Extraction of Galphimines from Galphimia glauca with Supercritical Carbon Dioxide.

The anti-depressive and anxiolytic effect of galphimine B (isolated from Galphimia glauca) has been demonstrated by researchers. Therefore, it is necessary to explore extraction techniques that produce materials with adequate quality for pharmaceutical applications. In this work, supercritical extractions of galphimines from Galphimia glauca were performed in the presence of carbon dioxide. Pressure, temperature, particle diameter, and flow rate effects were examined to explore the conditions with the highest yield and the concentration profile of galphimines in the studied interval. The identification of the nor-seco triterpenoids and galphimine B and E was carried out by HPLC analyses. The mathematical modeling of the extraction curves was attained by the approaches proposed by Sovová and Papamichail et al. According to results, the highest yield 2.22% was obtained at 323.15 K, 326 µm, 3 L/min, and 33.75 MPa. Meanwhile, the content of galphimine B in the extract was, on average, 19.5 mg·g-1.

Multidisciplinary Investigations on Galphimia glauca: A Mexican Medicinal Plant with Pharmacological Potential.

Galphimia glauca (Cav.) Kuntze is an important endemic plant species, which possesses many medicinal properties and has been used in the Mexican traditional medicine for its sedative, anxiolytic, anticonvulsant, antiasthmatic and antiallergic properties. The therapeutic properties of this plant are mainly due to the presence of diverse bioactive compounds such as flavonoids, triterpenoids, and phenolics. Several triterpenoids and flavonoids compounds have been isolated and identified. Modern studies have demonstrated many biological activities such as anti-inflammatory, antidiarrheal, gastroenteritis, antimalarial and cytotoxic activities. Nevertheless, many studies are restricted to the crude extract, and many bioactive compounds are yet to be identified and validated according to its traditional use. However, its commercial exploitation and use are highly limited due to the non-availability of enough plant material and lack of knowledge about its agronomical practices. Moreover, the misinterpretation and mislabeling of closely related species of the genus Galphimia Cav. as G. glauca or G. gracilis is a common problem for its rigorous scientific study and commercial exploitation. The present review provides comprehensive knowledge based on the available scientific literature. To the best of our knowledge, this is the first review on G. glauca. This comprehensive information will certainly provide a guide for the better understanding and utilization of G. glauca for its scientific and industrial exploitation.

Anti-inflammatory activity and chemical profile of Galphimia glauca.

Galphimia glauca, commonly known as "flor de estrella", is a plant species used in Mexican traditional medicine for the treatment of different diseases that have an acute or chronic inflammatory process in common. Aerial parts of this plant contain nor-seco-triterpenoids with anxiolytic properties, which have been denominated galphimines. Other compounds identified in the plant are tetragalloyl-quinic acid, gallic acid, and quercetin, which are able to inhibit the bronchial obstruction induced by platelet-activating factor. The objective of this work was to evaluate the anti-inflammatory effect of crude extracts from G. glauca and, by means of bioguided chemical separation, to identify the compounds responsible for this pharmacological activity. n-Hexane, ethyl acetate, dichloromethane, and methanol extracts showed an important anti-inflammatory effect. Chemical separation of the active methanol extract allowed us to identify the nor-seco-triterpenes galphimine-A (1) and galphimine-E (3) as the anti-inflammatory principles. Analysis of structure-activity relationships evidenced that the presence of an oxygenated function in C6 is absolutely necessary to show activity. In this work, the isolation and structural elucidation of two new nor-seco-triterpenes denominated as galphimine-K (4) and galphimine-L (5), together with different alkanes, fatty acids, as well as three flavonoids (17-19), are described, to our knowledge for the first time, from Galphimia glauca.

Pharmacokinetic study in mice of galphimine-A, an anxiolytic compound from Galphimia glauca.

