Preparation and In Vitro and In Vivo Evaluation of Rectal In Situ Gel of Meloxicam Hydroxypropyl-ß-cyclodextrin Inclusion Complex.

Meloxicam (MLX) is one of the most effective NSAIDs, but its poor water solubility and low bioavailability limit its clinical application. In this study, we designed a thermosensitive in situ gel of the hydroxypropyl-ß-cyclodextrin inclusion complex (MLX/HP-ß-CD-ISG) for rectal delivery to improve bioavailability. The best method for preparing MLX/HP-ß-CD was the saturated aqueous solution method. The optimal inclusion prescription was optimized using an orthogonal test, and the inclusion complex was evaluated via PXRD, SEM, FTIR and DSC. Then, MLX/HP-ß-CD-ISG was characterized regarding the gel properties, release in vitro, and pharmacokinetics in vivo. The inclusion rate of the inclusion complex obtained via the optimal preparation process was 90.32 ± 3.81%. The above four detection methods show that MLX is completely embedded in the HP-ß-CD cavity. The developed MLX/HP-ß-CD-ISG formulation has a suitable gelation temperature of 33.40 ± 0.17 °C, a gelation time of 57.33 ± 5.13 s, pH of 7.12 ± 0.05, good gelling ability and meets the requirements of rectal preparations. More importantly, MLX/HP-ß-CD-ISG significantly improved the absorption and bioavailability of MLX in rats, prolonging the rectal residence time without causing rectal irritation. This study suggests that the MLX/HP-ß-CD-ISG can have a wide application prospect with superior therapeutic benefits.

Perspectives of herbs and their natural compounds, and herb formulas on treating diverse diseases through regulating complicated JAK/STAT signaling.

JAK/STAT signaling pathways are closely associated with multiple biological processes involved in cell proliferation, apoptosis, inflammation, differentiation, immune response, and epigenetics. Abnormal activation of the STAT pathway can contribute to disease progressions under various conditions. Moreover, tofacitinib and baricitinib as the JAK/STAT inhibitors have been recently approved by the FDA for rheumatology disease treatment. Therefore, influences on the STAT signaling pathway have potential and perspective approaches for diverse diseases. Chinese herbs in traditional Chinese medicine (TCM), which are widespread throughout China, are the gold resources of China and have been extensively used for treating multiple diseases for thousands of years. However, Chinese herbs and herb formulas are characterized by complicated components, resulting in various targets and pathways in treating diseases, which limits their approval and applications. With the development of chemistry and pharmacology, active ingredients of TCM and herbs and underlying mechanisms have been further identified and confirmed by pharmacists and chemists, which improved, to some extent, awkward limitations, approval, and applications regarding TCM and herbs. In this review, we summarized various herbs, herb formulas, natural compounds, and phytochemicals isolated from herbs that have the potential for regulating multiple biological processes via modulation of the JAK/STAT signaling pathway based on the published work. Our study will provide support for revealing TCM, their active compounds that treat diseases, and the underlying mechanism, further improving the rapid spread of TCM to the world.

Preparation, In Vitro and In Vivo Evaluation of Nanoemulsion In Situ Gel for Transnasal Delivery of Traditional Chinese Medicine Volatile Oil from Ligusticum sinense Oliv.cv. Chaxiong.

Ischemic stroke is a difficult-to-treat brain disease that may be attributed to a limited therapeutic time window and lack of effective clinical drugs. Nasal-brain administration is characterized by low systemic toxicity and is a direct and non-invasive brain targeting route. Preliminary studies have shown that the volatile oil of Chaxiong (VOC) has an obvious anti-ischemic stroke effect. In this work, we designed a nanoemulsion thermosensitive in situ gel (VOC-NE-ISG) loaded with volatile oil of Chaxiong for ischemia via intranasal delivery to rat brain treatment of cerebral ischemic stroke. The developed VOC-NE-ISG formulation has a suitable particle size of 21.02 ± 0.25 nm and a zeta potential of -20.4 ± 1.47 mV, with good gelling ability and prolonged release of the five components of VOC. The results of in vivo pharmacokinetic studies and brain targeting studies showed that intranasal administration of VOC-NE-ISG could significantly improve the bioavailability and had excellent brain-targeting efficacy of nasal-to-brain delivery. In addition, the results of pharmacodynamics experiments showed that both VOC-NE and VOC-NE-ISG could reduce the neurological deficit score of model rats, reducing the size of cerebral infarction, with a significant effect on improving ischemic stroke. Overall, VOC-NE-ISG may be a promising intranasal nanomedicine for the effective treatment of ischemic stroke.

