The neutrophil-to-lymphocyte ratio and lactate dehydrogenase combined in predicting liver metastasis and prognosis of colorectal cancer.

Background: The neutrophil-to-lymphocyte ratio (NLR) and lactate dehydrogenase (LDH) level are inflammatory markers related to tumor growth and metabolism. This study investigated the value of preoperative NLR, LDH and the combination of NLR and LDH (NLR-LDH) for predicting colorectal cancer liver metastasis (CRLM) and tumor prognosis in the early stages of colorectal cancer (CRC). Materials and methods: Three hundred patients undergoing CRC resection were included. Logistic regression analysis was used to estimate the correlation between CRLM time and inflammatory markers, and Kaplan-Meier survival and Cox regression analyses were used to estimate overall survival (OS). Forest plots were prepared based on the multivariate Cox analysis model and evaluated by receiver operating characteristic (ROC) curve analysis. Results: The NLR cut-off value was 2.071 according to the ROC curve. The multivariate analysis showed that the elevated LDH level and a high NLR-LDH level were independent predictors of synchronous CRLM and OS (p < 0.05). The combination of a high NLR and elevated LDH and NLR-LDH levels suggested a poor prognosis and a significantly shorter median survival time than a low NLR and low levels of LDH and NLR-LDH. The ROC curve analysis results illustrated that the predictive value of the NLR-LDH score for synchronous CRLM [area under the curve (AUC) = 0.623, p < 0.001] and OS (AUC = 0.614, p = 0.001) was superior to that of the NLR or LDH score used alone. Conclusion: LDH and NLR-LDH are reliable, easy-to-use, independent biomarkers for predicting synchronous or metachronous CRLM and OS in CRC patients. The NLR is an important monitoring index for CRLM. Preoperative NLR, LDH and NLR-LDH may help to guide the use of therapeutic strategies and cancer surveillance.

Technical guidelines for risk assessment of heavy metals in traditional Chinese medicines.

BACKGROUND: Heavy metals are considered a global concern because they can deteriorate human health. This guideline aims to scientifically evaluate health risk of heavy metals in TCM and to propose a reference for decision making in developing TCM-related health policies. METHODS: Using a multidisciplinary approach, a steering committee oversaw the development of the guideline. To obtain a reasonable and accurate risk assessment, key exposure assessment parameters for TCM, e.g., exposure frequency (EF), exposure duration (ED), and daily ingestion rate (IR) were obtained from surveys. In addition, transfer rates for heavy metals from Chinese medicinal materials (CMM) to decoctions or preparations were examined. RESULTS: Based on the scientific theory of risk control, the guideline was designed systematically, and principles and procedures for the risk assessment of heavy metals in TCM were identified. The guideline can be utilized to assess the risk of heavy metals in CMM and Chinese patent medicines (CPM). CONCLUSION: This guideline may help standardize the risk assessment of heavy metals in TCM, advance regulatory standards for heavy metals in TCM, and ultimately improve human health through scientific TCM usage in clinic.

Novel Integrated Tiered Cumulative Risk Assessment of Heavy Metals in Food Homologous Traditional Chinese Medicine Based on a Real-Life-Exposure Scenario.

In order to serve population health better, the first integrated tiered decision tree for cumulative risk assessment of co-exposure of Pb-, Cd-, and As-associated health risks in food homologous traditional Chinese medicine (TCM) was designed, after measuring their concentrations by inductively coupled plasma-mass spectroscopy (ICP-MS). Basically, our three-step decision tree involving hazard quotient (HQ), hazard index (HI), and target-organ toxicity dose (TTD) modification of the HI method was developed to evaluate the potential risks of 949 batches of 15 types of food homologous TCM. To acquire a real-life exposure scenario, the cumulative risk assessment model was established by optimizing key parameters, such as ingestion rates, frequency, and duration of exposure to food homologous TCM based on questionnaire data. As a result, the mean concentrations of Pb, Cd, and As in 949 batches of food homologous TCM were 0.896, 0.133, and 0.192 mg/kg, respectively. The HQ values of As for Angelica sinensis (Oliv.) Diels and Houttuynia cordata Thunb. were 1.04 and 1.01, respectively, for females. Other HQs of Pb, Cd, or As in food homologous TCM were lower than 1 for both males and females. However, after rapid screening of the co-exposure health risks of heavy metals by the HI method, cumulative risk assessment results acquired by TTD modification of the HI method implied that the potential health risks associated with the co-exposure of Pb, Cd, and As in Lonicera japonica Thunb. and Houttuynia cordata Thunb. ingested as both TCM and food were of concern in the clinic. Additionally, the cumulative risks of Pb, Cd, and As in Mentha canadensis L., Chrysanthemum indicum L., and Zaocys dhumnades (Cantor) only used as food exceeded the human tolerance dose. Collectively, our innovation on the tiered strategy of decision tree based on a real-life exposure scenario provides a novel approach engaging in the cumulative risk assessment of heavy metals in food homologous TCM. All in all, such effort attempts to scientifically guide the rational use of TCM in the treatment of the complex diseases and the improvement of population health.

