Effects of dietary supplementation of cumin (Cuminum cyminum L.) essential oil on expression of genes related to antioxidant, apoptosis, detoxification, and heat shock mechanism in heat-stressed broiler chickens.

This study was carried out to evaluate the impact of cumin essential oil (CEO) supplementation on levels of certain gene expression related to antioxidant, apoptotic, detoxific, and heat shock mechanisms in the breast meat and ileum of heat-stressed broilers. The study was conducted on a 2 × 6 factorial design (heat stress + feed additive) on 600 day-old male broiler chicks for a period of 42 days. From day 7 to 42, although broilers in heat stress groups (HT) were exposed to constant chronic heat stress (36 °C), others were housed at thermoneutral ambient temperature (TN). The chicks in both conditions were fed with 6 experimental diets: C0 (basal diet with no additive), ANTIB (basal diet + 100 mg/kg chloramphenicol), VITE (basal diet + 50 IU α-tocopherol), C2 (basal diet + 200 mg/kg CEO), C4 (basal diet + 400 mg/kg CEO), C6 (basal diet+ 600 mg/kg CEO). The results showed that heat stress upregulated (except for Bcl-2) the genes related to antioxidant, apoptosis, detoxification, and heat shock mechanism. However, cumin essential oil increased the dose-dependently positive effect on certain genes in tissues of the heat-stressed broilers and downregulated (except for Bcl-2) these genes.

DNA barcoding for specific and sensitive detection of Cuminum cyminum adulteration in Bunium persicum.

BACKGROUND: Bunium persicum commonly called as Kala zeera, a very high value herbaceous spice used for medicinal purposes is often adulterated with Cuminum cyminum or Safed zeera, a closely related species. Lack of distinctive morphological features makes the identification of genuine kala zeera from its adulterant difficult, the problem is even exaggerated in case of powdered material. METHODOLOGY: Genomic DNA was extracted from all the plant materials by using CTAB-SDS method (Möller et al., 1992) with slight modifications. On the basis of reproducibility and high amplification ability, four universal barcoding loci viz. ITS2, rbcL-a, mat K and psbA-trnH and a specific locus Cum were used in the present study. The amplified PCR products were sequenced bidirectionally and assembled to obtain contigs. The sequences thus obtained were aligned using MUSCLE algorithm (Edgar, 2004) and information pertaining to conserved/ variable/ parsimony informative sites, number of transitions, transversions and Indels was obtained after analyzing the sequences. RESULTS AND CONCLUSION: Among the tested barcoding loci, psbA-trnH has proven to be best barcode in authentication of kala zeera as its amplification and sequencing success was high and it showed the presence of polymorphic sites to detect interspecific variation. This barcode could differentiate between safed zeera and kala zeera in a single reaction, simultaneously.

Development of a workflow for screening and identification of α-amylase inhibitory peptides from food source using an integrated Bioinformatics-phage display approach: Case study - Cumin seed.

The main objective of this study was to develop an efficient workflow to discover α-amylase inhibitory peptides from cumin seed. A total of 56 unknown peptides was initially found in the cumin seed protein hydrolysate. They were subjected to 2 different in silico screenings and 6 peptides were shortlisted. The peptides were then subjected to in vitro selection using phage display technique and 3 clones (CSP3, CSP4 and CSP6) showed high affinity in binding α-amylase. These clones were subjected to the inhibitory test and only CSP4 and CSP6 exhibited high inhibitory activity. Therefore, these peptides were chemically synthesized for validation purposes. CSP4 exhibited inhibition of bacterial and human salivary α-amylases with IC50 values of 0.11 and 0.04µmol, respectively, whereas CSP6 was about 0.10 and 0.15µmol, respectively. Results showed that the strength of each protocol has been successfully combined as deemed fit to enhance the α-amylase inhibitor peptide discovery.

Genetic linkage between protein and DNA polymorphisms and antioxidant capacity of Cuminum cyminum L. accessions.

