Abstract
The ethanol extracts of fresh apical stems of Phyllanthus niruri L. (Euphorbiaceae) cultured on Murashige and Skoog (MS) medium supplemented with IBA/BAP/Coco nucifera L. milk for 1, 2, 4, and 6 months were phytochemically and biologically investigated and compared to the intact plant part and whole plant extracts. A phytochemical screening revealed the presence of steroids, terpenes, and flavonoids as the major phytochemical groups, but not the same constituents belonging to these groups in all extracts. Alkaloids were detected in whole plant extract and were in traces in the intact apical stem extract and callus extracts. Results from the in vitro antiplasmodial testing indicated that the callus extract 1 month old (IC50 = 16.3±2.5 µg/ml) exhibited activity similar to the intact plant part extract (IC50 = 18.2±2.5 µg/ml) (p = 0.05). This activity was significantly higher (p < 0.001) than that exhibited by extracts from callus cultures 2, 4, and 6 months old (25 < IC50 < 40 µg/ml). The activity of these extracts is lower than that displayed by the same extract from the whole plant (IC50 = 2.5±0.2 µg/ml).
Introduction
The synthesis and the accumulation of secondary metabolites in plants are regulated in a coordinated fashion. The use of plant cell cultures has been reported to be a potential source for the production of secondary metabolites. Callus cultures are initiated for qualitative and quantitative comparative studies of secondary metabolites synthesis between the intact plant material and callus extracts (Spencer et al., Citation1993; Bahorun et al., Citation1994). They are also used to study the growth and productivity of some important constituents in plants that can be affected by the nature and concentrations of growth factors (Kitanaka et al., Citation1985; Steven et al., Citation1993; Stuhlemmer et al., Citation1993; Yamamoto et al., Citation1993; Saker & El Ashal, Citation1995; Balz et al., Citation1999; Cusido et al., Citation1999). This technique is widely used for the comparison of biological activities of extracts or isolated compounds from the intact plant material to that of cultured plant material obtained in some experimental conditions (Santos et al., Citation1994).
Phyllanthus niruri L. (Euphorbiaceae) is a medicinal plant widely used in different regions in the world for the treatment of various diseases. An aqueous infusion of the whole plant is a typical preparation employed as a stomachic, aperitive, antispasmodic, laxative, diuretic, carminative, against constipation, for fever including malaria, hepatitis, dysentery, gonorrhea, syphilis, tuberculosis, cough, diarrhea, and vaginitis (Kerharo & Adam, Citation1974; Oliver-Bever, Citation1986; Bharatiya, Citation1992; Nadkami, Citation1993).
The aim of the current investigation was to develop some callus culture conditions of fresh apical stems of this medicinal plant in order to compare the chemical composition of callus culture extracts to that of the intact plant part and whole plant. The in vitro antiplasmodial activity of all extracts was also examined.
Materials and Methods
Plant material
Whole plant and fresh apical stems of Phyllanthus niruri were collected in Kinshasa, Democratic Republic of the Congo, in January 1999. The plant was botanically identified by M.N. Nlandu of the Institut National d'Etudes et de Recherches en Agronomie (INERA) of the University of Kinshasa, where a voucher specimen has been deposited (INERA 994244). The whole plant was dried at 40oC and reduced to powder; the apical stems were used in fresh state.
