Toxicity Attenuation Mechanism on Processing Method for Aconiti Lateralis Radix Praeparata in Guilingji Based on Urine Metabolomics / 中国实验方剂学杂志

ObjectiveMetabolomics was used to reveal the mechanism of Aconiti Lateralis Radix Praeparata（ALRP） in attenuating toxicity by processing from the aspects of amino acid metabolism， oxidative stress and energy metabolism by analyzing multiple metabolic pathways. MethodTwenty-four rats were randomly divided into control group， raw group and processed group， 8 rats in each group. The raw and processed group were given with 0.64 g·kg-1 of raw ALRP and processed ALRP respectively every day， the control group was given with an equal amount of normal saline once a day. After continuous administration for 7 days， the urine， serum and heart tissue of rats were collected. Pathological examination of the heart was carried out using hematoxylin-eosin（HE） staining， and the activities of lactate dehydrogenase（LDH） and creatine kinase-MB（CK-MB） in serum and cardiac tissues were detected by microplate assay and immunoinhibition assay. The effects of ALRP on rat heart before and after processing were compared and analyzed. Ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS） was used to perform urine metabolomics analysis， and multivariate statistical analysis was used to screen for differential metabolites related to ALRP in attenuating toxicity by processing， and pathway enrichment analysis was carried out to explore the processing mechanism. ResultHE staining showed that no obvious pathological changes were observed in the heart tissue of the control group， while obvious infiltration of inflammatory cells such as plasma cells and granulocytes was observed in the heart tissue of the raw group， indicating that the raw ALRP had strong cardiotoxicity. There was no significant difference in HE staining of heart tissue between the processed group and the control group， indicating that the toxicity of ALRP was significantly reduced after processing. Compared with the control group， the activities of LDH and CK-MB were significantly increased in serum and heart tissue of the raw group， and those were significantly decreased in serum and heart tissue of the processed group， suggesting that the myocardial toxicity of processed ALRP was reduced. A total of 108 endogenous differential metabolites associated with the raw ALRP were screened using multivariate statistical analysis in positive and negative modes， of which 51 differential metabolites were back-regulated by the processed ALRP. Biological analysis of the key regulatory pathways and associated network changes showed that the pathways related to toxicity of ALRP mainly included tryptophan metabolism， arginine and proline metabolism， phenylalanine metabolism， aminoacyl-tRNA biosynthesis， alanine， aspartate and glutamate metabolism， etc. The metabolic pathways related to the attenuation of processed ALRP mainly included aminoacyl-tRNA biosynthesis， tryptophan metabolism， phenylalanine， tyrosine and tryptophan biosynthesis， phenylalanine metabolism and caffeine metabolism. ConclusionThe processing technology of ALRP in Guilingji can significantly attenuate the cardiotoxicity of raw products， the mechanism mainly involves amino acid metabolism， oxidative stress and energy metabolism， which can provide experimental bases for the research related to the mechanism of toxicity reduction of ALRP by processing and its clinical safety applications.

Guilingji capsule for Alzheimer's disease: secondary analysis of a randomized non-inferiority controlled trial.

OBJECTIVE: To investigate the effectiveness and safety of Guilingji capsule (, GLJC) in treatment of Alzheimer's disease (AD) patients with kidney-marrow deficiency pattern (KMDP) compared with gingko extract tablets. METHODS: This is a secondary analysis of a large-scale multicenter randomized non-inferiority clinical trial. A total of 120 AD patients with KMDP were enrolled in this study. The participants were randomly categorized into two groups: (a) GLJC group ( = 60) and (b) gingko group ( = 60). The GLJC group was treated with GLJC and gingko extract mimetic tablets, whereas the gingko group received gingko extract tablets and mimetic GLJC. The data on the Mini-Mental State Examination (MMSE), Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog), Activities of Daily Living (ADL), and Chinese Medicine Symptom Scale (CM-SS) was evaluated at 0, 12, and 24 weeks of treatment. The serum levels of acetylcholine (Ach), acetylcholinesterase (AchE), B-cell lymphoma-2 (Bcl-2), and Bcl-2-associated X protein (Bax) in the participants were measured before and after 24 weeks of treatment. The safety was based on the incidence of adverse events. RESULTS: Both interventions significantly increased the MMSE scores of the participants and decreased their ADAS-Cog, ADL, and CM-SS scores ( < 0.01). Compared with the gingko group, the GLJC group had a higher effective rate of improvement in the symptoms of "amnesia" and "dull expression and slow thinking" at the 12th week and 24th week ( < 0.05, < 0.01). In the GLJC group, serum Bcl-2 levels were significantly increased at the 24th week ( < 0.05). Serum Bax and AchE levels of the two groups were significantly decreased at the 24th week ( < 0.01). No treatment-related adverse events were reported in the two groups. CONCLUSIONS: GLJC is equivalent to the gingko extract tablets in terms of improving cognitive function and the quality of life in AD patients with KMDP and has good clinical efficacy and safety. When it comes to improving TCM symptoms and anti-aging, GLJC is even more advantageous.

Characterization of multiple chemical components of GuiLingJi by UHPLC-MS and 1H NMR analysis.

GuiLingJi (GLJ), a classic traditional Chinese medicine (TCM) formula, is composed of over 20 herbs, according to the Pharmacopeia of the People's Republic of China. Owing to its various activities, GLJ has been used in clinical settings for more than 400 years in China. However, the ambiguous chemical material basis limits the development of studies on the quality control and pharmacological mechanisms of GLJ. Therefore, comprehensive characterization of the multiple chemical components of GLJ is of great significance for the modernization of this formula. Given the great variety of herbs in GLJ, both UHPLC-MS and 1H NMR techniques were employed in this study. In addition, solvent extraction with different polarities was used to eliminate signal interference and the concentration of trace components. A variety of MS analytic methods were also used, including implementation of a self-built compound database, diagnostic ion filtering, mass defect filtering, and Compound Discoverer 3.0 analysis software. Based on the above strategies, a total of 150 compounds were identified, including 5 amino acids, 13 phenolic acids and glycosides, 11 coumarins, 72 flavones, 20 triterpenoid and triterpenoid saponins, 23 fatty acids, and 6 other compounds. Moreover, 13 compounds were identified by 1H NMR spectroscopy. The UHPLC-MS and 1H NMR results supported and complemented each other. This strategy provides a rapid approach to analyzing and identifying the chemical composition of Chinese herbal prescriptions. The current study provides basis for further research on the quality control and pharmacological mechanism of GLJ.

Characterization of multiple chemical components of GuiLingJi by UHPLC-MS and 1H NMR analysis / 药物分析学报

GuiLingJi(GLJ),a classic traditional Chinese medicine(TCM)formula,is composed of over 20 herbs,according to the Pharmacopeia of the People's Republic of China.Owing to its various activities,GLJ has been used in clinical settings for more than 400 years in China.However,the ambiguous chemical material basis limits the development of studies on the quality control and pharmacological mechanisms of GLJ.Therefore,comprehensive characterization of the multiple chemical components of GLJ is of great significance for the modernization of this formula.Given the great variety of herbs in GLJ,both UHPLC-MS and 1H NMR techniques were employed in this study.In addition,solvent extraction with different polarities was used to eliminate signal interference and the concentration of trace components.A variety of MS analytic methods were also used,including implementation of a self-built compound database,diagnostic ion filtering,mass defect filtering,and Compound Discoverer 3.0 analysis software.Based on the above strategies,a total of 150 compounds were identified,including 5 amino acids,13 phenolic acids and glycosides,11 coumarins,72 flavones,20 triterpenoid and triterpenoid saponins,23 fatty acids,and 6 other compounds.Moreover,13 compounds were identified by 1H NMR spectroscopy.The UHPLC-MS and 1H NMR results supported and complemented each other.This strategy provides a rapid approach to analyzing and identifying the chemical composition of Chinese herbal prescriptions.The current study provides basis for further research on the quality control and pharmacological mechanism of GLJ.

