Bark of the Stem of Libidibia Ferrea Associated with Mycorrhizal Fungi: An Alternative to Produce High Levels of Phenolic Acids

SHORT COMMUNICATION Bark of the Stem of Libidibia Ferrea Associated with Mycorrhizal Fungi: An Alternative to Produce High Levels of Phenolic Acids Francineyde Alves da Silva, Wliana Alves Viturino da Silva, Magda Rhayanny Assunção Ferreira, Luiz Alberto Lira Soares and Fábio Sérgio Barbosa da Silva Laboratório de Tecnologia Micorrízica – Universidade de Pernambuco – Campus Petrolina; Rodovia BR 203, Km 2, s/n Vila Eduardo, Petrolina, 56328-903, PE-Brazil. Laboratório de Farmacognosia, Departamento de Farmácia – CCS, Universidade Federal de Pernambuco, Av. Prof. Arthur Sá, S/N Cidade Universitária 50740521 – Recife, PE – Brazil. Laboratório de Análises, Pesquisas e Estudos em Micorrizas, Universidade de Pernambuco; Rua Arnóbio Marques, 310, Santo Amaro – 50100130 Recife, PE-Brazil. Programa de Pós-Graduação em Biologia Celular e Molecular Aplicada, Instituto de Ciências Biológicas, Laboratório de Análises, Pesquisas e Estudos em Micorrizas – LAPEM, ICB/Universidade de Pernambuco, Rua Arnóbio Marques, 310, Santo Amaro – 50100130, Recife, PE-Brazil.


INTRODUCTION
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 [1]. 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 [2].
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 [3], and the increase in the concentration of specific compounds of the secondary metabolism [4]. In this context, Mandal et al. [5] 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 [6], due to ethnopharmacological studies [7] and pharmacological properties related, such as antiinflammatory [8] and wound healing [9]. 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 [12], 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 [13]. In addition to the therapeutic function, these phenolic compounds are used as patterns for the quantitative research of phenolic compounds [14] as well as in the industry of dyes and inks [13].
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.

Field Experiment
The experimental field was set in February 2013 [15]  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 [16]. 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 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 [18]. 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.

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.

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.

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 C 18 (250 mm x 4.6 mm, 5 µm; Dionex ® ) protected by a security guard

Statistical Analysis
The data were subjected to ANOVA and the measures compared using Tukey test (5%) through Assistat 7.7 [19].

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 [4]. 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).

Variable
Inoculation treatment
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. [15] 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 [23]; 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. [24], 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. [5].
Vasconcelos et al. [25] 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 [26], that is, it would be extracted 25% more ellagic acid from the same amount of plant material.

CONCLUSION
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
Not applicable.

HUMAN AND ANIMAL RIGHTS
No animals/humans were used for studies that are the basis of this research.

CONSENT FOR PUBLICATION
Not applicable.

CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or otherwise