Callus culture conditions
Fresh apical stems of P. niruri rinsed with distilled water were sterilized in 70% ethanol for 1 min and then in calcium hypochloride 9% (w/v) solution for 30 min. They were finally washed five-times with sterile distilled water and aseptically cut in segments 0.5-cm long. These segments were cultured in sterile polystyrene flasks containing 40 ml of Murashige and Skoog (MS) medium (Murashige & Skoog, Citation1962) according to the nature of growth factors and the time of cultivation. The medium was supplemented separately with 2,4-dichlorophenoxyacetic acid (2,4-D; 4 mg/l), a mixture of indolebutyric acid (IBA) and benzylaminopurine (BAP; 2 mg/l each), a mixture of kinetine (4 mg/l) and albumen liquid of Coco nucifera L. (100 ml/l), and a mixture of IBA/BAP (2 mg/l each) and albumen liquid of C. nucifera milk (100 ml/l). Prior to autoclaving at 120°C (1.5 kg/cm2) for 30 min, the pH of all media was adjusted to 5.8 with 0.1 N NaOH. Some flasks were plugged with sterile cotton and incubated at 27°C in light and others in dark in a phytotron incubator for 12 h. The initiated calli were routinely subcultured onto a fresh medium at 2 to 3 weeks not only to produce large amounts of calli, but also to assure a normal growth and to prevent an eventual deterioration of cultured segments characterized by the appearance of a brown color. The adding of activated charcoal also prevents this phenomenon (CitationLuyindula, 1997).
Preparation of extracts
Fifty grams of fresh intact apical stems and powdered dry whole plant were separately macerated with 300 ml EtOH (3 × each 24 h). The mixture was filtered and the filtrate evaporated under reduced pressure, yielding corresponding dry extracts denoted PNT (2.873 g) and WP (5.37 6 g), respectively. Five grams of extract from callus culture 1, 2, 4, and 6 months old were treated in the same manner, yielding the corresponding dry extracts denoted as PNC1 (0.2887 g), PNC2 (0.3024 g), PNC4 (0.3081 g), and PNC6 (0.2379 g), respectively.
TLC analysis of extracts
Thin-layer chromatography (TLC) analysis of extracts WP, PNT, PNC1, PNC2, PNC4, and PNC6 was carried out on silica gel F254 plates (Merck; thickness layer 0.25 mm). Phytochemical groups were detected under UV (254 and 365 nm) before and after spraying with reagents in the following conditions: benzene/chloroform/ethanol 5:3:1 for terpenic or steroidal constituents revealed with Libermann-Bouchard; chloroform/methanol/ammonia 10% 6:4:1 for alkaloids detected with Dragendorff's reagent; n-butanol/acetic acid/water: 4:1:5 (top layer) for flavonoids using AlCl31% (MeOH solution) and FeCl3 0.1% (in 0.1 N HCl) as reagents. On the other hand, tannins and saponins were detected in aqueous macerates with a mixture of FeCl3 1%/K3Fe(CN)6 1% 1:1 and FeCl3 1% saturated with PbOAc and the froth test, respectively (Harborne, Citation1974).
Antiplasmodial testing
The in vitro antiplasmodial screening of extracts from intact plant and callus culture and intact plant material extracts was based on a modified Rieckman's version as reported by Tona et al. (Citation1999). Briefly, the test parasites were obtained from the blood of male and female adult subjects with acquired Plasmodium falciparum infection (malaria) confirmed after surgery at the Bondeko Clinic (Limete) and Medical Center Monkole (Mont-Ngafula) in Kinshasa. Donor selection was based on the following criteria: absence of antimalarial drugs use 1 week before performing the test; presence in blood of schizonts in the range of 1000 to 100,000 per mm3. The blood samples were obtained by venipuncture. A thin blood film stained with giemsa for 20 min was microscopically examined in order to determine the number of schizonts. The remaining blood samples were defibrinated in a sterile flask containing glass beads by rotation for 5 min. The in vitro antimalarial test was performed in microtiter plates. Two mg of each extract were dissolved separately in 6 ml of EtOH to give stock solutions of 0.3 mg/ml. Quinine dihydrochloride from Laboratoire d'Analyse et de Contrôle des Médicaments et des Denrées Alimentaires (LACOMEDA), Faculty of Pharmacy, University of Kinshasa, was used as an antimalarial reference product. Each test sample was applied in a series of two-fold dilution in concentrations between 500 and 0.05 µg/100 µl and were tested in triplicate. Fifty microliters of test samples were dispensed into microtiter plates and dried at 37°C. Next, 100 µl of RPMI 1640 culture medium (Difco) supplemented with 23 mM NaHCO3 and 25 mM HEPES [N-(2-hydroxyethyl)-piperrazine-N′-2-ethanesulfonic acid; Sigma) was added followed by 100 µl of aseptic infected blood suspension. In this way, the test concentrations of samples ranged from 11.65 to 187.5 µg/ml in the experimental medium. Controls consisted of infected blood in culture medium without test sample. The content of each microtiter plate was gently mixed and then incubated for 48 h at 37°C. Giemsa-stained thick blood films were prepared from each well. The number of schizonts with 3 or more nuclei per 200 asexual parasites were microscopically noted and compared with control plates for the determination of percentage inhibition of the parasite growth.
