Evaluation of Rhododendri Mollis Flos and its representative component as a potential analgesic.

Rhododendri Mollis Flos (R. mole Flos), the dried flowers of Rhododendron mole G. Don, have the ability to relieve pain, dispel wind and dampness, and dissolve blood stasis, but they are highly poisonous. The significance of this study is to explore the analgesic application potential of R. mole Flos and its representative component. According to the selected processing methods recorded in ancient literature, the analgesic activities of wine- and vinegar-processed R. mole Flos, as well as the raw product, were evaluated in a writhing test with acetic acid and a formalin-induced pain test. Subsequently, the HPLC-TOP-MS technique was utilized to investigate the changes in active components before and after processing once the variations in activities were confirmed. Based on the results, rhodojaponin VI (RJ-Vl) was chosen for further study. After processing, especially in vinegar, R. mole Flos did not only maintain the anti-nociception but also showed reduced toxicity, and the chemical composition corresponding to these effects also changed significantly. Further investigation of its representative components revealed that RJ-VI has considerable anti-nociceptive activity, particularly in inflammatory pain (0.3 mg/kg) and peripheral neuropathic pain (0.6 mg/kg). Its toxicity was about three times lower than that of rhodojaponin III, which is another representative component of R. mole Flos. Additionally, RJ-VI mildly inhibits several subtypes of voltage-gated sodium channels (IC50 > 200 µM) that are associated with pain or cardiotoxicity. In conclusion, the chemical substances and biological effects of R. mole Flos changed significantly before and after processing, and the representative component RJ-VI has the potential to be developed into an effective analgesic.

High toxin concentration in pollen may deter collection by bees in butterfly-pollinated Rhododendron molle.

BACKGROUNDS AND AIMS: The hypothesis that plants evolve features that protect accessible pollen from consumption by flower visitors remains poorly understood. METHODS: To explore potential chemical defenses against pollen consumption, we examined the pollinator assemblage, foraging behaviour, visitation frequency and pollen transfer efficiency in Rhododendron molle, a highly toxic shrub containing Rhodojaponin III. Nutrient (protein and lipid) and toxic components in pollen and other tissues were measured. KEY RESULTS: Overall in the five populations, floral visits by butterflies and bumblebees were relatively more frequent than visits by honeybees. All foraged for nectar but not pollen. Butterflies did not differ from bumblebees in the amount of pollen removed per visit, but deposited more pollen per visit. Pollination experiments indicated that R. molle was self-compatible, but both fruit and seed production were pollen limited. Our analysis indicated that the pollen was not protein-poor and had a higher concentration of the toxic compound Rhodojaponin III than petals and leaves, which compound was undetectable in nectar. CONCLUSION: Pollen toxicity in Rhododendron flowers may discourage pollen robbers (bees) from taking the freely accessible pollen grains, while the toxin-free nectar rewards effective pollinators, promoting pollen transfer. This preliminary study supports the hypothesis that chemical defense in pollen would be likely to evolve in species without physical protection from pollinivores.

Physical Characterization and Safety Evaluation of Folic Acid-conjugated Mesoporous Silica Nanoparticles Loaded with Rhodojaponin III.

BACKGROUND: Rhodojaponin III (RJ-III), a characteristic diterpene of Rhododendron molle G. Don, has a wide range of pharmacological activities including anti-inflammatory, antihypertensive, and analgesic effects. However, further research and development have been limited because of its intense acute toxicity and poor pharmacokinetic profile. OBJECTIVE: In this study, we propose the construction of folic acid-conjugated mesoporous silica nanoparticles (FA-MSNs) as carriers to deliver RJ-III in an attempt to reduce acute toxicity and improve biomedical applications by prolonging drug release and targeting delivery. METHODS: FA-MSNs were synthesized and characterized. RJ-III was then loaded into FA-MSNs (RJIII@ FA-MSNs), and the in vitro drug release profile was assessed. Subsequently, the RJ-III@FAMSNs' cytotoxicity and targeting efficiency were explored in lipopolysaccharide-activated RAW 264.7 cells, and their acute toxicity was investigated in mice. RESULTS: Spherical FA-MSNs were approximately 122 nm in size. Importantly, the RJ-III@FA-MSNs showed prolonged RJ-III release in vitro. Moreover, in lipopolysaccharide-activated RAW 264.7 cells, RJ-III@FA-MSNs not only reduced the cytotoxicity of RJ-III (P < 0.01), but also showed a good targeting effect from the results of cellular uptake. Additionally, the acute toxicity results demonstrated that RJ-III@FA-MSNs improved the LD50 value of RJ-III in mice by intraperitoneal injection 10-fold. CONCLUSION: This is the first study to use FA-MSNs as carriers of RJ-III to reduce the acute toxicity of RJ-III. The results confirm the potential for targeted delivery of RJ-III in inflammatory cells to enhance efficacy, as well as providing data for future investigations on anti-inflammatory activity.

