HPLC Method for Chemical Fingerprinting of Guggul (Commiphora wightii)--Quantification of E- and Z-Guggulsterones and Detection of Possible Adulterants.

A reversed-phase high-performance liquid chromatography method was developed for the chemical fingerprinting of Commiphora wightii gum resin (guggul). This method was also used for the quantification of E- and Z-guggulsterones in different C. wightii gum resin samples. The analysis was conducted on a high-performance liquid chromatography system with a reversed-phase column using a gradient elution comprised of water and acetonitrile with 0.1% acetic acid, and a run time of 50 min at 38 °C. The calibration curve of E- and Z-guggulsterones showed good linearity with a standard correlation coefficient >0.999, which is within the established range (0.5-250 µg/mL). Twelve marker compounds were selected and successfully analyzed by this method in 22 C. wightii gum resin samples along with 9 gum resin samples of possible adulterant species.

Fast Identification of 1,3-Dimethylamylamine Using Direct Analysis in Real Time-QToF-MS.

The central nervous system stimulant 1,3-dimethylamylamine (DMAA) has been found in preworkout products and dietary supplements. A fast direct analysis in real time-quadrupole time of flight-MS method was used for identification of DMAA in dietary supplements and to determine if this compound is present in geranium (Pelargonium graveolens) plants or oil. This method involved the use of [M+H]+ ions in the positive mode based on the exact mass of DMAA. The results of this investigation showed that DMAA was not detected from authentic samples of P. graveolens plant material or pelargonium oil or in multiple samples of commercially available pelargonium oil. DMAA was detected in three samples of dietary supplements. The LOD of DMAA was found to be 10 ng/mL.

Identification and Characterization of Indole and Oxindole Alkaloids from Leaves of Mitragyna speciosa Korth Using Liquid Chromatography-Accurate QToF Mass Spectrometry.

Alkaloids have been reported to be the major physiologically active constituents in Mitragyna. An analytical method was developed to provide an alternative, fast method for characterization of alkaloids from various M. speciosa samples. The separation was achieved using an RP octylsilyl (C8) column, MS detection, and a water-acetonitrile with formic acid gradient as the mobile phase. Ultra-HPLC/quadrupole time-of-flight MS analysis and characterization were performed on 12 corynanthe-type indole/oxindole alkaloids obtained from the leaves of M. speciosa Korth. The indoles and oxindoles had an open E ring with or without substitution occurring at the C9 position. The full single mass spectrum of alkaloids showed a strong signal for the protonated molecule [M+H]+. The product ion spectrum of mitragynine type of alkaloids showed strong response at m/z=174.0901 suggestive of an ion containing an odd number of nitrogen atoms corresponding to formula C11H12NO, which is characteristic of indole alkaloids. A multivariate statistical analysis technique, principal component analysis, was used to show discrimination between the M. speciosa samples. The results indicated that the analytical method is suitable for QC testing of various Mitragyna commercial samples and can be used to evaluate market products purported to contain M. speciosa.

Functional identification of valerena-1,10-diene synthase, a terpene synthase catalyzing a unique chemical cascade in the biosynthesis of biologically active sesquiterpenes in Valeriana officinalis.

Valerian is an herbal preparation from the roots of Valeriana officinalis used as an anxiolytic and sedative and in the treatment of insomnia. The biological activities of valerian are attributed to valerenic acid and its putative biosynthetic precursor valerenadiene, sesquiterpenes, found in V. officinalis roots. These sesquiterpenes retain an isobutenyl side chain whose origin has been long recognized as enigmatic because a chemical rationalization for their biosynthesis has not been obvious. Using recently developed metabolomic and transcriptomic resources, we identified seven V. officinalis terpene synthase genes (VoTPSs), two that were functionally characterized as monoterpene synthases and three that preferred farnesyl diphosphate, the substrate for sesquiterpene synthases. The reaction products for two of the sesquiterpene synthases exhibiting root-specific expression were characterized by a combination of GC-MS and NMR in comparison to the terpenes accumulating in planta. VoTPS7 encodes for a synthase that biosynthesizes predominately germacrene C, whereas VoTPS1 catalyzes the conversion of farnesyl diphosphate to valerena-1,10-diene. Using a yeast expression system, specific labeled [(13)C]acetate, and NMR, we investigated the catalytic mechanism for VoTPS1 and provide evidence for the involvement of a caryophyllenyl carbocation, a cyclobutyl intermediate, in the biosynthesis of valerena-1,10-diene. We suggest a similar mechanism for the biosynthesis of several other biologically related isobutenyl-containing sesquiterpenes.

