Development of an allele-specific PCR (AS-PCR) method for identifying high-methyl eugenol-containing purple Tulsi (Ocimum tenuiflorum L.) in market samples.

BACKGROUND: Ocimum tenuiflorum L. is a highly traded medicinal with several therapeutic values. Green Tulsi and purple Tulsi are two subtypes in O. tenuiflorum and both have the same medicinal properties. Recent reports have revealed that purple Tulsi contains higher quantities of methyl eugenol (ME), which is moderately toxic and potentially carcinogenic. Therefore, we developed an allele-specific PCR (AS-PCR) method to distinguish the green and purple Tulsi. METHODS AND RESULT: Using the green Tulsi as a reference, 12 single nucleotide polymorphisms (SNPs) and 10 insertions/deletions (InDels) were identified in the chloroplast genome of the purple Tulsi. The C > T SNP at the 1,26,029 position in the ycf1 gene was selected for the development of the AS-PCR method. The primers were designed to amplify 521 bp and 291 bp fragments specific to green and purple Tulsi, respectively. This AS-PCR method was validated in 10 accessions from each subtype and subsequently verified using Sanger sequencing. Subsequently, 30 Tulsi powder samples collected from the market were subjected to molecular identification by AS-PCR. The results showed that 80% of the samples were purple Tulsi, and only 3.5% were green Tulsi. About 10% of the samples were a mixture of both green and purple Tulsi. Two samples (6.5%) did not contain O. tenuiflorum and were identified as O. gratissimum. CONCLUSION: The market samples of Tulsi were predominantly derived from purple Tulsi. The AS-PCR method will be helpful for quality control and market surveillance of Tulsi herbal powders.

Chloroplast genome of Ocimum basilicum var. purpurascens Bentham 1830 (Lamiaceae).

Ocimum basilicum var. purpurascens Bentham 1830 (Red Rubin Basil) is an aromatic herb belonging to the family Lamiaceae and is known for its medicinal uses. It is commonly used in traditional medicine to treat cardiovascular diseases and obesity. It possesses anti-inflammatory, antioxidant, antifungal, and anti-spasmodic properties. In our recent study, we assembled the chloroplast genome sequence of O. basilicum var. purpurascens using Illumina paired-end sequencing technology. The assembled chloroplast genome was 152,407 base pairs (bp), inclusive of a large single-copy (LSC) region accounting for 83,409 bp and a small single-copy (SSC) region spanning 17,604 bp. Two inverted repeats (IRs) interspersed these regions, each 25,697 bp long. The chloroplast genome harbored 132 genes, comprising 88 protein-coding genes, 36 transfer RNA (tRNA), and eight rRNA genes. Among these, nine genes encompassed a single intron, two presented with two introns, with the remaining devoid of any introns. The overall GC content of the chloroplast genome was determined to be 38%. The GC content in the LSC, SSC, and IR regions was 35.9%, 31.6%, and 43.1%, respectively. Our phylogenetic exploration of the chloroplast genomes elucidated that O. basilicum var. purpurascens exhibits close genetic affinity with O. basilicum var. basilicum and other constituents of the Ocimum genus within the Lamiaceae family.

The complete chloroplast genome of Ocimum basilicum L. var. basilicum (Lamiaceae) and its phylogenetic analysis.

Ocimum basilicum L. var. basilicum (Sweet Basil) is an aromatic herb belonging to the family Lamiaceae and is known for its medicinal uses. It is commonly used in traditional medicine for its therapeutic value, including anti-allergic, anti-inflammatory, antioxidant, antitumor, and antimicrobial properties. In this study, we generated the complete chloroplast genome sequence of O. basilicum var. basilicum using Illumina paired-end sequencing data. The chloroplast genome was 152,407 bp in length, containing a large single-copy (LSC) region of 83,409 bp and a small single-copy region (SSC) of 17,604 bp, separated by a pair of inverted repeats (IRs) of 25,697 bp. The genome contained 134 genes, including 89 protein-coding, 37 tRNA, and eight rRNA genes. Nine genes had one intron, two genes had two introns, and others did not have any intron. Overall GC content of the chloroplast genome was 38%, while that of LSC, SSC, and IR regions was 35.9%, 31.6%, and 43.1%, respectively. Phylogenetic analysis of the chloroplast genomes revealed that O. basilicum var. basilicum was closely related to Ocimum basilicum from the Ocimum species.

