Synonym name: Mitrinermine
Catalogue No.: BP1216
Cas No.: 76-66-4
Formula: C₂₂H₂₈N₂O₄
Mol Weight: 384.476
Botanical Source: Alkaloid from Uncaria rhyncophylla, some other Uncaria spp., several Mitragyna spp. and Cephalanthus occidentalis (Naucleaceae)

Purity: 95%~99%
Analysis Method: HPLC-DAD or/and HPLC-ELSD
Identification Method: Mass, NMR
Packing: Brown vial or HDPE plastic bottle
Can be supplied from milligrams to grams.

For Reference Standard and R&D, Not for Human Use Directly.

Inquire for bulk scale.

Description:

Rhynchophylline, a noncompetitive antagonist of the NMDA receptor, which has anti-inflammatory, anti-hypertension, cardiacprotective and neuroprotective activities. Rhynchophylline can markedly inhibit rabbit platelet aggregation induced by ADP or thrombin possibly by depressing the inflow of Ca2+ and the rise of the cytoplasmic free calcium level in platelet.Rhynchophylline can reduce the systolic blood pressure (SBP) of spontaneously hypertensive rats (SHR) significantly, decrease plasma Ang II, ADMA, and AT1R levels, and promote serum NO and NOS levels, which has the protection of vascular endothelial function.

References:

Fitoterapia. 2014 Oct;98:166-73.    

Effects of rhynchophylline on GluN1 and GluN2B expressions in primary cultured hippocampal neurons.   

N-methyl-d-aspartate (NMDA) receptor subunits GluN1 and GluN2B in hippocampal neurons play key roles in anxiety. Our previous studies show that Rhynchophylline, an active component of the Uncaria species, down-regulates GluN2B expression in the hippocampal CA1 area of amphetamine-induced rat. 

METHODS AND RESULTS:

The effects of Rhynchophylline on expressions of GluN1 and GluN2B in primary hippocampal neurons in neonatal rats in vitro were investigated. Neonatal hippocampal neurons were cultured with neurobasal-A medium. After incubation for 6h or 48 h with Rhynchophylline (non-competitive NMDAR antagonist) and MK-801 (non-competitive NMDAR antagonist with anxiolytic effect, as the control drug) from day 6, neuron toxicity, mRNA and protein expressions of GluN1 and GluN2B were analyzed. GluN1 is mainly distributed on neuronal axons and dendritic trunks, cytoplasm and cell membrane near axons and dendrites. GluN2B is mainly distributed on the membrane, dendrites, and axon membranes. GluN1 and GluN2B are codistributed on dendritic trunks and dendritic spines. After 48 h incubation, a lower concentration of Rhynchophylline (lower than 400 μmol/L) and MK-801 (lower than 200 μmol/L) have no toxicity on neonatal hippocampal neurons. Rhynchophylline up-regulated GluN1 mRNA expression at 6h and mRNA and protein expressions at 48h, but down-regulated GluN2B mRNA and protein expressions at 48 h. However, GluN1 and GluN2B mRNA expressions were down-regulated at 6h, and mRNA and protein expressions were both up-regulated by MK-801 at 48h. 

CONCLUSIONS:

These findings show that Rhynchophylline reciprocally regulates GluN1 and GluN2B expressions in hippocampal neurons, indicating a potential anxiolytic property for Rhynchophylline.    

Phytother Res. 2012 Oct;26(10):1528-33.    

Rhynchophylline attenuates LPS-induced pro-inflammatory responses through down-regulation of MAPK/NF-κB signaling pathways in primary microglia.    

Excessive activation of microglial cells has been implicated in various types of neuroinflammation. Suppression of microglial activation would have therapeutic benefits, leading to the alleviation of the progression of neurodegeneration. 

METHODS AND RESULTS:

In this study, the inhibitory effects of Rhynchophylline (RIN), a tetracyclic oxindole alkaloid component isolated from Uncaria rhynchophylla (Miq.) Jacks., on the production of pro-inflammatory mediators were investigated in lipopolysaccharide (LPS)-stimulated microglia. The results showed that RIN markedly reduced the production of nitric oxide (NO), prostaglandins E(2) (PGE(2) ), monocyte chemoattractant protein (MCP-1), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in LPS-activated microglia. The mRNA expression levels of iNOS and COX-2 were also depressed by RIN in a concentration-dependent manner. Further studies revealed that RIN blocked IκBα phosphorylation and degradation, inhibited the phosphorylation of mitogen-activated protein kinases (MAPKs).

