Sampa-Sampalukan

Sampa-Sampalukan 

Common Names:   Stone-breaker (Engl.)                                       Sampa- sampalukan (Tagalog)                                      Chanca Peidra (Spanish) Scientific Name:      Phyllantus nimuri Family Name:          Phyllantaceae Description of plant and parts:  Annual Herb: 30-60 cm high, quite glabrous, stem often branched at the base. Leaves: Numerous, sbsessile distichous often imbricating, elliptic oblong obtuse. Stipules present, very acute.  Flowers: Yellowish, very numerous, axillary. The male flowers are one to three in number while the female flowers are solitary in nature.  Capsules: 2.5mm in diameter, depressed globose, smooth scarcely lobed.    Phyllantus ninuri plant and leaves Phyllantus ninuri flowers  Phyllantus ninuri capsules ACTIVE CONSTITUENTS 1. Kaempferol-4-o-alpha-L-rhamnoside, Aerial plant 0.9%, Root Culture 2. (-) Limonine, Leaf Essential oils 4.5% 3. Ascorbic acid, Leaf 0.41% 4. Cymene, Leaf Essential Oil 11% 5. Hypophyllanthin, Plant 0.05-0.17% 6. Geranin, Plant .23% 7. Linoleic acid, Seed Oil 21% 8. Linolenic acid, Seed Oil 51.4% 9.Ricinoleic acid, Seed Oil 1.2% 10.Phyltetralin, Plant, Leaf 0.14% 11.&Phyllanthin, Leaf, Aerial plant The following are parts of the plant: 1. (-)-Nor-secrurinine 2. 4-Hydroxy-sesamin 3. Corilagin 4. Ellagic acid 5. Estradiol 6. Fisetin-41-O-beta-D-dlucoside 7. Hinokinin 8. Iso-lintetralin 9. Nirurin 10.Nirurinetin 11.Phyllanthus 12.Trans-phytol 13.Repandusinic acid A Following are parts of the Roots: 1. (+)-Catechin 2. (+)-Gallocatechin 3. (-)-Epi-catechin 4. (-)-Epi-catechin-3-gallate 5. (-) Epi-gallocatechin 6. Gallic acid 7. (-)-Epi-gallocatechin-3-O-gallate 8. Eriodictyol-7-o-alpha-L-rhamnosidet 9. Fisetin-41-O-alpha-L-rhamoniside 10.Lupeol acetate 11.Lupeol 12.Nor-securinine Following are parts of the Leaves: 1. 4-Hydroxy-lintetralin 2. 2,3-dimethoxy-iso-lintertralin 3. Astragalin 4. Beta sitosterol 5. Demethylenedioxy niranthin 6. Hydroxy niranthin 7. Hypophyllanthin 8. Iso-quercitin 9. Linnanthin 10.Lintetralin 11.Niranthin 12.Quercitrin 13.Salicylic acid methyl ester 4.Seco-4-hydroxy-lintetralin Following are parts of the aerial plant: 1.24- Isopropyl Cholestrol 2.Dotriacontanoic acid 3.Nirphyllin 4.Nirurine 5.Phyllanthenol 6.Phyllantheol 7.Phyllester 8.Phyllinurin 9.Phylltetrin 10.Triacontan-1-al 11.Triancontan-1-ol Following are present in all leaf, stem, aerial plant, roots 1.Nirtetralin, 2.4-Methoxy-nor-securinine 3.Rutin 4. Phyllanthine 5. Phyllochrysine 6. Quercetin TRADITIONAL USES - Decoction of entire plant used as tonic for the stomach. - Bitter fruit used for tubercular ulcers, wounds, sores, scabies, and ringworm.  - Used for kidney stones and gallstones. - Fresh root used as remedy for jaundice.  - Used as emmenagogue and febrifuge.  - Also used for genitourinary problems: renal colic, cystitis, prostate problems, jaundice, constipation, dyspepsia, gonorrhea. - Young leaves used for fevers. - Chewing of fresh leaves used for hiccups. - Used for baths in newborns. - Decoction used for coughs in infants. - Infusion of root and leaves used as tonic and cold, taken cold in repeated doses. BIMINI Hot water extract of the entire plant is administered orally, to reduce fevers, and as a laxative. 17 DOMINICAN REPUBLIC Hot water extract of leaves is administered orally as a popular fever remedy. 18 FIJI Decoction of dried leaves and roots is taken orally for fever, and for good health. Dried entire plant, grounded in buttermilk is administered orally for jaundice. Fresh leaf juice is used externally for cuts and bruises. For eye diseases the juice is mixed with castor oil and applied to the eye. Infusion of dried leaves is administered orally for dysentery and diarrhea. Infusion of green root is taken orally to treat heavy menstrual periods. 19 FRENCH GUYANA Hot water extract of leaves is administered orally as a cholagogue. 20 HAITI Decoction of dried leaves is taken orally for or used in bath for fever, and orally for indigestion. Decoction of roots are used for stomach aches. Hot water extract of dried entire plant is administered orally as a spasmolytic and is also against fever. 21 INDIA Fresh plant juice is taken orally for genito urinary disorders . The fruit is used externally for tubercular ulcers, scabies and ringworm . Hot water extract of dried entire plant is administered orally for diabetes . For asthma in bronchitis, leprosy, anemia hiccups and as diuretic ayurvedic medicine. 22 KONKAN Rubbed down with rice-water and used as a remedy for menorrhagia. 23 PAPAU-NEW GUINEA Fresh leaf juice or fresh root juice are taken orally for venereal diseases. Decoction of dried entire plant is administered orally to treat venereal diseases. Decoction of dried leaf when taken orally is a treatment for diarrhea. A cupful of leaf decoction is drunk daily. 24 PHILIPPINES Decoction of dried entire plant is used as a bath for newborns. It is believed to remove disease-causing elements from the skin. Orally the decoction is used for coughs in infants. 25 PUERTO RICO Hot water extract of leaf and stem is taken orally for fevers. Infusion of young shoots and leaves are given for dysentery. Salted poultice of leaves are used for scabby affections; without salt, applied to bruises and wounds; and made with rice water, poultice lessens edematous swellings and ulcers. 26   SIND Roots, leaves, and young shoots are much employed in gonorrhea and other genito-urinary affections. Bark is used as purgative. Milky juice is applied to offensive sores. 