Harpagide inhibits the TNF-α-induced inflammatory response in rat articular chondrocytes by the glycolytic pathways for alleviating osteoarthritis.

Osteoarthritis (OA) causes severe and functional dysfunction due to abnormal inflammation. The objective of this study was to evaluate the effect of Harpagide (HPG) on TNF-α-induced inflammation in vitro and in vivo. The effect of HPG on the proliferation of rat chondrocytes was studied. The anti-inflammatory effect of HPG and its molecular mechanisms were elucidated by qPCR, Western blotting, flow cytometry, metabolome analysis in vitro. In addition, the OA rat model was established, and the effect of HPG on OA was verified in vivo. We revealed 10 µM HPG demonstrated biocompatibility. The results demonstrated that HPG restored the upregulation of MMP-13, COX2, IL-1ß and IL-6 induced by TNF-α. Moreover, HPG reversed TNF-α induced degradation of the extracellular matrix of chondrocytes. TNF-α treatment induced down-regulation of the mRNA/protein levels of proliferative markers Bcl2, CDK1 and Cyclin D1 were also recovered. HPG can inhibit TNF-α-induced inflammatory response through glycolytic metabolic pathways. HPG can restore TNF-α-induced upregulation of GRP78/IRE1α, and downregulation of AMPK proteins. In vivo experiments demonstrated that after HPG treatment, the appearance and physiological structure of articular cartilage were more integrated with highly organized chondrocytes and rich cartilage matrix compared with OA group. Finally, the molecular docking of HPG and selected key factors in glycolysis results showed that HPG had good binding potential with PFKM, PFKP, PFKFB3, PKM, HK2, and PFKL. In conclusion, the results shown HPG protects and activates chondrocytes, inhibits TNF-α-induced inflammatory response by glycolysis pathway in rat articular chondrocytes, and plays a role in the treatment of OA.

Iridoids Isolation from a Phytochemical Study of the Medicinal Plant Teucrium parviflorum Collected in Iraqi Kurdistan.

Herbal medicines are still widely practiced in Kurdistan Region-Iraq, especially by people living in villages on mountainous regions. Among plants belonging to the genus Teucrium (family Lamiaceae), which are commonly employed in the Kurdish traditional medicine, we have analyzed, for the first time, the methanol and aqueous methanol extracts of T. parviflorum aerial parts. The plant is mainly used by Kurds to treat jaundice, liver disorders and stomachache. We aimed to determine the phytochemical profile of the extracts and the structures of the main components, so to provide a scientific rationale for the ancient use of the plant in the ethno-pharmacological field. TLC analysis of the two extracts on silica gel and reversed phase TLC plates, using different visualization systems, indicated similar contents and the presence of phenolics, flavonoids, terpenoids and sugars. The chlorophyll-free extracts exhibited weak/no antimicrobial activities against a panel of bacteria (MICs = 800-1600 µg/mL) and fungal strains (MICs ≥ 5 mg/mL). At the concentration of 600 µg/mL, the methanol extract showed moderate antiproliferative effects against A549 and MCF-7 cancer cell lines in the MTS assay. Moreover, both extracts exhibited a significant dose-dependent free radical scavenging action against the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (EC50 = 62.11 and 44.25 µg/mL, respectively). In a phytochemical study, a high phenolic content (77.08 and 81.47 mg GAE/g dry extract, respectively) was found in both extracts by the Folin-Ciocalteu assay. Medium pressure liquid chromatographic (MPLC) separation of the methanol extract on a reversed phase cartridge eluted with a gradient of MeOH in H2O, afforded two bioactive iridoid glucosides, harpagide (1) and 8-O-acetylharpagide (2). The structures of 1 and 2 were established by spectral data, chemical reactions, and comparison with the literature. Interestingly, significant amounts of hepatotoxic furano neo-clerodane diterpenoids, commonly occurring in Teucrium species, were not detected in the extract. The wide range of biological activities reported in the literature for compounds 1 and 2 and the significant antiradical effects of the extracts give scientific support to the traditional use in Iraqi Kurdistan of T. parviflorum aerial parts for the preparation of herbal remedies.

