Inhibition of Pro‐Inflammatory Functions of Human Neutrophils by Constituents of Melodorum fruticosum Leaves

Abstract

In an initial screening, the dichloromethane extract from the leaves of Melodorum fruticosum showed distinct inhibitory effects on the release of interleukin‐8 (IL‐8) in human neutrophils. Therefore, the aim of the present study was the phytochemical and pharmacological investigation of this extract, to better understand which compounds might be responsible for the anti‐inflammatory effect. Phytochemical analysis led to the isolation of 12 known compounds and two new natural products, 5‐hydroxy‐6‐(2‐hydroxybenzyl)‐4′,7‐dimethoxyflavanone (13) and 2′,4′‐dihydroxy‐3′‐(2‐hydroxybenzyl)‐4,6′‐dimethoxychalcone (14). The influence of the isolated compounds on the production and release of the pro‐inflammatory factors IL‐8, tumor necrosis factor alpha (TNF‐α), reactive oxygen species (ROS), and adhesion molecules (CD62L and CD11b) in human neutrophils was evaluated. Three constituents, melodamide A, 2′,4′‐dihydroxy‐4,6′‐dimethoxychalcone, and 2′,6′‐dihydroxy‐4′‐methoxychalcone, showed significant inhibition of IL‐8 release (IC₅₀=6.6, 8.6, and 11.6 μm, respectively) and TNF‐α production (IC₅₀=4.5, 13.3, and 6.2 μm, respectively).

Introduction

Inflammation is a complex immune response following tissue injury or infection. As part of an innate immune response, circulating neutrophils (polymorphonuclear leukocytes) are activated. They migrate towards the site of inflammation by adhesion to endothelial cells followed by migration across vascular walls to the site of infection, where they engulf invading pathogens through phagocytosis, generate reactive oxygen metabolites, and release microbicidal, chemotactic, and pro‐inflammatory substances and lytic enzymes.1,2 Neutrophils also contribute to the resolution of inflammation through the production of pro‐resolving lipid mediators, blocking chemokines and cytokines, and finally by entering into apoptosis.3 However, inappropriate or excessive activation of neutrophils cause host tissue damage and may lead to chronic inflammation which is the basis of many chronic diseases such as atherosclerosis, bowel diseases (IBD, Crohn's disease, and ulcerative colitis), and rheumatoid arthritis.4, 5, 6

The current treatment of inflammatory disorders in Western medicine involves the use of non‐steroidal anti‐inflammatory drugs (NSAIDs) or the use of corticosteroids but since adverse effects including gastrointestinal ulcers, hypertension, renal toxicity, and cardiovascular risks are associated with these drugs for pain management and inflammation,7 finding alternatives which retain anti‐inflammatory potency but with limited side effects is a high priority.

Traditional medicine plays an important role in the healthcare system of Vietnam.8 The World Health Organization (WHO) estimates that approximately 30 % of the Vietnamese population receives treatment with traditional medicines which is heavily influenced by traditional Chinese medicine.9 Melodorum fruticosum lour. (Annonaceae), which is also known in Vietnamese traditional medicine as Dũ dẻ trơn, is widely distributed in Southeast Asia,10 and more specifically indigenous to Vietnam, Laos, Cambodia, and Thailand. The plant has edible fruits, the flowers are fragrant and are used to make perfume, while dried flowers are slightly heart stimulating and in Thailand these are used as a blood tonic. In Vietnam, the leaves are boiled in water to aid digestion.11 Antifungal, antioxidant, and cytotoxic activities of bioactive components such as essential oils, heptenes, and butenolides isolated from the flowers and bark of M. fruticosum have previously been reported,10,12, 13, 14, 15, 16, 17 while there has only been one report of anti‐inflammatory activity of a phenolic amide isolated from a methanolic leaf extract.18

To the best of our knowledge, there is no ethnopharmacological literature describing the usage of M. fruticosum as an anti‐inflammatory agent. However, since the dichloromethane leaf extract of the plant showed promising anti‐inflammatory activity in an initial screening of plants used in Vietnamese traditional medicine (results not shown), the present study aimed to identify and isolate the main components of the bioactive extract and to perform pharmacological testing on the isolated compounds by means of inhibition of interleukin 8 (IL‐8), tumor necrosis factor alpha (TNF‐α), reactive oxygen species (ROS), and adhesion molecules (CD11b and CD62L) for attenuation of pro‐inflammatory function of human neutrophils.

Results and Discussion

Extraction and Bioactivity Localization

In the present study, the anti‐inflammatory potential of M. fruticosum leaf extracts was investigated. First, the plant material was extracted successively with dichloromethane and methanol. The obtained extracts were analyzed on their ability to inhibit the release of IL‐8 in lipopolysaccharide (LPS) stimulated human neutrophils. The dichloromethane extract was highly active with 88 % inhibition at a concentration of 10 μg/mL compared to the stimulated control (0 % inhibition), whereas the methanol extract showed only weak inhibition (10 %) at 10 μg/mL. To ensure that the inhibitory effect on IL‐8 release is not due to cytotoxicity, the extracts were initially investigated on potential cytotoxicity on neutrophils. Neither the dichloromethane nor the methanol extract showed any cytotoxic effects on human neutrophils at the investigated concentration (10 μg/mL). Therefore, the bioactive dichloromethane extract was chosen for further phytochemical and pharmacological investigations. It was separated by silica gel column chromatography (CC) to obtain 47 fractions (a1–a47), which were pooled according to their compound spectra and forwarded to pharmacological testing on the release of IL‐8 in human neutrophils for activity localization (results not shown). While most of the fractions were either inactive or weakly active, fraction a31 showed strong inhibitory activity on IL‐8 release.

