RESUMO
BACKGROUND/OBJECTIVES: This study aims to elucidate the genetic causes of congenital hypogonadotropic hypogonadism (CHH), a rare genetic disorder resulting in GnRH deficiency, in six families from Pakistan. METHODS: Eighteen DNA samples from six families underwent genome sequencing followed by standard evaluation for pathogenic single nucleotide variants (SNVs) and small indels. All families were subsequently analyzed for pathogenic copy number variants (CNVs) using CoverageMaster. RESULTS: Novel pathogenic homozygous SNVs in known CHH genes were identified in four families: two families with variants in GNRHR, and two others harboring KISS1R variants. Subsequent investigation of CNVs in the remaining two families identified novel unique large deletions in ANOS1. CONCLUSION: A combined, systematic analysis of single nucleotide and CNVs helps to improve the diagnostic yield for variants in patients with CHH.
Assuntos
Variações do Número de Cópias de DNA , Hipogonadismo , Linhagem , Polimorfismo de Nucleotídeo Único , Humanos , Hipogonadismo/genética , Paquistão , Masculino , Feminino , Receptores de Kisspeptina-1/genética , Sequenciamento Completo do Genoma , Receptores LHRH/genética , Adulto , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso , Proteínas da Matriz ExtracelularRESUMO
Primary microcephaly is a rare, congenital, and genetically heterogeneous disorder in which occipitofrontal head circumference is reduced by a minimum of three standard deviations (SDs) from average because of the defect in fetal brain development. OBJECTIVE: Mapping of RBBP8 gene mutation that produce autosomal recessive primary microcephaly. Insilco RBBP8 protein models prediction and analysis. METHODS: Consanguineous Pakistani family affected with non-syndromic primary microcephaly was mapped a biallelic sequence variant (c.1807_1808delAT) in the RBBP8 gene via whole-exome sequencing. The deleted variant in the RBBP8 gene in affected siblings (V:4, V:6) of primary microcephaly was confirmed by sanger sequencing. RESULTS: Identified variant c.1807_1808delAT that truncated the protein translation p. Ile603Lysfs*7 and impaired the functioning of RBBP8 protein. This sequence variant was only reported previously in Atypical Seckel syndrome and Jawad syndrome, while we mapped it in the non-syndromic primary microcephaly family. We predicted 3D protein models by using Insilco tools like I TASSER, Swiss model, and phyre2 of wild RBBP8 protein of 897 amino acids and 608 amino acids of the mutant protein. These models were validated through the online SAVES server and Ramachandran plot and refined by using the Galaxy WEB server. A predicted and refined wild protein 3D model was deposited with accession number PM0083523 in Protein Model Database. A normal mode-based geometric simulation approach was used through the NMSim program, to find out the structural diversity of wild and mutant proteins which were evaluated by RMSD and RMSF. Higher RMSD and RMSF in mutant protein reduced the stability of the protein. CONCLUSION: The high possibility of this variant results in nonsense-mediated decay of mRNA, leading to the loss of protein functioning which causes primary microcephaly.
Assuntos
Microcefalia , Humanos , Microcefalia/genética , Linhagem , Mutação , Proteínas Mutantes , Aminoácidos/genética , Endodesoxirribonucleases/genéticaRESUMO
OBJECTIVE: Autosomal recessive primary microcephaly (MCPH) is a rare condition characterized by a reduced cerebral cortex accompanied with intellectual disability. Mutations in 17 genes have been shown to cause this phenotype. Recently, mutations in CIT, encoding CRIK (citron rho-interacting kinase)-a component of the central spindle matrix-were added. We aimed at identifying novel MCPH-associated genes and exploring their functional role in pathogenesis. METHODS: Linkage analysis and whole exome sequencing were performed in consanguineous and nonconsanguineous MCPH families to identify disease-causing variants. Functional consequences were investigated by RNA studies and on the cellular level using immunofluorescence and microscopy. RESULTS: We identified homozygous mutations in KIF14 (NM_014875.2;c.263T>A;pLeu88*, c.2480_2482delTTG; p.Val827del, and c.4071G>A;p.Gln1357=) as the likely cause in 3 MCPH families. Furthermore, in a patient presenting with a severe form of primary microcephaly and short stature, we identified compound heterozygous missense mutations in KIF14 (NM_014875.2;c.2545C>G;p.His849Asp and c.3662G>T;p.Gly1221Val). Three of the 5 identified mutations impaired splicing, and 2 resulted in a truncated protein. Intriguingly, Kif14 knockout mice also showed primary microcephaly. Human kinesin-like protein KIF14, a microtubule motor protein, localizes at the midbody to finalize cytokinesis by interacting with CRIK. We found impaired localization of both KIF14 and CRIK at the midbody in patient-derived fibroblasts. Furthermore, we observed a large number of binucleated and apoptotic cells-signs of failed cytokinesis that we also observed in experimentally KIF14-depleted cells. INTERPRETATION: Our data corroborate the role of an impaired cytokinesis in the etiology of primary and syndromic microcephaly, as has been proposed by recent findings on CIT mutations. Ann Neurol 2017;82:562-577.