The aim of this study was to obtain pharmacokinetic data for the anxiolytic compound galphimine-A (G-A) from Galphimia glauca. G-A is the most abundant anxiolytic compound in this plant, while Galphimine-E (G-E) is the most abundant galphimine, but inactive. G-E was transformed chemically into G-A. The pharmacokinetic study was carried out in ICR mice, which were orally administered a single 200 mg/kg dose of G-A. Samples of blood and brain were taken at different times after administration of G-A. Previously, we established the validation of methods for determining the concentration of G-A. The G-A was detected in plasma 5 min after oral administration, and its concentration reached 2.47 µg/mL. Data from concentration-time curves allowed us to establish the main pharmacokinetic parameters in two models: one- and/or two-compartment. C(max) values were 3.33 and 3.42 µg/mL respectively, likewise AUC(0→1440 min) were 1,951.58 and 1,824.95 µg/mL·min. The G-A in brain tissue was noted to cross the blood-brain barrier, reaching C(max) 2.74 µg/mL, T(max) 81.6 min, and then drop gradually to 0.32 µg/mL detected at 24 h. The presence of G-A in brain tissue, confirmed that this anxiolytic compound can access the target organ and acts directly on the CNS.

Triterpenes G-A and G-E from Galphimia glauca with anti-inflammatory and anti-arthritic activity in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Galphimia glauca is a medicinal plant that treats inflammatory and anti-rheumatic problems. Its anti-inflammatory capacity has been reported pharmacologically, attributed to the triterpenes G-A and G-E. AIM: The objective of the present work was to measure the anti-inflammatory and immunomodulatory effect of the methanolic extract (GgMeOH) of Galphimia glauca and the isolated galphimines G-A and G-E, first in an acute test of plantar edema with carrageenan, and later in the model of experimental-induced arthritis with CFA. The effect was measured by quantifying joint inflammation, the concentration of pro- (TNF-α, IL-6, IL-17) and anti-inflammatory (IL-10, and IL-4) cytokines, and the ADA enzyme in joints, kidneys, and spleen from mice with experimental arthritis. METHOD: The extract and the active triterpenes were obtained according to established methods using different chromatographic techniques. Female ICR strain mice were subjected to intraplantar administration with carrageenan and treated with different doses of GgMeOH, G-A, and G-E; edema was monitored at different times. Subsequently, the concentration of TNF-a and IL-10 in the spleen and swollen paw was quantified. Meloxicam (MEL) was used as an anti-inflammatory control drug. The most effective doses of each treatment were analyzed using a complete Freunds adjuvant (CFA)-induced experimental arthritis model. Joint inflammation was followed throughout the experiment. Ultimately, the concentration of inflammation markers, oxidant stress, and ADA activity was quantified. In this experimental stage, methotrexate (MTX) was used as an antiarthritic drug. RESULTS: Treatments derived from G. glauca, GgMeOH (DE50 = 158 mg/kg), G-A (DE50 = 2 mg/kg), and G-E (DE50 = 1.5 mg/kg) caused an anti-inflammatory effect in the plantar edema test with carrageenan. In the CFA model, joint inflammation decreased with all natural treatments; GgMeOH and G-A inhibited the ADA enzyme in all organs analyzed (joints, serum, spleen, left and right kidneys), while G-E inhibited the enzyme in joints, serum, and left kidney. CFA caused an increase in the weight index of the organs, an effect that was counteracted by the administration of G. glauca treatments, which also modulate the response to the cytokines analyzed in the different organs (IL-4, IL-10, IL-17, IL-6, and TNF- α). CONCLUSION: It is shown, for the first time, that the GgMeOH extract and the triterpenes G-A and G-E of Galphimia glauca have an anti-arthritic effect (anti-inflammatory, immunomodulatory, antioxidant, and ADA inhibitor), using an experimental arthritis model with CFA. Therefore, knowledge of the plant as a possible therapeutic agent for this rheumatic condition is expanding.

Therapeutic effectiveness of Galphimia glauca vs. lorazepam in generalized anxiety disorder. A controlled 15-week clinical trial.

