A comparative study of the anti-fatigue activity of extracts from different parts of Cistanche tubulosa(Schenk)Wight

Objective:To evaluate the anti-fatigue effects of different extracts from Cistanche tubulosa(Schenk)Wight(C.tubulosa,Rou Cong Rong),focusing on central and exercise-induced fatigue in mice.This study investigated the pharmacological effects of the total oligosaccharides,polysaccharides,and phenylethanoid glycosides(CPhGs)extracted from C.tubulosa.Methods: Models of sleep deprivation and forced swimming fatigue were established to simulate central and exercise-induced fatigue.The mice were treated with different extracts of C.tubulosa,and their effects were assessed using behavioral tests to measure exercise capacity,learning,and memory function.Biochemical analyses were performed to evaluate the changes in serum and brain neurotransmitter levels,liver and muscle glycogen storage,and various fatigue-related biomarkers.Results: This study found that treatment with C.tubulosa extract improved exercise capacity,learning,and memory in mice.Total oligosaccharides from C.tubulosa enhanced adrenocorticotropic hormone,cholinesterase,and thyroid-stimulating hormone levels,reduced cortisol levels in central fatigue models,and ameliorated biochemical markers of exercise-induced fatigue,including lowering lactic acid,blood urea nitrogen,and malondialdehyde levels.Among the tested extracts,the total oligosaccharides showed the most comprehensive anti-fatigue effects.Conclusion: The anti-fatigue effects of C.tubulosa,particularly those of its total oligosaccharides,are pronounced in both central and exercise-induced fatigue.These effects are mediated by the regulation of neurotransmitter levels,enhancement of glycogen storage,and improvement of antioxidant enzyme activity,suggesting potential therapeutic benefits in fatigue-related conditions.

Effects of parasitic plant Cistanche deserticola on chlorophyll a fluorescence and nutrient accumulation of host plant Haloxylon ammodendron in the Taklimakan Desert

The parasitic plant Cistanche deserticola attaches to Haloxylon ammodendron, a perennial shrub with high tolerance to salinity and drought. However, little was known about the parasite-host relation between the two species. Effects of the parasite on chlorophyll a fluorescence and nutrient accumulation in the host plant （H. am- modendron） were investigated in the Taklimakan Desert. Some photosynthetic parameters of both host and non-host H. ammodendron plants were measured by in vivo chlorophyll a fluorescence technology in the field. The assimilating branches of host and non-host plants were collected and nutrient and inorganic ion contents were analyzed. The results from field experiments showed that the infection of C. deserticola reduced the non-photochemical quenching of the variable chlorophyll fluorescence （NPQ） and the potential maximum quantum yield for primary photochemistry （Fv/Fm） of the host. Compared with non-host plants, the host H. ammodendron had low nutrient, low inorganic ion contents （Na~ and K~） and low K~/Na~ ratios in the assimilating branches. It suggested that C. deserticola infection reduced the nutrient acquisition and caused damage to the photoprotection through thermal dissipation of the energy of the photosystem II in the host, resulting in a decrease in the tolerance to salinity and high radiation. It was concluded that the attachment of the parasite plant （C. deserticola） had negative effects on the growth of its host.

The Prospect of Application of Extractive Reference Substance of Chinese Herbal Medicines

The emerging development of Extractive Reference Substance (ERS) is a methodology that meets the needs for quality control for Chinese Herbal Medicines (CHM) and respects the holistic viewpoint of Traditional Chinese Medicine (TCM) and its clinical use of multiple ingredients with synergistic effects. The convention of using just a selected few Chemical Reference Substances (CRS) cannot adequately assess the quality of intact CHM. A validated chemical spectrum of an ERS provides the global characteristics in order to more specifically identify and assess targeted CHM. This paper describes the fundamental concepts, potential significance, and basic criteria of ERS, along with methods of preparation and calibration. Given the diversity of CHM, the various problems that will occur in establishing the proper process of ERS will need to be solved in a step by step manner. The ERSs of Ziziphi spinosae semen and ERS of Fritillaria thunbergii bulbus are given as examples of the development of ERS and demonstrate why we are optimistic about the utility of this approach.

