Three new alkaloids isolated from the stem tuber of Pinellia pedatisecta.

The present study was designed to determine the chemical constituents of the stem tuber of Pinellia pedatisecta. The chemical constituents were isolated and purified by various chromatographic techniques, and their structures were elucidated on the basis of physicochemical properties and spectral data. Three new alkaloids (compounds 1, 2, and 3) were obtained and identified as 9-((5-methoxypyridin-2-yl)methyl)-9H-purin-6-amine (1), 4-(2-(2, 5-dioxopyrrolidin-1-yl)ethyl)phenyl acetate (2), and N-(9-((5-methoxypyridin-2-yl)methyl)-9H-purin-6-yl)acetamide (3). These compounds were evaluated for their cytotoxicity against human cervical cancer HeLa cells. Compounds 1 and 3 significantly inhibited the proliferation of HeLa cells with IC50 values being 3.02 ± 0.54 and 7.16 ± 0.62 µmol·L-1, respectively.

Pharmacokinetic Studies of Ganoderic Acids from the Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes), by LC-MS/MS.

Ganoderma lucidum is a famous medicinal mushroom that has been widely used in clinical practice and as a dietary supplementa. The triterpenoid ganoderic acids are the main constituents of G. lucidum. To determine the pharmacokinetic characteristics of ganoderic acids, we developed and validated a sensitive and selective liquid chromatography-tandem mass spectrometry method to determine simultaneously the concentration of 4 representative ganoderic acids in rat plasma after oral administration of the extract from G. lucidum. Because of the similarity of their chemical structures, the 4 components exhibited similar pharmacokinetic behaviors in some aspects. However, some of the pharmacokinetic parameters and the reabsorption peaks in the plasma concentration-time curves of ganoderic acids B and E after oral administration of the extract were different from those of ganoderic acids D and A because of the metabolic transformation among the ganoderic acids. These results increase our knowledge about the use of G. lucidum.

Characterization and profiling of phenolic amides from Cortex Lycii by ultra-high performance liquid chromatography coupled with LTQ-Orbitrap mass spectrometry.

Cortex Lycii, the root bark of Lycium chinense Mill. or Lycium barbarum L., is a frequently used traditional Chinese medicine. Phytochemical studies have shown that phenolic amides are not only characteristic compounds but also abundant ones in this plant. In the present study, an effective method was developed for structural characterization of phenolic amides from Cortex Lycii by ultra-high performance liquid chromatography coupled with linear ion trap Orbitrap tandem mass spectrometry. The fragmentation of 14 compounds including six cinnamic acid amides, six neolignanamides, and two lignanamides were studied systematically for the first time. It was found that, in the positive ion mode, neutral loss of the tyramide moiety (137 Da) or N-(4-aminobutyl)acetamide moiety (130 Da) were characteristic for these compounds. At least 54 phenolic amides were detected in the extract and 48 of them were characterized, among which 14 known compounds were identified unambiguously by comparing the retention time and mass spectra with those of reference compounds, and 34 components were tentatively identified based on the fragmentation patterns, exact mass, UV spectra, as well as retention time. Fifteen compounds were characterized as potential new ones. Additionally, the developed method was applied to analyze eight batches of samples collected from the northwest of China, and it was found that cinnamic acid amides were the main type of phenolic amides in Cortex Lycii. In conclusion, the identification of these chemicals provided essential data for further phytochemical studies, metabolites identification, and the quality control of Cortex Lycii.

Anti-inflammatory diterpenoids from the root bark of Acanthopanax gracilistylus.

