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Extraction, isolation -93

Extraction, Isolation and Purification of Saponins from Herbal Plants Surendar. M , Sai Kumar.P , Shyam Prasad.T , Madhava Reddy. A , Nagulu.M , Hari Prasad.P , Shaik Shabber , 1.Nalanda College of Pharmacy,2.Swamy Ramananda Tirtha Institute of Pharmaceutical Sciences Abstract
Saponins are a class of chemical compounds, one of many secondary metabolites found in natural sources, with saponins found in particular abundance in various plant species. Specifically, they are amphipathic glycosides grouped phenomenologically by the soap-like foaming they produce when shaken in aqueous solutions, and structurally by their composition of one or more hydrophilic glycoside moieties combined with a lipophilic triterpene derivative. A ready and therapeutically relevant example is the cardio-active agent digoxin, from common foxglove.most of the saponins of official saponins drugs are triterpene glycosides.some drugs also or only containe steroidal saponins.generally triterpene saponins having acidic properties due to presence more carboxyl group in the aglycone and sugar moiety.steroidal saponins posses less sugar units than the triterpene saponins.
Generally these saponins are separation by preparative separation was performed by water extraction using reversed-phased C18 column chromatography,their structure were characterized by eletrospray ionization mass spectrometry (ESI-MS),1HMNR and 13C-NMR.
Introduction
combined with a lipophilic triterpene derivative[Zohar Kerem]. A ready and therapeutically relevant example is the cardio-active Saponins are a class of chemical compounds, one of many secondary metabolites found in natural sources, with saponins found in particular abundance in various plant species. Specifically, they are amphipathic glycosides grouped phenomenologically by the soap-like foaming they produce when shaken in aqueous solutions, and structurally by their composition of one or more hydrophilic glycoside moieties combined with a lipophilic triterpene derivative. A ready and therapeutically relevant example is the cardio-active agent digoxin, from common foxglove.most of the saponins of official saponins drugs are triterpene glycosides.some drugs also or only containe steroidal saponins.generally triterpene saponins having acidic properties due to presence more carboxyl group in Chemical Structure of the Solanine (Saponin) the aglycone and sugar moiety.steroidal saponins posses less sugar units than the triterpene saponins[David G. I. Kingston] Sources of Saponin
Generally these saponins are used for the anti-inflammation, Saponins have historically been understood to be plant-derived, anti-allergy, anti-tumour, anti-obesity and anti-hyperlipidemic but they have also been isolated from marine organisms. Saponins are indeed found in many plants, and derive their name from the soapwort plant (Genus Saponaria, Family Caryophyllaceae), the root of which was used historically as a Saponins are a class of chemical compounds, one of many soap.Saponins are also found in the botanical family secondary metabolites found in natural sources, with saponins Sapindaceae, with its defining genus Sapindus (soapberry or found in particular abundance in various plant species. soapnut), and in the closely related families Aceraceae (maples) Specifically, they are amphipathic glycosides grouped and Hippocastanaceae (horse chestnuts; ref. needed).[ Haijiang phenomenologically by the soap-like foaming they produce when shaken in aqueous solutions, and structurally by their composition of one or more hydrophilic glycoside moieties E-mail : devinirmala1980@yahoo.co.in 12 l Herbal Tech Industry l September 2011 Zhang] It is also found heavily in gynostemma pentaphyllum Natural sapogenins differ only in their configuration at carbon (Genus Gynostemma, Family Cucurbitaceae) in a form called atoms 3,5 and 25 and in the spirostane series the orientation at gypenosides, and ginseng (Genus Panax, Family Araliaceae) in a c-22 need be specified[Kalpana Mujoo ].
form called ginsenosides.[ Niramon Utama-ang ] Within these families, this class of chemical compounds are found in various parts of the plant: leaves, stems, roots, bulbs, Digitoninn (2-glucose,2-galactose,1-xylose) blossom and fruit.Commercial formulations of plant-derived Gitonin (1-glucose,2-galactose,1-xylose) saponins – e.g., from the soap bark (or soapbark) tree, Quillaja saponaria, and from other sources—are available via controlled 2. Saponin glycosides
manufacturing processes, which make them of use as chemical and biomedical reagents.
