Protective effect of the total alkaloid extract from Bulbus Fritillariae Pallidiflorae on cigarette smoke-induced Beas-2B cell injury model and transcriptomic analysis.

Background: Bulbus Fritillariae Pallidiflorae (BFP) is a traditional Chinese medicine that has long been used to treat lung diseases, but the active components and mechanism are still unclear. Objective: This study aimed to investigate the effect and mechanism of the total alkaloid extract from BFP (BFP-TA) on cigarette smoke extract (CSE)-induced Beas-2B cells injury. Design: The Beas-2B cells injury model was induced by 2% CSE, then the effect of BFP-TA on the levels of total antioxidant capacity (T-AOC), superoxide dismutase (SOD) and malondialdehyde (MDA) was detected according to the instructions of the T-AOC assay kit, the SOD detection kit and the MDA detection kit, and the production of ROS was detected by fluorescence microscopy. The effect of BFP-TA on Beas-2B cells apoptosis was detected by flow cytometry, and the effect of BFP-TA on related protein expression was detected by western blot. Subsequently, the effect of BFP-TA on differentially expressed genes (DEGs) in CSE-induced Beas-2B cells was studied by transcriptomic sequencing, and the expression of DEGs was verified by quantitative real-time polymerase chain reaction (qPCR). Results: The results showed that BFP-TA could attenuate CSE-induced oxidative damage in Beas-2B cells by elevating T-AOC and SOD levels while inhibiting ROS and MDA levels, and the mechanism was potentially related to the SIRT1/Nrf2/Keap1 signaling pathway. Furthermore, BFP-TA could inhibit CSE-induced apoptosis by inhibiting the protein expression of Bax, MST1 and FOXO3a, and exert anti-inflammatory effect by inhibiting the activation of MAPK signaling pathway. Subsequently, transcriptome analysis and qPCR validation showed that BFP-TA could alleviate inflammation, oxidative stress, apoptosis and lipid metabolism disorders by regulating the expression of DEGs in PPAR and PI3K-Akt signaling pathways, thereby exerting a protective effect against CSE-induced Beas-2B cell injury. Conclusion: This study is the first to demonstrate that BFP-TA could exert a protective effect on CSE-induced Beas-2B cell injury by exerting anti-inflammatory, antioxidant, anti-apoptotic and regulate lipid metabolism disorders.

Protective Effect of the Total Alkaloid Extract from Bulbus Fritillariae pallidiflorae in a Mouse Model of Cigarette Smoke-Induced Chronic Obstructive Pulmonary Disease.

Purpose: In recent years, the incidence of chronic obstructive pulmonary disease (COPD) has been increasing year by year, but therapeutic drugs has no breakthrough. The total alkaloid extract from Bulbus Fritillariae pallidiflorae (BFP-TA) is widely used in treating lung diseases. Therefore, this study aimed to investigate the protective effect and mechanism of BFP-TA in COPD mice. Methods: BFP-TA was prepared by macroporous adsorbent resin, and the material basis of BFP-TA was analyzed by HPLC-ELSD and UHPLC-MS/MS. Then, the COPD mouse model was induced by cigarette smoke (CS) for 12 weeks, administered at weeks 9-12. Subsequently, the body weight, lung-body ratio, pulmonary function, histopathology, and the levels of pro-inflammatory cytokines, matrix metalloproteinases (MMPs) and oxidative stress markers in the serum of mice were determined. The expressions of related protein of EMT and MAPK signaling pathways in the lung tissues of mice were detected by Western blot. Results: The alkaloid relative content of BFP-TA is 64.28%, and nine alkaloids in BFP-TA were identified and quantified by UHPLC-MS/MS. Subsequently, the animal experiment showed that BFP-TA could improve pulmonary function, and alleviate inflammatory cell infiltration, pulmonary emphysema, and collagen fiber deposition in the lung of COPD mice. Furthermore, BFP-TA could decrease the levels of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1ß), MMPs (MMP-9 and MMP-12) and MDA, while increase the levels of TIMP-1 and SOD. Moreover, BFP-TA could decrease the protein expressions of collagen I, vimentin, α-SMA, MMP-9, MMP-9/TIMP-1, Bax, p-JNK/JNK, p-P38/P38, and p-ERK/ERK, while increase the level of E-cadherin. Conclusion: This study is the first to demonstrate the protective effect of BFP-TA in CS-induced COPD mouse model. Furthermore, BFP-TA may improve airway remodeling by inhibiting the EMT process and potentially exert anti-inflammatory effect by inhibiting the MAPK signaling pathway.

