An efficient process for the extraction of lutein and chemical characterization of other organic volatiles from marigold (Tagetes erecta L.) flower.

Marigold (Tagetes erecta L.) petals are the primary industrial source of lutein, which is used as a colouring agent and nutrient supplement to foods. This research extracted marigold petals using different solvents, covering conventional and non-toxic green solvents. The oleoresin, free lutein, and recrystallized lutein yields varied from 8.47-16.67%, 2.56-9.62%, and 1.11-1.61%, respectively. The purity of lutein was achieved up to 92.57% and 97.64% in conventional and newly established green methods, respectively. The present study described an efficient green process to isolate lutein with significantly improved yield (2.56%) and purity (97.33%) over the conventional methods. Based on the results, 2-methyltetrahydrofurancould be a practical green alternative to the traditional toxic solvents for the processing of lutein. Further, the chemical analysis of the essential oil of the residual receptacles obtained after removing petals revealed the presence of important organic volatiles, including piperitone (54.7%) and piperitenone oxide (6.5%), indicating its usefulness for value-addition.

Decellularization of caprine esophagus using fruit pericarp extract of Sapindus mukorossi.

Biological detergents like sodium deoxycholate, sodium dodecyl sulphate and Triton X-100 impairs the collagenous and non-collagenous proteins, glycosaminoglycans and growth factors. Further, certain chemical and enzymes are responsible for residual cytotoxicity in the decellularized extracellular matrix. The main focus of this study was to explore the decellularization property of soap nut pericarp extract (SPE) for development of decellularized tubular esophageal scaffold. For this 2.5, 5.0 and 10% concentrations of SPE were used for decellularization of caprine esophageal tissues. Histological analysis of hematoxylin and eosin and Masson's trichrome stained tissue samples confirmed decellularization with preservation of extracellular matrix microarchitecture. Scanning electron microscopic images of luminal surface of decellularized esophageal matrix showed randomly oriented collagen fibres with large interconnected pores and cells were absent. However, the external surface was more textured with fibrous structures and collagen fibres were well preserved. DAPI stained decellularized tissues revealed complete removal of nuclear components, verified by DNA content measurement and SDS-PAGE. The FTIR spectra of decellularized esophagus shows absorption peaks of amide A, B, I, II and III. Elastic modulus of the decellularized esophagus scaffolds increased (P > 0.05) as compared to native tissues. Histological and scanning electron microscopic evaluation of in vitro seeded scaffolds showed attachment and growth of primary chicken embryo fibroblasts over and within the decellularized scaffolds. It was concluded that 5% SPE is ideal for preparation of cytocompatible decellularized caprine esophageal scaffold with well-preserved extracellular matrix architecture and, may be used as an alternative to biological detergents and other chemicals.

A Significant Role of Chemistry in Drug Development: A Systematic Journey from Traditional to Modern Approaches with Anti-HIV/AIDS Drugs as Examples.

BACKGROUND: Traditionally, various plant extracts having interesting biological properties were the main source of new drugs. In the last 30 years, the role of chemistry in combination with new technologies, like various computational techniques in chemistry, has witnessed a major upsurge in drug discovery and targeted drug delivery. OBJECTIVE: This article provides a succinct overview of recent techniques of chemistry that have a great impact on the drug development process in general and also against HIV/AIDS. It focuses on new methods employed for drug development with an emphasis on in silico studies, including identifying drug targets, especially the proteins associated with specific diseases. METHODS: The rational drug development process starts with the identification of a drug target as the first phase, which helps in the computer-assisted design of new drug molecules. Synthetic chemistry has a major impact on the drug development process because it provides new molecules for future study. Natural products based semisynthesis or microwave assisted synthesis is also involved in developing newly designed drug molecules. Further, the role of analytical chemistry involves extraction, fractionation, isolation and characterization of newly synthesized molecules. RESULTS: Chemistry plays a key role in drug discovery and delivery by natural process or with the help of synthetic nanoparticles or nanomedicines. So, nanochemistry is also deeply involved in the development of new drugs and their applications. CONCLUSION: The previous era of drug discovery was dominated only by chemistry, but the modern approaches involve a comprehensive knowledge of synthetic chemistry, medicinal chemistry, computational chemistry and the concerned biological phenomenon.

