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I J R H
ORIGINAL ARTICLE
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Homoeopathic preparation of Berberis DOI:

10.4103/0974-7168.166374

vulgaris as an inhibitor of Calcium oxalate Quick Response Code:

crystallization: An in vitro evidence

Thellamudhu Ganesan, Divya Bhavani Ravi, Jyothilakshmi Vasavan, Anil Khurana1,
Debadatta Nayak1, Kalaiselvi Periandavan

Department of Medical
Biochemistry, Dr. ALM Post
ABSTRACT Graduate Institute of Basic Medical
Sciences, University of Madras,
Chennai, Tamil Nadu, 1Deputy
Background: Berberis vulgaris is the most widely used drug in Homoeopathy for
Director (H), Central Council
treating urolithiasis. However, its mechanism of action in alleviating its consequences for Research in Homeopathy,
remains uncertain. New Delhi, India

Address for correspondence:

Objective: To explicate the potential role of Homoeopathic preparation of B. vulgaris
Dr. Kalaiselvi Periandavan,
on in vitro Calcium oxalate (CaOx) crystallization. Department of Medical
Biochemistry, University of Madras,
Materials and Methods: Spectrophotometric crystallization assay was carried out, Taramani, Chennai ‑ 600 113,
and the slopes of the nucleation (till the maximum) and aggregation (after the peak) Tamil Nadu, India.
phases were calculated using linear regression analysis, and the percentage inhibition E‑mail: [email protected]

exerted by the modifiers was calculated. Light microscopic observation of CaOx Received: 09-10-2014
Accepted: 06-07-2015
crystals formed in the presence or absence of modifiers was carried out to support the
outcome with spectrophotometric crystallization assays and to ascertain the potential
role of B. vulgaris in CaOx crystallization.
Results: The crystallization studies performed so far signifies B. vulgaris to be a potent
drug against CaOx crystallization both at the level of nucleation and aggregation.
Conclusion: Our present findings add up to the experimental evidence to support the
efficacy of the homeopathic preparation of the B. vulgaris in modulating the primary
events of stone formation.

Keywords: Berberis vulgaris, Calcium oxalate crystals, Crystallization, Urolithiasis,

Anti‑urolithic agent

This is an open access article distributed under the terms of the Creative
INTRODUCTION Commons Attribution‑NonCommercial‑ShareAlike 3.0 License, which allows
others to remix, tweak, and build upon the work non‑commercially, as long as the
author is credited and the new creations are licensed under the identical terms.
Urolithiasis has been a menace to mankind since
the distant past and continues to be on the rise For reprints contact: [email protected]

worldwide besides being an imperative issue

How to cite this article: Ganesan T, Ravi DB, Vasavan J,
because of its incidence, recurrence, and vicious Khurana A, Nayak D, Periandavan K. Homoeopathic preparation of
consequences.[1] In India, approximately 5–7 million Berberis vulgaris as an inhibitor of Calcium oxalate crystallization:
patients suffer from kidney stone disease and at least An in vitro evidence. Indian J Res Homoeopathy 2015;9:152-7.

152 © 2015 Indian Journal of Research in Homoeopathy | Published by Wolters Kluwer ‑ Medknow

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Ganesan, et al.: Berberis vulgaris as an inhibitor of CaOx crystallization