Phylogenetic Analysis of Elaeagnus L. in China: A Basis for Genetic Improvement of a Berry Crop.

Elaeagnus L. is found in wild or grown as ornamental plants and is increasingly regarded as underutilized berry shrubs by breeders. This genus has cosmopolitan distribution with various species widely distributed in China, Europe, the United States, and Canada. Interspecific hybrids, which have been reported several times, have attracted intense interest from plant breeders attempting to develop a fruit crop of Elaeagnus. Orthogonal projections to latent structures discriminant analysis (OPLS-DA) is a powerful statistical modeling tool that provides insights into separations between experimental groups. In this study, the molecular phylogeny of Elaeagnus species was first discussed using the ITS and matK sequences for guiding the construction of a genetic basis pool. A morphological OPLS-DA clustering model based on the genetic divergence was also constructed for the first time, which effectively realized the morphological grouping of Chinese Elaeagnus species. The results showed that a total of 10 wild species widely distributed in China have the potential to develop fruit crops. Particularly, Elaeagnus conferta has the potential to provide a founder species with a large fruit size, while Elaeagnus Gonyanthes has the potential to provide important genetic resources with long pedicel. Elaeagnus lanceolata and Elaeagnus delavayi could be used to domesticate hybrids without spines, and the other five climbing shrubs could be used to develop high-yield crown-type commercial cultivars for automated field management. The top five contributing morphological traits affecting the current clustering model were V9 (flower color), V1 (flowering), V5 (evergreen or deciduous), V3 (leaf size), and V2 (fruiting). Furthermore, the grouping analysis indicated that the V9 was the most important factor affecting morphological clustering. Thereafter, the temporally calibrated phylogeny inferred from the matK sequence was used to reconstruct the origin and evolution of the genus Elaeagnus, and the results inferred an interesting geographic distribution pattern and potential cross-species interactions of Elaeagnus species at low latitudes in China. Our study also highlighted dispersal pattern investigation and genetic background analysis to improve future practices and policies related to species introduction of genetic basis pool.

Neobaicalein Inhibits Th17 Cell Differentiation Resulting in Recovery of Th17/Treg Ratio through Blocking STAT3 Signaling Activation.

Huangqin is the dried root of Scutellaria baicalensis Georgi, which has been widely utilized for heat-clearing (Qingre) and dewetting (Zaoshi), heat-killed (Xiehuo) and detoxifying (Jiedu) in the concept of Traditional Chinese Medicine and is used for treating inflammation and cancer in clinical formulas. Neobaicalein (NEO) is of flavonoid isolated from Huangqin and has been reported to possess prominent anti-inflammatory effects in published work. Th17/Treg balance shift to Th17 cells is an essential reason for autoimmune inflammatory diseases. However, the role NEO plays in Th17 and Treg and the underlying mechanism has not been elucidated yet. Network pharmacology-based study revealed that NEO predominantly regulated IL-17 signaling pathway. Moreover, our result shown that NEO (3-30 µmol/L) down-regulated Th17 differentiation and cellular supernatant and intracellular IL-17A level and tumor necrosis factor α production in a concentration-dependent manner. The further mechanism research revealed that NEO also specifically inhibited phosphorylation of STAT3(Tyr725) and STAT4 (Y693) without influence on activation of STAT5 and STAT6 in splenocytes. Immunofluorescence results illuminated that NEO effectively blocked STAT3 translocated into nucleus. Interestingly, NEO at appreciated dose could only inhibit Th17 cell differentiation and have no effect on Treg differentiation. The present study revealed that NEO effectively inhibited Th17 cell differentiation through specifically blocking the activation of STAT3 signaling without inactivation of STAT5 and STAT6. Additional inhibitory effect on activation of STAT4 by NEO also suggested the potential for antagonism against Th1 differentiation. All work suggested that NEO may be a potential candidate for immunoregulation and treating autoimmune inflammatory diseases through inhibiting immune cell viability and T cell differentiation.

Discovery of chemical markers for improving the quality and safety control of Sinomenium acutum stem by the simultaneous determination of multiple alkaloids using UHPLC-QQQ-MS/MS.