Targeting the initiation and termination codons of SARS-CoV-2 spike protein as possible therapy against COVID-19: the role of novel harpagide 5-O-ß-D-glucopyranoside from Clerodendrum volubile P Beauv. (Labiatae).

The global spread of the coronavirus infections disease - 2019 (COVID-19) and the search for new drugs from natural products particularly from plants are receiving much attention recently. In this study, the therapeutic potential of a new iridoid glycoside isolated from the leaves of Clerodendrum volubile against COVID-19 was investigated. Harpagide 5-O-ß-D-glucopyranoside (HG) was isolated, characterised and investigated for its druglikeness, optimized geometry, and pharmacokinetics properties. Its immunomodulatory was determined by chemiluminescence assay using polymorphonuclear neutrophils (PMNs) in addition to T-cell proliferation assay. In silico analysis was used in determining its molecular interaction with severe acute respiratory syndrome coronavirus-2 (SARS-COV-2). HG displayed potent druglikeness properties, with no inhibitory effect on cytochrome P450 (1A2, 2C19, 2C9, 2D6 and 3A4) and a predicted LD50 of 2000 mg/kg. Its 1H-NMR chemical shifts showed a little deviation of 0.01 and 0.11 ppm for H-4 and H-9, respectively. HG significantly suppressed oxidative bursts in PMNs, while concomitantly inhibiting T-cell proliferation. It also displayed a very strong binding affinity with the translation initiation and termination sequence sites of spike (S) protein mRNA of SARS-COV-2, its gene product, and host ACE2 receptor. These results suggest the immunomodulatory properties and anti-SARS-COV-2 potentials of HG which can be explored in the treatment and management of COVID-19.Communicated by Ramaswamy H. Sarma.

Harpagide alleviate neuronal apoptosis and blood-brain barrier leakage by inhibiting TLR4/MyD88/NF-κB signaling pathway in Angiotensin II-induced microglial activation in vitro.

Angiotensin II, the effector peptide of the renin-angiotensin system, is not only a pivotal peptide implicated in the regulation of blood pressure but also a key mediator of the inflammatory processes that play an important role in the pathology of hypertension-related cSVD. Harpagide is the major bioactive constituent of Scrophulariae Radix widely used in traditional Chinese medicine for numerous diseases including hypertension. The present study aimed to investigate the effect of harpagide on Ang II-induced neuroinflammation and the potential mechanism. Pretreated with harpagide or resatorvid (the TLR4 pathway inhibitor), BV2 cells were treated with Ang II or LPS (the TLR4 activator). NO, pro-inflammatory cytokines, the proteins on TLR4/MyD88/NF-κB signaling pathway and the expression of CD86, CD206, TREM2 in BV2 cells were detected respectively. Subsequently, the effects of harpagide on neurotoxicity and BBB destruction triggered by Ang II-induced neuroinflammation were investigated in the co-cultures of BV2 microglia/HT22 hippocampal neurons, BV2 microglia/bEnd.3 endotheliocyte and BV2 microglia/BBB monolayer model. We found that Ang II converted microglia into M1 state and resulted in neuroinflammation through activating TLR4/MyD88/NF-κB signaling pathway. It also triggered the imbalance of TLR4/TREM2 in microglia. Ang II-mediated inflammation microglia further led to neuronal apoptosis and BBB damage. Harpagide showed the effect of alleviating Ang II-mediated neuroinflammation as well as the resulting neurotoxicity and BBB destruction through inhibiting the TLR4/MyD88/NF-κB pathway. The anti-inflammatory and neuroprotective effect of harpagide suggested that it might be a potential therapeutic strategy in hypertensive cSVD.

Changes in the Harpagide, Harpagoside, and Verbascoside Content of Field Grown Scrophularia lanceolata and Scrophularia marilandica in Response to Season and Shade.

Scrophularia lanceolata Pursh and Scrophularia marilandica L. are two common species within the Scrophulariaceae family that are endemic to North America. Historically, these species were used by indigenous peoples and colonialists to treat sunburn, sunstroke, frostbite, edema, as well as for blood purification, and in women's health. Several iridoid and phenylethanoid/phenylpropanoid glycosides detected in these species, such as harpagoside and verbascoside, possess anti-inflammatory and anti-nociceptive properties. Due to the presence of anti-inflammatory metabolites and the historical uses of these species, we performed a two-year field study to determine the optimal production of these important compounds. We subjected the plants to shade treatment and analyzed differences in the metabolite composition between the two species and each of their leaves, stems, and roots at various times throughout the growing seasons. We determined that S. lanceolata plants grown in full sun produced 0.63% harpagoside per dried weight in their leaves compared to shade-grown plants (0.43%). Furthermore, S. lanceolata accumulated more harpagoside than S. marilandica (0.24%). We also found that verbascoside accumulated in the leaves of S. lanceolata and S. marilandica as the growing season progressed, while the production of this metabolite remained mostly seasonally unchanged in the roots of both species.

[Study on scrophulariaceae radix reference extract and its application in quality control of Scrophulariae Radix].

By establishing the preparation process of Scrophulariaceae Radix reference extract(SRRE) and calibrating it, we discussed its feasibility as a substitute for single reference substance in the quality control of Scrophulariae Radix. The SRREs were prepared by solvent extraction method and chromatographic separation technology, and then calibrated with the reference substances of harpagide, angoroside C and harpagoside. The HPLC content determination method of Scrophulariae Radixl was established with SRREs of the known content and the reference substances of harpagide, angoroside C and harpagoside respectively as the control ones. Then the content of three components in Scrophulariae Radix was determined, and the t-test method was used to compare the results of the two methods. With SRRE as references, harpagide, angoroside C and harpagoside were in a good linear relationship(r≥0.999 8) within each range, and the average recovery rate was 98.55% to 100.6%. The t-test results showed that the P values of two determination methods were 0.493, 0.155 and 0.171 for harpagide, angoroside C and harpagoside respectively, indicating no significant diffe-rence between the two methods of content determination. The SRRE can be used as a substitute for the reference in the quality control of Scrophulariaceae Radix. The SRRE can replace the corresponding reference substance for the quality control of Scrophulariae Radix. The results of this study provide new methods and new ideas for the quality evaluation of Scrophulariae Radix, and provide a scientific basis for the application of reference extracts in the quality research of traditional Chinese medicine.

Harpagide exerts a neuroprotective effect by inhibiting endoplasmic reticulum stress via SERCA following oxygen-glucose deprivation/reoxygenation injury.

Cerebrovascular diseases endanger human health, and the physiological and pathological processes of cerebral ischemia/reperfusion injury (CIRI) are critical for the occurrence of these diseases and as targets for their treatment. Here, we evaluated the effects of harpagide-mediated pharmacological and genetic inhibition of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) in vitro in PC12 cells. The molecular mechanism by which harpagide protects PC12 cells against oxygen-glucose deprivation/reoxygenation (OGD/R) injury was investigated by evaluating the cell survival rate with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, assessing apoptosis by flow cytometry, determining the intracellular Ca2+ concentration ([Ca2+]i) by laser scanning confocal microscopy (LSCM), and measuring the expression of proteins related to SERCA and endoplasmic reticulum stress (ERS) by Western blotting. The results revealed that harpagide significantly decreased thapsigargin (TG)-induced apoptosis of PC12 cells, downregulated the expression of ERS-related markers, considerably improved the TG-induced expression of SERCA-related proteins and reduced the [Ca2+]i, suggesting that harpagide effectively inhibited ERS directly. Moreover, harpagide did not significantly reduce OGD/R-induced apoptosis but increased the expression of ERS markers in PC12/SERCA- cells, indicating that harpagide targets SERCA to protect against CIRI by suppressing ERS-mediated apoptosis.

8-O-(E-p-methoxycinnamoyl)harpagide Inhibits Influenza A Virus Infection by Suppressing Intracellular Calcium.

