Evidence-Based Complementary and Alternative Medicine Special Issue on
Medicinal Plants in the Prevention and Treatment of Chronic Diseases 2014
Research Article
An Emerging Translational Model to Screen Potential
Medicinal Plants for Nephrolithiasis, an Independent Risk
Factor for Chronic Kidney Disease
San-Yuan Wu,1 Jui-Lung Shen,2,3 Kee-Ming Man,4,5,6,7 Yuan-Ju Lee,8
Huey-Yi Chen,1,9 Yung-Hsiang Chen,1,9 Kao-Sung Tsai,1,9 Fuu-Jen Tsai,1,9 Wei-Yong Lin,1,9,* Wen-Chi Chen1,9,*
1 School of Chinese Medicine, Graduate Institute of Chinese Medicine, Graduate Institute of Integrated
Medicine, College of Chinese Medicine, Research Center for Chinese Medicine & Acupuncture, China Medical University, Taichung 40402, Taiwan
2 Center for General Education, Feng Chia University, Taichung 40724, Taiwan
3 Department of Dermatology, Taichung Veteran General Hospital, Taichung 40705, Taiwan
4 Department of Medicinal Botanicals and Health Applications, Da-Yeh University, Changhua 51591,
Taiwan
5 Department of Anesthesiology, Tungs’ Taichung Harbor Hospital, Taichung 43304, Taiwan 6 Department of Life Sciences, National Chung Hsing University, Taichung 40227, Taiwan 7 Graduate Institute of Geriatric Medicine, Anhui Medical University, Hefei 230032, China 8 Department of Urology, National Taiwan University Hospital, Taipei 10002, Taiwan
9 Departments of Medical Research, Obstetrics and Gynecology, Dermatology, and Urology, China
Medical University Hospital, Taichung 40447, Taiwan
Correspondence should be addressed to Wen-Chi Chen; [email protected] and Wei-Yong Lin; [email protected]
Abstract
Urolithiasis is a common urologic disorder with high recurrence. It has been reported that kidney stones are a significant and independent risk factor for chronic kidney disease in the general population. Pharmacological therapy for urolithiasis using medicinal plants has been increasingly adopted for the prevention of its recurrence. However, large number of different medicinal plants exist; the lack of rapid tools and large-scale screening for these abundant medicinal plants make their identification difficult. A Drosophila melanogaster model developed for translational research of urolithiasis was applied to evaluate agents with potential antilithic effects and calcium oxalate (CaOx) formation. Potential antilithic herbs were prepared in a mixture of food in a diluted concentration of 5,000 from the original extract with 0.5% ethylene glycol as the lithogenic agent. The control group was fed with food only. After 3 weeks, flies (n 150 for each group) were killed using CO2 narcotization, and the Malpighian tubules were dissected, removed, and processed for polarized light microscopy examination of the crystals. The crystal formation rate in the positive control group was 100.0%. In the first study, 16 tested herbal drugs reached the crystal formation rate of 0%, namely Salviae miltiorrhizae Radix (#11), Paeoniae alba Radix (#19), Carthami flos (#21), Rhizoma corydalis (#29), Prunus armeniaca L. (#42) Herba ecliptae eclipta prostrala L. (#43), Folium artemisiae Argyi (#46),
Semen plantaginis (#48, and its seed #50), Caulis lonicerae (#49), Rhizoma polygoni Cuspidati (#52), Poria (#70), and Radix angelicae Sinensis (#77). Semen cuscutae (#78) enhanced CaOx crystal formation in the Malpighian tubules (crystal formation rate = 100%). Two herbal drugs causing the death of all flies, Myrrha (#44) and Natrii sulfas (#47), were excluded from the potential antilithic drug list. In conclusion, our rapid screening methods provided evidence that some medicinal plants have potential antilithic effects. These useful medicinal plants can be further studied using other animal or human models to verify their effects.
1. Introduction
Urolithiasis is a common urologic disorder with high prevalence and recurrence . It has been reported that kidney stones are a significant and independent risk factor for chronic kidney disease in the general population. An overall prevalence from 9.6% to 6.5% and a 5-year recurrence rate of 34.7% are observed in Taiwan . Owing to high recurrence, various treatment modalities are available; however , currently, the prevention of the recurrence of urolithiasis remains a challenge .
Pharmacological therapy for urolithiasis using medicinal plants in traditional Chinese medicine (TCM) has been arising for the prevention of its recurrence . A single agent or formula such as Takusha, Wulingsan, and Desmodium styracifolium were reviewed by Miyaoka et al. They concluded that TCM has promising roles in urinary stone prevention . However, over 300 different types of medicinal plants exist according to the record of “Ben Cao Bei Yao (本草備要; Complete Essentials of the Materia Medica)”, a famous TCM pharmacology book. Few of them were studied for the antilithic effect, although potential effects were recorded. One of the possible causes for this is the lack of rapid tools to extensively study these abundant herbal drugs .