Isolation and Identification of Natural Products

Aiming at identifying the active principle of the bioactive dichloromethane extract, the crude extract and in particular the highly active fraction a31 was investigated in more detail. Analysis of fraction a31 using high performance liquid chromatography (HPLC) revealed the presence of one major compound. Purification of this compound using Sephadex® CC yielded a white powder (compound 1). In addition to this major compound, 13 other constituents of the bioactive extract (Figure  1) could be isolated from various fractions by means of a combination of chromatographic techniques, i. e., silica gel and Sephadex® CC as well as semi‐preparative HPLC. The isolated natural products were identified as melodamide A (1),18 4′,7‐dihydroxy‐5‐methoxyflavanone (2),19,20 haplamide (3),21,22 4′‐hydroxy‐5,7‐dimethoxyflavanone (4),23 (2S)‐5‐hydroxy‐4′,7‐dimethoxyflavanone (5),24 7‐hydroxy‐4′,5‐dimethoxyflavanone (6),25 7‐hydroxy‐5‐methoxyflavanone (7),26 N‐trans‐cinnamoyltyramine (8),27 (2S)‐4′,5,7‐trimethoxyflavanone (9),28 2′,4′‐dihydroxy‐4,6′‐dimethoxychalcone (10),29 2′,4′‐dihydroxy‐4,6′‐dimethoxydihydrochalcone (11),30 and 2′,6′‐dihydroxy‐4′‐methoxychalcone (12)31 by NMR, MS, and comparison of the spectral data with those in literature. In addition, two new natural products were identified as 5‐hydroxy‐6‐(2‐hydroxybenzyl)‐4′,7‐dimethoxyflavanone (13) and 2′,4′‐dihydroxy‐3′‐(2‐hydroxybenzyl)‐4,6′‐dimethoxychalcone (14) (Figure  2).

Figure 1.

HPLC analysis of the dichloromethane extract of Melodorum fruticosum. HPLC conditions: stationary phase: Phenomenex Luna phenyl hexyl (150×4.6 mm, 5 μm particle size); mobile phase: solvent A, water; solvent B, acetonitrile; detection: DAD 230 nm, temperature: 40 °C; injection volume: 10 μL; flow rate: 1 mL/min; gradient: 0 min, 20 % B; 20 min, 98 % B; 30 min, 98 % B. The last 13 min of the analysis are not shown.

Figure 2.

Chemical structures of compounds 1–14.

Compounds 5 and 9 turned out to be pure enantiomers. Their assignment was based on experimental optical rotation data and their comparison to literature data from Abbate et al.32 who used vibrational circular dichroism (VCD) to determine that the (R)‐enantiomer of naringenin is the one exhibiting (+) optical rotation, while the (S)‐enantiomer exhibits (−) optical rotation. Specific optical rotations ([α]_D ²⁰) of compounds 5 and 9 were [α]_D ²⁰=−1.90 (c=0.03, MeOH) and [α]_D ²⁰=−0.04 (c=0.225, MeOH), respectively. Therefore, they were identified as (2S)‐5‐hydroxy‐4′,7‐dimethoxyflavanone (5) and (2S)‐4′,5,7‐trimethoxyflavanone (9). Compounds 2, 4, 6, 7, and 13 were isolated as mixtures of (R)‐ and (S)‐enantiomers.

Out of the 14 isolated constituents, 1, 2, 6, and 12 have previously been reported from M. fruticosum.18 8, 10, and 11 have been reported as constituents of another Melodorum species, namely M. siamensis.33 3,21,22,34 4,23 5,24 7,26 and 9 28 have been reported as natural products but have not previously been isolated from Melodorum species. This is the first reported isolation of compounds 13 and 14.

Structure Elucidation of New Compounds

Compound 13 was obtained as colorless solid. The HR‐ESI‐MS spectrum displayed a quasimolecular ion at m/z 405.1298 ([M−H]⁻) consistent with the chemical formula C₂₄H₂₂O₆. The UV spectrum showed adsorptions at λ _max 204, 281, and 319 nm and the IR spectrum indicated free hydroxy (v _max 3039 cm⁻¹) and ketone (v _max 1604 cm⁻¹) groups. The ¹H‐NMR spectrum of compound 13 showed two doublets at δ(H) 7.25 (d, J=8.7, H(2′), H(6′)) and δ(H) 6.87 (d, J=8.8, H(3′), H(5′)) consistent with a p ‐substituted aromatic ring (ring B). The singlet at δ(H) 6.18 (s, H(8)) indicated a penta‐substituted aromatic ring (ring A). These signals along with the signals from the α‐carbon at δ(H) 2.89 (dd, J=16.4, 12.1, H(3)) and δ(H) 2.65 (dd, J=16.4, 3.2, H(3)) together with the signal from the β‐carbon at δ(H) 5.36 (dd, J=12.0, 3.0, H(2)) from ring C confirmed that the core structure of 13 was a flavanone. Substitution of two methoxy groups was confirmed with signals at δ(H) 3.74 (3H, s) and δ(H) 3.73 (3H, s). The position of the two methoxy groups were determined from the HMBC data which revealed correlations between the proton signal of 4′‐MeO (δ(H) 3.73) and C(4′) (δ(C) 158.97). The HMBC between the proton signal of 7‐MeO (δ(H) 3.74) and C(7) (δ(C) 160.02) and a long range COSY correlation between the proton signal of 7‐MeO (δ(H) 3.74) and the proton signal of H(8) at δ(H) 6.18 confirms the position of the second methoxy attached to C(7). Further aromatic proton signals, which suggested a further aromatic ring, were observed at δ(H) 6.93 (ddd, J=8.0, 7.9, 1.8, H(4′′)), δ(H) 6.74 (d, J=7.9, H(3′′)), δ(H) 6.64 (d, J=6.6, H(6′′)), and δ(H) 6.60 (ddd, J=7.5, 7.4, 1.1, H(5′′)). Proton signals from a CH₂ group at δ(H) 3.72 (s, H(7′′)) with corresponding HMBCs to the carbon (δ(C) 106.07) at position 6 of ring A and the carbon (δ(C) 126.72) at position 1′′ of the additional aromatic ring indicated that the substitution of ring A by an aromatic moiety was bridged over a CH₂ group. Therefore, compound 13 was identified as 5‐hydroxy‐6‐(2‐hydroxybenzyl)‐4′,7‐dimethoxyflavanone.

