In vitro antitrypanosomal activity,antioxidant property and phytochemical constituents of aqueous extracts of nine Nigerian medicinal plants

ObjectiveTo study the in vitro antitrypanosomal activity, antioxidant property and phytochemical constituents of aqueous extracts of nine Nigerian medicinal plants.MethodsIn vitro antitrypanosomal activity test was carried out on aqueous extracts of dried leaves of Acacia albida (A. albida), Artemisia absinthium, Bryophyllum pinnatum, Gongronema latifolium, Holarrhena floribunda, Leptadenia hastata, Pericopsis laxiflora (P. laxiflora) and dried stem barks of A. albida and P. laxiflora. The phytochemical constituents and composition of the extracts and the in vitro antioxidant activity of the extracts were subsequently measured using the α,α-diphenyl-β-picryl-hydrazyl (DPPH) radical scavenging assay, Ferric reducing antioxidant power (FRAP) assay, thiobarbituric acid (TBA) activity assay and H₂O₂ radical scavenging activity assay.ResultsFrom the study, it was discovered that the stem bark extracts of A. albida and P. laxiflora were most active against both Trypanosoma evansi and Trypanosoma congolense. There was complete cessation of motility in both trypanosomes within 5 min at 40 mg/mL of the stem bark extract of A. albida and complete cessation of motility within 25 min and 40 min at 40 mg/mL with P. laxiflora stem bark extract for Trypanosoma congolense and Trypanosoma evansi, respectively. Quantitative analysis of the phytochemical constituents of the aqueous extracts of the plant parts such as alkaloids, saponins, flavonoids and phenols revealed that the stem barks of A. albida, P. laxiflora and leaves of Leptadenia hastata contained relatively high amount of all the phytochemicals quantified. The stem bark extracts of A. albida, P. laxiflora and leaves of Gongronema latifolium possess more scavenging capacity when compared to other extracts in relation to vitamin C, the reference antioxidant.ConclusionsThis study provides scientific evidence for the use of A. albida, and P. laxiflora for the treatment of trypanosomosis and diseases associated with oxidative stress.     

In vitro bioactivity and phytochemical screening of Suaeda maritima (Dumort): a mangrove associate from Bhitarkanika, India

ObjectiveTo investigate the in vitro antioxidant and antimicrobial activities along with phytochemical screening of organic and aqueous extracts of leaf and stem of Suaeda maritima (Dumort), a mangrove associate from Bhitarkanika of Odisha, India.MethodsAntioxidant activity of the crude extracts was evaluated in terms of total antioxidant capacity, total phenol content, ascorbic acid content, DPPH radical scavenging, metal chelating, nitric oxide scavenging, and reducing power etc. The antimicrobial activity of the plant was determined by agar well diffusion method along with MIC and MBC carried out by microdilution techniques against 10 gram positive and gram negative human pathogenic bacteria. The qualitative and quantative phytochemical screening were carried out by standard biochemical assays.ResultsOut of the seven antioxidant bioassays, both the leaf and stem extracts were found to posses strong antioxidant properties of 70 % to 92 % for phenol, total antioxidant capacity, DPPH free radical scavenging activity and fairly good ascorbic acid content, metal chelating (1.33 %-22.55 %), reducing power (0.01-0.12) and nitric oxide scavenging (0.84 %-66.99 %) activities. Out of the four extracts evaluated for antimicrobial activity, two leaf extracts such as acetone and ethanol showed promising activity against four pathogenic bacteria and one stem methanol extracts against one pathogenic bacteria when compared with amoxcycillin as standard. The MIC and MBC values of the antimicrobial extracts ranged between 2.5 to 5.0 mg/mL. Screening of phytochemicals showed presence of carbohydrates, protein, tannins, alkaloids and flavonoids in comparatively higher amount than other phytochemicals tested.ConclusionsThe present study reveals the presence of potential antioxidants and antimicrobial properties in the plant extract which could be exploited for pharmaceutical application.     

