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 Author's personal copy

Journal of Ethnopharmacology 138 (2011) 286–313

Contents lists available at SciVerse ScienceDirect

Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Review

Phyllanthus amarus: Ethnomedicinal uses, phytochemistry and pharmacology:

A review
Jay Ram Patel a , Priyanka Tripathi b,1 , Vikas Sharma a , Nagendra Singh Chauhan a , Vinod Kumar Dixit a,∗
a
Department of Pharmaceutical Sciences, Dr. Harisingh Gour Vishwavidyalaya, Sagar 470003, M.P., India
b
Shri Ramnath Singh Mahavidyalaya (Pharmacy), Gormi, Bhind 477660, M.P., India

a r t i c l e i n f o a b s t r a c t

Article history: Ethnopharmacological relevance: Phyllanthus amarus Schum. & Thonn. belongs to the family Euphorbiaceae
Received 3 March 2011 is a small herb well known for its medicinal properties and widely used worldwide. P. amarus is an
Received in revised form important plant of Indian Ayurvedic system of medicine which is used in the problems of stomach,
20 September 2011
genitourinary system, liver, kidney and spleen. It is bitter, astringent, stomachic, diuretic, febrifuge and
Accepted 23 September 2011
antiseptic. The whole plant is used in gonorrhea, menorrhagia and other genital affections. It is useful in
Available online 29 September 2011
gastropathy, 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-
Keywords:
Phyllanthus amarus
mation collected from traditional Ayurvedic texts and published literature on ethanomedicinal uses of
Hepatoprotective Phyllanthus amarus in different countries worldwide.
Anti-inflammatory Results: Phytochemical studies have shown the presence of many valuable compounds such as lignans,
Anticancer flavonoids, hydrolysable tannins (ellagitannins), polyphenols, triterpenes, sterols and alkaloids. The
Diuretics extracts and the compounds isolated from P. amarus show a wide spectrum of pharmacological activ-
Nephroprotective ities including antiviral, antibacterial, antiplasmodial, anti-inflammatory, antimalarial, antimicrobial,
Antioxidant anticancer, antidiabetic, hypolipidemic, antioxidant, hepatoprotective nephroprotective and diurectic
Antiviral
properties.
Antibacterial
Conclusion: The present review summarizes information concerning the morphology, ecology, ethnophar-
Antihyperglycemic
Antihypercholesterolemic 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 order
to 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, transcription
factors; AST, aspartate transaminase; CAT, catalase; CD4, T-helper cell; CFA, complete freund’s adjuvant; COX-2, cyclooxygenase; CTX, cyclophosphamide; DHBV, duck
hepatitis 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, Kupffer
cells; LPO, lipid peroxidation; LPS, lipopolysaccharides; MDA, malondialdehyde; MDR, multi-drug resistance; MICs, minimum inhibitory concentrations; mRNA, messenger
ribo 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, Phyllanthus
niruri; P. urinaria, Phyllanthus urinaria; PAF, platelet activating factor; PCV, packed cell volume; PGE2 , prostaglandin E2 ; P.O., per oral; ROS, reactive oxygen species; SCGE, single
cell gel electrophoresis; SEF, supercritical-fluid extraction; SL, silymarin; SOD, superoxide dismutase; STG, serum triglyceride; STZ, streptozotocin; TBARS, thiobarbituric acid
reactive 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
2. Historical perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
3. Botanical description and vernacular names . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
4. Biogeography and ecology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
5. Pharmacognostic characters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
6. Phytochemical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
7. Analytical techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
8. Pharmacological activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
8.1. Antiamnesic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
8.2. Antibacterial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
8.3. Anticancer activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
8.4. Anti-diarrhoeal, gastroprotective and antiulcer activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
8.5. Antifungal activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
8.6. Analgesic, anti-inflammatory, anti-allodynic and anti-oedematogenic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
8.7. Antinociceptic activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
8.8. Antioxidant activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
8.9. Antiplasmodial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
8.10. Antiviral activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
8.11. Clinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
8.12. Aphrodisiac activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
8.13. Contraceptive effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
8.14. Diuretic and antihypertentive activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
8.14.1. Clinical study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
8.15. Hepatoprotective activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
8.16. Hypoglycemic and hypocholesterolemic activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
8.16.1. Clinical study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
8.17. Immunomodulatory activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
8.18. Nephroprotective activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
8.19. Radioprotective effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
8.20. Spasmolytic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
8.21. Effect on reproductive organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
9. Toxicological assessment and contraindications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
10. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309

1. Introduction whole plant is used in gonorrhea, menorrhagia and other genital

affections. It is useful in gastropathy, diarrhoea, dysentery, inter-
Bhumyaamalaki (Phyllanthus amarus Schum. & Thonn., Euphor- mittent fevers, ophthalmopathy, scabies, ulcers and wounds. It is
biaceae), which is widely spread throughout the tropical and also used as a good tonic. The Spanish name ‘chanca piedra’ means
subtropical countries of the world including India is most com- “stone breaker or shatter stone.” In South America, ‘chanca piedra’
monly used in the Indian Ayurvedic system of medicine in has been used to eliminate gall bladder and kidney stones, and to
problems of stomach, genitourinary system, liver, kidney and treat gall bladder infections (Foo and Wong, 1992), cardiovascular
spleen. P. amarus has been described in Ayurveda by the Sanskrit problems (Chevallier, 2000), and also a remedy around the world
name – Bhoomyaamalakee, Taamalakee and Bhoodhatree. It was for influenza (Foo, 1993a). P. amarus has a long history of use in
described to have the properties of Rasa, Guna, Veerya and Vipaaka. the treatment of liver, kidney and bladder problems, diabetes and
The Ayurvedic literature has shown its uses as Kaasahara (antitus- intestinal parasites. In Suriname (Northeastern part of South Amer-
sive), Shwaasahara (antispasmodic, antidyspnoic), Kaphapittahara ica), P. amarus is always sold as fresh and dry plant material in the
(which relieves the Kapha Pitta Dosha), Pipaasaaghna (which herb markets. Decoctions are used in herbal baths and after labor,
relieves Polydipsia), Raktapittahara (hemorrhage disease), Paan- cramps, asthma, uterus complaints and to treat stomachache (May,
duhara (antianemic), Kaamalaahara (which cures jaundice), 1982; Titjari, 1985; Heyde, 1990; Sedoc, 1992; Nanden, 1998). It is
Kushthaghna (indicated in leprosy), Daahaghna (refrigerant, a restoration herb and used as an appetizer and as tonic. It is also
relieves burning sensation), Kshatakshayaghna (indicated in used as colic. The plant, when boiled with the leaves, is considered
Trauma) and Mootrarogahara (which cures urinary disorders). The to be a diuretic and is used in treatment of diabetes, dysentery, hep-
use of P. amarus is gaining momentum because of its novel antiviral atitis, menstrual disorders, and skin disorders (Wessels Boer et al.,
activity against hepatitis B virus and for several other biological 1976; Tirimana, 1987; Heyde, 1968, 1990). Plant extracts are used
activities such as kidney and gallbladder stones, for cold, flu, tuber- as blood purifiers, for light malaria fevers and anemia. It helps to
culosis, and other viral infections; liver diseases and disorders release phlegm (Heyde, 1990) and to combat fever (Nanden, 1998).
including hepatitis, jaundice and liver cancer (Unander et al., 1993). This herb can be used for constipation also (Tjong and Young, 1989).
It also acts against liver cell toxicity and improves the immune P. amarus elaborates different classes of organic compounds of
system of patients and has been found effective against hepatitis medicinal importance including alkaloids, flavonoids, hydrolysable
A (Jayaram et al., 1997). P. amarus is often used in the traditional tannins (Ellagitannins), major lignans, polyphenols, triterpenes,
system of medicine for a variety of ailments including dropsy, dia- sterols and volatile oil. Many lignans were isolated from the
betes, jaundice, asthma and bronchial infections (Foo and Wong, plant viz., phyllanthin (a bitter constituent) and hypophyllan-
1992). In the Ayurvedic system of medicine it is used in problems thin (a non bitter constituent) (Row et al., 1967). The highest
of stomach, genitourinary system, liver, kidney and spleen. It is amounts of phyllanthin (0.7% w/w) and hypophyllanthin (0.3%
bitter, astringent, stomachic, diuretic, febrifuge and antiseptic. The w/w) have been reported in leaves whereas, in the stem these
 Author's personal copy

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

are in minor quantities present (Sharma et al., 1993). Lig- Table 1

Ethnomedicinal uses of P. amarus in different countries worldwide.
nans isolated from P. amarus are phyllanthin, hypophyllanthin,
niranthin, phyltetralin, nirtetralin, isonirtetralin, hinokinin, linte- Region Ethnomedicinal uses
tralin, isolintetralin, demethylenedioxy-niranthin, 5-demethoxy- Amazonia Anodyne, apertif, blennorrhagia, carminative,
niranthin etc., flavonoïds such as gallocatechin, rutin, quercetin- colic, diabetes, digestive, diuretic, dropsy,
3-O-glucopyranoside, phyllanthusiin, quercetin, kaempferol dysentery, dyspepsia, emmenagogue, fever, flu,
3-␤-d-glucopyranoside, kaempferol etc., ellagitannins include gallstones, gonorrhea, itching, jaundice, kidney
ailments, kidney stones, laxative, malaria,
geraniin, amariin, furosin, geraniinic acid B, amariinic acid, amaru-
proctitis, stomachache, tenesmus, tonic,
lone, repandusinic acid A, corilagin, isocorilagin, elaeocarpusin, tumor, vaginitis, vermifuge
phyllanthusiin A, B, C, D and melatonin; securinega-type alka- Aruba Blood purifier
loids such as isobubbialine and epibubbialine and sterol such as Bahamas/Caribbean Antihepatotoxic, cold, constipation, fever, flu,
stomachache, typhoid, flatulence, vermifuge,
amarosterol A, amarosterol B.
appetizer
P. amarus had been reported to have pharmacological Barbados Abortifacient
effects such as antimicrobial, antiviral activities against hepati- Brazil Abortifacient, ache (joint), albuminuria,
tis B, chemoprotective, antimutagenic and hypoglycaemic agent. analgesic, antibacterial, anticancerous,
Methanolic extract of P. amarus exhibited immunomodulatory antidiabetic, anti-inflammatory, antilithic,
antispasmodic, antiviral, aperient, arthritis,
activity. Ellagitannins (geraniin and corilagin) were shown to be
biliary conditions, bladder problems, bladder
the most potent mediators of the antiviral HIV activity. Phyllan- stones, calculi, catarrh (liver and kidney),
thin and hypophyllanthin present in P. amarus exhibited antitumor chologogue, cystitis, deobstruent, diabetes,
activities against EAC in Swiss albino mice, cytotoxic effects on diaphoretic, digestion stimulant, diuretic,
fever, gallbladder, gallstones, gastritis,
K-562 cells, and hepatoprotective and antioxidant effects. The
gastrointestinal problems, gout, hepatitis,
present review assesses the potential of P. amarus in relation to its hydropsy, hypertension, hypoglycemic,
traditional uses and in terms of findings based on modern bioscien- jaundice, kidney, colic, kidney stones, liver,
tific research. The link between conventional remedies and recent malaria, muscle relaxant, obesity, prostatitis,
research in various areas has been well established in other plants purgative, renal colic, renal problems,
stomachic, sudorific, tonic, uric acid excess,
which facilitate to determine effective mode of action of plant
urinary problems, uterine relaxant
derived products. The plant is known to contain several pharmaco- Cuba Oedema and malaria
logical important biomolecules whose efficacy is well established Haiti Carminative, colic, digestive, diuretic, fever,
by several biochemical and pharmacological studies. This review indigestion, malaria, spasmolytic,
stomachache, stomachic, tenesmus
intent to compile various studies on this plant and critically eval-
India Anemia, asthma, astringent, bronchitis,
uates the issues related to ethnopharmacology, phytochemistry, conjunctivitis, cough, deobstruent, dropsy,
pharmacology, clinical studies and toxicology of P. amarus. diabetes, diarrhoea, diuretic, dysentery, fevers,
Table 1 represents ethnomedicinal uses of P. amarus in differ- eye disorders, galactagogue, genitourinary
ent countries and Table 2 represents the ethnomedicinal uses of P. disorders, gonorrhea, hepatitis, jaundice,
leucorrhea, menorrhagia, oligogalactia,
amarus used by different tribes of countries worldwide.
itchness, skin ulcers, sores, swelling,
ringworm, scabies, stomachic, thirst,
tuberculosis, tumor (abdomen), urogenital
2. Historical perspectives tract infections, warts, appetizer, dyspepsia
Indonesia Colic, cough, diuretic, eye diseases (external),
kidney diseases, stomachache, toothache,
P. amarus has been indexed in majority of published phytochem- veneral diseases
ical, pharmacological and ethno-botanical reviews and research Island of North Caicos Fever, prevention of intestinal worms
articles till date with different named. This species, P. amarus has Jamaica Diabetes, dysentery, diuretic, oedema,
been confused with Phyllanthus niruri Linn. In the past, Linnaeus’s gonorrhea, jaundice, stomachache
Malaya Caterpillar sting, dermatosis, diarrhoea,
P. niruri, though well defined in the ‘Hortus Cliffortianus’, became
diuretic, emmenagogue, itching, miscarriage,
confused owing to his erroneous reduction of other species to the purgative, renosis, syphilis, vertigo
synonyms of the former and subsequent misidentification by other Nigeria Malaria, chronic stomach pains, oral or vaginal
authors. The commonest weedy species so mistaken for P. niruri by thrush, alcoholic liver disease, hyperglycaemia,
Muller and others was defined as Phyllanthus swartzii Kosteletzsky urinary tract infection and venereal disease.
Taken with honey as aphrodisiac
in 1836, based on P. niruri of Swartz; but the earliest name of this Peru Calculus, diuretic, emmenagogue, gallstones,
species appears to be P. amarus Schum. & Thonn. For a full discus- hepatitis, kidney pain, kidney problems,
sion on the botanical description and nomenclature of this species kidney stones, renal problems, urinary
reference has been made to the excellent discussion by Webster infections, vermifuge
Trinidad Diuretic, veneral diseases
(1957). The earliest botanical descriptions could be observed from
United States Analgesic, bronchitis, chologogue, deobstruent,
South India, Sri Lanka and Indonesia which have cited Phyllanthus diabetes, fever, gallbladder problems,
urinaria and P. niruri respectively (Van Rhede, 1690; Rumphius, gallstones, gout, hepatitis, hypertension,
1750). In the Ayurvedic system in India, no definitive botanical kidney problems, kidney stones, liver disease,
description exists in the original literature and the plants indicated uric acid excess, urinary tract infections
Elsewhere Analgesic, antipyretic, appetite stimulant,
by many of the old Sanskrit words have been lost in course of his- blennorrhagia, bruises, chologogue, cough,
tory (Handa et al., 1951; Chopra et al., 1958). It was the opinion cuts, diabetes, diarrhoea, diuretic, dropsy,
of Dymock (1886) and Dymock et al. (1893) that the same com- dysentery, dyspepsia, emmenagogue, eye
mon names in a number of Indian languages apply for both P. niruri diseases, fever, gallstones, gonorrhea, itch,
jaundice, kidney disease, kidney stones,
and P. urinaria. Similar reports have stated that even the rather
laxative, malaria, menorrhagia, menstrual
visually different P. urinaria was often used interchangeably with problems, poultice, purgative, rectitis,
those species earlier known as P. niruri in the traditional system stomachache, tonic, tuberculosis, urinary tract
of India (Van Rhede, 1690; Dymock, 1886; Kirtikar and Basu, 1975) infections, vaginitis, venereal diseases
and suggested that the practioners did not differentiate it according
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J.R. Patel et al. / Journal of Ethnopharmacology 138 (2011) 286–313 289

