ISSN: 0975-8585

Research Journal of Pharmaceutical, Biological and Chemical

Sciences
Studies on Diuretic Effect of Lagerstroemia Speciosa Linn. Leaf Extracts in
Normal Rats

Priya TThambi1,2*, Sabu M Chacko1,3 and Jolly I Chungath1

1
Amala Cancer Research Centre, Amala Nagar, Thrissur, Kerala, India
2
Department of Chemistry, Catholicate College, Pathanamthitta, Kerala, India
3
Faculty of Pharmaceutical Sciences, UCSI University, Cheras, Malaysia

ABSTRACT

Lagerstroemia speciosa L is a medicinal, ornamental, decidous small tree native of China, commonly
cultivated in gardens through out India for beautiful flowers. The leaves of this tropical plant have been used as a
folk medicine for treatment of diabetes and kidney diseases. Many pharmacological studies has been carried out in
L. speciosa but the diuretic activity of the plant has not been studied yet. Many herbal diuretics exert their action
by directly effecting electrolyte balance of minerals. The ethyl acetate, ethanol, methanol and water extract of
Lagerstroemia speciosa L was evaluated for diuretic activity. Diuretic effect was carried out in rats by measuring
the urine volume by 1, 2, 4, 6 hours and later at 24 hours. Positive controls (furosemide and mannitol (20mg/kg
and 100mg/kg) were given intraperitoneal and intravenous route respectively. The extracts were administered
+ + - + +
orally at the dose of 250mg/kg b.wt. Na , K and Cl concentrations and urine volumes were determined. Na / K
ratio was higher in aqueous extract and followed by ethanol, ethylacetate and methanol extracts. The aqueous
extracts show best diuretic effect when compared with other extracts. It can be concluded that all the extracts
showed diuretic effect and cation excretion outstandingly.
Keywords: Furosemide, Mannitol, electrolytes, proximal tubule, L.speciosa

*Corresponding author

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INTRODUCTION

Lagerstroemia speciosa Linn called as banaba, is a tropical plant found in many parts of
Southeast Asia including the Philippines, Vietnam, Malaysia, India and southern China. The
leaves and other parts of banaba are used widely by the Philippines, Taiwan, China and Japan as
a tea preparation. This tea is consumed as a natural means for a variety of reasons involving the
kidneys, such as dissolving kidney stones, kidney cleanses, and kidney health in general. L.
speciosa has become relatively popular in the form of health-promoting tea products in Eastern
Asia and the United States [1]. The leaves of the plants are used in the treatment of diabetes [2]
and also the tribal people for heart diseases use it. It is also used for abdominal pains, mouth
ulcers, stimulant and febrifuge [3].

The subject of phytochemistry has been developed in recent years as a distinct

discipline somewhere in between natural product organic chemistry and plant biochemistry
and it is closely related to both. It is concerned with enormous variety of organic substances
that are elaborated and accumulated by plants and deals with the chemical structures of these
substances, their metabolism, their natural distribution and their biological function.

In earlier studies the extract of this plant is reported to have an antioxidant, anti
inflammatory, hepatoprotective, nephroprotective agent [4,5] and anti-diabetic properties. A
number of pharmacological properties have been reported where the diuretic activity has not
been experimentally proved. The present investigation was undertaken to confirm the diuretic
activity of different extracts of L. speciosa leaves. The diuretics are drugs that act on the kidney
and are able to increase the volume of urine excreted, the reason why are used in cardiac
failure, chronic and moderate cardiac insufficiencies, acute oedema of the lung, nephritic
edema syndrome, arterial hypertension, diseases related with the retention of fluids etc [6,7].
In the present study we have used fursemide and mannitol as reference standards. Furosemide
is a sulphonamyl derivative which is a high efficacy diuretic which has its primary action on
medullary ascending limb of loop of henle and can produce substantial effect because of
limited capacity for salt absorption in distal tubule and collecting duct. Mannitol, which is a
sugar, is an osmotic diuretic, when administered intravenously, is not metabolized and rapidly
filtered by glomeruli but not reabsorbed. It causes water to be retained in the proximal tubule
and descending limb of henle.

MATERIALS AND METHODS

Plant material

The leaves of Lagerstroemia speciosa Linn were collected from Amala Ayurvedic
Hospital premises, Trichur, Kerala, India. Dr. C.N. Sunil, Department of Botany, S.N.M College,
Maliankara, Kerala, authenticated the plant materials and a voucher specimen (BSI No. 62373)
was kept at Fr. Gabriel Herbarium, Amala Ayurvedic Hospital, Trichur.

