Advances in Toxicity of Ramulus et Uncus Uncariae

Ramulus et Uncus Uncariae is one of the 31 regional characteristic Chinese medicinal materials identified by Guangxi Zhuang Autonomous Region in 2021,with antihypertensive,antiarrhythmic,anti-cerebral ischemia,sedative,antithrombotic,anti-tumor and other pharmacological effects,and is neurotoxic and hepatotoxic.In this article,the chemical constituents,pharmacological effects,toxic effects,toxic manifestations in whole animals and mechanism of toxicity of Ramulus et Uncus Uncariae are reviewed.

Effects of Tianma(Rhizoma Gastrodiae)and Gouteng(Ramulus Uncariae Rhynchophyllae cum Uncis)on cytochrome P450 enzyme activities in rats

OBJECTIVE:To investigate the efficacy of Tianma(Rhizoma Gastrodiae)and Gouteng(Ramulus Uncariae Rhynchophyllae cum Uncis)on cytochrome P450(CYP450)enzyme activities in rats.METHODS:A cocktail strategy was followed to evaluate the influence of Tianma(Rhizoma Gastrodiae)and Gouteng(Ramulus Uncariae Rhynchophyllae cum Uncis)on the activities of CYP450 isoforms(CYP1A2,CYP3A4,CYP2E1,CYP2C19,CYP2C9,CYP2D6),which were determined by changes in the pharmacokinetic parameters of six probe drugs,theophylline,dapsone,chlorzoxazone,omeprazole,tolbutamide and dextromethorphan.Study groups included,Control group(CG),Tianma(Rhizoma Gastrodiae)group(TM),Gouteng(Ramulus Uncariae Rhynchophyllae cum Uncis)group(GT)and Tianma Gouteng(Gastrodia Uncaria)group(TMGT).RESULTS:No significant differences between Tianma(Rhizoma Gastrodiae)and control groups were found.Compared with the control group,in the Gouteng(Ramulus Uncariae Rhynchophyllae cum Uncis)group both the AUC and t1/2 of dapsone and tolbutamide were reduced,whereas the CL(clearance rate)of dapsone and tolbutamide were increased.Compared with the control group,in the Tianma Gouteng group,the AUC and t1/2 of dapsone and tolbutamide were reduced,the CL of dapsone and tolbutamide were increased,and the AUC and t1/2 of chlorzoxazone were increased and the CL of chlorzoxazone was reduced.CONCLUSION:Tianma(Rhizoma Gastrodiae)has no significant effect on the six CYP450 subtypes.The activities of CYP3A4 and CYP2C9 were increased by Gouteng(Ramulus Uncariae Rhynchophyllae cum Uncis).The activities of CYP3A4 and CYP2C9 were increased,whereas the activity of CYP32E1 was reduced by combined Tianma(Rhizoma Gastrodiae)and Gouteng(Ramulus Uncariae Rhynchophyllae cum Uncis).

Rapid Analysis of Four Alkaloids in Uncaria rhynchophylla by Core-Shell Column HPLC and Quantitative Analysis of Multi-Components by Single Marker(QAMS)

As a traditional herbal medicine,the major alkaloids in Uncaria rhynchophylla have been proven to have blood pressure-lowering and sedative effects.It is essential to develop an effective method for the determination of the major alkaloids in U.rhynchophylla.In this research,a rapid quantitative analysis involving multi-components analysis by a single marker strategy coupled with core-shell column HPLC was adopted to analyse four alkaloids(corynoxeine,isocorynoxeine,isorhynchophylline,rhynchophylline)in U.rhynchophylla.Isorhynchophylline was selected as the internal reference substance,the content of which was determined by the traditional external standard method.Relative correction factors(RCF)between isorhynchophylline and the other three alkaloids were calculated respectively.The results showed that the QAMS method had good robustness under different HPLC instruments.Nineteen batches of U.rhynchophylla were tested.No significant difference was observed between the results by QAMS and EMS(Correlation coefficient>0.99,p>0.05).The QAMS method could be employed as a rapid,effective technique for the quality control of U.rhynchophylla.

