(Curcuma Caesia) Using Rbcl Gene

International Research Journal of Applied Sciences, Engineering and Technology Vol.6, No.7; July-2020; ISSN (1573-1405); p –ISSN 0920-5691 Impact factor: 3.81

MOLECULAR AUTHENTICATION AND DNA BARCODING OF NEPALI BLACK TURMERIC (CURCUMA CAESIA) USING RBCL GENE.

Deepak Sharma and Janardan Lamichhane Department of Biotechnology, School of Science, Kathmandu University, Nepal Abstract: High value medicinal plants from different altitudes of mountains and Himalayas of Nepal have been documented in earlier days to authenticate the existence of such plants in different specific regions of the country. In this project black turmeric (Curcuma caesia Roxb.) commonly known as “Kaalo haledo” in Nepali language is selected as it is widely used by local community for various purpose and have high medicinal value. To assure and identify the correct species on the basis of molecular study barcoding is used with its conserved sequence and detailed analysis is carried out. Black turmeric is a one of the most important high value medicinal plant of Nepal belonging to zingiberaceae family. It is a perennial herb with bluish-black rhizome. Along with rhizome of this plant leaves are also claimed to be useful in treating several disease like piles, leprosy, bronchitis, asthma, cancer, epilepsy, fever, wounds, impotency, fertility, toothache and vomiting. As rhizome of black turmeric has a highly economic importance owing to its medicinal properties, it is needed to differentiate with other species of turmeric found in Nepal. To authenticate it we have isolated its genomic DNA and performed sequence analysis by amplification of barcode candidate genes rbcL, matK and ITS collected from three location of Nepal. The most prominent samples from Palpa, a hilly area of Province 5 of Nepal, was found to have 100% sequence similarity in NCBI Blast search output with Curcuma caesia and MEGA X tools with the phylogenic output of 10 closely associated sequence samples also zeal with NJ Bootstrap 1000 value which supported the results of the sample identity. Other two barcode matK and ITS also showed the same pattern of similarity with other species too. Keyword: Black Turmeric, rbcL, matK, DNA Barcode, molecular study and Mega X.

Introduction: community and national development is equally important. Nepal occupies a large number of medicinal plants in In last few years, our research group is engaged to different altitudes. Large numbers of community people authenticate such high value medicinal plant by using still rely on these medicinal plants in their daily life as per different quantitative analysis tools [5] to address the value the traditional healer’s instructions [1] where medical of those medicinal plants and authenticate them by DNA supply is lacking due to the less accessibility of roads. barcoding [6]. Large number of these plants in terms of Exploration of such high value medicinal plants by their local medical uses, food and economic value in the screening their Phytochemicals, antibacterial and community level is explored and studied. Rhodedendron antioxidant properties [2], total phenolic and flavonoid arboreum [7], Myrica esculanta [8], Diplokhema content study for their medical value [3] and identification butyracea spp. [9] are barcoded by analyzing their of major chemical constituents [4] for the lead compounds phytochemicals, microbial and antioxidant analysis. in future drug development is very important. Besides, the Black turmeric is one of the most commonly used conservation of natural diversity and use them for the medicinal herbs [18], by the community people. Its

