Animal medicine is an important part in traditional Tibetan medicine. However, information about the chemical composition of animal medicine is very limited, and there is a lack of comprehensive chromatographic purification methods. In the present work, animal medicine Osteon Myospalacem Baileyi was taken as an example and a novel two-dimensional preparative chromatographic method was established for the preparation of single compounds with high purity from the extract of Osteon Myospalacem Baileyi. The first-dimension preparation was carried on a DAISO Silica prep column, and ten fractions were obtained from the 112.3 g crude sample within 12 injections. A diol prep column used in nonaqueous mobile phase was selected for the second-dimension preparation. The purity of the compounds isolated from the crude extract was >98%, which indicated that the method built in this work was efficient to manufacture single compounds of high purity from the extract of Osteon Myospalacem Baileyi. Additionally, this method showed great potential in the purification of weakly polar chemicals and it could act as a good example in the purification of other traditional animal medicines.
Abstract Lancea tibetica is an important traditional Tibetan medicinal plant that grows on the Qinghai-Tibet Plateau with great development potential in pharmaceutical industry. In this study, a combinative method using HPLC-DPPH and two-dimensional liquid chromatography has been developed to identify and separate antioxidants from Lancea tibetica. Under the target-guidance of HPLC-DPPH experiment, three antioxidant fractions from Lancea tibetica were recognized. Then, separation of the three fractions using two-dimensional semi-preparation liquid chromatography led to seven phenylpropanoids: (+)-pinoresinol-β-D-glucoside (1), isoacteoside (2), acteoside (3), tibeticoside (4),epipinoresinol (5), anthelminthicol (6) and phillygenol (7). As a result, seven major antioxidants in Lancea tibetica were isolated with more than 96% purity. Furthermore, in vitro bioassay against DPPH revealed compounds 1 – 7 with IC 50 values ranging from 6.16 ± 0.08 to 25.09 ± 0.11 (μM) and compounds 1 , 2 and 3 showed activities stronger than the two reference antioxidants (vitamin C, rutin), with IC 50 values of 6.16 ± 0.08, 8.93 ± 0.06 and 7.98 ± 0.05 (μM), respectively. Results of the present study indicated that the method was an efficient technique to systematically screen and isolate antioxidants from medicine crops. Graphical abstract Unlabelled Image Highlights • A novel screen and separation method for purification of antioxidants directly. • Seven antioxidants isolated from Lancea tibetica bioactivity-guided. • Isolated antioxidants with IC 50 values from 6.16 ± 0.08 to 25.09 ± 0.11 (μM). • The first report on antioxidant capacity of the compounds from Lancea tibetica.
We used the Box-Behnken design to optimize polysaccharide extraction from <b>Armillaria luteo-virens</b> (Alb. et Schw. Fr.) Sacc. The independent factors included extraction time (X1), microwave power (X2) and water to raw material ratio (X3). The experimental values were fitted to a second-order polynomial equation using multiple regression analysis and a statistical method. Analysis of Variance results indicated that all factors including X1 - X3 had an impact on <b>Armillaria luteo-virens</b> (Alb. et Schw. Fr.) Sacc. polysaccharide extraction. The optimal conditions for efficient yield of polysaccharide, giving a maximum yield of 8.43%, were: X1 = 30.24 min, X2 = 600.6 W and X3 = 40 mL/g. The model was verified by modifying the optimal conditions (X1 = 30 min, X2 = 601 W and X3 = 40 mL/g) for practical application. A pilot scale test was also carried out under optimal conditions. The obtained yields 8.40 ± 0.12% and 8.34 ± 0.25% were comparable with the optimized condition, which indicated that our model is accurate. Fourier transform infrared spectroscopy characterization revealed that the extracted polysaccharide produced typical absorption peaks. Oxygen radical absorbance capacity results showed the polysaccharides had good potential as an antioxidant. Moreover, the polysaccharide showed relatively strong inhibitory activity on the growth of NCI-H446 cells.<br>• Extraction of ALSP by DEAE technique for the first time. • DEAE method for the extraction of ALSP was built. • ALSP has possessed a good antioxidant activity. • ALSP exhibited antiproliferative activities and may be applied in therapy.