Assuntos
Citocinese/genética , Regulação da Expressão Gênica/genética , Cinesinas/genética , Microcefalia/genética , Mutação/genética , Proteínas Oncogênicas/genética , Caspase 7/metabolismo , Movimento Celular/genética , Células Cultivadas , Criança , Pré-Escolar , Saúde da Família , Feminino , Fibroblastos/fisiologia , Estudo de Associação Genômica Ampla , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Microcefalia/diagnóstico por imagem , Microcefalia/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
Turmeric, a recognized spice, is known for miscellaneous health benefits in addition to culinary uses. In this study, in vitro evaluation of turmeric ethanol, methanol and aqueous extracts were mediated by disc diffusion, agar well method and phytochemical analysis. Purification of curcumin from turmeric was assisted by silica gel, TLC and HPLC for evaluation of its antioxidant and DNA protection activity. The sensitivity of alcoholic extracts against bacterial species differed, yet Staphylococcus aureus sub sp. Aureus and Bacillus subtilis both exhibited pronounced inhibition in disc diffusion and agar well method respectively. Overall, the crude ethanol extract of turmeric has an enhanced inhibitory effect on the growth of different bacterial species with a mean of 9.4±1.00 mm compared to 8.8±0.58 mm in case of crude methanol extract. Phytochemical analysis confirmed the presence of carbohydrates, flavonoids, coumarins, steroids, saponnins, tannins and phenols. Purification of curcumin through HPLC gave the main peak with 55% of acetonitrile at a retention time of 61- 65 minutes. Lower concentration of purified curcumin has protective effects on human DNA but increased concentrations instigate damaging effects. Its percentage scavenging ability was highest (91.84%) at 45 µg and per unit increase in the concentration prompted 6 units increase in percentage inhibition with a linear regression, R2= 0.914. All these traits boost its significance in herbal medicine with varied antimicrobial and pharmacological activities.
Assuntos
Antibacterianos/farmacologia , Antioxidantes/farmacologia , Curcuma , Compostos Fitoquímicos/farmacologia , Especiarias , Antibacterianos/isolamento & purificação , Antioxidantes/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana/métodos , Compostos Fitoquímicos/isolamento & purificação , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/fisiologiaRESUMO
There is a growing interest in standardizing the biocompatible, cost-effective, and eco-friendly manufacturing techniques for metallic nanostructures due to their widespread applications in the industrial and medical sectors. In recent decades, green synthesis has been proven as the most suitable technique for synthesizing metal nanoparticles. The present research study investigates the use of Cassia angustifolia (senna) leaves and Pistacia vera (Pistachio) nuts to prepare crude aqueous extracts, ethanolic extracts, and biogenic silver nanoparticles (AgNPs). The prepared aqueous extracts were used as reducing, stabilizing, and capping agents for the production of silver nanoparticles. These AgNPs were characterized by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), and ultraviolet-visible (UV-Vis) spectroscopy. The outcomes validated the formation of stable AgNPs with bioactive functional components. In vitro antibacterial, anticancer, anti-inflammatory, and antioxidant potentials were assessed by Kirby-Bauer disk diffusion test, MIC test, MBC test, MTT assay, BSA denaturation inhibition assay, and DPPH antioxidant assay, respectively. Results confirmed that the tested plant extract possesses a variety of bioactive compounds with various biological activities and is therapeutically effective. These findings verified that C. angustifolia and P. vera are promising bioresources for the synthesis of therapeutic extracts and nanostructures with commendable therapeutic potency.
