89. Baer, J. E., In: Animal and Clinical Pharmacological Techniques in

Drug Evaluation (J. H. Nodine and P. E. Siegler eds. ) Year Book

Medical Chicago, (1964).

90. Ginsburg, M. n: Evaluation of Drug Activities Pharmacometrics.,

Vol. II (D. R. Lawrence and A. L. Bacharach, Eds. ) Academic Press,

New York, (1964).

91. Laza row, In: Methods in Medical Research., Vol. 6 Year Book

Medical Chicago, (1954).

92. Satoskar, R.S., Bhandarkar, S.D.and Ainapure, S.S., Pharmacology

and Pharmacotherapeutics., 1 9 th Eds. New Delhi, Popular Prakashan

pp 520— 544 (1998)

93. Lindquist, K.M. and Rowe, L.W. J.A. MA., 38, 227 (1949).

94. Larson, E. J. Pharmacol. & Exper. Therap., 62, 346 (1938).

95. Vasi, I.G. and Kalintha, V.P. Comp. Physiol. Ecol., 7 (2), 68 - 70

(1982).

96. Vetrichelvan, T. and Jegadeesan, M. Amala Research Bull., 21,

63 - 66 (2001).

97. Huang, K.C. The Pharmacology of Chinese Herbs., CRC Press,

Washington DC Pp 303-313 (1999).

98. Mounnissamy, V.M., Gopal, V., Gunasegaran, R. and Saraswathy, A.

Amala Research Bull., 21. 47 - 50 (2001).

99. Becker, H.M., Niedermaier, G. and Orend, K.H., Fortschr. Med., 103,

593-6 (1985).

100. Casley Smith, J.R., Morgan, R.G. and Piller, N.B., N. Engl. J. Med.,

329,1158 — 63 (1993).

182

101. Chang, T.S., Gan, J.L., Fu, K.D. and Huang, W.Y., Lymphology., 29,106-11 (1996).

102. Casley Smith, J.R., Wang, C.T., Zi —hal, C. BMJ., 307, 1037 - 41

(1993).

103. Wadworth, A.N. and Faulds, D. Drugs., 44, 1013-32(1992).

104. Renton, S., Leon, M., Belcaro, G. and Nicolaides, AX, mt. Anglo!.,

13,259-62 (1994).

105. Piller, N.B., Morgan, R.G. and Casley Smith, J.R., Br. J. Plast. Surg.,

41,20 -7(1988).

106. Struckmann, JR., J. Vasc. Res., 36(1), 37-41 (1999).

107. Griffith, J.Q., J. Am. Pharm. Assoc., 42, 68 - 9 (1953).

108. Shanno, R.L., Am. J. Med. Sd., 211, 539 —43 (1946).

109 APHA AWWA, WEF, Standard methods for the examination of water

and waste water. American Public Health Association, New York,

Washington DC (1998).