HPLC法测定钩藤提取物中钩藤碱的含量

[目的]测定钩藤提取物中有效成分钩藤碱的含量。[方法]采用HPLC法测定钩藤提取物中钩藤碱的含量,色谱柱为DiamonsilC18(250 mm×4.6 mm,5μm);流动相为无水甲醇-水(60∶40,V/V)并含10 mmol/L三乙胺,冰醋酸调pH 7.5;柱温为30℃;流速为1.0ml/min;紫外检测波长为254 nm。[结果]钩藤碱浓度在11.0～110.0μg/ml范围内与峰面积的线性关系良好(R=0.999 6),平均回收率为96.84%,RSD为1.46%。[结论]该方法简单、快速、准确,可用于钩藤提取物中钩藤碱的含量测定。

钩藤的化学成分研究

[目的]对钩藤的化学成分进行研究。[方法]利用柱层析对钩藤的化学成分进行分离纯化,然后根据化合物的理化性质和光谱数据鉴定所得到化合物的结构。[结果]从钩藤中分离得到了5个化合物,分别鉴定为乌苏酸、β-谷甾醇、β-胡萝卜苷、柯诺辛碱和钩藤碱。[结论]该方法分析研究了钩藤的化学成分,为钩藤的进一步开发和利用提供参考。

钩藤中钩藤碱的提取和含量测定

[目的]研究钩藤中钩藤碱的提取和含量测定方法。[方法]采用生物碱回流提取方法提取,采用高效液相色谱法对钩藤中钩藤碱的含量进行测定并计算提取率。[结果]专属性考察结果显示,钩藤碱的保留时间约为18.843 min,异钩藤碱的保留时间约在22.323min,两峰分离度良好,且钩藤药材中其他成分不干扰测定;含量测定结果显示,钩藤碱和异钩藤碱的含量分别为23.23、129.17μg/g,加样回收试验平均回收率分别为99.12%、101.33%,RSD分别为1.84%、1.56%。[结论]该方法稳定、有效、准确度高,可用于钩藤中钩藤碱的含量测定。

钩藤水提物Batch-2对6-羟多巴胺诱导的SH-SY5Y细胞损伤的神经保护作用(英文)

以前的药理学研究表明,钩藤水溶性提取物C-MED-100TM不仅具有抗氧化活性,而且还具有很好的DNA修复和免疫功能.Batch-2是一种新的水溶性钩藤提取物,而它的自由基清除能力及神经保护作用还未见报道.首先检测了batch-2对六羟多巴胺诱导的SH-SY5Y细胞损伤的神经保护作用机制,然后利用红外光谱、HPLC和分光光度技术对batch-2的组成成分进行了分析.结果表明,batch-2具有清除各种自由基的能力,尤其是对羟自由基的清除(25mg/L的batch-2对羟自由基的清除率为60%),batch-2可剂量依赖性地抑制6-羟多巴胺诱导的细胞凋亡、脂质过氧化水平、线粒体膜电位的降低和细胞内活性氧和一氧化氮的增加.同时,batch-2抑制了由6-羟多巴胺诱导的SH-SY5Y细胞内iNOS和NF-κB蛋白的上调.结果表明,batch-2对六羟多巴胺诱导的SH-SY5Y细胞损伤的神经保护作用是通过清除活性氧和一氧化氮、抑制iNOS和NF-κB表达实现的.成分分析表明,batch-2中的多酚和奎宁酸含量分别为6.43%和0.0958%.上述结果显示,batch-2的抗氧化机制部分类似于EGCG.对于帕金森病的预防,batch-2是一个潜在具有很好的神经保护作用的天然抗氧化剂.

HPLC法对钩藤超临界萃取物中钩藤碱的分析

通过超临界二氧化碳萃取技术(SFE-CO_2)和高效液相色谱法(HPLC),以甲醇-水(70∶30,V/V)为流动相,对钩藤碱进行分析。研究表明,方法回归方程为y=7.000 0×10^(-8)x-0.000 6(R^2=0.994),即钩藤碱浓度在0.015~0.022 mol/m L内呈良好的线性关系。该分析方法快速、简单、灵敏,具有良好的准确度和精密度,可用于钩藤碱常规分析检验。

野生中药材资源钩藤种子发芽研究(英文)

[Object] The research aimed to find out seed germination conditions and regulations.[Method] Seeds were soaked by clear water and 4 chemical agents of different concentrations(NaHCO3,KHCO3,K2CO3,NaClO)then conducted germination test in illumination box.[Result] Seed germination number and absolute germination rate(above 80%)in water treatment group was obviously higher than other treatments.[Conclusion] No dormancy phenomenon in mature embryo and no inhibition in seed coat,so conventional moist treatment can induct seed germination.

