Study on the antioxidant activity of tea flowers (Camellia sinensis)

Major chemical compounds in different extracts from tea flowers (Camellia sinensis) were analyzed. Distilled water or 70% ethanol extracts were then fractionated with chloroform, ethyl acetate and n-butanol, respectively. Each extract fraction was tested its scavenging activities on 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl free radicals. The results showed that ethyl acetate fraction of ethanol-extract of tea flower (EEA) exhibited the highest quenching activity to hydroxyl radicals (SC50 11.6 mug/ml), followed by ethanol-extract (EE) of tea flower (SC50 19.7 microg/ml). Same tea flower extract showed big different scavenging activities on different free radicals. EEA quenched 80% of hydroxyl radicals generated by Fenton's reaction, however, only 40% of DPPH radical was scavenged in the Fe (II)-H2O2 -luminol system. The contents of flavones, polyphenols and catechins in EE and EEA fractions were higher than those in other fractions. We suggest that the stronger scavenging abilities to free radicals might be due to polyphenols, EGCG, ECG and flavones. However, the water extracts of tea flower and their fractions showed lower antioxidant activity for their inhibitory effect on hydroxyl radicals and DPPH radicals.     

Assessment of Antioxidant Potential,Total Phenolics and Flavonoids of Different Solvent Fractions of Monotheca Buxifolia Fruit

ObjectivesThis study was conducted to investigate the antioxidant potential of methanol extract and its derived fractions (hexane, ethyl acetate, butanol, and aqueous) of fruits of Monotheca buxifolia (Falc.) Dc., a locally used fruit in Pakistan.MethodsDried powder of the fruit of M. buxifolia was extracted with methanol and the resultant was fractionated with solvents having escalating polarity; n-hexane, chloroform, ethyl acetate, n-butanol and the residual soluble aqueous fraction. Total phenolic and total flavonoid contents were estimated for the methanol and various fractions. These fractions were also subjected to various in vitro assays to estimate the scavenging activity for 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), superoxide, hydroxyl, hydrogen peroxide and reductive ability for ferric ions and phosphomolybdate assay.ResultsThe n-butanol, aqueous and methanol fractions possessed high amount of phenolics and flavonoids compared with other fractions, and subsequently showed a pronounced scavenging activity on DPPH, ABTS, superoxide, hydroxyl and hydrogen peroxide radicals and had a potent reductive ability on ferric ion and phosphomolybdate assay. There was a found significant correlation between total phenolic and flavonoid contents and EC₅₀ of DPPH, superoxide, hydrogen peroxide radical and phosphomolybdate assays, whereas a nonsignificant correlation was found with the hydroxyl radical and ABTS radical assay.ConclusionM. buxifolia fruit can be used as natural antioxidant source to prevent damage associated with free radicals.     

Cytotoxic effect of buckwheat (Fagopyrum esculentum Moench) hull against cancer cells

Buckwheat (Fagopyrum esculentum Moench) hull was extracted with 70% ethanol and then further fractionated with n-hexane, chloroform, ethyl acetate, and water stepwise. In the in vitro test (SRB assay), hexane and ethyl acetate fractions showed higher inhibition effects against MCF-7 cells than other samples at the 1 mg/mL level: 89% and 93.2%, respectively. They also displayed higher inhibition rates against Hep3B cells of 83.6% and 75.3%, respectively, at 1 mg/mL. The ethyl acetate fraction yielded the highest inhibition rate against A549 cells with the level of 0.25 mg/mL, but it showed a lower inhibition rate than the hexane and chloroform fractions at higher levels of sample, i.e., 0.75 and 1.0 mg/mL. All samples showed higher inhibition effects against AGS human gastric carcinoma than any other cancer cells. The inhibition rates against HeLa cells were 81.2% and 82.0% for the chloroform and butanol fraction with 0.5 mg/mL, respectively. However, all samples yielded an inhibition rate of less than 35% against normal cells, at all treatment levels, except the ethanol extract. All extracts at doses of 25 and 50 mg/kg showed decreases of more than 20% and 42%, respectively, in tumor formation in sarcoma-180 implanted mice except for the aqueous fraction. From these results, it is suggested that buckwheat hull possesses anticancer properties against a variety of different cancer cell lines.     

