Immunomodulatory, antibacterial and antineoplastic potential of Trichilia catigua A. Juss. (Meliaceae) bark extracts and isolated compound cinchonain Ib

Potencial imunomodulador, antibacteriano e antineoplásico dos extratos da casca de Trichilia catigua A. Juss. (Meliaceae) e do composto isolado cinchonain Ib

Abstract

Immunomodulatory, antibacterial and antineoplastic activity of crude extract (CE), aqueous (AqF), and ethyl-acetate fractions (EAF) from the barks of Trichilia catigua, and cinchonain Ib EAF-isolated were investigated. CE, AqF, and EAF treatment induced significantly decreased levels of IL-6, IL-17A and IL-22 cytokines. IFN-y and IL-10 showed significant decreases after CE treatment. Cinchonain Ib significatively reduced IL-17A, IFN-y, TNF-α, IL-2 and IL-6 levels. All tested extracts and fractions showed antibacterial activity against Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus (MRSA). Cinchonain Ib exhibited MIC (31.25 µg/mL) and MBC (250 µg/mL) against S. aureus, MRSA and S. epidermidis clinical isolate. CE and EAF treatments showed cytotoxicity in HL-60 and TOLEDO cells. EAF treatment was cytotoxic in MCF-7 cells. Cinchonain Ib presented cytotoxicity to K562 (IC50=46.21±3.9), HL-60 (IC50=51.83±3.17) and MOLT-4 (IC50=57.52±4.57) cells. These results evidenced the availability of uses of T. catigua and cinchonain Ib as immunomodulatory, antineoplastic, bacteriostatic and bactericidal agent.

Keywords:
catuaba; cinchonain Ib; bacteria; cancer; inflammation

Resumo

A atividade imunomoduladora, antibacteriana e antineoplásica do extrato bruto (EB), frações aquosas (AqF) e de acetato de etila (EAF) das cascas de Trichilia catigua e cinchonain Ib isolada por EAF foi investigada. O tratamento com EB, AqF e EAF induziu níveis significativamente reduzidos das citocinas IL-6, IL-17A e IL-22. IFN-γ e IL-10 apresentaram reduções significativas após o tratamento com EC. A cinchonain Ib reduziu significativamente os níveis de IL-17A, IFN-γ, TNF-α, IL-2 e IL-6. Todos os extratos e frações testados apresentaram atividade antibacteriana contra Staphylococcus aureus e Staphylococcus aureus resistente à meticilina (MRSA). A cinchonain Ib apresentou CIM (31,25 µg/mL) e CBM (250 µg/mL) contra isolados clínicos de S. aureus, MRSA e S. epidermidis. Os tratamentos com EB e EAF apresentaram citotoxicidade em células HL-60 e TOLEDO. O tratamento com EAF foi citotóxico em células MCF-7. A cinchonain Ib apresentou citotoxicidade para células K562 (IC50=46,21±3,9), HL-60 (IC50=51,83±3,17) e MOLT-4 (IC50=57,52±4,57). Esses resultados evidenciam a disponibilidade de uso de T. catigua e cinchonain Ib como agentes imunomoduladores, antineoplásicos, bacteriostáticos e bactericidas.

Palavras-chave:
catuaba; cinchonain Ib; bactérias; câncer; inflamação

1. Introduction

The use of medicinal plants currently plays a crucial role in inflammatory processes, bacterial infections, cancer, among others (Aguiar, 2015; Tan et al., 2021; Muthuramalingam et al., 2023). In addition, plants represent an inexhaustible source of bioactive molecules, with potential anticancer multi-targeted treatment mechanisms, many of them already in the clinic as therapeutic agents, such as vincristine, vinblastine, camptothecin, curcumin, podophyllotoxin, paclitaxel, and other (Khan and Gurav 2018; Pradhan et al., 2021; Muthuramalingam et al., 2023).

Trichilia catigua A. Juss. (Meliaceae) is popularly known as ‘Big Catuaba’ and some studies have reported their antidiabetic, antioxidant, anticholinesterase, antifatigue, anti-inflammatory,neuroactive and neuroprotective effects (Gomes et al., 2017; Bernardo et al., 2018; Godinho et al., 2018; Martins et al., 2018; Oliveira et al., 2023). Additionally, the search for new antimicrobial agents is currently investigated due to the increase of microorganism resistance to conventional drugs (Ritter et al., 2021). This species is widely distributed in South America, especially in Brazil, and is popularly used in folk medicine as an anti-inflammatory, tonic, and neurostimulant agent. Ethnobotanical studies have documented its traditional use and pharmacological potential (Bernardo et al., 2018; Bernardo et al., 2022; Oliveira et al., 2023).

Generally, extracts obtained from T. catigua stem bark contain phytochemical compounds such as flavan-3-ols (procyanidin B2, epicatechin, catechin), flavalignans (cinchonains Ia, Ib, IIa, IIb, apocynin E) and phenylpropanoid derivatives (chlorogenic acid) (Pizzolatti et al., 2002; Resende et al., 2011; Rabelo et al., 2013; Bernardo et al., 2018; Godinho et al., 2018; Ritter et al., 2021). Even though flavalignans plays a significant role on immunomodulatory, bacterial and cancer process (Godinho et al., 2017; Bernardo et al., 2021; Martins et al., 2018; Longhini et al., 2013), the studies related to immunomodulatory, antibacterial and antineoplastic Cinchonain Ib potential are still poorly explored. In this sense these biological activities of the crude extract (CE), aqueous (AqF) and EAF fractions from T. catigua stem barks were evaluated in this work, as well as the biological potential of the isolated cinchonain Ib from EAF.

2. Results and Discussion

2.1. Immunomodulatory effect

CE, AqF, and EAF treatment induced significative decreased levels of IL-6 cytokine at 50 (p=0.01), 100 (p=0.01), and 100 µg/mL (p=0.007), respectively (Figure 1A). CE and both fractions reduced in a dose-dependent manner, IL-1β levels (Figure 1B). CE significantly reduced IFN-y levels at 50 and 100 µg/mL (p=0.03 and p=0.01, respectively) (Figure 1C). For IL-22 cytokine, all extracts significantly decreased it levels at 100 µg/mL (p=0.003 - CE; p=0.003 – AqF; p=0.03 - EAF), however, EAF also decreased IL-22 levels at 50 µg/mL (p=0.02) (Figure 1D). All extracts showed greater ability to significantly reduce IL-17A levels (Figure 1E): CE reduced at 50 µg/mL (p= 0.03) and at 100 µg/mL (p=0.001), AqF at 50 µg/mL (p=0.0005) and 100 µg/mL(p=0.01), and EAF reduced at 50 µg/mL (p=0.004) and at 100 µg/mL (p=0.003). However, CE treatment at 10 and 50 µg/mL (p=0.02 and p=0.03, respectively) reduced levels of IL-10 (Figure 1F). Regarding cinchonain Ib immunomodulatory potential, Figure 2 presents the CBA results. This flavolignan significantly reduced IL-17A, TNF-α, IL-6, and IL-2 levels at 50 µg/mL (p=0.03 to all cytokines). However, it was able to decrease IFN-y levels at 10 and 50 µg/mL (p=0.03 for both concentrations).

Figure 1
Cytokines profile in BALB/c mice splenocytes culture supernatant treated with T. catigua CE, AqF and EAF at concentrations of 5, 10, 50 and 100 µg/mL. (A) IL-6; (B) IL-1β; (C) INF-y; (D) IL-22; (E) IL-17A; (F) IL-10. (CNT = Untreated control, ConA = Concanavalin A and MP = methylprednisolone at 100 µM). Significance is given with asterisk (*) and bars represent median, minimum and maximum. The asterisk (**) and (***) represents degree of significance in relation to ConA. Outliers are shown as black circles.

Figure 2
Graphics A-G represents cytokine levels (IL-17A, TNF-α, IFN-γ, IL-6, IL-4, IL-2, and IL-10 respectively) measured in cell culture supernatants after stimulation with ConA, treatment whit standard drug MP, or cinchonain Ib treatment at 10 and 50 µg/mL. Data are presented as box plots. Asterisks (*) indicate statistically significant differences compared to the ConA group. (CELL = Untreated control, ConA = Concanavalin A and MP = methylprednisolone at 100 µM). Significance is given with asterisk (*) and bars represent median, minimum and maximum. Outliers are shown as black circles.

2.2. Antibacterial activity

The antibacterial activity of CE, AqF and EAF is shown at Table 1. All the plant extracts tested exhibited the same MBC (MBC ≥ 250 µg/mL). The extracts also exhibit the same MIC values against Staphylococcus aureus ATCC 29213 and methicillin-resistant Staphylococcus aureus (MRSA) ATCC 33591. CE exhibits MIC= 250 µg/mL against Pseudomonas aeruginosa ATCC 27853. CE, AqF and EAF presented the same profile of activity against Escherichia coli ATCC 25922, Staphylococcus epidermidis clinical isolate and Methicillin-resistant Staphylococcus aureus clinical isolates (LMB) 02 to LMB 10 (MIC= 250 µg/mL; MBC > 250 µg/mL). All plant extracts had no antibacterial activity against Klebsiella pneumoniae ATCC 29665 and LMB 01. Table 2 presents antibacterial profile of cinchonain Ib against Staphylococcus aureus ATCC 29213, MRSA ATCC 33591, Staphylococcus epidermidis clinical isolate and Escherichia coli ATCC 25922. Cinchonain Ib did not show differences against E. coli as compared with CE, AqF, and EAF. However, MBC was quantified at all tested bacteria (MBC=250 µg/mL). In addition we evidenced MIC and MBC greater values after treat the Staphylococcus epidermidis clinical isolate with cinchonain Ib. To avoid misinterpretation, we clarified that the observed activity for extracts and cinchonain Ib is primarily bacteriostatic at MIC levels, with bactericidal effect detected only at higher concentrations where MBC values were obtained (MBC/MIC ≥ 4). This clarification follows accepted interpretive criteria commonly used in antibacterial susceptibility testing.

Table 1
MIC values to T. catigua CE, AqF, and EAF.

	CE	AqF	EAF
		MIC (µg/mL)
Escherichia coli ATCC 25922	250	250	250
Pseudomonas aeruginosa ATCC 27853	250	>250	>250
Staphylococcus epidermidis clinical isolate	250	250	250
Staphylococcus aureus ATCC 29213	125	62.5	31.25
Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 33591	125	62.5	31.25

• Values are expressed in µg/mL.

Table 2
MIC and MBC values after Cinchonain Ib treatment.

Bacteria	MIC (µg/mL)	MBC (µg/mL)
Staphylococcus aureus ATCC 29213	31 µg/mL	250 µg/mL
Staphylococcus aureus methicillin resistant (MRSA) ATCC 33591	31 µg/mL	250 µg/mL
Staphylococcus epidermidis clinical isolate	31 µg/mL	250 µg/mL
Escherichia coli ATCC 25922	250 µg/mL	>250µg/mL

• Values are expressed in µg/mL.