A Study on the Pharmacological Effects and Mechanism of Rhodojaponin III in Rheumatoid Arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic inflammatory disease with a high rate of disability accompanied by various complications. The pathogenesis of RA is complex with multiple targets and links. This study aims to investigate pharmacological effects and mechanism of Rhodojaponin III in RA. METHODS: The bovine type II collagen-induced arthritis (CIA) rat model and tumor necrosis factor-alpha (TNF-α) induced human umbilical vein endothelial cells (HUVECs) model were constructed. Different concentrations of Rhodojaponin III were utilized for intervention. The progression of CIA was assessed by the arthritis index (AI). Pathological changes in knee joints and synovium were observed. The expressions of angiogenesis-related cytokines were detected. The proliferation, migration, invasion, and angiogenesis of HUVECs were detected. The levels of pro-inflammatory cytokines were determined. The expressions of nuclear factor kappa B-inducing kinase (NIK) pathway-related proteins were analyzed. The binding of Rhodojaponin III to NIK was simulated by molecular docking. RESULTS: Rhodojaponin III suppressed cartilage damage and bone erosion in the knee joints. Rhodojaponin III inhibited expressions of platelet endothelial cell adhesion molecule-1 (CD31) and vascular endothelial cell growth factor (VEGF) to decrease vascular density. Rhodojaponin III suppressed the proliferation, migration, invasion, and angiogenesis of HUVECs, and decreased the levels of interleukin (IL)-6, IL-1ß, and TNF-α. Molecular docking showed that Rhodojaponin III could spontaneously bind to NIK. Rhodojaponin III decreased the expression of NIK, p52, and C-X-C motif chemokine ligand 12 (CXCL12) and the phosphorylation level of IκB kinase-alpha (IKKα) in the synovium of CIA rats and TNF-α-induced HUVECs. NIK overexpression reversed the inhibitory effect of Rhodojaponin III on activation of the NIK/NF-κB pathway, migration, invasion, and angiogenesis of HUVECs, and the secretion of pro-inflammatory cytokines. CONCLUSIONS: Rhodojaponin III affected the angiogenesis and inflammation of CIA rats and TNF-α-induced HUVECs by regulating the NIK/IKKα/CXCL12 pathway. These findings suggest that Rhodojaponin III has potential as a therapeutic agent for RA. Further studies are needed to explore its precise mechanism of action and evaluate its clinical efficacy.

Rhodojaponin III-Loaded Chitosan Derivatives-Modified Solid Lipid Nanoparticles for Multimodal Antinociceptive Effects in vivo.

Background: Rhodojaponin III (RJ-III) is a bioactive diterpenoid, which is mainly found in Rhododendron molle G. Don (Ericaceae), a potent analgesia in traditional Chinese medicine with several years of clinical applications in the country. However, its clinical use is limited by its acute toxicity and poor pharmacokinetic profiles. To reduce such limitations, the current study incorporated RJ-III into the colloidal drug delivery system of hydroxypropyl trimethyl ammonium chloride chitosan (HACC)-modified solid lipid nanoparticles (SLNs) to improve its sustained release and antinociceptive effects in vivo for oral delivery. Results: The optimized RJ-III@HACC-SLNs were close to spherical, approximately 134 nm in size, and with a positive zeta potential. In vitro experiments showed that RJ-III@HACC-SLNs were stable in the simulated gastric fluid and had a prolonged release in PBS (pH = 6.8). Pharmacokinetic results showed that after intragastric administration in mice, the relative bioavailability of RJ-III@HACC-SLNs was 87.9%. Further, it was evident that the peak time, half-time, and mean retention time of RJ-III@HACC-SLNs were improved than RJ-III after the administration. In addition, pharmacodynamic studies revealed that RJ-III@HACC-SLNs markedly reduced the acetic acid, hot, and formalin-induced nociceptive responses in mice (P < 0.001), and notably increased the analgesic time (P < 0.01). Moreover, RJ-III@HACC-SLNs not only showed good biocompatibility with Caco-2 cells in vitro but its LD50 value was also increased by 1.8-fold as compared with that of RJ-III in vivo. Conclusion: These results demonstrated that RJ-III@HACC-SLNs improved the pharmacokinetic characteristics of the RJ-III, thereby exhibiting toxicity-attenuating potential and antinociceptive enhancing properties. Consequently, HACC-SLNs loaded with RJ-III could become a promising oral formulation for pain management that deserves further investigation in the future.

Evaluation of Rhodojaponin III from Rhododendron molle G. Don on oral antinociceptive activity, mechanism of action, and subacute toxicity in rodents.