Compound 14 was obtained as yellow solid. The HR‐ESI‐MS spectrum displayed a quasimolecular ion at m/z 405.1335 ([M−H]⁻) consistent with the chemical formula C₂₄H₂₂O₆. The UV spectrum showed adsorptions at λ _max 202, 282, and 370 nm. The IR spectrum indicated free hydroxy (v _max 3358 cm⁻¹) and ketone (v _max 1602 cm⁻¹) groups. The ¹H‐NMR spectrum of compound 14 showed two doublets at δ(H) 7.57 (d, J=8.7, H(2), H(6)) and δ(H) 6.95 (d, J=8.8, H(3), H(5)), which are consistent with a p ‐substituted aromatic ring (ring B) and a singlet at δ(H) 6.07 (s, H(5′)) for a penta‐substituted aromatic ring (ring A). These signals along with the signals from the alkene β‐carbon δ(H) 7.83 (d, J=15.5, H(β)) and the α‐carbon δ(H) 7.69 (d, J=15.5, H(α)) confirmed a chalcone core structure. Substitution of two methoxy groups was confirmed by signals at δ(H) 3.90 (s, 6′‐MeO) and δ(H) 3.82 (s, 4‐MeO). The HMBC data showed correlations between the proton signal of 4‐MeO (δ(H) 3.82) and C(4) (δ(C) 163.01), and correlations between the proton of 6′‐MeO (δ(H) 3.90) and C(6′) (δ(C) 162.93). Long range COSY correlations between the 6′‐MeO proton at δ(H) 3.90 and the H(5′) proton at δ(H) 6.07 confirmed this position. A further long range COSY correlation between the proton of 4‐MeO (δ(H) 3.82) and δ(H) 6.95 (H(3), H(5)) was observed. Aromatic proton signals, which indicated a further aromatic ring, were observed at δ(H) 7.13 (dd, J=7.6, 1.4, H(6′′)), δ(H) 7.00–6.96 (m, H(4′′)), δ(H) 6.76 (dd, J=8.0, 1.0, H(3′′)), and δ(H) 6.70 (td, J=7.5, 1.1, H(5′′)). Proton signals from a CH₂ group at δ(H) 3.84 (s, H(7′′)) with corresponding HMBCs to the carbon (δ(C) 108.52) at position 3′ of ring A and the carbon (δ(C) 127.88) at position 1′′ of the additional aromatic ring indicated that the substitution of ring A by an aromatic moiety was bridged over a CH₂ group. The NMR shifts were compared to those described by Prawat et al.33 who isolated a similar chalcone, whereby the α and β carbons were connected by a single bond rather than a double bond as it is the case in compound 14. Therefore, compound 14 was identified as 2′,4′‐dihydroxy‐3′‐(2‐hydroxybenzyl)‐4,6′‐dimethoxychalcone.

Biological Activities of Isolated Compounds

To identify the constituents responsible for the IL‐8 inhibition activity of the dichloromethane extract of the M. fruticosum leaves, all isolated compounds were tested on their ability to inhibit IL‐8 release in human neutrophils. In particular, cells were stimulated with LPS to activate an inflammatory response whereby the levels of IL‐8 were measured by ELISA. Quercetin (10 μm), which was used as a positive control, decreased the release of IL‐8 from neutrophils to 50.9±3.5 % compared to the stimulated control (100 % IL‐8 production). At the same concentration (10 μm), compounds 1, 10, and 12 also showed statistically significant (p<0.001) decreases of IL‐8 release (6.1±0.5, 73.4±3.4, and 30.4±2.3 % IL‐8 production, respectively; Figure  3a). Since the observed activities of these compounds were in the range of the activity of the positive control, the active compounds 1, 10, and 12 were analyzed in more detail in concentration response studies. All three compounds showed inhibition of IL‐8 production in a concentration dependent manner with the IC₅₀ values of 6.6, 8.6, and 11.6 μm, respectively (Figure  3b).

Figure 3.

Inhibition of IL‐8 production in LPS stimulated human neutrophils, given as % of stimulated control (100 %). a) Inhibition by compounds 1–14 at a concentration of 10 μm. b) Concentration dependent inhibition of IL‐8 production by compounds 1, 10, and 12. Results shown are the mean±SEM of three independent experiments, assayed in triplicate. Experiments were performed using cells from different donors. Statistical significance: # p<0.001 compared to non‐stimulated control, *** p<0.001 compared to stimulated control (Dunnett's post hoc test). ST stimulated control, NST non‐stimulated control. Quercetin (Q) was used as a positive control (10 μm).