Five new ent-pimarane (1-3, 7, and 8) and three new ent-kaurane diterpenoids (4-6) and a new oleanane triterpene acid (9), together with 22 known compounds, were isolated from the root bark of the medicinal herb Acanthopanax gracilistylus. The structures of 1-9 were established based on the interpretation of high-resolution MS and 1D- and 2D-NMR data. The absolute configurations of 7 and 11 were determined by single-crystal X-ray diffraction and electronic circular dichroism analysis. Compounds 7 and 8 represent rare naturally occurring structures based on the devinyl ent-pimarane skeleton. Compounds 3, 10, 14, 16, and 17 exhibited potent inhibitory effects on the release of interleukin-1ß (IL-1ß), interleukin-8 (IL-8), and tumor necrosis factor (TNF-α) in lipopolysaccharide-stimulated peripheral blood mononuclear cells.

Activity-guided isolation of NF-κB inhibitors and PPARγ agonists from the root bark of Lycium chinense Miller.

ETHNOPHARMACOLOGICAL RELEVANCE: The root bark of Lycium chinense Miller, Lycii radicis cortex, has been used in traditional Chinese medicine (TCM) to treat different inflammation-related symptoms, such as diabetes mellitus. The pro-inflammatory transcription factor nuclear factor kappa B (NF-κB) is a key regulator of inflammation, while the transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) is a key modulator of genes involved in diabetes development. To identify putative active compound(s) from Lycii radicis cortex inhibiting NF-κB or activating PPARγ. MATERIAL AND METHODS: Using activity-guided fractionation, six extracts with different polarity, isolated fractions, and purified compounds from Lycii radicis cortex were tested for NF-κB inhibition and PPARγ activation in vitro. The structure of the purified compounds was elucidated by NMR and MS techniques. RESULTS: The ethyl acetate extract and the methanol extract of Lycii radicis cortex suppressed tumor necrosis factor alpha (TNF-α)-induced activation of NF-κB, while the dichloromethane extract activated PPARγ. Nine phenolic amide analogues, including trans-N-(p-coumaroyl)tyramine (1), trans-N-feruloyltyramine (2), trans-N-caffeoyltyramine (3), dihydro-N-caffeoyltyramine (4), three neolignanamides (5-7), and two lignanamide (8, 9), were isolated and their inhibitory potential on NF-κB was determined (1-4 were also contained in water decoction). Two of the nine isolated phenolic amides inhibited TNF-α-induced NF-κB activation. Trans-N-caffeoyltyramine was verified as the key component responsible for the NF-κB inhibition with an IC50 of 18.4µM in our cell-based test system. Activation of PPARγ was attributed to a palmitic-acid enriched fraction which displayed concentration-dependent effect ablated upon co-treatment with the PPARγ antagonist T0070907. CONCLUSIONS: Phenolic amides were confirmed as main components from Lycii radicis cortex responsible for NF-κB inhibition. Fatty acids were identified as the major plant constituent responsible for the PPARγ activation. Structure-activity relationship analysis suggests that the NF-κB inhibitory activity of trans-N-caffeoyltyramine may be attributed to its Michael acceptor-type structure (α,ß-unsaturated carbonyl group). The data of this study contribute to a better understanding of the molecular mechanism of action of Lycii radicis cortex extracts in the context of inflammation.

Metabolomic analysis of anti-hypoxia and anti-anxiety effects of Fu Fang Jin Jing Oral Liquid.

BACKGROUND: Herba Rhodiolae is a traditional Chinese medicine used by the Tibetan people for treating hypoxia related diseases such as anxiety. Based on the previous work, we developed and patented an anti-anxiety herbal formula Fu Fang Jin Jing Oral Liquid (FJJOL) with Herba Rhodiolae as a chief ingredient. In this study, the anti-hypoxia and anti-anxiety effects of FJJOL in a high altitude forced-swimming mouse model with anxiety symptoms will be elucidated by NMR-based metabolomics. METHODS: In our experiments, the mice were divided randomly into four groups as flatland group, high altitude saline-treated group, high altitude FJJOL-treated group, and high altitude diazepam-treated group. To cause anxiety effects and hypoxic defects, a combination use of oxygen level decreasing (hypobaric cabin) and oxygen consumption increasing (exhaustive swimming) were applied to mice. After a three-day experimental handling, aqueous metabolites of mouse brain tissues were extracted and then subjected to NMR analysis. The therapeutic effects of FJJOL on the hypobaric hypoxia mice with anxiety symptoms were verified. RESULTS: Upon hypoxic exposure, both energy metabolism defects and disorders of functional metabolites in brain tissues of mice were observed. PCA, PLS-DA and OPLS-DA scatter plots revealed a clear group clustering for metabolic profiles in the hypoxia versus normoxia samples. After a three-day treatment with FJJOL, significant rescue effects on energy metabolism were detected, and levels of ATP, fumarate, malate and lactate in brain tissues of hypoxic mice recovered. Meanwhile, FJJOL also up-regulated the neurotransmitter GABA, and the improvement of anxiety symptoms was highly related to this effect. CONCLUSIONS: FJJOL ameliorated hypobaric hypoxia effects by regulating energy metabolism, choline metabolism, and improving the symptoms of anxiety. The anti-anxiety therapeutic effects of FJJOL were comparable to the conventional anti-anxiety drug diazepam on the hypobaric hypoxia mice. FJJOL might serve as an alternative therapy for the hypoxia and anxiety disorders.

Metabolite identification of crude extract from Ganoderma lucidum in rats using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

The metabolism of traditional Chinese medicine is very complicated and has been a great challenge. In the present paper, a new strategy was established to study the metabolism of crude extract from Ganoderma lucidum using the highly separative and sensitive ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. Based on the investigation of the metabolism of five representative single compounds (ganoderic acid), a total of 90 metabolites were identified from the bile sample after oral administration of the crude extract. Among them, 21 compounds were identified by comparison with the reference standards, the other unknown metabolites were tentatively characterized by interpretation of the high resolution low collision energy and high collision energy mass spectra using the fragmentation rules. The metabolic characteristics and "soft spots" of the ganoderic acids were revealed. After being absorbed, the ganoderic acids from the extract could undergo extensive phases I and II metabolism in rat before excreted into the bile. The main ganoderic acids could transform from one to another through reduction, oxidation, deacetylation and desaturation reactions. Other metabolic transformation included hydroxylation, sulfation and glucuronidation. The total tendency was that the low polar ganoderic acids were transformed into the high polar metabolites to eliminate from the organism. The metabolic "soft spots" of the ganoderic acids were 3,7,15,23-carbonyl groups (or hydroxyl groups), angular methyl groups, 20(22)-double bond, 12-acetoxyl group and 26-carboxylic acid moiety. These results are considered to be important for the further investigation of G. lucidum.

An integrated exact mass spectrometric strategy for comprehensive and rapid characterization of phenolic compounds in licorice.

RATIONALE: Licorice (Gancao) is derived from the dried roots and rhizomes of Glycyrrhiza species (Leguminosae) and appears as a component herb in about 60% of traditional Chinese medicine (TCM) prescriptions. Modern pharmacological studies have shown that flavonoids are one class of the major components responsible for the bioactivities of licorice. Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/QTOF MS) has proven to be a powerful tool for rapid profiling and identification of natural products in complex herbal medicines. METHODS: A UPLC/QTOF MS method was established for the first time for profiling and structural characterization of the phenolic compounds (most of them flavonoids) in licorice. The combined use of data-independent acquisition (MS(E) ) and data-dependent acquisition (DDA) was illustrated. RESULTS: Fifteen flavonoid reference compounds were used to explore the fragmentation pathways. Compound identification was based upon the exact mass, general fragmentation behaviors, retention times, UV absorption, and the related botanical biogenesis. As a result, a total of 51 compounds were characterized, three of which were reported for the first time. CONCLUSIONS: The LC/MS analysis for each injection took less than 9 min. The developed method is fast, accurate and reliable due to its high resolution and high efficiency characteristics as a result of combining both UPLC separation and QTOF exact mass measurement.

Elution-extrusion counter-current chromatography separation of two new benzyl ester glucosides and three other high-polarity compounds from the tubers of Pleione bulbocodioides.

INTRODUCTION: The tubers of Pleione bulbocodioides (Franch.) Rolfe, with gastrodin and benzyl ester glucosides as main components, have been used in traditional Chinese medicine for the treatment of various cancers and bacterial infections. Up to now, their official quality control method is still inadequate, and the difficulty of obtaining these high-polarity compounds is one of the major reasons. OBJECTIVE: To develop a rapid and efficient method for preparative separation of the high-polarity compounds gastrodin and benzyl ester glucosides. METHODS: An optimised solvent system composed of n-butanol:ethanol:water (20:1:20, v/v/v) was applied for the elution-extrusion counter-current chromatography (EECCC) separation. The upper phase was used as the stationary phase, and the lower phase was used as the mobile phase at a flow rate of 1.5 mL/min, a rotation speed of 850 rpm and a temperature of 35°C. RESULTS: Five high-polarity glucosides, including two new compounds, (E)-4-ß-D-glucopyranosyloxycinnamic acid 9-(4-ß-D-glucopyranosyloxybenzyl) ester (4 mg) and (Z)-2-(2-methylpropyl)butenedioic acid bis(4-ß-D-glucopyranosyloxybenzyl) ester (9 mg), and three main components, gastrodin (87 mg), dactylorhin A (60 mg) and militarine (15 mg), with HPLC purities of 95.4%, 96.4%, 91.1%, 97.2% and 95.5% respectively, were yielded from 400 mg of the prepared sample. CONCLUSION: Elution-extrusion counter-current chromatography could be used as a useful tool for the separation of high-polarity compounds such as gastrodin and benzyl ester glucosides and the enrichment of the minor ones.

A rapid and convenient method for preparative separation of three indissolvable polyphenols from Euphorbia pekinensis by the flexible application of solvent extraction combined with counter-current chromatography.

A rapid and convenient method was established to preparatively isolate the three ellagic acid types of compounds, which were the main polyphenols in Euphorbia pekinensis, by flexibly applying solvent extraction combined with counter-current chromatography (CCC). The total extract (extracted using 95% ethanol) of E. pekinensis was pretreated by two simple steps before CCC isolation, following the procedure: the total extract was extracted by classical solvent extraction using petroleum ether and ethyl acetate, respectively, and then the ethyl acetate extract was suspended using 95% ethanol, after being allowed to stand overnight, the sediment was obtained. Partial sediment (100 mg) was then directly separated by CCC with a two-phase solvent system composed of chloroform-95% ethanol-water-85% formic acid (50:50:50:5, v/v/v/v). About 22 mg of 3,3'-dimethoxy ellagic acid (1), 12 mg of 3,3'-di-O-methyl-4-O-(ß-D-xylopyranosyl)ellagic acid (2), and 35 mg of ellagic acid (3) with purities of 96.0, 95.2, and 95.4% were obtained respectively in one step within 4 h. After being purified by washing with methanol, the purities of the three compounds obtained were all above 98%. The purities were determined by HPLC and their chemical structures were further identified by (1)H and (13)C NMR spectroscopy. The recoveries were calculated as 84.6, 85.7, and 89.5%, respectively. The result demonstrated that the present isolation method was rapid, economical and efficient for the preparative separation of polyphenols from E. pekinensis.

Simultaneous quantification of 18 bioactive constituents in Tripterygium wilfordii using liquid chromatography-electrospray ionization-mass spectrometry.

A liquid chromatography-electrospray ionization-mass spectrometry method with multiple reaction monitoring was established for simultaneous quantification of 18 bioactive constituents from the stem and root of Tripterygium wilfordii Hook. f. collected from different places in China and various commercial preparations. The chromatographic separations were achieved on an Agilent Poroshell SB-C18 column (150 × 4.6 mm, 3.5 µm) with gradient elution using acetonitrile and 0.03 % formic acid aqueous solution in 45 min. Detection was performed in the positive ionization mode by monitoring the precursor-product combination. The validation of the method included tests of linearity, sensitivity, precision, repeatability, stability, and accuracy. All calibration curves showed good linearity (r > 0.9990) within the test range. The established method showed good precision and accuracy with intraday and interday variations of 2-5 % and 1-4 %, respectively, and recoveries of 95.5-104.5 %.

