Isolation and characterisation of strychnine and brucine from strychnos nux vomica ABSTRACT:

Strychnos is a medicinally important belonging to the family Loganiaceae consisting of about 394 species, which were widely distributed in tropical and subtropical India. The main goal is being presented for the separation and detection of strychnine and brucine, alkaloids of Strychnos nux vomica A new gas chromatography-mass spectroscopy (GCMS) method is being presented for the separation and detection of strychnine and brucine, alkaloids of Strychnos nux vomica in a single run. The analysis was carried out using 5% phenyl methyl silicone capillary column, electron impact ionization mode and quadrupole mass analyzer. The extracts of the exhibits were analyzed using the new method. The peaks of the two alkaloids were found to be well resolved, and there was clear separation between the two. The retention time and mass fragmentation pattern, base peaks, molecular peaks of strychnine and brucine standard/NIST library and crime case exhibits matched, establishing the presence of the two active principles of Strychnos nux vomica. The new method has the advantage of better separation of the two alkaloid peaks over the conventional GC-MS methods, and is useful for the identification and confirmation of Strychnos nux vomica constituents in biological matrices of poisoning cases that have ended in death. Keywords: Strychnine; Brucine; Strychnos nux vomica; Gas chromatographymass spectroscopy; GC-MS

Introduction:
India is richly endowed with a wide variety of plants having medicinal value. These plants are widely used by all sections of the society either directly as folk remedies or indirectly as pharmaceutical preparation of modern medicine. Its medicinal usage has been reported in the traditional systems of medicine such as Ayurvedha, Siddha and Unani. Strychnos has been described as a rasayana herb and has been used extensively for various medicinal purposes The genus Strychnos, the largest genus of the family Loganiaceae, was first described by Linnaeus on the basis of S. nux-uomica, the type species, and S. colubrine (S. minor). It is pantropical and comprises about 200 species, which may be subdivided into three geographically separated groups: one in Africa with 75 species; one in America with 73 species; and one in Asia (including Australia) with 44 species. The only exception is S. potatorum which is found both in

Africa and Asia. Strychnos nux vomica grows throughout tropical India up to an altitude of 1360 meters.It is an evergreen or deciduous tree. The leaves are glabrous, 8-15 cm. long, broadly elliptic obtuse or acute, entire and with prominent central nerves. The flowers are greenish white in terminal compound cymes. Corolla tube is cylindrical. The Style is filiform and stigma is undivided. The berries are globose. Seeds are discoid (coin-like), covered with fine and silky hair, embedded in white, bitter pulp.The tree that yields this drug isof moderate size, and is indigenous to tropical India and adjacent islands. Its nearest relative in the United States is Spigelia Marilandica,the little plant which yields Pink-root. The wood of Nux Vomica is close-grained and bitter, and from a very early date has been used as a tonic by the natives of its habitat, by whom it is still employed in this manner. Under the old name Lignum Columbrinum,the wood of this and related trees is valued by the natives of India as an antidote to the bite of the venemous serpent, cobra,The trunk is short, thick, crooked. The branches are ash colored,the young shoots highly polished, The fruit is about the size of a small orange, yellow and acidulous,It contains a white edible pulp,which is eaten by children and many birds. In this pulp are imbedded,irregularly and vertically from one to five disc-like seeds, which are intensely poisonous in character.

FRUIT OF STRYCHNOS NUX VOMICA

TRANSVERSE SECTION OF FRUIT AND SEEDS OF STRYCHNOS NUX VOMICA

These button-like seeds (not nuts) are aboutan inch in diameter by one-fourth inch thick, the center portion being usually depressed. They are mouse colored or greenish, and lustrous by reason of a silky covering of adpressed glistening hairs. They are albuminous, very hard, tough, horny, and extremely bitter. They are not easily powdered, unless previously softened by steam. Maceration in water, especially hot water,softens the albumen, after which transverse slicing of the seed displays a mass of cartilaginous albumen, divided into two bean-like parts, between which lies the germ. These seeds are exported in immense quantities, thousands of tons being respectively consigned to England, the Continent, and America. From Nux Vomica is made the bulk of the Strychnine and Brucine of commerce, although St. Ignatius Bean yields a greater proportion of each alkaloid. In this connection it may be said that it has been