27 TANZANIA Hot water extract of fresh entire plant is administered orally for gonorrhoea. 28 THAILAND Hot water extract of commercial sample of the entire plant, is administered orally as an antipyretic. 29  Hot water extract of dried aerial parts administered orally is used as a diuretic, as an antipyretic, and for malaria.  Hot water extract of dried entire plant is administered orally as an anti-inflammatory agent. 30 VIRGIN ISLANDS Hot water extract of the plant is taken orally to increase the appetite. 31 WEST INDIES Hot water extract of roots together with hot water extract of Citrus aurantifolia roots is taken orally to increase appetite. Hot water extract of entire plant administered orally, is taken for malarial fever. The plant is boiled and the tea taken. Water extract of the leaves and roots is taken orally for diabetes, and as a diuretic . Phyllanthus niruri is used as a diuretic in dropsical affection, gonorrhoea and   other troubles of genito urinary tract. Herb is bitter, astringent, diuretic and febrifuge, antiseptic. Fresh root is a remedy for jaundice. Infusion of young shoots given in dysentery. Leaves are popular remedy against fever. It can be used to increase the appetite and locally to relieve inflammations. It can also be used in case of anorexia. 32 PHARMACOLOGICAL ACTIVITY WITH CLINICAL BASIS I.Hepatoprotective Effect – 1.        Hepatitis B is one of the major diseases inflicting human population. Conventional treatment with interferon – alpha is very expensive and has many serious side effects. Alternative herbal medicine using extracts of Phyllanthus niruri and Phyllanthus urinaria have been reported to be effective against Hepatitis B and other viral infections. A study reports quantitative determination of the antiviral effect of these herbs in well-defined in vitro systems. 3 2.    Phyllanthus niruri has been reported to exhibit marked antihepatitis B virus surface antigen activity in in-vivo and in-vitro studies. Infectious hepatitis is due to the inability of the bodies’ immune system to eliminate the virus from the liver cells: hence the “carrier state”. An infection with the virus is documented by detectable levels of various viral antigens in the blood, including HbaAg (the surface antigen of the virus) as well as antibodies to the core of virus (HBc antibodies). In one study, 37 patients with chronic viral hepatitis B were treated with a daily dose of 600mg of Phyllanthus niruri for 30 days. 59% of the patients lost the HBsAg two weeks after the end of the treatment. Furthermore, none of the cases followed for up to 9 months had any symptoms of HBsAg. The authors postulated that Phyllanthus nirurimight inhibit proliferation of the virus by inhibiting replication of the genetic material of the virus. 4 3.    Hepatoprotective effect of an ayurvedic medicine; herbal preparation HPN – 12 (containing Glycirrhiza glabr, Pichorhiza kurroa, Berberis aristata, Piper longum, Phyllanthus niruri, Solanum dulcamara, Zingiber officinale, Curculigo orchioides, Elettaria cardamomum, Tinospora cordifolia, Desmodium trifolium and Sacchrum officinarum) orally administered to male albino rats at 1ml/100g body weight was found to be effective against liver damage. 5 4.     Animals with Carbon Tetrachloride induced hepatopathy were treated with catliv (contains extracts of Swertia chirata, Eclipta alba, Fumaria vaillanti, Picorrhiza kurroa, Andrographis paniculata and Phyllanthus niruri) at 25ml twice daily orally for six days starting at 48 hours after administration of Carbon tetrachloride. On basis of result obtained it was concluded that the ingredients in catliv, effectively helped in regeneration of hepatic cells and is an effective liver tonic for calves.6 5.     Research in Japan and India in the 1980's has demonstrated the liver -healing properties of Phyllanthus niruri.  The primary compounds responsible are phyllanthin, hypophyllanthin and triacontanal. Glycosides found in Phyllanthus niruri demonstrated Aldose reductase (AR) inhibitory activity in studies conducted by a Japanese research group in 1988 and 1989.7   II.HIV Replication Inhibition– 1.      Aqueous extract of Phyllanthus niruri is reported to have inhibitory effect on human immunodeficiency virus. The investigation examines the anti-HIV effects of the alkaloidal extract of Phyllanthus niruri in human cell lines. The inhibitory effect on HIV replication was monitored in terms of inhibition of virus induced cytopathogenecity in MT-4 cells. The alkaloidal extract of Phyllanthus niruri showed suppressing activity on strains of HIV-1 cells cultured on MT-4 cell lines. The CC50 for the extract was found to be 279.85μgmL-1 whereas the EC50 was found to be 20.98μgmL-1. Interestingly the Selectivity Index (SI) was found to be 13.34, which showed a clear selective toxicity of the extract for the viral cells. The alkaloidal extract of Phyllanthus niruri was thus found to exhibit sensitive inhibitory response on cytopathic effects induced by both the strains of human immunodeficiency virus on human MT-4 cells in the tested concentrations.8 2.       Extracts of five medicinal plants: Aristolochia indica, Cassia occidentalis, Phyllanthus niruri, Withania somnifera and Tinospora cordifolia were administered to 10 HIV infected patients for a period of six months to one year. The clinical status of the patient and their CD4 cell counts were periodically monitored. The results indicate that in seven of the ten patients, their CD4 count increased and the patients remained either asymptomatic or their clinical well being improved. There was no change in the CD4 cell count in one of the patient and the other two progressed to full blown AIDS.9   III. Lipid Lowering Activity  - 1.      