Harpagoside Research Articles

Epidural Injection of Harpagoside for the Recovery of Rats with Lumbar Spinal Stenosis.

Epidural administration is the leading therapeutic option for the management of pain associated with lumbar spinal stenosis (LSS), which is characterized by compression of the nerve root due to narrowing of the spinal canal. Corticosteroids are effective in alleviating LSS-related pain but can lead to complications with long-term use. Recent studies have focused on identifying promising medications administered epidurally to affected spinal regions. In this study, we aimed to investigate the effectiveness of harpagoside (HAS) as an epidural medication in rats with LSS. HAS at various concentrations was effective for neuroprotection against ferrous sulfate damage and consequent promotion of axonal outgrowth in primary spinal cord neurons. When two concentrations of HAS (100 and 200 μg/kg) were administered to the rat LSS model via the epidural space once a day for 4 weeks, the inflammatory responses around the silicone block used for LSS were substantially reduced. Consistently, pain-related factors were significantly suppressed by the epidural administration of HAS. The motor functions of rats with LSS significantly improved. These findings suggest that targeted delivery of HAS directly to the affected area via epidural injection holds promise as a potential treatment option for the recovery of patients with LSS.

Harpagoside attenuates local bone Erosion and systemic osteoporosis in collagen-induced arthritis in mice

BackgroundRheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that causes local bone erosion and systemic osteoporosis. Harpagoside (HAR), an iridoid glycoside, has various pharmacological effects on pain, arthritis, and inflammation. Our previous study suggests that HAR is more deeply involved in the mechanism of bone loss caused by inflammatory stimuli than hormonal changes. Here, we identified the local and systemic bone loss inhibitory effects of HAR on RA and its intracellular mechanisms using a type 2 collagen-induced arthritis (CIA) mouse model.MethodsThe anti-osteoporosis and anti-arthritic effects of HAR were evaluated on bone marrow macrophage in vitro and CIA in mice in vivo by obtaining clinical scores, measuring hind paw thickness and inflammatory cytokine levels, micro-CT and histopathological assessments, and cell-based assay.ResultsHAR markedly reduced the clinical score and incidence rate of CIA in both the prevention and therapy groups. Histological analysis demonstrated that HAR locally ameliorated the destruction of bone and cartilage and the formation of pannus. In this process, HAR decreased the expression of inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-6, and IL-1β in the serum of CIA mice. Additionally, HAR downregulated the expression of receptor activator of nuclear factor-κB ligand and upregulated that of osteoprotegerin. HAR suppressed systemic bone loss by inhibiting osteoclast differentiation and osteoclast marker gene expression in a CIA mouse model.ConclusionsTaken together, these findings show the beneficial effect of HAR on local symptoms and systemic bone erosion triggered by inflammatory arthritis.

Harpagoside Protects Against Doxorubicin-Induced Cardiotoxicity via P53-Parkin-Mediated Mitophagy

Doxorubicin (DOX) is one of the most effective chemotherapeutic agents. However, its clinical use is limited due to the severe risk of cardiotoxicity. One of the hallmarks of doxorubicin-induced cardiotoxicity (DICT) is the cascade of mitophagy deficiency-mitochondrial oxidative injury-apoptosis, while so far, there is no preventive strategy for alleviating DICT by targeting this molecular mechanism. Excitedly, based on our previous drug screen in DICT zebrafish model, harpagoside (HAR) showed dramatic anti-DICT efficacy superior to dexrazoxane (DXZ) only cardioprotectant approved by FDA. Therefore, its pharmacological effects and molecular mechanism on DICT mouse and rat cardiomyocytes were further discussed. In vivo, HAR significantly improved cardiac function and myocardial structural lesions with concomitant of diminished mitochondrial oxidative damage and recovered mitophagy flux. In parallel, HAR protected mitophagy and mitochondria homeostasis, and repressed apoptosis in vitro. Intriguingly, both nutlin-3 (agonist of p53) and Parkin siRNA reversed these protective effects of HAR. Additional data, including fluorescence colocalization of Parkin and MitoTracker and mt-Keima for the detection of mitophagy flux and coimmunoprecipitation of p53 and Parkin, showed that HAR promoted Parkin translocation to mitochondria and substantially restored Parkin-mediated mitophagy by inhibiting the binding of p53 and Parkin. Importantly, the results of the cell viability demonstrated that cardioprotective effect of HAR did not interfere with anticancer effect of DOX on MCF-7 and HepG2 cells. Our research documented p53-Parkin-mediated cascade of mitophagy deficiency-mitochondrial dyshomeostasis-apoptosis as a pathogenic mechanism and druggable pathway and HAR as a cardioprotection on DICT by acting on novel interaction between p53 and Parkin.

