Bark of the Stem of Libidibia Ferrea Associated with Mycorrhizal Fungi: An Alternative to Produce High Levels of Phenolic Acids
Francineyde Alves da Silva1, *, Wliana Alves Viturino da Silva2, Magda Rhayanny Assunção Ferreira2, Luiz Alberto Lira Soares2, Fábio Sérgio Barbosa da Silva3, 4
Identifiers and Pagination:Year: 2018
First Page: 412
Last Page: 418
Publisher Id: TOMICROJ-12-412
Article History:Received Date: 13/09/2018
Revision Received Date: 09/12/2018
Acceptance Date: 10/12/2018
Electronic publication date: 31/12/2018
Collection year: 2018
open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
The use of microorganisms such as Arbuscular Mycorrhizal Fungi (AMF) may represent a sustainable biotechnological alternative for the cultivation of medicinal plants to facilitate plant growth, in addition to increasing the production of secondary compounds. These fungi are associated with Libidibia ferrea, a species which produces gallic and ellagic acid, compounds with preventive properties against cancer and diabetes complications.
The objective of this paper was to verify whether the stem bark of L. ferrea concentrates higher amounts of gallic and ellagic acids when inoculated with Claroideoglomus etunicatum, Gigaspora albida and Acaulospora longula.
The extractive methanolic solutions from the barks of L. ferrea were analyzed by RP-HPLC in order to establish the contents of gallic and ellagic acids.
The application of fungus Claroideoglumus etunicatum was more efficient at increasing the concentration of gallic acid (18%) and ellagic acid (45.2%) in the stem bark of L. ferrea in comparison to the control. In contrast, plants inoculated with Acaulospora longula benefited only with the increase in the amount of gallic acid if compared with the non-inoculated plants.
The mycorrhizal technology may be an alternative to the cultivation of L. ferrea with higher concentrations of both gallic and ellagic acids in the stem bark, providing a promissory strategy to produce high quality herbal materials for the production of herbal medicines.
The World Health Organization (WHO) periodically establishes recommendations for the countries to formulate national policies and regulations for the use of herbal medicines which were proven efficient at treating diseases. In Brazil, in 2008, the National Program of Medicinal Plants and Herbal Medicines was created to assure the access of population to herbal medicines and medicinal plants in a safe and sustainable way for biodiversity . However, the raw material used to manufacture herbal medicines is majorly obtained through extractivism. In addition, the plant can produce a low amount of some therapeutically important compounds. This scenario has developed the necessity to employ methods of cultivation of medicinal plants species with higher amount of phytochemicals to provide the herbal medicines industry with raw material .
Among the alternatives to increase the production of phytochemicals, we highlight the Arbuscular Mycorrhizal Fungi (AMF), microorganisms that are able to benefit from the nutritional status of the host, growth, protection against pathogens , and the increase in the concentration of specific compounds of the secondary metabolism . In this context, Mandal et al.  registered that a higher expression of key genes of the metabolic routes and intensive density of glandular trichomes in plants associated with Rhizophagus intraradices justified the raise in the production of artemisinin in Artemisia annua.
Libidibia ferrea (Mart. ex Tul.) L. P. Queiroz var. ferrea, popularly known as “pau-ferro”, was included in the National Listing of Medicinal Plants of interest of the Sistema Único de Saúde (SUS - Basic Health System) (RENISUS) in Brazil , due to ethnopharmacological studies  and pharmacological properties related, such as anti-inflammatory  and wound healing . Phytochemical studies with pau-ferro ascertain the presence of gallic and ellagic acids, compounds which have anticancer activity and work against diabetes complications [10, 11]. Even though pau-ferro forms mycorrhizal association , no register is found on the influence of the AMF on the production of gallic and ellagic acids in the stem bark, one of the parts used by the population for medicinal purposes.
Gallic acid is a phenolic acid produced from shikimic acid and the ellagic acid formed by the dimer condensation of the gallic acid . In addition to the therapeutic function, these phenolic compounds are used as patterns for the quantitative research of phenolic compounds  as well as in the industry of dyes and inks .