Guilingji Protects Against Spermatogenesis Dysfunction From Oxidative Stress via Regulation of MAPK and Apoptotic Signaling Pathways in Immp2l Mutant Mice.

Male infertility is a major health issue with an estimated prevalence of 4.2% of male infertility worldwide. Oxidative stress (OS) is one of the main causes of male infertility, which is characterized by excessive reactive oxygen species (ROS) or lack of antioxidants. Meanwhile, it is reported that oxidative stress plays an important role in the spermatogenic impairment in Inner mitochondrial membrane peptidase 2-like (Immp2l) mutant mice. In this study, we focused on the potential mechanism of Guilingji in protecting the spermatogenic functions in Immp2l mutant mice. The results revealed that Immp2l mutant mice exhibit impaired spermatogenesis and histology shows seminiferous tubules with reduced spermatogenic cells. After administration of Guilingji [150 mg/kg per day intragastric gavage], however, alleviated spermatogenesis impairment and reversed testis histopathological damage and reduced apoptosis. What's more, western blotting and the levels of redox classic markers revealed that Guilingji can markedly reduce reactive oxygen species. Moreover, Guilingji treatment led to inhibition of the phosphorylation of mitogen-activated protein kinase (MAPK), regulated apoptosis in the cells. In summary, Guilingji can improve spermatogenesis in Immp2l mutant mice by regulating oxidation-antioxidant balance and MAPK pathway. Our data suggests that Guilingji may be a promising and effective antioxidant candidate for the treatment of male infertility.

[Guilingji Capsules reduce 900 MHz collphone electromagnetic radiation-induced testicular oxidative damage and downregulate Prdx2 protein expression in the rat testis].

OBJECTIVE: To investigate the relationship of electromagnetic radiation (EMR) from 900 MHz cellphone frequency with testicular oxidative damage and its influence on the Prdx2 protein expression in the rat testis, and to explore the mechanism of Guilingji Capsules (GC) alleviating oxidative damage to the testis tissue. METHODS: Fifty healthy SD male rats were randomly divided into five groups of equal number, sham-EMR, 4-h EMR, 8-h EMR, 4-h EMR+GC and 8-h EMR+GC and exposed to 900 MHz EMR (370 µW/cm2) for 0, 4 or 8 hours daily for 15 successive days. The rats of the latter two groups were treated intragastrically with GC suspension and those of the first three groups with pure water after exposure to EMR each day. After 15 days of exposure and treatment, all the rats were sacrificed and their testis tissue collected for observation of the histomorphological and ultrastructural changes by HE staining and transmission electron microscopy, measurement of the levels of serum glutathione (GSH), superoxide dismutase (SOD) and malondialdehyde (MDA) with thiobarbiuric acid and determination of the Prdx2 protein expression by immunohistochemistry and Western blot. RESULTS: Compared with the rats in the sham-EMR group, those in the 4-h and 8-h EMR groups showed different degrees of histomorphological and ultrastructural changes in the testis tissue, significantly decreased levels of GSH (ï¼»80.62 ± 10.99ï¼½ vs ï¼»69.58 ± 4.18ï¼½ and ï¼»66.17 ± 8.45ï¼½ mg/L, P < 0.05) and SOD (ï¼»172.29 ± 10.98ï¼½ vs ï¼»158.92 ± 6.46ï¼½ and ï¼»148.91 ± 8.60ï¼½ U/ml, P < 0.05) and increased level of MDA (ï¼»7.51 ± 1.73ï¼½ vs ï¼»9.84 ± 1.03ï¼½ and ï¼»11.22 ± 2.13ï¼½ umol/ml, P < 0.05), even more significantly in the 8-h than in the 4-h EMR group (P < 0.05). In comparison with the sham-EMR group, the expression of the Prdx2 protein was markedly downregulated in the 4-h and 8-h EMR groups (0.56 ± 0.03 vs 0.49 ± 0.03, 0.21 ± 0.01, P < 0.05), but again upregulated in the 4-h and 8-h EMR+GC groups (0.55±0.03 and 0.37±0.04) (P < 0.05). CONCLUSIONS: Electromagnetic radiation from cellphones can cause ultrastructural damage to the testis tissue of male rats, while Guilingji Capsules can alleviate it, presumably by upregulating the Prdx2 protein expression in the testis tissue and reducing testicular oxidative damage.