1/1000 of Indian population need hospitalization cuvette containing 1 ml of Potassium oxalate
due to kidney stone diseases.[2] About 70 to 80% solution, 1 ml of Calcium chloride solution was
of the calculi are composed primarily of Calcium added to give a final concentration of 4.25 mmol/L
oxalate (CaOx) mixed with varying amounts of Calcium and 0.75 mmol/L oxalate. All the solutions
Calcium phosphate.[3] were prepared in deionized water containing
200 mmol/L Sodium chloride and 10 mmol/L Sodium
The major objective while treating renal stones is to
acetate (pH 5.7). The time course of the optical
accomplish utmost clearance of stone, while causing
density at 620 nm was measured automatically
the quite lowest amount of morbidity to the patient.
using a UVIKON 930 Spectrophotometer (Kontron
Various minimally invasive modalities are described
for this, like shockwave lithotripsy, percutaneous Instruments, Italy). The values were also measured in
nephrolithotomy, and retrograde intra renal surgery; the 20, 50, and 100 μl of B. vulgaris (Φ, 6C, 30C, and
nevertheless, the recurrence rates are estimated at 200C). OD620 increases initially during nucleation
50% over a 10‑year and 75% over 20‑year period, with phase and decreases during the aggregation phase.
some people experiencing 10 or more episodes over Slopes of the nucleation (till the maximum) and
the course of a lifetime besides exhibiting equally aggregation (after the peak) phases will be calculated
worse side effects.[4] As an alternative, other novel using linear regression analysis, and the percentage
treatment options such as Homoeopathy can be inhibition exerted by the proteins will be calculated
considered as a replacement for the invasive treatment using the formula:
strategies. Several epidemiological data suggest and
Percentage inhibition = (1 − Sm/Sc) ×100
support the role of Berberis vulgaris, the most widely
used drug in homoeopathic medicine for urolithiasis where, Sm is the slope in the presence of the protein
in alleviating the pain and stone formation.[5] and Sc the slope of the control.
The earlier studies performed in our lab have assessed Light Microscopic Studies
the efficacy of the Homoeopathic preparation of B. CaOx crystals for light microscopic studies were
vulgaris in treating urolithiasis in a rat model, and the prepared according to the method of Nakai
results have justified its prominent role in reducing et al. (1996). CaOx crystals were formed by mixing
oxalate deposition in the tissues, protecting renal 0.2 ml of 20 mM Calcium chloride with 0.1 ml of
cell membrane integrity and thereby might prove as 20 mM potassium oxalate. All the solutions were
a potential antilithiatic agent.[6] prepared in 10 mM sodium acetate containing
200 mM sodium chloride. Each solution was adjusted
Several observations that highlight the effectiveness
to the pH 6.5. Crystallization was carried out by
of this homoeopathic preparation of B. vulgaris with
mixing appropriate ratio of the solutions, and the
a characteristic display of biochemical parameters
suspension formed was spread on a glass plate and
exist. However, data analysis studies for its efficiency
a cover slip was placed on it and investigated under
on modulating the CaOx crystal structure and
light microscope (Eclipse E400, NIKON Microscope,
morphology are scant so far.
Japan) and was photographed at × 40 magnification.
Hence, the main objective of this study is to elucidate The same procedure was repeated by adding each
the potential role of homoeopathic preparation of concentration of B. vulgaris procured.
B. vulgaris on in vitro CaOx crystallization.
RESULTS AND DISCUSSION
MATERIALS AND METHODS
The majority of urinary calculi found in patients with
Drugs and Chemicals urolithiasis are predominantly of CaOx composition.
Homoeopathic preparation of B. vulgaris (Φ, 6C, 30C, Calcium and oxalate are the two urine substances
and 200C) was procured from Hahnemann Publishing responsible for CaOx crystallization. Hence, generally,
Co., Pvt., Ltd., Kolkata, India. All other chemicals and the inhibitors of CaOx crystallization have been used
reagents used were of analytical grade. as a prophylactic agent to prevent the recurrence.
Spectrophotometric Crystallization Assay The problem of calculating the supersaturation in
Spectrophotometric crystallization assay was carried urine (i.e., the driving force of this particular case of
out by the method of Hess et  al. (1995). In a quartz phase transformation) and finding out the possible