TPC in the leaves of 116 sweet potato (Ipomoea batatas L.) varieties and Pushu 53 leaf extracts

The total polyphenol contents (TPC) of leaf extracts from 116 varieties of sweet potato cultivated in China were determined by Folin-Ciocalteu method. In addition, the crude extract (CE) of Pushu 53 leaves, with relatively higher TPC and its fractions of chloroform, ethyl acetate, n-butanol and water, were prepared and subjected to the determination of TPC, evaluation of antioxidant activities in vitro by assays of DPPH, ABTS and FRAP, and analysis of phenolic constituents by high performance liquid chromatography (HPLC) and HPLC–mass spectrometry. Our results show that the extracts demonstrated potential antioxidant activity, and that a satisfactory correlation between TPC and antioxidant activity was observed. In addition, the main bioactive compounds for the antioxidant activity of the extract were polyphenols, especially derivatives of caffeoylquinic acid (CQA), such as 5-CQA, 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA. Their contents (8.95%) were 0.41, 3.41, 4.09, and 1.04%, respectively, accounting for 79.6% of TPC in CE (11.24%).     

Antioxidant activity, anti-inflammatory activity, and whitening effects of extracts of Elaeagnus multiflora Thunb

In the present study, we analyzed the antioxidant, antiplatelet aggregation, anti-inflammatory, and tyrosinase inhibitory activities of a variety of solvent extracts of Elaeagnus multiflora Thunb. (Family Elaeagnaceae). Among the solvent extracts of E. multiflora, the ethyl acetate extract (EE) exhibited the highest 1,1-diphenyl-2-picrylhydrazyl radical and xanthine oxidase inhibition activity, as well as the greatest tyrosinase inhibition activity. Only the chloroform extract (CE) inhibited platelet aggregation, and that was a weak effect with 19.29% inhibition at 250 microg/mL, as compared to controls. The CE was also the most potent inhibitor of nitric oxide production among the tested fractions, with almost 100% inhibition at 500 microg/mL. We also detected 2,4-bis(1,1-dimethylethyl)phenol in the CE and EE, via a gas chromatography-mass spectrometry assay. In conclusion, we found that E. multiflora Thunb. has antioxidant and antiplatelet aggregation effects to some extent, and its CE and EE possess potent inhibitory effects on nitric oxide production and tyrosinase activity.     

Effects of elm bark (Ulmus davidiana var. japonica) extracts on the modulation of immunocompetence in mice

The immunomodulative effects of elm bark extract were studied in vitro by the proliferation of splenocytes and the production capacity of three kinds of cytokines [interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha] by mouse peritoneal macrophages cultured with various fractions (methanol, hexane, chloroform, ethyl acetate, butanol, and water) of elm bark extract. Splenocyte proliferation and cell viability of peritoneal macrophages were increased with concentrations of polar fractions, such as butanol and water, in the range of 1-500 microg/mL. Significantly higher levels of the production of all three cytokines were detected with supplementation of methanol extract compared with other fractions. In order to elucidate its effect in vivo, elm bark water extract was orally administrated every other day for 2 weeks. Proliferation of splenocytes and the production capacity of cytokines (IL-1beta, IL-6, and TNF-alpha) by mouse peritoneal macrophages were used as indices for immune activity. Splenocyte proliferation induced by elm bark with lipopolysaccharide or concanavalin A stimulation was enhanced at 500 mg/kg of body weight concentrations compared to that of the control group. In the case of cytokines, the highest production of IL-6 and TNF was detected at 500 mg/kg of body weight concentrations. In conclusion, this study suggests through in vitro and in vivo experiments that Ulmus davidiana var. japonica (elm bark) extracts may enhance the immunocompetent properties such as splenocyte proliferation and cytokine production capacity by activated macrophages and have a protective effect in mice.     