International Research Journal of Applied Sciences, Engineering and Technology Official Publication of Center for International Research Development Double Blind Peer and Editorial Review International Referred Journal; Globally index Available www.cird.online/IRJASET: E-mail: [email protected] pg. 1 International Research Journal of Applied Sciences, Engineering and Technology Vol.6, No.7; July-2020; ISSN (1573-1405); p –ISSN 0920-5691 Impact factor: 3.81 rhizome is tuberous and has camphoraceous sweet odor provide relief from indigestion or stomach pain. It also and 2-6 cm in diameter, the shape and size is often variable. helps in easy digestion and the proper functioning of the It is sessile, and covered with adventitious roots, root scars liver and kidneys [2]. and warts and is laterally flattened. The nodal and inter Rhizome and leaves of C. caesia is found to be used in nodal zones present due to its circular wrinkles on the different parts of the world. It is used as a tonic for the brain surface. The surface of rhizome is dark brown, bluish and the heart. Rhizomes are often used for treatment of black, or buff in color; a false impression of growth rings leukoderma, piles, bronchitis, asthma, Tumors, is the circular arrangements of remnants of scaly leaves. tuberculous glands of the neck, enlargement of the spleen, The branching is more or less sympodral. The leaves are epileptic [15]. Rhizome of C. caesia is grounded in the found of 10-20 grouped with broad oblong lanceolate and form of a paste in rheumatic arthritis. Fresh rhizome glabrous. In the middle region the lamina shows deep decoction is used as anti-diarrhoeiaric and to get relief farraginous purple colored clouds. The petiole is ivory from stomachache. The fresh rhizome paste of C. caesia is color and unsheathing the petioles encircles each other applied during the snake bite and scorpion bite. In some forming a pseudo axis. The variation is parallel, typical parts of India fresh rhizome juice mixed with mustard oil characteristic of monocots. The inflorescence is 15-20 cm and is given to cattle in dysentery [20]. long dense spike, which arises much before the opening of DNA barcoding bloomed as a routine application in leaf, the bracts are green, and the bracts of coma are deep biodiversity assessment and species identification. Several red, when it is old it become crimson. The flower is smaller improvements have occurred in the DNA sequence than bracts with pale yellow and reddish border. Calyx is analysis approaches. DNA barcoding is a new an important 10-15 mm long, obtuse, 3 toothed, and Corolla is long key for species identification and has greatly carried out tubular with pale yellow lip - 3 lobed semi-elliptic [10]. the pace of species discovery. In this aspect, more effective C. caesia is a wonder herb and contains the highest content technique, a short DNA sequence from a standard region of curcumin and it is a chemical substance with many of mitochondrial called “barcode” is used for taxonomy curative properties [11]. It is used for treatment of field. Barcode sequence has been employed as a possible menstrual disorders, piles, impotency and epilepsy. DNA marker for species and a number of studies in a Externally, this plant has been used in the treatment of variety of taxa have accordingly been performed to wounds, white patches on the skin and other skin disorder examine its efficacy. In some studies, barcoding provided allergy along with leprosy sores [12]. It is also capable of a means of highlighting potential cryptic, synonymous or enhancing fertility levels and also toothache and vomiting extinct species as well as matching adults with immature [13]. It is also used for the treatment of enlargement of the specimens. Recent developments in high throughput spleen and certain types of tuberculosis. The rhizome as parallel sequencing technology combined with DNA well as the leaves of the plant is used in medical barcoding can bring together the fields of phylogenetic and formulations [14]. phlogeographic analysis to resolve any problem between Traditionally, the rhizome of the herb is traditionally used species. Such an approach could be standardized for for the treatment [15] of hemorrhoids, leprosy, asthma, analyses at any geographical scale for a range of taxonomic fever, wounds, vomiting, anthelmintic, aphrodisiac, groups to quantify the formation and composition of gonorrheal discharges and inflammation and cold catch in species assemblages. children and also have anti-tumor and anti-oxidant properties [1]. Chewing a small piece of rhizome will International Research Journal of Applied Sciences, Engineering and Technology Official Publication of Center for International Research Development Double Blind Peer and Editorial Review International Referred Journal; Globally index Available www.cird.online/IRJASET: E-mail: [email protected] pg. 2 International Research Journal of Applied Sciences, Engineering and Technology Vol.6, No.7; July-2020; ISSN (1573-1405); p –ISSN 0920-5691 Impact factor: 3.81

