•A method for screening mitochondrial ligands from complex matrixes was developed.•10 mitochondrial ligands were found from Polygoni Cuspidati Rhizoma et Radix.•9 mitochondrial ligands were found from Scutellariae Radix.•17 compounds were identified as new mitochondrial ligands.•The direct mitochondria-bound activity of 9 compounds was confirmed.Mitochondria are an important intracellular pharmacological target because damage to this organelle results in a variety of human disorders and because mitochondria are involved in complex processes such as energy generation, apoptosis and lipid metabolism. To expedite the search for natural bioactive compounds targeting mitochondria, we initially developed an efficient mitochondria-based screening method by combining centrifugal ultrafiltration (CU) with liquid chromatography/mass spectrometry (LC/MS), which is called screening method for mitochondria-targeted bioactive constituents (SM-MBC) and is compatible with the search of mitochondria-targeted compounds from complex matrixes such as herbal medicines extracts. Functionally active, structurally intact and pure mitochondria were obtained from rat myocardium using an optimized protocol for mitochondrial isolation comprising organelle release followed by differential and Nycodenz density gradient centrifugation. After evaluating the reliability of the method using thiabendazole (TZ), rotenone (RN), amiodarone (AR) and trimetazidine (TD) as positive controls, this method was successfully applied to screen bioactive constituents from extracts of Polygoni Cuspidati Rhizoma et Radix (PCRR) and Scutellariae Radix (SR). Nineteen active compounds were detected and identified by LC/MS, of which 17 were new mitochondria-targeted compounds. The activity of 9 of the 19 hit compounds was confirmed by in vitro pharmacological trials. These results demonstrate that SM-MBC can be used for the efficient screening of mitochondria-targeted constituents in complex preparations used to treat mitochondrial disorders, such as PCRR and SR. The results may be meaningful for an in-depth understanding of drug mechanism of action and drug discovery from medicinal herbs.

The dried root tubers of Stemona tuberosa, S. japonica and S. sessilifolia are the original sources of Stemonae Radix (SR) for antitussive and insecticidal activities. The products of SR which are available on the market are variable, and imitations exist. In order to characterize the overall chemical constituents of SR and evaluate its quality, a novel, binary high-performance liquid chromatographic fingerprinting method, describing the pattern of alkaloids (fingerprint I) and non-alkaloids (fingerprint II) of SR was developed. It was also applied to determine whether the medicinal parts and the processing methods affect the quality of SR. Similarity and high-performance liquid chromatography−mass spectrometry (HPLC–MSn) were utilized to compare or identify the chemical constituents of SR. The results indicate that the chemical constituents from different parts of the underground material of Stemona plants are diverse and that the processing methods affect certain constituents in the root tuber samples. The similarity and the resulting chemical consitituents obtained show that the binary chromatographic fingerprint method can be used to differentiate the three official Stemona species or the adulterants of SR, which is helpful for the identification and quality evaluation of SR.

The authentication of natural medical herbs has crucial impact on the clinical curative effect. Much of these herbs have been ground into powders in the market, leading to the difficulty of identification. Microscopic images of these powders contain important evidences for identification. Currently identification based on these microscopic images are mostly conducted by manual observation. Identification aided by computer vision technique is an important research subject recently. These microscopic images usually contain variety of substance, and most of them are noises, thus the target segmentation is necessary for identification. An effective automatic target segmentation algorithm based on texture is proposed in this paper. Our method consists of two steps: “Preliminary Segmentation” and “Further Segmentation”. Firstly, gradient transform and image fusion are conducted for image preprocessing, then each pixel is encoded into a feature vector based on texture and clustered into two groups: background and foreground. Secondly, taking the continuity of edge and the locality of target into consideration, energy equations are established, and maximum flow-minimum cut algorithm is applied to solve them. Three groups of images are tested to evaluate our method, and the experimental results show that our method achieves a better segmentation compared with Grab-Cut, and additionally user inter-action is not required in our method.

