[Improvement effect of cinnamaldehyde on reserpine-induced Parkinson's disease rat model].

In order to study the neuroprotective mechanism of cinnamaldehyde on reserpine-induced Parkinson's disease(PD) rat models, 72 male Wistar rats were randomly divided into blank group, model group, Madopar group, and cinnamaldehyde high-, medium-, and low-dose groups. Except for the blank group, the other groups were intraperitoneally injected with reserpine of 0.1 mg·kg~(-1) once every other morning, and cinnamaldehyde and Madopar solutions were gavaged every afternoon. Open field test, rotarod test, and oral chewing movement evaluation were carried out in the experiment. The brain was taken and fixed. The positive expression of dopamine receptor D1(DRD1) was detected by TSA, and the changes in neurotransmitters such as dopamine(DA) and 3,4-dihydroxyphenylacetic acid(DOPAC) in the brain were detected by enzyme-linked immunosorbent assay(ELISA). The protein and mRNA expression levels of tyrosine hydroxylase(TH) and α-synuclein(α-Syn) in substantia nigra(SN) were detected by RT-PCR and Western blot. The results showed that after the injection of reserpine, the hair color of the model group became yellow and dirty; the arrest behavior was weakened, and the body weight was reduced. The spontaneous movement and exploration behavior were reduced, and the coordination exercise ability was decreased. The number of oral chewing was increased, but the cognitive ability was decreased, and the proportion of DRD1 positive expression area in SN was decreased. The expression of TH protein and mRNA was down-regulated, and that of α-Syn protein and mRNA was up-regulated. After cinnamaldehyde intervention, it had an obvious curative effect on PD model animals. The spontaneous movement behavior, the time of staying in the rod, the time of movement, the distance of movement, and the number of standing times increased, and the number of oral chewing decreased. The proportion of DRD1 positive expression area in SN increased, and the protein and mRNA expression levels of α-Syn were down-regulated. The protein and mRNA expression levels of TH were up-regulated. In addition, the levels of DA, DOPAC, and homovanillic acid(HVA) neurotransmitters in the brain were up-regulated. This study can provide a new experimental basis for clinical treatment and prevention of PD.

Ginseng promotes the function of intestinal stem cells through the Wnt/ß-catenin signaling pathway in D-galactose-induced aging mice.

BACKGROUND: Intestinal stem cells (ISCs) are the reservoir source of various types of intestinal cells, and the decline of stem cell function in the gut may be a potential factor for aging-related disease. The present study aimed to explore the regulatory mechanisms of Panax ginseng C.A.Meyer (Araliaceae, Panax genus) that could restore gut aging by enhancing intestinal function and regulating ISCs in aging mice based on the Wnt/ß-catenin signaling pathway. METHODS: A total of 60 ICR male mice were randomly divided into control, model, metformin, and ginseng water decoction (GWD) 3.6, 1.8, and 0.9 g/kg groups. The aging model was induced by 1 % D-galactose (s.c. 0.1 mL/10 g) for 28 days. Moreover, GWD was given to aging mice intragastrically (i.g.) once a day for 28 successive days. The learning memory ability, pathological status, and function in the ileum tissue, the activity of digestive enzymes, and short-chain fatty acid (SCFA) content in the colon were evaluated, and the related mechanism was investigated. RESULTS: Ginseng can decrease the escape latency time and increase the swimming speed and the number of crossing platforms in aging mice. Moreover, the pathology of ileum tissue improved, the length of the intestinal villi increased, and the width of the villi and the depth of the crypts decreased. The activities of trypsin, α-amylase, and lipase increased in duodenal content and intestinal mucosa. In the colon, the content of SCFA, such as acetic acid, propionic acid and butyric acid, increased, indicating that ginseng significantly improves intestinal function impairment. The mRNA expressions and protein levels of ß-catenin, C-myc, GSK-3ß, Lgr5, and Olfm4 were upregulated in the ginseng group. CONCLUSIONS: Ginseng improves intestinal function and regulates the function of ISCs in order to protect intestinal health by activating the Wnt/ß-catenin signaling pathway in aging mice.

Prophylactic Effects of Hemp Seed Oil on Perimenopausal Depression: A Role of HPA Axis.