Baoyuan decoction alleviates myocardial infarction through the regulation of metabolic dysfunction and the mitochondria-dependent caspase-9/3 pathway

Objective:Baoyuan decoction(BYD)is a traditional Chinese formula with myocardial protection efficacy validated by modern pharmacological tests.The present study aimed to investigate the effect and mechanism of BYD on alleviating myocardial infarction(MI).Methods:Nuclear magnetic resonance-based serum and urinary metabolomics were employed to explore the metabolic regulation effects of BYD in rats with MI induced by left anterior descending ligation.Oxygen-glucose deprivation/recovery(OGD/R)model in H9c2 cells and multiple molecular biology approaches were used to clarify the underlying action mechanisms of BYD.Results:BYD treatment recovered the serum and urinary metabolite profiles of the MI rats toward normal metabolic status and significantly improved mitochondrial energy metabolism and apoptosis pathways perturbed by MI.Analysis of the molecular mechanism of BYD indicated that it suppressed OGD/R-induced H9c2 cell apoptosis in a concentration-dependent manner by inhibiting the mitochondria-dependent caspase-9/3-poly ADP-ribose polymerase pathway.Conclusions:Our results demonstrate that BYD protects against myocardial apoptosis via the mitochondrial metabolic and apoptosis pathways.They also provide novel insights into the clinical application of BYD for the treatment of ischemic heart diseases.

Molecular characterization and structure basis of a malonyltransferase with both substrate promiscuity and catalytic regiospecificity from Cistanche tubulosa

Enzymatic malonylation of natural glycosides provides a promising alternative method for drug-like malonylated glycosides supply.However,the catalytic potential and structural basis of plant malonyltransferase are far from being fully elucidated.This work identified a new malonyltransferase CtMaT1 from Cistanche tubulosa.It displayed unprecedented mono-and/or di-malonylation activity toward diverse glucosides with different aglycons.A“one-pot”system by CtMaT1 and a malonyl-CoA synthetase was established to biosynthesize nine new malonylated glucosides.Structural investigations revealed that CtMaT1 possesses an adequately spacious acyl-acceptor pocket capable of accommodating diverse glucosides.Additionally,it recognizes malonyl-CoA through strong electrotactic and hydrogen interactions.QM/MM calculation revealed the H167-mediated SN2 reaction mechanism of CtMaT1,while dynamic simulations detected the formation of stable hydrogen bonds between the glucose-6-OH group and H167,resulting in its high malonylation regiospecificity.Calculated energy profiles of two isomeric glycosides highlighted lower reaction energy barriers towards glucoside substrates,emphasizing CtMaT1's preference for glucosides.Furthermore,a mutant CtMaT1H36A with notably increased di-malonylation activity was obtained.The underlying molecular mechanism was illuminated through MM/GBSA binding free energy calculation.This study significantly advances the understanding of plant acyltransferases from both functional and protein structural perspectives,while also providing a versatile tool for enzymatic malonylation applications in pharmacology.

Chemical constituents from the aerial parts of Waltheria indica Linn.

A phytochemical investigation on the aerial parts of Waltheria indica Linn.led to the isolation of 16 compounds,including five terpenoids(1–5),four coumarins(6–9),six flavonoids(10–15),and one phenylpropanoid glycoside(16).The structures of these compounds were identified by spectroscopic data analysis and comparison with those reported in the literatures.Except for 12 and 15,all the compounds were isolated from W.indica for the first time.Moreover,coumarins were firstly reported to be obtained from this herb.The NO production inhibitory activities of all the isolated compounds in LPS-induced BV-2 cells were also presented.

Rapid Simultaneous Determination of Four Ganoderic Acids in Ganoderma(Chinese Name:Lingzhi)by Direct Infusion-Multiple Reaction Monitoring Cubed

