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Journal of Ethnopharmacology 138 (2011) 286– 313

Contents lists available at SciVerse ScienceDirect

Journal of Ethnopharmacology

jo ur nal homep age : www.elsev ier .com/ locate / je thpharm

Review

Phyllanthus amarus: Ethnomedicinal uses, phytochemistry and pharmacology:A review

Jay Ram Patela, Priyanka Tripathib,1, Vikas Sharmaa, Nagendra Singh Chauhana, Vinod Kumar Dixita,∗

a Department of Pharmaceutical Sciences, Dr. Harisingh Gour Vishwavidyalaya, Sagar 470003, M.P., Indiab Shri Ramnath Singh Mahavidyalaya (Pharmacy), Gormi, Bhind 477660, M.P., India

a r t i c l e i n f o

Article history:Received 3 March 2011Received in revised form20 September 2011Accepted 23 September 2011Available online 29 September 2011

Keywords:Phyllanthus amarusHepatoprotectiveAnti-inflammatoryAnticancerDiureticsNephroprotectiveAntioxidantAntiviralAntibacterialAntihyperglycemicAntihypercholesterolemic

a b s t r a c t

Ethnopharmacological relevance: Phyllanthus amarus Schum. & Thonn. belongs to the family Euphorbiaceaeis a small herb well known for its medicinal properties and widely used worldwide. P. amarus is animportant plant of Indian Ayurvedic system of medicine which is used in the problems of stomach,genitourinary system, liver, kidney and spleen. It is bitter, astringent, stomachic, diuretic, febrifuge andantiseptic. The whole plant is used in gonorrhea, menorrhagia and other genital affections. It is useful ingastropathy, diarrhoea, dysentery, intermittent fevers, ophthalmopathy, scabies, ulcers and wounds.Materials and methods: The present review covers a literature across from 1980 to 2011. Some infor-mation collected from traditional Ayurvedic texts and published literature on ethanomedicinal uses ofPhyllanthus amarus in different countries worldwide.Results: Phytochemical studies have shown the presence of many valuable compounds such as lignans,flavonoids, hydrolysable tannins (ellagitannins), polyphenols, triterpenes, sterols and alkaloids. Theextracts and the compounds isolated from P. amarus show a wide spectrum of pharmacological activ-ities including antiviral, antibacterial, antiplasmodial, anti-inflammatory, antimalarial, antimicrobial,anticancer, antidiabetic, hypolipidemic, antioxidant, hepatoprotective nephroprotective and diurecticproperties.Conclusion: The present review summarizes information concerning the morphology, ecology, ethnophar-macology, phytochemistry, biological activities, clinical applications and toxicological reports of P.amarus. This review aims at gathering the research work undertaken till date on this plant in orderto provide sufficient baseline information for future works and commercial exploitation.

© 2011 Elsevier Ireland Ltd. All rights reserved.

Abbreviations: 2AA, 2-aminoanthracene; 2NF, 2-nitrofluorene; 3D7, chloroquine-sensitive strain of Plasmodium falciparum; 4-NQO, 4-nitroquinolone-1-oxide; ABTS, 2,2′-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid); AF2, 2-aminofluorene; AH, aniline hydroxylase; ALP, alkaline phosphatase; ALT, alanine transaminase; AP-1, transcriptionfactors; AST, aspartate transaminase; CAT, catalase; CD4, T-helper cell; CFA, complete freund’s adjuvant; COX-2, cyclooxygenase; CTX, cyclophosphamide; DHBV, duckhepatitis B virus; DHHDP, 1-galloyl-2,3-dehydrohexahydroxydiphenyl; DLA, Dalton’s lymphoma ascites tumor; DMN, dimethylnitrosamine; DMSO, dimethyl sulphoxide;DNA, deoxy ribonucleic acid; DPPH, 2-diphenyl-1-picrylhydrazyl; EAC, Ehrlich ascites carcinoma; EC50, effective concentration 50%; ENNG, N-ethyl-N-nitrosoguanidine; FRAP,ferric reducing antioxidant power; FT-IR, Fourier transform infrared spectroscopy; GGT, �-glutamyl transpeptidase; GPX, glutathione peroxidase; GSH, cellular glutathione;GST, glutathione-S-transferase; HBeAg, hepatitis B effective antigen; HBsAg, hepatitis B suppresive antigen; HBV DNA, hepatitis B viral DNA; HBV, hepatitis B virus; HCC,hepatocellular carcinoma; HE, hexane extract; HepA, Hepatitis A; HepA2, Hepatitis A2; HIV, human immunodeficiency virus; HPLC, high-performance liquid chromatography;HPTLC, high-performance thin layer chromatography; HTG, hepatic triglyceride; i.p., intraperitoneal injection; IC50, inhibitory concentration 50%; ID50, inhibitory dose 50%;IFN-a/c, interferon; IL-1, interleukin-1; IL-10, interleukin-10; IL-12, interleukin-12; IL-18, interleukin-18; iNOS, endotoxin-induced nitric oxide; IR, infra red; KC, Kupffercells; LPO, lipid peroxidation; LPS, lipopolysaccharides; MDA, malondialdehyde; MDR, multi-drug resistance; MICs, minimum inhibitory concentrations; mRNA, messengerribo nucleic acid; NDEA, N-nitrosodiethylamine; NF-kB, NF-kappa �; NMR, nuclear magnetic rasonance; 4-NQO, 4-nitroquinolone-1-oxide; P. amarus, Phyllanthus amarus; P.debilis, Phyllanthus debilis; P. fraternus, Phyllanthus fraternus; P. kozhikodianus, Phyllanthus kozhikodianus; P. maderaspatensis, Phyllanthus maderaspatensis; P. niruri, Phyllanthusniruri; P. urinaria, Phyllanthus urinaria; PAF, platelet activating factor; PCV, packed cell volume; PGE2, prostaglandin E2; P.O., per oral; ROS, reactive oxygen species; SCGE, singlecell gel electrophoresis; SEF, supercritical-fluid extraction; SL, silymarin; SOD, superoxide dismutase; STG, serum triglyceride; STZ, streptozotocin; TBARS, thiobarbituric acidreactive substances synthase; TLC, thin layer chromatography; TNF-�, tumor necrosis factor �; UV, ultra violet; WBC, white blood carpuscles; WHV, Woodchuck hepatitis virus.

∗ Corresponding author. Tel.: +91 9425647546.E-mail address: vkdixit2011@rediffmail.com (V.K. Dixit).

1 Present address: Division of Pharmaceutics, Central Drug Research Institute, Lucknow (U.P.) 226001, India.

0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.jep.2011.09.040

Author's personal copy

J.R. Patel et al. / Journal of Ethnopharmacology 138 (2011) 286– 313 287

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2872. Historical perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2883. Botanical description and vernacular names . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2904. Biogeography and ecology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2915. Pharmacognostic characters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2916. Phytochemical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2927. Analytical techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2938. Pharmacological activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295

8.1. Antiamnesic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2958.2. Antibacterial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2958.3. Anticancer activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2998.4. Anti-diarrhoeal, gastroprotective and antiulcer activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3008.5. Antifungal activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3018.6. Analgesic, anti-inflammatory, anti-allodynic and anti-oedematogenic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3018.7. Antinociceptic activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3018.8. Antioxidant activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3028.9. Antiplasmodial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3028.10. Antiviral activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3038.11. Clinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3048.12. Aphrodisiac activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3058.13. Contraceptive effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3058.14. Diuretic and antihypertentive activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305

8.14.1. Clinical study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3058.15. Hepatoprotective activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3058.16. Hypoglycemic and hypocholesterolemic activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306

8.16.1. Clinical study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3078.17. Immunomodulatory activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3078.18. Nephroprotective activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3078.19. Radioprotective effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3078.20. Spasmolytic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3088.21. Effect on reproductive organs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308

9. Toxicological assessment and contraindications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30810. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309

1. Introduction

Bhumyaamalaki (Phyllanthus amarus Schum. & Thonn., Euphor-biaceae), which is widely spread throughout the tropical andsubtropical countries of the world including India is most com-monly used in the Indian Ayurvedic system of medicine inproblems of stomach, genitourinary system, liver, kidney andspleen. P. amarus has been described in Ayurveda by the Sanskritname – Bhoomyaamalakee, Taamalakee and Bhoodhatree. It wasdescribed to have the properties of Rasa, Guna, Veerya and Vipaaka.The Ayurvedic literature has shown its uses as Kaasahara (antitus-sive), Shwaasahara (antispasmodic, antidyspnoic), Kaphapittahara(which relieves the Kapha Pitta Dosha), Pipaasaaghna (whichrelieves Polydipsia), Raktapittahara (hemorrhage disease), Paan-duhara (antianemic), Kaamalaahara (which cures jaundice),Kushthaghna (indicated in leprosy), Daahaghna (refrigerant,relieves burning sensation), Kshatakshayaghna (indicated inTrauma) and Mootrarogahara (which cures urinary disorders). Theuse of P. amarus is gaining momentum because of its novel antiviralactivity against hepatitis B virus and for several other biologicalactivities such as kidney and gallbladder stones, for cold, flu, tuber-culosis, and other viral infections; liver diseases and disordersincluding hepatitis, jaundice and liver cancer (Unander et al., 1993).It also acts against liver cell toxicity and improves the immunesystem of patients and has been found effective against hepatitisA (Jayaram et al., 1997). P. amarus is often used in the traditionalsystem of medicine for a variety of ailments including dropsy, dia-betes, jaundice, asthma and bronchial infections (Foo and Wong,1992). In the Ayurvedic system of medicine it is used in problemsof stomach, genitourinary system, liver, kidney and spleen. It isbitter, astringent, stomachic, diuretic, febrifuge and antiseptic. The

whole plant is used in gonorrhea, menorrhagia and other genitalaffections. It is useful in gastropathy, diarrhoea, dysentery, inter-mittent fevers, ophthalmopathy, scabies, ulcers and wounds. It isalso used as a good tonic. The Spanish name ‘chanca piedra’ means“stone breaker or shatter stone.” In South America, ‘chanca piedra’has been used to eliminate gall bladder and kidney stones, and totreat gall bladder infections (Foo and Wong, 1992), cardiovascularproblems (Chevallier, 2000), and also a remedy around the worldfor influenza (Foo, 1993a). P. amarus has a long history of use inthe treatment of liver, kidney and bladder problems, diabetes andintestinal parasites. In Suriname (Northeastern part of South Amer-ica), P. amarus is always sold as fresh and dry plant material in theherb markets. Decoctions are used in herbal baths and after labor,cramps, asthma, uterus complaints and to treat stomachache (May,1982; Titjari, 1985; Heyde, 1990; Sedoc, 1992; Nanden, 1998). It isa restoration herb and used as an appetizer and as tonic. It is alsoused as colic. The plant, when boiled with the leaves, is consideredto be a diuretic and is used in treatment of diabetes, dysentery, hep-atitis, menstrual disorders, and skin disorders (Wessels Boer et al.,1976; Tirimana, 1987; Heyde, 1968, 1990). Plant extracts are usedas blood purifiers, for light malaria fevers and anemia. It helps torelease phlegm (Heyde, 1990) and to combat fever (Nanden, 1998).This herb can be used for constipation also (Tjong and Young, 1989).

P. amarus elaborates different classes of organic compounds ofmedicinal importance including alkaloids, flavonoids, hydrolysabletannins (Ellagitannins), major lignans, polyphenols, triterpenes,sterols and volatile oil. Many lignans were isolated from theplant viz., phyllanthin (a bitter constituent) and hypophyllan-thin (a non bitter constituent) (Row et al., 1967). The highestamounts of phyllanthin (0.7% w/w) and hypophyllanthin (0.3%w/w) have been reported in leaves whereas, in the stem these

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288 J.R. Patel et al. / Journal of Ethnopharmacology 138 (2011) 286– 313

are in minor quantities present (Sharma et al., 1993). Lig-nans isolated from P. amarus are phyllanthin, hypophyllanthin,niranthin, phyltetralin, nirtetralin, isonirtetralin, hinokinin, linte-tralin, isolintetralin, demethylenedioxy-niranthin, 5-demethoxy-niranthin etc., flavonoïds such as gallocatechin, rutin, quercetin-3-O-glucopyranoside, phyllanthusiin, quercetin, kaempferol3-�-d-glucopyranoside, kaempferol etc., ellagitannins includegeraniin, amariin, furosin, geraniinic acid B, amariinic acid, amaru-lone, repandusinic acid A, corilagin, isocorilagin, elaeocarpusin,phyllanthusiin A, B, C, D and melatonin; securinega-type alka-loids such as isobubbialine and epibubbialine and sterol such asamarosterol A, amarosterol B.

P. amarus had been reported to have pharmacologicaleffects such as antimicrobial, antiviral activities against hepati-tis B, chemoprotective, antimutagenic and hypoglycaemic agent.Methanolic extract of P. amarus exhibited immunomodulatoryactivity. Ellagitannins (geraniin and corilagin) were shown to bethe most potent mediators of the antiviral HIV activity. Phyllan-thin and hypophyllanthin present in P. amarus exhibited antitumoractivities against EAC in Swiss albino mice, cytotoxic effects onK-562 cells, and hepatoprotective and antioxidant effects. Thepresent review assesses the potential of P. amarus in relation to itstraditional uses and in terms of findings based on modern bioscien-tific research. The link between conventional remedies and recentresearch in various areas has been well established in other plantswhich facilitate to determine effective mode of action of plantderived products. The plant is known to contain several pharmaco-logical important biomolecules whose efficacy is well establishedby several biochemical and pharmacological studies. This reviewintent to compile various studies on this plant and critically eval-uates the issues related to ethnopharmacology, phytochemistry,pharmacology, clinical studies and toxicology of P. amarus.

Table 1 represents ethnomedicinal uses of P. amarus in differ-ent countries and Table 2 represents the ethnomedicinal uses of P.amarus used by different tribes of countries worldwide.

2. Historical perspectives

P. amarus has been indexed in majority of published phytochem-ical, pharmacological and ethno-botanical reviews and researcharticles till date with different named. This species, P. amarus hasbeen confused with Phyllanthus niruri Linn. In the past, Linnaeus’sP. niruri, though well defined in the ‘Hortus Cliffortianus’, becameconfused owing to his erroneous reduction of other species to thesynonyms of the former and subsequent misidentification by otherauthors. The commonest weedy species so mistaken for P. niruri byMuller and others was defined as Phyllanthus swartzii Kosteletzskyin 1836, based on P. niruri of Swartz; but the earliest name of thisspecies appears to be P. amarus Schum. & Thonn. For a full discus-sion on the botanical description and nomenclature of this speciesreference has been made to the excellent discussion by Webster(1957). The earliest botanical descriptions could be observed fromSouth India, Sri Lanka and Indonesia which have cited Phyllanthusurinaria and P. niruri respectively (Van Rhede, 1690; Rumphius,1750). In the Ayurvedic system in India, no definitive botanicaldescription exists in the original literature and the plants indicatedby many of the old Sanskrit words have been lost in course of his-tory (Handa et al., 1951; Chopra et al., 1958). It was the opinionof Dymock (1886) and Dymock et al. (1893) that the same com-mon names in a number of Indian languages apply for both P. niruriand P. urinaria. Similar reports have stated that even the rathervisually different P. urinaria was often used interchangeably withthose species earlier known as P. niruri in the traditional systemof India (Van Rhede, 1690; Dymock, 1886; Kirtikar and Basu, 1975)and suggested that the practioners did not differentiate it according

Table 1Ethnomedicinal uses of P. amarus in different countries worldwide.

Region Ethnomedicinal uses

Amazonia Anodyne, apertif, blennorrhagia, carminative,colic, diabetes, digestive, diuretic, dropsy,dysentery, dyspepsia, emmenagogue, fever, flu,gallstones, gonorrhea, itching, jaundice, kidneyailments, kidney stones, laxative, malaria,proctitis, stomachache, tenesmus, tonic,tumor, vaginitis, vermifuge

Aruba Blood purifierBahamas/Caribbean Antihepatotoxic, cold, constipation, fever, flu,

stomachache, typhoid, flatulence, vermifuge,appetizer

Barbados AbortifacientBrazil Abortifacient, ache (joint), albuminuria,

analgesic, antibacterial, anticancerous,antidiabetic, anti-inflammatory, antilithic,antispasmodic, antiviral, aperient, arthritis,biliary conditions, bladder problems, bladderstones, calculi, catarrh (liver and kidney),chologogue, cystitis, deobstruent, diabetes,diaphoretic, digestion stimulant, diuretic,fever, gallbladder, gallstones, gastritis,gastrointestinal problems, gout, hepatitis,hydropsy, hypertension, hypoglycemic,jaundice, kidney, colic, kidney stones, liver,malaria, muscle relaxant, obesity, prostatitis,purgative, renal colic, renal problems,stomachic, sudorific, tonic, uric acid excess,urinary problems, uterine relaxant

Cuba Oedema and malariaHaiti Carminative, colic, digestive, diuretic, fever,

indigestion, malaria, spasmolytic,stomachache, stomachic, tenesmus

India Anemia, asthma, astringent, bronchitis,conjunctivitis, cough, deobstruent, dropsy,diabetes, diarrhoea, diuretic, dysentery, fevers,eye disorders, galactagogue, genitourinarydisorders, gonorrhea, hepatitis, jaundice,leucorrhea, menorrhagia, oligogalactia,itchness, skin ulcers, sores, swelling,ringworm, scabies, stomachic, thirst,tuberculosis, tumor (abdomen), urogenitaltract infections, warts, appetizer, dyspepsia

Indonesia Colic, cough, diuretic, eye diseases (external),kidney diseases, stomachache, toothache,veneral diseases

Island of North Caicos Fever, prevention of intestinal wormsJamaica Diabetes, dysentery, diuretic, oedema,

gonorrhea, jaundice, stomachacheMalaya Caterpillar sting, dermatosis, diarrhoea,

diuretic, emmenagogue, itching, miscarriage,purgative, renosis, syphilis, vertigo

Nigeria Malaria, chronic stomach pains, oral or vaginalthrush, alcoholic liver disease, hyperglycaemia,urinary tract infection and venereal disease.Taken with honey as aphrodisiac

Peru Calculus, diuretic, emmenagogue, gallstones,hepatitis, kidney pain, kidney problems,kidney stones, renal problems, urinaryinfections, vermifuge

Trinidad Diuretic, veneral diseasesUnited States Analgesic, bronchitis, chologogue, deobstruent,

diabetes, fever, gallbladder problems,gallstones, gout, hepatitis, hypertension,kidney problems, kidney stones, liver disease,uric acid excess, urinary tract infections

Elsewhere Analgesic, antipyretic, appetite stimulant,blennorrhagia, bruises, chologogue, cough,cuts, diabetes, diarrhoea, diuretic, dropsy,dysentery, dyspepsia, emmenagogue, eyediseases, fever, gallstones, gonorrhea, itch,jaundice, kidney disease, kidney stones,laxative, malaria, menorrhagia, menstrualproblems, poultice, purgative, rectitis,stomachache, tonic, tuberculosis, urinary tractinfections, vaginitis, venereal diseases

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Table 2Ethnomedicinal uses of Phyllanthus amarus Schum. & Thonn.

