Urolithiasis DOI 10.1007/s00240-014-0654-3
Original Paper
Herbal preparations affect the kinetic factors of calcium oxalate crystallization in synthetic urine: implications for kidney stone therapy Allen L. Rodgers · Dawn Webber · Ronica Ramsout · Mayur Danny I. Gohel
Received: 8 December 2013 / Accepted: 4 March 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Herbal remedies are increasingly being considered as suitable long-term treatments for renal dysfunction. The objective of the present study was to investigate the effect of some herbal extracts, all previously identified in published studies as influencing kidney stone formation, on the crystallization characteristics of calcium oxalate (CaOx) in synthetic urine (SU). Five herbal extracts were selected for the study: Folium pyrrosiae, Desmodium styracifolium, Phyllanthus niruri, Orthosiphon stamineus and Cystone®. Concentrated stock solutions of each herbal extract were prepared and were tested at their recommended dosages in in vitro crystallization studies in SU. CaOx crystallization experiments were performed in which the metastable limit (MSL), average particle size, and nucleation and growth rates were determined. The CaOx MSL of SU was unaltered by the five herbal extracts. Three of the herbs (Desmodium styracifolium, Orthosiphon stamineus and Cystone®) significantly reduced the average particle size of precipitated crystals relative to undosed SU. All of the extracts increased the rate of nucleation and decreased the rate of growth significantly in SU. Cystone® showed the greatest effect on the measured risk factors. It is concluded that all of the herbs have the potential to serve as inhibitors of calcium oxalate stone formation and warrant investigation in clinical trials.
A. L. Rodgers (*) · D. Webber · R. Ramsout Department of Chemistry, University of Cape Town, Private Bag, Rondebosch, Cape Town 7701, South Africa e-mail: [email protected] M. D. I. Gohel Department of Medical Science, Tung Wah College, Homantin, Kowloon, Hong Kong
Keywords Calcium oxalate urolithiaisis · Kinetic factors · Thermodynamic factors · Herbal preparations · Crystallization inhibitors
Introduction Herbal remedies are increasingly being considered as suitable long-term treatments for renal stone disease [1, 2]. Although the number of published papers describing their effects in humans is limited, there is a large body of anecdotal evidence in support of their efficacies [3]. In addition, there are several in vitro studies in which various herbs and combinations thereof have been shown to modulate aspects of calcium oxalate (CaOx) crystallization processes [1, 2, 4–8] and an abundance of animal model studies which have demonstrated their capacity for in vivo inhibition of CaOx crystal deposition and/or stone formation [5, 8–14]. Relatively few have been tested in humans [15–18]. Five herbal extracts, Folium pyrrosiae (FP), Desmodium styracifolium (DS), Phyllanthus niruri (PN), Orthosiphon stamineus (OS) and Cystone® (Cys) appear to be common among those which have been tested in the aforementioned models. FP has been shown to promote CaOx crystal nucleation and inhibit its growth and aggregation in vitro in synthetic urine [2]. DS increases in vitro CaOx nucleation [2, 4]. PN (or Phyllanthus species) has been used traditionally in the treatment of urolithiasis [19, 20]. In vitro studies [6] and in vivo rat studies [10, 11] have demonstrated PN’s ability to inhibit CaOx growth and aggregation. It has also been used to normalise urinary calcium levels in stone patients [15] and decrease their stone recurrence following extracorporeal shock wave lithotripsy [16]. It is delivered as the drug Uriston® in Western countries. OS has a diuretic and hypouricemic effects in rats [12] and also inhibits
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Urolithiasis
in vitro CaOx crystal growth [7]. Cys is a polyherbal preparation which has been shown to dissolve the crystal matrix and reduce the deposition of calcium and phosphate ions in vitro [21], as well as to aid the expulsion of stones in patients [17]. Further scientific investigation of these herbal preparations is warranted before firm conclusions can be drawn regarding their potential as therapeutic agents for CaOx stone disease and before clinical trials are launched to test their efficacy. The present study was undertaken to examine the effects of these five herbal extracts on the crystallization characteristics of CaOx in synthetic urine.