Buyang Huanwu decoction (BYHWD), a famous traditional Chinese medicine prescription for the treatment of cerebrovascular diseases, is composed of seven commonly used Chinese herbs—Astragali Radix, Angelicae Sinensis Radix, Paeoniae Radix Rubra, Chuanxiong Rhizoma, Carthami Flos, Persicae Semen and Pheretima. To determine the main absorptive constituents and the metabolites of BYHWD in vivo, urine samples from Wuzhishan (WZS) miniature pigs orally administered with BYHWD (13.6 g crude drugs/kg) were collected to investigate the characteristic compounds. By comparing the high-performance liquid chromatography of a drug-containing urine sample with that of a drug-free sample, 17 characteristic compounds were isolated from the methanol extract of a drug-containing urine sample by column chromatography. Their structures, including 11 isoflavanoids, 2 pterocarpanoids and 4 isoflavonoids, were identified by spectroscopic means. Of the 17 compounds, 8 (1–8) were new compounds with the following structures: 3S-7,3′,4′-trihydroxyisoflavan-3′-O-β-d-glucuronide (1), 3S-7,3′,4′-trihydroxyisoflavan-4′-O-β-d-glucuronide (2), 3S-7,2′,4′-trihydroxyisoflavan-2′-O-β-d-glucuronide (3), 3R-7,2′-dihydroxy-3′,4′-dimethoxyisoflavan-2′-O-β-d-glucuronide (4), 3R-7,2′-dihydroxy-3′,4′-dimethoxyisoflavan-2′-O-β-d-glucuronide-6″-methyl ester (5), 3R-7,2′-dihydroxy-3′,4′-dimethoxyisoflavan-7-O-β-d-glucuronide-6″-methyl ester (6), 3R-7,2′,3′-trihydroxy-4′-methoxyisoflavan-3′-O-β-d-glucuronide-6″-methyl ester (7), and 3S-7,4′,5′-trihydroxy-2′,3′-dimethoxyisoflavan-5′-O-β-d-glucuronide (8). Based on the possible relationship and metabolic pathways of the 17 compounds in vivo, 3R-7,2′-dihydroxy-3′,4′-dimethoxyisoflavan (isomucronulatol, 11), 6aR,11aR-3-hydroxy-9,10-dimethoxypterocarpan (methylnissolin, astrapterocarpan, 13), 7,3′-dihydroxy-4′-methoxyisoflavone (calycosin, 16) and 7-hydroxy-4′-methoxyisoflavone (formononetin, 17) were thought to be the most important absorptive original isoflavonoid constituents of BYHWD in vivo, which underwent reactions of glucuronidation, hydroxylation, demethylation and reduction. The other 13 compounds were deduced to be their metabolites.

Astragali Radix is a widely and commonly used Chinese herbal medicine, which is derived from roots of Astragalus membranaceus var. mongholicus and Astragalus membranaceus. To find a quick and reliable method of distinguishing these two species of Astragali Radix and of determining the age of a sample, microscopic characteristics of the two species were compared using light microscopy. The results showed that the microscopic characteristics, such as number of layers of phellem, continuing lignified xylem bundles within spring wood and lignified parenchyma cells in the central part of the xylem could be used for the differentiation of the root of A. membranaceus from the root of A. membranaceus var. mongholicus. Growth rings (annual rings) were found for the first time in the roots of both species, and could determine the age of a sample. For the first time, radial fibers in both species of Astragali Radix and pipette-shaped fibers in A. membranaceus var. mongholicus were found. The structure of “rotten heart” cork tissue (decayed central xylem) and tubular cork tissue was carefully studied, and the arranged order of tissues in both “rotten heart” and tubular cork tissues is phelloderm and phellem from outside to inside, which is contrary to that in the periderm.

