Research on bioresponse of active compounds of Strychnos...

Xu YY et al. Asian Journal of Pharmacodynamics and Pharmacokinetics 2009; 9(3):179-201

179

Asian Journal of

Pharmacodynamics and Pharmacokinetics

ISSN 1608-2281

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Research on bioresponse of active compounds of Strychnos nux-vomica L. Yan-Yan Xu1,2, Duan-Yun Si2, Chang-Xiao Liu1* 1 State Key Laboratories of Pharmacokinetics and Pharmacodynamics, Tianjin Institute of Pharmaceutical Research, Tianjin 300193, China 2 School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China

Abstract The genus Strychnos is very well known as the plants providing one of the most famous poisons, which is logically called strychnine. The pantropical Strychnos genus comprises about 200 species in the world. In China, Semen Strychni is used generally after processing (such as parching in a sand bath) to reduce its toxicity. Ethnopharmacologically, the seeds of Strychnos nux-vomica L. is used in the traditional Chinese medicine. The dried seeds of this plant are used for relieving pain, promoting blood circulation and curing indigestion. They are included as an ingredient in many analgesic prescriptions of traditional Chinese medicine. In Africa and in Asia, Strychnos genus is important for its reputation as a remedy against snakebites and poisonings. In West and Central Africa, Strychnos species are a major group of arrow poison adjuvants. In Europe, S. nux-vomica came early as seeds and later its wood was imported as a ‘lignum colubrinum’; they were used in the treatment of a variety of ailments and subsequently the seeds of this Strychnos have been the main source of the alkaloid strychnine. Alkaloids are the main bioactive ingredients in Strychnos nux-vomica; they are also responsible for the pharmacological and toxic properties possessed by Strychnos nux-vomica. In this review paper, we introduced the ethnobotany and ethnopharmacology, chemical, pharmacological, pharmacodynamic, pharmacokinetic, metabolomic and toxicological studies, and clinical response of Strychnos nux-vomica L. and its alkaloids.

Key words alkaloids; chemistry; ethnobotany; ethnopharmacology; metabolomics; pharmacology; pharmacodynamics; pharmacokinetics; Strychnos nux-vomica L.; toxicology

Article history Received 26 August 2008; Accepted 24 March 2009 Publication data Pages: 23; Tables:2; Figures:8; References: 76; Paper ID 1608-2281-2009-09030179-23 *Corresponding author Professor Chang-Xiao Liu, Research Center for New Drug Evaluation, Tianjin Institute of

Pharmaceutical Research, Tianjin 300193, China. Tel:+86-22-23006863 E-mail: liuchangxiao @163.com

Introduction

The genus Strychnos is very well known as the plants providing one of the most famous poisons. Because of their

toxicity, many of these species have been used as arrow poisons or in ordeals. The Strychnos genus comprises about 200 species in the world and can be subdivided into three geographically separated groups of species:


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one in Central and South America (at least 73 species), one in Africa (75 species), and one in Asia including Australia and Polynesia (about 44 species).

In China, Semen Strychni， the dried seeds of Strychnos nux-vomica L. (Fig 1), S. hainanensis Merr et Chum, S. pierriana A. W. Hill or S. confertifora Merr et Chum is used, generally after processing to reduce its toxicity in the traditional Chinese medicine.[1] This herb was recorded in Bencao Gangmu edited by Li Shi-Zheng in Ming Dynasty. The drug was listed as the third category with the toxic herbs, its biological effects (pharmacology and toxicity effects) is a dose-dependent relationship. Ethnopharmacologically, the property and taste are bitter and cold. The meridians are into heart, liver and spleen. The functions are to invigorate blood, stop pain, move stagnation, and reduce edema. In Chinese medicine prescriptions (such as Biqi capsule), the processed Semen Strychni is frequently used as an important ingredient in traditional medicine to promote blood circulation and remove blood stasis. After this process, the original sixteen strychnos alkaloids decreased significantly in Semen Strychni. Now, Semen Strychni was listed in Pharmacopoeia of the People's Republic of China[1,2]. Modern studies showed that Strychnos nux-vomica is used in the therapy of liver cancer. In this review paper, we introduced the ethnobotany and ethnopharmacology, chemical, pharmacological, pharmacodynamic, pharmacokinetic, metabolomic and toxicological studies, and clinical response of Strychnos nux-vomica L. and its alkaloids. Ethnobotany and Ethnopharmacology

The genus Strychnos is very well known as the plants providing one of the most famous poisons, which is logically called strychnine. The pantropical Strychnos genus comprises about 200 species and can be subdivided into three geographically separated groups of species: one in Central and South America (at least 73 species), one in Africa (75 species), and one in Asia including Australia and Polynesia (about 44 species). Belonging to the Loganiaceae family, they are found as erect or climbing shrubs, lianas or trees.[3,4]

In China, the seeds of Strychnos nux-vomica L. is used, generally after processing (such as parching

in a sand bath) to reduce its toxicity. The dried seeds of this plant are used for relieving pain, promoting blood circulation and curing indigestion. They are included as an ingredient in many analgesic prescriptions of traditional Chinese medicine. Such a use can induce accidents, even after processing. In this country, strychnine poisoning should be included in the differential diagnosis in any patient with ‘unexplained’ muscle spasms or convulsions and such patients should be asked about the use of herbal medicines.[6] Note that a study has demonstrated the antinociceptive effects of crude alkaloid fractions of processed and unprocessed S. nux-vomica seeds in different analgesic tests in mice. This crude alkaloid fraction seemed to be about 1000 times more potent than morphine. [7]

Fig1. Strychnos nux-vomica L. and its seeds

http://www.yiyanfang.com/shipu/shicaigongxiao/maqianzi_13780/

In Southeast Asia, arrow and dart poisons have been extensively utilized. The major source of poison throughout much of the region, from Burma to China and Indonesia is the latex of the highly toxic cardenolide-bearing Antiaris toxicaria (Moraceae), but the roots or stem barks of various species of Strychnos are another important source, and among them are S. ignatii and S. nux-vomica. An analysis of some dart-poisons from Malaysia, the centre of poisoned arrows and darts in Southeast Asia, has confirmed that their main active compounds are cardenolides from A. toxicaria and Strychnos alkaloids, probably from S. ignatii, the most common Strychnos in such poisons . [8] The leaves and fruits of some species (S. nux-vomica and S. potatorum in India, S. ignatii in the Philippines) have been used as fish poisons.[9]

In Europe, S. nux-vomica came early as seeds


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and later its wood was imported as a ‘lignum colubrinum’; they were used in the treatment of a variety of ailments and subsequently the seeds of this Strychnos have been the main source of the alkaloid strychnine. The seeds of S. ignatii also reached Europe and were used like those of S. nux-vomica. For more details about Strychnos medical uses, one can consult.[3,9,10]

In West and Central Africa, Strychnos species are a major group of arrow poison adjuvants, but their place in the hierarchy of African hunting poisons is, however, secondary in comparison with that of some plants such as Strophantus, the use of Strychnos species being limited locally. [3] Their roots are used especially in the equatorial forest regions of Central Africa. Most of the African Strychnos-based arrow poisons contain the wellknown convulsant strychnine or related alkaloids. Generally, it is the distinctively red-coloured roots of S. icaja that are used. The plant, like other Strychnos species, such as S. densiflora Baill., S. samba Duvign. and S. angolensis Gilg, has also been widely used as an ordeal poison.[11] Indeed, the importance of the genus Strychnos as a trial by ordeal poison is higher than as a hunting poison. Until the latter part of the 19th century, ordeals were widely used for determining innocence or guilt. The general principle of this type of ordeal was that if, after being given the poison to eat or drink, the subject rejected it, the person was assumed to be innocent. S. usambarensis Gilg is also used as an arrow poison in Africa, but it does not contain strychnine and was found to behave just like a good curare. This latter plant will be described further in the curare part of this manuscript. The fruit of different species, and especially S. aculeata, are known for their use as fish poison . [12]