Hemp seed, the dried fruit of Cannabis sativa L. (Moraceae), has been extensively documented as a folk source of food due to its nutritional and functional value. This study evaluated the antidepressant effect of hemp seed oil (HSO) during its estrogen-like effect in Perimenopausal depression (PMD) rats induced by ovariectomy combined with chronic unpredictable mild stress (OVX-CUMS). Female SD rats (SPF, 10 weeks, sham operated group, ovariectomy (OVX) model group, ovariectomy - chronic unpredictable mild stress (OVX-CUMS) group, HSO + OVX-CUMS group, fluoxetine (FLU) + OVX-CUMS group, n=8) were subjected to treatment with HSO (4.32 g/kg) or fluoxetine (10 mg/kg) for 28 days (20 mL/kg by ig). Sucrose preference test (SPT), forced swimming test (FST), open field test (OFT), estrogen receptor α (ERα) and estrogen receptor ß (ERß) expression, estradiol (E2), follicle stimulating hormone (FSH), luteinizing hormone (LH), cortisol (CORT), adrenocorticotropic hormone (ACTH), corticotropin releasing hormone (CRH), norepinephrine (NE), 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5HIAA) levels are measured to evaluate the function of the hypothalamic-pituitary-ovarian (HPO) and hypothalamic-pituitary-adrenal (HPA) axis. The results showed that OVX-CUMS significantly decrease sucrose preference rate in SPT, increase immobility time in FST and OFT, and decrease movement distance and stand-up times in OFT. HSO treatment significantly improves depression-like behaviors, upregulates the expression of ERα and ERß, improves HPO axis function by increasing E2 levels and decreasing FSH and LH levels, reverses HPA axis hyperactivation by decreasing CORT, ACTH, and CRH levels, and upregulates NE, 5-HT, and 5HIAA levels in model rats. The findings suggested that HSO could improve depression-like behavior in OVX-CUMS rats by regulating HPO/HPA axis function and neurotransmitter disturbance.

[Neuroprotective effect of cinnamaldehyde in MPTP-induced mouse model of subacute Parkinson's disease].

This paper aims to explore the neuroprotective effect of cinnamaldehyde(CA) in mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced subacute Parkinson's disease(PD) and the mechanism. To be specific, male C57 BL/6 mice(n=72, SPF) were randomized into control group, model group, positive control(madopar 0.1 mg·g~(-1)) group, and low-dose, me-dium-dose, and high-dose CA groups(0.15, 0.30, 0.60 mg·g~(-1)). MPTP(intraperitoneal injection, 0.03 mg·g~(-1), once a day for 5 days) was used to induce subacute PD in mice except for the control group. The administration began from the day of modeling and lasted 19 days. On the 0 th, 12 th, and 19 th day, the open field test, pole test, and rotarod test were carried out. After the tests, the mice were killed and brains were separated. In addition, the organ index was measured. The number of cells in substantia nigra(SN) in the midbrain of MPTP-induced PD model mice was detected based on hematoxylin and eosin(HE) staining. The levels of tyrosine hydroxylase(TH)-and α-synuclein(α-Syn)-positive cells in SN were determined by immunohistochemical staining, and the protein levels of TH and α-Syn in SN by Western blot. The results showed that the MPTP-stimulated mice had abnormal behaviors such as erect hair, arched back, rigidity of the tail, slow movement, and tremor, decreased number of crossings and rearing, increased frequency of urination and defecation, longer time of pole climbing, and shorter time of staying on the rotating rod. In addition, the mice showed obvious damage of neurons in the SN and reduced neuron cells in irregular arrangement with some shrinking. In addition, the average optical density of TH in SN decreased and that of α-Syn increased. All these suggested the successful modeling. CA displayed obvious therapeutic effect on the PD mice, as manifested by the increased number of crossings and rearing, decreased frequency of urination and defecation, shorter time of climbing pole, longer time of staying on the rotating rod, and more neuron cells in the SN with a few pykno-tic cells. Moreover, CA significantly alleviated the decrease of TH and the overexpression of α-Syn in SN. As a result, the MPTP-induced injury of dopaminergic neurons was alleviated. The performance of 0.3 mg·g~(-1) CA was the best. This study is expected to lay a scientific basis for the development of CA products.

Panax ginseng root, not leaf, can enhance thermogenic capacity and mitochondrial function in mice.