S.N. Place Local name Plant partused

Disease Method of use Reference

1 Dharapuram Taluk,Tamil Nadu, India

Keelanelli Whole plant 1. Migraine2. Jaundice

(1) Whole plant is boiled ingingelly oil, filtered andapplied on the head(2) The fresh root is used withwater. Paste or fresh roots aregiven orally

Balakrishnan et al. (2009)

2 Paliyar tribals inTheni district ofTamil Nadu, India

Keelanelli Leaves Jaundice Leaf paste is given internally Ignacimuthu et al. (2008)

3 Eastern part ofRajasthan, India

Bhumiamla Whole plant,leaves

Gonorrhea andsyphilisSkin diseases,malaria

Decoction of leaves, sugar andcumin seeds are taken orally totreat gonorrhea and syphilis.Leaves are crushed with salt tomake paste and applied locallyagainst skin diseases. Plant iscrushed into paste, mixed withseed powder of pepper, candyand water and taken as arefrigerant. Decoction of wholeplant is taken as anantimalarial

Upadhyay et al. (2010)

4 Uttara kannada,Western Ghats,India

Nelli Whole plant Malaria Not stated Kuppusamy and Murugan (2010)

5 Eastern region ofShimoga district,Karnataka, India

Nelanelli Root juice Jaundice Root juice is taken orally withcow’s milk early in themorning for 1 week

Rajakumar and Shivanna (2009)

6 Dindigul District,Tamil Nadu, Indian

Kizhnelli Leaves Menstrual problem Leaf extract with milk andonion is given during night,three times once in 3 days

Samuel and Andrews (2010)

7 Buldhana district;Maharashtra,Indian

Bhui-awala Whole plant Jaundice Extract, one spoonful per dayfor 3 days

Ahirrao and Patil (2010)

8 North AndamanIsland, India

Nallesari Whole plant Jaundice Handful of leaves is crushedwith a pinch of turmeric andone teaspoonful of extract istaken orally for 3–5 days

Prasad et al. (2008)

9 Sivagangai district,Tamil Nadu, India

Keelaanelli Leaves 1. Diabetes2. Jaundice

Diabetes:(A) Leaves of Piper betle,Cynodon dactylon, Azadirachtaindica and P. amarus are driedand powdered with the stempark of Syzygium cumini. Thepowder is boiled in water andthe extract is given orally(B) Leaf extracts of A. indicaand P. amarus are mixed andgiven orallyJaundice:(A) Leaves of C. dactylon and P.amarus are grounded with thefruits of Piper nigrum andextracted. The extract is givenorally(B) Leaves of Eclipta alba, P.amarus and Leucas aspera aregrounded and extracted. Theextract is given orally(C) Leaf extracts of C. dactylonand P. amarus are mixed andgiven orally

Shanmugam et al. (2009)

10 Shimoga district ofKarnataka, India

Nela nelli(Bhumyamalaki)

Leaves 1. Jaundice2. Chronicdysentery

(1) Leaf paste with cardamomis taken internally, two teaspoons daily(2) Leaves are ground withAcacia Senegal leaves, addsugar and give orally, or tenderleaves ground with cow’s milkcurd given orally, for 2–5 days,before food

Mahishi et al. (2005)

11 Kattunaykas tribesof MudumalaiWildlife Sanctuary,Nilgiris districtTamil Nadu, India

Kila nelli Whole plant Jaundice 15 mL whole plant juice istaken internally in emptystomach along with onetumbler goat’s milk againstjaundice

Udayan et al. (2007)

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Table 2 (Continued)

S.N. Place Local name Plant partused

Disease Method of use Reference

12 Kancheepuramdistrict TamilNadu, India

Keezhanelli Leaves Jaundice Fresh leaves are ground andmixed with a cup of cow orgoat’s milk and takeninternally to cure jaundice

Muthu et al. (2006)

13 India Bhumi amalaki Whole plant Liver disease,dyspepsia,anorexia, moderateconstipation,chronic colitis,irritable bowelsyndrome, urinarytract infection

20% whole plant and 10%Plumbago zeylanica (roots). 4 gof powdered mixer is given tothe patient twice daily, half anhour before meals with water

Samy et al. (2008)

14 Northern India Bhui amla Whole Jaundice,aphrodisiac,dysentery

Not stated Kala et al. (2006)

15 Sitamata WildlifeSanctuary ofChittorgarh andUdaipur districtRajasthan, India

Not stated Leaves Syphilis,gonorrhea,jaundice

Leaf paste and decoction ofleaves

Jain et al. (2005)

16 Esan North Eastlocal govt. area ofEdo State, Nigeria

Abenaghe Leaves Stomachache Ground leaves with pepper andsalt. Half of cup is taken twicedaily

Idu et al. (2008a)

17 Delta State Nigeria Ibuko-oyeke Leaves Stomachache The leaves are infused inalcohol and drunk as a remedyfor stomachache

Idu et al. (2008b)

18 Akwa Ibom State inNigeria

Oyomokiso,aman keeden

Leaves Malaria Boiled in water as decoction.Use internally 4 times a day for5 days

Ajibesin et al. (2008)

19 South West Nigeria Eyin olobe Whole plant Diabetes Decoction Abo et al. (2008)20 West Africa Hlinvi Arial Diabetes, fever,

malariaNot stated Adjanohoun et al. (1986)

21 Semi-aridNortheasten Brazil

Quebra-pedra Leaves Kidney problems Decoction of leaves soakingdrink

Cartaxo et al. (2010)

22 Dangme Westdistrict of Ghana

Ofobi okpabi Whole plant Malaria Boil about 50 g of plant in 1 L ofwater and drink a cupful ofdecoction three times dailyafter meals until recovered.Children should take half of thedose. Sweeten with honey orsugar if desired. The decoctionmay cause dizziness

Asase et al. (2010)

23 Surinamesemigrants inNetherland

Fini bita Whole plant Stomach-ache,cleaning uterus,laxative, healthpromotion, diseaseprevention

Whole plant is boiled in wateror soaked in alcohol and drunkto purify the blood. Theseso-called BITAS were said topromote one’s health, purity ofblood and prevent and curediseases like malaria, skinsores, diabetes and pimples.Bitter tonics are also reportedto be popular amongHIV-positives to support theirbody function

Van Andel and Westers (2010)

24 Akha people inThailand and China

Yu Jae Leaves Rashes, itches Poultice Inta et al. (2008)

Number of ethnomedicinal uses of P. amarus.Jaundice = 14, malaria = 08, diabetes = 06, skin disease = 04, dysentery = 03, fever = 03, stomachache = 03, syphilis = 03, urinary tract infection = 03, gonorrhea = 02, constipa-tion = 02, anorexia = 01, aphrodisiac = 01, chronic colitis = 01, dyspepsia = 01, laxative = 01, menstrual problem = 01.

to the current taxonomical understanding. One of the most difficultproblems in organising ethnobotanical data on Phyllanthus was theidentification of the correct species for citations under P. niruri. Thetrue P. niruri Linnaeus type of the genus was collected in Barbadosand described in 1738 by Linnaeus (Linnaeus, 1738). Specimens ofP. niruri have never been confirmed outside the America (Webster,1957). According to Mitra and Jain (1985) of the Botanical Surveyof India, the P. niruri in Flora of British India is indeed a mixture ofthree distinct species viz. P. amarus Schum. & Thonn., Phyllanthusfraternus webster and Phyllanthus debilis Klein ex willd, the true P.niruri Linn being endemic to West Indies. The specimen of P. debiliswas collected from Madras in 1799 (Webster, 1957). However it is

observed that P. amarus predominates over P. debilis in Madras areasuggesting that it is an introduced species over the years. Samples ofthe plants collected from the Madras area in 1983–1988 as P. niruriwere predominantly P. amarus with occasional plants of P. debilisas identified by Grady webster, University of California (Unanderet al., 1991) during the collaborative studies with the group led byBaruch S. Blumberg, Fox Chase Cancer Centre, Philadelphia.U.S.A.

3. Botanical description and vernacular names

P. amarus are erect annual herbs, 10–60 cm tall; main stem sim-ple or branched, terrete smooth or scabridulous in younger parts.

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Fig. 1. P. amarus plant.

Cataphylls, stipules 1.5–1.9 mm long, deltoid acuminate blade1–1.5 mm long, subulate acuminate. Leaves 3–11 × 1.5–6 mm ellip-tic oblong obovate, oblong, or even obovate, obtuse, or minutelyapiculate at apex, obtuse or slightly inequilateral at base, petioles0.3–0.5 mm long, stipules 0.8–1.1 mm long triangular accuminate.Flowers minutes, proximal 2–3 axis with unisexual cymules, eachconsisting of 1 male and 1 female or 2–3 males and female or 1male and 2 females flower or combination thereof; male flowerspedicals at anthesis ca 1 mm long. Calyx lobe 5, subequal eachca 0.7 × 0.3 mm elliptic or oblong elliptic and abruptly acute atapex hyaline with unbranched mid ribs. Disc segments 5, roundishstames 3 (rarely 2): filaments connate into a column 0.2–0.3 mmhigh autheros sessile a top dehiscing longitudinally. Female flow-ers; pedicles 0.8–1 mm long, obtusely 4-gonous, dialated above,ca 1.5 mm in fruits, calyx five lobes, subequal. Lobes sometimestoothed at apex. Styles 3, free, more or less spreading, and shallowlybifid at apex; arms divergent (Mitra and Jain, 1985). The seed cap-sules on stalks are 1–2 mm long, round, smooth, 2 mm wide, withsix seeds. When the fruits burst open the seeds are hurled away.Seeds are triangular (like an orange segment); light brown, 1 mmlong, with 5–6 ribs on the back (Morton, 1981; Wessels Boer et al.,1976) (Fig. 1 P. amarus plant). The vernacular names of P. amarushas been given in Table 3.

4. Biogeography and ecology

The genus Phyllanthus (Euphorbiaceae) consists of about 6500species in 300 genera, of which 200 are American, 100 African,70 from Madagascar and the remaining Asian and Australasian(Webster, 1994). Numerous species of this family are native toNorth, Central and South America (Unander et al., 1995). The name‘Phyllanthus’ means “leaf and flower” because the flower, as wellas the fruit, seems to become one with the leaf (Cabieses, 1993).The taxonomic revision on this genus by Webster included closelyrelated genera P. amarus, under the sub-section Swartiziani of thesection Phyllanthus. The nomenclature, taxonomic distinctnessand close relatives of P. amarus were addressed in detail based onmorphology and geographical distribution (Chowdhury and Rao,2002). It is said to be related to P. abnormis, which is endemic tosandy areas in Texas and Florida of southern USA. It is thereforemost likely that P. amarus originated in the Caribbean area as avicarious species of P. abnormis of the southern United States andhas spread around the tropics by trading vessels (Webster, 1957). P.amarus is widely distributed in all tropical and subtropical regionsof the world. Paleobotanical studies have not found the exactgeographic origin of this plant. It is indigenous to the rainforestsof the Amazon and other tropical countries like India, China, and

Table 3Vernacular name of P. amarus worldwide (Medicinal Plants of India, 1987,http://knol.google.com/k/pankaj-oudhia/phyllanthus-amarus-l/3nerdtj3s9l79/20).

S.No. Language Vernacular names

1 Tamil Keelanelli (Keezhanelli)2 Hindi Bhuyiavla, Jangli amla3 Bengali Bhuiamala, Sadahazurmani4 Gujarathi Bhonya anmali5 Marathi Bhuivali6 Oriya Bhuiaola, badianala7 Bihari Muikoa, Kantara, Pirikantaru8 Telugu Nela uirika, Nelavusari9 Kanada Nela – nelli, Kirunelli

10 Malayalam Kizhkkayinelli, Kilanelli11 Rajasthani Gugario12 Sanskrit Bhumyaamlaki, Bhoodhatree, Thamalaki13 English Black catnip, Carry me seed, Child

pick-a-back, Gale of wind, Gulf leaf flower,Hurricane weed, Shatterstone, Stone breaker

14 French Poudre de plomb (Ivory coast)15 German Weisse Blattblume16 Spanish Yerba magica (Cuba)17 African names Ahlivi (Mina-Togo), Bomagua kene (Ivory

coast), Bounou (Ivory coast), Bounou honlin(Ivory coast), Hinlinew (West Africa),Mokichinento (Korokoro–East Africa),Tsekulemegbe (Ouatchi-Togo)

18 North, Central andSouth Americannames

Black catnip, carry-meseed, chanca piedra,djari-bita, egg woman, fini-bita, florescondida, gale-of-(the)-wind, hurricaneweed, quebra-pedra, quinine creole, quinineweed, seed-under-leaf, stone breaker andyerba de la nina (Morton, 1981)

Bahamas, (Morton, 1981; Tirimana, 1987), Philippines or India(Chevallier, 2000). P. amarus is a common pantropical weed thatgrows well in moist, shady and sunny places (Cabieses, 1993;Nanden, 1998). P. amarus is distributed all over India and is con-sidered as the most widely occurring Phyllanthus species in India(Chowdhury and Rao, 2002). The presence of dioceous cymules(Mitra and Jain, 1985) at the end of the branches is considered tobe a unique character, though it resembles in many respects itsclose relatives, P. debilis and P. fraternus of the same sub-sectionSwartziani (Webster, 1994). This is the only sub-section in the sec-tion Phyllanthus, which consists of most widespread herbaceousspecies throughout the tropics (Jain et al., 2003).

5. Pharmacognostic characters

Various species of Phyllanthus are being sold in India under thetrade name ‘Bhuiamlki’. During market surveillance of herbal drug,it was observed that almost all the commercial samples, eithercomprise of P. amarus Schum. & Thonn. or Phyllanthus maderas-patensis Linn. or mixture of P. amarus, P. fraternus Webster and P.maderaspatensis Linn. The species admixtures have been assessedin raw drug trade of Phyllanthus in southern India using morpho-taxonomical characters and molecular analysis. The morphologicalanalysis of these samples revealed six different species of Phyl-lanthus. Seventy-six percent of the market samples contained P.amarus as the predominant species (>95%) and thus were devoidof admixtures. The remaining 24% of the shops had five differentspecies namely P. debilis, P. fraternus, P. urinaria, P. maderaspatensis,and Phyllanthus kozhikodianus. Species specific DNA barcode signa-tures were developed for Phyllanthus species using the chloroplastDNA region, psbA-trnH. The trade sample identities were validatedand confirmed by these species specific DNA barcodes (Sriramaet al., 2010). The detailed botanical evaluation of three species wascarried out with the aim to establish the identification markers ofthis plant. Some reliable diagnostic characters were specified as,number of sepals five in P. amarus and six in P. fraternus and P.

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maderaspatensis; the female flowers of P. maderaspatensis are muchlarger in size as compared to P. amarus and P. fraternus, in whichthese are almost equal in size; TS of branchlet circular, leaf margincrenate in P. amarus and in P. fraternus branchlets have six notcheswith serrated leaf margins, while the leaf margins are crenate andbulbous in P. maderaspatensis. Preliminary phytochemical screen-ing of the extractives showed that the triterpenoids were noticed inthe hexane extract of P. amarus and P. fraternus and in chloroformextracts of P. maderaspatensis, however, glycosides in the alcoholicextract of P. amarus and P. fraternus only. Likewise, the percent-age of sugar and tannins were quite high in P. maderaspatensis andtannins were almost nil in P. fraternus. On the contrary, the com-parative thin layer chromatography (TLC) finger print profiles of P.amarus and P. fraternus were almost similar and showed eight com-mon bands at Rf 0.04, 0.28, 0.38, 0.44, 0.47, 0.55, 0.60, 0.63 of lightgreen, greyish green, grey, faint green, light green, faint pink, lightgreen, greyish green, respectively. However, in P. maderaspatensisonly three bands at Rf 0.04, 0.28 and 0.38 were observed while onemajor band of bright blue colour was observed at Rf 0.9 in P. frater-nus only. Therefore, all these three species could be differentiatedon the basis of macro and microscopic characters, physico-chemicalvalues, high pressure thin layer chromatography (HPTLC), finger-print profile, and detection of phyllanthin and hypophyllanthinas marker components (Khatoon et al., 2006). Macromorphology,micromorphology, histochemical and physical pharmacognosticstudies of P. amarus revealed certain diagnostic uncommon char-acters: basal submarginal venation formed by curving of almostunbranched lateral veins, 4–6 angled cortical fibres (TS), 1–2 seriatexylem rays, crystals concentrated along the veins (mostly rosette),combination of paracytic and anomocytic stomata, sinuous epider-mal cell walls, vessel members tailed on two ends; high frequencyof crystals in leaf (87.5 mm−2), stomatal index, palisade ratio, etc.Additionally, distribution of alkaloidal reaction and protein in thesecondary xylem, extractive values, ash values, UV fluorescencewere also distinctive characterstics (De and Datta, 1990).

