Acupuncture attenuates cognitive impairments in vascular dementia through inhibiting miR-143-3p.

BACKGROUND: Acupuncture can be used to treat vascular dementia (VD), but the underlying mechanism remains unclear. This study aimed to investigate the expression and clinical significance of microRNA-143-3p (miR-143-3p) in VD patients and explore whether acupuncture ameliorates VD by regulating miR-143-3p. METHODS: Cognitive function and daily living ability in VD patients were assessed by mini-mental state examination, Hasegawa's dementia scale and activities of daily living scale, respectively. VD model of male Wistar rats was established using permanent bilateral common carotid artery occlusion. The expression level of miR-143-3p was measured by quantitative real-time PCR. Morris water maze test was used to assess the cognitive function of VD rat model. Receiver operating characteristic analysis was used to assess the diagnostic value of miR-143-3p in VD patients. Correlations between variables were analyzed by Pearson's correlation analysis. RESULTS: Increased serum miR-143-3p expression in VD patients had a high diagnostic value to screen VD patients. Serum miR-143-3p level in VD patients after acupuncture treatment was decreased. After acupuncture treatment, serum miR-143-3p was negatively correlated with cognitive function and daily living ability in VD patients. miR-143-3p level was increased in VD rats, and the suppressive effects of acupuncture on miR-143-3p levels were relieved by miR-143-3p mimic. Overexpression of miR-143-3p reversed the ameliorative effect of acupuncture on cognitive functions of VD rats. CONCLUSION: Serum miR-143-3p expression is upregulated in VD patients and downregulated in VD patients after acupuncture treatment. Additionally, acupuncture treatment may attenuate cognitive impairments in VD by suppressing miR-143-3p.

Imidazolylacetophenone oxime-based multifunctional neuroprotective agents: Discovery and structure-activity relationships.

Alzheimer's disease (AD) possesses a complex pathogenetic mechanism. Nowadays, multitarget agents are considered to have potential in effectively treating AD via triggering molecules in functionally complementary pathways at the same time. Here, based on the screening (∼1400 compounds) against neuroinflammation, an imidazolylacetophenone oxime ether (IOE) was discovered as a novel hit. In order to obtain SARs, a series of imidazolylacetophenone oxime derivatives were constructed, and their C=N bonds were confirmed as the Z configuration by single crystals. These derivatives exhibited potential multifunctional neuroprotective effects including anti-neuroinflammatory, antioxidative damage, metal-chelating, inhibition of acetylcholinesterase (AChE) properties. Among these derivatives, compound 12i displayed the most potent inhibitory activity against nitric oxide (NO) production with EC50 value of 0.57 µM 12i can dose-dependently suppress the expression of iNOS and COX-2 but not change the expression of HO-1 protein. Moreover, 12i exhibited evidently neuroprotective effects on H2O2-induced PC12 cells damage and ferroptosis without cytotoxicity at 10 µM, as well as selectively metal chelating properties via chelating Cu2+. In addition, 12i showed a mixed-type inhibitory effect on AChE in vitro. The structure-activity relationships (SARs) analysis indicated that dioxolane groups on benzene ring and rigid oxime ester can improve the activity. Parallel artificial membrane permeation assay (PAMPA) also verified that 12i can overcome the blood-brain barrier (BBB). Overall, this is the first report on imidazolylacetophenone oxime-based multifunctional neuroprotective effects, suggesting that this type of compounds might be novel multifunctional agents against AD.

1,10-Seco-Eudesmane sesquiterpenoids as a new type of anti-neuroinflammatory agents by suppressing TLR4/NF-κB/MAPK pathways.