Context: Panax ginseng C. A. Meyer (Araliaceae) root and leaf have always been considered in the traditional theory as hot and cold properties, respectively.Objective: To clarify the hot and cold properties of ginseng root and leaf from a thermodynamic viewpoint.Materials and methods: Thirty ICR male mice were randomly assigned to control (water), ginseng root group (GRP) and ginseng leaf group (GLP) with a concentration of 0.075 g/mL; the volume was 0.1 mL/10 g (body mass) per day by intragastric administration for 20 days. Ultra-Performance Liquid Chromatography (UPLC) was used to determine quality control through seven ginsenosides contained in ginseng root and leaf. Rest metabolic rate (RMR) and energy expenditure were monitored every 9 days by TSE System. At the 20th day, serum T3 or T4, liver or brown adipose tissue (BAT) mitochondrial respiration were investigated.Results: The quality control of GRP and GLP were within requirements of 2015 China Pharmacopoeia. The RMR (mLO2/h) in GLP (47.95 ± 4.20) was significantly lower than control (52.10 ± 4.79) and GRP (55.35 ± 4.48). Mitochondrial protein concentration and respiration were significantly increased in GRP (BAT, 79.12 ± 2 .08 mg/g, 239.89 ± 10.24 nmol O2/min/g tissue; Liver, 201.02 ± 10.89, 202.44 ± 3.24) and decreased in GLP (BAT, 53.42 ± 3.48, 153.49 ± 5.58; Liver, 138.69 ± 5.69, 104.50 ± 6.25) compared with control.Conclusions: The hot and cold properties of ginseng root and leaf are correlated with thermogenic capacity and mitochondrial function of BAT and liver, which deserve to further research.

Metabolite identification of gut microflora-cassia seed interactions using UPLC-QTOF/MS.

Cassia seed is the dried ripe seed of Cassia obtusifolia L. or Cassia tora L., which is widely used as a food or traditional Chinese medicine. The aim of the present study was to detect the components and metabolites in the culture of human or rat intestinal microflora suspension with the water decoction of cassia seed in vitro, using an ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry system equipped with a negative ion scan mode. Initially, ellagic acid was identified in the cassia seed decoction. Subsequently, six different metabolites, including urolithin (uro)-A, uro-B, uro-D, uro-M6, uro-M7 and uro-B-glucuronide (glur), were detected after co-culture of the cassia seed decoction with intestinal microflora, but not in the cassia seed decoction alone. Uro-M6, uro-M7, uro-A and uro-B were common metabolites in the culture of human or rat intestinal microflora suspension with the water decoction of cassia seed. However, uro-D was only detected in the culture of rat intestinal microflora suspension with the water decoction of cassia seed, and uro-B-glur was only detected in the culture of human intestinal microflora with the water decoction of cassia seed. The uro and intermediate metabolites were produced by ellagic acid in the cassia seed decoction under the action of the intestinal microflora. The production of metabolites might be related to the abundance and diversity of the intestinal microflora in humans and rats. The present study provided rationale for further pharmacological and clinical studies on the mechanisms of action of cassia seeds.

[Screening bioactive compounds inhibiting influenza virus from isatidis radix by ultrafiltration mass spectrometry].

In vitro neuraminidase inhibition assays and ultrafiltration liquid chromatography with diodearray detector coupled to time of flight mass spectrometer (UPLC-DAD-TOF-MS) were combined to screen bioactive compounds inhibiting neuraminidase from Isatidis Radix. By comparing the compounds from Isatidis Radix before and after ultrafiltration, we found that arginine, goitrin and adenosinea can bind with neuraminidase, and the binding degree of the three compounds were (36.23 +/- 1.12)%, (32.54 +/- 1.02)% and (9.38 +/- 0.47)%, respectively. The IC50 of arginine and goitrin were (1.16 +/- 0.02), (1.20 +/- 0.02) g x L(-1), respectively. While the IC50 of adenosinea was higher than 500 g x L(-1). The results showed that arginine and goitrin might be the main compounds with antiviral activity of Isatidis Radix. This study may provide a useful method for the screening of bioactive compounds and quality control of Isatidis Radix.

[Hemagglutination activity of radix isatidis detected by microcalorimetry].

