Hindawi Journal of Tropical Medicine Volume 2018, Article ID 6854835, 8 pages https://doi.org/10.1155/2018/6854835

Research Article Cytokine Secretion of Peripheral Blood Mononuclear Cells by anthelminthicus Seeds

Surang Leelawat 1 and Kawin Leelawat2

1 Faculty of Pharmacy, Rangsit University, Amphoe Mueang, Pathum Tani 12000, Tailand 2Department of Surgery, Rajavithi Hospital, Bangkok 10400, Tailand

Correspondence should be addressed to Surang Leelawat; [email protected]

Received 6 January 2018; Revised 28 April 2018; Accepted 6 May 2018; Published 3 June 2018

Academic Editor: Marcel Tanner

Copyright © 2018 Surang Leelawat and Kawin Leelawat. 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.

Background. Hydnocarpus anthelminthicus is primarily used as a traditional medicine for the treatment of leprosy. Previous studies demonstrated that the clinical course of leprosy and the susceptibility to mycobacteria are recognized by the immune response of the host. Te current study aims to investigate the efect of H. anthelminthicus seed oil and extracts on the secretion of cytokines from PBMCs involved in immune regulation. Methods. PBMCs from healthy volunteers were cultured and treated with LPS and H. anthelminthicus seed oil or extracts. Cell viability was detected with WST-1 cell proliferation assay reagent. Proinfammatory cytokines were quantifed using ELISA with a specifc antibody. Results. LPS-treated PBMCs signifcantly increased IL6 and TNF-� secretion. H. anthelminthicus seed oil had a synergistic efect with LPS on TNF-� secretion. Te aqueous extract of H. anthelminthicus seed kernels and hulls signifcantly induced IL6 and TNF-� secretion. However, the ethanol extract of H. anthelminthicus seed kernels and hulls signifcantly decreased IL6, IL8, and TNF-� secretion in LPS-treated PBMCs. Conclusions. Extracts of H. anthelminthicus seeds demonstrated various efects on the proinfammatory cytokine secretion of PBMCs. Te application of these extracts should depend on the immune response of the host, which determines the manifestation of the disease.

1. Introduction cellular immune response but have high titers of inefective Hydnocarpus anthelminthicus Pierre ex Laness. is a native antibodies against M. leprae [6]. Southeast Asian tree belonging to . Hydno- However, there is no study of the efects of H. carpus oil or chaulmoogra oil is prepared from the seeds anthelminthicus on the immune response. In this study, of Hydnocarpus spp. and is primarily used as a traditional the secretion of cytokines that are associated with immune responses for many systemic complications of infections, medicine for the treatment of leprosy [1]. � Leprosy is a chronic disease caused by infection with including IL6, IL8, IL10, and TNF- ,ismeasuredfrom Mycobacterium leprae. Afer infection, the immune response peripheral blood mononuclear cells (PBMCs) treated with of the host determines the manifestation of the disease. H. anthelminthicus. � Leprosy causes a peripheral neuropathy with potentially A recent study demonstrated that TNF- induces the long-term disabilities [2, 3]. Its clinical spectrum ranges immune response toward the elimination of the pathogen from tuberculoid leprosy through borderline forms to lep- and/or mediates the pathologic manifestations of the disease. � romatous leprosy of the Ridley-Jopling classifcation [4]. TNF- was induced following stimulation of PBMCs with Tuberculoid leprosy is found in patients who have a strong total or components of M. leprae, namely, lipoarabinoman- cell-mediatedimmuneresponsetoM. leprae, limiting the nan [7]. A previous study found that lipoarabinomannan disease to a few well-defned skin lesions [5]. Lepromatous enhances LPS-induced TNF-� production by engaging scav- leprosy is the severe form of leprosy presented with multiple enger receptors of PBMCs [8]. skin involvements, neuropathy, blindness, and permanent To stimulate the secretion of TNF-� and other proinfam- disability. Patients with lepromatous leprosy have a poor matory cytokines, PBMCs were treated with LPS. In addition, 2 Journal of Tropical Medicine

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50 50 Cell viability (% of control) (% of Cell viability Cell viability (% of control) (% of Cell viability 0 0 LPS - - - - + + + + LPS----++++ HSO (g/ml) 0 5 10 20 0 5 10 20 AHK (g/ml) 0 5 10 20 0 5 10 20 (a) (b) 150 150

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50 50 Cell viability (% of control) (% of Cell viability Cell viability (% of control) (% of Cell viability

0 0 LPS - - - -++++LPS----++++ EHK (g/ml) 0 5 10 20 0 5 10 20 AHH (g/ml) 0 5 10 20 0 5 10 20 (c) (d) 150

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50 Cell viability (% of control) (% of Cell viability

LPS0 - - - - + + + + EHH (g/ml) 0 5 10 20 0 5 10 20 (e)

Figure 1: Te efect of HSO (a), AHK (b), EHK (c), AHH (d), and EHH (e) on PBMC proliferation. PBMCs were separated using Ficoll-Paque density centrifugation, seeded in 96-well plates and treated with 10 �g/ml LPS and extracts at the indicated concentrations. Te cells were then incubated for 24 h before the addition of WST-1 cell proliferation assay reagent.

the modulation efect of H. anthelminthicus on cytokine pressed using a hydraulic presser. Seed kernel marc was ∘ secretion in LPS-treated PBMCs was examined. then dried at 50 C for 30 min, ground into paste, and extracted with 80% ethanol. Marc from the ethanol extract 2. Materials And Methods was then extracted with water by heating in a water bath for 30 min, fltered, and evaporated using a freeze dryer. 2.1. Sample Preparation. Hydnocarpus anthelminthicus from H. anthelminthicus seed oil (HSO), ethanol extract of H. Lampang province, Tailand, was cleaned, dried, and then anthelminthicus seed kernels (EHK), and aqueous extract of ∘ separated into seed hulls and kernels. Seed kernels were H. anthelminthicus seed kernels (AHK) were stored at -80 C ∘ cleaned, dried at 50 C for 30 min, ground into paste, and for further study. Journal of Tropical Medicine 3

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400 400 ∗ ∗ ∗ ∗ 200 200 IL8 Concentration (pg/ml) IL8 Concentration IL6 Concentration (pg/ml) IL6 Concentration

0 0 LPS - - - - + + + + LPS----++++ HSO (g/ml) 0 51020051020 HSO (g/ml) 0 5 10 20 0 5 10 20 (a) (b) 800 800 ∗ ∗ ∗ ∗∗ ∗∗ ∗∗

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∗ 400 400 Concentration (pg/ml) Concentration 200 200 ∗ ∗ IL10 Concentration (pg/ml) IL10 Concentration ∗∗ ∗∗ ∗ ∗∗ TNF -

0 0 LPS----++++LPS - - - - + + + + HSO (g/ml) 0 51020051020 HSO (g/ml) 0 5 10 20 0 5 10 20 (c) (d) Figure 2: Te efect of HSO on IL6 (a), IL8 (b), IL10 (c), and TNF-� (d)productioninPBMCsandLPS-treatedPBMCs.PBMCswereseparated using Ficoll-Paque density centrifugation, seeded in 12-well plates, and treated with 10 �g/ml LPS and HSO at the indicated concentration for 24 h. Levels of IL6, IL8, IL10, and TNF-� secretion were determined using ELISA with specifc antibodies to each cytokine. ∗ p < 0.05 compared to control group; ∗∗ p < 0.05 compared to LPS-treated group alone.

∘ H. anthelminthicus hulls were dried at 50 Cfor30min, 2.3. Te Efect of H. Anthelminthicus Seed Oil and Extracts on ground into powder, and extracted with 80% ethanol. Marc PBMC Proliferation. PBMCs were seeded in 96-well tissue from the ethanol extract was then extracted with water by culture plates at a density of 10,000 cells per well in Ham’s ∘ heating on a water bath for 30 min, fltered, and evaporated F12 at 37 Cina5%CO2 humidifed atmosphere for 24 hours. using a freeze dryer. Ethanol extract of H. anthelminthicus Lipopolysaccharide (LPS; 10 �g/ml) was added to PBMC seed hulls (EHH) and aqueous extract of H. anthelminthicus culture with HSO at concentrations of 0, 5, 10, and 20 �l/ml ∘ seed hulls (AHH) were stored at -80 Cforfurtherstudy. or H. anthelminthicus extracts (EHH, EHK, AHH, or AHK) at concentrations of 0, 5, 10, and 20 �g/ml. Te cells were 2.2. Isolation and Culture of Peripheral Blood Mononuclear subsequently incubated for 24 hours before applying WST- Cells. Blood samples were obtained from healthy volunteers 1 cell proliferation assay reagent (Roche Diagnostics, Laval, at Rajavithi Hospital afer approval by the Rajavithi Ethics Quebec) according to the manufacturer’s instructions. Te Committee. Six healthy volunteers (three males and three percentage of cell proliferation was calculated relative to females) aged 20-40 years with no diseases involved in control PBMCs. Te proliferation of control PBMCs at 24 immune response including acquired immune defciency hourswasindicatedas100%. syndrome (AIDS), diabetes mellitus, and autoimmune dis- eases participated in the study. Peripheral blood mononuclear 2.4. Te Efect of H. Anthelminthicus Seed Oil and Extracts 5 cells (PBMCs) were separated from 25 ml blood using Ficoll- on Proinfammatory Cytokine Secretion. PBMCs (5 × 10 Paque density centrifugation (Pharmacia, Piscataway, NJ, cells/ml) in 12-well tissue culture plates were cultured in ∘ USA). PBMCs were washed with phosphate-bufered saline Ham’s F12 at 37 Cina5%CO2 humidifed atmosphere for (PBS) and then cultured in Ham’s F12 (Gibco, Grand Island, 24 hours. Lipopolysaccharide (LPS; 10 �g/ml) was added to NY, USA) treated with 10% heat-inactivated fetal calf serum the PBMC culture with HSO at concentrations of 0, 5, 10, and (Gibco), 100 U/ml penicillin, and 100 �g/ml streptomycin 20 �l/ml or H. anthelminthicus extracts (EHH, EHK, AHH, ∘ (Gibco) at 37 Cina5%CO2 humidifed atmosphere. and AHK) at concentrations of 0, 5, 10, and 20 �g/ml. PBMCs 4 Journal of Tropical Medicine

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200 200 IL8 Concentration (pg/ml) IL8 Concentration IL6 Concentration (pg/ml) IL6 Concentration

0 0 LPS - - - -++++LPS ----++++ AHK (g/ml) 0 5 10 20 0 5 10 20 AHK (g/ml) 0 5 10 20 0 5 10 20 (a) (b) 600 600

∗ ∗ ∗ ∗ ∗ 400 400 ∗ ∗

200 200 ∗ (pg/ml) Concentration ∗ ∗ ∗∗ ∗ ∗ ∗ ∗∗ ∗∗ IL10 Concentration (pg/ml) IL10 Concentration 0 TNF - 0 LPS----++++LPS - - - -++++ AHK (g/ml) 0 51020051020 AHK (g/ml) 0 5 10 20 0 5 10 20 (c) (d)

Figure 3: Te efect of AHK on IL6 (a), IL8 (b), IL10 (c), and TNF-� (d)productioninPBMCsandLPS-treatedPBMCs.PBMCswere separated using Ficoll-Paque density centrifugation, seeded in 12-well plates, and treated with 10 �g/ml LPS and AHK at the indicated concentrations for 24 h. Te levels of IL6, IL8, IL10, and TNF-� secretion were determined using ELISA with specifc antibodies to each cytokine. ∗ p < 0.05 compared to control group; ∗∗ p < 0.05 compared to LPS-treated group alone.

were subsequently incubated for 24 hours. Te supernatant were compared using one-way analysis of variance (ANOVA) was collected for quantitative analysis of proinfammatory followed by Dunnett’s post hoc test. A p value of less than cytokines (IL6, IL8, IL10, and TNF-�)usinganenzyme- 0.05 was considered to indicate a statistically signifcant linked immunosorbent assay kit with a specifc antibody to result. each cytokine (Quantikine5 Colorimetric Sandwich ELISA, R&D Systems, Minneapolis, MN) according to the manu- 3. Results facturer’s instructions. Briefy, supernatants from control and treated samples were added to each well of a 96-well plate and 3.1. Te Efect of H. Anthelminthicus Seed Oil and Extract on incubated for 2 hours at room temperature. Afer washing, PBMC Proliferation. PBMCs and LPS-treated PBMCs were human IL6 (IL8, IL10, or TNF-�) conjugate (horseradish cultured with 5-20 �g/ml HSO, AHK, EHK, AHH, or EHH. peroxidase) was added and incubated for 2 hours at room Te results demonstrated that LPS and all extracts of H. temperature. Ten, the samples were washed and incubated anthelminthicus seeds had no efect on PBMC proliferation with substrate solution [stabilized hydrogen peroxide and (Figure 1). stabilized chromogen (tetramethylbenzidine)] for 20 min at room temperature and protected from light. A 50 �lstop 3.2. Te Efect of H. Anthelminthicus Seed Oil and Extracts on solution (2N sulfuric acid) was added to each well. Te color Proinfammatory Cytokine Secretion inthewellschangedfrombluetoyellow.Teopticaldensity of each well was determined within 30 minutes, using a 3.2.1. Te Efect of HSO on Cytokine Secretion. PBMCs were microplate reader set to 450 nm. cultured with 5-20 �g/ml HSO alone or 5-20 �g/ml HSO with 10 �g/mg LPS. Te results demonstrated that LPS induced 2.5. Statistical Analysis. Te experiments were performed in IL6 and TNF-� secretionofPBMCsbuthadnoefecton triplicate, and quantitative data were described as the mean IL8 and IL10 secretion. HSO signifcantly induced TNF-� ± standard deviation. Data between three or more groups and IL10 secretion in LPS-treated PBMCs at concentrations Journal of Tropical Medicine 5

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200 200 ∗ ∗ ∗ ∗∗ IL8 Concentration (pg/ml) IL8 Concentration IL6 Concentration (pg/ml) IL6 Concentration ∗ ∗ ∗ ∗∗ 0 0 LPS----++++LPS----++++ EHK (g/ml) 0 5 10 20 0 5 10 20 EHK (g/ml) 0 51020051020 (a) (b) 600 600

∗ 400 400

200 200 Concentration (pg/ml) Concentration

∗ ∗

IL10 Concentration (pg/ml) IL10 Concentration ∗∗ ∗ ∗∗

TNF - ∗∗ 0 0 LPS----++++LPS - - - - + + + + EHK (g/ml) 0 5 10 20 0 5 10 20 EHK (g/ml) 0 5 10 20 0 5 10 20 (c) (d) Figure 4: Te efect of EHK on IL6 (a), IL8 (b), IL10 (c), and TNF-� (d)productioninPBMCsandLPS-treatedPBMCs.PBMCswereseparated using Ficoll-Paque density centrifugation, seeded in 12-well plates, and treated with 10 �g/ml LPS and EHK at the indicated concentrations for 24 h. Te levels of IL6, IL8, IL10, and TNF-� secretion were determined using ELISA with specifc antibodies to each cytokine. ∗ p < 0.05 compared to control group; ∗∗ p < 0.05 compared to LPS-treated group alone. of 5-20 �g/ml.However,HSOhadnoefectonthesecre- 3.2.4. Te Efect of AHH on Cytokine Secretion. Treatment tion of IL6 and IL8 in PBMCs and LPS-treated PBMCs with LPS signifcantly induced the levels of IL6 and TNF- (Figure 2). � in PBMCs, whereas IL8 and IL10 levels were not altered. AHH treatment signifcantly increased IL6, IL10, and TNF- � 3.2.2. Te Efect of AHK on Cytokine Secretion. LPS-treated secretion in PBMCs but had no efect on IL8 secretion. PBMCs showed signifcantly increased IL6 and TNF-� secre- Additionally, treatment with AHH and LPS signifcantly � tion, although the secretion of IL8 and IL10 was not diferent. induced IL10 and TNF- secretion of PBMCs but had no Treatment with AHK signifcantly induced IL6, IL10, and efectonIL6andIL8(Figure5). TNF-� secretion in PBMCs. AHK and LPS treatment signif- 3.2.5. Te Efect of EHH on Cytokine Secretion. LPS treatment icantly induced IL10 secretion but had no efect on IL6, IL8, alone signifcantly released IL6 and TNF-� in PBMCs. How- and TNF-� secretion (Figure 3). ever,LPShadnoinfuenceonIL8andIL10secretion.EHH treatment signifcantly decreased the secretion of IL6 and IL8 3.2.3. Te Efect of EHK on Cytokine Secretion. LPS treatment at concentrations of 5, 10, and 20 �g/ml in PBMCs. However, � alone signifcantly induced IL6 and TNF- secretion, while EHH had no efect on IL10 and TNF-� secretion. In LPS- the secretion of IL8 and IL10 was not diferent from control treated PBMCs, EHH signifcantly decreased the secretion of PBMCs. Treatment with EHK signifcantly decreased the IL6, IL8, and TNF-� at concentrations of 5, 10, and 20 �g/ml. secretion of IL6 and IL8 at concentrations of 10 and 20 However, EHH did not infuence the level of IL10 in LPS- � � g/ml. However, EHK had no efect on IL10 and TNF- treated PBMCs (Figure 6). secretion. EHK and LPS treatment signifcantly decreased the secretion of IL6 and IL8 at a concentration of 20 �g/ml 4. Discussion and the secretion of TNF-� at a concentration of 5-20 �g/ml. However,EHKhadnoefectonIL10secretioninPBMCsand Te pathogenesis and clinical manifestation of leprosy are LPS-treated PBMCs (Figure 4). determined by the polarization of the immune response 6 Journal of Tropical Medicine

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0 0 LPS - - - - + + + + LPS - ---++++ AHH (g/ml) 0 5 10 20 0 5 10 20 AHH (g/ml) 0 51020051020 (a) (b) ∗ ∗ 600 600 ∗∗ ∗∗ ∗ ∗∗

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∗ IL10 Concentration (pg/ml) IL10 Concentration ∗ ∗ ∗ ∗ ∗ ∗∗ ∗∗ ∗∗ TNF -

0 0 LPS----++++LPS- - - -++++ AHH (g/ml) 0 5 10 20 0 5 10 20 AHH (g/ml) 0 5 10 20 0 5 10 20 (c) (d)

Figure 5: Te efect of AHH on IL6 (a), IL8 (b), IL10 (c), and TNF-� (d)productioninPBMCsandLPS-treatedPBMCs.PBMCswere separated using Ficoll-Paque density centrifugation, seeded in 12-well plates, and treated with 10 �g/ml LPS and AHH at the indicated concentrations for 24 h. Te levels of IL6, IL8, IL10, and TNF-� secretion were determined using ELISA with specifc antibodies to each cytokine. ∗ p < 0.05 compared to control group; ∗∗ p < 0.05 compared to LPS-treated group alone. specifc to M. leprae [9]. Consequently, the efect of drugs inhibitory activities, which may be responsible for antidia- on cytokine secretion associated with the immunological betic properties [14]. response should play an essential role in leprosy treatment. In this study, LPS induced proinfammatory cytokine Presently, the components of H. anthelminthicus seed oil (IL6 and TNF-�) secretion of PBMCs but had no efect have not been reported. However, the composition of certain on chemoattractant cytokine IL8 and anti-infammatory chaulmoogra oil has been studied. Chaulmoogra oil from cytokineIL10.HSOhadnoefectontheproliferationand H. kurzii is composed of triglycerides [70-80% glycerides cytokine secretion of PBMCs. However, HSO showed syner- of cyclopentenyl fatty acid (hydnocarpic, chaulmoogric, and gism with LPS on TNF-� secretion of PBMCs. Consequently, gorlic acids)] and palmitic, stearic, and oleic acids [10]. the use of HSO might be imprudent for patients infected with Chaulmoogra oil from H. wightiana is composed of triglyc- Gram-negative bacteria due to the induction of proinfamma- erides (80-90% glycerides of cyclopentenyl fatty acid) and tory cytokines. palmitic, oleic, and steric acids [10]. Chaulmoogra oil from H. OurstudydemonstratedthatEHHandEHKinhibitthe wightiana contains 48% hydnocarpic acid, 27% chaulmoogric release of infammatory cytokines from PBMCs and LPS- acid, gorlic acid, and other acids [11]. Tree favonolignans treated PBMCs. A previous study demonstrated that hyd- (hydnowightin, hydnocarpin, and neohydnocarpin) isolated nocarpin, the main product among favonolignans, showed from H. wightiana seeds established potent hypolipidemic good anti-infammatory and antineoplastic activity in vivo activity in mice [12]. A new phenolic glycoside isolated from [12]. Te major cyclopentenyl fatty acids, including gorlic the bark of H. annamensis exhibited COX-2 inhibitory activ- acid, chaulmoogric acid, and hydnocarpic acid, from seeds ity in vitro [13]. Hydnocarpin and isohydnocarpin from the of Carpotroche brasiliensis demonstrated signifcant oral anti- seed hulls of H. wightiana exhibited free radical scavenging, infammatory and peripheral antinociceptive efects in vivo �-glucosidase, and moderate N-acetyl-�-D glucosaminidase [15]. Furthermore, silybin, the major favonolignan from Journal of Tropical Medicine 7

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200 200 IL8 Concentration (pg/ml) IL8 Concentration

IL6 Concentration (pg/ml) IL6 Concentration ∗ ∗ ∗ ∗ ∗∗ ∗ ∗ ∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗ ∗ ∗ ∗∗ ∗∗ 0 0 LPS - - - - + + + + LPS----++++ EHH (g/ml) 0 5 10 20 0 5 10 20 EHH (g/ml) 0 5 10 20 0 5 10 20 (a) (b) 600 600

∗ 400 400

200 200 (pg/ml) Concentration IL10 Concentration (pg/ml) IL10 Concentration TNF - ∗∗ ∗∗ ∗∗ 0 0 LPS----++++LPS - - - - + + + + EHH (g/ml) 0 51020051020 EHH (g/ml) 0 5 10 20 0 5 10 20 (c) (d)

Figure 6: Te efect of EHH on IL6 (a), IL8 (b), IL10 (c), and TNF-� (d)productioninPBMCsandLPS-treatedPBMCs.PBMCswere separated using Ficoll-Paque density centrifugation, seeded in 12-well plates, and treated with 10 �g/ml LPS and EHH at the indicated concentration for 24 h. Te levels of IL6, IL8, IL10, and TNF-� secretion were determined using ELISA with specifc antibodies to each cytokine. ∗ p < 0.05 compared to control group; ∗∗ p < 0.05 compared to LPS-treated group alone.

Silybum marianum seed extracts, demonstrated an inhibitory AHH and AHK induced proinfammatory cytokine. We efect on LPS-induced production of TNF-� and IL-1� and suggested that AHH and AHK will be a great beneft for activation of NF-�B and the NLRP3 infammasome in mice patients with lepromatous leprosy who have a poor cellular [16]. It is suggested that EHH and EHK might contain immune response against M. leprae. Meanwhile, the ethanol favonolignan-like compounds that inhibit the proinfamma- extracts of H. anthelminthicus seed hulls and kernels demon- tory cytokine secretion of PBMCs. Flavonolignan is soluble strated an inhibitory efect on proinfammatory cytokine in organic solvent. Te aqueous extract of H. anthelminthicus secretion. Tey might infuence the downregulation of innate seed should not contain favonolignan. However, AHH and immune and cell-mediated immune responses due to the AHK induce proinfammatory cytokine (IL6 and TNF-�) inhibition of IL6, IL8, and TNF. Tese extracts should be production in PBMCs similar to LPS stimulation. It is used depending on the immune response of the host, which implicit that AHH and AHK might induce infammation. determines the manifestation of the disease. Consequently, Consequently, the usage of H. anthelminthicus seed extract the inhibitory efect on proinfammatory cytokines should should be consciously considered. be considered as anti-infammatory agents for the treatment TisisthefrststudyofH. anthelminthicus for the of infammatory diseases, including systematic infamma- immune response. Our study explored the efect of H. tory response syndrome (SIRS), severe sepsis, cancers, and anthelminthicus seed kernel and hull extracts on proinfam- autoimmune diseases. matory cytokine (IL6, IL8, and TNF-�)production.However, Further studies should be performed to elucidate the the chemical constituents of H. anthelminthicus seed hulls chemical constituents of H. anthelminthicus seed hulls and and kernels are unknown. Fractions of H. anthelminthicus kernels. Because of the study design limitations, this research demonstrated various efects. Consequently, the solvent and was conducted with PBMCs from healthy volunteers. Con- extraction process should be verifed for the therapeutic sequently, further study should be performed using PBMCs application of H. anthelminthicus. from leprosy patients or PBMCs treated with M. leprae 8 Journal of Tropical Medicine

antigen to study the efect of H. anthelminthicus on cytokines from Hydnocarpus wightiana seeds,” Journal of Natural Prod- secretion. Moreover, an in vivo study is important to inves- ucts,vol.54,no.5,pp.1298–1302,1991. tigate the potential application of H. anthelminthicus for the [13] H.-M. Shi, J. Wen, C.-Q. Jia et al., “Two new phenolic glycosides clinical treatment of infammation in a variety of diseases from the barks of Hydnocarpus annamensis and their anti- caused by a severe immune response. infammatory and anti-oxidation activities,” Planta Medica,vol. 72, no. 10, pp. 948–950, 2006. [14]S.V.Reddy,A.K.Tiwari,U.S.Kumar,R.J.Rao,andJ.M.Rao, Data Availability “Free radical scavenging, enzyme inhibitory constituents from Tedatausedtosupportthefndingsofthisstudyare antidiabetic ayurvedic medicinal Hydnocarpus wightiana blume,” Phytotherapy Research,vol.19,no.4,pp.277–281,2005. available from the corresponding author upon request. [15]J.A.Lima,A.S.Oliveira,A.L.P.deMiranda,C.M.Rezende, and A. C. Pinto, “Anti-infammatory and antinociceptive activ- Conflicts of Interest ities of an acid fraction of the seeds of Carpotroche brasiliensis (Raddi) (Flacourtiaceae),” BrazilianJournalofMedicaland Te authors declare that there are no conficts of interest. Biological Research,vol.38,no.7,pp.1095–1103,2005. [16] B. Zhang, B. Wang, S. Cao, Y. Wang, and D. Wu, “Silybin Acknowledgments attenuates LPS-induced lung injury in mice by inhibiting NF- �B signaling and NLRP3 activation,” International Journal of Tis study was funded by the National Research Council of Molecular Medicine, vol. 39, no. 5, pp. 1111–1118, 2017. Tailand (no. 2553-123). Te manuscript underwent English language editing by editors at American Journal Experts.

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