Corydalis saxicolaBunting total alkaloid eliminates Porphyromonas gingivalis strain 33277 internalized into macrophages by inhibition of TLR2.

OBJECTIVE: This study aimed to investigate the impact of Corydalis Saxicola Bunting Total Alkaloid (CSBTA) on Porphyromonas gingivalis internalization within macrophages and explore the potential role of Toll-Like Receptor 2 (TLR2) in this process. METHODS: We established a P. gingivalis internalization model in macrophages by treating P. gingivalis-infected macrophages (MOI=100:1) with 200 µg/mL metronidazole and 300 µg/mL gentamicin for 1 h. Subsequently, the model was exposed to CSBTA at concentrations of 0.02 g/L or 1 µg/mL Pam3CSK4. After a 6 h treatment, cell lysis was performed with sterile water to quantify bacterial colonies. The mRNA expressions of TLR2 and interleukin-8 (IL-8) in macrophages were analyzed using RT-qPCR, while their protein levels were assessed via Western blot and ELISA respectively. RESULTS: P. gingivalis could internalize into macrophages and enhance the expression of TLR2 and IL-8. Activation of TLR2 by Pam3CSK4 contributed to P. gingivalis survival within macrophages and increased TLR2 and IL-8 expression. Conversely, 0.02 g/L CSBTA effectively cleared intracellular P. gingivalis, achieving a 90 % clearance rate after 6 h. Moreover, it downregulated the expression of TLR2 and IL-8 induced by P. gingivalis. However, the inhibitory effect of CSBTA on the internalized P. gingivalis model was attenuated by Pam3CSK4. CONCLUSION: CSBTA exhibited the ability to reduce the presence of live intracellular P. gingivalis and lower IL-8 expression in macrophages, possibly by modulating TLR2 activity.

Inhibitory Effects of Corydalis saxicola Bunting Total Alkaloids on Macrophage Pyroptosis.

This study investigated the effect of Corydalis saxicola Bunting total alkaloids (CSBTA) on pyroptosis in macrophages (Mϕ). In the Mϕ pyroptosis model, an inverted fluorescence microscope was used to assess cell pyroptosis, while a scanning electron microscope was used to observe morphological changes in Mϕ. NLR family pyrin domain-containing 3 (NLRP3), caspase-1, and gasdermin D (GSDMD) expression levels were detected by polymerase chain reaction and western blotting, whereas interleukin-1 (IL-1) and interleukin-18 (IL-18) expression levels were measured by an enzyme-linked immunosorbent assay. After pretreatment with CSBTA or the caspase-1 inhibitor, acetyl-tyrosyl-valyl-alanyl-aspartyl-chloromethylketone (Ac-YVAD-cmk), it was discovered that NLRP3, caspase-1, and GSDMD expressions were significantly reduced at both the mRNA and protein levels, as were IL-1 and IL-18 levels. The inhibitory effects of CSBTA and Ac-YVAD-cmk did not differ significantly. These findings indicate that CSBTA blocks Porphyromonas gingivalis-lipopolysaccharide-induced Mϕ pyroptosis.

Lethal and Sublethal Toxicity of Beta-Carboline Alkaloids from Peganum harmala (L.) against Aedes albopictus Larvae (Diptera: Culicidae).

Plant-derived agents are powerful bio-pesticides for the eco-friendly control of mosquito vectors and other blood-sucking arthropods. The larval toxicity of beta-carboline alkaloids against the Asian tiger mosquito, Aedes albopictus (Skuse) (Diptera: Culicidae), was investigated under laboratory conditions. The total alkaloid extracts (TAEs) and beta-carboline alkaloids (harmaline, harmine, harmalol, and harman) from Peganum harmala seeds were isolated and tested in this bioassay. All alkaloids were tested either individually or as binary mixtures, using the co-toxicity coefficient (CTC) and Abbott's formula analysis. The results revealed considerable toxicity of the tested alkaloids against A. albopictus larvae. When all larval instars were exposed to the TAEs at 48 h post-treatment, the mortality of all larval instars varied in a concentration-dependent manner. The second-instar larvae were the most susceptible to different concentrations of TAEs, and the fourth-instar larvae were more tolerant to TAEs than the second-instar larvae. Especially, the third-instar larvae exposed to all alkaloids also showed that all doses resulted in an increased mortality of the third-instar larvae at 48 h post-treatment, and the toxicities of the tested alkaloids in a descending order were TAEs > harmaline > harmine > harmalol, with the LC50 values of 44.54 ± 2.56, 55.51 ± 3.01, 93.67 ± 4.53, and 117.87 ± 5.61 µg/mL at 48 h post-treatment, respectively. In addition, all compounds were also tested individually or in a 1:1 ratio (dose LC25/LC25) as binary mixtures to assess the synergistic toxicity of these binary combinations against the third-instar larvae at 24 and 48 h post-treatment, respectively. The results demonstrated that when tested as a binary mixture, all compounds (especially TAEs, harmaline, and harmine) showed their synergistic effects, exceeding the toxicity of each compound alone. Interestingly, the obtained data further revealed that the TAEs at sublethal doses (LC10 and LC25) could significantly delay the larval development and decrease the pupation and emergence rates of A. albopictus. This phenomenon could be helpful in order to develop more effective control strategies for different notorious vector mosquitoes.

