Background: The purpose of these studies was to determine the safety of a botanical treatment for supporting healthy liver function developed in Peru. The formulation, A4+, contains extracts of Curcuma longa L. rhizome (A4R), Cordia lutea Lam. flower (A4F) and Annona muricata L. leaf (A4L). The tests were used to support an application for a non-traditional Natural Health Product Licence from the Natural Health Product Directorate of Health Canada and future clinical trials.
Methods: Besides reviewing the scientific and clinical information from Peru on the ingredients and conducting an initial Ames test for mutagenicity, we analysed A4+ for its chemical profile and tested genotoxicity (micronucleus test) and general toxicity (28-day repeated dose).
Results: A4+ and extracts from the three plants provided distinctive chemical fingerprints. A4L contained acetogenins, requiring a second chromatographic method to produce a specific fingerprint. The Ames test proved positive at the highest concentration (5,000 μg/mL) but A4+ showed no evidence of genotoxicity in the more specific mouse micronucleus test. The 28-day repeated dose (general toxicity) study in rats showed no toxicity at 2,000 mg/kg.
Conclusions: We conclude that under the conditions of these studies, A4+ shows no evidence of toxicity at the levels indicated. A no observed adverse effect level (NOAEL) of 2,000 mg/kg was assigned.
Author contributions: HS directed the toxicology studies and drafted the manuscript. DS conducted the analytical chemistry. FG coordinated the study and manuscript preparation. SA provided medical and herbal expertise. AC assisted with background research and coordination of the project. JC invented the A4+ formulation and provided the ingredients for testing. All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: We wish to acknowledge Sabell Corporation for financing these studies.
Employment or leadership: Drs José Cabanillas, Francis Green and Steven Aung are shareholders in Sabell Corp and members of Sabell’s scientific advisory committee. Drs. Hugh Semple and Duff Sloley are members of Sabell’s scientific advisory committee and contributed to the safety and efficacy PLA submission to Health Canada.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
1. Natural Medicines. Graviola: Available at: https://naturalmedicines.therapeuticresearch.com/databases/food,-herbs-supplements/professional.aspx?productid=1054. Accessed: 8 Jul 2015.Search in Google Scholar
2. Health Canada. NHPD monograph for Turmeric (Curcuma longa). Available at: http://webprod.hc-sc.gc.ca/nhpid-bdipsn/monoReq.do?id=216. Accessed: 23 Jul 2014.Search in Google Scholar
3. Bussmann RW, Glenn A, Sharon D. Antibacterial activity of medicinal plants of Northern Peru – can traditional applications provide leads for modern science? Indian J Tradit Know 2010;9:742–53.Search in Google Scholar
4. Cabanillas J, Fong F, Report to Sabell Corp., January 2012.Search in Google Scholar
5. Akram M, Shahab-Uddin, Ahmed A, Usmanghani K, Hannan A, Mohiuddin E, et al. Curcuma longa and curcumin: a review article. Rom J Biol Plant Biol 2010;55:65–70.Search in Google Scholar
6. Krup V, Prakash LH, Harini A. Pharmacological activities of turmeric (Curcuma longa Linn): a review. J Homeop Ayurv Med 2013;2:133.10.4172/2167-1206.1000133Search in Google Scholar