Statistical analysis
Data were expressed as mean±SD. Statistical analysis was performed using Student's t-test; p values of 0.05 or less were considered statistically significant.
Results
The ethanol extract from the callus cultures of fresh apical stems of Phyllanthus niruri cultured on MS media supplemented with IBA/BAP/Coco nucifera milk after 1 month exhibited a similar antiplasmodial (schizonticidal) activity in vitro (16.3±2.5 µg/ml) than the same extract from the intact plant material (IC50 = 18.2±2.4 µg/ml) (p = 0.05). Other callus extracts 2, 4, and 6 months old obtained in the same experimental conditions showed a lower activity (25 < IC50 < 40 µg/ml) than that of the two first ones. These activities were lower (p < 0.001) than that displayed by quininne dihydrochloride (IC50 < 1 µg/ml) used as a reference product and ethanol extract of the whole plant (2 < IC50 < 3 µg/ml). A phytochemical screening of these extracts revealed the presence of alkaloids, steroids, terpenes, and flavonoids as the common phytochemical groups in these extracts, but constituents belonging to these different groups were not the same, as indicated by TLC analysis (chromatograms not presented).
Discussion
The chemistry, the pharmacology, and the therapeutic potency of different Phyllanthus species are reported (Calixto et al., Citation1998). Those of Phyllanthus niruri have particularly shown that this species possesses some interesting biological activities related to its worldwide uses in traditional medicine. An aqueous extract of P. niruri whole plant has been found to reduce significantly the cytotoxic effect of lead nitrate and aluminum sulfate in albino mice (Dhir et al., Citation1990). It is also reported to inhibit the endogenous DNA polymerase of hepatitis B virus in vitro and in vivo tests (Venkaterswaran et al., Citation1987; Shead et al., Citation1992). On the other hand, bioassay-guided fractionations of active extracts of P. niruri whole plant resulted in the isolation and characterization of some active constituents. For example, the inhibition of angiotensin-converting enzyme was attributed to geraniin (Ueno et al., Citation1988); this compound was also isolated from the leaves of Phyllanthus sellowianus Muell. Arg. and showed a potent analgesic effect (Miguel et al., Citation1996). It can be considered as responsible for the same property exhibited by the intact P. niruri extract (Santos et al., Citation1995) but not for callus culture extracts (Santos et al., Citation1994), because it was not detected in the extract (Ishimaru et al., Citation1992). Other P. niruri constituents such as ellagic acid, brevifolin carboxylic acid, and ethylbrevifolin have been shown to be inhibitors of the aldose reductase (Shimizu et al., Citation1989); phyllanthin and hypophyllanthin isolated from a hexane extract have been reported to possess antihepatotoxic properties (Syamasundar et al., Citation1985; Prakash et al., Citation1995). Repandisinic acid A monosodium salt has been reported as an inhibitor of human immunodeficiency virus type 1–reverse transcriptase (HIV-1-RT) (Ogata et al., Citation1992), and niruriside has been shown to be an inhibitor of the binding of HIV-REV protein to [33P]-labeled RRE RNA (Qian-Cutrone et al., Citation1996).