Microbial spectrum and drug resistance of pathogens cultured from gallbladder bile specimens of patients with cholelithiasis: A single-center retrospective study.

BACKGROUND: Bacterial infection is an important cause of cholelithiasis or gallstones and interferes with its treatment. There is no consensus on bile microbial culture profiles in previous studies, and identified microbial spectrum and drug resistance is helpful for targeted preventive and therapeutic drugs in the perioperative period. AIM: To analyze the bile microbial spectrum of patients with cholelithiasis and the drug susceptibility patterns in order to establish an empirical antibiotic treatment for cholelithiasis-associated infection. METHODS: A retrospective single-center study was conducted on patients diagnosed with cholelithiasis between May 2013 and December 2018. RESULTS: This study included 185 patients, of whom 163 (88.1%) were diagnosed with gallstones and 22 (11.9%) were diagnosed with gallstones and common bile duct stones (CBDSs). Bile culture in 38 cases (20.5%) was positive. The presence of CBDSs (OR = 5.4, 95%CI: 1.3-21.9, P = 0.03) and longer operation time (> 80 min) (OR = 4.3, 95%CI: 1.4-13.1, P = 0.01) were identified as independent risk factors for positive bile culture. Gram-negative bacteria were detected in 28 positive bile specimens, and Escherichia coli (E. coli) (19/28) and Klebsiella pneumoniae (5/28) were the most frequently identified species. Gram-positive bacteria were present in 10 specimens. The resistance rate to cephalosporin in E. coli was above 42% and varied across generations. All the isolated E. coli strains were sensitive to carbapenems, with the exception of one imipenem-resistant strain. K. pneumoniae showed a similar resistance spectrum to E. coli. Enterococcus spp. was largely sensitive to glycopeptides and penicillin, except for a few strains of E. faecium. CONCLUSION: The presence of common bile duct stones and longer operation time were identified as independent risk factors for positive bile culture in patients with cholelithiasis. The most commonly detected bacterium was E. coli. The combination of ß-lactam antibiotics and ß-lactamase inhibitors prescribed perioperatively appears to be effective against bile pathogens and is recommended. Additionally, regular monitoring of emerging resistance patterns is required in the future.

Berberine Reverses Breast Cancer Multidrug Resistance Based on Fluorescence Pharmacokinetics In Vitro and In Vivo.

Exploring the mechanism through which berberine (Ber) reverses the multidrug resistance (MDR) of breast cancer is of great importance. Herein, we used the methyl thiazolyl tetrazolium assay to determine the drug resistance and cytotoxicity of Ber and doxorubicin (DOX) alone or in combination on the breast cancer cell line MCF-7/DOXFluc. The results showed that Ber could synergistically enhance the inhibitory effect of DOX on tumor cell proliferation in vitro, and the optimal combination ratio was Ber/DOX = 2:1. Using a luciferase reporter assay system combined with the bioluminescence imaging technology, the efflux kinetics of d-luciferin potassium salt in MCF-7/DOXFluc cells treated with Ber in vivo was investigated. The results showed that Ber could significantly reduce the efflux of d-luciferin potassium salt in MCF-7/DOXFluc cells. In addition, western blot and immunohistochemistry experiments showed that the expression of P-glycoprotein (P-gp/ABCB1) and multidrug resistance protein 1 (MRP1/ABCC1) in MCF-7/DOXFluc cells was downregulated upon Ber treatment. Finally, high-performance liquid chromatography was used to investigate the effect of Ber on DOX tissue distribution in vivo, and the results showed that the uptake of DOX in tumor tissues increased significantly when combined with Ber (P < 0.05). Thus, the results illustrated that Ber can reverse MDR by inhibiting the efflux function of ATP-binding cassette transporters and downregulating their expression levels.

