THE DETERMINATION OF THE ANTI-UROLITHIC PROPERTY OF THE AQUEOUS EXTRACT OF SAMPA-SAMPALUKAN (Phyllantus niruri) by: Joanna Rose A. Motol Liezel Angela Paulino Joyce Anne Kimberly Perez Raiza Marie A. Palentinos Geddah Grace O. Ranoco CHAPTER 1 Problem and Its Setting Introduction Kidney stone formation or urolithiasis is a complex process that results from a succession of several physicochemical events including supersaturation, nucleation, growth, aggregation, and retention within the kidneys. Urinary calculi are the third prevalent disorder in the urinary system. It is a recurrent renal disease which affects __% of Filipino, _______ age _____ gender and ______ % worldwide. Approximately 80% of these calculi are composed of calcium oxalate and calcium phosphate. It is formed by excessive salt in the blood-stream and then it crystallizes in the kidney. The size and shape of the gravel is vary, from the very tiny particle that flowing undetectably through the blood stream till the gravel’s size. However, even the tiniest stone which is attached in the renal cell and causes renal injury and severe pain. Minor bleeding can be seen when the calculi causes the wound inside urinary system. In most cases kidney stone are source of distress and inconvenience, progressive lost of renal function can occur after repeated episodes of stone disease. Urinary calculi may cause obstruction, hydronephrosis, infection and hemorrhage in the urinary tract system. The recurrence rate without preventive treatment is approximately 10% at 1 year, 33% at 5 year and 50% at 10 years. Among the treatments used to expel or prevent the formation of renal calculi involves both surgical and medical approaches, i.e., percutaneous nephrolithotomy (PCNL), extracorporealshock wave lithotripsy (ESWL) and antibiotics However, these procedures are highly costly and with these procedures recurrence is quite common. Compelling data now suggest that exposure to shock waves in therapeutic doses may cause acute renal injury, decrease in renal function and an increase in stone recurrence. In addition, persistent residual stone fragments and the possibility of infection after ESWL represent a serious problem in the treatment of stones. Furthermore, in spite of substantial progress in the study of the biological and physical manifestations of kidney stones, there is no satisfactory drug to use in clinical therapy. Many remedies have been employed through the ages to treat urolithiasis. However; these treatments are relatively costly, painful and require expert hands with availability of appropriate equipments. These facts have stimulated various researches on traditional remedies showing anti-urolithiatic activity. One of the promising plant being studied is a unrecognized weed from the Phyllantus species. Phyllanthus niruri L., (Syn. P. fraternus Webster), Euphorbiaceae, weed found in both cultivated fields and wastelands. In the Philippines it is known as sampa-sampalukan while the Spanish name of the plant, chanca piedra, means “stone breaker” or “shatter stone.” It was named for its effective use to generations of Amazonian indigenous peoples in eliminating gallstones and kidney stones. In Brazil, the plant is known as quebra-pedra or arranca-pedras (which also translates to “break-stone”). P. niruri, are widely used in traditional medicine for the management of renal disturbances, namely in the treatment of kidney stones. It has tiny fruiting bodies or capsules located on the surface of the unpinnately compound leaves (Figure 1). The plant grows to a height of 50 cm to 70 cm and bears very tiny pale green or white flowers (Figure 2). It is of great medicinal importance for numerous ailments like dysentery, influenza, vaginitis, tumors, diabetes, jaundice, dyspepsia, anti-hepatotoxic, anti-hepatitis-B, antihyperglycemic and also as antiviral and antibacterial. Recently it has attracted the attention of researchers, because of its hepatoprotective properties and also the alternative treatment it offers, no adverse effects and less cost, for the humankind. Background of Study The high cost and the adverse effects presented by the available treatment, both invasive and non-invasive treatment, for urolithiasis encouraged the researches to seek for the best, less cost and no toxicity homeopathic alternative that a health professional can provide. Also, the enthusiasm of the researchers for a small, unrecognized plant but with great therapeutic value fueled the desire to conduct this study. Setting of Study The plant of sampa-sampalukan was collected at roadside of Calamba, Laguna on the month of July. The maceration, extraction and phytochemical screening of the sample were done at Centro Escolar University, Makati City, Gil Puyat Building, room 803. Figure 1 Leaves of Phyllantus niruri Figure 2 Whole plant of Phyllantus niruri Conceptual Framework The study is involved in the determination of anti-urolithic property of the aqueous extract from the stem and leaves of Phyllantus niruri. The renal calculi were induced to the experimental animals by the use of 0. 