The antiplasmodial activity of some Phyllanthus species was previously reported. The aqueous extracts from the leaves and stem of Phyllanthus reticulatus Poir. were found to be active against Plasmodium falciparum chloroquine-sensitive K67 (20 < IC50 < 25 µg/ml) and chloroquine-resistant ENT36 strains (1 < IC50 < 10 µg/ml) in vitro, whereas the aqueous extract of the root was less active against both strains (IC50 > 100 µg/ml) (Omulokoli et al., Citation1997). The CHCl3 extract from the partition of the aqueous extract of the root bark of Phyllanthus decipiens var. antsihanakensis Leandri exhibited good antiplasmodial activity in vitro against Plasmodium falciparum FCM 29/Cameroun chloroquine-resistant strain (IC50 < 5 µg/ml) (Rasoanaivo et al., Citation1999). A preliminary study conducted by Dhar et al. (Citation1968) demonstrated that 50% ethanol extract of Phyllanthus niruri whole plant failed to inhibit Plasmodium berghei growth infected mice. Recently, CitationTona et al. (1999, 2000) reported good in vitro and in vivo antiplasmodial activity of the ethanol and dichloromethane extracts of Phyllanthus niruri whole plant; the latter extract being more active than the first one. Continuing our investigation on this medicinal plant, callus cultures were initiated from fresh apical stem explants for 1, 2, 4, and 6 months, aseptically growing on MS medium containing some plant growth factors as described above. Cultures were carried out in the presence of blank cultures.
Results from callus cultures elaborated in our experimental conditions showed that no development was observed in MS medium containing no growth factors, whereas in culture medium supplemented with different growth factors, a development and/or germination was observed at different levels (results not presented). Among these trial combinations of plant growth factors, MS medium containing IBA/BAP/Coco nucifera milk was the best callus intitiation in the light as well as in the dark. Fresh apical stem segments horizontally cultured showed a better development than those vertically cultured. The cellular multiplication started at the extremities before invading all explant surfaces. The proliferation was faster in the light than in the dark. All calli were friable and characterized by a yellowish-brown color. For calli cultured in the light, the presence of small green structures resembling plantlets that emerged individually was observed. This suggested the occurrence of photosynthesis that was not observed in callus cultured in the dark. For these reasons, all calli obtained with these growth factors after 1, 2, 4, and 6 months of cultivation were selected for phytochemical and biological investigation as reported in the current study. Calli containing other growth factors in MS medium were neglected not only for their poor development, but also for their low amount after the cultivation time.
Phytochemical analysis carried out on TLC indicated the presence of alkaloids, steroids, terpenes, polyphenolic compounds, and flavonoids in the ethanol extracts of the intact fresh apical stem and whole plant. Except for alkaloids that were detected in traces, the remaining phytochemical groups cited above were also detected in the ethanol extracts of cultured calli 1, 2, and 6 months old. Steroids, terpenes, and polyphenolic compounds other than flavonoids were detected in callus 4 months old. Tannins were only detected in the whole plant extract (). The analysis of these phytochemical results suggested that growth factors introduced in MS medium qualitatively affect the production of some metabolites according to the time of cultivation. This finding corroborates the observations deduced by other authors on the influence of some plant growth factors for the production of some secondary metabolites during callus culture of some plant medicinal parts (Kartnig et al., Citation1993; Bahorun et al., Citation1994; Saker & El Ashal, Citation1995). Anthocyanins, anthraquinones, and saponins were not detected in all extracts (). Ishimaru et al. (Citation1992) reported the presence of phenolic compounds in tissue culture of Phyllanthus niruri on MS media containing 30 g/l sucrose. They included gallic acid, ( + )-catechin, ( − )-epicatechin, ( − )-epigallocatechin-3-O-gallate, ( − )-epigallocatechin, ( − )-epigallocatechin 3-O-gallate, and ( + )-gallocatechin. In our experimental conditions, it was observed that the phytochemical composition of calli cultured in the light and in the dark on MS media supplemented with IBP/BAP/C. nucifera milk was similar. They were thus combined for biological testing according to their time of cultivation.
Table 1 Phytochemical screening of whole plant, intact fresh apical stem, and callus extracts of Phyllanthus niruri.