Antibacterial activity of crude ethanolic extract and solvent fractions of Ficus pseudopalma Blanco leaves

ObjectiveTo investigate the antimicrobial properties of Ficus pseudopalma (F. pseudopalma) leaf extracts.MethodsThe antibacterial properties of F. pseudopalma Blanco crude ethanolic leaf extract, and its solvent fractions chloroform (CF), ethylacetate (EF) and water fractions were evaluated through antibacterial agar disc diffusion method and the minimum inhibitory concentration (MIC) was determined. Five Gram-positive and five Gram-negative bacteria were used for the study.ResultsThe zone of inhibitions obtained from the antibacterial agar diffusion disc method showed that CF, and EF exhibited active (14-19 mm) antibacterial activity against Bacillus subtilis UST CMS 1011, and partially active (10-13 mm) antibacterial properties against both Staphylococcus aureus ATCC 25923 and Staphylococcus epidermidis ATCC 12228. Water exhibited no antibacterial properties against all microorgranisms tested. The MIC values observed for all Gram-positive bacteria tested were >5 mg/mL, except for Bacillus subtilis whose MIC value was 5 mg/mL for CF and EF fractions. All extracts exhibited no antibacterial activity against Gram-negative bacteria.ConclusionsFrom this study, it can be concluded that F. pseudopalma extracts may be a potential antibacterial agent against Gram-positive bacteria. The antibacterial property may be attributed to flavonoids and terpenoids present in the crude ethanolic extract, CF and EF.     

Phytochemical screening and antioxidant activity of methanolic extract of selected wild edible Nigerian mushrooms

ObjectiveTo elucidate the phytochemical content and antioxidant activity of selected wild edible Nigerian mushroom species.MethodsPhytochemical screening was carried out using standard methods while 1,1-Diphenyl picryl hydrazyl (DPPH) radical and reductive power assays were used to evaluate the in vitro antioxidant properties of the selected edible Nigerian mushroom species.ResultsThe result obtained revealed the presence of alkaloids, cardiac glycosides, saponins, flavonoids, terpenes, steroids, tannins and phenols in the selected mushrooms extracts. The extract of Pleutorus ostearus showed a significantly (P<0.05) higher total phenol and flavonoid content of (248.80±7.63) mg/g and (42.63±0.63) mg/g respectively compared to other mushroom extracts. Cantherale cibarus had the most significant (P<0.05) amount of alkaloids [(135.57±0.27) mg/g] and saponins [(150.41±0.50) mg/g] when compared to other extracts while the tannin content [(170.56±0.74)] mg/g was highest in the mushroom Temitomyces robustus. All mushroom extracts scavenged DPPH radical in a dose dependent manner. However, Lactarus deliciousus had the highest DPPH scavenging activity compared to the other mushroom extracts. Pleutorus ostearus and Lactarus deliciousus had better reductive power than other mushroom extracts concentrations used.ConclusionsThe mushroom species analysed have been shown to be good sources of antioxidants and other phytoconstituents, thus it can be used in the management of oxidative stress induced diseases.     

In vitro screening of traditionally used medicinal plants in China against enteroviruses

AIM: To search for new antiviral agents from traditional Chinese medicine, specifically anti-enterovirosuses agents. METHODS: The aqueous extracts (AE) of more than 100 traditionally used medicinal plants in China were evaluated for their in vitro anti-Coxsackie virus B3 activities with a MTT-based colorimetric assay. RESULTS: The test for AE of 16 plants exhibited anti-Coxsackie virus B3 activities at different magnitudes of potency. They can inhibit three steps (inactivation, adsorption and replication) during the infection. Among the 16 plants, Sargentodoxa cuneata (Oliv.) Rehd. et Wils., Sophora tonkinensis Gapnep., Paeonia veitchii Lynch, Spatholobus suberectus Dunn. and Cyrtomium fortunei J. sm. also have activity against other enterovirus, including Coxsackie virus B5, Polio virus I, Echo virus 9 and Echo virus 29. Cell cytotoxic assay demonstrated that all tested AE had CC50 values higher than their EC50 values. CONCLUSION: The sixteen traditionally used medicinal plants in China possessed antiviral activity, and some of them merit further investigations.     

Role of phytochemicals in colorectal cancer prevention

Although the incidence of colorectal cancer (CRC) has been declining in recent decades, it remains a major public health issue as a leading cause of cancer mortality and morbidity worldwide. Prevention is one milestone for this disease. Extensive study has demonstrated that a diet containing fruits, vegetables, and spices has the potential to prevent CRC. The specific constituents in the dietary foods which are responsible for preventing CRC and the possible mechanisms have also been investigated extensively. Various phytochemicals have been identified in fruits, vegetables, and spices which exhibit chemopreventive potential. In this review article, chemopreventive effects of phytochemicals including curcumin, polysaccharides (apple polysaccharides and mushroom glucans), saponins (Paris saponins, ginsenosides and soy saponins), resveratrol, and quercetin on CRC and the mechanisms are discussed. This review proposes the need for more clinical evidence for the effects of phytochemicals against CRC in large trials. The conclusion of the review is that these phytochemicals might be therapeutic candidates in the campaign against CRC.     