This study aimed to link the genetic variation observed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and random amplified polymorphic DNA (RAPD) analysis among 11 Cuminum cyminum L. accessions, collected from diverse ecogeographical areas in Saudi Arabia, with their antioxidant capacity to better identify potential genotypes for breeding programs for this medicinal spice. SDS-PAGE analysis revealed genetic variation among cumin germplasms and distinct polymorphisms (100%). Protein polymorphisms were identified based on the number of polypeptide bands (288) with molecular weights ranging from 13.85 to 350 kDa, band intensity, the appearance of new bands, and the absence of other bands. RAPD analysis revealed 363 amplified DNA products with a high polymorphism value (98.88%) based on DNA band type (unique, non-unique, and monomorphic), DNA 90 to 1085-bp long, and band intensity. The unweighted pair group method with arithmetic mean clustering based on SDS-PAGE or RAPD and Jaccard's similarity coefficient divided cumin accessions into similar but distinct clusters with respect to their location of collection. The antioxidant potential of cumin accessions based on 1, 1-diphenyl-2-picrylhydrazyl radical scavenging activity, the ß-carotene-linoleate model system, and total phenolic and flavonoid contents revealed distinct variability. These data indicate that cumin is a valuable genetic resource with high antioxidant activity. Additionally, clustering based on antioxidant activity was not identical to that based on SDS-PAGE and RAPD. Data and clustering of SDS-PAGE and RAPD, combined with the high antioxidant capacity of cumin accessions, are important for the efficient use of genetic resources of cumin in breeding strategies and genetic improvement programs.

In planta Transformed Cumin (Cuminum cyminum L.) Plants, Overexpressing the SbNHX1 Gene Showed Enhanced Salt Endurance.

Cumin is an annual, herbaceous, medicinal, aromatic, spice glycophyte that contains diverse applications as a food and flavoring additive, and therapeutic agents. An efficient, less time consuming, Agrobacterium-mediated, a tissue culture-independent in planta genetic transformation method was established for the first time using cumin seeds. The SbNHX1 gene, cloned from an extreme halophyte Salicornia brachiata was transformed in cumin using optimized in planta transformation method. The SbNHX1 gene encodes a vacuolar Na+/H+ antiporter and is involved in the compartmentalization of excess Na+ ions into the vacuole and maintenance of ion homeostasis Transgenic cumin plants were confirmed by PCR using gene (SbNHX1, uidA and hptII) specific primers. The single gene integration event and overexpression of the gene were confirmed by Southern hybridization and competitive RT-PCR, respectively. Transgenic lines L3 and L13 showed high expression of the SbNHX1 gene compared to L6 whereas moderate expression was detected in L5 and L10 transgenic lines. Transgenic lines (L3, L5, L10 and L13), overexpressing the SbNHX1 gene, showed higher photosynthetic pigments (chlorophyll a, b and carotenoid), and lower electrolytic leakage, lipid peroxidation (MDA content) and proline content as compared to wild type plants under salinity stress. Though transgenic lines were also affected by salinity stress but performed better compared to WT plants. The ectopic expression of the SbNHX1 gene confirmed enhanced salinity stress tolerance in cumin as compared to wild type plants under stress condition. The present study is the first report of engineering salt tolerance in cumin, so far and the plant may be utilized for the cultivation in saline areas.

Presence of Undeclared Food Allergens in Cumin: The Need for Multiplex Methods.

Beginning in the autumn of 2014, millions of dollars of food and over 675 products were recalled in the United States due to the presence of undeclared peanut, attributed to cumin used in the manufacture of the products. Initial analyses also indicated the presence of almond. Subsequent research showed that the presence of peanut and almond did not fully explain the analytical results for the cumin samples. Using a combination of mass spectrometry, DNA-based methods (i.e., PCR and Sanger DNA Sequencing), microscopy, and antibody-based technologies (i.e., ELISA, Western blot analysis, and a novel xMAP multiplex assay) the presence of peanut was confirmed. Screening for secondary sources of adulteration (e.g., tree nuts, mahleb, peach, and cherry) supported the assessment that the cumin contained multiple contaminants. These results demonstrate the limitations of single analyte-specific assays and the need for orthogonal multiplex methods to detect food allergens irrespective of varietal or other differences.

An efficient method for Agrobacterium-mediated genetic transformation and plant regeneration in cumin (Cuminum cyminum L.).

