Urolithiasis

Pharmacological Evaluation of Antiurolithic Activity of Leaves of
Adansonia digitata Linn.

SUBMITTED BY
Mr. Swaroop Singh

SUPERVISOR
Prof. (Dr.) Neelam Balekar

College of Pharmacy, IPS Academy,
Rajendra nagar, A.B. Road, Indore-452012
2010-2011
1

1. INTRODUCTION
 Urolithiasis denotes stones originating anywhere in the urinary tract, including the

kidney and bladder (Pearle & Nakada, 2009).
 Overall male to female ratio of 2.4:1 increased from 1977 (1.86:1) to 2006 (2.7:1)
(Knoll et al., 2011).

 Recurrence rate (10 to 20% within 1 to 2 years, 35% within 5 years, & 60% within
10 years) (Berner et al., 2011).
 Types of kidney stones (Dipiro et al., 1997)

• Calcium stones (CaOx and CaP)- 80%
• Uric acid- 9%
• Struvite-10%

• Others- 1%
College of Pharmacy, IPS Academy

Fig. 1. Stones in kidney
2

1.1. PATHOGENESIS
 Urine is a complex fluid that contains a number of minerals, inhibitors and

promoters of crystallization.
 Crystallization can not occurs whether promoters and inhibitors are present or
absent, if a state of supersaturation does not exist.

Fig. 2. Scheme of the precess of stone formation (Tsujihata, 2005).

3

1.2. Possible series of events of the normal and pathological crystallization in
urine (adopted from ‘Oxford Textbook of Clinical Nephrology’).

Fig. 3. Normal crystallization in urine.

Fig. 4. Pathological crystallization in urine.

4

1.3. Risk factor associated with kidney stones
 Low urine volume

 Hypercalciuria (most common)
 Hypocitraturia

Primary defect in
renal tubular
reabsorption of
calcium

 Hyperoxaluria
Ion activity product

Supersaturation

Excretion of Calcium
in urine

Plasma level of calcium

Renal hypercalciuria

PTH

Resorptive
hypercalciuria

Fig.5. Types of hypercalciuria

Production of vit. D3
in kidney

Calcium transport
from lumen of gut

Absorptive
hypercalciuria

5

2. MANAGEMENT OF KIDNEY STONES
Therapeutic approaches

 Removal of stones (surgical management & MET)
 Lowering supersaturation (fluid intake & dietary modification)
 Prevent crystal retention in kidneys

 Reduction of renal oxidative stress
2.1. SURGICAL MANAGEMENT (Pearle & Nakada, 2009)
1) ESWL
2) PCNL
3) URS (flexible ureteroscopy)


6

Cont…
2.2. MEDICAL EXPULSIVE THERAPY (Pearle & Nakada , 2009)

 A new clinical approach
 Urethral smooth muscle relaxation
 CCB & alpha-1 receptor blockers

2.3. FLUID INTAKE AND DIETARY MODIFICATION
 High fluid intake in order to maintain a urine output of at least 2 L/day
 Reduced supersaturation and dilutes promoters of CaOx crystallization

 Restriction of animal protein diet
 Restriction of Na intake (2-3gm)
 No restriction of calcium


7

Cont…
2.4. PHARMACOLOGICAL TREATMENT (Dipiro et al., 1997)

 Diuretics
 Alkali Potassium citrate, potassium magnesium citrate
 Pyridoxine
2.5. LIMITATION
 Persistent of stone fragments cause renal injury & an increase in stone recurrence
with ESWL & development of diabetes and hypertension on long term use
(Butterweck & Khan 2000).
 Long

term

use

of

thiazide

causes

K

depletion

(hypokalemia), Fatigue, dizziness, impotence & musculoskeletal symtoms.

8

3. PLANT PROFILE
3.1. BIOLOGICAL SOURCE

 Botanical name

: Adansonia digitata L.