CONCLUSIONS:

In summary, these data suggest that RIN suppresses inflammatory responses of microglia and may act as a potential therapeutic agent for various neurodegenerative diseases involving neuroinflammation.   

Am J Chin Med. 2009;37(2):351-60.    

Uncaria rhynchophylla and Rhynchophylline inhibit c-Jun N-terminal kinase phosphorylation and nuclear factor-kappaB activity in kainic acid-treated rats.    

Our previous studies have shown that Uncaria rhynchophylla (UR) can reduce epileptic seizures. 

METHODS AND RESULTS:

We hypothesized that UR and its major component Rhynchophylline (RH), reduce epileptic seizures in rats treated with kainic acid (KA) by inhibiting nuclear factor-kappaB (NF-kappaB) and activator-protein-1 (AP-1) activity, and by eliminating superoxide anions. Therefore, the level of superoxide anions and the DNA binding activities of NF-kappaB and AP-1 were measured. Sprague-Dawley (SD) rats were pre-treated with UR (1.0 g/kg, i.p.), RH (0.25 mg/kg, i.p.), or valproic acid (VA, 250 mg/kg, i.p.) for 3 days and then KA was administered intra-peritoneal (i.p.). The results indicated that UR, RH, and VA can reduce epileptic seizures and the level of superoxide anions in the blood. Furthermore, KA was demonstrated to induce the DNA binding activities of NF-kappaB and AP-1. However, these inductions were inhibited by pre-treatment with UR, RH, or VA for 3 days. Moreover, UR and RH were shown to be involved in the suppression of c-Jun N-terminal kinase (JNK) phosphorylation. 

CONCLUSIONS:

This study suggested that UR and RH have antiepileptic effects in KA-induced seizures and are associated with the regulation of the innate immune system via a reduction in the level of superoxide anions, JNK phosphorylation, and NF-kappaB activation.    

 HPLC of Rhynchophylline

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Storage conditions：Short-term storage at 2~8℃, long-term storage at -20 ~ -80℃

Introduction/Overview

Rhynchophylline (CAS number: 76-66-4) is derived from the traditional Chinese medicine Rhynchophylline（Uncaria rhynchophylla A major indole alkaloid isolated from Miq. ex Havil. Gouteng, as an important medicine for "calming the liver and calming the wind, clearing heat and calming the nerves", has a long history of being used in traditional Chinese medicine clinical practice to treat hypertension, seizures, headaches, dizziness and other symptoms. Modern pharmacological research has revealed that crocetine is one of the key active ingredients that enable crocetin to exert various pharmacological effects, exhibiting a wide range of biological activities, especially in the fields of cardiovascular and central nervous system diseases, which have attracted much attention. As an inhibitor of EphA4 receptor, crocetine has shown great potential in neuroprotection, anti-inflammatory, anti fibrotic and other aspects. Its clear antihypertensive activity and its regulatory effects on multiple targets such as angiotensin-converting enzyme (ACE) and endothelial nitric oxide synthase (NOS3) make it a star molecule for the development of new cardiovascular and neurological disease treatment drugs from natural products. This article aims to systematically review the chemical characteristics, pharmacological activity, mechanism of action, pharmacological evaluation, and clinical application prospects of crocetin, in order to provide comprehensive scientific references for the in-depth research and development of this compound.

Chemical structure and physicochemical properties

The molecular formula of crocetine is C22H28N2O4, with a molecular weight of 384.4760. Its chemical structure belongs to the tetracyclic indole alkaloids, with a core skeleton composed of an indole ring fused with a complex polycyclic system (including one tetrahydropyran ring and one cyclohexene ring). The structure contains multiple chiral centers, giving it a specific stereochemical configuration, which is crucial for maintaining its biological activity.