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Ganesan, et al.: Berberis vulgaris as an inhibitor of CaOx crystallization

natural or pharmaceutical regulators of this driving mainly reflects an increase in particle number in
force, in the process of investigating the kinetics function of time and thus crystal nucleation.[10,11]
of crystal nucleation and growth, as well as in the
In a while, an equilibrium is reached where the
investigation of the kinetics of the dissolution of
solution gets saturated, and the crystal mass has
already existing stones, appears to be of utmost
to remain stable. However, data support the view
significance.[7] In this context, we set into assessing
that the observed decrease in OD620 with time
the CaOx crystallization kinetics in the presence
reflects a decline in particle number due to crystal
of an established homoeopathic preparation of
aggregation, which indeed has been demonstrated
B. vulgaris. In vitro crystallization systems are widely
by scanning electron microscope.[12] Thus, this slope
used for different purposes in urolithiasis research so
of decrease of OD620 with time can be taken as a
as to understand the pathology and thus to emerge
measure of crystal aggregation and can be used as
with efficient treatment strategies.
the standard for all comparisons.
Several in vitro and also in vivo studies on medicinal
Figure 2 represents the in vitro CaOx crystallization
plants have proven them to play a crucial role in
that was carried out with 50% ethanol which served
delaying and/or preventing the early phases of
as a control in the present study. The alcohol was
crystallization and thus conferring a remedy as
found to influence the CaOx crystallization process.
antiurolithic agents.[8,9]
Figures 3‑6 show the effect of B. vulgaris homoeopathic
To analyze the effect of the homoeopathic
preparations of the mother tincture (Φ), 6C, 30C, and
preparation of B. vulgaris on CaOx crystal formation
200C, respectively, on the nucleation and aggregation
events, the crystallization studies were carried out.
of CaOx.
In Figure 1, the time from addition of Calcium
Table 1 represents the effect of different B. vulgaris
chloride until the first detectable increment of
homoeopathic formulations on in vitro CaOx
OD620, reflects the time required for CaOx crystal
nucleation and aggregation [Figure 7]. Surprisingly,
nuclei to appear and grow which allow for detection.
the mother tincture is shown to favor aggregation
The increase in slope of OD620 with time in turn
0.1
0.06
0.09
0.05 0.08
0.07
0.04
0.06
OD620
OD620

0.03 0.05
0.04
0.02
0.03
0.01 0.02
0.01
0
0 5 10 15 20 0
Time 0 5 10 15 20

Figure 1: Standard graph for in vitro Calcium oxalate crystallization Figure 2: In vitro Calcium oxalate crystallization with 50% ethanol control

0.07 0.08
0.06 0.07

0.05 0.06
0.05
OD620

OD620

0.04
0.04
0.03
0.03
0.02
0.02
0.01 0.01
0 0
0 2 4 6 8 10 12 14 16 0 5 10 15 20
Time Time

Figure 3: Effect of Berberis vulgaris (Ø‑50μl) on Calcium oxalate crystallization Figure 4: Effect of Berberis vulgaris (6C‑50μl) on Calcium oxalate crystallization

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Ganesan, et al.: Berberis vulgaris as an inhibitor of CaOx crystallization

of CaOx to a greater extent of about 229%. On the X‑shaped habit in the control (without any additives)
contrary, the 6C, 30C and 200C have been found to [Figure 8], which is generally observed in increased
be inhibitory in nature, that too, they inhibited the supersaturation.[13] The increase of crystal number is
aggregation by 100%. related to supersaturation, and hence, the observed
These suggest that the maximum inhibition (>100%) crystallization pattern in the control dictates the
was observed when 6C, 30C, and 200C preparation prevalent high supersaturation. In the citrated one,
were tested for their efficacy to modulate the CaOx only bipyramidal CaOx dihydrate (COD) crystals
crystal aggregation. somewhat intertwining were observed and scanty
in numbers. Similar but not the exact observations
Light microscopic observation studies were carried
were made in the 30C preparation. However, these
out to affirm our outcomes with spectrophotometric
crystals were not thermodynamically stable; they
crystallization assays. The CaOx crystals appear
tend to change to COM crystals. This reveals that it
as hemispherulitic clusters of crystals with the
could relieve the supersaturation and prevent crystal
0.06
Table 1: Effect of different Berberis vulgaris
0.05
homoeopathic formulation on in vitro Calcium
0.04 oxalate nucleation and aggregation
Particulars (100 μl each) Nucleation Aggregation
OD620

0.03
Mother tincture 49.78629 −228.7
0.02
6C 22.29685 >100
0.01 30C 35.53871 >100
200C 63.25563 >100
0
0 5 10 15 20
Time
0.07
Figure 5: Effect of Berberis vulgaris (30C‑50μl) on Calcium oxalate
0.06
crystallization
0.05

0.04
OD620

100 100 100

0.03
100
0.02
63.25563
49.78629 0.01
50 35.53871 0
22.29685 0 5 10 15 20
Time

0 Figure 6: Effect of Berberis vulgaris (200C‑50μl) on Calcium oxalate

Mother 6C 30C 200C crystallization
tincture

-50

-100

a b
-150

-200

-228.57 Nucelation Aggregation

-250
c d
Figure 7: Percentage inhibition of nucleation and aggregation by Figure 8: (a‑d) Pattern of Calcium oxalate crystals. (a) Non-Citrated, (b)
Berberis vulgaris Citrated, (c) 30C, (d) Ø

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Ganesan, et al.: Berberis vulgaris as an inhibitor of CaOx crystallization

aggregation. Thus, a negative supersaturation in Financial Support and Sponsorship

respect to CaOx‑precipitation can occur in the presence Funded by CCRH.
of the homoeopathic preparation, and even the direct
dissolution of CaOx calculi situated in the kidney pelvis, Conflicts of Interest
or elsewhere in the urinary tract, could be expected. There are no conflicts of interest.
Our present findings suggest that homoeopathic REFERENCES
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of stones in the kidney (Jyothilakshmi et  al., 2013). Berberis vulgaris in therapeutics. Pak J Pharm Sci 2007;20:83‑92.
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Hence, the formation of COD crystals forms a Nayak D, Kalaiselvi P. Preliminary investigation on ultra high
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Thus, our in vitro crystallization assays demonstrate oxalate stones by Herniaria hirsuta on experimentally induced
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9. Barros ME, Lima R, Mercuri LP, Matos JR, Schor N, Boim MA. Effect
the human urine, thereby safeguarding it from of extract of Phyllanthus niruri on crystal deposition in experimental
crystalluria and its resultant injury. Although the urolithiasis. Urol Res 2006;34:351‑7.
relative effect of this homoeopathic preparation 10. Hennequin C, Lalanne V, Estepa L, Drueke T, Daudon M, Lacour B.
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on the solubility of CaOx has been determined in Calcium oxalate crystallization. Clin Nephrol 1997;48:292‑9.
simple salt solutions, the exact combination of the 11. Hess B, Jordi S, Zipperle L, Ettinger E, Giovanoli R. Citrate
factors that is responsible for variations in CaOx determines Calcium oxalate crystallization kinetics and crystal
morphology‑studies in the presence of Tamm‑Horsfall protein of
solubility in urine is very yet insufficiently known. a healthy subject and a severely recurrent Calcium stone former.
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Simultaneous measurements of Calcium oxalate crystal
Acknowledgment nucleation and aggregation: Impact of various modifiers. Urol Res
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Ganesan, et al.: Berberis vulgaris as an inhibitor of CaOx crystallization