Total alkaloid fraction of Leonurus japonicus Houtt. Promotes angiogenesis and wound healing through SRC/MEK/ERK signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Leonurus japonicus Houtt., also known as motherwort, is a traditional Chinese medicine that was first identified in Shennong Bencao Jing, the first and essential pharmacy monograph in China. L. japonicus has been regarded as a good gynecological medicine since ancient times. It has been widely used in clinical settings for treatment of gynecological diseases and postnatal rehabilitation with good efficacy and low adverse effects. AIM OF THE STUDY: The main purpose of this study was to determine the angiogenic and wound healing effects of total alkaloid fraction from L. japonicus Houtt. (TALH) in vivo and in vitro. In addition, the main bioactive components of total alkaloids were to be identified and analyzed in this study. MATERIALS AND METHODS: First, the UHPLC/Q-TOF-MS method was used to identify and quantify the major components in the TALH extract. The wound healing activity was evaluated in vivo using a rat full-thickness cutaneous wound model. Histological study of wound healing in rat model was performed via immunohistochemistry and immunofluorescence. Cell proliferation was determined by MTT assay. Wound healing and transwell assays were used for detection of cell migration. The effect on tube formation was determined by tube formation assay in HUVECs. Western blot and RT-PCR were used to detect the expressions of relative proteins and genes respectively. Knock-down of SRC by siRNA was done to verify the crucial role of SRC in promotion of angiogenesis induced by TALH. RESULTS: Seven characteristic peaks were recognized in the UHPLC/Q-TOF-MS spectrum, while four of the main components were quantified. The wound model in rats showed that treatment of TALH promoted wound healing by stimulating cellular proliferation and collagen deposition. In vitro experiments showed that co-treatment of TALH and VEGF increased cell proliferation, migration and tube formation in HUVECs. Mechanistic studies suggested that the co-treatment increased gene expressions of SRC, MEK1/2 and ERK1/2, as well as the phosphorylation levels of these proteins. Furthermore, the effect of co-treatment was attenuated after SRC knockdown, suggesting that SRC plays an important role in angiogenesis and wound healing induced by TALH and VEGF co-treatment. CONCLUSION: Our results showed that TALH was one of the main active components of L. japonicus that promoted angiogenesis and wound healing by regulating the SRC/MEK/ERK pathway. Our study provided scientific basis for better clinical application of L. japonicas.

Comparison on Skin Permeability of Corydalis Rhizoma Total Alkaloid Patches at Shenque Acupoint and Non-acupoint Administration / 中国实验方剂学杂志

ObjectiveTo compare the differences in resistance and structure of skin between acupoints and non-acupoints， and to study the difference in skin permeability characteristics of Corydalis Rhizoma total alkaloid patches （CTTP） after administration at Shenque acupoint and non-acupoint， so as to provide experimental support for its clinical acupoint application to prevent and treat chronic pain. MethodTaking corydaline （CD）， tetrahydropalmatine （THP） and corydalis L （CDL） as evaluation indexes， and the quantitative analysis was carried out by high performance liquid chromatography （HPLC）. The mobile phase was methanol-0.04 mol·L-1 phosphoric acid aqueous solution （70∶30， pH 6.0 adjusted with triethylamine）， the detection wavelength was 281 nm. In vitro transdermal test in Franz diffusion cell and in vivo transdermal test were used to study the skin permeability characteristics of CTTP through Shenque acupoint and non-acupoint administration. At the same time， the skin resistance between Shenque acupoint and non-acupoint was measured before and after the administration， and the distribution of the drug in each layer of the skin was compared by freezing sectioning， and visual verification was performed with fluorescence inverted microscope. ResultAfter 24 h of administration， the results of in vivo and in vitro experiments showed that the cumulative permeation and retention of CD， THP and CDL at Shenque acupoint skin were higher than those at non-acupoint skin （P<0.05， P<0.01）， the skin resistance of Shenque acupoint was lower than that of non-acupoint at all time points. The fluorescence microscopic observation results showed that the drug content of each layer of the skin was all Shenque acupoint>non-acupoint， indicating that the skin of Shenque acupoint had better effect on drug penetration and storage than non-acupoint. ConclusionThe 24 h cumulative permeation and retention of CTTP in Shenque acupoint skin are higher than those in non-acupoint skin， and the mechanism may be related to the thin skin， low electrical resistance and large number of hair follicle bodies at Shenque acupoint.