Resolution of inflammation can be achieved by decreasing levels of pro‐inflammatory mediators and/or hindering the recruitment of leukocytes and their activation.35 IL‐8 is an important pro‐inflammatory chemokine produced by neutrophils and acts as a mediator of inflammation by recruiting neutrophils to the site of inflammation.36 Another important pro‐inflammatory cytokine, which has been identified as a key regulator of inflammatory processes, is TNF‐α. Therapeutic blockade of TNF‐α has been shown to be highly beneficial in the treatment of chronic inflammatory conditions.37, 38, 39

Therefore, the effects of the isolated compounds on the TNF‐α production by LPS stimulated human neutrophils were also determined using an ELISA assay. Compounds 1, 10, and 12 at 10 μm showed pronounced inhibitory effects on TNF‐α production of LPS stimulated human neutrophils (Figure  4a). The inhibition of TNF‐α production was concentration dependent as shown in Figure  4b. The IC₅₀ values of 1, 10, and 12 reached 4.5, 13.3, and 6.2 μm, respectively (the IC₅₀ value of the positive control quercetin: 10 μm).

Figure 4.

Inhibition of TNF‐α production in LPS stimulated human neutrophils, given as % of stimulated control (100 %). a) Inhibition by compounds 1–14 at a concentration of 10 μm. b) Concentration dependent inhibition of TNF‐α production by compounds 1, 10, and 12. Results shown are the mean±SEM of three independent experiments, assayed in triplicate. Experiments were performed using cells from different donors. Statistical significance: # p<0.001 compared to non‐stimulated control, *** p<0.001 compared to stimulated control (Dunnett's post hoc test). ST stimulated control, NST non‐stimulated control. Quercetin (Q) was used as a positive control (10 μm).

Stimulation of human neutrophils results in the rapid phosphorylation of proteins, including p38 mitogen‐activated protein kinase (MAPK), p42/44 extracellular signal‐regulated kinase (ERK), c‐Jun NH2‐terminal kinase (JNK), and Akt kinase.40, 41, 42 Therefore, the decrease in the release of IL‐8 as well as of TNF‐α by compounds 1, 10, and 12 might be related to MAPK activation inhibition. The study of Chan et al. revealed that melodamide A (1) inhibits the p38 MAPK pathway without affecting p42/44 and Akt kinases phosphorylation.18 However, other mechanisms of action, such as NF‐κB activation inhibition, might be involved.

To ensure that the observed inhibitory effects on IL‐8 and TNF‐α release by the active compounds 1, 10, and 12 are not due to cytotoxicity, they were additionally analyzed for potential cytotoxic effects. This could be excluded since at the investigated concentration (10 μm) the three compounds did not show any cytotoxic effect on neutrophils (results not shown).

Reactive oxygen species (ROS) are essential in the progression of inflammation, acting both as signaling molecules and as mediators of inflammation.1 Therefore, it was also evaluated whether the isolated compounds inhibit ROS production in N‐formylmethionyl‐leucyl‐phenylalanine (f‐MLP) stimulated neutrophils. The incubation of stimulated neutrophils with compounds 1–14 at 10 μm resulted in a statistically significant decrease in ROS production by compounds 4, 6, and 13 to 47.7±5.6, 48.8±5.9, and 48.3±2.6 %, respectively, when compared to the stimulated cells (100 % ROS production). However, they were less active when compared to the positive control quercetin (10 μm), which decreased ROS production to 28.5±5.7 %. Interestingly, all other compounds, including 1, 10, and 12, showed no significant inhibitory activity on ROS production (Figure  5).

Figure 5.

Inhibition of ROS production in f‐MLP stimulated human neutrophils by compounds 1–14 at a concentration of 10 μm, given as % of stimulated control (100 %). Results shown are the mean±SEM of three independent experiments, assayed in triplicate. Experiments were performed using cells from different donors. Statistical significance: # p<0.001 compared to non‐stimulated control, *** p<0.001 and ** p<0.05 compared to stimulated control (Dunnett's post hoc test). ST stimulated control, NST non‐stimulated control. Quercetin (Q) was used as a positive control (10 μm).

The interaction of leukocytes with vascular endothelial cells is crucial in inflammatory and immune responses. Adhesion molecules, which are expressed on the surface of neutrophils, are key mediators allowing for an accumulation of leukocytes at the site of inflammation. Several adhesion molecules are part of this process such as selectins, responsible for the recruitment of leukocytes to the site of inflammation, and integrins which are involved in adhesion of leukocytes to the endothelial cells.43 When neutrophils are stimulated with pro‐inflammatory LPS, downregulation of the l‐selectin CD62L and upregulation of the integrin CD11b occur. A compound which can inhibit firm adhesion and extravascular migration to the site of inflammation is thus able to alleviate local inflammation.44 Therefore, within this study, the effects of the isolated compounds on CD11b and CD62L expression in stimulated neutrophils were analyzed. However, no inhibition of CD11b surface expression triggered by cytochalasin A/f‐MLP was observed for compounds 1–14 at a concentration of 10 μm when compared to the stimulated control (Figure  6). Results depicted in Figure  7 show that compounds 1–14 at a concentration of 10 μm also did not prevent CD62L from shedding (values compared to stimulated control) after stimulation with cytochalasin A/f‐MLP.

Figure 6.

Effect of compounds 1–14 at a concentration of 10 μm on CD11b expression in cytochalasin A/f‐MLP stimulated human neutrophils, given as % of stimulated control (100 %). Results shown are the mean±SEM of three independent experiments using cells from different donors. Statistical significance: # p<0.001 compared to non‐stimulated control. ST stimulated control, NST non‐stimulated control. Quercetin (Q) was used as a positive control (50 μm).