UFLC‑PDA‑MS/MS Profling of Seven Uncaria Species Integrated with Melatonin/5‑Hydroxytryptamine Receptors Agonistic Assay

Uncariae Ramulus Cum Uncis(Gou-Teng),the dried hook-bearing stems of several Uncaria plants(Rubiaceae),is a wellknown herbal medicine in China.The clinical application of Gou-Teng is bewildered for the morphological and chemical similarity between diferent species.In order to discern their chemical and biological diference,an ultra-fast liquid chromatography equipped with ion trap time-of-fight mass spectrometry(UFLC-IT/TOF-MS)combining with melatonin(MT1 and MT2)and 5-hydroxytryptamine(5-HT1A and 5-HT2C)receptors agonistic assay in vitro was conducted on seven Uncaria species.As a result,57 compounds including 35 indole alkaloids,ten favonoids,fve triterpenoids,fve chlorogenic analogues,and two other compounds were characterized based on their MS/MS patterns and UV absorptions.Specifcally,cadambine-type and corynanthein-type alkaloids were exclusively present in U.rhynchophylla and U.scandens,whereas corynoxine-type alkaloids were commonly detected in all the seven Uncaria plants.Three Uncaria species,U.rhynchophylla,U.macrophylla,and U.yunnanensis showed obviously agnostic activity on four neurotransmitter receptors(MT1,MT2,5-HT1A,and 5-HT_(2C)).This frst-time UFLCMS-IT-TOF analyses integrated with biological assay on seven Uncaria plants will provide scientifc viewpoints for the clinical application of Gou-Teng.

钩藤不同部位、不同时期药用成分分布规律分析

为揭示钩藤(Uncaria tomentosa)不同部位、不同时期主要药用成分的积累分布规律,确定其适宜采收期,采用高效液相色谱法测定一个生长周期内钩藤不同部位、不同时期钩藤碱、异钩藤碱含量。结果表明,钩藤叶、钩、茎中异钩藤碱的含量均相对较低,钩藤碱的含量相对较高,钩、茎中二者含量均较高。通过采收期的比较来看,11月钩藤碱和异钩藤碱含量相对较高,应为最佳采收期,与实际采收期吻合。

Indole Alkaloids Inhibiting Neural Stem Cell from Uncaria rhynchophylla

Uncaria rhynchophylla is commonly recognized as a traditional treatment for dizziness,cerebrovascular diseases,and nervous disorders in China.Previously,the neuro-protective activities of the alkaloids from U.rhynchophylla were intensively reported.In current work,three new indole alkaloids(1–3),identified as geissoschizic acid(1),geissoschizic acid N4-oxide(2),and 3b-sitsirikine N4-oxide(3),as well as 26 known analogues were isolated from U.rhynchophylla.However,in the neural stem cells(NSCs)proliferation assay for all isolated compounds,geissoschizic acid(1),geissoschizic acid N4-oxide(2),isocorynoxeine(6),isorhynchophylline(7),(4S)-akuammigine N-oxide(8),and(4S)-rhynchophylline N-oxide(10)showed unexpected inhibitory activities at 10 μM.Unlike previous neuro-protective reports,as a warning or caution,our finding showcased a clue for possible NSCs toxicity and the neural lesions risk of U.rhynchophylla,while the structure–activity relationships of the isolated compounds were discussed also.