Calcium (Ca2+) dependent signaling circuit plays a critical role in influenza A virus (IAV) infection. The 8-O-(E-p-methoxycinnamoyl)harpagide (MCH) exhibits pharmacological activities that exert neuroprotective, hepatoprotective, anti-inflammatory and other biological effects. However, not have reports of antiviral effects. To investigate the antiviral activity of MCH on IAV-infected human lung cells mediated by calcium regulation. We examined the inhibitory effect of MCH on IAV infections and measured the level of viral proteins upon MCH treatment using Western blotting. We also performed molecular docking simulation with MCH and IAV M2 protein. Finally, we analyzed MCH's suppression of intracellular calcium and ROS (reactive oxygen species) in IAV-infected human lung cells using a flow cytometer. The results shown that MCH inhibited the infection of IAV and increased the survival of the infected human lung cells. The levels of IAV protein M1, M2, NS1 and PA were inhibited in MCH-treated human lung cells compared to that in infected and untreated cells. Also, docking simulation suggest that MCH interacted with M2 on its hydrophobic wall (L40 and I42) and polar amino acids (D44 and R45), which formed intermolecular contacts and were a crucial part of the channel gate along with W41. Lastly, MCH inhibited IAV infection by reducing intracellular calcium and mitochondrial Ca2+/ROS levels in infected human lung cells. Taken together, these data suggest that MCH inhibits IAV infection and increases the survival of infected human lung cells by suppressing calcium levels. These results indicate that MCH is useful for developing IAV treatments.

Determination of harpagide and harpagoside contents in Scrophularia ningpoensis from 15 origins / 药学实践杂志

Objective To establish an HPLC method for the simultaneous determination of harpagide and harpagoside content in Scrophularia ningpoensis (SN). Methods An Eclipse C18 column was used for determination of methanol extract of S. ningpoensis with a HPLC-PDA method and mobile phase of acetonitrile-0.03% phosphate solution in a gradient elution manner. The flow rate of mobile phase was 1.0 ml/min, and the detection wavelengths were 210 nm and 280 nm. Results Harpagide and harpagoside contents in SN showed good linear relationships within 0.1020-0.5100 mg/ml (r=0.9999) and 0.0340-0.1700 mg/ml (r=0.9999). Their average recovery rates were 97.44% and 97.08%.The RSDs were 0.93% and 1.24%.. There were significant differences in the contents of harpagide and harpagoside in SN from 15 origins (P<0.01). The content of harpagoside in Sichuan Long-dong, Zhejiang Lin-an, Zhejiang Pan-an and Henan Nan-feng is higher. Conclusion This method is stable, accurate and reproducible and can be used for the quality control of SN.

Study on scrophulariaceae radix reference extract and its application in quality control of Scrophulariae Radix / 中国中药杂志

By establishing the preparation process of Scrophulariaceae Radix reference extract(SRRE) and calibrating it, we discussed its feasibility as a substitute for single reference substance in the quality control of Scrophulariae Radix. The SRREs were prepared by solvent extraction method and chromatographic separation technology, and then calibrated with the reference substances of harpagide, angoroside C and harpagoside. The HPLC content determination method of Scrophulariae Radixl was established with SRREs of the known content and the reference substances of harpagide, angoroside C and harpagoside respectively as the control ones. Then the content of three components in Scrophulariae Radix was determined, and the t-test method was used to compare the results of the two methods. With SRRE as references, harpagide, angoroside C and harpagoside were in a good linear relationship(r≥0.999 8) within each range, and the average recovery rate was 98.55% to 100.6%. The t-test results showed that the P values of two determination methods were 0.493, 0.155 and 0.171 for harpagide, angoroside C and harpagoside respectively, indicating no significant diffe-rence between the two methods of content determination. The SRRE can be used as a substitute for the reference in the quality control of Scrophulariaceae Radix. The SRRE can replace the corresponding reference substance for the quality control of Scrophulariae Radix. The results of this study provide new methods and new ideas for the quality evaluation of Scrophulariae Radix, and provide a scientific basis for the application of reference extracts in the quality research of traditional Chinese medicine.

Harpagide: Occurrence in plants and biological activities - A review.

In this review article, the occurrence of harpagide in the plant kingdom and its associated biological activities are presented and detailed for the first time. The presence of harpagide has been reported in several botanical families within Asteridae, and harpagide has been observed to exert a wide number of biological activities such as cytotoxic, anti-inflammatory, and neuroprotective. These results show how harpagide can be recovered from several natural sources for several pharmacological purposes even if there is a lot to still be studied. Nowadays, the interest is related to its presence in phytomedicines. Threfore, these studies are useful to support and validate the large use of several plants in the folklore medicine.