In Africa and in Asia, Strychnos genus is important for its reputation as a remedy against snakebites and poisonings. The reputed emetic and tonic (bitter) properties no doubt play an important part in the use of Strychnos in stomach, abdominal and intestinal complaints as well as in the treatment of worms and parasites. Strychnos also finds a use against fevers in both Africa and Asia. Moreover, this genus was implicated in the treatment of ulcers, wounds, and swellings, in skin troubles including leprosy, and in the treatment of more specific diseases like cholera and rabies. S. icaja is used in

different regions in Africa to treat chronic malaria.[3] Chemical studies

Alkaloids are the main bioactive ingredients in

Strychnos nux-vomica; they are also responsible for the pharmacological and toxic properties possessed by Strychnos nux-vomica. Alkaloids are 1.5～5% of the seeds, Strychnine is about 35～50% in total alkaloids of the seed of Strychnos nux-vomica L.. Brucine is also 35～ 50% in total alkaloids. Colubrine and 16-hydroxycolubrine, pseudostrychnine, vomicine, and loganin are presentred in the seeds. Mavacurine, novacine, icajine, α-colubrine, β-colubrine, isostrychnine, pserdo- strychnine, seudobrucine, 16-hydroxy-β-colubrine, 18-hydroxy-sungucine, 18-hydroxy- isosungucine and vomicine. Isobrucine, isobrucine N-oxide, isostrychnine N-oxide, 2-hydroxy-3-methoxy- strychnine are isolated from processed Strychnos nux-vomica seeds. Additionally, cycloartenyl palmitate, fatty acid, protein and polysaccharides were isolated from the seed of Strychnos nux-vomica L. Fig 2A and 2B exhibited the chemical structures of major alkaloids.

The composition of the coagulant polysaccharide fraction from Strychnos potatorum seeds is described. This fraction comprises a 1:1.7 mixture of a galactomannan and a galactan. The structure of these polysaccharides is also discussed. In addition, the coagulant properties of the polysaccharide fractions of two other Strychnos species, innocua and nux-vomica, have been assayed.[13] This paper deals with the extraction, determination and identification of the alkaloids in differently processed products of the seeds of Strychnos nux-vomica. The relationship between processing methods and toxicities is discussed according to the comparison of acute toxicity.[14] The contents of strychnine, brucine, isostrychnine and isobrucine in different processed products of Strychnos nux-vomica were determined by TLC-densitometry. The relationship of the contents of strychnos alkaloids with processing methods was studied.[15] Thirteen alkaloids were isolated from the seeds of Strychnos nux-vomica. They were identified as strychnine, beta-colubrine, pseudostrychnine, strychnine N-oxide, brucime,


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brucine N-oxide, novacine, icajine, vomicine, isostrychnine, isobrucine, isobrucine N-oxide and isostrychnine N-oxide by chemical and spectroscopic analysis.[16]

The application of an on-line sweeping preconcentration method in micellar electrokinetic chromatography (MEKC) for the determination of Strychnos alkaloids, namely strychnine and brucine, has been investigated in this work. After experimental optimizations, the best separation was achieved in 50 mmol ·L-1 H3PO4 (pH 2.0) containing 100 mmol·L-1 SDS and acetonitrile in a ratio of 4:1 (v/v), with an applied voltage of -20 kV at 20 degrees C. The sample matrix consisted of 100 mmol ·L-1 H3PO4 (pH 2.0), and sample introduction was performed at 0.5 psi for 270 s, with photodiode array detection at 203 nm. Compared with the conventional MEKC injection method, up to 100-fold improvement in concentration sensitivity was achieved in terms of peak height by using this sweeping injection technique. In the method, the compound berberine was used as the internal standard for the improvement of the experimental reproducibility. The calibration curve was linear over a range of 0.5-15 µg·ml-1 for both strychnine and brucine, with a correlation coefficient of 0.998 and 0.997, respectively. The detection limits (S/N=3:1) for strychnine and brucine were 0.05 and 0.07µg·ml-1, respectively. The sweeping-MEKC method has been successfully applied to the analysis of strychnine and brucine in Strychnos nux-vomica L. and its Chinese medicinal preparations.[17]

Direct analysis of alkaloids in the tissues of crude and processed Strychnos nux-vomica seeds by MALDI-TOFMS was described. The alkaloid profiles of the herb drugs were obtained without the need of complicated sample preparation to avoid potential damage or change of the active components. Seed tissues that were optimally sliced to a thickness of 10-20mum from the crude and processed Strychnos nux-vomica seeds as well as various parts of tissue such as endosperm and epidermis were analyzed on MALDI target plate after the matrix was directly applied onto the tissue surface. The obtained alkaloid profiles provided valuable information for the differentiation of crude and processed Strychnos nux-vomica seeds and for

the explanation of the significantly different toxicity. Experimental results indicated that the direct MALDI-TOFMS analysis allowed rapid screening of the alkaloid components in Strychnos nux-vomica seeds.[18]

The fragmentations of four strychnos alkaloids have been investigated by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) in the positive ion mode. Experiments using multi-stage tandem mass spectrometry (ESI-FT-ICR-MSn) allowed us to obtain precise elemental compositions of product ions at high mass resolution. The experimental data demonstrated that the nitrogen bridge and the coordinated oxygen atom on the nitrogen bridge in the alkaloid compounds were the active sites in the MS2 fragmentations. The loss of CH3 or the OCH3 group in those alkaloids, which have an OCH3 substituent, was the dominant fragmentation mode in the MS3 fragmentations. Logical fragmentation schemes for strychnos alkaloids have been proposed and these should be useful for the identification of these compounds.[19]

An easy, rapid method for simultaneous determination of strychnine and brucine in Strychnos nux-vomica L. and its preparation was developed by nonaqueous capillary electrophoresis (NACE) without pretreatment for the first time. Optimum separation was achieved with a fused-silica capillary column (50 cm×75 microm id) and a running buffer containing 30 mM ammonium acetate, 1.0% acetic acid and 15 % acetonitrile (ACN) in methanol. The applied voltage was 30.0 kV. The analytes were detected by UV at 214 nm. The effects of concentration of ammonium acetate, acetic acid and organic modifier on electrophoretic behavior of the analytes were studied. The established method with sophoridine as internal standard was linear in the range of 5-1000 mg·mL-1 for both strychnine and brucine. The extracts of Strychnos nux-vomica and its preparation could be directly injected for determination with recoveries ranging from 94.5 to 104%.[20]


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Strychnine

Molecular Weight: 334.412g/mol

Molecular Formula: C21H22N2O2

brucine



Vomicine



Pseudostrychnine



α-Colubrine



β-Colubrine



Icajine



Novacine



Strychnine N-oxide



Fig 2A. The chemical structures of major alkaloids.


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Brucine N-oxide



Isostrychnine



Isostrychnine N-oxide II



Isobrucine



Isobrucine N-oxide



Isosungucine



Sungucine



loganin


Molecular Formula: C17H26O10

isostrychnine N-oxide


Molecular Formula: C21H24N2O3 Fig 2B. The chemical structures of major alkaloids.


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A capillary zone electrophoresis (CZE) method has been developed for investigating the physicochemical characteristics of five Strychnos alkaloids in Strychnos nux-vomica L. Firstly, the dissociation constants of the five Strychnos alkaloids were determined, based on the relation between the effective mobility of the solutes and the buffer pH. The mathematical relationship was strictly deduced from the fundamental electrophoretic theory and the dissociation equilibrium. Secondly, an equation describing the relation between the migration time of alkaloids of similar structure and their molecular weights was developed and used to predict the migration order and to calculate the electrosomotic velocity. The results predicted by the theory agreed with those from experiments.[21] Chen et al used a capillary zone electrophoresis method for the separation and determination of strychnine and brucine in Strychnos nux-vomica L. and its preparation. The factors that could affect the separation were studied, such as the types and concentrations of electrolytes, pH, ionic strength and organic modifier. The optimum running buffer was 20 mmol·L-1 of ammonium acetate containing 0.2 mol/L of glacial acetic acid (pH 3.64). The applied voltage was 25 kV and the wavelength of the UV detector was set at 214 nm. The established method with dopamine hydrochloride as internal standard was linear in the range of 5-100 µg·mL-1 for both strychnine and brucine. The recovery was 102.96% for strychnine and 98.56% for brucine. The extracts of Strychnos nux-vomica and its preparation could be directly injected for analysis.[22]