UNLABELLED: Galphimia glauca Cav. has demonstrated anxiolytic activity attributable to nor-seco-triterpenes denominated galphimines, the most active of which is galphimine-B. Galphimine-B inhibits ventral tegmental area dopaminergic neurons and interacts with the serotoninergic system of the dorsal hippocampus. A previous clinical study that administered a G. glauca herbal medicinal product for 4 weeks evidenced high percentages of therapeutic effectiveness and safety in patients with generalized anxiety disorder. Based on the previous findings, the goal of the present study was to evaluate the effectiveness, safety, and tolerability of G. glauca herbal medicinal product administered during 15 weeks in patients with generalized anxiety disorder. STUDY DESIGN: double-blind, randomized, lorazepam-controlled clinical trial. STUDY SUBJECTS: adult males and females, ambulatory, diagnosed with generalized anxiety disorder, with 20 or more points on the Hamilton anxiety scale, without data of depression, and without anxiolytic treatment in the previous month. Interventions were as follows. Experimental treatment: G. glauca herbal medicinal product in capsules containing the dry extract of G. glauca standardized in 0.175 mg of galphimine-B, one or two capsules twice a day, during 12 weeks plus 3 withdrawal weeks, and control treatment: lorazepam 0.5 mg with the same presentation and posology. PRIMARY OUTCOME: anxiolytic effectiveness (≥ 50 % reduction of initial Hamilton anxiety scale score). SECONDARY OUTCOMES: tolerability and safety. One hundred ninety-one patients initiated the study with 94 in the experimental group. One hundred four patients concluded the study, 51 of these in the experimental group. Anxiolytic effectiveness, measured as 0 in a negative case and as 1 in a positive case, was assessed 593 times in the experimental group and 631 in the control; the mean effectiveness observed was 0.686 ± 0.019 vs. 0.588 ± 0.019 (repeated-measures ANOVA; p = 0.0003). In the same way, G. glauca-herbal medicinal product diminished the score in the Hamilton anxiety scale to 11.51 ± 8.27 points and lorazepam to 12.40 ± 8.07 points (repeated-measures ANOVA; p = 0.05). The tolerability analysis, which comprised patients who concluded the treatment plus 11 patients who withdrew due to adverse reactions did not show differences between treatments (p = 0.35), nor did therapeutic safety demonstrate differences between groups (p = 0.21). There were no cases of tolerance, intoxication, dependence, or suppression syndrome. We concluded that G. glauca herbal medicinal product, standardized in 0.175 mg of galphimine-B and administered for 15 weeks to patients with generalized anxiety disorder, showed greater anxiolytic effectiveness than that obtained with lorazepam, with high percentages of therapeutic tolerability and safety.

Further galphimines from a new population of Galphimia glauca.

Both DNA barcoding and phylogenetic data of the studied botanical material suggested the existence a new population of Galphimia glauca. Their leaves afforded three new nor-3,4-seco-friedelanes named galphimines M-O, together with known galphimines D, E, G, and I. Galphimines M and N possess bicyclic orthoacetates which are the first examples of orthoesters found in the Malpighiaceae family, while galphimine O has a 27,20-δ-lactone moiety. The structures elucidation followed from spectroscopic means and the absolute configuration followed from single crystal X-ray diffraction analyses. Tests for antibacterial and antifungal activities of galphimines N and M showed no promising effects.

Glaucacetalin E and galphimidin B from Galphimia glauca and their anxiolytic activity.