Methods and Methodologies: Sample collection: The samples were collected in 2018 Figure 2: Black Turmeric Plant (leaf) from three most potent districts of Nepal, Rupandehi, Palpa and Gulmi and were identified at the Biotechnology Department in Kathmandu University by Prof. Janardan DNA Isolation and Sequencing: DNA was isolated from lamichhane. The vernacular names of C. caesia in different the plant sample by Zymo Minprep kit (50) using its parts of India are different [17]. In hindi it is called Kali manual. Its quality was evaluated on 1.0 % agarose gel haldi, Nar Kachura Krishna Kedar , In Marathi languate it electrophoresis, a single band of high-molecular weight is called Kala-haldi ,Kannada: Kariarishina, Naru Kachora DNA has been observed. ,In Telugu it is pronounces as Nalla Pasupu , whereas in Mitochondria, chloroplast and nuclear genes have been Nepal it is commonly known as Kaalo Haledo. amplified using universal markers such as rbcL, matK and ITS and amplified products were purified. Sanger sequencing of amplicons were carried out using BDT v3.1 Cycle sequencing kit on ABI (3730xl Genetic Analyzer in Barcode Bioscience sequencing Lab in Bangalore, India). The amplicon gene sequences were used to carry out BLAST with the ‘nr’ database of NCBI. Gene Bank database and phylogenetic analysis were done with Mega X [19] software with DNA barcode development in collaboration of Bio Kart scientific team.

Figure 1: Black turmeric Rhizome

Figure 3: Genomic DNA and rbcL gene amplicon QC data

The Sequencing of rbcL gene contig was developed using its reverse and forward sequence and submitted to NCBI for accession number (BankIt 2359233 :(1)). The Blast nr

Data output after blast is shown in the Figure 4 with MSA viewer.

Figure 4: MSA Viewer alignment of rbcL gene show 100% similarity with Curcuma caesia

Figure 5: Contig 1 sequence producing significant alignment in database of NCBI BLAST.

Inferred Ancestral Sequences Ancestral states were inferred using the Maximum Likelihood method and Kimura 2-parameter model [19]. The tree shows a set of possible nucleotides (states) at each ancestral node aced on their inferred likely hood at site [17]. The initial tree was inferred using the method. The rates among sites were treated as being uniform (Uniform rates option). This analysis involved eight nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. There were a total of 599 positions in the final data set. Evolutionary analyses were conducted in MEGAX [19]

Figure 6 : Ancenstral States analyis of contig 1 sequence by ML method using MegaX tools.

Figure 7: Phylogenetic analysis of contig 1 sequence with by using the Maximum Likelihood method and Tamura- Mega X. Molecular Phylogenetic analysis by Maximum Nei model [16]. The tree with the highest log likelihood (- Likelihood method. The evolutionary history was inferred 839.67) is shown. Initial tree(s) for the heuristic search International Research Journal of Applied Sciences, Engineering and Technology Official Publication of Center for International Research Development Double Blind Peer and Editorial Review International Referred Journal; Globally index Available www.cird.online/IRJASET: E-mail: [email protected] pg. 6 International Research Journal of Applied Sciences, Engineering and Technology Vol.6, No.7; July-2020; ISSN (1573-1405); p –ISSN 0920-5691 Impact factor: 3.81

were obtained automatically by applying Neighbor-Join Unique differences in Sequence B 0 and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood Unique differences in Sequence C 0 (MCL) approach, and then selecting the topology with NOTE: The equality of evolutionary rate between superior log likelihood value. The tree is drawn to scale, sequences A (Curcuma caesia rbcL KX758481) with branch lengths measured in the number of and B (Curcuma longa Suranjana NBU 09703 rbcL), with substitutions per site. This analysis involved 11 nucleotide sequence C (Curcuma neilgherrensis TMP114 rbcL sequences. Codon positions included were KF432038) used as an out group in Tajima's relative rate 1st+2nd+3rd+Noncoding. There were a total of 599 test [16]. The χ2 test statistic was 1.00 (P = 0.31731 with 1 positions in the final dataset. Evolutionary analyses were degree[s] of freedom) P-value less than 0.05 is often used conducted in MEGA X [19]. to reject the null hypothesis of equal rates between Table1. Results from the Tajima's test for 3 Sequences lineages. This analysis involved 3 nucleotide sequences. [16] Codon positions included were 1st+2nd+3rd+Noncoding. Configuration Count All positions containing gaps and missing data were Identical sites in all three sequences 572 eliminated (complete deletion option). There were a total of 573 positions in the final dataset. Evolutionary analyses Divergent sites in all three sequences 0 were conducted in MEGA X [19] Unique differences in Sequence A 1