The content of strychnine from Strychnos nux-vomica seeds was analyzed and compared to processed seeds by the HPLC-ESI/MS method. Using this technique, levels as low as 1 ng of strychnine were detected. In contrast to conventional UV detectors, this method also made it possible to discriminate brucine. This study resulted in finding the content of strychnine in detoxified seeds to be one tenth of unprocessed Strychnos seeds.[23]

From seeds of Strychnos nux-vomica three iridoids, 6'-O-acetylloganic acid, 4'-O-acetylloganic acid and 3'-O-acetylloganic acid were isolated together with two known iridoid glucosides, loganic

acid and 7-O-acetylloganic acid. The structures of the compounds were established by ESI-MS and by 1D and 2D NMR spectroscopic methods.[24]

A new capillary electrophoresis procedure with field-enhanced stacking concentration for the analysis of strychnine and brucine is established. After optimization of the separation and concentration conditions, the two alkaloids can be separated within 5 min and quantified with high sensitivity (The detection limits were 1.0 ng·mL-1 for strychnine and 1.4 ng·mL-1 for brucine). The method was useful for qualitative and quantitative analysis of strychnine and brucine in Strychnos nux-vomica with recovery of 105.1% for strychnine and 98.4% for brucine.[25]

A procedure for quantitative estimation of strychnine and brucine in the extracts of Strychnos nux-vomica seeds by capillary zone electrophoresis (CZE) was developed. The buffer solution used was 10mM phosphate buffer-MeOH (9:1), pH 2.5. The linear calibration range was 0.01-0.15 mg·mL-1. This method is useful for the qualitative and quantitative determination of strychnine and brucine in plant drug samples, as well as in human plasma.

[26] Jiang et al reported a HPLC method for determinition of strychnine and brucine in Semen Strychni and its processed products of Jiangxi method and innovated method. SiO2 was used as the stationary phase, n-hexane-dichloromethane-methanol-ammonia(47.5:47.5:5:0.35) as the mobile phase, with detection wavelength of 254 nm. The contents of strychnine and brucine in the processed products of Jiangxi are lower. This method is accurate, simple and reliable. [27]

Sha et al reported the determination of strychnine and brucine in different parts of Semen Strychni (the seeds of Strychnos nux-vomica and S. pierriana), and made a comparison of the contents between the crude forms and processed products of the two seeds. Based on the results, the authors have made some discussion about the relationship between the alkaloid distribution in Semen Strychni and its processing.[28] In the course of study on the drug-processing of the seeds of Strychnos nux-vomica, the alkaloid composition of the heat-treated seeds of S. nux-vomica was compared to that of the untreated seeds. On heat treatment, the


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contents of the major alkaloids such as strychnine and brucine declined significantly with increases in the amounts of isostrychnine, isobrucine, strychnine N-oxide and brucine N-oxide. The cleavage of an ether linkage and the occurrence of N-oxidation were demonstrated by heat treatment of authentic strychnine and brucine. [29] Contents of strychnine and brucine in dry seeds of Strychnos nux-vomica and its preparations were determined by gas chromatography. The determination conditions were:support Gas Chrom Q; liquid phase 3% OV-101; stainless steel column 0.5 m × 3 mm; column temperature 265 degrees C; FID detector.[30] Pharmacological studies

Inhibition of cyclooxygenase and lipoxygenase

Sandhika is a polyherbal formulation, (water soluble fraction of Commiphora mukul, Boswellia serrata, Semecarpus anacardium and Strychnos nux vomica), which has been in clinical use in India for last 20 years. Its modified formulation BHUx has shown specific inhibition of cyclooxygenase COX2 and lipoxygenase LOX and has prevented diet-induced atherosclerosis in rabbits. In order to explore the possibility of the use of Sandhika for the management of osteoporosis, the authors have examined its influence on MC3T3-E1 osteoblast-like cells in presence of lipopolysaccharide (1 µg·mL-1) in terms of calcium nodule formation and alkaline phosphatase activity. MC3T3-E1 osteoblast-like cells (80% confluence in 6-well plates) were treated with water extract of Sandhika, for 10 days, in the concentration range of 0.5 to 16 mg/ml final concentration, in presence of LPS. Media was changed on every third day and culture supernatant was collected after every change to assess the alkaline phosphatase activity and on the tenth day, cells were washed and stained with "Alizarin S" for visualization of calcium nodules by using Meta Morph software (Universal Imaging, Downingtown, PA). The results showed significant enhancement in calcium nodule formation in the dose dependent manner up to 2 mg·mL-1, followed by gradual decrease at higher concentrations. This change was accompanied with the increase in the alkaline phosphatase activity in these plates,

indicating a potential anabolic effect of this polyherbal formulation on osteoblast-like cells under inflammatory conditions induced by LPS[31] Strychnine, is responsible for its antilipid peroxidative property. The mechanism of action of this drug is through the chelation of the free iron in the system. It has also been observed that strychnine does not have any pro-oxidant-property, because it does not convert Fe3+ to Fe2+ and vice versa in the reaction system, as has been observed with several other antioxidants.[32]

BHUx is a polyherbal formulation consisting of water-soluble fractions of five medicinal plants (Commiphora mukul, Terminalia arjuna, Boswellia serrata, Semecarpus anacardium and Strychnos nux vomica). The present study was undertaken to evaluate its antioxidant and antiinflammatory effects. Under in vivo conditions, BHUx significantly reduced inflammation in the carrageenan-induced rat paw oedema model of inflammation, suggesting its anti-inflammatory properties. In order to test the mechanism of action of BHUx, further in vitro studies were undertaken on cumene- hydroperoxide-induced lipid peroxidation (CHP) in liver homogenate, LPS-induced NO production in peritoneal macrophages and on key enzymes of arachidonic acid cascade, involved in the mediation of inflammation. Under the conditions, BHUx showed concentration-dependent inhibition of CHP-induced lipid peroxidation in liver homogenate, suggesting its antioxidant properties. Similarly the potent anti-inflammatory effects of BHUx are evident by (a) preferential inhibition of COX2 (IC50 for COX2 = 80 µg·ml-1 and IC50 for COX1 = 169µg·mL-1), (b) low ratios in the IC50 values of COX2/COX1 (0.47), (c) decreased production of NO in LPS-induced peritoneal macrophages and (d) inhibition of 5-LOX (IC50 = 795µg·mL-1). BHUx also showed a preference for inhibiting 15-lipoxygenase (IC50 = 44µg·mL-1), a key enzyme implicated in LDL oxidation. These studies suggest that BHUx is acting mainly at three levels, i.e., as a potent natural antioxidant, by reduction of key inflammatory mediators of arachidonic acid cascade and by preventing 15-LOX-mediated LDL oxidations, to prevent atherosclerosis.[33]

To further understand the purpose of the


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traditional processing method of the seeds of Strychnos nux-vomica L. (Loganiaceae) as well as analgesic and anti-inflammatory activities of brucine and brucine N-oxide extracted from this medicinal plant, various pain and inflammatory models were employed to investigate their pharmacological profiles. Both brucine and brucine N-oxide revealed significant protective effects against thermic and chemical stimuli in hot-plate test and writhing test. However, on different phases they exerted analgesic activities in formalin test. Brucine N-oxide showed stronger inhibitory effect than brucine in carrageenan-induced rat paw edema, both of them significantly inhibited the release of prostaglandin E2 in inflammatory tissue, reduced acetic acid-induced vascular permeability and the content of 6-keto-PGF1a in Freund's complete adjuvant (FCA) induced arthritis rat's blood plasma. In addition, brucine and brucine N-oxide were shown to reduce the content of 5-hydroxytryptamine (5-HT) in FCA-induced arthritis rat's blood plasma, while increase the content of 5-hydroxytryindole-3-acetic acid (5-HIAA) accordingly. These results suggest that central and peripheral mechanism are involved in the pain modulation and anti-inflammation effects of brucine and brucine N-oxide, biochemical mechanisms of brucine and brucine N-oxide are different even though they are similar in chemical structure.[34]