Sinomenium acutum stem is a popular traditional Chinese medicine used to treat bone and joint diseases. Sinomenine is considered the only chemical marker for the quality control of S. acutum stem in mainstream pharmacopeias. However, higenamine in S. acutum stem is a novel stimulant that was banned by the World Anti-Doping Agency in 2017. Therefore, enhancing the quality and safety control of S. acutum stem to avoid potential safety risks is of utmost importance. In this study, a fast, sensitive, precise, and accurate method for the simultaneous determination of 11 alkaloids in S. acutum stem by ultrahigh-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC-QQQ-MS/MS) was established. This method successfully analyzed thirty-five batches of S. acutum stem samples. The average contents of sinomenine, magnoflorine, coclaurine, acutumine, higenamine, sinoacutine, palmatine, magnocurarine, columbamine, 8-oxypalmatine, and jatrorrhizine were 24.9 mg/g, 6.35 mg/g, 435 µg/g, 435 µg/g, 288 µg/g, 44.4 µg/g, 22.5 µg/g, 21.1 µg/g, 15.8 µg/g, 9.30 µg/g, and 8.75 µg/g, respectively. Multivariate analysis, including principal component analysis (PCA), orthogonal partial least square method-discriminant analysis (OPLS-DA), and hierarchical cluster analysis (HCA), were performed to characterize the importance and differences among these alkaloids in S. acutum stem samples. As a result, sinomenine, magnoflorine, coclaurine, acutumine, and higenamine are proposed as chemical markers for quality control. Higenamine and coclaurine are also recommended as chemical markers for safety control. This report provides five alkaloids that can be used as chemical markers for improving the quality and safety control of S. acutum stem. It also alerts athletes to avoid the risks associated with consuming S. acutum stem.

Discovery of chemical markers for identifying species, growth mode and production area of Astragali Radix by using ultra-high-performance liquid chromatography coupled to triple quadrupole mass spectrometry.

BACKGROUND: Astragali Radix (AR) is a well-known Chinese herbal medicine. The quality of AR can be affected by many factors such as species, growth mode and production area, but there are still no chemical markers to distinguish it. PURPOSE: To explore chemical markers for improving the quality assessment of AR and discover chemical markers for identifying species, growth mode and production area of AR. METHODS: A highly sensitive, efficient and accurate method based on ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QQQ-MS/MS) for simultaneous quantitative determination of 14 major chemical components (five flavonoids and nine triterpene saponins) in 94 batches of AR from China, Republic of Korea and Germany was developed for the first time. To explore chemical markers and assess changes in the contents of 14 compounds in the 94 batches of AR samples from different regions, hierarchical clustering analysis (HCA) and principal component analysis (PCA) were performed. RESULTS: Astragaloside III was not only an important chemical marker for distinguishing two species of AR, i.e.: Astragalus mongholicus and A. membranaceus, but also a potential chemical marker for the classification of cultivated and semi-wild AR. In addition, in the batches of cultivated AR, the content of isoastragaloside II and cyclocephaloside II were greater in batches from the region of Shaanxi Province than that of other Provinces in China, but the content of calycosin-7-O-ß-D-glucoside and astragaloside IV, which are the quality control markers of AR required by the Chinese Pharmacopoeia, were higher than that of other Provinces in China. In addition, the content of calycosin-7-O-ß-D-glucoside, ononin, calycosin and astragaloside I could be used to identify samples of AR collected from China, Republic of Korea and Germany. CONCLUSION: This UHPLC-QQQ-MS/MS method could be applied to the quantitative evaluation of AR and could be an important and meaningful reference to develop chemical markers for quality control of AR.

Discovery of Quality Markers in Hugan Qingzhi Formula by Integrating a Lipid-Lowering Bioassay with UHPLC-QQQ-MS/MS.

Nonalcoholic fatty liver disease (NAFLD) is a prevalent chronic liver disease. The Hugan Qingzhi formula (HGQZ) has been proven effective in treating NAFLD through clinical and pharmacological mechanism studies. A screening study of the chemical components was carried out to better control the quality of this formula. Current research has combined biological activity assessment with chemical analysis to screen and identify the bioactive compounds in HGQZ for use as potential quality markers (Q-markers) to control the quality of this herbal product. The HGQZ extracted by three different solvents was evaluated in a free fatty acid-induced hepatic steatosis LO2 cell model. Simultaneously, the twelve major chemical constituents of these extracts were quantitatively measured by ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QQQ-MS/MS). Extraction with 50% ethanol showed the most potent lipid-lowering effect in steatosis LO2 cells and the highest extraction rate of major chemical constituents. Correlation analysis was used to establish the relationship between the biological activities and chemical characteristics of these extracts. The results showed that the contents of typhaneoside, hyperoside, isoquercitrin, isorhamnetin-3-O-neohesperidoside, notoginsenoside R1, and alisol B 23-acetate were positively correlated to the lipid-lowering effect. The subsequent bioassay confirmed that typhaneoside, isoquercitrin, and alisol B 23-acetate played the role of reducing the lipid effect. In conclusion, 50% of ethanol extraction produced the most active extract of HGQZ. Typhaneoside, isoquercitrin, and alisol B 23-acetate could be considered potential Q-markers for the quality control of HGQZ.

