PHYTOCHEMICAL & IN VITRO ANTIUROLITHIATIC STUDIES ON THE LEAF EXTRACTS OF BAUHINIA VARIEGATA LINN.

PHYTOCHEMICAL & IN VITRO ANTIUROLITHIATIC STUDIES ON THE LEAF EXTRACTS OF BAUHINIA VARIEGATA LINN.

Vani Mamillapalli ^*1, Padma Latha Khantamneni ², Zabeena Mohammad ¹, Anitha Mathangi ¹, Navyanuradha Nandigam ¹, Sri Manasa Namburi ¹ and Vani Katta ¹

Department of Pharmacognosy and Phyto Chemistry ¹, Department of Pharmacology ², Vijaya Institute of Pharmaceutical Sciences for Women, Enikepadu, Viayawada, Pin: 521108, Krishna District, Andhra Pradesh, India.

ABSTRACT: The aim of the present study is to carry out fluorescence analysis, phytochemical extraction, preliminary phytochemical screening, estimation of total flavonoids, tannins, alkaloids, steroids saponins and in vitro antiurolithiatic studies on the leaf aqueous and ethanolic extracts of Bauhinia variegata. The results of fluorescence analysis indicate that the powder on treatment with 50% H₂SO₄ shows dark brown colour under UV. The results of preliminary phytochemical screening indicated the presence of saponin glycosides, tropane alkaloids and acidic compounds. The results of quantitative determination indicated that the aqueous extract contains highest amount of flavonoids expressed as 54.6 mg/gm equivalents of quercetin, maximum amount of tannins in ethanolic exract as 56.30 mg/gm equivalents of quercetin, equal amounts of alkaloids are present in both extracts as 25mg/gm equivalents of atropine sulphate, steroids and saponins in lowest amount. The in vitro antiurolithiatic activity was studied as percentage inhibition of stones by nucleation, growth and aggregation assays for aqueous and ethanolic extracts at 200-1000 µg/ml taking cystone tablets as standard. The results indicated that the inhibition of growth of crystals increased with increase in concentration of the extract. Therefore, the plant claimed to possess antiurolithiatic activity and further in vivo studies as well as isolation of individual compounds responsible for the activity are necessary.

Fluorescence,

Quantitative Determination,

In vitro, Antiurolithiatic Study

INTRODUCTION World Health Organization manifests that approximately 75% of the global population, of the developing world, depends on botanical medicines for their basic healthcare needs. ¹ Exact identification and quality of the starting materials is an essential prerequisite to ensure reproductive quality of herbal medicine which will contribute to its safety and efficacy. ²

Deposition or formation of stones in any part of the urinary system i.e the kidney, the ureters or the urinary bladder is called Urolithiasis. Stone formation is the culmination of a series of physiochemical events i.e. super saturation and nucleation, growth of the crystal and aggregation that occurs as the glomerular filtrate traverses through the tubules of nephron.

Urine remains supersaturated with most stone forming salt components as well as chemicals that prevent or inhibit the crystals from urinary tract. These crystals remain tiny enough. ³ They will travel through the urinary tract and pass out of the body in the urine without being noticed. ⁴ Calcium oxalate stones represent upto 80% of analyzed stones. ⁵ It is considered as the third most common affliction of the urinary tract. ⁶ However, the presence of certain molecules raise the level of supersaturation of salts needed to initiate crystal nucleation or reduce the rate of crystal growth or aggregation and prevents stone formation. ⁷

Though technological advancements have made dramatic improvement, still some of the drawbacks of the methods exist which includes their being too costly and recurrence of stone formation along with number of other side effects ⁸. The problem of urinary stones is an ancient one, and still remains a common problem worldwide.

Some medicinal plants contain chemical compounds like Glycosaminoglycans (GAGs) which themselves possess an inhibitory effect in the crystallization of calcium oxalate. Antioxidant constituents of the plants also help in ameliorating the crystal/oxalate induced renal cell injury. Thus, antiurolithiatic activity of plants or herbal formulation may be due to synergism of their diuretic activity, crystallization inhibition along with antioxidant activity ⁹. The activity of the extracts was evaluated by measuring the ability of the extracts to inhibit the formation of calcium and phosphate precipitates ¹⁰. In order to find new potential antiurolithiatics the plant selected was Bauhinea variegata belonging to the family Leguminosae as there were no scientific reports published on the leaves of the plant for antiurolithiatic activity.