Figure 7.

Effect of compounds 1–14 at a concentration of 10 μm on CD62L expression in cytochalasin A/f‐MLP stimulated human neutrophils, given as % of non‐stimulated control. Results shown are the mean±SEM of three independent experiments using cells from different donors. Statistical significance: # p<0.001 compared to non‐stimulated control. ST stimulated control, NST non‐stimulated control. Quercetin (Q) was used as a positive control (50 μm).

Although the use of M. fruticosum as a digestive aid and blood tonic in traditional medicine has been reported,11 there is no mention of any specific anti‐inflammatory application to which the activity of the dichloromethane extract or isolated compounds could be correlated. However, the anti‐inflammatory activity of compound 1 has been reported previously by Chan et al. who isolated it from the methanolic extract of the leaves of M. fruticosum. In particular, they reported the inhibition of superoxide anion generation in f‐MLP/cytochalasin B activated human neutrophils by compound 1 with an IC₅₀ value of 5.25 μm, which could be attributed to inhibition of the p38 mitogen‐activated protein kinases (MAPK) signaling pathway. Further substitutions with halide, NO₂, MeO, and Me groups on ring A of 1 did not further increase the anti‐inflammatory activity.18

Interestingly, all of the active compounds (1, 10, and 12), being able to inhibit the release of IL‐8 and TNF‐α production, possess either an α,β‐unsaturated carbonyl or an α,β‐unsaturated amide moiety. Michael acceptor systems such as these are able to inhibit cysteine proteases by forming covalent bonds with the active site thiol of cysteine proteases.45 Harikumar et al. suggested that the prenylated chalcone xanthohumol, with one Michael acceptor and an electrophilic carbonyl group, interacts directly with the sulfhydryl group of the cysteine residue of IκBα kinase (IKK) and the p65 subunit of nuclear factor‐κB (NF‐κB), leading to an adduct formation and therefore inhibiting the NF‐κB activation pathway.46 Further enhancement of chalcone thiol activity by modifying the α‐position of the α,β‐unsaturated carbonyl with strong electrophiles has also been reported.47 However, undesired toxic effects can also occur through reactions with electrophilic groups causing disruptive effects of biological macromolecules.48,49 Michael reactivity is strongly influenced by the substituents on the α‐ and β‐carbons, whereby electron attracting groups increase reactivity while electron donating groups reduce activity.50 A recent perspective by Jackson et al. focused on the reactivity of biological thiols with Michael acceptors concluding that the reactivity decreases in the order of enals>enones>α,β‐unsaturated esters>acrylamides>α,β‐unsaturated carboxylic acids.51 For the reactivity of chalcones, the presence of a hydroxy group at the 2′ position is crucial, whereby activation of the carbonyl group through the intramolecular H‐bond and stabilization of the conjugation in the system occurs.52 The activity of the chalcones 10 and 12, in which a nucleophilic attack is possible owing to the Michael acceptor, may be explained by this type of interaction, since the dihydrochalcone 11, which lacks this important Michael acceptor site, did not show any activity. This could also be the mechanism of action for compound 1 owing to the α,β‐unsaturated amide moiety. However, further studies are necessary to confirm this hypothesis. Furthermore, since compounds, which alkylate cysteinyl thiols via Michael addition, often possess undesired toxic effects, the identified bioactive compounds should be analyzed in further toxicity studies to exclude potential cytotoxicity for humans, even though the traditional usage of M. fruticosum as a medicinal plant in Vietnam suggests the lack of severe toxicity.

Although compounds 1, 10, and 12 actively inhibited the release of IL‐8 and TNF‐α, no inhibition of CD11b surface expression or CD62L shedding was observed. This observation is similar to those by Arshad et al. who investigated a series of α,β‐unsaturated carbonyl‐based compounds, namely curcumin analogs and chalcone derivatives, and although moderate to strong activity for migration of neutrophils, phagocytic, and ROS production was observed, less activity was observed for CD11b/CD18 expression of leukocytes.53 The interaction target of the active compounds 1, 10, and 12 appears to be specific to cytokine production rather than the interaction of leukocytes with vascular endothelial cells.

Conclusions

The anti‐inflammatory properties of the Vietnamese traditional medicinal plant M. fruticosum presented within this study could not be supported by any documented ethnopharmacological survey known to date. However, the initial bioactivity of the active dichloromethane M. fruticosum leaf extract could be traced back to the presence of compounds 1, 10, and 12, of which compound 1 represents a major constituent of the bioactive extract. The results suggest that the mode of action for compounds 1, 10, and 12, which actively inhibited release of IL‐8 and TNF‐α, is specifically attributed to those mechanisms rather than the generation of ROS or the interaction with other functions of leukocytes such as rolling, firm adhesion, or degranulation at the site of inflammation. The identification of the anti‐inflammatory effects of M. fruticosum might provide a basis for potential future usage of the plant against inflammatory conditions.