ETHNOPHARMACOLOGICAL RELEVANCE: Galphimia glauca is a Mexican medicinal plant used to treat anxiety, fear, phobia and stress as it possesses sedative properties which produce a calming effect. Although some chemical and pharmacological studies have already been carried out on G. glauca, there are still new chemical entities from this plant whose anxiolytic activity should be established. AIM OF THE STUDY: To validate the use of G. glauca growing in Cuernavaca, Morelos, as an anti-stress agent, through the purification and structural identification of its extracts' chemical constituents; the analysis of the biogenetic relationship of its chemical compounds, and its biological evaluation to demonstrate its traditional use as anxiolytic agents. MATERIALS AND METHODS: The structures of all isolated compounds were established based on their spectroscopic and spectrometric data. The structure of compound 2 was corroborated through X-Ray. The anxiolytic and sedative-like activities were assessed by the open-field, hole-board and exploration cylinder test. RESULTS: The nor-triterpenes glaucacetalin E (1) and galphimidin B (2) were isolated for the first time along with seven other known compounds, one of them galphimidin (3), from the CHCl3 fraction of the aerial parts of Galphimia glauca. The biogenesis of the natural nor-triterpenes isolated from Galphimia glauca is delineated for the first time starting from the taraxasteryl cation. Oral administration of CHCl3 fraction and 1-3 compounds produced significant attenuation in the anxiety-response in cylinder activity, decrease in the ambulatory activity and in head dipping when compared to the vehicle. However, only the extract enhanced the pentobarbital-induced hypnosis. Diazepam was used as a positive control. CONCLUSION: Our results suggest that G. glauca growing in Cuernavaca, Morelos, exerts anxiolytic-like activity due to the presence of the nor-triterpenes 1-3. These results reinforce the potential use of this species in the treatment of anxiety.

Galphimine-B Standardized Extract versus Alprazolam in Patients with Generalized Anxiety Disorder: A Ten-Week, Double-Blind, Randomized Clinical Trial.

Galphimine-B (G-B), a compound isolated from Galphimia glauca, has been shown to possess important anxiolytic activity. In this study, we evaluated the effectiveness and tolerability of a G-B standardized extract (experimental treatment) that was administered daily for 10 weeks in patients with moderate or severe Generalized Anxiety Disorder (GAD). Alprazolam was used as control treatment and administered under the same conditions. A total of 167 patients were included. At the start of the study, the severe anxiety condition prevailed, with an average on the Hamilton Anxiety Scale of 35.1 ± 8.8 and 35.8 ± 8.1 points in the control and experimental groups, respectively. After the 10 weeks of administration, the average was reduced in the control group to 4.6 ± 6.5 points and in the experimental group to 3.5 ± 5.5 points. Therapeutic success in the control group was 85.7% and in the experimental group, 92.0%. A high proportion of patients (22.2%) treated with Alprazolam manifested daytime sleepiness, while in the group treated with the G-B standardized extract, daytime sleepiness was found in 4.7%. In conclusion, a G-B standardized extract demonstrated therapeutic effectiveness in patients with GAD, without exhibiting significant difference with Alprazolam, but showing fewer cases of daytime sleepiness. The trial was registered at http://clinicaltrials.gov by identifier: NCT03702803.

DNA barcoding and TLC as tools to properly identify natural populations of the Mexican medicinal species Galphimia glauca Cav.

Galphimia glauca is a plant that is endemic to Mexico and has been commonly used since pre-Hispanic times to treat various illnesses, including central nervous system disorders and inflammation. The first studies investigating a natural population of G. glauca in Mexico showed that the plant has anxiolytic and sedative activities in mice and humans. The plant's bioactive compounds were isolated and identified, and they belong to a family of nor-secofriedelanes called galphimines. The integration of DNA barcoding and thin-layer chromatography analysis was performed to clarify whether the botanical classification of the populations in the study, which were collected in different regions of Mexico, as G. glauca was correct or if the populations consist of more than one species of the genus Galphimia. We employed six DNA barcodes (matK, rbcL, rpoC1, psbA-trnH, ITS1 and ITS2) that were analyzed individually and in combination and then compared each other, to indicate differences among the studied populations. In the phylogenetic analysis, ITS1 and ITS2 markers as well as the combination of all DNA regions were the most efficient for discriminating the population studied. The thin-layer chromatography analysis exhibited four principal chemical profiles, one of which corresponded to the populations that produced galphimines. DNA barcoding was consistent and enabled us to differentiate the populations that produce galphimines from those that do not. The results of this investigation suggest that the studied populations belong to at least four different species of the genus Galphimia. The phylogenetic analysis and the thin-layer chromatography chemical profiles were convenient tools for establishing a strong relationship between the genotype and phenotype of the studied populations and could be used for quality control purposes to prepare herbal medicines from plants of the genus Galphimia.