Dysregulation of neuroinflammation is a key pathological factor in the progressive neuronal damage of neurodegenerative diseases. An in-house natural products library of 1407 compounds were screened against neuroinflammation in lipopolysaccharide (LPS)-activated microglia cells to identify a novel hit 1,6-O,O-diacetylbritannilactone (OABL) with anti-neuroinflammatory activity. Furthermore, a 1,10-seco-eudesmane sesquiterpenoid library containing 33 compounds was constructed by semisynthesis of a major component 1-O-acetylbritannilactone (ABL) from the traditional Chinese medicinal herb Inula Britannica L. Compound 15 was identified as a promising anti-neuroinflammatory agent by nitrite oxide (NO) production screening. 15 could attenuate tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2) productions, and inhibit the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at a submicromolar level. Mechanistic study revealed that 15 significantly modulated TLR4/NF-kB and p38 MAPK pathways, and upregulated the anti-oxidant response HO-1. Besides, 15 promoted the conversion of the microglia from M1 to M2 phenotype by increasing levels of arginase-1 and IL-10. The structure-activity relationships (SARs) analysis indicated that the α-methylene-γ-lactone motifs, epoxidation of C5=C10 bond and bromination of C14 were important to the activity. Parallel artificial membrane permeation assay (PAMPA) also demonstrated that 15 and OABL can overcome the blood-brain barrier (BBB). In all, compound 15 is a promising anti-neuroinflammatory lead with potent anti-inflammatory effects via the blockage of TLR4/NF-κB/MAPK pathways, favorable BBB penetration property, and low cytotoxicity.

Study of the volatile constituents in radix flemingiae macrophyllae and a substitute by gas chromatography-mass spectrometry and chemometric methods.

A combined approach of subwindow factor analysis and spectral correlative chromatography was used to analyze the volatile components in Radix Flemingiae Macrophyllae and Flemingiae Latifolia Benth, one of its substitutes. After extraction by a water distillation method, the volatile components in Radix Flemingiae Macrophyllae and Flemingiae Latifolia Benth were detected by GC-MS. Then the qualitative and quantitative analysis of the volatile components in Radix Flemingiae Macrophyllae was completed with the help of subwindow factor analysis resolving two-dimensional original data into mass spectra and chromatograms. Sixty five of 82 separated constituents in the total ion chromatogram of the volatile components in Radix Flemingiae Macrophyllae were identified and quantified, accounting for about 88.79% of the total content. Then, spectral correlative chromatography was used to extract correlative constituents in Flemingiae Latifolia Benth. Fifty one correlative components were recognized in essential oil of Flemingiae Latifolia Benth. The result proves the combined approach is powerful in the analysis of complex herbal samples. The developed method can be used to compare the sameness and differences of Radix Flemingiae Macrophyllae and its substitutes and it can also be used for quality control of Radix Flemingiae Macrophyllae.

Identification and determination of flavonoids in astragali radix by high performance liquid chromatography coupled with DAD and ESI-MS detection.

A method for the analysis of flavonoids in Astragali Radix by high-performance liquid chromatography (HPLC) combined with photodiode-array detection (DAD) and an electrospray ionization (ESI)--mass spectrometry (MS) was developed. After the samples were extracted with ethanol, the optimum separation conditions for these analytes were achieved using a gradient elution system and a 2.0 x 150 mm Shimadzu VP-ODS column. Eight flavonoids were identified to exist in Astragali Radix based on their characteristic UV data and mass spectra. The concentrations of three major components in this herb--ononin, calycosin and formononetin--were determined by LC/ESI-MS in positive selective ion monitoring (SIM) mode. The calibration curves were linear in the range of 0.9~180.0 µg·mL⁻¹ for ononin, 1.8~360.0 µg·mL⁻¹ for calycosin and 1.4~280 µg·mL⁻¹ for formononetin, respectively. The limits of quantification (LOQ) and detection (LOD) were 0.9 µg· mL⁻¹ and 0.2 µg mL⁻¹ for ononin, 1.8 µg mL⁻¹ and 0.5 µg·mL-1 for calycosin, 1.4 µg mL⁻¹ and 0.5 µg·mL⁻¹ for formononetin, respectively. The standard recoveries were between 95.4~104.7%. The developed method was proven to be useful for the quantitative and qualitative analysis of flavonoid constituents in various resources of Astragali Radix.

[Rapid simultaneous determination of main nucleosides in Cordyceps sinensis with LC-ESI-MS].

