Structure characterization and identification steroidal saponins from Ophiopogon japonicus Ker-Gawler (Liliaceae) by high-performance liquid chromatography with ion trap mass spectrometry

Introduction – Steroidal saponins are the main active constituents in Ophiopogon japonicus Ker‐Gawler (Liliaceae). However, because of their high polarity, non‐chromophores and low content in plants, steroidal saponins are difficult to be isolated from O. japonicus by conventional phytochemical methods. Objective – To develop a sensitive and rapid approach towards the structural analysis of steroidal saponins using HPLC/ESI‐MSⁿ. Methodology – The fragmentation behaviors of six known steroidal saponins in negative ESI‐MSⁿ were used to deduce their mass spectral fragmentation mechanisms. By using HPLC/ESI‐MSⁿ, the important structural information on aglycone types, sugar types and saccharide sequences can be obtained. Results – According to the HPLC retention behaviour, the molecular structural characteristics provided by multistage mass spectrometry spectra and the literature, a total of 8 steroidal saponins were tentatively identified or characterized in O. japonicus rapidly. Conclusion – This work has shown that HPLC‐ESI‐MSⁿ may be used as an effective and rapid method for the characterization and identification of steroidal saponins from O. japonicus. Copyright © 2010 John Wiley & Sons, Ltd.     

Saponins in Tribulus terrestris – Chemistry and Bioactivity

Tribulus terrestris is a valuable herb known for its application in the folk medicine in many parts of the world. Furostanol and spirostanol saponins of tigogenin, neotigogenin, gitogenin, neogitogenin, hecogenin, neohecogenin, diosgenin, chlorogenin, ruscogenin and sarsasapogenin type are frequently found in this plant. Four sulphated saponins of tigogenin and diosgenin type are also isolated. Extracts and steroidal saponins have been found to possess various pharmacological activities. Preparations based on the saponin fraction of T. terrestris are used for treatment of infertility and libido disorders in men and women, as well as for treatment of cardiac diseases. Food supplements containing T. terrestris extracts are on sale in USA and Europe with claim of a general stimulating action. In this review the steroidal saponins found in T. terrestris are presented, covering the literature up to 2004. The data reveal clear difference in the saponin composition of samples from this plant species collected from different geographical regions. A comprehensive account on the biological activity of extracts and individual saponins is also included.     

The Quantitative Analysis and Identification of Steroidal Sapogenins of the Dioscorea Plant

The results for the quantitative analysis and identification of steroidal sapo-genins of 16 species of Dioscorea rhizomes are given in Table 1. The amount of steroidal sapogenin varies from 0.073%–5.93%, its highest content of diosgenin is 5.93% in D. zingiberensis C. H. Wright. 2. The results observed from paper chromatography, thin layer chromatography and gas-liquid chromatography revealed that except the sapogenin of D. chingii Pr. and Br. is similar to tokorogenin, all other sapogenins after recrystallization yield a spot identical to diosgenin or yamogenin. Meanwhile, in most Dioscorea sample, a spot identical to △ 3, 5-diene-25D-spirostane was shown by using thin layer chromatography and gas-liquid chromatography. (Table 2). 3. The results observed from infrared spectrum and gas-liquid chromatography revealed that the sapogenins of D. collettii, Hook. f; D. collettii. Hook, f. var. hypoglauca, Pei and Ting. and D. tokoro Makino. contained 25L-spirostane. (A900≤ A920 cm-1).     

Production of diosgenin from yellow ginger (<Emphasis Type="Italic">Dioscorea zingiberensis</Emphasis> C. H. Wright) saponins by commercial cellulase

A commercial cellulase was first assessed to be effective in hydrolyzing glycosyl at the C-3 and C-26 positions in steroidal saponins from yellow ginger (Dioscorea zingiberensis C. H. Wright) to diosgenin, a very important chemical in the pharmaceutical industry. The effect of different parameters on enzyme hydrolysis was further investigated by systematically varying them. The highest yield was achieved when the hydrolysis ran at 55°C and pH 5.0 with an enzyme to substrate ratio of 15 × 10³ U/g. The biotransformed products identified using TLC and HPLC confirmed that the cellulase was capable of releasing diosgenin from steroidal saponins. Moreover, the biotransformation process was explored by LC-MS and LC-MS/MS analysis. Enzymatic hydrolysis together with 40 % of the original sulphuric acid used increased the diosgenin yield by 15.4 ± 2.7% than traditional method. Therefore, the commercial cellulase may serve as a promising tool for industrial diosgenin production and for further use in saponin modification.     