Dynamic microwave-assisted extraction (DMAE) technique was employed for the extraction of polysaccharides from Lycium ruthenicum (LRP). The extracting parameters were optimized by using three-variable-three-level Box-Behnken design and response surface methodology (RSM) based on the single-factor experiments. RSM analysis indicated good correspondence between experimental and predicted values. The optimum extraction parameters for the yield of polysaccharide were ratio of water to raw material 31.5 mL/g, extracting time 25.8 min and microwave power 544.0 W. Polysaccharide was analyzed by chemical methods and Fourier-transform infrared (FT-IR). The antioxidant activities of LRP were investigated including scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydrogen peroxide and free radicals of superoxide anion in vitro. The results of antioxidant activity exhibited LRP had the potential to be explored as novel natural antioxidant for using in functional foods or medicine.
ETHNOPHARMACOLOGICAL RELEVANCE: Osteon Myospalacem Baileyi, known as Sai long gu (Tibetan language, means "blind rat bone"), is the whole skeleton of Tibet plateau rodentia animal Myospalacem Baileyi. Osteon Myospalacem Baileyi had been widely used in the Tibet region as an anti-osteoporosis drug and since 1991 Osteon Myospalacem Baileyi has been listed in the Pharmacopoeia of People's Republic of China as the first-class animal new medical material. However, the mechanism of its anti-osteoporosis activities is still unclear. It is very desirable to solve this problem for further study.MATERIALS AND METHODS: in this study, preparative chromatography was employed to produce the active fraction ET4 from Osteon Myospalacem Baileyi crude. Flow cytometry and MTT assay were used to evaluate the toxicities of ET4. BMM cells were separated from mouse bone marrow to test the inhibition effects of ET4 on osteoclastogenesis. Western blot was used to find out the pathways, through which ET4 could act on osteoclastogenesis. Q-PCR was used to test the osteoclastogenesis marker genes. At last, immunofluorescence confocal microscopy was used to test the osteoclastogenesis master protein NFATc1 nuclei translocation.
RESULTS: In this study we report that ET4, at the dose of 60μg/mL, significantly inhibited the formation of osteoclasts. Notably, ET4 did not affect the BMM viability at that dose. In addition, Osteon Myospalacem Baileyi could inhibit the expression of osteoclast marker genes, including cathepsin K (CTSK), nuclear factor of activated T cells cytoplasmic 1 (NFATc1), tartrate resistant acid phosphatase (TRAP, Acp5) dendrite cell-specific transmembrane protein (DC-STAMP), calcitonin receptor (CTR), osteoclast associated and immunoglobulin-like receptor (OSCAR). Mechanistically, ET4 dose- and time-dependently blocked the RANKL-induced activation of ERK and c-Fos as well as the induction of NFATc1 which is essential for OC formation.
CONCLUSIONS: These data suggest that ET4 might be a useful alternative therapy in preventing or treating osteolytic diseases.
A phytochemical investigation on Lagotis brevituba led to the isolation and characterisation of 11 phenolic compounds: p-hydroxy-benzoic acid 1, methyl 3,4-dihydroxybenzoate 2, vanillic acid 3, protocatechuic acid 4, caffeic acid 5, glucose ester of (E)-ferulic acid 6, p-coumaric acid 7, vanillin 8, diosmetin-7-O-β-d-glucoside 9, chrysoeriol 10 and luteolin 11. Their structures were elucidated using spectroscopic methods and by comparison with data in the literature. Compounds 1-6 were first obtained from the genus Lagotis, and compounds 1-9 were isolated from L. brevituba for the first time. Compound 4 and 11 displayed remarkable antioxidant activities against DPPH radical (IC50 = 5.60 ± 0.09, 27.5 ± 0.06 mg/L, respectively), which were superior to positive control rutin. And compound 11 was also superior to rutin in ABTS assay (IC50 = 2.04 ± 0.13 mg/L).