Mechanisms enforcing the estrogen receptor ß selectivity of botanical estrogens.

Because little is known about the actions of botanical estrogens (BEs), widely consumed by menopausal women, we investigated the mechanistic and cellular activities of some major BEs. We examined the interactions of genistein, daidzein, equol, and liquiritigenin with estrogen receptors ERα and ERß, with key coregulators (SRC3 and RIP140) and chromatin binding sites, and the regulation of gene expression and proliferation in MCF-7 breast cancer cells containing ERα and/or ERß. Unlike the endogenous estrogen, estradiol (E2), BEs preferentially bind to ERß, but their ERß-potency selectivity in gene stimulation (340- to 830-fold vs. E2) is enhanced at several levels (coregulator recruitment, chromatin binding); nevertheless, at high (0.1 or 1 µM) concentrations, BEs also fully activate ERα. Because ERα drives breast cancer cell proliferation and ERß dampens this, the relative levels of these two ERs in target cells and the BE dose greatly affect gene expression and proliferative response and will be crucial determinants of the potential benefits vs. risks of BEs. Our findings reveal key and novel mechanistic differences in the estrogenic activities of BEs vs. E2, with BEs displaying patterns of activity distinctly different from those seen with E2 and provide valuable information to inform future studies.

Octulosonic acid derivatives from Roman chamomile (Chamaemelum nobile) with activities against inflammation and metabolic disorder.

Six new octulosonic acid derivatives (1-6) were isolated from the flower heads of Roman chamomile (Chamaemelum nobile). Their structures were elucidated by means of spectroscopic interpretation. The biological activity of the isolated compounds was evaluated toward multiple targets related to inflammation and metabolic disorder such as NAG-1, NF-κB, iNOS, ROS, PPARα, PPARγ, and LXR. Similar to the action of NSAIDs, all the six compounds (1-6) increased NAG-1 activity 2-3-fold. They also decreased cellular oxidative stress by inhibiting ROS generation. Compounds 3, 5, and 6 activated PPARγ 1.6-2.1-fold, while PPARα was activated 1.4-fold by compounds 5 and 6 only. None of the compounds showed significant activity against iNOS or NF-κB. This is the first report of biological activity of octulosonic acid derivatives toward multiple pathways related to inflammation and metabolic disorder. The reported anti-inflammatory, hypoglycemic, antiedemic, and antioxidant activities of Roman chamomile could be partly explained as due to the presence of these constituents.

Characterization of steroidal saponins from Dioscorea villosa and D. cayenensis using ultrahigh performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry.

Yam (Dioscorea spp.) is an important edible tuber plant used for medicinal purposes to promote health and longevity in Chinese tradition. Steroidal saponins were reported to be the major physiologically active constituents in yams. In this current work, the structural characteristics of steroidal saponins in methanolic extracts from dried rhizomes of two Dioscorea species (D. villosa L. and D. cayenensis Lam.) and dietary supplements have been identified and analyzed using UHPLC/QTOF-MS in both negative and positive ion modes. The fragmentation patterns of reference standards were determined and the steroidal saponins in the extracts were identified or tentatively characterized from their retention times and mass spectra. The fragments produced by collision-induced dissociation (CID) revealed the characteristic cleavage of glycosidic bonds, and the fragmentation pattern provided structural information about the sugars. Twenty-one saponins, including four tentatively identified compounds, were detected in the crude extracts of two Dioscorea species. These saponins can be used to distinguish D. villosa from D. cayenensis. For example, asperin and gracillin are found only in D. cayenensis, and dioscoreavilloside A and B and parvifloside are only found in D. villosa. This can be used to determine the presence or absence of D. villosa in commercial products, which may help determine the spiking of plant material, and/or prevent the use of potentially mislabeled or misidentified "Dioscorea" material. The analytical method also provided an alternative, fast method for quality control of Dioscorea species in dietary supplements. Principal component analysis showed that Dioscorea species and commercial products were easily distinguished. From this a partial least squares model was constructed to determine what species are in different products.