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Preparation of plant extract

Shade dried leaves were powdered, subjected to successive soxhlet extraction using a
series of solvents of increasing polarity starting from petroleum ether (for defatting), ethyl
acetate, ethanol, methanol and water respectively. The solvents were concentrated
separately by vacuum evaporator to get the residue. The extracts were further dried in
desiccators and the yields of the extracts were 3.56, 4, 14.2, 3.19 and 17.47% respectively.

Animals

Male Wistar rats (175-200g) Male Balb/C mice (25-30g) were used for the
experiments. They were housed in environmental conditions and fed with standard rodent
diet and water ad libitum. All animal experiments conducted during the present study got
prior permission from Institutional Animal Ethics Committee (IAEC) and followed the
guidelines of IAEC.

Phytochemical analysis

Phytochemical analysis of the major phytoconstituents of the plant extracts were

undertaken using standard qualitative colour tests using the conventional protocols [8].

Determination of macro elements using flame photometry

Preparation of sodium, potassium and calcium standard solution- Analar quantity of NaCl and
KCl is accurately weighed and dissolved it in exactly 250 mL of double distilled water. It is
diluted (1:100) to get 1mg Na/100 mL and 1mg K/100 mL, which is equivalent to 10 ppm. Analar
quantity of CaCO3 is accurately weighed and is dissolved in minimum quantity 1:1 HCl and it is
made up exactly to 250 mL with double distilled water and it is diluted to get 10 mg Ca/100 mL
which is equivalent to 100ppm.

Preparation of test solutions-An accurately weighed amount of ash of the plant materials was
digested with 5 mL of 10% HCl. This was filtered through Whatman No. 4 filter paper and the
residue was washed with hot water, cooled and made to volume. The sample solutions were
then compared in the flame photometer against standard solutions of NaCl, KCl, and CaCO 3
containing the same amount of HCl. The concentrations of the sodium, potassium and calcium
ions were collected by extrapolation method.

Determination of heavy metals (Atomic Absorption Spectrophotometer - AAS)

Heavy metals include the elements arsenic, cadmium, mercury and lead were detected
at our lab using atomic absorption spectrometer (AAS), (Thermoelectron, UK) ‘S’ series model
with VP100 vapour generator [9].

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Preparation of Sample solutions

The leaves of L. speciosa were cleaned visually and dried at 150oc to a constant weight.
The dried materials were then grounded to a fine powder and were used for dry ashing. Pre-
cleaned silica crucibles were heated to 600o C till the weight of the crucibles was constant. The
determination of mercury, lead, arsenic and cadmium was conducted by Atomic Absorption
Spectrophotometry (AAS). Standard solutions of all the metal elements were prepared as per
the standard procedures reported in the operating manual of the instrument and standard
curves were prepared for the same [10].

Assessment of diuretic activity

Male Wistar rats (175-200g) were purchased from Small Animals Breeding Station,
Mannuthy, Thrissur, Kerala. They were maintained under standard conditions of temperature
and humidity. The method of Lipschitz et al [11] was employed for the assessment of diuretic
activity. The dose of the extract used in the present study was based on our toxicity studies
reported earlier [5]. Six groups of six rats each were fasted and deprived of water for eighteen
hours prior to the experiment. On the day of experiment, normal group of animals were given
normal saline orally (25 ml/kg body weight.) and the treated groups were given 250mg/kg
bodyweight of extracts of ethyl acetate, ethanol and water. The standard groups were given
furosemide (20mg/kg) intraperitoneally and mannitol (100mg/kg) intravenously. The rats
were placed in metabolic cages specially designed to separate feacal matter and urine. The
urine volume was registered at 1, 2, 4, 6 and 24 hours post administration. During this period
no food or water was given to the animals. The total urine volume was measured for both
control and treated animals. The sodium, potassium and chloride ion concentration in the
urine samples were determined.

STATISTICAL ANALYSIS

All data were analyzed through one way analysis of variance (ANOVA) followed by
Multiple Comparison Range Test (means and 95.0 Percent Tukey HSD). The difference
between the test groups and control was determined by least significant difference method
at p<0.05 confidence levels.
RESULTS

Determination of qualitative phytochemical analysis

The chemical test showed that L. speciosa contains saponins in ethanol, methanol and
water extracts. Tannins are reported in all extracts of the plant. But alkaloids showed positive in
EtOH and MeOH extract of L. speciosa and except water extract, flavonoids are presented in all
other extracts of L. speciosa.