 Family

: Bombacaceae

3.2. PHARMACOLOGICAL ACTIVITIES OF LEAVES OF PLANT

 Anti-ulcer activity (Karumani et al., 2008)
 Antimicrobial activity (Oloyede et al., 2010)
 Antioxidant activity (Vetuani et al., 2002)

 Antiviral activity (Hudson et al., 2000 ; Ananil et al., 2000)
 Diuretic activity (Kirubha et al., 2006)
 Anti-inflammatory activity (Selvarani & Hudson, 2009)

 Antisickling activity (Adesanya et al., 1988)

9

3.3. TRADITIONAL USES OF LEAVES OF Adansonia digitata (Sibibe &
Willians, 2002)
 Insect bite & gunia worm sores & otitis
 Diseases of the bladder and urinary tract
 Antiasthmatic
 Antipyretic
 Fatigue
 Dysentery
 Opthalmia
 Astrigent
 Diaphoretic
 Tonic

Fig.6. Leaf of Adansonia digitata.
10

3.4. PHYTOCHEMICAL CONSTITUENT OF LEAVES OF PLANT
 Excellent source of amino acids (Yazzie et al., 1994)
 Carbohydrate , fat, protein and fibre (Caluwe., 2010)
 Antioxidants such as catechins and adansonia flavonosides (Maranz et al., 2007;
SEPASAL database., 2007; Kunkel ., 1979)
 Minerals (iron, zinc, calcium, magnesium, manganese, copper, phosphorus &
sodium) (Glew et al., 1997)
 Provitamin A along with thiamine, riboflavin &niacin (Caluweet al., 2010)
 Mucilage (Woolfe et al., 1977)
 Alkaloids & Steroids (Oloyede et al., 2010)
 Tannis (Oloyede et al., 2010)
 Cardio active glycosides (Oloyede et al., 2010)

11

4. OBJECTIVE
 The objective of present study to assess the effectiveness of leaves of Adansonia
digitata L., a medicinal plant used to treat kidney and bladder disease as
traditionally, as a prophylactic agent against CaOx stones by in vitro and in vivo
study.

 The present study shall undertake
•

Collection of plant material from local area.

•

Identification and authentication of the plant from Botanical survey of India.

•

Preparation of suitable extracts of selected plant material.

•

Evaluation of the extract for antiurolithic activity by in vitro and in vivo study.


12

4.1. RATIONALE
 No satisfactory drug to use in clinical therapy for the prevention or the recurrence of

stones.
 Traditional claim of the plant(Sidibe & Willians, 2002; Caius, 2003).
 Absence of literatures on the systematic investigation of antiurolithic activity.

 Data from In vitro and In vivo & clinical trials reveal that phytotherapeutic agent
could be useful in the management of urolithiasis. In this regards many plant have
been used to treat stones like Dolichous biflorus, Berginia ligulata, Tribulus
terrestris, Orthosiphon grandiflorus, Phylanthus niruri (Butterweck & khan, 2000).
 Diuretic activity (Kirubha et al., 2006) & antioxidant activity (Vetuani et al., 2002).
 Antiurolithic activity of L-arginine amino acid (Pragasam et al., 2005).


13

5. PLAN OF WORK
PLAN OF WORK
Minor project
Phytochemical
investigations

and

Major project

pharmacognostic
Detailed pharmacological investigations
of antiurilithic activity

Collection of plant
Authentication of plant material
Preparation of aqueous extract of leaves

In vivo study
Sodium oxalate induced urolithiasis
model in male rats

In vitro study
A gel model for measuring crystallization
inhibitor activity
Nucleation and aggregation assay
Fig.7. Plan of work

14

6. EXPERIMENTAL WORKS AND RESULTS
6.1. PLANT MATERIALS AND EXTRACTION PROCEDURE
 Leaves of Adansonia digitata were collected from MHOW, Indore & authentication of
plant was done in BSI, Pune (a voucher specimen number BSI/WC/Tech./2011/900A).
 Aqueous extract was prepared by decoction method (temperature 90-95ºC) and was dried
at 37ºC. Yild was found 15.8 % (Pauly, 2001).