Isolation of four high-purity dammarane saponins from extract of Panax notoginseng by centrifugal partition chromatography coupled with evaporative light scattering detection in one operation

Introduction – Centrifugal partition chromatography (CPC), as a continuous liquid–liquid partition chromatography with no solid support matrix, combined with evaporative light scattering detection (ELSD) was employed for systematic separation and purification of weak‐chromophoric saponins from a highly valued and important traditional Chinese herbal medicine, Panax notoginseng. Objective – To separate and isolate high‐purity saponins from extract of Panax notoginseng using CPC‐ELSD with a simple and low toxicity solvent system. Methodology – Samples were preparaed by extracting the root material with acetone, treated with n‐butanol and then freeze‐dried. CPC‐ELSD was applied in the separation and detection of notoginsenoside and ginsenosides from extract of Panax notoginseng using a solvent system composed of ethyl acetate–n‐butanol–water (1:1:2, v/v/v). The saponins were analysed and identified by their retention time with high‐performance liquid chromatography (HPLC) coupled with ELSD, as well as electrospray ionisation tandem mass spectrometry (ESI‐MSⁿ ) in the negative and positive ion modes with the authentic standards. Results – A total of 9.6 mg of notoginsenoside R₁, 67.8 mg of ginsenoside Rg₁, 2.3 mg of Re and 286.5 mg of Rb₁ were purified from 487.2 mg of n‐butanol extract of P. notoginseng. The purities of obtained saponins in a single run were assessed to be over 98% by HPLC‐ELSD. Conclusion – CPC‐ELSD was proved to be a very fast and efficient tool for separation of high‐purity dammarane saponins. Copyright © 2011 John Wiley & Sons, Ltd.     

Sapogenins of Yucca glauca seeds

From the seeds of Yucca glauca Nutt., sarsasapogenin. markogenin. tigogenin. neo-tigogenin. neo-gitogenin, hecogenin and gloriogenin have been isolated and identified. Sarsasapogenin was the predominant sapogenin. Markogenin and gloriogenin have not been reported in leaves, roots or rhizomes of this species before. Small amounts of a sapogenin believed to be either diosgenin or yamogenin were also detected.     

Rapid validated HPTLC method for estimation of betulinic acid in Nelumbo nucifera (Nymphaeaceae) rhizome extract

Introduction – Betulinic acid (pentacyclic triterpenoid) is an important marker component present in Nelumbo nucifera Gaertn. rhizome. N. nucifera rhizome has several medicinal uses including hypoglycaemic, antidiarrhoeal, antimicrobial, diuretic, antipyretic, psychopharmacological activities. Objective – To establish a simple, sensitive, reliable, rapid and validated high‐performance thin‐layer chromatography method for estimation of betulinic acid in hydro‐alcoholic extract of N. nucifera Gaertn. rhizome. Materials and methods – The separation was carried out on a thin‐layer chromatography aluminium plate pre‐coated with silica gel 60F₂₅₄, eluted with chloroform, methanol and formic acid (49 : 1 : 1 v/v). Post chromatographic derivatisation was done with anisaldehyde–sulphuric acid reagent and densitometric scanning was performed using a Camag TLC scanner III, at 420 nm. Results – The system was found to produce a compact spot for betulinic acid (R_f = 0.30). A good linear precision relationship between the concentrations (2–10 µg) and peak areas were obtained with the correlation coefficient (r) of 0.99698. The limit of detection and limit of quantification of betulinic acid were detected to be 0.4 and 2.30 µg per spot. The percentage of recovery was found to be 98.36%. The percentage relative standard deviations of intra‐day and inter‐day precisions were 0.82–0.394 and 0.85–0.341, respectively. Conclusion – This validated HPTLC method provides a new and powerful approach to estimate betulinic acid as phytomarker in the extract. Copyright © 2010 John Wiley & Sons, Ltd.     