From the analysis of physicochemical parameters related to drug properties, crocetine exhibits good drug like characteristics. Its lipid water partition coefficient (LogP) is 2.5093, indicating that the compound has moderate lipophilicity, which is beneficial for transmembrane transport and absorption. The topological polar surface area (TPSA) is 67.8700 Å ², which is within a range favorable for oral absorption (typically<140 Å ²). The water solubility value is 0.4340 mg/mL, which is slightly soluble. This suggests that solubilization strategies may need to be considered in formulation development to improve its bioavailability. Of particular importance is that its blood-brain barrier (BBB) permeability is predicted to be "high", which is highly consistent with its significant central nervous system pharmacological activities such as neuroprotection and anticonvulsant effects, indicating that crocetine can effectively enter the central nervous system to exert its effects. In addition, preliminary in vitro safety evaluations showed that crocetin had no significant hERG potassium channel inhibitory activity (predicted as "no"), indicating a low potential risk of arrhythmia; The Ames test predicted a result of 0.0, indicating that it may not have a direct genetic toxicity risk. These physicochemical and preliminary safety parameters together outline the outline of crocetin as a lead compound with good development potential.

Plant sources and extraction methods

Goutenine mainly comes from the genus Gouteng in the Rubiaceae family（Uncaria）Various plants, among which the hooked vine（Uncaria rhynchophylla）As the main source, it belongs to the same plant species, the large leaved hooked vine（U. macrophylla）Mao Gouteng（U. hirsuta）It also contains this ingredient. The traditional medicinal parts of Gouteng are the hooked stems and branches. Modern research has shown that its leaves, roots, and other parts also contain Gouteng alkaloids.

The extraction and separation technology of crocetine is relatively mature and usually follows the following process:
1. Preprocessing and Extraction Grind the dried Houttuynia cordata medicinal materials and use polar organic solvents such as ethanol or methanol for reflux extraction or ultrasound assisted extraction. Alternatively, acid water (such as dilute hydrochloric acid or dilute acetic acid) can be used for percolation or soaking to dissolve the alkaloids into salts.
2. Enrichment and Purification After concentration, the extract is adjusted to alkaline with alkali (such as ammonia water) to allow the alkaloids to precipitate freely. Then, organic solvents such as chloroform and dichloromethane are used for extraction to obtain crude total alkaloids. Further purification mainly relies on column chromatography technology, often using silica gel, alumina or macroporous adsorption resin as the stationary phase, and gradient elution with different ratios of chloroform methanol, petroleum ether ethyl acetate and other solvent systems. High performance liquid chromatography (HPLC) and preparative thin layer chromatography (PTLC) are key steps in obtaining high-purity crocetin monomers.
3. Identification and Quality Control The purified crocetine can be structurally confirmed by melting point determination, optical rotation determination, ultraviolet spectroscopy (UV), infrared spectroscopy (IR), nuclear magnetic resonance spectroscopy (NMR, especially 1H-NMR and 13C-NMR), and mass spectrometry (MS) techniques. At present, high-performance liquid chromatography (HPLC) has become the standard method for determining the content of crocetine in crocetin medicinal materials and their preparations, used for quality control.

Pharmacological activity research

A large number of pharmacological experiments in vitro and in vivo have confirmed that crocetine has various biological activities, mainly concentrated in the cardiovascular and nervous systems.

1. Hypotensive effect The antihypertensive effect of crocetine is one of the earliest and most extensively studied pharmacological activities. Animal experiments (spontaneously hypertensive rats, renal hypertensive dogs, etc.) have shown that intravenous injection or gavage of crocetine can produce significant and long-lasting antihypertensive effects, characterized by a synchronous decrease in diastolic and systolic blood pressure, without a significant increase in heart rate. Its antihypertensive mechanism is complex, involving multiple pathways such as peripheral vasodilation, central sympathetic nervous system inhibition, and renin-angiotensin system regulation.

2. Neuroprotective and anticonvulsant effects Gouteng alkaloids have significant regulatory effects on the central nervous system. In various neurodegenerative diseases and brain injury models, such as Alzheimer's disease (AD), Parkinson's disease (PD), cerebral ischemia-reperfusion injury, etc., crocetine has shown clear neuroprotective effects, reducing neuronal apoptosis, inhibiting inflammatory reactions, and improving learning and memory disorders. In addition, its anticonvulsant activity has also been confirmed in various epilepsy models.