The development of novel translational research technologies and approaches is of central importance for successful complementary and alternative (CAM) research.
Recently, we have developed a fruit fly (Drosophila melanogaster) model for translational research of urolithiasis and applied this model to the evaluation of agents that may have potential antilithic effect . The Malpighian tubule of the fruit fly has a function similar to the human kidney .
Calcium oxalate (CaOx) crystal formation can be seen 3 weeks after the addition of lithogenic agents [ethylene glycol (EG)] to the food. The crystals were easily observed under polarized microscopy, and the crystal formation rate can then be evaluated . For example, a test for the effect of melamine on crystal formation in Drosophila was performed. The results indicate that the administration of melamine caused crystal formation in a mixture of CaOx, calcium phosphate, uric acid, and melamine crystals . In a previous study by Ho et al., it was found that potassium citrate could prevent crystal formation in EG-induced CaOx nephrolithiasis in Drosophila. No inhibitory capability of commercial citrate-containing juice was observed for CaOx crystal formation in the Malpighian tubules of the fly .. Therefore, this model can be further used as a rapid screening method to test any drugs that have a potential antilithic effect. Since kidney stones are a significant and independent risk factor for chronic kidney disease in the general population. In this study, we attempted to use this emerging translational model to rapidly screen the potential antilithic medicinal plants.
2. Materials and methods
2.1. Animal Model. We used male D. melanogaster fed with lithogenic agents as positive control and wild-type D. melanogaster fed without lithogenic agents as negative control in this study for the evaluation of CaOx crystal formation . In brief, flies were bred in plastic vials containing a medium of yeast, corn syrup, and agar. Flies were maintained under a condition of 25°C, 60% humidity, and a 12-h light– dark cycle. Potential antilithic herbs were prepared in a mixture of food in a diluted concentration of 5,000 from the original extract with 0.5% EG (Sigma, USA) as the lithogenic agent. The control group of flies was fed with food only. After 3 weeks, flies (n 150 for each group) were killed by CO2 narcotization, and the Malpighian tubules were dissected, removed, and processed for examination of the crystals by polarized light microscopy.
2.2. Screening of Potential Antilithic Medicinal Plants. The extracts of herbs were provided by Sun Ten Pharmaceutical Co. (Taichung, Taiwan). We then selected potential herbs according to the record of“Ben Cao Bei Yao”. A total of 80 herbs were tested in this study (Table 1). Tested herbal drugs were considered to have an antilithic effect if the crystal formation rate was zero. The total death of flies in the
tested group was not considered for the positive effect.
2.3. Observation of CaOx Crystal Formation. The Malpighian tubules were dissected and immediately observed under normal and polarized white light using an Olympus BX51 optical microscope (Japan) after crystal induction. We photographed the relevant crystal aspects using a Kodak ProImage 100 film with scales (USA).
2.4. Statistical Analysis. For each group, data are presented as the crystal formation rate (%). All calculations were performed using Statistical Package for Social Sciences (SPSS for Windows, version 8.0, SPSS Inc, Chicago, IL, USA).
3. Results
3.1. CaOx Crystal Formation Rate. The crystal formation rate in positive and negative control groups were 100.0% and 10.2%, respectively. Positive CaOx crystal formation can be seen in the Malphigian tubules of flies (Figure 1). In the first study, 16 tested herbal drugs reached the crystal formation rate of 0% (Table 1), namely Salviae miltiorrhizae Radix (#11, 丹參), Paeoniae alba Radix (#19, 白芍藥), Carthami flos (#21, 紅花), Rhizoma corydalis (#29, 延胡索), Rhizoma imperatae (#35, 白茅根), Prunus armeniaca L. (#42, 杏仁) Herba ecliptae eclipta prostrala L. (#43, 旱蓮草),
Folium artemisiae Argyi (#46, 艾葉), Semen plantaginis (#48, and its seed #50, 車前
子), Caulis lonicerae (#49, 忍 冬 藤 ), Rhizoma polygoni Cuspidati (#52, 虎 杖 ),
Astragali radix (#67, 黃耆), Poria (#70, 茯苓), Radix scutellariae (#76, 黃岑), and Radix angelicae Sinensis (#77, 當歸).
Two herbal drugs caused the death of all flies in this study. These were Myrrha (#44, 沒藥) and Natrii sulfas (#47, 芒硝), and death maybe due to their toxicities.
In contrast, Semen Cuscutae (#78, 菟絲子) enhanced CaOx crystal formation in the Malpighian tubules, reaching the crystal formation rate of 100%.
4. Discussion
In our survey, 16 herbs were determined to be successful antilithic herbs. Our results provide valuable information for future studies regarding antilithic herbs suitable for the prevention of urolithiasis. In an ongoing study, Carthami flos has been further studied for its potential antilithic effects, and a positive result was obtained when it was applied to a rat model .