retained as a control or dosed with the 20× concentrated herbal stock solutions at 5 % of the final sample volume in order to achieve their respective recommended physiological concentrations. CaOx metastable limit and particle size distribution
FP and DS were purchased individually under the brand name Nong’s powder from PuraPharm, Hong Kong. PN was purchased from Raintree Nutrition, Nevada, USA. OS was purchased as Java Tea from Midas Herbs, Indonesia. Cys (Himalaya Drug Company, Johannesburg, South Africa) was obtained from an ayurvedic practitioner, Dr R. Coopan (Durban, South Africa).
The CaOx metastable limit (MSL) is a measure of the ability of urine to resist spontaneous nucleation and hence crystallization. An elevated MSL will therefore indicate a reduced risk of stone formation. The MSL of each sample of SU was determined according to the method of Ryall et al. [23]. Briefly, aliquots of SU were titrated with a series of sodium oxalate standard solutions of increasing concentration and crystallization was measured in each aliquot using a Coulter Multisizer II (Beckman Coulter; 140 μm orifice, 2.8–90.0 μm particle size range). A standard oxalate load (30 μmol/100 ml in excess of the previously determined MSL) was then added to each urine to induce crystallization [24] followed by incubation at 37 °C in a shaking water bath (100 cycles per minute) for 120 min. The particle volume–particle size distribution was measured at 120 min using the Coulter Multisizer. These experiments were performed in triplicate and average values are reported.
Preparation of plant stock solutions
CaOx crystal nucleation and growth kinetics
Concentrated stock solutions of each herb were prepared by dissolving the dried powders in distilled water at 20 times their respective recommended dosage concentrations. Each preparation was gently heated and stirred for 30 min: FP and DS, 1.5 g/25 ml [2]; PN, 3.75 g/25 ml [10]; OS, 2.5 g/25 ml [12]; and Cys, 2 tablets dissolved in 25 ml distilled water (manufacturer’s information sheet). All stock solutions were microfiltered (0.22 μm) and stored at 4 °C prior to use. Crystallization experiments were performed in the absence (control samples) and presence (test samples) of the plant stock solutions.
A mixed product mixed product removal (MSMPR) crystallizer was used to model the continuous flow system of the kidney [25–28]. All experiments were carried out at 37 °C in a constant temperature chamber according to the conditions described by Nishio and co-workers [26]. The experiments themselves were performed in the laboratory of Dr MD Gohel (Hong Kong Polytechnic University, Hong Kong) as part of a collaborative study. In order to induce crystallization, SU and solutions of calcium chloride (150 mM) and sodium oxalate (30 mM) were pumped continuously through the system. A constant volume of 20 ml was maintained in the MSMPR chamber by controlling the SU flow rate at 2.3 ml/min, and calcium chloride and sodium oxalate flow rates at 0.1 ml/min. The composition of the feed stream was 92 % SU, 4 % calcium chloride and 4 % sodium oxalate, corresponding to final concentrations of 6 and 1.2 mM for calcium and oxalate, respectively. After the crystalliser had achieved steady state by allowing it to run for 6–8 residence times (τ ≈ 8 min), CaOx crystallization kinetics (nucleation and growth rates) were measured. CaOx crystals were then removed from the product stream and the total number of particles and its size distribution were measured using a Coulter Multisizer III (150 μm orifice, 2–60 μm particle size range). The nucleation and growth rates of each sample were calculated as
Methods Origin of plant material
In vitro study in synthetic urine Preparation of synthetic urine Synthetic urine (SU) was prepared according to the method of Walton et al. [22] and had a composition corresponding to urine in the distal part of the collecting duct. The final calcium and oxalate concentrations were adjusted to 2.14 and 0.143 mM, respectively, since these are approximately equal to the average normal urinary concentrations of the South African population (own data, unpublished). SU was adjusted to pH 6.0 using NaOH, heated to 37 °C and filtered (0.22 μm) immediately before use. It was either
13
Urolithiasis Table 1 Crystallization data for synthetic urine dosed with various plant extracts
* p
Original Paper
Herbal preparations affect the kinetic factors of calcium oxalate crystallization in synthetic urine: implications for kidney stone therapy Allen L. Rodgers · Dawn Webber · Ronica Ramsout · Mayur Danny I. Gohel