Toxicological and cytotoxic evaluation of standardized extracts of Galphimia glauca.

Galphimia glauca Cav (Malpighiaceae) has been widely used in Mexican traditional medicine as a remedy for the treatment of mental disorders, principally as a sedative and tranquilizer. The sedative activity of extracts obtained from this plant has been demonstrated with different neuropharmacological models. Different triterpenes, known as galphimines, have been identified from the active extract. Galphimine-B (G-B) possesses anxiolytic activity and is able to selectively inhibit discharges of dopaminergic neurons in the ventral tegmental area in rats. Nevertheless, there have been no toxicological investigations carried out with products obtained from this plant. In this work three different extracts (aqueous, ethanolic, and methanolic) of Galphimia glauca, standardized in the content of three galphimines, were evaluated for their behavioral and pharmaco-toxicological effects. After administering the extracts to mice for 28 days (2.5g/kg, p.o.), no deaths were found and the histopathological analysis of different organs did not show alterations; only the behavioral parameters analyzed showed a diminution of spontaneous activity. The administration of these extracts for 56 days (same doses and route) in mice did not cause any changes in the biochemical parameters that evaluate liver function. On the other hand, no cytotoxic effects were found on KB, UISO, and OVCAR-5 transformed cell lines, but all extracts specifically inhibited colon cancer cell line growth with an ED(50) lower than 2microg/ml. The extracts were also evaluated in genotoxicity tests in vitro (250, 100 and 50microg/ml), which demonstrate that none of the three extracts from Galphimia glauca showed a genotoxic effect.

Solid-phase extraction of galloyl- and caffeoylquinic acids from natural sources (Galphimia glauca and Arnicae flos) using pure zirconium silicate and bismuth citrate powders as sorbents inside micro spin columns.

Galloyl- and caffeoylquinic acids are among the most important pharmacological active groups of natural compounds. This study describes a pre-step in isolation of some selected representatives of these groups from biological samples. A selective solid-phase extraction (SPE) method for these compounds may help assign classes and isomer designations within complex mixtures. Pure zirconium silicate and bismuth citrate powders (325 mesh) were employed as two new sorbents for optimized SPE of phenolic acids. These sorbents possess electrostatic interaction sites which accounts for additional interactions for carbon acid moieties as compared to hydrophilic and hydrophobic sorbents alone. Based on this principle, a selective SPE method for 1,3,4,5-tetragalloylquinic acid (an anti-HIV and anti-asthamatic agent) as a starting compound was developed and then deployed upon other phenolic acids with success. The recoveries and selectivities of both sorbents were compared to most commonly applied and commercially available sorbents by using high performance liquid chromatography. The nature of interaction between the carrier sorbent and the acidic target molecules was investigated by studying hydrophilic (silica), hydrophobic (C18), mixed-mode (ionic and hydrophobic: Oasis(®) MAX) and predominantly electrostatic (zirconium silicate) materials. The newly developed zirconium silicate and bismuth citrate stationary phases revealed promising results for the selective extraction of galloyl- and caffeoylquinic acids from natural sources. It was observed that zirconium silicate exhibited maximum recovery and selectivity for tetragalloylquinic acid (84%), chlorogenic acid (82%) and dicaffeoylquinic acid (94%) among all the tested sorbents.

A homoeopathic proving of Galphimia glauca.