Shuxuetong for Prevention of recurrence in Acute Cerebrovascular events with Embolism (SPACE) trial: rationale and design.

BACKGROUNDS: Embolic stroke is one of the main mechanisms of ischaemic stroke. Even if treated with recommended antithrombotic agents, stroke recurrence remains high. The Shuxuetong injection, a purified extract of traditional Chinese medicine widely used for thrombus diseases in clinical practice in China, could be a promising agent to prevent stroke recurrence. AIMS: To describe the design of the Shuxuetong injection for prevention of recurrence in acute ischaemic stroke with embolism mechanisms. DESIGN: The Shuxuetong for Prevention of recurrence in Acute Cerebrovascular events with Embolism (SPACE) trial is a multicentre, randomised, double-blind, placebo-controlled, parallel-group, superiority trial to evaluate the efficacy and safety of Shuxuetong injection in reducing recurrence or silent new ischaemic lesions on patients with acute embolic stroke within 10 days. An estimated 2416 patients with embolic stroke within 72 hours of symptom onset from 80 hospitals will be randomly assigned to one of two groups receiving Shuxuetong injection or placebo injection for 10 days. The primary endpoint is symptomatic or asymptomatic new cerebral infarction within 10 days after randomisation. CONCLUSION: The SPACE Trial will provide valuable evidence for the efficacy and safety of Shuxuetong injection for the prevention of stroke recurrence in patients with imaging-defined embolic stroke. CLINICAL TRIAL REGISTRATION: NCT03090113.

[Determination and risk assessment of heavy metals and harmful elements residues in Niuhuang Qingwei Pills based on ICP-MS].

To achieve a comprehensive understanding of heavy metals and harmful elements residues in Niuhuang Qingwei Pills,49 samples from 18 manufactures were collected from 31 provinces in China.Risk assessment and control preparations were applied innovatively in evaluation of exogenous pollution in traditional Chinese Medicine.Determination methods for Pb,Cd,As,Hg and Cu were established by inductively coupled plasma mass spectrometry(ICP-MS).Based on the procedures including hazard identification,hazard characterization,exposure assessment and risk characterization,risk assessment was performed and residual limits for Pb,Cd,As,Hg and Cu in the drug were formulated.The results showed that the hazardous quotients(HQ) of the elements were decreased in the following order:Pb>As>Cu>Hg>Cd,and the total hazardous index(HI) of heavy metals and harmful elements in Niuhuang Qingwei Pills was above 1,implying health risk of the drug.Under the proposed limits,5 elements in the control preparation as well as Cd and Cu in the samples were within the limits range,but the excess rates of Pb,As and Hg in the samples were 12%,12% and 14%,respectively.For the first time,basic steps for risk assessment of Chinese patent medicine were established,which provided model and reference for risk assessment and limit formulation of other drugs.

[Purification and biochemical function of Astragalus membtanaceus pathogenesis-related protein 10].

Astragalus membranaceus pathogenesis-related protein 10 (AmPR-10) is largely expressed in case of environmental pressure and pathogen invasion. This study aims to explore the biochemical functions of AmPR-10. The dried root of Astragalus membranaceus was mechanically homogenized and extracted by Tris-HCl buffer to obtain its crude extract, which was then purified by anion exchange chromatography and gel filtration chromatography to obtain electrophoretically pure AmPR-10. The nuclease activity of AmPR-10 was tested with different RNAs by detecting the absorption value at 260 nm. The results demonstrated potent nuclease activity toward yeast tRNA, yeast RNA, Poly (A) and Poly (C). The optimum reaction temperature was 50 °C and pH was 7-8. EDTA showed no effect on its activity, while Mg²âº exhibited potent activation effect on the activity, and Co²âº, Ca²âº and Zn²âº manifested moderately inhibition of the activity. Since AmPR-10 had no sequence homology with other known nucleases, AmPR-10 was probably a novel nuclease. The inhibition kinetic data against papain was analyzed by Lineweaver-Burk plots, and the results showed that the inhibition of papain followed noncompetitive-type kinetics. AmPR-10 played an important role in Astragalus membranaceus defense mechanism against environmental pressure and pathogen invasion, which may be achieved by inhibiting cycteine enzymes activity.