Antiamoebic coumarins from the root bark of Adina cordifolia and their new thiosemicarbazone derivatives

In continuation of our search for potential antiamoebic agents from folklore Indian medicinal plants, we found that the benzene and ethyl acetate extracts from the root bark of Adina cordifolia exhibited strong antiamoebic activity with IC(50) values of 2.92 and 2.50 microg/ml, respectively. Bioassay-guided fractionation of benzene and ethyl acetate extracts led to the isolation of 7-hydroxycoumarin (umbelliferone 1) and 7-beta-D-glucosylcoumarin (skimmin 2), respectively. Umbelliferone 1 was converted into 7-acetoxycoumarin 1a, which on treatment with aluminium chloride afforded 7-hydroxy-8-acetylcoumarin 2a. A new series of thiosemicarbazones 3a-e of 7-hydroxy-8-acetylcoumarin with different thiosemicarbazides were synthesized. Umbelliferone was also converted into its methoxy derivative (7-methoxycoumarin 4). Subsequently, all the compounds were assessed for antiamoebic activity against HM1:IMMS strain of Entamoeba histolytica. Umbelliferone and skimmin were found to possess a very good activity with IC(50) values of 6.38 and 4.35 microM/ml, respectively. The activity drastically increased on converting compound 2a into its thiosemicarbazone derivatives 3a-e with IC(50) values ranging between 1.06 and 4.46 microM/ml. Compounds 3b,c and e with IC(50) values of 1.49, 1.56 and 1.06 microM/ml, respectively, exhibited even higher antiamoebic activity than the standard drug metronidazole (IC(50)=2.62 microg/ml). The activity of 7-methoxycoumarin (IC(50)=8.92 microM/ml) was less than umbelliferone. Compounds 3b, c and e were tested for toxicity using H9c2 cardiac myoblasts cell line. The compounds exhibit >80% viability at 3.125-200 microg/ml. It is apparent from these results that umbelliferone and skimmin may be a useful lead for the development of new antiamoebic drugs.     

Stimulation of activity and expression of antioxidant enzymes by solvent fractions and isolated compound from Cedrela sinensis leaves in HepG2 cells

Cedrela sinensis has been widely used in traditional Oriental medicine to treat a variety of diseases. However, little is known about the cellular actions by which this plant mediates its antioxidant effects. In this study, activity-guided fractionations of C. sinensis leaves were performed using column chromatographic techniques as well as biological assays with HepG2 cells. The ethanol (95%) extract of C. sinensis leaves was sequentially extracted with hexane, chloroform, ethyl acetate (EtOAc), butanol, and water, and the fractions were screened for their antioxidant potentials for scavenging radicals as well as inducing antioxidant enzyme activity and expression. The most potent antioxidant EtOAc fraction was further separated using chromatographic techniques including open column and high-performance liquid chromatography. Compound 1 from the EtOAc fraction showed strong radical scavenging activity with a 50% scavenging concentration value close to that of ascorbic acid and induced both the activity and expressions of antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase. Inhibitory effects on the phosphorylations of upstream mitogen-activated protein kinases such as c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p38 were also observed after treatments with compound 1. Compound 1 was identified as quercitrin by (1)H- and (13)C-nuclear magnetic resonance techniques. Taken together, our findings demonstrated for the first time that C. sinensis leaves appear to be a useful source of a cytoprotective and chemopreventive agent that can stimulate the activity and expression of crucial antioxidant enzymes in cells.     