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

S.N. Place Local name Plant part Disease Method of use Reference
used

1 Dharapuram Taluk, Keelanelli Whole plant 1. Migraine (1) Whole plant is boiled in Balakrishnan et al. (2009)
Tamil Nadu, India 2. Jaundice gingelly oil, filtered and
applied on the head
(2) The fresh root is used with
water. Paste or fresh roots are
given orally
2 Paliyar tribals in Keelanelli Leaves Jaundice Leaf paste is given internally Ignacimuthu et al. (2008)
Theni district of
Tamil Nadu, India
3 Eastern part of Bhumiamla Whole plant, Gonorrhea and Decoction of leaves, sugar and Upadhyay et al. (2010)
Rajasthan, India leaves syphilis cumin seeds are taken orally to
Skin diseases, treat gonorrhea and syphilis.
malaria Leaves are crushed with salt to
make paste and applied locally
against skin diseases. Plant is
crushed into paste, mixed with
seed powder of pepper, candy
and water and taken as a
refrigerant. Decoction of whole
plant is taken as an
antimalarial
4 Uttara kannada, Nelli Whole plant Malaria Not stated Kuppusamy and Murugan (2010)
Western Ghats,
India
5 Eastern region of Nelanelli Root juice Jaundice Root juice is taken orally with Rajakumar and Shivanna (2009)
Shimoga district, cow’s milk early in the
Karnataka, India morning for 1 week
6 Dindigul District, Kizhnelli Leaves Menstrual problem Leaf extract with milk and Samuel and Andrews (2010)
Tamil Nadu, Indian onion is given during night,
three times once in 3 days
7 Buldhana district; Bhui-awala Whole plant Jaundice Extract, one spoonful per day Ahirrao and Patil (2010)
Maharashtra, for 3 days
Indian
8 North Andaman Nallesari Whole plant Jaundice Handful of leaves is crushed Prasad et al. (2008)
Island, India with a pinch of turmeric and
one teaspoonful of extract is
taken orally for 3–5 days
9 Sivagangai district, Keelaanelli Leaves 1. Diabetes Diabetes: Shanmugam et al. (2009)
Tamil Nadu, India 2. Jaundice (A) Leaves of Piper betle,
Cynodon dactylon, Azadirachta
indica and P. amarus are dried
and powdered with the stem
park of Syzygium cumini. The
powder is boiled in water and
the extract is given orally
(B) Leaf extracts of A. indica
and P. amarus are mixed and
given orally
Jaundice:
(A) Leaves of C. dactylon and P.
amarus are grounded with the
fruits of Piper nigrum and
extracted. The extract is given
orally
(B) Leaves of Eclipta alba, P.
amarus and Leucas aspera are
grounded and extracted. The
extract is given orally
(C) Leaf extracts of C. dactylon
and P. amarus are mixed and
given orally
10 Shimoga district of Nela nelli Leaves 1. Jaundice (1) Leaf paste with cardamom Mahishi et al. (2005)
Karnataka, India (Bhumyamalaki) 2. Chronic is taken internally, two tea
dysentery spoons daily
(2) Leaves are ground with
Acacia Senegal leaves, add
sugar and give orally, or tender
leaves ground with cow’s milk
curd given orally, for 2–5 days,
before food
11 Kattunaykas tribes Kila nelli Whole plant Jaundice 15 mL whole plant juice is Udayan et al. (2007)
of Mudumalai taken internally in empty
Wildlife Sanctuary, stomach along with one
Nilgiris district tumbler goat’s milk against
Tamil Nadu, India jaundice
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290 J.R. Patel et al. / Journal of Ethnopharmacology 138 (2011) 286–313

Table 2 (Continued)

S.N. Place Local name Plant part Disease Method of use Reference
used

12 Kancheepuram Keezhanelli Leaves Jaundice Fresh leaves are ground and Muthu et al. (2006)
district Tamil mixed with a cup of cow or
Nadu, India goat’s milk and taken
internally to cure jaundice
13 India Bhumi amalaki Whole plant Liver disease, 20% whole plant and 10% Samy et al. (2008)
dyspepsia, Plumbago zeylanica (roots). 4 g
anorexia, moderate of powdered mixer is given to
constipation, the patient twice daily, half an
chronic colitis, hour before meals with water
irritable bowel
syndrome, urinary
tract infection
14 Northern India Bhui amla Whole Jaundice, Not stated Kala et al. (2006)
aphrodisiac,
dysentery
15 Sitamata Wildlife Not stated Leaves Syphilis, Leaf paste and decoction of Jain et al. (2005)
Sanctuary of gonorrhea, leaves
Chittorgarh and jaundice
Udaipur district
Rajasthan, India
16 Esan North East Abenaghe Leaves Stomachache Ground leaves with pepper and Idu et al. (2008a)
local govt. area of salt. Half of cup is taken twice
Edo State, Nigeria daily
17 Delta State Nigeria Ibuko-oyeke Leaves Stomachache The leaves are infused in Idu et al. (2008b)
alcohol and drunk as a remedy
for stomachache
18 Akwa Ibom State in Oyomokiso, Leaves Malaria Boiled in water as decoction. Ajibesin et al. (2008)
Nigeria aman keeden Use internally 4 times a day for
5 days
19 South West Nigeria Eyin olobe Whole plant Diabetes Decoction Abo et al. (2008)
20 West Africa Hlinvi Arial Diabetes, fever, Not stated Adjanohoun et al. (1986)
malaria
21 Semi-arid Quebra-pedra Leaves Kidney problems Decoction of leaves soaking Cartaxo et al. (2010)
Northeasten Brazil drink
22 Dangme West Ofobi okpabi Whole plant Malaria Boil about 50 g of plant in 1 L of Asase et al. (2010)
district of Ghana water and drink a cupful of
decoction three times daily
after meals until recovered.
Children should take half of the
dose. Sweeten with honey or
sugar if desired. The decoction
may cause dizziness
23 Surinamese Fini bita Whole plant Stomach-ache, Whole plant is boiled in water Van Andel and Westers (2010)
migrants in cleaning uterus, or soaked in alcohol and drunk
Netherland laxative, health to purify the blood. These
promotion, disease so-called BITAS were said to
prevention promote one’s health, purity of
blood and prevent and cure
diseases like malaria, skin
sores, diabetes and pimples.
Bitter tonics are also reported
to be popular among
HIV-positives to support their
body function
24 Akha people in Yu Jae Leaves Rashes, itches Poultice Inta et al. (2008)
Thailand and China

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 difficult
problems in organising ethnobotanical data on Phyllanthus was the
identification of the correct species for citations under P. niruri. The
true P. niruri Linnaeus type of the genus was collected in Barbados
and described in 1738 by Linnaeus (Linnaeus, 1738). Specimens of
P. niruri have never been confirmed outside the America (Webster,
1957). According to Mitra and Jain (1985) of the Botanical Survey
of India, the P. niruri in Flora of British India is indeed a mixture of
three distinct species viz. P. amarus Schum. & Thonn., Phyllanthus
fraternus webster and Phyllanthus debilis Klein ex willd, the true P.
niruri Linn being endemic to West Indies. The specimen of P. debilis
was collected from Madras in 1799 (Webster, 1957). However it is
observed that P. amarus predominates over P. debilis in Madras area
suggesting that it is an introduced species over the years. Samples of
the plants collected from the Madras area in 1983–1988 as P. niruri
were predominantly P. amarus with occasional plants of P. debilis
as identified by Grady webster, University of California (Unander
et al., 1991) during the collaborative studies with the group led by
Baruch 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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J.R. Patel et al. / Journal of Ethnopharmacology 138 (2011) 286–313 291

Table 3
Vernacular 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 amla
3 Bengali Bhuiamala, Sadahazurmani
4 Gujarathi Bhonya anmali
5 Marathi Bhuivali
6 Oriya Bhuiaola, badianala
7 Bihari Muikoa, Kantara, Pirikantaru
8 Telugu Nela uirika, Nelavusari
9 Kanada Nela – nelli, Kirunelli
10 Malayalam Kizhkkayinelli, Kilanelli
11 Rajasthani Gugario
12 Sanskrit Bhumyaamlaki, Bhoodhatree, Thamalaki
13 English Black catnip, Carry me seed, Child
pick-a-back, Gale of wind, Gulf leaf flower,
Hurricane weed, Shatterstone, Stone breaker
Fig. 1. P. amarus plant. 14 French Poudre de plomb (Ivory coast)
15 German Weisse Blattblume
16 Spanish Yerba magica (Cuba)
Cataphylls, stipules 1.5–1.9 mm long, deltoid acuminate blade 17 African names Ahlivi (Mina-Togo), Bomagua kene (Ivory
1–1.5 mm long, subulate acuminate. Leaves 3–11 × 1.5–6 mm ellip- coast), Bounou (Ivory coast), Bounou honlin
(Ivory coast), Hinlinew (West Africa),
tic oblong obovate, oblong, or even obovate, obtuse, or minutely
Mokichinento (Korokoro–East Africa),
apiculate at apex, obtuse or slightly inequilateral at base, petioles Tsekulemegbe (Ouatchi-Togo)
0.3–0.5 mm long, stipules 0.8–1.1 mm long triangular accuminate. 18 North, Central and Black catnip, carry-meseed, chanca piedra,
Flowers minutes, proximal 2–3 axis with unisexual cymules, each South American djari-bita, egg woman, fini-bita, flor
consisting of 1 male and 1 female or 2–3 males and female or 1 names escondida, gale-of-(the)-wind, hurricane
weed, quebra-pedra, quinine creole, quinine
male and 2 females flower or combination thereof; male flowers
weed, seed-under-leaf, stone breaker and
pedicals at anthesis ca 1 mm long. Calyx lobe 5, subequal each yerba de la nina (Morton, 1981)
ca 0.7 × 0.3 mm elliptic or oblong elliptic and abruptly acute at
apex hyaline with unbranched mid ribs. Disc segments 5, roundish
stames 3 (rarely 2): filaments connate into a column 0.2–0.3 mm Bahamas, (Morton, 1981; Tirimana, 1987), Philippines or India
high autheros sessile a top dehiscing longitudinally. Female flow- (Chevallier, 2000). P. amarus is a common pantropical weed that
ers; pedicles 0.8–1 mm long, obtusely 4-gonous, dialated above, grows well in moist, shady and sunny places (Cabieses, 1993;
ca 1.5 mm in fruits, calyx five lobes, subequal. Lobes sometimes Nanden, 1998). P. amarus is distributed all over India and is con-
toothed at apex. Styles 3, free, more or less spreading, and shallowly sidered as the most widely occurring Phyllanthus species in India
bifid at apex; arms divergent (Mitra and Jain, 1985). The seed cap- (Chowdhury and Rao, 2002). The presence of dioceous cymules
sules on stalks are 1–2 mm long, round, smooth, 2 mm wide, with (Mitra and Jain, 1985) at the end of the branches is considered to
six seeds. When the fruits burst open the seeds are hurled away. be a unique character, though it resembles in many respects its
Seeds are triangular (like an orange segment); light brown, 1 mm close relatives, P. debilis and P. fraternus of the same sub-section
long, with 5–6 ribs on the back (Morton, 1981; Wessels Boer et al., Swartziani (Webster, 1994). This is the only sub-section in the sec-
1976) (Fig. 1 P. amarus plant). The vernacular names of P. amarus tion Phyllanthus, which consists of most widespread herbaceous
has been given in Table 3. species throughout the tropics (Jain et al., 2003).