6. Phytochemical studies

The secondary metabolites present in P. amarus are alkaloids,flavonoids, hydrolysable tannins (Ellagitannins), major lignans,polyphenols, triterpenes, sterols and volatile oil. The main activeconstituents of P. amarus are lignans (phyllanthin, hypophyllan-thin, nirurin niranthin, phyltetralin, niranthine, nirtetralin etc.(Morton, 1981; Chevallier, 2000; Srivastava et al., 2008; Kassuyaet al., 2006; Huang et al., 2003; Maciel et al., 2007; Singhet al., 2009), flavonoids (quercetin, quercetrin, rutin, gallocat-echin, phyllanthusiin, kaempferol etc.), (Foo and Wong, 1992;Foo, 1993a; Londhe et al., 2008; Morton, 1981), Ellagitannins viz.geraniin, amariin, furosin, geraniinic acid, amariinic acid, amaru-lone, repandusinic acid, corilagin, isocorilagin, elaeocarpusin,phyllanthin D gallic acid, repandusinic acid A etc. (Foo and Wong,1992; Foo, 1993a; Foo, 1995), triterpenes (phyllanthenol, phyllan-thenone, phytllantheol etc.) (Maciel et al., 2007; Foo and Wong,1992), alkaloids (securinine, dihydrosecurinine, tetrahydrose-curinine, securinol, phyllanthine, allosecurine, nor-securinine,epibubbialine, isobubbialine, 4-methoxy dihydrosecurinine, 4-methoxytetrahydrosecurinine, 4-hydrosecurinine etc.) (Houghtonet al., 1996; Kassuya et al., 2006; Foo and Wong, 1992), sterol(amarosterol-A, amarosterol-B etc.) (Ahmad and Alam, 2003) andvolatile oil (linalool, phytol etc.) (Moronkola et al., 2009). A detailedextraction, isolation and characterization method was optimizedfor phyllanthin (Hamrapurkar et al., 2009).

The oils obtained from P. amarus were analyzed for itsconstituents by means of gas chromatography (GC) and gas chro-matography coupled with mass spectrometry (GC/MS). The GC

analysis was carried out using an Agilent 6890N GC system usingflame ionization detector (FID) at the temperature 300 ◦C. Resultsfrom analysis of the oil of P. amarus revealed that 82 identifiedcompounds were responsible for 87.6% of the oil content. The oilwas characterized by the dominance of linalool (36.4%) and phytol(13.0%). Other significant compounds were hexahydrofarnesyl ace-tone (3.4%), pentacosane (2.5%), naphthalene (2.4%), (E)-b-ionone(2.3%), nonacosane (2.1%), tetracosane and octacosane (ca. 1.7%).Eight of the components were in trace amount (less than 0.1%),but they are likely important in the characteristics of the oil. Twocomponents with relative retention index (RRI) of 2692 and 2700were tentatively identified as nonyl phenol isomers. The classes ofcompounds present in P. amarus oil are monoterpene hydrocar-bons (0.2%), oxygenated monoterpenoids (11.0%), sesquiterpenehydrocarbons (1.3%), oxygenated sesquiterpenoids (3.3%), diter-penoids (8.5%), aliphatic alcohols (51.2%), fatty acids (3.9%),aldehydes (8.0%), ketones (0.5%) and esters (0.3%) (Moronkolaet al., 2009). Two new lignans, 3-(3,4-dimethoxy-benzyl)-4-(7-methoxy-benzo[1,3]dioxol-5-yl-methyl)-dihydrofuran-2-oneand 4-(3,4-dimethoxy-phenyl)-1-(7-methoxy-benzo[1,3]dioxol-5-yl)-2,3-bis-methoxymethyl-butan-1-ol were isolated fromthe leaves of P. amarus (Singh et al., 2009). A phytochemicalinvestigation of methanolic extract obtained from the wholeplant of P. amarus, revealed the presence of six bioactive lig-nans [isolintetralin (2,3-demethoxy-seco-isolintetralin diacetate),demethylenedioxy-niranthin, 5-demethoxy-niranthin, niranthin,phyllanthin and hypophyllanthin] and one triterpene 2Z, 6Z, 10Z,14E, 18E, 22E-farnesyl farnesol (Maciel et al., 2007). Phytochemicalevaluation of P. amarus showed to contain high level of saponinsand tannins at 24.05 and 17.50%, respectively, but with lowcontent of cyanogenic glycosides (1.46%). The plant containedhigh percentage level of fibre (24.50%) and carbohydrate (45.52%),with approximate content of fat (6.03%), protein (6.10%) and ash(6.80%). The potassium and sodium contents were high at 150.30and 228.20 mg per 100 g dry weight, respectively, while magne-sium, calcium, iron and phosphorus were all low at 2.40, 1.60, 1.65and 1.00 mg per 100 g dry weight, respectively (Igwe et al., 2007).The crude extract of phyllanthin was obtained from P. amarususing solvents of varied polarity. The presence of pyrrolizidinetype of alkaloids was reported in extract of P. amarus. These aresecurinine, dihydrosecurinine, tetrahydrosecurine, securinol-B,phyllanthine, allosecurine, nor-securinine etc. (Kassuya et al.,2006). The whole plant of P. amarus has afforded new secosterolsnamed as amarosterol-A characterized as 13, 14-seco-stigma5(6), 14(15)-diene-3-�-ol and amarosterol-B characterized as 13,14-seco-stigma 9(11), 14(15)-diene-3-�-ol (Ahmad and Alam,2003). Polyprenols comprising 11 and 12 isoprene units were thedominant ones in the majority of the plants investigated. Out ofthe 19 species studied the highest concentration of polyprenolswas observed in P. amarus (0.1–0.3%, dry weight) (George et al.,2001). Two new securinega types of alkaloids, isobubbialineand epibubbialine were isolated from the leaves of P. amarus, aswell as the three known alkaloids, phyllanthine, securinine andnor-securinine (Houghton et al., 1996). Chemical examination ofthe polar extractives of the aerial parts of P. amarus led to theisolation of amariinic acid, a novel ellagitannin, together with1-O-galloyl-2, 4-dehydrohexahydroxydiphenoyl-glucopyranose,elaeocarpusin, repandusinic acid A and geraniinic acid B. Thestructure of amariinic acid was established as the ring-openedoxidized cyclohexentrione moiety of dehydrohexahydroxydiphe-noyl attached to 0–4 of the glucose core (Foo, 1995). A novelcyclic metabolite named amarulone was isolated from P. amarus(Foo, 1993a). Amariin, a novel hydrolysable tannin together withgeraniin, corilagin, 1,6-digalloylglucopyranoside as well as rutinand quercetin-3-O-glucopyranoside were isolated from the polarfraction of P. amarus. The structure of amariin was established as

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Table 4Phytoconstituents reported in P. amarus.

S. No. Secondary metabolites Phyto-constituents References

1 Lignans Phyllanthin, hypophyllanthin, niranthin,phyltetralin, nirtetralin, isonirtetralin,hinokinin

Morton (1981), Sharma et al. (1993),Chevallier (2000), Srivastava et al.(2008), Kassuya et al. (2006), Huanget al. (2003), Singh et al. (2009)

Lintetralin, isolintetralin,demethylenedioxy-niranthin,5-demethoxy-niranthin

Maciel et al. (2007)

(3-(3,4-dimethoxy-benzyl)-4-(7-methoxy-benzo[1,3]dioxol-5-yl-methyl)-dihydrofuran-2-one,4-(3,4-dimethoxy-phenyl)-1-(7-methoxy-benzo[1,3]dioxol-5-yl)-2,3-bis-methoxymethyl-butan-1-ol

Singh et al. (2009)

2 Flavonoids Rutin, astragalin, kaempferol,quercetin-3-O-glucoside, quercetin, quercitrin

Morton (1981), Foo and Wong (1992),Foo (1993a, 1995), Londhe (2008)

3 Hydrolysable tannin(Ellagitannins)

Tannin precursors Gallic acid, ellagic acid, gallocatechin Foo (1993a, 1995)

Simple tannins 1,6-digalloylglucopyranose, 4-O-galloylquinicacid

Foo (1993a, 1995)

Complex tannins Geraniin, amariin, furosin, geraniinic acid B,amariinic acid, amarulone, repandusinic acid A,corilagin, isocorilagin, elaeocarpusin,phyllanthusiin A, B, C and D, melatonin

Foo and Wong (1992), Foo (1993a,1995)

4 Alkaloids Securinine, dihydrosecurinine,tetrahydrosecurinine, securinol, phyllanthine,allo-securine, nor-securinine, epibubbialine,isobubbialine

Houghton et al. (1996), Kassuya et al.(2006)

4-methoxy-nor-securinine, 4-methoxydihydrosecurinine,4-methoxytetrahydrosecurinine, 4hydrosecurinine

Foo and Wong (1992)

Phenazine and phenazine derivatives Foo (1993a)5 Triterpenes 2Z, 6Z, 10Z, 14E, 18E, 22E-farnesylfarnesol Maciel et al. (2007)

Lupeol, phyllanthenol, phyllanthenone,phyllantheol, Oleanolic acid, ursolic acid

Foo and Wong (1992)

6 Sterols Amarosterol A, amarosterol B Ahmad and Alam (2003)7 Volatile oil Linalool, phytol Moronkola et al. (2009)

1-galloyl-2,4: 3,6-bis-dehydrohexahydroxydiphenoyl-glucopyranoside in which the cyclohexenetrione portion ofthe dehydrohexahydroxydiphenoyl moieties were linked to theO-3 and O-4 of the glucose moiety (Foo, 1993b). An unusualellagitannin, phyllanthusiin D was isolated from the biologicallyactive polar fraction of P. amarus. Its structure was establishedas 1-galloyl-2, 4-(acetonyl-dehydrohexahydroxydiphenoyl)-3,6-hexahydroxydiphenoyl-glucopyranoside (Foo and Wong, 1992).Table 4 represents the secondary metabolites with their respectivephytochemicals present in P. amarus.

7. Analytical techniques

A HPLC analysis method was developed and validated to obtainan easily performable and inexpensive method for the standard-ization of crude extract of P. amarus and ellagic acid. Ethanolicextract of whole plant of P. amarus was dissolved in DMSO, ultra-sonicated for 15 min, and diluted with 50% methanol. Analysis wasperformed using water and methanol containing 0.06% TFA andthe peaks were detected at 254 nm. Ellagic acid showed a linearrelationship in the range of 1.74–20.91 �g/mL and a single-pointcalibration was allowed. The method was shown to be precise withrespect to time (RSD of 1.84%, 3 days, n = 6) and concentration (RSDof 2.54%, three levels, n = 6). The overall mean content of ellagic acidwas 2.06%. A recovery experiment was performed and it showedan accuracy of 100.4% (Dhooghe et al., 2011). A simple, specific andprecise RP-HPLC method has been developed and validated for theestimation of phyllanthin, present in P. amarus. Furthermore, thedeveloped method was also used to successfully quantify the phyl-lanthin in plant extract. The mobile phase optimized for RP-HPLC

was methanol–water 66:34 (% v/v) which was very simple and costeffective. The detection was carried out using variable wavelengthUV–vis detector set at 229 nm. Linearity for the developed methodwas found over the concentration range 1–50 �g/mL with a correla-tion coefficient of 0.999 (Alvari et al., 2011). An online-hyphenatedhigh-performance liquid chromatography-photodiode array-massspectrometry (HPLC-PDA-MS) analytical method was developedfor the simultaneous determination of six lignans of therapeuticimportance in four Phyllanthus spp. (P. amarus, P. maderaspatensis,P. urinaria, and Phyllanthus virgatus). HPLC with monolithic reversephase silica column (4.6 × 100 mm) and simple isocratic elution ofmethanol–water mixed with dioxane facilitated the separation oflignans of diverse nature such as diarylbutyrolactone, tetrahydro-furan, isomeric aryltetralin, and diarylbutane type for quantitativeanalysis. Targeted lignans viz. heliobuphthalmin lactone, virga-tusin, hypophyllanthin, phyllanthin, nirtetralin, and niranthin wereconfirmed unambiguously in four Phyllanthus species by theirabundant molecular adduct ions, retention time, UV, and massspectra as compared with those of reference compounds. Advan-tages and limitations of both detection techniques for qualitative(fingerprinting) and quantitative analysis of the above mentionedlignans in four Phyllanthus spp. are discussed. The method was vali-dated following international guidelines. The described method canbe utilized for assays and stability tests of P. amarus extracts as wellas traditional Indian medicine based on Phyllanthus herb (Shankeret al., 2011). Phyllanthin was extracted from the plant P. amarusby Soxhlet and supercritical-fluid extraction (SFE) and isolated bycolumn chromatography. A HPTLC method was established andvalidated for analysis. The method was used for quantitative anal-ysis and macro and micro fingerprinting analysis of phyllanthin.

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Fig. 2. Structures of Lignans isolated form P. amarus.

This study also revealed that SFE enabled more efficient isola-tion of phyllanthin than Soxhlet extraction (Hamrapurkar et al.,2010; Annamalai and Laxmi, 2009). Phyllanthin was isolated fromthe aerial parts of P. amarus by silica gel column chromatographyemploying gradient elution with hexane–ethyl acetate solvent mix-ture. It was obtained in high yields (1.23%), compared to reportedprocedures and the purity was ascertained by HPTLC and reversedphase HPLC analysis (Krithika et al., 2009). A sensitive, selective,and robust HPTLC method using chiral TLC plates for qualitativeand quantitative analysis of phyllanthin, hypophyllanthin, niran-thin, and nirtetralin, the active lignans of Phyllanthus species, wasdeveloped and validated. The effectiveness and role of various sta-tionary phases viz TLC silica gel 60F254, HPTLC silica gel 60F254, andchiral TLC plates in the quantitation were evaluated. A precoatedchiral TLC plate was found suitable for the simultaneous analysis offour pharmacologically active lignans. For achieving good separa-tion, the optimized mobile phase of n-hexane-acetone-1, 4-dioxane(9:1:0.5 by volume) was used. A densitometric determination of theabove compounds was carried out in reflection absorption mode at620 nm. Optimized chromatographic conditions provided well sep-arated compact bands for the tested lignans. The calibration curveswere found linear in the concentration range of 100–500 ng/band(Srivastava et al., 2008). Sharma et al. (1993) have developed areversed phase HPLC procedure for standardizing P. amarus on thebasis of its two bioactive lignans, phyllanthin and hypophyllanthin.The method has been found to be sensitive, precise and efficient torecord more than 98% recovery of these two lignans. The leavesof P. amarus were found to contain the highest amount of phyl-lanthin (0.7% w/w) and hypophyllanthin (0.3% w/w) as comparedto other parts of the plant. A method for amount determinationof gallic acid in P. amarus capsules was established by HPLC (Guoet al., 2007). Methanolic extract of P. amarus and standard phyllan-thin and hypophyllanthin were developed in the chromatographicconditions. They showed the presence of standard phyllanthin and

hypophyllanthin at Rf 0.3 and 0.4 respectively, by densitometricscanning at 254 nm (Mukherjee et al., 2006). Separation of phyl-lanthin and hypophyllanthin was carried out on silica gel 60F254layers eluted with hexane:acetone:ethyl acetate (74:12:8), and theanalytes were visualised through colour development with vanillinin concentrated sulfuric acid and ethanol. Scanning and quantifica-tion of spots was performed at 580 nm. Recoveries of phyllanthinand hypophyllanthin were 98.7 and 97.3%, respectively (Tripathi etal., 2006). Dhalwal et al. (2006) developed a HPTLC densitometricmethod for simultaneous quantitation of phyllanthin, hypophyl-lanthin, gallic acid, and ellagic acid in the whole plant of P. amarus.They were found at levels of 0.37, 1.16, 0.36, and 0.17% (w/w),respectively. For estimation of phyllanthin and hypophyllanthin inP. amarus, an isocratic, reversed phase (RP) HPLC procedure hasbeen adopted using a mixture of pH 2.8 phosphate buffer and ace-tonitrile as mobile phase, Cyano (CN) column as stationary phaseand UV detector. The developed method showed high resolution(R = 1.9), accuracy and reproducibility (Murali et al., 2001). Theinvention concerns an improved procedure for the extraction andisolation of phyllanthin from P. amarus by pulverization of the driedleaves with calcium carbonate, percolating the ground componentusing a mixture of organic solvents at room temperature to 80 ◦C,removing the solvent by distillation, removing the grease by precip-itation using organic solvents and filtration, vacuum sepration of ann-hexane fraction, and subjecting the phyllanthin-rich fraction tosilica gel column chromatography, and further purifying the phyl-lanthin by crystallization (Chaudhuri et al., 2001). A HPTLC methodwas described for the simultaneous determination of the bioac-tive lignans, phyllanthin and hypophyllanthin from the dried wholeplant powder of P. amarus (Sane et al., 1997). A TLC – densitometricmethod has been developed for the estimation of the two lignansof P. amarus phyllanthin and hypophyllanthin (Deb and Mandal,1996). Air-dried leaves of P. amarus were extracted with 0.5 M HCland filtered. The filtrate was basified with 10% aqueous Na2CO3

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Fig. 3. Structures of flavonoids isolated from P. amarus.

and extracted with CHCl3 to yield an oily residue (165 mg). Exam-ination by TLC showed the presence of five Dragendorff-positivezones. The residue was then subjected to preparative TLC on sil-ica gel to yield five compounds (PA1–PA5). PAl (0.026% yield),PA2 (0.021% yield) and PA3 (0.018% yield w/w) were character-ized as phyllanthine, securinine and nor-securinine, respectivelyby comparison of their spectral data with standard values. Twonew securinega-type alkaloids, isobubbialine and epibubbialinewere isolated from the leaves of P. amarus, as well as the threeknown alkaloids, phyllanthine, securinine and nor-securinine. Thestructures of the unknown compounds were determined by meansof UV, IR, mass and NMR spectroscopy (Houghton et al., 1996).The 70% aqueous acetone extract of the aerial part of P. amaruswas fractionated over a column of Sephadex LH20 using aqueousmethanol to yield various fractions subjected to repeate chro-matographic treatment alternating between MCI-gel CHP-20 usingaqueous methanol and Sephadex LH20 with aqueous ethanol ledto the isolation of amariinic acid, a novel ellagitannin, togetherwith 4-O-galloylquinic acid, elaeocarpusin, furosin, geraniinic acidB, amariinic acid and potassium salt of repandusinic acid B and theirstructures were established on the basis of chemical and 13C NMRspectrum (Foo, 1995). An unusual ellagitannin was isolated fromthe biologically active polar fraction of aerial part of P. amarus. Thehydrolysable tannin fraction was obtained by column chromatog-raphy of the water soluble portion of the 70% aqueous acetoneextract of the aerial parts of the plant on Sephadex LH20 using aque-ous methanol. Further chromatography of the fraction on MCI-gelCHP-20 yielded phyllanthusiin D as an amorphous powder whichgave a [M-H] ion peak at m/z 991 with fast atom bombardment(FAB) mass spectrometry (Foo and Wong, 1992).