Abundant herbal drugs with antilithic effects that are used for treatment are available in TCM books . Some drugs have been studied using in vitro models such as nucleation, crystal aggregation, and crystal growth . Conventionally, rats have been used as animal models to study the crystal formation rate in the kidney. The
application of a large number of herbal drugs requires the use of a large number of animals, leading to high cost. Our study used a large number of animals and large-scale drug lists to reach significant results that provide valuable data for the study of antilithic effects. The use of the fruit fly has been cited as a potential new animal model to study urolithiasis and has been proven to be effective .
Semen cuscutae has several pharmacological effects, particularly in the genitourinary tract, including replenishing the kidney essence and improving sperm motility, kidney deficiency, urinary frequency, and erectile dysfunction . Its effectiveness was reported by Pan et al.; they showed that an ethanol extract of Semen cuscutae was effective on Th1 and Th2 cell functions, and that it could be safely used as an adjuvant in a mice model . A patent (publication number CN102579706 A; 2012) has been used for the application of Semen cuscutae as part of a formula for treating urolithiasis in China. This formula reached an effective rate of 70%. We also selected Semen cuscutae as a potential antilithic agent in this study. However, the treatment detail of this formula remains unclear because this formula is not composed of a single herb. Based on our results, Semen cuscutae exerted adverse effects that completely enhanced crystal formation. Therefore, further studies will need to be performed to clearly identify the effects of individual components by other means.
Glauber’s salt, which is crystalline hydrated form of sodium sulfate. It mainly
contains Na2SO4 • 10H2O. In a study on rats, Natrii culfas was suggested to have a
protective effect on ischemia-induced brain edema and improve the physiological symptoms . It has been recorded in the TCM books that Natrii culfas can cause the lysis of urinary stones, but it caused the death of flies in this study. Although these herbal drugs may have potential antilithic effects, they have been revealed to have toxic effects; therefore, they cannot be used as a single agent for the treatment of urolithiasis. Conventionally, TCM uses a formula with a combination of several drugs to reduce the toxic effect. These drugs should be carefully reconsidered if used clinically.
Myrrha is a resin made from the Commiphora molmol tree. Studies on Myrrha revealed analgesic and anti-inflammatory effects in the treatment of various diseases associated with inflammatory pain, such as arthritis, obesity, microbial infection, wound, pain, fractures, tumor, and gastrointestinal diseases . In this study, we selected Myrrha because it is used for spasm and pain relief. However, in our study, Myrrha caused the death of flies. Some side effects have been associated with Myrrha, such as bleeding tendency, damage to the kidney, or stomach pain . The use of smaller dosage as part of the herbal formula has been suggested. The limitation of this herb is that it caused the death of flies. Further studies using smaller dosage may elucidate the effect
of Myrrha on the treatment of urolithiasis.
An advantage of our study is that a large number of types of herbs could be rapidly screened within a short period using a large number of animals. The limitations of this study include crude water extracts only, limited dosage control, invertebrate animal model, which may have important anatomical differences compared with mammals. However, this model has proven to be reliable, since several scientific research articles have been published with this stable fly model.
5. Conclusion
The development of novel translational research technologies and approaches is of central importance for successful CAM research . For long, a huge disconnect has been observed between clinical and preclinical studies of CAM agents. Our rapid screening methods provide evidence that some traditional herbs have a potential antilithic effect. These useful crude extracts can be further studied using other animal or human models to verify their effects, which have been recorded in TCM books.
Conflict of Interests
Acknowledgments
This work was supported by China Medical University Hospital (DMR-103-046), CMU under the Aim for Top University Plan of the Taiwan Ministry of Education, Taiwan Ministry of Science and Technology (NSC102-2314-B-039-025), and in part by the Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (DOH102-TD-B-111-004). S.-Y. Wu and Y.-H. Chen contributed equally to this study. We thank Professors Mohamed Eddouks, Sara Azab, and an anonymous reviewer for his/her constructive comments, which helped us to improve the paper and solved the problem regarding the publication process.
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Figure Legends
Fig. 1. EG-induced CaOx crystal deposition in the Malpighian tubules. The images
show representative polarized microscopy for the (a) control flies and (b) the flies with 0.5% EG -induced crystal formation in Malpighian tubules.
Table1. Potential anti-lithic drug list and rate of calcium oxalate crystal formation in Malphigian tubules of male fruit flies.