6.2. AGAR GEL MODEL (Schneider et al., 1983)

Fig.8. Diagram of punch-hole pattern (all dimensions in mm) of agar gel model

15

6.2.1. Protocols For Agar Gel Model
Table 1:Protocols for agar gel model.
Group

First method

Second method

Modifier (70 µL)

Modifier (conc. in agar gel)

Control

Distilled water

Distilled water

Standard

Magnesium chloride (100mM) Magnesium chloride (100mM)
Trisodium citrate (100mM)
HA extract (1mg/mL)

HA extract (1mg/mL)

HA extract (10mg/mL)

Test

Trisodium citrate (100mM)

HA extract (10mg/mL)


16

6.2.2. Results of Agar Gel Model
Crystallization streak’s Pattern
of CaOx crystal produced by
starter solution of 0.3 M CaCl2

and ammonium oxalate in agar
gel with addition of
a. distilled water.
b. 200 mM of MgCl2.
c. 1 mg/mL AAD.
d. 10 mg/mL of AAD.
e. 25 mg/mL of AAD.
f. distilled water.
g. 200 mM of MgCl2.
h. 100 mM of MgCl2.
i. 1 mg/mL of AAD.
j. 10 mg/mL of AAD; n=6

Fig.9. Pattern of streak of caox cyrstal in agar gel

17

6.3. NUCLEATION AND AGGREGATION ASSAY (Kulaksizoglu et al., 2008 &
Hennequin et al.,1993)
 The nucleation and aggregation of CaOx were studied at pH 5.7 using turbidimetric

measurment at 620 nm of suspensions produced by mixing calcium chloride and
sodium oxalate (4 mM & 0.5 mM respectively).
 Stock solutions (pH 7.5)
•

Calcium chloride (8 mM, containing 200 mM NaCl & 10 Mm sodium acetate).

•

Sodium oxalate (1 mM, ontaining 200 mM NaCl & 10 Mm sodium acetate).

•

Trisodium citrate (0.30 mM) & test solutions (0.2, 2.0 & 20.0) in calcium chloride
stock solution.

 All experiments with modifiers of CaOx crystallization were performed at assay
conc. of 4 mM CaCl2, 0.5 mM Na2C2O4, 200 mM NaCl, 10 mM sodium acatate,
0.1, 1.0, 10.0 mg/mL of extract, pH 5.7.

18

SN

SA

Equilibrium

0.08
0.07

OD (620 nm)

0.06
0.05

0.04
0.03

0.02
0.01

0
0

2

4

6

8

10 12 14 16 18 20 22 24 26 28 30

Time (min)
Fig. 10. Time Course Measurements of OD at 620 nm At Standard Condition
of CaOx Crystallization

19

6.3.1. Results of Nucleation and Aggregation Assay.

Table 2: Effect of extract on calcium oxalate crystallization.
Parameters

Control

tmax (min)

Std

Test
0.1mg/mL 1.0 mg/mL

10.0 mg/mL

7.96 ± 0.31

12.47 ± 0.54 12.38 ± 0.79 13.22 ± 0.38*

23.68 ± 1.69***

SN ( 10-2)

1.05 ± 0.04

0.36 ± 0.03**

0.41 ± 0.02*

0.23 ± 0.04***

% Inhibition of SN

-

65.12 ± 3.70 57.71 ± 5.40 60.42 ± 2.20

77.93 ± 4.10

SA ( 10-3)

1.24 ± 0.05

0.64 ± 0.06**

0.70 ± 0.02 0.57 ± 0.05***

0.63 ± 0.05*

% Inhibition of SA

-

48.42 ± 5.54 43.41 ± 2.04 53.03 ± 4.95

49.19 ± 4.08

0.52 ± 0.08

*P < 0.05,** P < 0.01,***P < 0.001 vs control (Kruskal-Wallis variance analysis); n=6-9,

SEM
20

6.4. ANIMAL MODEL OF SODIUM OXALATE INDUCED UROLITHIATIC IN
MALE RATS (Gupta et al., 2006; Khan et al., 1981., Ramesh et al., 2010)
Table 3: Treatment Schedule

Groups

Treatment (daily for7 days)

Normal control

0.9% saline (1 mL/100g, orally & i.p.)

