Inhibitory activity of Alpinia galanga on struvite crystals

1. GROWTH CHARACTERIZATION OF
STRUVITE CRYSTALS INFLUENCED
BY ALPINIA GALANGA METHANOL
RHIZOMES EXTRACT
PRESENTED BY:
S.DHASARADHA BAI
I M.SC BIOCHEMISTRY

2. GROWTH CHARACTERIZATION OF STRUVITE CRYSTALS INFLUENCED BY ALPINIA GALANGA
METHANOL RHIZOMES EXTRACT
ABSTRACT
Objective: To investigate the inhibitory effect of methanol extract of Alpinia galanga rhizomes on the growth of
struvite crystals.
Methods: Struvite crystals were grown by the single diffusion gel growth technique and the inhibitory effect of
methanol extracts of Alpinia galanga rhizomes on the growth of struvite crystals has been studied. The grown
crystals were characterized by Fourier Transform Infrared Spectroscopy (FTIR) methods for further confirmations.
Results: With an increase in the concentration of methanol extract of Alpinia galanga rhizomes, the weight of the
formed crystals were gradually reduced from 2.66 g to 0.25 g in struvite crystals, respectively. The crystals
harvested from the struvite were characterized by Fourier Transform Infrared Spectroscopy (FTIR) to confirm the
functional groups. Results obtained indicated that Alpinia galanga rhizomes has the potential to inhibit the
formation of struvite crystals.
Conclusion: This study confirms that using methanol extract of Alpinia galanga rhizomes can promote the
formation of ammonium magnesium phosphate hexahydrate crystals and reduce the nucleation rate of struvite
crystals, a major component of urinary stone.
Key words: Struvite, Alpinia galanga rhizomes, Fourier Transform Infrared Spectroscopy (FTIR).

3. INTRODUCTION
 Urolithiasis have been found in the tombs of Egyptian mummies dating back to 4000BC
 Urolithiasis is a global problem and it is also called as nephrolithiasis or kidney stones
 Urolithiasis is the presence of uroliths or calculi in the urinary tract
 Genetic predisposition, metabolic disorders and poor intake of water are the causes of kidney stones
 It is commonly found in males than females due to a longer urethra
 Kidney stone is also called renal calculi
 Urolithiasis and nephrolithiasis refers to the condition of having calculi in the urinary tract and
kidneys respectively
 Crystals also form if the urine is too alkaline, too acidic or too concentrated.
 Calcium stones – The common variety is Calcium Oxalate Monohydrate [ COM ]
TYPES OF KIDNEY STONES
 Struvite stones – contains Magnesium
 Uric acid stones – forms when there is too much acid in the urine
 Cystine stones – occurrence is very rare

4. PATHOPHYSIOLOGY OF URINARY
STONE
 Alkaline urine
 The presence of urea or ammonia in the urine
 High concentration of minerals in the urine
Kidney stones contain calcium oxalate, phosphate, magnesium and uric acid and
the formation of kidney stone includes :
 Nucleation
 Growth
 Aggregation of kidney stone

5. REVIEW OF LITERATURE
 The treatment and removal of renal calculi is often dependent upon the composition
and hardness of the stone , as well as stone size and placement in the urinary tract .
( Otnes , 1983; Dietrich et al.,1990)
 In addition to the surgical management of the problem there are certain drugs and
other conventional therapies which are recommended for its control. ( Pearl et
al.,1990)
 Lemon juice and orange juice are used as natural substances to inhibit calcium
oxalate crystallization under invitro ( Kulaksizoglu et al.,2007)
 The aqueous extracts of Commiphora wightii dissolved struvite type of calculi to some
extent in an invitro model ( Chauhan et al., 2008)

6. OBJECTIVE
 The growth characteristics of the most common types of urinary stones like struvite
crystals and the effect of methanol extracts of rhizomes of Alpinia galanga has been
studied
 The identification of one Penta cyclic triterpenoid botulin from the rhizomes of Alpinia
galanga extract by HPTLC techniques
 KINGDOM – PLANTAE
 CLASS – ANGIOSPERMS
 ORDER – ZINGIBERALES
 FAMILY – ZINGIBERACEAE
 GENUS – ALPINIA
 SPECIES – GALANGA
 DESCRIPTION - The plant grows from the rhizomes in clumps of stiff stalks upto 2m in
height with long leaves and red fruit
 It is native to SOUTH ASIA and INDONESIA
 The red fruit has a smell similar to cardamom

7. MATERIALS AND METHODS
 Anhydrous methanol
 Ammonium dihydrogen ortho phosphate
 Magnesium acetate
 Sodium metasilicate
 Orthophosphoric acid
COLLECTION OF PLANT MATERIAL
 The rhizome of Alpinia galanga were collected in the month of June, 2017 from Kolli
hills, Trichy, Tamil Nadu, India
 The plant was identified and confirmed by Dr.S.John Britto , Director, Rapinat
Herbarium, St. Joseph’s College, Trichy

