Multivariate analysis of chemometric based aroma dynamics in small cardamom (Elettaria cardamomum Maton) during drying.

The aroma compositions and essential oil contents extracted from small cardamom (Elettaria cardamomum Maton) dried at three temperatures using a hot air convective dryer was studied by chromatographic methods. The major compounds in the dried samples were 1,8-cineole (7.23-11.76%) and α-Terpinyl acetate (61.65-68.19%) that were responsible for the specific aroma. The extracted essential oil was found to be majorly composed of alkenes (33-42%), alcohols (18-30%) and esters (18-22%) with acids, alkyls and ketones. The changes in esters and alcohols with drying time were more significant (p < 0.05) than acids, alkenes and aldehydes at all three temperatures. The data were analysed using Principal Component Analysis (PCA) and Hierarchical Cluster Analysis. The analyses indicated that the compounds varied significantly (p < 0.05) with drying temperatures. The PCA revealed that there was overall 66.47, 75.96 and 78.25% cumulative data variance among the constituents of essential oils of cardamom samples dried at 40, 50 and 60 °C respectively. The samples showed a significant (p < 0.05) reduction in aroma constituents after 26, 18 and 16 h of drying at 40, 50 and 60 °C respectively. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05560-8.

Supercritical carbon dioxide extracts of small cardamom and yellow mustard seeds have fasting hypoglycaemic effects: diabetic rat, predictive iHOMA2 models and molecular docking study.

In the present investigation, the supercritical carbon dioxide (SC-CO2) extracts of small cardamom (SC) and yellow mustard (YM) seeds have been investigated for their efficacies in combating type 2 diabetes in streptozotocin-induced Wistar albino rats. Fasting blood glucose (FBG) levels in the rats were monitored on days 8, 15 and 21. On day 15, FBG level reduced appreciably by 31·49 % in rats treated with SC seed extract and by 32·28 % in rats treated with YM seed extract, comparable to metformin (30·70 %) and BGR-34 (a commercial polyherbal drug) (31·81 %) administered rats. Either extract exhibited desirable effects on hepatic glucose-6-phosphatase, glucose-6-phosphate dehydrogenase (G6PD) and catalase activities in controlling diabetes. A molecular docking exercise was conducted to identify specific compounds in the extracts which possessed augmenting effect on G6PD. The results revealed that all the bioactive compounds in the extracts have binding affinities with the enzyme and contributed to the antidiabetic efficacies of the extracts as G6PD augmenters. The effects of the extracts on insulin sensitivity and glucose uptake were investigated using non-invasive modelling by iHOMA2 software. This in vitro approach indicated that extract administration resulted in increased both insulin sensitivity of the liver and glucose uptake in the gut. The findings of the present study attest these SC-CO2 extracts of the spices as safe alternatives of metformin and BGR-34 in combating type 2 diabetes and could be safely subjected to clinical studies. These extracts could also be employed in designing proactive food supplements in mitigating the metabolic disorder.

Plant virus interaction mechanism and associated pathways in mosaic disease of small cardamom (Elettaria cardamomum Maton) by RNA-Seq approach.

Small cardamom (Elettaria cardamomum), grown in limited coastal tropical countries is one of the costliest and widely exported agri-produce having global turnover of >10 billion USD. Mosaic/marble disease is one of the major impediments that requires understanding of disease at molecular level. Neither whole genome sequence nor any genomic resources are available, thus RNA seq approach can be a rapid and economical alternative. De novo transcriptome assembly was done with Illumina Hiseq data. A total of 5317 DEGs, 2267 TFs, 114 pathways and 175,952 genic region putative markers were obtained. Gene regulatory network analysis deciphered molecular events involved in marble disease. This is the first transcriptomic report revealing disease mechanism mediated by perturbation in auxin homeostasis and ethylene signalling leading to senescence. The web-genomic resource (SCMVTDb) catalogues putative molecular markers, candidate genes and transcript information. SCMVTDb can be used in germplasm improvement against mosaic disease in endeavour of small cardamom productivity. Availability of genomic resource, SCMVTDb: http://webtom.cabgrid.res.in/scmvtdb/.

