Ancient Science of Life, Jol. 3, Ao.

1 (21) Pages 14 - 17
Effect of Phenolic Compounds from Bergenia ciliata leaves on
Experimental kidney stones
(Haw.) Sternb.
14
Vivek V. Byahatti`, K. Vasantakumar Pai, Marina G. D'Souza
Department oI PG studies and Research in Industrial Chemistry, Kuvempu University, Karnataka, India.
be useIul. However, the rational behind their use is not well ABS1RAC1
established except Ior a Iew plants and are reported to be
Kidney stone formation or Urolithiasis is a complex process
eIIective with no side eIIects.
that results from series of several physicochemical events
including super-saturation, nucleation, growth, aggregation
Bergenia ciliata (Haw) sternb. (SaxiIragaceae), is a
and retention within the kidneys. Among the treatments
perennial herb with stout underground rhizomes and large
include Extracorporeal Shock Wave Lithotripsy (ESWL) and
hairy leaves, oIten called as MEGASEAS, Iound in
drug treatment. Even this ESWL treatment may cause acute
temperate Himalayas between altitudes 900-3000 mts. It is
renal infury, decrease in renal function and increase in stone
commonly known as Pashana bheda/ silabheda and is
recurrence. In addition, persistent residual stone fragments
endowed with phytoconstituents Ilavonoids, tannins, sterols
and possibility of infection after ESWL represent a serious
etc. This Ayurvedic and Unani herb has been documented
problem in the treatment of stones. Data from in-vitro, in-
earlier Ior its therapeutics in treating kidney stones, ulcers,
vivo and clinical trials reveal that phytotherapeutic agents
spleen enlargement, dysentery, Iever, cough, as an
could be useful as either alternative or an adfunct therapy in
astringent, antisorbutic, laxative.
the management of Urolithiasis. Medicinal plants / natural
products are more acceptable to the body because they
The rhizomes oI the herb have been reported to possess
promote the repair mechanismin natural way. Jarious plant
antiurolithiatic, anti-inIlammatory, antioxidant,
1-7]
species of the genus Bergenia, have been reported to posses
antibacterial properties . This study has been undertaken to
antiurolithiatic property. Bergenia ligulata s
evaluate Bergenia ciliata (Haw.) Sternb. leaves Ior their
one the ingredient of reputed herbal formulation Cystone for
possible potential to dissolve experimental urinary stones
the treatment of kidney stones. In this study alcohol, butanol,
using a modiIied invitro model and to isolate the chemical
ethyl acetate extracts and isolated phenolic compounds from
entity (s) responsible Ior the activity.
the Ayurvedic and Unani herb, Bergenia
for their
potential to dissolve experimentally prepared kidney stones-
calcium oxalate and calcium phosphate, by an in-vitro
model. Phenolic compound P , isolated from the ethyl
1
acetate fraction of the leaves, demonstrated highest
Bergenia ciliate (Haw.) Sternb leaves were collected in the
dissolution of both stones when compared to test extracts at
month oI July/August Irom the local Iorests oI Chota Shimla
10 mg concentration. However, it was more effective in
and authenticated by Dolphin Institute oI Biomedical and
dissolving calciumphosphate stones (67.74 ) than oxalate
Natural Sciences, Dehradun (Uttaranchal state) India. A
(36.95). Reference standard-formulation Cystone was
voucher specimen oI the plant (No. Vb-Bc-02) has been
found to be more effective (48.48) when compared to
deposited in the Pharmacognosy research laboratory oI
compound P .
1 KLESs College oI Pharmacy, Hubli, Ior Iuture reIerence.
Bergenia ciliata, kidney stones, Urolithiatic,
Calciumoxalate, Calciumphosphate.
Shade dried leaves pulverized, and about 100 gms oI powder
was extracted with ethanol (95) in a soxhlet. Another 100
gms oI the crude drug powder was extracted successively
Kidney stones, one oI the painIul disorders oI urinary tract,
with pet. Ether (40-60), ChloroIorm, n-butanol, ethyl acetate
are ancient one and still remain a common problem world-
and Iinally with alcohol. All extracts were concentrated in a
wide. The pathogenesis oI urinary calculi continues to be
rotary Ilash evaporator and the residue was dried in a
|8]
more or less an enigma. The incidence oI Urolithiasis is more
desiccator over sodium Sulphite .