ETHNOPHARMACOLOGICAL RELEVANCE: In Chinese traditional medicine, Rhododendron molle G. Don is a recognized herb to ease pain. Rhodojaponin III (RJ-III) has been identified as the main pharmacological activity and toxic component of the herb; however, oral antinociception and mechanism of RJ-III have not yet been investigated. AIM OF THE STUDY: The significance of this study is to evaluate the effects of RJ-III on nociceptive and neuropathic pain, and to preliminarily explore the underlying mechanisms and subacute toxicity. MATERIALS AND METHODS: The antinociception of RJ-III was evaluated by hot plate, tail-immersion, acetic acid writhing, formalin test and chronic constriction injury (CCI) model in rodents. An experimental validation was conducted using whole-cell patch clamp technique based on the most likely mechanisms of action after screening and prediction by molecular docking study. In addition, the oral subacute toxicity of RJ-III was assessed. RESULTS: Behavioral experiments showed that RJ-III (0.20 mg/kg) reduced the latency of the nociceptive response in the hot plate and tail-immersion tests. Acetic acid and formalin-induced pain were significantly inhibited by RJ-III (0.10 and 0.05 mg/kg, respectively). Furthermore, 0.30 mg/kg of RJ-III improved hyperalgesia in the CCI-induced rats. Based on molecular docking results, electrophysiological experiments were used to demonstrate mild inhibition of voltage-gated sodium channel-related subtypes. Additionally, oral subacute toxicity that may cause leukopenia and abnormal liver function requires further attention in subsequent studies. CONCLUSION: RJ-III mildly blocks voltage-gated sodium channel to inhibit nociceptive pain and peripheral neuralgia, but 0.375 mg/kg and above may cause side effect after long-term oral administration.

The Crystal Structure of the Spodoptera litura Chemosensory Protein CSP8.

Spodoptera litura F. is a generalist herbivore and one of the most important economic pests feeding on about 300 host plants in many Asian countries. Specific insect behaviors can be stimulated after recognizing chemicals in the external environment through conserved chemosensory proteins (CSPs) in chemoreceptive organs, which are critical components of the olfactory systems. To explore its structural basis for ligand-recognizing capability, we solved the 2.3 Å crystal structure of the apoprotein of S. litura CSP8 (SlCSP8). The SlCSP8 protein displays a conserved spherical shape with a negatively charged surface. Our binding assays showed that SlCSP8 bound several candidate ligands with differential affinities, with rhodojaponin III being the most tightly bound ligand. Our crystallographic and biochemical studies provide important insight into the molecular recognition mechanism of the sensory protein SlCSP8 and the CSP family in general, and they suggest that CSP8 is critical for insects to identify rhodojaponin III, which may aid in the CSP-based rational drug design in the future.

Pharmacokinetics, bioavailability and tissue distribution studies of rhodojaponin III in mice using QTRAP LC-MS/MS.

Rhodojaponin III is a bioactive diterpenoid isolated from the medicinal plant Rhododendron molle G. Don. Quantitative analysis of rhodojaponin III was challenging and the pharmacokinetics of oral rhodojaponin III remained to be investigated. Here, a rapid and sensitive liquid chromatography tandem mass spectrometric (LC-MS/MS) method was developed and validated. The calibration curve was linear over the concentration range of 1-200 ng/mL (r = 0.992). The method was further validated following internationally approved guidelines and all the issues including intra- and inter-day precision, accuracy, carryover, extraction recovery, matrix effects and stability met the recommended limits. The method was then applied to study the pharmacokinetics of rhodojaponin III in mice after intravenous (0.06 mg/kg) or oral (0.24 mg/kg) administration. The results showed that rhodojaponin III had fast oral absorption (time to peak concentration, 0.08 h) and good oral bioavailability (73.6%). In addition, rhodojaponin III was quickly eliminated after it was intravenously or orally administered, with half-life values of 0.19 and 0.76 h, respectively. After oral administration, it was widely distributed in tissues including kidney, lung, heart, spleen and thymus, but had extremely low concentrations in liver and brain. The data presented in this study is beneficial for the further study of rhodojaponin III.

Contacting is essential for oviposition deterrence of Rhodojaponin-III in Spodoptera litura.

In Lepidoptera, choosing the right site for egg laying is particularly important, because the small larvae cannot forage for alternate host plants easily. Some secondary compounds of plants have the ability to deter oviposition behaviors of insects. Rhodojaponin-III, a botanical compound, has been reported to have intense deterring-oviposition activity against many insects, which have important implications for agricultural pest management. This study provided evidence for elucidating the perception mechanism underlying Rhodojaponin-III as oviposition deterrent. In this study, the antennas of moths could not elicit notable electroantennogram responses to Rhodojaponin-III, which suggested the Rhodojaponin-III could not exert effects like those volatile compounds. The results of physiological experiments confirmed the Rhodojaponin-III could produce the oviposition deterrence effect against moths without depending on antennas, while the physical contact was essential for perceiving the compound, which suggested that the sensilla on tarsus and ovipositor could be chemoreceptor for Rhodojaponin-III. Therefore, these sensilla were investigated by scanning electron microscopy to explore their potential functions in detecting Rhodojaponin-III. This study highlighted the contacting mechanism in deterring oviposition behaviors of moths by Rhodojaponin-III and provided new insight for development of contact-based pest management.