7. Moghadamtousi S, Fadaeinasab M, Nikzad S, Mohan G, Ali HM, Kadir HA. Annona muricata (Annonaceae): a review of its traditional uses, isolated acetogenins and biological activities. Int J Mol Med 2015;16:15625–58.10.3390/ijms160715625Search in Google Scholar
8. Priyanka D, Shrikant DA. Review on medicinal fruit Bhokar of species Cordia dichotoma Forst. Int J Pharm Biol Arch 2010;5:41–7.Search in Google Scholar
9. Gee P, Maron DM, Ames BN. Detection and classification of mutagens: a set of base-specific Salmonella tester strains. Proc Natl Acad Sci U S A 1994;91:11606–10.10.1073/pnas.91.24.11606Search in Google Scholar
10. Flückiger-Isler S, Baumeister M, Braun K, Gervais G, Hasler-Nguyen N, Reimann R, et al. Assessment of the performance of the Ames II assay: a collaborative study with 19 coded compounds. Mutat Res 2004;558:181–97.10.1016/j.mrgentox.2003.12.001Search in Google Scholar
11. Environment Directorate, Organisation for Economic Co-operation and Development. OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring, Number 1, OECD Principles on Good Laboratory Practice (as revised in 1997). Available at: http://www.oecd-ilibrary.org/docserver/download/9760011e.pdf?expires=1406135926&id=id&accname=guest&checksum=BB3336A7ADDEAF494719029B8DA6EF92. Accessed: 23 Jul 2014.Search in Google Scholar
12. Working Group of Experts on Systemic Short-Term and (Delayed) Neurotoxicity, Organisation for Economic Co-operation and Development. OECD guideline on the testing of chemicals, #407: repeated dose 28-day oral toxicity study in rodents. Available at: http://www.oecd-ilibrary.org/docserver/download/9740701e.pdf?expires=1406136283&id=id&accname=guest&checksum=BC2F7C38C8A92ABB47AF544A022AEAB4. Accessed: 23 Jul 2014.Search in Google Scholar
13. Organisation for Economic Co-operation and Development. OECD guideline on the testing of chemicals, #474: mammalian erythrocyte micronucleus test. Available at: http://www.oecd-ilibrary.org/environment/test-no-474-mammalian-erythrocyte-micronucleus-test_9789264224292-en. Accessed: 8 Jul 2015.Search in Google Scholar
14. Arita M. Flavonoid Index. Available at: http://metabolomics.jp/wiki/Index:FL. Accessed: 23 Jul 2014.Search in Google Scholar
15. Kim GS, Zeng L, Alali F, Rogers LL, Wu FE, Sastrodihardjo S, et al. Muricoreacin and murihexocin C, mono-tetrahydrofuran acetogenins, from the leaves of Annona muricata. Phytochemistry 1998;49:565–71.10.1016/S0031-9422(98)00172-1Search in Google Scholar
16. Kim GS, Zeng L, Alali F, Rogers LL, Wu FE, McLaughlin JL, et al. Two new mono-tetrahydrofuran ring acetogenins, annomuricin E and muricapentocin, from the leaves of Annona muricata. J Nat Prod 1998;61:432–6.10.1021/np970534mSearch in Google Scholar PubMed
17. Wu FE, Zeng L, Gu ZM, Zhao GX, Zhang Y, Schwedler JT, et al. Muricatocins A and B, two new bioactive mono-tetrahydrofuran annonaceous acetogenins from the leaves of Annona muricata. J Nat Prod 1995;58:902–8.10.1021/np50120a013Search in Google Scholar PubMed
18. Wu FE, Zeng L, Gu ZM, Zhao GX, Zhang Y, Schwedler JT, et al. New bioactive monotetrahydrofuran annonaceous acetogenins, annomuricin C and muricatocin C, from the leaves of Annona muricata. J Nat Prod 1995;58:909–15.10.1021/np50120a014Search in Google Scholar PubMed
19. Wu FE, Zhao GX, Zeng L, Zhang Y, Schwedler JT, McLaughlin JL, et al. Additional bioactive acetogenins, annomutacin and (2,4-trans and cis)-10R-annonacin-A-ones, from the leaves of Annona muricata. J Nat Prod 1995;58:1430–7.10.1021/np50123a015Search in Google Scholar PubMed