A rapid and non-destructive ATR-FTIR spectroscopy method supported by chemometrics for discriminating between facial creams and the classification into herbal and non-herbal brands.

Facial creams are considered to be essential beauty items and are used by both females and males on an everyday basis. These can be encountered as an evidentiary material in criminal investigations, particularly in cases related to sexual and physical assaults against women. These are found in trace amounts and therefore their analysis is difficult and also, it must be through non-destructive methods. In the present work ATR-FTIR spectroscopy was employed for the discrimination of 57 samples of face creams out of which 31 were non-herbal and 26 were from herbal category. Visual analysis of the obtained Spectra was done for discrimination purposes but the method was prone to human error and laborious too. The spectroscopic results were analyzed with PCA (Principal Component Analysis) and PLS-DA (Partial least square discriminant analysis) methods. A segregation of samples was seen in the PCA plots to some extent. The class separation and prediction of the samples was performed using PLS-DA method. A good classification was achieved between herbal and non-herbal samples using PLS-DA method. Further, validation of the model was also performed by testing 10 unknown samples.

Neuroprotective and Neurorescue Mode of Action of Bacopa monnieri (L.) Wettst in 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-Induced Parkinson's Disease: An In Silico and In Vivo Study.

Ethnopharmacological Relevance: Parkinson's disease (PD) is characterized by progressive death of dopaminergic neurons. The presently used medicines only tackle the symptoms of PD, but none makes a dent on the processes that underpin the disease's development. Herbal medicines have attracted considerable attention in recent years. Bacopa monnieri (L.) Wettst (Brahmi) has been used in Indian Ayurvedic medicine to enhance memory and intelligence. Herein, we assessed the neuroprotective role of Bacopa monnieri (L.) Wettst on Parkinson's disease. Aim of the Study: Bacopa monnieri (L.) Wettst, a medicinal herb, is widely used as a brain tonic. We investigated the neuroprotective and neurorescue properties of Bacopa monnieri (L.) Wettst extract (BME) in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mice model of PD. Materials and Methods: The mice model of MPTP-induced PD is used in the study. In the neuroprotective (BME + MPTP) and neurorescue (MPTP + BME) experiments, the animals were administered 40 mg/kg body weight BME orally before and after MPTP administration, respectively. Effect of BME treatment was evaluated by accessing neurobehavioral parameters and levels of dopamine, glutathione, lipid peroxide, and nitrites. An in silico study was performed using AutoDock Tools 1.5.6 (ADT). Results: A significant recovery in behavioral parameters, dopamine level, glutathione level, lipid peroxides, and nitrite level was observed in BME-treated mice. Treatment with BME before or after MPTP administration has a protective effect on dopaminergic neurons, as evidenced by a significant decrease in GFAP immunostaining and expression of inducible nitric oxide synthase (iNOS) in the substantia nigra region; however, the degree of improvement was more prominent in mice receiving BME treatment before MPTP administration. Moreover, the in silico study revealed that the constituents of BM, including bacosides, bacopasides, and bacosaponins, can inactivate the enzyme monoamine oxidase B, thus preventing the breakdown of MPTP to MPP+. Conclusion: Our results showed that BME exerts both neuroprotective and neurorescue effects against MPTP-induced degeneration of the nigrostriatal dopaminergic neurons. Moreover, BME may slow down the disease progression and delay the onset of neurodegeneration in PD.

Development of decellularized aortic scaffold for regenerative medicine using Sapindus mukorossi fruit pericarp extract.

The aim of this study is to develop a novel decellularization method using aqueous extract of soap nut pericarp (SPE) and its evaluation using hematoxylin-eosin staining, scanning electron microscopy, diamidino-2-phenylindol (DAPI) staining, mechanical testing, sodium dodecyl sulfate polyacrylamide gel electrophoresis and DNA quantification. The presently available decellularization agent raises some concerns due to the potential for presence of residual cytotoxic agents in the extracellular matrix. Histological analysis of hematoxylin and eosin and masson's trichrome stained processed aortic samples shows complete decellularization with preservation of extracellular matrix microarchitecture at 120 h. Further, staining of tissue samples with DAPI demonstrates complete removal of DNA fragments. Quantitative evaluation of DNA in the decellularized aorta tissues demonstrated a significant (P < 0.01) decrease in DNA content as compared to native tissues. Collagen quantification assay indicate no significant (P> 0.05) difference in its content between native and decellularized caprine aorta. Tensile strength of the decellularized scaffolds decreased non-significantly (P > 0.05) when compared to native tissues. There was no significant (P > 0.05) difference in young's modulus of elasticity, stiffness and stretch ratio between native aortic tissues and decellularized aortic scaffolds. Histological and scanning electron microscopic examination of in vitro cultured scaffold demonstrated the cell viability and proliferation of primary chicken embryo fibroblasts. SPE treatment is thus capable of producing cytocompatible decellularized caprine aorta scaffold with preservation of extracellular matrix architecture for vascular tissue engineering and could be applied widely as one of the decellularization agent.