Chemical constituents isolated and characterized so far from P.amarus and their structures are given in Figs. 2–8.

8. Pharmacological activity

8.1. Antiamnesic activity

The effect of aqueous extract of leaves and stems of P. amaruswas evaluated on cognitive functions and brain cholinesteraseactivity in male Swiss albino mice. P. amarus (50, 100 and200 mg/kg) produced a dose-dependent improvement in memoryscores of young and older mice. P. amarus also reversed successfullythe amnesia induced by scopolamine (0.4 mg/kg, i.p.) and diazepam(1 mg/kg, i.p.). Interestingly, brain cholinesterase activity was alsoreduced. Piracetam 400 mg/kg, i.p. was used as positive control(Joshi and Parle, 2007). Nootropic activity of [6]-gingerol and phyl-lanthin was studied in mice using elevated plus maze and passiveavoidance paradigm. [6]-gingerol (25 and 50 mg/kg, p.o.) and phyl-lanthin (7.5 and 15 mg/kg, p.o.) significantly attenuated amnesticdeficits induced by diazepam, scopolamine (0.4 mg/kg, i.p.) andnatural aging. [6]-gingerol and phyllanthin increased step downlatencies significantly in the aged mice, diazepam and scopolamineinduced amnesic mice as compared with piracetam (200 mg/kg,i.p.). [6]-gingerol and phyllanthin significantly decreased wholebrain acetyl cholinesterase activity (Joshi and Parle, 2006).

8.2. Antibacterial activity

Hexane, methanol and water extracts of aerial parts of P.amarus were screened for antimicrobial activities against Bacil-lus subtilis, Escherichia coli, Pseudomonas aeruginosa, Salmonellatyphi, Staphylococcus aureus and Candida albicans using the agar-cup diffusion protocol. The aqueous and methanolic extracts ofP. amarus were active against all the test microorganisms. Themethanolic extract of P. amarus also showed a broad spectrum ofactivity with a minimum inhibitory concentration of 1.56 mg/mLagainst all the test microorganisms. The extracts were also screenedfor secondary metabolites and the result indicated the presenceof alkaloids, saponins, tannins and terpenoids (Alli et al., 2011).The 80% methanolic extracts obtained from seven Phyllanthus sp.were evaluated for antibacterial activity using the broth micro-dilution assay. Best antibacterial activity was obtained by P. amarusagainst S. aureus (gram-positive) with a MIC value of 17.7 �g/mL(Eldeen et al., 2011). The antibacterial activity of ethanolic extractsof the root and leaf of P. amarus was assessed against extendspectrum �-lactamase (ESBL) producing E. coli isolated from thestool samples of HIV sero-positive patients with or without diar-rhoea using Bauer disc diffusion method. The strains isolatedfrom both HIV sero-positive patients were susceptible to vari-ous concentrations of the extracts (5, 10, 20, 40 and 80 mg/mL).The mean zones of inhibition of the root extracts ranged from8.0 ± 0.33 to 25.0 ± 1.50 mm against ESBL E. coli while the meanzones of inhibition the of the leaf extracts ranged from 8.0 ± 0.50to 26.0 ± 1.00 mm against ESBL E. coli. The root extracts showedthe highest zone of inhibition (25 ± 1.50 mm) against ESBL E. coliat 80 mg/mL while the leaf extracts showed the highest zone ofinhibition highest (26 ± 1.00 mm) against ESBL E. coli at 80 mg/mL.The minimum inhibitory concentration (MIC) and minimum bac-terial concentration (MBC) of the plant extracts ranging from5–20 to 5–30 mg/mL respectively (Akinjogunla et al., 2010). P.amarus was examined against ocular infections causing bacteria P.aeruginosa, Micrococcus lylae, Bacillus licheniformis, Staphylococcushominis, S. aureus, Staphylococcus haemolyticus, Micrococcus luteus,Bacillus lentus, Bacillus firmus and Pseudomonas stutzeri using agar-well diffusion method. Results revealed that P. amarus exhibitedremarkable bioactivity against M. lylae, S. haemolyticus, B. lentus,B. firmus, P. stutzeri, P. aeruginosa and S. aureus (Koday et al., 2009).Antimicrobial properties aqueous and methanolic extracts of leavesof P. amarus at the concentration of 100 mg/mL were tested againstE. coli, Streptococcus spp, Klebsiella spp, Pseudomonas spp and Staphy-lococcus spp. aqueous and methanolic extract of P. amarus. Thecrude extracts were observed to inhibit the growth of E. coli, Strep-tococcus and S. aureus. Methanolic extract of the plant was moreeffective (6–11 mm) than aqueous extracts (5–10 mm) inhibitingthe growth of pathogenic bacteria but was less potent when com-pared to that of ofloxacin (19 mm) and ciprofloxacin (21 mm) usedas positive controls (Okoli et al., 2009). 80% methanolic extract ofwhole plant of P. amarus showed the least MIC on the tested bac-teria viz. B. stearothermophilus, S. aureus, B. subtilis, M. leuteus, S.typhi, Enterobacter aerogens, Proteus mirabilis, and Proteus vulgaris.The MIC and MBC were 30 and 40 �g/mL respectively. Ampicillinwas used as standard (Komuraiah et al., 2009). The essential oil andits fractions obtained from fresh leaves and seeds of P. amarus weretested on 12 microorganisms including yeast, gram-positive andgram-negative bacteria viz. B. subtilis, Citrobacter sp., E. coli (isolate),E. coli (ATCC 25922), Enterococcus faecalis, Klebsiella pneumoniae,P. aeruginosa, P. mirabilis, Staphylococcus albus, S. aureus (ATCC25923), S. aureus (isolate) and C. albicans. All the samples of essen-tial oil and fractions demonstrated activity (11–20 mm diameterzone of inhibition) against the microorganisms except P. aeruginosa.0.05% ciprofloxacin (≥21 mm diameter zone of inhibition) was usedas positive control (Ogunlesi et al., 2009). Crude aqueous (hot and

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Fig. 4. Strutures of hydrolysable tannins (Ellagitannins) isolated form P. amarus.

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Fig. 4. Continued.

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Fig. 5. Structures of alkaloids isolated from P. amarus.

cold water) and ethanolic extracts of leaves of P. amarus was inves-tigated for antimicrobial activity against S. typhi. Ethanolic extractsof P. amarus revealed the strongest activity against S. typhi with8.0 mm zone of growth inbibition followed by hot water (4.7 mm)and cold water (3.8 mm). Ciprofloxacin had the highest zone of inhi-bition (9.0 mm) against S. typhi followed by ofloxacin (6.0 mm) andamoxicillin (4.0 mm) (Oluwafemi and Debiri, 2008). Antimicrobialeffect of the plant extracts of P. amarus showed that the organicand aqueous extracts of P. amarus were inhibitory to Streptococ-cus faecalis, while the extracts were not inhibitory to C. albicans.Agar-well determined minimum inhibitory concentration (MIC)values ranged between 3.125 and 6.25 mg/mL while the disc diffu-sion determined MIC values ranged between 6.25 and 25.0 mg/mL.The agar-well determined MIC values for the ethanolic P. amarusextracts (3.12 mg/mL) were lower than the corresponding disc dif-fusion MIC determined values (6.25–25.00 mg/mL). Bacteriocidaland bacteriostatic effect varied with, solvent type of extract, con-centration and method of the test adopted. The active componentsof the plant have no antifungal effect on the tested yeast, C. albi-cans (Okigbo and Igwe, 2007). The antimicrobial potential of the

methanolic extract of whole plant of P. amarus at the concentrationof 5, 10, 25, 50, 100, 200, 400, 800, and 1000 mg/mL was studiedagainst drug resistant pathogenic bacterial strains, Shigella dysen-teriae 1, S. dysenteriae 2, S. boydii 8, S. aureus ML267, S. aureusNCTC 7447, Streptococcus pneumoniae 7465, E. coli ROW 7/12, E. coliCD/99/1, B. subtilis CD/99/1, Salmonella typhimurium ATCC 6539,Vibrio cholerae 8531, P. aeruginosa 7241, and K. pneumoniae RM 8/98by disc diffusion and agar dilution method. Shigella spp. were foundto be inhibited at the least concentration (25 mg/mL) and found toshow the maximum diameter of zone of inhibition at the largesttested concentration of 1000 mg/mL comparable with sparfloxacin.Methanolic extract posses significant antimicrobial activity againstall the tested strains; maximum inhibitory effect was noted againstShigella spp., E. coli, V. cholerae, and S. aureus. The extract was foundto be moderately active against S. typhimurium, P. aeruginosa, B.subtilis, and other pneumonia causing strains of Klebsiella and Strep-tococcus spp. The antibacterial action was mainly due to the isolatedphyllanthin (Mazumder et al., 2006). The ethanolic extracts of ninePeruvian medicinal plants including P. amarus were screened forantimicrobial activity against Bacillus cereus ATCC 11,778, B. subtilis

Fig. 6. Structures of triterpenes isolated from P. amarus.

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Fig. 7. Structures of steroids isolated from P. amarus.

Fig. 8. Structures of volatile oils isolated form P. amarus.

ATCC 6633, Bacteroides fragilis ATCC 25,285, E. faecalis ATCC 29,212,E. coli ATCC 25,922, P. aeruginosa ATCC 27,853, S. aureus ATCC25,923, Staphylococcus epidermidis ATCC 12,228, and Streptococ-cus pyogenes ATCC 19,615. Among the plants tested, 80% ethanolicextract of aerial parts of P. amarus showed the most promisingantibacterial properties, inhibiting all of the strains tested withminimum inhibitory concentrations (MICs) ranging from 0.25 to16 mg/mL (Kloucek et al., 2005).

8.3. Anticancer activity

Cytotoxicity of the crude extracts (aqueous and methanolic)and their two fractions of P. amarus, were screened using theMTS (3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) reduction assay. It was shownto inhibit MCF-7 (breast carcinoma) and A549 (lung carcinoma)cells growth with IC50 values ranging from 56 to 126 �g/mL and150–240 �g/mL for methanolic and aqueous extracts respectively.In comparison, they have lower toxicity on normal cells with cellviability of 50% when treated up to 1000 �g/mL for both aqueousand methanolic extracts. After determining the non-toxic effectivedose, several antimetastasis assays were carried out and P. amarusextracts were shown to effectively reduce invasion, migration, andadhesion of both MCF-7 and A549 cells in a dose-dependent man-ner. This was followed by an evaluation of the possible modesof cell death that occurred along with the antimetastatic activ-ity. P. amarus was shown to be capable of inducing apoptosis inconjunction with its antimetastastic action, with more than 3-foldincrease of caspases-3 and -7, the presence of DNA-fragmentationand terminal deoxynucleotidyl transferase mediated dUTP nick endlabeling assay (TUNEL)-positive cells. The ability of P. amarus toexert antimetastatic activities is mostly associated to the presenceof polyphenol compounds in its extracts (Lee et al., 2011). Themethanolic extract of P. amarus hairy roots revealed potent antipro-liferative activity in the MCF-7 cells through induction of apoptosismediated by increased intracellular reactive oxygen species (ROS)in conjunction with decreased mitochondrial membrane poten-tial (Abhyankar et al., 2010). The effects of aqueous extract ofwhole plant of P. amarus against Cr(VI)-induced oxidative toxicityin vitro in MDA-MB-435S human breast carcinoma cells revealeda distinct decline in Cr(VI)-induced cytotoxicity was noticed inMDA-MB-435S cells with an increase in extract dosage. Its phenolicconstituents simultaneously may inhibit Cr(VI)-induced oxidativetoxicity to MDA-MB-435S cells (Guha et al., 2010). In vitro, apop-totic effect of 75% methanolic extract of aerial parts (stem andleaves) of P. amarus against Dalton’s Lymphoma Ascites (DLA)cells in culture produced significant reduction in cell viability

as determined by the MTT assay. Treatment of cell lines withP. amarus produced cytotoxicity after 24 h. The maximum effectwas observed in DLA cell lines where treatment with P. amarusat a concentration of 500 �g/mL produced cytotoxicity IC50 valuewas 102 ± 1.38 �g/mL. It also induced the formation of apoptoticbodies with characteristic features like plasma membrane invagi-nation, elongation, fragmentation, and chromatin condensation.P. amarus at concentrations of 100 and 200 �g/mL has shown toinduce DNA-fragmentation (Harikumar et al., 2009). Oral adminis-tration of 75% methanolic extract of aerial parts (stem and leaves)of P. amarus was found to enhance the life span of leukaemiaharboring animals and decrease the incidence of anemia. Treat-ment with P. amarus (750 and 250 mg/kg) induced the expressionof p53 and p45NFE2 and decreased the expression of Bcl-2 inthe spleen of infected mice. Histopathological evaluations of thespleen demonstrated that administration of P. amarus decreasedthe infiltration of leukemic cells into the sinusoidal space whencompared with the vehicle treated group (Harikumar and Kuttan,2009). A mixture of phyllanthin and hypophyllanthin (1:1), iso-lated from P. amarus exhibited antitumor activities against EAC inSwiss albino mice. The decrement of tumor volume and packedcell volume and viable cell count were observed in lignan treatedmice when compared only to EAC tumor bearing mice. Treat-ment with test compounds increased the survival time and normalperitoneal cell count. Hematological parameters, PCV which werealtered by tumor volume inoculation, were restored consider-ably (Islam et al., 2008a). An alcoholic extract of aerial parts ofP. amarus was found to inhibit cytochrome P450 enzymes bothin vivo as well as in vitro. It was studied using specific resorufinderivatives, as substrate for isoenzymes in the P450 super family.Concentration needed for 50% inhibition of 7-ethoxyresorufin-O-deethylase (EROD), CYP1A1 was 4.6 �/mL while concentrationneeded for 7-methoxyresorufin-O-demethylase (MROD) CYP1A2was 7.725 �/mL and 7-pentoxyresorufin-O-depentylase (PROD),CYP2B1/2 was found to be 4.18 �/mL indicating that the extractinhibited the P450 enzymes at very low concentration. Extract alsoinhibited the activity of aniline hydroxylase (an indicator of CYP2E1 activity, IC50 50 �/mL) and aminopyrine demethylase (an indi-cator of CYP 1A, 2A 2B, 2D and 3A activity, IC50 > 1000 �g/mL).Oral administration of the extract was also found to reduce theelevated P450 enzyme activities produced by phenobarbitone by50% at 250 mg/kg body weight. Extracts of P. amarus has pre-vented or stopped the cells from mutation with the existence ofchemical agents (Hari Kumar and Kuttan, 2006). N-methyl N′-nitro-N-nitrosoguanidine (MNNG) induced stomach cancer in maleWistar rats was significantly inhibited by the administration of75% methanolic extract of aerial parts of P. amarus at a dose of150 and 750 mg/kg body weight. It also reduced the incidence ofgastric neoplasms in rats (44%) as well as their numbers. The ele-vated enzymes level in the stomach was also found to reduce byP. amarus treatment (Raphael et al., 2006). 70% ethanolic extractsof three Phyllanthus species, P. urinaria, P. amarus and P. debilisat the concentration of 10 �g/mL significantly inhibited the pro-liferation of the HepG2 cells. The extracts induced apoptosis byinducing caspase-3. Further confirmation of extract-induced apo-ptosis was obtained by demonstrating that the Bcl-2/Bax ratiodecreased in response to treatment with the extracts detected themechanism by which the Phyllanthus extracts induced apopto-sis. These findings demonstrated that Phyllanthus extracts induceTNF-� production from the hepatocellular carcinoma cells whileinhibiting production of potent anti-apoptotic genes IL-8 and COX-2. Untreated cells (control) received only 0.01% DSMO (Surebanet al., 2006). Two human leukaemia cell lines were employed:K-562 and its vincristine-resistant counterpart Lucena-1, and Pgp-overexpressing subline to evaluate the possible cytotoxic effect andmultidrug resistance (MDR) reversing properties of the extract and

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compounds isolated from P. amarus. It was reported that Lucena-1 was significantly more resistant to the cytotoxicity of P. amarusderivatives: the hexane extract (100 �g/mL), the lignans-rich frac-tion (LRF, 100 �g/mL) and the lignans nirtetralin (43.2 �g/mL),niranthin (43 �g/mL) or phyllanthin (43 �g/mL) exerted cytotoxiceffects on K-562 cells with 40.3, 66.0, 62.0, 61.0 or 24.1% of celldeath, respectively. The cellular toxicity observed on Lucena-1 was16.3, 40.4, 29.4, 30.2, or 24.8%, respectively. These results suggesteda potential action of P. amarus derivatives as MDR reversing agents,mainly due to their ability to synergize with the action of conven-tional chemotherapeutics (Leite et al., 2006). Administration of 75%ethanolic extract of P. amarus at doses 250 and 750 mg/kg bodyweight i.p. in mice for 14 days significantly reduced the myelosup-pression and improved the WBC count, bone marrow cellularityas well as the number of maturing monocytes. P. amarus admin-istration was found to decrease the activity of phase I enzyme.Administration of P. amarus also increased the cellular glutathione(GSH) and glutathione-S-transferase (GST), thereby decreasing theeffect of toxic metabolites of cyclophosphamide (CTX) on the cells.Administration of P. amarus did not reduce the tumor reducingactivity of CTX. In fact, there was a synergistic action of CTX and P.amarus in reducing the solid tumors in mice. It was indicated thatadministration of P. amarus can significantly reduce the toxic sideeffects of CTX and is not interfering with the antitumor efficiencyof CTX. When the aqueous extract of P. amarus was administeredto cancer bearing mice, it lowered the tumor incidents, level ofcarcinogen-metabolizing enzymes levels of liver cancer markersdose dependently (Kumar and Kuttan, 2005). The 75% methanolicextract of aerial parts of P. amarus has been administered orally (750and 250 mg/kg body weight) in the radiation (6 Gy) induced balb/cmice for its protective activity against carcinogenesis. The WBCcount, bone marrow cellularity and �-esterase activity increasedsignificantly as compared to only radiation exposed mice. Theantioxidant enzymes such as superoxided dismutase (SOD), cata-lase (CAT), (glutathione-S-transferase) GST, glutathione peroxidase(GPX), and glutathione reductase, both in blood and tissue, whichwas reduced by radiation induced. P. amarus possesses the abilityto inhibit the unusual enzymatic pathways peculiar to cancer cellsproliferation and growth rather than a direct toxic effect of killingthe different types of cancer cells (Kumar and Kuttan, 2004). Theaqueous extract of P. amarus 150 and 750 mg/kg body weight thriceweekly for 8 weeks treatment exhibited potent anticarcinogenicactivity against 20-methylcholanthrene (20-MC) induced sarcomadevelopment and increased the survival of tumor harboring balb/cmice. The extract administration (p.o.) was also found to prolongthe life span of DLA and EAC bearing mice and reduced the vol-ume of transplanted solid tumors (Rajeshkumar et al., 2002). Theaqueous extract of the entire plant of P. amarus, at the concentra-tion of 10, 25, 50 and 100 �g/plate showed an antimutagenic effectagainst induction by 2-aminofluorene (AF2), 2-aminoanthracene(2AA) and 4-nitroquinolone-1-oxide (4-NQO) in S. typhimuriumstrains TA98 and TA100, and in E. coli WP2 uvrA/pKM101. Theinhibition of N-ethyl-N-nitrosoguanidine (ENNG)-induced muta-genesis was observed only with S. typhimurium TA100. The extractalso exhibited activity against 2-nitrofluorene (2NF) and sodiumazide-induced mutagenesis with S. typhimurium TA98 and TA100,respectively. Based on the alkaline elution method, the plant extractprevented in vivo DNA single-strand breaks caused by dimethyl-nitrosamine (DMN) in hamster liver cells. When the extract wasadministered 30 min prior to the administration of DMN, the elu-tion rate constant decreased more than 2.5 times, compared to thecontrol (Sripanidkulchai et al., 2002). Methanolic extract of stemsand leaves of P. amarus was tested for its antimutagenic activityin S. typhimurium strains TA1535, TA100, and TA102 (Ames test).P. amarus extract 500 mg/kg body weight for 12 days was ableto inhibit the activation and mutagenicity of 2-acetaminofluorene