Efficacy and Safety of Guilingji Capsules () for Treating Mild-to-Moderate Cognitive Impairment: Study Protocol for A Randomized, Double-Blind, Positive-Controlled, Multicenter and Noninferiority Trial.

BACKGROUND: The incidence of cognitive impairment (CI) is gradually increasing, which has attracted more attention from medical researchers worldwide. Definitive mechanisms of pathogenesis remain elusive, and there are few medications that have been proven effective for CI. The utilization of Chinese herbal medicine has shown positive therapeutic effects for a broad spectrum of diseases, including CI. OBJECTIVE: The purpose of this study is to evaluate the safety and efficacy of Guilingji Capsules (GLJC, ) in treating mild-to-moderate CI with Shen (Kidney) and marrow deficiency syndrome. METHODS: This is a randomized, double-blind, positive-controlled, multicenter clinical trial with a noninferiority design that included 348 participants randomly divided into an experimental arm and an active comparator arm. Individuals in the experimental arm (174 cases) took 0.6 g of GLJC once a day and 19.2 mg of Gingko biloba extract mimetic 3 times a day. Individuals in the active comparator arm (174 cases) took 0.6 g of GLJC mimetic once a day and 19.2 mg of Gingko biloba extract in tablet form 3 times a day. The intervention period included two sessions over 24 weeks. The primary outcome be the effectiveness of GLJC on cognitive improvement after 24 weeks of treatment, which was defined as an increase in the Mini Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA) Scale. The secondary outcomes were improvement in independence, daily living ability, and Chinese medicine (CM) syndrome, which were measured with the Alzheimer's disease Rating Scale-Cognitive Project (ADAS-Cog), Clinical Dementia Rating (CDR) Total Score, Activities of Daily Living (ADL) Total Score and the Chinese Medicine Symptom Scale (CM-SS), respectively. Serum acetylcholine, acetylcholinesterase, bax and bcl-2 were monitored to explore the mechanism of GLJC on CI. In addition, safety measures, including vital signs, electrocardiography, laboratory indicators (full blood count, kidney and liver function tests, routine urine test and routine stool test) and adverse events, were also recorded. DISCUSSION: The purpose of this trial is to evaluate the efficacy and safety of GLJC in patients with mild-to-moderate CI with kidney and marrow deficiency syndrome. If successful, the results would provide a viable treatment for patients with mild-to-moderate CI. (Clinical Trials.gov. ID: NCT03647384. Registered on 23 August 2018).

[Study on difference of chemical compositions of Guilingji before and after alchemy based on mass spectrometry and multivariate statistical analysis].

UPLC-Q-Orbitrap MS/MS and ICP-MS coupled with multivariate statistical analysis was employed to explore the differences in chemical compositions of Guilingji(GLJ) before and after alchemy.The changes in organic chemical compositions and inorganic elements were observed and 39 differential organic compositions were found in GLJ after alchemy, 24 compounds of which were identified. The differential compositions of GLJ included violet ketones, chalcones, amides, and fatty acids whose contents were increased after alchemy, as well as flavones, isoflavones, dihydroflavones, flavonoid glycosides, and coumarins whose content were decreased after alchemy. This study showed 6 inorganic elements filtered out as markers for distinguishing GLJ before and after alchemy, including B, Si, Mg, K, Cr, and Ni.The contents of Mg, K, Cr and Ni were increased while the contents of B and Si were decreased after alchemy.The difference of the contents after alchemy changed the cold and hot properties of the compound, showing the decrease of dryness, and the hot property was changed to warm and neutral properties; in addition, the membrane permeability and absorption of the compound compositions were improved. In this study, we preliminarily investigated the changes of chemical compositions in GLJ before and after alchemy as well as the effects of alchemy on physical and chemical properties and cold-heat nature of GLJ, laying a foundation for further clarifying the scientific connotation of alchemy process.

