A new mixed inhibitor of adenosine deaminase produced by endophytic Cochliobolus sp. from medicinal plant seeds

  • Xin-guo ZhangEmail author
  • Qiao-Yun Sun
  • Peng Tang
  • Guo-Yan Ma
  • Guang-Jun Guo
  • Si-Jia Guo
  • Xiao-Di Ma
Original Article


Medicinal plants have been studied for potential endophytic interactions and numerous studies have provided evidence that seeds harbor diverse microbial communities, not only on their surfaces but also within the embryo. Adenosine deaminase (ADA) is known as a potential therapeutic target for the treatment of lymphoproliferative disorders and cancer. Therefore, in this study, 20 types of medicinal plant seeds were used to screen endophytic fungi with tissue homogenate and streak. In addition, 128 morphologically distinct endophyte strains were isolated and their ADA inhibitory activity determined by a spectrophotometric assay. The strain with the highest inhibitory activity was identified as Cochliobolus sp. Seven compounds were isolated from the strain using a chromatography method. Compound 3 showed the highest ADA inhibitory activity and was identified as 5-hydroxy-2-hydroxymethyl-4H-pyran-4-one, based on the results of 1H and 13C NMR spectroscopy. The results of molecular docking suggested that compound 3 binds to the active site and the nonspecific binding site of the ADA. Furthermore, we found that compound 3 is a mixed ADA inhibitor. These results indicate that endophytic strains are a promising source of ADA inhibitors and that compound 3 may be a superior source for use in the preparation of biologically active ADA inhibitor compounds used to treat cancer.


Seed endophytes ADA inhibitors Molecular docking 


Funding information

This research was supported by the program of National Natural Science Foundation of China under Grant (No. 31860004), Gansu province natural science fund (17JR5RA128), and the Fundamental Research Funds for Key Laboratory of Drug Screening and Deep Processing for Traditional Chinese and Tibetan Medicine of Gansu Province (No. 20180802).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Animal studies

This article does not contain any studies with animals performed by any of the authors.