Eg: shatavari (shatavari I,II ) Brahmi (Bcosides A and B).
Medicinal Uses
3. Pentacyclic triterpenoid saponins:
There is tremendous, commercially driven promotion of The pentacyclic triterpenoid saponins are rare in monocolyte saponins as dietary supplements and nutriceuticals. There is dons.these are present in caryphyllaceae, sapindaceae, evidence of the presence of saponins in traditional medicine preparations, where oral administrations might be expected to lead to hydrolysis of glycoside from terpenoid (and obviation of Eg: Aescin (2-glucose, 1-glucuronic acid) any toxicity associated with the intact molecule).[ Farah S] But as Aralin (2-arabinose, 1-glucuronic acid).
is often the case with wide-ranging commercial therapeutic claims for natural products: 4.Triterpenoid saponins:
the claims for organismal/human benefit are often based on very preliminary biochemical or cell biological studies; and mention is generally omitted of the possibilities of individual Acidic properties,due to presence of one carboxyl group in the chemical sensitivity, or to the general toxicity of specific agents, and high toxicity of selected cases. Steroidal saponins posses less sugars than the triterpene saponins.
While such statements require constant review it appears that there are very limited US, EU, etc. agency-approved roles for saponins in human therapy. In their use as adjuvants in the production of vaccines, toxicity associated with sterol A. Foam test:
complexation remains a major issue for attention. Even in the Shake the drug extract or dry powder vigorously with case of digoxin, therapeutic benefit from the cardiotoxin is a result of careful administration of an appropriate dose [D.A. RICKERT ]. Very great care needs to be exercised in evaluating or B.Heamolytic test:
acting on specific claims of therapeutic benefit from ingesting saponin-type and other natural products[Maggie P.K. Choia ].
Add drug extract or dry powder to one drop of blood placed on glass side.heamolytic zones appears.
Classification:
Identification:
Sapogenins give color test with sulphuric acid,Antimony 1. Steroidal saponins
trichloride,tri chloro acetic acid but these are not very specific.the IR spectrum of sapogenins shows several strong Steroidal saponins are great pharmaceutical importent of absorption bands between 1350 and 875cm-1 due to the because of their relationship to compounds such as the sex spiroketal side chain.Another characteristic features is the hormone, cortisome, diuretic steroids, vit.D and cardiac intense absorption at 920-915cm-1 in the 25â series whereas in the 25á series these intense absorption at 899-894cm-1 Some are used as starting material for the synthesis of these Extraction Process:
compounds,diosgenin is the principle sapogenin used by indentra but most yams.
13 l Herbal Tech Industry l September 2011 Ginseng radix is extracted under the same conditions ,but with 90% ethanol.
Liquiritiae radix :
An ethanol extacts (above solution) is evaporated to dryness. the residue is dissolved in 20ml chloroform: methanol (1:1); 20ml is used for the detection of glycyrrhin [P.B.MALLIKHARJUNA].
Separation and Identification Techniques:
Adsorbent: silica gel 60 F254-percorated TLC plates.
Chloroform: glacial acetic acid :methanol: water.
(64:32:12:8). This system is suitable for the separations of 1.Chloroform-methanol-water(70:30:8) for extractions of 2.Ethyl acetate-ethanol-water-ammonia (65:25:9:1) for extraction of glycyrrhetic acid from Liquritae radix.
Characterisation Of Saponins.
With the exception of glycyyhetic acid (Liquiriae radix), no saponins are detectable by exposure to uv-254 or uv-365nm.
Conventional open –column chromatography Hemolytically active saponins are detected as with zones on a reddish background[M. Sajjad Khan ].
Saponins form mainly blue, blue-violet and sometimes Avenue sativa herbs (poaceace): steroidal saponins; Triterpene Separation of Saponins by using TLC Method:
Centellac herb(apiaceae):Ester saponins.