Transcriptome analysis of five different tissues of bitter gourd (Momordica charantia L.) fruit identifies full-length genes involved in seed oil biosynthesis.

The bitter gourd seed oil, rich in conjugated fatty acids, has therapeutic value to treat cancer, obesity, and aging. It also has an industrial application as a drying agent. Despite its significance, genomics studies are limited, and the genes for seed oil biosynthesis are not fully understood. In this study, we assembled the fruit transcriptome of bitter gourd using 254.5 million reads (Phred score > 30) from the green rind, white rind, pulp, immature seeds, and mature seeds. It consisted of 125,566 transcripts with N50 value 2,751 bp, mean length 960 bp, and 84% completeness. Transcript assembly was validated by RT-PCR and qRT-PCR analysis of a few selected transcripts. The transcripts were annotated against the NCBI non-redundant database using the BLASTX tool (E-value < 1E-05). In gene ontology terms, 99,443, 86,681, and 82,954 transcripts were classified under biological process, molecular function, and cellular component. From the fruit transcriptome, we identified 26, 3, and 10 full-length genes coding for all the enzymes required for synthesizing fatty acids, conjugated fatty acids, and triacylglycerol. The transcriptome, transcripts with tissue-specific expression patterns, and the full-length identified from this study will serve as an important genomics resource for this important medicinal plant.

The complete chloroplast genome and phylogenetic analysis of Ocimum kilimandscharicum Gurke (Camphor Basil) from India.

Ocimum kilimandscharicum Gurke commonly known as Camphor Basil, is a medicinal plant species that belongs to the Lamiaceae family. Here, the sequencing and characterization of complete chloroplast genome sequence of O. kilimandscharicum is reported for the first time using Illumina paired-end sequencing data. The size of the chloroplast (cp) genome is 151,741 bp in length, with a large single-copy (LSC) region of 82,882 bp and a small single-copy (SSC) region of 17,587 bp, separated by a pair of 25,636 bp inverted repeat (IR) regions. There are 135 predicted genes, including 90 protein-coding genes, 37 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes in the genome, and the overall GC content of the genome is 37.9%. The phylogenetic analysis based on the chloroplast genome data indicated that O. kilimandscharicum is closer to O. tenuiflorum and clustered to other Ocimum species in Lamiaceae.

The complete chloroplast genome of Ocimum gratissimum from India - a medicinal plant in the Lamiaceae.

Ocimum gratissimum L. is an important medicinal species with several therapeutic applications. It is used in traditional medicine as a single drug and in formulations. We generated the complete chloroplast genome sequence of O. gratissimum by using Illumina paired-end sequencing data. The O. gratissimum chloroplast genome is 152,469 bp in length, containing a large single copy (LSC) region of 83,614 bp and a small single copy region (SSC) of 17,607 bp, separated by a pair of inverted repeats (IRs) of 25,624 bp. The genome contains 138 unique genes, including 85 protein-coding, 45 tRNA, and eight rRNA genes. Among them, six genes have one intron each, and two genes contain two introns. The overall GC content of the chloroplast genome is 37.8%, while the corresponding values of LSC, SSC, and IR regions are 35.6%, 31.7%, and 43.2%, respectively. Phylogenetic analysis with the complete chloroplast genomes of other related species revealed that O. gratissimum is fully resolved in a clade with other Ocimum species classified to the family Lamiaceae.

Protein extracts from Momordica charantia var. charantia and M. charantia var. muricata show anti-lipidemic and antioxidant properties in experimental type 2 diabetic rats.