erroneously reported that Nux Vomica is a medicinal product of the Philippines. The Philippine tree is, however, St. Ignatius. The dominating constituent of Nux Vomica is a complex compound which, in natural form, is an invaluable remedy. By means of chemical reagents it can be split into parts,embracing two intensely poisonous alkaloidal products, a glucosid,and acids. Study isolated 13 alkaloids from the seeds: strychnine, betacolubrine, pseudostrychnine, strychnine N-oxide, brucime, brucine N-oxide, novacine, icajine, vomicine, isostrychnine, isobrucine, isobrucine N-oxide and isostrychnine N-oxide. The main acid is Igasuric Acid, while the glucosid is named Loganin. These are all colorless bodies, the alkaloids being very bitter, and energetically poisonous, Brucine being a poison similar to Strychnine, acting with less violence and more slowly, but not less surely, than Strychnine. Strychnine(C21H22N2O2) This violently poisonous alkaloidis crystaline, slightly soluble in cold water , the solute on being alkaline and bitter. It dissolves in 7 parts of chloroform and 150 parts of 90% alcohol. Strychnine is a terrible tetanic poison, affecting the cerebro spinal system, but it kills without producing marked anatomical change, the muscles and nerves being scarcely altered, although the brain and spinal cord may be congested, stomach and limbs intensely congested, right side of heart gorged , and the lungs congested, The fatal dose of Strychnine is as low as 4 grain. Brucine,This related alkaloid is also a product of chemical action on Nux Vomica. It is known to chemists as dimethoxylstrychnine (C23H26N204). Brucine is very bitter, freely soluble in cold alcohol, soluble (U. S. P.) in 850 parts of cold water. It differs in reaction from Strychnine in that strong sulphuric or nitric acid strikes with it a blood-red color, whereas with Strychnine no coloration appears. If to a hydrochloric acid solution of Brucine and Strychnine, potassium ferrocyanide solution be added, the Brucine precipitates, while the Strychnine remains dissolved, The mixed alkaloids can be quite closely separated by alcohol 0.97 sp. g., which freely dissolves Brucine, but scarcely dissolves Strychnine, Brucine is a poison which has the physiological qualities of Strychnine, but in a markedly less degree. It is absorbed much slower, and although more energetic as concerns the sensory nerves, is decidedly less dangerous than Strychnine. The antidotes and treatment for poisoning by Brucine are the same as for Strychnine. Mixtures of Nux Vomica were used as gastrointestinal stimulants, increasing appetite by stimulation of peristalsis. Strychnine also increase the flow of gastric juice. Alternative medicine systems have suggested strychnos for a wide range of medical problems: liver cancer, various gastrointestinal complaints (abdominal pain, vomiting, constipation, intestinal

rritation,heartburn),insomnia, nervous conditions, menopausal problems. In Chinese medicine, used for liver cancer treatment. Powdered seeds used for atonic dyspepsia. Seeds used in rheumatism, paralysis, asthma, diabetes, piles. Although widely used in medicine before World War II, there are no present uses in modern western medicine. It is a deadly poison with a lethal dose of about 30 to 120 mg in humans. Doses of 5 mg or more (as little as one seed) can cause anxiety, restlessness, painful convulsions, respiratory difficulty and even death from suffocation or exhaustion. In case of poisoning, a stomach pump should be used immediately and potassium permanganate given to inactivate strychnine. There is no specific antidote. Treatment is supportive. Convulsions can be controlled by anesthetics. Strychnine is a deadly poison with a lethal dose to humans of about 30 to 120 mg. Survival of substantially higher doses has been reported. The most direct symptom caused by strychnine is violent convulsions due to a simultaneous stimulation of the motor or sensory ganglia of the spinal cord. During the convulsions there is a rise in blood pressure. Brucine closely resembles strychnine in its action, but is slightly less poisonous as it only causes paralysis of the peripheral motor nerves. Strychnos nux-vomica has shown to suppress allergen-specific Immunoglobulin E (IgE) antibody response in mice. Suggesting its possible application in allergic conditions.In vitro Strychnos nux-vomica inhibited the growth of AGS human gastric carcinoma cells.