(2-AAF) and aflatoxin B1 at concentrations of 0.25–2 mg/plate.It was also found to inhibit mutagenicity induced by directacting mutagens sodium azide (NaN3), MNNG, and 4-nitro-0-phenylenediamine (NPD), at concentrations of 1–0.25 mg/plate.Urinary mutagenicity produced in rats by benzo[a] pyrene wasfound to be significantly inhibited by the oral administration ofP. amarus extract (Raphael et al., 2002a). Administration of aque-ous extract of aerial parts of P. amarus increases the life spanof rats with hepatocellular carcinoma. The effect of P. amarusextract administration after induction of hepatocellular carcinoma(HCC) by N-nitrosodiethylamine (NDEA) was studied in Wistar rats.Administration of an aqueous extract of P. amarus was found tosignificantly increase the survival of hepatocellular carcinoma har-boring animals. All the untreated rats died of tumor burden by33,791.6 weeks. Administration of P. amarus extract (150 mg/kgbody weight orally, 5 days weekly continuously for 54 weeksor till they died) after tumor development, increased the sur-vival of animals to an average of 52,292.3 weeks (Rajeshkumarand Kuttan, 2000). The P. amarus aqueous extract at the dose of150 and 75 mg/kg body weight on male Wistar rats, 5 days priorto NDEA treatment for 20 weeks signifcantly inhibit hepatocar-cinogenesis induced by NDEA in a dose-dependent manner. Theanticarcinogenic activity of the extract was evaluated by its effecton tumor incidence, level of carcinogen-metabolizing enzymes,level of liver cancer markers and liver injury markers. Animalstreated with NDEA alone showed 100% tumor incidence and sig-nifcantly elevated tissue level of drug metabolizing enzymes suchas GST and aniline hydroxylase (AH). Treatment of extract signif-icantly reduced these levels. Level of �-glutamyl transpeptidase(GGT) were also found to be elevated both in serum and tissuesof tumor bearing animals, while they were significantly reducedin the treated group. Similar reduction was seen in tissue levelof reduced glutathione. Serum level of lipid peroxide (LPO), alka-line phosphatase (ALP) and glutamate pyruvate transaminase (GPT)were also elevated. Morphology of liver tissue and level of markerenzymes indicated that these extracts offered protection againstchemical carcinogen (Jeena et al., 1999). Aqueous extract of P.amarus was reported a potent inhibitor of the hepatocarcinogene-sis induced in rats by NDEA. None of the P. amarus extract treatedanimals developed any tumor even 32 weeks after NDEA admin-istration, whereas all of the control animals died due to tumorburden. Liver weight was increased in the NDEA-treated group,whereas it was not altered after treatment with NDEA and P.amarus. The liver markers g-glutamyl transpeptidase, glutamyl-S-transferase, reduced glutathione and the aniline-4-hydroxylasecytochrome P450 enzyme were elevated in NDEA-treated animals,whereas they were almost normal in animals treated with the car-cinogen plus P. amarus extract. Animals were elevated by NDEAtreatment, were also normal in the NDEA and P. amarus treatedgroup (Joy and Kuttan, 1998). Phyllanthus extract reduced the cyto-toxic action of lead nitrate and aluminum sulfate in bone marrowcells of mice. Nickel clastogenicity was also found to be reducedin mice fed with Phyllanthus extract and ascorbic acid. These pos-itive reports on the antigenotoxic efficacy of Phyllanthus extractwas confirmed (Dhir et al., 1990). Lignans Phyllanthin, nirtetralin,and niranthin were found to be very effective against cancer.

8.4. Anti-diarrhoeal, gastroprotective and antiulcer activity

Oral administration of absolute ethanol (1 mL/200 g bodyweight) induced multiple, elongated, reddish bands of hemor-rhagic erosions in rat gastric mucosa was studied. The ethanolcontrol group had the highest ulcer index of 45.20 ± 2.39. Pre-treatment with P. amarus leaves aqueous extract (500 mg/kg) andcimetidine (100 mg/kg) significantly inhibited (p < 0.001) ulcera-tion by 59.3 and 41.2% respectively. The acetone extract yielded a

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dose-dependent percentage ulcer inhibition and the lower dosageof the aqueous extract had a higher ulcer inhibition (Shokunbi andOdetola, 2008). Methanolic extract of leaves and stems of P. amarusat the dose of 50, 200, and 1000 mg/kg body weight p.o., on adultmale Wistar rats significantly inhibited gastric lesions, inducedby intragastric administration of absolute ethanol (8 mL/kg bodyweight). Mortality, increased stomach weight, ulcer index, andintraluminal bleeding were reduced significantly by P. amarus.Biochemical analysis indicated that reduced GSH of gastric mucosaproduced by ethanol administration was significantly elevated bytreatment with P. amarus (Raphael and Khuttan, 2003). Gradeddoses of the aqueous extract of P. amarus (100–800 mg/kg) admin-istered orally produced a dose related inhibition of gut meal traveldistance in normal mice. The highest intestinal transit inhibitionof 31.65% was obtained with 400 mg/kg. In castor oil induceddiarrhoea in mice, P. amarus extract (400 mg/kg) delayed the onsetof diarrhoea, reduced frequency of defecation and reduced gutmeal travel distance significantly resulting in intestinal transitinhibition of 79.94% compared to 86.92% produced by morphine(100 mg/kg). In addition, the activities of some intestinal mucosaenzymes (maltase, sucrase, lactase and alkaline phosphatase)in mice pretreated with extract before castor oil were not asseverely depressed as those in castor oil treated mice (control)(Odetola and Akojenu, 2000).

8.5. Antifungal activity

The effect of nor-securinine, an alkaloid isolated from P. amaruswas studied against spore germination of some fungi (Alternariabrassicae, Alternaria solani, Curvularia pennisetti, Curvularia sp.,Erysiphe pisi, Helminthosporium frumentacei) as well as pea pow-dery mildew (E. pisi) under glasshouse conditions. The sensitivityof fungi to nor-securinine varied considerably. Nor-securinine waseffective against most of the fungi. H. frumentacei was more sen-sitive even at the lowest concentration (1000 �g/mL). Conidia ofE. pisi were also inhibited in partially or completely appresso-rium formation. Pre-inoculation treatment showed greater efficacythan post-inoculation in inhibiting powdery mildew developmenton pea plants in a glasshouse. Maximum inhibition occurred at2000 �g/mL (Sahni et al., 2005). Antifungal bioassay in terms ofreduction in weight, colony diameter and sporulation of the targetfungal colony was carried out using Broth Dilution method. Rootpart of the P. amarus, extracted in various organic solvents havenot shown any noticeable antifungal activity. The percentage inhi-bition observed in different solvent extracts of aerial part was foundas reduction in weight. It was concluded that Chloroform fractionof the aerial part of the plant P. amarus have showed significantinhibitory effect against dermatophytic fungi M. gypseum (Agrawalet al., 2004).

8.6. Analgesic, anti-inflammatory, anti-allodynic andanti-oedematogenic activity

The aqueous leaves extract of P. amarus was investigated foranalgesic and anti-inflammatory activities using both thermal andchemical models of pain assessment in rats. The extract causeda significant (p < 0.05) dose related increased inhibition of thecarrageenan-induced paw oedema in the rats. The inhibition pro-duced by 200 mg/kg aqueous extract of P. amarus (70.20%) wassignificantly higher than that of the reference drug (acetylsali-cylic acid). The extract produced a marked analgesic activity byinhibiting both early and late phases of pain stimulus in formalin-induced paw licking rats and also a significant and dose relatedincrease in inhibition of the mean tail immersion duration at vary-ing water bath temperature (50, 55 and 60 ◦C) (Iranloye et al.,2011). The effects of 75% methanolic extract of whole plant of P.

amarus on different phases of inflammation were performed usingdifferent phlogistic agents-induced paw oedema, carrageenan-induced air-pouch inflammation and cotton pellet granuloma inmale Wistar rats. Methanolic extract at a dose of 250 mg/kg bodyweight p.o. significantly inhibited carrageenan, bradykinin, sero-tonin and prostaglandin E1-induced paw oedema, but failed toinhibit the histamine-induced paw oedema (Mahat and Patil, 2007).The local administration of nirtetralin, phyltetralin or niranthin(30 nmol/paw), similar to WEB2170 (a PAF receptor antagonist,30 nmol/paw), significantly inhibited PAF-induced paw oedemaformation in mice. The extracts of P. amarus (100 �g/mL) and niran-thin (30 �M), but not nirtetralin or phyltetralin (30 �M), decreasedthe specific binding of [3H]-PAF in mouse cerebral cortex mem-branes. The mean IC50 values from these effects were 6.5 and0.3 �M, respectively. Both niranthin and WEB2170 (30 nmol/paw)inhibited the increase of myeloperoxidase activity induced byPAF injection in the mouse paw (Kassuya et al., 2006). The hex-ane extract (HE), the lignan-rich fraction (LRF), or the lignansphyltetralin, nirtetralin, niranthin, but not hypophyllanthin orphyllanthin from P. amarus inhibited carrageenan-induced pawoedema and neutrophil influx. The HE, the LRF or nirtetralin alsoinhibited the increase of IL1-� tissue levels induced by Cg. Fur-thermore, bradykinin (BK)-, platelet activating factor (PAF) – andendothelin-1 (ET-1)-induced paw oedema were significantly inhib-ited by the HE or LRF while histamine- and substance P-inducedpaw oedema were unaffected. Finally, nirtetralin or phyltetralincaused inhibition of paw oedema induced by PAF or ET-1 (Kassuyaet al., 2005). P. amarus EtOH, H2O (0.25%) and hexane extracts(0.01%) showed an inhibition of LPS-induced production of NO andPGE2 in KC and in RAW264.7. The extracts also attenuated the LPS-induced secretion of TNF-� in RAW264.7 as well as in human blood.Both extracts reduced expression of iNOS and COX-2 and inhib-ited activation of NF-kappa �, but not of AP-1. P. amarus inhibitedinduction of interleukin (IL)-1�, IL-10, and interferon-� in humanblood and reduced TNF-� production in vivo (Kiemer et al., 2003).The anti-allodynic and anti-oedematogenic effects of the hexaneextract, lignan-rich fraction and purified lignans from aerial partsof P. amarus was studied in the inflammatory and neuropathicmodels of nociception. The hexane extract at a dose of 100 mg/kgbody weight orally in male swiss albino rats inhibited the allodyniaand the oedema induced by the intraplantar injection of completeFreund’s adjuvant (CFA). The inhibition observed was 76 ± 7% (ipsi-lateral paw), 64 ± 7% (contralateral paw), and 41 ± 2% (oedema).Otherwise, the lignan-rich fraction 100 mg/kg body p.o., or the purelignans 30–100 mg/kg body weight i.p. did not affect CFA-inducedallodynia. Administered chronically, the lignan fraction reducedCFA-induced paw oedema (39 ± 9%). When evaluated in the modelof neuropathic pain caused by partial ligation of sciatic nerve, thehexane extract inhibited the mechanical allodynia (77 ± 7%), with asimilar efficacy to the gabapentin (71 ± 10%) (Kassuya et al., 2003).The aqueous extracts of leaves and stems of P. amarus at the dose of500, 250 and 100 mg/kg body weight orally as a single dose, 1 h priorto experiment was administered to female stains of Balb/c mice.Extracts produced an inhibition of 26, 33 and 39% respectively at3 h while methanolic extract of P. amarus at a dose level of 100, 250and 500 mg/kg body weight produced an inhibition of 29, 37 and42% respectively at 3 h and significant inhibition of paw oedemawas observed throughout the course of experiment up to 8 h(Raphael and Khuttan, 2003).

8.7. Antinociceptic activity

The hydroalcoholic extract of four new species of Phyllanthus,given intraperitoneally, produced significant inhibition of aceticacid-induced abdominal constrictions. The hydroalcoholic extractof the Phyllanthus species elicited significant inhibition of the

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capsaicin-induced neurogenic pain. The extract of these Phyllan-thus species when given intraperitoneally, produced dose relatedand pronounced antinociception when assessed against chem-ical models of nociception, including acetic acid, formalin andcapsaicin-induced pain. Orally, these species were less potent andefficacious than given by intraperitoneally (Santos et al., 2000).

8.8. Antioxidant activity

Antioxidant activity of aqueous extract of whole plant of P.amarus at the dose of 200 mg/kg body weight/day was evalu-ated in streptozotocin (STZ)-induced diabetic male Wistar albinorats. Antioxidant enzymes; GR, GPX and GST, CAT and SOD werealso assayed. Diabetic treated animal group showed a signifi-cant decrease in renal LPO, protein oxidation and a significantincrease in GSH content and GR, GPX and GST activities when com-pared with STZ-induced diabetic rats. The activities of SOD andCAT decreased significantly in STZ-induced diabetic rats, but werenormalized in diabetic treated group (Karuna et al., 2011). Theaqueous extract of whole plant of P. amarus showed significant(p < 0.05) potential in scavenging free radicals, and in inhibitinglipid peroxidation. Furthermore, the extract proved to contain ahigh content of phenolic compounds which were found to havestrong and significant (p < 0.05) positive correlations to free-radicalscavenging potential, lipid peroxidation inhibition capacity andcyto-protective efficiency against Cr(VI)-induced oxidative cellu-lar damage (Guha et al., 2010). Aqueous extract of P. amarus ata dose of 200 mg/kg body weight/day for 8 weeks treated malealbino Wistar rats showed a significant decrease in plasma LPOand a significant increase in plasma vitamin C, uric acid, GSHlevel, GPX, CAT and SOD activities. Single cell gel electrophore-sis (SCGE) experiment revealed that aqueous extract of P. amaruswas devoid of genotoxicity and had a significant protective effectagainst H2O2, STZ and nitric oxide (NO) induced lymphocyte DNAdamage (Karuna et al., 2009). Free-radical scavenging activity of50% ethanolic extract of aerial parts of P. amarus extract andphyllanthin was examined using 2,2-diphenyl-2-picrylhydrazyl(DPPH) assay. The DPPH free-radical scavenging activity wasconcentration-dependent in both cases and reaches a maximum ata concentration of 300 �g/mL for P. amarus extract and 20 �mol/mLfor phyllanthin. No difference in inhibition was noted with fur-ther increase in concentration of either of the compounds. Resultsindicated that phyllanthin exhibited very high antioxidative prop-erty as compared to P. amarus extract which is clearly evidentby its low IC50 value of 7.4 �mol/mL (Krithika et al., 2009). Theantioxidant activity of some of its principal constituents, namelyamariin, 1-galloyl-2,3-dehydrohexahydroxydiphenyl (DHHDP)-glucose, repandusinic acid, geraniin, corilagin, phyllanthusiinD, rutin and quercetin 3-O-glucoside were examined fortheir ability to scavenge free radicals in a range of systemsincluding DPPH, 2,2-azobis-3-ethylbenzthiazoline-6-sulfonic acid(ABTS)/ferrylmyoglobin, ferric reducing antioxidant power (FRAP)and pulse radiolysis. The compounds showed significant antiox-idant activities with differing efficacy depending on the assaysemployed. Amariin, repandusinic acid and phyllanthusiin Dshowed higher antioxidant activity among the ellagitannins andwere comparable to the flavonoids, rutin and quercetin 3-O-glucoside (Londhe et al., 2008). Pretreatment with P. amarus leavesextracts on antioxidant enzymes in gastric mucosa homogenatewas studied. Significant reductions (p < 0.05) in the gastric mucosacatalase (CAT), super oxide dismutase (SOD) and glutathione-s-transferase (GST) activities were observed in the ethanol groupcompared with the normal control group. P. amarus acetoneextracts (1000 mg/kg) and cimetidine (100 mg/kg) caused an ele-vation by 53 and 52% for CAT, 8 and 14% for SOD and 33 and38% for GST respectively when compared with the ethanol group.