Phytochemical screening and antioxidant capacity of the aerial parts of Thymelaea hirsuta L.

Objective

To assess antioxidant activities of different aerial parts of Thymelaea hirsuta (T. hirsuta) from west Algeria, and to search for new sources of safe and inexpensive antioxidants.

Methods

Samples of leaves, stems and flowers from T. hirsuta were tested for total phenolic content, flavonoids content, and evaluation its total antioxidant activity, were done using the spectrophotometric analyses.

Results

Results of preliminary phytochemical screening of leaf, flower and stem of T. hirsuta revealed the presence of tannins, alkaloids, steroids, saponins, coumarins, reducteurs compound and anthraquinones. The total phenolics and flavonoids were estimated. The aqueous extracts of the aerial parts of T. hirsuta showed potent in vitro antioxydant activities using various models viz, DPPH scavenging assay, ferric reducing antioxidant power (FRAP) and ABTS radical scavenging activity.

Conclusions

On the basis of the results obtained, T. hirsuta extracts are rich sources of natural antioxidants appears to be an alternative to synthetic antioxidants and this justifies its therapeutic usage.     

萝卜硫素的体外抗氧化和抑菌活性

目的观察萝卜硫素(SFN)体外抗氧化和抑菌活性。方法试剂盒检测SFN总抗氧化能力(TAC)、抑制超氧阴离子(O2-)活力;水杨酸比色法和邻苯三酚自氧化法检测羟自由基(OH-)与超氧阴离子(O2-)清除能力;滤纸片法检测SFN的抑菌作用,连续二倍稀释法测定其最低抑菌浓度(MIC)、最低杀菌浓度(MBC)和对大肠杆菌和金色葡球菌生长的抑制。结果 SFN在体外模拟系统中总抗氧化活性和抑制抑制O2-生成活力较高,但对OH-和O2-的直接清除率较低,低于30%。抑菌结果表明SFN对大肠杆菌、金色葡萄球菌、绿脓杆菌、枯草杆菌、表皮葡萄球菌5种受试菌均有较强的抑制作用,MIC为0.78～6.25μg/ml之间。结论 SFN具有较好的抗氧化活性和较强的抑菌效果。     

In vitro antibacterial and antitumor activities of some medicinal plant extracts,growing in Turkey

ObjectiveTo investigate antibacterial and antitumor activities of 51 different extracts prepared with 3 types of solvents (water, ethanol and methanol) of 16 different plant species (Ajuga reptans (A. reptans) L., Phlomis pungens (P. pungens) Willd., Marrubium astracanicum (M. astracanicum) Jacq., Nepeta nuda (N. nuda) L., Stachys annua (S. annua) L., Genista lydia (G. lydia) Boiss., Nuphar lutea (N. lutea) L., Nymphaea alba (N. alba) L., Vinca minor (V. minor) L., Stellaria media (S. media) L., Capsella bursa-pastoris (C. bursa-pastoris) L., Galium spurium (G. spurium) L., Onosma heterophyllum (O. heterophyllum) Griseb., Reseda luteola (R. luteola) L., Viburnum lantana (V. lantana) L. and Mercurialis annua (M. annua) L.) grown in Turkey was conducted.MethodsAntibacterial activity was evaluated with 10 bacteria including Streptococcus pyogenes (S. pyogenes), Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), Escheria coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Salmonella typhimurium (S. typhimurium), Serratia marcescens (S. marcescens), Proteus vulgaris (P. vulgaris), Enterobacter cloacae (E. cloacea), and Klebsiella pneumoniae (K. pneumoniae) by using disc diffusion method. Antitumor activity was evaluated with Agrobacterium tumefaciens (A. tumefaciens)-induced potato disc tumor assay.ResultsBest antibacterial activity was obtained with ethanolic extract of P. pungens against S. pyogenes. Ethanolic and methanolic extract of N. alba and ethanolic extract of G. lydia also showed strong antibacterial activities. Results indicated that alcoholic extracts especially ethanolic extracts exhibited strong antibacterial activity against both gram-positive and gram-negative bacteria. Best antitumor activity was obtained with methanolic extracts of N. alba and V. lantana (100% tumor inhibition). Ethanolic extract of N. alba, alcoholic extracts of N. lutea, A. reptans and V. minor flowers, methanolic extracts of G. lydia and O. heterophyllum and ethanolic extract of V. lantana and aqueous extract of V. minor leaves exhibited strong tumor inhibitions.ConclusionsIn near future works, identification of active components can be studied for plant extracts having strong bioactivity.     