Received: 8 December 2013 / Accepted: 4 March 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Herbal remedies are increasingly being considered as suitable long-term treatments for renal dysfunction. The objective of the present study was to investigate the effect of some herbal extracts, all previously identified in published studies as influencing kidney stone formation, on the crystallization characteristics of calcium oxalate (CaOx) in synthetic urine (SU). Five herbal extracts were selected for the study: Folium pyrrosiae, Desmodium styracifolium, Phyllanthus niruri, Orthosiphon stamineus and Cystone®. Concentrated stock solutions of each herbal extract were prepared and were tested at their recommended dosages in in vitro crystallization studies in SU. CaOx crystallization experiments were performed in which the metastable limit (MSL), average particle size, and nucleation and growth rates were determined. The CaOx MSL of SU was unaltered by the five herbal extracts. Three of the herbs (Desmodium styracifolium, Orthosiphon stamineus and Cystone®) significantly reduced the average particle size of precipitated crystals relative to undosed SU. All of the extracts increased the rate of nucleation and decreased the rate of growth significantly in SU. Cystone® showed the greatest effect on the measured risk factors. It is concluded that all of the herbs have the potential to serve as inhibitors of calcium oxalate stone formation and warrant investigation in clinical trials.
A. L. Rodgers (*) · D. Webber · R. Ramsout Department of Chemistry, University of Cape Town, Private Bag, Rondebosch, Cape Town 7701, South Africa e-mail: [email protected] M. D. I. Gohel Department of Medical Science, Tung Wah College, Homantin, Kowloon, Hong Kong
Keywords Calcium oxalate urolithiaisis · Kinetic factors · Thermodynamic factors · Herbal preparations · Crystallization inhibitors
Introduction Herbal remedies are increasingly being considered as suitable long-term treatments for renal stone disease [1, 2]. Although the number of published papers describing their effects in humans is limited, there is a large body of anecdotal evidence in support of their efficacies [3]. In addition, there are several in vitro studies in which various herbs and combinations thereof have been shown to modulate aspects of calcium oxalate (CaOx) crystallization processes [1, 2, 4–8] and an abundance of animal model studies which have demonstrated their capacity for in vivo inhibition of CaOx crystal deposition and/or stone formation [5, 8–14]. Relatively few have been tested in humans [15–18]. Five herbal extracts, Folium pyrrosiae (FP), Desmodium styracifolium (DS), Phyllanthus niruri (PN), Orthosiphon stamineus (OS) and Cystone® (Cys) appear to be common among those which have been tested in the aforementioned models. FP has been shown to promote CaOx crystal nucleation and inhibit its growth and aggregation in vitro in synthetic urine [2]. DS increases in vitro CaOx nucleation [2, 4]. PN (or Phyllanthus species) has been used traditionally in the treatment of urolithiasis [19, 20]. In vitro studies [6] and in vivo rat studies [10, 11] have demonstrated PN’s ability to inhibit CaOx growth and aggregation. It has also been used to normalise urinary calcium levels in stone patients [15] and decrease their stone recurrence following extracorporeal shock wave lithotripsy [16]. It is delivered as the drug Uriston® in Western countries. OS has a diuretic and hypouricemic effects in rats [12] and also inhibits
13
Urolithiasis
in vitro CaOx crystal growth [7]. Cys is a polyherbal preparation which has been shown to dissolve the crystal matrix and reduce the deposition of calcium and phosphate ions in vitro [21], as well as to aid the expulsion of stones in patients [17]. Further scientific investigation of these herbal preparations is warranted before firm conclusions can be drawn regarding their potential as therapeutic agents for CaOx stone disease and before clinical trials are launched to test their efficacy. The present study was undertaken to examine the effects of these five herbal extracts on the crystallization characteristics of CaOx in synthetic urine.
retained as a control or dosed with the 20× concentrated herbal stock solutions at 5 % of the final sample volume in order to achieve their respective recommended physiological concentrations. CaOx metastable limit and particle size distribution
FP and DS were purchased individually under the brand name Nong’s powder from PuraPharm, Hong Kong. PN was purchased from Raintree Nutrition, Nevada, USA. OS was purchased as Java Tea from Midas Herbs, Indonesia. Cys (Himalaya Drug Company, Johannesburg, South Africa) was obtained from an ayurvedic practitioner, Dr R. Coopan (Durban, South Africa).
