Extracts Of Callus Cultures Research Articles

Влияние биологически активных веществ на тепловой и окислительный стресс модельных объектов Caenorhabditis elegans

Modern medicine strives to prevent age-related diseases. Oxidative stress is associated with development and progression of various diseases. Reactive oxygen species are part of vital physiological processes. High levels of reactive oxygen lead to stress and pathology whereas low ones are associated with healthy physiology. Plant-derived adaptogens demonstrate good results in stress tolerance and homeostasis. Plant materials are a pharmacologically optimal source of chemical compounds to treat various diseases, including those caused by oxidative stress. The research featured biologically active substances isolated from extracts of callus, suspension, and root cultures of medicinal plants. Baicalin and trans-cinnamic acid were obtained from Scutellaria baicalensis while ursolic acid came from Thymus vulgaris. The biologically active substances were tested for neuroprotective properties, as well as for the impact on the expression of SOD-3 and HSP-16.2. Caenorhabditis elegans served as a model to study the accumulation of carbonylated proteins and lipofuscin. The neuroprotective activity of all tested substances decreased as their concentration fell from 200 to 10 μmol/L. C. elegans proved more resistant to thermal stress if pretreated with the biologically active substances. In response to thermal stress, nematodes expressed SOD-3 more actively than HSP-16.2. At 100 μmol/L, the biologically active substances could reduce the level of carbonylated proteins. Ursolic acid was especially effective against protein carbonylation and lipofuscin accumulation in all concentrations. Baicalin, trans-cinnamic acid, and ursolic acid made it possible to reduce oxidative and thermal stress, thus demonstrating good prospects for further studies as part of adaptogenic prepa rations.

Antioxidant and Anticancer Activity of Tannins Isolated from Callus Cultures of Achyranthes aspera L.

In the current study, the impact of oxidative stress induced by free radicals generated during metabolic processes was investigated. It was found to be linked to a variety of diseases due to their detrimental effects on nucleic acids and proteins. The primary focus remained on investigating the free radical-scavenging properties and potential anticancer activities of tannins found in callus cultures of A. aspera. It was observed that the induction of callus formation was notably successful when using leaf and root explants, resulting in a callus index as high as 160, as compared to stem explants in the presence of auxins. The extraction yield of callus tannins was the most abundant in chloroform extracts, although the overall antioxidant activity was comparable among chloroform, methanol, and petroleum ether callus extracts. Among these extracts, the chloroform extract from stem callus cultures grown on the MSDN medium showed the highest total antioxidant activity. Notably, tannins extracted from A. aspera leaf callus extracts displayed significant anticancer effects against the Jurket cell line. These effects were evaluated through measures such as cell viability and colony formation. The anticancer activity was notably higher in callus culture extracts as compared to the control. In summary, the results showed that in vitro biomass production can be a valuable approach for augmenting the production of bioactive compounds, such as tannins, from selected medicinal plants including A. aspera. Tannins extracted from A. aspera hold promise for their potential role in the development of new medicines.

Induction of bioactive constituents and antioxidant enzyme activities in Achillea fragrantissima (Forskal) callus cultures using ZnO nanoparticles

The medicinally effective plant Achillea fragrantissima exhibits a magnitude of pharmacological activities. In this study, the effects of different ZnONP concentrations on antioxidant enzymes, bioactive secondary metabolites, redox potential, and molecular changes in A. fragrantissima callus cultures were investigated. First, the concentrations of the growth regulators 2,4-D and BA were optimized using Murashige and Skoog (MS) medium. The MS medium was then administered with 2,4-D and BA at its optimal dosage (1.0 mg.L−1); afterward, different ZnONP supplements (0.0, 5.0, 10.0, 15.0, and 20.0 mg.L−1) were added. ZnONPs resulted in many physiological and molecular responses. ZnONPs significantly increased POD, APX, and SOD activities. While 10.0 mg.L−1 ZnONPs significantly increased POD and APX activities, 15.0 mg.L−1 ZnONPs significantly increased SOD. However, CAT activity gradually decreased with ZnONPs. Metabolically, ZnONPs increased phenolics, flavonoids, alkaloids, and saponin levels. Phenolic levels peaked at 20.0 mg.L−1, flavonoids at 15.0 mg.L−1, and alkaloids and saponins at 10.0 mg.L−1. Terpenoids were more prevalent at lower levels of ZnONPs. With 15.0 and 10.0 mg.L−1 giving the maximum activity, ZnONPs enhanced the DPPH activity and TAC of the callus culture extracts, respectively. RAPD and ISSR fingerprinting were applied using 12 random and ISSR primers to evaluate the genetic stability of ZnONP-induced callus cultures. Six RAPD primers showed 83% polymorphism while the seven ISSR primers achieved 30% polymorphism. Consequently, DNA mutations may have been induced by ZnONPs and caused DNA fragments to either appear or disappear in RAPD and ISSR callus profiles. The dendrogram based on RAPD and ISSR combined data showed that by increasing ZnONP concentration the genetic differentiation among callus cultures was elevated. In conclusion, higher accumulation of secondary metabolites and redox activity were increased in A. fragrantissima callus cultures using low ZnONPs (10.0 mg.L−1) concentration.