Urolithiactic group

NaOx (7 mg/100g, i.p.) + 0.9% saline orally (1 mL/100g)

Standard group

NaOx (7 mg/100g, i.p.) + potassium citrate monohydrate
(0.25g/100g, orally)

Treated group

NaOx (7 mg/100g, i.p.) + aqueous extract (200mg/kg, orally)

Urine volume, pH, body weight, kidney weight (wet and dry), serum and urine
level of creatinine, urea, magnesium, and calcium were evaluated on day 7. In
addition, histopathological changes in kidney and oxalate in urine and kidney were
evaluated.

21

6.4.2. Results of Sodium Oxalate Induced Urolithiasis
Table 4: Effect of ADD on serum chemistry in control and experimental animals
Parameters

Urolithiactic

PCi treated/

ADD Treated

Control

Standard group

group

Control
(mg/dl)

Ca++

10.44 ± 2.36

11.99 ± 0.065

10.26 ± 2.68

12.68 ± 4.96

Mg++

2.48 ± 0.21

1.63 ± 0.21***

2.76 ± 0.19###

2.90 ± 0.090###

Urea

8.19 ± 1.03

27.36 ± 5.59***

14.21 ± 4.75###

11.78 ± 1.69###

Creatinine

0.59 ± 0.19

2.13 ± 0.77**

0.89 ± 0.13###

0.85 ± 0.07##

Data are expressed as mean ± SD of 5 animals per group; * p< 0.05,
versus control group; # p< 0.05,

## p<

0.01,

### p<

** p<

0.01,

*** p<

0.001

0.001 versus urolithiatic control; one-way

analysis of variance, ANOVA followed by Newman–Keuls test for multiple comparisons

22

Table 5: Effect of AAD on urinary chemistry in control and experimental animals
Parameters

Control group

Urolithiactic

PCi treated/

ADD treated

control

standard group

group

Vol. (ml/100 g)

6.34 ± 4.11

4.51 ± 0.48

7.78 ± 1.32

6.02 ± 2.06

pH

8.78 ± 0.24

7.30 ± 0.84*

8.97 ± 0.97#

8.85 ± 0.22#

Ca++ (mg/dl)

28.96 ± 4.99

8.31 ± 1.40***

4.84 ± 3.47***

3.35 ± 0.89***

Mg++ (mg/dl)

2.50 ± 0.13

2.09 ± 0.10

2.80 ± 0.17#

3.35 ± 0.43###

Ox (µg/dl)

23.94 ± 0.84

29.48 ± 1.15***

23.83 ± 0.86###

24.35 ± 0.46###

Urea (mg/dl)

1686 ± 667.7

543.3 ± 174.8*

1529.7 ± 261.5#

1830.1 ± 248.1#

Creatinine

57.04 ± 15.05

16.92 ±1.43**

55.27 ±9.44##

41.50 ±4.67#

(mg/dl)

23

Table 6: Effect of AAD on kidney weight and chemistry in control and experimental
animals
Parameters

Control

Urolithiactic

PCi treated/

ADD Treated

Control

Standard group group

Wet Weight (mg/100 g) 385.00 ± 17.65 674.60 ± 62.87*** 474.70 ± 75.02### 527.80 ± 36.19###
Dry Weight (mg/100 g) 104.90 ± 9.46 127.80 ± 9.79**

95.09 ± 12.73##

100.60 ± 13.47##

Width (mm)

7.40 ± 0.89

11.80 ± 0.83***

9.00 ± 0.70###

9.80 ± 0.83##

length (mm)

13.80 ± 1.30

19.00 ± 0.70**

14.00 ± 3.08##

14.80 ± 2.77##

Ox (µg/100 mg)

3.642 ± 0.22

6.12 ± 1.96*

4.41 ± 0.57#

3.59 ± 0.45#

Ca++ (mg/100 mg)

