Nephroprotective activity of aerial parts of bauhinia purpurea ijrpp

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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 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.

1

_________________________________________________________________________ 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 ______________________________________________________________________________________________

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 _________________________________ * Corresponding author:

Neelima Neelapu Department of Pharmaceutical Chemistry, Genba Sopanrao Moze College of Pharmacy, Pune, Maharashtra, India.

E-mail address: neelima.neelapu@gmail.com

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, antiinflammatory and antipyretic1, hypoglycemic2, antimalarial, antimycobacterial, antifungal and Antioxidant and cytotoxic activities3. hepatoprotective activities of Bauhinia species have also been reported 4.

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, reddishbrown precipitate forms if reducing sugars are 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 redcolored 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 0.690.73 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 gentamicintreated 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 1 H 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 1 H 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 Groups

Control Gentamicin Gentamicin+leaf extract Gentamicin+unripe pod extract One-way F ANOVA d.f

Serum Creatinine (mg/ml) 1.23±0.021 2.94±0.45* 2.17±0.51*

Serum uric acid (mg/ml) 3.52±0.91 7.7±0.09* 4.4±1.54*

Blood urea nitrogen (mg/ml) 19.24±0.95 45.40±1.24* 9.19±1.54*

Serum urea (mg/ml)

Weight of kidney (g)

39.22±3.54 97.03±2.98* 64.78±3.12*

0.89±0.34 1.28±0.92* 1.07±0.23*

1.60±0.071*

3.25±2.05*

22.43±2.12*

48.22±2.76*

1.18±0.98*

62.98 3, 20

191.48 3, 20

2239.58 3, 20

78.96 3, 20

4881.66 3, 20

TABLE 2: HISTOPATHOLOGICAL FEATURES OF THE KIDNEYS OF RATS OF DIFFERENT TREATMENT GROUPS

Histopathological feature Glomerular conjestion Blood vessel congestion Interstitial edema Inflammatory cells Necrosis Tubular casts

Control

Gentamicin treated

Gentamicin and leaves extract treated

-

+++ ++

++ +

-

++ ++ ++ +++

+ + -+

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.

Fig.-4: Kidney tissue of unriped pods extracttreated animals Showing normal arrangement.

Stain H and E, magnification 100X.

7.

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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