4. Biogeography and ecology 5. Pharmacognostic characters

The genus Phyllanthus (Euphorbiaceae) consists of about 6500
species 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 to
North, Central and South America (Unander et al., 1995). The name
‘Phyllanthus’ means “leaf and flower” because the flower, as well
as the fruit, seems to become one with the leaf (Cabieses, 1993).
The taxonomic revision on this genus by Webster included closely
related genera P. amarus, under the sub-section Swartiziani of the
section Phyllanthus. The nomenclature, taxonomic distinctness
and close relatives of P. amarus were addressed in detail based on
morphology and geographical distribution (Chowdhury and Rao,
2002). It is said to be related to P. abnormis, which is endemic to
sandy areas in Texas and Florida of southern USA. It is therefore
most likely that P. amarus originated in the Caribbean area as a
vicarious species of P. abnormis of the southern United States and
has spread around the tropics by trading vessels (Webster, 1957). P.
amarus is widely distributed in all tropical and subtropical regions
of the world. Paleobotanical studies have not found the exact
geographic origin of this plant. It is indigenous to the rainforests
of the Amazon and other tropical countries like India, China, and
Various species of Phyllanthus are being sold in India under the
trade name ‘Bhuiamlki’. During market surveillance of herbal drug,
it was observed that almost all the commercial samples, either
comprise 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 assessed
in raw drug trade of Phyllanthus in southern India using morpho-
taxonomical characters and molecular analysis. The morphological
analysis 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 devoid
of admixtures. The remaining 24% of the shops had five different
species 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 chloroplast
DNA region, psbA-trnH. The trade sample identities were validated
and confirmed by these species specific DNA barcodes (Srirama
et al., 2010). The detailed botanical evaluation of three species was
carried out with the aim to establish the identification markers of
this plant. Some reliable diagnostic characters were specified as,
number of sepals five in P. amarus and six in P. fraternus and P.
 Author's personal copy
292 J.R. Patel et al. / Journal of Ethnopharmacology 138 (2011) 286–313
maderaspatensis; the female flowers of P. maderaspatensis are much
larger in size as compared to P. amarus and P. fraternus, in which
these are almost equal in size; TS of branchlet circular, leaf margin
crenate in P. amarus and in P. fraternus branchlets have six notches
with serrated leaf margins, while the leaf margins are crenate and
bulbous in P. maderaspatensis. Preliminary phytochemical screen-
ing of the extractives showed that the triterpenoids were noticed in
the hexane extract of P. amarus and P. fraternus and in chloroform
extracts of P. maderaspatensis, however, glycosides in the alcoholic
extract of P. amarus and P. fraternus only. Likewise, the percent-
age of sugar and tannins were quite high in P. maderaspatensis and
tannins 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 light
green, greyish green, grey, faint green, light green, faint pink, light
green, greyish green, respectively. However, in P. maderaspatensis
only three bands at Rf 0.04, 0.28 and 0.38 were observed while one
major band of bright blue colour was observed at Rf 0.9 in P. frater-
nus only. Therefore, all these three species could be differentiated
on the basis of macro and microscopic characters, physico-chemical
values, high pressure thin layer chromatography (HPTLC), finger-
print profile, and detection of phyllanthin and hypophyllanthin
as marker components (Khatoon et al., 2006). Macromorphology,
micromorphology, histochemical and physical pharmacognostic
studies of P. amarus revealed certain diagnostic uncommon char-
acters: basal submarginal venation formed by curving of almost
unbranched lateral veins, 4–6 angled cortical fibres (TS), 1–2 seriate
xylem 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 frequency
of crystals in leaf (87.5 mm−2 ), stomatal index, palisade ratio, etc.
Additionally, distribution of alkaloidal reaction and protein in the
secondary xylem, extractive values, ash values, UV fluorescence
were 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 active
constituents of P. amarus are lignans (phyllanthin, hypophyllan-
thin, nirurin niranthin, phyltetralin, niranthine, nirtetralin etc.
(Morton, 1981; Chevallier, 2000; Srivastava et al., 2008; Kassuya
et al., 2006; Huang et al., 2003; Maciel et al., 2007; Singh
et 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.) (Houghton
et al., 1996; Kassuya et al., 2006; Foo and Wong, 1992), sterol
(amarosterol-A, amarosterol-B etc.) (Ahmad and Alam, 2003) and
volatile oil (linalool, phytol etc.) (Moronkola et al., 2009). A detailed
extraction, isolation and characterization method was optimized
for phyllanthin (Hamrapurkar et al., 2009).
The oils obtained from P. amarus were analyzed for its
constituents 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 using
flame ionization detector (FID) at the temperature 300 ◦ C. Results
from analysis of the oil of P. amarus revealed that 82 identified
compounds were responsible for 87.6% of the oil content. The oil
was 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. Two
components with relative retention index (RRI) of 2692 and 2700
were tentatively identified as nonyl phenol isomers. The classes of
compounds present in P. amarus oil are monoterpene hydrocar-
bons (0.2%), oxygenated monoterpenoids (11.0%), sesquiterpene
hydrocarbons (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%) (Moronkola
et al., 2009). Two new lignans, 3-(3,4-dimethoxy-benzyl)-4-
(7-methoxy-benzo[1,3]dioxol-5-yl-methyl)-dihydrofuran-2-one
and 4-(3,4-dimethoxy-phenyl)-1-(7-methoxy-benzo[1,3]dioxol-
5-yl)-2,3-bis-methoxymethyl-butan-1-ol were isolated from
the leaves of P. amarus (Singh et al., 2009). A phytochemical
investigation of methanolic extract obtained from the whole
plant 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). Phytochemical
evaluation of P. amarus showed to contain high level of saponins
and tannins at 24.05 and 17.50%, respectively, but with low
content of cyanogenic glycosides (1.46%). The plant contained
high 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 150.30
and 228.20 mg per 100 g dry weight, respectively, while magne-
sium, calcium, iron and phosphorus were all low at 2.40, 1.60, 1.65
and 1.00 mg per 100 g dry weight, respectively (Igwe et al., 2007).
The crude extract of phyllanthin was obtained from P. amarus
using solvents of varied polarity. The presence of pyrrolizidine
type of alkaloids was reported in extract of P. amarus. These are
securinine, dihydrosecurinine, tetrahydrosecurine, securinol-B,
phyllanthine, allosecurine, nor-securinine etc. (Kassuya et al.,
2006). The whole plant of P. amarus has afforded new secosterols
named as amarosterol-A characterized as 13, 14-seco-stigma
5(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 the
dominant ones in the majority of the plants investigated. Out of
the 19 species studied the highest concentration of polyprenols
was observed in P. amarus (0.1–0.3%, dry weight) (George et al.,
2001). Two new securinega types of alkaloids, isobubbialine
and epibubbialine were isolated from the leaves of P. amarus, as
well as the three known alkaloids, phyllanthine, securinine and
nor-securinine (Houghton et al., 1996). Chemical examination of
the polar extractives of the aerial parts of P. amarus led to the
isolation of amariinic acid, a novel ellagitannin, together with
1-O-galloyl-2, 4-dehydrohexahydroxydiphenoyl-glucopyranose,
elaeocarpusin, repandusinic acid A and geraniinic acid B. The
structure of amariinic acid was established as the ring-opened
oxidized cyclohexentrione moiety of dehydrohexahydroxydiphe-
noyl attached to 0–4 of the glucose core (Foo, 1995). A novel
cyclic metabolite named amarulone was isolated from P. amarus
(Foo, 1993a). Amariin, a novel hydrolysable tannin together with
geraniin, corilagin, 1,6-digalloylglucopyranoside as well as rutin
and quercetin-3-O-glucopyranoside were isolated from the polar
fraction of P. amarus. The structure of amariin was established as
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J.R. Patel et al. / Journal of Ethnopharmacology 138 (2011) 286–313
Table 4
Phytoconstituents reported in P. amarus.
S. No. Secondary metabolites
1 Lignans
2 Flavonoids
3 Hydrolysable tannin Tannin precursors
(Ellagitannins)
Simple tannins
Complex tannins
4 Alkaloids
5 Triterpenes
6 Sterols
7 Volatile oil
1-galloyl-2,4: 3,6-bis-dehydrohexahydroxydiphenoyl-
glucopyranoside in which the cyclohexenetrione portion of
the dehydrohexahydroxydiphenoyl moieties were linked to the
O-3 and O-4 of the glucose moiety (Foo, 1993b). An unusual
ellagitannin, phyllanthusiin D was isolated from the biologically
active polar fraction of P. amarus. Its structure was established
as 1-galloyl-2, 4-(acetonyl-dehydrohexahydroxydiphenoyl)-3,
6-hexahydroxydiphenoyl-glucopyranoside (Foo and Wong, 1992).
Table 4 represents the secondary metabolites with their respective
phytochemicals present in P. amarus.
7. Analytical techniques
A HPLC analysis method was developed and validated to obtain
an easily performable and inexpensive method for the standard-
ization of crude extract of P. amarus and ellagic acid. Ethanolic
extract of whole plant of P. amarus was dissolved in DMSO, ultra-
sonicated for 15 min, and diluted with 50% methanol. Analysis was
performed using water and methanol containing 0.06% TFA and
the peaks were detected at 254 nm. Ellagic acid showed a linear
relationship in the range of 1.74–20.91 ␮g/mL and a single-point
calibration was allowed. The method was shown to be precise with
respect to time (RSD of 1.84%, 3 days, n = 6) and concentration (RSD
of 2.54%, three levels, n = 6). The overall mean content of ellagic acid
was 2.06%. A recovery experiment was performed and it showed
an accuracy of 100.4% (Dhooghe et al., 2011). A simple, specific and
precise RP-HPLC method has been developed and validated for the
estimation of phyllanthin, present in P. amarus. Furthermore, the
developed method was also used to successfully quantify the phyl-
lanthin in plant extract. The mobile phase optimized for RP-HPLC
293
Phyto-constituents References
Phyllanthin, hypophyllanthin, niranthin, Morton (1981), Sharma et al. (1993),
phyltetralin, nirtetralin, isonirtetralin, Chevallier (2000), Srivastava et al.
hinokinin (2008), Kassuya et al. (2006), Huang
et al. (2003), Singh et al. (2009)
Lintetralin, isolintetralin, Maciel et al. (2007)
demethylenedioxy-niranthin,
5-demethoxy-niranthin
(3-(3,4-dimethoxy-benzyl)-4-(7-methoxy- Singh et al. (2009)
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
Rutin, astragalin, kaempferol, Morton (1981), Foo and Wong (1992),
quercetin-3-O-glucoside, quercetin, quercitrin Foo (1993a, 1995), Londhe (2008)
Gallic acid, ellagic acid, gallocatechin Foo (1993a, 1995)
1,6-digalloylglucopyranose, 4-O-galloylquinic Foo (1993a, 1995)
acid
Geraniin, amariin, furosin, geraniinic acid B, Foo and Wong (1992), Foo (1993a,
amariinic acid, amarulone, repandusinic acid A, 1995)
corilagin, isocorilagin, elaeocarpusin,
phyllanthusiin A, B, C and D, melatonin
Securinine, dihydrosecurinine, Houghton et al. (1996), Kassuya et al.
tetrahydrosecurinine, securinol, phyllanthine, (2006)
allo-securine, nor-securinine, epibubbialine,
isobubbialine
4-methoxy-nor-securinine, 4-methoxy Foo and Wong (1992)
dihydrosecurinine,
4-methoxytetrahydrosecurinine, 4
hydrosecurinine
Phenazine and phenazine derivatives Foo (1993a)
2Z, 6Z, 10Z, 14E, 18E, 22E-farnesylfarnesol Maciel et al. (2007)
Lupeol, phyllanthenol, phyllanthenone, Foo and Wong (1992)
phyllantheol, Oleanolic acid, ursolic acid
Amarosterol A, amarosterol B Ahmad and Alam (2003)
Linalool, phytol Moronkola et al. (2009)
was methanol–water 66:34 (% v/v) which was very simple and cost
effective. The detection was carried out using variable wavelength
UV–vis detector set at 229 nm. Linearity for the developed method
was found over the concentration range 1–50 ␮g/mL with a correla-
tion coefficient of 0.999 (Alvari et al., 2011). An online-hyphenated
high-performance liquid chromatography-photodiode array-mass
spectrometry (HPLC-PDA-MS) analytical method was developed
for the simultaneous determination of six lignans of therapeutic
importance in four Phyllanthus spp. (P. amarus, P. maderaspatensis,
P. urinaria, and Phyllanthus virgatus). HPLC with monolithic reverse
phase silica column (4.6 × 100 mm) and simple isocratic elution of
methanol–water mixed with dioxane facilitated the separation of
lignans of diverse nature such as diarylbutyrolactone, tetrahydro-
furan, isomeric aryltetralin, and diarylbutane type for quantitative
analysis. Targeted lignans viz. heliobuphthalmin lactone, virga-
tusin, hypophyllanthin, phyllanthin, nirtetralin, and niranthin were
confirmed unambiguously in four Phyllanthus species by their
abundant molecular adduct ions, retention time, UV, and mass
spectra as compared with those of reference compounds. Advan-
tages and limitations of both detection techniques for qualitative
(fingerprinting) and quantitative analysis of the above mentioned
lignans in four Phyllanthus spp. are discussed. The method was vali-
dated following international guidelines. The described method can
be utilized for assays and stability tests of P. amarus extracts as well
as traditional Indian medicine based on Phyllanthus herb (Shanker
et al., 2011). Phyllanthin was extracted from the plant P. amarus
by Soxhlet and supercritical-fluid extraction (SFE) and isolated by
column chromatography. A HPTLC method was established and
validated for analysis. The method was used for quantitative anal-
ysis and macro and micro fingerprinting analysis of phyllanthin.
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294 J.R. Patel et al. / Journal of Ethnopharmacology 138 (2011) 286–313
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 from
the aerial parts of P. amarus by silica gel column chromatography
employing gradient elution with hexane–ethyl acetate solvent mix-
ture. It was obtained in high yields (1.23%), compared to reported
procedures and the purity was ascertained by HPTLC and reversed
phase HPLC analysis (Krithika et al., 2009). A sensitive, selective,
and robust HPTLC method using chiral TLC plates for qualitative
and quantitative analysis of phyllanthin, hypophyllanthin, niran-
thin, and nirtetralin, the active lignans of Phyllanthus species, was
developed and validated. The effectiveness and role of various sta-
tionary phases viz TLC silica gel 60F254 , HPTLC silica gel 60F254 , and
chiral TLC plates in the quantitation were evaluated. A precoated
chiral TLC plate was found suitable for the simultaneous analysis of
four 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 the
above compounds was carried out in reflection absorption mode at
620 nm. Optimized chromatographic conditions provided well sep-
arated compact bands for the tested lignans. The calibration curves
were found linear in the concentration range of 100–500 ng/band
(Srivastava et al., 2008). Sharma et al. (1993) have developed a
reversed phase HPLC procedure for standardizing P. amarus on the
basis of its two bioactive lignans, phyllanthin and hypophyllanthin.
The method has been found to be sensitive, precise and efficient to
record more than 98% recovery of these two lignans. The leaves
of P. amarus were found to contain the highest amount of phyl-
lanthin (0.7% w/w) and hypophyllanthin (0.3% w/w) as compared
to other parts of the plant. A method for amount determination
of gallic acid in P. amarus capsules was established by HPLC (Guo
et al., 2007). Methanolic extract of P. amarus and standard phyllan-
thin and hypophyllanthin were developed in the chromatographic
conditions. They showed the presence of standard phyllanthin and
hypophyllanthin at Rf 0.3 and 0.4 respectively, by densitometric
scanning at 254 nm (Mukherjee et al., 2006). Separation of phyl-
lanthin and hypophyllanthin was carried out on silica gel 60F254
layers eluted with hexane:acetone:ethyl acetate (74:12:8), and the
analytes were visualised through colour development with vanillin
in concentrated sulfuric acid and ethanol. Scanning and quantifica-
tion of spots was performed at 580 nm. Recoveries of phyllanthin
and hypophyllanthin were 98.7 and 97.3%, respectively (Tripathi et
al., 2006). Dhalwal et al. (2006) developed a HPTLC densitometric
method 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 in
P. amarus, an isocratic, reversed phase (RP) HPLC procedure has
been adopted using a mixture of pH 2.8 phosphate buffer and ace-
tonitrile as mobile phase, Cyano (CN) column as stationary phase
and UV detector. The developed method showed high resolution
(R = 1.9), accuracy and reproducibility (Murali et al., 2001). The
invention concerns an improved procedure for the extraction and
isolation of phyllanthin from P. amarus by pulverization of the dried
leaves with calcium carbonate, percolating the ground component
using 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 an
n-hexane fraction, and subjecting the phyllanthin-rich fraction to
silica gel column chromatography, and further purifying the phyl-
lanthin by crystallization (Chaudhuri et al., 2001). A HPTLC method
was described for the simultaneous determination of the bioac-
tive lignans, phyllanthin and hypophyllanthin from the dried whole
plant powder of P. amarus (Sane et al., 1997). A TLC – densitometric
method has been developed for the estimation of the two lignans
of P. amarus phyllanthin and hypophyllanthin (Deb and Mandal,
1996). Air-dried leaves of P. amarus were extracted with 0.5 M HCl
and filtered. The filtrate was basified with 10% aqueous Na2 CO3
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J.R. Patel et al. / Journal of Ethnopharmacology 138 (2011) 286–313
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-positive
zones. 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, respectively
by comparison of their spectral data with standard values. Two
new securinega-type alkaloids, isobubbialine and epibubbialine
were isolated from the leaves of P. amarus, as well as the three
known alkaloids, phyllanthine, securinine and nor-securinine. The
structures of the unknown compounds were determined by means
of UV, IR, mass and NMR spectroscopy (Houghton et al., 1996).
The 70% aqueous acetone extract of the aerial part of P. amarus
was fractionated over a column of Sephadex LH20 using aqueous
methanol to yield various fractions subjected to repeate chro-
matographic treatment alternating between MCI-gel CHP-20 using
aqueous methanol and Sephadex LH20 with aqueous ethanol led
to the isolation of amariinic acid, a novel ellagitannin, together
with 4-O-galloylquinic acid, elaeocarpusin, furosin, geraniinic acid
B, amariinic acid and potassium salt of repandusinic acid B and their
structures were established on the basis of chemical and 13 C NMR
spectrum (Foo, 1995). An unusual ellagitannin was isolated from
the biologically active polar fraction of aerial part of P. amarus. The
hydrolysable tannin fraction was obtained by column chromatog-
raphy of the water soluble portion of the 70% aqueous acetone
extract of the aerial parts of the plant on Sephadex LH20 using aque-
ous methanol. Further chromatography of the fraction on MCI-gel
CHP-20 yielded phyllanthusiin D as an amorphous powder which
gave 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. amarus
was evaluated on cognitive functions and brain cholinesterase
activity in male Swiss albino mice. P. amarus (50, 100 and
200 mg/kg) produced a dose-dependent improvement in memory
scores of young and older mice. P. amarus also reversed successfully
the amnesia induced by scopolamine (0.4 mg/kg, i.p.) and diazepam
(1 mg/kg, i.p.). Interestingly, brain cholinesterase activity was also
reduced. 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 passive
avoidance paradigm. [6]-gingerol (25 and 50 mg/kg, p.o.) and phyl-
lanthin (7.5 and 15 mg/kg, p.o.) significantly attenuated amnestic
deficits induced by diazepam, scopolamine (0.4 mg/kg, i.p.) and
natural aging. [6]-gingerol and phyllanthin increased step down
latencies significantly in the aged mice, diazepam and scopolamine
induced amnesic mice as compared with piracetam (200 mg/kg,
i.p.). [6]-gingerol and phyllanthin significantly decreased whole
brain acetyl cholinesterase activity (Joshi and Parle, 2006).
295
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, Salmonella
typhi, Staphylococcus aureus and Candida albicans using the agar-
cup diffusion protocol. The aqueous and methanolic extracts of
P. amarus were active against all the test microorganisms. The
methanolic extract of P. amarus also showed a broad spectrum of
activity with a minimum inhibitory concentration of 1.56 mg/mL
against all the test microorganisms. The extracts were also screened
for secondary metabolites and the result indicated the presence
of 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. amarus
against S. aureus (gram-positive) with a MIC value of 17.7 ␮g/mL
(Eldeen et al., 2011). The antibacterial activity of ethanolic extracts
of the root and leaf of P. amarus was assessed against extend
spectrum ␤-lactamase (ESBL) producing E. coli isolated from the
stool samples of HIV sero-positive patients with or without diar-
rhoea using Bauer disc diffusion method. The strains isolated
from 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 from
8.0 ± 0.33 to 25.0 ± 1.50 mm against ESBL E. coli while the mean
zones of inhibition the of the leaf extracts ranged from 8.0 ± 0.50
to 26.0 ± 1.00 mm against ESBL E. coli. The root extracts showed
the highest zone of inhibition (25 ± 1.50 mm) against ESBL E. coli
at 80 mg/mL while the leaf extracts showed the highest zone of
inhibition 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 from
5–20 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, Staphylococcus
hominis, S. aureus, Staphylococcus haemolyticus, Micrococcus luteus,
Bacillus lentus, Bacillus firmus and Pseudomonas stutzeri using agar-
well diffusion method. Results revealed that P. amarus exhibited
remarkable 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 leaves
of P. amarus at the concentration of 100 mg/mL were tested against
E. coli, Streptococcus spp, Klebsiella spp, Pseudomonas spp and Staphy-
lococcus spp. aqueous and methanolic extract of P. amarus. The
crude extracts were observed to inhibit the growth of E. coli, Strep-
tococcus and S. aureus. Methanolic extract of the plant was more
effective (6–11 mm) than aqueous extracts (5–10 mm) inhibiting
the growth of pathogenic bacteria but was less potent when com-
pared to that of ofloxacin (19 mm) and ciprofloxacin (21 mm) used
as positive controls (Okoli et al., 2009). 80% methanolic extract of
whole 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. Ampicillin
was used as standard (Komuraiah et al., 2009). The essential oil and
its fractions obtained from fresh leaves and seeds of P. amarus were
tested on 12 microorganisms including yeast, gram-positive and
gram-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 (ATCC
25923), S. aureus (isolate) and C. albicans. All the samples of essen-
tial oil and fractions demonstrated activity (11–20 mm diameter
zone of inhibition) against the microorganisms except P. aeruginosa.
0.05% ciprofloxacin (≥21 mm diameter zone of inhibition) was used
as positive control (Ogunlesi et al., 2009). Crude aqueous (hot and
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296 J.R. Patel et al. / Journal of Ethnopharmacology 138 (2011) 286–313