Isolation and characterization of genomic microsatellite markers for small cardamom (Elettaria cardamomum Maton) for utility in genetic diversity analysis.

Microsatellite markers in small cardamom (Elettaria cardamomum Maton) were developed using the selective hybridization enrichment method. A total of 140 microsatellite repeats were identified from 270 clones. Primers were designed for 58 microsatellites and 44 primer pairs amplified products of expected size in cardamom. These markers were used for studying the diversity of 20 important small cardamom genotypes, and six markers were found to be polymorphic. The number of alleles ranged from 2 to 7 with an average of 3.6 per locus. Polymorphic information content values ranged from 0.14 to 0.38 based on dominant scoring. The two markers ECM 47a and ECMG 28 generated specific banding patterns for the genotypes MCC7 (Pink tiller) and APG434 (MA18) respectively. Dendrogram illustrated the genetic similarity between different genotypes of Kerala and Karnataka regions. It differentiated the closely related genotypes and released varieties into separate groups. Principal coordinate analysis revealed PV1 and ICRI 1 as the most divergent genotypes. The study demonstrated that these markers are informative and can be further utilized for generating reliable molecular data for assisting the crop improvement of small cardamom. Cross generic transferability (71.4 %) of the developed primers proved that they are useful for phylogenetic studies in the family Zingiberaceae. This is the first report of de novo isolation, characterisation and utilization of microsatellite markers for the genetic diversity analysis of small cardamom.

A Novel spice-antioxidant-based nano-vehicle as a putative green alternative of synthetic AChE inhibitor drugs.

The present treatment for Alzheimer's disease (AD) involves well known synthetic acetylcholine esterase (AChE) inhibitor drugs which besides having short duration of action also have deleterious impact on human health. Therefore, there is a need for natural plant-based biomolecule(s) with potential AChE inhibition activity (ies). The aim of the work is to design a spice-based nano-vehicle as a novel green alternative of synthetic AD drugs by nanoencapsulating a solvent-less supercritical CO2 extract of small cardamom seeds (SCE) having a synergistic consortium of five antioxidant molecules, using polyethylene glycol and emulsifiers, selected based on Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) analyses. Ellman's assay and enzyme inhibition kinetics of the antioxidant molecules as well as the extract and its nanoliposomal formulation (SCE-NL) were performed, followed by rigorous molecular docking and dynamics studies using MM-PBSA and umbrella sampling. The antioxidants exhibited significant AChE inhibition in vitro, individually with 1, 8-cineole having the least IC50 value of 65.53 ± 0.05 µg/mL. . Although SCE-NL had higher IC50 value (575.67 ± 0.5 µg/mL) vis-à-vis that of rivastigmine (67.52 ± 0.02 µg/mL), it is safer for usage being 'green'.The Lineweaver-Burk plots (Vmax ∼1.04 mM/min) revealed competitive mode(s) of inhibition of AChE with each of these antioxidants. Binding energy analyses suggested very good binding free energies and stable docking/binding complexes (between the antioxidants and AChE). This study has delivered a nanoliposomal vehicle of food antioxidants as a putative 'green' alternative of synthetic AChE inhibitor drugs.Communicated by Ramaswamy H. Sarma.

Small cardamom genome: development and utilization of microsatellite markers from a draft genome sequence of Elettaria cardamomum Maton.