Binding interaction of harpagoside and bovine serum albumin: spectroscopic methodologies and molecular docking

Scrophularia ningpoensis has exhibited a variety of biological activities and been used as a pharmaceutical product for the treatment of inflammatory ailment, rheumatoid arthritis, osteoarthritis and so on. Harpagoside (HAR) is considerer as a main bioactive compound in this plant. Serum albumin has important physiological roles in transportation, distribution and metabolism of many endogenous and exogenous substances in body. It is of great significance to study the interaction mechanism between HAR and bovine serum albumin (BSA). The mechanism of interaction between HAR and BSA was investigated using 2D and 3D fluorescence, synchronous florescence, ultraviolet spectroscopy and molecular docking. According to the analysis of fluorescence spectra, HAR could strongly quench the fluorescence of BSA, and the static quenching process indicated that the decrease in the quenching constant was observed with the increase in temperature. The magnitude of binding constants (KA) was more than 1×10⁵ L·mol⁻¹, and the number of binding sites(n) was approximate to 1. The thermodynamic parameters were calculated through analysis of fluorescence data with Stern-Volmer and Van't Hoff equation. The calculated enthalpy change (ΔH) and entropy change (ΔS) implied that the main interaction forces of HAR with BSA were the bonding interaction between van der Waals forces and hydrogen. The negative values of energy (ΔG) demonstrated that the binding of HAR with BSA was a spontaneous and exothermic process. The binding distance(r) between HAR and BSA was calculated to be about 2.80 nm based on the theory of Frster's non-radiation energy transfer, which indicated that energy is likely to be transfer from BSA to HAR. Both synchronous and 3D florescence spectroscopy clearly revealed that the microenvironment and conformation of BSA changed during the binding interaction between HAR and BSA. The molecular docking analysis revealed HAR is more inclined to BSA and human serum albumin (HSA) in subdomain ⅡA (Sudlow's site I). This study will provide valuable information for understanding the action mechanism of HAR.

Harpagoside-induced anaphylactic reaction in an IgE-independent manner both in vitro and in vivo

Background: Harpagoside (HAR) is an active component of Scrophularia ningpoensis (SN), which has anti-inflammatory and anti-immune effects. SN is used widely in China to treat various diseases. Recently, SN has been used as a traditional Chinese medicine injection and used clinically. However, allergic responses to these injections are frequently reported.Aim: We examined whether the main component of SN, HAR, is associated with the allergic reaction to SN.Methods: This study assessed the effects of HAR in mice and mast cell activation to characterize its anaphylactic effects and underlying mechanisms. Mice hindpaw swelling, serum allergy factor detection, enzyme-linked immunosorbent assays, and degranulation assays were performed to measure allergic mediators both in vivo and in vitro.Results: The present study indicated that HAR induced paw swelling, interleukin-6, inositol triphosphate, tumor necrosis factor-α, and histamine increases in mice. Our in vitro data also showed that HAR induced β-hexosaminidase, inositol triphosphate, and interleukin-6 release, leading to mast cell degranulation. In contrast, neither C48/80 nor HAR induced local anaphylaxis in STOCK KitW-sh/HNihrJaeBsmJNju mice.Conclusions: HAR is a potential sensitization compound in SN, and these results provide information for the safe clinical use of SN.