ewy ys[k
dSfY'k;e v‚Dt+ysV fØLVyhdj.k ds çkojksèkd ds :i esa csjcsfjl oYxsfjl dk gksE;ksiSfFkd ;ksx% ,d ik=s çek.k
lkj
i`"BHkwfe% csjcsfjl oYxsfjl ew=k'ejrk ¼;wjksfyFkk;fll½ ds mipkj gsrq gksE;ksiSfFkd fpfdRlk esa lokZfèkd O;kid :i ls ç;qä vkS"kfèk gSA gkykafd]
mlds ifj.kkeksa dh xaHkhjrk de djus dh dk;Z&fØ;kfofèk vHkh rd vfuf'pr cuh gqà gSA
mís';% ik=s dSfY'k;e v‚Dt+ysV (CaOx) fØLVyhdj.k esa ch-oYxsfjl ds gksE;ksiSfFkd ;ksx dh laHkkO; Hkwfedk dh O;k[;k djukA
lkefxz;k¡ ,oa fofèk;ka% LisDVªehçdk'kferh; fØLVyhdj.k vkekiu fd;k x;k ,oa ukfHkdu ¼vfèkdre rd½ ,oa leqPp;u ¼'kh"kZ ds ckn½ çkoLFkkvksa dh
ço.krkvksa dh x.kuk jSf[kd lekJ;.k fo'ys"k.k }kjk dh xà rFkk la'kksèkdksa }kjk yxk, x, çfr'kr çkojksèk dh x.kuk dh xÃA LisDVªehçdk'kferh;
fØLVyhdj.k vkekiuksa ds ifj.kkeksa dk leFkZu djus ,oa CaOx fØLVyhdj.k esa ch-oYxsfjl dh laHkkO; Hkwfedk dks fuf'pr djus ds fy, la'kksèkdksa
dh mifLFkfr ;k vuqifLFkfr esa fuÆer gq, CaOx fØLVyksa dk çdk'k lw{en'kÊ; çs{k.k fd;k x;kAA
ifj.kke% vc rd fd, x, fØLVyhdj.k vè;;u n'kkZrs gSa fd ch-oYxsfjl ukfHkdu vkSj leqPp;u] nksuksa Lrjksa ij CaOx fØLVyhdj.k ds
fo#) ,d çHkkodkjh vkS"kfèk gSA
fu"d"kZ% gekjs çLrqr tkap&ifj.kke v'ejh fuekZ.k dh çkFkfed ?kVukvksa ds e‚Mqyu esa ch-oYxsfjl ds gksE;ksiSfFkd ;ksx dh çHkkfodrk dk leFkZu
djus esa ç;ksxkRed çek.k dk xBu djrs gSaA
eq[; 'kCn% csjcsfjl oYxsfjl] dSfY'k;e v‚Dt+ysV fØLVy] fØLVyhdj.k] ew=k'ejrk] ew=k'ej&jksèkh vfHkdrkZ

Preparado homeopático de Berberis vulgaris como inhibidor de la cristalización del oxalato de calcio: Evidencia
in vitro
RESUMEN
Fundamentos: Berberis vulgaris es el medicamento más ampliamente utilizado en homeopatía para el tratamiento
de la litiasis renal. Sin embargo, todavía no se ha esclarecido su mecanismo de acción en aliviar las secuelas.
Objetivo: Explicar el papel potencial del preparado homeopático de B. vulgaris en la cristalización in vitro del oxalato
de calcio (CaOx).
Materiales y métodos: Se ha realizado un ensayo espectrofotométrico de la cristalización. ymediante un análisis de
regresión lineal, se calcularon las pendientes de las fases de nucleación (hasta el máximo) y agregación (después del
máximo). Asimismo, se calculó la inhibición porcentual ejercida por los modificadores. Se realizó una observación
por microscopio óptico de los cristales de CaOx formados en presencia o ausencia de los modificadores para apoyar
los resultados con los ensayos espectrofotométricos de cristalización y determinar la función potencial de B. vulgaris
en la cristalización del CaOx.
Resultados: Los estudios de cristalización realizados hasta la fecha muestran que B. vulgaris es un medicamento
potente contra la cristalización del CaOx tanto en la fase de nucleación como en la de agregación.
Conclusiones: Los resultados del presente estudio aportan evidencias experimentales adicionales que muestran la
eficacia del preparado homeopático B. vulgaris en la modulación de los eventos primarios de la formación de cálculos.

Indian Journal of Research in Homoeopathy / Vol. 9 / Issue 3 / Jul-Sep 2015 157