The plant Bauhinea variegata has been used traditionally, the root is carminative, used in dyspepsia, flatulence and as an antidote to snake poison. ¹¹ The bark, flower uice are used as astringent, tonic, anthelmintic, scrofula and skin diseases ¹².  

The leaves of the plant were evaluated for the pharmacognostic, powder microscopy and physiochemical studies ¹³. The non woody aerial parts contain  6 flavonoids, namely kaempferol, ombuin, kaempferol 7,4’-dimethylether-3-o-β-D-glucopyranoside, kaempferol – 3 – o - β-D-gluco pyranoside, isorhamnetin-3-o-β-D-glucopyranoside & hesperidin together with one triterpene caffeate, 3β trans-(3,4 dihydroxycinnamoyloxy) olean-12-en-28-oic acid. The root contains novel flavonol glycosides ¹⁴.

The root bark possesses a new flavonone (2S) -5, 7-dimethoxy-3, methylenedioxy flavonone and new dihydrodibenzoxepin 5, 6dihydro-1, 7-dihydro-1, 7-dihydroxy-3, 4-dimethoxy-2-methyldibenzoxepin,   together with three known flavonoids ¹⁵.The Stem contains an unknown compound naringenin 5, 7 dimethyl ether 4 – rhamnoglucoside, a new phenanthraquinone named Bauhinione has been isolated from B. variegata L. ¹⁶. The leaves contain two new long chain compounds heptatriacontane- 12, 13-diol 7 dotetracont-15-en-9-ol. Anti cancer activity was studied on the ethanolic extract of stem ¹⁷. Antimicrobial activity was studied on the ethanolic extract of leaf and bark. ¹⁸ Antiinflammatory avtivity for the flavonoid compounds isolated from the non woody aerial parts. ¹⁹ Hepatoprotective activity was studied on the ethanolic extract of stem.²⁰ Antiulcer activity was studied on the ethanolic extract of stem of Bauhinea variegata. ²¹ The aimof the present study was to carry out UV-Fluoresence analysis of the powder, preliminary phytochemical screening, to determine total amount of flavonoids, tannins, alkaloids, steroids and saponins and to evaluate in vitro antiurolithiatic studies by nucleation, growth and aggregation assays for the aqueous and ethanolic extracts of leaves of Bauhinea variegata.

MATERIALS AND METHODS:

Materials: The Plant material Bauhinia variegata was collected in the month of December during afternoon from the grounds of Vijaya institute of Pharmaceutical Sciences for Women, Enikepadu, Vijayawada. Herbarium was prepared and the sample was authenticated by Dr. D.T. P. Satyanarayana Raju, plant taxonomist, department of Botany and Microbiology, Acharya Nagarjuna University, Guntur. The photographs of the plant and leaves were depicted in Fig. 1 and Fig. 2 The authentication letter was also enclosed. The Chemicals used were purchased from Finar chemicals. The dried leaves were coarsely powdered and depicted in Fig. 3 and Fig. 4.

Powder Analysis:

Powder Analysis using Chemical Reagents with naked Eye: The leaf powder was studied with naked eyeby using the chemicals and the results were noted and given in Table 1 ²².

Fluorescence Analysis of the Leaf Powder:

The leaf powder was studied for fluorescence analysis under UV (Lab India) and the results were noted after treating with different chemical reagents and given in Table 2.²²

Extraction: The powder was subjected to Soxhlet extraction by using water and alcohol as solvents, produced in Fig 5. The crude extract was dried using vaccum pump and weighed.