Experimental Section

General

HPLC analysis was performed on a HP 1100 system (Agilent, Waldbronn, Germany) equipped with an autosampler, column thermostat, and DAD detector; stationary phase: Phenomenex Luna phenyl hexyl (150×4.6 mm, 5 μm particle size); mobile phase: solvent A, water; solvent B, acetonitrile; detection: DAD 230 nm, temperature: 40 °C; injection volume: 10 μL; flow rate: 1 mL/min; gradient: 0 min, 20 % B; 20 min, 98 % B; 30 min, 98 % B. Optical rotations were determined with a PerkinElmer 341 polarimeter (Wellesley, MA, USA) at 20 °C. The ultraviolet spectra were recorded on a Thermo Scientific Multiskan GO spectrophotometer (Waltham, MA, USA); λ _max (log ϵ) in nm. IR spectra were recorded on a Bruker ALPHA spectrometer (Bruker Optics, Ettlingen, Germany) equipped with a PLATINUM‐ATR unit (spectral range 4000–400 cm⁻¹, 4 scans per cm^–1, OPUS 7 software); $\tilde{\nu }$ in cm⁻¹. NMR spectra (1D‐ and 2D‐experiments) were acquired on a Bruker Advance II 600 NMR spectrometer (Bruker BioSpin, Rheinstetten, Germany) operating at 600 MHz (¹H) and 151 MHz (¹³C) at 300 K (chemical shifts δ in ppm, coupling constants Ј in Hz). NMR solvents used were CD₃OD or (D₆)DMSO with 0.03 % TMS (Eurisotop, Gif‐Sur‐Yvette, France) as an internal standard. Mass spectrometry was performed on an Esquire 3000^plus (Bruker Daltonics, Bremen, Germany) with the following parameters: ESI; temperature: 325 °C; dry gas: 8.00 L/min; nebulizer: 30 psi; full scan mode: m/z 50–1500. HR‐ESI‐MS data were obtained using a Bruker mikrOTOF‐QII mass spectrometer (Bruker Daltonics, Bremen, Germany); in m/z.

Plant Material

Leaves from M. fruticosum were collected in the Tam thành commune, Phú ninh district, Quàng Nam Province of Vietnam in August 2014 and identified by Dr. Võ Văn Chi from the Department of Pharmacognosy, Faculty of Pharmacy, University of Medicine and Pharmacy of Ho Chi Minh City. A voucher specimen (BW‐20141210‐03) of the air‐dried plant material was deposited at the Institute of Pharmacy/Pharmacognosy of the University of Innsbruck.

Chemicals and Reagents

All solvents used for isolation were purchased from VWR International (Darmstadt, Germany). Solvents used for HPLC were purchased from Merck (Darmstadt, Germany) and ultrapure water was produced onsite by a Sartorius Arium 611 UV water purification system (Göttingen, Germany). Luminol, f‐MLP, LPS, cytochalasin A, Hanks’ balanced salt solution (HBSS), RPMI 1640 medium, 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES), and l‐glutamine were purchased from Sigma–Aldrich GmbH (Steinheim, Germany). Quercetin (95 % purity) was purchased from Carl Roth (Karlsruhe, Germany). Propidium iodide was purchased from BD Biosciences (San Diego, CA, USA). Fetal bovine serum (FBS) and phosphate‐buffered saline (PBS) were purchased from Gibco (Carlsbad, CA, USA). Both CD62L‐(APC)‐conjugate and CD11b‐(PE)‐conjugate were purchased from Becton Dickinson (Franklin Lakes, NJ, USA).

Preparation of Extracts for Activity Screening

Air‐dried and ground M. fruticosum leaf material (5 g) was extracted with 30 mL dichloromethane by sonication for 10 min at room temperature. The plant material was recovered by filtration and the extraction process was repeated twice with 20 mL of dichloromethane, respectively. The filtrates were combined and the solvent was evaporated under reduced pressure to yield 236.9 mg of crude dichloromethane extract. The residual plant material was air‐dried and the extraction process was repeated as described with methanol to yield 677.17 mg of crude methanol extract. Both extracts were subjected to pharmacological testing on their inhibitory effects on the release of IL‐8 in human neutrophils.

Preparation of the Extract for Compound Isolation

Powdered dried leaves of M. fruticosum (952 g) were exhaustively extracted with dichloromethane by sonication and the solvent was evaporated under reduced pressure to yield 63.8 g of crude extract.

Fractionation and Compound Isolation

The crude dichloromethane extract (30 g) was subjected to silica gel CC (605 g of silica gel 60, grain size 40–63 μm, Merck, Darmstadt, Germany), eluting with a stepwise gradient of dichloromethane, ethyl acetate, and methanol. The eluate was monitored by TLC and combined to yield 47 fractions (fractions a1–a47), which were pooled according to their compound spectra and forwarded to pharmacological testing on the release of IL‐8 in human neutrophils for activity localization (results not shown).