Acetone extract produced a dose-dependent increase whereas500 mg/kg of the aqueous extract seems more effective (Shokunbiand Odetola, 2008). Different drying treatments led to significantreduction (p < 0.05) in antioxidant properties of P. amarus methano-lic extracts, with microwave drying causing the highest decreasein total phenolic compound and antioxidant activity exhibited bythe reduction in both radical scavenging activity and FRAP. Onthe other hand, boiling water extracts appeared to exhibit sig-nificantly stronger antioxidant potentials (p < 0.05) even in driedplant materials due to greater solubility of compounds, breakdownof cellular constituents as well as hydrolysis of tannins (Lim andMurtijaya, 2007). The antioxidant activity of methanolic extracts offive species including P. debilis, P. urinaria, P. virgatus, P. maderas-patensis, P. amarus from the genus Phyllanthus was evaluated byvarious antioxidant assays. All the extracts at the concentration of50 �g/mL showed strong antioxidant activity in all the tested meth-ods. Among the five plants, P. debilis has been found to possess thehighest activity and P. amarus posses the lowest activity in all testedmodels (Kumaran and Karunakaran, 2007). Methanolic extract ofleaves and stems of P. amarus was found to have potential antiox-idant activity as it could inhibit lipid peroxidation, and scavengehydroxyl and superoxide radicals in vitro. The amount required for50% inhibition of lipid peroxide formation was 104 �g/mL and theconcentrations needed to scavenge hydroxyl and superoxide rad-icals were 117 and 19 �g/mL respectively (Raphael et al., 2002a).Amariin, repandusinic acid, phyllanthusiin D, phyllanthin and phe-nolic compouds isolated from P. amarus showed remarkable highantioxidant activity.

8.9. Antiplasmodial activity

The aqueous and ethanolic extracts of the whole plant ofP. amarus were administered to Swiss albino mice at doses of200, 400, 800 and 1600 mg/kg/day to investigate the prophylac-tic and chemotherapeutic effect of the extract against Plasmodiumyoelii infection and compared with those of standard antimalarialdrugs pyrimethamine and chloroquine, artesunate/amodiaquinerespectively. Results showed that extracts demonstrated a dose-dependent prophylactic and chemotherapeutic activity. Theaqueous extract showed slightly higher effect than the ethanolicextract. The antiplasmodial effects of extracts were comparableto the standard prophylactic and chemotherapeutic drugs used inchloroquine resistant plasmodium infection. The extracts showedprophylactic effect by significant delay in the onset of infection withthe suppression of 79% at a dose of 1600 mg/kg/day. The resultsindicate that the extracts of the whole plant of P. amarus possessrepository and chemotherapeutic effects against resistant strainsof P. yoelii in Swiss albino mice (Ajala et al., 2011). ‘Saye’, a tra-ditional medicine used in Burkina Faso, which consists of extractsof Cochlospermum planchonii (rhizome), Cassia alata (leaves) andP. amarus (whole plant). Mice treated with 50, 100, 150, 200,250 mg/kg body weight for 0–3 days, orally showed a significanteffect against P. falciparum and Plasmodium berghei parasites grownin vivo (IC50 = 80.11 ± 3.40 �g/mL; ED50 = 112.78 ± 32.32 mg/kg). Invitro the activity was lower at the concentration of 100, 50, 25 and12.5 �g/mL and compared with chloroquine (Traore et al., 2008).The aqueous extract of leaves and stem of P. amarus at doses of108.33, 165 and 325 mg/kg in Swiss albino mice was found to causea significant dose-dependent suppression of P. berghei parasites[p < 0.05] sulfadoxine/pyrimethamine caused a similar significantsuppression of P. berghei parasites (Dapper et al., 2007). Ethano-lic, methanolic and methylene chloride extracts of entire plantof P. amarus showed significant activity against the chloroquine-sensitive strain of P. falciparum 3D7. The IC50 of methanolic extractand methylene chloride extract was 5 and 14.53 �g/mL respectively(Adjobimey et al., 2004).

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8.10. Antiviral activity

Inhibitory effect of methanolic extracts of root and leaves of P.amarus against the NS3 and NS5B enzymes of hepatitis-C virus werescreened by in vitro enzyme assays. Effect on viral RNA replicationwas investigated by using TaqMan Real time RT-PCR. P. amarus rootextract showed significant inhibition of HCV-NS3 protease enzyme;whereas P. amarus leaves extract showed considerable inhibition ofNS5B in the in vitro assays. Further, the P. amarus root and leavesextracts significantly inhibited replication of HCV monocistronicreplicon RNA and HCV H77S viral RNA in HCV cell culture system.However, both P. amarus root and P. amarus leaves extracts did notshow cytotoxicity in HuH-7 cells in the MTT assay. Furthermore,P. amarus root extract with IFN-� showed additive effect in theinhibition of HCV RNA replication. Results suggested the possiblemolecular basis of the inhibitory activity of P. amarus extract againstHCV which would help in optimization and subsequent develop-ment of specific antiviral agent using P. amarus as potent naturalsource (Ravikumar et al., 2011). The aqueous extract of P. amarusshowed partial antiviral activity against white spot syndrome virusin shrimp at the concentration of 150 mg/kg of animal body weightfor 30 days (Balasubramanian et al., 2007). The aqueous, butanol,and alcoholic extracts of P. amarus and P. maderaspatensis, was stud-ied for antiviral properties on duck hepatitis B virus infection. Onehundred and fourteen ducks infected posthatch with the duck hep-atitis B virus (DHBV) were divided into groups at 3 months of ageand treated intraperitoneally with the aqueous, butanol, and alco-holic extracts of these two plants at doses of 25, 50, or 200 mg/kgbody weight. There was no definite antiviral property observed inthe treated ducks (Munshi et al., 1993a). The therapeutic potentialof plant extracts of P. amarus and P. maderaspatensis for postexpo-sure prophylaxis against infection by Hepadnaviruses was studiedin ducklings infected by the DHBV. The observations suggested thataqueous extract of P. amarus (100 mg/kg body weight) and ethano-lic extract of P. maderaspatensis (100 mg/kg body weight) are notuseful as therapeutic agents for postexposure prophylaxis againstDHBV infection (Munshi et al., 1993b). The polyphenol containingextract of P. amarus and representatives of simple phenols (shikimicacid 3-and 5-O-gallate), flavan-3-ols (epigallocatechin 3-gallate),proanthocyanidins (a hexamer) and hydrolysable tannins (corila-gin, casuariin, geraniin) were studied in vitro for gene expressions(iNOS, IL-1, IL-10, IL-12, IL-18, TNF-�, IFN-� and �) by RT-PCR. Allextracts and compounds at the concentration of 50 �g/mL werecapable of enhancing the iNOS and cytokine mRNA levels in para-sitised cells when compared with those in non-infected conditions(Kolodziej et al., 2005). In vitro culture of hairy roots of P. amarusinduced by Agrobacterium rhizogenes was shown to possess 85%inhibition (in contrast to 15% in the control) in binding of HepatitisB Surface Antigen (HBsAg) to its antibody (anti-HBs) after 24 h ofincubation with HbsAg-positive sera in vitro at 37 ◦C (Bhattacharyyaand Bhattacharya, 2004). Water–alcohol extract of leaves of P.amarus in vitro blocks HIV-1 attachment and the HIV-1 enzymesintegrase, reverse transcriptase and protease to different degrees.A gallotannin containing fraction and the isolated ellagitanninsgeraniin and corilagin were shown to be the most potent mediatorsof these antiviral activities. P. amarus derived preparations blockedthe interaction of HIV-1 gp120 with its primary cellular recep-tor CD4 at 50% inhibitory concentrations of 2.65 (water–alcoholextract) to 0.48 �g/mL (geraniin). Inhibition was also evident forthe HIV-1 enzymes integrase (0.48–0.16 �g/mL), reverse transcrip-tase (8.17–2.53 �g/mL) and protease (21.80–6.28 �g/mL) (Notkaet al., 2004). Aqueous as well as alcohol-based P. amarus extractspotently inhibited HIV-1 replication in HeLa CD4+ cells with50% effective concentration (EC50) values ranging from 0.9 to7.6 �g/mL. A gallotannin enriched fraction showed enhanced activ-ity (0.4 �g/mL), and the purified gallotannins geraniin and corilagin

were most active (0.24 �g/mL). HIV-1 replication was also blockedin CD4+ lymphoid cells with comparable EC50 values. Applyinga cell-based internalization assay, it was demonstrated 70–75%inhibition of virus uptake at concentrations of 2.5 �g/mL for thewater–alcohol extract and geraniin. In addition, a concentration-dependent inhibition of HIV-1 reverse transcriptase (RT) wasdemonstrated in vitro. The 50% inhibitory concentration (IC50) val-ues varied from 1.8 to 14.6 �g/mL (Notka et al., 2003). Study on25 compounds isolated from P. amarus, P. multiflorus, P. tenel-lus and P. virgatus found that niranthin, nirtetralin, hinokinin andgeraniin at the non-cytotoxic concentration of 50 �m, suppressedeffectively both HBsAg and hepatitis B effective antigen (HbeAg)expression, of these, niranthin showed the best anti-HBsAg activ-ity, while the most potent anti-HBeAg activity was observed withhinokinin (Huang et al., 2003). Analysis in HuH-7 cells (human hep-ato cellular carcinoma cell line) with transfected plasmids using aluciferase reporter showed that DMSO solution of whole plant ofP. amarus at the concentration of 50, 100 or 200 �g/mL specificallyinhibited HBV enhancer I activity. To identify the mechanism ofthis HBV enhancer I inhibition, liver-enriched cellular transcrip-tion factors were co-expressed in HuH-7 cells. The C/EBP � and� as well as HNF-3� and � (hepatocyte nuclear factor), transcrip-tion factor significantly upregulated the HBV enhancer I activity.In contrast, co-transfection of HNF-I � or � had no effect uponthe HBV enhancer I activity. Exposure to P. amarus inhibited C/EBP� and �-mediated up-regulation of HBV enhancer I activity in adose-dependent manner, whereas HNF-3�- and �-mediated up-regulation of HBV enhancer I was unaffected. In vitro gel shiftsshowed that P. amarus inhibited complexing of C/EBP transcrip-tion factors to a consensus oligonucleotide sequence, whereas DNAbinding of AP-1 and SP-1 transcription factors was unaffected(Ott et al., 1997). P. amarus inhibited hepatitis B virus polymeraseactivity, decreased episomal hepatitis B virus DNA content and sup-pressed virus release into culture medium. When DSMO solution ofwhole plant of P. amarus 80 mg/kg body weight i.p. daily for 7 dayswas administered to transgenic mice, hepatic HBsAg mRNA levelwas decreased, indicating transcriptional or post-transcriptionaldown-regulation of the transgene. Increase in hepatitis B virusmRNA expression after stimulation of the glucocorticoid responsiveelement was also suppressed by P. amarus, suggesting involvementof the hepatitis B virus enhancer in this response. Disruption by P.amarus of hepatitis B virus polymerase activity, mRNA transcrip-tion and replication supports its role as an antiviral agent (Lee et al.,1996). The effect of an aqueous extract of whole plant of P. amarusat the concentration of 1 mg/mL on the cultured hepatoma cellline HepA2 has been reported. The cell line had been transfectedwith tandemly arranged HBV DNA and continued to synthesizeand secrete both HBsAg and HBeAg. Extract of P. amarus reversiblyinhibited cellular proliferation and suppressed HBsAg productionbut not HBeAg production in HepA2 cells. P. amarus also suppressedHBsAg gene expression at mRNA level in a time-dependent manner,and selectively abolished the HBsAg gene promoter driven chlo-ramphenicol acetyltransferase activity (Yeh et al., 1993). Ethanolicextract and subsequent fractions (hexane, chloroform, butanol andwater) were tested for in vitro effects on HBsAg, HBeAg and HBVDNA in serum samples positive for HBV antigens followed by thescreening of respective antigens by ELISA. HBV DNA was deter-mined by molecular hybridization. The extracts were effectiveagainst HBV antigens, the butanol extract being the most potent.The chromatographic fractions showed an enhanced activity. Theactive fractions inhibited the interaction between HBsAg/HBeAgand their corresponding antibodies suggesting anti-HBs, anti-Hbelike activity and also an effect on HBV DNA (Mehrotra et al., 1991).Extracts of P. amarus have been shown to inhibit the DNA poly-merase of HBV and Woodchuck hepatitis virus (WHV) in vitro.Woodchuck carriers of WHV were treated intraperitoneally with

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P. amarus extract. When three out of four recently infected WHVcarriers were treated with aqueous extract of P. amarus at a doseof 0.9 mL (9 mg dry weight), i.p., twice weekly, they lost the WHV.Animals infected for greater than or equal to 3 months showeda decrease in virus levels. Preliminary results in human carrierstreated orally with P. amarus for 1 month indicated that approxi-mately 60% of the carriers lost HBV during the observation period(Blumberg et al., 1989, 1990). The aqueous, butanolic and alcoholicextract of P. amarus were described for the treatment of chronichepatitis B virus infection on duck hepatitis B virus at the dosesof 25, 50, and 200 mg/kg body weight. Nine ducks congenitallyinfected with the DHBV were treated either orally (four ducks for10 weeks) or intraperitoneally (five ducks for 12 weeks) with P.amarus, compared to placebo-treated control ducks. These treat-ments did not result in a reduction of circulating viral DNA in theserum or in the level of viral DNA replication in the liver. In twoof the five intraperitoneal-treated ducks, a reduction in the level ofduck hepatitis B surface antigenaemia (DHBsAg) was observed. Thedata strongly suggested that P. amarus has no significant inhibitoryeffect on DHBV DNA replication and only a minor effect on DHB-sAg P. amarus production (Niu et al., 1990). Alexander cell line, ahuman hepatocellular carcinoma derived cell line which has theproperty of secreting HBsAg in the supernatant was used to studythe antiviral property of P. amarus. Aquous extract of P. amaruswas evaluated for its in vitro ability to inhibit HBsAg secretion ona dose-dependent manner. Aquous extract of P. amarus at the con-centration of 1 mg/mL as a single dose inhibited the secretion ofHBsAg for a period of 48 h. Observations revealed the anti hepatitisB virus property of P. amarus at cellular level and further confirmedits beneficial use in the treatment of acute and chronic hepatitis Band healthy carriers of HBV (Jayaram and Thyagarajan, 1996). Lig-nans geraniin, corilagin, niranthin and hinokinin isolated from P.amarus exhibited excellent antiviral activity.

8.11. Clinical studies

Study focuses on effect of P. amarus therapy for protection ofliver in hepatitis-C through investigating liver profile enzymes;antioxidant enzymes, antioxidant vitamins and lipid peroxidation.The study consists of 50 clinical diagnosed hepatitis-C patientsranging in between age group 25 and 60 years. The control groupincludes 50 ages and sex matched normal healthy persons. Oxida-tive stress was assessed by estimating LPO. The parameters likeserum bilirubin, total proteins and activity of liver profile enzymeswere done. Activity of enzymatic antioxidants; SOD, GPX and lev-els of non-enzymatic antioxidant vitamins E and C was measuredin plasma or erythrocytes. Methods used in the study are mainlyenzyme kinetics by autoanalyzer and by turbidimetry. Plasma LPOlevels were significantly high but activity of SOD, GPX, catalase andlevels of vitamins E and C were significantly lowered in hepatitis-Cin comparison with controls. After P. amarus therapy for 5 and 10weeks plasma LPO levels were significantly decreased and activ-ity of SOD, GPX, catalase and vitamins E and C were significantlyincreased in hepatitis-C. It was concluded that hepatitis-C increasedoxidative stress and might be playing an important role in hepaticcell damage and pathogenesis of hepatitis-C. It was suggested thatthe therapy with P. amarus increased antioxidants and reduced lipidperoxidation of hepatic cellular and intracellular membranes andprotected liver damage due to free radicals in hepatitis-C (Nikamet al., 2011). A total of 16 randomized trials with 1326 patientswere included. One trial with 42 participants compared phyllan-thus with placebo. The trial found no significant difference in HBeAgseroconversion after the end of treatment [risk ratio (RR) 0.9; 95%(confidence intervals) CI 0.73–1.25] or follow-up (RR 1.00; 95%CI 0.63–1.60). No other outcomes could be assessed. Fifteen tri-als were compared with phyllanthus plus and an antiviral drug

like IFN-�, lamivudine, adefovir dipivoxil, thymosin, vidarabine, orconventional treatment with the same antiviral drug alone. Phyl-lanthus significantly affected serum HBV DNA (RR 0.69; 95% CI0.52–0.91, p = 0.008; I(2) = 71%), serum HBeAg (RR 0.70; 95% CI0.60–0.81, p < 0.00001; I(2) = 68%), and HBeAg seroconversion (RR0.77; 95% CI 0.63–0.92, p = 0.005; I(2) = 78%), but the heterogene-ity was substantial. The result obtained regarding serum HBV DNAwas not supported by trial sequential analysis. None of the trialsreported mortality and hepatitis B related morbidity, quality oflife, or liver histology. Only two trials reported adverse events withnumbers without significant differences. No serious adverse eventswere reported (Xia et al., 2011). Volunteers received either 1200 mgof water extract from leaves of P. amarus or 450 mg lamivudine,and blood was collected before, and 1, 2 and 3 h after treatment.MAGI cells were inoculated with HIV-1 in the presence of pre-and post-treatment serum (1, 2 and 3 h post-administration, asindicated). Sera at a final concentration of 5% reduced HIV repli-cation by more than 30%. These results supported that P. amarushas inhibitory effects on HIV in vitro and in vivo (Notka et al.,2004). Fifty-five patients with chronic viral hepatitis B were ran-domly divided into two groups. Thirty patients were treated withPA compound capsule (Chief ingredients: P. amarus, Radix noto-ginseng, etc. each capsule contains P. amarus 275 mg), orally, threetimes daily, four capsule each time for 3 months in the treatmentgroup; another 25 patients were treated with domestic recombi-nant human interferon alpha-1b (IFN-�1�) for 3 months as control.The total effective rate in the treatment group was 83.3%, showedno significant difference from the control (p > 0.05). The normaliza-tion rates of ALT, A/G and SB in the treatment group were 73.3, 80.0and 78.2% respectively, which were significantly higher than thatin the control (p < 0.05). The negative conversion rates of HbeAgand HBV DNA in the treatment group were 42.3 and 47.8%, showedno significant difference from the control (p > 0.005) (Wang et al.,2001). The efficacy and safety of genus Phyllanthus for chronic hep-atitis B virus (HBV) infection was evaluated by a systematic reviewof randomized clinical trials. Randomized trials comparing genusPhyllanthus vs. placebo, no intervention, general nonspecific treat-ment, other herbal medicine, or interferon treatment for chronicHBV infection were identified by electronic and manual searches.Trials of Phyllanthus herb plus interferon (IFN) vs. IFN alone werealso included. No blinding and language limitations were applied.The methodological quality of trials was assessed by the Jadadscale plus allocation concealment. Twenty-two randomized trials(n = 1947) were identified. The methodological quality was high infive double-blind trials and low in the 17 remaining trials. The com-bined results showed that Phyllanthus species had positive effecton clearance of serum HBsAg (relative risk 5.64, 95% CI 1.85–17.21)compared with placebo or no intervention. There was no signifi-cant difference on clearance of serum HBsAg, HBeAg and HBV DNAbetween Phyllanthus and IFN. Phyllanthus species were better thannonspecific treatment or other herbal medicines for the clearance ofserum HbsAg, HBeAg, HBV DNA, and liver enzyme normalization.Analysis showed a better effect of the Phyllanthus plus IFN com-bination on clearance of serum HBeAg (1.56, 1.06–2.32) and HBVDNA (1.52, 1.05–2.21) than IFN alone. The efficacy and safety ofgenus Phyllanthus for chronic hepatitis B virus (HBV) infection hasbeen systematically reviewed the clinical trials (Liu et al., 2001). Thepowder of the plant P. amarus was given in capsule form (300 mgcapsules-3 capsules thrice daily) and an antacid powder in similarcapsule was used as placebo. Fifty-seven patients were random-ized to receive either the placebo (28 cases) or the drug (28 cases).The two groups were comparable at the time of entry. Two casesfrom the placebo and one from the placebo and one from the druggroup dropped out of the study. The duration of disease (time takenfor bilirubin to come to below 2 mg %) was taken as the outcomemeasure. The duration of disease in the two groups was compared