Neolignanamides, lignanamides, and other phenolic compounds from the root bark of Lycium chinense.

Seven new neolignanamides (1-7), including two pairs of cis- and trans-isomers, and a new lignanamide (8) were isolated from the EtOAc-soluble fraction of an EtOH extract of the root bark of Lycium chinense, together with 22 known phenolic compounds (9-30), four of which were obtained from the genus Lycium for the first time. Compounds 5, 6, and 7 are unusual dimers having a rare connection mode between the two cinnamic acid amide units, while compounds 6, 7, and 8 are the first naturally occurring dimers derived from two dissimilar cinnamic acid amides. The cinnamic acid amides, neolignanamides, and lignanamides possess moderate radical-scavenging activity against the DPPH (2,2-diphenyl-1-picrylhydrazyl) and superoxide radicals.

Rapid preparative isolation of a new phenylpropanoid glycoside and four minor compounds from Sparganium stoloniferum using high-speed counter-current chromatography as a fractionation tool.

A rapid high-speed counter-current chromatography (HSCCC) method was used to isolate five minor compounds from rhizome of Sparganium stoloniferum namely San Leng in Chinese, including two phenylpropanoid glycosides, sparganiaside A (1) and 1-O-feruloyl-3-p-coumaroylglycerol (2), and three aromatic acids, vanillic acid (3), p-hydroxylcinnamic acid (4), and p-hydroxybenzoic acid (5), of which, compound 1 was a new one. Five compounds were preparatively enriched at top efficiency by one-step HSCCC operation in the isolation procedure. A suitable solvent system composed of chloroform-methanol-water (4:3.5:1.8, v/v/v) was used. And the operation time was less than 4 h. The purities of compounds (1-5) in the enriched fractions were determined to be 75.8%, 66.3%, 90.6%, 79.9%, and 98.2%, respectively. The mean recoveries of the five compounds were 84.8%, 87.3%, 81.8%, 90.3%, and 92.7%, respectively. Compounds 1-4 were further purified by semi-preparative high-performance liquid chromatography (HPLC). This is the first report on the use of HSCCC as a fractionation tool for preparative isolation of minor compounds from S. stoloniferum. The method was proved to be rapid, convenient, high yield, and low cost. HSCCC was shown to be a quick and effective tool in isolation of natural products even though the compounds were not abundant.

The protective effects of ginsenoside Rg1 against hypertension target-organ damage in spontaneously hypertensive rats.

BACKGROUND: Although a number of medicines are available for the management of hypertension, the organ damage induced by hypertension is not resolved. The aim of this study was to investigate the protection of ginsenoside Rg1 (Rg1) against vascular remodeling and organ damage in spontaneously hypertensive rats (SHR). METHODS: Male SHR were treated with 5, 10 or 20 mg/kg Rg1 through intraperitoneal injection per day for 1 month. SHR or Wistar-Kyoto rats (WKY) receiving vehicle (saline) was used as control. Blood pressure detection and pathological stain, transmission electron microscope, immunohistochemical assay were used to elucidate the protection of Rg1. RESULTS: Blood pressures were not different between control SHR rats and Rg1 treated SHR rats, but Rg1 improved the aortic outward remodeling by lowering the lumen diameter and reducing the media thickness according the histopathological and ultrastructural detections. Rg1 also protected the retinal vessels against inward remodeling detected by immunohistochemical assay. Furthermore, Rg1 attenuated the target heart and kidney damage with improvement on cardiac and glomerular structure. CONCLUSIONS: These results suggested that Rg1 held beneficial effects on vascular structure and further protected against the organ-damage induced by hypertension. These findings also paved a novel and promising approach to the treatment of hypertensive complications.

Biotransformation of gambogenic acid by Chaetomium globosum CICC 2445.

Microbial transformation of gambogenic acid (1), a caged polyprenylated xanthone isolated from the resin of Garcinia hanburyi, was carried out with Chaetomium globosum CICC 2445, after screening forty-six strains of filamentous fungi. A new caged polyprenylated xanthone, 16,17-dihydroxygambogenic acid (2), was specifically obtained, as a result of hydroxylation at C-16, and C-17. Its structure was elucidated on the basis of spectroscopic methods. The cytotoxicity of compounds 1 and 2 against HeLa tumor cell line was evaluated, with both of them being modestly active.

Simultaneous determination of atractylenolide II and atractylenolide III by liquid chromatography-tandem mass spectrometry in rat plasma and its application in a pharmacokinetic study after oral administration of Atractylodes Macrocephala Rhizoma extract.

Atractylenolide II (AII) and atractylenolide III (AIII) are the major active components in Atractylodes Macrocephala Rhizoma (AMR). In this study, a sensitive, rapid and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of AII and AIII in rat plasma using loliolide as internal standard (IS). After protein precipitation with ethyl acetate, the analytes were injected into an LC-MS/MS system for quantification. Chromatography was performed using a C(18) column, eluting with water and acetonitrile (45:55, v/v) at 0.2 mL/min. All analytes including IS were monitored under positive ionization conditions by multiple reaction monitoring with an electrospray ionization source. The validated method was successfully applied to the pharmacokinetic study of AII and AIII in rat plasma after oral administration of AMR extract. The results provided a meaningful basis for evaluating the clinical applications of traditional Chinese medicine.

Chemical constituents of Spatholobus suberectus.

AIM: To investigate chemical constituents of Spatholobus suberectus Dunn. METHODS: Isolation and purification were carried out by column chromatographic methods. Compounds were characterized based on their physical characteristics and spectra data. RESULTS: Seventeen compounds were isolated from ethanol extract of S. suberectus. The structures were elucidated as prestegane B (1), (2R, 3R)-buteaspermanol (2), (+)-medioresinol (3), (2R, 3R)-3,7-dihydroxyflavanone (4), benzeneethanol (5), 4, 7, 2'-trihydroxy-4'-methoxyisoflavanol (6), naringenin (7), blumenol A (8), protocatechuic acid ethyl ester (9), liquiritigenin (10), 7, 4'-dihydroxy-8-methoxy-isoflavone (11), 3, 5, 7, 3', 5'-pentahydroxyflavanone (12), protocatechuic acid (13), glycyroside (14), 8-methylretusin-7-O-ß-D-glucopyranoside (15), 3, 3', 4', 5, 6, 7, 8-heptahydroxyflavan (16), and dulcisflavan (17). CONCLUSION: All compounds are firstly isolated from the title plant and compounds 1, 3 were isolated from the Spatholobus genus for the first time.

Macromolecular and small-molecule modulation of intracellular Aß42 aggregation and associated toxicity.

Aß (amyloid ß-peptide) has a central role in AD (Alzheimer's disease) where neuronal toxicity is linked to its extracellular and intracellular accumulation as oligomeric species. Searching for molecules that attenuate Aß aggregation could uncover novel therapies for AD, but most studies in mammalian cells have inferred aggregation indirectly by assessing levels of secreted Aß peptide. In the present study we establish a mammalian cell system for the direct visualization of Aß formation by expression of an Aß(42)-EGFP (enhanced green fluorescent protein) fusion protein in the human embryonic kidney cell line T-REx293, and use this to identify both macromolecules and small molecules that reduce aggregation and associated cell toxicity. Thus a molecular shield protein AavLEA1 [Aphelenchus avenae LEA (late embryogenesis abundant) protein 1], which limits aggregation of proteins with expanded poly(Q) repeats, is also effective against Aß(42)-EGFP when co-expressed in T-REx293 cells. A screen of polysaccharide and small organic molecules from medicinal plants and fungi reveals one candidate in each category, PS5 (polysaccharide 5) and ganoderic acid DM respectively, with activity against Aß. Both PS5 and ganoderic acid DM probably promote Aß aggregate clearance indirectly through the proteasome. The model is therefore of value to study the effects of intracellular Aß on cell physiology and to identify reagents that counteract those effects.