In this study, microcalorimetry was adopted to establish a novel method for detecting the hemagglutination process of Radix Isatidis (Banlangen in Chinese, BLG), and to evaluate the hemagglutination activity diversity of BLG from various habitats. The hemagglutination biothermokinetics curves of positive reagent (phytohemagglutinin, PHA) and 8 batches BLG from different regions of the hemagglutination with 20% rabbit erythrocyte were recorded by microcalorimetry, then biothermokinetics parameters were abstracted, the hemagglutination utility of samples were calculated and analyzed by principal component analysis (PCA) and cluster analysis (CA), meanwhile the results were authenticated by micro-plate agglutination. It showed that the hemagglutination was an exothermic reaction, the reaction rate constant (k: 0.039-73.6 min(-1)), maximum reaction power (Pmax: -1 140.2 - 988.2 microW) and reaction enthalpy (Hi: -529.9 - 717.9 microJ) had good linear correlation with BLG extraction concentration (0.2-1.0 g mL(-1), r > 0.97), and PCA showed Pmax (531-1 335 microW) and Ht (585.2-989.2 microJ) could represent the hemagglutination activity diversity of BLG samples, just confirming with the results of micro-plate agglutination (the agglutination dilution was 3-11 respectively). According to the hemagglutination utility, the BLG samples from Good Agriculture Practice (GAP) regions, main producing area and general regions could be clustered correctly; meanwhile, the biothermokinetics curves with perfect distinctive fingerprint and specificity could give out more information for the quality control and evaluation for BLG. In conclusion, the microcalorimetry method established for detecting the hemagglutination activity of BLG samples on rabbit erythrocyte is sensitive and reliable, and could be adopted as an effective technique in detection aggulatination precisely, quantitatively and consecutively; and provide a novel approach for examining and evaluating quality for Chinese herbal medicine with aggulatinative activity such as BLG.

[Agglutinated activity bioassay method for the determination of antivirus potency of Banlangen granula].

To establish a bioassay method and quality standard of Banlangen granula, agglutinated activity assay was used in the analysis of the traditional Chinese medicine, Banlangen granula. It showed that masculined effect could be picked up effectively and the products quality of different pharmaceutical factories and different batch numbers from the same factory could be revealed conveniently, accurately, quickly and directly with this method (valence value was between 2 and 11). The established bioassay method had a good reproducibility with RSD = 2%. The dependablity of the activity of red cell agglutination and restrainting influenza virus NA was conspicuous (r2 = 0.878 3). In conclusion, this bioassay method is suitable to control and evaluate the quality of Banlangen granula. Thus the method may provide a simple and effective technique in supervising and examining the quality of other traditional Chinese medicine.

[Study on selecting the suitable solubilizing excipients for Shengmai injection].

OBJECTIVE: To select the suitable solubilizing excipients for Shengmai injection by the mean of hemolysis. METHODS: The hemolysis ratio of Shengmai injection, solubilizing excipients and different proportion of them were determined by absorption spectrometry. RESULTS: In the experimental concentration, Shengmai injection itself had no hemolytic effect. Polysorbate 80, Polyethylene glycol 400, Polyethylene glycol 600, Poloxamer 188 solution had no hemolytic effect to appear, but all the experimental concentrations of Polysorbate 20 had hemolytic effect. When combining with different proportion of Polysorbate 20, Polyethylene glycol 400, Polyethylene glycol 600, Poloxamer 188 and Polysorbate 80 of high concentration (0.75% - 1%), Shengmai injection had hemolytic effect. CONCLUSIONS: The absorption spectrometry is more reliable for the judgement of hemolysis than macroscopic observation, and it is helpful to improve the accuracy to select the solubilizing excipients for the TCM injection. Polysorbate 80 can be used as solubilizing excipient of Shengmai injection, which is safe in the concentration range of 0.1% to 0.5%.

[Essential issues of bioassay methods for quality control of laxative traditional Chinese medicines].

The essential issues of bioassay methods for quality control of traditional Chinese medicines (TCM) were investigated and discussed through the instantiation of developing the bioassay methods for laxative drugs. For the relatively broad variation of the quality of TCM, which might be influenced much by many factors, the parallel lines model of quantitative response is preferred to control the quality of TCM for its relatively high accuracy. The parallel model of quantal response is alternative while the sample can not meet the reliability standard for quantitative response model. According to the requirement of homogeneity between reference and sample for bioassay, the extract from referenced crude meterial is suitable to be used as reference substance after standardizing and defining by chemical substances, and to give reference to the establishment and reproducibility of authorized standard substance. The results of determination of the purgative biopotency of different species of rhubarb and the compound preparations showed that the bioassay methods and self-made reference substance established in this study could be used to control the quality of laxative medicines.