Comparison on Skin Permeability of Corydalis Rhizoma Total Alkaloid Patches at Shenque Acupoint and Non-acupoint Administration / 中国实验方剂学杂志

ObjectiveTo compare the differences in resistance and structure of skin between acupoints and non-acupoints， and to study the difference in skin permeability characteristics of Corydalis Rhizoma total alkaloid patches （CTTP） after administration at Shenque acupoint and non-acupoint， so as to provide experimental support for its clinical acupoint application to prevent and treat chronic pain. MethodTaking corydaline （CD）， tetrahydropalmatine （THP） and corydalis L （CDL） as evaluation indexes， and the quantitative analysis was carried out by high performance liquid chromatography （HPLC）. The mobile phase was methanol-0.04 mol·L-1 phosphoric acid aqueous solution （70∶30， pH 6.0 adjusted with triethylamine）， the detection wavelength was 281 nm. In vitro transdermal test in Franz diffusion cell and in vivo transdermal test were used to study the skin permeability characteristics of CTTP through Shenque acupoint and non-acupoint administration. At the same time， the skin resistance between Shenque acupoint and non-acupoint was measured before and after the administration， and the distribution of the drug in each layer of the skin was compared by freezing sectioning， and visual verification was performed with fluorescence inverted microscope. ResultAfter 24 h of administration， the results of in vivo and in vitro experiments showed that the cumulative permeation and retention of CD， THP and CDL at Shenque acupoint skin were higher than those at non-acupoint skin （P<0.05， P<0.01）， the skin resistance of Shenque acupoint was lower than that of non-acupoint at all time points. The fluorescence microscopic observation results showed that the drug content of each layer of the skin was all Shenque acupoint>non-acupoint， indicating that the skin of Shenque acupoint had better effect on drug penetration and storage than non-acupoint. ConclusionThe 24 h cumulative permeation and retention of CTTP in Shenque acupoint skin are higher than those in non-acupoint skin， and the mechanism may be related to the thin skin， low electrical resistance and large number of hair follicle bodies at Shenque acupoint.

Standardization of crude drugs - herb and seeds of Sophora Alopecuroides L / Монголын эм зүй, эм судлал

Introduction@#<i>Sophora alopecuroides L</i> has broadly been utilized in traditional medicine and all crude drugs including root, herb, and seed are used to treat numerous diseases. This herb is included in 181 Tibetan-Mongolian medicinal prescriptions and ranks 8^th among Mongolian medicinal plants in terms of frequency of administration. The <i>S.alopecuroidesL . </i> root standard was developed by the Institute of Traditional Medicine and Technology in 2017 and approved by “ҮФӨ-0307-2017”. Herb and seed are still used in medicine. Therefore, their standard parameters need to be determined and verified.@*Materials and methods@#The quantitative pharmacognosy analysis of herb and seed was carried out in accordance with the methodology specified in the “General requirements for medicinal plant raw materials” of the National Pharmacopoeia of Mongolia. To determine the total alkaloid in standard matrine, a bromcresol green complex was formed, which was measured by spectrophotometer.@*Conclusion@#By developing, standards for the crude drugs of herb and seed of <i>S.alopecuroides L. </i> which are included in numerous medicinal prescriptions, will confirm the rationale for the use of medicinal raw materials and to expand the utilization’s possibilities.

Phytochemical analysis, antioxidant activities and optimized extraction of embelin from different genotypes of Embelia ribes Burm f.: a woody medicinal climber from Western Ghats of India.

