Research Article Organic Extract of Justicia Pectoralis Jacq. Leaf
Total Page：16
File Type：pdf, Size：1020Kb
Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2018, Article ID 5762368, 10 pages https://doi.org/10.1155/2018/5762368 Research Article Organic Extract of Justicia pectoralis Jacq. Leaf Inhibits Interferon-� Secretion and Has Bacteriostatic Activity against Acinetobacter baumannii and Klebsiella pneumoniae Tiago Rafael de Sousa Nunes,1 Marina Ferraz Cordeiro,1,2 Fernanda Gomes Beserra,1 Matheus Landim de Souza,1 Wliana Alves Viturino da Silva,3 Magda Rhayanny Assunção Ferreira ,3 Luiz Alberto Lira Soares,3 Sérgio Dias Costa-Junior,4 Isabella Macário Ferro Cavalcanti,4,5 Maira Galdino da Rocha Pitta ,1 Ivan da Rocha Pitta,1 and Moacyr Jesus Barreto de Melo Rêgo 1 1 Laboratory of Immunomodulation and New Terapeutical Approaches, Research Centre for Terapeutic Innovation-SuelyGaldino (NUPIT-SG), Federal University of Pernambuco, Recife, PE, Brazil 2Department of Medicine, Federal University of the Sao˜ Francisco Valley (UNIVASF), Paulo Afonso, Brazil 3Laboratory of Pharmacognosy, Department of Pharmaceutical Sciences, Federal University of Pernambuco, Recife, PE, Brazil 4Laboratory of Immunopathology KeizoAsami (LIKA), Federal University of Pernambuco, Recife, PE, Brazil 5Microbiology and Immunology Laboratory of Academic Center of Vitoria, Federal University of Pernambuco, Recife, PE, Brazil Correspondence should be addressed to Moacyr Jesus Barreto de Melo Rˆego; [email protected] Received 16 May 2018; Revised 10 July 2018; Accepted 1 August 2018; Published 23 August 2018 Academic Editor: Yuri Clement Copyright © 2018 Tiago Rafael de Sousa Nunes et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Justicia pectoralis Jacq. (Acanthaceae) leaves currently found in the Brazilian north-east are widely used to treat diabetes, menstrual pains, asthma, and other disorders. Tis work aimed to identify the phytochemical characterization and biological activities of J. pectoralis leaf extracts. Te plant material was ground and the crude extracts were obtained with water or acetone: water (7:3 v/v), yielding aqueous (JPA), and organic (JPO) extracts. Phytochemical characterization was performed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Cytotoxicity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) (MTT) assay and trypan blue (TB) exclusion assay in peripheral blood mononuclear cells (PBMCs), BALB/c splenocytes, and neoplastic cells (TOLEDO, K562, DU-145, and PANC-1) at 1, 10, and 100 �g/mL. Antibacterial activity was evaluated using the microdilution test to obtain the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Cytokines, IFN-�, and IL-17A from culture supernatants of BALB/c mice splenocytes were measured by sandwich ELISA. In the TLC analysis, both JPA and JPO extracts presented coumarin and favonoids. In addition, HPLC was able to identify coumarin, apigenin, and ellagic acid in both extracts. JPO IC50 was 57.59 ± 1.03 �g/mL (MTT) and 69.44 ± 8.08 �g/mL (TB) in TOLEDO. MIC value of JPO against Acinetobacter baumannii and Klebsiella pneumoniae was 500 �g/mL. JPO (100 �g/mL) signifcantly inhibited IFN-� levels (p=0.03). J. pectoralis is a potential candidate to be further investigated as an IFN-� inhibitory agent and against Acinetobacter baumannii and Klebsiella pneumoniae. 1. Introduction widespread, and its occurrence has been registered in several South American countries [2]. Tere are also reports of its Justicia pectoralis Jacq. belongs to the family Acanthaceae [1] traditional uses. One of the frst publications was a report by and in Brazilian north-east it is popularly known as chamba,´ MacRae and Towers [3] who pointed out its administration chachamba,´ anador, clover-tree, or clover-cumaru. It is quite against lung infections, stating it to be an ingredient of 2 Evidence-Based Complementary and Alternative Medicine “hallucinogenic” snuf. In the form of syrups, infusion, or afer saturation with the mobile phase (Supplementary Table ∘ mash, it was found that the leaves of this plant were used S1) for about 30 minutes at room temperature (25 ± 2 C). as an expectorant and to treat asthma, cough, bronchitis Te bands were applied with a width of 10 mm and a [2, 4], colds, menstrual pains [5], and diabetes and as an distance between them and the edges of plates of 5 mm. Te antibacterial and sedative agent [6]. width and length of the chromatographic plates were 5 cm Other studies indicate that aerial parts are also used and 10 cm, respectively. Te samples were applied at 5 mm against nervousness and sleeplessness, as a hypotensive [7, 8], from the origin and with 5 mm from the end of the plate. and against epilepsy [9]. However, few authors have described Afer elution, the plates were dried at room temperature and its antineoplastic activity, with most studies about this plant observed under ultraviolet light of 254 and 365 nm and visible concentrating on its use for treatment of