Preparations of extracts:
Ginseng radix(araliaceae):2-3% tetra cyclic triterpene glycosides.
powdered drugs (2g) is extracts by heating for 10mints under reflux with 10ml,70% ethanol.the filtrate is evaporate to about Liuirititiae radix (fabaceae):saponins; 8-12% glycyrrhizin,calcium 5ml, and 20-40cm of this solutions is used for TLC[GEORGE V ]. A total of 3ml of the ethanolic extracts (with above solutions) is shaken several times with 5ml water and saturated n-butanol. Saponariae radix (caryop phylaceace) :3-5% bidesmosidic the n-butanol phase is separated and concentrated to about 1ml ;but with 20ml is used for TLC [REBECCA M. CORBIT ].
Sarsaparillae radix: 1.8-3% steroidal saponins,mono desmosidic, Identification of Major Saponins from Jiaogulan Extract 14 l Herbal Tech Industry l September 2011 resistant form of Teflon) vessels were used at a time, with pressure and temperature monitoring capabilities, The microwave power was limited to 300 W[Devendra N. Kage]. After cooling to room temperature, the extract was collected Fresh GP leaves were dried by microwave dryer until the moisture content was below 10%. Dried GP was vacuum packed in aluminium foil and kept in -200C until used.
Defatted powder (10 g) was extracted with the solvent of choice (150 ml), for 3 h. After cooling to room temperature, the extract The chemicals used for GC-MS analysis; Trimethylchorosilance was collected and kept at - 20 ?C until analysis.
Trimethyl-silylimidazol and N,N-bistrimethylsiyl- trifluoro acetamide and standard ginsenoside Rb1 The solvent, Eg: Microwave-assisted extraction bioactive saponins from methanol, ethanol and butanol, were analytical grades chickpea (Cicer arietinum L).
Extraction from microwave dried GP used three methods, hot The filtrate was loaded onto a C-18 preparative column and water, methanol and ethanol extractions. The dried GP were impurities were eluted with 600 ml l- 1 methanol in water. The extracted with solvents at 1:30 proportions[Shahla Najafi]. The saponin containing fraction was eluted with methanol. The water extract method used double distilled water, heating in eluted fraction was diluted with water and was further purified water bath shaker at 900C for 10 min at 100 rpm. The methanol using HPLC to isolate DDMP-saponins. The HPLC system was extract method used 80% methanol with Soxhlet extraction for 6 equipped with a diode-array detector (UV6000) and a column A GCQ ion trap gas chromatography mass spectrometer The substance was collected and analyzed by 1H and 13C NMR . (electron impact ionization, 70 eV) was used in this study. A SPB 1H NMR and 13C NMR spectra identical to the data reported for 1701 column (column length 15 m., 0.25 mm. I.D., film thickness a DDMP moiety. The amount of DDMP-saponin was also 0.25 mm) was used. The column flow rate was 0.8 ml/min by determined2,3-dihydro-2,5-dihydroxy-6-methyl- 4H-pyran-4- helium gas. The conditions for the SPB1701 column were 1500C for 0.1 min to 2700C at 100C/min with a hold for 10 min [P.Devil]. Injector temperature was 2500C. Ion source temperature was Solid-phase extraction and liquid chromatography–electrospray 2000C. The aglycones obtained from the samples were mass spectrometric analysis of saponins in Salvia miltiorrhizae identified by comparing of the retention time, relative retention solid-phase extraction (SPE) and HPLC/ESI-MSn for the Microwave-assisted extraction of bioactive saponins from identification of the major saponins in “Danshen Dripping Pill”, a Chinese patent medicine consisting of Salvia miltiorrhizae and Panax notoginseng.
these saponins were characterized by HPLC/ESI-MSn analysis.
Prior to all extractions, chickpea seeds were ground in a Wiley mill to pass a 2-mm pore-size screen, and dried at 55 ?C for 72 h. A 2 g weight of “Danshen Dripping Pill” was dissolved using The dried powder was then extracted using a Soxhlet apparatus 20mL 4% ammonia in an ultrasonic bath at 25 ?C for 15 min. with hexane for 6 h to remove all fats.