中藥名稱 No. Latin name Rate of crystal
formation (%)
Control EG 100.00
山楂 CMU-1 Crataegus pinnatifida Bge. var major N. E. Br. 36.36
山茱萸 CMU-2 Cornus officinalis Sieb. et Zucc. 62.50
山藥 CMU-3 Dioscorea opposite Thunb. 45.45
川芎 CMU-4 Rhizoma chuanxiong 27.27
三七 CMU-5 Radix notoginseng 33.33
三菱 CMU-6 Sparganium stoloniferum Buch.-Ham. 45.45
川楝子 CMU-7 Fructus toosendan 30.00
大黃 CMU-8 Radix et Rhizoma Rhei 25.00
巴戟天 CMU-9 Radix Morindae officinalis. 33.33
五靈脂 CMU-10 Faeces trogopterori 7.14
丹參 CMU-11 Salviae miltiorrhizae Radix 0.00
王不留行 CMU-12 Semen vaccariae 20.00
牛膝 CMU-13 Radix achyranthis Bidentatae 30.00
木香 CMU-14 Radix aucklandiae 20.00
化石草 CMU-15 Clerodendranthus spicatus. 30.00
木通 CMU-16 Caulis akebiae. 10.00
甘草 CMU-17 Radix glycyrrhizae 40.00
白朮 CMU-18 Rhizoma atractylodis Macrocephalae 80.00
白芍藥 CMU-19 Paeoniae alba Radix 0.00
生地黃 CMU-20 Rehmanniae radix 30.00
紅花 CMU-21 Cathami flos. 0.00
烏梅 CMU-22 Fructus mume 60.00
柴胡 CMU-23 Radix bupleuri 54.55
厚朴 CMU-24 Magnolia officinalis Rehd. et Wils. 50.00
桃仁 CMU-25 Semen persicae 25.00
枳實 CMU-26 Fructus aurantii Immaturus 50.00
枳殼 CMU-27 Fructus aurantii 40.00
枸杞子 CMU-28 Fructus lycii 66.67
延胡索 CMU-29 Rhizoma corydalis 0.00
胡桃 CMU-30 Juglans regia Linn. 30.00
炮附子 CMU-32 Radix aconiti lateralis Preparata. 40.00
威靈仙 CMU-33 Radix clematidis 28.57
吳茱萸 CMU-34 Fructus evodiae 18.18
白茅根 CMU-35 Rhizoma imperatae 0.00
肉桂 CMU-36 Cortex cinnamomi 8.33
生薑 CMU-37 Rhizoma zingiberis Recens 10.00
冬葵子 CMU-38 Semen abutili. 41.67
白芷 CMU-39 Radix angelicae Dahuricae 25.00
白前 CMU-40 Rhizoma cynanchi Stauntonii 8.33
白薇 CMU-41 Radix Cynanchi atrati 9.09
杏仁 CMU-42 Prunus armeniaca L 0.00
旱蓮草 CMU-43 Herba ecliptae eclipta prostrala L 0.00
沒藥 CMU-44 Myrrha 0.00
赤芍藥 CMU-45 Radix Paeoniae rubra 64.00
艾葉 CMU-46 Folium Artemisiae argyi 0.00
芒硝 CMU-47 Natrii sulfas 0.00
車前子 CMU-48 Semen plantaginis 0.00
忍冬藤 CMU-49 Caulis lonicerae 0.00
車前草 CMU-50 Semen plantaginis 0.00
乳香 CMU-51 Olibanum 11.00
虎杖 CMU-52 Rhizoma polygoni Cuspidati 0.00
金銀花 CMU-53 Flos lonicerae 18.00
豬苓 CMU-54 Polyporus 75.00
滑石 CMU-55 Talcum. 40.00
蒲公英 CMU-56 Taraxacum 57.14
雞內金 CMU-57 Endothelium corneum gigeriae Galli 66.67
燈心草 CMU-58 Medulla junci 28.57
鱉甲 CMU-59 Carapax trionycis. 57.14
熟地黃 CMU-60 Radix rehmanniae Preparata 44.44
八正散 CMU-61 Formula 33.00
瞿麥 CMU-62 Herba dianthi 75.00
澤瀉 CMU-63 Rhizoma alismatis 25.00
薏苡仁 CMU-64 Semen coicis 75.00
黨蔘 CMU-65 Pilose asiabell. 33.33
續斷 CMU-66 Radix dipsaci 100.00
黃耆 CMU-67 Astragali radix 0.00
莪秫 CMU-69 Rhizoma curcumae Aeruginosae. 50.00
茯苓 CMU-70 Poria 0.00
路路通 CMU-71 Fructus liquidambaris 20.00
通草 CMU-72 Medulla tetrapanacis 100.00
烏藥 CMU-73 Radix linderae 40.00
番麥鬚 CMU-74 Zea mays L. 66.00
萹蓄 CMU-75 Polygonum aviculare L. 50.00
黃芩 CMU-76 Radix scutellariae 0.00
當歸 CMU-77 Radix angelicae Sinensis 0.00
菟絲子 CMU-78 Semen cuscutae 100.00
補骨脂 CMU-79 Fructus psoraleae 25.00