Quantitative determination of lanostane triterpenes in Fomes officinalis and their fragmentation study by HPLC-ESI

Introduction – The fruit bodies of Fomes officinalis are used for the treatment of coughs, gastric cancer, rheumatism and hydropsia; however, no method is currently available to assess the quality of this medicinal fungus based on quantitative profile of its main triterpenes. Objective – To develop a simple and accurate HPLC‐UV method for the simultaneous quantification of five lanostane‐type triterpenes in the fruit bodies of F. officinalis. Method – Separations were performed on an Agilent Zorbax Eclipse XDB‐C₁₈ column by gradient elution using acetonitrile : formic acid. Analytes were identified by HPLC coupled with electrospray ionisation mass spectrometry experiments. The quantitative HPLC‐UV method was validated for linearity, precision, accuracy and limits of detection and quantification. Results – Calibration curves presented good linear regression (r > 0.9996) within test ranges. The relative standard deviation of this method was less than 1.7% for intra‐ and inter‐day assays and overall recoveries were 96.4–104.1% for the five compounds analysed. The method was successfully applied to the quantification of five triterpenes in 16 samples of F. officinalis collected from different regions. Conclusion – The developed assay could be considered as a suitable quality control method for F. officinalis. Copyright © 2010 John Wiley & Sons, Ltd.     

Comparative quantitative analysis of artemisinin by chromatography and qNMR

Introduction – Since the discovery of artemisinin in the 1970s, many techniques based on diverse chromatography techniques have been developed to detect and quantify this important antiplasmodial compound. The accurate quantification of this compound in the Artemisia annua plant material is mainly needed for breeding purposes in order to cultivate higher yielding varieties. It is also important for the quality control of herbal preparations containing A. annua plant material. Objective – To evaluate the most common validated quantification techniques (LC‐MS, HPLC‐ELSD and TLC) and compare the results to quantitative nuclear magnetic resonance spectroscopy (qNMR) in eight different A. annua samples collected from around the world. Methodology – The leaf material were extracted according to standard procedures and analysed with the validated quantification techniques. For the qNMR analysis we did not employ a standard curve but instead used an internal standard (maleid acid) which is not chemically related to artemisinin. Results – We found a significant difference between the results in this study. Compared with the qNMR results the HPLC‐ELSD corresponded closely, followed by LC‐MS. Quantitation with TLC led to an estimation range of ?0.5 to +3.2 mg artemisinin/g of A. annua. Conclusion – These results imply that qNMR, with the addition of an internal standard, can be used to quantify artemisinin in A. annua samples in a rapid and reproducible manner. Copyright © 2010 John Wiley & Sons, Ltd.     

O‐glycoside sequence of pentacyclic triterpene saponins from Phytolacca bogotensis using HPLC‐ESI/multi‐stage tandem mass spectrometry

Introduction – The lack of pharmacopoeial methodologies for the quality control of plants used for therapeutic purposes is a huge problem that impacts directly upon public health. In the case of saponins, their great structural complexity, weak glycoside bonds and high polarity hinder their identification by conventional techniques. Objective – To apply high‐performance liquid chromatography–electrospray tandem mass spectrometry (HPLC‐ESI/MSⁿ) to identify the O‐glycoside sequence of saponins from the roots of Phytolacca bogotensis. Methodology – Saponins were isolated by preparative HPLC and characterised by NMR spectroscopic experiments. Collision‐induced dissociation (CID) of isolated saponins was performed producing typical degradation reactions that can be associated with several glycosidic bonds as empirical criteria. A method using solid‐phase extraction (SPE) and HPLC/ESI‐MSⁿ for the characterisation of saponins and identification of novel molecules is described. Results – Three saponins reported for the first time in P. bogotensis were isolated and characterised by NMR spectroscopy. Characteristic cross ring cleavage reactions have been used as empirical criteria for the characterisation of the glycosidic bonds most frequently reported for Phytolacca saponins. One new saponin was proposed on the basis of empirical criteria, and other five saponins were identified for the first time for P. bogotensis using HPLC‐ESI/MSⁿ. Conclusion – Electrospray ionisation in combination with tandem mass spectrometry has been established as a powerful tool for the profiling of saponins from roots of P. bogotensis. CID proved to be a useful tool for the characterisation and identification of known and novel saponins from the plant family Phytolaccaceae and can be used for quality control purposes of crude plant extracts. Copyright © 2009 John Wiley & Sons, Ltd.     

