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Quantitative Analysis of Phyllanthus Species for Bioactive Molecules Using High-Pressure Liquid Chromatography and Liquid Chromatography–Mass Spectrometry

  • A. Muthusamy
  • E. R. Sanjay
  • H. N. Nagendra Prasad
  • M. Radhakrishna Rao
  • B. Manjunath Joshi
  • S. Padmalatha Rai
  • K. Satyamoorthy
Research Article

Abstract

Phyllanthus amarus Schum. & Thonn. and P. urinaria Linn. are valuable medicinal plants which are widely used in several countries for the treatment of jaundice, diabetes, cancer and other important diseases. The Phyllanthus species are well-known for their important bioactive lignans (phyllanthin, hypophyllanthin and niranthin) and an antioxidant (ellagic acid). Qualitative and quantitative analyses by high performance liquid chromatography (HPLC) and liquid chromatography coupled to mass spectrometry (LC–MS) method revealed the contents of four bioactive molecules in Phyllanthus amarus and Phyllanthus urinaria from district Udupi, Karnataka, India. The phyllanthin, hypophyllanthin and niranthin were eluted out at specific retention time of 16.008, 14.838 and 21.106 min respectively by isocratic method and ellagic acid was eluted out at 28.656 min using gradient method in HPLC. The extracts of P. amarus from three taluks in district Udupi, Karnataka, India showed higher amounts of phyllanthin, hypophyllanthin and niranthin as compared to that of P. urinaria. The ellagic acid was found to be quantitatively higher than the three lignans of P. amarus and P. urinaria. The phytochemical profiling of bioactive compounds in these two species collected from district Udupi showed significant variations in their contents. The quantification of bioactive molecules using HPLC and LC–MS is consistent and reproducible for the future studies on somaclonal variant plants with varied amount of bioactive molecules, and transgenic plants with over-expression of lignans and phenolic compounds which has higher market value in the pharmaceutical industry.

Keywords

Phyllanthus amarus Phyllanthus urinaria Bioactive molecules High-pressure liquid chromatography Liquid chromatography–mass spectrometry analysis High yielding plant 

Notes

Acknowledgements

The authors are grateful to TIFAC-CORE Pharmacogenomics, DST, New Delhi and Manipal University for the facilities, and Indian Council of Medical Research (ICMR), Ministry of Health and Family Welfare, Government of India, New Delhi for the financial support in the form of research project (Ref. No. 59/22/2008/BMS/TRA) to AMS. They are indebted to Mrs. Shashikala Tantry and S. Annapoorna for their help in extraction, Dr. U Raghavendra, Ms. S. Supriya and Mr. Manoj for their help in HPLC and LC–MS analysis, Ms. Swathy PS and Mr. Kiran KR for their assistance in final format of the manuscript.