Traditional Tibetan medicine (TTM) has been valuable for the identification of new therapeutic leads. Nevertheless, reports about the chemical constituents of TTM are meager owing to the lack of suitable purification techniques. In this study, an off-line two-dimensional reversed-phase/hydrophilic interaction liquid chromatography (2D RP/HILIC) technique guided by on-line HPLC-DPPH has been established for the isolation of pure antioxidants from the extract of Dracocephalum heterophyllum . According to the chromatographic recognition outcome of the HPLC-DPPH system, the first-dimensional (1D) separation on the Megress C18 preparative column yielded 6 antioxidative fractions (61.4% recovery) from the ethyl acetate fraction (6.1 g). In the second-dimensional (2D) separation, a HILIC XAmide preparative column was employed. In total, 8 antioxidants were isolated from D. heterophyllum with a purity of >95%, which indicated the efficiency of the developed method to prepare antioxidative compounds with high purity from plant extracts. In addition, this method was highly efficient for the preparation of structural analogues of the antioxidative polyphenols and could be applied for the purification of structural analogues from other resources. [ABSTRACT FROM AUTHOR]
Yak milk cheese is one of the most important foods for the Tibetan people. <b>Lactobacillus plantarum</b> SLG1 isolated from yak cheese was shown to produce a novel bacteriocin, plantaricin SLG1. Production of plantaricin SLG1 in MRS medium was maximized after 24 h incubation at 37 °C (the stationary phase of growth). An innovative method, namely magnetic liposome adsorption combined with reversed-phase high performance liquid chromatography (RP-HPLC), was developed to screen for and efficiently purify bacteriocin compounds from the cell free supernatants from <b>Lb. plantarum</b> SLG1. The molecular mass of plantaricin SLG1 is 1083.25 Da and its amino acid sequence Tyr-Gly-Asn-Gly-Val-Phe-Ser-Val-Ile-Lys was determined by N-sequencing. Analyses by Circular dichroism (CD) spectra and predicted 3D structure suggested that the peptide maintains a well-defined conformation. Plantaricin SLG1 exhibited a wide range of antimicrobial activity against many food-borne spoilage and pathogenic bacteria, as well as some fungi. Results using scanning electron microscopy indicated that the mode of action was bactericidal and plantaricin SLG1 was able to damage the cell membrane integrity ultimately causing pathogen lethality.<br>• Bacteriocin-producer <b>Lb. plantarum</b> SLG1 isolated from yak cheese. • Bacteriocin, plantaricin SLG1, was obtained. • Magnetic liposomes adsorption & RP-HPLC were used to purification. • The active spectrum include Gram-positive and negative bacteria and fungi.
Herbal plants are significant for the reason that they have a great potential in discovering drug precursors. However, how to purify compounds with higher purity from them is a question which needs to be discussed. In present study, an offline 2D reversed-phase (RP) preparative liquid chromatography coupled with solid-phase extraction (SPE) method was successfully developed for the separation of flavonolignan diastereoisomers from Arenaria kansuensis. Based on the analysis of results, the major conclusion that we have drawn from it is a RP-SPE was selected for enriching target flavonolignan sample from A. kansuensis. After that, an ODS preparative column was used for 1D preparation, and the target sample (4.6 g) was divided into five fractions with a recovery of 83.9%. Then, a C18HCE preparative column, a polar-modified RP (polar-copolymerized) type, was used for isolating flavonolignan diastereoisomers in the 2D preparation. By establishing optimal 2D chromatography, hydrophilic interaction chromatography (HILIC) columns and normal-phase (NP) columns were tested simultaneously, and the result showed that diastereoisomers are not suitable for HILIC and NP chromatography mode. Our study resulted in a tricin and five analogous derivative flavonolignans with purity >98% were successfully purified from A. kansuensis. This is the initial report of Salcolin C, Salcolin B, Tricin 4'-O-(C-veratroylglycol) ether and 5'-methoxyhydnocarpin D from A. kansuensis. In addition, it tended to be the first time that Tricin 4'-O-(C-veratroylglycol) ether is isolated from natural resource. This method has great potential for efficiently isolating flavonolignan diastereoisomers from A. kansuensis, and it shows a great prospect for the separation of flavonolignans from complex samples.