The data presented in summarize the growth measurements expressed as fresh and dry weight yields of different calli after respective times of cultivation. Except for dried PNC6, these results indicate that the weights of fresh and dried calli significantly (p < 0.01) increased with the duration of cultivation. No significant difference (p > 0.05) in loss on drying was observed for all dried callus extracts.
Table 2 Weights of explants (g) and extracts (mg) of intact apical stem and callus culture after 1, 2, 4, and 6 months in MS supplemented with indolebutyric acid/benzylaminopurine/C. nucifera L. milk.
Results from the in vitro antiplasmodial testing are summarized in and are expressed as percent inhibition of Plasmodium falciparum growth by different extracts. All extracts exhibited a dose-dependent inhibitory effect on the development of trophozoits to schizonts. Tested at concentrations from 3 to 11.75 µg/ml, all extracts produced less than 50% inhibition of the parasite. No significant difference in activity between PNT and PNC2 could be statistically observed (p > 0.05). These two extracts produced more than 50% inhibition when tested at 23.5 µg/ml, whereas PNC2, PNC4, and PNC6 extracts showed the same effect at 47 µg/ml. No significant difference in activity of PNC4 and PNC6 was statistically observed at this test concentration (p > 0.05). To appreciate the antiplasmodial activity of these extracts, the IC50 values resulting in 50% inhibition of the parasite growth in vitro were derived by linear regression. Results show that the extract PNC1 (IC50 = 16.3±2.5 µg/ml) exhibited a similar antiplasmodial activity to PNT (IC50 = 18.2±2.4 µg/ml) (p = 0.05). PNC2, PNC4, and PNC6 extracts showed an activity with IC50 of 27.5.±3.1, 34.7±2.9, and 36.2 ± 4.5 µg/ml, respectively. This activity was lower than that displayed by the two first ones (p < 0.01). Results also show that the activity significantly decreased with the duration of cultivation. This suggested that the production of active constituents decreases not only with a long time of cultivation, but seems to be also influenced by the nature and the concentration of the growth factors incorporated to the medium. This finding is supported by results of phytochemical screening reported here. In general, the EtOH extract of Phyllanthus niruri whole plant exhibited a higher antiplasmodial activity in vitro (IC50 = 2.5±0.2 µg/ml) than all calli and intact fresh apical stem extracts.
Table 3 Inhibition (%) ofPlasmodium falciparum growth in the presence of intact apical stem and callus culture extracts.
In conclusion, the in vitro antiplasmodial activity exhibited by all callus culture extracts could be considered as a low activity compared to that shown by the whole plant extract. Taking account of the small amount of dried calli extracts obtained in our experimental conditions, the in vivo antiplasmodial activity was not carried out. Whether steroids, terpenes, and flavonoids as common phytochemical groups identified in these extracts account for their displayed antiplasmodial activity remains to be determined in further investigations. Also, it is possible to optimize callus culture conditions aiming to obtain extracts with high activity by the modification of concentrations of growth factors or by the incorporation of others factors in the experimental medium. This aspect of the study is in progress.
Acknowledgment
This paper is dedicated to the memory of Prof. Dr. Ngimbi, N.P.