Validated TLC method for simultaneous quantitation of kutkoside and picroside‐I from Kutki extract

Introduction – The two iridoid glycosides kutkoside and picroside‐I are the active hepatoprotective principles of Picrorhiza kurroa Royle ex Benth (Scrophulariaceae), commonly known as Kutki. Quantitation of these phytoconstituents is important for the routine quality control of Kutki extract. Objective – To develop and validate a simple, precise and rapid thin‐layer chromatography (TLC) method for the simultaneous quantitation of kutkoside and picroside‐I in Kutki extract. Methodology – The analysis was performed on a TLC precoated silica gel 60 F₂₅₄ plate with ethyl acetate:methanol:glacial acetic acid:formic acid (25:5:1:1, v/v/v/v) as mobile phase. Densitometric evaluation of kutkoside and picroside‐I was carried out at 265 nm and the mobile phase showed good resolution with R_f values 0.42 ± 0.03 and 0.61 ± 0.03 for kutkoside and picroside‐I, respectively. The method was validated in terms of specificity, linearity, accuracy and precision. Results – The content of kutkoside and picroside‐I was found to be 2.18 and 1.90%, respectively, and was comparable with those obtained by HPLC. The linearity was found to be in the range of 80–480 ng/spot for both kutkoside and picroside‐I. The average recovery values were found to be 96.5 and 96.0% for kutkoside and picroside‐I, respectively. Conclusion – The developed method was found to be relatively simple, precise and reproducible for the simultaneous quantitation of kutkoside and picroside‐I. The method does not employ any derivatisation procedure and can be used as a quality control tool for the routine analysis of commercial Kutki extracts. Copyright © 2010 John Wiley & Sons, Ltd.     

Biotransformation of steriodal saponins in Dioscorea zingiberensis C. H. Wright to diosgenin by Trichoderma harzianum

Diosgenin is an important starting material in the steroidal hormone industry. Traditionally, diosgenin is mainly produced by acid hydrolysis of Dioscorea zingiberensis C. H. Wright (DZW) tubers. This method yields numerous byproducts that can cause serious pollution. In this study, diosgenin was obtained by biotransformation of steroidal saponins in DZW afforded by Trichoderma harzianum CGMCC 2979. The medium was optimized for maximum diosgenin production. The addition of phosphate buffer, surfactant Tween-85, and Fe²⁺ increased the yield of diosgenin by 50.28%, 33.35%, and 22.07%, respectively. The optimum medium obtained by response surface methodology was composed of 60 mmol l⁻¹ phosphate buffer, 0.07% (w/v) Tween-85, and 0.93 mmol l⁻¹ Fe²⁺. Under these conditions, a maximum diosgenin yield of 30.05 ± 0.59 mg g⁻¹ was achieved, which was slightly higher than that obtained from traditional acid hydrolysis. By hydrolyzing the un-transformed steroidal saponins after biotransformation, the total diosgenin yield increased by 35% compared to traditional method. Moreover, chemical oxygen demand and residual reduced sugar in the wastewater produced by this integrated process were only 3.72% and 0.3%, respectively, that of the traditional acid hydrolysis method.     

Three-liquid-phase extraction of diosgenin and steroidal saponins from fermentation of Dioscorea zingibernsis C. H. Wright