Relief of osteoarthritis with a herbal-amino acid supplement: A randomized double-blind placebo controlled trial

A redox active medicinal plant and L-leucine mixture (HLM) was investigated in subjects with established osteoarthritis of the knee in a multi-center, rando- m-ized, placebo-controlled, double-blind clinical trial. A total of 96 subjects with osteoarthritis were enrolled and randomized to either placebo (n = 38) or HLM treatment group (n = 38). The HLM group re- ceived a combination of Uncaria tomentosa (300 mg), Boswellia serrata (200 mg), Lepidium meyenii (1000 mg) and L-Leucine (700 mg) given as 3 capsules once a day. The placebo group received matching capsules with carboxymethylcellulose. The treatment period was 8 weeks, with assessments made at days 7, 14, 28 and 56. The primary outcome was reduction in total WOMAC score. VAS pain, tolerability, investigator assessments, use of rescue medication (acetominophen), and safety assessments of vital signs and laboratory assessments were included. Subject randomization was effective for age, gender and disease severity. In the placebo group 32/38 subjects completed the trial and for HLM 35/38. WOMAC scores (pain, stiffness, physical performance and total) steadily declined over the course of the 8 week study in both groups, but the magnitude was significantly greater for HLM (P < 0.05). Total WOMAC was reduced 46.5% for HLM and 25.4 % for placebo. VAS pain was reduced 21.8% in the placebo group (p < 0.002) but the changes were significantly greater (37.8% p < 0.03) with HLM treatment. Investigator’s global assessment rating of good-excellent was 24/35 (69%) for HLM and 14/32 (44%) for placebo (P = 0.05). Rescue medication consumption and tolerability were comparable for HLM and placebo. No safety issues were evident with either group. As expected a placebo effect was observed, nevertheless HLM was clearly more effective in relieving the symptoms of osteoarthritis. This HLM represents a safe and effective new approach to the management of osteoarthritis symptoms.

Pseudotargeted metabolomics for exploring the changes of neurotransmitters profile in aging rat brain and the potential neuroprotective effect of alkaloids from Uncaria rhynchophylla

Background:Aging is an essential risk factor for most neurodegenerative diseases,including Alzheimer’s disease and Parkinson’s disease.However,changes in the levels of neurotransmitters that are associated with aging are not well understood.Methods:Methods such as liquid-liquid extraction,protein precipitation,and solid-phase extraction,using 20 different extraction solvents,were evaluated to optimize the extraction of neurotransmitters.A pseudotargeted metabolomics approach was developed to detect neurotransmitters in brain tissues using ultra-high-performance liquid chromatography coupled with tandem triple quadrupole mass spectrometry.Alkaloids that crossed into the brain were used to evaluate the effect of glutamic acid-induced excitatory neurotoxicity in SH-SY5Y cells.Results:The overall extraction efficiency using protein precipitation was high.The changes in neurotransmitters’levels in the brain exhibited changes during the different growth cycles.The levels of seven neurotransmitters(aspartic acid,tyrosine,isoleucine,leucine,tryptophan,valine,andγ-aminobutyric acid)were significantly different.Meanwhile,alkaloids could reduce the excitatory neurotoxicity of glutamic acid-induced SH-SY5Y cells via suppression of oxidative stress.Conclusion:Significant differences were observed in neurotransmitter profiling between 1-and 8-month-old rats,and the discrepant neurotransmitters were associated with aging.Seven indole alkaloids from Uncaria rhynchophylla,which could cross the blood-brain barrier,were screened and used to explore their protective effects against aging.Uncaria rhynchophylla alkaloids exhibited a neuroprotective effect by inhibiting oxidative stress,indicating that the alkaloid could be a potential therapeutic candidate for neurological disorders caused by glutamic acid toxicity.

Triterpenoids from Uncaria rhynchophylla and Their PTP1B Inhibitory Activity

Uncaria rhynchophylla(Gouteng)is a famous traditional Chinese medicine used for psychiatric and hypotensive purposes in China.In this study,the ethyl acetate(EtOAc)part of U.rhynchophylla was revealed with protein tyrosine phosphatase 1B(PTP1B)inhibitory activity.Subsequent investigation on the EtOAc part yielded one new triterpenoid,3β-formyloxy-6β,19α-dihydroxyurs-12-en-28-oic acid(1)and four known ones,3β,6β,19α-trihydroxyurs-12-en-28-oic acid(2),2-oxopomolic acid(3),3β,19α-dihydroxy-6-oxo-olean-12-en-28-oic acid(4)and sumaresinolic acid(5).The structure of compound 1 was determined by extensive HRESIMS,IR,1D and 2D NMR spectroscopic analyses.Two ursane-type triterpenoids(2 and 3)showed selective inhibition on PTP1B with IC50 values of 48.2 and 178.7μM.The enzyme kinetic study suggested that compounds 2 and 3 were mixtype inhibitors on PTP1B with Ki values of 15.6 and 132.5μM.This investigation manifests the antidiabetic potency of U.rhynchophylla with triterpenoids as the active constituents.