INTRODUCTION: Homoeopathic provings are a fundamental concept in homoeopathy. The aim of this study was to record the symptoms produced by a homoeopathic drug compared to placebo. METHODS: Randomised, double-blind, placebo-controlled trial with a 1-week baseline, 4-week proving, and 2-week post-observational period. SUBJECTS: 15 healthy physicians and medical students volunteered as provers; 11 were randomised to verum and 4 to placebo. Proving substance: Galphimia glauca C12 compared to placebo; maximum intake of 5 days. OUTCOME MEASURES: Proving symptoms according to ICCH definition and the number of proving symptoms. The proving symptoms were analysed qualitatively using the Boenninghausen method. RESULTS: A total of 682 symptoms were observed in both groups. Galphimia glauca provers experienced states of exhaustion, weakness, lack of concentration, feelings of confusion, dryness of mouth, lacrimation, and burning sensation in the eyes. Two provers experienced an amelioration of their allergic rhinitis. Proving symptoms were completely reversible. The statistical analysis showed more ICCH proving symptoms for placebo (mean 72.3 +/- SD 37.3) than for Galphimia (35 +/- 24.2), but the group difference was not significant (95% confidence interval, -78 to 1, p = 0.097). DISCUSSION: Although statistical analysis showed no significant group differences, we observed specific symptoms under Galphimia glauca that correspond to those seen in clinical studies of phytotherapeutic preparations, including relaxing, sedative, anxiolytic, and anti-allergic effects. CONCLUSION: Our results confirm the toxicological and clinical effects of Galphimia glauca compared to placebo, but the ICCH criteria for proving symptoms were not suitable to distinguish between specific and unspecific symptoms.

Metabolic profiling of the Mexican anxiolytic and sedative plant Galphimia glauca using nuclear magnetic resonance spectroscopy and multivariate data analysis.

Galphimia glauca is popularly employed in Mexico for the treatment of central nervous system disorders. Pharmacological and phytochemical studies have resulted in the identification of the anxiolytic and sedative principle consisting of a mixture of nor-secofriedelanes, named the galphimine series (1 - 9). These active constituents were found in plants collected in the vicinity of a restricted region in Central Mexico, where this species is abundant. A metabolic profiling carried out by means of 1H-NMR spectroscopy and multivariate data analysis was applied to crude extracts from wild plant populations, collected from six different locations as a quality control assessment, in order to differentiate their chemical profile. Principal component analysis (PCA) of the 1H-NMR spectra revealed clear variations among the populations, with two populations out of the six studied manifesting differences, when the principal components PC-1 and PC-2 were analyzed. These two PCs permitted the differentiation of the various sample populations, depending on the presence of galphimines. This information consistently correlated with the corresponding HPLC analysis. The neuropharmacological effects of the crude extracts were evaluated by using ICR mice in the elevated plus maze, as well as the sodium pentobarbital-induced hypnosis models. Both assays demonstrated anxiolytic and sedative responses only among those sample populations which had previously been differentiated by PC-1. Partial least square regression-discriminant analysis (PLS-DA) also confirmed a strong correlation between the observed effects and the metabolic profiles of the plants. The overall results of this study confirm the benefits of using metabolic profiling for the in silico analysis of active principles in medicinal plants.

Anxiolytic and antidepressant-like activity of a standardized extract from Galphimia glauca.

An infusion prepared with aerial parts from Galphimia glauca has been widely used in Mexican traditional medicine as a remedy for nervous excitement. The sedative activity of a methanolic extract from this plant has been demonstrated by neuropharmacological tests. This effect was attributed to the nor-secotriterpene named galphimine B (GB). In the present work, the anxiolytic and antidepressant-like effects of G. glauca methanolic extract (standardized on GB content, 8.3mg/g) were assayed by using the elevated plus-maze, light-dark test and the forced swimming paradigm, on ICR albino mice. This extract, administered orally, three times (24, 18 and 1h before the test), and in different doses (125, 250, 500, 1,000 and 2,000 mg/kg) was able to increase significantly (p<0.05) the number of entries, as well as the time spent in the open arms of the elevated plus-maze, indicating an anxiolytic-like effect. A similar effect was observed in the light-dark paradigm test, the time spent in the light box was increased in treated mice. Nevertheless, this treatment was unable to change any parameter in the forced swimming test. Altogether, these results suggest an anxiolytic-like effect to the methanolic standardized extract of G. glauca on ICR inbred mice.

Anxiolytic effect of natural galphimines from Galphimia glauca and their chemical derivatives.