Phytochemical Study:

Qualitative screening: The aqueous and ethanolic extracts of the leaves of Bauhinia variegata were screened for the phytochemical constituents according to the standard methods. The results were given in Table 3. ^{23, 24, 25, 26}

Quantitative Determination:

Flavonoids: The extract (1.5 mL) was added to 1.5 ml of 2% methanolic AlCl₃ solution. The mixture was vigorously shaken on Centrifuge for 5 minutes at 200 rpm and the absorbance was read at 367 nm after 10 minutes of incubation. Quercetin was used as a standard for the calibration curve, given in fig 6. The assay was carried out in triplicate, results were calculated using the given formula, produced in Table 4 and expressed as mg/g equivalent of quercetin ^{27, 28}.

C = c.V/m

C – Total phenolic compounds mg/g of plant extract;

c – The concentration of standard established from the calibration curve mg/ml

V – The volume of extract in ml

m -The weight of pure plant extract

Tannins: This was determined by Folin-Denis colorimetric method with slight modification. 0.5 gmof the sample was dispersed in 50 ml of distilled water and shaken. The mixture was left undisturbed for 30 minutes at 28°C and filtered through Whatmann No. 1 filter paper. The filtrate (2 ml) was dispersed into a 50ml volumetric flask and 2.5 ml of 10% Na₂ CO₃ solution was added. The content of each flask was made up to 50 ml with distilled water and incubated at 28°C for 90 minutes. Absorbance was read at 260 nm using the reagent blank. Tannic acid was used for the calibration curve, given in Table 7. The procedure was repeated three times results were calculated using the given formula and values were given in Table 4, expressed as mg/g equivalent of quercetin. ^{28, 29}

Alkaloids:

Preparation of Reagents: Bromocresol green solution was prepared by heating 69.8 mg bromocresol green with 3 ml of 2N NaOH and 5 ml distilled water until completely dissolved and the solution was diluted to 1000 ml with distilled water.Phosphate buffer solution (pH 4.7) was prepared by adjusting the pH of 2M sodium phosphate (71.6 g Na₂HPO₄ in 1 L distilled water) to 4.7 with 0.2 M citric acid (42.02 g citric acid in 1 L distilled water).

Separation of Alkaloids: A part of extract residue was dissolved in 2N HCl and then filtered. 1 ml of this solution was transferred to separating funnel and washed with 10 ml chloroform (3 times). The pH of this solution was adjusted to neutral with 0.1 N NaOH. Then 5 ml of BCG solution and 5 ml of phosphate buffer were added to this solution. The mixture was shaken and complex extracted with 1, 2, 3 and 4 ml chloroform by vigorous shaking, the extract was then collected in a 10 ml volumetric flask and diluted with chloroform.

Method: The sample (0.5 g) was dispersed in 10% acetic acid solution in ethanol in a ratio of 1:10 (10%). The mixture was left undisturbed for 4 hours at 28°C and filtered with Whatmann No. 1 filter paper. The filtrate was concentrated to one quarter of its original volume by evaporation and treated with 3 drops of conc. NH₄OH. The alkaloid precipitate was received in a weighed filter paper, washed with 1% ammonia solution, and oven dried at 80°C. Alkaloid content, expressed as a percentage of the weight of sample analyzed and converted to mg/ml. Atropine was used as standard for the calibration curve, produced in fig 8.This was done in triplicate results were calculated using the given formula, expressed as mg/g equivalents of atropine and the values were produced in Table 4. ^{28; 30}

C = c.V/m

Steroids: Preparation of Reagents: 20% MgSO₄: 20 g of magnesium sulphate was dissolved in 100 ml of distilled water. ³¹

Method: This was done by the alkaline precipitation gravimetric method described by Harborne. The sample (0.5g) was taken and dissolved in 50 ml of butanol and it was stirred for about 5 hours for homogenous mixing and the above solutions was filtered by using Whatmann filter paper and add 20 ml of 20% Magnesium sulphate and was filtered. The above solution was analysed by using UV spectrophotometer at 380nm and this procedure was repeated for triplicate. Diosgenin was used as standard for the calibration curve, depicted in fig 9, results were calculated using the given formula and expressed as mg/g equivalents of diosgenin, produced in table 4.^28;32

C = c.V/m

Saponins:

Preparation of Reagents: 40% Magnesium carbonate: 40 g of MgCO₃ was dissolved in 100 ml of water.5% FeCl₃ solution: 5 g of ferric chloride was dissolved in 100 ml of water.