20. Zeng L, Wu FE, Oberlies NH, McLaughlin JL, Sastrodihadjo S. Five new monotetrahydrofuran ring acetogenins from the leaves of Annona muricata. J Nat Prod 1996;59:1035–42.10.1021/np960447eSearch in Google Scholar PubMed
21. Organisation for Economic Co-operation and Development. OECD guideline on the testing of chemicals, #471: bacterial reverse mutation test. Available at: http://www.oecd-ilibrary.org/docserver/download/9747101e.pdf?expires=1406136533&id=id&accname=guest&checksum=3558F9E2E2441F70529DF6CE530A8788. Accessed: 23 Jul 2014.Search in Google Scholar
22. Alali FQ, Liu X, McLaughlin JL. Annonaceous acetogenins: recent progress. J Nat Prod 1999;62:504–40.10.1021/np980406dSearch in Google Scholar
23. Resende FA, Vilegas W, dos Santos LC, Varanda EA. Mutagenicity of flavonoids assayed by bacterial reverse mutation (Ames) test. Molecules 2012;17:5255–68.10.3390/molecules17055255Search in Google Scholar
24. International Agency for Research on Cancer (IARC) – Summaries and Evaluations. Quercetin. 1999;73:497.Search in Google Scholar
25. Harwood M, Danielewska-Nikiel B, Borzelleca JF, Flamm GW, Williams GM, Lines TC. A critical review of the data related to the safety of quercetin and lack of evidence of in vivo toxicity, including lack of genotoxic/carcinogenic properties. Food Chem Toxicol 2007;45:2179–205.10.1016/j.fct.2007.05.015Search in Google Scholar
26. European Medicines Agency Committee on Herbal Medicinal Products (HMPC). Overview of comments received on draft ‘Guideline on the Assessment of Genotoxic Constituents in Herbal Substances/Preparations’ (EMEA/HMPC/107079/2007). Available at: http://www.ema.europa.eu/docs/en_GB/document_library/Other/2009/09/WC500003570.pdf. Accessed: 23 Jul 2014.Search in Google Scholar
27. Nagabhushan M, Bhide SV. Nonmutagenicity of curcumin and its antimutagenic action versus chili and capsaicin. Nutr Cancer 1986;8:201–10.10.1080/01635588609513894Search in Google Scholar
28. European Medicines Agency Committee on Herbal Medicinal Products (HMPC): Assessment Report on Curcuma longa L. Rhizoma. Available at: http://www.ema.europa.eu/docs/en_GB/document_library/Herbal_-_HMPC_assessment_report/2010/02/WC500070700.pdf. Accessed: 23 Jul 2014.Search in Google Scholar
29. Kelber O, Steinhoff B, Kraft K. Assessment of genotoxicity of herbal medicinal products: a co-ordinated approach. Phytomedicine 2012;19:472–6.10.1016/j.phymed.2011.11.008Search in Google Scholar
30. Krishna G, Hayashi M. In vivo rodent micronucleus assay: protocol, conduct and data interpretation. Mutat Res 2000;455:155–66.10.1016/S0027-5107(00)00117-2Search in Google Scholar
31. Shelby MD, Gulati DK, Tice RR, Wojciechowski JP. Results of tests for micronuclei and chromosomal aberrations in mouse bone marrow cells with human carcinogens 4-aminobiphenyl, treosulphan, and melphalan. Environ Mol Mutagen 1989;13:339–42.10.1002/em.2850130410Search in Google Scholar PubMed
32. Gafner S, Blumenthal M. Perspectives on the potential hepatotoxicity of various herbs, including green tea extract. HerbalGram 2014;104:52–9.Search in Google Scholar
33. Giknis ML, Clifford CB. (Charles River Laboratories): clinical laboratory parameters for Crl:CD(SD) rats. Available at: http://www.criver.com/files/pdfs/rms/cd/rm_rm_r_clinical_parameters_cd_rat_06.aspx. Accessed: 23 Jul 2014.Search in Google Scholar
The online version of this article (DOI: 10.1515/jcim-2015-0054) offers supplementary material, available to authorized users.
©2016 by De Gruyter