Chemical fingerprint analysis and quantitative determination of steroidal compounds from Dioscorea villosa, Dioscorea species and dietary supplements using UHPLC-ELSD.

Ultra high-performance liquid chromatography (UHPLC) with evaporative light scattering detection was used for the quantification of steroidal saponins and diosgenin from the rhizomes or tubers of various Dioscorea species and dietary supplements that were purported to contain Dioscorea. The analysis was performed on an Acquity UPLC™ system with an UPLC™ BEH Shield RP18 column using a gradient elution with water and acetonitrile. Owing to their low UV absorption, the steroidal saponins were observed by evaporative light scattering detection. The 12 compounds could be separated within 15 min using the developed UHPLC method with detection limits of 5-12 µg/mL with 2 µL injection volume. The analytical method was validated for linearity, repeatability, accuracy, limits of detection and limits of quantification. The relative standard deviations for intra- and inter-day experiments were <3.1%, and the recovery efficiency was 97-101%. The total content of standard compounds was found to be in the ranges 0.01-14.5% and 0.9-28.6 mg daily intake for dry plant materials and solid commercial preparations, respectively. UHPLC-mass spectrometry with a quadrupole mass analyzer and ESI source was used only for confirmation of the identity of the various saponins. The developed method is simple, rapid and especially suitable for quality control analysis of commercial products.

Screening of medicinal plants for PPARα and PPARγ activation and evaluation of their effects on glucose uptake and 3T3-L1 adipogenesis.

Medicinal plants are a rich source of ligands for nuclear receptors. The present study was aimed to screen a collection of plant extracts for PPARα/γ-activating properties and identify the active extract that can stimulate cellular glucose uptake without enhancing the adipogenesis. A reporter gene assay was performed to screen ethanolic extracts of 263 plant species, belonging to 94 families, for activation of PPARα and PPARγ. Eight extracts showed activation of PPARγ, while 22 extracts showed activation of PPARα. The extracts of five plants (Daphne gnidium, Illicium anisatum, Juniperus virginiana, Terminalia chebula, and Thymelaea hirsuta) showed activation of both PPARα and PPARγ and out of them, D. gnidium and T. hirsuta markedly increased PPARα/γ protein expression. All five extracts showed an increase in cellular glucose uptake. Of the five dual agonists, T. chebula and T. hirsuta did not show any increase in differentiation of 3T3-L1 preadipocytes, but I. anisatum caused an increase in adipogenesis, while D. gnidium and J. virginiana were toxic to adipocytes. The adipogenic effect of rosiglitazone was antagonized by T. chebula and T. hirsuta. It was concluded that T. hirsuta and T. chebula retain the property of elevating glucose uptake as PPARα/γ dual agonists without the undesired side effect of adipogenesis. This is the first report to reveal the PPARα/γ dual agonistic action and glucose uptake enhancing property of T. hirsuta and T. chebula.

Characterization of in vitro ADME properties of diosgenin and dioscin from Dioscorea villosa.