Antioxidant activity of Potentilla fulgens: An alpine plant of western Himalaya

Antioxidant activities of the methanol extract, fractions and isolated compounds from the roots of Potentilla fulgens Lodd. were evaluated by three in vitro experiments, namely, ABTS, DPPH, and FRAP assays. PF-2 was characterized as a new biflavanoid and designated as Potifulgene on the basis of NMR and mass spectrum, whereas PF-1 was identified as epicatechin. The activities of aqueous methanolic extract and fractions could be correlated with their respective total phenolic content and compared with standard natural antioxidants such as quercetin, vitamin C and pyrogallol. The root powder of the plant was extracted with methanol/water (80:20) by cold extraction and its extract was further partitioned with ethyl acetate, butanol and water fractions. Among the three fractions (ethyl acetate, butanol and water fraction) and the total aqueous methanolic extract, the butanol fraction exhibited good scavenging response measured in terms of TEAC (mM Trolox equivalent/mg extract). The butanol fraction was found to possess strong antioxidant activity (2.54 ± 0.69, 2.41 ± 0.53, 3.57 ± 0.05 mM Trolox equivalent/mg extract) with ABTS, DPPH and FRAP assays, respectively. The chemical composition of extracts, studied in terms of total polyphenol content (TPC), was found in the range of 20.61 ± 0.38 to 33.28 ± 0.11 mg/g gallic acid equivalent. A significant correlation was observed between total polyphenolics and antioxidant activity, indicating participation of phenolics in antioxidant activities of extract and fractions. The antioxidant activity of new biflavanoid (Potifulgene) was found to be higher, i.e. 6.85 ± 0.38, 4.24 ± 0.41, 5.35 ± 0.53 than that of epicatechin, 2.13 ± 0.05, 1.50 ± 0.02, 1.57 ± 0.03 with ABTS, DPPH and FRAP assays.     

Antioxidant and antimicrobial activity of Cissus quadrangularis L

Extracts of Cissus quadrangularis L. were tested for antioxidant activity by beta-carotene linoleic acid model and also by 1,1-diphenyl-2-picrylhydrazyl model. The ethyl acetate fraction of both fresh and dry stem extracts at a concentration of 100 ppm showed 64.8% antioxidant activity in the beta-carotene linoleic acid system and 61.6% in the 1,1-diphenyl-2-picrylhydrazyl system. This fraction showed the presence of sterols, vitamin C, and tannins as phytoconstituents. The antioxidant activity of methanol extract and aqueous extract were comparatively less significant than that of ethyl acetate extract, and n-hexane extract showed the least activity. The ethyl acetate extract and methanol extract of both fresh and dry stems further exhibited antimicrobial activity against Gram-positive bacteria, including Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, and Streptococcus species. The results of the study have implications in the use of C. quadrangularis as an antibacterial agent and more so as an antioxidant in several applications requiring these properties.     

Corni fructus scavenges hydroxy radicals and decreases oxidative stress in endothelial cells

Corni fructus has been used as a tonic, analgesic, and diuretic in Korean herbal medicine. The present investigation was undertaken to evaluate the antioxidative effect of corni fructus and its capacity to protect cells against oxidative damage. The radical scavenging activity of corni fructus extracts was measured using 1,1-diphenyl-2-picrylhydrazyl (DPPH), and the antioxidant activity was determined by measuring the peroxide value in a linoleic acid emulsion system. In addition, human umbilical vein endothelial cells (HUVECs) were treated with corni fructus extracts and incubated with H(2)O(2) to investigate protection against apoptosis induction. Both ethanol and water extracts of corni fructus produced higher DPPH radical scavenging activity than hexane, chloroform, and ethyl acetate extracts. Strong antioxidative activities of both water and ethanol extracts were observed in an emulsion system containing linoleic acid and phosphate buffer. The incubation of HUVECs with the addition of ethanol extract significantly decreased H(2)O(2)-initiated damage of endothelial cells, but the water extract did not. The pretreatment with ethanol extract, but not with water extract, significantly decreased apoptotic damage of the H(2)O(2)-treated HUVECs and kept the morphological normality. This study demonstrates that corni fructus is a potent antioxidant substance, and suggests that further investigation is needed to characterize the difference between extract types and to identify its antioxidant compounds.     