3. Anti inflammatory and immune regulatory effects Gouteng alkaloids have inhibitory effects on both acute and chronic inflammation models. It can inhibit the overexpression of pro-inflammatory factors (such as TNF - α, IL-1 β, IL-6) in macrophages induced by lipopolysaccharides (LPS), and its anti-inflammatory mechanism is closely related to the inhibition of inflammatory signaling pathways such as NF - κ B and MAPK.

4. Other activities: The study also found that rhynchophylline has anti platelet aggregation, anti atherosclerosis, anti myocardial hypertrophy, anti liver fibrosis and pulmonary fibrosis and other activities, showing a wide spectrum of pharmacological effects.

Mechanism of action and molecular targets

The pharmacological effects of crocetin depend on its regulation of multiple molecular targets, forming the molecular basis for its multi-target action.

1. Blood pressure related targets：

   • Angiotensin converting enzyme (ACE)Gouteng alkaloids may inhibit ACE activity through competitive or non competitive means, reduce the production of angiotensin II (Ang II), thereby relaxing blood vessels and lowering blood pressure.
   • Endothelial nitric oxide synthase (NOS3)Research has shown that crocetine can upregulate the expression or activity of NOS3 in endothelial cells, promote the synthesis and release of nitric oxide (NO), which is a powerful endogenous vasodilator.
   • Angiotensin II type 1 receptor (AGTR1)Gouteng alkaloids may act as antagonists of AGTR1, blocking the binding of Ang II to its receptors, inhibiting vasoconstriction and aldosterone release.
   • Adrenergic receptors (ADRB1, ADRA1)Gouteng alkaloids may produce negative inotropic/frequency effects and vasodilatory effects by blocking the cardiac β 1 receptor (ADRB1) and vascular smooth muscle α 1 receptor (ADRA1), respectively, leading to a decrease in blood pressure. Its inhibition of central sympathetic nervous system output may also involve the regulation of these receptor pathways.

2. Core neuroprotective target: EphA4 receptor In recent years, breakthrough research has found that crocetin is a specific small molecule inhibitor of Ephrin receptor A4 (EphA4). EphA4 plays a critical role in neurological development and disease, and its excessive activation promotes synaptic plasticity abnormalities, neuroinflammation, and neuronal damage. Gouteng alkaloid inhibits the kinase activity of EphA4 by binding to its ligand binding domain, thereby downstream regulating signaling pathways such as RhoA/ROCK, PI3K/Akt, NF - κ B. This is considered to be the core molecular mechanism by which crocetine exerts neuroprotection, improves cognitive function, and promotes nerve regeneration in models such as Alzheimer's disease, stroke, and spinal cord injury.

3. Inflammation and fibrosis related pathways The anti-inflammatory and anti fibrotic effects of crocetin are mainly achieved by inhibiting classic signaling pathways such as NF - κ B, TGF - β/Smad, MAPK (p38, JNK, ERK). For example, inhibiting the nuclear translocation of NF - κ B and reducing the transcription of downstream inflammatory mediators; Inhibit TGF - β 1-induced Smad2/3 phosphorylation, block fibroblast activation and collagen deposition.

Evaluation of drug properties and pharmacokinetics

Although crocetin has shown excellent in vitro activity and clear targets, its pharmacological properties, especially pharmacokinetic properties, are the key to determining whether it can be successfully developed as a drug.

1. Absorption, distribution, metabolism, excretion (ADME)：

   • absorb The oral bioavailability of crocetin varies in different studies, with an overall low level (about 10% -30%), which may be related to its solubility in the gastrointestinal tract, first pass effect, and efflux of P-glycoprotein (P-gp).
   • distribution Thanks to its moderate LogP and high BBB permeability prediction, crocetine is widely distributed in the body and can quickly enter brain tissue, which is consistent with its central pharmacological effects. The plasma protein binding rate data still needs further clarification.
   • Metabolism Gouteng alkaloid is mainly metabolized by the cytochrome P450 (CYP) enzyme system in the liver, among which CYP3A4 and CYP2D6 may be key isoenzymes involved in its main metabolic pathway. Metabolites are mostly hydroxylated, demethylated and other products, and their activity needs to be further studied.
   • excretion The prototype drug and its metabolites are mainly excreted through the kidneys and bile.