REVIEW OF LITERATURE: Literature reveals that Thirteen alkaloids were isolated from the seeds of Strychnos nux-vomica. They were identified as strychnine, beta-colubrine, pseudostrychnine, strychnine N-oxide, brucime, brucine N-oxide, novacine, icajine, vomicine, isostrychnine, isobrucine, isobrucine N-oxide and isostrychnine N-oxide by chemical and spectroscopic analysis. 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 cyclooxygenase-2 and caspase-3 in the pro-apoptotic effects exerted by brucine. Therefore, this paper indicate 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. Strychons nuxvomica is widely used by clinic and individual owing to its officinal value. Since toxic dose and therapeutic dose are very close, the poisoning cases are reported frequently. In this review, based on the recent available papers published in the PubMed and CNKI about Strychons nuxvomica, a traditional Chinese herbal medicine, we present the major current approaches in the field of composition, toxicology, pharmacokinetics, decreasing toxicity and increasing efficacy, in order to guide the use in the clinic. it has been investigated that strychnine, the major active principle in the alcoholic extract of the seeds of Strychnos nux-vomica, 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. A quantitative analysis using (1)H-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.

This report investigates the antioxidant property of Strychnos nux vomica Linn, alcohol extract on FeS04-induced lipid peroxidation in vitro. The results have been compared to those of vitamin E, parabenzoquinone, Tris, mannitol and EDTA. Tris and mannitol, the standard hydroxyl trappers failed to block this process. Interestingly EDTA, a strong metal chelator, significantly blocks the process of lipid peroxidation. S. nux vomica inhibits the lipid peroxidation in the dose dependent manner. The inhibition of lipid peroxidation was reversed by adding high concentration of Fe2+. This suggests the mechanism of action of S. nux vomica through the chelation of Fe++/Fe+++ ion in the system and not by trapping the hydroxyl radicals. This extract significantly maintains the hepatic glutathione content also in the time and dose dependent manner even in the presence of FeSO4, which is not the case with EDTA. It also inhibits the process of lipid peroxidation once induced. Strychnos nux-vomica L. (Loganiaceae), widely used in Chinese folk medicine, is grown extensively in southern Asian countries. The dried seeds of this plant have been claimed to improve blood circulation and relieve rheumatic pain. Historically, this plant has been widely used in treating diseases, such as tumor and rheumatic arthritis. Phytochemical analysis has identified alkaloids as the major components of this species. A few related studies also show that these alkaloids are, to a great extent, responsible for the pharmacological properties of Strychnos nux-vomica, such as cytoprotective and antitussive activities. However, the toxic properties of these alkaloids have limited the wider use and the investigation of this plant as a medicine.In order to reveal the chemical foundation for pharmacological activity of Strychnos nux-vomica L.,22 compounds were isolated from the antitumor active fraction of Strychnos nuxvomica L. by means of silica gel column chromatography, ODS column chromatography, Sephadex LH-20, PHPLC. On the basis of physico-chemical characters and spectroscopic analysis, their structures were identified as:brucine (1), strychnine (2), brucine-N-oxide (3), strychnine-N-oxide (4), strychnine methiodide (5), pseudobrucine (6), pseudostrychnine (7), icajine (8), vomicine (9), novacine (10), ferulic acid (11), caffeic acid ethyl ester (12), cinnamic acid (13), protocatechuic acid (14), salicylic acid (15), gallic acid (16), vanillic acid (17), ursolic acid (18),-simiarenol (19),-sitosterol (20), daucosterol (21). Among them, compounds 11-17 were obtained from this genus for the first time.The cytotoxicity in vitro of some isolated compounds towards human lung cancer cell lines was tested. Compounds 4,5,6,8 and 18 showed inhibitory activity towards human lung cell line A549.

MATERIALS AND METHOD: