UPLC-DAD Assisted Phytochemical Quantitation Reveals a Sex, Ploidy and Ecogeography Specificity in the Expression Levels of Selected Secondary Metabolites in Medicinal Tinospora cordifolia: Implications for Elites’ Identification Program

Author(s): Rakesh Kr. Thakur, Vijay Rani Rajpal*, S.N. Raina, Pawan Kumar, Anand Sonkar, Lata Joshi

Journal Name: Current Topics in Medicinal Chemistry

Volume 20 , Issue 8 , 2020

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Graphical Abstract:


Abstract:

Background: Medicinal phytochemistry involving UPLC-DAD in an exhaustive analysis involving quantification of eight commercially important phytochemicals viz. syringin, cordifolioside A, magnoflorine, tinocordiside, palmatine, 20β-hydroxyecdysone, L-tetrahydropalmatine and berberine has been done in 143 accessions from eight states and the union territories of Delhi and Jammu & Kashmir of India representing three different ploidy levels viz. diploid (2x), triploid (3x) and synthetic tetraploid (4x). The study was done to assess the effect of sex, ploidy level and ecogeography on the expression level of secondary metabolites in stems of dioecious, medicinally important shrub Tinospora cordifolia.

Methods: Two different UPLC-DAD methods were used for the quantification of eight selected phytochemicals from the alcoholic stem extracts of T. cordifolia accessions. The Waters Acquity UPLC system hyphenated to the QTOF micromass system, equipped with PDA and ESI-Q-TOF detectors was utilized for the quantitative analysis, Mass Lynx v 4.0 software was used for data analysis.

Results: Significant quantitative changes were observed in the analysed secondary metabolites among different accessions of T. cordifolia. The triploid (3x) cytotypes revealed higher amounts of seven out of eight analysed secondary metabolites than diploids and only 20β-hydroxyecdysone was observed to be present in significantly higher amount in diploid cytotypes. Further, at the tetraploid level, novel induced colchiploid (synthetic 4x) genotypes revealed increase in the yield of all of the analysed eight phytochemicals than their respective diploid counterparts. The quantity of active principles in tetraploid cytotypes were also higher than the average triploid levels at multiple locations in five out of eight tested phytochemicals, indicating the influence of ploidy on expression levels of secondary metabolites in T. cordifolia. Additionally, at each of the three ploidy levels (2x, 3x and synthetic 4x), a significant sex specificity could be observed in the expression levels of active principles, with female sex outperforming the male in the content of some phytochemicals, while others getting overexpressed in the male sex. The manifestation of diverse ecogeographies on secondary metabolism was observed in the form of identification of high yielding accessions from the states of Madhya Pradesh, Delhi and Himachal Pradesh and the Union territory of Jammu & Kashmir. Two triploid female accessions that contained approximately two- to eight fold higher amounts of five out of the eight analysed phytochemicals have been identified as superior elites from the wild from the states of Delhi and Madhya Pradesh.

Conclusion: The paper shows the first observations of ploidy specificity along with subtle sex and ecogeography influence on the expression levels of secondary metabolome in T. cordifolia.

Keywords: Medicinal Phytochemistry, Giloy, UPLC-DAD, Metabolomics, Colchitetraploids, Triploids, Diploids, Elites, Secondary Metabolites.