Small cardamom (Elettaria cardamomum Maton), the queen of spices, is the third most expensive spice in the world after saffron and vanilla, valued highly for its aroma and taste. This perennial herbaceous plant is a native of coastal parts of Southern India and displays a significant amount of morphological diversity. Its genetic potential has not been exploited due to lack of genomic resources limiting our understanding of the genome and important metabolic pathways which give it the economic advantage in the spice industry. Here, we report upon the de novo assembled, draft whole genome sequence of cardamom variety, Njallani Green Gold. We used a hybrid assembly strategy using the reads from the Oxford Nanopore, Illumina and 10x Genomics GemCode sequencing chemistries. The assembled genome length was 1.06 Gb (gigabases) which is close to the estimated genome size of cardamom. More than 75% of the genome was captured in 8000 scaffolds with a N50 of 0.15 Mb. The genome appears to have a high repeat content and 68055 gene models were predicted. The genome is close to Musa species and displays an expansion and contraction in different gene families. The draft assembly was used for in silico mining of simple sequence repeats (SSRs). A total of 2,50,571 SSRs were identified of which 2,18,270 were perfect SSRs and 32,301 were compound SSRs. Among the perfect SSRs, trinucleotides were most abundant (1,25,329) and hexanucleotide repeats appear least (2,380). From the 2,50,571 SSRs mined, 2,27,808 primer pairs were designed based on flanking sequence information. Wet lab validation was performed for 246 SSR loci and based on their amplification profiles, 60 SSR markers were used for diversity analysis of a set of 60 diverse cardamom accessions. The average number of alleles detected per locus were 14.57 with a minimum of 4 and maximum of 30 alleles. Population structure analysis revealed the presence of high degree of admixtures which could primarily be due to cross-pollination prevalent in this species. The SSR markers identified would help in the development of gene or trait-linked markers which can be subsequently used for marker-assisted breeding for crop improvement in cardamom. The information on utilization of the SSR loci for generation of markers has been developed into a public database, 'cardamomSSRdb' that is freely available for use by the cardamom community.

Fortification of a Desert Using Nanoencapsulated Supercritical Carbon Dioxide Extract of Small Cardamom Seeds: A Nutraceutical Custard with Antioxidant Synergy.

BACKGROUND: 1,8 cineole-rich supercritical CO2 extract of small cardamom seeds of Alleppey green variety exhibiting prominent antioxidant property was microencapsulated and utilized in formulating an antioxidant-rich custard. However, the antioxidant potency of the prepared custard was not appreciable. To redress the phytochemical loss during custard preparation, custard using nanoliposomes was formulated. Patents related to 1,8 cineole-rich food products have been revised thoroughly. OBJECTIVE: The objective of the current study is to examine whether nanoencapsulationmediated entrapment of antioxidants is more effective in fortifying a dessert, namely custard, vis-à-vis microencapsulated (spray dried)-mediated enhancement of antioxidative potency in the same. METHODS: Our previous investigations have established that nanoliposome of 1,8 cineole- rich supercritical CO2 extract of small cardamom seeds effectively redresses type 2 diabetes and hypercholesterolemia. In the current investigation, this pre-characterized nanoliposome which exhibited appreciable in vitro and in vivo antioxidant efficacy has been utilized at varying concentrations for fortification of a custard. The designer custard samples have been characterized for their sensory and physicochemical properties, identification of the cardamom antioxidants therein and determination of the synergistic efficacy value of the identified antioxidants. RESULTS: The custard formulated with 0.3% nanoliposomes exhibited appreciable antioxidant potency in terms of DPPH radical scavenging activity (304.58±1.09 mg/ml) and reducing power (0.020±0.001 mg BHT/g custard), conferred by its total phenolic content (0.049±0.004 mg GAE/g custard). It also had relatively more stable textural attributes vis-à-vis the control sample (formulated with the non-encapsulated native extract). GCMS analysis of the nanoliposome-fortified custard confirmed retention of the spice antioxidants, namely1,8- cineole, α-terpinyl acetate, α-terpineol and linalool and its synergistic efficacy value being greater than unity, attested to the synergistic presence of the said antioxidants therein. The newly formulated custard retained more than 4.5 times of 1,8-cineole (5.05 mg/g custard) vis-à-vis the custard sample (1.12 mg/g custard) prepared with a microencapsulated (spray-dried) formulation of the extract. Additionally, the absence of heavy metals in the formulated custard confirmed it to be safe for human consumption. CONCLUSION: This is the first study on the application of nanoliposomes of spiceuticals in the formulation of a dessert, and more emphatically on use of a 'green' supercritical CO2 extract of spice antioxidants in fortification of a dessert to achieve antioxidant synergy.

Is 1,8-Cineole-Rich Extract of Small Cardamom Seeds More Effective in Preventing Alzheimer's Disease than 1,8-Cineole Alone?

The present study demonstrates the efficacies of synthetic 1,8-cineole and an 1,8-cineole-rich supercritical carbon dioxide (SC-CO2) extract of small cardamom seeds in preventing oligomerization of amyloid beta peptide (Aß42) and inhibiting iron-dependent oxyradical production in vitro. The oligomerization of Aß42 was monitored by thioflavin T assay and MALDI-TOF analysis of the oligomers. The iron-dependent production of oxygen free radicals was detected by fluorometric benzoate hydroxylation assay. We observed that both pure 1,8-cineole and 1,8-cineole-rich extract of small cardamom seeds at concentrations of 50 µM and 100 µM prevented the production of reactive hydroxyl radicals from a mixture of Fe2+ and ascorbate. However, the 1,8-cineole-rich extract of small cardamom seeds prevented in vitro Aß42 oligomerization more effectively vis-à-vis the synthetic (99% pure) 1,8-cineole. Additional study on SHSY5Y cells indicated that both pure 1,8-cineole and 1,8-cineole-rich SC-CO2 extract of small cardamom seeds prevented iron-dependent cell death. Since oxidative damage, Aß42 aggregation and loss of cell viability (iron-induced) are characteristics of onset of Alzheimer's disease pathology, our results suggest a putative therapeutic role of 1,8-cineole-rich extract of small cardamom seeds over pure 1,8-cineole in preventing this neurodegenerative disease.

Data on small cardamom transcriptome associated with capsule rot disease.

Small cardamom (Elettaria cardamomum (L.) Maton, also known as the 'Queen of Spices' is a rhizomatous herbaceous monocot from the family Zingiberaceae. In the present study, using HiSeq™ 2000 RNA sequencing technology, transcriptome sequencing was performed for both control and disease stressed small cardamom leaf tissues. RNA-seq generated 46,931,637 (101 base) and 31,682,496 (101 base) raw reads and totally 9.93GB and 6.63GB of sequence data for cardamom control and stressed samples respectively. The raw data were submitted to NCBI SRA database of under the accession numbers SRX2512359 and SRX2512358 for the control and diseased samples respectively. The raw reads were quality filtered and assembled using TRINITY de novo assembler which created 1,11,495 (control) and 91,096 (diseased) contigs with N50 values 3013 (control) and 2729 (stressed). The data was further used to identify significantly differentially expressed unigenes between control and stressed samples. Assembled unigenes were further annotated and evaluated in silico to predict the function using publicly available databases and gene annotation tools.

Nanoliposomes of Supercritical Carbon Dioxide Extract of Small Cardamom Seeds Redresses Type 2 Diabetes and Hypercholesterolemia.