After centrifugation at 5000×g for 10 min, a certain volume of the supernatant fluid was loaded and drawn through by gravity on SPE cartridge (5 mL, packed with 250 mg of 40µm octadecyl silica,Waters, USA), which was pretreated by passing through Defatted powder (4 g) was mixed with a solvent of choice 5mL of methanol followed by 5mLwater before loading, and (MeOH, EtOH or EtOH:H2O 7:3, butanol or butanol:water 1:1; drawn through by gravity. Then, the solidphase cartridge was 16ml) in 20-ml closed vials, which were placed in a mechanically washed with 10.0mLofwater to elute the phenolic compounds modified microwave oven and irradiated at 2450Mhz for 10 or entirely off. Finally, the cartridge was eluted with 1.0mL 20 min. The solvent temperature was kept constant at 60?C methanol, in which fraction most of the saponins were using an automatic temperature control device submerged into concentrated.A20 mL volume of the methanol eluent was solvent containing vessel. Twelve sample TFM (a thermally injected into the HPLC system HPLC–MS analysis was performed 15 l Herbal Tech Industry l September 2011 with Agilent LCMSD/ Trap System (Agilent Company) equipped with an electrospray interface. TheMSspectra were acquired in negative ion mode.N2 was used as both drying gas with a of 70?C and a gain of 7, and the nebuliser gas (nitrogen) was flowrate of 10 L/min and as nebulizing gas with a pressure of 60 psi. The nebulizer temperature was set at 350 ?C and the The HPLC conditions for the platycosides were as follows: eluent capillary voltage was set at 3500V. The mass spectra were A, water; eluent B, acetonitrile; gradient, 0–6 (10–15% B), 6–50 recorded in the range of 400–1500µm. A fragment amplification min (15–25% B), 50–60 min (25–47.5% B) and then equilibrated of1.5V was selected for MS2 analysis.
with 10% B for 8 min at a flow of 1mL/min. The ELSD system was HPLC grade methanol was used for SPE preparation.
Preparative Isolation of Six Major Saponins from Platycodi Radix Identification of HSCCC peak fractions.
Identification of the HSCCC peak fractions was carried out by ESI- MS, 1H-NMR and 13C-NMR spectra with references. A TBE-300A HSCC, with three serially connected multilayer coil The chemical structures of components present in each peak fraction purified by HSCCC were identified from ESI-MS, ESI-MS/MS, 1H-NMR and 13C-NMR data. The subsequent Acetonitrile, methanol, n-butanol, ethylacetate, hexane and structural identification of the peak fractions collected isopropanol (HPLC-grade) for the preparation of the crude sample and for HSCCC separation from the HSCCC was performed by comparison with previous 1H-NMR and 13C-NMR data.
Preparation of the two-phase solvent system and sample solution. A New Bioactive Steroidal Saponin from Agave attenuate Herbal plant.
A solvent system consisting of hexane–n-butanol–water (1:40:20, v/v) was used for the separation of platycoside E and deapio-platycoside E. Hexane–n-butanol–water (1:10:5, v/v) was The occurrence of steroidal saponins in Agave genus is well used as the solvent separation of platycodin D3, platycodin D Some species have an ethnopharmacological background, in particular A. sisalana which in the Bahama Islands, the central In conventional HSCCC experiments, a multi-layer coiled column bud is boiled with salt and the decoction given as a remedy for is first entirely filled with one phase of the two phase solvent jaundice; said to be effective within 24 hours, the aqueous system as a stationary phase, followed by elution with the other extract of A. attenuate was evaluated for activity against Bulinus phase. Here, the column was first filled with a mixture of the two africanus, Daphnia pulex, Anopheles arabiensis and phases, thus reducing the amount of time for hydrodynamic Oreochromis mossambicus demonstrating molluscicidal, equilibrium to be established (Slacanin et al., 1989). The ratio of two phases was also optimised at 70:30 (stationary phase– mobile phase, v/v) within the range 90:10 to 60:40 based on the amount of time required to reach hydrodynamic equilibrium Fresh leaves of Agave attenuata were obtained from the owing to retention on the stationary phase. In the present experiment, several solvent systems based on n-butanol water with added hexane or ethyl acetate were tested, and the results are summarized in Table 1. The table shows that platycoside E and deapio-platycoside E, when analysed in the reverse-mode The fresh leaves of the plant (3 kg) were extracted with 80% solvent system composed of n-butanol–water at volume ratios aqueous EtOH (6 l) followed by concentration to 600 ml and of 2:1 (v/v), have appropriate K values (0.5–5) for separation. extraction with an equal volume of n-BuOH gave a crude However, the K values of platyodin D and deapio-platycodin D, in material (12.5 g). It was roughly chromatographed on Sephadex the same solvent system, were smaller than expected. It was LH-20 with MeOH to give crude steroidal glycoside (2.5 g). difficult to separate platyodin D and deapio-platycodin D from Further purification by chromatography on a silica gel column the other compounds. Also, when n butanol– water (2:1, v/v eluted with CHCl3:MeOH:H2O (70:30:10 v/v/v) to afford one TLC used the retention of the stationary phase was poor (<30%), and so that system was deemed unsuitable for separation.