Integrated adrenal and testicular metabolomics revealed the protective effects of Guilingji on the Kidney-Yang deficiency syndrome rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Guilingji (GLJ) is a well-known traditional Chinese medicine (TCM) prescription for the treatment of Kidney-Yang deficiency syndrome (KYDS). AIM OF THE STUDY: This study aimed to address the protective effects of GLJ against KYDS in rats with pharmacodynamic indicators and target tissues (adrenal gland and testis) metabolomics. MATERIALS AND METHODS: The rats were injected intraperitoneally (i.p) hydrocortisone to simulate KYDS and administered orally of GLJ for 30 days. Traditional pharmacodynamic indicators (body weight, behavioral indicators, biochemical parameters and histological examination) were performed to evaluate the efficacy of GLJ. Furthermore, adrenal gland and testis metabolic profiles obtained by UHPLC-Q Exactive Orbitrap-MS coupled with multivariate analysis were conducted to explore the metabolic regulation mechanism of GLJ. RESULTS: After administration of GLJ, the weight, levels of behavioral indicators and biochemical parameters of rats were increased compared with those of the model group, and the abnormalities of morphology in adrenal and testicular tissues were improved. Furthermore, GLJ had recovering effects via the adjustment of vitamins metabolism, which was accompanied by lipids metabolism, amino acid metabolism and nucleotides metabolism. CONCLUSIONS: The study firstly integrated the target tissues metabolic profiles, which were complementary, and GLJ had protective effects on KYDS rats via the regulation of steroid hormone biosynthesis, oxidant-antioxidant balance and energy acquisition.

Effects of Guilingji on Aging Rats and Its Underlying Mechanisms.

Guilingji (GLJ), a traditional Chinese medicine, is of wide concern because of its remarkable antiaging effect with a long application history. It mainly consists of traditional Chinese herbs, that is, Ginseng radix et rhizoma rubra. This study focused on the anti-aging effects of GLJ on natural aging rats and its underlying mechanisms. Morris water maze was used to determine the learning and memory ability of rats. The levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), acetylcholine (ACh), and acetylcholinesterase (AChE) in serum were determined. Furthermore, a 1H-NMR-based serum metabolomics coupled with multivariate data analysis was used to identify potential biomarkers and corresponding metabolic pathways. The results showed that GLJ significantly improved the learning and memorial dysfunctions of natural aging rats. The mechanisms of the anti-aging and memory ameliorative effects of GLJ related to balancing oxidative stress, improving cholinergic system. Its specific mechanism of action may be through regulating pyruvate metabolism and arginine and proline metabolism.

Effects of Guilingji on serum metabolites in D-galactose-induced aging in rats / 药学学报

This work investigates the effects of Guilingji (GLJ) on D-galactose-induced aging and changes in serum metabolites by UHPLC-Q exactive orbitrap-MS in rats. The rat model of aging by subcutaneous injection of D-galactose (300 mg·kg-1) was used to analyze the effect of different concentrations of GLJ (37.5, 75, 150 mg·kg-1) on an open field test in aging rats. Rat serum was collected after 8 weeks and subjected to LC-MS to analyze the anti-aging effect of GLJ. Animal experimentation was approved according to the Committee on the Ethics of Animal Experiments of Shanxi University (SXULL2014032). GLJ significantly improved the autonomous activity of rats. Compared with the control group, 23 metabolites in the treated group changed significantly, involving three main pathways. The group that was given GLJ had altered regulation of 4 serum metabolites in two pathways. Our results indicate that GLJ can delay aging behavior in rats; the mechanism of this anti-ageing effect remains to be determined.