Risk Assessment via Metabolism and Cell Growth Inhibition in a HepG2/C3A Cell Line Upon Treatment with Arpadol and its Active Component Harpagoside.

Harpagophytum procumbens (Hp) has been used as antiinflammatory and analgesic agent for the treatment of rheumatic diseases. The principal active component of Hp is harpagoside (HA). We tested the toxicity of this new therapeutic agent in a hepatic cell line (HepG2/C3A). Hp was found to be cytotoxic, and HA was found to decrease the number of cells in S phase, increase the number of cells in G2/M phase and induce apoptosis. Neither Hp nor HA was genotoxic. The expression of CDK6 and CTP3A4 was reduced by Hp, and both HA and Hp caused a significant reduction of CYP1A2 and CYP3A4 expression. It is possible that the cytotoxicity caused by HA and Hp does not involve transcriptional regulation of the cyclins and CDKs tested but is instead related to the inhibition of metabolism. This is evidenced by the results of an MTT assay and changes in the expression of genes related to drug metabolism, leading to cell death. Indeed, the cells exhibited decreased proliferation upon exposure to Hp and HA. The data show that treatment with either Hp or HA can be cytotoxic, and this should be taken into consideration when balancing the risks and benefits of treatments for rheumatic diseases. Copyright © 2016 John Wiley & Sons, Ltd.

Extraction by solvent using microwave and ultrasound-assisted techniques followed by HPLC analysis of Harpagoside from Harpagophytum procumbens and comparison with conventional solvent extraction methods

Abstract This research paper presents a quick and ecofriendly technique for the extraction of harpagoside (HS), the active marker of Harpagophytum procumbens (HP), along with a comparison with conventional methods so as to propose an efficient HPLC method. HP is widely used as an anti-inflammatory in phytotherapy. The quality control of the herbal drug and extract calls for a time consuming method of conventional extraction, which involves a high consumption of solvents. In this study, HP has been extracted using conventional ultrasound (UAE) and microwave (MAE)-assisted methods. The effects have been examined based on several parameters of HS extraction efficiencies. An HPLC method with a core-shell column was developed in order to calculate the HS in HP. The flow rate was reduced by 4. The method of validation used is specific, linear, precise and accurate. MAE and UAE saved solvent consumption, time and energy. It has, therefore, been found that the combined UAE-HPLC process is convenient and appropriate for the quality control of HP.

Harpagoside Inhibits RANKL-Induced Osteoclastogenesis via Syk-Btk-PLCγ2-Ca(2+) Signaling Pathway and Prevents Inflammation-Mediated Bone Loss.

Harpagoside (HAR) is a natural compound isolated from Harpagophytum procumbens (devil's claw) that is reported to have anti-inflammatory effects; however, these effects have not been investigated in the context of bone development. The current study describes for the first time that HAR inhibits receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis in vitro and suppresses inflammation-induced bone loss in a mouse model. HAR also inhibited the formation of osteoclasts from mouse bone marrow macrophages (BMMs) in a dose-dependent manner as well as the activity of mature osteoclasts, including filamentous actin (F-actin) ring formation and bone matrix breakdown. This involved a HAR-induced decrease in extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) phosphorylation, leading to the inhibition of Syk-Btk-PLCγ2-Ca(2+) in RANKL-dependent early signaling, as well as the activation of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1), which resulted in the down-regulation of various target genes. Consistent with these in vitro results, HAR blocked lipopolysaccharide (LPS)-induced bone loss in an inflammatory osteoporosis model. However, HAR did not prevent ovariectomy-mediated bone erosion in a postmenopausal osteoporosis model. These results suggest that HAR is a valuable agent against inflammation-related bone disorders but not osteoporosis induced by hormonal abnormalities.

