NEPHROPROTECTIVE ACTIVITY OF AERIAL PARTS OF BAUHINIA PURPUREA

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Available Online at: www.ijrpp.com Print ISSN: 2278 - 2648
_________________________________
* Corresponding author:
Neelima Neelapu
Department of Pharmaceutical Chemistry,
Genba Sopanrao Moze College of Pharmacy,
Pune, Maharashtra, India.
E-mail address: neelima.neelapu@gmail.com
Online ISSN: 2278 - 2656
International Journal of
Research in Pharmacology and
Pharmacotherapeutics
(Research article)
PHYTOCHEMICAL INVESTIGATION AND EVALUATION OF
NEPHROPROTECTIVE ACTIVITY OF AERIAL PARTS OF
BAUHINIA PURPUREA
1* Neelima Neelapu, 2Sudhakar Muvvala
1 Department of Pharmaceutical Chemistry, Genba Sopanrao Moze College of Pharmacy, Pune,
Maharashtra, India.
2 Malla Reddy College of Pharmacy, Dhulapally (via Hakimpet), Maisammaguda, Secunderabad,
Andhra Pradesh, India.
_________________________________________________________________________
ABSTRACT
General phytochemical screening of the aerial parts of Bauhinia purpurea (Fabaceae) revealed the presence of
flavonoids, carbohydrates, glycosides, tannins and terpenoids. The aim of this study is to identify and
characterize the bioactive principle from the aerial parts of the plant. For isolation of the compound, the dried
aerial parts powder of Bauhinia purpurea was subjected to soxhlet extraction with ethanol; this extract was
subjected to chromatography. Isolated compound, a white crystalline powder was subjected to spectral
identification by extensive1 H-NMR studies. The compound was concluded as β-sitosterol. The ethanol extract
of leaves and unripe pods of Bauhinia purpurea was evaluated for its protective effects on gentamicin-induced
nephrotoxicity in rats. Nephrotoxicity was induced in Albino wistar rats by intraperitoneal administration of
gentamicin 100 mg/kg/d for eight days. Effect of concurrent administration of ethanol extract of leaves and
unripe pods of Bauhinia purpurea at a dose of 300 mg/kg/d given by oral route was determined using serum
creatinine, serum uric acid, blood urea nitrogen and serum urea as indicators of kidney damage. It was observed
that the ethanol extract of leaves and unripe pods of B. purpurea possessed potent nephroprotective activity. Of
the two extracts ethanol extract of unripe pods has significant activity as compared to leaves extract.
Key words: Bauhinia purpurea, β-sitosterol, gentamicin, nephrotoxicity
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INTRODUCTION
Ayurvedic, the system of Indian medicine and
science of life deals with the well being of
mankind. Sushruta, the father of Surgery explained
urinary calculus under the heading of Ashmari in
detail including etiological factors, classification,
symptomatology, pathology, complications, and its
management in a more scientific manner. This
disease is dreadful and hence considered as one of
the ‘Mahagadas’, by Sushruta may be owing to its
potentiality to disturb the anatomy and physiology
of urinary system, many a times leading to
impairment and damage to the kidney. In
Ayurveda, several drugs are used as
nephroprotectives and this group of drug's acts as

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good non-specific cytoprotective. In this
background, it was thought worthwhile to evaluate
the plant extract, which could be useful as
nephroprotective which could be administered to
decrease the potential nephrotoxicity of drugs like
gentamicin, cisplatin, cyclosporine, etc.
Bauhinia purpurea is a flowering plant (Family:
Fabaceae). Several species of this plant are known
to possess pharmacological activities. Aqueous
extract of leaves having antinociceptive, anti-inflammatory
and antipyretic1, hypoglycemic2,
antimalarial, antimycobacterial, antifungal and
cytotoxic activities3. Antioxidant and
hepatoprotective activities of Bauhinia species
have also been reported 4.
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MATERIALS AND METHODS
Plant material and its extract:
The plant materials (leaves and unripe pods) of
Bauhinia purpurea were collected from
Maharashtra state, India. The plant material was
authenticated by botanist and voucher specimen
was deposited in the department of Botany, Pune
University. The plant material was air dried at
room temperature and powdered to 40 mesh and
subjected to soxhlet extraction at 600C with
ethanol. The extracts were concentrated under
reduced pressure, kept in a petridish and stored in a
dessicator at room temperature.