Plant-derived compounds are used to manage dyslipidemia and oxidative stress in type 2 diabetic condition. In this study, anti-lipidemic and antioxidant properties of the protein extracts from "Charantia" (PEC) and "Muricata" (PEM) varieties of Momordica charantia were analyzed by quantifying lipids, hepatic, renal, and oxidative stress markers, and histopathological examination of liver and kidney tissues. Protein extracts were orally administered at 10 (PEC10, PEM10) or 20 mg/kg body weight (PEC20, PEM20). Levels of cholesterol, low-density lipoprotein, and triglycerides decreased but high-density lipoprotein increased significantly in treated rats as compared to untreated diabetic rats (p < .01), and attained normal physiological range in both doses. Levels of superoxide dismutase, catalase, glutathione peroxidase, and reduced glutathione increased but thiobarbituric acid reactive substances decreased significantly in treated rats as compared to untreated diabetic rats (p < .01), and attained normal physiological range in PEM20 only. Histopathological examinations supported a protective role for the protein extracts against oxidative stress. PRACTICAL APPLICATIONS: Momordica charantia, a well-known medicinal plant is traditionally used for treating diabetes in India as well as other countries. The whole plant was shown to have medicinal importance. Anti-diabetic potential of this plant was scientifically established largely using organic extracts and water extract mainly from the fruits of this plant. However, protein extracts from the seeds and fruit pulp of this plant were also proven to have anti-diabetic activity. The present study illustrates the anti-lipidemic and antioxidant effect of protein extracts from the fruit pulp of two varieties of M. charantia (Charantia and Muricata) in Streptozotocin-induced type 2 diabetic rats. This study provides experimental evidence in support of its use in the management of type 2 diabetes-related complications.

Authentication of the market samples of Ashwagandha by DNA barcoding reveals that powders are significantly more adulterated than roots.

ETHNOPHARMACOLOGICAL RELEVANCE: Ashwagandha, also known as Indian Ginseng, is a highly traded medicinal plant, which is used in Ayurveda, Siddha and Unani systems of medicine to improve cognitive function, decrease inflammation, and to counter the ill-effects of aging. Withanolide A and Withaferin A from Ashwagandha were shown to improve immunity and have anti-cancer property, respectively. AIM OF THE STUDY: Here, we aimed to create reference DNA barcodes for W. somnifera and to authenticate root and powder samples of Ashwagandha collected from markets. MATERIALS AND METHODS: Three plant specimen of W. somnifera were collected, and reference DNA barcodes were generated using rbcL, matK, trnH-psbA, and ITS2 DNA barcode markers. Market samples in the form of root (n = 33) and powder (n = 70) were collected and authenticated using ITS2 and trnH-psbA DNA barcodes. RESULTS: Genomic DNA was successfully isolated from all plant specimens and market samples. DNA barcoding showed that 77% of samples were authentic. About 22% of non-authentic samples were powder samples and only 1% were root samples. Among the non-authentic samples, 18% were completely substituted with single species (Mucuna pruriens (L.) DC., Trigonella foenum-graceum L., or Senna auriculata (L.) Roxb.) and 82% were mixed samples containing more than one species. About 63% of the mixed samples contained Ashwagandha as the major ingredient. Furthermore, we identified that six taxonomically divergent plant species from four families were present as adulterants in the mixed samples. CONCLUSION: DNA barcoding revealed that botanical adulteration in the market samples of Ashwagandha is significant. Powder samples are more prone to adulteration than root samples. The adulterated samples contained plant material that is not related to Ashwagandha, which warrants strict quantity control and market surveillance to derive the true medicinal benefits of this medicinal plant.

Inhibition of New Delhi Metallo-ß-Lactamase 1 (NDM-1) Producing Escherichia coli IR-6 by Selected Plant Extracts and Their Synergistic Actions with Antibiotics.

Improper use of antibiotics has led to a great concern in the development of pathogenic microbial resistance. New Delhi metallo-ß-lactamase 1 (NDM-1) producing bacteria are resistant to most of the ß-lactam antibiotics, and so far, no new compounds have been clinically tested against these bacteria. In this study, ethanol extracts from the leaves of 240 medicinal plant species were screened for antibacterial activity against an NDM-1 Escherichia coli strain. The extracts that showed antibacterial activity were then tested for minimum inhibitory concentrations (MICs) and zones of inhibition. The extract from Combretum albidum G. Don, Hibiscus acetosella Welw. ex Hiern, Hibiscus cannabinus L., Hibiscus furcatus Willd., Punica granatum L., and Tamarindus indica L. showed bactericidal activity between 5 and 15 mg/ml and the MIC was between 2.56 and 5.12 mg/ml. All six plant extracts inhibited activity of the NDM-1 enzyme in vitro, and the IC50 value ranged between 0.50 and 1.2 ng/µl. Disruption of bacterial cell wall integrity by the plant extracts was clearly visible with scanning electron microscopy. Increases in membrane permeability caused 79.4-89.7% bacterial cell deaths as investigated by fluorescence-activated cell sorting. All the plant extracts showed synergistic effects when combined with colistin [fractional inhibitory concentration (ΣFIC) = 0.125-0.375], meropenem (ΣFIC = 0.09-0.313), and tetracycline (ΣFIC = 0.125-0.313). Thus, the plant extracts can be fractionated for the identification of active compounds, which could be used as new antibacterial compounds for the development of drugs against NDM-1 E. coli in addition to their use in combination therapy.

Identification of species adulteration in traded medicinal plant raw drugs using DNA barcoding.

Plants are the major source of therapeutic ingredients in complementary and alternative medicine (CAM). However, species adulteration in traded medicinal plant raw drugs threatens the reliability and safety of CAM. Since morphological features of medicinal plants are often not intact in the raw drugs, DNA barcoding was employed for species identification. Adulteration in 112 traded raw drugs was tested after creating a reference DNA barcode library consisting of 1452 rbcL and matK barcodes from 521 medicinal plant species. Species resolution of this library was 74.4%, 90.2%, and 93.0% for rbcL, matK, and rbcL + matK, respectively. DNA barcoding revealed adulteration in about 20% of the raw drugs, and at least 6% of them were derived from plants with completely different medicinal or toxic properties. Raw drugs in the form of dried roots, powders, and whole plants were found to be more prone to adulteration than rhizomes, fruits, and seeds. Morphological resemblance, co-occurrence, mislabeling, confusing vernacular names, and unauthorized or fraudulent substitutions might have contributed to species adulteration in the raw drugs. Therefore, this library can be routinely used to authenticate traded raw drugs for the benefit of all stakeholders: traders, consumers, and regulatory agencies.

In vitro and in vivo α-amylase and α-glucosidase inhibiting activities of the protein extracts from two varieties of bitter gourd (Momordica charantia L.).

BACKGROUND: α-amylase and α-glucosidase digest the carbohydrates and increase the postprandial glucose level in diabetic patients. Inhibiting the activity of these two enzymes can control postprandial hyperglycemia, and reduce the risk of developing diabetes. Bitter gourd or balsam pear is one of the important medicinal plants used for controlling postprandial hyperglycemia in diabetes patients. However, there is limited information available on the presence of α-amylase and α-glucosidase inhibiting compounds. In the current study, the protein extracts from the fruits of M. charantia var. charantia (MCC) and M. charantia var. muricata (MCM) were tested for α-amylase and α-glucosidase inhibiting activities in vitro, and glucose lowering activity after oral administration in vivo. RESULTS: The protein extract from both MCC and MCM inhibited the activity of α-amylase and α-glucosidase through competitive inhibition, which was on par with Acarbose as indicated by in vitro percentage of inhibition (66 to 69 %) and IC50 (0.26 to 0.29 mg/ml). Both the protein extracts significantly reduced peak blood glucose and area under the curve in Streptozotocin-induced diabetic rats, which were orally challenged with starch and sucrose. CONCLUSIONS: Protein extracts from the fruits of the two varieties of bitter gourd inhibited α-amylase and α-glucosidase in vitro and lowered the blood glucose level in vivo on par with Acarbose when orally administrated to Streptozotocin-induced diabetic rats. Further studies on mechanism of action and methods of safe and biologically active delivery will help to develop an anti-diabetic oral protein drug from these plants.

Creation of reference DNA barcode library and authentication of medicinal plant raw drugs used in Ayurvedic medicine.