The anxiolytic effects of galphimine B (1), galphimine A (2), and galphimine E (3), natural nor-secofriedelanes isolated from Galphimia glauca, as well as derivatives obtained by acetylation (4), hydrogenation of the C-1/C-2 double bond (5), basic hydrolysis followed by hydrogenation of the C-1/C-2 double bond (6), and deacetylation (7) of galphimine E (3), were evaluated on ICR mice exposed to the elevated plus-maze test. This study also included the evaluation of a galphimines-rich fraction (GRF) with a known concentration of 1-3, obtained from the dry leaves of G. glauca. Intraperitoneal administration of 15 mg/kg of 1, 2, 6, and GRF (1 h before testing) caused an anxiolytic-like effect in the animals, increasing significantly (p <0.001) the percentage of time of permanence and the number of crossings toward the open arms of the plus-maze. No activity was detected after administration of compounds 3, 4, 5, and 7. These results showed that GRF had activity similar to the most active pure galphimines (1 and 2) and that, like for the spasmolytic activity previously reported, the main determining factor responsible for the anxiolytic activity of the compounds was the presence of free hydroxyl groups at C-4, C-6, and C-7 and the presence of the double bond in the A ring.

In vitro propagation of Galphimia glauca and content of the sedative compound galphimine-B in wild and micropropagated plants.

An in vitro micropropagation protocol is described for Galphimia glauca Cav. (Malpighighiaceae). Multiple shoots were formed in vitro from axillary bud explants inoculated on MS medium supplemented with indole-3-acetic acid (IAA) and kinetin (KN) combinations. A maximum of 20 shoots was obtained from a single bud in a 60-day culture period. In vitro-grown shoots were successfully rooted and transferred to field conditions (90 % survival). The sedative triterpenoid galphimine-B (1) content of micropropagated plants transferred to field conditions was similar to that of wild plants. Our results suggest that the in vitro propagation protocol described here will have positive effects on conservation of natural resources as well as on adequate techniques for multiplication of an important Mexican medicinal plant.

Isolation of nor-secofriedelanes from the sedative extracts of Galphimia glauca.

Preparative-scale recycling HPLC was used for the complete resolution of a complex mixture of nor-secofriedelanes into five major peaks (I-V) from the sedative methanolic extracts prepared from the aerial parts of Galphimia glauca. Argentation chromatography was used to show peaks I, II, IV, and V to be mixtures of isomers around the E-ring double bond, represented by the endocyclic C-20, C-21 double-bond isomers, galphimines A (3), B (1), D (4), and E (2), and the C-20, C-29 exocyclic forms, galphimines F-I (5-8). Galphimine C (9), isolated from peak III, corresponded to the C-19, C-20 double-bond isomer of the previously known major sedative constituent galphimine B. The characterization of all the new triterpenes (3-9) was performed primarily by high-field NMR spectroscopy. Comparison between experimental and calculated (1)H-(1)H vicinal coupling constants and the analysis of molecular mechanics structures revealed that the ring B of these compounds exists in a boatlike conformation. The absolute configuration for the stereogenic carbinol center at C-4 was established by the application of the Mosher ester derivatization technique carried out in NMR tubes.

Assessment of the antiprotozoal activity of Galphimia glauca and the isolation of new nor-secofriedelanes and nor-friedelanes.

Four new terpenoids, comprising three nor-secofriedelanes (1-3) and one nor-friedelane (4), were isolated from Galphimia glauca, together with the known flavonol quercetin and the sterols stigmasterol and sitosterol 3-O-beta-D-glucoside. The structure elucidation of the new isolates was conducted by 1D and 2D NMR techniques. Compounds 1-4 were given the trivial names galphin A, galphin B, galphin C, and galphimidin, respectively. All isolates were tested for in vitro antiprotozoal and cytotoxic activities. Quercetin was the only substance isolated that showed any antiprotozoal activity, and this was weak; the IC(50) values were 14 microM against Plasmodium falciparum K1, 13.2 microM against Trypanosoma brucei brucei, and 63.8 microM against Leishmania donovani. Quercetin was found to be inactive against KB cells (IC(50) = 295.8 microM).