Harpagide from Scrophularia protects rat cortical neurons from oxygen-glucose deprivation and reoxygenation-induced injury by decreasing endoplasmic reticulum stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Harpagide is the main ingredient in Scrophularia ningpoensis Hemsl which is used for the therapeutic purpose of treating encephalopathy. Harpagide has shown promise in the treatment of oxygen-glucose deprivation and reoxygenation (OGD/R)-induced brain injury. However, the underlying mechanisms remain unclear. AIM OF STUDY: In this study, we aimed to determine the neuroprotective effect of harpagide on rat cortical neurons under OGD/R conditions that induce the development of ischaemia-reperfusion (I/R). MATERIALS AND METHODS: To explore the biological function of harpagide in cerebral ischaemia-reperfusion injury (CIRI), The CIRI model was established by oxygen-glucose deprivation and reoxygenation (OGD/R) on rat cortical neurons. It tested cell survival rate by 3-(4,5-dimethylthiazol-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, apoptosis by flow cytometry, intracellular Ca2+ concentration [Ca2+] i by cofocal laser, and expressions related to endoplasmic reticulum stress (ERS) by RT-PCR and Western blot. RESULTS: We found that pretreatment with harpagide (50 µM) prevented OGD/R-induced apoptotic cell death. Harpagide also significantly decreased the gene expression levels and protein production of ERS-related proteins. We found that harpagide also exerted a neuroprotective effect on TG-induced apoptosis in rat cortical neurons and decreased the gene expression levels and protein production of GRP78, caspase-12 and CHOP. We also measured the intracellular calcium ion concentration ([Ca2+]i) in neurons and found that harpagide significantly decreased the [Ca2+]i induced by OGD/R and TG. CONCLUSION: These results suggest that harpagide protects against OGD/R-induced cell apoptosis, likely by decreasing ERS. Collectively, harpagide was demonstrated to be a prominent suppressor of ERS and prevented the apoptosis of rat cortical neurons. Based on the results, harpagide could potentially serve as a therapeutic agent of ischaemia-like injury associated with excessive ERS and apoptosis.

[Isolation and identification of endophytic fungi producing harpagoside and harpagide from Scrophularia ningpoensis].

The endophytic fungi from root,main stem,branch and leaf of Scrophularia ningpoensis were isolated from Zhejiang,whether these strains could yield harpagide or harpagoside were tested by HPLC and LC-MS. According to the morphological characteristic and the similarity of the nucleotide sequence of internal transcribed spacer( ITS) between r DNAs,the strains producing harpagide or harpagoside were identified. The results showed that 210 strains were isolated from the samples,which were classified into 9 orders,13 families and 17 genera by morphological study. Harpagide was detected in endogenous fungi ZJ17 and harpagoside was detected in endogenous fungi ZJ25 by HPLC coupled with LC-MS. ZJ17 was identified as Alternaria alternate and ZJ25 was identified as A.gaisen by its morphology and authenticated by ITS( ITS4 and ITS5 regions and the intervening 5. 8 S rDNA region).

Harpagide inhibits neuronal apoptosis and promotes axonal regeneration after spinal cord injury in rats by activating the Wnt/ß-catenin signaling pathway.

The neuronal apoptosis program associated with spinal cord injury (SCI) has a severe impact on spinal cord function, which leads to further secondary and permanent neuronal damage that may cause irreparable damage to the central nervous system. Activation of the Wnt/ß-catenin signaling pathway is effective in reducing apoptosis and preventing SCI. Harpagide is one of the main active constituents of the iridoid class of molecules, which have neuroprotective effects after SCI. In this study, we demonstrated that harpagide attenuated neuronal apoptosis via activation of the Wnt/ß-catenin signaling pathway. This resulted in a promotion of axonal regeneration and an inhibition of glial scar formation, which ultimately improved functional behavioral recovery after SCI in rats. Specifically, the administration of harpagide after SCI increased the expression levels of ß-catenin, c-myc and cyclin D1 proteins in spinal cord neurons, as well as increased the number of motor neurons and reduced the size of the SCI lesion area. In addition, the administration of harpagide after SCI also decreased the protein expression levels as well as the number of cells immuno-stained for the pro-apoptotic proteins Bax and cleaved-caspase 3. The expression level of the anti-apoptotic protein Bcl-2 was also increased. When the Wnt /ß-catenin signaling pathway was inhibited, a weakened anti-apoptotic effect of harpagide was observed. Additionally, the application of harpagide led to an increase in NF200 staining and a reduction in GFAP staining in the SCI injury site. In summary, our study suggested that harpagide may be a promising drug for the treatment of SCI.

Grade quality of Scrophulariae Radix based on fingerprints and quantitative analysis of multi-index / 中草药

Objective In order to provide a scientific basis for the grade quality standard criterion of Scrophulariae Radix, a HPLC-DAD method was established to acquire fingerprint and detect the content of multiple components. Methods The chromatographic separate was achieved on Elipse XDB-C18 (250 mm × 4.6 mm, 5 μm) column, with the temperature of 25 ℃, using acetonitrile (A)-0.03% phosphoric acid water (B) as the mobile phase gradient elution, a flow rate of 1.0 mL/min, and the detection wavelength was set at 207 nm for fingerprint, 210 nm for Harpagide, 280 nm for harpagoside, and 264 nm for cinnamic acid. The injection volume was 10 μL. The fingerprints of different grades of Scrophulariae Radix from 30 batches were evaluated with a chromatographic fingerprint similarity evaluation system (version 2012), and the content of harpagide, harpagoside, and cinnamic acid was also determined at the same time. Results There were seven common peaks in the fingerprints, the RSD values of relative retention time were lower than 2.0% and those of relative peak area were quite different, which indicated that the main chemical compounds can exist stably in Scrophulariae Radix with different content. Compared with control, the results of fingerprintsimilarity were presented as follows: 5% under 0.7, 12.5% arranged 0.7 to 0.8, 40% between 0.8 and 0.9, and another 42.5% exceed 0.9, demonstrated that there was qualitative difference in various batches of Scrophulariae Radix. Also, the quantitative analysis of multi-index showed the differences in three main compounds, the content respectively was 0.18%—2.89% in harpagide, 0.01%—0.35% in harpagoside, and 0.01%—0.24% in cinnamic acid. Conclusion Suggestions were provided in the formulation of new grade quality standard, such as adding the fingerprints and multi-index detection of main chemical components based on the original criterion of classification.

Optimization of steaming technology for decoction pieces of Scrophularia ningpoensis by D-optimal response surface methodology based on AHP- entropy weight method / 中草药

Objective :To determine the weight coefficient and optimize the steaming technology of decoction pieces of Scrophularia ningpoensis (SN). Methods The contents of harpagide, harpagoside, aucubin, acteoside, angoroside C, and cinnamic acid were simultaneously determined by HPLC. The weight coefficient of each component was evaluated by AHP-entropy (analyitc hierarchy process) method. With composite score as index, D-optimal response surface methodology was adopted to investigate the effects of soaking time, steaming time, and drying temperature on the quality of processed products and optimize the processing technology of decoction pieces of SN. Results :Optimal processing parameters were as follows: soaking time was 15.63 min, steaming time was 85 min, drying temperature was 60 ℃, and the synthetical mark was 97.20. Considering the actual situation, the optimum processing technology of SN was obtained by fine-turning the soaking time. The soaking time was 15 min, the steaming time was 85 min, and the drying temperature was 60 ℃. Also, the synthetical mark were 98.53, 99.39, 98.86, and its RSD was 0.47% through the obtained conditions in parallel with three batches of samples. Conclusion: The optimized steaming technology is simple and feasible, which can provide a reference for the steaming of decoction pieces of SN. The method established to simultaneously determine the contents of six components in decoction pieces of SN is rapid and reliable for controlling the quality of decoction pieces of SN.

Isolation and identification of endophytic fungi producing harpagoside and harpagide from Scrophularia ningpoensis / 中国中药杂志

The endophytic fungi from root,main stem,branch and leaf of Scrophularia ningpoensis were isolated from Zhejiang,whether these strains could yield harpagide or harpagoside were tested by HPLC and LC-MS. According to the morphological characteristic and the similarity of the nucleotide sequence of internal transcribed spacer( ITS) between r DNAs,the strains producing harpagide or harpagoside were identified. The results showed that 210 strains were isolated from the samples,which were classified into 9 orders,13 families and 17 genera by morphological study. Harpagide was detected in endogenous fungi ZJ17 and harpagoside was detected in endogenous fungi ZJ25 by HPLC coupled with LC-MS. ZJ17 was identified as Alternaria alternate and ZJ25 was identified as A.gaisen by its morphology and authenticated by ITS( ITS4 and ITS5 regions and the intervening 5. 8 S rDNA region).