2.3. Antineoplastic effect of human tumor cell lineages

Results show no cytotoxicity until 200 µg/mL to PBMCs (Table 3). CE exhibited an antineoplastic activity against TOLEDO (IC₅₀=98.09±2.91) and HL-60 (IC₅₀=65.93±31.79), which showed a greater selectivity (SI=3.03). At HEP-G2 cells, CE also displayed an antineoplastic profile. T47-D IC₅₀ values are higher than 200 µg/mL. AqF showed IC₅₀ values against TOLEDO and HL-60 (IC₅₀ = 100.71±0.007 µg/mL; IC₅₀ = 85.63±2.35 µg/mL, respectively). IC₅₀ of HEP-G2, MCF-7 and T47-D cells are higher than 200 µg/mL. EAF was cytotoxic on MCF-7, TOLEDO and HL-60 cells (IC₅₀=91.07±6.57; 94.42±9.11; 71.91±15.76 µg/mL, respectively). Figure 3 detailed a cytotoxicity profile of Cinchonain Ib against neoplastic cells. Based on SI, once the IC50 value of Vero cells was higher than 100 µg/mL, we cannot consider a precise SI value to cinchonain Ib. Thus, the SI of cinchonain Ib are “virtual” values. K562 (SI) = 2.16 / MOTL-4 (SI) = 1.73 / HL-60 (SI) = 1.92.

Table 3
Cytotoxicity of T. catigua CE, AqF, and EAF.

	PBMC	HEP-G2	MCF-7	T47-D	TOLEDO	HL-60
CE	>200	100.34 ± 0.18	> 200	>200	98.09 ± 2.91	65.93 ± 31.79
		(SI = 1.99)	(SI = 1.00)	(SI = 1.00)	(SI = 2.03)	(SI = 3.03)
AqF	>200	>200	>200	>200	100.71 ± 0,007	85.63 ± 2.35
		(SI = 1.00)	(SI = 1.00)	(SI = 1.00)	(SI = 1.98)	(SI = 2.33)
EAF	>200	>200	91.07 ± 6.57	>200	94.42 ± 9.11	71.91 ± 15.76
		(SI = 1.00)	(SI = 2.19)	(SI = 1.00)	(SI = 2.11)	(SI = 2.78)

• Values are expressed in µg/mL. The Selectivity Index (SI) are virtual values due to it can be higher if tested in elevated concentrations.

Figure 3
Antineoplastic potential of Cinchonain Ib against K562, MOLT-4, HL-60, PANC-1 and DU-145 neoplastic cell lineages.

2.4. Discussion

Condensed tannins found in T. catigua extracts represent a class of phenolic compounds that have anti-inflammatory, antibacterial, and anticancer activities already known (Pizzolatti et al., 2002; Barbosa et al., 2004; Ritter et al., 2021; Godinho et al., 2017). Longhini et al. (2013) previously identified condensed tannins on our CE of T. catigua such as procyanidin B2 (PB2) and epicatechin (EPC). However, Godinho et al. (2017) also identified procyanidin B2 and epicatechin, and documented the presence of other phytochemicals, such as, catechins and cinchonain IIa, IIb and a mixture of cinchonains Ia and Ib on EAF used in our work. Godinho et al. (2017) showed that the activity of these molecules reflects in an inflammation reduction, following induction of global cerebral ischemia in rats. Recently, Godinho et al. (2018) demonstrated the protective effect of EAF against neuroinflammation on a rat model of brain ischemia. Therefore, the bioactive components found by these authors in EAF composition (epicatechin, procyanidin B2 and cinchonains) contribute to understand the immunomodulatory behavior of the crude extract, fractions and cinchonain Ib from T. catigua observed in this work.

Despite it is used in traditional medicine as an anti-inflammatory and neurostimulator agent (Bernardo et al., 2022; Barbosa et al., 2004; Oliveira et al., 2023), there are few studies that address the immunomodulatory activity of T. catigua barks extracts. To our knowledge, this is the first work to demonstrate a reduction of proinflammatory cytokines IFN-y,IL-10, IL-6, IL-17A, and IL- 22 and the increase of IL-10, an anti-inflammatory cytokine, after treatment with T. catigua CE, AqF, and EAF. The CE presented more significatively results than AqF and EAF, presumably due to high amounts of different phytochemicals mixtures found at CE. In addition, cinchonain Ib was able to decrease the levels of inflammatory cytokines IL-17A, IL-6, IL-2, TNF-α and IFN-y in BALB/c mice splenocytes supernatant. This result corroborates the anti-inflammatory activity observed in hydroethanolic extract from T. catigua barks, where an inhibition of PLA2 was demonstrated (Barbosa et al., 2004; Yedgar et al., 2006).

Some substances that comprise the extracts and fractions of T. catigua barks are important due to their ability to exert antibacterial activity. In general, these molecules are flavolignans: cinchonain Ia and Ib found in EAF, which have bactericidal activity against E. coli, P. aeruginosa, Bacillus cereus and Staphylococcus aureus, or flavan-3-ols: catechin-3-O-α-L-rhamnoside found in AqF, which have bactericidal activity against Vancomycin ResistantEnterococcus faecium (Pizzolatti et al., 2002; Wang et al., 2023). Therefore, the MIC values of extracts against S. aureus and MRSA observed in this work can be associated with cinchonains and flavan-3-ols presence in its composition, since these molecules are commonly found in T. catigua stem barks extracts (Aboufaras et al., 2023; Wang et al., 2023).

Currently, there are several medicinal plants, which are traditionally used for cancer treatment (Aboufaras et al., 2023; Khan and Gurav 2018; Machana et al., 2012). Our data shows an antineoplastic activity of T. catigua extracts resulting in promissory molecules sources to be evaluated for leukemia treatment. Enhanced response of CE can be related to excessive amounts of molecules that comprises this extract. These compounds acting synergistically with phenols content in CE to inhibits proliferation, adhesion, migration and invasion of tumor cells (Fuzer et al., 2013), and downregulation of EGFR, β1-integrin and phospho-Akt, which could have resulted in a decrease of NFκB levels and MMP9 activity (Becceneri et al., 2020). At present, the studies of Fuzer and Becceneri (Fuzer et al., 2013; Becceneri et al., 2020; Gomes et al., 2021) is the only work about T. catigua antineoplastic activity. It is important to emphasize that our antibacterial and antineoplastic findings represent preliminary in vitro screening data. While they demonstrate promising activity profiles, further mechanistic studies are required, such as time–kill kinetics, apoptosis/necrosis assays, and in vivo models, to confirm the translational potential of T. catigua extracts and cinchonain Ib.