in men (recurrence rate 70-80) than women (47-60). The
most common type oI kidney stones contains calcium, in
All the prepared extracts were subjected to qualitative
combination with either oxalate or phosphate. These
chemical tests to detect the presence oI diIIerent class oI
chemicals are part oI a person's normal diet and make up
Phytoconstituents. TLC studies were done to substantiate
important parts oI the body, such as bones and muscles.
the presence oI constituents detected in chemical tests and to
9]
Cystinuria and Hyperoxaluria are two other rare, inherited
know how many compounds are present in each extract .
metabolic disorders that oIten cause kidney stones. Many
The ethyl acetate Iraction was column chromatographed
remedial measures have been employed during ages to treat
Over silicagel Ior column chromatography and eluted with
this condition. Most oI them were Irom plants and prove to
(Wall.) Engl. i
ciliata (Haw.)
Sternb. leaves (Saxifragaceae) were evaluated
Materials and methods:
Plant material:
Key words:
Extraction and isolation:
Introduction:
* Corresponding author
pages 14 - 17
ANCILNT SCILNCL OI LIIL
15
ethyl acetate: glacial acetic acid: Iormic acid: dist. water the contents squeezed out completely Irom the decalciIied
(126.8:5:5:12.2). Fractions showing the same number oI egg. Washed thoroughly with distilled water, and placed it in
compounds & same R values, were combined (based on ammonia solution, in the moistened condition Ior a while &
I
|4,5]
then rinsed it with distilled water. Stored in reIrigerator at a
TLC studies) , concentrated & evaporated to dryness and
pHoI 7- 7.4.
named as P , P & P . These separated compounds subjected
1 2 3
to physical, chemical & spectral (UV, FT-IR & FT-NMR)
studies. The compound P , which was obtained in relatively
1
high quantity (565 mgs) and responded more positively Ior
Weighed exactly 1mg oI the calcium oxalate and 10mg oI the
identity parameters, was taken-up Ior pharmacological
extract/compound/standard and packed it together in semi
evaluation.
permeable membrane by suturing as shown in Model design
Figure-I. This was allowed to suspend in a conical Ilask
containing 100ml 0.1 M TRIS buIIer. One group served as
negative control (contained only 1mg oI calcium oxalate).
Albino mice oI either sex weighing between 20 and 30 gm
Placed the conical Ilask oI all groups in a incubator, pre
were used. The animals were Iasted over night prior to the
0
heated to 37 CIor 2 hours, Ior about 7-8 hours. Removed the
experimental procedure. The Up and Down or 'Staircase'
|10]
contents oI semi-permeable membrane Irom each group into
method was adopted , and accordingly doses oI Alcoholic,
a test tube. Added 2 ml oI 1 Nsulphuric acid and titrated with
n-Butanol Iraction, Ethyl acetate Iraction, Successive
0.9494 N KMnO till a light pink color end point
4 alcoholic and water extracts were calculated.
obtained.1ml oI 0.9494 NKMnO equivalent to 0.1898mg oI
4
Calcium.
Two mice were injected with particular dose, and observed
Ior a period oI 24 hr Ior any mortality. The subsequent doses
Up to removal oI contents Irom semi-permeable membrane are then increased by a Iactor oI 1.5, iI the dose was tolerated
same as above. Removed the contents oI semi permeable or decreased by a Iactor oI 0.7 iI it was lethal.
membrane Irom each group into a test tube. Added 2ml oI
1N sulphuric acid, 2.5ml oI Molybdic-sulphuric acid
reagent, 1ml oI Reducing solution and made up the volume
to 10ml using distill water. Standard dilutions oI calcium Alcoholic extract, n-butanol Iraction, ethyl acetate Iraction
phosphate were prepared, (200, 400, 600, 800 and 1000 and isolated phenolic compound P , were evaluated Ior
1
g/ml) containing 2.5ml oI Molybdic-sulphuric acid
antilithiatic activity by an in-vitro model using calcium
reagent, 1ml oI Reducing solution and made up the volume
oxalate and calcium phosphate stones. Formulation Cystone
to 10ml using distilled water respectively. Measured the
was used as a reIerence standard.
optical density oI standard dilutions and Ior the groups under
study in colorimeter at 600-750 nm. The undissolved
calcium phosphate was determined Irom the standard
calibration curve by extrapolation.
Equimolar solution oI Calcium chloride dihydrate (AR) in
distilled water and Sodium oxalate (AR) in 10ml oI 2N
H SO were allowed to react in suIIicient quantity oI distilled
2 4
water in a beaker. The resulting precipitate was calcium
oxalate.