Isolation of the Main Biologically Active Substances and Phytochemical Analysis of Ginkgo biloba Callus Culture Extracts.

The work reveals the results of studying the content of biologically active substances in samples of extracts of Ginkgo biloba callus cultures. Callus cultures grown in vitro on liquid nutrient media were the objects of the study. Considering various factors affecting the yield of the target components during extraction, the volume fraction of the organic modifier in the extracting mixture, the temperature factor, and the exposure time were identified as the main ones. The maximum yield of extractive substances (target biologically active substances with a degree of extraction of at least 50%) from the samples of callus culture extracts was detected at a ratio of extragent of 70% ethanol, a temperature of 50 °C, and exposure time of 6 h. Flavonoids, such as luteolin, quercetin, isoramentin, kaempferol, and amentoflavone, were isolated in the extract samples. As a result of column chromatography, fractions of individual biologically active substances (bilobalide, ginkgolide A, B, and C) were determined. The proposed schemes are focused on preserving the nativity while ensuring maximum purification from associated (ballast) components. Sorbents (Sephadex LH-20, poly-amide, silica gel) were used in successive stages of chromatography with rechromatography. The degree of purity of individually isolated substances was at least 95%.

Каллусные культуры Spergularia marina (L.) Griseb.: получение и фитохимический анализ

In recent decades, interest in halophyte plants has increased due to their high content of biologically active substances with powerful antioxidant, antimicrobial, anti-inflammatory and antitumor properties and promising for the prevention of various diseases. Several species of halophytes grow on the territory of the Kaliningrad region, among which the rare species Spergularia marina (L.) Griseb. is of particular interest, the biological activity and content of secondary metabolites of which have not been studied sufficiently. The purpose of this study was to obtain callus cultures, to study the content of some groups of phenolic compounds and the antioxidant activity of extracts. The study carried out the selection of growth regulators and concentrations that induce callus formation. 19 nutrient media were selected for the induction of S. marina callus cultures. The conducted phytochemical analysis showed a significant content of phenolic compounds and hydroxycinnamic acids, as well as a high level of antioxidant activity of extracts of callus cultures. Out of 19 callus cultures, cultures obtained on Murashige and Skoog nutrient media containing the following combinations of growth regulators are promising for obtaining target secondary metabolites: 0.25 mg/l TDZ and 1 mg/l 2,4-D; 0.1 mg/l TDZ and 1.5 mg/l 2,4-D; 0.25 mg/l TDZ and 0.25 mg/l IBA; 0.5 mg/l TDZ and 0.25 mg/l IBA; 0.25 mg/l KinN and 0.5 mg/l 2,4-D.

Effects of bioactive substances isolated from Siberian medicinal plants on the lifespan of Caenorhabditis elegans