References
- Bahorun T, Trotin F, Vasseux J. 1994. Comparative polyphenolic productions in Crataegus monogyna callus cultures. Phytochemistry. 37: 1273–1276
- Balz JP, Courtois D, Drieu J, Drieu K, Reynoird JP, Sohier C, Teng BP, Touché A, Pétard V. 1999. Production of ginkgolides and bilobalide by Ginkgo biloba plants and tissue culture. Planta Med. 65: 620–626. [PUBMED], [INFOTRIEVE], [CSA], [CROSSREF]
- Bharatiya VB. 1992; Selected Medicinal Plants of India. Bombay, Tata Press. 253–237
- Calixto JB, Santos ARS, Cechinel Filho V, Yunes RA. 1998. A review of the plants of the genus Phyllanthus: Their chemistry, pharmacology, and therapeutic potential. Med Res Rev. 14: 225–258. [CROSSREF]
- Cusido RM, Palazzo´n, Pifriol MT, Bonfill M, Morales C. 1999; Datura metel: In vitro production of tropane alkaloids. Planta Med. 65: 144–148
- Dhar ML, Dhar MM, Dhawan BN, Mehrotra RC. 1968. Screening of Indian plants for biological activity. Part 1. J Exp Biol. 6: 232–247
- Dhir H, Roy AK, Sharma A, Talukder G. 1990. Protection afforded by aqueous extracts of Phyllanthus species against cytotoxicity induced by lead and aluminum salts. Phytother Res. 4: 172–176
- Harborne JB. 1974; Phytochemical Methods. London, Chapman and Hall. 52–190
- Ishimaru K, Yoshimatsu K, Yamakawa T, Kamada H, Shimomura K. 1992. Phenolic constituents in tissue cultures of Phyllanthus niruri. Phytochemistry. 31: 2015–2018. [CROSSREF]
- Kartnig T, Kögl G, Heydel B. 1993. Production of flavonoids in cell culture of Crataegus monogyna. Planta Med. 59: 537–538
- Kerharo G, Adam JG. 1974; La Pharmacopée Traditionnelle Sénégalaise. Plantes Médicinales et Toxiques. Paris, Vigot Frères. 427–428
- Kitanaka S, Igarashi H, Takido M. 1985. Formation of pigments by tissue culture of Cassia occidentalis. Chem Pharm Bull. 33: 971–974
- Luyindula N. (1997): Approche moléculaire du phénomène de la symbiose entre le Soja et le Rhizobium japonicum. Thèse de doctorat. Université de Liège, Belgique, p. 54.
- Miguel OG, Calixto JB, Santos AR, Messana I, Ferrari F, Cechinel Filho V, Pizzolatti MG, Yunes RA. 1996. Chemical and preliminary analgesic evaluation of geraniin and furosin isolated from Phyllanthus sellowianus. Planta Med. 62: 146–149. [PUBMED], [INFOTRIEVE], [CSA]
- Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 15: 473–497
- Nadkarmi KM. 1993; India Materia Medica. 1, Bombay, Popular Prakashan, Private, Ltd. 947–948
- Ogata T, Higuchi H, Mochida S, Matsumoto H, Kato A, Endo T, Kaji A, Kaji H. 1992. HIV-1 reverse transcriptase inhibitor from Phyllanthus niruri. AIDS Res. Hum Retroviruses. 8: 1737–1744
- Oliver-Bever B. 1986; Medicinal Plants in Tropical West Africa. London, Cambridge University Press. 163
- Omulokoli E, Khan B, Chhabra SC. 1997. Antiplasmodial activity of four Kenyan medicinal plants. J Ethnopharmacol. 56: 133–137. [PUBMED], [INFOTRIEVE], [CSA], [CROSSREF]
- Prakash A, Satan KS, Wahi SP, Singh RP. 1995. Comparative hepaprotective activity of three Phyllanthus species, P. urinaria, P. niruri and P. simplex, on carbon tetrachloride induced liver injury in the rat. Phytother Res. 9: 594–596
- Qian-Cutrone J, Huang S, Trimble J, Li H, Lin PF, Alam M, Klohr SE, Kadow KF. 1996. Niruriside, a new HIV REV/RRE binding inhibitor from Phyllanthus niruri. J Nat Prod. 59: 196–199. [PUBMED], [INFOTRIEVE], [CSA], [CROSSREF]