References

  1. 1.
    Mabberley DJ (2008) Mabberley’s plant-book. A portable dictionary of plants, their classification and used, 3rd edn. Cambridge University Press, CambridgeGoogle Scholar
  2. 2.
    Kathriarachchi H, Hoffmann P, Samuel R, Wurdack KJ, Chase MW (2005) Molecular phylogenetics of Phyllanthaceae inferred from five genes (plastid atpB, matK, 3′ndhF, rbcL, and nuclear PHYC). Mol Phylogenet Evol 36:112–134CrossRefPubMedGoogle Scholar
  3. 3.
    Gangopadhyay M, Chakrabarty T, Balakrishnan NP (2007) Phyllanthus. In: Balakrishnan NP, Chakrabarty T (eds) The family Euphorbiaceae in India: a synopsis of its profile, taxonomy and bibliography. Bishen Singh Mahendra Pal Singh, Dehra Dun, pp 361–391Google Scholar
  4. 4.
    Kuttan R, Harikumar K (2011) Phyllanthus species: scientific evaluation and medicinal applications. CRC Press, FloridaCrossRefGoogle Scholar
  5. 5.
    Tharakan ST (2011) Taxonomy of the Genus Phyllanthus. In: Kuttan R, Harikumar K (eds) Phyllanthus species: scientific evaluation and medicinal applications. CRC Press, Florida, pp 23–36Google Scholar
  6. 6.
    Unander DW, Webster GL, Blumberg BS (1995) Usage and bioassays in Phyllanthus (Euphorbiaceae): IV. Clustering of antiviral uses and other effect. J Ethnopharmacol 45:1–18CrossRefPubMedGoogle Scholar
  7. 7.
    Calixto JB, Santos AR, Yunes RA (1998) A review of the plants of the genus Phyllanthus: their chemistry, pharmacology, and therapeutic potential. Med Res Rev 18:225–258CrossRefPubMedGoogle Scholar
  8. 8.
    Joseph B, Raj SJ (2011) An overview: pharmacognostic properties of Phyllanthus amarus Linn. Int J Pharmacol 7:40–45CrossRefGoogle Scholar
  9. 9.
    Patel JR, Tripathi P, Sharma V, Chauhan NS, Dixit VK (2011) Phyllanthus amarus: ethnomedicinal uses, phytochemistry and pharmacology: a review. J Ethnopharmacol 138:286–313CrossRefPubMedGoogle Scholar
  10. 10.
    Kassuya CAL, Silvestre A, JrO Menezes-De-Lima, Marotta DM, Rehder VLG, Calixto JB (2006) Antiinflammatory and antiallodynic actions of the lignan niranthin isolated from Phyllanthus amarus: evidence for interaction with platelet activating factor receptor. Eur J Pharmacol 546:182–188CrossRefPubMedGoogle Scholar
  11. 11.
    Priya K, Yin WF, Chan KG (2013) Anti-quorum sensing activity of the traditional Chinese herb, Phyllanthus amarus. Sensors 13:14558–14569CrossRefPubMedGoogle Scholar
  12. 12.
    Lee SH, Jaganath IB, Manikam R, Sekaran SD (2013) Inhibition of Raf-MEK-ERK and hypoxia pathways by Phyllanthus prevents metastasis in human lung (A549) cancer cell line. BMC Complem Altern M 13:271CrossRefGoogle Scholar
  13. 13.
    Sukhaphirom N, Vardhanabhuti N, Chirdchupunseree H, Pramyothin P, Jianmongkol S (2013) Phyllanthin and hypophyllanthin inhibit function of P-gp but not MRP2 in Caco-2 cells. J Pharm Pharmacol 65:292–299CrossRefPubMedGoogle Scholar
  14. 14.
    Srirama R, Deepak HB, Senthilkumar U, Ravikanth G, Gurumurthy BR, Shivanna MB, Chandrasekaran CV, Agarwal A, Shaanker RU (2012) Hepatoprotective activity of Indian Phyllanthus. Pharm Biol 50:948–953CrossRefPubMedGoogle Scholar
  15. 15.
    Huang RL, Huang YL, Ou JC, Chen CC, Hs FL, Chang C (2003) Screening of 25 compounds isolated from Phyllanthus species for anti-human hepatitis B virus in vitro. Phytother Res 17:449–453CrossRefPubMedGoogle Scholar
  16. 16.
    Bakkalbasi E, Mentes O, Artik N (2008) Food ellagitannins-occurrence, effects of processing and storage. Crit Rev Food Sci 49:283–298CrossRefGoogle Scholar
  17. 17.
    Girish C, Pradhan SC (2008) Drug development for liver diseases: focus on picroliv, ellagic acid and curcumin. Fund Clin Pharmacol 22:623–632CrossRefGoogle Scholar
  18. 18.
    Annamalai A, Lakshmi P (2009) HPTLC and HPLC analysis of bioactive phyllanthin from different organs of Phyllanthus amarus. Asian J Biotech 1:154–162CrossRefGoogle Scholar
  19. 19.
    Edeoga HO, Okwu DE, Mbaebie BO (2005) Phytochemical constituents of some Nigerian medicinal plants. Afr J Biotech 4:685–688CrossRefGoogle Scholar
  20. 20.
    Gouvea DR, Gobbo-Neto L, Lopes NP (2012) The influence of biotic and abiotic factors on the production of secondary metabolites in medicinal plants. In: Cechinel-Filho V (ed) Plant bioactives and drug discovery: principles, practice, and perspectives. Wiley, HobokenGoogle Scholar