Effect of different polarities leaves crude extracts of Omani juniperus excels on antioxidant,antimicrobial and cytotoxic activities and their biochemical screening

ObjectiveTo prepare different polarities leave crude extracts of Juniperus excels (J. excels) and to determine their phytochemical screening, antioxidant, antimicrobial and cytotoxic activities.MethodsThe phytochemical screening of different crude extracts was determined by well-established methods. The antioxidant activity was determined by free radical scavenging (2,2-diphenyl-1-picrylhydrazyl, DPPH) method. The antimicrobial activity was evaluated by agar disc diffusion method and cytotoxic activity was determined through brine shrimp lethality assay.ResultsThe phytochemical tests showed that alkaloids, flavonoids, saponins, steroide, triterpenoids and tannins are present in all leave crude extracts except chloroform crude extract do not contain tannins. The antioxidant results of crude extracts were found to be in the order of hydro alcoholic > chloroform> ethyl acetate >hexane. All leave crude extracts showed moderate antibacterial against gram positive and gram negative food borne pathogen bacteria. The cytotoxicity results revealed that hexane and chloroform extracts have killed all the shrimp larvae at the concentration of 500 μg/mL.ConclusionMore in-vivo and in-vitro studies along with detailed phytochemical investigations are needed in order to potentially use this plant in the prevention and therapies of some oxidative damage related diseases.     

Effect of Mentha piperita (Labiatae) and Mentha spicata (Labiatae) on iron absorption in rats

AIM: The effect of Mentha piperita (Labiatae) and Mentha spicata (Labiatae) teas, which contain different phenol compounds, on iron metabolism was studied. These teas grow in different areas of the city of Isparta, Turkey. These herbals were given to the rats in tea. METHODS: Forty-eight male Wistar albino rats weighing 200-250 g were used for this study. The rats were divided into four groups of 12 animals: Group I received no herbal tea (control group); Group II received 20 g/L M. piperita tea; Group III received 20 g/L M. spicata tea; Group IV received 40 g/L M. spicata tea. Herbal teas were prepared daily and provided at all times to the rats over 30 days as drinking water. RESULTS: M. piperita tea caused a decrease in serum iron and ferritin levels (P < 0.05), and caused an increase in unsaturated iron-binding capacity (UIBC) (P < 0.01). M. spicata tea caused no significant change in serum iron, ferritin levels and UIBC (P > 0.05). CONCLUSION: Both herbal teas inhibited Fe absorption. Inhibition caused by M. spicata tea was dose dependent. Therefore, when drinking these teas, their effect should be considered, especially for children and anemic patients.     

In vitro study of the PLA2 inhibition and antioxidant activities of <Emphasis Type="Italic">Aloe vera</Emphasis> leaf skin extracts

Background  

In the present work we determined the total phenolic content of Aloe vera leaf skin (AVLS) extracts by using various solvents (hexane, chloroform-ethanol (1/1), ethyl acetate, butanol and water). We have also evaluated the antioxidant and the anti-PLA2 properties of these extracts by measuring their inhibition potency on the human pro-inflammatory phospholipase A2 (group IIA).     

Subcellular accumulation of beta-carotene and retinoids in growth-inhibited NCI-H69 small cell lung cancer cells.

Delivery of beta-carotene in tetrahydrofuran slowed the growth of NCI-H69 small cell lung cancer cells. Analysis of cells and cellular fractions revealed that beta-carotene-treated cells accumulated beta-carotene as well as some polar metabolites, primarily in the crude nuclei. Cells were grown at 1 x 10(5) cells/ml and treated with 20 microM beta-carotene. Growth monitoring up to 15 days indicated an inverse relationship between the duration of beta-carotene treatment and the rate of cell growth. Reverse-phase high-performance liquid chromatography analysis of treated cells showed the presence of beta-carotene, retinoic acid, retinol, and retinal, with beta-carotene accounting for the major material recovered. When cellular fractions were analyzed for beta-carotene, it was found to be located primarily in the crude nuclei. These results demonstrate that treatment of small cell lung cancer cells with beta-carotene results in a reduced growth of the cells. Further investigation is required to show a direct effect of beta-carotene or its intracellular polar metabolites on these cells. Accumulation of beta-carotene in the nucleus suggests a need for evaluating the nuclear role for beta-carotene.     