Effect of ß-elemene on the kinetics of intracellular transport of d-luciferin potassium salt (ABC substrate) in doxorubicin-resistant breast cancer cells and the associated molecular mechanism.

In order to explore the mechanism of the reversing multidrug resistance (MDR) phenotypes by ß-elemene (ß-ELE) in doxorubicin (DOX)-resistant breast cancer cells (MCF-7/DOX), both the functionality and quantity of the ABC transporters in MCF-7/DOX were studied. Bioluminescence imaging (BLI) was used to study the efflux of d-luciferin potassium salt, the substrate of ATP-binding cassette transporters (ABC transporters), in MCF-7/DOX cells treated by ß-ELE. At the same time three major ABC transport proteins and genes-related MDR, P-glycoprotein (P-gp, ABCB1) and multidrug resistance-associated protein 1 (MRP, ABCC1) as well as breast cancer resistance protein (BCRP, ABCG2) were analyzed by q-PCR and Western blot. To investigate the efflux functionality of ABC transporters, MCF-7/DOXFluc cell line with stably-overexpressed luciferase was established. BLI was then used to real-time monitor the efflux kinetics of d-luciferin potassium salt before and after MCF-7/DOXFluc cells being treated with ß-ELE or not. The results showed that the efflux of d-luciferin potassium salt from MCF-7/DOXFluc was lessened when pretreated with ß-ELE, which means that ß-ELE may dampen the functionality of ABC transporters, thus decrease the efflux of d-fluorescein potassium or other chemotherapies which also serve as the substrates of ABC transporters. As the effect of ß-ELE on the expression of ABC transporters, the results of q-PCR and Western blot showed that gene and protein expression of ABC transporters such as P-gp, MRP, and BCRP were down-regulated after the treatment of ß-ELE. To verify the efficacy of ß-ELE on reversing MDR, MCF-7/DOX cells were treated with the combination of DOX and ß-ELE. MTT assay showed that ß-ELE increased the inhibitory effect of DOX on the proliferation of MCF-7/DOX, and the IC50 of the combination group was much lower than that of the single DOX or ß-ELE treatment. In all, ß-ELE may reverse MDR through the substrates of ABC transporters by two ways, to lessen the ABC protein efflux by weakening their functionality, or to reduce the quantity of ABC gene and protein expression.

Dual face of axonal inhibitory inputs in the modulation of neuronal excitability in cortical pyramidal neurons.

Limited by the tiny structure of axons, the effects of these axonal hyperpolarizing inputs on neuronal activity have not been directly elucidated. Here, we imitated these processes by simultaneously recording the activities of the somas and proximal axons of cortical pyramidal neurons. We found that spikes and subthreshold potentials propagate between somas and axons with high fidelity. Furthermore, inhibitory inputs on axons have opposite effects on neuronal activity according to their temporal integration with upstream signals. Concurrent with somatic depolarization, inhibitory inputs on axons decrease neuronal excitability and impede spike generation. In addition, following action potentials, inhibitory inputs on an axon increase neuronal spike capacity and improve spike precision. These results indicate that inhibitory inputs on proximal axons have dual regulatory functions in neuronal activity (suppression or facilitation) according to neuronal network patterns.

[Determination of stachydrine hydrochloride and leonurine hydrochloride in Yimucao preparations by HPLC-MS].

To establish the quantitative method of stachydrine hydrochloride and leonurine hydrochloride in the preparations of Leonuri Herba. The contents of stachydrine hydrochloride and leonurine hydrochloride in the preparations of Leonuri Herba were determined by HPLC-MS. The chromatographic column was Waters XBridge Amide(4.6 mm×250 mm,5 µm). The mobile phase was acetonitrile-0.1% formic acid in gradient mode,at the flow rate of 1.0 mL• min⁻¹,with the split ratio of 1∶4. MS conditions for the ESI ion source,positive ion mode,selective ion scan(SIM) of stachydrine hydrochloride(m/z 144.0) and leonurine hydrochloride(m/z 312.0) was measured. The linear ranges of stachydrine hydrochloride was 0.562 8-281.4 µg•L-1（r=0.999 8）. The linear ranges of leonurine hydrochloride was 0.521 2-260.6 µg•L-1（r=0.999 8）. The method is accurate,simple,and reliable,and can be used to determine the contents of stachydrine hydrochloride and leonurine hydrochloride in the preparations of Leonuri Herba.