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by Cox’s proportional hazards analysis after adjusting for the vari-ables that influence the duration of jaundice. Only initial serumbilirubin was an independent predictor of duration of jaundice.The analysis showed that P. amarus powder did not significantlyreduce the duration of jaundice in persons with virus B hepati-tis (Narendranathan et al., 1999). The efficacy of P. amarus to treatacute viral hepatitis (AVH) was evaluated in parallel to another drugEssentiale (an essential phospholipid extracted from soybean oil)and compared with a group of patients who were treated symp-tomatically with vitamins as the controls. Serological profile of 93sporadic AVH cases of the study showed that 25.8% had an acuteinfection due to hepatitis A virus (HAV), 52.6% suffered due to hep-atitis B viral (HBV) infection, while 19.3% of cases were classifiedas non-A non-B hepatitis (NANB) by exclusion. On follow up of thepatients at the end of treatment period of 4 weeks with respec-tive drug regimen, it was seen that both P. amarus and Essentialebrought about significant biochemical and clinical normalcy amongthe HAV infected patients compared to control group (p < 0.001). Inacute HBV group, P. amarus treated patients recovered faster thanthe essentiale treated group and the controls (p < 0.001). Essentialewas found to help the non-A non-B hepatitis patients to resumeearlier biochemical normalcy than by P. amarus and control treat-ments. P. amarus seemed to accelerate the clearance of HBsAg in86.9% of convalescing AVH-B cases in 3 months time as against48.0% in the Essentiale treated group and 50.0% in the controls(Jayaram et al., 1997). The role of P. amarus in eradication of thevirus in hepatitis B carriers was evaluated by administering it to30 asymptomatic carriers of HBsAg in a dosage of 250–500 mgthrice daily for 4–8 weeks. It was found that none of the 30 sub-jects cleared HBsAg. P. amarus was well tolerated, with no clinicalside effects or changes in the organ profiles for safety evaluation.It was found that P. amarus was not effective in clearing HBsAgin asymptomatic carriers of the antigen (Doshi et al., 1994). Sixty-five adult asymptomatic chronic carriers of hepatitis B virus wereenrolled to the randomized controlled efficacy study of P. amarus.Thirty-four received P. amarus 600 mg per day for 30 days and31 received placebo in identical capsules. The conversion rate ofHBsAg was 6% in the study group at day 30. When 20 subjects inthe P. amarus group were given a further 30-day treatment and 22placebo recipients given P. amarus 1200 mg per day for 30 days,the conversion was observed in 1 (5%) in the higher dose group.The results indicated that P. amarus, whole plant except root, givenat the studied dosage and duration, had a very minimal effect oneradication of HBsAg asymptomatic chronic carriers (Thamlikitkulet al., 1991). A total of 79 human carriers of HBV, out of these, 40were given 200 mg of dried, powdered whole plant of P. amarus,three times daily. The remaining 39 carriers were given lactoseplacebo at the same dosage and frequency. Carriers were assignedrandomly to the treatment or control groups and neither the car-riers nor their physicians were told of the assignment. The drugor placebo was administered for 30 days then stopped; the carri-ers were then tested monthly for up to 9 months after cessation oftreatment or placebo. Thirty-seven of the treated carriers and 23of the controls returned for follow-up. Of the treated subjects 59%,but only 4% of 23 control subjects, became HBsAg-negative after30 days and remained so until the end of the follow-up period.Thirteen out of 14 carriers with HBsAg but without HBeAg becamenegative, while only five out of 17 HBsAg positive, HBeAg-positivesubjects lost the surface antigen. HBsAg did not reappear in anyof these subjects. The individuals who remained HBsAg-positiveare now being treated for longer periods to determine whetheractive replication of virus requires longer duration of therapy. Therewas no evidence of toxicity in the treated individuals, but theseobservations were based primarily on clinical examination(Blumberg et al., 1990). In a preliminary study, carriers of hep-atitis B virus were treated with a preparation of the plant

P. amarus (200 mg doses in gelatin capsule presterilized with ethy-lene oxide, three times daily) for 30 days. 22 of 37 (59%) treatedpatients had lost hepatitis B surface antigen when tested 15–20days after the end of the treatment compared with only 1 of 23(4%) placebo-treated controls. Some subjects have been followedfor up to 9 months. In no case has the surface antigen returned.Clinical observation revealed few side effects which include fatigue,malaise, fever, chills, urticaria, anorexia, nausea, abdominal pain,diarrhoea, headache, dizziness, disturbance of sleep and skin rash(Thyagarajan et al., 1988).

8.12. Aphrodisiac activity

The effect of methanolic extract of the leaves of P. amarus on thehormonal parameters of male guinea pigs was investigated. Thehormonal parameters investigated were testosterone, leutinizingand follicle stimulating hormone. Methanolic extract of P. amarusleaves (50–800 mg/kg) caused a statistically significant increase inthe level of testosterone of the male guinea pigs, from 2.3 ± 0.06 to3.9 ± 0.05, 4.3 ± 0.6 and 2.8 ± 0.6 after the 7th, 14th and 21st day ofthe administration of the extracts, respectively. Furthermore, themethanolic extract of P. amarus (800 mg/kg) caused an insignifi-cant change in the level of leutenizing (LH) and follicle stimulating(FSH) hormones from 3.1 ± 0.22 and 1.6 ± 0.50 to 3.0 ± 0.08 and1.5 ± 0.13, respectively (Obianime and Uche, 2009).

8.13. Contraceptive effect

Antifertility effect of an alcoholic extract of the whole plant of P.amarus at a dose of 100 mg/kg body weight for 30 days orally wasinvestigated in cyclic adult female mice. The results revealed nosignificant change in absolute body and organ weights in extractfed animals indicated no alteration in general metabolic status.Cohabited females with normal male mice were unable to becomepregnant as their cyclicity was affected. These factors are relatedto a change in the hormonal milieu that governs female reproduc-tive function. Upon withdrawal of feeding for 45 days, these effectswere reversible. Thus, this extract manifests a definite contracep-tive effect in female mice (Rao and Alice, 2001).

8.14. Diuretic and antihypertentive activity

8.14.1. Clinical studyDiuretic, hypotensive and hypoglycaemic effects of P. amarus

on human subjects were assessed. Nine mild hypertensives (fourof them also suffering from diabetes mellitus) were treated witha preparation of the whole plant of P. amarus for 10 days. Suit-able parameters were studied in the blood and urine samples ofthe subjects along with physiological profile and dietary patternbefore and after the treatment period. Significant increase in 24 hurine volume, urine and serum Na levels was observed. A significantreduction in systolic blood pressure in non-diabetic hypertensivesand female subjects was noted. Blood glucose level was also signif-icantly reduced in the treated group. Clinical observations revealedno harmful side effects (Srividya and Periwal, 1995).

8.15. Hepatoprotective activity

The effect of aqueous leaves extract of P. amarus on matrix met-alloproteinases and tissue inhibitors of matrix metalloproteinaseswas evaluated in alcohol and thermally oxidized polyunsaturatedfatty acid-induced hepatic fibrosis. The matrix metalloproteinaseexpression was found to be significantly decreased and the levelsof tissue inhibitors of matrix metalloproteinases and the colla-gen were significantly increased in alcohol and thermally oxidized

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polyunsaturated fatty acid treated male Wistar albino rats. Admin-istration of P. amarus extract 3 mL/day for 45 days significantlydecreased the levels of collagen and tissue inhibitors of matrix met-alloproteinases; and positively modulated the expression of matrixmetalloproteinases. It revealed that P. amarus effectively modifiedalcohol and thermally oxidized polyunsaturated fatty acid-inducedfibrosis (Surya Narayanan et al., 2011). The protective effect ofphyllanthin, a known principal constituent of P. amarus on ethanol-induced rat liver cell injury was evaluated. Primary cultures ofrat hepatocytes (24 h culturing) were pretreated with phyllan-thin (1, 2, 3 and 4 �g/mL) for 24 h. After 24 h pretreatment, cellswere treated with ethanol (80 �L/mL) for 2 h. Ethanol decreased%MTT, increased the release of ALT and AST with increase in theproduction of intracellular ROS and lipid peroxidation. Phyllan-thin demonstrated its role in protection by antagonizing the aboveeffect induced by ethanol. Phyllanthin also restored the antioxidantcapability of rat hepatocytes including level of total glutathione,and activities of SOD and glutathione reductase (GR) which werereduced by ethanol (Chirdchupunseree and Pramyothin, 2010). Thehepatoprotective activity of 50% ethanolic extract of aerial partsof P. amarus plant (100, 200, and 300 mg/kg body weight dailyfor 30 days) against CCl4-induced liver damage in Swiss strainfemale albino mice was determined. Carbon tetrachloride adminis-tration caused a significant increase in liver and ALT, AST, ALP andacid phosphatase (ACP), while total protein content significantlydecreased as compared to vehicle control. The effect was dose-dependent. Oral administration of aqueous extract of P. amaruscaused significant mitigation of CCl4 induced changes. P. amarusattenuated the toxic effects of carbon tetrachloride (CCl4) andcaused a subsequent recovery towards normalization. Adminis-tration of P. amarus at 300 mg/kg body weight offered maximumrecovery (98–100%) against CCl4 (Krithika and Verma, 2009a,b).The protective effect of P. amarus extract and phyllanthin wasstudied on CCl4-induced toxicity in human hepatoma HepG2 cellline. The results indicated that CCl4 treatment caused a significantdecrease in cell viability. It was observed that phyllanthin (4.18,8.36 and 12.54 �g/mL for 24 h) effectively alleviated the changesinduced by CCl4 in a concentration-dependent manner with muchsmaller strengths as compared to (200, 400 and 600 �g/mL) P.amarus water ethanol extract (Krithika et al., 2009). Silymarin andstandardized extract obtained from the whole plant of P. amarus100 mg/kg body weight against CCl4-induced hepatotoxicity in ratswere found effective as hepatoprotective as evidenced by plasmaand liver biochemical parameters. The combination of silymarinand P. amarus (50 mg + 50 mg/kg body weight for 6 days orally onmale Rattus norvegicus strain showed synergistic effect for hep-atoprotection and silymarin with ethanolic extract of P. amarusshowed better activity due to the higher concentration of phyl-lanthin in ethanolic extract in comparison to aqueous extractof the plant (Yadav et al., 2008). The hepatoprotective effect ofmethanolic extract of the leaves of P. amarus was evaluated againstethanol-induced oxidative damage in adult male Wistar albinorats. Methanolic extract of P. amarus (250 and 500 mg/kg bodyweight/day and ethanol 5 g/kg body weight/day 20% w/v) wereadministered orally to animals for 4 and 3 weeks respectively.It was reported that the ethanol treatment markedly decreasedthe level of reduced GSH, SOD, and CAT in the liver, which weresignificantly enhanced by P. amarus treatment. GST, which wasincreased after chronic ethanol administration, was significantlyreduced by P. amarus treatment in the liver (Faremi et al., 2008).In vitro study, P. amarus aqueous extract (1–4 mg/mL) increased%MTT reduction assay and decreased the release of AST and ALT inrat primary cultured hepatocytes being treated with ethanol. Hep-atotoxic parameters studied in vivo included serum AST and ALT,serum triglyceride (STG), hepatic triglyceride (HTG), TNF-�, inter-leukin 1� (IL-1�), together with histopathological examination. In

acute toxicity study, single dose of P. amarus (25, 50 and 75 mg/kg,p.o.) or SL (Silymarin, 5 mg/kg), 24 h before ethanol (5 g/kg, p.o.) onmale Wistar rats lowered the ethanol-induced levels of AST and/orALT. The 75 mg/kg P. amarus dose gave the best result similar toSL. Histopathological observations confirmed the beneficial rolesof P. amarus and SL against ethanol-induced liver injury in rats(Pramyothin et al., 2007). The hepatoprotective effect of ethano-lic extract of whole plant except root of P. amarus was evaluatedon aflatoxin B1-induced liver damage in mice using different bio-chemical parameters and histopathological studies. Albino micetreatment with P. amarus extract (300 mg/kg body weight for 3months and animals were sacrified with the interval of 30 daystill the completion of study) was found to show hepatoprotectiveeffect by lowering down the content of thiobarbituric acid reactivesubstances (TBARS) and enhancing the reduced glutathione leveland the activities of antioxidant enzymes, GPX, GST, SOD and CAT.Histopathological analysis of liver samples also confirmed the hep-atoprotective value of the ethanolic extract of P. amarus (Naaz et al.,2007). The plasma concentrations of the liver function parametersin aqeous extract of whole plant of P. amarus treated Wistar rats(4 mL/rat/day) showed that the ALT and AST activities decreasedsignificantly in the blood of the test animals (Igwe et al., 2007). The�-glucuronidase inhibitory action of 50% methanolic, and aqueousextracts as well as isolated actives constituents such as corilagin,brevifolin carboxylic acid, phyllanthin, and hypophyllanthin from P.amarus was demonstrated. The results revealed that 50% methano-lic and water extracts were highly active and corilagin, the phenolicprinciple isolated from 50% methanolic extract of P. amarus wasfound to be more potent at the concentration of 200 �g/mL whichis comparable with silymarin (Joshi and Priya, 2007). Hot waterextracts of P. amarus (0.8, 1.6 or 3.2 g/kg) were orally administeredb.i.d. for 7 days prior, 2 days after, or 7 days prior and 2 days aftersingle oral dose of paracetamol (3 g/kg). The results showed that theextract at 1.6 and 3.2 g/kg decreased the paracetamol-induced hep-atotoxicity as indicated by the decrease in SGOT and SGPT, bilirubinand histopathological score while the ALP did not change. It was evi-dent that the hepatoprotective mechanism of this plant was neitherrelated to inhibition on cytochrome P450, nor induction on sulfateand glucuronide conjugation pathways of paracetamol, but partlydue to the antioxidant activity and the protective effect on thedecrease of hepatic reduced glutathione (Wongnawa et al., 2005).Silymarin, fresh leaves juice and ethanolic extracts of P. amarus(50 mg/kg, 1 mL/kg and 300 mg/kg body weight respectively) pre-vented the CCl4-induced reduction of ascorbic acid excretion inurine. The results indicated that the measurement of ascorbic acidexcretion could be used as a non-invasive test for screening protec-tive substances against CCl4-induced hepatotoxicity in albino rats(Visweswaram et al., 1994).