[1]
Zhang, Y.; Shi, Q.; Shi, P.; Zhang, W.; Cheng, Y. Characterization of isoquinoline alkaloids, diterpenoids and steroids in the Chinese herb Jin-Guo-Lan (Tinospora sagittata and Tinospora capillipes) by high-performance liquid chromatography/electrospray ionization with multistage mass spectrometry. Rapid Commun. Mass Spectrom., 2006, 20(15), 2328-2342.
[http://dx.doi.org/10.1002/rcm.2593] [PMID: 16817243]
[2]
Chatterjee, S.; Srivastava, S.; Khalid, A.; Singh, N.; Sangwan, R.S.; Sidhu, O.P.; Roy, R.; Khetrapal, C.L.; Tuli, R. Comprehensive metabolic fingerprinting of Withania somnifera leaf and root extracts. Phytochemistry, 2010, 71(10), 1085-1094.
[http://dx.doi.org/10.1016/j.phytochem.2010.04.001] [PMID: 20483437]
[3]
Bala, M.; Verma, P.K.; Awasthi, S.; Kumar, N.; Lal, B.; Singh, B. Chemical prospection of important ayurvedic plant Tinospora cordifolia by UPLC-DAD-ESI-QTOF-MS/MS and NMR. Nat. Prod. Commun., 2015, 10(1), 43-48.
[http://dx.doi.org/10.1177/1934578X1501000113] [PMID: 25920217]
[4]
Bajpai, V.; Singh, A.; Chandra, P.; Negi, M.P.S.; Kumar, N.; Kumar, B. Analysis of phytochemical variations in dioecious Tinospora cordifolia stems using HPLC/QTOF MS/MS and UPLC/QqQLIT -MS/MS. Phytochem. Anal., 2016, 27(2), 92-99.
[http://dx.doi.org/10.1002/pca.2601] [PMID: 26627195]
[5]
Binns, S.E.; Arnason, J.T.; Baum, B.R. Phytochemical variation within populations of Echinacea angustifolia (Asteraceae). Biochem. Syst. Ecol., 2002, 30(9), 837-854.
[http://dx.doi.org/10.1016/S0305-1978(02)00029-7]
[6]
Sultan, P.; Shawl, A.S.; Ramteke, P.W.; Kour, A.; Qazi, P.H. Assessment of diversity in Podophyllum hexandrum by genetic and phytochemical markers. Sci. Hortic. (Amsterdam), 2008, 115(4), 398-408.
[http://dx.doi.org/10.1016/j.scienta.2007.10.035]
[7]
Cirak, C.; Radusiene, J.; Stanius, Z.; Camas, N.; Caliskan, O.; Odabas, M.S. Secondary metabolites of Hypericum orientale L. growing in Turkey: variation among populations and plants parts. Acta Physiol. Plant., 2012, 34(4), 1313-1320.
[http://dx.doi.org/10.1007/s11738-012-0928-8]
[8]
Raman, V.; Avula, B.; Galal, A.M.; Wang, Y.H.; Khan, I.A. Microscopic and UPLC-UV-MS analyses of authentic and commercial yohimbe (Pausinystalia johimbe) bark samples. J. Nat. Med., 2013, 67(1), 42-50.
[http://dx.doi.org/10.1007/s11418-012-0642-2] [PMID: 22402817]
[9]
Galal, A.M.; Raman, V.; Avula, B.; Wang, Y.H.; Rumalla, C.S.; Weerasooriya, A.D.; Khan, I.A. Comparative study of three Plumbago L. species (Plumbaginaceae) by microscopy, UPLC-UV and HPTLC. J. Nat. Med., 2013, 67(3), 554-561.
[http://dx.doi.org/10.1007/s11418-012-0717-0] [PMID: 23151906]
[10]
Bhatt, V.; Sharma, S.; Kumar, N.; Sharma, U.; Singh, B. Simultaneous quantification and identification of flavonoids, lignans, coumarin and amides in leaves of Zanthoxylum armatum using UPLC-DAD-ESI-QTOF-MS/MS. J. Pharm. Biomed. Anal., 2017, 132, 46-55.
[http://dx.doi.org/10.1016/j.jpba.2016.09.035] [PMID: 27693952]
[11]