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

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J.R. Patel et al. / Journal of Ethnopharmacology 138 (2011) 286–313 297

Fig. 4. Continued.
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298 J.R. Patel et al. / Journal of Ethnopharmacology 138 (2011) 286–313

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 extracts
of P. amarus revealed the strongest activity against S. typhi with
8.0 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) and
amoxicillin (4.0 mm) (Oluwafemi and Debiri, 2008). Antimicrobial
effect of the plant extracts of P. amarus showed that the organic
and 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. amarus
extracts (3.12 mg/mL) were lower than the corresponding disc dif-
fusion MIC determined values (6.25–25.00 mg/mL). Bacteriocidal
and bacteriostatic effect varied with, solvent type of extract, con-
centration and method of the test adopted. The active components
of 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 concentration
of 5, 10, 25, 50, 100, 200, 400, 800, and 1000 mg/mL was studied
against drug resistant pathogenic bacterial strains, Shigella dysen-
teriae 1, S. dysenteriae 2, S. boydii 8, S. aureus ML267, S. aureus
NCTC 7447, Streptococcus pneumoniae 7465, E. coli ROW 7/12, E. coli
CD/99/1, B. subtilis CD/99/1, Salmonella typhimurium ATCC 6539,
Vibrio cholerae 8531, P. aeruginosa 7241, and K. pneumoniae RM 8/98
by disc diffusion and agar dilution method. Shigella spp. were found
to be inhibited at the least concentration (25 mg/mL) and found to
show the maximum diameter of zone of inhibition at the largest
tested concentration of 1000 mg/mL comparable with sparfloxacin.
Methanolic extract posses significant antimicrobial activity against
all the tested strains; maximum inhibitory effect was noted against
Shigella spp., E. coli, V. cholerae, and S. aureus. The extract was found
to 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 isolated
phyllanthin (Mazumder et al., 2006). The ethanolic extracts of nine
Peruvian medicinal plants including P. amarus were screened for
antimicrobial activity against Bacillus cereus ATCC 11,778, B. subtilis