•Electronic nose could discriminate two species of Asari Radix et Rhizoma rapidly.•54 volatile components were identified in Asari Radix et Rhizoma by headspace GC–MS.•9 major components could be used as markers to distinguish these two species.Traditional Chinese medicines (TCM) can be identified by experts according to their odors. However, the identification of these medicines is subjective and requires long-term experience. In this paper, electronic nose, headspace gas chromatography–mass spectrometry (GC–MS) and chemometrics methods were applied to differentiate two species of Asari Radix et Rhizoma by their odors. The samples used were the dried roots and rhizomes of Asarum heterotropoides var. mandshuricum (AH) and Asarum sieboldii (AS). The electronic nose was used to determine the odors of the samples and enabled rapid differentiation of AH and AS when coupled with principal component analysis. Headspace GC–MS was utilized to reveal the differences between the volatile constituents of AH and AS. In all, 54 volatile constituents were identified, and 9 major constituents (eucalyptol, eucarvone, 3,5-dimethoxytoluene, 3,4,5-trimethoxytoluene, methyleugenol, 2,3,5-trimethoxytoluene, croweacin, pentadecane and asaricin) could be used as chemical markers to distinguish these two species. AH contained higher relative contents of eucarvone (1.79–16.76%), 3,5-dimethoxytoluene (6.64–26.52%), 3,4,5-trimethoxytoluene/methyleugenol (6.43–31.67%) and 2,3,5-trimethoxytoluene (1.64–6.66%), whereas AS had higher relative contents of eucalyptol (14.06–24.95%), croweacin (5.64–13.55%), pentadecane (8.44–20.82%) and asaricin (7.03–13.45%). Moreover, AH and AS could be distinguished according to the contents of either all 54 identified volatile constituents or only the 9 major constituents by employing cluster analysis. The proposed method is rapid, simple, eco-friendly and can successfully differentiate these two species of Asari Radix et Rhizoma by their odors.

Two aristololactams, aristololactam GI (1) and aristololactam GII (2), and three aporphines, fissistigamide A (3), fissistigamide B (4) and fissistigmine (5), together with nineteen known alkaloids, one flavone and one anthraquinone were isolated from the ethanol extracts of the stems of Fissistigma oldhamii (Annonaceae). Their structures were elucidated primarily by analysis of NMR, IR, UV, MS and CD data. Alkaloid 1 is a chiral aristololactam formed from a phenylpropanoid derivative attached to a 3,4-dihydroxy aristololactam scaffold. The absolute configuration of 1 was determined by comparing experimental and calculated ECD spectra. The anti-inflammatory activity of the crude extracts and the five alkaloids were tested by measuring the amount of TNF-α and IL-6 released from LPS stimulated RAW264 cell via ELISA. The results demonstrated that the CHCl3-soluble part and alkaloid 2 exhibited significant anti-inflammatory activity in vitro in both assays.Graphical abstractAristololactams (1 and 2) and aporphines (3–5) were isolated. Alkaloid 1 is a chiral aristololactam. Alkaloid 2 showed significant anti-inflammatory activity in vitro.Highlights► Two aristololactams and three aporphines were isolated from Fissistigma oldhamii. ► Aristololactam GI is a chiral aristololactam. ► Aristololactam GI is formed by the aristololactam skeleton combining with a phenylpropanoid moiety. ► The in vitro anti-inflammatory effects of the crude extract and individual alkaloids were evaluated. ► Aristololactam GII showed potential anti-inflammatory activity.

Two alkaloids, named sterculinine I and sterculinine II, together with thirteen known compounds were isolated from the ethanol extract of a well-known Chinese traditional drug, Pangdahai (the seeds of Sterculia lychnophora Hance). Their structures were elucidated by NMR, UV, IR and MS spectroscopic analysis.Two alkaloids, named sterculinine I and sterculinine II, together with thirteen known compounds were isolated from the ethanol extract of the seeds of Sterculia lychnophora.