Chemical composition and antibacterial, antifungal, allelopathic and acetylcholinesterase inhibitory activities of cassumunar-ginger.

BACKGROUND: Zingiber montanum (J.Koenig) Link ex A.Dietr. (Zingiberaceae), commonly known as cassumunar-ginger, is a folk remedy for the treatment of inflammations, sprains, rheumatism and asthma. The aim of the present study was to assess the chemical composition, and antibacterial, antifungal, allelopathic and acetylcholinesterase inhibitory activities of the essential oil of Z. montanum originating from India. RESULTS: The hydrodistilled essential oil of Z. montanum rhizome was analyzed using gas chromatography-flame ionization detection and gas chromatography-mass spectrometry. A total of 49 constituents, forming 98.7-99.9% of the total oil compositions, was identified. The essential oil was characterized by higher amount of monoterpene hydrocarbons (32.6-43.5%), phenylbutanoids (27.5-41.2%) and oxygenated monoterpenes (11.4-34.1%). Major constituents of the oil were sabinene (13.5-38.0%), (E)-1-(3',4'-dimethoxyphenyl)buta-1,3-diene (DMPBD) (20.6-35.3%), terpinen-4-ol (9.0-31.3%), γ-terpinene (1.1-4.8%) and ß-phellandrene (1.0-4.4%). The oil was evaluated against eight pathogenic bacteria and two fungal strains. It exhibited low to good antibacterial activity (minimum inhibitory concentration: 125-500 µg mL-1 ) and moderate antifungal activity (250 µg mL-1 ) against the tested strains. The oil reduced germination (69.8%) and inhibited the root and shoot growth of lettuce significantly (LD50 : 3.58 µL plate-1 ). However, it did not demonstrate acetylcholinesterase inhibitory activity up to a concentration of 10 mg mL-1 . CONCLUSIONS: The essential oil of Z. montanum can be used as a potential source of DMPBD, terpinen-4-ol and sabinene for pharmaceutical products. The results of the present study add significant information to the pharmacological activity of Z. montanum native to India. © 2017 Society of Chemical Industry.

Role of ethanolic extract of Bacopa monnieri against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced mice model via inhibition of apoptotic pathways of dopaminergic neurons.

Parkinson's disease (PD) is a neurodegenerative disease which causes rigidity, resting tremor and postural instability. The neuroprotective effects of an ethanolic extract of Bacopa monnieri (BM) were evaluated in a Parkinsonian mice model induced by the MPTP. The present study investigates the mechanisms of neuroprotection elicited by BM, an herb traditionally recognized by the Indian system of medicine, Ayurveda. An ethanolic extract of BM was co-treated with the MPTP induced mouse model of PD and was shown to significantly rescue the motor behaviour (Rotarod, Grip Strength and Foot Printing test). Furthermore, on biochemical parameters too BM significantly showed protective effect as Catalase, LPO, Nitrite, SOD, GR, GPx parameters showed marked improvement and levels of Dopamine, DOPAC and HVA were enhanced significantly. There was a significant reduction in tyrosine hydroxylase (TH) immunoreactivity in the substantia nigra (SN) in MPTP treated group, which was considerably restored by the use of BM extract. BM also facilitated neuroprotection by creating an anti-apoptotic environment indicated by reduced apoptotic (Bax and caspase-3) and increased levels of anti-apoptotic (Bcl2) protein expression, respectively. Altogether, the present study suggests that BM treatment provides nigrostriatal dopaminergic neuroprotection against MPTP induced Parkinsonism by the modulation of oxidative stress and apoptotic machinery possibly accounting for the behavioural effects.

Wernicke’s encephalopathy associated with liver abscess.

Wernicke's encephalopathy is a rare neurological disorder caused by thiamine deficiency, characterised by ocular motor dysfunction, ataxia and impairment in consciousness. It predominantly affects brain regions with a high metabolic rate such as mammillary bodies, medial thalamic nuclei, the tectal region and the cerebellum. Although chronic alcoholism is the most common cause of Wernicke's encephalopathy, various other conditions not related to alcohol consumption such as bariatric surgery, acute pancreatitis, hyperemesis gravidarum, prolonged fasting and gastrointestinal surgery have been implicated in its aetiology. We report the case of a patient who underwent surgery for liver abscess and subsequently developed Wernicke's encephalopathy; he showed a positive response to thiamine supplementation. This is the first report describing liver abscess as the cause of Wernicke's encephalopathy.

Chemical Composition and Allelopathic, Antibacterial, Antifungal, and Antiacetylcholinesterase Activity of Fish-mint (Houttuynia cordataThunb.) from India.

Fish-mint (Houttuynia cordataThunb.), belonging to family Saururaceae, has long been used as food and traditional herbal medicine. The present study was framed to assess the changes occurring in the essential-oil composition of H. cordata during annual growth and to evaluate allelopathic, antibacterial, antifungal, and antiacetylcholinesterase activities. The essential-oil content ranged from 0.06 - 0.14% and 0.08 - 0.16% in aerial parts and underground stem, respectively. The essential oils were analysed by GC-FID, GC/MS, and NMR (1 H and 13 C). Major constituents of aerial-parts oil was 2-undecanone (19.4 - 56.3%), myrcene (2.6 - 44.3%), ethyl decanoate (0.0 - 10.6%), ethyl dodecanoate (1.1 - 8.6%), 2-tridecanone (0.5 - 8.3%), and decanal (1.1 - 6.9%). However, major constituents of underground-stem oil were 2-undecanone (29.5 - 42.3%), myrcene (14.4 - 20.8%), sabinene (6.0 - 11.1%), 2-tridecanone (1.8 - 10.5%), ß-pinene (5.3 - 10.0%), and ethyl dodecanoate (0.8 - 7.3%). Cluster analysis revealed that essential-oil composition varied substantially due to the plant parts and season of collection. The oils exhibited significant allelopathic (inhibition: 77.8 - 88.8%; LD50 : 2.45 - 3.05 µl/plate), antibacterial (MIC: 0.52 - 2.08 µl/ml; MBC: bacteriostatic) and antifungal (MIC: 2.08 - 33.33 µl/ml; MFC: 4.16 - 33.33 µl/ml) activities. The results indicate that the essential oil from H. cordata has a significant potential to allow future exploration and exploitation as a natural antimicrobial and allelopathic agent.

Nitrogen treatment enhances sterols and withaferin A through transcriptional activation of jasmonate pathway, WRKY transcription factors, and biosynthesis genes in Withania somnifera (L.) Dunal.

The medicinal plant Withania somnifera is researched extensively to increase the quantity of withanolides and specifically withaferin A, which finds implications in many pharmacological activities. Due to insufficient knowledge on biosynthesis and unacceptability of transgenic approach, it is preferred to follow alternative physiological methods to increase the yield of withanolides. Prior use of elicitors like salicylic acid, methyl jasmonate, fungal extracts, and even mechanical wounding have shown to increase the withanolide biosynthesis with limited success; however, the commercial viability and logistics of application are debatable. In this investigation, we tested the simple nitrogeneous fertilizers pertaining to the enhancement of withaferin A biosynthesis. Application of ammonium sulfate improved the sterol contents required for the withanolide biosynthesis and correlated to higher expression of pathway genes like FPPS, SMT1, SMT2, SMO1, SMO2, and ODM. Increased expression of a gene homologous to allene oxide cyclase, crucial in jasmonic acid biosynthetic pathway, suggested the involvement of jasmonate signaling. High levels of WRKY gene transcripts indicated transcriptional regulation of the pathway genes. Increase in transcript level could be correlated with a corresponding increase in the protein levels for WsSMT1 and WsWRKY1. The withaferin A increase was also demonstrated in the potted plants growing in the glasshouse and in the open field. These results implicated simple physiological management of nitrogen fertilizer signal to improve the yield of secondary metabolite through probable involvement of jasmonate signal and WRKY transcription factor for the first time, in W. somnifera besides improving the foliage.

Comparative evaluation of extract of Bacopa monnieri and Mucuna pruriens as neuroprotectant in MPTP model of Parkinson's disease.

Parkinson's disease (PD) results primarily from the death of dopaminergic neurons in substantia nigra. Treatment of PD has been shifted recently towards herbal medicines.Bacopa monnieri (L.) Wettst. (BM) and Mucuna pruriens (L.) DC (MP) are traditional herbal plants known to have neuroprotective effects due to the presence of bacosides in whole plant extract of Bacopa monnieri (BME) and L-DOPA in MP seed extract (MPE). In this study, the comparative effect of BME and MPE in Parkinsonian mice induced by chronic exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was evaluated. Twenty four Swiss albino mice (35-45 g) were grouped into Control, MPTP, MPTP+BME and MPTP+MPE (6 mice in each). Experimental mice were given 40 mg/kg body wt. BME, 48 mg/kg body wt. MPE treatment was given orally for one month with prior use of 15 mg/kg body wt. of MPTP for 2 wk. After the treatment period, behavioral study was performed and assessment of neuroprotective effect was done via neurochemical analysis, Immunohistochemical parameters studied included functional viability of dopaminergic neurons in substantia nigra by Tyrosine hydroxylase (TH) using monoclonal antibody against TH and apoptotic study through caspase-3 and m-RNA expression of neurogenic gene in substantia nigra region of brain. Treatment with BME or MPE for one month significantly decreased the elevated levels of oxidative stress found in Parkinsonian mice. In behavioral tests, comparative analysis of BME and MPE showed a significant increase in spontaneous locomotor activity and grip strength test. Moreover, it was found that the use of BME considerably improved the tyrosine hydroxylase activity, caspase-3 and expression of neurogenic gene in the substantia nigra region of the brain. The results suggest that BME may provide a better platform for future drug discoveries and novel treatment strategies for PD as compared to MPE.

Vitamin D Deficiency After Oral Retinoid Therapy for Ichthyosis.

Oral retinoids are being increasingly used to treat ichthyotic disorders in children. We report on two children with ichthyotic disorders who developed unusual manifestations after they were started on oral retinoids. The first case is a 10-year-old girl with nonbullous ichthyosiform erythroderma and the second is a 2-year-old girl with lamellar ichthyosis. The child with ichthyosiform erythroderma developed features of rickets within months of initiation of systemic retinoids. Her baseline examination before initiation of oral retinoids was normal. The second patient with lamellar ichthyosis was found to have low vitamin D levels after 6 months of retinoid therapy, and prompt supplementation reversed the levels in 2 months. These cases are being reported to bring attention to the probable need for initiation of vitamin D supplementation with systemic retinoid therapy in ichthyotic disorders in children.

Diffusion tensor tractography and neuropsychological assessment in patients with vitamin B12 deficiency.

INTRODUCTION: Structural imaging of the brain does not demonstrate any changes in a vast majority of patients with vitamin B12 deficiency, even in advanced stages. In this study, we aimed to assess and correlate the functional integrity of the brain fiber tracts using diffusion tensor tractography with neuropsychological examination in patients with vitamin B12 deficiency. METHODS: The study was conducted at two tertiary care centers. Thirty-two patients with vitamin B12 deficiency were enrolled and subjected to diffusion tensor tractography, as an extension of diffusion tensor imaging, and neuropsychological assessment. Tests of significance were done to detect changes, pre- and post-vitamin B12 supplementation in the diffusivity parameters (fractional anisotropy and mean diffusivity) and the neuropsychological test scores. RESULTS: Statistically significant changes were observed in the diffusivity parameters and the neuropsychological test scores between the controls and the patients with vitamin B12deficiency in the pre- and post-treatment phases. CONCLUSIONS: This is the first study to evaluate the diffusion tensor tractography (DTT) parameters in the light of clinical neuropsychological assessment in patients with vitamin B12 deficiency. Utilization of DTT parameters may antedate structural changes and may quantify the neurocognitive deficits.

Etiological spectrum of hypokalemic paralysis: A retrospective analysis of 29 patients.