Antimicrobial activities of Psophocarpus tetragonolobus (L.) DC extracts

Consecutive chloroform, ethanol, and ethyl acetate partitions of extracts from winged bean [Psophocarpus tetragonolobus (L.) DC] root, stem, leaf, and pod extracts were tested for their antimicrobial activity against 19 microbial species, including 11 bacterial pathogens, four yeasts, and four molds using the disk diffusion assay technique. The pod extract was found to be most effective against all of the tested organisms, followed by the stem, root, and leaf extracts, and the ethanol fraction showed the most significant (p < 0.05) antimicrobial activity against all of the tests among three soluble fractions of extract, followed by the ethyl acetate and chloroform fractions. The minimum inhibitory concentrations (MICs) of extracts determined by the broth dilution method ranged from 1.25 to 10.0 mg/mL. The MIC of ethanol fraction of pod extracts was the lowest by comparison with the other two extracts. The MIC for fungi was at or below 2.5 mg/mL and for bacteria was at or above 2.5 mg/mL.     

Comparative study on the spermicidal activity of organic solvent fractions from hydroethanolic extracts of Achyranthes aspera and Stephania hernandifolia in human and rat sperm

Background

This study was conducted to determine the most effective fraction of the hydroethanolic (water:ethanol, 1:1) extracts of Stephania hernandifolia leaves and Achyranthes aspera roots (in a composite manner at a ratio of 1:3, respectively) that will provide maximum spermicidal activity in human and rat spermatozoa out of five different ratios (1:1, 1:3, 1:7, 3:1 and 7:1) that have been studied in pilot experiments.

Study Design

n-Hexane, chloroform and ethyl acetate fractions of the hydroethanolic (1:1) extracts of S. hernandifolia and A. aspera were mixed at 1:3. Different concentrations were tested for sperm immobilization, sperm viability, acrosome status, 5′-nucleotidase activity and nuclear chromatin decondensation using human and rat spermatozoa for the selection of the most effective concentration.

Results

Out of three fractions of the hydroethanolic (1:1) extracts of the said plants, the n-hexane fraction was most effective, and the chloroform fraction exhibited minimum activity for this purpose. At a concentration of 0.1 g/mL hexane fraction, all sperm of the human sample were immobilized immediately (within 20 s). In case of the rat sample, all epididymal spermatozoa were immobilized immediately (within 20 s) by treatment with hexane fraction at a concentration of 0.004 g/mL. All human sperm were found to be nonviable within 20 min. The activity of acrosome enzymes was reduced, and significant release of 5′-nucleotidase (a plasma membrane marker) into the surrounding medium was noted after treatment with 0.1 g/mL hexane fraction, indicating that the hexane fraction affected the cytoarchitecture of the sperm plasma membrane. The maximum number of human sperm failed to decondense when treated with 0.1 g/mL hexane fraction, and sperm motility was also irreversible. The hexane fraction was tested in rats as vaginal contraceptive and showed 100% efficacy, indicating its potential for development as vaginal contraceptive.

Conclusion

The findings indicate that, among the different fractions, the hexane fraction of the hydroethanolic extracts of the two plants produced the most effective spermicidal activity and can be considered as vaginal contraceptive.     