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TABLE 1: Qualitative phytochemical evaluations of the extracts L. speciosa

Constituents Observations

Ethyl acetate Ethanol Methanol Water

(EtOAc) (EtOH) (MeOH) (Aqueous)

Saponins - + + +

Tannins + + + +

Alkaloids - + + -

Sterols + - + +

Glycosides - + - -

Flavonoids + + + -

Determination of calcium, potassium and sodium levels of L. speciosa

The amount of macronutrients present in the plant parts were measured using flame
photometry. The amount of calcium, potassium and sodium present in the leaves of L. speciosa
were found to be 143.85, 85.1 and 12.3 µg/g respectively (data not shown).

Estimation of heavy metal content of L. speciosa

The heavy metal content was estimated in the plant parts after complete digition and
estimated by AAS and levels were compared with the WHO standard and tabulated (Table 2).
The mercury content was found to be 62.6 PPB in the leaves of L. speciosa. It was found that
the L. speciosa contains 2.02, 1.16 and 0.26 PPM of arsenic, lead and cadmium respectively. The
result showed that none of the heavy metals presented in the plant parts are not above the
WHO recommended level.
TABLE 2 Data showing the Heavy metal content of L. speciosa

Heavy metals L. speciosa Recommended level (WHO)

Mercury 0.626 PPM 1 PPM
Arsenic 2.02 PPM 3 PPM
Lead 1.16 PPM 10 PPM
Cadmium 0.26 PPM 0.3 PPM

Determination of diuretic activity:

It was found that the ethyl acetate, ethanol, methanol and aqueous extract showed
diuretic activity when compared with the standard furosemide and mannitol (Figure 1). In the
normal rats the diuresis began passed one hour of the administration, showing low volumes of
urine excreted until completing 43.2 mL at 24 hours. The group dealt with furosemide (positive

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control), the beginning of the diuretic action was at 60 minutes. A final volume of 76.4 mL was
reached being significantly different from the obtained in the negative control group (p< 0.05).
In group III dealing with mannitol, does not show significant increase in urine volume. The
beginning of urine for the watery extract of the L. speciosa was also at 60 minutes post
administration, but the volume was smaller (12.5 mL), differing significantly from the values
obtained with furosemide (p<0.001) being reached a total volume of 29.4 mL. The order of
activity of increase of urine output was slightly greater for aqueous extract (12.5 mL) than that
of ethanol extract (12.1 mL) at the end of first hour but the ethyl acetate and methanol showed
lesser effect after 60 minutes. But at the end of fourth hour the methanol extract showed
better activity that urine output was increased to 29.7 mL which is comparable to ethanol and
aqueous extract at the end of fourth hour, showing that the methanol extract is showing more
activity after this time interval. But the increase in the urine volume of ethanol extract after
four hours up to six hours was found to be very less when compared with all the other extracts.
The electrolytes, Na+, K+ and Cl- levels were significantly (P<0.001) high in standard drug
treatment when compared with normal group. The plant extracts increased the electrolytes
level in urine and it is not significant. The EtOH extract showed grater electrolytes level than the
EtOAc extracts. Similarly it was obtained an increase of the excretion of Na+ in the urine was
significantly superior to the one registered in the negative control group (p<0.001) and very
highly significantly superior compared with furosemide group (Table 3).

TABLE 3 : Effect of extracts of L. speciosa on electrolyte level of Wistar rats

Group Dose mg/kg Electrolyte concentration in m eq/L

+ + - + +
Na K Cl Na /K

Normal Saline 5ml/kg 178.25 87.20 113.21 2.04

a a a
± 12.8 ± 10.5 ± 8.9

Furosemide 20mg/kg 323.61 123.56 215.70 2.61

b* b* b*
± 17.4 ± 10.8 ± 11.4

Mannitol 100mg/kg 261.53 112.32 123.62 2.32

c*† bc* ac†
± 10.5 ± 6.2 ± 9.1
L. speciosa 250mg/kg 149.16 83.15 118.81 1.79
ad† ad† acd†
EtOAc extract ± 9.9 ± 6.4 ± 6.2

L. speciosa 250 mg/kg 186.11 99.54 156.12 1.87

ae†‡ acde† ae†‡
EtOH extract ± 12.5 ± 8.5 ± 7.7

L. speciosa 250mg/kg 192.64 109.14 134.81 1.77

af† acf† acf†
MeOH extract ± 8.0 ± 11.1 ± 12.5

L. speciosa 250 mg/kg 243.15 121.17 165.23 2.01

afg† abcf ag†‡
Aq extract ± 11.5 ± 10.14 ± 7.2
Values are mean ± S.D, n=6 animals
*P<0.001, Comparison between normal group with other groups
†
P<0.001, Comparison between control group with other groups
‡
P<0.01, Comparison between different solvent extracts.

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The K+ concentration in urine, was very high significantly superior compared with
negative control, furosemide and mannitol groups (p<0.001). The aqueous extract
administration increases the Na+ concentration than other extract treated group. The ethyl
acetate fraction showed lesser urinary excretion when compared with other extracts. Increased
ratio of Na+/K+ represents the potent activity of a drug. The standard drugs showed elevated
level when compared with normal group. The Na+/ K+ ratio of the aqueous extract treated
group showed higher level than other extracts treated groups.

DISCUSSION

Kidney, the excretory organ of our body serves the important function of excretion of
waste products, regulation of fluid volume and electrolyte content of the extracellular fluid.
Diuretics are drugs capable of increasing levels of urine. In the normal rats diuresis began with
low volumes of urine excreted until completing 24 hours. The level of excreted Na + and K+ in
urine was equally low. The furosemide (positive control) treated group, the diuretic action start
at 60 minutes and increased significantly (p< 0.05) from normal rats. In the mannitol
administered group showed lesser urine volume when compared with furosemide. The
beginning of urine for the watery extract of the L. speciosa was also at 60 minutes post
administration, but the volume was smaller than furosemide.

The presence of phytoconstituents like terpenoids, saponins, flavonoids has been

reported previously to be responsible for the diuretic activity in plants [12,13]. The maximum
volume of urine at the end of 24 hours was for EtOH extract may be due to the presence of
flavonoids, saponins, tannins [14] etc. The best diuretic effects could be associated to the

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flavonoid content, also it promote high levels of Na+ and K+ in urine. There are correspondences
between the volume of urine and the concentration of Na+, this aspect is logical because the
mechanism of action of diuretic drugs is to decrease the tubular reabsorption of this ion, it
produces the dragging of the osmotic equivalent of water, other explanation that can support
this, is the high ion concentrations in this medicinal plants. [15, 16].

Diuretics relieve pulmonary congestion and peripheral edema. These agents are useful
in reducing the syndrome of volume overload, including orthopnea and paroxysmal nocturnal
dyspnoea. They increase plasma volume and subsequently venous return to the heart. This
decreases cardiac workload, oxygen demand and plasma volume, thus decreasing blood
pressure. Thus diuretics play an important role in hypertensive patients. The electrolytes, Na+,
K+ and Cl- levels were significantly (P<0.001) high in standard drug treatment when compared
with normal group. The plant extracts increased the electrolytes level in urine and it is not
significant. Increased ratio of Na+/K+ represents the potent activity of a drug. The standard
drugs showed elevated level when compared with normal group.

The ethyl acetate fraction did not increase urinary excretion when compared with other
extracts. All extracts did not increase the Na+ concentration when compared with the positive
controls. It was reported earlier that 30 to 70% of K+ filtered by the glomerulus is reabsorbed by
the proximal convoluted tubule [17] by a combination of three processes: active transport,
paracellular diffusion and solvent drag [18]. The mechanism of action are complex and involve a
variety of energy dependent trans membrane pumps as well as channels in between the loose
fitting cells of the proximal tubule (PT). About 80% of the nephrons lie in outer cortex, having
short loops of Henle and low Na+ reabsorptive capacity where as 20% are juxta medullary
possessing long loops of Henle and are responsible for creating the cortico medullary osmotic
gradient. The redistribution of blood flow between these two types of nephrons can alter salt
and water excretion. The increase in the ratio of concentration of excreted sodium and
potassium ions indicates that the extract increases sodium ion excretion to a greater extent
than potassium, which is a very essential quality of a good diuretic with lesser hyperkalaemic
side effect [19]. The chloride ion excretion was not elevated significantly when compared with
the normal animals and the results are indicating that the extract is a potent natriuretic [20].

CONCLUSION

The extracts showed diuretic effects after the administration of 250mg/kg body weight
dose. Out of these extracts water extract showed better diuretic properties and also superior
urine excretions of of Na+ and K+. Further studies like isolation and characterization of diuretic
principle from the plant is needed to understand and confirm the exact mechanism of action.

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