Medicinal importance of Embelia ribes Burm f. is known since ancient time. Its berries are the main ingredient in Vidanga' or 'Baibidanga'-a component of ayurvedic formulations and possess medicinal properties such as antihelmintic, anticancer, neuroprotective and antidiabetic. Studies were conducted on phytochemicals, antioxidant activities, extraction efficiency of embelin from ten genotypes. Methanolic extract of berries from Nagavelli accession exhibited the highest total phenolic content (18.18 ± 0.14 mg GAE/g DW); whereas, ethanolic extract showed highest total flavonoid content (8.35 ± 0.20 mg RE/g DW). The antioxidant activities (AOA) were assessed and noted that ethanolic and methanolic extracts of berries from Nagavelli (NAG) accession revealed highest activities in terms of DPPH radical scavenging activity (67.48 ± 0.17%) and FRAP (66.73 ± 0.60 mg Fe(II)/g DW), respectively. In AOA analysis, berries extracted with different solvents were positively correlated with TPC. Principal component analysis revealed TPC and TFC were the most influencing components for strong antioxidant activities in E. ribes. Reverse phase high performance liquid chromatography (RP-HPLC) was used to quantify embelin content and its optimize extraction using various methods. In the preliminary studies, berries from NAG accession revealed highest (1.770%) embelin content. Further, berries from NAG accession were subjected to various extraction methods and found three fold increase (5.08%) in embelin content in microwave assisted extraction (90 s). Present study suggested that NAG accession found to be a promising source of natural antioxidants and embelin that can be used in pharmaceutical industries.

Corydalis edulis total alkaloids (CETA) ameliorates cognitive dysfunction in rat model of Alzheimer disease through regulation of the antioxidant stress and MAP2/NF-κB.

BACKGROUND: In recent years, the morbidity of Alzheimer's disease in the world has become more and more serious. Therefore, it is an important means to find new drugs for treating AD from traditional medicines. It was found that Corydalis edulis Maxim. has a significant effect in the treatment of Alzheimer's disease (AD) in traditional application. In this work, we evaluated the efficacy of Corydalis edulis Maxim. total alkaloids (CETA) in AD model rats. METHODS: In this work, CETA was prepared by alkali extraction and acid precipitation, 11 alkaloids were identified by UPLC-MS/MS from CETA. AD model rats induced with D-galactose (D-gal) for 7 weeks. In modeling, the different doses of CETA (5, 20 mg/kg/Day) were continuously administered. Firstly, the change of the cognitive function, behavior, brain tissue pathology, and the activity of ROS, MDA, SOD, IL-1ß, TNF-α and CAT in rat hippocampal homogenate was measurement. Finally, the protein expression of Aß, microtubule-associated protein 2 (MAP2) and nuclear factor (κBp65) in rat brain was measurement. RESULT: CETA was found to have the activity in regulating AD. Compared with the normal control group, the levels of SOD and CAT in the hippocampus of the AD model group were decreased, and the level of ROS, MDA, IL-1ß and TNF-α was increased. The protein expression of Aß, and NF-κB were increased, and MAP2 were decreased. After treatment by CETA, the levels of SOD and CAT in hippocampus of AD model rats was significantly increased, ROS, MDA, IL-1ß and TNF-α were significantly decreased. The protein expression of Aß, and NF-κB were decreased, and MAP2 were increased. CONCLUSION: CETA can improve the learning and memory ability in AD model. The mechanism may be achieved by regulating the oxidative stress and inflammatory of AD rats, inhibiting the protein expression levels of Aß, and NF-κB, and promote the protein expression the levels of MAP2. Among them, 5 mg/kg is more effective than 20 mg/kg of CETA. Therefore, the therapeutic potential of CETA has been confirmed by our research, which may be a valuable drug for the treatment of AD.

Maltol total alkaloid inhibits expression and secretion of prolactin by upregulating dopamine D2 receptor / 中草药

Objective: To study the role of dopamine D2 receptor (D2R) on the regulation of prolactin (PRL) secretion by malt total alkaloids. Methods: MMQ and GH3 cells of pituitary adenoma were divided into control group, bromocriptine (5 μg/mL), malt total alkaloids (4.4, 8.8, 35.2, 70.4 μg/mL), haloperidol (10, 20, 40 μg/mL), and combined administration group of total malt alkaloids and haloperidol. Cell viability was detected by CCK-8; The expressions of PRL and D2R were detected by western blotting; The level of PRL was detected by ELISA; The level of PRL and D2R mRNA were detected by qRT-PCR. Results: Compared with control group, malt alkaloids (35.2, 70.4 μg/mL) significantly reduced the expression levels of PRL protein and mRNA, and the level of PRL in the supernatant of MMQ cells (P < 0.05). Malt alkaloids (35.2, 70.4 μg/mL) significantly increased the expression levels of D2R protein and mRNA in MMQ cells. Haloperidol significantly inhibited the downregulation of malt alkaloids on the expression levels of PRL protein and mRNA, and the expression level of PRL in supernatant of MMQ cells (P < 0.05). Haloperidol significantly inhibited the upregulation of malt alkaloids on the levels of D2R protein and mRNA (P < 0.05). The level of PRL in GH3 cells had no change by malt alkaloids. Conclusion: Malt alkaloids could inhibit the expression and secretion of PRL in MMQ cell by upregulating D2R.