A study was undertaken to evaluate the effect of aqueous and methanolic plant extracts of Acorus calamus rhizome, Pongamia glabra leaves, Aegle marmelos unripe fruit and Strychnos nux-vomica root bark for their antidiarrhoeal potential against castor-oil induced diarrhoea in mice. The methanolic plant extracts were more effective than aqueous plant extracts against castor-oil induced diarrhoea. The methanolic plant extracts significantly reduced induction time of diarrhoea and total weight of the faeces. The result obtained establish the efficacy of these plant extracts as antidiarrhoeal agents.[35]

Cytotoxicity and anti-cancer activity To study the cytotoxicity of four alkaloids:

brucine, strychnine, brucine N-oxide and isostrychnine from nux vomica on SMMC 7721

cells and their possible mechanisms, MET assay was used to examine the growth inhibitory effects of these alkaloids. Brucine revealed the strongest growth inhibitory effect on SMMC-7721 cells. Furthermore, as directly observed under an inverted microscope, fluorescent microscope and transmission electronic microscope, brucine caused SMMC-7721 cell shrinkage, membrane blobbing, formation of apoptotic body as well as nucleus condensation, all of which are typical characteristics of apoptotic programmed cell death. In addition, brucine dose-dependently caused SMMC-7721 cells apoptosis via formation of subdipolid DNA and phosphatidylserine externalization, as evidenced by flow cytometry analysis. The brucine-induced apoptosis was partially attributed to the activation of caspase 3 as well as cyclooxygenase 2 （COX-2）inhibition, since neither caspase 3 specific inhibitor, z-DEVD-fmk nor was exogenous addition of prostaglandin E(2) able to completely abrogate the brucine-induced SMMC 7721 cell apoptosis. In sum, It was indicated that the major alkaloids present in the seed of Strychnos nux-vomica are effective against SMMC-7721 cells proliferation, among which brucine proceeds SMMC-7721 cells death via apoptosis, probably through the participation of caspase 3 and cyclooxygenase 2.[36]

To examine the cytotoxicities of 6 crude Strychnos alkaloid fractions from the seeds of Strychnos nux-vomica unprocessed or processed with various traditional processing methods and 13 pure Strychnos alkaloids from the fractions. Using cell culture, their inhibitory effects on Vero cell growth-inhibition assay, and host cell DNA synthesis by 3H-thymidine (3H-TdR) uptake assay. The IC50 of processed seeds were 155% and 212% of unprocessed ones in cell growth-inhibition assay and in 3H-TdR uptake assay, respectively. The IC50 of 13 compounds were 0.45-0.80 mmol·L-1 and 0.50-12 mmol·L-1, respectively. The processing method with sand bath exhibited a wide safety margin compared with other traditional processing methods or no processing. The isomers of Strychnos alkaloids and their N-oxides showed much lower cytotoxicities among these alkaloids. Isobrucine N-oxide showed the lowest cytotoxicity. The contents of isomers and N-oxides of Strychnos alkaloids were the highest in the sand processing.


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Processing of nux vomica plays a critical role in its toxicity.[37]

To screen the anti-tumor effects of the four alkaloids: brucine, strychnine, brucine N-oxide and isostrychnine from the seed of Strychnos nux-vomica, MTT assay was used to examine the growth inhibitory effects of these alkaloids on human hepatoma cell line (HepG2). Brucine, strychnine and isostrychnine revealed significant inhibitory effects against HepG2 cell proliferation, whereas brucine N-oxide didn't have such an effect. In addition, brucine caused HepG2 cell shrinkage, membrane blebbing, apoptotic body formation, all of which are typical characteristics of apoptotic programmed cell death. The results of flow cytometric analysis demonstrated that brucine caused dose-dependent apoptosis of HepG2 cells through cell cycle arrest at G0/G1 phase, thus preventing cells entering S or G2/M phase. Immunoblot results revealed that brucine significantly decreased the protein expression level of cyclooxygenase-2, whereas increased the expression caspase-3 as well as the caspase-3-like protease activity in HepG2 cells, suggesting the involvement of COX-2 and caspase-3 in the pro-apoptotic effects exerted by brucine. Therefore, it was indicated that the major alkaloids present in the seed of Strychnos nux-vomica are effective

against HepG2 cells proliferation, among which brucine proceed HepG2 cells death via apoptosis, probably through the participation of caspase-3 and cyclooxygenase-2.[38]

In an attempt to dissect the mechanism of Strychnos nux-vomica, a commonly used Chinese folk medicine in the therapy of liver cancer, the cytotoxic effects of four alkaloids in Strychnos nux-vomica, brucine, brucine N-oxide, strychnine, and isostrychnine, Deng et al carried out the study on the Apoptotic Effect of Brucine from the Seed of Strychnos nux-vomica in Human Hepatoma Cells on Mediated via Bcl-2 and Ca2+ Involved Mitochondrial Pathway by 3-(4,5-dimethylthiazol-2-yl)- 2,5- diphenyl-tetrasolium bromide (MTT) assay. As shown in Fig 3B, the HepG2 cells following exposure to 0.5 mM brucine for 36 h underwent significant shape and volume changes. The cells

after treatment detached from the culture plate and shrank, and small apoptotic bodies formed. Other typical characteristics of apoptosis such as nuclear condensation and fragmentation were also revealed by Hoechst 33258 staining, as shown in Fig 3D. On the contrary, these apoptosis-related morphological alterations were not found in control cells (Fig 3A and 3C). Dose-response effect of brucine on DNA fragmentations in HepG2 cells (Fig 3E).[39]


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Fig 3. Brucine induced HepG2 cell death via apoptosis. Control cells (A) and brucine-treated cells (B) were observed under an inverted microscope; the cells morphology was photographed with a LM microscopy. For Hoechst 33258 staining, the

control cells (C) and brucine- treated cells (D) were loaded with Hoechst 33258 (1 µg/ml) for 20 min and observed under a fluorescence microscope in less than 15 min; cells were treated with 0.5 mM brucine for 36 h (B, D). Dose-response effect of

brucine on DNA fragmentations in HepG2 cells (E) [39] Brucine, among the four alkaloids, exhibited

the strongest toxic effect, the mechanism of which was found to cause HepG2 cell apoptosis, since brucine caused HepG2 cell shrinkage, the formation of apoptotic bodies, DNA fragmentation, cell cycle arrest, as well as phosphatidylserine externalization, all of which are typical characteristics of apoptotic programmed cell death. Caspases are the central executioners of apoptosis. Until now, two major apoptotic signaling pathways have been defined. The mitochondria-dependent pathway responds to extracellular cues and internal insults such as DNA damage. The second apoptotic pathway is triggered by death-receptor superfamily members through the activation of caspase-8. The death-receptor and mitochondrial pathways converge at the level of caspase-3. Brucine-induced HepG2 cell apoptosis was caspase dependent, with caspase-3 activated by caspase-9. Brucine also caused the proteolytic processing of caspase-9. In addition, brucine caused depolarization of the mitochondrial membrane of

HepG2 cells, the inhibition of which by cyclosporine A completely abrogated the activation of casapses and release of cytochrome c in brucine-treated HepG2 cells. These findings suggested a pivotal role of mitochondrial membrane depolarization in HepG2 cell apoptosis elicited by brucine. Furthermore, brucine induced a rapid and sustained elevation of intracellular Ca2+, which compromised the mitochondrial membrane potential and triggered the process of HepG2 cell apoptosis. Finally, Bcl-2 was found to predominately control the whole event of cell apoptosis induced by brucine. The elevation of Ca2+ caused by brucine was also suppressed by overexpression of Bcl-2 protein in HepG2 cells. From the facts given above, Ca2+ and Bcl-2 mediated mitochondrial pathway were found to be involved in brucine-induced HepG2 cell apoptosis.[39] Other alkaloids such as brucine N-oxide, strychnine, or isostrychine, which

closely resemble brucine in chemical structure, should also produce such an effect.