Five new phenanthrene derivatives: 9-ethoxy-7-methoxy-aristololactam IV (1), norcepharadione A N-β-d-glucopyranoside (2), aristololactamoside I (3), aristololactamoside II (4) and aristothiolactoside (5) together with eleven known phenanthrene derivatives (6–16) were isolated from the ethanol extract of the roots and rhizomes of Asarum heterotropoides var. mandshuricum. The aristololactams with substitution of ethoxy at C-9 position (1, 9, and 10) and the sulfur-containing phenanthrene derivative (5) were reported in the genus Asarum for the first time. Furthermore, six phenanthrene glucoside derivatives (2–5, 13 and 14) were also found in this genus for the first time and compounds 7 and 9–15 were isolated from the genus Asarum for the first time. Six of them (1, 2, 9, 10, 13 and 14) were submitted to cytotoxicity test against human renal proximal tubular epithelial cell lines (HK-2) using MTT and LDH assays. Compounds 1 and 10 showed significant cytotoxic activity against HK-2 cell lines with IC50 values of 18.18 and 20.44 μmol/L in MTT assay and 84.36 and 35.06 μmol/L in LDH assay, respectively. Compound 9 showed moderate cytotoxicity in MTT assay with IC50 values of 95.60 μmol/L, but no cytotoxicity in LDH assay. Compounds 2, 13 and 14 showed cytotoxic effect in neither MTT assay nor LDH assay. Considering the other nephrotoxic phenanthrene derivatives (6, 8, 12, 15 and 16) previously tested, the results implied the potency of renal toxicity of this herb used as a medicine.Download high-res image (180KB)Download full-size image

The cytotoxic activity against some tumor cell lines of 16 commonly used species of Asarum was evaluated in this study. All of these plants were widely used in Asian countries as traditional medicines or folk medicines. Their inhibitory activities against four tumor cell lines (HL-60, BGC-823, KB and Bel-7402) were compared. It was observed that 10 of the tested extracts (eight ethanol extracts and two water extracts) among 32 extracts of these plants showed cytotoxic activity. Those 95% ethanol extractions from A. caudigerellum, A. forbesii, A. inflatum and A. maximum exhibited the highest cytotoxic activity, and 95% ethanol extracts or water extracts of A. sieboldii var. seoulense, A. himalaicum, A. splendens and A. crispulatum showed selective activity against one or two cells among the tested tumor cells. This is the first report of Asarum plants possessing cytotoxic activity against tumor cell lines.

Smilax glabra Roxb. (SGR), a member of the Smilacaceae family and a rhizome of the Liliaceae plant, has shown anti-inflammation and detoxification properties, and a few studies reported its anti-cancer effect. In this study, we showed that SGR inhibited growth of human breast cancer cell line MCF7, colon carcinoma cell line HT-29, and gastric cancer cell line BGC-823 in a dose-dependent manner. Furthermore, SGR could inhibit tumor growth of HT-29 in Balb/c nude mice and murine hepatoma H22 cells in ICR mice. SGR elicited apoptotic cell death, as confirmed by DNA ladder formation, changes in nuclear morphology, and the increased FITC-Annexin-V/PI staining. Permeabilization of mitochondrial membrane (MMP), production of reactive oxygen species (ROS), elevation of intracellular [Ca2+], relocation of cytochrome c, and the activation of caspase-3 were found to be associated with the initiation of apoptosis by SGR treatment. Using microarray analysis, we found the changes in expression profiles of genes related to apoptosis, proliferation and cell cycle control in the cells treated with SGR. Our results demonstrated the mitochondrial regulation of apoptosis by which SGR exerts the anti-cancer effect.Highlights► Herein we examined the anti-cancer effect of Smilax glabra Roxb. (SGR). ► SGR inhibited growth of cancer cells in vitro and in vivo. ► SGR induced apoptosis through mitochondrial pathway. ► SGR affected expression of genes related to cell apoptosis and proliferation.