In this context, we carried out a field testing of the hypothesis that the mycorrhizal association may benefit the production of gallic and ellagic acids in the stem bark of pau-ferro. Therefore, the objective was to verify whether the stem bark of pau-ferro accumulated higher amount of these molecules upon inoculation with AMF.
2. MATERIALS AND METHODS
2.1. Field Experiment
The experimental field was set in February 2013  at the University of Pernambuco - Campus of Petrolina, Brazil. The planted area was 2.400 m2 and was divided in six blocks including the distribution of 96 plants. In the experimental area 20 soil sample (composite sample of each depth) were collected that were analysed for chemical characteristics: layer 0-20 (P 10.38 mg/dm3; K 0.24 mg/dm3; Ca 1.4 mg/dm3; Mg 0.5 cmolc dm-3; Na 0.03 cmolc dm-3; Al 0.00 cmolc dm-3; organic matter 0.41 g/kg; pH 6.2 H2O (1:2,5); C.E 0.21 mS/cm) and layer 20-40 (P 9.61 mg/dm3; K 0.14 mg/dm3; Ca 0.7 mg/dm3; Mg 0.3cmolc dm-3; Na 0.01 cmolc dm-3; Al 0.00 cmolc dm-3; organic matter. 0.23 g/kg; pH 5.6 H2O (1:2.5); C.E 0,21 mS/cm). Each block had four mycorrhizal treatments [control, Claroideoglomus etunicatum (W.N. Becker & Gerd.) C. Walker & A. Schüßler (UFPE 06), Gigaspora albida N. C. Schenck & G. S. Sm. (UFPE 01), and Acaulospora longula Spain & N. C. Schenck (UFPE 21)].
The seeds of L. ferrea were set to germinate in small cups (50 mL) filled with vermiculite with mean granulation followed by a 20-minute treatment with concentrated sulfuric acid to break the numbness . The transference of seedlings to black polyethylene bags was carried out upon the presentation of four definitive leaves and was laid on the root or not, inoculum soil (containing 200 spores, hypha and colonized roots) of A. longula, C.etunicatum e G. albida. The bags contained 1.2 Kg of soil + 5% of vermicomposting (Chemical characteristics: P, 12.68 mg dm-3; K, 0.26 cmolcdm-3; Ca, 2.7 cmolcdm-3; Mg, 1.8 cmolcdm-3; Na, 0.49 cmolcdm-3; Al, 0.05 cmolcdm-3; organic matter, 3.21 g kg-1; e pHH2O (1:2,5) 5.2). The multiplication of FMA (obtained from UFPE) was observed in soil + 10% vermicompost, using Panicum miliaceum L. (host) andstored (-4ºC) for 22 months .
The seedlings (on avarage: 14 leaves, 5.1 mm of steam diameter, 72 cm of height) were transferred to the experimental field 225 days later, using experimental netting. The irrigation was been for semi-automatic dripe system 15 min on alternate days (8.4 L H2O plant-1 h-1).
The stem bark was removed from the material collected from the pruning of the two central plants of each plot, 13 months after inoculation with the AMF. The plants inoculated with C. etunicatum, G. albida, A. longula and control had stem diameter of 2.97 cm, 3.32 cm, 3.2 cm and 2.46 cm, respectively . Subsequently, the material was dried in an oven (Biopar, Porto Alegre, RS, Brazil) at 45ºC along three consecutive days, the contents of gallic and ellagic acids were established.
2.2. Quantification of Gallic Acid and Ellagic Acid
2.2.1. Sample Solution
We weighted 0.5 g of the stem bark samples, transferred to a round-bottom flask of 100 mL and added with 15 mL of distilled water. The solution was subjected to water reflux (85-90 ºC, during 30 min). Following, the flask was cooled and its content was transferred to a volumetric flask of 25 mL, filtered in cotton, and the volume was assessed with distilled water, constituting the Stock Solution (SS). From the SS, an aliquot of 5 mL was removed and transferred to a volumetric flask of 10 mL; subsequently, the volume was assessed with distilled water, constituting a sample solution (SaS). After this procedure, the SaS was filtered in a PVDF filter (25 mm; 0.45 µm) for the vials.