Medicinal plants are sources of natural antioxidants. Acting as reducing agents, these substances protect the human body against oxidative stress and slow down the aging process. We aimed to study the effects of bioactive substances isolated from medicinal plants on the lifespan of Caenorhabditis elegans L. used as a model organism.
 High-performance liquid chromatography was applied to isolate bioactive substances from the extracts of callus, suspension, and root cultures of meadowsweet (Filipendula ulmaria L.), ginkgo (Ginkgo biloba L.), Baikal skullcap (Scutellaria baicalensis L.), red clover (Trifolium pretense L.), alfalfa (Medicágo sativa L.), and thyme (Thymus vulgaris L.). Their effect on the lifespan of C. elegans nematodes was determined by counting live nematodes treated with their concentrations of 10, 50, 100, and 200 µmol/L after 61 days of the experiment. The results were recorded using IR spectrometry.
 The isolated bioactive substances were at least 95% pure. We found that the studied concentrations of trans-cinnamic acid, baicalin, rutin, ursolic acid, and magniferin did not significantly increase the lifespan of the nematodes. Naringenin increased their lifespan by an average of 27.3% during days 8–26. Chlorogenic acid at a concentration of 100 µmol/L increased the lifespan of C. elegans by 27.7%. Ginkgo-based kaempferol and quercetin, as well as red clover-based biochanin A at the concentrations of 200, 10, and 100 µmol/L, respectively, increased the lifespan of the nematodes by 30.6, 41.9, and 45.2%, respectively.
 The bioactive substances produced from callus, root, and suspension cultures of the above medicinal plants had a positive effect on the lifespan of C. elegans nematodes. This confirms their geroprotective properties and allows them to be used as anti-aging agents.

The role of poliphenols in formation of fungicidal activity and resistance to exometabolites of the fungus on the example of in vitro crops sunflower and savory

When studying the formation of secondary metabolites represented by phenolic compounds in callus cultures of sunflower plants, it was found that under in vitro conditions under the influence of a stress factor, the accumulation of polyphenols increases. It was found that the biosynthetic activity of callus cultures with respect to polyphenol increases in proportion to the concentration of the culture filtrate of the fungus (Sclerotinia Sclerotiorum) in the nutrient medium. This is confirmed by the qualitative and quantitative determination of the content of polyphenols in callus cultures. In savory plants, when introduced into culture in vitro, there is a sharp decrease in the biosynthesis of phenolic compounds. Ethanol extracts of microclones and callus cultures showed no fungicidal activity.

Ginsenoside and phenolic compounds in hydromethanolic extracts of American ginseng cell cultures and their antioxidant properties

The present study was performed to investigate the antioxidant properties of callus and suspension culture extracts of <em>Panax quinquefolium</em> (American ginseng). The ginsenoside content and the total phenolic content (TPC) in these cultures were also examined. The total amount of the nine studied saponins was found to be 2.08, 1.69, and 0.202 mg g<sup>−1</sup> dry weight in red callus line (RCL), green callus line (GCL), and suspension cultures, respectively, by HPLC analysis. The TPC was estimated using the Folin–Ciocalteu method. The TPC of the suspension culture extracts was approximately 36.7% and 17.6% higher than that of the RCL and GCL, respectively. The antioxidant activity of the extracts was evaluated using the in vitro ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] and FRAP (ferric reducing antioxidant power) assays; the methanolic <em>P. quinquefolium</em> suspension culture extracts demonstrated stronger antioxidant activity than that of the callus cultures.

Benzophenanthridine Alkaloids from Callus Cultures of Toddalia Asiatica

Benzophenenathridine alkaloids nitidine and chelerythrine production from callus cultures of Toddalia asiatica was investigated. Callus cultures were initiated on Murashige and Skoogs medium fortified with different hormonal combinations. Nitidine and chelerythrine levels in callus culture extracts was analyzed by high performance liquid chromatography and was found that medium supplemented with 2,4 (0.5 mg/l) + BA (1 mg/l) accumu-lated significant amount of nitidine and chelerythrine than any other phytomormonal combination employed. An attempt was also made to compare high performance liquid chromatography and HPTLC analysis for secondary metabolite estimation. Production of secondary metabolites through callus cultures may serve as a best alternate system and optimization of media will result in improved yield of secondary metabolites.

Antioxidant and antimicrobial activities of callus culture and leaf extracts of wild <i>Crotalaria retusa</i> (rattlepod)