Guilingji Capsules reduce 900 MHz collphone electromagnetic radiation-induced testicular oxidative damage and downregulate Prdx2 protein expression in the rat testis / 中华男科学杂志

Objective@#To investigate the relationship of electromagnetic radiation (EMR) from 900 MHz cellphone frequency with testicular oxidative damage and its influence on the Prdx2 protein expression in the rat testis, and to explore the mechanism of Guilingji Capsules (GC) alleviating oxidative damage to the testis tissue.@*METHODS@#Fifty healthy SD male rats were randomly divided into five groups of equal number, sham-EMR, 4-h EMR, 8-h EMR, 4-h EMR+GC and 8-h EMR+GC and exposed to 900 MHz EMR (370 μW/cm2) for 0, 4 or 8 hours daily for 15 successive days. The rats of the latter two groups were treated intragastrically with GC suspension and those of the first three groups with pure water after exposure to EMR each day. After 15 days of exposure and treatment, all the rats were sacrificed and their testis tissue collected for observation of the histomorphological and ultrastructural changes by HE staining and transmission electron microscopy, measurement of the levels of serum glutathione (GSH), superoxide dismutase (SOD) and malondialdehyde (MDA) with thiobarbiuric acid and determination of the Prdx2 protein expression by immunohistochemistry and Western blot.@*RESULTS@#Compared with the rats in the sham-EMR group, those in the 4-h and 8-h EMR groups showed different degrees of histomorphological and ultrastructural changes in the testis tissue, significantly decreased levels of GSH (［80.62 ± 10.99］ vs ［69.58 ± 4.18］ and ［66.17 ± 8.45］ mg/L, P < 0.05) and SOD (［172.29 ± 10.98］ vs ［158.92 ± 6.46］ and ［148.91 ± 8.60］ U/ml, P < 0.05) and increased level of MDA (［7.51 ± 1.73］ vs ［9.84 ± 1.03］ and ［11.22 ± 2.13］ umol/ml, P < 0.05), even more significantly in the 8-h than in the 4-h EMR group (P < 0.05). In comparison with the sham-EMR group, the expression of the Prdx2 protein was markedly downregulated in the 4-h and 8-h EMR groups (0.56 ± 0.03 vs 0.49 ± 0.03, 0.21 ± 0.01, P < 0.05), but again upregulated in the 4-h and 8-h EMR+GC groups (0.55±0.03 and 0.37±0.04) (P < 0.05).@*CONCLUSIONS@#Electromagnetic radiation from cellphones can cause ultrastructural damage to the testis tissue of male rats, while Guilingji Capsules can alleviate it, presumably by upregulating the Prdx2 protein expression in the testis tissue and reducing testicular oxidative damage.

Study on difference of chemical compositions of Guilingji before and after alchemy based on mass spectrometry and multivariate statistical analysis / 中国中药杂志

UPLC-Q-Orbitrap MS/MS and ICP-MS coupled with multivariate statistical analysis was employed to explore the differences in chemical compositions of Guilingji(GLJ) before and after alchemy.The changes in organic chemical compositions and inorganic elements were observed and 39 differential organic compositions were found in GLJ after alchemy, 24 compounds of which were identified. The differential compositions of GLJ included violet ketones, chalcones, amides, and fatty acids whose contents were increased after alchemy, as well as flavones, isoflavones, dihydroflavones, flavonoid glycosides, and coumarins whose content were decreased after alchemy. This study showed 6 inorganic elements filtered out as markers for distinguishing GLJ before and after alchemy, including B, Si, Mg, K, Cr, and Ni.The contents of Mg, K, Cr and Ni were increased while the contents of B and Si were decreased after alchemy.The difference of the contents after alchemy changed the cold and hot properties of the compound, showing the decrease of dryness, and the hot property was changed to warm and neutral properties; in addition, the membrane permeability and absorption of the compound compositions were improved. In this study, we preliminarily investigated the changes of chemical compositions in GLJ before and after alchemy as well as the effects of alchemy on physical and chemical properties and cold-heat nature of GLJ, laying a foundation for further clarifying the scientific connotation of alchemy process.