Method: A known mass (1 g) of finely ground sample was weighed into a 250 mL beaker and 100 ml of isobutyl alcohol was added. The mixture was shaken for 5 hours to ensure uniform mixing and filtered through Whatmann No. 1 filter paper into a 100mL beaker, after which 20 ml of 40% magnesium carbonate solution was added. The resulting mixture was again filtered through Whatmann No. 1 filter paper to obtain a clear colorless solution. A known volume (1 ml) of the colorless solution was pipetted into a 50-mL volumetric flask and 2 ml of 5% FeCl₃ solution was added and made up to the marked level with distilled water. It was left undisturbed for 30 minutes for blood red color to develop. The absorbance was read after color development at a wavelength of 380 nm. Standard Saponin diosgenin was used for calibration curve, depicted in Fig 10, results were calculated in same way as above, given Table 4.^{28, 33}

C = c.V/m

In vitro antiurolithiatic activity:

Nucleation assay Method:

Preparation of Reagents: 10% Trichloro acetic acid was prepared by dissolving 10g of Trichloroacetic acid in 1000 ml of water. ³⁴

Method: It is the classical model for the study of oxalate crystallization because of its simplicity and satisfactory reproducibility. This model includes the study of crystallization without inhibitor and with it, in order to assess the inhibiting capacity of any chemical species used. Solution of calcium chloride and sodium oxalate were prepared at the final concentrations of 5mmol/L and 7.5 mmol/L respectively in a buffer containing Tris 0.05 mol/L and NaCl 0.15 mol/L at pH 6.5. 950 μL of calcium chloride solution mixed with 100 μL of herb extracts at the different concentrations (100 μg/ml–1000 μg/ml). Crystallization was started by adding 950 μL of sodium oxalate solution.

The temperature was maintained at 37 °C. The OD of the solution was monitored at 620 nm. The rate of nucleation was estimated by comparing the induction time in the presence of the extract with that of control. The Cystone tablets are used as standard solution.³⁵ The results were given in Fig 11 and 12 and Table 5.

The growth of crystals was expected due to the following reaction:

CaCl₂+NaC₂O₄→2CaCO₄+2NaCl.CaCl₂+Na₂C₂O₄→CaC₂ O₄+2NaCl

Growth Assay:

Preparation of Reagents: 4mM CaCl₂ was prepared by 4 g of calcium chloride was dissolved in 1 litre of water.4mM of Sodium oxalate was prepared by dissolving 4 g of sodium oxalate was dissolved in 1 litre of water.10mM NaCl was prepared by dissolving 10 g of NaCl was dissolved in in 1 litre of water.¹⁴

Method: Newly formed crystals may combine to form a small hard mass, called calculus. The percentage inhibition of calcium oxalate crystal growth was evaluated in presence and absence of drug extracts. 4mM calcium chloride and 4mMsodium oxalate of 1ml each were added to a 1.5ml of solution containing sodium chloride (10mM) buffered with Tris (10mM) at pH 7.2. To this 30µl of calcium oxalate monohydrate crystal slurry (1.5mg/ml acetate buffer) was added. Consumption of oxalate begins immediately after calcium oxalate monohydrate crystal slurry addition and was monitored for 600 seconds for the disappearance of absorbance at 214 nm, with and without extract. The results were produced in Fig. 13 and 14 and Table 6.^{36, 37, 38}

The Cystone tablets are used as Standard drug solution.¹⁰

The relative inhibitory activity was calculated as follows:

% relative inhibitory activity = ((C-S)/C) × 100

Where ‘C’ is the rate of reduction of free oxalate without any extract and ‘S’ is the rate of reduction of free oxalate with drug extract .³⁹

Aggregation Assay:

Preparation of Reagents: 50 mM CaCl₂ was prepared by 50 g of calcium chloride was dissolved in 1 liter of water. 50 mM of Sodium oxalate was prepared by dissolving 50 g of sodium oxalate was dissolved in 1 liter of water. 0.05 mM NaCl was prepared by dissolving 0.05 g of NaCl was dissolved in in 1 liter of water.⁴⁰