BACKGROUND & OBJECTIVES: In our previous investigation, oral administration of 1,8- cineole-rich supercritical carbon dioxide extract of small cardamom seeds in Wistar albino rats resulted in achieving normal fasting blood glucose (FBG) and serum cholesterol levels. The objective of this study was to further protect the aforesaid extract and to enhance its in vivo therapeutic efficacies in redressing type 2 diabetes and hypercholesterolemia, by encapsulating it as nanoliposomes. Patents related to nanoliposomes have been revised thoroughly. METHODS: PEGylated nanoliposomes of the aforesaid extract were formulated using soya phosphatidylcholine and Tween 80 by probe-sonication. These nanoliposomes were subjected to in vitro characterizations and were orally administered to Wistar albino rats at three different doses viz. 550, 175 and 55 mg/kg b.w. for detailed investigation of their antidiabetic and hypocholesterolemic efficacies. RESULTS: FT-IR, DSC and XRD analyses, HLB value (16), entrapment efficiency (84%) and release kinetics (obeying Higuchi model) revealed that the nanoliposomes were o/w type and were hydrophilic. They exhibited appreciable in vitro antioxidant potency (59% DPPH scavenging activity) owing to a synergistic consortium of antioxidants present therein. Oral administration of the liposomes in rats at 550 mg/kg b.w. could restore their normal FBG levels and serum lipid profiles on day 35, with desirable up-down regulations of related key enzymes. The iHOMA2 model could successfully predict the effects of nanoliposomes on insulin sensitivity and glucose uptake in rat liver and brain, respectively. CONCLUSION: Nanoliposome of 1,8-cineole rich extract of small cardamom seeds is a new biotherapeutic in redressing type 2 diabetes and hypercholesterolemia.

Consortia of bioactives in supercritical carbon dioxide extracts of mustard and small cardamom seeds lower serum cholesterol levels in rats: new leads for hypocholesterolaemic supplements from spices.

Melatonin-rich and 1,8-cineole-rich extracts have been successfully obtained from yellow mustard (YM) and small cardamom (SC) seeds, respectively, employing green technology of supercritical CO2 (SC-CO2) extraction. Chemical profiling confirmed the presence of melatonin and 1,8-cineole and co-extractants in the respective extracts. Electron paramagnetic resonance spectroscopy attested strong antioxidant activities of the extracts foregoing pan-assay interference compounds involved in spectroscopic analysis. These extracts also exhibited synergistic efficacies greater than unity confirming antioxidant synergy among the co-extracted bioactives therein. To ascertain hypocholesterolaemic efficacies, these extracts were co-administered orally with Triton X (at the pre-optimised dose of 175 mg/kg body weight (BW)) to Wistar albino rats at doses of 550, 175 and 55 mg/kg BW. Serum total cholesterol levels in the rats were monitored on days 3, 7, 15 and 21. On day 21, total cholesterol level reduced appreciably by 49·44 % in rats treated with YM seed extract and by 48·95 % in rats treated with SC seed extract, comparable with atorvastatin-administered rats (51·09 %). Either extract demonstrated inhibitory effects on hepatic 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase activity. A molecular docking exercise identified specific compounds in the extracts which possessed binding affinities comparable with therapeutically used HMG-CoA reductase inhibitors. In silico and in vivo studies concertedly concluded that the consortium of bioactive components in the extracts cannot be considered as invalid metabolic panaceas and therefore these 'green' extracts could be safely subjected to clinical studies as preventive biotherapeutics for hypercholesterolaemia. These extracts could be consumed per se as hypocholesterolaemic supplements or could be ingredients of new spice-based therapeutic foods.

Survey and RT-PCR Based Detection of Cardamom mosaic virus Affecting Small Cardamom in India.

Mosaic or marble or katte disease caused by Cardamom mosaic virus (CdMV) is an important production constraint in all cardamom growing regions of the world. In the present study, 84 cardamom plantations in 44 locations of Karnataka and Kerala were surveyed. The incidence of the disease ranged from 0 to 85%. The incidence was highest in Madikeri (Karnataka) while no incidence was recorded in Peermade (Kerala). In general, incidence and severity of the disease was higher in cardamom plantations of Karnataka. A procedure for total RNA isolation from cardamom and detection of CdMV through reverse transcription-polymerase chain reaction (RT-PCR) using primers targeting the conserved region of coat protein was standardized and subsequently validated by testing more than 50 field cardamom samples originating from Karnataka and Kerala states. The method can be used for indexing the planting material and identifying resistant lines/cultivars before either they are further multiplied in large scale or incorporated in breeding.