8. PREPARATION OF METHANOL
EXTRACTS
 The rhizomes of Alpinia galanga were washed in running water, cut into small pieces
and then shade dried for a week at 35-40ºC.
 After that it was grinded to a uniform powder of 40 mesh size (Doughari et al., 2007).
 The methanol extracts were prepared by soaking 100 g of the dried powder plant
materials in 1 L of methanol by using a soxhlet extractor for 10 hours continuously.
 The extracts were filtered through Whatmann filter paper No. 42 (125mm).
 The filtered extract was concentrated and dried by using a rotary evaporator under
reduced pressure.
 The obtained residue 1.2 g (rhizomes) was used to prepare the series of (0.15, 0.25,
0.50, 0.75 and 1.0%) methanol supernatant concentrations for in vitro studies .

9. PHYTOCHEMICAL SCREENING
 Test for terpenoids – Salkowski test
 Test for flavonoids
 Foam test – saponin
 Braymer’s test – tannin
 Hager’s test – alkaloids
 Salkowski test – steroids
 Liebermann’s test – glycosides
 Precipitate test – phlobatannins
 Xantoproteic test – proteins
 Test for coumarin , emodin, antroquinone, anthocyanin and carbohydrates
 Keller Killiani test – cardiac glycosides
 Test for phenol , leucoanthocyanin and xanthoprotein

10. IDENTIFICATION OF ACTIVE
COMPOUNDS FROM RHIZOMES BY
HPTLC
 Sample preparation
 Thin layer chromatography
 Detection and estimation of betulin
 Isolation of active compounds by column chromatography
 Growth and characterization of struvite crystals
 Statistical analysis

11. RESULTS AND DISCUSSIONS
CRYSTALS GROUP TREATENTS Harvested
crystals
(gram)
PERCENTAGE
OF INHIBITION
Struvite A Control 2.86 0%
B Control+Distilled water 2.32 18.8%
C Control+0.15% extracts 1.87 34.6%
D Control+0.25% extracts 1.72 39.8%
E Control+0.50% extracts 1.63 43.0%
F Control+0.75% extracts 0.52 81.8%
G
H
I
J
Control+1.00% extracts
Control+ fraction 7
Control+ fraction 8
Control+ fraction 12
0.43
0.72
0.83
0.64
84.9%
74.8%
70.9%
77.6%
S. No Phytochemical
constituents
Methanol extracts
of rhizomes of Alpinia
galanga
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Tannin
Flavonoid
Terpenoid
Saponin
Phlobatanin
Steroid
Carbohydrate
Glycoside
Coumarin
Alkaloid
Protein
Emodin
Anthoquinone
Anthocyanine
Cardiac glycosides
Leucoanthocyanin
Phenol
Xanthoprotein
+
+
+
+
+
+
+
+
+
+
_
+
+
-
+
+
+
+

13. SUMMARY AND CONCLUSION
 Kidney stone disease is a common urinary stone disorder in humans and often cause severe pain,
which may lead to emergent hospitalization, shock wave lithotripsy and surgery.
 Calcium oxalate is a major component of kidney stone. It forms upon the supersaturation of the urine
with calcium and other salts especially oxalate. The size of the stone can increase and obstruct in the
urinary system. Current pharmacological treatment of kidney stone consists of Gallium nitrate.
Although the most effective treatment of kidney stone is extracorporeal shock wave lithotripsy, the
side effects of this method are grave and can lead to recurrence of kidney stones. Therefore alternative
treatments are of high interest by using medicinal plants or phytotherapy.
 With an increase in the concentration of methanol extract of rhizomes of Alpinia galanga ,the weight
of the formed crystals were gradually reduced from 2.43 g to 0.08 g in struvite crystals.
 One way ANOVA performed with treated and untreated crystal growth data obtained from struvite
crystals showed significant differences (p<0.05)
 The purpose of this dissertation is to investigate the beneficial effects of rhizomes of Alpinia galanga at
a different dose and single compounds for the prevention of kidney stone formation.

14. REFERENCES
 Aggarwal, S. Tandon, C.D. Forouzanveh, M. Simgla, S.K. Kiran, R. and Jethi, R.K. 2000. Role of
biomolecules from human renal stone matrix on COM crystal growth. Molecular and cellular
Biochemistry., Vol.210, pp.109 –119.
 Anantha Krishna Chaitanya, D. Santosh kumar, M. Monohar Reddy, A. Mukherjee, N.S.V. Sumanth,
N.H. and Ramesh, R. 2010. Antiurolithiatic activity of Macrotyloma uniflorum seed extract of ethylene
glycol induced urolithiasis in albino rats, Vol.1(5), pp.216-226.
 Anbu, J. Suman, S. Swaroop Kumar, K. Satheesh Kumar, R. Nithya, S, Kannadhasan. 2011.
Antiurolithiatic activity of ethyl acetate root extract of Ichnocarpus frutescene using ethylene glycol
induced method in rats. J. Pharma. Sci. and Research., Vol. 3(4), pp. 1182-1189.
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