BACKGROUND: Ayurveda is a system of traditional medicine that originated in ancient India, and it is still in practice. Medicinal plants are the backbone of Ayurveda, which heavily relies on the plant-derived therapeutics. While Ayurveda is becoming more popular in several countries throughout the World, lack of authenticated medicinal plant raw drugs is a growing concern. Our aim was to DNA barcode the medicinal plants that are listed in the Ayurvedic Pharmacopoeia of India (API) to create a reference DNA barcode library, and to use the same to authenticate the raw drugs that are sold in markets. METHODS: We have DNA barcoded 347 medicinal plants using rbcL marker, and curated rbcL DNA barcodes for 27 medicinal plants from public databases. These sequences were used to create Ayurvedic Pharmacopoeia of India - Reference DNA Barcode Library (API-RDBL). This library was used to authenticate 100 medicinal plant raw drugs, which were in the form of powders (82) and seeds (18). RESULTS: Ayurvedic Pharmacopoeia of India - Reference DNA Barcode Library (API-RDBL) was created with high quality and authentic rbcL barcodes for 374 out of the 395 medicinal plants that are included in the API. The rbcL DNA barcode differentiated 319 species (85 %) with the pairwise divergence ranging between 0.2 and 29.9 %. PCR amplification and DNA sequencing success rate of rbcL marker was 100 % even for the poorly preserved medicinal plant raw drugs that were collected from local markets. DNA barcoding revealed that only 79 % raw drugs were authentic, and the remaining 21 % samples were adulterated. Further, adulteration was found to be much higher with powders (ca. 25 %) when compared to seeds (ca. 5 %). CONCLUSIONS: The present study demonstrated the utility of DNA barcoding in authenticating medicinal plant raw drugs, and found that approximately one fifth of the market samples were adulterated. Powdered raw drugs, which are very difficult to be identified by taxonomists as well as common people, seem to be the easy target for adulteration. Developing a quality control protocol for medicinal plant raw drugs by incorporating DNA barcoding as a component is essential to ensure safety to the consumers.

Genome-wide DNA polymorphisms in Kavuni, a traditional rice cultivar with nutritional and therapeutic properties.

Although rice genome was sequenced in the year 2002, efforts in resequencing the large number of available accessions, landraces, traditional cultivars, and improved varieties of this important food crop are limited. We have initiated resequencing of the traditional cultivars from India. Kavuni is an important traditional rice cultivar from South India that attracts premium price for its nutritional and therapeutic properties. Whole-genome sequencing of Kavuni using Illumina platform and SNPs analysis using Nipponbare reference genome identified 1 150 711 SNPs of which 377 381 SNPs were located in the genic regions. Non-synonymous SNPs (62 708) were distributed in 19 251 genes, and their number varied between 1 and 115 per gene. Large-effect DNA polymorphisms (7769) were present in 3475 genes. Pathway mapping of these polymorphisms revealed the involvement of genes related to carbohydrate metabolism, translation, protein-folding, and cell death. Analysis of the starch biosynthesis related genes revealed that the granule-bound starch synthase I gene had T/G SNPs at the first intron/exon junction and a two-nucleotide combination, which were reported to favour high amylose content and low glycemic index. The present study provided a valuable genomics resource to study the rice varieties with nutritional and medicinal properties.

DNA barcoding for species identification from dried and powdered plant parts: a case study with authentication of the raw drug market samples of Sida cordifolia.

The majority of the plant materials used in herbal medicine is procured from the markets in the form of dried or powdered plant parts. It is essential to use authentic plant materials to derive the benefits of herbal medicine. However, establishing the identity of these plant materials by conventional taxonomy is extremely difficult. Here we report a case study in which the species identification of the market samples of Sida cordifolia was done by DNA barcoding. As a prelude to species identification by DNA barcoding, 13 species of Sida were collected, and a reference DNA barcode library was developed using rbcL, matK, psbA-trnH and ITS2 markers. Based on the intra-species and inter-species divergence observed, psbA-trnH and ITS2 were found to be the best two-marker combination for species identification of the market samples. The study showed that none of the market samples belonged to the authentic species, S. cordifolia. Seventy-six per cent of the market samples belonged to other species of Sida. The predominant one was Sida acuta (36%) followed by S. spinosa (20%), S. alnifolia (12%), S. scabrida (4%) and S. ravii (4%). Such substitutions may not only fail to give the expected therapeutic effect, but may also give undesirable effects as in case of S. acuta which contains a 6-fold higher amount of ephedrine compared to the roots of S. cordifolia. The remaining 24% of the samples were from other genera such as Abutilon sp. (8%), Ixonanthes sp., Terminalia sp., Fagonia sp., and Tephrosia sp. (4% each). This observation is in contrast to the belief that medicinal plants are generally substituted or adulterated with closely related species. The current study strongly suggests that the raw drug market samples of herbal medicines need to be properly authenticated before use, and DNA barcoding has been found to be suitable for this purpose.