OBJECTIVE: To establish an LC-MS method for rapid simultaneous determination of adenine,adenosine and cordycepin in Cordyceps sinensis. METHOD: An electrospray ionization (ESI) interface and selective ion monitoring (SIM) mode were used. The analytical column was a Shimadzu VP-ODS column (2.0 mm x 150 mm) and the mobile phase was water-methanol-formic acid (92:7:1). Methanol was used as extraction solvent and 2-chloroadenosine was used as internal standard for this assay. RESULT: The regression equations and coefficient were Y = 0.07264X + 0.00622 and r = 0.9987 for adenine, Y = 0.1597X + 0.0146 and r = 0.9991 for adenosine, Y = 0.1942X + 0.0186 and r = 0.9994 for cordycepin. The linear range was 0.8-130.0 mg x L(-1), 0.5 - 124.5 mg x L(-1) and 0.5-128.5 mg x L(-1) for adenine, adenosine and cordycepin, respectively. The average recovery was 98.76%, 99.37% and 99.26% for adenine, adenosine and cordycepin, respectively. CONCLUSION: This established method was highly sensitive, fast and selective, which can be used for rapid simultaneous determination of adenine, adenosine and cordycepin in C. sinensis. This method also can be applied for the quality control of C. sinensis.

[Headspace solid-phase microextraction-gas chromatography-mass spectrometry for analysis of volatile components from Atractlodes macrocephala Koidz].

Headspace solid-phase microextraction (HS-SPME) technique was employed to extract the volatile components from Chinese traditional medicine, Atractlodes macrocephala Koidz. The volatile components were isolated and identified successfully by gas chromatography-mass spectrometry (GC-MS). The results from HS-SPME-GC-MS were compared with those obtained from steam distillation-GC-MS (SD-GC-MS) with a good agreement. The volatiles were collected using a polydimethylsiloxane-divinylbenzene (PDMS-DVB) fiber by HS-SPME. The best response was obtained when the extraction temperature was 70 degrees C, the equilibrium time and extraction time were all 30 min and the desorption time was 4 min. Analysis was performed by GC-MS with a polysiloxane capillary column (30 m x 0.25 mm, film thickness 0.25 microm) using He as the carrier gas and a programmed temperature run. Forty-one components accounting for 90.81% were identified. The main components (relative amount more than 1%) of the samples by HS-SPME were atractylone (40.12%), gamma-elemene (14.73%), aromadendrene (13.05%), eudesma-4 (14), 11-diene (5.46%), caryophyllene (2.56%), patchoulene (2.55%), 6,10,11,11-tetramethyl-tricyclo [6.3.0.1(2,3)] undec-7-ene (2.11%), cedrene (1.48%), alpha-caryophyllene (1.48%) and selina-4 (14) -7 (11) diene-8-one (1.01%). By SD-GC-MS, 31 components accounting for 88.19% were identified and all these components could be extracted by SPME except trans-beta-ocimene which accounts only 0.10%. The results showed that the HS-SPME technique can be used to extract the the volatile components from Atractlodes macrocephala Koidz. in place of the traditional time-consuming SD.

Identification and determination of nucleosides in Cordyceps sinensis and its substitutes by high performance liquid chromatography with mass spectrometric detection.

Cordyceps sinensis (Cs) is a well-known traditional Chinese medicine (TCM) and Cordyceps mycelia (Cm), a cultured Cordyceps, is a substitute for Cordyceps sinensis. The most important active components in them are nucleosides. A high selective, sensitive and accurate high performance liquid chromatography method with photodiode array detection (DAD) and mass spectrometric detection has been developed for simultaneous separation, identification and quantification of nucleosides in Cs and Cm using a mobile phase including (A) ammonium acetate (40 mM, pH 5.2) and (B) methanol with a gradient system on a 2.0 mm x 150 mm Shimadzu VP-ODS column. The presence of each nucleoside in Cs and Cm was ascertained by comparison of MS data, UV spectra and retention time with standards. LC/ESI-MS in selective ion monitoring (SIM) mode were used for the quantification of nucleosides in Cs and Cm. 2-Chloroadenosine was used as internal standard for this assay. The precisions and accuracies were in the range of 1.5-5.3% and -3.5 to 5.0%, respectively. The limits of detection and quantification for nucleosides were in the order of 0.1-0.6 microg ml(-1) and 0.5-2.0 microg ml(-1), respectively. The recoveries were in the range of 92.0-107.0%. With the developed method, the concentrations of nucleosides in Cs and Cm from different sources were determined. Cs, characterized with far lower concentration of adenosine and cordycepin than Cm, can be very easy to distinguish from Cm. This reliable method would be useful for the study and quality control of Cordyceps sinensis and its substitutes.

Comparison of the volatile constituents of Artemisia capillaris from different locations by gas chromatography-mass spectrometry and projection method.