  21. 21.
    Annamalai A, Lakshmi P, Lalithakumari D, Murugesan K (2004) Optimization of biofertilizers on growth, biomass and seed yield of Phyllanthus amarus (Bhumyamalaki) in sandy loam soil. J Med Aromat Pl Sci 26:717–720Google Scholar
  22. 22.
    Ibrahim MH, Jaafar HZ, Karimi E, Ghasemzadeh A (2013) Impact of organic and inorganic fertilizers application on the phytochemical and antioxidant activity of kacip fatimah (Labisia pumila Benth). Molecules 18:10973–10988CrossRefPubMedGoogle Scholar
  23. 23.
    Lambert J, Srivastava J, Vietmeyer N (1997) Medicinal plants: rescuing a global heritage. World Bank Technical Paper; no. WTP 355. The World Bank, Washington, D.C., p 1Google Scholar
  24. 24.
    Grunwald J (1994) The european phytomedicines market figures, trends and analyses. HerbalGram 34:60–65Google Scholar
  25. 25.
    Lewington A (1993) A review of the importation of medicinal plants and plant extracts into europe. TRAFFIC International, CambridgeGoogle Scholar
  26. 26.
    Kamble MB, Dumbre RK, Rangari VD (2008) Hepatoprotective studies of herbal formulations. Int J Green Pharm 2:147–151CrossRefGoogle Scholar
  27. 27.
    Ved DK, Goraya G (2007) Demand and supply of medicinal plants in india. NMPB, New Delhi & FRLHT, Bangalore, p xvGoogle Scholar
  28. 28.
    Ramakrishna A, Ravishankar GA (2011) Influence of abiotic stress signals on secondary metabolites in plants. Plant Signal Behav 6:1720–1731CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Gouvea DR, Gobbo-Neto L, Lopes NP (2012) The influence of biotic and abiotic factors on the production of secondary metabolites in medicinal plants. In: Cechinel-Filho V (ed) Plant bioactives and drug discovery: principles, practice, and perspectives. Wiley, Hoboken, pp 419–440CrossRefGoogle Scholar
  30. 30.
    Gopalakrishna Bhat K (2003) Flora of Udupi, Indian Naturalist, Inchara, Chitpady, Udupi, pp 576–580Google Scholar
  31. 31.
    Tripathi AK, Verma RK, Gupta AK, Gupta MM, Khanuja SPS (2006) Quantitative determination of phyllanthin and hypophyllanthin in Phyllanthus species by high-performance thin layer chromatography. Phytochem Anal 17:394–397CrossRefPubMedGoogle Scholar
  32. 32.
    Negi AS, Kumar JK, Luqman S, Shanker K, Gupta MM, Khanuja SPS (2008) Recent advances in plant hepatoprotectives: a chemical and biological profile of some important leads. Med Res Rev 28:746–772CrossRefPubMedGoogle Scholar
  33. 33.
    Khatoon S, Rai V, Rawat AKS, Mehrotra S (2006) Comparative pharmacognostic studies of three Phyllanthus species. J Ethnopharmacol 104:79–86CrossRefPubMedGoogle Scholar
  34. 34.
    Srivastava V, Singh M, Malasoni R, Shanker K, Verma RK, Gupta MM, Gupta AK, Khanuja SPS (2008) Separation and quantification of lignans in Phyllanthus species by a simple chiral densitometric method. J Sep Sci 31:47–55CrossRefPubMedGoogle Scholar
  35. 35.
    Sharma A, Singh RT, Handa SS (1993) Estimation of phyllanthin and hypophyllanthin by high performance liquid chromatography in Phyllanthus amarus. Phytochem Anal 4:226–229CrossRefGoogle Scholar
  36. 36.
    Dhooghe L, Meert H, Cimanga RK, Vlietinck AJ, Pieters L, Apers S (2011) The quantification of ellagic acid in the crude extract of Phyllanthus amarus Schum. & Thonn. (Euphorbiaceae). Phytochem Anal 22:361–366CrossRefPubMedGoogle Scholar
  37. 37.
    Lam SH, Wang CY, Chen CK, Lee SS (2007) Chemical investigation of Phyllanthus reticulatus by HPLC–SPE–NMR and conventional methods. Phytochem Anal 18:251–255CrossRefPubMedGoogle Scholar
  38. 38.
    Sprenger RDF, Cass QB (2013) Characterization of four Phyllanthus species using liquid chromatography coupled to tandem mass spectrometry. J Chromatogr A 1291:97–103CrossRefGoogle Scholar
  39. 39.
    Ahmad B, Alam T (2003) Components from whole plant of Phyllanthus amarus Linn. Indian J Chem Sect 42 B:1786–1790Google Scholar
  40. 40.
    Sawant L, Prabhakar B, Pandita N (2010) Quantitative HPLC analysis of ascorbic acid and gallic acid in Phyllanthus Emblica. J Anal Bioanal Tech 1:111CrossRefGoogle Scholar
  41. 41.