The NCBI based phylogetics sequence analysis for species

Figure: 8 NCBI BLAST alignment output for species C. caesia with highly similar species (taxid:508682)

DNA Barcode development: In support with IPR secrecy for the coding of each nucleotides color frequency with Bio-Rad international laboratory, we develop the DNA barcode for rbcL gene loci of 599 ntd. International Research Journal of Applied Sciences, Engineering and Technology Official Publication of Center for International Research Development Double Blind Peer and Editorial Review International Referred Journal; Globally index Available www.cird.online/IRJASET: E-mail: [email protected] pg. 7 International Research Journal of Applied Sciences, Engineering and Technology Vol.6, No.7; July-2020; ISSN (1573-1405); p –ISSN 0920-5691 Impact factor: 3.81

Figure 9: DNA Barcode of Black turmeric (Curcuma caesia rbcL gene) of Nepal.

Result and discussion: Reference: The rbCL gene isolated from the DNA of black turmeric (Curcuma caesia Roxb) obtained from three different 1. Janardan Lamichhane, Surya B Chhetri, locations of Nepal on PCR amplification showed 577 bp in Mukund Bhandari, Sameer Pokharel, Anaya length (Fig. 3) which on NCBI data base analysis showed Pokharel, Jae Kyung Sohng; its 100% identity and alignments with Curcuma caesia Ethnopharmacologicaal survey, isolated by S.Mohanty. and K.M Panda. [21] from eastern Phytochemical screening and Antibacterial India (Fig. 4) which distinctly clarify the species variation activity measurements of high altitude among the same genus. The phylogenecity analysis of the medicinal plants of Nepal: A bioprospecting data generated from NCBI data base and sequence approach. Indian Journal of Traditional similarity within the subgenus (Fig 8) distinctly Knowledge, 2014; 13(3): 496-507. discriminates this study and it is prompt to be a novel 2. Deepak Sharma, Tara Shrestha, Naresh sequence of Nepali species and authenticate it by DNA Rapal, Satiz C Poudel, Sujan Shrestha, barcoding. The sequence similarity also enhances the Surekshya Sharma, Krishna P Paudel, distinction of species diversity beyond the genus within the Janardan Lamichhane. Screening and geographical limitation from sea level to 1250 meter bioactivity measurement of high altitude altitude. Also revealed distinct genetic identity between medicinal plants of Nepal. Vegetos- An species of Curcuma and Zingiber. High genetic diversity International Journal of Plant Research; documented in the present study provides a baseline data 2016; 29 (4); 41-45. for optimization of conservation programme of the 3. Prashant Neupane, Janardan Lamichhane. Curcuma caesia species. Further detailed study of Estimation of total phenolic content, total phytochemicals, composition of major chemical flavonoid content and antioxidant capacities constituents and detailed study of its genome will open the of five medicinal plants from Nepal. Vegetos; new avenue of drug discovery in future. 2020, 33, 360-366 Acknowledgement: The project has been financially 4. Janardan Lamichhane, Keshab Bhattarai, supported by University Grant Commission (UGC), Nepal, Amit K Shrivastava, Tirtha M Shrestha, SC 2017. Acknowledge goes to Nepal Academy of Science Jain. Chemical Constituents of and Technology (NAST) for providing PhD scholarship Rhododendron lepidotum from Nepal grant during the PhD work. Himalayas. Chemistry of Natural Conflict of Interest: The author has no conflict. Compounds; 2014; 50(4); 767-789.

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