Dioscorea villosa (wild yam) is native to North America and has been widely used as a natural alternative for estrogen replacement therapy to improve women's health as well as to treat inflammation, muscle spasm, and asthma. Diosgenin and dioscin (glycoside form of diosgenin) are reported to be the pharmacologically active compounds. Despite the reports of significant pharmacological properties of dioscin and diosgenin in conditions related to inflammation, cancer, diabetes, and gastrointestinal ailments, no reports are available on ADME properties of these compounds. This study was carried out to determine ADME properties of diosgenin and dioscin and their effects on major drug metabolizing enzymes (CYP 3A4, 2D6, 2C9, and 1A2). The stability was determined in simulated gastric and intestinal fluids (SGF, pH 1.2 and SIF, pH 6.8), and intestinal transport was evaluated in Caco-2 model. Phase I and phase II metabolic stability was determined in human liver microsomes and S9 fractions, respectively. Quantitative analysis of dioscin and diosgenin was performed by UPLC-MS system. Dioscin degraded up to 28.3 % in SGF and 12.4 % in SIF, which could be accounted for by its conversion to diosgenin (24.2 %. in SGF and 2.4 % in SIF). The depletion of diosgenin in SGF and SIF was < 10 %. Diosgenin was stable in HLM but disappeared in S9 fraction with a half-life of 11.3 min. In contrast, dioscin was stable in both HLM and S9 fractions. Dioscin showed higher permeability across Caco-2 monolayer with no significant efflux, while diosgenin was subjected to efflux mediated by P-glycoprotein. Diosgenin and dioscin inhibited CYP3A4 with IC50 values of 17 and 33 µM, respectively, while other CYP enzymes were not affected. In conclusion, dioscin showed better intestinal permeability. Conversion of dioscin to diosgenin was observed in both gastric and intestinal fluids. No phase I metabolism was detected for both compounds. The disappearance of diosgenin in S9 fraction indicated phase II metabolism.

Implementing a "quality by design" approach to assure the safety and integrity of botanical dietary supplements.

Natural products have provided a basis for health care and medicine to humankind since the beginning of civilization. According to the World Health Organization (WHO), approximately 80% of the world population still relies on herbal medicines for health-related benefits. In the United States, over 42% of the population claimed to have used botanical dietary supplements to either augment their current diet or to "treat" or "prevent" a particular health-related issue. This has led to the development of a burgeoning industry in the U.S. ($4.8 billion per year in 2008) to supply dietary supplements to the consumer. However, many commercial botanical products are poorly defined scientifically, and the consumer must take it on faith that the supplement they are ingesting is an accurate representation of what is listed on the label, and that it contains the purportedly "active" constituents they seek. Many dietary supplement manufacturers, academic research groups, and governmental organizations are progressively attempting to construct a better scientific understanding of natural products, herbals, and botanical dietary supplements that have co-evolved with Western-style pharmaceutical medicines. However, a deficiency of knowledge is still evident, and this issue needs to be addressed in order to achieve a significant level of safety, efficacy, and quality for commercial natural products. The authors contend that a "quality by design" approach for botanical dietary supplements should be implemented in order to ensure the safety and integrity of these products. Initiating this approach with the authentication of the starting plant material is an essential first step, and in this review several techniques that can aid in this endeavor are outlined.

Quantitative determination of 10 phenylpropanoid and lignan compounds in Lancea tibetica by high-performance liquid chromatography with UV detection.

An HPLC method was developed for simultaneous determination of one phenylpropanoid glycoside, verbascoside (1), and nine lignans, including lantibeside (2), phillyrin (3), lantibeside B (4), lantibeside C (5), tibeticoside A (6), styraxjaponoside C (7), sylvatesmin (8), (+)-piperitol (9), and horsfieldin (10), from the Tibetan medicinal plant Lancea tibetica Hook. F. et Thoms. The analysis was performed within 45 min. The extraction method was optimized with different solvent systems. The HPLC method was validated for linearity, repeatability, accuracy, limits of detection, and limits of quantification. The limits of detection and limits of quantification of 10 analytes were found to be less than 0.1 and 0.5 µg/mL, respectively. The RSD for intra- and inter-day analyses was less than 4.2 %, and the recovery efficiency was 90-105 %. The method was used to analyze different populations of L. tibetica collected in China. HPLC profiles showed that the concentrations of analytes were different in samples collected from different areas of China. Verbascoside was the dominant component in three out of five plant samples; compounds 2, 3, 6, and 8 accounted for over 62 % yields in total lignan contents. The method is useful for identification, quality assurance, and quality control of L. tibetica and its related products.