2.2.2. Standard Solutions
Gallic acid (98%, Sigma-Aldrich®): 1.0 mg of gallic acid was dissolved with water in a volumetric flask of 10 mL (100 µg/mL) followed by the performance of the required dilutions in order to obtain the calibration form.
Ellagic acid (96%, SigmaAldrich®): 1.0 mg of ellagic acid was dissolved with methanol:water (3:1) in a volumetric flask of 10 mL (100 µg/mL) followed by the performance of the required dilutions in order to obtain the calibration curve.
2.2.3. Chromatographic Analysis
The analysis was conducted using a liquid chromatograph (Ultimate 3000, Dionex®) with a detector of photodiode arrays (wavelength of 254 and 280 nm); column C18 (250 mm x 4.6 mm, 5 µm; Dionex®) protected by a security guard C18 (3.9 µm x 4.6 mm, 5 µm, Phenomenex®), maintained under room temperature (24°C); and the flow of the mobile phase of 0.8 mL/minute. The volume of injection was 20 µL.
The mobile phase was constituted of purified water (Purelab Classic UV, Elga®) as solvent A and methanol as solvent B both acidified with trifluoroacetic acid at 0.05%, and degassed in ultrasonic bath (Unique®) and filtered through the membrane of PVDF (47 mm; 0.45 µm). The separation was conducted through linear gradient: 0-10 min, 20-22.5% B; 10-20 min, 22.5-40% B; 20-25 min, 40-75% B; 25-28 min, 75-20% B; 28-32 min, 20% B. The results were expressed in g % of gallic acid and ellagic acid.
2.3. Statistical Analysis
The data were subjected to ANOVA and the measures compared using Tukey test (5%) through Assistat 7.7 .
3. RESULTS AND DISCUSSION
The inoculation with AMF benefited the accumulation of gallic and ellagic acids in the stem bark (Fig. 1). Plants inoculated with C. etunicatum had an increase in the concentrations of gallic acid and ellagic acid of 18% and 45.2%, respectively, in relation to the control (Table 1). In contrast, fungus A. longula provided an increase of 22% in the concentration of gallic acid in comparison with the control (Table 1). The inoculation with Gigaspora albida did not bring additional benefits to the concentration of these phenolic acids in relation to the control plants (Table 1). Similar results were observed in the aerial part of Hypericum perforatum L., which was inoculated with different species of AMF . The authors verified that the fungi influenced the increase in the concentration of hypericin and pseudohypericin in relation to the control. Benefits of the application of AMF on the production of bioactive compounds were also evidenced in other legumes [15, 20-22].
|Fig. (1). Chromatographic profile of samples from stem bark of L. ferrea. 1- 6 (control without AMF); 7-12 (inoculated wih Acaulospora longula); 13-18 (inoculated with Claroideoglomus etunicatum); 19-24 (inoculated with Gigaspora albida).|
|Control||Acaulospora longula||Claroideoglomus etunicatum||Gigaspora albida|
|Gallic acid||0.050 ± 0.000 b||0.061 ± 0.001 a||0.059 ± 0.002 a||0.056 ± 0.000 ab|
|Ellagic acid||0.0104 ± 0.000 b||0.0127 ± 0.001 ab||0.0151 ± 0.001 a||0.0122 ± 0.000 ab|
In plants inoculated with A. longula, the fact of the gallic acid is the precursor of the ellagic acid did not imply a direct relationship of the increase in the concentration of the latter in detriment of the former when compared with the non-inoculated control (Table 1). This demonstrates the necessity of conducting studies with molecules that have a relation in the biosynthetic route, such as gallic and ellagic acids, in plants associated with AMF. A similar result was verified by Silva et al.  in leaves of mycorrhizal pau-ferro established in field for seven months. Plants inoculated with C. etunicatum revealed an increase of 21% in the concentration of gallic acid, without difference in the concentration of total phenols and total tannins in relation to the control.