A promiscuous beta-glucosidase is involved in benzoxazinoid deglycosylation in Lamium galeobdolon.

In the plant kingdom beta-glucosidases (BGLUs) of the glycosidase hydrolase family 1 have essential function in primary metabolism and are particularly employed in secondary metabolism. They are essential for activation in two-component defence systems based on stabilisation of reactive compounds by glycosylation. Based on de novo assembly we isolated and functionally characterised BGLUs expressed in leaves of Lamium galeobdolon (LgGLUs). LgGLU1 could be assigned to hydrolysis of the benzoxazinoid GDIBOA (2,4-dihydroxy-1,4-benzoxazin-3-one glucoside). Within the Lamiaceae L. galeobdolon is distinguished by the presence GDIBOA in addition to the more common iridoid harpagide. Although LgGLU1 proved to be promiscuous with respect to accepted substrates, harpagide hydrolysis was not detected. Benzoxazinoids are characteristic defence compounds of the Poales but are also found in some unrelated dicots. The benzoxazinoid specific BGLUs have recently been identified for the grasses maize, wheat, rye and the Ranunculaceae Consolida orientalis. All enzymes share a general substrate ambiguity but differ in detailed substrate pattern. The isolation of the second dicot GDIBOA glucosidase LgGLU1 allowed it to analyse the phylogenetic relation of the distinct BGLUs also within dicots. The data revealed long periods of independent sequence evolution before speciation.

Evaluation of in vivo analgesic activity of Scrophularia kotscyhana and isolation of bioactive compounds through activity-guided fractionation.

The present study was undertaken to evaluate the in vivo analgesic activities of the extracts prepared from the aerial parts and roots of Scrophularia kotscyhana and to isolate the bioactive metabolites from the most active extract. Analgesic activities of all extracts and subextracts at the doses of 5, 10 and 30 mg/kg (i.p.) were examined using hot plate test in mice. Among the tested extracts, MeOH extract prepared from the aerial parts and the n-butanol subextract prepared thereof displayed the best analgesic activity at all doses. Phytochemical studies on n-butanol subextract led to the isolation of two new iridoid glycosides as an inseparable mixture, 8-O-acetyl-4'-O-(E)-(p-coumaroyl)-harpagide (1) and 8-O-acetyl-4'-O-(Z)-(p-coumaroyl)-harpagide (2) along with five known secondary metabolites, ß-sitosterol 3-O-ß-glucopyranoside (3), apigenin 7-O-ß-glucopyranoside (4), apigenin 7-O-rutinoside (5), luteolin 7-O-ß-glucopyranoside (6) and luteolin 7-O-rutinoside (7). The iridoid mixture (1 and 2), 3 and 4 elicited significant inhibition of pain at 5 mg/kg dose.

Application of QAMS in quality control of Scrophularia Radix / 中草药

Objective To establish a measurement method for the content of five compounds (acteoside, harpagide, harpagoside, angoroside-C, and cinnamic acid) in Scrophularia Radix quantitative analysis multi-components by single-marker (QAMS), and verify the accuracy and feasibility of QAMS in the quality control. Methods Taking harpagide as internal standard substance, the relative correlation factor (RCF) of acteoside, cinnamic acid, harpagoside, and angoroside C was established. And the content of each component in Scrophularia Radix was determined by the above-mentioned RCF. In order to prove the scientificity and feasibility of this method, the results were compared with the external standard method. Results The relative correction factors of acteoside, cinnamic acid, harpagoside, and angoroside-C were 0.068 (RSD = 0.53%), 0.060 (RSD = 0.81%), 0.142 (RSD = 1.17%), 0.197 (RSD = 1.82%). No significant differences were found among the quantitative results of four components in 25 batches of Scrophularia Radix determined by the two methods. Conclusion It is feasible and accurate to evaluate the quality of Scrophularia Radix by QAMS.