Song, B.H.; Varshney, V.K.; Mittal, N.; Ginwal, H.S. High level of diversity in the phytochemistry, ploidy and genetics of the medicinal plant Acorus calamus L. Med. Aromat. Plants, 2015, •••, S1-S002.http://dx.doi.org/doi:10.4172/2167-0412.S1-002
[12]
Lal, R.K.; Gupta, P.; Dubey, B.K. Genetic variability and associations in the accessions of Manduk Parni Centella asiatica (L.). Ind. Crops Prod., 2017, 96, 173-177.
[http://dx.doi.org/10.1016/j.indcrop.2016.11.056]
[13]
Kapil, A.; Sharma, S. Immunopotentiating compounds from Tinospora cordifolia. J. Ethnopharmacol., 1997, 58(2), 89-95.
[http://dx.doi.org/10.1016/S0378-8741(97)00086-X] [PMID: 9406896]
[14]
Singh, S.S.; Pandey, S.C.; Srivastava, S.; Gupta, V.S.; Patro, B.; Ghosh, A.C. Chemistry and medicinal properties of Tinospora cordifolia (Guduchi). Indian J. Pharmacol., 2003, 35(2), 83-91.
[15]
Panchabhai, T.S.; Kulkarni, U.P.; Rege, N.N. Validation of therapeutic claims of Tinospora cordifolia: a review. Phytother. Res., 2008, 22(4), 425-441.
[http://dx.doi.org/10.1002/ptr.2347] [PMID: 18167043]
[16]
Choudhary, N.; Siddiqui, M.B.; Azmat, S.; Khatoon, S. Tinospora cordifolia: ethnobotany, phytopharmacology and phytochemistry aspects. Int. J. Pharm. Sci. Res., 2013, 4(3), 891-899.
[17]
Phan, V.K.; Chau, V.M.; Nguyen, T.D.; La, V.K.; Dan, T.H.; Nguyen, H.N.; Nguyen, X.C.; Hoang, T.H.; Trinh, V.L. Aporphine alkaloids, clerodane diterpenes, and other constituents from Tinospora cordifolia. Fitoterapia, 2010, 81(6), 485-489.
[http://dx.doi.org/10.1016/j.fitote.2010.01.005] [PMID: 20080155]
[18]
Sankhala, L.N.; Saini, R.K.; Saini, B.S. A review on chemical and biological properties of Tinospora cordifolia. Int. J. Med. Arom. Plants, 2012, 2(2), 340-344.
[19]
Kakkar, A.; Verma, D.R.; Suryavanshi, S.; Dubey, P. Characterization of chemical constituents of Tinospora cordifolia. Chem. Nat. Compd., 2013, 49(1), 177-179.
[http://dx.doi.org/10.1007/s10600-013-0550-z]
[20]
Chi, S.; She, G.; Han, D.; Wang, W.; Liu, Z.; Liu, B. Genus Tinospora: ethnopharmacology, phytochemistry, and pharmacology. Evid-Based Compl. Alt. Med., 2016, 216, 9232593.http://dx.doi.org/doi.org/10.1155/2016/9232593
[21]
Tiwari, P.; Nayak, P.; Prusty, S.K.; Sahu, P.K. Phytochemistry and pharmacology of Tinospora cordifolia: A Review. Sys. Rev. Pharm., 2018, 9(1), 70-78.
[http://dx.doi.org/10.5530/srp.2018.1.14]
[22]
Maurya, R.; Manhas, L.R.; Gupta, P.; Mishra, P.K.; Singh, G.; Yadav, P.P.; Amritosides, A. Amritosides A, B, C and D: clerodane furano diterpene glucosides from Tinospora cordifolia. Phytochemistry, 2004, 65(14), 2051-2055.
[http://dx.doi.org/10.1016/j.phytochem.2004.05.017] [PMID: 15279971]
[23]
Singh, D.; Chaudhuri, P.K. Chemistry and pharmacology of Tinospora cordifolia. Nat. Prod. Commun., 2017, 12(2), 299-308.
[http://dx.doi.org/10.1177/1934578X1701200240] [PMID: 30428235]
[24]
National Medicine Plant Board. Available at:. www.nmpb.nic.in (Accessed April 4, 2014).
[25]
Sharma, U.; Bala, M.; Kumar, N.; Singh, B.; Munshi, R.K.; Bhalerao, S. Immunomodulatory active compounds from Tinospora cordifolia. J. Ethnopharmacol., 2012, 141(3), 918-926.