Preliminary phytochemical screening:
It involves testing of the extracts for the contents of
different classes of compounds. The methods used
for detection of various phytochemicals were
followed by qualitative chemical tests to give
general idea regarding the nature of constituents
present in crude drug5,6,7.
A.Test for Alkaloids
Mayer’s test:
Alkaloids give the cream color precipitate with
Mayer's reagent Potassium mercuric iodide
solution.
Dragendorff’s test:
Alkaloids give reddish brown precipitate with
Dragendorff’s reagent Potassium bismuth iodide
solution.
Wagner's test:
Alkaloids give a reddish brown precipitate with
Wagner's reagent Solution of iodine in potassium
iodide.
Hager's test:
Alkaloids give yellow color precipitate with
Hager's reagent saturated solution of Picric acid.
Tannic acid test:
Alkaloids give buff color precipitate with 10%
Tannic acid solution.
B. Test for Glycosides
The extracts were tested for free sugars. The
extract is hydrolyzed with mineral acid and then
tested for the glycone and aglycone moieties.
Raymond’s test:
Test solution when treated with dinitro- benzene in
hot methanolic alkali, gives violet color.
Legal’s test:
Treat the extract with pyridine and add alkaline
sodium nitroprusside solution, blood-red color
appears.
Bromine water test:
Test solution when treated with bromine water
gives yellow precipitate.
C. Test for Flavonoids:
Test solution with 2ml of Millon's reagent
(Mercuric nitrate in nitric acid containing traces of
nitrous acid), white precipitate appears, which turns
red upon gentle heating.
Ninhydrin test:
Amino acids and Proteins when boiled with 0.2%
solution of Ninhydrin (Indane 1, 2, 3 trione
hydrate), Violet color appears.
D. Test for Sterols & Triterpenoids
Libermann- Buchard test:
Extract is treated with few drops of acetic
anhydride, boil and cool, con. Sulfuric acid is
added from the sides of the test tube, shows a
brown ring at the junction of two layers and the
upper layer turns green, which shows the presence
of Steroids and formation of deep red color
indicates the presence of triterpenoids.

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Salkowski test:
Treat extract in Chloroform with few drops of
conc. Sulfuric acid, shake well and allow standing
for some time, red color appears at the lower layer
indicates the presence of Steroids and formation of
yellow colored lower layer indicates the presence
of Triterpenoids.
E. Test for Carbohydrates
Molisch's test:
Treat the test solution with few drops of alcoholic
alpha naphthol. Add 0.2 ml of con. Sulfuric acid
slowly through the sides of the test tube, a purple to
violet color ring appears at the junction.
Benedict's test:
Treat the test solution with few drops of Benedict's
reagent (alkaline solution containing cupric citrate
complex) and upon boiling on water bath, reddish-brown
precipitate forms if reducing sugars are
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present.
Camnelisation:
Carbohydrates when treated with strong sulfuric
acid, they undergo charring with the dehydration
along with burning sugar smell.
Selwinoff’s test;
Hydrochloric acid reacts with ketose sugar to form
derivatives of furfuraldehyde, which gives a red-colored
compound when linked with resorcinol.
Add compound solution to about 5ml of reagent
and boil. Fructose gives red color within half
minute. The test is sensitive to 5.5 mmol / liter if
glucose is absent, but if glucose is present, it is less
sensitive and in addition of the largeamount of
glucose can give similar color.
Fehling's test:
Equal volume of Fehling's A (Copper sulfate in
distilled water) and Fehling's B (Potassium tartarate
and Sodium hydroxide in distilled water) reagents
are mixed and few drops of sample is added and
Boiled, a brick-red precipitate of cuprous oxide
forms, if reducing sugars are present.
F.Tests for alcohol
Cerric ammonium nitrate (4g) was dissolved in
10ml of 2N HNO3, on mild heating. A few crystals
of isolated compound were dissolved in 0.5ml of
dioxane. The solution was added to 0.5ml of cerric
ammoinium nitrate reagent and diluted to 1ml with
dioxane and shaken well. The developed yellow to
red color indicates the presence of an alcoholic
hydroxyl group8.