Lipid lowering activity of Phyllanthus niruri alcoholic extracts in triton induced hyperlipidaemia was examined in rats. It was observed that administration of triton in rat caused increase in serum cholesterol by 3.5 fold, phospholipid 2 fold and triglyceride 1.2 fold. Administration ofPhyllanthus niruri at the dose of 200mg/kg simultaneously with triton lowered the level of total cholesterol, phospholipid and triglyceride by 27, 25 and 24 percent respectively. In an experiment with cholesterol fed rats, Phyllanthus niruri at a dose of 100 mg/kg lowered the elevated level of low-density lipoprotein lipids in hyperlipidemic and drug fed animals.10 IV.Anti – Diabetic Activity – 1.      An alcoholic extract of Phyllanthus niruri was found to reduce significantly the blood sugar in normal rats and in alloxan diabetes rats. In normal rats, administration of Phyllanthus niruri 200mg/kg body weight reduced the blood sugar by 34.5 percent and to 47.4 percent at the concentration of 1000mg/kg by weight at 1 hour. However at 6thhour, values are almost similar to normal value. Continuous administration of the drug produced significant reduction in normal blood sugar in rats, which on 15th day was also found to reduce the blood sugar in alloxan diabetic rats. In short term experiment, drug was found to reduce the blood sugar at 4th hour by 6.07 percent at dose level of 200mg/kg by weight and 18.7 percent at concentration of 1000mg/kg by weight. Continuous administration of drug produced significant reduction in blood sugar in alloxan diabetic rats. On 15th day values were almost similar to normal in the group taking 1000mg/kg by weight. Plant extract did not produce any toxicity as seen from liver and kidney function test and in hematological parameters. The results indicate potential antidiabetic action of Phyllanthus niruri.11   V. Anti Malarial Activity – 1.      The ethanolic, dichloromethane and lyophilized aqueous extracts of Cassia occidentalis root bark, Morinda morindoides leaves and whole plants of Phyllanthus niruri were evaluated for their antimalarial activity in vivo, in 4-day, suppressive assays against Plasmodium berghei ANKA in mice. No toxic effect or mortality was observed in mice treated, orally, with any of the extracts as a single dose, of 500 mg/kg body weight, or as the same dose given twice weekly for 4 weeks (to give a total dose of 4 g/kg). No significant lesions were observed, by eye or during histopathological examinations, in the hearts, lungs, spleens, kidneys, livers, large intestines or brains of any mouse. At doses of 200 mg/kg, all the ethanolic and dichloromethane extracts produced significant chemosuppressions of parasitaemia (of > 60% for C.occidentalis root bark and Phyllanthus niruri whole plant, and of 30% for M.morindoides leaves) when administered orally. The most active ethanolic extract, that of Phyllanthus niruri, reduced parasitaemia by 73%. The dichloromethane extracts of M.morindoides and Phyllanthus niruri produced similar reductions (74% and 72% chemosuppression, respectively), whereas that of C.occidentalis was slightly less active (60% chemosuppression). Each lyophilized aqueous extract was less active than the corresponding ethanolic extract.12   VI.Activity Against Filarial Mosquito (Culex quinquefasciatus) – 1.      18 plants were evaluated for juvenile hormone analogue activity against Culex   quinquefasciatus. Of these acetone extracts of 8 plants namely Commelina benghalensis , Ageratum conyzoides , Achyranthus aspera, Sida acuta, Euphorbia pulcherrina, Rivinia humilis, Ruellia tuberosa and Phyllanthus niruri possessed significant juvenile hormone activity. The LC50 values of 5 most active plants namely Phyllanthus niruri, Amaranthus spinosus, Antegonon leptopus, Corchorus aestuans, Corchorus benghalensis were determined to be 13,16,17,17,14ppm respectively.13   VII.Anti- spasmodic activity – 1.      Research done in Brazil at the Federal University of Santa Catarina in 1984 on   Phyllanthus niruri revealed an alkaloid (phyllanthoside) in the leaves and stem with strong antispasmodic activity.  It served as a relaxing agent for smooth muscles and they concluded that its spasmolytic action probably accounted for the efficacy of Phyllanthus niruri in expelling stones.14 VIII. Analgesic activity – 1.      Methanol extract of dried callus tissue at a concentration of 10mg/kg, administered  intraperitonially to mice was active vs. acetic acid induced writhin and vs. formalin – induced pedal edema. The extract, at 50mg/kg was inactive vs tail flick response to radiant heat. Ethanol/ water (1:1) extract of dried entire plant at a dose of 50mg/kg, administered intragastric to male mice was active. The extract also administered intraperitonially to male mice at a dose of 0.3mg/kg was active. In both cases antinociceptive effects were demonstrated using 5 different models of nociception. 15 IX. Chromosome Aberration Inhibition – 1.      Water extract of dried fruit and leaves, at a dose of 685.0 mg/kg, administered to mice   by gastric incubation was active vs. chromosome damage induced by lead nitrate and aluminium sulphate in bone marrow chromosomes. Dosing was for 7 days. 16 TOXICITY OF SAMPA-SAMPALUKAN Abstract Phyllanthus niruri is a plant with medicinal properties. It is often used to treat mild malaria and the elimination of renal stones. However, studies on its toxicity are scarce. The study was carried out to determine if the aqueous leaf extract of P. niruri administered to female Sprague-Dawley rats would illicit evidence of toxicity. Fifteen female rats weighing 150–200 g were divided into 3 groups. Rats in Group 1 were given a single low dose (LD) of 2000 mg/kg b.w. of the extract by oral gavage within 24 hrs. Rats in Group 2 were given a single high dose (HD) of 5000 mg/kg b.w. of the extract by oral gavage within 24 hrs. Rats in Group 3 were not given any extract but drinking water and served as the control group (C). All the rats were observed for signs of toxidromes for 14 days. On the 15th day, all the rats were sacrificed. Body organs were harvested for macroscopic examination. Urine and blood samples were drawn and analyzed. Hematological tests performed included full blood count and hemoglobin. Biochemical examinations included bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein, albumin, globulin, alkaline phosphatse (ALP), γ-glutamyltranspeptidase (GGT), urea, and creatinine. The results of the three groups were not significantly different. Examination of the various body organs did not show any abnormality. Thus no toxicity was observed at the levels administered. The LD50 of the aqueous extract is>5000 mg/kg. b.w. Introduction The Phyllanthus genus contains over 600 species distributed throughout the tropical and subtropical regions of the world. In the 1990s, a major reorganization of the Phyllanthus genus was conducted which classified P. amarusas a type of P. niruri (Taylor, 2003). P. niruri extract was demonstrated to block the formation of calcium oxalate crystals (Campos and Schor, 1999; Freitas et al., 2002) and stone formation in urolithiasis (Barros et al.,2003; Barros et al., 2006). Recently, antispasmodic activity of P. niruri (Iizuka et al., 2006) and hypoglycemic effects were reported (Raphael et al., 2002; Ali et al., 2006). The hypotensive effects of P. niruri have been attributed to geraniin (Srividya & Periwal, 1995), and confirmed by its cholesterol and triglyceride lowering effects (Adeneye et al.,2006). Geraniin also possesses antiulcer properties and is believed to be seven times more potent than aspirin or acetaminophen (Miguel et al., 1996; Santos et al., 1994). The anti-malarial activity of P. niruri in 20 crude extracts from nine African medicinal plants used in Kinshasa, Congo, was confirmed (Tona et al., 1999; Cimangaet al., 2004; Mustofa et al., 2007). P. niruri and amarus are said to offer protection against HBV (Mehrotra et al., 1990), chemical toxins (Lee et al., 2006; Chatterjee et al., 2006; Wang, 2000), liver cancer (Rajeshkumar and Kuttan, 2000) and tumorigenesis (Rajeshkumar et al., 2002; Sripanidkulchai et al., 2002), although the latter is still controversial (Milne et al., 1994; Doshi et al., 1994; Thamlikitkul et al., 1991). In this study, acute toxicity of P. niruri aqueous leaf extract was investigated because of limited information available on its toxicity, despite the widespread use of this medicinal plant. Methods The protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Noguchi Memorial Institute for Medical Research (NMIMR) according to the Guidelines for Animal Experimentation. Plant material P. niruri leaves were collected from the Kpando area in the Volta region of Ghana in the month of September. The plant was identified in its vernacular names by the farmers and confirmed to be the same as those previously authenticated by the herbarium at the University of Ghana Botany Department. A specimen was lodged at the herbarium with voucher number GC1009. Extract preparation The leaves were air dried at room temperature to a constant weight and ground to powder. The powder (1800 g) was boiled in 3000 ml of water for 15 minutes under atmospheric pressure and the solution was later filtered. The filtrate was lyophilized using a freeze-drying system, which yielded 33.27 g of freeze-dried material. The freeze-dried sample was stored in a cool dry place until ready for use. Animals and experimental design Fifteen (15) female Sprague-Dawley (S-D) rats (weighing 150–200 g) were obtained from the NMIMR. During the acclimatization period, clinical observations as well as body weight measurements of the animals were conducted and they were found healthy. The rats were assigned into groups including a control group by the stratified random method according to their body weight. S-D rats were fed standard chow diet (AIN-93G formulation, obtained from GAFCO – Ghana) ad libitum. Housing conditions S-D rats were housed in metal cages with stainless steel tops in the animal care facility of NMIMR, where room temperature, humidity and ventilation were controlled according to international standards. The rats were maintained in a 12-h light-cycle and were studied for 14 days. Prior to sacrifice, they were anesthetized with diethyl ether and later euthanized. All visible organs and tissues were macroscopically examined and harvested. Blood collection was by cardiac puncture. Route of administration The route of administration was by oral gavage in accordance with the main route of intake of P. niruri decoction by humans for medicinal purposes. Acute toxicity test Five S-D rats constituted a group. Thus three groups including the control group (C) were established. A single oral low dose (LD) of 2000 mg/kg b.w. and a single oral high dose (HD) of 5000 mg/kg b.w. P. niruri were reconstituted as aqueous homogenous suspensions. The administration volume was set at 900 µl/kg b.w. Group 1, the control group (C), fed normal chow diet, was gavaged 162 µl drinking water (once). Group 2, low dose group (LD) and group 3, high dose group (HD) were gavaged with the extract at a single administration with the doses indicated previously. Clinical observations The observation period was 14 days post administration. Clinical sings of toxidromes (rising fur, draping, tremors, excitability, miosis, mydriasis, twitching, salivation, morbidity, etc.) and mortality were observed while dosing. Thereafter, daily observations were made until the 14th day. Body weights were measured before dosing on the day of administration and weekly thereafter. Urinalysis Urinalysis was performed on the 15th day. Urine was collected in the morning and examined for pH, protein, glucose, ketone bodies, bilirubin, occult blood and urobilinogen. Hematological indices Hematological examinations were conducted on the 15th day at necropsy. Blood samples were collected into EDTA-2K tubes for immediate analysis using the SYSMEX hematology autoanalyzer (Kobe, Japan). Reagents for the hematology autoanalyzer were obtained from STROMATOLYZER (WH, USA). Leukocyte count, erythrocyte count, hemoglobin concentration, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), reticulocyte ratio, platelet count and differential leukocyte counts were determined. Biochemical analyses Biochemical examinations were performed using blood collected into plain tubes. Blood samples were centrifuged at 3000 rpm for 5 minutes. The serum was collected for assays. The following biochemical assays were performed using the SELECTRA JUNIOR Version 04 autoanalyzer (Vital Scientific, Spankeren, The Netherlands): total bilirubin (TBIL), direct bilirubin (DBIL), aspartate amino transferase (AST), alanine amino transferase (ALT), total protein (TP), albumin (ALB), globulin (GB), alkaline phosphatase (ALP), γ-glutamyltranspetidase (γ-GT), urea (URE), creatinine (CR). Statistical analysis The statistical analysis of the data was done using SPSS (Statistical Package for Social Sciences) version 17.0. All data was expressed as Mean±SD. Statistical difference was established using the independent Student's t-test for paired and unpaired data. A probability value of p ≤0.05 was considered statistically significant. For multiple groups, analysis of variance (ANOVA) was used to determine statistical differences. p-values ≤0.05 were considered significant. Multiple regression analysis was performed to determine predictive indicators of toxicity and their relationship with dependent variables. Results Urine analysis for pH, protein, glucose, ketone bodies, bilirubin, occult blood and urobilinogen were negative. Hematological parameters did not show significant differences between C and LD groups. Slightly greater differences were noted for WBC of the C group (8.78×103/µl) and LD group (6.44×103/µl). Similarly, the platelet count was slightly lower for the LD group (793.6×103/µl) compared to the C group (812.8×103/µl). These differences, however, were not statistically significant. Additionally, at 5000 mg/kg b.w., slight WBC differences were observed for the C group (8.78×103/µl) and HD group (5.94×103/µl) that were insignificant. Table 1 Table of hematological indices of the Control group, Low Dose group (LD=2000 mg/kg b.w.) and High Dose group (HD=5000 mg/kg b.w.) on day 15 after the administration of P. niruri aqueous leaf extract on Sprague-Dawley rats on day 1. Variable Control LD HD p-value WBC×103/ml 8.8±2.0 6.4±2.5 5.9±2.6 NS RBC×106/ml         6.2±0.4 6.3±1.7          6.1±1.6 NS HGB g/dl         12.1±0.6 12.2±3.2       11.9±3.2 NS HCT%        39.4±1.7 39.3±10.6       38.3±10.3 NS MCV fl       63.3±2.0 62.9±17.0       63.1±16.7 NS MCH pg       19.4±0.6 19.5±5.3 19.6±5.2 NS MCHC g/dl       30.7±0.4 31.0±8.5        31.1±8.3 NS PLT×103/ml        813±261 794±259      782.8±248.6 NS LYM%        86.5±3.1 87.3±23.4        87.5±23.1 NS LYM×103/ml          7.6±1.7         5.6±2.1          5.2±2.3 NS RDW-SD fl 31.8±0.6       31.5±8.7 30.9±8.4 NS RDW-CV% 12.6±0.3 12.3±3.4        12.0±3.3 NS PDW fl        7.3±0.3   6.9±1.9          7.4±2.0 NS MPV fl        6.4±0.1        6.1±1.7          6.4±1.7 NS P-LCR%        4.4±0.6        3.6±1.1          4.9±1.6 NS NS=Not Significant; WBC=White Blood Cells; RBC=Red Blood Cells; HGB=Hemoglobin; HCT=Hematocrit; MCV=Mean Corpuscular Volume; MCH=Mean Corpuscular Hemoglobin; MCHC=Mean Corpuscular Hemoglobin Concentration; PLT=Platelet; LYM%=Lymphocytes Percentage; LYM=Lymphocyte Count; RDW-SD=Standard Deviation in Red Cell Distribution Width; RDW-CV=Coefficient of Variation in Red Cell Distribution Width; PDW= Platelet Distribution Width; MPV= Mean Platelet Volume; P-LCR= Platelet Larger Cell Ratio Table 2 shows renal function determined by urea and creatinine levels. Urea values were 7.6±1.1 mmol/l (C group), 8.1±2.1 mmol/l (LD group) and 8.7±2.3 mmol/l (HD group). The differences between the groups were insignificant. Creatinine was reduced from 67.9±9.7µmol/l (C) to 59.4±17.2 µmol/l (LD) and to 56.5±17.3 µmol/l (HD). Creatinine differences were not significant. For the liver function test, total protein, albumin and globulin showed slight yet insignificant decreases in the LD and HD groups. Total bilirubin decreased from 2.1±0.7 µmol/l (C group) to 0.5±0.1 µmol/l (LD group). However, there was a slight increase to 2.2±1.4 µmol/l when the HD extract was administered. Nevertheless, changes were not significant. Similarly did the direct and indirect bilirubin levels decline non-significantly in the LD group. In the HD group, direct and indirect bilirubin levels were virtually unchanged. ALT levels were slightly reduced in the LD group. There was a further reduction after the HD administration. However, differences were not significant. AST declined in the LD group (144±44 U/l) compared to the C group (159±42 U/l) but remained unchanged in the HD group (159±46 U/l). Changes were not significant. Mean ALP level was 444±36 U/l in the C group, 388±136 U/l in the LD group and 420±127 U/l in the HD group. The differences were insignificant. Although γ-GT increased from 1.4±0.68 U/l (C group) to 1.9±0.71 U/l (LD group), the difference was not significant. Contrary to the increase in γ-GT observed after the LD administration, γ-GT decreased from 1.4±0.68 U/l (C group) to 1.1±0.68 U/l in the HD group. The differences were not significant. Table 2 Table of biochemical indices of the Control group, Low Dose group (LD=2000 mg/kg b.w.) and High Dose group (HD=5000 mg/kg b.w.) on day 15 after administration of P. niruri aqueous leaf extract on Sprague-Dawley rats on day 1. Variable Control LD HD p-value URE mmol/l 7.6±1.1 8.1±2.1 8.7±2.3      NS CR µmol/l 67.9±9.7     59.4±17.2     56.5±17.3      NS TP g/l 60.1±5.3   57.4±15.5 55.0±15.6 NS ALB g/l 36.6±2.8 35.0±9.4     33.8±9.5       NS GB g/l 23.5±2.6  22.4±6.1 21.2±6.1 NS DBIL µmol/l 1.2±0.6       0.7±0.5 1.1±0.6      NS IBIL µmol/l 0.9±0.4 0.3±0.1      1.1±1.3  NS TBIL µmol/l       2.1±0.7 0.5±0.1     2.2±1.4       NS GGT U/l        1.4±0.7       1.9±0.7      1.1±0.7       NS ALT U/l 129±20      114±34 107±36       NS AST U/l 159±42 144±44 159±46 NS ALPU/l 444±36  388±136 420±127 NS NS=Not Significant; URE=urea; CR= creatinine; TP=total protein; ALB=albumin; GB=globulin; DBIL=direct bilirubin; IBIL=indirect bilirubin; TBIL=total bilirubin; GGT=γ-glutamyltranspeptidase; ALT=alanine aminotransferase; AST=aspartate aminotransferase; ALP=alkaline phosphatase Discussion International opinion and regulations relating to human health necessitate that every new pharmaceutical drug be tested for its safety before it is administered to human volunteers and patients. Toxicity studies in appropriate animal models are therefore commonly used to assess the potential health risk to humans. Such toxicity studies assess the hazard, namely the basic toxicity of the substance, and the risk is determined by considering the probability of exposure to a particular hazard at certain levels (Klaassen & Eaton, 1991). This is a key stage in ensuring the safety of drugs and an acute toxicity study is just one of the batteries of toxicity tests that are used for such purposes. Acute toxicity tests provide preliminary information on the toxic nature of a material for which no other toxicological information is available. Such information can be used to: (i) deal with cases of accidental ingestion of a large amount of the material; (ii) determine possible target organs that should be scrutinized and/or special tests that should be conducted in repeated-dose toxicity tests; and (iii) select doses for short-term and sub-chronic toxicity tests when no other toxicology information is available (Gad & Chengelis, 1988). Furthermore, the majority of pharmaceutical companies use only acute toxicity studies to determine the minimum lethal or maximum non-lethal dose. In exceptional circumstances, the information from acute toxicity studies is used in dose-setting for other studies (NC3RS, 2007) and in such cases, the pathological examination is usually limited to macroscopic observations so that target organs are generally identified. Additionally, acute toxicity measurements help to determine the therapeutic index, i.e. the ratio between the pharmacologically effective dose and the lethal dose in the same strain and species, as well as accurately elucidate the toxicity of the medicinal plant (Klaassen & Eaton,1991). The incorporation of all available information can help in reducing the hesitation in deciding to use herbal medicinal products (HMP). Although HMPs are widely considered to be of lower risk compared with synthetic drugs, they are not completely excluded from the possibility of having toxic or other adverse effects (De Smet, 2004). There are, however, challenges unique to HMPs. Often, deficiencies such as under-reporting of adverse reactions, general lack of toxicological information on herbs, and the quality of the reported information present challenges when signals of safety concern arise. The lack of adequate scientific evidence on the safety of P. niruri is often a major issue to the acceptance and use of this medicinal plant. In this study, the plant was successfully identified as P. niruri and therefore the results are not extrapolated beyond this species. The absence of toxidromes was evident at the time of extract administration and thereafter. The biochemical data, mainly the hepatobiliary and renal systems, did not suggest any toxicity. Furthermore, there were no statistical differences between the low dose (2000 mg/kg b.wt.) and the high dose (5000 mg/kg b.wt.) extract administration. Hematologically, the present data did not show any adverse effect either at the low or the high dose. Thus the aqueous leaf extract of P. niruri can be considered non-toxic at the acute level and consequently, the LD50 of P. niruri aqueous leaf extract is more than 5000 mg/kg b. wt. Because the existing literature on the toxicity of Phyllanthus niruri is limited, coupled with environmental factors including climate, soil and water changes that may have modified the chemical composition of the plant, retesting after long periods is imperative to validate any existing data in the light of newer analytical tools available. With single-ingredient products, it is important that the plant part used be identified. Knowledge of the specific plant part, associated with suspected adverse reactions or toxicity, improves assessment of previously reported adverse effects. Additionally, it must be recognized that various extraction procedures of the same herb, or plant part, produce finished products of varying chemical composition (Williamson et al., 1996) and therefore data interpretation must be judiciously assessed. From this study it is concluded that the aqueous leaf extract of P. niruri has an LD50 greater than 5000 mg/kg b.w. with no adverse effect of this dose after a single administration. References: 1.Prajapati N.D., Purohit S.S., Sharma A.K., Kumar T.: A Handbook of Medicinal Plants- A Complete Source Book. Agrobios: India (Jodhpur); 1stEdi., 392. 2.Ross Ivan A.: Medicinal Plants of the World Chemical Constituents, Traditional & Modern Medicinal uses, Humana Press: New Jersey (Totowa); 249-252. 3.Meixa W, Haowei C, Yanjin L. et al (1995) Herbs of the genus Phyllanthus in the treatment of Chronic hepatitis B observation with three preparation from different geographic sites, J. Lab. Clin. Med.126(2): 350. 4.Thyagarajan SP, Subramanian S, Thirunalasundar T. et al (1988): Effect of Phyllanthus niruri on chronic carriers of hepatitis B virus, Lancet. 2: 764­6. 5.Latha U, Rajesh MG. (1999): Hepatoprotective effect of an Ayurvedic medicine, Indian Drugs. 36 (7): 470-473. 6.Pradhan NR. (2001): Therapeutic effect of catliv on induced hepatopalthy in calves, Indian veterinary journal. 79 (12): 1104-1106. 7.Shimizu M.(1989): Studies on aldose reductase inhibitors from natural products. II. Active components of a Paraguayan crude drug Para-parai mi, Phyllanthus niruri. Chem. Pharm. Bull. 37 (9): 2531-2532. 8.Naik AD, Juvekar AR. (2003): Effects of alkaloidal extract of Phyllanthus niruri on HIV replication, Indian J. Med. Sci.  57: 387-93. 9.Natarraj CG. (2000): Role of herbal extracts in HIV infected patients,Proceedings of International Congress on Ayurveda. 207. 10.Chandra R. (2000): Lipid lowering activity of  P. niruri, Journal of Medicinal & Aromatic Plant Sciences. 22 (1): 29-30. 11.Raphael KR, Sabu MC, Kuttan R. (2000): Antidiabetic activity ofPhyllanthus niruri, Amala research bulletin. 20: 19-25. 12.Neraliya S, Gaur R. (2004): Juvenoid activity in plant extracts against filarial mosquito Culex quinquefasciatus, Journal of Medicinal and Aromatic Plant Sciences. 26 (1): 34-38. 13.Calixto JB. (1984): Antispasmodic effects of an alkaloid extracted from Phyllanthus sellowianus: a comparative study with papaverine,Braz J.med. Biol. Res. 313-321. 14.Grewal RC.: Medicinal Plants. Campus Book International: New Delhi, 1st Edi. ,   298-304. 15.Santos AR. (1994): Analgesic effects of callus culture extracts from selected species of Phyllanthus in mice, J.Pharm. Pharmacol. 46(9): 755-759. 16.Holdsworth D. Wamoi B. (1982): Medicinal plants of the Admiralty Islands, Papau New Guinea. Part 1, Int. J.Crude Drug Res. 20(4): 169-181. 17. Halberstein RA, Saunders AB. (1978): Traditional Medicinal Practices and Medicinal plant usage on a Bahamian Island, Cul. Mad. Psychiat. 2: 177-203. 18.Ricardo MS. (1944): investigation of quinine in Phyllanthus niruri,ANALES Univ.Santo Domingo. 8: 295. 19.Singh YN. (1986): Traditional medicine in Fiji. Some herbal folk cures used by Fiji Indians, J. Ethnopharmacol. 15(1): 57 -88. 20.  Duke JA. (1975): Ethnobotanical observations on Cuna Indians, Econ. Bot. 29: 278. 21.  Weninger B, Rouzier R M, Henrys DD, Henrys JH, Anthon R. (1986): Popular medicine of Plateau of Haiti. 2 Ethnopharmacological inventory,J. Ethnopharmacol. 17(1): 13-30. 22.  Weninger B, Haag-Berrurier M, Anthon R.(1982): Plants of Haiti used as antifertility agents, J. Ethnopharmacol. 6(1): 67-84. 23.Sahu TR. (1984): Less known uses of weeds as medicinal plants,Ancient Sci. Life. 3(4): 245-249 24.Chauhan JS, Sultan M, Srivastava SK. (1977): Two new Glycoflavones from the roots of Phyllanthus niruri, Planta Med., 32, 217-222. 25.  Jain SR, Sharma SN. (1967): Hypoglycaemic Drugs of Indian Indigenous Origin, Planta Med. 15(4): 439-442. 26.  Sircar NN. (1984): Pharmacotherapeutics of Dasemani Drugs,Ancient Sci. Life. 3(3): 132-135. 27.  Holdsworth D, Gideon O, Pilokos B. (1989): Traditional medicine of New Ireland, Papua New Guinea part III, Int. J. Crude Drug Res.  27(1): 55-61. 28.  Holdsworth D, Balun L. (1992): Medicinal plants of the East and West Sepik Provinces, Papua New Guinea, Int. J. Pharmaco. 30(3): 218-222. 29.  Velazco EA. (1980): Herbal and traditional practices related to material and child health care, Rural Reconstruction Review. 35-39. 30.Loustalot AJ, Pagan C. (1949): Local “Fever” Plants tested for the presence of Alkaloids, EL Crisol.3(5): 3. 31.Khan MR, Ndaalio G, Nkunya MHH, Wevers H. (1978): Studies on the rationale of African traditional medicine Part II. Preliminary screening of medicinal plants for antigonococci activity Pak., J. Sci. Ind. Res. 27(516): 189-192. 32.Mokkhasmit MK, Swasdimongkol W, Ngarmwathana , Permphipat U.(1971): Study of toxicity of Thai medicinal plants, J. Med. Assoc. Thailand. 54(7): 490-504. 33.Kitisin T. (1952): Pharmacological studies III Phyllanthus niruri, Siriraj Hospital Gaz. 4: 641-649. 34.  Wasuwat S A. (1967): List of Thai Medicinal plants. Asrct Bangkok, Report No. 1 on Res Project. 17, A.S.R.C.T. Thailand .17: 22. 35.Oakes AJ,  Morris MP. (1958): The West Indian Weedwoman of the United States Virgin Islands, Bull Hist. Med. 32:164. 36.Asprey G F, Thornton P. (1955): Medicinal Plants of Jamaica.III, West Indian Med. J 4: 69-82. 37.Campos Alexander H, Schor Nester (1999): Phyllanthus niruri inhibits calcium oxalate endocytosis by renal tubular cells: its role in urolithiasis, Nephron. 81:393-397. 38.Matsuura Subeki H, Takahashi K, Kobayashi S, Trimurningsih C, Yoshihara T. (2005): Anti-babesial and anti-plasmodial compounds fromPhyllanthus niruri, Journal of Natural Products. 68 (4): 537-539. 39.Khatoon S, Rai V, Rawat A K, Mehrotra S. (2005): Comparitive pharmacognostic studies of 3 Phyllanthus species, J. Ethnopharmacol.Oct 14. 40.  O'Neil J. Maryadle, Annsmith, Heckelman E Patricia, Obenchain R. John Jr., Gallipeau R. Jo Ann, Dárecca Ann Mary: The Merck Index. Merck Research Laboratories: New Jersey, 139, 312, 483, 599, 624, 631, 737, 1428, 1456, 5515, 9142. 41.  Chawla J, Sane R. (1997): Thin layer chromatographic differentiation of Phyllanthus species and its implication in rational search for antihepatotoxic agents from them, Indian Drugs. 34(1): 37. 42. Adeneye AA, Amole OO, Adeneye AK(2006). Hypoglycemic and hypocholesterolemic activities of the aqueous leaf and seed extract of Phyllanthus amarus in mice. Fitoterapia. 77:511–514.  43. Ali H, Houghton PJ, Soumyanath A. (2006).Alpha-Amylase inhibitory activity of some Malaysian plants used to treat diabetes; with particular reference to Phyllanthus amarus. J Ethnopharmacol. 107:449–455.[ 44. Barros ME, Lima R, Mercuri LP, Matos JR, Schor N, Boim MA. (2006) Effect of Extract of Phyllanthus niruri on crystal deposition in experimental urolithiasis. Urol Res. 34:351–357.  45. Barros ME, Schor N, Boim MA(2003);. Effects of an aqueous extract from Phyllantus niruri on calcium oxalate crystallization in vitro. Urol Res.  30:374–379.  46. Chatterjee SPC. (2006) Hepatoprotective effect of aqueous extract of Phyllanthus niruri on nimesulide-induced oxidative stress in vivo. Indian J Biochem Biophys. 43:299–305.  47. Cimanga RK, Tona L, Luyindula N, Mesia K, Lusakibanza M, Musuamba CT, Apers S, De Bruyne T, Van Miert S, Hermans N, Totté J, Pieters L, Vlietinck AJ. (2004); In vitro antiplasmodial activity of callus culture extracts and fractions from fresh apical stems of Phyllanthus niruri L. (Euphorbiaceae). part 2. J Ethnopharmacol. 95:399–404.  48. De Smet PAGM. (2004); Health risks of herbal remedies: an update. Clin Pharmacol Ther.  76:1–17.  49. Doshi JC, Vaidya AB, Antarkar DS, Deolalikar R, Antani DH.(1994); A two-stage clinical trial of Phyllanthus amarus in hepatitis B carriers: failure to eradicate the surface antigen. Indian J Gastroenterol.  13:7–8. 50. Freitas AM, Schor N, Boim MA. The effect of Phyllanthus niruri on urinary inhibitors of calcium oxalate crystallization and other factors associated with renal stone formation. BJU Int. 2002;89:829–834. 51. Gad SC, Chengelis CP. Caldwell, N.J.: The Telford Press; 1988. Acute toxicity testing perspectives and horizons; pp. 2–4. 318, 156, 165–167, 159. 52. Iizuka T, Moriyama H, Nagai M. (2006);Vasorelaxant effects of methyl brevifolincarboxylate from the leaves ofPhyllanthus niruri . Biol Pharm Bull. ;29:177–179.  53. Klaassen CD, Eaton DL. (1991); Principles of toxicology. In: Amdur MO, Doull JD, Klaassen CD, editors. Casarett and Doull's Toxicology: The Basic Science of Poison. New York: Pergamon Press. pp. 32–33. 54. Lee KW, Lee HJ, Lee CY. (2006);Hepatoprotective effect of Phyllanthus in Taiwan on acute liver damage induced by carbon tetrachloride. Am J Chin Med. 34:471–482.  55. Mehrotra R, Rawat S, Kulshreshtha DK, Patnaik GK, Dhawan BN. (1990); In vitro studies on the effect of certain natural products against hepatitis B virus. Indian J Med Res. 92:133–138. 56 Miguel OG, Calixto JB, Santos AR, Messana I, Ferrari F, Cechinel Filho V, Pizzolatti MG, Yunes RA. (1996); Chemical and preliminary analgesic evaluation of geraniin and furosin isolated from Phyllanthus sellowianus . Planta Med. ;62:146–149.  57. Milne A, Hopkirk N, Lucas CR, Waldon J, Foo Y. (1994); Failure of New Zealand hepatitis B carriers to respond toPhyllanthus amarus . N Z Med J. ;107:243.  58. Mustofa , Sholikhah EN, Wahyuono S. (2007);In vitro and in vivo antiplasmodial activity and cytotoxicity of extracts ofPhyllanthus niruri L. herbs traditionally used to treat malaria in Indonesia. Southeast Asian J Trop Med Public Health. 38:609–615.  59. Williamson EM, Okpako DT, Evans FJ. (1996); Pharmacological methods in phytotherapy research. Vol 1. Chichester, England: John Wiley & Sons Ltd.; Selection, preparation and pharmacological evaluation of plant material; pp. 131–154. 60. Thomas, Melody Elaine, CHANCA PIEDRA http://www.herballegacy.com) Chanca Piedra / Phyllanthus niruri / Tropical Plant Database 61.  Phyllanthus amarus Shumacher et Thonning / Chinese names / Catalogue of Life, China 62.  Phyto-Pharmacology of Phyllanthus amarus, an overview / Pharmacologyonline 3: 202-209 (2008) Compiled By: Anne Camille G. Magpantay BS Pharmacy 3 Pharmacy Informatics