RESUMO
BACKGROUND: Congenital cataract is causing one-third of blindness worldwide. Congenital cataract is heterogeneous in its inheritance patterns. The current study is aimed to explore the unknown genetic causes underlying congenital cataracts. METHODS: Blood samples from affected and normal individuals of n = 25 Pakistani families identified with congenital cataracts were collected. Genomic DNA was extracted and Sanger sequencing was performed to identify novel pathogenic variants in the FYCO1 (MIM#607182) gene. Later structural bioinformatics tools and molecular dynamics simulations were performed to analyze the impact of these variants on protein structure and function. RESULTS: Sanger sequencing resulted in the identification of a novel splice site mutation (NM_024513.3: c.3151-29_3151-7del) segregating in an autosomal recessive manner. This novel variant was confirmed to be absent in the n = 300 population controls. Further, bioinformatics tools revealed the formation of a mutant protein with a loss of the Znf domain. In addition, we also found a previously known (c.4127 T > C; p.Leu1376Pro) mutation in four families. We also report a novel heterozygous variant (c.3419G > A; p.Arg1140Gln) in another family. CONCLUSIONS: In conclusion, we report a novel deletion (NM_024513.3: c.3151-29_3151-7del) in one family and a frequent homozygous missense mutation (c.4127 T > C; p.Leu1376Pro) in four Pakistani families. The current research highlights the importance of autophagy in lens development and maintaining its transparency.
Assuntos
Catarata , Proteínas Associadas aos Microtúbulos , Catarata/genética , Catarata/patologia , Humanos , Padrões de Herança , Proteínas Associadas aos Microtúbulos/genética , Mutação , Paquistão , LinhagemRESUMO
This study was conducted to determine the in vitro anti-malarial activity of three medicinal plants, Picrorhiza kurroa, Caesalpinia bonducella and Artemisia absinthium of Pakistan. Different extracts of various parts of these plants were prepared by maceration and percolation, and were evaluated for their antimalarial activity. Aqueous, cold alcoholic and hot alcoholic extracts of Picrorhiza kurroa showed 34%, 100% and 90% inhibition in growth of Plasmodium falciparum, respectively, at 2.00 mg/ml. While aqueous, cold alcoholic and hot alcoholic extracts of Caesalpinia bonducella showed 65%, 56% and 76% inhibition in growth of Plasmodium falciparum, respectively at same concentrations. In the case of Artemisia absinthium, aqueous, cold alcoholic and hot alcoholic extract of Artemisia absinthium showed 35%, 55% and 21% inhibition in growth of Plasmodium falciparum, respectively at 2.00 mg/ml. In our study, extracts of Picrorhiza kurroa were found good for traditional therapy with highly significant results.
Assuntos
Antimaláricos/farmacologia , Extratos Vegetais/farmacologia , Plantas Medicinais/química , Animais , Antimaláricos/isolamento & purificação , Artemisia absinthium/química , Caesalpinia/química , Proliferação de Células/efeitos dos fármacos , Etanol/química , Paquistão , Parasitos/efeitos dos fármacos , Picrorhiza/química , Extratos Vegetais/isolamento & purificação , Estruturas Vegetais/química , Plasmodium falciparum/efeitos dos fármacos , Água/químicaRESUMO
BACKGROUND: Homocystinuria is an autosomal recessive metabolic disorder occurring due to the defects in cystathionine-ß-synthase enzyme. The study was carried out to investigate a Pakistani family presenting bilateral congenital cataract with symptoms of classical homocystinuria at LRBT Free Eye Hospital, Lahore, Pakistan. METHODS: Three affected individuals of the family presented skeletal deformations, intellectual disability, speech delay, and myopia with bilateral congenital cataract. Genetic analysis on DNA samples from affected individuals was done through whole exome sequencing to identify underlying genetic variant causing disease phenotypes in the family. In silico analysis was done to predict the effect of variation on the structure of mutant protein. RESULTS: A missense allelic variant (NM_000071.3: c.253G>A) of the CBS gene was revealed which may affect the catalytic activity of the substituted (NP_000062.1: p.G85R) protein by disrupting the folding of the enzymatic protein. High levels of homocysteine were observed in the plasma of affected individuals. This is the first report of this genetic variant from Pakistan causing homocystinuria and congenital cataract in association. CONCLUSION: This variant was reported first time in association with congenital cataract instead of ectopia lentis. Congenital cataract was developed secondarily in these patients and provided a clue for the early diagnosis of metabolic disorders like homocystinuria to prevent further complications and morbidity.