[Study on saponins in shengmai injection].

OBJECTIVE: To investigate the saponin in Shengmai injection. METHOD: On the basic of studing the chemical constituents of red ginseng and Shengmai injection, 20 compositions had been identifided by LC-MS/MS. RESULT: Twenty identifided compositions were the common components of Shengmai injection and red ginseng extracts. CONCLUSION: The analytical method for saponins in Shengmai injection was established which could be used as the basis for further study and quality control.

Functional distinction of rat liver natural killer cells from spleen natural killer cells under normal and acidic conditions in vitro.

BACKGROUND: The microenvironment within solid tumors has often been shown to exhibit an acidic extracellular pH. Although the morphologic and functional differences in natural killer (NK) cells of the liver and spleen have been reported previously under physiological conditions, the difference under acidic conditions is still unclear. This study was to investigate the differences in the morphological and functional characteristics between rat liver and spleen NK cells under normal and acidic conditions in vitro. METHODS: Liver and spleen NK cells were isolated and purified from Sprague-Dawley rats by density gradient centrifugation and the Dynabeads(®) FlowComp(TM) Flexi system, and stimulated for 4 days with or without IL-2 or treated with low pH or control for different times. Morphology was examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), cell death and proliferation assays were performed by flow cytometry, IFN-gamma production was tested by ELISA, and cytotoxic activity was evaluated by lactate dehydrogenase (LDH) release assay. RESULTS: Liver NK cells had significantly higher levels of cytotoxic activity than spleen NK cells under normal and acidic conditions, and the maximum difference was observed at pH 5.6. Further analysis revealed that the cytotoxic activity of NK cells was correlated with morphology, cell death, proliferative activity and IFN-gamma production. By TEM, liver NK cells contained a greater number of electron-dense granules per cell at pH 5.6. Moreover, a modest elevation of cell death and reduction of proliferation of liver NK cells occurred within a range of 5.6-7.2. Interestingly, an acidic extracellular pH only marginally, and not significantly, suppressed IFN-gamma production by liver NK cells. CONCLUSION: The sharp morphological and functional differences shown by the two types of NK cells in vitro indicate that liver NK cells are unexpectedly resistant to pH shock.

Inflammatory myofibroblastic tumor of the liver following renal transplantation.

Inflammatory myofibroblastic tumor (IMT), previously named inflammatory pseudotumor, is a benign lesion, the exact etiology of which remains obscure; immunosuppression and infections have been speculated to be responsible for the development of pseudotumor. IMT associated with transplantation is rarely reported; we report the first case of IMT of the liver in a renal transplantation patient, who presented with symptoms of abdominal pain. The findings of computed tomography suggested hepatocellular carcinoma or liver abscess, and surgical resection was performed. The lesion was pathologically diagnosed as IMT.

[Management of postoperative chyle leak after surgery for digestive malignancies].

OBJECTIVE: To investigate the treatment of postoperative chyle leak after surgery for digestive malignancies. METHODS: From December 2008 to February 2012, in the Sun Yat-sen Memorial Hospital of Sun Yat-sen University, clinical data of 19 patients with chyle leak after digestive system cancer surgery were retrospective analyzed. RESULTS: Nineteen cases of chyle leak were all identified between the second and the fourth postoperative day and were all initially managed with conservative treatment including early fasting, parenteral nutrition(PN), 24-hour continuous infusion of somatostatin, and low pressure suction drainage. Eight patients were treated successfully for 6 to 10 days with a significant reduction of the daily drainage volume. Ten patients had enteral nutrition(EN) and their drain tubes were repeatedly washed with 30 ml of compound meglumine diatrizoate injection every day until the drainage volume decreased to 200 ml/day. The time to resolution of chyle leak in these ten patients ranged from 12 to 24 days. One patient had no significant decrease in fluid drainage and developed abdominal distension after one week of conservative treatment. Surgical closure of chyle leak was performed on the 11th postoperative day, abdominal cavity drainage tube was removed on the 4th postoperative day. The patient was discharged home in good condition. CONCLUSION: Most postoperative chyle leak after surgery for digestive malignancies can be successfully managed with conservative treatment. Somatostatin and the drainage are the main therapeutic approaches. When chyle leak is not resolved with conservative treatment, surgical treatment should be considered to prevent serious complications.

High incidence of biliary complications in rat liver transplantation: can we avoid it?