Method: CaOx monohydrate (COM) crystals were prepared by mixing calcium chloride and sodium oxalate at 50 mmol/L. Both solutions were equilibrated to 60 °C in a water bath for 1 h and then cooled to 37 °C overnight. The crystals were harvested by centrifugation and then evaporated at 37 °C. CaOX crystals were used at a final concentration of 0.8 mg/ml, buffered with Tris 0.05 mol/L and NaCl 0.15 mol/L at pH 6.5. Experiments were conducted at 37 °C in the absence or presence of the plant extract after stopping the stirring. The Cystone tablets are used as Standard drug solution .The results were given in Fig. 15 and Table 7.^{41, 42} The percentage aggregation inhibition rate (Ir) was then calculated by comparing the turbidity in the presence of the extract with that obtained in the control using following formula:

Ir= (1−Turbiditysample/Turbidity control) × 100

RESULTS AND DISCUSSION:

FIG: 1 BAUHINEA VARIEGATA PLANT

FIG.2: BAUHINEA VARIEGATA LEAVES UPPER AND LOWER SIDE

FIG.3: BAUHINEA VARIEGATA DRIED LEAVES UPPER AND LOWER SIDE

FIG. 4: DRIED LEAF POWDER OF BAUHINEA VARIEGATA

Powder Analysis:

Powder analysis using chemical reagents with nacked eye:

TABLE 1: POWDER ANALYSIS WITH CHEMICAL REAGENTS FOR THE LEAVES OF BAUHINIA VARIEGATA

 Fluorescence Analysis of the Leaf Powder:

 TABLE 2: FLUORESCENCE ANALYSIS OF BAUHINIA VARIEGATA LEAF POWDER:

The results of powder analysis indicate that when the powder was observed with naked eye on treatment with 5% KOH solution shows brownish yellow which on UV shows green florescence. When the powder is observed by adding ammonia shows dark brown colour which under UV observation shows light green florescence. This analysis was helped in powder material of Bauhinia variegata.

Phytochemical Study:

FIG. 5:  PHYTOCHEMICAL EXTRACTION BAUHINIA VARIEGATA LEAF POWDER USING WATER AND ETHANOL AS SOLVENTS

Qualitative Screening: The results of preliminary phytochemical screening show the presence of Acidic compounds, saponins glycosides, alkaloids, Tropane alkaloids.

 TABLE 3: QUALITATIVE PHYTOCHEMICAL SCREENING OF AQUEOUS AND ETHANOLIC EXTRACTS OF BAUHINIA VARIEGATA LEAVES

AqELBV- aqueous extract of leaves of Bauhinia variegata,

ELBVTA- ethanolic extract of leaves of Bauhinia variegata.

Quantitative Determination:

TABLE 4: QUANTITATIVE PHYTOCHEMICAL SCREENING OF AQUEOUS AND ETHANOLIC EXTRACTS OF BAUHINIA VARIEGATA LEAVES

Values represented mean ± S.D. of three parallel measurements. AqELBV, EELBV- aqueous extract of roots of Bauhinia variegata, ethanolic extract of leaves of Bauhinia variegate

FIG. 6: STANDARD CALIBRATION OF FLAVONOIDS FOR QUERCETIN

FIG. 7: STANDARD CALIBRATION OF TANNINS FOR QUERCETIN

FIG. 8:  STANDARD CALIBERATION CURVE OF ATROPINE FOR ALKALOIDS

FIG. 9: STANDARD CALIBRATION OF STEROIDS FOR DIOSGENIN

FIG.10: STANDARD CALIBRATION CURVE OF SAPONINS FOR DIOSGENIN

The results of quantitative determination indicated that the leaf powder of Bauhinia variegata contains highest amount of Saponins in Aqueous extract. It contains lower amounts of steroids in Aqueous and ethanolic extracts of Bauhinia variegata. The amounts of Alkaloids are equal in both aqueous and ethanolic extracts.