2. Pharmacokinetic challenges and strategies Low bioavailability is the main challenge facing the development of crocetin. The current research strategies include:

   • Structural modification By synthesizing derivatives or prodrugs, improve their solubility and metabolic stability.
   • New drug delivery system Develop formulation technologies such as liposomes, nanoparticles, solid dispersions, and self microemulsions to improve their solubility and intestinal permeability, and reduce first pass effects.
   • combination therapy Combining with CYP enzyme inhibitors or P-gp inhibitors (such as verapamil) theoretically can increase their blood drug concentration and bioavailability, but the risk of drug interactions needs to be carefully evaluated.

3. safety Current toxicology studies have shown that crocetine has low toxicity within the effective dose range. The acute toxicity test showed that its LD50 value was relatively high. Long term toxicity testing requires a systematic evaluation of its effects on important organs such as the liver, kidneys, and heart. The absence of hERG inhibition and Ames negativity provide preliminary positive information for its safety, but a complete preclinical safety evaluation is still needed.

Clinical application prospects and prospects

The clinical application prospects of crocetin are broad, but there are still many opportunities and challenges from laboratory research to successful drug launch.

1. Potential indications：

   • Hypertension and related cardiovascular and cerebrovascular diseases As a multi-target antihypertensive natural ingredient, it is expected to be developed into a new type of antihypertensive drug or adjuvant therapy, especially suitable for patients with mild to moderate hypertension accompanied by anxiety and insomnia.
   • Neurodegenerative diseases Based on its clear EphA4 inhibition and neuroprotective effects, crocetine has great potential in the treatment of diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS), possibly improving symptoms and delaying disease progression.
   • Stroke and nerve damage In the neural repair after ischemic stroke, as well as the treatment of spinal cord injury and traumatic brain injury, crocetine may exert its effects through anti-inflammatory, anti apoptotic, and promoting neural plasticity.
   • Organ fibrosis Its activity in liver fibrosis and pulmonary fibrosis models provides new candidate molecules for the treatment of these refractory diseases.

2. Current R&D stage and challenges At present, research on crocetine is mostly in the preclinical stage. The main challenges include: ① Pharmacokinetic properties need to be optimized Especially oral bioavailability; ② Although the mechanism of action has been partially elucidated, the network is extremely complex A more systematic and in-depth analysis of the precise spectrum of multi-target synergistic effects is needed; ③ Lack of high-quality clinical evidence Rigorous clinical trials need to be designed to verify its effectiveness and safety in humans.

3. Future research directions：

   • In depth research on target biology Further elucidate the interaction mode between crocetin and EphA4 and other targets, and explore its specific regulatory mechanism in the disease microenvironment.
   • Structure based drug design Design and synthesize derivatives with higher activity, stronger selectivity, and better pharmacokinetic properties using the crystal structure or computer simulation of crocetin.
   • Innovative formulation development Vigorously promote the application of modern formulation technologies such as nano drug delivery systems and targeted formulations to achieve brain targeted delivery or lesion specific release.
   • Conduct clinical translational research After completing the GLP toxicology and pharmacokinetic studies of the system, actively promote early (phase I/II) clinical trials to explore its safe dose window and preliminary efficacy in humans.

Conclusion

Gouteng alkaloid, as the active essence of traditional Chinese medicine Gouteng, is a successful example of modern natural product pharmacology research. Starting from traditional blood pressure lowering applications, its research has expanded to multiple cutting-edge fields such as neuroprotection, anti-inflammatory, and anti fibrosis. Especially the discovery of its molecular mechanism as an EphA4 inhibitor provides a key breakthrough for understanding its neuropharmacological effects. Despite facing challenges in drug development, especially in pharmacokinetics, its clear multiple pharmacological activities, good drug like parameters, and preliminary safety characteristics make it a highly valuable lead compound for development. With the continuous integration of modern medicinal chemistry, pharmacy, and molecular biology technologies, and through structural optimization, formulation innovation, and mechanism deepening, crocetine is expected to be successfully developed into a new type of drug for treating major diseases such as hypertension, neurodegenerative diseases, and stroke in the future, achieving a magnificent transformation from ancient Chinese medicine to modern innovative drugs and contributing unique value to human health.