0.49 ± 0.22

1.34 ± 0.48**

0.75 ± 0.16#

0.76 ± 0.13#


24

Renal tissue of a. Control group’s rats
showing normal kidney parenchyma with
glomerulus, peroximal and distal convoluted
tubules with normal brush-broder and intact
glomeruli (×100). b. Control group’s rats
showing no sign of crystallization (×400) . c
Urolithiatic rats showing focal areas of
necrosis, infiltration of inflammatory cells and
atrophy glomerulus (×100). d. Urolithiatic rats
showing deposition of numerous crystals and
presence of casts and extensive haemorrhages
in collecting tubules (×400). e. Urolithiatic
rats showing deposition of salt of calcium,
extensive atrophy of tubular lumen as well as
dialated tubules (×400). f. Urolithiatic rats
showing extensive haemorrhages and
infiltration of inflammatory cells (×400). g.
PCi
treated
rats
showed
moderate
haemorrhage and absence of CaOx crystals
(×100). h. PCi treated rats showing
regeneration
of
tubules
with
large
hyperchromic nuclei (×400). i. AAD treated
animals showing highly regenerating tubules
with normal brush-broder and absence of
CaOx crystals (×100). j. AAD treated animals
showing no deposition of crystals with rare
tissue injury (×400).

Fig .11. Light microscopic of H & E stained kidney section

25

7. CONCLUSIONS
7.1. CONCLUSIONS

 Extract of leaves of Adansonia digitata could act as antilithic agent , by inhibiting
nucleation and aggregation of calcium oxalate crystals.
 High binding of extract with oxalate could prevent dietary absorption of oxalate
form intestine.
 In conclusion, based on our present results it seems that AAD contains several active
compounds which in a multifunctional/synergistic approach act as antiurolithic. The
mechanism underlying this effect is mediated possibly through an antioxidant,
antimicrobial, and/or anti-inflammatory activities contained in ADD and presence of
inhibitors of CaOx crystallization like magnesium and acidic amino acids. In vitro
and in vivo experiments confirm that AAD could be beneficial in the management of

CaOx stone disease.

26

9. REFERENCES
1.

2.

3.

4.

5.

6.

Ananil, K, Hudson, JB, Souzal, CD, Akpaganal, K, Tower, GHN, Arason, JT &
Gbeassor, M 2000, ‘Investigation of medicinal plants of togo for antiviral and
antimicrobial activities’, Pharmaceutical Biology, vol. 38, no. 1, pp. 40-45.
Atmani, F, Slimani, Y, Mimouni, M & Hacht, B 2003, ‘Prophylaxis of calcium
oxalate stones by Herniaria hirsute on experimentally induced nephrolithiasis in
rats’, BJU International, vol. 92, pp. 137-140.
Brener, ZZ, Winchester, JF, Salman, H & Bergman, M 2011, ‘Nephrolithiasis:
evaluation and management’, Southern Medical Journal, vol. 104, no. 2, pp. 133139.
Butterweck, V & Khan SR 2000, ‘Herbal medicines in the management of
urolithiasis: alternative or complementary?, Planta Med, vol. 75, pp. 1095-1103.
Davison, AM, Cameron, JS, Grunfeld, JP, Ponticella, C, Ritz, E, Wineras, CG &
Ypersele, CV 2005, Oxford textbook of clinical nephrology, Oxford university
press, New York.
Dipiro, JT, Talbert, RL, Yec, GC, Matzke, GR, Wells, BG & Posey, LM
1997, Pharmacotherapy a pathophisiology approach, Applenton & Lange, USA.


27

7.