Phytochemical screening,anti-oxidant activity and in vitro anticancer potential of ethanolic and water leaves extracts of Annona muricata (Graviola)

ObjectiveTo determine the phytochemical composition, antioxidant and anticancer activities of ethanolic and water leaves extracts of Annona muricata (A. muricata) from the Eastern Uganda.MethodsPhytochemical screening was conducted using standard qualitative methods and a Chi-square goodness of fit test was used to assign the relative abundance of the different phytochemicals. The antioxidant activity was determined using the 2, 2-diphenyl-2-picrylhydrazyl and reducing power methods whereas the in vitro anticancer activity was determined using three different cell lines.ResultsPhytochemical screening of the extracts revealed that they were rich in secondary class metabolite compounds such as alkaloids, saponins, terpenoids, flavonoids, coumarins and lactones, anthraquinones, tannins, cardiac glycosides, phenols and phytosterols. Total phenolics in the water extract were (683.69±0.09) μg/mL gallic acid equivalents (GAE) while it was (372.92±0.15) μg/mL GAE in the ethanolic extract. The reducing power was 216.41 μg/mL in the water extract and 470.51 μg/mL GAE in the ethanolic extract. In vitro antioxidant activity IC₅₀ was 2.0456 mg/mL and 0.9077 mg/mL for ethanolic and water leaves extracts of A. muricata respectively. The ethanolic leaves extract was found to be selectively cytotoxic in vitro to tumor cell lines (EACC, MDA and SKBR3) with IC₅₀ values of 335.85 μg/mL, 248.77 μg/mL, 202.33 μg/mL respectively, while it had no cytotoxic effect on normal spleen cells. The data also showed that water leaves extract of A. muricata had no anticancer effect at all tested concentrations.ConclusionsThe results showed that A. muricata was a promising new antioxidant and anticancer agent.     

In vitro studies on phytochemical evaluation and antimicrobial activity of Borassus flabellifer Linn against some human pathogens

ObjectiveTo detect preliminary phytochemicals and antimicrobial activity of seed coat of Borassus flabellifer (B. flabellifer) against some human pathogens.MethodsThe antimicrobial activity of the organic solvent extracts of seed coat of B. flabellifer against various test microorganisms including bacteria and fungi was investigated using agar well diffusion technique.ResultsThe preliminary phytochemical screening of the aqueous, methanoic and ethanolic extracts of seed coat of B. flabellifer revealed the presence of certain phytochemicals like tannins, flavonoids, saponins, glycosides and terpenoids. The zone of inhibition of methanolic extracts varied from 16 to 23 mm where as with ethanol extracts from 14 to 23 mm and aqueous extracts from 10 to 15 mm at 50 mg/mL concentrations. Among all tested organisms, Aspergillus brasiliensis and Bacillus subtilis showed a higher rate of inhibition with ethanolic and methanolic extracts of B. flabellifer.ConclusionsB. flabellifer exhibited higher rate of growth inhibition against some human pathogens, so it can be used for treatment of some infectious diseases. Further studies are being carried out to separate and purify the individual compounds that are present in seed coat of B. flabellifer by using various chromatographic techniques.     

In vitro antimalarial activity and cytotoxicity of some selected cuban medicinal plants

Terrestrial plants have been demonstrated to be sources of antimalarial compounds. In Cuba, little is known about antimalarial potentials of plant species used as medicinals. For that reason, we evaluated the antimalarial activity of 14 plant species used in Cuba as antimalarial, antipyretic and/or antiparasitic. Hydroalcoholic extracts were prepared and tested in vitro for the antimalarial activity against Plasmodium falciparum Ghana strain and over human cell line MRC-5 to determine cytotoxicity. Parasite multiplication was determined microscopically by the direct count of Giemsa stained parasites. A colorimetric assay was used to quantify cytotoxicity. Nine extracts showed IC50 values lower than 100 μg/mL against P. falciparum, four extracts were classified as marginally active (SI < 4), one as partially active (Parthenium hysterophorus) exhibiting SI equal to 6.2 and two extracts as active (Bambusa vulgaris and Punica granatum), showing SI > 10. B. vulgaris showed the most potent and specific antiplasmodial action (IC50 = 4.7 μg/mL, SI = 28.9). Phytochemical characterization of active extracts confirmed the presence of triterpenoids in B. vulgaris and polar compounds with phenol free groups and fluorescent metabolites in both extracts as major phytocompounds, by thin layer chromatography. In conclusion, antimalarial use of B. vulgaris and P. hysterophorus was validated. B. vulgaris and P. granatum extracts were selected for follow-up because of their strong antimalarial activity.     