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

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J.R. Patel et al. / Journal of Ethnopharmacology 138 (2011) 286–313
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 ATCC
25,923, Staphylococcus epidermidis ATCC 12,228, and Streptococ-
cus pyogenes ATCC 19,615. Among the plants tested, 80% ethanolic
extract of aerial parts of P. amarus showed the most promising
antibacterial properties, inhibiting all of the strains tested with
minimum inhibitory concentrations (MICs) ranging from 0.25 to
16 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 the
MTS (3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-
2-(4-sulfophenyl)-2H-tetrazolium) reduction assay. It was shown
to inhibit MCF-7 (breast carcinoma) and A549 (lung carcinoma)
cells growth with IC50 values ranging from 56 to 126 ␮g/mL and
150–240 ␮g/mL for methanolic and aqueous extracts respectively.
In comparison, they have lower toxicity on normal cells with cell
viability of 50% when treated up to 1000 ␮g/mL for both aqueous
and methanolic extracts. After determining the non-toxic effective
dose, several antimetastasis assays were carried out and P. amarus
extracts were shown to effectively reduce invasion, migration, and
adhesion of both MCF-7 and A549 cells in a dose-dependent man-
ner. This was followed by an evaluation of the possible modes
of cell death that occurred along with the antimetastatic activ-
ity. P. amarus was shown to be capable of inducing apoptosis in
conjunction with its antimetastastic action, with more than 3-fold
increase of caspases-3 and -7, the presence of DNA-fragmentation
and terminal deoxynucleotidyl transferase mediated dUTP nick end
labeling assay (TUNEL)-positive cells. The ability of P. amarus to
exert antimetastatic activities is mostly associated to the presence
of polyphenol compounds in its extracts (Lee et al., 2011). The
methanolic extract of P. amarus hairy roots revealed potent antipro-
liferative activity in the MCF-7 cells through induction of apoptosis
mediated by increased intracellular reactive oxygen species (ROS)
in conjunction with decreased mitochondrial membrane poten-
tial (Abhyankar et al., 2010). The effects of aqueous extract of
whole plant of P. amarus against Cr(VI)-induced oxidative toxicity
in vitro in MDA-MB-435S human breast carcinoma cells revealed
a distinct decline in Cr(VI)-induced cytotoxicity was noticed in
MDA-MB-435S cells with an increase in extract dosage. Its phenolic
constituents simultaneously may inhibit Cr(VI)-induced oxidative
toxicity to MDA-MB-435S cells (Guha et al., 2010). In vitro, apop-
totic effect of 75% methanolic extract of aerial parts (stem and
leaves) of P. amarus against Dalton’s Lymphoma Ascites (DLA)
cells in culture produced significant reduction in cell viability
299
as determined by the MTT assay. Treatment of cell lines with
P. amarus produced cytotoxicity after 24 h. The maximum effect
was observed in DLA cell lines where treatment with P. amarus
at a concentration of 500 ␮g/mL produced cytotoxicity IC50 value
was 102 ± 1.38 ␮g/mL. It also induced the formation of apoptotic
bodies 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 to
induce 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 leukaemia
harboring animals and decrease the incidence of anemia. Treat-
ment with P. amarus (750 and 250 mg/kg) induced the expression
of p53 and p45NFE2 and decreased the expression of Bcl-2 in
the spleen of infected mice. Histopathological evaluations of the
spleen demonstrated that administration of P. amarus decreased
the infiltration of leukemic cells into the sinusoidal space when
compared 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 in
Swiss albino mice. The decrement of tumor volume and packed
cell volume and viable cell count were observed in lignan treated
mice when compared only to EAC tumor bearing mice. Treat-
ment with test compounds increased the survival time and normal
peritoneal cell count. Hematological parameters, PCV which were
altered by tumor volume inoculation, were restored consider-
ably (Islam et al., 2008a). An alcoholic extract of aerial parts of
P. amarus was found to inhibit cytochrome P450 enzymes both
in vivo as well as in vitro. It was studied using specific resorufin
derivatives, 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 concentration
needed for 7-methoxyresorufin-O-demethylase (MROD) CYP1A2
was 7.725 ␮/mL and 7-pentoxyresorufin-O-depentylase (PROD),
CYP2B1/2 was found to be 4.18 ␮/mL indicating that the extract
inhibited the P450 enzymes at very low concentration. Extract also
inhibited the activity of aniline hydroxylase (an indicator of CYP
2E1 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 the
elevated P450 enzyme activities produced by phenobarbitone by
50% at 250 mg/kg body weight. Extracts of P. amarus has pre-
vented or stopped the cells from mutation with the existence of
chemical agents (Hari Kumar and Kuttan, 2006). N-methyl N -
nitro-N-nitrosoguanidine (MNNG) induced stomach cancer in male
Wistar rats was significantly inhibited by the administration of
75% methanolic extract of aerial parts of P. amarus at a dose of
150 and 750 mg/kg body weight. It also reduced the incidence of
gastric neoplasms in rats (44%) as well as their numbers. The ele-
vated enzymes level in the stomach was also found to reduce by
P. amarus treatment (Raphael et al., 2006). 70% ethanolic extracts
of three Phyllanthus species, P. urinaria, P. amarus and P. debilis
at the concentration of 10 ␮g/mL significantly inhibited the pro-
liferation of the HepG2 cells. The extracts induced apoptosis by
inducing caspase-3. Further confirmation of extract-induced apo-
ptosis was obtained by demonstrating that the Bcl-2/Bax ratio
decreased in response to treatment with the extracts detected the
mechanism by which the Phyllanthus extracts induced apopto-
sis. These findings demonstrated that Phyllanthus extracts induce
TNF-␣ production from the hepatocellular carcinoma cells while
inhibiting production of potent anti-apoptotic genes IL-8 and COX-
2. Untreated cells (control) received only 0.01% DSMO (Sureban
et 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 and
multidrug resistance (MDR) reversing properties of the extract and
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300 J.R. Patel et al. / Journal of Ethnopharmacology 138 (2011) 286–313
compounds isolated from P. amarus. It was reported that Lucena-
1 was significantly more resistant to the cytotoxicity of P. amarus
derivatives: 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 cytotoxic
effects on K-562 cells with 40.3, 66.0, 62.0, 61.0 or 24.1% of cell
death, respectively. The cellular toxicity observed on Lucena-1 was
16.3, 40.4, 29.4, 30.2, or 24.8%, respectively. These results suggested
a 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 body
weight i.p. in mice for 14 days significantly reduced the myelosup-
pression and improved the WBC count, bone marrow cellularity
as 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 the
effect of toxic metabolites of cyclophosphamide (CTX) on the cells.
Administration of P. amarus did not reduce the tumor reducing
activity of CTX. In fact, there was a synergistic action of CTX and P.
amarus in reducing the solid tumors in mice. It was indicated that
administration of P. amarus can significantly reduce the toxic side
effects of CTX and is not interfering with the antitumor efficiency
of CTX. When the aqueous extract of P. amarus was administered
to cancer bearing mice, it lowered the tumor incidents, level of
carcinogen-metabolizing enzymes levels of liver cancer markers
dose dependently (Kumar and Kuttan, 2005). The 75% methanolic
extract of aerial parts of P. amarus has been administered orally (750
and 250 mg/kg body weight) in the radiation (6 Gy) induced balb/c
mice for its protective activity against carcinogenesis. The WBC
count, bone marrow cellularity and ␣-esterase activity increased
significantly as compared to only radiation exposed mice. The
antioxidant enzymes such as superoxided dismutase (SOD), cata-
lase (CAT), (glutathione-S-transferase) GST, glutathione peroxidase
(GPX), and glutathione reductase, both in blood and tissue, which
was reduced by radiation induced. P. amarus possesses the ability
to inhibit the unusual enzymatic pathways peculiar to cancer cells
proliferation and growth rather than a direct toxic effect of killing
the different types of cancer cells (Kumar and Kuttan, 2004). The
aqueous extract of P. amarus 150 and 750 mg/kg body weight thrice
weekly for 8 weeks treatment exhibited potent anticarcinogenic
activity against 20-methylcholanthrene (20-MC) induced sarcoma
development and increased the survival of tumor harboring balb/c
mice. The extract administration (p.o.) was also found to prolong
the life span of DLA and EAC bearing mice and reduced the vol-
ume of transplanted solid tumors (Rajeshkumar et al., 2002). The
aqueous extract of the entire plant of P. amarus, at the concentra-
tion of 10, 25, 50 and 100 ␮g/plate showed an antimutagenic effect
against induction by 2-aminofluorene (AF2), 2-aminoanthracene
(2AA) and 4-nitroquinolone-1-oxide (4-NQO) in S. typhimurium
strains TA98 and TA100, and in E. coli WP2 uvrA/pKM101. The
inhibition of N-ethyl-N-nitrosoguanidine (ENNG)-induced muta-
genesis was observed only with S. typhimurium TA100. The extract
also exhibited activity against 2-nitrofluorene (2NF) and sodium
azide-induced mutagenesis with S. typhimurium TA98 and TA100,
respectively. Based on the alkaline elution method, the plant extract
prevented in vivo DNA single-strand breaks caused by dimethyl-
nitrosamine (DMN) in hamster liver cells. When the extract was
administered 30 min prior to the administration of DMN, the elu-
tion rate constant decreased more than 2.5 times, compared to the
control (Sripanidkulchai et al., 2002). Methanolic extract of stems
and leaves of P. amarus was tested for its antimutagenic activity
in S. typhimurium strains TA1535, TA100, and TA102 (Ames test).
P. amarus extract 500 mg/kg body weight for 12 days was able
to 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 direct
acting 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 was
found to be significantly inhibited by the oral administration of
P. amarus extract (Raphael et al., 2002a). Administration of aque-
ous extract of aerial parts of P. amarus increases the life span
of rats with hepatocellular carcinoma. The effect of P. amarus
extract 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 to
significantly increase the survival of hepatocellular carcinoma har-
boring animals. All the untreated rats died of tumor burden by
33,791.6 weeks. Administration of P. amarus extract (150 mg/kg
body weight orally, 5 days weekly continuously for 54 weeks
or till they died) after tumor development, increased the sur-
vival of animals to an average of 52,292.3 weeks (Rajeshkumar
and Kuttan, 2000). The P. amarus aqueous extract at the dose of
150 and 75 mg/kg body weight on male Wistar rats, 5 days prior
to NDEA treatment for 20 weeks signifcantly inhibit hepatocar-
cinogenesis induced by NDEA in a dose-dependent manner. The
anticarcinogenic activity of the extract was evaluated by its effect
on tumor incidence, level of carcinogen-metabolizing enzymes,
level of liver cancer markers and liver injury markers. Animals
treated with NDEA alone showed 100% tumor incidence and sig-
nifcantly elevated tissue level of drug metabolizing enzymes such
as 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 tissues
of tumor bearing animals, while they were significantly reduced
in the treated group. Similar reduction was seen in tissue level
of 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 marker
enzymes indicated that these extracts offered protection against
chemical 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 treated
animals developed any tumor even 32 weeks after NDEA admin-
istration, whereas all of the control animals died due to tumor
burden. 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-hydroxylase
cytochrome 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 NDEA
treatment, were also normal in the NDEA and P. amarus treated
group (Joy and Kuttan, 1998). Phyllanthus extract reduced the cyto-
toxic action of lead nitrate and aluminum sulfate in bone marrow
cells of mice. Nickel clastogenicity was also found to be reduced
in mice fed with Phyllanthus extract and ascorbic acid. These pos-
itive reports on the antigenotoxic efficacy of Phyllanthus extract
was 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 body
weight) induced multiple, elongated, reddish bands of hemor-
rhagic erosions in rat gastric mucosa was studied. The ethanol
control group had the highest ulcer index of 45.20 ± 2.39. Pre-
treatment with P. amarus leaves aqueous extract (500 mg/kg) and
cimetidine (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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J.R. Patel et al. / Journal of Ethnopharmacology 138 (2011) 286–313
dose-dependent percentage ulcer inhibition and the lower dosage
of the aqueous extract had a higher ulcer inhibition (Shokunbi and
Odetola, 2008). Methanolic extract of leaves and stems of P. amarus
at the dose of 50, 200, and 1000 mg/kg body weight p.o., on adult
male Wistar rats significantly inhibited gastric lesions, induced
by intragastric administration of absolute ethanol (8 mL/kg body
weight). Mortality, increased stomach weight, ulcer index, and
intraluminal bleeding were reduced significantly by P. amarus.
Biochemical analysis indicated that reduced GSH of gastric mucosa
produced by ethanol administration was significantly elevated by
treatment with P. amarus (Raphael and Khuttan, 2003). Graded
doses of the aqueous extract of P. amarus (100–800 mg/kg) admin-
istered orally produced a dose related inhibition of gut meal travel
distance in normal mice. The highest intestinal transit inhibition
of 31.65% was obtained with 400 mg/kg. In castor oil induced
diarrhoea in mice, P. amarus extract (400 mg/kg) delayed the onset
of diarrhoea, reduced frequency of defecation and reduced gut
meal travel distance significantly resulting in intestinal transit
inhibition of 79.94% compared to 86.92% produced by morphine
(100 mg/kg). In addition, the activities of some intestinal mucosa
enzymes (maltase, sucrase, lactase and alkaline phosphatase)
in mice pretreated with extract before castor oil were not as
severely 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. amarus
was studied against spore germination of some fungi (Alternaria
brassicae, Alternaria solani, Curvularia pennisetti, Curvularia sp.,
Erysiphe pisi, Helminthosporium frumentacei) as well as pea pow-
dery mildew (E. pisi) under glasshouse conditions. The sensitivity
of fungi to nor-securinine varied considerably. Nor-securinine was
effective against most of the fungi. H. frumentacei was more sen-
sitive even at the lowest concentration (1000 ␮g/mL). Conidia of
E. pisi were also inhibited in partially or completely appresso-
rium formation. Pre-inoculation treatment showed greater efficacy
than post-inoculation in inhibiting powdery mildew development
on pea plants in a glasshouse. Maximum inhibition occurred at
2000 ␮g/mL (Sahni et al., 2005). Antifungal bioassay in terms of
reduction in weight, colony diameter and sporulation of the target
fungal colony was carried out using Broth Dilution method. Root
part of the P. amarus, extracted in various organic solvents have
not shown any noticeable antifungal activity. The percentage inhi-
bition observed in different solvent extracts of aerial part was found
as reduction in weight. It was concluded that Chloroform fraction
of the aerial part of the plant P. amarus have showed significant
inhibitory effect against dermatophytic fungi M. gypseum (Agrawal
et al., 2004).
8.6. Analgesic, anti-inflammatory, anti-allodynic and
anti-oedematogenic activity
The aqueous leaves extract of P. amarus was investigated for
analgesic and anti-inflammatory activities using both thermal and
chemical models of pain assessment in rats. The extract caused
a significant (p < 0.05) dose related increased inhibition of the
carrageenan-induced paw oedema in the rats. The inhibition pro-
duced by 200 mg/kg aqueous extract of P. amarus (70.20%) was
significantly higher than that of the reference drug (acetylsali-
cylic acid). The extract produced a marked analgesic activity by
inhibiting both early and late phases of pain stimulus in formalin-
induced paw licking rats and also a significant and dose related
increase 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.
301
amarus on different phases of inflammation were performed using
different phlogistic agents-induced paw oedema, carrageenan-
induced air-pouch inflammation and cotton pellet granuloma in
male Wistar rats. Methanolic extract at a dose of 250 mg/kg body
weight p.o. significantly inhibited carrageenan, bradykinin, sero-
tonin and prostaglandin E1 -induced paw oedema, but failed to
inhibit 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 oedema
formation in mice. The extracts of P. amarus (100 ␮g/mL) and niran-
thin (30 ␮M), but not nirtetralin or phyltetralin (30 ␮M), decreased
the specific binding of [3H]-PAF in mouse cerebral cortex mem-
branes. The mean IC50 values from these effects were 6.5 and
0.3 ␮M, respectively. Both niranthin and WEB2170 (30 nmol/paw)
inhibited the increase of myeloperoxidase activity induced by
PAF injection in the mouse paw (Kassuya et al., 2006). The hex-
ane extract (HE), the lignan-rich fraction (LRF), or the lignans
phyltetralin, nirtetralin, niranthin, but not hypophyllanthin or
phyllanthin from P. amarus inhibited carrageenan-induced paw
oedema and neutrophil influx. The HE, the LRF or nirtetralin also
inhibited the increase of IL1-␤ tissue levels induced by Cg. Fur-
thermore, bradykinin (BK)-, platelet activating factor (PAF) – and
endothelin-1 (ET-1)-induced paw oedema were significantly inhib-
ited by the HE or LRF while histamine- and substance P-induced
paw oedema were unaffected. Finally, nirtetralin or phyltetralin