Fig 4. Caspase-9 was specifically involved in brucine-induced HepG2 cell apoptosis. Effect of casapse-8 or caspase-9 inhibitor on brucine-induced HepG2 cell apoptosis (A). The apoptosis was determined by Annexin V/PI double staining


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method. Effect of casapse-8 or caspase-9 inhibitor on the caspase-3-like protease activity in HepG2 cells after treatment with brucine (B). Time-course effects of brucine (0.5 mM) on the activation of caspase-9 and caspase-8 in HepG2 cells (C). Effect of casapse-8 or caspase-9 inhibitor on the protein abundance of cleaved caspase-3 in HepG2 cells after treatment with brucine (D). 50 µg protein was separated on a 12% SDS–PAGE gel. GAPDH was included as an inner standard to normalize the loadings (C, D). Cells were preincubated with z-LEHD-fmk (4 µM) or z-IETD-fmk (4 µM) for 30 min and further incubated with 0.5 mM brucine for 36 h (A) or 12 h (B, D). *p < 0.05, **p < 0.01 as compared to medium alone control.[39]

Caspase-3 has frequently been reported to be activated by either caspase-8 from intracellular death complex or caspase-9 from mitochondria in the process of apoptosis. Therefore, a selective caspase-8 inhibitor, z-IETD-fmk (4 µM), and caspase-9 inhibitor, z-LEHD-fmk (4 µM), were adopted to investigate the role of caspase-8 or caspase-9 in brucine-induced HepG2 cell apoptosis. As shown in Fig 4A, brucine-induced cell apoptosis was greatly rescued in the presence of caspase-9

inhibitor, but not caspase-8 inhibitor. Furthermore, the enhancement of caspase-3-like activity induced by brucine was totally blocked by caspase-9 inhibitor other than caspase-8 inhibitor (Fig 4B).

Brucine also enhanced the proteolytic processing of caspase-9 in a time-dependent manner. Instead, the proteolytic processing of caspase-8 was not detected within the time examined (Fig 4C). Finally, the cleavage of pro-caspase-3 was suppressed by caspase-9 inhibitor but not caspase-8 inhibitor. Taken together, the activation of casapse-9, rather than caspase-8, was responsible for brucine-induced

HepG2 cell apoptosis and caspase-3 activation.[39]

The final goal of Deng et al’s study hoping to achieve is to find a more effective anti-tumor drug

by properly modifying the structure of brucine, since many alkaloids such as harringtonine, vincaleukoblastine, and vincristine, which have strong anti-tumor effects, share the indole ring in their chemical structures with brucine. Thus, the experimental model established in Deng et al’s paper will surely accelerate the understanding of the structure–activity relationships in brucine and pave the way for discovering new drugs.

Xanthine oxidase inhibitory activity

Xanthine oxidase inhibitory activity was assayed from six species belonging to different families traditionally used for the treatment of gout and related symptoms by indigenous people of India. The aqueous, methanol-water mixture and methanolic extract of these plants were used for the

experiment. Of the 18 extracts assayed, 14 extracts demonstrated xanthine oxidase inhibitory activity at 100 µg·mL-1, among which 10 extracts showed an inhibition greater than 50% and IC50 values below 100µg·mL-1. The methanolic extracts of Coccinia grandis, Datura metel, Strychnos nux-vomica and Vitex negundo showed more than 50% inhibition, hence, they were screened for their in vivo hypouricaemic activity against potassium oxonate-induced hyperuricaemia in mice. Methanolic extracts of Coccinia grandis and Vitex negundo showed a significant decrease in the serum urate level (3.90±0.07 mg/dl, P<0.001) and (6.26±0.06 mg/dl, P<0.01), respectively, when compared to hyperuricaemic control (11.42±0.14 mg/dl). This effect is almost similar to the serum urate level of allopurinol (3.89±0.07 mg/dl).[40]

Neurotransmitter receptors Strychnine and brucine from the plant Strychnos

nux vomica have been shown to have interesting pharmacological effects on several neurotransmitter receptors, including some members of the superfamily of ligandgated ion channels. In their study, Jensen AA et al have characterised the pharmacological properties of tertiary and quaternary analogues as well as bisquaternary dimers of strychnine and brucine at human α1 and α1β-glycine receptors and at a chimera consisting of the amino-terminal domain of the α 7 nicotinic receptor (containing the orthosteric ligand binding site) and the ion channel domain of the 5-HT3A serotonin receptor. Although the majority of the analogues displayed significantly increased Ki values at the glycine receptors compared to strychnine and brucine, a few retained the high antagonist potencies of the parent compounds. However, mirroring the pharmacological profiles of strychnine and brucine, none of the analogues displayed significant selectivity between the alpha1 and alpha1beta subtypes. The structure-activity relationships for the compounds at the


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alpha7/5-HT3 chimera were significantly different from those at the glycine receptors. Most strikingly, quaternization of strychnine and brucine with substituents possessing different steric and electronic properties completely eliminated the activity at the glycine receptors, whereas binding affinity to the alpha7/5-HT3 chimera was retained for the majority of the quaternary analogues. This study provides an insight into the structure-activity relationships for strychnine and brucine analogues at these ligandgated ion channels. [41] This report the case of a 35 year-old man suffering from Reiter's disease. He did not respond to Lycopodium or Nux vomica in medium dilutions, but did respond to Nux vomica in very high potency.[42]

Antiplasmodial activity In the course of their search for new

antiplasmodial alkaloids from Strychnos icaja, Philippe et al have isolated five alkaloids: three monomers, protostrychnine and genostrychnine, previously described in Strychnos nux-vomica, pseudostrychnine, already found in the leaves of the plant, a new bisindolic alkaloid, named strychnogucine C, and the first naturally occurring trimeric indolomonoterpenic alkaloid: strychnohexamine. This latter trimeric alkaloid presented an antiplasmodial activity against the FCA Plasmodium falciparum line near 1 microM.[43]

Antialcoholic effect and reduction of alcohol induced sleep time

To see whether Strychnos nux-vomica extract (mother tincture [MT]), its potency Nux 30, and its principal alkaloid, strychnine, could reduce voluntary ethanol intake in rats. To analyze the solution structure of Nux MT, Nux 30c, 90% ethanol, and ethanol 30c by means of electronic (ES) and nuclear nuclear magnetic resonance (NMR) spectra. Potentially alcoholic rats were first given 20% ethanol and then kept on a two-choice bottle, one with 20% ethanol and another with tap water. These rats were given the following oral treatments for 15 days: group 1, control; group 2, strychnine at 0.36 mg/kg per day; group 3, ethanolic extract of S. nux-vomica seeds (Nux MT) at 3.6 mg·kg-1 per day; and group 4, Nux 30c at 0.05 mL·d-1 per rat. Nux 30c was prepared by successive dilution of Nux MT

and 90% ethanol (1:100) and sonication at 20 kHz for 30 seconds in 30 steps. Both Nux MT and Nux 30c significantly reduced ethanol intake and increased water intake in rats. ES of two dilutions of Nux MT and Nux 30c showed intersections at more than one point suggesting existence of molecular complexes. ES of Nux MT in CCl4 showed a red shift when 90% ethanol was added indicating molecular complexation and charge transfer interaction between ethanol and Nux compounds. NMR spectra of Nux MT, 90% ethanol, ethanol 30c, and Nux 30c indicated a change in solution structure of the medium (90% ethanol) of Nux 30c. Nux MT and Nux 30c could reduce ethanol intake in rats. The altered solution structure of Nux 30c is thought to mimic Nux MT and produce ethanol aversion in rats.[44] Nux vomica 30c, 200c and 1000c were administered orally to three batches of albino mice for three days. Six hours after the last dose on the third day the mice were injected i.p. with ethanol 4g·kg-1 body wt. They lost their righting reflex and lay motionless apparently sleeping due to alcohol. Mice treated with three potencies of Nux vomica regained their righting reflex more quickly than the corresponding untreated controls. Each of the three batches of mice was tested twice for ethanol sedation, once with a potency of Nux vomica and another time with a placebo control. The time interval between drug treatment and control was 10 days. NMR spectra of Nux 30, Nux 200, Nux 1000, alcohol 30, alcohol 30 (unagitated) and 90% alcohol showed significant difference from each other with respect to the spin-lattice relaxation time (T1) of the deuterium nuclei. This gives a measurable physical basis of the effective high potencies of Nux vomica. [45]