Column chromatography of ethanol extract of
leaves was conducted using silica gel (Mesh
60‐120) that was packed using wet packing method
in toluene. The column was run using toluene,
ethyl acetate by gradient elution technique.
TLC was used to monitor the eluates. A total of 20
eluates was collected. Similar fractions were
pooled together. Further purification is carried out
using preparative TLC. Spots were identified,
scraped and eluates using petroleum ether and
chloroform as solvents. Finally, eluate ST yielded
a single spot when subjected to TLC using solvent
system n-hexane: ethyl acetate (7:3) and it showed
to be homogenous compound. ST, a white
crystalline powder (100mg) was subjected to
extensive 1H NMR spectral analysis as well as by
comparison of its spectral data with previously
reported values.
The 1H NMR spectrum (400 MHz, CDCl3) of
compound ST has revealed a one proton multiplet
at δ 3.2, the position and multiplicity of which was
indicative of H-3 of the steroid nucleus. The typical
H-6 of the steroidal skeleton was evident as a
multiplet at δ 5.26 that integrated for one proton.
The spectrum further revealed signals at δ 0.69-
0.73 and δ1.07-1.13 (3H each) assignable to two
tertiary methyl groups at C-13 and C-10,
respectively. The 1H NMR spectrum showed two
doublets centered at δ 0.87 (J=6.7 Hz) and 0.85
(J=6.7 Hz) which could be attributed to two methyl
groups at C-25 . The doublet at δ 0.96 (J=6.5Hz)
was demonstrative of a methyl group at C-20. On
the other hand, the triplet of three-proton
intensity at δ 0.89 could be assigned to the primary
methyl group attached to C-28. The above spectral
features are in close agreement to those observed
for β-sitosterol in literature9,10.
Healthy, male Wistar rats each weighing 150-200 g
were used for the study. The rats were housed in
polypropylene cages and maintained under
standard conditions (12 h light and dark cycles, at
25±30 and 35-60% humidity). Standard pelletized
feed and tap water were provided ad libitum.
The Institutional Animal Ethical Committee of
Genba Sopanrao Moze College of Pharmacy, Pune,
approved the study. Twenty-four male Wistar rats
were assigned to four groups, Group I was the
control group, and Group II was the gentamicin-treated
group. Group III was the gentamicin-as well
as ethanol extract of leaves-treated group (BPLE)

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and group IV was the gentamicin-as well as ethanol
extract of unripe pods of B. purpurea-treated group
(BPPE). Each group consisted of six rats. The
gentamicin-treated group received 100 mg/kg/day
gentamicin (Hi Media Laboratories, Mumbai,
India) by the intraperitoneal (i.p) route11. Group III
received 100 mg/kg/d gentamicin i.p. and 300
mg/kg/d of the BPLE p.o. for eight days and group
IV received 100 mg/kg/d gentamicin i.p. and 300
mg/kg/day of the BPPE p.o. for eight days. Rats in
the control group were given sterile saline solution
i.p. for the same number of days. After dosing on
the 8th day, blood samples were collected via
cardiac puncture method at the end of these 24 h.
The serum was rapidly separated and processed for
determination of serum creatinine, serum uric acid,
blood urea nitrogen (bun) and serum urea using
commercially available kits of Span Diagnostics
Ltd, Hyderabad, India12.
Three rats per group were sacrificed, and both
kidneys were isolated from each rat13. The kidneys
were weighed and processed for histopathological
examination14. The kidneys were sectioned
longitudinally in two halves and were kept in 10%
neutral formalin solution15. Both kidneys were
processed and embedded in paraffin wax and
sections were taken using a microtome. The
sections were stained with hematoxylin and eosin
and were observed under a computerized light
microscope.
Statistical analysis:
The data obtained was analyzed using one-way
ANOVA followed by Dunnet’s multiple
comparison test. P<0.01 was considered
significant.
RESULTS AND DISCUSSION
The qualitative phytochemical investigations on the
ethanolic extract of plant material revealed the
presence of flavonoids, carbohydrates, glycosides,
tannins and terpenoids. Chromatographic
separation provided a compound, the structure of
which was observed as β-sitosterol by extensive
1H NMR spectral analysis as well as by comparison
of its spectral data with previously reported values.
The 1H NMR spectrum (400 MHz, CDCl3) of
compound ST has revealed a one proton multiplet
at δ 3.2, the position and multiplicity of which was
indicative of H-3 of the steroid nucleus. The
spectrum further revealed signals at δ 0.69- 0.73
and δ1.07-1.13 (3H each) assignable to two tertiary
methyl groups at C-13 and C-10, respectively. The
1H NMR spectrum showed two doublets centered
at δ 0.87 (J=6.7 Hz) and 0.85 (J=6.7 Hz) which
could be attributed to two methyl groups at C-25.
On the other hand, the triplet of three-proton
intensity at δ 0.89 could be assigned to the primary
methyl group attached to C-28.
The ethanol extract of leaves and unripe pods of B.
purpurea possessed potent nephro protective
activity. Of the two extracts ethanol extract of
unripe pods has significant activity as compared to
leaves extract which may be due to the phyto
constituents such as flavonoids, carbohydrates,
glycosides, tannins, volatile oils, anthrocyanidins,
lactones and terpenoids present in the extract.
Serum creatinine, serum uric acid, blood urea
nitrogen, serum urea and the weights of the kidneys
were found to be significantly increased in rats
treated with only gentamicin; whereas treatment
with the BPLE and BPPE was found to protect the
rats from such effects of gentamicin as shown in
Table 1.
Control rats showed normal glomerular and tubular
histology (Fig 1),
Group II animals exhibited glomerular, peritubular
and blood vessel congestion and result in the
presence of inflammatory cells in kidney sections
(Fig 2).
Concurrent treatment with the ethanolic extract of
leaves (Fig 3) and unripe pods (Fig 4) were found
to reduce such changes in kidney histology induced
by gentamicin (Table 2).
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TABLE 1: PARAMETERS STUDIED FOR THE NEPHROPROTECTIVE ACTIVITY
OF ETHANOL EXTRACTS OF BAUHINIA PURPUREA
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Groups Serum
Creatinine
(mg/ml)
Serum uric
acid (mg/ml)
Blood urea
nitrogen
(mg/ml)
Serum urea
(mg/ml)
Weight of
kidney (g)
Control 1.23±0.021 3.52±0.91 19.24±0.95 39.22±3.54 0.89±0.34
Gentamicin 2.94±0.45* 7.7±0.09* 45.40±1.24* 97.03±2.98* 1.28±0.92*
Gentamicin+leaf
2.17±0.51* 4.4±1.54* 9.19±1.54* 64.78±3.12* 1.07±0.23*
extract
Gentamicin+unripe
pod extract
1.60±0.071* 3.25±2.05* 22.43±2.12* 48.22±2.76* 1.18±0.98*
One-way F 62.98 191.48 2239.58 78.96 4881.66
ANOVA d.f 3, 20 3, 20 3, 20 3, 20 3, 20
TABLE 2: HISTOPATHOLOGICAL FEATURES OF THE KIDNEYS OF RATS OF
DIFFERENT TREATMENT GROUPS
Histopathological
feature
Control Gentamicin treated
Gentamicin and leaves
extract treated
Glomerular conjestion - +++ ++
Blood vessel
congestion
- ++ +
Interstitial edema - ++ +
Inflammatory cells - ++ +
Necrosis - ++ --
Tubular casts - +++ +
Fig-1: Kidney tissue of control animal showing
normal Histology. Stain H and E,
magnification 40X
Fig.-2: kidney tissue of animal treated with
Gentamicin showing Interstitial Round cell
collection. Stain H and E, magnification 200X

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Fig.-3: Kidney tissue of leaves extract-treated
animals Showing Diffuse Round cell collection.
Stain H and E, magnification 800X.
ACKNOWLEDGEMENTS:
The authors are thankful to the management of
Genba Sopanrao Moze College of Pharmacy for
providing the required facilities to carry out the
research work.
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