16 l Herbal Tech Industry l September 2011 Conventional open –column chromatography Hemolytically active saponins are detected as with zones on a reddish background[M. Sajjad Khan ].
Saponins form mainly blue, blue-violet and sometimes Avenue sativa herbs (poaceace): steroidal saponins; Triterpene saponins.3-4% free sugars.
SEPARATION OF SAPONINS BY USING TLC METHOD: Centellac herb(apiaceae):Ester saponins.
Ginseng radix(araliaceae):2-3% tetra cyclic triterpene glycosides.
powdered drugs (2g) is extracts by heating for 10mints under reflux with 10ml,70% ethanol.the filtrate is evaporate to about Liuirititiae radix (fabaceae):saponins; 8-12% glycyrrhizin,calcium 5ml, and 20-40cm of this solutions is used for TLC[GEORGE V ].A total of 3ml of the ethanolic extracts (with above solutions) is shaken several times with 5ml water and saturated n-butanol. Saponariae radix (caryop phylaceace) :3-5% bidesmosidic the n-butanol phase is separated and concentrated to about 1ml ;but with 20ml is used for TLC[REBECCA M. CORBIT ].
Sarsaparillae radix: 1.8-3% steroidal saponins,mono desmosidic, Identification of Major Saponins from Jiaogulan Extract Ginseng radix is extracted under the same conditions ,but with By GC-MC ANALYSIS.
90% ethanol.
Fresh GP leaves were dried by microwave dryer until the moisture An ethanol extacts (above solution) is evaporated to dryness. the content was below 10%. Dried GP was vacuum packed in residue is dissolved in 20ml chloroform: methanol (1:1); 20ml is aluminium foil and kept in -200C until used.
used for the detection of glycyrrhin [P.B.MALLIKHARJUNA].
The chemicals used for GC-MS analysis; Trimethylchorosilance Adsorbent: silica gel 60 F254-percorated TLC plates.
Trimethyl-silylimidazol and N,N-bistrimethylsiyl- trifluoro acetamide and standard ginsenoside Rb1 The solvent, methanol, ethanol and butanol, were analytical grades Chloroform: glacial acetic acid :methanol: water.
(64:32:12:8). This system is suitable for the separations of Extraction from microwave dried GP used three methods, hot water, methanol and ethanol extractions. The dried GP were extracted with solvents at 1:30 proportions[Shahla Najafi]. The Chloroform-methanol-water(70:30:8) for extractions water extract method used double distilled water, heating in water bath shaker at 900C for 10 min at 100 rpm. The methanol Ethyl acetate-ethanol-water-ammonia (65:25:9:1) for extract method used 80% methanol with Soxhlet extraction for 6 extraction of glycyrrhetic acid from Liquritae radix.
A GCQ ion trap gas chromatography mass spectrometer (electron impact ionization, 70 eV) was used in this study. A SPB With the exception of glycyyhetic acid (Liquiriae radix), no 1701 column (column length 15 m., 0.25 mm. I.D., film thickness saponins are detectable by exposure to uv-254 or uv-365nm.
0.25 mm) was used. The column flow rate was 0.8 ml/min by 17 l Herbal Tech Industry l September 2011 helium gas. The conditions for the SPB1701 column were 1500C Solid-phase extraction and liquid chromatography–electrospray for 0.1 min to 2700C at 100C/min with a hold for 10 min [P.Devil]. mass spectrometric analysis of saponins in Salvia miltiorrhizae Injector temperature was 2500C. Ion source temperature was 2000C. The aglycones obtained from the samples were identified by comparing of the retention time, relative retention solid-phase extraction (SPE) and HPLC/ESI-MSn for the time and mass authentic saponinMicrowave-assisted extraction identification of the major saponins in “Danshen Dripping Pill”, of bioactive saponins from chickpea (Cicer arietinum) a Chinese patent medicine consisting of Salvia miltiorrhizae and Panax notoginseng.
these saponins were characterized by HPLC/ESI-MSn analysis.
Seed powder:
Solid-phase extraction:
Prior to all extractions, chickpea seeds were ground in a Wiley mill to pass a 2-mm pore-size screen, and dried at 55 ?C for 72 h. A 2 g weight of “Danshen Dripping Pill” was dissolved using The dried powder was then extracted using a Soxhlet apparatus 20mL 4% ammonia in an ultrasonic bath at 25 ?C for 15 min. with hexane for 6 h to remove all fats.
After centrifugation at 5000×g for 10 min, a certain volume of the supernatant fluid was loaded and drawn through by gravity Microwave-assisted extraction
on SPE cartridge (5 mL, packed with 250 mg of 40µm octadecyl silica,Waters, USA), which was pretreated by passing through Defatted powder (4 g) was mixed with a solvent of choice 5mL of methanol followed by 5mLwater before loading, and (MeOH, EtOH or EtOH:H2O 7:3, butanol or butanol:water 1:1; drawn through by gravity. Then, the solidphase cartridge was 16ml) in 20-ml closed vials, which were placed in a mechanically washed with 10.0mLofwater to elute the phenolic compounds modified microwave oven and irradiated at 2450Mhz for 10 or entirely off. Finally, the cartridge was eluted with 1.0mL 20 min. The solvent temperature was kept constant at 60?C methanol, in which fraction most of the saponins were using an automatic temperature control device submerged into concentrated.A20 mL volume of the methanol eluent was solvent containing vessel. Twelve sample TFM (a thermally injected into the HPLC system HPLC–MS analysis was performed resistant form of Teflon) vessels were used at a time, with with Agilent LCMSD/ Trap System (Agilent Company) equipped pressure and temperature monitoring capabilities, The with an electrospray interface. TheMSspectra were acquired in microwave power was limited to 300 W[Devendra N. Kage]. negative ion mode.N2 was used as both drying gas with a After cooling to room temperature, the extract was collected flowrate of 10 L/min and as nebulizing gas with a pressure of 60 psi. The nebulizer temperature was set at 350 ?C and the capillary voltage was set at 3500V. The mass spectra were Soxhlet extraction:
recorded in the range of 400–1500µm. A fragment amplification Defatted powder (10 g) was extracted with the solvent of choice of1.5V was selected for MS2 analysis.
(150 ml), for 3 h. After cooling to room temperature, the extract HPLC grade methanol was used for SPE preparation.
was collected and kept at - 20 ?C until analysis.