The CaOx metastable limit (MSL) is a measure of the ability of urine to resist spontaneous nucleation and hence crystallization. An elevated MSL will therefore indicate a reduced risk of stone formation. The MSL of each sample of SU was determined according to the method of Ryall et al. [23]. Briefly, aliquots of SU were titrated with a series of sodium oxalate standard solutions of increasing concentration and crystallization was measured in each aliquot using a Coulter Multisizer II (Beckman Coulter; 140 μm orifice, 2.8–90.0 μm particle size range). A standard oxalate load (30 μmol/100 ml in excess of the previously determined MSL) was then added to each urine to induce crystallization [24] followed by incubation at 37 °C in a shaking water bath (100 cycles per minute) for 120 min. The particle volume–particle size distribution was measured at 120 min using the Coulter Multisizer. These experiments were performed in triplicate and average values are reported.
Preparation of plant stock solutions
CaOx crystal nucleation and growth kinetics
Concentrated stock solutions of each herb were prepared by dissolving the dried powders in distilled water at 20 times their respective recommended dosage concentrations. Each preparation was gently heated and stirred for 30 min: FP and DS, 1.5 g/25 ml [2]; PN, 3.75 g/25 ml [10]; OS, 2.5 g/25 ml [12]; and Cys, 2 tablets dissolved in 25 ml distilled water (manufacturer’s information sheet). All stock solutions were microfiltered (0.22 μm) and stored at 4 °C prior to use. Crystallization experiments were performed in the absence (control samples) and presence (test samples) of the plant stock solutions.
A mixed product mixed product removal (MSMPR) crystallizer was used to model the continuous flow system of the kidney [25–28]. All experiments were carried out at 37 °C in a constant temperature chamber according to the conditions described by Nishio and co-workers [26]. The experiments themselves were performed in the laboratory of Dr MD Gohel (Hong Kong Polytechnic University, Hong Kong) as part of a collaborative study. In order to induce crystallization, SU and solutions of calcium chloride (150 mM) and sodium oxalate (30 mM) were pumped continuously through the system. A constant volume of 20 ml was maintained in the MSMPR chamber by controlling the SU flow rate at 2.3 ml/min, and calcium chloride and sodium oxalate flow rates at 0.1 ml/min. The composition of the feed stream was 92 % SU, 4 % calcium chloride and 4 % sodium oxalate, corresponding to final concentrations of 6 and 1.2 mM for calcium and oxalate, respectively. After the crystalliser had achieved steady state by allowing it to run for 6–8 residence times (τ ≈ 8 min), CaOx crystallization kinetics (nucleation and growth rates) were measured. CaOx crystals were then removed from the product stream and the total number of particles and its size distribution were measured using a Coulter Multisizer III (150 μm orifice, 2–60 μm particle size range). The nucleation and growth rates of each sample were calculated as
Methods Origin of plant material
In vitro study in synthetic urine Preparation of synthetic urine Synthetic urine (SU) was prepared according to the method of Walton et al. [22] and had a composition corresponding to urine in the distal part of the collecting duct. The final calcium and oxalate concentrations were adjusted to 2.14 and 0.143 mM, respectively, since these are approximately equal to the average normal urinary concentrations of the South African population (own data, unpublished). SU was adjusted to pH 6.0 using NaOH, heated to 37 °C and filtered (0.22 μm) immediately before use. It was either
13
Urolithiasis Table 1 Crystallization data for synthetic urine dosed with various plant extracts
* p