The volatile chemical constituents of Artemisia capillaries (an important traditional Chinese medicine) were determined by gas chromatography-mass spectrometry (GC-MS) and sub-window factor analysis (SFA). Seventy-five components were separated and 43 of them were qualitatively and quantitatively determined, which represented about 89.03% of the total content. This profile was then used to identify and assess the consistency of the herb by using an orthogonal projection method. Four different sources of A. capillaries were analyzed and compared with each other. Among the components determined, there were 51 components coexisting in all samples although the relative peak areas of a few showed variations. It is the first time to apply orthogonal projection method to the comparison of different samples, and it reduces the burden of qualitative analysis as well as the subjectivity. The results showed a fair consistency in their GC-MS fingerprint. A. capillaris was distinguished from Artemisia sacrorum L., a possible substitute in traditional Chinese medicine by comparing the fingerprints with each other.

[Determination of adenosine and cordycepin in Cordyceps sinensis and C. militarris with HPLC-ESI-MS].

OBJECTIVE: HPLC-ESI-MS to establish a method for simultaneous determination of adenosine and cordycepin in Cordyceps sinensis and C. militarris. METHOD: HPLC-ESI-MS method. An electrospray ionization (ESI) interface and selective ion monitoring (SIM) mode were used. The analytical column was a 2.0 mm x 150 mm Shimadzu VP - ODS column and the mobile phase was water (94%), methanol (5%) and formic acid (1%). 2-Chloroadenosine was used as internal standard for this assay. RESULT: The regression equations and coefficient were Y = 0.134 6X + 0.001 29 (r = 0.998 4) for adenosine, Y = 0.216 4X + 0.021 5 (r = 0.999 1) for cordycepin. The liner range was 0.5 approximately 124.5 microg x mL(-1) and 0.5 approximately 136.5 microg x mL(-1) for adenosine and cordycepin, respectively. The average recoveries of adenosine and cordycepin were 95.8% and 98.1%, respectively. CONCLUSION: This method is highly sensitive, fast and selective, which can be used for the determination of nucleosides in C. sinensis and its substitutes. This method can also be applied for the quality control of above herbs.

Spectral correlative chromatography and its application to analysis of chromatographic fingerprints of herbal medicines.

A signal-processing method known as spectral correlative chromatography (SCC) for two-dimensional data obtained from hyphenated chromatography is developed and applied to chemical chromatographic fingerprint data sets of herbal medicine under specific experimental conditions. The method can judge the presence or absence of a spectral correlative peak among the spectrochromatograms. A local least squares regression model (LLS) is constructed in a piecewise manner to correct the shifts of retention time of some peaks of interest in the chromatograms of various test samples. The results compare favorably with those obtained by a two-point calibrated algorithm. It is shown that performing SCC and LLS on the piecewise clusters of various chromatographic fingerprints is more helpful in practice in revealing their common nature and for characterizing the chemical constituents. This approach holds great potential for facilitating quality control of herbal medicines.

Analyzing of the volatile chemical constituents in Artemisia capillaris herba by GC-MS and correlative chemometric resolution methods.

The volatile chemical constituents in traditional Chinese medicine (TCM), Artemisia capillaris herba, were determined by gas chromatography-mass spectrometry (GC-MS). The acquired two-dimensional data were resolved by correlative chemometric resolution methods. The noise in the raw data is pretreated by roughness penalty smoothing method. With the data denoised, heteroscedastic noise and signal-to-noise ratio were decreased apparently, which was favorable to the determination of component number. The selective range can be extracted from rankmap obtained by fixed size moving window evolving factor analysis (FSMWEFA) conveniently. The overlapped chromatographic peaks were resolved into pure chromatograms and pure spectra with evolving window orthogonal projection (EWOP). The purity of the resolved pure spectra were improved furthermore with the heteroscedastic noise decreased through roughness penalty smoothing method, although the basic structure of the raw data changes little. Qualitative analysis was performed by similarity search in NIST147 and Willey library. Pure chromatograms are in favor of quantitative analysis, which was obtained by total volume integration. Forty-two of seventy-five separated constituents in essential oil, accounting for 89.03% of the total content, were identified. The result proves the combined approaches to be powerful for the analysis of complex herbal samples.