Diosgenin is an important starting material in the steroidal hormone industry. The yield of diosgenin obtained from the fermentation of Dioscorea zingibernsis C. H. Wright (DZW) by Trichoderma harzianum is higher than that typically obtained from acid hydrolysis. In this paper, the extraction of steroids in the culture broth was studied. A novel three-liquid-phase system (TLPS) consisted of petroleum ether, ethanol, ammonium sulphate and water was used to separate diosgenin and steroidal saponins in the culture broth. The partition behaviors of various steroidal saponins, diosgenin and glucose were investigated. From this, an optimized TLPS was obtained, which composed of 30% ethanol (w/w), 17% (NH₄)₂SO₄ (w/w) and 40% (w/w) petroleum ether. In the optimized TLPS, almost all of the diosgenin was extracted into the top phase giving a recovery of 97.24%, whereas the steroidal saponins were mainly extracted into the middle phase, with recoveries of zingibernsis newsaponin, deltonin and diosgenin-diglucoside reaching almost 100%. The recoveries of trillin and diosgenin-triglucoside were 96.03% and 98.82%, respectively. Glucose tended to remain in the bottom phase, giving a recovery of 72.01%. The three-liquid-phase extraction (TLPE) successfully resulted in the simultaneous separation of diosgenin, untransformed steroidal saponins and glucose.     

Prolamellar body and saponins: Avenacosides are not constituents of Avena etioplasts

Etiolated Avena leaf cells were homogenized, then fractionated into four fractions in the presence of salts by differential centrifugation, and intact etioplasts were prepared by Percoll or sucrose density gradient centrifugation. Using thin layer chromatography steroidal saponins, avenacoside A and B were found in the leaf cells and heavy cell fractions which were rich in other cell structures besides intact etioplasts, but not detected in the purified etioplasts. We concluded that saponins are not constituents of the prolamellar bodies in etioplasts.     

Identification of some Bioactive Metabolites in a Fractionated Methanol Extract from Ipomoea aquatica (Aerial Parts) through TLC,HPLC, UPLC‐ESI‐QTOF‐MS and LC‐SPE‐NMR Fingerprints Analyses

Introduction

The plant species Ipomoea aquatica contains various bioactive constituents, e.g. phenols and flavonoids, which have several medical uses. All previous studies were executed in Asia; however, no reports are available from Africa, and the secondary metabolites of this plant species from Africa are still unknown.

Objective

The present study aims finding suitable conditions to identify the bioactive compounds from different fractions.

Methodology

Chromatographic fingerprint profiles of different fractions were developed using high‐performance liquid chromatography (HPLC) and then these conditions were transferred to thin‐layer chromatography (TLC). Subsequently, the chemical structure of some bioactive compounds was elucidated using ultra‐performance liquid chromatography‐quadrupole time of flight‐tandem mass spectrometry (UPLC‐QTOF‐MS) and liquid chromatography‐solid phase extraction‐nuclear magnetic resonance (LC‐SPE‐NMR) spectroscopy.

Results

The HPLC fingerprints, developed on two coupled Chromolith RP‐18e columns, using a gradient mobile phase (methanol/water/trifluoroacetic acid, 5:95:0.05, v/v/v), showed more peaks than the TLC profile. The TLC fingerprint allows the identification of the types of chemical constituents, e.g. flavonoids. Two flavonoids (nicotiflorin and ramnazin‐3‐O‐rutinoside) and two phenolic compounds (dihydroxybenzoic acid pentoside and di‐pentoside) were tentatively identified by QTOF‐MS, while NMR confirmed the structure of rutin and nicotiflorin.

Conclusion

The HPLC and TLC results showed that HPLC fingerprints give more and better separated peaks, but TLC helped in determining the class of the active compounds in some fractions. Bioactive constituents were identified as well using MS and NMR analyses. Two flavonoids and two phenolic compounds were tentatively identified in this species for the first time, to the best of our knowledge. Copyright © 2017 John Wiley & Sons, Ltd.     

Cell death induction and nitric oxide biosynthesis in white poplar (Populus alba) suspension cultures exposed to alfalfa saponins

The present work reports on the biological activity of alfalfa (Medicago sativa) saponins on white poplar (Populus alba, cultivar ‘Villafranca’) cell suspension cultures. The extracts from alfalfa roots, aerial parts and seeds were characterized for their saponin content by means of thin layer chromatography (TLC) and electrospray ionisation coupled to mass spectrometry. The quantitative saponin composition from the different plant extracts was determined considering the aglycone moieties and determined by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) analyses. Only soyasapogenin I was detected in the seed extract while several other saponins were found in the root and leaf extracts. Actively proliferating white poplar cell cultures were challenged with the different saponin extracts. Only alfalfa root saponins, at 50 µg ml^?1, induced significant cell death rates (75.00 ± 4.90%). Different cell subpopulations with peculiar cell death morphologies were observed and the programmed cell death (PCD)/necrosis ratio was reduced at increasing saponin concentrations. Enhancement of nitric oxide (NO) production was observed in white poplar cells treated with root saponins (RSs) at 50 µg ml^?1 and release of reactive oxygen species (ROS) in the culture medium was also demonstrated. Saponin‐induced NO production was sensitive to sodium azide and N^G‐monomethyl‐l ‐arginine, two specific inhibitors of distinct pathways for NO biosynthesis in plant cells.     