Fraction a8 (216 mg) was separated by Sephadex® LH‐20 CC with dichloromethane/acetone (85 : 15, v/v) as mobile phase to yield compound 14 (6.75 mg). Fraction a9 (219 mg) was also subjected to Sephadex® LH‐20 CC with dichloromethane/acetone (85 : 15, v/v) as mobile phase to yield compound 12 (12.34 mg). Separation of fraction a10 (237 mg) using Sephadex® LH‐20 CC with dichloromethane/acetone (85 : 15, v/v) as mobile phase led to the isolation of compounds 10 (66.84 mg) and 11 (19.91 mg), respectively. Fraction a11 (402.3 mg) was first separated by Sephadex® LH‐20 CC with dichloromethane/acetone (85 : 15, v/v) as mobile phase to yield seven sub‐fractions (a11.b1–a11.b7). Separation of sub‐fraction a11.b1 (330 mg) using semi‐preparative HPLC (column: Phenomenex Synergi polar RP, 250×10 mm, 80 Å, 4 μm particle size; detection: DAD 230 nm; temperature: 40 °C; mobile phase: water (solvent A), acetonitrile (solvent B); gradient composition: 0 min, 40 % B; 8 min, 80 % B; 12.5 min, 98 % B; flow rate: 2 mL/min) led to the isolation of compound 9 (5.74 mg). Fraction a16 (202 mg) was separated by Sephadex® LH‐20 with dichloromethane/acetone (85 : 15, v/v) as mobile phase to yield compounds 4 (37.84 mg), 7 (15.55 mg), and 8 (7.79 mg), respectively. Fraction a21 (201 mg) was separated by Sephadex® LH‐20 CC with dichloromethane/acetone (85 : 15, v/v) as mobile phase to give eight sub‐fractions (a21.b1–a21.b8). Sub‐fraction a21.b7 (24.68 mg) was further separated by semi‐preparative HPLC (column: Phenomenex Synergi polar RP, 250×10 mm, 80 Å, 4 μm particle size; detection: DAD 230 nm; temperature: 40 °C; isocratic 50 % water/50 % acetonitrile, 17 min; flow rate: 2 mL/min) to yield compounds 6 (11.51 mg) and 13 (4.24 mg), respectively. Fraction a26 (171 mg) was separated by Sephadex® LH‐20 CC with methanol as mobile phase to give eight sub‐fractions (a26.b1–a26.b8). Sub‐fraction a26.b7 (10.72 mg) was subjected to semi‐preparative HPLC (column: Phenomenex Synergi polar RP, 250×10 mm, 80 Å, 4 μm particle size; detection: DAD 230 nm; temperature: 40 °C, isocratic 50 % water/50 % acetonitrile, 15 min; flow rate: 2 mL/min) to yield compounds 2 (6.07 mg) and 5 (3.90 mg), respectively. Fraction a29 (205 mg) was separated by Sephadex® LH‐20 CC with dichloromethane/acetone (85 : 15, v/v) as mobile phase to yield compound 3 (16.74 mg). Fraction a31 (208 mg), which was the most active fraction, was separated by Sephadex® LH‐20 CC with methanol as mobile phase to yield compound 1 (19.19 mg). Purity was determined by HPLC‐DAD analysis at 205 nm and calculated as >95 % for all isolated compounds.

5‐Hydroxy‐6‐(2‐hydroxybenzyl)‐4′,7‐dimethoxyflavanone (13). Colorless solid. [α]_D ²⁰=−1.90 (c=0.03, MeOH). UV (MeOH): 204 (8.13), 281 (3.72), 319 (3.52). IR (ATR): 3039, 2918, 2849, 2724, 2615, 1337, 1604, 1567, 1515, 1488, 1455, 1410, 1354, 1327, 1283, 1247, 1201, 1174, 1146, 1101, 1083, 1032, 967, 933, 893, 874, 826, 782, 747, 697, 643, 616, 598, 577, 548, 535, 451, 418. ¹H‐NMR (600 MHz, (D₆)DMSO): 7.25 (d, J=8.7, H(2′), H(6′)), 6.93 (ddd, J=8.0, 7.9, 1.8, H(4′′)), 6.87 (d, J=8.8, H(3′), H(5′)), 6.74 (d, J=7.9, H(3′′)), 6.64 (d, J=6.6, H(6′′)), 6.60 (ddd, J=7.5, 7.4, 1.1, H(5′′)), 6.18 (s, H(8)), 5.36 (dd, J=12.0, 3.0, H(2)), 3.74 (s, 7‐MeO), 3.73 (s, 4′‐MeO), 3.72 (s, H(7′′)), 2.89 (dd, J=16.4, 12.1, H(3)), 2.65 (dd, J=16.4, 3.2, H(3)). ¹³C‐NMR (151 MHz, (D₆)DMSO): 187.86 (C(4)), 161.76 (C(5), C(9)), 160.02 (C(7)), 158.97 (C(4′)), 154.93 (C(2′′)), 131.25 (C(1′)), 127.81 (C(6′′)), 127.43 (C(2′), C(6′)), 126.72 (C(1′′)), 126.11 (C(4′′)), 118.48 (C(5′′)), 114.42 (C(3′′)), 113.67 (C(3′), C(5′)), 106.07 (C(6)), 104.45 (C(10)), 92.94 (C(8)), 77.35 (C(2)), 55.29 (7‐MeO), 55.03 (4′‐MeO), 44.42 (C(3)), 22.00 (C(7′′)) (see Supporting Information).

2′,4′‐Dihydroxy‐3′‐(2‐hydroxybenzyl)‐4,6′‐dimethoxychalcone (14). Yellow solid. UV (MeOH): 202 (4.05), 282 (3.28), 370 (3.74). IR (ATR): 3358, 2961, 2923, 2850, 1602, 1510, 1488, 1454, 1422, 1346, 1290, 1258, 1235, 1172, 1138, 1106, 1052, 1031, 980, 866, 826, 800, 755. ¹H‐NMR (600 MHz, CD₃OD): 7.83 (d, J=15.5, H(β)), 7.69 (d, J=15.5, H(α)), 7.57 (d, J=8.7, H(2), H(6)), 7.13 (dd, J=7.6, 1.4, H(6′′)), 7.00–9.96 (m, H(4′′)), 6.95 (d, J=8.8, H(3), H(5)), 6.76 (dd, J=8.0, 1.0, H(3′′)), 6.70 (td, J=7.5, 1.1, H(5′′)), 6.07 (s, H(5′)), 3.90 (s, 6′‐MeO), 3.84 (d, J=1.7, H(7′′)), 3.82 (s, 4‐MeO). ¹³C‐NMR (151 MHz, CD₃OD): 193.94 (C=O), 166.24 (C(2′), 164.55 (C(4′)), 163.01 (C(4)), 162.93 (C(6′)), 155.52 (C(2′′)), 143.13 (C(α)), 131.21 (C(6′′)), 131.09 (C(2), C(6)), 129.57 (C(1)), 128.48 (C(4′′)), 127.88 (C(1′′)), 126.51 (C(β)), 120.79 (C(5′′)), 116.02 (C(3′′)), 115.45 (C(5), C(3)), 108.52 (C(3′)), 106.44 (C(1′)), 92.30 (C(5′)), 56.22 (6′‐MeO), 55.86 (4‐MeO), 23.11 (C(7′′)) (see Supporting Information).