AIM: To investigate how to reduce the incidence of biliary complications in rat orthotopic liver transplantation. METHODS: A total of 165 male Wistar rats were randomly divided into three groups: Group A, orthotropic liver transplantation with modified "two-cuff" technique; Group B, bile duct was cut and reconstructed without transplantation; and Group C, only laparotomy was performed. Based on the approaches used for biliary reconstruction, Group A was divided into two sub-groups:A1 (n = 30), duct-duct reconstruction, and A2 (n = 30), duct-duodenum reconstruction. To study the influence of artery reconstruction on bile duct complication, Group B was divided into four sub-groups: B1 (n = 10), duct-duct reconstruction with hepatic artery ligation, B2 (n = 10), duct-duct reconstruction without hepatic artery ligation, B3 (n = 10), duct-duodenum reconstruction with hepatic artery ligation, and B4 (n = 10), duct-duodenum reconstruction without hepatic artery ligation. The samples were harvested 14 d after operation or at the time when significant biliary complication was found. RESULTS: In Group A, the anhepatic phase was 13.7 ± 1.06 min, and cold ischemia time was 50.5 ± 8.6 min. There was no significant difference between A1 and A2 in the operation duration. The time for biliary reconstruction was almost the same among all groups. The success rate for transplantation was 98.3% (59/60). Significant differences were found in the incidence of biliary complications in Groups A (41.7%), B (27.5%) and C (0%). A2 was more likely to have biliary complications than A1 (50% vs 33.3%). B3 had the highest incidence of biliary complications in Group B. CONCLUSION: Biliary complications are almost inevitable using the classical "two cuff" techniques, and duct-duodenum reconstruction is not an ideal option in rat orthotopic liver transplantation.

Donor liver natural killer cells alleviate liver allograft acute rejection in rats.

BACKGROUND: Liver enriched natural killer (NK) cells are of high immune activity. However, the function of donor liver NK cells in allogeneic liver transplantation (LTx) remains unclear. METHODS: Ten Gy of whole body gamma-irradiation (WBI) from a 60Co source at 0.6 Gy/min was used for depleting donor-derived leukocytes, and transfusion of purified liver NK cells isolated from the same type rat as donor (donor type liver NK cells, dtlNKs) through portal vein was performed immediately after grafting the irradiated liver. Post-transplant survival observation on recipients and histopathological detection of liver grafts were adoptive to evaluate the biological impact of donor liver NK cells on recipients' survival in rat LTx. RESULTS: Transfusion of dtlNKs did not shorten the survival time among the recipients of spontaneous tolerance model (BN to LEW rat) after rat LTx, but prolonged the liver graft survival among the recipients depleted of donor-derived leukocytes in the acute rejection model (LEW to BN rat). Compared to the recipients in the groups which received the graft depleted of donor-derived leukocytes, better survival and less damage in the allografts were also found among the recipients in the two different strain combinations of liver allograft due to transfusion of dtlNKs. CONCLUSIONS: Donor liver NK cells alone do not exacerbate liver allograft acute rejection. Conversely, they can alleviate it, and improve the recipients' survival.

Selaginellin A and B, two novel natural pigments isolated from Selaginella tamariscina.

Two new unusual natural pigments were first isolated from the whole herbs of Selaginella tamariscina. The structure of selaginellin A (1) was established as (R,S)-4-[(4'-hydroxy-3-((4-hydroxyphenyl)ethynyl)biphenyl-2-yl)(4-hydroxyphenyl)methylene]-2,5-cyclohexadien-1-one and selaginellin B (2) as (R,S)-4-[(4'-methoxy-4-(methyl)-3-((4-methoxyphenyl)ethynyl)biphenyl-2-yl)(4-methoxyphenyl)methylene]-2,5-cyclohexadien-1-one, along with four known biflavonoids, amentoflavone (3), hinokiflavone (4), heveaflavone (5), and 7''-O-methylamentoflavone (6). Their chemical structures were elucidated by spectral analysis of electrospray ionization mass spectroscopy (ESI-MS), one-dimensional nuclear magnetic resonance spectroscopy (1D-NMR) and two-dimensional-nuclear magnetic resonance spectroscopy (2D-NMR) including (1)H-NMR, (13)C-NMR, distortionless enhancement by polarization transfer (DEPT) and heteronuclear multiple bond coherence (HMBC), and single-crystal X-ray diffraction techniques.