Study on Inhibitory Effects of Total Alkaloids of Gelsemium elegans on the Proliferation and Angiogenesis of Human Colon Cancer Cells / 中国药房

OBJECTIVE：To investigate the inhibitory effects of total alkaloids of Gelsemium elegans （TAG） on the proliferation and angiogenesis of human colon cancer cells. METHODS ：Human colon cancer cell line HT- 29 and HUVEC were cultured in vitro . After the intervention of low- ，medium-，high-dose TAG （40，80，120 μg/mL），the morphology of the two cells was observed by fluorescence inversion microscope. The survival rate of HT- 29 cells and HUVEC was detected by CCK- 8 assay. Flow cytometry was used to detect HT- 29 cell cycle. The migration rate ，invasion rate and tube number of HUVEC were observed by scratching test ，Transwell invasion experiment and tube formation experiment. RESULTS ：Compared with blank group ，HT-29 cells and HUVEC were decreased to different extents in TAG groups ；dead cells were observed ，and the survival rate of both decreased significantly （P＜0.05 or P＜0.01）. The proportion of HT- 29 cells at G 2/M phase in TAG groups as well as those at G 0/G1 phase in medium-dose group were increased significantly ；the proportion of HT- 29 cells at S phase in TAG groups as well as those at G 0/G1 phase in high-dose group were decreased significantly （P＜0.05 or P＜0.01）. Survival rate ，migration rate and invasion rate of HUVEC were decreased significantly in TAG groups ，and tube number was also decreased significantly at each time point during 4-24 h（P＜0.01）. CONCLUSIONS ：TAG have inhibitory effect on the proliferation of human colon cancer HT- 29 cells and HUVEC，can change HT- 29 cell cycle ，inhibit the migration ，invasion and tube formation of HUVEC.

[Correlation between rhizospheric microorganisms distribution and alkaloid content of Fritillaria taipaiensis].

In order to reveal the relationship between the amount of soil microorganisms and the quality of Fritillaria taipaiensis, both cultivated and wild F. taipaiensis were collected from Chongqing, Wuxi at different stages of their growth as objects of the research. The mycorrhizal infection rate and colonization intensity, peimisine and total alkaloid content in bulbs, the amount of microorganisms and biomass carbon content in rhizospheric soil were all determined using common methods. The results showed that the typical arbuscular-vesicle roots were formed after the AM fungi infected the F. taipaiensis roots which were collected from different origins. The mycorrhizal infection rates were ranged from 78.74% to 98.68% and the colonization intensities were ranged from 13.29% to 37.06%. The rhizospheric microorganisms of F. taipaiensis showed abundant resources. The distribution rule of them in the rhizospheric soil was as follows: the amount of bacteria>the amount of actinomycetes>the amount of fungi. The rhizospheric bacteria, decomposition inorganic phosphorus bacteria, decomposition organic phosphorus bacteria, actinomycetes amount and the total number of microbes increased first and then decreased with the increase of years, while decomposition potassium bacteria showed decreasing trend and fungi showed gradual increasing trend. The soil microbial flora content in the soil changed from "bacterial type" with a high fertility to "fungal type" with a low fertility. The mass fraction of peimisine and total alkaloid content increased first and then decreased with the increase of over the years, the same trend of culturable rhizosphere soil bacteria and actinomycetes indicated that the growth years affected the quality of soil and medicinal materials on different levels. Therefore, the diversity of microbial communities in rhizosphere soil reduced with the increase of years leading to the continuous cropping obstacles and the destruction of medicinal quality of F. taipaiensis.

Correlation between rhizospheric microorganisms distribution and alkaloid content of Fritillaria taipaiensis / 中国中药杂志