Evaluation of the lemongrass plant (Cymbopogon citratus) extracted in different solvents for antioxidant and antibacterial activity against human pathogens

ObjectiveTo test antibacterial and antioxidant activity of the lemongrass plant Cymbopogon citratus (C. citratus) leaves extracted serially by the solvents (chloroform, methanol and water).MethodsThe plant leaves extracts were used for antibacterial activity on Bacillus subtilis, Pseudomonas aeruginosa, Proteus vulgaris, Staphylococcus aureus, Nocardia sp., Serratia sp., and Enterobacter aeruginosa microorganisms by the Kirby Bauer agar disc diffusion method. This study was carried out on lemongrass plant leaf extracts in different concentration of all solvents. The leaf extracts from different solvents were tested for their scavenging activity against the stable free radical DPPH in quantization using a spectrophotometric assay. Oxidative damage was induced in vitro by treating blood DNA and analyzing the effects of the leaf extracts.ResultsThe results showed that C. citratus extracts exhibited maximum zones of inhibition in chloroform, methanol and water extracts. It was Observed that the C. citratus extracts exhibited maximum zone of inhibition against Bacillus subtilis, Pseudomonas aeruginosa and Proteus vulgaris. Analyzed data in the present work suggested that antibacterial activity of C. citratus plant leaf extracts showed good results for Gram-positive and Gram-negative organisms. DPPH scavenging activity was highly elicited by the extract of C. citratus. Chloroform, methanol and water extracts of C. citratus leaves effectively decreased the extent of DNA damage.ConclusionsThe present study suggested that the lemongrass plant extracts could offer various health benefits.     

In vitro Antimicrobial Activity of Citrus aurantifolia and its Phytochemical screening

ObjectiveTo evaluate the antimicrobial efficacy of Citrus aurantifolia Linn (CA) against some microorganisms – bacteria and fungus were Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia, Pseudomonas spp, Aspergillus niger, Aspergillus fumigates, Mucor spp and Pencillium.Methods100 μl of 10 mg CA were assessed against eight test microorganisms by agar well Diffusion Method. Gentamicin and Ketoconazole 10 mg/ml were used as standards. A different solvent was used to obtain CA leaf extract by using maceration technique.Results%yield obtained for dried leaf extract of CA with chloroform, ethanol, acetone, petroleum ether and aqueous ethanol was approximately 15%, 18%, 09%, 11% and 24% respectively. Due to its high yield value hydroalcoholic extract of CA was used for estimating the antimicrobial activity and its phytochemical screening. Phytochemical screening of CA plant reveals the presence of Alkaloids, carbohydrates, flavonoids, steroids and tannins.ConclusionsThe study demonstrates that the hydroalcoholic extract of CA leaf exhibit antibacterial activity on Klebsiella pneumonia, Pseudomonas sp, Staphylococcus aureus and antifungal activity among Aspergillus niger, Aspergillus fumigates, Mucor species. These recognized a good support to the use of this plant in herbal medicine and as base for the development of new drugs and phytomedicine.     

In vitro antioxidant activity and total phenolic content of endophytic fungi isolated from Eugenia jambolana Lam.

ObjectiveTo elucidate the antioxidant activity and total phenolic content (TPC) of ethyl acetate extracts of endophytic fungi isolated from Eugenia jambolana by three different antioxidant assays.MethodsTwenty one different endophytic fungal extracts were screened for presence of various phytochemicals, TPC and in vitro antioxidant activity. TPC was tested by Folin-Ciocalteau reagent based assay. DPPH free radical scavenging, hydrogen peroxide scavenging and reducing power assays were used to evaluate the antioxidant activity.ResultsAlkaloids, phenols, flavonoids, saponins, and terpenes were the main phytochemicals presents in all 21 endophytes. A significant positive correlation was found between antioxidant activity and TPC in fungal extracts. There is 36% endophytic extracts having high phenolic content exhibited potent antioxidant activity. Chaetomium sp., Aspergillus sp., Aspergillus peyronelii and Aspergillus niger strain showed the highest antioxidant activity ranging from 50% to 80% having 58 mg/g to 60 mg/g GAE total phenolics. Ascorbic acid used as a standard showed 90% reducing potential.ConclusionsThe results reveal that metabolites produced by endophytic fungi isolated from Eugenia jambolana can be a potential source of novel natural antioxidant compounds.     