[http://dx.doi.org/10.1016/j.jep.2012.03.027] [PMID: 22472109]
[26]
Mittal, J.; Sharma, M.M.; Batra, A. Tinospora cordifolia: a multipurpose medicinal plant-A review. J. Med. Plants Stud., 2014, 2(2), 32-47.
[27]
Handique, P.J. In vitro propagation and medicinal attributes of Tinospora cordifolia: A Review. Austin J. Biotechnol. Bioeng., 2014, 1(5), 1-5.
[28]
Abhijeet, R.; Mokat, D. On vegetative propagation through stem cuttings in medicinally lucrative Tinospora species. J. Pharmacogn. Phytochem., 2018, 7(2), 2313-2318.
[29]
Ahmed, S.M.; Manhas, L.R.; Verma, V.; Khajuria, R.K. Quantitative determination of four constituents of Tinospora sps. by a reversed-phase HPLC-UV-DAD method. Broad-based studies revealing variation in content of four secondary metabolites in the plant from different eco-geographical regions of India. J. Chromatogr. Sci., 2006, 44(8), 504-509.
[http://dx.doi.org/10.1093/chromsci/44.8.504] [PMID: 16959127]
[30]
Paliwal, R.; Kumar, R.; Choudhury, D.R.; Singh, A.K.; Kumar, S.; Kumar, A.; Bhatt, K.C.; Singh, R.; Mahato, A.K.; Singh, N.K.; Singh, R. Development of genomic simple sequence repeats (g-SSR) markers in Tinospora cordifolia and their application in diversity analyses. Plant Gene, 2016, 5, 118-125.
[http://dx.doi.org/10.1016/j.plgene.2016.02.001]
[31]
Kim, Y.S.; Hahn, E.J.; Murthy, H.N.; Paek, K.Y. Effect of polyploidy induction on biomass and ginsenoside accumulations in adventitious roots of ginseng. J. Plant Biol., 2004, 47(4), 356-360.
[http://dx.doi.org/10.1007/BF03030551]
[32]
Sattler, M.C.; Carvalho, C.R.; Clarindo, W.R. The polyploidy and its key role in plant breeding. Planta, 2016, 243(2), 281-296.
[http://dx.doi.org/10.1007/s00425-015-2450-x] [PMID: 26715561]
[33]
Lavania, U.C. Genomic and ploidy manipulation for enhanced production of phytopharmaceuticals. Plant Genet. Resour., 2005, 3(2), 170-177.
[http://dx.doi.org/10.1079/PGR200576]
[34]
Van de Peer, Y.; Maere, S.; Meyer, A. The evolutionary significance of ancient genome duplications. Nat. Rev. Genet., 2009, 10(10), 725-732.
[http://dx.doi.org/10.1038/nrg2600] [PMID: 19652647]
[35]
Banyai, W.; Sangthong, R.; Karaket, N.; Inthima, P.; Mii, M.; Supaibulwatana, K. Overproduction of artemisinin in tetraploid Artemisia annua L. Plant Biotechnol., 2010, 27(5), 427-433.
[http://dx.doi.org/10.5511/plantbiotechnology.10.0726a]
[36]
Chen, Z.J. Molecular mechanisms of polyploidy and hybrid vigor. Trends Plant Sci., 2010, 15(2), 57-71.
[http://dx.doi.org/10.1016/j.tplants.2009.12.003] [PMID: 20080432]
[37]
Caruso, I.; Lepore, L.; De Tommasi, N.; Dal Piaz, F.; Frusciante, L.; Aversano, R.; Garramone, R.; Carputo, D. Secondary metabolite profile in induced tetraploids of wild Solanum commersonii Dun. Chem. Biodivers., 2011, 8(12), 2226-2237.
[http://dx.doi.org/10.1002/cbdv.201100038] [PMID: 22162160]
[38]
Dehghan, E.; Hakkinen, S.T.; Oksman-Caldentey, K.M.; Ahmadi, F.S. Production of tropane alkaloids in diploid and tetraploid plants and in vitro hairy root cultures of Egyptian henbane (Hyoscyamus muticus L.). Plant Cell Tissue Organ Cult., 2012, 110(1), 35-44.