Cryopreservation of Cat’s Claw (<i>Uncaria tomentosa</i>)

Uncaria tomentosa presents tomentum that resembles cat’s claws, hence its common name, is a plant that produces various secondary metabolites that are traditionally used in alternative medicine. The natural distribution of this species has been affected by indiscriminate harvesting from its habitat. In the present research, cryopreservation (liquid nitrogen, LN, -196&degC) was evaluated as an option for ex situ conservation of this species. The following techniques were evaluated: vitrification and encapsulation-dehydration of apices, vitrification of cell suspensions, and seed desiccation and vitrification. Preculture conditions and exposure times to LS and PVS2 were evaluated. Apex survival was the highest (82%) with preculture in 0.25 M sucrose followed by incubation for 20 and 30 min in LS and PVS2, respectively, prior to cooling in LN. The encapsulation-dehydration technique was evaluated by using sucrose preculture and different capsule moisture contents. Survival of apices cooled in LN was not significantly different between treatments and varied from 31.8% to 52.9% for capsule moisture contents between 22.7% and 20.3%. For cell suspensions precultured in 0.5 M sucrose, cell multiplication and formation of calli with very good appearance were observed in 61.1% of the cultures following vitrification. For cryopreservation of seeds, germination was 89.5% using the desiccation technique and 67.6% to78.1% using vitrification.

Zhang Hongxing's Experience in Exogenous Cough Treatment

Cough is a common clinical symptom,throughout history the medical experts have different discussions on the diagnosis and treatment of cough and put forward different theories on the treatment of cough.Chief physician Zhang Hongxing is a famous old doctor of traditional Chinese medicine in Dezhou city with rich experience in clinical practice and unique academic thoughts.In the treatment of exogenous cough,Director Zhang stressed that the differentiation of syndromes should be focused on‘wind' and pay attention to the role of liver‘wind’in cough.The prescription of medicines should emphasize on dispelling the‘wind’first,to dispel the external‘wind',but also to calm the internal‘wind’and making good use of Uncaria in medicine.Valuable experience for clinical diagnosis and treatment of exogenous cough was provided.

Two new ortho benzoquinones from Uncaria rhynchophylla

The present study was designed to determine the chemical constituents of the stems and hooks of Uncaria rhynchophylla. The chemical constituents were isolated and purified from CH_2Cl_2 fraction by chromatography. Their structures were elucidated by spectroscopic analyses. Their cytotoxicity was tested using MTT method. Two new ortho benzoquinones, 3-diethylamino-5-methoxy-1, 2-benzoquinone(1) and 3-ethylamino-5-methoxy-1, 2-benzoquinone(2), together with a known compound isorhynchophyllic acid(3) were isolated from U. rhynchophylla. These compounds were evaluated for their cytotoxicity against cancer cells A549, Hep G2 and A2780. Compounds 1 and 2 were new ortho benzoquinones and showed weak antiproliferative activities on A549, Hep G2 and A2780 cells. Compound 3 significantly inhibited the proliferation of A549, Hep G2 and A2780 cells with IC_(50) values being 5.8, 12.8 and 11.8 μmol·L^(–1), respectively.

Two folate-derived analogues from an aqueous decoction of Uncaria rhynchophylla

Two new folate-derived analogues,named uncarophyllofolic acids A(1)and B(2),respectively,were isolated from the Uncaria rhynchophylla hook bearing stem(Gouteng in Chinese).The distinct stereochemical structures of 1 and 2 were determined by spectroscopic data analysis in combination with acidic hydrolysis and Marfey’s derivatization,along with comparison of their specific rotation and Cotton effect(CE)data with those of the biogenetically related known derivatives as well as theoretical calculations of electronic circular dichroism(ECD)spectra.A plausible biosynthetic pathway of 1 and 2,associating to folate metabolism and the previously reported orychophragines A-C from Orychophragmus violaceus,is discussed.