Phytochemical and biological activities of Bituminaria bituminosa L. (Fabaceae)

ObjectiveTo investigate the phytochemical composition, the antioxidant and antibacterial activities of Bituminaria bituminosa L. (Fabaceae) (B. bituminosa).MethodsThe aerial parts of B. bituminosa yielded two compounds. The structures of these compounds were determinated using UV, ¹H-NMR and ¹³C-NMR experiments and comparison of their spectroscopic properties with literature data. The antibacterial activity of the extracts (CH₂Cl₂, ethyl acetate and n-BuOH) was determinated using disk diffusion method against standard and clinical strains. Antioxidant potential of n-BuOH extract was evaluated through two methods: DPPH and cupric ion reducing antioxidant capacity assay.ResultsThe n-BuOH extract from B. bituminosa yielded the isolation of isoflavone and flavone. The extracts CH₂Cl₂, ethyl acetate and n-BuOH demonstrated significant antibacterial activities. CH₂Cl₂ extract showed the maximum antibacterial activity with high concentration of 2 mg/mL against Staphylococcus aureus ATCC 29213, Klebsiella pneumonia and Escherichia coli ATCC 25922 (20.45 mm, 16.41 mm and 15.74 mm inhibition zone, respectively). The value IC₅₀ was 0.26 μg/mL for n-BuOH extract using DPPH method. Whereas the E% value was 0.10 L/mg every centimeter for cupric ion reducing antioxidant capacity assay.ConclusionsThe phytochemical study of B. bituminosa revealed the presence of isoflavone (daidzin) and flavone (isoorientin) and identified for the first time in this specie. The antibacterial activity of the plant B. bituminosa is certainly related to its chemical content. The n-BuOH extract showed a significant antioxidant activity.     

In vitro analysis of boundary identification techniques used in quantification of two-dimensional echocardiograms

Echocardiographic cross-sectional areas of 10 formalin-fixed animal left ventricles were determined by 5 independent observers using black-white (B-W) and mid-spot (M-S) endocardial boundary identification techniques. The echocardiographic cross-sectional areas were compared with the true anatomic cross-sectional areas of the same 10 hearts to determine the accuracy, variability and reliability of each technique. The results of these comparisons revealed that the M-S technique was more accurate than the B-W technique (3.3 +/- 7.2 vs 34.9 +/- 8.6% error). However, the B-W technique was more reliable in that it had a smaller interobserver and estimated intraobserver variability. The M-S technique had a 6% greater intraobserver variability.     

Conformational stability and in vitro bioactivity of porcine luteinizing hormone

Temperature-dependent dissociation of porcine luteinizing hormone (pLH) and of two of its glycoforms was studied by a combination of SDS-PAGE and micro-scale size-exclusion HPLC in parallel with the study of co-operative folding by high-sensitivity differential scanning calorimetry (HS-DSC). The transition temperature of dissociation of pLH at pH 7.0 as quantified by SDS-PAGE, HPLC and residual activity in radioreceptor assay was found to match exactly the transition temperature of its unfolding as measured by HS-DSC. Free alpha- and beta-subunits did not exhibit any unfolding transition in the same conditions. The microcalorimetric data for two pLH isoforms exhibiting different glycosylations were identical to those of a preparation of non-separated isoforms. It is concluded that: (a) free subunits exhibit no co-operative folding (i.e. no stable three-dimensional structure) and co-operative folding occurs only in alphabeta heterodimers; (b) the co-operative folding is responsible for the stability of the association of subunits; and (c) the heterogeneity of carbohydrate chains does not affect the stability of folding and association of subunits. The fastening of the "seat-belt" of the beta-subunit embracing the alpha-subunit by the Cysbeta26-beta110 disulfide bridge had been postulated to play a role in the preservation of the dimeric structure of gonadotropins. The present work shows that dissociation of subunits is directly related to their loss of common co-operative folding.