BACKGROUND: Hypokalemic paralysis is characterized by episodes of acute muscle weakness associated with hypokalemia. In this study, we evaluated the possible etiological factors in patients of hypokalemic paralysis. MATERIALS AND METHODS: We reviewed the records of 29 patients who were admitted with a diagnosis of hypokalemic paralysis. Modified Guillain-Barre´ Syndrome disability scale was used to grade the disability. RESULTS: In this study, 15 (51.7%) patients had secondary causes of hypokalemic paralysis and 14 patients (42.3%) had idiopathic hypokalemic paralysis. Thyrotoxicosis was present in six patients (20.6%), dengue infection in four patients (13.7%), distal renal tubular acidosis in three patients (10.3%), Gitelman syndrome in one patient (3.4%), and Conn's syndrome in one patient (3.4%). Preceding history of fever and rapid recovery was seen in dengue infection-induced hypokalemic paralysis. Approximately 62% patients had elevated serum creatinine phosphokinase. All patients had recovered completely following potassium supplementation. Patients with secondary causes were older in age, had significantly more disability, lower serum potassium levels, and took longer time to recover. CONCLUSION: In conclusion, more than half of patients had secondary causes responsible for hypokalemic paralysis. Dengue virus infection was the second leading cause of hypokalemic paralysis, after thyrotoxicosis. Presence of severe disability, severe hypokalemia, and a late disease onset suggested secondary hypokalemic paralysis.

Anti-mycobacterial screening of five Indian medicinal plants and partial purification of active extracts of Cassia sophera and Urtica dioica.

OBJECTIVE: To find out the anti-mycobacterial potential of Cassia sophera (C. sophera), Urtica dioica (U. dioica), Momordica dioica, Tribulus terrestris and Coccinia indica plants against multi-drug resistant (MDR) strain of Mycobacterium tuberculosis (M. tuberculosis). METHODS: Plant materials were extracted successively with solvents of increasing polarity. Solvent extracts were screened for anti-mycobacterial activity against fast growing, non-pathogenic mycobacterium strain, Mycobacterium semegmatis, by disk diffusion method. The active extracts were tested against MDR and clinical isolates of M. tuberculosis by absolute concentration and proportion methods. The active extracts were subjected to bio-autoassay on TLC followed by silica column chromatography for isolation of potential drug leads. RESULTS: Hexane extract of U. dioica (HEUD) and methanol extract of C. sophera (MECS) produced inhibition zone of 20 mm in disc diffusion assay and MIC of 250 and 125 µ g/mL respectively in broth dilution assay against Mycobacterium semegmatis. Semipurified fraction F2 from MECS produced 86% inhibition against clinical isolate and 60% inhibition against MDR strain of M. tuberculosis. F18 from HEUD produced 81% inhibition against clinical isolate and 60% inhibition against MDR strain of M. tuberculosis. Phytochemical analysis indicated that anti-mycobacterial activity of MECS may be due to presence of alkaloids or flavonoids and that of HEUD due to terpenoids. CONCLUSIONS: C. sophera and U. dioica plant extracts exhibited promising anti-mycobacterial activity against MDR strain of M. tuberculosis. This is the first report of anti-mycobacterial activity form C. sophera. This study showed possibility of purifying novel anti-mycobacterial compound(s) from C. sophera and U. dioica.

Study of micronutrients (copper, zinc and vitamin B12) in posterolateral myelopathies.

BACKGROUND: Vitamin B12 deficiency is a well recognized cause of posterolateral myelopathy. In Indian subcontinent, it may coexist with nutritional copper deficiency producing partial response of patients to B12 supplementation. Hence the study was planned to look for association of hypocupremia and B12 deficiency. METHODS: Twenty-three patients with posterolateral myelopathy (Romberg sign positive) were enrolled and investigated for levels of vitamin B12, copper and zinc and followed up for six months. RESULT: In three patients, copper deficiency alone was found to be the cause. In another three, both copper and vitamin B12 were deficient. In all these six patients, ceruloplasmin and 24h urinary copper were found to be low suggesting dietary copper deficiency. Hyperzincemia was found in four of these patients. Magnetic resonance imaging of spine was normal in lone Cu deficient patients but showed T2 hyperintensity of posterior column in lone B12 or combined B12 and copper deficiency. CONCLUSION: In cases of B12 deficiency myelopathy not responding to supplementation, copper deficiency must be sought at the earliest to avoid and treat persistent neurological disability.