Attributing to the rapid demand expansion for the edible medicinal materials in the market,the limited throughput of highperformance liquid chromatography-multiple reaction monitoring(HPLC-MRM)cannot fully address the measurement workload for a huge number of testing samples.Hence,it is urgent to pursue more efficient approaches for the quality evaluation.Because of the greater selectivity of MRM cubed(MRM^(3))over MRM,there might be a chance to omit the time-intensive LC separation.In current study,we attempted to develop a direct infusion(DI)-MRM^(3) program,and the applicability was thereafter assessed through simultaneous determination of four ganoderic acids(GAs)in one of the most famous tonic herbal medicines namely Ganoderma(Chinese name:Lingzhi).Primary parameters such as Q1>Q3>QLIT ion transitions,collision energy(CE),and excitation energy were optimized by programming online energy-resolved mass spectrometry with authentic compounds.A single DI-MRM measurement merely costed four minutes,and in spite of the wide occurrences of isomers,satisfactory selectivity was achieved.Method validation assays demonstrated the method to be sensitive,precise,accurate,and reproducible.The quantitative results from DI-MRM^(3) were also justified by conducting LC-MRM measurements in parallel.Significant differences occurred for the content patterns between the two original sources namely Ganoderma lucidum and G.sinense,and,moreover,either cultivar or harvest time showed dramatical influence on the quantitative features of the four targeted GAs.More importantly,DI-MRM3 is a meaningful analytical option for rapid quantitative analysis of herbal medicines,because of the comparable reliability,nonetheless,less consumptions of both measurement time and solvent,compared with LC-MRM.

Triple three-dimensional MS/MS spectrum facilitates quantitative ginsenosides-targeted sub-metabolome characterization in notoginseng

Although serving as the workhorse,MS/MS cannot fully satisfy the analytical requirements of quantitative sub-metabolome characterization.Because more information intrinsically correlates to more structural and concentration clues,here,efforts were devoted to comprehensively tracing and deciphering MS/MS behaviors through constructing triple three-dimensional(3×3D)-MS/MS spectrum.Ginsenosides-targeted metabolomics of notoginseng,one of the most famous edible medicinal plants,was employed as a proof-of-concept.Serial authentic ginsenosides were deployed to build the correlations between 3×3D-MS/MS spectra and structure/concentration features.Through assaying ginsenosides with progressive concentrations using QTOF-MS to configure 1st 3D spectrum,the generations of MS1 spectral signals,particularly multi-charged multimer anions,e.g.,[2Me2H]^(2-) and[2M+2HCOO]^(2-) ions,relied on both concentration and the amount of sugar chains.By programming progressive collision energies to the front collision cell of Qtrap-MS device to gain 2^(nd) 3D spectrum,optimal collision energy(OCE)corresponding to the glycosidic bond fission was primarily correlated with the masses of precursor and fragment ions and partially governed by the glycosidation site.The quantitative relationships between OCEs and masses of precursor and fragment ions were utilized to build large-scale quantitative program for ginsenosides.After applying progressive exciting energies to the back collision chamber to build 3^(rd) 3D spectrum,the fragment ion and the decomposition product anion exhibited identical dissociation trajectories when they shared the same molecular geometry.After ginsenosides-focused quantitative metabolomics,significant differences occurred for sub-metabolome amongst different parts of notoginseng.The differential ginsenosides were confirmatively identified by applying the correlations between 3×3D-MS/MS spectra and structures.Together,3×3D-MS/MS spectrum covers all MS/MS behaviors and dramatically facilitates sub-metabolome characterization from both quantitative program development and structural identification.

Author correction to‘Pharmacologically targeting molecular motor promotes mitochondrial fission for anti-cancer’[Acta Pharm Sin B 11(2021)1853–1866]

The authors regret that the representative images of tumor tissue staining for the intraperitoneal injection of J13 in the left panel of Fig.5E and G(Page 1862)were incorrect due to an inadvertent mistake of copying and pasting in the process of assembling figures with Adobe Photoshop software.In our studies,at least three biological replicates were included in each treatment group and at least three images were taken for different fields of each sample.The corrected version of Fig.5E and G have been provided below,and the change did not affect the results and conclusions of this study.The original data of these figures have been provided to the Editorial Office,and the corresponding authors or the Editorial Office can be contacted for original data access.

Comparative analysis of four edible mushrooms based on HPLC fingerprint and pattein recognition analysis

A high performance liquid chromatography-ultraviolet(HPLC-UV)fingerprint method for the overall chemical analysis of edible mushrooms was established based on Auricularia heimuer for the first time,and then applied to analyze 60 batches of A.heimuer,Auricularia cornea.Auricularia cornea*Yu Muer’and TremeUa fuciformis.A total of 9 characteristic peaks of A.heimuer.11 characteristic peaks of A.cornea,6 characteristic peaks of A.cornea‘Yu Muer’,and 9 characteristic peaks of I fuciformis were designated.Then,a combinatory analysis,including similarity evaluation,hierarchical cluster analysis and principal component analysis,revealed the chemical consistency and difference between samples from the same and different species.The HPLC fingerprint method established in this paper could be used to characterize the components of A.heimuer,A.cornea,A.cornea‘Yu Muer’,and T.fuciformis and discriminate the 4 edible mushrooms effectively in combination with pattern recognition analysis.

异源物代谢的表观遗传学变异影响抗癫痫药3,4-DCPB药物代谢动力学表型个体差异

抗癫痫药物治疗是控制癫痫的主要方法,但由于个体间对药物处置的差异性,患者对目前治疗的反应性并不一致。本研究通过在健康志愿者中进行的临床Ⅰ期剂量递增试验,考察了遗传和表观遗传变异是否影响抗癫痫药物氯桂丁胺(3,4-DCPB)的药代动力学表型。采用液相色谱-串联质谱(LC-MS/MS)法测定血浆中3,4-DCPB母药及其主要代谢物M1的浓度。通过基因分型和DNA甲基化水平分析细胞色素P4502D6(CYP2D6)、CYP2C9、CYP1A2、CYP2C19、CYP3A5、转运体ABCB1(C1236T)、核受体AhR、CAR和PXR的单核苷酸多态性(SNPs)。与野生型CYP2D6*1/*1纯合子(广泛代谢型,EMs)相比,变异等位基因CYP2D6*10携带者(中间代谢型,IMs)中,代谢产物M1与3,4-DCPB母药的药时曲线下面积(AUC0–t)的比值更低,血浆半衰期(t1/2)更久,DNA甲基化水平更高。这些数据表明胞嘧啶的丢失(CYP2D6*10,C>T)所诱导的表观基因突变可能解释3,4-DCPB基因型、表观基因型和药代动力学表型在个体差异之间的关系,为癫痫的个性化治疗提供新的思路。

Application of ~1H NMR-based metabolomics for discrimination of different parts and development of a new processing workflow for Cistanche deserticola

Cistanche deserticola(CD) is one of the two authoritative source plants of Cistanches Herba, a well-known medicinal plant. Herein,~1H NMR spectroscopy was employed to characterize the chemical profile and to distinguish the different parts, as well as to propose a new processing workflow for CD.Signal assignment was achieved by multiple one and two dimensional NMR spectroscopic techniques in combination with available databases and authentic compounds. The upper parts of the plant were distinguished from the lower parts by combining ~1H NMR spectroscopic dataset with multivariate statistical analysis. A new processing method that hyphenated steaming with freeze-drying, was demonstrated to be superior to either steaming coupled with oven-drying or direct freeze-drying via holistic ~1H NMR-based metabolomic characterization. Phenylethanoid glycosides, mainly echinacoside and acteoside, were screened out and confirmed as the chemical markers responsible for exhibiting the superiority of the new processing workflow, whereas serial primary metabolites, especially carbohydrates and tricarboxylic acid cycle metabolites, were found as the primary molecules governing the discrimination between the upper and lower parts of the plant. Collectively,~1H NMR spectroscopy was demonstrated as a versatile analytical tool to characterize the chemical profile and to guide the indepth exploitation of CD by providing comprehensive qualitative and quantitative information.

Schisandrol A protects AGEs-induced neuronal cells death by allosterically targeting ATP6V0d1 subunit of V-ATPase

Diabetes have been shown to cause progressive neuronal injury with pain and numbness via advanced glycation end-products(AGEs)-induced neuronal cell apoptosis;however, the valuable drug targets for diabetic neuropathy have been poorly reported so far. In this study, we discovered a natural small-molecule schisandrol A(SolA) with significant protective effect against AGEs-induced neuronal cell apoptosis. ATP6V0D1, a major subunit of vacuolar-type ATPase(V-ATPase) in lysosome was identified as a crucial cellular target of SolA. Moreover, SolA allosterically mediated ATP6V0D1 conformation via targeting a unique cysteine 335 residue to activate V-ATPase-dependent lysosomal acidification.Interestingly, SolA-induced lysosome pH downregulation resulted in a mitochondrial-lysosomal crosstalk by selectively promoting mitochondrial BH3-only protein BIM degradation, thereby preserving mitochondrial homeostasis and neuronal cells survival. Collectively, our findings reveal ATP6V0D1 is a valuable pharmacological target for diabetes-associated neuronal injury via controlling lysosomal acidification, and also provide the first small-molecule template allosterically activating V-ATPase for preventing diabetic neuropathy.

Authentic compound-free strategy for simultaneous determination of primary coumarins in Peucedani Radix using offline high performance liquid chromatography–nuclear magnetic resonance spectroscopy–tandem mass spectrometry

Herein, a strategy is proposed for the simultaneous determination of primary coumarins in Peucedani Radix(Chinese name: Qianhu). The methodology consists of three consecutive steps: 1) Semi-preparative LC in combination with a home-made automated fraction collection module to fragment the universal metabolome standard into ten fractions(Frs. I–X); 2) LC–accurate MS/MS and quantitative1 H NMR spectroscopy conducted in parallel to acquire the qualitative and quantitative data of each fraction; 3) Robust identification and quantification of components by use of LC coupled to multiple reaction monitoring. In this final step, the most significant fractions(Frs. III–X) were pooled to serve as the pseudo-mixed standard solution. Meticulous online parameter optimization was performed to obtain the optimal parameters, including ion transitions and collision energies. Concerns were particularly paid onto pursuing the parameters being capable of monitoring regiospecific isomers, notably praeruptorin E vs. 3′-isovaleryl-4′-angeloylkhellactone. The quantitative performance of the method was validated according to diverse assays. Eleven primary coumarins(1–11) were unambiguously identified and absolutely quantified, even though no external reference compound was used. Above all, the integrated strategy not only provides a feasible pipeline for the quality assessment of Peucedani Radix, but more importantly, shows the potential for authentic compound-free quantitative evaluation of traditional Chinese medicines.

Simultaneous determination of five phenylethanoid glycosides in Cistanches Herba using quantitative analysis of multi-components by single marker

In the present study, a method for the quantitative analysis of multi-components by single marker(QAMS) has been developed and validated for the simultaneous determination of echinacoside(ECH), tubuloside A, acteoside, isoacteoside, and2’-acetylacteoside in Cistanches Herba. ECH was used as the internal standard(IS) to obtain the relative correction factors(RCFs) of the other four phenylethanoid glycosides(PhGs);meanwhile, various influencing factors on RCFs were investigated under different conditions. The content of each component was calculated with RCF. The results were compared with those obtained by the external standard method(ESM) to verify the feasibility and accuracy of the established QAMS method. No significant difference was found in the quantitative results of 10 batches of Cistanches Herba between QAMS and ESM. The proposed QAMS method for simultaneous determination of PhGs in Cistanches Herba is accurate and feasible, providing an efficient and economical approach for the quality control of Cistanches Herba.

Lignans and flavonoids from Artemisia brachyloba

A phytochemical investigation on the aerial parts of Artemisia brachyloba led to the isolation of 13 compounds（1-13） for the first time, including eight lignans（1-8） and five flavonoids（9-13）. The structures of the isolated compounds were elucidated by analysis of the NMR spectroscopic data and comparison with the literature. The 1 H and 13 C NMR data of 1 were fully assigned for the first time by the 2 D NMR analysis. All the isolates were evaluated for their inhibitory effects on nitric oxide production in lipopolysaccharide-challenged BV-2 microglial cells.

Pharmacologically targeting molecular motor promotes mitochondrial fission for anti-cancer

Mitochondrial shape rapidly changes by dynamic balance of fusion and fission to adjust to constantly changing energy demands of cancer cells.Mitochondrial dynamics balance is exactly regulated by molecular motor consisted of myosin and actin cytoskeleton proteins.Thus,targeting myosin eactin molecular motor is considered as a promising strategy for anti-cancer.In this study,we performed a proof-of-concept study with a natural-derived small-molecule J13 to test the feasibility of anti-cancer therapeutics via pharmacologically targeting molecular motor.Here,we found J13 could directly target myosin-9(MYH9)eactin molecular motor to promote mitochondrial fission progression,and markedly inhibited cancer cells survival,proliferation and migration.Mechanism study revealed that J13 impaired MYH9 eactin interaction to inactivate molecular motor,and caused a cytoskeleton-dependent mitochondrial dynamics imbalance.Moreover,stable isotope labeling with amino acids in cell culture(SILAC)technology-coupled with pulldown analysis identified HSPA9 as a crucial adaptor protein connecting MYH9 eactin molecular motor to mitochondrial fission.Taken together,we reported the first natural small-molecule directly targeting MYH9 eactin molecular motor for anti-cancer translational research.Besides,our study also proved the conceptual practicability of pharmacologically disrupting mitochondrial fission/fusion dynamics in human cancer therapy.

Pharmacodynamic comparison of two different source plants of Murrayae Folium et Cacumen

A comparison of the pharmacodynamic effects of two source plants of Murrayae Folium et Cacumen(MFC),Murraya exotica L.and Murraya paniculata(L.)Jack,was performed in order to supply reference for its multi-source rationality and interchangeability in clinical practice.According to the traditional efficacy of MFC,the effects of promoting Qi,relieving pain,promoting blood circulation and removing blood stasis were systematically evaluated by the models of writhing response in mice,foot swelling in rats,gastric emptying and small intestine propulsion in mice,and acute blood stasis in rats,respectively.The results showed that both M.exotica and M.paniculata could significantly inhibit the writhing reaction induced by acetic acid in mice and the paw swelling induced by carrageenan in rats,reduce IL-6,TNF-αand PGE2 levels in plasma of paw-swelling rats and increase gastric empty rate and intestinal propulsive rate.The above-mentioned effects were dose-dependent,and there was no significant difference between M.exotica and M.paniculata at the same doses.Therefore,M.exotica and M.paniculata had the similar anti-inflammatory,analgesic and gastrointestinal motility promotion effects,which provided a support for the pharmacodynamic equivalence of the multi-source plants of MFC.

Cardioprotective effects of combination of notoginseng total saponins and safflower total flavonoids against myocardial infarction in rats

In this study, the cardioprotective mechanism of the combination of notoginseng total saponins and safflower total flavonoids（CNS） was investigated due to its excellently efficacy against myocardial infarction（MI） in rats. After the left anterior descending coronary artery（LADCA） ligation, rats were orally administered with CNS for 7 consecutive days. CNS prevented MI-induced pathophysiological changes and significantly decreased plasma levels of myocardial enzymes, including creatine kinase MB isoenzyme（CK-MB）, lactate dehydrogenase（LDH） and aspartate aminotransferase（AST）. Further investigation revealed that CNS attenuated the production of inflammatory factors in plasma, including tumor necrosis factor-alpha（TNF-α）, interleukin-6（IL-6） and interleukin-1β（IL-1β）. Moreover, CNS treatment decreased the expression of caspase-3 at the mR NA level in infarct tissue. Our findings demonstrated that the anti-inflammatory and anti-apoptotic properties of CNS might confer its cardioprotection against MI in rats.

Small molecule induces mitochondrial fusion for neuroprotection via targeting CK2 without affecting its conventional kinase activity

Mitochondrial fusion/hission dynamics plays a fundamental role in neuroprotection;however,there is till a severe lack oftherapeutic targets for this biological process.Here,we found that the naturally derived small molecule echinacoside(ECH)signifhcantly promotes mitochondrial fusion progression.ECH selectively binds to the previously uncharacterized casein kinase 2(CK2)α'subunit(CK2a)as a direct cellular target,and genetic knockdown of CK2α'abolishes ECH-mediated mitochondrial fusion.Mechanistically,ECH allosterically regulates CK2α'conformation to recruit basic transcription factor 3(BTF 3)to form a binary proteincomplex.Then,the CK2α'/BTF3 complex facilitatesβ-catenin nuclear translocation to activate TCF/AEF transcription factors andstimulate transcription of the mitochondrial fusion gene Mfn2.trikingly,in a mouse middle cerebral artery occlusion(MCAO)model,ECH administration was found to significantly improve cerebral injuries and behavioral deficits by enhancing Mfn2expression in wild-type but not CK2α'^(+/-)mice.Taken together,our findings reveal,for the first time,that CK2 is essential forpromoting mitochondrial fusion in a Wnt/B-catenin-dependent manner and suggest that pharmacologically targeting CK2 is apromising therapeutic strategy for ischemic stroke.