Chemical profiling of Euphorbia fischeriana Steud. by UHPLC-Q/TOF-MS.

Ultra-high-performance liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry (UHPLC-Q/TOF-MS) in positive ion mode was used to profile and identify the major constituents in Euphorbia fischeriana Steud. (E. fischeriana). An Agilent ZORBAX SB-C18 column was used to separate the crude extract of E. fischeriana, and the mobile phases were acetonitrile and 0.1% aqueous formic acid (v/v). A total of 40 peaks were detected, and 37 components, including phloroglucinol derivatives, diterpenoids and nitrogenous compounds, were identified based on their accurate masses and fragmentation characteristics. The chemical structures of 7 of the detected compounds were determined by comparing standard compounds to the compounds that were isolated from E. fischeriana. Reasonable fragmentation mechanisms and key fragment ions of the four jolkinolide components were determined to aid in the identification of the other diterpenoids in E. fischeriana. In addition, the compound corresponding to peak 18, namely, jolkinomethylide, was determined to be new, and its chemical structure was determined and elucidated based on MS and NMR spectroscopic data. The present study provided valuable information on the new components in E. fischeriana and established a practical method for evaluating the quality of E. fischeriana.

A new approach for identification of medicinal almonds by fourier transform infrared spectroscopy and systematic clustering of characteristic peaks.

Medicinal almonds have been used for over 2 000 years and its clinical efficacy includes relieving cough and asthma. The domestic market in China is flooded with different kinds of dried almonds, such as bitter almond (Armeniacae Semen Amarum, AAS), sweet almond (Armeniacae Semen Dulce, ADS), salted almond (Armeniacae Semen Salsa, ASS), and their sulfur-fumigating products (Armeniacae Semen Sulphur Fumabat, ASFS). Wide varieties of almonds may lead to uncertain efficacy, aberrant quality, and even increased safety risk. However, the authentication method for medicinal almonds has not been reported, although imposters may lead to ineffective medical response. In the present study, Fourier transform infrared spectroscopy (FTIR) and the 2-dimensional infrared (2D-IR) spectroscopy were used to identify different almonds, which were extracted with different solvents including water, methanol, ethanol, chloroform and ethyl acetate, respectively. A new simple FTIR method was developed in the present study. According to the gradient solvent polarity, a new 2D IR method was first developed, and the commodities of almonds in China were analyzed by using the FTIR spectroscopy supported by hierarchical clustering of characteristic peaks. Moreover, 5-hydroxymethyl-2-furfural could be used as a detection index and control target in the quality control of medicinal almonds.

Simultaneous quantification of nine aconitum alkaloids in Aconiti Lateralis Radix Praeparata and related products using UHPLC-QQQ-MS/MS.

Aconiti Lateralis Radix Praeparata (Fuzi) is obtained from processed daughter roots of Aconitum carmichaeli, a toxic plant with a high medical value well known in Chinese medicine. In addition to the known toxic alkaloids (aconitine, mesaconitine, and hypaconitine) and bioactive alkaloids (benzoylaconine, benzoylmesaconine, and benzoylhypaconine), three rarely found alkaloids have been previously reported in Fuzi, i.e., yunaconitine, 8-deacetyl-yunaconitine, and crassicauline A, and they were reported in recent years to cause potential risk to patients who took Fuzi or related products. To better control the quality of this herb and its related products and ensure safe use, developing a method to simultaneously determine these 9 alkaloids is important. In this research, sensitive and accurate ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry method was established and used to examine 51 Fuzi and 27 Fuzi-containing products. Unexpectedly, 8-deacetyl-yunaconitine was detected in 17 Fuzi samples (33.3%) and 3 Fuzi-containing products (11.1%); yunaconitine in 10 Fuzi samples (19.6%) and 10 Fuzi-containing products (37.0%); and crassicauline A in 3 Fuzi samples (5.8%). Industry and clinics should be aware of the unusually high detection rate of these three toxic alkaloids in the Fuzi herb and its related products and take the necessary precautions to protect patients from any potential risk.