75 % ethylene glycol and 0.75 % ammonium chloride. The aqueous extract of Phyllantus niruri were fed orally to the rats. Urine samples were collected to the animals weekly to undergo several urine chemistry tests. The results obtained to the urine analysis determined the changes happened in the urinary system of the animal and detected the possible formation of the calcium oxalate crystal. At the end of the experiment the animals were sacrificed to obtain their kidneys for further examination and complete analysis of the crystals formed in the organs. The whole plant P. niruri was collected after proper identification of the Philippine Nationl Library located at T.M Kalaw. Root free whole plants were cleaned and dried in air oven at 400C. The roots of the Phyllantus niruri were discarded since some of the results from the various researches conducted showed that there is a very small amount of phytochemicals present in this area. The remaining parts, leaves and stems, according to the study made by Dastur JF. confirmed that there is a high concentration of different classes of organic compounds with various medical interests, such as lignans, tannins, polyphenols, alkaloids, flavonoids, terpenoids and steroids (Calixto et al., 1998). The primary solvent used for the extraction is 90 % methanol. The sample was powdered in a mixture and the hydro-alcoholic extract was prepared by refluxing with the double distilled water (DDW) and alcohol (3:1) in a round bottom flask for 36 hrs (3x12 hrs) at 60ºC. The liquid extract was filtered, cooled and concentrated by evaporating its liquid contents in oven and collected. The powdered extract was redissolved in DDW prior for the oral administration in mice. The required dose for treatment was prepared by dissolving the extract in DDW at a dose level of 300 and 500 mg/kg body weight. The extracts were also submitted to the different phytochemical test. The urolithiasis was induced in the rats by ethylene glycol and ammonium chloride. The researches preferred to use the 0.75 % concentration of the said substances to prevent the premature death of the animals with in the period of the study. Preliminary studies conducted by the urology department of University of Colorado Health Sciences Center in Denver, showed that the administration of >0.75 % of ethylene glycol and ammonium chloride lead to the death of 6 out of 9 rats (67%) within two weeks, and it is all due to extensive kidney crystal depositions. Most of the renal calculi contain calcium oxalate and therefore the presence of ethylene glycol, which is the metabolic precursor of oxalate, promotes hyperoxaluria. Ammonium chloride administration provides an acidic environment in the kidney which favors for the crystallization of calcium oxalate. At the end of every week in the study duration, urine samples were collected to go through several urine biochemistry analyses. The researchers conduct several test to determine level of creatinine, uric acid and BUN. BUN, uric acid and creatinine are important markers for assessment of effect on kidney function and a slight alteration on it can mark a problem in the urinary system, more specifically the formation of renal calculi. The tests for the presence of calcium, oxalates and phosphates were also done in the urine sample. At the end of the study the rats were sacrificed to obtain their fresh kidney. The right kidney cell were subjected to be analyze for the formation of crystal under a light polarizing microscope at low power fields. The left kidney was used for kidney tissue Ca, Ox and PO4 measurements. The kidney tissue was homogenized by a standard homogenizer and dissolved in 1M HCl overnight. After centrifugation, the supernatant was removed and neutralized with 1M NaOH. The centrifugate was subjected for the determination of Calcium, Oxalate and Phosphate. Statement of the Problem The study dealt with the determination of the anti-urolithic property of the aqueous extract of sampa-sampalukan, Phyllantus niruri. Specifically, this study aimed to answer the following questions: 1. How was the plant sample prepared and collected? 2. What method of extraction of crude extract was done? 3. What are the possible active constituents of the aqueous extract of Phyllantus niruri? 4. How was the anti-urolitic effect of the aqueous extract of Phyllantus niruri determined? Hypothesis The researchers aimed to prove the following hypotheses: Ho: There are no significant active components in the aqueous extract of Phyllantus niruri that have anti-urolitic property. Ha: There are significant active components in the aqueous extract of Phyllantus niruri that have anti-urolitic property. Significance of the study This study aims to investigate the anti-urolithic property of the aqueous extract of Phyllantus niruri commonly known as sampa-sampalukan. For the academician, the study essentially introduces, Phyllantus niruri, another nature’s healing wonder and its important property that leads to its therapeutic effect. As, a professional, the power to share knowledge and reliable facts about this plant will somehow help to improve the quality of life the people in the society they belong. For the students, this study could help open their eyes and learn to appreciate the God’s abundance gift for human. A plant like sampa-sampalukan, which mainly unrecognized but proven with multiple therapeutic value, will encourage them to find and discovered another unknown herbal medicine here in the Philippines and make its value known to the country. For pharmacist, the study could help them improve the quality of life of the patient suffering from urolithiasis by promoting and introducing sampa-sampalukan. The study reveal significant facts that tells that sampa-sampalukan was indeed one of the best alternative medicine which shows no adverse effect on human and was economically affordable. The study reveals how plants in the environment takes part to support and improve the quality of health of the mankind. Scope and Delimitations This research is confined in determining the anti-urolithic property of sampa-sampalukan. The study covered the following: 1. Collection and preparation of crude extract. 2. Determination of the possible active constituents of the plant sample. 3. Inducing kidney stone using 0.75 % ethylene glycol and 0.75 % ammonium chloride to promote hyperoxaluria. 4. Determination of the anti-urolithic property based on the test performed in the urine sample and microscopic analysis of kidney tissue. 5. The doses of aqueous extract of Phyllantus niruri used in the determination of anti-urolithic property are 300mg/kg/wt and 500 mg/kg/wt.