[http://dx.doi.org/10.1007/s11240-012-0127-8]
[39]
Lavania, U.C.; Srivastava, S.; Lavania, S.; Basu, S.; Misra, N.K.; Mukai, Y. Autopolyploidy differentially influences body size in plants, but facilitates enhanced accumulation of secondary metabolites, causing increased cytosine methylation. Plant J., 2012, 71(4), 539-549.
[http://dx.doi.org/10.1111/j.1365-313X.2012.05006.x] [PMID: 22449082]
[40]
Cohen, H.; Tel-Zur, N. Morphological changes and self-incompatibility breakdown associated with autopolyploidization in Hylocereus species (Cactaceae). Euphytica, 2012, 184(3), 345-354.
[http://dx.doi.org/10.1007/s10681-011-0536-5]
[41]
Wang, X.; Cheng, Z.M.; Zhi, S.; Xu, F. Breeding triploid plants: A Review. Czech J. Genet. Plant Breed., 2016, 52(2), 41-54.
[http://dx.doi.org/10.17221/151/2015-CJGPB]
[42]
Pradhan, S.K.; Gupta, R.C.; Goel, R.K. Differential content of secondary metabolites in diploid and tetraploid cytotypes of Siegesbeckia orientalis L. Nat. Prod. Res., 2018, 32(20), 2476-2482.
[http://dx.doi.org/10.1080/14786419.2017.1423298] [PMID: 29334256]
[43]
Das, M. Chamomile: medicinal, biochemical, and agricultural aspects, 1st ed; CRC Press: New York, 2014.
[http://dx.doi.org/10.1201/b17160 ]
[44]
Dhawan, O.P.; Lavania, U.C. Enhancing the productivity of secondary metabolites via induced polyploidy: a review. Euphytica, 1996, 87(2), 81-89.
[http://dx.doi.org/10.1007/BF00021879]
[45]
Brar, R.; Gupta, R.C. Phytochemical analysis of two cytotypes (2x & 4x) of Physalis angulata an important medicinal plant, collected from Rajasthan. Biochem. Mol. Biol. J., 2017, 3(3), 15.
[http://dx.doi.org/10.21767/2471-8084.100043]
[46]
Thakur, R.K.; Rajpal, V.R.; Rao, S.R.; Singh, A.; Joshi, L.; Kaushal, P.; Raina, S.N. Induction and evaluation of colchitetraploids of two species of Tinospora Miers, 1851. Comp. Cytogenet., 2020. In Press
[47]
Sivasubramanian, A.; Gadepalli Narasimha, K.K.; Rathnasamy, R.; Campos, A.M.O. A new antifeedant clerodane diterpenoid from Tinospora cordifolia. Nat. Prod. Res., 2013, 27(16), 1431-1436.
[http://dx.doi.org/10.1080/14786419.2012.722088] [PMID: 22946632]
[48]
Tripathi, S.; Singh, N.; Shakya, S.; Dangi, A.; Misra-Bhattaccharya, S.; Dube, A.; Kumar, N. Landrace/gender-based differences in phenol and thiocayanate contents and biological activity in Piper betle L. Curr. Sci., 2006, 91(6), 746-749.
[49]
Krishnan, S.S.C.; Subramanian, I.P.; Subramanian, S.P. Isolation, characterization of syringin phenylpropanoid glycoside from Musa paradisiaca tepal extract and evaluation of its antidiabetic effect in streptozotocin-induced diabetic rats. Biomed. Prev. Nutr., 2014, 4(2), 105-111.
[http://dx.doi.org/10.1016/j.bionut.2013.12.009]
[50]
Mishra, P.; Jamdar, P.; Desai, S.; Patel, D.; Meshram, D. Phytochemical analysis and assessment of in vitro antibacterial activity of Tinospora cordifolia. Int. J. Curr. Microbiol. Appl. Sci., 2014, 3(3), 224-234.
[51]