Male adult albino mice were administered potentized Nux vomica 30 c (Nux v). The drug was mixed with sterile distilled water at 0.05 ml/2 ml water and given at 0.05 ml/individual. Control consisted of blank ethanol solution. Ethanolic extract from the seeds of Strychnos nuxvomica L was mixed with 90% ethanol 1:100 and sonicated for 30 s at 20 KHz. This was further diluted and sonicated in 30 steps to produce Nux v 30 c. Six hours after treatment, mice were given 25% ethanol i.p. at 4 g·kg-1 body wt. The duration of sleep time


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starting from the loss of righting reflex until its restoration was recorded for each mouse. The duration of sleep time with ethanol was recorded in four sessions for the same group of mice with an interval of 10 d between sessions. Treatments: session 1 with control solution, 2 with Nux v orally, 3 with control solution and 4 with Nux v (i.p.). Nux v orally produced the shortest sleep time as compared to other treatments which did not differ from each other significantly with respect to sleep time. In another experiment Nux v 30 c was prepared with distilled water and pure absolute ethanol by the above process of successive dilution and sonication. These two preparations together with Nux v 30 c, prepared with 90% ethanol, were tested on mice for their effect on alcohol-induced sleep time. Only Nux v 30 c prepared with 90% ethanol was effective in reducing the sleep time in mice. It is concluded that the solution structure of ethanol/water mixture carries the specificity of the Nux v at ultra high dilution. It is further concluded that the effect is mediated through oral receptors.[46]

Homeopathic treatment In a monocenter prospective randomized

double-blind clinical trial the efficacy of homeopathic treatment was investigated on children with adenoid vegetations justifying an operation. Patients were treated with either homeopathic remedies such as Nux vomica D200, Okoubaka D3, Tuberculinum D200, Barium jodatum D4 and Barium jodatum D6 or with placebo. The duration of the study for each patient was 3 months. Examination of the ears using a microscope, rhinoscopy, stomatoscopy and pharyngoscopy, as well as tympanometry and audiometry were performed after 4, 8 and 12 weeks. Out of a total of 97 children studied between the ages of 4 to 10 years 82 could be analyzed. At the end of the study no operation was required in 70.7% of the placebo-treated children and in 78.1% of the children treated with homeopathic preparations. These results show no statistical significance.[47]

Antinociceptive effects Cai et al examined the antinociceptive effects

of the crude alkaloid fractions (CAF) of nux vomica and the influences of various processing methods

upon their antinociception in three analgesic tests in mice. In the tail-pressure test, the CAF (0.01-1 µg·kg-1, i.p.) of nux vomica that was unprocessed or treated with sand-, licorice-, oil- or vinegar and sand-processing showed clear antinociception. The CAF (1µg·kg-1, i.p.) of vinegar-processed nux vomica showed antinociception, without effects at lower doses of 0.01 and 0.1 µg·kg-1 and those treated with urine- or urine and sand-processing were without effects at doses of 0.01-1 µg·kg-1. Morphine (2 mg·kg-1, s.c.) showed short-lasting antinociception, without effects at a dose of 1 µg·kg-1. In the hot-plate test, the CAF (100µg·kg-1, i.p.) of nux vomica having undergone sand-processing produced a significant antinociception, without effects at lower doses of 0.01 and 1 µg·kg-1. The CAF (0.01-100 µg·kg-1, i.p.) of nux vomica that was unprocessed or treated with oil- or vinegar and sand-processing and morphine (1 and 100 µg·kg-1, s.c.) were without effects. In the acetic acid-induced writhing test, the CAF (1 µg·kg-1, i.p.) of nux vomica that was treated with sand-processing significantly inhibited the writhing behavior, while those of nux vomica that was unprocessed or treated with oil- or vinegar and sand-processing and morphine were without effects at a dose of 1 µg·kg-1. The results demonstrate the antinociceptive effects of the CAF of nux vomica and suggest that sand-processing is good for the analgesic potency of nux vomica. It is also suggested that the CAF of nux vomica has distinct antinociceptive potency, even after treatment with licorice-, oil-, vinegar and sand-processing.[48] Drug metabolism studies

Several analytical methods for quantitative

determination of strychnine and brucine have been described, including HPLC with UV detection[49,50], liquid chromatography coupled with mass spectrometry (LC-MS) [51] or tandem mass spectrometry (LC-MS/MS)[52,53], thin layer chromatography (TLC) [54], GC-MS [55,56], 1H nuclear magnetic resonance spectroscopy (1H-NMR) [57] and capillary electrophoresis (CE) [58], etc. These methods were developed mainly for determination of one or both of the two strychnos alkaloids in the plants or medicinal preparations, for analysis of


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strychnine in human urine samples for doping control, or for forensic analysis of human biological fluids. However, few reports are available regarding determination of strychnine and brucine in biological matrix for pharmacokinetic studies of Semen Strychni. A fluorescence spectrophotometric method [59,60] was established for determination of brucine in mouse plasma after i.v. or p.o. administration of pure brucine standard. Another HPLC-UV method [49] was described to investigate pharmacokinetic properties of strychnine and brucine after an i.v. dose of a Semen Strychni preparation to rats. These two methods were not sensitive enough and required complex sample preparation. Furthermore, oral pharmacokinetic studies of strychnine and brucine are indispensable, because most medicinal Semen Strychni preparations are oral formulations. Up to now, no LC-ESI-MS method has been developed for simultaneous determination of strychnine and brucine in biological samples such as plasma for oral pharmacokinetic studies. A simple, sensitive and selective liquid chromatography-electrospray mass spectrometric (LC-ESI-MS) method was developed and validated for simultaneous determination of strychnine and brucine in rat plasma, using tacrine as the internal standard (IS). Sample preparation involved a simple liquid-liquid extraction of the analytes with n-hexane, dichloromethane and isopropanol (65: 30: 5, v/v/v) from 0.1 mL of plasma. Chromatographic separation was carried out on a Waters SymmetryTM

C18 column using a mobile phase of methanol-20 mM ammonium formate-formic acid (32: 68: 0.68, v/v/v) at a flow rate of 0.4 mL·min-1. Positive selected ion monitoring mode was used for detection of strychnine, brucine and the IS at m/z 335.2, m/z 395.2 and m/z 199.2, respectively. Calibration curves were linear over the concentration range of 0.5-500 ng·mL-1 for strychnine and 0.1-100 ng·mL-1 for brucine. The lower limit of quantification was 0.5 ng·mL-1 and 0.1 ng·mL-1 for strychnine and brucine, respectively. The intra- and inter-day precision for both strychnine and brucine was less than 7.74%, and accuracy ranged from -4.38% to 2.21% at all QC levels. Recoveries of both analytes and the IS from rat plasma were greater than 88%. No significant

matrix effect was observed and both analytes were stable during sample storage and preparation procedures. The method has been successfully applied to a pharmacokinetic study of Semen Strychni pulveratum after oral administration to rats (Fig 5).[61]

Fig 5. Mean plasma concentration-time profiles of strychnine and brucine after a single oral dose of 150 mg/kg of Semen Strychni pulveratum to rats. (n = 6, Mean ± S.D.)