Assuntos
Catarata/genética , Cistationina beta-Sintase/genética , Homocistinúria/genética , Fenótipo , Catarata/patologia , Criança , Cistationina beta-Sintase/química , Feminino , Homocistinúria/patologia , Humanos , Lactente , Masculino , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , LinhagemRESUMO
Tuberculosis (TB), a chronic disease caused by Mycobacterium tuberculosis (Mtb), is a global health issue across the world. Pakistan ranks fifth among the countries, which are facing, a significantly great number of mortalities and morbidities due to TB. Unfortunately, all previously reported treatments are not successful for the eradication of TB. Here in this study, we report an emerging treatment option for this disease. We have applied immunoinformatics to predict highly conserved B and T-cell epitopes from Mtb, showing significant binding affinities to the frequent HLA alleles in the Pakistani population. A total of ten highly referenced and experimentally validated epitopes were selected from the Immune Epitope Database (IEDB), followed by their conservancy analysis using weblogos. The consensus sequences and variants derived from these sequences were examined, for their binding affinities, with prevalent HLA alleles of Pakistan. Moreover, the antigenic and allergenic natures of these peptides were also evaluated via Vaxijen and AllerTOP, respectively. Consequently, all potentially allergenic and non-antigenic, peptide fragments, were excluded from the analysis. Among all putative epitopes, three CD8 + T-cell epitopes were selected, as ideal vaccine candidates and, population coverage analysis revealed that the combination of these three peptides was covering, 67.28% Pakistani Asian and 57.15% mixed Pakistani populations. Likewise, eleven linear and six conformational or discontinuous B-cell epitopes were also marked as potential vaccine candidates based on their prediction score, non-allergenic nature, and antigenic properties. These epitopes, however, need the final validation via wet-lab studies. After their approval, these epitopes would be effective candidates for the future designing of epitope-based vaccines against Mtb infections in Pakistan.
RESUMO
BACKGROUND: Primary microcephaly (MCPH) is a congenital neurodevelopmental disorder manifesting as small brain and intellectual disability. It underlies isolated reduction of the cerebral cortex that is reminiscent of early hominids which makes it suitable model disease to study the hominin-specific volumetric expansion of brain. Mutations in 25 genes have been reported to cause this disorder. Although majority of these genes were discovered in the Pakistani population, still a significant proportion of these families remains uninvestigated. METHODS: We studied a cohort of 32 MCPH families from different regions of Pakistan. For disease gene identification, genome-wide linkage analysis, Sanger sequencing, gene panel, and whole-exome sequencing were performed. RESULTS: By employing these techniques individually or in combination, we were able to discern relevant disease-causing DNA variants. Collectively, 15 novel mutations were observed in five different MCPH genes; ASPM (10), WDR62 (1), CDK5RAP2 (1), STIL (2), and CEP135 (1). In addition, 16 known mutations were also verified. We reviewed the literature and documented the published mutations in six MCPH genes. Intriguingly, our cohort also revealed a recurrent mutation, c.7782_7783delGA;p.(Lys2595Serfs*6), of ASPM reported worldwide. Drawing from this collective data, we propose two founder mutations, ASPM:c.9557C>G;p.(Ser3186*) and CENPJ:c.18delC;p.(Ser7Profs*2), in the Pakistani population. CONCLUSIONS: We discovered novel DNA variants, impairing the function of genes indispensable to build a proper functioning brain. Our study expands the mutational spectra of known MCPH genes and also provides supporting evidence to the pathogenicity of previously reported mutations. These novel DNA variants will be helpful for the clinicians and geneticists for establishing reliable diagnostic strategies for MCPH families.