The present study describes the in vitro antimicrobial and antioxidant activity of methanol extracts of callus culture and leaves of wild plants of Crotalaria retusa Linn. Callus cultures from leaves were initiated on MS media supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6 Benzyloaminopurine (BAP). Optimum callus regeneration was obtained on MS medium supplemented with 0.5 mg/L BAP and 1.0 mg/L 2,4-D with 50% cultures response at 20 days. The wild plant extracts (WPE) showed the highest antibacterial activity with zone of inhibition (ZI) of 13.83±0.33 mm on Pseudomonas aeruginosa (Gram negative), as the most affected organism. The callus culture extract (CCE) gave the lowest MIC value of 0.78 mg/ mL for most of the bacteria and fungi and the lowest MBC values of 0.78 mg/ mL and 1.56 mg/ mL against bacteria and fungi, respectively. The WPE showed a higher antioxidant activity (91%) than the CCE (58%). Comparable total phenol content was observed in the WPE and CCE, whereas the WPE showed a higher flavonoids content (6.56±1.52 mg equivalent of quercetin/g of extract) than the CCE (3.22±1.01 mg equivalent of quercetin/g of extract). The in vitro propagation protocol employed in this study supported the rapid development of calluses from Crotalaria retusa seed explants which may offer an alternative to the exploitation of this valuable medicinal plant for its antimicrobial and antioxidant properties via optimization of the tissue culture technique.Keywords: Crotalaria retusa; In vitro propagation; Callus culture; Antimicrobial activity; Antioxidant activity

English

The aim of this study was to evaluate the antibacterial activity of Mucuna pruriens (Velvet beans) callus culture with different hormonal combination in culture medium and leaf extracts was investigated for the maintenance of antimicrobial activity in in vitro level. The antibacterial activity was determined by the agar-well diffusion method against four different plants pathogenic bacterial species; they were Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumoniae and Pseudomonas aeruginosa. Various solvents such as chloroform, methanol and ethanol were used. The leaf, node and internode segments of the plant were cultured on Murashige and Skoog (MS) medium supplemented with various growth regulators. Maximum callus formation was observed in MS medium amended with 1mg/L α-Naphthalene acetic acid (NAA). Plant cell callus culture extracts obtained from culture conditions were also bioactive and it can be a potential source of natural antioxidant and antimicrobial agent. Thus, the positive results suggest that the M. pruriensextracts should be further studied to determine the bioactive chemical compounds.   Key words: Mucuna pruriens, callus culture, NAA (α-Naphthalene acetic acid), antimicrobial activity.

Preliminary Phytochemical Analysis of Methanol and Chloroform Extracts of Crude Whole Plant and Dried Callus cultures of Munronia pinnata

Munronia pinnata (Meliacea) is considered as an endangered plant in Sri Lanka due to over exploitation of wild populations for medicinal purposes. Propagation through seeds is a rather difficult task and there is no program for its large-scale cultivation. Therefore the market value of this is high in Sri Lanka. Hence, there is an urgent need to adopt ex situ conservation methods via in vitro propagation techniques. Due to this, in vitro propagation of Munronia pinnata has gained much attention at present [1, 2]. This study is based on the investigation of preliminary phytochemical analysis of natural plants and callus cultures of M. pinnata. Methanol and chloroform extracts of crude powder and callus cultures of M. pinnata were analyzed by Thin Layer Chromatography (TLC). Both methanol and chloroform extracts gave positive results for terpenoids, and negative results for alkaloids. It was observed that there are three chemical compounds with similar Rf for both extracts in different combinations of the solvent system. Tetranor triterpenoids, triterpenoids and musinisins have been reported on M. delavayi, M. henryi and M. sinica in China [3] and they are described as synonyms for M. pinnata in Flora of China [4]. To date no chemical research has been done about crude whole plant and dried callus cultures of Munronia pinnata. These terpenoids were further analyzed in order to characterize the nature of the compounds. Acknowledgements: University of Grant commission, Sri Lanka for Research Grants- 2008, The National Center for Advanced Studies in Humanities and Social Sciences (NCAS), Sri Lanka. The authors are thankful to NCNPR, UM, USA for supportive assistance to laboratory training for this research project.

Acute and Chronic Toxicity Studies of the Water and Ethanol Extracts of Natural and Callus Cultures of Munronia pinnata in Healthy Wistar Rats

Acute and Chronic Toxicity Studies of the Water and Ethanol Extracts of Natural and Callus Cultures of Munronia pinnata in Healthy Wistar Rats

In Vitro Cultures of Schisandra chinensis (Turcz.) Baill. (Chinese Magnolia Vine)—a Potential Biotechnological Rich Source of Therapeutically Important Phenolic Acids