The mean maximum plasma concentrations (Cmax) of strychine and brucine detected in the rats were 54.93 ng·mL-1 and 4.95 ng·mL-1, respectively.[61]

A simple, fast and sensitive method for the quantitative determination of strychnine residues in urine has been developed and validated. The method consists of a liquid-liquid extraction step with ethyl acetate at pH 9.2, followed by LC-MS/MS in positive atmospheric pressure chemical ionization (APCI)-mode. The method is linear in the range of 1-100 ng·mL-1 and allows for the determination of strychnine at sub-toxicological concentrations. The accuracy of the method ranged from 1.3% to 4.4%. The method was used to determine the excretion profile of strychnine after the ingestion of an over-the-counter herbal preparation of Strychnos nux-vomica. Each volunteer ingested a dose equivalent to 380 micro g of strychnine. This dose is lower than the prescription dose but results in the detection of strychnine for over 24-h post administration. Maximum detected urinary concentrations ranged from 22.6 to 176 ng·mL-1. The results of this study show that the use of this type of preparation by athletes can lead to a positive doping case.[62]

To study the pharmacokinetic process about the concentration in rat plasma of the alkaloids from processed seeds of Strychnos nux-vomica with

0

1

10

100

0 4 8 12Time (h)

Con

cent

ratio

n (n

g/m

L)

StrychnineBrucine


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RP-HPLC method. Hypersil BDS C18 column was used and the mobile phase consisted of acetonitrile-water at the flow rate of 0.8 mL.min-1. The UV detection wave length was 254 nm. The concentration-time data of strychnine, brucine, strychnine N-oxide and brucine N-oxide were all in accordance with an open two-compartment model after i.v. alkaloids. Their parameters were as follows: T1/2α were (8 ±5), (4 ±3), (6.2 ± 1.7) and (3.0 ±0.8) min, T1/2 β were (262±125), (416±131), (285 ±50) and (342±141) min, CL were (17± 4), (21±12), (1.9±1.8) and (2.8 ±1.1) mL.min-1, Vc were (1.4 ±0.5), (1.7 ±1.1), (0.24±0.16) and (0.23± 0.06) L.kg-1, Vd were (6.0 ±1.2), (12 ±7), (0.8 ±0.6) and (1.5 ± 0.6) L.kg-1, AUC were (57,578±25,578), (35,240± 15,616), (93,088 ±22,375) and (177,712±120,110) h.µg.L-1, respectively. The method is a good reference for pharmacokinetics in human bodies.[64]

A sensitive method for the identification and quantitation of the toxic alkaloids strychnine and brucine from postmortem specimens has been established. After solid-phase extraction using Oasis MCX cartridges the extracts were analyzed by high-performance liquid chromatography with diode-array detection. The limit of detection was 0.5 ng·mL-1 blood for strychnine and brucine, and the limit of quantitation was 5 ng·mL-1 blood for strychnine and brucine. The method was applied for the analysis of blood and gastric contents of a 34-year-old female who died after ingestion of a packet of herbal medicine powder containing the seeds of Strychnos nux-vomica L. Strychnine and brucine were detected in all the samples. The concentration in the case is consistent with that in previous reports.[65]

A quantitative analysis using 1H-NMR (Q-NMR) has been developed for the determination of strychnine and brucine in Strychnos nux-vomica seeds and stems. The advantages of the method are that no reference alkaloids are needed for calibration curves, the quantification could be directly realized on a crude extract, strychnine and brucine could easily be distinguished, an overall profile of the preparation (including non alkaloid compounds) could be directly obtained, and a very significant time-gain could be achieved, in comparison to conventional HPLC methods, for

instance.[66] Strychnine is rapidly absorbed from the

gastrointestinal tract and nasal mucosa as well as from muscle if injected. This alkaloid is very lipophilic and is thus rapidly distributed throughout the body. Most of the agent is detoxified by metabolism through hepatic microsomal enzymes, mostly the cytochrome P450 system, the major metabolite being strychnine N-oxide. The halflife of elimination is about 10 h. A small but apparently variable amount of strychnine is eliminated in urine. [67] Inhibitory effect of CYP450

The present study investigated the inhibition of

brucine and strychnine which are two main active constituents of principal drug Semen strychni in Biqi capsule on five main CYP450 enzymes with the screening method of Gentest recombination CYP450 enzyme inhibitor. At the same time effects of glycyrrhetic acid and glycryyhetinic acid which are the active constituents of messenger drug in Biqi capsule on the active constituents of principal drug concerning CYP450 enzyme activity and the inhibition of Biqi capsule on CYP450 were also investigated in vitro. Their study determined the in vitro inhibitory effects of Biqi capsulae extract, active constituents of Semen strychni (brucine and strychnine), brucine and strychnine combined with active constituents of Radix Glycyrrhiza uralensis (glycyrrhetic acid and glycyrrhetinic acid) on CYP1A2, 2C9, 2C19, 2D6 and 3A4. The results showed that brucine and strychnine had no in vitro inhibitory effect on 5 CYPs. It is possible that groups of brucine and strychnine combined with glycyrrhetic acid and glycyrrhetinic acid had in vitro inhibitory effects on CYP2C9 and CYP2C19. The present empirical method was reliable because five known specific inhibitors all significantly inhibited the activity of corresponding specific enzyme. Brucine and strychnine had almost no inhibition on five enzymes (CYP1A2, 2C9, 2C19, 2D6 and 3A4). Brucine and strychnine mixed with glycyrrhetic acid and glycyrrhetinic acid respectively were possible to have the inhibition on CYP2C9 and CYP2C19, but almost had no inhibition on the other three enzymes. It was


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reported that glycyrrhetic acid was a weak inhibitor of CYP2C9 in vitro, so the inhibition of brucine and strychnine mixed with glycyrrhetic acid and glycyrrhetinic acid respectively on CYP2C9 may be due to glycyrrhetic acid. However, glycyrrhetic acid was hydrolyzed to glycyrrhetinic acid by gastric acid in vivo, absorbed into the blood through small intestine, and finally 18β- glycyrrhetinic acid played an important role in the drug efficacy. But there is no report on the effect of glycyrrhetinic acid on CYP450, it is uncertain that brucine and strychnine mixed with glycyrrhetic acid and glycyrrhetinic acid respectively also have the inhibition on CYP450 in vivo.[68] Metabolomics study

In a study on Metabolomic analysis of Strychnos

nux-vomica, Strychnos icaja and Strychnos ignatii extracts by 1H nuclear magnetic resonance spectrometry and multivariate analysis techniques, [63] 1H-Nuclear magnetic resonance spectrometry and multivariate analysis techniques were applied for the metabolic profiling of three Strychnos species: Strychnos nux-vomica (seeds, stem bark, root bark), Strychnos ignatii (seeds), and Strychnos icaja (leaves, stem bark, root bark, collar bark). The principal component analysis (PCA) of the 1H NMR spectra showed a clear discrimination between all samples, using the three first components. The key compounds responsible for the discrimination were brucine, loganin, fatty acids, and Strychnos icaja alkaloids such as icajine and sungucine. The method was then applied to the classification of several ‘‘false angostura’’ samples. These samples were, as expected, identified as S. nux-vomica by PCA, but could not be clearly discriminated as root bark or stem bark samples after further statistical analysis.

Fig 6. Score plot of principal component analysis of Strychnos extracts obtained by covariance (a) and correlation (b) method using PC1 and PC2. The ellipse represents the Hotelling T2 with 95% confidence in score plots. (1) Stem barks of S. nux-vomica, (2) seeds of S. nux-vomica, (3) roots of S. nux-vomica, (4) stem barks of S. icaja, (5) root barks of S. icaja, (6) collars of S. icaja, (7) leaves of S. icaja, (8) seeds of S. ignatii. [63]

In this study, both methods were evaluated and

the covariance method showed better separation results (Fig. 6). The seeds of S. nux-vomica are clearly separated from other samples by PC1 (Fig. 6(a)). The seeds of S. nuxvomica are located in the higher PC1 region. Lipids play an important role in this discrimination. The discriminating metabolites are distinguishably shown in loading plot of PC1 and PC2 (Fig. 7).

For more separation, PC3 was additionally used (Fig 8). In this case, the root extracts of S. nux-vomica are obviously separated from other samples by PC3 (Fig. 4a) but the differentiating metabolite was identified by loading plot of PC3 and found to be brucine almost alone (Fig. 4b). Two positive distinguishable 1H NMR signals in the loading plot of PC3 were identified as H-12 and H-9 of brucine, respectively. Their study showed that it was possible to discriminate three different Strychnos species from various origins by multivariate analysis of 1H NMR spectra of crude extracts. Seeds of S. ignatii and S. nux-vomica are easily distinguishable. S. icaja and S. nux-vomica stem barks are much more similar, but could be distinguished using the brucine content (mostly influencing PC3).