Efficacy and Safety of Guilingji Capsules () for Treating Mild-to-Moderate Cognitive Impairment: Study Protocol for A Randomized, Double-Blind, Positive-Controlled, Multicenter and Noninferiority Trial / 中国结合医学杂志

BACKGROUND@#The incidence of cognitive impairment (CI) is gradually increasing, which has attracted more attention from medical researchers worldwide. Definitive mechanisms of pathogenesis remain elusive, and there are few medications that have been proven effective for CI. The utilization of Chinese herbal medicine has shown positive therapeutic effects for a broad spectrum of diseases, including CI.@*OBJECTIVE@#The purpose of this study is to evaluate the safety and efficacy of Guilingji Capsules (GLJC, ) in treating mild-to-moderate CI with Shen (Kidney) and marrow deficiency syndrome.@*METHODS@#This is a randomized, double-blind, positive-controlled, multicenter clinical trial with a noninferiority design that included 348 participants randomly divided into an experimental arm and an active comparator arm. Individuals in the experimental arm (174 cases) took 0.6 g of GLJC once a day and 19.2 mg of Gingko biloba extract mimetic 3 times a day. Individuals in the active comparator arm (174 cases) took 0.6 g of GLJC mimetic once a day and 19.2 mg of Gingko biloba extract in tablet form 3 times a day. The intervention period included two sessions over 24 weeks. The primary outcome be the effectiveness of GLJC on cognitive improvement after 24 weeks of treatment, which was defined as an increase in the Mini Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA) Scale. The secondary outcomes were improvement in independence, daily living ability, and Chinese medicine (CM) syndrome, which were measured with the Alzheimer's disease Rating Scale-Cognitive Project (ADAS-Cog), Clinical Dementia Rating (CDR) Total Score, Activities of Daily Living (ADL) Total Score and the Chinese Medicine Symptom Scale (CM-SS), respectively. Serum acetylcholine, acetylcholinesterase, bax and bcl-2 were monitored to explore the mechanism of GLJC on CI. In addition, safety measures, including vital signs, electrocardiography, laboratory indicators (full blood count, kidney and liver function tests, routine urine test and routine stool test) and adverse events, were also recorded.@*DISCUSSION@#The purpose of this trial is to evaluate the efficacy and safety of GLJC in patients with mild-to-moderate CI with kidney and marrow deficiency syndrome. If successful, the results would provide a viable treatment for patients with mild-to-moderate CI. (Clinical Trials.gov. ID: NCT03647384. Registered on 23 August 2018).

1H NMR-based metabolomics revealed the protective effects of Guilingji on the testicular dysfunction of aging rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Guilingji (GLJ), a famous and classical traditional Chinese medicine (TCM) prescription, has been used to extend the lifespan and improve the life qualities of the elderly for hundreds of years in China. AIM OF THE STUDY: We aimed to explore the protective effects of GLJ on the testicular dysfunction of aging rats, as well as the regulating effects of GLJ on the metabolic disturbance and metabolite changes in natural aging rats. MATERIALS AND METHODS: Forty 23-month-old rats were divided randomly into four groups, including the old control group and three groups of GLJ treatment at 37.5, 75, and 150 mg/kg doses, respectively. Additionally, 10 four-month rats were included as the youth control group. Testicular dysfunction was first evaluated by measuring the changes in the wet weights of the testicles, concentration of serum testosterone (T), and morphologic changes of the testis. Subsequently, an 1H NMR-based metabolomics approach coupled with multivariate analysis, including partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) was applied to monitor the metabolite changes. RESULTS: Compared with the old control group, the wet weights of the testicles and T concentration were significantly increased, while the morphologic abnormality of testicular tissues was improved by a 4-week treatment course with GLJ. Furthermore, compared with the old control group, the urinary levels of alanine, pantothenate, phenylalanine, ß-hydroxybutyrate and pyruvate were significantly decreased after a 4-week treatment course with GLJ. Additionally, we found that amino acid metabolism and pyruvate metabolism were significantly involved in the regulatory effect of GLJ. CONCLUSIONS: The current findings provided, for the first time, sound evidence of the protective effects of GLJ on testicular dysfunction from both biochemical and metabolomics perspectives. The mechanisms of GLJ could be related to regulating amino acid metabolism and pyruvate metabolism. The current study lays an important foundation for further research and for the broad clinical application of GLJ.