Quantitative determination of triperpene saponins and alkenated-phenolics from Labisia pumila using an LC-UV/ELSD method and confirmation by LC-ESI-TOF.

This study describes the first analytical method for the determination of four triterpene saponins (ardisicrenoside B, ardisiacrispin A, 3- O- α- L-rhamnopyranosyl-(1 → 2)-ß-D-glucopyranosyl-(1 → 4)-α-L-arabinopynanosyl cyclamiretin A and ardisimamilloside H) and three alkenated-phenolics (irisresorcinol, belamcandol B, and demethylbelamcandaquinone B) from the leaves, leaves/stems, and roots of LABISIA PUMILA using an HPLC-UV-ELSD method. The separation was achieved using a reversed-phase (C-18) column, PDA and ELS detection, and a water/acetonitrile gradient as the mobile phase. The major triterpenoid (ardisiacrispin A) and irisresorcinol compounds were detected at a concentration as low as 10.0 and 0.2 µg/mL, respectively. Analysis of various samples showed considerable variation of 0.11-2.46 % for the major triterpenoid compound, ardisiacrispin A. LC-mass spectrometry method coupled with electrospray ionization (ESI) is described for the identification of compounds in plant samples. This method involved the use of the [M + Na]+ and [M + NH4]+ ions for compounds 1-4 in the positive ion mode with extractive ion chromatogram (EIC).

NMR fingerprinting for analysis of hoodia species and hoodia dietary products.

Hoodia gordonii, a succulent plant growing in African arid regions, is used as a botanical dietary supplement for weight loss. The increasing concerns on the quality and safety of Hoodia products call for the needs of more science-based information, as well as objective and efficient tools for inspection. In the present study, NMR fingerprinting and multivariate analysis techniques were applied for the identification, discrimination, and quality analysis of Hoodia plant materials and commercial products. Four Hoodia species, namely H. gordonii (five authenticated samples), H. currorii (one authenticated sample), H. parviflora (three authenticated samples), and H. rushii (one authenticated sample), were investigated; the chemicals and characteristic spectral signals that made most contributions for their differentiations were revealed. With the aid of NMR fingerprint analysis, ten Hoodia products sold on the dietary supplement market were assessed for their chemical composition and quality. The study demonstrated that the NMR fingerprinting approach could be a promising and efficient tool for the authentication of botanicals.

Automated high-throughput system to fractionate plant natural products for drug discovery.

The development of an automated, high-throughput fractionation procedure to prepare and analyze natural product libraries for drug discovery screening is described. Natural products obtained from plant materials worldwide were extracted and first prefractionated on polyamide solid-phase extraction cartridges to remove polyphenols, followed by high-throughput automated fractionation, drying, weighing, and reformatting for screening and storage. The analysis of fractions with UPLC coupled with MS, PDA, and ELSD detectors provides information that facilitates characterization of compounds in active fractions. Screening of a portion of fractions yielded multiple assay-specific hits in several high-throughput cellular screening assays. This procedure modernizes the traditional natural product fractionation paradigm by seamlessly integrating automation, informatics, and multimodal analytical interrogation capabilities.

Two new triterpene glycosides from Centella asiatica.

Phytochemical investigation of the leaves of Centella asiatica resulted in the isolation and characterization of eight triterpenes and/or saponins [which were characterized as 23- O-acetylmadecassoside (1), asiatic acid (2), madecassic acid (3), asiaticoside C (4), asiaticoside F (5), asiaticoside (6), madecassoside (7), and 23- O-acetylasiaticoside B (8)] together with sitosterol 3-O-beta-glucoside ( 9), stigmasterol 3- O- beta-glucoside (10), and querectin-3- O-beta-D-glucuronide (11). A new ursane-derived saponin (23- O-acetylmadecassoside) and a new oleanane-derived saponin (23- O-acetylasiaticoside B) were found as well. Structure elucidation was done by using spectroscopic techniques (HR-ESI-MS, 1D and 2D NMR), chemical methods, and comparative literature studies.