The antioxidant (Gomes et al., 2021; Oliveira et al. 2023) and antimicrobial activities (Pizzolatti et al., 2002; Wang et al., 2023) of some flavolignans isolated from T. catigua barks have been described, but the antineoplastic activity of these molecules has not yet better elucidated. Tabanca et al. (2007) showed the antineoplastic potential of cinchonains Ia and IIa in hepatocarcinoma, melanoma, ovarian carcinoma, breast cancer, squamous cell carcinoma and promyelocytic leukemia cancer cells lines, but the compounds were isolated from the barks of Anemopaegma arvense.This study corroborate the results obtained from our work, where Cinchonaina Ib showed antineoplastic potential against MOLT-4 and K562 leukemia cancer cell lines. It should be noted that the SI found are considered "virtual" since no IC₅₀ values were found for the PBMCs and Vero. Thus, these values are even higher, showing that CE, AqF, EAF, and Cinchonain Ib are even more selective for hematological malignancies than for normal cells, therefore considered promising agents (Kamdem et al., 2012).

3. Experimental

3.1. Plant materials

The stem bark of T. catigua was acquired in Caetité, Bahia, Brazil (14º 04' 10" S - 42º 28' 30" W, h=562 m), in August 2008 and a voucher specimen was deposited in the Herbarium of Curitiba Town Hall (# 306253) (Rocha-Junior et al., 2013). CE, AqF, and EAF were sent to LINAT-UFPE by Prof. Dr. João Carlos Palazzo de Melo for biological activities evaluation. Cinchonain Ib isolated from EAF (Resende et al., 2011) was also sent to immunomodulatory, antibacterial and antineoplastic evaluation. The collected material was dried at room temperature, powdered, and subjected to hydroethanolic extraction to obtain the crude extract (CE). The CE was then fractionated with solvents of increasing polarity, yielding aqueous (AqF) and ethyl acetate (EAF) fractions. Cinchonain Ib was isolated from the ethyl acetate fraction (EAF) by chromatographic procedures, including Sephadex LH-20 column chromatography and preparative HPLC, and its identity was confirmed by NMR and MS analyses, in agreement with Resende et al. (2011).

3.2. Immunomodulatory activity

3.2.1. Citotoxicity evaluation in splenocytes

BALB/c mice (female, n=9, 45 days old) were obtained from LIKA-UFPE. Subsequently, BALB/c mice were sacrificed at the CO2 chamber after approval from the Ethics Committee of Experimental Animals at UFPE (process number 23076.041556/2015-62). Mice had their spleens removed and transferred to Petri dishes containing 4 mL RPMI 1640 medium (Gibco) to splenocytes obtention (Carvalho et al., 2016). The effect of each extract and fractions tested was quantified by the MTT method according to the procedure described by Franklin et al. (2012).

3.2.2. Cell culture and cytokine quantification

Splenocytes were cultured in 24-well plates (2x10⁶/mL in each well) in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 10 mM HEPES (Gibco) and penicillin and streptomycin 200 U/mL (Gibco). Cells were stimulated with ConA at 100 ng/mL. As controls, wells were used with untreated cells and one with methylprednisolone at 100 µM. CE, AqF, and EAF were added to different wells at concentrations of 5, 10, 50, and 100 µg/mL. The plates were incubated at 37.0 °C and 5.0% CO₂ during 24 and 48 h.

After incubation time, 1.0 mL of culture supernatants were collected from each well and stored at -30 °C until use. Cytokine determination was performed by ELISA kits following the manufacturer's information. The lower detection limits of ELISA kits were 9.3 pg/mL for IFN-y (BD Biosciences), 7.8 pg/mL for IL-1β (eBiosciences), 7.8 pg/mL for IL-6 (BD Biosciences), 15.6 pg/mL for IL-17A (eBiosciences), 15.6 pg/mL for IL-22 (eBiosciences), and 31.2 pg/mL for IL-10 (BD Biosciences).

IL-17A, TNF-α, IFN-y, IL-6, IL-4, IL-2, and IL-10 cytokines quantification after treatment with cinchonain Ib was determinate by CBA using the kit (BD Biosciences, San Jose, CA, USA) on the BD™ Accuri Fllow cytometer according to the manufacturer’s recommendations, and quantification was performed using the BD™ FCAP Array Software v3.0.

3.3. Antibacterial evaluation

3.3.1. Bacterial strains

Methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis clinical strains were obtained at the Hospital das Clinicas-UFPE and they were preserved at the Laboratory of Microbiology and Immunology (LMB) of the Academic Center of Vitória of -UFPE. The resistant profile MRSA of the clinical isolates were performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines (Bèzivin et al., 2003). Reference strains were obtained from American Type Culture, such as MRSA ATCC 33591, methicillin-sensitive Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 29665 and Pseudomonas aeruginosa ATCC 27853.

3.3.2. Antibacterial activity

Briefly, the antibacterial activity of T. catigua extracts, fraction and cinchonain Ib was assessed by the broth microdilution method according to CLSI guidelines (Bèzivin et al., 2003). Minimal Inhibitory Concentration (MIC) was defined as the lowest concentration of extracts that inhibited visible growth as indicated by the triphenyl tetrazolium chloride aqueous solution (TCC) staining (Franklin et al., 2012) and Minimal Bactericidal Concentration (MBC) was considered as the lowest concentration of the extracts associated with no bacterial growth. DMSO (0.5%) was used as a negative control.