Simultaneous Determination of Multiple Components in Guanjiekang in Rat Plasma via the UPLC-MS/MS Method and Its Application in Pharmacokinetic Study.

Guanjiekang (GJK) that is formed by five medicinal herbs including Astragali Radix, Aconiti Lateralis Radix Praeparaia, Glycyrrhizae Radix et Rhizoma, Corydalis Rhizoma and Paeoniae Radix Alba was used for the treatment of rheumatoid arthritis (RA). However, the pharmacokinetic (PK) profile of active components in GJK remains unclear. This study aims to evaluate the pharmacokinetic behavior of seven representative active constituents in GJK (i.e., benzoylhypaconine, benzoylmesaconine, paeoniflorin, tetrahydropalmatine, calycosin-7-glucoside, formononetin and isoliquiritigenin) after oral administration of GJK in rats. A rapid, sensitive and reliable ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) method has been successfully developed for the simultaneous determination of these seven constituents in rat plasma. Chromatographic separation was achieved on a C18 column with a gradient elution program that consists of acetonitrile and water (containing 0.1% formic acid) at a flow rate of 0.35 mL/min. Detection was performed under the multiple reaction monitoring (MRM) in the positive electrospray ionization (ESI) mode. The calibration curves exhibited good linearity (R² > 0.99) over a wide concentration range for all constituents. The accuracies ranged from 92.9% to 107.8%, and the intra-day and inter-day precisions at three different levels were below 15%. Our PK results showed that these seven compounds were quickly absorbed after the administration of the GJK product, and Tmax ranged from 30 min to 189 min. The in vivo concentrations of paeoniflorin and isoliquiritigenin were significantly higher than the reported in vitro effective doses, indicating that they could partly contribute to the therapeutic effect of GJK. Therefore, we conclude that pharmacokinetic studies of representative bioactive chemicals after administration of complex herbal products are not only necessary but also feasible. Moreover, these seven compounds that were absorbed in vivo can be used as indicator standards for quality control and for determining pharmacokinetic behavior of herbal medicines in clinical studies.

Simultaneous determination of thirteen major active compounds in Guanjiekang preparation by UHPLC-QQQ-MS/MS.

An ultra high performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QQQ-MS/MS) method has been developed to evaluate the quality of a pharmaceutical herbal preparation, Guanjiekang (GJK), through a simultaneous determination of 13 major active compounds with a huge difference in level of content. Chromatographic separation was achieved on a Waters Acquilty UPLC C18 column (2.1 × 100 mm, 1.7 µm) with a mobile phase consisting of acetonitrile and buffer solution (10mM ammonium acetate containing 0.1% acetic acid) under a gradient elution manner. A triple quadrupole mass spectrometer was operated in positive ionization mode with multiple reaction monitoring for the detection of the 13 compounds. All calibration curves showed excellent linear regressions (R(2)>0.999) within the test range. The precision, repeatability and stability of the 13 compounds were below 5.0% in terms of RSD. The recoveries were 99.2-103.9% with RSD of 0.23-3.30% for GJK samples. The method was successfully used for the analysis of samples of GJK preparation and showed that the lowest level was in aconitine (0.582 ± 0.143 ng/g) and the highest was in paeoniflorin (16.80 ± 0.886 mg/g), with a 41800 folds of difference. In conclusion, a rapid, sensitive, precise, accurate, and reliable UHPLC-QQQ-MS/MS method has been developed for the simultaneous detection of 13 active compounds with massive difference in level of content in the pharmaceutical samples of GJK preparation, which can be applied for the quality control of GJK product.

Characterization of phloroglucinol derivatives and diterpenes in Euphorbia ebracteolata Hayata by utilizing ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

Euphorbia ebracteolata Hayata (E. ebracteolata) is a Chinese herbal medicine used for the treatment of tumor diseases. An ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) based chemical profiling approach was established for the rapid separation and characterization on phloroglucinol derivatives and diterpenes in E. ebracteolata. Three phloroglucinol derivatives and nine diterpenes were identified by exact mass measurement and were further confirmed by Ms2 data. In addition, the chemical profiles of six compounds were acquired by reference standards. Furthermore, the fragmentation rules of phloroglucinol derivatives and diterpenes of E. ebracteolata were analyzed, and each chromatographic peak was classified.