The contents of free phenolic acids and cinnamic acid were determined using an HPLC method in methanolic extracts from biomass of Schisandra chinensis (Turcz.) Baill. (Chinese magnolia vine) at different stages of organogenesis, cultured in vitro on a few variants of Murashige and Skoog (MS) medium, containing different concentrations of plant growth regulators 6-benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA) (from 0.1 to 3.0 mg/l) and in extracts from overground parts of plants growing in vivo. Six of 12 analysed compounds were detected in all extracts: chlorogenic, p-coumaric, p-hydroxybenzoic, protocatechuic, salicylic and syringic acids. Total contents of the examined metabolites in biomass of shoot-differentiating callus culture cultivated on six MS medium variants were dependent on concentrations of growth regulators in the media and ranged from 14.90 to 60.05 mg/100 g d.w. Total contents of the compounds in biomass extracts from undifferentiating callus culture maintained only on two of six MS medium variants were higher and amounted to 74.54 and 78.24 mg/100 g d.w. Maximum total contents of phenolic acids in both types of in vitro cultures were greater than in fruits (55.73 mg/100 g d.w.) and leaves (4.55 mg/100 g d.w.) of plants gowning in vivo. Chlorogenic acid and salicylic acid were the main compounds identified in biomass extracts of shoot-differentiating callus cultures (max 22.60 and 21.17 mg/100 g d.w., respectively), while chlorogenic acid (max 38.43 mg/100 g d.w.) and protocatechuic acid (max 20.95 mg/100 g d.w.) prevailed in the extracts from undifferentiating callus cultures. Other compounds dominated in fruits, namely p-coumaric acid (23.36 mg/100 g d.w.) and syringic acid (14.96 mg/100 g d.w.). This is the first report on biochemical potential of cells from S. chinensis in vitro cultures to produce the biologically active phenolic acids. These are the first results on the analysis of this group of metabolites in overground parts of plants growing in vivo, too.

Bioactive Chemical Constituents and Comparative Antimicrobial Activity of Callus Culture and Adult Plant Extracts from Alternanthera tenella

Crude extracts of a callus culture (two culture media) and adult plants (two collections) from Alternanthera tenella Colla (Amaranthaceae) were evaluated for their antibacterial and antifungal activity, in order to investigate the maintenance of antimicrobial activity of the extracts obtained from plants in vivo and in vitro. The antibacterial and antifungal activity was determined against thirty strains of microorganisms including Gram-positive and Gram-negative bacteria, yeasts and dermatophytes. Ethanolic and hexanic extracts of adult plants collected during the same period of the years 1997 and 2002 [Ribeirão Preto (SP), collections 1 and 2] and obtained from plant cell callus culture in two different hormonal media (AtT43 and AtT11) inhibited the growth of bacteria, yeasts and dermatophytes with inhibition halos between 6 and 20 mm. For the crude extracts of adult plants bioassay-guided fractionation, purification, and isolation were performed by chromatographic methods, and the structures of the isolated compounds were established by analysis of chemical and spectral evidences (UV, IR, NMR and ES-MS). Steroids, saponins and flavonoids (aglycones and C-glycosides) were isolated. The minimum inhibitory concentration (MIC) of the isolated compounds varied from 50 to 500 microg/mL.

Antitumoral and antileishmanial dioncoquinones and ancistroquinones from cell cultures of Triphyophyllum peltatum (Dioncophyllaceae) and Ancistrocladus abbreviatus (Ancistrocladaceae)

From the methanolic extracts of solid callus cultures from two species of the closely related palaeotropical plant families Dioncophyllaceae and Ancistrocladaceae seven new natural naphthoquinones were isolated, dioncoquinones A ( 4) and B ( 5) from Triphyophyllum peltatum, and ancistroquinones B ( 6), C ( 7), D ( 9), E ( 10), and F ( 12) from Ancistrocladus abbreviatus. Their structures were elucidated by spectroscopic, chemical, and computational methods. Furthermore, the already known naphthoquinones plumbagin ( 2), droserone ( 3), malvone A ( 8), and nepenthone A ( 11) were found in the extract of A. abbreviatus. Dioncoquinones A ( 4) and B ( 5) showed good – and specific – activity against Leishmania major, while they were not active against other protozoic parasites. Moreover, treatment with 4 and 5 strongly induced apoptosis in human tumor cells derived from two different B cell malignancies, B cell lymphoma and multiple myeloma, without any significant toxicity towards normal peripheral mononuclear blood cells.