Antiurolithiatic Aactivity:

Nucleation Assay Method:

FIG.11: STUDY OF CALCIUM OXALATE CRYSTALS IN THE AQUEOUS AND ETHANOLIC LEAF EXTRACTS OF BAUHINIA VARIEGATA

TABLE 5:  ANTIUROLITHIATIC EFFECT OF DIFFERENT CONCENTRATIONS OF AQUEOUS AND ETHANOLIC EXTRACTS OF BAUHINIA VARIEGATA LEAVES BY NUCLEATION ASSAY METHOD

The values are expressed as Mean ±SEM, n= 6. The values are significant,^* p< 0.05;**p < 0.01 when compared with standard.  AqELBV, EELBV, STD– aqueous extract of leaves of Bauhinia variegata, ethanolic extract of leaves of Bauhinia variegata, Standard.

 FIG. 12: NUCLEATION ASSAY ON THE LEAF EXTRACTS OF BAUHINIA VARIEGATA

 Growth Assay Method:

FIG.13: FORMATION OF CALCIUM OXALATE CRYSTALS

 TABLE 6: ANTIUROLITHIATIC EFFECT OF DIFFERENT CONCENTRATIONS OF AQUEOUS AND ETHANOLIC EXTRACTS OF BAUHINIA VARIEGATA LEAVES BY GROWTH ASSAY METHOD

The values are expressed as Mean ±SEM, n= 6. The values are significant,^* p< 0.05;^** p < 0.01 when compared with standard.  AqLBV, EELBV, STD – Aqueous extract of leaves of Bauhinia variegata, Ethanolic extract of leaves of Bauhinia variegata, Standard.

FIG. 14: GROWTH ASSAY ON THE LEAF EXTRACTS OF BAUHINIA VARIEGATA

Aggregation Assay:

TABLE 7: ANTI UROLITHIATIC EFFECT OF DIFFERENT CONCENTRATIONS OF AQUEOUS AND ETHANOLIC EXTRACTS OF BAUHINIA VARIEGATA LEAVESBY AGGREGATION ACTIVITY

The values are expressed as Mean ± SEM, n= 6. The values are significant,^* p< 0.05; ^** p < 0.01 when compared with standard. AqELBV, EELBV, STD – Aqueous extract of leaves of Bauhinia variegata, ethanolic extract of leaves of Bauhinia variegata, Standard.

FIG. 15: AGGREGATION ACTIVITY OF BAUHINIA VARIEGATA

The results of Anti urolithiatic activity by nucleation assay indicated that % inhibition was more in aqueous extract at 1000µg/ml when compared to standard drug Cystone tablets. The results of Anti urolithiatic activity by growth assay indicated that % inhibition was more in ethanolic extract at 1000µg/mlwhen compared to standard drug Cystone tablets. The results of Anti urolithiatic activity by aggragation assay indicated that % inhibition is equivalent to standard drug Cystone tablets. The leaves of the plant exhibited significant antilithiatic activity due to the presence of flavonoids and tannns in rich amount (Surendra et al., 2011). The statistical analysis was performed using one way Annova, Dunnetts test through Insat graph pad software.

CONCLUSION: The aqueous and ethanolic extracts of Bauhinia variegata leaf have inhibitory effect on CaOx crystallization thus may be beneficial in the treatment of urolithiasis. But there is a need of detailed investigation in elaborated preclinical experimentations and clinical trials to establish the use of plant as antiurolithiatic agent.

ACKNOWLEDGEMENT: The authors are thankful to Viaya institute of Pharmaceutical Sciences for Women for providing facilities to carry out the research work.

REFERENCES:

1. Khan M A, Pradhan D M R. Antiurolithic activity of Ceropegia bulbosa Der Pharmacia Sinica.2012; 3(1):148-152.
2. Lalitharani S, Mohan V R, Maruthupandian A V S R. Pharmacognostic investigation on Bulbophyllum albidum (weight) Hook F. Int j of pharm tech Research. 2011; 3(1):556.
3. Singh S K, Karthik, Pavankumar S. Disease monograph-5: urolithiasis; central council for research in homoeopathy. CSIR. 2011; 453: 3.
4. Shashi A, Jain S K, Verma A, Kumar M, Sabharwal M. Pathophysiology of kidney, gallbladder and urinary stones treatment with herbal and allopathic medicine: A review. Asian Pacific Journal of Tropical Disease. 2013; 3(6): 496-504.
5. Awari M D, Mute V, Babhale S P, Chaudhar P S. Anti-Urolithiatic effect of Achyranthus aspera Linn. Leaves extract on ethylene glycol induced nephrolithiasis. J Pharma Res.2009; 2(5): 994-997.
6. Khan M A, Pradhan D M R. Antiurolithic activity of Ceropegia bulbosa Der Pharmacia Sinica.2012; 3(1):148-152.
7. Singh S K, Karthik, Pavankumar S. Disease monograph-5: urolithiasis; central council for research in homoeopathy. CSIR. 2011; 453: 3.
8. Prasad KVSRG, Sujatha D, Gharathi K. Herbaldrugs in Urolithiasis – A review. Phcog rev.2007; 1(1):175-179.
9. Surendra K. Pareta, Kartik C Patra, Papiya M Mazumder and Dinakar Sasmal. Establishing the Principle of Herbal Therapy for Antiurolithiatic Activity: A Review. Journal of Pharmacology and Toxicology. 2011; 6: 321-332.
10. Garimella T S, Jolly C I, Narayanan S. In Vitro studies on AntiUrolitiatic Activity of seeds of Dolichos biflorus Linn and Rhizomes of Begenialigulata Phytotherapy Research. 2001; 15: 351-355.
11. The Wealth of India. A Dictionary of Indian Raw Material and Industrial Products CISR New Delhi 1952; 2: 56-57.
12. Ghaisas MM, Shaikh SA, Deshpande AD. Evaluation of immunomodulatory activity of ethanolic extract of the stem bark of Bauhinia variegata Linn, Int J of Green Pharmacy. 2009; 70-74.
13. Kamal M Modh, Purvi T Parmar, Bibhuranjan Panigrahi, Indermeet Singh Anand. Pharmacognostical and Phytochemical Evaluation of Leaves of Bauhinia variegata Pharmacognosy Journal. 2011; 3(24):45–49.
14. Yadava R, Reddy Madhusudhan. Anti-inflammatory activity of novel flavonol glycosides from Bauhinia variegata Linn. Nat Prod Res. 2002; 17(3):165-169.
15. Reddy Mopuru V B, Reddy Muntha K, Gunasekar Duvvuru, Caux and Cristelle, Bodo Bernard. A flavanone & a dihydrodibenzoxepin from Bauhinia variegata. Phytochemistry 2003; 64:879-88
16. Zhao YY, Cui CB, Cai B, Han B, Sun QS. A new Phenanthraquinone from the stem of Bauhinia variegata L. Jl Asian Nat Prod Res. 2005; 7(6):835-838.
17. Pandey Sonam, agrawal Rc. Effects of Bauhinia variegata bark extract on DMBA induced mouse skin carcinogenesis. A preliminary study. 2009; 3(3):158-162.
18. Pokhrel Nabu Raj, Adhikari RP, Baral. In vitro evaluation of antimicrobial activity Bauhinia variegata, locally known as Koiralo. World Journal of Microbiology & Biotechnology.2002; 18:69-71.
19. Rao, Yerra Koteswara, Fang Shih-Hua, Tzeng Yew-Min. Anti-inflammatory activity of flavanoids and a triterpene caffeate isolated from Bauhinia variegata. Phytotherapy Research 2008; 22:957-962.
20. Bodakhe Surendra H, Ram Alpana., Hepatoprotective propertiesof Bauhinia variegata barks extract. The Pharmaceutical Society of Japan 2007; 127(9):1503-1507.
21. Rajkapoor B, Jayaka rB, Anandar R, Kavimani S., Antiulcer effect of Bauhinia variegata Linn. In rats. Journal of Natural Remedies. 2003; 3(2):215-217.