The interest of the pharmaceutical industry in the secondary compounds produced by medicinal plants has encouraged studies involving AMF seeking an increase in the concentration of these molecules, especially specific ones, such as recorded in this study (Table 1). There is a register of higher concentration of anticancer alkaloids vinblastine and vincristine in Catharanthus roseus in response to the inoculation with G. mosseae ; increased production of caffeic acid, an antioxidant compound, on the aerial part of Ocimum basilicum L. associated with fungi Glomus caledonium and Glomus mosseae was recorded by Toussaint et al. , finally, a higher concentration of artemisinin, an antimalarial compound, in the species Artemisia annua inoculated with Rhizophagus intraradices had also been documented by Mandal et al. .
Vasconcelos et al.  quantified, using HPLC, 112.76 mg gallic acid/g stem bark and 12.00 mg ellagic acid/g stem bark of L. ferrea. The data obtained in this paper reveal the highest concentrations of gallic acid as 59 and 61 mg/g of stem bark, induced by fungi C. etunicatum and A. longula, respectively. In contrast, the production of ellagic acid was 15.1 mg of ellagic acid/g of stem bark when inoculated with C. etunicatum. Based on these data, the use of the stem bark of pau-ferro of mycorrhizal plants with C. etunicatum (three-month cultivation) may be more profitable for the herbal medicines industry, considering that a 25% increase will occur in the contents of ellagic acid. Similarly, the use of a technology employing C. etunicatum would be profitable to the chemical industry that commercializes 1 g of ellagic acid from the biological source (powder of the stem bark of the tree) for R$ 838.00 , that is, it would be extracted 25% more ellagic acid from the same amount of plant material.
A technology applying C. etunicatum may be an alternative to increase the production of gallic and ellagic acids in the stem bark of L. ferrea by using a sustainable system which may encourage the establishment of plantations, contributing to reduce the extractive use of this legume.
ETHICS APPROVAL AND CONSENT TO PARTICIPATE
HUMAN AND ANIMAL RIGHTS
No animals/humans were used for studies that are the basis of this research.
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or otherwise
The authors would like to thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq – National Council for Scientific and Technological Development) for the financial support (308386/2015-9) and for having granted the Research Productivity Scholarships to FSB Silva (307749/2015-0) and LA Lira, and finally to the Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE – Foundation of Support to Science and Technology of the State of Pernambuco) (APQ-0493-4.03/14).
|||http://www.brasil.gov.br/saude/2012/07/saude-repassa-r-6-7-milhoes-para-programa-de-plantas-medicinais-e-fitoterapicos [Access 2016 december 29];|
|||Reis MS, Mariot A, Steenbock W. Diversidade e domesticação de plantas medicinais.Farmacognosia: da planta ao medicamento 5 ed. 2003; 45-74.|
|||Smith SE, Read DJ. Mycorrhizal symbiosis 3rd ed. 2008.|
|||Zubek S, Mielcarek S, Turnau K. Hypericin and pseudohypericin concentrations of a valuable medicinal plant Hypericum perforatum L. are enhanced by arbuscular mycorrhizal fungi. Mycorrhiza 2012; 22(2): 149-56.
|||Mandal S, Upadhyay S, Wajid S, et al. Arbuscular mycorrhiza increase artemisinin accumulation in Artemisia annua by higher expression of key biosynthesis genes via enhanced jasmonic acid levels. Mycorrhiza 2015; 25(5): 345-57.
|||Relação de Plantas Medicinais de Interesse ao SUS 2009.|
|||de Albuquerque UP, Muniz de Medeiros P, de Almeida AL, et al. Medicinal plants of the caatinga (semi-arid) vegetation of NE Brazil: a quantitative approach. J Ethnopharmacol 2007; 114(3): 325-54.
|||de Araújo AA, Soares LAL, Assunção Ferreira MR, et al. Quantification of polyphenols and evaluation of antimicrobial, analgesic and anti-inflammatory activities of aqueous and acetone-water extracts of Libidibia ferrea, Parapiptadenia rigida and Psidium guajava. J Ethnopharmacol 2014; 156: 88-96.
|||Pereira LdeP, Mota MRL, Brizeno LAC, et al. Modulator effect of a polysaccharide-rich extract from Caesalpinia ferrea stem barks in rat cutaneous wound healing: Role of TNF-α, IL-1β, NO, TGF-β. J Ethnopharmacol 2016; 187: 213-23.