  1. Agarwal RP (1982) Inhibitors of adenosine deaminase. Pharmacol Therapeut 17:399–429. CrossRefGoogle Scholar
  2. Ansari MM, Ahmad J, Ali M, Ansari SH (2006) 10-methyl-n-heptacosane and diglucosyldirhamnoside from the stem bark of Balanites aegyptiaca Delile. Indian J Chem Sect B 45:2154–2156. Google Scholar
  3. Arthaud ID, Rodrigues FA, Jimenez PC et al (2012) Studies on the secondary metabolites of a Pseudoalteromonas sp. isolated from sediments collected at the northeastern coast of Brazil. Chem Biodivers 9:418–427. CrossRefGoogle Scholar
  4. Azimi R, Baharfar R (2014) ChemInform abstract: DABCO-functionalized mesoporous SBA-15: an efficient and recyclable catalyst for the synthesis of Spiro-Pyranoxindoles as antioxidant agents. Can J Chem 46(19):1163–1168(6). CrossRefGoogle Scholar
  5. Basu M, Sanyal AK, Banerjee AB (2002) A new method of colorimetric assay of beta-lactamase suitable for estimation of beta-lactamase inhibition in crude microbial culture filtrates. Folia Microbiol 47:32–36. CrossRefGoogle Scholar
  6. Bezerra JDP, Nascimento CCF, Barbosa RDN et al (2015) Endophytic fungi from medicinal plant Bauhinia forficata : diversity and biotechnological potential. Braz J Microbiol 46:49–57. CrossRefGoogle Scholar
  7. Bransova J, Brtko J, Uher M, Novotny L (1995) Antileukemic activity of 4-pyranone derivatives. Int J Biochem Cell Biol 27:701–706. CrossRefGoogle Scholar
  8. Brtko J, Bransova-Bobalova JE, Eyblo V, Ivielnix M, Uheru M (2001) Kojic acid: a superior source for preparation of biological active compounds (current experience). Biomarker Environ 4:62–73Google Scholar
  9. Brtko J, Rondahl L, Ficková M, Hudecová D, Eybl V, Uher M (2004) Kojic acid and its derivatives: history and present state of art. Cent Eur J Public Health 12(Suppl):S16–S18Google Scholar
  10. Cristalli G, Costanzi S, Lambertucci C, Lupidi G, Vittori S, Volpini R, Camaioni E (2001) Adenosine deaminase: functional implications and different classes of inhibitors. Med Res Rev 21:105–128.<105::AID-MED1002>3.0.CO;2-U CrossRefGoogle Scholar
  11. Dobson RC, Griffin MD, Roberts SJ, Gerrard JA (2004) Dihydrodipicolinate synthase (DHDPS) from Escherichia coli displays partial mixed inhibition with respect to its first substrate, pyruvate. Biochimie 86:311–315. CrossRefGoogle Scholar
  12. Dong PJ, Zhang XG, Liu YJ, Kou F, Tang P, Su Y (2015) Rapid drug model for screening adenosine deaminase inhibitor. Chin J Mod Appl Pharm 32:1301–1305Google Scholar
  13. Fickova M, Pravdova E, Rondhal L, Uher M, Brtko J (2008) In vitro antiproliferative and cytotoxic activities of novel kojic acid derivatives: 5-benzyloxy-2-selenocyanatomethyl- and 5-methoxy-2-selenocyanatomethyl-4-pyranone. J Appl Toxicol 28:554–559. CrossRefGoogle Scholar
  14. Glader BE, Backer K, Diamond LK (1983) Elevated erythrocyte adenosine deaminase activity in congenital hypoplastic anemia. New Engl J Med 309:1486–1490. CrossRefGoogle Scholar
  15. Glazer RI (1980) Adenosine deaminase inhibitors: their role in chemotherapy and immunosuppression. Cancer Chemother Pharmacol 4:227–235. CrossRefGoogle Scholar
  16. Häberlein H, Tschiersch KP (1994) Triterpenoids and flavonoids from Leptospermum scoparium. Phytochemistry 35:765–768. CrossRefGoogle Scholar
  17. Hudecová D, Jantová S, Melník M, Uher M (1996) New azidometalkojates and their biological activity. Folia Microbiol 41:473–476. CrossRefGoogle Scholar
  18. Iaroshenko VO, Ostrovskyi D, Petrosyan A, Mkrtchyan S, Villinger A, Langer P (2011) Synthesis of fluorinated purine and 1-Deazapurine glycosides as potential inhibitors of adenosine deaminase. J Organomet Chem 76:2899–2903. CrossRefGoogle Scholar
  19. Irene D, Chung TY, Chen BJ, Liu TH, Li FY, Tzen JT, Wang CI, Chyan CL (2012) Solution structure of a phytocystatin from Ananas comosus and its molecular interaction with papain. PLoS One 7:e47865. CrossRefGoogle Scholar
  20. Kinoshita T, Nishio N, Sato A, Murata M (1999) Crystallization and preliminary analysis of bovine adenosine deaminase. Acta Crystallogr D Biol Crystallogr 55:2031–2032. CrossRefGoogle Scholar
  21. Li J, Zhang J, Lai B, Zhao Y, Li Q (2015) Cloning, expression, and characterization of Capra hircus Golgi α-mannosidase II. Appl Biochem Biotechnol 177:1241–1251. CrossRefGoogle Scholar
  22. Mardanyan S, Sharoyan S, Antonyan A, Armenyan A, Cristalli G, Lupidi G (2001) Tryptophan environment in adenosine deaminase. I. Enzyme modification with N-bromosuccinimide in the presence of adenosine and EHNA analogues. BBA-Protein Struct Mol 1546:185–195. CrossRefGoogle Scholar
  23. Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ (2009) AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem 30:2785–2791. CrossRefGoogle Scholar
  24. Nelson EB (2004) Microbial dynamics and interactions in the spermosphere. Annu Rev Phytopathol 42:271–309. CrossRefGoogle Scholar
  25. Ni H, Li YH, Hao RL, Li H, Hu SQ, Li HH (2016) Identification of adenosine deaminase inhibitors from tofu wastewater and litchi peel and their synergistic anticancer and antibacterial activities with cordycepin. Int J Food Sci Technol 51:1168–1176. CrossRefGoogle Scholar