respiratory tract light. Ten, the plates were derivatized with reagents specifc disorders and consequently its anti-infammatory potential for each metabolite (Supplementary Table S1). Te bands [10]. Some of these anti-infammatory studies include clinical visualized on the samples were compared to bands of the trials with J. pectoralis syrups for treatment of asthmatic corresponding standards. patients [11–13]. However, only one study has reported the cytokines modulation, showing exclusively the modulations 2.3. High-Performance Liquid Chromatography (HPLC) Anal- of IL-1� and TNF-� cytokines [2]. ysis. JPA and JPO (5 mg) were weighed and transferred Regarding the types of extract, most researchers have to a volumetric fask (5 mL) with ultrapure water (Purelab used aqueous or hydroalcoholic extracts. However, the most Classic UV,Elga). Te samples were brought to the ultrasonic efcient extraction of phenolic compounds is acquired using bath (Ultracleaner, Unique) for 15 minutes for complete an organic solvent such as acetone [14]. A range of biological dissolution. Te volume was then completed with ultrapure activities performed by medicinal plants are due to the water. Afer dilutions, the samples were fltered into vials presence of phenolic compounds and other phytochemicals through a PVDF membrane (25 mm, 0.45 �m). [15, 16] and, in this sense, acetone was the organic solvent used Te analysis was conducted in an HPLC (Ultimate 3000, in this work to extract a high amount of these compounds. Termo Fisher Scientifc) equipped with photodiode array Tus, considering the traditional knowledge and the use of detector (PDA–3000 [RS]; Termo Fisher Scientifc), a binary organic solvents that extract a high number of compounds, pump (HPG3x00RS, Termo Fisher Scientifc), a degasser, the present study aimed to explore the biological activities of and an autosampler. Te analysis was performed in a C18 col- the aqueous and organic extract of J. pectoralis leaves. umn (250 mm x 4.6 mm, 5 �m; NST) equipped with a precol- ∘ umn C18 (4 mm, 3.9 �m; Phenomenex), maintained at 24 C. 2. Materials and Methods Te mobile phase consisted of purifed water as solvent A 2.1. Plant Material and Extracts Preparation. Specimens were and methanol as solvent B, both acidifed with trifuoroacetic collected at the Training Center of the Agricultural Research acid (0.05%), with the fow adjusted at 0.8 mL/minute. Both Institute (CETREINO/IPA), located in Carpina, Pernam- were degassed in an ultrasonic bath and fltered through a ∘ � �� ∘ � �� � buco, Brazil (07 51 03 S35 15 17 W), under controlled membrane (45 mm, 0.45 m). Te injection volume used was � growth conditions. Afer collection, a voucher specimen (# 20 l. Te wavelength of the analyses was set at 280 nm. Te 91413) was identifed and deposited in the IPA Herbarium. separation was conducted using the following gradient: 0- Aferwards, the leaves were removed and dried in an oven 10 min, 15-30% B; 10-20 min, 30-50% B; 20-25 min, 50-75% for 48 hours. Te dried material was then milled using B; 25-28 min, 75-15% B; 28-32 min, 15% B. Te standards a knife mill (TE-680, Tecnal). Te extracts were obtained coumarin (98% of purity), apigenin (analytical standard), and by 10% (w/v) turbo-extraction (Metvisa) using water or an ellagic acid (96% of purity) were purchased from Sigma- acetone:water mixture (7:3, v/v). Te extracts were fltered Aldrich (Sao˜ Paulo, Brazil). Te experiments were performed by vacuum and the organic extract was concentrated under in triplicate. reduced pressure (RV10 Basic, IKA) to remove the acetone. ∘ Te fltrates were frozen (3 days, T = -80 C) and the 2.4. Cytotoxicity Evaluation. Cytotoxicity was initially eval- remaining solid material was lyophilized for 48 hours (L101, uated in nontransformed cells (peripheral blood mononu- Liotop) to yield the aqueous extract (JPA) and organic extract clear cells [PBMCs] from healthy donors and Balb/c mice (acetone:water - JPO) from J. pectoralis. splenocytes) and then in neoplastic cell lines. Both evalu- ations were performed with MTT (3-(4,5-Dimethylthiazol- 2.2. Phytochemical Characterization by TLC. Te crude 2-yl)-2,5-Diphenyltetrazolium Bromide) assay according to extracts were dissolved in methanol and agitated in a vortex Carvalho et al. [17] and with the trypan blue exclusion (LabDancer, IKA) to complete solubilization, obtaining a method. Te assays were only initiated afer approval from the fnal concentration of 1 mg/mL. For thin-layer chromatogra- Human Ethics Committee CAAE: 46976315.9.0000.5208 and phy (TLC) screening, all standards were used at 1 mg/mL. Te the Ethics Committee for the Use of Experimental Animals of samples and standards were applied, 20 �land5�l, respec- the UFPE (process number 23076.041556/2015-62). PBMCs tively, on silica gel 60 - F254 (Macherey-Nagel) chromato- were used from four clinically healthy individuals who graphic plates, using a semiautomatic applicator (Camag) and did not meet the exclusion criteria.