Highland barley is one of the most important industrial crops in Tibetan plateau. Previous research indicated that highland barley has many medical functions. In this work, the antibacterial abilities of highland barley were investigated. The protein solutions hydrolyzed by trypsin for 4 h exhibited the highest antibacterial activity. An antibacterial peptide, barleycin, was screened and purified by magnetic liposome extraction combining with the protein profiles of reversed-phase high-performance liquid chromatography (RP-HPLC). Structure, characterization, and safety evaluation of barleycin were further investigated. Amino acids sequence was determined as Lys-Ile-Ile-Ile-Pro-Pro-Leu-Phe-His by N-sequencing. Circular dichroism spectra indicated the a-helix conformation of barleycin. The activity spectrum included <i>Bacillus subtilis, Staphylcoccus aureus, Listeria innocua and Escherichia coli</i> and the MICs were from 4 to 16 μg/mL. Safety evaluations with cytotoxicity and hemolytic suggested this antibacterial peptide could be considered as safe at MICs. Finally, mode of action of barleycin on sensitive cells was primarily studied. The results suggested the damage of cell membrane.
Four iridoid glucosides, shanzhiside methyl ester, phloyoside II, chlorotuberside, and penstemonoside, were isolated and purified from an herbal medicinal plant for the first time by high-speed counter-current chromatography (HSCCC) using a two-phase solvent system composed of ethyl acetate-n-butanol-water (5:14:12, v/v/v). A total of 37mg of shanzhiside methyl ester, 29mg of phloyoside II, 27mg of chlorotuberside, and 21mg of penstemonoside with the purity of 99.2%, 98.5%, 97.3%, and 99.3%, respectively, were obtained in one-step separation within 4h from 150mg of crude extract. To the best of our knowledge, this is the first report of separation and purification of iridoid glucosides from natural sources by HSCCC. The chemical structures of all the four compounds were identified by ESI-MS, (1)H NMR, and (13)C NMR.
A new flavonoid, along with 16 known ones, was separated from the aerial parts of Asterothamnus centrali-asiaticus. Their structures were elucidated by extensive spectroscopic methods, including 1D and 2D NMR techniques and HRESIMS. To confirm the structure of the new compound, computational prediction of its 13C chemical shifts was performed. All of the 17 flavonoids were reported from A. centrali-asiaticus for the first time. In addition, all flavonoids were evaluated for their antioxidant and α-glucosidase inhibitory activities. The results showed that 10 of them exhibited antioxidant activity. Meanwhile, four flavonoids displayed α-glucosidase inhibitory effect with IC50 values ranging from 38.9 to 299.7 μM.
An orthogonally (80.3%) preparative two-dimensional hydrophilic interaction chromatography/reversed-phase liquid chromatography method has been established for the isolation and purification of flavonoids from Saxifraga tangutica. Initially, flavonoids were enriched by means of a middle-pressure chromatographic tower (containing middle chromatogram isolated gel). In the first dimension, a XION preparative column was used to separate the flavonoid fractions under the guidance of characteristic ultraviolet absorption spectra of flavonoids and nine flavonoid fractions were obtained. Then, the coeluted flavonoid fractions were selected for further purification via reversed-phase liquid chromatography with the parent ion peak of quercetin (303), kaempferol (287), or isorhamnetin (317). Several flavonoids could be separated from each hydrophilic interaction chromatography fraction; furthermore, flavonoids with poor resolution in one-dimensional liquid chromatography were isolated in two-dimensional liquid chromatography due to the orthogonality. In addition, this technique was valuable for trace flavonoids, which were concentrated in the first stage and separated in the second stage. In total, 18 flavonoids with either quercetin, kaempferol, or isorhamnetin parent nuclei were targetedly obtained, and 15 flavonoids were obtained for the first time from S. tangutica. These results established that the off-line two-dimensional hydrophilic interaction chromatography/reversed-phase liquid chromatography technique was efficient for the isolation of flavonoids from Saxifraga tangutica.