Antimutagenic potential of harpagoside and Harpagophytum procumbens against 1-nitropyrene.

Background:1-nitropyrene (1-NPy) is one of the most abundant nitro-polycyclic aromatic hydrocarbons particularly in diesel exhausts. It is a mutagenic and carcinogenic pollutant very widespread in the environment. So the discovery of antimutagenic agents is essential. Harpagophytum procumbens (HP) is traditionally used as anti-inflammatory and analgesic particularly against painful osteoarthritis. Harpagoside (HS), its major iridoid glycoside, is considered as the main active component.Objective:The aim of the present study was to evaluate the antimutagenic activity of HS and HP extracts against mutagenic activity of 1-NPy.Materials and Methods:The antimutagenic activity was investigated using the in vitro cytokinesis-block micronucleus assay in cultured human lymphocytes. Cells were exposed to HS or HP extracts before (pretreatment), during (co-treatment), and after (posttreatment) treatment with 1-NPy.Results:Results showed that HS significantly reduced the mutagenicity of 1-NPy in pretreatment and particularly in co-treatment, whereas all HP extracts significantly reduced the genotoxicity in the three protocols.Conclusion:These results suggested that HS was strongly involved in antimutagenic activity of HP extracts in co-treatment, but other components in HP extracts participated in this activity in pre- and post-treatment.

Rapid determination of cinnamic acid and harpagoside in a traditional Chinese medicine of Scrophularia ningpoensis by microwave-assisted extraction followed by high performance liquid chromatography (HPLC)

Cinnamic acid and harpagoside are important active components present in Scrophularia ningpoensis, which has been used in the treatment of several diseases such as pharyngalgia. In the present study, a novel method based on high performance liquid chromatography (HPLC) following microwave-assisted extraction (MAE) was developed for the determination of cinnamic acid and harpagoside in a traditional Chinese medicine (TCM) of S. ningpoensis. Effective chromatographic separation was achieved on a C18 column at the detection wavelength of 278 nm. Microwave power of 400 W and irradiation time of 4 min were found to be the optimum conditions for the MAE process. In addition, to demonstrate the feasibility of the MAE-HPLC method, the conventional ultrasound extraction (USE) method was used for the analysis of cinnamic acid and harpagoside in the TCM. The results indicated that MAE-HPLC is a simple, rapid, efficient, and low cost method for the determination of cinnamic acid and harpagoside in TCM as well as a potential tool for the assessment of TCM quality. Key words: Scrophularia ningpoensis, Cinnamic acid, harpagoside, microwave-assisted extraction, liquid chromatography, traditional Chinese medicine.

Optimization of Solid-Phase Extraction for the Liquid Chromatography--Mass Spectrometry Analysis of Harpagoside, 8-para-Coumaroyl Harpagide, and Harpagide in Equine Plasma and Urine

Solid-phase extraction cartridges among those usually used for screening in horse doping analyses are tested to optimize the extraction of harpagoside (HS), harpagide (HG), and 8-para-coumaroyl harpagide (8PCHG) from plasma and urine. Extracts are analyzed by liquid chromatography coupled with multi-step tandem mass spectrometry. The extraction process retained for plasma applies BondElut PPL cartridges and provides extraction recoveries between 91% and 93%, with RSD values between 8 and 13% at 0.5 ng/mL. Two different procedures are needed to extract analytes from urine. HS and 8PCHG are extracted using AbsElut Nexus cartridges, with recoveries of 85% and 77%, respectively (RSD between 7% and 19%). The extraction of HG involves the use of two cartridges: BondElut PPL and BondElut C18 HF, with recovery of 75% and RSD between 14% and 19%. The applicability of the extraction methods is determined on authentic equine plasma and urine samples after harpagophytum or harpagoside administration.