|||Nakamura ES, Kurosaki F, Arisawa M, et al. Cancer chemopreventive effects of a Brazilian folk medicine, Juca, on in vivo two-stage skin carcinogenesis. J Ethnopharmacol 2002; 81(1): 135-7.
|||Ueda H, Tachibana Y, Moriyasu M, Kawanishi K, Alves SM. Aldose reductase inhibitors from the fruits of Caesalpinia ferrea Mart. Phytomedicine 2001; 8(5): 377-81.
|||Gattai GS, Pereira SV, Costa CMC, Lima CEP, Maia LC. Microbial activity, arbuscular mycorrhizal fungi and inoculation of woody plants in lead contaminated soil. Braz J Microbiol 2011; 42(3): 859-67.
|||Carvalho JCT, Gosmann G, Schenkel EP. Compostos fenólicos simples e heterosídicos.Farmacognosia: da planta ao medicamento 5 ed. 2003.|
|||Ferreira MRA, Fernandes MT, da Silva WA, et al. Chromatographic and spectrophotometric analysis of phenolic compounds from fruits of Libidibia ferrea Martius. Pharmacogn Mag 2016; 12(Suppl. 2): S285-91.
|||Silva FA, Ferreira MRA, Soares LAL, Sampaio EVSB, Silva FSB, Maia LC. Arbuscular mycorrhizal fungi increase gallic acid production in leaves of field grown Libidibia ferrea (Mart. ex. Tul.) L. P. Queiroz. J Med Pl Res 2014; 8: 1110-5. b
|||Biruel RP, Aguiar IB, Paula RC. Germinação de sementes de pau-ferro submetidas a diferentes condições de armazenamento, escarificação química, temperatura e luz. Rev Bras Sementes 2007; 29: 134-41.
|||Silva FSB. Fase assimbiótica, produção, infectividade e efetividade de fungos micorrízicos arbusculares (FMA) em substratos com adubos orgânicos Tese de doutorado 2006.|
|||Dos Santos EL, Alves da Silva F, Barbosa da Silva FS. Arbuscular mycorrhizal fungi increase the phenolic compounds production concentration in the bark of the stem of Libidibia Ferrea in field conditions. Open Microbiol J 2017; 11: 283-91.
|||Assis F. Universidade Federal de Campina Grande, Campina Grande (http://www.assistat.com)|
|||Lima CS, Campos MAS, Silva FSB. Mycorrhizal Fungi (AMF) increase the content of biomolecules in leaves of Inga vera Willd. Seedlings. Symbiosis 2015; 65: 117-23.
|||Pedone-Bonfim MVL, Lins MA, Coelho IR, Santana AS, Silva FSB, Maia LC. Mycorrhizal technology and phosphorus in the production of primary and secondary metabolites in cebil (Anadenanthera colubrina (Vell.) Brenan) seedlings. J Sci Food Agric 2013; 93(6): 1479-84.
|||Silva FA, Silva FSB, Maia LC. Biotechnical application of arbuscular mycorrhizal fungi used in the production of foliar biomolecules in ironwood seedlings. J Med Pl Res 2014; 8: 814-9. a [Libidibia ferrea (Mart. ex Tul.) L.P.Queiroz var. ferrea].
|||Ratti N, Verma HN, Gautam SP. Effect of Glomus species on physiology and biochemistry of Catharanthus roseus. Indian J Microbiol 2010; 50(3): 355-60.
|||Toussaint JP, Smith FA, Smith SE. Arbuscular mycorrhizal fungi can induce the production of phytochemicals in sweet basil irrespective of phosphorus nutrition. Mycorrhiza 2007; 17(4): 291-7.
|||Vasconcelos CFB, Maranhão HML, Batista TM, et al. Hypoglycaemic activity and molecular mechanisms of Caesalpinia ferrea Martius bark extract on streptozotocin-induced diabetes in Wistar rats. J Ethnopharmacol 2011; 137(3): 1533-41.
|||http://www.sigmaaldrich.com/catalog/product/sigma/e2250?lang=pt®ion=BR [december 29, 2016];|