In order to reveal the relationship between the amount of soil microorganisms and the quality of Fritillaria taipaiensis, both cultivated and wild F. taipaiensis were collected from Chongqing, Wuxi at different stages of their growth as objects of the research. The mycorrhizal infection rate and colonization intensity, peimisine and total alkaloid content in bulbs, the amount of microorganisms and biomass carbon content in rhizospheric soil were all determined using common methods. The results showed that the typical arbuscular-vesicle roots were formed after the AM fungi infected the F. taipaiensis roots which were collected from different origins. The mycorrhizal infection rates were ranged from 78.74% to 98.68% and the colonization intensities were ranged from 13.29% to 37.06%. The rhizospheric microorganisms of F. taipaiensis showed abundant resources. The distribution rule of them in the rhizospheric soil was as follows: the amount of bacteria>the amount of actinomycetes>the amount of fungi. The rhizospheric bacteria, decomposition inorganic phosphorus bacteria, decomposition organic phosphorus bacteria, actinomycetes amount and the total number of microbes increased first and then decreased with the increase of years, while decomposition potassium bacteria showed decreasing trend and fungi showed gradual increasing trend. The soil microbial flora content in the soil changed from "bacterial type" with a high fertility to "fungal type" with a low fertility. The mass fraction of peimisine and total alkaloid content increased first and then decreased with the increase of over the years, the same trend of culturable rhizosphere soil bacteria and actinomycetes indicated that the growth years affected the quality of soil and medicinal materials on different levels. Therefore, the diversity of microbial communities in rhizosphere soil reduced with the increase of years leading to the continuous cropping obstacles and the destruction of medicinal quality of F. taipaiensis.

Effects of Capparis spinosa Total Alkaloid on Notch Pathway in Mice with Systemic Sclerosis / 中国药房

OBJECTIVE： To study the effects of Capparis spinosa total alkaloid on Notch pathways related protein Notch2， Delta-like 3 （DLL3）， Jagged1 and Notch intracellular domain 1 （NICD1） in mice with systemic sclerosis （SSc）. METHODS： BALB/c mice were randomly divided into blank control group， model group， positive control group （penicillamine 125 mg/kg）， C. spinosa total alkaloid low-dose， medium-dose and high-dose groups （225， 450， 900 mg/kg）， with 16 mice in each group. Except for blank control group， other groups were given bleomycin subcutaneously for 4 weeks to induce SSc model. C. spinosa total alkaloid groups were given relevant dose of C. spinosa total alkaloid cream for external use. Positive control group was given relevant dose of penicillamine intragastrically. Blank control group and model groups were given cream matrix without drug， once a day， for consecutive 8 weeks. 4 h after last administration， the skin of the administration area of each group of mice was collected. mRNA expression of Notch2 and NICD1 was detected by real-time PCR； the content of DLL3 was measured by ELISA； the protein expression of Jagged1 in skin tissue was detected by immunohistochemstry. RESULTS： Compared with blank control group， mRNA expression of Notch2 and NICD1， DLL3 content， protein expression of Jagged1 were markedly increased， with statistical significance （P＜0.01）. Compared with model group， mRNA expression of NICD1， DLL3 content and protein expression of Jagged1 were decreased significantly in C. spinosa total alkaloid medium-dose and high-dose groups， positive control group， mRNA expression of Notch2 in skin tissue were decreased significantly in C. spinosa total alkaloid high-dose group and positive control group， with statistical significance （P＜0.05 or P＜0.01）. CONCLUSIONS： C. spinosa total alkaloid can inhibit the abnormal expression of Notch2， NICD1， DLL3 and Jagged1 in skin tissue of SSc model mice， and inhibit over activation of Notch pathway in SSc model mice.

Optimization of the Ethanol Extraction Technology of Total Alkaloids from Shuanghu Capsules by Orthogonal Design / 中国药房

OBJECTIVE： To optimize the ethanol extraction technology of total alkaloids from Shuanghu capsules. METHODS： Using dendrobine as control， the contents of total alkaloids from Dendrobium nobile and Dendrobium officinale in Shuanghu capsules were determined by acidic dyes colorimetry. Using comprehensive scores calculated by the yield of the extract and the contents of total alkaloids as evaluation indexes， the effects of soaking time， ethanol volume fraction， extraction time， solid-liquid ratio and extraction times were investigated with single factor tests. L9（34） orthogonal test was used to optimize ethanol volume fraction， extraction time， solid-liquid ratio and extraction times according to the results of single factor test. The optimized technology was validated. RESULTS： The linear range of dendrobine were 4.16-14.56 μg/mL （r＝0.999 2）. RSDs of repeatability and precision tests were all lower than 5％. Average recovery tests were 93.01％ （RSD＝1.97％， n＝6）. The optimal ethanol extraction technology included soaking for 12 h， ethanol volume fraction of 70％， solid-liquid ratio of 1 ∶ 12 （g/mL）， extracting for 28 min， extracting 3 times. Results of validation test showed that the average yield of extract was 12.80％ （RSD＝4.39％， n＝3）， and the content of alkaloids was 0.359 0 mg/g（RSD＝0.66％， n＝3）. CONCLUSIONS： Established acidic dyes colorimetry is simple， precise and accurate， which can be used for the content determination of total alkaloids. The optimized ethanol extraction technology is stable and feasible， and can be used for the extraction of total alkaloids from Shuanghu capsules.