Simultaneous analysis of retinol,beta-carotene and tocopherol levels in serum of Vietnamese populations with different incomes

In this study, we clarified the status of the fat-soluble vitamins retinol and tocopherol, as well as beta-carotene, as antioxidants in the prevention of cardiovascular disease in middle-aged Vietnamese populations with different incomes. In order to measure simultaneously the serum concentrations of retinol, beta-carotene and tocopherol, we carried out high-performance liquid chromatography analysis with three separate detectors. The analytical method was modified, omitting the saponification process, and used a multi-evaporating system with dry ice. This allowed the analysis to proceed more rapidly, use a small amount of serum (40 microL) and be free of hexane contamination to the environment. The analyses reflected an adequate status of vitamin A (serum retinol = 20 microg/dL), but inadequate status of beta-carotene and vitamin E (serum beta-carotene <40 microg/dL; serum tocopherol < 600 microg/dL) in all three Vietnamese populations. As large numbers of Vietnamese subjects were observed with very low serum concentrations of beta-carotene and tocopherol, higher consumptions of green and yellow vegetables, fruits, vegetable oils and other foods rich in vitamin E are recommended for these Vietnamese populations.     

Studies for analyzing prohibited ingredients such as tetracaine hydrochloride in cosmetics

Tetracaine hydrochloride (TH) is nominated as the prohibited ingredients in cosmetics in Japanese Pharmaceutical Affairs Act. So the analytical method for TH was investigated by HPLC. After adding 5 ml of TH solution at 10 microg/ml and 2 ml of salicylic acid solution at 75 microg/ml as the internal standard to 0.5 g of the lotion, the mixture was made up to 10 ml with a mixture of water and methanol (1:1) as the testing solution. Milky lotion was procedured as follows: After adding 5 ml of TH solution at 10 microg/ml and 2 ml of internal standard solution to 0.5 g of the milky lotion, the mixture was made up to 10 ml with a mixture of water and methanol (1:1). Two milliliter of this mixture was placed into a centrifuging tube with a cap and 2 ml of hexane was added. After shaking vigorously and centrifuging, the lower layer was used as the testing solution. In the case of the cream, the other procedures were used: 0.5 g of cream was placed into a 10-ml volumetric flask and 1 ml of tetrahydrofuran was added. After dissolving, the mixture of methanol and water (1:1) was added to make up 10.0 ml. Two milliliter of this mixture was placed into a centrifuging tube with a cap and 2.0 ml of hexane was added. After shaking vigorously and centrifuging, the lower layer was used as the testing solution. The testing solution of 20 microl was analyzed by HPLC using the ODS column (CAPCELL PAK C18 column, 4.6 x 250 mm), the mixture of acetonitrile and 50 mmol/l phosphate buffer(pH 2.0)(7:3) and the detection wavelength of 303 nm. The working curves from 0.5 to 6.0 microg/ml showed a linear line between the concentrations of TH and the peak area ratio. There was no interference of peak of TH from the lotion, milky lotion and cream.     

Antioxidant and antimycobacterial activities of Tabernaemontana catharinensis extracts obtained by supercritical CO2 + cosolvent

In the present work the antioxidant and antimycobacterial activities were determined for extracts from Tabernaemontana catharinensis. The extracts' global yields were obtained using supercritical CO2 plus cosolvent. The cosolvents ethanol, isopropyl alcohol, methanol, and water and their mixtures were used. The extracts were fractionated and analyzed by thin-layer chromatography and gas chromatography/flame ionization detection. The antimycobacterial activity was measured against Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium kansasii. The antioxidant activity was determined by the coupled reaction of beta-carotene and limonene acid. The average global yield was approximately constant (2.4 +/- 0.1%) for the alcoholic cosolvents and significantly larger (15 +/- 1%) for the cosolvent water and its alcoholic mixtures. The content of alkaloids in the extracts was strongly affected by the cosolvent. The antioxidant activity of the extracts ranged from 53% to 95%. The highest antimycobacterial activity was detected in the alkaloidal fraction (minimum inhibitory concentration = 128 microg/mL), while the lowest was verified in the aqueous fraction (minimum inhibitory concentration >512 microg/mL).