caused inhibition of paw oedema induced by PAF or ET-1 (Kassuya
et al., 2005). P. amarus EtOH, H2 O (0.25%) and hexane extracts
(0.01%) showed an inhibition of LPS-induced production of NO and
PGE2 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 inhibited
induction of interleukin (IL)-1␤, IL-10, and interferon-␥ in human
blood and reduced TNF-␣ production in vivo (Kiemer et al., 2003).
The anti-allodynic and anti-oedematogenic effects of the hexane
extract, lignan-rich fraction and purified lignans from aerial parts
of P. amarus was studied in the inflammatory and neuropathic
models of nociception. The hexane extract at a dose of 100 mg/kg
body weight orally in male swiss albino rats inhibited the allodynia
and the oedema induced by the intraplantar injection of complete
Freund’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 pure
lignans 30–100 mg/kg body weight i.p. did not affect CFA-induced
allodynia. Administered chronically, the lignan fraction reduced
CFA-induced paw oedema (39 ± 9%). When evaluated in the model
of neuropathic pain caused by partial ligation of sciatic nerve, the
hexane extract inhibited the mechanical allodynia (77 ± 7%), with a
similar efficacy to the gabapentin (71 ± 10%) (Kassuya et al., 2003).
The aqueous extracts of leaves and stems of P. amarus at the dose of
500, 250 and 100 mg/kg body weight orally as a single dose, 1 h prior
to experiment was administered to female stains of Balb/c mice.
Extracts produced an inhibition of 26, 33 and 39% respectively at
3 h while methanolic extract of P. amarus at a dose level of 100, 250
and 500 mg/kg body weight produced an inhibition of 29, 37 and
42% respectively at 3 h and significant inhibition of paw oedema
was 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 acetic
acid-induced abdominal constrictions. The hydroalcoholic extract
of the Phyllanthus species elicited significant inhibition of the
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302 J.R. Patel et al. / Journal of Ethnopharmacology 138 (2011) 286–313
capsaicin-induced neurogenic pain. The extract of these Phyllan-
thus species when given intraperitoneally, produced dose related
and pronounced antinociception when assessed against chem-
ical models of nociception, including acetic acid, formalin and
capsaicin-induced pain. Orally, these species were less potent and
efficacious 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 albino
rats. Antioxidant enzymes; GR, GPX and GST, CAT and SOD were
also assayed. Diabetic treated animal group showed a signifi-
cant decrease in renal LPO, protein oxidation and a significant
increase in GSH content and GR, GPX and GST activities when com-
pared with STZ-induced diabetic rats. The activities of SOD and
CAT decreased significantly in STZ-induced diabetic rats, but were
normalized in diabetic treated group (Karuna et al., 2011). The
aqueous extract of whole plant of P. amarus showed significant
(p < 0.05) potential in scavenging free radicals, and in inhibiting
lipid peroxidation. Furthermore, the extract proved to contain a
high content of phenolic compounds which were found to have
strong and significant (p < 0.05) positive correlations to free-radical
scavenging potential, lipid peroxidation inhibition capacity and
cyto-protective efficiency against Cr(VI)-induced oxidative cellu-
lar damage (Guha et al., 2010). Aqueous extract of P. amarus at
a dose of 200 mg/kg body weight/day for 8 weeks treated male
albino Wistar rats showed a significant decrease in plasma LPO
and a significant increase in plasma vitamin C, uric acid, GSH
level, GPX, CAT and SOD activities. Single cell gel electrophore-
sis (SCGE) experiment revealed that aqueous extract of P. amarus
was devoid of genotoxicity and had a significant protective effect
against H2 O2 , STZ and nitric oxide (NO) induced lymphocyte DNA
damage (Karuna et al., 2009). Free-radical scavenging activity of
50% ethanolic extract of aerial parts of P. amarus extract and
phyllanthin was examined using 2,2-diphenyl-2-picrylhydrazyl
(DPPH) assay. The DPPH free-radical scavenging activity was
concentration-dependent in both cases and reaches a maximum at
a concentration of 300 ␮g/mL for P. amarus extract and 20 ␮mol/mL
for phyllanthin. No difference in inhibition was noted with fur-
ther increase in concentration of either of the compounds. Results
indicated that phyllanthin exhibited very high antioxidative prop-
erty as compared to P. amarus extract which is clearly evident
by its low IC50 value of 7.4 ␮mol/mL (Krithika et al., 2009). The
antioxidant activity of some of its principal constituents, namely
amariin, 1-galloyl-2,3-dehydrohexahydroxydiphenyl (DHHDP)-
glucose, repandusinic acid, geraniin, corilagin, phyllanthusiin
D, rutin and quercetin 3-O-glucoside were examined for
their ability to scavenge free radicals in a range of systems
including 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 assays
employed. Amariin, repandusinic acid and phyllanthusiin D
showed higher antioxidant activity among the ellagitannins and
were comparable to the flavonoids, rutin and quercetin 3-O-
glucoside (Londhe et al., 2008). Pretreatment with P. amarus leaves
extracts on antioxidant enzymes in gastric mucosa homogenate
was studied. Significant reductions (p < 0.05) in the gastric mucosa
catalase (CAT), super oxide dismutase (SOD) and glutathione-s-
transferase (GST) activities were observed in the ethanol group
compared with the normal control group. P. amarus acetone
extracts (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 and
38% for GST respectively when compared with the ethanol group.
Acetone extract produced a dose-dependent increase whereas
500 mg/kg of the aqueous extract seems more effective (Shokunbi
and Odetola, 2008). Different drying treatments led to significant
reduction (p < 0.05) in antioxidant properties of P. amarus methano-
lic extracts, with microwave drying causing the highest decrease
in total phenolic compound and antioxidant activity exhibited by
the reduction in both radical scavenging activity and FRAP. On
the other hand, boiling water extracts appeared to exhibit sig-
nificantly stronger antioxidant potentials (p < 0.05) even in dried
plant materials due to greater solubility of compounds, breakdown
of cellular constituents as well as hydrolysis of tannins (Lim and
Murtijaya, 2007). The antioxidant activity of methanolic extracts of
five species including P. debilis, P. urinaria, P. virgatus, P. maderas-
patensis, P. amarus from the genus Phyllanthus was evaluated by
various antioxidant assays. All the extracts at the concentration of
50 ␮g/mL showed strong antioxidant activity in all the tested meth-
ods. Among the five plants, P. debilis has been found to possess the
highest activity and P. amarus posses the lowest activity in all tested
models (Kumaran and Karunakaran, 2007). Methanolic extract of
leaves and stems of P. amarus was found to have potential antiox-
idant activity as it could inhibit lipid peroxidation, and scavenge
hydroxyl and superoxide radicals in vitro. The amount required for
50% inhibition of lipid peroxide formation was 104 ␮g/mL and the
concentrations 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 high
antioxidant activity.
8.9. Antiplasmodial activity
The aqueous and ethanolic extracts of the whole plant of
P. amarus were administered to Swiss albino mice at doses of
200, 400, 800 and 1600 mg/kg/day to investigate the prophylac-
tic and chemotherapeutic effect of the extract against Plasmodium
yoelii infection and compared with those of standard antimalarial
drugs pyrimethamine and chloroquine, artesunate/amodiaquine
respectively. Results showed that extracts demonstrated a dose-
dependent prophylactic and chemotherapeutic activity. The
aqueous extract showed slightly higher effect than the ethanolic
extract. The antiplasmodial effects of extracts were comparable
to the standard prophylactic and chemotherapeutic drugs used in
chloroquine resistant plasmodium infection. The extracts showed
prophylactic effect by significant delay in the onset of infection with
the suppression of 79% at a dose of 1600 mg/kg/day. The results
indicate that the extracts of the whole plant of P. amarus possess
repository and chemotherapeutic effects against resistant strains
of P. yoelii in Swiss albino mice (Ajala et al., 2011). ‘Saye’, a tra-
ditional medicine used in Burkina Faso, which consists of extracts
of Cochlospermum planchonii (rhizome), Cassia alata (leaves) and
P. amarus (whole plant). Mice treated with 50, 100, 150, 200,
250 mg/kg body weight for 0–3 days, orally showed a significant
effect against P. falciparum and Plasmodium berghei parasites grown
in vivo (IC50 = 80.11 ± 3.40 ␮g/mL; ED50 = 112.78 ± 32.32 mg/kg). In
vitro the activity was lower at the concentration of 100, 50, 25 and
12.5 ␮g/mL and compared with chloroquine (Traore et al., 2008).
The aqueous extract of leaves and stem of P. amarus at doses of
108.33, 165 and 325 mg/kg in Swiss albino mice was found to cause
a significant dose-dependent suppression of P. berghei parasites
[p < 0.05] sulfadoxine/pyrimethamine caused a similar significant
suppression of P. berghei parasites (Dapper et al., 2007). Ethano-
lic, methanolic and methylene chloride extracts of entire plant
of P. amarus showed significant activity against the chloroquine-
sensitive strain of P. falciparum 3D7. The IC50 of methanolic extract
and 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 were
screened by in vitro enzyme assays. Effect on viral RNA replication
was investigated by using TaqMan Real time RT-PCR. P. amarus root
extract showed significant inhibition of HCV-NS3 protease enzyme;
whereas P. amarus leaves extract showed considerable inhibition of
NS5B in the in vitro assays. Further, the P. amarus root and leaves
extracts significantly inhibited replication of HCV monocistronic
replicon RNA and HCV H77S viral RNA in HCV cell culture system.
However, both P. amarus root and P. amarus leaves extracts did not
show cytotoxicity in HuH-7 cells in the MTT assay. Furthermore,
P. amarus root extract with IFN-␣ showed additive effect in the
inhibition of HCV RNA replication. Results suggested the possible
molecular basis of the inhibitory activity of P. amarus extract against
HCV which would help in optimization and subsequent develop-
ment of specific antiviral agent using P. amarus as potent natural
source (Ravikumar et al., 2011). The aqueous extract of P. amarus
showed partial antiviral activity against white spot syndrome virus
in shrimp at the concentration of 150 mg/kg of animal body weight
for 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. One
hundred and fourteen ducks infected posthatch with the duck hep-
atitis B virus (DHBV) were divided into groups at 3 months of age
and treated intraperitoneally with the aqueous, butanol, and alco-
holic extracts of these two plants at doses of 25, 50, or 200 mg/kg
body weight. There was no definite antiviral property observed in
the treated ducks (Munshi et al., 1993a). The therapeutic potential
of plant extracts of P. amarus and P. maderaspatensis for postexpo-
sure prophylaxis against infection by Hepadnaviruses was studied
in ducklings infected by the DHBV. The observations suggested that
aqueous extract of P. amarus (100 mg/kg body weight) and ethano-
lic extract of P. maderaspatensis (100 mg/kg body weight) are not
useful as therapeutic agents for postexposure prophylaxis against
DHBV infection (Munshi et al., 1993b). The polyphenol containing
extract of P. amarus and representatives of simple phenols (shikimic
acid 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. All
extracts and compounds at the concentration of 50 ␮g/mL were
capable 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. amarus
induced by Agrobacterium rhizogenes was shown to possess 85%
inhibition (in contrast to 15% in the control) in binding of Hepatitis
B Surface Antigen (HBsAg) to its antibody (anti-HBs) after 24 h of
incubation with HbsAg-positive sera in vitro at 37 ◦ C (Bhattacharyya
and Bhattacharya, 2004). Water–alcohol extract of leaves of P.
amarus in vitro blocks HIV-1 attachment and the HIV-1 enzymes
integrase, reverse transcriptase and protease to different degrees.
A gallotannin containing fraction and the isolated ellagitannins
geraniin and corilagin were shown to be the most potent mediators
of these antiviral activities. P. amarus derived preparations blocked
the interaction of HIV-1 gp120 with its primary cellular recep-
tor CD4 at 50% inhibitory concentrations of 2.65 (water–alcohol
extract) to 0.48 ␮g/mL (geraniin). Inhibition was also evident for
the 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) (Notka
et al., 2004). Aqueous as well as alcohol-based P. amarus extracts
potently inhibited HIV-1 replication in HeLa CD4+ cells with
50% effective concentration (EC50 ) values ranging from 0.9 to
7.6 ␮g/mL. A gallotannin enriched fraction showed enhanced activ-
ity (0.4 ␮g/mL), and the purified gallotannins geraniin and corilagin
303
were most active (0.24 ␮g/mL). HIV-1 replication was also blocked
in CD4+ lymphoid cells with comparable EC50 values. Applying
a cell-based internalization assay, it was demonstrated 70–75%
inhibition of virus uptake at concentrations of 2.5 ␮g/mL for the
water–alcohol extract and geraniin. In addition, a concentration-
dependent inhibition of HIV-1 reverse transcriptase (RT) was
demonstrated in vitro. The 50% inhibitory concentration (IC50 ) val-
ues varied from 1.8 to 14.6 ␮g/mL (Notka et al., 2003). Study on
25 compounds isolated from P. amarus, P. multiflorus, P. tenel-
lus and P. virgatus found that niranthin, nirtetralin, hinokinin and
geraniin at the non-cytotoxic concentration of 50 ␮m, suppressed
effectively 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 with
hinokinin (Huang et al., 2003). Analysis in HuH-7 cells (human hep-
ato cellular carcinoma cell line) with transfected plasmids using a
luciferase reporter showed that DMSO solution of whole plant of
P. amarus at the concentration of 50, 100 or 200 ␮g/mL specifically
inhibited HBV enhancer I activity. To identify the mechanism of
this 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 upon
the HBV enhancer I activity. Exposure to P. amarus inhibited C/EBP
␣ and ␤-mediated up-regulation of HBV enhancer I activity in a
dose-dependent manner, whereas HNF-3␣- and ␤-mediated up-
regulation of HBV enhancer I was unaffected. In vitro gel shifts
showed that P. amarus inhibited complexing of C/EBP transcrip-
tion factors to a consensus oligonucleotide sequence, whereas DNA
binding of AP-1 and SP-1 transcription factors was unaffected
(Ott et al., 1997). P. amarus inhibited hepatitis B virus polymerase
activity, decreased episomal hepatitis B virus DNA content and sup-
pressed virus release into culture medium. When DSMO solution of
whole plant of P. amarus 80 mg/kg body weight i.p. daily for 7 days
was administered to transgenic mice, hepatic HBsAg mRNA level
was decreased, indicating transcriptional or post-transcriptional
down-regulation of the transgene. Increase in hepatitis B virus
mRNA expression after stimulation of the glucocorticoid responsive
element was also suppressed by P. amarus, suggesting involvement
of 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. amarus
at the concentration of 1 mg/mL on the cultured hepatoma cell
line HepA2 has been reported. The cell line had been transfected
with tandemly arranged HBV DNA and continued to synthesize
and secrete both HBsAg and HBeAg. Extract of P. amarus reversibly
inhibited cellular proliferation and suppressed HBsAg production
but not HBeAg production in HepA2 cells. P. amarus also suppressed
HBsAg 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). Ethanolic
extract and subsequent fractions (hexane, chloroform, butanol and
water) were tested for in vitro effects on HBsAg, HBeAg and HBV
DNA in serum samples positive for HBV antigens followed by the
screening of respective antigens by ELISA. HBV DNA was deter-
mined by molecular hybridization. The extracts were effective
against HBV antigens, the butanol extract being the most potent.
The chromatographic fractions showed an enhanced activity. The
active fractions inhibited the interaction between HBsAg/HBeAg
and their corresponding antibodies suggesting anti-HBs, anti-Hbe
like 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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304 J.R. Patel et al. / Journal of Ethnopharmacology 138 (2011) 286–313
P. amarus extract. When three out of four recently infected WHV
carriers were treated with aqueous extract of P. amarus at a dose
of 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 showed
a decrease in virus levels. Preliminary results in human carriers
treated 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 alcoholic
extract of P. amarus were described for the treatment of chronic
hepatitis B virus infection on duck hepatitis B virus at the doses
of 25, 50, and 200 mg/kg body weight. Nine ducks congenitally
infected with the DHBV were treated either orally (four ducks for
10 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 the
serum or in the level of viral DNA replication in the liver. In two
of the five intraperitoneal-treated ducks, a reduction in the level of
duck hepatitis B surface antigenaemia (DHBsAg) was observed. The
data strongly suggested that P. amarus has no significant inhibitory
effect on DHBV DNA replication and only a minor effect on DHB-
sAg P. amarus production (Niu et al., 1990). Alexander cell line, a
human hepatocellular carcinoma derived cell line which has the
property of secreting HBsAg in the supernatant was used to study
the antiviral property of P. amarus. Aquous extract of P. amarus
was evaluated for its in vitro ability to inhibit HBsAg secretion on
a dose-dependent manner. Aquous extract of P. amarus at the con-
centration of 1 mg/mL as a single dose inhibited the secretion of
HBsAg for a period of 48 h. Observations revealed the anti hepatitis
B virus property of P. amarus at cellular level and further confirmed
its beneficial use in the treatment of acute and chronic hepatitis B