Isolation of Human Neutrophils

Peripheral venous blood was taken from healthy human donors at the Warsaw Blood Donation Center. Neutrophils were isolated using a standard dextran sedimentation followed by hypotonic lysis of erythrocytes and centrifugation in a Ficoll Hypaque gradient. Neutrophils were suspended in the appropriate medium for the respective assay and stored at 4 °C prior to use.

Cytotoxicity

Cytotoxicity of the crude extracts and pure compounds was determined by standard flow cytometric probe using propidium iodide (PI) staining to distinguish cells with diminished membrane integrity. Neutrophils (2×10⁵/mL) were incubated in a 24‐well plate in RPMI 1640 medium (without calcium and magnesium) with 10 % FBS, 10 mm HEPES, and 2 mm l‐glutamine for 24 h at 37 °C with 5 % CO₂, in the presence of an extract at a concentration of 10 μg/mL or a pure compound at a concentration of 10 μm, and stimulated with 10 μL LPS (100 ng/mL). After 24 h, the neutrophils were harvested and centrifuged (1500 RPM, 10 min, 4 °C). The neutrophils were suspended in 400 μL PBS with 5 μL propidium iodide (50 μg/mL) and left in the dark for 15 min. Cells were analyzed by flow cell cytometry (BD FACSCalibur flow cytometer, BD biosciences, San Jose, CA, USA) and the data from 10,000 events were recorded. Cells, which displayed high permeability to PI, were expressed as a percentage of PI(+) cells.

IL‐8 and TNF‐α Production

Neutrophils were incubated in a 24‐well plate in RPMI 1640 medium with 10 % FBS, 10 mm HEPES, and 2 mm l‐glutamine for 24 h at 37 °C with 5 % CO₂, in the presence of an extract at a concentration of 10 μg/mL or a pure compound at a concentration of 10 μm, and stimulated with 10 μL LPS (100 ng/mL). Quercetin (10 μm) was used as a positive control. The amount of IL‐8 or TNF‐α released into the cell supernatants was measured by ELISA following the manufacturer's instructions (BD Biosciences). The effect on IL‐8 or TNF‐α production was calculated as a percentage of released agents in comparison with the stimulated control. IC₅₀ values were determined from experiments, in which compounds 1, 10, and 12 were added at concentrations of 1, 2.5, 5, 7.5, 10, 15, 20, and 25 μm.

Expression of Adhesion Molecules CD62L and CD11b

Cell suspensions (400 μL) of neutrophils (1×10⁶/mL) in PBS buffer were incubated with 100 μL of pure compounds (10 μm) for 30 min at 37 °C before a stimulation solution containing 60 μL f‐MLP (0.1 μg/mL) and 30 μL cytochalasin A (5 μg/mL) was added. After a further incubation time of 30 min at 37 °C, neutrophils were marked with monoclonal antibody against CD62L‐(APC)‐conjugate and CD11b‐(PE)‐conjugate and left in the dark for 15 min prior to analysis by flow cell cytometry (BD FACSCalibur flow cytometer, BD biosciences, San Jose, CA, USA), on which the data from 10,000 events were recorded. Quercetin (50 μm) was used as a positive control.

Evaluation of ROS Production by Human Neutrophils

The evaluation of the ROS production by f‐MPL stimulated neutrophils was determined using luminol dependent chemiluminescence. Following isolation, 70 μL of the neutrophil suspension (2×10⁵/mL) in Hanks balanced salt solution was incubated with 50 μL of each compound (10 μm) together with 50 μL luminol (100 μm). ROS production was stimulated by the addition of 30 μL f‐MLP (0.1 μg/mL). Chemiluminescence was measured immediately at 37 °C at 2‐min intervals over a 45‐min period. Quercetin (10 μm) was used as a positive control.

Statistical Analysis

GraphPad Prism (version 5.01) was used to plot data and to calculate statistical significance. The results are expressed as mean±SEM. Nonlinear regression (log inhibitor vs. normalized response, variable slope) was used to calculate IC₅₀ values. Statistical significance was determined by ANOVA with Dunnett's post hoc test. P values below 0.05 were considered statistically significant.

Author Contribution Statement

N. S. E. isolated and characterized the compounds and drafted the manuscript. L. H. collected the plant material and performed phytochemical experiments. N. S. E. and B. M. performed the biological experiments. B. W. and A. K. K. analyzed the data and supported planning and interpretation of the experiments. H. T., A. K. K., and H. S. contributed reagents, materials, and analysis tools. B. W., H. T., A. K. K., and H. S. conceived and supervised the study. All the authors approved the final manuscript and declare no conflict of interest associated with this work.

Supporting information

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Supplementary

N. S. Engels, B. Waltenberger, B. Michalak, L. Huynh, H. Tran, A. K. Kiss, H. Stuppner, C&B 2018, 15, e1800269.