Assuntos
Loci Gênicos , Microcefalia/genética , Mutação , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Consanguinidade , Feminino , Efeito Fundador , Frequência do Gene , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Microcefalia/patologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas do Tecido Nervoso/genética , LinhagemRESUMO
OBJECTIVES: To evaluate any association between the frequency of hereditary hemochromatosis (HFE) gene mutation (H63D and C282Y) and iron overload in beta-thalassemia major (BTM) patients. METHODS: The case-control study was conducted from June 2016 to February 2018. Blood samples from 204 BTM patients and 204 normal controls were taken from the Sundas Foundation Blood Bank. These samples were analyzed for serum ferritin assay and HFE mutation. Ferritin level was measured on the ARCHITECT 1000SR. Both patient and control samples were analyzed for mutations using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: Serum ferritin levels for all patients were greater than 1000ng/mL. The p.H63D mutation was observed in 23 (11.3%) cases, out of which 19 cases were heterozygous for p.H63D and 4 cases were homozygous. In control samples, 4 cases (2%) were found heterozygous for the p.H63D, and no homozygous mutation was found. Significantly high serum ferritin levels were found in BTM patients with the H63D mutation (p=0.00). In the case of p.C282Y, neither homozygous nor heterozygous mutation was found in patients or in controls. CONCLUSION: H63D polymorphism is associated with iron overload in BTM patients. Larger-scale research is required to give an elaborated view of the association of the HFE mutation with iron overload in these patients and to confirm our conclusion.
Assuntos
Ferritinas/sangue , Proteína da Hemocromatose/genética , Hemocromatose/sangue , Talassemia beta/sangue , Adulto , Transfusão de Sangue , Estudos de Casos e Controles , Criança , Pré-Escolar , Comorbidade , Feminino , Hemocromatose/epidemiologia , Hemocromatose/genética , Humanos , Sobrecarga de Ferro/sangue , Sobrecarga de Ferro/epidemiologia , Sobrecarga de Ferro/etiologia , Sobrecarga de Ferro/genética , Masculino , Mutação , Paquistão/epidemiologia , Prevalência , Reação Transfusional , Talassemia beta/epidemiologia , Talassemia beta/genéticaRESUMO
Tephrosia purpurea (L.) Pers., commonly known as "sarpunkha" and "wild indigo", is being used in traditional systems of medicine to treat liver disorders, spleen and kidney. In the present study, a validated High Performance Thin Layer Chromatography (HPTLC) method was established for the estimation of lupeol, ß-sitosterol and rotenone in various extracts of T. purpurea with the aim to see the effect of seasons on the quantity of aforesaid phytoconstituents. The plant material was collected in summer (April), rainy (August) and winter (December) during 2013-2014 from Lucknow, India. The method was validated in terms of precision, repeatability, specificity, sensitivity linearity and robustness. The method permits reliable quantification and showed good resolution on silica gel with toluene-ethyl acetate-formic acid (9:1:1 v/v/v) as mobile phase, and characteristic bands of ß-sitosterol, rotenone and lupeol were observed at Rf 0.38, 0.45 and 0.52, respectively. The content of aforesaid phytoconstituents varies from season to season and extract to extract. Our finding indicated that winter season (December) may not be appropriate for collection of T. purpurea for the preparation of therapeutic formulations because of the high content of rotenone, a known insecticide that is responsible for Parkinson's disease and associated with heart failure, fatty liver and liver necrosis.