Application of combined approach to analyze the constituents of essential oil from Dong quai.

A combined approach of sub-window factor analysis and spectral correlative chromatography has been employed to analyze the constituents of essential oils of Dong quai. Essential oils are the main pharmacological active individuals of Dong quai. Some constituents in the main root of Dong quai have been identified by GC-MS with the help of sub-window factor analysis resolving two-dimensional original data into mass spectra and chromatograms. Correlative constituents in another part of the root fiber have been recognized by spectral correlative chromatography. Seventy six of 97 separated constituents in the essential oil of main root were identified and quantified, accounting for about 91.36% of the total content. Sixty seven correlative components in the essential oil of root fiber were recognized. The result proves that the combined approach is powerful enough for the analysis of complex herbal samples.

Identification of structures of nitrogen-containing compounds in crude oils in conjunction with chemometric resolution.

A universal method was established for the systematically structural identification of nitrogen-containing compounds in crude oils. Pre-fractionation of the non-hydrocarbons in a crude oil sample into 7 fractions was performed by di-adsorption column chromatography using neutral aluminum oxide and silica gel; subsequent high-resolution separation of individual components was achieved by using capillary column gas chromatography, and compound types were detected by mass spectrometer. The two-dimensional data from the compounds in the fractions were further resolved by a chemometric method to obtained the deconvoluted chromatogram and mass spectrum of every compound, and then, the nitrogen-containing compounds were identified in combination with the retention indices. This method could relieve the difficulty of classical analysis in identifying those species with very low contents or incompletely separation, particularly in the cases where the authentic standards were not available for addition into the unknown samples in order to reveal what indeed existed in them. The structures of 168 nitrogen-containing compounds in a crude oil sample were determined by this method with satisfactory results.

Simultaneous separation and determination of active components in Cordyceps sinensis and Cordyceps militarris by LC/ESI-MS.

A simple and rapid isocratic LC/MS coupled with electrospray ionization (ESI) method for simultaneous separation and determination of adenine, hypoxanthine, adenosine and cordycepin in Cordyceps sinensis (Cs) and its substitutes was developed. 2-Chloroadenosine was used as internal standard for this assay. The optimum separation for these analytes was achieved using the mixture of water, methanol and formic acid (85:14:1, v/v/v) as a mobile phase and a 2.0 x 150 mm Shimadzu VP-ODS column. Selective ion monitoring (SIM) mode ([M+H]+ at m/z 136, 137, 268, 252 and 302) was used for quantitative analysis of above four active components. The regression equations were liner in the range of 1.4-140.0 microg ml(-1) for adenine, 0.6-117.5 microg ml(-1) for hypoxanthine, 0.5-128.5 microg ml(-1) for adenosine and 0.5-131.5 microg ml(-1) for cordycepin. The limits of quantitation (LOQ) and detection (LOD) were, respectively 1.4 and 0.5 microg ml(-1) for adenine, 0.6 and 0.2 microg ml(-1) for hypoxanthine, 0.5 and 0.1 microg ml(-1) for adenosine and cordycepin. The recoveries of four constituents were from 93.5 to 107.0%. The nucleoside contents of various types of natural Cs and its substitutes were determined and compared with this developed method.

Determination of the volatile chemical constituents of Notoptergium incium by gas chromatography-mass spectrometry and iterative or non-iterative chemometrics resolution methods.

The qualitative and quantitative determination of the chemical constitutes in traditional Chinese medicine (TCM) is an important task, which builds the foundation of the theory of pharmacological activity. The hyphenated chromatography instruments combined with the related chemometric methods provide powerful tools for the resolution of such complex systems. The familiar chemometrics methods can be roughly divided into two different kinds, the iterative one such as orthogonal projection approach (OPA) and non-iterative one representing by evolving window orthogonal projection (EWOP). One can use different kinds of methods according to overlapping condition, and then the measured data matrix can be resolved into pure concentration profiles and mass spectra of the chemical components with relative high efficiency and acceptable accuracy. One kind of TCM, named Notoptergium incium (NI) was analyzed by gas chromatography-mass spectrometry (GC-MS) and resolved by above chemometric approach. Experiment results show the efficiency and convenience of the proposed approach. 65 of the 98 separated constituents in essential oil, accounting for 92.13%, were identified by mass spectroscopy (MS).