In vitro antiplasmodial activity of callus culture extracts and fractions from fresh apical stems of Phyllanthus niruri L. (Euphorbiaceae): part 2

The ethanolic extracts from 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 with intact plant part and whole plant extracts. Results from the in vitro antiplasmodial testing indicated that the EtOH extract of a 1-month-old callus culture (IC 50 = 16.3 ± 2.5 μg/ml) exhibited a higher activity than the ethanolic extracts of the fresh apical stem (IC 50 = 18.2 ± 2.4 μg/ml) and callus cultures of 2-, 4- and 6-months-old (25 μg/ml < IC 50 < 40 μg/ml). These activities were however lower than that displayed by the ethanolic extract of the whole plant (IC 50 < 3 μg/ml). The EtOH extract of 1-month-old callus culture (the most active) was fractionated with solvents of different polarities. Its CH 2Cl 2 fraction rich in terpenic constituents (IC 50 = 9.2 ± 3.4 μg/ml) exhibited a higher antiplasmodial activity than its isoamylic alcohol fraction obtained at pH 2–3 (IC 50 = 25.6 ± 2.3 μg/ml) rich in flavonoids. The activity of these two fractions was lower than that displayed by the same fractions from the whole plant (2 μg/ml < IC 50 < 3 μg/ml). Alkaloidic fractions from the whole plant and 1-month-old callus culture of fresh apical stem were considered as inactive (IC 50 > 100 μg/ml).

Comparative study of antibacterial and antifugal activity of callus culture and adult plants extracts from Alternanthera maritima (Amaranthaceae)

The aim of this study was to evaluate the antibacterial and antifungal activity of callus culture (two different hormonal combination culture medium) and adult plants (two collect) extracts from Alternanthera maritima (Amaranthaceae) investigating the maintenance of antimicrobial activity in vivo and in vitro. The antibacterial and antifungal activity was determined by the agar-well diffusion method against thirty strains of microorganisms including Gram-positive and Gram-negative bacteria, yeasts and dermatophytes. All the organic crude extracts studied were bioactive. Extracts of aerial parts and roots of adult plants collected during the same period of years of 1995 and 1998 (Restinga de Marica (RJ), collect 1 and 2) inhibited the growth of several microorganisms (bacteria, yeasts and dermatophytes) with inhibition halo between 6 and 20 mm. Plant cell callus culture extracts obtained from two culture conditions were also bioactive. Thus, the positive results suggest that the A. maritima extracts should be further studied to determine the bioactive chemical compounds as well as to understand the possible mechanisms of action and evaluate their toxicity looking toward a pharmaceutical employment.

Single step Al2O3 chromatography for improved separation and recovery of Taxol

An improved method that uses a single alkaline Al2O3 chromatography step was developed both for separating Taxol from the extracts of Taxus cuspidate callus cultures, and meanwhile converting other taxanes to Taxol catalyzed by the Al2O3 adsorbent. Under the optimized operating conditions, a Taxol recovery of 170% was obtained. The final Taxol content was 29% starting from less than 1% in the initial extracts.

Analgesic effects of callus culture extracts from selected species of Phyllanthus in mice.

The aim of this study was to evaluate the analgesic effect of the methanolic extract from callus culture of Phyllanthus tenellus, P. corcovadensis and P. niruri in several models of pain in mice. The extracts (medium containing 2,4-dichlorophenoxyacetic acid) of P. corcovadensis, P. niruri and P. tenellus (3-90 mg kg-1, i.p.) caused graded inhibition of abdominal constrictions induced by acetic acid (0.6%), with ID50 (i.e. dose that reduced response of control by 50%) values of about 30, 19 and > 30 mg kg-1, respectively. The extract of callus of Phyllanthus obtained in indole-3-butyric acid and indole-3-acetic acid media (3-90 mg kg-1, i.p.) caused a similar analgesic effect. In the formalin test, the extract of P. tenellus obtained in indole butyric acid medium (3-100 mg kg-1, i.p.) inhibited only the second phase of formalin-induced pain with an ID50 value of about 100 mg kg-1. Both the indole acetic acid and indole butyric acid methanolic extracts of P. tenellus and P. corcovadensis (10-100 mg kg-1, i.p.) dose-dependently inhibited both phases of formalin-induced pain (ID50 values for the second phase were approx. 100 and 52 mg kg-1, respectively). However, the extract of callus from Phyllanthus failed to affect formalin-induced paw oedema, as well as the response to radiant heat in the tail-flick test. In addition, the analgesic effect of morphine, but not the analgesic effects caused by Phyllanthus callus extract, was fully antagonized by naloxone.(ABSTRACT TRUNCATED AT 250 WORDS)