22. Trease & Evans Pharmacognosy, 15^th edition, 2001; ELBS publications, New Delhi, Page no-138.
23. Qureshi S, DiabAl A, Al-Anazi FA, Al-Hassan MI, Ansari M M,Qureshi MF, Qureshi VF, et al., Negative aspects of the beneficial herbs: An over view. J Herb Med Toxicol. 2012; 6:1–14.
24. Saper BR, Phillips SR, Sehgal A, Khouri N, Davis BR, Paquin J, et al. Lead, mercury, and Arsenic in US- and Indian-manufactured Ayurvedic medicines sold via the internet.J Herb Med Toxicol. 2012; 6: 1-14.
25. Kokate C.K., Purohit AP, Gokhale SB. Text book of Pharmacognosy 46 edition, Niraliprakashan publications.
26. Anees A Siddiqui, Seemi Siddiqui. Natural products chemistry. Practical manual. 2008; First edition. J .S. Offset printers, Delhi.
27. Saper BR, Kales NS, Paquin J, Burns JM, Eisenberg MD, Davis BR, et al., Heavy metal content of Ayurvedic herbal medicine products. 2004; 214: 67-78.
28. Kim D, Jeong S, Lee C H. Antioxidant capacity of phenolic phytochemicals from various cultivars of plums. Food Chemistry. 2003; 81:321-326.
29. Bradley, R L. Moisture Content. In Introduction to the chemical analysis of food. United States: Jones and Barletts Publisher.1994; 234: 123-234.
30. Qureshi S, DiabAl A, Al-Anazi FA, Al-Hassan MI, Ansari M M,Qureshi MF, Qureshi VF, et al., Negative aspects of the beneficial herbs: An over view. J Herb Med Toxicol. 2012; 6:1–14.
31. Singh RS, Pandey HS, Ghanshyam. Two new long chain compounds from Bauhinia variegata lin Indian J Chem. 2006; 45B:2151-2153.
32. Armin G, Manfred H, editors. Alkaloids: The University of Zurisch Publication. 2009; 47:9431-9444.
33. Brouckere & Gillet. The determination of phytic acid. New York, NY: Springer.1933; 453: 234-236.
34. Trease GE, Evans WC. Pharmacognosy. 15th ed. Philadelphia: WB Saunders, Elsevier Science Limited.2002; 336: 564.
35. Chernyseva NN, Abdullin IF, Bundikov GK. Coulometric determination of purine alkaloids series with electro generated chlorine. J Anal Chem. 2002; 56; 663–5.
36. Chaudhary A., Tandon, C., and Singla, S.K. Calcium oxalate crystal growth inhibition by aqueous extract of Tamarindus indica, Indian J. Urol. 2009; 24:105-111.
37. Pak, H.Y., Ohata, and Holt, K. K. Effect of diphosphonate on crystallization of calcium oxalate in vitro, Kidney International. 1975; 7:154-160.
38. Farooq, A., Shiekh. E., Madhu Khullar, E S. K., and Singh. Lithogenesis: induction of renal calcifications by nanobacteria, Res. 2006; 240-254.
39. Sharma RK, editor. “15^thAdhyaya in chikitsastana” of Charaka Samhita 1st ed. Vol. 4. Varanasi: Chaukhambha Sanskri Sansthan, Varanasi (UP); 2005; 345: 689-876.
40. Saha S, Verma RJ. Inhibition of calcium oxalate crystallization in vitroby an extract of Bergenia ciliata. Arab Journal of Urology 2013; 11: 187-192.
41. Atmani F, Khan S.R. Effects of an extract from Herniaria hirsuta on calcium oxalate crystallization in vitro. Br J Urol Int. 2000; 85: 621–625.
42. Masao T., Osamu M., Kazuhiro Y., Ken-Ichi K.,. Shiro T, Akihiko O. Fibronectin as a potent inhibitors of calcium oxalate urolithiasis. J Urol; 2000; 164: 1718–1723
    

    ---

How to cite this article:

Mamillapalli V, Khantamneni PL, Mohammad Z, Mathangi A, Nandigam N, Namburi  SM and Katta V: Phytochemical and in vitro antiurolithiatic studies on the leaf extracts of Bauhinia variegata L.. Int J Pharm Sci Res 2016; 7(10): 4074-84.doi: 10.13040/IJPSR.0975-8232.7(10).4074-84.

All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.