Preparative Isolation of Six Major Saponins from Platycodi Radix Eg: Microwave-assisted extraction bioactive saponins from Apparatus:
Purification of saponins
A TBE-300A HSCC, with three serially connected multilayer coil The filtrate was loaded onto a C-18 preparative column and impurities were eluted with 600 ml l- 1 methanol in water. The saponin containing fraction was eluted with methanol. The Reagents and materials:
eluted fraction was diluted with water and was further purified using HPLC to isolate DDMP-saponins. The HPLC system was Acetonitrile, methanol, n-butanol, ethylacetate, hexane and equipped with a diode-array detector (UV6000) and a column isopropanol (HPLC-grade) for the preparation of the crude sample and for HSCCC separation Preparation of the two-phase solvent system and sample solution. Identification:
A solvent system consisting of hexane–n-butanol–water The substance was collected and analyzed by 1H and 13C NMR . (1:40:20, v/v) was used for the separation of platycoside E and 1H NMR and 13C NMR spectra identical to the data reported for deapio-platycoside E. Hexane–n-butanol–water (1:10:5, v/v) was a DDMP moiety. The amount of DDMP-saponin was also used as the solvent separation of platycodin D3, platycodin D determined2,3-dihydro-2,5-dihydroxy-6-methyl- 4H-pyran-4- 18 l Herbal Tech Industry l September 2011 In conventional HSCCC experiments, a multi-layer coiled column particular A. sisalana which in the Bahama Islands, the central is first entirely filled with one phase of the two phase solvent bud is boiled with salt and the decoction given as a remedy for system as a stationary phase, followed by elution with the other jaundice; said to be effective within 24 hours, the aqueous phase. Here, the column was first filled with a mixture of the two extract of A. attenuate was evaluated for activity against Bulinus phases, thus reducing the amount of time for hydrodynamic africanus, Daphnia pulex, Anopheles arabiensis and equilibrium to be established (Slacanin et al., 1989). The ratio of Oreochromis mossambicus demonstrating molluscicidal, two phases was also optimised at 70:30 (stationary phase– mobile phase, v/v) within the range 90:10 to 60:40 based on the amount of time required to reach hydrodynamic equilibriumowing to retention on the stationary phase. In the present experiment, several solvent systems based on n- Fresh leaves of Agave attenuata were obtained from the butanol water with added hexane or ethyl acetate were tested, Ornamental Plant Garden and the results are summarized in Table 1. The table shows that platycoside E and deapio-platycoside E, when analysed in the reverse-mode solvent system composed of n-butanol–water at volume ratios of 2:1 (v/v), have appropriate K values (0.5–5) for The fresh leaves of the plant (3 kg) were extracted with 80% separation. However, the K values of platyodin D and deapio- aqueous EtOH (6 l) followed by concentration to 600 ml and platycodin D, in the same solvent system, were smaller than extraction with an equal volume of n-BuOH gave a crude expected. It was difficult to separate platyodin D and deapio- material (12.5 g). It was roughly chromatographed on Sephadex platycodin D from the other compounds. Also, when n butanol– LH-20 with MeOH to give crude steroidal glycoside (2.5 g). water (2:1, v/v used the retention of the stationary phase was Further purification by chromatography on a silica gel column poor (<30%), and so that system was deemed unsuitable for eluted with CHCl3:MeOH:H2O (70:30:10 v/v/v) to afford one TLC homogeneous compound 1 (635 mg), Rf 0.43 which gave a dark green color with orcinol and H2SO of 70?C and a gain of 7, and the nebuliser gas (nitrogen) was adjusted to 2.5 The filtrate was loaded onto a C-18 preparative column and impurities were eluted with 600 ml l- 1 methanol in The HPLC conditions for the platycosides were as follows: eluent water[John Michael Berger]. The saponin containing fraction A, water; eluent B, acetonitrile; gradient, 0–6 (10–15% B), 6–50 was eluted with methanol. The eluted fraction was diluted with min (15–25% B), 50–60 min (25–47.5% B) and then equilibrated water and was further purified using HPLC to isolate DDMP- with 10% B for 8 min at a flow of 1mL/min. The ELSD system was saponins. The HPLC system was equipped with a diode-array Identification of HSCCC peak fractions.
Identification of the HSCCC peak fractions was carried out by ESI- The substance was collected and analyzed by 1H and 13C NMR . MS, 1H-NMR and 13C-NMR spectra with references. 1H NMR and 13C NMR spectra identical to the data reported for a DDMP moiety. The amount of DDMP-saponin was also determined2,3-dihydro-2,5-dihydroxy-6-methyl- 4H-pyran-4-one (DDMP) moiety on C-22.
The chemical structures of components present in each peak fraction purified by HSCCC were identified from ESI-MS, ESI- MS/MS, 1H-NMR and 13C-NMR data. The subsequent structural identification of the peak fractions collected Generally these saponins are used for the anti-inflammation from the HSCCC was performed by comparison with previous A New Bioactive Steroidal Saponin from Agave attenuate Herbal plant.
saponins used as dietary supplements and nutriceuticals The occurrence of steroidal saponins in Agave genus is well Some species have an ethnopharmacological background, in 19 l Herbal Tech Industry l September 2011 these are used for the preparation of cosmetics,mainly used in GEORGE V. ODELL aDd CBUEN-emN BSU, Isolation and Purification of the Saponin. of Glottidium vesicarium, BIOLOGICAL SCIENCES. References:
REBECCA M. CORBIT, JORGE F. S. FERREIRA, Simplified Extraction David G. I. Kingston, ChairNeal Castagnoli, jr. James Tanko of Ginsenosides from American Ginseng (Panax quinquefolius L.) Richard Gandour Paul Deck CHARACTERIZATION saponins. for High-Performance Liquid Chromatography-Ultraviolet Analysis, J. Agric. Food Chem. 2005, 53, 9867-9873 9867 Young Wan Ha and Yeong Shik Kim* Isolation of saponins from Platycodi Radix by hsccc M. Sajjad Khan, Nitin Nema, M.D. Kharya, Salma Khanam, Chromatographic estimation of maturity based phytochemical Zohar Kerem, Hilla German-Shashoua and O ded Yarden, Journal p r o f i l i n g o f I p o m o e a m a u r i t i a n a , of the Science of Food and Agriculture, Microwave-assisted http://www.arjournals.org/ijop.html. P.B.MALLIKHARJUNA, L.N.RAJANNA, Phytochemical Studies of Haijiang Zhang, Yiyu Cheng , Solid-phase extraction, Journal of Pharmaceutical and Biomedical Analysis 40 (2006) 429–432 Shahla Najafi and S. S. Deokule, Pharmacognostic study of Niramon Utama-ang, Penkwan Chompreeda, Vichai Tylophora dalzellii Hook. Journal of Medicinal Plants Research Haruthaithanasan, Identification of Major Saponins from Vol. 4(5), pp. 403-406, 4 March, 2010. P.Devil ,R.Meera, P.Muthumani, B.Kameswari, Phyto-Physico Farah S. Hosseinian and Trust Beta, Patented Techniques for the chemical evaluation and Antioxidant activities of leaves of Extraction and Isolation of Secoisolariciresinol Diglucoside from Naphellium lappaceum, Journal of Pharmaceutical Sciences and Flaxseed, University of Manitoba, Department of Food Science, Winnipeg, Manitoba, R3T 2N2 Canada Devendra N. Kage , Vijaykumar B. Malashetty, Y. N. Seetharam; P. D.A. RICKERT, M.A. MEYER, J. HU, AND P.A. MURPHY, Effect of Suresh & Saraswati B. Patil. Effect of Ethanol Extract of Whole Extraction pH and Temperature on Isoflavone and Saponin Plant of Trichosanthes cucumerina var. cucumerina L. Int. J. Partitioning. JFS C: Food Chemistry and Toxicology Maggie P.K. Choia, Kelvin K.C. Chanb, Hei Wun Leungb, Carmen John Michael Berger, ISOLATION, CHARACTERIZATION, AND W. Huiea, P ressurized liquid extraction of active ingredients SYNTHESIS OF BIOACTIVE NATURAL PRODUCTS FROM (ginsenosides) from medicinal plants. Journal of RAINFOREST FLORA, Copyright 2001, John M. Berger. Chromatography A, 983 (2003) 153–162 Kalpana Mujoo, Valsala Haridas, Joseph J. Hoffmann, Triterpenoid Saponins from Acacia victoriae (Bentham) Decrease Tumor Cell Proliferation and Induce Apoptosis. 20 l Herbal Tech Industry l September 2011

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Dear Fresh Breeze customer, You have just purchased a sophisticated product. We place great importance on the workmanship and the high quality of the materials used. If you have any questions which are not answered in this manual, please do not hesitate to contact us directly, or your Fresh Breeze dealer. from Fresh Breeze Table of Contents 1. Disclaimer and exclusion of liabili

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