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Fig 7. Loading plot of principal component analysis of Strychnos extracts obtained by covariance method. (a) PC1, (b) PC2.[63]

Their study then showed that the major compounds responsible for the discrimination were brucine, fatty acids, loganin and several S. icaja alkaloids (mainly icajine and sungucine). Strychnine, though present in various amounts in all extracts analysed, was not the key compound for the discrimination of samples. Toxicity

Toxicity of Strychnos species differs according to

the part used of the plant and is mainly related to the presence of strychnine. The fruit pulp of different African and Asian species, and among them S. nux vomica and lots of African species, is eaten by animals as well as by human beings, but seeds are often poisonous. Animals have been seen feeding or browsing on different species; this is presumably an indication of the presence of little or no alkaloid, or at least of relatively nontoxic alkaloids. In India, cows are known to graze the leaves of S. nux vomica.[69,70] Roots, the known site of biosynthesis of strychnine, have the richest content in alkaloids and have a greater pharmacological activity than other plant parts in the Strychnos species.

Fig 8. Score plot of principal component analysis of Strychnos extracts obtained by covariance method using PC1 and PC3 (a) and loading plot of PC3 (b). The ellipse represents the Hotelling T2 with 95% confidence in score plots. (1) Stem barks of S. nux-vomica, (2) seeds of S. nux-vomica, (3) roots of S. nux-vomica, (4) stem barks of S. icaja, (5) root barks of S. icaja, (6) collars of S. icaja, (7) leaves of S. icaja, (8) seeds of S. ignatii. [63]

Table 1. Mean LD50 of some dimeric and other Strychnos alkaloids in the mouse iv injection

Alkaloids LD100 mg.kg-1

Duration of Paralysisa (min)

Symmetric dimeric alkaloids

Toxiferine 2.3 12 Dihydrotoxiferine chloride 60 5.5 C-Alkaloid H chloride 24 3.7 Curarine chloride 50 4 C-Alkaloid G chloride 1-8 7 C-Alkaloid E chloride 7-12 18 Calebassine chloride 320 3 C-Alkaloid F chloride 120 1.3 C-Alkaloid A chloride 150 2 (t)-Tubocurarine 120 Assymetric dimeric alkaloids

Guiaflavine 3000 Guiachrysine 5000 Monomeric base Fluorocurine iodide 8500

a Dose about 30% inferior to DL50. The toxicities (LD50, i.v., mouse) for the main natural

curarizing alkaloids are reported in Table 1.[71] The whole seed extract of Strychnos nux-vomica

(in low doses) effectively neutralized Daboia


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russelii venom induced lethal, haemorrhage, defibrinogenating, PLA2 enzyme activity and Naja kaouthia venom induced lethal, cardiotoxic, neurotoxic, PLA2 enzyme activity. The seed extract potentiated polyvalent snake venom antiserum action in experimental animals. An active compound (SNVNF) was isolated and purified by thin layer chromatography and silica gel column chromatography, which effectively antagonised D. russelii venom induced lethal, haemorrhagic, defibrinogenating, oedema, PLA2 enzyme activity and N. kaouthia induced lethal, cardiotoxic, neurotoxic, PLA, enzyme activity. Polyvalent snake venom antiserum action was significantly potentiated by the active compound. Spectral studies revealed it to be a small, straight chain compound containing methyl and amide radicals. Detailed structure elucidation of the compound (SNVNF) is warranted before its clinical trials as a snake venom antagonist. [72]

The primary biomolecular target of a homeopathic potency is unknown. If it is a plasma membrane protein such as water-channel protein, the drug would alter water permeation in cells. Therefore, the objective is to see if potentized homeopathic drugs like Mercuric chloride 30c and Nux vomica 30c could alter permeation of water through the erythrocytes of a fresh water fish under acute ethanol intoxication. Red blood cells (RBCs) from ethanol-injected fish permeated more water than those from normal fish. Water permeation was enhanced with Merc cor 30c and Nux vom 30c. RBCs from fish pretreated with Nux vom 30c imbibed more water in in vitro treatments than those from fish pretreated with Ethanol 30c. Because water channel proteins or aquaporins are mainly responsible for water transport through the plasma membrane of RBCs, it is thought that potentized drugs interact with these proteins, thereby facilitating water influx in the cells.[73]

Clinical studies

The dried ripe seed of Strychnos nux-vomica L

('Maqianzi' in Chinese). contains 1.0-1.4% each of strychnine and brucine. After processing to reduce its toxicity, 'Maqianzi' was used as herbal remedy for rheumatism, musculoskeletal injuries and limb

paralysis. A 42-year old woman with neck pain was prescribed 15 g of 'Maqianzi' to be taken in two doses at 7 hours apart, although the recommended dose was 0.3-0.6 g. She was apparently well after drinking the first of two bowls of 'Maqianzi' decoction. One hour after she drank the second bowl of herbal decoction, she suddenly developed tonic contractions of all her limb muscles and carpopedal spasm lasting 5 min, difficulty in breathing, chest discomfort and perioral numbness. The second bowl of decoction probably became more concentrated because of evaporation of water during continued boiling and contained a larger amount of 'maqianzi'. On arrival in the hospital 1 hour later, she complained of muscle pain and tiredness. She was found to have hyperventilation and weakness of four limbs, with muscle power of grade 5(-)/5. All her symptoms gradually subsided over the next few hours. This case illustrated that 'maqianzi' can cause strychnine poisoning even after processing, especially when the recommended dose is greatly exceeded. In any patient with 'unexplained' muscle spasms or convulsions, strychnine poisoning should be included in the differential diagnosis and they should be asked about the use of herbal medicines.[74]

The effect of Asa foetida (0.1% alcoholic dilution) and a combination of Asa foetida and Nux vomica (0.01% alcoholic dilution) was tested in a multicenter placebo-controlled double-blind trial. Both groups with active medication differed trivially but showed a marked superiority to the placebo group. The differences, however, just miss the conventional 5%-level of statistical significance. For the interpretation of this result it must be considered that only about half of the total of patients could be reached who in the planning phase of the study had been regarded necessary for a statistical confirmation of a therapeutic effect. The diagnosis "Colon irritabile" is discussed in its historical context and the psychological structure of the patient sample is described by means of the scores of a standard personal inventory test (FPI). The trial shows that it is possible to obtain objective results on the basis of the patients' judgment (rating scales with low information content).[75] Strychnine is an alkaloid which is mainly extracted from the seeds of Strychnos nux vomica, a tree native to India. This


198

compound is highly toxic to humans. It is rapidly absorbed from the gastrointestinal tract and acts upon the central nervous system (CNS), causing excitation of all parts of the CNS. Strychnine poisoning was

quite frequent in the last century and in the first half of this century. Table 2 shows the strychnine distribution in biological samples from published investigations of fatalities.[75]

Table 2. Published data on strychnine tissue distribution from fatal cases (mg/ml or mg/g) [75]

Blood Liver Kidney Lung Urine Small Gastric

intestine contents

or n or n or n or n or n or n or n

range range range range range range range

1979 0–61 5 0–299 8 0.07–90 6 – – 1.0–7.7 3 – – 1–480 6

1982 0.5–61 – 5–257 – 0.07–

106

– – – 1–3 – – – 7.5–

100

–

1985 2.3–9.7 2 18.5–

80.4

2 12.9–32.

0

2 – – 2.0 1 39.0–

109.0

2 160.0–

1030.0

2

1985 3.3 1 6.2 1 3.2 1 – – 1.4 1 4.1 1 213.5 1

175.0

1986 3.30 1 6.20 1 3.20 1 – – 1.40 1 4.10 – 175.00 1

1989 1.0–20.3 68 1 – – – 6.9 1 1.0–3.2 – – – – –

1994 – – – – 6016 1 – – – – – – – –

1995 7.7 1 515 1 46 1 – – 3.2 1 – – 3000.0 1

In the fatal case described in this paper there was

no family or clinical information concerning to the cause of death. The autopsy result was not specific, although the pathological findings agree with those in published fatalities from acute poisonings which do not mention a specific pathology. Strychnine poisoning was only diagnosed from the results of the toxicological analysis performed in the FTL. The clinical picture of strychnine poisoning can be confused with epilepsy, tetanus and hysteria, but the clinicians strongly suspected strychnine poisoning. In the clinical case, the patient showed typical symptoms of strychnine poisoning. The clinical picture of strychnine poisoning can be confused with epilepsy, tetanus and hysteria, but the clinicians strongly suspected strychnine poisoning.[76]

Acknowledgements This review paper was

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