DNA barcode authentication of wood samples of threatened and commercial timber trees within the tropical dry evergreen forest of India.

BACKGROUND: India is rich with biodiversity, which includes a large number of endemic, rare and threatened plant species. Previous studies have used DNA barcoding to inventory species for applications in biodiversity monitoring, conservation impact assessment, monitoring of illegal trading, authentication of traded medicinal plants etc. This is the first tropical dry evergreen forest (TDEF) barcode study in the World and the first attempt to assemble a reference barcode library for the trees of India as part of a larger project initiated by this research group. METHODOLOGY/PRINCIPAL FINDINGS: We sampled 429 trees representing 143 tropical dry evergreen forest (TDEF) species, which included 16 threatened species. DNA barcoding was completed using rbcL and matK markers. The tiered approach (1st tier rbcL; 2nd tier matK) correctly identified 136 out of 143 species (95%). This high level of species resolution was largely due to the fact that the tree species were taxonomically diverse in the TDEF. Ability to resolve taxonomically diverse tree species of TDEF was comparable among the best match method, the phylogenetic method, and the characteristic attribute organization system method. CONCLUSIONS: We demonstrated the utility of the TDEF reference barcode library to authenticate wood samples from timber operations in the TDEF. This pilot research study will enable more comprehensive surveys of the illegal timber trade of threatened species in the TDEF. This TDEF reference barcode library also contains trees that have medicinal properties, which could be used to monitor unsustainable and indiscriminate collection of plants from the wild for their medicinal value.

De novo assembly and transcriptome analysis of five major tissues of Jatropha curcas L. using GS FLX titanium platform of 454 pyrosequencing.

BACKGROUND: Jatropha curcas L. is an important non-edible oilseed crop with promising future in biodiesel production. However, factors like oil yield, oil composition, toxic compounds in oil cake, pests and diseases limit its commercial potential. Well established genetic engineering methods using cloned genes could be used to address these limitations. Earlier, 10,983 unigenes from Sanger sequencing of ESTs, and 3,484 unique assembled transcripts from 454 pyrosequencing of uncloned cDNAs were reported. In order to expedite the process of gene discovery, we have undertaken 454 pyrosequencing of normalized cDNAs prepared from roots, mature leaves, flowers, developing seeds, and embryos of J. curcas. RESULTS: From 383,918 raw reads, we obtained 381,957 quality-filtered and trimmed reads that are suitable for the assembly of transcript sequences. De novo contig assembly of these reads generated 17,457 assembled transcripts (contigs) and 54,002 singletons. Average length of the assembled transcripts was 916 bp. About 30% of the transcripts were longer than 1000 bases, and the size of the longest transcript was 7,173 bases. BLASTX analysis revealed that 2,589 of these transcripts are full-length. The assembled transcripts were validated by RT-PCR analysis of 28 transcripts. The results showed that the transcripts were correctly assembled and represent actively expressed genes. KEGG pathway mapping showed that 2,320 transcripts are related to major biochemical pathways including the oil biosynthesis pathway. Overall, the current study reports 14,327 new assembled transcripts which included 2589 full-length transcripts and 27 transcripts that are directly involved in oil biosynthesis. CONCLUSION: The large number of transcripts reported in the current study together with existing ESTs and transcript sequences will serve as an invaluable genetic resource for crop improvement in jatropha. Sequence information of those genes that are involved in oil biosynthesis could be used for metabolic engineering of jatropha to increase oil content, and to modify oil composition.