Assuntos
Cromatografia em Camada Fina/métodos , Triterpenos Pentacíclicos/análise , Extratos Vegetais/análise , Rotenona/análise , Sitosteroides/análise , Tephrosia/química , Índia , Estações do AnoRESUMO
BACKGROUND: Clopidogrel (CLOP) is an antiplatelet drug with poor solubility in intestinal fluid, which limits its bioavailability after oral administration. OBJECTIVES: Current study focuses on developing site-specific floating microcarriers of CLOP using solvent diffusion evaporation method (SDEM) for retaining the drug in the stomach, thus improving the solubility of drug for better absorption. METHODS: SDEM was employed to formulate floating microcarriers using lipidic excipients, namely Gelucires (GL) to impart floating properties, in combination with ethyl cellulose as release retarding polymer. RESULTS: Prepared particles were 169 ± 6 µm to 375 ± 13 µm in size, whilst encapsulation efficiency was ranged from 39.6 ± 0.60% to 96.50 ± 3.50%. Electron micrographs depicted discrete spherical microcarriers with porous structure, which amplified with increasing HLB value of GL and concentration of Eudragit E100. FTIR study confirmed absence of major drug polymer interactions while DSC and XRD studies revealed the presence of non-crystalline nature of drug in all formulations. Drug release at pH 1.2 enhanced more than 2-folds with increasing HLB value with 32% cumulative drug release for GL 43/01 and 69% for GL 50/13. More interestingly, adding various proportions of Eudragit E100 to GL 43/01 based formulations resulted in increased drug release as high as 71%. In all formulations, the drug release followed diffusion dependent process. CONCLUSION: It is envisaged that this formulation strategy for CLOP is promising and could possibly be tested in future for its in vivo performance. Graphical abstract Lipid based floating microcarriers of clopidogrel.
Assuntos
Clopidogrel/farmacocinética , Excipientes/química , Triglicerídeos/química , Disponibilidade Biológica , Cápsulas , Clopidogrel/química , Microscopia Eletrônica de Varredura , Tamanho da Partícula , Solubilidade , SolventesRESUMO
Though many hearing impairment genes have been identified, only a few of these genes have been screened in population studies. For this study, 168 Pakistani families with autosomal recessive hearing impairment not due to mutations in the GJB2 (Cx26) gene underwent a genome scan. Two-point and multipoint parametric linkage analyses were carried out. Twelve families had two-point or multipoint LOD scores of 1.4 or greater within the transmembrane cochlear expressed gene 1 (TMC1) region and were subjected to further screening with direct DNA sequencing. Five novel putatively functional non-synonymous sequence variants, c.830A>G (p.Y277C), c.1114G>A (p.V372M), c.1334G>A (p.R445H), c.2004T>G (p.S668R), and c.2035G>A (p.E679K), were found to segregate within seven families, but were not observed in 234 Pakistani control chromosomes. The variants c.830A>G (p.Y277C), c.1114G>A (p.V372M), and c.1334G>A (p.R445H) occurred at highly conserved regions and were predicted to lie within hydrophobic transmembrane domains, while non-synonymous variants c.2004T>G (p.S668R) and c.2035G>A (p.E679K) occurred in extracellular regions that were not highly conserved. There is evidence that the c.2004T>G (p.S668R) variant may have occurred at a phosphorylation site. One family has the known splice site mutation c.536 -8T>A. The prevalence of non-syndromic hearing impairment due to TMC1 in this Pakistani population is 4.4% (95%CI: 1.9, 8.6%). The TMC1 protein might have an important function in K(+) channels of inner hair cells, which would be consistent with the hypothetical structure of protein domains in which sequence variants were identified.
Assuntos
Genes Recessivos , Variação Genética , Perda Auditiva/genética , Proteínas de Membrana/genética , Conexina 26 , Conexinas , Consanguinidade , Feminino , Ligação Genética , Humanos , Escore Lod , Masculino , Mutação , Paquistão , Análise de Sequência de ProteínaRESUMO
Identification of medicinal plants by their molecular signature is a fast growing tool. The identification of Desmodium gangeticum (L.) DC. (Shalparni, a constituent of Ayurvedic formulation "Dashmoolarishtha") was carried out using genomic approach. Authentic samples of D. gangeticum(L.) DC., D. velutinum (Willd.) DC. and D. triflorum (L.) DC. were analyzed and compared to commercial samples of various origin. Within twenty primers used, eleven gave 223 RAPD fragments. RAPD profiles of three species showed very low similarity index (0.21-0.39), whereas market samples showed high similarity of 0.82-0.89 with authenticated D. gangeticum.