3.4. Antineoplastic potential

3.4.1. Cytotoxicity in Peripheral blood mononuclear cells (PBMC) and Vero Cells

Cytotoxicity assay was performed with PBMC obtained from heparinized blood from three healthy donors who had not taken any drugs for at least 15 days prior to sample collection, who had not consumed alcohol (at least 3 days) and nonsmoking individuals. All donors gave informed consent and the biological material was collected after TCLE signature. This study was approved by the Human Research Ethics Committee of UFPE in the Health Sciences Center with CAAE number: 46976315.9.0000.5208. Cells were isolated through a standard method of density-gradient centrifugation over Ficoll- Hypaque solution (GE Healthcare). The PBMC were counted in a Neubauer chamber and viability was determined by the trypan blue exclusion method. Cells were used only when the viability was more than 98%. Cells were plated in 96-well plates (5.5x10⁵ cells/well). After plated, CE, AqF, and EAF were added ranging from 10 to 200 µg/mL and incubated for 48 h. After this point the cells were subjected to the MTT assay as described previously.

MTT assays were also performed to evaluate cytotoxicity of Cinchonain Ib at Vero Cells (ATCC^®–CCL-81) (normal epithelial kidney cells from Cercopithecus aethiops). Vero were plated in 96-well plates with 10⁴ cells/well. After 24 h, a dimethyl sulfoxide solution of Cinchonain Ib (0.1, 1, 10 and 100 µg/mL) was added to each well with DEMEM high glucose supplemented with 10% BFS, and the cells were incubated for 72 h at 37.0°C with 5.0% of CO₂.

3.4.2. Cytotoxicity in human neoplastic cell lines

Cytotoxicity of CE, AqF, and EAF were tested against five human neoplastic cell lines: HEP-G2 (hepatocarcinoma), MCF-7 (breast cancer), T47-D (breast cancer), TOLEDO (lymphoma non-Hodgkin's B cell) and HL-60 (Acute Promyelocytic Leukemia). Antineoplastic potential of Cinchonain Ib were assessed against K562 (chronic myeloid leukemia), HL-60, MOLT-4 (acute lymphoblastic leukemia), PANC-1 (pancreas Epithelioid carcinoma), and DU-145 (prostate carcinoma) cells. All cells were cultured in RPMI-1640 or DMEM (PANC-1 and DU-145) medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 mg/mL streptomycin, and 100 U/mL penicillin at 37.0°C with 5.0% CO₂. All neoplastic cell lineages were obtained from Rio de Janeiro Tissue Cell Bank. Cells were plated in 96-well plates according to each cell doubling time provided from NCI60 guidelines. After 24 h, CE, AqF, and EAF solutions (10, 50, 100, and 200 µg/mL) and cinchonain Ib with concentrations ranged from 0.1 to 100 µg/mL, was added to each well, and the cells were incubated for 72 h at 37.0°C with 5.0% of CO₂. Control groups were treated with the same amount of DMSO at final concentration of 0.1%. With the viability of four tested concentrations were quantified IC₅₀ values and then calculated the selectivity index (SI) by the relation PBMC IC₅₀/Neoplastic Cell IC₅₀. Extract, fractions, and cinchonain Ib were considered as promisors when values of SI were higher than 3 (Kamdem et al., 2012).

3.5. Statistical analysis

Normal variable distribution was evaluated by Kolmogorov-Smirnov test. Variables not following normal distribution were represented in the graphs as median, maximum and minimum. Statistical analysis was performed using GraphPad PRISM^® software version 8. Statistical non-parametric test used in the analysis of cytokines in the culture supernatant was Wilcoxon and the results were considered significant when p < 0.05. OringPro8 Software was used for IC50 determination.

4. Conclusions

Extract, fractions, and cinchonain Ib from T. catigua barks have immunomodulatory activity proving the traditional knowledge and uses of this specie. These plant materials also have bacteriostatic properties against MSSA and MRSA, and bactericidal activity against MSSA, MRSA, and S. epidermidis clinical isolate. The antineoplastic activity of CE, AqF, and EAF, most identified against hematological neoplasias, was also evidenced. Moreover, cinchonain Ib presented cytotoxicity against K562, HL-60 and MOLT-4 cells. Further studies related to T. catigua isolated compounds should be conducted to elucidate the major components that exhibited these three properties. These results should be interpreted as preliminary and exploratory, providing justification for deeper mechanistic investigations and future validation of the biological potential of T. catigua extracts and cinchonain Ib.

Acknowledgements

Authors thanks to the Post-graduation Programme of Therapeutic Innovation of Pernambuco Federal University. This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil), Instituto de Ciência e Tecnologia para Inovação Farmacêutica (INCT_if) [INCT_if, 15/2015 - INCTsCNPq/MCT/FNCT/FINEP/CAPES/FAPESP/FAPEMIG/FAPERG], and Fundação de Amparo à Pesquisa do Estado de Pernambuco (FACEPE, Brazil, project no. APQ-0898-4.03/15).

Data Availability Statement

The research data analyzed in this study are not publicly available by any means.

References

• ABOUFARAS, M., SELMAOUI, K. and OUZENNOU, N., 2023. Efficacies and side effects of medicinal plants used by patients with cancer in Morocco: a retrospective treatment-outcome study. Journal of Ethnopharmacology, vol. 301, pp. 115783. https://doi.org/10.1016/j.jep.2022.115783 PMid:36209952.
  » https://doi.org/10.1016/j.jep.2022.115783
• AGUIAR, B.A.A., 2015. Estudos de controle de qualidade fitoquímica de exratos e frações obtidas de sementes de Paullinia cupana Kunth e sua atividade sobre Helicobacter pylori Maringá: Universidade Estadual do Paraná, 70 p. Master’s thesis.
• BARBOSA, N.R., FISCHMANN, L., TALIB, L.L. and GATTAZ, W.F., 2004. Inhibition of platelet phospholipase A2 activity by catuaba extract suggests antiinflammatory properties. Phytotherapy Research : PTR, vol. 18, no. 11, pp. 942-944. https://doi.org/10.1002/ptr.1579 PMid:15597313.
  » https://doi.org/10.1002/ptr.1579
• BECCENERI, A.B., FUZER, A.M., POPOLIN, C.P., CAZAL, C.M., DOMINGUES, V.C., FERNANDES, J.B., VIEIRA, P.C. and COMINETTI, M.R., 2020. Acetylation of cedrelone increases its cytotoxic activity and reverts the malignant phenotype of breast cancer cells in 3D culture. Chemico-Biological Interactions, vol. 316, pp. 108920. https://doi.org/10.1016/j.cbi.2019.108920 PMid:31857088.
  » https://doi.org/10.1016/j.cbi.2019.108920
• BERNARDO, J., FERRERES, F., GIL-IZQUIERDO, Á., VIDEIRA, R.A., VALENTÃO, P., VEIGA, F. and ANDRADE, P.B., 2018. In vitro multimodal-effect of Trichilia catigua A. Juss. (Meliaceae) bark aqueous extract in CNS targets. Journal of Ethnopharmacology, vol. 211, pp. 247-255. https://doi.org/10.1016/j.jep.2017.09.039 PMid:28970152.
  » https://doi.org/10.1016/j.jep.2017.09.039
• BERNARDO, J., MALHEIRO, I., VIDEIRA, R.A., VALENTÃO, P., SANTOS, A.C., VEIGA, F. and ANDRADE, P.B., 2021. Trichilia catigua and Turnera diffusa extracts: in vitro inhibition of tyrosinase, antiglycation activity and effects on enzymes and pathways engaged in the neuroinflammatory process. Journal of Ethnopharmacology, vol. 271, pp. 113865. https://doi.org/10.1016/j.jep.2021.113865 PMid:33485975.
  » https://doi.org/10.1016/j.jep.2021.113865
• BERNARDO, J., SANTOS, A.C., VIDEIRA, R.A., VALENTÃO, P., VEIGA, F. and ANDRADE, P.B., 2022. Trichilia catigua and Turnera diffusa phyto-phospholipid nanostructures: physicochemical characterization and bioactivity in cellular models of induced neuroinflammation and neurotoxicity. International Journal of Pharmaceutics, vol. 620, pp. 121774. https://doi.org/10.1016/j.ijpharm.2022.121774 PMid:35489602.
  » https://doi.org/10.1016/j.ijpharm.2022.121774
• BÉZIVIN, C., TOMASI, S., LOHÉZIC-LE DÉVÉHAT, F. and BOUSTIE, J., 2003. Cytotoxic activity of some lichen extracts on murine and human cancer cell lines. Phytomedicine: International Journal of Phytotherapy and Phytopharmacology, vol. 10, no. 6-7, pp. 499-503. https://doi.org/10.1078/094471103322331458 PMid:13678234.
  » https://doi.org/10.1078/094471103322331458
• CARVALHO, L.V.N., CORDEIRO, M.F., LINS, T.U.L., SAMPAIO, M.C.P.D., MELLO, G.S.V., COSTA, V.C.M., MARQUES, L.L.M., KLEIN, T., MELLO, J.C.P., CAVALCANTI, I.M.F., PITTA, I.R., PITTA, M.G.R. and RÊGO, M.J.B.M., 2016. Evaluation of antibacterial, antineoplastic, and immunomodulatory activity ofevidence-based complementary and alternative medicinePaullinia cupanaseeds crude extract and ethyl-acetate fraction. Evidence-Based Complementary and Alternative Medicine, vol. 2016, no. 1, pp. 1203274. https://doi.org/10.1155/2016/1203274
  » https://doi.org/10.1155/2016/1203274
• FRANKLIN, R., MATTHEUW, A., ALDER, J., MICHAEL, N., GEORGE, M., FERRARO, M.J., SWIGHT, J., DAVID, W., JANET, A. and JEAN, B., 2012. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; Approved standard Wayne: CLSI.
• FUZER, A.M., FILHO, J.C., BECCENERI, A.B., SANTOS, D.A., SILVA, M.F., VIEIRA, P.C., FERNANDES, J.B., SELISTRE-DE-ARAUJO, H.S., CAZAL, C.M. and COMINETTI, M.R., 2013. Effects of limonoid cedrelone on MDA-MB-231 breast tumor cells in vitro. Anti-cancer Agents in Medicinal Chemistry, vol. 13, no. 10, pp. 1645-1653. https://doi.org/10.2174/18715206113139990314 PMid:23869780.
  » https://doi.org/10.2174/18715206113139990314
• GODINHO, J., DE OLIVEIRA, R.M.W., DE SA-NAKANISHI, A.B., BACARIN, C.C., HUZITA, C.H., LONGHINI, R., MELLO, J.C.P., NAKAMURA, C.V., PREVIDELLI, I.S., DAL MOLIN RIBEIRO, M.H. and MILANI, H., 2017. Ethyl-acetate fraction of Trichilia catigua restores long-term retrograde memory and reduces oxidative stress and inflammation after global cerebral ischemia in rats. Behavioural Brain Research, vol. 337, pp. 173-182. https://doi.org/10.1016/j.bbr.2017.08.050 PMid:28919157.
  » https://doi.org/10.1016/j.bbr.2017.08.050
• GODINHO, J., DE SA-NAKANISHI, A.B., MOREIRA, L.S., DE OLIVEIRA, R.M.W., HUZITA, C.H., MELLO, J.C.P., DA SILVA, A.O.F., NAKAMURA, C.V., PREVIDELLI, I.S., RIBEIRO, M.H.D.M. and MILANI, H., 2018. Ethyl-acetate fraction of Trichilia catigua protects against oxidative stress and neuroinflammation after cerebral ischemia/reperfusion. Journal of Ethnopharmacology, vol. 221, pp. 109-118. https://doi.org/10.1016/j.jep.2018.04.018 PMid:29660468.
  » https://doi.org/10.1016/j.jep.2018.04.018
• GOMES, A.F., MOREIRA, B.O., YATSUDA, R., DE SOUZA, É.P., DAVID, J.M., ALVES, C.Q., MACEDO, G.E.L., CORREIA, S.J. and DE PAULA, V.F., 2021. Chemical constituents, antioxidant, anti-inflammatory and, antinociceptive activities ofTrichilia ramalhoi Natural Product Research, vol. 35, no. 22, pp. 4789-4793. https://doi.org/10.1080/14786419.2020.1723088 PMid:32011167.
  » https://doi.org/10.1080/14786419.2020.1723088
• GOMES, R.M., DE PAULO, L.F., BONATO PANIZZON, C.P.D.N., NEVES, C.Q., CORDEIRO, B.C., ZANONI, J.N., FRANCISCO, F.A., PIOVAN, S., DE FREITAS MATHIAS, P.C., LONGHINI, R., DE MELLO, J.C.P., DE OLIVEIRA, J.C., PEDRINO, G.R., DA SILVA REIS, A.A., CECCHINI, A.L. and MARÇAL NATALI, M.R., 2017. Anti-diabetic effects of the ethyl-acetate fraction of trichilia catigua in streptozo-tocin-induced Type 1 diabetic rats. Cellular Physiology and Biochemistry, vol. 42, no. 3, pp. 1087-1097. https://doi.org/10.1159/000478761 PMid:28662504.
  » https://doi.org/10.1159/000478761
• KAMDEM, J.P., STEFANELLO, S.T., BOLIGON, A.A., WAGNER, C., KADE, I.J., PEREIRA, R.P., PRESTE, A.S., ROOS, D.H., WACZUK, E.P., APPEL, A.S., ATHAYDE, M.L., SOUZA, D.O. and ROCHA, J.B.T., 2012. In vitro antioxidant activity of stem bark of Trichilia catigua Adr. Juss. Acta Pharmaceutica: A Quarterly Journal of Croatian Pharmaceutical Society and Slovenian Pharmaceutical Society, Dealing with All Branches of Pharmacy and Allied Sciences, vol. 62, no. 3, pp. 371-382. https://doi.org/10.2478/v10007-012-0026-x PMid:23470349.
  » https://doi.org/10.2478/v10007-012-0026-x
• KHAN, T. and GURAV, P., 2018. PhytoNanotechnology: enhancing delivery of plant based anti-cancer drugs. Frontiers in Pharmacology, vol. 8, pp. 1002. https://doi.org/10.3389/fphar.2017.01002 PMid:29479316.
  » https://doi.org/10.3389/fphar.2017.01002
• LONGHINI, R., KLEIN, T., BRUSCHI, M.L., SILVA JUNIOR, W.V., RODRIGUES, J., LOPES, N.P. and DE MELLO, J.C., 2013. Development and validation studies for determination of phenylpropanoid-substituted flavan-3-ols in semipurified extract of Trichilia catigua by high-performance liquid chromatography with photodiode array detection. Journal of Separation Science, vol. 36, no. 7, pp. 1247-1254. https://doi.org/10.1002/jssc.201200911 PMid:23576366.
  » https://doi.org/10.1002/jssc.201200911
• MACHANA, S., WEERAPREEYAKUL, N., BARUSRUX, S., THUMANU, K. and TANTHANUCH, W., 2012. Synergistic anticancer effect of the extracts from Polyalthia evecta caused apoptosis in human hepatoma (HepG2) cells. Asian Pacific Journal of Tropical Biomedicine, vol. 2, no. 8, pp. 589-596. https://doi.org/10.1016/S2221-1691(12)60103-8 PMid:23569977.
  » https://doi.org/10.1016/S2221-1691(12)60103-8
• MARTINS, N.O., DE BRITO, I.M., ARAÚJO, S.S.O., NEGRI, G., CARLINI, E.A. and MENDES, F.R., 2018. Antioxidant, anticholinesterase and antifatigue effects of Trichilia catigua (catuaba). BMC Complementary and Alternative Medicine, vol. 18, no. 1, pp. 172. https://doi.org/10.1186/s12906-018-2222-9 PMid:29866157.
  » https://doi.org/10.1186/s12906-018-2222-9
• MUTHURAMALINGAM, P., AKASSH, S., RITHIGA, S.B., PRITHIKA, S., GUNASEKARAN, R., SHIN, H., KUMAR, R., BASKAR, V. and KIM, J., 2023. Integrated omics profiling and network pharmacology uncovers the prognostic genes and multi-targeted therapeutic bioactives to combat lung cancer. European Journal of Pharmacology, vol. 940, pp. 175479. https://doi.org/10.1016/j.ejphar.2022.175479 PMid:36566006.
  » https://doi.org/10.1016/j.ejphar.2022.175479
• OLIVEIRA, D.V., GODINHO, J., SA-NAKANISHI, A.B., COMAR, J.F., OLIVEIRA, R.M.W., BONATO, J.M., CHINEN, L.Y., PAULA, M.N., MELLO, J.C.P., PREVIDELLI, I.S., PEREIRA, O.C.N. and MILANI, H., 2023. Delayed administration of Trichilia catigua A. Juss. ethyl-acetate fraction after cerebral ischemia prevents spatial memory deficits, decreases oxidative stress, and impacts neural plasticity in rats. Journal of Ethnopharmacology, vol. 306, pp. 116176. https://doi.org/10.1016/j.jep.2023.116176 PMid:36682600.
  » https://doi.org/10.1016/j.jep.2023.116176
• PIZZOLATTI, M.G., VENSON, A.F., SMÂNIA JUNIOR, A., SMÂNIA, E.F. and BRAZ-FILHO, R., 2002. Two epimeric flavalignans from Trichilia catigua (Meliaceae) with antimicrobial activity. Zeitschrift für Naturforschung. C, A Journal of Biosciences, vol. 57, no. 5-6, pp. 483-488. https://doi.org/10.1515/znc-2002-5-614 PMid:12132689.
  » https://doi.org/10.1515/znc-2002-5-614
• PRADHAN, D., BISWASROY, P., SAHU, A., SAHU, D.K., GHOSH, G. and RATH, G., 2021. Recent advances in herbal nanomedicines for cancer treatment. Current Molecular Pharmacology, vol. 14, no. 3, pp. 292-305. https://doi.org/10.2174/1874467213666200525010624 PMid:32448111.
  » https://doi.org/10.2174/1874467213666200525010624
• RABELO, D.S., PAULA, J.R. and BARA, M.T.F., 2013. Quantificação de fenóis totais presentes nas cascas de Trichillia catigua A. Juss. (Meliaceae). Revista Brasileira de Plantas Medicinais, vol. 15, no. 2, pp. 230-236. https://doi.org/10.1590/S1516-05722013000200010.
• RESENDE, F.O., RODRIGUES-FILHO, E., LUFTMANN, H., PETEREITD, F. and MELLO, J.C.P., 2011. Phenylpropanoid substituted flavan-3-ols from Trichilia catigua and their in vitro antioxidative activity. Journal of the Brazilian Chemical Society, vol. 22, no. 11, pp. 2087-2093. https://doi.org/10.1590/S0103-50532011001100010
  » https://doi.org/10.1590/S0103-50532011001100010
• RITTER, M.R., OLIVEIRA, M.T., MAKIMORI, R.Y., SEREIA, A.L., SIMIONATO, A.S., CHIERRITO, D., ANDRADE FILHO, G., OLIVEIRA, A.G., SILVA, D.B., NOVELLO, C.R., MEDEIROS, D.C., DIAS FILHO, B.P. and MELLO, J.C.P., 2021. Dimeric glycosylated flavan-3-ol and antimicrobial in vitro evaluation ofTrichilia catiguaextracts. Natural Product Research, vol. 35, no. 19, pp. 3293-3300. https://doi.org/10.1080/14786419.2019.1698569 PMid:31813285.
  » https://doi.org/10.1080/14786419.2019.1698569
• ROCHA JUNIOR, L.F., RÊGO, M.J.,CAVALCANTI, M.B., PEREIRA, M.C., PITTA, M.G., OLIVEIRA, P.S., GONÇALVES, S.M., DUARTE, A.L., LIMA, M.C., PITTA, I.R. and PITTA, M.G., 2013. Synthesis of a novel thiazolidinedione and evaluation of its modulatory effect on IFN- γ, IL-6, IL-17A, and IL-22 production in PBMCs from rheumatoid arthritis patients. BioMed Research International, vol. 926060, pp. 926060. https://doi.org/10.1155/2013/926060 PMid:24078927.
  » https://doi.org/10.1155/2013/926060
• TABANCA, N., PAWAR, R.S., FERREIRA, D., MARAIS, J.P., KHAN, S.I., JOSHI, V., WEDGE, D.E. and KHAN, I.A., 2007. Flavan-3-ol-phenylpropanoid conjugates from Anemopaegma arvense and their antioxidant activities. Planta Medica, vol. 73, no. 10, pp. 1107-1111. https://doi.org/10.1055/s-2007-981563 PMid:17642036.
  » https://doi.org/10.1055/s-2007-981563
• TAN, W., PAN, T., WANG, S., LI, P., MEN, Y., TAN, R., ZHONG, Z. and WANG, Y., 2021. Immunometabolism modulation, a new trick of edible and medicinal plants in cancer treatment. Food Chemistry, vol. 376, pp. 131860. https://doi.org/10.1016/j.foodchem.2021.131860 PMid:34971892.
  » https://doi.org/10.1016/j.foodchem.2021.131860
• WANG, Q.Q., SUN, Q.R., JI, X.Y., TANG, Y., ZHANG, K., WANG, X.Q., LI, H.R., HUANG, X.Z. and ZHANG, B., 2023. The combined analgesic, sedative, and anti-gastric cancer mechanisms of Tinospora sagittata var. yunnanensis (S. Y. Hu) H. S. Lo based on integrated ethnopharmacological data. Journal of Ethnopharmacology, vol. 303, pp. 115990. https://doi.org/10.1016/j.jep.2022.115990 PMid:36509262.
  » https://doi.org/10.1016/j.jep.2022.115990
• YEDGAR, S., COHEN, Y. and SHOSEYOV, D., 2006. Control of phospholipase A2 activities for the treatment of inflammatory conditions. Biochimica et Biophysica Acta, vol. 1761, no. 11, pp. 1373-1382. https://doi.org/10.1016/j.bbalip.2006.08.003 PMid:16978919.
  » https://doi.org/10.1016/j.bbalip.2006.08.003

Edited by

• Editor:
  Marcelo A. M. Esquisatto

Publication Dates

• Publication in this collection
  09 Jan 2026
• Date of issue
  2025

History

• Received
  15 May 2025
• Accepted
  17 Oct 2025