Flavonol glycosides from the south African medicinal plant Sutherlandia frutescens.

Phytochemical investigation of the leaves of Sutherlandia frutescens led to the isolation of four new 3-hydroxy-3-methylglutaroyl-containing flavonol glycosides, sutherlandins A-D ( 1- 4). Their structures were elucidated by chemical and spectroscopic methods as quercetin 3- O- beta- D-xylopyranosyl(1 --> 2)-[6- O-(3-hydroxy-3-methylglutaroyl)]- beta- D-glucopyranoside ( 1), quercetin 3- O- beta- D-apiofuranosyl(1 --> 2)-[6- O-(3-hydroxy-3-methylglutaroyl)]- beta- D-glucopyranoside ( 2), kaempferol 3- O- beta- D-xylopyranosyl(1 --> 2)-[6- O-(3-hydroxy-3-methylglutaroyl)]- beta- D-glucopyranoside ( 3), and kaempferol 3- O- beta- D-apiofuranosyl(1 --> 2)-[6- O-(3-hydroxy-3-methylglutaroyl)]- beta- D-glucopyranoside ( 4).

Scuteflorins A and B, dihydropyranocoumarins from Scutellaria lateriflora.

Two new dihydropyranocoumarins, scuteflorins A (1) and B (2), together with the known compounds decursin (3), chrysin (4), oroxylin A (5), wogonin (6), 5,7-dihydroxy-8,2'-dimethoxyflavone, dihydrochrysin, dihydrooroxylin A, lupenol, scutellaric acid, pomolic acid, ursolic acid, beta-sitosterol, daucosterol, and palmitic acid, were isolated from the aerial parts of Scutellaria lateriflora, commonly used as a dietary supplement. The structures of 1 and 2 were established by means of 1D and 2D NMR spectra as well as HRMS data. The absolute configuration of coumarins 1 and 2 was determined by comparison of experimental and theoretical calculated CD spectra. The cytotoxicity and antioxidant effects of the methanol extract of this plant and some of the constituent flavonoids were evaluated in vitro.

Diterpenoids from Casearia sylvestris.

Two highly oxygenated clerodane diterpenes casearins U (1) and V (2) and two ent-kaurane diterpene glucosides sylvestrisides A (3) and B (4) were isolated from the leaves of Casearia sylvestris, together with 13 known compounds. Their structures were established on the basis of 1D and 2D NMR spectroscopic analyses and comparison with literature data.

RP-HPLC determination of phenylalkanoids and monoterpenoids in Rhodiola rosea and identification by LC-ESI-TOF.

An HPLC method permitting the simultaneous determination of fourteen analytes (phenylalkanoids and monoterpenoids) from the roots of Rhodiola rosea was developed. A separation was achieved within 35 min using C(18) column material and a water-acetonitrile mobile phase, both containing a 0.05% phosphoric acid gradient system and a temperature of 53 degrees C. The method was validated for linearity, repeatability, limits of detection and limits of quantification. The limits of detection and limits of quantification of 14 phenylalkanoids and monoterpenoids were found to be 0.20-1.0 and 0.5-3.5 microg/mL, respectively. The wavelengths used for quantification of phenylalkanoids and monoterpenoids with a diode array detector were 205, 220 and 251 nm. The method was used to analyze the roots of two species of Rhodiola and commercial extracts of R. rosea and provides preliminary evidence of phytochemical differences between North American and Eurasian populations of R. rosea. LC-mass spectrometry coupled with electrospray ionization (ESI) interface method is described for the identification of phenylalkanoids and monoterpenoids in various Rhodiola samples. This method involved the use of the [M + H](+), [M + NH(4)](+) and [M + Na](+) ions in the positive ion mode with extractive ion monitoring (EIM).