Equimolar solution oI Calcium chloride dihydrate (AR) in
distilled water and Disodium hydrogen phosphate (AR) in
10ml oI (2N H SO ), was allowed to react in suIIicient
2 4
quantity oI distilled water in a beaker. The resulting
precipitate was calcium phosphate. Both precipipitates Ireed
Irom traces oI sulphuric acid by Ammonia solution .Washed
0
with distilled water and dried at 60 CIor 4 hours.
The semi - permeable membrane oI eggs lies in between the
outer calciIied shell and the inner contents like albumin &
yolk. Shell was removed chemically by placing the eggs in
2M HCl Ior an overnight, which caused complete
decalciIication. Further, washed with distilled water, and
careIully with a sharp pointer a hole is made on the top and
13]
iii)Estimation of Calcium oxalate by Titrimetry :

AcuteToxicity Studies:
Procedure:
6, 12]
iv)Estimation of Calcium phosphate by colorimetry :
6,11,14,15]
Evaluation forAntilithiaticActivity (In- vitro) :
i) Preparation of experimental kidney stones (Calcium
oxalate & Calcium phosphate stones) by homogenous
11]
precipitation :
ii) Preparation of semi-permeable membrane from farm
eggs:
Figure-I Invitro Experimental model set- up to
evaluate antiurolithiatic activity
Ancient Science of Life, Jol. 3, Ao.1 (21) Pages 14 - 17
pages 14 - 17
Effect of Phenolic Compounds from Bergenia ciliata leaves on
Experimental kidney stones
(Haw.) Sternb.
16
The amount oI undissolved calcium oxalate/phosphate is system when sprayed with 5 alcoholic Ierric chloride
then subtracted Irom the total quantity used in the reagent.
experiment in the beginning, to knowhowmuch quantity oI
Acute toxicity studies oI alcohol and butanol extracts oI
calcium oxalate/phosphate actually the test substance(s)
Bergenia ciliata leaves showed that, up to
could dissolve.
5000 mg/kg body wt. they are practically non-toxic. Hence,
th
1/10 oI which i.e., 500 mg/kg b. wt., was considered as
eIIective dose. However, the ethyl acetate Iraction at
2000mg/kg b.wt. produced lethal eIIect and was Iound to be
Qualitative chemical tests indicated the presence oI phenolic
toxic.
compounds, steroids, glycosides, proteins & amino acids
and carbohydrates in diIIerent extracts oI Bergenia ciliata
The crude phenolic compound P at 10mg concentration
1
leaI. Isolated compound P responded
1
produced highest dissolution oI both calcium oxalate &
positively Ior Ierric chloride test (green color) & shinoda test
phosphate stones in comparison to alcohol extract, butanol
(yellow color), and showed a single black spot (R 0.92) in
I
& ethyl acetate Iraction. Cystone was Iound to be more
ethyl acetate:GAA: FA: dist. Water (126.8:5:5:12.2) solvent
eIIective when compared to compound P .
1
Results:

(Haw.) Sternb
(Haw.) Sternb
Table no. 1
Calcium oxalate dissolution by extract, phenolic compound P-1 & Cystone
Group
Vol. of standard
KMnO
4
Wt. of Calcium
Estimated
Wt. of Calcium
Reduced
Dissolution
Control 4.6 ml 0.8730 mg ---- ----
Standard*
(Cystone)
2.6 ml 0.4934 mg 0.3796 mg 48.48
Alcoholic extract* 3.8 ml 0.7212 mg 0.1518 mg 17.38
Butanol Iraction* 4.0 ml 0.7592 mg 0.1138 mg 13.03
Ethyl acetate
Iraction*
3.2 ml 0.6073 mg 0.2657 mg 30.43
Compound P

* 2.9 ml 0.5504 mg 0.3226 mg 36.95

Table no.2
Dissolution of Calcium phosphate by extract, Phenolic compound P-1 & Cystone
` Correspond to 10mg
` Correspond to 10mg
Ancient Science of Life, Jol. 3, Ao.1 (21) Pages 14 - 17
Group Wt. of Calcium Estimated Wt. of Calcium Reduced Dissolution
Control 0.8730 mg ---- ----
Standard* (Cystone) 0.4934 mg 0.3796 mg 67.74
Alcoholic extract* 0.7212 mg 0.1518 mg 40.86
Butanol Iraction* 0.7592 mg 0.1138 mg 26.88
Ethyl acetate Iraction* 0.6073 mg 0.2657 mg 53.76
Compound P
1
* 0.5504 mg 0.3226 mg 67.74

pages 14 - 17
ANCILNT SCILNCL OI LIIL
17
Discussion:
References:
5. Rakesh K Sharma, Rajesh Arora. Herbal Drugs. A twenty
Iirst century perspective. NewDelhi: Jaypee Brothers; 2006:
221-228
This study evaluates antiurolithiatic activity oI diIIerent
6. Garimella T.S., Jolly C.I., Narayanan S. Invitro studies on
extracts oI Bergenia ciliate leaves and crude
antilithiatic activity oI seeds oI Dolichos biflorus Linn. and
phenolic compound isolated Irom the same. The study oI the
rhizomes oI Bergenia ligulata Wall. Phytother Res.