[Establishment of bioassay method for antivirus potency of radix isatidis based on chemical fluorometric determination].

In the present study, the in vitro inhibitory activity of extracts from radix isatidis on neuraminidase (NA) was investigated by the chemical fluoremetic determination to establish the quality control method for antivirus action of radix isatidis. The initial study indicated that radix isatidis had obvious in vitro inhibitory activity on NA with IC50 = (0.90 +/- 0.20) mg (herb) x mL(-1). The correlation between logarithmic dose and reaction rate showed a "S" shape and a linear curve (linear equation, y = 8.7259 + 1.2169 x log(D), R = 0.9992) when the reaction rate was converted to probit-quite similar to Tamiflu's reaction curve, which hinted that radix isatidis had the same inhibitory function on NA as Tamiflu. According to the reaction type and the regularity of "parallel lines of qualitative effect", the experimental condition was optimized and a statistic method was confirmed based on the principle of bioassay statistic. Then the bioassay method for antivirus potency of radix isatidis based on fluorometric determination was established. The results of bio-potency assay showed the qualitative differences of radix isatidis samples sensitively and quantitatively. Meanwhile, this method has good reproducibility with RSD = 5.78% and reliability. The quality bioassay control method based on chemical fluorometric determination can reflect the pharmaco-dynamic features of Chinese medicine herb.

[Biological evaluation of Radix Isatidis based on neuraminidase activity assay].

Radix Isatidis (Banlangen in Chinese) is a traditional Chinese medicinal (TCM) herb, and is frequently used for treating influenza. However, the current quality control method for Radix Isatidis should be developed since it has little correlation to the pharmacodynamic action. In this paper, the in vitro inhibitory action of Radix Isatidis on neuraminidase (NA) was investigated by fluorometric assay with 4-methylumbelliferyl-D-N-acetylneuraminate (FL-MU-NANA) method. Based on the method, the experimental condition was optimized and a bioassay statistic method was established according to the reaction type and the regularity of "parallel lines of qualitative effect". Then the bioassay method of Radix Isatidis was established. This study indicated that Radix Isatidis had obvious in vitro inhibitory activity on NA with IC50 = (0.90 +/- 0.20) mg x mL(-1) (herb). The correlation between logarithmic dose and reaction rate showed an "S" shape--is quite similar to Tamiflu's reaction curve, which hinted that Radix Isatidis had the same inhibitory function on NA as Tamiflu. The established bioassay method of "parallel lines of qualitative effect" had a good reproducibility (RSD = 5.78%). The results of potency determination of Radix Isatidis were reliable (reliability test: deviation from straight line P > 0.05, deviation from parallel line P > 0.05) and well regular. As a conclusion, this bioassay method is suitable to control and evaluate the quality of Radix Isatidis.

Spectrum-effect relationships between ultra performance liquid chromatography fingerprints and anti-bacterial activities of Rhizoma coptidis.

The fingerprints of Rhizoma coptidis from various sources were established by ultra performance liquid chromatography (UPLC) and the anti-bacterial activities of R. coptidis on Escherichia coli (E. coli) growth was studied by microcalarimetry. The UPLC fingerprints were evaluated using similarity analysis (SA) and hierarchical clustering analysis (HCA). Some quantitative parameters obtained from the thermo-genic curves of E. coli growth affected by R. coptidis were analyzed using principal component analysis (PCA). The spectrum-effect relationships between UPLC fingerprints and anti-bacterial activities were investigated using canonical correlation analysis (CCA). The results showed that close correlation existed between the spectrum-effect relationships. Berberine, jateorrhizine and palmatine in the UPLC fingerprints might be the main anti-bacterial components. The anti-bacterial activities of R. coptidis were related with the main active constituents, along with the production place and the harvesting time of this herb, the latitude and longitude of the place. This work provides a general model of the combination of UPLC and microcalorimetry to study the spectrum-effect relationships of R. coptidis, which can be used to discover principle components of it on bioactivity.