8.16. Hypoglycemic and hypocholesterolemic activities

The hypoglycemic potential of aqueous extract of whole plantof P. amarus was investigated in alloxan-induced diabetic Wistaralbino rats. The extract at a dose of 260 mg/kg produced a signifi-cant (p < 0.05) reduction in blood glucose level by 112% at 24 h oforal administration. A significant reduction (p < 0.01) in blood glu-cose level of 81 and 61% (day 7) at doses of 130 and 260 mg/kgof extract were observed respectively. The extract also showed ahighly significant (p < 0.001) decrease in blood glucose level of 38and 30% (day 14) at doses of 130 and 260 mg/kg respectively. Onthe administration of 390 mg/kg dose of extract, significant reduc-tion (p < 0.001) in blood glucose level of 41% on day 7 and 16%on day 14 was observed (Mbagwu et al., 2011). The antihyper-glycemic and hypolipidemic activities of aqueous extract of wholeplant of P. amarus were evaluated in streptozotocin (STZ)-induceddiabetic male Wistar albino rats. Aqueous extract of P. amarus was

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administered at 200 mg/kg body weight/day to normal treated andSTZ-induced diabetic treated rats by gavage for 8 weeks. Duringthe experimental period, blood was collected from fasted rats at 10days intervals and plasma glucose level was estimated. The plasmalipid profile was estimated at the end of experimental period. Afterthe treatment period, kidney LPO, protein oxidation and GSH wereestimated. The significant decrease in the body weight, hyper-glycemia and hyperlipidemia was observed in STZ-induced diabeticrats with the treatment of aqueous extract of P. amarus in dia-betic treated group. STZ-induced diabetic rats showed increasedrenal oxidative stress with increased LPO and protein oxidation(Karuna et al., 2011). The �-amylase inhibitory activity of ethanol,chloroform, and hexane extracts of P. amarus against porcine pan-creatic amylase in vitro was evaluated. Different concentrations(10, 20, 40, 60, 80, and 100 �g/mL) in DMSO were subjectedto �-amylase inhibitory assay using starch azure as a substrate.The ethanol and hexane extracts of P. amarus exhibited apprecia-ble �-amylase inhibitory activity with an IC50 values 36.05 ± 4.01and 48.92 ± 3.43 �g/mL, respectively, when compared with acar-bose (IC50 value 83.33 ± 0.34 �g/mL). However, the chloroformextract failed to inhibit �-amylase activity (Tamil et al., 2010).Hydro-alcoholic extract of leaves of P. amarus (HAEPA) was stud-ied for its in vivo anti-hyperlipidemic potential using cholesteroldiet induced hyperlipidemia model in rats. Results indicated thatHAEPA possessed significant hypolipidemic activity at doses of 300and 500 mg/kg (Umbare et al., 2009). Oral administration of aque-ous extract of whole plant of P. amarus to Wistar albino rats at dosesof 50, 100 and 200 mg/kg body weight promotes glucose uptake. Inthe oral glucose tolerance test P. amarus extract showed significant(p < 0.05) reduction of serum glucose level based on the hypo-glycemic effect in normal rats. Daily administration of the extractsfor 14 days showed significantly (p < 0.05) reduced AST and ALT andurea at 100 mg/kg body weight when compared with other concen-tration doses and that of the control. However significant (p < 0.05)elevation of AST was observed with animals treated 200 mg/kgbody weight extract. Creatinine of treated animal did not show anysignificant (p > 0.05) difference. Significant (p < 0.05) reduction wasobserved with animal treated 100 mg/kg body weight for urea. Theextract did not produce significant (p > 0.05) effect on heamatolog-ical parameters except that the animals receiving the highest dose(200 mg/kg body weight) of the extracts had significant (p < 0.05)lowered PCV and Hb. All the animals gained some weight, while fortreated animal (50 and 100 mg), the weight gain are significantly(p < 0.05) lower compared to the control (James et al., 2009). Theaqueous extracts of leave and seed of P. amarus at oral dose of 150,300 and 600 mg/kg produced a dose-dependent decrease in thefasting plasma glucose and cholesterol, and reduction in weightsin treated male Swiss mice. The results suggested that the extractscould be enhancing the peripheral utilization of glucose (Adeneyeet al., 2006). Hexane extract of P. amarus had �-amylase inhibitoryproperties. Extraction and fractionation of P. amarus hexane extractled to the isolation of dotriacontanyl docosanoate, triacontanol anda mixture of oleanolic acid and ursolic acid. The compounds weretested in the �-amylase inhibition assay and the results revealedthat the oleanolic acid and ursolic acid (2:1) mixture was a potent�-amylase inhibitor with IC50 = 2.01 �g/mL (4.41 �M) and it con-tributed significantly to �-amylase inhibition activity of the extract.Three pure pentacyclic triterpenoids, oleanolic acid, ursolic acidand lupeol isolated from P. amarus were also shown to inhibit �-amylase (Ali et al., 2006). The methanolic extract of leaves and stemof P. amarus was found to reduce the blood sugar in alloxan diabeticrats at 4th hour by 6% at a dose level of 200 mg/kg body weight and18.7% at a concentration of 1000 mg/kg body weight. Continuedadministration of extract for 15 days produced significant(p = 0.001) reduction in blood sugar. On 18th day after alloxanadministration values were almost same to normal in the group

taking 1000 mg/kg body weight (Raphael et al., 2002b). Aqueousextract of aerial parts of P. amarus, 0.1 and 1 g/kg body weight,significantly enhanced clearance of glucose from the blood as com-pared to controls during an oral glucose tolerance test (OGTT), usingnormal fasted albino rabbits. Both doses had no effect on blood glu-cose in the unfed rabbits. A methanolic extract of the aerial parts,1 g/kg body weight, worsened glucose tolerance causing a signif-icant increase in area under the OGTT and fasting blood glucosecurves (Moshi et al., 1997).

8.16.1. Clinical studyThe glycaemic response to 124.5 ± 9.3 (mean ± SD) g of pancakes

was monitored in 21 non-insulin dependent diabetic (NIDDM)patients while on oral hypoglycaemics, after a week washout periodand after a week twice daily treatment with 100 mL of an aque-ous extract from 12.5 g of powdered aerial parts of P. amarus. Afterthe week washout period, the fasting blood glucose (FBG) andpostprandial blood glucose increased significantly compared withtreatment on oral hypoglycaemics (p < 0.05). After a week herbaltreatment no hypoglycaemic activity was observed (Moshi et al.,2001).

8.17. Immunomodulatory activity

The methanolic extract of P. amarus was investigated for itseffects on the respiratory burst of human whole blood, iso-lated human polymorphonuclear leukocytes and isolated micemacrophages using a luminol/lucigenin-based chemiluminescenceassay. The effect of the extract on chemotactic migration of poly-morphonuclear leukocytes at the concentration of 10, 5, 2.5, 1.25and 0.625 �g/mL was also tested using the Boyden chamber tech-nique. The extract of P. amarus produced the strongest oxidativeburst of polymorphonuclear leukocytes with luminol-based chemi-luminescence, with IC50 values of 0.7 �g/mL (Jantan et al., 2011).Administration of 75% methanolic extract of P. amarus at doses 250and 750 mg/kg body weight significantly reduced the myelosup-pression and improved the WBC count, bone marrow cellularityas well as the number of maturing monocytes. CTX treatment alsoreduced the activity of glutathione system and increased the activ-ity of phase I enzyme that metabolize CTX to its toxic side products.P. amarus administration was found to decrease the activity ofphase I enzyme. P. amarus also increased the cellular GSH and GST,thereby decreasing the effect of toxic metabolites of CTX on thecells. P. amarus did not reduce the tumor reducing activity of CTX. Infact, there was a synergistic action of CTX and P. amarus in reducingthe solid tumors in balb/c mice (Kumar and Kuttan, 2005).

8.18. Nephroprotective activity

Single oral dose (100–400 mg/kg/day) of the leaves and seedaqueous extracts of P. amarus were studied for their protectiveeffects in acetaminophen and gentamicin-induced nephrotoxicWistar rats for 14 days. The acetaminophen nephrotoxic rats,100–400 mg/kg/day significantly (p < 0.05, p < 0.01, p < 0.001) atten-uated elevations in the serum creatinine and blood urea nitrogenlevels in dose related fashion. Similar effects were also recorded inthe gentamicin model of acute renal injury (Adeneye and Adokiye,2008).

8.19. Radioprotective effect

The radioprotective activity of pure compounds isolated fromthe plant P. amarus was studied using rat liver mitochondria andpBR322 plasmid DNA as an in vitro model system. Ellagitannins(amariin, 1-galloyl-2, 3-dehydrohexahydroxydiphenyl (DHHDP)-glucose, repandusinic acid, geraniin, corilagin, phyllanthusiin D)

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and flavonoids (rutin, and quercetin 3-O-glucoside) at the con-centration of 0.1, 0.2, 0.3 and 0.4 nM effectively prevented lipidperoxidation and protein oxidation in mitochondria. The com-pounds also prevented radiation induced single-strand breaks inpBR322 plasmid DNA (Londhe et al., 2009). 75% methanolic extractof aerial parts of P. amarus at concentrations of 250 and 750 mg/kgbody weight on balb/c mice were found to elevate the antioxidantenzymes in the intestine and decrease the lipid peroxidation levels.Histopathological evaluations of the intestine revealed decreaseddamage to intestinal cells. P. amarus was found to protect theclastogenic effects of radiation as seen from decreased numberof micronuclei. The administration of P. amarus was also found todecrease the percentage of chromosomal aberrations (Harikumarand Kuttan, 2007). The radioprotective effect of 75% methanolicextract of aerial parts of P. amarus was investigated in adult balb/cmice. P. amarus extract (750 and 250 mg/kg body weight) signif-icantly increased the total WBC count, bone marrow cellularity,and �-esterase activity as compared to untreated radiation exposedanimals. P. amarus treatment also increased the activity of variousantioxidant enzymes, such as SOD, CAT, GST, GPX, and GR, bothin blood and tissue, which were reduced by radiation treatment.There was also a significant increase in GSH levels of blood and tis-sue. Lipid peroxidation levels, which were increased after radiation,were significantly reduced by P. amarus treatment, both in serumand liver (Kumar and Kuttan, 2004).

8.20. Spasmolytic activity

Potential spasmolytic activity of the extracts of P. amarus wasjudged by their ability to reduce forces of smooth muscle con-traction of a 2 cm long piece of guinea pig ileum induced byEC50 acetylcholine (27 ± 5 �g/L) or EC50 histamine (102 ± 13 �g/L)(Mans et al., 2004).

8.21. Effect on reproductive organs

The effect of a carbamate insecticide, carbofuran was studiedon estrous cycle and follicular growth in virgin female Wistar ratsas well as recovering from the damaged estrous cycle with treat-ment of P. amarus lignans viz. phyllanthin and hypophyllanthin.Since, phyllanthin and hypophyllanthin at the dose of 100 mg/kgbody weight have been found to be systemically transformed intoenterolignan(s), which is known to be responsible for augmentingestrous cycle in rats (Islam et al., 2008b). The aqueous crude extractsof P. amarus were administered to 38-week old sexually maturemale albino to determine the effect of extract on the male repro-ductive organs of these animals. The results from the study revealedthat the aqueous crude extracts of P. amarus caused varying degreesof testicular degeneration as well as reduction in the mean semi-niferous tubular diameter (STD) in the treated rats (Adedapo et al.,2003).

9. Toxicological assessment and contraindications

Histological studies to know the effects of oral administration ofaqueous extract of P. amarus on the kidney of adult Wistar albinorats were carried out at the doses of 500 and 1000 mg/kg bodyweight respectively for 28 days, while the control rats receivedequal volume of distilled water. The rats were sacrificed on day29 of the experiment and kidneys were dissected and quickly fixedin 10% formal saline for routine histological study. The histolog-ical findings indicated that the treated sections of the kidneysshowed hypertrophy of blood vessels, mild-severe infiltrate ofchronic inflammatory cells and varying degrees of tubular necrosiswhen compared to the control sections. The findings indicated thatthe administration of P. amarus extract has some adverse effects

on the kidneys of adult Wistar rats (Andrew and Enogieru, 2011).The effects of chronic administration of aqueous extract of leaves ofP. amarus on histology of kidney of adult Wistar rats indicated thatrats in the treated groups showed some varying degree of distortionand disruption in microanatomy of the kidney including intersti-tial oedema and tubular necrosis. It provides further evidence thatmedicinal use of P. amarus has a potential adverse effect on kid-ney (Adjene and Nwose, 2010). The aqueous extract of whole plantof P. amarus was found to be more cytotoxic than hydroalcoholicextract (IC50 being 89.6 �g/mL vs. 277 �g/mL). Acute and sub-acutetoxicity of the extract in Swiss mice and Wistar rats respectivelyshowed that no significant differences were observed in bodyweight gain and blood glucose level between control and treatedgroups. Clinical biochemistry revealed no toxic effect. Neither grossabnormalities nor histopathological changes in liver, kidney andpancreas were observed. Extract of P. amarus could then be consid-ered to be safe in animals by oral route (LD50 > 5 g/kg) even though itis slightly cytotoxic to the human adenocarcinoma cell line Caco-2(Lawson-Evi et al., 2008). Chemical and cytotoxicity examinationsof the crude methanol extract of the aerial parts of P. amaruswere investigated. The cytotoxicity property of the P. amarus wasevaluated in vitro, using the human ovarian A2780 cancer cell.Bioassay-guided fraction of the crude extract of the P. amarus (IC50value of 31.2 �g/mL), showed that the dichloromethane fractionwas most toxic with an IC50 value of 22.7 �g/mL, whereas thepolar methanol fraction was least cytotoxic with an IC50 value of31.2 �g/mL. This led to the isolation of a new chroman derivativefrom the dichloromethane fraction. The compound exhibited verylittle or no in vitro cytotoxicity with an IC50 value of 16.2 �g/mL, rel-ative to actinomycin, the reference compound, with an IC50 valueof 2.0 �g/mL (Ajaiyeoba and Kingston, 2006). Acute oral admin-istration of P. amarus leaves extract is non-toxic to the rat liver,even at a dose of 5 g/kg body weight. The chronic toxicity stud-ies of P. amarus extracts administration (of 100–800 mg/kg bodyweight) showed the absence of cumulative toxicity as reflected bythe non-significant change in the parameters studied as well asfrom the results of the histological studies (Sirajudeen et al., 2006).Chromatographic fractions obtained from fresh leaves extract ofP. amarus were tested for toxicity on the serum biochemistry ofrats. The six fractions were administered orally to albino rats at thedoses of 400, 800 and 1600 mg/kg body weight for 14 days. Theanimals in the controlled experiment received only distilled waterfor the same number of days. The results revealed that some frac-tions of P. amarus had potentially deleterious effects on the bloodand caused a significant increase in level of ALT, ALP, total bilirubi-nand blood urea nitrogen when compared with control (Adedapoet al., 2005a). The aqueous crude extract was administered orallyto male rats of the Sprague Dawley strain in three groups receiv-ing doses of 400, 800 and 1000 mg/kg but the fourth group servedas a control and received distilled water only. The pathologicalchanges by the aqueous crude extract of the leaves of P. amaruscaused a decrease in the red blood cell (RBC) count, packed cellvolume (PCV), haemoglobin concentration (Hb), but an increase inthe white blood cell (WBC) count. The extract also resulted in anincrease in the levels of aspartate amino transferase (AST), total andconjugated bilirubin, total protein and albumin. The study, how-ever, caused a decrease in the level of alanine amino transferase(ALT). Histopathologically, there were cases of protein casts in thekidney tubules with tubular nephrosis, foci of lymphocytic infil-tration at the portal areas of the liver as well as marked testiculardegeneration with severe disorganization of seminiferous tubules,which were devoid of spermatic cells. A reduction in the weight ofthe experimental animals was also recorded. The results revealedthat P. amarus has potential toxic properties (Adedapo et al., 2005b).P. amarus has demonstrated hypotensive effects in animals andhumans. People with a heart condition and/or taking prescription

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of heart medications should consult their doctor before taking thisplant. It may potentiate insulin and antidiabetic drugs. It containsa naturally occurring phytochemical geraniin. This chemical hasbeen documented with negative chronotropic, negative inotropic,hypotensive and angiotensin-converting enzyme inhibitor effectsin animal studies with frogs, mice and rats (Ueno et al., 1988). Assuch, this plant may potentiate antihypertensive drugs, �-blockersand other heart medications (including chronotropic and inotropicdrugs). It has been considered in herbal medicine to be abortive (athigh dosages) as well as an emmenagogue. Although it is not stud-ied in humans but animal studies do indicated that it has uterinerelaxant effects. It is therefore contraindicated during pregnancy. Ithas been documented with female antifertility effects in mice (theeffect was reversed in 45 days after cessation of dosing) (Rao andAlice, 2001). While this effect has not been documented in humans,the use of the plant is probably contraindicated in women seek-ing pregnancy or taking fertility drugs. Chronic and acute use ofthis plant may be contraindicated in various other medical con-ditions where diuretics are not advised. Chronic long-term use ofany diuretic can cause electrolyte and mineral imbalances; how-ever, human studies with P. amarus (for 3 months of chronic use)have not reported any side effects.

10. Conclusion

The scientific research on P. amarus suggests a huge biolog-ical potential of this plant. It is strongly believed that detailedinformation as presented in this review on the phytochemical andvarious biological properties of the plant might provide detailedevidence for the use of this plant in different diseases. It hasvarious traditional uses that differ from one country to anotherwhereas some important uses for the treatment of jaundice, dia-betes, dysentery, fever, gonorrhea, syphilis and stomachache andskin diseases are almost common. P. amarus, a potent herbalmedicine is attracting researchers since many decades due to itshigh therapeutic value. There is a demand to standardize the prop-erties of P. amarus and their detailed clinical trials. Pharmacologicaland chemical studies have demonstrated that the extracts of theplant possess various pharmacological actions viz. antiviral, anti-inflammatory antimalarial, antimicrobial, anticancer, antidiabetic,hypolipidemic, hepatoprotective and nephroprotective. Owing tothe impressive preclinical therapeutic potential, the plant extractshave been evaluated in human trials for the treatment of HIV, jaun-dice, hypertension and diabetes.

P. amarus is reported to contain lignans, flavonoids, hydrolysabletannins (ellagitannins), polyphenols, triterpenes, sterols andalkaloids. The phytochemicals exhibited different structural char-acteristics with various pharmacological actions. The lignansnirtetralin, phyltetralin or niranthin isolated from P. amarus sig-nificantly inhibited PAF-induced paw oedema formation in mice.Niranthin decreased the specific binding of 3[H]-PAF in mousecerebral cortex membranes. Phyltetralin, nirtetralin and niranthinalso inhibited carrageenan-induced paw oedema and neutrophilinflux. Niranthin also showed the best anti-HBsAg activity whilethe most potent anti-HBeAg activity was observed with henokinin.The presence of high contents of phenolic compounds in the aque-ous extract of P. amarus was found to have strong and significantantioxidant activity. Phyllanthin isolated from aqueous extractof leaves of P. amarus exhibited very high antioxidant activity.Amariin, repandusinic acid and phyllanthusiin D showed higherantioxidant activity. A gallotanin containing fraction and the iso-lated elllagitanins geraniin and corilagin were shown to be mostpotent mediators of antiviral activities. The purified gallataninsgeraniin and corilagin were most active to inhibit HIV-1 replica-tion in Hela CD4+ cells. Mixture of phyllanthin and hypophyllanthin

(1:1) exhibited antitumor activity against EAC in Swiss albinomice. Lignans nirtetralin, niranthin or phyllanthin exerted cyto-toxic effects on K-562 cells. Phyllanthin demonstrated its role inprotection by antagonizing rat liver cell injury induced by ethanol.It effectively alleviated the changes induced by CCl4 in a concen-tration dependence manner. Three pure pentacyclic triterpenoids,oleanolic acid, ursolic acid and lupeol isolated from hexane extractof P. amarus were shown to inhibit �-amylase. More importantly,there have been no side effects or toxicity reports from many yearsof research on this herb. Thus activity guided phytochemical andphytoanalytical information appears to be very useful and mightled to development of novel agents for various disorders and couldbe explored further for commercial purposes. However, there aremany aspects, which need to be explored like well-controlled clin-ical trials using large sample size (large number of patients) for theefficacy and toxicity, the mechanism of biological activity of activeconstituents present in the plant. On the basis of biological activitiesof P. amarus, crude extract and derived phytochemicals and theiruses as pharmacological agents in traditional and modern researchare possible but will first require more clinical trials and productdevelopment. The current evidence is large limited to correlationbetween identified phytochemicals and mode of action for anypharmacological activity. Mechanism of action studies are expectedto lead the way in the discovery of new agents with improvedand intriguing pharmacological properties. This could be achievedby molecular modeling studies involving interaction of bioactivephytochemicals from P. amarus with their respective molecular tar-gets and the extract of P. amarus could be further explored in thefuture as a source of useful phytochemicals for the pharmaceuticalindustry.

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