and 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 of
liver in hepatitis-C through investigating liver profile enzymes;
antioxidant enzymes, antioxidant vitamins and lipid peroxidation.
The study consists of 50 clinical diagnosed hepatitis-C patients
ranging in between age group 25 and 60 years. The control group
includes 50 ages and sex matched normal healthy persons. Oxida-
tive stress was assessed by estimating LPO. The parameters like
serum bilirubin, total proteins and activity of liver profile enzymes
were done. Activity of enzymatic antioxidants; SOD, GPX and lev-
els of non-enzymatic antioxidant vitamins E and C was measured
in plasma or erythrocytes. Methods used in the study are mainly
enzyme kinetics by autoanalyzer and by turbidimetry. Plasma LPO
levels were significantly high but activity of SOD, GPX, catalase and
levels of vitamins E and C were significantly lowered in hepatitis-C
in comparison with controls. After P. amarus therapy for 5 and 10
weeks plasma LPO levels were significantly decreased and activ-
ity of SOD, GPX, catalase and vitamins E and C were significantly
increased in hepatitis-C. It was concluded that hepatitis-C increased
oxidative stress and might be playing an important role in hepatic
cell damage and pathogenesis of hepatitis-C. It was suggested that
the therapy with P. amarus increased antioxidants and reduced lipid
peroxidation of hepatic cellular and intracellular membranes and
protected liver damage due to free radicals in hepatitis-C (Nikam
et al., 2011). A total of 16 randomized trials with 1326 patients
were included. One trial with 42 participants compared phyllan-
thus with placebo. The trial found no significant difference in HBeAg
seroconversion 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, or
conventional treatment with the same antiviral drug alone. Phyl-
lanthus significantly affected serum HBV DNA (RR 0.69; 95% CI
0.52–0.91, p = 0.008; I(2) = 71%), serum HBeAg (RR 0.70; 95% CI
0.60–0.81, p < 0.00001; I(2) = 68%), and HBeAg seroconversion (RR
0.77; 95% CI 0.63–0.92, p = 0.005; I(2) = 78%), but the heterogene-
ity was substantial. The result obtained regarding serum HBV DNA
was not supported by trial sequential analysis. None of the trials
reported mortality and hepatitis B related morbidity, quality of
life, or liver histology. Only two trials reported adverse events with
numbers without significant differences. No serious adverse events
were reported (Xia et al., 2011). Volunteers received either 1200 mg
of 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, as
indicated). Sera at a final concentration of 5% reduced HIV repli-
cation by more than 30%. These results supported that P. amarus
has 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 with
PA compound capsule (Chief ingredients: P. amarus, Radix noto-
ginseng, etc. each capsule contains P. amarus 275 mg), orally, three
times daily, four capsule each time for 3 months in the treatment
group; 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%, showed
no 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, 80.0
and 78.2% respectively, which were significantly higher than that
in the control (p < 0.05). The negative conversion rates of HbeAg
and HBV DNA in the treatment group were 42.3 and 47.8%, showed
no 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 review
of randomized clinical trials. Randomized trials comparing genus
Phyllanthus vs. placebo, no intervention, general nonspecific treat-
ment, other herbal medicine, or interferon treatment for chronic
HBV infection were identified by electronic and manual searches.
Trials of Phyllanthus herb plus interferon (IFN) vs. IFN alone were
also included. No blinding and language limitations were applied.
The methodological quality of trials was assessed by the Jadad
scale plus allocation concealment. Twenty-two randomized trials
(n = 1947) were identified. The methodological quality was high in
five double-blind trials and low in the 17 remaining trials. The com-
bined results showed that Phyllanthus species had positive effect
on 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 DNA
between Phyllanthus and IFN. Phyllanthus species were better than
nonspecific treatment or other herbal medicines for the clearance of
serum 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 HBV
DNA (1.52, 1.05–2.21) than IFN alone. The efficacy and safety of
genus Phyllanthus for chronic hepatitis B virus (HBV) infection has
been systematically reviewed the clinical trials (Liu et al., 2001). The
powder of the plant P. amarus was given in capsule form (300 mg
capsules-3 capsules thrice daily) and an antacid powder in similar
capsule 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 cases
from the placebo and one from the placebo and one from the drug
group dropped out of the study. The duration of disease (time taken
for bilirubin to come to below 2 mg %) was taken as the outcome
measure. The duration of disease in the two groups was compared
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J.R. Patel et al. / Journal of Ethnopharmacology 138 (2011) 286–313
by Cox’s proportional hazards analysis after adjusting for the vari-
ables that influence the duration of jaundice. Only initial serum
bilirubin was an independent predictor of duration of jaundice.
The analysis showed that P. amarus powder did not significantly
reduce the duration of jaundice in persons with virus B hepati-
tis (Narendranathan et al., 1999). The efficacy of P. amarus to treat
acute viral hepatitis (AVH) was evaluated in parallel to another drug
Essentiale (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 93
sporadic AVH cases of the study showed that 25.8% had an acute
infection due to hepatitis A virus (HAV), 52.6% suffered due to hep-
atitis B viral (HBV) infection, while 19.3% of cases were classified
as non-A non-B hepatitis (NANB) by exclusion. On follow up of the
patients at the end of treatment period of 4 weeks with respec-
tive drug regimen, it was seen that both P. amarus and Essentiale
brought about significant biochemical and clinical normalcy among
the HAV infected patients compared to control group (p < 0.001). In
acute HBV group, P. amarus treated patients recovered faster than
the essentiale treated group and the controls (p < 0.001). Essentiale
was found to help the non-A non-B hepatitis patients to resume
earlier biochemical normalcy than by P. amarus and control treat-
ments. P. amarus seemed to accelerate the clearance of HBsAg in
86.9% of convalescing AVH-B cases in 3 months time as against
48.0% in the Essentiale treated group and 50.0% in the controls
(Jayaram et al., 1997). The role of P. amarus in eradication of the
virus in hepatitis B carriers was evaluated by administering it to
30 asymptomatic carriers of HBsAg in a dosage of 250–500 mg
thrice daily for 4–8 weeks. It was found that none of the 30 sub-
jects cleared HBsAg. P. amarus was well tolerated, with no clinical
side effects or changes in the organ profiles for safety evaluation.
It was found that P. amarus was not effective in clearing HBsAg
in asymptomatic carriers of the antigen (Doshi et al., 1994). Sixty-
five adult asymptomatic chronic carriers of hepatitis B virus were
enrolled to the randomized controlled efficacy study of P. amarus.
Thirty-four received P. amarus 600 mg per day for 30 days and
31 received placebo in identical capsules. The conversion rate of
HBsAg was 6% in the study group at day 30. When 20 subjects in
the P. amarus group were given a further 30-day treatment and 22
placebo 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, given
at the studied dosage and duration, had a very minimal effect on
eradication of HBsAg asymptomatic chronic carriers (Thamlikitkul
et al., 1991). A total of 79 human carriers of HBV, out of these, 40
were given 200 mg of dried, powdered whole plant of P. amarus,
three times daily. The remaining 39 carriers were given lactose
placebo at the same dosage and frequency. Carriers were assigned
randomly to the treatment or control groups and neither the car-
riers nor their physicians were told of the assignment. The drug
or placebo was administered for 30 days then stopped; the carri-
ers were then tested monthly for up to 9 months after cessation of
treatment or placebo. Thirty-seven of the treated carriers and 23
of the controls returned for follow-up. Of the treated subjects 59%,
but only 4% of 23 control subjects, became HBsAg-negative after
30 days and remained so until the end of the follow-up period.
Thirteen out of 14 carriers with HBsAg but without HBeAg became
negative, while only five out of 17 HBsAg positive, HBeAg-positive
subjects lost the surface antigen. HBsAg did not reappear in any
of these subjects. The individuals who remained HBsAg-positive
are now being treated for longer periods to determine whether
active replication of virus requires longer duration of therapy. There
was no evidence of toxicity in the treated individuals, but these
observations 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
305
P. amarus (200 mg doses in gelatin capsule presterilized with ethy-
lene oxide, three times daily) for 30 days. 22 of 37 (59%) treated
patients had lost hepatitis B surface antigen when tested 15–20
days after the end of the treatment compared with only 1 of 23
(4%) placebo-treated controls. Some subjects have been followed
for 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 the
hormonal parameters of male guinea pigs was investigated. The
hormonal parameters investigated were testosterone, leutinizing
and follicle stimulating hormone. Methanolic extract of P. amarus
leaves (50–800 mg/kg) caused a statistically significant increase in
the level of testosterone of the male guinea pigs, from 2.3 ± 0.06 to
3.9 ± 0.05, 4.3 ± 0.6 and 2.8 ± 0.6 after the 7th, 14th and 21st day of
the administration of the extracts, respectively. Furthermore, the
methanolic 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 and
1.5 ± 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 was
investigated in cyclic adult female mice. The results revealed no
significant change in absolute body and organ weights in extract
fed animals indicated no alteration in general metabolic status.
Cohabited females with normal male mice were unable to become
pregnant as their cyclicity was affected. These factors are related
to a change in the hormonal milieu that governs female reproduc-
tive function. Upon withdrawal of feeding for 45 days, these effects
were 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 study
Diuretic, hypotensive and hypoglycaemic effects of P. amarus
on human subjects were assessed. Nine mild hypertensives (four
of them also suffering from diabetes mellitus) were treated with
a preparation of the whole plant of P. amarus for 10 days. Suit-
able parameters were studied in the blood and urine samples of
the subjects along with physiological profile and dietary pattern
before and after the treatment period. Significant increase in 24 h
urine volume, urine and serum Na levels was observed. A significant
reduction in systolic blood pressure in non-diabetic hypertensives
and female subjects was noted. Blood glucose level was also signif-
icantly reduced in the treated group. Clinical observations revealed
no 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 metalloproteinases
was evaluated in alcohol and thermally oxidized polyunsaturated
fatty acid-induced hepatic fibrosis. The matrix metalloproteinase
expression was found to be significantly decreased and the levels
of tissue inhibitors of matrix metalloproteinases and the colla-
gen were significantly increased in alcohol and thermally oxidized
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306 J.R. Patel et al. / Journal of Ethnopharmacology 138 (2011) 286–313
polyunsaturated fatty acid treated male Wistar albino rats. Admin-
istration of P. amarus extract 3 mL/day for 45 days significantly
decreased the levels of collagen and tissue inhibitors of matrix met-
alloproteinases; and positively modulated the expression of matrix
metalloproteinases. It revealed that P. amarus effectively modified
alcohol and thermally oxidized polyunsaturated fatty acid-induced
fibrosis (Surya Narayanan et al., 2011). The protective effect of
phyllanthin, a known principal constituent of P. amarus on ethanol-
induced rat liver cell injury was evaluated. Primary cultures of
rat hepatocytes (24 h culturing) were pretreated with phyllan-
thin (1, 2, 3 and 4 ␮g/mL) for 24 h. After 24 h pretreatment, cells
were treated with ethanol (80 ␮L/mL) for 2 h. Ethanol decreased
%MTT, increased the release of ALT and AST with increase in the
production of intracellular ROS and lipid peroxidation. Phyllan-
thin demonstrated its role in protection by antagonizing the above
effect induced by ethanol. Phyllanthin also restored the antioxidant
capability of rat hepatocytes including level of total glutathione,
and activities of SOD and glutathione reductase (GR) which were
reduced by ethanol (Chirdchupunseree and Pramyothin, 2010). The
hepatoprotective activity of 50% ethanolic extract of aerial parts
of P. amarus plant (100, 200, and 300 mg/kg body weight daily
for 30 days) against CCl4 -induced liver damage in Swiss strain
female albino mice was determined. Carbon tetrachloride adminis-
tration caused a significant increase in liver and ALT, AST, ALP and
acid phosphatase (ACP), while total protein content significantly
decreased as compared to vehicle control. The effect was dose-
dependent. Oral administration of aqueous extract of P. amarus
caused significant mitigation of CCl4 induced changes. P. amarus
attenuated the toxic effects of carbon tetrachloride (CCl4 ) and
caused a subsequent recovery towards normalization. Adminis-
tration of P. amarus at 300 mg/kg body weight offered maximum
recovery (98–100%) against CCl4 (Krithika and Verma, 2009a,b).
The protective effect of P. amarus extract and phyllanthin was
studied on CCl4 -induced toxicity in human hepatoma HepG2 cell
line. The results indicated that CCl4 treatment caused a significant
decrease in cell viability. It was observed that phyllanthin (4.18,
8.36 and 12.54 ␮g/mL for 24 h) effectively alleviated the changes
induced by CCl4 in a concentration-dependent manner with much
smaller strengths as compared to (200, 400 and 600 ␮g/mL) P.
amarus water ethanol extract (Krithika et al., 2009). Silymarin and
standardized extract obtained from the whole plant of P. amarus
100 mg/kg body weight against CCl4 -induced hepatotoxicity in rats
were found effective as hepatoprotective as evidenced by plasma
and liver biochemical parameters. The combination of silymarin
and P. amarus (50 mg + 50 mg/kg body weight for 6 days orally on
male Rattus norvegicus strain showed synergistic effect for hep-
atoprotection and silymarin with ethanolic extract of P. amarus
showed better activity due to the higher concentration of phyl-
lanthin in ethanolic extract in comparison to aqueous extract
of the plant (Yadav et al., 2008). The hepatoprotective effect of
methanolic extract of the leaves of P. amarus was evaluated against
ethanol-induced oxidative damage in adult male Wistar albino
rats. Methanolic extract of P. amarus (250 and 500 mg/kg body
weight/day and ethanol 5 g/kg body weight/day 20% w/v) were
administered orally to animals for 4 and 3 weeks respectively.
It was reported that the ethanol treatment markedly decreased
the level of reduced GSH, SOD, and CAT in the liver, which were
significantly enhanced by P. amarus treatment. GST, which was
increased after chronic ethanol administration, was significantly
reduced 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 in
rat 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.) on
male Wistar rats lowered the ethanol-induced levels of AST and/or
ALT. The 75 mg/kg P. amarus dose gave the best result similar to
SL. Histopathological observations confirmed the beneficial roles
of 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 evaluated
on aflatoxin B1 -induced liver damage in mice using different bio-
chemical parameters and histopathological studies. Albino mice
treatment with P. amarus extract (300 mg/kg body weight for 3
months and animals were sacrified with the interval of 30 days
till the completion of study) was found to show hepatoprotective
effect by lowering down the content of thiobarbituric acid reactive
substances (TBARS) and enhancing the reduced glutathione level
and 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 parameters
in aqeous extract of whole plant of P. amarus treated Wistar rats
(4 mL/rat/day) showed that the ALT and AST activities decreased
significantly in the blood of the test animals (Igwe et al., 2007). The
␤-glucuronidase inhibitory action of 50% methanolic, and aqueous
extracts 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 phenolic
principle isolated from 50% methanolic extract of P. amarus was
found to be more potent at the concentration of 200 ␮g/mL which
is comparable with silymarin (Joshi and Priya, 2007). Hot water
extracts of P. amarus (0.8, 1.6 or 3.2 g/kg) were orally administered
b.i.d. for 7 days prior, 2 days after, or 7 days prior and 2 days after
single oral dose of paracetamol (3 g/kg). The results showed that the
extract at 1.6 and 3.2 g/kg decreased the paracetamol-induced hep-
atotoxicity as indicated by the decrease in SGOT and SGPT, bilirubin
and histopathological score while the ALP did not change. It was evi-
dent that the hepatoprotective mechanism of this plant was neither
related to inhibition on cytochrome P450, nor induction on sulfate
and glucuronide conjugation pathways of paracetamol, but partly
due to the antioxidant activity and the protective effect on the
decrease 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 in
urine. The results indicated that the measurement of ascorbic acid
excretion 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 plant
of P. amarus was investigated in alloxan-induced diabetic Wistar
albino 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 of
oral 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/kg