Effects of Guilingji on improving learning and memory dysfunction caused by aging / 中草药

Objective To explore the effects of Guilingji on improving learning and memory dysfunction caused by aging. Methods The mouse model of subacute aging caused by D-galactose and the rat model of natural aging were used respectively to imitate learning and memory dysfunction caused by aging. The effects of Guilingji on improving of learning and memory function index were focused in the diving platform experiment and the Morris water maze experiment. At the same time, the effect of that on rat organ index and blood biochemical index were investigated. Results Guilingji can significantly prolong step down latency (P < 0.05) and reduce the number of errors within 5 min (P < 0.05, 0.01) of the model mice. It can shorten positioning navigation escape incubation period (P < 0.01), extend the space exploration quadrant retention time (P < 0.05, 0.01), and increase the number of access to the platform (P < 0.05). Guilingji can increase testicular, thymus and spleen index (P < 0.05) and reduce the ALT content in serum (P < 0.05). Conclusion Guilingji can obviously improve the impairment of learning and memory function caused by aging. It also has some good effects on enhancing immunity, improving reproductive capacity, and protecting liver.

Research progress on material basis of Guilingji based on pharmacodynamical effect / 中草药

The functional components of Guilingji in nervous system diseases, reproductive system diseases, and digestive system diseases were found by the study of functional components and modern pharmacological of main medicinal materials in Guilingji. The relationship among them was analyzed, which provided scientific basis and research ideas for explaining pharmacodynamic material basis of Guilingji, formulating Guilingji quality evaluation criteria, and carrying out the second development.

Metabolomic studies on special processing of Asari Radix et Rhizoma in Guilingji / 中草药

Objective: To explore the underlying reasons why Asari Radix et Rhizoma (ARR) should be processed by Shanxi aged vinegar when used in Guilingji. Methods: 1H-NMR-based metabolomic approach combined with multivariate statistical analysis was used to investigate the differential chemical components among the raw and two vinegar-baked ARR. Results: More than 30 metabolites were identified in the 1H-NMR spectra of ARR. Raw and two vinegar-baked ARR could be separated obviously, and the rice vinegar-baked ARR and the Shanxi aged vinegar-baked ARR also differed to each other, in which the priamary metabolites showed bigger difference than that of secondary metabolites. Conclusion: This study laid a basis for the modern research of the special processing technology for Guilingji.

Second Development Thoughts of Gui-Ling-Ji Based on Development Strategies of Traditional Chinese Medicine Secret Varieties / 世界科学技术-中医药现代化

Gui-Ling-Ji (GLJ), a classical prescription of traditional Chinese medicine (TCM), is the only existing compound of refining agent after 400-year testing. It is also one of 4 kinds of TCM secret varieties of the first batch after liberation. However, due to the lack of in-depth modern research, the magical effect of GLJ has not been fully understood and the clinical application has also been influenced. This article analyzed the situation and problems of secret varieties and GLJ. Experiences and strategies of the successful redevelopment of secret varieties were re-ferred to. Then, this paper proposed modern research thoughts of GLJ in order to realize the actively protection and the second development of the prescription.