A rapid screening technique for detection of diosgenin through in situ cytophotometry

Costus speciosus (Koenig) Sm. contains diosgenin, an important drug in family planning programs in India and underdeveloped countries. A simple, rapid method has been developed for in situ quantitation of diosgenin in this plant. The method is based on the formation of a suitable colored product of diosgenin in frozen sections of fresh material followed by determination of its optical density by in situ cytophotometry. The staining reagent is a combination of anisaldehyde, sulfuric acid and acetic acid. A positive correlation has been observed between cytophotometric diosgenin estimates and those derived from thin layer chromatography of extracts. The method is convenient for routine screening of plants for steroidal sapogenins such as diosgenin.     

Structure-function relationship for saponin effects on cell cycle arrest and apoptosis in the human 1547 osteosarcoma cells: a molecular modelling approach of natural molecules structurally close to diosgenin

In this paper, eight natural molecules structurally close to diosgenin (five saponins: diosgenin, hecogenin, tigogenin, sarsasapogenin, smilagenin; two steroidal alkaloids: solasodine, solanidine; one sterol: stigmasterol) have been tested for their biological activities on human 1547 osteosarcoma cells. Differences in activity were studied in term of proliferation rate, cell cycle distribution and apoptosis induction. By using molecular modelling, two structural characteristics were calculated: spatial conformation and electron transfer capacity. The second property has been investigated by the HOMO repartition and the corresponding energy. Correlation between the experimental and the theoretical data permit us to highlight the importance of the hetero-sugar moiety and the 5,6-double bond in the biological activity (apoptosis and cell cycle arrest) on the human 1547 cell line. The importance of conformation at C-5 and C-25 carbon atoms was also discussed.     

Steroidal alkaloids in tissue cultures and regenerated plants of Solanum dulcamara

Callus and cell suspension cultures were established with shoots of the soladulcidine variety of the bittersweet Solanum dulcamara L. Plantlets were regenerated from undifferentiated callus. From mixotrophic callus as well as mixotrophic suspension cultures soladulicidine, solasodine and the corresponding neutral spirostanes tigogenin and diosgenin were isolated and identified by thin layer chromatography and mass spectrometry. Total alkaloid concentrations were about 0.2 mg/g dry weight (callus) and 0.1 mg/g dry weight (green suspension cultures). In the heterotrophic cell line only the neutral sapogenins could be detected. Alkaloid accumulation in callus of Solanum dulcamara could be enhanced by the induction of organogenesis. The shoots of the regenerated plants from the mixotrophic callus contained soladulcidine (1.6 mg/g dry weight) and tigogenin. Thus, in concentration and composition the regenerated plants equalled the source plant.Abbreviations 2.4-D 2.4-dichlorophenoxyacetic acid - NAA naphthylacetic acid - HPLC high performance liquid chromatography - TLC thin layer chromatography     

A Rapid Screening Technique for Detection of Diosgenin Through In Situ Cytophotometry

Costus speciosus (Koenig) Sm. contains diosgenin, an important drug in family planning programs in India and underdeveloped countries. A simple, rapid method has been developed for in situ quantitation of diosgenin in this plant. The method is based on the formation of a suitable colored product of diosgenin in frozen sections of fresh material followed by determination of its optical density by in situ cytophotometry. The staining reagent is a combination of anisaldehyde, sulfuric acid and acetic acid. A positive correlation has been observed between cytophotometric diosgenin estimates and those derived from thin layer chromatography of extracts. The method is convenient for routine screening of plants for steroidal sapogenins such as diosgenin.