of extract were observed respectively. The extract also showed a
highly significant (p < 0.001) decrease in blood glucose level of 38
and 30% (day 14) at doses of 130 and 260 mg/kg respectively. On
the 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 whole
plant of P. amarus were evaluated in streptozotocin (STZ)-induced
diabetic male Wistar albino rats. Aqueous extract of P. amarus was
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J.R. Patel et al. / Journal of Ethnopharmacology 138 (2011) 286–313
administered at 200 mg/kg body weight/day to normal treated and
STZ-induced diabetic treated rats by gavage for 8 weeks. During
the experimental period, blood was collected from fasted rats at 10
days intervals and plasma glucose level was estimated. The plasma
lipid profile was estimated at the end of experimental period. After
the treatment period, kidney LPO, protein oxidation and GSH were
estimated. The significant decrease in the body weight, hyper-
glycemia and hyperlipidemia was observed in STZ-induced diabetic
rats with the treatment of aqueous extract of P. amarus in dia-
betic treated group. STZ-induced diabetic rats showed increased
renal 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 subjected
to ␣-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 ± 4.01
and 48.92 ± 3.43 ␮g/mL, respectively, when compared with acar-
bose (IC50 value 83.33 ± 0.34 ␮g/mL). However, the chloroform
extract 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 cholesterol
diet induced hyperlipidemia model in rats. Results indicated that
HAEPA possessed significant hypolipidemic activity at doses of 300
and 500 mg/kg (Umbare et al., 2009). Oral administration of aque-
ous extract of whole plant of P. amarus to Wistar albino rats at doses
of 50, 100 and 200 mg/kg body weight promotes glucose uptake. In
the 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 extracts
for 14 days showed significantly (p < 0.05) reduced AST and ALT and
urea 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/kg
body weight extract. Creatinine of treated animal did not show any
significant (p > 0.05) difference. Significant (p < 0.05) reduction was
observed with animal treated 100 mg/kg body weight for urea. The
extract 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 for
treated animal (50 and 100 mg), the weight gain are significantly
(p < 0.05) lower compared to the control (James et al., 2009). The
aqueous extracts of leave and seed of P. amarus at oral dose of 150,
300 and 600 mg/kg produced a dose-dependent decrease in the
fasting plasma glucose and cholesterol, and reduction in weights
in treated male Swiss mice. The results suggested that the extracts
could be enhancing the peripheral utilization of glucose (Adeneye
et al., 2006). Hexane extract of P. amarus had ␣-amylase inhibitory
properties. Extraction and fractionation of P. amarus hexane extract
led to the isolation of dotriacontanyl docosanoate, triacontanol and
a mixture of oleanolic acid and ursolic acid. The compounds were
tested in the ␣-amylase inhibition assay and the results revealed
that 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 acid
and lupeol isolated from P. amarus were also shown to inhibit ␣-
amylase (Ali et al., 2006). The methanolic extract of leaves and stem
of P. amarus was found to reduce the blood sugar in alloxan diabetic
rats at 4th hour by 6% at a dose level of 200 mg/kg body weight and
18.7% at a concentration of 1000 mg/kg body weight. Continued
administration of extract for 15 days produced significant
(p = 0.001) reduction in blood sugar. On 18th day after alloxan
administration values were almost same to normal in the group
307
taking 1000 mg/kg body weight (Raphael et al., 2002b). Aqueous
extract 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), using
normal 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 glucose
curves (Moshi et al., 1997).
8.16.1. Clinical study
The 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 period
and 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. After
the week washout period, the fasting blood glucose (FBG) and
postprandial blood glucose increased significantly compared with
treatment on oral hypoglycaemics (p < 0.05). After a week herbal
treatment no hypoglycaemic activity was observed (Moshi et al.,
2001).
8.17. Immunomodulatory activity
The methanolic extract of P. amarus was investigated for its
effects on the respiratory burst of human whole blood, iso-
lated human polymorphonuclear leukocytes and isolated mice
macrophages using a luminol/lucigenin-based chemiluminescence
assay. The effect of the extract on chemotactic migration of poly-
morphonuclear leukocytes at the concentration of 10, 5, 2.5, 1.25
and 0.625 ␮g/mL was also tested using the Boyden chamber tech-
nique. The extract of P. amarus produced the strongest oxidative
burst 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 250
and 750 mg/kg body weight significantly reduced the myelosup-
pression and improved the WBC count, bone marrow cellularity
as well as the number of maturing monocytes. CTX treatment also
reduced 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 of
phase I enzyme. P. amarus also increased the cellular GSH and GST,
thereby decreasing the effect of toxic metabolites of CTX on the
cells. P. amarus did not reduce the tumor reducing activity of CTX. In
fact, there was a synergistic action of CTX and P. amarus in reducing
the 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 seed
aqueous extracts of P. amarus were studied for their protective
effects in acetaminophen and gentamicin-induced nephrotoxic
Wistar 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 nitrogen
levels in dose related fashion. Similar effects were also recorded in
the gentamicin model of acute renal injury (Adeneye and Adokiye,
2008).
8.19. Radioprotective effect
The radioprotective activity of pure compounds isolated from
the plant P. amarus was studied using rat liver mitochondria and
pBR322 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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308 J.R. Patel et al. / Journal of Ethnopharmacology 138 (2011) 286–313
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 lipid
peroxidation and protein oxidation in mitochondria. The com-
pounds also prevented radiation induced single-strand breaks in
pBR322 plasmid DNA (Londhe et al., 2009). 75% methanolic extract
of aerial parts of P. amarus at concentrations of 250 and 750 mg/kg
body weight on balb/c mice were found to elevate the antioxidant
enzymes in the intestine and decrease the lipid peroxidation levels.
Histopathological evaluations of the intestine revealed decreased
damage to intestinal cells. P. amarus was found to protect the
clastogenic effects of radiation as seen from decreased number
of micronuclei. The administration of P. amarus was also found to
decrease the percentage of chromosomal aberrations (Harikumar
and Kuttan, 2007). The radioprotective effect of 75% methanolic
extract of aerial parts of P. amarus was investigated in adult balb/c
mice. 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 exposed
animals. P. amarus treatment also increased the activity of various
antioxidant enzymes, such as SOD, CAT, GST, GPX, and GR, both
in 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 serum
and liver (Kumar and Kuttan, 2004).
8.20. Spasmolytic activity
Potential spasmolytic activity of the extracts of P. amarus was
judged by their ability to reduce forces of smooth muscle con-
traction of a 2 cm long piece of guinea pig ileum induced by
EC50 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 studied
on estrous cycle and follicular growth in virgin female Wistar rats
as 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/kg
body weight have been found to be systemically transformed into
enterolignan(s), which is known to be responsible for augmenting
estrous cycle in rats (Islam et al., 2008b). The aqueous crude extracts
of P. amarus were administered to 38-week old sexually mature
male albino to determine the effect of extract on the male repro-
ductive organs of these animals. The results from the study revealed
that the aqueous crude extracts of P. amarus caused varying degrees
of 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 of
aqueous extract of P. amarus on the kidney of adult Wistar albino
rats were carried out at the doses of 500 and 1000 mg/kg body
weight respectively for 28 days, while the control rats received
equal volume of distilled water. The rats were sacrificed on day
29 of the experiment and kidneys were dissected and quickly fixed
in 10% formal saline for routine histological study. The histolog-
ical findings indicated that the treated sections of the kidneys
showed hypertrophy of blood vessels, mild-severe infiltrate of
chronic inflammatory cells and varying degrees of tubular necrosis
when compared to the control sections. The findings indicated that
the 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 of
P. amarus on histology of kidney of adult Wistar rats indicated that
rats in the treated groups showed some varying degree of distortion
and disruption in microanatomy of the kidney including intersti-
tial oedema and tubular necrosis. It provides further evidence that
medicinal use of P. amarus has a potential adverse effect on kid-
ney (Adjene and Nwose, 2010). The aqueous extract of whole plant
of P. amarus was found to be more cytotoxic than hydroalcoholic
extract (IC50 being 89.6 ␮g/mL vs. 277 ␮g/mL). Acute and sub-acute
toxicity of the extract in Swiss mice and Wistar rats respectively
showed that no significant differences were observed in body
weight gain and blood glucose level between control and treated
groups. Clinical biochemistry revealed no toxic effect. Neither gross
abnormalities nor histopathological changes in liver, kidney and
pancreas 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 it
is slightly cytotoxic to the human adenocarcinoma cell line Caco-2
(Lawson-Evi et al., 2008). Chemical and cytotoxicity examinations
of the crude methanol extract of the aerial parts of P. amarus
were investigated. The cytotoxicity property of the P. amarus was
evaluated in vitro, using the human ovarian A2780 cancer cell.
Bioassay-guided fraction of the crude extract of the P. amarus (IC50
value of 31.2 ␮g/mL), showed that the dichloromethane fraction
was most toxic with an IC50 value of 22.7 ␮g/mL, whereas the
polar methanol fraction was least cytotoxic with an IC50 value of
31.2 ␮g/mL. This led to the isolation of a new chroman derivative
from the dichloromethane fraction. The compound exhibited very
little or no in vitro cytotoxicity with an IC50 value of 16.2 ␮g/mL, rel-
ative to actinomycin, the reference compound, with an IC50 value
of 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 body
weight) showed the absence of cumulative toxicity as reflected by
the non-significant change in the parameters studied as well as
from the results of the histological studies (Sirajudeen et al., 2006).
Chromatographic fractions obtained from fresh leaves extract of
P. amarus were tested for toxicity on the serum biochemistry of
rats. The six fractions were administered orally to albino rats at the
doses of 400, 800 and 1600 mg/kg body weight for 14 days. The
animals in the controlled experiment received only distilled water
for the same number of days. The results revealed that some frac-
tions of P. amarus had potentially deleterious effects on the blood
and caused a significant increase in level of ALT, ALP, total bilirubi-
nand blood urea nitrogen when compared with control (Adedapo
et al., 2005a). The aqueous crude extract was administered orally
to male rats of the Sprague Dawley strain in three groups receiv-
ing doses of 400, 800 and 1000 mg/kg but the fourth group served
as a control and received distilled water only. The pathological
changes by the aqueous crude extract of the leaves of P. amarus
caused a decrease in the red blood cell (RBC) count, packed cell
volume (PCV), haemoglobin concentration (Hb), but an increase in
the white blood cell (WBC) count. The extract also resulted in an
increase in the levels of aspartate amino transferase (AST), total and
conjugated 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 the
kidney tubules with tubular nephrosis, foci of lymphocytic infil-
tration at the portal areas of the liver as well as marked testicular
degeneration with severe disorganization of seminiferous tubules,
which were devoid of spermatic cells. A reduction in the weight of
the experimental animals was also recorded. The results revealed
that P. amarus has potential toxic properties (Adedapo et al., 2005b).
P. amarus has demonstrated hypotensive effects in animals and
humans. People with a heart condition and/or taking prescription
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J.R. Patel et al. / Journal of Ethnopharmacology 138 (2011) 286–313
of heart medications should consult their doctor before taking this
plant. It may potentiate insulin and antidiabetic drugs. It contains
a naturally occurring phytochemical geraniin. This chemical has
been documented with negative chronotropic, negative inotropic,
hypotensive and angiotensin-converting enzyme inhibitor effects
in animal studies with frogs, mice and rats (Ueno et al., 1988). As
such, this plant may potentiate antihypertensive drugs, ␤-blockers
and other heart medications (including chronotropic and inotropic
drugs). It has been considered in herbal medicine to be abortive (at
high dosages) as well as an emmenagogue. Although it is not stud-
ied in humans but animal studies do indicated that it has uterine
relaxant effects. It is therefore contraindicated during pregnancy. It
has been documented with female antifertility effects in mice (the
effect was reversed in 45 days after cessation of dosing) (Rao and
Alice, 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 of
this plant may be contraindicated in various other medical con-
ditions where diuretics are not advised. Chronic long-term use of
any 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 detailed
information as presented in this review on the phytochemical and
various biological properties of the plant might provide detailed
evidence for the use of this plant in different diseases. It has
various traditional uses that differ from one country to another
whereas some important uses for the treatment of jaundice, dia-
betes, dysentery, fever, gonorrhea, syphilis and stomachache and
skin diseases are almost common. P. amarus, a potent herbal
medicine is attracting researchers since many decades due to its
high therapeutic value. There is a demand to standardize the prop-
erties of P. amarus and their detailed clinical trials. Pharmacological
and chemical studies have demonstrated that the extracts of the
plant possess various pharmacological actions viz. antiviral, anti-
inflammatory antimalarial, antimicrobial, anticancer, antidiabetic,
hypolipidemic, hepatoprotective and nephroprotective. Owing to
the impressive preclinical therapeutic potential, the plant extracts
have been evaluated in human trials for the treatment of HIV, jaun-
dice, hypertension and diabetes.
P. amarus is reported to contain lignans, flavonoids, hydrolysable
tannins (ellagitannins), polyphenols, triterpenes, sterols and
alkaloids. The phytochemicals exhibited different structural char-
acteristics with various pharmacological actions. The lignans
nirtetralin, 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 mouse
cerebral cortex membranes. Phyltetralin, nirtetralin and niranthin
also inhibited carrageenan-induced paw oedema and neutrophil
influx. Niranthin also showed the best anti-HBsAg activity while
the 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 significant
antioxidant activity. Phyllanthin isolated from aqueous extract
of leaves of P. amarus exhibited very high antioxidant activity.
Amariin, repandusinic acid and phyllanthusiin D showed higher
antioxidant activity. A gallotanin containing fraction and the iso-
lated elllagitanins geraniin and corilagin were shown to be most
potent mediators of antiviral activities. The purified gallatanins
geraniin and corilagin were most active to inhibit HIV-1 replica-
tion in Hela CD4+ cells. Mixture of phyllanthin and hypophyllanthin
309
(1:1) exhibited antitumor activity against EAC in Swiss albino
mice. Lignans nirtetralin, niranthin or phyllanthin exerted cyto-
toxic effects on K-562 cells. Phyllanthin demonstrated its role in
protection 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 extract
of P. amarus were shown to inhibit ␣-amylase. More importantly,
there have been no side effects or toxicity reports from many years
of research on this herb. Thus activity guided phytochemical and
phytoanalytical information appears to be very useful and might
led to development of novel agents for various disorders and could
be explored further for commercial purposes. However, there are
many aspects, which need to be explored like well-controlled clin-
ical trials using large sample size (large number of patients) for the
efficacy and toxicity, the mechanism of biological activity of active
constituents present in the plant. On the basis of biological activities
of P. amarus, crude extract and derived phytochemicals and their
uses as pharmacological agents in traditional and modern research
are possible but will first require more clinical trials and product
development. The current evidence is large limited to correlation
between identified phytochemicals and mode of action for any
pharmacological activity. Mechanism of action studies are expected
to lead the way in the discovery of new agents with improved
and intriguing pharmacological properties. This could be achieved
by molecular modeling studies involving interaction of bioactive
phytochemicals from P. amarus with their respective molecular tar-
gets and the extract of P. amarus could be further explored in the
future as a source of useful phytochemicals for the pharmaceutical
industry.
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