  26. Nohynek GJ, Kirkland D, Marzin D, Toutain H, Leclerc-Ribaud C, Jinnai H (2004) An assessment of the genotoxicity and human health risk of topical use of kojic acid [5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one]. Food Chem Toxicol 42:93–105. CrossRefGoogle Scholar
  27. Noonan M, Brady TG (1969) Purification of adenosine deaminase from bovine lung and spleen. Biochem J 115:15P–1515P. CrossRefGoogle Scholar
  28. Ono M, Honda F, Karahashi A, Kawasaki T, Miyahara K (1997) Resin glycosides. XXV. Multifidins I and II, new jalapins, from the seed of Quamoclit x multifida. Chem Pharm Bull 45:1955–1960. CrossRefGoogle Scholar
  29. Qiu P, Feng ZX, Tian JW, Lei ZC, Wang L, Zeng ZG, Chu YW, Tian YQ (2015) Diversity, bioactivities, and metabolic potentials of endophytic actinomycetes isolated from traditional medicinal plants in Sichuan, China. Chin J Nat Med 13:942–953. Google Scholar
  30. Reller LB, Weinstein MP, Petti CA (2007) Detection and identification of microorganisms by gene amplification and sequencing. Clin Infect Dis 44:1108–1114. CrossRefGoogle Scholar
  31. Saboury AA (2009) Enzyme inhibition and activation: a general theory. J Iran Chem Soc 6:219–229. CrossRefGoogle Scholar
  32. Sanner MF (1999) Python: a programming language for software integration and development. J Mol Graphics Modell 17:57–61. Google Scholar
  33. Segel IH (1975) Enzyme kinetics : behavior and analysis of rapid equilibrium and steady state enzyme systems. Wiley.
  34. Shen YC, Wang T, Chen L, Yang T, Wan C, Hu QJ, Wen FQ (2013) Diagnostic accuracy of adenosine deaminase for tuberculous peritonitis: a meta-analysis. Arch Med Sci 9:601–607. CrossRefGoogle Scholar
  35. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729. CrossRefGoogle Scholar
  36. Tellenbach C, Grunig CR, Sieber TN (2011) Negative effects on survival and performance of Norway spruce seedlings colonized by dark septate root endophytes are primarily isolate-dependent. Environ Microbiol 13:2508–2517. CrossRefGoogle Scholar
  37. Tritsch GL (1983) Validity of the continuous spectrophotometric assay of Kalckar for adenosine deaminase activity. Anal Biochem1 29:207–209. CrossRefGoogle Scholar
  38. Trott O, Olson AJ (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31:455–461. Google Scholar
  39. Truyens S, Weyens N, Cuypers A, Vangronsveld J (2015) Bacterial seed endophytes: genera, vertical transmission and interaction with plants. Environ Microbiol Rep 7:40–50. CrossRefGoogle Scholar
  40. Uher M, Chalabala M, Cizmarik J (2000) [Kojic acid and its derivatives as potential therapeutic agents]. Ceska Slov Farm 49:288–298Google Scholar
  41. Veverka M, Dubaj T, Gallovič J, Jorík V, Veverková E, Danihelová M, Šimon P (2015) Cocrystals of quercetin: synthesis, characterization, and screening of biological activity. Monatsh Chem Chem Mon 146:99–109. CrossRefGoogle Scholar
  42. Wang S-D, Ma Q, Wang K, Ma P-B (2018a) Strong and biocompatible three-dimensional porous silk fibroin/graphene oxide scaffold prepared by phase separation. Int J Biol Macromol 111:237–246. CrossRefGoogle Scholar
  43. Wang G, Qiu J, Xiao X, Cao A, Zhou F (2018b) Synthesis, biological evaluation and molecular docking studies of a new series of chalcones containing naphthalene moiety as anticancer agents. Bioorg Chem 76:249–257. CrossRefGoogle Scholar
  44. Wei J (1979) Fungal identification manual. Shang-hai Sci Technol PressGoogle Scholar
  45. Wei S, Ji Z (2016) Isolation, synthesis, and antifungal activity of Kojic acid and its derivatives. Chem Nat Compd 52:1–2. CrossRefGoogle Scholar
  46. Yon C, Suh JW, Chang JH, Lim Y, Lee CH, Lee YS, Lee YW (1995) ChemInform abstract: AL072, a novel anti-legionella antibiotic produced by Streptomyces sp. J Antibiot 48:773–779. CrossRefGoogle Scholar
  47. Zhang XG, Peng YN, Li XR, Ma GD, Chen XQ (2015) Screening of iron-enriched fungus from natural environment and evaluation of organically bound iron bioavailability in rats. Food Sci Technol 35:58–65. CrossRefGoogle Scholar
  48. Zhang XG, Li XR, Wang QL, Wang WN, Chen XQ (2016) Study on effects of endophytes on growth and production of Z-Ligustilide and ferulic acid in Angelica sinensis. Braz J Bot 39:417–426. CrossRefGoogle Scholar
  49. Zhang XG, Liu JW, Tang P, Liu ZY, Guo GJ, Sun QY, Yin JJ (2017) Identification of a new uncompetitive inhibitor of adenosine deaminase from endophyte Aspergillus niger sp. Curr Microbiol 75:1–9. CrossRefGoogle Scholar
  50. Zhang Q, Song C, Zhao J, Shi X, Sun M, Liu J, Fu Y, Jin W, Zhu B (2018) Separation and characterization of Antioxidative and angiotensin converting enzyme inhibitory peptide from jellyfish gonad hydrolysate. Molecules 23:E94. CrossRefGoogle Scholar

Copyright information

© Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2019

Authors and Affiliations

  • Xin-guo Zhang
    • 1
    Email author
  • Qiao-Yun Sun
    • 1
  • Peng Tang
    • 1
  • Guo-Yan Ma
    • 1
  • Guang-Jun Guo
    • 1
  • Si-Jia Guo
    • 1
  • Xiao-Di Ma
    • 1
  1. 1.School of Life Science and Engineering, Key Laboratory of Herbal-Tebitan Drug Screening and Deep Processing of Gansu ProvinceLanzhou University of TechnologyLanzhouChina

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