Cumin is an annual herbaceous medicinally important plant having diverse applications. An efficient and reproducible method of Agrobacterium-mediated genetic transformation was herein established for the first time. A direct regeneration method without callus induction was optimised using embryos as explant material in Gamborg's B5 medium supplemented with 0.5-µM 6-benzyladenine and 2.0-µM α-naphthalene acetic acid. About 1,020 embryos (a mean of 255 embryos per batch) were used for the optimisation of transformation conditions. These conditions were an Agrobacterium cell suspension of 0.6 OD600, a co-cultivation time of 72 h, 300-µM acetosyringone and wounding of explants using a razor blade. Pre-cultured elongated embryos were treated using optimised conditions. About 720 embryos (a mean of 180 embryos per batch) were used for transformation and 95 % embryos showed transient ß-glucuronidase expression after co-cultivation. Putative transformed embryos were cultured on B5 medium for shoot proliferation and 21 regenerated plants were obtained after selection and allowed to root. T0 plantlets showed ß-glucuronidase expression and gene integration was confirmed via PCR amplification of 0.96 and 1.28 kb fragments of the hygromycin-phosphotransferase II and ß-glucuronidase genes, respectively. In this study, a transformation efficiency of 1.5 % was demonstrated and a total of 11 transgenic plants were obtained at the hardening stage, however, only four plants acclimatised during hardening. Gene copy number was analysed by Southern blot analysis of hardened plants and single-copy gene integration was observed. This is the first successful attempt of Agrobacterium-mediated genetic transformation of cumin.

Loop-mediated isothermal amplification (LAMP): methods for plant species identification in food.

Loop-mediated isothermal amplification (LAMP) is a DNA-based analytical method that can be used as an isothermal alternative to polymerase chain reaction (PCR). In comparison to PCR, the advantage of LAMP is the possibility to perform the isothermal reaction without any sophisticated technical equipment; only a water bath is needed, and naked eye detection is sufficient. Up to now, an application of LAMP methods for the detection of even closely related plant species in food or feed matrices has not been described, whereas a large number of PCR methods for that topic are cited in the literature. The aim of the study was the evaluation of LAMP-based methods for plant species identification with respect to method parameters such as R(2), LOD, and LOQ. An existing (real-time) PCR method (for the detection of spices) was used for comparison. It could be shown that the developed LAMP methods have potential as alternative strategies to PCR in DNA-based analysis.

Combined direct regeneration protocols in tissue culture of different cumin genotypes based on pre-existing meristems.

Rapid and genotype-independent protocols for two direct in vitro morphogenesis pathways including direct shoot organogenesis from embryo and direct shoot proliferation from node have been developed in cumin (Cuminum cyminum L.). Direct regenerations occurring without passing callus phase are important since fewer somaclonal variation and genotype-dependency are likely to arise from these methods in comparison with regenerations trough callus. After embryo culture, shoots with single-cellular origin were regenerated from the meristematic zone of embryo without any intermediate callus phase. In contrast, proliferated shoots with multi-cellular origin were directly regenerated from the axillary buds (meristems) of node explants. Effects of different concentrations of 6-Benzylaminopurine (BAP), alpha-Naphthaleneacetic Acid (NAA) and Indole-3-kcetic Acid (IAA) on B5 medium of embryo and node cultures as well as subculture were studied in detail. In direct organogenesis pathway from embryo explant, 0.1 mg L(-1) NAA + 1 mg L(-1) IAA resulted the highest shoot regeneration response (89.5 shoots per regenerated explant), whereas 0.1 mg L(-1) BAP + 1 mg L(-1) NAA was the most effective combination in direct shoot proliferation from node explant (42 shoots per regenerated explant). BAP (cytokinin) revealed the inhibitory effect on induction of direct shoot organogenesis pathway from embryo explant, while low concentration of BAP (0.1 mg L(-1)) had positive effect on direct shoot proliferation pathway from node explant. Subculturing was not necessary for shoot multiplication and elongation in embryo culture, whereas multiplication and elongation of shoots in node culture were associated to subculture on growth regulator-free medium. In other part of study, the behavior of different cumin genotypes in direct regeneration pathways was studied. Both direct organogenesis and direct proliferation pathways were applicable to different cumin genotypes and regenerated plants were phenotypically normal. This study supports the feasibility of combined direct regenerations protocols from embryo and node of cumin in germplasm conservation by in vitro cloning and genetic improvement programs.

[ITS sequence identification of Radix Bupleuri].

OBJECTIVE: To provide the basis of molecular authentication of Radix Bupleuri by the comparison of the internal transcribed spacer (ITS) sequences of five kinds of Radix Bupleuri in common use. METHOD: Firstly, total DNA of five kinds of Radix Bupleuri was extracted. Secondly, the ITS sequence was amplified by PCR with universal primer of ITS and PCR product was directly sequenced after purification. RESULT: The length of ITS1 and ITS2 sequence was 214-220 bp and 230-231 bp respectively. There were distinct variation sites between the ITS sequences of the five kinds of Radix Bupleuri. CONCLUSION: ITS sequence may be the evidence for the molecular authentication of Radix Bupleuri.