Effects of Capparis Spinosa Total Alkaloid on Transforming Growth Factor-β1/Smad4 Signaling Pathways in Systemic Sclerosis / 医药导报

Objective To evaluate the effects of capparis spinosa total alkaloid on transforming growth factor-β( TGF-β)/Smad4 signalling pathways in systemic sclerosis (SSc). Methods A total of 90 BALB/c mice were randomly divided into normal control group, model control group, penicillamine ( 125 mg·kg-1) group and Capparis spinosa total alkaloid low (225 mg·kg-1),medium (450 mg·kg-1) and high ( 900 mg·kg-1) dose group. Except for the normal control group,SSc mouse model was established by daily subcutaneous injection of bleomycin in the back of the mice.After the establishment of the model,Capparis spinosa total alkaloid emulsifiable paste was externally applied to Capparis spinosa total alkaloid group,ground substance was externally applied to the mice in normal control group and model control groups, and penicillamine was intragastrically administrated in the penicillamine group for 60 days,once daily.After the treatment,The expression of TGF-β1in skin tissue was detected by Western-blotting and the levels of actin / in receptor-like kinase / Smad4, nuclear factor-κB in skin tissue were measured by ELISA. Results The expression of TGF-β1was significantly decreased after administration of 225,450 and 900 mg·kg-1capparis spinosa total alkaloid, and the levels of ALK1 and Smad4 were significantly decreased after administration of 900 mg·kg-1capparis spinosa total alkaloid as compared with model control group (P<0.05 or P<0.01),but the content of NF-κB was not influenced (P>0.05). Conclusion Capparis spinosa total alkaloid can accommodate abnormal expression of TGF-β1/Smad4 signalling pathways in SSc.

In vitro antiviral effects of Peganum harmala seed extract and its total alkaloids against Influenza virus.

This research was aimed to evaluate the in vitro antiviral effect and the mechanism of the effect of Peganum. harmala seeds extract against influenza A virus infection using Madin-Darby canine kidney (MDCK) cells. In this research, ethyl alcohol extract of P. harmala seeds and its total alkaloids was prepared. The potential antiviral activity of the extract and its total alkaloids against influenza A/Puerto Rico/8/34 (H1N1; PR8) virus was assessed. The mode of action of the extract to inhibit influenza replication was investigated using virucidal activity, hemagglutination inhibition assay, time of addition assays, RNA replication, western blot analysis and RNA polymerase blocking assay. The crud extract of P. harmala seed and its total alkaloids showed the best inhibitory effect against influenza A virus replication in MDCK cells using MTT assay, TCID50 method and hemagglutination assay. Our results indicated that the extract inhibits viral RNA replication and viral polymerase activity but did not effect on hemagglutination inhibition and virucidal activity. This study showed that, in vitro antiviral activity of P. harmala seed extract against influenza virus is most probably associated with inhibiting viral RNA transcription. Therefore, this extract and its total alkaloid should be further characterized to be developed as anti-influenza A virus agent.

Content determination of alkaloids in different parts of Papaver nudicaule from different populations / 中草药

Objective To determine the qualitities Papaver nudicaule from different populations, a total alkaloid and three main kinds of alkaloids (reframidine, nudicauline, and amurensinine) from different parts, in order to provide a scientific basis for rational utilization and quality evaluation of P. nudicaule. Methods The qualitative and quantitative analysis of P. nudicaule has been conducted based on the method of LCMS-IT-TOF, Bromocresol green acid dye colorimetry and UPLC-MS, respectively. Results From the quantitative analysis, 13 kinds of alkaloids have been analyzed, in which eight kinds of chemical structures have been identified, and the results of quantitative analysis showed that there were greater variabilities in total alkaloid and three main kinds of alkaloids. Comprehensively, the plants grown in WLBT, HGL, and DL were better than other populations, in which the plants grown in WLBT were the best. In addition, the contents of total alkaloid and three main kinds of alkaloids were highly existed in the entire plants and flowers. Conclusion Based on the evaluation of the quality characteristics of P. nudicaule, it is suggested that total alkaloid and nudicauline can be seen as main reference indexes. Additionally, the result suggests that WLBT, HGL, and DL can be chosen to select and breed excellent quality of P. nudicaule, develop the quality standard and make layout of source area. The best harvest time should be chosen in late July to early August, and harvest site should be chosen flowers and whole plants.