- Rasoanaivo P, Ratsimamanga-Uverg S, Ramanitrahasimbola D, Rafatro H, Ratoko-Ratsimamanga A. 1999. Criblage d'extraits de plantes de Madagascar pour la recherche d'activité antipaludique et d'effet potentialisateur de la chloroquine. J Ethnopharmacol. 64: 117–126. [CROSSREF]
- Saker MM, El Ashal HA. 1995. Stimulating effect of salt strees on alkaloid production of cultured Hyoscyamus cells. Fitoterapia. 64: 360–365
- Santos AR, Filho VC, Niero R, Viana AM, Moreno FN. 1994. Analgesic effects of callus culture extracts from selected species of Phyllanthus in mice. J Pharm Pharmacol. 46: 755–759. [PUBMED], [INFOTRIEVE]
- Santos AR, Filho VC, Yunes RA, Calixto JB. 1995. Analysis of the mechanisms underlying the antinoceptive effect of the extracts of plants from the genus. Phyllanthus Gen Pharmacol. 26: 1499–1506
- Shead A, Vickey K, Pajkos A, Medhurst R, Freiman J, Dixon R, Cossart Y. 1992. Effect of Phyllanthus plant extracts on duck hepatitis B in vivo. Antiviral Res. 18: 127–138. [PUBMED], [INFOTRIEVE], [CSA], [CROSSREF]
- Shimizu M, Hories S, Terashima S, Ueno H, Hayashi T, Atisawa M, Suzuki S, Yoshizaki M, Morita N. 1989. Studies on aldose reductase inhibitors from natural products. II. Active components of a Paraguayan crude drug “Parapari mi,” Phyllanthus niruri. Chem Pharm Bull. 37: 2531–2532. [PUBMED], [INFOTRIEVE]
- Spencer A, Hamil JD, Rhodest MJC. 1993. In vitro biosynthesis of monoterpenes by Agrobactrium transformed shoot cultures of two Mentha species. Phytochemistry. 32: 911–919. [CROSSREF]
- Steven LH, Giroud C, Pennings ED JM, Verpoorte R. 1993. Purification and characterization of strictosidine synthetase from a suspension culture of Cinchona robusta. Phytochemistry. 33: 99–106. [CROSSREF]
- Stuhlemmer U, Kreis W, Eisenbeiss M, Reinhard E. 1993. Cardiac glycosides in partly subemerged shoots of Digitalis lanata. Planta Med. 59: 539–546. [PUBMED], [INFOTRIEVE]
- Syamasundar KV, Singh B, Thakur RS, Husain A, Kiso Y, Hikino H. 1985. Antihepatotoxic principles of Phyllanthus niruri herbs. J Ethopharmacol. 14: 41–44. [CROSSREF]
- Tona L, Ngimbi NP, Tsakala M, Mesia K, Cimanga K, Apers S, De Bruyne T, Pieters L, Totté J, Vlietinck AJ. 1999. Antimalarial activity of 20 crude extracts from nine African medicinal plants used in Kinshasa, Congo. J Ethnopharmacol. 68: 193–203. [PUBMED], [INFOTRIEVE], [CSA], [CROSSREF]
- Tona L, Mesia K, Ngimbi NP, Chiriwami B, Okond'ahoka A, Cimanga K, De Bruyne T, Apers S, Hermans N, Pieters L, Totté J, Vlietinck AJ. 2000. In-vivo antimalarial activity of Cassia occidentalis, Morinda morindoides and Phyllanthus niruri. Ann Trop Med Parasitol. 95: 47–57. [CSA]
- Ueno H, Hories S, Nishi Y, Shogawa H, Kawasaki M, Suzuki S, Ayashi T, Arisawa M, Shimizu M, Yoshizaki M, Morita N, Berganza LH, Ferro E, Baswaldo I. 1988. Chemical and pharmaceutical studies on medicinal plants in Paraguay. Geraniin, an angiontensin-converting enzyme inhibitor from “paraparai mi,” Phyllanthus niruri. J Nat Prod. 51: 357–359. [PUBMED], [INFOTRIEVE], [CROSSREF]
- Venkateswaran PS, Millman I, Blumberg BS. 1987. Effects of an extract from Phyllanthus niruri on hepatitis B and woodchuck hepatitis viruses: in vitro and in vivo studies. Proc Natl Acad Sci USA. 84: 274–278. [PUBMED], [INFOTRIEVE]
- Yamamoto Y, Yan K, Ieda K, Tanaka T, Inuma M, Mizuno M. 1993. Flavonol glycosides production in cell suspension cultures of Vancouveria hexandra. Phytochemistry. 33: 841–846. [CROSSREF]