Separation of four homoisoflavonoids from Caesalpinia sappan by high-speed counter-current chromatography.

INTRODUCTION: The main chemical constituents of Caesalpinia sappan are homoisoflavonoids. Conventional column chromatographic techniques used for isolation of this type of compounds are tedious, time-consuming and waste solvents. High-speed counter-current chromatography (HSCCC) could be a suitable alternative for the enrichment and purification of these target compounds from traditional Chinese medicine (TCM). OBJECTIVE: To establish a method to isolate four homoisoflavonoids in one-step separation from C. sappan by HSCCC. METHODOLOGY: The crude extract of C. sappan was fractionated by HSCCC using a two-phase solvent system consisting of chloroform-methanol-water (4:3:2, v/v/v). The separation conditions were: flow rate, 1.0 mL/min; revolution speed, 900 rpm; detection wavelength, 280 nm; separation temperature, 25 °C; sample size, 120 mg crude sample dissolved in a mixture of the upper and lower phases (10 mL each). The retention of the stationary phase was 83%. RESULTS: Five milligrams of 3'-deoxysappanol, 8 mg of 3-deoxysappanone B, 20 mg of 4-O-methylsappanol and 18 mg of brazilin were obtained in one-step separation from 120 mg of an ethyl acetate extracted fraction of C. sappan. Their purities were 99%, 97%, 90% and 85% by HPLC analysis. The mean recoveries of the four compounds were 83%, 86%, 93% and 85%, respectively. CONCLUSION: The study has shown that HSCCC is effective for the separation and enrichment of the target compounds at a large scale.

Study of Luan-Pao-Prescription on ovarian dysfunction in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Luan-Pao-Prescription (LPP) has been clinically proven to be effective on infertility. In the present study we explored the improvement and underlying mechanism of LPP on ovarian dysfunction in rats. MATERIALS AND METHODS: 13 month old female rats were randomly divided into 4 groups: the Saline group, the LPP groups treated by low (1.67 g/kg), and high-dose (5 g/kg) LPP respectively, and the hormone group treated by pregnant mare gonadotrophin serum and chorionic gonadotrophin (PMSG/hCG). The estrous cycle was determined by daily observation of vaginal smears; serum estradiol and testosterone were estimated by enzyme-linked immunosorbent assay; ovarian morphology, ovary volume and fertility of female rats were all detected during the study. RESULTS: During 21 days of LPP treatment, about 20% increase of rats with regular estrous cycle of 4-6 days was found, but no change was detected on serum estradiol and testosterone at the dose of 1.67 g/kg and 5 g/kg LPP. Both ovary index and uterus index were up-regulated significantly at the dose of 5 g/kg LPP, but no regulation on oviduct index, adrenal gland index, pancreatic gland index and spleen index was observed at the two LPP groups. 5 g/kg LPP increased total number of pregnant mothers and the offspring; however there are no offspring in PMSG/hCG group. The offspring exhibited similar body weight in each treatment, and no apparent malformation was found for the cubs. While PMSG/hCG treatment increased the ovary index, serum estradiol and testosterone concentration considerably, but no improvement was found on estrous cycle, oviduct index, uterus index, and reproduction. CONCLUSION: Administration of LPP may have comparable benefits for ovarian dysfunction, but with fewer side effects. Oral LPP have a better overall influence on rats than PSMG/hCG; it may be more effective in improvement of estrous cycle, ovary function and reproduction.