Simultaneous determination of four active constituents in the roots of Scrophularia buergeriana by HPLC‐DAD and LC‐ESI‐MS

The chemical profile of four main bioactive constituents including harpagoside (HS), 8-O-(E-p-methoxycinnamoyl)harpagide (HG), E-cinnamic acid (CA), and E-p-methoxycinnamic acid (MCA), in the extract of Scrophularia buergeriana was studied using HPLC-diode array detector-electrospray-MS (HPLC-DAD-ESI-MS). The HPLC-DAD conditions were optimized for the simultaneous analysis of these four compounds. This method was validated in terms of specificity, linearity (r(2) >0.9998), precision (< 2.0% RSD), and recoveries (94.4-115.1%). The LOD of these compounds were ranged from 5.9 to 37.8 ng. In addition, the main compounds in S. buergeriana and S. ningpoensis were quantified using this validated method, which resulted in significant difference in the contents of these compounds between the species.

Validation of A Fast‐HPLC Method for the Separation of Iridoid Glycosides to Distinguish Between the Harpagophytum Species

A fast high‐performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of the iridoid glycosides harpagoside (HS) and 8‐p‐coumaroyl‐harpagide (8pCHG) in extracts and preparations of Harpagophytum procumbens and H. zeyheri. The ratio between 8pCHG and the sum of HS and 8pCHG can be used to distinguish between both species. Quantitation was accomplished with the internal standard (IS) method. The separation was performed on a monolithic silica column (Chromolith Performance RP‐18e), under gradient conditions using a mobile phase of water (pH 2.0 adjusted with phosphoric acid) and acetonitrile. The elution of the analytes was monitored at 278 nm and conducted at a column temperature of 30°C. Because of the high porosity of the monolithic column the mobile phase was able to be pumped at a flow rate of 5.0 mL/min. The retention time of 8pCHG, HS, and the IS was 1.9 min, 2.1 min, and 3.0 min, respectively, and the total run time of the assay was 5 min. The method was validated by specificity, linearity, accuracy, and precision. For the determination of method robustness a number of chromatographic parameters were varied.

Rapid Measurement and Evaluation of the Effect of Drying Conditions on Harpagoside Content inHarpagophytum procumbens(Devil's Claw) Root

The effect of drying conditions on harpagoside (HS) retention, as well as the use of near-infrared spectroscopy (NIRS) for rapid quantification of the iridoids, HS, and 8-rho-coumaroyl harpagide (8rhoCHG) and moisture, in dried Harpagophytum procumbens (devil's claw) root was investigated. HS retention was significantly (P < 0.05) lower in sun-dried samples as compared to tunnel-dried (60 degrees C, 30% relative humidity) and freeze-dried samples. The best retention of HS was obtained at 50 degrees C when evaluating tunnel drying at dry bulb temperatures of 40, 50, and 60 degrees C and 30% relative humidity. NIRS can effectively predict moisture content with a standard error of prediction (SEP) and correlation coefficient (r) of 0.24% and 0.99, respectively. The HS and 8rhoCHG NIRS calibration models established for both iridoid glucosides can be used for screening purposes to get a semiquantitative classification of devil's claw roots (for HS: SEP = 0.236%, r = 0.64; for 8rhoCHG: SEP = 0.048%, r = 0.73).

Determination of Harpagide, 8-Para-Coumaroyl Harpagide, and Harpagoside by High Performance Liquid Chromatography in Harpagophytum Procumbens Drugs and in a Commercial Extract

Abstract A selective method for the determination of HARPAGIDE (HG), 8-para-coumaroyl HARPAGIDE (8PCHG) and HARPAGOSIDE (HS) is described. HS and 8PCHG are the two main compounds extracted from commercial dry extract of Harpagophytum secondary roots. Analysis of iridoids was performed using a linear gradient system of methanol/water as mobile phase, on RP 18 column. For HG Light-Scattering and UV detection were used for the quantification. Assays were linear in the concentration ranges studied. Statistic evaluation of the method was also realised to improve its security. In a total run time of 60 mn this procedure permitted to quantify simultaneously these three iridoids in the drug including HG for the first time in HPLC.