|Year : 2015 | Volume
| Issue : 3 | Page : 152-157
Homoeopathic preparation of Berberis vulgaris as an inhibitor of Calcium oxalate crystallization: An in vitro evidence
Thellamudhu Ganesan1, Divya Bhavani Ravi1, Jyothilakshmi Vasavan1, Anil Khurana2, Debadatta Nayak2, Kalaiselvi Periandavan1
1 Department of Medical Biochemistry, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Chennai, Tamil Nadu, India
2 Deputy Director (H), Central Council for Research in Homeopathy, New Delhi, India
|Date of Submission||09-Oct-2014|
|Date of Acceptance||06-Jul-2015|
|Date of Web Publication||30-Sep-2015|
Department of Medical Biochemistry, University of Madras, Taramani, Chennai - 600 113, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Background: Berberis vulgaris is the most widely used drug in Homoeopathy for treating urolithiasis. However, its mechanism of action in alleviating its consequences remains uncertain.
Objective: To explicate the potential role of Homoeopathic preparation of B. vulgaris on in vitro Calcium oxalate (CaOx) crystallization.
Materials and Methods: Spectrophotometric crystallization assay was carried out, and the slopes of the nucleation (till the maximum) and aggregation (after the peak) phases were calculated using linear regression analysis, and the percentage inhibition exerted by the modifiers was calculated. Light microscopic observation of CaOx 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
|How to cite this article:|
Ganesan T, Ravi DB, Vasavan J, Khurana A, Nayak D, Periandavan K. Homoeopathic preparation of Berberis vulgaris as an inhibitor of Calcium oxalate crystallization: An in vitro evidence. Indian J Res Homoeopathy 2015;9:152-7
|How to cite this URL:|
Ganesan T, Ravi DB, Vasavan J, Khurana A, Nayak D, Periandavan K. Homoeopathic preparation of Berberis vulgaris as an inhibitor of Calcium oxalate crystallization: An in vitro evidence. Indian J Res Homoeopathy [serial online] 2015 [cited 2018 Dec 10];9:152-7. Available from: http://www.ijrh.org/text.asp?2015/9/3/152/166374
| Introduction|| |
Urolithiasis has been a menace to mankind since the distant past and continues to be on the rise worldwide besides being an imperative issue because of its incidence, recurrence, and vicious consequences.  In India, approximately 5-7 million patients suffer from kidney stone disease and at least 1/1000 of Indian population need hospitalization due to kidney stone diseases.  About 70 to 80% of the calculi are composed primarily of Calcium oxalate (CaOx) mixed with varying amounts of Calcium phosphate. 
The major objective while treating renal stones is to accomplish utmost clearance of stone, while causing the quite lowest amount of morbidity to the patient. Various minimally invasive modalities are described for this, like shockwave lithotripsy, percutaneous nephrolithotomy, and retrograde intra renal surgery; nevertheless, the recurrence rates are estimated at 50% over a 10-year and 75% over 20-year period, with some people experiencing 10 or more episodes over the course of a lifetime besides exhibiting equally worse side effects.  As an alternative, other novel treatment options such as Homoeopathy can be considered as a replacement for the invasive treatment strategies. Several epidemiological data suggest and support the role of Berberis vulgaris, the most widely used drug in homoeopathic medicine for urolithiasis in alleviating the pain and stone formation. 
The earlier studies performed in our lab have assessed the efficacy of the Homoeopathic preparation of B. vulgaris in treating urolithiasis in a rat model, and the results have justified its prominent role in reducing oxalate deposition in the tissues, protecting renal cell membrane integrity and thereby might prove as a potential antilithiatic agent. 
Several observations that highlight the effectiveness of this homoeopathic preparation of B. vulgaris with a characteristic display of biochemical parameters exist. However, data analysis studies for its efficiency on modulating the CaOx crystal structure and morphology are scant so far.
Hence, the main objective of this study is to elucidate the potential role of homoeopathic preparation of B. vulgaris on in vitro CaOx crystallization.
| Materials and methods|| |
Drugs and Chemicals
Homoeopathic preparation of B. vulgaris (Φ, 6C, 30C, and 200C) was procured from Hahnemann Publishing Co., Pvt., Ltd., Kolkata, India. All other chemicals and reagents used were of analytical grade.
Spectrophotometric Crystallization Assay
Spectrophotometric crystallization assay was carried out by the method of Hess et al. (1995). In a quartz cuvette containing 1 ml of Potassium oxalate solution, 1 ml of Calcium chloride solution was added to give a final concentration of 4.25 mmol/L Calcium and 0.75 mmol/L oxalate. All the solutions were prepared in deionized water containing 200 mmol/L Sodium chloride and 10 mmol/L Sodium acetate (pH 5.7). The time course of the optical density at 620 nm was measured automatically using a UVIKON 930 Spectrophotometer (Kontron Instruments, Italy). The values were also measured in the 20, 50, and 100 μl of B. vulgaris (Φ, 6C, 30C, and 200C). OD620 increases initially during nucleation phase and decreases during the aggregation phase.
Slopes of the nucleation (till the maximum) and aggregation (after the peak) phases will be calculated using linear regression analysis, and the percentage inhibition exerted by the proteins will be calculated using the formula:
Percentage inhibition = (1 − Sm/Sc) ×100
where, Sm is the slope in the presence of the protein and Sc the slope of the control.
Light Microscopic Studies
CaOx crystals for light microscopic studies were prepared according to the method of Nakai et al. (1996). CaOx crystals were formed by mixing 0.2 ml of 20 mM Calcium chloride with 0.1 ml of 20 mM potassium oxalate. All the solutions were prepared in 10 mM sodium acetate containing 200 mM sodium chloride. Each solution was adjusted to the pH 6.5. Crystallization was carried out by mixing appropriate ratio of the solutions, and the suspension formed was spread on a glass plate and a cover slip was placed on it and investigated under light microscope (Eclipse E400, NIKON Microscope, Japan) and was photographed at × 40 magnification. The same procedure was repeated by adding each concentration of B. vulgaris procured.
| Results and discussion|| |
The majority of urinary calculi found in patients with urolithiasis are predominantly of CaOx composition. Calcium and oxalate are the two urine substances responsible for CaOx crystallization. Hence, generally, the inhibitors of CaOx crystallization have been used as a prophylactic agent to prevent the recurrence.
The problem of calculating the supersaturation in urine (i.e., the driving force of this particular case of phase transformation) and finding out the possible natural or pharmaceutical regulators of this driving force, in the process of investigating the kinetics of crystal nucleation and growth, as well as in the investigation of the kinetics of the dissolution of already existing stones, appears to be of utmost significance.  In this context, we set into assessing the CaOx crystallization kinetics in the presence of an established homoeopathic preparation of B. vulgaris. In vitro crystallization systems are widely used for different purposes in urolithiasis research so as to understand the pathology and thus to emerge with efficient treatment strategies.
Several in vitro and also in vivo studies on medicinal plants have proven them to play a crucial role in delaying and/or preventing the early phases of crystallization and thus conferring a remedy as antiurolithic agents. ,
To analyze the effect of the homoeopathic preparation of B. vulgaris on CaOx crystal formation events, the crystallization studies were carried out.
In [Figure 1], the time from addition of Calcium chloride until the first detectable increment of OD620, reflects the time required for CaOx crystal nuclei to appear and grow which allow for detection. The increase in slope of OD620 with time in turn mainly reflects an increase in particle number in function of time and thus crystal nucleation. ,
In a while, an equilibrium is reached where the solution gets saturated, and the crystal mass has to remain stable. However, data support the view that the observed decrease in OD620 with time reflects a decline in particle number due to crystal aggregation, which indeed has been demonstrated by scanning electron microscope.  Thus, this slope of decrease of OD620 with time can be taken as a measure of crystal aggregation and can be used as the standard for all comparisons.
[Figure 2] represents the in vitro CaOx crystallization that was carried out with 50% ethanol which served as a control in the present study. The alcohol was found to influence the CaOx crystallization process.
|Figure 2: In vitro Calcium oxalate crystallization with 50% ethanol control|
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[Figure 3],[Figure 4],[Figure 5] and [Figure 6] show the effect of B. vulgaris homoeopathic preparations of the mother tincture (Φ), 6C, 30C, and 200C, respectively, on the nucleation and aggregation of CaOx.
|Figure 3: Effect of Berberis vulgaris (Ø-50μl) on Calcium oxalate crystallization|
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|Figure 4: Effect of Berberis vulgaris (6C-50μl) on Calcium oxalate crystallization|
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|Figure 5: Effect of Berberis vulgaris (30C-50μl) on Calcium oxalate crystallization|
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|Figure 6: Effect of Berberis vulgaris (200C-50μl) on Calcium oxalate crystallization|
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[Table 1] represents the effect of different B. vulgaris homoeopathic formulations on in vitro CaOx nucleation and aggregation [Figure 7]. Surprisingly, the mother tincture is shown to favor aggregation of CaOx to a greater extent of about 229%. On the contrary, the 6C, 30C and 200C have been found to be inhibitory in nature, that too, they inhibited the aggregation by 100%.
|Figure 7: Percentage inhibition of nucleation and aggregation by Berberis vulgaris|
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|Table 1: Effect of different Berberis vulgaris homoeopathic formulation on in vitro Calcium oxalate nucleation and aggregation|
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These suggest that the maximum inhibition (>100%) was observed when 6C, 30C, and 200C preparation were tested for their efficacy to modulate the CaOx crystal aggregation.
Light microscopic observation studies were carried out to affirm our outcomes with spectrophotometric crystallization assays. The CaOx crystals appear as hemispherulitic clusters of crystals with the X-shaped habit in the control (without any additives) [Figure 8], which is generally observed in increased supersaturation.  The increase of crystal number is related to supersaturation, and hence, the observed crystallization pattern in the control dictates the prevalent high supersaturation. In the citrated one, only bipyramidal CaOx dihydrate (COD) crystals somewhat intertwining were observed and scanty in numbers. Similar but not the exact observations were made in the 30C preparation. However, these crystals were not thermodynamically stable; they tend to change to COM crystals. This reveals that it could relieve the supersaturation and prevent crystal aggregation. Thus, a negative supersaturation in respect to CaOx-precipitation can occur in the presence of the homoeopathic preparation, and even the direct dissolution of CaOx calculi situated in the kidney pelvis, or elsewhere in the urinary tract, could be expected.
|Figure 8: (a-d) Pattern of Calcium oxalate crystals. (a) Non-Citrated, (b) Citrated, (c) 30C, (d) Ø|
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Our present findings suggest that homoeopathic preparation of the B. vulgaris in appropriate concentrations proves to be effective against the early events of crystallization.
CaOx stones are found in two different varieties, CaOx monohydrate (COM) or Whewellite, and COD or weddellite. COM, the thermodynamically most stable form, is observed more frequently in clinical stones than COD, and it has a greater affinity for renal tubular cells, thus responsible for the formation of stones in the kidney (Jyothilakshmi et al., 2013). Hence, the formation of COD crystals forms a favorable environment of harmless excretion of CaOx. In this regard, B. vulgaris tends to form COD crystals.
| Conclusion|| |
Thus, our in vitro crystallization assays demonstrate even a possibility to dissolve CaOx stones in the human urine, thereby safeguarding it from crystalluria and its resultant injury. Although the relative effect of this homoeopathic preparation on the solubility of CaOx has been determined in simple salt solutions, the exact combination of the factors that is responsible for variations in CaOx solubility in urine is very yet insufficiently known. Hence, further studies are warranted to conclude its modus operandi in in vivo systems.
The authors are highly indebted to the Central Council for Research in Homoeopathy (CCRH), New Delhi for their financial assistance.
Financial Support and Sponsorship
Funded by CCRH.
Conflicts of Interest
There are no conflicts of interest.
| References|| |
Ngo TC, Assimos DG. Uric acid nephrolithiasis: Recent progress and future directions. Rev Urol 2007;9:17-27.
Kaladhar DS, Apparao RK, Varahalarao V. Statistical and data mining aspects on kidney stones. Statistical and data mining aspects on kidney stones: A systematic review and meta-analysis. Open Access Scientific Reports; 2012;1:543.
Tiselius HG. A hypothesis of Calcium stone formation: An interpretation of stone research during the past decades. Urol Res 2011;39:231-43.
Srivastava A, Chipde SS. Management of 1-2 cm renal stones. Indian J Urol 2013;29:195-9.
Arayne MS, Sultana N, Bahadur SS. The berberis story: Berberis vulgaris
in therapeutics. Pak J Pharm Sci 2007;20:83-92.
Jyothilakshmi V, Thellamudhu G, Kumar A, Khurana A, Nayak D, Kalaiselvi P. Preliminary investigation on ultra high diluted B. vulgaris in experimental urolithiasis. Homeopathy 2013;102:172-8.
Atanassova SS, Gutzow IS. Hippuric acid as a significant regulator of supersaturation in Calcium oxalate lithiasis: The physiological evidence. Biomed Res Int 2013;2013:374950.
Atmani F, Slimani Y, Mimouni M, Hacht B. Prophylaxis of Calcium oxalate stones by Herniaria hirsuta
on experimentally induced nephrolithiasis in rats. BJU Int 2003;92:137-40.
Barros ME, Lima R, Mercuri LP, Matos JR, Schor N, Boim MA. Effect of extract of Phyllanthus niruri
on crystal deposition in experimental urolithiasis. Urol Res 2006;34:351-7.
Hennequin C, Lalanne V, Estepa L, Drueke T, Daudon M, Lacour B. Validation by image analysis of a turbidimetric method to study Calcium oxalate crystallization. Clin Nephrol 1997;48:292-9.
Hess B, Jordi S, Zipperle L, Ettinger E, Giovanoli R. Citrate determines Calcium oxalate crystallization kinetics and crystal morphology-studies in the presence of Tamm-Horsfall protein of a healthy subject and a severely recurrent Calcium stone former. Nephrol Dial Transplant 2000;15:366-74.
Hess B, Meinhardt U, Zipperle L, Giovanoli R, Jaeger P. Simultaneous measurements of Calcium oxalate crystal nucleation and aggregation: Impact of various modifiers. Urol Res 1995;23:231-8.
Carvalho M, Vieira MA. Changes in Calcium oxalate crystal morphology as a function of supersaturation. Int Braz J Urol 2004;30:205-8.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]