    Preeti Sagar N, Anubha U, Sunil Kumar D, Sathrupa R (2010) Quantitative determination of phyllanthin in Phyllanthus amarus by high-performance thin layer chromatography. Bol Latinoam Caribe Plant Med Aromat 9:353–358Google Scholar
  42. 42.
    Wang CY, Lee SS (2005) Analysis and identification of lignans in Phyllanthus urinaria by HPLC–SPE–NMR. Phytochem Anal 16:120–126CrossRefPubMedGoogle Scholar
  43. 43.
    Yang CM, Cheng HY, Lin TC, Chiang LC, Lin CC (2007) Hippomanin a from acetone extract of Phyllanthus urinaria inhibited HSV-2 but not HSV-1 infection in vitro. Phytother Res 21:1182–1186CrossRefPubMedGoogle Scholar
  44. 44.
    Yang CM, Cheng HY, Lin TC, Chiang LC, Lin CC (2007) The in vitro activity of geraniin and 1,3,4,6-tetra-O-galloyl-β-d-glucose isolated from Phyllanthus urinaria against herpes simplex virus type 1 and type 2 infection. J Ethnopharmacol 110:555–558CrossRefPubMedGoogle Scholar
  45. 45.
    Cheng HY, Yang CM, Lin TC, Lin LT, Chiang LC, Lin CC (2011) Excoecarianin, isolated from Phyllanthus urinaria Linnea, inhibits Herpes Simplex Virus Type 2 infection through inactivation of viral particles. Evid-Based Complenent Altern 259103:1–10Google Scholar
  46. 46.
    Mamza UT, Sodipo OA, Khan IZ (2012) Gas chromatography-mass spectrometry (GC-MS) analysis of bioactive components of Phyllanthus amarus leaves. Int Res J Plant Sci 3:208–215Google Scholar
  47. 47.
    Guo J, Chen Q, Wang C, Qiu H, Liu B, Jiang ZH, Zhang W (2015) Comparison of two exploratory data analysis methods for classification of Phyllanthus chemical fingerprint: unsupervised vs. supervised pattern recognition technologies. Anal Bioanal Chem 40:1389–1401CrossRefGoogle Scholar
  48. 48.
    Pavarini DP, Pavarini SP, Niehues M, Lopes NP (2012) Exogenous influences on plant secondary metabolite levels. Anim Feed Sci Tech 176:5–16CrossRefGoogle Scholar
  49. 49.
    Silva RRD, da Câmara CA, Almeida AV, Ramos CS (2012) Biotic and abiotic stress-induced phenylpropanoids in leaves of the mango (Mangifera indica L., Anacardiaceae). J Braz Chem Soc 23:206–211Google Scholar
  50. 50.
    Osbourn AE, Qi X, Townsend B, Qin B (2003) Dissecting plant secondary metabolism—constitutive chemical defences in cereals. N Phytol 159:101–108CrossRefGoogle Scholar
  51. 51.
    Wink M (2003) Evolution of secondary metabolites from an ecological and molecular phylogenetic perspective. Phytochemistry 64:3–19CrossRefPubMedGoogle Scholar
  52. 52.
    Hanudin E, Wismarini H, Hertiani T, Sunarminto BH (2012) Effect of shading, nitrogen and magnesium fertilizer on phyllanthin and total flavonoid yield of Phyllanthus niruri in Indonesia soil. J Med Plant Res 6:4586–4592Google Scholar
  53. 53.
    Ferreira JFS (2007) Nutrient deficiency in the production of artemisinin, dihydroartemisinic acid, and artemisinic acid in Artemisia annua L. J Agric Food Chem 55:1686–1694CrossRefPubMedGoogle Scholar
  54. 54.
    Stewart AJ, Chapman W, Jenkins GI, Graham I, Martin T, Crozier A (2001) The effect of nitrogen and phosphorous deficiency on flavonol accumulation in plant tissues. Plant Cell Environ 24:1189–1197CrossRefGoogle Scholar
  55. 55.
    Ozguven M, Sener B, Orhan I, Sekeroglu N, Kirpik M, Kartal M, Pesin I, Kaya Z (2008) Effects of varying nitrogen doses in yield components and artemisinin content of Artemisia annua L. Ind Crop Prod 27:60–64CrossRefGoogle Scholar
  56. 56.
    Babalar M, Mumivand H, Hadian J, Tabatabaei SMF (2010) Effects of nitrogen and calcium carbonate on growth, rosmarinic acid content and yield of Satureja hortensis L. J Agric Sci 2:92–98Google Scholar

Copyright information

© The National Academy of Sciences, India 2017

Authors and Affiliations

  • A. Muthusamy
    • 1
  • E. R. Sanjay
    • 1
  • H. N. Nagendra Prasad
    • 1
  • M. Radhakrishna Rao
    • 1
  • B. Manjunath Joshi
    • 2
  • S. Padmalatha Rai
    • 2
  • K. Satyamoorthy
    • 3
  1. 1.Department of Plant Sciences, School of Life SciencesManipal UniversityManipalIndia
  2. 2.Department of Biotechnology, School of Life SciencesManipal UniversityManipalIndia
  3. 3.School of Life SciencesManipal UniversityManipalIndia

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