<br>Display Omitted<br>• Three new monoterpene glycosides (<b>1</b>-<b>3</b>) were isolated from <b>Sibiraea laevigata</b> (L.) Maxim. • Fourteen known compounds (<b>4</b>-<b>17</b>) were also obtained from the title plant. • All of the isolated compounds were evaluated for their anti-oxidant and α-glucosidase inhibitory activities. • Compounds <b>7</b> and <b>17</b> exhibited α-glucosidase inhibitory effect with IC50 values of 220.0 and 113.0 μM, respectively.<br>Three new compounds, 3,7-dimethy-7-methoxy-3-octene-5-one-1-<b>O</b>-<b>β</b>-d-glucopyranoside (1), 3,7-dimethy-7-methoxy-3(<b>Z</b>)-octene-5-one-1-<b>O</b>-<b>β</b>-d-glucopyranoside (2) and 3,7-dimethy-3-hydroxy-6-octene-5-one-1-<b>O</b>-<b>β</b>-d-glucopyranoside (3), together with fourteen known compounds (4-17) were isolated from the leaves and shoots of <b>S. laevigata</b>. The structures of the new compounds were elucidated on the basis of extensive spectroscopic analysis, including one- and two-dimensional NMR, as well as mass spectral data. All isolates were evaluated for their α-glucosidase inhibitory and antioxidant activities. The results demonstrated that 3,7-dimethyl-3(Z),6-ocatdien-5-one-1-<b>O</b>-<b>β</b>-d-glucoside (7) and sitosteryl <b>β</b>-d-glucoside (17) exhibited α-glucosidase inhibitory effects with IC50 values of 220.0 and 113.0 μM, respectively.
Traditional Tibetan medicine is important for discovery of drug precursors. However, information about the chemical composition of traditional Tibetan medicine is very limited due to the lack of appropriate chromatographic purification methods. In the present work, A. kansuensis was taken as an example and a novel two-dimensional reversed-phase/hydrophilic interaction liquid chromatography(HILIC) method based on on-line HPLC-DPPH bioactivity-guided assay was developed for the purification of analogue antioxidant compounds with high purity from the extract of A. kansuensis. Based on the separation results of many different chromatographic stationary phases, the first-dimensional (1D) preparation was carried on a RP-C18HCE prep column, and 2 antioxidant fractions were obtained from the 800mg crude sample with a recovery of 56.7%. A HILIC-XAmide prep column was selected for the second-dimensional (2D) preparation. Finally, a novel antioxidant β-carboline Alkaloids (Glusodichotomine AK) and 4 known compounds (Tricin, Homoeriodictyol, Luteolin, Glucodichotomine B) were purified from A. kansuensis. The purity of the compounds isolated from the crude extract was >98%, which indicated that the method built in this work was efficient to manufacture single analogue antioxidant compounds of high purity from the extract of A. kansuensis. Additionally, this method showed great potential in the preparation of analogue structure antioxidant compounds and can serve as a good example for the purification of analogue structure antioxidant carboline alkaloids and flavonoids from other plant materials.
Traditional Tibetan medicine is important for discovery of drug precursors. However, knowledge of the chemical composition of traditional Tibetan medicines is very limited due to the lack of appropriate chromatographic purification methods. In the present work, Salvia prattii was taken as an example, and an off-line hydrophilic interaction liquid chromatography/reversed-phase liquid chromatography preparative method was developed for the purification of phenylpropanoids with high purity from a crude sample of Salvia prattii. Based on the separation results of four different chromatographic stationary phases, the first-dimensional preparation was performed on an XAmide preparative column with the crude sample concentration of 62.0 mg/mL, and five main fractions were obtained from the 12.4 g crude sample with a recovery of 54.8%. An XCharge C18 preparative column was applied in the second-dimensional preparation to further isolate the phenylpropanoids from the redissolved first-dimensional fractions with concentration of approximately 50.0 mg/mL. The purities of the phenylpropanoids isolated from the crude sample of Salvia prattii were higher than 98%, indicating that the method was efficient for the purification of phenylpropanoids with high purity from Salvia prattii. Additionally, this method showed great potential in the preparation of phenylpropanoids and can serve as a good example for the purification of phenylpropanoids from other plant materials.
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