Zhao, L.C.; He, Y.; Deng, X.; Xia, X.H.; Liang, J.; Yang, G.L.; Li, W.; Wang, H. Ultrasound-assisted extraction of syringin from the bark of Ilex rotunda thumb using response surface methodology. Int. J. Mol. Sci., 2012, 13(6), 7607-7616.
[http://dx.doi.org/10.3390/ijms13067607] [PMID: 22837716]
[52]
Patel, A.; Bigoniya, P.; Singh, C.S.; Patel, N.S. Radioprotective and cytoprotective activity of Tinospora cordifolia stem enriched extract containing cordifolioside-A. Indian J. Pharmacol., 2013, 45(3), 237-243.
[http://dx.doi.org/10.4103/0253-7613.111919] [PMID: 23833365]
[53]
Maurya, R.; Wazir, V.; Kapil, A.; Kapil, R.S. Cordifoliosides A and B, two new phenypropene disaccharides from Tinospora cordifolia possessing immunostimulant activity. Nat. Prod. Lett., 1996, 8(1), 7-10.
[http://dx.doi.org/10.1080/10575639608043231]
[54]
Joshi, G.; Kaur, R. Tinospora cordifolia: A phytopharmacological review. Int. J. Pharm. Sci. Res., 2016, 7(3), 890-897.
[55]
Li, C.; Wang, M.H. Potential biological activities of magnoflorine: A compound from Aristolochia debilis Sieb. et Zucc. Korean J. Plant Res., 2014, 27(3), 223-228.
[http://dx.doi.org/10.7732/kjpr.2014.27.3.223]
[56]
Morris, J.S.; Facchini, P.J. Isolation and characterization of reticuline N-methyltransferase involved in biosynthesis of the aporphine alkaloid magnoflorine in Opium Poppy. J. Biol. Chem., 2016, 291(45), 23416-23427.
[http://dx.doi.org/10.1074/jbc.M116.750893] [PMID: 27634038]
[57]
Chang, Y.L.; Usami, S.; Hsieh, M.T.; Jiang, M.J. Effects of palmatine on isometric force and intracellular calcium levels of arterial smooth muscle. Life Sci., 1999, 64(8), 597-606.
[http://dx.doi.org/10.1016/S0024-3205(98)00602-X] [PMID: 10069523]
[58]
Sangeetha, M.K.; Priya, C.D.; Vasanthi, H.R. Anti-diabetic property of Tinospora cordifolia and its active compound is mediated through the expression of Glut-4 in L6 myotubes. Phytomedicine, 2013, 20(3-4), 246-248.
[http://dx.doi.org/10.1016/j.phymed.2012.11.006] [PMID: 23290487]
[59]
Trenin, D.S.; Volodin, V.V. 20-hydroxyecdysone as a human lymphocyte and neutrophil modulator: In vitro evaluation. Arch. Insect Biochem. Physiol., 1999, 41(3), 156-161.
[http://dx.doi.org/10.1002/(SICI)1520-6327(1999)41:3<156:AID-ARCH7>3.0.CO;2-Q] [PMID: 10398339]
[60]
Wang, J.B.; Mantsch, J.R. l-tetrahydropalamatine: a potential new medication for the treatment of cocaine addiction. Future Med. Chem., 2012, 4(2), 177-186.
[http://dx.doi.org/10.4155/fmc.11.166] [PMID: 22300097]
[61]
Han, Y.; Zhang, W.; Tang, Y.; Bai, W.; Yang, F.; Xie, L.; Li, X.; Zhou, S.; Pan, S.; Chen, Q.; Ferro, A.; Ji, Y. l-Tetrahydropalmatine, an active component of Corydalis yanhusuo W.T. Wang, protects against myocardial ischaemia-reperfusion injury in rats. PLoS One, 2012, 7(6)e38627
[http://dx.doi.org/10.1371/journal.pone.0038627] [PMID: 22715398]


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VOLUME: 20
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Year: 2020
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DOI: 10.2174/1568026620666200124105027
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