Glew, RH, Vanderjagt, DJ, Lockett, C, Grivetti, LE, Smith, GC, Pastuszyn, A &
Millson M 1997, ‘Amino acid, fatty acid, and mineral composition of 24
indigenous plants of Burkina Faso’, Journal of Food Composition and Analysis,
vol. 10, pp. 205-217.
8. Hennequin, C, Lalanne, V, Daudon, M, Lacour, B & Drueke, T 1993, ‘A new
approach to studying inhibitors of calcium oxalate crystal growth’, Urol Res, vol.
21, pp. 101-108.
9. Kirubha, TSV, Nagavalli, D, Hemalatha, S & Karunambigai, K 2006, ‘Diuretic
activity of the extracts of Adansonia digitata leaves’, Hamdard Medicus, vol. 49,
no. 4, pp. 53-54.
10. Knoll, T, Schubert, AB, Fahlenkamp, D, Leusmann, DB, Wendt-Nordahl, G &
Schubert G 2011, ‘Urolithiasis through the ages:data on more than 200,000
urinary stone analyses’, J Urol, vol. 185, no. 4, pp. 1304-11.
11. Kulaksizoglu, S, Sofikerim, M & Cevik C 2008, ‘In vitro effect of lemon and
orange juices on calcium oxalate crystallization’, Int Urol Nephrol, vol. 40, pp.
589-594.


28

12. Okada, A, Noumura, S, Higashibata, Y, Hirose, M, Gao, B, Yoshimura, M, Itoh, T,
Tozawa, K & Kohri, K 2007, ‘Successful formation of calcium oxalate crystal
deposition in mouse kidney by intraabdominal glyoxylate injection’, Urol Res, vol.
35, pp. 89-99.
13. Oloyede, GK, Onocha, PK, Soyinka, I, Oguntokun, O & THonda, E 2010,
‘Phytochemical screening, antimicrobial and antioxidant activies of four Nigerian
medicinal plants’, Annals of Biological Research, vol. 1, no. 2, pp. 114-120.
14. Pauly, G 2001, ‘Use of an extract of the genous Adansonia, United State Patent
6,274,123 B1.
15. Pearle, MS & Nakada, SY 2009, Urolithiasis medical and surgical management,
Informa healthcare, London.
16. Pragasam, V, Periandavan, K, Sumitra, K, Srinivasan, S & Varalakshmi, P 2005,
‘Oral L-arginine supplementation ameliorates urinary risk factors and kinetics
modulation of Tamm-Horsfall glyprotein in experimental hyperoxaluric rats’,
Clinica Chimica Acta, vol. 360, no. 1-2, pp. 141-150.
17. Schneider, HJ, Rohrborn, C & Rugendroff EW 1983, ‘A gel model for measuring
crystallization inhibitor activities in calcium oxalate urolithiasis’, World Journal of
Urology, vol. 1, pp. 155-158.

29

18. Selvarani, V & James HB 2009, ‘Multiple inflammatory and antiviral
activities in Adansonia digitata leaves, fruits and seeds’, Journal of
Medicinal Plants Research, vol. 3, no. 8, pp. 576-582.
19. Selvarani, V & James HB 2009, ‘Multiple inflammatory and antiviral
activities in Adansonia digitata leaves, fruits and seeds’, Journal of
Medicinal Plants Research, vol. 3, no. 8, pp. 576-582.
20. Sidibe, M & Williams, JT 2002, ‘Baobab Adansonia digitata L.’,
International centre for underutilised crops, UK.
21. Vertuani, S, Bracciol, E, Buzzoni, V & Manfredini, S 2002, ‘Antioxidant
capacity of Adansonia digitata fruit pulp and leaves’, Acta
Phytotherapeutica, vol. 5, no. 2, pp. 2-7.
22. Woolfe, ML, Chaplin, MF & Otcher G 1977, ‘Studies on the mucilages
extracted from okra fruits (Hibiscus esculentus L.) and baobao leaves
(Adansonia digitata L.)’, Journal of Science, Food and Agriculture, vol. 28,
pp. 519-529.
23. Yazzie, D, Vanderjagt, DJ, Pastuszyn, A, Okolo, A & Glew, RH 1994, ‘The
amino acid and mineral content of Baobab (Adansonia digitata L.) leaves’,
Journal of Food Composition and Analysis, vol. 7, pp. 189-193.

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