urinary chemistry with respect to the stone-Iorming minerals
(England) 2001; 15(4): 351-5.
7. Sinha S., Murugesan T., Pal M., Saha B.P. Evaluation oI anti- will provide a good indication oI the risk oI stone Iormation.
tussive activity oI Bergenia ciliata Sternb. rhizome extract in From the study results it is observed that isolated crude
mice. Phytomedicine. 2001; 8 (4):298-301.
phenolic compound P produced highest dissolution oI both
1
8. Harborne J.B. Phytochemical Methods- A guide to modern
calcium oxalate & phosphate stones in comparison to
techniques oI plant analysis, 3 rd edition. New Delhi :
alcoholic extract, butanol & ethyl acetate Iractions.
Springer (India) private limited ; 1998 : 5-32.
However, it was more eIIective in dissolving calcium
9. Wagner H. Bladet S., Zgainski E.M. Plant Drug Analysis - A
st
phosphate stone than oxalate. The dissolution capacity oI
TLC Atlas; 1 edition. New York : Springer Verlag Berlin
phenolic compound P can be Iurther enhanced by
1 Heidelberg ; 1996 : 195-214.
puriIication. Further, it is observed that isolated compound 10. Ghosh M.N. Fundamentals oI Experimental Pharmacology ,
nd
2 edition. Culcutta : ScientiIic Book Agency ; 1998 : 174- P is more potent in dissolving stones than ethyl acetate
1
179.
extract, Irom which it has been isolated. This study has given
11. Saso l, Valentini G, Leone MG, Grippa E. Development oI an
primary evidence Ior Bergenia ciliata why it is commonly
Invitro assay Ior the screening oI substances capable oI
known as shailagarbhafa / pashaanabhedha, meaning
dissolving calcium oxalate crystals. Urology International.
plants grow between rocks, appearing to break them or they
1998; 61(4): 210.
possess lithotriptic property. This in vitro study has given
12. Sakakura T, Fujita K, Yasui T, Sasaki and et al. Calcium
lead data, and shown that compound P is quite promising Ior
1 phosphate stones produced by Madin -Darby canine kidney
Iurther work in this regard. (MDCK) cells inoculated in nude mice. Urology Research.
1999 ; 27(93): 200-5.
13. http://www.olympiades-de-chimie.org/pdI/30tp.pdI
14. A.J.M.Christianaa, M.Priyamole, Pmuthumani and
S.Sthyanarayana. Antilithiatic study oI Chicorium intybus
1. Anonymous. The Wealth oI India - Adictionary oI Indian raw
linn. against glycolic acid induced stone Iormation. Adv.
materials & industrial products, revised edition. New Delhi :
Pharmacol. Toxicol. 2004 ; 5(1) : 33-6.
Publication & InIormation Directorate, CSIR; 1988 : Vol-2 B:
15. Khan ZA, Assiri AM, AL-AIghani HM, Maghrabi TM.
119-120.
Inhibition oI oxalate nephrolithiasis with Ammi visnaga (Al-
2. Kirtikar K.Rand Basu B.DIndian Medicinal Plants. Dehradun
Khillah). Int. Urol. Nephorol. 2001; 33(4) : 605-8.
: International bookdistributors; 2006: 993-994.
3. Anonymous. Indian herbal pharmacopoeia. Revised newedn.
Mumbai : Indian drug manuIacturer's association ; 2002 : 79-
87.
4. Warrier PK, Nambiar VPK, Raman Kutty C. Indian medicinal
plants. A compendium oI 500 species. Madras : Orient
Longman Ltd.; 1994: Vol- I : 95-97
(Haw.) Sternb
Ancient Science of Life, Jol. 3, Ao.1 (21) Pages 14 - 17
pages 14 - 17